INTRODUCTION:

Anemia is a condition in which there is a lack of healthy red blood cells (RBC) to hold adequate oxygen to the body’s tissues^1,2,3. There are many types of anemia such as iron deficiency anemia, sickle cell anemia, anemia of inflammation, aplastic anemia, hemolytic anemia, and vitamin deficiency anemia that affect people, each with their own causes^4,5,6. Iron deciency anemia is the most common type of anemia⁷. Adult male and females of average height have about 4 and 3.5 grams of iron in their body⁸. Almost two-thirds of the body’s iron is found in the haemoglobin, which serves a vital role in carrying oxygen from the lungs to body cells^9,10.

The body cannot produce iron and must acquire it from food^11,12. Therefore, we must take foods that are loaded with iron such as red meat, poultry, pork, seafood, dark green leafy vegetables, beans, cereals, dried fruit and peas^13,14. Heme iron is found in animal products and is generally absorbed easily^15,16,17. But, non-heme iron presented in vegetarian foods is not as easily absorbed¹⁸. Therefore, vegetarian people have a much higher risk of iron deficiency anemia than their nonvegetarian counterparts^19,20. Here, the recent research suggested that vitamin-C facilitates non-heme iron absorption by making a chelate or complex with ferric iron at an acid pH that remains soluble at the alkaline pH of the small intestine^21-22. It is the only food constituent other than the animal product that has been shown to facilitate the absorption of iron^23-24. It is an effective way to manage iron deficiency in vegetarian people. Later, the blood plasma protein called transferrin acts a central role in iron metabolism and is responsible for the transport of iron in the spleen, liver and bone borrow etc., Elimination of iron is commonly taking place through the shedding of intestinal endothelial cells into the faeces²⁵. In view of the above study and continuation of our interest to prove the function of vitamin-C in iron absorption, the study was performed. To the best of our knowledge, the study demonstrated the effectiveness of vitamin-C in iron absorption from non-heme iron food such as Pumpkin seeds, Lentils, and Soybeans. A combination of non-heme and vitamin-C food supplements increased iron absorption in the group 5 animals. Therefore the blood cells, haemoglobin content and blood cell indices level were increased as like a group 3 animals. It is a significant milestone in the management of iron deficiency anemia.

MATERIALS AND METHOD:

Materials:

Excellent quality chicken, meat, fish, pumpkin seeds, lentils, soya beans, oranges, kiwi and lemon were procured from a local shop in Rajamahendravaram, AP, India.

Chemicals and drugs:

Needed chemicals such as Phenyl hydrazine and Heparin have been purchased from Sigma-Alrich to perform the experimental study.

Preparation of food:

Heme food:

Chicken, meat and fish were collected and dried and then made into coarse powder separately (Figure 1)^26-28.

Figure 1: A) Chicken B) Meat C) Fish D) Chicken powder E) Meat powder F) Fish powder.

Non-heme food:

Pumpkin seeds, lentils, and soybeans were collected and dried under sunlight for 24 hours and then made into fine powder separately (Figure 2)^29-31.

Figure 2: A) Pumpkin seeds B) Lentils C) Soybeans seed D) Pumpkin seeds powder E) Lentils powder F) Soybeans powder.

Vitamin-C:

The vitamin-rich foods such as kiwi, lemon and orange were collected and prepared as juices. The juices were made every days (Figure 3)³².

Figure 3: A) Kiwi fruits B) Orange fruits C) Lemon fruits D) Kiwi fruit juice E) Orange fruit juice F) Lemon fruit juice.

Experimental animals:

The study was conducted on 30 certified 6 week-old male and female Wistar albino rats with a mean initial weight of 175±25g. All animals were placed in standard polypropylene cages and maintained in a controlled environment of (55±5) percentage humidity, (25±2)°C temperature and a 12-hour dark/light cycle. They were fed a standard rat diet and water was provided ad libitum³³. The food was provided every 3 hours during the 12-hour feeding window. The experiment protocol was approved by IAEC (GSP/IAEC/2019/12/01).

Study design:

The study consists of 30 Wistar albino rats equally divided into five groups designated as negative control (Group 1), positive control (Group 2), Heme food (Group 3), Non-heme food (Group 4) and Non-heme food along with Vitamin-C food supplements (Group 5) etc., Groups (3, 4 and 5) served as treatment groups. All the rats except Group 1 were intraperitoneally administered phenylhydrazine 60mg/kg once daily for 2 days. The animals were fed with the heme, non-heme and non-heme along with Vitamin-C rich food supplements for 28 days according to the designated group. Later, the blood was collected from rats by retro-orbital puncture and examined³⁴.

Analysis of blood sample:

On 28th day, the rats were weighed and blood samples were collected into different Eppendorf tubes containing heparin sodium and used to analyse different blood parameters using with an automatic hematology analyzer (Sysmex 2100) through the following formulas³⁵. The parameters evaluated with the collected blood samples are haemoglobin, red blood cell count, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH) and mean corpuscular haemoglobin concentration (MCHC)^36-40.

MCV [fl] = HCT/RBC

MCH [pg] = hemoglobin /RBC

MCHC [g/dl] = hemoglobin/HCT

Due to the calculated values of HCT and RBC by the device, the above parameters are impacted.

Statistical data:

Mean values of the chosen blood parameters were compared separately by using one-way anova. The value of the test rats (Group 3,4 and 5) were compared to negative control groups.

RESULTS:

Haemoglobin content:

The results obtained from the study after 28 days of treatment were shown in Table 1. It is observed that the hemoglobin content of all the test groups 3, 4 and 5 were found to be improved as compared to the initial values and also compared to the negative control group. The rats from group 5 gained a haemoglobin count of 15.36± 0.09g/dl from 8.31±0.12g/dl.

Table 1: Effect of heme food, non-heme food, and the combination of non-heme along with Vitamin-C food supplements on hemoglobin in anemic adults rats

S No	Group	Day 0 (Before induction) g/dl	Day 2 (After induction) g/dl	Day 28 (After treatment) g/dl
1	Negative control (Group 1)	13.29± 0.16	13.34± 0.06	13.40± 0.17
2	Disease control (Group 2)	13.16± 0.19	8.61± 0.17	8.72± 0.19
3	Group 3 (Heme food)	13.52± 0.24**	8.38± 0.21**	15.53± 0.22***
4	Group 4 (Non-heme food)	13.27± 0.13**	8.27± 0.13**	14.42± 0.18**
5	Group 5 (Non-heme food along with Vitamin-C)	13.41± 0.26***	8.31± 0.12**	15.36± 0.09**

**P < 0.05 and ***P < 0.001 as compared to negative control group. Statistical analysis One-way ANOVA.

Red blood cell count:

The results obtained from the study were shown in Table 2. The RBC count of all the treatment groups 3, 4 and 5 were found to be improved as compared to the after-induction of anemia and also compared to the negative control group. Animals treated with non-heme iron along with vitamin-C rich foods gained a red blood cell count of 9.12±0.07 million/mm³ from 4.07±0.03 million/mm³.

Table 2: Effect of heme, non-heme and combination of non-heme along with vitamin-C food supplements on red blood cells in anemic adult rats

S No	Group	Day 0 (Before induction) (million/mm³)	Day 2 (After induction) (million/mm³)	Day 28 (After treatment) (million/mm³)
1	Negative control (Group 1)	7.26± 0.09	7.27±0.10	7.28± 0.09
2	Disease control (Group 2)	7.37±0.07	3.88±0.07	3.80± 0.09
3	Group 3 (Heme food)	7.30±0.09**	4.12±0.10***	9.50± 0.06**
4	Group 4 (Non-heme food)	7.28±0.10***	3.91±0.06**	7.93± 0.12**
5	Group 5 (Non-heme food along with Vitamin-C)	7.35±0.10**	4.07±0.03***	9.12± 0.07**

**P < 0.05 and ***P < 0.001 as compared to negative control group. Statistical analysis One-way ANOVA.

Table 3: The values of red blood cell indices in anemic Wistar albino rats.

S No	Group	MCH (picogram/ cell)		MCHC (gm/dcl)		MCV (femtoliter)
S No	Group	0 day	28^th day	0 day	28^th day	0 day	28^th day
1	Negative control (Group 1)	30.57± 0.54	30.61± 0.31	33.74± 0.25	33.5± 0.78	72.58± 1.36	72.21± 1.19
2	Disease control (Group 2)	31.23± 0.29	19.96± 0.19	33.74± 0.58	22.2± 0.68	73.05± 2.65	45.33± 0.41
3	Group 3 (Heme food)	29.47± 0.25**	32.12± 0.25***	32.36± 0.14*	34.26± 0.44**	71.05± 1.09**	74.33.± 1.19***
4	Group 4 (Non-heme food)	21.72± 0.18*	22..63± 0.43***	28.28± 0.53*	29.82± 0.45**	62.34± 3.42**	63.01± 2.74***
5	Group 5 (Non-heme food along with Vitamin C)	30.13± 0.26**	32.18± 0.41**	32.74± 0.53*	34.12± 0.78***	72.34± 3.42**	74.46± 0.84**

**P < 0.05 and ***P < 0.001 as compared to negative control group. Statistical analysis One-way ANOVA.

Red blood cell indices:

The results obtained from the red blood cell indices (MCH, MCHC, MCV) study shown in Table 3. It is shown that the red blood cell indices were found to have improved to 32.18±0.41 picograms/ cell, 34.12±0.78 gms/dl and 74.46±0.84 femtoliters for all the treatment groups 3, 4 and 5 as compared to the initial values.

DISCUSSION:

The study was designed to assess the effect of Vitamin-C rich foods on non-heme iron absorption. This concept becomes quite reasonable in facilitating the non heme iron absorption which is required only in vegetarian subjects as the required amount of iron is not offered to them even though there is complete availability of nutrition provided to them. As the vegetarian subjects are more prone for anemia due to poor absorption of non heme iron according to the mechanism explained below, In this case the authors are trying to scientifically prove that the co administration of Vitamin C can enhance the non heme iron absorption.Vitamin-C effectively increases the absorption of ferrous ions (Fe³⁺) and ferric ions (Fe²⁺)⁴¹. In this study, Phenylhydrazine was used to induce anemia in the experimental animals. The phenylhydrazine-induced anemia is well-recognized and often used in hematological study. All the experimental animals were placed in the cage and grouped (1 to 5). Groups 1 & 2 were treated as negative and disease control. The remaining animals were fed with the heme (group 3), non-heme (group 4) and non-heme along with Vitamin-C rich food supplements (group 5) for 28 days according to the designated group. Later the blood was withdrawn from all the groups and analysed for blood parameters using automatic hematology analyzer (Sysmex 2100). It is observed that the non-heme food along with Vitamin-C food supplements group after treatment has increased the haemoglobin, red blood cells and blood cell indices level as like heme food treated animals due to the increased level of absorption of iron substantially. The key role of Vitamin-C in the absorption of dietary non-heme iron due to the facilitate iron absorption by making a chelate or complex with ferric iron at acid pH that remains soluble at the alkaline pH of the small intestine and reducing ferric (Fe³⁺) to ferrous iron (Fe²⁺), which seems to be a requirement for the uptake of iron into the mucosal cells. Therefore, animals gained a hemoglobin count of 15.36±0.09 gms/dl from 8.31±0.12gms/dl, red blood cell count of 9.12±0.07million/mm³ from 4.07±0.03 million/mm³ and red blood indices 32.18±0.41 (MCH), 34.12±0.78 (MCHC) and 74.46±0.84 (MCV) etc., The above-mentioned result showed a strong correlation with Li et al in hemoglobin content, red blood cell count and red blood indices level. In previously conducted intervention studies in anemic women, it was revealed that the amount of vitamin C which is effective in improving iron status was 164mg of ascorbic acid in the intervention meal or 174.6mg as a daily intake^42-43. Some scientists noticed the similar results from vitamin C supplements in the iron absorption^44-45. It is noted that rats treated with non-heme food along with vitamin-C were observed to be more.

CONCLUSIONS:

Our study confirmed that Vitamin-C rich foods such as kiwi, orange and lemon enhanced the absorption of non-heme iron present in the vegetable food items. Hence, the level of hemoglobin, red blood cells count and blood cell indices were increased in the animals treated with non-heme containing vegetable foods along with Vitamin-C rich food supplements. The elevated level of these parameters in the animals treated with non-heme containing vegetable foods along with Vitamin-C rich food supplements is equivalent to that of animals treated with heme food. Therefore, increasing vitamin C intake from supplementation along with vegetable food remains a practical measure to minimize the incidence of iron deficiency anaemia. It is a significant milestone in the management of iron deficiency anemia.

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Received on 17.08.2023 Modified on 08.11.2023

Research J. Pharm. and Tech 2024; 17(5):2224-2228.

DOI: 10.52711/0974-360X.2024.00350