Natural Products Chemistry & Research

ISSN - 2329-6836

Research Article - (2018) Volume 6, Issue 6

Isolation and Analytic Characterization of β-Sitosterol and GC-MS Analysis of Methanolic Leaves Extract of Pongamia pinnata (L.) pierre

Savita Sangwan*
Department of Chemistry, Jaipur Engineering College and Research Center University, Jaipur, India
*Corresponding Author: Savita Sangwan, Department of Chemistry, Jaipur Engineering College and Research Center University, Jaipur, India, Tel: + 9928195351 Email:

Abstract

In the course of present research venture, a phytosterol viz., β-sitosterol was secluded from the in vivo leaves of P. pinnata and identification of the secluded compound was accomplished through TLC, Rf value, IR spectroscopy and NMR. Furthermore, the leaf methanolic extract was also analyzed by GC/MS spectroscopy in P. pinnata. It was observed that 64 secondary compounds were isolated in P. pinnata, respectively.

Keywords: β-sitosterol; TLC-Thin Layer Chromatography; IR-InfraRed; NMR-Nuclear Magnetic Resonance; P. pinnata (L.)

Introduction

From the past few decennary, various records are available as well as investigations have been accomplished, showing the relevance of extracting, isolating, identifying, utilizing and exploring various medicinally bioactive compounds, from many plant species in in vivo and in vitro conditions such as in Sericocalyx schomburgkii , Nerium oleander , Piper nigrum and Piper longum , Pavetta indica , Momordica charantia etc. [1-5]. Hence, this field has opened up new vistas in the field of plant sciences and medicinal world. In the present research work, β-sitosterol has been extracted from the leaves of P. pinnata , and then purified and identified by some spectroscopic methods like IR, NMR etc. Besides this, GC/MS studies have been done using crude methanolic extracts of P. pinnata.

Pongamia pinnata (L.) belongs to family Leguminosae and subfamily Papilionaceae is a medium sized glabrous, perennial tree grows in South Eastern Asia and Australia [6]. This tree is 15-25 m in height, with straight or crooked trunk 50-80 cm or more in diameter and broad crown of spreading or drooping branches. Its bark is smooth and grey-brown; leaves are alternate, imparipinnate, hairless, pinkish-red when young, glossy dark green above and dull green with prominent veins beneath when mature. Flowers short-stalked, pea-shaped, 2-4 together and 15-18 mm long. Pods are smooth, oblique oblong to ellipsoid, 3-8 × 2-3.5 × 1-1.5 cm, flattened but slightly swollen, slightly curved with short, curved point (beaked), brown, thick-walled, thick leathery to subwoody, hard, indehiscent, 1-2 seeded, short stalked. Seed compressed ovoid or elliptical, bean-like, 1.5-2.5 × 1.2-2 × 0.8 cm, with a brittle coat long, flattened, dark brown, oily.

Root, bark, leaves, flower and seeds of this plant have been used as crude drug for the treatment of tumors, piles, skin diseases, wounds and ulcers [7]. In the traditional system of medicines, such as Ayurveda and Unani, this plant is used for anti-plasmodial, antiinflammatory, anti-nonciceptive, anti-hyperglycamic, antidiarrhoeal, antioxidant, anti-hyperammonic and anti-ulcer [8].

Materials and Methods

Extraction of phytosterol

Shade dried leaves and the powder (100 gm) of Pongamia pinnata was defatted in a soxhlet apparatus in petroleum ether (60-80°C) for 24 hours on a water bath and extraction of steroids was done by using the protocol developed by Tomita et al. [9]. For the quantitative estimation TLC was done. Identification of the extracted compounds was done by using Rf value, NMR and IR spectroscopy.

GC-MS analysis

Shade dried leaves and the powder (50 gm) of Pongamia pinnata was taken into thimble and 750 ml of methanol was taken into the flask of soxhlet apparatus and cycled 10-15 times, then it was decanted into the beaker and was left open, so that the methanol gets evaporated. The qualitative and quantitative compositions of the methanol fraction were studied by GC/MS (QP 2010 Plus Shimadzu) at AIRF, JNU, Delhi. Mass was scanned in the range 38-300 amu. Compounds were identified by comparison of mass spectra with those in the Wiley 275 and NIST 02 libraries and mass spectra of standards.

Thin-layer chromatography

TLC Glass plates were coated with silica gel (30 gm/60 ml distilled water) dried at room temperature and activated before use at 100°C for 30 minutes in an oven and cooled at room temperature. Crude extract of leaf was applied 1.0 cm above from the lower edge of the silica gel plates along with the reference standard compound of β-sitosterol. Then plate was developed in an air tight chamber containing chloroform: methanol: water (60:25:15). The developed plates were air dried, sprayed with 50% sulphuric acid and heated (at 100°C for 15 min), it showed spots which harmonize with that of the reference standard β-sitosterol. When this plate placed in a chamber saturated with I2 vapors, it also showed dull red color of β-sitosterol. Rf value come out with the Rf value of the standard β-sitosterol 0.83-0.86 [10]. The marked spots were scrapped and collected along with the silica gel and wash with ethanol then crystallized with chloroform. Finally, this purified material was subjected to its IR and NMR spectral analysis.

Results And Discussion

In the present research assay, phytosterol β-sitosterol was isolated from the leaves of P. pinnata . The characteristic IR, 1H NMR and 13C NMR spectral peaks of compound was found to be super imposable with those of their respective standard reference compound of β- sitosterol. Its IR and NMR spectra showed different peaks in Figure 1 and Table 1.

Name of Compound UV light absorption band IR : vcm-1/max KBr 1H NMR 13C NMR
β-sitosterol 206 sh, 268 sh, 356 sh, 540 sh 3400 (O-H), 2700(C-H), 1700 (C=O), 1640, 1610, 1570, 1510, 1450, 1400 (aromatic), 1385, 1310, 1270, 1180, 1010, 815 0.94 (H1), 1.39 (H2), 1.18 (H3), 1.56 (H4), 1.07 (H5), 1.86 (H6), 1.06 (H7), 1.27 (H8), 1.29 (H9), 1.04 (H10), 1.67 (H11), 1.77 (H12), 1.16 (H13), 1.25 (H14), 1.45 (H15), 1.25 (H16), 1.29 (H17), 1.46 (H18), 1.25 (H19), 1.65 (H20), 1.06 (H21), 1.21 (H22), 1.46 (H23), 0.86 (H24), 0.86 (H25), 1.26 (H26) 13.3 (C1), 21.6 (C2), 34.5 (C3), 42.7 (C4), 30.5 (C5), 20.1 (C6), 20.1 (C7), 30.0 (C8), 35.7 (C9), 29.7 (C10), 18.5 (C11), 48.2 (C12), 20.3 (C13), 20.4 (C14), 40.1 (C15), 27.4 (C16), 295.35 (C17), 36.9 (C18), 36.8 (C19), 32.1 (C20), 26.9 (C21), 36.2 (C22), 364.81 (C23), 269.86 (C24), 25.2 (C25), 257.24 (C26), 34.5 (C27), 19.3 (C28), 23.4 (C29)

Table 1: Spectral studies of isolated phytosterol from Pongamia pinnata (L.).

natural-products-chemistry-research-sitosterol

Figure 1: 1H NMR and 13C NMR of β-sitosterol.

A range of compounds have been detected in P. pinnata including Saturated fatty acid (pentadecanoic acid), phytosterols (cholesterol, stigmasterol, β-sitosterol), tridecane, dodecane (alkane hydrocabon), glycerin (alcohol), tri-terpene (squalene, lupeol, betulin), sesquiterpenes (valerenol, alpha-caryophyllene, sclareolide), aromatic compound (myo-inositol), evonine (alkaloid), hexadecen-1ol (terpene alcohol), hexadecanoic acid (palmitic acid), phytol (diterpene), octadecadienoic acid (linoleic acid), and tocopherol (vitamin-E). In the methanolic leaf extract of P. pinnata the highest peak area (%) of 15.53 was obtained by 2-Thiopheneacetic acid, morpholide (retention-time 12.610) and the lowest peak area (%) of 0.12 was obtained by pentadecane (retention-time 17.252) (Figure 2). The detailed tabulation of the GC-MS analysis of the above plant species has been given in Table 2.

S.No R.T Name of the Compound Peak Area % M.F M.Wt Compound Nature
1. 8.181 2,3-Dihydrobenzofuran 0.47 C8H8O 120  
2. 8.432 N,1-Dimethyl-4-piperidinamine 1.84 C7H16N2 128  
3. 9.185 p-Vinylguaiacol 0.41 C9H10O2 150.18 Aromatic flavoring agent
4. 9.366 2-Thiopheneacetic acid 0.13 C6H6O2S 142.17 Organic acid
5. 9.771 Pentadecane 0.10 C15H32 212 Fragrance agents
6. 9.875 1-Ethyl-2-methylpyrrolidine 0.41 C7H15N 113  
7. 10.159 l-4-Hydroxylysine 0.50 C6H14N2O3 162.18  
8. 10.652 Trimethylpiperazine 1.42 C7H16N2 128  
9. 11.333 2(4H)-benzofuranone, 5,6,7,7a-tetrahydro-4,4,7a-trimethyl-, (r)- 0.19 C11H16O2 180.24 Flavoring agent
10. 11.395 Butyl hydroxy anisole 1.04 C11H16O2 180.24 Antioxidant organic compound
11. 11.584 1-cyclohexyl-1-butanone 0.31 C10H18O 154.25 Flavoring agent
12. 12.610 2-Thiopheneacetic acid, morpholide 15.53 C10H13NO2S 211.28  
13. 12.723 Tetradecanoic acid
(Myristic acid)
0.16 C14H28O2 228.37 Saturated fatty acid
14. 12.842 4-((1E)-3-Hydroxy-1-propenyl)-2-methoxyphenol 0.29 C10H12O3 180.20 Alcohol
15. 13.158 2(4h)-benzofuranone, 5,6,7,7a-tetrahydro-6-hydroxy-4,4,7a-trimethyl-, (6s-cis)- 0.66 C11H16O3 196.10 Flavoring agent
16. 13.298 Tetradecanal 7.08 C14H28O 212.37 Flavor and fragrance agents
17. 13.599 3,7,11,15-Tetramethyl-2-hexadecen-1-ol 0.40 C20H40O 296.54 Acyclic terpenoid
18. 13.866 Hexadecanoic acid, methyl ester 0.52 C17H34O2 270.45 Saturated fatty acid.
19. 14.131 Pentadecanoic acid 5.97 C15H30O2 242.4 Saturated fatty acid
20. 14.540 Benzenepropanoic acid, 2,5-dimethoxy 0.71 C11H14O4 210.23 Fatty acid derivative
21. 14.753 Heptadecanoic acid
(Margaric acid)
0.13 C17H34O2 270.45 Saturated fatty acid
22. 15.014 8-Octadecenoic acid, methyl ester 0.30 C19H36O2 296 fatty acid
23. 15.121 2-Hexadecen-1-ol,3,7,11,15-tetramethyl-, [R-[R*,R*-(E)]]-; 2.79 C20H40O 296.60 Fatty acid
24. 15.277 9-Octadecenoic acid (Oleic acid) 5.44 C18H34O2 282.46 Unsaturated fatty acid
25. 15.382 Octadecanoic acid
(Stearic acid)
3.54 C18H36O2 284.47 Saturated fatty acid
26. 15.702 Neophytadiene 0.17  C20H38 278.52 Hydrocarbon
27. 16.676 4,8,12,16-Tetramethylheptadecan-4-olide 0.70 C21H40O2 324.54  
28. 17.252 2-methyl-3-hydroxy-2,3- dihydrobenzofuranone 0.12 C10H10O2 162.18 Flavoring agent
29. 17.340 1,3,5-Trisilacyclohexane 0.19 C3H12Si3 132.38  
30. 17.430 Trichloroacetic acid, hexadecyl ester 0.35 C18H33Cl3O2 387.81 Fatty acid
31. 17.637 Hexadecanoic acid, 2-hydroxy-1-(hydroxymethyl)ethyl ester 0.58 C19H38O4 330 fatty acid
32. 17.928 1,2-Benzenedicarboxylic acid (Phthalic acid) 0.17 C8H6O4 166.13 Aromatic dicarboxylic acid
33. 18.299 Dichloroacetic acid, undec-2-enyl ester 0.22 C13H22Cl2O2 281.219 Fatty acid
34. 18.813 1,3,5-Trisilacyclohexane 0.75 C3H12Si3 132 Organic Compound
35. 19.021 2-phenyl-furo[b]benzopyran-4(4h)-one (Karanjin) 1.78 C18H12O4 292.29 Flavonoid
36. 19.101 2-[5-(2-Methyl-benzooxazol-7-yl)-1H-pyrazol-3-yl]-phenol 4.15 C17H13N3O2 291 Organic compound
37. 19.231 Octadecanoic acid, 2,3-dihydroxypropyl ester 4.30 C21H42O4 358.55 Fatty acid
38. 19.486 2-phenyl-5-hydroxy-furo[b]benzopyran-4(4h)-one 0.60 C17H10O4 278  
39. 19.720 2-propenoic acid, 3-(3-nitrophenyl)-, ethyl ester 0.29 C11H11NO4 221 Fatty acid
40. 20.239 Squalene 2.27 C30H50 410.72 Triterpene
41. 20.399 Phenanthro[3,2-b]furan-7,11-dione, 1,2,3,4,8,9-hexahydro-4,4,8-trimethyl-, (+)- 0.14 C19H20O3 296  
42. 20.619 2,5-Cyclohexadiene-1,4-dione, 2,5-dihydroxy-3,6-diphenyl- 1.44 C18H12O4 292  
43. 20.877 1-Eicosanol 0.64 C20H42O 298.55 Fatty alcohol
44. 21.435 Sericic acid 0.76 C30H48O6 504 Phenolic acid
45. 21.857 δ-Tocopherol 3.97 C27H46O2 402 Vitamin- E
46. 22.978 Humulane-1,6-dien-3-ol 0.38 C15H26O 222.36 Monocyclic sesquiterpene
47. 23.117 Beta-tocopherol 1.18 C28H48O2 416.68 Vitamin- E
48. 23.226 Boldine 0.45 C19H21NO4 327.37 Alkaloid
49. 23.349 Tocopherol 0.51 C28H48O2 416 Vitamin- E
50. 23.460 Solanesol 0.54 C45H74O 630 Terpene
51. 23.567 1-Triacontanol 0.86 C30H62O 438.81 Fatty alcohol
52. 24.022 Tetra-O-methylfisetin 0.24 C19H18O6 342 Flavonoid
53. 24.339 Labetalol diacetate 1.51 C23H28N2O5 412 Anti hypertensive agent
54. 24.700 α-Tocopherol 0.46 C29H50O2 430.71 Vitamin- E
55. 25.153 Cholesterol 0.38 C27H46O 386.65 Phytosterol
56. 25.242 3,5,6-Trimethoxyfurano-(7,8,2",3")-flavone 0.37 C20H16O6 352.33 Flavonoid
57. 27.311 Campsterol 0.31 C28H48O 400.68 Phytosterol
58. 27.980 Stigmasterol 2.10 C29H48O 412.69 Phytosterol
59. 28.813 Tris(3-fluorophenyl)boroxin 0.70 C18H12B3F3O3 365.71 Organic compound
60. 29.413 β-Sitosterol 6.01 C29H50O 414.71 Phytosterol
61. 30.802 Β- amyrin 2.00 C30H50O 426.73 Phytosterol
62. 31.346 Lupeol 2.03 C30H50O 426.73 Triterpenoid
63. 32.038 Betulin 5.54 C30H50O2 442.72 Triterpene
64. 34.789 3,7,11,15-Tetramethyl-2-hexadecen-1-ol (Phytol) 0.49 C20H40O 296.53 Diterpene alcohol

Table 2: Activity of phytocomponents identified in the methanolic leaf extract of Pongamia pinnata

natural-products-chemistry-research-methanolic-leaf

Figure 2: GC/MS spectrogram for the methanolic leaf extract of Pongamia pinnata.

Furthermore, the identification of (unknown) plant constituents is still of interest [10,11], as it is the first step to explain the benefits of traditionally used medicinal plants by scientific means [12]. GC-MS method used for the analysis of the obtained extracts can be an interesting tool for testing the amount of some active principles in herbs used in cosmetic, drugs, pharmaceutical or food industry.

The present research study states that, methanolic leaf extract sample of P. pinnata was first time analyze. The comparison of the mass spectrum with the NIST database library gave more than 90% match as well as a supportive compound structure match. This study will help to identify the compounds, which may be used in body products, drugs, pharmaceutical and therapeutic value. Based on the present inventory of biochemical compounds we conclude that it may be used for medical purposes. This knowledge can be used for the further development of phytomedicines from this plant species.

Conclusion

The present research study states that, methanolic leaf extract sample of P. pinnata was first time analyze. The comparison of the mass spectrum with the NIST database library gave more than 90% match as well as a supportive compound structure match. This study will help to identify the compounds, which may be used in body products, drugs, pharmaceutical and therapeutic value. Based on the present inventory of biochemical compounds we conclude that it may be used for medical purposes. This knowledge can be used for the further development of phytomedicines from this plant species.

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Citation: Sangwan S (2018) Isolation and Analytic Characterization of β-Sitosterol and GC-MS Analysis of Methanolic Leaves Extract of Pongamia pinnata (L.) pierre. Nat Prod Chem Res 6:348.

Copyright: © 2018 Sangwan S. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.