Phytochemical Study of Aerial Parts of Leea asiatica.

Leea asiatica (L.) Ridsdale (Leeaceae) is found in tropical and subtropical countries and has historically been used as a traditional medicine in local healthcare systems. Although L. asiatica extracts have been found to possess anthelmintic and antioxidant-related nephroprotective and hepatoprotective effects, little attention has been paid toward the investigation of phytochemical constituents of this plant. In the current study, phytochemical analysis of isolates from L. asiatica led to the identification of 24 compounds, including a novel phenolic glucoside, seven triterpenoids, eight flavonoids, two phenolic glycosides, four diglycosidic compounds, and two miscellaneous compounds. The phytochemical structures of the isolates from L. asiatica were elucidated using spectroscopic analyses including 1D- and 2D-NMR and ESI-Q-TOF-MS. The presence of triterpenoids and flavonoids supports the evidence for anthelmintic and antioxidative effects of L. asiatica.

1. Introduction

Plant species of the genus Leeaceae (Leea) are widely distributed among tropical and subtropical regions, including Eastern Africa, Australia, China, India, Indonesia, Laos, Malaysia, Myanmar, and Thailand [1,2]. Around 70 different species of the genus Leea (L.) are found in these regions, with some being used in local traditional healthcare systems to treat bone fractures, liver disorders, physical wounds, skin diseases, and worm infections [1]. Various biological studies have reported the antibacterial, antioxidative, anthelmintic, cytotoxic, hepatoprotective, and nephroprotective effects of the Leea species. Several phytochemicals, including coumarin, essential oils, flavonoids, hydrocarbons, and triterpenoids have been determined from the extracts of diverse Leea species, such as L. thorelii [1,3], L. indica [4,5,6,7,8,9], L. macrophylla [10,11], and L. guineense [12,13]. Although several Leea species have been evaluated from both a biological and phytochemical perspective, relatively few studies have included L. asiatica (L.) Ridsdale in their analyses. Leea asiatica has also been used as a folk medicine, especially in India, to treat worm infection, bone fracture, liver disorder, and oxidative stress-related diseases. Up to date, three published reports have described the biological effects of L. asiatica extracts, including anthelmintic and antioxidant-related nephroprotective and hepatoprotective activities [14,15,16], and no phytochemical study on this species has been attempted yet even though L. asiatica has been used as a traditional medicine, as have other Leea species. The identification of the phytochemicals of herbal medicine is very important because it can be used as a primary data for the prediction of biological effects, safety information, and clarification of medicinal use. From this point of view, the current study was designed to identify chemical constituents of L. asiatica and to provide their spectroscopic information. We describe here a novel compound (1) along with seven triterpenoids (2–8), eight flavonoids (9–16), two phenolic gluocosides (17–18), four diglycosidic compounds (19–22), and two miscellaneous compounds (23–24) (Figure 1).

Figure 1

The chemical structures of compounds 1–24 from the aerial parts of Leea asiatica.

2. Results and Discussion

2.1. Elucidation of Chemical Structures of Compounds –

The molecular formula of compound 1 was identified as C30H34O14, according to its ESI-Q-TOF-MS spectrum, which showed the quasi-molecular ion peak at m/z 617.1884 [M − H]− −(calcd. for C30H33O14, 617.1870). The 1H-NMR spectrum of compound 1 showed resonances characteristic for a 1,4-disubstituted benzene ring [δH 6.91 (2H, d, J = 9.0 Hz, H-2, 6), 6.56 (2H, d, J = 9.0 Hz, H-3, 5)], an anomeric proton of a sugar group at δH 4.73 (1H, d, J = 7.4 Hz, H-1′), two sets of 1,3,4-trisubstitued benzene moieties [δH 7.57 (1H, dd, J = 8.5, 2.0 Hz, H-6′′), 7.51 (1H, d, J = 2.0 Hz, H-2′′), and 7.06 (1H, d, J = 8.5 Hz, H-5′′); δH 7.07 (1H, o, H-2′′′), 6.87 (1H, dd, J = 8.1, 2.2 Hz, H-6′′′), and 6.71 (1H, d, J = 8.1 Hz, H-5′′′)], two oxygenated methine signals at δH 4.84 (1H, o, H-7′′′) and 4.64 (1H, m, H-8′′′), a hydroxylated methylene resonance at δH 3.86 (2H, brd, J = 4.9 Hz, H-9′′′), and two methoxy signals [δH 3.79 (3H, s, 3′′-OCH) and 3.80 (3H, s, 3′′′-OCH)]. Exhaustive 1D- and 2D-NMR (1H-1H COSY, HSQC, and HMBC) interpretations suggested that compound 1 contained a guaiacylglycerol 8-yl, a 3-methoxy-4-hydroxyphenoxy, a 4-hydroxy-benzene, and a β-glucopyranosyl moiety, as shown in Figure 1. The sugar unit was identified as D-glucose by acid hydrolysis. The connectivity of each functional group was established using the HMBC spectrum. Crossed peaks were observed at δH 4.73 (H-1′)/ δC 152.3 (C-1), δH 4.68, 4.37 (H-6′a and H-6′b)/ δC 167.7 (C-7′′), and δH 4.64 (H-8′′′)/ δC 154.2 (C-4′′) (Figure 2a). The 7′′′,8′′′-erythro configuration of compound 1 was deduced from the ΔδC8′′′- C7′′′ value (11.0 ppm), and a negative Cotton effect at 229 nm in its circular dichroism (CD) spectrum indicated the 8R configuration, according to the findings of previous reports (Figure 2b) [17,18]. Based on the spectroscopic evidence (Supplementary Information SI 7–SI 8), and searching the confirmed structure of compound 1 through Scifinder® and Reaxys® databases, compound 1 was identified as a new compound with a chemical structure of 4-hydroxyphenol-β-D-{6-O-[4-O-(7S,8R-guaiacylglycerol-8-yl)-3-methoxybenzoyl]}-β-D-glucopyranoside. The molecular structure of 1 was very similar to that of (−)-4-hydroxy-3-methoxyphenol β-D-{6-O-[4-O-(7S,8R)-(4-hydroxy-3-methoxyphenylglycerol-8-yl)-3-methoxybenzoyl]}-glucopyranoside, which was determined in a previous phytochemical study [16]. The comparison of 1H- and 13C-NMR data of both compounds was described in Supplementary Information SI 8.

Figure 2

(a) 1H-1H COSY and HMBC correlation of compound 1; (b) circular dichroism (CD) spectrum of compound 1.

The following known compounds were also identified: Oleanolic acid (2) [19], ursolic acid (3) [19], maslinic acid (4) [20,21], chebuloside ii (5) [22,23], corosolic acid (6) [21], hederagenin-3-O-arabinopyranoside (7) [24], oleanolic acid 3-O-glucopyranosyl-(1→2)-arabinopyranoside (8) [25], (+)-catechin (9) [26], (−)-epicatechin (10) [26], (−)-epiafzelechin (11) [27], juglanin (12) [28], mearnsetin 3-O-rhamnopyranoside (13) [29], myricitrin (14) [30], afzelin (15) [30], quercitrin (16) [30], 4-hydroxyphenol-[6-O-(4′′-hydroxy-3′′,5′′-dimethoxy-benzoate)]-β-d-glucopyranoside (17) [31], breynioside A (18) [32], phenylethyl-rutinoside (19) [33], icariside D1 (20) [34], hexenyl-rutinoside (21) [35], everlastoside C (22) [36], bergenin (23) [37], and citroside A (24) [38]. To the best of our knowledge, compounds 2 [9] (L. macrophylla), 3 (L. indica) [5], 16 [3,5,9] (L. indica and L. thorelii), 9, 10, 14, 16, and 24 (L. inidca and L. thorelii) [3] have been previously isolated from the several other Leea species, whereas compounds 4–8, 11–13, 15, and 17–22 were here isolated from the Leea species, especially from L. asiatica, for the first time. However, the phytochemical profile of the plant is mainly affected by the genetic and environmental factors (e.g., soil, climate conditions, and seasonality) during plant growth, extraction, and isolation method. This phytochemical study does not represent all chemical profiles of L. asiatica. Therefore, further phytochemical study on L. asiatica from different regions should be performed to achieve a full understanding of the chemical composition of L. asiatica.

As mentioned in the Introduction, L. asiatica possessed anthelmintic [14] and antioxidative activity-related nephroprotective and hepatoprotective effects [15,16]. The current study demonstrated the presence of triterpenoids, including oleanolic acid, ursolic acid, the representative pentacyclic triterpenes, and their derivatives (1–8). The anthelmintic activity of L. asiatica is thought to be exerted by triterpenoids because several reports demonstrated that oleanolic acid and ursolic acid possessed anthelmintic activity [39,40], and saponins were also associated with the ovicidal action of the plant extract [41]. Thus, this study supports the evidence for clarifying the anthelmintic effect of L. asiatica. The antioxidant properties of the L. asiatica extract were well-proven in previous literatures and, especially the ethyl acetate-soluble extract, showed the most potent antioxidative effects [16]. In this study, eight flavonoids (9–16) were determined specifically from the ethyl acetate-soluble extract (Supplementary Information SI 9), which indicated that flavonoids were enriched in an ethyl acetate-soluble extract. Therefore, it is suggested that the antioxidant potency of the ethyl acetate-soluble extract of L. asiatica was directly related to the flavonoid contents because flavonoids such as catechins, quercetin, kaempferol, and myricetin and their derivatives are the most important natural antioxidants and exhibit numerous antioxidant-related beneficial effects [42,43,44,45,46]. Consequently, this study provides reasonable evidence for previous biological effects of L. asiatica as well as its phytochemical profile.

2.2. Characterisation of Compounds –

4-Hydroxyphenol-{6-O-[4-O-(7S,8R-guaiacylglycerol-8-yl)-3-methoxybenzoyl]}-β-D-glucopyra- noside (1): Brown amorphous powder; ESI-Q-TOF-MS: m/z = 617.1884 [M − H]− (calcd. for C30H33O14, 617.1870); [α]22D -4.4 (c = 0.05, MeOH); CD(MeOH) Δε −5.35 (229 nm), −0.3 (252 nm), −0.14 (264 nm), −1.02 (281 nm), 0.05 (298 nm); UV (MeOH) λmax (log ε) 204 (6.15), 219 (5.90), 266 (5.54), 285 (5.46) nm; 1H-NMR (500 MHz, MeOH-d4): δ 7.57 (1H, dd, J = 8.5, 2.0 Hz, H-6′′), 7.51 (1H, d, J = 2.0 Hz, H-2′′), 7.07 (1H, o, H-2′′′), 7.06 (1H, d, J = 8.5 Hz, H-5′′), 6.91 (2H, d, J = 9.0 Hz, H-2, 6), 6.87 (1H, dd, J = 8.1, 2.2 Hz, H-6′′′), 6.71 (1H, d, J = 8.1 Hz, H-5′′′), 6.56 (2H, d, J = 9.0 Hz, H-3, 5), 4.84 (1H, o, H-7′′′), 4.73 (1H, d, J = 7.4 Hz, H-1′), 4.68 (1H, dd, J = 11.7, 2.1 Hz, H-6′a), 4.64 (1H, m, H-8′′′), 4.37 (1H, dd, J = 11.7, 7.6 Hz, H-6′b), 3.86 (1H, brd, J = 4.9 Hz, H-9′′′), 3.80 (3H, s, 3′′′-OCH), 3.79 (3H, s, 3″-OCH), 3.71 (1H, m, H-5′), 3.45 (1H, o, H-3′), 3.44 (1H, o, H-2′), 3.39 (1H, brt, H-4′), m: Multiplet, o: Resonance was overlapped; 13C-NMR (125 MHz, MeOH-d4): δ 167.7 (C-7″), 154.2 (C-4″), 154.0 (C-4), 152.3 (C-1), 151.2 (C-3″), 148.8 (C-3″′), 147.2 (C-4″′), 133.9 (C-1″′), 124.8 (C-6″), 124.2 (C-1″), 121.4 (C-6″′), 119.6 (C-2), 119.6 (C-6), 116.7 (C-3), 116.7 (C-5), 116.4 (C-5″), 115.7 (C-5″′), 114.5 (C-2″), 112.2 (C-2″′), 103.7 (C-1′), 85.3 (C-8″′), 78.1 (C-3′), 75.6 (C-5′), 75.1 (C-2′), 74.3 (C-7″′), 72.2 (C-4′), 65.4 (C-6′), 62.6 (C-9″′), 56.7 (3″-OCH3), 56.5 (3″′-OCH3).

Oleanolic acid (2): White amorphous powder; ESI-Q-TOF-MS: m/z 455.3532 [M − H]− (calcd. for C30H47O3, 455.3525); 1H-NMR (500 MHz, pyridine-d5): δ 5.52 (1H, s, H-12), 3.47 (1H, dd, J = 10.6, 6.4 Hz, H-3), 1.31 (3H, s, H-27), 1.27 (3H, s, H-23), 1.05 (6H, s, H-26, H-30), 1.03 (3H, s, H-24), 0.97 (3H, s, H-29), 0.92 (3H, s, H-25); 13C-NMR (125 MHz, pyridine-d5): δ 180.6 (C-28), 145.2 (C-13), 122.9 (C-12), 78.4 (C-3), 56.1 (C-5), 48.5 (C-9), 47.0 (C-17), 46.8 (C-19), 42.5 (C-14), 42.4 (C-18), 40.1 (C-8), 39.7 (C-4), 39.3 (C-1), 37.7 (C-10), 34.6 (C-21), 33.6 (C-22, 29), 33.5 (C-7), 31.3 (C-20), 29.1 (C-23), 28.6 (C-15), 28.4 (C-2), 26.5 (C-27), 24.2 (C-11), 24.1 (C-16), 23.2 (C-30), 19.1 (C-6), 17.8 (C-26), 16.9 (C-24), 15.9 (C-25).

Ursolic acid (3): White amorphous powder; ESI-Q-TOF-MS: m/z 455.3529 [M − H]− (calcd. for C30H47O3, 455.3525); 1H-NMR (500 MHz, pyridine-d5): δ 5.51 (1H, s, H-12), 3.48 (1H, dd, J = 10.4, 5.7 Hz, H-3), 1.27 (6H, s, H-23, 26), 1.25 (3H, s, H-27), 1.05 (3H, s, H-24), 1.03 (3H, s, H-29), 0.98 (3H, s, H-30), 0.92 (3H, s, H-25); 13C-NMR (125 MHz, pyridine-d5): δ 180.4 (C-28), 139.6 (C-13), 125.9 (C-12), 78.4 (C-3), 56.1 (C-5), 53.9 (C-18), 48.4 (C-9), 48.4 (C-17), 42.8 (C-14), 40.3 (C-8), 39.7 (C-4, 19, 20), 39.4 (C-1), 37.7 (C-10), 37.6 (C-22), 33.9 (C-7), 31.4 (C-21), 29.1 (C-23), 29.0 (C-15), 28.4 (C-2), 25.3 (C-16), 24.2 (C-27), 23.9 (C-11), 21.7 (C-30), 19.1 (C-6), 17.8 (C-26), 16.9 (C-29), 16.0 (C-24), 14.6 (C-25).

Maslinic acid (4): White amorphous powder; ESI-Q-TOF-MS: m/z 471.3477 [M − H]− (calcd. for C30H47O4, 471.3474); 1H-NMR (500 MHz, pyridine-d5): δ 5.49 (1H, s, H-12), 4.12 (1H, m, H-2), 3.42 (1H, d, J = 9.4 Hz, H-3), 1.30 (3H, s, H-29), 1.29 (3H, s, H-23), 1.23 (3H, s, H-27), 1.10 (3H, s, H-26), 1.04 (3H, s, H-30), 0.96 (3H, s, H-25); 13C-NMR (125 MHz, pyridine-d5): δ 180.7 (C-28), 145.3 (C-13), 122.7 (C-12), 84.1 (C-3), 68.9 (C-2), 56.2 (C-5), 48.5 (C-8), 48.5 (C-9), 48.1 (C-1), 47.0 (C-17), 46.8 (C-19), 42.5 (C-14), 42.3 (C-18), 40.2 (C-4), 38.9 (C-10), 34.6 (C-21), 33.6 (C-7), 33.5 (C-22, 29), 31.3 (C-20), 29.7 (C-23), 28.6 (C-15), 26.5 (C-27), 24.3 (C-30), 24.1 (C-11), 24.0 (C-16), 19.2 (C-6), 18.0 (C-26), 17.8 (C-25), 17.2 (C-24).

Chebuloside ii (5): White amorphous powder; ESI-Q-TOF-MS: m/z 689.3886 [M + Na]+ (calcd. for C36H58O11Na, 689.3877); 1H-NMR (500 MHz, pyridine-d5): δ 6.34 (1H, d, J = 8.1 Hz, H-1′), 5.55 (1H, t, J = 3.4 Hz, H-12), 5.12 (1H, s, H-6), 4.44 (2H, m, H-2, H-23), 4.26 (1H, m, H-3), 4.09 (1H, d, J = 10.6 Hz, H-23), 1.84 (3H, s, H-26), 1.79 (3H, s, H-23), 1.77 (3H, s, H-25), 1.22 (3H, s, H-27), 0.89 (6H, s, H-29, H-30); 13C-NMR (125 MHz, pyridine-d5): δ 176.7 (C-28), 143.8 (C-13), 123.4 (C-12), 96.2 (C-1′), 79.1 (C-3′), 78.7 (C-3, 5′), 74.5 (C-2′), 71.5 (C-4′), 69.4 (C-2), 67.9 (C-6), 66.6 (C-23), 62.5 (C-6′), 50.1 (C-1), 49.2 (C-9), 49.1 (C-5, 19), 47.3 (C-17), 43.2 (C-4, 14), 42.2 (C-18), 41.5 (C-7, 8), 38.6 (C-10), 34.4 (C-21), 33.4 (C-29), 32.9 (C-22), 31.1 (C-20), 28.6 (C-15), 26.5 (C-27), 23.9 (C-16, 30), 23.9 (C-30), 23.2 (C-11), 19.4 (C-26), 19.2 (C-25), 16.4 (C-24).

Corosolic acid (6): White amorphous powder; ESI-Q-TOF-MS: m/z 471.3483 [M − H]− (calcd. for C30H47O4, 471.3474); 1H-NMR (500 MHz, pyridine-d5): δ 5.48 (1H, s, H-12), 4.12 (1H, m, H-2), 3.42 (1H, d, J = 9.4 Hz, H-3), 1.30 (3H, s, H-29), 1.23 (3H, s, H-27), 1.10 (3H, s, H-24), 1.06 (3H, s, H-25), 1.00 (3H, d, J = 6.3 Hz, H-30), 0.97 (3H, d, J = 6.3 Hz, H-29); 13C-NMR (125 MHz, pyridine-d5): δ 180.4 (C-28), 139.7 (C-13), 125.8 (C-12), 84.1 (C-3), 68.9 (C-2), 56.2 (C-5), 53.9 (C-18), 48.4 (C-1, 17), 48.3 (C-9), 42.9 (C-14), 40.3 (C-8), 40.2 (C-4), 39.8 (C-20), 39.7 (C-19), 38.8 (C-10), 37.8 (C-22), 33.8 (C-7), 31.4 (C-21), 29.7 (C-23), 29.0 (C-15), 25.2 (C-16), 24.2 (C-27), 24.1 (C-11), 21.7 (C-30), 19.2 (C-6), 18.0 (C-24), 17.8 (C-26, 29), 17.3 (C-25).

Hederagenin-3-O-arabinopyranoside (7): White amorphous powder; ESI-Q-TOF-MS: m/z 663.3887 [M − H]− (calcd. for C35H55O8, 603.3897); 1H-NMR (500 MHz, pyridine-d5): δ 5.49 (1H, s, H-12), 5.00 (1H, d, J = 7.1 Hz, H-1′), 4.31 (2H, m, H-3, H-23a), 3.75 (1H, d, J = 10.4 Hz, H-23b), 1.25 (3H, s, H-27), 1.04 (3H, s, H-26), 1.02 (3H, s, H-30), 0.96 (3H, s, H-25), 0.94 (6H, s, H-24, H-29); 13C-NMR (125 MHz, pyridine-d5): δ 180.7 (C-28), 145.2 (C-13), 122.8 (C-12), 107.0 (C-1′), 82.2 (C-3), 75.0 (C-2′), 73.4 (C-3′), 70.1 (C-4′), 67.3 (C-5′), 64.8 (C-23), 48.5 (C-9), 47.9 (C-5), 47.0 (C-17), 46.8 (C-19), 43.8 (C-4), 42.5 (C-14), 42.3 (C-18), 40.1 (C-8), 39.1 (C-1), 37.3 (C-10), 34.6 (C-21), 33.6 (C-22, 29), 33.2 (C-7), 31.3 (C-20), 28.7 (C-15), 26.5 (C-2, 27), 24.2 (C-11), 24.1 (C-16), 24.0 (C-30), 18.5 (C-6), 17.8 (C-26), 16.4 (C-25), 13.9 (C-24).

Oleanolic acid 3-O-glucopyranosyl-(1→2)-arabinopyranoside (8): White amorphous powder; ESI-Q-TOF-MS: m/z 749.4456 [M − H]− (calcd. for C41H65O12, 749.4476); 1H-NMR (500 MHz, pyridine-d5): δ 5.49 (1H, s, H-12), 5.22 (1H, d, J = 7.7 Hz, H-1′′), 4.99 (1H, d, J = 5.9 Hz, H-1′), 3.22 (1H, dd, J = 11.6, 4.2 Hz, H-3), 1.30 (3H, s, H-27), 1.25 (3H, s, H-23), 1.06 (3H, s, H-24), 1.03 (6H, s, H-26, H-30), 0.97 (3H, s, H-29), 0.84 (3H, s, H-25); 13C-NMR (125 MHz, pyridine-d5): δ 180.6 (C-28), 145.2 (C-13), 122.8 (C-12), 106.4 (C-6′), 105.2 (C-1′), 89.1 (C-3), 81.4 (C-2′), 78.5 (C-2′′, 4′′), 76.8 (C-1′′), 73.8 (C-3′), 71.8 (C-3′′), 68.6 (C-4′), 65.3 (C-5′), 62.9 (C-5′′), 56.1 (C-5), 48.3 (C-9), 46.8 (C-17, 19), 42.5 (C-14), 42.3 (C-18), 40.0 (C-8), 39.8 (C-4), 39.0 (C-1), 37.3 (C-10), 34.5 (C-21), 33.6 (C-7, 22, 29), 31.3 (C-20), 28.6 (C-15, 23), 26.5 (C-2, 27), 24.1 (C-16), 24.0 (C-11, 30), 18.8 (C-6), 17.7 (C-26), 17.1 (C-24), 15.8 (C-25).

(+)-Catechin (9): Brown amorphous solid; ESI-Q-TOF-MS: m/z 291.0858 [M+H]+ (calcd. for C15H15O6, 291.0869); 1H-NMR (500 MHz, DMSO-d6): δ 6.84 (1H, s, H-2′), 6.76 (1H, d, J = 8.0 Hz, H-5′), 6.72 (1H, d, J = 8.0 Hz, H-6′), 5.93 (1H, d, J = 1.9 Hz, H-8), 5.85 (1H, d, J = 1.9 Hz, H-6), 4.56 (1H, d, J = 7.5 Hz, H-2), 3.97 (1H, dd, J = 13.2, 7.6 Hz, H-3), 2.85 (1H, dd, J = 16.1, 5.3 Hz, H-4a), 2.50 (1H, dd, J = 16.1, 8.1 Hz, H-4b); 13C-NMR (125 MHz, DMSO-d6): δ 156.4 (C-7), 156.1 (C-5), 155.3 (C-9), 144.8 (C-3′, 4′), 130.5 (C-1′), 118.4 (C-6′), 115.0 (C-5′), 114.5 (C-2′), 99.0 (C-10), 95.0 (C-6), 93.8 (C-8), 80.9 (C-2), 66.3 (C-3), 27.8 (C-4).

(−)-Epicatechin (10): Brown amorphous solid; ESI-Q-TOF-MS: m/z 291.0860 [M+H]+ (calcd. for C15H15O6, 291.0869); 1H-NMR (500 MHz, DMSO-d6): δ 6.88 (1H, d, J = 1.6 Hz, H-2′), 6.66 (1H, d, J = 8.1 Hz, H-5′), 6.64 (1H, dd, J = 8.1, 1.6 Hz, H-6′), 5.89 (1H, d, J = 2.3 Hz, H-6), 5.85 (1H, d, J = 2.3 Hz, H-8), 4.73 (1H, s, H-2), 4.00 (1H, s, H-3), 2.67 (1H, dd, J = 16.3, 4.5 Hz, H-4a), 2.49 (1H, dd, J = 16.3, 3.4 Hz, H-4b); 13C-NMR (125 MHz, DMSO-d6): δ 156.5 (C-5), 156.2 (C-7), 155.7 (C-9), 144.4 (C-3′, 4′), 130.5 (C-1′), 117.9 (C-6′), 114.8 (C-5′), 114.7 (C-2′), 98.4 (C-10), 95.0 (C-6), 94.0 (C-8), 78.0 (C-2), 64.8 (C-3), 28.1 (C-4).

(−)-Epiafzelechin (11): Yellow amorphous powder; ESI-Q-TOF-MS: m/z 297.0735 [M + Na]+ (calcd. for C15H14O5Na, 297.0739); 1H-NMR (500 MHz, DMSO-d6): δ 7.22 (2H, d, J = 8.5 Hz, H-2′, 6′), 6.71 (2H, d, J = 8.5 Hz, H-3′, 5′), 5.89 (1H, d, J = 2.3 Hz, H-8), 5.72 (1H, d, J = 2.3 Hz, H-6), 4.80 (1H, s, H-2), 4.02 (1H, dd, J = 8.3, 4.5 Hz, H-3), 2.67 (1H, m, H-4a), 2.46 (1H, m, H-4b); 13C-NMR (125 MHz, DMSO-d6): δ 156.5 (C-7), 156.2 (C-5, 9), 155.7 (C-4′), 129.9 (C-1′), 128.2 (C-2′, 6′), 114.3 (C-3′, 5′), 98.4 (C-10), 95.0 (C-8), 94.0 (C-6), 77.9 (C-2), 64.7 (C-3), 28.1 (C-4).

Juglanin (12): Yellow amorphous powder; ESI-Q-TOF-MS: m/z 441.0796 [M + Na]+ (calcd. for C20H18O10Na, 441.0798); 1H-NMR (500 MHz, DMSO-d6): δ 7.96 (2H, d, J = 8.7 Hz, H-2′, 6′), 6.92 (2H, d, J = 8.7 Hz, H-3′, 5′), 6.40 (1H, d, J = 1.6 Hz, H-8), 6.21 (1H, d, J = 1.6 Hz, H-6), 5.50 (1H, s, H-1′′); 13C-NMR (125 MHz, DMSO-d): δ 180.0 (C-4), 166.2 (C-7), 163.2 (C-5), 161.7 (C-4′), 159.5 (C-9), 158.7 (C-2), 135.1 (C-3), 132.1 (C-2′, 6′), 122.9 (C-1′), 116.6 (C-3′, 5′), 109.8 (C-1′′), 105.8 (C-10), 100.0 (C-6), 94.9 (C-8), 88.1 C-4′′), 83.5 (C-2′′), 78.8 (C-3′′), 62.7 (C-5′′).

Mearnsetin 3-O-rhamnopyranoside (13): Yellow amorphous powder; ESI-Q-TOF-MS: m/z 501.1012 [M + Na]+ (calcd. for C22H22O12, 501.1009); 1H-NMR (500 MHz, DMSO-d6): δ 6.81 (2H, s, H-2′, 6′), 6.36 (1H, brs, H-6), 6.19 (1H, brs, H-8), 5.15 (1H, brs, H-1′′), 3.98 (1H, dd, 3.5, 1.2 Hz, H-2′′), 3.74 (1H, s, 4′-OCH), 3.51 (1H, dd, J = 8.4, 3.0 Hz, H-3′′), 3.32 (1H, m, H-5′′), 3.15 (1H, m, H-4′′), 0.82 (3H, d, J = 5.4 Hz, H-6′′); 13C-NMR (125 MHz, DMSO-d6): δ 177.7 (C-4), 164.9 (C-7), 161.3 (C-5), 157.1 (C-9), 156.5 (C-2), 150.7 (C-3′, 5′), 137.7 (C-4′), 134.8 (C-3), 124.8 (C-1′), 108.1 (C-2′, 6′), 103.9 (C-10), 102.1 (C-1′′), 98.9 (C-6), 93.7 (C-8), 71.1 (C-4′′), 70.5 (C-3′′), 70.3 (C-2′′), 70.0 (C-5′′), 59.7 (4′-OCH3), 17.4 (C-6′′).

Myricitrin (14): Yellow amorphous powder; ESI-Q-TOF-MS: m/z 487.0852 [M + Na]+ (calcd. for C21H20O12, 487.0852); 1H-NMR (500 MHz, DMSO-d6): δ 6.90 (2H, s, H-2′, 6′), 6.38 (1H, d, J = 1.9 Hz, H-8), 6.21 (1H, d, J = 1.9 Hz, H-6), 5.21 (1H, d, J = 1.0 Hz, H-1′′), 3.99 (1H, brs, H-2′′), 3.57 (1H, dd, J = 9.2, 3.0 Hz, H-3′′), 3.40 (1H, m, H-5′′), 3.17 (1H, m, H-4′′) 0.86 (3H, d, J = 6.2 Hz, H-6′′); 13C-NMR (125 MHz, DMSO-d6): δ 177.7 (C-4), 164.2 (C-7), 161.2 (C-5), 157.4 (C-2), 156. 3(C-9), 145.7 (C-3′, 5′), 136.4 (C-4′), 134.2 (C-3), 119.5 (C-1′), 107.8 (C-2′, 6′), 103.9 (C-10), 101.9 (C-1′′), 98.6 (C-6), 93.4 (C-8), 71.2 (C-4′′), 70.5 (C-5′′), 70.3 (C-3′′), 69.9 (C-2′′), 17.5 (C-6′′).

Afzelin (15): Yellow amorphous powder; ESI-Q-TOF-MS: m/z 455.0954 [M + Na]+ (calcd. for C21H20O10, 455.0954); 1H-NMR (500 MHz, DMSO-d6): δ 7.75 (2H, d, J = 8.7 Hz, H-2′, 6′), 6.90 (2H, d, J = 8.7 Hz, H-3′, 5′), 6.33 (1H, s, H-8), 6.14 (1H, s, H-6), 5.29 (1H, s, H-1′′), 3.97 (1H, brs, H-2′′), 3.47 (1H, dd, J = 9.0, 2.9 Hz, H-3′′), 3.38 (1H, o, H-5′′), 3.13 (1H, t, J = 9.2 Hz, H-4′′), 0.79 (3H, d, J = 6.0 Hz, H-6′′); 13C-NMR (125 MHz, DMSO-d6): δ 177.4 (C-4), 164.7 (C-7), 161.2 (C-5), 159.9 (C-4′), 158.8 (C-9), 156.6 (C-2), 134.0 (C-3), 130.5 (C-2′, 6′), 120.5 (C-1′), 115.3 (C-3′, 5′), 104.3 (C-10), 101.7 (C-1′′), 99.0 (C-6), 93.9 (C-8), 70.5 (C-3′′), 70.3 (C-2′′), 70.1 (C-4′′), 70.0 (C-5′′), 17.4 (C-6′′).

Quercitrin (16): Yellow amorphous powder; ESI-Q-TOF-MS: m/z 447.0931 [M − H]− (calcd. for C21H19O11, 447.0927); 1H-NMR (500 MHz, DMSO-d6): δ 7.30 (1H, d, J = 2.1 Hz, H-2′), 7.26 (1H, dd, J = 8.3, 2.1 Hz, H-6′), 6.87 (1H, d, J = 8.3 Hz, H-5′), 6.38 (1H, s, H-6), 6.20 (1H, s, H-8), 5.27 (1H, s, H-1′′), 3.99 (1H, brs, H-2′′), 3.52 (1H, dd, J = 9.1, 3.3 Hz, H-3′′), 3.40 (1H, o, H-5′′), 3.16 (1H, t, J = 9.4 Hz, H-4′′), 0.83 (3H, d, J = 6.1 Hz, H-6′′); 13C-NMR (125 MHz, DMSO-d6): δ 177.6 (C-4), 164.7 (C-7), 161.2 (C-5), 157.1 (C-9), 156.4 (C-2), 148.5 (C-4′), 145.2 (C-3′), 134.1 (C-3), 121.0 (C-6′), 120.6 (C-1′), 115.5 (C-5′), 115.4 (C-2′), 103.9 (C-10), 101.7 (C-1′′), 98.8 (C-6), 93.6 (C-8), 71.1 (C-4′′), 70.5 (C-3′′), 70.3 (C-2′′), 70.0 (C-5′′), 17.4 (C-6′′).

4-Hydroxyphenol-[6-O-(4′′-hydroxy-3′′,5′′-dimethoxy-benzoate)]-β-D-glucopyranoside (17): Brown amorphous solid; ESI-Q-TOF-MS m/z 475.1217 [M+H]+ (calcd. for C21H25O11, 475.1216); 1H-NMR (500 MHz, MeOH-d4): δ 7.35 (2H, s, H-2′′, 6′′), 6.91 (2H, d, J = 8.9 Hz, H-2, 6), 6.58 (2H, d, J = 8.9 Hz, H-3, 5), 6.69 (1H, J = 12.2 Hz, H-6′a), 4.74 (1H, d, J = 7.4 Hz, H-1′), 4.38 (1H, d, J = 12.1 Hz, H-6′b), 3.86 (6H, s, 3′′-OCH and 5′′-OCH), 3.39-3.72 (4H, m, H-2′-5′),; 13C-NMR (125 MHz, MeOH-d4): δ 168.0 (C-7′′), 154.0 (C-4), 152.4 (C-1), 149.1 (C-3′′, 5′′), 142.5 (C-4′′), 125.4 (C-1′′), 119.6 (C-2, 6), 116.7 (C-3, 5), 108.5 (C-2′′, 6′′), 103.8 (C-1′), 78.0 (C-3′), 75.7 (C-5′), 75.1 (C-2′), 72.2 (C-4′), 65.4 (C-6′), 57.1 (3′′-OCH3 and 5′′-OCH3)

Breynioside A (18): Brown amorphous solid; ESI-Q-TOF-MS m/z 391.1043 [M − H]− (calcd. for C19H20O9, 391.1029); 1H-NMR (500 MHz, MeOH-d4): δ 7.90 (2H, d, J = 8.7 Hz, H-2′, 6′), 6.94 (2H, d, J = 8.9 Hz, H-2, 6), 6.86 (2H, d, J = 8.7 Hz, H-3′, 5′), 6.60 (2H, d, J = 8.9 Hz, H-3, 5), 3.41-3.87 (6H, m, H-2′-6′); 13C-NMR (125 MHz, MeOH-d4): δ 168.0 (C-7′′), 163.8 (C-4′′), 154.0 (C-4), 152.4 (C-1), 133.0 (C-6′′), 133.0 (C-2′′), 122.3 (C-1′′), 119.7 (C-6), 119.7 (C-2), 116.7 (C-3, 5), 116.4 (C-5′′), 116.4 (C-3′′), 103.8 (C-1′), 78.1 (C-3′), 75.7 (C-5′), 75.1 (C-2′), 72.2 (C-4′), 65.2 (C-6′).

Phenylethyl-rutinoside (19): White amorphous powder; ESI-Q-TOF-MS: m/z 453.1748 [M + Na]+ (calcd. for C20H30O10Na, 453.1737); 1H-NMR (500 MHz, MeOH-d4): δ 7.27 (2H, m, H-2, 6), 7.26 (2H, o, H-3, 5), 7.17 (1H, o, H-4), 4.74 (1H, d, J = 1.3 Hz, H-1′′), 4.29 (1H, d, J = 7.8 Hz, H-1′), 4.04 (1H, m, H-8a), 3.77 (1H, m, H-8b), 2.94 (2H, td, J = 7.3, 3.0 Hz, H-7), 1.25 (1H, d, J = 6.2 Hz, H-6′′); 13C-NMR (125 MHz, MeOH-d4): δ 140.1 (C-1), 130.1 (C-3, 5), 129.5 (C-2, 6), 127.3 (C-4), 104.7 (C-1′), 102.4 (C-1′′), 78.3 (C-3′), 77.1 (C-5′), 75.3 (C-2′), 74.2 (C-4′′), 72.5 (C-3′′), 72.3 (C-2′′), 71.9 (C-8), 71.8 (C-4′), 69.9 (C-5′′), 68.4 (C-6′), 37.4 (C-7), 18.5 (C-6′′).

Icariside D1 (20): White powder; ESI-Q-TOF-MS: m/z 439.1581 [M + Na]+ (calcd. for C19H28O10Na, 439.1580); 1H-NMR (500 MHz, pyridine-d): δ 7.29 (2H, o, H-2, 6), 7.27 (2H, o, H-3, 5), 7.17 (1H, o, H-4), 5.82 (1H, d, J = 2.5 Hz, H-1′′), 4.85 (1H, d, J = 7.8 Hz, H-1′), 4.35 (1H, m, H-8a), 3.91 (1H, m, H-8b), 3.00 (2H, t, J = 7.3 Hz, H-7); 13C-NMR (125 MHz, pyridine-d): δ 139.7 (C-1), 129.8 (C-3, 5), 129.8 (C-5), 129.0 (C-2, 6), 126.7 (C-4), 111.5 (C-1′′), 105.0 (C-1′), 80.8 (C-3′′), 78.9 (C-3′), 78.1 (C-2′′), 77.5 (C-5′), 75.4 (C-2′), 75.4 (C-4′′), 70.9 (C-8, 4′), 69.3 (C-6′), 65.9 (C-5′′), 37.0 (C-7).

Hexenyl-rutinoside (21): White amorphous powder; ESI-Q-TOF-MS: m/z 431.1892 [M + Na]+ (calcd. for C18H32O10Na, 431.1893); 1H-NMR (500 MHz, MeOH-d4): δ 5.46 (1H, o, H-4), 5.40 (1H, o, H-3), 4.74 (1H, d, J = 1.3 Hz, H-1′′), 4.25 (1H, d, J = 7.8 Hz, H-1′), 3.81 (1H, m, H-1a), 3.54 (1H, m, H-1b), 2.38 (2H, m, H-2), 2.08 (2H, m, H-5), 1.26 (3H, d, J = 6.3 Hz, H-6′′); 13C-NMR (125 MHz, MeOH-d4): δ 134.7 (C-4), 126.1 (C-3), 104.6 (C-1′), 102.4 (C-1′′), 78.2 (C-3′), 77.0 (C-5′), 75.2 (C-2′), 74.2 (C-4′′), 72.5 (C-3′′), 72.4 (C-2′′), 71.8 (C-4′), 70.7 (C-1), 69.9 (C-5′′), 68.3 (C-6′), 28.9 (C-2), 21.6 (C-5), 18.1 (C-6′′), 14.7 (C-6).

Everlastoside C (22): Black powder; ESI-Q-TOF-MS: m/z 405.1741 [M + Na]+ (calcd. for C16H30O10Na, 405.1737); 1H-NMR (500 MHz, pyridine-d5): δ 5.83 (1H, d, J = 2.3 Hz, H-1′′), 4.81 (1H, d, J = 7.8 Hz, H-1′), 4.19 (1H, m, H-1a), 3.68 (1H, m, H-1b), 1.72 (1H, m, H-3), 1.52 (2H, dd, J = 13.4, 6.7 Hz, H-2), 0.82 (3H, d, J = 5.4 Hz, H-4), 0.80 (3H, d, J = 5.4 Hz, H-5); 13C-NMR (125 MHz, pyridine-d5): δ 111.5 (C-1′′), 105.0 (C-1′), 80.8 (C-3′′), 78.9 (C-3′), 78.1 (C-2′′), 77.5 (C-5′), 75.4 (C-2′), 75.4 (C-4′′), 70.9 (C-4′), 69.3 (C-6′), 68.5 (C-1), 65.9 (C-5′′), 39.3 (C-2), 25.4 (C-3), 23.0 (C-5), 22.9 (C-4).

Berganin (23): Brown powder; ESI-Q-TOF-MS: m/z 351.0701 [M + Na]+ (calcd. for C14H16O9Na, 351.0692); 1H-NMR (500 MHz, DMSO-d6): δ 7.01 (1H, s, H-7), 4.98 (1H, d, J = 10.8 Hz, H-10b), 3.99 (1H, dd, J = 10.8, 9.6 Hz, H-4a), 3.85 (1H, brd, J = 11.4 Hz, H-11a), 3.78 (1H, s, OMe), 3.64 (1H, brd, J = 10.3 Hz, H-4), 3.58 (1H, m, H-2), 3.44 (1H, o, H-11b), 3.19 (1H, t, J = 8.7 Hz, H-3); 13C-NMR (125 MHz, DMSO-d6): δ 163.8 (C-6), 151.4 (C-8), 148.5 (C-10), 141.0 (C-9), 118.5 (C-6a), 116.3 (C-10a), 109.9 (C-7), 82.2 (C-2), 80.2 (C-4a), 74.1 (C-4), 72.5 (C-10b), 71.1 (C-3), 61.5 (C-11).

Citroside A (24): Brown amorphous powder; ESI-Q-TOF-MS: m/z 409.1842 [M + Na]+ (calcd. for C19H30O8Na, 409.1838); 1H-NMR (500 MHz, MeOH-d4): δ 5.89 (1H, s, H-8), 4.52 (1H, d, J = 7.7 Hz, H-1′), 4.32 (1H, m, H-3), 2.48 (1H, ddd, J = 13.4, 4.0, 2.1 Hz, H-4a), 2.19 (3H, s, H-11), 1.92 (1H, d, J = 1.4 Hz, H-2a), 1.47 (3H, s, H-10), 1.38 (3H, s, H-12), 1.36 (3H, d, J = 2.3 Hz, H-4b), 1.34 (3H, d, J = 1.5 Hz, H-2b), 1.15 (3H, s, H-13); 13C-NMR (125 MHz, MeOH-d4): δ 213.1 (C-9), 200.8 (C-7), 119. 2(C-6), 101.5 (C-8), 98.8 (C-1′), 78.8 (C-5), 78.7 (C-3′), 77.9 (C-5′), 75.4 (C-2′), 71.8 (C-4′), 63.9 (C-3), 63.0 (C-6′), 50.0 (C-2), 48.2 (C-4), 37.1 (C-1), 32.6 (C-13), 30.2 (C-12), 26.8 (C-11), 26.7 (C-10).

3. Materials and Methods

3.1. General Experiments

1H-NMR (500 MHz) and 13C-NMR (125MHz) data were obtained using a Bruker Avance III 500 Spectrometer (Bruker, Karlsruhe, Germany). ESI-Q-TOF-MS spectra were acquired using an Agilent 6530 Accurate-Mass Q-TOF LC/MS system (Agilent Technologies, Santa Clara, CA, USA). Preparative-scale high performance liquid chromatography (HPLC) was performed using a Gilson HPLC system (Middleton, WI, USA) composed of a binary pump, a liquid handler, and a UV/VIS detector. Gas chromatography (GC) was performed using a GC353B-FSL (GL Sciences Inc., Tokyo, Japan) and a flame ionization detector. A Jasco P-2000 polarimeter and a J-815 CD spectrometer (Jasco, Tokyo, Japan) were used to record optical rotation and circular dichroism (CD) spectra, respectively. UV absorbance was recorded using a UV-1800 spectrophotometer (Shimadzu, Kyoto, Japan). Silica gel (40-63 μm, Merck, Germany), ODS resin (S-5 μm, YMC Co., Tokyo, Japan), and SephadexTM LH-20 (GE Healthcare, Chicago, IL, USA) were used in liquid column chromatography, and thin layer chromatography was performed using Silicagel 60 F254 (Merck, Darmstadt, Germany). The preparative HPLC column was a Luna C18 (21.2 × 250 mm I.D., 5 μm; Phenomenex, Torrance, CA, USA). A BPX50 column (0.25 mm × 30 m; Trajan Scientific and Medical, Victoria, Australia) was used to perform GC analysis. Authentic D-glucose was purchased from Sigma-Aldrich Korea (Yongin, Korea). Organic solvents used in extraction, partition, and column chromatography were of analytical grade and were purchased from Dae-Jung Chemical Co. Ltd. (Seoul, Korea). Methanol and acetonitrile for HPLC were provided by Thermo Fisher Scientific Korea (Seoul, Korea).

3.2. Plant Material

The aerial parts of Leea asiatica were collected from Popa Mountain National Park (Mandalay, Myanmar) in August 2011. Khin Myo Htwe (Popa Mountain National Park) identified specimens of L. asiatica and a voucher specimen (#PopaLeeaA082011) was deposited at the Herbarium of the College of Pharmacy, the Catholic University of Korea.

3.3. Extraction and Isolation

The dried L. asiatica (651.4 g) samples were treated with methanol and extracts were obtained through immersion in an ultrasonic bath (3 h × 3 times). This process yielded methanol-soluble extracts (35.3 g). The methanol-soluble extracts were suspended in water and partitioned sequentially with organic solvents to yield n-hexane- (11.2 g), ethyl acetate- (9.1 g), and n-butanol-soluble extracts (6.1 g). The detailed isolation schemes for compounds 1–24 from organic solvent soluble extracts are described in Supplementary Information SI 9.

3.4. Acid Hydrolysis of Compound

Acid hydrolysis of compound 1 was carried out based on the previous report [47]. Derivatized authentic D-glucose gave GC peaks at tR 11.84 min and the tR of hydrolysate of 1 was similar to that of authentic D-glucose.