Unique guanidine-conjugated catechins from the leaves of Alchornea rugosa and their autophagy modulating activity Author Doan, Thi-Phuong , Eun-Jin Park &, Byeol Ryu &, Hyo-Moon Cho &, Sang-Jun Yoon &, &, Phuong-Thien Thuong & * & Korea Bioactive Natural Material Bank, Research Institute of Pharmaceutical Sciences, College of Pharmacy, Seoul National University, Seoul, 08826, Republic of &, Eun-Jin Park &, Byeol Ryu Author Park, Eun-Jin Author Ryu, Byeol Author Cho, Hyo-Moon Author Yoon, Sang-Jun Author Jung, Gwan-Young Author Thuong, Phuong-Thien Author Oh, Won-Keun text Phytochemistry 2023 113521 2023-02-28 206 113521 113521 http://dx.doi.org/10.1016/j.phytochem.2022.113521 journal article 56273 10.1016/j.phytochem.2022.113521 d37bcf61-e44a-48bf-8733-1bd80b306d94 1873-3700 8160626 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. Fig. 1. Chemical structures of 1–9 from the leaves of A. rugosa . 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. Fig. 2. Key 1 H– 1 H COSY (bold) and HMBC (red arrow) correlations for 1–8 . (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.) 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. Fig. 3. Key NOESY correlations for sugar moieties of compounds 1–3 and 7 . 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.

Position	1b	2 a	3 c
δ H (J in Hz )	δ C	δ H (J in Hz )	δ C	δ H (J in Hz )	δ C
2	4.71,	81.2	4.67, d (7.3)	81.1	4.70,	81.2
d (7.3)	d (7.3)
3	3.97, q	75.4	3.93, q (7.3)	75.2	3.96, td	75.3
(7.3)	(5.4, 7.7)
4	2.86, dd	27.3	2.83, dd	27.3	2.86, dd	27.4
(5.5, 16.5)	(5.5, 16.3)	(5.4, 16.2)
2.75, dd	2.69, dd	2.72, dd
(7.7, 16.5)	(7.8, 16.3)	(5.4, 16.2)
5	158.8	157.2	158.7
6	6.10, s	96.0	6.06, s	95.9	6.09, s	95.8
7	157.3	155.7	157.3
8	95.9	95.7	95.8
9	155.5	155.4	157.3
10	100.8	100.7	100.8
1′	131.6	131.5	131.6
2′	6.76,	115.0	6.73, d (2.0)	114.9	6.76,	115.0
d (2.0)	d (2.1)
3′	146.2	146.1	146.2
4′	146.4	146.3	146.4
5′	6.73,	116.1	6.69, d (8.1)	115.9	6.72,	116.0
d (7.8)	d (8.1)
6′	6.66, dd	119.6	6.62, dd	119.5	6.65, dd	119.6
(2.3, 8.3)	(2.0, 8.1)	(2.1, 8.1)
1′′	115.0	114.9	114.9
2′′	131.3	131.2	131.2
3′′	2.78, q	26.2	2.75, q (7.3)	26.2	2.76, p	26.2
(7.3)	(7.0)
4′′	1.11,	21.5	1.07, d (7.1)	21.4	1.10,	21.5
d (7.0)	d (7.0)
5′′	1.13,	21.6	1.10, d (7.1)	21.5	1.12,	21.6
d (7.0)	d (7.0)
C= NH	11.93d	147.6	11.31	147.6	11.43	147.6
1’’’	3.81,	41.9	3.77, d (7.0)	41.8	3.34,	40.4
d (6.9)	d (6.9)
2’’’	5.26, t	120.1	5.23, t (7.3)	120.1	1.75, dd	42.4
(6.9)	(6.9, 8.2)
3’’’	138.5	138.4	70.9
4’’’	1.69, s	18.0	1.66, s	17.9	1.23,	29.5
d (5.0)
5’’’	1.73, s	25.7	1.70, s	25.6	1.23,	29.5
d (5.0)
1’’’’	4.35,	102.0	4.33, s	102.0	4.35,	102.0
d (1.5)	d (1.7)
2’’’’	3.54,	72.0	3.51, br s	71.9	3.53, q	72.0
d (3.4)	(1.7)
3’’’’	3.57, dd	72.3	3.55, br s	72.1	3.56,	72.2
(3.4, 9.4)	d (9.5)
4’’’’	3.33,	73.9	3.28,	73.8	3.33,	73.9
d (9.4)	d (10.0)	d (7.4)
5’’’’	3.65, dq	70.3	3.62, dt	70.3	3.64, dd	70.3
(6.3, 12.4)	(3.4, 6.1)	(6.2, 9.5)
6’’’’	1.25,	17.9	1.21, d (6.1)	17.8	1.24,	17.9
d (6.3)	d (6.2)
1′′ -NH	11.65d	11.23	11.31

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 compounds 4–7 at a concentration of 20 μM ( Fig. 4 ). 2.4. Compounds 4–7 inhibit autophagic flux To investigate autophagy regulation by the tested compounds, the expression levels of LC3B and p62 proteins were detected. The LC3B level was calculated as the LC3B II/I level, indicating the transition of LC3B I to II in autophagy. The p62 protein level was detected to monitor the degradation level in autophagosomes. CQ and RAPA were used as an autophagy inhibitor and activator, respectively. Under the CQ treatment, both the protein levels of LC3B and p62 were increased. Since CQ blocks the autophagic system, p62 could not be degraded, and LC3B II and I could not be used properly; therefore, they accumulated in the cells. In contrast, in the RAPA-treated group, autophagy was induced, leading to a strong LCB I to II transition; hence, a decrease in the LC3B I level and an increase in the LC3II level were observed in the Western blot results. Moreover, the p62 level was decreased due to the degradation of autophagolysosomes ( Wang et al., 2015 ). As a result, compounds 4–7 at various concentrations of 5 and 20 μM exhibited the same effects in the CQ-treated group, which showed increases in the LC3B and p62 levels ( Fig. 5A ). Furthermore, the p62 and LC3 II proteins expression levels showed an increase in a dose-dependent manner in comparison to the control group ( Fig. 5A ). This finding suggested that these compounds inhibited protein degradation by the autolysosome by blocking the fusion of autophagosomes and lysosomes. To prove this hypothesis, GFP-mRFP-LC3 was transfected into HEK293 cells, and autophagosomes were examined using confocal microscopy. GFP-mRFP-LC3 transfected cells were treated with compounds 4–7 at a concentration of 20 μM, and DAPI staining was performed on glass slides. GFP and mRFP were activated in the autophagosome, but when the autophagosome became an autolysosome, GFP was deactivated due to the autolysosome’ s pH. Therefore, in the RAPA-treated group in which autophagy was induced, GFP activation was lost, but mRFP puncta were activated and expressed. Thus, the combined image showed red fluorescence puncta that indicated the increased autophagy flux and increased numbers of autolysosomes under RAPA treatment compared to the control group ( Fig. 5B ). In contrast, the CQ treatment group had a higher quantity of yellow puncta than the control group in the merged image, suggesting that autophagosome activation was prevented by CQ treatment, and autophagosomes accumulated in the cytosol, revealing a large number of GFP- and mRFP-activated autophagosomes ( Fig. 5B ). Based on this result, the effects of compounds 4–7 on autophagosomes were also analyzed, and the yellow puncta in these treatment groups appeared strongly. According to these findings, guanidine-conjugated catechins ( 4–7 ) isolated from A. rugosa leaves have an inhibitory effect on autophagic flux. Based on the structures and activities of 1–9 , a gross SAR for this guanidine type can be delineated as follows: (1) the core skeleton of guanidine-conjugated flavan-3-ol is crucial for autophagy modulation activity since the activities were observed in 4–7 but not 8 , which has no hydroxy functional group at C-3 and showed no effect in the autophagy assay ( Fig. 5A ); (2) the addition of one substituent, such as one sugar moiety or a side chain, does not affect the activities, but two or more substituents have an effect, as indicated by the activities of 6 and 7 and the lack of activities observed for 1–3 ( Fig. 1 ); (3) the different configurations at C-2 and C-3 did not affect the autophagy inhibition effects, as shown for 4 and 5 , which differed in the C-2 and C-3 absolute configurations but both showed activity. Contrary to other types of alkaloids, guanidines are less frequently found in natural products, particularly plant materials, but their significant hydrophilicity makes them attractive for drug development, and some have even been applied in clinical such as streptomycin ( Berlinck et al., 2017 ). By using HRESI-MS/MS-based molecular networking, the interesting cluster of guanidines from A. rugosa leaves has been quickly investigated in order to isolate undescribed natural guanidine derivatives. Notably, the finding of autophagy inhibition activities in guanidine-catechine conjugations ( 4–7 ) contributes to the potential activities of natural products in general and guanidine derivatives in particular, as well as explores the traditional use of A. rugosa . Firstly, compounds 4–7 showed autophagy inhibitory effects as same as the other guanidine derivative (DBeQ) suggesting that the guanidine functional group may be important for autophagy modulatory activity, which is promising for targeted therapy in cancer ( Sohn and Park, 2017 ), or muscle atrophy treatment ( Sartori et al., 2021 ). Secondly, the relationship between autophagy inhibitors and malarial management has explored the next target for malaria treatment ( Coppens, 2011 ; Ghartey-Kwansah et al., 2020 ) even artemisinin-resistant Plasmodium falciparum malaria ( Ray et al., 2022 ). CQ was used as an antimalarial for a long time and was also a well-known autophagy inhibitory agent. Compounds 4–7 showed autophagy inhibitory effects as CQ could contribute to the traditional use of A. rugosa in malaria treatment. Fig. 4. Screening for autophagy regulation by compounds ( 1–9 ) isolated from A. rugosa in HEK293 cells. HEK293 cells stably expressing GFP-LC3 were treated with 20 μM compounds for 24 h, and confocal imaging was used to examine the production of LC3-GFP puncta. 3. Conclusions In summary, the application of molecular networking allowed us to investigate eight undescribed guanidine-fused flavan derivatives from the leaves of A. rugosa . The structures of all isolated compounds were comprehensively elucidated by NMR and CD spectral analysis. These compounds were therefore tested, and compounds 4–7 showed potential autophagy inhibitory activities in HEK293 cells. The results showed the advantage of HRESI-qTOF-MS/MS-based molecular networking for targeting the isolation of guanidine derivatives from A. rugosa as well as their inhibitory activities on autophagy in HEK 293 cells, which could be promising for future drug discovery for the treatment of malaria, cancer and of muscle atrophy. 4. Experimental 4.1. General experimental procedures Optical rotation ([ α ] D 25 ) was measured by a polarimeter (JASCO P-2000, International Co. Ltd., Tokyo , Japan ). Fourier Transform Infrared Spectroscopy (FT-IR) was recorded by a FT-IR spectrometer (Nicolet 6700, Thermo Electron Corp., Waltham, MA, USA ). Circular dichroism (CD) data were conducted on a Chirascan CD spectrophotometer (Applied Photophysics, Leatherhead, UK ). Experimental CD data processing was done by Pro-Data Viewer software version 4.4.2.0. All NMR spectra were measured on Bruker Advance 500 and 600 MHz spectrometers (Bruker, Rheinstetten, Germany ). High-resolution electrospray ionization mass spectrometry (HRESIMS) data were acquired from a Waters XEVO G2 Q-TOF MS (Waters MS Technologies, Manchester , UK ) coupled with electrospray ionization (ESI), and a quadrupole Timeof-Flight (Q-TOF) Agilent 6530 spectrometer (Agilent Technologies, Inc., Santa Clara, CA, USA ). Various column chromatographies (CC) were applied for separation and purification steps, including Diaion HP-20 resin (by Mitsubishi Chemical Co., Tokyo , Japan ), Sephadex LH-20 (from Sigma–Aldrich, St. Louis, MO, USA ), medium pressure liquid chromatography (MPLC) equipped with a C 18 -PREP column (COSMOSIL 40, Nacalai Tesque Inc., Kyoto , Japan ). Thin-layer chromatography (TLC) separations were performed on RP-18 F254 (Merck KGaA, Darmstadt, Germany ) and visualized by TLC reagent (vanillin/sulfuric acid). A semipreparative high-performance liquid chromatography (HPLC, Gilson), an Optima Pak C18 column (5 μm, RS Tech, Seoul , Korea , i. d. 10 × 250 mm ), and UV detection (201 and 280 nm ) were used for purification. All of the fractionation and isolation processes used extra pure grade solvents (Daejung Chemicals & Metals Co. Ltd., Siheung, Korea ). Fig. 5. ( A ) The level of autophagy marker protein expression in cells treated with compounds 4–7 . LC3B was determined using the LC3B II/I ratio, which was then standardized against the control group (nontreated group). The p62 protein level was detected, and the expression level was normalized by dividing by the protein level in the control group. (CQ: chloroquine treatment group at 25 μM, Rapa: rapamycin treatment group at 0.25 μM). ( B ) GFP-mRFP-tagged LC3 puncta after treatment with the test compounds. HEK293 cells were treated with autophagy-regulating compounds 4–7 , and LC3 puncta were detected by using confocal microscopy. 4.2. Plant material Leaves of Alchornea rugosa (Lour.) Müll.Arg. (Euphorbiaceae) were collected in May 2016 (muggy and hot season) at Quang Trung , Ngoc Lac , Thanh Hoa , Vietnam ( 20 ◦ 8 ′ 3 ′′ N , 105 ◦ 24 ′ 15 ′′ E ). The sample was authenticated by Dr. P. T. Thuong , Vietnam-Korea Institute of Science and Technology (VKIST), Hanoi, Vietnam. The sample voucher specimen (accession number: VKIST-HP-01 ) was deposited in the VKIST’ s Herbarium .