Evaluation of antioxidant and antimicrobial activity of various extracts of Buxus wallichiana Baill wood

 

Nandeesh R, Manjunatha E, Vijay Kumar S

Sree Siddaganga College of Pharmacy, B. H. Road, Tumkur - 572 102, Karnataka, India.

 

ABSTRACT:

The present study was designed to evaluate the antioxidant and antimicrobial activity of various extracts of Buxus wallichiana Baill wood. Extraction of wood was done by hot continuous percolation process by using suitable solvents and the concentrated extract was used for the study. Anti oxidant Activity is done by in vitro DPPH (2,2-diphenyl-1- picryl hydrazyl ) method and Nitric oxide radical inhibition activity method, where Antibacterial Activity is done by Cup Plate Method. The MEBW (Methanolic extract of Buxus wallichiana ) showed an IC₅₀ value of 29.2±2.10, where as ABBW (Aqueous extract of Buxus wallichiana ), CEBW(cholroform extract of Buxus wallichiana ) and PEBW (Petroleum ether extract of Buxus wallichiana ) produced IC₅₀ values of 190±5.33, 647.5±16.43 and 747.5±10.2 respectively in DPPH method. When assayed for Antibacterial Activity MEBW at 500 μg/ml produced zone of inhibition of 26mm against Klebsiella aerogenes organism, against proteus vulgaris organism, CEBW, AEBW at 500 μg/ml produced zone of inhibition of 19mm.Against bacillus premiles, b. aerogenes and b.coacoagulans MEBW at 500 μg/ml shown zone of inhibition of 17, 19 and 24 mm respectively. The result of this study reveal that Buxus wallichiana Baill could be considered as potential source of natural anti oxidant and anti bacterial plant.

 

 

 

INTRODUCTION:

The Buxus wallichiana Baill wood is commonly called as Himalayan Box Wood belongs to family Buxaceae. Box Wood is an evergreen monoecious shrub growing to an height of six meters with variable form. The green branches are initially pubescent, later glabrous, oleogreeen angular and densely covered with ovate leaves, which are usually opposite. Leaves are nearly sessile, opposite, nearly lanceolate to ovate 2-7 cms long. Flowers are clusters, auxiliary open in early spring.

 

The fruit is capsule oblong, 5-6mm long seed. (Fleming 1990, Chopra et al 1992)  Box Wood traditionally used as antihelmentic, analgesic, bitter tonic, anti rheumatic, purgative anti syphilitic, vermifuge and as hair growth stimulant.(Husain et al 1992, Chopra et al 1992).  The plant contains large number of alkaloids, the main alkaloids have been isolated including buxemenol E, buxaltine H, n-3-bezoyl cyclohexobuxidine F, buxatine, buxepidine (Kvaltinova et al 1991, Husain et al 1992). Steroidal alkaloid buxemenol E was found to have hypotensive effect in rats attributed by central and peripheral activation of muscranic receptor and also by partial inhibition of acetylcholineesterase enzyme (Kvaltinova et al 1991). This plant is widely used for the treatment of different ailments, hence its anti oxidant (Yogesh Shivhare et al 2010) and antibacterial   (Venkatachalam et al 2009) effects of various extracts were performed.  

 

MATERIAL AND METHODS:

Chemicals, Ethanol, petroleum ether, chloroform, were purchased from SD fine chemicals, India. DPPH, (2,2-diphenyl-1- picryl hydrazyl) NEDD (napthyl ethylene diamine dihydrochloride) DMSO (dimethyl sulfoxide), sodium bicarbonate, potassium hydrogen phosphate, diortho sodium phosphate, and glacial acetic acid were purchaced from E Merk India. All chemicals used were of analytical grade purity.

 

Plant material, wood of B. wallichiana was collected from Doddabetta region of Nilgiris district of Tamilnadu, India. The sample of the wood was made into herbarium and deposited in the department of pharmacognosy, Sree Siddaganga college of Pharmacy Tumkur and coded as PP-13

 

Extraction of Plant Material,  The leaves from the wood were separated and the wood was washed clean and shade dried. The dried wood was grinded to a coarse powder. Successive extraction was done with petroleum ether, chloroform, methanol and water respectively by hot percolation process.The extract obtained was concentrated using rotavac evaporator and the yield value of B. wallichiana wood of pet ether, chloroform, methanol and water was found to be 0.62, 0.75, 5.37, and 2.59 % w/w respectively.

 

Anti oxidant Activity (in vitro test)

DPPH method      

21 mg of pet ether chloroform, methanol and aqueous extracts of Buxus wallichiana wood was dissolved using 100 μM DMSO to obtain a solution of 21 mg/ml concentration and further diluted to appropriate concentrations (500, 125, 62.5, 31.25, 15.625 and 7.5 mg/ml) before transferring to 96 well microtitre plate. To 200 ml of DPPH (100μM) solution, 10ml of the test extract was added separately in wells of microtitre plate. The plates were incubated at 37⁰ C for 30 min and the absorbance of each solution was measured at 490 nm, using ELISA reader (Compaq). Each well contained 200 μl of methanol solution of DPPH (100μM) and 10 μl of sample solutions of different concentration (7- 250μg/ml). Controls were prepared with 200μl methanol and DMSO.  Rutin (10.5 mg/ml in 10μM of DMSO solution) was used as a positive control. The % inhibition by sample exposure was determined by comparing with DMSO treated control.  IC₅₀ value is the concentration of the sample required to inhibit 50% DPPH free radical. The data obtained were used to determine the concentration of the sample required to scavenge 50% of the DPPH free radical (IC₅₀). The % of the inhibition was plotted against the concentration and IC₅₀ obtained from the fitted arm. The results are expressed as the mean ± SEM of 5 replicates . (Rekha Rajendran et al 2009, Smith et al 1987)

 

Nitric oxide radical inhibition activity           

The reaction mixture (6ml) containing sodium nitro prusside (10μM, 4ml), Phosphate buffer saline (PBS, 1ml) and 1 ml pet ether, chloroform, methanol and aqueous extract solution (strengths of 500, 125, 62.5, 31.25, 15.625 and 7.5 mg/ml diluted with 100μM DMSO) was incubated at 25^oC for 150 minutes. After incubation, 0.5ml of the reaction mixture-containing nitrate was removed by adding 1ml of sulphanilic acid reagent (0.33% in warm 20 % glacial acetic acid), mixed well and allowed to stand for 5 min for completion of diazotisation.

 

Then 1ml of NEDD (0.1% Naphthyl Ethylene Diamine Dihydrochloride - NEDD was prepared by dissolving 100mg of NEDD in 60ml 50% glacial acetic acid by heating and made up the volume to 100ml in a volumetric flask with distilled water) was added, mixed and allowed to stand for 30 min in diffused light.

 

A pink colored chromophore was formed.  The absorbance of these solutions was measured at 540 nm against corresponding blank solution.  IC₅₀ value is the concentration of the sample required to inhibit 50% nitric oxide radical. The data obtained were used to determine the concentration of the sample required to scavenge 50% of the nitric oxide free radical (IC₅₀). Ascorbic acid  (1 mg/ml in 10μM of DMSO solution) was used as a positive control. The % inhibition was plotted against the concentration and IC₅₀ obtained from the fitted arm. The results are expressed as the mean ± SEM of 5 replicates (Sreejayan et al 1997).

 

Antibacterial Activity (Cup Plate Method)

Standard glass petridishes and prepared nutrient agar media were sterilized by autoclaving. Nutrient agar media was poured in to sterilized petridishes to give a depth of 3 to 4 mm under aseptic condition (under laminar flow bench). The prepared petridishes were stored in incubator for 24 h to ensure no growth of any microorganisms. The standardized suspensions of selected bacterial strain were spread on the surface of the agar media of the petridish under aseptic condition. By using stainless steel borer around 5 mm diameter cup were bored and residues from the bored area was removed, in each petridish four cups were bored, one for standard antibiotic, one for test sample, one for solvent blank and another with out any treatment. Standard antibiotic (corresponding to micro organisms) and test solutions were serially diluted, to each cup 100ml of serially diluted antibiotic solution, test solution and solvent used for dilution were added separately under aseptic condition. These petridishes were placed in an incubator for 24 h, growth of microorganism and zone of inhibition around the cup were measured, the zone inhibition of sample were compared with standard zone of inhibition of standard antibiotic used (I P1996, Esther Lydia et al 2016, PL Ladda, et al 2010)

 

RESULTS AND DISCUSSIONS:

Antioxidant Activity (in vitro test)

When assayed for antioxidant potential by DPPH and NO inhibition models, the MEBW showed an IC₅₀ value of 29.2 ± 2.10 in DPPH model, whereas rutin in the same model showed IC₅₀ value of 3.41 ± 0.11.  The AEBW, CEBW and PEBW produced IC₅₀ values of 190 ± 5.33, 647.5 ± 163.43, and 747.5 ± 102.2 respectively (Table 1).

 

Antibacterial Activity

When assayed the antibacterial activity by cup-plate method, PEBW did not produce any zone of inhibition in all the bacterial strains tested, whereas MEBW at 500mg level produced the zone of inhibition of 26mm when compared to standard chloramphenicol (70mg) showed 30.5 mm against Klebsiella aerogenes organism.  Against Proteus vulgaris organism CEBW, AEBW at 500mg level produced zone of inhibition of 19cm, which were comparable to the standard, chloramphenicol (70mg) produced 28mm inhibition. Against gram –ve bacteria such as Bacillus premiles, B. aerogenes and B. coagulans MEBW at 500mg level produced zone of inhibition of 17, 19 and 24 respectively, whereas standard penicillin G (40mg) produced 28, 26 and 32 respectively for these microorganisms (Table 2).

 

Table 1-Antioxidant activity of various extract of B.wallichiana wood in DPPH and NO inhibition model (in vitro study)

Treatment	IC50 (µg/ml)
	DPPH method(n=6)	NO inhibition method(n=6)
PEBW	747.5  ± 102.2	380.0 ±   9.5
CEBW	647.5  ± 163.5	184.0 ± 22.0
MEBW	29.2  ±     2.1	110.0 ± 11.6
AEBW	190.0  ±     5.3	189.0 ±13.3
Ascorbic acid	-	39.2 ±  0.49
Rutin	3.41 ±    0.11	-

Values are mean ± SEM.;    n= number tests per group

In NO inhibition model MEBW, CEBW, AEBW and PEBW produced IC₅₀ values of 110 ± 11.6, 184 ± 22, 189 ± 13.3 and 380 ± 9.5 respectively where as ascorbic acid showed IC₅₀ value of 39.2 ± 4.5

 

Table 2- Effect of various extract of B.wallichiana wood on bacterial growth by cup plate method.

Zone of inhibition (mm)
Bacteria strain	PEBW		CEBW		MEBW		AEBW		ChloramPhenicol    (70µg)	Penicillin    (40 µg)
	250 µg	500 µg	250 µg	500  µg	250 µg	500 µg	250 µg	500 µg
Klebsiella aerogenus	NA	NA	12	19	23	26	12	24	30.5	NT
E coli	NA	NA	NA	18	12	18	14	20	26	NT
Seerptococcus Epidermis	NA	NA	NA	15	NA	20	NA	NA	28	NT
Proteus vulgaris	NA	NA	13	18	13	19	12	19	22	--
Bacillus premiles	NA	NA	NA	14	12	17	NA	14	NT	28
Bacillus aerogenes	NA	NA	NA	NA	11	19	NA	NA	NT	26
Bacillus coagulans	NA	NA	NA	15	17	24	17	21	NT	32

NT-Not Tested; NA- No Activity

 

 

 

CONCLUSION:

As an ongoing process on blind screening of antibacterial property of herbs in our department, The MEBW, ABBW, CEBW and PEBW  showed potent antioxidant activity in DPPH method. The preliminary antibacterial activity by cup plate method results indicates that MEBW has potential components against Gram^-ve and Gram^+ve bacteria tested in the present study.  Whereas CEBW and AEBW had potential activity against  Proteus vulgaris only.  Further studies by bioassay guided fractionation could yield the potential antibacterial activity from Buxus wallichiana wood.

 

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Received on 21.02.2017          Modified on 27.02.2017

Accepted on 22.03.2017      ©AandV Publications All right reserved