Lignan (+)-Piperitol-γ,γ-Dimethylallylether from Stem Bark of Zanthoxylum rhetsa (Roxb.) DC (Rutaceae)

Lignans are a group of phenylpropanoid dimers in which the phenylpropane units are linked by their propyl side chains' central carbon (C 8 ). Lignans have various biological activities, including antiviral, anticancer, cancer preventive, and cytotoxic. Zanthoxylum rhetsa (Roxb.) DC is a tree with prickly branchlets belonging to the Zanthoxylum genus, commonly known as panggal buaya in Indonesia. Asian tribes have used this plant as traditional medicine. In this study, (+)-piperitol-γ,γ-dimethylallylether, a furofuran lignan, was successfully isolated. The chemical structure of compound 1 was determined based on spectroscopic data, including 1D- and 2D-NMR, mass spectroscopy, and by comparing with previous spectral data. In addition, compound 1 was tested for its cytotoxic activity against MCF-7 breast cancer cell lines in vitro and showed weak activity with the IC 50 value of 261.37 µg/mL.

Lignans can be found in more than 70 plant families and have been isolated from several plant parts (Pan et al., 2009), especially the Rutaceae family. One of the genera in the Rutaceae family with abundant lignan content is the Zanthoxylum genus (Wang et al., 2022). Zanthoxylum rhetsa is a big "Panggal buaya" tree in Indonesia. In Indian tribes, the plant treats many infirmities, such as diabetes, inflammation, rheumatism, toothache and diarrhea (Santanam et al., 2016). The methanol extract of the seed and root of Z. rhetza showed antioxidant and antibacterial activity (Zohora et al., 2019;Hayat & Vandna, 2018). In addition, the Z. rhetsa stem extract gave significant anti-inflammatory activity (Parthiban et al., 2017). The previous phytochemical studies reported the presence of alkaloids, flavonoids, glycosides, saponin, tannins and terpenoids in Z. rhetsa (Kyaw et al., 2020;Mallya and Bhitre, 2020). Furthermore, other secondary metabolites found in the Z. rhetsa bark are lignans which have a skeleton of furofuran, asarinin and horsfieldine and showed strong antibacterial activity against Staphylococcus aureus (Tantapakul et al., 2012).
As part of our research on Indonesian Rutaceae plants, herein, we report the isolation, and characterization of a furofuran lignan, established as (+)-piperitol-γ,γdimethylallylether (1), and its cytotoxic activity assay against breast cancer MCF-7 cell lines.

General
UV spectra were measured using a TECAN Infinite M200 pro with MeOH, and IR spectra were recorded on a SHIMADZU IR Prestige-21 in KBr. The mass spectra were obtained with Waters Q-TOF Xevo mass spectrometer instrument. NMR spectra were obtained on a Bruker Topspin spectrometer at 500 MHz for 1 H and 125 MHz for 13 C using tetramethylsilane (TMS) as an internal standard. Silica gel G 60 (Merck) 70-230 mesh for crude separation and 200-400 mesh for purification using column chromatography. Thin Layer Chromatography (TLC) plates were precoated with silica gel GF 254 (Merck, 0.25 mm), and detection was achieved by spraying with 10% H 2 SO 4 in ethanol, followed by heating and irradiated under UV light at wavelength 254 and 365 nm.

Plant Material.
The stem bark of Z. rhetsa (Roxb) D.C. was collected in September 2018 at Bogor Botanical Garden, Bogor, West Java Province, Indonesia. The plant was identified by the staff of the Bogoriense Herbarium, Bogor,.

Extraction and Isolation
The air-dried and powdered Z. rhetsa stem bark (5.5 kg) was extracted with methanol (36 L) at room temperature. The methanol (MeOH) extract was evaporated in a vacuum rotary evaporator to yield a concentrated MeOH extract (593.40 g). The MeOH extract was successfully dissolved in water and partitioned with n-hexane, methylene chloride (CH 2 Cl 2 ), and n-butanol (n-BuOH). Evaporation of the solvent resulted in the concentrated extract of n-hexane (87.90 g), CH 2 Cl 2 (73.30 g), and n-BuOH (121.50 g).

Cytotoxic Activity
Cell viability was assessed with Presto Blue reagent (Thermo Fisher Scientific, Uppsala, Sweden) to rapidly evaluate the viability and quantitatively proliferation of various resazurin-based cell types using live cell reduction capabilities. When cells are alive and healthy, they maintain a reduced environment in their cytosol. The reduction of resazurin (blue) works as a cell viability indicator by using absorbance or utilizing fluorescent outputs to reduce resorufin (purple). The conversion is proportional to the number of metabolically active cells. Briefly, MCF-7 cell lines were grown in 70% confluent, harvested and counted, and then diluted with complete culture RPMI medium. The cells were then transferred into 96 well plates with 170.000 cells/well. After overnight growth, the cells were treated with increasing compound 1 concentration (7.81, 15.63, 31.25, 62.50, 125, 250, 500, 1000 ppm) with cosolvent 2% (v/v) DMSO in PBS. Cisplatin was used as the positive control. All samples were incubated at 37 °C in a 5% CO 2 incubator for 24 hours. After incubation, the medium was immediately replaced by 10 µL PrestoBlue reagent in 90 µL RPMI medium. The plates were incubated for 1-2 hours until resorufin was formed (colour changes from blue to purple). The absorbance was measured at 570 nm using a microplate reader. The IC 50 value is the concentration for 50% growth inhibition. The percentage of cytotoxicity compared to untreated cells was determined with the equation below. A plot of % cytotoxicity versus sample concentrations was used to calculate the concentration, which showed 50% cytotoxicity (IC 50 ) (Camarillo et al., 2014;Machana et al., 2011). All assays and analyses were each runs in duplicate, and all were averaged.

RESULTS AND DISCUSSION
The concentrated MeOH extract (593.40 g) from the stem bark of Z. rhetsa was fractionated with n-hexane, methylene chloride (CH 2 Cl 2 ), and n-butanol (n-BuOH) successively to yield the concentrated extract of n-hexane (87.90 g), CH 2 Cl 2 (73.30 g), and n-BuOH (121.50 g). The CH 2 Cl 2 fraction (73.30 g) was subjected to silica gel 60 by vacuum liquid chromatography (VLC) with nhexane-EtOAc-MeOH as a solvent system to afford five fractions (A-E). The B fraction (14.20 g) was further separated according to the Thin Layer Chromatography (TLC) profile suggesting the presence of aromatic compounds, whose structurally gave fluorescence under UV light 254 nm. Separation with gradient technique 5% using VLC and isocratic elution of n-hexane-EtOAc (7.5:2.5) using preparative thin layer chromatography (PTLC) on silica gel GF 254 were applied to give compound 1 (9.5 mg).
Previously, the furofuran lignans were isolated from the roots of Z. planispinum showed the strongest inhibitory effect on the growth of HL-60 and PC-3 (Su et al., 2015). Furofuran lignans have a variety of structures due to different substituents at aryl groups and diverse configurations at furofuran ring. They exhibit a wide range of significant biological activities, including antioxidant, anti-inflammatory, and cytotoxic (Xu et al., 2018).

CONCLUSIONS
In summary, we report the known lignan furfuran-type (+)-piperitol-γ,γdimethylallylether (1). Compound 1 was isolated from the methylene chloride (CH 2 Cl 2 ) extract of Z. rhetsa stem bark. The elucidation stucture of compound 1 was determined based on the 1D-and 2D-NMR, as well as comparison with the previous spectral data. Furthermore, the (+)-stereochemistry of compound 1 was established based on the reported positive optical rotation values. In this study, compound 1 was evaluated for its cytotoxic activity against the MCF-7 breast cancer cell line, showing weak inhibition with an IC 50 value of 261.37 µg/mL. This compound was first reported from the Zanthoxylum rhetsa species to the best of our knowledge.