1. INTRODUCTION:

Degenerative diseases or non-communicable diseases are caused by age-related changes and still take place chronically and progressively in world health problems because they have the potential to cause morbidity and mortality. The liver, which is the main metabolizing organ in humans, was not exempt from the progression of degenerative diseases that start from liver damage due to alcohol consumption, viral infections, and side effects of drugs. Numerous hazardous compounds, such as certain antibiotics, chemotherapeutic drugs, carbon tetrachloride (CCl4), thioacetamide (TAA), etc., excessive alcohol intake, and microorganisms have all been shown to induce liver cell damage¹.

The mortality rate due to liver damage increased by 65% to cirrhosis and liver cancer². The prevalence of patients with liver disorders in Indonesia, both viral and non-viral infections, tends to increase rapidly^3,4, The high cost of treatment has resulted in people switching to using medicinal plants to overcome them⁵. Indonesia's biological wealth is a potential natural resource that can be developed for medicinal raw materials. Several plants have shown activity as anti-hepatitis A (Ocimum basilicum) and antihepatitis B (Phyllanthus amarus and Morinda citrifolia)^6,7. These plants contain secondary metabolites that can protect and repair liver damage. Mechanisms that are traversed include reduction of oxidative stress, suppression of inflammation, and increased immune response⁸.

The peel of red dragon fruit (Hylocereuspolyrhizus) has rich in natural antioxidant compounds in the form of phenolic compounds, flavonoids, carotenoids, and anthocyanins. Red dragon fruit peel also contains levels of anthocyanins, pectin compounds, and other compounds such as galacturonic acid, mannose, galactose, xylose and ramnose^9,10. Wahdaningsih., et al.'s (2017) research results showed that the total phenolic content of the methanol extract, the soluble fraction of ethyl acetate and the insoluble fraction of ethyl acetate were 0.1994, 0.0196 and 0.4020ugGAE/g extract and the total flavonoid content of the methanol extract, the soluble fraction of ethyl acetate and the insoluble fraction, respectively. ethyl acetate 0.5139, 46.54 and 11.3811ugQE/g extract, respectively¹¹, The antioxidant activity of the methanol extract, the soluble ethyl acetate fraction and the insoluble ethyl acetate fraction had antioxidant activities of 241.19g/mL, 8.34 ug/mL, 46.84ug/mL, respectively. Red dragon fruit peel extract can increase phagocytosis, increase the number of cells and the total number of leukocytes and affect the relative weight of the spleen¹². Currently, no scientific publications have been found on the antihepatitic activity of red dragon fruit peel and prototype preparations for phytopharmaceuticals. Searching the patent database has not found any patent publications regarding hepatitis C antiviral compounds from red dragon fruit peel. Patent CN105524482A extracts flesh and red pigment from red dragon fruit, the invention CN107183296A discloses a method of dry powder of red dragon fruit peel and does not arrive at the recovery of pure compounds. The discovery of a class of compounds that have anti-degenerative activity will contribute to the prevention of the development of liver disease. In the degenerative process, there is a gradual reduction in the function of nerve cells so that cells that previously functioned normally become abnormal and may even not function at all, resulting in a decrease in cell resistance, resulting in cell death. With this background information, we, therefore, intended to investigate the hepatoprotective as well as anti-HCV activity of Hylocereuspolyrhizus peel extract.

2. MATERIALS AND METHODS:

2.1 Material:

The dragon fruit used in the study was collected from Pontianak, West Borneo.

Extraction and Preparation of Hylocereuspolyrhizus Peel Extract:

The dried and powdered Hylocereuspolyrhizus Peel were soaked in methanol at room temperature for 24 hours. Then, hours the filtrate was separated by filtration using a Buchner funnel assisted by a vacuum. The residue was extracted again in the same way and the treatment was carried out 3 times. The filtrate was collected and evaporated with the help of a fan to produce an extract of 75.95 grams with a yield of 5.73%

Animals and Treatment:

25 rats were weighed first and grouped based on similar weight. Then animals were were randomized and divided into five groups of 5 animal each. grouped for negative control, positive control, extract group 100 mg/kgBW, 200 mg/kgBW, and 300 mg/kgBW. The negative control rat group were fed 1% CMC-Na solution, the positive control rat group were fedby mixture of 1% CMC-Na solution and Paracetamo (l:20 ratio), the 100 mg/kgBW extract group received 60 ml mixture of 1% CMC Na solution and the extract 600 mg of extract, the 200 mg/kgBW extract group received 60 ml mixture of 1% CMC Na solution and the extract 1200 mg of extract, and the 300 mg/kg BW extract group received 60 ml mixture of 1% CMC Na solution and the extract 600 mg of extract. The dose of each rat in each group was recalculated according to the rat's body weight. After 4 days of induction treatment was completed, termination was carried out on day 5. The rats were terminated and blood samples were taken by cardiac puncture of the rat's organs in the form of liver. Blood samples were coagulated and serum was taken, and the liver was preserved with 10% formalin for hispathological studies¹³.

3. RESULT:

3.1 Phytochemical Screening:

The qualitative phytochemical screening of the Hylocereuspolyrhizus crude extract and organic fractions showed the presence of alkaloids, triterpenoids flavonoids, saponin and phenolic (Table 1.)

Table 1: Phytochemical screening result of Hylocereuspolyrhizus methanol extract.

Screening	Alkaloids	Triterpenoids	Flavonoids	Saponins	Phenolic
Methanol Extract	+	+	-	+	+

3.2 Hepatoprotective Effect:

Hepatoprotector test results were obtained in the form of data from SGPT (Serum Glutamic Pyruvic Transaminase) and SGOT (Serum Glutamic Oxaloacetic Transaminase) from the liver of rats as shown in Table 2

Serum Glutamate Pyruvate Transaminase (SGPT) and Serum Glutamate Oxaloacetate Transaminase (SGOT) is enzymes commonly found in liver, muscle, kidney, heart and liver cytoplasm cells. However, SGPT is more commonly found in the liver while SGOT is mostly found in the heart. This is why, compared to the SGPT enzyme, SGOT enzyme levels do not provide specific results for liver damage. The SGPT enzyme is needed to form amino acids which will later make up the protein needs of the liver¹⁴. Examination of SGPT levels can provide specific information if there is acute hepatocellular damage. Increased SGPT enzyme levels are the most sensitive sign of damage to liver cells. The normal SGPT concentration of mice are 2.1 – 23.8 U/L meanwhile SGOT is 23.2 – 48.8 U/L¹⁵.

Table 2: Data Analysis of SGPT

Groups	SGPT Level (mg/dL)		P Value		SGOT Level (mg/dL)		P Value
Groups	Mean	SD	Sig. Between Group	Sig. to Negatif Group	Mean	SD	Sig. Between Group	Sig. to Negatif Group
Negative Control	201.2400	29.44451	0,124^a	0.534	223.5640	28.92218	0,011^b	0.81
Positive Control	237.9260	29.10252			291.4780	42.85387
100 mg/KgBW	190.5680	32.52064		0.291	223.6260	23.43841		0.81
200 mg/KgBW	177.9780	39.40292		0.117	205.6500	30.09655		0.18
300 mg/KgBW	183.9520	50.26873		0.184	200.8260	59.06172		0.12

^a p > 0,05 no difference significance

^bp < 0,05 difference significance

The test results of SGPT enzyme levels (Table 2) of rats showed that the administration of red dragon fruit peel extract was likely potential hepatoprotective effect. Considering the SGPT levels in the treated group (doses of 100 mg/kg, 200 mg/kg, and 300 mg/kg) were lower than the positive control group, especially the dose of 200 mg/kg reducing the lowest level of SGPT compared to other group. It can be said administration red dragon fruit peel extract can reduce SGPT enzyme levels in mice given toxic doses of paracetamol. However, the onewayanova results show that there is no significant difference in SGPT levels between groups, this is indicated by the test result (sig) which is greater than 0.05 (significance level).Even so, judging from the mean and SD values, it can be seen that there are differences in SGPT levels between the test groups, where the lowest SGPT levels are shown by the 200mg/Kg bodyweight dose group.

A significant change also seen at SGOT concentration levels in the treated group (Doses of 200 mg/kg, and 300 mg/kg), which significantly decreased compared to the positive control group (paracetamol). contrast to SGPT levels, the one wayanova test shows that there are differences in SGPT levels between groups, which is indicated by the test results (sig) which are smaller than 0.05 (significance level). Owing to the fact that SGOT data is less specific for detecting liver damage, the authors can conclude that the dose that has the most optimal hepatoprotective effect is a dose of 200 mg/Kg bodyweight. This was indicated by the very low levels of the SGPT enzyme produced compared to the other groups. Generally, all of SGPT and SGOT levels resulted from doses 100; 200; and 300 mg/Kg were decreased or lower than negative control and positive control group.

The variation of decreasing liver enzyme by medicine plants especially red dragon fruit are arising from the variability of chemical compounds of plants. Approximately 40 commercial of mix-herbal formulations are available and recommended by doctors in India to treat hepatic problems, but the quest for a straightforward and exact herbal medication continues to be an exciting challenge. Additionally, some of these plant medicines have been said to have potent antioxidant properties. Various chemical components, including phenols, coumarins, lignans, fundamental oil, monoterpenes, carotinoids, glycosides, flavanoids, natural acids, lipids, alkaloids, and derivatives of xanthones, are present in domestic hepatoprotective plants^16,17.

3.3 Histopathological Observations:

Paracetamol or N-acetyl-p-aminophenol (also called acetaminophen (APAP)) is extensively used as an over-the-counter drug in fever, pain,and inflammation at its normal therapeutic doses, however, the overdoses of it is evident to cause liver failure. The overdose of acetaminophen depletes the level ofthe glutathione (GSH) which ultimately results in the hepatocellular damage. Currently, it is commonly used as a hepatotoxic agent inassessing hepatoprotective effects of wide varieties of substances,including crude extracts, isolated compounds, and synthetic chemicals¹⁸.

Paracetamol was used as a hepatoxicity agent which affects widely areas of acute liver parenchymal necrosis, together with an appearance of dilated vasculature and sinusoids around the necrotic zones. In large dosages, paracetamol causes hepatic necrosis by covalently binding its poisonous metabolite N-acetyl-p-benzoquinone imine to protein sulphadryl groups. This causes cell necrosis due to lipid peroxidation, which is triggered by a decline in glutathione levels in the liver. Because serum transaminases are cytoplasmic in nature and are released into the circulation as a result of cellular injury, an increase in their levels indicates structural damage to the liver. Free radicals harm biological processes, which in turn causes cellular damage that can result in conditions like cancer, liver damage, heart disease, etc. A metabolite called imidazole, which binds covalently in endoplasmic reticulum, reduces the toxicity¹⁹.

Figure 1: Histopathology of experimental rat liver. (a) Histrogram showing (a) normal rat liver (negative control group) with healthy tissues with normal hepatocytes, central veins, fat and glycogen accumulation in the cells, (b) acetaminophen-injured rat liver (positive control group) with large necrosis.

The liver from animals in the negative control group had normal hepatic cells with well-preserved cytoplasm, a prominent nucleus, nucleolus and visible central veins, but there is also fat and glycogen accumulation in the cells. Fatty is indicated by white cells that look round and have clear boundaries that separate them from other normal cells, while the presence of glycogen is indicated by white cells that are round but do not have clear boundaries like fat, the presence of glycogen in cells also occurs due to rats that were not fasted beforehand. termination and organ harvesting. Whereas the liver collected from acetaminophen-treatedmice showed extensive, mainly pericentral necrosis with loss of hepatic architecture, and vacuolar fatty change (Figure 1).

The 200mg/kg bodyweight dose extract group did not fully prevent the toxic effect of acetaminophen with a small necrotic area still present. On the other hand, the higher dose, namely the 300mg/kg bodyweight dose extract group showed a better prevention of the acetaminophen toxic effect with a smaller necrotic area present than the 200mg/kg bodyweight dose (Figure 2).

Figure 2: Histopathology of experimental rat liver. Histogram showing (a) a rat liver from 200 mg/kg bodyweight extract dose group, showing a small area of necrosis, (b)a rat liver from 300 mg/kg bodyweight extract dose group, showing even smaller area of necrosis compared to other groups

Rats' liver lesions showed histological alterations and biomarker enzymes indicative of hepatocellular injury brought on by paracetamol. The serum enzymes SGPT and SGOT are sensitive indicators of liver damage, and elevated levels of these enzymes are a sign of cellular leakage and a loss of the functional integrity of the cell membrane in the liver, which were caused by hepatocellular injury brought on by drug toxicity and xenobiotic exposure²⁰.

3.4 Anti-HCV activities of methanol extracts of Hylocereuspolyrhizus Peel:

Numerous studies recently showed that plant extracts exhibit an inhibitory effect on HCV enzyme and replication.They have demonstrated their utility in eradicating chronic hepatitis C through a multitude of hepatoprotective functions in initial trials. Therefore, plant extracts have been considered as potential sources of new bioactive compounds against HCV infection²¹.

Table 3.Anti-HCV activity (IC₅₀), cytotoxicity (CC₅₀) of methanol extracts of Hylocereuspolyrhizus Peel

Sample	IC₅₀(µg/mL)	CC₅₀(µg/mL)
Methanol Extract	>300	> 1000

However, the Hylocereuspolyrhizus methanol extract appear to have weak anti-HCV activities. This is shown by (IC₅₀) and cytotoxicity (CC₅₀) results, with an IC₅₀value above 300µg/mL ( no inhibition showed ) and CC₅₀value above 1000µg/mL.It can be concluded that the level of toxicity in the cells that have been tested is high compared to the normal limit (427.7µg/ml), namely > 1000µg/ml, so that the effect caused by Hylocereuspolyrhizus peel extract is less visible and there is no significance on results.The antioxidant and cytotoxicity effect might come from anthocyanin of Hyelocereus plant²². Other group compound of H. polyrhizus is flavonoid, which revealed in Patil et. Al (2015) has the antioxidant and anticancer activity. Moreover it is coming from compound shows good interaction like hydrogen and hydrophobic binding. The flavonoids also showed had a good cytotoxicity by Brine shrimp lethality assay²³.

CONCLUSION:

Administration of red dragon fruit peel extract (Hylocereuspolyrhizus) have a potential of hepatoprotection. The extract was able to protect the rats liver from exposure to toxic doses of paracetamol as indicated by decreased levels of SGPT and SGOT, and histopathological examination of the rat liver microscopically. However, the results of the Anti-HCV activity test showing that the red dragon fruit peel extract (Hylocereuspolyrhizus) appear to have a weak effect to prevent HCV. Based on the results of reading the SGPT and SGOT analysis data, it can be concluded that the effective dose to provide a hepatoprotector effect at dose of 200 mg/kg bodyweight.

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

ACKNOWLEDGMENTS:

The authors would like to thank the Ministry of Education and Culture of Indonesia for financial support and research analysis.

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Received on 07.12.2022 Modified on 24.05.2023

Research J. Pharm. and Tech 2023; 16(11):5096-5100.

DOI: 10.52711/0974-360X.2023.00826