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Antimicronbial activity of the crude root extract of Berberis lycium Royle.

Introduction

The importance, necessity and potentiality of medicinal plants in the practice of medicine today is well established and can not be overlooked. The Indo-Pak subcontinent is very rich in having resources of medicinal plants. A large number of these plants are used in the form of powder, decoction DECOCTION, med. jurisp. The operation of boiling certain ingredients in a fluid, for the purpose of extracting the parts soluble at that temperature. Decoction also means the product of this operation.
     2.
 and infusion for the treatment of various diseases including the infection caused by microbes with fair amount of success by hakims and vaids. Several workers throughout the world have carried out anti-microbial studies on some medicinal plants including Datura metel, Ageratum houstonianum [5]. A number of other studies on anti-microbial activity of plants have been carried out in different parts of the world [4,17,10]. Many plants have been used because of their antimicrobial traits, which are due to compounds synthesized in the secondary metabolism of the plant. These products are known by their active substances, for example, the phenolic compounds which are part of the essential oils [13] as well as in tannin tannin, tannic acid, or gallotannic acid, astringent vegetable product found in a wide variety of plants. Sources include the bark of oak, hemlock, chestnut, and mangrove; the leaves of certain sumacs; and plant galls.  [23]. The antimicrobial properties of plants have been investigated by a number of researchers world wide. In Argentina, researcher tested 122 known plant species used for therapeutic treatments [1]. It was documented that among the compounds extracted from these plants, twelve inhibited the growth of Staphylococus aureus, ten inhibited Escherichia coli, and four inhibited Aspergillus niger and also reported that the most potent compound was one extracted from Tabebuia impetiginosa. Many studies have been conducted in Brazil. The inhibitory activity of Vatairea macrocarpa macrocarpa

cupressusmacrocarpa.
 on Klebsiella spp. and S. aureus was observed [20] and the inhibitory activity of extracts from Eucaliptus spp. against soil fungi [6]. A more detailed study on antimicrobial compounds was done evaluating extracts from 120 plant species from 28 different families [22]. It was documented that 81 extracts obtanained from 58 plants were active against S. aureus, and five extracts from four other plants inhibited the growth of P. aeruginosa. Another study [18] detected the antibacterial and antifungal (C. albicans) activity of essential oils obtained from Croton croton, in botany
croton (krō`tən), any of several species of Codiaeum that are widely cultivated as ornamentals and houseplants. The most popular species is C.
 triangularis leaves. Extracts from Lippia gracilis and Xylopia sericea showed antifungal activity. The antimicrobial activity from Mikania triangularis, known as "thin leaf guaco", was tested against five genera of bacteria and three genera of yeast, and showed it had activity against Bacillus cereus, E. coli, P. aeruginosa, S. aureus and S. epidermidis [9]. Effects of phytochemical phy·to·chem·i·cal
n.
A nonnutritive bioactive plant substance, such as a flavonoid or carotenoid, considered to have a beneficial effect on human health.
 were conducted [12,13] and it was observed the antimicrobial activity of anacardic acid on S. aureus, Brevibacterium ammoniagenes, Streptococcus mutans and Propionibacterium acnes. Later, it was tested the bactericidal bactericidal /bac·te·ri·ci·dal/ (bak-ter?i-si´d'l) destructive to bacteria.
Bactericidal
An agent that destroys bacteria (e.g.
 activity of anacardic acid and totarol on methicillin resistant strains of S. aureus and the synergistic effect of these compounds associated with methicillin [21].

Berberis Berberis

genus in the plant family Berberidaceae; contains berberine, a pyridine alkaloid; causes cardiomyopathy and congestive heart failure. Called also barberries.
 lyceum Royle, (Berberidaceae) commonly known as Barberry barberry (bär`bĕr'ē), common name for the family Berberidaceae, and specifically for the spiny barberries (Berberis species). The family includes perennial herbs and shrubs found in the Northern Hemisphere.  is an evergreen shrub usually 1.2-1.8 m. high, but attaining 3.6m. height and 10cm. diam. Twigs pale yellowish, glabrous glabrous /gla·brous/ (gla´brus) smooth and bare.

gla·brous
adj.
Having no hairs or projections, especially on body parts that normally have hair; smooth.
 or minutely pubescent pubescent /pu·bes·cent/ (pu-bes´int)
1. arriving at the age of puberty.

2. covered with down or lanugo.


pu·bes·cent
adj.
1.
. Bark rough and rather deeply furrowed. Blaze 5mm, bright yellow with coarse reticulate re·tic·u·late  
adj.
Resembling or forming a net or network: reticulate veins of a leaf.

v. re·tic·u·lat·ed, re·tic·u·lat·ing, re·tic·u·lates

v.tr.
1.
 fiber. Leaves 2.5-7.5 by 0.7-1.8 cm., lanceolate Lanceolate
Narrow, leaf shape that is longer than it is wide, and pointed at the end.

Mentioned in: Echinacea
 or narrowly obovate-oblong, coriaceous, entire or with a few large spinous spinous /spi·nous/ (spi´nus) pertaining to or like a spine.

spi·nous
adj.
Relating to, shaped like, or having a spine or spines.



spinous

pertaining to or like a spine.
 teeth, dull green above, pale and glaucous glau·cous  
adj.
1. Of a pale grayish or bluish green.

2. Botany Covered with a grayish, bluish, or whitish waxy coating or bloom that is easily rubbed off: glaucous leaves.
 beneath, secondary nerves not prominent on the upper surface. Petiol are distinct up to 2.5mm. Inflorescence inflorescence

Cluster of flowers on one or a series of branches, which together make a large showy blossom. Categories depend on the arrangement of flowers on an elongated main axis (peduncle) or on sub-branches from the main axis, and on the timing and position of flowering.
 a simple raceme ra·ceme
n.
An optically inactive chemical compound.
 13-38mm. Long, often with a few long--stalked flowers at the base.

Root bitter with an unpleasant taste; used in spleenic trouble; tonic, a good febrifuge feb·ri·fuge
n.
An agent that acts to reduce a fever; an antipyretic.

adj. or fe·brif·u·gal
Acting to reduce fever.


Febrifuge
A plant substance that acts to prevent or reduce fever.
; intestinal astringent; good for cough, chest and throat troubles, eye-sores and itching of the eye, piles, and manorrhagia; useful in chronic diarrhea; always thrist; as a gargle gargle /gar·gle/ (gahr´g'l)
1. a solution for rinsing mouth and throat.

2. to rinse the mouth and throat by holding a solution in the open mouth and agitating it by expulsion of air from the lungs.
 strengthens the gums; a good application to boils.

An extract prepared by digesting in water sliced pieces of the root, stem and branches is called rusot, and is used in cases of ophthalmia ophthalmia /oph·thal·mia/ (of-thal´me-ah) severe inflammation of the eye.

Egyptian ophthalmia  trachoma.

gonorrheal ophthalmia  gonorrheal conjunctivitis.
.

The leaves are administered in Baluchistan as a cure for Jaundice. Due to above mentioned fact that this plant is very useful and the fact that little information is available on its biological activity, there is a need to find out more about the potential of this plant as antimicrobial agent.

The present study is therefore, designed to assess the potency of the plant extract on selected microorganisms.

Material and methods

The plant was collected from Rawalakot Azad Kashmir. This was authenticated by Department of Botany University of Azad Jammu & Kashmir Muzaffarabad. Root were separated and dried under shade at room temperature. The dried root were ground into powder and stored in glass bottle.

The dried powdered 50gms of Berberis lycium were soaked in to 250ml in each solvent for 7-10 days. Each mixture was shaked after 24 hours. After ten days each extract was filtered with Whatman filter paper in separate bottles and were preserved until required.

Pure cultures of Escherichia coli, Staphylococcus aureus, Bacillus subtilis, Staphylococcus epidermidis, Solmonella typhi and a fungus Candida albicans were obtained from Microbiology Section, Drug control and Traditional Medicine Division, NIH Islamabad.

The culture media for bacteria was prepared by dissolving 20mgs of dehydrated nutrient agar in 1000ml of distilled water, boiled and stirred. The prepared media was autoclaved for 15 minutes at 121[degrees]C and 15psi. The PH of media was maintained at 7.0 [+ or -] 0.2 at 25[degrees]C.

The culture media for fungus were prepared for dissolving 65gms of dehydrated sabourand's dextrose dextrose: see glucose.  agar medium in 1000ml of distilled water by with constant stirring autoclaved for 15 minutes at 121[degrees]C and 15psi. The PH of medium was adjusted 5.6 [+ or -] 0.2 at 25[degrees]C.

Antimicrobial Activity:

The antimicrobial activities of the three extracts were determined by using the paper-disc method, describing by Casal [7] and Chung et al. [8]. Zones of inhibition for each extract on each organism were measured and recorded in millimeter.

Preparation of Inoculum inoculum /in·oc·u·lum/ (-ok´u-lum) pl. inoc´ula   material used in inoculation.

in·oc·u·lum
n. pl.
:

A 24 hours old culture of each bacterium and 72 hours old culture of fungus was used as inoculum for the test.

The culture was prepared on slants of their respective media. Each test tube was labeled with the name of bacterium and fungus.

These test tubes were incubated for 24 hours at 37[degrees]C and 72 hours at 25[degrees]C respectively.

Preparation of Petri Dishes:

The sterilized Petri dishes were also labeled with the bacterial and fungal names. 1ml of dilution of each bacterium and fungus was poured into previously labeled Petri dishes. The sterile nutrient agar at a temperature not more than 45[degrees]C was poured in the Petri dishes containing bacterial suspension and sabourand's dextrose agar medium in molten state was poured in Petri dishes containing fungal suspension. All the Petri dishes were rotated gently to mix the inoculum and the media were allowed to become solidified at room temperature.

Uniform filter paper discs were made. These discs were soaked in the solution of aqueous, petroleum ether and ethanolic crude extracts of the root of Berberis lyceum and were placed in the Petri dishes at their labeled position. Another set of Petri dishes were prepared in the same way in which commercially available antibiotic disc i.e. Tetracycline 50ug were placed on the top of the medium.

Incubation of Petri Dishes:

The Petri dishes containing the bacterial culture were incubated at 37[degrees]C for 24 hours while plates with fungal suspension were incubated at 25[degrees]C for 72 hours. After the incubation time, all the Petri dishes were examined for the presence of zones of inhibition as an indication of antimicrobial activity. The zones were measure in millimeter and were tabulated in table.

Statistical Analysis:

At least three replicates were measured for all assays, and all assays were performed twice. The mean values, standard deviations and statistical differences were evaluated through analysis of variance [24].

Results and discussion

In the present study the antimicrobial activity of Berberis lycium roots extracts were observed against Gram +, Gram- bacteria and a fungal strain. The medicinal plant (Berberis lycium) was selected on the basis of their local medicinal uses and collected from Dreak Rawalakot distt. Poonch Azad Kashmir.

In this investigation the zones of inhibition produced by crude extracts were recorded against the microorganisms used. For the extraction of plant material a broad solvent extraction was carried out. Berberis spp. contain Berberine berberine /ber·ber·ine/ (bur´bur-en) an alkaloid from species of Berberis and related plants, and from Hydrastis canadensis;  and other chemical compounds [14]. The compounds may active against test organisms.

The antimicrobial activities of ethanolic, aqueous and petroleum ether crude extracts of root of Berberis lycium were summarized in table.

Aqueous Extract:

The maximum zone of inhibition were observed against Candida albicans, Styphlococcus epidermidis and E. coli (27.00 [+ or -] 0.31 mm, 24.00 [+ or -] 0.25 mm, and 23.00 [+ or -] 0.30 mm) while minimum zone of inhibition against Salmonella typhi, B. subtilis and Staphylococcus aureus (22.00 [+ or -] 0.38mm, 20.00 [+ or -] 0.28mm and 20.00 [+ or -] 0.33mm). Anjum and Khan [2] study the antimicrobial activity of crude extracts of Cuscuta reflexa against Gram +, Gram-and fungal strains. On the whole all the crude extracts were active against tested microorganisms. Chloroform and petroleum ether extracts were found to be more effective against the microorganisms used.

Comparison of the present research work with the work of Anjum and Kahn showed that the polar extracts were more active than the non-polar.

Ethanolic Extract:

The crude ethanolic extract of Berberis lycium root show high activity against Candida albicans, S. aureus and E. coli (27.00 [+ or -] 0.65mm, 25.00 [+ or -] 0.74mm and 23.00 [+ or -] 0.50mm) and low activity against S. epidermidis, Salmonella typhi and B. subtilis (15.00 [+ or -] 0.12mm, 16.00 [+ or -] 0.12mm and 21.00 [+ or -] 0.23mm). Kokoska et al. [16] studied antimicrobial activity of crude ethanolic extracts of 16 Siberian medicinal plants that tested against 5 species of microorganisms. Out of 16 plants tested, 12 showed antimicrobial activity against one or more species of microorganisms. Present work was also be compared with the work of Kokoska under same laboratory condition which were very closed and identical in magnitude with the results discuss above.

Petroleum Ether Extract:

There was no significant activity shown by the petroleum ether extract of root of Berberis lyceum.

Conclusion:

Berberis lycium root possess antimicrobial activity. This can explain the rationale for the use of the plant in treating infections in traditional medicine. The plant could be a veritable and cheaper for conventional drugs since the plant is easily obtainable and the extract can easily be made via a simple process of maceration mac·er·a·tion
n.
1. Softening by soaking in a liquid.

2. Softening of the tissues after death by autolysis, especially of a stillborn fetus.
 or infusion. On considering the present work, it is necessary to isolate the active principle from the plant extracts and to carryout pharmaceutical studies.

References

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[2.] Anjum, N. and Z. Khan, 2003. Antimicrobial activity of crude extracts of Cuscuta reflexa. Pak. J. Bot; 35: 999-1007.

[3.] Alonso-Paz, E., M.P. Cerdeiras, J. Fernandez, F. Ferreira, P. Moyna, M. Soubes, A. Vazquez, S. Veros and L. Zunno, 1995. Screening of Uruguayan medicinal plants for antimicrobial activity. J. Ethnopharmacology, 45: 67-70.

[4.] Belachew, K.H. Desta, 1993. Antimicrobial activity of Plumbago zeylanica. Journal of Ethnophormocology, 39: 129-139.

[5.] Bowers, W.S., 1976. Discovery of insect juvenile hormones in plants. Science, 195: 542-547.

[6.] Bruna, E.P., B. Fernandes, A.C. Borges, J. Almeida and N.F. Barros, 1989. Effects of Eucalyptus litter extracts on microbial growh. Pesq. Agrop. Bras., 24: 1523-1528.

[7.] Casal, K.B., 1979. Tablet sensitivity testing of pathogenic fungi. J. Chem pathol., 32: 719-722.

[8.] Chung, K.T., V.R. Thamasson, C.D. Wu Chung, 1990. Growth inhibition of selected food borne bacteria, particularly Listeria monocytogenes

by plants extracts. J. Appl. Bacteriol., 69: 498-503.

[9.] Cruz, F.G., N.F. Roque, A.M. Giesbrecht and S.C. Davino, 1996. Antibiotic activity of diterpenes from Mikania triangularis. Fitoterapia, 67: 189-190.

[10.] Didry, N., L. Dubreuil, M. Pinkas, 1994. Activity of anthraquinonic and naphthaquinonic compound on oral bacteria. Pharmazei, 49(9): 681-683.

[11.] Didry, N., L. Dubreuil, F. Trotin, M. Pinkas, 1998. Antimicrobial activity of aerial parts of Drosera Drosera

genus of insectivorous plants in family Droseraceae; some species may cause cyanide poisoning. Called also sundews.
 pellata Smith on oral bacteria. J. of Ethanopharmacology., 60: 91-96.

[12.] Izzo, A.A., G. Di Carlo, D. Biscardi, R. Fusco, N. Mascolo, F.Borreli, F. Capasso, M.P. Fasulo and G. Autore., 1995. Biological screening of Italian medicinal plants for antibacterial activity. Phytother. Res., 9: 281-286.

[13.] Jansen, A.M., J.J.C. Cheffer and A..B. Svendsen., 1987. Antimicrobial activity of essencial oils: a 1976-1986 literature review. Aspects of test methods. Planta Med., 40: 395-398.

[14.] Joshi, S.G., 1997. Medicinal plants. Oxford IBH Publishing Co. Pvt. Ltd. New Delhi.

[15.] Kirtikar, K., B.D. Basu, 1987. Indian medicinal plants, 2nd ed; Vol. 1. international book distributors. Rajpur road, Dehradun, India.

[16.] Kokoska, L., Z. Polesry, V. Rada, A. Nepovim and T. Vanek, 2002. Screeining of some Siberian medicinal plants for antimicrobial activity. J. Ethnopharmacol, 82: 51-53.

[17.] Lajubutu, B.A., R.J. Pinney, M.F. Roberts, H.A. Odelola and B.A. Oso, 1995. Antimicrobial activity of disiquinone and plumbagin for the root of Diospyru mespiliformis. Phytotherapy, 14: 2717.

[18.] Lemos, T.L.G., F.J.Q. Monte, F.J.A. Matos, J.W. Alencar, A.A. Craveiro, R.C.S.B. Barbosa and E.D. Lima., 1992. Chemical composition and antimicrobial activity of essencial oils from Brazilian plants. Fitoterapia 63: 266-268.

[19.] Martinez, M.J., S.M. Vasquez, C. Espinosa-Perez, M. Dias and M. Herrera-Sanchez. 1994. Antimicrobial properties of argentatine A isolated from Parthenium argentatum. Fitoterapia, 65: 371-372.

[20.] Matos, F.J.A., L.M.B.A. Aguiar and M.G.A. Silva., 1988. Chemical constituents and antimicrobial activity of Vatairea macrocarpa Ducke. Acta Amazonica, 18: 351-352.

[21.] Muroi, H. and I. Kubo., 1996. Antibacterial activity of anacardic acids and totarol, alone and in combination with methicillin, against methicillin-resistant Staphylococcus aureus methicillin-resistant Staphylococcus aureus Methicillin-aminoglycoside resistant Staphylococcus aureus, MRSA An organism with multiple antibiotic resistances–eg, aminoglycosides, chloramphenicol, clindamycin, erythromycin, rifampin, tetracycline, . J. Appl. Bacteriol., 80: 387-394.

[22.] Santos Filho, D., S.J. Sarti, J.K. Bastos, H.F. Leitao Filho, J.O. Machado, M.L.C. Araujo, W.D. Lopes and J.E. Abreu, 1990. Atividade antibacteriana deextratos vegetais. Rev., Cien. Farm., 12: 39-46.

[23.] Saxena, G., A.R. McCutcheon, S. Farmer, G.H.N. Towers and R.E.W. Hancock., 1994. Antimicrobial constituents of Rhus glabra. J. Ethnopharmacol., 42: 95-99.

[24.] Steel, R.G.D. and J.H. Torrie, 1980. Principles and Procedures of Statistics. Mc Graw Hill Book Co. Inc. New York., 134-145.

(1) Muhammad Altaf Hussain, (2) Muhammad Qayyum Khan, (3) Tariq Habib and (4) Nazar Hussain (1,2,3,4) Department of Botany, University of Azad Jammu & Kashmir Muzaffarabad.

Corresponding Author

Muhammad Altaf Hussain, Department of Botany, University of Azad Jammu & Kashmir Muzaffarabad.

E-mail: scholar.altaf@gmail.com
Table 1: Antimicrobial activity of Berberis lyciun
root extracts. (Zones of inhibition in mm)

S. No.   Extracts            S. aureus             S. typhi

1        Aqueous             20.00 [+ or -] 0.33   22.00 [+ or -] 0.38
2        Ethanolic           25.00 [+ or -] 0.74   16.00 [+ or -] 0.12
3        Petroleum Ether     0.00 [+ or -] 0.00    0.00 [+ or -] 0.00
4        Tetracycline 50ug   26.00 [+ or -] 0.61   28.00 [+ or -] 0.47

S. No.   E.coli                S. epidermidis

1        23.00 [+ or -] 0.30   24.00 [+ or -] 0.25
2        23.00 [+ or -] 0.50   15.00 [+ or -] 0.12
3        0.00 [+ or -] 0.00    0.00 [+ or -] 0.00
4        27.00 [+ or -] 0.31   26.00 [+ or -] 0.50

S. No.   B. subtilis           C. albicans

1        20.00 [+ or -] 0.28   27.00 [+ or -] 0.31
2        21.00 [+ or -] 0.23   27.00 [+ or -] 0.65
3        0.00 [+ or -] 0.00    0.00 [+ or -] 0.00
4        26.00 [+ or -] 0.45   25.00 [+ or -] 0.32
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Title Annotation:Original Article
Author:Hussain, Muhammad Altaf; Khan, Muhammad Qayyum; Habib, Tariq; Hussain, Nazar
Publication:Advances in Environmental Biology
Article Type:Report
Geographic Code:9PAKI
Date:Mar 1, 2011
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