Evaluation of xanthine oxidase inhibitory activity of sinapyl alcohol diacetate and stigmasterol compounds and phytochemical screening stem of Etlingera

Etlingera rubroloba A.D Poulsen is a genus Etlingera found in Southeast Sulawesi. This plant is widely used in local communities to relieve joint pain and increase stamina. Previous researchers isolated and identified this plant to obtain the compounds sinapyl alcohol diacetate and stigmasterol. However, these two compounds have never been reported on the Xanthine Oxidase (XO) inhibitory activity in vitro. The results of phytochemical screening have also not been reported in this plant. Therefore, in this study, a phytochemical screening test was conducted using colour reagents, and an in vitro test for XO inhibitory activity was carried out using the XO Kit. The results showed that E. rubroloba contains secondary metabolites of flavonoids, alkaloids, tannins, saponins, terpenoids, and the value of the percent XO inhibitory activity at a concentration of 320 µg/mL, namely sinapyl alcohol diacetate 85.52%, stigmasterol 90.42%, and allopurinol 97.63%, as a positive control. This research will be a reference in the development of anti - hyperuricemia medicine of nature.


Introduction
Etlingera is a family of Zingiberaceae, which has many species globally, around 150 to 200 species. This plant grows in Indonesia, including Java, Sulawesi, and Kalimantan. In Kalimantan, this plant is consumed daily as vegetables and traditional medicines, such as the species E. elatior used by local people as a cooking spice and headache medicine (Poulsen, 2012). In Sulawesi, Wawonii people use it as a nausea medicine (Rahayu, 2006).
One of the synthetic drugs used to relieve joint pain and reduce uric acid (hyperuricemia) is allopurinol in health services. Allopurinol works by inhibiting XO, which catalyzes the formation of uric acid (Lullmann, 2000) because it has side effects of causing allergies and hepatotoxicity. Researchers are trying to find other XO inhibitors that are more potent and safer (Bomalaski and Clark, 2004). One of the Etlingera plant species studied is E. rubroloba. This plant has been isolated, identified, and generated synaphyl alcohol diacetate and stigmasterol compounds ( Figure 1) (Jabbar et al., 2021b). These two compounds have never been tested for their activity as XO inhibitors. The aim of this study was to evaluate the activity of synaphyl alcohol diacetate and stigmasterol towards Xanthine oxidase inhibition and phytochemical screening of E. rubroloba A.D. Poulsen stem.

Sample preparation and extraction
The stems of E. rubroloba were collected from Konawe District, Southeast Sulawesi, and then determined at the LIPI Research Center for Biology, Cibinong, Bogor, Indonesia. The sample was cleaned, dried at 50 o C, and powdered, and 5120 g was obtained.
Samples were extracted using the maceration method with 12 L of methanol as a solvent (3 × 24 hrs). The filtrate was obtained by evaporating at 50°C to obtain 150 g of methanol extract.

Phytochemical screening
The screening test takes several crude extracts and then adds an appropriate solvent to test the secondary metabolite compound, namely flavonoids, alkaloids, saponins, tannins, terpenoids, and steroids (Amaral et al., 2021). Table 1 shows specific reagents used in each class of secondary metabolite compounds.

In vitro Xanthine Oxidase inhibitory activity
The XO inhibitory activity test was carried out in this study using the XO assay kit protocol (Abcam, 2018; Jabbar et al., 2021a)

Standard curve preparation
Approximately 4 μL of 0.88 M H 2 O 2 standard was diluted into 348 μL dH 2 O to generate 10 mM H 2 O 2 standard, then 20 μL of 10 mM H 2 O 2 standard was diluted into 980 μL dH 2 O to generate 0.2 mM H 2 O 2 standard.

Reaction mix
The reaction mixture used contains 50 μL (Assay Buffer 44 μl, Substrate Mix 2 μL, Enzyme Mix 2 μL, OxiRed Probe 2 μL). Furthermore, a 50 µL reaction mixture was added to each well containing standard H2O2, positive control, and test sample, then mixed well. Then, the plate was measured (OD = 570 nm/ colorimetric test) before incubation (T1 read A1), and then measured again after incubation at 25°C for 10-20 mins protected from light (T2 read A2). Selatan of Indonesia. Then the sample was extracted by maceration using methanol as a solvent (3 × 24 hrs) to obtain a methanol extract of E. rubrolaba. Furthermore, this extract was tested for phytochemical screening according to the established procedure, and the results can be seen in Table 2. Phytochemical screening was carried out on the methanol extract of E. rubroloba and generated secondary metabolites of flavonoids, alkaloids, tannins, saponins, and triterpenoids.

Results and discussion
The XO inhibitory activity test was conducted in vitro using the XO Abcam 1022522 kit (Abcam, 2018). Activity testing was carried out on sinapyl alcohol diacetate, stigmasterol, and allopurinol compounds as positive controls with various concentrations (20, 40, 80, 160, 320 μL/mL). Figure 2 shows the value of the percentage of xanthine oxidase inhibitory activity.
The XO inhibitory activity showed different effects at various concentrations (20,40,80,160, and 320 µL/ mL) of sinapyl alcohol diacetate, stigmasterol compared with allopurinol as a standard control. All samples have different abilities to activate the XO enzyme at different concentrations. Its activity increased concentration with inhibition that is, allopurinol as a standard control was more significant than sinapyl alcohol diacetate and stigmasterol.
In this study, allopurinol had the best effect on XO inhibition both in vitro and in vivo. XO has an essential role in regulating uric acid and preventing overuse, such as hyperuricemia (Shukor et al., 2018). Gout and hyperuricemia can be treated by increasing uric acid excretion of uric acid production (Kostic et al., 2015). At this time, allopurinol is widely used in the treatment of hyperuricemia or gout, but it has side effects such as hypersensitivity, and intolerance (Lü et al., 2013). Therefore, alternative XO inhibitors are recommended from natural compounds that have fewer side effects (Wong et al., 2014).

Conclusion
Etlingera rubroloba extract contains flavonoids, alkaloids, tannins, saponins, and terpenoids. Sinapyl alcohol diacetate and stigmasterol compounds have XO inhibitory activity and can be developed as antihyperuricemia.

Conflict of interest
The authors declare no conflict of interest.