Toxicity and recovery studies of two ayurvedic preparations of iron

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1 Indian Journal of Experimental Biology Vol. 47, December 2009, pp Toxicity and recovery studies of two ayurvedic preparations of iron P K Sarkar *1, P K Prajapati 2, V J Shukla 3 & B Ravishankar 3, A K Choudhary 4 1 Department of Rasashastra, J. B. Roy State Ayurvedic Medical College & Hospital, , Raja Dinendra Street, Kolkata , India 2 Department of Rasashastra and Bhaishajya Kalpana including Drug Research, 3 Pharmacology Laboratory, Institute for Post Graduate Teaching and Research in Ayurveda, Gujarat Ayurved University, Jamnagar , India 4 Department of Rasashastra, Faculty of Ayurveda, Institute of Medical Sciences, Banaras Hindu University, Varanasi , India Received 23 February 2009; revised 23 September 2009 Lauha Bhasma and in 55 mg/kg dose (5 times the therapeutic effective dose) for 60 days exhibited no serious toxic effects in Charles Foster albino rats. Both the drugs showed significant recovery from chronic toxic effect after 45 days of recovery period. Keywords: Iron, Lauha Bhasma,, Recovery, Toxicity Therapeutic effectiveness of the Ayurvedic drugs has been established and well documented by the great Acharyas in the form of classics attributed to them. However lots of changes have occurred in our living milieu since the time these classics were written and the impact of these changes on the therapeutic efficacy of the preparations formulated has not been ascertained 1. Further the art of preparing the formulations requires certain amount of expertise and no information is available about the likely impact of changes in the manufacture techniques or improper preparation on the expression of biological activity including possibility of production of undesirable effects. Lauha Bhasma and are the two most commonly used preparations of incinerated iron. These are indicated for the same as well as different diseases like in anaemia, oedema, etc. and are also used as immunomodulators (Rasayana) 2. All iron preparations are probably equally toxic per unit mass of soluble iron 3. They produce mild gastrointestinal disturbances characterized by colicky pain, nausea, vomiting, diarrhoea and gastric distress in about 6 to 12% individual 4. Many studies have so far been carried out on both of the preparations. Pandit et al. 5 investigated Lauha Bhasma for haematinic activity *Correspondent author Telephone: Mobile: prasantaksarkar@yahoo.com prasantsarkar@indiatimes.com and hemoglobin regeneration efficacy on agar gel diet and phlebotomy induced iron deficiency anemia in rats and reported significant haematinic and hemoglobin regeneration efficiency in comparison to vehicle control and ferrous sulphate control. Kanase et al. 6 studied curative effects of on liver and kidney of albino rats and noticed total recovery in two weeks. In a recent study Sarkar et al. 7 evaluated Lauha Bhasma and as haematinic on HgCl 2 induced anaemia in rats, and reported significant haematinic and cytoprotective activity of both the Bhasma preparations. The World Health Organization (WHO) guidelines for conducting toxicity study recommend to observe recovery of the experimental animals from toxicity after a certain interval of toxicity study 8. Excess iron causes iron overloading syndrome in the body 9. Both the test drugs contain iron. The present toxicity study of Lauha Bhasma and has been undertaken to evaluate the toxicity profiles of both the Bhasma preparations and to observe recovery from the toxic effects of Lauha Bhasma and Mandura Bhasma, if any during toxicity study. The present study confirms to the guidelines laid by the Institutional Animals Ethics Committee. Materials and Methods Preparation of test drugs Lauha Bhasma and were prepared in the laboratory of Department of Rasashastra and Bhaishajya Kalpana, I.P.G.T. & R.A., Gujarat Ayurved University. Scraps

2 988 INDIAN J EXP BIOL, DECEMEBER of wrought iron and old rusted iron collected from Mahabaleswara were procured from local market, taken as raw materials of Lauha and Mandura, and made into coarse powder by hammering 10. These were then subjected to Shodhana (purification) according to traditional Ayurvedic procedure. For this purpose, these were heated to red hot and quenched in sesame oil, butter milk, cow s urine, sour gruel, decoction of Kulottha seeds (Dolichos biflorus) and decoction of Triphala, 7 times in each media, total 42 times 11. The purified materials were subjected to Marana (incineration) according to traditional procedure. For this purpose, one part of these materials were mixed with 1/12th part of purified cinnabar (Hingula, HgS) and was levigated by Kumari Swarasa (aloe gel) for 6 h 12. Pellets were prepared from this levigated doughy mass and taken into earthen crucibles faced together, and the junction was sealed by mud smeared clothes. This apparatus, called as Sarava Samputam was subjected for heating in electric muffle furnace. Heating of materials confined to this apparatus is called Putapaka in the parlance of Ayurveda. Burning was continued approximately for 3 h, when cooled down (approx. after 24 h) the apparatus was taken out and opened to get the incinerated iron powder. These procedures were repeated for 7 times and finally the prepared test drugs (Lauha Bhasma and Mandura Bhasma) were collected separately in glass container for further pharmacological studies. Animal, diet, drug and dose Charles Foster strain albino rats of either sex weighing between g obtained from the animal house were housed in breeding cases at an ambient temperature with a natural day and night cycles. The animals had free access of Amrut brand rat pellet feed supplied by Pranav Agro Industries and tap water. The dose for experimental study of the test drugs was calculated by extrapolating the human dose (125 mg/day) to animal dose (11 mg/kg) based on the body surface area ration. Drug suspension was prepared in 3% gum acacia solution (1 ml in 50 ml distilled water). Experimental design This study was divided into two phases, toxicity study and recovery study. In toxicity study, toxicity of Lauha Bhasma and were assessed by administering 55 mg/kg dose (5 therapeutic effective dose) of test drugs suspension through oral route by No. 3 simple rubber catheter for 60 days. Some rats of toxicity study from each test drug treated group were kept for recovery study for a period of 45 days. During this recovery period no drug was given, only normal diet and tap water was provided to the rats. At the end of this study any recovery from the toxicity was observed. Study protocol for toxicity study Total 26 rats were divided into 3 groups, group I and II were comprised of 10 rats each and group III was comprised of 6 animals. Each group contained equal number of male and female rats. Animals in Group I and II were treated with Lauha Bhasma and Mandura Bhasma respectively. Group III was designated as control group and received vehicle (1 ml 3% gum acacia solution in 50 ml distilled water) used for preparation of the suspension of test drugs. Suspension was prepared in 11 mg/ml concentration. The schedule was continued for 60 days with daily doses of test drugs and vehicle. Gross behaviour was observed throughout the study period. On 61st day 6 rats from each group were scarified by stunning, blood was collected from jugular vein for hematological and biochemical tests. All vital organs were collected for histopathological study and weight of rats before sacrificing was recorded. Study protocol for recovery study Rats (4), equal number of male and female from each Group I and Group II were kept for recovery study. This schedule was continued for 45 days. Gross behaviour was observed throughout the period of study. On 46th day rats were sacrificed by stunning, blood was collected from jugular vein for haematological and biochemical tests, all vital organs were collected for histopathological study. Statistical analysis All the values were expressed as mean ± SE. The data were analyzed by unpaired 't' test. A level of P<0.05 and P<0.01 was considered as statistically significant and highly significant respectively. Level of significance was noted and interpreted accordingly. Results Effects on body weight Body weight of rats increased in all the groups. In toxicity study, body weight increase was comparatively less in Lauha Bhasma treated group than treated and control groups. Changes were found to be highly significant (P<0.01) in both treated group and control group. In recovery study, a significant increase (P<0.02) in body weight was found in Lauha Bhasma recovery group and a statistically highly significant (P<0.01) increase in

3 SARKAR et al.: TOXICITY STUDY OF AYURVEDIC IRON PREPARATIONS 989 body weight was observed in recovery group (Table 1). Effects on biochemical parameters In toxicity study, a statistically significant increase (P<0.02) in blood sugar level was found in treated group, whereas a mild increase in blood sugar level observed in Lauha Bhasma treated group was found to be statistically non-significant. A mild decrease (P>0.1) in serum urea level was observed in both the test drug treated groups. A mild increase (P>0.1) in serum creatinine level was found in both the treated groups in comparison to control group. In recovery study, a statistically highly significant (P<0.001) increase in blood sugar level was observed in Lauha Bhasma recovery group, whereas a mild non-significant (P>0.1) increase in blood sugar level was found in recovery group in comparison to their respective toxicity group. Statistically highly significant (P<0.01) decrease in SGOT activity was observed in both the recovery Table 1 Changes in body weight & g in control and treated rats [Values expressed in g are mean ± SE of 6 animals in control and toxicity groups and 4 animals in recovery groups] Groups Dose (mg/kg) Initial Final Change (%) Control ± ± ± 2.7** Lauha Bhasma ± ± ± 5.4 toxicity Lauha Bhasma ± ± ± 2.3* recovery toxicity ± ± ± 2.1** recovery ± ± ± 1.6** *P 0.05; **P 0.01 by the student s t test. group in comparison to their toxicity group. Serum alkaline phosphatase activity was increased highly significantly (P<0.01) in recovery group, whereas the elevation was non-significant in Lauha Bhasma recovery group in comparison to respective toxicity group. Other biochemical parameters were not affected to significant extent (Table 2). Effects on haematological parameters In the toxicity study haematological parameters (Hb%, total RBC, total WBC, platelet count, etc,) were not changed to significant level in both the test drug administered groups. But in recovery study, significant (P<0.05) and non significant (P>0.05) decreases in haemoglobin content was found in Lauha Bhasma recovery group and recovery group respectively. Significant (P<0.05) decrease was also observed in haematocrit value, red cell distribution width and mean corpuscular volume, but mean corpuscular haemoglobin concentration was increased highly significantly (P<0.01) in both the recovery groups. The data of total WBC count of both recovery groups were almost similar to the data of control group of toxicity study. Mean platelet volume was decreased highly significantly (P<0.01) in both the recovery groups. Other haematological findings of the recovery study do not alter to significant level (Table 3). Effects on histopathology of vital organs In liver, even obtained from control group, mild to moderate fatty changes occurred. Moderate fatty degenerative changes, diffused necrosis, periportal necrosis, central vein congestion, and sinusoidal dilatation observed in Lauha Bhasma treated group. Mild fatty changes and sinusoidal dilatation were observed in Mandura Bhasma treated group. Other vital organs like spleen, Table 2 Biochemical observations after treatment and recovery period in control and treated rats [Values are mean ± SE of 6 animals in control and toxicity groups and 4 animals in recovery groups] Parameters Groups (dose mg/kg) Control Lauha Bhasma Lauha Bhasma Blood sugar (mg/dl) ± ± ±4.82** ±3.80* ±3.50 S. urea (mg/dl) 39.9± ± ± ± ±3.15 S. creatinine (mg/dl) 0.86± ± ± ± ±0.17 S. cholesterol (mg/dl) 52.08± ± ± ± ±12.0 S. triglyceride (mg/dl) ± ± ±06.94* ± ±44.0 S. total protein (g/dl) 7.0± ± ± ± ±0.23 SGOT (IU/L) ± ± ±13.16** 445.8± ±108.6** S. alkaline phos. (IU/L) 40.53± ±4.42* 83.45± ± ±9.10** *P 0.05; **P 0.01 by the student s t test; toxicity groups are compared with control group; recovery groups are compared with their toxicity groups.

4 990 INDIAN J EXP BIOL, DECEMEBER heart, lungs, kidney, brain, etc. exhibited almost normal cytoarchitecture in all the groups. Microscopic examination of sections of liver from both the recovery groups was observed. Almost normal cytoarchitecture with mild micro-fatty changes in hepatic cells was observed in Lauha Bhasma recovery group, whereas sinusoidal dilatation with micro-fatty changes in hepatic cells was observed in Mandura Bhasma recovery group (Fig 1). Discussion Body weight indicates health status of any living being. So here increase in body weight in albino rats indicate normal progressive health status of the animals and it is also indicative of the fact that no degenerative changes are occurring during drug administration. So, increase in body weight in both toxicity and recovery study could suggest that there is no harmful effect of the test drugs on body function as a whole. Blood sugar level is dynamic in nature and is influenced by several factors, some of which favour elevation and some favour lowering. In the above background it can be suggested that the test drugs may be interfering with the secretion of insulin or may be enhancing the release of hyperglycemia favouring factors like glucagons and corticosteroids 13. This hyperglycemia may be due to decrease in the secretion of insulin. In recovery study the blood sugar level was increased further in both the recovery groups. Its statistically highly significant increase (P<0.01) in Lauha Bhasma recovery group indicates existing of underlying persisting pathological condition in this group. In contrast, the significant blood sugar elevation observed during toxicity study in administered group was found to be attenuated to greater extent and almost reaches to normal level. This clearly indicates strong recovery from the hyperglycemia producing proclivity of the test drugs. Liver is the main organ for carbohydrate metabolism. Decreased hepatic glucose uptake leads to hyperglycemia 14. In the toxicity study, injury in liver tissue was found, this injury also existed, though less severe, during the recovery period. This persisting hepatic functional derangement may be the cause of the persisting hyperglycemia observed in Lauha Bhasma administered group. The SGOT level was decreased highly significantly (P<0.01) in both recovery groups. The level of SGOT activity was quite high during toxicity study and the decrease indicates return towards normalcy. However, it may be noted that the level of SGOT was high in control group also. Taking all the factors in consideration it can be suggested that lowering of elevated SGOT level is indicator of recovery from early derangement. The highly significant (P<0.01) increase in serum alkaline phosphatase level in recovery group may indicate that during the toxicity study marked decrease in serum alkaline phosphatase activity was observed and the elevation observed during the recovery phase is a definite indicator of recovery from whatever derangement that might have occurred during the toxicity study. The observed changes in haemoglobin level and red cells indices may be attributed to the stoppage of Table 3 Haematological observations after treatment and recovery period in control and treated rats [Values are mean ± SE of 6 animals in control and toxicity groups and 4 animals in recovery groups] Parameters Groups (dose mg/kg) Control Lauha Bhasma Lauha Bhasma Hb (g %) 14.53± ± ±0.27* 14.96± ±0.84 RBC (10 12 /L) 8.25± ± ± ± ±0.24 Haematocrit (%) 47.80± ± ±1.54** 50.22± ±1.45* RDW (%) 12.98± ± ±0.12** 14.86± ±0.20** MCV (fl) 57.95± ± ±1.19* 57.44± ±0.30** MCH (pg) 17.65± ± ± ± ±0.05 MCHC (g/dl) 30.47± ± ±0.69** 29.80± ±0.05** TLC (10 9 /L) 6.25± ± ± ± ±0.90 Platelet (10 9 /L) 969.0± ± ± ± ±55.00 MPV (fl) 7.7± ± ±0.11** 8.2± ±0.20** PDW (%) 9.05± ± ±0.21* 8.78± ±0.40 *P 0.05; **P 0.01 by the Student s t test; toxicity groups are compared with control group; recovery groups are compared with respective toxicity groups.

5 SARKAR et al.: TOXICITY STUDY OF AYURVEDIC IRON PREPARATIONS 991 Fig. 1 Effect of Lauha Bhasma and toxicity and recovery period on liver. [(A) Lauha Bhasma treated moderate fatly changes, sinusoidal dilatation, central vein congestion, diffusion necrosis; (B) Lauha Bhasma recovery almost normal cytoarchitecture with sinusoidal dilatation; (C) treated moderate fatty changes, sinusoidal dilatation and mild diffusion necrosis ; (D) recovery mild micro fatty changes ; CV: Central vein; FC: Fatty changes; HC: Hepatic cell; KC: Kuffer cell; N: Necrosis; S: Sinusoid; SD: Sinusoidal dilatation 1-4: 400]. iron supplement in the form of test preparations in these groups in comparison to control group. Non availability of additional iron might have induced withdrawal effect in these test drug administered groups leading to the above observed changes. From this observation it may be suggested that the drug administration involving the test drugs should not be stopped abruptly because of the possibility of the reversal of the observed efficacy as a result of withdrawal effects. It would be advisable to stop the drugs gradually rather than stopping abruptly. Mean platelet volume was decreased highly significantly (P<0.01) in both the recovery groups may associate with low platelet count hence the changes may be considered as a consequence of platelet count lowering effect of test drugs. The observed pathological changes in liver of treated groups in toxicity study may be due to over dose leading to higher availability of free iron that exceeds the iron binding capacity of transferrin leading to liver injury. Almost normal cytoarchitecture with mild fatty changes in hepatic cells observed in both the recovery groups, were suggestive of partial recovery from the hepatotoxicity of both the test drugs, which was found in toxicity study. Impairment in the hepatic functions and cytoarchitecture in toxicity study can be explained as ferrous iron is absorbed into mucosal cells of the duodenum and jejunum and is oxidized to ferric iron and bound to ferritin. It is then slowly released into the plasma, where it is bound to transferrin, a specific iron binding globulin, and transported to tissues. Iron bound to transferrin is nontoxic with over dosage free iron that exceeds the iron binding capacity of transferrin, and high ferritin levels cause tissue damage. Free iron also injures mitochondria, causes lipid per oxidation, and may result in renal, hepatic, myocardial and pulmonary injury 15. In this study both

6 992 INDIAN J EXP BIOL, DECEMEBER Lauha Bhasma and were given in five times (55 mg/kg) more than therapeutic dose (11 mg/kg) for a long duration (60 days). And this overdose may be the cause for the observed pathological changes. Analysis of the data of the recovery study reveals that rats of both the recovery groups have shown partial to significant recovery with respect to ponderal, biochemical, haematological and histopathological parameters. Haematological changes especially in Lauha Bhasma treated group were indicative of pseudo-iron-deficiency induced by sudden stoppage of iron supplement in the form of test drugs. However no signs of iron overloading syndrome was observed in biochemical and haematological parameters in both the recovery groups. Acknowledgement Thanks are due to the Director, I. P. G. T. & R. A., Gujarat Ayurved University for facilities. References 1 Singh R H, Tradicional medicine and the new patent regime, Seminar proceeding, National seminar on Ayurveda for better health, (Gujarat Ayurved University, Jamnagar) Sharma S P, Rasatarangini, edited by K N Shastri, (Motilal Banarasi Das, Varanasi) 2000, Clark W M, Jurow S S, Walford R L & Warthen R O, Ferrous sulfate poisoning, Amer J Dis Child, 88 (1954) Satoskar R S, Bhandarkar S D & Ainapure S S, Pharmacology and pharmacotherapeuties, Vol 1 (Popular Prakashan, Mumbai) 1997, Pandit S, Biswas T K, Debnath P K, Saha A V, Chowdhury U, Shaw B P & Mukherji B P, Chemical and pharmacological evaluation of different ayurvedic preparations of iron, J Ethnopharmacol, 65 (1999) Kanase A, Patil S & Thorat B, Curative effects of Mandura Bhasma on liver and kidney of albino rats after induction of acute hepatitis by CCl 4, Indian J Exp Biol, 35 (1997) Sarkar P K, Prajapati P K, Choudhary A K, Shukla V J & Ravishankar B, Haematinic evaluation of Lauha Bhasma and on HgCl 2 induced anaemia in rats, Indian J Pharm Sci, 69 (2007) Anonymous, Research guidelines for evaluating the safety and efficacy of herbal medicine (The Office of Publications, World Health Organization, Geneva), 1993, Chaudhuri S K, Concise medical physiology (New Control Book Agency (P) Ltd, Calcutta) 1993, Upadhyay Madhav, Ayurved Prakash, edited by G S Mishra, (Chaukhamba Bharati Academy, New Delhi) 1994, Rasavagbhat, Rasa Ratna Samucchaya, edited by D A Kulkarni, (Meherchand Lacchaman Das, New Delhi) 1999, Bhatta K G, Rasendra Sara Samgraha, edited by I D Tripathi, (Chaukhamba Orientalia, Varanasi) 2000, Das P C, Text book of medicine (Current Books International, Calcutta) 2000, Braunwald E, Fauci A S, Kasper D L, Hauser S L, Longo D L, Jameson J L, editors Harrison's principles of internal medicine, Vol 2 (The McGraw Hill Companies, New Delhi) 1998, Chalterjee C C, Human physiology, Vol 1 (Medical Allied Agency, Calcutta) 1994, 161.