Vol.:(0123456789) 1 3 Journal of Natural Medicines https://doi.org/10.1007/s11418-019-01355-y ORIGINAL PAPER LC–MS analysis of saponins of Achyranthes root in the Japanese market Kohei Kuwada 1  · Satoshi Kawase 1  · Karin Nakata 1  · Nodoka Shinya 1  · Yuji Narukawa 1  · Hiroyuki Fuchino 2  · Nobuo Kawahara 2  · Fumiyuki Kiuchi 1 Received: 10 June 2019 / Accepted: 5 August 2019 © The Japanese Society of Pharmacognosy 2019 Abstract LC–MS analyses of saponin fractions of Achyranthes roots in the Japanese market revealed that there were three patterns for the saponin fraction of their water extracts, i.e., the saponins with a sugar moiety at position 28 [achyranthosides B (3), C (4) and D (5)] were the major constituents, the saponins without sugar moiety at position 28 [betavulgarosides II (10) and IV (11)] were the major constituents, and mixtures of these saponins. In a decoction prepared from the sample which con- tained 10 and 11 as the major saponins, their amounts were largely decreased compared with those of the water extract. As large amounts of these saponins were found in the precipitates formed by heating of the water extract, these saponins were seemed to precipitate out under heating. When hot water was used for the extraction, 3, 4 and 5 were detected even from the samples whose water extract did not contain these saponins. This was attributed to inhibition of endogenous esterase which hydrolyzes the ester linkage at position 28. When saponins were extracted with reagent grade 1-butanol, in addition to the decrease of the amounts of highly polar saponins, oxidative decarboxylation of 3 and 10 occurred resulting in formation of achyranthoside E (6) and spinacoside D (12), respectively. As these changes were not observed with HPLC grade 1-butanol, which contain not more than 5 ppm of peroxide impurities, the change was attributable to the peroxide impurities contained in the reagent grade 1-butanol. Keywords Achyranthes root · Saponin composition · Achyranthoside · Betavulgaroside · Achyranthes bidentata · LC–MS Introduction Achyranthes root is a crude drug used as diuretic, tonic, and remedy for blood stasis. In the Japanese Pharmaco- poeia, the origin of this crude drug is defned as the root of Achyranthes fauriei Leveillé and Vaniot or A. bidentata Blume (Amaranthaceae) [1]. From the methanol extract of A. fauriei, Ida et al. isolated oleanane saponins, named achyranthosides (ASs) A–H as their methyl esters, which have characteristic dicarboxylic acids bound through an acetal linkage to the glucuronic acid moiety at the 3-posi- tion of oleanolic acid [2–6] (Fig. 1). Achyranthosides B (3), C (4) and D (5) were also isolated from sugar beet (Beta vulgaris) and named betavulgarosides (BVSs) I, III and V, respectively, by Yoshikawa et al. [7–9]. Achyranthoside E (6) was also isolated from spinach (Spinacia oleracea) and named spinacoside C by Yoshikawa’s group [10]. Achyran- thosides B (3) and D (5) were also isolated from A. bidentata and named bidentatosides I and II, respectively [11, 12]. We isolated sulfated achyranthosides named sulfachyran- thosides (SASs) B (8) and D (9) from a water extract of Achyranthes root [13]. These saponins having characteristic dicarboxylic acid moiety show extensive peak broadening under ion-suppression conditions of reversed phase HPLC. In the previous paper, we reported an LC–MS condition with a volatile ion-pair reagent for simultaneous analysis of the saponins of Achyranthes root [14]. Under this con- dition, the retention time is dependent on the number of carboxylic acids. Saponins with more carboxylic acids have larger retention times, i.e. saponins with a free carboxylic acid (without a sugar moiety) at position 28 of oleanolic acid (group II saponins: 7, 10, 11) moved slower than those * Fumiyuki Kiuchi kiuchi-fm@pha.keio.ac.jp 1 Faculty of Pharmacy, Keio University, 1-5-30 Shibakoen, Minato-ku, Tokyo 105-8512, Japan 2 Research Center for Medicinal Plant Resources, National Institutes of Biomedical Innovation, Health and Nutrition, 1-2 Hachimandai, Tsukuba, Ibaraki 305-0843, Japan