Iron deficiency anaemia (IDA) is one of the most common health problems during pregnancy . Presently available antianaemic preparations to treat IDA in pregnancy have many side effects like nausea, vomiting, heart burn and constipation  . So, it would be better to use a natural remedy which has been traditionally used for a long time with less or no side effects. The fruit Emblicaofficinalis (amloki) is one of the most frequently used ayurvedic herbs   .
Though iron deficiency anaemia can be diagnosed by patient’s history and clinical examination, some laboratory examinations are required to establish the diagnosis. Complete blood count documents the severity of the anaemia. In chronic IDA, the cellular indices show a microcytic and hypochromic erythropoiesis. In 40% cases, IDA can show normocytic normochromic blood picture . Platelet count may be increased (>450,000/µL); which becomes normal after iron therapy. The white blood cell count may be elevated than the normal range (4500 - 11,000/µL) .
Several studies have been done about IDA in pregnancy in Bangladesh and worldwide. WHO has recommended iron and folic acid supplementation as standard treatment for IDA in pregnancy . But it has several side effects, like heart burn, nausea, upper gastric discomfort, constipation and diarrhea. Some studies have shown to generate free radicles, which cause damage to the intestine . But, amloki has the ability to increase iron absorption and decreases the side effects .
Researchers observed the haematological effects of Amloki on both experimental animals and humans. Haque and Sinha (2015) experimented that aqueous extracts of Emblicaofficinalis fruit administration significantly increases red blood cell, hemoglobin concentration and WBC count in wister albino rats .
Amloki is considered one of the richest sources of vitamin C (ascorbic acid). Presence of low molecular weight hydrolysable tannins, prevents the oxidization of ascorbic acid even in dried fruit. So, nutritional qualities of fruit remain unaltered. Scartezzini et al. (2016) found that dried and frozen fruit contains about the same (0.4%) ascorbic acid, while Ayurvedically processed fruit preparation contains 3 times more (1.28%) ascorbic acid  .
Non-haem iron predominates in all diets. About 90% - 95% of total daily iron intake derived from non-haem iron. The majority of dietary non-haem iron remains in Fe3+ form, which must be reduced to Fe2+ form for its absorption. Vitamin C present in amloki causes this reduction and increases iron absorption. Combination of amloki with FeSO4 can enhance iron absorption 3 fold over the control iron preparation of FeSO4 alone . Phytates found in cereal products and polyphenolic compounds in all plant products are the most potent dietary inhibitor of non-haem iron absorption. Dietary ascorbic acid can counteract the inhibitory effect of phytates and polyphenol . Adding vitamin C to a meal increases non-heme iron absorption up to six-fold which makes the absorption of non-heme iron as good as or better than that of heme iron .
2. Material and Methods
This prospective comparative study was carried out on patients of Outpatient Department of Obstetrics and Gynecology, Dhaka Medical College Hospital from July 2016 to June 2017 recruiting 43 pregnant women between the 13th to the 20th weeks of gestation with IDA. All of them were in between 18 - 36 years of age.
1) Study design: Prospective, open labeled interventional study.
2) Study location: This was a tertiary care teaching hospital based study done in Department of Physiology, Dhaka Medical College and Hospital, Dhaka.
3) Sample size calculation: The sample size was estimated on the basis of the difference between two means from previous study. We assumed that the power of the study was 90% and the level of significance was 5%. The sample size actually obtained for this study was 21 patients for each group. We planned to include 25 patients (Group I-study, Group II-Control of 25 patients for each group) with 16% drop out rate.
4) Subjects & selection method: Sample size was divided into two groups.
a) Group A (study group) consisted of 24 pregnant women with IDA, were supplemented with oral amloki capsule (1.072 gm) thrice daily and iron tablet [ferrous fumarate (200 mg) + folic acid (0.02 mg)] once daily for 45 days. This group was studied two times designated as,
Group A1: At baseline i.e. before starting intervention.
Group A2:.After 45 days i.e. after 45 days of intervention.
b) Group B (control group) consisted of 19 pregnant women with IDA, treated with only iron tablet [ferrous fumarate (200 mg) and folic acid (0.20 mg)] once daily for 45 days. This group was studied two times designated as,
Group B1: At baseline i.e. before starting intervention.
Group B2: After 45 days i.e. after 45 days of intervention.
5) Diagnostic criteria
· Signs and symptoms of iron deficiency anaemia.
· Haemoglobin concentration (Hb) ≥ 8 to <11 g/dl.
· Blood pictures with micocytic hypochromic and normocytic normochromic RBC.
6) Inclusion criteria:
· Pregnant women between the 13th wk to the 20th wk of gestation.
· Age 18 to 36 yrs.
· Not taking any other drugs.
7) Exclusion criteria:
· Patients having Hb < 8 g/dl.
· Patients with a history of anemia due to any conditions like, bleeding piles, PUD, cardiac/renal/liver disorders, DM, HTN, thalassaemia, sickle cell anaemia, rheumatoid arthritis, worm infestation, hemoglobinopathies and malignancy.
· Defective absorption like patients of gastrectomy, gastrojejunostomy, sprue syndrome etc.
· Pregnancy induced complications like hyperemesis gravidarum, antepartum hemorrhage, pre-eclamptic toxaemia, eclampsia, GDM, jaundice. Patients with a history of drug or alcohol abuse.
8) Authentification of amloki capsule
The amloki capsule (Amlahills) was authentified by the Department of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Dhaka, which was manufactured by Isha Agro Developers PVT.LTD, India.
9) Procedure methodology
After selection of the subjects, the nature, purpose and benefit of the study were explained to each subject in detail. They were encouraged for voluntary participation. They were allowed to withdraw from the study whenever they feel like. Informed written consent was taken from the participants. The research work was carried out after obtaining ethical clearance from concerned departments, Research Review Committee and Ethical Review Committee of Dhaka Medical College, Dhaka. Before taking blood, detailed family and medical history were taken. Anthropometric measurement of the subjects was done and blood pressure was measured. All the informations were recorded in a prefixed questionnaire. Haematological parameters were estimated in the Department of Haematology & BMT Unit, Dhaka Medical College Hospital, Dhaka by using automated haematology analyzer Model XT-2000i. These parameters were studied 2 times in all subjects of control and study groups, i.e. at the beginning of the study (baseline) and after 45 days of study period. Compliance to the supplementation was monitored by regular telephonic communications. Amloki capsules were given in boxes for 45 days and participants were encouraged to continue the supplied medicine daily. After 2 weeks of study period one subject was excluded from study group, because she was advised by someone not to take amloki capsule. Within 6 weeks of study, 6 subjects from control group left Dhaka. So, finally 24 subjects of study and 19 subjects of control groups completed the study.
10) Statistical analysis
All the parameters were expressed as mean ± SD (standard deviation). Paired Student’s “t” test and Unpaired Student’s “t” test were used as the test of significance as applicable. The p value < 0.05 was accepted as level of significance. Statistical analyses were performed by using a computer based statistical program SPSS (Statistical package for social science) Version 22.0.
Figures 1-3 show different levels of RBC, WBC and Platelet respectively in different groups.
Table 1 shows, mean RBC count (p < 0.05) was found significantly increased in both groups, in comparison to that of their baseline values. There were also
Figure 1. Mean total count of RBC in different groups (n = 43). A1: Study group (at baseline); A2: Study group (after 45 days of supplementation with amloki and iron tablet); B1: Control group (at baseline); B2: Control group (after 45 days of supplementation with iron tablet).
Figure 2. Mean total count of WBC in different groups (n = 43). A1: Study group (at baseline); A2: Study group (after 45 days of supplementation with amloki and iron tablet); B1: Control group (at baseline).
Figure 3. Mean total count of platelet in different groups (n = 43). A1: Study group (at baseline); A2: Study group (after 45 days of supplementation with amloki and iron tablet); B1: Control group (at baseline); B2: Control group (after 45 days of supplementation with iron tablet).
Table 1. Haematological parameters in different groups (n = 43).
Results are expressed as mean ± SD. Paired “t” test was performed for comparison within groups and Unpaired “t” test was performed to compare between groups. The test of significance was calculated & p value < 0.05 was accepted as level of significance. n = number of subjects; ns = non significant; */** = significant. A1: Study group (At baseline); A2: Study group (After 45 days of supplementation with amloki and iron tablet); B1: Control group (At baseline); B2: Control group (After 45 days of supplementation with iron tablet).
significant increase in RBC count (p = 0.007) in pregnant women supplemented with amloki and iron tablet in comparison to only iron supplemented group.
Almost similar types of results were observed by different researchers   . The increase in total count of WBC (p = 0.387) and decrease in total count of platelet (p < 0.198) in amloki and iron supplemented group was not statistically significant in comparison to only iron supplemented group.
Almost similar types of results were observed by different researchers   .
Iron is found in ferrous/ferric form at the site of absorption of the intestine. Reduction of ferric to ferrous form is necessary for the absorption of iron. Ferric reductase (Dcytb) present on the brush border of enterocytes causes this reduction. Other dietary factor like vitamin C can also cause this reduction and enhance iron absorption. Vitamin C present in amloki is the most potent enhancer of iron absorption by its reduction to ferrous form . Moreover, phytates present in cereals and legumes impair absorption of iron from foods as well as from soluble iron salts. Phytates form diferric and tetraferric phytates, which are insoluble in gastric environment . Vitamin C can counteract the inhibitory effect of phytates by replacing it from iron and make iron soluble for better absorption . This increased absorption of iron can cause increased erythropoiesis.
Other phytochemical properties of Emblicaofficinalis may play a role in erythropoiesis. It also contains other vitamins (vitamin-A, K, B2, B5), different minerals like iron, magnesium, potassium, zinc, copper, phenolic compounds (gallic acid, methyl gallate, gallic acid, trigallay glucose), amino acids (glutamic acid, proline, aspartic acid, alanine, cystine, lysine)  . These phytochemicals are well known hemopoietic factors that have direct influence on the production of blood cells in the bone marrow .
Increasing in number of WBC may be a normal reaction to foreign substances. Amloki preparations may act as an antigenic substance that stimulates immune system and increase WBC count .
Moderate iron deficiency anaemia (IDA) is usually associated with reactive thrombocytosis. There are different possible causes of reactive thrombocytosis. In IDA there is increased rate of influx and efflux of precursor cells into the megakaryocyte compartment and shortening of megakaryocyte maturation. There is also stem-cell shunt due to inhibition of erythropoiesis, resulting in increased production of other pluripotent cells (haemostatic compensatory mechanism). In IDA free transferrin concentration is increased, which have stimulator effect on megakaryopoiesis. Thrombocytosis may disappear after iron supplementation as iron has an inhibitor effect on platelet counts . Moreover, amloki is useful in reducing platelet counts. Flavonoids present in amloki, increase the cAMP levels in platelet, which is known to inhibit platelet activation by lowering intracellular Ca2+ levels  . In this study, there was also decrease in platelet count which were not statistically significant.
After analyzing the results of the study, it can be concluded that combination of oral iron and Emblicaofficinalis (amloki) supplementation can cause significant increase in RBC count than that of only iron supplementation in pregnant women with iron deficiency anaemia.
The authors acknowledge Department of Pharmaceutical Chemistry, Faculty of Pharmacy and Department of Soil, Water and Environment, University of Dhaka for their kind cooperation.
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