A randomized controlled trial of sweet basil leaf powder‐enriched cookies for anemia management in adolescent girls

Abstract The study aimed to evaluate the effectiveness of sweet basil leaf powder as a natural source of iron for the treatment of anemia in adolescent girls. Purposive sampling technique of two‐stage sampling; part of the nonprobability sampling approach. Out of 2400 approached adolescent girls, 1645 agreed to participate and their nutritional status was assessed. Of these, 89.95% had clinical signs and symptoms of anemia, and 59.79% were found to be anemic based on Hb levels. From the anemic group, 65.18% were randomly selected to receive either B0 (Control), B1 (12.699 g FeSO4.7H2O/100 g), and B3 (16 g SBLP/100 g) cookies for 4 months. At the end of the intervention, the assessment of nutritional status, complete blood count, serum iron, serum ferritin, serum total iron‐binding capacity (TIBC), and transferrin saturation was explored. Hematological parameters such as Hb, Hct, TIBC, MCV, MCH, MCHC, serum iron, and serum ferritin were significant (p ≤ .05). The result showed that the serum Fe was highest in group B3 while a significant decline was noted for group B0. Serum ferritin for B1 was better than B3. The entire treatment for transferrin saturation showed a highly significant increasing trend in B3 and B1, regardless of the control. TIBC levels raised in the control group while in all other treatments, it declined. The study demonstrated that SBLP‐fortified cookies can be an effective treatment option for anemia, as evidenced by significant improvements in key hematological parameters.


| INTRODUC TI ON
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.

| MATERIAL S AND ME THODS
Approval for the research work was granted by the Biosafety and Ethical Review Committee at the University of Sargodha, Sargodha, Pakistan.
Treatment (B 0 ) involves no specific additions of either agent and was prepared according to the method listed in AACC (2000) with some modifications, sealed in wraps of bi-oriented polypropylene (BOPP), packed in containers, and stored at room temperature.

| 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, B 3 , Ferrous sulfate, that is, B 1 , and along with B 0 (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.
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, B 0 (Control), B 1 (12.699FeSO 4 .7H 2 O/100 g), and B 3 (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.

| Assessment of demographics and anthropometrics of anemic adolescent girls
Demographic details such as the name of the volunteer, ethnicity, religion, marital status, education, and socioeconomics were collected.The girl's body measurements like height (Gordon, 1983), weight, composition of the body, and body mass index (BMI) were recorded (Baumgartner et al., 1998).

| Anemia assay biomarkers
Volunteers' blood was obtained and tested to hematological parameters such as serum iron, serum ferritin, Hb, MCV, MCH, and TIBC and identifying anemia (Gibney et al., 2008).

| Assessment of dietary intakes
Each of the baseline volunteers filled proforma of dietary consumption data to compare the food diary at the baseline and completion of study (Rockett et al., 1995).A diet plan was also prepared for anemic adolescent girls focusing on incorporating iron-rich foods into their meals and snacks, along with foods that help to increase the absorption of iron and better their eating habits (Table 1).It also included fortified cookies which were approximately 2100-2650 calories.

| Statistical analysis
Quantitative data were collected according to factorial layout under CRD and analyzed using SPSS.Variance analysis (ANOVA) methodology (Steel et al., 1997) was used to analyze the data, and the Tukey's test was used to evaluate significance among groups (Abdi et al., 2009).The significance level was set at p ≤ .05.Descriptive statistics were used to evaluate the distribution and frequency of qualitative data, which helped to reach the inferences and conclusions.
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,  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, B 0 (control group), B 1 (FeSO 4 .7H 2 O group), and B 3 (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 B 0 at 50.018%, B 1 at 50.265%, and B 3 at 50.239%.At day 120, the mean percentage of body water was highest in the B 3 group at 51.072%, followed by B 1 at 50.481% and B 0 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 B 0 at 21.74%, B 1 at 21.81%, and B 3 at 21.32%.At day 120, the mean percentages were B 0 at 21.96%, B 1 at 21.79%, and B 3 at 21.59%.For muscle mass, the mean percentages were similar across the groups TA B L E 1 Diet plan with iron-rich food.at day 0, with B 0 at 30.296%, B 1 at 30.467%, and B 3 at 30.003%.At day 120, the mean percentage of muscle mass was highest in the B 3 group at 31.000%, followed by B 1 at 30.747% and B 0 at 29.187%.
The changes in muscle mass between days 0 and 120 were small for It was revealed from Figure 2a-h presents information on the mean values of various dietary variables for three different groups (B 0 , B 1 , and B 3 ) 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 B 1 and B 3 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 ironfortified 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.

| DISCUSS ION
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 ironfortified 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 sulfatefortified 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 (B 0 , B 1 , and B 3 ) 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 B 1 and B 3 , 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 TA B L E 5 Impact of treatments on the biomarkers of anemic adolescent girls.Note: Capital letter superscripts show the statistically significant differences of means within the same treatment group and small letter superscripts show the statistically significant differences of means among the different treatment groups.
the anthropometrics of volunteers in all treatment groups were 16.797 ± 0.2043, 16.831 ± 0.1925, and 16.831 ± 0.1968 for B 0 , B 1 , and B 3 , respectively.There was no significant relationship observed among adolescent girls in terms of F I G U R E 1 Assortment of anemic adolescent girls.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/m 2 ) at both the baseline and end of the study.

B
0 and B 1 , but larger for B 3 .For bone mass, the mean values were similar across the groups at both time points.At day 0, the mean values were B 0 at 4.160 kg, B 1 at 4.135 kg, and B 3 at 4.133 kg.At day 120, the mean values were B 0 at 4.102 kg, B 1 at 4.200 kg, and B 3 at 4.9635 kg.The changes in bone mass between day 0 and 120 were small for B 0 and B 1 , but larger for B 3 .The results suggest that the SBLP treatment (B 3 ) may have a positive impact on muscle and bone mass among anemic adolescent girls, while the FeSO 4 .7H 2 O treatment (B 1 ) did not show significant differences compared to the control group (B 0 ) for any of the variables measured.
Table 5 that the iron-fortified treatment groups (B 1 , ferrous sulfate-fortified group and B Impact of treatments on the anthropometrics of anemic adolescent girls.Impact of treatments on body composition of anemic adolescent girls.All means in the table are non-significant at (p > .05).to B 3 (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 B 3 (390.71± 0.31 to 376.61 ± 0.61 μg/dL) and B 1 (390.44 ± 0.82 to 374.84 ± 0.59 μg/dL).The final mean of transferrin saturation (%) in B 1 was 13.016 ± 0.0983%, in B 3 it was 11.039 ± 0.0434%, and in B 0 it was 10.566 ± 0.0953%.The Hb level increased gradually in both treatment groups fortified by iron B 1 , that is, ferrous sulfate, B 3 , that is, SBLP and decreased in control group, that is, B 0 at the end of 4-month study.
3 , 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 B 3 , while the mean RBC level decreased from 25.455 ± 0.0344 to 24.053 ± 0.1219 g/dL in the control group (B 0 ).The study also showed that the serum ferritin levels were highest in the B 1 group (10.827 ± 0.1921 to 20.611 ± 0.6079 ng/mL) comparedTA B L E 3Note: All means in the table are non-significant at (p > .05).TA B L E 4 3 .The highest rise was (35.522 ± 0.0552 to 33.856 ± 0.0397 g/dL) in B 3 group and B 0 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 SBLPfortified treatments had a positive effect on anemic adolescent girls.