The beneficial effect of the ethanolic extract Etlingera elatior fruit

Etlingera elatior In sepsis, endothelial dysfunction can occur due to oxidative stress and hyperinflammation. This study evaluated the effect of E. elatior (EE) ethanolic extract in reducing IL-1ß and caspase-3 levels in sepsis model mice. Thirty-two n = 32) were randomly distributed into four groups ( n = 8). Induction of sepsis was performed through intraperitoneal injection of lipopolysaccharide (LPS) 0.3 mg/kgBW. Ethanol extract of EE 4.2 mg/20 g, p.o. was given 5 days before (EE-1), 5 days after (EE-2), and simultaneously during LPS induction (EE-3). ELISA was used to measure IL-1ß and caspase-3 levels. The ANOVA was performed to test the differences of each group. Mean levels of IL-1ß ( p < 0.05) and caspase-3 ( p < 0.05) differed in the control group with each intervention group. The EE significantly reduced levels of IL-1ß and caspase-3 ( p < 0.05), with the best results being before LPS induction. This study provides that EE may have a role in averting endothelial dysfunction in sepsis by lowering levels of IL-1ß and


INTRODUCTION
Sepsis is a life-threatening emergency due to multiple organ dysfunction resulting from systemic host dysregulation in response to infection (Gyawali et al., 2019). The central mechanism of sepsis is acute inflammation involving multiple organs (Hotchkiss et al., 2017). Data from the World Health Organization (WHO) showed that the global incidence of sepsis was 189 per 100,000 people/year with a mortality rate of 26.7%, and the mortality of sepsis patients in intensive care units was 42% (WHO, 2020). Fifteen percent of sepsis patients experienced septic shock, with a mortality rate of more than 50% (Dugar et al., 2020).
Sepsis pathophysiology and pathogenesis come from the reaction of infection, and bacterial infection is still the main cause (Prescott and Angus, 2018). The occurrence of infection can also be influenced by environmental factors such as air, water, food, or changes in climate and seasons. In sepsis, excessive inflammation and oxidative stress cause degranulation and release of proteases, resulting in local and systemic endothelial damage. Complement released as an immune response is known to induce endothelial dysfunction (Chang, 2019;Jacobi, 2021). Endothelial dysfunction can be used as a predictor of patients' mortality in sepsis, where endothelial dysfunction plays a role in the pathophysiology of septic shock and organ dysfunction (Boisramé-Helms et al., 2013). Current management of sepsis does not specifically target inflammation and oxidative stress. Therefore, effective adjuvant therapy is needed to reduce inflammation and oxidative stress (Lin et al., 2018).
Sepsis is still a health problem with high mortality rates. Prompt and appropriate treatment can improve the prognosis in sepsis patients (Cecconi et al., 2018). An in vivo study is needed to assess the potential anti-inflammatory and antioxidant effects of the ethanol extract of EE fruit in the treatment of sepsis. The aim of this study was to identify and evaluate the potential of local plants, namely ethanol extract of kecombrang fruit in reducing IL-1ß and caspase-3 levels in experimental animal models.

MATERIAL AND METHODS
The use of experimental animals in this study has complied with the 3R principles according to the provisions of the National Center for the Replacement, Refinement, and Reduction of Animals in Research (NC3Rs) and has been ethically approved by the Health Research Ethics Committee of Dr. Moewardi General Hospital (serial number: 477/IV/HREC/2021). This study was conducted at the Laboratory of Food and Nutrition Study of PAU, Universitas Gadjah Mada, Yogyakarta, and the Biomedical Laboratory of Universitas Sebelas Maret, Surakarta.

Experimental animals
and weighed 20-30 g were used in this research. Male mice were obtained and kept in experimental animal care at PAU Universitas Gadjah Mada, Yogyakarta. Throughout the observation, all animals were kept at room temperature of 32°C with light controlled in 12-hour light:12-hour dark cycle, fed standard with BR 1 diet according to body weight, and given water ad libitum for 7 days.

Preparation of extract
EE fruits were obtained from Langkaplancar, Pangandaran, and Jawa Barat. Extraction is conducted by maceration methods. The chemicals used are 70% ethanol. The fruits were peeled, washed, and dried, then crushed with a blender. The first maceration process was conducted for 24 hours, followed by another 2 hours at room temperature. The sample was centrifuged at 4°C at a 4,200 rpm speed for 20 minutes. The filtrate obtained was put into an extraction flask and evaporated with a rotary evaporator at 50°C. The extract was then tested using highperformance liquid chromatography examination (Farida and Maruzy, 2016). According to an analysis of the levels of vanillic acid and ethanol extract, 1 gram of ethanol extract of E. elatior fruit contained 255 mg of vanillic acid).

Experimental design
The research laboratory with post-test only group design study was calculated using the Federrer formula to get at least 5 mice per group for 4 groups. The mice were divided into four groups at random (n = 8 per group). Each group of mice received an intraperitoneal injection of 0.3 mg/kgBW lipopolysaccharide (LPS) to induce sepsis.
Control group: sepsis control mice received LPS alone. EE-1 group: given ethanol extract of EE starting from 5 days before LPS induction. EE-2 group: given ethanol extract of EE 5 days after LPS induction. EE-3 group: given ethanol extract of EE simultaneously LPS induction.
Each experimental animal received a daily dose of 4.2 mg/20 g ethanol extract of EE. All treatments were administered orally every day until 7 days before the sacrifice. The IL-1β level was measured before the mice were killed and caspase-3 expression was examined after mice were sacrificed.

Measurement of IL-1β levels and caspase-3 expression
The venous blood sample of each mice was collected into a separated serum tube before the sacrificed day. The research protocol of IL-1β and Caspase-3 levels (Sigma Aldrich, USA) used the ELISA method and were conducted as a manufacturer's instruction. Each plate contained 100μl of mice serum that has been diluted with serum diluent (1:50) and incubated at 37°C for 45 minutes. A conjugate solution and 100μl of ABTS peroxidase were also added to the plate. Each plate then received a 25 μl stop solution treatment. If the difference in optical density (OD) between the (+) and (−) is ≥0.300 at a wavelength of 405 nm, the result is positive.

Statistical analysis
Statistical analysis in this study was done using SPSS version 22.0 for Windows. A descriptive analysis was conducted to determine the characteristics of the research sample. The numerical data were tested for normality with Shapiro-Wilk and homogeneity of variance with Levene's test. The ANOVA F-test was used to determine differences in each group of data with a normal and homogeneous distribution, followed by the post-hoc least significant difference test to determine the difference in mean between groups. Meanwhile, for data that is not normally distributed, the Kruskal-Wallis test is carried out, followed by the Mann-Whitney test.  Table 1 shows the ANOVA test results for differences in caspase-3 levels in the EE-1, EE-2, EE-3, and control groups. According to Table 1, an ethanol extract of kecombrang fruit at 4.2 mg/20 g significantly (p = 0.001) reduced caspase-3 levels ( Fig. 1), with the best results in the EE-1 group. Table 2 shows significant difference in caspase-3 levels in the EE-1, EE-2, and EE-3 groups, and partially in the control group (p < 0.05). Based on the description above, it is clear that the ethanol extract of kecombrang fruit is effective in reducing caspase-3, with the best results in the group (EE-1) treated with the extract 5 days before LPS. Table 1 shows the effect of the ethanol extract on the IL-1ß levels in the EE-1, EE-2, EE-3, and control groups. The ethanol extract significantly (p = 0001) reduced IL-1ß levels in all the groups (Fig. 2), with the best results in the EE-1 group. Table 2 shows significant differences in IL-1ß levels in the EE-1, EE-2, and EE-3 groups and partially in the control group (p < 0.05). Based on the result, it is clear that the ethanol extract of kecombrang fruit can reduce the IL-1ß levels and that it can do so more effectively when administered prior to LPS (EE-1).

DISCUSSION
Sepsis is a medical emergency induced by a systemic immune response to infection that can lead to septic shock, where bacterial infections are still the main cause of sepsis (Dugar et al., 2020;Gyawali et al., 2019;Hotchkiss et al., 2017;Prescott and Angus, 2018). A mortality rate above 50% is associated with sepsis and septic shock (Dugar et al., 2020). In sepsis, severe endothelial cell dysfunction may lead to multiorgan disorders' progression (Ince et al., 2016). The release of proinflammatory cytokines such as TNF-a, IL-1, and Il-6 from macrophages may exacerbate systemic infection and endothelial dysfunction in sepsis (Aziz et al., 2012).
In septic conditions, endothelial apoptosis is involved in endothelial injury and dysfunction. The Shioiri et al. (2009) research found that LPS in sepsis induces a significant increase in caspase-3 activity (Shioiri et al., 2009). The main executor of the apoptotic process is caspase-3. Nonsurvivor septic patients were recorded to have higher serum caspase-3 concentrations than survivors. Compared to healthy ones, septic patients have higher caspase-3 in their lymphocytes, spleen samples, and plasma (Lorente et al., 2018).
IL-1ß is known to cause vasodilation and hypotension and increase integrin synthesis, leading to the adherence of leukocytes and also stimulates the expression of intercellular adhesion molecules to promote neutrophil adhesion to the endothelial cells (Namas et al., 2012;Nasronudin, 2011). This phenomenon may trigger endothelial leakage and lead to septic shock. Thus, IL-1ß and caspase-3 can be the apparent markers of endothelial dysfunction in sepsis (Aziz et al., 2012;Luan et al., 2015;Namas et al., 2012). In this study, the level of caspase-3 expression was reduced in subjects who received the ethanol extracts treatment at various times, with treatment group 1 (EE-1) having the lowest caspase-3 value. IL-1ß also significantly decreased compared to the baseline value (control group). The EE-1 had the lowest IL-1ß values. It shows that administering EE 5 days prior to mice-induced sepsis protects against endothelial dysfunction by inhibiting caspase-3 and IL-1ß expression. In sepsis, decreased caspase-3 expression and IL-1ß levels reduce the likelihood of endothelial dysfunction. This is consistent with the findings of Juwita et al. (2020), who found that a 1,000 mg/kgBW dose of EE extract reduced the expression of nuclear factor-B (NF-kB) in gastric ulcer model rats (Juwita et al., 2020).
Kecombrang fruit extract contains bioactive compounds such as vanillic acid that plays a role in fighting inflammation.   The inflammatory process is believed to be inhibited through the suppression of cyclooxygenase-2 expression and the inhibition of transcriptional activation (NF-kB) and caspase-1 (Ernilasari et al., 2021;Hu et al., 2022). In addition, the alkaloids, phenolics, and flavonoids of kecombrang fruit extract can inhibit the growth of Staphylococcus aureus and Escherichia coli bacteria at a concentration of 2% through cell wall destruction (Ernilasari et al., 2021;Ghasemzadeh et al., 2015). Based on the results of the study, the potential of EE to inhibit bacterial growth is shown. Administration of EE before the induction of sepsis may have a protective effect on the endothelium by suppressing bacterial growth. Sepsis will affect various organs, and endothelial cells are the first to be exposed due to the inflammatory cascade by various cytokines (Dolmatova et al., 2021). It was reported that 48.4% of septic patients based on the Sepsis-3 criteria had bacteremia (Yu et al., 2022) In sepsis, there is also an increase in the activity of the enzyme NADPH (nicotinamide adenine dinucleotide phosphate) oxidase, which synthesizes reactive oxygen species (ROS) in blood vessels (Heerebeek et al., 2002;Wu et al., 2007). Increased ROS triggers an increase in caspase-3 and causes apoptosis. Flavonoids can inhibit the action of the NADPH oxidase enzyme and capture ROS (Yousefian et al., 2019). Inhibition of the expression of NADPH oxidase, NF-kB, and IL-1β can reduce the apoptotic process which is characterized by a decrease in caspase-3 levels and prevent endothelial damage. Bacterial infection can trigger apoptosis in endothelial cells (Dolmatova et al., 2021;Ince et al., 2016). EE plant-containing flavonoids may have an anti-inflammatory effect and inhibit endothelial cells apoptosis which can be the target of therapeutic development of sepsis.
Further study awaits to determine the exact mechanism of EE suppressing the level of caspase-3 and IL-1β and also the effect of the daily routine of EE administration. To determine the effective dose, studying different doses of EE is necessary to prevent endothelial damage. This study will benefit the development and utilization of EE ethanolic extract and provide reliable evidence for its future clinical applications for sepsis treatment.

CONCLUSION
The intervention of kecombrang fruit ethanol extract on caspase-3 and IL-1β levels in various preparation treatments showed that kecombrang fruit ethanol extract (4.2 mg/20gr) can reduce caspase-3 and IL-1β, with the best results in the group given ethanol extract before LPS induction. This demonstrates that kecombrang extract given before induction can significantly reduce caspase-3 and IL-1β expression levels, demonstrating the potential benefits of kecombrang extract in averting endothelial dysfunction in sepsis. As a result, kecombrang extract may be utilized as an adjuvant therapy in treating sepsis.