Naturally Occurring Saponins: Chemistry and Biology

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Naturally occurring saponins: Chemistry and biology

J. S. Negi 1*, P. S. Negi 2, G. J. Pant 2, M. S. M. Rawat 2, S. K. Negi 3

1Herbal Research and Development Institute, Mandal, Gopeshwar (Chamoli) - 246 401, Uttarakhand, India. 2Department of Chemistry, HNB Garhwal University, Srinagar (Garhwal)- 246 174, Uttarakhand, India. 3Department of Botany, HNB Garhwal University, Srinagar (Garhwal) - 246 174, Uttarakhand, India.

Accepted 2 April, 2013

Naturally occurring saponins are glycosides of steroids, alkaloids and triterpenoids. They are widely distributed in nature and reported to be present in 500 genera of plants. A wide variety of plants belonging to family Liliaceae, Dioscoreaceae, Solanaceae, Sapindaceae and Agavaceae are the major source of saponins. They are amorphous substances having high molecular weight and are soluble in water and alcohol to produce foam but organic solvents inhibit their foaming property. Plants saponins are generally extracted into butanol through liquid-liquid partition and separated through column chromatography using silica gel as adsorbent and chloroform: methanol as mobile phase. HPLC, GC, Sephadex LH-20 Chromatography, DCCC, preparative paper chromatography and TLC were also used for the separation and isolation of saponins. The structures of saponins were determined by several spectroscopic techniques, viz., UV, IR, 1H NMR, 13 C NMR and Mass spectroscopy. Saponins possess several biological activities such as antioxidant, immunostimulant, antihepatotoxic, antibacterial, anticarcinogenic, antidiarrheal, antiulcerogenic, antioxytoxic, antihypoglycemic, anticytotoxic and antimolluscicidal. Saponins are biologically synthesized by C5 isoprene units through cytosolic mevalonate pathway. 2,3-Oxidosqualene gives βββ-amyrin or triterpenoid skeletons on cyclization through isoprenoid pathway. The triterpenoid backbone then undergoes various modifications to form saponins.

Key words: Saponin, triterpenoid, isoprene, aglycone

INTRODUCTION

Saponins are recognized by their ability to produce a aubergine, tomato seed, alliums, asparagus, yam, soapy lather when shaken with water. They are widely fenugreek, yucca and ginseng. In Alkaloids saponins, distributed in nature and reported to be present in 500 aglycone carry N atom as a bridge between two rings e.g. genera of plants. All saponins are polar in nature and are solanidine (Ripperger and Schreiber, 1981) or ring F freely soluble in water but insoluble in non polar solvents. carring –NH or –NCH 3 e.g. hapepunine. Triterpenoid Saponins on hydrolysis yield an aglycone known as saponins are triterpene based glycosidic compounds, "sapogenin" and glycone known as sugar. A wide variety most of the triterpenoid compounds in adaptogenic plants of plants belonging to family Liliaceae, Dioscoreaceae, are found as saponin glycosides which refers to the Solanaceae, Sapindaceae and Agavaceae are the major attachment of various sugar molecules to the triterpene source of saponins, however, a few neutral saponins unit. The sapogenins of these glycosides are tetracyclic have also been isolated and characterized from animal or pentacyclic triterpenoids which on selenium source. Steroidal saponins are widely distributed in dehydrogenation gives naphthalene and phenanthrene nature and exhibit various biological activities. The hydrocarbons, mainly sapotalene (1,2,7- aglycone of steroidal saponins is usually a spirostanol or teimethoxynaphthalene). Triterpenoids generally occur in its modification. They are found in oats, peppers, family Leguminosae, Hippocastanaceae, Ranunculaceae, Symplocaceae, Euphorbiaceae, Verbenaceae and Araliaceae. Most of the triterpenoid sapogenins, with few exceptions, belong to β-amyrin group and are usually *Corresponding authour. E-mail: [email protected]. Tel: simple alcohol and acids. Occasionally sapogenin is +919997239903. Fax 01372254273 . encountered having aldehydic and lactone functional 002 J. Poisonous. Med. Plants. Res.

29 29

20 OH 20 22 22

11 11 16 16 COOH 15 COOH HO 15 OH 2 2 H 5 H 27 5 27 6 6 HO HO Acacic acid CH OH Arjunolic acid 23 2 29 29

20 20 22 22 11 11 16 COOH 16 COOH HO 15 15 HO 2 2 5 H 27 5 H 27 6 6 HO HO HOH C HOOC Barringtogeneol 2 Bassic acid 29 29

20 20 22 22 11 11 16 15 COOH 16 COOH 15 2 2 5 H 27 5 H COOH 6 6 HO HO 23 Oleanolic acid 23 Cincholic acid 29 COOH 20 20 22 22 11 11 16 COOH 15 16 28 15 2 5 H 27 2 6 5 H 27 HO 6 HO OHC Gypsogenin CH OH Hederagenin 2

Figure 1. Structure of some β-amyrin group sapogenins.

groups. Some examples of β-amyrin group triterpenoid of physical, chemical and biological properties. Saponins aglycones are cincholic acid, oleanolic acid, gypsogenin, are usually amorphous substances having high molecular hederagenin, gummosogenin, erythrodiol and cochalic weight. They are soluble in water and produce foam but acid (Figure 1). The tetracyclic triterpenoid genins have organic solvent like chloroform, acetone and ether inhibit also been reported, there are five examples namely their foaming property. Solubility of saponins is also panaxadiol, bryogenin, bacogenins, gratiogenin and affected by the properties of the solvent (as affected by cucurbitacins (Basu and Rastogi, 1967; Figure 2). temperature, composition, and pH), while water and alcohols (methanol, ethanol) are the most common extraction solvents for saponins. Due to the presence of a PROPERTIES lipid-soluble aglycone and water soluble sugar chain in their structure (amphiphilic nature), saponins are surface The structural complexity of saponins results in a number active compounds. In aqueous solutions Negi et al 003

OH OH O O

HO HO

H OH

Panaxadiol Panaxatriol

O OH O O OH O H OH

HO HO

Gratiogenin Hydroxygratiogenin OH

O O O

CH2OH

HO HO Bryogenin Bacogenin A 1 O O HO HO

O OAc O OAc OH OH CH OH H C CO 2 CO 3

HO HO

Cucurbitacin A Cucurbitacin B

Figure 2. Structure of some tetracyclic triterpenoid sapogenins.

surfactants form micelles above a critical concentration phytoanticipins or phytoprotectants. called critical micelle concentration (cmc). The micelle forming property is affected by temperature, salt concentration and pH of the aqueous phase (Mitra and EXTRACTION AND CHARACTERIZATION Dangan, 1997). Saponins possess a variety of biological activities such as antioxidant, immunostimulant, Traditionally, saponins are extracted into water/ethanol antihepatotoxic, anticarcinogenic, antidiarrheal, mixtures, after which the ethanol is removed by antiulcerogenic, antioxytoxic, hypocholesterolemic, anti- distillation and the saponins extracted from the water coagulant, hepatoprotective, hypoglycemic, phase into 1-butanol through liquid-liquid partition. neuroprotective, antiinflammatory, inhibition of dental Supercritical CO 2 extraction in combination with modifiers caries and platelet aggregation (Guclu-ustundag and such as methanol, ethanol or aqueous methanol has Mazza, 2007 ; Rao and Gurfinkel, 2000) and also useful proven successful (Guclu-ustundag and Mazza, 2007). in diabetic retinopathy and reproduction. Many saponins High performance liquid chromatography is the most are known to be antimicrobial to inhibit mould and to important method of choice for the separation of protect plants from insects. They may be considered as saponins. Both normal phase and reverse phase columns defense system and have been included in a large group have been used. However, RP-HPLC with C 18 columns of protective molecules found in plants named and gradient elution seems to be the most preferred 004 J. Poisonous. Med. Plants. Res.

method. There are several strategies available for the electronegativity of oxygen in tigogenin. The presence of isolation of saponins. They are separated by column double bond at C-5 has remarkable effect on the chromatography on silica gel with mobile phase chemical shift of C-5 and C-6 in diosgenin and tigogenin. composed of CHCl 3-MeOH with increasing polarity. The signal intensity of the carbonyl carbon is always very Sephadex LH-20 has successfully been used for the low and it is recorded in the range of 200-220 ppm. This separation of steroidal saponins. The carbonyl group in can be explained by comparing hecogenin with tigogenin. steroidal saponins absorbs UV light in the range of 280- The C-12 signal is recorded dowfield at δ 213 ppm in 300 nm and ethylenic double bond appears at 195-198 hecogenin due to the influence of the doubly bonded nm. Due to lack of strong chromophore in some oxygen. The C-11 and C-13 signals are also recorded sapogenins they do not absorb UV light. IR spectra of downfield by 16.4 and 14.4 ppm, respectively, in saponins and sapogenins provided valuable information hecogenin. Acetylated C-3 OH group causes a downfield about the various functional groups and also shift of about 2.2 ppm for C-3 ( α-effect) and upfield shift stereochemistry of molecules to some extent. Spirostane of 4 ppm for C-2 and C-4 signals ( β-effect). Electron derivatives showed absorptions between 1350-875 cm -1. ionization mass spectroscopy (EI-MS) has been shown to The relative intensities of absorptions around 920-950 be a very useful method for identification and structure cm -1 and 900-884 cm -1 permit a choice of 25 R or 25 S elucidation of saponins (Kasai et al., 1977). Saponins compounds. In 25 S the former is more intense than the containing more than four sugars do not give molecular latter whereas it is vice versa in 25 R configuration. In ions, even when derivatized. However, MS has limited Hopane triterpenoids, IR spectra is useful for the application in the field of underivatized oligosaccharides determination of the substitution patterns of hopane 6 α-, because it required volatilization and ionization of the 15 α-, 22 α-, 7 β-, 22, 24-triol. Peak at 1700-1702 cm -1 sample. Ionization and volatilization are coupled in one indicates the presence of C-12 carbonyl group whereas process in field desorption mass spectrometry (FD-MS). at 1660-1680 cm 1 suggest the conjugated carbonyl FD-MS of underivatized steroidal and triterpenoidal group. 1H NMR spectrum of saponin peracetate or saponins have been reported. The spectra showed the permethylate is helpful in determination of mode of sugar intense ions formed by attachment of alkali cation to the linkages. The signals of anomeric proton in the spectrum neutral molecule. A new technique of plasma desorption are assignable to that of a D-glucopyranose (or L- mass spectrometry (PD-MS) has also been used for arabinopyranose) unit, the sugar may be regarded as molecular weight determination of underivatized steroidal having β-configuration. The J value suggests trans-diaxial saponins (Hostettmann et al., 1978). The positions of relationship of the proton at C1 and C2 of the pyranose various substituents in the sapogenin are determined by residues. When anomeric proton signal appears with J= estimating the shifts in the masses of the characteristic 1-3 Hz suggesting the equatorial-equatorial or axial- fragments with relation of the peaks of the standard. equatorial orientation of C1 and C2 proton. However, the anomeric proton signal of α-D-glucoside, α-D-manoside, α-L-rhamnoside and β-L-arabinoside appears generally at BIOSYNTHESIS lower field ( δ 5.0-6.0) than those of corresponding β and α anomers respectively ( δ 4.5-5.0). This difference is also The sequence of enzyme catalyzes reactions by which helpful in the differentiation of the anomeric structure complex molecules in living cells are formed from (Mahato et al., 1981). In both 25 R and 25 S series, the nutrients with relatively simple structures are known as 27-methyl protons resonated upfield than the 21-methyl biosynthesis. Triterpenes belong to a large group of protons. Moreover, 27-methyl signal in 25 R appears compounds arranged in four or five ring configurations of upfield than in 25 S, hence these two isomers can be 30 carbons attached with several oxygens. These are distinguished (Kutne 1963). formed by assembly of C5 isoprene units through the 13 C NMR spectroscopy is very useful tool for the cytosolic mevalonate pathway to make C30 compounds. structure elucidation of saponins. The chemical shift They are synthesized via the isoprenoid pathway by values for sugar moieties and for few steroidal cyclization of 2,3-oxidosqualene to give primarily sapogenins have been reported. The points of linkages oleanane ( β-amyrin) or dammarane triterpenoid are confirmed by the glycosylation shift rules (Kasai et al., skeletons. The triterpenoid backbone then undergoes 1979), according to which α- and β- carbon of the various modifications (oxidation, substitution and aglycones as well as sugar moieties undergo glycosylation), mediated by glycosyltransferases and characteristic shifts on glycosylation. The α-C is shifted 6- other enzymes. The cyclization of 2,3-oxidosqualene to 9 ppm downfield whereas the β-C signals move slightly lanosterol and cycloartebik skeleton is initiated by upfield. If 27-CH 3 is axial a small α effect is observed in participation of a neighbouring π-bond via protosteryl C- C-25 signal. This signal appears 3-4 ppm upfield in 25 S 20 cation. This cation then undergoes a series of 1,2- neoyonogenins than that of 25 R yonogenin. The methyl and hydride shifts with proton elimination to yield difference of deoxytigogenin and tigogenin is reflected in either lanosterol or cycloartenol skeleton. The cyclisation the C-3 signal appearing 45 ppm downfield due to the of 2,3-oxidosqualene to sterols and triterpenoids Negi et al 005

22 24 27 22 26 22 24 27 CH 2O 25 25 23 25 1 27 16 26 26 CH 2OH 6 16

H H HO O O 27-Hydroxycholesterol Cholesterol 26-Hydroxycholesterol

26 27 O O 22 22 25 27 25 26 16 O 16 O

HO HO O Diosgenin Yemogenin Cholestenone

O O

HO HO HO H H H Neotigogenin Cholestanol Tigogenin

Figure 3. Biosynthesis of C 27 sapogenins.

represents a bridge point between primary and as antioxidant, immunostimulant, antihepatotoxic, secondary metabolism (Henry et al., 1992) antibacterial, anticarcinogenic, antidiarrheal, In lanosterol biosynthesis two 1,2-hydride shifts take antiulcerogenic, antioxytoxic, antihypoglycemic, place from protosteryl cation from C-17 and then two 1,2- anticytotoxic and antimolluscicidal. Saponins are methyl shifts occur from 14 β to 13 β and from 8 α to 14 α biologically synthesized by C5 isoprene units through accompanied by elimination of C-9β proton. The cytosolic mevalonate pathway. They may be considered mechanism of cyclization of oxidosqualene into as defense system and have been included in a large cycloartenol is the same as lanosterol except the final 9 β, group of protective molecules named phytoanticipins or 19-cyclopropane ring closure instead of C-9 hydrogen phytoprotectants. migration. The steroidal sapogenins are spiroketals having same configuration at C-22 but stereoisomerism at C-25 as in cholesterol. Hydroxycholesterol was shown REFERENCES to be the first intermediate which was converted to diosgenin (Figure 3). And diosgenin on reduction is Basu N, Rastogi RP (1967). Triterpenoid saponins and converted to tigogenin (Tschesche et al., 1968; sapogenins. Phytochemistry , 6: 1249-1270 . Tschesche et al., 1970) but not to yamogenin, however, Guclu-ustundag O, Mazza G (2007). Saponins: yamogenin obtained from (25S)-hydroxycholesterol. Properties, Applications and Processing . Food. Sci. Yamogenin converted into neotigogenin on reduction and Nutr., 47 : 231-258 . (25S)-hydroxycholesterol was shown to be the key Henry M, Rahier A, Taton M (1992). Effect of gypsogenin intermediate in the formation of neotigogenin (Ronchetti 3,O-glucuronide pretreatment of Gypsophila paniculata et al., 1975). and Saponaria officinalis cell suspension cultures on the activities of microsomal 2,3-oxidosqualene cycloartenol and amyrin cyclases. Phytochemistry , 31: CONCLUSION 3855 -3859. Hostettmann K, Hostettmann Kaldas M, Nakanishi K Saponins possesses a variety of biological activities such (1978). Molluscicidal saponins from Cornus florida L. 006 J. Poisonous. Med. Plants. Res.

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