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2.2. Structure elucidation of compounds 1–8 from Alchornea rugosa 25 Compound 1 was isolated as an amorphous powder with [α] D = + 54.9 (c 0.20, MeOH). The molecular formula, C 32 H 41 N 3 O 10, was deduced from its HRESIMS ion peak at m / z 628.2859 [M + H] + (calcd for C 32 H 42 N 3 O 10, 628.2865). The IR spectrum of 1 showed absorption bands characterized by hydroxyl or amine (3704 cm 1), C–H in heteroaromatic rings (2967 cm 1), C––NH or aromatic C (1682 cm 1), benzofuran (1203 cm 1), and C–O (1032 cm 1). The 1 H NMR spectrum of 1 showed two N–H protons (δ H 11.93, 11.65 ppm, Fig. S14), four aromatic signals (δ H 6.76, 6.73, 6.66, and 6.10), one olefinic proton at δ H 5.26 (t, J = 6.9 Hz), an anomeric signal (δ H 4.35 ppm), one N -methylene group at δ H 3.81 (d, J = 6.9 Hz), six protons on oxygenated carbons (δ H 3.33–4.71 ppm), one methylene group (δ H 2.86/2.75 ppm), one methine group (δ H 2.78 ppm), and five methyl groups (δ H 1.73, 1.69, 1.25, 1.13, and 1.11 ppm). The 13 C NMR spectrum of 1 showed 32 carbon signals, including a guanidine carbon (δ C 147.6), fourteen aromatic signals (δ C 95.9–158.8), two olefinic carbons (δ C 120.1/138.5), one anomeric carbon at δ C 102.0 ppm, six oxygenated carbons (δ C 70.3–81.2), an N - methylene group (δ C 41.9), a methylene group (δ C 27.3), one methine group (δ C 26.2), and five methyl group signals (δ C 17.9–25.7). The HMBC correlations from H-2 (δ H 4.71) to C-1’ (δ C 131.6), C-3 (δ C 75.4), C-4 (δ C 27.3), and C-9 (δ C 155.5); from H-6 (δ H 6.10) to C-5 (δ C 158.8), C-7 (δ C 157.3), C-10 (δ C 100.8), and C-8 (δ C 95.9); and from H 2 -4 (δ H 2.86/ 2.75) to C-5, C-9, and C-10 suggested the presence of a C 6 –C 3 –C 6 unit. A hexose sugar moiety was revealed by the mass loss of 146 Da in HRMS/ MS data as well as signals of an anomeric signal (δ H 4.35/ δ C 102.0), four oxygenated methine groups (δ H 3.33–3.65; δ C 70.3–73.9), and a doublet methyl group (δ H 1.25 (d, J = 6.3 Hz)/ δ C 17.9). This rhamnose unit was proven by the 1 H– 1 H COSY spin system (Fig. 2). The HMBC cross peak from H-1’’’’ (δ H 4.35) to C-3 indicated that the rhamnose was linked to the catechin moiety of 1 at C-3. The small coupling constant of H-1’’’’ (d, J = 1.5 Hz), as well as the large 1 J C-H (169.6 Hz), indicated that the relative configuration of the sugar moiety was α -oriented. In addition, the NMR data of 1 exhibited a guanidine unit characterized by the carbon signal at δ C 147.6 with two N–H signals at δ H 11.93 and 11.65 that shared similarities to those of guanidine derivatives reported in the Alchornea genus (Barrosa et al., 2014; Tapondjou et al., 2016). An isoprenyl unit in 1 elongated from the guanidine group was indicated by the presence of the N -methylene group (δ H 3.81/ δ C 41.9), a double bond (δ H 5.26/ δ C 120.1, 138.5), and two methyl groups (δ H 1.69/ δ C 18.0; δ H 1.73/ δ C 25.7), and the HMBC cross-peaks from H 2 -1 ′′′ to guanidine carbon (δ C 147.6). Moreover, the HMBC correlations from two doublet methyls (Me-4 ′′, δ H 1.11, and Me-5 ′′, δ H 1.13) to methine C-3’’ (δ C 26.2) and the other olefinic carbon (δ C 131.3, C-2 ′′), as well as from 1 ′′ -NH (δ H 11.93) and C––NH (δ H 11.65) to the double bond C-1’’/C-2 ′′, suggested that the other five-carbon chain was linked to the guanidine moiety (Fig. 2). As suggested by HRESIMS, the molecular formula of 1 was C 32 H 41 N 3 O 10, which consisted of 14 double bond equivalents (DBE); however, only 13 out of 14 DBEs had been assigned. Therefore, an additional ring of 1 through C-8/C-1 ′′ and C-1’’/7-OH was suggested because of the consistency with the conjugation reported in alchornealaxine (Tapondjou et al., 2016). The relative orientations of the rhamnose moiety were determined by the coupling constants; in particular, 2 J H-H of H-1’’’’ and H-2’’’’ indicated the equatorial orientation of H-2’’’’ while the large coupling constants of H-3’’’’, H-4’’’’, and H-5’’’’ suggested axial orientations of those protons in the sugar unit. Moreover, the NOESY correlations between H-1’’’’/H-2’’’’, H-3’’’’/H-5’’’’, and H-4’’’’/Me-6’’’’ demonstrated the relative configuration of rhamnose sugar in 1 (Fig. 3). The absolute configuration of rhamnose was established by acid hydrolysis, followed by conversion to the corresponding thiocarbamoyl-thiazolidine carboxylate derivative with L- cysteine methyl ester and o -tolyl isothiocyanate (Tanaka et al., 2007). According to the consistent retention times on HPLC chromatography between derivatives of sugar in 1 and the authentic L- rhamnose, the sugar was identified as α -L- rhamnose. The stereocenters at C-2 and C-3 of 1 were determined to be 2 R, 3 S based on their large coupling constants of H-2 (d, J = 7.3 Hz)/H-3 (q, J = 7.3 Hz), which suggested a 2,3- trans flavan-3-ol, and its CD data with negative CEs of approximately 290 and 240 nm (Fig. S73A) (Slade et al., 2005). Thus, the structure of 1 was identified as (2 R,3 S)-rugonine A. Compound 2 was isolated as an amorphous powder with [α] 25 = D 78.0 (c 0.10, MeOH). The molecular formula, C 32 H 41 N 3 O 10, was deduced from its HRESIMS ion peak at m / z 628.2870 [M + H] + (calcd for C 32 H 42 N 3 O 10, 628.2865). The IR spectrum of 2 exhibited the absorption bands of hydroxyl or amine (3252 cm 1), C–H in heteroaromatic rings (2976 cm 1), C––NH or aromatic C (1668 cm 1), benzofuran (1200 cm 1), and C–O (1072 cm 1). The 1 H and 13 C NMR spectra of 2 shared similarities to those of 1, suggesting a similar planar structure. The configuration of the sugar moiety in 2 was also determined by analyzing its NMR coupling constants and NOESY correlations and by comparing the HPLC retention time with the derivative of authentic L- rhamnose, suggesting the presence of an α -L- rhamnose. Moreover, the 2,3- trans flavan-3-ol, which was identified in 1, was also seen in 2 based on its NMR data for the same positions (δ H 4.67, d, J = 7.3 Hz/ δ C 81.1 for C-2 and δ H 3.93, d, 7.3 Hz/ δ C 75.2 for C-3). The different features in 1 and 2 were observed in their optical rotations (+ 55 and 78, respectively) and the opposite CE at 290 nm in the CD spectra. The CD data of 2 showed a positive CE at 290 nm and a negative CE at 240 nm (Fig. S78B), indicating that the absolute configuration of 2 was 2 S, 3 R (Slade et al., 2005). Therefore, the structure of compound 2 was identified as (2 S,3 R)-rugonine B. Compound 3 was acquired as an amorphous powder with [α] D 25 = + 199.6 (c 0.10, MeOH). The molecular formula, C 32 H 43 N 3 O 10, was deduced from its HRESIMS ion peak at m / z 646.2974 [M + H] + (calcd for C 32 H 44 N 3 O 11, 646.2976). The IR spectrum of 3 displayed the absorption bands of hydroxyl or amine (3704 cm 1), C–H in heteroaromatic rings (2922 cm 1), C––NH or aromatic C (1682 cm 1), benzofuran (1195 cm 1), and C–O (1057 cm 1) functional groups. The NMR data of 3, which were similar to those of 1 and 2 (Table 1), indicated that 3 shared a similarity in the planar structure to 1 and 2 excluding the absence of an olefinic bond at δ H 5.26/ δ C 120.1 and 138.5, an additional methylene group (δ H 1.75/ δ C 42.4), and an oxygenated quaternary carbon signal (δ C 70.9 ppm) in 3. The COSY correlation between H 2 -1 ′′′ (δ H 3.34) and H 2 -2 ′′′ (δ H 1.75), as well as HMBC cross-peaks from H 2 -2 ′′′ to C-3 ′′′ (δ C 70.9), C-4 ′′′, and C-5 ′′′ (δ C 29.5) revealed the presence of a 4-hydroxyl-4-methyl pentyl moiety in 3 instead of the isoprenyl moiety in 1. The rhamnose moiety in 3 was also determined by the same method as in 1 and 2 by analyzing NMR coupling constants, NOESY correlations, and comparing the retention times to the derivative of authentic L- rhamnose suggesting that the sugar moiety of 3 was α - L-rhamnose. The absolute configuration at C-2 and C-3 was identified based on the large coupling constants of H-2 (δ H 4.70, J = 7.3 Hz)/H-3 (δ H 3.96, J = 7.7, 5.4 Hz), indicating a 2,3- trans flavan-3-ol, and the CD data showed negative CEs at approximately 290 and 240 nm similar to those of 1 (Fig. S78A). Therefore, the structure of compound 3 was identified as (2 R,3 S)-rugonine C. Compound 4 was isolated as an amorphous powder with [α] D 25 = + 27.5 (c 0.20, MeOH). The chemical formula, C 21 H 23 N 3 O 6, was deduced from its HRESIMS ion peak at m / z 414.1649 [M + H] + (calcd for C 21 H 24 N 3 O 6, 414.1665). The 1 H and 13 C NMR spectra of 4 showed a similar pattern to the core guanidine-fused catechin skeleton of 1 without sugar and isopentenyl moieties. The relative configurations at C-2 and C-3 of 4 were assigned based on the large J coupling constants of H-2 (δ H 4.54, J = 6.9 Hz), H-3 (δ H 3.83, J = 12.6, 6.9 Hz) that indicated a 2,3- trans flavan-3-ol as those of 1–3. The absolute configuration of 4 was determined to be 2 R, 3 S by experimental CD data that showed negative CEs at approximately 290 nm and 240 nm (Fig. S78A), which is typical for (+)-catechin (Slade et al., 2005). Thus, the structure of 4 was determined to be (2 R,3 S)- rugonine D. 25 Compound 5 was isolated as an amorphous powder with [α] D = 16.4 (c 0.20, MeOH). The molecular formula of 5 was the same as that of 4, C 21 H 23 N 3 O 6, which was deduced from its HRESIMS ion peak at m/z 414.1675 [M + H] + (calcd for C 21 H 24 N 3 O 6, 414.1665). The 1 H and 13 C NMR spectra of 5 shared similarity to those of 4, excluding signals at δ H 4.79 (s) (H-2) and 4.02 (s) (H-3). These features and the opposite optical rotation between 4 and 5 suggested that they might have different orientations at C-2 and C-3. In particular, based on the small coupling constants at C-2 and C-3 of 5, the relative configurations were identified as 2,3- cis flavan-3-ol (Slade et al., 2005). Moreover, the negative CEs at 290 and 240 nm in the CD spectrum of 5 (Fig. S78C) suggested that the absolute configurations of 5 were 2 S, 3 S (Slade et al., 2005). Hence, compound 5 was identified as (2 S,3 S)- rugonine E. Compound 6 was acquired as an amorphous powder with [α] D 25 = + 34.1 (c 0.20, MeOH). The molecular formula, C 26 H 31 N 3 O 6, was deduced from its HRESIMS ion peak at m / z 482.2316 [M + H] + (calcd for C 26 H 32 N 3 O 6, 482.2291). The 1 H and 13 C NMR spectra of 6 shared a pattern similar to those of 1 except for the absence of sugar moiety. This led to the conclusion that 6 is another derivative of guanidine-catechin with an isoprenyl substituent that included two methyl groups at C-4 ′′′ (δ H 1.64, δ C 25.3) and C-5 ′′′ (δ H 1.68, δ C 17.8), an olefinic bond at C-3 ′′′ (δ C 135.0), C-2 ′′′ (δ H 5.22, δ C 120.2), and methylene at C-1 ′′′ (δ H 3.75, δ C 44.1). The connection between the isopentyl group and guanidine moiety was confirmed via the HMBC correlation from H-1 ′′′ (δ H 3.75 ppm) to δ C 146.8 ppm. Similar to 1, the absolute configuration of 6 was elucidated as 2 R, 3 S by the large coupling constants of H-2 (J = 5.8 Hz), H-3 (J = 6.2 Hz) indicating a 2,3- trans flavan-3-ol, and negative CEs at approximately 280–290 nm and 240 nm in CD spectra (Fig. S78A). Therefore, compound 6 was determined to be (2 R,3 S)-rugonine F. Compound 7 was isolated as an amorphous powder with [α] D 25 = + 47.5 (c 0.20, MeOH). The molecular formula, C 27 H 33 N 3 O 10, was deduced from its HRESIMS ion peak at m / z 560.2272 [M + H] + (calcd for C 27 H 34 N 3 O 10, 560.2244). The 1 H and 13 C NMR data of compound 7 shared similarity with compound 1, excluding the absence of the isoprenyl substituent. The sugar moiety was determined by an anomeric signal (δ H 4.30, δ C 99.9 ppm), four oxygenated carbons (δ H 3.15–3.47 ppm, and δ C 69.0–72.0 ppm), and an additional methyl at δ H 1.14, δ C 17.9 ppm, which indicated the presence of a rhamnose unit in 7. Based on the HMBC correlations from anomeric proton H-1’’’’ (δ H 4.30) to C-3 (δ C 72.04 ppm), the sugar moiety of 7 was identified as 3-O-rhamnoside. The relative configuration of the sugar unit was determined by analyzing J coupling constants and NOESY correlations. The small coupling constant of H-1’’’’ (δ H 4.30, s) and large 1 J C-H (170.0 Hz) suggested an α orientation of C-1’’’’, and the observed large coupling constants 3 J H-H of H-4’’’’ (9.1 Hz) and H-5’’’’ (12.4, 6.1 Hz) indicated that H-3’’’’/H-4’’’’/H-5’’’’ were in axial orientation. Furthermore, the relative configuration of the sugar, which was established by its J values, was supported by the NOESY correlations of 7 (Fig. 3). The absolute configuration of rhamnose moiety was also determined to be α -L-rhamnose by Tanaka’ s method as the same as in 1–3 (Tanaka et al., 2007). The stereocenters at C-2 and C-3 of 7 were determined to be 2 R, 3 S based on their large J values at H-2 (J = 6.7 Hz) and H-3 (J = 12.7, 6.5 Hz), which indicated a 2,3- trans flavan-3-ol, and negative CEs at approximately 290 nm and 240 nm in experimental CD data (Fig. S78A). Finally, the structure of 7 was identified as (2 R,3 S)- rugonine G. 25 Compound 8 was obtained as an amorphous powder with [α] D = + 20.3 (c 0.20, MeOH). The molecular formula, C 21 H 23 N 3 O 5, was deduced from its HRESIMS ion peak at m / z 396.1581 [M H] (calcd. for C 21 H 22 N 3 O 5, 396.1565). The 1 H NMR and 13 C NMR data of 8 showed similarities to those of 4 and 5, and the mass was different by 16 Da, suggesting that the structure of 8 differed from those of 4 and 5 by less than one hydroxy group. The differences were also indicated by the presence of methylene at δ C 28.6/ δ H 2.00, 1.82 instead of an oxygenated group as in 4 and 5 and the greater upfield shift of C-4 (δ C 18.8) in 8 compared to the other compounds. Correlations from H-2 (δ H 4.80) to C-3 (δ C 28.6) and C-4 on the HMBC spectrum and the 1 H– 1 H COSY spin system of H-2/H 2 -3/H 2 -4 suggested that 8 contained a C 6 –C 3 –C 6 ring similar to that of luteoliflavan (Roemmelt et al., 2003). Hence, the planar structure of 8 was identified as shown in Fig. 2. The configuration of C-2 was determined by comparing its ECD and NMR data with references. To date, only a few flavans have been reported to occur naturally and all of them have the 2 R absolute configuration that would be expected from the flavanone origin (Slade et al., 2005). Flavans showed a low specific rotation, making firm conclusions difficult, and their configuration could be determined only by studying their CD data (Slade et al., 2005). Experimentally, the CD spectrum of 8 showed a negative cotton effect (CE) at a 1 L b of 285 nm (Fig. S78A), which indicated the 2 R absolute configuration (Slade et al., 2005). Moreover, the NMR data of 8 shared similarity with those of luteoliflavan 5-glucoside (Roemmelt et al., 2003), suggesting a 2 R configuration. Therefore, compound 8 was identified as (2 R)-rugonine H. Table 1 1 H and 13 C NMR data for compounds 1–3 in methanol- d 4. a 1 H and 13 C NMR spectra were acquired at 500 and 125 MHz, respectively. b 1 H and 13 C NMR spectra were acquired at 600 and 150 MHz, respectively. c 1 H and 13 C NMR spectra were acquired at 800 and 200 MHz, respectively. d Data recorded in DMSO‑ d (Fig. S14). 6 2.3. Biological activities of compounds 1–9 in autophagy modulation To screen the autophagy regulatory activities of compounds 1–9, HEK293 cells stably expressing GFP-LC3 were administered. In HEK293 cells, the formation of puncta could be observed by using chloroquine (CQ) and rapamycin (RAPA), which are known to inhibit and induce autophagy, respectively. In the confocal microscopic image, the tested CQ and RAPA showed the formation of puncta, and a GFP signal was detected in the cell cytosol. The results indicated that the formation of puncta in the HEK293 cells stably expressing GFP-LC revealed autophagy regulation of the compounds. Nine isolated compounds from A. rugosa (1–9) were treated at a concentration of 20 μM for 24 h, and the cytosol were checked under a confocal microscope. Compared with the control groups, a significant increase in LC3 puncta in HEK293-GFP-LC3 cells was observed in the cells treated with c, Published as part of Doan, Thi-Phuong, Park, Eun-Jin, Ryu, Byeol, Cho, Hyo-Moon, Yoon, Sang-Jun, Jung, Gwan-Young, Thuong, Phuong-Thien & Oh, Won-Keun, 2023, Unique guanidine-conjugated catechins from the leaves of Alchornea rugosa and their autophagy modulating activity, pp. 113521 in Phytochemistry (113521) (113521) 206 on pages 2-8, DOI: 10.1016/j.phytochem.2022.113521, http://zenodo.org/record/8160626, {"references":["Barrosa, K., Pinto, E., Tempone, A., Martins, E., Lago, J., 2014. Alchornedine, a new antitrypanosomal guanidine alkaloid from Alchornea glandulosa. Planta Med. 80, 1310 - 1314. https: // doi. org / 10.1055 / s- 0034 - 1382994.","Tapondjou, L. A., Kristina, J., Siems, K., 2016. Alchornealaxine, an unusual prenylguanidinyl-epicatechin derivative from alchornealaxine from Alchornea laxiflora (Benth) Pax and Hoffman. Record Nat. Prod. 10, 508 - 512.","Tanaka, T., Nakashima, T., Ueda, T., Tomii, K., Kouno, I., 2007. Facile discrimination of aldose enantiomers by reversed-phase HPLC. Chem. Pharm. Bull. 55, 899 - 901. https: // doi. org / 10.1248 / cpb. 55.899.","Slade, D., Ferreira, D., Marais, J. P. J., 2005. Circular dichroism, a powerful tool for the assessment of absolute configuration of flavonoids. Phytochemistry 66, 2177 - 2215. https: // doi. org / 10.1016 / j. phytochem. 2005.02.002.","Roemmelt, S., Zimmermann, N., Rademacher, W., Treutter, D., 2003. Formation of novel flavonoids in apple (Malus domestica) treated with the 2 - oxoglutarate-dependent dioxygenase inhibitor prohexadione-Ca. Phytochemistry 64, 709 - 716. https: // doi. org / 10.1016 / S 0031 - 9422 (03) 00389 - 3.","Wang, H., Liu, T., Li, L., Wang, Q., Yu, C., Liu, X., Li, W., 2015. 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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., Published as part of 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, pp. 112181 in Phytochemistry (112181) (112181) 170 on pages 2-7, DOI: 10.1016/j.phytochem.2019.112181, http://zenodo.org/record/8160259, {"references":["Wu, Z. Y., Raven, P. H., 1995. Flora of China, vol. 16 (Gentianaceae through Boraginaceae). Science Press, Beijing, and Missouri Botanical Garden Press, St. Louis.","Deokule, S. S., Kavade, S. P., Lakshminarasimhan, P., Berde, V. B., 2013. An endemic and critically endangered species, Gymnema khandalense Santapau (Apocynaceae: Asclepiadoideae) - a new record to Goa State, India. J. Threat 5, 4598 - 4600. https: // doi. org / 10.11609 / JoTT. o 3438.4598 - 600.","Ramachandran, A., Viswanathan, M. B. G., 2009. A new species of Gymnema (asclepiadaceae) from the Kollihills in Peninsular India. Adansonia 31, 407 - 411. https: // doi. org / 10.5252 / a 2009 n 2 a 10.","Meve, U., Alejandro, G. J., 2011. Taxonomy of the asian Gymnema latifolium (Apocynaceae, Asclepiadoideae, Marsdenieae), including lectotypification of the synonymous. G. khandalense. Will. 43, 81 - 86. https: // doi. org / 10.3372 / wi. 43.43108.","Liu, H. - M., Kiuchi, F., Tsuda, Y., 1992. Isolation and structure elucidation of gymnemic acids, antisweet principles of Gymnema sylvestre. Chem. Pharm. Bull. 40, 1366 - 1375. https: // doi. org / 10.1248 / cpb. 40.1366.","Yoshikawa, K., Nakagama, M., Yamamoto, R., Arihara, S., Matsuura, K., 1992. Antisweet natural products. V. Structures of gymnemic acids VIII-XII from Gymnema sylvestre R. Br. Chem. Pharm. Bull. 40, 1779 - 1782. https: // doi. org / 10.1248 / cpb. 40.1779.","Yoshikawa, K., Amimoto, K., Arihara, S., Matsuura, K., 1989. Structure studies of new antisweet constituents from Gymnema sylvestre. Tetrahedron Lett. 30, 1103 - 1106. https: // doi. org / 10.1016 / S 0040 - 4039 (01) 80371 - 3.","Pham, H. T. T., Hoang, M. C., Ha, T. K. Q., Dang, L. H., Tran, V. O., Nguyen, T. B. T., Lee, C. H., Oh, W. K., 2018. Discrimination of different geographic varieties of Gymnema sylvestre, an anti-sweet plant used for the treatment of type 2 diabetes. Phytochemistry 150, 12 - 22. https: // doi. org / 10.1016 / j. phytochem. 2018.02.013.","Yoshikawa, K., Taninaka, H., Kan, Y., Arihara, S., 1994. Antisweet natural products. X. Structures of sitakisosides I-V from Stephanotis lutchuensis Koidz. var. japonica. Chem. Pharm. Bull. 42, 2023 - 2027."]}

A screening of Sudanese medicinal plants for antiprotozoal activities revealed that the chloroform and water fractions of the ethanolic root extract of Haplophyllum tuberculatum exhibited appreciable bioactivity against Leishmania donovani. The antileishmanial activity was tracked by HPLC-based activity profiling, and eight compounds were isolated from the chloroform fraction. These included lignans tetrahydrofuroguaiacin B (1), nectandrin B (2), furoguaiaoxidin (7), and 3,3&prime, dimethoxy-4,4&prime, dihydroxylignan-9-ol (10), and four cinnamoylphenethyl amides, namely dihydro-feruloyltyramine (5), (E)-N-feruloyltyramine (6), N,N&prime, diferuloylputrescine (8), and 7&prime, ethoxy-feruloyltyramine (9). The water fraction yielded steroid saponins 11&ndash, 13. Compounds 1, 2, and 5&ndash, 13 are reported for the first time from Haplophyllum species and the family Rutaceae. The antiprotozoal activity of the compounds plus two stereoisomeric tetrahydrofuran lignans&mdash, fragransin B2 (3) and fragransin B1 (4)&mdash, was determined against Leishmania donovani amastigotes, Plasmodium falciparum, and Trypanosoma brucei rhodesiense bloodstream forms, along with their cytotoxicity to rat myoblast L6 cells. Nectandrin B (2) exhibited the highest activity against L. donovani (IC50 4.5 &micro, M) and the highest selectivity index (25.5).