ANTIMICROBIAL ACTIVITY OF METHANOLIC EXTRACT FROM ...
ANTIMICROBIAL ACTIVITY OF METHANOLIC EXTRACT FROM ...
ANTIMICROBIAL ACTIVITY OF METHANOLIC EXTRACT FROM ...
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Dynashwar K.Mhaske et al. / International Journal on Pharmaceutical and Biomedical Research (IJPBR)<br />
Vol. 2(4), 2011, 107-111<br />
<strong>ANTIMICROBIAL</strong> <strong>ACTIVITY</strong> <strong>OF</strong><br />
<strong>METHANOLIC</strong> <strong>EXTRACT</strong> <strong>FROM</strong><br />
RHIZOME And ROOTS <strong>OF</strong><br />
VALERIANA WALLICHII<br />
Dynashwar K.Mhaske 1. Dinanath D. Patil 2 , Gurumeet C. Wadhawa 3<br />
1 Principal R.B.N.B .College Shrirampur .<br />
2 Head department of chemistry, R.B.N.B. College shrirampur,<br />
3 Post-Graduate Department of Chemistry and Research Centre, R.B.N.B. College, Shrirampur- 413 709(MS)<br />
E-mail address: wadhava_gc2004@rediffmail.com<br />
Abstract – The plant root and stem occurs in short, irregular pieces about 5 cm long and 6-12 cm in diameters<br />
marked with transverse ridges and bearing numerous, prominent, circular tubercles, to some of which on the<br />
under surface, thick rootlets are attached. The upper surface bears the remains of leaves. The rhizome is hard<br />
and tough internally, it is greenish-brown in color. The odour is powerfully valerianaceous both root and<br />
raizome are studied for antibacterial and antifungal activity in plants, root shows excellent antibacterial and<br />
antifungal activity.<br />
Keywords – Antibacterial activity, Polarity, valeriana wallichii, methanol<br />
Introduction –<br />
Valeriana wallichii is an extremely polymorphous complex of sub-species with natural populations dispersed<br />
throughout temperate and sub-polar Eurasian zones. The species is common in damp woods, ditches, and along<br />
streams in Europe, and is cultivated as a medicinal plant, especially in Belgium, England, Eastern Europe,<br />
France, Germany, India, the Netherlands, the Russian Federation, and the United States of America.<br />
Rhizome, with epidermis of polygonal cells, having the outer walls slightly thickened; cork, immediately below<br />
the epidermis, of up to 7 layers of slightly suberized, brownish, large polygonal cells; cortex, parenchymatous<br />
with rather thick-walled parenchyma, containing numerous starch granules and traversed by numerous roottraces;<br />
endodermis of a single layer of tangentially elongated cells containing globules of volatile oil; pericycle,<br />
parenchymatous; vascular bundles, collateral, in a ring and surrounding a very large parenchymatous pith,<br />
containing starch granules and occasional scattered groups of sclereids with thick pitted walls and narrow<br />
lumen; xylem, with slender, annular, spiral, and pitted vessels, in small numbers. Branches similar to rhizome<br />
but with a prominent endodermis and a well-defined ring of vascular bundles, showing second-ary thickening.<br />
Root, with piliferous layer, of papillosed cells, some developed into root hairs; exodermises, or a single layer of<br />
quadrangular to polygonal cells, with suberized walls, and containing globules of volatile oil; cortex,<br />
parenchyma-tous, with numerous starch granules, the outermost cells containing globules of volatile oil;<br />
endodermis, of 1 layer of cells with thickened radial walls; primary xylem, of 3–11 arches surrounding a small<br />
central parenchymatous pith con-taining starch granules, 5–15....m in diameter, sometimes showing a cleft or<br />
stellate hilum; the compound granules, with 2–6 components, up to 20....m in diameter. Older roots show a pith<br />
of starch-bearing parenchyma, vascular bundles with secondary thickening and a periderm originating in the<br />
piliferous layer.<br />
Plant Material and Preparation of Extracts:<br />
Preparatin of various extract of VALERIANA WALLICHII bark:<br />
Around 1kg of fresh shade dried bark was powdered and around 800gm were extracted by hot<br />
percolation method by soxhalate apparatus. With five liter of each pet-ether (40-60 0 c) chloroform, acetone and<br />
methanol successively. All the extracts finally reduced to dryness at 40degree c by rotary evaporator<br />
(Rotavapour Buchhi Switzerland).<br />
The quantity of each extract after the extraction was pet. Ether (15gm), chloroform (20gm), Acetone<br />
(12gm), Methanol (40gm), aqueous extracts (28gm).
Dynashwar K.Mhaske et al. / International Journal on Pharmaceutical and Biomedical Research (IJPBR)<br />
Vol. 2(4), 2011, 107-111<br />
Preparation of various extract of VALERIANA WALLICHII Root:<br />
In present study we use dry stem of the plant collected from western ghat region Maharashtra. Dried stems<br />
are cut into small pieces these pieces are then grinded. The grinded sample is dark brown in colour with a<br />
special smell.<br />
This powder stirred in non-polar solvent i. e. CCl4, for 1/2 hour & then it is refluxed for 1/2 hour this is<br />
performed for extraction of non-polar component from powder. After extraction the CCl4 layer is distilled to<br />
recover solvent & to get a brown colored liquid fraction which shows single spot on thin layer chromatography.<br />
The residue of CCl4 extraction is used for further study. This residue is mixed with CHCl3 & stirred for 1/2 hour<br />
& then refluxed for 1 hour. After filtration the filtrate is distilled to get CHCl3 Fraction which is Red-brown<br />
colored liquid.<br />
Then the Residue of CHCl3 is used for extraction with Ethyl acetate stirred well & refluxed for 1 hour<br />
then filtered. Filtrate is then distilled & fraction of Ethyl acetate is collected it shows no spot on TLC plate.<br />
Conclusion is that no organic compound is present.<br />
The Ethyl acetate residue is further mixed with methanol & stirred for 1/2 hr & refluxed for 1hr. Then<br />
it is filtered & filtrate is distilled out.Methanol fraction is yellow brown in colour & show single spot On TLC<br />
platte<br />
The remaining residue also have smell & it is observed that residue is insect repellant.<br />
Microorganisms:<br />
The test microorganism used for antimicrobial activity screening were & bacteria (2 gram +ve) - Enterococcus<br />
faecalis, Staphylococcus onerous, (2gram –ve) - Klebsiallea pneumaniae, Escherichia coli, and two fungi-<br />
Candida albicans and Aspergillus fumigants.<br />
These organisms were identified a procured from Nikhil analytical laboratory Sangli, Maharashtra.<br />
Antimicrobial Activity:<br />
The agar diffusion method [11] was used to evaluate the antimicrobial activity. Bacteria were cultured overnight<br />
at 37 o C in Mueller Hinton 10 µl Broth (MHB, Oxoid) and fungi at 28 o C for 72h in Potato Dextrose Broth<br />
(PDB, Oxide) and used as inoculums. A final inoculums, using 100 µl of suspension containing 10 8 CFV/ml of<br />
bacteria 10 4 spore/ml of fungi spread on Mueller Hinton Agar (MHA) and Potato Dextrose Agar (PDA)<br />
medium respectively.<br />
The disc (6 mm in diameter) was impregnated with 10 µl of 75 µl/ml, 50 µl/ml, 25 µl/ml, 10 µl/ml and 5 µl/ml<br />
of each extracts and for each organism placed on seeded agar. Ciprofloxacin and Fluconazole (75 µl/ml, 50<br />
µl/ml, 25 µl/ml, 10 µl/ml and 5 µl/ml) were used as positive control bacteria and fungi respectively. The test<br />
plates were incubated at 37 o C for 24h for bacteria and at 28 o C for 72h for fungi depending on the incubation<br />
time required for a visible growth.<br />
MIC values were also studied for microorganisms by turbidimetric method, which were determined as sensitive<br />
to the extracts in cup plate method. MIC was defined as the lowest concentration of extract that inhibit visible<br />
growth.
Dynashwar K.Mhaske et al. / International Journal on Pharmaceutical and Biomedical Research (IJPBR)<br />
Vol. 2(4), 2011, 107-111<br />
Table-1 Antibacterial activity of methanolic extract from extract from Rhizome of Valeriana wallichii<br />
Bacterial Extract<br />
ethanolic<br />
Extract CCl4 Cefotax Penicil Tetrax<br />
G (+)<br />
In mm In mm In mm In mm In mm<br />
1. Staphylococcus<br />
epidermidis<br />
2. Staphylococcus<br />
oureus<br />
3. Bacillus paludis<br />
4. Bacillus subtilis<br />
G (-)<br />
1. Escherichia Coli<br />
2. Pseudomonus<br />
aeruginosa<br />
3. Shigella flaxinely<br />
4. Enterobacter aero<br />
genes<br />
9 8 7 10 9<br />
7 12 10 8 8<br />
12 8 11 10 8<br />
7 8 11 10 7<br />
3 2 4 3.5 5.5<br />
4 2 4 3 3.5<br />
3 4 4.5 3.5 4<br />
3 4 5 3 2.<br />
Table-II; Antifungal activity of ethanilic extract from <strong>EXTRACT</strong> <strong>FROM</strong> Rhizome of Valeriana wallichii<br />
Fungus Extract<br />
ethanolic<br />
Extract CCl4 Cefotax Penicil Tetrax<br />
1. Candida albicans 7 6 7 5 9<br />
2. Aspergillus fumigatus 6 8 5 7 4<br />
3. Aspergillus niger 4 3 8 4 5<br />
Table-III Antibacterial activity of methanolic extract from root of Valeriana wallichii plant.<br />
Bacteria Extract<br />
ethanolic<br />
Extract CCl4 Cefotax Penicil Tetrax<br />
G (+)<br />
In mm In mm<br />
1. Staphylococcus<br />
epidermidis<br />
2. Staphylococcus<br />
oureus<br />
3. Bacillus paludis<br />
4. Bacillus subtilis<br />
G (-)<br />
1. Escherichia Coli<br />
2. Pseudomonus<br />
aeruginosa<br />
3. Shigella flaxinely<br />
4. Enterobacter aero<br />
genes<br />
12 11 10 10 10<br />
12 10 10 8 8<br />
10 9 10 11 9<br />
12 5 11 10 9<br />
8 9 4 3.5 5.5<br />
10 9 4 3 3.5<br />
11 9 4.5 3.5 4<br />
12 4 5 3 2.
Dynashwar K.Mhaske et al. / International Journal on Pharmaceutical and Biomedical Research (IJPBR)<br />
Vol. 2(4), 2011, 107-111<br />
Table-IV; Antifungal activity of methanolic extract from root of delonix regia plant.<br />
Fungus Extract<br />
mthanolic<br />
Extract CCl4 Cefotax Penicil Tetrax<br />
1. Candida albicans 13 5 7 5 4<br />
2. Aspergillus<br />
Fumigatus<br />
3. Aspergillus niger<br />
10 9 5 6 4<br />
11 8 6 4 5<br />
(Value represents MIC values expressed in ug/ml.)<br />
A comparison is shown between the antibiotic activities of the plant extract with reference antibiotics<br />
(Cefotaxime). Na-cefotaxime, (Pencil) benzyl penicillin sodium and (tetrac) tetracycline R=absence of<br />
inhibition even at the highest concentration used (100 ug/ml)<br />
1. Tested material: Methanolic extract was obtained after the degatting with petroleum ether (40º-60º) and<br />
CHCl3.<br />
2. Studied activities: Antibacterial activity was studied using the MIC broth dilution method. (8, 9)<br />
3. Organism used: for the antibacterial activity standard bacterial strains, obtained from the Nikhil Analytical<br />
Laboratory, Sangli (Maharashtra).<br />
Result:<br />
The results of Antimicrobial activity were done for all the five, pet ether, chloroform, acetone, and methanol and<br />
aqueous extracts. During antimicrobial study methanolic extracts showed maximum zone of inhibition against<br />
almost all organisms in cup plate method.<br />
Conclusion:<br />
The methanolic extract from roots of Valeriana wallichii<br />
showed a good inhibition against all the bacterial Strains tested (MIC between 10&80 ug/ml). The gram (+)<br />
bacteria were sensitive with gram (-) bacteria and some common fungi.<br />
Acknowledgement:<br />
We are grateful to the Principal Dr. D.K.Mhaske , R.B.N.B.College, Shrirampur for inspiring us and<br />
providing laboratory facility . Nikhil analytical and research laboratory, Sangli (M.S.) India for their technical<br />
assistance.<br />
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