Free radical scavenging, α -amylase, α -glucosidase, and lipase inhibitory activities of metabolites from strawberry kombucha: Molecular docking and in vitro studies

, α-amylase, α-glucosidase


Introduction
Fermented beverages are gaining popularity due to their potential as probiotic beverages, enriched with bioactive compounds, antioxidants, and significant health benefits (Selvaraj and Gurumurthy 2023).One prominent example is kombucha, which has become a focal point in functional food research.Kombucha is a fermented beverage produced using black or green tea as a substrate, along with sucrose and a symbiotic culture of bacteria and yeast (SCOBY) (Jakubczyk et al. 2020).The microorganisms present in SCOBY consist of a mixture of acetic bacteria, lactic acid bacteria, and osmophilic yeast, which, during the fermentation process, generate new compounds from the substrate, leading to the production of various metabolites (Miranda et al. 2022).
Several studies have demonstrated that kombucha exhibits significant therapeutic effects as an antioxidant, anti-inflammatory, anticancer, and antimicrobial agent.Moreover, it possesses the ability to bolster the immune system and prevent various diseases, including diabetes, hypertension, and cardiovascular diseases (Kitwetcharoen et al. 2023).As a fermented beverage, kombucha is a rich source of nutrients and phytonutrients, and thus, it holds the potential for further development (Ferruzzi et al. 2020).The exploration of kombucha's health-beneficial effects as a beverage is ongoing.Additionally, it is noteworthy that kombucha is no longer exclusively derived from tea; it can also be produced using other basic ingredients, such as strawberries.In a systematic review study, strawberries have several functional metabolites as a functional food (Basu et al. 2014).In addition, strawberries also contain various biologically active non-nutrient compounds, mainly represented by polyphenolic phytochemicals (Giampieri et al. 2017).These strawberry phenolics have wide clinical potential for humans as an antioxidant, anti-inflammatory action, and inhibition of metabolic enzymes and receptors, alleviating oxidative stress-related conditions (Afrin et al. 2016).
Recently, kombucha is well-known as a beverage made through the fermentation process of tea and sugar with SCO-BY (Villarreal-Soto et al. 2018).Recent studies have shown that because of fermentation, kombucha drink has anti-inflammatory, antioxidant, antidiabetic, cholesterol-lowering, and hepatoprotective effects (Kapp and Sumner 2019;Júnior et al. 2022).In addition, multiple studies have consistently shown that the chemical properties of fermented beverages are enhanced compared to unfermented beverages (Jafari et al. 2020;Zofia et al. 2020;Değirmencioğlu et al. 2021).Incorporating strawberries (Fragaria ananassa) into a kombucha using the SCOBY fermentation method is expected to increase its bioactive properties.This study aimed to investigate bioactive compounds of strawberry kombucha using liquid chromatography high-resolution mass spectrometry (LC-HRMS) and its effect on the modulation of the immune system related anti-oxidative (DPPH and ABTS), markers of metabolic disorders (enzymes activity: lipase, alpha-amylase, alpha-glucosidase,) through in vitro enzymatic studies and biochemical analysis, as well as molecular docking approach.

Formulation of strawberry kombucha drink (SKD)
Strawberry (Fragaria ananassa; Fragaria X ananassa ssp.ananassa; Integrated Taxonomic Information System -Report and Taxonomic Serial Number: 837344) collected from Sarangan Strawberry Garden, Raya Sarangan Street No.47, Plaosan III, Sarangan, Plaosan District, Magetan Regency, East Java 63361, Indonesia (Google Maps Coordinates = -7.6743967, 111.2308193).Plant species were identified at the Biochemistry and Biomolecular Laboratory, Brawijaya University, Malang, Indonesia.Specimens were collected for future validation.The characteristic of strawberry fruit harvested and used is the fruit derived from the strawberry plant aged 2.5 months and has a chewy-tender texture when held; the skin is dark red, and the stalk is yellowish brown.To maintain its quality, strawberry is kept in a refrigerator with a temperature of 4-8 °C before being fermented into a kombucha using SCOBY.This method adopted the well-established protocol published in other papers (Permatasari et al. 2021(Permatasari et al. , 2022)).
Furthermore, the SKD drink was formulated using 2,000 mL of water, 24 g of strawberry pulp, 300 g of white sugar, 166 g v/v of SCOBY starter solution, and 10 g of SCOBY gel, all contributing to a total volume of 2,500 mL.The production was initiated by boiling 2 L of water (±80 °C) followed by the addition of 300 g of table sugar.The mixture was stirred until homogenous and then added with 24 g of strawberry flesh.After that, the water was stirred until the color turns dark brownish red, turn off the stove heat, cover the pot, and let it cool.The solution was then poured into a sterile 3 L jar along with the SCOBY starter solution and SCOBY gel.A clean gauze was placed over and tied to the bottle; then the bottle was kept in anaerobic conditions at 20-25 °C for 12 days.Right after the fermentation process finished, all beverage samples were kept in a 4-8 °C refrigerator for further studies.

Assay for lipase inhibition (%)
First, 1 mg/mL crude porcine pancreatic lipase (PPL) was solubilized in a 50 mM phosphate buffer, followed by the removal of insoluble materials through centrifugation (12,000 g).The process was then continued with the addition of buffer to the supernatants, resulting in a 10-times dilution.
The potential inhibition of lipase was determined using the method utilized by Permatasari et al. 2022.As much as 100 µL of SKD at all concentrations (50, 100, 150, 200, and 250 µg/mL along with 20 µL 10 mM p-nitrophenyl butyrate were added into the reaction buffer in a clear 96-well microplate and incubated for 10 min at 37 °C.The result was contrasted with the positive control (orlistat).The absorbance values were determined using a microplate reader at 405 nm.The unit of activity was calculated using the yield resulting from a one-minute reaction rate of 1 mol p-nitrophenol at 37 °C.When PPL activity was incubated in the test combination, the reduction percentage was used to calculate the inhibition activity of lipase inhibition.To ensure that the findings of the study are accurate, each sample was verified three times (in triplicate).The inhibitory data were obtained using the equation below: A = Lipase inhibition activity without any inhibitor; Ac = Negative control without any inhibitor; B = Lipase inhibition activity with inhibitor; Bc = Negative control with inhibitor.

Determining the α-amylase inhibition (%)
Diluted SKD (at all concentrations) were incubated for 10 min at room temperature with 500 L of 0.02 M pH 6.9 Na 3 PO 4 buffer, NaCl 0.006 M, and porcine pancreatic amylase 0.5 mg/mL.Then, each mixture in the assay buffer received 500 L of a 1% starch solution.After 10 minutes of incubation at 25 °C, 3,5-dinitrosalicylic acid was added to complete the process (1.0 mL).After 5 minutes in a 100 °C water bath, the test was continued and allowed to cool at 22 °C.The measurements were diluted with distilled water (10 mL) to make them readable within the permissible range (540 nm).Acarbose served as the positive control in this investigation.Enzymes and reagents are included in the reference sample but not the sample itself.This method referred to a similar methodology in Permatasari et al. 2022.

Investigation of α-glucosidase inhibition (%)
1.52 UI/mL α-glucosidase solution was created by combining 1 mg (76 UI) of the enzyme with 50 mL pH 6.9 of phosphate buffer, which was proceeded to be kept at -20 °C.Then, 0.35 mL each of the sucrose (65 mM), maltose solution (65 mM), and SKD (0.1 mL) at 50, 100, 150, 200, and 250 g/mL were added.Prior to adding the α-glucosidase solution (0.2 mL), the system was heated to 37 °C for 5 min and maintained at that temperature for 15 min.In a 100 °C water bath, the system was warmed up for 2 minutes.Acarbose was employed in this investigation as the positive control under identical conditions as SKD.The testing solution (0.2 mL), coloring reagent (3 mL), and α-glucosidase inhibitory test solution were then sequentially combined.After 5 minutes of system warming at 37 °C, the system was then evaluated at 505 nm absorbance.The amount of glucose produced during the response served as a sign of inhibitory activity.

DPPH inhibition activity assay (%)
The determination of antioxidant activity was based on the inhibition of DPPH, referring to Kaur et al. (Kaur et al. 2021).Glutathione (Sigma-Aldrich) was chosen as a positive control.The samples and the control (at all concentrations) were poured into the testing vials, followed by the addition of DPPH reagent (3 mL).The resulting DPPH extract mixture was then left undisturbed (30 min; dark cycle).The change was determined on 517 nm absorbance.The proportion of inhibition of DPPH was expressed and calculated using the following formula: A0 = Absorbance value of blank; A1 = Absorbance value of standard or sample.
The half maximal effective concentration (EC 50 ), a concentration measurement of a sample that results in a 50% reduction in the starting radical concentration, expressed the radical scavenging ability of SKD and GSH.

Quantification of the radical scavenging activity of ABTS (%)
The scavenging capability of 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS) was assessed with the protocol adhering to (Sancho et al. 2013).Potassium persulfate (2.4 mM) and 7 mM ABTS were mixed in a 1:1 ratio to form the stock solution.Aluminum foil was used to block the light from the combination, and it was then left to react for 14 hours at 22 °C.Afterward, 1 mL of ABST stock solution was added with 60 mL of ethanol to get an absorbance of 0.706 at 734 nm.For each test, a brand-new functioning solution was created.After allowing the samples at all concentrations to react for 7 minutes with 1 mL of the ABTS working solution, the absorbance at 734 nm was measured.As a positive control, Trolox was employed.A0 = Absorbance value of blank; A1 = Absorbance value of standard or sample.
The half maximal effective concentration (EC 50 ) is the amount of sample concentration that reduces the concentration of radical levels to 50% reduction in the starting radical concentration, which expressed the radical scavenging ability of SKD and Trolox.

Untargeted metabolomic profiling of strawberry kombucha drink
Testing service at the Central Laboratory of Life Sciences, Brawijaya University, Malang, Indonesia) was utilized to analyze an untargeted metabolomic profile test on SKD using the combination of a high-performance liquid chromatography system with a high-resolution mass spectrometer (LC-HRMS) as described according to the manufacturer's specification (Suppl.material 1).The concentration of 50 μl of SKD was reduced by 30 times using ethanol (96%) and vortexed (at 2,000 rpm for 2 min), followed by centrifugation (at 6,000 rpm for 2 min).The supernatants were accumulated and then filtrated using a 0.22 μm syringe filter prior to analysis.
Thermo Scientific Dionex Ultimate 3000 RSLC Nano High-Performance Liquid Chromatography (HPLC) and a micro-flow meter made up the LC-HRMS system.The analytical column was a Hypersil GOLD aQ with a particle size of 50 × 1 mm × 1.9 maintained at 30 °C, and the solvents A and B are 0.1% formic acid and 0.1% formic acid dissolved in water and acetonitrile, respectively.Next, they were kept apart with a 40 uL/min linear gradient for 30 minutes.HRMS utilized Thermo Scientific Q Exactive which has 70,000 resolution at its full scan capacity, a 17,500 resolution data-dependent MS2, and a 30 minute operating period in positive and negative modes.The successfully identified compounds were then analyzed in silico against the human pancreatic lipase (1LPB), α-glucosidase (2QV4), and α-amylase (3L4Y) enzymes.

Preparing ligands and targets
The compounds that were identified as a constituent of the SKD metabolomic profile were used as test ligands.Chem-Draw Ultra 12.0 was used to sketch the whole structure in 2D, which was then transformed to 3D and optimized using the MM2 algorithm.The selected target proteins were human pancreatic lipase (PDB ID: 1LPB), α-amylase (PBD ID: 2QV4), and α-glucosidase (PDB ID: 3L4Y).All proteins were acquired from the website of Protein Data Bank (https:// www.rcsb.org).Kollman charges were applied to the receptors while the ligands were added with a Gasteiger charge.

Validation of molecular docking
Redocking was used as the molecular docking validation approach.By utilizing AutoDock tools version 4.2, the original ligand was transferred to the target pocket with specific coordinates.After the re-docking method, the ligand position's RMSD (root-mean-square deviation) must be less than 2.0 Å.

Simulation of molecular docking
The docking parameters were developed using the findings of docking validation (Table 1).The outcome was recorded in a *dlg file for each docking's final conformation structure.Analysis was done on the ligand-receptor interaction using Discovery Studio 2016.

Statistical analysis
In the early phase of the study, in vitro data regarding antioxidants (DPPH and ABTS) were analyzed using an unpaired T-test CI 95% with the Windows version of Graph-Pad Prism 9.4.1 software (San Diego, California USA, www.graphpad.com).EC 50 datasets were each acquired from nonlinear regression models.GraphPad Prism 9.4.1 was used to present the graphic visualizations.

Metabolite profile of strawberry kombucha drink (SKD)
A total of 45 compounds were identified in SKD (Table 2).The majority of these compounds exhibited several health benefits, ranging from antioxidant, neuroprotective, and hepatoprotective to hypolipidemic and protection against cardiometabolic risk factors.The identification produced a spectrum that may be compared to those in the database by combining electrospray ionization and Fourier processing (Fig. 1A).An electrospray positive weak peak with 1.80 × 10 6 counts is transformed into an appropriate spectrum (m/z 50-750 Da) (Fig. 1B).Table 1 lists the detected chemicals in detail based on the findings of non-targeted metabolomic profiling using LC-HRMS.

In silico study of α-amylase, α-glucosidase, and lipase inhibitory activities
As shown in Table 2, identified compounds of SKD were validated by computational in silico or molecular docking assays on the enzymes lipase, a-glucosidase, and a-amylase.After the validation process was done, the molecular docking tests against lipase, α-amylase, and α-glucosidase enzymes were performed on 47 compounds (due to the availability of the databases), acarbose (as the control for α-amylase and α-glucosidase), and orlistat (as a control for lipase).This examination found that 1-Methyl-N-{[(2R,4S,5R)-5-(2-methyl-6-phenyl-4-pyrimidinyl)-1-azabicyclo[2.2.2]oct-2-yl]methyl}-4-piperidinamine had the best result based on the ligand test in each receptor when compared with other compounds and controls.This comparison is based on the value of ∆G (kcal/mol), and the results of molecular docking tests were listed in Table 3.

Lipase inhibition activity by SKD
The inhibition of lipase by SKD and orlistat are detailed in Fig. 2. Statistical analysis revealed that orlistat showed a significantly higher inhibition against lipase than SKD at the dose of 50 μg/mL (Fig. 2A).Interestingly, the lipase inhibition activity of SKD at 100, 150, 200, and 250 μg/ mL were equal to orlistat.However, further validation through the analysis of EC 50 values of SKD and orlistat concluded that the lipase inhibition of orlistat is without a doubt stronger than SKD (Fig. 2B).
α-Amylase inhibition activity of SKD Fig. 3 showed the α-amylase inhibition activity by SKD and acarbose.An α-amylase inhibition activity that was equal to acarbose was observed in SKD at 50 and 250 μg/ mL, which are the lowest and highest doses (Fig. 3A).However, at 100, 150, and 200 μg/mL, the α-amylase inhibition property of SKD differs significantly from orlistat.The EC 50 value of SKD was also lower than orlistat, suggesting greater α-amylase inhibition than the positive control (Fig. 3B).

α-Glucosidase inhibition activity by SKD
The antidiabetic property of SKD was compared to acarbose, which was shown in Fig. 4. The potential of SKD in inhibiting α-glucosidase was identical to acarbose at 50, 150, and 250 μg/mL while on the other doses, they differ notably from acarbose (Fig. 4A).The EC 50 value of SKD and acarbose were 15.03 μg/mg and 10.62 μg/mg, respectively (Fig. 4B).

Free radical scavenging activity of SKD
The free radical scavenging activity of SKD was observed through the inhibition of DPPH and ABTS, which were compared against glutathione (Figs 5,6).The statistical approach found that SKD showed a significantly lower DPPH inhibition than glutathione at the dose of 50 and 100 μg/mL (Fig. 5A).Interestingly, the free radical scavenging activity of SKD at 150, 200, and 250 μg/mL was equal to the control.On the other side, the ABTS inhibition activity of all doses of SKD still fell short of the   control, significantly (Fig. 6A).The determination of EC 50 values also revealed that DPPH inhibition by glutathione is notably stronger than SKD (Fig. 5B).The EC 50 values of SKD and glutathione regarding the inhibition of ABTS were 18.52 μg/mg and 19.61 μg/mg, respectively (Fig. 6B).

Discussion
In this study we demonstrated that SKD had 62 potential secondary metabolites that may be beneficial for metabolic health.In addition, we also showed inhibition activity of these metabolites on the activity of α-amylase, α-glucosidase, and lipase at least from in silico modelling.Furthermore, SKD had the ability to inhibit of α-amylase, α-glucosidase, and lipase in vitro.Interestingly, we also showed the free radical scavenging activity of SKD in vitro.
Clinical evidence showed that strawberries promoted health and prevented diseases due to several nutritive and non-nutritive bioactive compounds (Afrin et al. 2016;Miller et al. 2019).This work incorporated strawberries into a kombucha probiotic drink and evaluated its activity in silico and in vitro.The initial work successfully identified 62 secondary metabolites in the strawberry kombucha drink (SKD) (Table 1).These results also highlighted that SKD contained far more metabolites from the strawberry in the absence of fermentation, with a comparison of 62 to 12-20 metabolites (Zhang et al. 2011;Antunes et al. 2019).This is in line with other studies that the fermentation process -especially using kombucha or SCO-BY -can increase bioactive compounds in a food product (Leonard et al. 2021;Permatasari et al. 2022).Furthermore, we confirmed the potential biological significance of these metabolites to the biological properties of SKD using molecular docking (Table 3).Finally, in vitro examination also revealed the potential antiobesity, antidiabetic, and antioxidant activity of SKD.
The capability of SKD in inhibiting lipase was examined in this study.On a weight basis, lipase inhibition activity of SKD at doses of 100, 150, 200, and 250 μg/mL was similar to orlistat, a control that inhibits lipase in obesity (Son and Kim 2020).SKD had an EC 50 value of lipase inhibition of 39.70 μg/ mg (Fig. 2B) while strawberry extract had a reported EC 50 of around 5 μg/mL (McDougall et al. 2009).Lipase inhibition will improve lipid metabolism in obese individuals by reducing the accumulation of fatty acids, maintaining the HDL-to-LDL ratio, and preventing adipocyte growth (Liu et al. 2020).On the other hand, strawberry supplementation also demonstrated health benefit implications in obese adults (Basu et al. 2016).Sustaining a metabolically healthy condition in obesity may also decrease the risk of diabetes, NA-FLD, and metabolic syndrome (Godoy-Matos et al. 2020).
The α-amylase and α-glucosidase inhibition activities of SKD were observed.SKD at doses of 50 and 250 μg/mL showed similar α-amylase inhibition to acarbose while inhibition of α-glucosidase of SKD did not differ significantly from acarbose at doses of 50, 150, and 250 μg/mL.Acarbose is an α-amylase and α-glucosidase inhibitor with the potential as a calorie restriction mimetic and weightloss agent (Smith et al. 2021).SKD showed an EC 50 value of α-amylase inhibition of 5.39 μg/mg while strawberry extract had a reported EC 50 of 96.82-398.46μg/mL (Huneif et al. 2022).The same trend was observed in the inhibition of α-glucosidase, where SKD exhibited an EC 50 value of α-glucosidase inhibition of 15.03 μg/mg while strawberry extract had a reported EC 50 of 117.54-429.39μg/mL (Huneif et al. 2022).The health-promoting effects of strawberries may be contributed to the phenolics and antioxidant compounds contained in strawberries (Giampieri et al. 2012(Giampieri et al. , 2017)).Dietary strawberry was also shown to decrease the risk factors of obesity-related disorders (Zunino et al. 2012).These findings also support the fact that kombucha showed anti-diabetic potential based on the inhibition of α-amylase and α-glucosidase (Permatasari et al. 2021(Permatasari et al. , 2022)).
The antioxidant activity of plant-based fermented drinks was widely studied since fermentation will result in a high-quality beverage with enhanced antioxidant, total phenolic, and bioactive compounds (Hur et al. 2014;Yang et al. 2018).In this study, SKD showed DPPH inhibition similar to glutathione at 150, 200, and 250 μg/mL doses.Interestingly, at all doses, SKD had a similar ABTS inhibition to Trolox.The EC50 value of DPPH inhibition of SKD was also greater than strawberry crude extract (17.28 μg/mg to 59.55-349.35μg/mL (Huneif et al. 2022).
A diverse range of aspects, such as total phenolic, organic acids, vitamins, and microbial hydrolysis in fermentation can influence the antioxidant activity of kombucha drink (Massoud et al. 2022).Subsequently, the identified phenolic compounds (catechin and epicatechin) and flavonoids (such as rutin) have been proven to improve metabolic function and inflammation while also acting as an antioxidant (Simos et al. 2012;Muvhulawa et al. 2022).On the other hand, dietary strawberry supplementation has induced beneficial effects on lipid profile, antioxidant, and inflammatory markers in obese adults (Zunino et al. 2012;Basu et al. 2016).Inflammation and oxidative stress themselves have been linked with the pathophysiology of obesity and metabolic syndrome (Ruiz-Ojeda et al. 2018).Therefore, SKD may become a nutraceutical with antiobesity and antidiabetic activities along with antioxidant and anti-inflammatory properties.

Conclusion
Strawberry or Fragaria ananassa can be processed or innovated into a functional probiotic drink (SKD) with several secondary metabolites that inhibit the activity of lipase, α-glucosidase, and α-amylase as proved by in silico study.SKD also exhibited potential antiobesity, antidiabetic, and antioxidant properties which may play a role in attenuating metabolic and inflammatory disorders in vitro, further reinforcing the potential health benefits of SKD.However, our study has not studied the potential of SKD using cell lines, hence the limitation of our study.These findings suggest that SKD can be a promising therapeutic functional food in preventing metabolic disorders and obesity.

Figure 2 .
Figure 2. Lipase Inhibition Activity Test of SKD and Orlistat.The inhibition of lipase was presented in % activity (A) and EC 50 value (B).

Figure 3 .
Figure 3. α-Amylase Inhibition Activity Test of SKD and Acarbose.The inhibition of α-amylase was presented in % activity (A) and EC 50 value (B).

Figure 4 .
Figure 4. α-Glucosidase Inhibition Activity Test of SKD and Acarbose.The inhibition of α-glucosidase was presented in % activity (A) and EC 50 value (B).

Figure 5 .
Figure 5. DPPH Inhibition Activity Test of SKD and Glutathione.The inhibition of DPPH was presented in % activity (A) and EC 50 value (B).

Figure 6 .
Figure 6.ABTS Inhibition Activity Test of SKD and Trolox.The inhibition of ABTS was presented in % activity (A) and EC 50 value (B).

Table 2 .
Validation of molecular docking simulation.

Table 3 .
Molecular Docking Parameter of Identified Compounds of SKD.