Two new monoterpene esters from the pericarps of Alpinia zerumbet

Abstract Two new monoterpene esters (1 and 2) and four known compounds (3–6) were isolated from the pericarps of Alpinia zerumbet. Their structures were elucidated by extensive spectroscopic analyses and their anti-inflammatory activity was evaluated by monitoring their inhibitory effects on the interleukin-1β-induced production of nitric oxide in primary cultured rat hepatocytes. The new compound 1 and cardamonin 3 showed inhibitory activities with IC50 values of 17.6 ± 1.1 and 10.2  ±  1.3 µM, respectively, which are comparable to that of the positive control NG-methyl-L-arginine acetate salt. Graphical Abstract


Introduction
Alpinia zerumbet (Pers.)B.L.Burtt & R.M.Sm.(Zingiberaceae), also known as shell ginger, is an aromatic plant widely distributed in tropical and subtropical regions of the world that has been used as a flavour additive and a traditional medicine for centuries (Ji et al. 2019;Xiao et al. 2020).The common medicinal part of Zingiberaceae plants is rhizomes (Zahara et al. 2018), whereas in A. zerumbet, leaves, flowers, mature fruits, seeds and rhizomes are traditionally used as medicinal sources (Wei et al. 2020), whose therapeutic uses vary depending on the parts of the plant.Thus, the leaves are used in Brazil as a diuretic, antihypertensive and antiulcerogenic agent (De Moura et al. 2005).In Japan, the seeds are used as an alternative to the aromatic stomachic 'amomum seed' and the leaves are used for the wrapping of mu-chi, a traditional food in Okinawa, to prevent common cold (Tawata et al. 2008).In Guizhou province of China, the mature fruits (pericarps, seeds and placenta) are used for the treatment of cardiovascular diseases (Ji et al. 2019).
Previous phytochemical investigations have led to the isolation of bioactive secondary metabolites such as flavonoids, phenolic acids, phenylpropanoids, kavapyrones, sterols and terpenoids mainly from leaves, seeds and rhizomes (Chan et al. 2017).However, the chemical constituents of the pericarps and the placenta that contribute to the medicinal properties of mature fruits are still unclear.Therefore, a phytochemical investigation of the pericarps and the placenta of A. zerumbet was conducted, revealing kavalactone derivatives as the main constituents (Nishidono et al. 2020b).As a continuation of this investigation, this study led to the isolation of six compounds (1-6) from the methanol (MeOH) extract of the pericarps of A. zerumbet.Their structures were elucidated by HRESIMS and NMR spectroscopy and identified as two undescribed compounds (1 and 2) and four known ones (3-6) (Figure 1).Herein, we report the structure elucidation of compounds 1 and 2 and the investigation of their antiinflammatory activities.

General experimental procedures
UV spectra were obtained on a SHIMADZU UV-1800 spectrometer (Shimadzu, Kyoto, Japan).IR spectra were recorded on an IRAffinity-1S (Shimadzu) in chloroform solution in a cell with a fixed thickness of 0.20 mm (KBr).The NMR spectra were recorded on a JNM-ECZ500R spectrometer (JEOL Ltd., Tokyo, Japan) in chloroform-d with tetramethylsilane as an internal reference.The HRESIMS analysis was conducted on a Shimadzu LCMS-IT-TOF mass spectrometer (Shimadzu).Column chromatography was performed using silica gel (Wakogel C-200, FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan).A Yamazen Pump 540 (Yamazen, Osaka, Japan) with a UNIVERSAL ODS-SM column (Yamazen) was used for the MPLC analysis.Semi-preparative HPLC was performed on a COSMOSIL 5 C 18 MS-II column (10 mm i.d.Â 250 mm, 5 mm, Nacalai Tesque, Inc., Kyoto, Japan) using a Shimadzu Prominence HPLC system with an SPD-20A Prominence UV/Vis detector (Shimadzu).Pre-coated silica gel 60 F 254 plates and RP-18 F 254 plates for analytical and preparative TLC were supplied by Merck (Darmstadt, Germany).

Plant identification
The total DNA was extracted from the dried seeds of the samples using a NucleoSpin Plant II (MACHEREY-NAGEL, D€ uren, Germany) according to the manufacturer's instructions.The PCR test was performed in a mixture (50 mL) containing 10 Â Ex-Taq Buffer (TaKaRa-Bio, Shiga, Japan), 0.2 mM of each dNTP, 0.5 mM of each primer, 0.25 mg of anti-Taq high (TOYOBO, Osaka, Japan), 1.25 units of TaKaRa Ex Taq (TaKaRa-Bio) and sterilised purified water.The primers used for the amplification are listed in Table S4.The programme of the PCR cycle was as follows: 2 min at 94 C; 10 cycles of 1 min at 94 C, 2 min at 72 C; 15 cycles of 1 min at 94 C, 1.5 min at 65 C, 0.5 min at 72 C; 10 cycles of 1 min at 94 C, 1.5 min at 60 C, 0.5 min at 72 C; and 1 min at 72 C. The PCR products were sequenced by Eurofins Genomics K.K. (Tokyo, Japan).BLAST (nucleotide sequence comparison programme) of the GenBank database revealed that the rDNA ITS1 region sequence of the sample matched those of A. zerumbet (GenBank accession number: KX018020) and A. formosana (FJ496863).Additionally, the discrimination between the two species was then performed via the sequence analysis of the chloroplast trnH-psbA region and the sample used in the present study exhibited a high identity of 100% with GU180443 and was identified as A. zerumbet.

Anti-inflammatory and LDH activity
As an index of the anti-inflammatory activity, the effect of the compounds on nitric oxide (NO) production was evaluated in the presence of interleukin-1b in primary cultured rat hepatocytes.The LDH activity in the medium was measured as a cytotoxicity indicator.These studies were performed in accordance with our previous report (Nishidono et al. 2020a).In the present study, L-NMMA acetate salt was used as a positive control.When the compounds showed no cytotoxicity, the IC 50 values were calculated.The IC 50 values are presented as the means ± standard deviation of three independent experiments.

Conclusions
In conclusion, six compounds (1-6), including two new monoterpene esters with a kavalactone derivative as an acyloxy group (1 and 2), were isolated from the pericarps of A. zerumbet.This is the first report establishing the structure of the kavalactone derivative moiety of the new compounds.In addition, cardamonin 3 and uvangoletin 4 were isolated from the pericarps of A. zerumbet for the first time.The anti-inflammatory activity of the compounds in terms of the inhibitory activity for NO production showed that the new compound 1 and cardamonin 3 have similar inhibitory activity as the positive control L-NMMA.The discovery of these compounds deepens the knowledge on the chemical composition of the medicinally active pericarps of A. zerumbet.