12 l Herbal Tech Industry l September 2011

Review
Extraction, Isolation and Purification of Saponins from Herbal Plants
Abstract
Saponins are a class of chemical compounds, one of many secondary metabolites found in natural sources, with
saponins found in particular abundance in various plant species. Specifically, they are amphipathic glycosides
grouped phenomenologically by the soap-like foaming they produce when shaken in aqueous solutions, and
structurally by their composition of one or more hydrophilic glycoside moieties combined with a lipophilic
triterpene derivative. A ready and therapeutically relevant example is the cardio-active agent digoxin, from
common foxglove.most of the saponins of official saponins drugs are triterpene glycosides.some drugs also or
only containe steroidal saponins.generally triterpene saponins having acidic properties due to presence more
carboxyl group in the aglycone and sugar moiety.steroidal saponins posses less sugar units than the triterpene
saponins.
Generally these saponins are separation by preparative separation was performed by water extraction using
reversed-phased C18 column chromatography,their structure were characterized by eletrospray ionization
mass spectrometry (ESI-MS),1HMNR and 13C-NMR.
combined with a lipophilic triterpene derivative[Zohar Kerem]. A
ready and therapeutically relevant example is the cardio-active
agent digoxin, from common foxglove
Sources of Saponin
Saponins have historically been understood to be plant-derived,
but they have also been isolated from marine organisms.
Saponins are indeed found in many plants, and derive their name
from the soapwort plant (Genus Saponaria, Family
Caryophyllaceae), the root of which was used historically as a
soap.Saponins are also found in the botanical family
Sapindaceae, with its defining genus Sapindus (soapberry or
soapnut), and in the closely related families Aceraceae (maples)
and Hippocastanaceae (horse chestnuts; ref. needed).[ Haijiang
Introduction
Saponins are a class of chemical compounds, one of many
secondary metabolites found in natural sources, with saponins
found in particular abundance in various plant species.
Specifically, they are amphipathic glycosides grouped
phenomenologically by the soap-like foaming they produce
when shaken in aqueous solutions, and structurally by their
composition of one or more hydrophilic glycoside moieties
combined with a lipophilic triterpene derivative. A ready and
therapeutically relevant example is the cardio-active agent
digoxin, from common foxglove.most of the saponins of official
saponins drugs are triterpene glycosides.some drugs also or only
containe steroidal saponins.generally triterpene saponins
having acidic properties due to presence more carboxyl group in
the aglycone and sugar moiety.steroidal saponins posses less
sugar units than the triterpene saponins[David G. I. Kingston]
Generally these saponins are used for the anti-inflammation,
anti-allergy, anti-tumour, anti-obesity and anti-hyperlipidemic
effects. [Young Wan Ha]
Saponin
Saponins are a class of chemical compounds, one of many
secondary metabolites found in natural sources, with saponins
found in particular abundance in various plant species.
Specifically, they are amphipathic glycosides grouped
phenomenologically by the soap-like foaming they produce
when shaken in aqueous solutions, and structurally by their
composition of one or more hydrophilic glycoside moieties
*1 1 1 1 2 2 2
Surendar. M , Sai Kumar.P , Shyam Prasad.T , Madhava Reddy. A , Nagulu.M , Hari Prasad.P , Shaik Shabber ,
2
Vamshi Sharath Nath. K
1.Nalanda College of Pharmacy,
2.Swamy Ramananda Tirtha Institute of Pharmaceutical Sciences
*Corrsponding Author
E-mail : devinirmala1980@yahoo.co.in
Chemical Structure of the Solanine (Saponin)
13 l Herbal Tech Industry l September 2011
Review
Zhang] It is also found heavily in gynostemma pentaphyllum
(Genus Gynostemma, Family Cucurbitaceae) in a form called
gypenosides, and ginseng (Genus Panax, Family Araliaceae) in a
form called ginsenosides.[ Niramon Utama-ang ]
Within these families, this class of chemical compounds are
found in various parts of the plant: leaves, stems, roots, bulbs,
blossom and fruit.Commercial formulations of plant-derived
saponins e.g., from the soap bark (or soapbark) tree, Quillaja
saponaria, and from other sourcesare available via controlled
manufacturing processes, which make them of use as chemical
and biomedical reagents.
Medicinal Uses
There is tremendous, commercially driven promotion of
saponins as dietary supplements and nutriceuticals. There is
evidence of the presence of saponins in traditional medicine
preparations, where oral administrations might be expected to
lead to hydrolysis of glycoside from terpenoid (and obviation of
any toxicity associated with the intact molecule).[ Farah S] But as
is often the case with wide-ranging commercial therapeutic
claims for natural products:
?the claims for organismal/human benefit are often based on
very preliminary biochemical or cell biological studies; and
?mention is generally omitted of the possibilities of individual
chemical sensitivity, or to the general toxicity of specific
agents, and high toxicity of selected cases.
While such statements require constant review it appears that
there are very limited US, EU, etc. agency-approved roles for
saponins in human therapy. In their use as adjuvants in the
production of vaccines, toxicity associated with sterol
complexation remains a major issue for attention. Even in the
case of digoxin, therapeutic benefit from the cardiotoxin is a
result of careful administration of an appropriate dose [D.A.
RICKERT ]. Very great care needs to be exercised in evaluating or
acting on specific claims of therapeutic benefit from ingesting
saponin-type and other natural products[Maggie P.K. Choia ].
Classification:
Saponins are generally classified in to,
1. Steroidal saponins
Steroidal saponins are great pharmaceutical importent of
because of their relationship to compounds such as the sex
hormone, cortisome, diuretic steroids, vit.D and cardiac
glycosides.
Some are used as starting material for the synthesis of these
compounds,diosgenin is the principle sapogenin used by
indentra but most yams.
Natural sapogenins differ only in their configuration at carbon
atoms 3,5 and 25 and in the spirostane series the orientation at
c-22 need be specified[Kalpana Mujoo ].
Eg: Sarsaponin (3-glucose,1-rhamnose)
Digitoninn (2-glucose,2-galactose,1-xylose)
Gitonin (1-glucose,2-galactose,1-xylose)
2. Saponin glycosides
Eg: shatavari (shatavari I,II ) Brahmi (Bcosides A and B).
3. Pentacyclic triterpenoid saponins:
The pentacyclic triterpenoid saponins are rare in monocolyte
dons.these are present in caryphyllaceae, sapindaceae,
polygalaceae families.
Eg: Aescin (2-glucose, 1-glucuronic acid)
Aralin (2-arabinose, 1-glucuronic acid).
4.Triterpenoid saponins:
These are classified in to three groups
a.-amyrin b.amyrin c.lupeol
Acidic properties,due to presence of one carboxyl group in the
aglycone and sugar moiety
Steroidal saponins posses less sugars than the triterpene
saponins.
Tests for saponin glycoside:
A. Foam test:
Shake the drug extract or dry powder vigorously with
water.persistent foam observed.
B.Heamolytic test:
Add drug extract or dry powder to one drop of blood placed on
glass side.heamolytic zones appears.
Identification:
Sapogenins give color test with sulphuric acid,Antimony
trichloride,tri chloro acetic acid but these are not very
specific.the IR spectrum of sapogenins shows several strong
absorption bands between 1350 and 875cm-1 due to the
spiroketal side chain.Another characteristic features is the
intense absorption at 920-915cm-1 in the 25 series whereas in
the 25 series these intense absorption at 899-894cm-1
Extraction Process:
l Direct solvent extraction
14 l Herbal Tech Industry l September 2011
Review
l
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Soxhlet extraction
Sequential solvent extraction
Ultrasound-assisted extraction
Microwave-assisted extraction.
Separation and Identification Techniques:
Chromatographic technique
Ultra violet technique
Infra red techniques
NMR techniques
Mass spectrometry
Cappillary electrophoresis
Gel filteration
Centrifugal partition chromatography.
Characterisation Of Saponins.
Paper chromatography
Column chromatography
Open-column chromatography
Conventional open column chromatography
Dry-column chromatography
Thin-layer chromatography
Liquid-Liquid chromatography
Medium pressure liquid chromatography
Centrifugal partition chromatography
Gas-liquid chromatograph
Separation of Saponins by using TLC Method:
Preparations of extracts:
powdered drugs (2g) is extracts by heating for 10mints under
reflux with 10ml,70% ethanol.the filtrate is evaporate to about
5ml, and 20-40cm of this solutions is used for TLC[GEORGE V ]. A
total of 3ml of the ethanolic extracts (with above solutions) is
shaken several times with 5ml water and saturated n-butanol.
the n-butanol phase is separated and concentrated to about 1ml
;but with 20ml is used for TLC [REBECCA M. CORBIT ].
Exceptions:
Ginseng radix:
Ginseng radix is extracted under the same conditions ,but with
90% ethanol.
Liquiritiae radix :
An ethanol extacts (above solution) is evaporated to dryness. the
residue is dissolved in 20ml chloroform: methanol (1:1); 20ml is
used for the detection of glycyrrhin [P.B.MALLIKHARJUNA].
T L C:
Adsorbent: silica gel 60 F254-percorated TLC plates.
Solvents:
Chloroform: glacial acetic acid :methanol: water.
(64:32:12:8). This system is suitable for the separations of
saponins mixtures from the listed drugs;
1.Chloroform-methanol-water(70:30:8) for extractions of
gensenoside from Ginseng radix.
2.Ethyl acetate-ethanol-water-ammonia (65:25:9:1) for
extraction of glycyrrhetic acid from Liquritae radix.
Detection:
1. without chemical treatment:
With the exception of glycyyhetic acid (Liquiriae radix), no
saponins are detectable by exposure to uv-254 or uv-365nm.
Blood reagent:
Hemolytically active saponins are detected as with zones on a
reddish background[M. Sajjad Khan ].
2. vanillian-sulphuric acid reagent:
Saponins form mainly blue, blue-violet and sometimes
red/yellow-brown zones.
Drug List:
Avenue sativa herbs (poaceace): steroidal saponins; Triterpene
saponins.3-4% free sugars.
Centellac herb(apiaceae):Ester saponins.
Ginseng radix(araliaceae):2-3% tetra cyclic triterpene glycosides.
Liuirititiae radix (fabaceae):saponins; 8-12% glycyrrhizin,calcium
salt of glycyrrhizic acid.
Saponariae radix (caryop phylaceace) :3-5% bidesmosidic
triterpene saponins
Sarsaparillae radix: 1.8-3% steroidal saponins,mono desmosidic,
spirostanol saponins
Identification of Major Saponins from Jiaogulan Extract
15 l Herbal Tech Industry l September 2011
Review
By GC-MC ANALYSIS.
Gynostemma pentaphyllum
Fresh GP leaves were dried by microwave dryer until the moisture
content was below 10%. Dried GP was vacuum packed in
aluminium foil and kept in -200C until used.
Chemicals
The chemicals used for GC-MS analysis; Trimethylchorosilance
Trimethyl-silylimidazol and N,N-bistrimethylsiyl- trifluoro
acetamide and standard ginsenoside Rb1 The solvent,
methanol, ethanol and butanol, were analytical grades
Sample preparation
Extraction from microwave dried GP used three methods, hot
water, methanol and ethanol extractions. The dried GP were
extracted with solvents at 1:30 proportions[Shahla Najafi]. The
water extract method used double distilled water, heating in
water bath shaker at 900C for 10 min at 100 rpm. The methanol
extract method used 80% methanol with Soxhlet extraction for 6
hours
GC-MS analysis.
A GCQ ion trap gas chromatography mass spectrometer
(electron impact ionization, 70 eV) was used in this study. A SPB
1701 column (column length 15 m., 0.25 mm. I.D., film thickness
0.25 mm) was used. The column flow rate was 0.8 ml/min by
helium gas. The conditions for the SPB1701 column were 1500C
for 0.1 min to 2700C at 100C/min with a hold for 10 min [P.Devil].
Injector temperature was 2500C. Ion source temperature was
2000C. The aglycones obtained from the samples were
identified by comparing of the retention time, relative retention
time and mass authentic saponin
Microwave-assisted extraction of bioactive saponins from
chickpea (Cicer arietinum)
Extraction procedures
Seed powder:
Prior to all extractions, chickpea seeds were ground in a Wiley
mill to pass a 2-mm pore-size screen, and dried at 55 ?C for 72 h.
The dried powder was then extracted using a Soxhlet apparatus
with hexane for 6 h to remove all fats.
Microwave-assisted extraction
Defatted powder (4 g) was mixed with a solvent of choice
(MeOH, EtOH or EtOH:H2O 7:3, butanol or butanol:water 1:1;
16ml) in 20-ml closed vials, which were placed in a mechanically
modified microwave oven and irradiated at 2450Mhz for 10 or
20 min. The solvent temperature was kept constant at 60?C
using an automatic temperature control device submerged into
solvent containing vessel. Twelve sample TFM (a thermally
resistant form of Teflon) vessels were used at a time, with
pressure and temperature monitoring capabilities, The
microwave power was limited to 300 W[Devendra N. Kage].
After cooling to room temperature, the extract was collected
and kept at - 20 ?C until analysis.
Soxhlet extraction:
Defatted powder (10 g) was extracted with the solvent of choice
(150 ml), for 3 h. After cooling to room temperature, the extract
was collected and kept at - 20 ?C until analysis.
Eg: Microwave-assisted extraction bioactive saponins from
chickpea (Cicer arietinum L).
Purification of saponins
The filtrate was loaded onto a C-18 preparative column and
impurities were eluted with 600 ml l- 1 methanol in water. The
saponin containing fraction was eluted with methanol. The
eluted fraction was diluted with water and was further purified
using HPLC to isolate DDMP-saponins. The HPLC system was
equipped with a diode-array detector (UV6000) and a column
oven (35 ?C)
Identification
The substance was collected and analyzed by 1H and 13C NMR .
1H NMR and 13C NMR spectra identical to the data reported for
a DDMP moiety. The amount of DDMP-saponin was also
determined2,3-dihydro-2,5-dihydroxy-6-methyl- 4H-pyran-4-
one (DDMP) moiety on C-22.
Solid-phase extraction and liquid chromatographyelectrospray
mass spectrometric analysis of saponins in Salvia miltiorrhizae
and Panax notoginseng
solid-phase extraction (SPE) and HPLC/ESI-MSn for the
identification of the major saponins in Danshen Dripping Pill,
a Chinese patent medicine consisting of Salvia miltiorrhizae and
Panax notoginseng.
these saponins were characterized by HPLC/ESI-MSn analysis.
Solid-phase extraction:
A 2 g weight of Danshen Dripping Pill was dissolved using
20mL 4% ammonia in an ultrasonic bath at 25 ?C for 15 min.
After centrifugation at 5000g for 10 min, a certain volume of
the supernatant fluid was loaded and drawn through by gravity
on SPE cartridge (5 mL, packed with 250 mg of 40m octadecyl
silica,Waters, USA), which was pretreated by passing through
5mL of methanol followed by 5mLwater before loading, and
drawn through by gravity. Then, the solidphase cartridge was
washed with 10.0mLofwater to elute the phenolic compounds
entirely off. Finally, the cartridge was eluted with 1.0mL
methanol, in which fraction most of the saponins were
concentrated.A20 mL volume of the methanol eluent was
injected into the HPLC system HPLCMS analysis was performed
16 l Herbal Tech Industry l September 2011
Review
with Agilent LCMSD/ Trap System (Agilent Company) equipped
with an electrospray interface. TheMSspectra were acquired in
negative ion mode.N2 was used as both drying gas with a
flowrate of 10 L/min and as nebulizing gas with a pressure of 60
psi. The nebulizer temperature was set at 350 ?C and the
capillary voltage was set at 3500V. The mass spectra were
recorded in the range of 4001500m. A fragment amplification
of1.5V was selected for MS2 analysis.
HPLC grade methanol was used for SPE preparation.
Preparative Isolation of Six Major Saponins from Platycodi Radix
by High-speed Counter-current
Apparatus:
A TBE-300A HSCC, with three serially connected multilayer coil
separation column
Reagents and materials:
Acetonitrile, methanol, n-butanol, ethylacetate, hexane and
isopropanol (HPLC-grade) for the preparation of the crude
sample and for HSCCC separation
Preparation of the two-phase solvent system and sample
solution.
A solvent system consisting of hexanen-butanolwater
(1:40:20, v/v) was used for the separation of platycoside E and
deapio-platycoside E. Hexanen-butanolwater (1:10:5, v/v) was
used as the solvent separation of platycodin D3, platycodin D
and their deapiose forms.
HSCCC separation.
In conventional HSCCC experiments, a multi-layer coiled column
is first entirely filled with one phase of the two phase solvent
system as a stationary phase, followed by elution with the other
phase. Here, the column was first filled with a mixture of the two
phases, thus reducing the amount of time for hydrodynamic
equilibrium to be established (Slacanin et al., 1989). The ratio of
two phases was also optimised at 70:30 (stationary phase
mobile phase, v/v) within the range 90:10 to 60:40 based on the
amount of time required to reach hydrodynamic equilibrium
owing to retention on the stationary phase. In the present
experiment, several solvent systems based on n-butanol water
with added hexane or ethyl acetate were tested, and the results
are summarized in Table 1. The table shows that platycoside E
and deapio-platycoside E, when analysed in the reverse-mode
solvent system composed of n-butanolwater at volume ratios
of 2:1 (v/v), have appropriate K values (0.55) for separation.
However, the K values of platyodin D and deapio-platycodin D, in
the same solvent system, were smaller than expected. It was
difficult to separate platyodin D and deapio-platycodin D from
the other compounds. Also, when n butanol water (2:1, v/v
used the retention of the stationary phase was poor (<30%), and
so that system was deemed unsuitable for separation.
HPLC analysis:
of 70 C and a gain of 7, and the nebuliser gas (nitrogen) was
adjusted to 2.5
The HPLC conditions for the platycosides were as follows: eluent
A, water; eluent B, acetonitrile; gradient, 06 (1015% B), 650
min (1525% B), 5060 min (2547.5% B) and then equilibrated
with 10% B for 8 min at a flow of 1mL/min. The ELSD system was
set to a probe temperature bar.
Identification of HSCCC peak fractions.
Identification of the HSCCC peak fractions was carried out by ESI-
MS, 1H-NMR and 13C-NMR spectra with references.
Structural identification
The chemical structures of components present in each peak
fraction purified by HSCCC were identified from ESI-MS, ESI-
MS/MS, 1H-NMR and 13C-NMR data. The subsequent
structural identification of the peak fractions collected
from the HSCCC was performed by comparison with previous
1H-NMR and 13C-NMR data.
A New Bioactive Steroidal Saponin from Agave attenuate Herbal
plant.
Introduction:
The occurrence of steroidal saponins in Agave genus is well
documented
Some species have an ethnopharmacological background, in
particular A. sisalana which in the Bahama Islands, the central
bud is boiled with salt and the decoction given as a remedy for
jaundice; said to be effective within 24 hours, the aqueous
extract of A. attenuate was evaluated for activity against Bulinus
africanus, Daphnia pulex, Anopheles arabiensis and
Oreochromis mossambicus demonstrating molluscicidal,
piscicidal and larvicidal properties
plant:
Fresh leaves of Agave attenuata were obtained from the
Ornamental Plant Garden
Extraction and isolation:
The fresh leaves of the plant (3 kg) were extracted with 80%
aqueous EtOH (6 l) followed by concentration to 600 ml and
extraction with an equal volume of n-BuOH gave a crude
material (12.5 g). It was roughly chromatographed on Sephadex
LH-20 with MeOH to give crude steroidal glycoside (2.5 g).
Further purification by chromatography on a silica gel column
eluted with CHCl3:MeOH:H2O (70:30:10 v/v/v) to afford one TLC
Open-column chromatography
17 l Herbal Tech Industry l September 2011
Review
Conventional open column chromatography
Dry-column chromatography
Thin-layer chromatography
Liquid-Liquid chromatography
Medium pressure liquid chromatography
Centrifugal partition chromatography
Gas-liquid chromatograph
SEPARATION OF SAPONINS BY USING TLC METHOD:
Preparations of extracts:
powdered drugs (2g) is extracts by heating for 10mints under
reflux with 10ml,70% ethanol.the filtrate is evaporate to about
5ml, and 20-40cm of this solutions is used for TLC[GEORGE V ].A
total of 3ml of the ethanolic extracts (with above solutions) is
shaken several times with 5ml water and saturated n-butanol.
the n-butanol phase is separated and concentrated to about 1ml
;but with 20ml is used for TLC[REBECCA M. CORBIT ].
Exceptions:
Ginseng radix:
Ginseng radix is extracted under the same conditions ,but with
90% ethanol.
Liquiritiae radix :
An ethanol extacts (above solution) is evaporated to dryness. the
residue is dissolved in 20ml chloroform: methanol (1:1); 20ml is
used for the detection of glycyrrhin [P.B.MALLIKHARJUNA].
T L C:
Adsorbent: silica gel 60 F254-percorated TLC plates.
Solvents:
Chloroform: glacial acetic acid :methanol: water.
(64:32:12:8). This system is suitable for the separations of
saponins mixtures from the listed drugs;
1. Chloroform-methanol-water(70:30:8) for extractions
of gensenoside from Ginseng radix.
2. Ethyl acetate-ethanol-water-ammonia (65:25:9:1) for
extraction of glycyrrhetic acid from Liquritae radix.
Detection:
1. without chemical treatment:
With the exception of glycyyhetic acid (Liquiriae radix), no
saponins are detectable by exposure to uv-254 or uv-365nm.
Blood reagent:
Hemolytically active saponins are detected as with zones on a
reddish background[M. Sajjad Khan ].
2. vanillian-sulphuric acid reagent:
Saponins form mainly blue, blue-violet and sometimes
red/yellow-brown zones.
Drug List:
Avenue sativa herbs (poaceace): steroidal saponins; Triterpene
saponins.3-4% free sugars.
Centellac herb(apiaceae):Ester saponins.
Ginseng radix(araliaceae):2-3% tetra cyclic triterpene glycosides.
Liuirititiae radix (fabaceae):saponins; 8-12% glycyrrhizin,calcium
salt of glycyrrhizic acid.
Saponariae radix (caryop phylaceace) :3-5% bidesmosidic
triterpene saponins
Sarsaparillae radix: 1.8-3% steroidal saponins,mono desmosidic,
spirostanol saponins
Identification of Major Saponins from Jiaogulan Extract
By GC-MC ANALYSIS.
Gynostemma pentaphyllum
Fresh GP leaves were dried by microwave dryer until the moisture
content was below 10%. Dried GP was vacuum packed in
aluminium foil and kept in -200C until used.
Chemicals
The chemicals used for GC-MS analysis; Trimethylchorosilance
Trimethyl-silylimidazol and N,N-bistrimethylsiyl- trifluoro
acetamide and standard ginsenoside Rb1 The solvent,
methanol, ethanol and butanol, were analytical grades
Sample preparation
Extraction from microwave dried GP used three methods, hot
water, methanol and ethanol extractions. The dried GP were
extracted with solvents at 1:30 proportions[Shahla Najafi]. The
water extract method used double distilled water, heating in
water bath shaker at 900C for 10 min at 100 rpm. The methanol
extract method used 80% methanol with Soxhlet extraction for 6
hours
GC-MS analysis.
A GCQ ion trap gas chromatography mass spectrometer
(electron impact ionization, 70 eV) was used in this study. A SPB
1701 column (column length 15 m., 0.25 mm. I.D., film thickness
0.25 mm) was used. The column flow rate was 0.8 ml/min by
18 l Herbal Tech Industry l September 2011
Review
helium gas. The conditions for the SPB1701 column were 1500C
for 0.1 min to 2700C at 100C/min with a hold for 10 min [P.Devil].
Injector temperature was 2500C. Ion source temperature was
2000C. The aglycones obtained from the samples were
identified by comparing of the retention time, relative retention
time and mass authentic saponinMicrowave-assisted extraction
of bioactive saponins from chickpea (Cicer arietinum)
Extraction procedures
Seed powder:
Prior to all extractions, chickpea seeds were ground in a Wiley
mill to pass a 2-mm pore-size screen, and dried at 55 ?C for 72 h.
The dried powder was then extracted using a Soxhlet apparatus
with hexane for 6 h to remove all fats.
Microwave-assisted extraction
Defatted powder (4 g) was mixed with a solvent of choice
(MeOH, EtOH or EtOH:H2O 7:3, butanol or butanol:water 1:1;
16ml) in 20-ml closed vials, which were placed in a mechanically
modified microwave oven and irradiated at 2450Mhz for 10 or
20 min. The solvent temperature was kept constant at 60?C
using an automatic temperature control device submerged into
solvent containing vessel. Twelve sample TFM (a thermally
resistant form of Teflon) vessels were used at a time, with
pressure and temperature monitoring capabilities, The
microwave power was limited to 300 W[Devendra N. Kage].
After cooling to room temperature, the extract was collected
and kept at - 20 ?C until analysis.
Soxhlet extraction:
Defatted powder (10 g) was extracted with the solvent of choice
(150 ml), for 3 h. After cooling to room temperature, the extract
was collected and kept at - 20 ?C until analysis.
Eg: Microwave-assisted extraction bioactive saponins from
chickpea (Cicer arietinum L).
Purification of saponins
The filtrate was loaded onto a C-18 preparative column and
impurities were eluted with 600 ml l- 1 methanol in water. The
saponin containing fraction was eluted with methanol. The
eluted fraction was diluted with water and was further purified
using HPLC to isolate DDMP-saponins. The HPLC system was
equipped with a diode-array detector (UV6000) and a column
oven (35 ?C)
Identification:
The substance was collected and analyzed by 1H and 13C NMR .
1H NMR and 13C NMR spectra identical to the data reported for
a DDMP moiety. The amount of DDMP-saponin was also
determined2,3-dihydro-2,5-dihydroxy-6-methyl- 4H-pyran-4-
one (DDMP) moiety on C-22.
Solid-phase extraction and liquid chromatographyelectrospray
mass spectrometric analysis of saponins in Salvia miltiorrhizae
and Panax notoginseng
solid-phase extraction (SPE) and HPLC/ESI-MSn for the
identification of the major saponins in Danshen Dripping Pill,
a Chinese patent medicine consisting of Salvia miltiorrhizae and
Panax notoginseng.
these saponins were characterized by HPLC/ESI-MSn analysis.
Solid-phase extraction:
A 2 g weight of Danshen Dripping Pill was dissolved using
20mL 4% ammonia in an ultrasonic bath at 25 ?C for 15 min.
After centrifugation at 5000g for 10 min, a certain volume of
the supernatant fluid was loaded and drawn through by gravity
on SPE cartridge (5 mL, packed with 250 mg of 40m octadecyl
silica,Waters, USA), which was pretreated by passing through
5mL of methanol followed by 5mLwater before loading, and
drawn through by gravity. Then, the solidphase cartridge was
washed with 10.0mLofwater to elute the phenolic compounds
entirely off. Finally, the cartridge was eluted with 1.0mL
methanol, in which fraction most of the saponins were
concentrated.A20 mL volume of the methanol eluent was
injected into the HPLC system HPLCMS analysis was performed
with Agilent LCMSD/ Trap System (Agilent Company) equipped
with an electrospray interface. TheMSspectra were acquired in
negative ion mode.N2 was used as both drying gas with a
flowrate of 10 L/min and as nebulizing gas with a pressure of 60
psi. The nebulizer temperature was set at 350 ?C and the
capillary voltage was set at 3500V. The mass spectra were
recorded in the range of 4001500m. A fragment amplification
of1.5V was selected for MS2 analysis.
HPLC grade methanol was used for SPE preparation.
Preparative Isolation of Six Major Saponins from Platycodi Radix
by High-speed Counter-current
Apparatus:
A TBE-300A HSCC, with three serially connected multilayer coil
separation column
Reagents and materials:
Acetonitrile, methanol, n-butanol, ethylacetate, hexane and
isopropanol (HPLC-grade) for the preparation of the crude
sample and for HSCCC separation Preparation of the two-phase
solvent system and sample solution.
A solvent system consisting of hexanen-butanolwater
(1:40:20, v/v) was used for the separation of platycoside E and
deapio-platycoside E. Hexanen-butanolwater (1:10:5, v/v) was
used as the solvent separation of platycodin D3, platycodin D
and their deapiose forms.
HSCCC separation.
19 l Herbal Tech Industry l September 2011
Review
In conventional HSCCC experiments, a multi-layer coiled column
is first entirely filled with one phase of the two phase solvent
system as a stationary phase, followed by elution with the other
phase. Here, the column was first filled with a mixture of the two
phases, thus reducing the amount of time for hydrodynamic
equilibrium to be established (Slacanin et al., 1989). The ratio of
two phases was also optimised at 70:30 (stationary phase
mobile phase, v/v) within the range 90:10 to 60:40 based on the
amount of ti me requi red to reach hydrodynami c
equilibriumowing to retention on the stationary phase. In the
present experiment, several solvent systems based on n-
butanol water with added hexane or ethyl acetate were tested,
and the results are summarized in Table 1. The table shows that
platycoside E and deapio-platycoside E, when analysed in the
reverse-mode solvent system composed of n-butanolwater at
volume ratios of 2:1 (v/v), have appropriate K values (0.55) for
separation. However, the K values of platyodin D and deapio-
platycodin D, in the same solvent system, were smaller than
expected. It was difficult to separate platyodin D and deapio-
platycodin D from the other compounds. Also, when n butanol
water (2:1, v/v used the retention of the stationary phase was
poor (<30%), and so that system was deemed unsuitable for
separation.
HPLC analysis:
of 70 C and a gain of 7, and the nebuliser gas (nitrogen) was
adjusted to 2.5
The HPLC conditions for the platycosides were as follows: eluent
A, water; eluent B, acetonitrile; gradient, 06 (1015% B), 650
min (1525% B), 5060 min (2547.5% B) and then equilibrated
with 10% B for 8 min at a flow of 1mL/min. The ELSD system was
set to a probe temperature bar.
Identification of HSCCC peak fractions.
Identification of the HSCCC peak fractions was carried out by ESI-
MS, 1H-NMR and 13C-NMR spectra with references.
Structural identification
The chemical structures of components present in each peak
fraction purified by HSCCC were identified from ESI-MS, ESI-
MS/MS, 1H-NMR and 13C-NMR data. The subsequent
structural identification of the peak fractions collected
from the HSCCC was performed by comparison with previous
1H-NMR and 13C-NMR data.
A New Bioactive Steroidal Saponin from Agave attenuate Herbal
plant.
Introduction:
The occurrence of steroidal saponins in Agave genus is well
documented
Some species have an ethnopharmacological background, in
particular A. sisalana which in the Bahama Islands, the central
bud is boiled with salt and the decoction given as a remedy for
jaundice; said to be effective within 24 hours, the aqueous
extract of A. attenuate was evaluated for activity against Bulinus
africanus, Daphnia pulex, Anopheles arabiensis and
Oreochromis mossambicus demonstrating molluscicidal,
piscicidal and larvicidal properties
plant:
Fresh leaves of Agave attenuata were obtained from the
Ornamental Plant Garden
Extraction and isolation:
The fresh leaves of the plant (3 kg) were extracted with 80%
aqueous EtOH (6 l) followed by concentration to 600 ml and
extraction with an equal volume of n-BuOH gave a crude
material (12.5 g). It was roughly chromatographed on Sephadex
LH-20 with MeOH to give crude steroidal glycoside (2.5 g).
Further purification by chromatography on a silica gel column
eluted with CHCl3:MeOH:H2O (70:30:10 v/v/v) to afford one TLC
homogeneous compound 1 (635 mg), Rf 0.43 which gave a dark
green color with orcinol and H2SO
General Purification of Saponins:
The filtrate was loaded onto a C-18 preparative column and
impurities were eluted with 600 ml l- 1 methanol in
water[John Michael Berger]. The saponin containing fraction
was eluted with methanol. The eluted fraction was diluted with
water and was further purified using HPLC to isolate DDMP-
saponins. The HPLC system was equipped with a diode-array
detector (UV6000) and a column oven
Identification:
The substance was collected and analyzed by 1H and 13C NMR .
1H NMR and 13C NMR spectra identical to the data reported for
a DDMP moiety. The amount of DDMP-saponin was also
determined2,3-dihydro-2,5-dihydroxy-6-methyl- 4H-pyran-4-
one (DDMP) moiety on C-22.
Applications;
Generally these saponins are used for the anti-inflammation
anti-allergy,
anti-tumour,anti-obesity
anti-hyperlipidemic effects.
saponins used as dietary supplements and nutriceuticals
production of vaccines,
used in anti-cancer drugs formulations
20 l Herbal Tech Industry l September 2011
Review
these are used for the preparation of cosmetics,mainly used in
the soap preparation and perfumes.
References:
David G. I. Kingston, ChairNeal Castagnoli, jr. James Tanko
Richard Gandour Paul Deck CHARACTERIZATION saponins.
Young Wan Ha and Yeong Shik Kim* Isolation of saponins from
Platycodi Radix by hsccc
Zohar Kerem, Hilla German-Shashoua and O ded Yarden, Journal
of the Science of Food and Agriculture, Microwave-assisted
extraction
Haijiang Zhang, Yiyu Cheng , Solid-phase extraction, Journal of
Pharmaceutical and Biomedical Analysis 40 (2006) 429432
Niramon Utama-ang, Penkwan Chompreeda, Vichai
Haruthaithanasan, Identification of Major Saponins from
(Gynostemma pentaphyllum)
Farah S. Hosseinian and Trust Beta, Patented Techniques for the
Extraction and Isolation of Secoisolariciresinol Diglucoside from
Flaxseed, University of Manitoba, Department of Food Science,
Winnipeg, Manitoba, R3T 2N2 Canada
D.A. RICKERT, M.A. MEYER, J. HU, AND P.A. MURPHY, Effect of
Extraction pH and Temperature on Isoflavone and Saponin
Partitioning. JFS C: Food Chemistry and Toxicology
Maggie P.K. Choia, Kelvin K.C. Chanb, Hei Wun Leungb, Carmen
W. Huiea, P ressurized liquid extraction of active ingredients
(ginsenosides) from medicinal plants. Journal of
Chromatography A, 983 (2003) 153162
Kalpana Mujoo, Valsala Haridas, Joseph J. Hoffmann,
Triterpenoid Saponins from Acacia victoriae (Bentham) Decrease
Tumor Cell Proliferation and Induce Apoptosis.
GEORGE V. ODELL aDd CBUEN-emN BSU, Isolation and
Purification of the Saponin. of Glottidium vesicarium,
BIOLOGICAL SCIENCES.
REBECCA M. CORBIT, JORGE F. S. FERREIRA, Simplified Extraction
of Ginsenosides from American Ginseng (Panax quinquefolius L.)
for High-Performance Liquid Chromatography-Ultraviolet
Analysis, J. Agric. Food Chem. 2005, 53, 9867-9873 9867
M. Sajjad Khan, Nitin Nema, M.D. Kharya, Salma Khanam,
Chromatographic estimation of maturity based phytochemical
p r o f i l i n g o f I p o m o e a m a u r i t i a n a ,
http://www.arjournals.org/ijop.html.
P.B.MALLIKHARJUNA, L.N.RAJANNA, Phytochemical Studies of
Strychnos potatorum
Shahla Najafi and S. S. Deokule, Pharmacognostic study of
Tylophora dalzellii Hook. Journal of Medicinal Plants Research
Vol. 4(5), pp. 403-406, 4 March, 2010.
P.Devil ,R.Meera, P.Muthumani, B.Kameswari, Phyto-Physico
chemical evaluation and Antioxidant activities of leaves of
Naphellium lappaceum, Journal of Pharmaceutical Sciences and
Research
Devendra N. Kage , Vijaykumar B. Malashetty, Y. N. Seetharam; P.
Suresh & Saraswati B. Patil. Effect of Ethanol Extract of Whole
Plant of Trichosanthes cucumerina var. cucumerina L. Int. J.
Morphol., 27(1):173-182, 2009.
John Michael Berger, ISOLATION, CHARACTERIZATION, AND
SYNTHESIS OF BIOACTIVE NATURAL PRODUCTS FROM
RAINFOREST FLORA, Copyright 2001, John M. Berger.