Antioxidant and Sensory Properties of Herbal Teas Formulated from Dried Moringa (Moringa Stenopetala) and Stevia (Stevia Rebaudiana Bertoni) Leaves

Herbal teas are gaining popularity and acceptance due to their sensory and health benefits. The demand for moringa tea currently increased in Ethiopia due to its nutritional and medicinal values. However, using moringa alone is difficult due to its poor sensory appeal and adding sugar to enhance the sensory has implications for health. The purpose of this study was to optimize the sensory properties (taste and aroma) of formulated herbal teas in addition to evaluating the antioxidant properties of the formulated herbal tea from dried moringa and stevia leaves. Seven moringa-based herbal teas were brewed with stevia ranging from 0 to 35% with five-level (5) and compared for their sensory and antioxidant properties. The moringa tea infusion and commercial green tea were considered as control. The results of sensory analysis showed that herbal tea brewed with 20-35% stevia in the formulation results in  higher sweetness compared to 100%-moringa and green tea. Herbal tea brewed with 20-35% stevia in the formulation results in the highest in antioxidant (DPPH scavenging capacity, ferric reducing power and total antioxidant activities) values comparable to 100%-moringa. This study provides evidence that adding stevia to moringa improves the sensory and antioxidant properties without compromising its health-promoting compounds. Keywords: Moringa, stevia, phenolic content, antioxidant activity, herbal tea, sensory, herbal infusion. DOI: 10.7176/FSQM/102-01 Publication date: November 30 th 2020

sweeteners are considered as best substitutes of cereals and sugar to enhance the sensory apples of moringa. Stevia is assumed as the main substitution of natural sugar, due to the presence of steviol glycosides that has great potential in the food industry as a strategy to reduce sugar consumption (Ahmad & Ahmad, 2018). Partial replacement of sugar with stevia and modifying the sensory appeal of moringa tea has not been studied. In this study, alternative herbal tea development from moringa and stevia leaves blend was investigated for its nutraceutical and sensory properties. The general objective of the study was to evaluate the sensory and antioxidant properties of herbal teas formulated from dried moringa and stevia leaves to optimize the sensory appeal of moringa tea and partially replace sugar with stevia (i.e. natural low caloric sweetener).

MATERIALS AND METHODS 2.1. Sample Collection and Preparation
Fresh moringa and stevia leaves were obtained from Arba Minch and Wondo Genet Agriculture research centers (Ethiopia), respectively. The samples were packed in polyethylene (plastic) bags and transported to Wondo Genet Natural Product Research Laboratory.
The fresh leaves of uniform shape, color and size were selected and subjected to shade drying at ambient temperature for about one week according to Killedar et al., (2017). The leaves were spread thinly on paper-lined wooden trays and protected from direct sunlight to prevent the loss of volatile aroma compounds and also photooxidation The dried samples were separately milled using an electric Blender (Model BLG401, Zhejiang YiLi Tool Co., Ltd., China). The milled samples were sieved using 2 mm sieve size to separate the milled leaves. From the sieved samples, formulations were prepared and kept in an air-tight container and stored at room temperature until further analysis.

Preparation of Herbal Tea
The herbal tea was prepared according to the method developed by Horẑić et al., (2009); formulated samples 2 g were infused in 200 ml boiling water for 5 minutes at 97 o C to mimic normal tea preparation till it becomes an appealing fragrance. Herbal tea was prepared from all formulated herbal teas and controls. The formulated herbal teas were unsweetened using sugar and considered for the analysis in this experiment.

Preparation of Extracts from Herbal Tea
The extract was prepared according to Koh et al., (2009) andMingarro et al., (2003). The decoction was boiled for 5 min at 97 o C as usual for normal tea prepared. The decoction was filtered through a double-layered muslin cloth to get rid of the large particles and filtered through a filter paper (Whatman no.1). The filtered product was then allowed to concentrate at 45 o C for three consecutive days by evaporate excess water and obtain the dried. The extract was weighed and its respective percentage yield was recorded. The crude extract 1 g was dissolved in 50 ml of respective solvent (methanol) to make a stock solution of 20 mg/mL. The prepared stock solution was kept at 4°C in a refrigerator, to serve as the working solution for all the phytochemicals and antioxidant tests.

Descriptive sensory analysis
The descriptive sensory analysis of formulated herbal tea infusions was conducted using trained sensory panels of 9 people. Only panelists who voluntarily accepted and signed a consent form after they have been informed about 3 the nature of study materials and the activity involved were considered. The sensory profiling of the products was performed using generic descriptive analysis method Einstein, (1991). The panelists were trained in two sessions of 3 hrs per day using reference samples for green tea and lexicon terms were developed Lee and Chambers, (2007). Profiles of aroma, appearance tastes, aftertaste and overall evaluation sensory properties of the formulated herbal tea infusions were generated with their definition, reference standard, and anchors (Table 3). The attributes were evaluated on 10-scale anchored with verbal descriptions at extreme ends Munoz and Civille, (1998).
2.6. Antioxidant activities 2.6.1. DPPH radical scavenging activity The 2, 2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity of the herbal extract was determined as described by Brand-Williams et al., (1995). Different concentrations (50 to1000 µg/mL) of the extracts were taken in different test tubes. Freshly prepared DPPH solution (2 mL, 0.06%, and w/v) in methanol was added in each of the test tubes containing 1 mL of the extract. The reaction mixture and the reference standards (ascorbic acid and BHT) were vortexed and left to stand at room temperature in the dark for 30 min. The absorbance of the solutions was measured using a UV-visible spectrophotometer (JANEWAY, 96500, UK) at 520 nm. Methanol (100%) was used as a blank. The ability to scavenge the DPPH radical was calculated using equation 1 Radical scavenging effect (%) = ×100……………………………………………….1 Where Ac = Absorbance of the control; As = Absorbance of the sample The antioxidant activity of the extract was expressed as EC50 and the EC50 value is the concentration in (µg/mL) of extracts that scavenges the DPPH radical by 50%.

Ferric reducing antioxidant power
This assay was carried out according to Safdar et al., (2016). One milliliter of the herbal extract with a concentration of 1 mg/mL was mixed with 2.5 mL sodium phosphate buffer (0.2 M, pH 6.6) and 2.5 mL of 1% potassium ferricyanide. Then the mixture was incubated at 50 o C for 20 min. Trichloroacetic acid (2.5 mL, 10%) was added to the mixture. Finally, 2.5 mL of the supernatant solution was mixed with 2.5 mL of distilled water and 0.5 mL FeCl3 (0.1%) and the absorbance of the solutions was measured using a UV-visible spectrophotometer (JANEWAY, 96500, UK) at 700 nm. The reducing power was expressed as mg of ascorbic acid equivalents/g of dried extract (mg AAE/g) using the calibration curve (y=0.0063x+0.148, R 2 =0.99(p<0.01)).

Total antioxidant activity using phosphomolybdenum assay
The total antioxidant activity of formulated herbal tea was determined by phosphomolybdenum assay according to Prieto et al., (1999). Sample of 0.3 mL of extract (1 mg/ mL) in the solution was mixed with 3 mL phosphomolybdenum reagent (28mM sodium phosphate and 4mM ammonium molybdate in 0.6 M sulphuric acid) in capped test tubes. The samples were incubated for 90 min in a water bath at 95°C. After cooling to room temperature, the absorbance of the solutions was measured using a UV-visible spectrophotometer (JANEWAY, 96500, UK) at 695 nm against a blank (3 mL methanol without plant extract). The total antioxidant activity was expressed as milligram butylated hydroxytoluene equivalent/gram of dried extract (mgBHTE/g) using a calibration curve (y=0.0094x+0.112, R 2 =0.99(p<0.001)).
Evaluation of formulated herbal tea infusions was performed on single products in replicated sessions. The actual product evaluation was done in the Wondo Genet Research center hall room. The formulated herbal tea infusions were presented in a transparent teacup with random three-digit codes. A glass of drinking water was provided for rinsing between tastes.

Statistical Analysis
All data were analyzed using one-way ANOVA with traits as an independent variable. The means were separated using Tukey's HSD test at p<0.05. Principal Component Analysis (PCA) for all numerical results was performed using XLSTAT version 2016.03.30882 (Addinsoft, New York).

RESULTS AND DISCUSSIONS 3.1. Antioxidant Activities of the Formulated Herbal Teas
The antioxidant activities g for formulated herbal tea were determined using DPPH Scavenging activity, Ferric reducing power (FRAP) and total antioxidant activity assay. DPPH Scavenging activity, FRAP and total antioxidant activity measured the hydrogen, electron-donating abilities of primary antioxidants and reduction of Mo (Molybdenum) (VI) to Mo (Molybdenum) (V) in the presence of antioxidant compound and subsequent formation of a green phosphate/ Mo (V) complex at acidic pH and at higher temperature were studied, respectively according to Lim et al., (2007) and presented in Figure 1 and Table 2.

DPPH scavenging activity
The concentration of an antioxidant needed to decrease the initial DPPH concentration by 50% (IC50) is a Food Science and Quality Management www.iiste.org ISSN 2224-6088 (Paper) ISSN 2225-0557 (Online) Vol.102, 2020 4 parameter widely used to measure antioxidant activity (Sánchez- Moreno et al. 1998). The lower the IC50 the higher is the antioxidant activity (Brand-Williams et al., 1995). All the formulated herbal teas had considerable variation and ranged from 0.05 to 0.73 g/ml ( Table 2). The IC50 of herbal teas of TF6, TF7 and TF8 were lower (p<0.05) compared to the infusions of 100% moringa (TF1) ( Table 2). These samples had comparable (p>0.05) IC50 to the references ascorbic acid (AA). All other formulations had higher (p<0.05) IC50 compared to samples of TF6, TF7, and TF8. This is indicative of the samples with a higher level of stevia (25-35%) improved the DPPH Scavenging Activity of the herbal tea formulations. This can be attributed to the high level of phytochemicals in the dried stevia leaves ( Table 2).
As shown from the dose dependence curve for the DPPH radical scavenging activity, the concentration of the sample increased, the percent inhibition of DPPH radical scavenging activity also increased (Figure 1). At a concentration of 1 mg/ml, the scavenging effect of ascorbic acid and formulated herbal tea from dried moringa and stevia, the DPPH radical scavenging decreased in the order of the ascorbic acid, stevia and formulated herbal tea. The DPPH radical scavenging activity (IC50) of formulated herbal tea infusions was higher than the moringa leaves tea infusion. The variations in the results of the present study could be due to the difference in addition to stevia to the moringa.

Ferric reducing antioxidant power (FRAP)
The ferric reducing antioxidant powered (FRAP) of all the formulated herbal tea ranged between 1.01 and 4.24 mg AAE/g with a considerable variation (Table 2). Herbal tea samples with a relatively high level of stevia (TF7 and TF8) had higher (p<0.05) FRAP compared to the 100% moringa (TF1). Whereas the samples with a lower level of stevia (TF2 and TF3) had lower FRAP compared to the 100% moringa, this is due to a stable proteinantioxidant activity interaction, resulting in a decrease or increasing of antioxidant activities (Gallo et al., 2013). Indeed, binding to food proteins may have implications in terms of bioavailability, as the antioxidant capacity of polyphenols can be modified by the presence of proteins (Yildirim-Elikoglu & Erdem, 2018). Herbal teas of TF4, TF5 and TF6 had similar (p>0.05) FRAP to the reference sample 100% moringa ( Table 2). The increased FRAP in the herbal tea formulations that have a higher level of stevia is due to the high level of phytochemicals in the dried stevia leaves (Table 2).

Descriptive Sensory Analysis of the Formulated Herbal Tea
The sensory panels generated twenty-five moringa-stevia herbal tea quality descriptors and their definition, reference standards and anchors are given (Table 3). The aroma and appearance attributes were not affected (p<0.05) by the formulation (Table 4). However, taste attributes (sweetness and bitterness), after-taste attributes (sweet aftertaste) and overall sensory appeal (sweetness and sweet after-taste) properties of the herbal teas were affected (p<0.05) by the formulations. Furthermore, taste attributes (astringency, grassy taste, leafy taste and sour), after-taste attributes (leafy aftertaste, astringent aftertaste, grassy aftertaste, and lingering aftertaste) and overall sensory appeal (tea/herbal aroma) of the herbal teas were not significantly affected (p<0.05) by the formulation (Table 4).
The herbal teas formulated showed considerable variation in sweetness (Table 5). Herbal tea of TF8, TF7, and TF6 was sweeter (p<0.05) compared to reference samples TF1 (100% moringa) and GT (green tea) with no significant difference among them. Herbal teas of TF2, TF3, TF4, and TF5 were lower (p<0.05) in sweetness compared to TF8 (teas with a higher level of stevia) and were sweeter (p<0.05) than 100% moringa. Furthermore, TF2, TF3, TF4, and TF5 herbal teas were lower (p<0.05) in sweetness compared to the sample having a high level of stevia (TF8). This revealed that the addition of stevia to moringa tea infusion improved its sweetness.
The principal component analysis (PCA) of the herbal teas formulated showed that formulations with a relatively high level of stevia TF4, TF5, TF6, TF7, TF8 were aligned with sweetness ( Figure 2). These herbal teas were on the same PC1 quadrant as sweetness. Furthermore, the principal component analysis (PCA) showed that teas with a low level of stevia TF2, TF3, TF1(100% moringa) and GT (green tea) were negatively associated with sweetness. Hence, the addition of stevia in herbal tea is highly associated with the sweetness of the product. The finding of this study is in agreement with Abdo, (2016) that found the sweetness equivalence ratio of stevia with sugar adopted in Ethiopia.
The herbal teas formulated showed considerable variation in bitterness (Table 5). All the formulated herbal teas were less (p<0.05) in bitterness compared to GT (green tea) and the TF1 (100% moringa) was similar (p>0.05) in bitterness with all the formulated herbal teas and green tea (GT). This shows that the addition of stevia to moringa tea infusion does significantly affect the bitterness of the herbal teas in the limit of the study. However, principal component analysis (PCA) of the herbal teas formulated revealed that formulations with relatively high level of moringa TF1 (100% moringa), TF2 and TF3 were aligned with bitterness ( Figure 2). These herbal teas were on the same PC1 quadrant as bitterness. Furthermore, the PCA showed that teas with a high level of moringa TF1, TF2 and TF3 were negatively associated with bitterness. No sweet aftertaste, intense Hence, the presence of high level moringa in herbal tea is highly associated with the bitterness of the product. It has been described that the presence of moringa is responsible for the increasing bitterness.
The herbal teas formulated showed considerable variation in the sweet aftertaste (Table 5). Herbal tea of TF6 TF7 and TF8 had a sweeter aftertaste (p<0.05) compared to reference samples TF1 (100% moringa) and GT (green tea) with no significant difference among them. Herbal teas of TF5 had higher (p<0.05) sweet after taste compared to TF2 and it was similar (p>0.05) in sweet after taste to TF4, TF3 and the controls (TF1 and GT). Herbal teas of TF2 and TF3 had lower (p<0.05) sweet aftertaste compared to TF6, TF7, and TF8 (teas with a higher level of stevia) and were similar (p>0.05) in the sweet aftertaste to TF1 (100% moringa). This also showed that the addition of stevia to moringa results increased the sweet aftertaste of herbal tea infusions.

Conclusions
This study to investigate the sensory and chemical composition of moringa (moringa stenopetala) leave tea with partial replacement of stevia (stevia rebaudiana bertoni) leaves. The result of sensory properties all the formulated herbal teas have the similar in aroma and appearance, especially herbal teas brewed from TF5, TF6, TF7 and TF8 were the most preferred in sweetness and sweetness after taste compared to that of TF2, TF3, TF4, TF1 and GT (Green tea). The blending of moringa and stevia produced herbal teas with the most appealing sensory characteristics as compared with herbal tea brewed from only moringa and green teas which are regularly consumed. The results of the antioxidants show that the herbal tea brewed from 20-35% of stevia had the highest DPPH scavenging, ferric reducing power and total antioxidant activities than the herbal tea brewed from 100% moringa herbal tea infusion. This implies that the formulated teas could have better sensory and nutraceutical benefits. It concludes that using these formulated herbal teas due to their sensory, nutraceutical, and economical advantage to the consumer rather than using moringa alone or with table sugar is evident from this work.
Based on this finding of this study the optimization of aroma and taste of moringa (moringa stenopetala) leave tea with partial replacement of stevia (stevia rebaudiana bertoni) leaves and the characterization chemical composition of dried moringa and stevia leaves, formulation of herbal teas and analyzing of their sensory properties and antioxidant (DPPH scavenging activity, ferric reducing assay power and total antioxidant) activity. However, there is a need to carry out further herbal tea composition profile using GS-MS, HPLC and UPLC to explore the potential chemicals present in the formulated herbal tea. The determination of steviol glycoside, antimicrobial activities, the commercialization and promotion of herbal tea including this product. 5. Acknowledgments: The authors would like to thanks, the Ethiopian Institute of Agricultural Research, for allowing as to pursue the work and financial support and Hawassa University for allowing us the laboratories and other facilities for doing this work. 6. Conflicts of Interests: -The authors declare that there are no conflicts of interests regarding the publication of this paper.