Global J Res. Med. Plants & I ndigen. Med.

| Volume 3, Issue 2 | February 2014 | 3339

Global J ournal of Research on Medicinal Plants & I ndigenous Medicine || GJRMI ||


ISSN 2277-4289 | www.gjrmi.com | International, Peer reviewed, Open access, Monthly Online Journal

CONVENTIONAL METHOD FOR SAPONIN EXTRACTION FROM
CHLOROPHYTUM BORI VI LI ANUM Sant. et Fernand
Sharma Rohit
1*
, Saxena Nidhi
2
, Thakur Gulab S
3
,
Sanodiya Bhagwan S
4
, Jaiswal Pallavi
5

1, 2, 3, 4, 5
Plant Biotechnology Laboratory, R&D Division, Tropilite Foods Pvt. Ltd., Davars Campus, Tansen
Road, Gwalior-474002 (M.P.), India.
*Corresponding Author: accessrohit25@gmail.com; Mob: +919755594040
Received: 07/12/2013; Revised: 25/01/2014; Accepted: 31/01/2014
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.








Short communication
Cite this article:
Sharma Rohit, Saxena Nidhi, Thakur Gulab S,
Sanodiya Bhagwan S, Jaiswal Pallavi (2014), CONVENTIONAL METHOD FOR
SAPONIN EXTRACTION FROM CHLOROPHYTUM BORI VI LI ANUM Sant. et
Fernand, Global J Res. Med. Plants & I ndigen. Med., Volume 3(2): 3339

Global J Res. Med. Plants & I ndigen. Med. | Volume 3, Issue 2 | February 2014 | 3339

Global J ournal of Research on Medicinal Plants & I ndigenous Medicine || GJRMI ||


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
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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, 8089% protein, 3
4% fiber and 217% 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 100C for
45 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 F
254
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
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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 F
254
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%, H
2
SO
4
). After spraying the
reagents, the plates were kept at 110C for 10
15 minutes in hot air oven and results were
observed later (Fig 1).

Fig 1: Thin layer chromatography results of In vivo, in vitro and standard samples developed
in mobile phase of chloroform: methanol: water (65:35:10 v/v/v)


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.
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Global J ournal of Research on Medicinal Plants & I ndigenous Medicine || GJRMI ||


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 23
times.
2. Delipidization: Ethanol was evaporated by
heating the collected supernatant at 45
55
o
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 4555
o
C in hot water bath and
leading to formation of a crude residue. The
residue was again dissolved in methanol
and heated at 4555
o
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.



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