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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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