Studies on Phytochemical Constituents of Medicinal Plants

Sciknow Publications Ltd. AJPPS 2014, 1(4):61-74 American Journal of Pharmacy and Pharmaceutical Sciences DOI: 10.12966/ajpps.12.01.2014 ©Attribution 3.0 Unported (CC BY 3.0)

Studies on Phytochemical Constituents of Medicinal Plants

Gaurav Kumar Sharma1,*

, Abhishek Yogi2, Bhavesh Joshi

1, Kalpesh Gaur

1 and Ashok Dashora

1

1Geetanjali Institute of Pharmacy, Geetanjali University, Hiranmagri Extension, Manwakhera, NH-8 Bypass, Near Eklingpura Chouraha, Udaipur-313002, Rajasthan (India)

2Theon Pharmaceuticals Ltd., Saini Mazra, Nalagarh- 174 101, Dist. Solan, Himachal Pradesh (India)

*Corresponding author (Email: [email protected])

Abstract - Tannins, phlobatannins, saponins, flavonoids, terpenoids, cardiac glycosides and alkaloids distribution in poly herbal

preparation, where each medicinal plant belongs to different families were examined and compared. The plants used are Justica

adhatoda Linn, Momordica charantia, Nordostachys jatamansi and Tephrosia purpurea. Qualitative analysis carried out on each

plant shows that terpenoids, steroids, cardiac glycosides, saponins, flavonoids, tannins and phenolic compound, alkaloids were

present in all the plants. Coumerin glycosides were found to be present in Nordostachys jatamansi and Justica adhatoda only and

were absent in the rest of the plants. Anthraquinone glycosides were present in Justica adhatoda Linn, Momordica charantia and

Nordostachys jatamansi and found to be absent in Tephrosia purpurea. The significance of the phytochemical constituents with

the respect to the role of these plants in traditional medicine treatment is discussed.

Keywords - Medicinal Plants, Traditional Medicine, Phytochemical Constituents, Soxhlet Apparatus

1. Introduction

The medicinal plants contains bioactive phytochemical constituents that produce definite physiological action on the human

body [1].The most important bioactive phytochemical constituents are alkaloids, essential oils, flavonoids, tannins, terpenoid,

saponins, phenolic compounds and many more [2].

Phytochemical is a natural bioactive compound found in plants, such as vegetables, fruits, medicinal plants, flowers, leaves

and roots that work with nutrients and fibers to act as an defense system against disease or more accurately, to protect against

disease. Phytochemicals are divided into two groups, which are primary and secondary constituents; according to their functions

in plant metabolism. Primary constituents comprise common sugars, amino acids, proteins and chlorophyll while secondary

constituents consists of alkaloids, terpenoids and phenolic compounds [3] and many more such as flavonoids, tannins and so on.

Justica adhatoda Linn, Momordica charantia, Nordostachys jatamansi and Tephrosia purpurea are chosen to study because

they come in abundant source, easily available, and some of them are already being utilized in traditional medicine. By studying

the presence of phytochemical in these plants, the uses of these plants in traditional treatment can be explained scientifically.

Fruit of Momordica charantia contains terpenoids, steroids, cardiac glycosides, anthraquinine glycosides, saponins,

flavonoids, tannins and phenolic compound, alkaloids which possess anti-fertility, uterine stimulant, hypoglycemic, inhibit

prostate tumor growth and analgesic activity.

Rhizomes of Nardostachys jatamansi contains terpenoids, steroids ,cardiac glycosides, anthraquinine glycosides, saponins,

coumarin glycosides, flavonoids, tannins and phenolic compound, alkaloids Constituents which possessantiarrythmic,

hypotensive,stimulant,antispasmodic,diuretic,deobstruent,emmenagogue, stomachic, laxative, tranquilizer, anticonvulsant,

hypolipidamic activity.

Justica adhatoda contains terpenoids, steroids, cardiac glycosides, anthraquinone glycosides, saponins, coumaringlycosides,

flavonoids, tannins, phenolic compound and alkaloids which possess antibacterial, anticholinesterase, wound healing,

hypoglycemic, abortifacient, antitussive, digestive, cardio protective, hepatoprotective, anti-inflammatory, antimutagenic and

antituberculer activity.

Tephrosia purpurea contains terpenoids, steroids, cardiac glycosides, saponins, flavonoids, tannins and phenolic compound

and alkaloids which possess anti-inflammatory, anti ulcer, hepatoprotective, antidiarrhoeal, anthelmintic, alexeteric, antipyretic,

antibacterial, antimicrobial, antihyperglycemic, immunomodulatory and antiallergic activity.

62 American Journal of Pharmacy and Pharmaceutical Sciences (2014) 61-74

2. Materials and Methods

2.1. Collection of plant samples

Fruit of Momordica charantia, rhizomes of Nardostachys jatamansi, Justica adhatoda and Tephrosia purpurea were procured

from Local market Jaipur.

2.1.1. Processing of plant samples

All the materials were shade dried at room temperature and powdered mechanically and passed through a sieve # 40. The

powdered form of these plants is stored in airtight glass containers, protected from sunlight until required for analysis.

2.1.2. Preparation of aqueous extract of plant samples Extraction [4]

The air-dried parts of the plants were powdered and extracted with 95% ethanol, chloroform, pet ether (40º-60

º) and aqueous

solvent systems by hot percolation method by using Soxhlet apparatus assembly at a controlled temperature. After complete

extraction, marc was pressed to collect the micelle, mixed with the contents of RBF, filtered and concentrated to get the extract.

The color and consistency of the extract was noted. This extract was further subjected to phytochemical investigation.

2.2. Phytochemical analysis

Chemical tests are conducted on the extract of each plant sample and also of the powdered form of the plant samples using

standard methods.

2.2.1. Qualitative analysis on phytochemical constituents- Qualitative Chemical Tests [5]

2.2.1.1. Tests for Carbohydrates:

Preparation of test solution: The test solution was prepared by dissolving the test extract with water. Then it was hydrolyzed with

1 volume of 2N HCl and subjected to following chemical tests.

a) Molish's test (General test): To 2-3 ml aqueous extract, added few drops of -naphthol solution in alcohol, shaken and added

concentrated H2SO4 from sides of the test tube was observed for violet ring at the junction of two liquids.

b) Fehling's test: 1 ml Fehling's A and 1ml Fehling's B solutions was mixed and boiled for one minute. Equal volume of test

solution was added. Heated in boiling water bath for 5-10 min was observed for yellow, then brick red precipitate.

c) Benedict's test: Equal volume of Benedict's reagent and test solution in test tube were mixed. Heated in boiling water

bath for 5 min. Solution may appear green, yellow or red depending on amount of reducing sugar present in test solution.

d) Barfoed's test: Equal volume of Barfoed's reagent and test solution were added. Heated for 1-2 min, in boiling water bath

and cooled. Observed for red precipitate.

e) Cobalt-chloride test: 3 ml of test solution was mixed with 2 ml cobalt chloride, boiled and cooled. Added FeCl3 drops on

NaOH solution. Solution observed for greenish blue (glucose), purplish (Fructose) or upper layer greenish blue and

lower layer purplish (Mixture of glucose and fructose).

f) Tests for Non-Reducing Sugars: Test solution does not give response to Fehling's and Benedict's test.

g) Tannic acid test for starch: With 20% tannic acid, test solution was observed for precipitate.

2.2.1.2. Tests for Proteins:

Preparation of Test Solution: The test solution was prepared by dissolving the extract in water.

a) Biuret test (General test): To 3 ml T.S added 4% NaOH and few drops of 1% CUSO4 solution observed for violet or pink

colour.

b) Million's test (for proteins): Mixed 3 ml T.S. with 5 ml Million's reagent, white precipitate obtained. Precipitate warmed

turns brick red or precipitate dissolves giving red colour was observed.

c) Xanthoprotein test (For protein containing tyrosine or tryptophan): Mixed 3ml T.S. with 1 ml concentrated H2SO4 observed

for white precipitate.

d) Precipitation test: The test solution gave white colloidal precipitate with following reagents: Absolute alcohol, 5% HgCl2

solution, 5% CUSO4 solution, 5% lead acetate, 5% ammonium sulphate.

2.2.1.3. Tests for Steroids:

Preparation of test extracts solution: The extracts were refluxed separately with alcoholic solution of potassium hydroxide till

complete saponification. The saponified extract was diluted with water and unsaponifiable matter was extracted with diethyl

ether. The ethereal extract was evaporated and the residue (unsaponifiable matter) was subjected to the following test by dissolving

the residue in the Chloroform.

American Journal of Pharmacy and Pharmaceutical Sciences (2014) 61-74 63

a) Salkowski reaction: To 2 ml of extract, 2 ml chloroform and 2 ml concentrated H2SO4 was added. Shook well, whether

chloroform layer appeared red and acid layer showed greenish yellow fluorescence was observed.

b) Libermann-Burchard test: Mixed 2ml extract with chloroform. Added 1-2 ml acetic anhydride and 2 drops concentration

H2SO4 from the side of test tube observed for first red, then blue and finally green colour.

c) Libermann's test: Mixed 3 ml extract with 3 ml acetic anhydride. Heated and cooled. Added few drops concentrated H2SO4

observed for blue colour.

2.2.1.4. Tests for Amino Acids:

a) Ninhydrin test (General test): 3 ml T.S. and 3 drops 5% Ninhydrin solution were heated in boiling water bath for 10 min.

Observed for purple or bluish colour.

b) Test for Tyrosine: Heated 3 ml T.S. and 3 drops Million's reagent. Solution observed for dark red colour.

c) Test for tryptophan: To 3 ml T.S. added few drops glycoxalic acid and concentrated H2SO4 observed for reddish violet ring

at junction of the two layers.

2.2.1.5. Tests for Glycosides:

Preparation of test solution: The test solution was prepared by dissolving extract in the alcohol or hydro-alcoholic solution. Tests for Cardiac Glycosides:

a) Baljet's test: A test solution observed for yellow to orange colour with sodium picrate.

b) Bromine water test: Test solution dissolved in bromine water giving yellow precipitate

c) Legal's test (For cardenoloids): To aqueous or alcoholic test solution, added 1ml pyridine and 1 ml sodium nitroprusside

observed for pink to red colour.

d) Test for deoxysugars (Kellar Killani test): To 2 ml extract added glacial acetic acid, one drop of 5% FeCl3 and

concentrated H2SO4 observed for reddish brown colour at junction of the two liquid and upper layers bluish green.

e) Libermann's test (For bufadenolids): Mixed 3 ml extract with 3 ml acetic anhydride. Heated and cooled. Added few drops

concentrated H2SO4 observed for blue colour.

Test for anthraquinone glycosides:

a) Modified Borntrager's test: C-glycosides of anthraquinones require more drastic conditions for hydrolysis.Hydrolysis of

the drug was carried out with 5 ml of dilute HCl and 5 ml of 5% solution of FeCl3.

b) Borntrager's test: Boiled powdered drug with 5 ml of 10% sulphuric acid for 5 mins. Filtered while hot, cooled the filtrate

shaken gently with equal volume of benzene. Benzene layer was separated and then treated with half of its volume

solution of ammonia (10%). Allowed to separate it. The ammonical layer acquired rose pink colour due to the presence

of anthraquinones.

Cyanogenetic glycosides:

Grignard's test: Strips of sodium picrate filter paper were inserted between split cork stopper which was fitted in to the neck of

the test tube containing a small amount of powdered drug in water. Care was exercised that the paper didn't touch the inner side

of the test tube. The content was warmed for half an hour. The red colour of the strips indicated the presence of cyanogenetic

glycosides.

Tests for Saponin Glycosides:

a) Foam test: The drug extract or dry powder was shaken vigorously with water. Persistent foam was observed.

b) Foaming index: Weigh 1 gm of finely powdered drug accurately and transfer to a 500 ml conical flask containing 100ml

of boiling water. Maintain at moderate boiling for 30 min. Cool and filter into a 100 ml volumetric flask and add

sufficient water to make the volume to 100 ml.

Place the above decoction into 10 stoppered, graduated test-tubes in a series of successive portions of 1, 2, 3 upto 10 ml and

adjust the volume of the liquid in each test tube water to 10 ml. Stopper the tubes and shake them vertically for 15 seconds, 2

frequencies/ sec. Allow to stand for 15 min and measure the height of the foam.

The results assed as follows:

a) If the height of the foam in every tube is less than 1 cm, the foaming index is less than 100.

b) If a height of foam of 1 cm is measured in any tube, the volume of the plant material decoction in this tube (a) is used to

determine the index. If this tube is the first or second tube in the series, prepare an intermediate dilution in a similar

manner to obtain a more precise result.

c) If the height of the foam is more than 1 cm in every tube, the foaming index is over 1000. In this case repeat the

determination using a new series of dilution of the decoction in order to obtain a result.

Foaming Index = 100/a (1)

a = volume in ml of the decoction used for preparing dilution in the tube where foaming to a height of 1 cm is observed.

c) Haemolytic test: Added test solution to one drop of blood placed on glass slide. Haemolytic zone whether appeared was


observed.

Tests for Coumarin Glycosides: Test solution when made alkaline, observed for blue or green fluorescence.

2.2.1.6. Tests for Alkaloids:-

a) Dragendorff's test: To 2-3 ml filtrate added few drops Dragendorff's reagent observed for orange brown precipitate.

b) Mayer's test: 2-3 ml filtrate with few drops Mayer's reagent observed for precipitate.

c) Hager's test: 2-3 ml filtrate with Hagers reagent observed for yellow precipitate.

d) Wagner's test: 2-3 ml filtrate with few drops of Wagner's reagent observed reddish brown precipitate.

2.2.1.7. Tests for Flavonoids:-

The flavonoids are all structurally derived from the parent substance called flavone. The flavonoids occur in the free form as

well as bound to sugars as glycosides. For this reason, when analyzing flavonoids it is usually better to examine the flavonoids in

hydrolyzed plant extracts.

Preparation of test solution:

a) To a small amount of extract added equal volume of 2M HCl and heated in a test tube for 30 to 40 min. at 100ºC.

b) The cooled extract was filtered, and extracted with ethyl acetate.

c) The ethyl acetate extract was concentrated to dryness, and used to test for flavonoids.

a) Shinoda test: To dried powder or extract, added 5 ml 95% ethanol, few drops concentrated HCl and 0.5 g magnesium

turnings. Pink colour was observed.

To small quantity of residue, added lead acetate solution observed for Yellow coloured precipitate. Addition of

increasing amount of sodium hydroxide to the residue whether showed yellow colouration, which was decolorized after

addition of acid was observed.

b) Ferric chloride test: Test solution, added few drops of ferric chloride solution observed for intense green colour.

2.2.1.8. Test for Vitamins:

a) Test for Vitamin A: Dissolve a quantity equivalent to 10-15 units in 1ml chloroform and add 5ml of antimony trichloride

solution, a transient blue colour is produced immediately.

b) Test for vitamin C (Ascorbic acid):- Dilute 1 ml of 2% w/v solution with 5 ml of water and added 1 drop of freshly

prepared 5% w/v solution of sodium nitroprusside and 2 ml dilute NaOH solution. Added 0.6 ml of hydrochloric acid

dropwise and stir, the yellow color turns blue.

c) Test for Vitamin D: Dissolved a quantity equivalent to about 100 units of Vitamin D, activating in chloroform and

added 10 ml of antimony tricohloride solution, a pinkish-red colour appeared at once.

2.2.1.9. Saponins

Preparation of test solution: The test solution was prepared by dissolving extract in the water.

a) Foam test: Test solution when shaken showed the formation of foam, which was stable for at least 15 min.

b) Haemolysis test: 2 ml of 18% sodium chloride in 2 test tubes was taken, to one test tube added distilled water and to other

2 ml test solution. Few drops of blood were added to both the test tubes. Mixed and observed for haemolysis under

microscope.

c) Test for steroidal saponins: The extract was hydrolyzed with dilute sulphuric acid and extracted with chloroform. The

chloroform layer was tested for sterols.

d) Test for triterpenoid and saponins: The extract was hydrolyzed with dilute sulphuric acid and extracted with chloroform.

The chloroform layer was tested for triterpenoids.

2.2.1.10. Tannins and phenol compounds

To 2-3 ml of alcoholic or aqueous extract, added few drops of following reagents:

a) 5% FeCl3 solution: Deep blue-black colour.

b) Lead acetate solution: White precipitate.

c) Bromine water: Discoloration of bromine water.

d) Acetic acid solution: Red colour solution.

e) Dilute iodine solution: Transient red colour.

One drop NH4OH, excess 10% AgNO3 solution. Heated for 20 min in boiling water bath. White precipitate was observed,

then dark silver mirror deposited on wall of test tube.

2.3. Abbreviations and Tables


Table 1. Summary of plants and solvent used for extraction

S. No. Drug Weight of drug Taken Solvent Volume of Solvent Taken

1. M. charantia 900 grams Petroleum ether 2.5 lit.

2. 900 grams Chloroform 2.5 lit.

3. 900 grams Ethanol 2.5 lit.

4. 900 grams Aqueous 2.5 lit.

5. N. jatamansi 500 grams Petroleum ether 2.5 lit.




9. J. adhatoda 400 grams Petroleum ether 2.0 lit.




13. T. purpurea 500 grams Petroleum ether 2.5 lit.




Table 2. The percentage (%) yield after extraction

S.No. Drug Solvent % Yields

1. M. charantia Petroleum ether 6.78

2. Chloroform 04.44

3. Ethanol 06.30

4. Aqueous 25.00

5. N. jatamansi Petroleum ether 06.11

6. Chloroform 04.33

7. Ethanol 03.80

8. Aqueous 11.60

9. J. adhatoda Petroleum ether 07.50

10. Chloroform 11.75

11. Ethanol 06.75

12. Aqueous 13.75

13. T. purpurea Petroleum ether 07.50

14. Chloroform 04.50

15. Ethanol 06.00

16. Aqueous 15.00

Table 3. Qualitative Analysis on Phytochemical Constituents

Test

Pet. Ether Extract Chloroform Extract

M.

charantia N. jatamansi J. Adhatoda T. purpurea M.charantia

N.

jatamansi J. Adhatoda T. purpurea

I Test for Carbohydrate

A Molish Test - - - + - - - +

B

Test for

reducing sugars

Fehling

Test - - - + - - - +

Benedict test

- - - + - - + +

C Test for

Monosaccharide

Barfoeds Test

- + - - - + - -

D

Test For

Hexose

Sugars

Cobalts

Chloride test - - - - - - - +

E Test for Non- Reducing Sugars

- - - - - - - +


F

Test for Non-

Reducing

polysaccharide

Iodine test - + - + - + - +

Tannic acid test - + - + - + - +

II Test for Proteins

Biuret test - - + + - - - -

Millon’s test - - + - - - - -

Xanthoprotein + + + - + + + +

Test for protins

containing Sulphur - - - - - - - -

Precipitation test

+ + + + + + + +

III Test for

Amino Acid

Ninhydrin test + + + + + + + +

Test for tyrosin

- - + - + - -

Test for tryptophan - - + - + - - -

Test for

cysteine - + + - + - + -

IV Test for Steroids

Liebermann-

Buchard + - + - - - + +

Liebermann reaction + - + - + + + +

V Test for Terpenoids

Liebermann-

Buchard + - + - + + + +

Liebermann reaction + - + - + + + +

VI Test for Glcosides

A Test for

Cardiac Glycoside

Baljet test - + + - + + + +

Legal’s test - - - - - - - -

Test for deoxy sugar

(Keller killani test) - + - - - + - +

Liebermann’s test (Bufadienolides)

- + + - - - + +

B

Test for

Anthraquinone

glycoside

- - + - + - - -

C Test for

Saponin Glycoside - + - - + + - +

D Test for Coumarin

Glycoside - - - - - - + -

VII Test For Flavanoids

Ferric chloride test - - - - - + + +

Shinoda test + + + - + + + +

Alkaline

reagents + + - - + - - +

Lead acetate test + + + - + + + +

VIII Test for alkaloids + - + + + + + -

IX

Test for

Tannins & Phenolic cpd.

- + + - + + + +

X Test For Lipids + - - - - - - -

Test

Alcohol Extract Aqueous Extract

M. charantia

N. jatamansi J. Adhatoda T. purpurea M. charantia

N. jatamansi

J. Adhatoda T. purpurea

I Test for

Carbohydrate

A Molish Test - + - + + + + +

B

Test for reducing

sugars

Fehling Test

- - + + + + - +

Benedict

test - - + + + + + +

C Test for


Monosaccharide

Barfoeds

Test - - + + + - - +

D

Test For

Hexose

Sugars

Cobalts Chloride test

- - + + + + + +

E Test for Non-

Reducing Sugars - - + + + + + +

F

Test for Non- Reducing

polysaccharide

Iodine test - + + + + + - +

Tannic acid test - + + + + + - +

II Test for Proteins

Biuret test - + + + + + + +

Millon’s test - + + + + - - -

Xanthoprotein - + + + + + + -

Test for protins containing Sulphur

- - - - - - - -

Precipitation

test + + + + + + + +

III Test for Amino Acid

Ninhydrin test + + + + + + + +

Test for

tyrosin - + + + + - + -

Test for tryptophan + + + - + - + -

Test for

cysteine + + + - + + + -

IV Test for Steroids

Liebermann- Buchard

+ - - - - - + +

Liebermann reaction + - - - - - + +

V Test for Terpenoids

Liebermann- Buchard

+ - - - - - + +

Liebermann reaction - - - - - - + +

VI Test for

Glcosides

A Test for Cardiac Glycoside

Baljet test - + + - - + + +

Legal’s test - + + - - - + -

Test for deoxy sugar (Keller killani test)

- + + - - + - +

Liebermann’s

test (Bufadienolides) - + + - - + + +

B

Test for Anthraquinone

glycoside

- + + - - - + -

C Test for Saponin Glycoside

- + + - - + - -

D Test for Coumarin

Glycoside - + + - - - - -

VII Test For Flavanoids

Ferric chloride test - + + - - - - -

Shinoda test - + + - + + + -

Alkaline

reagents - + + - + + - -

Lead acetate test - + + - + + + -

VIII Test for alkaloids - + + + + + + -

IX

Test for

Tannins & Phenolic

cpd.

- + + + - + + +

X Test For Lipids + + + + - - - -

Presence of phytochemical constituents: +; Absence of phytochemical constituents: -.


3. Results and Discussion

3.1. Qualitative analysis

All the four plants and their parts selected for the present studies were powdered after shade drying. The powdered plant material

was placed into a five liter conical flask with different solvents viz. petroleum ether, chloroform, ethanol and distilled water for

extraction. The quantities of solvent and powdered drug used are shown in Table 1. The dried extracts were weighed, percent

yield is calculated the results of which is given in Table 2. Adopting standard procedures carried out preliminary phytochemical

investigation to assess presence or absence of various phytoconstituents like carbohydrates, proteins, amino acids, sterols,

triterpenes, cardiac glycosides, flavonoids and lipids. The results of phytochemical studies are shown in Table 3.

3.1.1. Justica adhatoda Linn:

Effect of terpenoids, steroids, cardiac glycosides, anthraquinine glycosides, saponins, coumarin glycosides, flavonoids, tannins

and phenolic compound, alkaloids

N

N

O

HO

N

N

OH

HO

N

N

HO Vasicinone (a) 6-Hydroxy peganine (b) Vasicine (c)

N

N

OH

O

OMe

N

N

OHOMe

HN

N

OH

HO

O

OH

Adhavasinone (d) Adhavasine (e) Vasicol (f)

N

N

O

N CH3

CH3

N

N

N CH3

CH3

N

N

NHCOOCH3

NHCH3

Vasicolinone (g) Vosicoline (h) Adhatodine (i)

N

N

NHCOOCH3

NHCH3

O

N

N

OH

HO

O Anisotine (j) Vasicinolone (k)

Fig. 1. Quinazoline ring containing Constituents of Adhatoda zeylanica

Pharmacological activities: Water and alcoholic extracts of Vasaka exerts significant pharmacological actions due to the

presence of active constituents like vasicine and vasicinone. The pharmacological activities of vasicine and vasicinone are much

more as compared to their racemic forms. Various activities reported are:

Antibacterial: Alcoholic extract of leaves and roots showed antibacterial activity against Staphylococcus aureus and

Escherichia coli, whereas water extract showed activity against S. aureus only.

Anticholinesterase: Vasicinone obtained from the roots produced transient hypotension in cats, contraction of isolated


intestine and depression of isolated heart in guinea pigs, thus showing good anticholinesterase activity.

Wound healing: The rate of healing was found to be higher in the plant extract treated wounds in buffaloes as compared to

pancreatic tissue extracts.

Hypoglycaemic: Ethanolic extract of the leaves exhibited hypoglycaemic activity in the rats. Modak and Rao found that when

non-nitrogenous principle of the leaves in suspension form administrated orally at the dose of 25 mg/kg, lowered the blood sugar

level of rabbits for a short period of time.

Abortifacient/Oxytocic: Vasicine possesses uterine stimulating and oxytocic activity and causes abortifacient effect by the

release of prostaglandins under the influence of oestrogens. The activity was found almost similar to oxytocin. In a study

conducted on rats, rabbits, hamsters and guinea pigs it was found that vasicine has uterotonic and abortifacient effects possibly

by enhancing the synthesis and release of prostaglandins. In this study dose dependent effect was observed with effective doses

ranging between 2.5-10 mg/kg. However, administration of estradiol dipropionate potentiated the abortifacient effect in guinea

pigs whereas treatment with aspirin inhibited the abortifacient activity due to inhibition of release of prostaglandins.

Antitussive: Vasicine and vasicinone showed bronchodilatory activity in vitro and bronchoconstrictory activity in vivo,

however, combination of both the alkaloids (1:1) showed bronchodilatory activity in vivo as well as in vitro. Evaluation of

antitussive effect of vasaka on anesthetized guinea pigs and rabbits was confirmed electrically and mechanically and found to be

1/20th

and 1/40th

as active as codeine intravenously and almost similar in activity as by oral administration. Kanjang, a fixed

combination of A. zeylanica, Echinacea purpurea (Linn.) Moench and Eleuthrococcus senticosus Maxim showed a significant

efficacy in acute upper respiratory infections.

Digestive: Decoction of the leaves activated the trypsin enzyme in in vitro studies and thus stimulated the digestion process.

Cardioprotective :In combination of vasicine and vasicinone, a significant reduction in cardiac depressant effect was

observed. No effect was shown by vasicinone (dl-form), however l-form was found to be weakly effective in stimulating cardiac

muscles. Anti-inflammatory :Anti-inflammatory activity of methanolic extracts (a non alkaloid fraction, saponins and the alkaloids)

were evaluated by using modified hens egg chorioallantoic membrane showing potent activity at a dose of 50 μg/pellet

equivalent to that of hydrocortisone whereas methanolic extract showed lesser activity [6].

Hepatoprotective:The leaves showed significant hepatoprotective effect at a dose of 50-100 mg/kg on the liver damage

induced by d-galactosamine in rats.

Antiulcer:Leaf powder of this plant showed considerable antiulcer activity in experimental rats in ethanol Induced ulceration

model.

Antimutagenic: Antimutagenic and antioxidant status have also been attributed for A. zeylanica. It exerts antioxidant effect

against lipid peroxide and xanthine oxidase induced oxidation.

Radiomodulation: Leaf extract showed a radiomodulatory influence against radiation induced hematological alterations in

the peripheral blood of swiss albino mice. A significant increase in serum alkaline phosphatase activity and decrease in acid

phosphatase activity was observed in leaf extract pretreated irradiated animals during the entire period of study.

Antituberculer: Growth of Mycobacterium tuberculosis was found to be inhibited by benzyl amine, ambroxol, bromhexine

(semi synthetic derivatives of vasicine) due to their mucolytic action. These have ability to concentrate in the macrophages and

enhance the level of lysozyme in bronchial secretions along with level of Rifampicin in the lung tissue and sputum, acting as

adjunctive for the therapy of tuberculosis.

3.1.2. Momordica charantia:

Effect of terpenoids, steroids, cardiac glycosides, anthraquinine glycosides, saponins, flavonoids, tannins and phenolic

compound, alkaloids Constituents:

A bitter glucoside soluble in water, insoluble in ether; a yellow acid resin and ash 6 p.c. Fresh vegetable contains 88.75 p.c.

moisture, and the completely dried material contains Ether extract 2.93 p.c., Albuminoids 1.62 (cont'g. Nitrogen 0.26 p.c.);

soluble carbohydrates 85.41 p.c., woody fiber 1.51 p.c. and Ash 8.53 p.c. (cont'g. Sand 0.17 p.c.) respectively.

Worldwide Ethano botanical Uses [7]:

The fruit and leaf of bitter melon has demonstrated an in vivo anti-fertility effects of female animals. In male animals, it was

reported to affect the production of sperm negatively. The seeds, however, have demonstrated the ability to induce abortions in

rats and mice, and the root has been documented with a uterine stimulant effect in animals [8-12].

Bitter melon contains an array of novel and biologically active phytochemicals including triterpenes, proteins and

steroids. In numerous studies, at least three different groups of constituents found in all parts of bitter melon have clinically

demonstrated hypoglycemic properties (blood sugar lowering) or other actions of potential benefit against diabetes mellitus.

These hypoglycemic chemicals include a mixture of steroidal Saponins known as Charantin, insulin like peptides and

alkaloids [13-22].

The fruit has also shown the ability to enhance cells uptake of glucose [23], to promote insulin release and potentiate the

effect of insulin [24-25].


In other in vivo studies, bitter melon fruit and/or seed has been shown to reduce total cholesterol and triglycerides [26-27]

in both the presence and absence of Dietary cholesterol. A chemical analog of bitter melon proteins was developed and named

MAP-30 and its inventors reported that it was able to inhibit prostate tumor Growth [28]. The phytochemical momordin has

clinically demonstrated cytotoxic activity against Hodgkin's lymphoma in vivo [29].

In one study, a water extract blocked the growth of rat prostate Carcinoma [30]; another study reported that a hot water

extract of the entire plant inhibited the development of mammary tumors in mice [31]. Numerous in vitro studies have also

demonstrated the anti-cancerous and anti-leukemic activity of bitter melon against numerous cell lines including liver cancer,

human leukemia, melanoma and solid sarcomas. In an in vivo study, a leaf extract demonstrated the ability to increase

resistant to viral infections as well as to provide an immunostimulant effect in humans and animals (increasing interferon

production and natural killer cell activity) [32].

Two proteins known as alpha- and beta- momocharin (which are present in the seeds, fruits and leaves) have been reported

to inhibit the HIV virus in vitro [33]. The analgesic activity of the methanol extract of the seeds of Momordica charantia in

rats and mice is described. The results demonstrate that the extract exhibited a dose-related analgesic response in mice. Only an

equivocal analgesic response was shown in rats [34].

3.1.3. Nordostachys jatamansi:

Effect of terpenoids, steroids ,cardiac glycosides, anthraquinine glycosides, saponins, coumarin glycosides, flavonoids, tannins

and phenolic compound, alkaloids Constituents-Essential oil, resin, sugar, starch, gum, bitter matter K = rhizomes and roots

contain volatile essential oil .5% oleum jatamansi, resin, sugar, starch, bitter matter, gum, ketone called jatamansone,

sesquiterpee seychelane, and beta-sitosterol. Roots have many compounds viz. valeranone, valeranal, nardolcalarenol,

nardostechone, n-hexacosanyl arachidate, n - hexaconsanol, calarene, n-hexacosane, h-hexacosanyl isovalerate, acosanyl

arachidate, n-hexaconsanol, calarene, norseychelanone, seychellen, patchouli alcohol, hydrocarbons, beta-eudesmol, elemol,

beta-sitosterol, angelicin, jatamansinol.

Worldwide Ethanobotanical Uses

This drug possess antiarrhythmic activity with possible therapeutically usefulness in cases of auricular flutter; it is less

effective than quinidine but has the advantage of being less toxus; oil exerts hypotensive effect and in moderate doses it has a

distinct depressant action on the central nervous system; lethal doses cause deep narcosis and death within a few hours; rhizome

is considered tonic, stimulant, antispasmodic, diuretic, deobstruent, emmenagogue, stomachic, and laxative; infusion of rhizome

is reported to be useful in epilepsy, hysteria, palpitation of heart and chorea; tincture given in intestinal colic and flatulence;

rhizome used as aromatic adjunct in preparation of medicinal oils; reported to promote growth of hair and impart blackness. Folk

and traditional medicines: In India roots are well know tranquilizer and may be used alone or along with Valerian jatamansi (V.

wallichi); infusion of root given in hysteria, palpitation of heart, menopause, and various nervous diseases; in infusion, ammonia,

camphor, or cinnamon (Cinnamomum verum) may be added; for treatment of diseases associated with indigestion a compound

preparation consisting of cinnamon, fennel (Foeniculun vulgare), ginger (Zingiber officinalie) and sugar in addition to jatamansi

is used; essential oil from rhizome used in perfumery and has a tranquilizing effect when massaged on head; also applied on

leprous wounds. In ayurveda used in "Mansyadi Kwath" and "Rakshoghna Ghrita" while in Unani. However, one of the most

used in the clinic essential oil is the essential oil of Jatamansi (Spikenard), which is obtained by steam distillation of dried

rhizomes of Nardostachys jatamansi D.C. It is a slightly viscous liquid in appearance and varies from amber to deep blue or

greenish blue colour. The aroma of Jatamansi oil is lingering, heavy, sweet woody and spicy-animal when used in treatment. In

clinical aromatherapy, Jatamansi oil can be employed in the treatment of epilepsy, hysteria and in many varieties of convulsive

affections. It is used in cases of heart palpitations (even as a substitute for Valerian). Its antispasmodic characteristics are

unsurpassed. Tibetan herbalists use Jatamansi oil for the purpose of hair growth and colour restoration. The herb is used in tribal

medicine for incurable skin conditions and is believed to contain anti-ageing properties.

The decoction of the roots and tubers are excellent antidote to all poisons. A paste of the fresh tubers applied to the breasts

acts as an effective galactagogue. The root is often used for developing high memory. This herb also harmonizes the liver, spleen,

and pancreas.

Rao V. S, et al. studied anticonvulsant activity and neurotoxicity of ethanolic extract of the roots of Nardostachys jatamansi

DC. (Valerianaceae) for its, alone and in combination with phenytoin in rats. The results demonstrated a significant increase in

the seizure threshold by Nardostachys jatamansi root extract against maximal electroshock seizure (MES) model as indicated by

a decrease in the extension/flexion (E/F) ratio. The extract was ineffective against pentylenetetrazole (PTZ)-induced seizures,

also showed minimal neurotoxicity against rotarod test and synergistic action with phenytoin [35].

Salim S, et al. studied the protective effect of Nardostachys jatamansi (NJ) on neurobehavioral activities, thiobarbituric acid

reactive substance (TBARS), reduced glutathione (GSH), thiol group, catalase and sodium-potassium ATPase activities, in

middle cerebral artery (MCA) occlusion model of acute cerebral ischemia in rats. MCA occlusion caused significant depletion in

the contents of glutathione and thiol group and a significant elevation in the level of TBARS. The study provides first evidence of

effectiveness of NJ in focal ischemia most probably by virtue of its antioxidant property [36].


Chatterjee A, et al. studied the structure and stereochemistry of a new terpenoid ester, nardostachysin (1), isolated from the

rhizomes of Nardostachys jatamansi. And those were established as the 7', 8'-dihydroxy-4'-methylene

hexahydrocyclopenta[c]pyran-1'-one-8'-methyl ester of 7, 9-guaiadien-14-oic acid, by spectral and chemical studies [37].

Effects of Nardostachys jatamansi on biogenic amines and inhibitory amino acids in the rat brain were studied. The acute oral

administration of the extract did not change the level of nor epinephrine (NE) and dopamine (DA) but resulted in a significant

increase in the level of serotonin (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA). A significant increase in the level of

gamma-amino butyric acid (GABA) and taurine was observed in the drug-treated groups when compared to the controls. A

15-day treatment resulted in a significant increase in the levels of all biogenic amines. These data indicate that the alcoholic

extract of the roots of N. jatamansi causes an overall increase in the levels of central monoamines and inhibitory amino acids

[38].

Hypolipidaemic effects of Curcuma longa L and Nardostachys jatamansi, DC in triton-induced hyperlipidaemic rats were

measured. Fifty per cent ethanolic extract of Curcuma longa (tuber) and Nardostachys jatamansi (whole plant) feeding elevates

HDL-cholesterol/total cholesterol ratio. The extracts also caused a significant reduction in the ratio of total

cholesterol/phospholipids. Curcuma longa exhibited better cholesterol and triglyceride lowering activity [Ch = -85%; Tg = -88%]

as compared to N. jatamansi in triton-induced hyperlipidaemic rats. In view of the protective action of HDL against heart disease

and atherogenecity, C. longa consumption is recommended [39].

Efficacy of some essential oils and their constituents on few ubiquitous molds was demonstrated. Zentralbl Bakteriol

Naturwiss. Six essential oils of Mentha arvensis, Mentha piperita, Anethum sowa, Cymbopogon winterianus, Nardostachys

jatamansi, and Commiphora mukul were selected and tested for their efficacy against Aspergillus flavus, A. fumigatus, A.

sulphureus, Mucor fragilis, and Rhizopus stolonifer. These oils were fungistatic or fungicidal to one or the other molds,

depending upon the concentrations [40].

Isolated and studied pharmacodynamic activity of the sesquiterpene valeranone from Nardostachys jatamansi DC. In some

experiments, typical for tranquilizers, certain activities could be demonstrated such as the prolongation of barbiturate hypnosis,

the impairment of rotarod performance, an anticonvulsive activity on electric shock and potentiation of the body-temperature

lowering activity of reserpine. In three other pharmacological models an anti-ulcer action was detected. In general, the activity of

valeranone was lower than those of the standard substances used. As regards the hypotensive property, only a weak activity was

demonstrated. In toxicological studies on rats and mice an oral LD50 of greater than 3160 mg/kg was found, which suggests the

possibility of a therapeutically useful dose ratio [41].

3.1.4. Tephrosia purpurea:

Effect of terpenoids, steroids, cardiac glycosides, saponins , flavonoids, tannins and phenolic compound, alkaloids Traditional uses:

The root has a bad bitter taste; enriches the blood, useful in bronchitis, wounds, boils, pimples, liver and spleen diseases,

asthma, inflammation, antiulcer, hepatoprotective, used in poisoning due to snakebite, useful in enlargement of spleen,

antidiarrhoeal. Given in tympanitis, dyspepsia chronic diarrhea and diuretic.

In French Guiana it is used as fish poison. Seeds are used in poisoning due to rat bite. Leaves are useful in Diseases of lungs

and of the chest, tonic to intestines, improve the appetite, good in piles, syphilis, and gonorrhoea.

Whole plant is Digestible, Anthelmintic, Alexeteric, Antipyretic, Cures diseases of liver, spleen, heart, blood, and cures

tumors, ulcers, leprosy, asthma, bronchitis, piles, and caries of the teeth, laxative, and blood purifier.

The white flowered variety is more effective than the red variety. Fresh root bark, ground and made into a pill, with a little

black pepper, is frequently given in cases of obstinate colic. The drug is useful in cough and in derangement of the kidneys. A

decoction of the 1 in 10 was administered in one ounce doses to cases of Blight’s disease with dropsy.

Scientifically validated uses:

Antibacterial activity: Murganathan G et al shows that alcoholic extract of the roots of Tephrosia purpurea have been found to

possess mild antibacterial activity. Deshpande et al shows that antibacterial activity was significant in fresh juice of Tephrosia

purpurea roots but not in aqueous extract of Tephrosia purpurea roots [42]. Mahajan R T et al studied the antibacterial activity of

Tephrosia purpurea on ethanolic and methanolic extracts.

Antimicrobial activity: It has been reported by G S Kumar et al that Tephrosia purpurea (roots) had the greatest antimicrobial

effect against acne-inducing bacteria among many plant species selected. M Gupta et al studied the Antimicrobial activity of

methanol extracts of Plumeria acuminata Ait. leaves and Tephrosia purpurea (Linn.) Pers. roots. Vandana Singh et al show that

antimicrobial effect of Tephrosia purpurea is due to presence of flavonoides. Study of Antimicrobial activity Tephrosia purpurea

(roots) was done by Deshpande shrikalp et al which shows that fresh extract shows antibacterial activity not antifungal activity

by agar well diffusion and R & B agar well diffusion method.

Hepatoprotective activity: Tephrosia purpurea (aerial parts) was evaluated by Ramamurthy M Sree et al for its efficacy in rats

by inducing hepatotoxicity with D-galactosamine HCl (acute) and carbon tetrachloride (chronic). The results of the study

indicated that the administration of Tephrosia purpurea along with the hepatotoxins offered a protective action in both acute (D-


galactosamine) and chronic (CCl4) models [11]. A Jain et al reported that Ethanol extract of leaves and flavonoid (isolated from

leaves extract) from Tephrosia purpurea were evaluated for hepatoprotective activity in rats by inducing hepatotoxicity with

carbon tetrachloride and conclude that the hepatoprotective activity was more in ethanolic extract of leaves than isolated

flavonoid. Hepatoprotective activity of a benzopyrone from Tephrosia purpurea Pers. was reported by Shankar, M. B et al [13].

The hepatoprotective activity of the aerial parts of Tephrosia purpurea and stem bark of Tecomella undulata against

thioacetamide-induced hepatotoxicity was proved by Amit Khatria et al [14]. Mitra S K et al reported the protective effect of

HD-03(an herbal formulation) against Tephrosia purpurea in rats. And anticholestatic activity of HD-03(an herbal formulation)

in thioacetamide induced experimental cholestasis. Effect of T purpurea, an herbal hepatoprotective on drug metabolism in

patients of cirrhosis and hepatic enzyme function in experimental liver damage has been reported by Chauhan C K et al.

Antiulcer activity : S S Deshpande et al reported that Pylorus ligation and ethanol induced ulcers study on rats shows

significant protection of Tephrosia purpurea roots in aqueous extract in dose range of 100 – 500 mg/kg p.o. for gastric ulcers and

gastric cytoprotection. Satyabrata Mohapatrab et al reported that the methanolic extract of Tephrosia purpurea showed promising

activity against clinical isolates and standard strains of Helicobacter pylori, including metronidazole-resistant strains.

Free radical scavenging activity: The dried alcoholic extracts of the plant Tephrosia purpurea Linn. was investigated by K

Soni et al for its free radical scavenging activity, hydroxyl radical scavenging activity.

Antihyperglycemic and Antilipid Peroxidative activity: S. Sethupathy et al evaluated the antihyperglycemic and

antilipidperoxidative effects of ethanolic seed extract of Tephrosia purpurea (TpEt) in streptozotocin induced diabetic rats. Oral

administration of “TpEt” at a dose of 300mg/kg BW showed significant antihyperglcemic and antilipidperoxidative effects as

well as increased the activities of enzymatic antioxidants and levels of nonenzymatic antioxidants [21]. K Soni et al found that

the Tephrosia purpurea dried alcoholic extract showed significant inhibition of lipid peroxidation.

Chemo preventive Potential and Antilipidperoxidative activity: Kavitha, et al. investigates the chemopreventive potential and

antilipidperoxidative effects of ethanolic root extract of Tephrosia purpurea (Linn.) Pers. (TpEt) on 7, 12-

dimethylbenz(a)anthracene (DMBA)- induced hamster buccal pouch carcinoma.

Immunomodulatory Activity: The flavonoid fraction of Tephrosia purpurea (FFTP) was studied by A. S. Damre et al for its

effect on cellular and humoral functions and on macrophage phagocytosis in mice. Oral administration of FFTP significantly

inhibited sheep red blood cells (SRBC)-induced delayed-type hypersensitivity reactions. It also produced a significant,

dose-related decrease in sheep erythrocyte-specific haemagglutination antibody titre [24].

Antioxidant Activity: The ethanol extract of Tephrosia purpurea Linn. Was studied by M N Saraf et al and found that ethanol

extract shows significant inhibition of the carbon tetrachloride-induced lipid per oxidation in vivo and superoxide generation in

vivo and the ethyl acetate soluble fraction has improved antioxidant potential than the extract.

Role in Haemopoetic injury: Taraphdar A K et al studied the role of Tephrosia in Haemopoetic injury in Swiss albino mice.

Wound healing Activity: A.K. Singhai et al studied the wound healing potential of ethanolic extract of Tephrosia purpurea

(aerial part) in the form of simple ointment using three types of wound models in rats as incision wound, excision wound and

dead space wound. The results were comparable to standard drug Fluticasone propionate ointment, in terms of wound

contraction, tensile strength, histopathological and biochemical parameters such as hydroxyproline content, protein level, etc.

Antiallergic Activity: The inhibitory effect of ethanolic extract of the aerial parts of T. purpurea was studied by A Gokhale et

al on late-phase allergy by the inhibition of leukotriene synthesis.

Antileishmanial Activity: Y. Guru et al found that Tephrosia purpurea have significant antileishmanial activity, and has been

extensively fractionated to locate the abode of activity. A fraction (F062) obtained from N-butanol extract of T. purpurea showed

consistent antileishmanial activity at 50 mg/ kg × 5 days by oral route against Leishmania donovani infection in hamsters.

Antibiotic Activity: C.L. Abayasekara et al studied the antibiotic activity of the water extracts of roots, leaves, pods and a

combination of these three plant parts of T. purpurea they concluded that the ethanolic root extract of T. purpurea shows

significant activity against Pseudomonas aeruginosa, two other Pseudomonas strains and two coliform strains.

Antilithiatic Activity: Aqueous extract of the roots of Tephrosia purpurea was evaluated by K.V.S.R.G. Prasad et al for its

antilithiatic activity, in two models of urolithiasis. They report that the effect of aqueous extract of T. purpurea on the excretion

and deposition of various calculi forming constituents like calcium, oxalate, magnesium and phosphate in urine, kidney and

foreign body.

Anti-Asthmatic Activity : Deshpande et al reported that ethyl acetate extract of Tephrosia purpurea roots shows a significant

protection of rat mesenteric mast cells from disruption caused by compound 48/80 was offered by Ethyl acetate extract. The

extract also offered significant protection against mast cell disruption caused by antigen. Ethyl acetate extract did not produce

any significant difference in the count of all the types of WBC detected in the bronchial fluid of sensitized animals compared

with untreated sensitized animals.

Antihyperglycemic Activity: S. Sethupathy et al studied the Antihyperglycemic Activity on ethanolic seed extract of

Tephrosia purpurea (TpEt) in streptozotocin induced diabetic rats. They also studied the Hyperglycemia associated with an

altered hexokinase and glucose 6 phosphatase activities, elevated lipid peroxidation, disturbed enzymatic and non-enzymatic

antioxidants status were observed in streptozotocin induced diabetic rats. H B N Swift et al studies on the hypoglycemic effect of


plant Tephrosia purpurea variety pumila in which rutin was administered by stomach tube to normal and alloxan induced diabetic

rabbits. P Pavana et al reported the Effects of Tephrosia Purpurea Aqueous Seed Extract on Blood Glucose and Antioxidant

Enzyme Activities in Streptozotocin Induced Diabetic Rats.

Antitumor Activity: Saleem et al. have shown that Tephrosia purpurea ameliorates benzoyl peroxide-induced oxidative stress

in murine skin. They investigated a chemopreventive efficacy of T. purpurea against N-diethylnitrosamine-initiated and

potassium bromate-mediated oxidative stress and toxicity in rat kidney. They further assessed the effect of Tephrosia purpurea

on 12-O-tetradecanoyl phorbal-13-acetate (TPA; a well-known phorbol ester) induced cutaneous oxidative stress and toxicity in

murine skin. The pre-treatment of Swiss albino mice with Tephrosia purpurea prior to application of croton oil (phorbol ester)

resulted in a dose-dependent inhibition of cutaneous carcinogenesis.

Anthalmintic Activity: Surve Suvidha S et al studied the Anthalmintic activity of seed part of plant Tephrosia purpurea Linn

using Adult Indian earthworms, Pheretima posthuma.

Insecticidal and Repellent Properties was studied by B. N. Saxena et al on seed extract of Tephrosia purpurea (linn.) pers.

Kiuchi F et al identified the nematocidal principles in the roots of Tephrosia purpurea and showed that rotenoides have strong

nematocidal activity.

4. Conclusion

This research work has revealed further potentials of these four plants in the area of pharmacology as potential source of useful

drugs. This study therefore has provided some biochemical basis for ethno pharmacological uses of these plants in the treatment

and prevention of various diseases and disorders. The phytochemical screening on qualitative analysis shows that the whole plant

of Justica adhatoda Linn , Tephrosia purpurea ,fruits of Momordica charantia and Roots & Rhizomes of Nordostachys jatamansi

are rich in terpenoids, steroids ,cardiac glycosides, saponins, flavonoids, tannins and phenolic compound, alkaloids, which are

popular phytochemical constituents.

Acknowledgment

On the occasion of presenting this article, it is my privilege to express my sincere thanks to my supervisor Dr. Kamal Kumar

Goyal, Department of Pharmacognosy & Phythochemistry, Sri Balaji College Of Pharmacy, Jaipur who has provided excellent

guidance, valuable advices, and shared intelligent thoughts, criticisms and inculcated discipline. I am highly indebted to him for

his valuable presence even in his busy schedule, which helped me to complete this work successfully. I owe a huge debt of

gratitude to Principal Dr. Vikram Sharma for providing me all the facilities and encouragement for the successful completion of

my project work. I extend my profound respect and heartful gratitude to my beloved Parents Specially my father Late. Rajendra

kumar Sharma, and also express my affection to my brother Kapil for their constant love, support, and encouragement

throughout my life.

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