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Introduction

Iron deficiency anaemia arises when the iron supply is insufficient to meet the needs of haemoglobin production. Its prevalence as per the National Family Health Survey 5 (2019-21), is 59.1 percent in adolescent girls, 31.1 percent in adolescent boys (15-19 yrs),  57.0 percent in women (15-49 years) and 25.0 percent in men (15-49 years).[1] Clinical signs of iron deficiency anaemia include oedema, weakness, anorexia, pallor, and irritability, pica etc. Several Iron contained medicines are conventionally used for the management of IDA whereas some of them have adverse reactions such as constipation, abdominal cramps, nausea, vomiting, diarrhoea etc.[2] IDA is managed with so many formulations mentioned under Panduroga chikitsa(Treatment of pallor/clinical features of p???u) in Ayurveda also.  Harithakiyogam is one among such formulation which has limited number of drugs clinically useful, but not yet scientifically proven. Hence, the mixture of Harithaki(Terminalia chebula Retz.) and Bhadra(Aerva lanata Juss.) mentioned in Yogamritham Pandurogachikitsa adhyayam[3] which provides a scientific validation to its haematinic activity can definitely become beneficial to the society as it makes less chance of adulteration, less expensive and easy home remedy for anaemia. This study was done to analyse the effectiveness of this combination using quasi experimental trial and is discussed here.

Materials And Methods:

Inclusion criteria 

• Male participants having the range of Haemoglobin level 8-12.9 g/dl and female participants having the range of Haemoglobin level 8-11.9 g/dl.
• Participants with microcytic hypochromic anaemia from peripheral smear.
• Participants with RDW-CV greater than 14.
• Participants who are willing to give consent

Exclusion Criteria

• Known cases of Anaemia due to other illnesses like peptic ulcer disease (ulcers in the stomach or small intestine), chronic kidney disease, cancer (especially gastrointestinal cancers), iron deficiency due to blood loss from the intestine or other sites.
• Pregnant, intend to become pregnant, breastfeeding, within 2 weeks postpartum, having a positive serum or urine pregnancy test.
• Participant currently receiving iron supplementation.

Intervention:

Harithakiyogam comprising of Harithaki (Terminalia chebula Retz.) and Bhadra (Aerva lanata Juss.) are the study formulation which were dispensed as tablet. The enrolled participants were given Albendazole 400-mg single dose orally for deworming before dispensing the study tablet. Then they were advised to take 1 tablet weighing 500 mg thrice daily after food. The trial Ayurveda intervention is manufactured from College Pharmacy and the punching of tablets were done from GMP Certified Pharmaceutics.

Preparation and Standardisation of Intervention:

The raw drugs were carefully cleaned, shade dried and kept in airtight containers. Equal parts of the two drugs (1:1 ratio) were separately powdered and made into coarse powder. Similarly, equal number of drugs were powdered and made it fine after passing through the mesh of nominal aperture size 180 micrometre and kept separately in the same type container. Then these fine powders of both drugs mixed together and prepared Harithakiyogachurna. Then the coarse powder of Harithakiyogam was taken and 8 times of water was added and reduced to ? as per the method mentioned in the classical Ayurvedic literature Bhaishajya ratnavali.[4] The fine powder of Harithakiyogam was subjected to grinding in the kashaya obtained till the complete absorption of the liquid into the powder and the paste obtained was subjected to drying. It takes about 6-7 hours to become soft and fine.  Same process repeated 2 times for the potentiation of the churna and for dose reduction. The resultant grinded, potentiated and dried powder of Harithakiyogam churnam was made into tablets each weighing 500mg after adding 0.2% preservative and 5% binders.

Standardisation of Harithakiyogam Tablet:

Individual drugs procured and ensured to meet the authentic standardisation parameters after pharmacognostical analysis, physicochemical and phytochemical analysis, ICP-MS Analysis, TLC and HPTLC Analysis. All the parameters were on a par with the API limit for individual drugs. Standardisation parameters of tablet shown in Table 1.

From the data, it showed that major drawback of the preparation of medicine after the procedure is the highest disintegration time and hardness. It makes the tablet a long time to dissolve in water. Administration of tablet to the participants is advised after powdering it and mixed with lukewarm water.

HPTLC Analysis: HPTLC analysis of Harithakiyogam and its individual component drugs showed best separation in Toluene: Ethyl acetate: Chloroform: Formic acid (4:8:1:3) shown in Figure 1

Track 1 - Gallic acid (Marker compound)

Track 2 - Terminalia chebula Retz. (2.5 μL) (Raw Drug)

Track 3 - Harithakiyogam (2.5 μL) (Formulation)

Track 4 - Aerva lanata Juss. (2.5 μL) (Raw Drug)

From the data obtained after the graphical representation (Figure 2) of 4 tracks at 254 nm, there are common Rf values shared by the Gallic acid standard and other components which is 0.979, 0.981, 0.982 and 0.984 for Gallic acid, Terminalia chebula Retz., Harithakiyogam, Aerva lanata Juss. respectively. While comparing the graphical representation of 4 tracks at 366 nm (Figure 3), common Rf value obtained were 0.982 for Gallic acid and Terminalia chebula Retz., 0.985 for Harithakiyogam and 0.987 for Aerva lanata Juss. From this, we can analyse that almost Rf values of all the single drugs and combination is similar to that of reference standard gallic acid and can infer the presence of gallic acid standard in the individual drugs and the combination.

Outcome measures:

The primary outcome measure is the change in hemoglobin level which was assessed at baseline and at 90th day. The secondary outcome measures are changes in red blood cell count, red cell indices, Peripheral smear and RDW-CV were assessed at baseline and 90th day. Secondary outcome of the study is improvement in Subjective parameters [Table 2] at baseline and 90th day.

Fatigue was assessed based on Fatigue Assessment Scale [8]

Participant Timeline

A total of 35 participants were screened and 5 partipants were excluded as they failed to meet the inclusion criteria. 30 participants were met with inclusion criteria but 3 of them refused to take part. However, 27 participants were included but 4 participants were withdrawn from the study. Consequently, 23 participants were considered for statistical analysis. [Figure 4]

Sample Size

Sample size calculated was 25 from Previous study.[9] By considering mean difference in Hb as 1.19, and average standard deviation of Hb from previous study is 1.455  with 90% power and considering an expected dropout rate of 10%, a total of 25 participants was calculated as sample size in the study.

Statistical Analysis

To find out the impact of the intervention, paired t-tests were performed to compare pre-treatment and post-treatment measurements for each outcome. A significance level of P?<?0.01 was established to determine statistical significance.

Results:

Demographic Profile of the Study Participants:

Total 23 participants completed the trial. Among which, 78.3% were females which proves the prevalence of iron deficiency anaemia in women. 47.8% participants were in age group 40-50. This may be due to the unsatisfactory and irregular food habits in both sexes whereas in females, there may be a chance of premenopausal symptoms also where heavy bleeding persists which were prevalent in this age group. Among the domicile distribution, 82.6% participants were belonged to rural area and 78.3% were from middle class in which there may be an inadequate intake of dietary iron supplements and fruits which enhances iron absorption leading that IDA more specifically caused due to nutrient deficiency. While analysing the occupation, 39.1% are of student category and 39.12% are working in public and private sector. Due to the work habit or nature of work, there might be a chance of irregular food intake or skipping of 1 or 2 meals per day which may lead to IDA. Demographic profile depicted in Table 3.

Effect of Treatment in trial group:

In trial group, the mean Hb increased from 9.51 to 11.54 with an increase of 2.03(g/dl) after the treatment. The mean RBC increased from 3.96 to 4.36 with an increase of 0.40(million/mm3) after the treatment. The mean MCV increased from 70.24 to 81.74 with an increase of 11.5(fl) after the treatment. The mean MCH increased from 22.65 to 27.93 with an increase of 5.28(pg/cell) after the treatment. The mean MCHC increased from 30.58 to 33.98 with an increase of 3.4(g/dl) after the treatment. The mean RDW-CV decreased from 16.7 to 13.55 with a decrease of 3.14(%) after the treatment. All the parameters were significant at 1% level of significance (p-value<.01) (Table 4). Graphically represented in Figure 5.

Effect of Treatment in Peripheral Smear:

Before treatment all cases were microcytic hypochromic, 73.9% became normal after the treatment and only one remains microcytic hypochromic. (Table 5) (Figure 6)

Discussion

Iron deficiency anaemia has variable causes such as impaired production of red blood cells, excessive destruction of cells, inadequate nutritional iron, excess blood loss, parasite infection leading to the non-absorption of iron etc. T. chebula ethanol extract acts as a natural anthelmintic that has produced inhibitory effects on the action of Acetylcholinesterase (AChE) by blocking AChE activity and also decreases the motor activity of Cotylophoron cotylophorum in a time-dependent concentration manner. Halting the motility of worms and a repercussion from the intestinal peristalsis of the host also occurred.[10] Also, Aqueous and Alcoholic extracts obtained from the leaf and stem of plant Aerva lanata Juss. possess good anthelmintic activity as compared to albendazole. [11] This anthelminthic effect of both drugs is able to prevent the further formation of worms in intestine and thereby increasing iron absorption.

Hepcidin is an Iron regulator hormone in which the release of hepcidin increases during inflammatory condition. It can able to bind with ferroportion and cause degradation which results in anaemia. Total and Reducing Sugar present in the drugs have anti-inflammatory action thereby reducing the excess release of hepcidin and in turn correcting the metabolism and may cure anaemia.[12] [13]

While coming to the ayurvedic treatment, diagnosis of IDA with respect to Ayurveda is important. Pandu (pallor/clinical features of p???u) altogether or its specific types simply cannot be directly correlated to Iron Deficiency Anaemia (IDA) because the concising of this broad term not easily possible. Although,while going through the samprapthi(pathogenesis) of panduroga(pallor/clinical features of p???u) and pathogenesis of IDA, rakthakshaya(depletion of blood) or reduction in haemoglobin is a common factor and while comparing the symptoms of both, reduced appetite, dryness of skin, fatigue, weakness, dyspnoea on exertion, palpitation & pallor of the skin, mucous membranes & sclera, tinnitus, unusual dietary cravings such as pica[14] can be correlated with Agnimandya(diminution of agni), Twakrukshatha, Angamarda, Dourbalya, Srama(exhaustion/fatigue), hridayaspandana, Panduvarnata(Pallor), Karnanada(ringing in the ears/ tinnitus), Mritbhakshanaecha etc. respectively. Hence, considering all these, IDA can be considered in the spectrum of Panduroga(pallor/clinical features of p???u). Panduroga(pallor/clinical features of p???u) is a pittapradhana tridoshaja vyadhi in which Tridoshas(three regulatory functional factors of the body), Rasa(primary product of digested food), Raktha(blood tissue), Medodhathus and Rasavaha(channels carrying nutrient fluids) - Rakthavaha srothas(channels carrying blood tissue) are involved in the samprapthi(pathogenesis) and has affecting jataragni(metabolic factors located in digestive tract) and rasa-rakthadhatwagni(metabolic factors located in dh?tu) also. Due to ahara - viharaja nidana(aetiology) of Panduroga(pallor/clinical features of p???u) and vishamasana, adhyasana causing jataragnimandyathwam(diminution of metabolic factors located in digestive tract) and thereby vitiating rasa raktha dhathus(primary product of digested food and blood tissue) and formation of ama which got sthanasamsraya( stage of localization) in the srotasas(structural or functional channels) and causing srothorodha(obstructive pathology occurring in channels) which ultimately cause impairment in the dhathupaka. Here, a quantitative increase of pittadosa( do?a responsible for regulating body temperature and metabolic activities) with which it reduces the tikshna(sharpness) usna(hotness) swabhava and increases drava(fluidity) sara(instability/mobility) guna of pittadosha( do?a responsible for regulating body temperature and metabolic activities) which causes kaphavilayanatwa thereby rasadushti turns to the inappropriate dhathu(major structural components of the body) transformation and reduction in quantitative and qualitative formation of rakthadhathu(blood tissue). Due to the loss of specific gunas of pitta (do?a responsible for regulating body temperature and metabolic activities), it will be equivalent to kapha (do?a responsible for regulating body fluids and keeping the body constituents cohesive) and vikrtavarna or pandutwa(Pallor) occurs. And there is qualitywise diminished pittavardhana (Swagunahanipitta) is happening and results in rakthakshaya(depletion of blood tissue). The term Rakthakshaya indicates the reduction in viscosity and increase in the fluidity or sidhilatha of rakthadhathu(blood tissue). The rakthakshaya(depletion of blood tissue) causing vatakopa. As the rasavahasrothomoola is hridaya, the rasadushti causes vyanavayudushti (vitiation of a subtype of v?ta, that is seated in h?daya) also and results in pratilomagati of vyanavayu(a subtype of v?ta, that is seated in h?daya) and circulates throughout the body which gives rise to loss of integrity of that dhathu (sidhilatha), Gourava (heaviness of the body), vaivarnya (abnormal change of complexion), bala kshaya (loss of strength), sneha kshaya (loss of unctuousness/depletion of normal fat) and ultimately ojokshaya(depletion of essence of all seven dh?tu). Thus, the drug of choice should have apakwapitta sodhana, agnideepana(increase in power of agni), pachana(enhancing digestion), srotosodhana(cleanliness of srotas/ felling of cleanliness of srotas), rasayana(rejuvenation) properties.

Harithaki (Terminalia chebula Retz.) is one of the best anulomaka(mild purgative action/regularizing physiological movement) Dravya (material or substance used for therapeutic purpose or health benefits). Harithaki with its Deepana (digestion and metabolism enhancing), anulomana karma can capable of normalise the agni and pratilomagati of vyanavayu and results in anulomana of vayu and purisha also. Also, it is having the karma of Yakrtuttejaka, hence potential to regularize rakthavahasrothomoola which ultimately improve the process of raktha formation.[15] Bhadra is having tiktha, kashaya rasa can contradict the gunas (attribute/ property) of pitta( do?a responsible for regulating body temperature and metabolic activities) and Tikshna(sharpness) ,usna(hotness) guna(attribute/ property) of Bhadra  act as srothosodhana(cleanliness of srotas/ felling of cleanliness of srotas) and improving the jatharagni(metabolic factors located in digestive tract) thereby evacuating the apakwapitta. Also, plant possess less toxicity towards erythrocyte membrane which has been proved by in-vitro research and also have anti-microbial, anti-parasitic, anthelmintic activity.[16] The study participants were having irregular food habits which paves to the formation of ama. Correction of jatharagni(metabolic factors located in digestive tract) is very important in the proper formation of pitta (do?a responsible for regulating body temperature and metabolic activities). Agnimandya (diminution of metabolic factors located in digestive tract) which is the ultimate cause of Pandu (Pallor) which leads to amatva, srotorodha(obstructive pathology occurring in channels) and improper rasadhatu formation. Usnavirya(hot potency), Deepana (increase in power of agni), Pachana(enhancing digestion) properties of the study drugs will correct the jatharagni(metabolic factors located in digestive tract). Also, both the drugs having kashaya rasa which exhibit the properties like asravisodhana and atitwakprasadana. Thus, the quality of raktadhatu(blood tissue) could be improved.

Conclusion

The Study drug Harithakiyogam tablet (Combination of Harithaki & Bhadra) 500 mg thrice daily after food for 3 months in 23 study participants is effective in improving Haemoglobin count in Iron Deficiency Anaemia and was statistically significant also. The tablet is effective in improving red cell indices parameter of study participants. Peripheral Smear of 73.9% of study participants became normal after the trial.

The Study drug also shows improvement in the clinical symptoms of IDA like Pallor, loss of appetite, fatigue, shortness of breath etc. which are the most affected symptoms of study participants. The Study drug is also found to be a safe combination in management of IDA as no adverse effects were observed during the study.