Development of Mussaenda frondosa sepal infused functional tea with enhanced antioxidant and alpha amylase inhibitory activities

Development of Mussaenda frondosa sepal infused functional tea with enhanced antioxidant and alpha amylase inhibitory activities | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Development of Mussaenda frondosa sepal infused functional tea with enhanced antioxidant and alpha amylase inhibitory activities M. F.F. Nafla, T. C. Kananke, M. G.A.N. Perera This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4602535/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 17 You are reading this latest preprint version Abstract Mussaenda frondosa (MF) is an edible species of the genus Mussaenda (Rubiaceae) that contains a wide array of medicinal compounds. The present study was conducted to evaluate the physicochemical and functional properties of the MF sepals, to develop a novel herbal tea with improved functional properties. Remarkably high antioxidant and α-amylase inhibitory activities were exhibited by water and ethanol extracts of MF, subsequent to their high phenolic and flavonoid contents. None of the extracts showed a toxicity, as evaluated by brine shrimp lethality assay. A tea was formulated by blending different proportions of dry sepals of MF with black tea. The sensory analysis showed a significantly high level of acceptancy for the formula that containing 40% MF (MFT-40) with augmented phenolic contents, antioxidant and hypoglycemic activities. This study revealed the potential use of MF as a source for the development of new functional teas with enhanced health benefits. Antioxidant Herbal tea Hypoglycemic Mussaenda frondosa Value addition Figures Figure 1 Figure 2 1. Introduction Tea is one of the major sources of exports for Sri Lanka. In 2018, Sri Lanka contributed 5.1% (303 million kilograms) of the world’s tea production [ 1 ]. While the global tea market is rapidly growing with emerging customer needs, currently, it has become one of the most competitive beverage markets in the world. Sri Lanka must compete with many other tea-exporting countries around the world to sustain its global market. Value-added tea production is a successful strategy for facing international competitiveness in the tea industry. By adding value to its tea products, such as through unique blends, packaging, or organic certifications, Sri Lanka can attract tea consumers and differentiate itself in the global market. The production of new tea blends to augment the health benefits of tea while preserving its original organoleptic properties has become a popular value-adding strategy. The genus Mussaenda L. is one of the largest genera of the Rubiaceae family, which has approximately 200 species that are primarily distributed in the forests of the tropical old world [ 2 ], [ 3 ]. This genus is native to West Africa through the Indian subcontinent, Southeast Asia and South China. The genus Mussaenda is a significant source of medicinal natural products, particularly iridoids, triterpenes and flavonoids [ 2 ], [ 3 ]. Mussaenda frondosa (MF) is an edible species of the genus Mussaenda . The whole plant is used to treat various ailments in traditional medicine. A weak decoction of dry shoots is given to children to relieve cough, while root juice is used to heal tongue blemishes [ 4 ]. Green leaves are used in traditional medicine for stomach problems. Sepals are diuretics and are considered a food that enhances memory and is beneficial against many liver-associated diseases [ 5 ]. The deep-oil-fried sepals of this plant are popular snacks among Sri Lankan villagers. Green leaves are used to prepare herbal porridge, which is believed to have good liver-protecting power. Preliminary phytochemical investigations of different extracts of MF have shown the presence of flavonoids, saponins, glycosides, steroids, mucilage, phenols and proteins. MF has also been reported to show a broad spectrum of antibacterial activity [ 6 ]. In traditional medicine, sepals are diuretic, while root juice is used to heal tongue blemishes. Phytoconstituents such as rutin, quercetin, hyperin, singapic acid, ferulic acid and stigluside have been isolated from the methanolic extract of MF sepals [ 5 ]. The sepals of MF were found to have antimicrobial activity against Saccharomyces cerevisiae, Ustilago mayadis, Escherichia coli, Micrococcus luteus, Bacillus subtilis and Bacillus cereus . The methanolic extract of MF leaves was found to possess hypolipidemic activity in high-fat diet-fed rats. The aqueous and alcoholic extracts of MF leaves showed significant hepatoprotective activity in a paracetamol-induced liver damage model in Wistar rats [ 5 ]. Despite its medicinal and nutritional value, MF is still an underutilized plant and unfortunately has been subjected to eradication because it is considered a weed. The present study was conducted to investigate the health and nutritional benefits of MF sepals as a nutraceutical source that can be used to develop novel value-added herbal tea products. 2. Materials and Methods 2.1 Materials Matured good-quality M. frondosa (MF) sepals were collected from the Belihuloya area and authenticated at the National Herbarium of Sri Lanka, Peradeniya. Voucher specimens were deposited at the Department of Physical Sciences and Technology, Faculty of Applied Sciences, Sabaragamuwa University of Sri Lanka. DPPH, gallic acid, quercetin, Folin-Ciocalteu reagent, 3,5-dinitrosalicylic acid, and α-amylase ( Aspergillus oryzae ) were purchased from Sigma Aldrich (USA). All the other solvents and chemicals used in this study were of analytical grade or higher. 2.2 Preparation of the MF leaves and sepals powders The freshly collected sepals and leaves were washed with tap water, shade-dried until a constant weight was obtained, and ground into a fine powder using a kitchen grinder. 2.3 Preparation of extracts 2.3.1 Water extraction (without ultrasonication) Water extracts were prepared as described previously by Hui et al. [ 7 ], with some modifications. Briefly, dried sepal powder was mixed with distilled water (ratio of 1:50) and extracted using a magnetic stirrer (600 rpm; 30 minutes) at three different temperatures, 60°C, 70°C and 80°C. The extracted samples were filtered, lyophilized and stored at -20°C until further use. 2.3.2 Water extraction (with ultrasonication) Ultrasonic water bath extraction was conducted according to the method described previously by Gadjalova & Mihaylova [ 8 ], with some modifications. Dried sepal powder was mixed with distilled water at a ratio of 1:50 and extracted at 60°C for 30 minutes in an ultrasonic water bath. The same procedure was used to extract the samples at 70°C and 80°C. The resulting extracts were filtered, lyophilized and stored at -20°C until further use. 2.3.3 Ethanol extraction The dried herbs were defatted with petroleum ether using a Soxhlet apparatus. The defatted plant materials were then divided into two equal portions, and one part was subjected to hot ethanol extraction using a Soxhlet apparatus (with a sample-to-ethanol ratio of 1:50). The second part of the sample was extracted with cold ethanol (24 h at room temperature). The filtered extracts were then concentrated under reduced pressure to obtain crude ethanol extracts. A non-defatted sepal powder sample was also extracted with cold ethanol at room temperature for 24 hours. 2.4 Formulation of tea blends incorporated in MF sepals and preparation of tea extracts Dried MF sepal powder was blended with BOPF (Broken Orange Pekoe Fanning) grade black tea powder in the proportions of 0:100, 30:70, 40:60, and 50:50 (w/w %) to formulate the tea (Table S1 – supplementary information). Tea bags containing 2 g of each blend were brewed in hot water (120 ml) at 95°C for 3 minutes, filtered while hot, lyophilized and stored at -20°C until further use. 2.5 Sensory evaluation to select the best formulation of the MF-incorporated tea samples A sensory evaluation was conducted to select the most suitable formulation for MF-incorporated tea samples. A 5-point hedonic ranking test was conducted using 30 untrained panelists to evaluate the sensory attributes, such as appearance, aroma, color, bitterness, astringency, after taste and overall acceptability of the tea samples. The sensory data were analyzed using the nonparametric Freidman test and Tukey pairwise comparison test (one-way ANOVA) with Minitab 19 statistical software. 2.6 Determination of the physicochemical properties of the MF sepal powder extracts and the selected MF sepal- incorporated tea sample (MFT-40) 2.6.1 Proximate analysis The moisture (AOAC 990.19), crude fat (AOAC 920.39), dry ash (AOAC 900.02), crude protein (AOAC 991.20) and crude fiber (AOAC 991.43) contents of selected MF-incorporated tea (MFT-40) and BOPF black tea (control) samples were determined according to the AOAC standard methods. 2.6.2 Determination of total phenolic content (TPC) The total phenolic content was determined by the Folin-Ciocalteu method as described previously by Perera et al. [ 9 ]. Gallic acid was used as the standard. The total phenolic content of the samples was expressed as mg of gallic acid equivalents (GAE) in one gram of sample. $$\:\text{T}\text{o}\text{t}\text{a}\text{l}\:\text{p}\text{h}\text{e}\text{n}\text{o}\text{l}\text{i}\text{c}\:\text{c}\text{o}\text{n}\text{t}\text{e}\text{n}\text{t}=\frac{\text{X}\times\:\text{V}\times\:\text{D}\text{F}}{\text{W}}$$ where x = concentration (ppm), V = volume of sample solution (extract) (ml), DF = dilution factor of the sample solution and w = sample weight (g). 2.6.3 Determination of total flavonoid content (TFC) The total flavonoid content was determined by the aluminum chloride method as described previously by Sumaiyah et al. [ 10 ]. Quercetin was used as the standard flavonoid to construct the standard calibration plot. The total flavonoid content was expressed in mg equivalent quercetin/g samples (mg Q/g) and calculated based on the following equation: $$\:\text{T}\text{o}\text{t}\text{a}\text{l}\:\text{F}\text{l}\text{a}\text{v}\text{o}\text{n}\text{o}\text{i}\text{d}\:\text{c}\text{o}\text{n}\text{t}\text{e}\text{n}\text{t}=\frac{\text{X}\times\:\text{V}\times\:\text{D}\text{F}}{\text{W}}$$ where x = concentration (ppm), V = volume of sample solution (extract) (ml), DF = dilution factor of the sample solution and w = sample weight (g). 2.6.4 Determination of antioxidant properties The antioxidant potential of the MF sepal extracts and MFT samples was evaluated by a DPPH radical scavenging assay and an iron reducing power assay as previously described by Perera et al. [ 9 ]. Ascorbic acid was used as the standard antioxidant. 2.6.5 Determination of α-amylase inhibitory activity The alpha amylase inhibitory activity of the samples was assayed using the dinitrosalicylic acid (DNS) method as described by Keharom et al. [ 11 ]. Acarbose was used as the positive control. The inhibition of α-amylase activity was determined by measuring the absorbance at 540 nm using a microplate reader. All samples were assayed in triplicates, and the results are expressed as the IC 50 . 2.6.7 Determination of the cytotoxicity of the aqueous MF extracts and MFTs A lethality assay of brine shrimp ( Artemia salina ) was conducted to determine the toxicity of water extracts of MF sepals and MFTs according to the method described previously by Karim et al. [ 12 ], with slight modifications. DMSO (1%) was used as the negative control. The LC 50 is the concentration of the sample required to kill 50% of the brine shrimp population, which was calculated from the plot of % inhibition against the log concentration of sample extract. 2.7 Microbial analysis of selected MF-incorporated tea samples (MFT-40) The total plate count and yeast and mold counts were analyzed using the methods described by Dharmarathna et al. [ 13 ] . 2.8 Statistical analysis All the experiments were carried out in replicates in 4–6 separate experiments. The 50% inhibitory concentrations (IC 50 ) and 50% lethal concentrations (LC 50 ) were determined by using Compusyn 1.0 software (ComboSyn Inc., accessed from www.combosyn.com ). The results are expressed graphically as the mean ± standard error of the mean (SEM) unless otherwise specified. 3. Results and Discussion The therapeutic benefits of many medicinal plants are often attributed to their antioxidant properties. Tea ( Camellia sinensis ), the world’s most widely consumed beverage, has been extensively studied in the past few years for its antioxidant and radical scavenging activities. The unique taste and flavor of tea can be attributed to its composition of color and flavor-generating compounds, such as dietary polyphenols, benzotrpolone compounds (theaflavins, thearubigins), tea catechins such as epigallocatechin-3-gallate (EGCG), epigallocatechin (EGC), epicatechin-3-gallate (ECG), epicatechin (EC), methylxanthines (such as caffeine, theobromine, theophylline), and some amino acids [ 14 – 17 ]. However, it has been reported that the bioaccessibility of these active components is often suboptimal to produce the desired response from conventional tea consumption [ 18 ]. In this context, increasing the nutraceutical value of tea while maintaining its unique and original organoleptic properties is a promising product diversification strategy in the tea manufacturing industry [ 1 ]. The present study investigated the nutraceutical properties of an underutilized edible plant material, MF, and its potential for use as a key ingredient in the development of a novel herbal tea formulation (MFT-40) with enhanced functional properties. 3.1 Extraction yields, total phenol and total flavonoid contents of ethanol and water extracts of MF sepals Edible herbs possess a wide array of phenolic substances and are reported to have many useful functional and nutraceutical properties. Many health challenges that we are facing today are directly or indirectly connected with oxidative stress. Phenolic compounds act as antioxidants due to their capacity to scavenge free radicals. In this context, dietary phenol-rich edible plants have great value in the development of novel nutraceutical formulations. The total phenolic and flavonoid contents of the hot and cold ethanol extracts of native and defatted MF sepals were analyzed. The extraction yields of the three ethanol extracts and their total phenol and flavonoid contents are shown in Table 01 . The extraction yields of the three ethanol extracts ranged from 15.88–46.71% w/w dry matter. The hot ethanol extract of defatted MF sepals (defatted HEMF) had a greater extraction yield than the other two extracts. This inferred that the prior defatting process and higher solvent temperatures positively affected the extraction yield. The total phenolic content (TPC) of each extract was measured using Folin–Ciocalteu reagent. The results were derived from a calibration curve (y = 0.0978x + 0.0443; R² = 0.9922) of gallic acid (0–10 ppm final concentration) and expressed in gallic acid equivalents (GAE) per gram dry extract weight. Flavonoids are polyphenol compounds that have a wide array of therapeutic potential [ 10 ], [ 19 ]. The flavonoid contents (TFC) of three ethanol extracts were determined using aluminum chloride via a colorimetric method. The results were derived from the calibration curve (y = 0.0444x + 0.2065; R² = 0.9835) of quercetin (0–25 µg/mL) and expressed in quercetin equivalents (QE) per gram dry extract weight. Table 01 Extraction yield, total phenolic content (as gallic acid equivalents), total flavonoid content, DPPH scavenging activity (as IC 50 µg/ml) and α amylase inhibitory activity (as IC 50 µg/ml) of ethanol extracts of MF sepals. Type of extract/ Standard Compound (%) Extraction yield Total phenolic content (TPC) mg (GAE)/g Total flavonoid contents (TFC) mg QE/g DPPH radical scavenging activity EC 50 (µg/ml) α amylase inhibitory activity (as IC 50 µg/ml) Cold ethanol extract of native MF sepals (native CEMF) 26.33 ± 0.16 b 108.05 ± 0.67 b 64.29 ± 0.69 b 25.00 ± 0.12 f - Cold ethanol extract of defatted MF sepals (defatted CEMF) 15.88 ± 0.02 c 93.87 ± 0.56 c 61.63 ± 0.17 c 26.37 ± 0.23 e - Hot ethanol extract of defatted MF sepals (defatted HEMF) 46.71 ± 0.23 a 114.42 ± 0.56 a 66.47 ± 0.31 a 24.06 ± 0.16 g 87.61 ± 0.19 Ascorbic acid * − − − 1.625 ± 0.020 − Acarbose − − − − 12.35 ± 0.047 The values represent the means ± standard deviations of 3 replicates. The values indicated by different superscript letters in the same column are significantly different at P<0.05. GAE−gallic acid equivalent, QE−quercetin equivalent The highest TPC was 114.42 mg (GAE)/g from defatted HEMF, and the lowest TPC was 93.87 mg (GAE)/g from defatted CEMF extract. A similar pattern of results was observed for the total flavonoid contents of the three extracts. The highest total flavonoid content (64.29 mg QE/g) was detected in the defatted HEMF, and the lowest TFC (61.63 mg QE/g) was detected in the defatted CEMF extract. The highest total phenolic and flavonoid contents in defatted HEMF may be attributed to its high extraction yield. Water extracts of MF sepals were prepared at three different temperatures, 60, 70 and 80°C, with or without ultrasonication, and their phenolic and flavonoid contents are given in Table 02 . Table 02 Total phenolic content (as gallic acid equivalents), total flavonoid content, DPPH scavenging activity (as IC 50 µg/ml) and α amylase inhibitory activity (as IC 50 µg/ml) of water extracts of MF sepals Type of extract/ Standard Compound Total phenolic content (TPC) mg (GAE)/g Total flavonoid contents (TFC) mg QE/g DPPH radical scavenging activity IC 50 (µg/ml) a-amylase inhibitory activity (as IC 50 µg/ml) Water extract of MF sepals at 60 o C, Without sonication (WMF-60) 74.51 ± 0.57 e 41.51 ± 0.10 c 33.33 ± 0.48 a - Water extract of MF sepals at 60 o C, With sonication (WMF-S60) 86.35 ± 0.36 c 41.88 ± 0.17 c 29.84 ± 0.09 c - Water extract of MF sepals at 70 o C, Without sonication (WMF-70) 80.75 ± 0.84 d 42.83 ± 0.32 c 32.23 ± 0.27 b - Water extract of MF sepals at 70 o C, With sonication (WMF-S70) 92.95 ± 0.97 b 45.51 ± 0.54 b 28.48 ± 0.63 d - Water extract of MF sepals at 80 o C, Without sonication (WMF-80) 90.22 ± 0.96 b 42.97 ± 0.30c 30.21 ± 0.15 c - Water extract of MF sepals at 80 o C, With sonication (WMF-S80) 102.12 ± 0.57 a 47.06 ± 0.07 a 27.04 ± 0.30 e 133.09 ± 0.98 a Ascorbic acid * − − 1.625 ± 0.020 − Acarbose − − − 12.35 ± 0.047 The values represent the means ± standard deviations of 3 replicates. The values indicated by different superscript letters in the same column are significantly different at P<0.05. GAE−gallic acid equivalent, QE−quercetin equivalent Both the TPC and TFC increased with increasing temperature from 60°C to 80°C. The extract obtained from sonication at 80°C for 30 minutes (WMF-S80) had the highest TPC (102.12 ± 0.57 ppm), TFC (47.06 ± 0.06 ppm) and antioxidant activity (IC 50 of 27.04 ± 0.30 ppm), and these values were significantly greater than those of the other two extracts prepared at 60°C and 70°C with sonication (Table 02 ). It was apparent that a high solvent temperature plus sonication improved the extraction of phenolics into water. However, the ethanol extracts had a greater TPC and TFC than did the water extracts. It is not surprising that many phenolic compounds are more soluble in polar organic solvents such as ethanol than in water. 3.2 DPPH radical scavenging activity of ethanol and water extracts of MF sepals The free radical scavenging activities of three ethanol extracts, native CEMF, defatted CEMF and defatted HEMF, at increasing concentrations are shown in Table 01 . Ascorbic acid was used as the positive control. The reduction of alcoholic DPPH by all three extracts was considerably high, and the scavenging potential increased with increasing concentrations of the extracts. The greatest DPPH radical scavenging potency with a minimum IC 50 value was recorded for the defatted hot ethanol extract (24.06 ± 0.16 ppm). All the data were compared with the IC 50 value of standard ascorbic acid (1.625 ± 0.02 ppm), as presented in Table 01 . The results demonstrated that the sonication process significantly enhanced the TPC and TFC in the water extracts of the MF sepals, leading to greater antioxidant activity than that of the unsonicated samples (Table 02 ). Scientific information on the phytochemical composition of MF sepals have not been previously reported. However, it has been reported that the methanol extract of MF leaves contains important phenolic compounds such as flavonoids, hydroxyl benzoic acid derivatives, cinnamic acid derivatives and stilbenes [ 20 ]. These compounds have been identified to possess good antioxidant properties; therefore, the high DPPH radical scavenging activities of the ethanol extracts of MF sepals may be attributed to the presence of these chemical entities. 3.3 Alpha-amylase inhibitory activity of ethanol and water extracts of MF sepals Alpha-amylase is a carbohydrate digestion enzyme that plays a vital role in controlling glucose levels in blood. The inhibition of α-amylase has been identified as a potential approach for controlling diabetes mellitus [ 21 ], [ 22 ]. Dietary polyphenols have been widely studied for their antidiabetic potential, and many such phenolic compounds exert their hypoglycemic effects through the inhibition of carbohydrate digestion enzymes such as α-amylase and α-glucosidase [ 23 ]. The alpha amylase inhibitory activity of the hot ethanol extract of defatted MF sepals (defatted HEMF) and the hot water extract obtained from sonication at 80°C for 30 minutes (WMF-S80) were determined using the dinitro salicylic acid method. The IC 50 values were calculated by plotting the % α-amylase inhibition as a function of extract concentration (Figure S1 – supplementary information). Defatted HEMF extract and hot water extract at 80°C (with sonication) showed considerable α-amylase inhibition, with IC 50 values of 87.61 ± 0.19 ppm (Table 01 ) and 133.09 ± 0.98 ppm (Table 02 ), respectively. Acarbose was extracted from commercially available acarbose tablets (Glucobay, 50 mg, Bayer Pharmaceuticals Pvt. Ltd.) was used as the positive control and had an IC 50 value of 12.35 ± 0.047 ppm. Figure S1 (supplementary information) comparatively demonstrates the α-amylase inhibitory potentials of acarbose, the WMF-S80 hot water extract and the defatted ethanol extract (defatted HEMF). By comparing these values with the phenolic contents of the two extracts, it can be inferred that phenolic compounds are responsible for the α-amylase inhibitory effects of MF sepals. The hypoglycemic activity of MF sepals has not been previously studied. However, alcoholic extracts of M. macrophyla (roots) and M. roxburghii (leaves) have been reported to possess α-amylase and α-glucosidase inhibitory activity [ 4 ]. 3.4 Cytotoxicity of water extracts of MF sepals using a brine shrimp lethality assay MF sepals are well-known edible herbs that are used in the preparation of snacks and porridge. However, as a basic requirement, the water extracts were investigated for any possible toxic effects prior to sensory analysis. The brine shrimp lethality assay is a useful tool for preliminary assessment of toxicity, and all the extracts were subjected to the above assay at concentrations up to 2000 ppm. The lyophilized extracts were dissolved in 1% DMSO in deionized water to obtain the required concentrations. None of the extracts were lethal to brine shrimp even at the highest concentration (2000 ppm) tested after 72 h of exposure. 3.5 Formulation of MF sepal-incorporated tea blends and sensory evaluation to determine the best tea formulation Sensory analysis is an important component of food quality control. It provides a comprehensive and direct measurement of the perceived intensity of sensory attributes, such as appearance, color, aroma, taste and texture. Considering the biochemical potential of MF sepals, a series of MF-tea blends were formulated, and their sensory properties were evaluated. Dried MF sepal powder at different proportions was blended with BOPF-grade tea powder to give rise to three different tea formulations, namely, MFT-30, MFT-40 and MFT-50. BOPF Tea (100%) was used as the control formulation. The compositions of the four formulations are given in Table S1 (supplementary information). A sensory evaluation (5-point hedonic ranking test; 30 untrained panelists) was conducted to characterize and quantify the sensory qualities of the product. Statistical analysis using the Friedman test revealed significant differences among the four tea samples across all evaluated attributes. Sample 174 (MFT-40) had the highest sum of ranks and mean values for attributes such as appearance, color, aroma, bitterness, astringency, aftertaste, and overall acceptability. Conversely, Sample 253 (MFT-30) displayed the lowest mean values for attributes including appearance, bitterness, astringency, and overall acceptability (Fig. 1 ). Accordingly, Sample 174, which was formulated with 40% sepal powder (MFT-40), was chosen as the most preferred sample among the panelists for further analysis. 3.6 Proximate analysis of MFT-40 and black tea (control) samples Proximate analysis of tea samples is important for determining their nutritional quality. The moisture content of tea should be between 3 and 5% [ 24 ]. proximate compositions of the MFT-40 and black tea samples were given in Table S2 (supplementary information). Accordingly, significant differences (P < 0.05) were observed in all proximate components between the MFT-40 and black tea samples. The determined nutritional values of both tea samples were in the order of crude fat < moisture < ash < crude fiber < crude protein < total carbohydrate. The moisture contents of the MFT-40 sample and black tea sample were 4.88 ± 0.100% and 4.30 ± 0.020%, respectively. These tea samples also contained moisture contents between the standard ranges. Both tea samples contained high amounts of carbohydrates, with more carbohydrate content in MFT-40 (33.91 ± 0.58%) than in black tea (29.45 ± 0.06%). The crude fiber content of MFT-40 was 19.38 ± 0.07%, and that of the black tea sample was 20.15 ± 0.02%. Both tea samples contained relatively similar amounts of fiber. The protein content was slightly greater in the MFT-40 sample (22.14 ± 0.23%) than in the black tea sample (21.01 ± 0.29%). The ash content provides the amount of minerals present in the food product. According to this analysis, both tea samples contained more than 8% mineral content. The crude fat content of the MFT-40 sample was 3.74 ± 0.05%, which was greater than the crude fat content of the black tea sample (2.87 ± 0.12%). According to the proximate analysis results, differences between proximate compositions of the black tea and MFT-40 samples were small, but statistically significant. 3.7 Evaluation of the TPC, TFC and antioxidant capacity of lyophilized MFT-40 Based on the sensory analysis results, 40% of the MF-sepals-incorporated Tea, MFT-40, was selected as the blend with the best organoleptic properties. Therefore, MFT-40 was subjected to TPC, TFC and antioxidant capacity analyses. The 100% Broken Orange Pekoe Fannings (BOPF) grade of black tea was selected as the control. Table 03 TPC, TFC, and antioxidant capacity of lyophilized MFT-40 MFT-40 BOPF Grade Black Tea (Control) Total Phenolic Content in mg GAE/g 138.82 ± 0.21 a 128.47 ± 0.13 b Total Flavonoid Content in mg QE/g 77.08 ± 0.08 a 69.78 ± 0.10 b DPPH Radical Scavenging Activity (IC 50 ) in mg/ml 12.23 ± 0.45 b 18.70 ± 0.68 a The values represent the means ± standard deviations of 3 replicates. The values indicated by different superscript letters in the same row are significantly different at P≤0.05. GAE−gallic acid equivalent, QE−quercetin equivalent Lyophilized tea extracts were dissolved in deionized water to obtain the desired concentrations. The TPC, TFC, and antioxidant capacity of MFT-40 and control black tea are shown in Table 03 . The TPC of MFT-40 (138.82 ± 0.21 mg GAE/g) was significantly greater (p < 0.05) than that of black tea (128.47 ± 0.13 mg GAE/g). The TFCs of the MF-incorporated tea (MFT-40) and black tea control groups were 77.08 ± 0.08 and 69.78 ± 0.10 mg QE/g, respectively. Statistical analysis revealed that the TFC of MFT-40 was significantly greater than that of black tea (p < 0.05). The antioxidant capacity of MFT-40 was evaluated using two antioxidant models, namely, the DPPH scavenging assay and the iron reducing power assay. As shown in Table 03 , DPPH scavenging capacity of MFT-40 (IC 50 value of 12.23 ± 0.45 ppm) was significantly greater (p < 0.05) than that of black tea (18.70 ± 0.68 ppm). The positive control, ascorbic acid, had an IC 50 of 1.63 ± 0.02 µg/ml. The reducing capacity of a compound may serve as a significant indicator of its potential antioxidant activity [ 25 ]. A comparative demonstration of the reducing power of MFT-40, black tea and ascorbic acid is given in Fig. 2 . Although the reducing power of the two tea extracts increased with increasing concentration, the values remained lower than that of ascorbic acid. The mean absorbance ± SD (at 700 nm) of ascorbic acid, MET-40 and black tea at 200 ppm was 2.94 ± 0.05, 1.33 ± 0.03, and 1.02 ± 0.05, respectively. The results clearly indicated that the incorporation of Mussanda sepals into black tea enhances the reducing power of black tea. 3.8 Evaluation of the antidiabetic activity of MFT-40 by an alpha-amylase inhibition assay . The alpha-amylase inhibition activity of MFT-40 and black tea was investigated. Acarbose extracted from glucobay-50 tablets was used as the positive control. Based on the IC 50 values of the tea samples, significantly greater enzyme inhibition (p < 0.05) was shown by MFT-40 (104.80 ± 0.59 ppm) than by the black tea sample (153.07 ± 0.61 ppm). The positive control acarbose had an IC 50 value of 12.35 ± 0.05 ppm (Table 04 ). Table 04 α-Amylase inhibitory activity of tea samples Tea samples IC 50 value of samples (µg/ml) MFT 40 lyophilized extract 104.80 ± 0.59 b Black tea lyophilized extract 153.07 ± 0.61 a Acarbose (Positive control) 12.35 ± 0.05 The values represent the means ± standard deviations of 3 replicates. The values indicated by different superscript letters in the same column Many plant extracts, including tea extracts, have been studied for their inhibitory activity against α-amylase, and it has been demonstrated that the main active components that have inhibitory effects are phenolic compounds [ 23 ], [ 26 ]. Previous studies have revealed that Mussanda species are rich in phenolics. In silico studies have shown that dietary polyphenols interact with alpha amylase active site residues, forming H-bonds between the hydroxyl and carbonyl moieties of their structures [ 27 ]. Therefore, our findings suggest that the incorporation of MF sepals into black tea results in a blend of tea with a relatively high phenolic content and increased alpha-amylase inhibitory activity. 3.9 Microbial analysis of MFT-40 The total plate count measures the total number of viable microorganisms, including bacteria, yeast, and molds, present in a tea sample. It is an indicator of the overall quality and safety of a product for human consumption. Microbial analysis of MFT-40 for up to three months revealed the presence of very few microbial colonies, indicating that the developed herbal tea was of good quality and not contaminated during processing (Table S3 – supplementary information). 4. Conclusions M. frondosa is an underutilized green leafy food source with remarkable nutritional potential. It can be effectively blended with green or black tea to develop new value-added tea formulations that can further fortify the health effects exerted by a cup of traditional tea. Cellular oxidative stress is considered a crucial step in the onset and development of many inflammatory and noncommunicable diseases. M. frondosa -incorporated tea (MFT-40) has high antioxidant potential and can alleviate cellular oxidative stress, while enhancing its health benefits. MFT-40 possesses palatable organoleptic properties, including taste, flavor and aroma, which we believe are the most important characteristics of a marketable tea product. Toxicity assays, proximate composition analyses and microbiological evaluations confirmed the safety of MFT-40 as a consumer-friendly tea product. Currently, our research team is investigating further biological benefits of MFT-40 and its toxicological effects in vivo . Abbreviations Abbreviation Full name CEMF Cold Ethanol extract of MF sepals DNS Dinitrosalicylic acid GAE Gallic Acid Equivalence HEMF Hot Ethanol extract of MF sepals MF Mussaenda frondosa MFT Mussaenda frondosa blended tea QE Quercetin Equivalence TPC Total phenolic Content TFC Total flavonoid Content WMF Water extract of MF sepals WMF-S Water extract of MF sepals -sonicated Declarations Competing Interests The authors declare that they have no competing interests. Ethics waived off statement The Sabaragamuwa University of Sri Lanka Research Ethics Committee has confirmed that no ethical approval is required. Informed Consent Informed consent was obtained from all individual participants included in the study. Author Contribution NP conceived the idea, supervised the work and wrote the manuscript. NF designed and carried out experiments. TK supervised the work, wrote and edited the manuscript. All authors read and approved the final manuscript. Acknowledgement The authors thankfully acknowledge the technical staff of the research laboratory of the Department of Physical Sciences and Technology, Faculty of Applied Sciences, Sabaragamuwa University of Sri Lanka, for providing technical supports. Data Availability All data generated or analyzed during this study are included in this article. References Pilapitiya HMCG, Silva SD, Miyazaki H. Innovative value addition in tea industry: Sri Lanka vs. Japan. 2020;https://doi:10.21203/rs.3.rs-27492/v1. Astalakshmi N, Sundara Ganapathy R. A comprehensive review on the genus: Mussaenda. Int J Pharm Sci Res. 2023;8:534–541. Shimpale VB, Yadav SR, Babu CR. A review of the genus Mussaenda ( Rubiaceae ) from great Nicobar Island, India, including a new species. Rheedea. 2009;19:53–57. Vidyalakshmi KS, Vasanthi HR. Ethnobotany, Phytochemistry and Pharmacology of Mussaenda Species (Rubiaceae). Ethnobotanical Leaflets. 2008;12:469–475. Shanthi S, Radha R. Antimicrobial and phytochemical studies of Mussaenda frondosa Linn. Leaves. Pharmacognosy Journal. 2020;12:630–635. Jayasinghe U. Antimicrobial activity of some Sri Lankan Rubiaceae and Meliaceae . Fitoterapia. 2002;73:424–427. Hui CK, Majid NI, Zainol MKM, Mohamad H, Zin ZM. Preliminary phytochemical screening and effect of hot water extraction conditions on phenolic contents and antioxidant capacities of Morinda citrifolia leaf. Malaysian Applied Biology. 2018;47:13–24. Gadjalova AV, Mihaylova DS. Ultrasound-assisted extraction of medicinal plants and evaluation of their biological activity. Food Res. 2019;3: 530–536. Perera, MGAN, Soysa SSSBDP, Abeytunga DTU, Ramesh R. Antioxidant and cytotoxic properties of three traditional decoctions used for the treatment of cancer in Sri Lanka. Pharmacogn Mag. 2008;4:172–181. Sumaiyah M, Dalimunthe A. Determination of total phenolic content, total flavonoid content, and antimutagenic activity of ethanol extract nanoparticles of Rhaphidophora pinnata (l.f) schott leaves. RASAYAN Journal of Chemistry. 2018;11(2):505–510. Keharom S, Mahachai R, Chanthai S. The optimization study of α-amylase activity based on central composite design-response surface methodology by dinitrosalicylic acid method. Int Food Res J. 2016;23:10–17. Karim MA, Islam MA, Islam MM, Hosen MJ, Mazumder K, Hasan MNH, Biswas S. Biswas. Evaluation of antioxidant, anti-hemolytic, cytotoxic effects and antibacterial activity of selected mangrove plants ( Bruguiera gymnorrhiza and Heritiera littoralis ) in Bangladesh. Clin Phytosci. 2020;6(1):8.https://doi:10.1186/s40816-020-0152-9 Dharmarathna EKGPU, Liyanawickramasinghea TR, Aruppala ALYH, Abeyrathne EDNS. A study on level of microbiological contamination in made tea as a raw material in commercial tea bagging factory and its workers’ hand hygiene. Journal of Agriculture and Value Addition. 2018;1:49–59. Parveen A, Qin CY, Zhou F, Lai G, Long P, Zhu M, Ke J, Zhang L. Zhang. The chemistry, sensory properties and health benefits of aroma compounds of black tea produced by Camellia sinensis and Camellia assamica . Horticulturae. 2023;9:1253. https://doi.org/10.3390/horticulturae9121253. Wong M, Sirisena S, Ng K. Phytochemical profile of differently processed tea: A review. J Food Sci. 2022;87(5):1925-194. https://doi: 10.1111/1750-3841.16137. Zhang L, Cao QQ, Granato D, Xu YQ, Ho CT. Association between chemistry and taste of tea: A review. Trends Food Sci Technol. 2020;101:139-149. Tounekti T, Joubert E, Hernández I, Munné-Bosch S. Improving the polyphenol content of tea. Crit Rev Plant Sci. 2013;32:192–215. Da Silva Pinto M. Tea: A new perspective on health benefits. Food Res Int. 2013;53: 558-567. Graf BA, Milbury PE, Blumberg JB. Flavonols, Flavones, Flavanones, and Human Health: Epidemiological Evidence. J Med Food. 2005;8:281–290 Manasa D, Chandrashekar K, Bhagya N. Rapid in vitro callogenesis and phytochemical screening of leaf, stem and leaf callus of Mussaenda frondosa linn.: a medicinal plant. Asian J Pharm Clin Res. 2017;10:81-86. https://doi:10.22159/ajpcr.2017. v10i6.17527. Kaur N, Kumar V, Nayak SK, Wadhwa P, Kaur P, Sahu SK. Alpha-amylase as molecular target for treatment of diabetes mellitus: A comprehensive review. Chem Biol Drug Des. 2021;98: 539–560. Kashtoh H, Baek KH. New insights into the latest advancement in α-amylase inhibitors of plant origin with anti-diabetic effects. Plants. 2023;12:2944. https://doi:10.3390/ plants12162944 Sun C, Zhao C, Guven EC, Paoli P Gandara JS, Ramkumar KM, Wang S, Buleu F, Pah A, Turi V, Damian G, Dragan S, Tomas M, Khan W, Wang M, Delmas D, Portillo MP, Dar P, Chen L, Xiao J. Dietary polyphenols as antidiabetic agents: Advances and opportunities. Food Frontiers. 2020;1: 18–44. Zou H, Shen S, Lan T, Sheng X, Zan J, Jiang Y, Du Q, Yuan H. Prediction Method of the Moisture Content of Black Tea during Processing Based on the Miniaturized Near-Infrared Spectrometer. Horticulturae. 2022;8(12):1170. https://doi.org/10.3390/ horticulturae8121170 Meir S, Akiri B, Kanner J, Hadas S. Determination and involvement of aqueous reducing compounds in oxidative defense systems of various senescing leaves. J Agric Food Chem. 1995;43:1813–1817. Lin D, Xiao M, Zhao J, Li Z, Xing B, Li X, Kong M, Li L, Zhang Q, Liu Y, Chen H, Qin W, Wu, H, Chen S. An overview of plant phenolic compounds and their importance in human nutrition and management of type 2 diabetes. Molecules. 2016;21:1374. https://doi: 10.3390/molecules21101374 Riyaphan J, Pham DC, Leong MK, Weng CF. In silico approaches to identify polyphenol compounds as α-glucosidase and α-amylase inhibitors against type-II diabetes. Biomolecules. 2021;11:1877. https://doi: 10.3390/biom11121877. Additional Declarations No competing interests reported. Supplementary Files SupplimentaryInformationDF.docx Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 08 Aug, 2024 Reviews received at journal 07 Aug, 2024 Reviews received at journal 07 Aug, 2024 Reviews received at journal 06 Aug, 2024 Reviews received at journal 06 Aug, 2024 Reviews received at journal 05 Aug, 2024 Reviewers agreed at journal 04 Aug, 2024 Reviewers agreed at journal 01 Aug, 2024 Reviewers agreed at journal 01 Aug, 2024 Reviewers agreed at journal 01 Aug, 2024 Reviewers agreed at journal 31 Jul, 2024 Reviewers agreed at journal 31 Jul, 2024 Reviewers agreed at journal 31 Jul, 2024 Reviewers invited by journal 12 Jul, 2024 Editor assigned by journal 12 Jul, 2024 Submission checks completed at journal 11 Jul, 2024 First submitted to journal 18 Jun, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4602535","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":335804294,"identity":"b9ec3ce6-3a28-4328-9690-31dfffc22f26","order_by":0,"name":"M. F.F. Nafla","email":"","orcid":"","institution":"Sabaragamuwa University of Sri Lanka","correspondingAuthor":false,"prefix":"","firstName":"M.","middleName":"F.F.","lastName":"Nafla","suffix":""},{"id":335804295,"identity":"0d0d79fb-cf40-4873-8bf7-f977e244ab7e","order_by":1,"name":"T. C. Kananke","email":"","orcid":"","institution":"Sabaragamuwa University of Sri Lanka","correspondingAuthor":false,"prefix":"","firstName":"T.","middleName":"C.","lastName":"Kananke","suffix":""},{"id":335804296,"identity":"e7b8f357-0035-487f-9bf3-62dccab33800","order_by":2,"name":"M. G.A.N. Perera","email":"data:image/png;base64,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","orcid":"","institution":"Sabaragamuwa University of Sri Lanka","correspondingAuthor":true,"prefix":"","firstName":"M.","middleName":"G.A.N.","lastName":"Perera","suffix":""}],"badges":[],"createdAt":"2024-06-19 01:55:06","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4602535/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4602535/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":61764028,"identity":"45425b7e-1c40-43f6-ac2d-19f43f6cd91d","added_by":"auto","created_at":"2024-08-05 10:03:30","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":82942,"visible":true,"origin":"","legend":"\u003cp\u003eRadar diagram of the median sensory evaluation score\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-4602535/v1/3057281e4c3b215c74b9893e.png"},{"id":61763566,"identity":"e6cf36c9-fae2-45e7-863b-4f86e10b2140","added_by":"auto","created_at":"2024-08-05 09:55:30","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":41176,"visible":true,"origin":"","legend":"\u003cp\u003eTotal reduction power of different concentrations of ascorbic acid, MFT-40 and black tea. The values represent the means ± standard deviations of 3 replicates\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-4602535/v1/a5702fba349a5eabe4690e9b.png"},{"id":61764437,"identity":"c4a92394-53db-4612-9365-eca46e66bc3a","added_by":"auto","created_at":"2024-08-05 10:11:31","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1118031,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4602535/v1/b675bf8a-abc8-4b23-ab73-ccc43b254e6b.pdf"},{"id":61763565,"identity":"7f899be4-6700-4c19-a35e-0b9425d614a1","added_by":"auto","created_at":"2024-08-05 09:55:30","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":25115,"visible":true,"origin":"","legend":"","description":"","filename":"SupplimentaryInformationDF.docx","url":"https://assets-eu.researchsquare.com/files/rs-4602535/v1/66919170eaa0205533593186.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Development of Mussaenda frondosa sepal infused functional tea with enhanced antioxidant and alpha amylase inhibitory activities","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eTea is one of the major sources of exports for Sri Lanka. In 2018, Sri Lanka contributed 5.1% (303\u0026nbsp;million kilograms) of the world\u0026rsquo;s tea production [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. While the global tea market is rapidly growing with emerging customer needs, currently, it has become one of the most competitive beverage markets in the world. Sri Lanka must compete with many other tea-exporting countries around the world to sustain its global market. Value-added tea production is a successful strategy for facing international competitiveness in the tea industry. By adding value to its tea products, such as through unique blends, packaging, or organic certifications, Sri Lanka can attract tea consumers and differentiate itself in the global market. The production of new tea blends to augment the health benefits of tea while preserving its original organoleptic properties has become a popular value-adding strategy.\u003c/p\u003e \u003cp\u003eThe genus \u003cem\u003eMussaenda L.\u003c/em\u003e is one of the largest genera of the \u003cem\u003eRubiaceae\u003c/em\u003e family, which has approximately 200 species that are primarily distributed in the forests of the tropical old world [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e], [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. This genus is native to West Africa through the Indian subcontinent, Southeast Asia and South China. The genus \u003cem\u003eMussaenda\u003c/em\u003e is a significant source of medicinal natural products, particularly iridoids, triterpenes and flavonoids [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e], [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cem\u003eMussaenda frondosa\u003c/em\u003e (MF) is an edible species of the genus \u003cem\u003eMussaenda\u003c/em\u003e. The whole plant is used to treat various ailments in traditional medicine. A weak decoction of dry shoots is given to children to relieve cough, while root juice is used to heal tongue blemishes [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Green leaves are used in traditional medicine for stomach problems. Sepals are diuretics and are considered a food that enhances memory and is beneficial against many liver-associated diseases [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. The deep-oil-fried sepals of this plant are popular snacks among Sri Lankan villagers. Green leaves are used to prepare herbal porridge, which is believed to have good liver-protecting power.\u003c/p\u003e \u003cp\u003ePreliminary phytochemical investigations of different extracts of MF have shown the presence of flavonoids, saponins, glycosides, steroids, mucilage, phenols and proteins. MF has also been reported to show a broad spectrum of antibacterial activity [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. In traditional medicine, sepals are diuretic, while root juice is used to heal tongue blemishes. Phytoconstituents such as rutin, quercetin, hyperin, singapic acid, ferulic acid and stigluside have been isolated from the methanolic extract of MF sepals [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe sepals of MF were found to have antimicrobial activity against \u003cem\u003eSaccharomyces cerevisiae, Ustilago mayadis, Escherichia coli, Micrococcus luteus, Bacillus subtilis and Bacillus cereus\u003c/em\u003e. The methanolic extract of MF leaves was found to possess hypolipidemic activity in high-fat diet-fed rats. The aqueous and alcoholic extracts of MF leaves showed significant hepatoprotective activity in a paracetamol-induced liver damage model in Wistar rats [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eDespite its medicinal and nutritional value, MF is still an underutilized plant and unfortunately has been subjected to eradication because it is considered a weed. The present study was conducted to investigate the health and nutritional benefits of MF sepals as a nutraceutical source that can be used to develop novel value-added herbal tea products.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Materials\u003c/h2\u003e \u003cp\u003eMatured good-quality \u003cem\u003eM. frondosa\u003c/em\u003e (MF) sepals were collected from the Belihuloya area and authenticated at the National Herbarium of Sri Lanka, Peradeniya. Voucher specimens were deposited at the Department of Physical Sciences and Technology, Faculty of Applied Sciences, Sabaragamuwa University of Sri Lanka.\u003c/p\u003e \u003cp\u003eDPPH, gallic acid, quercetin, Folin-Ciocalteu reagent, 3,5-dinitrosalicylic acid, and α-amylase (\u003cem\u003eAspergillus oryzae\u003c/em\u003e) were purchased from Sigma Aldrich (USA). All the other solvents and chemicals used in this study were of analytical grade or higher.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Preparation of the MF leaves and sepals powders\u003c/h2\u003e \u003cp\u003eThe freshly collected sepals and leaves were washed with tap water, shade-dried until a constant weight was obtained, and ground into a fine powder using a kitchen grinder.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Preparation of extracts\u003c/h2\u003e \u003cdiv id=\"Sec6\" class=\"Section3\"\u003e \u003ch2\u003e2.3.1 Water extraction (without ultrasonication)\u003c/h2\u003e \u003cp\u003eWater extracts were prepared as described previously by Hui et al. [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], with some modifications. Briefly, dried sepal powder was mixed with distilled water (ratio of 1:50) and extracted using a magnetic stirrer (600 rpm; 30 minutes) at three different temperatures, 60\u0026deg;C, 70\u0026deg;C and 80\u0026deg;C. The extracted samples were filtered, lyophilized and stored at -20\u0026deg;C until further use.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section3\"\u003e \u003ch2\u003e2.3.2 Water extraction (with ultrasonication)\u003c/h2\u003e \u003cp\u003eUltrasonic water bath extraction was conducted according to the method described previously by Gadjalova \u0026amp; Mihaylova [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e], with some modifications. Dried sepal powder was mixed with distilled water at a ratio of 1:50 and extracted at 60\u0026deg;C for 30 minutes in an ultrasonic water bath. The same procedure was used to extract the samples at 70\u0026deg;C and 80\u0026deg;C. The resulting extracts were filtered, lyophilized and stored at -20\u0026deg;C until further use.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section3\"\u003e \u003ch2\u003e2.3.3 Ethanol extraction\u003c/h2\u003e \u003cp\u003eThe dried herbs were defatted with petroleum ether using a Soxhlet apparatus. The defatted plant materials were then divided into two equal portions, and one part was subjected to hot ethanol extraction using a Soxhlet apparatus (with a sample-to-ethanol ratio of 1:50). The second part of the sample was extracted with cold ethanol (24 h at room temperature). The filtered extracts were then concentrated under reduced pressure to obtain crude ethanol extracts. A non-defatted sepal powder sample was also extracted with cold ethanol at room temperature for 24 hours.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Formulation of tea blends incorporated in MF sepals and preparation of tea extracts\u003c/h2\u003e \u003cp\u003eDried MF sepal powder was blended with BOPF (Broken Orange Pekoe Fanning) grade black tea powder in the proportions of 0:100, 30:70, 40:60, and 50:50 (w/w %) to formulate the tea (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e \u0026ndash; supplementary information). Tea bags containing 2 g of each blend were brewed in hot water (120 ml) at 95\u0026deg;C for 3 minutes, filtered while hot, lyophilized and stored at -20\u0026deg;C until further use.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Sensory evaluation to select the best formulation of the MF-incorporated tea samples\u003c/h2\u003e \u003cp\u003eA sensory evaluation was conducted to select the most suitable formulation for MF-incorporated tea samples. A 5-point hedonic ranking test was conducted using 30 untrained panelists to evaluate the sensory attributes, such as appearance, aroma, color, bitterness, astringency, after taste and overall acceptability of the tea samples. The sensory data were analyzed using the nonparametric Freidman test and Tukey pairwise comparison test (one-way ANOVA) with Minitab 19 statistical software.\u003c/p\u003e \u003cp\u003e \u003cb\u003e2.6 Determination of the physicochemical properties of the MF sepal powder extracts and the selected MF\u003c/b\u003e \u003cb\u003esepal-\u003c/b\u003e\u003cb\u003eincorporated tea sample (MFT-40)\u003c/b\u003e\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section3\"\u003e \u003ch2\u003e2.6.1 Proximate analysis\u003c/h2\u003e \u003cp\u003eThe moisture (AOAC 990.19), crude fat (AOAC 920.39), dry ash (AOAC 900.02), crude protein (AOAC 991.20) and crude fiber (AOAC 991.43) contents of selected MF-incorporated tea (MFT-40) and BOPF black tea (control) samples were determined according to the AOAC standard methods.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section3\"\u003e \u003ch2\u003e\u003cb\u003e2.6.2 Determination of total phenolic content (TPC)\u003c/b\u003e\u003c/h2\u003e \u003cp\u003eThe total phenolic content was determined by the Folin-Ciocalteu method as described previously by Perera et al. [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Gallic acid was used as the standard. The total phenolic content of the samples was expressed as mg of gallic acid equivalents (GAE) in one gram of sample.\u003cdiv id=\"Equa\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equa\" name=\"EquationSource\"\u003e\n$$\\:\\text{T}\\text{o}\\text{t}\\text{a}\\text{l}\\:\\text{p}\\text{h}\\text{e}\\text{n}\\text{o}\\text{l}\\text{i}\\text{c}\\:\\text{c}\\text{o}\\text{n}\\text{t}\\text{e}\\text{n}\\text{t}=\\frac{\\text{X}\\times\\:\\text{V}\\times\\:\\text{D}\\text{F}}{\\text{W}}$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003ewhere x\u0026thinsp;=\u0026thinsp;concentration (ppm), V\u0026thinsp;=\u0026thinsp;volume of sample solution (extract) (ml), DF\u0026thinsp;=\u0026thinsp;dilution factor of the sample solution and w\u0026thinsp;=\u0026thinsp;sample weight (g).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section3\"\u003e \u003ch2\u003e2.6.3 Determination of total flavonoid content (TFC)\u003c/h2\u003e \u003cp\u003eThe total flavonoid content was determined by the aluminum chloride method as described previously by Sumaiyah et al. [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Quercetin was used as the standard flavonoid to construct the standard calibration plot. The total flavonoid content was expressed in mg equivalent quercetin/g samples (mg Q/g) and calculated based on the following equation:\u003cdiv id=\"Equb\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equb\" name=\"EquationSource\"\u003e\n$$\\:\\text{T}\\text{o}\\text{t}\\text{a}\\text{l}\\:\\text{F}\\text{l}\\text{a}\\text{v}\\text{o}\\text{n}\\text{o}\\text{i}\\text{d}\\:\\text{c}\\text{o}\\text{n}\\text{t}\\text{e}\\text{n}\\text{t}=\\frac{\\text{X}\\times\\:\\text{V}\\times\\:\\text{D}\\text{F}}{\\text{W}}$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003ewhere x\u0026thinsp;=\u0026thinsp;concentration (ppm), V\u0026thinsp;=\u0026thinsp;volume of sample solution (extract) (ml), DF\u0026thinsp;=\u0026thinsp;dilution factor of the sample solution and w\u0026thinsp;=\u0026thinsp;sample weight (g).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section3\"\u003e \u003ch2\u003e2.6.4 Determination of antioxidant properties\u003c/h2\u003e \u003cp\u003eThe antioxidant potential of the MF sepal extracts and MFT samples was evaluated by a DPPH radical scavenging assay and an iron reducing power assay as previously described by Perera et al. [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Ascorbic acid was used as the standard antioxidant.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section3\"\u003e \u003ch2\u003e2.6.5 Determination of α-amylase inhibitory activity\u003c/h2\u003e \u003cp\u003eThe alpha amylase inhibitory activity of the samples was assayed using the dinitrosalicylic acid (DNS) method as described by Keharom et al. [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Acarbose was used as the positive control. The inhibition of α-amylase activity was determined by measuring the absorbance at 540 nm using a microplate reader. All samples were assayed in triplicates, and the results are expressed as the IC\u003csub\u003e50\u003c/sub\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section3\"\u003e \u003ch2\u003e2.6.7 Determination of the cytotoxicity of the aqueous MF extracts and MFTs\u003c/h2\u003e \u003cp\u003eA lethality assay of brine shrimp (\u003cem\u003eArtemia salina\u003c/em\u003e) was conducted to determine the toxicity of water extracts of MF sepals and MFTs according to the method described previously by Karim et al. [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], with slight modifications. DMSO (1%) was used as the negative control. The LC\u003csub\u003e50\u003c/sub\u003e is the concentration of the sample required to kill 50% of the brine shrimp population, which was calculated from the plot of % inhibition against the log concentration of sample extract.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Microbial analysis of selected MF-incorporated tea samples (MFT-40)\u003c/h2\u003e \u003cp\u003eThe total plate count and yeast and mold counts were analyzed using the methods described by\u003c/p\u003e \u003cp\u003eDharmarathna et al. [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e] .\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e2.8 Statistical analysis\u003c/h2\u003e \u003cp\u003eAll the experiments were carried out in replicates in 4\u0026ndash;6 separate experiments. The 50% inhibitory concentrations (IC\u003csub\u003e50\u003c/sub\u003e) and 50% lethal concentrations (LC\u003csub\u003e50\u003c/sub\u003e) were determined by using Compusyn 1.0 software (ComboSyn Inc., accessed from \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e\u003ca href=\"http://www.combosyn.com\" target=\"_blank\"\u003ewww.combosyn.com\u003c/a\u003e\u003c/span\u003e\u003cspan address=\"http://www.combosyn.com\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The results are expressed graphically as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of the mean (SEM) unless otherwise specified.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results and Discussion","content":"\u003cp\u003eThe therapeutic benefits of many medicinal plants are often attributed to their antioxidant properties. Tea (\u003cem\u003eCamellia sinensis\u003c/em\u003e), the world\u0026rsquo;s most widely consumed beverage, has been extensively studied in the past few years for its antioxidant and radical scavenging activities. The unique taste and flavor of tea can be attributed to its composition of color and flavor-generating compounds, such as dietary polyphenols, benzotrpolone compounds (theaflavins, thearubigins), tea catechins such as epigallocatechin-3-gallate (EGCG), epigallocatechin (EGC), epicatechin-3-gallate (ECG), epicatechin (EC), methylxanthines (such as caffeine, theobromine, theophylline), and some amino acids [\u003cspan additionalcitationids=\"CR15 CR16\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. However, it has been reported that the bioaccessibility of these active components is often suboptimal to produce the desired response from conventional tea consumption [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. In this context, increasing the nutraceutical value of tea while maintaining its unique and original organoleptic properties is a promising product diversification strategy in the tea manufacturing industry [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe present study investigated the nutraceutical properties of an underutilized edible plant material, MF, and its potential for use as a key ingredient in the development of a novel herbal tea formulation (MFT-40) with enhanced functional properties.\u003c/p\u003e \u003cp\u003e \u003cb\u003e3.1 Extraction yields, total phenol and total flavonoid contents of ethanol and water extracts of MF sepals\u003c/b\u003e \u003c/p\u003e \u003cp\u003eEdible herbs possess a wide array of phenolic substances and are reported to have many useful functional and nutraceutical properties. Many health challenges that we are facing today are directly or indirectly connected with oxidative stress. Phenolic compounds act as antioxidants due to their capacity to scavenge free radicals. In this context, dietary phenol-rich edible plants have great value in the development of novel nutraceutical formulations.\u003c/p\u003e \u003cp\u003eThe total phenolic and flavonoid contents of the hot and cold ethanol extracts of native and defatted MF sepals were analyzed. The extraction yields of the three ethanol extracts and their total phenol and flavonoid contents are shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e01\u003c/span\u003e.\u003c/p\u003e \u003cp\u003eThe extraction yields of the three ethanol extracts ranged from 15.88\u0026ndash;46.71% w/w dry matter. The hot ethanol extract of defatted MF sepals (defatted HEMF) had a greater extraction yield than the other two extracts. This inferred that the prior defatting process and higher solvent temperatures positively affected the extraction yield. The total phenolic content (TPC) of each extract was measured using Folin\u0026ndash;Ciocalteu reagent. The results were derived from a calibration curve (y\u0026thinsp;=\u0026thinsp;0.0978x\u0026thinsp;+\u0026thinsp;0.0443; R\u0026sup2; = 0.9922) of gallic acid (0\u0026ndash;10 ppm final concentration) and expressed in gallic acid equivalents (GAE) per gram dry extract weight.\u003c/p\u003e \u003cp\u003eFlavonoids are polyphenol compounds that have a wide array of therapeutic potential [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e], [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. The flavonoid contents (TFC) of three ethanol extracts were determined using aluminum chloride via a colorimetric method. The results were derived from the calibration curve (y\u0026thinsp;=\u0026thinsp;0.0444x\u0026thinsp;+\u0026thinsp;0.2065; R\u0026sup2; = 0.9835) of quercetin (0\u0026ndash;25 \u0026micro;g/mL) and expressed in quercetin equivalents (QE) per gram dry extract weight.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 01\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eExtraction yield, total phenolic content (as gallic acid equivalents), total flavonoid content, DPPH scavenging activity (as IC\u003csub\u003e50\u003c/sub\u003e \u0026micro;g/ml) and α amylase inhibitory activity (as IC\u003csub\u003e50\u003c/sub\u003e \u0026micro;g/ml) of ethanol extracts of MF sepals.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eType of extract/\u003c/p\u003e \u003cp\u003eStandard Compound\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e(%)\u003c/p\u003e \u003cp\u003eExtraction yield\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTotal phenolic content (TPC)\u003c/p\u003e \u003cp\u003emg (GAE)/g\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eTotal flavonoid contents (TFC)\u003c/p\u003e \u003cp\u003emg QE/g\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eDPPH radical\u003c/p\u003e \u003cp\u003escavenging activity\u003c/p\u003e \u003cp\u003eEC\u003csub\u003e50\u003c/sub\u003e (\u0026micro;g/ml)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eα amylase inhibitory activity (as IC 50 \u0026micro;g/ml)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCold ethanol extract of native MF sepals (native CEMF)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e26.33\u0026thinsp;\u0026plusmn;\u0026thinsp;0.16\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e108.05\u0026thinsp;\u0026plusmn;\u0026thinsp;0.67\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e64.29\u0026thinsp;\u0026plusmn;\u0026thinsp;0.69\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e25.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12\u003csup\u003ef\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCold ethanol extract of defatted MF sepals (defatted CEMF)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e15.88\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e93.87\u0026thinsp;\u0026plusmn;\u0026thinsp;0.56\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e61.63\u0026thinsp;\u0026plusmn;\u0026thinsp;0.17\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e26.37\u0026thinsp;\u0026plusmn;\u0026thinsp;0.23\u003csup\u003ee\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHot ethanol extract of defatted MF sepals (defatted HEMF)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e46.71\u0026thinsp;\u0026plusmn;\u0026thinsp;0.23\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e114.42\u0026thinsp;\u0026plusmn;\u0026thinsp;0.56\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e66.47\u0026thinsp;\u0026plusmn;\u0026thinsp;0.31\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e24.06\u0026thinsp;\u0026plusmn;\u0026thinsp;0.16\u003csup\u003eg\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e87.61\u0026thinsp;\u0026plusmn;\u0026thinsp;0.19\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAscorbic acid *\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.625\u0026thinsp;\u0026plusmn;\u0026thinsp;0.020\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAcarbose\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e12.35\u0026thinsp;\u0026plusmn;\u0026thinsp;0.047\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"6\"\u003e\u003csup\u003eThe values represent the means \u0026plusmn; standard deviations of 3 replicates. The values indicated by different superscript letters in the same column are significantly different at P\u0026lt;0.05. GAE\u0026minus;gallic acid equivalent, QE\u0026minus;quercetin equivalent\u003c/sup\u003e\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eThe highest TPC was 114.42 mg (GAE)/g from defatted HEMF, and the lowest TPC was 93.87 mg (GAE)/g from defatted CEMF extract. A similar pattern of results was observed for the total flavonoid contents of the three extracts. The highest total flavonoid content (64.29 mg QE/g) was detected in the defatted HEMF, and the lowest TFC (61.63 mg QE/g) was detected in the defatted CEMF extract. The highest total phenolic and flavonoid contents in defatted HEMF may be attributed to its high extraction yield.\u003c/p\u003e \u003cp\u003eWater extracts of MF sepals were prepared at three different temperatures, 60, 70 and 80\u0026deg;C, with or without ultrasonication, and their phenolic and flavonoid contents are given in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e02\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 02\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eTotal phenolic content (as gallic acid equivalents), total flavonoid content, DPPH scavenging activity (as IC\u003csub\u003e50\u003c/sub\u003e \u0026micro;g/ml) and α amylase inhibitory activity (as IC \u003csub\u003e50\u003c/sub\u003e \u0026micro;g/ml) of water extracts of MF sepals\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eType of extract/\u003c/p\u003e \u003cp\u003eStandard Compound\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTotal phenolic content (TPC)\u003c/p\u003e \u003cp\u003emg (GAE)/g\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTotal flavonoid contents (TFC)\u003c/p\u003e \u003cp\u003emg QE/g\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eDPPH radical\u003c/p\u003e \u003cp\u003escavenging activity\u003c/p\u003e \u003cp\u003eIC\u003csub\u003e50\u003c/sub\u003e (\u0026micro;g/ml)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003ea-amylase inhibitory activity (as IC 50 \u0026micro;g/ml)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWater extract of MF sepals at 60 \u003csup\u003eo\u003c/sup\u003eC, Without sonication (WMF-60)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e74.51\u0026thinsp;\u0026plusmn;\u0026thinsp;0.57\u003csup\u003ee\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e41.51\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e33.33\u0026thinsp;\u0026plusmn;\u0026thinsp;0.48\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWater extract of MF sepals at 60 \u003csup\u003eo\u003c/sup\u003eC, With sonication (WMF-S60)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e86.35\u0026thinsp;\u0026plusmn;\u0026thinsp;0.36\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e41.88\u0026thinsp;\u0026plusmn;\u0026thinsp;0.17\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e29.84\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWater extract of MF sepals at 70 \u003csup\u003eo\u003c/sup\u003eC, Without sonication (WMF-70)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e80.75\u0026thinsp;\u0026plusmn;\u0026thinsp;0.84\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e42.83\u0026thinsp;\u0026plusmn;\u0026thinsp;0.32\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e32.23\u0026thinsp;\u0026plusmn;\u0026thinsp;0.27\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWater extract of MF sepals at 70 \u003csup\u003eo\u003c/sup\u003eC, With sonication (WMF-S70)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e92.95\u0026thinsp;\u0026plusmn;\u0026thinsp;0.97\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e45.51\u0026thinsp;\u0026plusmn;\u0026thinsp;0.54\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e28.48\u0026thinsp;\u0026plusmn;\u0026thinsp;0.63\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWater extract of MF sepals at 80 \u003csup\u003eo\u003c/sup\u003eC, Without sonication (WMF-80)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e90.22\u0026thinsp;\u0026plusmn;\u0026thinsp;0.96\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e42.97\u0026thinsp;\u0026plusmn;\u0026thinsp;0.30c\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e30.21\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWater extract of MF sepals at 80 \u003csup\u003eo\u003c/sup\u003eC, With sonication (WMF-S80)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e102.12\u0026thinsp;\u0026plusmn;\u0026thinsp;0.57\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e47.06\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e27.04\u0026thinsp;\u0026plusmn;\u0026thinsp;0.30\u003csup\u003ee\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e133.09\u0026thinsp;\u0026plusmn;\u0026thinsp;0.98\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAscorbic acid *\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.625\u0026thinsp;\u0026plusmn;\u0026thinsp;0.020\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAcarbose\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e12.35\u0026thinsp;\u0026plusmn;\u0026thinsp;0.047\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"5\"\u003e\u003csup\u003eThe values represent the means \u0026plusmn; standard deviations of 3 replicates. The values indicated by different superscript letters in the same column are significantly different at P\u0026lt;0.05. GAE\u0026minus;gallic acid equivalent, QE\u0026minus;quercetin equivalent\u003c/sup\u003e\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eBoth the TPC and TFC increased with increasing temperature from 60\u0026deg;C to 80\u0026deg;C. The extract obtained from sonication at 80\u0026deg;C for 30 minutes (WMF-S80) had the highest TPC (102.12\u0026thinsp;\u0026plusmn;\u0026thinsp;0.57 ppm), TFC (47.06\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06 ppm) and antioxidant activity (IC\u003csub\u003e50\u003c/sub\u003e of 27.04\u0026thinsp;\u0026plusmn;\u0026thinsp;0.30 ppm), and these values were significantly greater than those of the other two extracts prepared at 60\u0026deg;C and 70\u0026deg;C with sonication (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e02\u003c/span\u003e). It was apparent that a high solvent temperature plus sonication improved the extraction of phenolics into water. However, the ethanol extracts had a greater TPC and TFC than did the water extracts. It is not surprising that many phenolic compounds are more soluble in polar organic solvents such as ethanol than in water.\u003c/p\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e3.2 DPPH radical scavenging activity of ethanol and water extracts of MF sepals\u003c/h2\u003e \u003cp\u003eThe free radical scavenging activities of three ethanol extracts, native CEMF, defatted CEMF and defatted HEMF, at increasing concentrations are shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e01\u003c/span\u003e. Ascorbic acid was used as the positive control. The reduction of alcoholic DPPH by all three extracts was considerably high, and the scavenging potential increased with increasing concentrations of the extracts. The greatest DPPH radical scavenging potency with a minimum IC\u003csub\u003e50\u003c/sub\u003e value was recorded for the defatted hot ethanol extract (24.06\u0026thinsp;\u0026plusmn;\u0026thinsp;0.16 ppm). All the data were compared with the IC\u003csub\u003e50\u003c/sub\u003e value of standard ascorbic acid (1.625\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02 ppm), as presented in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e01\u003c/span\u003e. The results demonstrated that the sonication process significantly enhanced the TPC and TFC in the water extracts of the MF sepals, leading to greater antioxidant activity than that of the unsonicated samples (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e02\u003c/span\u003e). Scientific information on the phytochemical composition of MF sepals have not been previously reported. However, it has been reported that the methanol extract of MF leaves contains important phenolic compounds such as flavonoids, hydroxyl benzoic acid derivatives, cinnamic acid derivatives and stilbenes [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. These compounds have been identified to possess good antioxidant properties; therefore, the high DPPH radical scavenging activities of the ethanol extracts of MF sepals may be attributed to the presence of these chemical entities.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003e\u003cb\u003e3.3 Alpha-amylase inhibitory activity of ethanol and water extracts of MF sepals\u003c/b\u003e\u003c/h2\u003e \u003cp\u003eAlpha-amylase is a carbohydrate digestion enzyme that plays a vital role in controlling glucose levels in blood. The inhibition of α-amylase has been identified as a potential approach for controlling diabetes mellitus [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e], [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Dietary polyphenols have been widely studied for their antidiabetic potential, and many such phenolic compounds exert their hypoglycemic effects through the inhibition of carbohydrate digestion enzymes such as α-amylase and α-glucosidase [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. The alpha amylase inhibitory activity of the hot ethanol extract of defatted MF sepals (defatted HEMF) and the hot water extract obtained from sonication at 80\u0026deg;C for 30 minutes (WMF-S80) were determined using the dinitro salicylic acid method. The IC\u003csub\u003e50\u003c/sub\u003e values were calculated by plotting the % α-amylase inhibition as a function of extract concentration (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e \u0026ndash; supplementary information).\u003c/p\u003e \u003cp\u003eDefatted HEMF extract and hot water extract at 80\u0026deg;C (with sonication) showed considerable α-amylase inhibition, with IC\u003csub\u003e50\u003c/sub\u003e values of 87.61\u0026thinsp;\u0026plusmn;\u0026thinsp;0.19 ppm (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e01\u003c/span\u003e) and 133.09\u0026thinsp;\u0026plusmn;\u0026thinsp;0.98 ppm (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e02\u003c/span\u003e), respectively. Acarbose was extracted from commercially available acarbose tablets (Glucobay, 50 mg, Bayer Pharmaceuticals Pvt. Ltd.) was used as the positive control and had an IC\u003csub\u003e50\u003c/sub\u003e value of 12.35\u0026thinsp;\u0026plusmn;\u0026thinsp;0.047 ppm. Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e (supplementary information) comparatively demonstrates the α-amylase inhibitory potentials of acarbose, the WMF-S80 hot water extract and the defatted ethanol extract (defatted HEMF). By comparing these values with the phenolic contents of the two extracts, it can be inferred that phenolic compounds are responsible for the α-amylase inhibitory effects of MF sepals. The hypoglycemic activity of MF sepals has not been previously studied. However, alcoholic extracts of \u003cem\u003eM. macrophyla\u003c/em\u003e (roots) and \u003cem\u003eM. roxburghii\u003c/em\u003e (leaves) have been reported to possess α-amylase and α-glucosidase inhibitory activity [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003e3.4 Cytotoxicity of water extracts of MF sepals using a brine shrimp lethality assay\u003c/h2\u003e \u003cp\u003eMF sepals are well-known edible herbs that are used in the preparation of snacks and porridge. However, as a basic requirement, the water extracts were investigated for any possible toxic effects prior to sensory analysis. The brine shrimp lethality assay is a useful tool for preliminary assessment of toxicity, and all the extracts were subjected to the above assay at concentrations up to 2000 ppm. The lyophilized extracts were dissolved in 1% DMSO in deionized water to obtain the required concentrations. None of the extracts were lethal to brine shrimp even at the highest concentration (2000 ppm) tested after 72 h of exposure.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec23\" class=\"Section2\"\u003e \u003ch2\u003e3.5 Formulation of MF sepal-incorporated tea blends and sensory evaluation to determine the best tea formulation\u003c/h2\u003e \u003cp\u003eSensory analysis is an important component of food quality control. It provides a comprehensive and direct measurement of the perceived intensity of sensory attributes, such as appearance, color, aroma, taste and texture.\u003c/p\u003e \u003cp\u003eConsidering the biochemical potential of MF sepals, a series of MF-tea blends were formulated, and their sensory properties were evaluated. Dried MF sepal powder at different proportions was blended with BOPF-grade tea powder to give rise to three different tea formulations, namely, MFT-30, MFT-40 and MFT-50. BOPF Tea (100%) was used as the control formulation. The compositions of the four formulations are given in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e (supplementary information).\u003c/p\u003e \u003cp\u003eA sensory evaluation (5-point hedonic ranking test; 30 untrained panelists) was conducted to characterize and quantify the sensory qualities of the product. Statistical analysis using the Friedman test revealed significant differences among the four tea samples across all evaluated attributes. Sample 174 (MFT-40) had the highest sum of ranks and mean values for attributes such as appearance, color, aroma, bitterness, astringency, aftertaste, and overall acceptability. Conversely, Sample 253 (MFT-30) displayed the lowest mean values for attributes including appearance, bitterness, astringency, and overall acceptability (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Accordingly, Sample 174, which was formulated with 40% sepal powder (MFT-40), was chosen as the most preferred sample among the panelists for further analysis.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003e3.6 Proximate analysis of MFT-40 and black tea (control) samples\u003c/h2\u003e \u003cp\u003eProximate analysis of tea samples is important for determining their nutritional quality. The moisture content of tea should be between 3 and 5% [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. proximate compositions of the MFT-40 and black tea samples were given in Table S2 (supplementary information). Accordingly, significant differences (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) were observed in all proximate components between the MFT-40 and black tea samples. The determined nutritional values of both tea samples were in the order of crude fat\u0026thinsp;\u0026lt;\u0026thinsp;moisture\u0026thinsp;\u0026lt;\u0026thinsp;ash\u0026thinsp;\u0026lt;\u0026thinsp;crude fiber\u0026thinsp;\u0026lt;\u0026thinsp;crude protein\u0026thinsp;\u0026lt;\u0026thinsp;total carbohydrate. The moisture contents of the MFT-40 sample and black tea sample were 4.88\u0026thinsp;\u0026plusmn;\u0026thinsp;0.100% and 4.30\u0026thinsp;\u0026plusmn;\u0026thinsp;0.020%, respectively. These tea samples also contained moisture contents between the standard ranges. Both tea samples contained high amounts of carbohydrates, with more carbohydrate content in MFT-40 (33.91\u0026thinsp;\u0026plusmn;\u0026thinsp;0.58%) than in black tea (29.45\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06%). The crude fiber content of MFT-40 was 19.38\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07%, and that of the black tea sample was 20.15\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02%. Both tea samples contained relatively similar amounts of fiber. The protein content was slightly greater in the MFT-40 sample (22.14\u0026thinsp;\u0026plusmn;\u0026thinsp;0.23%) than in the black tea sample (21.01\u0026thinsp;\u0026plusmn;\u0026thinsp;0.29%). The ash content provides the amount of minerals present in the food product. According to this analysis, both tea samples contained more than 8% mineral content. The crude fat content of the MFT-40 sample was 3.74\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05%, which was greater than the crude fat content of the black tea sample (2.87\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12%). According to the proximate analysis results, differences between proximate compositions of the black tea and MFT-40 samples were small, but statistically significant.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec25\" class=\"Section2\"\u003e \u003ch2\u003e3.7 Evaluation of the TPC, TFC and antioxidant capacity of lyophilized MFT-40\u003c/h2\u003e \u003cp\u003eBased on the sensory analysis results, 40% of the MF-sepals-incorporated Tea, MFT-40, was selected as the blend with the best organoleptic properties. Therefore, MFT-40 was subjected to TPC, TFC and antioxidant capacity analyses. The 100% Broken Orange Pekoe Fannings (BOPF) grade of black tea was selected as the control.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 03\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eTPC, TFC, and antioxidant capacity of lyophilized MFT-40\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMFT-40\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBOPF Grade Black Tea (Control)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTotal Phenolic Content in mg GAE/g\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e138.82\u0026thinsp;\u0026plusmn;\u0026thinsp;0.21\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e128.47\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTotal Flavonoid Content in mg QE/g\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e77.08\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e69.78\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDPPH Radical Scavenging Activity (IC\u003csub\u003e50\u003c/sub\u003e) in mg/ml\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e12.23\u0026thinsp;\u0026plusmn;\u0026thinsp;0.45\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e18.70\u0026thinsp;\u0026plusmn;\u0026thinsp;0.68\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"3\"\u003e\u003csup\u003eThe values represent the means \u0026plusmn; standard deviations of 3 replicates. The values indicated by different superscript letters in the same row are significantly different at P\u0026le;0.05. GAE\u0026minus;gallic acid equivalent, QE\u0026minus;quercetin equivalent\u003c/sup\u003e\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eLyophilized tea extracts were dissolved in deionized water to obtain the desired concentrations. The TPC, TFC, and antioxidant capacity of MFT-40 and control black tea are shown in Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e03\u003c/span\u003e. The TPC of MFT-40 (138.82\u0026thinsp;\u0026plusmn;\u0026thinsp;0.21 mg GAE/g) was significantly greater (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) than that of black tea (128.47\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13 mg GAE/g). The TFCs of the MF-incorporated tea (MFT-40) and black tea control groups were 77.08\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08 and 69.78\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10 mg QE/g, respectively. Statistical analysis revealed that the TFC of MFT-40 was significantly greater than that of black tea (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003eThe antioxidant capacity of MFT-40 was evaluated using two antioxidant models, namely, the DPPH scavenging assay and the iron reducing power assay. As shown in Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e03\u003c/span\u003e, DPPH scavenging capacity of MFT-40 (IC\u003csub\u003e50\u003c/sub\u003e value of 12.23\u0026thinsp;\u0026plusmn;\u0026thinsp;0.45 ppm) was significantly greater (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) than that of black tea (18.70\u0026thinsp;\u0026plusmn;\u0026thinsp;0.68 ppm). The positive control, ascorbic acid, had an IC\u003csub\u003e50\u003c/sub\u003e of 1.63\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02 \u0026micro;g/ml.\u003c/p\u003e \u003cp\u003eThe reducing capacity of a compound may serve as a significant indicator of its potential antioxidant activity [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. A comparative demonstration of the reducing power of MFT-40, black tea and ascorbic acid is given in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAlthough the reducing power of the two tea extracts increased with increasing concentration, the values remained lower than that of ascorbic acid. The mean absorbance\u0026thinsp;\u0026plusmn;\u0026thinsp;SD (at 700 nm) of ascorbic acid, MET-40 and black tea at 200 ppm was 2.94\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05, 1.33\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03, and 1.02\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05, respectively. The results clearly indicated that the incorporation of \u003cem\u003eMussanda\u003c/em\u003e sepals into black tea enhances the reducing power of black tea.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec26\" class=\"Section2\"\u003e \u003ch2\u003e\u003cb\u003e3.8 Evaluation of the antidiabetic activity of MFT-40 by an alpha-amylase inhibition assay\u003c/b\u003e.\u003c/h2\u003e \u003cp\u003eThe alpha-amylase inhibition activity of MFT-40 and black tea was investigated. Acarbose extracted from glucobay-50 tablets was used as the positive control. Based on the IC\u003csub\u003e50\u003c/sub\u003e values of the tea samples, significantly greater enzyme inhibition (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) was shown by MFT-40 (104.80\u0026thinsp;\u0026plusmn;\u0026thinsp;0.59 ppm) than by the black tea sample (153.07\u0026thinsp;\u0026plusmn;\u0026thinsp;0.61 ppm). The positive control acarbose had an IC\u003csub\u003e50\u003c/sub\u003e value of 12.35\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05 ppm (Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e04\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 04\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eα-Amylase inhibitory activity of tea samples\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"2\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTea samples\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eIC\u003csub\u003e50\u003c/sub\u003e value of samples (\u0026micro;g/ml)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMFT 40\u003c/p\u003e \u003cp\u003elyophilized extract\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e104.80\u0026thinsp;\u0026plusmn;\u0026thinsp;0.59\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBlack tea\u003c/p\u003e \u003cp\u003elyophilized extract\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e153.07\u0026thinsp;\u0026plusmn;\u0026thinsp;0.61\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAcarbose\u003c/p\u003e \u003cp\u003e(Positive control)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e12.35\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"2\"\u003e\u003csup\u003eThe values represent the means \u0026plusmn; standard deviations of 3 replicates. The values indicated by different superscript letters in the same column\u003c/sup\u003e\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eMany plant extracts, including tea extracts, have been studied for their inhibitory activity against α-amylase, and it has been demonstrated that the main active components that have inhibitory effects are phenolic compounds [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e], [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Previous studies have revealed that \u003cem\u003eMussanda\u003c/em\u003e species are rich in phenolics. In silico studies have shown that dietary polyphenols interact with alpha amylase active site residues, forming H-bonds between the hydroxyl and carbonyl moieties of their structures [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Therefore, our findings suggest that the incorporation of MF sepals into black tea results in a blend of tea with a relatively high phenolic content and increased alpha-amylase inhibitory activity.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec27\" class=\"Section2\"\u003e \u003ch2\u003e3.9 Microbial analysis of MFT-40\u003c/h2\u003e \u003cp\u003eThe total plate count measures the total number of viable microorganisms, including bacteria, yeast, and molds, present in a tea sample. It is an indicator of the overall quality and safety of a product for human consumption. Microbial analysis of MFT-40 for up to three months revealed the presence of very few microbial colonies, indicating that the developed herbal tea was of good quality and not contaminated during processing (Table S3 \u0026ndash; supplementary information).\u003c/p\u003e \u003c/div\u003e"},{"header":"4. Conclusions","content":"\u003cp\u003e \u003cem\u003eM. frondosa\u003c/em\u003e is an underutilized green leafy food source with remarkable nutritional potential. It can be effectively blended with green or black tea to develop new value-added tea formulations that can further fortify the health effects exerted by a cup of traditional tea. Cellular oxidative stress is considered a crucial step in the onset and development of many inflammatory and noncommunicable diseases. \u003cem\u003eM. frondosa\u003c/em\u003e-incorporated tea (MFT-40) has high antioxidant potential and can alleviate cellular oxidative stress, while enhancing its health benefits. MFT-40 possesses palatable organoleptic properties, including taste, flavor and aroma, which we believe are the most important characteristics of a marketable tea product. Toxicity assays, proximate composition analyses and microbiological evaluations confirmed the safety of MFT-40 as a consumer-friendly tea product. Currently, our research team is investigating further biological benefits of MFT-40 and its toxicological effects \u003cem\u003ein vivo\u003c/em\u003e.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"449\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"32.962138084632514%\" valign=\"top\"\u003e\n \u003cp\u003eAbbreviation\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"67.03786191536749%\" valign=\"top\"\u003e\n \u003cp\u003eFull name\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"32.962138084632514%\" valign=\"top\"\u003e\n \u003cp\u003eCEMF\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"67.03786191536749%\" valign=\"top\"\u003e\n \u003cp\u003eCold Ethanol extract of MF sepals\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"32.962138084632514%\" valign=\"top\"\u003e\n \u003cp\u003eDNS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"67.03786191536749%\" valign=\"top\"\u003e\n \u003cp\u003eDinitrosalicylic acid\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"32.962138084632514%\" valign=\"top\"\u003e\n \u003cp\u003eGAE\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"67.03786191536749%\" valign=\"top\"\u003e\n \u003cp\u003eGallic Acid Equivalence\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"32.962138084632514%\" valign=\"top\"\u003e\n \u003cp\u003eHEMF\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"67.03786191536749%\" valign=\"top\"\u003e\n \u003cp\u003eHot Ethanol extract of MF sepals\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"32.962138084632514%\" valign=\"top\"\u003e\n \u003cp\u003eMF\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"67.03786191536749%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003eMussaenda frondosa\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"32.962138084632514%\" valign=\"top\"\u003e\n \u003cp\u003eMFT\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"67.03786191536749%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003eMussaenda frondosa\u0026nbsp;\u003c/em\u003eblended tea\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"32.962138084632514%\" valign=\"top\"\u003e\n \u003cp\u003eQE\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"67.03786191536749%\" valign=\"top\"\u003e\n \u003cp\u003eQuercetin Equivalence\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"32.962138084632514%\" valign=\"top\"\u003e\n \u003cp\u003eTPC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"67.03786191536749%\" valign=\"top\"\u003e\n \u003cp\u003eTotal phenolic Content\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"32.962138084632514%\" valign=\"top\"\u003e\n \u003cp\u003eTFC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"67.03786191536749%\" valign=\"top\"\u003e\n \u003cp\u003eTotal flavonoid Content\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"32.962138084632514%\" valign=\"top\"\u003e\n \u003cp\u003eWMF\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"67.03786191536749%\" valign=\"top\"\u003e\n \u003cp\u003eWater extract of MF sepals\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"32.962138084632514%\" valign=\"top\"\u003e\n \u003cp\u003eWMF-S\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"67.03786191536749%\" valign=\"top\"\u003e\n \u003cp\u003eWater extract of MF sepals -sonicated\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e"},{"header":"Declarations","content":"\u003ch2\u003eCompeting Interests\u003c/h2\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003ch2\u003eEthics waived off statement\u003c/h2\u003e\n\u003cp\u003eThe Sabaragamuwa University of Sri Lanka Research Ethics Committee has confirmed that no ethical approval is required.\u003c/p\u003e\n\u003ch2\u003eInformed Consent\u003c/h2\u003e\n\u003cp\u003eInformed consent was obtained from all individual participants included in the study.\u003c/p\u003e\n\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\n\u003cp\u003eNP conceived the idea, supervised the work and wrote the manuscript. NF designed and carried out experiments. TK supervised the work, wrote and edited the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003ch2\u003eAcknowledgement\u003c/h2\u003e\n\u003cp\u003eThe authors thankfully acknowledge the technical staff of the research laboratory of the Department of Physical Sciences and Technology, Faculty of Applied Sciences, Sabaragamuwa University of Sri Lanka, for providing technical supports.\u003c/p\u003e\n\u003ch2\u003eData Availability\u003c/h2\u003e\n\u003cp\u003eAll data generated or analyzed during this study are included in this article.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003ePilapitiya HMCG, Silva SD, Miyazaki H. Innovative value addition in tea industry: Sri Lanka vs. Japan. 2020;https://doi:10.21203/rs.3.rs-27492/v1.\u003c/li\u003e\n \u003cli\u003eAstalakshmi N, Sundara Ganapathy R.\u0026nbsp;A comprehensive review on the genus: Mussaenda. Int J Pharm Sci Res. 2023;8:534\u0026ndash;541.\u003c/li\u003e\n \u003cli\u003eShimpale VB, Yadav SR, Babu CR. A review of the genus Mussaenda (\u003cem\u003eRubiaceae\u003c/em\u003e) from great Nicobar Island, India, including a new species. Rheedea. 2009;19:53\u0026ndash;57.\u003c/li\u003e\n \u003cli\u003eVidyalakshmi KS, Vasanthi HR. Ethnobotany, Phytochemistry and Pharmacology of \u003cem\u003eMussaenda\u003c/em\u003e Species (Rubiaceae). Ethnobotanical Leaflets. 2008;12:469\u0026ndash;475.\u003c/li\u003e\n \u003cli\u003eShanthi S, Radha R. Antimicrobial and phytochemical studies of \u003cem\u003eMussaenda frondosa\u003c/em\u003e Linn. Leaves. Pharmacognosy Journal. 2020;12:630\u0026ndash;635.\u003c/li\u003e\n \u003cli\u003eJayasinghe U. Antimicrobial activity of some Sri Lankan \u003cem\u003eRubiaceae\u003c/em\u003e and \u003cem\u003eMeliaceae\u003c/em\u003e. Fitoterapia. 2002;73:424\u0026ndash;427.\u003c/li\u003e\n \u003cli\u003eHui CK, Majid NI, Zainol MKM, Mohamad H, Zin ZM. Preliminary phytochemical screening and effect of hot water extraction conditions on phenolic contents and antioxidant capacities of \u003cem\u003eMorinda citrifolia\u003c/em\u003e leaf. Malaysian Applied Biology. 2018;47:13\u0026ndash;24.\u003c/li\u003e\n \u003cli\u003eGadjalova AV, Mihaylova DS. Ultrasound-assisted extraction of medicinal plants and evaluation of their biological activity. Food Res. 2019;3: 530\u0026ndash;536.\u003c/li\u003e\n \u003cli\u003ePerera, MGAN, Soysa SSSBDP, Abeytunga DTU, Ramesh R. Antioxidant and cytotoxic properties of three traditional decoctions used for the treatment of cancer in Sri Lanka. Pharmacogn Mag. 2008;4:172\u0026ndash;181.\u003c/li\u003e\n \u003cli\u003eSumaiyah M, Dalimunthe A. Determination of total phenolic content, total flavonoid content, and antimutagenic activity of ethanol extract nanoparticles of \u003cem\u003eRhaphidophora pinnata\u003c/em\u003e (l.f) schott leaves. RASAYAN Journal of Chemistry. 2018;11(2):505\u0026ndash;510.\u003c/li\u003e\n \u003cli\u003eKeharom S, Mahachai R, Chanthai S. The optimization study of \u0026alpha;-amylase activity based on central composite design-response surface methodology by dinitrosalicylic acid method. Int Food Res J. 2016;23:10\u0026ndash;17.\u003c/li\u003e\n \u003cli\u003eKarim MA, Islam MA, Islam MM, Hosen MJ, Mazumder K, Hasan MNH, Biswas S. Biswas. Evaluation of antioxidant, anti-hemolytic, cytotoxic effects and antibacterial activity of selected mangrove plants (\u003cem\u003eBruguiera gymnorrhiza\u003c/em\u003e and \u003cem\u003eHeritiera littoralis\u003c/em\u003e) in Bangladesh. Clin Phytosci. 2020;6(1):8.https://doi:10.1186/s40816-020-0152-9\u003c/li\u003e\n \u003cli\u003eDharmarathna EKGPU, Liyanawickramasinghea TR, Aruppala ALYH, Abeyrathne EDNS.\u0026nbsp;\u0026nbsp;A study on level of microbiological contamination in made tea as a raw material in commercial tea bagging factory and its workers\u0026rsquo; hand hygiene. Journal of Agriculture and Value Addition. 2018;1:49\u0026ndash;59.\u003c/li\u003e\n \u003cli\u003eParveen A, Qin CY, Zhou F, Lai G, Long P, Zhu M, Ke J, Zhang L. Zhang. The chemistry, sensory properties and health benefits of aroma compounds of black tea produced by \u003cem\u003eCamellia sinensis\u003c/em\u003e and \u003cem\u003eCamellia assamica\u003c/em\u003e. Horticulturae. 2023;9:1253. https://doi.org/10.3390/horticulturae9121253.\u003c/li\u003e\n \u003cli\u003eWong M, Sirisena S, Ng K. Phytochemical profile of differently processed tea: A review. J Food Sci. 2022;87(5):1925-194. https://doi: 10.1111/1750-3841.16137.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eZhang L, Cao QQ, Granato D, Xu YQ, Ho CT. Association between chemistry and taste of tea: A review. Trends Food Sci Technol. 2020;101:139-149.\u003c/li\u003e\n \u003cli\u003eTounekti\u0026nbsp;T, Joubert E, Hern\u0026aacute;ndez I, Munn\u0026eacute;-Bosch S. Improving the polyphenol content of tea. Crit Rev Plant Sci. 2013;32:192\u0026ndash;215.\u003c/li\u003e\n \u003cli\u003eDa Silva Pinto M. Tea: A new perspective on health benefits. Food Res Int. 2013;53: 558-567.\u003c/li\u003e\n \u003cli\u003eGraf BA, Milbury PE, Blumberg JB. Flavonols, Flavones, Flavanones, and Human Health: Epidemiological Evidence. J Med Food. 2005;8:281\u0026ndash;290\u003c/li\u003e\n \u003cli\u003eManasa D, Chandrashekar K, Bhagya N. Rapid in vitro callogenesis and phytochemical screening of leaf, stem and leaf callus of \u003cem\u003eMussaenda frondosa\u003c/em\u003e linn.: a medicinal plant. Asian J Pharm Clin Res. 2017;10:81-86. https://doi:10.22159/ajpcr.2017. v10i6.17527.\u003c/li\u003e\n \u003cli\u003eKaur N, Kumar V, Nayak SK, Wadhwa P, Kaur P, Sahu SK. Alpha-amylase as molecular target for treatment of diabetes mellitus: A comprehensive review. Chem Biol Drug Des. 2021;98: 539\u0026ndash;560.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eKashtoh H, Baek KH. New insights into the latest advancement in \u0026alpha;-amylase inhibitors of plant origin with anti-diabetic effects. Plants. 2023;12:2944. https://doi:10.3390/ plants12162944\u003c/li\u003e\n \u003cli\u003eSun C, Zhao C, Guven EC, Paoli P Gandara JS, Ramkumar KM, Wang S, Buleu F, Pah A, Turi V, Damian G, Dragan S, Tomas M, Khan W, Wang M, Delmas D, Portillo MP, Dar P, Chen L, Xiao J. Dietary polyphenols as antidiabetic agents: Advances and opportunities. Food Frontiers. 2020;1: 18\u0026ndash;44.\u003c/li\u003e\n \u003cli\u003eZou H, Shen S, Lan T, Sheng X, Zan J, Jiang Y, Du Q, Yuan H. Prediction Method of the Moisture Content of Black Tea during Processing Based on the Miniaturized Near-Infrared Spectrometer. Horticulturae. 2022;8(12):1170. https://doi.org/10.3390/ horticulturae8121170\u003c/li\u003e\n \u003cli\u003eMeir S, Akiri B, Kanner J, Hadas S. Determination and involvement of aqueous reducing compounds in oxidative defense systems of various senescing leaves. J Agric Food Chem. 1995;43:1813\u0026ndash;1817.\u003c/li\u003e\n \u003cli\u003eLin D, Xiao M, Zhao J, Li Z, Xing B, Li X, Kong M, Li L, Zhang Q, Liu Y, Chen H, Qin W, Wu, H, Chen S. An overview of plant phenolic compounds and their importance in human nutrition and management of type 2 diabetes. Molecules. 2016;21:1374. https://doi: 10.3390/molecules21101374\u003c/li\u003e\n \u003cli\u003eRiyaphan J, Pham DC, Leong MK, Weng CF. In silico approaches to identify polyphenol compounds as \u0026alpha;-glucosidase and \u0026alpha;-amylase inhibitors against type-II diabetes. Biomolecules. 2021;11:1877. https://doi: 10.3390/biom11121877.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"discover-food","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"discoverfood","sideBox":"Learn more about [Discover Food](https://www.springer.com/44187)","snPcode":"","submissionUrl":"","title":"Discover Food","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Discover Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Antioxidant, Herbal tea, Hypoglycemic, Mussaenda frondosa, Value addition","lastPublishedDoi":"10.21203/rs.3.rs-4602535/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4602535/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e \u003cem\u003eMussaenda frondosa\u003c/em\u003e (MF) is an edible species of the genus \u003cem\u003eMussaenda\u003c/em\u003e (Rubiaceae) that contains a wide array of medicinal compounds. The present study was conducted to evaluate the physicochemical and functional properties of the MF sepals, to develop a novel herbal tea with improved functional properties. Remarkably high antioxidant and α-amylase inhibitory activities were exhibited by water and ethanol extracts of MF, subsequent to their high phenolic and flavonoid contents. None of the extracts showed a toxicity, as evaluated by brine shrimp lethality assay. A tea was formulated by blending different proportions of dry sepals of MF with black tea. The sensory analysis showed a significantly high level of acceptancy for the formula that containing 40% MF (MFT-40) with augmented phenolic contents, antioxidant and hypoglycemic activities. This study revealed the potential use of MF as a source for the development of new functional teas with enhanced health benefits.\u003c/p\u003e","manuscriptTitle":"Development of Mussaenda frondosa sepal infused functional tea with enhanced antioxidant and alpha amylase inhibitory activities","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-08-05 09:55:26","doi":"10.21203/rs.3.rs-4602535/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-08-08T09:41:39+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-08-07T09:29:21+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-08-07T06:57:01+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-08-06T14:03:46+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-08-06T09:54:54+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-08-05T18:56:11+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"106456686091177018717973078767487359579","date":"2024-08-05T03:44:17+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"338175949932990560429513487792575423285","date":"2024-08-01T13:57:55+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"132899291890906687444969435594539649690","date":"2024-08-01T11:46:37+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"69233272292421071051807986814999298021","date":"2024-08-01T05:29:47+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"135166676536088688213175795520969736423","date":"2024-07-31T17:25:41+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"280071972384480620511435742034692731072","date":"2024-07-31T08:55:04+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"8717001466769624236443562125879320314","date":"2024-07-31T08:51:14+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-07-12T05:31:15+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-07-12T05:19:33+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-07-11T05:20:26+00:00","index":"","fulltext":""},{"type":"submitted","content":"Discover Food","date":"2024-06-19T01:53:50+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"discover-food","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"discoverfood","sideBox":"Learn more about [Discover Food](https://www.springer.com/44187)","snPcode":"","submissionUrl":"","title":"Discover Food","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Discover Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"87d6a4e1-dc90-461f-ac24-ba7454c95817","owner":[],"postedDate":"August 5th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2024-10-21T05:38:09+00:00","versionOfRecord":[],"versionCreatedAt":"2024-08-05 09:55:26","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4602535","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4602535","identity":"rs-4602535","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}