|Year : 2016 | Volume
| Issue : 3 | Page : 159-166
Toxicity study of Lauha Bhasma (calcined iron) in albino rats
Namrata Joshi1, Manoj Kumar Dash2, Laxmikant Dwivedi3, GD Khilnani4
1 Department of Rasa Shastra, Faculty of Ayurveda, Institute of Medical Sciences, Banaras Hindu University, Varanasi, Uttar Pradesh, India
2 Department of Rasa Shastra and Bhaishajya Kalpana, Government Ayurveda College, Raipur, Chhattisgarh, India
3 Department of Rasa Shastra and Bhaishajya Kalpana, G.J. Patel Institute of Ayurvedic Studies and Research, Anand, Gujarat, India
4 Department of Pharmacology, S.M.S Medical College, Jaipur, Rajasthan, India
|Date of Web Publication||8-Apr-2016|
Department of Rasa Shastra, Faculty of Ayurveda, Institute of Medical Sciences, Banaras Hindu University, Varanasi, Uttar Pradesh - 221 005
Source of Support: None, Conflict of Interest: None
Background: Lauha Bhasma(LB) is a complex herbomineral preparation widely used as an Ayurvedic hematinic agent. It is an effective remedy for chronic fever (jīrṃa jvara), phthisis (kşaya), Breathlessness (śvāsa) etc., and possesses vitality enhancing (vājīkara), strength promoting and anti aging (rasāyana) properties.
Objectives: The present work was conducted to establish the safety aspects of the use of Lauha bhasma.
Setting and Design: LB was prepared by Ayurvedic procedures of purification (śodhana), sun drying (bhānupāka), sthālīpāka, followed by repeated calcination (māraṃa) and “nectarization” (amṃṛtīkaraṃa). The resultant product was subjected to acute and sub acute toxicity studies.
Materials and Methods: Acute and subacute toxicity study of LB was conducted in albino rats. Criteria for assessment included ponderal changes, change in biochemical parameters viz., LFT and KFT and hematological parameters. Histopathological studies of different organs including liver, kidney, spleen, testis etc., were also conducted to observe pathological changes if any.
Results: In the acute toxicity study, the animal group did not manifest any signs of toxicity and no mortality was observed up to 100 times the therapeutic dose (TD). Significant increase in blood urea (27.83%, P< 0.01), serum creatinine (30.92%, P< 0.05), Aspartate aminotransferase (15.09%, P< 0.05), and serum alkaline phosphatase (27.5%, P< 0.01) was evident in group IV (10 TD). A significant increase in serum total protein (6.04%, P< 0.05) level was observed in group III (5 TD). Histopathological examination of livers in group IV (10 TD) showed mild inflammation in terms of bile stasis, peri-portal hepatic inflammation and sinusoidal congestion; lymphocyte infiltration in kidney and intracellular deposits in the splenic tissue.
Conclusion: Lauha Bhasma was found to be safe at the therapeutic dose and also at five times the therapeutic dose levels. However, alteration in some of the biochemical and haematological parameters along with histopathological findings were evident at the highest dose level.
Keywords: Biochemical, histopathology, Lauha bhasma, liver, spleen Triphala, toxicity
|How to cite this article:|
Joshi N, Dash MK, Dwivedi L, Khilnani G D. Toxicity study of Lauha Bhasma (calcined iron) in albino rats. Ancient Sci Life 2016;35:159-66
| Introduction|| |
Traditional medicines are used in the treatment of various chronic disorders and for the improvement of well-being of individuals. In Ayurveda, metals such as Iron, Copper, Zinc, and Lead, etc., are used in many preparations, after transforming the metals into nonmetallic forms. The medicines so prepared are classified under a groupcalled Rasauşadhis. Rasauşadhis have been prescribed by Ayurvedicphysicians since long with rare mentions of toxicity. It is observed that herbomineral complexes are more stable and more interactive compared to plain herbs as these result in faster therapeutic action and have a longer shelf life.Bhasmas are unique preparations involving metallic/mineral preparations calcined using heat to transform metals into non-toxic organometallic forms. In preparations of these Bhasmas, the preliminary process which causes detoxification without harming its medicinal properties (guṃas) is called as 'Śodhana'., The process of śodhana was well accepted by the pioneers of Rasaśāstra for the purification of herbomineral drugs. These methods were developed to detoxify the raw material by chemical transformations and enhance the properties and therapeutic potential., There have been concerns raised regarding the use of metals and minerals in therapeutics. Raising safety concerns about traditional preparations, the WHO has issued guidelines regarding toxicity studies of herbal drugs. The present study was designed to establish the toxicity profile of Lauha Bhasma (LB) in experimental animals.
| Materials and Methods|| |
Iron flakes were procured from a local market of Jaipur. Raw Triphalā used in the pharmaceutical processing was procured from pharmacy of NIA Jaipur and was made into a decoction. Sesamum oil (tila tailam), buttermilk (takra), seeds of Dolichos biflorus (kullatha) used for śodhana were purchased from local market and authenticated at Pharmacognosy Laboratory, National Institute of Ayurveda, Jaipur. Cow urine (gomūtra) was procured from a local gośālā.
Preparation of Lauha bhasma
Iron flakes were subjected to the Ayurvedic process of purification which included generalpurification (sāmānyaśodhana), specificpurification(viśeşaśodhana), followed by sun drying (bhānupāka),sthālīpāka, levigation and calcination (puṃapāka) and “nectarization” (amṃṛtīkaraṃa) as per the classical references mentioned in the Ayurvedic Formulary of India (AFI). The final product was named as LB and was subjected to acute and sub-chronic toxicity studies.
Adult albino rats of Wistar strain of both sex weighing between 150 – 200 g were used in this study. They were procured from the animal house attached to the Department of Zoology, Rajasthan University, Jaipur. The animals were kept in standard conditions of 22°C ± 2°C and relative humidity 55% ±15%. The rats had free access to food (Pranav agro mills “Amrut” brand rat pellets) and water ad libitium with 12 hours light and dark cycle. All animals were acclimatized for at least 5 days before the start of the study. All the experimental protocols were approved by Institutional Animal Ethics Committee (IAEC) and performed according to the CPCSEA guidelines for the care and use of animals. Doseof Lauha bhasma was escalated as per conversion of human dose to experimental animal dose.
Acute toxicity study of Lauha bhasma
Totally, 15 Wistar albino rats of either sex, weighing 150–250 g were divided randomly into five groups, containing three animals each. All animals in group I were treated with Lauha bhasma orally at the dose of 10 times the therapeutic dose. Animals of Group II were given 20 times the therapeutic dose while, group III were given 40 times, Group IV were given 80 times, and Group V were given 100 times the therapeutic dose of Lauha Bhasma respectively. Single doses of drugs was administered orally according to the stated dosage schedule. Gross behaviour and exitus (death) were recorded for 14 consecutive days.
Sub-acute toxicity study of Lauha bhasma
Twenty four Wistar rats (weighing 150–250 g) were randomly assigned into four groups of six each as shown in [Table 1]. First group was treated as control whereas other three groups were administered therapeutic dose (TD) i.e., 4.16 mg/kg, five times of TD (20.80 mg/kg) and ten times of TD (41.60 mg/kg) along with 0.5 ml of honey as vehicle respectively. Initial body weights of all the animals was recorded and blood was drawn from supraorbital plexus  for laboratory testing. The same procedure was repeated at the end of study. At the end of study the rats were sacrificed by stunning and severing the neck vessels.
All the vital organs were carefully dissected, cleaned, weighed and transferred to Bouin's solution for preservation and sent to a commercial laboratory for preparation of histopathological slides. The slides were scanned in trinocular Carl Zeiss's microscope (Germany) under different magnifications. Changes if any, in cytoarchitecture were noted down.
All the hematological, histopathological as well as biochemical studies were conducted in a private well equipped laboratory in Jaipur.
Hematological and biochemical analysis
Hematological analysis was performed using an automatic hematological analyser. Samples of blood were drawn at the beginning and termination of the experiment. Parameters studied included red blood cell (RBC) count which was done by Haymes method, white blood cell (WBC) count by Turkish method, haemoglobin (Hb) by Sahil's method. Packed cell volume (PCV), mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC) and platelets count were investigated by complete blood count (CBC). For biochemical analysis, blood sugar, total cholesterol, triglyceride, high-density lipoproteins (HDL), urea, creatinine, total protein, bilirubin, aspartate aminotransferase (AST), alanine aminotransferase (ALT) and alkaline phosphatase (ALP) were analysed by rapid photoelectric method.
Values reported are mean ± standard error. The data were analysed by unpaired Student's t-test and ANOVA. A level of 0.001 was considered to be highly significant whereas P < 0.05, 0.01 were considered to be significant. Level of significance was noted and interpreted accordingly. All the statistical analyses were performed using SPSS, version 15 (SPSS Inc., Chicago).
| Results and Discussion|| |
Iron based drugs are widely used in modern medicine as haematinics. These drugs are known to induce adverse drug reactions including gastro intestinal symptoms such as nausea, vomiting, epigastric pain, eructation, pyrosis, meteorism, borborygami, colic pain, flatulence, constipation, black faeces, and diarrhoea. The haematinics market in India is currently worth around Rs. 900 crore and is growing at 15% per annum.Lauha Bhasma can be an effective hematinic provided its safety can be established. In the acute toxicity study, none of the animal groups manifested any signs of toxicity and no death was observed even at a dose which was 100 times the therapeutic dose. There was only mild behavioural hyperactivity noticed. From this it can be concluded that the approximate LD50 value is more than 416 mg/100 g of the weight of the experimental animal.
In sub-acute toxicity study, in all groups at varying test doses, a considerable non-significant increase in body weight was evident. Body weight is indicative of increased appetite and food intake and adverse effects of a drug may become expressed as decrease in body weight. As there was no decrease in body weight observed, it may be presumed that LB at even its highest dose level did not have any deleterious effect. A non-significant increase or decrease in organ weight was evident in all groups as shown in [Table 2]. Eleven biochemical parameters [as shown in [Table 3] and [Table 4] were measured in all three test groups, out of which significant increase in blood urea (27.83%, P < 0.01), serum creatinine (30.92%, P < 0.05), Aspartate aminotransferases (15.09%, P < 0.05), and serum alkaline phosphatase (27.5%, P < 0.01) was evident in group IV (highest dose level). A significant increase in serum total protein (6.04%, P < 0.05) level was observed in group III. Urea contributes most of the body's non-protein nitrogen. It is the major end product of protein catabolism synthesized in liver, released in blood and excreted by the kidneys. It is a chief indicator of renal and hepatic integrity. Elevated serum urea level may be due to pre renal, renal or post renal etiology. Similarly, serum creatinine is a product of creatine and phosphocreatine, which are important components of muscle. Creatinine is freely filtered and therefore the serum creatinine level depends on the glomerular filtration rate. Renal dysfunction diminishes the ability to filter creatinine and raises its serum level. The only condition that causes a significant increase in serum creatinine level is damage to a large number of nephrons. The increase in serum urea and creatinine in animals which received higher dose of Lauha bhasma with evident inflammatory histopathological changes indicate that as the dose is increased from intermediate dose level (i.e., 5 times to T.D.) there is a likelihood of kidney damage even when absolute values were still within normal range. However, utmost precautions are needed in prescribing higher than equivalent doses in human beings. This feature is totally dose dependent and result of excess of free iron, which acts as a free radical and is tissue toxic. Aspartate aminotransferases (APT) is a liver enzyme that aids in producing proteins. Besides liver, it is also found in other organs such as heart, muscle, brain and kidney. Injury to any of these tissues can cause an elevated blood level. It also helps in detecting hepatocellular necrosis but is considered a less specific biomarker enzyme for hepatocellular injury  as it can also signify abnormalities in heart, muscle, brain or kidney. On the other hand serum alkaline phosphatase (ALP) is an important parameter in distinguishing hepatobiliary disease. It is particularly present in the cells which line the biliary ducts of the liver. Cholestatic injury is characterized by predominantly initial alkaline phosphatase level elevations. Significant but not marked increase in APT at the highest dose (H.D.) which was 10 times the Therapeutic Dose (T.D.) level with simultaneous increase in ALP, as well as histopathological evidence of bile stasis reflects a stage of hepatobiliary damage, although again it is a dose dependent finding as increase in alkaline phosphatase and/or bilirubin with little or no increase in ALT is primarily a biomarker of hepatobiliary effects and cholestasis., The estimation of total proteins in the body is helpful in differentiating between normal and damaged liver functions. This is because the majority of plasma proteins such as albumins and globulins are produced in the liver. Low total protein level is suggestive of kidney disorder or disorder where protein is not absorbed or digested properly. Thus, increase in total body protein without any untoward histopathological findings is suggestive of anabolic effect of test drugs. Analysis of blood parameters is relevant to risk evaluation of alterations of the haematological system in humans. A highly significant increase in haemoglobin percentage at T.D (5.25%, P < 0.001) and significant increase in other two tested groups shows haematinic potential. Just as haemoglobin concentration, the total R.B.C. count is an indicator of ready bioavailability of iron. At therapeutic dose level, an increase in total R.B.C. count was observed thus suggesting better absorbability and ready availability of iron for R.B.C. formation. Other red cell indices as shown in [Table 5] were not much affected except for a change in haematocrit which showed a significant increase (8.68%) at therapeutic dose level. None of the groups showed any effect on W.B.C. and platelet count except for their raised levels at 10 T.D. Increased WBC count is indicative of inflammatory conditions of certain organs, especially of liver. Histopathological findings showing normal tissue structures after administration of Lauha Bhasma is shown in [Figure 1], however histopathological examination showed some notable changes in liver, kidney and spleen at highest dose treated L.B. group as shown in [Table 6]. Liver findings of the 10 T.D. group were suggestive of inflammatory infiltrate [Figure 2]c, mild inflammation in terms of bile stasis [Figure 2]d, and sinusoidal congestion. [Figure 2]f, mild inflammation with peri-portal ballooning [Figure 2]e was also seen in 5 T.D. group. Similarly, 10 T.D. group showed lymphocyte infiltration [Figure 2]a in kidney which in all of the other dose was found to be of normal cytostructure. The same group showed intracellular deposits in the splenic tissue that might be due to deposition of excess of iron that failed to get metabolized [Figure 2]g. Increase in some of the indicators for liver and kidney functions i.e. urea, creatinine, ALT etc., along with histopathological findings may contribute to elevation of WBC count and was only seen in group treated with highest dose. None of the findings could be attributed to toxic effect of the drug as most of the findings observed are mild in nature and thus are self-limiting. Critical analysis of the observations mentioned above reveals that Lauha bhasma did not impart any untoward effect when administered at therapeutic dose level but on increasing the dose level further, features suggestive of altered physiology (not toxicity) were evident. So, it may be concluded that the untoward effect of Lauha is totally a dose dependant phenomenon. The famous phrase of Paracelsus “all things are poisonous and there is nothing that is harmless, the dose alone determines that something is no poison” is true for Ayurvedic metallic preparations. Use of Lauha Bhasma alone at high doses is not recommended in Ayurvedic texts. Wherever Lauha bhasma is administered in high doses, it is administered in one of the following ways: (i) Kalpa Krama i.e., administration in gradual increasing order so as to make the body adapt to the large dose or (ii) as a kalpa i.e., with sufficient amount of adjuvant drugs that help in counteracting the ill effect of metal. Further, these manoeuvers are chiefly employed for rasāyanakarma of kşetrīkaraṃ a (making body suitable for excess amounts of metal) as necessary. But for routine therapeutic application, such a large amount of metal is not usually prescribed. Moreover, Lauha kalpas are administered along with suitable kramaṃa dravyas (vehicle or anupāna) rather than the Lauha bhasma being administered alone. These kramaṃa ~ Anupāna drugs act as on adjuvants to Lauha and exert synergistic effects to potentiate the therapeutic action and also eliminate the innate toxic effects of the metal. A great emphasis is laid on dosage and anupāna (vehicle) with which a bhasma should be administered. In anupāna may possibly lie the key to the safety of the bhasma. In absence of such caution, adverse reaction is likely.Madhu (honey) as anupāna (vehicle) brings about quick action due to its Yogavāhi (super-advenient) property.
|Figure 1: Normal cytostructure of histopathological findings of toxicological study of Lauha Bhasma. (a) Photograph of liver showing normal cytostructure of portal tract control group (H and E, ×10) (portal vein hepatic artery, bile duct hepatocyte). (b) Photograph of spleen showing normal follicular cytostructure control group (H and E, ×10). (1) Central sinusoid (2) red pulp space (3) white pulp space. (c). Photograph of heart showing normal cytostructure control group (H and E, ×40). (d) Photograph of kidney showing cortical and medullary areas control group (H and E, ×10). (1) Cortex (2) medulla (3) glomerulus (4) renal tubule. (e) Photograph of kidney showing normal cytostructure of glomerulus control group (H and E, ×40). (f) Photograph of brain showing normal cytostructure control group (H and E, ×40). (1) Grey matter (2) white matter (3) astrocyte (4) oligodendrocyte|
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|Figure 2: Histopathological findings in toxicoogical study of Lauha Bhasma. (a) Photograph of kidney showing periportal lymphocytic infiltrations L.B. 10 T.D. (H and E, ×40). (b) Photograph of renal tubule showing normal cytostructure L.B. 5 T.D. (H and E, ×40). (c) Photograph of liver showing inflammatory infiltrate L.B. 10 T.D. (H and E, ×40). (d) Photograph of liver showing bile stasis L.B. 10 T.D. (H and E, ×10). (e) Photograph of liver showing mild inflammation with periportal ballooning L.B. 5 T.D. (H and E, ×10). (f) Photograph of liver showing sinusoidal congestion L.B. 10 T.D. (H and E, ×40). (g) Photograph of spleen showing intra cytoplasmic pigments (normal cytostructure) L.B. 10 T.D. (H and E, ×40)|
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Metals, while being made into bhasma, get chelated with organic molecules (ligands) present in the herbs which leads to better assimilation. Chelation therapies have been used to bring down the levels of toxic metals in patients. During pharmaceutical processing of Lauha bhasma, right from śodhana till amṃṛtīkaraṃa, Triphalā is used as an organic media to convert metal Iron (Lauha) into a herbomineral complex. Triphalā mainly consists of tannins, gallic acid, ascorbic acid (Vitamin C), and phenolics. Ascorbic acid increases the bioavailability of iron by converting Fe 3+ to Fe 2+, while phenolics can reduce the iron by binding to it. The presence of ascorbic acid or a lack of dietary tannins has both been suggested as contributing to clinical/pathological iron storage disease. Excess Iron causes Iron overloading in the body  and can damage the liver, heart, and pancreas and irritate the stomach and gut, causing constipation and diarrhoea. In other words, the various constituents of Triphalā have antagonizing activity and thereby too much iron absorption is prevented. InAyurvedictexts, terms like Vişaghna (antitoxic), Giridoşa nāśaka (remover of metallic properties) are abundantly used for Triphalā, and this is an indication of the above phenomenon. Triphalā is a mild laxative and thereby counteracts the constipating property of iron and thus is beneficial. This may be the reason due to which ācāryas might have recommended Triphalā in a maximum number of Lauha formulations.
The findings from one study suggested that Triphalā and its individual constituents have an inhibitory effect on metabolic enzymes when consumed along with therapeutic products. Further the inhibitory effects were relatively comparable to all the constituents tested, despite the variability of the content of biomarker. Triphalā and its ingredients are likely to inhibit drug metabolizing enzymes, but less likely to produce significant drug interactions. Certain major factors of metabolism such as competition between coadministered drugs, unspecific interactions with proteins, and enzyme induction due to chronic intake are not addressed in that in vitro assay. However, one study clearly suggested that herbal products containing gallic acid may have the potential to inhibit the metabolism of certain coadministered drugs. The present study clearly shows that use of Lauha Bhasma does not cause any adverse reaction, provided the drug is properly processed in suitable herbal media (as in the present case, Triphalā), taken in suitable dose, for a limited period of time and that too with proper anupāna (vehicle).
| Conclusion|| |
Observations of the present study did not show any untoward effect of Lauha bhasma at therapeutic dose and at a dose five times the therapeutic dose, thus proving its safety. However, alteration in some of the biochemical and haematological parameters along with histopathological findings at highest dose level were evident. As these changes were dose dependent, and none of the Ayurvedic text suggests taking great amounts of Lauha bhasma, precautions are needed in prescribing higher than equivalent doses in human beings. Moreover, more detailed studies are still required to come to a final conclusion about the safety of Lauha bhasma.
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[Figure 1], [Figure 2]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6]