Micronutrient deficiencies can impair cognitive performance in children of all ages. Deficiencies adversely affect many aspects of a child's cognitive functions including lower IQ, poor memory, impaired verbal, and non-verbal learning, depression, attention deficit, and delayed processing speed (Sachdev et al., 2005; Eilander et al., 2010; Khor and Misra, 2012). These deficiencies can also lead to poor academic outcomes, lower employment success and even lower life expectancies (Mendez and Adair, 1999; Shariff et al., 2000; Demment et al., 2003; Grantham-McGregor, 2007). School children are at a critical stage of cognitive development as several areas of the brain that are involved in higher-order cognitive function continue to develop and mature until mid-teenage years (Sowell et al., 2004; Lam and Lawlis, 2016). As such, children and adolescents at risk of micronutrient deficiency are particularly vulnerable to impaired cognitive development and function, which can adversely impact their later adult life.

Several studies have suggested that diet may affect a child's neurocognitive development and learning (Mendez and Adair, 1999; Shariff et al., 2000; Grantham-McGregor, 2007). The developing brain requires a range of proteins, vitamins and energy sources to function effectively. These dietary constraints may also affect learning and cognitive processes particularly in environments which may be deficient in any of these substrates (Sachdev et al., 2005; Eilander et al., 2010; Khor and Misra, 2012). Nutritional deficiency is a highly recognized public health issue affecting over 2 billion people worldwide (Initiative, 2004). The most commonly discussed malnutrition issues throughout the world are vitamin A deficiency (VAD), iodine deficiency disorders (IDD), and iron deficiency anemia (IDA) (Ramakrishnan, 2002). Children deficient in Vitamin A are associated with higher mortality rates than those children supplementing vitamin A in their diet (Awasthi et al., 2013). A study showing increases in school attendance following vitamin A supplementation highlights a secondary effect of treatment via not only reducing illness but enabling active learning (Mahawithanage et al., 2007). Iodine deficiency is the leading cause of brain damage as well as a host of other neurological and cognitive disorders (Meletis, 2011; WHO, 2017). Recent randomized clinical trials have demonstrated that iodine supplementation in children and adolescents' leads to improvements in cognitive outcomes in even mildly deficient subjects (Zimmermann et al., 2006; Gordon et al., 2009). A recent meta-analysis regarding oral iron supplementation in iron-deficient children revealed improvements in attention, concentration and IQ (Falkingham et al., 2010). In such cases, iron-dependent dopamine D2 receptors are thought to become impaired in iron-deficient subjects (Pollitt et al., 1985). Studies using animal models have found that dopaminergic, cholinergic, and opiate system interactions may all play a part in cognitive deficiencies (Youdim, 2008). Previous research has demonstrated improvements in cognitive functioning (Kashyap and Gopaldas, 1987), learning (Soemantri, 1989), and memory (Bruner et al., 1996) following iron supplementation in children with IDA (Sanghvi et al., 2007). Despite the positive findings of individual nutrient supplementation, multiple micronutrients have shown to not only reduce the severity and prevalence of common illnesses but also improve the cognitive abilities of developing children to a greater extent than individual nutrients alone (Solon et al., 2003; Vazir et al., 2006; Muthayya et al., 2009; Nga et al., 2011). A recent meta-analysis demonstrated the positive effects of a multi-micronutrient intervention, as opposed to singular micronutrient interventions, on the physical growth of malnourished children (Ramakrishnan et al., 2004). This highlights the importance of considering several nutrients when targeting micronutrient deficient populations. Previous research investigating the effects of food and beverages fortified with micronutrients has reported significant improvements in a number of cognitive domains including non-verbal intelligence (Schoenthaler et al., 2000), mental abilities (Solon et al., 2003), visual recall (Kumar and Rajagopalan, 2007; Manger et al., 2008), measures of intelligence (Nga et al., 2011), short-term working memory (van Stuijvenberg et al., 1999; Kumar and Rajagopalan, 2007; Muthayya et al., 2009), and attention and concentration (Vazir et al., 2006). A recent chronic (12 month) study investigating the effects of a micronutrient intervention alone (iron, zinc, folate, and vitamins A, B-6, B-12, and C) in well-nourished children from Australia and Indonesia found significant improvements in verbal learning and memory (Osendarp et al., 2007). These results were replicated by a research group in India who also found improvements in the short-term memory and fluid reasoning of children aged 6–10 years taking high and low doses of a micronutrient treatment (Muthayya et al., 2009). Research is also investigating the benefits of micronutrient interventions in pre- and post-natal care (Ramakrishnan et al., 2014; Vanhees et al., 2014) on cognition and neurodevelopment in healthy children (Anjos et al., 2013). This research suggests that nutrition influences neurodevelopment and may be modified to improve cognitive development (Bryan et al., 2004). The current treatment intervention contains vitamins A, D, E, B1, B2, B3, B12, B9, C, Biotin, and Pantothenic Acid and minerals iron, iodine, zinc, selenium, copper, magnesium, and calcium which covers a broad spectrum of vitamin and mineral deficiencies.

India has recently experienced a nutrition transition (Griffiths and Bentley, 2001; Misra et al., 2011), resulting in a 7% decrease in carbohydrate-derived and a 6% increase in fat-derived energy. Furthermore, in addition to the historical problem of under-nutrition in India, rising urbanization and economic development have led to both under- and over-weight individuals coexisting in the same population (Griffiths and Bentley, 2001). In addition, because Indian diets are predominantly cereal-based with little variety, the risk of micronutrient deficiency is high, irrespective of bodyweight (Swaminathan et al., 2013). Deficiencies of iron, vitamin A, iodine, B vitamins, and zinc constitute the common types of micronutrient malnutrition affecting millions of children in low-income countries (Khor and Misra, 2012). In 2004, two billion people worldwide were estimated to be at risk of iodine deficiency (Andersson et al., 2007). In 2011, anemia was estimated to affect 42.6% of school-aged children globally with around 50% of cases linked to an iron deficiency (WHO, 2015).

Nutrition can be modified to optimize cognitive development (Bryan et al., 2004), and micronutrient supplementation assists in preventing deficiencies in those at risk of impaired cognitive performance (Huskisson et al., 2007; Lam and Lawlis, 2016). The micronutrients most closely associated with cognitive function are vitamins B1, B2, B3, B6, B12, C, folic acid and minerals such as iron, iodine, and zinc (Huskisson et al., 2007; Lam and Lawlis, 2016). A recent review assessed the role of micronutrient interventions on cognitive performance of children aged 5–15 years in developing countries and reported a beneficial effect of micronutrient interventions for a duration of 3 months to 1 year, using food fortified with multiple micronutrients (MMN). Many of the studies were conducted in rural communities of low socio-economic status, with a high prevalence of undernutrition, including underweight, stunting, and anemia. Although there was a lack of consistency in the impact of micronutrient supplementation on intelligence, long-term mental functions and school examination grades of the children, a beneficial effect of MMN supplementation on short-term memory was consistently reported (Khor and Misra, 2012).

The impact of micronutrients on cognitive functions in school aged children remains unclear. It has been proposed that multiple micronutrients may have a synergistically positive influence on the hippocampus, an area known to play a key role in the encoding and retrieval of novel information in short term memory (Kumaran, 2008). Although a review of micronutrient intervention studies reported a beneficial effect of using food fortified with multiple micronutrients on short term memory, it was suggested that additional RCTs be conducted in developing countries to provide more definitive evidence of the impact of micronutrients on improving cognition in school-aged children (Khor and Misra, 2012) particularly in environments in which deficiencies occur. The botanical product, BM, has been used in traditional medicine as a “memory tonic” (Russo and Borrelli, 2005) although its mechanism of action in children is unknown. The cognitive benefits of chronic administration of MMN or extracts of BM, administered separately, have been reported following 4 months' intervention in three studies in children (Negi et al., 2000; Solon et al., 2003; Nga et al., 2011) and in numerous studies in adults.

The current study compared the effects of a 4-month supplementation with a combination of MMN and EBM fortified beverage with a non-fortified beverage on the cognitive functions of healthy school children aged 7–12 years in India. In our study, we showed that the beverage powder fortified with MMN and EBM extract did not significantly affect either short term memory or any of the secondary outcomes tested. Although the SWM strategy score showed significant improvement on Day 60, there was no statistical significance when tested on Day 121. With regard to rapid visual information processing, although there were no significant treatment differences observed on either Day 60 or Day 121, disparities in “Prime” and “Total false alarm” were seen across the age stratums. Improved “prime” function with the EBM and MMN fortified beverage was observed in the youngest (age ≥7 and < 9 years) and oldest (age ≥11 and ≤ 12 years) stratums, while the reverse was seen in stratum 2 (age ≥9 and < 11 years), suggesting an interesting interaction. For “Total false alarm,” after 4 months of treatment, younger children in the control group made more errors than those who received the MMN/EBM-fortified beverage; in contrast, in older children, error rates were higher in the active treatment vs. the control group. This divergent pattern suggests a significant interaction for “Total false alarm;” however, the magnitudes of treatment responses were not considered to be clinically relevant. Therefore, it is likely that the two interactions detected were due to chance (type 1 error) rather than indicative of a true underlying treatment-by-age interaction.

While micronutrient interventions have been found to improve children's mental development in participants deficient in certain micronutrients, such as iron (Sachdev et al., 2005) and iodine (Bleichrodt and Born, 1994), in our study, the failure of the combined MMN and EBM supplement to significantly improve cognitive function is consistent with some but not all previous reports in healthy children. A meta-analysis of MMN supplementation in healthy children aged 5–16 years failed to show a significant effect on short term memory (Eilander et al., 2010). In their recent meta-analysis of RCTs, Lam and colleagues reported inconsistent findings regarding the impact of micronutrient supplementation and the cognitive domains of short-term memory and attention and that micronutrient supplementation had no significant effect on school performance (Lam and Lawlis, 2016). While the specific interrelationship of diet, brain development and cognition is complex, micronutrients are known to have a direct influence on cognitive function through their involvement in the energy metabolism of neurons and glia cells, neurotransmitter synthesis, receptor binding etc (Simeon and Grantham-McGregor, 1990; Huskisson et al., 2007). Current preclinical research has demonstrated various central nervous system actions of BM including antioxidant (Bhattacharya et al., 2000), anti-depressant (Sairam et al., 2002), and nootropic (Hota et al., 2009) effects; along with direct and indirect links to changes in dopamine (DA), serotonin (5-HT), noradrenaline (NA) and acetylcholine (ACh) neurotransmitter systems (Charles et al., 2011). It is possible that the dosage of EBM used in this study might have been too low to effects these central nervous system actions that could ultimately have a positive impact on attention and memory.

In our study, the choice of tests was guided by two principles: (i) tests that were validated in Indian children and (ii) were translated into Indian languages. The CANTAB system was employed to evaluate cognitive performance. It is a computerized test battery that consists of a series of neuropsychological tests specific to particular aspects of sustained attention, working memory, executive function (planning), and episodic memory. This battery was developed to identify both the neural and neurochemical substrates of cognitive parameters in children. The CANTAB tests have been widely used in interventional drug trials and nutrition studies and have been validated in various populations: children, young adults and the elderly (Sheppard and Cheatham, 2013). The CANTAB tests are available in Hindi, and Marathi. The Raven's Progressive Colored Matrices (Raven, 2007) was used to test for other executive functions (problem solving and reasoning ability) and is a well-validated measure of basic cognitive functioning (Raven, 2000).

The beverage powder fortified with MMN and BME was well-tolerated by the children, with no serious adverse events observed. Our observations are in agreement with studies in which high dosages of BM have been administered in children with no major side effects (Sharma et al., 1987; Dave et al., 2008).

Any conclusions drawn from our findings must be made within the context of the study's limitations and this is particularly true for non-significant treatment effects. A range of potential methodological issues may have contributed to the lack of an effect due to the treatment: (1) The dose of BM extract used in our study (260 mg/day) was lower than that used in previous studies that have reported significant cognitive effects (Sharma et al., 1987; Ramarao et al., 2011). Future studies could include higher doses of Bacopa, such as those used by Sharma et al. (1,050 mg Bacopa per day; Sharma et al., 1987) and Ramarao et al. (2,000 mg of Bacopa per day; Ramarao et al., 2011); (2) There is also a possibility that the bioavailability of Bacopa may have been reduced in a food supplement matrix or in a beverage powder rather than a capsule. Previous studies have used Bacaopa extract in the form of a capsule (Negi et al., 2000) or syrup (Ramarao et al., 2011), and it would be of interest to compare whether the efficacy of Bacopa differs according to method of administration as this may affect bioavailability; (3) Another shortcoming related to the potential confounding effect of the overall diet of the children which may have significantly differed between participants. In addition, a general lack of energy and protein in the diets of children in developing countries such as India, could have overridden the effect of micronutrients; (4) It is also possible that the duration of the trial may have been too short to demonstrate beneficial effects. longer term studies are needed to further elucidate the role of micronutrient supplementation on children's cognitive function. In addition, in a healthy child population, the impact of supplementation with MMN and EBM is likely to be less discernible and without a food frequency questionnaire at baseline or blood samples that are able to determine micronutrient deficiencies it is not possible to know whether the children were deficient or otherwise healthy in key nutrients supplemented in this study; (5) lastly it is unknown how MMN and EBM interacts and whether the simultaneous administration of these substances interact in some unknown manner. Future studies may wish to address some of these shortcomings and evaluate whether there are sub-groups related to such variables as diet, SES, scholastic performance, diet, and BMI.

In summary, this study showed that when compared with a non-fortified formulation, a beverage powder fortified with multiple micronutrients and B. monnieri extract did not have a significant impact on either short term memory or any of the secondary outcomes tested in the Indian school children tested.

TM-G, CS, JK, SB, and SK were responsible for study design and inputs in protocol writing. TM-G wrote the paper, while NW performed the statistical analyses. VS was responsible for the overall conduct of the study. As the Principal Investigator, AK was responsible for the conduct of the study.