Initial Studies on Astragalus Species
The notable improvements observed in the blood profile of a leukemia patient who utilized an extract derived from a folkloric medicinal plant initiated our research on Turkish Astragalus species. In the early 1990 s, the first plant material, which consisted of the roots of a plant used for this purpose, was provided to us by the Gülhane Military Medical Academy (GMMA) for examination. The origin of these roots was unknown, and the user referred to it as Gune root (Turkish name: Gune Kökü).
At the end of the study conducted on three pieces of roots, three compounds were isolated as the major constituents of the methanolic extract. The structure elucidation studies revealed that these compounds were glycosides with a novel skeleton containing 24S-cycloartane-1α,3β,7β,24,25-pentol aglycone. Despite the absence of the isolated compounds in chromatographic studies, the morphological structure of the roots, the high polysaccharide content, and the plant samples sent by the patient from Şanlıurfa, a city in Southeastern Turkey, along with the structures of the compounds identified as cycloartane-type glycosides, suggested that the roots belong to an Astragalus species. The novel aglycone structures of these three glycosides, which were previously unknown in the scientific world, prompted us to conduct further research on the genus Astragalus.
Our phytochemical studies on the genus of Astragalus focused on 14 species belong to 8 sections, viz, Macrophyllium Bunge. (A. oleifolius DC.), Christiana Bunge. (A. melanophrurius Boiss.), Rhacophorus Bunge. (A. microcephalus, A. cephalotes Banks & Sol, A. zahlbruckneri Hand.-Mazz, A. prusianus Boiss.), Pterophorus Bunge. (A. brachypterus Fischer, A. trojanus Stev, A. baibutensis Bunge.), Dissitiflori DC. (A. elongatus Willd, A. campylosema (Syn. A. pendulus DC.), Vulneraria Bunge. (A. vulneraria DC.), Stereocalyx Bornm. (A. stereocalyx Bornm.), and Erophaca Boiss. (A. lusitanicus Lam.).
Astragalus oleifolius DC. (Sect. Macrophyllium Bunge.)
The three glycosides obtained from the drug sent by GATA were found only in the methanol extract of A. oleifolius collected from Ahlatlıbel, Ankara, among the many Astragalus species collected from various regions of our country. Subsequently, two more glycosides were isolated and named macrophyllosaponins A – E (25 – 29) ([Fig. 5]) [36], [37]. Studies conducted by the NCI (National Cancer Institute–Maryland, USA) revealed that these compounds did not exhibit any significant cytotoxic activity against the 60 human cancer cell lines.
Fig. 5 Structures of compounds 25 – 44.
In continuation of our studies on this species, two new cycloartane-type glycosides oleifoliosides A (30) and B (31), along with three known compounds cyclocanthoside E (32), astragaloside II (33) and IV (34), were obtained [29], [38] from the lower stem parts of A. oleifolius, which was collected from Şırnak: Uludere-Habur junction toward Hakkari ([Fig. 5]) [39]. The potential cytotoxicity of the isolated compounds on primary mammalian (L6) cells was evaluated along with their in vitro trypanocidal, leishmanicidal, and antiplasmodial activities. All the compounds, with the exception of astragaloside IV, exhibited a significant growth inhibitory activity against Leishmania donovani with IC50 values in the range of 13.2 to 21.3 µg/ml. Weak activity against Trypanosoma brucei rhodesiense
was observed with the known compounds astragaloside II (4, IC50 66.6 µg/ml) and cyclocanthoside E (3, IC50 85.2 µg/ml), whereas all compounds were inactive against Trypanosoma cruzi and Plasmodium falciparum. None of the compounds showed toxicity to mammalian cells. In this study, the leishmanicidal and trypanocidal activity of cycloartane-type triterpene glycosides were reported for the first time.
Astragalus melanophrurius Boiss. (Sect. Christiana Bunge.)
The studies conducted on A. melanophrurius, an endemic species collected from Ankara, Ahlatlıbel, resulted in the isolation of eight known saponins: astragalosides I (35) [29], II (33) [29], IV (34) [29], and VI (36) [40]; astrasieversianins II (37) [41] and X (38) [41]; and cyclocanthosides E (32) [38] and G (39) [38] ([Fig. 5]) [42]. Notably, the majority of these compounds are cycloastragenol glycosides, many of which have been reported previously from A. membranaceus, a plant used in Far Eastern traditional medicine [29], [40]. In subsequent in vitro
bioassays, these isolates were evaluated for cytotoxicity, estrogenic-antiestrogenic properties, antimalarial activity, antimicrobial activity, and immunomodulatory effects. The screening studies revealed that all compounds showed significant immunomodulatory activity, in addition to moderate antibacterial activity. Furthermore, in the lymphocyte stimulation test, all compounds were found to stimulate proliferation in human lymphocytes at a concentration of 0.01 to 10 µg/ml. These findings were confirmed in a similar study conducted by another research group using cycloartane and oleanane-type saponins obtained from the Astragalus species [43].
Astragalus microcephalus Willd. (Sect. Rhacophorus Bunge.)
A. microcephalus is one of the species used for the production of tragacanth in Turkey. This species was collected from Mucur-Avanos, Nevşehir, Central Anatolia. Cycloastragenol (4) [29], cyclocantoside E (32) [38], astragaloside IV (34) [29], and two new compounds, cyclocephaloside I (40) and II (41), were isolated from the roots of A. microcephalus
[44], [45]. Notably, cyclocephaloside I (40) ([Fig. 5]) was a novel cycloartane-type glycoside with a structure of 20,25-epoxy,3β-(β-D-xylopranosyl)oxy-6α-(β-D-glucopranosyl)oxy-cycloartane-16β,24α-diol. It was the first structure bearing an epoxide group between the 20th and 25th carbons in the side chain [44].
Astragalus brachypterus Fischer (Sect. Pterophorus Bunge.)
A. brachypterus was collected from Mucur-Avanos, Nevşehir, Central Anatolia. In addition to astragalosides I (35) [29], II (33) [29], IV (34) [29], and cyclocantoside E (32) [38], three new compounds named brachyosides A (42), B (43), and C (44) were isolated from this species ([Fig. 5]) [45].
Astragalus trojanus Stev. (Sect. Pterophorus Bunge.)
In another study, both the roots and aerial parts of A. trojanus, an endemic species collected from Hacıbozlar Village, Burhaniye-Balıkesir, West Anatolia, were studied [46], [47], [48]. Six novel cycloartane type glycosides (45 – 50), together with a new oleanane glycoside (astrojanoside A) (51) and tryptophan derivative (52) [(Achillamide)=N-(3-hydroxy-3-methyl-glutaroyl)-tryptophan], were obtained from the roots of the plant. Trojanoside A (45) and B (46) were found to contain (20R,24S)-epoxy-3β,6α,16β,25 tetrahydroxycycloartane as the aglycone, whereas trojanosides C (47), D (48), E (49), and F (50) had 3β,6α,16β, (24S),25-pentaahydroxycycloartane as the aglycone [47]. In addition, four new compounds [trojanosides H
(53), I (54), J (55), and K (56)] along with the known cycloartane glycosides astragalosides I (35) [29], II (33) [29], IV (34) [29], VII [49], astrasiversianins IX (49) [41], XV (50) [41], [50], and brachyosides B (43) [45] and C (44) [45], and a pterocarpane derivative macianin (maackianin) (59) [51] were isolated from the aerial parts of the plant ([Fig. 6]) [47], [48].
Fig. 6 Structures of compounds 45 – 60.
Astragalus cephalotes Bangs & Sol. var. brevicalyx Eig. (Sect. Rhacophorus Bunge.)
A. cephalotes var. brevicalyx, traditionally used for wound healing in southeastern Anatolia, was collected from Borgaç village, Hilvan, Şanlıurfa. Mono- (cyclocantoside A) [52], bi-(cyclocantosides D and E) [38] and tridesmosidic (cephalatoside A (60)) glycosides of cyclocantogenin were obtained in this study [53]. Cephalotoside A (60) ([Fig. 6]), a new tridesmosidic cycloartane type glycoside, was isolated from the roots of A. cephalotes var. brevicalyx. Tridesmosidic glycosides are rarely encountered in nature and have only been isolated from the Astragalus species.
Astragalus zahlbruckneri Hand.-Mazz. (Sect. Rhacophorus Bunge.)
The study on the roots of A. zahlbruckneri, collected from Sivrice, Elâzığ, Eastern Anatolia, resulted in the isolation of six compounds (61 – 66) ([Fig. 7]). The apolar fractions of the ethanolic extract afforded two cycloartane derivative triterpenes, 20(R),25-epoxy-3β,6α,16β,24α-tetrahydroxycycloartane (61) and 20(R),24(S)-epoxy-3β,6α,25-trihydroxycycloartan-16-one (62), together with cycloastragenol [29]. Compound 62 was previously reported as a cycloartane derivative obtained by chemical oxidation of cycloastragenol [29], [40], [49]. A new lignan [(+)-neo-olivil-4-O-β-apiofuranosyl-(1 → 2)-β-glucopyranoside (63)] and three phenolic glycosides [7,8-dihydro-7-hydroxyconiferyl alcohol
4-O-β-apiofuranosyl-(1 → 2)-β-glucopyranoside (64), 2-methoxyphenol-4-O-β-apiofuranosyl-(1 → 2)-β-glucopyranoside (65), and 3-hydroxy-5-methoxyphenol-2-O-β-apiofuranosyl-(1 → 2)-β-glucopyranoside (66)] were isolated from the polar fractions of A. zahlbruckneri
[54].
Fig. 7 Structures of compounds 61 – 71.
Astragalus prusianus Boiss. (Sect. Rhacophorus Bunge.)
Continuing our research on the genus Astragalus, A. prusianus was collected from Kale, Muğla, West Anatolia. In this study, two novel cycloartane-type triterpene glycosides, 16-O-β-D-glucopyranosyl-20(S),24(R)-5α,9-diepoxy,2α,3β,16β,25-tetrahydroxy-9,10-seco-cycloarta-1(10),6(7)-diene (67) and 3-O-β-D-xylopyranosyl-16-O-β-D-glucopyranosyl-20(S),24(R)-epoxy-3β,16β,25-trihydroxycycloartane (68), were obtained ([Fig. 7]). The 5α,9-epoxy structural feature in prussianoside A (67) was reported for the first time in triterpene chemistry [55].
Astragalus vulneraria DC. (Sect. Vulneraria DC.)
A. vulneraria was the only species from which no cycloartane derivative has been isolated in our studies. However, two flavonol glycosides were isolated from the aerial parts of the plant material collected from Polatlı, Ankara, Central Anatolia. One of the glycosides was a new compound (69) [isorhamnetin 3-O-β-D-apiofuranosyl-(1 → 2)-[α-L-rhamnopyranosy-(1 → 6)]-β-D galactopyranoside], and the other was a known isorhamnetine derivative (70) [56], isorhamnetin 3-O-β-D-apiofuranosyl-(1 → 2)-β-D-galactopyranoside ([Fig. 7]) [57].
Astragalus baibutensis Bunge (Sect. Pterophorus Bunge)
As a result of our studies on the chemistry of A. baibutensis, (20R,24S)-3-O-[β-D-apiofuranosyl-(1 → 2)-β-D-xylopyranosyl]-6-O-β-D-glucopyranosyl-3β,6α,16β,25-tetrahydroxy-20,24-epoxycycloartane, named baibutoside (71) ([Fig. 7]), a new cycloartane-type glycoside together with four known glycosides, acetylastragaloside I [29], astragaloside I (35) [29], II (33) [29], and IV (34) [29], were reported [58]. The antiprotozoal activities of the isolated compounds were also evaluated against some parasites, including Trypanosoma brucei rhodesiense, Trypanosoma cruzi, Leishmania donovani, and Plasmodium falciparum. All the tested compounds were inactive against L. donovani and P.
falciparum. In addition, the selective toxicity tests on primary L6 mammalian cells (rat skeletal myoblasts) demonstrated that only acetylastragaloside I had a cytotoxic effect with narrow selectivity index values of 2.5 and 4.8.
Astragalus campylosema Boiss. ssp. campylosema (Astragalus pendulus DC. (Sect. Dissitiflori DC.), Astragalogia: 232 (1802) [11])
In the course of studies on the Turkish Astragalus species, four new cycloartane glycosides, 3-O-[α-L-arabinopyranosyl-(1 → 2)-β-D-xylopyranosyl]-3β,6α,16β,23α,25-pentahydroxy-20(R),24(S)-epoxycycloartane (72), 3-O-[α-L-arabinopyranosyl-(1 → 2)-β-D-xylopyranosyl]-16-O-hydroxyacetoxy-23-O-acetoxy-3β,6α,25-trihydroxy-20(R),24(S)-epoxycycloartane (73), 3-O-[α-L-arabinopyranosyl-(1 → 2)-β-D-xylopyranosyl]-25-O-β-D-glucopyranosyl-3β,6α,16β,25-tetrahydroxy-20(R),24(S)-epoxycycloartane (74), and 3-O-[α-L-arabinopyranosyl-(1 → 2)-β-D-xylopyranosyl]-3β,6α,23α,25-tetrahydroxy-20(R),24(S)-16β,24;20,24-diepoxycycloartane (75) ([Fig. 8]), together with three previously
isolated cycloartane glycosides, namely, 3-O-[α-L-arabinopyranosyl-(1 → 2)-β-D-xylopyranosyl]-3β,6α,16β,25-tetrahydroxy-20(R),24(S)-epoxycycloartane [59], askendoside C [60], and askendoside G [61], were obtained from the MeOH extract of the roots of A. pendulus, collected from Tutak, Ağrı, East Anatolia [62].
Fig. 8 Structures of compounds 72 – 82.
Astragalus elongatus (Sect. Dissitiflori DC.)
Continuing of our work on the genus Astragalus, the roots of Astragalus elongatus, collected from Central Anatolia, Ahlatlıbel, Ankara, were also studied. In this study, a new monodesmosidic cycloartane-type glycoside, elongatoside (76) (3-O-[α-arabinopyranosyl-(1 → 2)-β-xylopyranosyl]-cycloastragenol) ([Fig. 8]), was isolated in addition to two known cycloartane-type glycosides: askendosides D (3-O-[α-arabinopyranosyl-(1 → 2)-β-xylopyranosyl]-6-O-β-xylopyranosyl-cycloastragenol [59] and G (3-O-[α-arabinopyranosyl-(1 → 2)-β-xylopyranosyl]-16-O-β-glucopyranosyl-3β,6α,16β,24(R),25-pentahydroxycycloartane) [61]. These compounds were assessed for their effects on cell proliferation and ICAM-1 expression using the human microvascular endothelial cell line
HMEC-1. The results showed that compound 76 exhibited weak activity in the ICAM-1 assay [63].
Astragalus stereocalyx Bornm. (Sect. Stereocalyx Bornm.)
As part of our ongoing studies on the Turkish Astragalus species, 3-O-[α-L-arabinopyranosyl-(1 → 2)-β-D-xylopyranosyl]-16-O-β-D-glucopyranosyl-3β,6α,16β,20(S),24(R),25-hexahydroxycycloartane (77), 3-O-[α-L-arabinopyranosyl-(1 → 2)-β-D-xylopyranosyl]-3β,6α,16β,20(S),24(R),25-hexahydroxycycloartane (78), 3-O-[α-L-arabinopyranosyl-(1 → 2)-β-D-glucopyranosyl]-3β,6α,16β,20(S), 24(R),25-hexahydroxycycloartane (79), 3-O-[α-L-arabinopyranosyl-(1 → 2)-β-D-glucopyranosyl]-24-O-β-D-glucopyranosyl]-3β,6α, 16β,24(R),25-pentahydroxycycloartane (80),
3-O-[α-L-arabinopyranosyl-(1 → 2)-β-D-glucopyranosyl]-16-O-β-D-glucopyranosyl-3β,6α,16β,24(R),25-pentahydroxycycloartane (81), and 3-O-{α-L-rhamnopyranosyl-(1 → 4)-[α-L-arabinopyranosyl-(1 → 2)-β-D-glucopyranosyl]}-3β,6α,16β,24(R),25-pentahydroxycycloartane (82) were isolated from the MeOH extract of A. stereocalyx ([Fig. 8]). Additionally, the known compounds askendoside C [60], askendoside F [64], askendoside G [61], 3-O-β-D-glucopyranosyl-16-O-β-D-glucopyranosyl-3β,6α,16β,24(R),25 pentahydroxycycloartane [43], elongatoside (76) [63], and trojanoside H (53) [47] were
also obtained from the roots of A. stereocalyx. In addition, the isolated compounds were evaluated for their cytotoxicity against different cell lines including human cervical cancer (Hela), human colon cancer (HT-29), human leukemia (U937), and human lung cancer (H446). Only a few compounds exhibited a weak cytotoxic activity in the concentration of 1 – 50 µM [65].
Astragalus lusitanicus Lam. (Sect. Erophaca Boiss.)
Ongoing studies are being conducted on A. lusitanicus, a species known for its toxicity in Turkey and the countries bordering the Mediterranean Sea. So far, only kersetol and kaempferol derivative flavonol glycosides have been obtained. It is believed that aliphatic nitro compounds are responsible for the toxicity in animals caused by this species [14].
In 2000, due to the well-known immunostimulatory activity of saponins together with our earlier investigation on some of the compounds for their bioactivity [42], 19 cycloartane-type triterpene glycosides were tested for their immunostimulatory effects on macrophage activation and expression of inflammatory cytokines. Macrophlyllosaponins B – D (25 – 29) [36], [37], askendoside G [61], cyclocanthoside D [38] and E (32) [38], cephalotoside A (60) [53], astrasieversianin II (37) [41] and X (38) [41], astragaloside I (35) [29], II (33) [29], IV (34) [29], VI (36) and VII
[40], trojanoside A (45) [46] and H (46) [47], cycloastragenol (4) [29], brachyoside B (43) [45], and cyclocephaloside I (40) [44] were evaluated using a transcription-based bioassay for nuclear factor kappa B (NF-kappa B) activation in THP-1 human monocyte cells. Only astragaloside I was active at 100 µg/ml, which increased NF-kappa-B-directed luciferase expression up to 65% compared with maximal stimulation by Escherichia coli lipopolysaccharide (LPS) at 10 µg/ml. At low concentrations, all the compounds were inactive in the presence of 50 ng/ml LPS. In addition, astragaloside I increased mRNA expression of the inflammatory cytokines interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) [66].
In 2005, another study was conducted to evaluate the immunostimulating activity of cycloartane- and oleanane-type saponins, namely, brachyoside A (42) [45], brachyoside B (43) [45], brachyoside C (44) [45], cyclocephaloside I (40) [44], cyclocephaloside II (41) [45], cycloastragenol (4) [29], astragaloside I (35) [42], astragaloside II (33) [47], astragaloside IV (34) [42], astragaloside VII [47], trojanoside A (45) [47], trojanoside H (53) [47], and astrojanoside A (51) [47], isolated from Turkish Astragalus
species. Additionally, methanol extracts from the roots of three Astragalus species (Astragalus cephalotes Banks and Sol. var. brevicalyx Eig, Astragalus oleifolius DC. and Astragalus trojanus Stev.) were also examined. Cytokine concentrations of interleukins IL-1 and IL-8, and TNF-α after bacterial lipopolysaccharide (LPS) stimulation, and IL-2, IL-4, and INF-γ after phorbolacetate (PHA) stimulation were determined via commercially available enzyme-linked immunosorbent assay (ELISA) kits. All of the compounds tested in this study exhibited a significant IL-2-inducing activity between 35.9% for brachyoside A and 139.6% for astragaloside VII. Among the extracts tested, Astragalus oleifolius DC. showed the highest activity score, at 141.2%. In general, glycosides of 20,24-epoxy and 20,25-epoxy cycloartanes exhibited higher IL-2-inducing activity compared to those of acyclic cycloartanes [67].
The evaluation of the gastroprotective effect of astragaloside IV (34), obtained from Astragalus zahlbruckneri, was studied [68]. Ulceration was induced by intragastric instillation of ethanol (1 ml/rat). The rats were orally administered with astragaloside IV, which was found to reduce gastric hemorrhagic lesions in a dose-dependent manner when compared to the control group. The maximum percentage inhibition of ulcers (% gastroprotection) obtained with 30 mg/kg astragaloside IV following oral administration was 52%. Furthermore, the results demonstrate that endogenous NO (nitric oxide) plays an important role in the gastroprotective mechanism of astragaloside IV on ethanol-induced gastric lesions.
In a recent study, the antitumor properties of five Astragalus cycloartanes, namely, astragaloside IV (34) [29], cyclocanthoside E (32) [38], astrasieversianin X (38) [41], and macrophyllosaponin B (26) [36] and D (28) [36], were evaluated in MCF-7 and MDA-MB-231 breast cancer cell lines. This was the first study to investigate the antitumor properties of different saponin extracts from Astragalus species in breast cancer. The results demonstrated that Astragalus saponins can inhibit the proliferation of breast cancer cells in a dose- and time-dependent manner. These findings indicated that saponins obtained from Astragalus species have important antiproliferative and antiapoptotic effects in the MCF-7 cell line [69].
Apart from our studies on the Turkish Astragalus species, there have been several reports by other research groups.
Six known cycloartane-type saponins, astrasieversianins I [41], II (37) [41], VI [41], VIII [41], X (38) [41], and astragaloside IV (34) [29], were isolated from the roots of Astragalus gilvus Boiss. (Sect. Christiana). In addition to A. gilvus, only Astragalus melanophrurius was found to be rich in acylated cycloartane-type glycosides [49]. It is noteworthy that A. gilvus and A. melanophrurius were both members of the Christiana section. This suggests that the presence of acylated glycosides found in the Christiana section could be of taxonomic importance [70].
Phytochemical investigation of the roots of Astragalus flavescens (Sect. Eustales) resulted in the isolation of six new triterpene saponins [3-O-α-L-rhamnopyranosyl-(1 → 2)]-β-D-glucopyranosyl-(1 → 2)]-β-D-glucuronopyranosyl-21-epi-kudzusapogenol A; 3-O-α-L-rhamnopyranosyl-(1 → 2)]-β-D-xylopyranosyl-(1 → 2)]-β-D-glucuronopyranosyl-22-O-β-D-glucopyranosyl-21-epi-kudzusapogenol A; 3-O-α-L-rhamnopyranosyl-(1 → 2)]-β-D-glucopyranosyl-(1 → 2)]-β-D-glucuronopyranosyl-22-O-β-D-glucopyranosyl-21-epi-kudzusapogenol A; 3-O-α-L-rhamnopyranosyl-(1 → 2)]-β-D-xylopyranosyl-(1 → 2)]-β-D-glucuronopyranosyl-22-O-α-L-arabinopyranosyl-21-epi-kudzusapogenol A; 3-O-α-L-rhamnopyranosyl-(1 → 2)]-β-D-glucopyranosyl-(1 → 2)]-β-D-glucuronopyranosyl-22-O-α-L-arabinopyranosyl-21-epi-kudzusapogenol A]
[71] along with five previously isolated compounds (trojanoside B (46) [46], azukisaponin V [72], astragaloside IV (34) [29], astragaloside VII [49], and VIII [49]).
Another phytochemical study was performed on Astragalus amblolepis Fischer (Sect. Rhacophorus), which resulted in the isolation and structural elucidation of five new cycloartane-type triterpene glycosides, including 3-O-β-D-xylopyranosyl-3β,6α,16β,24(S),25-pentahydroxycycloartane (83), 3-O-[β-D-glucuronopyranosyl- (1 → 2)-β-D-xylopyranosyl]-25-O-β-D-glucopyranosyl-3β,6α,16β,24(S),25-pentahydroxycycloartane (84), 3-O-β-D-xylopyranosyl-24,25-di-O-β-D-glucopyranosyl-3β,6α,16β,24(S),25-pentahydroxycycloartane (85), 6-O-α-L-rhamnopyranosyl-16,24-di- O-β-D-glucopyranosyl-3β,6α,16β,24(S),25-pentahydroxycycloartane (86), and
6-O-α-L-rhamnopyranosyl-16,25-di-O-β-D-glucopyranosyl-3β,6α,16β,24(S),25-pentahydroxycycloartane (87), together with a known compound, 3-O-β-D-xylopyranosyl-16-O-β-D-glucopyranosyl-3β,6α,16β,24(S),25-pentahydroxycycloartane [73] ([Fig. 9]). The researchers noted that cycloartane glycosides without a sugar residue at the C-3 position, such as compounds 86 and 87, are quite uncommon in nature. In addition, the presence of a rhamnosyl unit at the C-6 position in the cyclocanthogenol skeleton was reported for the first time, which is one of the most common aglycons in the genus Astragalus, along with cycloastragenol. The glucuronic acid moiety in cycloartanes was encountered for the first time in this study [74].
Fig. 9 Structures of compounds 83 – 101.
Polat et al. (2010) reported on the isolation and structural elucidation of three new cycloarte-type saponins, 3-O-[α-L-rhamnopyranosyl-(1 → 2)-β-D-glucopyranosyl]-25-O-β-D-glucopyranosyl-20(R),24(S)-epoxy-3β,6α,16β,24(S),25-tetrahydroxycycloartane, 3-O-[α-L-rhamnopyranosyl-(1 → 2)-β-D-xylopyranosyl]-6-O-β-D-glucopyranosyl-24-O-α-(4′-O-acetoxy)-L-arabinopyranosyl-16-O-acetoxy-3β,6α,16β,24(S),25-pentahydroxycycloartane, and 3-O-[α-L-rhamnopyranosyl-(1 → 2)-β-D-xylopyranosyl]-6-O-β-D-glucopyranosyl-24-O-α-L-arabinopyranosyl-16-O-acetoxy-3β,6α,16β,24(S),25-pentahydroxycycloartane, from Astragalus wiedemannianus Fischer (Sect. Pterophorus) [75], along with eight known compounds (cycloastragenol
[30], cycloascauloside B [76], astragaloside IV (34) [29], astragaloside VIII [49], brachyoside B (43) [45], astragaloside II (33) [29], astrachrysoside A [50], and astrasieversianin X (38) [41]). The authors stated that an arabinose moiety on the acyclic side chain was reported for the first time.
In another study, six new cycloartane-type triterpene glycosides were isolated from the MeOH extract of the whole plant of A. icmadophilus (Sect. Acanthophace) together with eight known secondary metabolites, namely, oleifolioside B (31) [39], astragaloside I (35) [29], azukisaponin V [72], azukisaponin V methyl ester [77], astragaloside VIII [49], astragaloside VIII methy ester [33], 22-O-[β-D-glucopyranosyl-(1 → 2)-O-α-L-arabinopyranosyl]-3β,22β,24-trihydroxy-olean-12-ene [78], and narcissin [79]. The structures of the new compounds were established as
3-O-[α-L-arabinopyranosyl-(1 → 2)-O-3-acetoxy-α-L-arabinopyranosyl]-6-O-β-D-glucopyranosyl-3β,6α,16β,24(S),25-pentahydroxycycloartane; 3-O-[α-L-rhamnopyranosyl-(1 → 2)-O-α-L-arabinopyranosyl-(1 → 2)-O-β-D-xylopyranosyl]-6-O-β-D-glucopyranosyl-3β,6α,16β, 24(S),25-pentahydroxycycloartane; 3-O-[α-L-arabinopyranosyl-(1 → 2)-O-3,4-diacetoxy-α-L-arabinopyranosyl]-6-O-β-D-glucopyranosyl-3β,6α,16β,24(S),25-pentahydroxycycloartane; 3-O-[α-L-arabinopyranosyl-(1 → 2)-O-3-acetoxy-α-L-arabinopyranosyl]-6-O-β-D-glucopyranosyl-3β,6α,16β,25-tetrahydroxy-20(R),24(S)-epoxycycloartane;
3-O-[α-L-arabinopyranosyl-(1 → 2)-O-β-D-xylopyranosyl]-6-O-β-D-glucopyranosyl-3β,6α,16β,24α-tetrahydroxy-20(R),25-epoxycycloartane, and 3-O-[α-L-rhamnopyranosyl-(1 → 2)-O-α-L-arabinopyranosyl-(1 → 2)-O-β-D-xylopyranosyl]-6-O-β-D-glucopyranosyl-3β,6α,16β,24α-tetrahydroxy-20(R),25-epoxycycloartane [80].
Phytochemical investigation of A. ptilodes Boiss. var. cariensis Boiss. (Sect. Pterophorus) resulted in the isolation of five previously isolated compounds [81], i.e., astragaloside VII (36) [49], cyclosiversioside E [82], cyclosiversioside F [82], astragaloside I (35) [29], and cyclosiversioside A [83].
Studies on A. aureus Willd (Sect. Adiaspastus) resulted in the isolation of eight new cycloartane-type triterpene glycosides. The structures of the new compounds were established as 3-O-[α-L-rhamnopyranosyl-(1 → 2)-α-L-arabinopyranosyl-(1 → 2)-β-D-xylopyranosyl]-6-O-β-D-xylopyranosyl-3β,6α,16β,24(S),25-pentahydroxycycloartane (88), 3,6-di-O-β-D-xylopyranosyl-3β,6α,16β,24(S),25-pentahydroxycycloartane (89), 3,6-di-O-β-D-xylopyranosyl-25-O-β-D-glucopyranosyl-3β,6α,16β,24(S),25-pentahydroxycycloartane (90), 3-O-β-D-xylopyranosyl-6,25-di-O-β-D-glucopyranosyl-3β,6α,16β,24(S),25-pentahydroxycycloartane (91), 6-O-β-D-glucopyranosyl-3β,6α,16β,24(S),25-pentahydroxycycloartane (92),
3-O-[α-L-arabinopyranosyl-(1 → 2)-β-D-xylopyranosyl]-3β,6α,16β,24α-tetrahydroxy-20(R),25-epoxycycloartane (93), 6-O-β-D-glucopyranosyl-3β,6α,16β,24α-tetrahydroxy-20(R),25-epoxycycloartane (94), and 6-O-β-D-xylopyranosyl-3β,6α,16β,24α-tetrahydroxy-20(R),25-epoxycycloartane (95) ([Fig. 9]), in addition to 10 known compounds, 3-O-[α-L-rhamnopyranosyl-(1 → 2)-O-α-L-arabinopyranosyl-(1 → 2)-O-β-D-xylopyranosyl]-6-O-β-D-glucopyranosyl-3β,6α,16β,24(S),25-pentahydroxycycloartane [78], oleifolioside B (31) [39], cyclocanthoside E (32) [38], cyclocanthoside G (39) [38], 3-O-[α-L-rhamnopyranosyl-(1 → 2)-O-α-L-arabinopyranosyl-(1 → 2)-O-β-D-xylopyranosyl]-6-O-β-D-glucopyranosyl-3β,6α,16β,24α-tetrahydroxy-20(R),25-epoxycycloartane [80], 3-O-[α-L-arabinopyranosyl-(1 → 2)-O-β-D-xylopyranosyl]-6-O-β-D-glucopyranosyl-3β,6α,16β,24α-tetrahydroxy-20(R),25-epoxycycloartane [80], cyclocanthoside F [84], cyclocephaloside I (40) [44], cyclotrisectoside [85], and macrophyllosaponin B (26) [36]. According to the authors, aminoglycosides of cyclocanthogenin (84) and cyclocephalogenin (94,95) were reported for the first time. In addition, the isolated compounds were tested for their cytotoxic
activity against different cancer cell lines. Compound 95 was the only one that showed moderate activity against the human breast cancer cell line (MCF7) at a concentration of 45 µM [86].
In another study on the Turkish Astragalus species, four new cycloartanes (hareftoside A – D) and a new oleanane-type triterpenoid (hareftoside E) were isolated and characterized from the MeOH extract of the whole plant of Astragalus hareftae (Sect. Acanthophace), along with 11 known cycloartane-type glycosides [87], namely, cyclocanthoside E (32) [38], macrophyllosaponin B (26) [36], cyclocephaloside I (40) [44], oleifolioside B (31) [39], astrasieversianin X (38) [41], trojanoside B (46) [46], cycloastragenol (4) [29], astragaloside IV (34) [29], brachyoside B (43) [45], cyclodissectoside [85], and 3-O-β-D-xylopyranosyl-6,25-di-O-β-D-glucopyranosyl-3β,6α,16β,24(S),25-pentahydroxycycloartane (91) [86].
Phytochemical investigation of A. schottianus Boiss. (Sect. Rhacophorus) resulted in the isolation of three new cycloartane type glycosides. Their structures were determined as 20(R),25-epoxy-3-O-β-D-xylopyranosyl-24-O-β-D-glucopyranosyl-3β,6α,16β, 24α-tetrahydroxycycloartane, 20(R),25-epoxy-3-O-[β-D-glucopyranosyl-(1 → 2)]-β-D-xylopyranosyl-24-O-β-D-glucopyranosyl-3β,6α,16β,24α-tetrahydroxycycloartane, and 3-O-β-D-xylopyranosyl-3β,6α,16β,20(S),24(S),25-hexahydroxycycloartane (96) ([Fig. 9]). The authors stated that compound 96 was the second cycloartane-type compound in the genus Astragalus that possesses a 20-OH functional group [88].
A new cycloartane-type saponin, namely, 3-O-[β-D-xylopyranosyl-(1 → 2)-β-D-xylopyranosyl]-6-O-β-D-glucuronopyranosyl-3β,6α,16β,24(S),25-pentahydroxycycloartane, was obtained from A. erinaceus (Sect. Rhacophorus) together with five known compounds. According to the authors, this new compound represents the second example of a cycloartane-type compound that possesses a glucuronic acid moiety [89]. Known compounds were identified as cyclodissectoside [85], cycloastragenol (4) [29], oleifolioside B (31) [39], 3,6-di-O-β-D-xylopyranosyl-3β,6α,16β,24(S),25-pentahydroxycycloartane (89) [86], and 6-O-β-D-glucopyranosyl-3β,6α,16β,24(S),25-pentahydroxycycloartane
(92) [86].
Gülcemal et al. (2012) reported on the isolation and characterization of six new cycloartane-type triterpenoids, 3-O-[α-L-rhamnopyranosyl-(1 → 2)-β-D-glucopyranosyl]-16-O-hydroxyacetoxy-3β,6α,16β,25-tetrahydroxy-20(R),24(S)-epoxycycloartane, 3-O-[α-L-rhamnopyranosyl-(1 → 2)-β-D-glucopyranosyl]-16-O-hydroxyacetoxy-3β,6α,16β, 23α,25-pentahydroxy-20(R),24(S)-epoxycycloartane, 3-O-[α-L-rhamnopyranosyl-(1 → 2)-β-D-glucopyranosyl]-3β,6α,25-trihydroxy-20(R),24(S)-epoxycycloartane-16-one, 3-O-[α-L-rhamnopyranosyl-(1 → 2)-β-D-glucopyranosyl]-3β,6α,16β,25-tetrahydroxy-20(R),24(R)-epoxycycloartane (97),
3-O-β-D-xylopyranosyl-6-O-α-L-rhamnopyranosyl-3β,6α,16β,25-tetrahydroxy-20(R),24(R)-epoxycycloartane (98), and 6-O-α-L-rhamnopyranosyl-3β,6α,16β,25-tetrahydroxy-20(R),24(R)-epoxycycloartane (99), from A. angustifolius (Sect. Melanocercis) ([Fig. 9]), along with four oleanane-type triterpenoids, namely, 3-O-[α-L-rhamnopyranosyl-(1 → 2)-β-D-xylopyranosyl-(1 → 2)-β-D-glucuronopyranosyl]-3β,21β,22α,24,29-pentahydroxyolean-12-ene, 3-O-[α-L-rhamnopyranosyl-(1 → 2)-β-D-xylopyranosyl-(1 → 2)-β-D-glucuronopyranosyl]-3β,22β,24-trihydroxyolean-12-en-29-oic acid,
3-O-[α-L-rhamnopyranosyl-(1 → 2)-β-D-xylopyranosyl-(1 → 2)-β-D-glucuronopyranosyl]-22-O-α-L-arabinopyranosyl-3β,22β,24-trihydroxyolean-12-ene, and 29-O-β-D-glucopyranosyl-3β,22β,24,29-tetrahydroxyolean-12-ene, and five known triterpene glycosides (astrojanoside A (51) [47], astragaloside VIII [49], 25-O-glucopyranosylcycloastragenol [49], 3-O-[α-L-rhamnopyranosyl-(1 → 2)-β-D-glucopyranosyl]-25-O-β-D-glucopyranosyl-20(R),24(S)-epoxy-3β,6α,16β,24(S),25-tetrahydroxycycloartane [75], and cycloaraloside D [90]). According to the authors, compounds 89 – 91 possessed the C-24 epimer of cycloastragenol as their aglycone, which was reported for the first time. The
compounds were evaluated for their ability to inhibit cell growth in cell lines including Hela, H-446, HT-29, and U937. Of these compounds, only one compound (3-O-[α-L-rhamnopyranosyl-(1 → 2)-β-D-xylopyranosyl-(1 → 2)-β-D-glucuronopyranosyl]-3β,22β,24-trihydroxyolean-12-en-29-oic acid) showed a weak inhibitory effect with IC50 values of 36 and 50 µM in the Hela and HT-29 cell lines, respectively [91].
A new cycloartane-type glycoside (20R,24S)-3-O-[α-L-arabinopyranosyl-(1 → 2)-β-D-xylopyranosyl]-20,24-epoxy-16-O-β-D-glucopyranosyl-3β,6α,16β,25-tetrahydroxycycloartane, and a new glycoside (100) ([Fig. 9]), 3-O-[β-D-apiofuranosyl-(1 → 2)-β-D-glucopyranosyl]maltol, were isolated from the whole plant of A. halicacabus (Sect. Halicacabus), together with seven known cycloartane-type glycosides, namely, cyclocanthoside D [38], askendoside D [59], askendoside F [64], askendoside G [61], elongatoside (76) [63], cyclosieversioside G [92], and cyclostipuloside A [73]. Authors reported that a maltol glycoside (100) was encountered for the
first time in the Leguminosae family [93].
In 2013, the results of an online screening by HPLC-ESIMSn led to the isolation of 22 oleanane-type triterpene glycosides from A. tauricolus (sect. Malacothrix), including 10 new compounds, namely, 3-O-[α-L-rhamnopyranosyl-(1 → 2)-β-D-xylopyranosyl-(1 → 2)-β-D-glucuronopyranosyl]-29-O-β-D-glucopyranosyl-3β,22β, 24-trihydroxyolean-12-ene-29-oic acid, 3-O-[α-L-rhamnopyranosyl-(1 → 2)-β-D-glucopyranosyl-(1 → 2)-β-D-glucuronopyranosyl]-29-O-β-D-glucopyranosyl-3β,22β,24,29-tetrahydroxyolean-12-ene, 3-O-[α-L-rhamnopyranosyl-(1 → 2)-β-D-xylopyranosyl-(1 → 2)-β-D-glucuronopyranosyl]-21-O-α-L-rhamnopyranosyl-3β,21β,22α,24-tetrahydroxyolean-12-ene,
3-O-[α-L-rhamnopyranosyl-(1 → 2)-β-D-glucopyranosyl-(1 → 2)-β-D-glucuronopyranosyl]-21-O-α-L-rhamnopyranosyl-3β,21β,22α,24-tetraydroxyolean-12-ene, 3-O-[α-L-rhamnopyranosyl-(1 → 2)-β-D-glucopyranosyl-(1 → 2)-β-D-glucuronopyranosyl]-29-O-β-D-glucopyranosyl-3β,22β,24-trihydroxyolean-12-ene-29-oic acid, 3-O-[α-L-rhamnopyranosyl-(1 → 2)-β-D-xylopyranosyl-(1 → 2)-β-D-glucuronopyranosyl]-22-O-α-L-rhamnopyranosyl-3β,22β,24-trihydroxyolean-12-ene, 3-O-[α-L-rhamnopyranosyl-(1 → 2)-β-D-glucopyranosyl-(1 → 2)-β-D-glucuronopyranosyl]-3β,24-dihydroxyolean-12-ene-22-oxo-29-oic acid, 3-O-[α-L-rhamnopyranosyl-(1 → 2)-β-D-glucopyranosyl-(1 → 2)-β-D-glucuronopyranosyl]-3β,21β,22α,24,29-pentahydroxyolean-12-ene,
3-O-[β-D-glucopyranosyl-(1 → 2)-β-D-glucuronopyranosyl]-29-O-β-D-glucopyranosyl-3β,22β,24-trihydroxyolean-12-ene-29-oic acid, and 3-O-[β-D-xylopyranosyl-(1 → 2)-β-D-glucuronopyranosyl]-29-O-β-D-glucopyranosyl-3β,22β,24-trihydroxyolean-12-ene-29-oic acid. Known compounds were identified as astrojanoside A (51) [47], astragaloside VIII [49], azukisaponin II [72], azukisaponin V [72], 3-O-[α-L-rhamnopyranosyl-(1 → 2)-β-D-xylopyranosyl-(1 → 2)-β-D-glucuronopyranosyl]-3β,21β,22α,24,29-pentahydroxyolean-12-ene [91], melilotus-saponin O2 [94], wistariasaponin B1 [95], wistariasaponin B2 [95],
wistariasaponin D [96], cloversaponin IV [97], dehydroazukisaponin V [98], and 3-O-β-D-glucuronopyranosyl-soyasapogenin B [99]. It is noteworthy that cycloartane-type triterpene glycosides, which are the main constituents of Astragalus spp., were not found. This unique feature is present only in a small group of Astragalus species, including A. hamosus
[100], A. sinicus
[32], A. complanatus
[33], and A. corniculatus
[34]. Moreover, the antiproliferative activity of the isolated compounds was evaluated against four human cell lines: MCF-7 (breast cancer), A549 (lung adenocarcinoma), PC-3 (prostate cancer), and U937 (leukemia). Only one compound
(3-O-[α-L-rhamnopyranosyl-(1 → 2)-β-D-glucopyranosyl-(1 → 2)-β-D-glucuronopyranosyl]-3β,21β,22α,24,29-pentahydroxyolean-12-ene) exhibited moderate activity, with an IC50 of 22 µM against the U937 cell line at concentrations ranging from 1 to 50 µM [101].
Sixteen cycloartane glycosides were obtained from the methanol extract of A. plumosus var. krugianus Chamb. & Matthews (Sect. Rhacophorus). Among them, krugianoside A, 3-O-[α-L-rhamnopyranosyl-(1 → 2)-α-L-arabinopyranosyl-(1 → 2)-β-D-glucuronopyranosyl]-25-O-β-D-xylopyranosyl-3β,6α,16β,24(S),25-pentahydroxycycloartane, was a new compound. Known compounds were characterized as oleifolioside B (31) [39], cyclocephaloside I (40) [44], cyclocanthoside E (32) [38], cycloastragenol (4) [29], brachyoside B (43) [45], elongatoside (76) [63], astragaloside IV (34) [29], cycloaraloside D [90], cycloaraloside A [102], cyclogaleginoside B [103], 3-O-[α-L-arabinopyranosyl-(1 → 2)-O-β-D-xylopyranosyl]-6-O-β-D-glucopyranosyl-3β,6α,16β,24α-tetrahydroxy-20(R),25-epoxycycloartane [80], 3-O-[α-L-rhamnopyranosyl-(1 → 2)-O-α-L-arabinopyranosyl-(1 → 2)-O-β-D-xylopyranosyl]-6-O-β-D-glucopyranosyl-3β,6α,16β,24(S),25-pentahydroxycycloartane [80], 3-O-[α-L-arabinopyranosyl-(1 → 2)-β-D-xylopyranosyl]-3β,6α,16β,24α-tetrahydroxy-20(R),25-epoxycycloartane (93) [86], and 6-O-β-D-glucopyranosyl-3β,6α,16β,24α-tetrahydroxy-20(R),25-epoxycycloartane (94) [86]. In
this study, the cytotoxic activity of the isolated compounds was evaluated in human skin fibroblast cells (WS1). Compounds that did not significantly affect WS1 viability were tested for their antioxidant potential. Krugianoside A and oleifolioides B prevented the elevation of reactive oxygen species (ROS) induced by t-BOOH, indicating their potential to protect fibroblasts from oxidative stress [104].
Phytochemical investigation of A. pennatulus (Sect. Rhacophorus) resulted in isolation of four new cycloartane-type glycosides, 3-O-β-D-xylopyranosyl-6-O-β-D-glucopyranosyl-3β,6α,16β-trihydroxy-24-oxo-20(R),25-epoxycycloartane (101) ([Fig. 9]), 3-O-[β-D-glucuronopyranosyl-(1 → 2)-β-D-xylopyranosyl]-3β,16β,24α- trihydroxy-20(R),25-epoxycycloartane, 3-O-[β-D-glucuronopyranosyl-(1 → 2)-β-D-xylopyranosyl]-3β,16β,25-trihydroxy-20(R),24(S)-epoxycycloartane, 3,25-di-O-β-D-glucuronopyranosyl-6-O-β-D-xylopyranosyl-3β,6α,16β,25-tetrahydroxy-20(R),24(S)-epoxycycloartane, and a new oleanane-type glycoside, 29-O-α-L-rhamnopyranosyl-abrisapogenol B, along with five previously isolated cycloartane-type compounds
(6-O-β-D-glucopyranosyl-3β,6α,16β,24α-tetrahydroxy-20(R),25-epoxycycloartane (94) [86], cyclodissectoside [85], hareftoside C [87], cyclocephaloside I (40) [44], and astragaloside IV (34) [29]). According to the authors, the aglycone of compound 101 was encountered for the first time. In addition, the cytotoxic activity of the compounds was tested on three cell lines including A549 (human lung adenocarcinoma), A375 (human melanoma), and DeFew (human B lymphoma) cells. The results showed that none of the tested compounds exhibited significant cytotoxicity [105].
