Physicochemical, antioxidant, and enzyme inhibitory properties of Bignay ( Antidesma bunius L. Spreng) and Duhat ( Syzygium cumini L.) extracts microencapsulated with β - cyclodextrin

Seasonal tropical fruits with health - promoting properties are commonly dehydrated to preserve their bioactive potential. Despite the commercial and industrial appeal of spray drying, the significant capital outlay may deter small - scale applications associated with small volumes of seasonal fruits. In this study, inclusion complexation using β cyclodextrin (bCD) was applied to ethanolic extracts of two common seasonal fruits, duhat and bignay, and the resulting powders were characterized using spectrophotometric, electron microscopy, colourimetric, and gravimetric methods. Electron microscopy images of microcapsules and changes in FTIR spectra indicate successful encapsulation. Despite having different concentrations of monomeric anthocyanins, phenolic contents and radical scavenging activities, the inclusion complexes possess equal encapsulation efficiencies and retention of bioactive components with antioxidant and enzyme inhibitory activities. The results of this study can be used to design small - scale encapsulation strategies for indigenous, seasonal fruits.


Introduction
Syzygium cumini L. ("Duhat") and Antidesma bunius L. Spreng ("Bignay") are tropical, fruit-bearing trees endemic to South and Southeast Asia. Some varieties of these fruits are found in the wild and possess ethnobotanical importance; cultivated varieties find increasing use in the processing of jams, candies, and wine. Recently, many authors reported the healthpromoting potential for these two fruits arising from anthocyanins and other phenolic compounds. For instance, Quiming et al. (2016) reported that A. bunius fruits have flavonoids and tannins that impart aglucosidase, iron chelating and free radical scavenging activities. The in vivo bariatric potential of S. cumini and A. bunius seed extracts that contain saponins, tannins, polyphenols, phlobatannins, steroids and terpenoids were proven with reduced serum insulin and improve lipid profiles after ingestion (Sampath et al., 2013;Grijaldo et al., 2019).
The fruit extracts can also be encapsulated to extend the availability of the fruits and their bioactive compounds. Although spray drying is a commercially viable encapsulation technique, the costs of capital outlay may limit small-scale processors from using this technology. Thus, less energy-intensive encapsulation techniques, such as inclusion complexes may be more suitable. Inclusion complexes form when hydrophobic molecules replace the water molecules present in the cyclodextrin cavity even in highly aqueous systems (Abarca et al., 2016). However, previous studies suggest that the colourless hemiketal, rather than the reddish forms, are complexed with cyclodextrin and that complexation of cyanidin-3-O-glucoside with αcyclodextrin is not favoured (Fernandes et al., 2013). More recently, however, the capacity of β-cyclodextrin (bCD) to protect crude fruit extracts from gastrointestinal and thermal degradation was demonstrated (Fernandes et al., 2018).
Inclusion complexes prepared with bCD have been reported to stabilize phenolic compounds and anthocyanins present in fruit extracts (Fernandes et al., 2013;Shao et al., 2014;Aguilera et al., 2016) with low to intermediate encapsulation efficiencies (Barba et al., 2015;Abarca et al., 2016). However, data regarding the retention of antioxidant and enzyme inhibitory activities of the inclusion complexes are scarce. Thus, this present investigation aimed to characterize the microcapsules prepared with bCD and fruit extracts and quantify the efficiency of encapsulation in terms of anthocyanins, phenolics and antioxidant activity. The results of this eISSN: 2550-2166 © 2022 The Authors. Published by Rynnye Lyan Resources FULL PAPER study may impact current approaches to developing costeffective food ingredients or health-promoting supplements.

Materials
Duhat "Giant", Accession Number 6375, Pangasinan (1990) and bignay "Jumbo", Accession Number 6328, Batangas (1990) samples were collected from the National Plant Genetics Research Laboratory (NPGRL) of the Institute of Plant Breeding (IPB), University of the Philippines Los Baños (UPLB). Three batches of each fruit were obtained in a single fruiting season (May-July). Fresh fruits (at least 80% maturity index per bunch, i.e., >80% red mature and fully mature) were cleaned of stems and leaves and blanched for 5 mins by immersing in boiling water. Seeds were removed using a fabricated pulper/finisher.

Preparation of bCD-fruit extract inclusion complexes
Based on preliminary tests, 2 hrs of extraction with 80% (v/v) aqueous ethanol were found sufficient to recover anthocyanins and phenolic compounds. Subsequently, each fruit pulp was extracted using those conditions at a ratio of 1:10 (fruit solids: solvent) in a water bath maintained at 40 o C. The extracts were decanted and filtered using a Whatman No. 1 under 33 kPa to 50 kPa vacuum pressure. The fruit extract (5 mL) and 5 g of bCD were placed in a Petri dish and kneaded continuously for 15 mins to make a paste, and subsequently dried at 60°C in a convection oven until constant weight. The dry powder was stored in glass containers and kept in a freezer at -20 o C to minimize any physicochemical change until further analyses.

Water activity, moisture content and colour properties of the inclusion complex powders
Water activity was evaluated for each 1 g powder sample using Novasina LabShift (Lachen, Switzerland). Readings were obtained after the constant temperature was achieved. Moisture content was evaluated using oven drying at 105°C and gravimetric analysis. The colour values of the textured proteins were determined using a Konika Minolta BC-10 colourimeter. The instrument was calibrated with a white tile, and colour values were expressed in CIE-Lab parameters as L* (0 = black and 100 = white), a* (-a/+a denotes greenness or redness), and b* (-b/+b denotes blueness or yellowness). Values were expressed as the mean of the measurements taken from three random surface locations. Chroma and hue angle were determined using equations (1) and (2):

Fourier Transform Infrared (FTIR) analysis of the inclusion complex powders
The Fourier Transform Infrared (FTIR) spectra of bCD and bCD-fruit extract complexes were obtained using an Infrared Fourier Transform spectrometer (ABB model MB 3000, USA). The spectral range used was 485 -8500 cm -1 with a resolution of 0.7 cm -1 . The samples were pelletized before measurement.

Scanning electron microscopy of the inclusion complex powders
Particle morphology was determined using FEI Teneo (FEI Co., Hillsboro, OR) field emission scanning electron microscopy with 2 kV acceleration voltage and Everhart-Thornley secondary electron detector.

Encapsulation efficiency and retention of total monomeric anthocyanins, phenolic content and radical scavenging activity of rehydrated powders relative to extracts
The ethanolic extracts were diluted appropriately and powder samples were rehydrated with distilled water at a ratio of 1:5 mass: volume, vortexed for 60 s and centrifuged at 1066×g for 15 mins. The supernatant fluid was used as a test sample. Equations (3) and (4) were used to calculate individual encapsulation efficiencies (where X = monomeric anthocyanins or phenolics content) and individual retention values (where X = monomeric anthocyanins, phenolics content or antioxidant activity), respectively.
Where X = total monomeric anthocyanins (ppm cyanidin-3-O-glucoside equivalents), total phenolic content (ppm gallic acid equivalent) or antioxidant activity (ppm ascorbic acid equivalent) The total monomeric anthocyanin content of extracts and powder samples was measured using the pH differential method (Flores et al., 2013). The A modified Folin-Ciocalteu method (Flores et al., 2013) was used to measure total phenolics content with modifications. Briefly, 0.5 mL of the test sample was mixed with 0.5 mL of 2 N Folin-Ciocalteu's phenol reagent along with 1 mL aqueous sodium carbonate solution (20%). After 5 mins, 8 mL of distilled water were added. The final mixture was vortexed for 1 min and stored for 60 mins to allow sufficient colour development. The absorbance was read at λ = 765 nm. The TPC was calculated from a standard curve of gallic acid (0-100 ppm). Values were reported as ppm gallic acid equivalent (GAE).
The DPPH radical scavenging activity of the extracts was determined using an adapted method (Dela Cruz and Flores, 2020). Approximately 2 mL of 0.150 mM DPPH methanolic solution were mixed with 2 mL of the extract, ascorbic acid (100 μg/mL) or methanol (blank). The mixture was then vortexed for 15 s and stood for 30 mins at room temperature in the dark before its absorbance was measured at λ = 517 nm. For powders, 50 mg of each sample were mixed with 5 mL of 50 % (w/v) methanol. The solution was vortexed for 10 mins and centrifuged at 1066×g for 15 mins. One millilitre of the supernatant fluid was diluted 5-fold using distilled water and mixed with 1 mL of freshly prepared 0.150 mM DPPH methanolic solution. The resulting solution was stood for 30 mins at room temperature in the dark before its absorbance was measured at λ = 517 nm. A calibration curve was generated with ascorbic acid. The DPPH antioxidant activity was calculated as the percentage rate relative to the blank and reported as ppm ascorbic acid equivalent (AAE).

Alpha-glucosidase inhibitory assay of rehydrated powders relative to extracts
A modified colourimetric method was also used (Flores et al., 2013). Crude extracts were diluted 2-fold, while rehydrated powders were diluted 5-fold. Type I αglucosidase from Saccharomyces cerevisiae (100 μL of 0.35 U/mL in 100 mM phosphate-buffered saline or PBS, pH 6.9) were added to 50 μL of the diluted extracts and incubated at 37°C for 10 mins in a Taitec Personal-10, Tokyo, Japan water bath. Next, 100 μL of 1.5 mM pnitrophenyl-α-D-glucopyranoside (dissolved in 100 mM PBS, pH 6.9) were added and incubated for 20 mins. The reaction was terminated by the addition of 1 mL of 1 M sodium carbonate. Absorbance was read at λ = 400 nm. Results are presented as per cent inhibition of reactants with test sample relative to without.

Statistical analysis
Extractions and preparation of inclusion complexes were conducted twice and the resulting samples were pooled. Means of triplicate measurements were analyzed using PROC TTEST of SAS University Edition (Cary, NC) at α = 0.05.

Results and discussion
Bignay extract contained less monomeric anthocyanin equivalents than Duhat (366 vs. 879 mcg C3G equivalents/g fruit), but the resulting bCD complex was significantly darker with a bluer component than bCD-Duhat (Table 1). These results are consistent with the redder duhat extract and bluish black bignay extract, suggesting different anthocyanin profiles in the fruits.
Morphological examination of bCD showed irregularly shaped particles and sheets of various sizes (Figure 1). In contrast, the inclusion complex particles appear as dented spheres with maximum dimensions of 10 mm. The particles appear different from spray-dried mixtures of bCD and saffron anthocyanin extracts, which morphologically resemble the host material (Ahmad et al., 2018). Simulated molecular interactions between anthocyanins and bCD usually result in flat to globular shapes (Fernandes et al., 2014). Changes in the drying rate and final moisture content between convection and spray drying may modulate the hydrophobic and hydrophilic interactions between the guest and host molecules. The FTIR spectrum of the host material ( Figure 1) shows six characteristic peaks associated with bCD (Xie et al., 2019): O-H stretching (3294 cm -1 ), C-H stretching (2916 cm -1 ), H-O-H bending (1636 cm -1 ), C-C stretching (1149 cm -1 ), antisymmetric C-O-C stretching (1018 cm -1 ) and symmetric C-O-C stretching (941 cm -1 ) that was also associated with skeletal vibration of the α-1,4 glycosidic bond (Gomes et al., 2014). Changes in intensity and shifts in wavenumbers are generally regarded as evidence of inclusion complexation. For instance, the intensity of the peaks observed at 3294, 2916 and 1018 cm -1 was attenuated for both bCD-fruit  (Ahmad et al., 2018;Xie et al., 2019). In contrast, the relative intensity of the peak observed at 941 cm -1 for the mixtures was significantly greater than the native bCD, indicative of changes in the toroid structure of bCD due to the presence of the phenolic compounds. Shifts in wavenumbers were observed for 3294 (bCD-Duhat), 1636 (bCD-Duhat and bCD-bignay), and 1018 cm -1 (bCD-Duhat). Thus, mixtures of bCD and fruit extracts can form inclusion complexes.
The C3G values were comparable to 547 mcg/g bignay and ~200 mcg/g duhat reported in the literature (Jorjong et al., 2015;Branco et al., 2016). However, the phenolic content of bignay and duhat extracts were lower than reported in similar studies (Banerjee et al., 2005;Lim, 2012). This is more likely attributable to the slightly nonpolar extraction solvent and the cultivar used in this study. Ethanol was chosen because it can extract higher concentrations of phenolic compounds that are sparingly soluble in water. Regardless, the radical scavenging activities of the extracts were within the range of 80-100% (Banerjee et al., 2005;Quiming et al., 2016) and the extracts even had greater α-glucosidase Figure 1. Scanning electron microscopy images and FTIR spectra of microcapsules prepared with bCD and fruit extracts of duhat and bignay Figure 2. Retention, encapsulation efficiencies and glucosidase inhibitory activities of extracts and microcapsules prepared with bCD and fruit extracts of duhat and bignay. For each fruit sample, the bars refer to (from left to right): retention of total monomeric anthocyanins (Retention TMAC), retention of total phenolic content (Retention TPC), retention of DPPH radical scavenging activity (Retention AOX), encapsulation efficiency of total monomeric anthocyanins (EE TMAC), encapsulation efficiency of total phenolic content (EE TPC), α-glucosidase inhibitory activity of extracts (Gluc Extrac) and α-glucosidase inhibitory activity of the microcapsules (Gluc caps). eISSN: 2550-2166 © 2022 The Authors. Published by Rynnye Lyan Resources FULL PAPER inhibitory activity than similar samples (20-40%) used in the same study (Quiming et al., 2016). Fruit extracts can thus be incorporated into inclusion complexes without significant loss in enzyme inhibitory activity. This can impact current efforts in controlling postprandial glucose concentrations.

Conclusion
Inclusion complexation was successfully applied to duhat and bignay extracts with high retention of bioactive properties, especially in terms of enzyme inhibition. Results of this study can impact current approaches in microencapsulation that may be commercially feasible but require significant capital outlay. Future studies can be performed to determine the in vitro release profiles of the bioactive host molecules and evaluate the impact of different extraction methods. These may lead to more food and health-promoting applications of anthocyanin-containing inclusion complexes.

Conflict of interest
The authors declare no conflict of interest.