Der Pharma Chemica
Journal for Medicinal Chemistry, Pharmaceutical Chemistry, Pharmaceutical Sciences and Computational Chemistry

- Der Pharma Chemica ( 2019) Volume , Issue

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

Gurrala Jayalakshmi1, Vejendla Anuradha2*, Setty Ratna Kumari2, Sakamuri Sivarambabu3 and Sk. Rabbani Basha1
 
1Department of Chemistry, Vignan Degree and P.G. College, Guntur, Andhra Pradesh, India
2Department of Basic Sciences and Humanities VNITSW, Guntur, Andhra Pradesh, India
3Department of Chemistry, J.K.C. College, Guntur, Andhra Pradesh, India
 
*Corresponding Author:
Vejendla Anuradha, Department of Basic Sciences and Humanities VNITSW, Guntur, Andhra Pradesh, India, Email: [email protected]

Abstract

From the root of Caralluma attenuata belonging to the family Asclepiadaceae, a novel pentacyclic triterpenoid was isolated. Its structure was elucidated on the basis of spectroscopic data. This is the first report of such novel pentacyclic triterpenoid from C. attenuata root.

Keywords

Caralluma attenuata, Asclepiadaceae, Pentacyclic triterpenoid

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 image, 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 Rf–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 image

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.

Results and Discussion

The compound was isolated as white crystalline needles with MP 240ºC and analysed for C37H64O2 [M+]m/z 538 [M-2H]. The compound showed positive test for Libermann-Burchard reaction, Salkowski test indicating it to be a steroid/terpenoid.

The IR spectrum (Table 1) showed strong absorption at image 3264.10 cm-1 as a broad peak indicating the presence of –OH group. Absence of strong absorption between image 1650-1800 cm indicates the absence of >C=O. Thus from the molecular formula presence of two –OH groups can be expected which is supported by (M-2H) peak in mass spectra. A strong absorption at image 1641.65 cm-1 indicates the presence of olefinic double bond.

Table 1: IR spectral data

The important absorption peaks are shown below.

S. No. Absorption cm-1  Group Assignment
1 3264.1 O-H stretching vibration, the peak is broad
2 2956.08, 2844.25 C-H stretching in CH2
3 1412.24  =CH2 absorbs due to scissoring vibration of the terminal methylene group
4 1641.65 C=C stretching mode of unconjugated alkenes

The 1H-NMR spectrum showed the characteristic ring protons ranging from δ 0.6-1.8 indicating the presence of pentacyclic triterpenoid [40]. A septet at δ 1.602(1H) and a doublet at δ 0.756 (6H) indicates the presence of isopropyl group, loss of 43 units in mass spectra [M+ m/z- 43] also gives support for the presence of isopropyl group.

A two proton doublet at δ 5.156 indicates the presence of a disubstituted olefenic bond, weak C-H absorption in IR at image 1412.24 cm-1 indicates the presence of terminal double bond which is supported by 13C-NMR spectra. The 13C-NMR signal and δ 151 and δ 104.9 indicates the presence of olefenic carbons, 13C-NMR signal at δ 151 is not observed in DEPT clearly indicates this olefenic carbon to be a quaternary carbon. The signal at δ 104.9132 is seen in DEPT as CH2 carbon indicating a disubstituted terminal olefinic double bond [> C=CH2] .

A two protons downfield signal at δ 3.379d indicates that CH2 is attached to electronegative group i.e., presence of 1o alcoholic group -CH2OH. This is further supported by 13C-NMR and DEPT at δ 65.04 ppm and is further supported by [M+- CH2OH] at 509 in mass spectra.

A one proton signal at δ 2.302(m) indicates the allylic proton. A singlet signal at δ 1.432 accounting for 3 protons indicates –CH3 attached to a tertiary carbon with electronegative group accounting for a tertiary hydroxy group at C-3 is further supported by 13C-NMR signal at δ 79.2 which is not seen in DEPT. A signal at δ 1.2 accounting for 12[H] indicating the presence of 4 angular methyl groups. It is evidenced in 13C-NMR and DEPT at (δ 17.3015, 15.6035, 15.8027, 22.1138) and is supported by mass spectra through loss of 15 units.

1H-NMR shows a doublet signal corresponding to 3H at δ 0.925 indicating presence of a methyl attached to methine [-CH-] carbon.

It also shows a quartet at δ 1.054(2H) and a triplet δ 0.79 (3H) indicating an ethyl group attached to a quarternary carbon. Thus, based on IR, NMR (1H-NMR and 13C-NMR) (Table 2) partial structure of the compound can be as shown below (Figure 1).

Carbon Number 1H-NMR Data 13C-NMR Data Carbon Number 1H-NMR Data 13C-NMR Data
1   a-1.381  
b-1.534
35.3611 2   a-1.436  
b-1.674
36.9137
3 ----- 79.2 4 1.68 41.0862
5 1.18 44.3537 6 a-1.367
b-1.678
29.3801
7 a-1.42
b-1.69
30.0668 8 ---- 45
9 1.329 50.5015 10 ----- 38
11 a-1.13
b-1.396
21.4534 12  a-1.412
b-1.712
26.8097
13 1.17 47.663 14 ------ 42.4
15 a-1.396
b-1.495
27.6043 16 a-1.36
b-1.742
39.362
17 ------ 35 18 1.14 47.8396
19 a-1.496
b-1.702
41.391 20  ------ 42.4
21 a-1.343
b-1.742
37.2731 22 a-1.367
b-1.762
36.9137
Carbon Number 1H-NMR Data 13C-NMR Data Carbon Number 1H-NMR Data 13C-NMR Data
23 0.925 18.9442 24 1.432 27.3659
25 0.87 17.3015 26 1.015 15.6035
27 1.315 15.8027 28 0.882 22.1138
29 1.054 27.4312 30 0.79 14.9319
31 5.156 104.9 32  ---- 151
33 2.302 40.187 34 1.602 22.9581
35 0.756 14.5294 36 0.756 14.407
37 3.379 65.04      

Table 2: 1H-NMR and 13C-NMR spectral analysis

derpharmachemica-Furan-Partial-structure

Figure 1: Partial structure of the compound

Based on the triterpenoids isolated from the same plant [16,17], the position of angular methyls and tertiary –OH can be given as shown in Figure 2.

derpharmachemica-Furan-methyls-teritiary

Figure 2: Placement of angular methyls and teritiary –OH

Based on 1H-NMR, 13C-NMR and DEPT (Tables 3 and 4), the structure of the compound can be given as shown below (Figure 3).

Table 3: Mass spectral analysis

The mass spectral data is given below.

S. No. m/z Ion formed
1 538  [ M-2H]
2 539  [M-H]
3 523  [M - OH ], [M-2H-CH3]
4 510  [M-2CH3 ], [M-H-C2H5]
5 509  [M-CH2OH]
6 495  [M-2H-CH(CH3)2]
7 481  [M-2CH3-C2H5]
8 466 [M-2CH3-C2H5-CH3]
Carbon No.  -CH 0  -CH3  C
1  -- 35.36  --  --
2  -- 36.91  --  --
3  --  --  -- 79.2
4 41.086  --  --  --
5 44.333  --  --  --
Carbon No.  -CH 0  -CH3  C
6  --- 29.3801  --  --
7  -- 30.668  --  --
8  --  ----  -- 45
9 50.501  ---  --  --
10  --  ----  -- 38
11  --- 21.4534  --  --
12  -- 26.8097  --  --
13 7.663  ---  --  --
14  --  --  -- 42.4
15  -- 27.6043  ---  --
16  -- 39.362  --  --
17  --  --  -- 35
18 47.8396  ---  --  --
19  -- 41.39  --  --
20  --  --  --- 42.4
21  -- 37.2731  --  --
22  -- 36.9137  --  ---
23  --  -- 18.9442  ---
24  --  -- 27.3659  --
25  --  -- 17.3015  --
26  --  -- 15.6035  --
27  --  -- 15.8027  --
Carbon No.  -CH 0  -CH3  C
28  --  -- 22.1188  --
29  -- 27.4312  --  --
30  --  -- 14.9319  --
31  -- 104.39  ---  --
32  --  --  -- 151
33 40.187  --  --  --
34 22.9581  --  --  --
35  --  -- 14.4  --
36  --  -- 14.5294  --
37  -- 65.04  --  --

Table 4: DEPT spectral analysis

derpharmachemica-Furan-compound-based

Figure 3: Structure of the compound based on the analysis of 1H-NMR, 13C-NMR and DEPT

This structure is further supported by the mass spectral fragmentation as given in Scheme 1. This terpenoid is isolated and reported for the first time from this plant as well as from nature.

derpharmachemica-Furan-Spectral-Fragmentation

Scheme 1: Mass Spectral Fragmentation of Compound

The mass spectral data is given below.

DEPT spectrum showed carbon resonances relating to –CH, -CH2 and –CH3 protons present in the compound which are in agreement with 13C-NMR resonances.

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.

References

bornova escort karşıyaka escort osmangazi escort buca escort anne porno fetiş porno anal porno bartın escort burdur escort eskişehir escort escort izmir bursa escort porno escort bayan