Long term toxicity study of a Siddha formulation Vetpalai thailam (VT) in Rats

Mr. Ganesh Shenoy¹, Dr. Smita Shenoy²*, Dr. Sathish Pai B³, Dr. Nitesh Kumar⁴,

Dr. Arul Amuthan¹, Mr. Manjunath Shetty¹, Dr. Mohandas Rao⁵, Bharath Rao K⁶

¹Selection Grade Lecturer, Department of Pharmacology, Melaka Manipal Medical College, Manipal,

Manipal Academy of Higher Education, Manipal, Karnataka, India-576104.

²Professor and Head, Department of Pharmacology, Kasturba Medical College, Manipal, Manipal Academy of Higher Education, Manipal, Karnataka, India-576104.

³Professor, Department of Dermatology, Kasturba Medical College, Manipal, Manipal Academy of Higher Education, Manipal, Karnataka, India-576104.

³Assistant Professor, Department of Pharmacology and Toxicology, NIPER, Hajipur.

¹Assistant Professor, Department of Pharmacology, Melaka Manipal Medical College, Manipal,

Manipal Academy of Higher Education, Manipal, Karnataka, India-576104.

¹Senior Grade Lecturer, Department of Pharmacology, Melaka Manipal Medical College, Manipal,

Manipal Academy of Higher Education, Manipal, Karnataka, India-576104.

⁵Professor, Department of Anatomy, Melaka Manipal Medical College, Manipal, Manipal Academy of Higher Education, Manipal, Karnataka, India-576104.

⁶Associate Professor, Department of Oral Biology, Melaka Manipal Medical College, Manipal,

Manipal Academy of Higher Education, Manipal, Karnataka, India-576104.

*Corresponding Author E-mail: smita.shenoy@manipal.edu

ABSTRACT:

Vetpalai thailam, a Siddha formulation is used topically as well as orally in the treatment of Psoriasis. Vetpalai thailam is prepared from the leaves of Wrightia tinctoria by insolation with coconut oil as the base. The adverse effect profile of VT has not been addressed and hence this study was taken up to assess the long term toxicity of VT in rats. A 90-day repeated dose toxicity study was performed by modifying the protocol described by OECD guideline 408 for testing chemicals. The study constituted of six groups consisting of six rats in each group. There was no mortality among the six study groups. No signs of toxicity and behavioural changes were observed. The histopathological examination of vital organs did not show any change in the architecture. Our study demonstrates that Vetpalai thailam does not cause any toxicity on long term use in rats.

KEYWORDS: Vetpalai thailam, Wrightia tinctoria, long term toxicity.

INTRODUCTION:

Siddha system of medicine is a form of medicine that has been widely in practice in the Indian state of Tamil Nadu with its origin going back to B.C 10,000 to B.C 4,000. It is practised by Sidhars also known as vaithiyars in Tamil. The current form of Siddha medicine has scientifically evolved over the years as a result of scientists' efforts and India has been successful in promoting Siddha mecine through more research and a science-based approach, more extensive research and evidence base are still required¹^,²^,³^,⁴^,⁵. Siddha Vaidya classifies medicinal ingredients based on the source into three types: Thavaram are medicines which are derived from plants, Janaganam from animals, earth, and organic toxins. Thavaram comprises of thousands of plants and its products ⁶.

Plant products have played an important role in the management of skin diseases in traditional medicine and are amongst the economical of sources for several popular and established drugs ⁷^,⁸. Medicinal plants are considered to be the backbone of the traditional system of medicine such as Siddha ⁹^,¹⁰^,¹¹. A number of traditional formulations are showing promising results in preventing and treating diseases ¹². One-third of the traditional herbal medicines are used to treat dermatologic disorders in comparison to 1-3% of modern drugs ¹³^,¹⁴.

Psoriasis is a chronic immune modulated inflammatory skin disease that could involve the nails, skin, and joints. It affects approximately 2-3% of the world population ¹⁵^,¹⁶^,¹⁷. Psoriasis presents in various forms which include plaque psoriasis, pustular psoriasis, erythrodermic psoriasis, guttate psoriasis, inverse psoriasis, scalp psoriasis and nail psoriasis¹⁸. Even though the aetiology of the disease is not known, defective proliferation and differentiation of the keratinocytes with infiltration of inflammatory cell infiltrate comprising of neutrophils, macrophages, and T lymphocytes are seen¹⁹^,²⁰. A sustained phase of inflammation leads to the uncontrolled proliferation of the keratinocytes and dysfunctional differentiation. The histopathological features of psoriasis include epidermal hyperplasia also called acanthosis overlying the inflammatory infiltrate comprised of neutrophils, macrophages, T cells, and dermal dendritic cells. Neovascularization is also an associated predominant feature ²¹^,²².

Long term treatment is a necessity in psoriasis due to the chronicity and relapsing nature of the disease. The treatment for the disease is based on the severity, presence of associated comorbidities, and health care access. The patients are categorized into mild or moderate to severe based on the clinical severity of the lesion, the percentage of the surface area of the body affected as well the quality of life ²³. The current treatment modalities fall into three categories i.e., topical therapy, phototherapy and systemic therapy. ²⁴.

Siddha's form of medicine is gaining popularity in the world of alternate medicine. The mediation and maintenance of the body function are due to Vali, Azhal, and AIyam (Vatham, Piththam, and Kapam in Ayurveda). Aggravation of any one of the forces leads to an imbalance and thus disease. The treatment aims at the restoration of the balance of the three forces which mediate the physiological body function ²⁵^,²⁶. One of the dermatologic condition in which Siddha drugs have been quite effective is Psoriasis. Herbal as well as herbomineral drugs have been used in the treatment of Psoriasis. There are various Siddha formulations used in the treatment of psoriasis-like Vetpalai thailam, Sivanar vembu kuzhi thailam/capsules, Sarvanga rasayanam, Karbogi tablets, Ganthaga Chooranam/tablets, Neeradimuthu Vallathi Legiyam/capsules, and Ganthaga Rasayanam ²⁷. Among these, Vetpalai thailam and Sivanar vembu kuzhi thailam/capsule are commonly prescribed for psoriasis.

Vetpalai thailam ²⁸ is prepared from the leaves of Wrightia tinctoria by insolation with coconut oil as the base ²⁹^,³⁰. This process of preparation in Siddha medicine is called “Sooriya Pudam” ³¹. Wrightia tinctoria plant is very useful as stomachic, it is also used in the treatment of abdominal pain, skin diseases, as an antidiarrhoeal and as an antihaemorrhagic ³². It is applied topically as well as used orally in the treatment of psoriasis. A patented Siddha drug consisting of Wrightia tinctoria and coconut oil (777 oil) has been shown to have anti-inflammatory, analgesic, and antipyretic property in the animal model ³³. However, the mechanistic action, adverse effects, prophylactic role, therapeutic role, and remission time have not been addressed and the physicochemical standardization of this classical drug had not been done. Pre-clinical toxicity studies are essential to determine a safe dose for human trial and also is of utmost importance while screening drugs ³⁴^,³⁵^, ³⁶. Before the initiation of human clinical trials of novel drugs, the safety of their application is to be proved. Generally, this is accomplished by the implementation of extensive preclinical toxicity experiments to uncover potential poisonous effects of any drug in animals ³⁷,³⁸. There is no reported literature on the preclinical toxicity of Vetpalai thailam. Hence, an effort has been made to explore the chronic oral toxicity of Vetpalai thailam in experimental rats to authenticate its safety behind its traditional use.

MATERIALS AND METHODS:

1. Procurement: Vetpalai thailam was procured from SKM Siddha and ayurveda (GMP certified) Company (India) Ltd, Saminathapuram (Post). Modakkurichi, Erode District- 638 104, Tamilnadu.

2. Chemicals and reagents: The clinical diagnostic kits were purchased from Agappe Diagnostics Limited, India

3. Study site: The study was conducted at the central animal research facility, Manipal.

4. IAEC approval: Approval for the study was been obtained from the Institutional animal ethics committee, Kasturba Medical College, Manipal (IAEC/KMC/78/ 2017 dated 28.10.2017)

5. Study design:

5.1 Dose selection:

Three different doses of VT (40 mg/kg, 130 mg/kg and 400 mg/kg body weight (BW) of rat) were used. 40 mg/kg BW was Animal equivalent dose (AED) i.e. human dose of VT was converted to rat dose based on surface area ratios. ¹⁵ The second dose selected was approximately 3 times higher than the AED and the third dose was approximately 10 times higher than AED.

5.2 Experimental Animals:

Healthy adult female Sprague Dawley rats, 12–13 weeks old were used in the study. The females selected were nulliparous and non-pregnant. All the animals were housed individually in polypropylene cages and maintained at a temperature of 27 ± 3°C, relative humidity of 60 ± 10% and 12 hours light /dark cycle with paddy husk as the bedding material. Animals were maintained on standard laboratory diet, rat pellet food and water ad libitum. Animal care and handling were done according to the CPCSEA (Committee for the Purpose of Control and Supervision of Experiments on Animals) guidelines. The body weight was recorded before the commencement of the experiment. A total of 30 rats were used in this experiment and were divided into 5 groups with 6 rats in each. The groups were as follows: The groups were as follows:

The control and test drug control group consisted of 30 rats which were divided into 5 groups (n = 6/group): Group 1 - Normal control group-Distilled water – 10 mL/kg body weight; Group 2 – Oral VT 40 mg/kg body weight; Group 3- Oral VT 130 mg/kg body weight; Group 4 - Oral VT 400 mg/kg body weight; Group 5 – Satellite group - Oral VT 400 mg/kg body weight

A 90-day repeated dose toxicity study was performed in accordance with OECD Test Guideline 408. Rats were randomly assigned to 5 groups. Group 5 was used as the satellite group to observe the reverse sign of any toxicity.

5.3 Drug administration:

Doses were administered by oral gavage once daily for 90 consecutive days at a similar time each day to minimize variations. The control animals were administered vehicle only. The satellite group animals received test drug orally for 90 days, and there was no further treatment for the following 28 days in satellite group. The experiment was terminated on day 118 for satellite group and day 90 for other groups. All the experimental animals were observed for mortality daily throughout the study duration. Visual observations for the behavioural pattern (salivation, fur, lethargy, and sleep), changes in physical appearance, injury, pain and signs of illness were done once daily during that period. Bodyweight of the animals was recorded once in a month.

5.4 Parameters assessed

At the end of the 90 days treatment period in group 1 – 4 and at the end of 118 days in group 5, the overnight fasted (water provided ad-libitum) rats were anaesthetized with pentobarbital (150 mg/kg), blood samples were collected by a retro-orbital puncture in the EDTA vacutainers (for hematological) and serum vacutainers (for biochemical and electrolyte analysis) as reported earlier. Hemoglobin, Total red blood cells, Packed cell volume, Mean corpuscular volume, Mean corpuscular Hb, Mean corpuscular Hb conc, Red blood cells Distribution width, Total white blood cells, Neutrophils, Lymphocytes, Monocytes and Platelet count were analyzed using fully automated hematology analyzer.

Serum biochemical parameters such as urea, creatinine, uric acid, alkaline phosphatase, aspartate aminotransferase, and alanine transaminase were analyzed using a fully automatic analyzer. Serum electrolytes such as sodium, potassium and chloride were estimated in a fully automated electrolyte analyzer. At the end of the study duration, all animals were subjected to gross necropsy. Organs like Heart, Liver, Spleen, Kidneys, Lungs, Skin and Brain were isolated, trimmed and weighed. These organs were preserved in 10 % formalin and subjected to histological examination.

Statistical Analysis:

Data expressed as mean ± standard error of mean (SEM). Mean difference between the control and treatment group was analyzed by ANOVA followed by Tukey’s test using SPSS (licensed version 20). P-value ≤ 0.05 was considered as statistically significant.

RESULTS:

Table 1: Effect of VT on haematological and biochemical parameters in Rats

PARAMETERS	GROUPS
PARAMETERS	NORMAL	VT (40 mg/kg)	VT (130 mg/kg )	VT (400 mg/kg )	VT satellite
WBC (10³/µl)	4.8 ± 0.9	4.7 ± 0.8	4.8 ± 0.3	4.9 ± 0.2	4.8 ± 0.3
RBC (10⁶/µl)	8 ± 0.7	7.8 ± 0.5	7.9 ± 0.6	8.0 ± 0.4	7.8 ± 0.5
Hb (g/dl)	14 ± 1	13.4 ± 1.0	13.9 ± 0.7	13.8 ± 0.5	14.0 ± 0.6
PLT (10³/µl)	406.8 ± 152.5	430.5 ± 4.5	422.2 ± 18.9	418.3 ± 11.8	422.2 ± 11.9
AST (IU/L)	160.7 ± 44.7	134.2 ± 13.4	135.4 ± 25.2	135.8 ± 5.2	133.9 ± 15.3
ALT (IU/L)	89.6 ± 9.5	81.8 ± 11.9	85.4 ± 16.4	84.3 ± 13.5	85.2 ± 9.9
ALP (IU/L)	204.3 ± 25.3	210.0 ± 23.5	217.0 ± 76.7	213.8 ± 21.3	206.7 ± 21.5
UREA (mg/dL)	11.1 ± 0.5	11.3 ± 0.6	11.4 ± 0.7	11.4 ± 0.6	11.2 ± 0.8
CREATININE (mg/dL)	0.7 ± 0.04	0.7 ± 0.1	0.7 ± 0.3	0.8 ± 0.3	0.8 ± 0.07
URIC ACID (mg/dL)	2.8 ± 0.3	2.5 ± 0.6	2.5 ± 0.3	2.4 ± 0.4	2.5 ± 0.3

One-way ANOVA followed by post hoc TUKEY test was performed. Values are expressed asmean ±SDWBC: Total white blood cells; RBC: Total red blood cells; Hb: Haemoglobin; PLT: Platelet count; AST: Aspartate aminotransferase; ALT: Alanine transaminase; ALP: Alkaline phosphatase

Figure 1: Qualitative assessment of Haematoxylin and Eosin (H and E) stained organ tissues

Skin tissue

a,b,c,d:

Epithelium

was normal.

Collagen bundle

was organised

and no inflammatory

cells were seen.

Liver

a,b,c,d: Hepatocytes arrangement and structure, central vein (CV) and portal triad looked normal

Kidney

a,b,c,d : Cortex looked normal with a cut section of Bowman’s capsule, glomeruli, proximal convoluted tubule and distal convoluted tubule.

There was no statistically significant difference seen in hematological parameters like packed cell volume, mean corpuscular volume, mean corpuscular Hb, mean corpuscular Hb conc, and red blood cells distribution width; and biochemical parameters such as total protein, albumin, globulin, sodium, potassium and chloride between the groups. Also, histopathological examination of the heart, spleen, lungs and brain showed no significant changes in all the groups. Body weight changes and relative organ weights did not show any statistically significant difference.

Clinical signs:

Daily oral administration of Vetpalai thailam (all three doses) for 90 days did not induce any obvious symptom of toxicity in rats. No deaths or obvious clinical signs were found in any groups throughout the experimental period. Physical observation of the treated rats throughout the study indicated that none of them showed signs of toxicity in their skin, fur, eyes, mucus membrane, or behavioural changes, diarrhoea, tremors, salivation, sleep, and coma.

DISCUSSION:

Toxicity studies are important in order to ascertain the safety of herbal products as well as to establish the bioactive component of these herbal medicines ³⁹. Also, traditional or folk healing herbal medicines are used approximately by 60% of the world population both in the developing and developed countries ⁴⁰. This toxicology study focused to determine and evaluate the chronic oral toxicity and the safety of Vetpalai thailam in Sprague Dawley rats. The effect of the drug on the biochemical and haematological parameters as well as organ toxicity was assessed. The toxicity induced by a drug is determined by clinical biochemistry values and haematological data. An increase in these enzyme levels indicates damage to the parenchymal cells of the liver. The levels of bilirubin in the serum and tissue increase due to increased haemolysis or damage to the liver in conditions such as obstruction of the bile duct or hepatitis³⁸. The possible toxicity of drugs can be predicted by the evaluation of the levels of the liver enzymes such as AST, ALT, and ALP. The liver releases AST and ALT and an increase in their plasma concentration indicates liver damage. ⁴¹. Creatinine is derived endogenously due to the breakdown of tissue creatinine. Muscle mass, high protein diet and catabolic state are some of the factors that affect the levels of plasma creatinine in normal individuals; thus the concentration of serum urea is a reliable indicator of renal function², ⁴². In the present chronic oral toxicity study, VT at three dose levels (40, 130, and 400 mg/kg body weight) in the experimental groups as well as in the satellite group did not show any significant differences (P>0.05) in the values of serum bilirubin, liver enzymes such as AST, ALT, ALP, total protein, creatinine, uric acid and electrolytes such as sodium, potassium and chloride ions as compared to control group.

Risk evaluation is done by the analysis of the blood parameters due to the increased predictive value for toxicity in humans by the hematological system in assays involving rodents and nonrodents ⁴³. Drugs and xenobiotics are transported in the body by blood thus exposing the components of blood such as RBCs, WBCs, platelet and hemoglobin to varying concentration of toxic compounds. The destruction of cells in the blood is detrimental to the functioning of the human body²^,⁴⁴. In a healthy individual, the turnover rate of red blood cells, white blood cells and platelets range between 1 to 3 million per second which could be altered due to various physiological as well as pathological conditions. In our study, there was no alteration in the hematological parameters post-treatment with VT thus indicating no adverse effect on haematopoiesis. According to the society of toxicologic pathology, in toxicology studies, the organ/body weight has to be calculated and evaluated as the test drug may cause organ damage, lead to changes in the metabolism of phospholipids, cause a decrease or increase in the secretion of enzymes as well as hormones and lead to hypo/hyperplasia of the organs ⁴⁵^,⁴⁶. There was no significant difference in relative organ weights between the control group and the VT treated as well as the satellite treated group. There were some histological alterations observed only in brain (pyknotic neurons), kidney (edematous cortex in some areas) and lungs (aggregation of inflammatory cells) of experimental animals as well as in satellite group. However, similar changes were also observed in the control group and hence cannot be attributed to the administration of the VT. All the remaining organs were normal histologically.

CONCLUSION:

There was no toxicologically significant observation with respect to body weight, relative organ weight, haematology and clinical biochemistry in VT treated rats at 40, 130 and 400 mg/kg body weight respectively. The histopathological finding did not reveal any treatment related toxicologically significant changes. Hence VT can be considered safe and non-toxic up to a dose of 400 mg/kg body weight in rats.

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Received on 02.12.2020 Modified on 04.05.2021

Research J. Pharm.and Tech 2021; 14(12):6653-6658.

DOI: 10.52711/0974-360X.2021.01149