Bioactive Compounds and Antioxidant Properties of Banana Inﬂorescence in a Beverage for Maternal Breastfeeding

: Banana inﬂorescence is consumed as a traditional Thai cuisine for milk lactation in maternal breastfeeding. In this study, the inﬂorescence of banana ( Musa x paradisiaca ) was extracted in various solvents to determine the bioactive compounds and antioxidant activity in 2,2 (cid:48) -azino-bis 3-ethylbenzthiazoline-6-sulfonic acid (ABTS) radical scavenging. A suitable extract was developed into a beverage. We compared the results for the amount of total phenolic compounds and the capability of antioxidants obtained in polar and non-polar solvents. The extract in a high-polarity solvent demonstrated high total phenolic compounds and ﬂavonoids. The bioactive compounds of banana inﬂorescence contained β - sitosterol, ﬂavonoids, saponin, and other phenolic compounds such as catechin and isoquercetin. The aqueous extract of banana inﬂorescence was developed to act as a primary beverage ingredient. The beverage containing the aqueous extract of banana inﬂorescence (BAB) exhibited a brownish-yellow color and displayed high acidity and high total phenolic compounds, which are responsible for the antioxidant activity. The food processing of BAB showed no contamination of microbial pathogens. From our results, we concluded that banana inﬂorescence is a beneﬁcial health food supplement for general consumers. Additionally, the beverage provides convenience and an alternative drink for postpartum mothers who breastfeed for their infants.


Introduction
Breastmilk contains all the optimal nutrients for infants. Breastmilk can promote sensory and cognitive development and protect the infant against infectious and chronic diseases. The World Health Organization (WHO) recommends that infants be breastfed for the first six months of life, and up to the second year. Additionally, breastfeeding can reduce infant mortality and common childhood illnesses [1]. However, breastfeeding still faces problems, especially in whether the breast milk quantity is enough for infant growth. Therefore, postpartum mothers often explore herbal and pharmaceutical galactagogues to improve breastfeeding for infants who are breastfed exclusively.
A well-known herb in Thailand for breastfeeding is the banana flower and inflorescence. This has been used in Thai food for postpartum mothers [5]. Natural galactagogues are usually botanical or other food agents influenced by familiarity and local customs [6,7]. The flower of Musa x paradisiaca (M. x paradisiaca) was extracted using various solvents, and these crude extracts contain alkaloids, saponins, glycosides, tannins, flavonoids, and steroids. The extracts also showed anti-oxidant activity, which can contribute towards healthy lactation [8]. Aqueous extracts of M. x paradisiaca flowers were demonstrated to act as a significant galactagogue in rats [9]. In addition, the banana flower flour of Musa x paradisiaca was significantly increased breast milk production [10]. Previous reports showed the potential of bananas in promoting milk production [8][9][10]; thus, in this study, banana inflorescence was extracted to determine the bioactive compounds and antioxidants and develop a maternal beverage for breastfeeding.

Plant Sample and Extraction
Fresh banana inflorescence (Musa x paradisiaca) was purchased from a community enterprise, Chiang Mai Province, Thailand, in February 2020. A voucher specimen (number AI2059) has been deposited at the herbarium of the Center of Excellence in Agricultural Innovation for Graduate Entrepreneur, Maejo University, Chiang Mai, Thailand. The fresh banana inflorescences were rinsed several times with tap water and shade dried at room temperature. Dried plant materials were pulverized to a fine powder using a dry grinder. The dried sample (530.08 g) was extracted successively following maceration with 1.5 L of hexane, ethyl acetate, and ethanol for three days (three times). Each solvent solution was filtered, concentrated by rotary evaporation, and then lyophilized using a freeze dryer to obtain the crude hexane, ethyl acetate, and ethanol extracts of the banana inflorescence. These crude extracts were stored in a refrigerator at 4 • C prior to the determination of the active compounds and biological activities.

Determination of Total Phenolic Compounds of Crude Extracts
The total phenolic contents of the solvent extracts were measured via the Folin-Ciocalteu method. We added 200 µL of different concentrations of the extracts to a test tube, followed by 1000 µL of 10% (v/v) Folin-Ciocalteu's reagent and 800 µL of sodium carbonate (7.5% w/v). The mixture was incubated for 60 min at an ambient temperature. Next, the absorbance was measured at 765 nm using a spectrophotometer (Thermo Scientific TM Evolution 260 Bio). The total phenolic content was expressed as the gallic acid equivalent (GAE) in milligrams per gram of dried extract (mg GAE/g of extract).

Determination of Phenolic Composition
The dried banana inflorescence was assessed and compared to a standard of phenolic compounds (Sigma-Aldrich, USA) using liquid chromatography and mass spectrometry (LC-MS) using an Agilent Technologies 1100 series equipped with a LiChroCART RP-18e column (4.6 × 150 × 5 mm, diameter 5 mm) and diode array detector (Agilent Technologies, Waldbronn, Germany) recording at 270, 330, 350, and 370 nm. The mobile phases were a binary solvent system consisting of acetonitrile (A) and 10 mM ammonium formate at pH 4 (B) (acetonitrile: Sigma-Aldrich, Saint Louis, MO, USA; ammonium formate: Sigma-Aldrich, Darmstadt, Germany). The gradient used was 5 min 100% B, 5 min 20% A, 10 min 20% A, and 40 min 40% A [11]. The sample amount was calculated from the peak area of the chromatogram.

Isolation of Chemical Composition of Banana Inflorescence
We extracted the dried banana inflorescence (1.5 kg) using ethanol (4 L) with maceration at room temperature for 2 days. We repeated this two times, then filtered and evaporated the product using a vacuum evaporator. An ethanolic extract of 35.28 g (2.35%) was then fractionated with a chromatography column (column height 12 cm, diameter 10 cm) with a step gradient of ethyl acetate (EA): hexane (0:10) to EA: hexane (10:0) and MeOH: EA (10:0). The fraction was further separated by a combination of preparative thin layer chromatography (TLC) and compared with a standard.

Determination of Antioxidant Activity
The ABTS radical scavenging activity was determined from the in vitro antioxidant activity according to Re et al. [12]. The ABTS reagent was prepared by mixing 7 mM ABTS (2,2 -azino-bis 3-ethylbenzthiazoline-6-sulfonic acid) with 140 mM potassium persulfate. The mixture was then kept in the dark at room temperature for 16 h to allow the completion of radical generation. The mixture was then diluted with deionised water to give an absorbance of 0.70 ± 0.05 at 734 nm before use. To determine the scavenging activity, 1 mL of the ABTS reagent was mixed with 10 µL of sample, and the absorbance was measured at 734 nm, six minutes after the initial mixing. The scavenging activity of the ABTS radicals was calculated using Equation (1). This experiment was conducted in triplicate. The IC 50 value is the inhibitory concentration at which the ABTS radical scavenged at 50%.

Formulation of Beverage Containing Banana Inflorescence
The dried banana inflorescence was pulverized and then boiled with distilled water at a ratio of 2:10 for 30 min. The discard was separated and the aqueous was formulated as a beverage that consisted of 95% banana inflorescence solution and 5% flavor. The physico-chemical properties of the banana inflorescence beverage were measured for the standardization of the product, including the color, pH, solubility, and the amount of citric acid. The color of the beverage was evaluated with a colorimeter (Color CF-18, China) and determined in terms of the L*, a*, and b* values. The pH was measured using a pH-meter (SI Analytics ® , Mainz, Germany). In addition, the nutritional value and the contamination of the pathogenic microbes were sent for analysis at the Central Laboratory (Thailand) Co. Ltd., Chiang Mai Branch, following an in-house method. The tested beverages were analyzed for pathogens, with the notification of Thai Ministry of Public Health standards (No. 356 Re: Beverages in Sealed Container), such as Coliform, Escherichia coli, Salmonella spp. The methods of analysis for the concerned pathogens complied with the methods specified in the Bacteriological Analytical Manual (BAM) (online) of the U.S. Food and Drug Administration. According to standards of the Thai Ministry of Public Health, the beverage shall be approved by the Food and Drug Administration, as follows: (1) The odor and flavor shall be inherent to the specific characteristics. (2) It shall be free of sediment, except sedimentation naturally occurring from ingredients. (3) The water to be used in production shall be in accordance with qualities or standards. (4) Coliform bacteria shall be found to be less than 2.2 per 100 mL of beverage. (5) It shall be free of Escherichia coli.  7) It shall be free of toxic substances released by microorganisms or other toxic substances in quantities that may be hazardous to health.
The methods of this study were approved by the Research Ethics Committee, Faculty of Nursing, Chiang Mai University, Thailand, with the protocol title: Effectiveness of herb beverage on breast milk production among pre-term mothers and post-cesarean mothers

Data Analyses
The data from the total phenolic content and antioxidant activity were produced in triplicate and expressed as the mean ± standard deviation. The statistical analysis was conducted using the Statistical Package for the Social Sciences (SPSS), version 17.0 for Windows (SPSS Inc., Chicago, IL, USA). Significant differences were examined using independent-samples t-test. The R-square and regression line were performed in Microsoft Excel for Office 365.

Yield and Total Phenolic Compound of Crude Extract
The yields of crude hexane, ethyl acetate, and ethanolic extracts from banana inflorescence were 0.0141%, 0.0093%, and 0.0088%, respectively. The phenolic contents are shown in Table 1. We found that the total phenolic content of the ethanolic extract was two times higher than in the ethyl acetate (EA) extract. The results indicate that the ethanolic extract demonstrated higher antioxidant activity than the EA extract. The IC 50 of the ethanolic extract represented a higher efficiency than the hexane extract, and its concentration was lower than the other extract (Table 1 and Figure 1). The hexane extract was insoluble in the phenolic and ABTS assays. However, the bioactive compound in the hexane extract was determined in another phytochemical study. tochemistry from TLC showed the main component was β-sitosterol, followed by flavonoid and saponin in a gradient of EA:hexane solvents. The compound in the MeOH:EA solvent will be determined in a further study.

Antioxidant Activity
As shown in Figure 1, the crude ethanolic and hexane extracts of banana inflorescence exhibited a concentration-response relationship on the inhibitory effect of ABTS radicals with the presence R 2 as 0.9801 and 0.9686. The ethanolic extract demonstrated a higher efficacy than the ethyl acetate extract.

Physico-Chemical Properties
Based on the above results obtained for the phenolic contents and antioxidant activity, the high-polarity solvents, including ethanol and water, displayed more suitable bioactivity compared with the non-polar solvents. Therefore, water was used instead of ethanol due to the non-toxicity for a maternal beverage and the low cost. The physical and chemical properties of the beverage containing the aqueous extract of banana inflorescence (BAB) are shown in Table 3. We determined the value of the colour, including the lightness (L *), redness (a *), and yellowness (b *). The pH value showed an acidic beverage, with a citric acid content of 0.35. The solid content of BAB was 9.6 °Brix. In addition, the total phenolic content of BAB was a high amount per bottle.

Phenolic Composition of Dried Banana Inflorescence
From LC-MS analysis, we determined that the phenolic compounds of dried banana inflorescence contained gallic acid, isoquercetin, quercetin, rutin, catechin, and tannic acid. Catechin and isoquercetin were the primary phenolic components in the banana inflorescence. These results are displayed in Table 2. Values are expressed as means ± standard deviation.

The Chemical Composition of Banana Inflorescence
Each solvent extract of banana inflorescence was subjected to TLC, which was compared with a standard using a one-dimensional ascending method. All fractions were separated by chromatograms of TLC and depicted in 11 main fractions. The preliminary phytochemistry from TLC showed the main component was β-sitosterol, followed by flavonoid and saponin in a gradient of EA:hexane solvents. The compound in the MeOH:EA solvent will be determined in a further study.

Antioxidant Activity
As shown in Figure 1, the crude ethanolic and hexane extracts of banana inflorescence exhibited a concentration-response relationship on the inhibitory effect of ABTS radicals with the presence R 2 as 0.9801 and 0.9686. The ethanolic extract demonstrated a higher efficacy than the ethyl acetate extract.

Physico-Chemical Properties
Based on the above results obtained for the phenolic contents and antioxidant activity, the high-polarity solvents, including ethanol and water, displayed more suitable bioactivity compared with the non-polar solvents. Therefore, water was used instead of ethanol due to the non-toxicity for a maternal beverage and the low cost. The physical and chemical properties of the beverage containing the aqueous extract of banana inflorescence (BAB) are shown in Table 3. We determined the value of the colour, including the lightness (L *), redness (a *), and yellowness (b *). The pH value showed an acidic beverage, with a citric acid content of 0.35. The solid content of BAB was 9.6 • Brix. In addition, the total phenolic content of BAB was a high amount per bottle.  Table 4 shows the results of the nutritional analysis and the Thai RDIs (recommended daily intakes). The volume of 350 milliliters (mL) per bottle provided energy of 140 kcal, 36 g of carbohydrates, and 70 mg of sodium. Total fat 0 g * Percentage of nutrients for the Thai recommend daily intakes (Thai RDI) for the age of 6 years and up are based on the energy demand of 2000 kcal/day Protein 0 g Values are expressed as means ± standard deviation. * refers to Percentage of nutrients for the Thai recommend daily intakes (Thai RDI) for the age of 6 years and up are based on the energy demand of 2000 kcal/day.

Analysis of Pathogen
The results of the microbial contamination of the banana inflorescence beverage are shown in Table 5. The amount of yeast and mold, Bacillus cereus, and Clostridium perfringens in the beverage were found to be less than 1 CFU (colony forming unit)/mL with Coliform at less than 1.1 MPN (most probable number)/100 mL. In addition, Escherichia coli, Salmonella spp., and Staphylococcus aureus were not found in the product. The results of the microbial analysis demonstrated that the BAB obtained the quality standards for beverages according to the Thai Food and Drug Administration (Thai FDA), Ministry of Public Health.

Discussion
The flower and bract (inflorescence) of Musa x paradisiaca are consumed as Thai medicinal plants, and they are recommended in foods for breastfeeding mothers. Thus, a beverage containing the aqueous extract of banana inflorescence was developed in this study. This beverage provides convenience for working mothers and is intended to increase milk lactation. Mahmood et al. [7] reported that active compounds, such as saponins and tannins, in the Musa flower's aqueous extract demonstrated significant galactagogue properties in rats. Ahmad et al. [13] reported that banana flowers contained tannins, saponins, reducing and non-reducing sugars, sterols, and triterpenes. Banana bracts were found to contain anthocyanins, including delphinidin, pelargonidin, peonidin, and malvidin, which exhibited potent antioxidant and pharmacological activity.
In this study, we extracted banana inflorescence, including the flowers and bracts, to investigate the bioactive compounds. The banana inflorescence extract contained βsitosterol, flavonoids, saponin, and other phenolic compounds in accordance with the above reports. The main phenolic components in banana inflorescence were catechin and isoquercetin.
Polyphenols are one of the largest groups of phytochemicals, and they contain a class of flavonoids. The flavonoids consist of flavones, isoflavones, flavanones, catechins, and anthocyanins. The banana inflorescences are rich in anthocyanins [14] and demonstrated a high total presence of phenolic compounds, especially flavonoid, catechin, and isoquercetin in this study. Regarding the research of Tsopmo, whether flavonoids caused an increase in milk lactation in mothers who were breastfeeding still remains unclear; however, they were demonstrated to protect mothers from oxidative damage, which leads to chronic diseases. The consumption of fruits and vegetables is recommended during both pregnancy and lactation [15].
The efficacy of the antioxidant properties of banana inflorescences was found in polar rather than non-polar solvents. The ethanolic extract of banana inflorescences showed higher phenolic contents and antioxidant activity than the ethyl acetate extract. Michele et al. demonstrated that the total phenolic compounds and flavonoids with antioxidant potential in banana inflorescence were found in high-polarity solvents. The antioxidant activity of banana inflorescence may be due to the total phenolic compounds and flavonoids [16]. Thus, the banana inflorescence plays a beneficial role as a health food supplement for breastfeeding mothers, including postpartum mothers and post-cesarean mothers.
In Thailand, banana inflorescence and flowers are a traditional cuisine to promote lactation in maternal breastfeeding. A clinical study demonstrated that the consumption of banana inflorescence was significantly correlated with increased milk lactation in 36 maternal breastfeeding cases and suggested that flavonoids and saponins were the main active compounds of lactation by acting as dopamine antagonists [17]. However, Thai traditional cuisine containing banana inflorescence requires skill and time for food processing. Therefore, a beverage containing banana inflorescence was developed as an alternative food for breastfeeding mothers. The beverage is ready to drink and has an increased shelf life. The beverage is suitable for pre-term mothers and post-cesarean mothers who desire to breastfeed their infants.
We produced a beverage containing the aqueous extract of banana inflorescence (BAB) and evaluated the physico-chemical properties to standardize the product from the above results. BAB produced a brownish-yellow beverage that displayed high acidity and 392 mg GAE total phenolic compounds per serving size (350 mL/bottle). No contamination of microbial pathogens was seen. Thus, BAB demonstrated the quality standards for beverages according to the Thai FDA. These findings can be used to determine standardization for commercialization. The beverage specification includes the color, pH, and solid and total phenolic contents, which makes it possible to be used in industrial applications. However, further studies might be needed for evaluating the organoleptic properties on subjects and the efficiency of this beverage on breast milk production among pre-term mothers and post-cesarean mothers.

Conclusions
The inflorescence of bananas was extracted to analyze the bioactive compounds and antioxidant activity. In a high-polarity solvent, the extract demonstrated high total phenolic compounds and flavonoids, which are responsible for the antioxidant activity. Therefore, the aqueous extract was selected and produced in a beverage. This beverage, which contained the aqueous extract of banana inflorescence, demonstrated the presence of phenolic compounds and antioxidant activity, which would benefit maternal breastfeeding and the general consumer. Additionally, the beverage provides both convenience and an alternative drink for mothers with both typical and atypical labor, including preterm mothers, postpartum mothers, and post-cesarean mothers.