1 INTRODUCTION

Iron, a vital micronutrient, plays a central role in maintaining human health and well-being. Adequate iron intake is crucial for various physiological functions, including oxygen transport, energy production, and immune system maintenance. Anemia is prevalent in adolescent girls, characterized by insufficient red blood cells or hemoglobin to transport oxygen to the body's tissues. Iron deficiency is the most common cause, resulting from factors like poor diet, heavy menstrual bleeding, and rapid growth during puberty. Public health programs have historically neglected this vulnerable period in human life, with girls being more likely to become anemic due to limited resources, lack of good food and education, and extra household work. Signs of anemia in adolescent girls may include weakness, reduced appetite, fatigue, dizziness, headaches, pale skin, and shortness of breath during exercise. In this context, the consumption of plant-based foods rich in iron holds paramount importance for ensuring optimal iron nutrition, particularly within the framework of balanced diets and sustainable food systems. Plant-based foods offer a valuable solution to iron deficiency due to their inherent iron content, as well as the presence of other nutrients that enhance iron absorption. While animal sources like red meat are traditionally associated with high iron content, various plant-based foods can provide a substantial amount of this essential mineral (Wang & Scrimgeour, 2021). These foods not only supply iron but also contribute to improved iron absorption. Beyond their nutritional benefits, these foods align with the principles of sustainability and environmental conservation. The production of plant-based foods generally requires fewer resources, including water and land, compared to animal agriculture. As the world grapples with the challenges of climate change and resource scarcity, transitioning toward diets rich in plant-based foods becomes a pivotal step toward a more sustainable future. While it also offers a promising solution to iron deficiency, it is important to acknowledge potential challenges. Treatment involves a diet rich in iron-containing foods, fortification of staple foods, and the use of medicinal plants rich in minerals. Sweet Basil (Ocimum basilicum L.) is a nutrient-rich herb with several health benefits, including vitamins and minerals such as vitamin C, calcium, and potassium (Şen et al., 2019). Mostly fresh leaves are used but dry leaves are rich source of nutrients as compared to the fresh. Its components have traditionally been used to flavor food, dental care, and oral drugs (Singletary, 2018). Iranian basils are used for managing fevers, inflammation of the throat, and stomachache (Babaeian et al., 2015; Kulkarni et al., 2012). According to El Khouly (2017), antianemic activity of sweet basil remains unknown although it showed high content of Fe (89.80 mg/100 g). Incorporating dried basil leaves into cookies can be a practical way to address nutrient deficiencies, specifically in adolescent girls. A study was designed to evaluate the effectiveness of sweet basil leaf powder as a natural source of iron for the treatment of anemia in adolescent girls. SBLP (Fe)-fortified cookies have been administered in adolescent girls to overcome anemia. It has been a very slow yet effective process to control anemia in young girls. Therefore, it is also a better and cost-effective option to utilize sweet basil (SB) in cookies through the fortification process for meeting the partial requirement of iron, especially in adolescents.

2 MATERIALS AND METHODS

Approval for the research work was granted by the Biosafety and Ethical Review Committee at the University of Sargodha, Sargodha, Pakistan.

2.2 Experimental design and data collection

For the study, multiple schools and colleges within the Sargodha region were selected as study sites. To collect data from adolescent girls, a two-stage sampling technique was used, which is classified as a nonprobability sampling approach according to Muhammad (2000). One from the best treatment; SBLP, that is, B₃, Ferrous sulfate, that is, B₁, and along with B₀ (control) were selected based on physicochemical, sensory, and stability analysis results as shown in Figure 1. Considering the RDA of iron (13.6 mg) for anemic adolescent girls, fortified cookies were prepared. Of those who met the inclusion criteria, 38.83% (n = 313) had Ferritin levels < 12 μg/L, s. Fe < 60 μg/dL, TIBC > 350 mg/dL, TS < 15%, MCV < 80 fL. Out of these, 108 were excluded from the study due to hepatitis B and C, renal anomalies, and hemorrhoids. Finally, 204 volunteers were randomly selected into three treatment groups, B₀ (control/placebo group), B₁ (FeSO₄.7H₂O group), and B₃ (SBLP group), with 68 volunteers in each group.

Each group was provided with three different iron treatments for 120 days to explore their impacts. After the preliminary trial, best cookie treatments, that is, B₀ (Control), B₁ (12.699 FeSO₄.7H₂O/100 g), and B₃ (16 g SBLP/100 g) were selected for efficacy studies to furnish 50% of RDA of Fe (13.6 mg). The selected cookie treatments were prepared in the lab of IFSN University of Sargodha, with five cookies (50 g) providing 50% RDA of anemic adolescent girls (7 mg). Cookies were provided to schools/colleges on a weekly basis for distribution to the target girls.

3 RESULTS

All participants were between the ages of 13 and 19. The largest ethnic group was Punjabi, accounting for 139 volunteers (71.3%), followed by Saraiki with 49 volunteers (25.1%). The religious affiliation of the volunteers consisted of 186 Muslims (95.4%), 1 Christian (1.5%), and 8 Qadiani/Ahmadi (4.1%). In terms of education, 115 volunteers (59%) were pursuing graduation, 60 (30.8%) were pursuing intermediate studies, 14 (7.2%) were pursuing matriculation, and 6 (3.1%) were pursuing other courses. According to the marital status, 193 volunteers (99%) were unmarried while only two (1%) were married, as evident from the frequency distribution table (Table 2). In terms of income, the frequency distribution table shows that 89 volunteers (45.6%) belonged to the low-middle class, 103 (52.8%) were from the middle-income bracket, while three (1.5%) had income above PKR ≥ 2,071,992.

According to Table 3, the anthropometrics of volunteers in all treatment groups were 16.797 ± 0.2043, 16.831 ± 0.1925, and 16.831 ± 0.1968 for B₀, B₁, and B₃, respectively. There was no significant relationship observed among adolescent girls in terms of their body weight (46.190 ± 1.3076 to 45.624 ± 1.6150 kg), height (154.35 ± 1.0651 to 155.81 ± 1.0421 cm), and BMI (19.25 ± 0.4128 to 19.26 ± 0.4116 kg/m²) at both the baseline and end of the study.

Table 4 presents the impact of different treatments on the body composition of anemic adolescent girls including body water, fat, muscle mass, and bone mass among all three groups, namely, B₀ (control group), B₁ (FeSO₄.7H₂O group), and B₃ (SBLP group), with measurements taken at two time points, 0 and 120 days. For body water, the mean percentages were similar across the groups at day 0, with B₀ at 50.018%, B₁ at 50.265%, and B₃ at 50.239%. At day 120, the mean percentage of body water was highest in the B₃ group at 51.072%, followed by B₁ at 50.481% and B₀ at 49.126%. The changes in body water between days 0 and 120 were not large for any of the groups. For fat, the mean percentages were similar across the groups at both time points. At day 0, the mean percentages were B₀ at 21.74%, B₁ at 21.81%, and B₃ at 21.32%. At day 120, the mean percentages were B₀ at 21.96%, B₁ at 21.79%, and B₃ at 21.59%. For muscle mass, the mean percentages were similar across the groups at day 0, with B₀ at 30.296%, B₁ at 30.467%, and B₃ at 30.003%. At day 120, the mean percentage of muscle mass was highest in the B₃ group at 31.000%, followed by B₁ at 30.747% and B₀ at 29.187%. The changes in muscle mass between days 0 and 120 were small for B₀ and B₁, but larger for B₃. For bone mass, the mean values were similar across the groups at both time points. At day 0, the mean values were B₀ at 4.160 kg, B₁ at 4.135 kg, and B₃ at 4.133 kg. At day 120, the mean values were B₀ at 4.102 kg, B₁ at 4.200 kg, and B₃ at 4.9635 kg. The changes in bone mass between day 0 and 120 were small for B₀ and B₁, but larger for B₃. The results suggest that the SBLP treatment (B₃) may have a positive impact on muscle and bone mass among anemic adolescent girls, while the FeSO₄.7H₂O treatment (B₁) did not show significant differences compared to the control group (B₀) for any of the variables measured.

It was revealed from Table 5 that the iron-fortified treatment groups (B₁, ferrous sulfate-fortified group and B₃, SBLP-fortified group) showed a significant increase in red blood cell (RBC) levels. The mean RBC level increased from 25.333 ± 0.0503 to 41.511 ± 0.0327 g/dL in group B₃, while the mean RBC level decreased from 25.455 ± 0.0344 to 24.053 ± 0.1219 g/dL in the control group (B₀). The study also showed that the serum ferritin levels were highest in the B₁ group (10.827 ± 0.1921 to 20.611 ± 0.6079 ng/mL) compared to B₃ (10.631 ± 0.0471 to 20.016 ± 0.1033 ng/mL) and the control group. Total iron binding capacity (TIBC) levels decreased the most in B₃ (390.71 ± 0.31 to 376.61 ± 0.61 μg/dL) and B₁ (390.44 ± 0.82 to 374.84 ± 0.59 μg/dL). The final mean of transferrin saturation (%) in B₁ was 13.016 ± 0.0983%, in B₃ it was 11.039 ± 0.0434%, and in B₀ it was 10.566 ± 0.0953%. The Hb level increased gradually in both treatment groups fortified by iron B₁, that is, ferrous sulfate, B₃, that is, SBLP and decreased in control group, that is, B₀ at the end of 4-month study. The mean values of Hb for B₀ were 9.252 ± 0.0503 g/dL at baseline and 7.234 ± 0.0327 g/dL at the end of the study. For B₁, the mean values were 9.416 ± 0.0503 g/dL at baseline and 12.513 ± 0.0327 g/dL at the end of the study. For B₃, the mean values were 9.438 ± 0.0503 g/dL at baseline and 14.786 ± 0.0327 g/dL at the end of the study. The means of Hct indices in control group, that is, B₀ were significantly reduced from 31.269 ± 0.0446 to 30.946 ± 0.1449% while highly significant improvement from 31.365 ± 0.0333 to 33.314 ± 0.2151% in B₁ group and 31.250 ± 0.0211 to 33.590 ± 0.1819% in B₃ was observed. Treatment B₃ of SBLP-fortified group (77.064 ± 0.2325 to 78.408 ± 0.2100 fL) was better than B₁ (ferrous sulfate-fortified group). In the control group, a highly significant drop (27.173 ± 0.0956 to 26.916 ± 0.1238 pg) was noted and improvement (27.545 ± 0.1956 to 28.688 ± 0.0592 pg) was observed in B₃. The highest rise was (35.522 ± 0.0552 to 33.856 ± 0.0397 g/dL) in B₃ group and B₀ group showed a drop (33.448 ± 0.1203 to 29.692 ± 0.6239 g/dL) that differs significantly from other treatments. Overall, the study demonstrated that SBLP-fortified treatments had a positive effect on anemic adolescent girls.

Figure 2a-h presents information on the mean values of various dietary variables for three different groups (B₀, B₁, and B₃) before and after a study. The variables included water consumption and servings of different food groups (cereal, bread, rice and pasta, vegetables, fats/oils/sweets, fruits, milk/yogurt/cheese, junk food, and meat/fish/poultry/beans/eggs/nuts). By comparing the means before and after the study, one could evaluate the change in dietary behavior among the different groups. It was observed that the mean water consumption increased for all three groups after the study. Similarly, the mean servings of cereal, bread, rice, and pasta decreased for groups B₁ and B₃ after the study compared to before the study. The mean servings of vegetables increased for all three groups after the study. The changes observed in the eating behavior of the girls before and after this study were due to the diet plan and personal motivation that was given to them during the study. The diet plan included different types of iron-rich foods and iron-fortified cookies which may have influenced the girls' iron deficiency as well as the diet plan may have been designed to improve the girls' iron deficiency, overall health, and wellness, which could have led to positive changes in their eating habits.

4 DISCUSSION

The results of the study provided demographic information about the participants, such as their age, ethnicity, religion, education, marital status, and income. The majority of the participants were Punjabi, Muslim, and pursuing higher education. This demographic profile is consistent with Pries et al. (2019) studies conducted in Pakistan, which showed that the majority of the population is Muslim and that education is highly valued. However, the study did not provide any analysis of how these demographic factors may have influenced the results of the study. The results of this study indicate that there was no significant difference in anthropometric measurements (body weight, height, and BMI) among the three treatment groups at the beginning and end of the study. This finding was consistent with some studies that have found no significant relationship between iron supplementation and anthropometric measurements in adolescent girls (Ahmad et al., 2015; Salam et al., 2016).

However, there are also studies that have reported a positive effect of iron supplementation on anthropometric measurements in adolescent girls. For example, a study conducted by Dad et al. (2017) in Pakistan found that iron supplementation for 6 months resulted in significant improvements in weight and height of adolescent girls. It is important to note that the duration and dosage of iron supplementation may play a role in the effect on anthropometric measurements. Dostal et al. (2014) mentioned that the duration of iron supplementation was 8 weeks, which may not have been sufficient to observe significant changes in anthropometric measurements.

The results of this study were consistent with previous research demonstrating that iron-fortified treatments can effectively improve RBC levels and increase serum ferritin levels in anemic individuals. In a study conducted by Glinz et al. (2017), it was found that iron-fortified foods increased RBC levels and serum ferritin levels in women with iron deficiency anemia. Another study by Kumar et al. (2022) revealed that iron-fortified biscuits improved the hemoglobin levels in anemic adolescent girls.

The results of this study indicate that fortified food with SBLP and ferrous sulfate had a significant positive effect on the hemoglobin, hematocrit, and serum iron levels of anemic adolescent girls compared to the control group. These findings are consistent with the results of several other studies that have shown that iron-fortified foods can improve the iron status of anemic individuals. A study conducted by Hess et al. (2016) showed that iron-fortified soy sauce had a significant positive effect on the iron status of anemic women in China. Kuong et al. (2016) demonstrated that iron-fortified rice was effective in improving the iron status of anemic women in Cambodia. Similarly, in a study conducted by Abdalla (2018), it was found that iron-fortified bread had a positive effect on the iron status of anemic women in Korea.

The current study also found that SBLP-fortified food had a similar effect on the iron status of anemic adolescent girls as ferrous sulfate-fortified food. This is consistent with the findings of a study conducted by Manggul et al. (2021), which showed that iron-fortified biscuits containing Moringa oleifera leaves had a similar effect on the hemoglobin levels of anemic adolescent girls as ferrous sulfate-fortified biscuits. Present study analyzed changes in dietary behavior among three groups (B₀, B₁, and B₃) before and after the study, including water consumption and servings of different food groups. Mean water consumption increased for all three groups, while mean servings of cereal, bread, rice, and pasta decreased for groups B₁ and B₃, and mean servings of vegetables increased for all three groups. The changes were attributed to a diet plan and personal motivation, including iron-rich foods and iron-fortified cookies, designed to improve the girls' iron deficiency and overall health and wellness, leading to positive changes in their eating habits. A similar finding by Association (2007) showed that 91% of college students had expectations that they had good health status but their total consumption was 7%. In the present situation, fewer than 10% of U.S. college students say that they eat five required regular portions of fruits and vegetables. Croezen et al. (2009) recorded that the rise of unhealthy eating habits and the growing consumption of sweetened drinks by young people played an important role in obese and atherosclerosis children. In a similar study by Collison et al. (2010), it was shown that current malnutrition is the result of growing unhealthy eating habits among women. Younger part of a population often stays away from breakfast which adds to the risk of being either undernourished or obese. However, increasing water intake and consumption of fruits, vegetables, and whole grains are recommended as part of a healthy diet. Additionally, limiting consumption of foods high in added sugars, saturated fats, and sodium is recommended to improve overall health outcomes.

5 CONCLUSION

In conclusion, all treatment groups showed significant changes in anemia indices, while safety indices remained normal. The group that received 16% SBLP (B₃) demonstrated the highest increase in Hb (11.8%), serum Fe (63.88%), serum ferritin (31.84%), and transferrin saturation (17.91%), along with a decrease in TIBC (4.0%) while reverse of all was explored in B₀ (control group). the findings of this study support the use of iron-fortified foods, including those fortified with plant-based sources of iron like SBLP, as a strategy to improve the iron status of anemic adolescent girls. Additionally, the implementation of a diet plan and personal motivation, which included iron-rich foods and iron-fortified cookies, was successful in improving the iron deficiency and overall health and wellness of adolescent girls and also led to positive changes in their eating habits.

AUTHOR CONTRIBUTIONS

Felix Kwashie Madilo: Data curation (equal); formal analysis (equal); methodology (equal); writing – review and editing (equal). Farah Naz Akbar: Conceptualization (equal); data curation (equal); investigation (equal). Shahid Mahmood: Data curation (equal); formal analysis (equal); funding acquisition (equal); investigation (equal). Ghulam Mueen-ud-din: Conceptualization (equal); formal analysis (equal); software (equal); writing – original draft (equal). Waseem Khalid: Software (equal); supervision (equal); writing – original draft (equal); writing – review and editing (equal). Muhammad Zubair Khalid: Conceptualization (equal); data curation (equal); investigation (equal); software (equal); supervision (equal); writing – original draft (equal); writing – review and editing (equal). Zaira Aziz: Formal analysis (equal); funding acquisition (equal); investigation (equal); validation (equal); writing – original draft (equal). Saleh Alfarraj: Data curation (equal); investigation (equal); writing and editing (equal); writing – original draft (equal); funding acquisition (equal). Mohammad Javed Ansari: Formal analysis (equal); funding acquisition (equal); investigation (equal); writing – review and editing (equal).

FUNDING INFORMATION

Not applicable.

CONFLICT OF INTEREST STATEMENT

No potential conflict of interest declared by the author(s).