Pharmacognostical Evaluation and HPTLC Analysis of Two Medicinally important Secondary Metabolites in Bauhinia variegata Leaves from Gorakhpur District in Summer Season

 

Prakash Deep¹*, Dr. Anurag Mishra², Dr. Neeraj Verma³

¹Amity Institute of Pharmacy, Amity University, Lucknow

²Ashoka Institute of Technology and Management, Varanasi

³J K Institute of Technical Education, Lucknow

 

ABSTRACT:

 

 

INTRODUCTION:

Since immemorial time, India has been the center of origin of various tropical and temperate plant species, most of which are still growing in wild or semi wild state. Among gigantic variety of medicinal plants only some important plants have been accepted and are being cultivated. A lot of native, tropical and temperate plants are still underutilized due to lack of knowledge, awareness and market demand. These species have various uses as fruits, vegetables and have therapeutic and medicinal properties also.

 

Bauhinia variegata (Kachnar) is one of them. In the tropical regions of the world, near about 600 species of genus bauhinia, grow.¹ Generally plants of genus bauhinia are planted for their beautiful flowers and ornamental foliage. Bauhinia variegata has emerged from south eastern Asia and grows throughout India and China.^2,3

 

Bauhinia variegata Linn (Fabaceae) also known as  Kanchanara grows upto 10-12 metres, this is deciduous tree.⁴ The tree and bears beautiful, white to pinkish flowers in early spring in February to April.⁵ Bauhinia is known for its partially opened inflorescence which are used as vegetables. Buds are collected 2 to 3 times in a season. In general tree yields 20-25kg flowers. Young buds are used in the preparation of soup and pickles. The buds are rich in phenol and are having very good antioxidant activity.⁶ It is traditionally used in the treatment of piles, bronchitis, dysentery, tumors and ulcer, leprosy.⁷

Different name of Bauhinia variegata in India:

Common English name: Mountain ebony

Hindi: Kaniar, Gurial, Kachnar, Kandan, Khairwal, Padrian, Barial, Khawairaal, Gwiar, Koliar,

Marathi: Kanchan, Raktakanchan, Thaur

Bengali: Raktakanchan

Mundari: Buruju, Burju

Konkani: Kanchan

Malayalam: Kovidaram, Suvannamandaram, Unna

Sanskrit: Asphota, Ashmantaka, Chamari, Chamarika, Champavidala, Girija, Gandari, Kovidara, Kanakaprabha, Kanchanala, Kanchana, Kanchanara, Kanchanaraka, Kantar, Karaka, Karbudara, Karbudara, Kuddala, Raktapushpa, Shonapushpaka, Suvarnara, Svalpakkesara, Tamrapushpa, Uddalaka, Yamalachada, Yugapatraka, Yugmapatra

Uriya: Kosonaro, Ronga-Konjono, Kanjoni, Boroda.

Telugu: Devakanjanamu, Kanjanamu, Bodanta, Mandari, Mandara,

Tamil: Vellaippuvatti, Segappumandarai, Semmandarai, Mandarai.

Urdu: Kachnal

 

Different names in various parts of world:

French: Bauhinie panache, Arbe de saint Thomas.

Canarese: Ayata, Bilikanjivala, Arisinantige,Irkubalitu, Karalabhogi, Kanjivala, Kempumandara, Kempukanjivala, Ulipe, Mandara

Burmese: Bwechin, Bwaycheng

 

Taxonomic hierarchy

Kingdom: Plantae

Division: Tracheophyta

Class: Magnoliposida

Order: Fabales

Family: Fabaceae

Genus: Bauhinia

Species: variegata

 

Phytochemical constituent Main constituents of B. variegata are (Figure 1):

1)    Kaempferol, Kaempferol-3-o-β-D-glycopyranoside, kaempferol 7, 4-dimethyl ether- 3-o- β-D-2 glycopyranoside, isorhamenetin-3-oβ-D-glycopyranoside and triterpene, ombuin, hesperidin, are reported in aerial part of bauhinia.

2)    Kaempferol glycosides, kaempferol-3- glucoside, lupeol, β sitosterol, quercitroside, isoquercitroside, rutoside, myricetol glycoside and Bauhinione (2,7-dimethoxy-3-methyl-9,10 dihydrophenanthrene-1-4, dione) were reported in Stem bark.

3)    Lupeol, kaempferol-3- glucoside, rutin, quercitin alkaloids, lignin, saponins, terpenoids, β-sitosterol, tannins, apigenin, carbohydrates, apigenin-7- o-glucoside amides, reducing sugar, vitamin C, protein, fiber, phosphorus and calcium were reported in leaves.

4)    Fatty oil containing linoleic acid, oleic acid, stearic acid and palmitic acid and protein were reported in seeds.

5)    (2s)- 5,7-dimethoxy-3’-4’- methylene flavone was found in root.^{8, 9}

 

MATERIALS AND METHODS:

Plant Material:

Leaves of Bauhinia variegata were collected from Gorakhpur district, U.P., India, during month of May. Identification and authentication of the plant was done by herbarium department, CSIR-NBRI. A voucher specimen has been submitted in the LWG herbarium.

 

Chemicals:

Analytical grade chemicals used in experimental work and obtained from E. Merck Limited, India and Hi-Media Laboratories, Mumbai, India.

 

 

Kaempferol                             Ombuin

 

β sitosterol  

 

Lupeol

 

Hesperidin

Figure 1 Important chemical constituents of B. variegata

 

Macro and Microscopic Evaluation:

Macroscopical study involves study of morphological characters and organoleptic properties like colour, odour, taste, texture etc. Microscopic evaluations were performed under Magnus MLM microscope.

HPTLC:

HPTLC analysis was performed on 20cm × 10cm HPTLC silica gel 60 F₂₅₄ plates obtained from Merck. lupeol and quercetin were supplied by Sigma-Aldrich (Steinheim, Germany). All the analytical grade chemical used in the experiment and were supplied by Merck (Darmstadt, Germany).

 

Samples and standard compound solutions of known concentrations were prepared with methanol and applied 15 mm above from the bottom, in layers as 6 mm-wide bands using Camag Linomat V automated TLC applicator coupled with nitrogen flow at the rate of 150 nl/s from application syringe. Throughout the experiment these conditions were kept constant.

 

RESULT AND DISCUSSION:

Pharmacognostical evaluation of Bauhinia variegate:

It is 10-15m high with a short, dark trunk and hairy branches, fast growing, flowering tree. It usually grows on a soil with rocks on hill slopes and loamy soil in the valley. It requires slightly acidic soil as it is unable to tolerate salty conditions. The bark externally is grayish brown, rough  (due to presence of fissures transverse cracks, exfoliations) and internally cream colour, smooth surface, (the colour of the inner surface becomes reddish brown on drying).

 

 

Figure 2 Leaf, flower and pod of B. variegata

Morphology:

Leaves of B. variegata are green colored, linear-lanceolate, entire margin, alternate, smooth and glabrous surface, with parallel venation, broad, rounded and bilobed apex, length is up to 15 cm,  9-15 nerved, cleft 1/4 to 1/3 into two obtuse lobes. On drying the colour of leaf changes to brown. Sleep movements are found in leaves. It has minute stipules 1-2 mm, size of petiole is 3-4 cm and it is puberulous to glabrous, lamina is ovate to circular, breadth of lamina is more than length, 11-13 nerves are found, apex is bifurcated and broadly rounded, base is heart shaped, upper surface is glabrous. (Figure 2).

 

Flowers are ornamental, rosy purple coloured, bisexual, fragrant and with five petals (4-6 cm long and 2-3 cm wide). Fruits are brownish in colour, pod like elongated, 6-12 inches in size, with dry/hard covering. Seeds are 15-16 mm in length and 11-13 mm in diameter.¹⁰

 

Microscopy (Figure 3)

 

Figure 3 TS of B. variegata leaf

 

Lamina:

Upper and lower epidermis of lamina is made up of thin walled, single layered, rectangular cells, covered with thin cuticle. Palisade layer consists of columnar cells in double layer, spongy tissues are loosely arranged.

 

Midrib:  

Midrib also contains upper and lower epidermis which consists of thin walled, single layered, rectangular cells having fine cuticle over it. Epidermis is followed by two-three layers of collenchyma cells, which is followed by two-four layers of parenchyma cells of thin walled, it also contains large number of calcium oxalate crystals, vascular bundles are well developed and are shield shaped. Vascular bundles are surrounded by sclerenchymatous cell.

 

Petiole:

Petiole is divided into epidermis which is made up of thin walled, single layered, rectangular cells, which is covered with fine cuticle, two-three layered collenchymatous tissues are present below epidermis, after this two to four layers of parenchyma cells, with large amount of calcium oxalate crystals. Well developed xylem and phloems are present. Underneath xylem phloem two-five layer of sclerenchymatous cells are found.¹¹

 

Table 1: Phytoconstituents in different extracts of Bauhinia variegata:

 

Phytoconstituents of different extracts of Bauhinia variegata:

On the basis of results obtained from preliminary phytochemical screening, presence or absence of certain phytochemicals in the extract, was determined. The tests performed using petroleum ether, chloroform, methanol and aqueous extract. Phytochemical test showed the presence of Tannin, Alkaloid, Steroid, Cardiac glycosides, anthraquinone, saponins, flavonoids, coumarin (results are given in Table 1).

 

Physicochemical parameters Bauhinia variegata:

Different physico-chemical parameters were checked like total ash, water-soluble ash, acid insoluble ash, alcohol soluble extractive and water soluble extractives were carried out as per standard procedure, Figure 4.

 

Figure 4: Physicochemical parameters of Bauhinia variegata

 

Leaf constants:

Different parameters of leaf constants were performed as per the standard procedures, during the study palisade ratio was found 7.6, stomatal index was 5.27, vein termination no. was 6.6and vein islet no. was 8.3.

 

Figure 5 (A) HPTLC profiling of B. variegata extract with standard quercetin and lupeol at 366nm; (B) Chromatogram of B. variegata extract; (C) Chromatogram of standards (peak 1-quercetin and peak 2-lupeol)

 

Table 2: Instrumentation and Chromatographic Conditions

 

HPTLC analysis:

Characteristic HPTLC fingerprinting of  B. variegata extract was developed with the identification and quantification of two medicinally important biomarkers including lupeol and quercetin as shown in (Figure 5) chromatographic conditions and instrumentation details are given in Table 2

 

HPTLC profile of B. variegata extract showed distinct spots at Rf value 0.5 and 0.77 for quercetin and lupeol respectively.

 

Solvent system toluene: ethyl acetate: formic acid (8.5:1.5:0.5 v/v/v) demonstrated the compact spots with good resolution for standard compounds between other peaks of extract. At six point calibration curve separation and estimation of different compounds in B. variegata leaf extract was reported, in this lupeol and quercetin were quantified with method validation (Table 3).

 

By analyzing inter and intra day variations, precision studies have been performed for the determinations of these phenolic compounds. This has been carried out at three different concentration levels of 200, 400 and 600 ng per band, the mean percent RSD values of intra and inter day variations for lupeol and quercetin were presented in Table 4.

 

For recovery studies the pre-analyzed B. variegata extract was spiked with extra 50, 100 and 150% of the standard marker compounds and the mixtures were re analyzed showing a good recovery ranges from 97.04% to 99.06% for lupeol and 98.61% to 100.06% for quercetin, experimental data are expressed as mean percentage of recovered analyte; standard deviation and relative standard deviation were also determined during experiment as shown in Table 5.

 

The methanolic extract of B. variegata leaves showed the presence of lupeol (0.35%) and quercetin (0.03%), quantified via HPTLC.

 

Table 3: Summary of validation parameters

 

 

Table 4 Intra-day and inter day precisions

Standard Markers	Conc. (ng/band)	Intraday		Interday
		% RSD	Mean RSD	%RSD	Mean RSD
Lupeol	200	0.61	0.51	1.32	1.71
	400	0.56		1.89
	600	0.37		1.91
Quercetin	200	0.89	0.76	1.9	1.66
	400	0.86		1.31
	600	0.52		1.78

 

 

Table 5 Recovery analysis of lupeol and quercetin

Standard	Amount added (%)	Amount Recovered (%) Mean	SD	RSD
Lupeol	50	98.25	2.24	2.28
	100	99.06	2.93	2.96
	150	97.04	1.89	1.95
Quercetin	50	98.61	1.99	2.02
	100	100.06	1.87	1.86
	150	98.65	1.02	1.03

 

CONCLUSION:

Bauhinia variegata commonly known as kachnar is tropical ornamental plant, under this study pharmacogostic parameters were developed for Bauhinia variegata leaves, collected from Gorakhpur. Under the study pharmacognostic parameters were performed including macroscopic characters, microscopic characters, physicochemical parameters (extractive values, ash values, pH analysis, LOD) and preliminary phytochemical screening. HPTLC analysis was also done for B. variegata leaf extract which showed the presence of different phytochemicals in the form of spots (on HPTLC plate) and peaks (in chromatogram). At 366nm distinct peaks for lupeol and quercetin were observed in the leaf extract which shows the presence of two secondary metabolite in leaf extract. On quantifying lupeol was found 0.35% and quercetin 0.03%.

 

Pharmacognostic studies and monographic development of Bauhinia variegata  leaves from district Gorakhpur, (U.P.) provides a collective data for the plant which can be utilized for the determination of purity of drug material available from various sources, data is useful for the detection of adulterants and other impurities and the results of various parameters are useful for the proper standardization of herbal formulations containing Bauhinia variegata species as an ingredient.

 

REFERENCES:

1.      Larson SS, Pollen. Morphology of Thai species of Bauhinia (Fabaceae). Grana 1974; 14: 114-131.

2.      Bailey LH. The Standard Cyclopedia of Horticulture. New York: MacMillan. 1941.

3.      Neal MC. In gardens of Hawaii Honolulu. London: Bishop Museum Press, 1965; 924.

4.      Nadkarni AK, Nadkarni KM. Indian Materia Medica Vol. 1. Bombay: Popular Prakashan; 1996.

5.      Noatay KL. Asia’s useful trees, plants, The Tribune. 22 January, 2001.

6.      Latif A, Shinwari ZK, Sustainable market development of non-timber forest products in Pakistan, 2002.

7.      Kirtikar KR, Basu BD, Indian Medicinal Plants, Vol. 2. Dehradun: International Book Publisher 1993.

8.      Zhao YY, Cui CB, Cai B, Han B, Sun QS. A new phenanthraquinone from the stems of Bauhinia variegata L. Journal of Asian Natural Product Research, 2005; 7: 835-838.

9.      Reddy MV, Reddy MK, Gunasekar D, Caux C, Bodo B, A flavanone and a dihydro dibenzoxepin from Bauhinia variegate, Phytochemistry, 2003; 64: 879-882.

10.   Kirtiker KR, Basu AD, Indian medicinal plants, Oriental Enterprises, Dehradun, India, 1935.

11.   Gupta A, Paarakh PM, Gavani U, Pharmacognostical and Phytochemical Screening of Bauhinia variegata Linn leaves. Pharmaceutical Research, 2009; 2(7): 1196-1198.  

 

Accepted on 13.12.2019           © RJPT All right reserved

Research J. Pharm. and Tech 2020; 13(6): 2667-2671.