Quassinoids from Twigs of Harrisonia perforata (Blanco) Merr and Their Anti-Parkinson’s Disease Effect

Six new C-20 and one new C-19 quassinoids, named perforalactones F-L (1–7), were isolated from twigs of Harrisonia perforata. Spectroscopic and X-ray crystallographic experiments were conducted to identify their structures. Through oxidative degradation of perforalactone B to perforaqussin A, the biogenetic process from C-25 quassinoid to C-20 via Baeyer–Villiger oxidation was proposed. Furthermore, the study evaluated the anti-Parkinson’s disease potential of these C-20 quassinoids for the first time on 6-OHDA-induced PC12 cells and a Drosophila Parkinson’s disease model of PINK1B9. Perforalactones G and I (2 and 4) showed a 10–15% increase in cell viability of the model cells at 50 μM, while compounds 2 and 4 (100 μM) significantly improved the climbing ability of PINK1B9 flies and increased the dopamine level in the brains and ATP content in the thoraces of the flies.


Introduction
Quassinoids are a group of structurally diverse natural products found in Simaroubaceae family plants, with over 500 ones identified to date [1].These compounds can be categorized into six groups: C-26, C-25, C-22, C-20, C-19, and C-18 types, based on their carbon backbone [2].Although their biogenetic pathway is not yet fully understood, it is believed that they originate from tetracyclic tirucallane triterpene and undergo a series of oxidation, rearrangement, and recyclization steps [3].Additionally, these quassinoids have been found to exhibit a wide range of biological and pharmacological effects, including antiinflammatory, anti-tumor, and anti-malarial properties [1,[3][4][5][6][7].Therefore, this type of compounds has become an area of significant interest in the scientific community [8,9].
Parkinson's disease (PD) is a common neurodegenerative condition [10].Unfortunately, there is currently no cure available, and the drugs that are available only provide symptomatic relief and can cause side effects [11].As a result, there is a pressing need for new anti-PD drugs [12,13].The PTEN-induced putative kinase 1 (PINK1) is a promising drug target [11], as mutations in these gene have been linked to early-onset PD [6,14,15].
drug target [11], as mutations in these gene have been linked to early-onset PD [6,14,15].Researchers have been exploring a variety of natural products for potential anti-PD compounds, and one such product is ruthenine A from black wolfberry (Figure 1) [16].PINK1 loss-of-function flies (PINK1 B9 ), a PD animal model, have exhibited PD-like symptoms, such as degeneration of flight muscle and dopaminergic neurons, shortened lifespan, and impaired locomotor abilities [17,18].Harrisonia perforata (Bl.)Merr.belongs to the Simarubaceae family and is a shrub plant [19].A number of chemical components have been identified in previous studies, including quassinoids, limonoids, chromones, and polyketides [4,6,[20][21][22].Our previous research found that some of these compounds exhibit biological activities against neurodegenerative disease through various mechanisms (Figure 1).For example, harpertrioate A reduced Aβ42 and Aβ40 production and shifted APP processing toward a nonamyloidogenic pathway, while perforalactones D and E significantly induced lysosomal biogenesis through transcriptional activation of lysosomal genes [6,22].As part of our ongoing work to discover [23] bioactive molecules from plant [14,20,21,[24][25][26][27], we isolated seven undescribed quassinoids (1)(2)(3)(4)(5)(6)(7), including six C-20 and one C-19 quassinoids from the twigs of this plant.We also verified the oxidative degradation of C-25 quassinoid, perforalactone B (Figure 1), to the C-20 quassinoid, perforaquassin A, through chemical transformation, and then the crucial "link" between C-25 type and C-20 type quassinoids was proposed.Additionally, these quassinoids were tested for their anti-PD potential using 6-OHDA-induced PC12 cells and the Drosophila Parkinson's disease model of PINK1 B9 .The results showed promising bioactive properties, making these newly discovered quassinoids potential candidates for further development as anti-PD drugs.

Structure Elucidation
Seven undescribed quassinoids, perforalactones F-L (1-7), were isolated from the twigs of H. perforata plant using solvent partitioning and various types of chromatography techniques, including normal, reverse, and molecular-exclusion (Figure 2).Harrisonia perforata (Bl.)Merr.belongs to the Simarubaceae family and is a shrub plant [19].A number of chemical components have been identified in previous studies, including quassinoids, limonoids, chromones, and polyketides [4,6,[20][21][22].Our previous research found that some of these compounds exhibit biological activities against neurodegenerative disease through various mechanisms (Figure 1).For example, harpertrioate A reduced Aβ42 and Aβ40 production and shifted APP processing toward a nonamyloidogenic pathway, while perforalactones D and E significantly induced lysosomal biogenesis through transcriptional activation of lysosomal genes [6,22].As part of our ongoing work to discover [23] bioactive molecules from plant [14,20,21,[24][25][26][27], we isolated seven undescribed quassinoids (1-7), including six C-20 and one C-19 quassinoids from the twigs of this plant.We also verified the oxidative degradation of C-25 quassinoid, perforalactone B (Figure 1), to the C-20 quassinoid, perforaquassin A, through chemical transformation, and then the crucial "link" between C-25 type and C-20 type quassinoids was proposed.Additionally, these quassinoids were tested for their anti-PD potential using 6-OHDA-induced PC12 cells and the Drosophila Parkinson's disease model of PINK1 B9 .The results showed promising bioactive properties, making these newly discovered quassinoids potential candidates for further development as anti-PD drugs.
Perforalactone H (3) has a molecular formula of C20H26O5, which is 14 mass units less than that of perforalactone G (2), according to HRESIMS [m/z 347.1840 (M + H) + ].The NMR data of perforalactone H (3) are similar to those of perforalactone G (2) (Table 1, Figures S13 and S14), indicating their structural similarity.However, signals corresponding to tertiary methyl in the NMR spectra of perforalactone H (3) were observed, instead of the signals for hydroxymethyl at C-10 and O-bear methyl at C-2 in perforalactone G (2).This was further confirmed via 2D NMR experiments (Figures S15-S18), and its planar structure and relative configuration are shown in Figures 3 and 4, respectively.
According to HRESIMS analysis, perforalactone I (4) has a molecular formula of C21H26O6 [m/z 379.1511 (M + Na) + (calculated for C21H24O5Na, 379.1516)], which is 42 mass units less than perforalactone C (8).Further analysis of its NMR data (Table 2, Figures S19  and S20) revealed that its structure closely resembles that of the latter, except for the absence of acetyl at C-19.This was confirmed via the HMBC correlations of H2-19 to C-1, C-5, and C-9 (Figures S21-S24).Thus, the structure of perforalactone I (4) was depicted as shown in Figures 3 and 4.
A white powder identified as perforalactone K ( 6) was found to have the same molecular formula, C21H25O5, as perforalactone J (5) according to HRESIMS [m/z 357.1704, (M + H) + ].However, the 1 H and 13 C NMR spectroscopic data of perforalactone K (6) (Table 2, Figures S31 and S32) showed the presence of exomethylene (δC 113.0, δH 4.94, 5.18) instead   S7 and S8) of perforalactone F (1) and perforalactone G (2), it was found that the absence of acetyl at C-19 in the latter is the only difference.The structural assignment of perforalactone G (2) was confirmed via 2D NMR spectra (Figures S9-S12), as illustrated in Figures 3 and 4.
Perforalactone H (3) has a molecular formula of C 20 H 26 O 5 , which is 14 mass units less than that of perforalactone G (2), according to HRESIMS [m/z 347.1840 (M + H) + ].The NMR data of perforalactone H (3) are similar to those of perforalactone G (2) (Table 1, Figures S13 and S14), indicating their structural similarity.However, signals corresponding to tertiary methyl in the NMR spectra of perforalactone H (3) were observed, instead of the signals for hydroxymethyl at C-10 and O-bear methyl at C-2 in perforalactone G (2).This was further confirmed via 2D NMR experiments (Figures S15-S18), and its planar structure and relative configuration are shown in Figures 3 and 4, respectively.
A white powder identified as perforalactone K (6) was found to have the same molecular formula, C 21 H 25 O 5 , as perforalactone J (5) according to HRESIMS [m/z 357.1704, (M + H) + ].However, the 1 H and 13 C NMR spectroscopic data of perforalactone K (6) (Table 2, Figures S31 and S32) showed the presence of exomethylene (δ C 113.0, δ H 4.94, 5.18) instead of sp 2 quaternary carbon in perforalactone J (5).These data suggest that a C(4)-C(18) double bond in perforalactone K (6) replaced the C(4)-C(5) double bond in perforalactone J (5), which was confirmed by the HMBC correlations from H 2 -18 to C3, C-4, and C-5.The remaining part of the structure and the relative configuration of perforalactone K ( 6) was similar to that of perforalactone J (5) S37).Except for three carbonyls and two trisubstituted double bonds, the remaining four degrees of unsaturation indicated the presence of a four-ring system in perforalactone L (7).All the data suggested that perforalactone L ( 7) is a C-19 quassinoid.

Chemical Transformation from C 25 Quassinoids to C 20 and Their Proposed Biogenetic Pathway
As shown in Scheme 1A, quassinoids are known to be derived from tetracyclic triterpene precursors through a series of oxidative degradation processes.To date, several biogenetic intermediates have been found suggesting how C 30 triterpenoids are degraded to C 20 quassinoids; however, no chemical transformation was accomplished [1].Since C 25 quassinoid perforalactone B structurally resembles the C 20 quassinoids perforalactones F-L, along with the fact that both types of quassinoids coexist in H. perforata, we surmised that Baeyer-Villiger oxidation would be a straightforward gateway to the fission of the C-13/C-17 bond in perforalactone B [28].After trying a number of reaction conditions, we found that perforalactone B in the presence of H 2 O 2 and NaOH in MeOH at 70 • C overnight could be converted to a product with 83% yield (Table S2).The product displayed a peak at m/z 359 [M + H] + .Further analysis of its 13 C NMR spectra (Table 2) revealed 21 signals, including 5 methyls (1 oxygenated), 2 methylenes, 6 methines (including 2 olefinic and 1 oxygenated), and 7 non-hydrogenated carbons (including 3 carbonyls and 2 olefinic).Its structure was identified to be a C 20 quassinoid, perforaquassin A (9), by comparison of the NMR data with the literature [29].On the basis of these results and the literature precedent, we propose a plausible route for this reaction, as shown in Scheme 1B.Perforalactone B could be further transformed to a key intermediate i via a mechanism similar to that of chemical Baeyer-Villiger oxidation.The insertion of an oxygen atom could occur between C-13 and C-17 because the fully substituted C-13 carbon has a migratory aptitude comparable to that of the γ-lactone group.The intermediate i would then undergo hydrolysis/dehydration to produce perforaquassin A (9). Thus, chemical transformation from perforalactone B to perforaquassin A (9) provides new insight into understanding the oxidative degradation pathway of quassinoids.

Neuroprotective Activity of the Isolated Compounds on 6-OHDA-Induced Injury of PC12 Cells
6-Hydroxydopamine (6-OHDA) is an oxidative neurotoxin and a redox cycling dopamine analog that was used to generate Parkinson's disease (PD) models in cells and animals.Based on our previous report, a dose of 6-OHDA was determined for the C12 cells.The main mechanism of the neurotoxicity of 6-OHDA includes the induction of oxidative stress [30], while rasagiline has a neuroprotective effect in vitro in PC12 cells against a number of toxins such as 6-hydroxydopamine (6-OHDA), MPTP, etc. [31].Therefore, rasagiline has been usually used as the positive control in this model for screening compounds of anti-PD potential [32,33].In the preliminary experiment testing, four compounds (1, 2, 4, and 5) at 50 µM improved the cell viability of 6-OHDA-induced PC12 cells by 10-15% compared to the model group (Figure 6A).Further, different concentrations (25, 50, and 100 µM) of 1, 2, 4, and 5 were tested, and all the samples improved the cell viability except for compounds 1 and 2 at 100 µM (Figure 6B).It is worth noting that 50 µM of compound 2 exhibited the strongest cytoprotective effect (92.84% of cell viability), surpassing even the positive control rasagiline (90.48%).Furthermore, the cytotoxicity of compounds 1, 2, 4, and 5 at different concentrations (25,50, and 100 µM) is extremely low, as demonstrated in Figure 6C.This study is the first report on the neuroprotective effects of compounds from H. perforata, providing a fresh perspective for the development and design of anti-PD drugs [34].

Effect of the Isolated Compounds on Locomotor Ability of PINK1 B9 Flies
Out of the seven compounds tested, 1, 2, 4, and 5 displayed superior neuroprotective effects in cell experiments.Consequently, we proceeded to investigate their impact on the Drosophila PD model of PINK1 B9 .Following a six-day diet containing 100 µM of the compounds, we evaluated the locomotor ability through a climbing experiment.Figure 7A shows that the PINK1 B9 flies' climbing ability was only 18.46% that of the WT flies.However, administering compounds 2 and 4 significantly improved their performance to 40.01 and 47.33%, respectively.

Neuroprotective Activity of the Isolated Compounds on 6-OHDA-Induced Injury of PC12 Cells
6-Hydroxydopamine (6-OHDA) is an oxidative neurotoxin and a redox cycling dopamine analog that was used to generate Parkinson's disease (PD) models in cells and animals.Based on our previous report, a dose of 6-OHDA was determined for the C12 cells.The main mechanism of the neurotoxicity of 6-OHDA includes the induction of oxidative stress [30], while rasagiline has a neuroprotective effect in vitro in PC12 cells against a number of toxins such as 6-hydroxydopamine (6-OHDA), MPTP, etc. [31].Therefore, rasagiline has been usually used as the positive control in this model for screening compounds of anti-PD potential [32,33].In the preliminary experiment testing, four compounds (1, 2, 4, and 5) at 50 µM improved the cell viability of 6-OHDA-induced PC12 cells by 10-15% compared to the model group (Figure 6A).Further, different concentrations (25, 50, and 100 µM) of 1, 2, 4, and 5 were tested, and all the samples improved the cell viability except for compounds 1 and 2 at 100 µM (Figure 6B).It is worth noting that 50 µM of compound 2 exhibited the strongest cytoprotective effect (92.84% of cell viability),

Effect of the Isolated Compounds on ATP Contents of PINK1 B9 Flies
The role of PINK1 in preserving mitochondrial homeostasis cannot be overstated; when it is absent, ATP production is significantly reduced.ATP levels are a widely accepted measure of mitochondrial function, and in this study, the thoracic ATP content of the PINK1 B9 flies was only 42.41% that of the WT flies at 6 days of age (as depicted in Figure 7B).However, the administration of compounds 2 and 4 (at a concentration of 100 µM) led to a substantial increase in ATP content for the PINK1 B9 flies, with levels reaching 66.26 and 67.70%, respectively, at this age.

Effect of the Isolated Compounds on Dopamine Content of PINK1 B9 Flies
A decrease in dopamine levels is a typical characteristic of Parkinson's disease patients and PINK1 B9 flies.Figure 7C illustrates that the dopamine content in PINK1 B9 flies was notably lower (54.8%)than that of WT flies at 18 days.However, administering compounds 2 and 4 (100 µM) resulted in a significant increase in dopamine levels, with relative levels reaching 71.99% and 89.45% at 18 days, respectively.
surpassing even the positive control rasagiline (90.48%).Furthermore, the cytotoxicity of compounds 1, 2, 4, and 5 at different concentrations (25, 50, and 100 µM) is extremely low, as demonstrated in Figure 6C.This study is the first report on the neuroprotective effects of compounds from H. perforata, providing a fresh perspective for the development and design of anti-PD drugs [34].Drosophila PD model of PINK1 B9 .Following a six-day diet containing 100 µM of the compounds, we evaluated the locomotor ability through a climbing experiment.Figure 7A shows that the PINK1 B9 flies' climbing ability was only 18.46% that of the WT flies.However, administering compounds 2 and 4 significantly improved their performance to 40.01 and 47.33%, respectively.

Discussion
While past reports focused mainly on the antifeedant effect of quassinoids, [4] we found, for the first time, the anti-PD potential of these compounds.The toxicity of 6-OHDA in PC12 cells has been shown to be directly related to its auto-oxidation and induction of oxidative stress, thus causing mitochondrial dysfunction and cell apoptosis [35].Recently, a new alkaloid named chaetonigrisin G, isolated from the fungus Chaetomium nigricolor YT-2, showed significant neuroprotective activity through the upregulation of anti-oxidative (SOD1) and anti-apoptosis gene (Bcl-2) expression [36].The new quassinoids (1, 2, 4, and 5) in this work showed an equal or better effect on the 6-OHDA-induced PC12 cells compared to chaetonigrisin G.This suggests that these new quassinoids (1, 2, 4, and 5) might also exert neuroprotective effects through anti-oxidative and anti-apoptosis mechanisms.
PINK1 is a mitochondria-localized serine-threonine kinase which plays important roles in combating oxidative stress and mitochondrial dysfunction.The PINK1-knock-out fruit flies (PINK1 B9 flies) exhibit PD-like phenotypes including impaired motor ability, reduction of dopamine and ATP levels, shortened lifespan, and degeneration of dopaminergic neurons [18].Thus, PINK B9 flies are an excellent PD animal model in the discovery of anti-PD drugs.Recently, ginseng protein was found to delay the PD-like phenotype of PINK B9 flies, and up-regulate the key markers (Hsp60, mtHsp70, and CG5045) of the UPR mt pathway to maintain the mitochondrial homeostasis [37].Another study reported that grape skin extract exhibited the likely anti-PD activity in PINK B9 flies and enhanced the mitochondrial autophagy pathway by upregulating the LC3-II/LC3-I ratio and reducing p62 accumulation [38].Compared to previous studies, the new quassinoids (2 and 3) improved the motor ability in PINK1 B9 flies more than grape skin extract and increased the level of ATP in PINK1 B9 flies to a much greater extent than ginseng protein.Therefore, we speculate that these two new quassinoids might regulate the anti-oxidative and mitochondrial autophagy-related pathway.
Overall, seven new quassinoids (1-7) were discovered in the twigs of H. perforata during this study.The structural diversity of these quassinoids is extensive and contributes to our understanding of H. perforata's chemical composition.This research also revealed the transformation of C-25-type quassinoids into C-20-type quassinoids via chemical means, a first-time demonstration.Additionally, we discovered the remarkable anti-PD potential in C-20 quassinoids, another first.This work lays the groundwork for further investigation into H. perforata's properties and provides a potential avenue for developing Parkinson's disease medication.

Plant Material, Extraction, and Isolation
The plant source was the twigs of H. perforata from Hainan Province, China, obtained in January 2018.The samples were collected and identified by Dr. Shengzhuo Huang, assistant professor at Hainan Institute of Tropical Biotechnology.The specimens (NO.20180104) are kept in the State Key Laboratory of Phytochemistry and Sustainable Utilization of Western Plant Resources, Kunming Institute of Botany, Chinese Academy of Sciences.

X-ray Diffraction Analysis
X-ray Crystallographic Analyses of 7. Perforalactone L were crystallized from a mixed solvent system (acetone/H 2 O, 20:1) at rt.The X-ray crystallographic data for 7 have already been retrieved at the Cambridge Crystallographic Data Centre, CCDC number 2285369.

Cell Culture and Cell Viability Assays
The CCK-8 assay was used to determine the neuroprotective effect of the isolated compounds, as described previously [17].The PC12 cells were cultured in Dulbecco's Modified Eagle's medium (DMEM, TransGen Biotech, FS101-02) with 10% (v/v) of fetal bovine serum (FBS) and 1% (v/v) of penicillin/streptomycin at 37 • C in 5% CO 2 in a humid atmosphere.First, the test compound was dissolved in dimethyl sulfoxide (DMSO) and diluted with DMEM to a concentration of 50 µM.PC12 cells were seeded in 96-well microplates at a density of 10 4 cells/well for 24 h.Second, the cells were pre-treated with the test compounds at 37 • C for 2 h, followed by cultivation with 300 µM of 6-OHDA for 24 h.Finally, 10 µL of CCK-8 was added and incubated for 1 h before the UV absorbance was measured by a multimode microplate reader (Thermo Fisher, Waltham, MA, USA) at 450 nm.The flies were maintained in an incubator with a regular fly medium at 25 ± 1 • C, 60% relative humidity, and a 12 h light/dark cycle.The regular fly medium was prepared using a mixture of 90 g yeast, 240 g corn, 32 g agar, 189 g glucose, 98 g sucrose, and 25 mL preservative per 3.5 L boiling water.Male flies of both PINK1 B9 and WT were collected for the following experiments.Experimental diets were prepared by adding 100 µM test compounds in Formula 4-24 Instant Drosophila Medium (Carolina Biological Supply Company, Burlington, NC, USA), which was changed every 2-3 days.The flies were divided into three groups: group I, WT flies fed with an unmedicated diet; group II, PINK1 B9 flies fed with unmedicated food; and group III, PINK1 B9 flies fed with the test compounds (100 µM).

Climbing Assay
The locomotor ability of the flies was evaluated using an infrared behavioral recorder (Yihong Technology Co., Ltd., Wuhan, China).Cohorts of 20 flies (6 days old) from each group were subjected to the assay.Flies were first anesthetized using CO 2 , transferred into a vertical glass tube (15 cm in length and 1.5cm in diameter), and then acclimated at room temperature for 30 min.The glass tube was inserted into the recorder, and the locomotor ability of the flies was recorded every 5 min for at least 4 h at the same time of day.

ATP Measurement
The contents of ATP in the thoraces of flies were measured by a luciferin-luciferase system using the ATP Assay Kit (Biyuntian Co., Ltd., Shanghai, China).Protein content was determined using the BCA method (BCA protein assay, Elabscience, Wuhan, China).Thoraces of five 6-day-old flies were collected and homogenized in the lysis buffer, which was centrifugated at 12,000× g at 4 • C for 5 min.The supernatant was mixed with a luminescent solution and detected by a Luminometer (Molecular Devices, San Jose, CA, USA) [18].ATP content was calculated as a percentage of total protein for each sample.Relative ATP contents were determined, and experiments were repeated at least three times.

Dopamine Measurement
The dopamine content in the flies' brains was determined via a Dopamine Research ELISA kit (Enzyme Link Biotechnology Co., Ltd., Dongguan, China).Protein content was determined via the same method as in the above ATP measurement section.First, flies (30 flies in each group) were anesthetized and transferred into EP tubes which were frozen in liquid nitrogen.Second, the flies were decapitated, and the collected heads were homogenized in 50 µL of ice-chilled citrate acetate buffer (50 mM pH = 6.5) and then centrifugated at 12,000× g at 4 • C for 15 min.Finally, the supernatant was used to determine the dopamine level according to the manufacturer's instructions.Optical density was measured at 450 nm by a multimode microplate reader (Thermo Fisher, USA).Dopamine content was calculated as a percentage of total protein for each sample.4.6.5.Data Analysis Each experiment was repeated at least three times.Statistical significance was measured using one-way analysis of variance (ANOVA) analyses with Dunnett's multiple comparisons test.The significance is indicated as follows: * p < 0.05, ** p < 0.01, and *** p < 0.001.All statistics analyses were performed by GraphPad Prism 7 (GraphPad Software, San Diego, CA, USA).

Scheme 1 .
Scheme 1. (A) Updated hypothesis for the biosynthetic pathway of C20 quassinoids, and proposed step in this work in blue.(B) Chemical transformation (in plain) and proposed mechanism (in dashed) of quassinoid from C25 quassinoid to C20 one.

Scheme 1 .
Scheme 1. (A) Updated hypothesis for the biosynthetic pathway of C 20 quassinoids, and proposed step in this work in blue.(B) Chemical transformation (in plain) and proposed mechanism (in dashed) of quassinoid from C 25 quassinoid to C 20 one.