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 Table of Contents  
ORIGINAL ARTICLE
Year : 2017  |  Volume : 11  |  Issue : 1  |  Page : 48-57

Safety studies of homoeopathic drugs in acute, sub-acute and chronic toxicity in rats


1 Department of Pharmacology, All Institute of Medical Sciences, New Delhi, India
2 Central Council for Research in Homeopathy, Ministry of AYUSH, Government of , New Delhi, India

Date of Web Publication23-Feb-2017

Correspondence Address:
Surender Singh
Department of Pharmacology, All India Institute of Medical Sciences, Ansari Nagar, New Delhi - 110 029
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ijrh.ijrh_68_16

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  Abstract 

Background: Homoeopathic drugs are frequently recommended in day to day life as therapeutic agents by homoeopathic practitioners. However, safety of homoeopathic drugs remains a challenge because of the high variability of chemical components involved. Aim: The objective of the present study was to investigate the acute, subacute, and chronic oral toxicity of different homoeopathic drugs (Ferrum phosphoricum 3X, Ferrum phosphoricum 6X, Calcarea phosphoricum 6X, and Magnesium phosphoricum 6X) in experimental models. Materials and Methods: In acute oral toxicity study, homoeopathic drugs were administered orally at 2000mg/kg body weight, and animals were observed for toxic symptoms till 10 days as per the OECD guidelines. For subacute and chronic toxicity study, homoeopathic drugs were administered for 28 and 180 days, respectively, as per the OECD guidelines. At the end of 28 and 180 days, the animals were sacrificed and toxicity parameters were assessed. Histopathological evaluation of different organs was also performed to assess any toxicity. Results: In acute toxicity study, no mortality was found at a dose of 2000 mg/kg which indicates that oral LD50of homoeopathic drugs were more than 2000 mg/kg. The administration of drugs at a dose of 70 mg/kg body weight for 28 and 180 days did not produce any significant change in haematological and biochemical parameters of male and female rats as compared to normal control group. No pathological changes were observed in histology of various organs of treated rats as compared to normal control animals. Conclusion: These homoeopathic drugs are safe & produce no toxicity when administered for longer duration.

Keywords: Acute toxicity, Calcarea phosphoricum 6X, Chronic toxicity, Ferrum phosphoricum 3X, Ferrum phosphoricum 6X, Magnesium phosphoricum 6X, Subacute toxicity


How to cite this article:
Singh S, Kalra P, Karwasra R, Khurana A, Manchanda RK, Gupta YK. Safety studies of homoeopathic drugs in acute, sub-acute and chronic toxicity in rats. Indian J Res Homoeopathy 2017;11:48-57

How to cite this URL:
Singh S, Kalra P, Karwasra R, Khurana A, Manchanda RK, Gupta YK. Safety studies of homoeopathic drugs in acute, sub-acute and chronic toxicity in rats. Indian J Res Homoeopathy [serial online] 2017 [cited 2019 Nov 13];11:48-57. Available from: http://www.ijrh.org/text.asp?2017/11/1/48/200849


  Introduction Top


Homoeopathy is practiced in about eighty countries either as independent or as complementary drug to modern system. Homoeopathy is one of the most frequently used complementary and alternative drugs, which uses highly diluted preparations prepared in a specific way unique to Homoeopathy. Homoeopathic preparations have been commonly used for the treatment of various ailments because they are economical, effective, and accessible.[1] About 70%–80% of the world population, particularly in the developing countries, relies on nonconventional drugs in their primary health care as reported by the World Health Organization.[2]

Ferrum phosphoricum is also known as Ferrum phosphoricum or iron phosphate and is prepared by amalgamating the minerals such as iron and phosphorus. It is formulated by blending three solutions - iron sulfate, sodium acetate, and phosphorus. The resultant product iron phosphate is pulverized using large amounts of sugar lactose (also known as milk sugar) to make it nontoxic. Ferrum phosphoricum is primarily used for beginning of inflammations. This remedy is most suitable for fevers, rheumatic pain, haemorrhage, nose bleed, headache, sore throat, etc.[3]Calcarea phosphoricum is prepared using white calcium phosphate precipitate which is filtered, diluted, dried, and triturated with lactose sugar. Calcium is found naturally within our bodies in our bones and teeth. Within Homoeopathy, Calcarea phosphoricum was discovered centuries ago and has been used to treat bone and tooth problems ever since. Calcarea phosphoricum is used for the treatment of pain and stiffness in arthritis, neck, and fractures. Calcarea phosphoricum works well if the teething process is slow or and the teeth that form are weak and may decay easily. It is also useful in headaches, heartburn, indigestion, and pain after eating. Magnesium phosphoricum (Magnesium phos) is found inside the cells of muscles, nerves, bones, brain, and spine. It is a great anti-spasmodic remedy in Homoeopathy. It is useful in cramping of muscles with radiating pain, neuralgic pains, toothache, vertigo, angina pectoris, and soreness of throat.[4] These three medicines are used in lower trituration as boichemic medicines.

It is believed that Homoeopathic formulations are safe without performing any safety study; however, there is no scientific rationale to assume that plants or other medicinal sources, their parts, or derived products, including those of long-standing popular use, are intrinsically safe or beneficial,[5] and it has been reported that many of the drugs used in Homoeopathy with approved pharmacological activity have been rejected as their safety profile is not evaluated, so they would require fewer and simpler preclinical and clinical studies for validation.[6] A scientifically carried out screening is, therefore, important to ascertain safety and efficacy of products used traditionally in Homoeopathy.

Therefore, the present study was carried out to evaluate the safety profile of these homoeopathic medicines i.e. as Ferrum phosphoricum 3X, Ferrum phosphoricum 6X, Calcarea phosphoricum 6X, Magnesium phosphoricum 6X which are used for treating various disorders.[7]


  Materials and Methods Top


Animals

The study was carried out in the Department of Pharmacology with the approval of the Institutional Animal Ethics Committee, All India Institute of Medical Sciences (AIIMS), New Delhi (698/IAEC/12). Adult Wistar albino rats (150–200 g) from the Central Animal Facility, AIIMS, were used in the study. Animals were housed under standard laboratory conditions at 25 ± 20°C, and humidity levels were in the range of 30%–70% in groups with free access to food and water ad libitum. They were acclimatized to the laboratory conditions for a period of 5 days before the study.

Drugs and chemicals

The homoeopathic drugs in the form of tablets were provided by the Central Council for Research in Homoeopathy, Ministry of AYUSH, New Delhi (manufactured by Kerala State Homoeopathic Corporate Pharmacy Limited, Kerala, India).

Acute toxicity study

Acute oral toxicity test was performed as per the OECD-425 guidelines (OECD, 2001). All the animals were randomly distributed into different treatment groups. Following the fasting period, the female Wistar rats (n = 5) were weighed, and the dose was calculated in reference to the body weight. For the main test, a single dose of 2000 mg/kg of each drug was administered to rats in the treatment groups by oral route on day 1. Groups 1, 2, 3, and 4 were given Ferrum phosphoricum 3X, Ferrum phosphoricum 6X, Calcarea phosphoricum 6X, and Magnesium phosphoricum 6X administered orally on day 1 and observed till 10 days, respectively. Food was provided to the rats approximately an hour after treatment. The animals were observed 30 min after dosing, followed by hourly observation for 8 h till 10 days.[8]

Subacute toxicity study

Evaluation of 28-day oral toxicity of homoeopathic drugs was carried out in 5 male and 5 female Wistar rats in each group according to the OECD guidelines for testing of chemicals - 407 (OECD, 2008). Group 1 was given normal saline and served as normal control; Groups 2, 3, 4, and 5 were given Ferrum phosphoricum 3X, Ferrum phosphoricum 6X, Calcarea phosphoricum 6X, and Magnesium phosphoricum 6X administered orally for 28 days, respectively. Body weight were recorded on days 0, 7, 14, 21, 28 and statistical analysis was carried out by one way ANOVA as compared to normal control. At the end of 28 days (subacute toxicity), rats were sacrificed by cervical dislocation. Blood samples were collected for haematological and biochemical analysis under diethyl ether anesthesia through the retro-orbital sinus. The liver, kidney, heart, brain, and ovaries also harvested immediately and organ weights measured. These organs were fixed in 10% formalin for histopathological examination.[9] Biochemical and haematological parameters were measured and analysis of all treatment groups (Ferrum phosphoricum 3X, Ferrum phosphoricum 6X, Calcarea phosphoricum 6X, Magnesium phosphoricum 6X) was carried out as compared to normal control.

Chronic toxicity study

Chronic toxicity tests determine toxicity from exposure for a substantial portion of a subject's life. They are similar to the subchronic tests except that they extend over a longer period of time (180 days) according to the OECD guidelines for testing of chemicals - 452 (OECD, 2008) Chronic toxocity study was conducted on in 5 male and 5 female Wistar rats in each grooup. Group 1 was given normal saline and served as normal control; Groups 2, 3, 4, and 5 were given Ferrum phosphoricum 3X, Ferrum phosphoricum 6X, Calcarea phosphoricum 6X, and Magnesium phosphoricum 6X administered orally for 180 days, respectively. The animals were observed daily for clinical signs of toxicity. The body weight, biochemical and hematological parameters were measured and analysis of all treatment groups (Ferrum phosphoricum 3X, Ferrum phosphoricum 6X, Calcarea phosphoricum 6X, Magnesium phosphoricum 6X) was carried out as compared to normal control.[10]

A flowchart has been added describing the detailed experimental protocol for all the treatment groups in acute, subacute, and chronic toxicity studies [Figure 1].
Figure 1: Flowchart describing the detailed experimental protocol for acute, subacute, and chronic oral toxicity studies

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  Results Top


Acute oral toxicity study

Acute oral toxicity of the homoeopathic drugs was determined using the limit test at 2000 mg/kg. All animals were observed for mortality for 10 days. All animals survived for 10 days as shown in [Table 1], indicating that 2000 mg/kg of homoeopathic drugs was considered safe in rats.
Table 1: Effect of administering different homoeopathic drugs on acute oral toxicity of Homeopathic drugs

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Subacute oral toxicity study (28 days)

Effect of administering different homoeopathic drugs on Wistar rats for 28 days

All the animals survived on regular administration of Homoeopathic drugs for 28 days. The results indicated that no significant changes were observed in body weight [Table 2], relative organ weight [Table 3], biochemical parameters [Table 4], and haematological parameters [Table 5] as compared to control. No histopathological changes were observed in the tested animals as compared to normal control.
Table 2: Effect of administering different Homoeopathic drugs on body weight of rat over a period of 28 days

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Table 3: Effect of administering different homoeopathic drugs on relative organ weight of Wistar rats on 28th day

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Table 4: Effect of administering different homoeopathic drugs on biochemical parameters (serum glutamic oxaloacetic transaminase, serum glutamate pyruvate transaminase, blood glucose, serum creatinine, triglycerides, and high-density lipoprotein) of Wistar rats on 28th day

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Table 5: Effect of administering different Homoeopathic drugs on hematological parameters of Wistar rats on 28th day

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Effect on body weight change

No significant changes in body weights of treated rats were observed when compared to untreated control groups. The body weight changes of control and treated groups are shown in [Table 2].

Effect on relative organ weight

After administration of homoeopathic drugs for 28 days, there were no significant changes observed in relative organ weight of different organs. Treatment with homoeopathic drugs do not cause any change in organ weight. No signs of gastric ulceration or erosion were observed. Relative organ weight of different organs in control and treated groups is presented in [Table 3].

Effect on biochemical parameters

After administration of homoeopathic drugs for 28 days, there were no significant changes observed in biochemical parameters (hepatic transaminase level, triglycerides level, high-density lipoprotein level, and serum creatinine level) as compared to control as shown in [Table 4].

Effect on haematological parameters

After administration of homoeopathic drugs for 28 days, there were no significant changes observed in haematological parameters (haemoglobin [Hb], red blood cell [RBC], white blood cell [WBC], platelets, blood glucose, and clotting and bleeding time) as compared to normal control animals. The effect of Homoeopathic drugs on the haematological parameters is presented in [Table 5].

Histopathological evaluation

No histopathological changes were observed in kidney, liver, brain, testis, ovary, and heart of various treated animals in comparison to normal control animals after administration of homoeopathic drugs for 28 days.

Chronic toxicity study (180 days)

Effect on body weight change

No significant changes in body weights of treated rats were observed when compared to untreated control groups. The body weight changes of control and treated groups are shown in [Table 6].
Table 6: Effect of administering different Homoeopathic drugs on body weight of rat over a period of 180 days

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Effect on relative organ weight

After administration of homoeopathic drugs for 180 days, there were no significant changes observed in relative organ weight of different organs. Treatment with homoeopathic drugs does not cause any change in organ weight. No signs of gastric ulceration or erosion were observed. Relative organ weight of different organs in normal control and treated groups is presented in [Table 7].
Table 7: Effect of administering different Homoeopathic drugs on relative organ weight of Wistar rats on 180th day

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Effect on biochemical parameters

After administration of Homeopathic drugs for 180 days, there were no significant changes observed in biochemical parameters (hepatic transaminase level, triglycerides level, high-density lipoprotein level, and serum creatinine level) as compared to normal control as shown in [Table 8].
Table 8: Effect of administering different Homoeopathic drugs on biochemical parameters (serum glutamic oxaloacetic transaminase, serum glutamate pyruvate transaminase, blood glucose, serum creatinine, triglycerides, and high-density lipoprotein) of Wistar rats on 180th day

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Effect on haematological parameters

After administration of homoeopathic drugs for 180 days, there were no significant changes observed in haematological parameters (Hb, RBC, WBC, platelets, blood glucose, and clotting and bleeding time) as compared to normal control. The effect of Homoeopathic drugs on the haematological parameters is presented in [Table 9].

Histopathological evaluation

No histopathological changes were observed in kidney, liver, brain, testis, ovary, and heart of various treated animals in comparison to normal control animals after administration of homoeopathic drugs for 180 days as shown in [Figure 2] and [Figure 3].
Figure 2: (A1–A3) Normal control (kidney, liver, and heart), (B1–B3) Ferrum phosphoricum 3X (kidney, liver, and heart), (C1–C3) Ferrum phosphoricum 6X (kidney, liver, and heart), (D1–D3) Magnesium phosphoricum 6X (kidney, liver, and heart), and (E1–E3) Calcarea phosphoricum 6X (kidney, liver, and heart). No histopathological changes were observed in kidney, liver, and heart tissues of treated group when compared to control group in chronic toxicity study

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Figure 3: (A1–A3) Normal control (brain, testis, and ovary), (B1–B3) Ferrum Phosphoricum 3X (brain, testis, and ovary), (C1–C3) Ferrum Phosphoricum 6X (brain, testis, and ovary), (D1–D3) Magnesium Phosphoricum 6X (brain, testis, and ovary), and (E1–E3) Calcarea Phosphoricum 6X (brain, testis, and ovary). Photomicrographs were taken at magnification (×20). No histopathological changes were observed in brain, testis, and ovary tissues of treated group when compared to control group in chronic toxicity study

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  Discussion Top


In the present study, the safety profile of various homoeopathic drugs, that is, Ferrum phosphoricum 3X, Ferrum phosphoricum 6X, Calcarea phosphoricum 6X, and Magnesium phosphoricum 6X was evaluated. Homoeopathic drugs were used in day to day life and considered safe. However, there are very limited data available for the safety evaluation of these homoeopathic drugs. Hence, in the present study, it was evaluated that these homoeopathic drugs were safe in acute, subacute, and chronic toxicity studies. These studies were performed according to the OECD guidelines.

The acute toxicity test was conducted according to the OECD guideline 425.[8] The test is a sequential test that uses a maximum of 5 animals. A test dose of 2000, or exceptionally 5000 mg/kg, may be used. The procedures for testing at 2000 and 5000 mg/kg are slightly different. The selection of a sequential test plan increases the statistical power and also has been made to intentionally bias the procedure toward rejection of the limit test for compounds with LD50 near the limit dose, that is, to be on the side of safety. As with any limit test protocol, the probability of correctly classifying a compound will decrease as the actual LD50 more nearly resembles the limit dose. In the acute toxicity studies, oral LD50 of homoeopathic drugs (Ferrum phosphoricum 3X and 6X, Calcarea phosphoricum 6X, and Magnesium phosphoricum 6X) in wistar rats was >2000 mg/kg body weight. The 10-day observation period during the acute oral toxicity study and body weight measurements did not reveal any toxic effect in Wistar rats (data not shown). Necropsy at the end of the study did not reveal any gross pathological abnormality in rats. These observations from oral acute toxicity study suggest that the homoeopathic drugs are practically nontoxic. Hence, there were no mortality or any sign of toxicity observed after oral administration of drugs up to the dose level of 2000 mg/kg in rats. In addition, the LD50 was found to be >2000 mg/kg.

The subacute toxicity studies were conducted according to the OECD guidelines 407.[9] The test substance is orally administered daily to several groups of experimental animals, one dose level per group for 28 days. During the period of administration, the animals were observed closely each day for signs of toxicity. Animals that die or are euthanized during the test are necropsied and at the conclusion of the test surviving animals are euthanized and necropsied. A 28-day study provides information on the effects of repeated oral exposure and can indicate the need for further long-term studies. It can also provide information on the selection of concentrations for long-term studies. The data derived from these test guidelines should allow for the characterization of the test substance toxicity, for an indication of the dose-response relationship and the determination of the no observed adverse effect level (NOAEL).

The administration of Homoeopathic drugs such as Ferrum phosphoricum 3X, Ferrum phosphoricum 6X, Calcarea phosphoricum 6X, and Magnesium phosphoricum 6X for 28 days did not produce any significant change in haematological and biochemical parameters of male and female rats as compared to those in normal control group. All animals survived until the scheduled necropsy and their physical and behavioral examinations did not reveal any treatment-related adverse effect. No pathological changes were observed in histological section of heart, kidney, liver, testis, ovaries, and brain of homoeopathic drugs (Ferrum phosphoricum 3X and 6X, Calcarea phosphoricum 6X, and Magnesium phosphoricum 6X) treated male and female rats as compared to normal control animals. These observations from oral acute and subacute toxicity study suggest that the extract is practically nontoxic, found to be safe, and the NOAEL of the extract was found to be 70 mg/kg/day.

The chronic long-term administration of homoeopathic drugs (Ferrum phosphoricum 3X and 6X, Calcarea phosphoricum 6X, and Magnesium phosphoricum 6X) for 180 days was further explored to confirm the safety of homoeopathic medications. As these homoeopathic drugs (Ferrum phosphoricum 3X and 6X, Calcarea phosphoricum 6X, and Magnesium phosphoricum 6X) are mineral based, so their long-term safety studies are needed and it was done according to the OECD guideline 452.[10] The test substance is administered daily to several groups of experimental animals, normally for 6 months. This duration is chosen to be sufficiently long to allow any effect of cumulative toxicity to become manifest, without the confounding effects of geriatric changes. The test substance is normally administered by the oral route. The study design may also include one or more interim kills, for example, at 3 months, and additional groups of animals may be included to accommodate this. During the period of administration, the animals are observed closely for signs of toxicity. Animals that die or are killed during the test are necropsied, and at the conclusion of the test, surviving animals are killed and necropsied. The body weight and the relative organ weights of experimental animals did not show any significant change after administration of the different homoeopathic drugs (Ferrum phosphoricum 3X and 6X, Calcarea phosphoricum 6X, and Magnesium phosphoricum 6X) for 180 days when compared to the control group. Comparison of relative organ weights between treated and control group of animals has conventionally been used to evaluate the toxic or adverse effects of test compounds or drugs. Relative organ and change in body weight is also used as an assessment of therapeutic response to drugs. This means that these homoeopathic drugs did not have any adverse effect on experimental animals that would cause them to decrease appetite. This signifies that the organ weights did not indicate any toxic or adverse effect from Homoeopathic drugs.

Haematological parameters analyzed included the complete blood count of experimental and control group animals. Analysis of blood parameters in animal studies is relevant to evaluate the risk of alterations of the haematopoietic system in toxicity studies, for necessary application to humans. In this study, administration of the different homoeopathic drugs after 180 days induces no significant change in all haematological parameters as compared to control group. The effect on Hb concentration and WBC count indicated the unlikelihood of the homoeopathic drugs to induce anemia even after long use. Assay of biochemical parameters was performed to evaluate the liver, renal, lipid, and glycemic profiles of experimental compared to control animals, in order to give insight into pathological changes and nature of disease. In this study, assay of the liver profile parameters (serum glutamic oxaloacetic transaminase, and serum glutamic pyruvic transaminase) revealed normal functioning of the liver after 180 days of administration at different homoeopathic drugs (Ferrum phosphoricum 3X and 6X, Calcarea phosphoricum 6X, and Magnesium phosphoricum 6X), with normal values in experimental animals as compared to control group. The bleeding time in rats was measured by tail tip amputation method and is usually defined as the time of the first cessation of bleeding. No significant changes were observed in bleeding time of Homoeopathic drugs as compared to normal control animals and the data obtained is in correlation to the previous studies.[5],[11] Creatinine is indicator of glomerular filtration rate, which is an indicator of the renal function. The renal profile parameters (creatinine and blood urea nitrogen) are in normal range in all experimental animals for 180 days, when compared to control group. Histopathological examinations of the liver, kidney, heart, brain, testis, and ovaries did not reveal any morphological change after administration of the homoeopathic test compounds.

Thus, we corroborate that the mineral-based homoeopathic drugs (Ferrum phosphoricum 3X and 6X, Calcarea phosphoricum 6X, and Magnesium phosphoricum 6X) are safe and produce no toxicity even administered for longer durations. These can be invariably used for clinical purposes. Further, this study can be elaborated and effect of homoeopathic drugs on DNA damage and genotoxicity can further be evaluated to study the scope of these drugs on molecular levels.


  Conclusion Top


These homoeopathic drugs are considered safe as no adverse effect on biochemical and hematological parameters and histopathology of heart, kidney, liver, brain, ovaries, and testis was observed even after administering these drugs for 180 days.

Financial support and sponsorship

Financial assistance provided by Central Council for Research in Homoeopathy, Ministry of AYUSH, Government of India in the form of collaborative research project.

Conflict of interest

None declared.

 
  References Top

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Prakash P, Gupta N. Therapeutic uses of Ocimum sanctum Linn (Tulsi) with a note on eugenol and its pharmacological actions: A short review. Indian J Physiol Pharmacol 2005;49:125-31.  Back to cited text no. 1
    
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Larrey D. Liver involvement in the course of phytotherapy. Presse Med 1994;23:691-3.  Back to cited text no. 2
    
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Patil AS, Jadhav AB, Arya MP. Effect of biochemic preparation of Frrum phos 3X on blood neutrophils. Pharm Lett 2014;6:169-71.  Back to cited text no. 3
    
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Boericke W. Pocket Manual of Homeopathic Materia Medica and Repertory. Delhi: B Jain Publishers; 2004. p. 421-65.  Back to cited text no. 4
    
5.
Singh S, Kumar R, Karwasra R, Kalra P, Rani S, Nayak D, et al. Evaluation of safety profile of homeopathic mother tincture. Indian J Res Homeopthy 2014;8:81-6.  Back to cited text no. 5
    
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Ernst E. The efficacy of herbal medicine – An overview. Fundam Clin Pharmacol 2005;19:405-9.  Back to cited text no. 6
    
7.
Government of India, Ministry of Health and Family Welfare. Homoeopathic Pharmacopoeia of India. 1st ed., Vol. II. New Delhi: The Controller of Publications; 1984.  Back to cited text no. 7
    
8.
OECD. Guidelines for the Testing of Chemicals, N-425. Adopted; 17th December, 2001. Available from :http://www.oecd.org/chemicalsafety/risk-assessment/1948378.pdf. [Last accessed on 2017 Jan 30].  Back to cited text no. 8
    
9.
OECD (2008), Test No. 407: Repeated Dose 28-day Oral Toxicity Study in Rodents, OECD Publishing, Paris. DOI: http://dx.doi.org/10.1787/9789264070684-en. [Last accessed on 2017 Jan 30].  Back to cited text no. 9
    
10.
OECD (2009), Test No. 452: Chronic Toxicity Studies, OECD Publishing, Paris. DOI: http://dx.doi.org/10.1787/9789264071209-en. [Last accessed on 2017 Jan 30].  Back to cited text no. 10
    
11.
Liu Y, Jennings NL, Dart AM, Du XJ. Standardizing a simpler, more sensitive and accurate tail bleeding assay in mice. World J Exp Med 2012; 2: 30-36.  Back to cited text no. 11
    


    Figures

  [Figure 1], [Figure 2], [Figure 3]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6], [Table 7], [Table 8]



 

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