IL-6 Inhibitory Compounds from the Aerial Parts of Piper attenuatum and Their Anticancer Activities on Ovarian Cancer Cell Lines

Piper attenuatum Buch-Ham, a perennial woody vine belonging to the Piperaceae family, is traditionally used in Southeast Asia for treating various ailments such as malaria, headache, and hepatitis. This study described the isolation and identification of three new compounds, piperamides I-III (1–3), which belong to the maleimide-type alkaloid skeletons, along with fifteen known compounds (4–18) from the methanol extract of the aerial parts of P. attnuatum. Their chemical structures were elucidated using spectroscopic methods (UV, IR, ESI-Q-TOF-MS, and 1D/2D NMR). All the isolates were evaluated for their ability to inhibit IL-6 activity in the human embryonic kidney-Blue™ IL-6 cell line and their cytotoxic activity against ovarian cancer cell lines (SKOV3/SKOV3-TR) and chemotherapy-resistant variants (cisplatin-resistant A2780/paclitaxel-resistant SKOV3). The compounds 3, 4, 11, 12, 17, and 18 exhibited IL-6 inhibition comparable to that of the positive control bazedoxifene. Notably, compound 12 displayed the most potent anticancer effect against all the tested cancer cell lines. These findings highlight the importance of researching the diverse activities of both known and newly discovered natural products to fully unlock their potential therapeutic benefits.


Introduction
Piper attenuatum Buch-Ham is a perennial woody vine belonging to the Piperaceae family and is mainly distributed in tropical Southeast Asian regions, such as India, Indonesia, Myanmar, and Laos.Piper is the largest genus within the family Piperaceae and encompasses over 700 species [1,2].Renowned for its distinctive taste and scent, the Piper species holds widespread acclaim as a popular spice with considerable economic significance [3], and P. attenuatum is a plant called oval-leaved pepper, which can be used as an alternative to black pepper (P.nigrum) [4,5].The leaves and roots of P. attenuatum are traditionally used to treat malaria, headache, hepatitis, mechanical injury, muscular pain, and pulmonary and urinary disorders [6][7][8].
The studies of the chemical composition of P. attenuatum have identified alkaloids, amides lignans, neolignans, and terpenes [7,9].A few research groups have investigated the biological activities of P. attenuatum extracts, but there have been no systematic studies on the chemical identification of its biological activities.However, several studies have shown that P. attenuatum exhibits antibacterial, anticancer, antidiabetic, anti-inflammatory, antipyretic, diuretic, hepatoprotective, and hyperlipidemic activities [7].
Ovarian cancer is the eighth most common gynecological malignancy and the fifth most common cause of cancer-related deaths in women [10].Interleukin (IL)-6 is a major immunoregulatory cytokine present in the ovarian cancer microenvironment [11].It induces tumor angiogenesis, which plays an important role in the development of ascites and the metastasis of ovarian cancer, leading to rapid cancer progression and poor prognosis [12,13].Preclinical evidence has shown that IL-6 improves ovarian cancer tumor cell survival and increases resistance to chemotherapy through the Janus kinase/signal transducer and the activator of transcription 3 (STAT3) signaling in tumor cells and the IL-6 receptor translocation in tumor endothelial cells [14].IL-6 regulates some of its functions through the IL-6 receptor, which is a complex of IL-6Rα and glycoprotein (gp) 130 [11].This makes the IL-6/IL-6Rα/gp130 signaling pathway an attractive target for therapeutic or preventive interventions.Currently, the application of IL-6/IL-6Rα/gp130 blockers as anticancer agents has not been extensively studied, especially for ovarian cancer.
In this study, we identified IL-6-targeting and -inhibitory compounds using isolates from P. attenuatum and evaluated their antitumor activity, particularly in ovarian cancer.The chemical investigation of P. attenuatum determined nine alkaloids (1-3, 7-10, 13-14), three lignans (4-6), a sesquiterpenoid (11), a monoterpenoid lactone (12), a simple phenolic compound (15), and three flavonoids (16)(17)(18) (Figure 1).Among the isolates, compounds 1-3 were previously undescribed, and six of the isolates exhibited IL-6 inhibitory activity and antitumor potential in human ovarian cancer cell lines.These findings suggest the potential of P. attenuatum as an effective resource against ovarian cancer and lay the groundwork for the development of novel therapeutic strategies against ovarian cancer.
the biological activities of P. attenuatum extracts, but there have been no systematic studies on the chemical identification of its biological activities.However, several studies have shown that P. attenuatum exhibits antibacterial, anticancer, antidiabetic, anti-inflammatory, antipyretic, diuretic, hepatoprotective, and hyperlipidemic activities [7].
Ovarian cancer is the eighth most common gynecological malignancy and the fifth most common cause of cancer-related deaths in women [10].Interleukin (IL)-6 is a major immunoregulatory cytokine present in the ovarian cancer microenvironment [11].It induces tumor angiogenesis, which plays an important role in the development of ascites and the metastasis of ovarian cancer, leading to rapid cancer progression and poor prognosis [12,13].Preclinical evidence has shown that IL-6 improves ovarian cancer tumor cell survival and increases resistance to chemotherapy through the Janus kinase/signal transducer and the activator of transcription 3 (STAT3) signaling in tumor cells and the IL-6 receptor translocation in tumor endothelial cells [14].IL-6 regulates some of its functions through the IL-6 receptor, which is a complex of IL-6Rα and glycoprotein (gp) 130 [11].This makes the IL-6/IL-6Rα/gp130 signaling pathway an attractive target for therapeutic or preventive interventions.Currently, the application of IL-6/IL-6Rα/gp130 blockers as anticancer agents has not been extensively studied, especially for ovarian cancer.
In this study, we identified IL-6-targeting and -inhibitory compounds using isolates from P. attenuatum and evaluated their antitumor activity, particularly in ovarian cancer.The chemical investigation of P.attenuatum determined nine alkaloids (1-3, 7-10, 13-14), three lignans (4-6), a sesquiterpenoid (11), a monoterpenoid lactone (12), a simple phenolic compound (15), and three flavonoids (16-18) (Figure 1).Among the isolates, compounds 1-3 were previously undescribed, and six of the isolates exhibited IL-6 inhibitory activity and antitumor potential in human ovarian cancer cell lines.These findings suggest the potential of P.attenuatum as an effective resource against ovarian cancer and lay the groundwork for the development of novel therapeutic strategies against ovarian cancer.

Structure Elucidation
The dried and powdered P.attenuatum were extracted with MeOH and the concentrated extract was then partitioned with n-hexane, EtOAc, and n-BuOH.The EtOAc soluble fraction was resolved by various column chromatography on normal-and reversedphase silica gel, Sephadex LH-20, and semi-preparative HPLC to yield compounds 1-18.

Structure Elucidation
The dried and powdered P. attenuatum were extracted with MeOH and the concentrated extract was then partitioned with n-hexane, EtOAc, and n-BuOH.The EtOAc soluble fraction was resolved by various column chromatography on normal-and reversed-phase silica gel, Sephadex LH-20, and semi-preparative HPLC to yield compounds 1-18.
The HMBC spectrum supported the above information, with a cross peak from 9-OCH 3 to C-9 and H-9 to C-9 ′ /C-8/C-8 ′ , which indicated the presence of a 3,4-disubstituted-5methoxypyrrolin-2-one group (Figure 2).Additionally, the correlations between H-7 and C-8/C-9/C-8 ′ , H-7 ′ and C-8/C-9 ′ /C-8 ′ provide evidence that a 3,4-methylenedioxybenzyl group and a 3 ′ ,4 ′ -dimethoxybenzyl group were connected to the positions at H-8 and H-8 ′ , respectively (Figure 2).Based on the spectroscopic data, compound 2 was determined to be piperimide II.Compound 3 was obtained as a yellow amorphous powder and its molecular formula was determined as C 22 H 23 NO 6 by ESI-Q-TOF-MS at m/z 398.1603 [M + H] + .In the IR spectrum, the amine group (3390 cm −1 ), the amide group (1707 cm −1 ), and the methylenedioxy group (924 cm −1 ) were observed.A positive reaction was observed when tested using Dragendorff's reagent.The 1D NMR spectrum of compound 3 was nearly identical to that of 2 (Table 1).The only difference between compounds 2 and 3, as evidenced by the HMBC correlations (Figure 2), was that the 3 ′ ,4 ′ -dimethoxybenzyl group was attached to C-8, and the 3,4-methylenedioxybenzyl group was linked to C-8 ′ in 3. From the spectroscopic analysis, compound 3 was confirmed as piperimide III.
Compounds 2 and 3 were considered racemates, as indicated by their close-to-zero optical rotation values and the absence of Cotton effects in the CD spectrum.Consequently, the absolute configuration of C-9 in these compounds was not determined.
The main scaffold of 1-3 is maleimide, which possesses a five-membered heterocyclic structure belonging to the imide group and is called a lignan alkaloid.Maleimide is a natural compound mainly found in marine microorganisms and fungi [16] and its wide range of biological activities has prompted its synthesis based on structure-activity relationships (SARs) [17,18].To date, maleimide derivatives have not been found in plants, and this study reports the first isolation and characterization of a maleimide derivative from a plant, thus expanding the natural sources of this significant structure.
However, compounds 1-3 represent structures with a maleimide moiety, a benzene ring bearing a methylenedioxy group, and fully methylated hydroxy groups.Interestingly, (-)-kusunokinin (5), a naturally occurring compound, shares structural similarities with compounds 1-3, including the presence of a methylenedioxy group and fully methylated hydroxy groups [19,20].While this suggests the potential natural origin of compounds 1-3, the presence of methyl ether groups raises concerns about the possibility of artificial methylation during the extraction and isolation process.To definitively determine whether the methyl ether groups in compounds 1-3 are a result of natural occurrence or artificial methylation during the extraction and isolation process, further analysis using an alternative extraction solvent, such as ethanol instead of methanol, is warranted.This additional experiment will help elucidate the origin of the methyl ether groups and provide conclusive evidence regarding the natural or synthetic nature of these compounds.

Screening of Compounds Using Human Embryonic Kidney (HEK)-Blue™ IL-6 Cells
The use of HEK-Blue reporter cell lines to screen for small-molecule drugs has recently gained popularity in cancer research [34,35].We isolated eighteen compounds from P. attenuatum and assessed their inhibitory effects on IL-6 activity using the HEK-Blue™ IL-6 cell line.As the HEK-Blue™ IL-6 cells were transfected with a reporter gene expressing STAT3-inducible secreted embryonic alkaline phosphatase (SEAP), STAT3 was activated, and SEAP was produced when the cells were stimulated with IL-6.The extent of the inhibition of IL-6 downstream signaling by these compounds was assessed in vitro using bazedoxifene (gp130 inhibitor) as a positive control in the HEK-Blue™ IL-6 cells.Bazedoxifene is a newly developed third-generation indole-based estrogen receptor (ER) ligand used in the treatment of postmenopausal osteoporosis, also known as gp130 inhibitor.In our previous study, we demonstrated the anticancer effects of GP130 in ovarian cancer through combined administration of conventional anticancer drugs bazedoxifene and paclitaxel [36].

Anticancer Activity
Various chemotherapeutic drugs, particularly cisplatin and paclitaxel, have been used to treat ovarian cancer after surgical tumor removal.However, despite chemotherapy, many patients die of ovarian cancer recurrence.Inflammatory mediators such as IL-6 and several other cytokines influence drug resistance and recurrence in ovarian cancer [37].The six compounds (3, 4, 11, 12, 17, and 18) that inhibited IL-6 activity were screened for their antitumor effects in the A2780, SKOV3, cisplatin-resistant (A2780-cis), and paclitaxelresistant (SKOV3-TR) ovarian cancer cell lines using a Cell Counting Kit-8 (CCK-8) assay.
These compounds showed anti-proliferative activity in the ovarian cancer cell lines, but relatively weak activity in drug-resistant cell lines (Table 3).However, compounds 11 and 12 significantly reduced the viability of the A2780 and SKOV3 cells showing their half-maximal inhibitory concentration (IC 50 ) values at around 10 µM.In addition, both the compounds showed more potent anti-proliferative activities against the A2780-cis and SKOV3-TR ovarian cancer cell lines than the positive control, bazedoxifene, indicating a stronger anticancer activity against the drug-resistant cancer cell lines.Compound 18 showed the most potent IL-6 inhibitory effect but exhibited weak anticancer activity.Upon treatment with compound 3, maleimide derivative, the viability of the A2780 and SKOV3 cells was moderately inhibited with the IC 50 values of 17.54 ± 0.19 and 15.76 ± 0.15 µM, respectively.Compound 12, (-)-loliolide exhibited the most potent anticancer activity, comparable to that of bazedoxifene.(-)-Loliolide is a C11-terpene lactone, which arises from the breakdown of carotenoids by light or heat and is found in diverse marine algae and terrestrial plants [38].In plants, (-)-loliolide serves as a defensive molecule against pathogens and herbivores and possesses allelopathic effects, such as germination and growth inhibition in other plants [39].(-)-Loliolide has been shown to have several biological activities [40][41][42][43], but no studies on its anticancer effects have been reported.While research on the biological activities of Piper species has focused heavily on alkaloids, (-)-loliolide highlights the importance of investigating other secondary metabolites for potential benefits.Therefore, research on the diverse range of the activities of both known and newly discovered compounds is crucial to fully unveil the potential of natural products.

Plant Material
The aerial parts of P. attenuatum were collected from Popa Mountain National Park (Mandalay, Myanmar) in August 2011 and identified by Khin Myo Htwe (Popa Mountain National Park, Mandaley, Myanmar).A voucher specimen (#M-PA20110812) was deposited at the herbarium of the College of Pharmacy, the Catholic University of Korea.

Extraction and Isolation
The dried aerial parts of P. attenuatum were stored in a freezer at -20 • C before use, and extraction, fractionation, compound isolation, and structural elucidation were carried out from December 2013 to August 2015.The isolated compounds had been kept in a deep freezer at -70 • C until the bioactivity screening which was performed from April to December 2023.Prior to the bioactivity screening, the purity of the isolated compounds was evaluated through the HPLC analysis using their relative peak area (%) (Tables S1 and S2).

Cell Culture
The human epithelial ovarian cancer cell lines SKOV3 and A2780 were purchased from the Korean Cell Line Bank (KCLB, Seoul, Republic of Korea) and the European Collection of Cell Cultures (ECACC, Wiltshire, UK), respectively.The cisplatin-resistant A2780 (A2780cis) and paclitaxel-resistant SKOV3 (SKOV3-TR) cells were obtained from Yonsei University (Institute of Women's Life Medical Science, Division of Gynecological Oncology, Republic of Korea).SKOV3, SKOV3-TR, A2780, and A2780-cis cells were cultured in the RPMI-1640 medium (Welgene, Daegu, Republic of Korea).SKOV3-TR and A2780-cis cells were cultured in 100 nM paclitaxel (Cayman Chemical Company, Ann Arbor, MI, USA) and 1 µM cisplatin (Dong-A Pharmaceutical Co., Seoul, Republic of Korea), respectively.All the culture media were supplemented with 10% (v/v) fetal bovine serum and 1% penicillin or streptomycin (Gibco-BRL, Gaithersburg, MD, USA), and the cells were maintained at 37 • C in a humidified atmosphere of 5% CO 2 and 95% air.Each culture medium was replaced with a fresh medium every 2-3 days.

IL-6 Inhibition Bioassay Using HEK-Blue™ IL-6 Cells
The HEK-Blue™ IL-6 cells purchased from InvivoGen (San Diego, CA, USA) were used to seed 3 × 10 4 cells/200 µL in a 96-well plate.Each well was treated with different concentrations (0, 0.1, 1, 5, 10, 50, and 100 µM) of the isolated compounds.Recombinant human IL-6 (BioLegend, San Diego, CA, USA) was administered at 10 ng/mL.After 24 h, 20 µL of the cell suspension was added to 180 µL of QUANTI-Blue™ solution (In-vivoGen, San Diego, CA, USA) in a fresh 96-well plate.After incubation at 37 • C for 2 h, the absorbance was measured spectrophotometrically (Epoch, BioTek Instruments, Winooski, VT, USA) at 635 nm.The EC 50 values were calculated from a log([drug]) versus normalized response curve fit using GraphPad Prism version 5.00 for Windows (Graph-Pad Software, San Diego, CA, USA).The CC 50 values were calculated from extrapolated concentration-response curves plotted with GraphPad Prism version 5.0 for Windows (GraphPad Software, San Diego, CA, USA) using inhibitory percentages versus the drug concentration logarithm.

Cytotoxicity Assay
A CCK-8 assay was performed to assess cytotoxicity.After seeding 5 × 10 5 cells/300 µL in 48-well plates, the cells were treated with the isolated compounds at 1, 5, 10, 20, 40, and 60 µM concentrations for 24 h.For the CCK-8 analysis, 30 µL of the CCK-8 solution (Dojindo, Kumamoto, Japan) was added to each well, allowed to react for 1 h, and then, the optical density value was measured at 450 nm using an ELISA microplate reader (Epoch, BioTek Instruments, Winooski, VT, USA).All the experiments were performed in triplicates.The IC 50 values were calculated from a log([drug]) versus normalized response curve fit using GraphPad Prism version 5.00 for Windows (GraphPad Software, San Diego, CA, USA).

Statistical Analysis
All the graphs were constructed using the GraphPad Prism 8 software (GraphPad Software, San Diego, CA, USA).Statistical analyses were performed using one-way ANOVA with Tukey's multiple comparison test and a Student's t-test.The data are mean ± standard deviation (SD).Statistical significance was defined as p < 0.05.The levels of significance are indicated by * p < 0.05, ** p < 0.01, and *** p < 0.001.

Conclusions
In this study, we identified a total of eighteen compounds in the methanol extract of the aerial parts of P. attenuatum.Among these, three previously undescribed compounds 1-3, piperamides I-III, have maleimide scaffolds.It was the first isolation of maleimide derivatives from plants, presenting a particularly intriguing discovery in phytochemistry.We evaluated the isolated compounds for their IL-6 inhibitory activity in the HEK-Blue IL-6 cell line.The six compounds exhibited IL-6 inhibition comparable to that of the positive control (bazedoxifene).Furthermore, we screened the IL-6 inhibitory compounds using an in vitro cytotoxicity test against the ovarian cancer cell lines (A2780/SKOV3) and drug-resistant variants (A2780-Cis/SKOV3-TR) to assess their anticancer potential.Among the isolates, (-)-loliolide (12) demonstrated the most potent anticancer activity against both the A2780 and SKOV3 cells, with the IC 50 values of 8.62 ± 0.10 and 10.71 ± 0.31 µM, respectively, even exceeding the effects of bazedoxifene against drug-resistant cell lines.Although the ability of (-)-loliolide to inhibit IL-6 activity and its potential as an anticancer agent for ovarian cancer is promising, further research is necessary to elucidate the mechanisms underlying its anticancer effects.

, 4, 11, 12, 17, and 18.
b 50% cytotoxic concentration (CC 50 ) is the concentration that reduces the number of viable cells by 50% compared with the control; c therapeutic index was calculated as CC 50 /EC 50 ; d positive control.

Table 3 .
Cytotoxicity of the compounds 3
a 50% inhibitory concentration; the values were expressed as the means (±SD) of logIC 50 ; b positive control.