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Adhav M, Patel B, Gharia A. Evaluation of Antimicrobial Agent from Leaves of Synadenium Grantii Hk.F.Bot.Mag. Biosci Biotech Res Asia 2010;7(2)
Manuscript received on : August 11, 2010
Manuscript accepted on : September 20, 2010
Published online on:  28-12-2010
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Evaluation of Antimicrobial Agent from Leaves of Synadenium Grantii Hk.F.Bot.Mag

Madhavi Adhav, Bhavesh Patel and Anil Gharia

Department of Botany, Department of Microbiology, Department of Chemistry P.M.B. Gujarati Science College, Indore India.    

ABSTRACT: Higher plants have played a dominant role in the introduction of new therapeutic agents. In India, there are over 15000 kinds of naturally occuring higher plants1. About 2000 species of these are reported to posses medicinal values. Discovery and development of new therapeutic agents is a continuing process. In spite of the fact that at present we have our command a formidable array of modern drugs, the need to discover and invent new agents is geniune and urgent. Many of the members of family Euphorbiaceae are known because of their medicinal uses in tribals as well as in Ayurveda2,3,4. Present paper deals with one of such medicinally important plant Synadenium grantii. Antimicrobial studies of leaves extract of S. grantii shows strong antibacterial activity against gram positive bacteria viz. S. aureus and Streptococcus sp. and gram negative bacteria viz. E. coli, S. dysentrae, S. typhi, P. aeruginosa and K. pneumoniae. The IR of active fraction shows the presence of secondary amine, amide groups, Co-NH2-C-N ring vibration. This proves its correlation with antimicrobial study.

KEYWORDS: Synadenium grantii; antimicrobial activity; therapeutic agents

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Introduction

Syndaneium grantii locally called as Badi Dudhi. This plant is recent introduction and found only on hedges Flrs. and Frts – Nov. – Feb. The plant is used as stimulant of central nervous system, and is used by tribals in many diseases viz. urinary tract infection, diarrhoea, dysentery, pains also used in bronchial affections.

The present work yielded useful information which can be exploited for the successful treatment of many diseases.

Material and Methods 

The material for the present study was collected from Indore and its surrounding. 100 gm. of the shade dried plant material is extracted in “Soxhlet Extraction Appartus” successively with 500 ml. of each of the following solvents – P. ether, Benzene, Chloroform, Acetone, Ethanol (95%). Each time before extracting with next solvent the plant material is dried. Finally, the drug is macerated with water for 24 hrs. to obtain the aquous extract5,6,7. Each extract is concentrated by distilling off the solvent. The extract obtained with each solvent is weighted and its percentage is calculated in terms of the shade dried weight of the plant material. The colour and consistency of each extract is noted.

The extracts thus obtained are then subjected to qualitative test for identification of various plant constituents by the methods suggested by Finar (1962)7, Farnsworth (1966)8 and Harborne et.al. (1979)9. Each extract sample was then tested for antimicrobial activity againste human pathogenic bacteria by “Cup Borrer Method”.

The components of the ethanolic plant extract were seperated by column chromotography using silica gel column where Benzene and Hexane are used as the solvent. The purity of each component was checked by Thin Layer Chromotography.

Each fraction are then again subjected for testing the antibacterial activity by “Disc Diffusion Method”.10,12

Table 1: Successive solvent extraction of Synadenium grantii (leaves) which show the colour and consistancy of each extract.

S. No. Solvent Used Colour Average Value of Extractive
1. Petroleum ether (60-80)0C Dark Yellow 19.0%
2. Benzene Light Yellow 21.0%
3. Chloroform Light Green 23.5%
4. Ethanol (95%) Light Brown 17.5%
5. Water Light Green 24.0%

Table 2: Qualitative chemical examination of various extracts of Synadenium grantii (leaves)

S. No. Plant Constituents Test/Reagents P.ether extract Benzene extract Chloroform extract Ethanol extract Water extract
1. Alkaloids
1. Mayer’s Reagent +
2. Dragendraff Reagent
2. Carbohydrates
1. Molish’s test + +
2. Fehling Soln. test
Glucose +
Fructose
Galactose +
Starch
3. Glycosides
1. Borntrager’s test + +
2. Legal test + +
4. Phytosterols
1. Liebermann’s test
2. Liebermann’s

Burchard’s test

5. Phenolic Compound
1. Ferric chloride test +
2. Lieberman test +
6. Proteins
1. Xanthoproteic test +
2. Biuret test +
7. Flavonoids +

Note : Test for oil, saponin, aminoacids, gum and mucilage were also performed but found to be negative.

Table 3: Antimicrobial testing of each extract of Synadenium grantii (leaves) against gram positive bacteria.

S. No. Extract Used Quantity of extract used S. aureus Streptococcus Sp. Bacillus subtilis
1 Ethanolic 0.05 ml 10 mm 12 mm No Zone
0.08 ml 13 mm 15 mm No Zone
0.11 ml 15 mm 17 mm No Zone
0.13 ml 18 mm 20 mm No Zone
0.16 ml 21 mm 23 mm No Zone
r 0.97 0.92
  Zone Colour –   Light Brown  
2 Water 0.05 ml 09 mm No Zone No Zone
0.08 ml 11 mm 10 mm No Zone
0.11 ml 14 mm 13 mm No Zone
0.15 ml 17 mm 15 mm No Zone
0.16 ml 19 mm 17 mm No Zone
r 0.92 0.93
Zone Colour –   Light Brown

Note : * Antimicrobial testing of P. ether, Benzene and Chlorform extract

were also performed but there was no zone of inhibition found.

*  r = Correlation coefficient

Table 3: (b)Antimicrobial testing of each extract of Synadenium grantii (leaves) against gram negative bacteria

S. No. Extract used Quantity extract used E.coli Shigella dysenterae Salmo-nella typhi Pseudo-monas aeruginosa k. pneum-oniae
1. Ethanolic 0.05 ml 15 mm 17 mm 12 mm 10 mm No Zone
0.08 ml 17 mm 19 mm 15 mm 12 mm 8 mm
0.11 ml 20 mm 22 mm 17 mm 14 mm 10 mm
0.13 ml 23 mm 25 mm 20 mm 17 mm 13 mm
0.16 ml 25 mm 28 mm 22 mm 20 mm 15 mm
r 0.99 0.99 1.00 0.96 0.93
Zone Colour –  Light Brown
2. Water 0.05 mm 12 mm 14 mm 10 mm No Zone No Zone
0.08 mm 14 mm 17 mm 12 mm 8 mm No Zone
0.11 mm 16 mm 19 mm 14 mm 10 mm 8 mm
0.13 mm 18 mm 22 mm 17 mm 12 mm 11 mm
0.16 mm 21 mm 25 mm 19 mm 15 mm 14 mm
r 0.98 0.99 0.94 0.96 0.92
Zone Colour –  Light Brown

Note :* Antimicrobial testing of P. ether, Benzene and Chlorform extract were also performed but there was no zone of inhibition found.

*  r = Correlation coefficient

Table 4(a): Antimicrobial testing of each fraction of benzene and hexane against gram positive bacteria

S. No. Fractions S. aureus Streptococcus spp. Bacillus subtilis
1. B1 No Zone No Zone No Zone
2. B2 No Zone No Zone No Zone
3. B3 20 mm 17 mm No Zone
4. B4 No Zone No Zone No Zone
5. B5 No Zone No Zone No Zone
6. H1 15 mm 16 mm No Zone
7. H2 12 mm 14 mm No Zone
8. H3 10 mm 12 mm No Zone
9. H4 No Zone No Zone No Zone
10. H5 No Zone No Zone No Zone
11. H6 No Zone No Zone No Zone
12. H7 No Zone No Zone No Zone
13. H8 No Zone No Zone No Zone
14. H9 No Zone No Zone No Zone
15. H10 No Zone No Zone No Zone
16. St. Ben. & Hex No Zone No Zone No Zone

Table  4(b): Antimicrobial testing of each fraction of benzene and  hexane against gram negative bacteria

S. No. Fractions E.coli Shigella dysen-terae Salmonella typhi Pseudo-monas aeruginosa K. pneum-oniae
1. B1 No Zone 22 mm 16 mm 18 mm 12 mm
2. B2 No Zone No Zone No Zone No Zone No Zone
3. B3 8 mm 25 mm 17 mm 21 mm 10 mm
4. B4 No Zone No Zone No Zone No Zone No Zone
5. B5 No Zone No Zone No Zone No Zone No Zone
6. H1 16 mm 20 mm 19 mm 13 mm No Zone
7. H2 11 mm 12 mm 18 mm No Zone 14 mm
8. H3 23 mm 20 mm 13 mm 16 mm 15 mm
9. H4 15 mm 14 mm No Zone No Zone No Zone
10. H5 No Zone No Zone No Zone No Zone No Zone
11. H6 No Zone No Zone No Zone No Zone No Zone
12. H7 No Zone No Zone No Zone No Zone No Zone
13. H8 No Zone No Zone No Zone No Zone No Zone
14. H9 No Zone No Zone 10 mm No Zone No Zone
15. H10 No Zone No Zone No Zone No Zone No Zone
16. St. Ben. & Hex No Zone No Zone No Zone No Zone No Zone

Results and Discussion

Antimicrobial studies of these extracts (P.ether, Benzene, Chloroform, Ethanol and water) is performed which includes various disease causing gram positive and gram negative organisms. Table No. 3(a) and 3(b) shows that ethanolic extract is having strong antibacterial activity against gram positive and gram negative organisms. In case of gram positive bacteria extract shows antibacterial activity against S. aureus and Streptococus spp. but it does not show any response against B. subtilis. Extract shows strong antibacterial activity against gram negative organism viz E.coli, S.dysenterae, S.typhi, P.aeruginosa and K. pneumoniae.

Water extract also shows antibacterial activity against gram positive and gram negative bacteria. It shows strong antibacterial activity against S. aureus and Streptococcus spp. while extract is inactive against B. subtilis. In case of gram negative bacteria extract shows strong antibacterial activity against S.dysenterae, E.coli and S.typhi while extract shows positive response against P. aerugionsa and K. pneumonia.

Antimicrobial testing of P.ether, Benzene, Chlorform extract were also performed but there was no zone of inhibition found.

The ethanolic extract is the most effective than the extract of water. Fractionation of extract by column chromotography and antimicrobial susceptibility testing of individual fraction further suggest that not all the fractions are equally effective against micro ogranisms. Table No. 4(a)  and 4(b) indicates that B1, B2, B4, H5, H6, H7, H8, H9, H10 does not show antibacterial activity against gram positive bacteria while B3, H1 shows strong antibacterial activity against S. aureus and Streptococcus spp., H2 and H3 fractions also shows antibacterial activity against S. aureus and Streptococcus spp. while all the fractions B1 to B5 and H1 to H10 does not show any response against B. subtilis.

Table No. 4(b) shows that B2, B4, B5, H5, H6, H7, H8, H9, & H10 are inactive fractions against gram negative bacteria. Fraction No. B1, B3, H1, H2, H3 & H4 shows antibacterial activity against gram negative bacteria viz. E. coli, S. dysenterae, S. typhi, P. aeruginosa and K. pneumoniae. Out of all the studied fraction B3 and H3 shows strong antibacterial activity. In B3 fraction shows strong antibacterial activity against S. dysenterae, P. aeruginosa, S. typhi while fraction shows positive response against K. pneumoniae and E. coli. H3 fraction shows strong antibacterial activity in B3 fraction shows strong antibacterial activity against S. dysenterae, P. aeruginosa, S. typhi while fraction shows positive response against K. pneumoniae and E. coli. H3 fraction shows strong antibacterial activity against E. coli, S. dysenterae, P. aeruginosa while fraction H3 shows positive response against K. pneumoiae and S. typhi.

Thus overall conclusion suggested that only B3 and H3 fractions are most effective among all the studied fractions. This fraction shows strong antibacterial activity against all the tested gram positive as well as gram negative organisms. However, the antimicrobial effect of a crude extract may be a combine action of all thee fractions.

Figure 1 shows that there is a sharp and strong peak (broad) hump like at 3443 cm-1. This peak is most probably due to secondary amine and nitrogen is in the ring. This is might be due to residual moiety of some alkaloid molecule as usual test also show the positive result (as seen in the Table No. 1) about the presence of alkaloids. The peak is broaden which is might be due to some moisture in the compound.

Further strong peak at 2981 cm-1 is due to asymmetic stretching of    C-H. Another strong peak at 1643 cm-1 is most probably due to –CONH– and also it is due to –CO stretching in –CONH– The presence of amide group is well reported in many of the alkaloids and the extract under study also shows the positive test for alkaloids.

The broader peak at 1393 cm-1 is most probably due –C-N- ring stretching vibration conclusively we can draw that extract under study have any of the compound which contain hetrocyclic ring with nitrogen as hetrocyclic atom.

The I.R. of the active fraction Synadenium grantii shows the presence of secondary amines and amide groups –CO-NH-C-N ring structural vibration. Amines and amides play important roles in our day-to-day lives. In many drugs viz. Amphetamines, Barbiturate, Analgesics, Anesthetics, Decongestants and antibiotics are such medicinal compounds which are amines or amine derivatives. Amphetamines stimulate the central nervous system and are also used to treat psychological disorders such as severe depression12. Many of the medicinal amines are also used as analygsis and anesthetics Novocaine and related compounds e.g. are used as local anesthetic while Demerol is a very strong pain reliever11. Phenacetin and accetaminophen which are common substitutes of aspirine and Dopa and Dopamine, the related compounds Epinephrine and Norenephrine are also amine and amide derivatives 5,12.

Sulfa drugs an important class of antibiotics are also systehsized from amine and amine related compounds11. This proves its correlation with antimicrobial activity. Thus, the results of present study not only confirm the correct usage of the plant by the tribals but also enhances the creditability of ethnobotanical explorations.

Acknowledgement 

The authors are grateful to University Grants Commission, Bhopal for providing the financial support and also thankful to Dr. O.P. Joshi Principal Prof. Santosh Nagar, Head Department of Botany P.M.B. Gujarati Science College, Indore and Dr. N.S. Sapre Department of Chemical Sciences.     U.T. D. D.A.V.V., Indore for giving the Laboratory facilities and valuable suggestions.

Reference

  1. Jain S.K. (1996) Medicinal Plants Pub. National Book Trust India, New DelhiCaius, J.E. (1986)
  2. The Medical and Poisonous Plants of India  Scientific Publishers, Jodhpur 264-313. Jain S.K. (1987)
  3. A Manual of Ethnobotany Scientific Publishers, Jodhpur
  4. Jain S.K. (1981)
  5. Observation On Ethnobotany of the Tribals of Central Indian, Glimes of India, Ethnobotany. Oxford and SBH Publishers New Delhi. Kokate, C.K. (1999)
  6. Practical Pharmacognosy Nirali Prakashan Kokate C.K., A.P. Purohit & B.B. Gokhale (2000)
  7. Pharmocognosy, Eights Edition Nirali Prakashan Finar L.L. (1962)
  8. Organic Chemistry Vol. II Longman, Green and Grosvent Street, London, WI Fanswarth, N.F. (1966)
  9. Biological and Phytochemical Screening of Plants. Pharm. Sci. 55-225-276 Harborne, J.B., J.M. Mabry & H. Mabry (1979)
  10. The Flavonoides Chapman and HallBauer, A.W., Kirby W.M.N., Sherris J.C. and Turek, M., Antibiotic susceptibility Testing by Standardized Single disc Method,Am J. Clin. Pathol 45(196)493.Agarwal, K.C. (1974)
  11. Antibiotic Sensitivity Test by disc diffusion Method Standardization and Interpretation Indian J. Pathol, Bact. 17, 149-159.Drug Today (July-Sept. 1999)
  12. Ready Reckonar of Current Medical Formulations. Nicholas Piramal (India) Limited, Mumbai. 84-85, 367-376.
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