CONVENTIONAL METHOD FOR SAPONIN EXTRACTION FROM CHLOROPHYTUM BORIVILIANUM Sant. et Fernand

Abstract

Saponins are imperative non-volatile chemical compounds valued for several medicinal properties. The pharmaceutical use of saponins for semi-synthesis of steroidal drugs makes it an essential element of life with a diverse range of properties including antimicrobial, insecticidal, haemolytic, aphrodisiac, foaming and emulsification. The tuberous roots of Chlorophytum borivilianum always remains a major source for isolation of saponin. A conventional efficient method was developed for saponin isolation from in-vivo and in-vitro samples of C. borivilianum by delipidization and deproteinization with petroleum ether and chloroform leading to development of a whole new process for saponin isolation. Protocol was tested with saponin confirmatory test, followed by thin layer chromatography.

Headnote

ABSTRACT

Saponins are imperative non-volatile chemical compounds valued for several medicinal properties. The pharmaceutical use of saponins for semi-synthesis of steroidal drugs makes it an essential element of life with a diverse range of properties including antimicrobial, insecticidal, haemolytic, aphrodisiac, foaming and emulsification. The tuberous roots of Chlorophytum borivilianum always remains a major source for isolation of saponin. A conventional efficient method was developed for saponin isolation from in-vivo and in-vitro samples of C. borivilianum by delipidization and deproteinization with petroleum ether and chloroform leading to development of a whole new process for saponin isolation. Protocol was tested with saponin confirmatory test followed by thin layer chromatography.

KEY WORDS: Saponin, Chlorophytum borivilianum, delipidization, steroid.

INTRODUCTION

Saponins are generally known as non- volatile, surface-active compounds that are widely distributed in nature, occurring primarily in the plant kingdom (Hostettmann et al., 2005). The name 'saponin' is derived from the Latin word sapo, which means 'soap', because saponin molecules form soap-like foams when shaken with water. They are structurally diverse molecules that are chemically referred to as triterpene and steroid glycosides. They consist of nonpolar aglycones coupled with one or more monosaccharide moieties (Oleszek, 2002). This combination of polar and non-polar structural elements in their molecules explains their soap-like behaviour in aqueous solutions. Saponins are the important chemical compounds from tubers of C. borivilianum. They are used in the indigenous systems of medicine as a well known health tonic, aphrodisiac and galactogogue (Chopra et al., 1956; Marais et al., 1978; Nadkarni, 1996; Oudhia, 2001). Pharmaceutical industries buy saponins in large quantities because of their use for the semi-synthesis of steroidal drugs for phyto-therapy and in cosmetic industry (Sharma et al., 2012; Haque et al., 2011; Ksouri et al., 2011). They are believed to form the main constituents of many plant drugs and folk medicines responsible for numerous pharmacological properties (Marais et al., 1978; Estrada et al., 2000; Debnath et al., 2006; Katoch et al., 2010). Therefore, it is a category of phyto-nutrients (plant nutrients) found abundantly in many beans, and other plants such as Ginseng, Alfalfa, Yucca, Aloe, Quinoa seed and also in Safed Musli (Chopra et al., 1956; Nadkarni, 1996).

Saponins have a diverse range of properties from sweetness to bitterness (Grenby, 1991; Kitagawa, 2002; Heng et al., 2006; Thakur et al., 2009), foaming and emulsification (Price et al., 1987), pharmacological and medicinal (Attele et al., 1999; Debnath et al., 2007), haemolytic (Oda et al., 2000; Sparg et al., 2004), and antimicrobial, insecticidal, and molluscicidal activities (Sparg et al., 2004; Sundaram et al., 2011) and finds some place in beverages, confectionery and cosmetic industry (Price et al., 1987; Petit et al., 1995; Uematsu et al., 2000). Saponins consist of a sugar moiety, usually containing glucose, galactose, glucuronic acid, xylose, rhamnose or methylpentose, glycosidically linked to a hydrophobic aglycone (sapogenin) which may be triterpenoid or steroid (Abe et al., 1993; Haralampidis et al., 2002); derived from the 30 carbon atoms containing precursor oxidosqualene (Haralampidis et al., 2002). The difference between the two classes lies in the fact that the steroidal saponins have three methyl groups removed (i.e. they are molecules with 27 C-atoms), whereas in the triterpenoid saponins all 30 C-atoms are retained. Saponins were classified into three classes, namely, the triterpenoid saponins, the spirostanol saponins and the furostanol saponins. However, due to secondary biotransformation such a classification emphasizes incidental structural elements and does not reflect the main biosynthetic pathways (Sparg et al., 2004). There are some other classes of compounds that have been considered as saponins, such as the glycosteroid alkaloids (Haralampidis et al., 2002). Baumann et al., (2000) reported that saponins have hemolytic properties that generally are attributed to the interaction between the saponins and the sterols of the erythrocyte membrane. As a result erythrocyte membrane bursts, causing an increase in permeability and a loss of haemoglobin. A study was made to establish the relationship between the adjuvant and haemolytic activity of saponins derived and purified from 47 different food and medicinal plants. However, the results indicated that the adjuvant activity does not relate with haemolytic activity (Oda et al., 2000).

Chlorophytum borivilianum Sant. et Fernand commonly known, as Safed Musli is a traditional rare Indian medicinal herb having many therapeutic applications in Ayurvedic, Unani, Homeopathic and Allopathic medicine system. It is an herbaceous plant with fasciculated tuberous root found naturally in forests and its shoots can be seen during the rainy seasons (Kothari et al., 2003). Research studies on Chlorophytum conducted in India and elsewhere indicate that saponins (viz. neohecogenin, neotigogenin, stigmasterol and tokorogenin) are responsible for medicinal properties (Jat et al., 1990). Safed musli is among the few medicinal plants witnessing steady growth in pharmaceutical, phytopharmaceutical and nutraceutical products (Debnath et al., 2006, 2007; Thakur et al., 2009). Due to the many therapeutic applications and several bioactive compounds, C. borivilianum is also called 'The white gold for biopharmaceuticals and neutraceuticals' (Thakur et al., 2009).

It contains steroidal and triterpenoidal saponins, sapogenins, fructans and flavonone glycosides, which are powerful uterine stimulants. Dried roots of Chlorophytum contain 42% carbohydrate, 80-89% protein, 3- 4% fiber and 2-17% saponin (Wagle et al., 2000). It is useful in curing impotency with spermatogenic property and is considered as an alternative to 'Viagra. It is a rich source of over 25 alkaloids, vitamins, proteins, carbohydrates, steroids, saponins, potassium, calcium, magnesium, phenol, resins, mucilage and polysaccharides with high content of simple sugars mainly sucrose, glucose, fructose, galactose, mannose and xylose (Ramawat et al., 2000; Debnath et al., 2006, 2007; Thakur et al., 2009). Due to their high medicinal value, several medicinal herbs are being indiscriminately collected before they could reach phenological maturity and vegetative regeneration capacity (Biswas et al., 2003). This has led to the depletion of natural source of several valuable plants like Safed musli. The restricted distribution and indiscriminate over- exploitation of this plant coupled with low seed set and viability and poor seed germination rates has made its status rare in the wild (Debnath et al., 2006). Among all the species of Chlorophytum present in India, C. borivilianum produces the maximum root tuber along with the highest saponin content (Attele et al., 1999). Traditionally, roots of these species are reputed to posses various pharmacological utilities having saponins as one of the important phyto-chemical constituents (Marais et al., 1978). The objective of the manuscript is to develop a brisk protocol for extraction of saponin from tubers of C. borivilianum with special attention on the screening of extracted metabolite.

MATERIAL AND METHODS

1. Plant Material:

Plants and roots of Chlorophytum borivilianum were collected from plant herbarium, Plant biotechnology laboratory at Tropilite foods Pvt. Ltd., Gwalior, India. Plants were available in vitro (in test tubes) and in vivo (in pots) conditions in laboratory. Plants (both roots and shoots) were washed thoroughly and were sliced into pieces followed by drying in hot air oven at 100°C for 4-5 days. On complete drying, the plant material was grinded uniformly with the help of mortar-pestle and stored in an airtight container.

2. Chemicals used:

Chemicals used for isolation purposes were 95% Ethanol (Merck Millipore), Petroleum ether (Sigma-Aldrich), Ethyl acetate (Sigma- Aldrich), Chloroform (Ultra pure, HiMedia), Methanol (Merck Millipore), Acetone (LR grade, HiMedia), Distilled water. Quality of isolated saponin was tested on TLC plates Silica gel 60 F254 plates (Merck) with Sulphuric acid (Rankem) as spraying agent.

3. Saponin Extraction Procedure:

The extraction process was carried out with both in vivo and in vitro samples by soaking the dried plant material in ethanol 95% overnight. The extraction was done with Petroleum ether, Ethyl acetate, Chloroform, Methanol and Acetone. Petroleum ether was used for delipidization and chloroform for deproteinization of dried mixture. On extraction of crude saponin, methanol was used to mellow the developing mixture followed by drop wise addition into acetone solution leading to precipitation. The precipitated material was extracted and dried in hot air oven leading to formation of whitish brown crystals (Lakshmi et al., 2012).

4. Saponin Confirmatory Test

Froth test: 0.5 gm of the alcoholic extract was dissolved in 10 ml of distilled water in a test tube. The test tube was shaken vigorously for about 30 seconds .The test tube was allowed to stand in vertical position and was observed over a 30 min period of time. Thick persistent froth was observed on the surface of the liquid indicating presence on saponin.

5. Thin layer Chromatography

TLC technique was used for purification of saponins isolated from C. borivilianum. Samples (crude saponin) and the reference standards (Saponin, Sigma) were loaded on the pre-coated TLC plates silica gel 60 F254 plates. Mobile phase chloroform: methanol: water (65:35:10 v/v/v) was used for the separation. Two drops of standard and sample were loaded up on TLC plates with the help of a micropipette. The loaded plates were placed in the TLC jar which contained the solvent system. After the completion of the run the plates were taken out and kept at room temperature to get dried for 10 minutes. The plates were developed with the spraying reagent (5%, H2SO4). After spraying the reagents, the plates were kept at 110°C for 10- 15 minutes in hot air oven and results were observed later (Fig 1).

RESULT AND DISCUSSION

The phytochemical extraction of in vivo root tubers and in vitro plant body of Chlorophytum borivilianum was carried out using six different solvent systems (Ethanol, petroleum ether, ethyl acetate, chloroform, methanol and acetone). Whitish brown crystals were obtained as end product of the process.

Experimental procedure:

1. Powder Soaking: In vivo and in vitro dried plant samples with a quantity of 30 gm each were mixed in 95% ethanol (180 ml) solution separately in conical flasks. After uniform mixing the solutions were placed in orbital shaker for stirring at 100 RPM for 12 hours. The supernatant was collected by filtration and the process was repeated 2-3 times.

2. Delipidization: Ethanol was evaporated by heating the collected supernatant at 45- 55°C in hot water bath to concentrate the solution. Petroleum ether was added to the concentrated solution and heated for around 30 minutes. After complete evaporation of the solvent, the residue was collected on a filter paper. Petroleum ether was used to remove lipid and fatty acids from plant and tuber of C. borivilianum.

3. Deproteinization: Residue was treated with equal ratio of Ethyl acetate- Chloroform and stirred the mixture for 15 minutes. Chloroform is deproteinizing agent used to remove proteins from plant and tuber of C. borivilianum.

4. Precipitation: In this step, ethyl acetate- chloroform was evaporated by heating the mixture at 45-55°C in hot water bath and leading to formation of a crude residue. The residue was again dissolved in methanol and heated at 45-55°C. The remaining warm residue was dropped in acetone solution drop by drop. White colored powder was obtained as precipitate in acetone. The precipitate was filtered and oven dried to obtain white crystals. Saponin in form of small crystals was collected on filter paper and preserved in air tight container for further testing.

CONCLUSION

The commercial promotion of saponin as dietary and nutraceutical supplement and evidences of presence of saponins in traditional medicine preparations also propagating a need for efficient method saponin isolation. The developed protocol is economic and less time consuming as well which only includes soaking, delipidization and deproteinization and avoiding the steps of water as mixing solvent and overnight stirring in water bath followed by dipping in organic solvent. The final quantity of product obtained depends upon the quality of ex-plant cultured. The final product obtained from the protocol was tested on froth confirmatory test and on thin layer chromatographic against the standard saponin.

Sidebar

Cite this article: Sharma Rohit, Saxena Nidhi, Thakur Gulab S, Sanodiya Bhagwan S, Jaiswal Pallavi (2014), CONVENTIONAL METHOD FOR SAPONIN EXTRACTION FROM CHLOROPHYTUM BORIVILIANUM Sant. et Fernand, Global J Res. Med. Plants & Indigen. Med., Volume 3(2): 33-39

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Source of Support: NIL

Conflict of Interest: None Declared