INTRODUCTION:

Standardization and authentication of medicinal plants is the major problem for traditional medical practitioners. Pharmacognosy, in recent years has gained immense importance as it is an efficient tool for the authentication and identification of plant raw materials^[¹^]. The traditional medicine all over the world is nowadays revalued by an extensive activity of research on different plant species and their therapeutic principles. In the past few years, natural antioxidants have generated considerable interest in preventive medicine.

The human body has a complex system of natural enzymatic and non-enzymatic antioxidant defenses which counteract the harmful effects of free radicals and other oxidants. Free radicals, from both endogenous and exogenous sources, are implicated in the etiology of several degenerative diseases^[²^]. The intake of natural antioxidants or other compounds that can neutralize free radicals may be of central importance in the prevention of vascular diseases, some forms of cancer^[³^] and oxidative stress responsible for DNA, protein and membrane damage^[⁴^]. Plants (fruits, vegetables, medicinal herbs, etc.) may contain a wide variety of free radical scavenging molecules, such as phenolic compounds (e.g. phenolic acids, flavonoids, quinones, coumarins, lignans, stilbenes, tannins), nitrogen compounds (alkaloids, amines, betalains), vitamins, terpenoids (including carotenoids), and some other endogenous metabolites, which are rich in antioxidant activity^[⁵^,⁶^]. Therefore, much attention has been focused on the use of natural antioxidants to protect the damage of free radicals.

The genus Bauhinia consists of approximately 300 species, are widely distributed in most tropical countries, including Africa, Asia and South America. Their leaves and stem-bark have been used frequently in folk medicine as a remedy for different kinds of pathologies, particularly diabetes, infections, pain and inflammatory processes. The biological properties of different Bauhinia spp. phytopreparations and pure metabolites have been investigated in numerous experimental in vivo and in vitro models. The secondary metabolites produced by this genus, particularly the poly-phenols, make the plants under this genus an important source of potential phytotherapeutic and medicinal agents.

Bauhinia variegata Linn. (family: Leguminosae) is a medium sized, deciduous tree, found throughout India. Also called as Rakta Kanchan (Sanskrti), Mandarai (Tamil), Kanchnar (Hindi) and Mountain Ebony (English)^[⁷^]. The leaves are 10-15 cm long and broad, subcoriaceous and deeply cordate. The flowers are large, fragrant, white or purplish, appearing when the tree is leafless. The pods are 15-30 by 1.8-2.5 cm hard, flat, dehiscent and 10-15 seeded^[⁷^,⁸^]. The various parts of the plant viz., flower buds, flowers, stem, stem bark, leaves, seeds and roots are practiced in various indigenous systems of medicine and popular among the various ethnic groups in India for the cure of variety of ailments.

The bark was previously studied for astringent, tonic to the liver^[⁹^] antileprotic, antigoitrogenic^[¹⁰^,¹¹^], antitumour^[¹²^] and used in fever, skin disease^[¹³^] and wound healing^[¹⁴^]. It is also reported to be used in obesity.^[¹⁵^] The leaves are used in treatment of skin disease, ulcers ^[¹⁶^] and stomatitis^[¹⁷^]. The roots of the plant were traditionally used as an antidote for snake poisoning, in dyspepsia, flatulence and as carminative^[¹⁸^]. The dried buds are used for the treatment of diarrhea and dysentery, worms, piles and tumors ^[¹⁹^]

Keeping the above into consideration a study was carried out to evaluate and compare antioxidant activity of successive extracts of bark powder such as as n-hexane, dichloromethane, chloroform, ethyl acetate, methanol and hydro alcoholic. Also this work was undertaken to assess photochemical, pharmacognostic parameters and physicochemical properties of the stem bark of plant.

MATERIALS AND METHODS:

Chemicals and reagents:

Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2- carboxylic acid), ABTS (2, 2’-azinobis (3-ethyl benzothiazoline-6-sulfonic acid) diammonium salt, gallic acid (GA) and potassium persulfate were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). Ferric chloride was purchased from Merck, India. Folin-ciocalteu reagent was purchased from SD Fine Chemicals, Mumbai, India. All solutions were freshly prepared in distilled water. Other reagents and solvents used were of analytical grade and purchased from local vendors.

Sample collection and extraction:

Stem bark of B. variegata was collected from botanical garden of Saurashtra University, Rajkot, Gujarat, India. The bark was washed with distilled water and cut into small pieces and then air dried under shade. The plant was taxonomically identified and authenticated by Dr. Sunita Garg, National Institute of Science Communication and Information Resources (NISCAIR), New Delhi, India. A voucher specimen (SU/DPS/HERB/61) was submitted in the depository of Department of Pharmaceutical Sciences, Saurashtra University, Rajkot, Gujarat, India for future reference. The stem bark of B. variegata was coarsely powdered and passed through sieve (#60). The obtained powder (100 g) was then sequentially extracted with different solvents by increasing in polarity order i.e. n-Hexane (BNE), dichloromethane (BDE), chloroform (BCE), ethyl acetate (BEE), methanol (BME), hydro alcohol (BHE) for 48 hr at ≤ 40 °C using soxhlet apparatus^[²⁰^]. The powdered material was dried each time before extracting with next solvent in oven below 50 °C. After filtration, the resultant extracts were concentrated under reduced pressure at room temperature using rotary flash vacuum evaporator. The final obtained extracts were weighed with the solvent and calculate its percentage in terms of the air-dried weight of the plant material. Further, the concentrated extract was dried in desiccator and stored in vacuum sealed air tight containers at -20 °C until further use.

Pharmacognostic evaluation:

The fresh stem bark of B. variegata was studied for external visible characters such as size, shape, surface, fracture and organoleptic properties. The microscopic study was performed as per basic micro-technique and staining was done as per safranin-fast green protocol^[²¹^]. The microscopic studies were carried out using microscope camera from leica microsystem, Germany. The section was then viewed under 10X and 40X.

The powder of stem bark was subjected to powder microscopy. Powder was first cleared with chloral hydrate and then stained with staining reagents phloroglucinol and concentrated HCl and then viewed under 10X and 40X.

Physicochemical evaluation:

Physicochemical evaluation like total ash value, acid insoluble ash value, water soluble ash value, loss on drying were determined as describe in official books^[²²^].

Preliminary phytochemical screening:

Phytochemical screening of B. variegate stem bark extracts was performed for the qualitative detection of various phyto-constituents like alkaloids, glycosides, carbohydrates, phenolic and tannins, phytosterols, fixed oils, amino acids, flavonoids, saponins, gums and mucilage using reported method.^[²³^]

Determination of the total phenolic content:

The total phenolic content in various extracts were quantified by colorimetric assay using Folin-ciocalteau reagent (FCR) as described by Singleton and coworker^[²⁴^]. All dried different solvent extracts were dissolved in dimethylsulfoxide (DMSO) to obtain a concentration of 1 mg/mL. The 100 µL of this solution was taken into 25 mL volumetric flask, to which 10 mL of water and 1.5 mL of FCR were added. The resulting mixture was then kept for 5 min at room temperature, followed by addition of 4 mL of sodium carbonate (20% w/v) solution. The final volume was made up to 25 mL with double distilled water. The mixture was kept for 30 min until blue color was developed. The absorbance of samples was then measured at 765 nm in UV- visible spectrometer (Shimadzu, UV-1700, Japan). All the estimations were done in triplicate. Phenolic content was calculated with gallic acid as the standard and expressed as mg of gallic acid equivalent per gm of dry extract.

Antioxidant assays:

ABTS radical cation decolorization assay:

The free radical scavenging activity of different extracts were estimated by ABTS radical cation decolorization assay as described by Miller^[²⁵^] with minor modifications. Briefly, ABTS and potassium persulfate were dissolved in distilled water to a final concentration of 7 mM and 2.45 mM respectively and kept for 12 to 15 hrs in the dark at room temperature to produce ABTS radical (ABTS^•+). For the study of different extracts the ABTS radical solution was diluted with distilled water to an absorbance of 0.7 (± 0.02) at 734 nm. An appropriate solvent blank reading was taken (A_B). After the addition of 100 μL of all different solvent extract to 3 mL of ABTS^•+ solution and the absorbance were taken at 6 min after through mixing using the spectrophotometer. All solutions were freshly prepared, and all determinations were carried out in triplicate. The percentage of inhibition of ABTS^•+ was calculated using below formula. Calibration curve was obtained with Trolox (0-33 µM).

A_B - A_E

% Inhibition =--------------- X 100

A_B

Where

A_B = absorbance of blank sample and

A_E= absorbance of standard/plant extracts at 6 min.

FRAP assay:

The ferric reducing antioxidant potential (FRAP) of the plant extract was measured by the method described by Benzie and Strain.^[²⁶^] The working FRAP reagent was freshly prepared by mixing 200 mL acetate buffer (300mM, pH 3.6), 20 mL TPTZ (2,4,6-tri(2-pyridyl)-s-triazine) (10mM) in 40mM HCl, 20 mL ferric chloride (FeCl₃) (20mM) solution and 24 mL distilled water. A standard curve was prepared using various concentrations of FeSO₄× 7H₂O (0.1–1mM). An aliquot (25 µL) of the appropriately diluted extract was added to 700 µL of the freshly prepared working FRAP reagent and the absorbance was measured at 593 nm at 0 min and after 30 min standing at room temperature. An intense blue color complex was formed when ferric tripyridyl triazine (Fe³⁺ TPTZ) complex was reduced to ferrous (Fe²⁺) form against a reagent blank (700 µL FRAP reagent + 25 μL distilled water) after 30 min incubation at 37 °C. All the determinations were performed in triplicates. The FRAP values were obtained by comparing the absorbance change in the test mixture with those obtained from increasing concentrations of Fe³⁺ and expressed as µMol of Fe (II) equivalent per gram of sample (y = 0.853x - 0.012; R² = 0.999).

Statistical analysis:

Experimental results from the present study were expressed as means ± SD of three parallel measurements. Linear regression analysis was done to findout the correlation coeﬃcient. Correlation and regression analysis was carried out using Microsoft Excel and Graph Pad Prism (Version 5.0) software.

RESULTS AND DISCUSSION:

Macroscopic evaluation of bark:

A curved or channeled bark pieces of about 1 to 10 cm in length, 3 to 5 cm in width and 5 to 10 mm in thickness. B. variegata stem bark is grayish brown externally while reddish yellow internally. The outer surface is rough, longitudinally and transversely fissured and cracked, exposing the inner reddish bark. The inner surface is hard, smooth to faintly longitudinally striate with outer short and inner fibrous fracture (Figure 1). Odor is characteristic and has astringent taste. All the above macroscopic characters were used to identify and authenticate the bark and also helpful to distinguish from adulterants.

Figure 1: Macroscopy of Bauhinia variegata stem bark

Microscopic evaluation of bark:

Transverse section of the bark shows multilayered cork, cortex and very wide phloem traversed with sclereids, and medullary rays (Figure 2). Tangentially running alternate bands of fibers and parenchymatous phloem tissue runs throughout the inner zone of the phloem. Pinkish powder of the bark under microscope showed abundant crystals of calcium oxalate, bits of fibers, cork and secondary cortex cells, containing colored content (Figure 3). Both transverse section and powder microscopy further help to distinguish both bark and its powder form adulterants.

Figure 2: Transverse section of Bauhinia variegara stem bark. Section was stained with double staining technique. Main section magnification =50X and magnified view = 400X.

Physicochemical parameters:

The physicochemical characters are important parameters for detecting adulteration or improper handling of drugs. Various physicochemical parameters like different ash values and loss on drying were determined. The results were summarized in Table 1. These qualities help in the evaluation of purity of drugs.

Table 1: Physicochemical parameters of B. variegata stem bark

Sr. No.	Determinations	Value obtained (%w/w)
1	Total ash	9
2	Acid insoluble ash	0.16
3	Water soluble ash	7
4	LOD	7.26

Figure 3: Powder microscopy of Bauhinia variegata stem bark.

Preliminary phytochemical screening:

Different phytochemical have different physicochemical properties, such as solubility, type of nature, etc. It was found that different solvent extracts from stem bark of B. variegata contained different chemical constituents. Results were described in Table 2. The flavonoid, alkaloids, phenol and tannin were observed in polar extracts (BEE, BME and BHE) whereas glycoside and phytosterol were observed in most polar extracts (BME and BHE), while terpenoids was present in BDE and BCE extracts. All the extracts tested positive for carbohydrates. None of the extracts showed presence of all the phytoconstituents tested.

Table 2: Phytochemical screening of different extracts of stem bark of B. variegata.

	n- Hexane	DCM	CHCl₃	EA	MeOH	HA
Alkaloids	˗	˗	+	+	+	-
Glycoside	˗	˗	˗	˗	+	+
Saponin	˗	˗	˗	˗	˗	˗
Phytosterol	˗	˗	˗	˗	+	+
Phenol and Tannin	˗	˗	˗	+	+	+
Protein	˗	˗	˗	˗	+	+
Fixed Oil	+	+	+	+	+	-
Carbohydrate	-	-	+	+	+	+
Gum and Mucilage	-	˗	˗	˗	˗	+
Flavanoids	˗	˗	˗	+	+	+
Terpenoids	˗	+	+	˗	˗	˗

+ present, - absent

DCM: Dichloromethane, CHCl₃: Chloroform, EA: Ethyl acetate, MeOH: Methanol, HA: Hydro-alcohol

Total phenolic content and recovery percent of B. variegata extracts:

Table 3 depicts the total phenolic contents as well as % yield of the different solvent extracts of B. variegata. The % yield of extract obtained from 100 g of dry plant material was estimated for each extract. The amount of extractable compounds, expressed as percentage by weight of dried powder. The lowest and highest % yield was found to be 0.13% of the chloroform extract and 4.78% of the methanol extract, respectively. The total phenolic content was ranged from 1.24 – 15.44 mg GA/g (y = 0.015x + 0.027; R² = 0.994). The highest concentration of phenols was measured in methanolic, hydroalcoholic and ethyl acetate extracts. Particularly, chloroform, dichloromethane and n-hexane extracts contained considerably less concentration of phenols.

Table 3: The yields extraction and total phenolic contents in different extracts of stem bark of B. variegata

*B. variegata* Extracts	Yields (%)	Total Phenolic content^a (mg GA/g)^b	ABTS (IC₅₀ µg/mL)^a	FRAP^a [Fe(II) equivalent µMol /g of extract]
BNE	0.13	1.24 ± 0.398	>600	188.75 ± 12.90
BDE	0.44	3.10 ± 0.270	>600	227.04 ± 11.14
BCE	0.75	5.11 ± 0.413	571.06 ± 19.10	411.49 ± 16.12
BEE	1.03	9.16 ± 0.331	520.29 ± 16.23	527.55 ± 10.02
BME	4.78	15.44 ± 0.572	269.57 ± 14.89	705.74 ± 24.62
BHE	3.28	10.18 ± 0.375	446.65 ± 15.24	577.96 ± 18.20
Trolox			14.24 ± 1.5

^aEach value represents the mean ± SD of three experiments.

^bmg of gallic acid equivalent per g of dry stem bark extract.

Antioxidant activity of B. variegata extracts:

ABTS assay:

ABTS assay is better to assess the antiradical capacity of both hydrophilic and lipophilic antioxidant because it can be used in both organic and aqueous solvent system as compared to other antioxidant assay. The decolorization of the ABTS^•+, through measuring the reduction of the radical cation as the percentage inhibition of absorbance at 734 nm^[²⁷^]. ABTS^•+ was generated by incubating ABTS^•+ chromophore through the reaction^[²⁸^]. The presence of specific chemical compounds in the extracts of B. variegata may inhibit the potassium persulfate activity and hence reduced the production of ABTS^•+. In the present work, different solvent extracts of B. variegata were evaluated for their ABTS radical cation scavenging activity. Trolox was used as standard and itʹs IC₅₀ values was 0.552 µMol. From Table 3, the order of free radical scavenging ability of the extracts was found to be as follows, BME > BHE > BEE > BCE > BDE > BNE, which were consistent with FRAP assay. The BME possess a significant free radical scavenging ability. IC₅₀ values ranged from 0.270 to 2.209 mg/mL. Methanolic extract showed good ABTS radical cation scavenging activity, Hydroalcoholic, ethyl acetate and chloroform extracts showed moderate radical cation scavenging activity where as hexane and dichloromethane extracts showed poor ABTS radical cation scavenging activity.

B. variegata extracts was found to be effective in scavenging the ABTS radical. The percentage inhibition of this radical was concentration-dependent. Lower IC₅₀ value in ABTS assay showed positive correlation between scavenging power and higher phenolic content in B. variegata extracts (Fig. 4a)

FRAP assay:

This assay measures the reducing power of a potential antioxidant which is based on the reduction of Fe³⁺ TPTZ complex (Yellow/colorless complex) to Fe²⁺ -tripyridyltriazine (blue colored complex) formed by the action of electron donating antioxidants at low pH^[²⁹^]. The reducing properties associated with the presence of compounds exert their action by breaking the free radical chain through donating a hydrogen atom^[³⁰^]. The ability of extract to reduce iron (FRAP) suggests that they contain compounds that are electron donors, which can react with free radicals to convert them to more stable products and terminate radical chain reaction.

Figure 5a shows the range of differences in the antioxidant activity of the extracts from B. variegata by the FRAP assay. The order of the antioxidant activity was as follows: BME>BHE>BEE> BCE> BDE> BNE. From the results, the methanolic extract contained substantial ferric reducing activities compared to chloroform, dichloromethane and n-hexane extracts (Table 3). Thus, it was inferred that highest amount of the antioxidant components from B. variegata were soluble in methanol.

FRAP assay showed positive correlation between reducing power and phenolic content in B. variegata extracts (Fig. 5a). It was reported by Rice-Evans et al. that phenolic compounds have redox properties, which allow them to act as reducing agents, hydrogen donators, and singlet oxygen quenchers^[³⁰^]. The redox potential of phenolic compounds played an important role in determining the antioxidant potential.

Correlation study:

Different phytoconstituents and condition of testing greatly influence the antioxidant potential of plant extracts. Antioxidant assay can be broadly classified in to two types: assays based on electron transfer (ET) and assays based on hydrogen atom transfer (HAT). HAT-based assays (ORAC assay) apply a competitive reaction scheme, in which antioxidant and substrate compete for thermally generated peroxyl radicals. ET-based assays measure the capacity of an antioxidant to reduce an oxidant, which changes color when reduced. The degree of color change is correlated with the sample’s antioxidant concentration. ET-based assays include the total phenols assay by Folin-Ciocalteu reagent, DPPH and ABTS radical scavenging capacity assays, the SOD assay, and the FRAP assay. In current study we have studied total phenol, ABTS radical scavenging and FRAP assay. Pearson correlation study indicates a relationship between total phenol v/s ABTS (Figure 4b) and total phenol v/s FRAP assay (Figure 5b). Both the correlation studies are in agreement with previous studies^[³¹^]. Most of the phenolics are composed of one (or more) aromatic rings bearing one or more hydroxyl groups and are therefore potentially able to quench free radicals by forming resonance-stabilized phenoxyl radicals which make them great antioxidant^[³²^,³³^]. Asignificant correlation between antioxidant properties and total phenolic content was found, indicating that phenolic compounds are the major contributor to the antioxidant properties of B. variegata.

Figure 4: Correlation between radical scavenging capacity assay (ABTS) and total phenolic content. A) Direct correlation of total phenolic content v/s ABTS radical scavenging activity; B) Correlation between ABTS radical scavenging activity and total phenolic content. Pearson R = -0.8438

Figure 5: Correlation between FRAP and total phenolic content. A) Direct correlation of total phenolic content v/s FRAP; B) Pearson Correlation between ABTS radical scavenging activity and total phenolic content. Pearson R = 0.9774

CONCLUSION:

All antioxidants have a chemical element referred to as a “redox” potential, which is the measurement of their ability to be oxidized. Considering the fact that the redox equilibrium is important to the body’s coping mechanism, it follows that antioxidants can influence many health conditions^[³⁴^]. In the present investigations, the pharmacognostical and physicochemical characteristics of the stem bark of B. variegata were studied. Phytochemical parmeters will help in controlling the standards and quality of the raw material of stem bark of B. variegata. The study adds a systematic comparative record on the relative free radical scavenging activity in different solvent extract of the stem bark of B. variegata. Our findings suggest that methanol, hydroalcoholic and ethyl acetate extracts have the property to reduce the production of ABTS^•+ and Ferric reducing power. Also the methanolic extract showed an edge better activity than hydroalcoholic and ethyl acetate extract even at very low concentrations which can be attributed to the phenolic compounds present in the extract. Further, detailed work on isolation and characterization of specific chemical moieties from the B. variegata bark extracts and their biological testing can provide us with effective nontoxic antioxidants agents in future.

CONFLICT OF INTEREST:

The authors declare that there are no any conflicts of interest. The authors alone are responsible for the content and writing of the paper.

ACKNOWLEDGEMENTS:

Authors are highly thankful to UGC, New Delhi for providing financial assistance in the form of research fellowship and contingency grant. The authors are thankful to Mr. Samir Rabadiya, Assistant Professor at Department of Pharmaceutical Sciences, Saurashtra University, Rajkot for their continuous technical support.

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Received on 04.04.2018 Modified on 19.05.2018

Research J. Pharm. and Tech 2018; 11(8): 3250-3256.

DOI: 10.5958/0974-360X.2018.00597.8