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 IC50 µg/ml) and α amylase inhibitory activity (as IC50 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 EC50 (µg/ml) | α amylase inhibitory activity (as IC 50 µg/ml) |
Cold ethanol extract of native MF sepals (native CEMF) | 26.33 ± 0.16b | 108.05 ± 0.67b | 64.29 ± 0.69b | 25.00 ± 0.12f | - |
Cold ethanol extract of defatted MF sepals (defatted CEMF) | 15.88 ± 0.02c | 93.87 ± 0.56c | 61.63 ± 0.17c | 26.37 ± 0.23e | - |
Hot ethanol extract of defatted MF sepals (defatted HEMF) | 46.71 ± 0.23a | 114.42 ± 0.56a | 66.47 ± 0.31a | 24.06 ± 0.16g | 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 IC50 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 IC50 (µg/ml) | a-amylase inhibitory activity (as IC 50 µg/ml) |
Water extract of MF sepals at 60 oC, Without sonication (WMF-60) | 74.51 ± 0.57e | 41.51 ± 0.10c | 33.33 ± 0.48a | - |
Water extract of MF sepals at 60 oC, With sonication (WMF-S60) | 86.35 ± 0.36c | 41.88 ± 0.17c | 29.84 ± 0.09c | - |
Water extract of MF sepals at 70 oC, Without sonication (WMF-70) | 80.75 ± 0.84d | 42.83 ± 0.32c | 32.23 ± 0.27b | - |
Water extract of MF sepals at 70 oC, With sonication (WMF-S70) | 92.95 ± 0.97b | 45.51 ± 0.54b | 28.48 ± 0.63d | - |
Water extract of MF sepals at 80 oC, Without sonication (WMF-80) | 90.22 ± 0.96b | 42.97 ± 0.30c | 30.21 ± 0.15c | - |
Water extract of MF sepals at 80 oC, With sonication (WMF-S80) | 102.12 ± 0.57a | 47.06 ± 0.07a | 27.04 ± 0.30e | 133.09 ± 0.98a |
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 (IC50 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 IC50 value was recorded for the defatted hot ethanol extract (24.06 ± 0.16 ppm). All the data were compared with the IC50 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 IC50 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 IC50 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 IC50 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 ± 0.21a | 128.47 ± 0.13b |
Total Flavonoid Content in mg QE/g | 77.08 ± 0.08a | 69.78 ± 0.10b |
DPPH Radical Scavenging Activity (IC50) in mg/ml | 12.23 ± 0.45b | 18.70 ± 0.68a |
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 (IC50 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 IC50 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 IC50 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 IC50 value of 12.35 ± 0.05 ppm (Table 04).
Table 04
α-Amylase inhibitory activity of tea samples
Tea samples | IC50 value of samples (µg/ml) |
MFT 40 lyophilized extract | 104.80 ± 0.59b |
Black tea lyophilized extract | 153.07 ± 0.61a |
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).