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CHAPTER – 4

Preliminary phytochemical analysis of

in vitro and in vivo extracts

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INTRODUCTION:

Historically, plants have provided novel drug compounds, as plant derived

medicines contribution to human health. These plants often exhibit a wide range of

biological and pharmacological activities, such as anti-inflammatory, anti-bacterial,

antifungal and insecticidal properties (Okwute and Yakubu, 1998; Okwu and Ekeke,

2003). Extracts from the roots, bark, seeds and fruits of these medicinal plants are

used in the preparation of syrups and infusions in traditional medicine as cough

suppressant and in the treatment of liver cirrhosis and hepatitis (Ogu and Agu, 1995).

It is generally assumed that the active constituents contribute to these protective

effects are the phytochemicals (Okwu and Ekeke, 2003). Phytochemicals are present

in a variety of plants utilized as important components of both human and animal

diets. These include fruits, seeds, herbs and vegetables. Diets containing abundance of

fruits and vegetables are protective against a variety of diseases, particularly

cardiovascular diseases (Uruquiaga and Leghton, 2000).

Medicinal plants contain some organic compounds which provide definite

physiological action on the human body and these bioactive substances include

tannins, alkaloids, carbohydrates, terpenoids, steroids and flavonoids (Edoga, et

al.2005).These compounds are synthesized by primary or rather secondary

metabolism of living organisms. Secondary metabolites are chemically extremely

diverse compounds with obscure function. They are widely used in the human

therapy, veterinary, agriculture, scientific research and countless other areas (Vasu et

al.2009).A large number of phytochemicals belonging to several chemical classes

have been shown to have inhibitory effects on all types of microorganisms

(Cowan,1999).

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Plant products have been part of phytomedicines since time immemorial. This

can be derived from barks, leaves, flowers, roots, fruits, seeds (Criagg and David, 2001).

Knowledge of the chemical constituents of plants is desirable because such

information will be of value for the synthesis of complex chemical substances (Parekh

and Chanda, 2008) .Phytochemicals are major source of dyes, flavors, sweeteners,

aromas, perfumes, insecticides, antiparasitic drugs and many more substances. Further

research on plants will provide, apart from drugs, additional sources of industrial raw

materials. All these potentials justify the broadest and most exhaustive phytochemical

research.

Phytochemicals exhibit a wide range of biological effects. Several types of

polyphenols (phenolic acid, hydrolysable tannins and flavonoids) show anti-

carcinogenic and anti-mutagenic effects, which is of major importance in present

scenario (Uruquiaga and Leighton, 2000). Polyphenols might interfere with several of

the steps that lead to the development of malignant tumors, inactivating carcinogens,

inhibiting the expression of mutagens. However several studies have shown that in

addition to their antioxidant protective effects, polyphenols, particularly flavonoids

inhibit the initiation, promotion and progression of tumors (Salah et al., 1995;

Uruquiaga and Leighton, 2000 and Okwu, 2004). In recent times, plant flavonoids

have attracted attention as potentially importantly dietary cancer chemo-protective

agents. In addition, the possible anti-tumor action of certain flavonoids has also

generated interest (Kandaswamy et al., 1991; Elangovan et al., 1994 and Okwu and

Okwu, 2004).

Phytochemical information of medicinal plants form a basis for chemical

analysis, followed by in vitro chemical and clinical studies. Almost every species of

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medicinal plant contains more than one active compound and it is necessary to know

the composition before other studies are being undertaken. Phytochemical study helps

in discovering alternative source of therapeutic chemicals of importance. For example

camptothecin, the anticancer drug was originally discovered in the Chinese species

Camptotheca acuminata for which it was later discovered in an Indian alternative

Nothapodytes foetida (Rao, 2000).Certain classes of chemical compounds have been

diverse effects in different therapeutic contents. A phytochemical survey would

provide information on the distribution of these compounds in different species to

offer a wide choice of materials (Roja & Rao, 2000).

In Ayurvedic system, different parts of the plant are utilized in the form of

crude extracts. Care is never taken to store intact plants with characteristic flowers

and fruits. Many a time these crude drugs are collected and stored for very long

periods. Long storage leads to deterioration of the physico-chemical characteristics.

Sometimes controversies with regard to the origin of botanical sources of the same

drug arise, this makes it imperative to furnish phytochemical parameters which helps

in standardization as well as differentiation of Ayurvedic drugs besides providing

adequate data for laying pharmacopoeia standards pertaining to Ayurvedic drugs

(Bhutani,2000) .

Plant chemical constituents may be therapeutically active or inactive. The ones

which are active are called active constituents and the inactive ones are called inert

chemical constituents (Iyengar, 1995). Many higher plants accumulate extractable

organic substances in quantities sufficient to be economically useful as chemical feed

stock or raw materials for various scientific technological and commercial

applications (Balandrin et al., 1985). It has been estimated that only 5 to 15% of the 2,

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170

50, 000 to 7, 50,000 existing species of higher plants has been surveyed for

biologically active compounds (Farnsworth and Morris, 1976; Balandrin et al., 1993;

Verpoorte, 2000). Among the estimated 3, 50,000 plant species on the earth, only a

small percentage has been phytochemically investigated. The plant kingdom thus

represents an enormous reservoir of pharmacologically valuable molecules to be

discovered (Hostettmann et al., 2000).

The chemical diversity of plants is greater than any chemical library made by

humans and thus the plant kingdom represents an enormous reservoir of

pharmacologically valuable substances waiting to be discovered (Oksman-Caldentey

and Inzé, 2004). In modern pharmacy, about 25% of drugs still contain active

compounds from natural sources, which are primarily isolated from plants (Oksman-

Caldentey and Hiltunen, 1996).During the screening of plants for secondary

metabolites and for their activities only one activity will be considered. Thus it

appears that the plant kingdom has received little attention as a resource of potentially

useful bioactive compounds. Many secondary metabolites are genus or species

specific, the chances are good to excellent that many plant constituents with

potentially useful biological properties remain undiscovered, undeveloped and unused

(Balandrin et al. 1985).

The occurrences of active principles in plants are generally very low and

isolation of these compounds becomes uneconomical. Most of these plants are slow

growing and the accumulation pattern of active substances show significant variation

among and within population and species (Wheeler et al. 1992).

Rapid progress has been made possible to obtain specific and valuable

medicinal substances of plant origin from plant specific and valuable medicinal

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substances of plant origin from plant tissue culture. Tropane alkaloids have been

reported from tissue culture of Datura species (Khanna & Nag, 1972). Chopra et al.

(1923) investigated the pharmacology and therapeutics of Boerhaavia diffusa.

Buchner and Staba (1964) did a preliminary phytochemical analysis of tissue culture

of Digitalis species. Zenk et al. (1977) gave the formulation for alkaloid production

by manipulation of media and by refinement of culture conditions.

REVIEW OF LITERATURE:

Tissue culture protocol has been extensively used for the in vitro propagation,

germplasm conservation, and production of pharmaceutically important bioactive

compounds. Genetically homogenous plants with uniform contents of secondary

metabolites can be obtained by in vitro propagation of plants or shoot organogenesis.

Many reviews available on the studies of secondary metabolites produced in in

vitro regenerated plants or in regenerated shoots and also in callus culture

(Ravishankar and Ramachandra Rao, 2000; Nalawade et al., 2004; Mulabagal and

Tsay, 2004; Sarin, 2005).

Lee et al. (2001) reported the formation of protoberberine type alkaloids in the

tubers of somatic embryo derived plants of Cordylis yanhusuo. They have compared

the potential of obtaining pharmaceutically important metabolites such as D, L-

tetrahydropalmatine and D-cordaline from the tubers of somatic embryo derived

plants. Kulkarni and Decodhar (2002) have reported the hydroxycitric acid production

in both callus and in vitro regenerated shoots and it was detected by HPLC method in

Garcinia indica.

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Casado et al. (2002) achieved the micropropagation of Santolina canescens

and compared the in vitro volatile production in shoot explants of regenerated plants.

The composition of oil obtained from the shoots, oil yield and qualitative and

quantitative changes in oil composition of the plantlets was tested. Levieille and

Wilson (2002) have analysed iridoid in 18th

month old plants of Harpagophytum

species. Analysis of the tuber tissue of the micropropagated plants showed the

presence of iridoids, harpagoside and hapagide and concentration compared with

those found in the wild material. Santarem and Astarita (2003) compared hypericin

content between the in vitro and field grown plants of Hypericum perforatum. Leaves

and shoots showed similar concentration of hypericin of field grown plants.

Yadav and Agarwala (2011) investigate the phytochemicals like proteins,

carbohydrates, phenols, tannins, flavonoids, saponins, in seven medicinal plants of

North-eastern India such as Bryophyllum pinnatum, Ipomea aquatica, Oldenlandia

corymbosa, Ricinus communis, Terminalia bellerica, Tinospora cordifolia and

Xanthium strumarium. Eman and Alam (2012) screened the calli of plants like

Fagonia indica and Fagonia bruguieri and revealed a variation in the presence/

amount of carbohydrates and / or glycosides, saponins, tannins, unsaturated sterols

and/or triterpenoids, alkaloids, cardiac glycosides, cyanogenic glycosides, flavonoids,

coumarins , chlorides and sulphates.

The preliminary phytochemical analysis of Centella asiatica leaf and callus

extracts showed the presence of alkaloids, glycosides, terpenoids, steroids, flavonoids,

tannins, saponins and reducing sugars (Arumugham et al.2011). The medicinal plants

like Camellia sinensis, Glycyrrhiza glabra and Calendula officinalis which are used

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in the treatment of acne were phytochemically screened by Nand et al., (2012) and

reported the presence of various phytoconstituents.

Savithramma et al (2011) carried out the study of phytochemical screening of

nearly eighteen important medicinal Plants which are used to cure various ailments in

different parts of country. Qualitative phytochemical analysis of these plants confirm

the presence of various phytochemicals like saponins, terpenoids, steroids,

anthocyanins, coumarins, fatty acids, tannins, leucoanthocyanins and emodins.

The biosynthetic ability of callus to produce secondary metabolites present in

wild medicinal plants have been reported in many callus cultures of medicinal plants

and callus culture also has been used to produce different types of secondary

metabolites such as alkaloids, terpenoids, steroids, sterols and flavonoids. When

valuable product is found in a wild or scarce plant species, intensive cell culture is

practical alternative to wild collection of fruits or other plant material. The

protoberine alkaloids including berberine formation or production in in vitro

regenerated shoots or callus culture has been reported in Coptis japonica (Furuya et

al., 1972) Thalictrum minus (Ikuta and Itokawa, 1982) Berberis sp., (Cassels et al.,

1987) Tinospora cordifolia (Chintalwar et al., 2003) Thalictrum flavum ( Samanani et

al., 2002).

In vitro culture techniques have been proven to be useful tool for the

production of tropane alkaloid. Lai (2003) had proven that tropane alkaloids could be

obtained via micropropagated plantlets and the root cultures of Hyoscyamus niger.

Lin et al (2003) analyzed the content of anthraquinones, emodinn and physcion in, in

vitro grown shoots and in vitro propagated plants of Polygonum multiflorum. Analysis

revealed that the contents of the major medicinal compounds emodin and physcion in

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the 6-week old in vitro grown shoots and 3-month old in vitro propagated plants

grown in greenhouse higher than those of the marketed crude drugs.

Ravishankar and Grewal (1991) indicted the importance of media constituents

and the stress influenced by nutrients in the production of diosgenin from callus

culture of Dioscorea deltoidea. Parisi et al. (2002) reported high yield of proteolytic

enzymes from the callus tissue culture of Allium sativum L. on MS medium

supplemented with NAA and BAP. Pradel et al. (1997) achieved maximum amount of

biosynthesis of cardenolides from hairy root cultures than leaf culture of Digitalis

lanata and also it was reported that the production of azadirachtin and nimbin has

been shown to be higher in cultured shoots and roots of Azadirachta indica compared

to field grown plant (Prakash et al. 2002). Pande et al. (2002) reported that the yield

of lepidine from Lepidium sativum Linn depends upon the source and type of

explants.

A novel alkaloid dichlorocetumine was reported from Menispermum dauricum

cultured roots by Sugimota et al., (1998). Highest amount of catechin ever produced

in vitro has been reported in Polygonum hydropiper cell cultures (Ono et al., 1998).

Podophyllotoxin content in Juniperus chinensis callus cultures was shown to be twice

the amount present in intact plant (Muranaka et al., 1998). The production increased

fifteen-fold by the addition of chito-oligosaccharides an elicitor to the calli. Eleven-

fold increase was observed on addition of phenylalanine precursor.

A preliminary phytochemical constituent of both in vivo and in vitro extracts

of T.involucrata and O.secamone were analysed.

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Materials and Methods:

The plant material was cut into small pieces, air dried and powdered

separately. The calli proliferated on the induction medium was stored at 4 ⁰C in a

refrigerator for 2-3 weeks when the biosynthetic process will be at stand still. Then

the callus was brought to room temperature and further dried at 60⁰C over night.

Dried callus was powdered and calculated quantity of powder was weighed and

subjected to extraction.

The extraction of in vivo and in vitro was carried out by following methods:

1. Aqueous extraction

2. Soxhlet extraction

3. Prollius fluid extraction

1. Aqueous extraction:

Leaf extract: About 25gms of leave powder was macerated with 50ml of sterile

distilled water in a blender for 10-15 mins. The homogenized extract was filtered

through a double layer of muslin cloth and the extract was collected (Gupta et

al.1996)

Callus extract: In the same way 25 gms of leaf calli obtained on the proliferating

MS medium supplemented with individual hormones were harvested at their

maximum growth indices. The calli was dried at 60⁰C over night in oven. The dried

calli were powdered. To this 50 ml of sterli distilled water was added and

homogenized in a blender for 10-15 mins. The extract was collected by filtering the

solution through a muslin cloth.

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The extracts were collected, concentrated and preserved at 5°C in refrigerator for

further analysis.

2. Soxhlet extraction:

About 25 gms of leaf and leaf calli powder was filled in a thimble separately.

The individual thimble was placed in extractor region of Soxhlet apparatus and

subjected to extraction with 100ml of petroleum ether, chloroform, acetone and

methanol successively up to 48 hrs in each solvent.

Each of solvent extract was concentrated separately using rotary evaporator

and preserved at 5°C extracts were collected and concentrated and preserved at 10°C

in refrigerator for further phytochemical analysis.

3. Prollius fluid extract:

Twenty five grams of finely powdered leaf and leaf calli was digested for 24

hrs in Prollius fluid (ether: chloroform: alcohol: ammonia solution in 25:8:2.5:1.0)

with occasional shaking. The fluid was then completely drained into a separating

funnel, the aqueous layer was run off, and the mixed solvent was extracted with 1%

HCl to remove any alkaloid present. Test for alkaloids was done by means of one of

the normally used reagents.

I. Extractive value determination experiments: (Anonymous, 2002)

1. Alcohol soluble extractive values: Five gram, each of dried plant material and

callus powder were macerated in 100ml alcohol (90%) in a closed flask for 24 hours

with frequent shaking. The extraction was allowed to stand for 18 hours. The extract

obtained was filtered rapidly to avoid loss of alcohol. 25ml of filtrate was evaporated

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in tarred flat bottom shallow dish, dried at 105 C and weighed. The percentage of

alcohol soluble extract was calculated with reference to shade dried plant and callus

material.

2. Water soluble extractive values: The above described procedure was followed

except when chloroform-water was used in place of alcohol for extraction.

II. Ash values determination: (Anonymous, 2002)

Quality and purity of crude powdered materials can be determined by use of ash

values.

1. Total ash: Three gram each of powdered plant material and callus were taken in a

previously ignited and weighed silica crucible. The powdered material was evenly

spread at the bottom of tarred crucible. Gradual incineration of the crucible was

carried to make it dull red hot to free it from carbon. It was allowed to cool in a

desiccator and weighed. This was repeated until constant weight was obtained. The

percentage of total sash was calculated.

2. Acid insoluble ash%: The ash obtained as outlined above was boiled with 25ml of

2N HCL for 3 minutes. Insoluble ash was collected on ash less filter paper and

washed with hot water. The insoluble ash matter was transferred to tarred silica

crucible, which was ignited and weighed as described above. The percentage of acid

insoluble ash was calculated with reference to air dried material.

3. Water soluble ash%: The total ash was boiled with 25ml of water for 5 minutes.

Insoluble material was collected on ash less filter paper, washed with hot water and

ignited for 15 minutes (<450◦C). The weight of insoluble material was subtracted

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from the weight of ash. Difference represents the water soluble ash. The percentage of

water soluble ash was calculated with respect to air dried material.

III. Detection of Chemical Components: (Harborne

The extracts obtained above were tested for the following phytochemicals using

the methods mentioned below.

1. Alkaloids: Small amount of extract was dissolved with few drops of dilute HCl

and filtered. Following reagents were used to detect the presence of alkaloids.

a. Mayer’s reagent (potassium mercuric iodide): Filtrates were treated with this

reagent formation of cream colour precipitate indicated the presence of alkaloids.

b. Dragendorff’s reagent: When treated with the filtrate, results in the formation of

reddish brown precipitate indicate presence of alkaloids.

2. Carbohydrates: Small amounts of extracts were taken in 5 ml of distilled water

and filtered. Filtrates were used for various tests.

a. Molish’stest: Filtrate(2ml) was treated with 2 drops of 5% ethanol solution of

alpha- naphthal and few drops of H2SO4 was added through the sides of the test

tube. Formation of coloured ring (violet) at the junction of the two liquids indicates

the presence of carbohydrates.

3. Glycosides: Extracts were hydrolyzed using dilute Hcl for few hours in a water

bath and hydrolysed extracts were subjected to various glycosidal tests.

a. Libermann-Burchard’s test: To the hydrolysate few drops of acetic anhydride

was added,soluution was boiled and cooled. Few drops of conc.H2SO4 was added

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179

through the sides of the test tube. Formation of a brown ring at lower layer and green

colour at upper layer indicated the presence of steroids.

b. Ligal’s test: The hydrolysate was treated with nitropruside in pyridine and

methanolic alkali. Formation of a reddish colour is a positive test of ketones.

4. Sugars: Extracts taken in water were also tested for the presence of different

sugars.

a. Fehling’s test: To 2ml of Fehling A and B solution few drops of test solution was

added and boiled for few minutes. Formation of rusty brown colour indicated

presence of reducing sugars.

b. Benedicts test: The filtrates were treated with 2 ml of Benedicts reagent boiled in

water bath. Orange red precipitate formed in a minute indicated the presence of

reducing sugars.

5. Detection of Phytosterols: Aqueous and methanolic extract were refluxed

separately with alcoholic KOH, till saponification. Then the saponified mixture was

diluted with dilute water and extracted with ether. The extracts obtained on

evoparation, were subjected to sterol test by Libermann-Burchard’s test as described

earlier

6. Phenolics and tannins: Alcoholic and aqueous extracts were taken and tested

separately for the phenolics compounds and tannins.

a. FeCl3 test: To test solution few drops of neutral FeCl3 solution was added.

Formation of bluish black colour indicated the presence of phenolics.

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b. Gelatin test: To test solution 1% gelatin containing NaCl solution was added.

White precipitate formation indicates the presence of tannins.

7. Saponins: To the alcoholic and aqueous extracts,1ml of alcohol was added and

diluted with water to get 20ml of solution . The mixture was shaken for 15mins. A

froth formation in upper layer indicated the presence of saponin.

Preparation TLC plates for monitoring purpose:

Glass plates (5x20 cm) were coated with a uniform layer (0.5mm) of slurry of

silica gel G prepared in distilled water using an applicator. After initial drying, the

plates were activated in a hot air oven at 110⁰C for 30 min. Samples were applied to

the adsorbent surface at about 2 cm from the edge using a capillary tube and

developed in glass chambers (6x25 cm) preciously saturated with the vapors of the

respective solvent system .

The solvent systems like Butanol: Acetic acid: Water (4:1:5); Chloroform:

Acetone (7:1); Chloroform: Methanol (9:1); Chloroform: Ethanol (9:1); Chloroform:

Acetic acid (7:1) were the solvent systems used. The plate was removed when the

solvent travelled four fifth of the length of the adsorbent on the plate. Visualization

was done initially with iodine vapors and observed for any coloration developed and

sprayed with suitable chromatographic reagent. Again the plates are activated at 100

C for 10 min and observed for any color development, finally visualized under UV

light (366-254 nm). Three replicates of each sample were examined and mean Rf

values were taken (Harborne. 1998).

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Characterization of Methanol extract of T.involucrata

Methanol extract of both aqueous and callus samples were dissolved in

methanol and were subjected to HPLC (model LC-6A, Shimadzu) analysis on a

SphereClone 5µ ODS 2 column (4.6 mm x 150 mm, Phenomenox) using UV-

detection system. Chromatographic run was done with mobile phase consisting of an

isocratic solvent mixture of water: acetic acid: methanol (80: 5: 15 v/v/v) with a flow

rate of 1 mL/min the chromatograms were monitored at 280 nm (Sureshkumar et al.

2006). The standard phenolic acids dissolved in methanol (1 mg/mL) were also run on

the same HPLC column under similar conditions. The retention time of the samples

were compared with that of standard phenolic acids to identify the respective

phenolics.

RESULTS:

Preliminary phytochemical analysis with respect to the purity of crude

powdered materials of both plants is being presented in table 4.1 and 4.2.

Preliminary phytochemical analysis of T.involucrata leaf and callus extracts

was shown in Table 4.3 It showed varied types of phytochemicals in different

extracts. The petroleum ether extract of both leaf and callus gave positive result for

phenolics and tannins only. Chloroform extract reveals the presence of phytosterols

and sugars. But the acetone and methanol extracts showed the presence of alkaloids,

Glycosides, Carbohydrate Saponins, Phytosterols, Phenolic and tannins. Both the

solvents gave negative result for sugars.

Aqueous extract when subjected to standard testing method showed positive

result in all the methods. This revealed the presence of tested phytochemicals such as

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alkaloids, glycosides, Carbohydrates, saponins, phyosterols,phenolics and tannins and

Sugars.

Testing of Prollius’s fluid extract revealed the presence of alkaloids,

carbohydrates, Saponins and phyosterols.

The various extracts of O.secamone when subjected to preliminary

phytochemical analysis showed the presence of various compounds. The results are

presented in table 4.4.

Analysis of aqueous extract of both leaf and callus revealed the presence of

glycosides, carbohydrates, phyosterols, phenolics and tannins and sugars. While the

Prollius fluid extract gave positive result for glycosides, carbohydrates, phyosterols,

phenolics and tannins only.

Different solvent extracts showed varied results. Petroleum ether was positive

for only Saponins but Chloroform extracts revealed presence of phyosterols and

sugars. The other two extracts like acetone and methanol indicate the presence of all

tested compounds except saponins and sugars.

TLC for alkaloids, steroids, and phenol compounds:

Methanol and acetone extracts of both leaf and leaf calli of Tragia involucrata

was run with different solvent systems.

After running of compounds on plates, the plates were sprayed with

Dragendorff’s reagent for Alkaloids, Libermann-Burchard’s reagents for steroids and

FeCl3 (2% in ethanol) for phenolics. After spraying the plates were activated at 100⁰

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183

C for 10 mins. When colour develops Rf values were recorded and presented in

table 4.5.

All the plates revealed colour of pink, and violet which indicate the presence

of alkaloids, and steroids. The plates concentrated in acetone and methanol extract

showed blue coloured spot which indicates presence of phenolics besides alkaloids

and steroids.

The HPLC analysis of methanol extract of T.involucrata leaf and calli

revealed the presence of different phenolic compounds. The results indicated presence

of same compound in both in vivo and in vitro extracts.

The peak graph of HPLC analysis clearly reveals the presence of phenolics

like Gallic acid, Chlorogenic acid, Protocatechic acid and vanillic acid (Graph.3.1) in

both leaf extract (in vivo) and leaf callus extracts (in vitro). Ferullic acid was found in

tracer quantity in leaf extract, whereas in callus extract (in vitro) prominent peak was

observed when compared to in vivo extract (Graph.3.2).

DISCUSSION:

The plant kingdom has proved to be the most useful in the treatment of

diseases and they provide an important source of all the world’s pharmaceuticals. The

most important of these bioactive constituents of plants are steroids, terpenoids,

carotenoids, flavanoids, alkaloids, tannins and glycosides. Plants in all facet of life

have served a valuable starting material for drug development (Edeoga et al. 2005).

For the pharmacological as well as pathological discovery of novel drugs, the

essential information regarding the chemical constituents are generally provided by

the qualitative phytochemical screening of plant extracts.

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184

The result of present study revealed that the selected plants possess almost all

important bioactive compounds. The presences of these phytochemicals are the basis

for their recognition as medicinal plant.

The presence of alkaloids, saponins and tannins in the plants play a major role

in pharmaceutical field because Kubmarawa et al., (2007) and Mensah et al., (2008)

reported the importance of these compounds in various antibiotics, used in treating

common pathogenic strains. It should be noted that steroidal compounds are of

importance and interest in pharmacy due to their relationship with such compounds as

sex hormones (Edeoga et al., 2005; Okwu. 2001). Steroids have been reported to

possess anti-inflammatory activities (Chawla. 1987).

The second important phytochemical obtained was glycosides which have

been long used as cardio tonic, also in nephrological diseases. They have also been

shown to be useful in managing infections. Cardiac glycosides are cardioactive

compounds belonging to triterpenoids class of compounds. Their inherent activity

resides in the aglycone portions of their sugar attachment. Their clinical effects in

cases of congestive heart failure are to increase the force of myocardiac contraction

(Brian et al., 1985).

The presence of phenols in both in vivo and in vitro extracts indicates the

antimicrobial nature of plants. Because Phenols are considered as antioxidants and

antibacterial compounds. Many plant phenols exhibit antibacterial properties (Kefeli

et al., 2003) and a relatively fair correlation between phenolic content of plant

extracts and their antioxidant activities has been also reported (Kaur and Kapoor,

2002; Ivanova et al., 2005; Farrukh et al., 2006). These compounds have significant

application against human pathogens, including those that cause enteric infections and

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185

are reported to have curative properties against several pathogens and therefore could

suggest their use in the treatment of various diseases (Hassan et al., 2004). In general,

the total phenolic compounds found in the leaf, root and petiole are the major

contributions to the antioxidant activities of the plant (Zainol et al., 2003). In support

to our present antimicrobial study, also reveals the antimicrobial property of plant and

callus extracts.

Tannins are inhibitory to fungi, yeasts and bacteria however very few studies

have been carried out with purified tannins of known molecular structures (Scalbert,

1991). Several tannins or related phenolic compounds have been reported to possess

antiviral activity. For example, corilagin, previously isolated in the studies from

Acalypha species, has been reported to inhibit HIV reverse transcriptase (Singh et al,

2005). They have also been used for treating intestinal disorders such as diarrhea and

dysentery (Dharmananda 2003)

Flavonoids extracted have shown to exhibit wide range of biological activities

like antimicrobial, anti-inflammatory, anti allergic, anti analgesic cytostatic, and anti

oxidant properties

Efforts to produce large quantities of active secondary compounds by plant

tissue culture techniques have been developed for the rapid, large scale production of

cells and their secondary compounds (Lee et al., 2011). Through this approach we can

isolate active components through callus without exploiting the plants from natural

resources. Therefore, the present study has been carried out to evaluate the

preliminary screening of bioactive compounds and antimicrobial activity of leaf and

in vitro developed callus from the leaves of T.involuvrata.

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Table 4.1: Preliminary phytochemical analysis of plant T.involucrata

Sl,

No. Parameter Plant Powder Callus powder

1 Ash value 8.2% 9.05%

2 Water soluble ash 0.05% 0.07%

3 Acid insoluble ash 0.57% 0.64%

4 Moisture content 12.05% 95.10%

5 Water solubility 8.62% 9.85%

6 Alcohol soluble ash 15.39% 11.04%

Table 4.2: Preliminary phytochemical analysis of plant O.secamone

Sl,

No. Parameter Plant Powder Callus powder

1 Ash value 9.05% 10.45%

2 Water soluble ash 0.04% 0.09%

3 Acid insoluble ash 0.66% 0.91%

4 Moisture content 11.85% 89.2%

5 Water solubility 9.67% 15.4%

6 Alcohol soluble ash 7.85% 6.92%

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Table 4.3: PRELIMINARY PHYTOCHEMICAL ANALYSIS OF T.INVOLUCRATA LEAF AND ITS CALLUS EXTRACTS

+ = presence of compounds - = absence of compounds

Tests for

Solvent Extracts Aqueous

extract

Prollius fluid

extract Petroleum

ether Chloroform Acetone Methanol

Leaf Callus Leaf Callus Leaf Callus Leaf Callus Leaf Callus Leaf Callus

Alkaloids - - - - + + + + + + + +

Glycosides - - - - + + + + + + - -

Carbohydrates - - - - + + + + + + + +

Saponins - - - - + + + + + + + +

Phytosterols - - + + + + + + + + + +

Phenolics and tannins + + - - + + + + + + - -

Sugars - - + + - - - - + + - -

Chapter - 4

188

Table 4.4: PRELIMINARY PHYTOCHEMICAL ANALYSIS OF O.SECAMONE LEAF AND ITS CALLUS EXTRACTS

Tests for

Solvent Extracts

Aqueous

extract

Prollius fluid

extract Petroleum

ether Chloroform Acetone Methanol

Leaf Callus Leaf Callus Leaf Callus Leaf Callus Leaf Callus Leaf Callus

Alkaloids - - - - + + + + + + + +

Glycosides - - - - + + + + + + - -

Carbohydrates - - - - + + + + + + + +

Saponins + + - - - - - - - - - -

Phytosterols - - + + + + + + + + + +

Phenolics and tannins + + - - + + + + + + + +

Sugars - - + + - - - - + + - -

+ = presence of compounds - = absence of compounds

Chapter - 4

189

TABLE 4.5: TLC Profile of Plant and Callus Extracts of T.involuctara

Extracts Source Solvent

system

Spraying

reagents No. of Spots Colour Alkaloid Steroid Phenolics

Acetone

Plant Chloroform:

methanol

(7:3)

D L

FeCl3

1 1

2

Pink Violet

Blue

+ -

-

- +

-

- -

+

Callus

Chloroform:

methanol (7:3)

D

L FeCl3

1

1 2

Pink

Violet Blue

+

- -

-

+ -

-

- +

Methanol

Plant

Chloroform:

acetic acid

(7:1)

D

L

FeCl3

1

1

2

Pink

Violet

Blue

+

-

-

-

+

-

-

-

+

Callus

Chloroform:

acetic acid

(7:1)

D

L

FeCl3

1

1

2

Pink

pink

Blue

+

-

-

-

+

-

-

-

+

Prollius fluid

Plant

Chloroform:

acetic acid

(7:1)

D

L FeCl3

1

1 1

reddish

Violet -

+

- -

-

+ -

-

- -

Callus

Chloroform:

acetic acid

(7:1)

D

L

FeCl3

1

1

2

reddish

Violet

-

+

-

-

-

+

-

-

-

-

+ =presence of compound D = Dragendorff” reagent FeCl3 in (2% in ethanol)

- =absence of compound L = Libermann-Burchard reagent

Chapter - 4

190

GRAPHS: 4.1: Methanol extract of T. involucrata leaf (in vivo)

Retention time Compounds

2.33 Gallic acid

2.923 Chlorogenic acid

4.357 Protocatechuic acid

10.477 Vanillic acid

Chapter - 4

191

GRAPHS: 4.2: Methanol extract of T. involucrata leaf callus (in vitro)

Retention time Compounds

2.457 Gallic acid

3.05 Chlorogenic acid

4.48 Protocatechic acid

10.7 Vanillic acid

27.46 Ferulic acid

Chapter - 4

192

Plate 4.1: In vivo and In vitro Acetone extract of T. involucrata tested for

phenolics on TLC plate showing the common spots

Plate 4.2: In vivo and In vitro Methanol extract of T. involucrata tested for

phenolics on TLC plate showing the common spots