Bioactive triterpenoids from Indonesian medicinal plant Syzygium aqueum

: A large common species, Syzygium aqueum , belonging to the genus Syzygium possesses numerous bioactive phytochemical constituents. Moreover, the dif ferent parts of this species have been used as folk med icine since centuries ago. In this study, a phytochemical exploration was carried out on the plant ’ s stem bark. Isolation of the compounds was carried out through the extraction step with some organic solvents, followed by separation and puri ﬁ cation using chromatography tech niques until the two triterpenoids were isolated from nonpolar and semipolar extracts. Structure elucidation was done using spectroscopic methods. These compounds were identi ﬁ ed as alphitolic acid and arjunolic acid. Subsequently, these two compounds were used in anti cancer tests against human cancer cells HeLa, T47D, and A549 using colorimetric assay. The result showed that both compounds showed more inhibition of the growth of HeLa and T47D than A549 cancer cells, with the highest activity shown by arjunolic acid against HeLa cell lines.


Introduction
One of the probable medicament manufacturing countries, Indonesia, between the Pacific and Indian Oceans, is commonly known as a country with the second richest biodiversity of medicinal plants on the earth. Medicinal plants have therapeutic properties or provide beneficial pharmacological effects for the human body. Some examples are given here. The chemical composition of the essential oils extracted from Elaeagnus umbellata Thunb fruit demonstrated antioxidant, anticholinesterase, and antidiabetic activities that could be used as an alternative drug to treat oxidative stress-related diseases [1]. The essential oil of Teucrium stocksianum possesses a strong antinociceptive potential, which is further used as a topical analgesic [2]. The study of Woodwardia unigemmata (Makino) Nakai plant extracts, which were rich in polyphenolic compounds (total phenolic compounds and total flavonoid compounds) with efficient antioxidant activity, also showed remarkable antibacterial activity against plant and animal pathogenic bacteria [3]. The diverse Indonesian medicinal plants have been used as the sources of herbal medicines for the inhibition and treatment of human diseases for several thousand years [4].
The sixteenth largest genus of flowering plants in the Myrtaceae family, Syzygium, comprises about 1,200-1,800 species. Moreover, Syzygium species of tree and shrub types are distributed in tropical areas worldwide, especially Indonesia, Malaysia, Philippines, and Thailand. Some species of Syzygium are widely cultivated for economic importance, edible fruits, spices, flavoring agents, and pharmacological proprieties. Syzygium contains abundant diversity of bioactive phytochemical constituents such as terpenoids, steroids, chalcones, flavonoids, lignans, alkyl phloroglucinols, hydrolysable tannins, and chromone derivatives [5].
A medicinal species of Syzygium found in Indonesia, Syzygium aqueum, is a large common species in this genus found in Malaysia [6]. Fruits, leaves, and roots of this species have been used as folk medicine, especially for antibiotic properties, diabetic treatment, the treatment for a cracked tongue, relieving itches, stomach aches, dysentery, and reducing of swelling, respectively [7][8][9][10][11].
Based on the literature study, the stem bark of S. aqueum was designated for this research. Primarily, some extracts of stem bark of S. aqueum were separated to collect bioactive constituents with vacuum liquid chromatography and column chromatography. The structure of isolated compounds was identified with spectroscopic methods, including UV, Fourier transform infrared (FTIR), nuclear magnetic resonance (NMR [1D and 2D]), and HR-DART-MS. After this, their anticancer activity was determined with colorimetric assay (MTT and XTT assays). The isolated bioactive constituents were triterpenoid compounds, first isolated from S. aqueum.

Plant materials
The stem bark of S. aqueum was collected in December 2018 from Wage, Taman, Sidoarjo, East Java, Indonesia. The plant material was identified at the Department of Biology, Faculty of Science and Technology, Universitas Airlangga, and the voucher specimen (UA-MSa050918) was deposited at the Herbarium of Universitas Airlangga, Laboratory of Biosystematic, Department of Biology, Faculty of Science and Technology, Universitas Airlangga. The collected stem bark was cleaned, chopped, and crushed into small pieces of coarse powder.

Instrumentations
A polarimeter with φ 3.5 mm diameter (cell size) × 100 mm (cell length) was used to measure the specific rotation. For structure elucidation, some of the instruments used are a UV-vis 1800 spectrometer (Shimadzu), FTIR spectra on a Tracer-100 spectrophotometer (Shimadzu), NMR spectra on Bruker Avance III HD 600, and HR-DART-MS on an Exactive Plus Orbitrap DART Mass Spectrometer. Enzymelinked immunosorbent assay (ELISA) reader was used for the anticancer test.

General procedure
For extraction, fractionation, isolation, and purification of a sample, organic solvents such as n-hexane (n-Hex), ethyl acetate (EtOAc), dichloromethane (DCM), and methanol (MeOH) were used. In analytical thin-layer chromatography (TLC), a precoated silica gel 60 F 254 (Merck) was used and an anisaldehyde-sulfuric acid reagent was sprayed on a TLC plate for visualization. Silica gel 60 (700-200 mesh ASTM) was applied for column chromatography. Then, vacuum liquid chromatography (VLC) was performed using Kieselgel 60 (F 254 , Merck). The melting point of pure compounds was measured on a Fisher-Johns melting point apparatus (Stuart SMP30). Spectroscopic methods, including UV-vis, IR, NMR, and MS, were used to determine the structure of isolated compounds. An anticancer test was carried out using MTT and XTT reagents (colorimetric assay), and the absorbance for an anticancer test was recorded on an ELISA reader.

Extraction and isolation
At room temperature, 2 kg of coarse powder of stem bark was extracted with a polar solvent (MetOH, 40 L) for 3 × 24 h. This crude extract (450 g) was then mentioned to partition with a nonpolar solvent (n-hexane). It was separated into a nonpolar fraction and a methanol fraction. Then, 400 mL of aqueous was added to methanol fractions before partitioning with a semipolar solvent, EtOAc. After this, this mixture was separated into a semipolar fraction and an aqueous methanol residue. First, the nonpolar fraction was chromatographed with gradient polarity solvents, n-Hex:EtOAc. Separation procedure was carried out for three times using column chromatography to obtain a pure compound. Compound 1 (30 mg) was acquired from a fraction eluted with n-Hex:EtOAc = 7:3. The semipolar fraction was separated by VLC using gradient solvent mixtures of n-Hex:DCM and DCM:EtOAc. Separation procedure using column chromatography was carried out twice to obtain a pure compound, Compound 2 (50 mg), that was eluted with DCM:EtOAc = 6:4.

Anticancer activity 2.5.1 Cell culture
Three cancer cell lines, HeLa (cervical cancer), T47D (breast cancer), and A459 (lung cancer), were obtained from American Type Culture Collection. Cells were cultured at 37°C in a CO 2 incubator for 24 h and 100% humidity in medium supplemented with 10% FBS, 1% L-glutamine, and 1% penicillin/streptomycin. The cell culture process was carried out in the Cancer Chemoprevention Research Center, Faculty of Pharmacy at Universitas Gadjah Mada, Indonesia and in Physic Laboratory, Faculty of Science of University Putra Malaysia (UPM), Malaysia.

MTT assay
An anticancer test of pure compounds was performed using MTT assay. Cells were cultured in 96 well plates at 213 × 10 4 cell/well density and were incubated at 37°C in a CO 2 incubator for 24 h. The cells were then treated with 100 µL of the prepared sample with different concentration series (1.5625-100 µg/mL) at 37°C in a CO 2 incubator for 24 h. As much as 100 µL of the MTT reagent (50 mg in 10 mL of PBS) was filled in each well after incubation, and it was incubated again at 37°C in a CO 2 incubator for 2-4 h until purple formazan crystals are formed. After the crystal formation, cells were observed with a microscope, and 100 µL of 10% SDS stopper in 0.1 N HCl was added, and it was kept in a dark place overnight (room temperature). Next, the plate's absorbance value was read at a wavelength of 560 nm by using an ELISA reader [18,19].

XTT assay
The anticancer test was carried out using XTT assay. Cells were seeded (10 4 -10 5 cells/well) into 96 well plates and were incubated at 37°C in a CO 2 incubator for 24 h. As much as 100 µL of the prepared sample with various concentrations (1.5625-100 µg/mL) was inserted to treat cancer cells. The plate was incubated at 37°C in a CO 2 incubator for 24 h. And then, PMS solution (10 mM PMS solution and PBS (3 mg PMS + 1 mL PBS)) was prepared and 10 µL of this solution was added in 4 mL of XTT (4 mg of XTT dissolved in 4 mL of 37°C cells culture medium). After incubation, 25 µL of the XTT reagent (XTT/PMS solution) was filled in each well. After this, it was incubated for 2-4 h in a CO 2 incubator at 37°C until the changing of the orange formazan. The next step was to read the absorbance value at a wavelength of 450 nm using an ELISA reader [19,20]. Each experiment was carried out in triplicate, and the number of viable cells was calculated using the following formula:   Table 1. Bioactive triterpenoids from Indonesian medicinal plant S. aqueum  207    (C-29), and 22.6 ppm (C-30) were suggested as primary carbons. The presence of eight quaternary carbons, six methine carbons, ten methylene carbons, and six methyl carbons was also confirmed by the DEPT experiment. According to the above data, the structure of Compound 2 was verified as arjunolic acid, and its molecular formula was C 30 H 48 O 5 . DQF-COSY ( 1 H-1 H), NOESY ( 1 H-1 H), and HMBC ( 1 H-13 C) correlations of the structure are demonstrated in Figure 2. And then, the spectral data of both 1 H NMR (600 MHz, methanol-d 4 ) with their coupling constants and 13 C NMR (150 MHz, methanol-d 4 ) are shown in Table 2, completed with the reported literature data.

Anticancer activity
Isolated compounds in this study were evaluated their anticancer activity against the human cancer cells as HeLa (cervical cancer), T47D (breast cancer), and A549 (lung cancer) cell lines using MTT and XTT assays, which are the colorimetric measurement of cell viability. In the present anticancer activity test, doxorubicin was used as the standard. The pure compound is deemed highly toxic if the half-maximal inhibitory concentration (IC 50 ) is ≤4 µg/mL and toxic if the IC 50 is ≤20 µg/mL. The compound is moderately toxic if the IC 50 is 20-100 g/mL and if IC 50 above 100 g/mL is recommended as nontoxic [23,24]. Based on anticancer activity data obtained, alphitolic acid was suggested as a toxic compound against HeLa and T47D cell lines, with IC 50 being 16.12 µg/mL and 7.37 µg/mL, respectively. However, its activity against the A549 cell line was presented as moderate inhibition (IC 50 = 84.41 µg/mL). Moreover, arjunolic acid with an IC 50 of 6.74 µg/mL was considered a toxic compound against HeLa cell lines. Arjunolic acid was a moderate compound against the T47D cancer cell line with an IC 50 of 27.15 µg/mL, although it was a nontoxic compound against the A549 cancer cell line. According to the data mentioned earlier, the two types of triterpenoids inhibited higher toxicity against HeLa and T47D cell lines than the A549 cell line, and their activities may improve to obtain the natural origin drug from this species. The IC 50 values of the two compounds are listed in Table 3.
Many terpene compounds have interesting bioactivities, so it is necessary to carry out other activity tests such as agar-well diffusion methods to evaluate the minimum inhibitory concentrations, minimum bactericidal concentration, IC 50 , and zone of inhibitions that could determine the potential antimicrobial and antifungal efficacy. The significant antioxidant potential could be evaluated through 1,1-diphenyl-2-picryl-hydrazyl and 2,2'-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) assays. The inhibiting of the α-glucosidase represents an antidiabetic activity that also could be visualized through molecular docking simulations. The effective anti-inflammatory agent could be determined via the carrageenan-induced assay, while an appreciable analgesic activity could be observed through the acetic acid-induced writhing bioassay [25].

Conclusion
This study reports the success of isolating two bioactive triterpenoid compounds from stem bark of S. aqueum, an Indonesian medicinal plant, namely alphitolic acid and arjunolic acid. These two isolated triterpenoids were significantly toxic against cervical cancer and breast cancer, shown by small IC 50 values. Based on this result, we recommended continuing exploring the potential of this plant so that it can be used as a source of secondary metabolite compounds with interesting bioactivity.
Acknowledgments: All authors express big thanks to the Indonesian Ministry of Education and Culture for the Research Grant (Penelitian Dasar, 2021), to the Faculty of Pharmacy, Meijo University, Tempaku, Nagoya, Japan, for NMR and MS data, to the Faculty of Pharmacy, Gajah Mada University, Indonesia, and the Faculty of Science, UPM, Malaysia for an anticancer test. Author contributions: Alfinda Novi Kristanti: conceptualization, formal analysis, funding acquisition, investigation, resources, supervision, and writingreview and editing; Ei Ei Aung: data curation, formal analysis, investigation, project administration, validation, visualization, and writingoriginal draft; Nanik Siti Aminah: conceptualization, formal analysis, funding acquisition, methodology, resources, and supervision; Yoshiaki Takaya: data curation, resources, software, validation, and visualization; Hnin Thanda Aung: data curation, formal analysis, methodology, and supervision; and Rico Ramadhan: data curation, formal analysis, software, and validation.

Conflict of interest: Authors state no conflict of interest.
Ethical approval: The conducted research is not related to either human or animal use.