Structural Elucidation of a New Novel Pentacyclic Triterpenoid Isolated from Caralluma Attenuata Root

Abbreviations

TLC: Thin Layer Chromatography, CC: Column Chromatography, MS: Mass Spectrometry, EI: Electron Ionisation, DEPT: Distortionless Enhancement by Polarisation Transfer, MPs: Melting point, NMR: Nuclear Magnetic Resonance

Introduction

Recently, triterpenoids are known to exhibit several pharmacologically activities and extensively discussed in the literature. These compounds can be used as anticancer agents [1-8], anti HIV [9,10], antiinflammatory [11], antiviral [5] and against neurodegenerative disorders [12]. Triterpenoids are the class of compounds which include squalene derivatives, lanostanes, holostanes, cycloartanes cucurbitanes, dammaranes, euphanes, triucallanes, tetranortriterpenoids, quassoids, luphanes, oleananes, friedelanes urasanes, hopanes, serratanes, isomalabaricanes and saponins.

Various medicinal uses of Caralluma species have been reported in Indian traditional medicine system. Most of the Caralluma species are used as anticancer [13], antitubercular and anthelmintic [14], antibacterial [15], healing of ulcers [16], appetite suppressant [17], nootropic [18], nociceptive [19], antioxidant actions [20], ability to lower blood sugar [21]. Species like Caralluma tuberculata, Caralluma Fimbriata, and Caralluma attenuata are the most popular widely utilized species in the genus. Isolation and characterization of oxypregnane glycosides [22,23], pregnane gylcosides [24-31], triterpene saponins [32], flavonoids [33], pregnane esters [34], bisdesmosidic glycosides [35,36], flavone glycosides [37], were earlier reported from the same genus. In the course of our investigation on chemical constituents of Carallumas we have isolated two novel triterpenoids [38,39]. In this paper we report the structural elucidation of another new novel pentacyclic triterpenoid derivative from C. attenuata.

Materials and Methods

Experimental

The plant material of C. attenuata was collected in Tirumala forests during January-2017. MPs uncorrected IR , Proton Nuclear Magnetic Resonance (¹H-NMR) δ ppm, 300 MHz CDCl3, Carbon 13 Nuclear Magnetic Resonance (¹³C-NMR) 150 MH, distortion less Enhancement by Polarization Transfer (DEPT) 135, Mass spectrometry (SHIMADZU 2000) Column Chromatography (CC) and Thin Layer Chromatography (TLC) on silica gel, TLC Chamber (Sigma Aldrich).

Preparation of TLC plates

A homogenous suspension of silica gel is prepared by mixing 20 g of 200 mesh silica gel G (Qualigens) in about 45 ml distilled water. This suspension is poured into TLC (UNDPLAN model) spreader, which was adjusted to 0.25 mm thickness. Glass plates (20 cm × 5 cm) are coated with this gel using spreader. These plates are air dried and activated in the oven at 110ºC for 30 min and then kept in a desiccator.

Extraction and isolation

The roots of Carallluma attenuata were air dried, powdered (1.8 kg) and extracted with 3 l of hexane, 3 l of benzene, 3 l of acetone and 3 l of methanol respectively using SOXHLET extractor. n-Hexane extract 50 g was subjected to column chromatography using silica gel 10-40 mesh. It is eluted with various fractions of benzene, acetone and methanol with increasing polarity. The various eluted fractions were observed time to time using TLC plate, benzene fractions (95-100) a yielded a white solid.

Detection by TLC

This white solid is dissolved in chloroform, spotted on TLC plate. The chromatograph was developed using benzene as a mobile phase. The dried plates were then sprayed with methanol-sulphuric acid reagent (98: 2) and heated in an oven for about 45 min, single spot (red colour) with R_f–0.1875 is observed. The solid obtained was recrystallised using benzene and acetone mixture and melting point was determined. MPs 240ºC. This was further analyzed by spectral data.

Spectral Data IR

3264.10, 2956.08, 2844.25, 1641.65, 1412.24, 1112.06, 1015.02.

¹H-NMR (δ ppm)

1.381, 1.534, 1.436, 1.674, 1.68, 1.18, 1.367, 1.678, 1.42, 1.69, 1.329, 1.13, 1.396, 1.412, 1.712, 1.17, 1.396, 1.495, 1.36, 1.742, 1.14, 1.496, 1.702, 1.372, 1.762, 0.925, 1.432, 0.87, 1.015, 1.315, 0.882, 1.054, 0.79, 5.156, 2.302, 3.379, 1.602, 0.756.

¹³C-NMR (δ ppm)

35.3611, 36.9137, 79.2, 41.0862, 44.3537, 29.3801, 30.0668, 45.0, 50.5015, 38.0, 21.4534, 26.8097, 47.6693, 42.4, 27.6043, 39.3620, 35.0, 47.8396, 41.391, 42.4, 37.2731, 36.9137, 18.9442, 27.3659, 17.3015, 15.603, 15.8027, 22.1138, 27.4352, 14.9319, 104.9132, 151, 40.187, 22.9581, 14.5294, 14.4079, 65.04.

DEPT

δ 35.3611, 36.9137, 41.0862, 44.3537, 29.3801, 30.0668, 50.5015, 21.4534, 26.8097, 47.66927.6043, 39.3620, 47.8396, 41.319, 37.2731, 18.9442, 27.3659, 17.3015, 15.6035, 15.8027, 22.1138, 27.4352, 14.9319, 104.9132, 40.187, 22.9581, 14.5294, 14.4079, 65.04.

MS EI+

[M+] m/z 538, 523, 537, 523, 510, 509 (100%), 495, 481, 466, 452, 410, 395, 353, 339, 227, 185, 171, 129, 116, 87, 73, 61.

Conclusion

In this paper, we report the isolation, purification and characterization of a new triterpenoid from C. attenuata. The available scientific research on Caralluma signifies its importance as medicinal plant used in a wide range of ethnomedicinal treatment especially for diabetes, obesity, gastro intestinal problems, blood disorders, skin problems and cancer. The medicinal properties of Caralluma are attributed to the presence of chemicals like pregnane glycosides, flavonoids, steroids, steroidal glycosides and terpenoids. Very few species of Caralluma are of these genus have been subjected to stringent scientific evaluation. The scientific evaluation of the triterpenoid isolated may help in exploring the medicinal importance of various Caralluma species. Since no sufficient information is available for the toxicity of the genus or its derivative medicines that need some extensive research. The diverse uses of Caralluma have made it susceptible to over exploitation and it may become threatened due to high trade. However Carallumas is not an invasive species, so modern cultivation techniques should be developed for commercial exploration. Tissue culture techniques must be developed to enhance the production and preserve natural germplasms.

Acknowledgement

I extend my sincere thanks to my research guide, Chairman and Principal of Vignan Degree and P.G. College for providing facilities to carry out my research work. I am also thankful to all my colleagues and family members for their support.

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