Kluai Hin (Musa sapientum Linn.) peel as a source of functional polyphenols identified by HPLC-ESI-QTOF-MS and its potential antidiabetic function

To date, information on the polyphenolic composition of Kluai Hin banana peel and pulp and the potential antidiabetic activity of its major active compounds is limited. This study aimed to identify polyphenols in extracts of fresh and freeze-dried Kluai Hin banana peel and pulp (methanol:water; M:W, 80:20 for flavonoids and acetone:water:acetic acid; A:W:A, 50:49:1 for phenolic acids) by RP-HPLC-DAD and HPLC-ESI-QTOF-MS. Additionally, inhibition of α-amylase and α-glucosidase activities was investigated with crude extracts from Kluai Hin banana peel and pulp, and compared with its major polyphenols ((+)-catechin, (−)-epicatechin and gallic acid) and the antidiabetic drug acarbose. (−)-Gallocatechin was the most abundant polyphenol and was detected in all fresh and freeze-dried pulp and peel extracts by RP-HPLC-DAD. Furthermore, unidentified polyphenol peaks of Kluai Hin were further explored by HPLC-ESI-QTOF-MS. The A:W:A fresh peel extract contained more total phenolic content (811.56 mg GAE/100 g) than the freeze-dried peel (565.03 mg GAE/100 g). A:W:A extraction of the fresh and freeze-dried peel of exhibited IC50 values for α-amylase activity 2.66 ± 0.07 mg/ml and 2.97 ± 0.00 mg/ml, respectively, but its inhibitory activity was lower than acarbose (IC50 = 0.25 ± 0.01 mg/ml). Peel extracts inhibited α-glucosidase activity, whereas pulp extracts had no effect. In addition, all standards, except gallocatechin, activated α-amylase activity, while, gallocatechin inhibited α-glucosidase activity better than acarbose. Therefore, we propose a further investigation into the use of Kluai Hin banana peel as a potential functional food for the management of postprandial glycaemic response to reduce diabetes risk and in the management of diabetes with a commercial drug.

Determination of total phenol content. The total phenol content (TPC) was determined according to Sulaiman et al. 24 . Each extracts (500 µl, 1 mg/ml) was oxidized using 1.0 ml of 10% Folin-Ciocalteau reagent (v/v). After that, the reaction mixture was neutralized by 3.0 ml of 7.5% sodium carbonate. The reaction mixture was then incubated at room temperature for 2 h in the dark. Finally, the absorbance was measured at 760 nm using a spectrophotometer and TPC was calculated and expressed as gallic acid equivalent (GAE). α-Amylase inhibition assay. The α-amylase activity was performed with a slight modification according to two different studies(Oboh et al. 14 Tan et al. 25 ). To study the inhibitory activity of banana extracts, 250 µl of each sample (0-10 mg/ml of banana extracts or a positive control, acarbose) were mixed with porcine pancreatic α-amylase (EC 3.2.1.1; 0.5 mg/ml) in 0.02 M sodium phosphate buffer (pH 6.9 with 0.006 M NaCl at 250 µl volume). For the analysis of standard polyphenols, 250 µl of each sample (0-350 µg/ml of standard polyphenols or acarbose) was mixed with 250 µl of porcine pancreatic α-amylase. Consequently, all of the samples were incubated at 37 °C for 10 min. Then, 250 µl of 1% starch solution in the same solution buffer was added to the reaction mixture. Thereafter, the reaction mixture was incubated at 37 °C for 10 min. The reaction was stopped by adding 250 µl of 3,5-dinitrosalicylic acid (DNS) reagent, incubated in a boiling water bath for 5 min, and cooled to room temperature. 10 ml of distilled water was added to reaction mixture and measured at the wavelength of 540 nm using a spectrophotometer. The α-amylase inhibitory activity was expressed as inhibition (%).
where Asample is the absorbance of the mixture of phenolic sample, starch solution, enzyme and DNS colour reagent solution; A control is the absorbance of the mixture of buffer, starch solution and DNS colour reagent without enzyme; A test is the absorbance of the mixture of buffer (instead of the sample), starch solution, enzyme and DNS colour reagent; A blank is the absorbance of the mixture of phenolic sample, starch solution and DNS colour reagent without enzyme. α-glucosidase inhibition assay. The α-glucosidase activity was measured using a method of Oboh et al. 14 and Tan et al. 25 with a slight modification. Firstly, using 0.1 M phosphate buffer (pH 6.9) solution the reaction mixture was prepared as follows; 100 µl of appropriate dilutions of the extracts (0-10 mg/ml) or the standard polyphenols (0-350 µg/ml) and 100 µl of α-glucosidase (EC 3.2.1.20, 1U/mL) were mixed. Then the mixture was incubated at 37 °C for 10 min. Subsequently, 50 µl of 5 mM p-nitrophenyl-a-D-glucopyranoside, prepared with 0.1 M phosphate buffer (pH 6.9) solution was added to the mixture and incubation was repeated at the same conditions. Finally, the absorbances was read at 405 nm. The α-glucosidase inhibitory activity was expressed as a percentage of inhibition and calculated the same as the equation of α-amylase inhibition assay as previously mentioned.
Statistical analysis. In this study, three replicates were conducted in each experiment. The results of enzyme inhibition were expressed as mean ± standard deviation (SD). The data were analysed using paired T-test and significance differences were accepted at p ≤ 0.05. The inhibitory concentration of 50% (IC 50 ) was determined using linear regression analysis. Previous studies investigated the polyphenol content of different kinds of bananas, but their (−)-gallocatechin, (+)-catechin and (−)-epicatechin contents were detected in the pulp but were not quantified [3][4][5][6][7] . In another study, (−)-gallocatechin was identified in fresh Musa Cavendish banana peel (158 mg/100 g dry wt) and pulp (29.6 mg/100 g dry wt) 21 . In agreement with Huang et al. 3 and Someya et al. 21 , the present study revealed that (−)-gallocatechin was the major compound analyzed by RP-HPLC-DAD with higher content. Other polyphenols in Thai banana, KluaiHom, were determined using isocratic HPLC-UV, and ferulic acid was found to be the main insoluble phenolic acid 26 . A local banana that grows in Malaysia (Pisang) was extracted with different solvents and the analysis method 27 . The extraction method and solvent types affected the composition and polyphenol content of bananas. For example, methanol worked better for flavonoids extraction 3 while acetone was better for phenolic acids 5 . The current results indicated that Kluai Hin is a natural source of polyphenols, and its peel contains more phenolics than pulp. In addition, the type of polyphenols obtained from freeze-dried banana extracts was higher than that obtained from fresh banana extracts. It is possible due to freeze-dried process preserved the polyphenol content and antioxidant activity better than fresh 14 .

Results and discussion
In this study, the total phenolic content of crude extracts, expressed as the GAE value (gallic acid equivalent, mg gallic acid/g fresh weight), was evaluated using the Folin-Ciocalteu method (Supplementary material 3). The phenolic content of fresh samples was higher than that of freeze-dried bananas. In contrast, Guine et al. 23 found that freeze-dried bananas preserved the phenolic content better than a heat-treated banana. In this study, the preparation process of freeze-dried samples through 100 mesh sieves probably caused a significant loss of other polyphenols. Thus, the preparation step should be improved for future studies. Furthermore, peel samples contained more total phenolic content than the pulp of Kluai Hin, except in freeze-dried sample extraction with M:W (80:20) (p value < 0.05). The A:W:A (50:49:1) extraction solution presented a higher yield of total phenolic content than the extracted M:W (80:20). Alothman et al. 27 revealed that acetone provided the highest yield of total flavonoid content in banana extracts due to the solubility of the polyphenols when compared with other solvent systems. Therefore, the A:W:A solvent was more effective in extracting Kluai Hin polyphenols.
However, some of the peaks were not identified in the RP-HPLC-DAD chromatogram, and unidentified peaks might be a source of polyphenols. After that, freeze-dried peel extracts, the richest polyphenolic profile  (Table 2).
In another study, the polyphenol composition of plantain pulp and peel were analysed with HPLC-ESI-HR-MS and HPLC-DAD, and hydroxycinnamics was the major compound in pulp, and peel predominantly contained flavonol glycosides 5 . The difference in polyphenol composition might be due to genetic variation, species, area of growth or environmental conditions, harvest time and storage conditions 3,19,28,29 . Although the types and the content of polyphenols found in Kluai Hin banana were different from those in other banana types, due to its rich bioactive components, it is still beneficial to human health.

Samples Extraction solution
Content of polyphenols (mg/100 g FW) www.nature.com/scientificreports/ www.nature.com/scientificreports/ from Kluai Hin were analyzed for their inhibitory activity on digestive enzymes. The α-amylase activity of the extracts was reduced in a concentration-dependent manner ( Table 3).

Gallic acid (−)-Gallocatechin (+)-Catechin (−)-Epicatechin Fresh
The IC50 values of the fresh and freeze-dried peel of banana extracts were 2.66 ± 0.07 mg/ml and 2.97 ± 0.00 mg/ml, respectively. There were approximately 10 times higher than the positive control (0.25 mg/ ml). Another study reported that the inhibitory effect of Kluai Hom Thong (Musa AAA group), Kluai Nam Wa (Musa ABB group) and Kluai Khai (Musa AA group) bananas on α-amylase ranged between 7.22 ± 0.41 mg/ml and 15.5 ± 0.02 mg/ml, and the purified α-amylase inhibitor had similarities to the plant chitinase family 30 . The findings of our study revealed that Kluai Hin shows a stronger inhibitor of α-amylase activity than other types of Thai bananas.
In this study, Kluai HIn peel inhibited α-glucosidase activity; however, pulp did not exhibit any inhibitory activity of α-glucosidase. It ranged from 1.86 ± 0.02 mg/ml to 1.99 ± 0.01 mg/ml or 13 times higher than acarbose (0.14 ± 0.01 mg/ml). Pulp and peel extracts showed lower inhibition of α-amylase and α-glucosidase than the therapeutic inhibitor acarbose. Thus, Kluai Hin can be used as a source of functional food for the prevention of diabetes but not as a therapeutic drug to treat diabetic patients. The result from this study may encourage health-conscious consumers to consume more Kluai Hin bananas.
Furthermore, Kluai Hin consumption probably provides an alternative health benefit similar to another type of Thai banana called Kluai Khai. For example, Leerach et al. 31 reported that the intake of Kluai Khai prevented UVB-induced skin damage in mice. Hence, it possibly provides a low risk of premature skin ageing and carcinomas. In addition, the resistant starch content, the in vitro starch digestibility and physico-chemical properties of flour and starch from Thai bananas, including Kluai Hin, were analysed, and it was stated that four starches from these bananas are possibly suitable for creating healthy food products 32 . Not only Kluai Hin pulp but an active compound from its peel will be used as natural antioxidants in food products [33][34][35] .
Furthermore, several studies reported that there are only polyphenols but not the other secondary metabolites (phytosterols, terpenoids) are explored in a different part of banana (such as flower, pseudostem and peel) 8,17 . These compounds can inhibit enzymes responsible for the α-glucosidase and insulin levels. Additionally, some phytosterols can enhance the glycolytic activities of enzymes in diabetic rats, resulting in hepatic glucose utilization 36 . Thus, Kluai Hin peel probably contains similar types of phytosterols and is capable to control blood glucose levels with a similar mechanism of action. Therefore, the value of local banana, Kluai Hin, will be added and higher utilized for food development instead of discarded as waste. Huang and colleagues 3 reported that (−)-gallocatechin, (+)-catechin and (−)-epicatechin were detected in banana pulp, but no study of α-amylase and α-glucosidase inhibitory effects was performed. Therefore, the effects of (−)-gallocatechin, (+)-catechin, (−)-epicatechin and gallic acid on α-amylase and α-glucosidase inhibitory activities were studied and are presented in Supplementary material 4.
Although authentic standards in this study showed that they were not effective in treating diabetes at the same amount as acarbose, these standards may affect indirect mechanisms of diabetes or other mechanisms 13,16 . For example, gallocatechin exhibited anti-inflammatory activity by decreasing NOS2 in LPS + IFN-γ-treated RAW 264.7 cells 39 . Thus, the pathogenesis of several disorders, including diabetes, could not be caused by inflammation.