34

2.1. CARISSA CARANDAS

The karanda has attracted more interest as a source of fruit and as a medicinal

plant than as an ornamental. Its botanical name was in recent years changed to Carissa

congesta Wight (syn. C. carandas Auct, formerly widely shown as C. carandas L.). It is

called kerenda in Malaya, karaunda in Malaya and India; Bengal currant or Christ's thorn

in South India; nam phrom, or namdaeng in Thailand; caramba, caranda, caraunda and

perunkila in the Philippines.

Table2.1: Scientific classification of Carissa carandas

Kingdom Plantae – Plants

Subkingdom Tracheobionta – Vascular plants

Superdivision Spermatophyta – Seed plants

Division Magnoliophyta – Flowering plants

Class Magnoliopsida – Dicotyledons

Subclass Asteridae

Order Gentianales

Family Apocynaceae – Dogbane family

This species is a rank-growing, straggly, woody, climbing shrub, usually growing

to 10 or 15 ft (3-5 m) high, sometimes ascending to the tops of tall trees; and rich in

white, gummy latex. The branches, numerous and spreading, forming dense masses, are

set with sharp thorns, simple or forked, up to 2 in (5 cm) long, in pairs in the axils of the

leaves. The leaves are evergreen, opposite, oval or elliptic, 1 to 3 in (2.5-7.5 cm) long;

dark-green, leathery, glossy on the upper surface, lighter green and dull on the underside.

The fragrant flowers are tubular with 5 hairy lobes which are twisted to the left in the bud

instead of to the right as in other species. They are white, often tinged with pink, and

borne in terminal clusters of 2 to 12. The fruit, in clusters of 3 to 10, is oblong, broad-

ovoid or round, 1/2 to 1 in (1.25-2.5 cm) long; has fairly thin but tough, purplish-red skin

turning dark-purple or nearly black when ripe; smooth, glossy; enclosing very acid to

fairly sweet, often bitter, juicy, red or pink, juicy pulp, exuding flecks of latex. There may

be 2 to 8 small, flat, brown seeds.

Medicinal Uses: The unripe fruit is used medicinally as an astringent. The ripe fruit is

taken as an antiscorbutic and remedy for biliousness. The leaf decoction is valued in

35

cases of intermittent fever, diarrhea, oral inflammation and earache. The root is employed

as a bitter stomachic and vermifuge and it is an ingredient in a remedy for itches. The

roots contain salicylic acid and cardiac glycosides causing a slightly decrease in blood

pressure. Also reported are carissone; the D-glycoside of B-sitosterol; glucosides of

odoroside H; carindone, a terpenoid; lupeol; ursolic acid and its methyl ester; also

carinol, a phenolic lignan. Bark, leaves and fruit contain unnamed flavonoids.

Other Uses:

Root: Pruritis; Gonorrhoea: Pyrexia; Indigestion; Chronic ulcer.

Unripe fruit: Haematemesis; Appetizer; Mucolytic; to allay thirst.

Ripe fruit: Carminative; Expectorant; Biliousness; Haematemesis; Antidote for poisons;

Appetizer; easily digested.

Chemical constituents

Various fatty acids such as palmitic, stearic, Oleic and linoleic acids were found in seed of

Carissa carandas. A terpinic alcohol carisol, amino acids serine, glutamine, alanine, valine,

phenylalanine and glycerin reported in fresh fruits extract. Leaf part of Carissa Carandas

Linn. Show the radical scavenging activity and inhibitory effects towards the in‐vitro

reaction of hypoxanthine and xanthin oxidase (XO) due to presence of plant extract, which

contain the aglycones quercetin, kaempferol and apigenin along with allopurinol .(1, 2, 3)

Substituents and Adulterants: No substituents and adulterants of Carrisa Carandas.

36

2.2. CORDIA DICHOTOMA

Cordia is a genus of shrubs and trees in the borage family Boraginaceae. a number of the

tropical species have edible fruits, called sebesten or clammy cherries. In India, the fruits

of local species are used as a vegetable, raw, cooked, or pickled, and are known by many

names, including lasora in Hindi. One such species is Cordia Dichotoma (fragrant

manjack), which is called gunda in Hindi.

Table2.2: Scientific classification of C. dichotoma

Kingdom: Plantae

(unranked): Angiosperms

(unranked): Eudicots

(unranked): Asterids

Order: (unplaced)

Family: Boraginaceae

Genus: Cordia

Species: C. dichotoma

Cordia Dichotoma L. is a medium sized tree with short crooked trunk; leaves simple,

entire and slightly dentate, elliptical-lanceolate to broad ovate with a round and cordate

base; flower white, small in lax terminal or axillary cyme; fruits drupe, yellowish brown,

pink or nearly black when ripe with viscid sweetish transparent pulp surrounding a

central stony part. The plant part used is bark, leaves and fruits, in India (Western

Rajasthan) ripe fruits are eaten, green fruits are eaten as vegetable, in curry and pickle.

Medicinal uses: The bark is bitter astringent, acrid after digestion, constipating,

antihelmenthic, cooling and is useful in dyspepsia, fever, and diarrhoea, burning

sensation, vitiated conditions of cough and pitta, helminthiasis, leprosy and skin diseases.

The leaves are aphrodisiac and are useful in gonorrhoea and ophthalmodynia. The fruits

are sweet, cooling, emollient, anti-helminthic, purgative, diuretic, expectorant, vulnerary,

depurative and febrifuge and are useful in vitiated conditions of vata and pitta, ulcers,

leprosy, skin diseases and burning sensation, bronchitis, dry cough, pectoral diseases,

37

urethritis, chronic fever, arthralgia, pharyngopathy, splenopathy and ring worm

infestations(4,5,6).

38

2.3. STANDARDIZATION OF PLANTS

2.3.1. Plant collection and identification:

Leaf part of Cordia Dichotoma & Carissa Carandas was collected from Jalandhar Cant.,

Jalandhar in the month January 2010. The plant material was identified by Dr. H. B.

Singh of Raw Material Herbarium & Museum, NISCAIR, New Delhi where a voucher

specimen (No.NISCAIR/RHMD/Consult/-2009-10/326/128) has been deposited at the

herbarium unit.

2.3.2. Material:

2.3.2.1 Solvent: Acetic acid, acetone, benzene, n-Butanol, chloroform, Dichloromethane,

ethanol, n-Hexane, methanol, pyridine, petroleum ether, tween 80, toluene, Xylene are

used. All the solvents were purchased from Sd fine chemicals Pvt Ltd.

2.3.2.2 Chemicals: anisaldehyde, alloxan, bismuth carbonate, calcium chloride, copper

sulphate, ferric chloride, hydrochloric acid, Iodine, Lead acetate, Magnesium chloride,

mercuric chloride, Ninhydrin, Niric acid, Phloroglucinol, Potassium iodide, Potassium

dichromate, Potassium sodium tartarate, Ruthenium red, safranine, sodium acetate,

sodium iodode, sodium hydroxide, sodium nitroprusside, suden red III, Sulphuric acid,

Tannic acid, vanillin. All the chemicals were purchased from Sd fine chemicals Pvt Ltd.,

Mimbai.

2.4. Pharmacognostic Studies (7)

2.4.1. Macroscopical evaluation: Medicinal plant materials are categorized according to

sensory, microscopical characteristics. Taking into consideration the variations in sources

of crude drug and their chemical nature, they are standardized by using different

techniques including the methods of estimation of chief active constituents.

Organoleptic evaluation of drugs refers to the evaluation of drugs by colour, odour, size,

shape, taste and special features including touch and texture etc. They are of primary

importance before any further testing can be carried out. Organoleptic evaluations can be

done means of organs of sense which includes the above parameters and thereby define

some specific characteristics of material which can be considered as a first step towards

39

establishment of identity and degree of purity. The following organoleptic investigations

were done.

Colour: The untreated sample was properly was properly examined under diffused

sunlight or artificial light source with wavelengths similar to that of daylight.

Shape and Size: The length, breadth and thickness of the drug are of great importance

while evaluating a crude drug. A graduating ruler in millimeter is adequate for the

measurement. Small fruit & leaves were measured by aligning ten of them on a sheet of a

calibrated paper 1 mm apart between the line and the result was divided 10. Average

length, breadth and thickness were determined.

2.4.2. Surface characteristics, texture and fracture: The texture was best examined by

taking a small quantity of material and rubbing it between the thumb and forefingers, it

was usually described as smooth, rough and gritty. The touch of the material determined

the softness or hardness. Bends or rupture caused to the samples provided information of

brittleness and appearance of the fractured plan as fibrous, smooth, rough, granular etc.

All these characteristics were valuable in indicating the general type of material and

presence of more than one compartment.

The study of morphology of leaves was done by taking ten samples and observed for

various qualitative and quantitative macroscopical characters viz. shape of lamina, type

of apex and base, surface texture, margins, petiole structure, organoleptic characters, type

of floral arrangements and length width of the same.

2.4.3. Macroscopical evaluation (7): In macroscopical evaluation determined the

presence of any crystal structures.

2.4.3.1 Powder Study:

Leaves of Cardio Dichotoma/ Carissa Carandas were powdered and fine powder was

taken up for microscopical evaluation as follows:

1. A small quantity was put on a slide and after mounting on glycerin, 10 min were

provided as spread out time. Finally, it was observed for microscopical characters.

2. Another small quantity was stained using safranin, mounted with glycerin and

observed for various microscopical characters.

40

3. Another small quantity was stained using Sudan red III, mounted with glycerin and

observed for various microscopy characters.

2.4.4. Physiochemical studies: Physiochemical investigations were done to identify the

amount of inorganic and moisture content and estimate dry weight of drug:

2.4.4.1. Determination of ash values:

a. Total Ash Value

Method: Weighed accurately 2 to 3 g of air-dried leaves in a tarred platinum or silica

dish and incinerated at a temperature not exceeding 4500C until free from carbon, cooled

and weighted. When a carbon free could not be obtained in this way exhausted the

charred mass with hot water, collected the residue on an ash less filter paper incinerated

the residue and filter paper until the ash was white or nearly so, added the filtrate,

evaporated to dryness and ignited at a temperature not exceeding 4500C. Calculated the

percentage of ash with reference to the air dried drug.

b. Determination of Acid Insoluble Ash

Method: boiled the ash with 25 ml of 2M Hydrochloric acid for 5 min, collected the

insoluble matter in a Gooch crucible or on an ash less filter paper, washed with hot water,

ignited, cooled in desiccator and weighed. Calculated the percentage of acid insoluble ash

with reference to air-dried drug.

c. Determination of water soluble ash:

Method: Boiled the ash for 5min. with 25 ml of water, collected the insoluble in a Gooch

crucible or on an ash less filter paper, washed with hot water, and ignited for 15min. at a

temperature not exceeding 4500C. Subtracted the weight of insoluble matter from the

weight of the ash, the difference in weight represents the water soluble ash. Calculated

the percentage of water soluble ash with reference to the air dries drug.

2.4.4.2. Loss on drying (8): Loss on drying of the air-dried leaves was analyzed by using

procedure as described in I.P. 1996.

41

Accurately weighted quantity of sample was taken in a tarred L.O.D. Bottle and initial

weight was taken. The sample was heated at 1050C in an oven and weighed. This

procedure was repeated until a constant weight was obtained. The moisture content of

sample was calculated with reference air dried drug.

2.4.5. Determination of extractive values: The water soluble and alcohol soluble

extractive values of air dried sample were evaluated using the procedure described in IP

1996.

Water soluble extractives

Alcohol soluble extractives

Water soluble extractives

5 grams of air dried plant material was added to 50 ml of boiled water at 800C in a

stoppered flask. It was shaken well and allowed to stand for 10 minutes so as to cool it

and then it was filtered. 5 ml of filtrate was transferred to a tarred evaporating dish,

which is 7.5 cm in diameter, the solvent was evaporated on water bath, allowed to dry for

30 minutes, finally dried in an oven for 2 hours at 1000C and residue was weighed.

Percentage of water soluble extractives was calculated with reference to the air dried

drug.

Alcohol soluble extractives

5 grams of air dried plant material was macerated with 100 ml of methanol in a closed

flask, shaking frequently during the first 6 hours and allowed to stand for 18 hours.

Thereafter it was filtered rapidly taking precaution against loss of methanol. Evaporate 25

ml of filtrate to dryness in a tarred flat bottom shallow dish dried at 1050C and weighed.

Percentage methanol soluble extractive was calculated with reference to the air-dried

plant material. (I.P., 1996)

2.4.6. Determination of systematic solvent extractive values:

A definite weight of crude drug powder was taken in a soxhlet apparatus thimble after

making moderately coarse and then continuous hot soxhlet extraction was done by

various solvent in a sequence of increasing polarity as follows:

42

1. Petroleum ether

2. Acetone

3. Chloroform

4. Methanol

Before starting extraction with subsequent solvent, the powder was dried of all its

previous solvent content by spreading out on paper. The weight of the extracts obtained

was determined after concentration in buchi rota evaporator and the percentage of

extractive value expressed on dry weight basis.

2.4.7. PRELIMINARY PHYTOCHEMICAL SCREENING (8)

Chemical tests: Presence of type of constitutes was determined by using various chemical

diagnostic agents as follows:

A. Test for alkaloids :

a. Mayer’s test: Extract/fraction on treatment with Mayer‟s reagent [Potassium mercuric

iodide solution] should give cream coloured precipitate if alkaloids are present.

b. Dragendorffs test: Extract/fraction on treatment with Dragondroff‟s reagent

[Potassium bismuth iodide solution] should give reddish brown precipitate if alkaloids

are present.

c. Wagner’s test: Extract or fraction on treatment with Wagner‟s reagent [solution of

iodine in potassium iodide] should give reddish brown precipitate if alkaloids present.

d. Hager’s test: Extract or fraction on treatment with Hager‟s reagent [saturated solution

of Picric acid] should give yellow precipitate if alkaloids are present.

e. Tannic acid test: Extract or fraction on treatment with 10% Tannic acid solution

should give buff coloured precipitate if alkaloids are present.

B. Test for Glycosides: 1) General test for the presence of glycosides:

Part A: Extracted 200 mg of the drug by warming in test tube with 5ml of dilute (10%)

sulfuric acid on a water bath at 1000C for 2 min, centrifuged or filtered, pipette off

supernatant or filtrate. Neutralized the acid extract with 5% solution of sodium hydroxide

43

(noted the volume of NaOH added). Added 0.1 ml of fehling‟s solution A and B until

alkaline (test with pH paper) and heated on water bath for 2 min. noted the quantity of red

precipitate formed and compared with that formed in Part-B.

Part B: Extracted 200mg of the drug using 5 ml of water instead of sulfuric acid. After

boiling added volume of water equal to the volume of NaOH used in the above test.

Added .01 ml of Fehling‟s solution A and B until alkaline (test with pH paper) and heated

on water bath for 2 min. noted the quantity of red precipitate formed.

Compared the quantity of precipitate formed in Part B with that of formed in Part-A. If

the precipitate in Part-A greater than in Part-B then Glycoside may be present. Since

Part-B represents the amount of free reducing sugar already present in the crude drug.

Whereas Part-A represents free reducing sugar plus those related on acid hydrolysis of

any sides in the crude drug.

1. Test for free sugars: after complete removal of free sugars, the extract was

hydrolyzed with mineral acid and then tested for the glycone and aglycones moieties.

a. Raymond’s test: extract fraction when treated with dinitro benzene in hot methanolic

alkaline, should give violet color.

b. Legal’s test: extract fraction when treated with pyridine and added alkaline sodium

nitroprusside solution, blood red color should appear.

c. Bromine water test: extract fraction when treated with bromine water should give

precipitate.

2. Chemical test for specific glycosides:

I.Test for saponin glycosides:

a) Froth Test: Placed solution of drug in water in a semi micro tube shaken well and

noted the stable froth.

b) Haemolysis test: added 0.2 ml solution of saponin (prepared in 1% normal saline) to

0.2 ml of v/v blood in normal saline and mixed well, centrifuged and noted the red

supernatant compared with control tube containing 0.2 ml of 10% blood in normal saline

diluted with 0.2 ml of normal saline.

44

II. Test for anthraquinone glycosides:

a). Borntrager’s test: Boiled extract or fraction with 1 ml of dil. Sulphuric acid in test

tube for 5 min (anthracene glycosides are hydrolyzed to aglycone and sugars by boiling

with acids) centrifuged or filtered while hot (if centrifuged hot, the plant material can be

removed while anthracene aglycones are still sufficiently soluble in hot water, they are

however insoluble in cold water), pipette out the supernatant or filtrate, cooled and

shaken with an equal volume of dichloromethane (the aglycones will dissolve preferably

in dichloromethane) separated the lower dichloromethane layer and shaken with half its

volume with dilute ammonia. A rose pink to red color is produced the ammonical layer

(aglycones based on anthraquinones give red color in the presence of alkali) if

anthraquinone is present.

b). Modified Brontrager’s test: Boiled 200 mg of the extract with 2ml of dilute

sulphuric acid, 2ml of 5% aqueous ferric chloride solution for 5min and continued the

test as above. As some plant contains anthracene aglycone in reduced form, if ferric

chloride is used during the extraction, oxidation to anthraquinones takes place, which

shows response to the Borntrager‟s test if anthraquinone is present.

III.Test for cardiac glycosides:

a) Kedde’s test: Extracted the drug with chloroform, evaporated to dryness and added

one drop of 90% alcohol and 2 drops of 2% 3,5-dinitro benzoic acid (3, 5-dinitro benzene

carboxylic acid-kedde‟s reagent) in 90% alcohol. Made alkaline with 20% sodium

hydroxide solution. If purple color is produced it shows the color reaction with 3, 5-

dinitrobenzoic acids depends upon the presence of an α,β-unsaturated-o lactones in

glycone.

b) Keller-Killiani test [test for Deoxy sugars]: Extracted the drug with chloroform

and evaporated it to dryness. Added 0.4ml of glacial acetic acid containing a trace

amount of ferric chloride. Transferred to a small test tube and added carefully 0.5 ml of

concentrated sulphuric acid by the side of the test tube, blue color appears in the acetic

acetic acid layer if cardiac glycosides are present.

45

IV.Test for cyanogenetic glycosides: placed 200mg of drug in a conical flask and

moistened with few drops of water, (there should be no free liquid at the bottom of the

flask the test will not work if there is any liquid in flask as the hydrogen cyanide

produced will dissolve in the water rather than, come off as a gas to react with the paper).

Moistened a piece of picric acid paper with sodium carbonate solution (5% aqueous) and

suspended by means of cork in the neck of the flask, warmed gently at about 370C.

Observed the change in color. Hydrogen cyanide is liberated from cyanogenetic

glycoside by the enzyme activity and reacts with sodium picrate to from the reddish

purple sodium isopicrate if cyanogenetic glycoside is present.

V.Test for tannins & phenolic compounds:

a. Gelatin test: Extract or fraction with 1% gelatin solution containing 10% sodium

chloride gives white precipitate if tannins are present.

b. Ferric chloride test: Extract or fraction gives blue green color with ferric chloride if

phenols are present.

c. Vanillin Hydrochloride test: Extract or fraction when treated with few drops of vanillin

hydrochloride reagent gives purplish red color if the test is positive.

d. Extract or fraction when treated with heavy metals precipitates tannins if present.

e. Extract or fraction yield bulky precipitate with phenazone especially in the presence of

sodium and phosphate provided tannis are present.

f. Alkaline reagent test: Extract or fraction with sodium hydroxide solution gives yellow

to red precipitate within short time if tannins are present.

g. Mitchell‟s test: with iron and ammonium citrate or iron and sodium tartarate extract or

fraction gives a water-soluble iron-tannin complex, which is insoluble in soluble of

ammonium acetate if tannins are present.

46

VI.Test for flavonoids:

a. Shinoda test (Magnesium Hydrochloride reduction test): To the extract or fraction,

added few fragments of magnesium ribbon and added cone. Hydrochloric acid drop wise,

pink scarlet, crimson red or occasionally green to blue color appears few minutes if

flavonoids are present.

b. Zinc Hydrochloride reduction test: To the extract or fraction, added a mixture of

zinc dust and conc. Hydrochloric acid. It gives red color after few minutes if flavonoids

are present.

c. Alkaline reagent test: To the extract or fraction, added few drops of sodium

hydroxide solution; formation of an intense yellow color, which turns to colorless on

addition of few drops of dil. acid , indicates presence of flavonoids.

VII.Test for proteins & amino acids:

a). Millons test: Extract or fraction when treated with 2 ml of millons reagent (Mercuric

nitrate in nitric acid containing traces of nitrous acid), gives a white precipitate which

turns red upon gentle heating indicates presence of proteins and amino acids.

b). Ninhydrin test: Extract or fraction when boiled with 0.2% solution of ninhydrin

(Indane 1, 2.3 trione hydrate), gives violet color if proteins and amino acids are present.

VIII.Test for sterols & triterpenoids:

a). Liebermann–Burchard test: Extract or fraction when treated with few drops of

acetic anhydride and then boiled and cooled, Conc. Sulfuric acid was then added from the

sides of the test tube, shows brown ring at the junction of two layers and the upper layer

turns green which shows the presence of steroids and formation of deep red color

indicates the presence of triterpenoids.

b). Salkowski test: Extract or fraction when treated with chloroform and few drops of

conc. sulfuric acid, shaken well and allowed to stand for some time, red color appears at

lower layer indicates the presence of steroids and formation of yellow colored lower layer

indicates the presence of triterpenoids.

47

IX.Test for carbohydrates:

a. Molisch’s test: treated the extract or fraction with few drops of alcoholic alpha

naphthol and added 0.2 ml of conc. Sulfuric acid slowly through the sides of the test tube

when a purple to violet color ring at the junction indicates presence of carbohydrates.

b. Benedict’s test: Treated the extract or fraction with few drops of Benedict‟s reagent

(alkaline solution containing cupric citrate complex) and upon boiling on water bath,

reddish brown precipitate forms if reducing sugars are present.

c. Barfoed’s test: it is a general test for monosaccharide. Heated the test tube containing

1 ml of reagent and 1 ml of extract fraction in a beaker of boiling water, if red cuprous

oxide is formed within 2 min, monosaccharide id present. Disaccharide on prolonged

heating (about 10 min) may also cause reduction, owing to partial hydrolysis to

monosaccharide.

d. Camnelisation: Extract or fraction when treated with strong sulfuric acid, undergoes

charring with the dehydration along with burning sugar smell indicating presence of

carbohydrates.

e. Selwinoff’s test: hydrochloric acid reacts with ketose sugar to form derivatives of

furfur aldehyde, which gives red colored compound when linked with resorcinol. Added

extract or fraction to about 5 ml of reagent and boiled. Fructose gives red color within

half minute. The test is sensitive to 5.5 mmol/ ltr if glucose is absent, but if glucose is

presents it is less sensitive and in addition of large amount of glucose can give similar

color.

f. Tollen’s test: to 100 ml of extract or fraction, added 2 ml of Tollen‟s reagent and

heated gently, a silver mirror is obtained onside the wall of the test tube, indicates the

presence of aldose sugar.

g. Bromine water test: It gets decolorized by aldose but not by ketoses because bromine

water oxidizes selectively the aldehyde group to carboxylic group, giving raise to general

class of compounds call aldonic acid.

h. Fehling’s test: Equal volume of Fehling‟s A (Copper sulfate in distilled water) and

Fehling‟s B (Potassium tartarate and sodium hydroxide in distilled water) reagents are

mixed and few drops of extract or fraction were added and boiled, brick red precipitate of

cuprous oxide forms, if reducing sugars are present.

48

C. Test for fats & fixed oils:

a). Stain test: Pressed the small quantity of extract between two filter papers, the stain on

Ist filter paper indicates the presence of fixed oils.

b). Saponification test: Added a few drops of 0.5 N of alcoholic potassium hydroxide to

small quantities of various extracts along with a drop of Phenolphthalein separately and

heated in water bath for 1-2 hours. The formation of soap or partial neutralization of

alkali indicates the presence of fixed oils and fats.

2.4.8. Chromatographic Studies

2.4.8.1. HPTLC Study for Cordia Dichotoma & Carissa Carandas (9, 10, 11, 12)

The HPTLC was carried out using a Hamilton 100 μl HPTLC syringe, Camag Linomat

V automatic spotting device, Camag twin trough chamber, Camag TLC Scanner-3,

WINCAT integration software, aluminum sheet precoated with Silica Gel 60F254

(Merck), 0.2 mm thickness. Development of plates, chromatograms and calibration

curve. Use for both plant extracts and formulation. From the calibration curve

determination of Quercetin in respective extracts and formulation can be done.

Materials and Methods:

a. Preparation of extract

The leaves were dried under the shade and ground into coarse powder. The powder (500

g) was macerated in 205 L of distilled water at room temperature for 24 h. it was then

filtered using a filter paper (Whatman size no. 1 ) and the filtrate evaporated to dryness in

the water bath at 600C. A brownish residue weighing 20.6 g/ 20.16 g (yield of 4.12 &

4.02 % w/w) was obtained. This was kept in air tight bottles in a refrigerator until used.

b. Reagents and other materials

Quercetin [Natural remedies, Bangalore], toluene, acetone, ethyl acetate,

dichloromethane, formic acid, glacial acetic acid and methanol [all Reagents of

analytical grade, Sigma] and silica gel 60F254 precoated TLC aluminum plates [E-

Merck].

49

c. Preparation of standard solution

A stock solution of quercetin was prepared by dissolving 10 mg of accurately

weighed quercetin in distilled water and making up the volume to 10 ml with

distilled water to get the final concentration of 1 mg/ml and passed through 0.45

Millipore filters. Calibration curve from 1-5 µg/spot was prepared.

d. Preparation of Sample solution

Accurately weighted 10 mg of aqueous extract of cordia dichotoma / Carissa

Carandas, dissolved in 10 ml distilled water (1000 µg/ml)and passed through 0.45

Millipore filters.

e. Application of sample

In HPTLC analysis sample application is the most critical step for obtaining good

resolution. For this Camag microliter syringe (2µl) used for the extracts and marker

solution.

f. Chromatographic conditions Development

The plate was developed in CAMAG glass twin-through chamber (10-10 cm)

previously saturated with the solvent Toluene: Ethyl acetate: Acetone: Formic acid

(10:5:15:1) for 60 min (Temperature 25.2°C, Relative Humidity 40%).

g. Detection

The plate was scanned at UV 366 nm and 254 nm using CAMAG TLC Scanner-3 and

LINOMAT-V. Rf value of each compound which were separated on plate and data of

peak area of each band was recorded.

h. Validation of the method

ICH guidelines were (CPMP/ICH/381/95; CPMP/ICH/281/95) followed for the

validation of the analytical procedure. The method was validated for precision,

repeatability and accuracy. The repeatability of the method was checked by repeated

scanning of the same spot of quercetin, seven times and was expressed as coefficient of

50

variance (% CV). Accuracy of the method was tested by performing recovery

studies at three levels (50%, 100% and 150% addition). The percent recovery as

well as average percent recovery was calculated. For the determination of limit of

detection and limit of quantification, different dilutions of the standard solutions of

quercetin were applied along with methanol as well as the blank and determined on

the basis of signal to noise ratio.

51

2.4. Results:

2.4. Pharmacognostical Studies:

2.4.1. Morphological Evaluation:

The leaves showed the following characters on morphological examination

Figure 2.1: Cardio Dichotoma morphology of leave.

52

Figure 2.2: Carissa Carandas morphology of leave.

Table 2.3: Morphological Characters of leaves Carissa carandas

Characters Description

Cardio Dichotoma Carissa carandas

Color Green Green

Odour characteristics characteristics

Size 12.5 cm long, 10.01 cm wide 5.1 cm long, 1.9 cm wide

Shape Fan shaped Oval shaped

Venation Unicostate Reticulate

53

Apex Round Round

Margin Entire Entire

2.4.3.1. Powder Microscopy:

The leaf powder was observed microscopically after staining and then glycerin

mounting:

Table 2.4: powder microscopy of cardio Dichotoma and Carissa carandas.

Sr. No. POWDER MICROSCOPY

Cordio chichotoma Carissa carandas

1 Calcium Oxalate crystals Calcium Oxalate crystals

2 Fibers Fiber like fragments

3 Epidermis of the peptide in surface

view showing cuticular striations

Parenchymatous tissue (ground

tissue)

(a) (b)

Figure 2.3 : Calcium Oxalate crystals (a) Cordio chichotoma & (b) Carissa

carandas.

54

Figure 2.4: (a) Fibers of Cordio chichotoma & (b) Fiber like fragments

Carissa carandas.

Figure 2.5: (a) Epidermis of the peptide in surface view showing cuticular

striations of Cordio chichotoma & (b) Parenchymatous tissue (ground tissue)

of Carissa carandas.

2.4.4. PHYSIOCHEMICAL STUDIES:

The leaf powder was evaluated for determination of physiochemical parameters,

namely ash value, moisture content etc.

55

Table2. 5: Physiochemical results of Carissa Carandas & Cordia Dichotoma

Quantitative standards Results in %w/w

Carissa carandas Cordia Dichotoma

Total Ash Value 8.6 9.0

Acid insoluble ash 3.1 4.21

Water soluble ash 0.95 1.02

Moisture content 9.91 11.81

Alcohol soluble extractive 2.69 4.042

Water soluble extractives 4.02 4.12

Ether soluble extractives 1.422 1.160

2.4.4.4. DETERMINATION OF SYSTEMIC SOLVENT EXTRACTIVE

VALUES:

The air dried leaves are extracted by successive extraction (exhaustive) with

various solvents of different polarity namely Pet. Ether (600-800C), chloroform

and acetone, and methanol in a soxhlet extractor. Each extract was concentrated to

a small volume in buchirota evaporator and allowed to dry. After drying, the

respective extracts were weighed and percentage extractive values were

determined.

Table 2.6: Showing colour and yield of various extracts.

Sr.

No

Extracts Colour % Yield

Carissa

carandas

Cordia

Dichotoma

Carissa

carandas

Cordia

Dichotoma

1 Petroleum

ether

Dark green Green 4.85 3.90

56

2 Chloroform

extract

Dark green Green 8.40 6.70

3 Methanol

extract

Dark green Green 5.66 5.74

4 Acetone

extract

Light Brown Brown 7.11 8.65

5 Water extract Brown Brown 22.67 25.82

2.4.4.5. PHYTOCHEMICAL SCREENING: The chemical tests on various

extracts and fractions gave following results.

Table 2.7: Chemical examination of various extract of leaves of Carissa

carandas

Sr.

No.

Phytoconstituent Diethyl

ether

extract

Chloroform

extract

Water

extract

Acetone

extract

Methanol

extract

1 Carbohydrates -ve -ve +ve +ve +ve

2 Proteins and

amino acids

-ve -ve -ve -ve -ve

3 Flavonoids -ve -ve +ve -ve +ve

4 Tannin &

Phenolic

Compounds

-ve -ve +ve +ve +ve

5 Alkaloids +ve +ve +ve +ve +ve

6 Glycoside -ve -ve -ve +ve +ve

7 Sterols &

Triperpenoid

-ve +ve -ve +ve +ve

57

Table 2.8: Chemical examination of various extract of leaves of Cordia

Dichotoma.

Sr.

No.

Phytoconstituent Diethyl

ether

extract

Chloroform

extract

Water

extract

Acetone

extract

Methanol

extract

1 Carbohydrates -ve -ve +ve -ve -ve

2 Proteins and

amino acids

-ve +ve -ve -ve -ve

3 Flavonoids -ve -ve -ve +ve +ve

4 Tannin &

Phenolic

Compounds

-ve -ve +ve -ve +ve

5 Alkaloids +ve +ve +ve +ve +ve

6 Glycoside -ve -ve +ve -ve -ve

7 Sterols &

Triperpenoid

-ve -ve -ve -ve -ve

2.4.8. Chromatographic Studies

HPTLC Study for Cordia Dichotoma & Carissa carandas:

58

Figure 2.6: HPTLC chromatogram for Cordia Dichotoma extract.

Figure 2.7: HPTLC chromatogram of Quercetin (cordia dichotoma).

59

Figure 2.8: HPTLC chromatogram for Carissa Carandas extract.

Table 2.9: method validation parameters for estimation of marker compound

by HPTLC.

Sr No. Parameters Quercetin

1 Linearity (correlation-coefficient) 0.994

2 Repeatability (% CV) 0.5972

3 Limit of quantification (µg/spot) 68.72

4 Limit of detection (µg/spot) 19.66

5 Range (µg/spot) 200-1500

6 Specificity specific

60

Table 2.10: Recovery study of marker compound by proposed HPTLC method.

Marker Amount

in sample

(µg)

Amount

added

(µg)

Peak area Amount

found

Recovery Average

Recovery

Quercetin

100 50 1671.78±11.22 147.64 98.42%

98.70% 100 100 2463.01±17.33 197.88 98.94%

100 150 3198.24±24.16 246.90 98.76%

Table 2.11: Value are expressed as mean ± SD (n=3)

Sr. No. Plant Extarct Area Quercetin content (µg/ml)

1 Aqueous extract of Cordia dichotoma 1962.05 2.67

2 Aqueous extract of Carissa carandas 1535.92 1.87

61

Discussion:

The Cordia Dichotoma and Carissa Carandas are belongs from the respective

Boraginaceae & Apocynaceae families. The both plants are study for pharmacognostical

characterization and standardization of plants. In pharmacognostical parameter study the

microscopical evaluation, macroscopical evaluation, physicochemical studies,

phytochemical screening and chromatographic studies.

In microscopy studies, it was found that leaves of both plants in green colour,

characteristic odour, size of leaves was 12.5 cm long, 10.01 cm wide & 5.1 cm long, 1.9

cm wide, fan shaped & oval shaped, unicostate & reticulate venation, both were round in

apex along with entire margin.

In macroscopy studies, both plants were contains Calcium Oxalate crystals, Fiber.

Epidermis of the peptide in surface view showing cuticular striations in Cordia

Dichotoma and Parenchymatous tissue (ground tissue) found in carissa carandas.

In physiochemical studies, Cordia Dichotoma & Carissa Carandas were evaluated

for total ash value, acid insoluble ash, water soluble ash, moisture content, alcohol

soluble extractive, water soluble extractive and ether soluble extractive values; 8.6 w/w,

3.1 w/w, 0.95 w/w, 9.91 w/w, 2.69 w/w, 7.66 w/w, 1.422 w/w & 9.0 w/w, 4.21 w/w, 1.02

w/w, 11.81 w/w, 4.042 w/w, 9.012 w/w, 1.160 w/w. Extractive values for Cordia

Dichotoma & Carissa Carandas in different solvents such as Petroleum ether, Chloroform

extract, Methanol extract, Acetone extract and Water extract were 3.90 %, 6.70%, 5.74%,

8.65%, 25.82% & 4.85%, 8.40%, 5.66%, 7.11%, 22.67%.

In phytochemical screening studies, various extract of Cordia Dichotoma &

Carissa Carandas were studies for carbohydrates, proteins & amino acids, flavonoids,

tannin & phenolic compounds, alkaloids, glycoside and sterols etc. It was found that

Carissa Carandas was contains carbohydrate ( water, acetone & methanol extracts),

flavonoids (water, methanol extracts), tannin & phenolic compounds (water, acetone &

methanol extracts), alkaloids (all extracts), glycosides (acetone & methanol extracts) and

sterols & triterpenoids (water & methanol extracts) and Cordia Dichotoma was contains

carbohydrate ( water & methanol extracts), protein & amino acids (chloroform extract),

62

flavonoids (water, methanol extracts), tannin & phenolic compounds (water & methanol

extracts), alkaloids (all extracts), glycosides (water extract). Protein & amino acids &

sterols & triterpenoids were absent in Carissa Carandas and Cordia Dichotoma in all

extracts.

Chromatographic studies used the HPTLC analysis technique in which different

concentrations of standard solution of quercetin were applied in triplicate on HPTLC

plates and aqueous extract of Cordia Dichotoma applied on HPTLC plate for estimation

of quercetin. The HPTLC plates were developed in a solvent system Toluene: Ethyl

acetate: Acetone: Formic acid (10:5:15:1) for 60 min (temperature 25.2°C, relative

humidity 40%).and scanned densitometrically at 366 nm for quercetin.

The peak areas were recorded and validate the method. The relationship between

the concentration and peak response was linear with range of 1 µg/ml-5 µg/ml for

quercetin with correlation coefficient of 0.9929. The percentage recovery was found to be

98.70 % for marker compound. The aqueous extracts of Cordia Dichotoma and Carissa

Carandas contain 2.67 µg/ml and 1.87 µg/ml.

63

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