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The Hepatoprotective effect in vivo and Antioxidant effects in vitro of Tinospora cripa stem extract Group 6 Biochemistry Mark Manguerra, John Christian Aniban, Apple Balbiran, Roscel Cadeliña, Hazel Cappleman, Lowe Chiong, Genevieve Cruz, Jeffrey de los Santos, Vienna Encila, Veronica Flores, Carmela Gonzales, Darnel Hurtado, Joseph Anthony Lachica, Ronald Luna, Charles Mercado, Christopher Ocampo, Pamela Patdu, Grace Quilloy, Carlo Rubio, Arunee Siripunvarapon, Diana Tamondong, Roland Joseph Tan, Ma-am Joy Tumulak, and Diane Zaragoza College of Medicine, University of the Philippines Manila Adviser: Dr. Mariluz Mojica-Henshaw Department of Biochemistry, College of Medicine, University of the Philippines Manila Rifampicin is used prevalently in the treatment of tuberculosis, but it usually results in hepatotoxic effects such as jaundice. This study investigated the hepatoprotective effect of Tinospora crispa root extract against rifampicin-induced liver toxicity in mice and its free radical scavenging activity compared to ascorbic acid. Groups of 7 mice given a single 400 mg/kg dose of rifampicin were treated with either the extract or silymarin, a common hepatoprotective drug. Mortality of mice given rifampicin alone was similar to the treatment groups but the serum ALT and bilirubin levels for both groups were decreased. Histopathological studies revealed that all groups except for those given rifampicin alone did not have fatty metamorphosis of hepatocytes. The free radical scavenging activity of the extract was comparable to silymarin, but both had about 1/200 the antioxidant activity of ascorbic acid. Tinospora crispa may have a hepatoprotective effect in mice but its mechanism is still unknown. Keywords: Tinospora crispa, hepatoprotection, antioxidant INTRODUCTION Background of the Study There is an estimated 1.9 billion people worldwide afflicted with tuberculosis. In 1999, the total number of TB cases registered in the Philippines was 144,932. The World Health Organization ranks the Philippines fourth among all countries in number of tuberculosis cases, with up to 39% of children aged between 5 and 9 already infected (Easton 1998, Wallerstein 1999). Among the countries in the Western Pacific Region, the Philippines ranks third with a high case notification rate of 198.1 per 100,000 population. Rifampicin is included in the first-line combination therapy against tuberculosis along with isoniazid (INH), pyrazinamide (PZA) and ethambutol (Chan and Iseman 2002). However, jaundice and other manifestations of hepatotoxicity usually occur during treatment. The risk is increased when rifampicin is given concomitantly with INH and PZA (Saraswathy et al. 1998). Liver diseases may also occur due to excess consumption of alcohol, infections, and autoimmune disorders (Visen et al. 1996, Zhang et al. 2002). Despite advances in allopathic medicine, there is no available effective hepatoprotective medicine, which has encouraged the development of herbal extracts that have shown a positive hepatoprotective effect (Murthy and Srinivasan 1993). This study serves as a preliminary screening of hepatoprotective compounds in Tinospora crispa which, in the future, may be included in the TB regimen. Studies have validated Tinospora cordifolia extract as a possible treatment for hepatotoxicity (Singh et al. 1984). Pyridoxine (Vitamin B6), for example, is given to prevent peripheral neuropathy associated with INH. In this experiment, we will assess the hepatoprotective activity of the fractions of ethanolic extract of Tinospora in rifampicin-treated albino mice by measuring the levels of various biochemical parameters. We will also attempt to propose a possible mechanism of activity by determining radical scavenging activity of the fractions. Objectives The general objective of the study is to determine if crude extracts of Tinospora crispa possess a hepatoprotective activity in rifampicin induced hepatotoxicity in male Swiss albino mice. The specific objectives are: 1) To determine serum ALT and bilirubin levels in mice treated with rifampicin alone, Silymarin and the plant extract. 2) To determine if crude extracts of Tinospora crispa possess an antioxidant property Hypotheses Ho1: There is no difference in serum ALT and bilirubin levels between the mice treated with Silymarin (the positive control), plant extract and rifampicin alone (the negative control). Ha1: There is a difference in serum ALT and bilirubin levels between the mice treated with Silymarin (the positive control), plant extract and rifampicin alone (the negative control). Ho2: The plant extract possesses no free radical scavenging activity. Ha2: The plant extract possesses a free radical scavenging activity Significance of the Study Due to the high prevalence of tuberculosis in the country, rifampicin has been used as a common medication in treatment of the disease. This drug, however, has hepatotoxic effects. Due to the high cost of living, impoverished Filipinos turn to herbal medicine as an alternative to commercially produced drugs for many of their health complications. This study, therefore, will not only make a valuable contribution to the growing number of studies devoted to hepatotoxicity and hepatoprotection but will also add to the investigations currently being undertaken to find medicinal plants with hepatoprotective functions. Results of the study may confirm local information regarding the hepatoprotective potential of T. crispa. This may add to the knowledge o the already established medicinal uses of T. crispa. Finally, the study could pave the way to provide a much cheaper source of medicine of liver damage. Scope and Limitations This study assessed the hepatoprotective potentials of T. crispa on male Swiss Albino mice. This was done by measuring the serum levels of ALT and bilirubin in the different treatment groups. In addition, the free radical scavenging activity of the various treatment groups was also compared. Histopathological analysis was also performed. The study utilized mice of which died due to the toxic effects of rifampicin. This study is limited to the rifampicin induced hepatotoxicity model. The extracts were derived from the stem and not from the other parts of the plant. REVIEW OF RELATED LITERATURE Rifampicin-Induced Liver Damage Rifampicin is a broad-spectrum antibiotic effective against both gram (+) and gram (-) bacteria. It inhibits RNA synthesis by combining with RNA polymerase. It is active on tubercle bacilli of lower metabolism and slower growth such as those persisting in the closed lesions of lower O2 tension. Moreover, it is also useful in the treatment of leprosy. Rifampicin (RIF) along with isoniazid (INH), pyrazinamide (PZA), and ethambutol is the first-line combination therapy against tuberculosis. However, many studies have revealed the hepatotoxic activity of rifampicin with significant increase in risk when given concomitantly with isoniazid. In an experiment on male albino mice, combination of isoniazid with rifampicin resulted in a higher activity of transaminase and alkaline phosphatases and a higher rate of inhibition of biliary secretion and synthesis and excretion of bile acids, bilirubin, and cholesterol with bile. Moreover, an increase was observed in the level of lipid peroxidation products of the hepatocyte membranes in liver homogenates and blood. The increased hapatotoxicity of isoniazid, was evident from a more pronounced decrease in the number of sulfhydryl groups accompanied by an increase in the number of disulfide ones in the liver and blood. Potentiation of isoniazid hepatotoxicity under the action of rifampicin was due to its inducing activity with respect to the microsomal oxidation enzymes. According to Vavricka et. al. (2001), the antibiotics rifamycin SV and rifampicin has been shown to interfere with hepatic organic anion uptake systems in the liver with the inhibition of the organic anion transporting polypeptides or OATPs. These polypeptides are responsible for the elimination of sulfobromophthalein (BSP) and served as indicator of potent liver organic anion uptake system since it is a natural substrate of the OATPs as well as bile salts, steroid conjugates, thyroid hormone and unconjugated bilirubin among others. According to one study, the antituberculosis therapy induces acute or chronic liver damage in some individuals. Liver injury was characterized as being mild to moderate and the type of injury associated was represented by pure cholestasis and hepatocanalicular lesions. Other effects include rise in serum bilirubin and transaminase levels. Furthermore, rifampicin may cause epigastric distress, CNS side effects, and immunological disturbances. It also imparts a reddish-orange color to body fluids. Some parallel studies have sought to test the protective effect of certain substances against the hepatotoxicity of rifampicin and isoniazid as well. Oral treatment with the ethanol extract of Hemidesmus indicus roots significantly prevented rifampicin and isoniazid-induced hepatotoxicity in mice. Berberine compound from Berberis aristata was studied for its possible antihepatotoxic action in rats. Pretreatment of animals with berberine prevented the acetaminophen- or CCl4-induced rise in serum levels of alkaline phosphatase and transaminases, suggestive of hepatoprotection. Post-treatment with three successive oral doses of berberine reduced the hepatic damage induced by acetaminophen, while CCl4-induced hepatotoxicity was not modified suggesting a selective curative effect against acetaminophen. Furthermore, the hepatoprotective activity of N-acetylcysteine (NAC), a glutathione (GSH) precursor was investigated in young Wistar rats. The oxidative hepatic injury in INH-RIF co-exposed animals which showed histological lesions ranging from intralobular inflammation to patchy necrosis was closely associated with significant decline of GSH and related thiols as well as with compromised antioxidant enzyme system. The oxidative stress was furthered by increased lipid peroxidation. The co-administration of NAC which supported the cellular antioxidant defense mechanism, prevented the induction of oxidative stress. The use of rifampicin in other diseases also manifested signs of hepatotoxicity. A case of a patient with borderline tuberculoid Hansen’s disease (leprosy) who developed the diaminodiphenylsulphone syndrome after multi-drug therapy comprising dapsone and rifampicin demonstrated features consistent with drug-induced hepatitis, tubulo-interstitial nephritis and myocarditis. In addition, rifampicin monotherapy is considered an effective second line therapy for controlling pruritus in patients with chronic cholestatic liver disease. It is used as an antipruritic agent in the autoimmune cholestatic liver disease, primary biliary cirrhosis (PBC). The patients experienced significant hepatitis or impairment of liver synthetic function. Tinospora crispa Tinospora crispa, known as Makabuhay plant in the Philippines is a climbing, dioecious vine reaching a height of 4-10 meters. The stem is about 1 centimeter thick, somewhat fleshy, with scattered protuberances. The leaves are thin, ovate, 6-12 centimeters long and 7-12 centimeters wide. The petiole is 3-6 centimeters long. Racemes are solitary or in pairs arising from the axis of the leaves, pale green, and short-pedicelled. The fruit is 7-8 millimeters long (Merrill 1974). This plant is widely distributed in the Philippines usually flowering from March to May. It also occurs in Malaya. (Merrill 1974). Other local names of Tinospora crispa include paliaban, panauan, pangiauan, pangiauban and taganagtagtua in the Visayas. It is also known as sangaunau in Baguio (Quisumbing 1978). The Filipinos and Malays in general regard this vine as a universal medicine. The plant has long been used traditionally in the treatment of a number of varied diseases. It is commonly prescribed as an aqueous extract in the treatment of stomach trouble, indigestion, and diarrhea. It is also an effective remedy in the treatment of tropical ulcers. In powder form, it is prescribed in fevers. A preparation with coconut oil is an effective cure for rheumatism and flatulence of children. This preparation starts with chopping the stem into pieces of 1 or 2 inches long, and placing them in a jar with coconut oil. The jar is put aside and not opened until a year has elapsed. Prior to treatment, the preparation is first "cooked" under the sun. Other uses include the decoction of the stem as an excellent vulnerary for itches, ordinary and cancerous wounds. It is also used as a hot foot bath in the treatment of athlete's foot by boiling chopped 1 foot long portion of the vine in 5 glasses of water for 15 minutes and a tub bath for scabies by boiling chopped 1 meter long portion of the vine in 1 gallon of water for 15 minutes. Internally, it is used as a tonic and antimalaria and externally, as a parasiticide (Quisumbing 1978). More recent studies have verified the efficacy of Tinospora crispa for the treatment of diabetes in animal models. Moreover, these studies have provided sufficient biochemical evidence that support the traditional claims for the hypoglycemic effect of the plant. The antihyperglycemic effect of T. crispa is not due to interference with intestinal glucose uptake but rather is associated with increased insulin secretion (Noor et al. 1989, Noor and Ashcroft 1998). In addition, current studies have also revealed the usefulness of T. crispa for the treatment of malaria (Rahman et al. 1999) and various types of inflammation (Higashino et al. 1992). Chemical studies on the plant report that the whole plant contains a bitter principle, traces of an alkaloid, berberine and a glucoside. Further studies, however, have identified that the bitter principle is glucosidal in nature. This bitter principle occurs as a white, crystalline powder which is freely soluble in alcohol but very slightly soluble in other organic solvents, such as ether and chloroform. It dissolves slowly in water. The bitter principle consists of 41.15% carbon, 11.67% hydrogen and 47.18% oxygen (Marañon 1927). More recently, two triterpenes have been isolated from the stems of T. crispa, namely, cycloeucalenol and cycloucalenone. Both of these triterpenes possess cardiotonic activities (Konghathip et al. 2002). The hepatoprotective effect of T. cordifolia extract has been studied in carbon tetrachloride-induced liver damage in rats. While acute damage was enhanced by prior exposure to the drug, it proved effective in the prevention of fibrosis, and in stimulating regeneration in hepatic tissue. Clinically, this drug has been tried as a therapeutic modality in rheumatoid arthritis, jaundice and in diabetes (Singh et al. 1984). Measurement of Serum Alanine Aminotransferase Activity Transamination is the process in which an amino group is transferred from amino acid to an -keto acid (Murray et al. 2000). The glutamic transaminase enzyme, serum alanine aminotransferase (ALT), catalyzes the formation of glutamate and pyruvate from the reaction of L-alanine and -ketoglutaric acid (Murray et al. 2000). Measuring blood levels of serum alanine aminotransferase (ALT), is the most common way to test for liver disease. Blood enzyme levels above a certain value indicate liver disease (ACP-ASIM 2002). Elevated serum ALT levels are found in hepatitis cirrhosis and obstructive jaundice. Levels of ALT are only slightly elevated in patients following a myocardial infarction (Biotron Diagnostics 2001). Many methods and modifications have been proposed for the determination of serum ALT. The various methods generally fall into two categories: colorimetric and spectrophotometric (Biotron Diagnostics 2001). The Reitman-Frankel colorimetric method involves the addition of 2,4-dinitrophenyl hydrazine (DNPH) which reacts with pyruvate, the product of transamination to form a hydrozone complex (Reitman and Frankel 1957). Subsequent addition of sodium hydroxide breaks down the hydrozone complex and produces a reddish coloration on the solution (Biotron Diagnostics 2001). The intensity of the color is proportional to the enzymatic activity (Reitman and Frankel 1957). With the advent of spectrophotometry, measurement of serum ALT has become much more sensitive and accurate (Biotron Diagnostics 2001). The absorbance of the product from the Reitman-Frankel colorimetric method is read at 550 nm. Increase in pyruvate production results to a higher absorbance reading (Rafei 2001). However the presence of the co-substrate, -keto glutarate in the assay contributes to the final absorbance reading such that the change in absorbance is not linearly related to the theoretical value of pyruvate produced and hence the enzyme activity (Rafei 2001). Total Bilirubin Determination Bilirubin is a breakdown product of hemoglobin. When hemoglobin is destroyed in the body, the iron porphyrin or heme portion is catabolized by a complex enzyme system, heme oxygenase. Hemin, which constitutes the ferric form of the oxidized iron, is reduced to heme with NADPH, and with the aid of more NADPH, oxygen is added to the alpha-methenyl bridge between pyrrole I and II of the porphyrin ring. Further addition of oxygen releases ferric iron and produces carbon monoxide. Also splitting of the tetrapyrrole ring results in an equimolar quantity of biliverdin IX-alpha. In mammals, a soluble enzyme called biliverdin reductase reduces the methenyl bridge between pyrrole III and pyrrole IV to a methylene group to produce bilirubin IX-a, a yellow pigment (Harper 2000). Bilirubin metabolism occurs primarily in the liver. It is taken up by liver parenchymal cells, conjugated in the smooth endoplasmic reticulum, and secreted into the bile (Harper 2000). A low level of bilirubin (about 1 mg/dL) circulates throughout our bodies, too faint to be visible. However, excessive levels of bilirubin cause the yellow pigment to be visible. This condition is known as jaundice (Greene 1998). Jaundice, the discoloration of skin and sclera of the eye, occurs when bilirubin accumulates in the blood at a level greater than approximately 2.5 mg/dL. This is due to red blood cells being broken down too fast for the liver to process, liver diseases, bile duct blockage, viral hepatitis, and liver scarring or cirrhosis (MedlinePlus Encyclopedia). Drugs like allopurinol, anabolic steroids, diuretics, some antibiotics, antimalarials, azathioprine, chlorpropamide, cholinergics, codeine, epinephrine, meperidine, methyldopa, methotrexate, MAO inhibitors, morphine, nicotinic acid, oral contraceptives, phenothiazines, quinidine, salicylates, steroids, sulfonamides, theophylline, and rifampicin cause a significant rise on bilirubin levels (MedlinePlus Encyclopedia). At very high levels of bilirubin in the bloodstream, permanent damage to certain areas of the brain of newborn infants (kernicterus) occurs which causes a characteristic form of crippling known as athetoid cerebral palsy (EncyMaster). There is also an increased risk for permanent hearing loss, mental retardation, spastic quadriplegia, or even death (Greene 1998). Conjugation is the process by which bilirubin is converted from a nonpolar form to a polar one, which is readily excreted in the bile, by adding glucoronic acid molecules to it (Harper 2000). This conjugated bilirubin is called total bilirubin. On the other hand, unconjugated bilirubin is called direct bilirubin. When this is elevated, the cause is usually outside the liver, typically gallstones. Total bilirubin testing measures the amount of bilirubin in the bloodstream. Normal total bilirubin levels range from 0.20 mg/dL to 1.5 mg/dL. Measurement of Antioxidant Activity The ability of several plant extracts such as Solanum hainanense (Phuc 1998) and ‘Banzhi-lian’ (Lin 1997) to prevent drug-induced hepatotoxicity has been correlated with their free radical scavenging property. Antioxidative activity (AOA) is conventionally used to indicate the ability of an antioxidant to scavenge some radicals. Free radical colorimetry relies on the reaction of an antioxidant with the stable free radical 2,2-diphenyl-1-picrylhydrazyl (DPPH, C18H12N5O6, MW= 394.33 g/mol) dissolved in methanol (Buijnsterns 2001). DPPH is a relatively stable paramagnetic free radical that accepts electrons or H+ radicals to become stable diamagnetic molecule (Brand-Williams et al. 1995). The reduction of DPPH by an antioxidant results in the formation of purple-blue colored solution. The activity is measured through conventional spectrophotometry at 514 nm (Buijnsterns 2001). MATERIALS AND METHODS Preparation of extract. Tinospora crispa (Makabuhay) stems were procured from a public market in Quiapo. These were homogenized in absolute ethanol and left to macerate for 3 days at 25ºC. The mixture was then filtered using filter paper and the resulting liquid was concentrated under reduced pressure at 40ºC. The extract was dissolved using distilled water to concentrations of 10 g/L and 20 g/L. Test animals. A single strain of albino mice were maintained under standard husbandry conditions and acclimatized for 10 days. The rats are given standard laboratory feed and water ad libitum. Groups consisted of 7 rats each. For all tests, a double blind procedure was followed wherein neither the data observer nor the data collector were aware of the particular treatment of the mice. Rifampicin-induced hepatotoxicity. The control group received distilled water 0.2 mL four times at 12h intervals and 0.2 mL distilled water 30 mins after the first administration of distilled water. The rifampicin group received 0.2 mL distilled water four times at 12h intervals and a single 0.2 mL dose of rifampicin suspension (400 mg/kg) 30 mins after the first administration of distilled water. The extract low group received crude extract solution 0.2 mL (100 mg/kg) four times at 12h intervals and a single 0.2 mL dose of rifampicin suspension (400 mg/kg) 30 mins after the first dose of the crude extract solution. The extract high group received crude extract solution (200 mg/kg) four times at 12h intervals and a single 0.2 mL dose of rifampicin suspension (400 mg/kg) 30 mins after the first dose of the crude extract solution. The silymarin group received silymarin solution (100 mg/kg) four times at 12h intervals and a single 0.2 mL dose of rifampicin suspension (400 mg/kg) 30 mins after the first dose of silymarin solution. Forty-eight hours after rifampicin administration, blood was collected using the cardiac extraction method from all groups. The blood samples were placed in calcium-EDTA tubes at room temperature. Plasma was separated by centrifugation at 2500 rpm at 37° for 15 mins and analyzed for ALT (SGPT) and direct bilirubin. Total Bilirubin (Jendrassik method, Randox kit). The following solutions were pipetted into a cuvette (mL): Blank Sample Sulfanilic acid 0.20 0.20 Sodium nitrite - 0.05 ml Caffeine 1.00 1.00 Sample 0.20 0.20 The contents were mixed and allowed to stand for 10 mins at 22ºC. To each cuvette, 1 mL of the tartrate solution was added, and the solutions were mixed and allowed to stand for 5 mins at 22ºC and absorbance of the sample was read against the blank at 578 nm. The total bilirubin was calculated using the following equations: Total Bilirubin (umol/L)= 185 x Abs or Total Bilirubin (mg/dL)= 10.8 x Abs ALT/SGPT (Young-Karmen method, Randox kit). L-alanine was reconstituted with a lactate dehydrogenase, NADH and -ketoglutarate solution. Into a cuvette, 1 mL of the reconstituted solution and 0.1mL of the sample was pipetted and mixed. Initial absorbance at 340 nm was read and again after 1, 2 and 3 mins. The activity of alanine aminotransferase present in the sample was calculated using the following equation: U/L= 1746 x ΔA 340 nm/min DPPH Scavenging activity. A 1 mg/mL DPPH solution, a 10 mg/mL T. crispa solution, a 10 mg/mL silymarin solution, and a 1 µg/mL ascorbic acid solution were prepared using methanol as a solvent. In a 10 mL volumetric flask, 0.5 mL of the DPPH solution and 1, 2, 3, 4, 5, and 6 mL of the test solutions (either silymarin and T. crispa extract) were mixed and diluted to 10 mL using methanol. The procedure was repeated for ascorbic acid using 0.25, 0.5, 1, 2, 3, and 4 mL volumes. Twenty minutes were allowed to elapse before the spectrophotometry readings were taken. Initial absorbance (without sample) and final absorbance (with sample) was read at 517 nm using methanol as blank. The EC50 or effective concentration which reduces the absorbance by 50% using linear regression was determined using ascorbic acid as control. Statistical analysis. The results of the serum alanine aminotransferase and bilirubin assays were subjected to continuous summary descriptives and comparatives analyses since the sample size was insufficient to analyze using analysis of variance. RESULTS The table reflects the number of deaths after 48 hours of rifampicin treatment. The silymarin and extract treated groups showed higher mortality compared to rifampicin alone. Table 1. Number of deaths observed per treatment group following administration of rifampicin Treatment Doses Deaths out of 7 Control 0 Rifampicin 400 mg/kg 5 Ethanolic Extract 100 mg/kg 7 Ethanolic Extract 200 mg/kg 5 Silymarin 100 mg/kg 5 As shown in the table and figures below, 48 hours after intoxication with a single dose of rifampicin (400 mg/kg) an increase in serum ALT was observed relative to the control (without rifampicin). The treatment with the crude ethanolic extract of T. crispa (200 mg/kg) induced a decrease of serum ALT levels greater than that of silymarin (100 mg/kg). The difference was not statistically significant (p=0.10). The concentration of bilirubin in the serum was also markedly elevated when rifampicin was administered alone, but with T. crispa the increase was suppressed but at a lesser extent compared to silymarin. The results for this test were not statistically significant (p=0.10). Table 2: Bilirubin serum concentrations Trial Bilirubin (mg/dL) Control Rifampicin 1 7.95 15.29 2 6.31 105.41 3 10.97 4 21.51 5 9.16 6 7.60 7 24.71 Average 12.60 60.35 Standard deviation 7.38 63.72 Silymarin 10.37 1.22 Extract 17.88 17.11 5.79 6.47 17.50 0.55 Table 3: Alanine Aminotransferase serum concentrations Trial Alanine Aminotransferase (U/I) Control Rifampicin Silymarin 1 146.66 290.53 2 205.33 108.95 10.13 3 22.35 4 0.00 5 11.17 6 5.59 7 12.57 Average 42.84 127.81 150.33 Standard Deviation 79.96 26.67 198.28 Extract 29.33 12.57 20.95 11.85 Figure 1: Comparison of ALT serum concentrations in the different treatment groups 400 350 300 Serum ALT, U/L 250 200 150 100 50 0 Control Rifampicin Silymarin Extract -50 -100 The table and figure show the scavenger activity and their correlation coefficients. Ascorbic acid showed the maximum activity, followed by silymarin and the crude extract. The scavenger activity shown by T. crispa is comparable to silymarin, however both possess only about 1/200 the antioxidant power of ascorbic acid. All the samples tested were at a concentration of 25 ug/mL of the reactive medium, DPPH. Figure 2: Comparison of Bilirubin serum concentrations in the different treatment groups 140 120 total bilirubin, mg/dl 100 80 60 40 20 0 Control Rifampicin Silymarin Extract -20 Table 4: Percent Scavenging Activity of the Different Treatment Groups Samples Tinospora crispa extract Silymarin Acorbic acid Effective Concentration 5.9 x 10-2 2.00 x 10-1 2.92 x 10-4 % Scavenging Activity 0.50 0.15 100 Fig. 3 Histopathological Findings of Mice Liver in Various Treatments Rifampicin (400x) Control (400x) Extract, 200 mg/mL (400x) Silymarin (100x) DISCUSSION In the present study, serum ALT and bilirubin levels were measured as indicators of hepatotoxicity. Elevated levels of serum enzymes indicate cellular leakage and loss of functional integrity of the liver cell membrane (Phuc et al. 1998). Results show that mice treated with rifampicin alone showed highly significant increase in serum ALT and bilirubin. This result is consistent with previous studies concerning the harmful effects of this drug. Several mechanisms have been proposed regarding the role of rifampicin in inducing liver damage. rifampicin is converted to its active metabolite 25desacetylrifampin, which in turn reduces drug-metabolizing enzymes and actively and specifically binds to RNA polymerases thereby inhibiting nucleic acid and protein synthesis (Rao, et al. 1998). Also the active metabolite can also induce liver injury when it is converted to free radicals via liver enzymes such as cytochrome P450 which would cause lipid peroxidation, disrupting the integrity of the hepatocellular membrane (Lin et al. 1997). On the other hand, subjects treated with crude extracts of T. crispa (200 mg/kg) had lower levels of serum ALT compared to those treated with rifampicin and Silymarin. Also, these subjects exhibited reduced levels of bilirubin compared to those treated with rifampicin alone. These results suggest that a hepatoprotective activity may be present in the T. crispa extracts. Since rifampicin induced toxicity relies on cytochrome P450 to produce reactive free radical metabolites, the hepatoprotective actions of T. crispa ethanolic extracts may be due to inhibition of cytochrome P450, prevention of lipid peroxidation and stabilizing hepatocellular membrane. Results of the experiment indicate that crude extract of T. crispa possesses only about 1/200th the antioxidant activity of ascorbic acid but is three times more potent than silymarin. The negligible scavenger activity T.crispa suggests that its hepatoprotective mechanism may not be due to its ability to directly combine with free radicals but other mechanisms such as inhibition of radical formation or membrane stabilization (Lin et. al. 1997). Histopathologic examination of the liver of rifampicin-treated group revealed lipid vacuoles within hepatocytes. The lipid accumulates when lipoprotein transport is disrupted and/or when fatty acids accumulate. rifampicin is a hepatotoxin that interferes with mitochondrial and microsomal function in hepatocytes, leading to an accumulation of lipid (Lin et al. 1997). This was not evident in the control, silymarin and extract-treated groups. These results, however, are not statistically significant due to the high mortality among the test mice brought about by the toxic side effects of rifampicin. Symptoms, manifested twelve hours after administration of rifampicin include red-orange discoloration in the urine and feces and tremors in the subjects. Such side effects were also observed in previous studies (Skakun and Shman’ko 1985). In addition, the researchers were not able to determine if the crude extract or silymarin alone was toxic. Further studies, however, may focus on this matter by determining the LD50 of both the crude extract or silymarin. RECOMMENDATIONS The researchers recommend that further studies be made regarding the hepatoprotective properties of Tinospora crispa using a larger sample size and various test dosages that are to be administered to the test subjects. Different extraction procedures and solvents may also be utilized to further track down the possible active component of Tinospora crispa involved in its role in hepatoprotection. Other hepatotoxicity induced models may also be explored such as CCl4, Trinitrotoluene, and Paracetamol in testing the plant extract. Enzymatic assays may also be performed to further investigate the role of cytochrome P450 systems in free radical formation. CONCLUSIONS Tinospora crispa ethanolic extract at 200 mg/kg possesses hepatoprotective activity against rifampicin-induced toxicity. Its activity is comparable to silymarin, a known hepatoprotective agent. Also, since the ethanolic extract posseses only about 1/200 the antioxidant activity of ascorbic acid, its hepatoprotective activity cannot be correlated with its ability to scavenge free radicals. Thus, further studies on its mechanism of action are warranted, as well as isolation of compounds responsible for the activity. LITERATURE CITED American College of Physicians-Annals of Internal Medicine (ACP-ASIM). 2002. Should Blood Enzyme Levels for Liver Disease Be Changed? http://www.acponline.org/college/ pressroom/tipsheets/02jul02.htm. Attri S., S. V. Rana, et al. 2000. Isoniazid- and rifampicin-induced oxidative hepatic injury— protection by N-acetylcysteine. Hum. Exp. Toxicol. 19: 517-522. Biotron Diagnostics 2001. Alanine Aminotransferase (ALT) Reagent Set. http://www.biotrondiagnostics.com/ALT_SGPT_REAGENT_COLORIMETRIC_ENDP OINT_METHOD.htm. Brand-Williams, W., M. E. Cuvelier, and C. Berset. 1995. Use of a free radical method to evaluate antioxidant activity. Lebensm. Wiss. Technol. 28: 25-30. Buijnsters, M., D. Bicanic, M. Chirtoc, M. C. Nicoli and Y. Min-Kuo. 2001. Evaluation of antioxidative activity of some antioxidants by means of a combined optothermal window and a DPPH free-radical colorimetry. Anal. Sci. 17: s544-s546. Chan, E. D. and M. D. Iseman. 2002. Current medical treatment for tuberculosis. BMJ 325: 1281-1286. De Souza, A. F., A. de Oliveira e Silva, J. Baldi, T. N. de Souza, and P. M. Rizzo. 1996. Hepatic functional changes induced by the combined use of isoniazid, pyrazinamide, and rifampicin in the treatment of pulmonary tuberculosis. Arq. Gastroenterol. 33: 194-200. Easton, A. 1998. Tuberculosis controls in Philippines have failed so far. BMJ 317: 557. Guo J. T., H. L. Lee, S. S. Chiang, F. I. Lin, and C. Y. Chan. 2001. Antioxidant properties of the extracts from different parts of Broccoli in Taiwan. J. Food Drug Anal. 9: 96-101. Higashino, H., A. Suzuki, Y. Tanaka, and K. Pootakham. 1992. Inhibitory effects of Siamese Tinospora crispa extracts on the carrageenin-induced foot pad edema in rats. Nippon Yakurigaku Zasshi 100: 339-344. Janbaz, K. H. and A. H. Gilani. 2000. Studies on preventive and curative effects of berberine on chemical-induced hepatotoxicity in rodents. Fitoterapia 71: 25-33. Kagaya, N., M. Kawase, H. Maeda, Y. Tagawa, H. Nagashima, H. Ohmori, and K. Yagi. 2002. Enchancing effect of zinc on hepatoprotectivity of epigallocatechin gallate in isolated rat hepatocytes. Biol. Pharm. Bull. 25: 1156-1160. Kanai, K. 1991. Introduction to tuberculosis and mycobacteria. Tokyo, Japan: SEAMIC/IMF. p.15. Kongkathip, N., P. Dhumma-upakorn, B. Kongkathip, K. Chawananoraset, P. Sangchomkaeo, and S. Hatthakitpanichakul. 2002. Study on cardiac contractility of cycloeucalenol and cycloeucalenone isolated from Tinospora crispa. J. Ethnopharmacol. 83: 95-99. Lau G. 1995. A fatal case of drug-induced multi-organ damage in a patient with Hansen’s disease: dapsone syndrome or rifampicin toxicity? Forensic Sci. Int. 73: 109-115. Lin, C., Shieh, D. and Yen, M. 1997. Hepatoprotective Effect of the Fractions of Ban-Zhi-Lian on Experimental Liver Injuries in Rats. J. Ethnopharmacology. 56: 193-200. Marañon, J. 1927. The bitter principle of makabuhay, Tinospora rumphii Boerlage. Philipp. J. Sci. 33: 357-361. Merrill, E. D. 1912. Flora of Manila. Manila: Bureau of Printing. p204. Murray et al. 2000. Harper’s Biochemistry. Stanford. Appleton and Lange. Murthy, M. S. R. and M. Srinivasan. 1993. Hepatoprotective effect of Tephrosia purpurea in experimental animals. Indian J. Pharmacol. 25: 34-36. Noor H. and S. J. Ashcroft. 1998. Pharmacological characterization of the antihyperglycaemic properties of Tinospora crispa extract. J. Ethnopharmacol. 62: 7-13. Noor, H., P. Hammonds, R. Sutton, and S. J. Ashcroft. 1989. The hypoglycaemic and insulinotropic activity of Tinospora crispa: studies with human and rat islets and HITT15 B cells. Diabetologia 32: 354-359. Prabakan, M., R. Anandan, and T. Devaki. 2000. Protective effect of Hemidesmus indicus against rifampicin and isoniazid-induced hepatotoxicity in rats. Fitoterapia 71: 55-59. Prince M. I., A. D. Burt, and D. E. Jones. 2002. Hepatitis and liver dysfunction with rifampicin therapy for pruritus in primary biliary cirrhosis. Gut 50: 436-439. Quisumbing, E. 1978. Medicinal Plants of the Philippines. Quezon City: Katha Publishing. p300301. Rafei, U. M. 2001. Transaminases - Colorimetric end-point method. http://w3.whosea.org/micro/ 10.htm. Rahman, N., T. Furuta, S. Kojima, K. Takane, and M. A. Mohd. 1999. Antimalarial activity of extracts of Malaysian medicinal plants. J. Ethnopharmacol. 64: 249-254. Rao, K.S. and Mishra, S. H. 1998. Antihepatotoxic Activity of Monomethyl Fumarate Isolated from Fumaria indica. J. Ethnopharmacology. 60: 207-213. Reitman, S. and S. Frankel. 1957. A colorimetric method for the determination of glutamic oxaloacetic and glutamic pyruvic transaminases. Am. J. Clin. Pathol. 28: 56-63. Saraswathy, S. D., V. Suja, P. Gurumurthy, and C. S. S. Devi. 1998. Effect of Liv.100 against antitubercular drugs (isoniazid, rifampicin, and pyrazinamide) induced hepatotoxicity in rats. Indian J. Pharmacol. 30: 233-238. Singh, B., M. L. Sharma, D. K. Gupta, C. K. Atal, and R. K. Arya. 1984. Protective effect of Tinospora cordifolia Miers on carbon tetrachloride induced hepatotoxicity. Indian J. Pharmacol. 16: 139-142. Skakun, N. P. and V. V. Shman’ko. 1985. Synergistic effect of rifampicin on hepatotoxicity of isoniazid. Antibiot. Med. Biotekhnol. 30: 185-189. Vavricka, S. R., J. V. Montfoort, H. R. Ha, P. J. Meier, and K. Fattinger. 2002. Interactions of rifamycin SV and rifampicin with organic anion uptake systems of the human liver. Hepatology 36: 164-172. Visen, P. K. S., B. Saraswat, G. K. Patnaik, D. P. Agarwal, and B. N. Dhawan. 1996. Protective activity of picroliv isolated from Picrorhiza kurrooa against ethanol toxicity in isolated rat hepatocytes. Indian J. Pharmacol. 28: 98-101. Wallerstein, C. 1999. Tuberculosis ravages Philippine slums. BMJ 319: 402. Zhang, G. L., Y. H. Wang, W. Ni, H. L. Teng, and Z. B. Lin. 2002. Hepatoprotective role of Ganoderma lucidum polysaccharide against BCG-induced immune liver injury in mice. World J. Gastroenterol. 8: 728-733.