FORM 2 THE PATENTS ACT 1970 (39 OF 1970) & THE PATENT RULES, 2003 COMPLETE SPECIFICATION [See section 10 & Rule 13] A novel herbomineral formulation useful for effective management of iron overload initiated complications including steatohepatitis Benmoon Pharma Research Private Limited, 403, Jeet Complex, Opp. Municipal Market, near Jain Derasar, C.G.Road, Navarangpura, Ahmedabad, Gujarat, India A novel herbomineral formulation useful for effective management of iron overload initiated complications including steatohepatitis. FIELD OF THE INVENTION The present invention relates to a novel herbomineral formulation useful for effective management of iron overload initiated complications including steatohepatitis. BACKGROUND OF INVENTION Iron is an essential nutrient, it's breadth of life. The ability of iron to carry oxygen more efficiently over copper and vanadium because of their chemical limits making iron as a versatile catalyst for living organisms has evolutionarily led iron to be the predominant REDOX agent in most cells and organisms [ Nielsen FH, 2000 ]. Virtually all cells and organisms utilize iron as a cofactor in a range of biochemical activities - such as oxygen transport (hemoglobin), electron transport (mitochondrial heme and nonheme iron proteins essential for energy production), and DNA synthesis amongst other. Too little iron results in anemia and deficiency of iron enzymes whereas iron present in excess poses a threat to cells and tissues, and therefore iron homeostasis is tightly controlled. There are reasons why the homeostasis fails and body then faces excess iron. Excess iron, i.e. free iron, promotes the generation of reactive radicals, which attack and damage cellular macromolecules and promote cell death and tissue injury. This condition of oxidative stress is encountered in diseases of hereditary and secondary iron overload [ Andrews NC, 1995]. Hemochromatosis and transfusional siderosis are examples where excessive iron accumulation eventually results in liver damage and heart failure. Oxidative stress is also a hallmark of neurodegenerative disorders [Sipe JC et al, 2002], It is commonly observed that iron accumulates pathologically in substantia nigra [Berg D et al, 2001] or in senile plaques [Bishop GM e al, 2002], respectively, in Parkinson's and Alzheimer's diseases. It is not clear if the iron accumulation is a primary cause or a secondary effect. Iron is increasingly being considered as a pathogenic cofactor [Rouault TA, 2001; Ke Y and Ming Qian Z, 2003]. In order to consider pathophysiologygical mechanisms of organ injury by iron overload, and the need to prevent the same, an understanding of molecular mechanisms of body iron metabolism is essential. IRON METABOLISM Unlike most metals, iron is not excreted from the body. The only way body looses iron is through bleeding or cell desquamation (e.g. in sloughed mucosal cells). This non-controlled iron loss is compensated by dietary iron absorption, which takes place in the proximal small intestine, near the gastro-duodenum junction. The real challenge for the human physiology and practically all forms of life is to acquire adequate amount of iron for critical biological processes and yet avoid excess iron, which is free iron, associated with toxicity. IRON OVERLOAD Iron Overload is caused by the excess of accumulation of iron in the body tissues. The commonly affected organs are liver, heart, spleen and endocrine glands. Liver being the major storage site for iron it will be affected to a greater extent. Iron overload and anemia are two extremes of iron status in mammals. Both the conditions have implication in the normal iron metabolism. Primary Iron overload is due to uncontrolled iron absorption, while in the anemic condition, induction of transfusion leads to the (secondary) iron overload. These variations in the iron homeostasis leads to the several pathogenic conditions such as, in heart cardiac cells are damaged with iron overload [Buja LM and Roberts WC, 1971]. Also iron overload cardiomyopathy [Kumfu S et al., 2009], which could be because of the functional impairment of the mitochrondrial respiratory chain and abnormal energy metabolism [ Hershko C et al., 1998]. Further injury to myocytes causes congestive heart failure [ Liu P and Olivieri N, 1994] and damage to myocardium and conducting system of heart leads to the arrhythmias f Schellhammer PF et al., 1967]. Iron overload also affects the liver, as a liver is being the major site for iron storage. Excess of iron accumulation can lead to the oxidative stress and chronic hepatic iron overload results in, fibrosis and cirrhosis, porphyria cutanea tarda, and hepatocellular carcinoma [Bonkovsky HL, 1991]. Another major organ affected by iron overload is the spleen. In case of iron overload there is destruction of splenic architecture and splenomegaly. Further the "free" iron (which is not bound to the ferretin) leads to the formation of hydroxyl radical (OH) [Gutteridge JM et al., 1981]. This hydroxyl radical is highly reactive, and attacks lipids, proteins and DNA and can lead to the formation of peroxide molecule, which further interacts with other molecules to form cross links whose functions is totally altered [Bacon BR, and Britton RS, 1990]. Iron overloading also affects the lipid metabolism which can lead to the increases in serum triacylglycerols levels and can alter the glucose homeostasis [Rodriguez A et al., 2007;, Silva M etal., 2008]. Many treatment modalities have been used with the various successes in the iron overload condition. Amongst them iron chelators and siderophores are very commonly used. Chelators: Chelators are small molecules that bind tightly to metal ions. Iron chelators are those which bind to the metallic Iron, making them inactive. Iron chelators can be classified using a number of criteria such as their origin, whether they are hydrophobic or hydrophilic and also their stoichiometric interaction [bidentate like LI, or hexadentate like Desferrioxamine (DF).] Some of the most common iron chelators are reviewed below. Siderophores: Siderophores are natural product iron chelators, secreted by microbes in response to the insoluble nature of iron in the environment. They convert this insoluble form to the soluble form, allowing microorganisms to take up these complexes via specific transport systems. The agents which fall under this group are Desferrioxamine, Desferrithiocin, Desferri-exochelin, Synthetic Iron Chelators: As the name suggests these molecules are not naturally derived as in case of siderophores. They are synthetically obtained to meet the need of iron chelation. Some of them are 1CL670A, Deferiprone and Hydroxypyridinone Analogs, Tachpyridine, Aroylhydrazones, Thiosemicarbazon. Phlebotomy technique: One of the emerging modality in the treatment of the iron overload is phlebotomy. Here blood is drawn at regular intervals from the patient who is suffering from iron overload disease. Chrobak L [ Chrobak L., 2006] in his study has successfully shown that phlebotomy conducted regularly, at months interval, can be of great help in reducing iron overload disease. Activity of iron chelator depends upon the degree of it being absorbed from the G.I. Tract. One of the drawbacks of most effective iron chelator, desferrioxamine, is that it has very poor absorption from the gastrointestinal tract. Another major complication in the therapeutic use of chelators is the propensity of chelators to affect not only the desired metal but also many other essential metals and their associated metabolic pathways and other processes. For example, the treatment with DF and LI requires zinc supplementation to prevent the occurrence of zinc deficiency diseases [Al-Refaie FN et al, 1992; De Virgiliis S et al, 1988]. The problem associated with using DFO is that it has to be given via parental route, for which it requires specified instruments and skilled person. Phlebotomy is of limited use because the method itself is cumbersome to the patient and requires skilled person for conducting the procedures. Available treatments, work well in removing and controlling iron overload, but have side effects. Besides that, there are shortcomings associated with the commercially available iron chelating agents like poor oral bioavailability, short plasma half-life and severe side effects. Thus there is still a need for optimal agents for treatment of iron overload which obviate the drawbacks listed above. Some earlier studies have been carried out to find alternative sources of chelating agents with lower side effects. For example, in one study on iron chelating activity of some medicinal plants from Iran, highest chelating activity was found in Mellilotus arvensis (Ebrahimzadeh, M. A., 2008). In another study, correlation between the in vitro iron chelating activity and poly phenol and flavonoid contents of some medicinal plants was studied. The highest chelating activity was found in aerial parts of Leonurus cardiana and Grammosciadium platycarpum which had high amount of phenol and flavonoid contents. Weak correlations were found between phenolic and flavonoids contents and iron chelatory activity in this study (Ebrahimzadeh, M. A., 2009). Andreu et al. reported that in vitro antioxidant activity of mangiferin was related to its iron-chelating properties and not merely due to the scavenging activity of free radicals (Andreu, G.P., 2005). Interaction of Mangifera indica L. extract with Fe (III) was found to improve its antioxidant and cytoprotecting activity and it was proposed as a potential therapy against the deleterious action of reactive oxygen species generated during iron-overload, such as that occurring in diseases like β-thalassemia, Friedreich's ataxia and haemochromatosis (Pardo-Andreu, G. L., 2006). In addition, protective effects of Mangifera indica L extract, and its major component mangiferin, on iron-induced oxidative damage to rat serum and liver has been looked into where besides acting as antioxidants, The mangiferin component was found to decrease liver iron.by increasing its excretion (Pardo-Andreu, G. L., 2008). Extracts of Tinospora cordifolia stem, Terminalia arjuna bark, Withania somnifera root, and juice of Allium sativum cloves, separately as well as together, were found to cause amelioration of iron overload (Bhatt P., 2006). Due to the local predominance of iron overload population in Jamnagar as well as lack of well tolerated treatments, the applicants in the present invention have disclosed effective, novel and non-obvious formulation to reduce and control iron overload. OBJECTS OF THE INVENTION: Main object of the present invention is to provide a novel herbomineral formulation useful for effective management of iron overload initiated complications including steatohepatitis. Another object of the invention is to provide an oral, economical and non-toxic herbomineral formulation for development of an iron chelator drug which can provide protection against iron overload. Still another object of the invention is to provide an herbomineral formulation useful for improvement of various biochemical, liver function parameters and chronic fatigue symptoms in the iron overload patients. Yet another object is to provide the method for preparation of the novel herbomineral formulation provided by the present invention. DEFINITIONS As used herein, the term "DCYTB" is duodenal cytochrome b As used herein, the term "DMT1" is divalent metal transporter I As used herein, the term "DNA" is Deoxyribonucleic acid As used herein, the term "FPN" is ferroprotein As used herein, the term Fe denotes the Iron. As used herein, the term, HAMP, stands for Hepcidin As used herein, the term "HCP1" is heme carrier protein As used herein, the term "Hemoglobin" is the oxygen-carrying pigment and predominant protein in the red blood cells. "As used herein, the term Hephaestin" is membrane-bound ferrooxidase protein As used herein, the term, HFE stands for Human hemochromatosis protein As used herein, the term, HH stands for hereditary haemochromatosis As used herein, the term "HO- 1" is heme oxygenase As used herein, the term HS stands for Herbosulfur formulation. As used herein, the term, HJV stands for hemojuvelin As used herein, the term, JH stands for juvenile haemochromatosis As used herein, the term "Hypoxia" is a pathological condition in which the body as a whole (generalized hypoxia) or region of the body (tissue hypoxia) is deprived of adequate oxygen supply. As used herein, the term, IOF stands for Iron Overload Formulation. As used herein, the term MSM stands for methyl sulfonyl methane. As used herein, the terms "Neurodegenerative disorders" are the group of disorders where there is degeneration of the neurons, found in diseases like Parkinson's and Alzheimer's diseases. As used herein, the term "NTBI" is Non-transferrin-bound iron As used herein, the term "STEAP3" is Six - Transmembrane epithelial antigen of the prostate 3 As used herein, the term "TFR1" is TF receptor 1 BRIEF DESCRIPTION OF THE ACCOMPANYING TABLES AND FIGURES: Table 1. Quantity of Plant extract used Table 2: Iron- sorbitol injections were given intra muscular at various dose levels- the injections lead to the following observations Table-3.1: Effect of test drugs on weight of Liver in iron sorbitol overloaded albino rats Tabfe -32: Effect of test drugs on weight of Heart in iron sorbitol overloaded albino rats: Table -3.3: Effect of test drugs on weight of Kidney in iron sorbitol overloaded albino rats Table -3.4: Effect of test drugs on weight of Spleen in iron sorbitol overloaded albino rats Table -3.5 Effect of test drugs on blood sugar level in iron sorbitol overloaded albino rats Table -3.6: Effect of test drugs on SGOT activity in iron sorbitol overloaded albino rats Table -3.7: Effect of test drugs on SGPT activity in iron sorbitol overloaded albino rats Table -3.8: Effect of test drugs on blood urea level in iron sorbitol overloaded albino rats Table -3.9: Effect of test drugs on serum creatinine level in iron sorbitol overloaded albino rats Table-3.10: Effect of test drugs on serum alkaline phosphatase activity in iron sorbitol overloaded albino rats Table -3.11: Effect of test drugs on serum ferritin level in iron sorbitol overloaded albino rats Table- 3b Standardization of iron-sorbitol dose in rats for causing chronic iron-overload symptoms Table 4.1: Distribution of patients registered for the study of Iron overload in Thalassemia Table 4.2: Side effects observed after one month administration of the herbomineral formulation / Kelfer Table 4.3: Effect of hebomineral formulation / Kelfer therapy on symptoms of iron overload. Table 4.4: Effect of hebomineral formulation / Kelfer therapy on RBS Table 4.5: Effect of hebomineral formulation / Kelfer therapy on S. cholesterol Table 4.6: Effect of hebomineral formulation / Kelfer therapy on SGOT. Table 4.7: Effect of hebomineral formulation / Kelfer therapy on SGPT. Table 4.8: Effect of hebomineral formulation / Kelfer therapy on S. alkaline phosphatase. Table 4.9: Effect of hebomineral formulation / Kelfer therapy on S. Billirubin Table 4.10:Effect of hebomineral formulation / Kelfer therapy on S. Ferritin Table 4.11 :Effect of hebomineral formulation / Kelfer therapy Photomicrographs: Plate-1: Sections of liver from iron overload control group Plate-2: Sections of liver from Herbomineral compound group Plate-3: Sections of liver from kelfer treated group Plate-4: Sections of Heart from iron overload group PIate-5: Sections of Heart from Kelfer given group Plate-6: Spleen sections from iron overload control group Plate-7: Spleen sections from Herbomineral compound treated group Plate-8: Heart sections from iron overload group Plate-9: Heart sections from herbomineral compound treated group Plate-10: Hear sections from Kelfer treated group showing iron deposits Plate-11: Kidney sections from iron overload control rats and herbomineral compound treated group Plate-12: Kidney sections from iron overload control rats and Kelfer treated group Plate-13: Liver sections from iron overload control group Plate-14: Liver sections from iron overload control rats and herbomineral compound treated group Plate-15: Liver sections from Kelfer treated group Plate-16: Spleen sections from iron overload control group Plate-17: Spleen sections from herbomineral compound treated group Plate-18: Spleen sections from Kelfer treated group Details of the abbreviations used in the photomicrographs: Liver: VC=Vacuolization, Hc=Hepatic cells, FC = fatty changes, CV = Central vein, S=sinusoidal dilatation, Hg=Hemorrhage, CI=Cell infiltration, Kc=Kupper cells, SD=sinusoidal dilatation, Heart: Ep=epicardium, Myc- myocaridtis, Mc=Myocardium, En=Endocardium, FC=fatty changes Spleen: RP= red pulp, WP= white pulp, Cp-Capsule Kidney: G= Glomerulus, Cp. = Capsule, CT= Convoluted tubule SUMMARY OF THE INVENTION: Present invention describes a novel herbomineral formulation useful for reduction of iron overload. Liver and spleen are the organs which are affected most due to iron overload in the body and their functioning gets affected. Various Biochemical parameters of liver and complications are caused. The novel herbomineral formulation of the present invention, prepared from various herbs, has use in control and reduction of iron overload remarkably. The novel herbomineral formulation of the invention is useful for various therapeutic treatments, including iron overload, liver health in steatohepatitis, both alcoholic and non-alcoholic. The novel herbomineral formulation has been shown to be organoprotective as shown by normalization of various organ weights including that of liver and spleen in iron overload. The herbomineral formulation has also shown to cause significant recovery on iron overload induced change in biochemical parameters. In Thalassemia major patients, with a history of blood transfusions, the herbomineral formulation has shown significant improvements in quality of life, specifically in chronic fatigue. Accordingly, present invention provides a novel herbomineral formulation useful for effective management of iron overload initiated complications including steatohepatitis. In an embodiment to the invention, the formulation comprises processed sulphur (sulphurion, 52% wt), Embelica officinalis (6% wt), Terminalia Arjuna (6% wt), Cassia fistula (6% wt), Tinospora cordifolia (6% wt), Terminelia chebula (6% wt), Picrorhiza kurroa (6% wt), Tacoma undulate (6% wt) and Boerhhavia diffusa (6% wt). In another embodiment to the invention, the formulation is useful for treating liver ailments.. In yet another embodiment to the invention, the formulation is useful for treating iron overload condition. In still another embodiment to the invention, the formulation is useful for treating steatohepatitis conditions.. In still another embodiment to the invention, the formulation is nontoxic and its LD50 value is greater than 1600 mg/kg. In yet another embodiment to the invention, the formulation can be given either alone or in combination with other pharmaceutically active agents. In still another embodiment to the invention, the formulation is administered orally to iron overloaded rats at a dose of 90 mg/kg body wt.. In still another embodiment to the invention, the formulation causes improvement of 47 % in regaining normal liver weight in iron overloaded rats. In still another embodiment to the invention, the formulation causes improvement of 50 % in regaining normal spleen weight in iron overloaded rats. In still another embodiment to the invention, the formulation is therapeutically useful in organ weight normalcy in iron overload rats.. In yet another embodiment to the invention, the formulation causes 9.85 folds decrease in serum ferritin level as compared to iron overloaded control rats . In still another embodiment to the invention, the formulation is organoprotective in iron overloaded rats. In still another embodiment to the invention, the optimal dose for human patients is 8.33 mg/Kg body weight, In still another embodiment to the invention, the formulation causes improvement in liver health and function in iron overload patients. In still another embodiment to the invention, the formulation causes lowering of serum ferritin in iron overload patients. In still another embodiment to the invention, the formulation causes improvement in chronic fatigue in iron overload patients. DETAILED DESCRIPTION OF THE INVENTION: Several places for example the city of Jamnagar (India) and surrounding area has high incidence of iron overload. Iron overload is very common in some of the communities residing in the area, Use of herbomineral formulation in iron overload is totally novel and safe. Several natural products routinely used in medicinal system, but not in iron overload, were carefully chosen and one or more of them were used along with the herbomineral formulation to form an integrated approach to iron overload diseases. Preparation of the herbal extracts. A) Preparation of fresh extracts. Fresh extracts of Tinospora cordifolia Cassia fistula were prepared. The fresh leaves and stem of Tinospora cordifolia and fruit pulp of Cassia fistula respectively were ground with distilled water in a mixer grinder. The fresh juice was then filtered through double layered muslin cloth and this was designated as the fresh extract. B) Preparation of decoctions: The decoction was prepared in case of dried plant material. The respective part of Embelica officinalis, Terminalia Arjuna, Terminelia chebula, Picrorhiza kurroa, Tacoma undulate or Boerhhavia diffusa was heated at 55-85°C with 16 times weight of distilled water till the volume reduced to half. Preparation of herbomineral formulation Step 1: Preparation of processed sulfur: One Kg commercial sulfur was powdered with the help of mortar and pestle and was taken in a 5 L steel vessel and heated at a temperature of around 250°C. One litre Eclipta alba leaves extract was taken in another steel vessel and a piece of muslin cloth was tied on the mouth of the steel vessel. When sulfur was totally melted it was poured into the vessel containing Eclipta alba leaves extract through the muslin cloth and contents were mixed. The solidified mass was then taken out from the steel vessel and melted again. Whole procedure was repeated thrice to obtain processed sulfur which was dried, powdered and kept in an air tight jar. Step 2: Processed sulfur was taken in the granite mortar and extracts of various herbs such as Embelica officinalis, Terminalia Arjuna, Cassia fistula, Tinospora cordifolia, Terminelia chebula, Picrorhiza kurroa, Tacoma undulate and Boerhhavia diffusa (table 1) were added to the powdered processed sulfur and trituration was completed. The paste thus obtained was shade dried. Trituration was carried out thrice with each extract. In each case, after drying of the . previous paste, next plant extract in the order mentioned in table 1 was used for trituration. Table 1. Quantity of Plant extract used Plant material Part used Quantity Processed Sulfur Mineral 1kg Embelica officinalis Fruit 700 ml Terminalia Arjuna Stem 650 ml Terminelia chebula Fruit 640 ml Picrorhiza kurroa Rhizomes 630 ml Tinospora cordifolia Leaves & stem 680 ml Cassia fistula Fruit pulp 680ml Tacoma undulata Bark 630 ml Boerhhavia diffusa Whole part 650 ml For example, Embelica officinalis extract was followed by extracts of Terminalia arjuna, after which came Terminalia chebula, Picrorhiza Kurroa, Tinospora cardifolia, Cassia fistula, Tacoma undulateand Boerhhavia diffusa extracts. Finally, after whole procedure, the mass was powdered and was sieved through 80 no. mesh and the resulting powder was stored in a non reacting glass bottle. ANIMAL STUDIES The animal study involved study of efficacy and safety of the prepared herbo-mineral formulation and comprised: 1. Standardizing existing experimental model [Ozguner M and Sayin N, 2002] to study the iron-sorbitol induced chronic iron overloading in Charles Foster albino rats. 2. To evaluate the safety and efficacy of the herbomineral formulation on iron sorbitol induced chronic iron overloading resulting in changes in bio chemical and histological parameters. To check if the herbomineral formulation has cytoprotective activity. To compare the efficacy of the herbomineral formulation with known iron chelator, i.e., Kelfer (deferiprone). STANDARDIZATION OF ANIMAL MODEL: Iron sorbitol injections were administered to Charles Foster albino rats (150-250 g). through intra muscular route at various dose levels as follow as: ■ 0.4 mg/g for two days per week, for eight weeks ■ 0.2 mg/g for two days per week, for eight weeks ■ 0.1 mg/g for two days per week, for eight weeks ■ 0.04 mg/g for two days per week, for eight weeks Results The dose of iron sorbitol- 0.04 mg/g/day per week, for eight weeks in Charles Foster rats (150-250 g), was found to be ideal to produce chronic iron overloading toxicity (Table 2), taking into consideration the ratio between mortality and the magnitude of tissue damage induced observed in the histo-pathological studies. SAFETY Acute Toxicity Study The purpose of this study was to determine the nature and extent of untoward reaction, which might follow after the administration of a single dose. Eighteen animals were allotted in 6 groups, 3 numbers in each group. Six groups were: A1 (100 mg/kg), A2 (200 mg/kg), A3 (400 mg/kg), A4 (800 mg/kg), A5 (1600 mg/kg) and a control group. Doses were derived from the human dose for Kelfer, i.e. deferiprone (Cipla), as lg per day. Dose of the herbomineral formulation taken was also lg per day. Considering adult human dose of both drugs as 1 g, the dose for experimental study was calculated 0.09 g or 90 mg/ kg body weight of rat based on the body surface areas ratio as following. Dose Calculation for rat: Suitable rat dose of commercial preparation Kelfer (deferiprone) (standard control) was calculated in accordance to Paget and Barnes (Ghosh, M.N. 2005) Human adult dose x Body surface area ratio convertible factor - 1gx0.018 = 0.018 g/200 g body weight of rat. = 0.018 g x 5 (converting to mg/kg by multiplying with suitable factor 5) = 0.09 g or 90 mg/ kg body weight of rat. This gives the dose for experimental study of both the drugs (Kelfer and the formulation of the resent invention) for rats. the animals were observed for gross behavior and activity continuously for every hour for 5 hours on the first day and once in a day for the next three days and general observations for 7 days. The parameter observed after 30 min of drug administration were, exitus, hypo activity, passivity, relaxation, ataxia, narcosis, ptosis and exopthalmus. The parameter observed after 60 minutes of drug administration were: hyperactivity, irritability, stereotypy, tremors, convulsion, straub tail, analgesia and vocalization. Behavioral observations: On treatment with the herbomineral formulation, rats were found to be hypoactive during the initial part of the study which was followed by a period of mild to moderate hyperactivity especially at lower dose level thereafter the behavior of the rats was almost normal. The animals were also observed for gross behaviors and exitus (death) for 7 days in all the groups. No behavioral changes and mortality were noticed in all the groups treated with the herbomineral formulation. During the acute toxicity study, the animals in the test drug group (herbomineral formulation) did not manifest any signs of toxicity, behavioral changes and no exitus (death) was observed up to approximately 16 times more than the therapeutic dose. From this it can be mentioned that the approximate LD50 value is much more than 1600mg/kg. EFFICACY This study evaluated the efficacy of the herbomineral formulation in chronic iron overloading induced by iron sorbitol. The herbomineral formulation was compared against standard control drug Kelfer (Cipla). The animals selected were 'Charles Foster strain albino rats' of both sexes weighing 150 to 250 g. The test drugs were administered according to the body weight of the animals by oral route. The selected animals were grouped into 4 groups as follows randomly as, irrespective of sex, Group-1 - Normal control - only tap water was given, Group- II - Iron sorbitol control Group - only iron sorbitol injection was given, Group- III - Herbomineral formulation + iron sorbitol injection, Group- IV- Kelfer + iron sorbitol injection. The selected animals were weighed. Then they were allotted to different groups as mentioned above. Test drug and vehicles were administered to respective groups for 56 consecutive days. The dose of the test drug and reference standard is 90 mg/kg (per oral). Equal volume of vehicle was administered to control group rats. Iron sorbital injection was given intramuscularly to hind leg twice weekly. On the 57th day animals were weighed again and sacrificed by stunning and . severing the jugular veins. Blood samples were collected and sent to laboratory for estimation of serum biochemical parameters. The vital organs like, liver, heart, spleen and kidney were dissected out carefully, cleaned for extraneous tissue and weighed, and transferred to 10% formalin solution (including lymph node stomach) and sent for histopathological studies. Bone marrow smear was also prepared for histopathological studies. Parameters studied were, blood glucose, S.G.P.T., S.G.O.T., serum creatinine, serum alkaline phosphatase, blood urea and serum ferret in. Histopathological Study: Histopathological studies were carried out on the tissues from liver, kidney, heart and spleen from various animal test groups comprising normal control, Iron sorbitol control, Herbomineral formulation + iron sorbitol and Kelfer + iron sorbitol administered. The tissues were excised out immediately after sacrificing, cleaned of extraneous tissue cut into pieces of appropriate thickness and sections of the 5-6μm thickness cut with the help of Spencer type rotating microtome and were processed for Haematoxylin-Eosin and Prussian blue staining The slides were viewed under binocular research Carl-Zeiss's microscope (Germany) at various magnifications to note down the changes in the microscopic features of the tissues studied. Expected staining results: 1. FERRIC IRON : BRIGHT BLUE stained 2 .NUCLIE + CYTOPLASM: LIGHT PINK stained Observations and results: Impact of test drugs on iron sorbitol induced chronic iron overload toxicity was as follows. * Ponderal changes: The data pertaining to the effect of test drugs on weights of liver, heart, kidney and spleen, in iron sorbitol treated albino rats, have been summarized in Tables - 3.1 to 3.4. Liver: The herbomineral formulation showed an improvement of 47 % in regaining normal weight from the iron sorbitol induced change in the liver weight, whereas Kelfer (deferiprone) showed an improvement of 37.2 % (Table 3.1). Heart: There was insignificant change in the weight of the heart with iron sorbitol. Similarly there was negligible change observed with either the herbomineral formulation or Kelfer (deferiprone) (Table 3.2). Hence, percent improvement from the iron sorbitol induced change was not calculated. Kidney: There was practically no change in weight of the organ with either iron sorbitol or the herbomineral formulation plus iron sorbitol. Surprisingly Kelfer (deferiprone) plus iron sorbitol showed detrimental weight increase of 55 % from normal (Table 3.3). Thus, the percentage improvement was not calculated. Spleen:. The herbomineral formulation showed improvement of 50 % in regaining normal weight from the iron sorbitol induced change in the spleen weight, whereas Kelfer (deferiprone) showed improvement of 20 % (Table 3.4). Thus, the herbomineral formulation was found to be therapeutically useful in organ weight normalcy Biochemical Parameters Test drug the herbomineral formulation when compared with iron sorbitol treated control group showed statistically significant improvements in some of the biochemical parameters. The herbomineral formulation showed statistically significant decrease in SGOT activity (32.08 %l) as compared to iron sorbitol treated control group which shows significant increase in SGOT activity (20.64% ↑) [Table 3.6]. The herbomineral formulation showed remarkable decrease in SGPT activity (72.45 %|) when compared with iron sorbitol treated control group where there was marked increase in SGPT activity (194.16% ↑). This is an important and novel finding [Table 3.7]. The herbomineral formulation also showed highly significant decrease in serum ferritin level (45.91 %|) as compared to iron sorbitol treated control group (452.15 % ↑) [Table 3.11]. These findings suggest that the herbomineral formulation has a significant action against iron overload as reflected by serum ferritin level and also has the cytoprotective effect on liver as found out by SGOT and SGPT activity. The herbomineral formulation also produced decrease in the blood sugar level (2.670 % ↓), blood Urea level, serum creatinine level (15.65 % ↓) and serum alkaline phosphatase activity (1.38 %i) but the decrease was non statistically significant [Table 3.5, 3.8. 3.9, 3.10]. CLINICAL STUDY: To evaluate the therapeutic effect as Iron chelator of both trial drugs herbomineral compound and deferiprone (Kelfer) comparatively, a clinical study was undertaken. The selected patients were randomly placed & studied under three groups. 1. Group A were subjected to Herbomineral compound. 2. Group B (standard control group) received deferiprone (Kelfer (deferiprone)). Dose: Adult dose 500 mg b.i.d. each of Herbomineral formulation & deferiprone (Kelfer (deferiprone)). • Duration: One month. Observations: Special emphasis was made to observe side effect of test drug as well as standard control group was also made. There was no any serious side effect observed in test drugs as shown in the table. Arthritic joint pain was observed in standard control group as major side effect. As many as 88.23 % patients had arthritic joint pain which was the main cause of LAMA in Kelfer (deferiprone) treated group. Moderate improvement was obtained in 26.66 % in the herbo-mineral compound treated group A while 20% had marked and mild improvement. In the Kelfer treated group B, moderate improvement was obtained in 36.36% and 27.27 % of patients had mild and marked improvement. Tables 4.0 to 4.11 show the observations on effect of treatment to patients by administering herbomineral compound / Kelfer (standard drug) on various parameters such as side effects, relief from iron-overload symptoms, serum biochemistry (Bilirubin, ferritin, cholesterol, alkaline phosphatase, SGOT and SGPT etc). The data reveals that the test Herbomineral formulation provided in the present invention is an economical non¬toxic drug having very less side effects and is orally administrable. Human study: Human subjects of various age groups exhibiting the iron overload symptoms suffering with Thalassemia major were evaluated for symptoms of iron overload and biochemical parameters before and after administration of the herbomineral formulation. Significant relief in symptoms and biochemical parameters such as serum ferritin, SGPT, SGOT, blood urea or serum creatinin etc was observed in patients treated with therapeutic dose of the herbomineral formulation. Patients' Case Studies: Case 1 Child age 5yrs, male, suffering from Thalassemia major, had already taken 38 settings of blood transfusion. He was also experiencing chronic fatigue from last few months. Chronic iron overload was observed in him with serum ferritin level 3980 ng/ml before treatment. He was then given the herbomineral formulation 500 mg two times a day, for 45 days. After 45 days of treatment with the herbomineral formulation declining trend was observed in serum ferritin level. Also chronic fatigue was reduced remarkably with the herbomineral formulation treatment. No negative results were observed in SGPT, SGOT, blood urea or serum creatinin. Case 2 Female, age 11 yrs, has been on blood transfusion for Thalassemia major. She had already taken 184 settings of blood transfusion. She was suffering from mild jaundice, chronic fatigue and remarkable spleenomegaly (24.5 cm in length). Her serum ferritin level was 6283 ng/ml. She was given the herbomineral formulation 500 mg two times a clay, for 45 days. After the 45 days of treatment period her serum ferritin reduced to 5422 ng/ml. Her chronic fatigue was reduced remarkably in 45 days of treatment. Size of spleen was also reduced to 19 cm. No negative results were observed in SGPT, SGOT, blood urea or serum creatinin. Case 3 Female, age 15 yrs, suffering from Thalassemia major, had already taken 310 settings blood transfusion. She was complaining of chronic fatigue and shortness of breath. Her serum ferritin level was 4190 ng/ml before treatment. She was given the herbomineral formulation 500 mg two times a day, for 45 days. After the 45 days of treatment period her serum ferritin level declined to 3510 ng/ml. Her chronic fatigue was reduced remarkably. Her breathing was also improved with the herbomineral formulation. No negative results were observed in SGPT, SGOT, blood urea or serum creatinin. Case 4 Female, age 12 yrs, had been on blood transfusion for Thalassemia major. She had already taken 238 settings of blood transfusion. She was complaining of chronic fatigue and severe knee joint pain. Her serum ferritin level was 3980 ng/ml, before the treatment. She was given the herbomineral formulation, 500 mg two times a day, for 60 days. After administering the herbomineral formulation for 60 days serum ferritin level declined up to 3330 ng/ml. She also found improvement in her chronic fatigue, with the treatment. The following examples are given by way of illustration and therefore should not be construed to limit the scope of the present invention. Example 1: Preparation of the herbal extracts. A) Preparation of fresh extracts: Fresh extracts of Tinospora cordifolia Cassia fistula were prepared. The fresh leaves and stem of Tinospora cordifolia and fruit pulp of Cassia fistula respectively were ground with distilled water in a mixer grinder. The fresh juice was then filtered through muslin cloth and this was designated as the fresh extract. B) Preparation of decoctions: A decoction was prepared in case of dried plant material. The respective part of Embelica officinalis, Terminalia Arjuna, Terminelia chebula, Picrorhiza kurroa, Tacoma undulate and Boerhhavia diffusa was taken and heated at 55-85°C with 16 times weight of distilled water till the amount of water remaining was half of the initial amount. Example 2: Preparation of herbomineral formulation Step 1: Preparation of processed sulfur: One Kg commercial sulfur was powdered with the help of mortar and pestle and was taken in a 5 L steel vessel and heated at a temperature of around 250°C. One litre Eclipta alba leaves extract was taken in another steel vessel and a piece of double layered muslin cloth was tied on the mouth of the steel vessel. When Sulfur was totally melted it was poured into the vessel containing Eclipta alba leaves extract through the muslin cloth and contents were mixed. The solidified mass was then taken out from the steel vessel and melted again in the sulfur steel vessel. Whole procedure was repeated for three times to obtain processed sulfur which was dried, powdered and kept in an air tight jar. Step 2: Processed sulfur was taken in granite mortar and extracts of various herbs as mentioned in table 1 were added into the powdered sulfur and trituration was completed.. The paste thus obtained was shade dried dried. Each extract was triturated three times. In each case, after drying of the previous paste, next extract in the order mentioned in table 1 was used for trituration. For example, Embelica officinalis extract was followed by extracts of Terminalia arjuna, after which came Terminalia chebula, Picrorhiza Kurroa, Tinospora cardifolia, Cassia fistula, Tacoma undulate and Boerhhavia diffusa extracts. Finally, after whole procedure, the mass was powdered and was sieved through 80 no. mesh and the resulting powder (composition mentioned in table 1) was stored in a non reacting glass bottle. Example 3: ANIMAL STUDIES STANDARDIZATION OF ANIMAL MODEL: Iron sorbitol injections were administered to Charles Foster albino rats (150-250 g) through intra muscular route at various dose levels as follow as: ■ 0.4 mg/g for two days per week, for eight weeks ■ 0.2 mg/g for two days per week, for eight weeks ■ 0.1 mg/g for two days per week, for eight weeks ■ 0.04 mg/g for two days per week, for eight weeks Initial pilot studies with emphasis on histopathological changes showed that the administration of iron sorbitol in the dose of 0.04 mg/g/day per week, for eight Weeks in Charles Foster rats (150-250 g), is ideal to produce chronic iron overloading toxicity. The dose was fixed taking in to consideration the ratio between mortality and the magnitude of tissue damage induced as observed with histo-pathological examination. Iron sorbitol injections were given intra muscular at various dose levels- the injections lead to the follow types of observations: Table 3b: Standardization of iron-sorbitol dose in rats for causing chronic iron-overload symptoms ■ No ■ Dose of iron sorbitol/days per week, ■ for eight weeks to 10 Charles foster rats (150-250 g). ■ Result observed ■ 1 ■ 0.4 mg/g/day ■ 6 rats died within 72 hours/ all rats died within 120 hours ■ 2 ■ 0.2 mg/g/day ■ 6 rats died within 72 hours/ all rats died within 150 hours ■ 3 ■ 0.1 mg/g/day ■ 4 rats died within 72 hours/ all rats died within 200 hours ■ 4 ■ 0.04 mg/g/day ■ No mortality observed up to 15 days The dose of iron sorbitol- 0.04 mg/g/day per week, for eight weeks in Charles Foster rats (150-250 g). was found ideal to produce chronic iron overloading toxicity taking in to consideration the ratio between mortality and the magnitude of tissue damage induced observed with histo-pathological studies. Example 4: SAFETY Acute Toxicity Study The purpose of this study was to determine the nature and extent of untoward reaction, which might follow after the administration of a single dose. Eighteen animals were allotted in 6 groups, 3 numbers in each group. Six groups were: A (100 mg/kg), A2 (200 mg/kg), A3 (400 mg/kg), A4 (800 mg/kg), A5 (1600 mg/kg) and a control group. Doses were derived from the human dose for Kelfer i.e. deferiprone, as lgm per day. Dose of the herbomineral formulation taken was also lgm per day. Considering adult human dose of both drugs as 1 g, the d ose for experimental study was calculated 0.09 g or 90 mg/ kg body weight of rat based on the body surface areas ratio as following. Dose Calculation for rat: Suitable rat dose was calculated by referring the table of Paget and Barnes (Ghosh, M.N. 2005) Human adult dose x Body surface area ratio convertible factor - lg x 0.018 - 0.018 g / 200 g body weight of rat. = 0.018 g x 5 (converting to mg/kg by multiplying with suitable factor 5) = 0.09 g or 90 mg/ kg body weight of rat which gives the dose for experimental study of both the drugs for rats. The animals were observed for gross behavior and activity continuously for every hour for 5 hours on the first day and once in a day for the next three days and general observations for 7 days. The parameter observed after 30 min of drug administration were, exitus, hypo activity, passivity, relaxation, ataxia, narcosis, ptosis and exopthalmus, The parameter observed after 60 min of drug administration were, hyperactivity, irritability, stereotypy, tremors, convulsion, straub tail, analgesia and vocalization. Example 5: EFFICACY This study evaluated the efficacy of the herbomineral formulation in chronic iron overloading induced by iron sorbitol. The herbomineral formulation was compared against standard control drug Kelfer (Cipla). Animals selected were 'Charles Foster strain albino rats' of both sexes weighing 150 to 250 g. The test drugs were administered according to the body weight of the animals by oral route as per above formula, i.e., 90 mg/Kg body weight. The selected animals were grouped into 4 groups randomly, irrespective of sex, as follows Group- 1 - Normal control - only tap water was given, Group- II - Iron sorbitol control Group -only iron sorbitol injection was given, Group- III - herbomineral formulation + iron sorbitol injection, Group- IV- Kelfer + iron sorbitol injection. The selected animals were weighed and were allotted to different groups as mentioned above. Test drug and vehicles were administered to respective groups for 56 consecutive days. The dose of the test drug and reference standard is 90 mg/kg (per oral). Equal volume of vehicle was administered to control group rats. Iron sorbital injection was given intramuscularly to hind leg twice weekly. On the 57th day animals were weighed again and sacrificed by stunning and severing the jugular veins. Blood samples were collected and sent to laboratory for estimation of serum biochemical parameters. The vital organs like, liver, heart, spleen and kidney were dissected out carefully, cleaned for extraneous tissue and weighed, and transferred to 10% formalin solution (including lymph node stomach) and sent for histopathological studies. Bone marrow smear was also prepared for histopathological studies. Parameters studied were, blood glucose, S.G.P.T., S.G.O.T., serum creatinine, serum alkaline phosphatase, blood urea and serum ferretin. Table-3.1 Effect of test drugs (Kelfer and herbomineral formulation) on weight of Liver in iron sorbitol overloaded albino rats Group DOSE (g/kg) Weight of liver % impro vemen t Absolute (g) % Chang e Relative (g/lOOg body wt) % Change Normal control Q.S. 7.72 ± 0.22 - 3.07 ±0.17 - Iron Sorbitol control (Water +iron sorbitol) 0.04 11.05±0.51** 43.13 5.22 ± 0.26a** 70.03 ↑ the herbominera 1 formulation +iron sorbitol 0.09 + 0.04 09.96 ±0.82 9.86*b 4.21±0.34*b 19.34*b↓ 47 % # Deferipron (kelfer) +iron sorbitol 0.09 + 0.04 07.70 ±0.24*b 30.37*b 4.42 ± 0.24 15.32*b↓ 37.2 % u Table -3.2 Effect of test drugs on weight of Heart in iron sorbitol overloaded albino rats: DOSE (g/kg) Weight of Heart Group Absolute (g) Mean ± SEM % Change Relative (g/lOOg body wt) Mean ± SEM % Change Normal control Q.S. 0.82 ±0.02 - 0.33 ± 0.02 - Iron Sorbitol control (Water +iron sorbitol) 0.04 0.68±0.04*a 17.074 0.31±0.02 6.06↓ the herbomineral formulation +iron sorbitol 0.09+ 0.04 0.73±0.05 7.35↑*b 0.31±0.04 0.00*b Deferipron (kelfer) +iron sorbitol 0.09+ 0.04 0.57±0.04 16.17↓*b 0.33 ± 0.03 6.54↑*b Data: Mean± SEM ↑- Increase ↓ - Decrease *aP <0.05 - in comparison with normal control group *b in comparison to Iron sorbitol control group Table -3.3 Effect of test drugs on weight of Kidney in iron sorbitol overloaded albino rats: DOSE (g/kg) Weight of Kidney Group Absolute (g) Mean ± SEM % Relative (g/lOOg body wt)Mean ± SEM % Change Normal control Q.S. 1.53± 0.04 - 0.60 ± 0.02 - Iron Sorbitol control Water +iron sorbitol) 0.04 1.25 ± 0.12 18.30↓a 0.60 ±0.08 00.00a the herbomineral formulation +iron sorbitol 0.09+ 0.04 1.39±0.15 11.2↑*b 0.59± 0.05 1.664↓*b Deferipron (kelfer) +iron sorbitol 0.09+ 0.04 1.58±0.36 26.4↑ *b 0.93 ± 0.25 55↑*b Data: Mean ± SEM ↑- Increase ↓- Decrease a in comparison to normal control, * b in comparison to Iron sorbitol control group Table -3.4 Effect of test drugs on weight of Spleen in iron sorbitol overloaded albino rats Weight of SPLEEN % Improvement Group DOSE Absolute Relative (g/kg) (g) % (g/lOOg body /o Mean ± SEM Change wt) Mean ± SEM Change Normal control Q.S. 0.57 ± 0.06 - 0.23 ± 0.03 - Iron Sorbitol control 0.04 0.69± 0.10 21-05↑a 0.33 ± 0.06 43.47↑a (Water +iron sorbitol) the herbomineral formulation 0.09+ 0.04 0.65 ±0.06 5.79↓b 0.28 ± 0.04 15.15↓ *b 50 % # +iron sorbitol Deferipron (kelfer) +iron 0.09+ 0.04 0.54 ±0.02 21.73↓ *b 0.31 ±0.005 6.06 ↓*b 20 % # sorbitol Table -3.5 Effect of test drugs on blood sugar level in iron sorbitol overloaded albino rats Group Dosage (g/kg) S. Glucose (mg/dl) Mean ± SEM % Change Normal control Q.S. 082.5 ± 04.55 Iron Sorbitol control (Water +iron sorbitol) 0.04 112 ±9.27*a 24.60 ↑ the herbomineral formulation +iron sorbitol 0.09+ 0.04 109.4 ± 8.84 2.670↓ Deferipron (kelfer) +iron sorbitol 0.09+ 0.04 110.75 ±5.39 1.780↓ Data: Mean ± SEM ↑ - Increase ↓ - Decrease a** P <0.001 - in comparison with normal control group *b in comparison to Iron sorbitol control group Table -3.6 Effect of test drugs on SGOT activity in iron sorbitol overloaded albino rats Group Dosage (g/kg) SGOT activity (IU/dl) Mean ± SEM % Change Normal control Q.S. 310.00 ±0.97 Iron Sorbitol control (Water +iron sorbitol) 0.04 374.5+ 19.41*a 20.64T Herbomineral formulation +iron sorbitol 0.09+ 0.04 254.333 + 39.22 32.08↓, Deferipron (kelfer) +iron sorbitol 0.09+ 0.04 296.5 ± 52.57 20.85↑ Data: Mean ± SEM ↑ - Increase ↓. - Decrease a* - In comparison with normal control group *** h: p<0.001 in comparison to iron sorbitol treated control group Table -3.7 Effect of test drugs on SGPT activity in iron sorbitol overloaded albino rats Group Dosage (g/kg) SGPT activity (IU/dl) Mean ± SEM % Change Normal control Q.S. 137.00106.87 Iron Sorbitol control (Water +iron sorbitol) 0.04 403.00±21.91 *a 194.16↑ the herbomineral formulation +iron sorbitol 0.09+ 0.04 111.8 ± 10.61** b 72.45↓ Deferipron (kelfer) +iron sorbitol 0.09+ 0.04 313.25 ±54.47 22.33↓ Data: Mean ± SEM ↑ - Increase ↓- Decrease a* - In comparison with normal control group **a; PO.01 in comparison to normal control ** b: P<0.01 in comparison to iron sorbitol treated control Table-3.8 Effect of test drugs on blood urea level in iron sorbitol overloaded albino rats Group Dosage (g/kg) Blood urea (mg/dl) Mean ± SEM % Change Normal control Q.S. 55.83 ±03.83 Iron Sorbitol control (Water +iron sorbitol) 0.04 55.67 ±4.177 a 0.2865↓ the herbomineral formulation +iron sorbitol 0.09+ 0.04 52.50 ±3.80 5.694↓ Deferipron (kelfer) +iron sorbitol 0.09+ 0.04 55.75 ± 7.61 0.143 ↑ Data: Mean ± SEM ↑ - Increase ↓- Decrease a - In comparison with normal control group Table-3.9 Effect of test drugs on serum creatinine level in iron sorbitol overloaded albino rats Group Dosage (g/kg) Serum Creatinine (mg/dl) Mean ± SEM % Change Normal control Q.S. 0.98 ± 0.06 Iron Sorbitol control (Water +iron sorbitol) 0.04 1.07 ±0.03 a 9.18↑ the herbomineral formulation +iron sorbitol 0.09+ 0.04 0.90 ± 0.20 15.65↓ Deferipron (kelfer) +iron sorbitol 0.09+ 0.04 1.00 ±0.11 6.27↓ Data: Mean ± SEM ↑ - Increase ↓- Decrease a - In comparison with normal control group Table-3.10 Effect of test drugs on serum alkaline phosphatase activity in iron sorbitol overloaded albino rats Group Dose (g/kg) S. Alkaline Phosphatase activity (H phenol released /mg/protein/mm) Mean ± SEM % Change Normal control Q.S. 139.50+13.90 Iron Sorbitol con(Water +iron sorbitol) 0.04 96.33 ±6.12 a* 30.93↑ the herbomineral formulation +iron sorbitol 0.09+ 0.04 95.00 ±4.53 1.38↓ Deferipron (kelfer) +iron sorbitol 0.09+ 0.04 96.25 ± 2.84 0.08,↓ Data: Mean ± SEM | - Increase ↓ - Decrease a* - P <0.01 in comparison with normal control group Table-3.11 Effect of test drugs on serum ferritin level in iron sorbitol overloaded albino rats Serum ferritin Group In dosage (g/kg) (ng/ml) Mean ± SEM % Change Iron Sorbitol Control 0.04 4595.45 ±313.81 452.15↑ a* the herbomineral formulation + iron sorbitol inj 0.09 + 0.04 2485.11 ±207.05** 45.9↓ Kelfer ± iron sorbitol inj 0.09 + 0.04 2575.36± 265.10** 43.96↓ Data: Mean ± SEM↑- Increase ↓, - Decrease a* - In comparison with normal control group ** P <0.001 in comparison to Iron sorbitol control Histopathological Study: 1. Fixation : The tissues were excised out immediately after sacrificing, cleaned of extraneous tissue cut into pieces of appropriate thickness and were transferred into 10% formalin solution. The tissues were allowed to remain in it till they are taken up for processing. 2. Tissue processing: Tissue processing involves dehydration, clearing and infiltration of the tissue with paraffin. Tissue were thoroughly washed by placing them under running tap water and then covered through a series of following solvents as per schedule for dehydration, clear and paraffin infiltration. Alcohol 70% - 20 minutes Alcohol 80% - 20 minutes Alcohol 90% - 20 minutes Alcohol 95% (2 changes)- 20 minutes each Isopropyl alcohol - 20 minutes Acetone (2 changes) - 20 minutes each Chloroform (3 changes) - 20 minutes each vlelted paraffin wax (600 °C) (2 changes) - 30 minutes each next the tissues were embedded in paraffin wax to prepare tissue blocks. Tissue blocks were fixed to metal object holder after trimming them to suitable size. 1. Section cutting: The tissue sections of the 5-6 μm thickness cut with the help of Spencer type otating microtone and floated in a water bath between 50-550 °C for 30 minutes. Then they were nounted on clear glass slides with a drop of Mayer's egg albumin dried on hot plate at about 500 °C for 30 minutes. 4. Staining: Procedure: 1: H.E, staining: After fixing the section on slide, the sections were stained by serially placing them in the following reagents. Xylol (2 changes) - 3 minutes each Acetone - 3 minutes Alcohol 95% - 3 minutes Haematoxylin stain - 20 minutes Running water - 20 minutes Alcohol 95% (3 changes) - 3 minutes each Acetone (2 changes) - 3 minutes each Xylol (2 changes) - 3 minutes each After passing though all the above reagents and stains, the slides were covered with D.P.X. (Diphenyl phthalein Xylene) and cover slip were placed. Care was taken to avoid the air bubble formation during mounting the slide. The slides were viewed under binocular research Carl-Zeiss's microscope (Germany) at various magnifications to note down the changes in the microscopic features of the tissues studied. 2: Prussaian blue staining (Pearl et al, 1958): The Prussian blue staining was carried out to ascertain the extent of iron deposits in different organs such as liver, heart, kidney and spleen. It comprised of steps as given below: 1 10 % potassium ferrocynide solution (stock) 2 10 %HC1 solution (stock) 3 70 ml potassium ferrocynide+ 30 HC1 solutions (to be mixed just before use) 4 eosin solution (for counter staining) Staining procedure: deparafinaze and hydrate in distilled water stock solution of 70 ml potassium ferrocynide+ 30 HC1 solutions for 20 mins after 25 minutes rinse well in distilled water counter stain with eosin for 3 minutes and subsequently wash well in tap water De-hydrate in 95 % alcohol, abs alcohol and clear it in xylin 2 changes each. After passing though all the above reagents and stains, the slides were covered with D.P.X. (Diphenyl phthalein Xylene) and cover slip were placed. Care was taken to avoid the air bubble formation during mounting the slide. The slides were viewed under binocular research Carl-Zeiss's microscope (Germany) at various magnifications to note down the changes in the microscopic features of the tissues studied. Two sets of slides were prepared for histopathological examination. The first set comprosed of the slides prepared with normal Hematoxylin and Eosin staining. The second set comprised of the slides prepared with Prussian blue staining to ascertain the extent of iron deposits in different organs. Expected staining results: 1 Ferric Iron : Bright Blue stained 2. Nuclei + Cytoplasm: Light Pink stained OBSERVATIONS: Liver: Liver is both a secretory and an excretory gland and is made up of polygonal cells radiating from the center. Periphery is delineated by portal triads. Centre is occupied by interlobular vein, through the connective tissue sheath i.e. Glisson's capsule; the portal vein, hepatic artery, bile duct and hepatic vein pass. It has a honeycomb or sponge like structure in which these polygonal cells plated are tunneled by a communicating system of cavities or lacunae. They contain hepatic sinusoids formed by endothelial cells and kupffer cells. The narrow spaces separating the sinusoids wall from the liver cells plates is called the Disshe's space. Each radiating arm of hepatic cells on one side has vascular capillary and bile capillary. These drain into bigger channels. Capillaries contain number of mast cells. The parenchyma is demarcated by clear cell membrane and bi nuclei. Microscopic examination of liver sections from different test groups was carried out at different magnifications to ascertain test preparations induced alterations in the organ cytoarchitecture. Norma! H and E staining: In normal control rats receiving Iron Sorbitol, extensive fatty changes, cell infiltration, multinucleated cell formation, focal necrosis, sinusoidal dilatation and hemorrhage at many sites (Plate-1) were observed in the liver sections. In the Kelfer (deferiprone) treated group, in the Iron sorbitol receiving rats, the above changes were much less (Plate- 3). In the Herbosulfur formulation treated group, the liver exhibited normal cytoarchitecture in majority of the rats; mild to moderate changes were observed in one rat (Plate-2 panel A and B ). Heart: Consists of-pericardium, pericardial cavity, myocardium, atria, ventricles, endocardium, valve cusps, valvular openings, and coronary arteries. Visceral pericardium may be enlarged by hypertrophy of the wall or dilatation of cavities, the endocardium lining the inside of the cavities, damage to this by infections, and stasis may lead to thrombosis and subsequent embolism. Valve cusps are very thin silk like with the commissures of aortic valve jointed together with out much thickness in cases such as syphilis. The Purkinje system makes the skeleton of the heart they are sin atrial node, atrio ventricular node of Tawara and bundle of His. The myocardium of the heart is supplied by the capillaries of the coronary arteries. Normal HE staining: Examination of the sections of heart from different groups was carried out under microscope at different magnifications. The heart sections from the iron sorbitol administered groups exhibited moderate to marked degenerative changes in the form of fatty degeneration, myocaridtis and oedema (Plate-4 panel A and B), in Kelfer (deferiprone) treated group these changes were comparatively less (Plate-5 panel C and D). In the Herbosulfur formulation administered group the changes were less in comparison to control and Kelfer (deferiprone) treated group (Plate-4 Panel C and D). Spleen: The color of the organ is reddish purple indicating the high content of blood. It has no ducts unlike other abdominal organs. It is reticulo-endothelial sponge with a supporting framework of trabeculae and reticulum and certain amount of lymphoid tissue super added. Large arteries are clothed with a sheath of lymphoid tissue, malpighian bodies. Each arteriole ends abruptly in a globular mass of cells known as ellipsoids represented by elongated collection of reticulo-endothelial cells. The capillaries end in the wide mesh of splenic pulp and then gather into the sinusoids. the sections of spleen from different groups were subjected to microscopic examination under lifferent magnifications. The cytoarchitecture of the sections from control group was compared o the cytoarchitecture of the drug treated groups to note down the changes that might have occurred due to drug administration. Normal H and E staining: Examination of the sections of spleen from different groups was carried out under microscope at different magnifications. In the spleen sections obtained from the iron sorbitol control group, an increased proportion of white pulp was observed (Plate-6). In the Herbosulfur formulation administered group, almost similar cytoarchitecture was observed (Plate-7 panel A and B). PART- II- PRUSSIAN BLUE STAINING: Heart: In the heart sections from vehicle control group in some sections heavy focal iron deposits were observed, in some sections moderate accumulation was observed and in some section diffused accumulation was observed. In the Herbosulfur formulation administered group a few minor patches of iron deposits were observed. In Kelfer (deferiprone) administered group, a few minor deposits were observed. However, in all the three groups fatty changes were observed, Plates-8 to 10 depict the photomicrographs of representative sections from different groups. Kidney: Heavy iron deposits were found in sections obtained from the iron sorbitol control group (plate-11- panels A and B). In the Herbosulfur formulation treated group, moderate iron deposits were observed (Plate-11, panels C and D). In Kelfer (deferiprone) treated group (Plate 12 B, panels C and D) a few moderate deposits were observed in comparison to the control group (Plate-12B, panels A and B). Liver: in the liver sections from iron sorbitol control group moderate to heavy focal and diffused iron deposits were observed. In the Herbomineral formulation treated group moderate deposits were observed. Plates 13 to 15 depict photomicrographs of representative sections from different groups. Spleen: Very heavy focal iron deposits were observed in spleen sections obtained from iron sorbitol treated control group. In comparison to this moderate to heavy deposits were observed in the spleen sections obtained from the Herbomineral formulation treated group but the deposits were of comparatively less magnitude. In the Kelfer (deferiprone) treated groups, moderate iron deposit in some rats and moderate to heavy iron deposits in others was noted. However, the magnitude of deposits was much less in comparison to iron sorbitol control group. This shows that both the test herbomineral formulation and Kelfer (deferiprone) decrease iron deposit in this organ. Plates 16 to 18 depict photomicrographs of representative sections of tissue from different groups. Example 6: Clinical study: To evaluate the therapeutic effect as Iron chelator of both trial drugs herbo- mineral compound and deferiprone (Kelfer) comparatively, a clinical study was undertaken. The selected patients were randomly placed & studied under three groups. 3. Group A were subjected to Herbomineral compound. 4. Group B (standard control group) received deferiprone (Kelfer (deferiprone)). Dose: Adult dose 500 mg b.i.d. each of Herbomineral formulation & deferiprone (Kelfer (deferiprone)). • Duration: One month. CLINICAL OBSERVATIONS: (Human Studies) Table 4.1: Distribution of patients registered (79 No.) for the study of Iron overload in Thalessemia Gp. A: Herbomineral treated Gp. C: Kelfer treated LAMA: Left Against Medical Advice Group Completed LAMA Total patients registered A 30 01 31 B 11 07 18 Total 41 08 49 Table 4.2: Side effects observed after one month administration of the herbomineral formulation / Kelfer Gp. A: Herbomineral treated Gp. C: Kelfer treated Side effects noted GpA GpB Total Tiredness 00 12 12 Depression 00 03 03 Loose motion 02 04 09 Loss of appetite 00 05 05 Lack of energy 00 03 03 Vomiting 00 03 03 Nausea 00 03 03 Arthritic Joint Pain 00 15 15 Special emphasis was made to observe side effect of test drug as well as standard control group was also made. There was no any serious side effect observed in test drugs as shown in the table. Arthritic joint pain was observed in standard control group as major side effect. As many as 88.23 % patients had arthritic joint pain which was the main cause of LAMA (left against medical advice) in Kelfer (deferiprone) treated group. Table 4.3 Effect of herbomineral formulation / Kelfer therapy on symptoms of iron-overload. Gp. A: Herbomineral treated Gp. B: Kelfer treated BT: before treatment AT: after treatment Symptoms Gp No. BT AT Mean % SD SE T P Chronic Fatigue GpA 30 2.16 1.36 0.73 33.79 0.58 0.10 6.88 <0.001 GpB 11 2.23 1.46 0.77 34.52 0.44 0.14 5.29 <0.001 Arthritic joint pain GpA 17 2.53 1.94 0.59 23.32 0.50 0.12 4.78 <0.001 GpB 05 2.36 2.11 0.25 0.10 0.44 0.09 2.00 <0.1 Loss Of Libido (Sex Drive) Or Impotence GpA 07 3.14 2.85 0.28 9.09 0.48 0.18 1.54 <0.1 GpB 04 3.75 3.50 0.25 6.66 0.54 0.25 1.00 <0.1 Abdominal pain GpA 14 1.35 1.07 0.28 21.05 0.47 0.12 2.28 <0.01 GpB 05 1.2 1.0 0.20 0.33 0.54 0.24 1.63 <0.1 Enlargement Of Spleen GpA 07 1.66 1.50 0.16 17.14 0.48 0.18 1.54 <0.1 GpB 02 1.5 1.5 00 00 00 00 00 00 Irritable Bowel Syndrome GpA 07 1.66 1.50 0.16 17.14 0.48 0.18 1.54 <0.1 GpB 05 1.2 1.00 0.20 1.66 0.44 0.20 1.00 <0.1 Table 4.4: Effect of herbomineral formulation / Kelfer therapy on symptoms of iron-overload. Gp. A: Herbomineral treated Gp. B: Kelfer treated BT: before treatment AT: after treatment Symptoms Gp N BT AT Mean % SD SE T P Jaundice GpA 04 1.2 0.4 0.8 66.00 0.44 0.20 4.00 <0.01 GpB 02 1.5 1.5 00 0000 00 00 00 00 Bronze Or Gray-Olive Colored Skin GpA 07 1.57 1.14 0.43 37.59 0.53 0.20 2.12 <0.05 GpB 03 1.33 0.66 0.66 50 0.57 0.33 2.00 <0.1 Skin tanning without being In The Sun GpA 06 2.00 1.66 0.33 16.66 0.51 0.21 1.58 <0.1 GpB 03 1.33 0.66 0.66 50 0.57 0.33 2.00 <0.1 Redness In the Palms of the hands GpA 02 1.5 1.00 0.50 33.00 0.70 0.50 1 <0.1 GpB 00 00 00 00 00 00 00 00 00 Tables 4.5 to 4.11: EFFECT OF HERBOMINERAL FORMULATION / KELFER THERAPY ON SERUM BIOCHEMISTRY: Table 4.5: Effect of therapy on RBS RBS B.T. A.T. Mean % relief SD SE t P Group A 88.24 88.75 2.53 2.87 27.53 5.02 0.50 <0A Group B 94 93.6 0.36 0.38 2.65 0.80 0.45 <0.1 Gp. A: Herbomineral treated Gp. C: Kelfer treated Table 4.6: Effect of therapy on S CHOLESTEROL S. CHOLESTEROL B.T. A.T. Mean % relief SD SE T P Group A 119.45 107.18 9.00 7.53 32.94 6.01 1.50 <0.1 Group B 120.17 120.8 10.64 8.85 49.10 14.80 0.72 <0.1 Gp. A: Herbomineral treated Gp. C: Kelfer treated Table 4.7: Effect of therapy on SGOT SGOT B.T. A.T. Mean % relief SD SE T P Group A 54.68 45 14.07 25.73 . 21.47 3.92 3.59