K. Ravi Shankar¹*, Chandralekha Gurjar², V. G. Rajalakshmi² and Narasimhaachar H. Joshi²

¹Sri Sai Aditya Institute of Pharmaceutical Sciences and Research, Surampalem (India).

²Sarada Vilas College of Pharmacy, Mysore (India).

Abstract

Various extracts of the plant Trichodesma indicum (Boraginaceae) were subjected to preliminary Phytochemical screening and it was shown that Flavanoids, Triterpenes, Tannins and Saponins were present. Flavanoids and Triterpenes were present in alchoholic extract, Tannins and saponins in aqueous extract, steroids and saponins in petroleum ether and chloroform extract. Further these various extracts were confirmed by thin layer chromatographic study.

Keywords

Phytochemical screening; TLC; Trichodesma indicum

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Copy the following to cite this article: Ravi Shankar K, Gurjar C, Rajalakshmi V. G, Joshi N. H.Phytochemical Investigations of Plant Trichodesma Indicum.Biomed. Pharmacol. J.;1(2)

Copy the following to cite this URL: Ravi Shankar K, Gurjar C, Rajalakshmi V. G, Joshi N. H.Phytochemical Investigations of Plant Trichodesma Indicum.Biomed. Pharmacol. J.;1(2). Available from: http://biomedpharmajournal.org/?p=510

Materials and Methods 

Collection and extraction of plant

The plant was collected in Mysore and taxonomical identification of the plant specimen was authenticated by Prof. Shashikala, HOD of Botany, Sarada Vilas Science College, Mysore. The plant was removed and dried under shade and powdered in a mechanical grinder.  The powder was extracted with various solvents.  All chemicals and reagents used are of analytical grade.

Sample taken—Trichodesma indicum whole plant extract

Solvent—Petroleum ether,Benzene,Chloroform,Alcohol and Water.

Method used—Reflux method.

Apparatus—Heating mantle,beaker, round bottom flask,measuring cylinder, double walled condenser,stand etc.

Proportion of  the drug with the solvent—1:4

Quantity of the drug taken 100g

Time-16hrs

Drying temperature—–<50⁰ C

Extraction Procedure ¹

The preparation of various extracts was by successive solvent extraction. The material to be extracted was subjected to extraction with solvents in ascending order of polarity successively. The constituents which are soluble in both polar and non polar solvents can be extracted separately by adopting this approach. For each and every extraction the extract was evaporated under reduced pressure until the solvent had been removed to give an extract sample with a yield of w/w.

All the extracts were examined for their colour and consistency. Their percentage weights were calculated with reference to the dried sample. These data are compiled in table no.1         

Table 1: 

Sl No.	Na         Name of the extract	Nature	Colour	Wt. of  Extract
1.	Pet.Ether	Resinous	Yellowish green	2g
2.	Chloroform	Resinous	Greenish	2.5g
3.	Alcohol	Resinous	Greenish brown	4g
4.	Aqueous	Resinous	Brownish yellow	3.5g

 

Qualitative chemical tests for various chemical   Phyto constituents.

Qualitative tests were conducted for all the extracts to identify the various phyto constituents .The various tests for steroids, flavonoids and  tannins were observed and the tests are recorded in table no.2 which shows the qualitative chemical  tests conducted for various extracts.

Table 2:

Sl.No.	Chemical test	Pet.Ether	CHCl3	Alcohol	Aqueous
1.	Steroids	+ve	+ve	-ve	-ve
2.	Triterpenes	-ve	-ve	+ve	+ve
3.	Saponins	-ve	-ve	+ve	+ve
4.	.Alkaloids	-ve	-ve	-ve	-ve
5.	Flavonoids	-ve	-ve	+ve	+ve
6.	Tannins	-ve	-ve	+ve	+ve

 

Purification and separation of Flavanoidal compounds by column chromatography ²

Attempts were made to purify and separate the Flavanoidal compound by taking alcoholic extracts (prepared by successive solvent extraction) and mixed with silica gel and kept overnight to get adsorbed on the silica gel. Then the column was packed with silica gel using petroleum ether for building the column.  Then the silica gel along with alcoholic extract was packed at the top of the column and the column was developed using different solvents. The solvent mixtures were collected in the decreasing order of their polarity as shown in the table no.3 :

Different fractions were collected at the rate of 18 to 20 drops per minute and each fraction was subjected to TLC examination. The course of chromatogram is given below:

Table 3:

Sl.no	Fractions	Eluate	Nature	Color
1.	1-4	PetEther(100%)	colorless	-ve
2.	5-10	Pet.Ether+Benzene (50:50)	V.light yellow	-ve
3.	11-15	Benzene+EtOH (75:25)	light brownish yellow	+ve
4.	16-20	Benzene+EtOH (50:50)	yellowish brown	+ve
5.	21-25	Benzene+EtOH (25:75)	Light orange yellow	+ve
6.	26-30	EtOH(100%)	greenish yellow	+ve
7.	31-37	Acetone+MeOH (50:50)	brownish yellow	+ve
8.	38-42	Acetone+MeOH (25:75)	light yellow	+ve
9.	43-48	MeOH(100%)	brownish yellow	+ve
10.	49-54	EtOH(100%)	greenish yellow	+ve

 

Examination of the Eluates 

Fraction 1-10 

All the fractions showed –ve  test for Flavoniods .Hence further  investigation was not carried out.

Fraction 15-54

All the fractions gave +ve test for Flavoniods and subjected to TLC examination. These fractions showed a single spot having same Rf value. Hence all the fractions were pooled together and the solvents were removed under reduced pressure.

The residue was dissolved in alcohol and left overnight. The light brownish yellow compound was obtained which was soluble in EtOH, Acetone, MeOH, dil H₂SO₄ and DMSO.

The isolated Flavonoidal compounds gave +ve test for the following color reactions:

Fecl₃ test: Dark green color

Magnesium ribbon Hcl reductiontest-Crimson red color (Shinoda test)

Zinc-Hcl reduction test –yellow ppt

Ammonium Hydroxide solutes test-yellow flouresnce (UV)

TLC study of isolated compounds

Materials used- Glass plate 20X5cm size

Adsorbent- Silica gel-G

Solvent system used-    a) Ethyl acetate: AA (9:1)

b) Pet.Ether : ethanol (1:9)

c) Toluene: Ethyl alcohol: acetone (7:2:2)

Sample:                         Isolated flavonoids

Detecting agent:            Antimony trichloride

Rf value:                       0.9

Further it needs spectral studies to confirm the findings. In a similar fashion steroids (obtained from Pet.Ether- CHCl₃ extract) were subjected to column chromatography and  fractions were tested for solvents collected at regular intervals of time and each fraction was subjected to TLC study which showed two different Rf values i.e, 0.9 & 0.75 with different solvent systems which are shown in the Table no.4

Similarly triterpenoids were isolated by taking alcoholic extract with the help of column chromatography & fractions were collected, tested for triterpenoids and gave positive response and later it was subjected to TLC study. It has also shown two different Rf values (0.9& 0.72) with different nature of their residues. Further studies like UV, IR & Mass, NMR spectral studies are required to confirm the findings.

Table 4: TLC study of the various extracts of Trichodesma indicum.

Sl.No	Chemical group	Solvent system	Spray  reagent	Rf value
1	Steroids	a)      CHCl3:MeOH (7:3)   b)      Toluene: EtoH:diethylamine     c)      CHCl3:EtOH (7:3)	Liebermann Burchard Reagent	0.9     0.9&0.75       0.9&0.72
2	Triterpenoids	CHCl3: MEOH (7:3)	Antimony Trichloride Reagent	0.87&0.72
3	Flavonoids	a) EA:HAc (9:1)   b)CHCl3: MeOH (1:1)   c)Toluene: EtOH:acetone (2:4:4)   d)EtOH: formic acid: HAc:water (100:11:11:26)		0.9,0.81&0.54   0.92&0.75   0.92&0.85     0.93&0.86)

 

Discussion and results

Trichodesma indicum is a plant containing steroids, triterpinoidal saponins & Flavanoids.The qualitative tests & chromatographic studies substantiate the presence of Steroids, triterpenoidal saponins & flavanoids. The presence of these phytochemical constituents may have different biological activities.  The Pharmacological screening of various extracts that are taken up is under progress in our laboratories.

Acknowledgements

The authors thank Prof.Shashikala, H.O.D.,Dept.of Botany, SaradaVilas Science College, for taxanomical identification of the plant specimen. The authors wish to thank the management for encouraging and providing laboratory facilities to pursue this work.

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