Ancient Science of Life

ORIGINAL ARTICLE
Year
: 2018  |  Volume : 38  |  Issue : 1  |  Page : 3--11

Acute oral toxicity evaluation of Hridayarnava Rasa (A Herbo-Mineral Ayurvedic Formulation) prepared from aśodhita and śodhita tāmra bhasma


Chandrashekhar Yuvaraj Jagtap1, Mukesh Nariya2, Vinay J Shukla3, Pradeep Kumar Prajapati4,  
1 Central Ayurved Research Institute, Jhansi, Uttar Pradesh, India
2 Pharmacology Laboratory, Institute of Teaching and Research in Ayurveda, Jamnagar, Gujarat, India
3 Pharmaceutical Chemistry Laboratory, Institute of Teaching and Research in Ayurveda, Jamnagar, Gujarat, India
4 Department of Rasashastra and Bhaishajya Kalpana, All India Institute of Ayurved, New Delhi, India

Correspondence Address:
Dr. Chandrashekhar Yuvaraj Jagtap
Central Ayurved Research Institute, Jhansi, Uttar Pradesh
India

Abstract

Background: Herbo-mineral preparations used in Ayurveda are seen as a matter of concern nowadays; especially for containing metals such as mercury, lead, and copper. One of such formulations is hṛdayārṇava rasa (HR) which contains black sulfide of mercury and tāmra bhasma. Till date, no safety profile of this formulation is available. In the present study, acute oral toxicity of HR prepared from tāmra bhasma in aśodhita and śodhita form was evaluated to provide the safety profile on acute administration. Materials and Methods: HR prepared from śodhita tāmra bhasma (STBHR) and HR prepared from aśodhita tāmra bhasma (ATBHR) was prepared as per the classical reference. Acute toxicity test was evaluated as per OECD 425 guidelines with 2000 mg/kg as a limit test. Fifteen Wistar strain albino female rats (Rattus norvegicus) were randomly divided into three groups of five animals each. One group served as control, and other two served as experimental groups. Test formulation was administered orally to overnight fasted female rats, and detailed behavioral changes and mortality were recorded for 14 days. Parameters such as body weight, hematological and biochemical parameters, and histopathological study of some important organs were assessed. Results: No significant changes in behavior, mortality, body weight, and hematological parameters were observed in all the animals. Some biochemical parameters such as blood urea, alkaline phosphatase, serum glutamic oxaloacetic transaminase, and serum glutamic pyruvic transaminase were affected in both the test drugs. In histopathological study, sections of liver and kidney showed some degenerative changes; comparatively more in ATBHR. Conclusion: The results of the study demonstrate that LD50 of both the test drugs are higher than 2000 mg/kg. It was concluded that both the test drugs at higher dose levels can cause hepatorenal toxicity. However, further chronic toxicity evaluation is necessary to establish the safety profile on chronic administration.



How to cite this article:
Jagtap CY, Nariya M, Shukla VJ, Prajapati PK. Acute oral toxicity evaluation of Hridayarnava Rasa (A Herbo-Mineral Ayurvedic Formulation) prepared from aśodhita and śodhita tāmra bhasma.Ancient Sci Life 2018;38:3-11


How to cite this URL:
Jagtap CY, Nariya M, Shukla VJ, Prajapati PK. Acute oral toxicity evaluation of Hridayarnava Rasa (A Herbo-Mineral Ayurvedic Formulation) prepared from aśodhita and śodhita tāmra bhasma. Ancient Sci Life [serial online] 2018 [cited 2022 Nov 28 ];38:3-11
Available from: https://www.ancientscienceoflife.org/text.asp?2018/38/1/3/358112


Full Text



 Introduction



Rasashastra is an integral part of the Indian system of medicine, which mainly deals with various alchemical procedures and pharmaceutical preparations made from mercury along with other metals, minerals, gemstones, sea and animal products, organic poisonous substances, etc., Ancient seers were very well aware of the harmful effects of such drugs and thus advocated great caution while using them. They made use of these things very smartly in therapeutics only after converting them into form which is safe for humans. For this purpose, various procedures such as śodhana māraṇa, and bhāvana are given in different rasaśāstra classics.[1] Nowadays, it is a curiosity among the Ayurved practitioners to know about the toxicity profile of the medicines which they intend to use in clinical settings, especially the formulations which contain metals and minerals. Even the concerns have been raised recently regarding the heavy metal contents of such herbomineral formulations which are being successfully used in subcontinent since centuries.[2]

As per rasaśāstra classics, impure copper or improperly prepared Tamra Bhasma is toxic and has hazardous effects on the human body when ingested. To indicate the toxic potential, eight major ill effects have been told in classics and due emphasis has been given to its śodhana procedure.[3] Previously, in acute toxicity study, both aśodhita and śodhita tāmra bhasma did not show any mortality up to 14 days when given dose up to 2000 mg/kg to Wistar albino female rats.[4] Furthermore, aśodhita tāmra bhasma has shown cardiotoxic properties in animal experimentation.[5] However, in practice, tāmra bhasma is neither used singularly nor in nondetoxified form but is always used in compound and śodhita form. Furthermore, compound formulations of rasaśāstra are not mere mixtures of different ingredients, but they are again treated in different ways like heating, roasting, melting and levitating with different juices, decoctions etc., thus making them safer and potent. Hridayarnava Rasa (HR) is one of such herbomineral compound formulations of rasaśāstra, which contains equal amount of black sulfide of mercury and tāmra bhasma triturated with a decoction of three myrobalans and juice of Solanum nigrum L.[6] It is being extensively used by Ayurvedic physicians in the treatment of cardiac disorders.[7] But being a preparation containing mercury, copper, and sulfur it may not be considered safe to be used internally in human beings according to modern thinking. Genotoxic potential of this formulation has been evaluated previously in which 14 days administration did not produced any chromosomal aberrations and sperm morphological abnormalities in mice, thus proving its safety from a genotoxicity point of view.[8] However, acute and chronic toxicity studies of HR are not reported to date.

In the present study, acute oral toxicity of HR prepared from tāmra bhasma in aśodhita and śodhita form was evaluated on Wistar strain albino rats (Rattus norvegicus) to establish the safety profile on acute administration. The present study also tries to find out whether tāmra bhasma prepared from aśodhita copper produces toxicity when used in the compound formulation.

 Materials and Methods



Test drugs and chemicals

Test drug 1: Hridayarnava Rasa prepared using śodhita tāmra bhasma (STBHR).

Test drug 2: Hridayarnava Rasa prepared using aśodhita tāmra bhasma (ATBHR).

Both the test drugs were prepared in the laboratory of Rasashastra and Bhaishajyakalpna, Institute for Post Graduate Teaching and Research in Ayurveda (I. P. G. T. and R. A.), Gujarat Ayurved University, Jamnagar. śodhana of mercury and sulfur, preparation of black sulfide of mercury were done as per the classical references [Figure 1]a, [Figure 1]b, [Figure 1]c.[9],[10] For the preparation of śodhita tāmra bhasma, copper scrap (99.89% purity) was subjected to general and specific śodhana procedures [Figure ]2a and [Figure 2]b. For general śodhana, copper scrap was heated in iron pan to red hot state and then quenched seven times in each of these media– sesame oil, buttermilk, cow urine, sour gruel and decoction of Dolichos biflorus L.[11] Specific śodhana of thus obtained copper was carried out by boiling in cow urine for three hours.[12] After that it was washed with hot water and dried called as śodhita copper. This copper was then subjected to māraṇa by mixing equal amount of black sulfide of mercury, śodhita sulfur, and juice of Citrus medica Watt. in electrical muffle furnace [Figure 2]c.[13] Ashodhita tāmra bhasma was prepared by the same manner, but the procedures of general and specific śodhana of copper were excluded in it. The process of Amritikarana was carried out in both the bhasmas using corm of Amorphophyllus campanulatus L. as per classical reference.[14] HR was prepared as per the reference from rasendrasārasaṅgraha [Figure 3].[7]{Figure 1}{Figure 2}{Figure 3}

(Voucher numbers of herbs used in the preparation of formulation: S. nigrum L.: IPGTandRA/PHM 6217; C. medica Watt.: IPGTandRA/PHM 1582; A. campanulatus L.: IPGTandRA/PHM 1359; Dolichos biflorus L.: IPGTandRA/PHM 6894) All chemicals used in the study were of analytical grade.

Animals

Healthy, female, nulliparous, and nonpregnant Wistar strain albino rats weighing 200 ± 20 g were obtained from animal house (Registration No. 548/2002/Committee for the Purpose of Control and Supervision on Experiments on Animals [CPCSEA]) attached to Pharmacology laboratory, I. P. G. T. and R. A., Jamnagar. Five animals per group were housed in each poly-propylene cage with stainless steel top grill. The dry wheat (posthulled) husk was used as bedding material and was changed every morning. The animals were acclimatized for 7 days before commencement of the experiment in standard laboratory conditions; 12 ± 01 h day and night rhythm, maintained at ambient temperature (25°C ± 3°C) and humidity (40%–60%). Animals were fed with Amrut brand rat pellet feed supplied by Pranav Agro Mills Pvt. Limited and reverse osmosis purified water was provided ad libitum. The experimental protocols used in this study were approved by Institutional Animal Ethics Committee (Approval number: IAEC/10/2012/12) and the care of animals was taken as per the guidelines of CPCSEA, India.

Grouping and schedule of acute oral toxicity study

Acute oral toxicity studies for both the test drugs were carried out following OECD guideline 425 (modified, adopted March 23, 2006) with 2000 mg/kg as a limit test.[15] The animals were randomized into three groups consisting of five animals in each. Group I was kept as control group. Group II and III were administered with test drug 1 (STBHR) and test drug 2 (ATBHR), respectively, in the form of suspension made in honey, orally, with the help of rubber catheter attached to a disposable syringe. For the preparation of suspension, test drug was taken in requisite quantity (2000 mg/kg) in small porcelain mortar, and honey (10 ml/kg body weight of rat) was added, the formed mixture was further grounded for 5 min to make it homogenous. Food, but not water, was withheld overnight before the experiment and further two hours after administration of test drugs.

Examination of physical and behavioral changes

The animals were observed continuously for 8 hours after the dosing. The careful cage side observation was done without disturbing the animal attention, and at the end of every hour, the animals were individually exposed to open arena for recording the behavioral changes like increased or decreased motor activity, convulsions, straub's tail, muscle spasm and relaxation, catatonia, spasticity, hyperesthesia, arching and rolling, lacrimation, salivation, diarrhea, writhing, changes in skin color, ataxia, narcosis, etc.

Mortality

All the animals were observed at ½, 1, 2, 3, 4, 5, 6, 8, 24 h after dosing and thereafter daily once for mortality during the entire period of the study (14 days).

Body weight

The body weight of each animal was recorded just before dosing on day one and thereafter on 7th and 14th day.

Hematological and serum biochemical parameters

On 14th day, all animals were kept for overnight fasting. On 15th day, animals were anaesthetized with diethyl ether. Supraorbital plexus was punctured under light anesthesia and blood was collected by capillary in two different types of tubes, one containing anticoagulant fluid (0.02 ml of Ethylenediaminetetraacetic acid) for hematological parameters and another plain tube for serum biochemical investigations. Then the rats were sacrificed, and the abdomen was opened through midline incision to record the autopsy changes followed by dissecting out the important organs such as thymus, heart, liver, spleen, kidney, uterus, lymph node, stomach, and ovary.

Hematological analysis was performed using an automatic hematological analyzer (Swelab, Sweden). Total red blood cell, hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, total white blood cells, percentages of neutrophils, lymphocytes, eosinophils and monocytes, packed cell volume, and platelet count were measured from the blood samples.

Serum biochemical parameters were carried out using fully automated biochemical random access analyzer (BS-200, Lilac Medicare Pvt. Ltd., Mumbai). The parameters were blood glucose,[16] serum urea,[17] serum creatinine,[18] serum total cholesterol,[19] serum triglyceride,[20] serum serum total protein,[21] serum albumin, serum globulin,[22] serum alkaline phosphatase,[23] serum glutamic oxaloacetic transaminase (SGOT),[24] serum glutamic pyruvic transaminase (SGPT),[25] serum uric acid,[26] serum direct bilirubin,[26] serum total bilirubin,[27] and serum calcium.[28]

After noting any sign of gross lesion and ponderal changes of major organs such as liver, heart, kidenys, stomach, thymus, and uterus were transferred to 10% phosphate buffered formalin solution for fixation and later on subjected to dehydrating, wax embedding, sectioning, and staining with aematoxylin and eosin for histological evaluation.[29] The slides were viewed under trinocular research Carl-Zeiss's microscope at various magnifications to note down the changes in the microscopic features of the tissues.

Statistical analysis

The results were presented as mean ± standard error of mean for five rats per experimental group. Statistical comparisons were performed by both unpaired student's t-test and one way analysis of variance with Dunnets' multiple t-test as post hoc test by using Sigma stat software (version 3.1) to determine significant difference between groups at P < 0.05.

 Results



During the course of the study no mortality was observed in treated rats and no toxic effect was observed at the given dose of 2000 mg/kg of both the test drugs. Gross behavior of all the animals was found to be normal in both the test drugs treated groups.

The changes in body weight of the treated and control rats are shown in [Table 1]. Normal progressive body weight gain was observed in normal control rats during the course of the study. A uniform increase in the body weight was observed in the ATBHR administered group. However, these changes were found to be similar in fashion as shown in control group. STBHR did not produce any changes in body weight of rats.{Table 1}

None of the hematological parameters was affected to significant levels in both the test drugs treated groups in comparison to the control group [Table 2]. Both the test drugs at the given dose increased blood urea levels, but only the increase in ATBHR treated group was statistically significant compared to the control group [Table 3]. Increase in serum alkaline phosphatase, SGPT, and SGOT levels were seen in both the test groups in comparison to control group, but it was not statistically significant. Other parameters were not affected at significant level in treated group in comparison to the control group.{Table 2}{Table 3}

ATBHR produced marked decrease in weight of spleen, thymus, and uterus; however, the results were not significant in comparison to control group [Table 4]. No significant difference was observed in all studied organs of albino rats in STBHR treated group except uterus in comparison to the control group.{Table 4}

Microscopic examination of the sections of heart and stomach exhibited normal cytoarchitecture in both the treated groups [Figure 4]. Degenerative changes such as congestion and hemorrahge in cortical and medulla part were observed in some sections of kidney from STBHR treated group and almost all sections of ATBHR treated group. Mild fatty changes were observed in some sections of liver of STBHR treated group and moderate to severe fatty changes with infiltration are observed in all sections of ATBHR treated group [Figure 5] and [Figure 6].{Figure 4}{Figure 5}{Figure 6}

 Discussion



Safety and toxicity profile of a drug is very important because it provides data for the physician to decide whether he is justified in taking a certain amount of risk in treating a disease. This mainly can be generated through laboratory animal experimentation. Since HR is herbomineral formulation containing metals such as mercury and copper, in the present study, acute toxicity was carried out to record immediate adverse signs and symptoms after the administration of single dose of drugs at dose levels that are several folds higher than the therapeutic equivalent dose.

For the purpose of reducing the variability and as a means of minimizing the number of animals used, guideline 425 of OECD was conducted in the present study using single sex (preferably females). Also, the literature surveys of conventional LD50 tests have found that usually there is slight difference in sensitivity between the sexes but, where the differences were observed, females were generally slightly more sensitive.[30] Hence, in the present study, acute toxicity was evaluated in female rats. Changes in body weight are an important factor to monitor the health of an animal. Loss of body weight is frequently the first indicator of the onset of an adverse effect. The dose, at which body weight loss is by 10% or more, is considered to be a toxic dose, irrespective of whether or not it is accompanied by any other changes.[31] In the present study, the body weight was not found affected at a significant level by administration of test drugs in comparison to the control group. Further, both the test drugs at the given dose (2000 mg/kg) did not produce any mortality during entire duration of study, no significant behavioural changes were observed and no haematological parameters were significantly affected. This implicates that LD50 of both the test drugs are higher than 2000 mg/kg which means that the test preparations may not likely to produce drastic degenerative changes at the therapeutic doses administered in clinical conditions.

The matters of concern in the present study which can be considered as toxic effects are the affected levels of blood urea, serum alkaline phosphatase, SGPT, and SGOT. These observations are suggestive of liver and kidney damage which are in corroboration with the findings of histopathological studies of these organs. This indicates the mild to moderate liver and kidney damage potential of test drugs at a very high dose of 2000 mg/kg in rats. On comparison, STBHR produced less damage than ATBHR. The toxic potential of the test drugs at higher dosage towards kidneys can be attributed to the presence of mercury in them since all forms of mercury have toxic effects in a number of organs, especially in the kidneys. There is ample evidence of the renal damage associated with mercury.[32] Acute doses of inorganic mercury (Hg2+) induce cellular necrosis that is most severe in the inner cortex and outer medulla.[33] Mercury-induced cellular degeneration leads to the loss of enzymes in the proximal tubule that can be detected as an increase in serum alkaline phosphatase levels. With further cellular and tubular necrosis intracellular enzymes (like lactate dehydrogenase) are excreted in urine and impaired reabsorption of water and solutes in the proximal tubule leads to diuresis, glucosuria, and proteinuria.[34] Although mercury may not pose serious damage to the liver, the copper from the tāmra bhasma at a higher dosage may lead to acute hepatorenal failure. Ingestion of toxic amounts (1–10 g) of copper usually results in to acute hepatotoxicity which is characterized by marked elevations in serum aminotransferase levels, increase in serum alkaline phosphatase and hepatocyte damage leading to cellular degeneration or necrosis.[35]

The mercury and copper used in HR go through procedures of śodhana, māraṇa, etc., It can be opined that ancient scholars of rasashastra were very well aware of the adverse effects of these metals at such high dose levels and that's why they developed the tedious alchemical procedures and advised to use these herbomineral formulations in lower doses. This fact can be established by the present study in which aśodhita tāmra bhasma containing HR group showed more damage potential to liver and kidneys than its counterpart group. Although further research is needed in line with the importance and necessity of śodhana in preparation of bhasmas. The metals used in herbomineral formulations are not in inorganic form, but they are converted to specific compound and organometallic complexes of different forms and sizes (micro to nano) by variuos alchemical techniques such as śodhana māraṇa, and bhāvana with different herbal juices, decoctions, etc., These procedures lead to the formation of various organo-metallic complexes in them. The differences between these complexes in śodhita and aśodhita metallic bhasma may provide some leads towards their effects and ill effects. Moreover these formulations are levitated with different herbal decoctions and juices to biologically produce nanoparticles which are found to be chelated with organic ligands derived from the herbs. This leads to the cumulative effect of the compound formulation, which makes it easily assimilable, eliminating their harmful effects and enhancing their biocompatibility.[36]

In the present study, the administered doses of test drugs are several times higher than generally advocated in clinical practice. This means that the test preparations may be safely administered at therapeutic doses in clinical conditions but only after ascertaining their chronic toxicity profiles. It is also imperative from the study that these formulations have potential to damage kidneys and liver at higher doses, so physician should take utmost care regarding their doses while administering them.

From the present study, certain leads for future studies could be generated. In order to compare two samples of same compound formulation and to find out toxicity of specific ingredient in them samples should be withdrawn at each step of manufacture and animal studies carried out at each stage. Detection and identification of organo-metallic complexes formed in herbomineral formulations by advanced analytical tools is certainly a herculean task but not impossible. Toxicity studies could also be performed with and without bhāvana to study its effect on compound formulation.

 Conclusion



From the observations recorded in acute oral toxicity study for behavioral changes, hematological and biochemical parameters, body weight changes, histopathological findings and mortality, it is clear that LD50 of both the samples of HR is higher than 2000 mg/kg and thus are safe for clinical use. From the results of biochemical parameters of liver and kidney and histopathological findings of these organs, it can be concluded that both the test drugs at such a high dose can impaire their functions. However, on comparison, hṛdayārṇava rasa prepared from śodhita tāmra bhasma (STBHR) showed relatively less significant changes in these parameters than the one which was prepared from aśodhita tāmra bhasma (ATBHR). This may reveal the importance of śodhana procedure in Rasashastra formulations. Detection of different organo-metallic complexes formed in śodhita and aśodhita bhasma samples with the help of advanced analytical techniques may provide some leads. Further, chronic toxicity profiles are necessary to establish the safety of these test drugs on chronic administration.

Acknowledgment

Authors are thankful to Ex-Director, Prof. M.S Baghel, IPGT and RA, Gujarat Ayurved University, Jamnagar, for sanctioning the necessary funds required for the study.

Financial support and sponsorship

This study was financially supported by Institute for Post Graduate Teaching and Research in Ayurveda, Gujarat Ayurved University, Jamnagar.

Conflicts of interest

There are no conflicts of interest.

References

1Acharya YT. Rasamrutam; Rasayoga Vigyaneeyam, Translated to English by Joshi Damodar. Varanasi: Chaukhamba Amarbharati Prakashan; 2003. p. 284.
2Kohli KR. Ayurvedic medicines and heavy metals issue. Ayurveda Herit 2005;1:5-6.
3Upadhyay M. Ayurved Prakasha, 3/115. Varanasi: Chaukhamba Bharatiya Academy; 1999. p. 365.
4Jagtap CY, Ashok BK, Patgiri BJ, Prajapati PK, Ravishankar B. Acute and sub chronic toxicity study of Tamra Bhasma (Incinerated copper) prepared from Ashodhita (unpurified) and Shodhita (purified) Tamra in rats. Indian J Pharm Sci 2013;75:346-52.
5Jagtap CY, Ashok BK, Patgiri BJ, Prajapati PK, Ravishankar B. Evaluation of antihyperlipidemic activity of Tamra Bhasma prepared from Shodhita (purified) and Ashodhita (unpurified) Tamra. Indian J Nat Prod Resour 2013;4:205-11.
6Anonymous. Ayurved Formulary of India. Part 1., 2nd ed., Vol. 55. New Delhi: Govt of India, Ministry of Health and Family Welfare, Dept of I.S.M. and H; 2003. p. 279.
7Krishnagopala B. Rasendrasarasamgraha, Hridroga Chikitsa, Indradeo Tripathi, 'Rasavidyotini' Hindi Commentary. 2nd ed. Varanasi: Chaukhamba Orientalia; 1998. p. 380.
8Jagtap CY, Chaudhari SY, Thakkar JH, Galib R, Prajapati PK. Assessment of genotoxic potential of hridayarnava rasa (a herbo-mineralo-metallic ayurvedic formulation) using chromosomal aberration and sperm abnormality assays. Toxicol Int 2014;21:242-7.
9Sharma SN. Rasa Tarangini, 5/27-30, Shastri KN, Hindi Commentary. Delhi: Motilal Banarasi Das; 2004. p. 79.
10Vagbhattacharya. Rasaratna Samuchchaya, 3/20, Kulkarni DA, Hindi Commentary. New Delhi: Meherchand Laxmandas publication; 1998. p. 176.
11Vagbhattacharya. Rasaratna Samucchaya, 5/13, Kulkarni D A, Hindi Commentary. New Delhi: Meherchand Laxmandas Publication; 1998. p. 93.
12Vagbhattacharya. Rasaratna Samucchaya, 5/52, Kulkarni D A, Hindi Commentary. New Delhi: Meherchand Laxmandas Publication; 1998. p. 101.
13Vagbhattacharya. Rasaratna Samucchaya, 5/53, Kulkarni D A, Hindi Commentary. New Delhi: Meherchand Laxmandas Publication; 1998. p. 101.
14Sharma SN. Rasa Tarangini, 17/40-42, Shastri KN, Hindi Commentary. Delhi: Motilal Banarasi Das; 2004. p. 418.
15Organization for Economic Cooperation and Development (OECD). Test No. 425: Acute Oral Toxicity: Up-and-Down Procedure, OECD Guidelines for the Testing of Chemicals, Section 4. Paris: OECD Publishing; 2008. https://doi.org/10.1787/9789264071049-en.
16Pennock CA, Murphy D, Sellers J, Longdon KJ. A comparison of autoanalyser methods for the estimation of glucose in blood. Clin Chim Acta 1973;48:193-201.
17Talke H, Schubert GE. Enzymatic urea determination in the blood and serum in the warburg optical test. Klin Wochenschr 1965;43:174-5.
18Slot C. Plasma creatinine determination. A new and specific Jaffe reaction method. Scand J Clin Lab Invest 1965;17:381-7.
19Roeschlau P, Bernt E, Gruber WA. Enzymatic determination of total cholesterol in serum. J Clin Chem Clin Biochem 1974;12:226.
20Fossati P, Prencipe L. Serum triglycerides determined colorimetrically with an enzyme that produces hydrogen peroxide. Clin Chem 1982;28:2077-80.
21Tietz NW, editor. Text Book of Clinical Chemistry. Philadelphia (PA): WB Saunders; 1986. p. 579.
22Doumas BT, Arends RL, Pinto PC. In Standard Methods of Clinical Chemistry. Vol. 7. Chicago: Academic Press; 1972. p. 175-89.
23Wilkinson JH, Boutwell JH, Winsten S. Evaluation of a new system for the kinetic measurement of serum alkaline phosphatase. Clin Chem 1969;15:487-95.
24Tietz NW, editor. Clinical Guide to Laboratory Tests. 3rd ed. Philadelphia (PA): WB Saunders and Company; 1995. p. 76.
25Burtis CA, Ashwood ER, editors. Tietz Textbook of Clinical Chemistry. 3rd ed. Philadelphia (PA): WB Saunders and Company; 1999. p. 652, 1136.
26Kabasakalian P, Kalliney S, Westcott A. Determination of uric acid in serum, with use of uricase and a tribromophenol-aminoantipyrine chromogen. Clin Chem 1973;19:522-4.
27Pearlman PC, Lee RT. Detection and measurement of total bilirubin in serum with use of surfactants as solubilising agents. Clin Chem 1974;20:447.
28Moorehead WR, Biggs HG. 2-Amino-2-methyl-1-propanol as the alkalizing agent in an improved continuous-flow cresolphthalein complexone procedure for calcium in serum. Clin Chem 1974;20:1458-60.
29Raghuramulu N, Nair KM, Kalyanasundaram S, editors. A Manual of Laboratory Techniques. Hyderabad: National Institute of Nutrition (NIN); 1983. p. 246.
30Lipnick RL, Cotruvo JA, Hill RN, Bruce RD, Stitzel KA, Walker AP, et al. Comparison of the up-and-down, conventional LD50, and fixed-dose acute toxicity procedures. Food Chem Toxicol 1995;33:223-31.
31Timbrell JA, editor. Principles of Biochemical Toxicology. London: Taylor and Francis Ltd.; 1982. p. 446.
32Liu J, Shi JZ, Yu LM, Goyer RA, Waalkes MP. Mercury in traditional medicines: Is cinnabar toxicologically similar to common mercurials? Exp Biol Med (Maywood) 2008;233:810-7.
33Diamond GL, Zalups RK. Understanding renal toxicity of heavy metals. Toxicol Pathol 1998;26:92-103.
34Zalups RK, Knutson KL, Schnellmann RG. In vitro analysis of the accumulation and toxicity of inorganic mercury in segments of the proximal tubule isolated from the rabbit kidney. Toxicol Appl Pharmacol 1993;119:221-7.
35Chen Z, Meng H, Xing G, Chen C, Zhao Y, Jia G, et al. Acute toxicological effects of copper nanoparticles in vivo. Toxicol Lett 2006;163:109-20.
36Kumar A, Nair AG, Reddy AV, Garg AN. Availability of essential elements in Bhasmas: Analysis of Ayurvedic metallic preparations by INAA. J. Radioanal Nucl Chem 2006;270:173-80.