Extraction of Mangiferin and Chemical Characterization and Sensorial Analysis of Teas from Mangifera Indica L. Leaves of the Ubá Variety

Mangiferin is present in various parts of Mangifera indica L. and has proven biological activities, such as antioxidant capabilities. The aim of this work was to evaluate the chemical composition of teas prepared from M. indica leaves, their potential use as a source of mangiferin and their total phenolic compounds. Teas were prepared with young and mature leaves of M. indica at three (medicinal plant: solvent) ratios utilizing three different preparation techniques. The mangiferin content was analyzed via high-performance liquid chromatography (HPLC). The tea with the highest mangiferin content was characterized for its total phenolic content and antioxidant activity. The oxidative stability was also evaluated by quantifying mangiferin, total phenolics and antioxidant activity using two preservation treatments for 0, 24 and 48 h. Sensory analysis was performed to measure the acceptance of the tea. The type of leaf, preparation technique and concentration influenced the mangiferin content in the teas. The highest concentration of mangiferin was obtained through decoction at a 5% (w/v) medicinal plant concentration. This tea exhibited stability up to 48 h after preparation under both preservation treatments and provided a positive sensory acceptance for consumers with flavors added. In conclusion, teas made from M. indica leaves have great potential as sources of mangiferin and phenolic compounds.


Introduction
Tea is among the most consumed natural beverages worldwide due to its great benefits [1,2].Chinese medicine, for instance, suggests the ingestion of plant parts in the form of tea for different purposes, such as energy improvement and sedative, stimulant, antihypertensive, stomachic and detoxifying actions, as well as depression treatment and the promotion of longevity [3][4][5].In traditional medicine, mango leaf tea is popularly used for therapeutic purposes to treat influenza, diarrhea and throat disorders [6].Thus, tea is a beverage choice with the potential to be consumed as a coadjuvant in the treatment or prevention of chronic non-communicable diseases [7].
In Brazil, statistics show that in recent years, the consumption of teas prepared from medicinal plants has increased by 16% [8].The growing consumer interest in teas is mainly due to their functional properties, as most of them are sources of bioactive compounds and a myriad of substances with potential benefits for human health.These bioactive compounds include simple phenols and polyphenols, flavonoids, anthocyanins, tannins and others [3,[9][10][11].
Mangifera indica leaves contain phenolic secondary metabolites, including gallic acid, quercetin 3-β-D glucoside, α tocopherol, 3-methyl-gallate, propyl gallate, propyl benzoate (+) catechin, (−) epicatechin, benzoic acid and D-glucose [12,13].Mangiferin (1,3,6,7-tetrahydroxy-2-[(2S,3R,4R,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]xanthen-9-one), of the xanthone group (Figure 1), is a major constituent of the leaves and stem bark of Mangifera indica L. (Anacardiaceae) and has antioxidant, immunomodulatory and anti-inflammatory activities [14].For example, Cuba has a product commercialized as a nutritional supplement named VIMANG ® , whose major component is mangiferin [15].Due to its biological effects as an antioxidant and antiinflammatory agent, it is interesting to consider the development of beverages containing mangiferin to incorporate its functional properties into the diet of individuals with chronic, non-communicable diseases that manifest subclinical inflammation and oxidative stress [16,17].Brazilian sanitary legislation considers teas to be traditional herbal products, which are defined as "plant drugs with medicinal purposes that are prepared by infusion, decoction or maceration with water" [18].This legislation currently allows M. indica extracts to be utilized for the development of beverages enriched with bioactive compounds.Because consumer opinion is important in this scenario, sensory testing is fundamental to gathering knowledge for the launch of appealing beverages with a high level of acceptance.Blind tests and expectation tests are widely used for this purpose [19][20][21].
Research on the use of herbal beverages and medicinal plants has been gaining importance due to their therapeutic and healing properties, low price and availability [22][23][24][25].This is a positive development, considering the vast biodiversity of plants in Brazil and the need for deeper knowledge concerning the potential use of teas as adjuvants in the control and treatment of chronic noncommunicable diseases.
This work aimed to evaluate M. indica leaf tea as a source of mangiferin and total phenolics.The teas were prepared by three techniques at three different concentrations of the medicinal plant.The tea with the highest mangiferin content was chosen for analysis of its antioxidant activity stability and consumer acceptance.The results provide data for future studies with an emphasis on the development of new beverages with health claims and the evaluation of their functional effects.

Plant
Mature leaves (dark green, located far from the branch tips) and young leaves (light green, located at the branch apex) were collected from different M. indica trees (Ubá variety) in the Zona da Mata region, southeast of Minas Gerais State, Brazil.(20°60′ S, 43°06′ W, 183 m).The specimens were Brazilian sanitary legislation considers teas to be traditional herbal products, which are defined as "plant drugs with medicinal purposes that are prepared by infusion, decoction or maceration with water" [18].This legislation currently allows M. indica extracts to be utilized for the development of beverages enriched with bioactive compounds.Because consumer opinion is important in this scenario, sensory testing is fundamental to gathering knowledge for the launch of appealing beverages with a high level of acceptance.Blind tests and expectation tests are widely used for this purpose [19][20][21].
Research on the use of herbal beverages and medicinal plants has been gaining importance due to their therapeutic and healing properties, low price and availability [22][23][24][25].This is a positive development, considering the vast biodiversity of plants in Brazil and the need for deeper knowledge concerning the potential use of teas as adjuvants in the control and treatment of chronic non-communicable diseases.
This work aimed to evaluate M. indica leaf tea as a source of mangiferin and total phenolics.The teas were prepared by three techniques at three different concentrations of the medicinal plant.The tea with the highest mangiferin content was chosen for analysis of its antioxidant activity stability and consumer acceptance.The results provide data for future studies with an emphasis on the development of new beverages with health claims and the evaluation of their functional effects.

Plant
Mature leaves (dark green, located far from the branch tips) and young leaves (light green, located at the branch apex) were collected from different M. indica trees (Ubá variety) in the Zona da Mata region, southeast of Minas Gerais State, Brazil.(20 • 60 S, 43 • 06 W, 183 m).The specimens were identified, and an authentication certificate (No.VIC37611) was issued by the botanical survey of the Federal University of Viçosa herbarium.The leaves were washed and sanitized with chlorine solution (200 mg•mL −1 ) and then dried at 55 ± 2 • C for 38 h in an oven with air circulation (Marconi MA035, Piracicaba, SP, Brazil).The samples were crushed (Marconi Rotor Mill MA090/CFT, Piracicaba, SP, Brazil) to obtain a fine powder, and the resulting "medicinal plant" was packaged in polypropylene bags (Selovac 200B, São Paulo, SP, Brazil).

Preparation of Teas
Eighteen teas were prepared using two types of leaves, three preparation techniques and three (medicinal plant: solvent) ratios (Table 1).The techniques were decoction (boiling for 5 min at 100 • C using direct flame), infusion (letting stand for 5 min in boiling water) and ultrasound (sonication at 26 • C, for 15 min) using UNIQUE ® ultrasonic equipment (model USC1600A).The sonication conditions were as follows: 135 W ultrasonic power, 40 kHz excitation frequency and 3.8 L (300 × 151 × 100 mm) in the water bath.To establish the concentration range of the medicinal plant utilized in this study, individual portions of teas available in the Brazilian market were considered (2.5 g•100 mL −1 ).The samples were filtered (Melitta paper filter N • 4), vacuum packaged (Tecnal pump, TE-058 model) and stored in sealed amber glass bottles.

Physical and Chemical Characterization of Tea
The method that presented the highest concentration of mangiferin was selected to prepare the tea for physical and chemical characterizations, which included analyses of pH (pH meter 966 Plus, BEL), total phenolic content (TP) and antioxidant activity by radical scavenging activity (RSA) as follows.

Total Phenolic (TP) Content
The phenolic compound content was estimated colorimetrically in the aqueous extract (tea) using the Folin-Ciocalteu reagent [28].Aliquots of 100 µL of tea and 1 mL of Folin-Ciocalteu reagent (0.25 N) were mixed and left standing for 3 min.Then, 1 mL of sodium carbonate (1 M) was added, and the reaction was left for 7 min.Next, 7 mL of water was added, and the material was stirred for 2 min.The samples were protected from direct exposure to light and allowed to stand for 30 min at room temperature.The absorbance was measured in a microplate UV/visible spectrophotometer (Term Scientific MultiSkan™ GO) at 726 nm.The results were expressed as milligram equivalents of gallic acid (GAE) per milliliter of sample using a standard curve of gallic acid with concentrations varying from 0.05 to 0.35 mg•mL −1 .

Antioxidant Activity (RSA)
The antioxidant activity was analyzed by the radical scavenging activity method using the 2,2-diphenyl-1-picrilhidrazil-DPPH assay [29].A 100 µL aliquot of the tea was diluted with 900 µL of deionized water.Then, 100 µL of this solution was mixed with 1.5 mL of the DPPH solution at 0.1 M. The mixture was shaken, homogenized, and kept for 30 min in the dark.The absorbance was read using a microplate UV/visible spectrophotometer (ELISA; Scientific MultiSkan™ Term GO) at 517 nm against a blank (100 µL of water and 1.5 mL of 0.1 M solution of DPPH).The data were expressed as the percentage of radical scavenging activity (RSA).The calculation was performed using the following equation: where A is the absorbance.

Oxidative Stability of the Tea
The oxidative stability of the tea was evaluated by variation analysis of the phenolic compound content (Folin-Ciocalteu reagent method), the radical scavenging activity (DPPH test) and the mangiferin content (HPLC) according to the methodologies described in Sections 2.3.2 and 2.3.3.Two preservation conditions were evaluated (cooling at 4 ± 2 • C and room temperature of 20 ± 2 • C) at 0, 24 and 48 h after preparation of the beverage.

Preparation of the Tea
Three samples of tea were prepared with the processed leaf of M. indica and drinking water at a final M. indica concentration of 5% (w/v) using the decoction technique (as described in Section 2.3.1).In two of the samples, artificial flavoring of fennel or orange was added (five drops per 50 mL to each tea).

Sensorial Test
The acceptance of the teas was evaluated by 50 untrained adult panelists of both genders, including university students and officials.Tea samples of 30 mL (55 ± 3 • C) were monadically served in white porcelain cups capped with saucers, each receiving a random 3-digit code number along with the evaluation form and one glass of filtered water.A mixed 9-point hedonic scale (9 = like extremely; 1 = dislike extremely) was used for aroma, flavor and overall impression [30].The sensory analysis was conducted in an appropriately designed and lit room and was approved by the Brazilian Ethics Committee under number 845-894/2014.

Statistical Analysis
The design was completely randomized in a 2 × 3 × 3 factorial model (two types of leaves × three (medicinal plant: solvent) ratios × three extraction methods).All experiments were performed in triplicate.The data were subjected to analysis of variance (ANOVA) followed by the Tukey test.The results were expressed as the mean ± standard deviation.The tea stability over time was analyzed by regression.The results of the acceptance test were evaluated by ANOVA using a randomized block design (RBD), followed by the Tukey test.The significance level for all analyses was 5%.

Ethical Statements
All subjects gave their informed consent for inclusion before they participated in the study.The study was conducted in accordance with the Declaration of Helsinki, and the protocol was approved by the Brazilian Ethics Committee under number 845 894/2014.

Mangiferin Concentration in Teas
Overlaid chromatograms obtained from injections of the M. indica tea samples for the different treatments are shown in Figure 2. Comparison among the chromatograms indicated that the tea made with 5% young M. indica leaves using the decoction technique presented the highest mangiferin concentration.The same figure showed that mangiferin presented a retention time of approximately 7.75 min, which was compatible with the mangiferin standard.
The mangiferin concentration of the teas was directly proportional to the medicinal plant: solvent ratio, with statistically significant differences among the concentrations (Figure 3).For all three tea preparation techniques, the highest concentration of medicinal plant (5%) was the most effective for mangiferin extraction.The mangiferin concentration values found in the teas of M. indica leaves prepared by the three techniques, at the three (medicinal plant: solvent) ratios and for the two types of leaves, are presented in Table 2.
Significant differences were observed in the mangiferin concentrations among the tea preparation techniques.The tea prepared by decoction had the highest values of mangiferin (0.717 ± 0.08 and 0.573 ± 0.01 mg•mL −1 using young and mature leaves, respectively), indicating that the mangiferin extraction by this technique is the most efficient.There was a significant difference in the mangiferin concentrations between the types of leaves for all three concentrations and three techniques (p < 0.05), and the highest concentration of mangiferin was found in the teas prepared with young leaves by decoction.The use of mature leaves favored mangiferin extraction in the tea prepared by infusion.
Mangiferin extraction by ultrasound was less efficient due to the influence of time and temperature and the saturation of this compound [31].Chan et al. [32] reported that young leaves of Camellia sinensis had a higher total phenolic content and antioxidant activity than mature leaves, claiming that the young leaves are characterized by the highest amounts of bioactive compounds and nutrients, which are therefore more extractable.
The mangiferin concentration increased proportionately with the amount of medicinal plant in the teas, as expected.This finding corroborates the results of Zaleta et al. [33], who commented that mangiferin presents low solubility in water and that the teas of M. indica leaves prepared with the highest (medicinal plant: solvent) ratio have the highest contents of this compound in the preparation.
The decoction method was the most efficient for the extraction of mangiferin, which is important considering that the method is simple and commonly used and therefore accessible by the consumer.In this study, a cup (240 mL) of M. indica leaf tea provides approximately 2.88 mg•kg −1 mangiferin, considering a human adult with a 60 kg body weight.Studies with rats utilized mangiferin concentrations that varied from 15 to 30 [34], 10 to 30 and 15 to 25 [35] mg•kg −1 of body weight to obtain anti-diabetes, anti-inflammatory and hepatoprotective effects.Converting the dose used in the mentioned experiments in rats to a dose based on surface area for humans (formula for dose translation based on BSA [36]), the values are 2.4 to 4.9, 1.6 to 4.9 and 2.4 to 4.1 mg•kg −1 respectively, for a 60 kg person.The mangiferin contained in a portion of M. indica leaf tea is within these ranges, suggesting that this tea has potential as a functional beverage.Additionally, there are other bioactive compounds in the human diet that can exert synergistic biologic activity with the phytochemicals contained in M. indica leaf tea, increasing the beneficial health effects.
With respect to toxicity, a study with rats showed that the level of mangiferin required to trigger myocardial infarction is above 1000 mg•kg −1 per day [37].In this study, the mangiferin levels are low, indicating that the tea could be consumed without producing toxic effects.

Physicochemical Characterization of the Tea
Taking into account that decoction was the optimal method for mangiferin extraction, a tea was prepared with young M. indica leaves at 5% concentration using this technique for physicochemical characterization.The results are presented in Table 3.The pH value of the M. indica leaf tea was similar to the result described for green tea (pH = 5.3).Commercial plant-based teas are slightly acidic, with pH values ranging from 4.0 to 6.0 [38].Tannins are found in foods and beverages, especially in green and black tea.They confer acidity and astringency to wine and a variety of drinks [39,40].Although the tannin content was not determined in this study, M. indica has been reported to contain gallotannins [41], suggesting that teas made from its leaves contain certain amounts of these compounds, which may be related to the acidity, astringency and bitterness of these beverages.
Regarding the total phenolic content, the evaluated tea contains high amounts of this phytochemical group and is capable of providing approximately 381 mg of phenolic compounds, expressed as GAE, in 240 mL.Considering that the average human ingestion of phenolic compounds is 1123 mg per day [42], tea consumption can enhance the intake of this phytochemical group by approximately 30%, increasing the antioxidant capacity in the diet.The antioxidant activity of tea, evaluated by radical scavenging activity, was 80%, indicating that the EC 50 value is less than 0.007 µg•µL −1 .EC 50 represents the antioxidant concentration that inhibits 50% of the oxidation reaction under the assay conditions.Some authors have reported that herb infusions have potential applications as sources of total phenolic and antioxidant activity.However, it is inadequate to compare the phenolic contents and antioxidant activity observed in this work with the results obtained in other studies, especially as there have been a limited number of tests performed with M. indica leaves under different preparation techniques and assay conditions [43,44].
The scientific literature contains reports of the beneficial effects of bioactive compounds in teas on the reduction of chronic disease risk.A study demonstrated the suppressing effect of Goishi and green tea at 5% (w/v) on the adipose cell growth of obesity-induced rats [45].Total phenolics are potential agents in the prevention of obesity and diabetes [46].Favorable anti-obesity effects [47] and improvement of cardiovascular disease [48] have been attributed to the total phenolics present in green and black tea extracts.The low intake of bioactive compounds is a risk factor for chronic, non-communicable diseases [49].

Stability
There was no significant variation in the mangiferin concentration and total phenolic content up to 48 h after the preparation of the teas (Figure 4a,b).
Even though there was a negative variation in the antioxidant activity (% RSA) of the teas under both preservation methods at 24 h, the activity was stable at 48 h, exhibiting a value statistically equal to the one found at the initial time (Figure 4c).This variation is observed in processed vegetable raw materials and can be attributed to the influence of the phenolic compound levels [43,50], to the preparation method of teas [51,52], or to interactions associated with other compounds present in M. indica leaf tea, which were not evaluated in this study.

Stability
There was no significant variation in the mangiferin concentration and total phenolic content up to 48 h after the preparation of the teas (Figure 4a,b).Even though there was a negative variation in the antioxidant activity (% RSA) of the teas under both preservation methods at 24 h, the activity was stable at 48 h, exhibiting a value statistically equal to the one found at the initial time (Figure 4c).This variation is observed in processed vegetable raw materials and can be attributed to the influence of the phenolic compound levels [43,50], to the preparation method of teas [51,52], or to interactions associated with other compounds present in M. indica leaf tea, which were not evaluated in this study.
A stability analysis of fermented soy milk-added teas (green and black) reported variations in the content of phenolic compounds and antioxidants influenced by the storage time at 4 °C [53].In A stability analysis of fermented soy milk-added teas (green and black) reported variations in the content of phenolic compounds and antioxidants influenced by the storage time at 4 • C [53].In this study, the tea was stable for up to 48 h after preparation.The oxidative stability of these functional compounds in the tea suggests that the functional properties were preserved during the evaluation period.In industrial terms, this result is a positive factor for the development of new beverages that could be commercialized as sources of phytochemicals.
Microbiological analysis and sensory stability were also performed.For the preparation of tea, at the household level, stability ensures convenience to the consumer.This study suggests that during the first 48 h, M. indica leaf tea does not require refrigeration to preserve its mangiferin content and its antioxidant activity.These results corroborate the results from Rubio-Perez et al. [54], who showed that the antioxidant compounds of green tea blended with apple are stable at room temperature and do not require refrigeration.

Sensory Analysis
The results of the sensory evaluations of aroma, flavor and overall impression showed differences between the natural teas and the flavored teas.The mean overall impression of the natural teas (5.7 ± 1.9) was similar to the results reported for ginseng tea with sugar added [55].Sugar or sweeteners can influence the consumer preference for foods and beverages [56].In terms of health, high sugar consumption has been associated with increased risk of developing chronic diseases [57,58].The teas from M. indica leaves did not include any additional sugar-sweeteners.
The flavor scores for teas from M. indica leaves were similar to the scores for canned green tea [59].The scores for overall acceptability were considerably higher than those in mate and green tea studies [60,61].Mango is accepted by the consumers due to its delicious taste, flavor and aroma.This fruit contains several volatile compounds that contribute to the aroma, which is so strong that it can influence the flavor perception [62,63], which may explain the higher scores for natural M. indica leaf tea.The teas with flavors added presented higher levels of acceptance (p < 0.05) in all evaluated attributes, indicating the positive effect of adding flavoring agents to M. indica leaf tea (Table 4).On the other hand, the tea without flavor added fell essentially in the 'neither like nor dislike' range.Sensory quality is regarded as a marketing and production strategy when it influences food quality and is a key to food perception acceptance by the consumer [60,64].The addition of flavoring agents does not alter the nutritional properties of foods and can improve consumer perception, helping to improve acceptance.There is evidence that when taste and odor are encoded together as flavor, they interact to modify perception [65].This study showed that M. indica leaf tea was well accepted by the consumers, and this acceptance was due to the addition of a flavoring agent.This finding suggests that the development of an M. indica leaf beverage through the addition of an appropriate flavor for sensory improvement is promising.However, these consumer results should be considered preliminary because of the small sample size and the nature of the consumer sample (only students and university officials were used).Further research will need to be conducted to confirm these results.Data are expressed as means ± s.d.a-c p < 0.05.Significant differences between tastes for the same attribute (analysis of variance followed by the Tukey test).Evaluated with a mixed, 9-point hedonic scale (9 = like extremely; 1 = dislike extremely).N = 50.* Teas with the addition of orange or fennel flavors.

Conclusions
This study showed the development of M. indica leaf tea as well as its chemical characterization, stability and sensory acceptance.Our results indicated that M. indica leaf tea can be consumed as a source of bioactive compounds, mainly mangiferin.This tea presented 80% radical scavenging activity, suggesting its potential as an antioxidant beverage.The intake of M. indica leaf tea is a strategy to supply phytochemicals to the human diet that offers an opportunity to develop a new functional beverage.Further studies on the toxicological and biological effects of M. indica leaf tea are required to guarantee the safety of this beverage.

Table 1 .
Preparation methods of the teas from Mangifera indica leaves.

Figure 2 .
Figure 2.Overlaid HPLC chromatograms of the mangiferin peaks obtained from teas of M. indica prepared using young and mature leaves at three different concentrations (5, 2.5 and 1.25 g/100 mL) through decoction (black peaks), infusion (magenta peaks) and ultrasound (blue peaks) techniques.For the chromatographic conditions, see Section 2.3.2.

Figure 3 .Figure 2 .of 13 Figure 2 .
Figure 3. Correlation coefficient and linear regression between the mangiferin content and the medicinal plant: solvent ratio of teas prepared with (a) young; and (b) mature M. indica leaves.

Figure 3 .Figure 3 .
Figure 3. Correlation coefficient and linear regression between the mangiferin content and the medicinal plant: solvent ratio of teas prepared with (a) young; and (b) mature M. indica leaves.

Figure 4 .
Figure 4. Stability of the contents of (a) mangiferin; and (b) total phenolics; and of the (c) radical scavenging activity of teas for both preservation methods (cooling 4 ± 2 °C and room temperature 20 ± 2 °C) at different times (0, 24 and 48 h).* p < 0.05.Significant differences between times for the same temperatures (analysis of variance followed by the Tukey test).

Figure 4 .
Figure 4. Stability of the contents of (a) mangiferin; and (b) total phenolics; and of the (c) radical scavenging activity of teas for both preservation methods (cooling 4 ± 2 • C and room temperature 20 ± 2 • C) at different times (0, 24 and 48 h).* p < 0.05.Significant differences between times for the same temperatures (analysis of variance followed by the Tukey test).

Table 2 .
Mangiferin concentration of teas prepared using two types of M. indica leaves, three preparation methods and three (medicinal plant: solvent) ratios.Data are expressed as the means ± s.d.The differences between values are analyzed with ANOVA followed by Tukey's test.A-D p < 0.05.Concentration of medicinal plant (5%) vs. preparation techniques.a-c p < 0.05.Concentration of medicinal plant (2.5%) vs. preparation techniques.I-III p < 0.05.Concentration of medicinal plant (1.25%) vs. preparation techniques.

Table 3 .
Mean values of the pH, total phenolic content and antioxidant activity of M. indica leaf tea at a 5% (medicinal plant: solvent) ratio.

Table 4 .
Average values of the scores obtained by the sensory analysis of M. indica leaf tea.