INVESTIGATION OF HEPATOPROTECTIVE AND

ANTIOXIDANT ACTIVITIES OF INDIAN MEDICINAL

PLANTS

Final Report Submitted to University Grants Commission in response to completion of Major

Research Project

By

Dr. S. Raja

INSTITUTE OF PHARMACY

GITAM UNIVERSITY

VISAKHAPATNAM

MARCH, 2017

Annexure –VIII

UNIVERSITY GRANTS COMMISSION

BAHADUR SHAH ZAFAR MARG

NEW DELHI – 110 002

Annual/Final Report of the work done on the Major Research Project.

(Report to be submitted within 6 weeks after completion of each year)

1. Project report No. 1st /2nd /3rd/Final : Final

2. UGC Reference No. F. : F. No. 42-691/2013 (SR) dated 25.03.2013

3. Period of report : From 1st April 2013 to 31st March 2017

4. Title of research project :

Investigation of hepatoprotective and

antioxidant activities of Indian medicinal plants

5. (a) Name of the Principal Investigator : Dr. S. Raja

(b) Department : Pharmacy

(c) University/College where work has

progressed : GITAM University

6. Effective date of starting of the project : 01.04.2013

7. Grant approved and expenditure incurred during the period of the report:

a. Total amount approved : ₹ 10,21,348/-

b. Total expenditure : ₹ 10,21,348/-

c. Report of the work done : Complete Project Report submitted in bound

(Please attach a separate sheet) book format

I. Brief objective of the project: Enclosure-I

II. Work done so far and results achieved and publications, if any, resulting from the work

(Give details of the papers and names of the journals in which it has been published or

accepted for publication. Enclosure-II

III. Has the progress been according to original plan of work and towards achieving the

objective. If not, state reasons. YES

IV. Please indicate the difficulties, if any, experienced in implementing the project. NO

V. If project has not been completed; please indicate the approximate time by which it is

likely to be completed. A summary of the work done for the period (Annual basis) may

please be sent to the Commission on a separate sheet. COMPLETED

VI. If the project has been completed, please enclose a summary of the findings of the

study. One bound copy of the final report of work done may also be sent to University

Grants Commission. ENCLOSED A COPY OF BOUND BOOK

VII. Any other information that would help in evaluation of work done on the project. At

the completion of the project, the first report should indicate the output, such as:

a) Manpower trained : - 1

b) Ph. D. awarded : - Ph.D. Registered, about to submit

c) Publication of results : - 10

d) Other impact, if any : - Nil

PRINCIPAL INVESTIGATOR REGISTRAR

CHAPTER CONTENTS Pg. No

Chapter I Introduction

1-22

Chapter II

Literature review of selected Indian medicinal plants- Couroupita guianensis, Limnophila

heterophylla and Michelia champaca

23-41

Chapter III Plants description 42-49

Chapter IV

Standardization of Couroupita guianensis, Limnophila heterophylla and Michelia

champaca

50-56

Chapter V

Extraction, Phytochemical analysis and TLC studies of Couroupita guianensis, Limnophila

heterophylla and Michelia champaca

57-71

Chapter VI

Isolation and Characterization of phytoconstituents from Couroupita guianensis,

Limnophila heterophylla and Michelia

champaca

72-91

Chapter VII Toxicity study of Couroupita guianensis, Limnophila heterophylla and Michelia champaca

92-95

Chapter VIII

Invitro hepatoprotective activity of Couroupita guianensis, Limnophila heterophylla and

Michelia champaca

96-101

Chapter IX Invivo hepatoprotective activity of Couroupita guianensis, Limnophila heterophylla and

Michelia champaca

102-113

References 114-133

Enclosure-I

The main objective of the proposed work as follows

Selection of Indian medicinal plants based on ethnomedical information.

Authentication of selected medicinal plants

Extraction of plant material by successive solvents techniques.

Qualitative phytochemical analysis of extracts.

Isolation of phytoconstituents from different extracts by chromatography techniques.

Characterization of isolated compounds by UV, IR, NMR and Mass spectrum

analysis.

Toxicity study of extracts and active isolated compounds by OECD guidelines.

Invitro and invivo hepatoprotective activities by standard methods.

Analysis of different biochemical parameters from blood and liver tissues.

Chapter-I

Introduction

1

CHAPTER-I

INTRODUCTION

1.1. INTRODUCTION TO LIVER

The liver is among the most complex and important organs in the human body, weighs

1200– 1500 gm and comprises one-fiftieth of the total adult body weight. It is relatively

larger in infancy, comprising 1/18th of the birth weight. The liver is occupying a large

region mostly on the right side of the body, below the diaphragm and behind the ribs

(Sherlock and Dooley, 2002). It has a pivotal role in regulation of physiological

processes and it involved in several vital functions such as metabolism, secretion and

storage. Furthermore, detoxification of a variety of drugs and xenobiotics occurs in liver.

The bile secreted by the liver plays an important role in digestion (Guyton and Hall,

2011).

1.2. FUNCTIONS OF LIVER

The main function of the liver is synthesis of proteins viz., albumin, coagulation factors,

α-antitrypsin, very low density lipoprotein and many others that circulate in the blood.

Stores glucose as glycogen and converts it back to glucose as needed. The liver can also

synthesize glucose from amino acids, lactate and glycerol, although this is less efficient

than breaking down glycogen into glucose (Young et al., 2006). Additionally, the liver

metabolizes fatty acids, cholesterol and amino acids. The liver can convert excess

amount of glucose and amino acids into fatty acids for storage. The liver synthesizes

cholesterol and removes it from circulation. The liver can synthesize non-essential

amino acids. Toxins are detoxified by the liver’s ability to metabolize lipophillic

compounds. These compounds (bound to albumin) enter the liver sinusoids and then the

area of disease. Enzymes in the hepatocytes (CYP-450 enzymes) are involved in the

metabolism of the lipophillic compounds, which include toxins and many drugs.

The liver cells can produce bile juice that can act as a detergent and breaks fats down

into smaller components so they can be digested in the small intestine (Young et al.,

2006).

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1.3. LIVER ENZYMES

1.3.1. SGOT and SGPT

The aminotransferases (formerly transaminases) are the most frequently utilized and

specific indicators of hepatocellular necrosis. These enzymes, aspartate

aminotransferase (AST, formerly serum glutamate oxaloacetic transaminase-SGOT) &

alanine amino transferas (ALT, formerly serum glutamic pyruvate transaminase-SGPT)

(Rosalki and Mcintyre, 1999). Both these enzymes are associated with liver

parenchymal cells. The ratio of AST to ALT is mostly useful in differentiating between

causes of liver damage (Nyblom et al., 2004). ALT is primarily localized to the liver but

the AST is present in a wide variety of tissues like the heart, skeletal muscle, kidney,

brain and liver (Friedman et al., 2003).

1.3.2. Alkaline Phosphatase

Alkaline phosphatase (ALP) is an enzyme in the cells lining the biliary ducts of the liver.

ALP levels in plasma rise with large bile duct obstruction, intrahepatic cholestasis, or

infiltrative diseases of the liver. ALP is also present in bone and placental tissue, so it is

higher in growing children and elderly patients with Paget's disease. ALP is associated

with the plasma membrane of hepatocytes adjacent to the biliary canaliculus.

Obstruction or inflammation of the biliary tract results in an increased concentration of

the ALP in the circulation (Kaplan, 1972).

1.3.3. Bilirubin

Bilirubin (previously referred to as haematoidin and discovered by Rudolf Virchow in

1847) is a yellow compound that occurs in the normal catabolic pathway that breaks

down heme in vertebrates (Lightner, 2013). The production of biliverdin from heme is

the first major step in the catabolic pathway, after which the enzyme biliverdin reductase

performs the second step, producing bilirubin from biliverdin (Stocker, 1987). Bilirubin

is excreted in bile and urine, and elevated levels may indicate certain liver diseases. It

is responsible for the yellow color of bruises and the yellow discoloration in jaundice.

The liver is responsible for clearing the blood of unconjugated bilirubin and about 30%

of it is taken up by a normal liver on each pass of the blood through the liver.

3

1.3.4. Superoxide Dismutase

Superoxide dismutase (SOD) was first isolated in 1938 and thought to be a copper

storage protein. Subsequently, the enzyme was identified by a number of names,

erythrocuprein, indophenol oxidase, and tetrazolium oxidase until its catalytic function

was discovered (McCord and Fridovich, 1969). SOD catalyses the dismutation of two

superoxide radicals into hydrogen peroxide and oxygen. The hydrogen peroxide is

further oxidised by other enzymes. These enzymes obey first order reaction kinetics and

the forward rate constants are almost diffusion limited. These results suggest that

concentration of superoxide in tissue that varies directly with rate of superoxide

generation and inversely with the tissue concentration of scavenging enzymes (Enghild

et al., 1999). SOD plays an important role in the elimination of ROS and protects cells

against the deleterious effects of super oxide anion derived from the peroxidative

process in liver and kidney tissues. SOD is biologically necessary because superoxide

reacts even faster with certain targets such as NO radical, which makes peroxynitrite.

Superoxide is one of the main reactive species in the cell and such, SOD serves a key

antioxidant role.

1.3.5. Catalase

Catalase was first noticed by Louis Jacques Thenard in 1811. In 1900 Oscar Loew was

the first to give it the name catalase. Catalase is present predominantly in the

peroxisomes of liver and kidney and also in the micro peroxisomes of other tissues.

Catalase (CAT) serves several biochemical functions, but the principal purpose of CAT

is to catalyze the breakdown of H2O2 into H2O and O2. Catalase was one of the first

enzymes to be isolated in a highly purified state. Careful examination of the structure of

beef liver catalase has shown to possess four NADPH binding sites per catalase

tetramer, but these sites were not in close association with the hydrogen peroxide

reaction centre. Instead, NADPH functions in animal catalase to protect against

inactivation by hydrogen peroxide (Kirkman, 1987). Iron is required cofactor attached

to the active site of the enzyme (Kirkman and Gaetani, 2007). Although CAT and GPx

share common substrates, CAT has a much lower affinity for H2O2 at low concentrations

compared with GPx (Sies, 1985). H2O2 is a powerful oxidizing agent and is potentially

damaging to cells. By preventing excessive H2O2 build up, catalase allows important

cellular process which produce H2O as a byproduct to take place safely.

4

1.3.6. Glutathione Peroxidase

Analysis of the selenoproteome has identified five glutathione peroxidases in mammals

(Brigelius-Flohe, 2006; Drevet, 2006). All of these GPx enzymes catalyze the reduction

of H2O2 or organic hydroperoxide (ROOH) to water (H2O) and alcohol (R-OH),

respectively, using reduced glutathione GSH) or in some eases thioredoxin or

glutaredoxin as the electron donor (Bjomstedt et al., 1994; Bjomstedt et al., 1997;

Holmgren et al., 2005). Although the reaction catalyzed by all GPxs appears to be the

same, individual GPx’s differ in substrate specificity and cellular localization

(Brigelius-Flohe, 2006). Many GPx isoenzymes will diminish a wide range of

hydroperoxides extending from H2O2 to complex organic hydroperoxides (ROOH)

makes GPx an important intracellular antioxidant to protect against ROS mediated

damage to membrane lipids, proteins, and nucleic acids (Ji and La, 2000). To function,

most GPx isoforms require a supply of GSH to provide electrons. Since GSH is oxidized

by GPx to form GSSG, cells must hold a pathway capable of regenerating GSH. The

reduction of GSSG back to GSH is accomplished by glutathione reductase (GR), a flavin

containing enzyme whereby NADPH provides the reducing power (Meister and

Anderson, 1983).

1.3.7. Glutathione Reductase

Glutathione reductase (GR) is not directly involved in removing ROS, it serves an

important role in converting GSSG to GSH, thereby maintaining GSH-Px catalytic

function and a reduced intracellular redox status. Glutathione reductase is an ancillary

enzyme to limit the amounts of ROS via its reduction of GSSG in the presence of an

adequate supply of NADPH thus, the ration of GSH/GSSG is maintained at high level

so that the cell maintains the capacity to combat oxidative stress (Halliwell and

Gutteridge, 1989).

1.3.8. Glutathione-S-Transferase

The mammalian GST super-family comprises cytosolic dimeric isoenzymes of 45-55 k

Da size which have been assigned to at least four generic classes: α, µ, π and Ø. GST

family of enzymes comprises a long list of cytosolic, mitochondrial and microsomal

proteins which are capable of multiple reactions with a multitude of substrates, both

endogenous and xenobiotic. These enzymes catalyze the conjugation of a molecule of

GSH to an electrophilic or other reactive species (Jakoby and Habig, 1980; Kodavanti,

5

1999). This activity is useful in the detoxification of endogenous compounds such as

peroxidised lipids as well as the metabolism of xenobiotics. GST’s may also bind toxins

and function as transport proteins, because of this, an early term for GSTs was ligandin

(Litwack et al., 1971). GST catalyzes the conjugation GSH with a wide variety of

organic compounds, including certain species of hydroperoxides thereby shares

peroxidase activity with GSH- Px (Habig et al., 1974). Unlike GSH-Px, GST activity is

not affected by selenium deficiency; however, GSH concentration is critical for the

enzyme’s catalytic function (Ji and Leeuwenburgh, 1996).

1.4. LIVER DESTRUCTION

Liver diseases are among the most serious ailments. They may be classified as acute or

chronic hepatitis (inflammatory liver diseases), hepatitis (non-inflammatory diseases)

and fibrosis. Liver diseases are mainly caused by toxic chemicals [certain antibiotics,

chemotherapeutics, peroxidised oil, aflatoxin, carbon tetrachloride, chlorinated

hydrocarbons, etc.], excess consumption of alcohol, infections and autoimmune

disorders. Most of the hepatotoxic chemicals damage the liver cells mainly by inducing

lipid peroxidation and other oxidative damages in the liver (Brattin et al., 1985).

Enhanced lipid peroxidation produced during the liver microsomal metabolism of

ethanol may result in hepatitis and cirrhosis (Smuckler, 1975). Liver damage refers to

any disorder of the liver and includes the steatosis or fatty deposits in the liver, fibrosis

or scarring of the liver, hepatitis or inflammation of the liver, cirrhosis where scarring

and inflammation spread through the liver and irreversibly disrupt its shape or function

causing permanent cell damage and ultimately liver failure and leading to liver cancer.

The majority of liver deaths are due to cirrhosis and it has recently been reported that,

there are about 4000-5000 deaths from cirrhosis in the UK each year (Iredale, 2003;

Ryder, 2006). The other main cause of death from liver disease is due to liver cancer. It

should be noted that, cirrhosis is a specific precursor of liver cancer (Perz et al., 2006).

Liver cancer can either arise in the hepatobiliary system itself (primary liver cancer) or

metastasize from a tumor elsewhere in the body (secondary liver cancer). Most liver

cancer (95%) is a secondary cancer (Williams et al., 2007). Liver cancer is more

common in men compared to women and it predominately occurs in older people (Khan

et al., 2005). Moreover, primary liver cancer consists of either hepatocellular carcinoma

(HCC) which arises in liver cells (hepatocytes) or cholangiocarcinoma (CCA) which

arises in the bile ducts either within (intrahepatic) or outside of the liver (extra-hepatic).

6

HCC is responsible for the majority (70-85%) of primary liver cancer worldwide (Perz

et al., 2006). However, recent rises in primary liver cancer have been attributed to a

rapid rate of increase in CCA (Khan et al., 2005). CCA is the commonest type of primary

liver cancer in women and HCC increases have only the commonest type of primary

liver cancer in men (West et al., 2006). According to Kaner et al. (2007) a large number

of people die from liver disease each year, therefore drugs with novel mode of action

required.

1.5. LIVER DISEASES

1.5.1. Cirrhosis

Cirrhosis is a condition in which, normal liver parenchyma cells replaced by largely

spread fibrosis with regenerative nodules. Alcohol and viral hepatitis are the major

causes of cirrhosis (Li et al., 1999).

1.5.2. Congenital Metabolic Disorder

Deficiency of α–1–antitrypsin may result in liver injury and led to cirrhosis. The best

known congenital jaundice syndromes are Gsilbert syndrome, Rotor syndrome, and

Dubin – Johnson syndrome.

1.5.3. Acquired Metabolic disorder

Certain drugs, alcohol, food and beverages are the main causative agents for this

disorder. Liver diseases induced by alcohol include cirrhosis, hepatomegaly and

hepatitis.

1.5.4. Acute Hepatitis

Death of hepatocytes leads to the development of necrosis at certain areas of liver and

the eventual outcome depends on the size and number of these areas. The main causes

include viral infections, toxic substances and circulatory disturbances.

1.5.5. Viral Hepatitis

Viral infections are the most common cause of acute liver injury, which include Type

A, Type B and Type C. These types are distinguished on the basis of antibodies

production to combat the infection. Type A virus (infectious hepatitis) occurs

endemically, causing mild illness mainly in children (Levo et al., 1977). Infection is

7

spread by hands, water, food, and infected faeces. Type B virus (serological hepatitis)

occurs at any age, but mostly affects the adults (Seto and Gerety, 1985). Virus spread

through blood and blood products and may result in massive liver necrosis and death.

Type C (hepatitis C) spread by blood and blood products. When hepatitis develops, it is

often recurrant and may result in chronic liver diseases like cirrhosis (Jemal et al., 2006).

1.5.6. Neoplasia

Liver tumors may arise both from liver cells or bile ductules. At times kupffer cells,

hepatic capsule and portal tracts are also common sites for the tumors. The most

common primary malignant liver tumor is hepatocellular carcinoma (malignant

hepatoma).

1.6. OXIDATIVE STRESS

Oxidative stress is the condition that occurs when the steady-state balance of

prooxidants to antioxidants is shifted in the direction of the former, creating the potential

for organic damage. Pro-oxidants are by definition free radicals, atoms or clusters of

atoms with a single unpaired electron. Physiologic concentrations of pro-oxidants are

determined both by internal and external factors. Pro-oxidant reactive oxygen species

(ROS), for example, are normal products of aerobic metabolism. However, under

pathological conditions ROS production can increase, surpassing the body’s

detoxification capacity and thus contribute to molecular-level organic pathology.

Oxidative stress contributes too many pathological conditions and diseases, including

cancer, neurological disorders, atherosclerosis, hypertension, ischemia/perfusion,

diabetes acute respiratory distress syndrome, idiopathic pulmonary fibrosis, chronic

obstructive pulmonary disease, and asthma (Birben et al., 2012). Most cells can tolerate

a mild degree of oxidative stress, because they have sufficient antioxidant defense

capacity and repair systems, which recognize and remove molecules damaged by

oxidation. The imbalance can result from a lack of antioxidant capacity caused by

disturbances in production and distribution or by an overabundance of reactive oxygen

species (ROS) from other factors (Ha et al., 2010).

There are increasing evidences that free radicals and reactive oxygen species

play a crucial role in the various steps that initiate and regulate the progression of liver

diseases independently of the agent in its origin. Oxidative stress is also involved in

liver damage induced by alcohol abuse, viral infection, alteration of lipid and

8

carbohydrate metabolism and xenobiotics (Loguercio and Federico, 2003; Vitaglione et

al., 2004). Besides the formation of ROS and RNS, other reactive species can also be

formed by the metabolism of drugs and toxins. Carbon tetrachloride (CCU), for

example, is metabolised forming high reactive free radicals such as CCl3 and CCl3OO*

(Weber et al., 2003). Carbon tetrachloride (CCI4), a hepatotoxin, has been used

extensively for decades to induce liver injury in various experimental models to

elucidate the mechanisms behind hepatotoxicity (Hardin, 1954). Experimentally

induced cirrhotic response in the rat by CCl4 is shown to be superficially similar to

human cirrhosis of the liver (Tamayo, 1983).

1.7. ROLE OF ANTIOXIDANTS

Antioxidants are our first line of defense against free radical damage, and are critical for

maintaining optimum health and wellbeing. Free radicals are electrically charged

molecules, i.e., they have an unpaired electron, which causes them to seek out and

capture electrons from other substances in order to neutralize themselves. Although the

initial attack causes the free radical to become neutralized, another free radical is formed

in the process, causing a chain reaction to occur. And until subsequent free radicals are

deactivated thousands of free radical reactions can occur within seconds of the initial

reaction. Antioxidants are capable of stabilizing, or deactivating, free radicals before

they attack cells. An antioxidant is capable of slowing or preventing the oxidation of

other molecules. In a biological system they may protect cells from damage caused by

unstable molecules known as free radicals. Antioxidants terminate these chain reactions

by removing free radical intermediates and inhibit other oxidation reactions by being

oxidized themselves (Prithviraj et al., 2009).

1.8. SYNTHETIC DRUGS USED FOR LIVER PROTECTION

Liver diseases constitute a major medical problem of worldwide proportions. In Africa

and Asia, their main causes are viral and parasitic infections, while in Europe and

America alcohol abuse is the major cause of liver diseases, although viral hepatitis has

increased recently. There is now general agreement among hematologists that the

number of useful liver drugs currently available is far from sufficient, and that there is

a need for a range of safe and efficient therapeutic agents. In contrast, may be dozens of

compounds (most of them of herbal origin) have been studied to protect or cure the liver

from several kinds of injuries; however, most of these compounds are not well

9

characterized or need to be better studied in animal models and/or clinical trials; others

have recently been discovered, and some others possess a real potential benefit in human

liver diseases. Treatment for liver damage depends with synthetic drugs depends on the

cause and extent of damage. The goals of treatment are to slow the progression of scar

tissue in the liver and to prevent or treat symptoms and complications of cirrhosis. The

following synthetic drugs are generally used for liver diseases.

Aldactone: Aldactone removes excess fluid from the body in congestive heart failure,

cirrhosis of the liver, and kidney disease.

Boceprevir: Boceprevir is a man-made antiviral medication that targets hepatitis C virus

(HCV). Similar drugs include simeprevir (Olysio) and telaprevir (Incivek). They block

the replication of hepatitis C virus in human cells by binding to and inhibiting protease

enzymes that HCV use for reproducing. Inhibiting viral replication reduces HCV viral

load in the body to undetectable levels in some patients.

Cholestyramine: Cholestyramine is used for reducing cholesterol levels in the blood,

to relieve the itching of liver and biliary disease, and to treat overdoses of digoxin

(Lanoxin), or thyroid hormone. Cholestyramine also is recommended for the rapid

elimination of leflunomide (Arava).

Colestipol: It is used in the treatment of diarrhea due to increased intestinal bile acids

after some types of intestinal surgery and treatment of itching associated with partial

obstruction to the flow of bile due to liver disease.

Epclusa: Epclusa is used to treat chronic hepatitis C virus genotype 1, 2, 3, 4, 5 or 6

infection in adults without cirrhosis or with compensated cirrhosis. It also may be

combined with ribavirin (Rebetol) for treatment of adults with decompensated cirrhosis.

L-Ornithine L-Aspartate: L-Ornithine L-Aspartate is a stable salt of the amino acids

ornithine and aspartic acid, prescribed for the treatment of high ammonia levels or

severe liver impairment. It is also used for end-stage cirrhosis.

10

Ondansetron: Ondansetron is a 5-HT3 receptor antagonist, prescribed for nausea and

vomiting caused by cancer chemotherapy, radiation therapy and surgery. It blocks

serotonin receptors in the vomiting center and on nerves supplying the digestive system.

Harvoni: Harvoni contains ledipasvir and sofosbuvir. Sofosbuvir is converted to an

active form in the body before it is effective. The active form of sofosbuvir directly

blocks replication of the HVC by interfering with a hepatitis C virus enzyme called

NS5B. Ledipasvir is an inhibitor of another hepatitis C virus enzyme called NS5A,

which also is needed for viral replication.

Lactulose: Lactulose is used to treat hepatic encephalopathy, a loss of brain function

and change in mentation that occurs when the liver is unable to remove toxins from the

blood. Bacteria in the colon digest lactulose into chemicals that bind ammonia that is

believed to be the toxin that causes hepatic encephalopathy. The binding of ammonia

prevents ammonia from moving from the colon into the blood and also draws ammonia

from the blood and into the colon. The bound ammonia then is removed from the body

in the stool.

Viekira Pak: It contains four medicines, dasabuvir, ombitasvir, paritaprevir, and

ritonavir. They block the effect of proteases which are enzymes that HCV uses for

making new virus, leading to reduced numbers of HCV copies in the body.

Technivie: Technivie is a combination oral medication containing ombitasvir,

paritaprevir, and ritonavir. They block the effect of proteases which are enzymes that

HCV uses for making new virus, leading to reduced numbers of HCV copies in the

body.

Telaprevir: Telaprevir is a man-made antiviral medication that targets hepatitis C virus

(HCV). It blocks the replication of hepatitis C virus in human cells by binding to and

inhibiting protease enzymes that HCV use for reproducing.

For people with severe alcoholic hepatitis, treatment in hospital may be necessary.

Specific treatment with corticosteroids or pentoxifylline medication may be used to

reduce inflammation of the liver in some people with this condition. Other medications

11

that have been used to treat liver damage include: anabolic steroids and ropylthiouracil

(medicine originally designed to treat overactive thyroid glands).

1.9. HERBAL THERAPY FOR LIVER DISEASES

Due to usage of chemical based medicinal preparation in allopathic results in toxic

effects in increased dose or on long term usage and also has many side effects. However

herbal forms of medication are more advantageous comparison and are have less no of

side effect. Natural herbal remedies are constituted primarily from plant extracts. These

plant extracts and natural compounds contain the essential nutrients needed to respond

to your body’s natural recovery process and improve your overall health (Yadav et al.,

2011). Herbal remedies are less expensive than those remedies manufactured with

synthetic compounds and chemicals. There’s a really simple reason why natural herbal

remedies cost less than manufactured drugs. It’s because natural ingredients and plant

extracts are used to make herbal remedies while expensive chemicals are used to create

manufactured medicine in a lab. Because alternative herbal remedies are 100% natural,

you don’t have to be concerned about allergic reactions or counter-indications with any

foods that you consume (Morton and Malone, 1972). However, there are no guarantees

that each person taking herbal medicine will reap the same benefits or effects as another

person as people are different and the results vary. It’s always a good idea to inform

your doctor of any herbal remedies that you are taking so that if necessary, he can order

any tests that may be needed to check for compatibility or contraindications (Kamath et

al., 2003).

1.10. MEDICINAL PLANTS USED FOR THE LIVER DISEASES

For the prevention and treatment of ailments, seasoning plants extracts are

extraordinarily a prosperous supply everywhere the planet as and eightieth of the planet

population majorly within the developing countries for primary health care they're

exploitation seasoning drugs solely. As a result of eco-friendly nature of seasoning drug

merchandise from precedent days for a few age-related diseases particularly cognitive

state, pathology, diabetic wounds, immune and liver disorders, they used seasoning

drugs solely. From many years, before the event fashionable drugs ancient drugs

(including seasoning drugs) as therapeutic practices that are breathing and are still in

use nowadays additionally. The pity nature has bestowed some seasoning plants that

have helpful result on liver disorders with fewer facet effects. These hepatoprotective

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plants (Table 1.1) (agents) shield liver from injury or facilitate in regeneration of hepatic

cells. Healthful herbs are important supply of hepatoprotective medicine. There's

tremendous analysis goes on currently on a daily basis for management of liver disorders

in a very precise manner on seasoning medicines that are claimed to possess

hepatoprotective activity. The incredible complexness of liver chemistry and its

essential role in biotransformation of medicine is therefore discouraging to researchers

that they visualize that maybe easy seasoning plants remedies can be helpful in treatment

for hepatotoxicity.

1.11. PHYTOCONSTITUENTS USED FOR LIVER DISEASES

Phytoconstituents isolated from different plants belongs to fifty-five families do possess

hepatoprotective activity (Handa, 1991) (Table 1.2). Liver protecting seasoning

medicine contain a selection of chemical constituents like alkaloid (Wang, 2008),

flavonoids (Souza, 1997), glycoside (Bhandari et al., 2008), lignan (Upadhyay et al.,

2007), organ sulphur compounds (Sabayan et al., 2007), phenolic acids (Liu et al.,

2013), phenyl ethanoid organic compound (Jin et al., 2004), polyphenols (Fonseca et

al., 2013), polyprenols (Yang et al., 2011), resins (Arteaga et al., 2005) and triterpenoid

(Kumari et al., 2012).

Organosulphar compounds (Allium sativum): The fresh bulbs of Allium sativum

comprises of volatile oils, alliin and allicin. In addition to that, garlic comprises of wide

variety of organosulphar compounds like; thioethers, thioesters, thioacetals, sulfones

and thiosulfinates, sulfimides, sulfoximides, and sulfonediimines. These compounds

have an optimistic role in prevention of valproicacid induced hepatotoxicity (Sabayan

et al., 2007).

Acteoside (Buddleia officinalis): Buddleia consists of numerous phytoconstituents

namely; flavonoids, terpenoids, iridoids and phenylethanoids. Acteoside a

phenylethanoid glycoside is an important constituent plays a vital role in carbon

tetrachloride induced hepatotoxicity (Lee et al., 2006)..

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Table 1.1: List of Hepatoproytective Plants Used for Liver Diseases

Name of the plant Family Mechanism References Allium sativum Lilliaceae Prevention and Alteration of GSH dependent enzymes Sabayan et al., 2007

Buddleia officinalis Loganiaceae Decreased levels of AST,ALP Lee et al., 2006

Camellia sinensis Theaceae Inhibited hepatocellular apoptosis and up-regulated BCl-2

protein expression. Sharangi, 2009

Cistus laurifolius L. Cistaceae MDA, AST, GSH levels decreased. Esra et al., 2006

Corydalis saxicola Fumariaceae Decreased levels MDA, SOD, GPx Li et al., 2006

Curcuma longa Zingerberaceae Decreased levels SGOT, SGPT, ALP Akram et al., 2010

Egletes viscosa L. Asteraceae Decrease lipid peroxidation Souza, 1997

Gardenia jasminoides Rubiaceae Antioxidant Tseng et al., 1995

Ginkgo biloba L Ginkgoaceae ALT, AST, ALP, ALB, TP, TG and CHO levels decreased Yang et al., 2011

Gossypium herbaceum Malvaecae Antioxidant Randel et al., 1992

Hibiscus sabdariffa L. Malvaceae LDH, AST, ALP, MDA levels Decreased Seng et al., 1998

Larrea tridentate Zygophyllaceae Antioxidant Arteaga et al., 2005

Magnolia officinalis Magnoliaceae Antioxidant Lo et al., 1994

Mangifera indica Anacardiaceae Decreased levels SGOT, SGPT, ALP, TB Scartezzini & Speroni, 2000

Nigella sativa Ranunculaceae scavenger of free radicals Houghton et al., 1995

Phyllanthus amarus Euphorbiaceae SGOT, SGPT, ALKP, SBLN & total protein levels decreased. Pramyothin et al., 2007

Picrorhiza kurroa Scrophulariaceae Antioxidant Bhandari et al, 2008

Pinus maritama Pinaceae SOD, GSH-Px, GSH-reductase and TBARS levels decreased Rohdewald, 2002

Rubia cordifolia Rubiaceae Decreased levels SGOT, SGPT, SALP and gamma-GT Tripathi and Sharma, 1998

Silybum marianum Asteraceae Antioxidant Upadhyay et al., 2007

14

Poly Phenols (Camellia sinensis): Tea leaves consists of different active constituents

like polyphenols (catechins and flavonoides), alkaloids (caffeine, theobromine,

theophylline), amino acids, volatile oils, polysaccharides, lipids, vitamin C (Sharangi,

2009). Green tea comprises of catechin and its related compounds (epicatechin,

gallocatechin, epigallocatechin, epigallocatechin gallate and epicatechin gallate) and

flavonoids. The protective effect against hepatotoxicity was due to presence of various

poly phenols.

Tetrahydro curcuminoid (Curcuma longa): Various active constituents present in

turmeric are curcumin (diferuloylmethane), volatile oils, tumerone, atlantone,

zingiberone, sugars, proteins, and resins. Curcumin, which comprises 0.3-5.4% of raw

turmeric, is the principal curcuminoid widely used in India for various pharmacological

activities. Tetrahydro curcuminoid is metabolite obtained from the curcumin play a

protective role in Acetaminophen induced hepatotoxicity.

Dehydrocavidine (Corydalis saxicola): This plant consists of alkaloids in higher

proportion. Dehydrocavidine is one among the organic compound plays a key role in

hepatotoxicity/hepatoprotective.

Ternatin (Egletes viscosa): Ternatin is an energetic ingredient obtained from this plant

shows variety of pharmacological activities such as anti-inflammatory,

antianaphylactic, antithrombotic and antihepatotoxic (Souza, 1997).

Geniposide (Gardenia jasminoides): The Gardenia nuts contain carbohydrates,

minerals, fats, vitamins, picrocrocin and volatile oils. Inhibitory effect on the

hepatotoxicity and hepatic DNA binding of aflatoxin B1 in rats was mainly due to

presence of geniposide.

Ginkgolide (Ginkgo biloba): Different active constituents present in Ginko biloba such

as flavonoids, glycosides, diterpenes, bioflavones, quercitin, isorhamnetine,

kaempferol, proanthocyanidins, sitosterols, lactones and anthocyanin. Ginkgolide the

active component of Ginkgo biloba somehow inhibits a chemical found in the body

called platelet activating factor (PAF) which plays a role in organ rejection. Laterally

15

Ginkgo biloba poly phenols have antivirus, antitumor, antioxidant and hepatoprotective

effects.

Protocatechuic acid (Hibiscus sabdariffa): Protocatechuic acid is a phenolic

compound obtained from the roots of Hibiscus sabdariffa how strong anti-oxidant and

anti-tumour activities (Seng et al., 1998). Protocatechilic acid is an important lead

molecule shows protective effect in hepatotoxicity.

Nordihydroguaiaretic acid (Larrea tridentate): This plant consists of wide variety of

resins, monoterpenoids, aromatic sesquiterpenoids, glycosylated flavonoids,

sapogenins, essential oils and halogenic alkaloids (Mabry and Bohnstedt, 1981). Even

though it contains so many compounds, a resin called Nordihydroguaiaretic acid is

present in excess amount (50%), shows promising effect against hepatotoxicity

(Arteaga et al., 2005).

Magnolol (Magnolia officinalis): Magnolol (5, 5’-diallyl-2, 2’ dihydroxybipheny) and

Honokiol (5, 3’-diallyl-2, 4’-dihydroxybiphenyl) are two major active constituents

obtained from stem bark of Magnolia officinalis. Chemical constituents present in this

plant are isoquinolines, lignans, neolignans, alkaloids, mono and sesquiterpenes. The

magnolol showed anti-hepatotoxic activity on N-acetyl-p-aminophenol/APAP-induced

hepatic toxicity (Lo et al., 1994).

Lupeol (Mangifera indica): Different types of active constituent’s present mango bark

are mangiferin, protocatechicacid, catechin, alanine, glycine, γ- kinic acid,

shikimicacid, aminobutyricacid and tetracyclic triterpenoids (Scartezzini and Speroni,

2000). Mango peel and pulp contains carotenoids, polyphenols and omega-3 and 6

polyunsaturated fatty acids, triterpinoids and lupeol (Saeed and Sabir, 2002). Lupeol

shows anti-hepatotoxic activity (Bhandari et al., 2008).

Thymoquinone (Nigella sativa): Thymoquinone is a major active constituent of Nigella

sativa and shows strong anti-oxidant properties. Along with these alkaloids, steroids,

phenolic compounds are also present in Nigella sativa. Protective effect of

thymoquinone on sodium fluoride-induced hepatotoxicity and oxidative stress

(Houghton et al., 1995).

16

Kutkin (Picrorhiza kurroa): Hepatoprotective effect of this plant is mainly due to

presence of kutkin. Kutkin is a mixture of both picroside-I and picroside-II (Bhandari

et al, 2008).

Phyllanthin (Phyllanthus amarus): It consists of number of dynamic constituents such

as lignans, glycosides, flavonoids, alkaloids and phenylpropanoids. Phyllanthus amarus

and its related Indian plants show excellent antiviral properties due to presence of

phyllanthin, hypophyllanthin, and polyphenols. Phyllanthus amarus shows protective

effect against CCl4 induced hepatotoxicity due to presence of above mentioned

chemical constituents (Pramyothin et al., 2007).

Pycnogenol (Pinus maritima): It is a French maritime pine, native to the western and

south-western Mediterranean area and it contains a standardized extract called

pycnogenol. Catechin, epicatechin, and taxifolin and polyphenol are the active

principles present in pycnogenol shows protective effects against liver toxicity

(Rohdewald, 2002).

Rubaidin (Rubia cordifolia): It consists of energetic elements like phenolics,

triterpenoids, anthraquinones and cyclopeptides. Rubaidin showed defending effect on

CCl4 encouraged hepatotoxicity (Tripathi et al., 1998).

Silymarin (Silybum marianum): It consists of assorted phytoconstituents such as

apigenin, betainehydrochloride, dehydrosilybin, deoxysilycristin, deoxysildianin,

neosilyhermin, silymarin, silidianin, silychristin, siliandrin, silybinome, silyhermin and

stearic acids. Historically milk weed is employed to protect the liver, secretion of

digestive juice and shelter against oxidative injuries like radiation. So far it is widely

used as hepatoprotective agents because of its antioxidant property (Upadhyay et al.,

2007).

However, as a result of lack of refined outfit’s small portion of hepatoprotective plants

solitary utilized in ancient drugs are pharmacologically evaluated for its effectiveness.

From ancient time, varied plants and plant derived compounds are utilized in the

treatment of hepatotoxicity by reducing levels of assorted SGOT, SGPT, LDH, AST,

17

ALT and bilirubin. Asian nation features a made history of exploitation varied effective

herbs and seasoning parts for treating hepatotoxicity. Several ethnomedicine

information is available for the use of Couroupita guianensis, Limnophila heterophylla

and Michelia champaca for the variety of diseases. Even in the folklore medicine both

the selected plant species are being used for several ailments. To substantiate these

claim and thereby to validate the therapeutic uses and the phytoconstituents present

there in the project work was undertaken.

The main objective of this work was to explore the bioactive principle present in plant

species, so also to evaluate their hepatoprotective and antioxidant activities. The work

has been represented in this thesis with the following broad heading in different

chapters.

Selection of Indian medicinal plants based on ethnomedical information

Literature review of selected plants

Plants description

Standardization of selected Indian medicinal plants

Extraction, phytochemical analysis and Thin Layer Chromatography (TLC)

studies of plants extracts

Isolation and Characterization (UV, IR, NMR & Mass) of phytoconstituents

Toxicity study of extracts by OECD guidelines.

Invitro hepatoprotective activity

Invivo hepatoprotective and invivo antioxidant activities by standard

methods.

18

Table 1.2: Phytoconstituents Proved for Hepatoprotective Activity

Phytoconstituents

constituent IUPAC Name Structure

Acteoside

[(2R,3R,4R,5R,6R)-6-[2-(3,4-dihydroxyphenyl)ethoxy]-5-

hydroxy-2-(hydroxymethyl)-4-[(2S,3R,4R,5R,6S)-3,4,5-

trihydroxy-6-methyloxan-2-yl]oxyoxan-3-yl](E)-3-(3,4-

dihydroxyphenyl)prop-2-enoate

HO

OH

O

O

O

OH

O O

OH

OH

O

HO

OH

HO

OH

Quercetin

3,5,7-trihydroxy-2-(3,4-dihydroxyphenyl)-4H-chromen-4-

one O

O

HO

OH

HO

OH

OH

Catechin

(2R,3S)-3,4-dihydro-2-(3,4-dihydroxyphenyl)-2H-

chromene-3,5,7-triol

OHO

OH

OH

OH

OH

Curcumin

(1E,6E)-1,7-bis(4-hydroxy-3-methoxyphenyl)hepta-1,6-

diene-3,5-dione

O O

O

HO

O

OH

H3C CH3

19

Dehydrocavidine

8,9-Dimethoxy-6-methyl[1,3]dioxolo[4,5-h]isoquino[2,1-

b]isoquinolin-13-ium N

H

O

O

O

O

Ternatin

4',5-dihydroxy-3,3',7,8-tetramethoxyflavone

O

O

O

O

O

O

OH

OH

Gossypol

2,2′-bis-(Formyl-1,6,7-trihydroxy-5-isopropyl-3-

methylnaphthalene)

OH

HO

HO O

O OH

OH

OH

Poly prenols

butan-1-ol

H

OHn

Geniposide

(1R)-methyl 1-((3R,4S,5S)-tetrahydro-3,4,5-trihydroxy-6-

(hydroxymethyl)-2H-pyran-2-yloxy)-1,4a,5,6,7,7a-

hexahydro-7-(hydroxymethyl)cyclopenta[c]pyran-4-

carboxylate

O

OH

HO

HO

O O

HO

O

O

OH

20

Proto catechulic

acid

3, 4-dihydroxyBenzoic acid

OH

OH

COOH

Nor

dihydroguaiaretic

acid

4-(4-(3,4dihydroxyphenyl)-2,3-dimethylbutyal)benzene-1,2-

diol.

HO

HO

OH

OH

Magnolol

4-Allyl-2-(5-allyl-2-hydroxy-phenyl)phenol

OH

HO

Lupeol

(1R,3aR,5aR,5bR,7aR,9S,11aR,11bS,13aR,13bR)-

icosahydro-3a,5a,5b,7a,8,8,11a-heptamethyl-1-(prop-1-en-2-

yl)-1H-cyclopenta[a]chrysen-9-ol HO

H3C CH3

CH3 CH3

H

CH3

H CH3

H

CH2

H3C

Thymoquinone

2-Isopropyl-5methylcyclohexa-2,5-diene-1,4-di one

O

O

21

Kutkin

2,3-dihydro-2-(2,3-dihydro-2-(4-hydroxy-3-

methoxyphenyl)-3-(hydroxymethyl)benzo[b][1,4]dioxin-7-

yl)-3-methylchromen-4-one

O

O

H

H

O

OH2C

H

H

OH

OH

O

CH3

Phyllanthin

(2R,3R)-2,3-diethyl-2-methyl-1,4-bis(3,4-

dimethylphenyl)butane

H

Pycnogenol

(2R,3R)-3,4-dihydro-2-(3,4-dihydroxyphenyl)-2H-

chromene-3,5,7-triol

O

OH

HO

OH

OH

OH

Rubaidin

2-hydroxy- 3-methylanthracene-9,10-dione

O

O

OH

Silymarin

2-(2,3-Dihydro-2-(4-hydroxy-3-methoxyphenyl)-3-

(hydroxymethyl)-1,4-benzodioxin-6-yl)-2,3-dihydro-3,5,7-

trihydroxy-4H-1-benzopyran-4-one

HO

O

O

OH

O CH3

HO

O OH

22

Rosamarinic acid

2-((E)-3-(3,4-dihydroxyphenyl)acryloyloxy)-3-(3,4-

dihydroxyphenyl)propanoic acid

O

O

OHO

OH

OH

OH

HO

Organo sulphur

compound 1,2-diphenyldisulfane

S

S

Chapter-II

Literature review

23

CHAPTER-II

REVIEW OF LITERATURE

Limnophila heterophylla

Limnophila heterophylla (Syononyms: Columnea heterophylla Roxb., Limnophila reflexa

Benth., Limnophila heterophylla var. reflexa (Benth.) Hook. f., Limnophila roxburghii G.

Don) is an aquatic herb, mainly submerged, but with shoots that often emerge above the

water surface, rooting at nodes. About 40 species of the genus Limnophila are mentioned in

(Table 2.1) (Shi, 1998).

Table 2.1: Different Species of the Genus Limnophila

Genus Species

Limnophila aromatica (Lamarck) Merrill.

(Syn. L.aromaticoides Yang &

Yen; gratissima Blum)

Limnophila australis Wannan & Waterh.

Limnophila balsamea (Benth.) Benth. (Syn.

Limnophila thorelii Bonati )

Limnophila borealis Y. Z. Zhao & Maf.

Limnophila laotica Bonati

Limnophila laxa Bentham

Limnophila micrantha (Benth.) Bentham

Limnophila parviflora Yamazaki

Limnophila polyantha Kurz ex Hook.f.

Limnophila brownii Wannan

Limnophila chinensis (Osbeck) Merill (Syn.

Limnophila chevalieri Bonati; L. hirsuta

(Heyne ex Benth.) Benth.

Limnophila connata (Buchanan-Hamilton

ex D. Don) Handel-Mazzetti

Limnophila erecta Bentham

Limnophila fragrans Seem

Limnophila geoffrayi Bonati

Limnophila hayatae Yamazaki

heterophylla (Linnaeus) Druce

(Syn. reflexa Bentham)

Limnophila indica (Linnaeus) Druce (Syn.

Limnophila gratioloides R. Brown; racemosa

Bentham; aquatic Roxburgh) (Syn.

polyantha Yamazaki )

24

Limnophila repens (Bentham) Bentham

(Syn. conferta Bentham;

Limnophila dubia Bonati; sessilis

(Bentham) Fischer

Limnophila rugosa (Roth) Merill (Syn. roxburghii

G. Don)

Limnophila sessiliflora (Vahl) Blume

Limnophila siamensis Yamazaki

Limnophila taoyuanensis Yang & Yen

Limnophila verticillata Yamazaki

Limnophila villifera Miq

Limnophila Xludoviciana Thieret

Limnophila dasyantha Skan

Limnophila glabra (Benj.) Kerr

Limnophila hottonoides Druce

Limnophila gigantean

2.1. LITERATURE REVIEW OF LIMNOPHILA HETEROPHYLLA

“Limnophila” is a genus of flowering plants in the plantain family, Scrophulariaceae.

It is distributed in tropical and subtropical regions of Africa, Asia, Australia and the

Pacific Islands (Brown, 1998).

Species are known commonly as marshweeds. These are annual or perennial herbs.

Some species are glandular and aromatic. Plants of the genus vary in form, from erect

to prostrate, and with branching or unbranched stems.

Submerged leaves are whorled; aerial leaves are whorled or oppositely arranged. The

leaves are lance-shaped or pinnate, and the blades have smooth or serrated edges.

Some species have flowers solitary in the leaf axils, and others have flowers in

inflorescences. The sepals are arranged in a tubular calyx, and the corolla is tubular

or funnel-shaped.

The corolla has a lower lip with three lobes and an upper lip that is unlobed or double-

lobed (Brown, 1998).

2.1.1. Ethnomedical Information of Limnophila heterophylla

The plant finds lot of applications in the Traditional system of medicine (TSM)

against various complaints (Kapil et al., 1983).

25

The plant leaves are crushed with coconut oil and applied on the wound to quicken

healing (Arul Manikandan, 2005).

2.1.2. Pharmacological Information of Limnophila heterophylla

Different parts of Limnophila heterophylla with pharmacological information was mentioned

in Table 2.2.

COX Inhibitor Activity

The flavone, Nevadensin, obtained from the aerial parts and roots of the plant was found to

exhibit in vitro cyclooxygenase-1 and 2 (COX-1 and COX-2) inhibitory efficacy measured by

COX catalyzed prostaglandin biosynthesis assay method (Brahmachari et al., 2008).

Antimicrobial Activity

Padiya et al. (2013) reported significant antibacterial as well as antifungal activities of the

ethanol extract of the whole plant of Limnophila heterophylla. The extract at different

concentrations of 5, 25, 50, 100 and 250 µg/mL exhibited remarkable inhibition activity

against pathogenic bacterial strains such as Bacillus subtilis, Staphylococcus aureus (gram-

positive) and Escherichia coli, Klebsiella pneumonia (gram-negative) and two fungal strains

such as Aspergillus flavus and Candida albicans.

Wound Healing Activity of Limnophila heterophylla

Alcoholic extract of Limnophila heterophylla was found to possess wound healing activity on

rats as investigated by Reddy et al. (1991).

Table 2.2: Pharmacological Information of Limnophila heterophylla

Plant part Solvent Uses Reference

Aerial parts & roots Petrol (60-800) COX Inhibitor activity Brahmachari et al., 2008

Whole plant Ethanol Antimicrobial activity Padiya et al., 2013

Whole plant Ethanol Wound healing activity Reddy et al., 1991

2.1.3. Phytoconstituents Information of Limnophila heterophylla

Many chemical constituents are widely isolated from the Limnophila heterophylla

phytoconstituents with their IUPAC names and structures are illustrated in Table 2.3 and 2.4.

26

2.2. LITERATURE REVIEW OF MICHELIA CHAMPACA

Michelia” is a genus of flowering plants belonging to the family Magnoliaceae, about

50 species of the genus Michelia are reported, some common species are mentioned

in Table 2.5.

The genus includes about 50 species of evergreen trees and shrubs, native to tropical

and subtropical south and southeast Asia (Indomalaya), including southern China.

The Magnoliaceae are an ancient family; fossil plants identifiably belonging to the

Magnoliaceae date back 95 million years.

A primitive aspect of the Magnolia family is that their large, cup-shaped flowers lack

distinct petals or sepals. The large non-specialized flower parts, resembling petals,

are called tepals.

The leaves, flowers, and form of Michelia resemble Magnolia, but the blossoms of

Michelia generally form clusters among the leaves, rather than singly at the branch

ends as Magnolia does.

2.2.1. Ethnomedical Information

Conventionally it is widely used in both Ayurveda and Siddha medicine. It is being

used in fever, colic, leprosy, post-partum protection (Khan et al., 2002) and in eye

disorders (Nayak et al., 2004).

Juice of the leaves of Michelia champaca is given with honey in cases of colic (Mehla

et al., 2010). The flower oil is useful in cephalalgia, opthalmia and gout (Gupta et al.,

2011).

They are useful as a diuretic in renal diseases and in gonorrhoea. The flower buds of

Michelia champaca are commonly used by many traditional healers in most of herbal

preparations for diabetes (Rajagopalan, 2000).

The flowers and fruits are considered stimulant, antispasmodic, tonic, stomachic,

bitter and cool remedies and are used in dyspepsia, nausea and fever.

The bark is used as a stimulant, pectorant, astringent and febrifugal properties

(Varier, 2003). The dried root and roots bark, mixed with curdled milk, is useful as

an application to absecesses, clearing away or maturing the inflammation.

In the form of an infusion it is valuable emmenagogue. It is also considered purgative.

Root and bark are used as purgative and in the treatment of inflammation,

constipation and dysmenorrhea.

27

Flower and flower buds, fruits are useful in ulcers, skin disease wounds (Nadkarni,

1954). The flowers mixed with sesamum oil forms an external application in vertigo

and also applied to foetid discharges from the nostrils.

The flowers and fruits in combination with other drugs are recommended as an anti-

dote to snake and scorpion venoms (Kirtikar and Basu, 1984).

It finds mention as one of the ingredients of the sarvasugandhi group and is used in

psychoneurosis by traditional healers. Different parts of Michelia champaca with

ethnomedical information is stated in Table 2.6.

Table 2.3: Phytoconstituents Information of Limnophila heterophylla

Compound name Part of the plant References

5-Hydroxy-7,8,2',4'-

tetramethoxyflavone Aerial parts & roots

Mukherjee et

al., 1994

5,7-Dihydroxy-6,8,4'-

trimethoxyflavone Aerial parts & roots

Brahmachari

et al., 2008

5,2'-Dihydroxy-7,8,4'-

trimethoxyflavone Aerial parts & roots

Mukherjee et

al., 1998

5,7-Dihydroxy-6,8,3',4'-

tetramethoxyflavone Aerial parts & roots

Reddy et al.,

1991

1β-Hydroxy-3-keto-olean-12-

en-28-oic acid Aerial parts & roots

Mukherjee et

al., 1995

Methyl-olean-12-ene-3α-

benzoyloxy-29-carboxylate Aerial parts & roots

Mukherjee et

al., 1995

Hydroxyolean-12-ene-29-oic

acid Aerial parts & roots

Mukherjee et

al., 1997

Ursolic acid Aerial parts & roots Mukherjee et

al., 1994

(+)-Limonene, (+)-Cadinene,

α-Pinene, p-Cymene, α-

Eudesmol

Essential oil

Rastogi &

Mehrotra,

1988

2.2.2. Pharmacological Activities

Different parts of Michelia champaca with pharmacological information is mentioned in

Table 2.7.

Cytotoxic Activity of Michelia champaca

The ethanolic extract of bark of Michelia champaca showed activity against human

epidermoid carcinoma of the nasopharynx test system was studied by Hoffmann et al.

(1977).

28

Antihyperglycemic Activity of Michelia champaca

Various extracts of flower buds of Michelia champaca for antidiabetic activity was

reportable by Jarald et al. (2008). Results advised that among all the extracts the ethanolic

extract of Michelia champaca exhibited vital dose-dependent antihyperglycemic activity

however didn't produce hypoglycemia in fasted normal rats.

Anti-inflammatory activity of Michelia champaca

The anti-inflammatory drug activity in methyl alcohol (95%) extract of Michelia champaca

leaves by varied carrageenan-induced inflammation rat models was highlighted by Gupta et

al. (2011). Results showed highly significant maximum inhibition concluding anti-

inflammatory activity in pro-inflammatory conditions. This study put together disclosed the

presence of some phytoconstituents like flavanoids (Vimala et al., 1997) jointly showed anti-

inflammatory drug property of methyl alcohol extract of Michelia champaca flowers.

Table 2.5: Different Species of the Genus Michelia

Genus Species

Michelia aenea

Michelia alba

Michelia angustioblonga

Michelia balansae

Michelia baillonii

Michelia braianensis

Michelia calcicola

Michelia caloptila

Michelia chapensis

Michelia compressa

Michelia cavaleriei

Michelia coriacea

Michelia crassipes

Michelia doltsopa

Michelia elegans

Michelia elliptilimba

Michelia figo

Michelia flaviflora

Michelia floribunda

Michelia foveolata

Michelia fujianensis

Michelia fulgens

Michelia fuscata

Michelia guangxiensis

Michelia hedyosperma

Michelia hypolampra Dandy

29

Michelia ingrate

Michelia iteophylla

Michelia kisopa

Michelia koordersiana Noot.

Michelia lacei

Michelia laevifolia

Michelia lanuginose

Michelia leveilleana

Michelia longipetiolata

Michelia longistamina

Michelia longistyla

Michelia macclurei

Michelia martinii

Michelia masticata

Michelia maudiae

Michelia mediocris

Michelia microtricha

Michelia montana Blume

Michelia nilagirica Zenker.

Michelia odora

Michelia pachycarpa

Michelia platypetala

Michelia polyneura

Michelia punduana

Michelia rajaniana

Michelia salicifolia

Michelia scortechinii

Michelia wilsonii

Michelia shiluensis

Michelia skinneriana

Michelia sphaerantha

Michelia subulifera

Michelia szechuanica

Michelia xanthantha

Michelia yunnanensis

Leishmanicidal Activity of Michelia champaca

Timber extracts of Michelia champaca showed potent leishmanicidal activity (Takahashi et

al., 2004).

Table 2.6: Ethnomedical Information of Michelia champaca

Part of the plant Uses Reference

Dried root & bark Absecesses, purgative Kirtikar et al., 1984

Flower & flower

buds Ulcers, skin disease wounds Nadkarni, 1954

Flower buds Herbal preparation for diabetes Rajagopalan, 2000

30

Flower oil Cephalalgia, oetipthalmia and gout Gupta et al., 2011

Flowers

Stimulant, antispasmodic, tonic,

stomachic, bitter and cool remedies and

are used in dyspepsia, nausea & fever.

Gupta et al., 2011

Flowers Anti-dote to snake and scorpion venoms. Kirtikar et al., 1984

Flowers Foetid discharges from the nostrils. Gupta et al., 2011

Flowers Vertigo, foetid discharges from the

nostrils. Nadkarni, 1954

Fruits Ulcers, skin disease wounds. Gupta et al., 2011

Leaves Colic Mehla et al., 2010

Root and bark Purgative, inflammation, constipation and

dysmenorrhea. Varier, 2003

Stem bark Stimulant, expectorant, astringent and

febrifuge. Kirtikar et al., 1984

Anti-infective Activity of Michelia champaca

The dichloromethane extract of Michelia champaca possess antiinfective activity.

Dichloromethane extract of Michelia champaca and A madagascarienjse showed the most

variety of growth inhibiting compounds against Cladosporium cucumerinum; the crude

extracts showed activity against many phytophathogenic thread like fungi (Rangasamy et

al., 2007).

Radical Scavenging Activity of Michelia champaca

Ethyl acetate and hexane extracts of Michelia champaca possesses a sturdy in vitro inhibitor

activity (Kumar et al., 2011). This study was centered on invitro activity by victimization

fully completely different parameters like 2, 2-diphenyl-1-picrylhydrazyl (DPPH) assay,

reducing power and in-vitro lipoid peroxidation. Results prompt that each extracts of

Michelia champaca were found to be considerably effective in scavenging DPPH.

Antibacterial Activity of Michelia champaca

The bactericide activity in ester extract of Michelia champaca flowers was highlighted by

Umadevi and Deepthi (2012). The bactericide activity of Michelia champaca ester extract

was studied against gram-positive organism (Staphylococcus aureus, Bacillus subtilis) and

gram-negative bacteria (Escherichia coli, Pseudomonas aeruoginosa). The ester extract was

simpler against all microorganism strains tested.

Wound Healing Activity of Michelia champaca

The wound healing activity in ethyl alcohol (95%) extract of Michelia champaca flowers by

burn wound healing methodology was highlighted by Shanbhag et al. (2011). Several

31

parameters like incision wound, epithelization quantity, scar area, enduringness and amino

acid (hydroxyl proline) measurements beside wound contraction, were accustomed assess

the impact of Michelia champaca on wound healing. The results indicated that Michelia

champaca hurries the wound healing methodology by declining the expanse of the wound

and increasing the permanency (Dwajani et al., 2009).

Diuretic Activity of Michelia champaca

Aqueous extracts of stem bark and leaves of Michelia champaca has been investigated for

diuretic activity (Ahamad et al., 2011). Results clearly advised that aqueous compound

extracts of stem bark exhibited higher diuretic drug potential as compared to leaves extract,

with the upper dose evoking pronounced symptom even larger than standard furosemide

drug (Lasix) in terms of Na+ and K+ concentration, and approximating diuretic drug in terms

of excretory product volume.

Antiulcer Activity of Michelia champaca

Alcoholic and aqueous extracts of leaves and flowers were evaluated for anti ulcerogenic

property against NSAID-aspirin induced lesion (Mullaicharam and Surendra, 2011). Various

parameters like reduction in internal organ volume, free acidity and lesion index were down

upon administration of alcoholic and aqueous extract of Michelia champaca. Flower binary

compound extract showed most effectiveness followed by leaf alcoholic, flower alcoholic,

and leaf binary compound extracts.

Antifertility Activity of Michelia champaca

The antifertility activity of a hydroalcoholic leaf extract of Michelia champaca in feminine

rats was illustrated by Taprial et al. (2013). Results showed vital anti-fertility impact which

can ensure to inhibition of implantation and steroid hormone impact due to presence of some

phytoconstituents.

Antihelmintic Activity of Michelia champaca

The methanolic and aqueous extracts of leaves of Michelia champaca showed robust

antihelmintic activity against test worms Pheretima posthuma (Dama et al., 2011).

Parameters like paralysis time (PT) and death time (DT) were increased upon administration

of each extracts.

32

Cardioprotective Activity of Michelia champaca

The cardio protecting potential of methanolic extract of Michelia champaca flowers on

isoproterenol-induced cardiac muscle anemia in male albino wistar rats was studied by

Kulkarni, (2012). Results indicated that retreatment with varied doses showed dose-

dependent cardioprotective edges with restoration of biochemical parameters and

histopathological confirmation of biochemical findings.

2.2.3. Phytoconstituents Information of Michelia champaca

Michelia champaca Linn comprises michelia- A, liriodenine, parthenolide and

guaianolides (Hoffmann et al., 1977; Toshiyuki et al., 1982).

Methanol extract of flowers of Michelia champaca found to have phytoconstituents

viz; alkaloids, saponins, tannins, sterols, flavonoids and triterpenoids (Khan et al.,

2002).

The plant could be an excellent supply of esters of carboxylic acid, benzaldehyde,

group alcohol, isoeugenol and sesquiterpene lactones.

Polyphenolic compounds like gallic acid was isolated from the leaves and stem bark

of Michelia champaca Linn.

Methyl linoleate, methyl anthranilate were different esters isolated from Michelia

champaca Linn (Ahamad et al., 2011).

Stigmasterol and 3β-16α- dihydroxy- 5-cholestene-21-al were additionally isolated

from stem bark of Michelia champaca Linn (Makhija et al., 2010).

Phytoconstituents with their IUPAC names and structures are given in Table 2.8 and

2.9.

Table 2.7: Pharmacological Information of Michelia champaca

Plant part Solvent for

Extraction Uses References

Bark Ethanol Antitumor Hoffmann et al., 1977

Flower Methanol Anti-inflammatory Vimala et al., 1997

Leaves --------- Anti-inflammatory Gupta et al., 2011

Flower Ethanol Anti-diabetic Jarald et al., 2008

Plant ------------ Leishmanicidal

activity

Takahashi et al., 2004

Plant ------------ Wound healing Dwajani et al., 2009

Flower Ethanol Wound healing Shanbhag et al., 2011

Different

parts

Dichloromethane Anti-infective Rangasamy et al 2007

33

Flower ------------ Antioxidant Kumar et al., 2011

Stem bark Aqueous Diuretic Ahmad et al., 2011

Leaves/

flowers

Aqueous/alcoholic Anti-ulcer Mullaicharam &

Surendra, 2011

Plant Hexane/ ethyl

acetate

Antibacterial Umadevi and Deepthi,

2012

Leaf Hydro alcoholic Anti-fertility Taprial et al., 2013

Leaves ------------- Antihelmintic Dama et al., 2011

Flowers Methanol Cardioprotective Kulkarni, 2012

Table 2.8: Phytoconstituents Information of Michelia champaca

Plant part Solvent Use Reference

Flower Methanol

alkaloids, saponins,

tannins, sterols,

flavonoids &

triterpenoids

Khan et al., 2002

Plant -----

alkaloids, saponins,

tannins, and

triterpenoids

Khan et al., 2002

Various

parts of the

plant

-----

michelia-A, liriodenine,

parthenolide &

guaianolides

Hoffmann et al,

1977; Toshiyuki et

al, 1982

Leaves,

stem bark Ethanol Gallic acid

Hoffmann et al,

1977; Toshiyuki et

al, 1982

Plant ----- Methyl linoleate and

methyl anthranilate Ahamad et al., 2011

Stem bark Petroleum

ether

Stigmasterol and 3β-

16α- dihydroxy- 5-

cholestene-21-al

Makhija et al., 2010

2.3.1. Ethnomedical Information of Couroupita guianensis

Fruits of Couroupita guianensis unit edible and sometimes eaten, however attributable to

dangerous smell of white flesh, it discourages the general public. The fruit pulp, bark and

flowers area unit used for varied medicative applications. The pulp of the fruit of the cannon

ball tree is rubbed on the infected skin of animal disease dog (Sanz et al., 2009). The within

of the fruit will make clean wounds and young leaves cure odontalgia (Kumar et al., 2011).

Traditionally leaves as used as antiseptic and odontalgia. Juice made up of the leaves is

employed to cure skin ailments, and shamans of South America have even used tree

components for treating protozoal infection. Historically, the leaves of this plant are utilized

34

in the treatment of skin diseases, stomach ache, and enteral gas formation, antithrombotic

and vasodilatory actions (Golatkar et al., 2001; Elumalai et al., 2012). Historically, the leaves

of this plant are utilized in the treatment skin diseases (Satyavathi et al., 1976). Leaves and

flowers of Couroupita guianensis unit used for healthful applications like upset, tumors, pain

and inflammatory processes (Sanz et al., 2009), cold, enteric gas formation and abdomen

ache (Elumalai et al., 2012). The trees unit accustomed cure colds and abdomen aches. The

volatile oils from the flowers show antibacterial and antifungal properties. It's one in every

of the ingredients within the several preparations that cure redness, hemorrhage, piles,

scabies, dysentery, scorpion poison (Shah et al., 2012). Different parts of Couroupita

guianensis with ethnomedical information are stated in Table 2.9.

Table 2.9: Ethnomedical information of Couroupita guianensis

Parts Uses References

Fruit Skin infections Sanz et al., 2009

leaves Skin infections Satyavathi et al., 1976

Fruit odontalgia Kumar et al., 2011

leaves Skin diseases, stomach ache, and

enteral gas formation, antithrombotic

and vasodilatory actions

Golatkar et al., 2001;

Elumalai et al., 2012

Leaves and

flowers

Upset, tumors, pain and inflammatory

processes

Sanz et al., 2009

Flowers Hemorrhage, piles, scabies,

dysentery, scorpion poison

Shah et al., 2012

2.3.2. Pharmacological information

Different parts of Couroupita guianensis with pharmacological information are mentioned

in Table 2.10.

Analgesic and anti-inflammatory activity of Couroupita guianensis

According to the Geetha et al., (2004) analgesic and anti-inflammatory activities in benzene,

ethyl alcohol (95%) extract of Couroupita guianensis flowers and barks by victimization tail

flick methodology and carrageenan induced hind paw swelling methodology severally.

Numerous parameters like tail flick latency (TFL) for physiological condition and reduction

35

in carrageenan induced hind paw swelling for medicament was measured. Potent to

paracetamol in its analgesic activity and to in its anti-inflammatory activity was discovered.

Pinheiroa et al., 2013 additionally explicit that ethanolic extract of Couroupita guianensis

possess anti-inflammatory activity.

Antibacterial activity of Couroupita guianensis

The antibacterial activity in ethyl alcohol (95%) extract of Couroupita guianensis fruit pulp

by maceration methodology was delineated by Shah et al., (2012). The antibacterial activity

of Couroupita guianensis ethyl alcohol extract was studied against gram-positive

microorganism (Staphylococcus aureus, Bacillus subtilis) and gram-negative bacteria

(Escherichia coli, Pseudomonas aeruoginosa). Compared to doxycyclin, ciprofloxacin and

fluconazole, vital activity was found against B. subtilis at concentration 4mg as compare to

further tested organisms. This study conjointly disclosed the presence of some

phytoconstituents like tannins, sugars and polyphenols. Azimi et al., 2012 collectively

showed antibacterial property of ethanolic extract of Couroupita guianensis oil.

Antiulcer activity of Couroupita guianensis

The antiulcer activity of Couroupita guianensis leaves in ethanolic extract was studied by

Elumalai et al., (2012). Numerous parameters like reduction in internal organ volume, free

acidity and lesion index were lowered upon administration of ethanolic extract of

Couroupita guianensis (150mg/kg and 300mg/kg).

Antidepressant activity of Couroupita guianensis

Wankhede et al., (2009) showed antidepressant activity in methanolic extract of Couroupita

guianensis root. This study focused on measure of assorted parameters like tail suspension

check (TST), forced swim check (FST) and antihypertensive antagonism in mice. Results of

this study indicated that considerably decrease within the immobility time in TST and FST,

almost like that of the imipramine (10 mg/kg). In antihypertensive antagonism exhibited

deeply decline in period of hypersomnia & degree of ptosis in tested mice.

Antifertility activity of Couroupita guianensis

Benzene, ethyl alcohol and water extracts of bark and flowers of C. guianensis showed

antifertility activity was studied for their impact on period of assorted stages of estrus cycle

in female person rats and on the number implantation sites within the pregnant rats (Geetha

36

et al., 2005). The ethyl alcohol extract of C. guianensis bark and every one the extracts of its

flower condensed the quantity of implantations. Supported the on top of criteria Couroupita

guianensis extract shows protective activity in a very therapeutic vary.

Antimicrobial, antimycobacterial and antibiofilm properties of Couroupita guianensis

Al-Dhabi et al., (2012) showed antimicrobial, antimycobacterial and antibiofilm properties

in chloroform extract of fruit of Couroupita guianensis. Chloroform extract of Couroupita

guianensis fruit showed sensible antimicrobial and antibiofilm forming activities however it

showed less antimycobacterial activity. The zones of inhibition by chloroform extract ranged

from zero to twenty six millimeter. Chloroform extract showed effective antibiofilm activity

against gram-negative microorganism referred to as genus Pseudomonas aeruginosa ranging

from two mg/mL biofilm repressive concentration (BIC), with 52 inhibition of biofilm

formation. From the HPLC-DAD analysis, it absolutely was established that indirubin was

one amongst the key compounds during this plant (0.0918% dry weight basis). Ramalakshmi

et al., 2013 conjointly showed antimicrobial property of methanolic extract Couroupita

guianensis flowers. The results of the antimicrobial activity showed effective repressing

activity against Plesiomonas Shigelloides, Cocci aureus, Vibrio mimicus, and Proteus

vulgaris. Moderate antimicrobial activity was recorded against E.coli, Klebsiella pneumonia

and Salmonella typhi. Regina et al., 2012 additionally incontestable that chloroform,

hexaneane and ethanol extract of fruit rind of Couroupita guianensis Aubl. showed its vital

antibacterial and antifungal activity at the assorted conc.(10 mg/ml) during which the

fermentation ethanol extract showed sensible restrictive activity against S. aureus, E. coli,

C. diptheriae and Micrococcus sp. among the alternative tested extracts whereas chloform

extracts showed sensible restrictive activity against C. albicans.

Antipyretic activity of Couroupita guianensis

Antipyretic activity of flower and bark a part of Couroupita guianensis in chloroform,

ethanol, water, ether, petroleum ether extracts was done by victimization yeast induces

febrility methodology (Usman et al., 2012). This yeast induces febrility methodology

suggesting that the antipyretic action of all the extracts was reflective; chloroform, ethanol,

water extracts have vital onset of action on reduction of temperature (within 30 minutes)

almost like that of paracetmol (30 minutes). On alternative hand petroleum ether and ether

extract are showing somewhat late response.

37

Anxiolytic impact of Couroupita guianensis

Vinod et al., (2013) showed anxiolytic impact in aqueous and methanolic extract of

Couroupita guianensis flowers. Elevated plus maze (EPM), light and dark (LD), and open

field test (OFT) models were measured. From the results each the extracts (aqueous associate

degreed methanolic) of Couroupita guianensis at a dose of 500mg/kg showed an anxiolytic

activity associated with vehicle management in LD, EPM and open field test in mice.

Immunomodulatory activity of Couroupita guianensis

Immunomodulatory activity (Invitro polymorpho nuclear white corpuscle operate test) in

acetone, benzene, petroleum ether, chloroform, methanol and water extracts of Couroupita

guianensis flowers by victimization rat as an animal model was given by Pradhan et al.,

2009. Hypersensitivity, hemagglutinations reactions were calculated by victimization sheep

red blood cells (SRBC) as matter. Within the in-vivo studies, the continual fuel extract was

found to exhibit a dose connected increasing within the hypersensitivity, to the SRBC matter

at concentration of one hundred and two hundred mg/kg in animal studies. This study

conjointly according that methanolic extract was found to stimulate cell mediate and

antibody mediate immune responses in rats.

Neuropharmacological action of Couroupita guianensis

Methanolic extract of Couroupita guianensis flowers in mice showed numerous

neuropharmacological actions (Vinod et al., 2012). Spontaneous motor activity, rotarod

performance and sodium thiopental sleeping time in mice were measured. Beside medicine

actions some phytoconstituents conjointly (alkaloids, glycosides, tannins and flavonoids)

known. From the results methanolic extract (100, 250 and 500 mg/kg) of Couroupita

guianensis showed vital reduction in spontaneous motor activity however no impact had on

motor coordination. It conjointly leads to reduction of the onset and period of pentobarbitone

evoked psychological state. Finally this study declared that extract contained associate

degree agent that has pivotal role on each central and peripheral nervous system.

Wound healing activity of Couroupita guianensis

Umachigi et al., (2007) showed wound healing activity in ethanolic extract of Couroupita

guianensis whole plant (barks, leaves, flowers and fruits). Many parameters like incision

wound, epithelization amount, scar area, enduringness and aminoalkanoic acid (hydroxyl

proline) measurements beside wound contraction, were accustomed assess the impact of

38

Couroupita guianensis on wound healing. The results indicated that Couroupita guianensis

hurries the wound healing method by declining the expanse of the wound and increasing the

enduringness.

Antiarthritic activity of Couroupita guianensis

Elumalai et al., (2012) by victimization invitro technique showed antiarthritic activity of

Couroupita guianensis leaves in methanolic extract. Protein denaturation methodology was

assessed. The activity of extract was principally reckoning on concentration (dose dependent

manner). Protein denaturation was found to be 87.41% at a dose of 500 μg /ml.

Antistress activity of Couroupita guianensis

Couroupita guianensis possess sturdy antistress activity in methanolic extract was studied

by Vinod et al., (2013) by victimization cold restrain stress (RS). During this they measure

parameters like levels of glyceride, sterol and glucocorticoid to live the capability of

methanolic extract on antistress. Animals treated with methanolic extract of Couroupita

guianensis 100mg/kg and 250 mg/kg, 500 mg/kg doses considerably lowered in the least the

3 doses in a very dose dependent manner as compared to stress control. Cold restrain stress

caused an increase within the weight of adrenal glands at advanced dose.

Antidiarrheal action of Couroupita guianesis

Antidiarrheal action of Couroupita guianensis leaves on Castrol oil evoked diarrhea in

unusual person rats was disclosed by Elumalai et al., (2013). In Castrol oil evoked diarrhea

each the methanolic and liquid extracts beside common place loperamide showed vital

reduction in diarrheic episodes. 100mg/kg of methanolic extract and 100mg/kg of liquid

extract of Couroupita guianensis dried leaves are used for antidiarrheal activity.

Ovicidal activity of Couroupita guianensis

Baskar et al., (2013) showed ovicidal activity in hexane, chloroform and ester extracts of

Couroupita guianensis plant on the eggs of Helicoverpa armigera. All the extracts showed

ovicidal activity, and among them alkane extract showed additional (64.28%) ovicidal

activity with LC50 worth of two.62% and regression (r2) worth of 83.5%.

39

Antinociceptive activity of Couroupita guianensis

Ethanol extract of Couroupita guianensis leaves exhibited sturdy antinociceptive activity

was illustrated by Pinheiro et al., (2010) by victimization 3 analgesic models (acetic acid-

induced contortions, tail flick, and hot plate). Results are clearly showed that ethyl alcohol

extract of Couroupita guianesis all fractions showed antinociceptive activity within the tail

flick model whereas within the hot plate methodology the best impact discovered was at the

dose of 100 mg/kg and eventually extract considerably restrained the quantity of contortions

evoked by ethanoic acid.

Antifeedent and larvcidal activity of Couroupita guianensis

Ethyl acetate extract of Couroupita guianensis leaves exhibited Antifeedent and larvcidal

activity was studied by Baskar et al., 2012 and n-Hexane extract of Couroupita guianensis

leaves exhibited Antifeedent and larvcidal activity was illustrated by Lingathurai et al. 2011.

Table 2.10. Pharmacological Information of Couroupita guianensis

Plant Part Solvent used for

Extraction Uses References

Flowers and

Bark

Benzene, ethanol

(95%)

Analgesic & Anti-

inflammatory

Geetha et al., 2004

Leaves Ethanol Anti-inflammatory Pinheiroa et al., 2013

Fruit pulp Alcohol (95%) Antibacterial Shah et al., 2012

Oil Ethanol Antibacterial Azimi et al., 2012

Flowers Ethyl acetate fraction

of water

Antioxidant Bafna et al., 2011

Leaves Ethanol Antiulcer Elumalai et al., 2012

Dried flowers Not mentioned Anticancer &

antioxidant

Premanathan et al.,

2010

Root Methanol Antidepressant Wankhede et al., 2009

Bark &

flowers

Benzene, ethanol and

water

Antifertility Geetha et al., 2005

Fruits

Chloroform Antimicrobial, anti-

mycobacterial,

antibiofilm

Al-dhabi et al., 2012

Flowers Methanol Antimicrobial Ramalakshmi et al.,

2013

Fruit Ethanol Antifungal and

Antimicrobial

Regina et al., 2012

Flower, bark Not mentioned Antipyretic Usman et al., 2012

Flower Methanol Anxiolytic Vinod et al., 2013

Flowers Methanol Immunomodulatory Pradhan 2009

40

Plant Methanol Neuropharmacological Vinod et al., 2012

Whole plant Not mentioned Wound healing Umachigi et al., 2007

Leaves Methanol Antiarthritic Elumalai et al., 2012

Plant Methanol Antistress Gupta et al., 2013

Leaves Not mentioned Antidiarrheal Elumalai., 2013

Leaves Hexane, ethyl acetate,

chloroform

Ovicidal Bhasker et al., 2013

Leaves Ethanol Antinociceptive Pinheiroa et al., 2010

Leaves Ethyl acetate Antifeedent, larvcidal Baskar et al., 2012

Leaves n-Hexane Antifeedent &

larvicidal

Lingathurai et al., 2011

2.3.3. Active constituents Information of Couroupita guianensis

Few chemical studies discovered to this species had proved the presence of α-amirin, β-

amirin, β-sitosterol, tannins (Row et al., 1966; Bergman et al., 1985), ketosteroids

(Anjaneyulu and Rao, 1998) and terpenoids, alkaloids, carbohydrates, proteins

(Ramalakshmi et al., 2013). Among the flowers, it completely was getable to recognize

eugenol, volatile oil and (E, E)-farnesol whereas triterpenoid esters of fatty acids as β-amirin

palmitate were categorized among the leaves of Couroupita guianensis (Eknat and

Shivchandraji, 2002) and dyes like indigo and indirubin (Tayade, 2013). Associate in nursing

compound stigma sterol and camp sterol were isolated from fruit of Couroupita guianensis

(Rastogi and Mehrotra 1995). Devaraj et al., (2013) synthesized and characterized silver

nano particles from leaves of Couroupita guianensis. Bergman et al., (1985) isolated linoleic

acid, nerol, tryptanthrin etc., from flowers, seeds, fruits, and leaves of Couroupita

guianensis. Active constituent’s information was given in Table 2.11.

Table 2.11. Active constituents Information of Couroupita guianensis

Plant part Solvent used for

Extraction

Use Reference

Flowers Methanol Carbohydrates, alkaloids,

terpenoids, phenolics,

reducing sugars &

triterpenoids.

Ramalakshmi et al.,

2013

Leaves Ethanol, acetone,

and chloroform.

Silver nano particles Devaraj et al., 2013

41

Flowers

Not mentioned a) α-amirin, β-amirin, β-

sitosterol, tannins

b) ketosteroids

Row et al., 1966;

Bergman et al.,

1985, Anjaneyulu

and Rao, 1998

Leaves Not mentioned Eugenol, farnesol and

triterpenoid esters of fatty

acids as β-amirin palmitate

Eknat and

Shivchandraji, 2002

Fruit and flowers Not mentioned Indigo and Indirubin Tayade 2013

Flowers, seeds, Not mentioned Linoleic acid, and nerol Bergman et al., 1985

Fruit Not mentioned stigmasterol and campesterol Rastogi and

Mehrotra 1995

Chapter-III

Plants description

42

CHAPTER-III

PLANT DESCRIPTION

3.1.1. BOTANICAL DESCRIPTION OF LIMNOPHILA HETEROPHYLLA

It is an aquatic herb, mainly submerged, but with shoots that often emerge above

the water surface (Figure 3.1), rooting at nodes (Pancho and Soerjani, 1978).

1-2 Leaves are arranged in whorls of four to ten, sessile, 2–3 cm long. Below

water, they are finely twice pinnatifid. Above water, they are undivided but

shallowly toothed.

Flowers occur singly, sessile, in the axils of the upper leaves, above water. The

corolla tube is 3–4 mm long with four lobes, the upper bifid, spreading to about

3 mm across. Calyx tubular, 1–2 mm long, with five teeth; capsule ovoid, with

many very small seeds irregularly ovoid in shape and less than 1 mm long

(Pancho and Soerjani, 1978).

The plant occurs mainly in still or slowly moving water, at the edges of streams

and irrigation channels, and in rice and jute fields. It flowers and fruits from

October to March in India. It may propagate from stem fragments as well as

from seeds.

3.1.2. GEOGRAPHICAL DISTRIBUTION OF LIMNOPHILA HETEROPHYLLA

The plant occurs mainly in still or slowly moving water, at the edges of streams

and irrigation channels, and in rice and jute fields.

It flowers and fruits from October to March in India (Ambasta, 1986). It may

propagate from stem fragments as well as from seeds. While this plant appears

to be restricted to the true tropics, it poses a significant threat to tropical and

possibly subtropical regions and islands of United States.

It is native in Asia including India, Laos, Malaysia, Philippines, Sri Lanka,

Thailand and Vietnam (Holm et al., 1979).

This plant is listed as a “serious” weed in India and a “common” weed in

Thailand and regarded as among the most problematic weeds of deep-water rice

in West Bengal, India (Sahu, 1992).

Dense growth of this plant can restrict the flow of water in irrigation channels

and choke ponds. The taxonomical classification of Limnophila heterophylla is

illustrated in Table 3.1.

43

Figure 3.1: Limnophila heterophylla

Table 3.1: Taxonomical Classification of Limnophila heterophylla

Kingdom Plantae

Sub-kingdom Tracheobionta

Division Magnoliophyta

Class Magnoliopsida

Sub-class Asteridae

Order Scrophulariales

Family Scrophulariaceae

Genus Limnophila

Species heterophylla

Binomial name Limnophila heterophylla

(Roxb.)Benth.

3.2.1. BOTANICAL DESCRIPTION OF MICHELIA CHAMPACA

Michelia champaca, known by the scientific name Michelia champaca is a very

tall tree (Figure.3.2) that grows up to 30m tall. The young branches are covered

with grey hairs.

44

The leaves (Figure.3.3), are ovate in shape and are up to 30.5cm long and

10.2cm wide narrowing to a fine point at the apex. Small bracts, known as

stipules, are present on the leaf stalk of the alternately arranged leaves.

The flowers are pale yellow to orange and fairly large growing up to 5.1cm in

diameter. They are also very fragrant and when a Michelia tree is in flower the

fragrance produced is noticeable some distance from the tree.

The flowers have 15 tepals that curve up towards the tips and many stamens.

The fruit of Michelia champaca is made up of up to 3-20 brown follicles that

are dry at maturity Michelia champaca and split open at one side. Each follicle

contains 2-6 reddish seeds.

Michelia champaca Linn. known as champaca is belonging to the family of

Magnoliaceae (Rout et al., 2006).

The taxonomical classification, common names and vernacular names of

Michelia champaca are mentioned in Table 3.2, Table 3.3 and Table 3.4

respectively.

3.2.2. GEOGRAPHICAL DISTRIBUTION OF MICHELIA CHAMPACA

There are three species of Michelia available in Malaysia. They are Michelia

Alba (white chempaka), Michelia champaca (orange chempaka) and

Michelia figo (dwarf chempaka) with Michelia champaca and Michelia Alba

being the most popular species within the family (Ibrahim et al., 2005).

The genus Michelia contains about 40 species with a distribution in from

India, to Malaysia and Indonesia, and in southern Japan and Taiwan.

Michelia champaca is native to India, where it occurs in humid tropical

evergreen forests from 250-1500 m in elevation.

It is found throughout Indo-China, Malaysia, Sumatra, Java and

southwestern China. Outside of India the native range of this species is

difficult to determine as it has been dispersed extensively by humans

throughout Southeast Asia and Indonesia on account of the use of the trees

(Raja and Ravindranadh, 2015).

The authentication certificates of Limnophila heterophylla and Michelia

champaca was illustrated in Figure 3.4 and 3.5.

45

Table 3.2: Taxonomical Classification of Michelia champaca

Kingdom Plantae

Subkingdom Tracheobionta

Super division Spermatophyta

Division Magnoliophyta

Class Magnoliopsida

Subclass Magnoliidae

Order Magnoliales

Family Magnoliaceae

Genus Michelia

Species champaca

Table 3.3: Common Names of Michelia champaca

Bengali Champa, Swarnachampa

Hindi Campa, Campaka, Champ, Champa, Champac,

Champaca, Champe-ke-phul, Champaka

Kannada

Champaka, Kendasampige, Kolasampige,

Sampage-huvvu, Sampige, Gandhaphali, Kolu

sampige

Malayalam

Campakam, Cempakam, Champacam,

Champakam,Chempakap-pu, Chembagam,

Chembakam, Champa, Champaca, Champaga,

Champak, Chempacam, Chempakam

Marathi Sonchampa, Champa, Kudchampa,

Pivalachampa,Sonachampa, Sona champa, Chamfo

Sanskrit

Anjana, Atigandhaka, Bhramaratithi, Bhringmohi,

Campaca, Campaka, Campakah, Campakam,

Campeya, Chambunala, Champaka,

Champakapushpam, Champeya, Deepapushpa,

Gandhaphali, Hemanga, Hemapushpa,

Hemapushpika, Hemapuspaka,

Hemavha,Kamabana, Kancana

Tamil

Amariyam, Sambagam, Sembagam, Sempakam,

Sempuga, Shampangi, Vandumarmalar,

Canpakam, Shampangi-pushpam, Shanbagapoo,

Campakam, Canpakappu, Campanki, Ilai

campanki, Shampang, Shenbagam,

Shanbagam, Sanbagam, Champakam,

Chembagam, Akacampanki, Akantakaram,

Ancanam, Atikantam, Shenbagapoo.

46

Table 3.4: Vernacular Names of Michelia champaca

Java Chempaka, Chepaka, Pechari,

Lochari, Kantil, Semendara

Malaysia

Chempaka, cempaka merah,

Chempa, Cempaka kuning, Jampaka

Sundanese Champaka.

Sumatra Champaga

Thai Champah, Champi

Figure 3.2: Michelia champaca Tree

47

Figure 3.3: Michelia champaca Leaf

3.3.1. BOTANICAL DESCRIPTION COUROUPITA GUIANENSIS

Cannonball Tree is a deciduous tree. Native to tropical South America (particularly

Guyana and Surinam) it has large, apricot-pink and gold flowers with an unusual,

lopsided arrangement of central stamens and a penetrating fragrance. The taxonomical

classification of Couroupita guianensis was mentioned in Table 3.5. It is a really

wonderful tree doesn't grow branches that reach out from the straight trunk; it bears

vast, showy flowers, with 3" to 5" waxy aromatic smelling growing directly on the bark

of the trunk (cauliflower). In Buddhist culture in country these flowers (Figure 3.4) had

a special significance. The tree additionally produces orbicular brown woody,

indehiscent; double fleshy fruits of associate degree astonishing size, adequate to the

scale of an individual's head. The fruit includes of little seeds in an exceedingly white,

unpleasant smelling edible jelly. Size of a mature fruit is 24 cm in diameter, weight of

a mature fruit-1450gms, and weight of the shell (fruit rind) from a fruit-545 gms. It's

wide planted in tropical and sub-tropical biology gardens as a decorative throughout the

tropics and sub tropics, it will well below cultivations. This plant is listed as a rare tree

and flower in Republic of India, by a preferred decorative in Caribbean and SE Asian

biology gardens. The origin and growing conditions of Couroupita guianensis was

illustrated in Table 3.6.

48

Table 3.5. Taxonomical classification of Couroupita guianensis

Kingdom Plantae

Sub kingdom Tracheobionta

Division Magnoliophyta

Class Magnoliopsida

Order Lecythidales

Family Lecythidaceae

Genus Couroupita

Species Couroupita guianensis Aubl

Synonyms Couratori pedicellaris, Couroupita acreensis,

Couroupita antillana, Couroupita froesii,

Couroupita surinamensis, Couroupita idolica,

Couroupita membranacea, Couroupita peruviana,

Couroupita saintcroixiana, Couroupita

surinamensis, Couroupita venezuelensis, Lecythis

bracteata, Pekea couroupita.

Other names Arbre a bombes (French), Bala de canon (Spanish),

Boesi (Dutch), Carrion tree, Kanonenkugelbaum

(German) and Taparon (German).

Table 3.6. Origin and growing conditions of Couroupita guianensis

Origin Honduras to Northern South America to Peru

Zone 10a - 12b, 28°F minimum

Growth rate Fast

Flowering month March - September

Flowering days Not identified

Leaf persistence Briefly deciduous

Messiness High

Salt tolerance Low

Drought tolerance Medium

Nutritional requirements Medium

Typical dimensions 70’x45’

Uses Park, Shade, Specimen

3.3.2. GEOGRAPHICAL DESCRIPTION COUROUPITA GUIANENSIS

Couroupita guianensis (Aubl) belongs to family called Lecythidaceae, could be a

massive deciduous tropical tree 90' tall and autochthonous to the Amazon timberland.

49

Figure 3.4: Couroupita guianensis flower

3.4. COLLECTION AND AUTHENTICATION

Couroupita guianensis, Limnophila heterophylla and Michelia champaca were

collected from the vicinity of Tirumala hills, Chittor district of Andhra Pradesh, India.

Further, plants were distinguished, affirmed and validated by Dr. Madavchetty,

Professor, Botany office, Sri Venkateswara University, Tirupati. Voucher specimen of

these plants (GIP-Plant No-001, GIP006/2013-2014 and GIP005/2013-2014) have been

kept in the GITAM Institute of Pharmacy, GITAM University, Visakhapatnam, Andhra

Pradesh, India.

Chapter-IV

Standardization of plant materials

50

CHAPTER-IV

STANDARDIZATION OF PLANT MATERIALS

4.1. INTRODUCTION

The use of herbal medicines continues to expand rapidly across the world. Many

countries now turn towards herbal medicines or herbal products for their health care in

national health care settings. According to World Health Organization (WHO), 80% if the

rural population in developing countries depend on traditional medicines to meet their

primary healthcare needs (Bannerman et al., 1993). Authentication and standardization

are prerequisite steps while considering source materials for herbal formulation in any

system of medicine (Ahmad et al., 2009). In traditional systems of medicine (TSM), the

drugs are primarily dispensed as water decoction or ethanol extract. Fresh plant parts,

juice, or crude powders are a rarity rather than a rule. Thus medicinal plant parts should

be authentic and free from harmful materials like pesticides, heavy metals, microbial or

radioactive contamination, etc. (Kamboj, 2000). It is very important that a system of

standardization should establish for every plant medicine in the market because the scope

for variation in different batches of medicine is enormous.

WHO encourages, recommends and promotes traditional / herbal remedies in national

health care programmes because these drugs are safe, people have faith in them and easily

available at low cost. The WHO is continuously emphasizing to ensure quality control of

medicinal plant products by using modern techniques and applying suitable standards

(Raina, 2003). India has a rich heritage of traditional medicine constituting with its

different components like Ayurveda, Siddha, Unani, Homoeopathy and naturopathy.

Traditional health care has been flourishing in this country for many centuries (Kumar,

2011). The growing use of botanicals by the public is forcing moves to evaluate the health

claims of these agents and to develop standards of quality and manufacture. Various

traditional medicine systems, especially Indian system of medicine attracted the global

attention due to their long historical clinical use and reliable therapeutic efficacy. Many

big pharmaceutical companies are using traditional medicine as an excellent pool for

discovering natural bioactive compounds. With the growing need for safer drugs,

51

attention has drawn to their quality, efficacy, and standards of the traditional Indian

medicine (Marwick, 1995). Each traditional system of medicines has their own method of

standardization for assuring quality most in human linguistic terms. This method of

evaluation has to be taken into consideration in standardization of herbal medicine/drugs

(Kumar, 2011). The basic requirements to establish herbal medicine in modern’s

scientific era includes;

Well documented ethno-botanical information

Medicinal plants free from pesticides, heavy metals

Standardization, based on chemical and activity profile, and Safety and stability

data. However, mode of action studies in animals and efficacy in human will be

supportive.

Such scientifically generated data will project herbal medicine in a proper perspective and

such herbal medicine will be sustained in modern scientific world. There is a strong

demand and need to accelerate the research in phytomedicine. Development of authentic

analytical methods, which can reliably profile the phytochemical composition, including

quantitative analysis of marker/bioactive compounds and other major constituents, is a

major challenge to scientists. Without consistent quality of a phytochemical mixture, a

consistent pharmacological effect is not expected. Standardization is the first step for the

establishment of a consistent biological activity, a consistent chemical profile, or simply a

quality assurance program for production and manufacturing (Ulrich-Merzenich et al.,

2007). As there is no enough evidence for detailed physicochemical and phytochemical

evaluation on whole plants of Couroupita guianensis, Limnophila heterophylla and

Michelia champaca was reported.

The present study was an effort to standardize the selected Indian medicinal plants

(Couroupita guianensis, Limnophila heterophylla and Michelia champaca). Plants were

subjected for determination of physicochemical parameters such as loss on drying, ash

values, pH value in 1% and 10% solution, aqueous and alcoholic extractive values were

carried out according to the methods recommended by the WHO.

52

4.2. MATERIALS AND METHODS

Loss on drying / Moisture content (Gravimetric determination)

Separately place about 1.0g of leaves powder, in an accurately weighed moisture disc. For

estimation of loss on drying, it was dried at 105°C for 5 hours in an oven, cooled in a

desiccator for 30 minutes, and weighed without delay. The loss of weight was calculated

as the content of in mg per g of air -dried material.

Determination of total ash

Two grams of the leaves powder, was placed in a previously ignited (350°C for 1 hour)

and tarred crucible accurately weighed. Dried material was spread in an even layer in the

crucible and the material ignited by gradually increasing the heat to 550°C for 5 hours in

a muffle furnace (Nabertherm) until it is white, indicating the absence of carbon. Cooled

in desiccators and weighed. Total ash content was calculated in mg per g of air-dried

material.

Determination of water-soluble ash

Twenty- five (25) ml of water was added to the crucible containing the total ash, covered

with a watch glass and boiled gently for 5 minutes. Insoluble matter was collected on an

ash less filter -paper. Washed with hot water and ignited in a crucible for 15 minutes at a

temperature not exceeding 450°C in a muffle furnace. Allowed the residue to cool in

suitable desiccators for 30 minutes, and then weighed without delay. The weight of the

residue was subtracted in mg from the weight of total ash. Water - soluble ash content

was calculated as mg per g of air-dried material.

Determination of acid-insoluble ash

Twenty- five (25) ml of hydrochloric acid (70g/l) TS was added to the crucible containing

the total ash, covered with a watch-glass and boiled gently for 5 minutes. The watch-glass

was rinsed with 5ml of hot water and this liquid added to the crucible. The insoluble

matter was collected on an ash less filter -paper (Whatmann -41) and washed with hot

water until the filtrate was neutral. The filter -paper containing the insoluble matter was

transferred to the original crucible, ignited by gradually increasing the heat to 550°C for

53

3hours in a muffle furnace (Nabertherm) to constant weight. Allowed the residue to cool

in a suitable desiccator for 30 minutes, and then weighed without delay. Acid-insoluble

ash content was calculated as mg per g of air dried material.

Determination of sulfated ash

Ignited a suitable crucible (silica) at 550°C to 650°C for 30 minutes, cooled the

crucible in a desiccators (silica gel) and weighed it accurately. One gram of the whole

plant powder of the Limnophila heterophylla was placed in a previously ignited crucible,

ignited gently at first, until the substance was thoroughly white. Cooled and moistened the

sample with a small amount (usually 1 ml) of sulfuric acid (1760 g/l) TS, heated gently at

a temperature as low as practicable until the sample is thoroughly charred. After cooling,

moistened the residue with a small amount (usually 1 ml) of sulfuric acid (1760 g/l) TS,

heated gently until white fumes were no longer evolved, and ignited at 800°C + 25°C

until the residue is completely incinerated. Ensure that flames were not produced at any

time during the procedure. Cooled the crucible in a desiccator (silica gel), weighed

accurately. This was repeated until the sample reaches a constant weight and calculated

the percentage of residue.

Determination of pH range

The pH of different formulations in 1% w/v (1g: 100ml) and 10% w/v (10g: 100ml) of

water soluble portions of leaves powders were determined using standard simple glass

electrode pH meter [Choudhary & Sekhon, 2011].

Determination of hot water and ethanol-extractable matter

Separately place about 4.0g of leaves powder in an accurately weighed, glass toppered

conical flask. For estimation of hot water -extractable matter, 100ml of distilled water was

added to the flask and weighed to obtain the total weight including the flask. The contents

were shaken well and allowed to stand for 1 hour. A reflux condenser was attached to the

flask and boiled gently for 1 hour; cooled and weighed. The flask was readjusted to the

original total weight with distilled water and it was shaken well and filtered rapidly

through a dry filter. Then 25ml of the filtrate was transferred to an accurately weighed,

54

tarred flat-bottomed dish (Petri dish) and evaporated to dryness on a water-bath. Finally,

it was dried at 105°C for 6 hours in an oven, cooled in desiccators for 30 minutes, and

weighed without delay. Same procedure was followed using ethanol instead of distilled

water to determine extractable matter in ethanol. The extractable matter was calculated as

the content of in mg per gm of air -dried material.

4.3. RESULTS AND DISCUSSION

The physicochemical evaluation of the plant powder was an important parameter in

detecting adulteration or improper handling of drugs. Physicochemical parameters of

whole plant powder of Couroupita guianensis, Limnophila heterophylla and Michelia

champaca were estimated separately based on the methods recommended by WHO.

The percentage of active chemical constituents in any crude drugs was used to

mention on air dried basis. As apparent from Table 4.1, loss on drying or moisture content

values were found to be 10.25 ± 0.33, 9.25 ± 0.33 and 10.25 ± 0.33 respectively for

Couroupita guianensis, Limnophila heterophylla and Michelia champaca. The less value

of moisture content of drugs could prevent content bacterial, fungal or yeast growth

through storage (Pandey et al., 2010; Bhattacharya and Zaman, 2009). Ash value was

particularly important in the evaluation of purity of drugs, i.e. the presence or absence of

foreign inorganic matter such as metallic salts and/or silica (Musa et al., 2006; Swamy et

al., 2012). The ash values like total ash (08.16 ± 0.09); water soluble ash (02.75 ± 0.08),

acid insoluble ash (01.89 ± 0.07) and sulfated ash value (01.30 ± 0.10) were estimated for

Couroupita guianensis. Total ash; water soluble ash; acid insoluble ash and sulfated ash

values for Limnophila heterophylla were found to be 8.36 ± 0.07, 1.75 ± 0.08, 1.85 ± 0.06

and 1.20 ± 0.10. Similarly, the above mentioned values for Michelia champaca were

found to be 7.36 ± 0.07, 1.75 ± 0.07, 1.55 ± 0.06 and 1.20 ± 0.10 respectively.

The pH of 1% w/ v and 10% w/v solutions of all three plant powders were found

to be 05.12 ± 0.02; 04.87 ± 0.04, 5.12 ± 0.02; 3.87 ± 0.04 and 4.12 ± 0.02; 2.87 ± 0.04

respectively. These values were showed not much difference in the pH of water soluble

portions of whole plants of Limnophila heterophylla and Michelia champaca. The

solubility percentage of Couroupita guianens in aqueous hot extraction is higher (37.21 ±

55

1.27) when compared with ethanolic hot extraction (24.2 ± 0.64). Whereas Limnophila

heterophylla aqueous hot extraction was higher (34.21 ± 1.27) when compared with

ethanoic hot extraction (24.52 ± 0.61). Likewise, the percentage solubility of Michelia

champaca hot extraction was higher (34.21 ± 1.17) when compared with ethanol (22.52 ±

0.61) hot extraction. The extractive values were useful to evaluate the chemical

constituents present in the crude drug and also help in estimation of specific constituents

soluble in a particular solvent (Shweta et al., 2011).

4.4. CONCLUSION

Therefore, present work was taken up in the view to completely standardize the plants in

accordance to parameters of World Health Organization (WHO) guidelines and standard

laboratory procedures. The present study on the pharmacognostic standardization of the

Couroupita guianensis, Limnophila heterophylla and Michelia champaca plant materials

might be useful to supplement information with regard to its identification parameters,

which are assumed significant for the acceptability of herbal drugs in the present

consequence, which lacks regulatory laws to control the quality of herbal drugs.

Therefore, the result generated from this study would be useful in identification and

standardization of the plant material towards quality assurance.

56

Table 4.1. Physicochemical parameters of leaves of Couroupita guianensis,

Limnophila heterophylla and Michelia champaca

Parameters Standard

values

Couroupita

guianensis Limnophila

heterophylla

Michelia

champaca

Loss on drying 12.15 ± 0.33 10.25 ± 0.33 9.25 ± 0.33 10.25 ± 0.33

Total ash value 9.16 ± 0.09 08.16 ± 0.09 8.36 ± 0.07 7.36 ± 0.07

Water soluble ash 3.35 ± 0.09 (02.75 ± 0.08 1.75 ± 0.08 1.75 ± 0.07

Acid insoluble ash 3.79 ± 0.07 01.89 ± 0.07 1.85 ± 0.06 1.55 ± 0.06

Sulfated ash value 1.29 ± 0.10 01.30 ± 0.10 1.20 ± 0.10 1.20 ± 0.10

pH of 1% w/v

solution 6.12 ± 0.02 05.12 ± 0.02 5.12 ± 0.02 4.12 ± 0.02

pH of 10% w/v

solution 3.67 ± 0.04 04.87 ± 0.04 3.87 ± 0.04 2.87 ± 0.04

Water soluble

(hot) extractive

value

39.11 ± 1.27 37.21 ± 1.27 34.21 ± 1.27 34.21 ± 1.17

Ethanol soluble

(hot) extractive

value

25.12 ± 0.64 24.52 ± 0.61 24.52 ± 0.61 22.52 ± 0.61

Chapter-V

Extraction, Phytochemical analysis and

TLC Study of Couroupita guianensis,

Limnophila heterophylla & Michelia

champaca

57

CHAPTER-V

EXTRACTION, PHYTOCHEMICAL ANALYSIS AND TLC

STUDY OF COUROUPITA GUIANENSIS, LIMNOPHILA

HETEROPHYLLA AND MICHELIA CHAMPACA

5.1. INTRODUCTION

Quality can be defined as the status of a drug that is determined by identity, purity, content and

other chemical, physical, biological properties, or by the manufacturing processes. Quality control

is a term that refers to processes involved in maintaining the quality and validity of a

manufactured product. Generally, phytochemical investigation of a plant may thus involve the

following stages: Extraction of plant materials, preliminary phytochemical screening, separation

and isolation of the constituents and characterization of the isolated compounds. These plant-

based traditional medicine systems continue to play an essential role in health care, with about

80% of the world’s inhabitants relying mainly on traditional medicines for their primary health

care. Plant products also have an important role in the health care systems of the remaining 20%,

who reside in developed countries. Indeed, analysis for alkaloids, flavonoids, steroids,

carbohydrates, tannins, phenolics, quinines and terpenoids has been successfully carried out on

herbarium plant tissues dating back many years (Harborne, 1973). They act as a natural defense

system for host plants and provide colour, aroma and flavor (Ahmed and Urooj, 2010). The

flavonoids are also widely distributed in plants both as co-pigment to anthocyanins in petals and

also in leaves of higher plants. The flavonols occur most frequently in glycosidic combination like

anthocyanins. Alkaloids, steroids, triterpenoids, saponins are also present in plants (Harborne,

1973). The present chapter deals with extraction, qualitative analysis and thin layer

chromatography (TLC) study of leaves of Couroupita guianensis, Limnophila heterophylla and

Michelia champaca.

5.2. MATERIALS

Plant Materials

Leaves of Couroupita guianensis, Limnophila heterophylla and Michelia champaca

(described in chapter-III) were used in this examination.

58

Chemicals and Reagents

All chemicals used in the study were of analytical grade and they were procured from

Coastal Enterprises Pvt. Ltd., Visakhapatnam, Andhra Pradesh, India.

Preparation of TLC Plates

TLC plates were ready by using silica Gel-GF 254 as adsorbent. 20gm silica gel-GF was

mixed with 40ml of distilled water (1:2) to create suspension. The suspension was straight

off poured into the plates. Plates were then allowed to air dry for one hour and layer was

mounted by drying at 1100C for one and half hour.

Phytochemical Screening

Extracts of three distinct plants were subjected to the preliminary phytochemical tests so

as to examine the existence of different phytoconstituents namely; alkaloids, glycosides,

starches, terpinoids, phenolic, tannins, sugars, steroids, flavonoids, amino acids and oils

(Saxena et al., 2012).

Test for Steroids

Libermann-Burchard Test

10 mg of extract was dissolved in 1ml of chloroform (CHCl3). 1 ml of acetic anhydride

was added following the addition of 2ml of concentrated sulphuric acid, a reddish violet

color developed, indicating the presence of steroids (Mukherjee, 2002; Singh, 1995).

Salkowski Test

1 ml of concentrated sulphuric acid (Conc. H2SO4) was added to 10 mg of extract

dissolved in 1 ml of chloroform. A reddish-blue color exhibited by chloroform layer and

green fluorescence by the acid layer suggested the presence of steroids.

Test for Triterpinoids

In the test tube, 2 or 3 granules of tin was added, and dissolved in a 2 ml of thionyl

chloride solution and test solution was added. Pink color was produced which indicates

the presence of triterpinoids (Kokate, 1991).

59

Test for Flavonoids

5 ml of extract solution was hydrolyzed with 10% v/v sulphuric acid and cooled. Then it

was extracted with diethyl ether and divided into three portions in three separate test

tubes. 1 ml of dilute ammonia, 1 ml of dilute sodium bicarbonate and 1 ml of 0.1 (N)

sodium hydroxide were added to the first, second and third test tubes respectively. In each

test tube, development of yellow color indicated the presence of flavonoids (Trease and

Evans, 1972; Trease and Evans, 1983).

Test for Alkaloids

1.2 ml of extract was taken in a test tube. 0.2 ml of dilute hydrochloric acid and

0.1 ml of Mayer’s reagent were added. Formation of yellowish buff colored

precipitate gives positive test for alkaloid (Mukherjee, 2002).

1.0 ml of dilute hydrochloric acid and 0.1 ml of Dragendroff's reagent were added

in 2 ml solution of extract in a test tube. Development of orange brown colored

precipitate suggested the presence of alkaloid (Trease and Evans, 1972).

Test for Protein and Amino Acids (Kokate, 1991; Singh, 1995)

Biuret Test: 1 ml of 40% NaOH mixed with 2 drops of 1% copper sulphate to the extract,

a violet color indicated the presence of proteins.

Ninhydrin Test: Freshly prepared 0.2% Ninhydrin reagent (2 drops) was treated with

extract and heated. A blue color developed indicating the presence of proteins or peptides

or amino acids.

Xanthoprotein Test: The extract treated with 1 ml of conc. nitric acid, White precipitate

formed. Further, boiled and cooled. Sodium hydroxide (20%) or ammonia was added to

the solution. Orange color indicated presence of aromatic amino acid.

Test for Reducing Sugars (Mukherjee, 2002; Singh, 1995)

5 ml of the extract solution, mixed with 5 ml of Fehling’s solution was boiled for 5

minutes. Formation of brick red colored precipitate demonstrated the positive test for

60

reducing sugars. To 5 ml of the extract solution, 5 ml of Benedict’s solution was added in

a test tube and boiled for few min. Development of brick red precipitate confirmed the

presence of reducing sugars.

Test for Deoxy Sugars

Keller-Kiliani Test: To 1gm of the sample, 10 ml of 70% ethyl ethanol were added and

boiled for 2-3 min. it was filtered and to the 5 ml of the filtrate, 5 ml of distilled water and

0.5 ml strong lead acetate solution were added. It was filtered and 5 ml of chloroform

were added to the filtrate. Excess chloroform was pipetted off and gentle evaporation of

chloroform was done on a porcelain dish. It was cooled and to the residue, 3 ml of glacial

acetic acid and 2 drops of 5% ferric chloride were added. The solution was transferred to

the surface of 2 ml concentrated sulphuric acid. Reddish brown color (which changed to

bluish green to dark on standing) at the junction confirmed the presence of deoxy sugars

in the sample (Kokate, 1991).

Test for Glycosides (Vinod, 2002)

Legal Test: Extract was dissolved in pyridine; sodium nitroprusside solution was added

to it and made alkaline. Pink red color was produced.

Baljet Test: To a drug extract, sodium picrate solution was added, yellow to orange color

was produced.

Borntrager’s Test: Few ml of dil. sulphuric acid added to the test solution. Boiled,

filtered and extracted the filtrate with ether or chloroform. Then organic layer was

separated to which ammonia was added, pink red color was produced in organic layer.

Test for Pentose

Few ml of extract was dissolved in conc. hydrochloric acid and phloroglucinol (1:1) were

added and heated. Red coloration confirmed the presence of pentose (Kokate, 1991).

61

Test for Tannins

5 ml of extract solution was allowed to react with 1 ml of 5% ferric chloride

solution. Greenish black coloration indicated the presence of tannins.

5 ml of the extract was treated with 1 ml of 10% aqueous potassium dichromate

solution. Formation of yellowish brown precipitate suggested the presence of

tannins (Tyler et al., 1988).

5 ml of extract was treated with 1 ml of 10% lead acetate solution in water.

Yellow color precipitation gave the test for tannins.

Test for Saponins

1 ml solution of the extract was diluted with distilled water to 20 ml and shaken in a

graduated cylinder for 15 min. Development of stable foam suggested the presence of

saponins (Kokate, 1991).

Test for Gums (Kokate, 1991)

2ml of concentrated sulphuric acid was added to 2ml of extract solution. Then it was

treated with 15% ethanolic α-naphthol (Molisch’s reagent). Formation of a reddish violet

ring at the junction of two layers indicated the positive test for gums (Molisch’s test).

5.3. METHODS

5.3.1. Preparation of Extracts

Leaves (Couroupita guianensis, Limnophila heterophylla and Michelia champaca) were

dried in shade and pulverized independently into coarse powder by a mechanical

processor. The subsequent course powders of plants were utilized independently for

further studies. Leaves were separately refluxed successively with the four different

solvents like petroleum ether, chloroform, ethyl acetate and methanol in a soxhlet

extractor for 72 hrs in batches of 500gm each. Unfailingly, the marc was dried before

extracting with the next solvent. The extracts were dried and stored in desiccators for

phytochemical, hepatoprotective and antioxidant examines.

62

5.3.2. TLC Study of Different Extracts of Couroupita guianensis

Employing a micropipette, regarding 10μl of extracts were loaded step by step over the

plate and air dried. The plates were developed in numerous solvent systems such as;

dioxane: ammonia 25% (9:1); Chloroform: acetone: diethyl amine (5:4:1); toluene:

chloroform: ethanol (40:40:10) ethyl acetate: acetic acid: formic acid: water

(100:11:11:26). The different solvent systems showed different Rf value for the same

plant extract. The chromatograms were observed under visible light and were

photographed. The Rf value was obtained by using the following formula.

5.3.3. TLC Study of Different Extracts of Limnophila heterophylla

Employing a micropipette, regarding 10μl of extracts were loaded step by step over the

plate and air dried. The plates were developed in numerous solvent systems such as; ethyl

acetate: formic acid: acetic acid: water (100:11:11: 26); toluene: dioxane: acetic acid

(50:40:10); ethyl acetate: toluene: formic acid (50:50:15) and toluene: chloroform:

ethanol (40:40:10) for determination of flavonoid and terpene respectively. The different

solvent systems showed different Rf value for the same plant extract. The chromatograms

were observed under visible light and were photographed. The Rf value was obtained by

using the following formula.

5.3.4. TLC Study of Different Extracts of Michelia champaca.

Employing a micropipette, regarding 10μl of extracts were loaded step by step over the

plate and air dried. Dioxane: ammonia 25% (9:1); chloroform: acetone: diethyl amine

(5:4:1); Toluene: chloroform: ethanol (40:40:10) and ethyl formate: toluene: formic acid

(50:50:15) solvent systems were used for detection of alkaloid and terpene respectively.

The different solvent systems showed different Rf value for the same plant extract. The

chromatograms were observed under visible light and were photographed. The Rf value

was obtained by using the following formula.

Distance traveled by the substance (cm)

Rf = --------------------------------------------------------

Distance traveled by the solvent (cm)

63

5.4. RESULTS AND DISCUSSION

5.4.1. Successive extraction

Couroupita guianensis

The air dried powder of Couroupita guianensis leaves were extracted by successive

extraction with a variety of solvents. The average yield (% w/w) obtained during

extraction with petroleum ether, chloroform, ethyl acetate, and methanol was found to be

3.8, 4.2, 6.0 & 8.2 respectively. The average yield during successive extraction of

Couroupita guianensis plant with four different solvents was tabulated as Table No.5.1

Table: 5.1. Successive extraction of Couroupita guianensis

Type of extract Amount of extract

(gm)

Yield (%

w/w) Appearance

Petroleum ether 19 3.8 Yellowish black

Chloroform 21 4.2 Greenish brown

Ethyl acetate 30 6.0 Brownish black mass

Methanol 41 8.2 Brownish mass

Limnophila heterophylla

The air dried powder of Limnophila heterophylla leaves extracted by successive

extraction with a variety of solvents. The average yield (% w/w) obtained during

extraction with petroleum ether, chloroform, ethyl acetate, and methanol was found to be

3.0, 5.5, 2.6 & 4.8 respectively. The average yield during successive extraction of

Limnophila heterophylla plant with four different solvents was tabulated as Table No.5.2

Table: 5.2 Successive extraction of Limnophila heterophylla

Type of extract Amount of extract

(gm)

Yield (%

w/w) Appearance

Petroleum ether 15 3.0 Yellowish black

Chloroform 22 5.5 Greenish brown

Ethyl acetate 13 2.6 Brownish black mass

Methanol 24 4.8 Brownish mass

64

Michelia champaca

The air dried powder of Michelia champaca leaves were extracted by successive

extraction with a variety of solvents. The average yield (% w/w) obtained during

extraction with petroleum ether, chloroform, ethyl acetate, and methanol was found to be

3.8, 3.6, 3.0 & 7.0 respectively. The average yield during successive extraction of

Michelia champaca plant with four different solvents was tabulated as Table No.5.3.

Table: 5.3 Successive extraction of Michelia champaca

Type of extract Amount of extract

(gm)

Yield (%

w/w) Appearance

Petroleum ether 19 3.8 Yellowish black

Chloroform 28 3.6 Greenish brown

Ethyl acetate 15 3 Brownish black mass

Methanol 35 7 Brownish mass

5.4.2. Phytochemical Analysis

It was observed that the preliminary phytochemical screening (Table No 5.4) of

Couroupita guianensis showed the presence of carbohydrates, glycosides, triterpinoids

and saponins in petroleum ether extract. Chloroform extract revealed the presence of

carbohydrates, proteins, alkaloids, steroids, phenolics, glycosides, steroids and tannins.

Ethyl acetate extract showed the presence of carbohydrates, proteins, alkaloids,

glycosides, steroids and flavonoids, while the methanolic extract showed the presence of

proteins, alkaloids, glycosides, steroids, triterpinoids, saponins, tannins and flavonoids.

Similarly, it was observed that the preliminary phytochemical screening of

Limnophila heterophylla showed the presence of triterpinoids, flavonoids and saponins in

petroleum ether extract. Chloroform extract showed the presence of alkaloids, steroids,

flavanoids, phenolics and tannins. Ethylacetate extract showed the presence of alkaloids,

flavanoids and steroids while the methanolic extract showed the presence of steroids,

triterpinoids, saponins and flavonoids. Phytochemical constituents present in Limnophila

heterophylla was illustrated in below Table No 5.5.

65

Table 5.4: Phytochemical constituents present in Couroupita guianensis

Tests Petroleum

ether

extract

Chloroform

extract

Ethyl

acetate

extract

Methanol

extract

Test for carbohydrates

Molisch’s test + + + -

Felhing’s test + + + -

Test for proteins and amino acids

Ninhydrin test + + + +

Biuret test + + + +

Test for alkaloids

Mayer’s test - + + +

Wagner’s test - + + +

Test for fixed oils and fats

Spot test - - _ -

Test for glycosides

Borntrager’s test + + + +

Legal test + + + +

Test for Steroids

Liebermann burchard

test - + + +

Salkowski’s test - + + +

Test for Triterpinoids

Tin+thionyl chloride + - + +

Test for phenolics and

tannins

Ferric chloride test - + - +

Gelatin test - + - +

Lead acetate test - + - +

Alkaline reagent test - + - +

Test for Saponins

Foam test + - - +

Haemolysis test + - - +

Test for Flavones and

flavonoids

Shinoda test - - + +

With NaOH - - + +

66

Likewise, preliminary phytochemical screening of Michelia champaca showed the

presence of triterpinoids, flavonoids and saponins in petroleum ether extract. Chloroform

extract showed the presence of alkaloids, steroids, flavonoids, phenolics and tannins.

Ethylacetate extract showed the presence of alkaloids, flavonoids and steroids while the

methanolic extract showed the presence of steroids, triterpinoids, saponins and

flavonoids. Phytochemical constituents present in Michelia champaca was mentioned in

below Table No 5.6.

5.4.3. TLC study of Couroupita guianensis

Identification of alkaloid from Couroupita guianensis

It was observed that the thin layer chromatography analysis of Couroupita guianensis

chloroform extract showed the presence of alkaloids (Table 5.7) with Rf values of 0.79 &

0.38 in dioxane: ammonia 25% (9:1) & Chloroform: acetone: diethyl amine (5:4:1)

solvent systems respectively. Ninhydrin & Marquis reagents were applied for the

detection of alkaloids. Appearance of gray colour and Violet fluorescence indicated the

presence of alkaloids in chloroform extract.

Table 5.7: TLC Studies for chloroform extract of Couroupita guianensis

Solvent system Spraying

reagent

Colour of

spots

Rf

value Inference

Chloroform: acetone:

diethylamine (5:4:1) Marquis reagent

Violet

fluorescence 0.38

Presence of

Alkaloid

Dioxane: ammonia 25%

(9:1) Ninhydrin Gray colour 0.79

Presence of

Alkaloid

Identification of alkaloid from Couroupita guianensis

The thin layer chromatography analysis of ethyl acetate extract of Couroupita guianensis

showed the presence of triterpenes with Rf values of 0.92 & 0.67 in toluene: chloroform:

ethanol (40:40:10) ethyl acetate: acetic acid: formic acid: water (100:11:11:26) solvent

systems correspondingly. Anisaldehyde-sulphuric acid reagent was applied for the

detection of triterpenes. Appearance of dark colour indicated the presence of triterpenes

67

(Table 5.8) in ethyl acetate extract. TLC Studies for ethyl acetate extract of Couroupita

guianensis were mentioned below table No: 5.8.

Table 5.5: Phytochemical constituents present in Limnophila heterophylla

Tests Petroleum

ether extract

Chloroform

extract

Ethyl

acetate

extract

Methanol

extract

Test for proteins and amino acids

Ninhydrin test - - - -

Biuret test - - - -

Test for alkaloids

Mayer’s test - + + +

Wagner’s test - + + +

Test for fixed oils and fats

Spot test - - - -

Test for glycosides

Borntrager’s test - - - -

Legal test - - - -

Test for Steroids

Liebermann burchard

test - + + +

Salkowski’s test - + + +

Test for Triterpinoids

Tin+thionyl chloride + + + +

Test for phenolics and tannins

Ferric chloride test - + - -

Gelatin test - + - -

Lead acetate test - + - -

Alkaline reagent test - + - -

Test for Flavones and flavonoids

Shinoda test + + + +

With NaOH + + + +

68

Table 5.6: Phytochemical constituents present in Michelia champaca

Tests Petroleum

ether extract

Chloroform

extract

Ethyl

acetate

extract

Methanol

extract

Test for proteins and amino acids

Ninhydrin test - - - -

Biuret test - - - -

Test for alkaloids

Mayer’s test - + + +

Wagner’s test - + + +

Test for fixed oils and fats

Spot test - - - -

Test for glycosides

Borntrager’s test - - - -

Legal test - - - -

Test for Steroids

Liebermann burchard - + + +

Salkowski’s test - + + +

Test for Triterpinoids

Tin+thionyl chloride + + + +

Test for phenolics and tannins

Ferric chloride test - + + -

Alkaline reagent test - + + -

Test for flavonoids

Shinoda test + + + +

With NaOH + + + +

Table 5.8: TLC Studies for ethyl acetate extract of Couroupita guianensis

Solvent system for

compound

Spraying

reagent

Colour of spots Rf

value

Inference

Toluene: chloroform:

ethanol (40:40:10)

Anisaldehyde-

sulphuric acid Blue-violet colour 0.92

Presence of

triterpene

Ethyl acetate: acetic

acid: formic acid: water

(100:11:11:26)

Anisaldehyde-

sulphuric acid Red-violet colour 0.67

Presence of

triterpene

69

TLC study of Limnophila heterophylla

Identification of Flavonoid from Limnophila heterophylla

The thin layer chromatography analysis of ethyl acetate extract of Limnophila

heterophylla showed the presence of flavonoids with Rf values of 0.82 & 0.98 in toluene:

dioxin: acetic acid (90:25:4) & ethyl acetate: formic acid: gla. acetic acid: water

(100:11:11:26) solvent systems correspondingly. Iodine vapours & Natural products- poly

ethylene glycol reagent (NP/PEG) were applied for the detection of flavonoids.

Appearance of orange yellow colour and intense fluorescence colour indicated the

presence of flavonoids in ethyl acetate extract. TLC Studies for ethyl acetate extract of

Limnophila heterophylla were illustrated in below table No: 5.9.

Table 5.9: TLC Studies for ethyl acetate extract of Limnophila heterophylla

Solvent system Spraying reagent Colour of

spots Rf value Inference

Toluene: dioxin: acetic

acid (90:25:4) Iodine vapours

Orange-

yellow 0.82

Presence

of

Flavonoid

Ethyl acetate: formic

acid: gla. acetic acid :

water (100:11:11:26)

Natural products-

poly ethylene

glycol reagent

(NP/PEG)

Intense

fluorescence

colour

0.98

Presence

of

Flavonoid

Identification of triterpene from Limnophila heterophylla

The thin layer chromatography analysis of ethyl acetate extract of Limnophila

heterophylla showed the presence of triterpenes with Rf values of 0.72 & 0.56 in toluene:

chloroform: ethanol (40:40:10) ethyl acetate: acetic acid: formic acid: water

(100:11:11:26) solvent systems correspondingly. Anisaldehyde-sulphuric acid reagent

was applied for the detection of triterpenes. Appearance of dark colour indicated the

presence of triterpenes (Table 5.10) in ethyl acetate extract.

70

TLC study of Michelia champaca

Identification of triterpene from Michelia champaca

The thin layer chromatography analysis of ethyl acetate extract of Michelia champaca

showed the presence of triterpenes (Table 5.11) with Rf values of 0.88 & 0.78 in toluene:

chloroform: ethanol (40:40:10) ethyl acetate: acetic acid: formic acid: water

(100:11:11:26) solvent systems correspondingly. Anisaldehyde-sulphuric acid reagent

was applied for the detection of triterpenes. Appearance of dark colour indicated the

presence of triterpenes in ethyl acetate extract.

Table 5.10: TLC Studies for ethyl acetate extract of Limnophila heterophylla

Solvent system for

compound

Spraying reagent Colour of

spots

Rf value Inference

Toluene: chloroform:

ethanol (40:40:10)

Anisaldehyde-

sulphuric acid

reagent

Blue-

violet

colour

0.72 Presence of

triterpene

Ethyl acetate: acetic

acid: formic acid:

water (100:11:11:26)

Anisaldehyde-

sulphuric acid

reagent

Red-violet

colour 0.56

Presence of

triterpene

Table 5.11: TLC Studies for ethyl acetate extract of Michelia champaca

Solvent system for

compound

Spraying reagent Colour of

spots

Rf value Inference

Ethyl formiate: toluene:

formic acid (50:50:15)

Anisaldehyde-

sulphuric acid

reagent

Blue-violet

colour 0.88

Presence of

triterpene

Ethyl acetate: acetic

acid: formic acid: water

(100:11:11:26)

Anisaldehyde-

sulphuric acid

reagent

Red-violet

colour 0.78

Presence of

triterpene

Identification of phenolic compound from Michelia champaca

The thin layer chromatography analysis of ethyl acetate extract of Michelia champaca

showed the presence of phenolic compounds with Rf values of 0.62 & 0.67 in chloroform:

ethyl-acetate: formic acid (5:4:1), ethyl acetate: acetic acid: formic acid: water

(100:11:11:26) solvent systems correspondingly. FeCl3 (2% in ethanol) & Anisaldehyde-

sulphuric acid reagents were applied for the detection of phenolic compounds.

71

Appearance of dark colour indicated the presence of phenolic compounds in ethyl acetate

extract. TLC Studies for ethyl acetate extract of Michelia champaca were illustrated in

below table No: 5.12.

Table 5.12: TLC Studies for ethyl acetate extract of Michelia champaca

Solvent system for

compound

Spraying

reagent

Colour of

spots

Rf value Inference

Chloroform: ethyl-acetate:

formic acid (5:4:1)

FeCl3 (2% in

ethanol) Blue colour 0.62

Phenolic

compound

Ethyl acetate: acetic acid:

formic acid: water

(100:11:11:26)

Anisaldehyde-

sulphuric acid

reagent

Red-violet

colour 0.67

Phenolic

compound

Chapter-VI

Isolation and characterization of

phytoconstituents from Couroupita

guianensis, Limnophila heterophylla &

Michelia champaca

72

CHAPTER-VI

ISOLATION AND CHARACTERIZATION OF PHYTOCONSTITUENTS FROM

DIFFERENT EXTRACTS BY CHROMATOGRAPHY TECHNIQUES

6.1 INTRODUCTION

Natural products from medicinal plants, either as pure compounds or as standardized

extracts, provide unlimited opportunities for new drug leads because of the unmatched

availability of chemical diversity (Veeresh kumar et al., 2015). Botanicals and herbal

preparations for medicinal usage contain various types of bioactive compounds.

According to the World Health Organization (WHO), nearly 20,000 medicinal plants

exist in 91 countries (Sudhakar et al., 2016). The premier steps to utilize the biologically

active compound from plant resources are extraction, pharmacological screening,

isolation and characterization of bioactive compound, toxicological evaluation and

clinical evaluation. Isolation is a process or fact mainly useful for the separation of active

constituents from crude mixture. Due to the fact that plant extracts usually occur as a

combination of various type of bioactive compounds or phytochemicals with different

polarities, their separation still remains a big challenge for the process of identification

and characterization of bioactive compounds. A number of different separation techniques

like TLC, column chromatography, flash chromatography, paper chromatography and

HPLC are in common practice in isolation of bioactive compounds such as like alkaloids,

flavonoids, terpenes, resins, oils, glycosides, phenolic and amino acids (Sasidharan et al.,

2010). Among these isolation techniques, column chromatography, is a very compatible

technique and the most commonly used technique for the isolation of bioactive

components. Column chromatography in chemistry is a method used to purify individual

chemical compounds from mixtures of compounds. It is devised on the basis of

differential adsorbance of substances on solid adsorbent (silica or alumina) to an extent

that depends on the substance polarity and other chemical properties and structural

properties (Clark Still et al., 1978). Some compounds adsorb more strongly to the

stationary phase than others, as they elute (wash down) the column at a very slow rate.

Thus they can be separated on the basis of their elution rate accordingly. The fractions

with potent biological activity is proceed for isolation and identification of

73

phytoconstituents with the help of physical and spectral analysis such as Mass

spectroscopy, Nuclear magnetic resonance (NMR) and Infrared (IR) spectroscopic

analysis. Majority of the biologically active natural products have been isolated using

bioactivity-guided fractionation (Pezzuto et al., 1997). This investigation deals with

isolation and structural elucidation (UV, IR, NMR and Mass) of phytoconstituents from

various extracts of leaves of Couroupita guianensis, Limnophila heterophylla and

Michelia champaca.

6.2. MATERIALS

Chemicals

All chemicals used in the study were of analytical grade and they were procured from

Coastal Enterprises Pvt. Ltd., Visakhapatnam, Andhra Pradesh, India.

Reagents

The reagents (described in chapter-V) used in the study were of analytical grade and they

were procured from Coastal Enterprises Pvt. Ltd., Visakhapatnam, Andhra Pradesh, India.

Instruments

The absorbance was measured using UV-VIS double beam spectro photometer

[SHIMADZU-UV-1800]. IR was taken in BRUKER-ALPHA-FT-IR spectrometer. 1H-

NMR spectra were recorded at 400 MHz NMR spectrometer (Bruker DPX – 400) with

solutions in DMSO using 1% TMS (tetra methyl silane) as internal standard. Mass spectra

were recorded on AGILENT QQQ LCMS-6410 spectrometer.

6.3. Isolation of alkaloid from methanol extract of Couroupita guianensis

Methanol extract of leaves of Couroupita guianensis was dissolved in 2% sulfuric acid

and defatted with diethyl ether. Then, the acidic solution was made alkaline with 25%

ammonia to pH 9-10, and it was extracted with chloroform. A crude mixture was obtained

after evaporation of the organic solvent and it was subjected to column chromatography

on silica gel, eluting with hexane: chloroform (5:0, 4.5:0.5, 4.0:1.0, 3.5:1.5, 3.0:2.0,

2.5:2.5, 2.0:3.0, 1.5:3.5, 1.0:4.0, 0.5:4.5), chloroform: ethyl acetate (5.0:0, 4.0:1.0,

74

3.0:2.0, 2.0:3.0, 1.0:4.0, 0.5:4.5, 0:5.0) & ethyl acetate: methanol (4:1, 3:2, 2.5:2.5,

2:3,1:4, 5:45, 0:5) to obtain 251fractions. Each and every fraction of 50 ml was collected

from different solvent systems. Fractions were subjected to TLC analysis and the fraction

having same Rf value were combined. Fraction number from 204 to 211 having same Rf

values, which was obtain with ethyl acetate: methanol (2:3). After evaporation of the

solvent at reduced pressure, brown amorphous powder (compound-I) was formed. The

flow chart diagram of the isolation of alkaloid from methanolic extract of Couroupita

guianensis as mentioned in Figure 6.1.

Plant extracted with methanol

Residue was dissolved in 2% sulfuric acid, and defatted with Diethyl ether

Acidic soln. was made alkaline with 25% ammonia to pH 9-10

Extracted with CHCl3

Crude mixture was subjected to column chromatography

Hexane gradually enriched with chloroform (5:0, 4.5:5, 4:1, 3.5:1.5, 3:2, 2.5:2.5, 2:3,

1.5:3.5, 1:4, 0.5:4.5), and chloroform: ethyl acetate (5:0, 4:1, 3:2, 2:3, 1:4, 0.5:4.5, 0:5.0)

Rechromatographed column was run with ethyl acetate :methanol (4:1, 3:2, 2.5:2.5,

2:3,1:4, 5:4, 0:5)

50ml of 251 fractions were collected

Fractions were subjected to TLC studies

Fraction number from 204 to 211 having same Rf values were combined

After evaporation of the solvent at brown amorphous powder was formed

Figure 6.1: Flow chart diagram of the isolation of alkaloid from Couroupita guianensis

75

Qualitative test for compound-I

The compound-I was tested for alkaloid, glycoside, terpene, tannins, carbohydrates,

steroid and flavonoids with different chemical reagents as discussed as discussed in

chapter-V.

Thin layer chromatography of compound-I

The compound-I (10μl) was loaded step by step over the plate by using a micropipette,

and air dried. The chromatogram was developed under TLC chamber saturation

conditions with ethyl acetate: methanol (9:1) & ethyl acetate: methanol: water

(100:13.5:10) as the solvent systems.

Spray reagent for alkaloid

Dragendorff reagent: 0.85 gm of basic bismuth nitrate was dissolved in 40 ml water and

10 ml glacial acetic acid, followed by addition of 8 gm potassium iodide dissolved in 20

ml water. Brown zone appears immediately on spraying. The colour was not stable.

Marquis reagent: 3 ml of formaldehyde was diluted to 100ml with concentrated sulphuric

acid. The plate is evaluated in Vis, immediately after spraying.

6.4. Isolation of triterpenes from ethyl acetate extract of Couroupita guianensis

The ethyl acetate extract was extracted with dichloromethane for 5hrs in a Soxhlet

apparatus. The extract was filtered and concentrated in vacuum to yield a residue. The

residue (dichloromethane extract) was subjected to column chromatography on silica gel,

eluting with hexane: chloroform (5:0, 4.5:0.5, 4.0:1.0, 3.5:1.5, 3.0:2.0, 2.5:2.5, 2.0:3.0,

1.5:3.5, 1.0:4.0, 0.5:4.5) and chloroform: methanol (5.0:0, 4.5:0.5, 4.0:1.0, 3.5:1.5,

2.5:2.5, 2.0:3.0, 1.0:4.0, 0.5:4.5, 0:5.0) to obtain 180 fractions. Each and every fraction of

50 ml was collected from different solvent systems. Fractions were subjected to TLC

analysis and the fraction having same Rf value were combined. Fraction number from 160

to 168 having same Rf values, which was obtain with chloroform: methanol (1:4). After

evaporation of the solvent at reduced pressure, white amorphous solid (compound-II) was

76

formed. The flow chart diagram of the isolation of triterpenes from ethyl acetate extract of

Couroupita guianensis as mentioned in Figure.6.5.

Plant extracted with ethyl acetate

Marc was dried and extracted with dichloromethane

Crude mixture was subjected to column chromatography

Hexane gradually enriched with chloroform (5:0, 4.5:0.5, 4.0:1.0, 3.5:1.5, 3.0:2.0, 2.5:2.5,

2.0:3.0, 1.5:3.5, 1.0:4.0, 0.5: 4.5)

Eluted with chloroform & methanol (5.0:0, 4.5:0.5, 4.0:1.0, 3.5:1.5, 2.5:2.5, 2.0:3.0,

1.0:4.0, 0.5:4.5, 0:5.0)

50ml of 180 fractions were collected

Fractions were subjected to TLC studies

Fraction number from 160 to 168 having same Rf values were combined

After evaporation of the solvent a white amorphous solid was formed

Figure.6.5. Flow chart diagram of the isolation of triterpene from Couroupita guianensis

Qualitative test for compound-II

The compound-II was tested for alkaloid, glycoside, terpene, tannins, carbohydrates,

steroid and flavonoids with different chemical reagents as discussed qualitative analysis

tests.

77

Thin layer chromatography of compound-II

The compound-II (10μl) was loaded step by step over the plate by using a micropipette

and air dried. The chromatogram was developed under the TLC chamber saturation

conditions with toluene: chloroform: ethanol (40:40:10) & ethyl formiate: toluene: formic

acid (50:50:15) as the solvent systems.

Spray reagent for triterpene

Anisaldehyde-sulphuric acid reagent: 0.5ml of Anisaldehyde was mixed with 10ml of

glacial acetic acid, followed by 85ml of methanol and 5ml conc. sulphuric acid in that

order.

6.5. Isolation of flavonoids from ethyl acetate extract of Limnophila heterophylla

The ethyl acetate extract was submitted to alkaline hydrolysis according to usual

procedure, KOH-H2O-EtOH (1:1) under reflux for 30 min. The crude mixture (ethyl

acetate) was subjected to column chromatography on silica gel, eluting with chloroform:

ethyl acetate (5:0, 4.5:0.5, 4.0:1.0, 3.5:1.5, 3.0:2.0, 2.5:2.5, 2.0:3.0,1.5:3.5, 1.0:4.0,

0.5:4.5) and ethyl acetate: methanol (5.0:0, 4.5:0.5, 4.0:1.0, 3.5:1.5, 2.5:2.5, 2.0:3.0,

1.0:4.0, 0.5:4.5, 0:5.0) to obtain 130 fractions. Each and every fraction of 50ml was

collected from different solvent systems. Fractions were subjected to TLC analysis and

the fraction having same Rf value were combined. Fraction number from 71 to 80 having

same Rf values, which was obtain with ethyl acetate: methanol (4:1). After evaporation

of the solvent at reduced pressure, brown amorphous powder (compound-III) was formed.

The flow chart diagram of the isolation of flavonoids from ethyl acetate extract

Limnophila heterophylla as mentioned in Figure.6.9.

Qualitative test for compound-III

The compound-III was tested for alkaloid, glycoside, terpene, tannins, carbohydrates,

steroid and flavonoids with different chemical reagents as discussed qualitative analysis

tests.

78

Plant extracted with ethyl acetate

Extract was submitted to alkaline hydrolysis

Crude mixture was subjected to column chromatography

Chloroform : ethyl acetate (5:0, 4.5:0.5, 4.0:1.0, 3.5:1.5, 3.0:2.0, 2.5:2.5, 2.0:3.0,1.5:3.5,

1.0:4.0, 0.5:4.5) and Ethyl acetate & methanol (5.0:0, 4.5:0.5, 4.0:1.0, 3.5:1.5, 2.5:2.5,

2.0:3.0, 1.0:4.0, 0.5:4.5, 0:5.0)

50 ml of 130 fractions were collected

Fractions were subjected to TLC studies

Fraction number from 71 to 80 having same Rf values were combined

After evaporation of the solvent a brown amorphous powder was formed

Figure.6.9. Flow chart diagram of the isolation of flavonoid from Limnophila heterophylla

Thin layer chromatography of compound-III

The compound-III (10μl) was loaded step by step over the plate by using micropipette

and air dried. The chromatogram was developed up to 80mm under chamber saturation

conditions with ethyl acetate: formic acid: acetic acid: water (100:11:11: 26) and toluene:

dioxane: acetic acid (50:40:10) as the solvent systems.

79

Spray reagent for flavanoid

Liebermann-Burchard reagent: 5ml of acetic anhydride and 5 ml of conc. sulphuric acid

are added carefully to 50ml absolute ethanol, while cooling in ice.

Natural products- poly ethylene glycol reagent (NP/PEG): The plate was sprayed with

1% ethanolic diphenyl boric acid-β-ethyl amino ester, followed by 5% ethanolic

polyethylene glycol-4000 (10ml and 8ml, respectively).

6.6. Isolation of terpene from methanol extract of Limnophila heterophylla

Methanol extract was concentrated and chloroform is added to concentrate and equal

volume of water is added to the separating funnel and shaked well. Allow to settle and

separate the chloroform layer. Repeat the water treatment two to three times and collect

chloroform layer. Concentrate the chloroform layer. The crude mixture (chloroform

extract) was subjected to column chromatography on silica gel, eluting with benzene:

methanol (5:0, 4.5:0.5, 4.0:1.0, 3.5:1.5, 3.0:2.0, 2.5:2.5, 2.0:3.0,1.5:3.5, 1.0:4.0, 0.5:4.5,

0:5.0). Each and every fraction of 50ml was collected from different solvent systems.

Fractions were subjected to TLC analysis and the fraction having same Rf value were

combined. Fraction number from 61 to 68 having same Rf values, which was obtain with

benzene: methanol (1.5:3.5). After evaporation of the solvent at reduced pressure, a

reddish brown to brown colour powder (compound-IV) was obtained. The flow chart

diagram of the isolation of terpene from methanol extract of Limnophila heterophylla as

mentioned in Figure. 6.13.

Qualitative test for compound-IV

The compound-IV was tested for alkaloid, glycoside, terpene, tannins, carbohydrates,

steroid and flavonoids with different chemical reagents as discussed qualitative analysis

tests.

Thin layer chromatography of compound-IV

The compound-IV (10μl) was loaded step by step over the plate by using a micropipette

and air dried. The chromatogram was developed under TLC chamber saturation

80

conditions with ethyl acetate: formic acid: acetic acid: water (100:11:11: 26) and toluene:

chloroform: ethanol (40:40:10) as the solvent systems.

Plant extracted with methanol

Added CHCl3:H20 (1:1)

Using separating funnel

Separate the chloroform layer

Repeat the water treatment 2-3 times

Collected CHCl3 layer

Crude mixture (CHCl3 layer) was subjected to column chromatography

Benzene :methanol (5:0, 4.5:0.5, 4.0:1.0, 3.5:1.5, 3.0:2.0, 2.5:2.5, 2.0:3.0,1.5:3.5, 1.0:4.0,

0.5:4.5, 0:5.0)

50ml of 100 fractions were collected

Fractions were subjected to TLC studies

Fraction number from 61 to 68 having same Rf values were combined

After evaporation of the solvent a reddish brown to brown colour powder was formed

Figure 6.13. Flow chart diagram of the isolation of terpene from Limnophila heterophylla

81

Spray reagent for Terpene

Anisaldehyde-sulphuric acid reagent: 0.5ml of anisaldehyde was mixed with 10ml of

glacial acetic acid, followed by 85ml of methanol and 5ml conc. sulphuric acid in that

order.

6.7. Isolation of phenolic compound from hexane extract of Michelia champaca

The chloroform extract was dried and extracted with n-hexane for 5hrs in a Soxhlet

apparatus. n-hexane fraction, was subjected to the silica gel column chromatography,

eluting with benzene: ethylacetate (5:0, 4.5:0.5, 4.0:1.0, 3.0:2.0, 2.5:2.5, 2.0;3.0, 1.0:4.0,

0.5:4.5, 0:5.0) to yield several fractions of 1-90. Each and every fraction of 50 ml was

collected from different solvent systems. Fractions were subjected to TLC analysis and

the fraction having same Rf value were combined. Fraction number from 52 to 58 having

same Rf values, which was obtain with benzene: ethylacetate (2:3). After evaporation of

the solvent at reduced pressure, orange colored crystals (compound-V) were obtained.

The flow chart diagram of the isolation of phenolic compound from hexane extract of

Michelia champaca as mentioned in Figure 6.17.

Qualitative test for compound-V

The compound-V was tested for alkaloid, glycoside, terpene, tannins, phenolics,

carbohydrates, steroid and flavonoids with different chemical reagents as discussed

qualitative analysis tests.

Thin layer chromatography of Compound-V

The compound-V (10μl) was loaded step by step over the plate by using a micropipette

and air dried. The chromatogram was developed under TLC chamber saturation

conditions with ethyl acetate: formic acid: acetic acid: water (100:11:11:26) and

chloroform: ethyl-acetate: formic acid (5:4:1) as the solvent systems.

Spray reagent for Phenolic compound

Anisaldehyde-sulphuric acid reagent: 0.5ml of Anisaldehyde is mixed with 10ml of

glacial acetic acid, followed by 85ml of methanol and 5ml conc. sulphuric acid in that

order.

82

FeCl3 (2% in ethanol): Dissolve few ml of FeCl3 solution in 2% in ethanol.

Plant extracted with CHCl3

Chloroform extract

Extracted with n-hexane

n-hexane extrct

Crude mixture (n-hexane) was subjected to column chromatography

Benzene :ethyl acetate (5:0, 4.5:0.5, 4.0:1.0, 3.0:2.0, 2.5:2.5, 2.0;3.0, 1.0:4.0, 0.5:4.5,

0:5.0)

50 ml of 90 fractions were collected

Fractions were subjected to TLC studies

Fraction number from 52 to 58 having same Rf values were combined

After evaporation of the solvent an orange colour crystals was formed

Figure 6.17. Flow chart diagram of the isolation of phenolic compound from Michelia champaca.

6.8. Isolation of triterpene from chloroform extract of Michelia champaca

The defatted methanolic extract was concentrated, dissolved in water, and extracted

sequentially with chloroform and n-butanol. Chloroform fraction, was subjected to the

silica gel column chromatography, eluting with hexane: dichloromethane (5:0, 4.5:0.5,

4.0:1.0, 3.0:2.0, 2.5:2.5, 2.0;3.0, 1.0:4.0, 0.5:4.5, 0:5.0) and dichloromethane: methanol

(5.0:0, 4.5:5, 3.0:2.0, 2.5:2.5, 1.0:4.0, 0.5:4.5: 0:5.0) to yield 170 fractions. Fraction 66-72

was rechromatographed over a silica gel column (20g), eluting with hexane: ethylacetate

83

(2:0, 1.8:0.2, 1.6:0.4, 1.5:0.5, 1.2:0.8, 1.0:1.0, 0.8:1.2, 0.6:1.4, 0.4:1.6, 0.2:1.8, 0:2.0) to

obtain 44 fractions. Each and every fraction of 50 ml collected from different solvent

systems. Fractions were subjected to TLC analysis and the fraction having same Rf value

were combined. Fraction number from 25 to 30 having same Rf values, which was obtain

with hexane: ethyl acetate (0.8:1.2). After evaporation of the solvent at reduced pressure,

orange colored mass (compound-VI) was obtained. The flow chart diagram of the

Isolation of triterpene from chloroform extract of Michelia champaca as mentioned in

Figure 6.21.

Qualitative test for compound-VI

The Compound-VI was tested for alkaloid, glycoside, terpene, tannins, Phenolics,

carbohydrates, steroid and flavonoids with different chemical reagents as discussed

qualitative analysis tests. The results of the study showed that the presence of triterpene

compound in Compound-VI.

Thin layer chromatography of compound-VI

Compound-VI (10μl) was loaded step by step over the plate by using a micropipette and

air dried. The chromatogram was developed under TLC chamber saturation conditions

with ethyl acetate: formic acid: acetic acid: water (100:11:11:26) and toluene: chloroform:

ethanol (40:40:10) as the solvent systems.

Spray reagent for compound-VI

Anisaldehyde-sulphuric acid reagent: 0.5ml of Anisaldehyde is mixed with 10ml of

glacial acetic acid, followed by 85ml of methanol and 5ml conc. sulphuric acid in that

order.

7.4. RESULTS AND DISCUSSION

Qualitative Analysis and TLC of Compound-I

These qualitative phytochemical analysis and TLC studies clearly demonstrated that the

presence of alkaloid in the compound-I. A brown, odorless, crystalline powder was

obtained from methanol. It was practically insoluble in water, slightly soluble in alcohol,

84

and sparingly soluble in aqueous buffers. The material has melting range 283-285○C

(Table. 6.1).

Plant extracted with methanol

Partitioned b/n aqueous methanol &

hexane

Defatted methanolic extract was concentrated, dissolved in water, and extracted

sequentially with chloroform and n-butanol

Chloroform extrct

Crude mixture (CHCl3 extract) was subjected to column chromatography

Hexane : dichloromethane (5:0, 4.5:0.5, 4.0:1.0, 3.0:2.0, 2.5:2.5, 2.0;3.0, 1.0:4.0, 0.5:4.5,

0:5.0) & dichloromethane: methanol (5.0:0, 4.5:5, 3.0:2.0, 2.5:2.5, 1.0:4.0, 0.5:4.5: 0:5.0)

50 ml of 170 fractions were collected

Fractions were subjected to TLC studies

Fractions 66-72 were rechromatographed on silica gel, eluting with hexane: ethyl acetate

(2:0, 1.8:0.2, 1.6:0.4, 1.5:0.5, 1.2:0.8, 1.0:1.0, 0.8:1.2, 0.6:1.4, 0.4:1.6, 0.2:1.8, 0:2.0)

Fraction number from 25 to 30 having same Rf values were combined

After evaporation of the solvent a orange colour crystals was formed

Figure. 6.21. Flow chart diagram of the isolation of triterpene from Michelia champaca

85

Table. 6.1: Thin layer chromatography study of compound-I

Solvent system for

compound

Spraying

reagent

Colour of

spots Rf value Inference

Ethyl acetate:

Methanol: water

(100:13.5:10)

Dragendorff

reagent Brown colour 0.70

Presence of

alkaloid

Ethyl acetate:

Methanol (9:1) Marquis reagent Purple colour 0.64

Presence of

alkaloid

Spectral Analysis of Compound-I

IR and mass spectrum of compound-1

The IR spectra were recorded in the region of 4000 cm-1 to 500 cm-1. From the IR

spectrum, (Figure 6.2) compound-I showed the presence aromatic stretching at 717.06

cm-1, 30 amine stretching at 1054 cm-1, amine stretching at 3053.95cm-1, vibration of

phenolic OH at 3423.85 cm-1, NH bond stretching at 1611.44cm-1, and vibrational

stretching of alkaline CH bond at 1467.59cm-1. In mass spectrum, a molecular ion peak at

m/z 161, corresponding to the molecular formulae C10H15NO (Figure 6.3). Other

fragmentation and prominent peaks at m/z 149, 87, 58. It was concluded that the isolated

compound may be hordenine, from the fragmentation pattern.

NMR Spectrum of compound-I

Thus, the skeleton of compound –I (Figure-6.4) contains 15 hydrogen atoms with the

following characteristic signals. The signal at δ 5.0 was assigned to proton on a methine

carbon bearing a hydroxy group for C-1. The doublet signals at δ 6.651, 6.658 for the 1H

group at H-2 position. The signal at δ 6.953 was due to the 1H group present at H-3

position. The δ value 6.960 indicate 1H group present at H-5 position. The triplet peaks at

δ 2.555 assigned to 2H in H-1' position. A doublet peaks at δ 2.573 indicates 2H present

in H-2' position. The other signals at δ 2.349 and 2.331, corresponding to the protons

present in methyl amine (N-CH3) position. The above data confirmed that the compound-I

was hordenine, which is comparable to the reported value of Berkov et al (2007) isolated

from Galanthus elwesii.

86

Qualitative analysis and TLC of Compound-II

These qualitative phytochemical analysis and TLC studies clearly demonstrated that the

presence of terpene in the compound-II. A white, odorless, crystalline solid was obtained

from methanol. It was practically insoluble in water, slightly soluble in alcohol, and

sparingly soluble in aqueous buffers. The material has melting range 285-288○C (Table

6.2).

Table.6.2 Thin layer chromatography study of compound-II

Solvent system for

compound

Spraying

reagent Colour of spots Rf value Inference

Toluene:

chloroform: ethanol

(40:40:10)

Anisaldehyde-

sulphuric acid

reagent

Blue-violet

colour

0.78 Presence

of terpene

Ethyl formiate:

toluene: formic acid

(50:50:15)

Anisaldehyde-

sulphuric acid

reagent

Red-violet

colour

0.64 Presence of

terpene

Spectral Analysis of Compound-II

IR and mass spectrum of compound-II

The IR spectra were recorded in the region of 4000 cm-1 to 500 cm-1. From the IR

spectrum (Figure 6.6) of compound showed the presence of C-O group at 1188.48 cm-1,

carbonyl group at 1350.65cm-1, 1647.58 cm-1, vibration of methyl C-H at 2943.52 cm-1,

2880.96 cm-1, stretching of methoxy group at 1238.09 cm-1 and vibrational stretching of

hydroxyl group at 3387.91cm-1. In mass spectrum, a molecular ion peak at m/z 455,

corresponding to the molecular formulae C30H48O3 (Figure 6.7). Other fragmentation and

prominent peaks were shown at m/z 439, 248, 191 and 99. From the fragmentation pattern

it was concluded that the isolated compound may be ursolic acid.

NMR Spectrum of compound-II

From 1H NMR spectrum (Figure No 6.8) exhibits singlet peaks at δ 11.0 and δ 1.992 due

to presence of carboxyl group for H-28 position. A doublet peak occurs at δ 3.550 due to

H-3 hydroxy group and δ 2.368 for 1H at H-18 position. The typical multiplet peaks at δ

5.731 for 1H at H-12 position and δ 3.540 for 1H for H-3 position, respectively. The

broad multiplet peak at δ 2.028 showed the presence of CH2 proton in H-22 position.

87

The signals at δ 0.76 – 1.06 indicated that presence of proton at H-21 position and 7

methyl groups. The above data confirmed that the compound- II was ursolic acid, which

is comparable to the reported value of Suhagia et al (2013) isolated from Alstonia

scholaris.

Qualitative analysis and TLC of compound-III

These qualitative phytochemical analysis and TLC studies clearly demonstrated that the

presence of flavonoid in the compound-III. A brown, odorless, amorphous powder was

obtained from methanol. It was practically soluble in ethanol, Dimethyl sulfoxide, and

water. The material has melting range 216-220 °C (Table.6.3).

Table.4.3 Thin layer chromatography study of compound-III

Solvent system for

compound Spraying reagent

Colour of

spots

Rf

value Inference

Ethyl acetate: formic

acid: acetic acid: water

(100:11:11: 26)

Liebermann-

Burchard reagent Dark colour 0.98

Presence of

flavonoid

Toluene: dioxane: acetic

acid (50:40:10)

Natural products-

poly ethylene glycol

reagent (NP/PEG)

Intense

fluorescence

colour

0.74 Presence of

flavonoid

Spectral analysis of compound-III

IR and mass spectrum of compound-III

The IR spectra were recorded in the region of 4000 cm-1 to 500 cm-1. From the IR

spectrum (Figure. 6.10) of compound showed the presence of aromatic OH at 3418.27

cm-1, carbonyl group at 1616.69 cm-1, aromatic C=C stretching at 1475.14 cm-1, 1265.59

cm-1, stretching of carbonyl OH group at 3198.91 cm-1, C-O stretching at 1082.61 cm-1

and vibrational stretching of aromatic C-H at 2924.21cm-1. In mass spectrum, a molecular

ion peak at m/z 302, corresponding to the molecular formulae C15H12O7 (Figure 6.11).

Other fragmentation and prominent peaks were shown at m/z 191, 147, and 99. From the

fragmentation pattern it was concluded that the isolated compound may be Taxifolin.

NMR Spectrum of compound-III

In NMR spectrum (Figure 6.12), A doublet signal at δ 5.728 and 5.738 was due to

presence of proton in 2H and 3H positions. Additionally, the signal peak at δ 11.882 was

88

a typical one for a C-5 hydrogen bonded hydroxyl group. A singlet peak at δ 2.00

indicates hydroxyl proton at H-3 position and δ 6.712 for proton present in H-2' position.

The hydroxyl proton present in H-5, H-7, H-3' and H-4' position of substitution resonates

at δ 4.979. Further, it also showed singlet peak at 5.895 indicates 1H proton present in H-

8 position. The above data suggests that the compound-III may be taxifolin and it was

earlier isolated by Chhagan Lal et al., 2011 from Juglans nigra.

Qualitative analysis and TLC of compound-IV

These qualitative phytochemical analysis and TLC studies clearly demonstrated that the

presence of terpene in the fraction. A reddish brown to brown, odorless, powder was

obtained. It was practically soluble in dimethyl sulphoxide, ethanol, methanol and

chloroform. The material has melting range 230-232 °C (Table 6.4).

Table.6.4 Thin layer chromatography study of compound-IV

Solvent system for

compound

Spraying

reagent Colour of spots Rf value Inference

Toluene: chloroform:

ethanol (40:40:10)

Anisaldehyde-

sulphuric acid

reagent

Blue-violet

colour 0.82

Presence of

terpene

Ethyl acetate: acetic

acid: formic acid:

water (100:11:11:26)

Anisaldehyde-

sulphuric acid

reagent

Red-violet

colour 0.45

Presence of

terpene

Spectral analysis of compound-IV

IR and mass spectrum of compound-IV

The IR spectra were recorded in the region of 4000 cm-1 to 550 cm-1. From the IR

spectrum (Figure 6.14) of compound showed the presence of OH stretching at 3446.61

cm-1, aromatic C-H stretching at 3011.75 cm-1, carbonyl group at 1698.39 cm-1, C=C

stretching at 1463.71 cm-1, C-O stretching at 1273.11 cm-1, methyl group stretching at

1217.65 cm-1 and vibrational stretching of C-H at 2866.41cm-1. In mass spectrum, a

molecular ion peak at m/z 409, corresponding to the molecular formulae C22H34O7

(Figure 6.15). Other fragmentation and prominent peaks at m/z 349, 279, 139 and 99.

From the fragmentation pattern it was concluded that the isolated compound may be

forskolin.

89

NMR Spectrum of compound-IV

The 1H NMR spectrum (Figure No 6.16) exhibit signals characteristic to the benzo

chromen derivative. Two protons at H-2 and H-3 resonated at δ 1.659 and 1.623 as

singlet. Another singlet peak at δ 1.600 indicates 1H at H-5 position. Additionally, singlet

peak occurs at 1.945 and 1.939 for presence of hydroxyl proton in 6th and 9th position. A

proton present in H-7 position of substitution resonates at δ 4.300. The doublet peaks

occurs at δ 2.397, 5.106 and 5.116 were as a typical proton at H-12, H-15 and H-16

positions. Further, it also showed singlet peak at 1.330 and 5.738 indicates 1H proton

present in H-13 and H-14 positions. The above data suggests that the compound-IV may

be forskolin and it was earlier isolated by Saleem et al., (2006) from Coleus forskohlii.

Qualitative analysis and TLC of compound-V

These qualitative phytochemical analysis and TLC studies clearly demonstrated that the

presence of phenolic compound in the fraction. An orange colored, odorless, crystals were

obtained from hexane. It was practically soluble in dimethyl sulphoxide, ethanol. The

material has melting range 142-143°C (Table 6.5).

Table.6.5 Thin layer chromatography study of compound-V

Solvent system for

compound Spraying reagent

Colour of

spots Rf value Inference

Chloroform: ethyl-

acetate: formic acid

(5:4:1)

FeCl3 (2% in ethanol) Blue colour 0.92 Presence of

phenolics

Ethyl acetate: acetic

acid: formic acid:

water

(100:11:11:26)

Anisaldehyde-

sulphuric acid reagent

Red-violet

colour 0.67

Presence of

phenolics

Spectral analysis of compound-V

IR and mass spectrum of compound-V

The IR spectra were recorded in the region of 4000 cm-1 to 550 cm-1. From the IR

spectrum (Figure.6.18) of compound showed the presence of vibrational stretching of

methyl C-H at 2849.33 cm-1, 2921.47 cm-1, vibrational stretching of α, β-unsaturated C=O

group at1343.84cm-1, 1614.54 cm-1, C-O stretching at 1194.62 cm-1, vibrational stretching

90

of OH groups at 3311.17 cm-1. In mass spectrum, a molecular ion peak at m/z 294,

corresponding to the molecular formulae C17H26O4 (Figure 6.19). Other fragmentation

and prominent peaks were occurs at m/z 151, and 141. From the fragmentation pattern it

was concluded that the isolated compound may be Embelin.

NMR Spectrum of compound-V

In 1H NMR spectrum (Figure 6.20), showed signals at δ 6.8 ppm integrated for one proton

present in the ring (C6 position). The –OH proton comes to resonate at δ 5.734 ppm as

small broad singlet. There was a triplet appeared at δ 2.284 ppm corresponds to –CH–

proton in the ring, remaining all aliphatic hydrogen atoms appeared at δ 1.29 integrated

for CH2 protons. In addition, the presence of triplet peak at δ 0.816 suggested that proton

present at side chain of H-11 position. Similarly, singlet peak occurred at 1.96, 1.330

and 1.347 for CH2 proton present at H-1', H-2' and H-10'position. The above data

suggests that the compound V may be Embelin and it was earlier isolated by Kumara

swamy et al., 2007 from Embelia ribes.

Qualitative analysis and TLC of compound-VI

These qualitative phytochemical analysis and TLC studies clearly demonstrated that the

presence of triterpene in the fraction (Table.6.6). An orange colored, odorless, crystals

were obtained. It was practically Soluble in dimethyl sulphoxide, ethanol. The material

has melting range 256-257°C.

Table. 6.6: Thin layer chromatography study of compound-VI

Solvent system for

compound

Spraying

reagent Colour of spots

Rf

value Inference

Toluene: chloroform:

ethanol (40:40:10)

Anisaldehyde-

sulphuric acid

reagent

Blue-violet

colour 0.92

Presence of

triterpene

Ethyl acetate: acetic

acid: formic acid:

water (100:11:11:26)

Anisaldehyde-

sulphuric acid

reagent

Red-violet

colour 0.67

Presence of

triterpene

91

Spectral analysis of compound-VI

IR and mass spectrum of compound-VI

The IR spectra were recorded in the region of 4000 cm-1 to 550 cm-1. From the IR

spectrum (Figure.6.22) of compound showed the presence of asymmetric CH2 stretching

at 2942 cm-1 , bending vibrations of methyl group at 1375.39 cm-1, C=C stretching at

1642.22 cm-1, C-O stretching at 1029.06 cm-1, bending vibrations of methyl & CH2

groups in the rings at 1453.34 cm-1. In mass spectrum, a molecular ion peak at m/z 442,

corresponding to the molecular formulae C30H50O2 (Figure 6.23). Other fragmentation

and prominent peaks at m/z 427, 189, 135 and 87. From the fragmentation pattern it was

concluded that the isolated compound may be betulin.

NMR Spectrum of compound-VI

In 1H NMR spectrum (Figure 6.24), the presence of singlet peak at δ 1.556 and 3.059

suggested that proton present at H-1 and H-3 position. In addition, two doublet peaks at δ

1.462, 1.431 and 1.556 for two protons present in H-2, H-6 and H-15. The methyl proton

present in H-23, H-24, H-26 and H-30 position of substitution resonates at δ 1.251, 1.355

and 4.651 as doublet peaks. Further, it also showed singlet peak at 1.904 indicates OH

proton present in H-3 position. Apart from these signals were observed to CH2 proton at δ

1.579, 3.497, 2.364, 1.846 and 4.523 for H-7, H-16, H-19, H-21 and H-29. The above

data suggests that the compound VI may be Betulin and it was earlier isolated by Joshi et

al 2013 from Betula utilis.

6.2. FT-IR Spectrum of isolated Compound-I from Coroupita guianensis

6.3 Mass spectrum of isolated Compound-I from Coroupita guianensis

6.4 1H-NMR spectrum of isolated Compound-I from Coroupita guianensis

6.6. FT-IR Spectrum of isolated Compound-II from Coroupita guianensis

6.7. Mass spectrum of isolated Compound-II from Coroupita guianensis

6.8. 1H-NMR spectrum of isolated Compound-II from Coroupita guianensis

6.10. FT-IR Spectrum of isolated Compound-III from Limnophila heterophylla

6.11. Mass-spectrum of isolated Compound-III from Limnophila heterophylla

6.12. 1H-NMR-spectrum of isolated Compound-III from Limnophila heterophylla

6.14. FT-IR Spectrum of isolated Compound-IV from Limnophila heterophylla

6.15. Mass-spectrum of isolated Compound-IV from Limnophila heterophylla

6.16. 1H-NMR-spectrum of isolated Compound-IV from Limnophila heterophylla

6.18. FT-IR-spectrum of isolated Compound-V from Michelia champaca

6.19. Mass-spectrum of isolated Compound-V from Michelia champaca

6.20. 1H-NMR-spectrum of isolated Compound-V from Michelia champaca

6.22. FT-IR-spectrum of isolated Compound-VI from Michelia champaca

6.23. Mass-spectrum of isolated Compound-VI from Michelia champaca

6.24. 1H-NMR-spectrum of isolated Compound-VI from Michelia champaca

Chapter-VII

Toxicity study of Couroupita

guianensis, Limnophila heterophylla

and Michelia champaca

92

CHAPTER-VII

TOXICITY STUDY

7. 1. INTRODUCTION

Drugs that survive the initial screening and profiling procedure must be carefully

evaluated for potential risks before clinical testing begun. While no chemicals can be

certified as completely “safe” (free of risks), since every chemical is toxic at some level

of dosage, it is usually possible to estimate the risk associated with exposure to the

chemical under specified conditions, if appropriate tests are performed (Katzung, 1998).

Toxicology is the science that deals with the adverse effect of chemicals on living

organisms. Traditional uses of herbal drugs may be broadly divided into three categories

as follows

Which are well known and have been widely used for many years.

Which are not well known in the country but for which international experience is

available.

Those represent a new compound hitherto not evaluated as to its safety and

efficacy.

The first category consists chiefly of foodstuff(s) which have been in use for a long

time as traditional herbal remedies and the requirements are limited. In general, it seems

unnecessary to require the proof of safety of these products. For the second category,

views concerning the type of documents required to be presented may differ from country

to country. So it is necessary that varieties of requirements will be elaborated for these

products covering anything from reference in scientific literature confirming that the

product is safe. To satisfy the demands for limited or shortened toxicological testing of

these products, an investigation must be carried out on toxicity profile. The third group,

where the authority is faced with a product not previously screened for its toxicological

properties, toxicity studies of those product must have to be undertaken.

The index of the acute toxicity is LD50 (50 percent or median lethal dose), which

should not be regarded as a biological constant, since differing results are observed on

reception or when the investigations are carried out in different laboratories. This has

been indicated very clearly in multicentric study carried out in the European community

93

with five substances (Kingk, 1979). Historically, the LD50 was determined with high

degree of precision and was used to compose toxicities of compounds relative to their

therapeutic doses. It is now realized that high precision may not be necessary to compare

toxicities (Malmford and Teiling, 1983). Therefore, the median lethal dose is now

estimated from the smallest number of animals possible (Katzung, 1998). Median lethal

dose or LD50 is the dose (mg kg-1), which would be expected to kill one-half of an

unlimited population of the same species and strain. The median effective dose or ED50 is

the dose (mg kg-1), which produces a desired reaction in 50 percent of the test population

(Satoskar and Bhandarkar, 1978). In the present study toxicity study of the methanol

extracts of leaves of Couroupita guianensis, Limnophila heterophylla and Michelia

champaca was determined.

7.2. MATERIALS AND METHODS

Plant materials

Methanol extracts of leaves of Couroupita guianensis, Limnophila heterophylla and

Michelia champaca (described in chapter-V) were used as test drug in these experiments

and propylene glycol was used as control vehicle.

Animals

Albino wistar rats of either sex, weighing 200-250 gm were procured and maintained in

standard laboratory conditions. The animals were fed with standard pellet diet and water

ad libitum. They were allowed to acclimatize for one week before experimentation. All

experiments were carried out according to the guidelines for care and use of experimental

animals and approved by Committee for the Purpose of Control and Supervision of

Experiments on Animals (CPCSEA).

Methods of evaluation

The animals were grouped (ten in each group) and administered with different doses,

ranging from 250 mg/kg to 2000 mg/kg, of methanol extract of Couroupita guianensis,

Limnophila heterophylla and Michelia champaca individually by oral route. In each case

control group was included which received propylene glycol as control vehicle. The acute

94

toxicity testing was done by the method of Chandan et al. (2007). The animals were under

observation in open field condition for 72 hrs after the administration of extract of

Couroupita guianensis, Limnophila heterophylla and Michelia champaca, and the number

of deaths and signs of clinical toxicity were recorded (Litchfield and Wilcoxon, 1949).

7.3. Results and Conclusion

From acute toxicity studies it was observed that the administration of methanol extracts of

Couroupita guianensis, Limnophila heterophylla and Michelia champaca to rats did not

induce drug related toxicity and mortality in the animals. The results of the LD50 of

methanol extract of Couroupita guianensis, Limnophila heterophylla and Michelia

champaca were presented in Table 7.1, 7.2 and 7.3. The LD50 was found to be more than

2000 mg/kg body weight for Couroupita guianensis, Limnophila heterophylla and

Michelia champaca, by oral route. Therefore, it was concluded that the methanol extract

of all the three plants as did not have any toxic manifestation up to 2000 mg/kg body

weight.

Table 7.1: Determination of LD50 of methanol extract of Couroupita guianensis

(Oral route)

Treatment Dose

(mg/kg)

Number of

animals

Number of

Survival

Number

of Death LD50

Methanol extract 250 10 10 0

2000

mg/kg

,, 500 10 10 0

,, 1000 10 10 0

,, 2000 10 10 0

Control

(propylene glycol) 1 ml/kg 10 10 0

95

Table 7.2: Determination of LD50 of methanol extract of Limnophila heterophylla

(Oral route)

Treatment Dose

(mg/kg)

Number of

animals

Number of

Survival

Number

of Death LD50

Methanol extract 250 10 10 0

2000

mg/kg

,, 500 10 10 0

,, 1000 10 10 0

,, 2000 10 10 0

Control

(propylene glycol) 1 ml/kg 10 10 0

Table 7.2. Determination of LD50 of methanol extract of Michelia champaca

(Oral route)

Treatment Dose

(mg/kg)

Number of

animals

Number of

Survival

Number

of Death LD50

Hydroalcohol

extract 250 10 10 0

2000

mg/kg

,, 500 10 10 0

,, 1000 10 10 0

,, 2000 10 10 0

Control

(propylene glycol) 1 ml/kg 10 10 0

Chapter-VIII

Invitro hepatoprotective activity of

Couroupita guianensis, Limnophila

heterophylla & Michelia champaca

96

CHAPTER-VIII

IN VITRO HEPATOPROTECTIVE ACTIVITY

8.1. INTRODUCTION

Liver plays a major role in detoxification and excretion of many endogenous and

exogenous compounds, any injury or impairment of its functions may lead to many

implications on the health. Liver disease is a broad term describing a number of diseases

affecting the liver, such as hepatitis, cirrhosis, cancer, and non-alcoholic fatty liver

disease (Adwusi and Afolayan, 2010). Plant based herbal medicines are viable alternative

to synthetic drugs have been used since the dawn of civilization. Herbal based therapeutic

for liver disorder has been in use in India for a long time and has been popularized world

over by leading pharmaceutical. Despite the significant popularity of several herbal

medicines in general and for liver disease in particular they are still unrespectable

treatment modalities for liver disease (Handa et al., 1986). Therefore, important has been

safe given globally develop plant based hepatoprotective drugs effective against a variety

of liver diseases. The In-vivo studies require a large number of animals and needs up to

several days of drug administration for significant effect to be produced. It needs large

amount of drug. On the other hand, the invitro model is rapid and requires fewer amounts

of test substances (drug). Bioactive compounds obtained from the plant extracts are

usually available in the small quantities. Therefore, invitro models can be more useful in

assessment of activity. The present study was undertaken to evaluate an invitro

hepatoprotective effect of methanol extracts of Couroupita guianensis, Limnophila

heterophylla and Michelia champaca.

8.2. MATERIALS AND METHODS

Plant extracts

The methanol extracts of leaves of Couroupita guianensis, Limnophila heterophylla and

Michelia champaca (described in chapter-V) were used in this investigation.

Invitro Hepatoprotective Activity

The rat hepatocytes were isolated according to Seglen (1975) with slight modifications

(Visen et al. 1991) by recirculating enzymatic perfusion technique (in situ). The viability

of the cells was determined by trypan blue exclusion method.

97

The petri dishes will be divided into different groups of three petri dishes each. Group 1

shall be kept as normal. Group 2 will be given CCl4 treatment (10 mL) and remaining

groups will be taken for extracts (10 mL) and isolated compounds (10 mL). After

incubation, contents of the petri dishes will be centrifuged. The supernatant will be

removed to determine the levels of transaminases of GOT and GPT assay (Reitman and

Frankel 1957).

Cell viability

To each well of monolayer cultures, 50 μl of carbon tetrachloride (10 mM) was added

followed by the addition of 50 μl of methanol extracts of Couroupita guianensis,

Limnophila heterophylla and Michelia champaca at different concentrations (250 and

500 μg/ml) individually and incubated at 37ºC for 24 hours. After incubation, the cell

viability was determined by trypan blue exclusion method.

Trypan Blue Staining

Trypan blue was one of the several stains recommended for use in dye exclusion

procedures for viable cell counting. This method was based on the principle that live

cells do not take up dye unlike the dead cell. After 24h incubation with the extracts and

carbon tetrachloride, cells were trypsinised and resuspended in Minimum Essential

Medium (MEM). A cell suspension containing approximately 2.5×105 cells/ ml was

prepared in MEM & 0.2 ml of cell suspension was added, and mixed thoroughly with

0.4% trypan blue. The mixture was allowed to stand for 5 min. The suspension was

viewed in a hemocytometer and analysed for viable cells. Viable cell count was

determined as per the method described previously (Freshney, 2005) by using the

following calculations.

Cells / ml = Average cell count per square × dilution factor × 104

Total cells = Cells / ml original volume of fluid from which cell sample was

removed.

% Cell viability = Total viable (Unstained) / Total cells (stained and unstained) ×

100

98

8.3. RESULTS

Invitro hepatoprotective activity by recirculating enzymatic perfusion technique

Invitro hepatoprotective activity The results obtained in the experiments were reported

in Table 8.1. Incubation of rat hepatocytes with 10 mL CCl4 resulted in an induction of

hepatotoxicity, which was indicated by significant (p<0.01) increase in the GOT and

GPT levels in the toxin control group (CCl4-treated group) in comparison to control

group. The moderate significant (p<0.01) decrease in SGOT and SGPT levels was

observed in the incubation of the rat hepatocytes with methanol extracts of all three plants

at a dose of 250 µg/ml and CCl4. Incubation of the rat hepatocytes with methanol extracts

and CCl4 resulted in more significant (p<0.01) decrease in SGOT and SGPT levels,

indicating a more protective effect offered by the methanol extracts of three plants at 500

µg/ml. Based on the above results, methanol extract of Couroupita guianensis,

Limnophila heterophylla and Michelia champaca was found to be more effective, hence,

further invivo hepatoprotective activity was carried out by methanol extract.

Determination of Effect of Couroupita guianensis, Limnophila heterophylla and

Michelia champaca extracts on the cell viability by trypan blue assay

The changes in the cell viability status following exposure of hepatocytes to CCl4 alone

and CCl4 with Couroupita guianensis, Limnophila heterophylla and Michelia champaca

were investigated. After 24 hr of exposure to 10mM CCl4, the cell viability percentage

decreased (P>0.01 versus control) Table 8.2. depict the cell viability by trypan blue

assay in terms of percentage of cell death. The percentage of cell death significantly

increased (P<0.001) in CCl4 induced group (Group 2) when compared to control (Group

1). Of the two concentrations of the Couroupita guianensis methanol extract, 500 µg/ml

(Group-V) was found to be more effective (P<0.01) than other concentrations (250 µg/ml

(Group-IV) in reducing the rate of cell death. Similarly, with other two plants

(Limnophila heterophylla and Michelia champaca) methanol extract 500 µg/ml (Group-

VII) was found to be more effective (P<0.01) than other concentrations (250 µg/ml)

(Group-VI). Among, both the plant methanol extracts, Couroupita guianensis showed

better effect in decreasing the cell death by CCl4.

99

8.4. DISCUSSION

The In-vivo studies require a large number of animals and needs up to 3-5 days of drug

administration for significant effect to be produced. It needs large amount of drug. On

the other hand, the invitro model is rapid and requires fewer amounts of test substances.

Bioactive fractions obtained from the plant extracts are usually available in the small

quantities. Therefore, invitro models can be more useful in assessment of activity.

Isolated hepatocytes have become a powerful model for pharmacological, toxicological,

metabolic and transport studies of xenobiotics since the development of techniques for

high yield isolation of rat hepatocytes (Suresh kumar and Mishra, 2008). Freshly isolated

rat hepatocytes are also very useful and common tool for study of cytotoxicity and

metabolic studies in this area as they keep enzymatic activity similar to in vivo for several

hours. Various hepatotoxins like carbon tetrachloride, paracetamol, alcohol,

thioacetamide and various drugs have been shown to result in reduction of viability of

hepatocytes and leakage of enzymes which are considered to be the markers of cellular

damage.

Table 8.1: Effects of CCl4 and Methanol Extracts of Limnophila

heterophylla & Michelia champaca on the Biochemical Parameters

Treatment SGOT (IU/dl) SGPT (IU/dl)

Control 19.26±1.22 17.32±0.57

CCl4 72.17±1.84 63.47±1.72

Silymarin+ CCl4 22.16± 1.37 19.31±0.42

MECG (500 μg/ml) 26.23±0.23 25.83±0.13

MECG (250 μg/ml) 32.49±1.79 31.23±1.86

MELH (500 μg/ml) 27.85±0.27 28.83±0.46

MELH (250 μg/ml) 34.55±1.44 33.47±1.43

MEMC (500 μg/ml) 28.74±1.73 29.34±0.62

MEMC (250 μg/ml) 36.56±0.79 34.12±1.43

Values were expressed as mean ± SD for triplicate in each group

100

Table 8.2.: Effects of CCl4 and methanol extracts of Limnophila

heterophylla & Michelia champaca on cell viability by Trypan blue assay in Hep

G2 cell line.

Carbon tetrachloride (CCl4) is one of the oldest and most widely used toxins for

experimental induction of liver injury in laboratory animals (Brautbar and Williams,

2002). It is generally accepted that the hepatotoxicity of CCl4 was the result of reductive

dehalogenation, which is catalyzed by hepatic cytochrome P-450, and which forms

unstable trichloromethyl (CCl3) and trichloromethyl peroxyl (CCl3O2) radicals (Brattin

et al., 1985). Both trichloromethyl and its peroxy radical are capable of binding to

proteins or lipids, or of abstracting a hydrogen atom from an unsaturated lipid, initiating

lipid peroxidation and liver damage (Recknagel et al., 1989). It was observed that

methanol extracts of both the plants showed significant activity in carbon tetrachloride

induced liver damage. Results of our findings are comparable to that of silymarin.

8.5. CONCLUSION

A number of pharmacological and chemical agents act as hepatotoxin and produce

variety of liver ailments. Carbon tetrachloride intoxication in rats was an experimental

model widely used to study necrotic and steatonic changes in hepatic tissue. Accordingly,

cytotoxicity screening and invitro hepatoprotective activity of Limnophila heterophylla

and Michelia champaca leaves on CCl4 induced hepatotoxicity was investigated in

fleshly isolated rat hepatocytes. Cytotoxic screening indicated that the Limnophila

heterophylla and Michelia champaca leaves significantly reduced cell viability against

S. No Groups Incubation time (hrs)

12 24

1 Control 34 44

2 CCl4 17 22

3 Silymarin+ CCl4 32 39

4 MECG (250μg/ml)+ CCl4 24 30

5 MECG (500μg/ml)+ CCl4 29 36

6 MELH (250μg/ml)+ CCl4 23 28

7 MELH (500μg/ml)+ CCl4 27 35

8 MEMC (250μg/ml)+ CCl4 22 26

9 MEMC (500μg/ml)+ CCl4 26 32

101

CCl4 induced toxicity. This indicates that, the potential invitro hepatoprotective activity

of both plants may be due to the presence of various phytochemical compounds. Further

investigation is needed to confirm through invivo experiment. Over all, Couroupita

guianensis, Limnophila heterophylla and Michelia champaca leaves as a source of

natural hepatoprotective agent that can be important in oxidative stress mediated diseases

diabetic, cancer, arthritis.

Chapter-IX

Invivo hepatoprotective Couroupita

guianensis, Limnophila heterophylla &

Michelia champaca

102

CHAPTER-IX

IN-VIVO HEPATOPROTECTIVE ACTIVITY

9.1. INTRODUCTION

Hepatic injury is associated with distortion of the metabolic functions. Hepatic fibrosis

is a common condition in which major amounts of liver parenchyma cells are replaced

by fibrous connective tissue. Experimentally hepatic fibrosis is formed by the

administration of CCl4, paracetamol, thioacetamide etc. Despite remarkable advances in

modern medicine, hepatic disease remains a worldwide health problem, thus the search

for new medicines is still ongoing. Liver diseases remain one of the serious health

problems and it is well known that free radicals cause cell damage through mechanisms

of covalent binding and lipid peroxidation with subsequent tissue injury (Babu et al.,

2001). As many synthetic antioxidants have been shown to have one or the other side

effects (Musk et al., 1994), there has been an upsurge of interest in the therapeutic

potential of medicinal plants as antioxidants in reducing free radical induced tissue injury

(Siddique et al., 2000; Koleva et al., 2002). The literature has constantly shown that

hepatoprotective effects are associated with plant extracts rich in antioxidants. Numerous

medicinal plants and their formulations are used for liver disorders in ethnomedical

practice as well as in traditional systems of medicines in India (Subramonium et al.,

1996). Free radicals may also be a contributory factor in a progressive decline in the

function of the immune system (Pike and Chandra, 1995). Cooperative defense systems

that protect the body from free radical damage include the antioxidant nutrients and

enzymes. The antioxidant enzymes include superoxide dismutase (SOD), catalase

(CAT), glutathione peroxidase (GPx) and indirectly glutathione reductase (GRD). Their

roles as protective enzymes are well known and have been investigated extensively

invivo model systems. The present study was undertaken to evaluate the hepatoprotective

and antioxidant effects of methanol extracts of Couroupita guianensis, Limnophila

heterophylla and Michelia champaca on CCl4, intoxicated liver grievance in rats.

9.2. MATERIALS

Plant Extracts

The methanol extracts of leaves of Couroupita guianensis, Limnophila heterophylla and

Michelia champaca (described in chapter-V) were used in this investigation.

103

Animals

In this experiments fourty two healthy Wistar albino strains rats, weighing 200 – 250 gm

were selected for acclimation for a period of two weeks in laboratory animal house and

maintained under standard conditions of temperature 27±20C, relative humidity of 60 ±

5% and 12: 12-hour light: dark cycle prior to experimentation. The animals were fed with

standard pellet diet and water ad libitum. All experiments were carried out according to

the guidelines for care and use of experimental animals and approved by Committee for

the Purpose of Control and Supervision of Experiments on Animals (CPCSEA).

9.3. METHODS

Experimental Design

The hepatoprotective activity of methanol extracts of Couroupita guianensis, Limnophila

heterophylla and Michelia champaca was determined by using carbon tetrachloride

induced hepatotoxic rat model (Singh et al., 1999). Group-I served as vehicle control and

received normal saline (5ml/kg p.o.) for seven days. The group-II served as toxic control

and was administered carbon tetrachloride in olive oil (1:1 v/v, 0.5ml/kg, i.p.) daily for

seven days. Group-III served as standard and was administered silymarin (25mg/kg, p.o.

daily) along with carbon tetrachloride in olive oil (1:1 v/v, 0.5ml/kg, i.p.) daily for seven

days. Group-IV and V were administered with methanol extract of Limnophila

heterophylla at a dose of 250 & 500mg/kg, p.o. daily along with carbon tetrachloride in

olive oil (1:1 v/v, 0.5ml/kg, ip) daily for seven days respectively. Similarly, group VI

and VII were administered with the methanol extract of Michelia champaca (250 & 500

mg/kg, p.o, daily) along with carbon tetrachloride in olive oil (1:1 v/v, 0.5ml/kg, ip) daily

for seven days. Likewise, group VIII and IX were administered with the methanol extract

of Couroupita guianensis (250 and 500 mg/kg, p.o, daily) along with carbon tetrachloride

in olive oil (1:1 v/v, 0.5ml/kg, ip) daily for seven days.

Biochemical Assays

Preparation of Serum from Blood

After 24 hr, animals were sacrificed by chloroform anesthesia. Blood was collected by

heart puncture. The blood samples of each animal were taken and allowed to clot for 45

minutes at room temperature. Serum was separated by centrifugation at 3500 rpm at 370C

for 15 min and analysed for various biochemical parameters like; SGOT, SGPT, ALP,

LDH bilirubin and TP (Ahamed et al., 2003).

104

Preparation of Liver Homogenate

Hepatic tissues were carefully exercised and homogenized in cold 0.15M Tris-HCl buffer

with ethylene diamine tetra acetic acid (EDTA, pH 7.4) and centrifuged at 1500 rpm for

15 min at 40C. The supernatant was used for the assay of marker enzymes (glutathione

peroxidase, glutathione reductase, superoxide dismutase and catalase), thiobarbituric

acid reactive substances (TBARS) content, reduced glutathione content and protein

estimation.

Biochemical assays

At the end of the drug treatment period, all the animals were anaesthetized by application

of light chloroform and blood samples were collected from a group of animals from

dorsal aorta by heparinized syringe in vacutainer tubes. Separate blood samples were

collected from another group of anaesthetized animals in glass test tubes and allowed to

coagulate for 30 min. Serum was separated by centrifugation at 600×g for 15 mina and

it shall be analysed for various biochemical parameters including serum glutamate

oxaloacetate transaminases (SGOT), serum glutamate pyruvate transaminases (SGPT),

alkaline phosphatase, bilirubin, lactate dehydrogenase and total protein.

Preparation of liver homogenate

Hepatic tissues will be homogenized in KCl [10 mM] phosphate buffer (1.15%) with

ethylene-diamine tetra acetic acid (EDTA; pH 7.4) and shall be centrifuged at 12,000×g

for 60 min. The supernatant will be used for assay of the marker enzymes (glutathione

peroxidase, glutathione reductase, superoxide dismutase and catalase), reduced

glutathione, thiobarbituric acid reactive substances (TBARS) content and protein

estimation.

Biochemical parameters

SGOT & SGPT

Serum transaminases (GOT and GPT) were determined by the method of Reitman and

Frankel (1957). Each substrate (0.5mL) [either α -L-alanine (200mM) or L-aspartate

(200mM) with 2mM α - ketoglutarate] was incubated for 5 min at 37°C. A 0.1mL of

serum was added and the volume shall be adjusted to 1.0mL with sodium phosphate

buffer (pH 7.4; 0.1M). The reaction mixture was incubated for 30 and 60 min for GPT

105

and GOT, respectively. A 0.5mL of 2, 4-dinitrophenyl hydrazine (1mM) was added to

the reaction mixture and left for 30 min at room temperature. Finally, the color was

developed by the addition of 5mL NaOH (0.4 N) and the product formed was read at

505nm. Data were expressed as IUL-1

.

Alkaline phosphatase

Alkaline phosphatase (ALP) was assayed by the method of Kind and King (1954). The

reaction mixture of 3.0 ml containing 1.5 ml of buffer (carbonate-bicarbonate buffer,

0.1M, pH 10.0), 1 ml of substrate and requisite amount of the enzyme sources was

incubated at 37oC for 15minutes. The reaction was arrested by the addition of 1.0 ml of

Folins phenol reagent. The control tubes were received the enzyme after arresting the

reaction. The contents was centrifuged and to the supernatant, 1.0 ml of 15% sodium

carbonate solution, 1.0ml of substrate and 0.1ml of magnesium chloride (0.1M), was

added and mixture shall be incubated for 10 minutes at 370C. The colour was read out

640 nm against the blank.

Bilirubin

Bilirubin content was estimated by method of Malloy and Evelyn (1937). The two test

tubes were taken and each into was added 0.2ml of serum sample and 1.8 ml of distilled

water. To the unknown, 0.5 ml of diazo reagent and to the blank, 0.5 ml of 1.5%

hydrochoric acid was added. Finally, to each tube, 2.5 ml of methanol was added and

then allowed to stand for 30 minutes in ice and absorbance was read at 540nm. For a

standard curve, the above standard was diluted 1in 5ml methanol. The amount of direct

reacting bilirubin was determined similarly by substituting 2.5ml of water for 2.5ml of

methanol. The values were expressed as mg/dl.

Lactate dehydrogenase activity

Lactate dehydrogenase (LDH) activity was estimated in serum by the standard method

(Kornberg, 1955; Raja et., 2007). The reaction mixture consisted of 0.1mL of

nicotinamide adenine dinucleotide (NADH)-reduced disodium salt (0.02 M), 0.1mL of

sodium pyruvate (0.01 M), 0.1mL of serum, and made up to 3mL with sodium

phosphate buffer (0.1M; pH 7.4). The changes in the absorbance was recorded at 340nm

at 30s interval each for 3 min and the enzyme activity was calculated using a molar

106

extinction coefficient of 6.220M-1

cm-1

and it was expressed as nanomoles NADH

oxidized min-1

mg-1

protein.

Antioxidant Parameters

Lipid peroxidation assay

Lipid peroxidation (LPO) was measured by the method of Liu et al (1990). Acetic acid

1.5mL (20%; pH 3.5), 1.5 of TBA (0.8%), and 0.2mL of sodium dodecyl sulfate (8.1%)

was added to 0.1ml of supernatant and heated at 100oC for cooled and 60 min. Mixture

was cooled, and 5mL of n-butanol : pyridine (15 : 1) mixture and 1mL of distilled water

was added and vortexed vigorously. After centrifugation at 1200g for 10min, the organic

layer was separated and the absorbance was measured at 532nm using a

spectrophotometer. Malonyldialdehyde (MDA) is an end product of LPO, which reacts

with TBA to form pink chromogen–TBA reactive substance. It was calculated using a

molar extinction coefficient of 1.56 X 105M

-1 cm

-1 and shall be expressed as nanomoles

of TBARS mg-1

of protein.

Superoxide dismutase assay

Superoxide dismutase (SOD) activity was analyzed by the method described by Rai et

al. (2006). Assay mixture contain 0.1mL of supernatant, 1.2mL of sodium pyrophosphate

buffer (pH 8.3; 0.052M), 0.1mL of phenazine methosulfate (186 mM), 0.3mL of

nitroblue tetrazolium (300 mM), and 0.2mL of NADH (750 mM). Reaction was started

by the addition of NADH. After Incubation at 30oC for 90s, the reaction was stopped

by the addition of 0.1mL of glacialacetic acid. Reaction mixture was stirred vigorously

with 4.0mL of n-butanol. Color intensity of the chromogen in the butanol was

measured spectrophotometric ally at 560nm and the concentration of SOD was expressed

as Umg-1 of protein.

Reduced glutathione assay

Reduced glutathione (GSH) was measured according to the method of Ellman (1959).

The equal quantity of homogenate was mixed with 10% trichloroacetic acid and

centrifuged to separate the proteins. To 0.01 ml of this supernatant, 2ml of phosphate

107

buffer (pH 8.4), 0.5 ml of 5’5-dithio, bis (2-nitrobenzoic acid) and 0.4ml double distilled

water was added. Mixture was vortexed and the absorbance read at 412nm within 15

min. The concentration of glutathione was expressed as µ g/mg of protein.

Catalase assay

Catalase activity (CAT) was measured by the method of Aebi (1974). A 0.1mL of

supernatant was added to cuvette containing 1.9mL of 50mM phosphate buffer (pH 7.0).

Reaction was started by the addition of 1.0mL of freshly prepared 30mM H2O2.

The rate of the decomposition of H2O2 was measured spectrophotometrically at 240 nm.

Activity of CAT was expressed as Umg-1

of protein.

Glutathione peroxidase assay

Glutathione peroxidase (GPx) activity was determined by the method described by

Wendel (1981). The reaction mixture consist of 400μL of 0.25M potassium phosphate

buffer (pH- 7.0), 200 mL supernatant, 100 μL GSH (10 mM), 100 μL NADPH (2.5mM),

and 100μL GRD (6UmL-1

). Reaction was started by adding 100μL hydrogen peroxide

(12mM) and absorbance was measured at 366nm at 1min intervals for 5 min using a

molar extinction coefficient of 6.22X 103M

-1cm

-1. Data was expressed as mU mg

-1 of

protein.

Glutathione reductase assay

Glutathione reductase (GRD) activity was assayed by the method of Mohandas et al.

(1984). The assay system consist of 1.65mL sodium phosphate buffer (0.1M; pH 7.4),

0.1mL EDTA (0.5 mM), 0.05mL oxidized glutathione (1mM), 0.1mL NADPH (0.1

mM), and 0.05mL supernatant in a total mixture of 2mL. The enzyme activity was

quantified by measuring the disappearance of NADPH at 340nm at 30s intervals for

3min. The activity was calculated using a molar extinction coefficient of 6.22 X 103

M-

1cm

-1 and was expressed as nanomoles of NADPH oxidized min

-1 mg

-1 protein. Protein

content in the tissue was determined by earlier method reported (Lowry et al., 1951),

using bovine serum albumin (BSA) as the standard.

108

Histopathology

The livers were excised out, were washed with normal saline and weighed. The materials

were fixed in 10% buffered neutral formalin for 48 hrs. Liver slices were then embedded

in paraffin and sections of 5µ were taken using Microtome. The sections were stained

with Hematoxylin and Eosine (H/E) and were observed under microscope for

architectural changes, inflammation, congestion, steatosis and necrosis (Galigher etal

1971).

9.4. RESULTS

Effect on the Levels of SGOT, SGPT and Lactate Dehydrogenase Activities

CCl4 administration (group-II) resulted in significant (p< 0.01) rise in the levels of

SGOT, SGPT and LDH when compared to the control (group-I) (Table 9.1). Oral

administration of methanol extract of Couroupita guianensis, Limnophila heterophylla

and Michelia champaca at two different doses and standard drug, silymarin (group-III)

were seen to lower significantly (p< 0.01) the levels of marker enzymes namely SGOT,

SGPT and LDH.

Effect on the Levels of ALP, TB and TP Activities.

Total protein levels were diminished considerably in the toxic control (group-II). Oral

administration of methanol extract of Couroupita guianensis, Limnophila heterophylla

and Michelia champaca at two dissimilar doses significantly elevated the levels of total

proteins. Bilirubin levels were increased in the toxic control (group-II) and significantly

reduced in both plants treated groups at 250 & 500mg/kg (p<0.05 for 250 and p<0.01

500 mg/kg) (Table 9.1). Alkaline phosphatase levels were significantly (p< 0.01)

elevated in the toxic control (group-II) and was significantly reduced in MECG, MELH

(p<0.05 for 250 and p<0.01 for 500 mg/kg) and MEMC treated groups (p<0.05 for 250

and p<0.01 500 mg/kg).

Effect on the level of superoxide dismutase and catalase activities

As shown in Table 9.2, administration of CCl4 caused a significant (p< 0.01) decrease in

SOD and CAT levels in rats when compared with control animal (group-I). The plant

extract of three plants (Couroupita guianensis, Limnophila heterophylla and Michelia

champaca) at 500 mg/kg (group-V, VII and IX) showed significant (p< 0.01) increase in

SOD and CAT when compared to CCl4 treated rats; plant extracts at 250 mg/kg (group-

109

IV, VI and VIII) showed only less significant (p< 0.05) increase in SOD and CAT in

liver homogenate as compared with CCl4 treated rats (group-II). The standard drug,

silymarin treated (group-III) rats also showed significant (p< 0.01) increase in SOD and

CAT in comparison to CCl4 treated rats.

Effect on the Level of Glutathione Reductase Activities

GRD levels were significantly (p< 0.01) decreased in all animals treated with CCl4 when

compared to control rats (group-I). The plant extract of Couroupita guianensis,

Limnophila heterophylla and Michelia champaca at 250 mg/kg showed less significant

(p< 0.05) increase GRD in liver homogenate compared with CCl4- intoxicated rats

(group-II). The results were indicated in Table 9.2. Administration of methanol extract

of 3 plants at 500 mg/kg (group-V and VI) dose and silymarin were found to significantly

(p< 0.01) increase the GRD levels in the CCl4 intoxicated rats and restored the value to

that of control animals.

Effect on the Levels of Thiobarbituric Acid Reactive Substance

Lipid peroxidation (LPO) level of liver homogenates significantly increased (p< 0.01) in

CCl4 treated rats (group-II) when compared to control rats was presented in Table 9.2.

The methanol extract of Couroupita guianensis, Limnophila heterophylla and Michelia

champaca at 250 mg/kg dose showed less significant (p< 0.05) decrease in LPO in liver

homogenate when compared to CCl4 treated rats. Treatment with methanol extract of

Couroupita guianensis, Limnophila heterophylla and Michelia champaca at 500 mg/kg

dose and silymarin were showed significant (p< 0.01) decrease in LPO when compared

to CCl4 treated rats.

Effect on the Level of Reduced Glutathione

The CCl4- treatment caused significant (p< 0.01) decrease in the level of GSH in liver

homogenate tissue, when compared with control group (Table 9.2). The pre-treatment of

three plant extracts (Couroupita guianensis, Limnophila heterophylla and Michelia

champaca) at the dose of 500 mg/kg resulted in significant (p< 0.01) increase in GSH

content when compared to CCl4 treated rats. However, extract of 3 plants at 250 mg/kg

showed less significant (p< 0.05) increase in GSH level when compared to CCl4 treated

110

rats. The standard drug, silymarin treated animals also showed significant (p< 0.01)

increase in GSH level in liver compared with CCl4 treated rats.

Effect on the Level of Glutathione Peroxidase

GPx level in liver was significantly (p< 0.01) reduced in CCl4 treated rats when compared

with control (group-I). However, the extract of all three plants at 250 mg/kg (group-IV,

VI and VIII) showed less significant (p< 0.05) increase of GPx. Treatment with extract

of 3 plants (Couroupita guianensis, Limnophila heterophylla and Michelia champaca)

at 500 mg/kg and silymarin showed significant (p< 0.01) increase in GPx level when

compared to CCl4 treated rats. The result was shown in Table 9.2.

Histopathological Examination

The liver of normal control (group-I) revealed normal architecture. The hepatocytes,

sinusoids, central veins and portal tracts appear within normal limits. CCl4 treated group

(group-II) revealed loss of normal liver architecture with fibrosis, congested sinusoids,

ballooning degeneration, lymphocytic infiltration, haemorrhage and centrilobular

necrosis (Fatty liver). In silymarin treated group (group-III), liver architecture was

preserved with portal tract, minimal fibrosis and lesser extent of focal degeneration of

hepatocytes. In case of methanol extracts of all three plants treated groups (IV, VI &

VIII), the liver architecture was normal with moderate lymphocytic infiltration, light

regeneration of hepatocytes, and ballooning degeneration associated with haemorrhage.

Likewise, methanol extracts of Couroupita guianensis, Limnophila heterophylla and

Michelia champaca treated groups (V, VII & IX) at a dose of 500 mg/kg showed mild

lymphatic infiltration, regeneration of hepatocytes and ballooning degeneration

associated with haemorrhage. The liver photomicrographs were presented in the Figure

12.7.

111

Table 9.1. Biochemical parameters of methanol extracts of Limnophila

heterophylla and Michelia champaca against CCl4 induced liver damage

Treatment SGOT

(IUL-1)

SGPT

(IUL-1)

ALP

(IUL-1)

LDH (nM

NADH oxidized

min-1 mg-1

protein)

TB

(mg/dl)

TP

(mg/dl)

Control 46.9±7.4 106±9.5 42.8±1.18 375.6±11.4 2.7±0.04 9.7±0.15

Toxic

Control 186.8±11.4 254±15.4 74.6±0.99 524.8±14.5 4.24±0.14 4.28±0.06

Standard 52.7±76 102±9.5 45.2±1.25 404.7±0.13 2.2±0.05 8.2±0.13

MELH

250 mg/kg 121.4±5.6 196 ±12.6 44.7±0.65 446.8±2.5 2.0±0.17 6.5±0.02

MELH

500 mg/kg 66.8±11.6 147±8.5 42.4±1.39 446.2±0.05 3.2±0.24 8.7±0.37

MEMC

250 mg/kg 191 ±9.06 119.3±4.2 43.2±0.55 439.8±2.1 1.8±0.56 5.9±0.62

MEMC

500 mg/kg 139±6.21 65.7±10.5 41.4±1.28 445.2±1.15 3.12±1.24 7.91±0.55

MECG

250 mg/kg 204±11.6 111.4±8.6 54.7±0.55 486.8±8.5 3.0±0.07 7.5±0.04

MECG

500 mg/kg 157±8.5 66.8±11.6 40.4±1.39 446.2±0.05 3.1±0.04 8.7±0.37

Data are given as mean SD of six animals. * Significant difference (p < 0.05) from control or CCl4 -

treated rats. ALP = alkaline phosphatase; BIL=bilurubin, LDH= Lactate dehydrogenase; SGOT

=serum glutamic oxaloacetic transaminase; SGPT = serum glutamic pyruvic transaminase

112

Table. 9.2. Antioxidant parameters of methanol extract of Limnophila heterophylla

against CCl4 induced liver damage

Treatment

SOD

(u/mg of

protein)

CAT

(u/mg of

protein)

GRD (nM

of NADPH

oxidized

min-1 mg-1

protein)

GPx (mU

mg-1 of

protein)

GSH

(µ g/mg

of

protein)

MDA (nM

of TBARS

mg-1 cm of

protein)

Control 59.4±6.5 189.6±0.06 18.6±1.39 276.6±0.99 9.2±07 4.6±0.3

Toxic

Control 24.6±0.34 46.5±07 7.4±1.16 167.4±0.85 3.4±09 13.6±0.2

Standard 59.8±0.45 179.4±15 15.2±0.04 259.2±18 8.8±08 5.8±0.3

MELH 250

mg/kg 43.8± 03 132.6±1.3 11.4±0.37 211.6±0.17 6.8±2.3 9.4±0.05

MELH 500

mg/kg 54.6±015 159.8±11 13.8±0.11 224.8±0.18 7.4±1.4 6.4±0.4

MEMC 250

mg/kg 41.5± 0.3 131.5±1.05 9.4±2.7 207.4±1.7 6.1±3.4 8.9±1.05

MEMC 500

mg/kg 53.7±0.5 157.9±1.1 11.9±1.2 219.8±0.11 6.2±5.9 6.4±0.4

MECG

250 mg/kg 43.8± 0.3 134.6±1.5 10.4±0.37 201.6±0.07 6.8±1.3 9.2±0.5

MECG

500 mg/kg 54.6±0.15 169.8±1.3 14.8±0.13 244.8±0.08 8.4±1.2 7.4±0.4

Data are given as mean SD of six animals. * Significant difference (p < 0.05) from control or CCl4 -

treated rats. SOD= Super oxide dismutase; CAT= Catalase, GPx= Glutathione peroxidase; GRD

=glutathione reductase; MDA = Malonyldialdehyde; RD = Reduced glutathione

113

Normal Toxic control

CCl4 and Olive oil (1:1, 1 ml/kg)

Standard

(Silymarin) Low dose (250 mg/kg) L.H

Low dose (250 mg/kg) M.C High dose (500 mg/kg) L.H High dose (500 mg/kg) M.C Low dose (250 mg/kg) CG High dose (500 mg/kg) C.G

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PUBLICATIONS Enclosure –II

S.No Authors Title Journal Vol, Pg. No

& year

1 S. Raja &

K. Ravindranadh

A complete profile on

Couroupita guianensis –

traditional uses, pharmacological

activities and phytoconstituents

Pharmacophore 5 (1), 147-

159, 2014

2 S. Raja &

K. Ravindranadh

Phyto, physicochemical

standardization and TLC finger

printing of medicinal plant

Couroupita guianensis

International

journal of

phytomedicine

6, 587-594,

2015.

3 S. Raja &

K. Ravindranadh

Hepatoprotective and

antioxidant activities of

Couroupita guianensis on CCl4

induced liver damage.

International

journal of

phytomedicine

7, 281-289,

2015.

4 S. Raja &

K. Ravindranadh

Preliminary phytochemical

screening of various extracts of

Limnophila heterophylla

International

journal of

biological &

pharmaceutical

research..

5(12), 950-

957, 2014.

5 S. Raja &

K. Ravindranadh

Hepatoprotective and

antioxidant activities of

Limnophila heterophylla

Der pharmacia

lettre,

7 (7), 241-

249, 2015.

6 S. Raja &

K. Ravindranadh

In vitro antioxidant activity on

roots of Limnophila

heterophylla

Free radicals

and

antioxidants,

6(2), 178-

185, 2016.

7 S. Raja &

K. Ravindranadh

A complete profile on Michelia

champaca - traditional uses,

pharmacological

Activities and phytoconstituents

International

journal for

pharmaceutical

Research

scholars

3 (2),

432-440,

2014

8 S. Raja &

K. Ravindranadh

Preliminary phytochemical

screening and TLC

fingerprinting of whole plant

extracts of Michelia champaca

World journal of

pharmaceutical

research

3 (10), 631-

645, 2014

9 S. Raja &

K. Ravindranadh

Hepatoprotective and

antioxidant activities of

methanol extract of

Michelia champaca on carbon

tetra chloride induced liver

damage

Am. J.

Pharmtech res.

5(4), 574-

586, 2015

10 S. Raja &

K. Ravindranadh A review on hepatoprotective

activity leads

Am. J. Pharm

Tech res.

4(2), 32-59,

2014