Assessment of Antioxidant and Anti-inflammatory Activities of Stem Bark of Bauhinia acuminata L

Medicinal plants have been used from prehistoric times as first source of health care to fight infectious and non infectious diseases. Most of these medicinal plants may have scientific evidence to be considered in general practice. Various parts of Bauhinia acuminata is employed in the treatment of glandular swelling, skin diseases and ulcer etc. The aim of this work was to investigate the antioxidant capacities and anti-inflammatory activities of the methanol extract of stem bark of B. acuminata and its different fractions. Crude Methanol Extract (CME) of stems and its various fractions such as Chloroform (CHF), Ethyl Acetate (EF), and Aqueous (AQF) were subjected to assay for antioxidant activity using various methods of assay like 2, 2-Di Phenyl Picryl Hydrazyl (DPPH), hydrogen peroxide and hydroxyl radical-scavenging activity. Total phenolics and flavonoids were determined by Folin-Ciocalteu and colorimetric methods. Anti-inflammatory activity was evaluated by carrageenan and formalin induced hind paw edema model.


Introduction
Reactive Oxygen Species (ROS) results from oxidative stress due to the improper balance between ROS and antioxidant is an important factor in the pathogenesis of various diseases, such as cardiovascular disorders, neurological conditions, Parkinson's disease, rheumatoid arthritis, and ageing [1]. Common free radical or Reactive Oxygen Species (ROS) are the hydroxyl radical, the superoxide anion radical, hydrogen peroxide, singlet oxygen, nitric oxide radical and hypochlorite radical. Antioxidants are agents and molecules that can reduce and limit oxidative damage to biological structures by scvenging the free radicals, neutralize the harmful free radicals before ROS can attack the cells and prevent damage living cells, proteins, enzymes and DNA etc. They break off the chain reactions via the removal of free radical and inhibit other oxidation reactions. Antioxidants synthesized in the body, including all antioxidant proteins and various small molecules, form a defense against oxidative stress, and their levels in the body cannot be altered by simple means.
On the other hand, the levels of antioxidant vitamins such as ascorbate, α-tocopherol, β-carotene and phytochemicals viz.
polyphenols, flavonoids, can be increased easily by supplementation [2]. Enzymatic antioxidants include Superoxide Dismutase (SOD), Glutathione Peroxidase (GPx), Catalase (CAT), and micronutrients such as, selenium in glutathione, and copper and zinc in SOD as integral components of metallo enzymes [3]. Synthetic antioxidants such as Butylated Hydroxytoluene (BHT), Butylated Hydroxyanisole (BHA), tertiary butylated hydroquinone and gallic acid esters which are used in food industries now have restricted use in foods due to their prompt negative health effects [4]. So, there has been a growing interest in replacing commercial antioxidants with natural ingredients. Moreover, natural antioxidants are considered to be safe and environmentally friendly and they play a very important role in the physician's therapeutic armamentarium. Currently, the use of plants and herbs with antioxidants is gaining importance.
The anti-oxidative activity has been confirmed contributing inflammatory preventions for its multiple functional roles. The production of oxidants is a typical event associated with aerobic metabolism. When oxygen is supplied in excess or its reduction is insufficient, reactive oxygen species or free radicals such as superoxide anions, hydroxyl radicals and hydrogen peroxide are generated [5]. Accumulation of the free radicals in body organs or tissues can cause oxidative damage to biomolecules and membranes of cell, eventually leading to many chronic diseases, such as inflammatory, cancer, diabetes, aging, cardiac dysfunction and other degenerative diseases [6]. In the last 50 years, antioxidant and anti-inflammatory activities of extracts from medicinal or food plants have been extensively investigated. Many pharmacological studies have shown that extracts of some antioxidant plant possess anti-inflammatory, anti-allergic, anti-tumor, anti-bacterial, antimutagenic and anti-viral activities to a greater or lesser extent.

Trouillas et al. investigated the antioxidant, anti-inflammatory
and anti-proliferative properties of sixteen French herbal tea and found some herbs exhibited high antioxidant, anti-inflammatory and anti-proliferative activities [7]. Antioxidant activities in twenty traditional anti-inflammatory herbs extracts were investigated.
The results suggested that the anti-inflammatory activities of these extracts could be explained, at least in part, by their antioxidant properties [8]. Free radicals liberated from phagocyte cells are important in inflammatory processes, because they are implicated in the activation of nuclear factor kB, which induces the transcription of inflammatory cytokines and cyclooxygenase-2 [9].
B. acuminata is a member of the Fabaceae family locally known as Shwet Kanchan. The plant is used in the treatment of common cold and cough [10]. The bark and leaves in a decoction can relieve biliousness and asthmatic attack [11]. The bark and leaves in a decoction also used to treat stones in the bladder, venereal diseases and leprosy.
The paste of the leaves applied on the throat is beneficial for throat troubles and to treat skin diseases. The root is boiled in oil and applied to burns [12]. Khammuang et al. [13] reported that crude extract of B. acuminata seeds showed strong antibacterial activity against various pathogenic bacteria. Anti-inflammatory activity of the leaves of this plant was reported by Mohammed et al. [14]. Therefore, B. acuminata is one of the famous plant based drugs used in traditional medicine. In the present study, we evaluated the antioxidant and anti-inflammatory effect of stem bark extract for the first time using different assay system to characterize its significance as a drug in traditional medicine.

In vitro Antioxidant Activity
The radical scavenging effects of B. acuminata stem bark extract/fractions were evaluated against DPPH, hydrogen peroxide and hydroxyl radicals.

1, 1-Diphenyl-2-Picryl Hydrzyl (DPPH) Radical-Scavenging
Assay: The radical scavenging capacity was determined by the modified DPPH method [17]. A methanol solution containing 0.008% DPPH was prepared. After adjusting the blank with methanol, an aliquot of extracts and standard concentration ranging from 10 to100 µg was added to 2.5 ml of this solution and volume was adjusted to 5 ml with distilled water. The decrease in absorbance at 517 nm was measured after 30 minutes. The free radical scavenging capacity was expressed as the concentration of the samples required to reduce the original amount of free radicals by 50% (IC 50 ). The mixture was heated at 95°C in water bath for 15 min followed by the addition of 1 ml each of TCA (2.8%) and TBA (1%). Finally the reaction mixture was cooled on ice and centrifuged at 5000 rpm for 15 min. Absorbance of supernatant was measured at 532 nm.

Hydrogen Peroxide Scavenging Activity
All readings were corrected for any interference from brown color of the extract or antioxidant by including appropriate controls.
The negative control without any antioxidant or samples was considered 100% deoxyribose oxidation. Ascorbic acid was taken as the positive control. The % hydroxyl radical scavenging activity was calculated by the following equation.
Where, V t = is the average paw edema circumference of each extract treated group, as well as diclofenac sodium group; V c = is the paw circumference of the negative control group that only received the vehicle.
Formalin Induced Edema Assay: The formalin induced rat paw edema was carried out as described by Chau et al. [21] to evaluate acute anti-inflammatory activity of stem bark extract of Bauhinia acuminata Paw edema was induced by injecting 0.1 ml of 2.5% (w/v) formalin suspension in 0.9% (w/v) sterile saline into the plantar tissue of the right hind paw of experimental rats, one hour following oral administration of standard drug or test samples. Increase in paw circumference was measured at 0.5hr, 1hr, 2hrs, 3hrs and 4hrs after the challenge, using the method described by Tjolsen et al. [22]. The percentage inhibition of inflammation was calculated as follows: Where, V t = is the average paw edema circumference of each extract treated group, as well as diclofenac sodium group; V c = is the paw circumference of the negative control group that only received the vehicle.

Total Phenolic and Flavonoid Content
The total phenolic content (TPC) of the samples were calculated on the basis of the regression calibration curve, y = 0.

In vitro Antioxidant Assay
Effect on DPPH Radical-Scavenging Activity: The results of DPPH radical scavenging activity of CME and various fractions were shown in Figure 1 as comparable with reference standard ascorbic acid (AA  (Figure 2). Standard AA had higher scavenging activity than CME and all the fractions but EAF showed DPPH scavenging activity which was close to standard AA.

Figure 1:
The scavenging effects of CME and its various fractions on DPPH radical.

In vivo Anti-inflammatory Activity
Carrageenan-Induced Paw Edema Test:

Discussion
The present study showed the antioxidant and anti- Several studies proposed that phenolic compounds are responsible for effective free radical scavenging and antioxidant activities [23].
Therefore, determination of the quantity of phenolic compounds is very important in order to determine the antioxidant capacity of plant extracts [24]. In the present study, higher concentration of polyphenols was found in EAF and CHF, suggesting the importance of EAF and CHF for effective antioxidant activities in traditional medicine. A number of methods are used to determine the radical scavenging effects of antioxidants. The DPPH method is a preferred method because it is fast, easy and reliable and does not require a special reaction and device. DPPH is a stable free radical, capable of accepting an electron or hydrogen radical to become a stable diamagnetic molecule [25].
Antioxidant compounds specially, phenolics present in the extract lose hydrogen which is received by DPPH and discoloration occurs due to the decreasing quantity of DPPH radicals in the environment. The discoloration of the DPPH therefore reflects the radical scavenging activity of the analyzed extract [26]. The higher content of total phenolic compounds and flavonoid in the extracts might have contributed towards the scavenging of free radicals [27]. CHF, which showed 2 nd highest for the content of

Conclusion
On the basis of results in this study, it can be concluded that the methanol extracts specially, EAF/CHF of B. acuminata stem bark is capable of scavenging a wide range of synthetic and naturally occurring free radicals and could be utilized as a good natural source of antioxidants. In addition, the results indicate that phenolic compounds might be major contributors to the antioxidant and anti-inflammatory activities of stem bark. Hence, this study supports the idea that B. acuminata stem bark can be a promising sources of potential antioxidants that can participate in fighting inflammation through the scavenging of reactive oxygen species. This preliminary study also warrants a need for further research to isolate, identify and characterize the specific phenolic compounds which may contribute in the therapy of diseases related to oxidative stress.