Field Survey of Glycyrrhiza Plants in Central Asia (5). Chemical Characterization of G. bucharica Collected in Tajikistan

Abstract

One triterpene and five triterpene glycosides, including four new compounds, have been identified in the underground parts of Glycyrrhiza bucharica, which was shown to be closely related to Glycyrrhizin-producing Glycyrrhiza species, G. uralensis, G. glabra and G. inflata, based on their chloroplast rbcL sequences. Two known compounds were identified squasapogenol and macedonoside C. The structures of four new compounds, bucharosides A, B, C, and D, were determined to be 3-O-α-L-rhamnopyranosyl-(1→2)-β-D-glucuronopyranosyl-(1→2)-β-D-glucuronopyranosyl-22-O-α-L-rhamnopyranosyl squasapogenol, 3-O-α-L-rhamnopyranosyl-(1→2)-β-D-glucuronopyranosyl-(1→2)-β-D-glucuronopyranosyl-macedonic acid, 3-O-α-L-rhamnopyranosyl-(1→2)-β-D-glucuronopyranosyl-(1→2)-β-D-glucuronopyranosyl-squasapogenol, and 22-O-α-L-rhamnopyranosyl squasapogenol, respectively. Contents of these triterpene glycosides were less than 0.5% of dry weight, and no main saponin, like glycyrrhizin or macedonoside C found in other Glycyrrhiza species, was found in the underground parts of G. bucharica.

Introduction

Licorice, the roots and stolons of Glycyrrhiza plants (Fabaceae), is one of the most important crude drugs in the world. Its main triterpene glycoside, glycyrrhizin, is used as a natural sweetener and a pharmaceutical agent because of its anti-inflammatory and hepatoprotective properties.¹⁾ Three Glycyrrhiza species, G. glabra L., G. uralensis FISCH., and G. inflata BATAL., are the major glycyrrhizin-producing species in the world, and other Glycyrrhiza species, such as G. macedonica BOISS. et ORPH, G. echinata L., G. pallidiflora MAXIM., G. lepidota (NUTT.) PURSH, and G. yunnanensis S. H. CHENG et L. K. DAI, produce other triterpene saponins as major constituents.^2–5)

Tajikistan, a landlocked country in Central Asia, is one of the world’s main habitats of Glycyrrhiza species. Six Glycyrrhiza species (G. glabra, G. uralensis, G. aspera PALL., G. kulabensis T. MASL., G. gontscharovii T. MASL., and G. bucharica RGL.) are distributed in Tajikistan.⁶⁾ In our previous report, two Glycyrrhiza species, G. glabra and G. bucharica, were collected in field surveys in Tajikistan.⁷⁾ G. bucharica is an endemic plant of Tajikistan⁶⁾ and is alternatively classified into genus Meristotropis (Meristotropis bucharica).⁸⁾ G. bucharica is also known to hybridize with G. glabra,⁸⁾ and G. gontscharovii, another endemic Glycyrrhiza species in Tajikistan, is suggested to be a hybrid between G. glabra and G. bucharica.⁶⁾ Notably, the phylogenetic tree constructed from the chloroplast rbcL sequences indicates that G. bucharica is closely related to G. uralensis and G. inflata mentioned in our previous report.⁷⁾ However, HPLC analysis indicated that glycyrrhizin was not detected in the underground parts of G. bucharica. Although some triterpenes were reported from G. bucharica,⁹⁾ triterpene glycoside has not yet been found in this plant. It is of interest to characterize triterpene glycosides of G. bucharica from a viewpoint of the evolution of glycyrrhizin biosynthesis. Thus, the characterization of triterpene saponins from the underground parts of G. bucharica was examined in the present study.

Results and Discussion

Isolation of Triterpene and Triterpene Glycosides from Underground Parts of Glycyrrhiza bucharica Collected in Tajikistan

Roots and stolons of G. bucharica were collected at two collection sites in Tajikistan (Fig. 1). These roots and stolons were extracted with ethyl acetate. The dried residue of ethyl acetate extraction was further extracted with acetonitrile–water (2 : 8). The ethyl acetate soluble fraction was subjected to a series of reverse-phase column and silica gel column chromatography to afford a known triterpene, squasapogenol (1, 20 mg),^10,11) and a new triterpene glycoside 2 (36 mg). The extract with acetonitrile–water (2 : 8) was subjected to another series of reverse-phase column and Sephadex LH20 column chromatography to isolate a known triterpene glycoside, macedonoside C (3, 26 mg),²⁾ and three new triterpene glycosides 4 (68 mg), 5 (34 mg), and 6 (14 mg).

Fig. 1. Collection Sites of Glycyrrhiza bucharica in Tajikistan

1: Dangara (38°19′38″N, 69°25′31″E); 2: Khuroson (38°11′17″N, 68°39′30″E). Solid lines in the map indicate the route of field surveys in Tajikistan.

Compound 2 was obtained as a colorless amorphous powder and showed an ion [M + HCOO]⁻ at m/z 631.4175 in high-resolution electrospray ionization (HR-ESI)-MS, which corresponds to the molecular formula C₃₆H₅₈O₆. Acid hydrolysis of compound 2 with water containing 4 M trifluoroacetic acid yielded L-rhamnose. The sugar configuration was determined using a previously described method.¹²⁾ The ¹H- and ¹³C-NMR spectra were similar to those of squasapogenol (1),¹¹⁾ except compound 2 had a rhamnopyranosyl moiety (Table 1). Its connectivity with aglycone in compound 2 was confirmed by a heteronuclear multiple bond connectivity (HMBC) experiment. Long-range correlations were observed between the signals of H-1″″ of L-rhamnose and C-22 of squasapogenol, and of H-22 of squasapogenol and C-1″″ of L-rhamnose. The anomeric configuration of L-rhamnose was determined as α from the ¹³C–¹H coupling constant (167 Hz) of the anomeric carbon signal.¹³⁾ Therefore, the structure of compound 2, called bucharoside D, was determined to be 22-O-α-L-rhamnopyranosyl–squasapogenol (Fig. 2).

Table 1. ¹³C-NMR Data for Bucharosides A (4), B (5), C (6), D (2). (in Pyridine-d₅)

	4	5	6	2		4	5	6	2
1	38.1	38.1	38.1	38.4	GlcUA 1′	105.3	105.3	105.3
2	26.5	26.5	26.5	28.0	2′	79.3	79.3	79.4
3	90.1	90.1	90.1	78.0	3′	78.8	78.8	78.8
4	39.8	39.8	39.8	39.5	4′	73.6	73.6	73.6
5	55.4	55.4	55.5	55.3	5′	77.5	77.6	77.5
6	18.6	18.5	18.6	18.9	6′	172.7	172.3	172.6
7	32.6	32.5	32.7	32.7	GlcUA 1″	102.8	102.8	102.8
8	40.6	40.7	41.1	40.7	2″	78.3	78.2	78.2
9	54.4	54.6	54.5	54.6	3″	78.9	79.0	78.9
10	36.6	36.6	36.6	37.0	4″	73.6	73.6	73.6
11	126.2	126.5	126.5	126.3	5″	77.5	77.6	77.6
12	127.0	126.7	126.4	127.0	6″	172.3	172.8	172.2
13	136.0	134.5	135.6	136.0	Rha 1‴	102.3	102.3	102.3
14	42.2	42.5	42.4	42.2	2‴	72.4	72.4	72.4
15	24.4	24.6	24.6	24.4	3‴	72.7	72.8	72.8
16	33.7	36.2	33.8	33.8	4‴	74.4	74.4	74.4
17	40.2	36.8	40.7	40.2	5‴	69.6	69.7	69.7
18	136.5	135.2	137.5	136.7	6‴	19.0	19.1	19.0
19	37.7	35.2	37.8	37.7	Rha 1″″	98.4			98.4
20	32.0	49.8	32.3	32.0	2″″	73.2			73.3
21	39.4	73.3	44.9	39.5	3″″	73.1			73.1
22	81.3	49.5	76.6	81.4	4″″	73.9			73.9
23	28.2	28.2	28.2	28.5	5″″	70.5			70.5
24	16.4	16.3	16.4	16.1	6″″	18.6			18.6
25	18.2	18.2	18.2	18.4
26	16.7	16.8	16.8	16.8
27	20.2	20.3	20.3	20.3
28	19.3	26.3	18.6	19.3
29	25.0	179.5	25.5	25.0
30	32.4	25.1	32.6	32.4

Fig. 2. Structures of Triterpene and Triterpene Glycosides Isolated from the Underground Parts of Glycyrrhiza bucharica Collected in Tajikistan

Compound 4 was obtained as a colorless amorphous powder and showed an ion [M–H]⁻ at m/z 1083.5380 in HR-ESI-MS, which corresponds to the molecular formula C₅₄H₈₄O₂₂. Acid hydrolysis of compound 4 with water containing 4 M trifluoroacetic acid yielded L-rhamnose and D-glucuronolactone. The sugar configuration was determined using a previously described method.¹²⁾ The ¹H- and ¹³C-NMR spectra were similar to those of bucharoside D (2), except that compound 4 had two rhamnopyranosyl moieties and two glucuronopyranosyl moieties (Table 1). The oligoglycoside structure and its connectivity with aglycone in compound 4 were confirmed by an HMBC experiment. Long-range correlations were observed between the signals of H-1′ and C-3, H-1″ and C-2′, H-1‴ and C-2″, and H-1″″ and C-22. The anomeric configuration was determined as β for two D-glucuronic acids from the coupling constants of anomeric proton signals. The anomeric configuration of L-rhamnose was determined as α from the ¹³C–¹H coupling constants of the C-1‴ signal (176 Hz) and C-1″″ signal (169 Hz).¹³⁾ Therefore, the structure of compound 4, called bucharoside A, was determined to be 3-O-α-L-rhamnopyranosyl-(1→2)-β-D-glucuronopyranosyl-(1→2)-β-D-glucuronopyranosyl-22-O-α-L-rhamnopyranosyl-squasapogenol (Fig. 2).

Compound 5 was obtained as a colorless amorphous powder and showed an ion [M–H]⁻ at m/z 967.4510 in HR-ESI-MS, which corresponds to the molecular formula C₄₈H₇₂O₂₀. Acid hydrolysis of compound 5 with water containing 4 M trifluoroacetic acid yielded L-rhamnose and D-glucuronolactone. The sugar configuration was determined using a previously described method.¹²⁾ The ¹H- and ¹³C-NMR spectra were similar to those of macedonoside C (2),²⁾ except that compound 5 had a rhamnopyranosyl moiety (Table 1). The oligoglycoside structure and its connectivity with aglycone in compound 5 were confirmed by an HMBC experiment. Long-range correlations were observed between the signals of H-1′ and C-3, H-1″ and C-2′, and H-1‴ and C-2″. The anomeric configuration was determined as β for two D-glucuronic acids from the coupling constants of anomeric proton signals. The anomeric configuration of L-rhamnose was determined as α from the ¹³C–¹H coupling constant (173 Hz) of the anomeric carbon signal.¹³⁾ Therefore, the structure of compound 5, called bucharoside B, was determined to be 3-O-α-L-rhamnopyranosyl-(1→2)-β-D-glucuronopyranosyl-(1→2)-β-D-glucuronopyranosyl-macedonic acid (Fig. 2).

Compound 6 was obtained as a colorless amorphous powder and showed an ion [M–H]⁻ at m/z 937.4754 in HR-ESI-MS, which corresponds to the molecular formula C₄₈H₇₄O₁₈. Acid hydrolysis of compound 6 with water containing 4 M trifluoroacetic acid yielded L-rhamnose and D-glucuronolactone. The sugar configuration was determined using a previously described method.¹²⁾ The ¹H- and ¹³C-NMR spectra were similar to those of bucharoside A (4), except that compound 6 had only one rhamnopyranosyl moiety (Table 1). The oligoglycoside structure and its connectivity with aglycone in compound 6 were confirmed by an HMBC experiment. Long-range correlations were observed between the signals of H-1′ and C-3, H-1″ and C-2′, and H-1‴ and C-2″. The anomeric configuration was determined as β for two D-glucuronic acids from the coupling constants of anomeric proton signals. The anomeric configuration of L-rhamnose was determined as α from the ¹³C–¹H coupling constant (173 Hz) of the anomeric carbon signal.¹³⁾ Therefore, the structure of compound 6, called bucharoside C, was determined to be 3-O-α-L-rhamnopyranosyl-(1→2)-β-D-glucuronopyranosyl-(1→2)-β-D-glucuronopyranosyl-squasapogenol (Fig. 2).

The aglycone of bucharosides A (4), C (6) and D (2) is squasapogenol (1, olean-11(12),13(18)-diene-3β,22β-diol), which was reported from Glycyrrhiza squamulosa and Hedysarum gmelinii.^10,11) The aglycone of bucharoside B (5) and macedonoside C (3) is macedonic acid (olean-11(12),13(18)-diene-3β,21α-diol-29-ic acid), which was isolated from many Glycyrrhiza species as an aglycone of triterpene glycosides.^2,3,9) These olean-11(12),13(18)-diene triterpenes are major aglycones of triterpene glycosides isolated from glycyrrhizin-non-producing Glycyrrhiza species, such as G. macedonica, G. echinata, G. pallidiflora and G. yunnanensis.^2,5,9) Although G. bucharica is closely related to G. uralensis and G. inflata based on their chloroplast rbcL sequences,⁷⁾ the structures of the triterpene glycosides of G. bucharica have been shown to be similar to those of glycyrrhizin-non-producing Glycyrrhiza species by the present study.

HPLC Analysis of Triterpene Glycosides in Underground Parts of G. bucharica

HPLC analysis of roots and stolons of G. bucharica collected at two collection sites was performed to determine the contents of these triterpene glycosides. The content of bucharosides A–D and macedonoside C in the underground parts of G. bucharica was less than 0.5% of dry weight (Table 2). As shown in Fig. 3, there is no single main triterpene glycoside in the underground parts of G. bucharica, like glycyrrhizin or macedonoside C in other Glycyrrhiza species.³⁾ Glycyrrhizin was not detected in the underground parts of G. bucharica.

Table 2. Contents of Bucharosides A (4), B (5), C (6), D (2), and Macedonoside C (3) in the Roots and Stolons of Glycyrrhiza bucharica Collected in Tajikistan

Collection site	Plant number	Root/Stolon	Diameter (mm)	Contents (% of dry weight) of
				4	5	6	2	3
Dangara	13A04	Stolon	9.4–9.7	0.06	0.10	0.01	0.00	0.08
	13A05	Root	11.6–12.1	0.05	0.07	0.01	0.00	0.05
	13A06	Stolon	8.1–9.5	0.22	0.41	0.03	0.00	0.20
Khuroson	13A24	Stolon	18.3–18.6	0.21	0.30	0.08	0.02	0.10
	13A24	Stolon	17.3–19.9	0.40	0.44	0.27	0.03	0.17
	13A24	Stolon	8.4–8.7	0.15	0.18	0.05	0.00	0.09
	13A25	Stolon	15.5–18.1	0.25	0.27	0.14	0.01	0.09
	13A25	Root	18.1–21.5	0.32	0.30	0.16	0.00	0.07

Fig. 3. HPLC Profile of the Underground Parts of G. bucharica (13A24) Collected in Tajikistan

Absorbance at 250 nm. 2, bucharoside D; 3, macedonoside C; 4, bucharoside A; 5, bucharoside B; 6, bucharoside C.

Although the structure of the sugar moiety of glycyrrhizn and macedonoside C was β-D-GlcA-(1→2)-β-D-GlcA, bucharosides A (4), B (5) and C (6) have the sugar moiety α-L-Rha-(1→2)-β-D-GlcA-(1→2)-β-D-GlcA. The same sugar moiety was observed in triterpene glycosides isolated from other Glycyrrhiza species.^14,15) These triterpene glycosides, such as licorice-saponin D3 and rhaoglycyrrhizin, were isolated as minor constituents of G. glabra and G. uralensis.^14,15) In contrast, triterpene glycosides, having the sugar moiety of α-L-Rha-(1→2)-β-D-GlcA-(1→2)-β-D-GlcA, were major constituents of G. bucharica.

Experimental

General Methods

¹H- and ¹³C-NMR spectra were recorded using an ECA500 (JEOL, Tokyo, Japan) spectrometer. Chemical shifts are given on a δ (ppm) scale with tetramethylsilane as an internal standard. TLC aluminum sheets 20 × 20 cm Silica gel 60 F₂₅₄ (Merck, Darmstadt, Germany) were used for TLC analysis. Biotage^® SP system (Biotage AB, Uppsala, Sweden) was used for flash column chromatography. Prominence HPLC system (Shimadzu, Kyoto, Japan) was used for preparative HPLC. Specific rotation was measured on a SEPA-300 (Horiba, Kyoto, Japan) spectrometer. Quantitative HPLC analysis and HR-ESI-MS were measured on a Shimadzu LCMS-IT-TOF (Kyoto, Japan). Acetic acid, ethyl acetate, formic acid, D-glucuronolactone, n-hexane, isopropanol, and n-propanol were of Special Grade; and L-cysteine methyl ester hydrochloride and o-toryl isothiocyanate were of First Class Grade, purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). D-Cysteine methyl ester hydrochloride of First Class Grade was purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). L-Rhamnose of Special Grade was purchased from Nacalai Tesque Inc. (Kyoto, Japan). Acetonitrile for separation was of Special Grade (Wako Pure Chemical Industries, Ltd.), and acetonitrile for LC-MS analysis was of LC-MS Grade (Thermo Fisher Scientific, Waltham, U.S.A.). Pyridine-d₅ was purchased from Cambridge Isotope Laboratories (Andover, U.S.A.).

Plant Materials

The underground parts of Glycyrrhiza bucharica Rgl. (Fabaceae) used in the present study were collected at two collection sites in Tajikistan (Fig. 1). These G. bucharica plants were identified based on phenotypic change of fruits and leaves by Hiroaki Hayashi. All plant specimens used in the present study were deposited into the Herbarium of the Institute of Botany, Plant Physiology and Genetics, Academy of Science of Tajikistan.

Isolation of Triterpene and Triterpene Glycosides

Dried underground parts (240 g) of G. bucarica were extracted twice with 1200 mL of ethyl acetate by ultrasonication for 2 h. The dried ethyl acetate extract (1.8 g) was dissolved in isopropanol–water (3 : 7). The solution was applied on Biotage SNAP cartridge KP-C18-HS 60g column (E1), and eluted with a gradient of isopropanol–water (from 3 : 7 to 10 : 0) in 21-mL fractions (fractions 1–32), repeatedly. Fractions 15–19 (305 mg) of the KP-C18-HS 60g column (E1) were applied on Biotage SNAP Ultra 10g column, and eluted with a gradient of n-hexane-ethyl acetate (10 : 0–0 : 10) in 21-mL fractions (fractions 1–43) to isolate a new triterpene glycoside 2 (36 mg, fractions 35–37), named bucharoside D. Fractions 20–25 (242 mg) of the KP-C18-HS 60g column (E1) were applied on Biotage SNAP Ultra 10g column, and eluted with a gradient of n-hexane–ethyl acetate (10 : 0–0 : 10) in 21-mL fractions (fractions 1–43) to isolate squasapogenol¹¹⁾ (1, 20 mg, fractions 14–16).

Bucharoside D (2): Colorless amorphous powder. ¹H-NMR (pyridine-d₅) δ: 0.81 (3H, s, H-29), 0.86 (3H, s, H-26), 0.96 (3H, s, H-30), 0.99 (3H, s, H-25), 1.07 (3H, s, H-24), 1.10 (3H, s, H-27), 1.17 (3H, s, H-28), 1.27 (3H, s, H-23), 1.72 (3H, d, J = 5.8 Hz, rhamnosyl methyl-6″″), 2.04 (1H, br s, H-9), 2.48 (1H, d, J = 14.3 Hz, H-19α), 3.51 (1H, dd, J = 5.2 Hz, 10.3 Hz, H-3), 3.75 (1H, dd, J = 4.6 Hz, 12.0 Hz, H-22), 5.46 (1H, br s, H-1″″), 5.73 (1H, br d, J = 10.9 Hz, H-11), 6.50 (1H, dd, J = 2.9 Hz, 10.3 Hz, H-12). ¹³C-NMR (pyridine-d₅) δ: Table 1. UV λ_max (MeOH) nm (ε): 242 (9200), 250 (10390), 260 (6730). HR-ESI-MS m/z: 631.4175 (M + HCOO)⁻ (Calcd for C₃₇H₅₉O₈: 631.4210). [α]_D²²−46.3° (c = 0.04, MeOH).

The dried residue of ethyl acetate extraction was further extracted twice with 2 L of acetonitrile–water–formic acid (20 : 80 : 1) at 60°C for 1 h. The extract (3.6 L) was applied on Biotage SNAP cartridge KP-C18-HS 120 g column (A1), and eluted with a gradient of acetonitrile–water containing 0.2% formic acid (from 2 : 8 to 9 : 1) in 21-mL fractions (fractions 1–48). Fractions 20–25 (879 mg) of the KP-C18-HS 120 g column (A1) were dissolved in 50 mL of acetonitrile–water containing 0.2% formic acid (3 : 7). The solution was applied on Sephadex LH-20 column (180 mL), and eluted with acetonitrile–water containing 0.2% formic acid (3 : 7) in 50-mL fractions (fractions 1–25). Fractions 6–8 (340 mg) of the Sephadex LH-20 column were dissolved in 50 mL of acetonitrile–water containing 0.2% formic acid (2 : 8). The solution was applied on Biotage SNAP cartridge KP-C18-HS 120 g column, and eluted with a gradient of acetonitrile–water containing 0.2% formic acid (2 : 8–9 : 1) in 21-mL fractions (fractions 1–48). The fractions 21–22 (170 mg) were dissolved in 34 mL of acetonitrile–water (4 : 6) containing 0.2% formic acid, and were repeatedly chromatographed using preparative HPLC to isolate compounds 4 (68 mg) and 5 (34 mg). The conditions used for preparative HPLC were as follows: column, Inertsil PREP-ODS (20 mm i.d. × 250 mm, GL Sciences, Japan); solvent, 40% acetonitrile–water containing 0.2% formic acid; and flow rate, 5 mL/min; retention time (t_R), 17.4 min (compound 4) and 21.2 min (compound 5). Fractions 12–20 (163 mg) of the Sephadex LH-20 column were dissolved in 50 mL of acetonitrile–water containing 0.2% formic acid (2 : 8). The solution was applied on Biotage SNAP cartridge KP-C18-HS 120 g column, and eluted with a gradient of acetonitrile–water containing 0.2% formic acid (2 : 8–9 : 1) in 21-mL fractions (fractions 1–48). The fractions 22–24 (66 mg) were dissolved in 12 mL of acetonitrile–water (4 : 6) containing 0.2% formic acid, and were repeatedly chromatographed using preparative HPLC, as mentioned above, to isolate macedonoside C²⁾ (3, 26 mg, t_R: 24.8 min).

Bucharoside A (4): Colorless amorphous powder. ¹H-NMR (pyridine-d₅) δ: 0.75 (3H, s, H-26), 0.79 (3H, s, H-29), 0.80 (3H, s, H-25), 0.95 (3H, s, H-30), 1.08 (3H, s, H-27), 1.14 (3H, s, H-28), 1.23 (3H, s, H-24), 1.46 (3H, s, H-23), 1.72 (3H, d, J = 5.8 Hz, rhamnosyl methyl-6″″), 1.85 (3H, d, J = 6.3 Hz, rhamnosyl methyl-6‴), 1.97 (1H, br s, H-9), 2.43 (1H, d, J = 14.3 Hz, H-19α), 3.36 (1H, dd, J = 4.0 Hz, 11.5 Hz, H-3), 3.72 (1H, dd, J = 4.6 Hz, 11.5 Hz, H-22), 5.14 (1H, d, J = 7.5 Hz, H-1′), 5.44 (1H, br s, H-1″″), 5.60 (1H, br d, J = 10.3 Hz, H-11), 5.97 (1H, d, J = 8.0 Hz, H-1″), 6.41 (1H, br d, 8.6 Hz, H-12), 6.46 (1H, br s, H-1‴). ¹³C-NMR (pyridine-d₅) δ: Table 1. UV λ_max (MeOH) nm (ε): 242 (11064), 250 (12393), 260 (8138). HR-ESI-MS m/z: 1083.5380 (M−H)⁻ (Calcd for C₅₄H₈₃O₂₂: 1083.5376). [α]_D²²−28.4° (c = 0.1, MeOH).

Bucharoside B (5): Colorless amorphous powder. ¹H-NMR (pyridine-d₅) δ: 0.74 (3H, s, H-26), 0.80 (3H, s, H-25), 0.98 (3H, s, H-27), 1.21 (3H, s, H-28), 1.23 (3H, s, H-24), 1.46 (3H, s, H-23), 1.69 (3H, s, H-30), 1.86 (3H, d, J = 6.3 Hz, rhamnosyl methyl-6‴), 1.92 (1H, br s, H-9), 3.33 (1H, dd, J = 4.6 Hz, 12.1 Hz, H-3), 3.47 (1H, d, J = 14.9 Hz, H-19α), 4.04 (1H, dd, J = 4.6 Hz, 11.5 Hz, H-21), 5.12 (1H, d, J = 7.5 Hz, H-1′), 5.61 (1H, d, J = 10.3 Hz, H-11), 5.98 (1H, d, J = 7.5 Hz, H-1″), 6.47 (1H, s, H-1‴), 6.89 (1H, dd, J = 2.4 Hz, 10.3 Hz, H-12). ¹³C-NMR (pyridine-d₅) δ: Table 1. UV λ_max (MeOH) nm (ε): 241 (6620), 249 (7280), 259 (5200). HR-ESI-MS m/z: 967.4510 (M−H)⁻ (Calcd for C₄₈H₇₁O₂₀: 967.4539). [α]_D²² −33.0° (c = 0.1, MeOH).

Fractions 26–28 (216 mg) of the KP-C18-HS 120 g column (A1) were dissolved in 50 mL of acetonitrile–water containing 0.2% formic acid (3 : 7). The solution was applied on Sephadex LH-20 column (180 mL), and eluted with acetonitrile–water containing 0.2% formic acid (3 : 7) in 50-mL fractions (fractions 1–25). Fractions 14–17 (59 mg) of the Sephadex LH-20 column were dissolved in 12 mL of acetonitrile–water (4 : 6) containing 0.2% formic acid. The solution was repeatedly chromatographed using preparative HPLC, as mentioned above, to isolate a compound 6 (14 mg, t_R: 44 min).

Bucharoside C (6): Colorless amorphous powder. ¹H-NMR (pyridine-d₅) δ: 0.78 (3H, s, H-26), 0.82 (3H, s, H-25), 0.90 (3H, s, H-29), 1.02 (3H, s, H-30), 1.08 (3H, s, H-27), 1.24 (3H, s, H-24), 1.33 (3H, s, H-28), 1.47 (3H, s, H-23), 1.85 (3H, d, J = 6.5 Hz, rhamnosyl methyl-6‴), 1.98 (1H, br s, H-9), 2.48 (1H, d, J = 14.2 Hz, H-19α), 3.35 (1H, dd, J = 4.0 Hz, 11.5 Hz, H-3), 3.80 (1H, dd, J = 5.0 Hz, 12.0 Hz, H-22), 5.13 (1H, d, J = 7.3 Hz, H-1′), 5.60 (1H, br d, J = 10.7 Hz, H-11), 5.97 (1H, d, J = 7.7 Hz, H-1″), 6.46 (1H, br s, H-1‴), 6.47 (1H, dd, J = 2.0 Hz, 10.7 Hz, H-12). ¹³C-NMR (pyridine-d₅) δ: Table 1. UV λ_max (MeOH) nm (ε): 242 (7060), 250 (7830), 260 (5400). HR-ESI-MS m/z: 937.4754 (M−H)⁻ (Calcd for C₄₈H₇₃O₁₈: 937.4797). [α]_D²² −34.1° (c = 0.04, MeOH).

Acid Hydrolysis of Saponins

Each saponin (1 mg) was dissolved in 1 mL of water containing 4 M trifluoroacetic acid, and the solution was refluxed for 2 h. After the reaction mixture was cooled, it was dried in vacuo. The residue was dissolved in 50% acetonitrile–water, and sugars were analyzed by TLC (n-propanol–water–acetic acid, 170 : 30 : 5). The spots were detected by spraying of 50% H₂SO₄ followed by heating.

Determination of Sugar Configuration

The sugar configuration was determined using a modified method described previously.¹²⁾ Each saponin (1 mg) was dissolved in 1 mL of water containing 4 M trifluoroacetic acid, and the solution was refluxed for 2 h. After cooling the reaction mixture, it was dried in vacuo. The residue was dissolved in 0.2 mL of pyridine containing L-cysteine methyl ester hydrochloride (1 mg) and heated at 60°C for 1 h, following which 0.2 mL of pyridine containing o-torylisothiocyanate (1 mg) was added to the mixture and heated further at 60°C for 1 h. The reaction mixture (2 µL) was directly analyzed by HPLC; column: Inertsil ODS-SP (3 µm, 2.1 mm i.d. × 250 mm, GL Sciences, Japan); solvent: 25% acetonitrile–water containing 0.1% formic acid (isocratic elution); column temp.: 40°C; detector: UV absorption at 250 nm. The peaks were compared with those of derivatives prepared from D-glucuronolactone and L-rhamnose. The t_R of L-glucuronolactone, obtained by reaction of D-glucuronolactone with D-cysteine methyl ester hydrochloride, was different from that of D-glucuronolactone.

HPLC Analysis of Underground Parts

Dried underground parts were powdered with a mortar and pestle, and 50 mg of each powdered sample was extracted with 5 mL of 80% methanol at 60°C for 2 h. An aliquot (2 µL) of the extract was analyzed by LC-MS; column: Inertsil ODS-3 (3 µm, 2.1 mm i.d. × 250 mm, GL Sciences, Japan); solvent: gradient of H₂O containing 0.1% formic acid (solvent A) and acetonitrile containing 0.1% formic acid (solvent B), 0 min, 40% B, 25 min 90% B, 40 min 90% B, 41 min 40% B, 70 min 40% B; flow rate: 0.1 mL/min; column temp.: 40°C; detector: photodiode array detector. The quantities of the constituents were determined on the basis of their peak area of UV absorption at 250 nm. Each constituent was identified by comparing its retention time, UV spectrum, and HRESI-MS with a respective authentic sample.

Acknowledgments

This research was supported by a Japan International Cooperation Agency (JICA) & Japan Society for the Promotion of Science (JSPS) Program, Dispatch of Science and Technology Researchers. This work was also supported in part by a Grant-in-Aid for Scientific Research (Grant Number JP15K07999) from the Ministry of Education, Culture, Sports, Science and Technology of Japan. We are grateful to Prof. Hikmat Hisoriev for depositing the plant specimens in the Herbarium. We are also grateful to Ms. Musavvara Shukurova, Ms. Zumrad Sharopova, Mr. Shoh Sharipov, and Mr. Mirzokarim Sobirov for their assistance in the field surveys.

Conflict of Interest

The authors declare no conflict of interest.

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