SPECTRAL ANALYSIS OF METHANOLIC ROOT BARK EXTRACT OF SEMAL (BOMBAX CEIBA) USING GAS CHROMATOGRAPHY-MASS SPECTROMETRY

SPECTRAL ANALYSIS OF METHANOLIC ROOT BARK EXTRACT OF SEMAL (BOMBAX CEIBA) USING GAS CHROMATOGRAPHY-MASS SPECTROMETRY

Akash Saini * and Sangeeta Bhargava

Department of Chemistry, University of Rajasthan, Jaipur, Rajasthan, India.

ABSTRACT: Bombax ceiba L. is an important medicinal plant belonging to the family Bombacaceae. Root bark samples of the plant have been subjected to phytochemical investigation through gas chromatography-mass spectrometry (GC-MS). Twenty phytochemical compounds have been identified in the methanolic extract of B. ceiba root bark. The identification of phytochemical compounds is based on the peak area, retention time, molecular weight and molecular formula.

Keywords: Bombax ceiba, Bombacaceae, Lupeol, GC-MS, Bioactivity

INTRODUCTION: Bombax ceiba L. of family Bombacaceae which contain about 26 genera and nearly 140 pantropical species ¹.  It is commonly known as semal or silk cotton tree or Indian kapok ². B. ceiba is widely distributed throughout Nepal, India, West China, Malaysia, and the hotter part of India ³. Semal is a lofty, deciduous tree that is up to 40 m tall and 6 m in girth with horizontally spreading branches and a young stem covered with stout, hard prickles. The bark is pale ash to silver-grey in color. Flowers are large in diameter, red in colour and numerous with copions nectar. The fruits are brown capsule-like up to 15 mm long, filled with numerous black seeds which are irregular obovoid in shape, smooth and oily with dense silky hair. The fruit pulp is sweet and edible. Semal tree have compound leaves which are palmate in appearance.

It is digitate, large, spreading, glabrous, which has common petioles, and the size of the leaf is 15-30 cm long. The size of leaflets varies from 10 cm to 20 cm. new leaves usually do not appear until flowering is over. The bark of this plant is used in wound healing, flower and fruits are used in revitalizing sexual impotency and gum are used as a remedy of diarrhea, dysentery, influenza, menorrhagia, and disease of CNS⁴. Methanolic extract of bark of B. ceiba showed α-glucosidase inhibitory activity. Cadinane sesquiterpenoids isolated from B. ceiba root bark were found to possess hypoglycaemic activity ⁵, antifungal ⁶, cytotoxic ⁷, anti-HIV activity ⁸. It is used as folk medicine because of its demulcent, diuretics, restorative, aphrodisiac and emetic properties ^9-11.

Root bark sample of the plant was subjected to phytochemical investigation through Gas Chromatography-Mass Spectrometry (GC-MS). B.ceiba is an important medicinal plant; the phytochemical study revealed that it is rich in phenolic compounds while mangiferin a xanthone is present in large amounts in the leaves and obtained directly from the extract.

It has a significant effect as a hypotensive agent and possesses hypoglycemic activity with negligible toxicity. Twenty phytochemicals compounds were identified in the methanolic extract of B. ceiba root bark Table 1. The identification of phytochemical compounds is based on the peak area, retention time, molecular weight and molecular formula. Gas Chromatography-Mass Spectrometry (GC-MS) analysis of B. ceiba root bark Fig. 1 revealed the existence of lupeol, squalene, lup-20(29)-ene-3-ol, palmitic acid, vitamin E, alpha tocospiro B, etc Table 2.

MATERIALS AND METHODS:

Plant Material: Samples of B. ceiba root bark were collected from the nursery of the University of Rajasthan, Jaipur city Rajasthan (India). The plant was identified by the department of botany, University of Rajasthan, Jaipur. The root bark was thoroughly washed using deionized water and was transferred to our laboratory. The root bark sample was washed, cleaned, and dried. B. ceiba root bark is also packed in sealed polythene bags for further experimental purposes.

Extraction: The shade dried root bark (1 Kg) was finely powdered using a grinder. Then the powdered root bark (approximately 500 gm) was placed in a Soxhlet apparatus and then extracted with 2 L Methanol for 20 h on a water bath. Excess solvent was removed under vacuum in a rotary evaporator (Rotary Vacuum Evaporator N. N. Series equipped with an aspirator and a digital water bath SB-651; Eyela, Tokyo, Japan) at 45 °C and further made moisture free with sodium sulphate. The resulting extract was stored at 4 °C until further analysis.

GC-MS Analysis: Gas Chromatography combined with Mass Spectrometry is a preferred methodology for routine analysis of compounds. The GC-MS analysis of the above-mentioned extracts was performed with a Gas Chromatography unit Shimadzu GCMS-QP2010 Plus comprising AOC-20i+s autosampler. Various components were identified by different retention times, which were detected by a mass spectrophotometer. The chromatogram plot of intensity against retention time was recorded by the software attached to it. From the graph, the compounds are identified, comparing the data with the existing software libraries like WILEY8.lib, NIST11.lib, NIST11s.lib, FFNSC2.lib and mass spectra of standard. The name, molecular weight, and structure of the components of the test materials were ascertained.

RESULTS AND DISCUSSION: The GC spectrum of the methanolic extract shows total 20 compounds present in the methanol extract were determined by the chromatographic method with the help of NIST and WILLEY library, as shown in Table 1. Compound Lupeol was found to be in the highest concentration (23.08%) followed by squalene (8.34%), lup-20(29)-en-3-ol (7.67%), and palmitic acid (5.70%) Scheme I. Other compounds were found in trace amount Table 1. Either one or all the identified compounds may be responsible for the biological activity of the methanolic extract. Further separation of the identified compounds will be done in due course.

Lupeol (1), a phytoconstituent belonging to the triterpene class, is found in several fruit plants and medicinal plants that have been the object of study in the treatment of various diseases, including skin wounds. Various medicinal properties of lupeol have been reported in the literature, including anti-inflammatory, antioxidant, anti-diabetic, and anti-mutagenic effects. Investigation effects of lupeol (0.1, 1, 10 and 20 µg/mL) on in-vitro wound healing assays and signaling mechanisms in human neonatal foreskin keratinocytes and fibroblasts showed that, at high concentrations, Lupeol reduced cell proliferation of both keratinocytes and fibroblasts, but increased in-vitro wound healing in keratinocytes and promoted the contraction of dermal fibroblasts in the collagen gel matrix.

This triterpene positively regulated matrix metalloproteinase (MMP)-2 and inhibited the NF-κB expression in keratinocytes, suggesting an anti-inflammatory effect. Lupeol also modulated the expression of keratin 16 according to the concentration tested. Additionally, in keratinocytes, lupeol treatment resulted in the activation of Akt, p38, and Tie-2, signaling proteins involved in cell proliferation and migration, angiogenesis, and tissue repair. These findings suggest that lupeol has therapeutic potential for accelerating wound healing.

TABLE 1: PHYTOCHEMICALS IDENTIFIED IN THE METHANOL EXTRACT OF THE ROOT BARK OF B. CEIBA L. BY GC-MS

TABLE 2: MAJOR PHYTOCHEMICALS IDENTIFIED IN THE METHANOL EXTRACT OF THE ROOT BARK OF B. CEIBA L.

Experimental Section: Characterization data. Compound 1 Lupeol:  White powder; ¹H NMR (300 MHz, CDCl₃): δH 0.75, 0.78, 0.82, 0.93, 0.95, 1.02, 1.25 (each, 3H, s, CH₃ × 7), 3.22 (1H, dd, J = 6.8 Hz, H-3), 3.20 (1H, dd, J = 7.6, H-3); 4.58 (3H, s, J = 2.0 Hz, H - 29), 0.76 (3H, s, H-23), 0.79 (3H, s, H-24), 0.83 (3H, s,H-25), 0.86 (3H, s, H-26), 0.88 (3H, s, H-27), 0.87 (3H, s, H-28), 1.68 (2H, d, H-30), 1.39 (2H, d, H- 1), 1.32 (2H, m, H-6), 1.36 (m, 2H, H-7), 1.39 (d, 1H, H-9), 1.36 (m, 2H, H-15), 1.36 (2H, m, H-16), 1.39 (2H, m, H-12), 1.39 (1H, d, H-13), 1.32 (2H, m, H-11), 1.42 (2H, m, H-22), 1.53 (2H, m, H-21), 1.59 (2H, m, H-2), 4.58 (3H, s,H-29), 1.36 (1H, d H-5), 1.42 (, 1H, d H-18), 2.37 (1H, d, H-19) and 3.18 (1H, d, H-3); ¹³C NMR (300 MHz, CDCl₃): δC 19.2 (C- 23),19.3 (C-24), 29.1 (C-25), 20.9 (C-26), 27.4 (C-27), 25.1 (C-28), 109.3 (C-30), 35.2 (C-1), 19.2(C-6), 35.5 (C-7), 50.4  (C-9), 27.4 (C-15), 40.0 (C-16), 34.2 (C-12), 40.0 (C-13), 29.83 (C-11), 42.81 (C-22), 43.0 (C-21), 34.2 (C-2), 29.1 (C- 29), 50.4 (C-5), 48.2 (C-18), 151.0 (C-20), 55.25 (C-19), 79.0 (C-3).

Compound 2: Squalene: Colourless oil; ¹H NMR (400 MHz, CDCl₃): δH 1.61-1.69 (3H, s, J = 5.17 Hz, H-19'; 1H, m, J = 5.12 Hz, H-3), δ 1.30 (s, 3H, H-1), 2.04 (s, 3H, H-21), 5.12 (m, 1H, H- 3), 1.32 (m, 2H, H-4), 2.10 (m, 2H, H-5), 1.70 (s, 3H, H-6'), 5.10 (m, 1H, H-7), 1.32 (m, 2H, H-8), 2.10 (m, 2H, H-9), 1.70 (s, 3H, H-10'), 5.10 (m, 1H, H-11), 2.04 (m, 2H, H-12), 1.70 (m, 2H, H-13), 5.10 (m, 1H, H-14), 1.71 (s, 3H, H-15'), 2.10 (m, 2H, H-16), 1.62 (m, 2H, H-17), 5.10 (m, 1H, H-18), 5.17 (s, 3H, H-19'), 2.08 (m, 2H, H-20), 1.32 (m, 2H, H-21), 5.10 (m, 1H, H-22), 1.30 (s, 3H, H-23') 1.80 (s, 3H, H-24). ¹³C NMR (400 M Hz, CDCl₃): δC 16.0 (C-1), 134.8 (C-2), 17.6 (C-2'), 124.2 (C-3), 28.2 (C-4), 39.73 (C-5), 135.0 (C-6), 15.9 (C-6'), 124.2 (C-7), 28.2 (C-8), 39.73 (C-9), 135.0 (C-10), 15.9 (C-10'), 124.2 (C-11), 29.67 (C-12), 29.67 (C-13), 124.28 (C-18), 135.0 (C-19, 39.7 (C-20), 28.2 (C-21), 124.3 (C-22), 134.8 (C-23), 16.0 (C-23), 17.6 (C-24).

Compound 3: Lup-20(29)-en-3-ol: White crystalline powder (needles). M.p. 214–216 °C. IR-FT (KBr): 3550, 3400, 3295, 2920, 2850, 1640 (weak), 1455, 1380, 1040, 1015, 880. ¹ H-NMR (400 MHz; CDCl₃)  4.69 (br. d, 2.5 Hz, H-29b), 4,57 (dt, 2.5, 2.5 e 1.3 Hz, H-29a), 3.19 (dd, 11.0 e 5.0 Hz, H-3), 2.38:d (dt, 11.0 e 5.8 Hz, H-19), 1.68 (br. dd, 1.3 e 0.8 Hz, H-30), 1.03 (s, H-26), 0.97 (s, H-23), 0.94 (br. d, :d0.8 Hz, H-27), 0.83 (br. d, 0.8 Hz, H-25), 0.79 (s, H-28), 0.76 (s, H-24). ¹³C-NMR (100 MHz; CDCl₃)  0.96 (C-20), 109.32 (C-29), 79.01 (C-3), 55.32 (C-5), 50.46 (C-9), 48.33 (C-18), 47.78 (C-19), 43.01 (C-17), 42.85 (C-14), 40.85 (C-8), 40.02 (C-22), 38.87 (C-4), 38.73 (C-1), 38.07 (C-13), 37.19 (C-10), 35.60 (C-16), 34.30 (C-7), 29.87 (C-21), 28.00 (C-23), 27.46 (C-15), 27.43 (C-2), 25.17 (C-12), 20.95 (C-11), 19.32 (C-30), 18.33 (C-6), 18.01 (C-28), 16.12 (C-25), 15.99 (C-26), 15.37 (C-24), 14.56 (C-27). MS, m/z (%): 69 (100), 55 (72), 41 (64), 135 (52), 133 (34), 197 (28), 203 (26), 218 (26), 121 (44), 189 (40), 409 (1), 408 (1), 426 (1, M+, C₃₀H₅₀O).

Compound 4 Palmitic Acid: White solid; ¹H NMR (400 MHz, CDCl₃): δH 2.35 (2H, t, J = 7.6 Hz, H-2), 0.89 (3H, t, J = 6.8 Hz, H-16), 1.66 (2H, q, J = 7.6 Hz, H-3), 1.26 (2H, m, H-4, H-5, H-6, H- 7, H-8, H-9, H-10, H-11, H-12, H-13, 14, 15). ¹³C NMR (400 MHz, CDCl₃): δC 180.1(C-1), 33.9 (C-2), 24.6 (C-3), 29.0 (C- 4), 29.2 (C-5), 29.3 (C-6), 29.4 (C-7), 29.5 (C-8), 29.6 (C-9), 29.6 (C-10), 29.6 (C-11), 31.9 (C-12), 29.6 (C-13), 29.6 (C- 14), 22.7 (C-15), 14.0 (C-16).

CONCLUSION: Present study of the methanol extract of B. ceiba root bark indicated that it contains biologically active compounds. The properties of these compounds probably contribute, at least to some extent, to the pharmacological and traditional medicinal uses of the B. ceiba. Further separation and identification of compound present in it may give new biologically active compounds, which can be used as lead compounds in the future.

ACKNOWLEDGEMENT: Financial support from UGC, New Delhi, India, is highly acknowledged. Further, the authors are grateful to JNU Delhi for providing GC-MS spectral analysis/data of plant extract.

CONFLICTS OF INTEREST: Nil

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    How to cite this article:

    Saini A and Bhargava S: Spectral analysis of methanolic root bark extract of semal (Bombax ceiba) using gas chromatography-mass spectrometry. Int J Pharm Sci & Res 2021; 12(11): 6067-71. doi: 10.13040/IJPSR.0975-8232.12(11).6067-71.

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