Hepatoprotective activity of the ethanol extract of Curcuma heyneana rhizome on isoniazid and rifampin-induced liver injury rats

Introduction Based on the global tuberculosis report of the World Health Organization (WHO) in 2013, Indonesia was in position 9 with the incidence of 185 cases per 100 000 population and ranked 3rd in the south-east region with the incidence of 82, 799 new cases in 2012 (1). Isoniazid (INH) and rifampin (RIF) are the first lines in the therapy of tuberculosis (2). The utilisation of INH along with RIF produces toxic metabolites or oxidants such as acetylhydrazine (AcHz) and hydrazine (Hz) which can cause serious liver injury (3). DILI or drug-induced liver injury is an important issue nowadays. DILI may occur to the tuberculosis patients who consume INH for 6 to 9 months, RIF for 4 months, or a combination of INH and RIF for 4 months (4). This problem limits the use of antituberculosis agents and may cause treatment failure or even resistance. Therefore, this problem must be figured out in order to make patients safe and comfortable in using antituberculosis agents without risk of liver damage. Curcuma heyneana Val. & Zijp. or temu giring is one of the medicinal plants that are grown in Indonesia. This plant belongs to Zingiberaceae family (5). People in Indonesia usually use the rhizome as a food ingredient http://www.herbmedpharmacol.com doi: 10.34172/jhp.2020.42


Introduction
Based on the global tuberculosis report of the World Health Organization (WHO) in 2013, Indonesia was in position 9 with the incidence of 185 cases per 100 000 population and ranked 3 rd in the south-east region with the incidence of 82, 799 new cases in 2012 (1). Isoniazid (INH) and rifampin (RIF) are the first lines in the therapy of tuberculosis (2). The utilisation of INH along with RIF produces toxic metabolites or oxidants such as acetylhydrazine (AcHz) and hydrazine (Hz) which can cause serious liver injury (3).
DILI or drug-induced liver injury is an important issue nowadays. DILI may occur to the tuberculosis patients who consume INH for 6 to 9 months, RIF for 4 months, or a combination of INH and RIF for 4 months (4). This problem limits the use of antituberculosis agents and may cause treatment failure or even resistance. Therefore, this problem must be figured out in order to make patients safe and comfortable in using antituberculosis agents without risk of liver damage. Curcuma heyneana Val. & Zijp. or temu giring is one of the medicinal plants that are grown in Indonesia. This plant belongs to Zingiberaceae family (5). People in Indonesia usually use the rhizome as a food ingredient or to cure many ailments. Even though it has been used traditionally for years, there is still lack of scientific information about the efficacy of this plant.

Animals
Adult male Wistar albino rats (150-200 g), used in this study, were obtained from the Animal House Faculty of Pharmacy, Universitas Sumatera Utara. The animals were acclimatized for at least two weeks prior to the experiment. The rats had access to standard food and water ad libitum.
Extraction of Curcuma heyneana leaves The fresh rhizomes of C. heyneana were dried in drying cabinet and continuously weighed until constant.
Extraction was done in n-hexane, ethyl acetate and ethanol by maceration method. The filtrates were evaporated at 40°C under reduced pressure. The dried extract was stored at 2-8°C and re-constituted in carboxymethylcellulose sodium to be administrated to the experimental animals.

Phytochemical screening
The sample was screened for the following components: tannins, saponins, flavonoids, alkaloids, glycosides, steroids and triterpenoids.

Antioxidant activity
The extract of C. heyneana was tested for antioxidant activity by using DPPH method. DPPH solution was made in the concentration of 40 ppm, then maximum wavelength was measured. The extract solution was made in the concentrations of 20, 40, 60 and 80 ppm. The ascorbic acid solution was made in the concentrations of 1, 2, 4 and 8 ppm. Afterwards, the extract was measured at 516 nm. The activity of radical scavenging was calculated by using the equation below: The activity of radical scavenging

Control absorbance Sample absorbance
Control absorbance = − × Experimental design isoniazid-rifampin induced hepatotoxicity test The rats were designated into 7 groups of 5 each. Treatments were done for 15 days as indicated in Table 1.
Collecting of blood samples On the 16 th day of experiment, blood samples were collected from the rats by cardiac puncture under anaesthesia into a microtube. The blood samples were allowed to clot for 45 minutes at room temperature. Serum Tabel  Animals received standard rodent feed and pure drinking water ad libitum was separated by centrifugation at 3000 r/min for 15 minutes. Afterwards, animals were sacrificed by cervical dislocation and laparotomy for collecting the liver. Livers were then placed into neutral buffered formalin 10% for histological assessment.
Evaluation of various serum biochemical parameters Serum samples were analysed for ALT and AST using reagent kits from DiaSys ® .

Histological assessment
The liver was isolated, fixed in neutral buffered formalin 10%, embedded in paraffin and cut into 4-5 µm. Afterwards, the liver was deparafined, rehydrated and stained with hematoxylin subsequently eosin, after that, dehydrated with graded concentration alcohol and ultimately mounted (13).

Statistical analysis
Values were presented as mean ± standard error (n=5). Statistical analysis was carried out using one-way analysis of variance (ANOVA) followed by Tukey's multiple comparison tests using SPSS 19. Values were statistically significant at P < 0.05.  Figure 1. Based on Figure 1, administration of INH-RIF significantly increased AST and ALT compared to normal control group (P<0.01). C. heyneana at the doses of 5, 25, 125 and 625 mg/kg significantly prevented the elevation ALT and AST level compared to negative control group (P <0.01) in a dose-dependent manner.

Phytochemical constituent
At the doses of 25, 125 and 625 mg/kg, the effect of the extract toward ALT and AST level were the same as the positive control group as well as normal control group. It means that at the minimum dose of 25 mg/kg, the extract could totally prevent the liver from injury.
After the surgical operation, macroscopic appearance of the liver was observed. The livers belong to treatment groups obviously had different colours compared to negative control. The livers of negative control group were pale red with white spot while tested groups were red-brownish. At the dose of 5 mg/kg, the liver was pale red without white spot. As the dosage was increased, the intensity of the colour of the liver became darker and closer to the colour of positive control and normal control that was red-brownish. There was a strong correlation between AST and ALT levels with the macroscopic appearance of the liver. The pictures can be seen in Figure 2.
After macroscopic analysis, histological analysis was carried out. The results can be seen in Figure 3. as well as the dose of 25 mg/kg underwent injury in hepatocytes which was marked with sinusoidal dilatation and congestive of the central vein caused by endothelium cells lysis. The death nucleus appeared small and solid (pycnotic nucleus) (14).
In groups 125 and 625 mg/kg, there was no significant injury compared to negative control. It proved that the administration of C. heyneana could protect the liver from damage.

Discussion
INH is a drug choice in treatment of tuberculosis. INH undergoes metabolism in the liver and produces metabolites such as AcHz, isonicotinic acid, Hz, diacetylhydrazine (DiAcHz), and acetylisoniazid (AcINH) (15). Next, AcHz and Hz are oxidized by microsomal enzymes P450 especially CYP2E1 into radical metabolites. These metabolites may cause hepatotoxicity. In this study, INH was administrated with RIF. Pretreatment with P450 inducers, such as RIF, raised the yield of radical metabolites dramatically. These radical metabolites later bind to the acetyl group of liver protein covalently and cause liver injury (16).
AST and ALT are the enzymes that can be found in the liver. ALT is more specific than AST in detecting liver damage whereas AST can be found not only in the liver but also in the heart, muscle, kidney as well as brain. Injury in these organs, release these enzymes and travel into blood. Therefore these enzymes are used as biomarkers in liver injury (17). In this research, administration of INH and RIF generated elevation of AST and ALT more than two fold. However, administration of ethanol extract of C. heyneana inhibited the elevation of AST and ALT. This result showed that the extract could prevent the damaging in the liver.
Administration of INH and RIF concomitantly, usually causes hepatic oxidative stress. This effect is protected by glutathione, the endogenous antioxidant with thiols group as reducing agent (18). The injury in hepatocytes is associated with significant decline of glutathione as well as antioxidant enzyme system (19). Later, the cell undergoes inflammation and death. In this research, administration of C. heyneana prevented the congested central vein, necrotic hepatocytes and dilated sinusoid. Co-administration of ethanol extract of C. heyneana prevented the induction of oxidative stress in INH-RIF  co-exposed animals. The prevention of oxidative stress by extract was depicted as normal morphology in the animals liver. In contrast, the animals co-exposed to INH-RIF alone (negative control) showed histological lesions which ranged from congested central vein, necrosis hepatocyte as well as dilated sinusoid. These results suggest that INH-RIF-induced liver injury can be prevented by supporting the cellular antioxidant defense mechanism activated by ethanol extract of C. heyneana. Based on the phytochemical screening of ethanol extract of C. heyneana, revealed the presence of flavonoids, saponins, tannins, glycosides and steroids/triterpenoids. In vitro antioxidant test using DPPH method exhibited the ethanol extract of C. heyneana possessed strong antioxidant activity. Flavonoid was suspected to be responsible for the potency of antioxidant. Functional hydroxyl groups in flavonoids mediate their antioxidant effects by scavenging free radicals. In addition to that, flavonoids also activate glutathione peroxidase system as protective enzyme and inhibit the enzymes which are involved in reactive oxygen species generation (20,21). Therefore, in this research, administration of extract for 2 weeks is suggested to induce protective enzyme that can tackle reactive metabolites generated from metabolism of RIF and INH. Ultimately, the extract serves as hepatoprotective and anti-inflammatory agent. Curcuma heyneana also contains curcuminoid (22). Curcuminoid comprises curcumin, demethoxycurcumin and bisdemethoxycurcumin. Curcumin is main polyphenol found in the rhizome of Zingiberaceae. The synonym of curcumin (1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6heptadiene-3,5-dione) is diferuloymethane. Polyphenol has a property as antioxidant related to reducing agent, acting as donor of electron or hydrogen which stabilizes or delocalizes unpair electron (23)(24)(25). In addition, curcumin may also stimulate antioxidant enzyme activity like glutathione peroxidase, superoxide dismutase and catalase. Curcumin is capable of scavenging oxygen free radicals such as superoxide anions and hydroxyl radicals which are important in initiation of lipid peroxidation (26,27). The radicals AcHz and Hz may cause toxicity in the liver by lowering the existing antioxidant. Therefore, availability of curcumin in the rhizome of C. heyneana may protect the liver cells from oxidation by two mechanisms, directly by stabilizing or delocalizing unpair electron or indirectly by stimulating antioxidant activity (28).

Conclusion
The ethanol extract of C. heyneana rhizome at doses of 25, 125 and 625 mg/kg may possess activity to protect the liver. For that reason, C. heyneana has potential to be developed as adjunctive therapy in prevention and treatment of antitubercular drug-induced liver injury. Further investigations are needed to isolate, identify and characterize the active compounds and to determine the exact mechanisms of actions.