**1. Introduction**

Arising from traditional dietary and alternative medicine practices across Asia (e.g., traditional Chinese medicine, Ayurveda) there have been a plethora of food-based components claimed as beneficial in improving the risk of diabetes and other cardiometabolic conditions. Unfortunately, the majority of these traditional practices have been based on historical pretext rather than on solid scientific evidence. In fact, robustly controlled clinical trials have been sparse, and most of the research claims came from in vitro studies and/orin vivo animal studies. In general, these traditional ingredients are all plant-based ingredients, rich in polyphenols and other phytonutrients with a wide range of biological functions including α-amylase and α-glucosidase enzyme inhibition, anti-inflammatory and antioxidant properties, insulin secretagogues, etc. [1–3]. In Asia, over centuries these ingredients have been integrated as part of daily diets of individuals into various culinary preparations and thereby used simultaneously as traditional "medicines" to maintain health and reduce disease risk. Three such commonly used traditional ingredients include corn silk, cumin, and tamarind, and some of the prior

evidence for their biological e ffects related to improving glycemic control and/or type 2 diabetes risk are outlined below.

Corn silk (*Stigma maydis*) is a waste by-product of corn and has been long used in traditional Chinese medicine to treat diabetes and associated conditions [4]. Corn silk is rich in various phytonutrients (e.g., polyphenols, terpenoids, etc.), vitamins, and minerals, as detailed elsewhere [5]. There are several mechanisms through which corn silk is thought to improve glycemic control, which include inhibition of α-amylase and α-glucosidase enzymes [6–8], inhibition of inflammation [9], reduced formation of advanced glycation end products (AGEs), reduced oxidative stress, and the enhancement of glucose-stimulated insulin secretion [6,10,11]. Therefore, corn silk has the potential to improve postprandial glycemic control, although to our best knowledge this has not been tested using a randomized controlled trial (RCT) in normoglycemic, non-diabetic individuals.

Similarly, both cumin and tamarind have been used as traditional food ingredients across various Asian cuisines and are considered to have glycemic-improving properties. For example, it has been previously shown that the consumption of cumin seeds by streptozotocin-induced diabetic rats led to significant reductions in hyperglycemia and glucosuria as well as improvements in renal function [12]. Another study showed reductions in both fasted blood glucose and glycated hemoglobin when alloxan-induced diabetic rats were administered cumin over a 6-week period [13], whereas a separate study showed a reduction in postprandial glycemic responses in rabbits fed cumin [14]. More recently, it has also been demonstrated that the cuminaldehyde found in cumin has significant α-glucosidase as well as aldose reductase inhibition [15]. On the other hand, tamarind pulp extract has been shown to improve glycemic, insulinemic, as well as lipidemic responses upon feeding to high-fat-induced obese rats for 10 weeks [16]. Several other studies using rat/mice models of diabetes have also shown improvements in glycemic parameters with various extracts of tamarind [17–20]. While the collective evidence in animal studies for both cumin and tamarind having the potential to improve glycemic control is rather substantial, there have been hardly any randomized controlled trials in humans.

Therefore, in this "Phytochemical Rich Ingredients as Functional Foods (PRIFF)" study, the primary aims were to assess the three ingredients, viz., corn silk, cumin, and tamarind as listed above, in terms of their e ffects on postprandial glycemia and insulinemia, when consumed with a carbohydrate-rich meal principally consisting of a high-glycemic-index (GI) white rice. The secondary aim was to also measure the e ffects of these three ingredients on postprandial blood pressure. The study used aqueous extracts which can be obtained using household preparation methods. For cumin and tamarind, we used dietary doses, whereas for corn silk we used two separate doses—the "high dose" consisting of an amount commonly used in traditional Chinese medicine, and a lower (one-half of the high) dose to assess whether this lower, "dietary" dose would still be e ffective in improving glycemic parameters. Moreover, there is now considerable evidence that the food form plays an important role in determining glycemic and/or insulinemic responses [21–23]. We therefore investigated all the test ingredients in two separate forms: (i) extracts consumed as a drink (D) with plain rice or (ii) extracts added into rice (R) during cooking and consumed with plain water.

#### **2. Materials and Methods**

#### *2.1. Recruitment and Ethics*

Eighteen volunteers were enrolled in this study through flyers placed around The National University of Singapore and from our center's recruitment database of volunteers who had previously given consent to be contacted for research. Inclusion criteria for this study were: Chinese, male, age between 21 to 60 years old, body mass index (BMI) between 18.5 and 25.0 kg/m2, waist circumference ≤90 cm, fasting blood glucose <7.0 mmol/L, blood pressure <140 mmHg and <90 mmHg for systolic and diastolic, respectively. Interested volunteers attended a screening visit after an overnight fast (no food or drink except water for a minimum of 10 h). At the screening visit, height was measured using a stadiometer (Seca 217, Hamburg, Germany), weight and body fat percentage were measured

using the Tanita analyzer (BC-418, Tokyo, Japan), seated resting blood pressure was measured using an automated sphygmomanometer (Omron HEM907, Singapore). Fasting glucose was measured in whole blood using a HemoCue 201 RT analyzer (Angelholm, Sweden) upon obtaining capillary blood using a single-use lancet device (Abbott Diabetes Care, Alameda, CA, USA). Volunteers were excluded if they met any one of the following criteria: taken part in sports at the competitive and/or endurance levels; smoking; being allergic to common food (e.g., eggs, shellfish, dairy, nuts, gluten) and test ingredients; intentionally restricting food intake; have glucose-6-phosphate deficiency (G6PD) or metabolic diseases, cardiovascular diseases, or disorders involving the gut, liver, or kidney; taking prescribed medication or dietary supplement that a ffects glycemia or interferes with other study measurements; consume ≥6 alcoholic drinks per week; donated blood within 4 weeks of study participation; had poor veins impeding venous access; had a history of severe vasovagal syncope following blood draws; had or was a carrier of a chronic infection (e.g., hepatitis B or C, human immunodeficiency virus); su ffering from active tuberculosis (TB) or receiving treatment for TB; or was related to a member of the study team. All volunteers provided written informed consent. All volunteers were given a choice of attending five or nine test sessions. This study was reviewed and approved by the National Healthcare Group (NHG) Domain-Specific Review Board, Singapore (DRSB Ref: 2018/00258). The study has been registered on clinicaltrials.gov under study ID No. NCT03685916.

#### *2.2. Preparation of Aqueous Extracts and Study Meals*

Dried, mature corn silk, commonly sold as an ingredient to treat diabetes by traditional Chinese medicine (TCM) practitioners, was purchased locally. Aqueous extracts were obtained by adding 552 g of corn silk to 2.3 L of tap water and boiling for 15 min. A total of three batches of extraction were undertaken. After pooling, the total volume of the corn silk extract (CS) was made up to 6.9 L and filtered through a fine mesh strainer to remove corn silk particles. Then, 50 mL of this concentrated extract (24 g/100 mL) was aliquoted and stored frozen in a −20 ◦C freezer until further use. An aqueous extract of cumin was prepared by adding 552 g of ground cumin powder (Pattu brand, Sabi Foods (India) Pvt. Ltd., Tamin Nadu, India) into 2.4 L of tap water and allowing the mixture to boil for 15 min. A further 2.4 L of tap water was added to the viscous mixture and boiled for an additional 15 min. Finally, the cumin extract (CU) was filtered through a fine mesh filter and the volume made up to 2.3 L and filtered through a fine mesh strainer to remove cumin particles. Then, 50 mL of this concentrated extract (24 g/100 mL) was aliquoted and stored frozen in a −20 ◦C freezer until further use. For the tamarind extract (TAM), 460 g of deseeded tamarind pulp (Lion Dates Impex Pte Ltd., Trichy, India) was added to 1.2 L of tap water and boiled for 15 min. The first aqueous extract was obtained by filtering the mixture through a fine mesh filter and the residual tamarind pulp re-extracted with another 1.2 L of tap water via boiling for another 15 min. The mixture from the second extraction was filtered and both tamarind aqueous extracts were pooled and the volume made up to 2.3 L. Then, 50 mL of this concentrated extract (20 g/100 mL) was aliquoted and stored frozen in a −20 ◦C freezer until further use. All stored concentrated extracts were thawed and diluted either four-fold for CS-12, CU, and TAM, or diluted two-fold for CS-24 with filtered water on the day of the test session, to make up to a total of 200 mL of "diluted aqueous extract".

The study meals (including rice) were prepared fresh every morning, and the test extracts were either added into rice (R) during cooking or made up into a drink (D). The study meals were rich in carbohydrates, providing approximately 50 g of available carbohydrates mainly from high-GI glutinous rice (Thai glutinous rice, Fairprice, Singapore) and 20 g of cooked garden peas (Frozen garden peaks, Fairprice, Singapore). The control meal (CON) was made up of 65 g of glutinous rice cooked in 200 mL of filtered water in a household rice cooker, 20 g of peas (steamed for 2 min in microwave), and a glass (200 mL) of filtered water. The rice (R) test meals consisted of 60 g of glutinous rice cooked in 200 mL of the diluted aqueous extract (detailed above), 20 g of peas, and 200 mL of filtered water, whereas the drink (D) test meals consisted of 60 g of glutinous rice cooked in 200 mL of plain filtered water, 20 g of peas, and 200 mL of diluted aqueous extract. The food ingredients used and the energy content and nutrient composition of each test meal as obtained from food packet labels or nutrient databases (US Department of Agriculture) are shown in Table 1.

#### *2.3. Total Polyphenol Content (TPC Analyses) of Aqueous Extracts*

Total polyphenol contents (TPCs) were determined by the Folin–Ciocalteu (FC) assay in triplicates using a method adopted from Medina-Remón et al. [24]. Briefly, 1 mL of the aqueous concentrated extract was transferred to a microfuge tube and centrifuged at 13,000× *g* for 5 min at room temperature to obtain the supernatant. Subsequently, 100 μL of tap water (blank), supernatant, or gallic acid standard was added to 200 μL of 10% FC reagen<sup>t</sup> in a 96-well clear polystyrene microplate followed by the addition of 800 μL of 700 mM sodium carbonate. After a 2-h incubation at room temperature in the dark, the absorbance at 765 nm was measured in a microplate reader (Tecan Infinite 200, Mannedorf, Switzerland). The TPC was calculated using the regression equation derived from a gallic acid standard curve and expressed as gallic acid equivalents (GAE).
