Search

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. Tetrandrine is a potent cell autophagy agonist via activated intracellular reactive oxygen species. Cell Biosci. 5 https: // doi. org / 10.1186 / 2045 - 3701 - 5 - 4.","Berlinck, R. G. S., Bertonha, A. F., Takaki, M., Rodriguez, J. P. G., 2017. The chemistry and biology of guanidine natural products. Nat. Prod. Rep. 34, 1264 - 1301. https: // doi. org / 10.1039 / C 7 NP 00037 E.","Sohn, E. J., Park, H. T., 2017. Natural agents mediated autophagic signal networks in cancer. Cancer Cell Int. 17, 110. https: // doi. org / 10.1186 / s 12935 - 017 - 0486 - 7.","Sartori, R., Romanello, V., Sandri, M., 2021. Mechanisms of muscle atrophy and hypertrophy: implications in health and disease. Nat. Commun. 12, 330. https: // doi. org / 10.1038 / s 41467 - 020 - 20123 - 1.","Coppens, I., 2011. Metamorphoses of malaria: the role of autophagy in parasite differentiation. Essays Biochem. 51, 127 - 136. https: // doi. org / 10.1042 / bse 0510127.","Ghartey-Kwansah, G., Aboagye, B., Adu-Nti, F., Opoku, Y. K., Abu, E. K., 2020. Clearing or subverting the enemy: role of autophagy in protozoan infections. Life Sci. 247, 117453 https: // doi. org / 10.1016 / j. lfs. 2020.117453.","Ray, A., Mathur, M., Choubey, D., Karmodiya, K., Surolia, N., 2022. Autophagy Underlies the proteostasis mechanisms of artemisinin resistance in P. falciparum Malaria. mBio 13, 1 - 19. https: // doi. org / 10.1128 / mbio. 00630 - 22."]}