Original Article. Acute and subchronic toxicity study of the water extract from the fruits of Piper chaba Hunter in rats

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1 International Journal of Applied Research in Natural Products Vol. 3 (4), pp , Dec 2010-Jan 2011 Directory of Open Access Journals IJARNP-HS Publications Original Article Acute and subchronic toxicity study of the water extract from the fruits of Piper chaba Hunter in rats Jaijoy K 1, Vannasiri S 2, Piyabhan P 2, Lerdvuthisopon N 3, Boonraeng S 4, Khonsung P 5, Lertprasertsuke N 6, Sireeratawong S 7* 1 Department of Pharmacology, Faculty of Medicine, Chiang Mai University, Muang, Chiang Mai 50200, Thailand. 2 Division of Physiology, Department of Preclinical Science, Faculty of Medicine, Thammasat University, Rungsit Campus, Klongloung, Pathumthani 12120, Thailand. 3 Division of Biochemistry, Department of Preclinical Science, Faculty of Medicine, Thammasat University, Pathumthani 12120, Thailand. 4 Division of Agro-industry, Faculty of Agricultural Technology, Chiang Mai Rajabhat University, Chiang Mai, Thailand. 5 Department of Pathology, Faculty of Medicine, Chiang Mai University, Muang, Chiang Mai 50200, Thailand. 6 Department of Pathology, Faculty of Medicine, Chiang Mai University, Muang, Chiang Mai 50200, Thailand. 7 Division of Pharmacology, Department of Preclinical Science, Faculty of Medicine, Thammasat University, Rungsit Campus, Klongloung, Pathumthani 12120, Thailand. Summary: The water extract from the fruits of Piper chaba Hunter was evaluated for acute and subchronic toxicity in both male and female rats. For the study of acute toxicity, a single oral dose of 5,000 mg/kg body weight was administered in rats (five females, five males). The results showed no signs of toxicity such as general behavior change, mortality, or change in gross appearance of internal organs. Subchronic toxicity was studied by daily oral doses (ten females, ten males) of 300, 600 and 1,200 mg/kg body weight for consecutive 90 days. The satellite group was treated with the extract at the dose of 1,200 mg/kg/day for 90 days and kept for other 28 days after treatment. The results showed no abnormalities in treated groups as compared to the controls. Although significantly different, all of the values were within normal limits. Neither gross abnormalities nor histopathological changes were observed. The results suggest that P. chaba extract does not produce acute or subchronic toxicity in either female or male rats. Industrial relevance: Herbal medicines are popular and extensively used in the developing world. In many places, they offer a more wide available and more affordable alternative to pharmaceutical drugs and natural food supplements. The data of the acute and subchronic toxicity studies on medicinal plants or preparations derived from them should be obtained in order to increase the confidence in its safety to human, particularly for use in the development of pharmaceutical. Keywords: Piper chaba Hunter; acute toxicity; subchronic toxicity Introduction Piper chaba Hunter is a plant of Piperaceae family, locally known as pepper, long pepper, and also known as Dee Plee. The dried fruit are extensively used for flavouring a variety of foods. In Thai traditional medicine, the decoction of dried mature unripe fruits (1 handful or fruits) are used to reduce gas and motion sickness, as well as help improving appetite and stopping diarrhea. Due to oxytocic effect for post-labor, it is not recommended for women during pregnancy (Saralamp et al., 1996). Chemical studies of P. chaba fruit reveal *Corresponding Author: Sireeratawong S: seewaboon@gmail.com Tel: Available online

2 Jaijoy et al several bioactive compounds such as piperine, piperlongumine (Wu et al., 2004; Park et al., 2007), piperrolein B, piperchabamide D (Lee et al., 2008), piperchabamide B, piperundecalidiene (Zhang et al., 2008), piperchabamide C, piperchabamide E (Matsuda et al., 2008). Pharmacological importance of P. chaba fruit extract have been reported as gastroprotective (Morikawa et al., 2004), cytotoxicity, antitumor (Sunila & Kuttan 2004), analgesic, anti-inflammatory, antidiarrhoeal (Taufiq-Ur-Rahman et al., 2005), chemopreventive (Selvendiran et al., 2006), antiangiogenesis (Sunila & Kuttan 2006), immunomodulator (Devan et al., 2007), hepatoprotective (Matsuda et al., 2008), and adipogenesis (Zhang et al., 2008). Despite long record of usage for various purposes, little information on P. chaba toxicity is currently available. The purposes of the present study are to investigate acute and subchronic oral toxicities of the water extract from fruits of P. chaba in rats. Materials and methods Plant material: The fruits of Piper chaba were collected from Songkhla, Thailand. The voucher specimen (SBK 0013) was kept and identified by the National Park, Wildlife and Plant Conservation Department, Ministry of Natural Resources and Environment, Bangkok, Thailand. Preparation of plant extract: One of the effective methods in Ayurvedic and Thai traditional medicine is water decoction of herb (Farnsworth et al., 1992). In the present study, a decoction of P. chaba dried fruit was prepared. Dried fruit powder of P. chaba 500 grams was wrapped in a calico bag and put into a stainless boiler. Ten liters of water were added, then boiled for 3-4 h to extract the substance in P. chaba, and filtered when it cooled down. The residue from the filtration was boiled and filtered again with the same ratio. The filtrates were collected and evaporated in a rotary evaporator until concentrated. P. chaba water extract was stored at -20 C after preparation. Experimental animals: Male and female Sprague-Dawley rats, weighing g were obtained from the National Laboratory Animal Center, Nakorn Pathom, Thailand. Animals were randomly assigned to control and treated groups. They were housed under standard environmental conditions of temperature at 24 ± 1 C under a 12 h dark-light cycle, and allowed free access to drinking water and standard pellet diet. Rats were acclimated to holding facilities for 1 week prior to dosing. All experimental protocols were approved by the Animal Ethics Committee of Faculty of Medicine, Thammasat University, Pathumthani, Thailand (No. 0004/2005). Acute toxicity: Acute toxicity test was performed according to the World Health Organization (WHO) guideline (WHO 2000) and the Organization of Economic Co-operation and Development (OECD) guideline for testing of chemicals (OECD 2001). Five rats per sex were administrated a single oral dose of 5,000 mg/kg body weight while the control group received water vehicle. Body weight, signs of toxicity and mortality were observed after the administration at the first, second, fourth and sixth hour and once daily for next 14 days. On the 15 th day, all rats were kept fasted for h, and then sacrificed for necropsy examination. The internal organs were excised and weighed. The gross pathological observations of the tissues were performed. Subchronic toxicity: According to WHO guideline (WHO 2000) and the OECD (OECD 1981), rats were divided into 5 groups of 20 animals (10 male and 10 female). According to Ayurvedic medicine, 500 mg to 1 g of powdered fruits are used for treatment of the several symptoms including anti-diarrheal, carminative, etc. In the present study, the doses of P. chaba water extract were 300, 600 and 1,200 mg/kg/day, which are equivalent to times of the normal human dose (10-20 mg/kg). The extracts were dissolved in distilled water and orally given to each group of rats daily for 90 days, while the control group received the water vehicle. In order to assess reversibility effect, the extract at the dose of 1,200 mg/kg was given once daily to the fifth group of rats for 90 days, and kept for another 28 days post treatment (satellite group). Toxic manifestations such as signs of toxicity, mortality and the body weight changes were monitored daily. At the end of the study, all animals were fasted for h and then anesthetized with intraperitoneal injection of pentobarbital sodium at a dose of 50 mg/kg on day 91 st and 118 th (satellite groups). Blood samples for hematological and blood chemical analyses were taken from common carotid artery. All rats were sacrificed after the blood collection. The internal organs and some tissues were weighed to determine relative organs weights, and observed for gross lesions. All tissues were preserved in 10% neutral buffered formaldehyde solution for histopathological examination. Statistical analysis: Results were expressed as mean ± standard error of mean (S.E.M.). Statistical significance was determined by one-way analysis of variance (ANOVA) and post hoc least-significant difference (LSD) test. The data obtained from acute toxicity studies were analyzed using Student s paired t-test. P values less than 0.05 were considered significant. Results Acute Toxicity: The water extract from fruits of P. chaba at a single dose of 5000 mg/kg was orally given to rats. Neither sign of toxicity nor death of rats was observed during the 14 days of the experimental period. Toxicity evaluation was further carried out by observing both body weight gain and internal organ weight. In male rats, P. chaba extract caused a significant difference in body weight gain when compared with their control group. In addition, the liver and testis weight were slightly difference from the control group (p<0.05). 30

3 Acute and subchronic toxicity of Piper chaba Furthermore, gross examinations of the internal organs of treated rats revealed no pathological abnormality as compared with those of the control (data not shown). Subchronic toxicity: Neither changes in animal behaviors nor toxic signs were detected in the treatment rats. The body weight and body weight gain on day 90 of female and male treatment groups were significantly lower than those of the control group (Table 1). As shown in Table 2, the female treatment group with the extract at the dose of 300 mg/kg/day had the heart and kidney weight significantly lower than the control. The dose of 600 mg/kg caused a significant decrease in the weights of lung, heart and kidney. At the dose of 1,200 mg/kg, a significant lower of kidney weight was detected. Moreover, the satellite female group showed a significant decline in the kidney and ovary weights when compared with the control (p<0.05). In the male group (Table 3), the weights of heart, spleen, and kidney were significantly decreased in the group treated with 300 mg/kg/day, while those of lung, heart, spleen, kidney and testis decreased in the treatment group with 600 mg/kg/day. At the dose of 1,200 mg/kg/day, a significant weight decrease was found not only in lung, heart and liver, but also spleen and kidney as compared with those of the controls. The weight of heart was significantly changed in the male satellite group. Necropsy and histopathology examinations were further confirm whether or not the organs or tissue had been damaged. The results showed no macroscopic or microscopic changes in the internal organs of any of the treatment rats. Table 4 listed the hematological values of female and male rats. In the female treatment group with 300 mg/kg/day, mean corpuscular volume (MCV) was significantly higher than the control values. At the dose of 600 mg/kg, a significant weight decrease was found in mean corpuscular hemoglobin concentration (MCHC). In the satellite female and 300 mg/kg male treatment group, a slight but significant different of MCV and MCHC were shown. The differential white blood cell count values of female and male treated groups are shown in Table 5. A significant decrease in monocyte was observed in the female treatment with 300 mg/kg/day. In the female satellite group, the monocyte and eosinophil were significantly changed from the control group. In the male satellite rats, eosinophil was significantly decreased from the control values. Blood chemistry values of the female and male rats are summarized in Table 6. The data indicates a significant increase in total protein and albumin in the female satellite rats. Moreover, the concentrations of alkaline phosphatase (ALP) was significantly decreased as compared with those of the controls (p<0.05). In addition, the results of the histopathological assessment showed no significant histopathological change in the internal, especially vital, organs. Table 1. Body weights of rats in the subchronic toxicity test of the water extract from fruit of P. chaba. Body weight (g) Day 0 Day 90 Day 118 Weight gain on day 90 Female Control P. chaba 300 mg/kg * P. chaba 600 mg/kg * P. chaba 1,200 mg/kg a * P. chaba 1,200 mg/kg b Male Control P. chaba 300 mg/kg * * P. chaba 600 mg/kg * * P. chaba 1,200 mg/kg a * * P. chaba 1,200 mg/kg b * *. 31

4 Jaijoy et al Table 2. Organ weights of female rats in the subchronic toxicity test of the water extract from fruit of P. chaba. Dose Organ weight (g) mg/kg Lung Heart Liver Spleen Adrenal Kidney Ovary gland * * * * * ,200 a * ,200 b * *. Table 3. Organ weights of male rats in the subchronic toxicity test of the water extract from fruit of P. chaba. Dose Organ weight (g) mg/kg Lung Heart Liver Spleen Adrenal Kidney Testis gland * * * * * * * * 1,200 a * * * * * ,200 b * Table 4. Hematological values of female and male rats in the subchronic toxicity test of the water extract form fruit of P. chaba. Dose Red blood cell Hemoglobin Hematocrit MCV MCH MCHC Platelet (mg/kg) (x10 6 /µl) (g/dl) (fl) (pg) (g/dl) (x10 5 /µl) * * ,200 a ,200 b * * * * ,200 a ,200 b

5 Acute and subchronic toxicity of Piper chaba Table 5. Differential white blood cell count values of female and male rats in the subchronic toxicity test of the water extract from fruit of P. chaba. Group Dose (mg/kg) White blood cell (x10 3 /µl) Neutrophil Lymphocyte Monocyte Eosinophil Basophil Female Control P. chaba * ,200 a ,200 b * * Male Control P. chaba ,200 a ,200 b * Table 6. Clinical blood chemistry values of female and male rats in the subchronic toxicity test of the water extract from fruit of P. chaba. Female Glucose mg dl BUN mg dl Creatinine mg dl Total protein g dl Albumin g dl Total bilirubin mg dl Direct bilirubin mg dl SGOT U l SGPT U l ALP U l Male Glucose mg dl BUN mg dl Creatinine mg dl Total protein g dl Albumin g dl Total bilirubin mg dl Direct bilirubin mg dl SGOT U l SGPT U l ALP U l Control P. chaba (mg/kg),200 a,200 b Discussion In acute toxicity study, P. chaba extract at a single dose of 5,000 mg/kg did not show any toxicity signs (body weight, internal organ weight, and general behaviors). The results suggest that P. chaba extract is practically not toxic after an acute exposure in rats. Commonly, P. chaba has been used over long time periods; little toxicity information is available regarding safety following repeated exposure. From the evaluation of its subchronic toxicity at the doses of 300, 600 and 1,200 mg/kg/day for 90 days, both female and male rats treated doses presented no signs of behavior changes and toxic signs as shown by the normal appearance of respiration pattern, color of body surfaces, frequency and nature of movement, both involuntary and voluntary (Chan et al., 1982; Auletta 2002). The differences in body 33

6 Jaijoy et al weight and body weight gain may have resulted from physiological variation in rats such as food intake, and metabolism. Furthermore, neither morbidity nor disease was observed during the entire experimentation period. In hematological examinations, the significant changes are within normal ranges (Feldman et al., 2000; Inala et al., 2002). Likewise, the significant changes of blood chemical values fall within the normal ranges (Caisey & King 1980; Levine 2002; Angkhasirisap et al., 2002). Besides, the physical examination during the experimental period indicated that all animals are healthy. In the last experiment, necropsy and histopathological examinations were performed to further confirm whether or not the internal organs or tissues had been damaged. The results showed no macroscopic or microscopic changes in these internal organs or tissues in any treated rats. Thus, these results indicated the healthy status of liver and kidney in the treatment rats. In summary, the water extract from the fruit of P. chaba administered orally did not cause acute and subchronic toxicities in female or male rats. A chronic toxicity study should be further carried out to assess the long-term safety of the extract. Acknowledgements The authors are thankful to the Department for Development of Thai Traditional and Alternative Medicine, Ministry of Public Health, Bangkok, Thailand, and Dr. Somboon Kietinun for financial support. Deep appreciation is especially extended to the National Park, Wildlife and Plant Conservation Department, Ministry of Natural Resources and Environment, Bangkok, Thailand, for the plant material identification. Bibliography Angkhasirisap W, Inala P, Sirimontaporn A, Inpunkaew R, Rungrojejinda K, Kengkoom K, Ratanasak W, Lawson BD Blood chemistry profiles of outbred Sprague-Dawley rat in The Facility of National Laboratory Animal Centre. 28th Congress on Science and Technology of Thailand. Auletta CS Acute, Subchronic and Chronic Toxicology. In: Derelanko MJ, Hollinger MA, editors. Handbook of Toxicology. USA: CRC Press Inc. p Bailey SA, Zidell RH Perry RW Relationships between organ weight and body/brain weight in the rat: what is the best analytical endpoint? Toxicol Pathol 32(4): Caisey JD, King DJ Clinical chemical values for some common laboratory animals. Clin Chem 26: Chan PK, O Hara GP, Hayes AW Principles and methods for acute and subchronic toxicity. In: Hayes AW, editor. Principles and Methods of Toxicology. New York: Raven Press. p Devan P, Bani S, Suri KA, Satti NK, Qazi GN Immunomodulation exhibited by piperinic acid through suppression of proinflammatory cytokines. Int Immunopharmacol 7: Inala P, Sirimontaporn A, Inpunkaew R, Rungrojejinda K, Kengkoom K, Ratanasak W, Buripakdi Lawson D Hematological analysis of outbred Sprague-Dawley rat in The Facility of National Laboratory Animal Centre. 28th Congress on Science and Technology of Thailand. Lee SW, Kim YK, Kim K, Lee HS, Choi JH, Lee WS, Jun CD, Park JH, Lee JM, Rho Mc Alkamides from the fruits of Perper longum and Piper nigrum displaying potent cell adhesion inhibition. Bioorg Med Chem Lett 18: Matsuda H, Ninomiya K, Morikawa T, Yasuda D, Yamaguchi I, Yoshikawa M Protective effects of amide constituents from the fruit of Piper chaba on D-galactosamine/TNF-alpha-induced cell death in mouse hepatocytes. Bioorg Med Chem Lett 18(6): Morikawa T, Matsuda H, Yamaguchi I, Pongpiriyadacha Y, Yoshikawa M New amides and gastroprotective constituents from the fruit of Piper chaba. Planta Med 70(2): Park BS, Sonb DJ, Park YH, Kim TW, Lee SE Antiplatelet effects of acidamides isolated from the fruits of Piper longum L. Phytomedicine 14: Saralamp P, Chuakul W, Temsiririrlkul R, Clayton T Medicinal Plant in Thailand Volume I. Department of Pharmaceutical Botany, Faculty of Pharmacy, Mahidol University, Thailand. Selvendiran K, Singh JPV, Sakthisekaran D In vivo effect of piperine on serum and tissue glycoprotein levels in benzo(a)pyrene induced lung carcinogenesis in Swiss albino mice. Pulmonary Pharmacology & Therapeutics 19: Sunila ES, Kuttan G Immunomodulatory and antitumor activity of Piper longum Linn. And piperine. J Ethnopharmacol 90(2-3): Sunila ES, Kuttan G. Piper longum inhibits VEGF and proinflammatory cytokines and tumor-induced angiogenesis in C57BL/6 mice. Int Immunopharmacol. 2006; 6(5): Taufiq-Ur-Rahman M, Shilpi JA, Ahmed M, Faiz Hossain C Preliminary pharmacological studies on Piper chaba stem bark. J Ethnopharmacol 99(2): The Organization of Economic Co-operation and Development (OECD) The OECD guideline for testing of chemical: 408 Subchronic Oral Toxicity-Rodent: 90-day Study. France. 34

7 Acute and subchronic toxicity of Piper chaba The Organization of Economic Co-operation and Development (OECD) The OECD guideline for testing of chemical: 420 Acute Oral Toxicity. France. World Health Organization (WHO) General guidelines for methodologies on research and evaluation of traditional medicine. Switzerland. Wu S, Sun C, Pei S, Lu Y, Pan Y Preparative isolation and purification of amides from the fruits of Piper longum L. by upright counter-current chromatography and reversed-phase liquid chromatography. J Chromatogr A 1040: Zhang H, Matsuda H, Nakamura S, Yoshikawa M Effects of amide constituents from pepper on adipogenesis in 3T3-L1 cells. Bioorg Med Chem Lett 18: