Oktavianus 11 *Pharmacy Department, Faculty of Medicine and Health Sciences Jenderal Soedirman University **Medical Department, Faculty of Medicine and Health Sciences Jenderal Soedirman University Correspondence Heny Ekowati, M.Sc., Apt. Pharmacy Department Medicine and Health Sciences Faculty Jenderal Soedirman University Jl. Dr. Soeparno Karangwangkal Purwokerto 53123 Fax. +62281-642840 E-mail : heny240377@gmail.com Univ Med 2013;32:11-9 ABSTRACT UNIVERSA MEDICINA January-April, 2013January-April, 2013January-April, 2013January-April, 2013January-April, 2013 Vol.32 - No.1 Vol.32 - No.1 Vol.32 - No.1 Vol.32 - No.1 Vol.32 - No.1 BACKGROUND Doxorubicin as an anticancer drug has hepatotoxic side effects. Curcuma xanthorrhiza, Curcuma longa and Zingiber officinale are commonly used as herbals in Indonesia and around the world. Several compounds in these plants have antioxidant activities and are known to exhibit protection against doxorubicin-induced toxicities. This study aimed to observe the hepatoprotective effect of a combination of C. xanthorriza, C. longa, and Z. officinale extract on doxorubicin-induced hepatotoxicity in rats. METHODS A total of 28 Wistar male rats were divided into four groups: 1) control group (0.9% NaCl); 2) doxorubicin 5 mg/kg intraperitoneally (ip) four times in 14 days (days 1, 5, 9, 13); 3) doxorubicin + combination of C. xanthorriza, C. longa, and Z. officinale (temulawak, kunyit, and jahe merah, designated as Tekuja) 250 mg/kg/day orally for 14 days; and 4) doxorubicin + Tekuja extract 500 mg/kg/day orally for 14 days. Measurements of parameters based on liver histopathology and the parameters of serum alanine amino transferase (ALT) and aspartate amino tranferase (AST). RESULTS Doxorubicin caused significant elevation in serum ALT and AST enzymes after 14 days of treatment. Rats treated with doxorubicin + Tekuja extract 250 mg/kg/ day showed no histological changes, but had decreased levels of ALT and AST. CONCLUSION This study indicates that the combination of C. xanthorriza, C. longa, and Z. officinale has a protective effect in rats against liver damage induced by doxorubicin. Key words : Doxorubicin, Curcuma xanthorriza, Curcuma longa, Zingiber officinale, hepatotoxicity, rats Combination of three species of Zingiberaceae prevents doxorubicin-induced hepatotoxicity Heny Ekowati*, Sarmoko*, and Retno Widiastuti** 12 Ekowati, Sarmoko, Widiastuti Zingiberaceae prevent hepatotoxicity Kombinasi tiga spesies Zingiberacea mencegah hepatotoksitas yang diinduksi doksorubisin LATAR BELAKANG Doksorubisin adalah antikanker yang memiliki efek samping hepatotoksik. Curcuma xanthorrhiza (Temulawak), Curcuma longa (kunyit), dan Zingiber officinale (jahe), adalah herbal yang biasa digunakan di Indonesia dan di dunia. Senyawa-senyawa yang terkandung dalam ketiga herbal tersebut dilaporkan dapat menurunkan toksisitas yang disebabkan penggunaan doksorubisin. Penelitian ini bertujuan untuk menilai efek hepatoprotektif dari kombinasi ekstrak C. xanthorriza, C. longa, and Z. officinale (Temulawak, kunyit dan jahe merah) pada tikus yang diinduksi doksorubisin. METODE Hewan tikus galur Wistar jantan sejumlah 28 ekor dibagi dalam empat kelompok: 1) kelompok kontrol (0.9% NaCl); 2) doksorubisin 5 mg/kg intraperitoneal, empat kali selama 14 hari (hari 1, 5, 9, 13); 3) doksorubisin + kombinasi ekstrak temulawak, kunyit dan jahe merah (Tekuja) 250 mg/kg/hari per oral selama 14 hari; dan 4) doksorubisin + kombinasi ekstrak Tekuja 500 mg/kg/hari per oral selama 14 hari. Pengukuran parameter berdasarkan gambaran histopatologi organ hati dan parameter alanine amino transferase (ALT) dan aspartate amino tranferase (AST). HASIL Doksorubisin menyebabkan peningkatan yang signifikan pada enzim ALT dan AST setelah 14 hari perlakukan. Hewan yang diberikan ekstrak tidak menunjukkan perbedaan pada gambaran histopatologi namun memberikan gambaran penurunan level ALT dan AST. Doksorubisin menyebabkan kenaikan jumlah yang signifikan enzim ALT dan AST serum. KESIMPULAN Studi ini menunjukkan bahwa kombinasi ekstrak Tekuja memiliki efek protektif pada organ hati yang diinduksi doksorubisin. Kata kunci : Doksorubisin, Curcuma xanthorriza, Curcuma longa, Zingiber officinale, hepatotoksisitas, tikus ABSTRAK INTRODUCTION N a t u r a l p r o d u c t s a n d t h e i r a c t i v e constituents as a source of new drugs for the treatment of disease have attracted attention in recent years. Medicinal applications of spices or herbs have been gradually increasing in developed countries. Curcuma xanthorriza, Curcuma longa and Zingiber officinale are commonly used as spices as well as herbals in Indonesia and around the world. These spices are an indispensable component of curry, and belong to the Zingiberaceae family.(1) Several compounds in these plants have antioxidant activities and are known to exhibit protection against doxorubicin-induced toxicities.(1,2) C. xanthorrhiza is known as temulawak or Javanese turmeric and its extract reportedly possesses anti-inflammatory and antitumor activities, particularly one of its active principles, xanthorrhizol,(3) Z. officinale or ginger, is one of the most commonly used spices in Indonesia and around the world. The active compounds of Z. officinale are gingerol, paradol, shogaol, and 13 zingerone.(4) The major chemical constituent of C. longa rhizomes is a yellow pigment, 1,7-bis (4-hydroxy-3-methoxyphenyl) -1, 6-heptadiene- 3 , 5 - d i o n e , k n o w n a s c u r c u m i n (diferulolylmethane). Curcumin has shown a n t i o x i d a n t , a n t i - n e o p l a s t i c a n d a n t i - inflammatory properties.(5) Doxorubicin (DOX) is an anthracycline glycoside antibiotic that possesses a potent and broad spectrum antitumor activity against a variety of human solid tumors and hematological malignancies.(6) The DOX antitumor effects include mechanisms related to alterations of DNA and the production of free radicals.(7) However its use in chemotherapy has been limited, largely due to its diverse toxicities, including cardiac, hepatic, hematological and testicular toxicity.(6) Several studies have shown that the combination of the inflammatory process, f r e e r a d i c a l o x i d a t i v e s t r e s s , a n d l i p i d peroxidation is frequently associated with liver damage, induced by toxic agents such as DOX.(7) Persistent and irreversible liver damage has been a well-known side effect of DOX therapy. It has been observed that there is an increase in the apoptotic processes in liver tissues after a single dose of DOX.(8,9) It was confirmed that the therapeutic dose of DOX enhanced lipid peroxidation in microsomes and mitochondria in the liver, especially in the presence of Fe3+ ions.(6) DOX-mediated hepatotoxicity includes focal damage in hepatocytes, vascular damage and steatosis.(10) Although the antioxidant activities of C. xanthorrhiza, Z. officinale and C. longa were well-known, their protective effect against DOX- induced hepatotoxicity has not yet been reported. Several studies have shown that using a c o m b i n a t i o n o f p l a n t s i n c r e a s e s t h e i r effectiveness because of synergy with minimal s i d e e ff e c t s . ( 11 ) I n o u r s t u d y, w e u s e d a combination of three herbs, C. xanthorriza (temulawak), C . l o n g a (kunyit) a n d Z . officinale ( j a h e m e r a h ) ( w h i c h w e w i l l designate as Tekuja). A study on the combination of C. longa and C. xanthorrixa showed that it had a protective effect against doxorubicin- induced liver damage in rats.(3,5) An earlier study, using Z.officinale only, suggested that Z. officinale protected against nephrotoxicity either by enhancing the renal antioxidant status that had been reduced by DOX, or by exerting a direct antioxidant activity.(12) Therefore the current study was aimed at evaluating the hepatoprotective activity of an aqueous ethanolic extract of a combination of C. xanthorrhiza, Z. officinale and C. longa (Tekuja) on doxorubicin-induced hepatotoxicity in rats. METHODS Study design An experimental laboratory study was conducted from March to October 2012. A total of 28 male rats was randomized into 4 groups (control and experimental groups) of seven rats each. The minimum group size as derived from the formula (t-1) (r-1) >15, where t = number of treatments and r = number of rats, was 4 rats per group. Animals from group I (control group) received NaCl 0.9%; animals from group II r e c e i v e d d o x o r u b i c i n ( D O X ) ( 5 m g / k g ) intraperitoneally (ip) 4 times in 14 days (on days 1, 5, 9,13); group III was given Tekuja extract 250 mg/kg/day orally for 14 days and DOX (5 mg/kg) intraperitoneally (on days 1, 5, 9,13); animals from group IV were given Tekuja extract 500 mg/kg/day orally for 14 days and DOX (5 mg/kg) intraperitoneally (on days 1, 5, 9, 13). DOX-treated groups received the drug every fourth day, while the other groups instead of DOX received 0.9% sodium chloride (10 mL/ kg BW) intraperitoneally. The time intervals between two DOX administrations were similar to the most frequently-used treatment schedules in humans. On day 15 (1 day after the last dose of DOX) all animals were sacrificed.(12,13) Experimental animals Laboratory-bred Wistar albino rats (8- week-old and weighing 140 ± 30 g) were Univ Med Vol. 32 No.1 14 Ekowati, Sarmoko, Widiastuti Zingiberaceae prevent hepatotoxicity obtained from Medical Faculty, Gadjah Mada University, Yogyakarta, Indonesia and approved by the Animal Care Committee of Gadjah Mada University. The animals were maintained under standard laboratory conditions of 25o C and a photoperiod of 12 h dark and 12 h light. Commercial pellet diet (Clinical Pharmacy Laboratory rat and mouse feed, Purwokerto, Indonesia) and water were provided ad libitum. The animals were allowed to acclimatize for 10 days before beginning the experiments. Extract preparation C. xanthorriza, C. longa and Z. officinale rhizomes were purchased from and authenticated by the Center for Research and Development of Medicinal Plants and Traditional Medicine/ B2P2TO2T, Tawangmangu, Indonesia. The sundried rhizomes were ground separately to a powder, then 500 g each of the powders were combined and macerated in ethanol for 3 days at room temperature. The filtrate was air-dried and concentrated under reduced pressure. Laboratory determination Blood samples were withdrawn from the orbital vein using a capillary tube. The blood samples were left to clot at room temperature. The clotted blood was then centrifuged at 3,000 rpm for 15 min to obtain blood serum, and analyzed for the biochemical parameters serum alanine amino transferase (ALT) and aspartate amino tranferase (AST). All serum biochemical testing was conducted at Biofit Laboratory, Purwokerto, Indonesia using a SYSMEX KX 21 serum analyzer. H i s t o p a t h o l o g i c e x a m i n a t i o n o f l i v e r tissues At autopsy, the liver of each rat was removed and fixed in 10% buffered formalin. After 12–24 h of fixation, 3–5 mm tissue slices were embedded in paraffin, and stained with H e m a t o x y l i n ( H ) a n d E o s i n ( E ) f o r histopathologic examination. Ethical Clearance Animals were handled according to the rules and regulations of the Animal Care Committee of the Faculty of Medicine, Gadjah Mada University. Data analysis All results were expressed as mean and standard deviation. Differences between groups were assessed by one-way analysis of variance (ANOVA), followed by Tukey HSD. Statistical significance was defined as p<0.05. RESULTS Observation was terminated at 14 days a f t e r t h e l a s t D O X t r e a t m e n t . Macroscopic observation of the livers showed no perceptible differences between groups (Figure 1). The histopathological appearance of the livers in rats treated with DOX and DOX + Tekuja extract also showed no differences between groups (Figure 2). However, we found a significant increase in ALT and AST levels (p<0.05) in the group treated with DOX only, compared with the control group (Table 1). Administration of Tekuja (250 and 500 mg/kg BW) plus DOX significantly (p < 0.05) attenuated the decrease in ALT and AST levels observed after administration of DOX alone. The ALT levels were restored to normal in the Tekuja plus DOX groups (Tukey HSD Test, p<0.05) (Table 2). DISCUSSION The results of the present study indicate that the aqueous ethanolic extract of the combination of C. xanthorrhiza, C. Longa and Z. officinale (Tekuja) significantly protected rats against DOX–induced hepatotoxicity. A study on the combination of C. longa and C.xanthorrixa showed that it had a protective effect against DOX-induced liver damage in rats.(3,5) Another study using C. longa alone 15 Figure 1. Absence of morphological differences in livers of rats treated with C. xanthorrhiza, Z. officinale and C. longa combination and doxorubicin (A) NaCl control group; (B) Doxorubicin group; (C) Doxorubicin+250 mg/kgBW C. xanthorrhiza, Z. officinale, and C. longa combination; (D) Doxorubicin+500 mg/kgBW C. xanthorrhiza, Z. officinale, and C. longa combination Figure 2. Histological evaluation in liver tissues of control and experimental group. (A) NaCl control group; (B) Doxorubi- cin group; (C) Doxorubicin + 250 mg/kgBW C. xanthorrhiza, Z. officinale, and C. longa extract combination; (D) Doxorubicin + 500 mg/kgBW C. xanthorrhiza, Z. officinale, and C. longa extract combination Univ Med Vol. 32 No.1 16 Ekowati, Sarmoko, Widiastuti Zingiberaceae prevent hepatotoxicity Table 1 Mean of on ALT and AST bases on Treatment groups in rats Each value represents mean ± S.D. of seven animals Table 2. Multiple comparison test (Tuckey HSD) of ALT (U/L) and AST (U/L) levels between treatment groups showed that C. longa has multiple therapeutic activities by blocking cardiac, hepatic,and renal toxicities induced by doxorubisin.(14) An earlier study using Z.officinale only, suggested that Z. officinale protected against nephrotoxicity either by enhancing the renal antioxidant status that had been reduced by DOX, or by exerting a direct antioxidant activity.(12) The present study explored the effect of a combination of C. xanthorrhiza, C. longa and Z. officinale extract (Tekuja) on DOX- induced hepatotoxicity in rats. The Tekuja extract was given during the DOX induction period. Treatment with the combination of C. xanthorrhiza , Z. officinale and C. longa extract was designed to prevent metabolic activation of DOX and suppress liver damage. The liver is the central organ of metabolism and acts as a storage organ. Liver cells metabolize toxic agents, including DOX. The liver is extremely vulnerable to damage by chemical agents, presumably as a result of its central role in the metabolism of foreign substances.(15) In our study, histopathological e x a m i n a t i o n o f t h e l i v e r s s h o w e d n o appreciable differences between groups. These results may be interpreted as indicating that administration of DOX does not result in liver damage, although the serum transaminase was elevated. DOX in the form of DOX semiquinone has been suggested to play a major role in its hepatotoxic action. Semiquinones are unstable under aerobic conditions, thereby generating superoxide anion radicals by reacting with molecular oxygen.(16) Hepatocytes are the likely targets of attack by reactive oxygen species (ROS) in the failing liver. It is conceivable that free radicals cause damage at their formation. Consequently the mitochondria as the major source of ROS production, could also be the major target susceptible to attack by ROS.(16) 17 Serum transaminases have long been considered a sensitive indicator of hepatic injury. Injury to the hepatocytes alters their transport function and membrane permeability, leading to leakage of enzymes from the cells, causing a decrease in the levels of ALT and AST in hepatic cells but an increase of these enzymes in serum.(17) In this study, administration of DOX to rats significantly increased serum ALT and AST levels. AST is a more liver- specific enzyme. On the other hand, an increase in ALT activity is usually proportional to the extent of cardiac damage. Treatment with the combination of C. xanthorrhiza, Z. officinale and C. longa (250 and 500 mg/kg) resulted in a significant decrease in enzyme activities in DOX-treated animals, thus offering considerable protection against hepatotoxicity. The data presented in this study demonstrate that DOX increased serum indices of liver function including ALT and AST. These elevations of ALT and AST are attributable to hepatocellular damage and decreased liver functions.(15,18) These elevated levels of serum indices for hepatocellular damage have been p r e v i o u s l y r e p o r t e d i n a D O X - i n d u c e d hepatoxicity model.(18,19) The effectiveness of C. xanthorrhiza in lowering the serum enzyme levels of AST, ALT, and glutamate transferases demonstrates the hepatoprotective effect of this plant against cisplastin induced hepatotoxicity.(20,21) The plant also acts to prevent fatty degeneration of the liver, which can cause irreversible functional breakdown, inevitably leading to death.(22) Yemitan et al.(23) tested the effect of an ethanol extract Z. officinale rhizomes against carbon tetrachloride (CCl 4 ) and acetaminophen- i n d u c e d l i v e r t o x i c i t i e s i n r a t s . C a r b o n tetrachloride and acetaminophen induced many histopathological changes and increased the activities of ALT, AST, ALP, LDH and SDH in serum. The protective effect of Z. officinale extract against carbon tetrachloride- and acetaminophen-induced damage was confirmed by histopathological examination of the liver. C . x a n t h o r r i z a , C . l o n g a a n d Z . officinale all contain the active compounds curcumin, xanthorrizhol and oleoresin.(3-5) Curcumin has shown antioxidant, anti-neoplastic and anti-inflammatory activity.(5,24,25) Many of the activities associated with curcumin relate to its ability to suppress acute and chronic inflammation.(26) An in vivo study showed that rats fed curcumin for 7 days prior to being treated with cyclophosphamide to induce lung injury, exhibited an increase in antioxidant defense mechanisms. Thus, curcumin exhibits substantial antioxidant properties in a wide variety of experimental settings.(25) The antioxidant properties of [6]-gingerol, which is a very effective agent for anticipation of ultraviolet B (UVB)-induced reactive oxygen species production and COX-2, and a promising therapeutic agent against UVB-induced skin disorders, have been studied both in vitro and in vivo. The compound also has a protective role against the toxicity and lethality induced by a g e n t s s u c h a s c a r b o n t e t r a c h l o r i d e a n d cisplatin.(27,28) The present study provides the information that Tekuja extract protected against the hepatotoxicity of DOX. Tekuja was shown to p r o t e c t l i v e r t i s s u e , a s i n d i c a t e d b y t h e histopatological profile, and ALT and AST levels. These results suggest the possible use of Tekuja as a novel agent against DOX-induced organ toxicities and a potential candidate to be further evaluated. However, the parameters in our present study could not explore the activity of DOX in DOX-induced decline in hepatic antioxidant status or its direct antioxidant activity. Therefore further studies should be conducted to explore hepatic antioxidant status and direct antioxidant activity. CONCLUSION The results of the present study indicate that the combination of C. xanthorriza, C. longa, and Z. officinale has a protective effect against liver damage induced by DOX. Univ Med Vol. 32 No.1 18 Ekowati, Sarmoko, Widiastuti Zingiberaceae prevent hepatotoxicity ACKNOWLEDGEMENTS The authors wish to express their sincere gratitude to the Directorate of Higher Education, Ministry of Education and Culture, and to the Jenderal Soedirman University for financial support by Institutional Research Grant 2011- 2012. REFERENCES 1. Ali BH, Blunden G, Tanira MO, Nemmar A. 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