Gymnema latifolium wall ex. Wight

Pham, Ha Thanh Tung, Ryu, Byeol, Cho, Hyo Moon, Lee, Ba-Wool, Yang, Woo Young, Park, Eun Jin, Tran, Van On & Oh, Won Keun, 2020, Oleanane hemiacetal glycosides from Gymnema latifolium and their inhibitory effects on protein tyrosine phosphatase 1 B, Phytochemistry (112181) 170, pp. 112181-112181 : 2-7

2.1. Authentication of Gymnema latifolium wall ex. Wight

The whole plant contains bright yellow latex and the lianas are up to 6 m high. The stem was corky lenticellate, and old stem was changed basally wing-like corky. The young branchlets are yellowish green and densely pubescent, and stalks have densely hairy. Leaves opposite with broadly ovate is 8–13 cm × 5–8 cm. Apex and base truncate have the rounded, and margin entire is orange-yellow pubescent abaxially with 5–7 lateral veins per side. Petiole with 1.5–4 cm long is densely pubescent. Inflorescences are paired at node, multi-flowered, umbrellashape cymes and pubescent. Flowers are yellow with 3 mm × 3 mm and corolla is a yellowish campanulate with a dense pubescent inside without glabrous. Gynostegium is concealed in corolla, slightly swollen base in cylindrical form, and has ten oval nectary patches. Pollinia is a oblong, erect and top enlargement, and ovary has dense pubescent. Stigma conical divided into two. Follicles are in pair or solitary, lanceolate cylindrical, beaked, 7–10 cm long, 0.3–0.6 cm in diameter, apex acuminate, base dilated and dense pubescent. There are many seeds, oblong-lanceolate and winged with a thin edge ( Fig. 1 ). All of these external morphology are closely matched the description of Gymnema latifolium by Flora of China ( Wu and Raven, 1995) with a slight difference on the fruit shape (7–10 cm × 0.3–0.6 cm) compared with 4.5–5.5 × 1.5–2 cm. The morphological characteristic of wing-like cork was not mentioned in Flora of China, but the descriptions of Gymnema khadalense ( Deokule et al., 2013) and Gymnema kollimalayanum ( Ramachandran and Viswanathan, 2009) are found. These two species, Gymnema khandalense and Gymnema kollimalayanum , were also reported as synonyms of Gymnema latifolium ( Meve and Alejandro, 2011) . Thus, by comparing the descriptive morphological characteristics with the specimens of syntype K000872841 ( Fig. S1 ) and lectotype specimen K000872839 ( Fig. S2 ) of Gymnema latifolium stored in the herbarium at the Royal Botanic Gardens Kew and the description by Meve and Alejandro (2011), the studied sample was finally authenticated as Gymnema latifolium Wall. ex Wight. In addition, a DNA sequence of the ITS1-5.8S-ITS2 internal transcribed spacer of the sample was also deposited at Genbank (National Institutes of Health) with the accession number KP163979 .

2.2. Isolation and structural elucidation of compounds from G. latifolium

A 70% EtOH extract of G. latifolium was subjected to various chromatographic columns and then to purification by preparative highperformance liquid chromatography (HPLC) to afford seven previously undescribed oleanane triterpenoid glycosides, gymlatinosides GL1-GL7 (1–7) and two known compounds, gymnemic acid IX (8) and gymnemagenin (9) ( Liu et al., 1992) ( Fig. 2 ). The structures of the previously undescribed compounds were elucidated by 2D-NMR, while known compounds were determined using 1 H and 13 C NMR analysis and comparing the physical and spectroscopic data with those in the literature.

Gymlatinoside GL1 (1), obtained as a white amorphous powder, with [] D 25 +15.2 (c 0.2, MeOH), was found to possess a molecular formula of C 49 H 76 O 19 and twelve indices of hydrogen deficiency (IHDs) based on the high-resolution electrospray ionization mass spectrometry (HRESIMS) ion peak at m / z 967.4896 [M − H] − (calcd for C 49 H 75 O 19, 967.4903). The broad IR absorption at 3389 cm −1 indicated the presence of hydroxy groups, and the absorption at 1641 cm −1 revealed the existence of carboxylic moieties. The 1 H NMR spectrum exhibited seven methyl singlets at δ H 1.94, 1.27, 1.20, 1.05, 1.04, 0.92 and 0.90 (each 3H). The most downfield resonance (δ H 1.94) is the signal of an acetoxy, and the others suggested an oleanane backbone. Two other methyls can be observed at δ H 1.23 (3H, d, J = 7.0 Hz) and δ H 0.97 (3H, t, J = 7.0 Hz) ( Table 1 ). The 13 C NMR spectrum showed signals for 49 carbons, including three carboxylic groups at δ C 176.8, 171.6 and 171.1; two olefinic carbon signals at δ C 141.5 and 124.9; two anomeric carbons at δ C 106.8 and 97.4; fifteen oxygenated carbons in the range from δ C 62.6 to 94.1, and others are methine, methylene and methyl signals ( Table 2). The COSY, HSQC and HMBC spectra indicated a planar structure of oleanane-type triterpene possessing six hydroxy groups substituted at C-3, 16, 21, 22, 23 and 28 for the main aglycone of 1 ( Fig. 3 ). Comparing chemical shifts of 1 with gymnemic acid VIII and gymnemic acid X suggested gymnemagenin to be the main aglycone ( Liu et al., 1992). A series of NOESY correlations between H-3 (δ H 4.26 dd, J = 12.3, 4.5 Hz), H-23 [(δ H 3.70 d, J = 10.5 Hz); δ H 4.32 d, J = 10.5 Hz)], H-5 (δ H 1.64, overlap), H-9 (δ H 1.64, overlap), H-27 (δ H 1.27, s), H-16 (δ H 5.09, dd, J = 11.0, 5.2 Hz), H-21 (δ H 5.67, d, J = 10.5 Hz) and H-29 (δ H 1.04, s) confirmed the α configuration for these protons. Meanwhile, the series of NOESY cross peaks between H-24 (δ H 0.92, s), H-25 (δ H 0.92, s), H-26 (δ H 1.05, s), H-28 (δ H 4.57, overlap; δ H 4.99 d, J = 10.5 Hz), H-18 (δ H 2.84, dd, J = 13.7, 3.9 Hz), H-22 (δ H 4.50, overlap) and H-30 (δ H 1.20, s) demonstrated their β configurations ( Fig. 4 ). Furthermore, the acid hydrolysis of 1 afforded an aglycone which exhibited the same retention time and mass fragmentation with the standard gymnemagenin on LC-MS analysis. Two carboxylic groups were identified to be a 2-methylbutyryl (δ C 176.8) and an acetyl (δ C 171.1) substitutions by comparing with those reported in literature ( Yoshikawa et al., 1992). Compared with gymnemagenin, the acylation shifts can be observed at C-28 (δ C 62.6, +4.1 ppm) and C-21 (δ C 78.6, +1.4 ppm) ( Liu et al., 1992). The protons at C-21 and C-28 also showed significant downfield shifts from those of gymnemagenin [(H-21: δ H 4.04 → δ H 5.67) and (H-28: δ H 4.07 → δ H 4.57; δ H 4.71 → δ H 4.99)]. The HMBC correlations from H-21 (δ H 5.67, d, J = 10.5 Hz) to C-1 M−21 (δ C 176.8), and H-28 [(δ H 4.99, d, J = 10.5 Hz); (δ H 4.57, overlap)] to C-1 A−28 (δ C 171.1) confirmed the linkages of the 2-methylbutyryl to C-21 and the acetyl to C-28. NMR chemical shifts of the 2-methylbutyryl portion in compound 1 were similar to the data reported by Yoshikawa, where the absolute configuration of 2-methylbutyryl in gymnemic acid II isolated from G. sylvestre is 2(S) ( Yoshikawa et al., 1989). Since the plants G. latifolium and G. sylvestre have been classified in the same genus, it is suggested that they possess similar biosynthesis pathway for 2(S)-methylbutyryl. In addition, the configuration of the 2(S)-methylbutyryl moiety in compound 1 was further confirmed by the clear NOESY correlation between H-21 (δ H 5.67, d, J = 10.5 Hz) to H-2 M−21 (δ H 2.56, sextet, J = 6.0 Hz) and the absence of NOESY correlation between H-21 and H-5 M−21 (δ H 1.23, d, J = 7.0 Hz). The distance 2.016 Å between H-21 and H-2 M−21 observed in a 3D geometry model optimized using MM2 minimized energy force field also supported this configuration (Fig. S10B). The COSY and dimensional (2D) J -resolved NMR spectra allowed the detection of two sugar chains, and the precise assignment of the coupling constants of their sugar protons ( Table 1 ). A doublet signal at δ H 5.17 (d, J = 7.5 Hz) can be assigned to a β -isomer anomeric proton H-1′, corresponding to the anomeric carbon signal at C-1′ (δ C 106.8). The appearance of proton H-5′ (δ H 4.51) as a doublet (J = 10.5 Hz) and its HMBC cross peak with C-6′ (δ C 171.6) suggested that the first sugar moiety is a glucuronic acid. The second anomeric proton H-1′′ (δ H 5.32) which correlated with C-1′′ (δ C 97.4) on HSQC spectrum and the signal of the quaternary carbon C-2′′ (δ C 94.1) are the characteristics of a 2-oxo-hexose which is forming an intramolecular hemiacetal with another hydroxy group ( Liu et al., 1992). The existence of this 2-oxo-glucose portion was also recognizable by a fragment loss of 160 amu in the positive mass fragmentation. Furthermore, the 13 C-NMR data of the sugar portions were almost identical to those of gymnemic acid VIII ( Liu et al., 1992). The HMBC correlation from H-4′ (δ H 5.21, t, J = 10.5 Hz) to C-2″, which was not detected by Liu, but finally could be observed in a high resolution HMBC NMR experiment. This HMBC correlation further confirmed the existence dioxane ring between two sugar moieties ( Fig. 3 ). Coupling constants and NOESY correlations confirmed the β configurations for H-2′ and 4′, and α orientations for H-1′, 3′, 5′, 1″, 3″ and 5′′. The 3 J coupling constant (J = 10.0 Hz) observed by dimensional (2D) J -resolved NMR indicated the antiperiplanar conformation between H-3″ and H-4″ and thus verified the axial orientation of H-4′′. Taken together, compound 1 was deduced as 21- O -2(S)-methylbutyryl-28- O -acetyl-gymnemagenin 3- O -β -D-arabino-2-hexulopyranosyl-(1 → 3)- β -D-glucuronopyranoside.

Gymlatinoside GL2 (2) was obtained as a white amorphous powder, with [] D 25 +9.6 (c 0.2, MeOH). Its HRESIMS showed a pseudo molecular ion peak at m / z 965.4752 [M − H] − (calcd for C 49 H 73 O 19, 965.4746), indicating a molecular formula of C 49 H 74 O 19 and twelve IHDs. The 1 H and 13 C NMR spectroscopic data of 2 ( Tables 1 and 2) showed similar resonances to those of 1, apart from the signals of the 2(S)-methylbutyryl moiety which were replaced by the chemical shifts of a tigloyl moiety ( Pham et al., 2018). A neutral fragmentation loss of 82 amu observed in positive mode of mass spectrum also established the occurrence of the tigloyl, and its linkage to C-21 was demonstrated by the HMBC correlation from H-21 [δ H 5.76, d, J = 10.5 Hz) to C-1 T−21 (δ C 168.4). Therefore, compound 2 was defined as 21- O -tigloyl-28- O -acetyl-gymnemagenin 3- O -β -D-arabino-2-hexulopyranosyl-(1 → 3)- β -D-glucuronopyranoside.

Gymlatinoside GL3 (3) was obtained as a white amorphous powder, with [] D 25 +10.1 (c 0.2, MeOH). Its HRESIMS showed a pseudo molecular ion peak at m / z 987.4581 [M − H] − (calcd for C 51 H 71 O 19, 987.4590), indicating a molecular formula of C 51 H 71 O 19 and sixteen IHDs. The 1 H and 13 C NMR spectroscopic data of 3 ( Tables 1 and 2) exhibited generally similar resonances to those of 1, except for the replacement of signals of the 2(S)-methylbutyryl moiety with the chemical shifts of a benzoyl moiety ( Pham et al., 2018). The positive mass fragment loss Δ m/z = 104 amu observed in 3 confirmed the existence of benzoyl. The downfield shift of C-21 (δ C 80.3) and the HMBC cross peak from H-21 (δ H 5.77, d, J = 10.5 Hz) to C–1 B−21 (δ C 167.2) established the connection of the benzoyl to C-21. Therefore, compound 3 was elucidated as 21- O -benzoyl-28- O -acetyl-gymnemagenin 3- O -β -D-arabino-2-hexulopyranosyl-(1 → 3)- β -D-glucuronopyranoside.

Gymlatinoside GL4 (4) was obtained as a white amorphous powder, with [] D 25 + 22.1 (c 0.2, MeOH), was found to possess a molecular formula of C 51 H 76 O 20 and fourteen IHDs based on the HRESIMS ion peak at m / z 1007.4826 [M − H] − (calcd for C 51 H 75 O 20, 1007.4852). Mass fragmentation in the positive mode showed the neutral losses of two acetyls (2 × 42 amu) and one tigloyl (82 amu). The 1 H and 13 C NMR spectroscopic data of 4 ( Tables 1 and 2) showed similar resonances to those of 2 in the aglycone and glycosyl moieties with some differences occurred in the positions of acyl substitutions. Further investigation of the acylation shifts and HMBC spectra revealed that the tigloyl was attached to C-21 (δ C 76.7) and two acetyls were substituted to C-16 (δ C 68.7) and C-22 (δ C 72.0), respectively. Therefore, compound 4 was determined as 21- O -tigloyl-16,22- O -diacetyl gymnemagenin 3- O - β -D-arabino-2-hexulopyranosyl-(1 → 3)- β -D-glucuronopyranoside.

Gymlatinoside GL5 (5) was obtained as an amorphous powder with [] D 25 +13.4 (c 0.2, MeOH). The molecular formula C 42 H 70 O 13 was determined by a quasimolecular ion peak at m/z 781.4746 [ M – H] – (calcd for C 42 H 69 O 13 , 781.4738 ) in HRESIMS. 1 H, 13 C and HSQC NMR spectroscopic data of the aglycone ( Tables 1 and 2) exhibited signals for seven methyl groups: (δ H 0.87, δ C 16.1 ), (δ H 0.93, δ C 33.3 ), (δ H 0.95, δ C 24.4 ), (δ H 0.98, δ C 17.4 ), (δ H 1.03 , δ C 17.4 ), (δ H 1.32 , δ C 28.6 ), and (δ H 1.35 , δ C 27.5 ). The double bond at C 12-13 was demonstrated by the signals of olefinic group [δ H-12 5.24 (br s), δ C-12 123.3] and δC-13 143.8. Signals of fifteen oxygenated carbons can be observed including two anomeric carbons at δ C 107.3 and 106.2, ten other glycosyl carbons and three others of the main skeleton. These NMR data suggested the structure of a longispinogenin moiety with two attached sugars ( Pham et al., 2018). Sugar analysis and NMR data suggested the sugar type of this structure is glucose which is identified to be in β -configuration by the coupling constant J = 7.5 Hz of their anomeric protons. Their positions at C-3 and C-28 were evident by the HMBC correlations from H-1′ (4.97, d, J = 7.5 Hz) to C-3 (δ C 89.2 ) and H-1′′ (4.94, d, J = 7.5 Hz) to C-28 (δ C 78.7 ) ( Fig. 3 ). Consequently, compound 5 was elucidated as 3- O -β -D-glucopyranosyl longispinogenin 28- O -β -D-glucuronopyranoside .

a 1

H NMR (500 MHz).

b 1 H NMR (600 MHz).

a 13

C NMR (125 MHz).

b 13 C NMR (150 MHz).

Gymlatinoside GL6 (6), obtained as an amorphous powder with [] D 25 +5.4 (c 0.2, MeOH). The HRESIMS of this compound revealed an ion peak at m / z 791.4225 [M – H] – (calcd for C 42 H 63 O 14, 791.4218). It suggested a molecular formula C 42 H 64 O 14 and indicated the presence of 11 IHDs. The 1 H-NMR spectrum of 6 showed seven methyl groups (δ H 0.78, 0.86, 0.96, 0.99, 1.20, 1.32 and 1.41), and twelve oxymethine protons ( Table 1 ). The signals in the 13 C NMR spectrum together with HSQC analysis could be assigned as eleven quaternary carbons (four carboxylic acids at δ C 173.3, 170.8, 170.5, 170.3 and one olefinic at δ C 140.8), thirteen tertiary carbons (nine oxygenated methines, one olefinic carbon at δ C 125.0), eight secondary carbons (one oxygenated methylene at δ C 68.6) and ten methyl carbons ( Table 2). Comparing its resonances with literature, together with the NOESY experiment ( Fig. 4 ), suggested that 6 has a marsglobiferin aglycone which possesses two β -oriented hydroxyl group substituted at C-16, C-21 and one α - oriented hydroxyl group at C-22 ( Yoshikawa et al., 1994). Furthermore, positive fragment ions exhibited a glucuronic fragment loss (176 amu) and three acetyl substitutions (3 × 42 amu). The HMBC correlations from H-16 (δ H 6.31, overlap) to C-1 A−16 (δ C 179.5), H-21 (δ H 5.61, d, J = 11.0 Hz) to C-1 A−21 (δ C 170.3), and H-22 [δ H 5.16, d, J = 11.0 Hz] to C-1 A−22 (δ C 170.8) confirmed the acylated linkage positions at C-16, 21 and 22. The connection of the β glucuronic acid to C-3 was determined by the coupling constant J = 7.5 Hz of the anomeric proton, the glycosylated chemical shift of C-3 (δ C 89.1, +11.1) and the HMBC correlation from H-1′ (δ H 4.97) to C-3 ( Fig. 3 ). Therefore, compound 6 was elucidated as 16,21,22- O -triacetyl marsglobiferin 3- O -β -D-glucuronopyranoside.

Gymlatinoside GL7 (7), obtained as an amorphous powder with [] D 25 +12.4 (c 0.2, MeOH), possessed a molecular formula of C 45 H 68 O 15 based on HRESIMS ion peaks at m/z 847.4508 [M – H] – (calcd for C 45 H 67 O 15, 847.4480). The LC-MS experiment in positive mode showed key ions of gymnemagenin (489, 471, 453 and 435) and neutral losses of a glucuronic acid (176 amu), a tigloyl (82 amu) and two acetyls (2 × 42 amu). The 1 H, 13 C and HSQC NMR spectroscopic data confirmed the structure of aglycone gymnemagenin similar with compound 1 ( Tables 1 and 2). Meanwhile, its 13 C-NMR showed similar acylation chemical shifts for C-16 (δ C 68.7), C-21 (δ C 76.7) and C-22 (δ C 72.0) compared with 4, and the sugar portion showed was superimposable with compound 6. The glycosylated chemical shift of C-3 (δ C 82.2) and HMBC cross peak from anomeric proton H-1′ (δ H 5.27, d, J = 7.5 Hz) indicated the presence of a β -D-glucuronic acid substituted at C-3 ( Fig. 3 ). Accordingly, compound 7 was determined as 21- O -tigloyl-16,22- O -diacetyl gymnemagenin 3- O -β -D-glucuronopyranoside.

Figures

Fig. 1. Morphological characteristics of Gymnema latifolium Wall ex. Wight. (a) Old stem showing wing-like cork (b) Living form; (c) Young branch with pairs of inflorescensces; (d) Adaxial leaf; (e) Abaxial leaf; (f) Inflorescence; (g) Dense bronze hairs on the young leaf; (h) A flower; (i) Calyx; (j) Corolla; (k) Gynostegium cylindric; (l) Pollinarium; (m) Stigma head; (n) Follicles.

Fig. 2. Chemical structure of isolated compounds 1–9 isolated from Gymnema latifolium.

Fig. 3. Key COSY and HMBC correlations of compounds 1, 5, 6 and 7.

Fig. 4. Key NOESY correlations of compounds 1 (a: the aglycone, b: sugar moiety), 5 and 6. Fig. 4 c showed the chemical shifts and coupling constants of the protons on the glycosides. HMBC correlations bridging two sugar moieties were measured. All the discussions about COSY, NOESY and the coupling constant rules to identify the relative configuration of the sugar moiety were suggested at Results and discussion of compound 1.