Steroidal saponins from the genus Allium

Steroidal saponins are widely distributed among monocots, including the Amaryllidaceae family to which the Allium genus is currently classified. Apart from sulfur compounds, these are important biologically active molecules that are considered to be responsible for the observed activity of Allium species, including antifungal, cytotoxic, enzyme-inhibitory, and other. In this paper, literature data concerning chemistry and biological activity of steroidal saponins from the Allium genus has been reviewed. Electronic supplementary material The online version of this article (doi:10.1007/s11101-014-9381-1) contains supplementary material, which is available to authorized users.


Introduction
The genus Allium (Amaryllidaceae) is one of the largest monocot genera comprising more than 800 species (Li et al. 2010;APG 2009). It is widely distributed in nature and has adapted to diverse habitats across the Holarctic region, with the exception of A. dregeanum, which is native to South Africa (Li et al. 2010). Some Allium species, such as garlic, onion and leek, are widely cultivated as vegetable products, spices and for medical purposes. The most characteristic constituents in Allium plants are sulfur compounds, which are the most important substances both in terms of chemotaxonomic value and biological activity (Rose et al. 2005). However, various researchers tend to attribute the potential pharmacological benefits of Allium plants to constituents other than sulfur compounds, such as steroidal saponins. Also, polyphenolic compounds, especially flavonoids, as well as fructans, N-cynnamic amides, and antioxidative enzymes are considered to be equally important (Matsuura 2001;Lanzotti 2005;Štajner et al. 2006;Amagase 2006;Lanzotti 2012).
There are numerous reports referring to pharmacological activities of steroidal saponins. Some of them showed promising antifungal, cytotoxic, anti-inflammatory, antithrombotic, and hypocholesterolemic effects (Sparg et al. 2004;Lanzotti 2005;Güçlü-Ü stündag and Mazza 2007). Most importantly, these compounds are used as substrates in the production of steroid hormones and drugs.
Steroidal sapogenins and saponins have been identified so far in over 40 different Allium species. The earliest reports on Allium saponins date back to the 1970s and dealt with identification of diosgenin in A. albidum (Kereselidze et al. 1970) and alliogenin in the bulbs of A. giganteum (Khristulas et al. 1970). Further studies performed worldwide in the following years led to the isolation of a large number of new compounds. The first chemical survey of saponins from the genus Allium was published by Kravets in 1990, and this was followed by an update by Lanzotti in 2005(Kravets et al. 1990Lanzotti 2005). Since then, a large number of new compounds has been discovered, and there were also some that have not been included in the previous surveys.
A recent review by Lanzotti et al. (2014) compiled data on various compounds identified in Allium species with a reported cytotoxic and antimicrobial activity.
The present review is predominantly focused on the chemistry of Allium steroidal saponins and their biological activities.

Chemistry of Allium saponins
Steroidal saponins from the genus Allium can be divided into three groups on the basis of the sapogenin structure: spirostanols, furostanols, and open-chain saponins. The latter group is often referred to in the literature as ''cholestane saponins'' (Challinor and De Voss 2013). Allium saponins are mostly mono-or bidesmosides, however a tridesmodic cholestane glycoside has been reported in the bulbs of A. macleanii (Inoue et al. 1995). The sugar residue in Allium saponins consists of linear or branched chains made up most often of glucose (Glc), rhamnose (Rha), galactose (Gal), xylose (Xyl), and arabinose (Ara) units.

Spirostane-type saponins
A vast structural diversity of Allium spirostanols is associated with the differences in the structure of aglycones, especially their oxygenation patterns and stereochemistry (Table 1). In spirostane-type sapogenins, the steroid A/B ring junction is found mostly in a trans (5a), or more rarely in a cis (5b) fusion (e.g. anzurogenin A [48] and C [58]). D 5(6) unsaturation is considered to be a quite common feature (diosgenin [4], ruscogenin [17], yuccagenin [19], lilagenin [20], cepagenin [44], karatavigenin C [45]). However, a double bond located at C25(27) was reported in the aglycones of saponins present in A. macrostemon and in one of the sapogenins identified in A. ursinum bulbs (He et al. 2002;Sobolewska et al. 2006;Cheng et al. 2013). The C-25 methyl group is found with either S or R absolute configuration. In many cases the isolated sapogenins appear to be a mixture of diastereomers R and S.
Until now, over 130 spirostanol glycosides have been identified in various Allium species. It should be mentioned however that some of these compounds were obtained as a result of enzymatic hydrolysis of furostanol saponin fraction by b-glucosidase (Ikeda et al. 2000).

Cholestane-type (open-chain) saponins
A review of available literature data shows that as much as 18 cholestane-type compounds have been identified in ten different Allium species.
Allium open-chain aglycones possess D 5(6) unsaturation with an exception of schubertoside A [329]-D 4(5) , and one of the glycosides found in A. albopilosum with a saturated aglycone (Kawashima et al. 1991b;Mimaki et al. 1993). Glycosides based on alliosterol-(22S)-cholest-5(6)-ene-1b,3b,16b,22-tetrol ( Fig. 1 [196]), or related sapogenins showing the same oxygenation pattern at C-1, C-3, C-16 and C-22 are most common (Challinor and De Voss 2013). Sugar units are attached at one, two or, more seldom, at three separate positions (in A. macleanii) (Inoue et al. 1995). Most of these compounds are glycosylated at C-16, whereas in contrast to spirostanol and furostanol saponins, the attachment of sugar chain at position C-3 is almost unique (tuberoside U [353]) (Sang et al. 2003). Table 2 lists steroidal saponins/sapogenins identified in Allium species. Plant names are cited exactly as they were referred to in the original report. It is almost certain that some of them are synonyms but as the authors of the present review are not specialists in plant taxonomy no amendments have been made.
Biological and pharmacological properties of Allium saponins Saponins are considered responsible for numerous pharmacological properties of many plants, and they are recognized as active constituents of Allium species as well. It should be mentioned, however, that Allium plants are not rich sources of these compounds. Results from quantitative studies indicate that saponin content is usually very low, for example A. nigrum total saponin content in different parts of the plant was determined as: 19.38 mg/g dw in the roots, 15.65 mg/ g dw-bulbs, and 10.48 mg/g dw-leaves (Mostafa  (Akhov et al. 1999). It should be emphasized however that the results from many pharmacological in vitro and in vivo studies revealed several interesting activities of Allium saponins, for example antifungal, cytotoxic, antispasmodic, hypocholesterolemic, and other.
Other compounds isolated from this latter species were considered to be inactive. The authors concluded that the presence of an additional OH group at C-6 in gitogenin skeleton is detrimental to activity, while cholestane glycosides showed no effect. It is probable that the presence of a carbonyl at C-6 in a laxogenin glycoside [158] isolated by Timité et al. (2013) from the whole plant of A. schoenoprasum could be responsible for the loss of activity against two cancer cell lines HCT 116 and HT-29, an effect similar to that seen by Mimaki et al. when an additional OH group was introduced at C-6 of gitogenin (Timité et al. 2013;Mimaki et al. 1999c). In accordance with the studies of Mimaki et al. (1999a, b, c) were also the results obtained for cholestane glycosides, nigrosides C [303] and D [304] isolated from the bulbs of A. nigrum, which showed no effect ( (Fattorusso et al. 2000).
Results of cytotoxicity assays of several spirostanol sapogenins indicated their weak activity or lack of it.

Antifungal activity
Numerous steroidal saponins isolated from different plant sources have been reported to have antifungal/ antiyeast activity, particularly against agricultural pathogens. Antifungal saponins require particular attention as there is a constant need for new agents that would be effective against opportunistic fungal infections and could provide an alternative to chemical fungicides used in the fight against plant pathogens. Unfortunately, only a few studies have been performed so far on Allium steroidal glycosides.
Antifungal activity of Allium saponins was modulated by both the sapogenin type and the number and structure of the sugar residue. Generally saponins with spirostanol skeleton exhibited higher antifungal activity than furostanols. Yu et al. (2013) observed several biochemical changes which could be involved in the possible mechanism of antimicrobial activity of saponins, such as reduced glucose utilization rate, decrease of catalase activity and protein content in microorganisms.
Also, saponins isolated from A. giganteum bulbs inhibited cAMP phosphodiesterase (Mimaki et al. 1994) and in concordance with previously cited results, an acetyl derivative-3-O-acetyl-(24S,25S)-5a-spirostane-2a,3b,5a,6b,24-pentaol 2-O-b-D-Glc [193] exhibited inhibitory activity almost equal to that of papaverine (IC 50 4.1 9 10 -5 and 3.0 9 10 -5 M respectively). In the same study, furostanol saponins were revealed to be much more potent than the corresponding spirostanol glycosides. The results were in contrast to the previous studies of these authors which showed that furostanol glycosides were less active, exhibiting only weak inhibitory activity or none. The authors concluded that the anti-enzyme activity could be dependent on the number of hydroxyls in the A and B rings as in the present study the tested furostanol saponins contained several OH groups.
Saponins isolated from the fruits of A. karataviense and A. cepa as well as the products of chemical modifications of karatavioside A, were studied on a highly purified porcine kidney Na ? /K ? ATP-ase, in the concentration range from 1 9 10 -4 to 1 9 10 -7 M (Mirsalikhova et al. 1993). All the compounds affected the enzyme activity being capable of its inhibition, and/or activation. As was showed, the presence of a hydroxyl group in the F-ring at C-24 led to a decrease in the percentage inhibition of Na ? / K ? ATP-ase. At the concentration of 1 9 10 -4 M the inhibitory effect of karatavioside A Drugs acting via inhibition of the activity of this transport enzyme may be of potential use in the treatment of many diseases of the cardiovascular system, the kidneys, the immune system, which are connected with disturbances in the active transport of ions.

Cardioprotective activity
Three saponins from A. chinense and their aglycones were tested for the protective effects against oxidative stress-induced cardiac damage (Ren et al. 2010). Their activities were evaluated on H 2 O 2 -injured cardiac H9C2 cells. The cytotoxicity was measured using MTT assay while the oxidative damage by determination of MDA and NO contents. All tested compounds protected cultured H9C2 cells from death in the concentration range of 5-20 lL. It was shown that glycosides exhibited less protective efficacy than sapogenins. Among these, laxogenin [6] and tigogenin [1] displayed stronger effects than furostane-type aglycones. The authors concluded that the presence of F ring in spirostanols may enhance their protective activity whereas oxidation in the B ring might be detrimental as laxogenin was less active than tigogenin.
In animal studies, alloside B [334], isolated from fruits of A. suvorovii and A. stipitatum, exhibited a statistically reliable hypotriglyceridemic activity in experimental hyperlipidemia caused by 1-day starvation, Triton WR-1339 and vitamin D 2 -cholesterol, when compared with lipanthyl (Aizikov et al. 1995).
The hypocholesterolemic activity of saponins was reported in many animal studies.
The cholesterol-lowering effect of garlic is probably partially due to the steroid saponin presence. In a rat model of experimental hyperlipidemia induced by feeding a 0.5 % cholesterol-enriched diet saponin-rich fraction from raw garlic administrated at 10 mg/kg/ day led to a decrease of plasma total and LDL cholesterol concentration level without affecting HDL cholesterol levels after 16 weeks (Matsuura 2001). It was claimed that the reduction of concentration of plasma cholesterol concentration is the result of inhibition of cholesterol absorption by saponins in the intestine or a direct effect on cholesterol metabolism.  [31], from A. elburzense, were subjected to biological assays on the guinea-pig isolated ileum in order to evaluate their possible antispasmodic activity (Barile et al. 2005). Apart from the agapanthagenin glycoside, all the tested compounds were able to reduce induced contractions, as measured by the reduction of histamine release, in a concentration-dependent manner.
The authors concluded that the positive effect is associated with the presence of a hydroxyl group at position C-5 and of a glucose unit at position C-26. On the other hand, hydroxylation at C-6 and glucose attachment at C-3 seem to be structural features responsible for the loss of activity. Furostane-type saponins that were isolated from A. cepa var. tropea, namely tropeosides A1/A2 [213,214] and B1/B2 [215,216] were able to dose-dependently relieve acetylocholine-and histamine-induced contractions (50 % inhibition of contractions was seen at the concentration of 10 -5 M) (Corea et al. 2005). Interestingly, other furostanols identified in this plant, such as ascalonicosides A1/A2 [217,218], were inactive.

Other activities
Macrostemonoside A [65] inhibited ADP-induced rabbit ertythrocyte aggregation with IC 50 = 0.065 mM (Peng et al. 1992). An in vitro inhibitory activity of ADP-induced platelet aggregation was also reported for macrostemonosides E [272], F [273] and G [274] (IC 50 = 0.417; 0.020; 0.871 mM, respectively) (Peng et al. 1993(Peng et al. , 1995  , isolated from the bulbs of A. ampeloprasum var. porrum, demonstrated in vivo antiinflammatory and gastroprotective effects in a carrageenan-induced oedema assay and by measuring acute gastric lesions induced by acidified ethanol (Adão et al. 2011a). Saponin administrated orally (100 mg/kg) inhibited oedema formation similar to dexamethasone (25 mg/kg). Cytoprotective activity of b-chlorogenin glycoside resulted in a significant reduction in gastric hyperemia and also in the severity and number of lesions.
Macrostemonoside A [65] increased the synthesis and release of visfatin in 3T3-L1 adipocytes and elevated mRNA levels in this cytokine in a dose-and time-dependent mode (Zhou et al. 2007). In a study on C57BL/6 mice fed on a high-fat diet, this saponin when administered at the dose of 4 mg/kg/day for 30 days moderately inhibited glucose level, glycogen hepatic content, total plasma cholesterol level and abdominal adipose tissue (Xie et al. 2008).
In  (Chen and Snyder 1989). The authors observed that the molluscicidal activity of isolated compounds increased with an increasing number of monosaccharides in a sugar moiety.
Aginoside [93] was found to be toxic to leek-moth larvae Acrolepiopsis assectella (Harmatha et al. 1987). The compound caused mortality and ecdysial failures 56 ± 10 and 19 % respectively in larvae of A. assectella reared on semisynthetic diet at a concentration of 0.9 mg/g of diet.

Conclusions
In this paper steroidal saponins reported in various Allium species from early 1970 to March 2014 are reviewed, including their skeletal structures and sugar chains.
Until now, as many as 290 saponins have been identified, including a certain number of methoxyl derivatives originating from furostanol compounds, that should be considered as artifacts resulting from the use of methanol in the extraction/isolation procedures.
Allium genus is characterized by a great diversity of structures. Apart from spirostane-and furostane-type compounds, a rare group of open-chain saponins has been identified in several species. Allium genus is also a source of unique steroidal sapogenins, such as 25(S)-5b-spirostane-1b,3b-diol [8] and 2,3-seco-porrigenin [64]. Despite a relatively low content of steroidal glycosides in Allium species, they are considered to contribute, in addition to sulfur compounds, to the overall biological activity of these plants. Undoubtedly, stability of saponins is their advantage as compared to fairly unstable sulfur compounds, thus, they in fact may be predominant active constituents of Allium products. Bearing this aspect in mind it seems highly feasible to develop antifungal Allium preparations against animal and plant pathogens. Also, reports on high in vitro cytotoxic activity of steroidal saponins from Allium species makes them potential candidates for further development as anti-cancer agents.
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