Development & Evaluation of Polyherbal Formulation Used For The Treatment & Management of Anaemia

Anemia treatment and management are based on the underlying cause, focusing on addressing the deficiency or condition causing low red blood cell count. Common treatments include iron supplements for iron deficiency anemia, vitamin supplements for deficiencies like B12 and folate, and addressing the underlying disease in chronic disease-related anemia. Blood transfusions may be necessary in severe cases or when other treatments are insufficient (1, 2). Specific treatment strategies include oral iron supplementation for iron deficiency anemia, dietary supplements and injections for vitamin deficiencies, managing chronic conditions like chronic kidney disease and inflammatory conditions, aplastic anemia, hemolytic anemias, blood loss, and bone marrow disease. General management principles include addressing the underlying cause, monitoring hemoglobin levels, considering individual patient factors, providing nutritional support, and collaborating with healthcare professionals in complex cases. (3, 4) Regular assessment of hemoglobin levels helps track treatment effectiveness and adjust strategies as needed. A balanced diet rich in iron, vitamin B12, and folate can help prevent and manage anemia. Anemia, affecting one-fourth of the world's population, is primarily caused by iron deficiency. It is linked to chronic fatigue, impaired cognitive function, and reduced well- being. Gastroenterologists often treat patients with iron deficiency anemia due to its gastrointestinal origin (5, 6). Proper management improves quality of life, alleviates symptoms, and reduces blood transfusions. Treatment options include oral and intravenous iron therapy, but oral iron's efficacy is limited in certain gastrointestinal conditions. This article provides a summary of diagnosis and treatment, along with a management algorithm for identifying patients needing further gastrointestinal evaluation (7-9). The study focuses on the development and evaluation of a polyherbal formulation for treating and managing anemia. The objective is to enhance iron absorption, boost red blood cell production, provide essential nutrients, reduce anemia symptoms, minimize side effects, improve hemoglobin levels, improve overall blood health, support bone marrow function, enhance vitality and energy, strengthen the immune system, promote better nutrient utilization, and address underlying anemia causes.

MATERIALS AND METHODS

Selection, Collection, authentication and processing of the Crude Drugs

All the crude drugs were collected from Chikhli, Maharastra in the month of July-August when the plants bear flower and authenticated from the Shri Shivaji Science and Arts College, Chikhli, Buldana, Maharastra. The collected plant materials were shade dried. The dried materials were coarsely powdered individually by means of mechanical grinder. Then they were passed though sieve no.40 (aperture size-425mm) to get moderately coarse powder. The resulting powdered materials were used for further studies.

Pharmacognostical Evaluation of Plant Material

Macroscopic characters like appearance, taste, colour, and odour were evaluated. Plant part sections were cut by free hand sectioning and numerous sections were examined microscopically. For powder microscopic analysis about 2 gm of powder in a small beaker and wash thoroughly with water, pour out the water without loss of material, mount a small portion in glycerine; warm a few mg with chloral hydrate solution, wash and mount in glycerine; treat a few mg with iodine in potassium iodide solution and mount in glycerine. Observed the characteristics in the various mounts

Extraction of the Plant Materials

All the plant parts (tuber of ginseng, leaves of Moringa oleifera, roots of the Beta vulgaris, fruits of Phyllanthus emblica, roots of Withania somenifera and Asparagus racemosus) were shade dried and grind coarsely and extracted using methanol through Cold Maceration technique.

TLC examination of the Polyherbal Syrup (PHS)

20 µl of the each volatile sample extract was applied on E. Merck aluminium plate precoated with silica gel 60 F254 of 0.2 mm thickness and the plate was developed in Toluene: Ethyl acetate (9: 1) dried the plate and kept under UV 254 nm and 366 nm and TLC profiles were documented. The TLC plate developed above, before dipping in vanillin-sulphuric acid was scanned at 254 nm using Scanner-3, Camag HPTLC instrument using Deuterium lamp. The plate was then dipped in Vanillin-sulphuric acid reagent and heated in a hot air oven at105oC until the colour of the spots appeared and profile photo was documented under white light

Formulation of Polyherbal Formulation

We selected 2.5:1.5:1:3:2.5:1.5 ratio of methanolic extracts of tuber of ginseng, leaves of Moringa oleifera, roots of the Beta vulgaris, fruits of Phyllanthus emblica, roots of Withania somenifera and Asparagus racemosus for polyherbal syrup. For preparation of polyherbal syrup, sugar base was prepared by mixing of sucrose and 50 ml of water, heated to boiling point. The liquid was strained and volume made up to 100 ml with distilled water. The preservatives were dissolved in few milliliter of boiled and cooled water and added to a sugar base. Extracts were dissolved in propylene glycol at 45–50°C and this glycerin and sorbitol were added. The remaining sweetening agents were added and mixed thoroughly. Adjust the pH between 5.5 and 7.0 with, if necessary (10).

Characterization of the Polyherbal Formulation

The Polyherbal formulation was evaluated for the color, taste, density, pH and Viscosity (11-13).

Acute Toxicity Study

Total 6 rats of 10-12 weeks age were selected and randomly divided into 2 groups. Group I was vehicle control group which received vehicle (gum acacia 1% w/v in distilled water) while group II was test group that received PHS. Each group consisted of 3 animals (females). Females were nulliparous and non-pregnant. Dose selected for the present study is limit test dose as mentioned in the guidelines. The starting dose for limit test 2000 mg/kg was selected on the basis of dose suggested in OECD guideline (14).

Evaluation of Anti Anaemia activity of Polyherbal Formulation Syrup (PHS)

The rats were grouped into four groups with 5 female rats in a group. Group I was kept as control group and the remaining 15 rats were given Phenyl hydrazine (PHZ) by daily oral administration at the dose of 10 mg/kg for 8 days (15). On 9th day, blood was withdrawn from retro orbital sinus and analysed for haemoglobin concentration. The other parameters like RBC, MCH, MCV, MCHC, PCV, and Serum Iron were also noted. Haemoglobin concentration lower than 12 gm/dl were recruited for the study. They were grouped as Group II, III and IV (Table 1).

Table 1: Animal Grouping and Dosing

Parameters to be evaluated

At the end of 28 days, rats were over night fasted, blood samples were collected from the retro- orbital puncture under anaesthesia with and without anticoagulant and used for haematological and biochemical parameters. Hematological parameters like Hb, RBC, MCH, MCV, MCHC, PCV, and Serum Iron were analyzed (16).

RESULTS AND DISCUSSION

Pharmacognostical Study of Plant Material

The macroscopic characteristics of all the plan parts are consolidated in Table 6 while microscopic features are presented in Figure.

Table 1: Macroscopic Characteristics

Percentage (%) Yield

The percentage yield of each extract alongwith the texture is mentioned in Table 2.

Table 2: Percentage Yield

TLC Fingerprint Profiles of the Extracts

The TLC plate was developed in Toluene: Ethyl acetate (9: 1) mobile phase, air dried, observed under visualize and documented the fingerprint profiles of UV 254 nm, 366 nm and under white light for the derivatized plate in vanillin in sulphuric acid (Table 9; Figure 11).

Table 9: TLC Fingerprint Profile of the Formulation

Characterization of the Polyherbal Formulation

Organoleptic characteristics were examined physically by means of sensory organs which are mentioned in Table 3, while other evaluations were done by using appropriate laboratory methods mentioned in table 4 and stability study mentioned in table 5.

Table 3: Organoleptic Characteristics

Table 4: Characteristics of the Polyherbal Formulation

Table 5: Stability Study according to ICH guidelines

The Polyherbal Formulation Syrup (PHS) was found to be safe at a dose level of 2000 mg/kg, BW of the animals and no clinical signs were observed other than normal activity (Table 9). During necropsy, no gross morphological changes were observed in the organs from test groups compared to organs from vehicle control rats. On conclusion, we can say that Polyherbal Formulation Syrup (PHS) so prepared is safe for the further animal study.

Evaluation of Anti Anaemia Activity of Polyherbal Formulation Syrup (PHS)

Phenylhydrazine administration induces anemia in rats, leading to focal degenerative changes in cardiac muscle and dilation of cardiac fibers. The decrease in Hb content was reversed by treatment with Polyherbal Formulation Syrup (PHS) and Iron Syrup. Iron Syrup was found to be more effective in increasing Hb content in anemic rats. The HCT count also decreased in anemia-induced rats compared to normal control rats. However, this decrease was reversed by treatment with PHS and Iron Syrup, which were found to be equipotent in this regard (Table 6).

Figure 9: Effect of Polyherbal Formulation Syrup (PHS) on blood levels of a) WBCs, b) RBCs and c)  hemoglobin in anemia-induced rats