Benzylisoquinoline Alkaloids from the Stems of Limacia scandens and Their Potential as Autophagy Inhibitors

Limacia scandens is traditionally used to treat depression and affective disorders in Malaysia. The chemical compositions have been reported to include bisbenzylisoquinoline and aporphine-type alkaloids in the genus Limacia Lour., but studies on the components of L. scandens have rarely been reported. Therefore, this study was conducted to determine new benzylisoquinoline alkaloid derivatives with autophagy regulation activity from this plant. Bioactivity-guided isolation was applied to various column chromatography methods using RP-18, Sephadex LH-20 open column chromatography, and preparative HPLC. The chemical structures of the isolated compounds were elucidated through spectroscopic data analysis, including NMR, HR-ESI-MS, and ECD data. In addition, isolated compounds were tested for autophagy-regulating activity in HEK293 cells expressing GFP-L3. Three new dimeric benzylisoquinoline alkaloids (1−3), one new 4-hydroxybenzoic acid-conjugated benzylisoquinoline alkaloid (4), and six known compounds (5−10) were isolated from the stems of L. scandens. All compounds (1–10) were screened for autophagy regulation in HEK293 cells stably expressing the GFP-LC3 plasmid. Among the isolated compounds, 1, 2, and 4 showed autophagic regulation activity that blocked the process of combining autophagosomes and lysosomes. They also inhibit the protein degradation process from the autolysosome as inhibitors of autophagy. Novel benzylisoquinoline alkaloids from L. scandens showed potent potency for the inhibition of autophagic flux. This study provides potential candidates for developing natural autophagy inhibitors for disease prevention and treatment.

harmaceuticals 2022, 14, x FOR PEER REVIEW 4 of 14 between C-11 and C-12′ of the two benzylisoquinoline moieties [13]. The absolute configuration of 1 was calculated using TDDFT at the 6-31G/B3LYP level according to the Boltzmann distribution. The experimental ECD spectrum of 1R,α′R closely matches the calculated ECD spectrum ( Figure 3A). Therefore, the structure of limaciascadine A was assigned as shown.  between C-11 and C-12′ of the two benzylisoquinoline moieties [13]. The absolute configuration of 1 was calculated using TDDFT at the 6-31G/B3LYP level according to the Boltzmann distribution. The experimental ECD spectrum of 1R,α′R closely matches the calculated ECD spectrum ( Figure 3A). Therefore, the structure of limaciascadine A was assigned as shown.   , 581.2288) from a positive ion peak of HRESIMS. The 1 H and 13 C NMR data were similar to those of 1, suggesting that 2 is an isomer of 1. The absolute configuration of 2 was determined as 1R,α S by comparing the experimental ECD spectrum that calculated the ECD spectrum for 1R,α S and 1S,α R ( Figure 3B).

Screening of Autophagy Regulation in HEK293 Cells Stably Expressing GFP-LC3
Many human diseases, including cancer and neurodegenerative diseases, are linked to autophagy. To identify potential autophagy-regulating compounds, HEK293 cells stably expressing GFP-LC3 were used to screen for autophagy regulation. In HEK293 cells, chloroquine and rapamycin, which are known to inhibit and induce autophagy drugs, were used as positive controls, showing the formation of punctuates [20,21]. In the confocal microscopic image, the tested chloroquine and rapamycin exhibited the formation of puncta, and a strong GFP signal was detected in the cell cytosol. These results indicated that the formation of puncta in HEK293 cells stably expressing GFP-LC3 reveals the autophagy regulation of the compounds. Cells were treated with the isolated compounds (1−10) at 20 µM concentrations for 24 h, and the cytosol was observed under a confocal microscope (Figure 4). Compound 1 showed the strongest formation of LC3 puncta among the treated compounds, and this result was higher than that in the control group. Compound 2, which is only different at α-OH or β-OH compared to that of 1, showed autophagic regulation activity similar to that of 1. Interestingly, 3 with a ketone functionality instead of α-OH or β-OH in 1 and 2 disappeared the formation of the punctuates at the same concentration. Similarly, 4 with a carboxyl group also showed strong punctuates. Thus, 1, 2 and 4 were selected for further investigation into whether the compounds were autophagy inhibitors or inducers.

Upregulating the Protein Expression Levels of LC3B and p62 in HEK293 Cells
The autophagic system is involved in both the bulk degradation of cytosolic proteins and the selective degradation of cytoplasmic organelles. Autophagosomes fuse the engulfed substrates with the lysosomes, and the formed autolysosomes, which are active at an acidic pH, degrade the cytoplasm-derived compartments, including the inner membrane of autophagosomes [22]. During autophagy, a cytosolic form of LC3 (LC3-I) is changed to LC3-phosphatidylethanolamine conjugate (LC3-II) on the autophagosomal membranes. p62 is a ubiquitin-binding protein that is recruited into autophagosomes due to its direct interaction with LC3-II. As autophagy progresses, p62 is degraded, but when autophagy is inhibited, p62 is not degraded and appears to increase [23]. To determine whether the isolated compounds based on autophagy marker proteins induce or inhibit autophagy, changes in p62, LC3B-I, and LC3B-II as marker proteins were detected by Western blot. LC3B levels were calculated as LC3B II/I levels, which indicate the level of transition from LC3B I to II. Protein levels of p62 were also detected to monitor the degradation tendency of autophagosomes. Chloroquine and rapamycin were treated as controls for autophagy of the inhibitor and activator, respectively. The expression levels of LC3B and p62 increased in the chloroquine-treated group, an autophagy inhibitor, and these results indicated that p62, LC3B II and I were not degraded but accumulated in the cells. In the rapamycin-treated group, Western blot results showed that the levels of LC3B-I and p62 were decreased, whereas the concentration of LC3B-II was increased. Interestingly, LC3B II/I levels were increased, indicating that conversion from LC3B I to II was actively processed as autophagy was activated [24]. When the isolated 1, 2, and 4 were treated at concentrations of 5 μM and 20 μM, the results, which are similar to those of the chloroquine-treated group, are shown. Compound 1 showed the highest intensity at the p62 protein level, and the LC3 II level also increased strongly compared to the control group in a dose-dependent manner. Compounds 2 and 4 also showed an increase in ac-

Upregulating the Protein Expression Levels of LC3B and p62 in HEK293 Cells
The autophagic system is involved in both the bulk degradation of cytosolic proteins and the selective degradation of cytoplasmic organelles. Autophagosomes fuse the engulfed substrates with the lysosomes, and the formed autolysosomes, which are active at an acidic pH, degrade the cytoplasm-derived compartments, including the inner membrane of autophagosomes [22]. During autophagy, a cytosolic form of LC3 (LC3-I) is changed to LC3-phosphatidylethanolamine conjugate (LC3-II) on the autophagosomal membranes. p62 is a ubiquitin-binding protein that is recruited into autophagosomes due to its direct interaction with LC3-II. As autophagy progresses, p62 is degraded, but when autophagy is inhibited, p62 is not degraded and appears to increase [23]. To determine whether the isolated compounds based on autophagy marker proteins induce or inhibit autophagy, changes in p62, LC3B-I, and LC3B-II as marker proteins were detected by Western blot. LC3B levels were calculated as LC3B II/I levels, which indicate the level of transition from LC3B I to II. Protein levels of p62 were also detected to monitor the degradation tendency of autophagosomes. Chloroquine and rapamycin were treated as controls for autophagy of the inhibitor and activator, respectively. The expression levels of LC3B and p62 increased in the chloroquine-treated group, an autophagy inhibitor, and these results indicated that p62, LC3B II and I were not degraded but accumulated in the cells. In the rapamycin-treated group, Western blot results showed that the levels of LC3B-I and p62 were decreased, whereas the concentration of LC3B-II was increased. Interestingly, LC3B II/I levels were increased, indicating that conversion from LC3B I to II was actively processed as autophagy was activated [24]. When the isolated 1, 2, and 4 were treated at concentrations of 5 µM and 20 µM, the results, which are similar to those of the chloroquine-treated group, are shown. Compound 1 showed the highest intensity at the p62 protein level, and the LC3 II level also increased strongly compared to the control group in a dose-dependent manner. Compounds 2 and 4 also showed an increase in activity similar to that of 1, but the activity of 4 was weaker than that of 1 and 2 ( Figure 5). These results strongly suggested that 1, 2, and 4 blocked the process of combining autophagosomes and lysosomes and inhibited the protein degradation process by the autolysosomes.

Blocking Autophagic Flux in HEK293 Cells Transfected with GFP-mRFP-LC3
To determine whether isolated 1, 2, and 4 inhibited autophagy at the cellular level, HEK293 cells were transfected with GFP-mRFP-LC3, and autophagosomes were directly analyzed by confocal microscopy. When autophagosomes are generated, both GFP and mRFP are activated, allowing the identification of yellow puncta. Rapamycin, an autophagy activator, increased the formation of autolysosomes, and the green signal of GFP was lost due to the acidic environment of the autolysosomes. As a result, the red mRFP signal strongly appeared, and these results suggested a relationship between autophagosomes and autolysosomes induced by the compounds [25]. GFP-mRFP-LC3-transfected cells were treated with the compounds at a concentration of 20 μM, and nuclear counterstaining was detected with DAPI staining. The formation of autophagosomes by the autophagy inhibitor chloroquine was detected with strong green fluorescence of GFP-LC3 and red Figure 5. The autophagy-related protein expression level of compound-treated cells. LC3B was calculated by the LC3B II/I ratio, and the ratio was normalized to the control group (nontreated group). The p62 protein level was detected, and the expression level was divided by the control group's protein level for normalization. (Rapa: rapamycin treatment group; CQ: chloroquine treatment group).

Blocking Autophagic Flux in HEK293 Cells Transfected with GFP-mRFP-LC3
To determine whether isolated 1, 2, and 4 inhibited autophagy at the cellular level, HEK293 cells were transfected with GFP-mRFP-LC3, and autophagosomes were directly analyzed by confocal microscopy. When autophagosomes are generated, both GFP and mRFP are activated, allowing the identification of yellow puncta. Rapamycin, an autophagy activator, increased the formation of autolysosomes, and the green signal of GFP was lost due to the acidic environment of the autolysosomes. As a result, the red mRFP signal strongly appeared, and these results suggested a relationship between autophagosomes and autolysosomes induced by the compounds [25]. GFP-mRFP-LC3-transfected cells were treated with the compounds at a concentration of 20 µM, and nuclear counterstaining was detected with DAPI staining. The formation of autophagosomes by the autophagy inhibitor chloroquine was detected with strong green fluorescence of GFP-LC3 and red fluorescence of mRFP-LC3 in the cytoplasm. The chloroquine-treated group showed more yellow puncta than the control group in the merged image; thus, the result demonstrated that autophagosomes were blocked by chloroquine treatment and stacked in the cytoplasm as yellow fluorescence. In contrast, the rapamycin-treated group destroyed the green fluorescence of GFP-LC3 in autolysosomes, and strong mRFP puncta were detected, which means that rapamycin treatment exerted autophagic flux. The increase in yellow puncta after treatment with isolated 1, 2, and 4 clearly showed an inhibitory effect on autophagic flux ( Figure 6). icals 2022, 14, x FOR PEER REVIEW 9 of 14 puncta after treatment with isolated 1, 2, and 4 clearly showed an inhibitory effect on autophagic flux ( Figure 6). Figure 6. The GFP-mRFP-tagged LC3 punctuates images with the treatment of compounds. HEK293 cells were treated with autophagy-regulating 1, 2, and 4. The LC3 puncta were detected using confocal microscopy.

Discussion
In this study, stems and roots of L. scandens are used in traditional medicine to have therapeutic intestinal disease activity and sympathomimetic activity [12], but there are few reports of the chemical composition and biological activity of L. scandens. The four Figure 6. The GFP-mRFP-tagged LC3 punctuates images with the treatment of compounds. HEK293 cells were treated with autophagy-regulating 1, 2, and 4. The LC3 puncta were detected using confocal microscopy.

Discussion
In this study, stems and roots of L. scandens are used in traditional medicine to have therapeutic intestinal disease activity and sympathomimetic activity [12], but there are few reports of the chemical composition and biological activity of L. scandens. The four novel benzylisoquinoline alkaloids along with six known compounds were isolated by various column chromatography methods from the stems of L. scandens. Three new dimeric benzylisoquinoline alkaloids (1−3) and one new 4-hydroxybenzoic acid conjugated benzylisoquinoline alkaloid (4) are novel substances isolated from this plant. Compounds 1, 2, and 4 had autophagic regulatory activity that prevented the process of autophagosomes and lysosomes. Additionally, these compounds acted as autophagy inhibitors in HEK293 cells expressing GFP-L3, preventing the autolysosome from degrading proteins (Figure 7). Compounds 1−3 are derivatives of similar structures having different structures only in the α position. Compounds 1 and 2, which differ only in α-OH and β-OH at α position, showed similar activity, but compound 3, having a ketone functionality at the same position, did not. These results revealed that compounds with similar derivatives exhibited different biological activities depending on the functional group.
tures only in the αʹ position. Compounds 1 and 2, which differ only in α-OH and β-OH at αʹ position, showed similar activity, but compound 3, having a ketone functionality at the same position, did not. These results revealed that compounds with similar derivatives exhibited different biological activities depending on the functional group.
The regulation of autophagy is an essential mechanism in research on disease treatment. It has been reported that autophagy inhibitors enhance the effectiveness of cancer treatment. The efficacy of TRAIL was boosted by blocking the autophagic flux after treatment with 6-gingerol. Cotreatment of ovarian cancer cells with wortmannin, a wellknown autophagy inhibitor, and cisplatin increased the apoptosis-inducing effect of anticancer medications and decreased resistance to chemotherapy [26]. The aging muscles in patients with muscle atrophy or sarcopenia significantly enhanced autophagic flux and increased protein degradation in the muscles [27]. Docosahexaenoic acid (DHA) has been suggested as a treatment for sarcopenia, but few related studies have been reported. There is currently no drug development in which the exact target mechanism for sarcopenia has been identified. In this regard, the inhibitory action of autophagy could be an important drug target. Tetrandrine, a bisbenzylisoquinoline alkaloid isolated from Stephania tetrandra, inhibited autophagy by deacidification of lysosomes in the late stage of bladder, prostate, cervical, and pancreatic cancers [28]. Additionally, neferine isolated from Nelumbo nucifera promoted apoptosis through the accumulation of p62/SQSTM1 by inhibiting autophagic flux in head and neck squamous cell carcinoma [29]. We isolated and reported different types of bisbenzylisoquinoline alkaloids in this manuscript and confirmed their autophagy inhibitory activity. This result suggests the possibility of a new skeleton of a treatment candidate for anticancer drugs or sarcopenia-related drugs.

General Experimental Procedures
Optical rotations were recorded on a JASCO P-2000 polarimeter (JASCO International Co., Ltd., Tokyo, Japan). IR data were collected using a Nicolet 6700 FT-IR spectrometer (Thermo Electron Corp., Waltham, MA, USA). ECD spectra were obtained using Chirascan Plus (Applied Photophysics Ltd., Surrey, United Kingdom). A Waters Xevo G2 QTOF MS spectrometer (Waters Co., Milford, MA, USA) was used for high-resolution electrospray ionization mass spectrometry (HRESIMS) values. Semipreparative HPLC experiments were performed using a Gilson HPLC system with a 321 pump and a UV/VIS-155 detector. A Phenomenex Phenyl-Hexyl column (10 × 250 mm, 5 μm particle size, USA) was used as the HPLC column. The NMR spectra for 1D ( 1 H and 13 C) and 2D (HSQC, The regulation of autophagy is an essential mechanism in research on disease treatment. It has been reported that autophagy inhibitors enhance the effectiveness of cancer treatment. The efficacy of TRAIL was boosted by blocking the autophagic flux after treatment with 6-gingerol. Cotreatment of ovarian cancer cells with wortmannin, a well-known autophagy inhibitor, and cisplatin increased the apoptosis-inducing effect of anticancer medications and decreased resistance to chemotherapy [26]. The aging muscles in patients with muscle atrophy or sarcopenia significantly enhanced autophagic flux and increased protein degradation in the muscles [27]. Docosahexaenoic acid (DHA) has been suggested as a treatment for sarcopenia, but few related studies have been reported. There is currently no drug development in which the exact target mechanism for sarcopenia has been identified. In this regard, the inhibitory action of autophagy could be an important drug target. Tetrandrine, a bisbenzylisoquinoline alkaloid isolated from Stephania tetrandra, inhibited autophagy by deacidification of lysosomes in the late stage of bladder, prostate, cervical, and pancreatic cancers [28]. Additionally, neferine isolated from Nelumbo nucifera promoted apoptosis through the accumulation of p62/SQSTM1 by inhibiting autophagic flux in head and neck squamous cell carcinoma [29]. We isolated and reported different types of bisbenzylisoquinoline alkaloids in this manuscript and confirmed their autophagy inhibitory activity. This result suggests the possibility of a new skeleton of a treatment candidate for anticancer drugs or sarcopenia-related drugs.