ANTIDIABETIC EFFICACY OF LEAVES AND CALLUS OF PEDALIUM

MUREX L. ON ALLOXAN INDUCED DIABETIC ALBINO RATS

1

INTRODUCTION

Diabetes is a serious metabolic disorder with micro and macro vascular

complications that results in significant morbidity and mortality. The increasing number

of ageing population, consumption of calories rich diet, obesity and sedentary life style

have led to increase the number of diabetes worldwide. The current treatment, although

provide a good glycemic control but do a little in preventing complications (Vats et al.,

2004). Besides, these drugs are associated with side effects (Rang et al., 1991). There is

an increased demand to use natural products with antidiabetic activity due to the side

effects associated with the use of insulin and oral hypoglycemic agents (Holman et al

1991& Kameswara Rao et al 1997).The World Health Organization (WHO) (1980) has

also recommended the evaluation of the effectiveness of plants in condition where we

lack safe modern drugs (Upathaya et al.,1991 ). The pharmaceutical drugs are either too

expensive or have undesirable side effects. Treatment with sulphonylureas and

biguanides are also associated with side effects. (Rang et al., 1991). The term is derived

from Greek words "diabetes" means to pass through," Mellitus" means honey or related

to sugar(Akhtar and Hussain,1992).

Diabetes is defined as a state in which homeostasis of carbohydrate and lipid

metabolism is improperly regulated by insulin. This results primarily in elevated fasting

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and postprandial blood glucose levels. If this imbalanced homeostasis does not return to

normalcy and continues for a protracted period of time, it leads to hyperglycemia that in

due course turns into a syndrome called diabetes mellitus. There are two main categories

of this disease. Type 1 diabetes mellitus also called insulin-dependent diabetes mellitus

(IDDM) and Type 2, the noninsulin dependent diabetes mellitus (NIDDM). IDDM

represents a heterogenous and polygenic disorder, with a number of non-HLA loci (about

20) contributing to the disease susceptibility Lernmark and ott (1998). Though this form

of diabetes accounts for 5 to 10% of all cases, the incidence is rapidly increasing in

specific regions. It is estimated that incidence of Type 1 diabetes will be about 40%

higher in the year 2010 than in 1997 (Onkamo et al.,1999) and yet there is no identified

agent substantially capable of preventing this type of disease(Rabinovitch et al., 1998 ;

Schatz et al., 2000). NIDDM is far more common and results from a combination of

defects in insulin secretion and action. This type of disease accounts for 90 to 95% of all

diabetic patients. Treatment of Type 2 diabetes is complicated by several factors inherent

to the disease process, typically, insulin resistance, hyperinsulinemia, impaired insulin

secretion, reduced insulin-mediated glucose uptake and utilization (De Fronzo, 1997;

Polonsky ,1996 )

Diabetes mellitus is a metabolic disorder in which the body does not produce or properly

use insulin . It causes disturbances in carbohydrate , protein , and lipid metabolism and

complications such as retinopathy , microangiopathy , and nephropathy (Rotshteyn and

Zito, 2004) In practical terms diabetes mellitus is condition in which cells are starting in

the set of glucose. During diabetes a profound alteration in the concentration and

3

composition of lipid occurs. The global figure of people with Diabetes was estimated to

affect 177 million people worldwide in 2000 and this figure is projected to increase to

300 million by 2025 (Porter and Barret, 2005).

Diabetes Mellitus is a heterogenic metabolic disorder characterized by chronic

hyperglycemia and disturbance of carbohydrate, fat and protein metabolism.The term is

derived from Greek words "diabetes" means to pass through," Mellitus" means honey or

related to sugar(Akhtar and Hussain, 1992).

Diabetes is characterized by hyperglycemia resulting in various short term metabolic

changes in lipid and protein and long term irreversible vascular changes .These include

diabetes specific complication of the micro vascular system (retinopathy,

nephropathy,and neuropathy)and complications of macrovascular system (atherosclerosis

leading to heart diseases, stroke and peripheral vascular disease) which are present in the

non-diabetic population but have a 2-5 fold increase in diabetic subjects (zimmet and

Alberti., 1997)

Diabetes mellitus is a depilating and often life-threatening disorder with

increasing disorder with increasing incidence throughout the world (WHO-

1985).Diabetes is a chronic disease without a cure, however, with proper management

and treatment diabetics can live normal, healthy lives .Normally, the body gets a major

source of energy from glucose ,a single sugar that comes from foods high in simple

carbohydrates or from the breakdown of complex carbohydrates such as starch. After

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sugar and starch are digested in stomach, they enter the bloodstream in the form of

glucose .The glucose in the blood stream becomes a potential source of energy for the

entire of body. In diabetes, there is too much glucose in the blood .When glucose builds

in the blood, instead of going into the cells, it can cause two problems.

1. Cells may become starved for energy

2. Overtime, high blood glucose levels may harm kidney, heart, eyes or

nerves

A complex disease like diabetes mellitus, where little is talked about in

aspects of prevention and curation, but rather management, there is an increased focus on

plants in the search for appropriate hypoglycemic/antihyperglycemic agents. Firstly,

because of leads provided by traditional medicine to natural products that may be better

treatments than currently used conventional drugs (Rates , 2001). Secondly the plants by

secondary metabolic means contain a variety of herbal and non-herbal ingredients that are

thought to act on a variety of targets by various modes and mechanisms (Tiwari et

al.,2002)-given the multi-factorial pathogenicity of the disorders.

Diabetes is becoming something of a pandemic and despite the recent

surge in new drugs to treat and prevent the condition, its prevalence continues to soar.

Perhaps the most worrying aspect of all is that the rise is even reflected in children ( Yost

et al.,2001; Ludwig and Ebbeling 2001). Although several drugs targeted for

carbohydrate hydrolysing enzymes (pseudosaccharides), release of insulin from

pancreatic b-cells (sulphonyl urea), glucose utilization (biguanides), insulin sensitizers,

5

PPARg agonists (glitazones) are in clinical practice, the growing diabetes market

observes a number of changes. The glitazones are meant to target the problem

of insulin resistance and enhance insulin action at the cellular level; however, some of

these drugs are linked to liver toxicity (troglitazone), including a number of deaths from

hepatic failure( Krische and West, 2000; Stern, 1999 )and raising the symptoms and risk

factors of heart disease leading to heart failure (rosiglitazone) (Gale, and Lancet., 2001).

Therefore, as the long term of risk and effect on the complications of diabetes related

with these drugs are not yet clear, UK Drug and Therapeutic Bulletin warrants that

patients taking glitazones be monitored for signs of heart failure (Scrip, 2001 ) On the

other hand, traditional medicinal plants with various active principles and properties as

discussed in this article have been used since ancient times by physicians and laymen to

treat a great variety of human diseases such as diabetes, coronary heart disease and

cancer . ( Middleton et al., 2000; Havsteen 1984) . The beneficial multiple activities like

manipulating carbohydrate metabolism by various mechanisms, preventing and restoring

integrity and function of b-cells, insulin-releasing activity, improving glucose uptake and

utilization and the antioxidant properties present in medicinal plants offer exciting

opportunity to develop them into novel therapeutics. The multifactorial pathogenicity of

diabetes demands multi-modal therapeutic approach. Thus, future therapeutic strategies

require the combination of various types of multiple agents. (Gale and Lancet, 2001)

laments that ‘. . the rise of modern medicine has largely been based on new drugs, and

most of us can expect to hobble to our graves on the crutch of polypharmacy’. However,

medicatrix naturae – the power of self-preservation or adjustment has been the motto of

traditional medicinal practice, which prescribes polyherbal formulations. The theories of

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polyherbal formulation have the synergistic, potentiative, agonistic/antagonistic

pharmacological agents within themselves due to incorporation of plant medicines with

diverse pharmacological actions. These pharmacological principles work together in a

dynamic way to produce maximum therapeutic efficacy with minimum side effects.

Traditional medicinal preparations therefore, should not be considered just as a collection

of therapeutic recipes. They are formulated and prepared keeping in mind the conditions

of sickness and the healing properties of individual ingredients. It is important therefore,

that herbal medicines and preparations should be taken with the consideration of their

holistic therapeutic approach. The multiple activities of plant-based medicinal

preparations meant for diabetes offer enormous scope for combating the threat of the

diabetic epidemic. To achieve a blockbuster status, clear evidence of the advantage over

the existing therapy is the most important requirement of the day. The ability of modern

medicine and health care systems to adequately manage symptoms of chronic and

terminal disease is a central theme. The systematic reviews and Meta analysis of clinical

trials are the foundation of their success. Unfortunately, despite the apparent supremacy

in terms of multiple therapeutic approaches of herbal medicines, well-organized, rigorous

clinical trail evidences are not adequately available in order to advocate their scientific

merit and supremacy over the existing drugs. Though the markets for herbal medicines

are booming (Brevoort and Herbalgram, 1998) and evidence for effectiveness is

growing, it is also being simultaneously counterbalanced by inadequate regulation (Ernst,

2000).

Ayurveda is a traditional system of medicine using a wide range of modalities

to create health and well being. The primary aim of Ayurveda health care is to restore the

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physicalmental and emotional balance in patients, thereby improving health, preventing

disease and also treating any current illness. The number of patients seeking alternate and

herbal therapy is growing exponentially. Herbal medicines are now in great demand in

the developing world for primary healthcare not because they are inexpensive but also for

better cultural acceptability, better compatibility with the human body and minimal side

effects. Herbal medicine is still the mainstay of about 75–80% of the world population,

mainly in the developing countries for primary healthcare . However among the

estimated 250,000-400,000 plant species, only 6% have been studied for biological

activity, and about 15% have been investigated phytochemically (Balandrin et al., 1985;

Cragg et al .,1997 ).

Pedalium murex L. (pedaliaceae) commenly known as "yanai nerungi" /

"peru nerungi" is an erect ' much branched , foetid smelling succulent annual herb . The

leaves contain pedalitin , diosmetin , dianatin,pedalin , dianatin-7-glucoronide and

diosmetin-7-glucuronide(Subramanian and Nair, 1972).Decoction of the root is used an

antibilious agent , while the juice of the fruit is used as an emmenagogue and to promote

lochial discharge (Satyavathi et al., 1987). Leves are applied for healing ulcers. The fruits

and leaves of pedalium murex yield a number of phenolic acids.(Das et al ., 1996). The

decoction of pedalium murex and glycoside obtained from it showed mild diuretic

activity (Harvey, 1996). Diuretic activity was also reported in ethanolic

extract(50%) .The extracted was devoid of anthelmintic, and anti cancer activities (Dhar

et al., 1974)

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Pedalium murex, commenly called Gukhru in India belonging to the

family Pedaliaceae is distributed in the coastal areas of south India (Nadkarani, 1982). An

infusion or extract prepared from the leaves, stem and fruits in cold water is demulcent

and a diuretic found useful in the disorders of urinary systems such as gonorrhea, dysuria

and incontinence of urine etc (Chopra et al., 1999; Shukla and Khanuja, 2004). An

aqueous and antipyretic activities (Muralidharan and Balamurugan, 2008)

The medicinal and culinary uses of members of the family are well ‐documented

in literatures. Bhakuni et., al. (1992) reported that fruits of Pedalium murex Linn, possess

flavonoids and other constituents with diuretic, antispasmodic and aphrodisiac

properties. Also, Kothari & Moorthy (1994) stated that Pedalium murex is used for the

treatment of urinogenital system diseases in India while Shah et al., (1997) reported that

P. murex contains male contraception properties hence it can be used for fertility

regulation. Ecologically, the plant is a saline soil indicator in coastal regions (Hutchinson

and Dalziel 1964).

Medicinal plants are of great interest to the researchers in the

field of biotechnology as most of the drug industries depend in part, on plants for the

production o pharmaceutical compounds (Chand et al., 1997). The endeavor is to adopt

the method to multiply the medicinal herbs and monitor their secondary metabolites.

Conservation of endangered medicinal plants has also been achieved through cell cultures

with significance (Rao et al., 1996). Reports of in vitro plant regeneration from tissues of

medicinal plants are available (Gupta et al., 1997; Verma and Kant, 1996; Hoque et al.,

2000; Nichol et al., 1991 ; Palai et al., 2000). Plant tissue culture is a boon and can help

9

produce large quantities of the herbal material. However, it is speculated that plant

materials produced through tissue culture are deficient in secondary chemicals of

therapeutic importance

The use of medicinal plants as a source for relief from illness can be traced back

over five millennia to written documents of the early civilization in India ,china, and the

near east ,but it is doubtless an art as old as mankind.Plants are still widely used in

ethanomedicine around the world ,(Thomson, 1978;Stockwell, 1988). The multidrug

resistant strain of many microorganisms has revealed exploration of alternative

antimicrobial agent. Medicinal plants have become the focus of intense study in terms of

validation of their traditional uses through the determination of their actual

pharmacological effects. Synthetic drugs are not only expensive and inadequate for the

treatment of diseases but also often with adulterations and side effects. Therefore, there is

need to search new infection fighting strategies to control microbial infections

(Sieradzki et al., 1999)

In our present study is standard protocol for Pedalium murex in

tissue culture technique and anti microbial ,anti diabetic activity of invitro and invivo

plant extract of Pedalium murex

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.

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Review

Diabetes mellitus is a metabolic disorder featured by hyperglycemia and

alterations in carbohydrate, fat and protein metabolism associated with absolute or

relative deficiency of insulin secretion and /or insulin action, (Kameswara Rao et

al.,2003) It is one of the oldest diseases affecting millions of people all over the world.

(Andallu et al., 2002). Different types of oral hypoglycaemic agents such as biguanides

and sulphonylurea are available along with insulin for the treatment of diabetes mellitus

(Holman et al.,1991), but the side effects associated are unavoidable with their uses

(Kameshwara Rao, et al., 1997). Herbal drugs are widely prescribed today because of the

biologically active compounds are having minimal adverse effects and low costs.

Valiathan,(1998). In recent years, numerous traditional medicinal plants were tested for

their antidiabetic potential in the experimental animals, ( Srivastava et al.,1993). World

Health Organization (WHO), also permites the use of plant drugs for different disease,

including diabetes mellitus, (Gupta et al., 2005).

There are two main categories of this disease. Type 1 diabetes mellitus is

called insulin-dependent diabetes mellitus (IDDM) and Type 2 is the non insulin

dependent diabetes mellitus (NIDDM). IDDM represents a heterogenous and polygenic

12

disorder, with a number of non-HLA loci (about 20) contributing to the disease

susceptibility (Lernmark, 1998). Though this form of diabetes accounts for 5 to 10% of

all cases, the incidence is rapidly increasing in specific regions. It is estimated that

incidence of Type 1 diabetes will be about 40% higher in the year 2010 than in 1997

(Onkamo et al.,1999), and yet there is no identified agent substantially capable of

preventing this type of disease (Rabinovitch, and Skyler,1998;Schatz et al., 2000

;Atkinson et al.,2001). NIDDM is far more common and results from a combination of

defects in insulin secretion and action. This type of disease accounts for 90 to 95% of all

diabetic patients. Treatment of Type 2 diabetes is complicated by several factors inherent

to the disease process, typically, insulin resistance, hyperinsulinemia, impaired insulin

secretion, reduced insulin-mediated glucos uptake and utilization ( De Fronzo , 1997;.

Polonsky et al.,1996; Groop et al., 1989).

Despite the great strides that have been made in understanding and management

in this disease, serious problems like diabetic retinopathy (Ferris et al., 1999), diabetic

nephropathy (Ritz et al., 1999) and lower extremity amputation (Reiber et al., 1995)

continue to confront patients and physicians. The graph of diabetes-related mortality is

rising unabated (Olefsky, 2000). The level of serum lipids is usually raised in diabetes

and such an elevation represents a risk factor for cardiovascular disease

(shamaony et al., 1994). The chronic hyperglycemia of diabetes is associated with long

term damage, dysfunction and failure of various organs (Lyra et al., 2006).

The elevated levels of blood glucose in diabetes produce oxygen-free

radicals that cause membrane damage due to peroxidation of membrane lipids and protein

glycation (Baynes 1991). Glucose auto-oxidize in the presence of transition metal ions

generates oxygen-free radicals, which make the membrane vulnerable to oxidative

damage (Hunt et al. 1990). The oxidative stress and resultant tissue damage are important

component in the pathogenesis of diabetic complications (Baynes 1991). The free

radicals react with biomembrane causing oxidative destruction of polyunsaturated fatty

acids forming cytotoxic aldehydes by a process known as Lipid Peroxidation (LPO)

(Wolff 1993). The extent of LPO was measured in terms of ThioBarbituric Acid Reactive

Substances (TBARS) and lipid hydroperoxides (HPX), which are the end products of

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LPO. Several studies on human and animal models, using TBARS assay have shown

increased LPO in membranes and lipoproteins in the diabetic state, (Griesmacher et al.

1995; Krishnakumar et al., 1999).

HPX formed by LPO have direct toxic effects on endothelial cells and also

degrade to form hydroxyl radicals (OH-) (Testafamariam 1993). The action of

streptozotocin and alloxan produces reactive free radicals, which have been shown to be

cytotoxic to the B-cells of the pancreas (Ivorra et al., 1989; Lenzen, and Panten, 1988).

As the diabetogenic action of streptozotocin is preventable by SuperOxide Dismutase

(SOD), Catalase (CAT) and other OH- scavengers such as ethanol and dimethyl urea,

there is evidence to suggest that the action of streptozotocin and alloxan involve a

superoxide anion and OH (Asplund et al. 1984). Thus, alloxan-induced diabetes could

elicit changes in the antioxidant defense systems in response to increased oxidative stress.

The deleterious effects of superoxide radicals (O.− 2) and OH- in oxidative

stress can be counteracted by antioxidant enzymes such as SOD, CAT and glutathione

peroxidase (GPx). In addition to these enzymes, glutathione-S-transferase (GST)

provides glutathione (GSH) and help to neutralize toxic electrophiles. There is an

evidence to show the role of free radicals in diabetes and studies indicate that tissue

injury in diabetes may be due to free radicals (Wohaieb and Godin 1987; Kakkar et al.

1995). Diabetes is becoming pandemic and despite the recent surge in new drugs to treat

and prevent the condition, its prevalence continues to soar ( Tiwari and Madhusudana

Rao, 2002).

Diabetes mellitus is associated with augmented oxidative stress

(Menon et al., 2004) which leads major chronic complications namely retinopathy,

neuropathy, nephropathy, atherosclerotic coronary artery disease, and peripheral

atherosclerotic vascular disease (Kaczmar ,1998). Hyperglycemia increases the

production of reactive oxygen species (ROS) inside the aortic endothelial cells. ROS-

induced activation of protein kinase-C isoforms, increased formation of glucose-derived

advanced glycation end products, increased glucose flux through aldose reductase

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pathways, and activation of cytokines are some of the known biochemical mechanisms of

hyperglycemia-induced tissue and cell injury (Koya et al., 1998; Brownlee, 1995).

The mammalian cells are operational with both enzymatic and nonenzymatic

antioxidant defenses which minimize ROS mediated cellular damage (Haliwell and Gutteridge,1994). The majority of plasma antioxidants are depleted in diabetes

patients (Valabhji et al.,2001). Thus antioxidant therapy in diabetes may be helpful in

relieving symptoms and complications observed in diabetes patients. As plants often

contain a substantial amount of antioxidants, so herbal hypoglycemic coupled with

antioxidant property may serve as a wonderful antidiabetic agent (Larson, 1998).

Different medicinal systems are using the active plant constituents, which

discovered as natural hypoglycemic medicine, came from the virtue of traditional

knowledge. Herbal drugs are considered free from side effects than synthetic one and

they are less toxic, relatively cheap and popular (Moming, 1987). In India, medicinal

plants have been used as natural medicine since the days of Vedic glory. Many of these

medicinal plants and herbs are part of our diet as spices, vegetables and fruits.

Historically, in ‘Atharva-Veda’ (about 200 B.C.) description of medicinal plants was

made under a separate chapter ‘Ayurveda’. Sushruta (about 400 B.C.) compiled

classification of 700 herbal drugs under 37 classes in ‘Sushruta Samhita’ (A compendium

of ancient Indian surgery). Charak (about 600 B.C.) made the scientific classification of

herbal drugs based on remedial properties in his renowned treatise ‘Charaka Samhita’ (A

compendium of general medicine). In which, it described 50 classes of herbal remedies

comprising 500 crude drugs (Mukherjee, 1983; Saxena et al., 2006). The medicinal

values of plants have been tested by trial and error method for a long time by different

workers. Indian medicinal plants having blood sugar lowering potentials (Mukherjee et

al., 1981; Grover et al., 2002; Saxena et al., 2004; Mukherjee et al., 2006).

Aegle marmelos is widely used in Indian Ayurvedic medicine for the treatment

of diabetes mellitus (Kamalakkanan et al., 2003). Hypoglycemic effect of Aegle

marmelos root bark decoction (Karunanayake et al., 1984), leaf extract of Aegle

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marmelos produced anti hyperglycemic activity in alloxan diabetic rats (Ponnachan et al.,

1993),and produced hypoglycemic effect and increased plasma insulin level of STZ-

diabetic rats, (Sharma et al., 1996).

Allium cepa significantly reduced blood glucose level of alloxan induced

diabetic rats (Sheela etal., 1995). Alloxan induced diabetic rats fed a diet containing

Allium sativum (12.5%) for 15 days were able to reduce blood glucose as compare to

control group (Jelodar et al., 2005). Herbal extract of garlic produced hypoglycemia,

probably by interfering with food intake of both normal and STZ-diabetic rats

(Musabayane et al., 2006).

Aloe vera leaf pulp extract showed hypoglycemic activity on type 1 and type 2

diabetic rats, the effect being enhanced in type 2 diabetes as compared with

glibenclamide (Okyar et al., 2001). Oral administration of ethanolic extract to STZ-

diabetic rats for 21 days resulted in a prominent reduction of fasting blood glucose along

with improved plasma insulin level of diabetic rats (Rajsekaran et al., 2005). Oral

administration of Aloe vera gel extract to STZ-diabetic rats resulted in a significant

reduction of fasting blood glucose and improved the plasma insulin level

(Rajsekaran et al., 2006).

Andrographis paniculata extract effectively produced hypoglycemic and

anti-hyperglycemic activity in normal rats (Borhanuddin et al., 1994),and different doses

of Andrographis paniculata extract effectively reduced the fasting serum glucose level

of STZ-diabetic rats (Zhang et al., 2000a). Significant reduction in blood glucose level

(52.90%) observed when hyperglycemic rats treated with aqueous extract of

Andrographis paniculata, (Husen et al. 2004).

Annona squamosa aqueous leaf extract produces hypoglycemic activity

in streptozotocin-nicotinamide induced diabetic rats (Shirwaikar et al., 2004), and the

fruit pulp extract has been observed to improve the glucose tolerance of alloxan diabetic

rats, (Gupta et al., 2005). Oral administration of aqueous leaf-extract to diabetic rats for

16

30 days significantly reduced the levels of blood glucose and increased the activity of

plasma insulin and antioxidant enzymes (Kaleem et al., 2006).

Aqueous extract Azadirachta indica to produce antihyperglycemic and

hypoglycemic activity in diabetic dogs (Satyanarayan et al., 1978), fresh leaves decoction

induced antihyperglycemic activity (Chattopadhyay et al., 1987a) and increased the

peripheral glucose utilization in normal rats (Chattopadhyay et al., 1987b). Leaf extract

of Azadirachta indica has been reported to produce the hypoglycemic activity in normal

rats (Chattopadhyay et al., 1999) and the crude ethanol extract of Azadirachta indica

potentially lowered the blood sugar level of alloxan diabetic rats , (Kar et al., 2003), and

also it produce anti-hyperglycemic activity in streptozotocin diabetic rats without altered

serum cortisol level (Gholap et al., 2004).

Ethanolic extract of Cinnamomum tamala leaves induced potential

hypoglycemic effect in 18 hours fasted albino rats (Tripathi et al., 1990),and it produced

hypoglycemic activity in alloxan induced diabetic rats when administered orally for two

weeks at a dose of 250mg/kg (Kar et al., 2003). Ethanol extract of Coccinia indica

produced hypoglycemic activity in fasted, glucose fed and diabetic albino rats ,

(Mukherjee et al., 1988), and hypoglycemic and hypercholesterolemic effect of aqueous

Ficus bengalensis bark extract was observed in alloxan induced mild and severe diabetic

rabbits, (Gupta et al., 2002).

Alcoholic leaf extract of Gymnema sylvestre lowered maximum blood sugar in

fasted, glucose fed and diabetic rats along with insulin released from pancreatic ß-cells

(Chatopadhyay et al., 1993), and Gymnemic acid isolated from leaves Gymnema

sylvestre to produced potent hypoglycemic effect in STZ-diabetic mice (Sugihara et al.,

2000). Gymnema sylvestre leaf extract shown anti-hyperglycemic activity (Gholap et

al., 2003) and hypoglycemic (Gholap et al., 2004) effects of in corticosteroid-induced

diabetes mellitus,

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Oleanolic acid and momordin from Momordica charantia plant, produced

antihyperglycemic effect by inhibiting glucose transport in intestine of rat (Matsuda et

al., 1988). Fruit aqueous extract (200mg/kg, orally for 6 weeks), and exercise potentially

lowered blood sugar of type 2 diabetic and hyperinsulinemic (insulin resistance) rats

(Miura et al., 2004). Oral administration of alcoholic extract of leaves of Ocimum

sanctum lowered blood sugar level in normal; glucose fed hyperglycemic and STZ-

diabetic rats, (Chattopadhyay, 1993). Aqueous extract of Pterocarpus marsupium (1g/kg,

orally) bark has been observed to produce antidiabetic activity in alloxan diabetic rats

(Vats et al., 2004). Alcoholic extract of Swertia chirayita exhibited hypoglycemic effect

in alloxan induced diabetic rats (Kar et al., 2003).

Aqueous and ethanolic extracts of Syzygium cumini fruit-pulp has been

produces antihyperglycemic effect in alloxan induced diabetic rats, (Sharma et al., 2006).

The methanol extract of Mallotus roxburghianus leaves have the antidiabetic properties on streptozocin induced diabetic rats (Lalhlenmawia et al., 2007). Eucalyptus globulus Labill. (Tasmanian Bleu Gum) when given to streptozotocin-

diabetic mice reduced the level of hyperglycaemia.(Swatson-Flatt et al.,1990).

Tournefortia hirsutissima Linn. decreased the hyperglycaemic level in rabbits,( Alarcon-

Aguilar et al.,1998).

Similarly the plant Guazuma ulmifolia Wall. also significantly decreased the

hyperglycaemic peak in rabbits.( Roman-Ramos et al., 1991) The root mucilages of

Glossostemon bruguieri Desf. (Moghat) had remarkable hypoglycaemic activity

decreasing the blood glucose levels in diabetic rats by 54.5% within 15 days.

(Ibrahim et al.,1997). The aqueous extract of Camellia sinensis L. (black tea)

significantly reduced the blood glucose levels of streptozotocin-induced diabetic rats

(Gomes et al., 1995).

Janapati et al.,(2008) reported that alcoholic extract of the Talinum

cuneifolium leaves have significantly lower the blood sugar level of hyperglycemic

rats.Aqua solution of Artemisia herba-alba, possessed antidiabetic activity in alloxan

induced diabetic rats,(Didem Tastenkin et al.,2006). Tanko et al .,(2008) reported that

18

methanolic extract of Adansonnia digitata stem bark have a anti-diabetic activity in

streptozotocin-induced rats.

Ethyl acetate and ethanol extracts of the Momordica dioica fruits have

been shown significant anti diabetic activity (Reddy et al., 2006). Oral administration of

the extract of Asteracantha longifolia Nees. can significantly improve glucose tolerance

in healthy human subjects and diabetic patients,(Fernando et al.,1991). Methanol and

water extract of the Achyranthes aspera L produced a significant dose-related

hypoglycaemic effect in normoglycaemic and alloxan-induced diabetic rabbits. (Akhtar

and Iqbal,1991). Antidiabetic activity of Mangifera indica leaves in rat (Aderibigebe and

Lawal ,1999) . Nymphaea stellata flower extracts exhibited antihyperglycaemic and

antihyperlipidaemic effects of on alloxan-induced diabetic rats, (Rajagopal and Sasikala,

2008).

The diabetogenic effects of alloxan are attributed to a specific cytotoxic action

mediated by hydroxyl radical generation on pancreatic β-cells. This damages a large

number of β-cells resulting in a decrease in endogenous insulin release. Alloxan

administered rats therefore become hyperglycaemic in a short period of time, followed by

hepatic glucose over production. (Milgro and Martinez, 2000)

The increase in oxygen free radicals in diabetes could be due to increase in

blood glucose levels, which upon autooxidation generate free radicals. Streptozotocin has

been shown to produce oxygen free radicals (Ivorra et al., 1989). Lipid peroxide

mediated tissue damages have been observed in the development of type I and type II

diabetes mellitus (Feillet-Coudray et al., 1999). Previous studies have reported that there

was an increased LPO in liver and kidney of diabetic rats (Pari and Latha 2002;

Venkateswaran and Pari 2002). Peroxidation of membrane lipids associated with increased membrane

rigidity, and reduced cells survival has been implicated in diabetes mellitus (Selvam and Anuradha, 1988).

GSH acts as an antioxidant and its decrease was reported in diabetes mellitus

(Baynes and Thorpe 1999). The decrease in GSH levels represents increased utilization

due to oxidative stress (Anuradha and Selvam 1993). The depletion of GSH content may

also lower the GST activity as GSH is required as a substrate for GST activity

19

(Rathore et al., 2000). Depression in GPx activity was also observed in liver and kidney

during diabetes. GPx has been shown to be an important adaptive response to condition

of increased peroxidative stress (Matkovics et al., 1982).

SOD and CAT are the two major scavenging enzymes that remove toxic free

radicals in vivo. Previous studies have reported that the activity of SOD is low in diabetes

mellitus (Vucic et al., 1997). Reduced activities of SOD and CAT in liver and kidney

have been observed during diabetes and this may result in a number of deleterious effects

due to the accumulation of O.− 2 and H2O2 (Searle and Wilson, 1980). Scoparia dulcis

possessed an antidiabetic effect in addition to antioxidant activity, which may be

attributed to its protective action on LPO and to the enhancing effect on cellular

antioxidant defense contributing to the protection against oxidative damage in

streptozotocin diabetes, (Pari and Latha, 2005). Diabetes represents a state of increased lipid

peroxidation and reduced antioxidant reserve (Panneerselvam and Govindasamy, 2004) .

Phyllanthus fraternus possessed antidiabetic and antioxidant activity (Munish

Garg et al.,2008). Annona squamosa leaf extract prevents diabetic complications from

lipid peroxidation and antioxidant systems in experimental diabetic rats. Anthocephalus

indicus, root as a potent sugar, lipid lowering and antioxidant (Vishnu Kumar et al.,

2009). vitamin E prevents stress-induced elevation of lipid peroxidation.

(Olanlokun ,2008). Decreased lipid peroxides and tissue lipids clearly showed the

antihyperlipidemic and antiperoxidative effect of Diasulin apart from its antidiabetic

effect (Ramalingam Saravanan and Leelavinothan Pari 2005). Diospyros peregrine

possessed considerable antioxidant activity in alloxan induced diabetic rats. (Dewanjee et

al., 2007). Scoparia dulcis, possessed an antidiabetic effect in addition to antioxidant

activity, (Pari and Latha,2005). Nymphaea stellata flower extracts exhibited

antihyperglycaemic and antihyperlipidaemic effects of on alloxan-induced diabetic rats.(

Rajagopal and Sasikala ,2008),an Tephrosia purpurea has potent antihyperglycemic and

antilipidperoxidative effects in streptozotocin induced diabetic rats. (Pavana et al., 2007).

20

Methanolic extracts of Cleome droserifolia exhibited a significant antidiabetic and

antiperoxidative activity in alloxan diabetic rats, (Naggari et al., 2005).

A significant increase in lipid peroxidation in diabetic rats suggests that

increased generation of free radicals by hyperglycemia related to glucose auto-oxidation

(Woeff and Dean, 1987). The antioxidant activity of olive oil has been reported that its

elevates the activities of hepatic antioxidant enzymes such as catalase, superoxide

dismutase and glutathione peroxidase, (Ruiz-Gutierrez et al.,1999; (Aguilera et al.,

2003)). Olive oil is highly enriched in oleic acid (unsaturated fatty acid) which results in

a decrease of LDL, cholesterol and triglycerides levels of hypercholesterolemic patients,

(Sirtori et al., 1992). Polyunsaturated fatty acids have a hypocholesterolemic activity

(Reaven et al., 1993).

The level of serum lipids is usually raised in diabetes and such an elevation

represents a risk factor for cardiovascular disease (shamaony et al.,1994). Lowering of

serum lipid levels through dietary or drug therapy seems to be associated with a decrease

in risk of vascular disease (Rhoads et al., 1976). Anthocephalus indicus root possessed

hypoglycemic and hyperlipidemic activity in alloxan induced diabetic rats. (Vishnu

Kumar et al.,2009). Hypoglycemic and hypolipidemic action of alcohol extract of

Tinospora cordifolia roots in chemical induced diabetes in rats were studied.

(Stanley et al., 2003)

Experimental diabetes, induced by streptozotocin provoked hyperglycemia

accompanied by symptoms like loss of weight, polydipsia and polyphagia (Szkudelski

and Szkudeska ,2002 ) . Sundaram et al. (1996) have reported that the concentration of

lipid peroxides increases in the kidney of diabetic rats. An increased level of TBARS is

an index of lipid peroxidation Casearia esculenta extract decreased level of TBARS

diabetic kidney and liver (Prakasam et al., 2005)

21

In the liver, the enzyme is an important regulator of glucose storage and

disposal (Robert and Christopher, 1999). Insulin decreases gluconeogenesis by

decreasing the activities of key enzymes such as glucose-6-phosphatase, fructose 1,6,

bisphosphatase, phosphoenolpyruvate carboxykinase, and pyruvate carboxylase ( Murray

et al.,2000). Glucose 6-phosphatase, one of the key enzymes in the homeostatic

regulation of blood glucose level, catalyzes the terminal step in both gluconeogenesis and

glycogenolysis (Beaudet et al 1991; Hers et al.,1989) and fructose 1,6-bisphosphatase,

catalyzes one of the irreversible step in gluconeogeneis, and serves as a site for the

regulation of process (Jejwani and Horecker,1976). Ethanolic extract (200mg/kg) of

Momordica charantia was produced hypoglycemic activity in normal and streptozotocin

diabetic rats; this was occurred possibly due to inhibiting glucose-6-phosphatase and

fructose-1,6-biphosphatase in liver, and stimulating hepatic glucose-6- phosphate

dehydrogenase activities (Shibib et al., 1993). Alcoholic leaf extract produced

hypoglycemic effect in normal fed and 48 hours fasted rats, response mediated by

suppression of gluconeogenic enzyme glucose-6-phosphatase (Hossain et al., 1992). Ethanol (60%) leaf extract (200mg/kg, orally) lowered the blood sugar level of diabetic

rats due to suppressed glucose synthesis, through depression of glucose-6-phosphatase,

fructose-1-6-biphosphatase and enhanced glucose oxidation by shunt pathway through

activation of glucose-6-phosphate dehydrogenase (Shibib et al., 1993).

Liver and kidney exhibits numerous morphological and functional

alterations during diabetes (Sochar et al.,1985). Trigonella foenum-graecum L. helps to

recover the pathological effects of diabetes on liver and kidney of streptozotocin induced

diabetic rats(Naveen et al.,2007). Ayesha Noor et al .,(2008) reported that changes in the

histology of kidney and stomach sections after feeding with A. vera extract to diabetic

rats.

During diabetes the excess of glucose present in blood react with

heamoglobin to form glycosylated haemoglobin. (Alyassin and Ibrahim,1981;Sheela and

Augusti,1992).Glycosylated haemoglobinhas been found to be increased over a long

period time in the diabetes mellitus.(Bunn et al.,1978). There is an evidence that

22

glycation may it self induce the generation of oxygen derived free radicals in diabetic

condition. (Gupta et al., 1997).

Many active compounds have been isolated from the plant and herb species

of India. These active principles are dietary fibres, alkaloids, flavonoids, saponins, amino

acids, steroids, peptides and others. These have produced potent hypoglycemic, anti

hyperglycemic and glucose suppressive activities (Saxena et al., 2006). The above effects

achieved by either insulin release from pancreatic ß-cells, inhibited glucose absorption in

gut, stimulated glycogenesis in liver or increased glucose utilization by the body (Grover

et al., 2002; Saxena et al., 2004). These compounds also exhibited their antioxidant,

hypolipidemic, anticataract activities, restored enzymatic functions, repair and

regeneration of pancreatic islets and the alleviation of liver and renal damage (Mukherjee

et al., 2006). Some active constituents have been obtained from plants possess insulin

like activity and could be provide alternate for insulin therapy.

Plant-based antimicrobials and antibacterials represent a vast untapped source

for medicines and hence have enormous therapeutic potential (Phillipson, 1994). They

are effective in the treatment of infections while mitigating many of the side effects

associated with synthetic antimicrobial and antibacterial (Mathews et al., 1999; Bagghi,

2000). Lauric, palmitic, linolenic, linoleic, oleic, stearic and myristic acids are known to

have potential antibacterial and antifungal agents. (McGaw et al.,2002; Seidel et

al.,2004).

Aqueous extract of roots of the Hemidesmus indicus exhibited

bacteriostatic activity in mice infected with Mycobacterium leprae, Pmethoxy salicylic

aldehyde present in the extract was considered to be responsible for the activity (Gupta,

1981). Essential oil of H .indicus exhibited marked antibacterial activity against both

gram positive and gram negative bacteria even at concentration of 0.2%. The oil however

failed to show appreciable antifungal activity against fungi tested (Prasad et al., 1983).

Chloroform and ethanol (95%) extracts of H .indicus showed antifungal activity against

A. niger (Hiremath et al.,1997). The methanolic extract of root was proved to possess

anti-diarrhoeal activity in in-vivo and in-vitro studies (Das et al., 2003).

23

Terminalia chebula exhibited antibacterial activity against a number of bacterial

species (Ahmad et al.,1998). One group of researchers found that it is effective in

inhibiting the urease activity of Helicobactor pyroli (H. pyroli), an ubiquitous bacterium

implicated in the development of gastritis, ulcers and stomach cancers, (Malckzadeh et

al.,2001). Antibacterial activity of Terminalia chebula against both Gram positive and

Gram negative human pathogenic bacteria has also been reported (Phadke and

Kulkarni ,1989). Gallic acid and its ethyl ester isolated from ethanolic extract of

Terminalia chebula showed antimicrobial activity against methicillin-resistant

Staphylococcus aureus (Sato et al.,1997). Diffusate of Terminalia chebula showed an

inhibitory effect against strain XC-100 of the bacterium Xanthomonas Campestris pv.

Citri indicating its usefulness for the management of citrus canker disease (Afzalakhtar

et al.,1997). It has also growth inhibitory action against Salmonella typhi (Rani, and

Khullar ,2004) and intestinal bacteria (Kim et al.,2005).Nayeemulla et al .,(2006)

reported that Rauvolfia tetrophylla and Physalis minima leaf and callus extracts inhibited

bacterial and fungal growth. Y. Rajeshwar et al., (2005) reported the antimicrobial

activity of the methanolic extract of Mucuna pruriens.

In vitro propagation of medicinal plants could help in raising disease free health

clones in a large scale for extraction of pure drug. To date only one report available on

regeneration for this important medicinal plant (Thulaseedharan and Vaidyanathan 1990)

reported callus induction and plant regeneration in Vicoa indica. callus and cell

suspension culture of several plant species and extraction of medicinally important

compounds (Mulabagal et al., 2004). In vitro callus culture of Aegle marmelos has as

much potential in diabetes management ( Sevugan Arumugam et al.,2008). Allium cepa

callus cultures showed greater hypoglycemic potential over natural onion bulb (Kelkar et

al., 2001).

The medicinal and culinary uses of members of the Pedalaceae family are

well-documented in literatures. Kothari and Moorthy (1994),stated that Pedalium murex

is used for the treatment of urinogenital system diseases in India .While Shah et al.,

24

(1997) reported that P. murex contains male contraception properties hence it can be used

for fertility regulation. Ecologically, the plant is a saline soil indicator in coastal regions

(Hutchinson & Dalziel 1963).Pedalium murex leaves extract possessed antimicrobial

activity.(Nagaraj et al.,2008). Sahayaraj et al., (2008). Reported anti hyperlipidemic

activity of Pedalium murex fruits.Aqueous extract from Pedalium murex has been

evaluated for its analgesic and antipyretic activities (Muralidharan and

Balamurugan,2008).An infusion or extract prepared from leaves ,stem, and fruits in cold

water is demulcent and diuretic.

Present study we are investigated anti microbial activity and antidiabetic

activity of pedalium murex leaves and leaves derived callus on Alloxan induced rats.

25

26

27

INTRODUCTION

Medicinal plants are gifts of nature to cure limitless number of diseases

among human beings (Bushra and Ganga, 2003). The abundance of plants on the

earth’s surfaces has led to an increasing interest in the investigation of different

extracts obtained from traditional medicinal plants as potential sources of new

antimicrobial agents. (Bonjar and Farrokhi, 2004). The potential as a source for

new drugs is still largely unexplored. Among the estimated 250,000-500,000 plant

species ,only a small percentage has been investigated phytochemically and the

fraction submitted to biological or pharmacological screening is even smaller

(Mahesh and Sathish,2008).Thus,any phytochemical investigation of a given

plant will reveal only a very narrow spectrum of its constituents . Historically

pharmacological screening of compounds of natural or synthetic origin has been

the source of innumerable therapeutic agents. Random screening as tool in

discovering new biologically active molecules has been most productive in the

28

area of antibiotic. Even now, contrary to common belief, drugs from higher plants

continue to occupy an important niche in modern medicine. On a global basis, at

least 130 drugs, all single chemical entities extracted from higher plants, or

modified further synthetically for economic reasons .Medicinal plants represent a

rich source of antimicrobial agents.

Plants are used medicinally in different countries and are

source of many potent and powerful drugs. One of such resources is folk

medicine and systematic screening of them may result in the discovery of novel

effective compounds (Janovska et al., 2003).

Infectious diseases are the leading cause of death world-wide.

Antibiotic resistance has become a global concern (Westh et al., 2004). The

clinical efficacy of many existing antibiotics is being threatened by the emergence

of multidrug-resistant pathogens (Bandow et al., 2003). Many infectious diseases

have been known to be treated with herbal remedies throughout the history of

mankind. Natural products, either as pure compounds or as standardized plant

extracts, provide unlimited opportunities for new drug leads because of the

unmatched availability of chemical diversity. There is a continuous and urgent

need to discover new antimicrobial compounds with diverse chemical structures

and novel mechanisms of action for new and re-emerging infectious diseases

(Rojas et al ., 2003). Therefore, researchers are increasingly turning their attention

to folk medicine, looking for new leads to develop better drugs against microbial

infections (Benkeblia , 2004).

29

The increasing failure of chemotherapeutics and antibiotic

resistance exhibited by pathogenic microbial infectious agents has led to the

screening of several medicinal plants for their potential antimicrobial activity

(Colombo and Bosisio ,1996; Iwu et al.,1996). India is a varietal emporium of

medicinal plants and is one of the richest countries in the world in regard to

genetic resources of medicinal plants. It exhibits a wide range in topography and

climate, which has a bearing on its vegetation and floristic composition.

Moreover, the agro-climatic conditions are conducive for

introducing and domesticating new exotic plant varieties (Martins et al., 2001). In

recent years, secondary plant metabolites (phytochemicals), previously with

unknown pharmacological activities, have been extensively investigated as a

source of medicinal agents (Krishnaraju et al., 2005).

Thus, it is anticipated that phytochemicals with adequate antibacterial

efficacy will be used for the treatment of bacterial infections (Balandrin et al.,

1985). Since time immemorial, man has used various parts of plants in the

treatment and prevention of various ailments (Tanaka et al., 2002).

In many developing countries, a large proportion of the

population relies heavily on traditional practitioners, who are dependent on

medicinal plants to meet the primary health care needs. Although, modern

medicines are available, herbal medicines have often retained popularity for

historical and cultural reasons. Since the usage of these herbal medicines has

increased, the issues regarding their safety, quality, and efficacy in industrialized

30

and developing countries are cropped up (Anonymous, 1999). Growing interest

has also prompted researcher to screen scientifically various claims regarding

properties and uses of medicinal plant materials.

Presently, both, common consumers and healthcare professionals

seek updated, authoritative information towards safety and efficacy of any

recommended medicinal plant as drug prior to its use. (Manish et

al.,2007).Medicinal plants are of great interest to the researchers in the field of

biotechnology as most of the drug industries depend in part , on plants for the

production of pharmaceutical compounds .Tissue culture techniques are being

used globally for the exist conservation of plants . (Amutha et al., 2008).

Medicinal plants are of great interest to the researchers in the field

of biotechnology as most of the drug industries depend in part, on plants for the

production o pharmaceutical compounds (Chand et al., 1997).

Many higher plants are major sources of natural products used as

pharmaceuticals, agrochemicals, flavours and fragrances ingredients, food

additives, and pesticides (Balandrin and Klocke, 1988). It has been mentioned that

natural habitats for medicinal plants are disappearing fast and together with

environmental and geopolitical instabilities; it is increasingly difficult to acquire

plant derived compounds.

This has prompted industries, as well as scientists to consider the

possibilities of investigation into cell cultures as an alternative supply for the

production of plant pharmaceuticals (Mulabagal and Tsay, 2004).

31

In the search for alternative to production of desirable medicinal

compounds form plants, biotechnological approaches, especially, plant tissue

cultures, are found to have potential as a supplement to traditional agriculture in

the industrial production of bioactive plant metabolites (Dicosmo and Misawa,

1995; Ramanchandra Rao and Ravishankar, 2002).

Pedalium murex is a valuable plant source of medically useful

compounds that has been used in several trational ailments preparations. Leaf part

of the plant extract in organic solvents showed good source for the bioactive

compounds and good antibacterial properties .particularly the gram positive

organisms ( Nagaraj et al.,2008).The present investigation is callus induction of

the Pedalium murex leaves used MS media ,Phytochemical analysis and anti

microbial activiy of leaf callus and field grown leaves of the Pedalium murex.

32

MATERIALS AND METHODS

MATERIALS

CALLUS INDUCTION

Plant material

. Field grown plants were used as source of explants. Leaf, of four weeks

old seedlings were selected as explants for callus induction. The explants were

washed in running tap water for 30 minutes. Then they were washed in an agitated

solution of liquid detergent 2 %( v/v) (Teepol) for two minutes and rinsed in

distilled water three times. Surface sterilization was performed by immersion of

the explants in 70% (v/v) aqueous ethanol for 40 seconds followed by 0.1% (w/v)

mercuric chloride for five minutes. Finally, the materials were thoroughly rinsed

with sterile distilled water five times to remove the traces of mercuric chloride. All

the explants were cut into pieces approximately 10–15 mm long for inoculation.

Glassware:

The glassware used for culture work comprised of 6”x1” Riviera

and Borosil test tubes, 100 ml, 250 ml, 500 ml, and 1000 ml corning and Borosil

flasks, pipettes, and measuring cylinders (100 ml, 500 ml). Before use, glassware

were thoroughly brushed with alkaline detergent teepol and then washed in

33

running water. These were then treated with hot Chromic acid (mixture of

K2Cr2O7 + H2SO4 + H2O) followed by thorough washing with tap water. The

glasswares were then inverted in a clean tray and left to dry in the oven. Plugs for

the tubes and flasks were made out of absorbent surgical cotton wrapped in

muslin. 5 - 10 ml water was then poured into every culture vessel which was

tightly plugged. The glasswares were then steam sterilized in an autoclave at a

pressure of 15 lb/in at 121o C for 15 - 20 minutes.

Culture Medium:

The media formulation described as Murashige and Skoog

(1962) referred as MS medium was selected as the optimal culture medium. Stock

solutions of generally 4 times major elements, 1000 times minor elements, 100

times organic constituents were prepared. These stock solutions were stored in a

freeze chest at - 4oC and were mixed in desired proportions only before use. None

of the stock solutions were stored for more than 15 days.

34

Table 1. Composition of MS medium (Murashige and Skoog’s, 1962).

Constituents Molecular formula Concentration (mg/l)

Macronutrients Ammonium nitrate Potassium nitrate Calcium chloride Magnesium sulphate Potassium dihydrogen ortho Phosphate

NH4NO3 KNO3

CaCl2.2H2O MgSO4.7H2O KH2PO4

1650 1900 440 370 170

Micronutrients Ferrous Sulphate Disodium Ethylene Diamine Tetra Acetic Acid Boric acid Potasium iodide Manganese sulphate Zinc sulphate Sodium molybdate Cobalt chloride Copper sulphate

FeSO4. 7H2O Na2EDTA

H3BO3

KI MnSO4. 4H2O ZnSO4.7H2O Na2MoO4. 2H2O CoCl2. 6H2O CuSO4.5H2O

27.6 37.4

6.2

0.83 22.3 8.6

0.25 0.025 0.025

Vitamins Nicotinic acid Pyridoxine HCl Thiamine HCl Myo-inositol

100 0.5 0.5 0.1

Amino acids : Glycine 2.0

35

Sucrose 30,000

Agar 80,000

Table 2. Preparation of stock solutions of MS medium

Stock ingredients Quantity (mg)

Volume of stock/L of medium (ml)

Stock A (20X)

NH4NO3 KNO3

CaCl2. 2H2O MgSO4.7H2O

For 500 ml.

16500 19000 4400 3700

50

Stock B (100X)

MnSO4. 4H2O ZnSO4. 7H2O H3BO3 KI Na2MoO4.2H2O CuSO4.5H2O CoCl2.6H2O

For 100 ml.

223 86 62 8,3 2.5 0.25 0.25

10

Stock C(100 X)

Nicotinic Acid Pyridoxine HCl Thiamine HCl Glycine

For 200 ml.

10 10 2 40

10

Stock D (100X) For 100 ml 10

36

KH2PO4 1700

Stock E (200X)

FeSO4. 7H2O Na2EDTA

For 100 ml.

556 746

5

Table 3. Preparation of plant growth regulator stock solution

Plant growth regulator (PGR)

Common abbreviation

Quantity (mg) for 10 ml.stock

Solvents Concentration of stocks

Auxins Indole -3- acetic acid α-Naphthalene acetic acid 2,4–Dichlorophenoxy acetic Acid Indole -3- butyric acid

IAA NAA 2,4 – D

IBA

10

Ethanol /1N NaOH 1 N

NaOH 1m1 = 1 mg/l

Cytokinins 6-Benzyl amino purine kinetin

BAP KIN

101 N HCl 1 ml = 1 mg/l

Gibberellic acid GA3 10 1 N NaOH 1 ml = 1 mg/l

The medium was supplemented with growth harmones

such as α-Naphthalene Acetic Acid (NAA), Indole Acetic Acid (IAA) , 2,4-

37

Dichlorophenoxy Acetic Acid (2,4-D), 6-Benzyl Amino Purine (BAP) and

Kinetin (KIN) either alone or in combinations at various concentrations.

The reagents used were of Analytical Reagent Grade. Each salt

was dissolved separately one after one to avoid precipitation. All the constituents

except agar were mixed and then the pH of the solution was adjusted to 5.5 - 5.8.

Later, agar was added and the medium was heated to boil so as to homogenize

agar. Following are some of the supplements which were used either singly or in

combination for the induction of callus, differentiation and multiple shoot

formation.

After the preparation of the medium, water was poured out of the

autoclaved glassware. Definite aliquots of the medium were then added depending

upon the capacity of the culture vessel. Generally 25 ml, 50 ml, 100 ml of the

medium was distributed into the test tubes, 100 ml and 250 ml flasks respectively.

After plugging the glassware with cotton plugs, media ware steam-sterilized at 15

lb/in (121oC) for 15 - 20 minutes. After Autoclaving, tubes were placed in stands

to prepare the slants. These were then left to cool and solidify.

Inoculations:

All the experimental manipulations were carried out under strictly

aseptic conditions in laminar air flow bench fitted with a bactericidal U. V. tube

(15 W, peak emission 2637 Ao). The floor of the chamber was thoroughly

scrubbed with cotton dipped in alcohol.

The surface of all the vessels and other accessories such as

instruments (spatula, forceps, scalpels, blade etc.), gas burner, lighter, tube

containing absolute alcohol etc were also cleaned with alcohol. The fresh material

to be inoculated was kept in a Petri dish covered with a piece of black paper in

order to protect it from the harmful effects of U. V. rays. Alcohol was then

38

sprayed in the chamber with the help of an atomizer. The chamber was then

sterilized with U.V. rays continuously on for one hour. The explant like leaves

and were taken from the plants growing under the in vivo conditions. The leaves,

were placed in different bottles and covered with net and washed for 30 minutes

under running tap water to remove all the adhering dust particles and microbes

from the surface. The explants were then washed with liquid detergent (teepol) for

another 15 minutes and then washed properly to remove the detergent. The

explants were then treated with Bavistin (fungicide) for another 20-30 minutes to

remove the fungus and then washed properly to remove the fungicide.

Hands and arms which were to be used inside the inoculation

chamber were scrubbed with alcohol before inoculation. The rims of the test tubes

and the sides of the plugs were flame sterilized. Instruments (like forceps, scalpels,

spatula etc.) were all sterilized by dipping in the alcohol and flaming a number of

times. Care was taken to cool the instruments before putting into operation. The

explants taken from field borne plants were treated with 0.01 - 0.1% mercuric

chloride solution for 5-10 minutes respectively depending upon the explants.

Shoot apices of Nasturtium were treated with 0.1% mercuric chloride for 4 - 5

minutes.

The explants like stems and leaves were treated with 0.1% Hgcl 2 for 5 –

6 minutes. The explants were then thoroughly washed (4 - 5 washings) with

sterilized distilled water to remove the traces of Hgcl2. Fresh cuts were given to

the stem explants after sterilization to remove undesirable or dead portions. The

explants were then planted on variously augmented MS medium. Seeds were

surface sterilized with 0.1% Hgcl2 for 7 - 8 minutes. Constant shaking was done

during this period to get thorough sterilization. Rinsing with sterile distilled water

4 - 5 times was necessary for the removal of sterilant from the seeds. These were

then planted on Basal MS medium for germination. Leaves were excised from 4

weeks old seedlings and transferred separately to different experimental media.

39

Cultural conditions:

All the cultures were maintained in an air conditioned culture room

at a temperature of 25 ± 4oC. The source of illumination consisted of 2.5 feet wide

fluorescent tubes (40 watt) and incandescent bulb (25 watt). The intensity of

illumination was 3500 lux at the level of cultures and a 12 hour light regime was

followed by 12 hour darkness.

GC-MS Programme

Column:Elite-1 (100% Dimethyl poly siloxane),30m χ 0.25mm ID ×1µm df

Equipment:GC Clarus 500 Perkin Elmer

Carrier gas : Helium 1ml/min

Detector :Mass detector-Turpo mass gold –Perkin Elmer, Software-Turpo mass

5.1

Sample injected:2 µl

Split: 10:1

Oven Temperature programme:

110○C-2min hold

Up to 200 ○C at the rate of 10○C/min

Up to 280 ○C at the rate of 5 ○C/min-9 min hold

Total GC time: 36 min

Injector temp: 250 ○C

MS Programme

Library used : NIST Ver.2.0-year 2005

40

Inlet line temperature: 200 ○C

Source temperature: 200○C

Electron energy: 70 eV

Mass scan: (m/z) 45-450

MS Time: 36 min

Antimicrobial activity

Microorganisms

The activity of the Pedalium murex leaves and leaves derived callus extract was

tested against following organisms: Escherichia coli ,Pseudomonas

aeruginosa ,Salmonella typhi Enterococcus facalis ,Klebsiella

Pneumoniae ,Basillus species Staphylococcus aures , Streptococcus epidermidis.

These culture were collected from the Depatment of microbiology ,JJ college of Arts and

Science ,Pudukkottai , Tamilnadu,S.India.

Media:

Nutrient broth, Nutrient agar, Malt extract broth and Sabouraud dextrose agar, all

product of Himedia Laboratories Mumbai (India) were used in this study.

Preparation of inoculum

41

Each organism was recovered by sub-culturing on fresh media. A loopful

inoculum of each bacterium was suspended in 5 ml of nutrient broth and incubated

overnight at 37○C .These overnight cultures were used as inoculums.

Sub-culturing of microorganisim

The pure cultures of microorganism were maintained on nutrient agar slants by

frequent sub-culturing. These cultures were stored at 4○C.

Antibacterial assay

Agar well diffusion

The assay was conducted as described by Perez et al., (1990).

Procedure

Microorganisms from growth on nutrient agar incubated at 37○C for 18 h were

suspended in saline solution o.85% NaCl and adjusted to a turbididy of 0.5 Mac

farland standards(108 ctu/ml).The suspension was used to inoculate 90mm

diameter Petri plates with a sterile non toxic cotton swab on a wooden applicator.

Six millimeter diameter wells were punched in the agar and filled with 50µl of

2000µg/ml extract. Plates were incubated in air at 37○C for 24 hours. Antibacterial

activities were evaluated by measuring inhibition zone diameters. The experiments

conducted thrice.

RESULTS

Initially callus was initiated form leaf explants Of Pedalium murex on

basal MS medium supplemented with 2,4-D and IAA at different concentrations

42

(1,1.5,2,2.5,and 3.0 mg/l). In order to study the effects of plant growth regulators

(PGRs) on callus culture, different PGRs were tried at different concentration and

various combinations. Based on the results the growth of callus was obtained in

MS medium individually amended PGRs such as IAA and 2,4 D the callus

response was low .The combination of auxins and cytokinins in MS medium were

tested for initiate callus from the leaves . The combination of IAA and BAP in MS

medium produced white friable callus and MS medium supplemented with IAA

and kinetin produced brown callus.2,4 D was tested for the same .

Based on the results the maximum response and well growth of

callus were produced by the combination of MS medium supplemented with IAA

and BAP growth regulators .The high percentage of callus growth was found in

auxin such as 2,4-D (2.5 mg/l )with BAP (0.5 mg/l) among the other combination

and concentration of growth regulators .This combination producing callus was

taken for the further pharmacological studies .

Percentage of the main types of compound which were identified

by GC-MS in ethanol extract from Pedalium murex leaves and leaves derived

callus .GC chromatogram as shown on Figure 1 yielded two major peaks and three

minor peaks identified from the field grown leaf extract .Two major compounds

are n-Hexadecanoic acid (retention time (RT):17.46 and a- linolenic acid

(RT :20.24),and three minor compounds are 2,3 dihydro benzofuran (RT:7.16),

2,propenoic acid ,3,phenyl (RT :10.10) ,and octadeconoic acid (RT:20.56). GC

chromatogram as shown on Figure 2 yielded two major peaks and two minor

43

peaks identified from the leaf derived callus ethanol extract. Two major

compounds are n-Hexadecanoic acid (RT:17.47), and oleic acid (RT:20.29), two

minor compounds are 2-Furancarboxaldehyde,5-methyl (RT: 7.09) , octadecanoic

acid (RT:20.58)

Alcohol extract of the leaf and leaf derived callus of Pedalium murex

showed a well profound activity against Escherichia coli ,Pseudomonas

aeruginosa ,Salmonella typhi Enterococcus facalis ,Klebsiella

Pneumoniae ,Basillus species Staphylococcus aures , Streptococcus

epidermidis.Results obtained in the present study relieved that the tested Pedalium

murex extract posses potential anti microbial activity against pathogenic bacteria .

The field grown leaves and leaves derived callus extract of Pedalium murex high

effective inhibition activity in Escherichia coli , Pseudomonas

aeruginosa ,salmonella typhi ,and staphylococcus aureus among the ten bacterial

culture .

DISCUSSION

Based on the results of the previous experiments, the high biomass yielding

concentration of 2,4-D (2.5 mg/l) with BAP (0.5 mg/l) were selected for the

synergistic effects of auxins wih cytokinin on callus culture .The culture grew very

fast within 15 days in all combinations tested . in order to determine the active

principles present, callus/cell was extracted by using ethanol and compared with

44

leaf extract of Pedalium murex through GCMS analysis. The result of

chromatogram of callus/cell samples showed most of compounds present in leaf

extract chromatogram .Interestingly, additional compounds were found in ethanol

extract .The accumulation of active principles in cultured cells at higher level than

those in native plants through optimization of cultural conditions has been

observed in Panax ginseng (Ushiyama, 1991).Rosmarinic acid by Colleus blumei

(Ulbrich et al.,1983) . Shikonin by Lithospermum erythrorhizon (Takahashi and

Fujita 1991),diosgenin by Dioscorea (Rokem et al .,1984),ubiquinone-10 by

Nicotiana tabacum (Matsumoto et al.,1980) were accumulated in much higher

levels in cultured cells than intact plants .

Sometimes cultured plant cells often produce reduced quantities and

different profiles of secondary metabolites when compared with the intact plant

(Whitaker et al.,1986).This report coincides with our where there were additional

peaks in GCMS chromatogram of callus ethanol extract sample which was not

seen in leaf ethanol extract sample.

Plants are important source of potentially useful structures for

the development of new chemotherapeutic agents. The first step towards this goal

is the in vitro antibacterial activity assay (Tona et al., 1998). Many reports are

available on the anti microbial activity plants (Bylka et al., 2004.). Some of these

observations have helped in identifying the active principle responsible for such

activities and in the developing drugs for the therapeutic use in human beings.

45

Infection associated with Proteus sp. (Madigan et al.,

2000). Both leaves and leaves derived callus ethanol extracts of the Pedalium

murex can be used in the treatment of boils, sores and wounds, since

Staphylococcus aureus and P. aeruginosa have been implicated as causative

agents of these diseases (Braude, 1982). Since the demand in pharmaceutical

industries for plant based raw materials is ever increasing .

The present study is a stepping stone for in vitro production Of

required active principles of Pedalium murex .So far, there is no known report of

active principles of Pedalium murex by callus .

Organic solvent extracts exhibited a higher degree of antimicrobial

activity. Several workers have reported that many plants possess antimicrobial

properties including the parts which include; flower, bark, stem, leaf, e.t.c. It has

been shown that when solvents like ethanol, hexane and methanol are used to

extract plants, most of them are able to exhibit inhibitory effect on both gram

positive and gram negative bacteria (Bushra and Ganga, 2003). Similar work by

Omonkhelin et al., showed that ethanolic extract of- - Kigelia africana has

minimum inhibitory concentration of 6.25 + 1.07 mg/ml and 7.92 + 1.52 mg/ml

for S. aureus and C. albicans .

Antibacterial effects of these plants on Staphylococcus aureus, E. coli,

and Pseudomonas aeruginosa showed that the plants can be used in the treatment

of gastrointestinal infection and diarrhea in man and skin diseases (Rogger et al.,

1990) and they can also be used in the treatment of urinary tract

46

Pedalium murex is a valuable plant source of medically useful

compounds that has been used in several trational ailment preparation .Leaf and

leaf derived callus extract in organic solvent showed good source for the bioactive

compounds and good antibacterial properties .However, a detailed study is

required to find out the specific bioactive compounds responsible for the

antimicrobial properties through various advanced techniques.

47

INTRODUCTION

Diabetes mellitus is a group of metabolic disorders characterized by

hyperglycemia and defective metabolism of glucose and lipids. Diabetes was estimated to

affect 177 million people world wide in 2000 and this figure is projected to increase to

300 million by 2025 (Porter and Barret, 2005). Diabetes is not a single disease rather it is

a heterogeneous group of syndromes characterized by an elevation of blood glucose

caused by relative or absolute deficiency of insulin. Diabetes can be divided into two

main groups based on their requirements of insulin: insulin dependent diabetes mellitus

(Type 1), and non-insulin dependent diabetes mellitus (Type 2). However, other types of

diabetes have also been identified. Maturity Onset Diabetes of the Young (MODY) is

now classified as Type 3 and gestational diabetes classified as Type 4. NIDDM type 2

diabetes account for about 90 percent of diabetic cases (WHO, 2002). Insulin resistance

and β-Cell dysfunction are the metabolic abnormalities in the type 2 diabetes (Sa’ad et

48

al.,1991). Glycemic control is one of the targets for managing diabetes mellitus. Studies

have confirmed that for the type 2 diabetes, effective control of blood glucose

substantially decrease the risk of developing diabetic complications (Ohkubo et al., 1995;

UKPDS, 1997). Orthodox treatment of diabetes mellitus includes a modification of life

style, such as diet and exercise and the use of insulin and/or oral hypoglycaemic drugs.

These pharmacologic agents target increased insulin secretion, decreased hepatic glucose

production and increased sensitivity to insulin (Kelly and Mandarino, 2000).

Management of this disease with insulin and/or oral hypoglycaemic agents have certain

drawbacks (University Group Diabetes Program, 1974; Knatterud et al., 1978). For

insulin such drawbacks include ineffectiveness on oral administration, short shelf life,

requirement of constant refrigeration and in the event of excess dosage-fatal

hypoglycaemia. The use of oral hypoglycaemic drugs like sulfonylureas and biguanides

is also associated with side effects such as propensity to gain weight (Rang and Dale,

1991).

Throughout the world many traditional plants have been found successful

antidiabetic activity .further, most of marketed medicine are distillations, combinations,

reproductions or variations of substances that are found in nature. Our forefathers

recommended some of the substances, which are abundantly found in nature. Long before

their value was demonstrated and understood by scientific methods. How ever , few have

received scientific or medical scrutiny and the World Health Organization (WHO) has

recommended the trational plant treatment for diabetes warrant further evaluation (WHO

, 1980) .More ever today it is justified to use a plant or its active principles for treatment

(Singh et al.,2006).

49

Insulin therapy affords glycemic control in IDDM yet its short comings include

ineffectiveness on oral administration, fatal hypoglycemia in the event of exess dosage

(Senthilkumar et al.,2006) .Diabetes is one of the oldest known diseases of the man

whose devastating effect is increasing by the day and severity almost at epidemic level. It

is a disease of disordered metabolism of carbohydrate, protein and fat which is caused by

the complete or relative insufficiency of insulin secretion and /or insulin action (Balkau et

al., 2000).

Experimental diabetes in animals has provided considerable insight into the physiologic

and biochemical derangement of the diabetic state. Many of this derangement were in the

form of significant changes in lipid metabolism and structure (Sochar et al., 1985). These

structural changes are clearly oxidative in nature and are associated with development of

vascular disease (Baynes et al., 1999) .In diabetic rats, increased lipid peroxidation was

also associated with hyperlipidemia (Morel and Chisolm ,1989). During diabetes, a

profound alteration in the concentration and composition of lipids occurs. Liver and

kidney are important for glucose and lipid homeostasis, they participates in the uptake,

oxidation and metabolic conversion of free fatty acids, synthesis of cholesterol,

phospholipids and triglycerides. Thus it is expected to have changes in liver and kidney

during diabetes (Seifter and England, 1982).

MATERIALS AND METHODS

50

Animals

Healthy male adult albino rats (Wistar strain) of 6-7 weeks old, weighing

150 ± 20 g was procured from “Sri Venkateswara Enterprises”, Bangalore, India.

They were housed in clean sterile polypropylene cages with proper aeration and

lighting (12 ± 1 hr day / night rhythm) throughout the experimental period. During

the course of the experiments, the temperature was maintained between 27ºC ±

2ºC. The animals were fed with commercially available pelleted rat feed (Sri Sai

Durga feeds Bangalore, India.Under the trade name “Sri Sai Durga feed and

food”) and water ad libitum. The usage and handling of experimental rats was

followed as per the rules and regulations given by the Institutional Ethics

Committee for the purpose.

Chemicals

Alloxan was purchased from sigma chemicals company, St .Louis Mo.,

USA Thiobarbituric acid (TBA), pyrogallol, hydrogen peroxide, 1-chloro 2,4-

dinitro benzene(CDNB), dithio dinitro benzoic acid (DTNB), glutathione,trichloro

acetic acid, cholesterol,thio urea,palmitic acid and O-toluidine were purchased

from E.Merck,India.All other chemicals and reagents used in this study are

analytical grade purchased from Fine chemicals (P).Ltd., Mumbai

Induction of Diabetes mellitus in rats

The rats were injected alloxan monohydrate dissolved in sterile

normal saline at a dose of 150mg/kg body weight , intraperitoneally .Since alloxan

is capable producing fatl hypoglycemia as a result of massive insulin release rats

were treated with 20% glucose solution bottles in their cages to prevent

hypoglycemia(Stanely Mainzen Prince,1998).After a fornight ,rats with moderate

51

diabetes having glycosuria (indicated by Benedicts test for urine) and hyper

glycemia with blood glucose range of 250±30mg/dl were used for the treatment.

Collection and identification of plant

Fresh whole plants of Pedalium murex were collected from Sivapuram

Pudukkottai District, during the months of September-December 2006 and

identified by Dr.P.Jayaraman (Director), Plant Anatomy Reasearch centre,

Chennai.

Preparation of the Pedalium murex extract (PME)

The leaves were collected from Sivapuram, Pudukkottai District. The leaves and

leaves derived callus were collected and dried in shade for 15 days and made to

coarse powder. The power was passed through sieve No.40 to achieve uniform

particle size and then used for extraction process. A weighed quantity of the

powder was subjected to continuous hot extraction in soxhlet apparatus with 99%

ethanol.The extract was evaporated under reduced pressure using rotovac

evaporator until all solvent was removed to give a molten extract with a yield of

36% w/w.The ethanolic extract of of Pedalium murex was used for the study

Acute toxicity study:

Albino rats of either sex weighing 230-250 g selected by random sampling

technique were used in the study. Acute oral toxicity was performed as per OECD-

423 guidelines (Ecobichon,1997).The animals were fasted overnight ,provided

only water ,after which the drug PME was administered to the respective groups

orally at the dose level of 5 mg/kg body weight by gastric intubations and the

groups observed for 14 days .If mortality was observed in 2 or 3 animals ,then the

52

dose administered was assigned as a toxic dose. If mortality was observed in one

animal,then the same dose was repeated again to confirm the toxic dose .If

mortality was not observed ,the procedure was repeated for further higher doses

such as 50,300,and 2000 mg/kg body weight .The animals were observed for toxic

symptoms such as behavioral changes, locomotion ,convulsions and mortality for

72 h.

Experimental designs

Experimental design

The rats were divided into seven groups, each group consists of six animals.

Group I :Served as control and received normal feed and water ad libitum .

Group II : served as a normal rats received 200 mg/kg /bw Pedalium murex

leaves extract and water ad libitum.

Group III : served as normal rats received 200 mg/kg /bw Pedalium murex

leaves derived callus extract and water ad libitum.

Group IV :Served as diabetic control and received feed and water ad libitum

Group V : Diabetic rats and were treated orally with ethanol extract of

Pedalium murex leaves at the dose of 200 mg/kg body weight daily

for 21 days, once a day.

Group VI : Diabetic rats and were treated orally with ethanol extract of

Pedalium murex leaves derived callus at the dose of 200 mg/kg

body weight daily for 21 days, once a day.

Group VII :Diabetic rats given glibenglamide orally at the dose of 0.6 mg/kg

53

body weight daily for 21 days, once a day.

The animals were carefully monitored everyday and weighted every

week and blood glucose of all rats were determined .animals described as fasted

were deprived of food for at least 12h but allowed free access to drinking

water .After 3 weeks of treatment the rats were sacrificed by cervical

dislocation.Blood was collected and processed for the estimation of blood

glucose and glycosylated haemoglobin in separate tubes.Blood collected in

another set of tubes without anticoagulant was allowed to stand for 30minutes and

centrifuged at 3000rpm for 15minutes to separate the serum. Liver and kidney

were dissected out, washed in ice cold saline , patted dry and weighed.

Estimation of Blood glucose

Blood glucose was estimated by O-toluidine method (Sasaki et al., 1972).

Procedure

0.1ml of freshly drawn blood was immediately mixed with 1.9ml of 10%

TCA to precipitate the proteins and then centrifuged.To 1ml of the

supernatant ,added 4.0ml of O-toluidine reagent and kept in a boiling water bath

for 15 minutes .The green colour developed was read colorimatrically at 620

nm .A set of standard glucose(20-100g) were treated simultaneously using reagent

blank.

Glucose concentration was expressed as mg/dL of blood

54

Estimation of Glycosylated Haemoglobin (HBA1c)

The estimation of glycosylated haemoglobin was done by the method of

Sudhakar Nayak and Pattabiraman (1971) with modification according to Bannon

(1982).

Procedure

5ml of blood was collected with EDTA and plasma was separated .To

0.5ml of packed cell,5 ml of citrate buffer was added .mixed and incubated at

37○ C for 15 minutes .After centrifugation the supernatant was discarded. To the

aliquot ,4.0ml of (1M) oxalate in (2M) HCl solution was added,mixed and heated

at 100○C for 4 hours,cooled and precipitated with 2.0 ml of 40% TCA.The mixture

was then centrifuged .To the aliquot,0.05 ml of 80% phenol and 3.0ml of

conc.H2SO4 were added .A set of standards (10-50mg) were also treated in the

similar manner. The colour developed was read at 480 nm after 30 minutes.

The values were expressed as g/dL.

Estimation of liver Glycogen

Hepatic glycogen content was estimated by the method of Morales

et al.,(1973).

Procedure

A weighted amound of the tissue was subjected to alkali digestion in a

boiling water bath for 20 minutes after addition of 5ml of 30% potassium

55

hydroxide.The tubes were cooled and 3ml of absolute ethanol and a drop of

ammonium acetate were added .The tubes were then placed in a freezer overnight

to precipitate the glycogen .The precipitated glycogen was collected after

centrifugation at 3000 g for 10 minutes . The precipitate was washed thrice with

alcohol and dissolved in 3ml of water .Aliquotes were taken and made up to 1ml

with water .4ml of anthrone reagent was added to the tubes kept in an ice

bath,mixed and heated in a boiling water bath for 20 minutes.The green colour

developed was read at 640 nm .Working standard glucose and a blank were treated

similarly.

The values were expressed as mg/g tissue

Determination of the activity of Hexokinase

Hexokinase (ATP: D-hexose-6-phosphotransferase) was determined by the

method of Brandstrup et al., (1957).

Procedure

The reaction mixture contains 1.0 ml of glucose solution, 0.5 ml of ATP,

0.1 ml of magnesium chloride, 0.4 ml of dipotassium hydrogen phosphate, 0.4

ml of potassium chloride, 0.1 ml sodium fluoride and 2.5 ml of Tris-HCl buffer

(pH 8.0), was pre incubated at 37ºC for 5 minutes. The reaction was initiated by

the addition of 2.0 ml of tissue homogenate. 1.0 ml aliquot of the reaction mixture

was taken immediately (zero time) to tube containing 1.0 ml of 10% TCA. A

second aliquot was removed after 30 minutes of incubation at 37ºC. The

precipitated protein was removed by centrifugation and the residual glucose in the

supernatant was estimated by the O-toluidine method of Hyvarinen and Nikkila

(1962) as described previously. A reagent blank was run with each test. The

difference between the two values gave the amount of glucose phosphorylated.

56

The enzyme activity was expressed as nM of glucose-6-phosphate formed

per min per mg of protein.

Determination of activity of Glucose-6-Phosphatase

The enzyme activity was determined by the method of Fiske and subbarow(1925)

Procedure

Incubation mixture contains 0.7ml of citrate buffer ,0.3ml of substrate and

0.3ml tissue homogenate.The reaction mixture was incubated at 37○ C for 1

hour .Addition of 1ml of 10% TCA to the reaction tubes terminates the reaction of

the enzyme .The suspension was centrifuged and phosphorus content of the

supernatant was estimated by the method of Fiske and Subbarow.The supernatant

was madeup to known volume.To this 1ml of ammonium molybdate was added

followed by 0.4ml ANSA.The blue colour developed after 20 minutes was read at

640nm colorimetrically.

The enzyme activity was expressed as µM of phosphate liberated /min/mg protein

Determination of activity of Fructose-1, 6-diphosphatase

Fructose-1, 6-diphosphatase was determined by the method of Fiske and

subbarow(1925)

Procedure

The incubation mixture contains 1.5 ml buffer, 0.1 ml substrate, 0.25 ml MgCl2,

0.1 ml KCl, 0.25 ml EDTA, and 0.1 ml tissue homogenate.The reaction mixture

57

was incubated at 37ºC for 15 minutes. The reaction was terminated by the addition

of 1.0 ml of 10% TCA. The suspension was centrifuged and the supernatant was

made upto known volume. To this 1ml of ammonium molybdate was added

followed by 0.4ml ANSA.The blue colour developed after 20 minutes was read at

640nm colorimetrically.

The enzyme activity was expressed as µM of phosphate liberated /min/mg protein

Estimation of Total Cholesterol

Cholesterol was estimated by the method of Parekh and Jung (1970).

Procedure

About 0.1 ml of aliquot was taken and it was evaporated to dryness. The

dried extract and standard were made up to 3.0 ml with ferric chloride- uranyl

acetate reagent. Then 2.0 ml of sulphuric acid ferrous sulphate reagent was added

to all the tubes and the contents were mixed well. After 20 minutes the colour was

read at 540nm in a spectrophotometer.

Total cholesterol level was expressed as mg per dL for serum and mg per g

of wet tissue for liver and kidney.

Estimation of Triglycerides

Triglycerides were estimated by the method of Rice (1970) based on the

method of Van Handel (1961).

Procedure

About 0.1 ml of the lipid extract was mixed with 1.0 ml of chloroform-

methanol mixture and 50 mg of activated silicic acid was added, shaken

58

vigorously, allowed to stand for 30 minutes and centrifuged. To 0.5 ml of

supernatant, as well as standard and blank, 0.5 ml of alcoholic potassium

hydroxide was added and the mixture was saponified in a 60-70ºC water bath for

20 minutes. To this 0.5 ml of 0.2 N sulphuric acid was added and kept in a boiling

water bath for 10 minutes. After cooling the tubes, 0.1 ml of sodium meta

periodate was added and allowed to stand for 10 minutes.

The excess periodate was reduced by the addition of 0.1 ml of sodium meta

arsenite. Then 5.0 ml of chromotrophic acid was added, mixed thoroughly, and

kept in a boiling water bath for 30 minutes. After cooling 0.5 ml of thiourea

solution was added and the colour developed was read at 570nm using

spectrophotometer.

The triglyceride level was expressed as mg per dL for serum and mg per g

of wet tissue.

Estimation of phospholipids

Phospholipids in tissue was estimated by the method of Rouser et al.,

(1970)

Procedure

0.1ml of sample was diluted to 2.0ml with 10%TCA.The precipitated proteins

were sedimented by centrifugation .The supernatant was discarded.1.0ml of 70%

perchloric acid was added to the residue and digested on a sand bath till the

solution become colourless .After cooling ,the solution was made up to 5.0ml with

distilled water.Standard phosphate solution and blank containing water were

mixed with 0.8ml of perchloric acid and the final volume was made up to 5.0ml

distilled water .0.5ml of ammonium molybdate and ascorbic acid were added and

the mixture was kept in a boiling water bath for 6 minutes.The blue colour

developed was read at 710nm .

59

Phospholipids was expressed as mg/g in wet tissue

Estimation of HDL cholesterol

HDL cholesterol was estimated by the method of Allin (1974)

Procedure

1. Precipitation

0.5ml of serum was mixed with 1.0ml of precipitant and it was allowed to stand

for 10 minutes at 4000 rpm. After centrifugation ,the clear supernatant containing

HDL cholesterol was separated .

2. Estimation

0.1ml of the supernatant was mixed with 2.0ml of diluted reagent (1:1)

and incubated for 5 minutes at 37○C .The absorbance of sample against reagent

blank was read at 505nm. HDL cholesterol levels were expressed as mg/g in wet

tissue

Estimation of Protein

The Protein content of serum and the tissue homogenates of liver and

kidney were estimated by the method of Lowry et al., (1951).

Procedure

To 0.1 ml of suitably diluted serum/homogenate, 0.9 ml of water and 4.5 ml

of alkaline copper reagent were added and kept at room temperature for 10

minutes. Then 0.5 ml of Folin’s phenol reagent was added and the blue colour

developed was read after 20 minutes at 640nm using a spectrophotometer.

60

The level of protein was expressed as g per dL in serum and mg per g wet

tissue of liver and kidney.

Estimation of serum Albumin

Albumin in serum was estimated by Biuret method(Reinhold,1953).

Procedure

0.5 ml of sample was layered on to 9.5 ml of sodium sulphite in a centrifuge

tube and it was inverted to mix.2ml of mixture was taken immediately and marked

as total protein .The rest of the solution was allowed to stand for 10-15minutes for

precipitation of globulin and filtered using Whatman filter paper.The filterate

contains the albumin and 2ml of filtrate was taken and marked as total

albumin .The contents of all the tubes were made up to 2.5ml of distilled

water .About 2.5ml of distilled water served as blank.Then 5ml of Biuret reagent

was added to all the tubes .Mixed the contents and kept for 10 minutes.A series of

standard were prepared and treated as the test .The purple or violet colour

developed was read colorimatrically at 540nm

The serum albumin levels were expressed as g/dL

Aspartate amionotransferase (2-oxyglutrate amino transferase) (AST)

The activity of aspartate aminotransferase was estimated by the method of

King (1965a).

Procedure

About 1.0 ml of substrate was incubated at 37oC for 10 minutes. Then 0.2

ml of enzyme solution was added and the mixture was incubated at 37oC for one

61

hour. To the control tubes, enzyme solution was added after the reaction and it was

arrested by the addition of 1.0 ml of DNPH reagent. The tubes were kept at room

temperature for 30 minutes. Then 5.0 ml of 0.4N sodium hydroxide was added. A

set of standard pyruvate solution was also treated in a similar manner. The colour

developed was read at 540 nm using spectrophotometer.

The enzymes activity was expressed as IU per L in serum and in tissue as

µM of pyruvate liberated per min per mg of protein.

Alanine aminotransferase (L-Alanine: 2-oxyglumate amino

transferase) (ALT)

The activity of alanine aminotransferase was assayed by the method of

King (1965a).

Procedure

About 1.0 ml of substrate was incubated at 37oC for 10 minutes. Then 0.2

ml of enzyme solution was added. The tubes were incubated at 37oC for 30

minutes. To the control tubes enzymes were added after arresting the reaction with

1.0 ml of DNPH reagent. The tubes were kept at room temperature for 20 minutes.

Then 5.0 ml of 0.4 N sodium hydroxide was added and the colour developed was

read at 540nm using spectrophotometer.

The enzymes activity was expressed as IU per L in serum and in tissues as

µM of pyruvate liberated per min per mg of protein.

Estimation of Urea

62

Urea in serum was determined by Diacetyl Monoxime method

(Natelson, 1957).

Procedure

0.1ml of serum and 3.3ml of water was taken ; 0.3ml of 10% sodium

tungstate and 0.3ml of 2/3N sulphuric acid was added and centrifuged .To 1ml of

supernatant fluid added 1ml of water ,0.4ml diacetyl monoxime and 1.6ml of the

sulphuric acid –phosphoric acid mixture ,cooled ,and read against water blank at

480nm .

The serum urea levels were expressed as mg/dl

Estimation of Uric acid

The level of uric acid in serum was estimated by the method of Caraway(1963).

Procedure

4.5ml of tungstic acid was added 0.5ml of samples were added and

centrifuged .3ml of the supernatant was added to 0.6 ml of sodium carbonate and

followed with 0.6ml of diluted phosphotungstate .Mixed and placed in water bath

at 25 oC for 30min.Then read within 15 minutes at 700nm.

The values expressed as mg/dL

Estimation of creatinine

Serum creatinine was estimated by the method of Slot(1965).

Procedure

63

1.0ml of serum was mixed with 7.0ml of distilled water .1.0ml of sodium

tungstate and 1.0 ml of sulphuric acid were added and centrifuged. From this

4.0ml of supernatant was mixed with 1.0ml of sodium hydroxide and 1.0ml of

picric acid .The tubes were kept in a boiling water bath for 15minutes.The colour

developed was read at 470nm in a spectrophotometer.

Serum creatinine level was expressed as mg/dL

Determination of activity of Alkaline Phophatase (ALP)

(Orthophosphoric-monoester phosphohydrolase) (EC. 3.1.3.1)

The activity of alkaline phosphatase was assayed by the method of Moog

(1946) as modified by King (1965c).

Procedure

The assay mixture containing 1.5 ml of buffer, 1.0 ml of substrate and 0.1

ml of magnesium chloride were pre-incubated at 37oC for 10 minutes. Then 0.1 ml

of enzyme was added and incubated at 37oC for 15 minutes. The reaction was

arrested by 1.0 ml of 10% TCA. Control without enzyme was also incubated and

the enzyme was added after the addition of TCA solution. Then 1.0 ml of sodium

carbonate and 0.5 ml of Folin’s phenol reagent were added. After 10 minutes the

blue colour developed was read at 640nm using a spectrophotometer.

The enzyme activity was expressed as IU per Lit in serum

Determination of activity of acid phospatase (ACP)

The activity of acid phospatase was determined by the method of gutman

and gutman (1938,1940)

Procedure

64

To 6.0 ml of buffered substrate 0.3 ml of serum was added and incubated for

1 hr .at 37 0C . After 1 hr the tube was taken out , at the same time maintained a

separate tube containing 6 ml buffered substrate, 0.3 ml of serum was added to

which after incubation 2.7 ml of folin ciocataue reagent was added ,mixed and

centrifuged. About 4 ml supernatant from each tube (test,control , and standard ) in

a boiling water bath for 15 min ,cooled and the color developed was read at 680

nm

The activity of enzyme is expressed in IU/L

Estimation of LPO

LPO in tissues were estimated colorimetrically by TBARS and HPX by the method

of Nehius and Samuelson (1968) and Jiang et al. (1992), respectively. In brief, 0.1 ml of

tissue homogenate (Tris-HCl buffer, pH 7.5) was treated with 2 ml of (1 : 1 : 1 ratio)

TBA-TCA-HCl reagent (TBA 37%, TCA 15% and 0.25 N HCl) and placed in water bath

for 15 min, cooled and centrifuged at room temperature for 10 min at 1,000 rpm. The

absorbance of clear supernatant was measured against reference blank at 535 nm and

expressed as mmol/100 g tissue.

HPX were expressed as mmol/100 g tissue. 0.1 ml of tissue homogenate

was treated with 0.9 ml of fox reagent (88 mg butylated hydroxytoluene, 7.6 mg xylenol

orange and 9.8 mg ammonium ion sulphate were added to 90 ml of methanol and 10 ml

250 mmol/l sulphuric acid) and incubated at 37◦C for 30 min. The colour developed was

read colorimetrically at 560 nm.

Statistical analysis

Statistical analysis was performed using the SPSS software package (Statistical Package

for the Social Sciences, United States). Data are presented as means with their standard

65

deviations, and the data were analyzed using analysis of variance (ANOVA). The group

means were compared with Duncan's Multiple Range Test (DMRT).

RESULTS

In all groups prior to Alloxan administration, the basal levels of blood glucose of the rats

were not significantly different. However, 48 h after streptozotocin administration, blood

glucose levels were significantly higher in rats selected for the study. In contrast, non-

diabetic controls remained persistently euglycaemic throughout the course of the study.

Table shows the change in body weight gain to control and experimental groups of

rats .There was a signifigant decrease in the body weight of diabetic rats compared with

control rats .Upon the treatment of Pedalium murex leaves and leaves derived callus and

glibenglamide .the body weight gain was improved but the effect was more pronounced

in Pedalium murex leaves callus treated rats then leaves and glibenglamide

Table 1 shows the effect of treatment with extracts on blood glucose levels.

In all the Pedalium murex leaves, callus ethanol extract -treated groups a significant

antihyperglycaemic effect was evident from first week onwards the decrease in blood

sugar being maximum on completion of the third week in the group receiving 200

mg/kg/day of Pedalium murex leaves and leaves derived callus.

Table shows plasma insulin glycosylated haemoglobin in normal

and experimental group. While the level of plasma inulin was decreased and

glycosylated heamoglobin was significant elevation during diabetes when compared to

66

control group .Oral administration of Pedalium murex leaves and leaves derived callus

brought back to near normal values as that of standard drug glibenglamide treatment.

Effects on the administration of Pedalium murex leaves ,leaves derived

callus and glibenclamide on hepatic hexokinase and glucose-6-phosphatase, fructose-1,

6-bisphosphatase of liver are presented in Table 3. table – shows the activity of these

enzymes in kidney of diabetic rats The activity of hepatic hexokinase is significantly

decreased while glucose-6-phosphatase and fructose- 1, 6-bisphosphatase are

significantly elevated in allaxon treated diabetic rats as compared to normal rats. Oral

administration of Pedalium murex leaves and leaves derived callus brought back to near

normal values as that of standard drug glibenglamide treatment.

Effects on the administration of Pedalium murex leaves ,leaves derived callus

and glibenclamide on protein,albumin and haemoglobin of serum are presented in Table

3.the activity of protein,albumin and haemoglobin are significantly decreased in allaxon

treated diabetic rats as compared to normal rats. Oral administration of Pedalium murex

leaves and leaves derived callus brought back to near normal values as that of standard

drug glibenglamide treatment.

Triglycerides are a group of lipids absorbed from the diet and produced

endogenously from carbohydrates .hyperlipidemia ,a common feature of diabetes ,is

evidenced by the increased serum cholesterol, triglycerides and phospholipids in diabetic

rats compared to normal control rats .table shows the result for the level of serum

67

cholesterol, triglycerides and phospholipids .the activity of serum cholesterol,

triglycerides and phospholipids significantly increased alloxan diabetic rats when

compare with control rats .administration of Pedalium murex leave and leaves derived

callus and glibenglamide to diabetic rats tends to bring the values to near normal.

Table shows the level of urea ,uric acid ,and creatinine in normal control and

diabetic control rats. The level of urea ,uric acid and creatinine were significantly

reduced in the Pedalium murex leaves and callus extract treated rats as compared with

the diabetic rats.

Table shows the level of AST ,ALT,ACP and ALP control and diabetic treated

rats .the diabetic rats showed a significant increase in AST ,ALT,ACP AND ALP as

compared to normal control rats. by supplementing Pedalium murex leaves and callus

extract maintained the level to near normal status.

Table shows the concentration of TBARS and hydro peroxides in tissues of normal

control and experimental animals. There was a significant elevation in tissues TBARS

and HPx during diabetes when compared to the corresponding control group.

Administration of Pedalium murex leaves and leaves derived callus and glibenglamide

tends to bring the values to near normal.

DISCUSSION

The use of traditional medicine and medicinal plants in most developing countries, as a

normative basis for the maintenance of good health, has been widely observed

(Bhattaram et al., 2002). Furthermore, an increasing reliance on the use of medicinal

68

plants in the society has been traced to the extraction and development of several drugs

and chemotherapeutics from these plants as well as from traditionally used rural herbal

remedy (Bhattaram et al., 2002). This study was under taken to asses the antidiabetic

effect of Pedalium murex leaves and leaves derived callus on ethanol extract in alloxan

induced diabetic rats.

Alloxan , a β cytotoxin induces chemical diabetes in a wide variety of animal

species by damaging the insulin secreting cells of the pancreas . This damages a large

number of β-cells resulting in decrease in endogenous insulin release .Alloxan

administered rats therefore become hyperglycemic in short period of time ,followed by a

hepatic glucose over production .(Matinez and Milagro, 2000). Numerous studies

demonstrated that a variety of plant extracts effectively lowered the glucose level in

alloxan-induced diabetic animal (Vijayvargia et al.,2000 ; Satyanarayana et al .,2005).

Hyperglycemia causes oxidative damage by the generation of reactive oxygen species

(Mohamed et al., 1999) and results in the development of diabetic complications

(Donnini et al.,1996; Baynes et al.,1999 ).

Oral administration of Pedalium murex leaves and callus (200mg/kg body

wt./day) resulted in a significant reduction in the blood glucose and improvement in body

weight. The decrease in body weight in diabetic rats clearly shows a loss or degradation

of structural proteins due to diabetes. The structural proteins are known to contribute for

the body weight (Rajkumar and Govindarajulu, 1991). Protein synthesis is decreased in

all tissues due to absolute or relative deficiency of insulin (an anabolic hormone) in

alloxan-induced diabetic rats. The ability of the Pedalium murex leaves and callus to

69

protect from maximum body weight loss seems to be due to its ability to reduce

hyperglycemia.

Further, the antihyperglycemic activity of Pedalium murex was associated with an

increase in plasma insulin level suggesting an insulinogenic activity of the Pedalium

murex leaves and callus extract. The observed increase in the level of plasma insulin

indicates that P.murex leaves and callus extract stimulates insulin secretion from

regenerated β-cells. In this context, a number of other plants have also been reported to

extent hypoglycemic activity through insulin release stimulatory effect .(Pari and latha,

2002; chattopadhyay, 1999).

The observed increase the levels of glycosylated haemoglobin in diabetic

control group of rats is due to the presence of excessive amount of blood glucose. During

diabetes the excess of glucose present in blood react with haemoglobin to form

glycosylated haemoglobin .(Alyassin and Ibrahim , 1981; sheela and Augusti , 1992).

glycosylated haemoglobin has been found to be increased over a long period of time in

the diabetes mellitus (Bunn et al .,1978). There is evidence that glycation may itself

induce the generation of oxygen derived free radicals in diabetic condition. (Gupta et

al.,1997). Treatment with P.murex leaves and callus extract showed a decrease in the

glycosylated haemoglobin with a concomitant increase in the level of haemoglobin in the

diabetic rats standard drug glibenglamide also showed the same results.

The liver is regarded as one of the central metabolic organs in the

body, regulating and maintaining homeostasis. Diabetes results in a decrease in glucose

utilization and an increase in glucose production in insulin-dependent tissues such as

liver.( Seifter et al.,1982) . Decreased glycolysis, impeded glycogenesis and increased

70

gluconeogenesis are some of the changes of glucose metabolism in the diabetic liver.

(Baquer ,1998) Hexokinase is an insulin-dependent and insulin-sensitive enzyme and are

almost completely inhibited or inactivated in diabetic rat liver in the absence of insulin.

(Gupta ,1997) Decreased enzymatic activity of hexokinase and phosphofructokinase has

also been reported in diabetic animals, resulting in depletion of liver and muscle

glycogen. (Laakso et al.,1995 ; Murray et al.,2000). In our study, we also have observed

decrease in hepatic as well as renal hexokinase activity in alloxandiabetic rats.

Administration of P.murex leaves and callus to alloxan treated rats resulted in an

increased activity of hexokinase in liver and kidney. This increased activity of

hexokinase can cause the increased utilization of glucose for energy production. P.murex

leaves and callus has been observed to decrease the level of blood glucose. The decrease

in the concentration of glucose in alloxan-treated rats given P.murex may be as a result of

increased glycolysis (increased liver hexokinase activity).

Two gluconeogenic enzymes, glucose-6-phosphatase and fructose-1,6-

bisphosphatase have been measured in the liver and kidney of diabetic animals and those

treated with P.murex. Both enzymes showed an increase in activity during diabetes in the

liver. Administration of P.murex leaves and callus was found to be more effective in

reversing both the enzymes to normal levels in the liver of alloxan-diabetic rats. The

increased hepatic as well as renal fructose1,6- bisphosphatase activity may be due to the

changes in the allosteric effectors of the enzymes namely fructose-2,6-bisphosphate,

ATP, AMP and citrate. In a diabetic state, there is more lipolysis than lipogenesis,

especially in liver, which will result in the formation of more AMP and lower utilization

of citrate for lipogenesis leading to high energy state in the cell, i.e. higher concentration

71

of ATP is more favorable for fructose-1,6-bisphosphatase activation. (Baquer ,1998) .

The reduction in the activities of these gluconeogenic enzymes can result in decreased

concentration of blood glucose. Administration of P.murex leaves and callus had

increased the activity of hexokinase and decreased the activities of both glucose-6-

phosphatase and fructose-1, 6-bisphosphatase in alloxan diabetic rats.

Reduction in plasma total protein and albumin level was observed in diabetic rats

and this is consistent with the results obtained by Bakris,1997 ; Tuvemo et al.,1997.The

decrease in protein and albumin may be due to microproteinuria and albuminuria, which

are important clinical markers of diabetic nephropathy, ( Mauer et al. ,1981) and/or may

be due to increased protein catabolism.( Almdal et al.,1988) The results of the present

study demonstrated that the treatment of diabetic rats with the aqueous extract of P.murex

leaves and callus caused a noticeable elevation in the plasma total protein and albumin

levels as compared with their normal levels. Such improvement of serum protein and

albumin was previously observed after the oral administration of Balanites aegyptiaca (B.

aegyptiaca) to experimentally diabetic rats.( Mansour et al .,2000) It has been established

that insulin stimulates the incorporation of amino acids into proteins.(Almdal et al.,1988).

Lipid profile ,which I altered in serum of diabetic patients

( orchard,1990 ;Betteridge ,1994) , appears to be a significant factor in the development

of premature atherosclerosis and includes an increase in triglycerides and total cholesterol

levels .In this study the extract significantly reduces the triglycerides ,phospholipids and

total cholesterol. This reduction could be beneficial in preventing diabetic complication

as well as improving lipid metabolism in diabetes (cho et al., 2002).

72

The levels of serum lipids is usually elevated in diabetes mellitus ,and such an

elevation represents the risk factor for coronary heart disease ( Davidson ,1981).

Lowering of serum lipids concentration through dietary with or drug therapy seems to

be associated with a decrease in the risk of vascular disease (Rhoads et al., 1976).Several

investigation demonstrated that near normalization of the blood glucose level resulted in

significant reduction in the levels of plasma total cholesterol, triglycerides ,and

phospholipids .the same results were obtained with the fruit ethanol extract of Pedalium

murex ,which showed hypolipidemic effect in diabetic rats.(Balasubramanian et

al.,2008). The result of this study show that continuous administration of P.murex

leaves and leaves derived callus extract at the dose of 200mg /kg/day significantly lower

the increased levels of serum total cholesterol, triglycerides , and phospholipids .

The plasma levels of urea, uric acid and creatinine levels were measured, as DM

also causes renal damage due to abnormal glucose regulation, including elevated glucose

and glycosylated protein tissue levels, haemodynamic changes within the kidney tissue,

and increased oxidative stress.( Aurell and Bjorck ,1992) The Alloxan-induced diabetic

rats exhibited significantly higher plasma urea, uric acid and creatinine levels compared

to the DM group. However, the P.murex leaves and callus supplement lowered these

plasma values to a control range. A significant elevation in serum creatinine and urea

levels indicate an impaired renal function of diabetic animals.( Shinde and Goyal ,2003))

Thus, it would appear that the P.murex leaves ,callus supplement lowered the plasma

urea, uric acid and creatinine levels by enhancing the renal function that is generally

impaired in diabetic rats.

73

The serum AST and ALT levels increase as a result of metabolic changes

in the liver, such as administration of toxin, cirrhosis of the liver, hepatitis and liver

cancer including diabetes.( Chalasani et al .,2004) Similarly in the present study, it was

observed that the levels of serum AST and ALT in alloxan induced diabetic rats were

elevated. It may be due to leaking out of enzymes from the tissues and migrating into the

circulation by the adverse effect of alloxan.( Stanely et al .,1999).AST and ALT were

used as markers to assess the extent of liver damage in streptozotocin induced diabetic

rats.( Hye-Jin Hwang et al .,2005). In this study, the administration of P.murex callus

ethanol extract to alloxan-induced diabetic rats reduces AST and ALT levels efficiently

than P.murex leaves extract treated rats. In addition to the assessment of AST and ALT

levels during diabetes, the measurement of enzymatic activities of phosphatases such as

acid phosphatase (ACP) and alkaline phosphatase (ALP) is of clinical and toxicological

importance as changes in their activities are indicative of tissue damage by toxicants.

Singh et al.,2001). In our study, serum ACP and ALP increased considerably in alloxan

induced diabetic rats. Elevated level of these enzymes in diabetes may be due to

extensive damage to liver in the experimental animals by alloxan. Treatment with

P.murex callus ethanol extract in alloxan-induced diabetic rats produces a more

significant decline in these levels than the leaves extract treated rats. From the present

observation, it was evident that P.murex leaves and callus ethanol extract protects the

adverse effects of lipid peroxide mediated tissue damage in alloxan induced diabetic rats.

A marked increase in the concentration of TBARS and

hydroperoxides are observed in liver and kidney of diabetic rats (Latha and Pari, 2003;

74

Sathish and Pari, 2004). In this study shows that P.murex leaves and callus and

glibenglamide tends to bring the increased concentration of lipid peroxidation products to

near normal level.

75

INTRODUCTION

Diabetes mellitus, a disease of metabolic disorders is associated with a number of

chronic complications like nephropathy, neuropathy, retinopathy and cardiovascular

diseases (Mahdi et al., 2003). Implication of oxidative stress in the pathogenesis of

diabetes is suggested not only by oxygen free-radical generation but also due to non

enzymatic protein glycosylation and alteration in antioxidant enzymes (Mullarkey et al.,

1990; Gillery et al., 2006). Several herbal drugs in different formulations have been

experimented in search of an effective treatment. However, hyperglycemia-induced

76

oxidative stress ultimately leads to tissue damage has advanced considerably in recent

years. Effective therapeutic strategies to prevent or delay the development of this damage

remain limited and the American Diabetes Association recommended that antioxidant

therapy needs to be improved either older antioxidants such as vitamin E, L. A. (lipoic

acid), and NAC (N-acetyl-L-cysteine) needs to be reformulated, or newer antioxidants

need to be identified (Evans et al. , 2003). Plants constitute an important source of active

natural products, which differ widely in terms of structure and biological properties. They

have a remarkable role in the traditional medicine in different countries. The protective

effects of plant products are due to the presence of several components, which have

distinct mechanisms of action; some of them are enzymes and proteins and others are low

molecular weight compounds such as vitamins, carotenoids, flavonoids (Zhang and Wang

,2002), anthocyanins and other phenolic compounds (Sanchez-Moreno et al., 1998).

In recent years, much attention has been focused on the role of oxidative

stress, and it has been reported that oxidative stress may constitute the key and common

event in the pathogenesis of secondary diabetic complications (Ceriello, 2000). Free

radicals are continuously produced in the body as a result of normal metabolic processes

and interaction with environmental stimuli. Oxidative stress results from an imbalance

between radical-generating and radical-scavenging systems that has increased free radical

production or reduced activity of antioxidant defenses or both. Implication of oxidative

stress in the pathogenesis of diabetes mellitus is suggested not only by oxygen free

radical generation but also due to non-enzymatic protein glycosylation, auto-oxidation of

glucose, impaired glutathione metabolism, alteration in antioxidant enzymes and

77

formation of lipid peroxides (Mullarkey et al .,1990; Lennan et al., 1991). In addition to

reduced glutathione (GSH), there are other defense mechanisms against free radicals,

such as the enzymes superoxide dismutase (SOD), glutathione peroxidase (GPx) and

catalase (CAT), whose activities contribute to eliminate superoxide, hydrogen peroxide

and hydroxyl radicals (Soto et al .,2003).

Many of the complications of diabetes mellitus, including retinopathy and

atherosclerotic vascular disease, the leading cause of mortality in diabetes mellitus, have

been linked to oxidative stress, and antioxidants have been considered as treatments

(Cunningham et al.,1998). Plants often contain substantial amounts of antioxidants,

flavonoids and tannins and the present study suggests that antioxidant action may be an

important property of plant medicines associated with the hypoglycemic effect on

diabetes mellitus (Larson, 1988).

Materials methods

Determination of the activity of Superoxide Dismutase (SOD)

The activity of superoxide dismutase was determined by the method of

Kakkar et al., (1984)

Procedure

Preparation of enzyme source

To 0.5 ml of tissue homogenate, 0.5 ml of distilled water was added followed

by the addition of 2.5 ml of ethanol and 1.5 ml of chloroform (all reagents were

78

chilled). The mixture was shaken for 1 minute at 4oC , centrifuged and the enzyme

activity of the supernatant was determined.

ii. Assay of the activity of SOD

The assay mixture contains 1.2 ml of sodium phosphate buffer, 0.1 ml of

phenazine methosulphate, 0.3 ml of nitroblue tetrazolium and 0.5 ml of enzyme

preparation. The reaction was started by the addition of 0.2 ml of NADH and

incubated at 30oC for 90 seconds. Then the reaction was arrested by the addition of

1.0 ml of glacial acetic acid. The contents were mixed and shaken with 4.0 ml of n-

butanol. The mixture was allowed to stand for 10 minutes, centrifuged and the colour

intensity of the chromogen in butanol layer was read at 560nm in a

spectrophotometer. A system devoid of enzyme source was maintained as control.

The activity of SOD was expressed as the amount of enzyme required to give

a 50% inhibition of the reduction of nitroblue tetrazolium per minute per mg of

protein.

Determination of the activity of Catalase (CAT)

The activity of catalase was determined by the method of Sinha (1972).

Procedure

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The assay mixture contains 4.0 ml of hydrogen peroxide, 5.0 ml of phosphate

buffer and 1.0 ml of homogenate. 1.0 ml portion of the reaction mixture was

withdrawn and blown into 2.0 ml of dichromate/acetic acid reagent at 1 minute

intervals. Then the mixture was heated for 10 minutes in a boiling water bath. After

cooling, the absorbance was measured at 570nm in spectrophotometer.

The activity of catalase was expressed as M of H2O2 utilized

per min per mg of protein.

Determination of the activity of Glutathione Peroxidase (GPx)

The activity of glutathione peroxidase was assayed by the method of Rotruck

et al., (1973).

Procedure

The reaction mixture consisted of EDTA, sodium azide and H2O2, (each 0.2

ml) 0.4 ml of phosphate buffer and 0.1 ml homogenate was incubated at 37C for 10

minutes. The reaction was arrested by the addition of 0.5 ml of 10%TCA and the

tubes were centrifuged at 2000 rpm. To 0.5 ml of supernatant, 3.0 ml of disodium

hydrogen phosphate and 0.5 ml DTNB were added and the colour developed was

read at 420 nm immediately.

80

The activity of GPx was expressed as M of glutathione oxidized per min per

mg of protein.

Determination of the activity of Glutathione-S-transferase (GST)

The activity of Glutathione-S-transferase was determined by the method of

Habig et al., (1974).

Procedure

To 1.0 ml of phosphate buffer, 0.1 ml of CDNB, 1.7 ml of water and 0.1 ml

of enzyme source were added. After 5 minutes of incubation at 37C, 0.1 ml of GSH

was added and the change in optical density was measured immediately with an

internal of 1 minute for 3 minutes at 340nm in a UV-vis spectrophotometer. A

complete assay mixture without enzyme was used a control.

Activity of glutathione S-transferase was expressed as nM of CDNB-GSH

conjugate formed per min per mg of protein.

Estimatima

tion of non-enzymatic antioxidants and glutathione

recycling enzymes

Estimation of reduced glutathione

Reduced glutathione was estimated by method of Moron et al., (1979).

Procedure

81

About 0.5 ml of sample (plasma/homogenate) was precipitated with 1.0

ml of 10% TCA and the precipitate was removed by centrifugation. To 0.5 ml

of the supernatant 1.0 ml of DTNB was added and the total volume was made up

to 3.0 ml with phosphate buffer. The absorbance was read at 412nm.

The level of glutathione was expressed as mg per dL in plasma and mg per

g tissue in liver and kidney.

Estimation of vitamin E (α-Tocopherol)

The level of vitamin E was estimated by the method of Baker et al., (1980).

Procedure

To 0.5 ml of sample (plasma/homogenate), 1.5 ml of ethanol was added,

mixed and centrifuged. The supernatant was dried at 80oC. To the tubes after

dried, 0.2 ml of 2,2'-dipyridyl and 0.2 ml of ferric chloride solutions were added.

Mixed well and 4.0 ml of butanol was added. The red colour developed was read

at 520nm.

The level of -tocopherol was expressed as mg per dL in plasma and mg

per g tissue in liver and kidney.

Estimation of ascorbic acid (Vitamin C)

82

The level of ascorbic acid was estimated by the method of Omaye et al.,

(1979).

Procedure

To 0.5 ml of sample (plasma/tissue), 0.5 ml of water and 1.0 ml of 5%TCA

were added, mixed thoroughly and centrifuged for 20 minutes. To 1.0 ml of the

supernatant, 0.2 ml of DTC reagent was added and incubated at 37C for 3 hrs. Then

1.5 ml of sulphuric acid was added, mixed well and the solutions were allowed to

stand at room temperature for another 30 minutes. The colour developed was read at

520nm using a spectrophotometer.

The level of ascorbic acid was expressed as mg per dL in plasma and mg

per g tissue in liver and kidney.

RESULT

The concentration of tissues SOD, CAT, GSH, GST, and GPx were significantly

decreased in diabetic rats when compared to the control group. Administration of

PEDALIU MUREX extract and insulin to diabetic rats tend to bring the activities of these

enzymes to near normal level . The extent of increase was higher in groups treated with

ethanol extract of Pedalium murex leaves and callus than glibenclamide treated groups.

Treatment with P.murex leaves and callus to normal animals did not show any significant

alterations.

83

Table – shows significant reduction in non-enzymatic antioxidants in

liver and kidney of Alloxan-diabetic rats when compared to controls. Administration of

P.murex leaves and callus extract (200 mg/kg body weight) and glibenclamide for a

period of 3 weeks decreased the glucose levels significantly and improved the tissue

antioxidant status significantly (p < .05). p.murex callus extract at a dose of 200 mg/kg

body weight was more effective then the other dose of leaves extracted 200 mg/kg body

weight.

DISCUSSION

Under in vivo conditions, GSH acts as an antioxidant and its decrease was

reported in diabetes mellitus (Baynes and Thorpe 1999). We have observed a significant

decrease in GSH levels in liver and kidney during diabetes. The decrease in GSH levels

represents increased utilization due to oxidative stress (Anuradha and Selvam 1993). The

depletion of GSH content may also lower the GST activity as GSH is required as a

substrate for GST activity (Rathore et al. 2000). Depression in GPx activity was also

84

observed in liver and kidney during diabetes. GPx has been shown to be an important

adaptive response to condition of increased peroxidative stress (Matkovics et al. 1982).

The increased GSH content in the liver and kidney of the rats treated with P.murex

leaves, callus and glibenclamide may be one factor responsible for inhibition of LPO.

SOD and CAT are the two major scavenging enzymes that remove toxic free radicals in

vivo. Previous studies have reported that the activity of SOD is low in diabetes mellitus

(Vucic et al. 1997). Reduced activities of SOD and CAT in liver and kidney have been

observed during diabetes and this may result in a number of deleterious effects due to the

accumulation of O.− 2 and H2O2 (Searle and Wilson 1980). Administration of Pedalium

murex leaves and callus increased the activity of enzymes and may help to control free

radical, which scavenge the free radicals generated during diabetes. Any compound,

natural or synthetic, with antioxidant properties, might contribute towards the partial or

total alleviation of this damage. Therefore, removing O.− 2 and OH∗ is probably one of

the most effective defenses against diseases (Lin et al., 1995). The result of the SOD

and CAT activity suggest that P.murex leaves and callus contains a free radical

scavenging activity, which could exert a beneficial action against pathological alterations

caused by the presence of O.− 2 , H2O2 and OH∗. This action could involve mechanisms

related to scavenging activity. Increased lipid peroxidation in diabetes can be

due to enhance oxidative stress in the cells as a result of depletion of

antioxidant scavenger system. Reduced glutathione is a major

endogenous antioxidant which counteracts free radical mediated

damage. Depletion of liver and kidney reduced glutathione levels

represents enhanced oxidative stress (Anuradha and Selvam ,1993).

85

Superoxide dismutase is an antioxidant enzyme which reduces superoxide

radicals to water and molecular oxygen (McCord et al., 1976) whilst catalase

reduces hydrogen peroxide (Gutteridge, 1995). Diminished activity of

these antioxidant enzymes result elevation of ROS and ROS mediated cell

destruction. Reduced activities of superoxide dismutase and catalase in

liver and kidney were observed in diabetic rats and these were reverted to

near normal status on extract treatment.

The decrease could have been due to increased utilization of ascorbic

acid as an antioxidant defense against increased reactive oxygen species or to a decrease

in the GSH level, since GSH is required for the recycling of ascorbic acid Hunt, (1996).

α-Tocopherol, a lipid soluble, chainbreaking antioxidant was significantly decreased in

liver and kidney of STZ-diabetic rats. P.murex leaves,callus and glibenclamide treatment

tends to bring the α tocopherol levels to near normal value. Higuchi (1982) observed a

decreased hepatic α-tocopherol in rats with STZ-induced diabetes. These results suggest

that the demand for the antioxidant vitamin E is increased due to the activation of free

radical related metabolism in diabetes. Impaired generation of naturally-occurring

antioxidants (GSH, ascorbic acid, and α-tocopherol) results in increased oxidative injury

by failure of protective mechanisms. There is increased flux of glucose through the

polyol pathway, which is hyperactive in hyperglycemia (Moncada and Higgs ,1993).

Vitamin E is one of the most important free radical scavenging chain-breaking

antioxidant within biomembrane (Parks and Traber, 2002). Reduced glutathione, a major

86

endogenous antioxidant, plays a crucial role in the antioxidant defense (Anuradha and

Selvam ,1993). Vitamin C, a major extra cellular non-enzymatic antioxidant, has crucial

role in scavenging several reactive oxygen species. The observed increase in antioxidant

status P.murex leaves and callus extact to treated diabetic rats suggests its potent

antioxidative effects. Furthermore the plant drug was found to be as effective as that of

the reference drug glibenclamide. Further studies are therefore needed to isolate and

characterize the bioactive antidiabetic principles from P.murex leaves and leaves derived

callus.

87

INTRODUCTION

Diabetes mellitus is the most common disease associated with

carbohydrate metabolism, affecting about 200 million people worldwide. Extracts of

88

various plant materials capable of decreasing blood sugar have been tested in

experimental animal models and their effects confirmed. Many unknown and lesser

known plants are used in folk and tribal medicinal practices in India. The medicinal

values of these plants are not much known to the scientific world.

Today, more than 200 traditional medicinal plants have been used for the

treatment of diabetes mellitus and widely practiced in South India. Plant drugs are

frequently considered to be less toxic and more free from side-effects than synthetic ones

[Momin , 1985]. Synthetic oral hypoglycemic agents can produce a series of side-effects

including hematological, gastro-intestinal reactions, hypoglycemic coma, and

disturbances in liver and kidney metabolisms. In addition, these preparations are not ideal

for use during pregnancy [Altan and Kilic .,1997).

Liver disease is one of the leading causes of death in persons with type 2

diabetes. The standardized mortality rate for death from liver disease is greater than that

of cardiovascular disease. The spectrum of liver disease in type 2 diabetes ranges from

nonalcoholic fatty liver disease to cirrhosis and hepatocellular carcinoma (Keith et al.,

2004). Experimental type 1 diabetes induced with streptozotcin or alloxan in rats display

many features seen in human subjects with uncontrolled diabetes mellitus(Chattopadhyay

et al ., 1997). liver and kidney in some cases (Ghosh, 2001). Patients depend on insulin

for management of IDDM. Without insulin, they develop degenerative complications

such as microangiopathy, nephropathy and retinopathy.

89

Diabetic nephropathy is the most important cause of death in type 1 diabetic patients,

of whom, 30 – 40% eventually develop end stage renal failure (Giorgino et al., 2004).

Liver disease is one of the leading cause of death in persons with type 2 diabetes. The

standardized mortality rate for death from liver disease is greater than that of

cardiovascular disease. The spectrum of liver disease in type 2 diabetes ranges from

nonalcoholic fatty liver disease to cirrhosis and hepatocellular carcinoma (Keith et al.,

2004).

Oxidative stress has been considered as a common pathogenetic factor in

diabetic nephropathy and other complications (Baynes ,1991; Larkins, and

Dunlop,1992,). Diabetic nephropathy is characterized by glomerular hypertrophy,

thickening of glomerular and tubular basement membranes, increased amounts of

extracellular matrix (ECM) in the mesangium, and increased glomerular permeability

(Zatz et al.,1986 ; Steffes et al.,1992). Excessive excretion of glycogen through the

glomeruli is reabsorbed into the cytoplasm of tubules. These phenomena are especially

prominent in the straight portion of proximal tubules. In histological preparations, these

glycogen inclusions are washed out and result in a clearing effect within the tubular

epithelial cells. This phenotype is referred to as Almanni- Ebstein cells and is a clear

morphological characteristic of a diabetic kidney (Watanabe and Hotta, 1997).

Lipid peroxidation is a free radical mediated process leading to oxidative

deterioration of polyunsaturated lipids. Under normal physiological conditions, low

90

concentrations of lipid peroxide are found in plasma and tissues. The possible source of

oxidative stress in diabetes includes shifts in redox balance resulting from altered

carbohydrate and lipid metabolism, increased generation of reactive oxygen species, and

decreased level of antioxidant defenses such as GSH and ascorbic acid (Baynes, 1991).

Increased levels of TBARS suggest increasing oxygen free radicals. Lipid peroxide-

mediated tissue damage has been observed in the development of Type II and Type I

diabetes.(Sundaram et al.,1996) have reported that the concentration of lipid peroxides

increases in the kidney of diabetic rats.

Liver plays an important role in the maintenance of blood glucose level by

regulating its metabolism, hexokinase ,which brings about the first phosphorilation.stepof

glues metabolism is reduced significantly in the diabetic rats (Nehal and Baquer,1989).In

the liver the enzymes is an important regulator of glucose storage and disposal . Attention

has long centered on the liver in diabetes mellitus because of the importance of this organ

in carbohydrate metabolism and regulation of blood sugar.

During diabetes, a profound alteration in the concentration and

composition of lipids occurs. Liver and kidney are important for glucose and lipid

homeostasis, they participates in the uptake, oxidation and metabolic conversion of free

fatty acids, synthesis of cholesterol, phospholipids and triglycerides. Thus it is expected

to have changes in liver and kidney during diabetes (Seifter and England ,1982).. Liver

during diabetes, showed a relatively severe impairment in antioxidant capacity than

kidney. The kidney exhibits a characteristic pattern of changes during diabetes (Sharma

91

et al., 2003). The present study demonstrates the efficacy of P.murex leaves and leaves

derived callus (ethanol extract) in reducing diabetes-induced functional and histological

alterations in the kidneys.

MATERIALS AND METHODS

Animals

Healthy male adult albino rats (Wistar strain) of 6-7 weeks old, weighing

150 ± 20 g was procured from “Sri Venkateswara Enterprises”, Bangalore, India.

They were housed in clean sterile polypropylene cages with proper aeration and

lighting (12 ± 1 hr day / night rhythm) throughout the experimental period. During

the course of the experiments, the temperature was maintained between 27ºC ±

2ºC. The animals were fed with commercially available pelleted rat feed (Sri Sai

Durga feeds Bangalore, India.Under the trade name “Sri Sai Durga feed and

food”) and water ad libitum. The usage and handling of experimental rats was

followed as per the rules and regulations given by the Institutional Ethics

Committee for the purpose.

Induction of Diabetes mellitus in rats

The rats were injected alloxan monohydrate dissolved in sterile

normal saline at a dose of 150mg/kg body weight , intraperitoneally .Since alloxan

is capable producing fatl hypoglycemia as a result of massive insulin release rats

were treated with 20% glucose solution bottles in their cages to prevent

hypoglycemia(Stanely Mainzen Prince,1998).After a fornight ,rats with moderate

diabetes having glycosuria (indicated by Benedicts test for urine) and hyper

glycemia with blood glucose range of 250±30mg/dl were used for the treatment.

Experimental designs

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Experimental design

The rats were divided into seven groups, each group consists of six animals.

Group I :Served as control and received normal feed and water ad libitum .

Group II : served as a normal rats received 200 mg/kg /bw Pedalium murex

leaves extract and water ad libitum.

Group III : served as normal rats received 200 mg/kg /bw Pedalium murex

leaves derived callus extract and water ad libitum.

Group IV :Served as diabetic control and received feed and water ad libitum

Group V : Diabetic rats and were treated orally with ethanol extract of

Pedalium murex leaves at the dose of 200 mg/kg body weight daily

for 21 days, once a day.

Group VI : Diabetic rats and were treated orally with ethanol extract of

Pedalium murex leaves derived callus at the dose of 200 mg/kg

body weight daily for 21 days, once a day.

Group VII :Diabetic rats given glibenglamide orally at the dose of 0.6 mg/kg

body weight daily for 21 days, once a day.

Histopathological studies

One portion of liver and kidney tissues were removed after sacrificed and rapidly

placed in 10% phosphate buffered-formalin for histological examination. Tissues were

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dehydrated in alcohol, embedded in paraffin wax, sectioned in 5m and stained with

haematoxylin and eosin for light microscopy. The sections were examined at 40x. in liver

,10x in kidney

Histopathology

The liver, kidney and pancreas were preserved in 20% formalin immediately after

removal from the animal.

Tissue processing

Liver, kidney and pancreatic tissues were placed in 10% formalin (diluted to 10%

with normal saline) for 1 hr to rectify shrinkage due to high concentration of formalin.

The tissues were dehydrated by ascending grades of isopropyl alcohol by immersing in

80% isopropanol overnight and 100% isopropyl alcohol for 1 hour. The dehydrated

tissues were cleared in two changes of xylene, 1 hour each. The wax impregnated tissues

were embedded in paraffin blocks using the same grade wax. The paraffin blocks were

morented and cut with rotary microtome at 3 micron thickness.

The sections were floated on a tissue floatation bath at 40°C and taken on glass

slides and smeared with equal parts of egg albumin and glycerol. The sections were then

melted in an incubator at 60°C and after 5 min the sections were allowed to cool.

Tissue staining

The sections were deparaffinised by immersing in xylene for 10 min in horizontal

staining jar. The deparaffinised sections were washed in 100% isopropyl alcohol and

stained in Ehrlich’s hematoxylin for 8 min in horizontal staining jar. After staining in

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hematoxylin, the sections were washed in tap water and dipped in acid alcohol to remove

excess stain (8.3% HCl in 70% alcohol).

The sections were then placed in running tap water for 10 min for

blueing (slow alkalization). The sections were counter stained in 1% aqueous eosin (1 gm

in 100 ml tapwater) for 1 min and the excess stain was washed in tap water and the

sections were allowed to dry.

Complete dehydration of stained sections was ensured by placing the

sections in the incubator at 60°C for 5 min. When the sections were cooled, they were

mounted in DPX mount having the optical index of glass (the sections were wetted in

xylene and inverted on to the mount and placed on the cover slip). The architecture was

observed low power objective under microscope. The cell injury and over aspects were

observed under high power dry objective (Dunn 1974).

RESULT

Kidney sections of diabetic animals showed thickening on the walls of nephrons

filling their lumen and glomerulopathy. The thickening of the walls was reversed by the

P.murex leaves and leaves derived callus (200 mg/kg) treatment, but the glomerulus

remain expanded Liver tissue of diabetic rat showed distortion in the arrangement of cells

around the central vein, enlargement and thickening of the walls of veins, capillaries, and

development of fibrosis in the degenerated cells. P.murex leaves and leaves derived

callus (200 mg/kg) treatment almost restored the cellular arrangement of hepatocytes

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around the central vein and reduced fibrosis. It also helped to bring the blood vessels to

normal condition.

In the normal liver tissue section shows sinusoidal cards of hepatocytes with

central vein and portal tracts. The portal tracts show portal triad with portal vein, hepatic

artery and bile duct, where as the diabetic rat liver tissue section shows distortion in the

arrangement of cells around the central vein, periportal fatty infiltration with focal

necrosis of hepatocytes (Figure 1 a and b). The leaf extract of Pedalium murex (100 and

200 mg / kg body weight) treated brought back the cellular arrangement around the

central vein and reduced necrosis. Also it helped to bring the blood vessels to normal

condition (Figure 1 c and d). The group V and VI did not show any significant change of

liver, when compared with group I (figure 1 e and f).

Kidney sections of diabetic animals showed thickening on the walls of nephrons

filling their lumen and glomerulopathy. Kidney sections of diabetic rat showed tubular

damage, proteinuria and haemorrhage. Haemorrhage is seen with in the Bowman’s space

due to glomerular damage (Figure 2 a and b). In Pedalium murex leaf extract and callus

extract (200 and 200 mg / kg body weight) treated diabetic kidney, the damaged capillary

loops with increase in the thickness of the wall, glomeruli and tubules without proteinuria

and haemorrhage (Figure 2 c and d) Group V and VI did not alter the structure of kidney,

when compared with group I (Figure 2 e and f).

DISSCUSION

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The main function of the kidneys is to excrete the waste products of metabolism

and to regulate the body concentration of water and salt. The morphological changes in

alloxan diabetic rats in the present investigation is associated with significant increased of

total protein excreted, albuminuria, glycosuria, and urinary urea levels, indicating

impaired renal function of diabetic rats. Alloxan-induced diabetes in rats had been shown

to be associated with functional and/or morphological changes in the kidney (Badole et

al., 2006).In our study, treatment of alloxan-diabetic rats with P.murex leaves and leaves

derived callus and glibenglamide induced a fall in the level of all these metabolic

parameters. However, the improvement in urinary protein, albumin, glucose and urea

excretion with P.murex extract were not sufficient to reach the levels observed in the

non-diabetic rats; moreover P.murex did not alter any biochemical kidney function

variables in non diabetic rats. Similar results were obtained with diabetic rabbits treated

with Eugenia jambolana (Kedar and chakrabarti, 1983). and non-diabetic rats treated

with Bauhinia forficata (Pepato et al, 2002). Albumin measurements are required, as

measurements of urinary total protein are insufficiently sensitive (Harycy, 2002).

Microalbuminuria and proteinuria typically reflect the presence of moderate and

advanced lesions, respectively, in kidney disease (Roy, 2004; Van den Born et al., 1995).

However, the development of diabetic nephropathy is characterised by a progressive

increase in urinary protein particularly albumin and a late decline in glomerular filtration

rate, leading eventually to end-stage renal failure (Salah et al., 2004). Histologically, the

kidneys section of STZ-diabetic control rats showed marked multifocal clarifications,

vacuolations and abundance of mucopolysaccharide in diabetic rats’ kidneys. Moreover,

it has been reported that streptozotocin does not possess any significant nephrotoxic

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potential (Floretto et al., 1998). All structural changes in kidneys resulting from STZ

administration in rats can thus be attributed to altered metabolism in diabetes (Rasch,

1980). Normoglycaemia with A. occidentale treatment could ameliorate the glomerular

and tubular lesions that characterise diabetic nephropathy. The improvement of renal

morphology and function associated with STZ-induced diabetes and P.murex treatment

in the present investigation could be attributed to its antidiabetic action resulting in

alleviation of altered metabolic status in animals. An infusion of extract prepared from

the leaves ,stem, and fruits of P.murex in cold water is a demulcent and a diuretic found

useful in the disorder of urinary ststems.such as gonorrhea , dysuria and incontinence of

urine etc.(Chopra et al.,1999; Shukla and khanuja,2004).

The action by which the extract lowered the blood glucose is not well

known; it may increase glycogen level in liver by an increase in glycogenesis and/or a

decrease in glycogenolysis. Since P.murex did not significantly reduce glycaemia in non-

diabetic animals, it is possible that its mechanism of action is similar to that of

glibenclamide and insulin. Similar results have been observed with the treatment of STZ-

induced diabetic rats with Cassia kleinii leaf extract and glibenclamide (Babu et al.,

2003). In another hand, the chemical substances therapeutic properties could be mediated

by the stimulation of regeneration process and revitalisation of remaining β cells

(Diatewa et al., 2004).

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The phytochemical analysis had revealed the presence of alkaloids,

polyphenols and saponins in the plant extract. Based on the increasing number of reports

on blood glucose reduction associated with some saponins (Diatewa, 2004) and alkaloids

(Bolkent et al., 2000) isolated from other medicinal plants, it is likely that the active

principle (s) could be present in one or the two families of chemical substances.

Accelerated chemical modification of proteins by glycosidation and accumulation of

AGE (Advanced Glycation End-products) are implicated in diabetic nephropathy and

Hennebele et al. (2004) suggested that these molecules can be inhibited with

polyphenols. Standard antidiabetic drugs such as insulin and sulfonylureas cause

hypoglycaemia when taken in excessive doses and hypoglycaemia is the most worrisome

effect of these drugs P.murex did not cause any hypoglycaemia, therefore, it could be an

effective treatment for early renal disease and possibly other diabetic complications.

In the liver of diabetic rats (group-III) shrunken nuclei, granular cytoplasm

(Figure 2 b), dilatation in the sinusoids and inflammation were noticed (Figure 2 c).

These changes were reduced in A. vera-fed rats of group-IV (Figure ). This may be due to

beneficial and protective effect of A. vera extract on liver tissues of diabetic rats. Our

histological findings are in agreement with the degenerative structural changes reported

in liver tissues as result of insulin depletion in neonatal STZ (100 mg/kg) - induced type-

II diabetic rat models.( Can et al.,2004 ).observed an increase in degeneration in central

veins to portal veins, excess vacuolization, granular appearance in the cytoplasm,

dilations in the sinusoids and moderate hyperemia.(Ayesha et al.,2008).P.murex leaves

and leaves derived callus appears to be an attractive material for further studies leading to

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possible drug development for diabetes. Development of phytomedicines is relatively

inexpensive and less time consuming; it is more suited to our economic conditions than

allopathic drug development which is more expensive and spread over several years.

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