Tribulin, a New Steroidal Saponin from the Aerial parts of Tribulus terrestris

Mamdouh N. Samy¹*, Mokhtar M. Bishr², Ahmed A. Ahmed³ and Mohamed S. Kamel¹

¹Department of Pharmacognosy, Faculty of Pharmacy, Minia University, Minia 61519, Egypt.

²Research and Development Department, Mepaco Company, Cairo, 11361, Egypt.

³Department of Organic Chemistry, Faculty of Sciences, Minia University, Minia 61519, Egypt.

ABSTRACT:

From the aerial parts of Tribulus terrestris, a new steroidal saponin, Diosgenin 3-O-[{a-L-rhamnopyranosyl-(1"3)} {a-L-rhamnopyranosyl-(1"4)}]-b-D-glucopyranosyl-(1"4)-b-D-glucopyranoside namely tribulin has been isolated together with six known compounds; scoparon, campesterol, stigmasterol, β-sitosterol, Diosgenin 3-O-[{a-L-rhamnopyranosyl-(1"4)-b-D-glucopyranoside and D-(+)-pinitol. The structure of the new saponin was elucidated on the basis of spectroscopic analyses including 1D-NMR and mass spectroscopy (EI-MS) and chemical reactions.

KEYWORDS: Tribulus terrestris, Zygophyllaceae, Steroidal saponin, Tribulin

INTRODUCTION:

Tribulus terrestris L.( Zygophyllaceae) is a herbaceous, annual, prostrate or semierect, diffusely branched herb ¹; native in dry and sandy districts in South Europe to Central Asia and in tropical and South Africa ², growing in India, other warm countries such as Ceylon ³, desert plains, waste ground, weed of cultivation and Mediterranean region ⁴. It is growing wildly in the green areas of Minia University campus. It is used in folk medicine to increase spermatogenesis, for treatment of eye troubles, edema, abdominal distension, leucorrhea and impotence, as aphrodisiac, galactagogue, anti-inflammatory, antidiarrheal and diuretic¹. The present study deals with the isolation and structure elucidation of a new steroidal saponin together with 6 known compounds from the aerial parts of T. terrestris (Fig. 1).

Fig. 1: Structure of compound 1

MATERIAL AND METHODS:

General Experimental Procedures

¹H- and ¹³C-NMR spectra were recorded on a JEOL LA. 500 MHz FT, 125 MHz, NMR spectrometer, (Japan) with tetramethylsilane as an internal standard. HR-EI mass spectrum was taken on Finnigan mat SSQ 7000 mass spectrometer, (USA). Silica gel column chromatography (CC) was performed on silica gel 60 [(E. Merck, Darmstardt, Germany) 70‒230 mesh]. Precoated silica gel 60 F₂₅₄ plates (E. Merck; 0.25 mm in thickness) were used for TLC analyses, visualized by spraying with a 10 % H₂SO₄ solution in ethanol and heating to around 150 °C on a hotplate.

Plant material: The aerial parts of T. terrestris were collected during the flowering and fruiting stage in October 2004 from the green areas of Minia University Campus, Minia, Egypt. A voucher specimen of the plant is deposited in the Herbarium of Faculty of Pharmacy, Minia University, Egypt (Minia-04-Mar-TT).

Extraction and isolation: The air-dried powdered aerial parts (3 kg) of T. terrestris was extracted with 70 % methanol (5 l × 5) till exhaustion and then concentrated under reduced pressure to yield a viscous gummy material. This residue (330 g) was dissolved in 250 ml of water and defatted with petroleum ether (1 l × 5). The aqueous layer was evaporated to remove a race amount of organic solvent, and then extracted with CHCl₃ and EtOAc, successively (1l ×5 each). The CHCl₃ and EtOAc fractions were concentrated under reduced pressure to give 45 g and 70 g of residues, respectively. The remaining aqueous layer was concentrated to furnish a water-soluble fraction (90 g).

The CHCl₃ fraction (45 g) was subjected to silica gel CC. The column was eluted initially with petroleum ether-CHCl₃ gradient elution, then with CHCl₃-MeOH gradient mixture, 500 ml fractions being collected. The similar fractions have been combined, affording 11 fractions. The second fraction II provided compound 2 (8 mg).The third fraction III was subjected to silica gel CC using petroleum ether-CHCl₃ then CHCl₃-MeOH gradient mixture to give compounds 3 (3 mg), 4 ( 3.5 mg), 5 (7 mg).

The EtOAc fraction (70 g) was subjected to silica gel CC and eluted with initially with CHCl₃, then with CHCl₃-MeOH stepwise gradient with increasing MeOH The effluents were collected in fractions (500 ml each). The similar fractions were combined to provide nine fractions.

The fifth fraction was repeatedly subjected to silica gel CC using CHCl₃-MeOH gradient mixture to give compound 6 (18 mg). The sixth fraction was repeatedly subjected to silica gel CC using CHCl₃-MeOH gradient mixture to give compound 1 (20 mg). The seventh fraction was repeatedly subjected to silica gel CC using then CHCl₃-MeOH gradient mixture to give compound 7 (8 mg).

RESULTS AND DISCUSSION:

The extract of the aerial parts of T. terrestris was subjected to repeated chromatographic purifications on silica gel column chromatography to afford 7 compounds. The isolated compounds were identified as tribulin (1), scoparon (2) ⁵, campesterol (3) ⁶, stigmasterol (4) ⁷, β-sitosterol (5) ⁸, Diosgenin 3-O-[{a-rhamnopyranosyl-(1" 4)-b-glucopyranoside (6) ⁹ and D-(+)-pinitol (7) ¹⁰. The structures of known compounds were determined by comparing their spectroscopic data with those reported in literature.

Compound 1 was obtained as an amorphous yellow powder and gave a pink colour after spraying the TLC plate with sulfuric acid followed by heating. It had the molecular formula of C₅₁H₈₂O₂₁ which was established from the positive-ion mode high-resolution (HR)-electron impact (EI)-mass spectrometry (MS). The ¹³C-NMR spectrum of compound 1 (Table 1) revealed the presence of 51 carbon signals, of which 24 carbons were assignable to the sugar moiety and 27 carbons to the aglycone. The signals at d_C 98.8, 99.0, 100.9 and 101.6 revealed the presence of 4 sugar units in the moiety ¹¹. The number of the sugar units confirmed from ¹H-NMR spectrum (Table 2), that showed 4 anomeric proton signals at d_H 4.34, 4.37, 4.55 and 4.58. The carbon signal at d_C 109.2 in the ¹³C-NMR spectrum indicated that the spirostanol nature of the aglycone ¹². The other carbon signals of the aglycone were superimposable with those reported for 3-O-substituted diosgenin ¹³. On the other hand, the sugar units were identified as one monosubstituted glucopyranose, one disubstituted glucopyranose and two terminal rhamnopyranoses ¹⁴.

Table 1: ¹³C-NMR Spectral Data of Compound 1 (125 MHz, CD₃OD):

Position	C	Position	C
1	37.2	Glucose
2	31.8	1'	101.6^a
3	77.7	2'	75.2
4	38.1	3'	78.2^b
5	140.5	4'	78.4^b
6	121.3	5'	77.9^b
7	31.7	6'	63.3^c
8	31.3	Glucose
9	50.3	1''	100.9^a
10	36.6	2''	72.5
11	20.6	3''	83.5
12	39.5	4''	76.6
13	40.0	5''	77.9^b
14	56.4	6''	62.6^c
15	31.4	Rhamnose
16	80.8	1'''	99.0^d
17	59.5	2'''	70.8^e
18	15.1	3'''	72.1^e
19	18.5	4'''	72.9^e
20	41.5	5'''	69.3^f
21	13.6	6'''	16.6^g
22	109.2	Rhamnose
23	30.1	1''''	98.8^d
24	28.5	2''''	70.6^e
25	29.3	3''''	71.1^e
26	66.5	4''''	72.4^e
27	16.2	5''''	68.4^f
28	--	6''''	16.5^g

a-g: interchangeable values

Table 2: ¹H-NMR Spectral Data of Compound 6 (500 MHz, CD₃OD):

Position	H (m, J in Hz)
18	0.21 (3H,s)
19	0.02 (3H,s)
21	0.12 (3H, d, 6.9)
27	0.04 (3H, d, 4.3)
1'	4.34 (1H, d, 10.7)
1''	4.58 (1H, d, 10)
1'''	4.37 (1H, br s)
1''''	4.55(1H, br s)
6'''	0.40 (3H, d, 6.1)
6''''	0.42(3H, d, 6.1)

The attachment sites of the sugar units were determined as 4-substituted glucopyranose from the downfield shift of C-4 at d_C 78.4 while those for the disubstituted glucopyranose were determined in positions 3 and 4 from the downfield shifts of their carbon signals at d_C 83.5 and 76.6 respectively.

The sequence of the sugar moiety was assigned on the bases of EI/MS analysis after acetylation of compound 1. It showed the presence of 2 terminal rhamnosyl units and 2 substituted glucosyl units from the peak at m/z=273 [2 acetyl rhamnose]⁺ indicate the presence of 2 terminal rhamnosyl units, the peak at m/z 791 [(2 acetyl rhamnose)+(acetyl disubstituted glucose)]⁺ indicated the presence of two terminal rhamnosyl units attached to one glucosyl unit, while the peak at m/z 1079 [2 acetyl rhamnose)+(acetyl disubstituted glucose)+(acetyl monosubstituted glucose)]⁺ indicated the presence of two terminal rhamnosyl units attached to one glucosyl unit which attached to another glucosyl unit (Fig. 2).

Fig. 2: EI/MS Fragmentation pattern of the acetylated compound 1

The β-configuration of the glucosyl units was determined from ¹H-NMR spectrum that revealed J= 10.7 Hz and 10 Hz for the anomeric signals at d_H 4.34 and 4.58 respectively.

Consequently the structure of compound 1 was determined as diosgenin 3-O-[{a-L-rhamnopyranosyl-(1"3)}-{a-L-rhamnopyranosyl-(1"4)}]-b-D-glucopyranosyl-(1"4)-b-D-glucopyranoside, namely tribulin.

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Received on 16.10.2012

Modified on 22.10.2012

Accepted on 27.10.2012

© A&V Publication all right reserved

Research Journal of Pharmacognosy and Phytochemistry. 4(5): September – October 2012, 237-239