CHAPTER II SCREENING AND ESTIMATION OF …shodhganga.inflibnet.ac.in/bitstream/10603/62592/9/09_chapter 2.pdf · screening of S. indicus showed the presence of alkaloids, flavonoids

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CHAPTER II

SCREENING AND ESTIMATION OF PHYTOCONSTITUENTS

OF FEW INDIAN MEDICINAL PLANTS

Introduction:

Medicinal plants have been used as a source of medicine to treat illness since

time immemorial. For a long time, plants have provided a source of emerging

modern medicines and drug compounds. Plant derived medicines have made large

contributions to human health. Their role is two fold in the development of new

drugs. They may become the base for development of a medicine, a natural blue

print for the development of the new drugs or a phytomedicine to be used for the

treatment of diseases. Active compounds present in the medicinal plants provide the

bountiful resource for the pharmaceutical, cosmetics and food industries and more

recently in agriculture for pest control [Han et al.,2007].

Herbal products from medicinal plants are preferred because of less testing

time, higher safety, efficiency, cultural acceptability and lesser side effects. The

chemical compounds present in herbal products are a part of the physiological

functions of living organisms and hence they are believed to have better

compatibility with the human body [Prasad et al., 2012].

Phytochemicals are bioactive nutrients derived from plants (Phyto is derived

from Greek, which means plants). They have a variety of human health effects such

as possessing reputed chemo-retentive properties (anticarcinogenic and

antimutagenic) [Surh, 2003]. It is estimated that more than 5000 individual

phytochemicals have been identified in fruits, vegetables, grains and other plants,

mainly classified as phenolics, carotenoids, vitamins, alkaloids, nitrogen – containing

compounds and organo sulphur compounds. Among the great structural diversity of

phytochemicals, phenolic compounds have attracted considerable interest and the

most attention for their wide variety of bioactivities [Han et al., 2007].

Phytochemical screening and quantitative estimation of secondary

metabolites of five plant species are aimed to be carried out in this study with the

intention of discovering the highest content of phytochemical in each species and

thereby assesing its therapeutic value.

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a. Sphaeranthus indicus Linn.

Division : Tracheophtya

Class : Magnoliopsida

Order : Asterales

Family : Asteraceae

Genus : Sphaeranthus

Species : indicus, Linn

Vernacular name : Utharrimurrippan

Pharmacological importance:

Spheranthus indicus is a herb whose stems, leaves and flowers contain a

bitter alkaloid „Sphaeranthine‟. The herb is bitter, promoting appetite, alterative,

pectoral and revealing inflammation and externally emollient. The herb is laxative

and digestive tonic; used in insanity, bronchitis, irritation at base of a bladder,

hemicrania and good for sore eyes. Root and seeds are used as a stomachic and

parasitic worm. Flowers are highly esteemed as alteratives, freeing from impurities,

refrigerants and tonics, useful as blood – purifiers in skin diseases. Root – bark

ground small and mixed with whey is a valuable remedy in bleeding piles; also used

as paste for local application. Oil prepared from the root by steeping it in water and

then boiling it in sesame oil until all the water is expelled, taken on empty stomach

is a valuable aphrodisiac. It is used in glandular swellings in the neck with benefit

and also a good remedy in jaundice. Leaves dried in the shade and powdered are

used in chronic skin diseases as antisyphilitic and nerving tonic. The drug is useful

in urethral discharge and jaundice.

Spheranthus indicus whole plant extracts have great potential as

antimicrobial compounds against micro organisms and they can be used in the

treatment of infectious diseases caused by micro organisms. Acetone and methanol

extracts of S. indicus was antibacterial against Gram +ve and insensitive to Gram -ve

bacteria. It is against vibrio cholera and micro coccus. The hydroalcoholic extract of

S. indicus whole plant has central nervous depressant activity and anticonvulsant

activity which leads pharmacological justification to the use of plant extract by

traditional medicine practitioners in the treatment of epilepsy [Galani and Patel,

53

2010]. The methanolic extract of whole plant of S. indicus exhibited significant

protection against bronchospasm induced by histamine in guinea pigs [Sarpate et al.,

2009] and could act as a hepatoprotective and antioxidant agent against CCl4

induced liver toxicity [Lizpy et al., 2012]. Ethanolic extract of S. indicus leaves

elicited significant anti-inflammatory activity in carrageenan induced paw edema

and cotton pellet granuloma model which is attributed to Brady kinin and

prostaglandin synthesis inhibition property of flavonoids present in leaves extract

[Meher et al., 2011]. The root of S. indicus exhibited antidiabetic, hypolipidemic and

in vivo antioxidant effects in STZ – induced diabetes which may be due to the

presence of biomarker compounds such as gallic acid and quercetin in the plant root

which indirectly helped to decrease the levels of glucose, prevent alteration of lipids

level, increase antioxidant status in diabetic condition [Ramachandran et al., 2011].

Ethanolic extract of underground portions of S. indicus exhibited free radical

scavenging potential [Annie Shirwaikar et al., 2006]. Preliminary phytochemical

screening of S. indicus showed the presence of alkaloids, flavonoids and terpeniods

[Venkata et al., 2010; Doss, 2009].

Various parts of S. indicus have been explored for anxiolytic activity,

neuroleptic, immunomodulatory activity, mast cell stabilizing action,

antihyperglycaemic activity, larvicidal action and Reno protective effect. It is

reported to contain rudesmanoids, eudesmanolides, sesquiterpene lactone, sterol

glycoside, flavonoids and essential oil [Galani et al., 2010].

Materials and Methods:

Plant Collection

Healthy flower petals of Spheranthus indicus were collected from the bank

of river Cauvery, Kutchippalayam, Namakkal District, Tamil Nadu, India and

authenticated by Taxonomist, Kandaswami Kandar‟s College, Velur, Namakkal

District, Tamil Nadu, India.

Chemicals and Reagents

All the chemicals and reagents used were of analytical grade obtained from

S. D. Fine Chemicals Ltd., Mumbai, India.

54

II.1. Phytochemical Screening:

Phytochemical screenings were performed as described below. Samples of

plant materials were extracted with Soxhlet extractor using petroleum ether, diethyl

ether and ethyl acetate solvents.

II.1.1. Test for alkaloids (Mayer‟s test):

One ml of different solvent extracts of flower petals of S. indicus was diluted

to 10 ml with acidic ethanol, boiled and filtered. To 5 ml of the filtrate, 2 ml of

dilute ammonia, 5 ml of chloroform was added and shaken gently to extract the

alkaloidal base. The chloroform layer was extracted with 10 ml of acetic acid and

Mayer‟s reagent was added. (Mayer's reagent: Dissolve 1.358 g of HgCl2 in 60ml of

water and pour into a solution of 5 g of KI in 10 ml of water, add sufficient water to

make 100 ml). The formation of reddish brown precipitate (with Mayer‟s reagent)

confirms the presence of alkaloids [Kumar et al., 2009].

II.1.2. Test for Flavonoids (Shinoda test):

The different solvent extracts of flower petals of S. indicus (1 ml) were

treated with few drops of concentrated HCl and magnesium turnings (0.5 g). The

presence of flavonoids is indicated by formation of magenta colour within 3 minutes

[Bot, 2007].

II.1.3. Test for Flavones:

The different solvent extracts of flower petals of S. indicus (1 ml) were

treated with few drops of concentrated sulphuric acid. The presence of flavones

is indicated by the colour change from yellow to orange within three minutes

[Finar, 2006].

II.1.4. Test for Flavonols:

The different solvent extracts of flower petals of S. indicus (1ml) were

treated with few drops of aqueous sodium hydroxide. The presence of flavonols is

indicated by the colour change from yellow to orange within three minutes [Finar,

2006].

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II.1.5. Test for Cardiac glycosides (Keller-Killiani test):

To one ml of different solvent extracts of flower petals of S. indicus diluted

to 5 ml using water 2 ml of glacial acetic acid containing one drop of ferric

chloride solution was added. To this 1 ml of concentrated sulphuric acid was added.

A brown ring at the interface indicated the presence of cardiac glycosides

[Sofowora, 1993].

II.1.6. Test for Tannins / Phenolics:

One ml of different solvent extracts of flower petals of S. indicus were stirred

with 10 ml of distilled water, filtered and a few milliliters of 5 % ferric chloride was

added to the filtrate. A deep blue colouration confirmed the presence of tannin

[Sofowora, 1982].

II.1.7. Test for Amino acids and Proteins:

One ml of the different solvent extracts of flower petals of S. indicus were

dissolved in 2 ml of 1% ethanolic Ninhydrin solution. The formation of violet colour

showed the presence of amino acids and proteins [Adamu et al., 2009].

II.1.8. Tests for Carbohydrates:

To 1ml of different solvent extracts of flower petals of S. indicus diluted to

5 ml using water Fehling's test (standard test for reducing sugar) was performed to

confirm the presence of carbohydrates. Fehling's "A" was prepared by using 7 g

CuSO4.5H2O dissolved in distilled water containing 2 drops of dilute sulfuric acid.

35 g of potassium tartrate and 12 g of NaOH in 100 ml of distilled water were used

for preparing Fehling's "B" [Odebiyi and Sofowora, 1978].

II.1.9. Test for Steroids (Salkowski test):

One ml of the different solvent extracts of flower petals of S. indicus were

dissolved in 10 ml of chloroform and an equal volume of concentrated sulphuric

acid was added by the sides of the test tube. The upper layer turning red and

sulphuric acid layer turning yellow green fluorescence indicates the presence of

steroids [Joao et al., 2008].

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II.1.10. Test for Saponins:

About 1 ml of different solvent extracts of flower petals of S. indicus were

diluted with distilled water to 20 ml and shaken in graduated cylinder for 15

minutes. One cm layer of foam indicated the presence of saponins [Odebiyi and

Sofowora, 1978].

II.1.11. Test for Terpenoids (Salkowski Test):

One ml of different solvent extracts of flower petals of S. indicus were mixed

with 2 ml of chloroform and concentrated H SO

(3 ml) was carefully added to form 2 4

a layer. A reddish brown coloration of the interface indicated the presence of

terpenoids [Sofowora, 1993].

II.1.12. Test for Fats and Oils:

One ml of extract was treated with 0.5 N alcoholic potassium hydroxide

using 1 drop of phenolphthalein as indicator. This solution was heated on waterbath

for 1 hr, a white colour soap was formed [Rangari, 2003].

II.1.13. Test for Anthocyanins and Leucoanthocyanins:

The different solvent extracts (1 ml) were treated with few drops of

concentrated HCl and magnesium turnings (0.5 g). The presence of anthocyanins is

indicated by formation of red colour. The presence of leucoanthocyanins is indicated

by formation of pink colour [Harbone, 1973].

II.2. Quantitative Analysis of Secondary Metabolites:

All the parameters were determined with the extract of flower petals of S.

indicus following the spectrophotometric principle.

II.2.1. Principle:

Beer-Lambertz law

When a beam of monochromatic radiation is passed through a solution of an

absorbing substance, the rate of decrease of intensity with thickness of the absorbing

solution is directly proportional to the intensity of incident radiation as well as to the

concentration of the solution.

The following parameters have been determined with the plant extract.

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II.2.2. Determination of Total Phenolics:

Principle:

Phenol reacts with phosphomolybdic acid in presence of Folin-ciocalteau

reagent in an alkaline medium and produce a blue coloured complex

(molybdenum blue) that can be estimated colorimetrically at 725 nm [Singleton and

Rossi, 1965].

Contents of total phenolics in the extract of flower petals of S. indicus were

estimated by a UV – Visible spectrophotometer (Systronics, model 108) array.

Basically, 1 ml of sample extract (concentration 1 mg / ml) was mixed with 1 ml

of Folin ciocalteau phenol reagent. After 3 minutes, 1 ml of saturated sodium

carbonate solution was added to the mixture and was made up to 10 ml with

distilled water. The reaction mixture was kept in dark for 90 minutes, after

which the absorbance was read at 725 nm. Gallic acid was used for constructing

the standard curve and the results were expressed as microgram of gallic acid

equivalent per gram of extract (GAEs).

II.2.3. Determination of Total Flavonoids:

Flavonoid content in the extract was determined by the UV – Visible

spectrophotometer array [Jia et al., 1999]. 250 µL of flower petals of S. indicus

extract (concentration 1 mg / mL) was mixed with 1.25 ml of distilled water and

75 µL of 5% NaNO2 solution. After 5 minutes, 150 µL of 10% AlCl3.H2O solution

was added. After 6 minutes, 500 µL of 1 M NaOH and 275 µL of distilled water

were added to the mixture. The solution was mixed well and the absorbance

was read at 510 nm. (±) – Catechin was used to calculate the standard curve and

the results were expressed as µg of (±) – Catechin equivalents per gram of extract

(CEs).

II.2.4. Estimation of Flavonol:

Principle:

The content of flavonols was determined by using rutin as a reference

compound. This method was based on the formation of complex with maximum

absorption at 440 nm. [Grubesic et al., 2005]

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Reagents:

1. Aluminium chloride solution (20 mg/mL)

2. Sodium acetate (30 mg/mL)

Procedure:

About 1 ml of each extract was mixed with 1 ml aluminium chloride and 3

ml sodium acetate. The absorbance at 440 nm was read after 2.5 hours. The

absorption of standard rutin solution in methanol was measured under the same

conditions. A duplicate was carried out for all the determinations.

Calculation:

The amount of flavonols in plant extracts in rutin equivalents was calculated

by the following formula.

Where,

X = (A-m0) / (A0-m)

X was the flavonoid content (µg/ml) in rutin equivalents.

A was the absorption of plant extract solution,

A0 was the absorption of standard rutin solution,

m was the weight of plant extract ( µg)

m0 was the weight of rutin in the solution (µg)

II.2.5. Estimation of Tannins:

Principle:

Spectrophotometric estimation of tannins is based on the measurement of

blue colour formed by the reduction of phosphotungstomolybdic acid by tannin like

compounds in alkaline solution [William, 1960].

Reagents:

1. Folin-Denis reagent:

To 750 ml of water, add 100 g of sodium tungstate (Na2WO4.2H2O), 20 g of

phosphomolybdic acid and 50 ml of 85% phosphoric acid (H3PO4). Reflux the

mixture for 2 hrs, cool to 25o

C and dilute to 1000 ml with water.

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2. Saturated sodium carbonate solution:

To 100 ml of water, add 35 g of anhydrous sodium carbonate dissolve at 70-

80o C and cool overnight and decant the clear liquid before use.

3. Tannic acid standard solution:

Dissolve 100 mg of tannic acid in 1 litre of water. Prepare fresh solution for

each determination (1 ml = 0.1 mg of tannic acid).

4. Preparation of standard curve:

Pipette out 0 to 10 ml aliquots of the standard tannic acid solution into 100-

ml volumetric flasks containing 75 ml of water. Add 5 ml Folin-Denis reagent and

10 ml Na2CO3 solution into each of the volumetric flasks and make up to 100 ml

with water. Mix well and measure the colour after 30 min at 700 nm against

experimental blank adjusted to absorbency.

5. Preparation of Sample:

Dissolve 1 g of sample with 80 ml of water, transfer to 100 ml volumetric

flask and dilute to mark, shake well and filter.

6. Determination:

Use an aliquot of the filtrate containing not more than 0.1 mg of tannic acid.

Proceed as in standard and obtain mg tannic acid from the standard curve.

Results and Discussion:

Results

Preliminary Qualitative Screening

The flower petals of S. indicus in different solvent extracts revealed the

presence of many secondary metabolites which is presented in table II-1.

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Table II-1. Phytochemical screening of S. indicus flower in various solvent

extracts

S.No.

Phytochemical Constituents

Various Extracts

Petroleum ether

Diethyl ether

Ethyl acetate

1. Alkaloids - ++ ++

2. Flavonoids + +++ +++

3. Flavones - ++ +

4. Flavanones - + ++

5. Flavonols + +++ ++

6. Isoflavones + ++ ++

7. Glycosides - ++ +

8. Tannins + +++ +++

9. Phenolics + +++ +++

10. Aminoacids - + +

11. Steroids - - +

12. Proteins - + +

13. Carbohydrates - +++ ++

14. Saponins ++ - +

15. Terpenoids + +++ ++

16. Fats/Oils +++ - +

17. Anthocyanins - - -

18. Leucoanthocyannins - ++ ++

+++ = Copiously present, ++ = Moderately present, + = Slightly present, - = Absent.

Determination of Phytoconstituents:

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Total phenolic content

Fig. II-1. Determination of phenolic content using Gallic acid as a standard

Table II-2 Total phenolic content in various solvent extracts (S. indicus)

Extracted Solvents (µg / mL of extract)*

Ethyl acetate 74.33 ± 0.56

Diethyl ether 141.43 ± 1.67

Petroleum ether 16.79 ± 0.93

*Each value is the Mean ± Standard deviation of three replicate experiments.

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Total flavonoid content

Fig. II-2. Determination of flavonoid content using Catechin as a standard

Table II-3 Total flavonoid content in various solvent extracts (S. indicus)






Total flavonol content

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Table II-4 Total flavonol content in various solvent extracts (S. indicus)

Extracted Solvent (µg / mL of extract)*

Ethyl acetate

58.67 ± 0.001

Diethyl ether

71.95 ± 0.001

Petroleum ether

22.36 ± 0.002


Total Tannin content

Fig. II-3. Determination of Tannin content using tannic acid as a standard

Table II-5 Total tannin content in various solvent extracts (S. indicus )





*Each value is the Mean ± Standard deviation of three replicate experiments

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The qualitative screening of phytochemical constituents of flower petals

of S. indicus invarious solvent extracts for the presence of alkaloids, flavonoids,

phenolics, saponins, tannins, terpenoids, carbohydrates etc., are carried out.

Alkaloids, flavonoids, phenolics, tannins, carbohydrate, terpenoids

and leucoanthocyannins are significantly present in diethyl ether and

ethylacetate extracts. Amino acids and proteins are slightly present in diethyl

ether and ethylacetate extracts. Saponins, Fats and oils are moderately present in

petroleum ether extract. Anthocyanins are absent in these solvent extracts.

The quantitative estimation of total phenolic content, total flavonoid content,

total flavonol content and total tannin content are carried out and the values are

shown in the tables II – 2, II – 3, II – 4 and II – 5.

Total phenolic content:

The total phenolic content of the flower petals of S. indicus extracts in gallic

acid equivalents is presented in the table II – 2. The highest value (141.43 µg/mL of

extract) is obtained in diethyl ether extract with petroleum ether the lowest value

(16.79 µg/mL of extract) is obtained. The total phenolic content in ethyl acetate

extract is 74.33 µg/mL of extract.

Total flavonoid content:

The total flavonoid content of the various solvent extracts in catechin

equivalents is shown in the table II – 3. Again the highest value (121.46 µg/mL of

extract) in diethyl ether extract and lowest value (18.82 µg/mL of extract) in

petroleum ether extract are found. In between these two, the flavonoid content

88.13 µg/mL of extract is obtained in ethylacetate extract.

Total flavonol content:

The total flavonol content is estimated by using rutin as a standard. The

diethyl ether extract has the highest content of 71.95 µg/mL of extract and petroleum

ether extract has the lowest content of 22.36 µg/mL of extract. The methanol extract

has moderately 58.67 µg/mL of extract.

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Total tannin content:

Using tannic acid as the equivalent, the total tannin contents are tabulated.

Again the same results are found in various solvent extracts.

Diehylether extract> ethyl acetate extract> petroleum ether extract

Discussion:

The bioactive chemical constituents like alkaloids, flavonoids, phenolics,

tannins, carbohydrates, terpenoids and saponins exhibit definite physiological and

biochemical actions in the human body [Hill, 1952; Edeoga, 2005].

All these green chemicals have potent pharmaceutical values. Alkaloids are

used as basic medicinal agents for their analgesic, antispasmodic and bacterial

effects [Stray, 1998; Okwu and Okwu, 2004]. Flavonoids are used as

chemopreventive „nutraceuticals‟ for reducing incidence of coronary heart and

liver diseases as well as some cancers. Tannins are water soluble plant polyphenols

that precipitate proteins. Tannins have been reported to prevent the development

of microganisms by precipitating microbial protein and making nutritional

protein unavailable for them [Sodipo et al., 1991]. Saponins are a special class

of glycosides which have soapy characteristics [Fluck, 1973]. It has the property

of precipitating and coagulating red blood cells. Some of the characteristics

of saponin include formation of foams in aqueous solution, haemolytic

activity, cholesterol binding properties and bitterness [Sodipo et al., 2000].

Terpenoids are a large class of natural isoprenoids present in higher plants

which exhibit a wide range of biological activities. Plant terpenoids are used

extensively for their aromatic qualities. They play a role in traditional herbal

remedies. The steroids in animals are biologically produced from terpenoid

precursors. Carbohydrates orginate as products of photosynthesis. They are major

source of metabolic energy for plants and animals. They play key roles in the

immune system, fertilization, preventing pathogenesis, blood clotting and

development.

The herb possesses a number of secondary metabolites like alkaloids,

flavonoids, phenols, tannins, saponins, sterols, glycosides etc. The qualitative

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screening tests are very useful in the detection of the bioactive components and they

lead to the discovery and development of drugs. Also the qualitative tests facilitate

separation of the medicinally important active chemical compounds [Ali and

Ghatak, 1975].

In the quantitative analysis, the diethyl ether extract of flower petals of

S. indicus shows the highest contents of total phenolics, total flavonoids and

total tannins. Flavonoids, flavonols and tannins are polyphenolic compounds

which are found to act as strong antioxidants. Hence the flower petals of S. indicus

possess pharmacological values; this species is to be considered as a medicinal herb.

b. Zanthoxylum tetraspermum

Division : Magnoliophyta


Order : Sapindales

Family : Rutaceae

Genus : Zanthoxylum

Species : Tetraspermum

Vernacular name : Virasingampattai

It is an aromatic, spiny, thicket-forming, deciduous shrub or small tree,

typically attaining a height of between 2.5 and 3.5 m, some specimens up to 7.5 m.

The alternate branches are armed with strong brown prickles, about 1 – 2 cm long,

cone shape with broad base and found irregularly throughout the tree, but frequently

at the base of young branches. It is a potent unidentified medicinal plant and it is

vernacularly called “Tooth ache tree”. The species has antirheumatic, antispasmodic,

diuretic, odontalgic activities. The bark and roots of the plant along with the fruit are

diaphoretic, stimulant and useful tonic in debilitated conditions of the

stomach and digestive organs. The stem bark of the species have shown the

presence of secondary metabolites such as alkaloids, flavonoids, glycosides,

phenols, sterols, saponins, tannins in higher levels in hydroethanolic and aqueous

extracts and some minerals in the whole plant extract [Narayanasamy and

Ragavan, 2012]. Two benzophenanthrene alkaloids, 8-acetonyldihydronitidine and

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8-acetonyldihydrovicine were isolated from the stem bark along with liriodenine,

sesamin, lichexanthrone and (+) piperitol – γ, γ – dimethyl allyl ether [Nissanka et al.,

2001]. A literature search revealed that there are no reported chemical and biological

studies on woodheart of the plant.


Plant collection

Healthy Z. tetraspermum woodhearts were collected from Kolli Hills,

Western Ghats, Namakkal District, Tamil Nadu, India, identified and authenticated

by Taxonomist, Kandaswami Kandar‟s College, Velur, Namakkal district, Tamil

Nadu, India.

Phytochemical screening and estimation:

The preliminary phytochemical screening tests and estimations of

phytoconstituents were carried out by standard methods as in II-1 and II-2.


Results

Phytochemical screening

The phytochemical screening of Z. tetraspermum woodheart in various

extracts evidenced their antioxidant properties by revealing the presence of

numerous secondary metabolites. The presence of phytochemical constituents

from copious, slightly and moderately present in all extracts has been listed in

Table II-6.

Table II-6. Phytochemical screening of Z. tetraspermum woodheart in various

solvent extracts

S.

No.

Phytochemical

Constituents

Various Extracts

Petroleum ether Diethyl ether Ethyl acetate

1. Alkaloids + + ++


3. Flavones - ++ ++

4. Flavanones - + -

68


6. Isoflavones - - -

7. Glycosides - + +

8. Tannins + +++ +++


10. Amino acids - + -

11. Steroids - - +

12. Proteins - + +

13. Carbohydrates - +++ ++

14. Saponins +++ - +

15. Terpenoids - ++ ++

16. Fats/Oils +++ - -


18. Leucoanthocyannins - - -


Determination of Phytoconstituents




69

Table II-7. Total phenolic content in various solvent extracts (Z. tetraspermum)

Extracted Solvent

Absorption at 725 nm*

(µg / mL of extract)*

Ethyl acetate

0.203 ± 0.003

66.75 ± 1.19

Diethyl ether 0.226 ± 0.002 75.89 ± 0.68

Petroleum ether

0.072 ± 0.002

13.84 ± 0.98



Fig . II-5. Determination of flavonoid content using Catechin as a standard

Table II-8. Total flavonoid content in various solvent extracts (Z. tetraspermum)

Extracted Solvent



Ethyl acetate

0.188 ± 0.001

49.98 ± 0.49

Diethyl ether 0.327 ± 0.004 115.39 ± 1.61

Petroleum ether

0.105 ± 0.001

10.70 ± 0.49


70


Table II-9. Total flavonol content in various solvent extracts (Z. tetraspermum)

Extracted Solvent



Ethyl acetate

0.316 ± 0.005

42.86 ± 0.001

Diethyl ether 0.455 ± 0.009 61.65 ± 0.001

Petroleum ether

0.154 ± 0.007

20.87 ± 0.001



Fig. II-6. Determination of tannin content using tannic acid as a standard

Table II-10. Total tannin content in various solvent extracts (Z. tetraspermum)

Extracted Solvent



Ethyl acetate

0.073 ± 0

74.682 ± 0.572

Diethyl ether 0.124 ± 0.001 144.143 ± 1.334

Petroleum ether

0.013 ± 0

14.421 ± 0.143

71

The phytoconstituents flavonoids, phenolics, carbohydrates and tannins are

significantly present in both diethyl ether and ethyl acetate extracts. Alkaloids are

moderately present in ethyl acetate extract and slightly present in diethyl ether

extract. In petroleum ether extract, saponins and fats / oils are copiously present and

flavonoids, tannins and phenolics are slightly present. Proteins are slightly present in

both diethyl ether and ethyl acetate extracts. Anthocyanins and leucoanthocyanins

are absent in these different solvent extracts.

The estimated results of the major phytochemicals Z. tetraspermum

woodheart in various solvent extracts are given in tables II – 7, II – 8, II – 9 and II –

10 and shown in the figures II – 4, II – 5 and II – 6.


The diethyl ether extract recorded the highest content of about 75.89 µg/mL

of extract. The next highest was observed in ethyl acetate extract and the least was

observed in petroleum ether extract of about 13.84 µg/mL


The diethyl ether extract recorded the highest content of flavonoid (115.39

µg/mL) followed by ethyl acetate extract (49.98 µg/mL) with the least value for

petroleum ether extract (10.70 µg/mL)


The trend continued with diethyl ether extract exhibiting maximum (61.65

µg/mL) followed by ethyl acetate extract (42.86 µg/mL) and petroleum ether extract

has the minimum value (20.87 µg/mL).


Diethyl ether extract recorded the highest value of 144.14 µg/mL. Ethyl

acetate extract recorded intermediate value of 74.68 µg/mL. Petroleum ether

recorded the lowest value of 14.42 µg/mL.

Discussion:

The curative properties of medicinal plants are mainly due to the presence of

various complex chemical substances of different composition which occur as

secondary metabolites [Karthikeyan et al., 2009]. Medicinal plants form a large

72

group of economically important plants that provide the basic raw materials for

indigenous pharmaceuticals [Augusti, 1996]. The medicinal value of plants lies in

some chemical substances that produce a definite physiologic action on the human

body. The phytochemical research based on ethno pharmacological information is

generally considered an effective approach in the discovery of new anti–infective

agents from higher plants [Duraipandiyan et al., 2006].

Knowledge of the chemical constituents of plant is desirable, not only for the

discovery of therapeutic drugs but also because such information may be of value

disclosing new sources of such economic materials as tannins,oils,gums, precursors

or the synthesis of complex chemical substances. In addition, the knowledge of the

chemical constituents of plants would further be valuable in discovering the actual

value of folklore remedies [Mojab et al., 2003].

Phytochemicals which possess many ecological and physiological roles

are widely distributed as plant constituents. Woody plants can synthesize

and accumulate in their cells, a great variety of phytochemicals including

alkaloids, flavonoids, tannins, cyanogenic glycosides, phenolic compounds

[Hill, 1952 and Edeoga et al., 2005], saponins, lignins and lignans [Okwu, 2004].

Phytochemicals exhibit a wide range of biological effects as consequences

of their antioxidant properties. Several types of Polyphenols (Phenolic acid,

Hydrolysable tannins and Flavonoids) show anticarcinogenic, anti mutagenic

effects [Uruguiaga and Leighton, 2000] and antitumour activity [Karthikumar

et al., 2007].

The extractive capability of phenolic components from herb material is

considerably dependent on the type of solvent. Observation from the present

study suggested that the phenolics, flavonoids and tannins are high in diethyl

ether extract as compared to petroleum ether extract. Thus the diethyl ether extract is

more potent in therapeutic value as compared to petroleum ether extract. This

work is in agreement with the finding of Narayanasamy and Ragavan (2012). As

rich source of phytoconstituents, the plant‟s parts can be a potential source of useful

drug.

73

c. Cuscuta reflexa Roxb

Devision : Magnoliophyta

Class : Magnoliopside

Order : Solanales

Family : Convolvulaceae

Genus : Cuscuta L

Species : reflexa Roxb

Vernacular name : Kodiyagundal

It is known in Hindi as Amarbela or Akashbela (sky twinner) and is a

phaneragamic stem parasite and is distributed throughout India.

Stem is thick, branched, glabrous, or slightly pubescent, mostly not

interlaced, succulent, light brown to dark brown.Leaves are persistent, cup-shaped,

fleshy, margin scarious, obovate obtuse, 1 – 2.0 × c. 1.0 mm. Flowers are arranged

in paniculate cymes, ivory white, pedicellate, 5.5 – 7.5 mm long; pedicel 0.5 – 2.0 (–

7) mm long; or obsolete; bracts leaf – like. There are 5 Calyx lobes, fleshy,

margin scarious, rounded ovate – obtuse, deeply divided overlapping at the base, 0.7

– 3.0 × 1.5 – 2.5 mm, tube c. 1 mm long, 5 Corolla lobes, ivory white, obtuse,

upright, spreading or reflexed, 1 – 1.5 × 0.5-0.7 mm; corolla tub, fringed, c. 1mm

long and 5 Stamens, filaments linear, 2 – 3 mm long, fused filaments prominent like

a vein in the petal; anther oblong, basifixed, 1 – 1.5 mm long. Ovary is conical,

fleshy, 1 – 2 mm long; style 1, thick, c. 0.5 mm long or obsolete stigmas 2, unequal,

conical to elongated, dark brown, 0.7 – 1.0 stun long; ovules 4, oblong – elliptical, c.

0.3 × c. 0.2 mm. Capsule is 1 – 3 × 2 – 3 mm, globose – conical, brown, not

depressed, without intrastylar opening, not circumscissile with a definite line of

cleavage, i.e., dehiscing irregularly with the remains of sepal lobes below, style and

stigma at the top.

The herb has a bitter sharp taste. Various parts of this plant are used in tribal

medicine for the diseases such as impotence, premature ejaculation, sperm leakage,

frequent urination, ringing in the ears, lower back pain, sore knees, leucorrbea, dry

eyes, blurred vision and tired eyes [Dandopani Chatterjee et al., 2011].

74

The species has been analyzed for antispasmodic, heamodynamic,

bradycardia [Hassan Gilani and Aftab, 1992], antisteroidogenic [Gupta et al., 2003],

antihypertensive, muscle relaxant, cardio tonic [Singh and Garg, 1973],

psychopharcological [Pal et al., 2003], antiviral and anticonvulsant [Gupta et al.,

2003 a] activities.

Many chemical constituents have been isolated from this species such as,

custin, amarbelin, beta-sterol, stigmasterol, kaempferol, dulcitol, myricetin,

quercetin, coumarin and olenolic acid [Mohammad Ali, 2004].

Phytochemical investigation of Cuscuta reflexa indicates the presence of

kaempferol-3-0-glucoside, astragalliin [Anis et al., 1999], myricetin, benzopyrone

[Kelker et al., 1984], glucopyranosides [Chemesova, 1990], propenamide, flavonols

[Niwa et al., 1988], quercetin and quercetin – 3 – O – glucoside, β – sitosterol and

bergenin [Pacheco, 1966].


Plant Collection

Healthy plants of Cuscuta reflexa were collected from the Kandaswami

Kandar‟s College, Velur, Namakkal District, Tamil Nadu, India and authenticated

by Taxonomist, Kandaswami Kandar‟s College, Velur, Namakkal District, Tamil

Nadu, India.





Results


Evidencing the antioxidant characteristic of C. reflexa plant its different

solvent extracts revealed the presence of many secondary metabolites which is

presented in table II -11.

75

Table II-11. Phytochemical screening of C. reflexa plant in various solvent

extracts

S.No.

Phytochemical

Constituents

Various Extracts


1. Alkaloids + ++ ++


3. Flavones - ++ +

4. Flavanones - ++ ++




8. Tannins ++ +++ +++


10. Aminoacids + + +

11. Steroids + ++ +

12. Proteins + + +

13. Carbohydrates ++ ++ ++

14. Saponins ++ + +

15. Terpenoids + ++ ++

16. Fats/Oils +++ + +


18. Leucoanthocyannins - - -


76

Determination of phytoconstituents



Table II-12. Total phenolic content in various solvent extracts (C. reflexa)

Extracted Solvent Absorption(725 nm)* (µg / mL of extract)*

Ethyl acetate 0.136 ± 0 39.10 ± 0.13

Diethyl ether 0.3 ± 0.001 104.94 ± 0.25

Petroleum ether 0.075 ± 0 15.63 ± 0.42


77



Table II-13 Total flavonoid content in various solvent extracts (C. reflexa)

Extracted Solvent Absorption (510 nm)* (µg / mL of extract)*

Ethyl acetate 0.117 ± 0.001 92.55 ± 2.29

Diethyl ether 0.135 ± 0.001 122.74 ± 1.16

Petroleum ether 0.069 ± 0 12.60 ± 0.95



Table II-14 Total flavonol content in various solvent extracts (C. reflexa)

Extracted Solvent Absorption (440 nm)*


Ethyl acetate 0.481 ± 0.003 65.13 ± 0

Diethyl ether 0.57 ± 0.013 77.28 ± 0.002

Petroleum ether 0.113 ± 0.006 15.31 ± 0.001


78



Table II-15. Total tannin content in various solvent extracts (C. reflexa)

Extracted Solvent Absorption (700 nm)* (µg / mL of extract)*

Ethyl acetate 0.032 ± 0.001 70.218 ± 2.158

Diethyl ether 0.043 ± 0.001 110.2 ± 0.679

Petroleum ether 0.02 ± 0 21.688 ± 0


The results of preliminary phytochemical tests indicate that flavonoids,

tannins and phenolics are highly present in both diethyl ether and ethyl acetate

extracts and alkaloids, carbohydrates, terpenoids are moderately present and amino

acids, proteins, saponins and fats/oils are slightly present in these two extracts.

Tannins, carbohydrates, saponins and fats / oils are significantly present in

petroleum ether extract and alkaloids, flavonoids, phenolics, amino acids, steroids,

proteins and terpenoids are slightly present in it. Glycosides and steroids are

moderately present in diethyl ether extract. Anthocyanins and Leucoanthocyanins

are absent in these three extracts.

79

The quantitative values of the most useful phytoconstituents of C. reflexa

plant extract of various solvents are calculated and shown in the tables II – 12, II –

13, II – 14 and II – 15.

Total Phenolic content:

The diethyl ether extract has the phenolic content of about 104.94 µg/mL and

petroleum ether has the least quantity of 15.63 µg/mL.


The highest value of total flavonoid content is obtained (122.74 µg/mL for

diethyl ether extract, next for ethyl acetate 92.55 µg/mL and the least content 12.60

µg/mL for petroleum ether extract.


The trend is again repeated in the estimation of total flavonol content.

Diethyl ether extract has 77.28 µg/mL, ethyl acetate extract possesses 65.13 µg/mL

and petroleum ether extract has the least value 15.31 µg/mL.


By comparing with tannic acid equivalent, as usual diethyl ether extract

exhibits the highest value of tannin content 110.2 µg/mL, ethyl acetate has the

moderate value 70.218 µg/mL and the least value is for petroleum ether extract,

21.688 µg/mL.

Discussion:

The presence of wide range of phytochemical constituents indicates that the

plant could be used in a multitude of ways which may be beneficiary to the

population [Jayanthi and Christy, 2011]. An important part of natural products from

plants, biomolecules and secondary metabolites usually exhibits some kind of

biological activities. They are widely used in the human therapy, veterinary,

agriculture, scientific research and in countless other areas [Kandukuri et al.,

2009].The usefulness of plant materials medicinally is due to the presence of

bioactive constituents such as alkaloids, tannins, flavonoids and phenolic

compounds. Alkaloids play some metabolic role and control development in living

system. They are also involved in protective function in animals and are used as

80

medicine especially the steroidal alkaloids. Tannins are known to inhibit pathogenic

fungi. Flavonoids are known to inhibit the initiation, promotion and progression of

tumours [Lata and Dubey, 2010].

The flavonoids and phenolic compounds in plants have been reported to

exert multiple biological effects including antioxidant, free radical scavenging

abilities, anti-inflammatory, anticarcinogenic etc., [Asha et al., 2011].

Anthraquinones are considered to be one of the most active agents in metastatic

breast cancer. The antimicrobial activity of the extracts could be explained by the

presence of tannins. The mechanism of action of tannins is based on their ability to

bind proteins thereby inhibiting cell protein synthesis [Brahim et al., 2010].

Triterpenoids are a large class of natural isoprenoids present in higher plants, which

exhibit a wide range of biological activities. Steroids (anabolic steroids) have been

observed to promote nitrogen retention in osteoporosis and in animals with wasting

illness [Jayanthi and Christy, 2011].

In the phytochemical screening of C. reflexa, important bioactive

constituents are significantly present in diethyl ether and ethyl acetate extracts. The

findings are in agreement with Anjana Roy et al., (2013). The total contents of

phenolics, flavonoids, flavonols and tannins are high in ethyl acetate extract of this

species. The presence of the above said phytochemical constituents could account

for the much medicinal properties of the species for the treatement of various

diseases / ailments.

d. Citrullus colocynthis (L) Schred

Devision : Magnoliophyta

Class : Magnoliopside

Order : Cucurbitales

Family : Cucurbitaceae

Genus : Citrullus

Species : Colocynthis

Vernacular name : Pey – komatti

Citrullus colocynthis (L.) Schrad. (Cucurbitaceae), grow in arid areas. It is

annual or perennial (in wild) herbaceous, stems are angular and rough, leaves rough,

81

3 to 7 lobed, 5 to 10 cm long, flowers monoecious, solitary, pedunded, axillary,

corolla 5 – lobed, ovary villous, fruit is a pepo, nearly globular, 4 – 10 cm in

diameter with somewhat elliptical fissures, about size of small orange, green and

yellow variegated becoming yellow when ripe, with hard rind, pulp light in weight,

spongy, easily broken, light yellowish – orange to pale yellow; seeds numerous,

smooth, dark brown to light yellowish orange, borne on parietal placenta.

This plant is well recognized in the traditional medicine and is used by rural

people. The fruits are useful in the treatment of tumors, antipyretic [Sunilkumar et

al., 2008], immunostimulant [Bendjeddou et al., 2003] anti – inflammatory

[Marzouk et al., 2010], antioxidant [Marzouk et al., 2010a] and it is used against

hepatic diseases [Gebhardt, 2003], antidiabetic [Atole et al., 2009], ulcers, urinary

diseases and rheumatism. It is also used to treat arterial hypertension [Ziyyat et al.,

1997]. The dried pulp of Citrullus colocynthis L. is used for constipation, edema,

bacterial infections, cancer [Ghaithi et al., 2004]. Oil from the seed is used for

snake-bites, scorpion-stings and bowel complaints (dysentery, diarrhoea), epilepsy

and also for the growth and blackening of hair. Root is useful in the treatment of

jaundice, ascites, urinary diseases, rheumatism etc, and is given for abdominal

enlargements and in cough and asthmatic attacks of children. A poultice of the root

is useful in the inflammation of breast of nursing mother [Nadkarni, 1954; Dastur,

1962].

The chemical constituents of Citrullus colocynthis previously reported are

docosan – 1 – 01 acetate, 0, 13 – dimethyl – pentadec – 13 – en – 1 – al, 11, 14 –

dimethyl hexadecane 14 – 01 2 – one, 10, 14 – dimethylhexadecane 14, 01 2 – one,

linoleic acid, oleic acid, carbohydrate, amino acid, organic acid, lipid, sterols and

phenols [Ayoub, 1981; Basalah et al., 1985; Habs et al., 1984 and Navot and Zamir,

1986]. Three flavone glucosides, isosaponarin, isovitexin and isoorientin 3' – O –

methyl ether and two cucurbitacin glucosides, 2 – O – β – D – glucopyranosyl

cucurbitacin 1 and 2 – O – β – D – glucopyranosyl cucurbitacin L are isolated and

identified [Abbas Delazar et al., 2006].

82


Plant collection

Healthy Citrullus colocynthis fruits were collected from the bank of river

Cauvery, Kutchipalayam, Namakkal District, Tamil Nadu, India and authenticated

by Taxonomist, Kandaswami Kandar‟s College, Velur, Namakkal District, Tamil

Nadu, India.





Results


Evidencing the medicinal values of C.colocynthis fruits, the different solvent

extracts revealed the presence of many secondary metabolites which is presented in

table II-16.

Table II-16. Phytochemical screening of C.colocynthis fruits in various solvent

extracts

S.No.

Phytochemical

Constituents

Various Extracts


1. Alkaloids - ++ ++

2. Flavonoids + ++ +++

3. Flavones - ++ +

4. Flavanones - + ++

5. Flavonols + ++ ++



8. Tannins + +++ +++

83


10. Aminoacids - + +

11. Steroids + - +

12. Proteins + + +

13. Carbohydrates + + ++

14. Saponins ++ + +

15. Terpenoids + ++ ++

16. Fats/Oils +++ + +



+++ = Copiously present, ++ = Moderately present, + = Slightly present, -=Absent.




84

Table II-17. Total phenolic content in various solvent extracts (C. colocynthis)

Extracted Solvent


Ethyl acetate

79.76 ± 2.42


Petroleum ether

19.46 ± 1.21




Table II-18. Total flavonoid content in various solvent extracts (C. colocynthis)






85


Table II-19. Total flavonol content in various solvent extracts (C. colocynthis)








Table II-20. Total tannin content in various solvent extracts (C.colocynthis)






86

Tannins and phenolics are copiously present, alkaloids, flavonoids,

glycosides, terpenoids and leucoanthocyannins are moderately present and amino

acids, proteins, carbohydrates, saponin and fats/oils are slightly present in diethyl

ether extract.

Flavonoids, tannins and phenolics are copiously present, alkaloids,

carbohydrates, terpenoids and leucoanthocyannins are moderately present and

glycosides, Amino acids, steroids, proteins, saponin and fats/oils are slightly present

in ethyl acetate extract.

In petroleum ether extract, fats/oils are copiously present, saponins are

moderately present and flavonoids, tannins, phenolics, steroids, proteins,

carbohydrates and terpenoids are slightly present. Anthocyanins are completely

absent in these three solvent extracts.

The results of estimation of total phenolic, total flavonoid, total flavonol and

total tannins of various solvent extracts are shown in tables II – 17, II – 18, II – 19

and II – 20.


The ethyl acetate extract has the highest content of about 79.76 µg/mL and

the diethyl ether extract possesses the content of about 63.62 µg/mL. The total

phenolic content has been calculated by using the standard curve of gallic acid

(standard curve equation: r = 0.025 x + 0.0378, R2 = 0.997).


The highest value recorded is 84.19 µg/mL in ethyl acetate and in diethyl

ether extract the content value is 53.46 µg/mL. The petroleum ether extract has the

least value 20.58 µg/mL. Total flavonoid content is calculated using the standard

curve of catechin (standard curve equation; Y = 20x, R2 = 1)


The total flavonol content which is rich in ethyl acetate extract is 58.90

µg/mL of rutin equivalent, whereas the diethyl ether and petroleum ether extracts

record 49.19 µg/mL and 19.33 µg/mL respectively.

87


All the extracts analysed for their tannin content reveal an appreciable record

of about 70.68, 52.99 and 29.77 µg/mL of tannic acid equivalent for ethyl acetate,

diethyl ether and petroleum ether extracts respectively. It can be noted that the

highest content is produced by ethyl acetate extract. Total tannin content is

calculated by using standard curve of tannic acid (standard curve equation:

Y=0.0003x + 0.0112; R2 = 0.9761).

Discussion

The knowledge of the chemical constituents of plants is desirable to

understand herbal drugs and their preparations. Most importantly, these studies will

be helpful to isolate and characterize the chemical constituents present in those plant

extracts. In addition, the knowledge of the chemical constituents of plants would

further be valuable in discovering the actual value of folkloric remedies

[Farnsworth, 1996].

Phenolic compounds and flavonoids are also widely distributed in plants

which have been reported to exert multiple biological effects, including antioxidant,

free radical scavenging abilities, anti-inflammatory, anticarcinogenic etc., [Miller,

1973]. As crude extracts of herbs are rich in phenolics they are of increasing interest

in the food industry because they retard oxidative degradation of lipids and thereby

improve the quality and nutritional value of food. While, flavonoids are a group of

polyphenolic compounds with known properties, which include free radical

scavenging, inhibition of hydrolytic and oxidative enzymes and anti-inflammatory

action [Frankel, 1995].

In this investigation, ethyl acetate extract of fruits of C. colocynthis exerts more

polyphenolic compounds such as flavonoids, tannins etc. This observation is in

agreement with the studies of Jeyanthi and Christy (2011). Prasanth Reddy et al., (2012)

reported that the fruit extracts (aqueous and ethanolic extracts) of C.colocynthis

contained alkaloids, flavonoids, saponins and sterols and confirmed the antiulcerogenic

efficacy of flavonoids present in the extracts. The results reveal that the species

C.colocynthis has efficient pharmaceutical values and behaves as a medicinal herb.

88

e. Argemone mexicana L.,

Division : Magnoliophyta


Order : Papaverales

Family : Papaveraceae

Genus : Argemone L.

Species : mexicana L.

Vernacular name : Ponnummuttai or Brahamadandu

Argemone mexicana L., is a species of poppy found in Mexico and now wide

spread in many tropical and sub – tropical countries. This plant is common

everywhere by roadsides and fields in India [Bhalke and Gosavi, 2009]. The plant

species is informally known as the poppy family; plants under this family are an

ethnopharmacologically important family of 44 genera and approximately 760

species of flowering plants. The plant is an erect prickly annual herb of about 1 m

high; leaves are usually 5 – 11 cm long, and more or less bdlotched with green and

white, glaucous broad at the base, half-lobed and spiny [Chopra et al., 1956]. The

flowers become 4 to 5 cm in diameter and are terminal, yellow and scentless. The

capsule is spiny, obovate of elliptic-onlong, and about 3 cm in length. The seeds are

spherical, shining, black and pitted.

Argemone mexicana L. is widely known as a medicinal plant all over the

World. In India, decoctions of the leaves are used for the treatment of bacterial

infections [Indrenil et al., 2006]. The yellow juice from the injured plant has been

used in India as traditional medicine for dropsy, jaundice, ophthalmia, scabies and

cutaneous affections [Chopra et al., 1956; Ambasta, 1986 and Sharma et al., 2012].

Various parts of this plant are used in chronic skin diseases, also as emetic,

expectorant, demulcent and diureric; the seeds and seed oil are employed as a

remedy for dysentery, ulcers, asthma and other intestinal infections [Bose et al.,

1963; Ambasta, 1986; Prajapati et al., 2003 and Savithramma et al., 2007]. Leaves

and seeds are also reported to find application in maintaining normal blood

circulation and cholesterol level in human body [Albuquerque et al., 2007]. These

plant parts also have anti-venom property [Makhija and Kharmar, 2010; Minu et al.,

89

2012]. Flowers are found to be expectorant and have been used in the treatment of

coughs [Brahmachare et al., 2010]. Seeds of the plant are used as purgative, laxative

and digestive while its latex is used against conjunctivitis [Agra et al., 2008]. The

pharmacological properties of the plant are anti-malarial activities [Willcox et al.,

2007] antimicrobial and anti-inflammatory activities [Sourabie et al., 2012], muscle

relaxant, antifungal, antioxidant, anti – helminthic and wound healing activities etc.,

[Lekhya et al., 2012].

The bioactive secondary metabolites such as saponins, tannins, glycosides,

anthracenosides and isoquinoline alkaloids type sanguninarin, dihydrosanguninarin,

berberin, protopin etc., have been repoted to be present in the plant [Bose et al.,

1963; Harbone and Williams, 1983; Upreti et al., 1989].

Materials and Methods

Plant collection

Healthy leaves of Argemone mexicana were collected from Mohanur,

Namakkal District, Tamil Nadu, India and were identified and authenticated by

Taxonomist, Kandaswami Kandar‟s College, Velur, Namakkal district, Tamil Nadu,

India.




Results and Discussion

Results


Evidencing the bioactive potential of A.mexicana leaf, the different solvent

extracts revealed the presence of many secondary metabolites which is presented in

table II-21.

90

Table II-21. Phytochemical screening of A.mexicana leaf in various solvent

extracts

S.No.

Phytochemical

Constituents

Various Extracts


1. Alkaloids + ++ ++


3. Flavones - ++ +

4. Flavanones - ++ ++




8. Tannins ++ +++ +++


10. Aminoacids + + +

11. Steroids + ++ +

12. Proteins + + +

13. Carbohydrates ++ ++ ++

14. Saponins ++ + +

15. Terpenoids + +++ ++

16. Fats/Oils +++ + +




91




Table II-22. Total phenolic content in various solvent extracts (A. Mexicana)

Extracted Solvent Absorption(725 nm)* (µg/mL of extract)*

Ethyl acetate 0.235 ± 0.003 79.30 ± 1.21

Diethyl ether 0.277 ± 0.004 95.92 ± 1.69

Petroleum ether 0.077 ± 0.003 16.22 ± 1.26


92



Table II-23. Total flavonoid content in various solvent extracts (A. Mexicana)

Extracted Solvent

Absorption (510 nm)*

(µg/mL of extract)*

Ethyl acetate 0.101 ± 0.002 65.25 ± 3.50

Diethyl ether 0.148 ± 0.002 143.83 ± 3.32

Petroleum ether 0.072 ± 0.001 17.83 ± 1.60



Table II-24. Total flavonol content in various solvent extracts (A. Mexicana)

Extracted Solvent Absorption (440 nm)* (µg/mL of extract)*

Ethyl acetate 0.443 ± 0.009 60.03 ± 0.001

Diethyl ether 0.530 ± 0.008 71.77 ± 0.001

Petroleum ether 0.129 ± 0.008 17.52 ± 0.001


93



Table II-25. Total tannin content in various solvent extracts (A.Mexicana)

Extracted Solvent Absorption (700 nm)* (µg/mL of extract)*

Ethyl acetate 0.049 ± 0.001 134.79 ± 3.27

Diethyl ether 0.058 ± 0 161.98 ± 2.01

Petroleum ether 0.02 ± 0 22.75 ± 1.508


The phytochemical screening results of leaves of A. Mexicana reveal that

alkaloids, flavonoids, tannins, phenolics, terpenoids and leucoanthocyanins are

highly present in diethyl ether extract and ethylacetate extracts. Carbohydrates are

moderately present, aminoacids and proteins are slightly present in all the three

extracts. Fats and oils are significantly present in petroleum ether extract slightly

present in diethyl ether and ethyl acetate extracts. Saponins are moderately present

in petroleum ether extract and slightly present in diethyl ether and ethylacetate

extracts. Anthocyanins are totally absent in all the solvent extracts. Due to the high

contents of bioactive secondary metabolites such as alkaloids, flavonoids, tannins,

phenolics etc., A. mexicana is used as a medicinal plant.

94

In the quantitative estimation of the major phytoconstituents, total phenolic

content, total flavonoid content, total flavonol content and total tannin content

exhibit the therapeutic values of the plant species.


The diethyl ether extract has shown a higher content of about 95.92 µg/mL

than ethylacetate extract 79.30 µg/mL and petroleum ether 16.22 µg/mL.


The diethyl ether extract has the highest content value 143.83 µg/mL and the

next highest content value is exhibited by ethylacetate extract (65.25µg/mL). The

least value (17.83µg/mL) is obtained for petroleum ether extract.


Both diethyl ether extract and ethylacetate extract have significant contents

of about 71.77 µg/mL and 60.03 µg/mL respectively. Petroleum ether extract has the

least value (17.52 µg/mL)


The highest value 161.98 µg/mL is observed in diethyl ether extract and

134.74 µg/mL for ethyl acetate extract. Again petroleum ether extract has the lowest

value 22.75 µg/mL.

Discussion:

Medicinal plants contain several active principles with specific therapeutic

effects. They represent a source of chemical compounds such as tannins, flavonoids,

saponins, resins and alkaloids with curative properties, often not provided by synthetic

chemical compounds [Fabricant and Farnsworth, 2001]. The diethyl ether and ethyl

acetate extracts of A. mexicana leaves possess dominantly medicinally valued secondary

metabolites like alkaloids, flavonoids, phenolics, tannins, terpenoids etc.

The diethyl ether and ethylacetate extracts of the plant show the highest

contents of total phenolic, flavonoid and tannins compounds. Previous literatures

provide information about the antioxidant efficacy of phenolic, flavonoid and tannin

compounds [Kathirvel and Sujatha, 2012; Jain et al., 2012]. The crude extracts of

95

herbs rich in phenolics are of increasing interest in the food industry because they

retard oxidative degradation of lipids and thereby improve the quality and nutritional

value of food. While, flavonoids are a group of polyphenolic compounds with

known properties, which include free radical scavenging and inhibition of hydrolytic

and oxidative enzymes [Patel et al., 2009].

Alkaloids are organic compounds that contain nitrogen and are physiologically

active with sedative and analgesic properties. They are used in relieving pains, anxiety

and depression. Alkaloids are toxic due to their stimulatory effects, leading to excitation

of cells and neurological dysfunction [Jisika et al., 1992].

Glycosides are compounds containing a carbohydrate residue (moiety) in the

same molecule. In these compounds, the carbohydrate moiety is attached by an

acetal linkage of C – 1 to the non – carbohydrate residue (aglycone). They mostly

contain steroid as aglycone component in combination with sugar molecules. They

are important in medicine because of their action on heart and are used in cardiac

insufficiency [Balch and Balch, 2000]. Thus, cardiac glycosides are drugs and can

be used in the treatment of congestive heart failure and cardiac arrhythmia.

Tannins and saponins are responsible for the antibacterial activity of the

plant seed extracts [Gloor, 1997]. Saponins are used in veterinary vaccines as

adjuvant (e.g. foot – and – mouth disease vaccines) helping to enhance immune

response. They are also mild detergents and can be used commercially as well as for

research. Also, because of its ability to permeate cells without destroying cell

morphology, it is used in laboratory applications to treat live cells in order to

facilitate peptide or reagents such as antibodies entering cells instead of the

detergents [Balch and Balch, 2000].

Photochemical screening of this study revealed many important chemical

constituents such as alkaloids, phenols, flavonoids, tannins, saponins and

carbohydrates which are present in A.mexicana leaf extracts of various solvents. The

generated data are in agreement with the earlier findings [Sourabie et al., 2012;

Vaghasiya et al., 2011; Kumar and Pradeep, 2011]. In tune with its pharmacological

potency as anti-inflammatory, antiallergenic and antipyretic activities the plant is

used to cure several illnesses such as jaundice, malaria, bacterial gastro-enteritis etc.,

So, A. mexicana occupies an important place in medicinal world.

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