Acute and Sub Chronic Toxicity of Mucuna pruriens, Cinnamomum zeylanicum, Myristica fragrans and their Effects on Hematological Parameters

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1 Australian Journal of Basic and Applied Sciences, 7(8): , 2013 ISSN Acute and Sub Chronic Toxicity of Mucuna pruriens, Cinnamomum zeylanicum, Myristica fragrans and their Effects on Hematological Parameters 1,2 Rafeeq Alam Khan, 2 Muhammad Arif, 3 Bushra Sherwani, 4 Mansoor Ahmed 1 College of Medicine-Jeddah, Department of Basic Medical Sciences, King Saud bin AbdulAziz University for Health Sciences, Kingdom of Saudi Arabia. 2 Department of Pharmacology, Faculty of Pharmacy, University of Karachi. 3 Islam Medical College, Sialkot, Pakistan. 4 Department of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Karachi, Karachi 75270; Pakistan. Abstarct: Use of herbal medicines prevails in rural and urban areas of the world; especially for treating ailments which allopathic medicines fail to treat. Present study was undertaken for the pharmacological and toxicological evaluation of Mucuna pruriens, Cinnamomum zeylanicum, and Myristica fragrans, alone and in combination. The acute toxicity was studied on mice and sub chronic toxicity and hematological parameters were evaluated on rabbits. The mice and rabbits were divided into control and treated groups and kept under observation, for specified time period. To determine acute toxicity mice were kept under observation up to 24 hours after dosing, for sub chronic gross toxicities and hematological parameters rabbits were treated daily for 90 days. The sub chronic gross toxicities were assessed on weekly basis and hematological parameters were tested on 91 st day after completion of dosing period. Statistical analysis was done using one-way analysis of variance (ANOVA) followed by post hoc. LD 50 in all the treated groups was found to be greater than 1600 mg/kg body weight and there were no significant sub chronic gross toxicities in most of the rabbits. Moreover no significant difference was found in most of the hematological parameters of treated groups as compared to control. The drugs were found to be safe in the doses used in this study both on mice and rabbits. However further preclinical and clinical studies are required to establish the safety of these drugs in herbal formulations. Key words: Toxicity, Hematology, Herbal drugs. INTRODUCTION Natural plant products have been used for therapeutic purposes since the time immemorial; and their use is of a greater demand nowadays (Calixto, 2000). Majority of the users rely on herbal medicines for health care because the other treatment options available are more expensive and often thought to be associated with serious adverse effects. Most of these plants have been ingested indiscriminately by many local populations for the management of various disease states without knowing how the relief is brought about or having adequate information on the safety and toxicity associated with their use. Hence for proper guidance of the populace, especially users of these natural products, there is need to document the safety and toxicity profile of these medicinal plants (Agbaje et al, 2009). In recent years, numerous efforts have been made to investigate the mechanism of action of herbal drugs (Hu et al, 2009; Yang et al, 2009; Yao et al, 2009), however majority of the research has been focused on the identification of the biological functions and active components of individual constituents. There are relatively few studies that consider the effects of interaction among herbs within a formulation. The use of highly complex multi component formulations increases the possibility of multiple biological interactions between herbal components and requires extensive preclinical systematic evaluation of safety, efficacy and mechanism of these multi-herbal formulas. (Yu et al, 2010). Use of herbal medicines is equally prevailed in rural and urban areas of the world especially for treating ailments which allopathic medicines fail to treat. There are many herbal products available in the market for the treatment of different diseases. These products contain varying number of herbal drugs in combinations, however the scientific basis for the use of these combinations has not been evaluated. Moreover, there is a lot of variation in the doses of individual herbal drugs among different herbal formulations. Hence comparative pharmacology, toxicology and drug-interactions of all such herbal drugs in every possible combination with every possible dose must be evaluated; otherwise their safety, efficacy and cost effectiveness cannot be justified. Mucuna pruriens, Cinnamomum zeylanicum, and Myristica fragrans are among the important constituents of different herbal formulations used for the treatment of male infertility; however the scientific basis for their use, especially in combination is scanty. This study has been undertaken for the pharmacological and toxicological evaluation of Mucuna pruriens, Cinnamomum zeylanicum, and Myristica fragrans, alone and in combination. Corresponding Author: Rafeeq Alam Khan, College of Medicine-Jeddah, Department of Basic Medical Sciences, King Saud bin AbdulAziz University for Health Sciences, Kingdom of Saudi Arabia. 641

2 MATERIALS AND METHODS Selection of Animals: In the present study mice and rabbits were selected as experimental animals. The mice were used to determine acute toxicity, while rabbits were used for sub chronic gross toxicities and hematological studies. The rabbits were selected as experimental animals because of several reasons like: physiological changes produced in rabbits are relatively similar as observed in humans, large quantity of blood samples can be obtained with ease, rabbits are easily available, easy to handle and economical (Feroz et al., 2010). The study was conducted on fifty healthy white male rabbits; weight ranging from gram. Animal were accommodated in separate cages, under controlled conditions of temperature (22 ± 2 C) and humidity (50-60%) with alternate periods of light and dark of 12 hour. All animals were given green leafy diet and water regularly. Animals were randomly and uniformly divided into five groups. Each group consisted of 10 animals; one group was treated as a control while the other remaining groups received the drugs singly and in combination. Before administration of the drugs, visible changes in health of these animals were observed during the conditioning period under the laboratory environments for a week; specifically noticing loss of hair, diarrhea, edema, ulceration and lack of activity. Dosing: Crude drugs were crushed to form fine powder and administered through oral route after forming suspension in 0.5% carboxy methylcellulose. Drugs were administered on daily basis for a period of 90 days in the doses given below: Mucuna pruriens: 70 mg/kg body weight Cinnamomum zeylanicum: 30 mg /kg body weight Myristica fragrans: 20 mg /kg body weight Groups of Animals: Rabbits were divided into five groups, each containing 10 animals in the following pattern: Group A: Control given 0.5% carboxy methylcellulose Group B: Mucuna pruriens Group C: Cinnamomum zeylanicum Group D: Myristica fragrans Group E: Combination of Mucuna pruriens-cinnamomum zeylanicum-myristica fragrans Acute Oral Toxicity: LD 50 was carried out on male mice weighing gm bred at animal house of Department of Pharmacology, University of Karachi. Before administration of drugs, visible health of these animals was observed during the conditioning period under the laboratory environments for a week. The animals were maintained under constant environmental conditions 22 2 o C. All animals were given standard diet prepared in the laboratory and water ad libitum. The LD 50 (dose of the drug that caused death in 50% of the animals within 24 hrs) was calculated using the method described by Lorke (1983). Drugs were given in doses of 10, 100, 1000 mg / kg orally to groups of three mice at each dose. Upon mortality in each group after 24 hrs, 4 mice were administered different dose of drugs in order to obtain the least and most toxic values and LD 50 was calculated by geometric mean of these values. Assessment of Sub Chronic Gross Toxicities: The gross toxicities were assessed on weekly basis for 90 days specifically noticing skin ulceration, loss of activity, hematuria, loss of hair, vomiting, diarrhea, edema, salivation, tremor, aggressive behavior, and average body weight variation. Mortality Rate: The number of animals died during the total period of experiment was also noted and percentage mortality was calculated. Hematological Parameters: Blood sample of about 5 cc were collected from rabbits through cardiac puncture technique after completion of dosing period on 91 st day to perform the laboratory tests for hematological parameters. Blood was transferred immediately from syringe to the test tubes with anticoagulant, so that blood does not clot for the hematological analysis. Laboratory tests for hematological parameters were performed on Sysmex automated hematology analyzer kx

3 Statistical Analysis: All values were compared with control by taking mean and standard error to the mean using one-way analysis of variance (ANOVA) followed by post hoc. The data was reported as mean ± standard error to the mean with 95% confidence interval and p-values were observed. p < 0.05, was considered as significant and < 0.01, as highly significant. Results: Acute Oral Toxicity: LD 50 in all the groups of animals was found to be greater than 1600 mg/kg body weight, since all the animals tolerated the doses of all 3 drugs up to 1600 mg/kg. Assessment of Gross Toxicities: The gross toxicities in animals were assessed on weekly basis during the dosing period of 90 days specifically noticing skin ulceration, hematuria, loss of hair, loss of activity, vomiting, diarrhea, edema, salivation, tremor and aggressive behavior and average weight variation, in all animal groups kept on different drugs as shown in Table 1. Table 1: Comparison of Gross Toxicities in Different Groups Against Control. Toxicities Time Interval Control MP CZ MF Combination (Weeks) Skin Ulceration 1st NIL NIL NIL NIL NIL 6th 2/10 NIL NIL NIL NIL 7th 2/10 1/10 NIL 1/10 NIL 8th 2/10 1/10 NIL 1/10 1/10 9th 2/10 1/10 NIL 1/10 2/10 10th 2/10 1/10 NIL 1/10 2/10 11th 2/10 1/10 NIL 1/10 2/10 12th 2/10 1/10 NIL 1/10 2/10 Hematuria 1st NIL NIL NIL NIL NIL Loss of Hair 1st NIL NIL NIL NIL NIL 5th 1/10 NIL NIL NIL 1/10 6th 1/10 NIL NIL NIL 1/10 7th 1/10 NIL NIL NIL 1/10 8th 1/10 NIL NIL NIL 1/10 Loss of 1st NIL NIL NIL NIL NIL Activity 643

4 Table 1 (Continued): Comparison of Gross Toxicities in Different Groups Against Control. Toxicities Time Interval Control MP CZ MF Combination (Weeks) Vomiting 1st NIL NIL NIL NIL NIL Diarrhea 1st NIL NIL NIL NIL NIL Edema 1st NIL NIL NIL NIL NIL Salivation 1st NIL NIL NIL NIL NIL Table 1 (Continued): Comparison of Gross Toxicities in Different Groups Against Control. Toxicities Time Interval Control MP CZ MF Combination (Weeks) Tremor 1st NIL NIL NIL NIL NIL Aggressive 1st NIL NIL NIL NIL NIL behavior 3rd NIL NIL NIL 1/10 NIL 4th NIL NIL NIL 1/10 NIL 5th NIL NIL NIL 1/10 NIL 644

5 Average Weight Variation (g) 6th NIL NIL NIL 1/10 NIL 7th NIL NIL NIL 1/10 NIL 8th NIL NIL NIL 1/10 NIL 9th NIL NIL NIL 1/10 NIL 10th NIL NIL NIL 1/10 NIL 1st nd rd th th th th th th th th th MP = Mucuna pruriens, CZ = Cinnamomum zeylanicum, MF = Myristica fragrans, Combination=Mucuna pruriens+cinnamomum zeylanicum+myristica fragrans Two animals in control group showed skin ulceration during 6 th to 12 th week of study. One animal each in Mucuna pruriens and Myristica fragrans groups showed skin ulceration during 7 th to 12 th weeks of study period. In combination group one animal showed skin ulceration from 7 th week and one animal from 8 th week to 12 th week of study period. One animal in control group and one animal in combination group showed loss of hair between 5 th to 8 th weeks of study. No animal in any group showed hematuria, loss of activity, vomiting, diarrhea, edema, salivation, and tremor. One animal in Myristica fragrans group showed aggressive behavior from 3 rd to 10 th week of study. There has been an overall insignificant increase in average body weight of all animals at the end of study period however maximum increase in body weight was in the animals received Cinnamomum zeylanicum, which was g as compared to control group (Table 1). Mortality Rate: Table 2 reveals mortality rate between animals groups against control following 90 days of drugs administration. No death was observed in control, Mucuna pruriens, Cinnamomum zeylanicum, and Myristica fragrans groups; however one animal died in combination group. Table 2: Comparison of Mortality Rate in Control and Treated Groups. Animal groups Mortality rate * Control 0/10 (00.00) Mucuna pruriens 0/10 (00.00) Cinnamomum zeylanicum 0/10 (00.00) Myristica fragrans 0/10 (00.00) Combination 1/10 (10.00 %) *Number of animals expired /total number of animals (Percentage) Hematological Parameters: Tables 3 reveals the comparison of hematological parameters i.e. hemoglobin concentration (g/dl of blood), red blood cells, white blood cells and platelets counts (per cubic millimeter of blood) following 90 days administration of different drugs against control. There was insignificant decrease in hemoglobin concentration in all the treated groups as compared to control. Similarly all treated groups showed insignificant decrease in red blood cells count as compared to control, however all these changes were in normal physiological limits. There was also insignificant decrease in white blood cells count in combination group as compared to control. Whereas in other treated groups increase was insignificant as compared to the control. However there was significant increase in white blood cells count in Myristica fragrans group. There was insignificant decrease in platelets count of Cinnamomum zeylanicum and combination groups as compared to control, but insignificant increase in Myristica fragrans and Mucuna pruriens groups as compared to control. However all these changes were in normal physiological limit. 645

6 Table 3: Comparison of Hematological Parameters Following 90 Days Administration of Drugs in Different Groups Against Control. PARAMETERS ANIMAL GROUPS Control MP CZ MF Combination Hemoglobin (g /dl) Red blood cells (x10 6 / mm 3 ) White blood cells * (x 10 3 / mm 3 ) Platelet (x10 3 / mm 3 ) n = 10 Average values + S.E.M. p< 0.05 significant as compared to control MP = Mucuna pruriens CZ = Cinnamomum zeylanicum MF = Myristica fragrans, Combination =Mucuna pruriens+cinnamomum zeylanicum+myristica fragrans Discussion: There are many herbal formulations available throughout the world used to treat male infertility. These formulations contain variety of herbs with varying doses but their safety, efficacy and cost-effectiveness has not been assessed on scientific basis. Drugs used in present study i.e. Mucuna pruriens, Cinnamomum zeylanicum, Myristica fragrans are among the important herbs used in different formulations for the treatment of male infertility. It is well established that many herbal drugs have various adverse effects and shows drug interactions (Dennehy and Tsourounis, 2012). There is a lack of scientific studies to compare the pharmacology, toxicology and drug-interactions of the individual herbal drugs and their different formulations. This is the novel study; carried out to evaluate the pharmacological and toxicological effects of three individual herbal drugs, Mucuna pruriens, Cinnamomum zeylanicum and Myristica fragrans and their combination. LD 50 in all groups of animals was found to be greater than 1600 mg/kg body weight. All the animals tolerated the doses of all 3 drugs up to 1600 mg/kg body weight. So these drugs showed wide margin of safety. In this study the doses used were quite safe, however another study of Mucuna pruriens in which hot water extract was used in male rats showed LD 50 at 1300 mg / kg body weight (Chukwadi et al, 2011). There are studies in which Mucuna pruriens was given up to 10 g/kg orally but no deaths and visible signs of toxicity were observed (Akindele and Busayo, 2011). Similarly ethanol extract of Cinnamomum zeylanicum was also tested at 0.5, 1.0, and 3 g/kg and results showed no acute toxicity in mice (Shah et al, 1998). Cinnamomum zeylanicum and Myristica fragrans are among the commonly used spices. One grated nutmeg (Myristica fragrans) is approximately one tablespoon and weighs about 6-7 g. Symptoms of toxicity do not usually develop following the ingestion of less than 10 g nutmeg (Forrest and Heacock, 1972). There is a lot of variation in the doses mentioned in Myristica fragrans intoxication. Variation in toxicity may result from the loss of essential oils from the ground nutmeg. No feature of toxicity, like nausea, vomiting, restlessness, agitation, tremor, ataxia, or anticholinergic signs (urinary retention, constipation, or mydriasis) has been noted in present study. Symptoms reported in literature usually begin about 3-6 hours after ingestion and resolve by hours (Forrest and Heacock, 1972; Sangalli and Chiang, 2000). Most of the animals in all treated groups showed no feature of sub chronic gross toxicities, however there has been an insignificant increase in average body weight of animals received Cinnamomum zeylanicum as compared to control group, which was in accordance to another study by Shah et al, There has been also an insignificant increase in average body weight of animals received Mucuna pruriens as compared to control, however, rats fed on raw mucuna bean flour led to significant (p < 0.05) weight loss after 28 days study period (Ngatchic et al, 2013). This discrepancy may be because we used the Mucuna pruriens in therapeutic doses and not as a diet. There was insignificant decrease in hemoglobin concentrations in all treated groups but it was in normal physiological range. Similarly there was also insignificant decrease of red blood cells counts in all treated groups but again in normal physiological range. These results are compatible with another study reported to show no significant difference in packed cell volume and hemoglobin concentration between Mucuna pruriens treated and control groups of male rats after 6 weeks of study period (Chukwadi et al, 2011). Results of present study showed insignificant decrease in white blood cells count in combination group as compared to control, however in other treated groups there was increase in white blood cells count, but mostly these changes were in normal physiological range. However, there was significant (p < 0.05) increase in white blood cells count in Myristica fragrans group as compared to control. In combination group decrease in white blood cells count may be due to neutralizing effects of these drugs to one another. In another study Mucuna pruriens seed extract produced a significant (p < 0.05) increase in white blood cells count in comparison with control rats after 6 weeks (Chukwadi et al, 2011). The difference in significance may be due to differences in 646

7 doses, animal species, and study period. Conclusion: Results of present did not reveal any acute and sub chronic toxicity of Mucuna pruriens, Cinnamomum zeylanicum, Myristica fragrans. These drugs are usually used in herbal formulations to treat infertility, hence found to have a wide margin of safety in acute toxicity and sub chronic gross toxicities. There is no significant difference found in most of the hematological parameters of the treated groups as compared to control. These drugs seem to be safe alone as well as in combination, however further preclinical and clinical studies are required to establish the safety of these drugs in different herbal formulations. REFERENCES Agbaje, E.O., A.A. Adeneye, A.O. Daramola, Biochemical and toxicological studies of aqueous extract of Syzigium aromaticum (L.) Merr. & Perry Myrtaceae) in rodents. Afr J Trad CAM., 6(3): Akindele, A.J., F.I. Busayo, Effects of the hydroethanolic extract of Mucuna pruriens (L.) DC (Fabaceae) on haematological profile in normal and haloperidol treated rats. Nig Q J Hosp Med., 21(2): Calixto, J.B., Efficacy, safety, quality control, marketing and regulatory guidelines for herbal medicines (phytotherapeutic agents). Braz J Med Biol Res., 33(2): Chukwudi, N.H., O. Simeon, A.J. Chinyere, Analysis of some biochemical and haematological parameters for Mucuna pruriens (DC) seed powder in male rats. Pak J Pharm Sci., 24(4): Dennehy, C.E. and C. Tsourounis, Dietary Supplements & Herbal Medications. In: Basic and Clinical Pharmacology, 12th edi. (Katzung BG ed.), pp: Feroz, Z., R.A. Khan, T. Mirza and S. Afroz,2010. Adverse effects of cumulative administration of antiepileptic, anti-hypertensive, anti-diabetic and anti-arrhythmic drugs on renal and cardiac parameters. Int J Med Res., 1(1): Forrest, J.E., R.A. Heacock, Nutmeg and mace. The psychotropic species from Myristica fragrans. Lloydia, 35: Hu, Y., X. Chen, H. Duan, X. Mu, Chinese herbal medicinal ingredients inhibit secretion of IL-6, IL- 8, E-selectin and TXB(2) in LPS-induced rat intestinal microvascular endothelial cells. Immunopharmacol Immunotoxicol (April Epub ahead of print]. Lorke, D., A new approach to practical acute toxicity testing. Archive of toxicology, 54: Ngatchic, J.T., S.D. Sokeng, N.Y. Njintang, T. Maoundombaye, J. Oben, C.M. Mbofung, Evaluation of some selected blood parameters and histopathology of liver and kidney of rats fed protein-substituted mucuna flour and derived protein rich product. Food Chem Toxicol., 57: Doi: /j.fct Epub 2013 Mar 6. Sangalli, B.C., W. Chiang, Toxicology of nutmeg abuse. Clin Toxicol., 38: Shah, A.H., A.H. Al-Shareef, A.M. Ageel, S. Qureshi, Toxicity studies in mice of common spices, Cinnamomum zeylanicum bark and Piper longum fruits. Plant Foods Hum Nutr., 52(3): Yang, M., C. Xiao, Q. Wu, M. Niu, Q. Yao, et al., Anti-inflammatory effect of Sanshuibaihu decoction may be associated with nuclear factor-kappab and p38 MAPKalpha in collagen-induced arthritis in rat. J Ethnopharmacol., 127: Yao, C.J., C.M. Yang, S.E. Chuang, J.L. Yan, C.Y. Liu, et al., Targeting PML- RAR{alpha} and Oncogenic Signaling Pathways by Chinese Herbal Mixture Tien-Hsien Liquid in Acute Promyelocytic Leukemia NB4 Cells. Evid Based Complement Alternat Med. Nov 23 [Epub ahead of print]. Yu, Y.B., L. Dosanjh, L. Lao, M. Tan, B.S. Shim, Y. Luo, Cinnamomum cassia bark in two herbal formulas increases life span in Caenorhabditis elegans via insulin signaling and stress response pathways. PLoS One, 5(2): e