Preclinical Appraisal of Hematinic Potential of Mandura Bhasma for Treating Anemia

Aims: To evaluate hematinic potential of mandura bhasma. Study Design: Experimental study. Place and Duration of Study: Institute of Pharmaceutical Education and Research, Wardha, Maharashtra, India. 6 Months. Methodology: The anti-anaemic potential of Mandura bhasma in Wistar rats was investigated. Anaemia was induced in rats with phenyl hydrazine hydrochloride at a dose of 10 mg kg body weight by oral administration. Anaemia was treated with mandura bhasma administered in three different doses based on body weight. Results: In vivo investigation showed that though the dose of 6mg kg body weight of mandura bhasm produced only a minimal antianaemic (hematinic) effect, oral daily dose of 11 mg kg -1 body weight and 22mg kg body weight a produced a significant (P < 0.05) antianaemic effect when compared to standard drug ferrous sulphate indicating dose dependent activity. Conclusions: The results indicate that Mandura Bhasm have very potential dose dependant hematinic activity and can be a safe and effective drug for treating anemia. Original Research Article Khobragade et al.; JPRI, 33(30B): 207-214, 2021; Article no.JPRI.68930 208


INTRODUCTION
Anemia is a one of the most common and serious blood disorder globally affecting health and social and economic development of one third of the population of developing and under developed countries (WHO 2005) due to inadequate and imbalanced food intake [1][2]. Anemia can affect people of all ages, but the elderly, young women and the infants but pregnant women are most susceptible due to high demand from the developing foetus [3].
Though anemia can be treated by many modern drugs, modern medicines and therapies are not affordable by poor population due to financial constrains or unavailability in rural areas. The patients in these areas use plants and herbal products for the treatment of diseases including anemia. Herbal drugs are very effective in treating anemia and are proved by many studies [12][13][14].
Anemia is likely to increase in future and costeffective and better treatment strategies for it are need of the hour. . Mandura bhasma is traditionally used in ayurveda for treating blood many disorders including anemia. The scientific evaluation of its effectiveness in treating anemia is desired. This study investigate the antianaemic potential of mandura bhasma in wistar rats administered in three different doses and compared with standard iron supplement drug. Various blood parameters such as hemoglobin, RBCs, WBCs PCV, MCH, MCV were evaluated as factors contributing improvement of anemia.

Animals
The albino rats (Wistar strain) of either sex weighing 150-200 g, were selected for the study.
The animals were housed in polypropylene cages at a temperature of 25 + 2ºC with relative humidity of 40-60 % and 12 hrs light dark cycle, with maximum of 6 animals per cage. Animals were fed with a balanced diet and water ad libitum during the complete experimental period.

Chemicals
Chemicals like EDTA sodium salt, phenylhydrazine, standard drug ferrous sulphate of analytical grades required for animal study were procured from institutional store.
Mandura bhasma was prepared in Institute laboratory using standard procedure. In the present study, the three different doses of mandura bhasma were decided to be as 6mg, 11mg and 22mg/kg of body weight. The doses were calculated by extrapolating the human dose to animal dose based on the body surface area ratio.

Study protocol
The albino rats (Wistar strain) of either sex weighing 150-200 g, were randomly divided into 6 groups, each containing 6 animals.

Induction of anaemia
In the present study phenyl hydrazine was used to induce anemia in rats. Solution of phenyl hydrazine (50mg / ml) was prepared by dissolving phenyl hydrazine in absolute alcohol and diluting with distilled water [15,16] to induce anemia. Solution of phenylhydrazine 10mg/kg was administered by oral route for period 8 days [17].
Drug suspension (mandura bhasma in 3 doses 6mg/kg, 11mg/kg, 22mg/kg ) was prepared in 3% gum acacia solution in distilled water [15,18]. All administrations of drug were done by oral intubation. The treatment schedule was kept for 21 days .Every 7 days the blood parameters were checked.
The treatment schedule was as follow.

Analysis of haematological parameters
Red blood cell count (RBC), haemoglobin (HB) concentration, packed cell volume (pcv) , mean cell volume (MCV), mean cell haemoglobin (MCH), mean cell haemoglobin concentration (MCHC), and white blood cells (WBC) count were analysed for evaluating hematinic potential of mandure bhasm Blood was collected (1-2 ml) from the retro-orbital plexus, under slight chloroform anaesthesia, in ctoppered vials with EDTA solution. The blood was sampled on day 0 before phenyl hydrazine administration, and on days 1, 7, 14 and 21, days after phenyl hydrazine administration and drug treatment [19,20]. All the aforementioned parameters were determined by using Auto cell counter 920 E , swelab ltd.

Statistical analysis
Statistic Results are expressed as the mean ± SEM, n=6. The data were statistically analyzed using a one-way ANOVA followed by Dunnett test. Statistical significance was determined at a level of p < 0.05.

RESULTS AND DISCUSSION
Lauha bhasma and mandura bhasma are the two most commonly used ayurvedic preparations. Apart from haematinic and astringent, they are used in disorders of liver, spleen and kidney disease. They are also useful in treating intestinal worms, nervous disease, and neuralgia. Being a powerful haematinic and general tonic they are very valuable in the treatment of haemolytic anaemia and jaundice [21,22].
The reported acute toxicity studies as per OECD guidelines and Globally Harmonized System (GHS) for the classification of acute toxic substances, the mandura bhasma was classified in the Category 5 with a LD 50 higher than 2000 mg/kg [23,24] Based on the earlier reports, a lower dose of 11 mg/kg and higher dose of 22 mg/kg was selected in the present study [25]. Haematinic activity of mandura bhasma was evaluated in vivo in phenyl hydrazine induced anaemic albino rats.
Changes in body weight, RBC, HGB, HCT, MCV, MCH, MCHC and WBC were evaluated to assess and compare the haematinic activity.

Evaluation of Changes in Body Weight
Anemic patients, generally suffer from the loss of the body weight which does not increase easily. Slight increment or at least maintenance of the body weight in can be a sign of better anemic control. The study shows the Mandura bhasm show significant improvement in body weight of anemic animals comparable to standard drug. The results are depicted in Fig. 1.

Evaluation of Changes in RBC Count
The changes in RBC count of experimental animals during course of study are given in Fig. 2.
Increased RBC count is considered as one of the most important goals of the anemia treatment. When RBC count of experimental animals was evaluated, as expected, the standard showed significant improvement (p<0.05) in RBCs count. Mandura bhasma also produced significant increase (p<0.01) in RBCs count when compared with anemic control groups. The results indicate strong anti anemic potential of Mandura bhasma.

Evaluation of Changes in HB Count
Anemia causes depletion in hemoglobin count and this is reason which makes anemia a very challenging health problem. Depletion in hemoglobin count is primary reason for the complications of anemia. Mandura bhasma increases HB content significantly (p<0.05 respectively) when compared with anemic control. The results are comparable with Standard. The results are graphically represented in Fig. 3.

Evaluation of Changes MCH
Standard drug and mandura bhasma produced a significant decrease (p<0.01) in MCH as compared to anemic control (Fig. 4). The results indicate that MB has good reducing effect on MCH indicating improved anemic condition.

Evaluation of Changes in MCV
Significant decrease in the mean cell volume after treatment indicates that the drug treatment is good enough for anemia. Mandura bhasma show significant decrease (p<0.05) in the mean cell volume when compared with anemic control. The decrease is significant compared to standard (Fig. 5). The results indicate that mandura bhasm is a as good as standard in treating anemia.

Evaluation of Changes in MCHC
Decreased Mean Cell Hemoglobin Concentration is a indication of the recovery of anemic condition. From Fig. 6, It can be observed that MB shows a significant decrease (p<0.05) in MCHC in experimental anemic animals. The results are comparable with standard indicating as good activity as that of standard.

Evaluation of changes in PCV
Packed cell volume or Hematocrit is decreased in anemia. Any viable treatment for anemia must restore hematocrit in anaemic condition. From the Fig. 7 it is clearly observed that mandura bhasma produced a significant increase (p<0.01) in PCV/HCT (%), when compared with anaemic control.

Evaluation of WBC
Increase in WBC count is caused by most of health challenges to body including anemic condition. Effective haematinic agent must be able to decrease increased WBC count in blood after anemia or at least it should prevent WBC count from increasing drastically. The mandura bhasma produced a significant decrease (p<0.05) in WBC count in anemic animals as compared to anaemic control. The effect on WBC was comparable to standard drug.

CONCLUSIONS
The study indicates that not only the standard drug but Mandura Bhhasm show significant haematinic activity with the significant increase in blood RBC, HB and the % HCT/PCV count. The results indicate and confirm the potential effect of mandura bhasma in treating anemia.

DISCLAIMER
The products used for this research are commonly and predominantly use products in our area of research and country. There is absolutely no conflict of interest between the authors and producers of the products because we do not intend to use these products as an avenue for any litigation but for the advancement of knowledge. Also, the research was not funded by the producing company rather it was funded by personal efforts of the authors.

CONSENT
It is not applicable.

ETHICAL APPROVAL
All the authors hereby declare that All animal experiments were approved by the Institutional Animal Ethical Committee (Registration No. 535/02/a/CPCSEA/Jan2002) of Institute of Pharmaceutical Education and Research, Wardha.