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Table of Contents
ORIGINAL ARTICLE
Year : 2019  |  Volume : 7  |  Issue : 1  |  Page : 20-27

In vivo anticancer activity and toxicity of Ayurveda compound W.S.R. to leukemia


Department of Agad Tantra, National Institute of Ayurveda, Jaipur, Rajasthan, India

Date of Web Publication27-Aug-2019

Correspondence Address:
Dr. Monika Sharma
Department of Agad Tantra, National Institute of Ayurveda, Amer Road, Jaipur 302002, Rajasthan.
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/JISM.JISM_2_19

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  Abstract 

Introduction: A diseased caused by an uncontrolled division of abnormal cells in any tissue of any organ of the body is called cancer. There are various types of cancers on the basis of primary abnormal growth in tissues. Leukemia is a type of cancer of blood, which is caused by the rapid production of abnormal white blood cell. In Ayurveda, the reference of the cancer and blood cancer is found indirectly under the heading of Arbuda (cancer) and Rakta Arbuda (leukemia), respectively. In Ayurveda, there are so many herbo-mineral drugs that are useful in treating cancer. Aims and Objectives:

  1. To study the in vivo anticancer (blood cancer) activity of Ayurveda compound.
  2. To compare the toxicity of Ayurveda compound with injection/tablet arsenic trioxide.
  3. To evaluate, elaborate, and discuss the etiology of leukemia as per Ayurveda.
Materials and Methods: An in vivo study was conducted in Institute of Biomedical and Industrial Research. The in vivo antileukemic effect was carried out by using benzene-induced (carcinogen) model in Swiss albino mice and the acute toxicity study was conducted according to fixed single-dose toxicity. The herbo‑mineral drugs has been prepared by using purified arsenic (arsenic trioxide), Vinca rosea, and Urginia indica to study in vivo anti-leukemic effect and toxicity. This study was conducted according to organisation for economic co-operation and development Guidelines 423 and the antileukemic activity was carried out by benzene-induced leukemia in albino mice after animal ethical clearance. Result: The highest dose of the test drug (2000mg/kg) in the acute toxicity study shows minimal adverse effect of toxicity on liver and no adverse effect was found on kidney and spleen. The result was found better in study group 1 having myelocytic leukemia than study group 2 having lymphocytic leukemia. Conclusion: The effect of study drug shows good antileukemic activity, although standard drug was found better than study drug. Overall, the study was found safe and effective on blood cancer.

Keywords: Ayurveda compound, lymphocytic leukemia, myelocytic leukemia, Rakta arbuda


How to cite this article:
Sharma M, Porte S, Charak S. In vivo anticancer activity and toxicity of Ayurveda compound W.S.R. to leukemia. J Indian Sys Medicine 2019;7:20-7

How to cite this URL:
Sharma M, Porte S, Charak S. In vivo anticancer activity and toxicity of Ayurveda compound W.S.R. to leukemia. J Indian Sys Medicine [serial online] 2019 [cited 2019 Oct 14];7:20-7. Available from: http://www.joinsysmed.com/text.asp?2019/7/1/20/265517


  Introduction Top


Cancer is an abnormal uncontrolled growth of cells that can lead to death. Cancer cells rapidly reproduce despite restriction of space, nutrients shared by other cells, or signals sent from the body to stop reproduction.[1] Leukemia is a cancer of the blood or bone marrow (which produces blood cells).[2] A person who has leukemia has an abnormal production of blood cells, generally leukocytes (white blood cells, WBC). The discretion of malignant disease is available in the text book of Indian medicine indirectly. It does not mean that ancient Indian clinicians were unaware about the malignant diseases. The text book of Ayurveda has been mention the cancer & its clinical feature under the heading of Arbuda which is somewhat resemble to cancer. They presented their views regarding cancer as a swelling superficially or situated in the deeper structure or sometimes as chronic ulcer. Such swellings or lumps have been categorized under the heading of Arbuda (cancer). The cancer and blood cancer are not new for Ayurveda and have been described under the heading of Arbuda (cancer) and Rakta Arbuda (blood cancer). Acharya Madhav[3] has well defined the definition of Arbuda and stated that the vitiated Dosha afflicts the Mamsa and Rakta both to produce a mass. Leukemia is the 11th most common cancer worldwide, with around 352,000 new cases diagnosed in 2012 (2% of the total). In 2014, it is estimated that there will be 52,380 new cases of leukemia and an estimated 24,090 people will die of this disease.[4] Though the treatment of leukemia in the form of chemotherapy and radiotherapy is available in modern science, the morbidity and mortality is still high. In Ayurveda, there are so many herbals, herbo-minerals, and mineral drugs that are useful in treating cancer (Arbuda), but there is a need to evaluate the anticancer effect of such drugs on the scientific parameter.

Injection arsenic trioxide (Trisonex) [Figure 1] has been proved as chemotherapeutic agent used for leukemia. The purified arsenic (Sudh Sankheya) is a Sthavar dhatu visha (inanimate poison), which can show anticancer effect on leukemia. It may be used in therapeutic doses and may show less toxic effect than the injection arsenic trioxide. Vincristine & Vinblastine these are the alkaloids of vinca rosea which are used as anticancer agents in leukaemia. He also mentioned the Rakta Arbuda Nashak (antileukemic) property of Urginia indica [Figure 2].[5] Hence, the hypothetical Ayurvedic compound prepared from purified arsenic powder (Sudha Somala Bhasm), aqueous extract of V. rosea, [Figure 3] and U. indica was selected for this anticancer study W.S.R. to leukemia.
Figure 1: First drug of the experimental trial is arsenic trioxide

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,
Figure 2: Second drug of the experimental trial is Urginia indica

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,
Figure 3: Third drug of the experimental trial is Vinca rosea

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  Materials and Methods Top


Materials: The items listed below were taken for experimental study.

Test sample. Preparation of test sample (Ayurveda compound) -4 mg arsenic mixed with 10 g of aqueous extract of Vinca rosea and 200 mg of aqueous extract of Urgenia indica [Figure 4] were mixed properly by using mortar and pestle to prepare test sample. The animals were purchased from Research Laboratory Animal, Hyderabad and Institutional animal ethical committee (approval no. IAC/ACA/2015/23). Three animals were used for each group. Group 1 received 50mg/kg test sample, group 2 received 300mg/kg test sample, and group 3 received 2000mg/kg test sample. The test substance had been administered in a single dose by gavages using an oral feeding needle. Animals were observed individually after dosing at least once during the first 4h, 24h, 30h, 48h, 1 week, and 2 week. Changes in skin and fur, eyes, mucous membranes, salivation, lethargy, sleep, coma, convulsions, tremors, diarrhea, morbidity, and mortality were observed. All test animals (including those that die during the test or were removed from the study for animal welfare reasons) were subjected to gross necropsy. All gross pathological changes were recorded for each animal. At the end of experimental period, one animal of each group was killed and observed for gross lesions of internal organs.
Figure 4: The hypothetical Ayurveda compound prepared from purified arsenic powder (Sudha Somala Bhasm), aqueous extract of Vinca rosea, and Urginia indica selected for this anticancer study W.S.R to leukemia

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Benzene-induced leukemia in albino mice: Test drug dose calculation: Dose fixation of test drug for mice was calculated on this basis of body surface area ratio by referring to table of Paget and Barnes.[6]

The Human dose of Arsenic, Vinca rosea and Urgenia indica is 4 mg,[7] 10 gm[8] and 200 mg[9] for adult respectively.

Human equivalent dose: 10204 mg

The dose of study drug in mice was calculated as follows:

Human equivalent dose × conversion factor (0.007)[10]



Route of drug administration: oral, once a day [Figure 5]
Figure 5: Effect of test and standard drug on lymphocytic leukemia on bone marrow pattern (study GP 2 and standard GP 2)

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Duration of administration: 21 days

Housing and feeding conditions was carried out according to organisation for economic co-operation and development (OECD) Guideline 423

Marking of Swiss albino mice for identification: The albino Swiss albino mice were marked with picric acid in each group as head (H), back (B), tail (T), head and back (HB), back and tail (BT), and head and tail (HT).


  Group Design Top


Twenty-four adult Swiss albino mice were divided into four groups having six Swiss albino mice in each. These groups received different treatment in the following manners:

  • Study group 1: In this group, the myelocytic leukemia was produced by giving carcinogenic agent and then Ayurvedic compound was given 21 days as per schedule.


  • Study group 2: In this group, the lymphocytic leukemia was produced by giving carcinogenic agent and then Ayurveda compound was given 21 days as per schedule.


  • Standard group 1: In this group, injection Trisonex (arsenic trioxide) was given after developing the myelocyte leukemia by carcinogenic agent.


  • Standard group 2: In this group, injection Trisonex (arsenic trioxide) was given after developing the lymphocytic leukemia by carcinogenic agent.


  • Experimental Procedure

    Twenty-four adult Swiss albino mice were used to induce myelocytic and lymphocytic leukemia.[11]

    Induction of myelocytic leukemia by benzene: Twelve albino Swiss mice were weighted before starting the experiment. Benzene was used to induce leukemia in female Swiss albino mice by using two doses of 0.2mL/kg for 4 months by two intraperitoneal (IP) injections per week.

    Induction of lymphocytic leukemia by benzene: Twelve albino Swiss mice were weighted before starting the experiment. Benzene was used to induce leukemia in female Swiss albino mice by using two doses of 0.4mL/kg for 4 months by IP injection per week.

    Evaluation of Leukemia after Experimental Study

    Blood collection: After the end of 21 days, the animals were killed and blood sample was collected by using a 5-mL disposable syringe. Then 1mL of blood sample was put in ethylene diamine tetra acetic acid tube for measuring the following hematological parameters by using a hematology analyzer: total WBCs, neutrophils, basophils, lymphocytes, monocytes, red blood cell (RBC), and hemoglobin (Hb).

    Collection of bone marrow: Bone marrow of mice was collected from spinal cord after anesthetization with ketamine and xylazine.

    Statistical analysis: The results were expressed as mean ± SE (standard error). A comparison between before and after treatment was performed by using Student’s t-test paired, and a comparison between the treatment and control groups was performed by analysis of variance (ANOVA) followed by Dunnett’s multiple test. In all the tests performed, a value of P < 0.05 was considered to be statistically significant.


      Observation and Results Top


    The observations and results of the present study has been presented in the following two points.




      Discussion Top


    Cancer is one of leading causes of death in all over the world and leukemia accounts for 4% of all cancer deaths. Ayurveda is an ancient medicine science, which has abundant of herbal, minerals, and herbo-minerals medicine for treating cancer and blood cancer. Ayurveda compounds containing U. indica, V. rosea, and arsenic trioxide are referential drugs of Ayurveda and has been used for various diseases including cancer and blood cancer since ancient times.

    The primary outcome of this research was to show the effect of this Ayurveda compound on leukemia scientifically and the secondary outcome was to compare the antileukemic effect of study drug with modern chemotherapeutic agent.

    The entire in vivo experiments were studied W.S.R. to leukemia into two parts: acute toxicity study and anticancer activity. The acute toxicity of Ayurveda compound was carried out according to OECD Guideline 423. The experiment was studied in three groups having three animals for each group. The test sample was given to group 1 (50mg/kg), group 2 (300mg/kg), and group 3 (2000mg/kg), respectively, for the acute toxicity study. The bone marrow of experimental mice of the study and standard groups after treatment was collected from spinal cord after anesthetization with ketamine and xylazine, and then it was sent for histopathology. No signs of toxicity were observed in these groups. In group 2, the toxicity was found till 24h of observation. One mouse was found to be in morbid conditions after 48h, which also died after 48h of the observation. In group 3, one mouse was found to be in morbid condition, which also died after 48h of the observation, whereas two mice were found to be in morbid conditions, which also died after one week of the observation. All gross histopathological changes were recorded for each animal after killing them.

    Minimal inflammatory cellular infiltration was observed in the histology of hepatic cells in group 1 of the acute toxicity study, which shows almost near normal hepatic and renal architecture. It means that both hepatic and renal tissues were safe in group 1 having dose 50mg/kg of test drug. Normal lobular architecture with central vein and radiating hepatic cords of hepatic tissues was seen in group 2 of acute toxicity, whereas severe degenerative alterations in the tubules were seen in group 2 of acute toxicity of test drug. It means that the hepatic tissue did not show any adverse effect of hepatic toxicity, whereas renal tissue showed a severe adverse effect of toxicity in this group. There was little evidence of fatty vacuoles and cellular necrosis along with normal arrangement of hepatic tissue in group 3 of acute toxicity of test drugs. The renal glomeruli, the proximal, and distal convoluted tubules showed normal structure of renal tissues in group 3 of acute toxicity of test drugs. At the highest dose of test drug in acute toxicity, the minimum toxicity was found but in medium dose of test drug in acute toxicity, the severe degenerative changes in the renal tubules were observed. It may be due to idiosyncrasy effect of test drug on those particular mice used in group 2. The term idiosyncratic drug reaction denotes an aberrant or bizarre reaction or hypersensitivity to a substance, without connection to the pharmacology of the drug. This phenomenon may be explained as an abnormal response of the living body and an allergic response to that particular substance with that particular living body.

    The study drug has been given as scheduled each to six leukemic models selected in both myelocytic and lymphocytic leukemia to analyze the effect. WBC was found to be increased in all the experimental mice in all the groups, including study group 1, study group 2, standard group 1, and standard group 2. However, after the treatment of test drug and standard drug in experimental mice, WBC was found to be decreased substantially. Nonetheless, both study group and standard group drugs were not found to be suitable to normalize the WBC count within the range. The mean level of WBC count was found to be decreased substantially in study group of after treatment than that of before treatment in each experimental mice marked H(mark on head), B (Mark on Back), T (mark on Tail), and BT stands for Back Tail of albino Mice, but the mean level of WBC count was found increased in the experimental mice marked Head Back and Head Tail. It means that the WBC count has been calm down in study group 1 of experimental mice of all marking except HB stands for Head back of albino Mice and HT stands for Head tail of albino Mice. Among all the mice marked in the study group 1 had a positive effect on leukemia. The experimental mice marked H had the highest result followed by the experimental mice marked T, B, and BT. The mean level of WBC count was also found decreased in study group 2 of after treatment in each experimental mice marked B, T, HB, BT, and HT but the mean level of WBC count was found increased slightly in study group 2 of after treatment than that of before treatment in experimental mice marked H. This suggests that lymphocytic leukemia decreased in this group of after treatment than that of before treatment in all the experimental mice except those marked H. Thus, it can be concluded that the result was found better in study group 1 having myelocytic leukemia than that in study group 2 having lymphocytic leukemia.

    Antileukemic effect of standard drug was found just better than study drug as it is a clinically proven drug but the test drug also showed some positive effects on leukemia overall as the ingredients of test drug such as arsenic, V. rosea, and U. indica.


      Conclusion Top


    No signs of toxicity were observed in hematological and histopathological study of drugs. The effect of standard drugs was found just better than study drugs on leukocyte count, although the study drugs also performed better on leukemia. In the bone marrow biopsy, no differences were found in the pattern of study group and standard group in both myelocytic and lymphocytic leukemia.

    Financial support and sponsorship

    Nil.

    Conflicts of interest

    There are no conflicts of interest.

     
      References Top

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    Paget GE, Barnes JM. Toxicity tests in evaluation of drug activities pharmacometries (Laurence, D. R. and Bacharach, A. L. eds) Academic Press: London and New York; 1964.  Back to cited text no. 6
        
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    Acharyap. Sharma, Hindi Commentary, Drayva Guna Vigyan. Vol. II. Reprint ed. Varanasi, India: Chaukhamba Bharti Academy; 2009. p. 832.  Back to cited text no. 8
        
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    Nair AB, Jacob S A simple practice guide for dose conversion between animals and human. J Basic Clin Pharm2016;7:27-31. Available at https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4804402/ [Last accessed on 2018 December 1].  Back to cited text no. 10
        
    11.
    Cook GJ, Pardee TS Animal models of leukemia: any closer to the real thing?. Cancer Metastasis Rev.2013;32:63-76.  Back to cited text no. 11
        


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