Isolation of Tiliroside from Tribulus terrestris

Madhavi T.^1*, Soosamma John², Bincy Raj³, G.H.Urmilla¹

¹Nargund College of Pharmacy, Bangalore-85

²East Point College of Pharmacy, Bangalore-49

³Dyananda Sagar College of Pharmacy, Bangalore-78

ABSTRACT:

Gokhru, the fruits of Tribulus terrestris Linn belonging to the family Zygophyllaceae. Large number of compounds has been isolated from the fruits of Tribulus terrestris Linn. The present investigation was to isolate and characterize the compounds. Attempt was made to fractionate the 50%bmethanolic extract to isolate the phytoconstituents using column chromatography. One among the isolated compound is Tiliroside. It has been confirmed by various physio-chemical investigations like (M.P, NMR, HPLC, TLC etc).

KEYWORDS: Tribulus terrestris, Zygophyllaceae, Tiliroside, Column chromatography.

INTRODUCTION:

Tribulus terrestris Linn is a herb distributed throughout India and is popularly known as gokhru belonging to family Zygophyllaceae. The entire plant herb, more particularly the fruits are used in medicine. The plant is reported to contain steroidal saponins¹, alkaloids², lignamides³, flavanoids⁴ and steroidal glycosides⁵.Major steroidal saponin terrestrosin A-K⁶, two new steroidal saponins terrestrinins AandB⁷ have been isolated from Tribulus terrestris recently. More steroidal saponins are isolated from Tribulus terrestris fruits⁸.Tribulusterine and terrestribisamide⁹are two alkaloids isolated from Tribulus terrestris. The new hepatoprotective lignanamides from the fruits are tribulusamide AandB¹⁰. Isoterrestrosin B and tribulosaponin A are two major glycosides⁵ obtained from Tribulus terrestris. Kaempferol, kaempferol-3-rutinosode, kaempferol-3-glucoside and tribulosides have been isolated from Tribulus terrestris¹³. Protodioscin¹¹, a new saponin and their respective sulfates were detected. The anthelmentic activity of Tribulosin and β-Sitosterol - D-glucoside was found. Tribulus terrestris was used as rejuvenation tonic. In china, Tribulus terrestris is used in a number of conditions affecting the liver and kidney as well as the cardiovascular and immune systems¹². It is known in ayurveda for its anti-urolithiatic, diuretic and aphrodisiac properties^14.Therefore, in present study, an attempt was made to isolate the chemical constituents from 50% methanolic extract of fruits of Tribulus terrestris by using column chromatography and analytical HPLC and characterize them.

MATERIALS AND METHODS

The authenticated fresh fruits of Tribulus terrestris were obtained from Natural remedies, Bangalore. The fruits were then sundried, powdered and stored in airtight containers.

Extraction of plant material

The powdered plant material was extracted successively with 50% water and methanol. The main target of the study was to isolate phytoconstituents from 50% methanolic extract.

Partition of methanolic extract:

2 kg of 50% methanolic extract was partitioned with three solvents viz., water, n-butanol and ethyl acetate in pilot scale given in table1. 15% petroleum ether in ethyl acetate and 20% methanol in chloroform were showing the maximum separation of phytoconstituents.

Isolation of phytoconstituents:

The constituents of ethyl acetate layer were isolated by column chromatography. The selected extract was subjected to systematic column chromatography using different solvents systems. The fractions collected were further chromatographed using different stationary phases. Two types’ stationary phases viz., silica gel G and diaion HP20 were used to isolate non-polar as well as polar constituents. Column chromatography was done using a glass column. The dimension of the columns was 1.2m in length and 0.1mm in diameter. The column was packed with silica gel by wet packing method. The charged column is eluted with different mobile phases with gradual increase in polarity/non polarity Fig 1. The fractions collected were dried in rotavapour. The dried fractions were then weighed and recovered using methanol. All the fractions were subjected to TLC for the identification of the desired brands.10% methanol in chloroform from the column IV has shown a single spot in TLC fig 2. Methanol was added to the semisolid obtained. The compound was completely soluble in methanol and kept for recrytallization. A yellow solid compound got crystallized which was dried in vacuum, weighed and stored in labeled container.

HPLC Characterization:

The compounds were subjected to analytical HPLC for assessing its purity. A new HPLC method was developed and standardized in the laboratory of natural remedies.fig3

Column : Chromosil C-18

Mobile phase : Water containing H₃PO₄and Acetonitrile

Elution : Gradient

Flow rate : 1.5ml/min

Detector : SPD- M10Avp

Injection volume : 100 microlitres

Instrument : Shimadzu

NMR spectroscopy of the isolated compounds:

The isolated compound was subjected to following NMR analysis using pyridine and methanol as the solvent. The analysis was done at sophisticated instrument facility, Indian institute of science, Bangalore (Figs. 4.1, 4.2, 4.3, 4.4).

¹³ C NMR

¹H NMR

HMBC NMR

HSQC NMR

RESULTS:

Partition of methanolic extract:

2 kg of 50% methanolic extract for pilot scale is partitioned with water, ethyl acetate and n-butanol. The ethyl acetate layer has been taken for isolation.

Table 1

PROCESS	SOLVENTS	METHOD	YIELD (%W/W)
PILOT SCALE	Water	Separation	900gm
	Ethyl acetate	Separation	144.0gm
	n-butanol	Separation	475gm

Fig 2, TLC Analysis of the compound from column III and IV

Fig 3 HPLC Analysis of the compound

The purity of the compound has found to be 97.5464%

Fig 4 NMR Spectroscopy of the isolated compound

¹H NMR (fig 4.1)

¹³ C NMR (fig 4.2)

HMBC NMR (fig 4.3)

HSQC NMR (fig 4.4)

DISCUSSION:

The present investigation was to isolate and characterize the constituents from the fruits of Tribulus terrestris.50% methanolic extract was prepared and partioned with water,ethyl acetate and n-butanol.The ethyl acetate layer is selected for isolation of the compound using analytical techniques the isolated compound has been identified as Tiliroside. The purity of the compound has found to be 97.5464%

Tiliroside

ACKNOWLEDGEMENTS:

We thank Dr. Amit Agarwal, Director and Dr. Deepak M Senior Manager, Natural Remedies Pvt. Ltd. Bangalore for the constant support and providing all the facilities to carry out the research work.

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Received on 23.07.2011

Accepted on 29.06.2011

Research Journal of Pharmacognosy and Phytochemistry. 3(6): Nov. - Dec. 2011, 281-285