Acute Toxicity for Combination Extractof Terminalia muelleri Benth. and Curcuma xanthorrhiza

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1 Acute Toxicity for Combination Extractof Terminalia muelleri Benth. and Curcuma xanthorrhiza Khairul Anam a, Dewi Kusrini a, Ratna Megawati Widharna b Abstract The aim of this study was to investigate the acute toxicity for combination of Terminalia muelleribenth and Curcuma xanthorrhiza extract. The extract combination oft. muelleri and C. xanthorrhiza were tested for acute toxicity towards Swiss Webster mice by method according to Organization for Economy Cooperation and Development (OECD 425). Toxic effect was evaluated based on the observation of behaviour, spinal reflex, central nerve system, urination, defecation on the first day of tested material in large single dose and the amount of death animals was observed throughout the observation. Then the Lethal Dose 50 (LD 50) combination extract of T. muelleri C. xanthorrhizawas determined. LD 50 for the combination extract of T. muelleri C. xanthorrhizais 5 g.kg -1 BW, which means practically nontoxic. Keywords:Combretaceae, fruit, bark, leaves, aqueous extract, acute toxicity a Departmentof Chemistry, Diponegoro University, Semarang, Indonesia, b Faculty of Pharmacy, Widya Mandala University, Surabaya, Indonesia Corresponding author address: k_anam@undip.ac.id Introduction Terminalia muelleri Benth. is classified in to genus Terminalia, family Combretaceae. This species was reported containing gallic acid, ellegic acid and showed that this plant was potential as antimicrobial (Anam et al, 2010a and 2010b), antioxidant (Bajpai et al., 2005) and inhibit -glucosidase (Anam et al., 2009). Acute toxicity test is a test of a material or compound that is suspected to be toxic in short term, usually less than 24 hours, administed in a large single dose, to be observed in a specific period of time, to find out the effect caused by the administration (Kram and Keller, 2006). The dose estimation which is used referred to the mortality dose of 50% of the tested animal (Lethal Dose 50, LD 50) which coded by Organization for Economic Cooperation and Development (OECD 425, 2006). in this case, maximal 5 tested animals in each determined dose that are 55, 175, 550, 1750 and 5000 mg/kg bw given per oral. The animals used usually mice or rabbit and it is not required that it must be male or female, although it was mentioned that female is more prone compared to male. in a specific period of time mice behaviour is observed, such as motoric activity, curiosity, the effect towards central and autonom nerve system, defecation, urination, and mortality. Then for the next continuous 14 days the weight gain and mortality was observed. On day 15 the 8 vital organs were observed such as lungs, heart, liver, kidney, adrenal gland, stomach, testes/ovarium and spleen. All the data was compared to control that was not given the tested material. from the mortality data, LD 50 values were calculated (Kram and Keller, 2006). OECD 425 method was chosen as it has the advantages over other methods (Weil C.S. method (Weil, 1952), Miller-Tainter method, Reed Muench method, Karber method and Farmakope Indonesia 3 rd edition method), that is the amount of mice used in the research is minimal (maximum 15 mice in each test), the dose of administration in mice was based on the guide in OECD 425, the LD 50 calculation can be done by Excel from Microsoft Windows and it does not need any specific graphical paper so that the distortion from the estimation of the marking can be avoided. In this opportunity will be reported acute toxicity of the three parts of Terminalia muelleri which previously proven very effective as antimicrobial agent (Anam et al, 2010a and 2010b) and to determine the LD 50 values from each part of this plant. Basically, the aim of this study was to investigate the acute toxicity properties of T. muelleri Benth extract. Methodology Plant material:terminalia muelleri Benth fruits were collected from Bogor Botanical Gardens, West Java, Indonesia, in November2013. The plant was determined by The Centre for Plant Conservation Bogor Botanical Garden, Indonesian Institute of Sciences. Curcuma xanthorrhiza were collected from Tembalang, Semarang, Central Java, March P a g e Green Chemistry Section 4: Organic Chemistry, Khairul Anam, et al.

2 ISBN Test Animals Used:Swiss Webster Mice were obtained from Faculty of Pharmacy, Airlangga University, Surabaya. Indonesia. Preparation of Extracts:The dried milled fruits of T. muelleriwere extracted with distilled water, at 90 C temperature for 3 hours. The extracted were evaporated to dryness with a freeze dryer. The dried milled of C. xanthorrhizawere extracted with ethanol 96% at room temperature and evaporatedby vacuum rotary evaporator. Ethanol extract was fractionated through Liquid Vacuum Chromatography, and n- hexane used as mobile phase and silica gel H60 as state phase. N-hexane fraction of C. xanthorrhiza was used to combination with T. muelleri extract (3:1) Acute Toxicity Test of Terminalia muelleri Before the test was conducted, mice was adapted and fasted. Before the treatment, mice were observed for their behaviour. After that, every fraction was dissolved in aquadest according to the dose which would be given. This fraction suspension was administered into mice stomach according to the weight with feeding needle aid. OECD 425 method (OECD, 2006) is and up and down method. in this research, down method was used, that was 1 mouse was tested at highest dose (5000 mg/kg bw). If the mouse died, the main test was conducted to determine the LD 50. If this animal survived, another 2 additional animals were given the same dose. If these 2 animals survived, the LD 50 value is bigger that the dose limit and test was stopped (observation was still be continued for 14 days without the administration of dose to the animals). LD 50 value is bigger than the tested dose (5000 mg/kg bw) when 3 or more animals survived. Animals was observed individually minimal once in 30 minutes after the administration of the dose, and periodically for 24 hours (with special attention for 4 first hours), and everyday, for 14 days in total, except when the animals needed to be removed from the research and humanly killed for the animal welfare or the animal was found dead. However, the duration of the observation was not strictly determined. The observation duration was determined by toxic reaction and the time of onset and the length of recovery period and might be prolonged if necessary. Time when toxicity appears and disappears is really important, especially if there is tendency to be a delayed toxicity sign. All the observation was recorded individually for each animal. As control group, aquadest was used and the research was conducted in triplicates. One, two, and four hours after the treatment, mice was observed for their behaviour and recorded. for 14 days, mice was given food and drink as usual but the development of body weight gain was calculated and the death was recorded. On day-15, mice were sacrificed and observed for their 8 vital organ, which were lungs, heart, liver, kidney, adrenal glands, stomach, testes/ovarium and spleen. Results and Discussion Acute toxicity research on female mice to determine the LD 50 values of the extract combination of T. muelleri and C. xanthorrhiza was conducted. The determination was conducted on female and male mice, although usually conducted only in female mice as female mice was more prone compared to male mice. The treatment towards female and male mice is aimed at finding out whether there are gender effects towards toxicity effect. OECD425 method conducted in this research is the downward method, and the highest dose chosen was 5000 mg/kg bw as the tested material was natural product. Two female mice which were given fruit aqueous extract 5000 mg/kg bw died on the second day and third day after the administration of the compound. from the organ section, it was found that the cause of death was the damage of the stomach (the presence of atrophy and colour change in stomach indicated there was toxicity toward the stomach. The observation was then continued by adding more female mice to achieve the minimal amount of highest dose administration, but using as minimal mice as possible. Both of the additional mice survived so that 3 female mice survived for 14 days after the administration of combination extract. Therefore, by the achievement of minimal 3 survived female mice at the administration of highest dose 5 g/kg bw, it could be concluded that the LD 50 of the combination extract for female mice is above 5 g/kg bw. A male mouse A male mouse which was given combination extract 5000 mg/kg bw died 2 days after the administration of the tested material so that another male mouse was added to fulfil the amount of 3 male mice which is needed for the conclusion of LD 50 value determination. The three male mice survived for 14 days after the administration of the tested materials. At the administration of combination extract at the dose of 5000 mg/kg bw, a male mouse died on the second day after the administration of the tested material, therefore the administration of the tested compound of the same dose was tried the next day to find out if there was toxicity. The male mouse given the same dose did not experience toxicity, therefore 3 mice were fulfilled as the requirement of the dose safety. The treatment for 14 days is aimed at observing any latent toxic effect. (Thompson, 1985). The observation conducted for 4 hours after the administration of the compound showed that there was a decrease in the amount and motoric activity at minute 30, 60, 120 and 240 for the combination extract towards male mice, but this was not found in female mice. This might be caused by the animals was getting sleepy or adapted (familiar) with the platform Green Chemistry Section 4: Organic Chemistry,Khairul Anam, et al. P a g e 299

3 so that the activity became less. There was no Straub effect, piloerection and ptosis. No Straub effect indicates that there was no toxicity in the neuromuscular system (Lu, 1995). No piloerection means that there were no toxic effect to skin and autonomic system toward the tested animals. Piloerection also means that there was compensation towards lower temperature or simpatomimetic activity (Turnerand Hebborn, 1971). There were no decreases in pineal and corneal reflexes. This means there was no toxicity towards sensoric system, or blocking towards the spinal synapse or the efferent pathway. There was no lacrimation and midriasis at all the animals at all doses. There was no decrease in grip reflex (hanging ability) and reestablishment at the administration of the combination extract 5 g/kg bw. This indicates that there was no muscle relaxant activity (myorelaxant activity), neuromuscular blocking or central depression. There was no decrease in flexi relax and Haffner test. Grooming was found in mice given aquadest as negative control and in the combination extracts.this meant that the tested extract autonomic nerves and gastrourinary (Lu, 1995), but in normal limit. Excessive grooming indicated there was central or simpatic stimulation (Turnerand Hebborn, 1971). There was no catalepsy at the administration of the combination extract indicated that there was no sedation effect. Urination was found in the first 4 hours of the observation towards mice that were given the tested materials. Excessive urination could be caused by muscarinic activity or irritation of the urine tract by the tested material or its metabolite. Defecation was found at different interval of test period (4 hours of observation on platform), but this was a normal variation of gastrointestinal system (Lu, 1995). However, there was an increase in defecation amount at the administration of combination extract at the dose of 5g/kg bw both in female and male mice compared to control group. From the body weight data for 14 days with controlled amount of food and drink is provided ad libitum, all mice gained weight (observed by positive regression), although there was tendency to decrease on the last days. Therefore, there was no effect of the tested material on body weight decrease. The data organ weight showed a normal result at all the tested materials at the dose of LD 50. Therefore, there was no effect of the tested materials towards the organ weight. From the research, it could be concluded that the LD 50 values for these combination extract of are 5 g/kg BW. Therefore, these extracts are categorized as practically nontoxic. appearing symptoms, acute non-lethal effect, affected organ, and reversibility of non-lethal effect. The result of the test also beneficial as a risk prediction in working and other benefit is that the test could be conducted in a short period of time (Hodgson and Levi, 2004). For the next research, sub acute and sub chronic toxicity could be conducted to see the effect of long term usage. References Anam, K., R.M. Widharna and D. Kusrini, α-glucosidase inhibitor activity of Terminalia species. Int. J. Pharmacol., 5: Anam, K., A.G. Suganda, E.Y. Sukandar and L.B.S. Kardono, 2010a, Antibacterial Agents ofterminalia muelleribenth. Leaves. Res. J. Med. Plant, 4: Anam, K., A.G. Suganda, E.Y. Sukandar and L.B.S. Kardono,. 2010b, Antibacterial Effect of Component ofterminalia muelleribenth. against Staphylococcus aureus, International Journal of Pharmacology, 6(4), Bajpai, M., A. Pande, S.K. Tewari and D. Prakash, : Phenolics contents and antioxidant activity of some food and medicinal plants, International Journal of Food Sciences and Nutrition, 56(4), Hodgson, E. and P.E. Levi, 2004, Hepatotoxicity, In: A Textbook of Modern Toxicology, Hodgson, E. (Ed.). 3 rd Edn., John Wiley and Sons, Inc. Hoboken, USA, pp: , Kram D.J and K.A. Keller, Toxicological Testing Handbook: Principles, Applications, and Data Interpretation. 2 nd Edn., Informa Healthcare, New York, ISBN: , Pages: 499. Lu, F.C., 1995, Toksikologi Dasar Asas, Organ Sasaran, dan Penilaian Resiko, ed 2, Terjemahan E. Nugroho, Penerbit University of Indonesia, Jakarta, 67-75, 86-99, Thompson, E.D., Drug Bioscreening: Fundamentals of Drug Evaluation Techniques in Pharmacology. Vol. 1, Graceway Publishing Co., Fresh Meadows, Queens, NY., USA., ISBN-13: , Pages: 164. Turner, R.A. and P. Hebborn, 1971, Screening Methods in Pharmacology. Vol. 2, Academic Press, New York, ISBN: , Pages: 288. Weil, C.S., Tables for convenient calculation of median-effective dose (LD 50 and ED 50) and instructions in their use. Biometrics 8, Acute toxicity test is beneficial to see the acute toxicity effect such as the cause of death, time of death, 300 P a g e Green Chemistry Section 4: Organic Chemistry, Khairul Anam, et al.

4 ISBN Appendix A. Pharmacological activity profile of female and male mice before and after the administration of a single dose aquadest (negative control) per oral on day 1 Female Dose mg/kg bw (n = 3) Male Dose mg/kg bw (n=3) T0 T30 T60 T120 T240 T0 T30 T60 T120 T240 Platform Motoric activity N =3 N =3 N = 3 N = 3 N = 3 N = 3 N = 3 N = 3 N = 3 N = 3 Straub - = 3 - = 3 - = 3 - = 3 - = 3 - = 3 - = 3 - = 3 - = 3 - = 3 Piloerection - = 3 - = 3 - = 3 - = 3 - = 3 - = 3 - = 3 - = 3 - = 3 - = 3 Ptosis - = 3 - = 3 - = 3 - = 3 - = 3 - = 3 - = 3 - = 3 - = 3 - = 3 R. Pineal + = 3 + = 3 + = 3 + = 3 + = 3 + = 3 + = 3 + = 3 + = 3 + = 3 R.Kornea + = 3 + = 3 + = 3 + = 3 + = 3 + = 3 + = 3 + = 3 + = 3 + = 3 Lacrimation - = 3 - = 3 - = 3 - = 3 - = 3 - = 3 - = 3 - = 3 - = 3 - = 3 Midriase - = 3 - = 3 - = 3 - = 3 - = 3 - = 3 - = 3 - = 3 - = 3 - = 3 Katalepsi - = 3 - = 3 - = 3 - = 3 - = 3 - = 3 - = 3 - = 3 - = 3 - = 3 Righting ability N =3 N =3 N = 3 N = 3 N = 3 N = 3 N = 3 N = 3 N = 3 N = 3 Grip reflex + = 3 + = 3 + = 3 + = 3 + = 3 + = 3 + = 3 + = 3 + = 3 + = 3 Reestablishment + = 3 + = 3 + = 3 + = 3 + = 3 + = 3 + = 3 + = 3 + = 3 + = 3 Flexi + = 3 + = 3 + = 3 + = 3 + = 3 + = 3 + = 3 + = 3 + = 3 + = 3 Hafner + = 3 + = 3 + = 3 + = 3 + = 3 + = 3 + = 3 + = 3 + = 3 + = 3 Cholic Ach - = 3 - = 3 - = 3 - = 3 - = 3 - = 3 - = 3 - = 3 - = 3 - = 3 Mortality - = 3 - = 3 - = 3 - = 3 - = 3 - = 3 - = 3 - = 3 - = 3 - = 3 Grooming - = 3 - = 2 - = 1 - = 3 - = 3 - = 1 - = 2 - = 2 - = 2 + = 3 + = 1 + = 2 + = 2 + = 1 + = 1 + = 1 Urination - = 2 - = 1 - = 1 - = 1 - = 2 - = 2 - = 3 - = 2 - = 2 - = 2 + = 1 + = 2 + = 2 + = 2 + = 1 + = 1 + = 1 + = 1 + = 1 Defecation + = 2 + = 0 + = 4 + = 0 + = 1 + = 3 + = 2 + = 4 + = 2 + = 3 Appendix B. Pharmacological activity profile of female and male mice before and after the administration of a single dose of combination extract T. muelleri C. xanthorrhiza 5000 mg/kg bw on day 1 Female Dose mg/kg bw (n = 3) Male Dose mg/kg bw (n=3) T0 T30 T60 T120 T240 T0 T30 T60 T120 T240 Platform Motoric activity N =3 N =3 N =3 N =3 N =3 N =3 N = 3 N = 1 N =1 N = 1 = 2 = 1 = 1 Still = 2 Still = 1 Still = 1 Straub - = 3 - = 3 - = 3 - = 3 - = 3 - = 3 - = 3 - = 3 - = 3 - = 3 Piloerection - = 3 - = 3 - = 3 - = 3 - = 3 - = 3 - = 3 - = 3 - = 3 - = 3 Ptosis - = 3 - = 3 - = 3 - = 3 - = 3 - = 3 - = 3 - = 3 - = 3 - = 3 R. Pineal + = 3 + = 3 + = 3 + = 3 + = 3 + = 3 + = 3 + = 3 + = 3 + = 3 R.Kornea + = 3 + = 3 + = 3 + = 3 + = 3 + = 3 + = 3 + = 3 + = 3 + = 3 Lacrimation - = 3 - = 3 - = 3 - = 3 - = 3 - = 3 - = 3 - = 3 - = 3 - = 3 Midriase - = 3 - = 3 - = 3 - = 3 - = 3 - = 3 - = 3 - = 3 - = 3 - = 3 Catalepsi - = 3 - = 3 - = 3 - = 3 - = 3 - = 3 - = 3 - = 3 - = 3 - = 3 Righting ability N =3 N =3 N = 3 N = 3 N = 3 N = 3 N = 3 N = 3 N = 3 N = 3 Grip reflex + = 3 + = 3 + = 3 + = 3 + = 3 + = 3 + = 3 + = 3 + = 3 + = 3 Reestablishment + = 3 + = 3 + = 3 + = 3 + = 3 + = 3 + = 3 + = 3 + = 3 + = 3 Green Chemistry Section 4: Organic Chemistry,Khairul Anam, et al. P a g e 301

5 Flexi + = 3 + = 3 + = 3 + = 3 + = 3 + = 3 + = 3 + = 3 + = 3 + = 3 Hafner + = 3 + = 3 + = 3 + = 3 + = 3 + = 3 + = 3 + = 3 + = 3 + = 3 Cholic Ach - = 3 - = 3 - = 3 - = 3 - = 3 - = 3 - = 3 - = 3 - = 3 - = 3 Mortality - = 3 - = 3 - = 3 - = 3 - = 3 - = 3 - = 3 - = 3 - = 3 - = 3 Grooming - = 1 - = 1 - = 2 - = 2 - = 1 - = 2 - = 3 - = 2 + = 3 + = 2 + = 2 + = 1 + = 1 + = 2 + = 1 + = 1 + = 3 Urination + = 1 - = 2 + = 1 - = 3 - = 3 - = 3 - = 2 - = 3 + = 2 + = 1 - = 3 - = 2 - = 3 Defecation + = 5 + = 2 + = 3 + = 3 + = 4 + = 4 + = 2 + = 0 + = 2 + = 3 Appendix C. Table of Organ Weight (g/10g bb) 14 days after the administration of Single dose of Aquadest, combination extract T. muelleri C. xanthorrhiza 5 g/kg bw Table C-1. Organ Weight(g/10g bb) female mice 14 days after the administration of a single dose of aquadest, combination extract T. muelleri C. xanthorrhiza of 5 g/kg bw No Organ Index Liver Spleen Heart Stomach ulcer Lungs Kidney Adrenal glands Ovarium, tuba falopii 1 Aquadest (n = 3) combination extract T. muelleri C. xanthorrhiza (5000 mg/kg bw (n = 3) Table C-2. Organ Weight (g/10g bb) male mice 14 days after the administration of a single dose of aquadest, combination extract T. muelleri C. xanthorrhiza of 5 g/kg bw No Index organ Liver Spleen Heart Stomach ulcer Lungs Kidney Adrenal glands Testes Vas deferens 1 Aquadest (n = 3) combination extract T. muelleri C. xanthorrhiza (5000 mg/kg bw (n = 3) Notes: * = Did not have stomach ulcer, but toxicity was inform of atrofi and colour change in stomach 302 P a g e Green Chemistry Section 4: Organic Chemistry, Mardiyah Kurniasih, et al.