MATERIALS AND METHODS

This research lasted for 4 months (August-November 2022) at Sumber Belajar Ilmu Hayati (SBIH) Ruyani, Bengkulu, Indonesia¹⁵.

Extract preparation: The identity of the Etlingera hemisphaerica (Blume) plant was verified by the Center for Plant Conservation and Botanical Gardens Research, LIPI, Bogor, Indonesia, (http://lipi.go.id/,NumberB-1750/IPH.3./KS/V/2019). The leaves/fruits of E. hemisphaerica were collected from Lebong District, Bengkulu Province, Indonesia. The leaves/fruits were cleaned and then sliced into small pieces of 3,000 g/6,260 g fresh leaves/fruits of E. hemisphaerica, then dried with the help of wind and ground into a powder of 800 g/1,360 g dried leaves/fruits. The powder was macerated for seven days in 96% ethanol, then concentrated using a rotary evaporator at 50°C and the filtrate was condensed to obtain a concentrated extract¹⁶. After the ethanol contained in the concentrated extract evaporates, the crude extract can be used as a test material in this study¹⁷.

Phytochemical screening of LE3H and FE3H: Sucrose level of LE3H and FE3H was analyzed quantitatively by High-Performance Liquid Chromatography (HPLC, Gadjah Mada University, Indonesia, https://lppt.ugm.ac.id/id/)¹⁸, while five test parameters (flavonoids, alkaloids, saponins, tannins and phenols) of LE3H and FE3H were analyzed quantitatively. The quantitative analysis was done with UV-vis Spectrophotometry (1800 Shimadzu)^19-23.

Fresh chicken liver juice (FCLJ): The purpose of making fresh chicken liver juice (FCLJ) is for mice to have a high purine diet to increase uric acid levels, achieving a hyperuricemia condition. The composition of this chicken liver juice is Leghorn chicken liver 20 g which was made into juice using up to 100 mL of water. The FCLJ was made by mashing chicken liver weighing 20 g and adding 100 mL of water, then in a blender¹⁰ and then FCLJ is ready to use. Induction of hyperuricemia was carried out by giving FCLJ at a dose of 0.3 mL/20 g BW for seven days.

Dosages: Hyperuricemia in mice was induced by giving 0.3 mL/20 g BW of (FCLJ) for 7 days¹⁰. Allopurinol is a generic drug used to lower uric acid levels in the blood. This drug works by inhibiting the enzyme xanthine oxidase thereby inhibiting the formation of uric acid and can also inhibit the synthesis of purines. This study used a 0.01 mg g^–1 body weight (BW) dose of allopurinol as a positive control¹⁰. According to the report, the three doses of LE3H and FE3H used in this study were 0.13, 0.26 and 0.39 mg g^–1 BW^19-26.

Group of experimental animals: The first stage (A) used 24 male mice which were divided into 6 groups (A0, A1, A2, A3, A4 and A5). On day (D)1, all groups of mice were satisfied, then the early determination of blood uric acid levels was measured using a glucose, cholesterol and uric acid (GCU) Meter Device. Five mice groups, A1, A2, A3, A4 and A5, were induced by giving 0.3 mL of fresh chicken liver juice (FCLJ) for seven days to achieve hyperuricemia. On day 8, further determination of blood uric acid levels was measured by the GCU Meter Device (Halodoc Building, Jl. HR Rasuna Said Kav. B32-33, South Jakarta 12940). The condition of hyperuricemia in A1 was not neutralized. Meanwhile, hyperuricemia conditions in A2, A3, A4 and A5 were neutralized by giving 0.01 mg g^–1 body weight (BW) allopurinol, 0.13, 0.26 and 0.36 mg g^–1 BW LE3H for seven days. On day 15, the final determination of blood uric acid levels was measured by the GCU Meter Device (Table 1).

The second stage (B) used 24 male mice which were divided into 6 groups (B0, B1, B2, B3, B4 and B5). On the day (D) 1, all groups of mice were satisfied, then the early determination of blood uric acid levels was measured using the GCU Meter Device. Five groups of mice, namely B1, B2, B3, B4 and B5 were induced by giving 0.3 mL of fresh chicken liver juice (FCLJ) for seven days to achieve hyperuricemia. On D 8, further determination of blood uric acid levels was measured by the GCU Meter Device. The condition of hyperuricemia in A1 was not neutralized. Meanwhile, B2, B3, B4 and B5 hyperuricemia conditions were neutralized for seven days by giving 0.01 mg g^–1 body weight (BW) allopurinol, 0.13, 0.26 and 0.36 mg g^–1 BW LE3H. On day 15, the final determination of blood uric acid levels was measured by the GCU Meter Device (Table 1).

Blood uric acid level determination: Blood uric acid concentration in M. musculus was measured using the glucose, cholesterol and uric acid (GCU) Meter Device (https://apotekalkes.com/product/EASY-TOUCH-GCU-181818) Produced by Bioptic Technology with Indonesia Ministry of Health Registration: AKL20101902214 (Halodoc Building, Jl. HR Rasuna Said Kav. B32-33, South Jakarta 12940, Phone: +62 21-5095-9900, Email: help@halodoc.com).

Previously, the GCU Meter Device as a uric acid meter was adjusted to the code printed on the strip packaging. After that, blood is dripped on the strip, waited for about ±5 sec to read the blood uric acid level. The level of accuracy shown by the GCU Meter Device is the blood uric acid concentration in units of mg dL^{–1 27}.

Statistical analysis: Multiple comparisons were used to generalize the data from this study, followed by a significant difference test at the 95% confidence level²⁸.

Ethical statement: This research focuses on the ethics of using animals as well as aspects of human treatment, following the principle of the five freedoms (F), which include (a) freedom from hunger and thirst, (b) freedom from discomfort, (c) free from pain, injury and disease, (d) free from fear and long-term pressure and (e) freedom to express standard patterns of behavior²⁹. This study follows the National Institutes of Health’s Guide for the Care and Use of Laboratory Animals. The working protocol was accepted by the Bengkulu University Animal Experiment Ethics Committee (No. 53/KEH-LPPM/EC/2022, April 18, 2022).

RESULTS

Phytochemical analysis of LE3H and FE3H was carried out simultaneously in the same way. Five of the six test parameters, namely flavonoids, alkaloids, tannins, sucrose and phenol levels in FE3H were higher than in LE3H. Meanwhile, the levels of saponins in LE3H (2.32% w/w) were higher than in FE3H (1.64% w/w) (Table 2).

Giving 0.3 mL of FCLJ for seven days significantly increased (104.23-134.94%) uric acid (A1, A2, A3, A4 and A5) compared to the control (A0), so hyperuricemia was achieved. The condition of hyperuricemia in one group was not neutralized (A1). Administration of 0.01mg g^–1 BW Allopurinol for seven days reduced hyperuricemia by 54.09% (A2). It was further noted that the administration of doses of 0.13, 0.26 and 0.36 mg g^–1 BW LE3H for seven days significantly recovered hyperuricemia as much as 54.09, 56.14 and 60.88%, respectively (A3, A4 and A5). The LE3H has the same or higher potential for reducing hyperuricemia than Allopurinol. The results of statistical tests showed that the administration of FCLJ significantly increased uric acid higher than the control, while the administration of LE3H was able to restore hyperuricemia not different from the control (Table 3).

Giving 0.3 mL of FCLJ for seven days significantly increased (30.30-121.78%) uric acid (B1, B2, B3, B4 and B5) compared to the control (B0), so hyperuricemia was achieved. The condition of hyperuricemia in one group is not neutralized (B1). Administration of 0.01mg g^–1 BW Allopurinol for 7 days reduced hyperuricemia by 55.00% (B2).

It was further noted noted that the administration of doses of 0.13, 0.26 and 0.36 mg g^–1 BW LE3H for seven days significantly recovered hyperuricemia as much as 60.37, 62.24 and 65.57%, respectively (B3, B4. and B5). The LE3H has the potential to reduce hyperuricemia higher than Allopurinol. The results of statistical tests showed that the administration of FCLJ significantly increased uric acid higher than the control, while the administration of LE3H was able to restore hyperuricemia not different from the control (Table 4).

The LE3H and FE3H at the same dose showed that FE3H had a higher potential to restore hyperuricemia than LE3H (Table 5).

DISCUSSION

Comparison of phytochemical content of the leaf ethanolic extracts E. hemisphaerica (LE3H) and the fruit ethanolic extract E. hemisphaerica (FE3H) revealed that five of the six test parameters (flavonoids, alkaloids, tannins, sucrose and phenol) levels in FE3H were higher than in LE3H. Meanwhile, the levels of saponins in LE3H (2.32% w/w) were higher than in FE3H (1.64% w/w). Flavonoids are the most prominent phytochemical content in LE3H and FE3H (Table 2). Flavonoids have been recognized as molecules with great potential as solutions for hyperuricemia and gout at preclinical and clinical levels^30,31. One non-toxic natural flavonoid, quercetin (QC), in a safe dosage range with antioxidant, anti-apoptotic and anti-inflammatory properties, plays an important role in the treatment of aging-related diseases³². The last report states that the QC level in LE3H was 7.47±0.2 μg mL^{–1 33}. If the presence of QS has been detected in LE3H, then FE3H which contains higher levels of flavonoids is suspected to have QC levels in line with the presence of these flavonoids. It was further proven that the percentage of FE3H potential for restoring hyperuricemia at a dose of 0.13 mg g^–1 BW was 60.37% (Table 4), while for LE3H at the same dose, it was 54.09% (Table 3).

There were several factors that can trigger an increase in uric acid levels in a person’s blood, including often eating foods with high purine content, such as red meat, animal offal and several types of seafood. Fresh chicken liver juice (FCLJ) is an offal animal that contains a lot of purines, so when FCLJ (0.3 mL) was given to mice in gavage for 1 week (A2/Table 3, B2/Table 4) it markedly increased blood uric acid levels compared to control (A0/Table 3, B0/Table 4). Several other studies have also used FCLJ to induce hyperuricemia in test animals^34-36. Some reports explain that allopurinol is one of the drugs to recover the body from hyperuricemia^37-39. The facts from this research showed that 0.13 mg g^–1 BW LE3H works equivalent to 01 mg g^–1 BW allopurinol in 54.09% restoring hyperuricemia, whereas, at the same dose, 0.13 mg g^–1 BW FE3H shows a restoring hyperuricemia effect of 60.37% (A3/B3 Table 5). Uric acid is the main substance that causes hyperuricemia (gout), the end product of purine metabolism⁴⁰. It is formed from purines primarily by oxidation catalyzed by xanthine oxidase (XO) of hypoxanthine and xanthine. One of the gout treatment mechanisms is to block the XO enzyme which will inhibit the oxidation of hypoxanthine and xanthine and further inhibit uric acid production⁴¹.

A study was conducted on hyperuricemia mice to assess the effect of five flavonoids genistein, apigenin, QC, rutin and astilbin which showed a significant decrease XO in vivo⁴².

It was further reported that the uric acid formation was reduced in hyperuricemia mice after oral administration of flavonoid-rich plant extracts such as rutin, QC, kaempferol and apigenin⁴³. In another study, morin, myricetin, kaempferol, apigenin and puerarin at 50 and 100 mg/kg body weight elicited a hypouricemic action in hyperuricemia rats. These compounds significantly reduce serum uric acid levels by inhibiting hepatic XO as well⁴⁴. The QC is effective against allergy, inflammation, arteriosclerosis and cancer due to its strong antioxidant properties and metal ion chelating capacity^45,46. It is one of the most potent flavonoids that can interact by modulating the activity of various enzyme systems including lipoxygenase, phosphodiesterase and tyrosine kinase. The QC is a potent anti-gout compound that exhibits its activity through inhibition of the XO enzyme and exerts strong synergistic activity with kaempferol⁴⁷.

The fruit of E. hemisphaerica is quite popular among Indonesian people (https://eol.org/pages/1119400), has a distinctive aroma and taste image so that it can be developed as a raw material for the production of fresh drinks⁴⁸ and also has anti-hyperuricemic properties.

This preclinical research is limited to showing that the leaf and fruit ethanolic extract of E. hemisphaerica can potentially restore hyperuricemia in mice. These preclinical data form the basis that dietary supplementation with the leaf and fruit ethanolic extract may be beneficial in individuals with hyperuricemia problems. If the fresh drink E. hemisphaerica with anti-hyperuricemia benefits is successful, this preclinical research will develop into clinical research that has high pharmaceutical and economic value.

CONCLUSION

Leave and fruit ethanolic extract Etlingera hemisphaerica can potentially restore hyperuricemia in mice. A dose of 0.13 mg g^–1 BW LE3H as the anti-hyperuricemic in mice is equivalent to a dose of 0.01 mg g^–1 BW Allopurinol. The FE3H is more effective than LE3H to reduce uric acid in cases of hyperuricemia in mice. Dietary supplementation with LE3H and FE3H may be beneficial in individuals with hyperuricemia problems.

SIGNIFICANCE STATEMENT

This preclinical research shows that the leaf and fruit ethanolic extract of Etlingera hemisphaerica can potentially restore hyperuricemia in mice. Dietary supplementation with the leaf and fruit ethanolic extract may be beneficial in individuals with hyperuricemia problems. The E. hemisphaerica has a distinctive taste image and is widely known by the people of Indonesia. It is possible to produce E. hemisphaerica fresh drink with anti-hyperuricemia benefits.

ACKNOWLEDGMENTS

We sincerely thank Muslih Muslihin, SE (SBIH Ruyani, Bengkulu) for his valuable advice and recommendations on conducting this study. We also sincerely thank Dr. Hevi Wihadmadyatami (Gadjah Mada University, Indonesia) for her phytochemical screening of LE3H and FE3H.