Exploring Efficacy of Bauhinia Variegata as Medicinal Herb in Combating Different Clinical Conditions: A Systematic Review

Introduction

Natural and herbal remedies are gaining huge attraction due to the presence of therapeutically active constituents. This traditional therapy has less toxic effects as compared to allopathy which endorses its use in various health ailments. Nowadays, the popularity of using herbal remedies in the treatment of diseases has been augmented severalfold. According to the recent report of the World Health Organization (WHO), 80% of the world population is shifting towards herbal approaches for treating diseases [1]. Phytoconstituents of the plants are responsible for all therapeutic activities. Due to India’s substantial biodiversity, it is one of the largest producers of herbs and their derived products. Bauhinia variegata is one such plant that finds therapeutic usefulness in the management of diabetes, leprosy, piles, asthma, skin disease, ulcers, wound healing, and obesity. The seeds of the plant contain proteins in the lectins and trypsin inhibitors [2, 3]. Polyphenols from the fruits of Bauhinia species represent a matchless class of antioxidants to counter reactive oxygen species (ROS) (Figure 1).

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B. variegata (orchid) belonging to the family Caesalpiniaceae is a leguminous, medium-sized deciduous tree that extends all over India and is found at the altitude of 1800 m in the Great Himalayas [4, 5]. Kachnar (Hindi) and Mountain ebony (English) are its vernacular names. It is a hermaphrodite flower-bearing plant that generally blooms from November till the end of March. It is cultivated by seeds that get easily dispersed by air. It has connate leaves which are rounded at the apex with a length of 10-15 cm, white or pink coloured flowers, flat dehiscent fruits, and 10-15 seeds [4]. It is one of the traditional plants consisting of various phytochemicals like tannins, saponins, carbohydrates, alkaloids, resins, terpenoids, steroids, proteins, flavonoids, cardiac glycosides, etc. [6].

Phytochemical screening is generally employed to confirm the presence of therapeutically active constituents in the alcoholic extract of plant parts. Different parts of the plant such as bark, seeds, leaves, and the type of solvent/solvent mixture used for extraction determine the phytochemicals present in the extract. These phytochemicals are utilized as anti-helminthic [7], hepatoprotective [8], antioxidant [9], anti-microbial [6], anti-inflammatory, anti-diabetic [10, 11], anti-cancer [12], wound-healing [4] and ulcer-protective [13].

With the advancement of various separation and characterization techniques like FTIR, NMR, LC-MS, 2D- LC, gel electrophoresis, digestion techniques, column chromatography, etc, it has become relatively easier to fractionate and characterize the crude plant material extracts and isolate the phytoactive in the purest form.

Lectin present in the seed of B. variegata is is a non- immune protein or glycoprotein which has bio-adhesive properties towards the cell membrane. It is a proven potent antibacterial agent and is being used in a variety of formulations. The plant also possesses antioxidants like kaempferol-3 glucoside, lupeol, beta-sitosterol, and other flavones. The ID7 (column fraction from the bark) fraction isolated from B. variegata has been evaluated for anti-tumor activity [14]. 21,000 plants were listed as per the World Health Organization (WHO) which is being used for various medicinal purposes, out of which 2500 species were found in India and 800 herbs were linked with anti-diabetic activity [15].

Phytoconstituents like glucokinins, inorganic ions sulphur compounds, coumarins, phenolic compounds (polyphenols, flavonoids), steroids, amines, peptides, terpenes, glycopeptides, and polysaccharides are associated with hypoglycemic effects. Glucokinins possess functional similarity with insulin [16].

Chemical Constituents and their Extraction

Certain quantification tests have been suggested for the presence of phytoconstituents. The determination of these compounds helps in the selection of plants for in-depth future investigation. Table 1 represents the various constituents in the extract of B. variegata plant parts such as leaves, bark, and flowers [12].

Table 1 clearly indicates the abundance of tannins, flavonoids, and polyphenols in B. variegata. The stem bark and flowers also contain alkaloids and saponins while the leaves do not have these constituents. The presence of these constituents can also be correlated to their therapeutic activities; for example, high antimicrobial activity has been detected to a greater extent in the aqueous-alcoholic extract of bark and flowers than in leaves. Also, some unique aqueous/organic ratios have been suggested to impart reproducibility in the extract and isolation outcomes of the B. variegata bark [17].

Extraction Procedures

Take 20 gm of Methanol extract of bark (MEB) and subject it to silica gel (60-120 mesh) column chromatography, eluting with a gradient of Hexane: EtOAc (100:0 to 0:100).

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Figure 2: Extraction procedure of Bauhinia variegate. MEB-Methanol extract of bark, BV- Bauhinia variegata, EtOAc: Ethyl Acetate MEB, MEB-I, and MEB-II are obtained from column chromatography of methanol extract of bark (MEB) of B. variegata whereas BV1, BV2, and BV3 are the pure isolates obtained (Figure 2). The ethanolic bark extract of B. variegata was explored against Cisplatin and was found to play a prime role in treating triple negative breast cancer (TNBC).

In the case of cancer, the continuous metastasis of the cancer cells is the cause of the spread of cancer and related deaths. In the research, the anti-tumor activity of ID7 (column fraction from the bark) was evaluated by performing in-vitro and in-vivo assays. In-vitro methods include cell viability assays like murine cell line assay, propidium iodide/acridine orange dual staining, and human cell viability assay wherein the absorbance for formazan crystals can be determined spectroscopically, and cell migration assay [18].

Similarly, a distinctive purification has been adopted for lectin fractionation from the seeds of B. variegata. The slurry formed by seed homogenization was subjected to centrifugation twice and the supernatant was collected as crude seed extract. This seed extract was then charged onto the affinity y gel blue gel column (preconditioned by Tris HCl buffer) after adjusting the pH to 7.6 by 10 mM Tris buffer.

The unretained or early eluting fraction was collected and loaded to another column called Q- Sepharose which was subsequently eluted with Tris HCl buffer containing 1N NaCl to obtain the bound fraction. The eluent obtained was lyophilized. The lyophilized powder was resuspended into a 10mM Tris buffer and loaded on the Mono Q column. The bound components were eluted by NaCl gradient. The last peak in the absorbance spectra containing the highest lectin content was collected and lyophilized. The powder was redissolved and loaded to Superdex 75 HR 10/30 column and purified lectins were recovered.

An ointment is a topical preparation that is generally medicated i.e., a pharmaceutical preparation in which the drug or Active Pharmaceutical Ingredient (API) is diffused in the medium. Depending upon the type of base used for ointment preparation there are different types of ointment such as hydrophilic based or water-miscible ointment, oleaginous (greasy) based ointment, and water repellent- based ointment. In a recently published study by Rajput R and Kashmira G [4], the plant has been explored for its wound healing ability in rat models. The crude drug was collected and cleaned with water followed by drying in a hot air oven at 35 0C to protect the phytoconstituents present in the plant parts. An extract in water was prepared using the maceration technique in which 300g of the powdered drug was placed in water for 7 days at a temperature of 37 0C. After 7 days the extract was filtered and concentrated. Herbomineral ointment was prepared by emulsifying the oleaginous phase in the aqueous phase.

Nowadays an increasing number of infectious agents are becoming resistant to commercial antimicrobial compounds [19]. Also, synthetic drugs have many side effects and are an emerging class of environmental contaminants. Mankind is facing challenges of emerging resistance in virtually all pathogens. Phytochemicals from natural sources like plants have fewer side effects and hence present a safer option for formulating various antimicrobial/anticancer drugs. Previous work has reported the isolation of quercetin, rutin, apigenin, and 7-O-glucoside from B. variegate [20], but none of them categorized the extracted phytoconstituents to specific aqueous/ organic ratio to impart better reproducibility for obtaining pure fractions.

Moreover, there are numerous natural lectins present in the seeds of B. variegate. It is not easy to separate them using the conventional extraction protocols. Two major challenges commonly faced during the extraction of these lectins from the homogenized fraction of B. variegata seeds are the separation of the lectin fraction from the rest of the seed constituents i.e., other proteins, carbohydrates, etc, and purifying of the protein fraction into individual lectins.

B. variegata Lectin (BVL-1), a single chain lectin, obtained from the seeds, has been employed for its anti- bacterial activity. BVL-1 interacts strongly with metastatic tumor cells and inhibits cell adhesion and proliferation. Lectins additionally recover wounds and injuries by increasing epithelial reconstruction, keratin deposition, and stimulating the growth of fibroblastic cells [21]. Due to limited native lectins, recombinant lectin proteins (rBVL-lp) using Pichia pastoris, a methylotrophic yeast were produced as a large-scale production alternative. Recombinant lectin proteins were expressed in Escherichia coli. It is also suitably used for avoiding and treating dental caries at early stages by blocking microbial adhesions. The mechanism of oral bacteria inhibition prevention is still not clear but the secretion of many extracellular polysaccharides by the bacteria mediates the adhesion process.

Characterization

The stem bark of B. variegata consists of constituents presented in Figure 3. β-Sitosterol has a melting point of 136-139○C. It gives red colour in Salkowski’s test and green colour in Liebermann-Burchard’s test. Kaempferol has a melting point of 275-277○C, it responds at λmax265 and 267 nm; Quercetin is a yellow amorphous powder, has a melting point of 311-313○C and responds at 369nm. Lupeol is a white amorphous solid having a melting point of 211-213○C and gives a positive response to Liebermann-Burchard’s test for triterpenoids.

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Applications of B. Variegata Extract in Various Clinical Conditions

Medicinal plants have been well reported for many indications, however, when a single plant consists of a mixture of active constituents in different parts, proper isolation of individual constituents is essential. Moreover, the isolated substance cannot be directly applied or ingested by an individual because of its physicochemical incompatibilities and palatability issues. The development of a formulation in view of its applicability and acceptability to the patient is the prime challenge for pharmaceutical formulation scientists.

The preformulation parameters such as physicochemical properties, stability parameters, and organoleptic behavior should also be considered before designing any formulation. These parameters decide the type of dosage form to be made for the drug or active constituents, whether they are derived synthetically or naturally. In the case of topical preparations, the parameters such as spread ability, wettability, angle of contact, and retaining ability are to be considered.

The stem bark of B. variegata has been reported for wound healing, fever-aid, astringent, goitre, dysmenorrhoea, menorrhagia, tumours, leukemia, etc. Leaf extract is also being used as an antioxidant and antidiabetic. B. variegata is being explored as an herbal alternative to synthetic or allopathic medications.

Discussion

The leaf extract of plant Bauhinia variegata was found to possess excellent and comparable antioxidant property with respect to synthetic antioxidants. The designed W/O emulsion had shown good and promising physical characteristics when stored at different storage conditions. The emulsion formulation tested for stability evaluation involving organoleptic evaluation, centrifugation assay, microscopy and rheology was found to be meeting the industry standards. The different protein fractions obtained at different purification procedures were further separated on the basis of their molecular weight by running on SDS- PAGE [37]. The species differentiation has been done on the basis of proteome characterization. The lysate extract was subjected to in solution trypsin digestion and subsequent analysis by High Resolution Mass Spectrometry to characterize and differentiate the proteins present in various herbal medicines with the hypoglycemic agents claim.

In a recently published study by Gupta P, Singh S and Tripathi A in Dec 2019 Bauhinia variegata nanoemulsion has been prepared and explored for its prophylactic effect in diabetes induced peripheral neuropathic pain on experimental rat model [38]. The flower extract was prepared in petroleum ether to remove impurities and 90% ethanol was used to extract the phytoconstituents using hot percolation method or Soxhlet extraction. The extract was filtered and concentrated under vacuum. The prepared emulsions were characterized for particle size, particle morphology, zeta potential, in-vitro release, stability studies and biological evaluation on STZ (streptozotocin) induced diabetes in rat model [39, 40] for assessing protective effect on diabetic neuropathic pain [41]. Plantar and Vonfrey filament stimulation test was performed and the results were compared. In another recently published study [42] the physical interactions of the lectins of Bauhinia variegata (BVL) with the phosphatidylcholine (PC) liposomes have been examined [43].

Liposomes are another type of formulation being used extensively nowadays for targeted drug delivery. A liposome is a tiny bubble (vesicle), made out of the same material as a cell membrane. They act as carriers for administration of various drugs and herbal remedies etc.

The yield of the ethanolic extract (BVE) prepared from the leaves of B.variegata was found to be 20.8%. In an in-vitro release by Franz Diffusion test, it was observed that a maximum of 92.19 % release from nanoemulsion was achieved in 8 hours. Nanoemulsions prepared using the extract had passed the stability parameters and the biological activity of nanoemulsion was found to be better when compared with results of Bauhinia variegata extract alone treated animal group.

BVN (Bauhinia variegata nanoemulsion) treated animals had shown marked decrease in blood glucose level and prevention in progression of neuropathic pain [42]. Hence BVN can be concluded to be an efficient means for preventing the further progression of the diabetic neuropathy. However further studies are essential to understand the proper mechanism of the Bauhinia variegata nanoemulsion for various pharmacodynamic effects.

Bauhinia variegata lectin and phosphatidylcholine liposomes complex that promoted hardening up of the proteins was determined by circular dichroism spectroscopy and also demonstrated to be very efficient macromolecule- drug carrier for local and systemic administration [44], whereas evaluation of the photo-physics parameters stated that the B. variegata does not affects its ground state electronic parameters in the presence of Phosphatidylcholine liposomes [45].

There are various drugs available in the market that are used for treating various disorders, but these marketed and synthetic drugs are associated with a number of side effects. B. variegata is reported to have anti-depression, antitumor, antioxidant and antibacterial properties. In spite of the availability of various antibacterial drugs, in a recent research it was concluded that native BVL-1 lectins and recombinant BVL-1 lectins were decreasing the agglutination of red blood cells.

The minimal concentration to exert hemagglutination activity for B. variegata lectin was found to be 96ng/ml. It can be a promising natural herbal approach employed for having anti- bacterial activity. It can also be suitably used for avoiding and treating dental caries at early stages by blocking microbial adhesions.

In all the four studies used to compile this review, various parameters have been evaluated for anti- diabetic, antioxidant and anti- hyper lipidemics properties justifying the herbal approach of the plant. B. variegata showed promising results in lowering blood glucose levels against STZ or alloxan induced diabetes.

Ethanolic extract was also found to be associated with decreasing cholesterol, triglycerides and BUN in the animal model. Hepatoprotective and renoprotective effect of the extract was confirmed by measuring serum levels of liver enzymes, creatinine and urea in diabetic rats. In- vitro assays for B. variegata extract, DPPH and hydrogen peroxide showed 86.60% and 68.47% scavenging activity respectively at 100µg/mL while BHT showed 91% scavenging results for DPPH and 73.34% for H2O2 assay. This confirms the antioxidant potential of plants.

The body weights of B. variegata extract treated animals with dose of 250 mg/kg [26] were observed to be reduced as compared to the diabetic and reference control groups so at low dose the plant has also been reported to possess obesity treatment properties related to obesity in type-I diabetes. The plant possessing antioxidant constituents were decreasing the generation of ROS, RNS which was decreasing the stress on the cell therefore preventing neuropathy, nephropathy, and cardiac-myopathy. At a glance, B. variegata leaf and flower extract had shown impressive results for hyper- glycaemia and its associated risk factors [23, 24].

Another outcome of the Hernandez et al was that the glucokinin, expected earlier for the antidiabetic activity, was found to be concentrated only in the chloroplast and not in any other specific structure. Therefore, the properties of lowering of blood glucose level of this plant species must not be correlated with glucokinin and with the chlorogenic acid and isorientin metabolites [17].

Plant derived materials are a rich source of the upcoming therapeutics. The work by Sonam, et al. Have compared the antimicrobial properties of the hydroalcoholic extracts of the stem bark, flowers and leaves of B. variegata quantitatively in terms of presence of various phytoconstituents. The work done by Neha, et al. Isolates purified fractions from the bark of B. Variegata. After characterization, these compounds were found to be Kaempferol, stigmasterol, protocatechuic acid-methyl ester and protocatechuic acid. These compounds were found to possess antioxidant and cytotoxic activities against various cancer cell lines.

In the study done by Pedrete, et al. a total of 131 proteins were identified by in-solution trypsin digestion and subsequent analysis of the surrogate peptides have revealed structures of various proteins exhibiting hypoglycaemic properties. These identified proteins can be of great value in species characterization for such category of the plants [46].

The study by Chan, et al. has demonstrated the extraction of a 64 -KDa protein by extensive column chromatography and was checked to have in vitro-antiproliferative activity on nasopharyngeal carcinoma cells [47].

Conclusion

In conclusion many phytoconstituents have been extracted from the various plant parts of B. variegata exhibiting antitumor, antioxidant and antidiabetic activities. With the advancement in the extraction technologies and characterization techniques even more complex phytoconstituents have been isolated from this plant. These active constituents are not only having potential to aid in the treatment of various indications, but they are also finding their usefulness in the species characterization and environmental condition’s influence studies.

Scope of Future Work

Bauhinia variegata is a very useful plant finding application in various herbal medicines in the form of extracts. Due to the advancement of better extraction and characterization techniques, the time has come to isolate a pure drug-able compound from such plants finding utilization in so many indications. This will not only reduce the consumption bulkiness of the herbal extract for direct consumption but open more routes for their better and effective formulations. In most of the previous studies, leaves bark and flowers have been used to isolate the medicinal phytoconstituents but none of such extraction work has been reported using the stem exudate of the plant. There is a plenty of scope to isolate and characterize the phytoconstituents of the stem exudate of B. variegata.