ISOLATION OF PHYTOCONSTITUENTS FROM THE STEM BARK OF BAUHINIA VARIEGATA LINN.

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ABOUT AUTHORS:
Vijay Kumar MMJ¹*, Eswarappa B², Yadav D. Bodke³, Jayadevaiah KV¹, Basavaraja HS^1
1PG Dept. of Pharm.Chemistry, SJM College of Pharmacy, SJMIT Campus, Chitradurga, Karnataka, INDIA.
²Dept. of PG Studies and Research in Industrial Chemistry, Sir MV Govt. Science College, Bommanakatte, Bhadravathi, Karnataka, INDIA.
³Dept. of PG Studies and Research in Industrial Chemistry, Kuvempu University, Shankaraghatta, Karnataka, INDIA
*vijaykumarmmj@yahoo.in

ABSTRACT
Fractionation of crude petroleum ether extract of the stem bark of Bauhinia variegata Linn (Caesalpiniaceae) led to the isolation of heptatricontan-12, 13-diol, friedelin, n-docosanoic acid, stigmasterol, β-sitosteryl, n-hexadecanoic acid, lupeol. Eriodictyol & Quercetin were isolated from methanolic extract. Their structures were elucidated by spectroscopic methods such as UV, IR, ¹H NMR and MASS. Eriodictyol was isolated for the first time from this plant.

REFERENCE ID: PHARMATUTOR-ART-2240

INTRODUCTION
Bauhinia variegata (Linn.), belonging to family caesalpiniaceae. It is known as mountain ebony in English, Kanchanar in Hindi. The plant is a medium-sized deciduous tree abundant in Sub-Himalayan tract extending eastwards to Assam, Eastern, Central and South India.^[1] The bark is grey or brownish with longitudinal cracks and light pink inside. Leaves are 10-12.5 cm long, 1- foliate, 2- lobed, not deeply cleft, rigidly subcoriaceous and deeply cordate. Flowers are pink coloured and sessile or born in short peduncled corymbs. Pods are 15-30 cm into 1.2-2.0 cm long, flat hard, compressed and debiscent. The seeds are 10-15 in pods.  The various parts of the plants viz., leaves, flower buds, flower, stem, stem bark, seeds and roots are used in fever, as tonic, astringent, diarrhoea, dysentery, hemorrhoids, piles, edema, laxative, anthelmintic, antileprotic, in skin diseases, wound healing, antigoitrogenic, antitumor, in obesity, stomatitis, antidote for snake poisoning, dyspepsia, flatulence and as carminative.^[2] The chemical constituents isolated so far from the plant are β-sitosterol, kaempferol-3-glucoside, tannins,^[3] carbohydrates, amides, reducing sugars, vitamin C, crude protein, fibers,^[4] calcium, phosphorus,^[5] quercetin, rutin, quercitrin, apigenin, apigenin- 7-O-glucoside,^[6] heptatriacontan-12, 13-diol and dotetracontan-15-en-9-ol.^[7] Despite the enormous medicinal properties mentioned in the traditional literature and a profound and systematic Phytochemical and pharmacological investigations of stem bark of Bauhinia variegata Linn., are not reported in the scientific arena, thus the present investigation is emphasizing on the investigation of phytochemical screening and evaluation of pharmacological prosperities of the bark.

In the present work, we have isolated heptatricontan-12, 13-diol (KA 01), n-Docosanoic acid (KA 02), Hexadecanoic acid (KA 03), β-Sitosterol (KA 04), Stigmasterol (KA 05), friedelin (KA 06) & Lupeol (KA 07) from petroleum ether extract.  Eriodictyol (KA 08) & Quercetin (KA 09) were obtained from ethanolic extract.

MATERIALS & METHODS
Collection and authentification of the plant:
The stem bark of Bauhinia variegata Linn., was collected from Chitradurga, Karnataka. It was authenticated by Prof. V.T. Hiremath, Department of Botany, SJM College of Arts, Commerce & Science, Chitradurga, Karnataka. A voucher specimen no 108-A is deposited in the herbarium of SJM College of Pharmacy, Chitradurga, Karnataka, India.

General chemicals & instrument details
AR grade solvents were purchased from Sigma–Aldrich chemicals.
UV: Shimadzu UV VIS-1700; IR: Bruker FTIR 5300; MASS-ESI.1cm^-1H-NMR (400 MHz): Bruker Avance II 400.

Extraction and isolation procedure
The shade dried stem bark of Bauhinia variegata (Linn.),were powdered (4Kg) and exhaustively extracted by soxhlet extractor, using 95 % ethanol as solvent. The ethanol was distilled off and the concentrate was evaporated on a water bath to a sticky mass (36g).

The Ethanolic extract of stem bark of Bauhinia variegata (Linn.),was subjected to systematic qualitative analysis for the identification of various phyto-constituents.

The Ethanolic extract (30g) was suspended in distilled water (1500 ml) and then extracted with petroleum ether (60 – 80°C, 8 X 500 ml), Benzene (8 X 500 ml), chloroform (8 X 500 ml), ethyl acetate (8 X 500 ml), and methanol (8 X 500 ml), All the fractions were then washed with distilled water (30 ml), dried over anhydrous sodium sulphate and freed of solvent by distillation. The ethanol extract was thus fractioned into petroleum ether soluble fraction (3.5g), Benzene soluble fraction (2.9g), chloroform soluble fraction (2.5g), ethyl acetate soluble fraction (3.2g), and methanol soluble fraction (6.3g).

The petroleum ether extract (3.1g) was dissolved in CHCl₃ (20 ml) and adsorbed onto silica gel (20 g). After evaporation of the solvent it was loaded onto silica gel column (150 g) prepared in petroleum ether (60–80°C). The column was eluted first with petroleum ether (60-80°C) 100% and continued with petroleum ether (60-80°C): benzene graded mixtures, benzene 100% and benzene: chloroform graded mixtures.

100% chloroform and chloroform: ethyl acetate graded mixtures, ethyl acetate and ethyl acetate: methanol graded mixtures and finally with 100% methanol. The elution was monitored by TLC (Silica gel G; visualization: vanillin-sulphuric acid reagent heated at 110°C and Iodine chamber). Each time 10 ml were collected in a test tube and identical eluates (TLC monitored) were combined and concentrated to 5 ml and kept in a desiccator.

Elution carried out with petroleum ether (100%), fractions 9-16 (KA-01, 45 mg); fractions 29-43 (KA-02, 52 mg); petroleum ether: benzene (95:5), fraction 21-30 (KA-03, 65 mg); petroleum ether: benzene (10:90), fractions 42-49 (KA-04, 57 mg); benzene: chloroform (90:10), fraction 142-145 (KA-05, 65 mg);benzene: chloroform (50:50), fraction 242-252 (KA-06, 70 mg); chloroform (100%), fraction 385-391 (KA-07, 60 mg); were obtained which on further purification by fractional crystallization. Further elution was carried out with various solvents.

The ethanolic extract (5g) was dissolved in CHCl₃ (20 ml) and adsorbed onto silica gel (20 g).After evaporation of the solvent it was loaded onto silica gel column (150 g) prepared in chloroform. Elution carried out with ethyl acetate (100%), fraction 54-63, compound (KA-08,  35 mg). ethyl acetate: methanol (90:10), fraction 115-121, compound (KA-09, 44 mg).

RESULT & DISCUSSION
The structures of isolated compounds were elucidated on the basis of R_f, Melting point and_Spectral data.

Compound KA 01: m.p.65°C,The compound was isolated as a pale green colour needle shape crystals. R_f value: 0.42 [solvent system; petroleum ether (60-80º C)]. The IR peak at 3447.51 cm^-1indicated the presence of OH group, peak at 2922.75 cm^-1indicates C-H stretching CH₃, peak at 1635.2cm^-1, indicate C-H stretching CH₂ and peak at 1019.65 cm^-1 indicates O-H bending.¹HNMR spectra conclude at δ3.42 for CH₂ and at δ2.5 for CH₃. The Mass spectra showed the molecular ion peak at m/z 552[M⁺] which corresponds to the molecular formulaC₃₇H₇₆O₂. With the above data and mass spectrum, the compound 01 is identified asHeptatricontan-12, 13-diol.

Compound KA 02: m.p.262-265°C, it showed positive test to Libermann-Burchard’s test and salkowski test. R_f value: 0.76 [solvent system- benzene: chloroform (90:10)]. The IR spectrum exhibited strong absorption at 2926 cm^-1 IR peak at 2929.0 cm^-1  and 2869.24 cm^-1  indicating the presence of C-H stretching in CH₃ and CH₂ respectively.IR peak at 1714.96 cm^-1 for C=O stretching, 1631.8 cm^-1 indicates CH₂-CH₂ stretching. Peak at 1462.6 cm^-1 indicates C-H deformation in gem dimethyl. The ¹H NMR spectra of this compound exhibited the presence of eight methyl group at position in between δ0.725 to δ1.179. The ¹H NMR spectra also showed at δ1.1957 to δ0.1586 for CH₂ protons. The Mass spectra showed the molecule ion peak at 426[M⁺] corresponding to the molecular formula C₃₀H₅₀O.With the above data and mass spectrum, the compound 02 is identified asFriedelin.

Compound KA 03: m.p. 74-78°C. The compound was white to cream colour crystal. R_f value: 0.6 [solvent system; benzene: chloroform (80:20)]. IR spectrum showed characteristic peak at 1708.76 cm^-1 indicated the presence of C=O, 1464.9 cm^-1 and 1017.8 cm^-1 indicating the presence of C-H deformation in CH₃ and CH₂ respectively. The peak at 3412.16 cm^-1 indicated –OH stretching. The¹H NMR signal at δ0.85 indicated terminal methyl protons δ1.2-1.3 indicated CH₂ protons. The Mass spectra showed the molecular ion peak at m/z 340 [M⁺] corresponding to the molecular formula C₂₂H₄₄O_2. With the above data and mass spectrum, the compound 03 is identified as n-Docosanoic acid.

Compound KA 04: Pearl white crystals, m.p.167-170°C.It gave a characteristic colour reaction of sterols. It gave red colour in Salkowski’s test and a green colour in Libermann Burchard’s test. The peaks at 3351, 2919 and 1665 cm^-1in the IR spectra indicated to presence of OH group, C=O stretching and C=C stretching respectively. The peaks at 1462 and 1054 cm^-1 indicated the presence of deformation of gem dimethyl group and stretching of secondary alcohol functional groups.¹H NMR spectra showed multiplet at  d0.714 to 1.046 corresponds to eighteen protons of six methyl groups. Further peaks at d1.04 to 2.28 corresponds to eighteen protons of nine -CH₂- groups and eight protons of -CH. The peaks at d5.05 to 5.14 corresponds to olefinic two protons. The broad peak at d3.53 and 5.36 corresponds to one proton for –OH group and vinylic proton respectively. ¹³C NMR spectra showed presence of 29 carbons in the compound.The Mass spectra showed the molecular ion peak at m/z 412 (M⁺). The Mass fragmentation was typically to that of Stigmasterol.

Compound KA 05: m.p63-64°C. The compound isolated as colourless crystals. R_f value: 0.79 [solvent system; petroleum ether: benzene (95: 5)]. The IR peak at 3391.1 cm^-1 indicates the presence of OH group, peak at 2928.0 cm^-1 indicates C-H stretching in CH₃, peak at 2860.5 cm^-1 indicate C-H stretching in CH₂ and peak at 1707.6 cm^-1 indicates C=O stretching and peak at 1461.0 cm^-1and1073.9 cm^-1 indicates C-H deformation in CH₃ and CH₂ respectively. ¹H NMR spectra at δ 0.7991 - δ 0.9690 indicates terminal methyl protons, a multiplet was observed in the range δ 1.0334 - δ 1.3929 of which corresponds to twenty four protons of CH_2, NMR signal at δ 2.0030- δ 2.05841 shows a multiplet corresponds to two protons of CH₂, a triplet was observed in the range of δ 2.3272 – δ 2.3648 which corresponds to two protons of CH₂.The Mass-ESI spectra showed the molecular ion peak at m/z 256 which corresponding to the molecular formulaC₁₆H₃₂O₂. With the above data and mass spectrum, the compound V is identified as n-Hexadecanoic acid.

Compound KA 06: m.p. 60-62^oC. It showed positive test to Libermann-Burchard’s test and salkowski test. R_f value: 0.71 [solvent system; benzene: chloroform 90:10)]. The peak at 3441.41 cm^-1 in the IR spectra indicates O-H stretching_, peak at 2935.42 cm^-1 indicates C-H stretching in CH₃, peak at 2851.86 cm^-1 indicates C-H stretching in CH₂, peak at 1736.85 cm^-1 and 1604.33 cm^-1  indicates C=O stretching and  C=C stretching  respectively. Peak at 1464.47 cm^-1 indicates C-H deformation in CH₃. In ¹HNMR spectra the signal at δ 0.6482 to 1.0110 indicates multiplet corresponds to eighteen protons of six CH₃ groups, signal at δ 1.076 to δ 1.5622 indicates multiplet which corresponds to twenty two protons of eleven CH₂ groups,signal at δ 1.8356 to δ 2.2871 indicates multiplet which corresponds to four protons of four CHgroups, signal at δ 3.515 to 3.538 indicates multiplet which corresponds to one proton of CHgroup, signal at δ 5.022 indicates doublet which corresponds to two protons of CH₂ groups, signal at δ 5.15 indicates quadralet which corresponds to two proton of CH₂ groups, signal at δ 5.34 indicates hump which indicates one proton of –OH group. The Mass-ESI spectra showed the molecular ion peak at m/z 597 corresponding to the molecular formula C₄₁H₇₂O₂. With the above data and mass spectrum, the compound 06 was identified as β-Sitosterol.

Compound KA 07: m.p. 213-215ºC. The compound isolated as white colour crystals. Positive test for Liebermann Burchardt test indicated the presence of tetracyclic triterpenoid compound. R_f value: 0.712 [Solvent system, chloroform: methanol (95:5)]. In the IR spectra the broad peak at 3423.44 cm^-1 indicated the presence of OH, IR peak at 2929cm^-1 and 2850cm^-1 indicating the presence of C-H stretching in CH₃ and CH₂ respectively, IR peak at 1458.14 cm^-1 represents CH deformation in gem dimethyl, further peak at 880.00 represents exocyclic CH_2. In ¹HNMR a multiplet was observed in the range of δ 0.7605 to 0.9213 corresponding to twenty one  protons of seven methyl groups, a multipletwas observed in the range of δ 0.9436 to 1.6811 corresponding to twenty protons of ten CH₂ groups, a singlet was observed at δ 2.1735  corresponding to one proton of CH, a multiplet  was observed in the range of δ 3.1645 to 3.2051 corresponding to one proton of CH group, a triplet was observed in the range of  δ 4.2024 to 4.2321 corresponding to one proton of CH group, a multiplet was observed in the range of δ 4.5604 to 4.5704 corresponding to one proton of CH group, a doublet was obseved in the range of δ 4.6840 to 4.6898 which corresponds to one proton of CH group, a doublet was observed in the range of  δ 1.6823 to 1.6908 corresponding to one proton of CH group_. The Mass spectra showed the molecular ion peak at m/z 427.4corresponding to the molecular formula C₃₀H₅₀O_2. With the above data and mass spectrum, the compound 07 was identified as Lupeol.

Compound KA 08: m.p. 95 – 97⁰c.The compound was Yellowish solid. R_f value: 0.85 (solvent system; (Methanol 100%). It gave reddish brown colour for Shinoda test. In the IR spectra the broad peak at 3425 cm^-1 indicates the presence of OH, peak at 2917 cm^-1 shows Ar-H str in aromatic ring, peak at 2848cm^-1shows C-H str in CH3, peak at 1690 cm^-1shows α, β unsaturated ketone, peak at 1373 cm^-1 shows C-H bending in –C(CH₃)₂, peak at 1249 cm^-1shows C-O-C stretching. In ¹H NMR spectra a singlet at δ 6.1, 6.3 and 7.7 corresponds for three  Ar-H, doublet at δ 6.8 & 7.5 indicates two proton of Ar-H, humps at δ 8.8, 10.4, 12.4 indicates OH groups corresponds for five protons. The Mass-ESI spectra showed the molecular ion peak at m/z 302 and the other peaks appeared at 261, 216, 170 and 133 corresponding to the molecular formula C₁₅H₁₀O_7. With the above data and mass spectrum, the compound KA 08 is identified as Quercetin.

Compound KA - 09: m.p. 256-258 ^oC. The compound wasbrown color crystals. It gave reddish brown colour for Shinoda test. Rf value : 0.71 (solvent system; petroleum ether : benzene 50:50).In the IR spectra the broad peak at 3422.41 cm^-1 indicates the presence of OH.The peak at 2958.08 cm^-1 indicates C-H stretching in CH₃, peak at 2854.0 cm^-1 indicates  C-H stretchingin CH_2, peak at  1729 cm^-1  and 1617.80 indicates C=O stretching and C=C stretching  respectively. The IR peak at 1453.50 cm^-1 indicates C-H deformation in CH₃.  In proton NMR spectra, a triplet at δ 4.0046  indicates two protons of CH, a doublet at δ 4.5151 indicates CH₂ which corresponds to two protons while multiplet ranging from δ 5.8522 to δ 6.3546 and δ 6.3939 to δ 6.5200 indicates aromatic protons corresponds to three hydrogen’s and aromatic protons corresponding to one hydrogen respectively.  The multiplet ranging from δ 6.6789 to δ 6.8694 and δ 6.9317 to δ 7.5619 represents aromatic protons corresponds to two hydrogens. A signal from δ 8.1878 to δ 8.9660 indicates four -OH group.The Mass-ESI spectra showed the molecular ion peak at m/z 288corresponding to the molecular formula C₁₅H₁₂O. With the above data and mass spectrum, the compound KA-09 is identified as Eriodictyol.

CONCLUSION
The phytochemical investigation of the petroleum ether and ethanolic extract of the stems of B. variegata belonging to the family Caesalpiniaceae was successfully carried out. The chemical constituents isolated from the extracts must be accounted for the biological activities exhibited by the crude petroleum ether and ethanolic extract of the plant. Therefore, it is now turn of the pharmacologists/biologists to explore the plant more systematically by carrying out individual bioactivity of the isolated chemical constituents. Therefore, the present work will boost the scientific communities to do more research work on this important medicinal plant to explore it in the drug development programme going on around the world.

Acknowledgement:
The authors are thankful to Dr. Shivamurthy Murugha Sharanaru, President, & The Executive Directors, SJM Vidyapeetha for providing all necessary facilities through the Principal & HOD of Dept. of Pharmaceutical chemistry, SJM College of Pharmacy, Chitradurga. The authors are also thankful to the students of Dept. of Pharmaceutical Chemistry, SJM College of Pharmacy, Chitradurga.

Structures of the isolated compounds from stem bark of Bauhinia variegata Linn.

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