Isolation of new indole alkaloid triglucoside from the aqueous extract of Uncaria rhynchophylla

Uncaria rhynchophylla (Miq.) Miq. (Rubiaceae), an evergreen liana growing in warm climates and distributed in the southern part of the Boso peninsula in Japan and in central and southern China, is widely used as a botanical raw material for traditional Japanese and Chinese medicines. For example, a Kampo formula consisting of U. rhynchophylla hooks (Yokukansan) is used to treat neurosis, anxiety, night-time crying, and the behavioral and psychological symptoms of dementia [1]. Although the compounds contained in these hooks (alkaloids, triterpenes, phenolic acids, and flavonoids) have been extensively profiled for many decades, novel constituents continue to be discovered [2‐6]. Given that the constant quality of traditional Japanese medicines can only be achieved when the quality of the corresponding botanical raw materials is secured, the identification of previously unreported U. rhynchophylla constituents is a matter of high practical significance. Herein, we profiled the aqueous extract of U. rhynchophylla hooks and and isolated one new alkaloid (1), nine known alkaloids (2‐10), and thirteen known nonalkaloids (11‐23) (Figs. 1 and 2). The structures of known compounds were determined by spectroscopic analysis (high-resolution electrospray mass spectrometry (HR-ESI–MS) and NMR spectroscopy). The absolute configuration of 1 was determined as rhynchophylloside L 11-O-β-D-glucopyranoside by single-crystal X-ray diffractometry (SC-XRD) using a micro-drop crystallization technique, the validity of which was confirmed by application to known compounds, namely rhynchophylloside G (2) [5] and vincosamide 11-O-β-D-glucopyranoside (3) [7].

U. rhynchophylla hooks (14.25 kg) were percolated with 60% (v/v) aqueous MeOH, and the aqueous phase obtained after the evaporation of MeOH was extracted with CHCl₃ to remove hydrophobic constituents and fractionated by column chromatography (DIAION HP-20, silica gel, reversed-phase C18, Sephadex LH-20, and MCI gel CHP-20P columns) to isolate one novel alkaloid (1, pale-brown powder, 68 mg), nine known alkaloids (rhynchophylloside G (2) [5], vincosamide 11-O-β-D-glucopyranoside (3) [7], vincosamide (4) [8], strictosamide (5) [9], 5b-carboxystrictosidine (6) [10], strictosidine (7) [11], cadambine (8) [11], 3a-dihydrocadambine (9) [12], and (E)-vallesiachotamine (10) [13]), and thirteen known non-alkaloids (8-epiloganic acid (11) [14], chlorogenic acid (12) [15], erigeside C (13) [16], (1S,2R,3S)-lyoniresinol-3a-O-β-glucoside (14) [17], (1R,2S,3R)-lyoniresinol-3a-O-β-glucoside (15) [17], ( +)-catechin (16) [18], (−) -epicatechin (17) [18], hyperin (18) [19], rutin (19) [20], procyanidin B1 (20) [21], procyanidin B2 (21) [21], procyanidin B3 (22) [21], and procyanidin B4 (23) [21]) (Figs. 1 and 2).

Compound 1 was obtained as a pale-brown powder and its molecular formula was estimated as C₃₈H₄₈N₂O₂₀ by high resolution electrospray ionization mass spectrometry (HR-ESI–MS) at m/z 875.2698 [M + Na]⁺ (calculated for C₃₈H₄₈N₂O₂₀Na, 875.2693). The ¹H, ¹³C and DEPT NMR spectra (Table 1, Figs. S4, S5) as well as HSQC spectra (Fig. S6) implied the presence of nineteen sp³ methines, four sp² methines, five sp³ methylenes, six sp² quaternary carbons, one vinyl group [δ_C 133.2 (C-19); δ_H 5.47 (1H, dt, J = 17.1, 8.6 Hz, H-19), δ_C 120.6 (C-18); δ_H 5.21 (1H, dd, J = 8.6, 1.9 Hz, H-18), and δ_H 5.33 (1H, dd, J = 17.1, 1.9 Hz, H-18)], two carbonyl groups [δ_C 162.1(C-22) and 172.9 (C-7)] and three anomeric protons [δ_H 4.43 (1H, d, J = 7.6 Hz, H-1′′), 4.69 (1H, d, J = 7.6 Hz, H-1′), 5.00 (1H, d, J = 7.3 Hz, H-1′′′)].

Chemical shift values in the ¹H and ¹³C NMR spectra of 1 well resembled those of rhynchophylloside L, except for positions 10 and 12 (Table 1 and Fig. 3), while the estimated molecular formula of 1 (C₃₈H₄₈N₂O₂₀) corresponded to an addition of C₆H₁₀O₅ to rhynchophylloside L (C₃₂H₃₈N₂O₁₅). The COSY and HSQC-TOCSY correlations also supported the presence of an additional spin system in 1 corresponding to an aldohexose moiety (Figs. 4, S8 and S9). The HMBC spectrum displayed long-range ¹H-¹³C correlations from H-1′ to C-21, H-1′′ to C-2′, and from H-1′′′ to C-11, suggesting that compound 1 shared the same disaccharide chain with rhynchophylloside L at position 21 while the additional sugar is attached to the hydroxy group at C-11 (Figs. 4 and S7). However, the sugar species and their locations in 1 could not be confirmed by the extensive NMR analysis. The nine known alkaloids 2–10 and the thirteen known non alkaloids 11–23 were identified by comparing their ¹H and ¹³C NMR spectra and MS data with those reported previously.

The absolute configurations of 1, 2, 3, 4, 8, and 10 were determined by SC-XRD (Cu K_α radiation) with a Flack parameter (Fig. 5). Given that the initial recrystallization of 1 (1 mg) from water (0.1 mL) afforded very small single crystals (Fig. S14), we reduced the volume of water and obtained better crystals using a micro-drop technique. Specifically, after compound 1 (50 µg) was charged at the center of a V-shaped vial, water (4 µL) was added, and the vial was capped. Use of V-shaped vials is preferred to keep the droplets spherical. The vial was heated to obtain a homogenous solution, with suitable single crystals of 1 subsequently obtained within a day (Fig. S15). This method was applied to grow single crystals of 2 and 3 as well as different solvent systems because absolute configurations of these compounds had been deduced from NMR and CD spectroscopic analyses in the previous reports [5, 7] while their three-dimensional structures remained elusive. The micro-drop crystallization technique allowed us to obtain the first crystallographic data of 2 and 3 only using 50 µg of materials and confirm their proposed stereochemistry were correct.

The micro-drop crystallization method developed in this study forms single crystals without oil; thus, crystallization studies are repeatable with various solvent systems in a single vial and compounds are easily recoverable after the structural analysis. Whereas state-of-the-art techniques such as the nanodrop crystallization method provides higher quality of crystals from smaller amount of molecules [22], our method is sufficient to elucidate the three-dimensional structures of rare natural products at a laboratory scale only using < 100 µg materials and a conventional XRD device, and thus does not require any expensive handling robot or specific environment.

The potential pharmacological activities of U. rhynchophylla extracts to treat Alzheimer’s disease are relevant to its inhibition of acetylcholine esterase (AChE) activities in the brain [23]. Monoterpenoid indole alkaloids such as geissoschizine methyl ether N-oxide and geissoschizine methyl ether in this plant exhibit AChE inhibitory activities [24, 25]. We examined in vitro AChE inhibitory activities of all isolated compounds, but no compound showed any inhibitory activity at 200 µM. This result is comparable with the previous report that the glycosylated derivatives of such indole alkaloids rarely show AChE inhibitory activities [5].

One new indole alkaloid triglucoside (1), nine known alkaloids (2–10), and thirteen known nonalkaloids (11–23) were isolated from the aqueous extract of U. rhynchophylla hooks. The typically difficult-to-obtain single crystals of alkaloid glucosides were prepared using a micro-drop crystallization method. Given the importance of knowing absolute configurations for medicinal applications, we further plan to investigate whether this method can be applied to other compounds reluctantly forming single crystals.