INTRODUCTION:

Now-a-days, herbalism is in trend as it effective, safe and cheaper¹. Bauhinia variegata belongs to family Leguminosae (Caesalpinioideae) is also called Mountain Ebony (English), Rakta kanchan (Marathi), Kachnar (Hindi). It is a medium-sized, deciduous tree found throughout India, at an altitude of 1800m in Himalayas. Leaves are broader, rigidly sub-coriaceous, deeply cordate with two leaflets, connate for about two-thirds up, leaflets are ovate, rounded at apex, 10-15cm long, pubescent beneath when young. Flowers are variously colored, lateral, sessile, stamens 5, staminodes absent, fruits flat; hard glabrous dehiscent pods, 10-15 seeded². It grows throughout India and China. It is a reliable greenhouse species which grows at an altitude of 1800 m in Himalayas³.

Bauhinia variegata Linn. is traditionally used in bronchitis, intestinal worms, leprosy, inflammation, bacterial infection, liver disorders, diarrhoea, dysentery, skin disease, leprosy, wounds, ulcer, fungal infection, ulcers and tumors^4,5.

Aims and Objectives:

In this research, the effects of the ethanolic extract of dried leaf extract of Bauhinia variegata on the catalase and superoxide dismutase (SOD) activity in swiss albino mice were studied.

MATERIAL AND METHODS:

Plant material:

The leaves of Bauhinia variegata L. were collected from the College Campus of Shri Ram Murti Smarak (College of Engineering and Technology), Bareilly (Uttar Pradesh) and identified (specimen number- RU/PS/2016/415) by Prof. A.K. Jaitly, Head, Department of Plant Science, Mahatma Jyotiba Phule Rohilkhand University, Bareilly, Uttar Pradesh. A voucher specimen of collected sample was deposited in the institutional herbarium for future reference.

Animals:

Animals were procured from Animal House, Department of Pharmacy, SRMS CET (Pharmacy), Bareilly, U.P. Animals were approved by Institutional Animal Ethic Committee (IAEC) of Department of Pharmacy, SRMS CET (Pharmacy), Bareilly, U.P. Approval number (715/PO/Re/S/02/CPCSEA) was given for this work. The preferred rodent species included mice. Swiss albino strains of young healthy adult of either sex animals in equal numbers per group (n=6) were taken. At the commencement of the study the weight variations of animals used was kept minimal and not exceeded ± 20% of the mean weight of each animal. Normal weight of mice was 25-30 gm.

The temperature of the experimental animal room was maintained to be 22ºC (±3ºC). Relative humidity was maintained between 50-60%. Lighting was artificial, the sequence being 12 hours light, 12 hours dark. For feeding, conventional laboratory diets were used with drinking aqueous supplied ad libitum. Animals of same group were caged together. Healthy young adult of either sex mice were randomly assigned to the control, standard and treatment groups. The animals were identified uniquely (i.e., via marking at the base of the tail) and acclimatized for not less than 5 days in their cages prior to the start of the study.

Preparation of extracts:

The leaves of Bauhinia variegata were washed thoroughly in tap water, shade dried and powdered. This powder was packed into Soxhlet column and extracted with petroleum ether (60º -80ºC) for 24 h. The same marc was successively extracted with chloroform (50º-60ºC) and later with ethanol (68º-78⁰C) for 24 h. The extracts were concentrated on water bath (50ºC). After concentrated preparation, the dried powder extract was stored at room temperature. The yield of the petroleum extract, chloroform extract, methanol extract, ethanolic extract and water extract and were found to be 9.50 % (w/w), 7.65 % (w/w), 8.95% (w/w), 8.50 % (w/w) and 0.30% (w/w) respectively. Ethanolic extract was used for the experimental study.

Animal Handling and Treatment:

Measurement of Weight of Animals:

The mice were acclimatized for about 7 days prior to the treatment. In order to calculate the volume of ethanolic extract of Bauhinia variegata to be given to the mice, the mice are weighed daily.

Animal Grouping:

The animals were placed in 8 groups with six mice in each group.

Administration of Sample (Extract) to the Mice:

The extract was administered orally. Doses of 200mg/kg and 400mg/kg body weight were given to groups 4, 5, 7 and 8 respectively for 28 consecutive days.

Estimation of biochemical parameters:

The biochemical parameters for oxidative stress like SOD and Catalase were estimated in brain of mice on the 28^th day after Colchicine injection.

Brain tissue preparation:

The mice were sacrificed by using ether anaesthesia. The brain was removed after cutting the skull. The brain was cleansed by using normal saline solution (chilled). 10% (w/v) homogenate of brain sample was obtained by homogenizer at 10 strokes at 2000 rpm with 0.03M sodium phosphate buffer (pH 7.4). SOD and Catalase could be measured by using homogenized brain tissue preparation.

Superoxide dismutase activity:

The SOD activity was estimated by method described by Kono, 1978 by observing the inhibitory rate of NBT (Nitroblue tetrazolium) reduction. One unit is referred to the quantity of enzyme which is responsible for half-maximal inhibition of NBT reduction⁶.

Reagents:

1. Solution A consists of 50 mM Na₂CO₃ (0.52g/100ml) in 0.1mM EDTA (0.003g/100ml) (pH=10.0).

2. Solution B consists of 96μM NBT in solution A (0.008g/100ml).

3. Solution C consists of 0.6% Triton X-100 (v/v) in solution A.

4. Solution D consists of 20mM Hydroxylamine HCl, adjust pH to 6 with 2 M NaOH.

Test Method:

Procedure of Superoxide dismutase estimation:

Test solution was prepared by adding 1.3 ml Solution A to 1.0 ml double distilled water, 0.5 ml Solution B and 0.1 ml Solution C, 0.1 ml Solution D and 0.1 ml Sample.

Standard solution was prepared by adding 1.3 ml Solution A to 1.1 ml double distilled water and 0.5 ml Solution B and 0.1 ml Solution C and 0.1 ml Solution D.

Reference (Blank) solution was prepared by adding 1.3 ml Solution A to 1.1 ml double distilled water, 0.5 ml Solution B and 0.1 ml Solution C and 0.1 ml Sample.

The change in absorbance was estimated at 560 nm at 30 sec intervals for 2 min by using spectrophotometer. The reaction was started by adding hydroxylamine to the reaction mixture. The reduction of NBT occurred which caused an increase in absorbance at 560 nm in aerobic conditions.

Catalase activity:

The catalase enzyme is found in all the aerobic cells as endogenous antioxidant which helps in the removal of hydrogen peroxide. The method of Aebi H, 1984 was used for the estimation of catalase activity. 0.1 ml of the supernatant was added to 1.9 ml of 50 mM phosphate buffer (pH 7.0) in the cuvette. The addition of 1 ml of 30 mM H₂O₂ (freshly prepared) initiated the reaction. Spectrophotometer was used to measure the rate of decomposition of H₂O₂and the alteration in absorbance was done for 2 min at 30 s at 240 nm.

The reaction started by the addition of 1 ml freshly prepared 30 mM H₂O₂. The rate of decomposition of H₂O₂ was measured spectrophotometrically from the changes in absorbance was recorded for 2 min at 30 s at 240 nm. The catalase activity was expressed as U/mg protein⁷.

Change in OD/min x Vol. of assay = µ moles of H₂O₂ decomposed/min/mg protein

0.071 x mg protein

(0.071=Molar extinction coefficient)

Protein estimation:

Protein was estimated in all brain samples according to the method of Lowry where bovine serum albumin (BSA) (1 mg/ml) was used as standard⁸.

Reagents:

1. Alkaline solution

a) 2% (w/v) Sodium carbonate in 0.1 M NaOH.

b) 1% (w/v) Copper Sulphate

c) 2% Sodium Potassium tartrate

Working alkaline solution: 48ml of A + 1ml of B + 1ml of C

2 Stock std. Bovine Serum Albumin (BSA) – 1mg/ml

3 Working standard BSA (1000μg/ml) diluted the stock 20 times.

4 Folin-Phenol reagent (ice-cold) diluted with equal amount of water at the time of use.

Test Method:

0.1ml of supernatant was added to 0.9 ml DDW and 5 ml alkaline working reagent. The mixture was mixed well and incubated for 10 min at room temperature. It was followed by the addition of 0.5 ml Folin-phenol reagent and incubated for 30 min at room temperature. The absorbance was measured at 750 nm against blank. Then a standard curve (50- 1000μg) was plotted followed by the estimation of protein content of sample as mg/ml⁸.

Statistical analysis:

The results are expressed as mean ± S.E.M. Statistical analysis of passive avoidance, Morris water maze and biochemical values were performed by one-way analysis of variance (ANOVA) followed by Tukey test.

RESULTS AND DISCUSSION:

Table 1: Effect of ethanolic leaf extract of Bauhinia variegata on SOD and Catalase level

Treatment	SOD (U/mg Protein)	Catalase (U/mg Protein)
Control	65.20±1.38	29.06±1.88
Standard	74.90±1.94**	14.24±1.66***
Colchicine	24.80±1.95***	5.50±1.04***
LD BV	68.39±1.89	8.51±0.58***
HDBV	70.42±1.88	11.58±1.14***
Pir+col	53.64±1.37***	17.93±0.78***
LdBV+col	66.46±1.66	17.55±0.77***
Hd BV+col	68.28±1.65	19.03±0.83***

SOD and Catalase was studied in swiss albino mice which were treated with ethanolic extract of leaves of Bauhinia variegata. Alzheimer’s disease is one of the neurodegenerative disorders which are symptomatised by apraxia, aphasia, dementia and agnosia. Vast research is going on to investigate the herbal drugs which can be used in the treatment of Alzheimer’s disease. In the present study BVE extract (400mg/kg) was administered orally for 28 days. It was observed that it improved learning and memory of mice significantly. The pretreatment of mice with BVE for 28 days protected the animals from colchicine induced memory impairment. The cognitive activity may be reduced due to the formation of excessive free radicals or reactive oxygen species which may ultimately result in Alzheimer’s disease. Bauhinia variegata possess antioxidant activity as well. The neuroprotective effect of BVE may be due to its antioxidant property i.e. the brain cells are exposed to fewer oxidative stresses hence, decreasing the neuronal damage. The symptoms of Alzheimer’s disease are directly linked to the impaired neurotransmission in the affected brain areas. Bauhinia variegata plant extract is composed of flavonoids which is responsible for free radical scavenging activity.

CONCLUSION:

The results of my research predicts that the ethanolic extract of Bauhinia variegata leaves can be used to manage and cure various diseases which are caused due to oxidative stress and excessive free radicals like Alzheimer’s disease, anxiety, depression, etc.

ACKNOWLEDGEMENT:

We are thankful to the Department of Pharmacy, Shri Ram Murti Smarak, College of Engineering and Technology (Pharmacy), Bareilly, U.P. for providing chemicals and other infrastructure for doing this research work. The work is dedicated to my guide and co-guide.

CONFLICT OF INTEREST:

Authors declare that they have no conflict of interest.

REFERENCES:

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3. Deswal G, Arora K. Ethnobotany and phytopharmacology of Bauhinia variegata. International Journal of Pharmaceutics and Drug Analysis. 2015; 3(9): 261-3.

4. Prashar Y, Kumar AS. Antiobesity activity of Bauhinia variegata Linn. in high fat diet induced obesity in female rats. Pharmacologyonline. 2010; 2: 1008-16.

5. Yadava RN, Reddy VM. Anti-inflammatory activity of a novel flavonol glycoside from the Bauhinia variegata Linn. Nat Prod Res. 2003; 7: 159-65.

6. Singh GK, Kumar V. Anxiolytic Activity of Standardized Extract of Fumaria indica. Annals of Neuro-sciences. 2009; 16: 81.

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8. Lowry OH, et al. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951; 193: 265–75.

Received on 14.03.2019 Modified on 21.04.2019

Research J. Pharm. and Tech 2019; 12(9):4259-4262.

DOI: 10.5958/0974-360X.2019.00732.7