alvina 123 ABSTRACT The kelakai fern (Stenochlaena palustris) contains bioactive substances, such as flavonoids, steroids, and alkaloids. The kelakai is used by the Kenyah Dayak communities for the treatment of anemia, fever, and cutaneous disorders. Fever is increased temperature regulation of the body, which process indirectly in- creases the production of peroxides, leading to peroxidative stress. The aim of this research study was to evaluate the influence of an aqueous extract of the kelakai fern on peroxidative stress in the hoary marmot (Marmota caligata) with experimentally-induced fever. This was a true experimental study, with post test-only and control group design, involving 6 groups, each made up of 4 animals. Peroxide concentration was measured by a modification of the FOX2 method and peroxidase activity by the Kanehira method. The kelakai extract was administered to the animals by gavage at weight-adjusted dosages between 7.75 x 10-4 mg/kg body weight to 1.24 x 10-2 mg/kg body weight. Data were analyzed using singe linear regression test. The results showed that kelakai ex- tract dose significantly reduced the peroxide levels, regression equation with the Y = 2.513-106.03 X (R2 = 0.8057), indicating that 80.5% variation of perox- ide levels is predicted by the kelakai extract dose. From the results we conclude that kelakai aqueous extract decreases peroxide levels in hoary marmots with experimentally-induced fever. Keywords: Fever, peroxidative stress, Stenochlaena palustris, hoary marmots *Department of Public Health, Medical Faculty, Lambung Mangkurat University **Department of Chemistry/ Biochemistry, Medical Faculty, Lambung Mangkurat University ***Study Group on Free Radicals and Utilization of Natural Substances Correspondence Drs. Eko Suhartono, MSi Department of Chemistry/ Biochemistry, Medical Faculty, Lambung Mangkurat University Jl. A Yani KM 36 Banjarbaru 70714 Email: ekoantioksidan@yahoo.com Univ Med 2010;29:123-8. Stenochlaena palustris aqueous extract reduces hepatic peroxidative stress in Marmota caligata with induced fever Adenan* and Eko Suhartono**,*** September-December, 2010September-December, 2010September-December, 2010September-December, 2010September-December, 2010 Vol.29 - No.3 Vol.29 - No.3 Vol.29 - No.3 Vol.29 - No.3 Vol.29 - No.3 UNIVERSA MEDICINA INTRODUCTION The potential of Indonesian wetlands, including those of South Kalimantan, is still u n d e r e x p l o i t e d . T h e p r o v i n c e o f S o u t h Kalimantan has relatively extensive areas of marshland (both freshwater and peat swamps) of 287,000 hectares. It is estimated that peat swamps account for 8-11% of the total area of this province, occupying extensive lowland areas of Kalimantan. The peat swamps have a distinctive type of forest formation with relatively limited flora, in which the kelakai fern [Stenochlaena palustris (Burm.f) Bedd] constitutes one of its species.(1) In South Kalimantan, the kelakai has few 124 Adenan, Suhartono Stenochlaena palustris reduces peroxidative uses. However, on the basis of empirical studies, the kelakai is used by the Kenyah Dayak communities for the treatment of anemia, fever, and cutaneous disorders. In spite of this, few scientific studies have confirmed this empirical evidence. Previous studies state that the plant contains bioactive substances, such as flavonoids, steroids, and alkaloids.(2) The study by Suhartono demonstrated that the kelakai fern may act as an analgesic as well as antiphlogistic; the mechanism of synthesis involves oxidative reactions by peroxide molecules and the enzyme peroxidase acting holistically.(3) When the peroxide concentration exceeds the activity of peroxidases by which it is catalyzed, peroxidative stress occurs, w h i c h i s m a r k e d b y a l o w p e r o x i d e t o peroxidase ratio. The results of the study conducted by Suhartono et al. revealed that hoary marmots with experimentally-induced fever have raised plasma peroxide levels, presumably as an immunological response of the body.(4) Since peroxides possess oxidant properties and are thus capable of forming hydroxyl radicals t h r o u g h F e n t o n a s w e l l a s H a b e r- We i s s reactions, administration of kelakai extract to hoary marmots with experimentally-induced fever may be expected to reduce peroxidative stress. Thus the objective of the present study was to explore the influence of kelakai aqueous extract on hepatic peroxidative stress in hoary marmot. METHODS Design of the study The present study was a true experimental study with post-test-only and control group design to examine the impact of kelakai extract on peroxidative stress in hoary marmots Subjects The study involved 6 groups of 4 male hoary marmots (Marmota caligata), where one group was the control group, while the other five constituted intervention groups. The test substance was an aqueous extract of kelakai ferns. Preparation of kelakai aqueous extract Kelakai ferns were collected in June 2009 from Gambut subdistrict, South Kalimantan. Species identification was performed by the Department of Biology, Pharmacy Study Program, Faculty of Mathematics and Natural S c i e n c e s (FMIPA), L a m b u n g M a n g k u r a t University. The active principles in the kelakai plants were extracted by maceration. Animal experiments The experimental animals used in this study were 3-4 months’ old hoary marmots weighing approximately 300-400 g, obtained from the Veterinary Research and Investigation Station (Balai Penelitian dan Penyidikan Veteriner, BPPV) at Banjarbaru. The animals were kept for one week prior to the experimental treatment to bring them to approximately equal physical and psychological conditions. In the adaptation period, the animals received animal feed and water ad libitum, the water being tap w a t e r s u p p l i e d b y t h e p r o v i n c i a l w a t e r purification plant. Complete random sampling was used to assign the animals into 6 groups, each consisting of 4 animals. Each group was kept in one small cage for adaptation. One group, the control group, was designated P0, while the remaining five intervention groups were designated P1-P5. The animals were subsequently weighed and their rectal temperature taken, after which all animals received 0.16 ml DPT vaccine by intramuscular injection in the right thigh, the dose having been determined in preliminary tests. The rectal temperature of the animals was again taken at the onset of fever (the time of onset of fever was determined in preliminary t e s t s t o b e 9 0 m i n u t e s p o s t - i n j e c t i o n ) . Afterwards kelakai extract was administered to the animals by gavage (stomach tube) at weight-adjusted dosages as follows: 125 22 bu bs OH mM 1x AA AA ionconcentrat Peroxide − − = 1-10 min 5 AA activity Peroxidase − = Experimental group Kelakai aqueous extract (mg/kg body weight) Distilled water P0 - 1 ml P1 7.75 x 10 -4 - P2 1.55 x 10 -3 - P3 3.10 x 10-3 - P4 6.20 x 10-3 - P5 1.24 x 10-2 - At the peak of fever (determined in preliminary tests to be 2 hours after onset), the animals were sacrificed by decapitation and their livers surgically removed. Subsequently the livers were washed in phosphate buffer at pH 7, then minced into a liquid, of which 5 ml was centrifuged at 3500 rpm for 10 minutes, and 200 µL of the supernatant was taken for determination of peroxide concentration by the modified FOX2 method, as described below. The standard and test solutions consisted of 1 M H2O2 200 µL and 200 µL plasma, respectively, with the addition of 160 µL PBS p H 7 . 4 , 1 6 0 µL F e C l 3 ( 2 5 1 . 5 m g F e C l 3 dissolved in 250 ml distilled water) and 160 µL o-fenantroline (120 mg o-phenantroline dissolved in 100 ml distilled water) for both solutions. The composition of the blank solution was identical to that of the test solution, except for absence of FeCl3 in the blank. Subsequent to preparation, all solutions w e r e i n c u b a t e d f o r 3 0 m i n u t e s a t r o o m temperature, then centrifuged at 12,000 rpm for 10 minutes, and the absorbance of the standard (As), test (Au) and blank (Ab) solutions measured at λ=505 nm, using the supernatant of each solution. Peroxide concentration was calculated from the following formula, Ab= absorbance of blank solution; and Au = absorbance of test solution. Peroxidase activity was determined by the Kanehira method.(5) One ml of the supernatant layer of the test solution was mixed with 45 µL FeCl3 , 45 µL phosphate buffer (pH 7) and 45 µL o-phenantroline, then the absorbance was measured at ë=505 nm (A0). Subsequently the mixture was incubated for one minute at room temperature and its absorbance (A1) measured at the same wavelength. Peroxidative stress is the ratio of peroxide concentration and peroxidase activity. Statistical analysis The relationship between kelakai aqueous extract dose and levels of peroxidative stress was analyzed by single linear regression. Data processing was done using Microsoft Excel. P values < 0.05 were considered statistically significant. RESULTS As shown in Figure 1, marmot hepatic peroxidative stress due to fever was inversely proportional to test substance concentration, decreasing with increasing concentration of the test substance. From Figure 1, it may be concluded that administration of kelakai extract is capable of inhibiting peroxidative stress. The correlation of the administered dose of kelakai extract with peroxidative stress is given in the equation: Ln Y = 2 . 5 1 3 - 1 0 6 . 0 3 X w i t h R 2 = 0 . 8 0 5 7 , indicating that 80.5% variation of peroxidative stress level is predicted by the kelakai extract dose. This indicates that there is a strong correlation between fever-induced hepatic Univ Med Vol.29 No.3 where As = absorbance of standard solution; 126 Adenan, Suhartono Stenochlaena palustris reduces peroxidative Figure 1. Correlation of hepatic peroxidative stress in marmots with experimentally-induced fever peroxidative stress and the administered dose of kelakai extract. DISCUSSION The febrile process indirectly leads to an increase in free radicals such as superoxide anions (•O2) which afterwards are converted t o p e r o x i d e s b y t h e e n z y m e s u p e r o x i d e dismutase (SOD). The peroxides are then catalyzed by the enzyme peroxidase into water and oxygen. Administration of distilled water (P0) to DPT-injected control animals resulted in very high peroxide concentrations in the liver, since administration of DPT vaccine induces a r e s p i r a t o r y b u r s t b y n e u t r o p h i l s a n d macrophages through concurrent formation of H2O2. At this stage there are a considerable number of phagocytosed bacteria, necessitating high H2O2 levels. In addition, arachidonic acids are ultimately transformed to prostaglandins, w h i c h a r e m e d i a t o r s o f p a i n a s w e l l a s inflammation,(6,7) the prostaglandin synthesis i n v o l v i n g o x y g e n a t i o n c a t a l y z e d b y cyclooxygenase to form peroxides.(8) The peroxides formed from these sources will in appropriate amounts be beneficial to the body that is being subjected to infection, acting as a line of defense, since peroxides possess microbicidal properties that aid in eradication of pathogenic bacteria. On the other hand, peroxides may also have a negative impact on the host, which may occur if the p r o d u c t i o n o f p e r o x i d e s e x c e e d s t h e c o u n t e r a c t i n g a n t i o x i d a n t a c t i v i t y. T h i s condition is called peroxidative stress, which may cause biomolecular damage comprising a m o n g o t h e r s c e l l u l a r d a m a g e , e n z y m e inactivation, and mutation. Additionally, peroxidative stress may lead to oxidation of DNA and proteins followed by membrane damage due to lipid peroxidation, causing m e m b r a n e p e r m e a b i l i t y c h a n g e s a n d alterations in protein structure and functions. Oxidative damage also occurs in mitochondrial m e m b r a n e s , t h u s c a u s i n g m e m b r a n e depolarization, disruption of the oxidative 127 phosphorylation chain, and altered cellular respiration. This finally causes damage to the m i t o c h o n d r i a , r e s u l t i n g i n s y n t h e s i s o f cytochrome C and activation of apoptosis (programmed cell death).(9,10) The results of the present study indicate that kelakai aqueous extract is capable of significantly decreasing hepatic peroxidative stress. This is presumably due to bioactive s u b s t a n c e s i n k e l a k a i f e r n s , n a m e l y flavonoids, steroids, and alkaloids, which are useful as antipyretics, antioxidants, and antiphlogistics.(11-13) The capture mechanism of free radicals b y f l a v o n o i d s i s i n i t i a t e d b y r e l e a s e o f hydrogen, forming reactive flavonoid radicals. The latter will bind to free radicals (•O2), causing the reactivity of the latter to diminish or even disappear,(14-16) leading to decreased formation of peroxides. Other bioactive substances contained in kelakai are alkaloids and steroids. In addition to being antipyretic, the alkaloids and steroids possess anti-inflammatory properties. The study by Sudjarwo revealed that the alkaloid piperine contained in kelakai can function as antipyretic through inhibition of prostaglandin synthesis,(17- 19) while the steroids inhibit phospholipase activity and arachidonic acid transformation to prostaglandins, reduce microvascular leakage, prevent direct migration of pyretic cells, and inhibit cytokine production.(17,20) CONCLUSION Administration of kelakai (Stenochlaena palustris) aqueous extract reduces the hepatic peroxidative stress in febrile hoary marmots (Marmota caligata). REFERENCES 1. Suhartono E, Yasmina A. Kajian farmakologis ekstrak air tumbuhan kelakai sebagai antiyeri dan antiradang dengan pendekatan mekanisme oksidatif. Laporan Penelitian Universitas Lambung Mangkurat; 2007. 2. Maharani, Haidah, Hainiyah. Studi potensi kelakai sebagai pangan fungsional. Kumpulan Makalah PIMNAS XIX, 26-29 Juli 2006;Universitas Muhamadiyah Malang; 2006. 3. Suhartono E. 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