Influence Of In Vitro Human Digestion Simulation On The Phenolics Contents And Biological Activities Of The Aqueous Extracts From Turkish Cistus Species Part 2

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To sum up

The aqueous extract of C.salvifolius exhibited a better digestive enzyme inhibitory activity than the extracts of other species. Additionally, IN samples of all aqueous extracts revealed lower enzyme inhibitory activities compared to ND samples.

2.4.AGEs Inhibitory Activity

As presented in Table 4, concentration-dependent AGE inhibitory activity was observed in all aqueous extracts. ND samples of CCA, CPA, and CSA exhibited better inhibitory activity than the reference compound quercetin in both 0.5 and 1mg/mL concentrations. However, only C.saluifolius extract displayed better inhibitory activity than quercetin among IN samples of the extracts. Bioavailable samples of aqueous extracts showed lower AGE inhibitory activities compared to non-digested samples. According to the results, the ND sample of aqueous extract of C.salvifolius possessed the highest AGE inhibitory activity. However, the IN sample of C.monspeliensis aqueous extract displayed the weakest AGE inhibition potential in tested concentrations.

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3. Discussion

Free radicals might attack proteins, lipids, or DNA to procure an electron, resulting in various health problems. For this reason, it is essential to balance the antioxidant system of the body and the free radicals formed by oxidation. In case of the deterioration of this balance, oxidative stress arises [29]. Oxidative stress is one of the main factors resulting in various chronic disorders such as cancer, diabetes, cardiovascular diseases, Alzheimer's disease, etc. While there are self-antioxidant systems in the human body to combat the oxidative damage to tissues and organs, these defense systems may lose their efficiency due to different conditions such as alcohol over-consumption, smoking, stress, chronic drug usage, and radiation, etc. [30]. Various scientific reports have indicated that secondary plant metabolites play a significant role in preventing oxidative stress and its harmful effects [13,14,31]Even though bioavailability is an important parameter affecting the bioactivity of these compounds, it has not been taken into account in most studies. However, it is well known that the secondary metabolites are subjected to structural transformations due to different pH conditions, enzymatic activity, and body temperature in the gastrointestinal system. In addition, the chemical characteristics of the secondary metabolites such as molecular weight, polarity, and degree of binding to macromolecules in the plant matrix are also significant factors affecting their bioavailability [13]. Accordingly, absorption of the compounds or their metabolites into the bloodstream once ingested is essential in order to exert their systemic physiological effects. In vitro digestion simulation models may provide evidence for the assessment of the possible bioavailability characteristics of substances. While confirmation in human trials is necessary to claim any functional property, in vitro simulation models are broadly used as alternatives to in vivo studies or human trials, which are often ethically debatable, resource-intensive, expensive, and time-consuming [32].

Cistanche can improve immunity

Several researchers have also investigated the antioxidant potential of extracts prepared from different organs of Cistus species. According to the literature survey, this is the first study on Cistus species regarding the bioavailability of phenolic substances and their impact on antioxidant activity by employing an in vitro digestion simulation model. Karas et al. [33] suggested that 10% of the polyphenolic components remain undigested in the plant matrix and 90% of them are subjected to digestion in the gastric or intestinal phase (approximately 48% and 52%, respectively). As mentioned earlier, all phenolic amounts in aqueous extracts were negatively affected by the in vitro human digestion simulation procedure. Thus, significant losses were detected in the phenolic contents of the bioavailable samples of all extracts. Moreover, total proanthocyanidin concentrations of IN samples were below the limit of detection in all aqueous extracts. Various studies also prompted the negative influence of digestion procedure on phenolic compounds in the plant extracts and thus related bioactivities. For instance, we reported the total phenolic content of Salvia virgata Jacq. was also adversely affected by the digestion procedure in our previous study. In addition, the amounts of major metabolites of the extract,i.e., rutin and rosmarinic acid, tended to decline [13]. In contrast, several studies have reported contradictory results. In the study by Celeb et al. [34], they observed the increment of the total phenolic acid, flavonoid, and phenolic amounts in the bioavailable samples of methanolic extract from Hypericum perfoliatum L. In fact, the main metabolites, quercitrin, chlorogenic and gallic acid, had bioaccessibility ratios over 100%. These studies showed that the effects of the digestion procedure might vary according to plant materials. In order to clarify these differences, it is essential to consider the mechanism of action of the digestion system on phenolic substances. Serra et al. [35] suggested that phenolic compounds are mainly found in glycosides, polymers, and ester forms in the plant matrix and are hydrolyzed in the digestive system before absorption. Various factors may influence the structural transformations of the phenolic compounds in the gastrointestinal tract. For instance, compounds with higher molecular weights, such as proanthocyanidins or procyanidins, need to be hydrolyzed before absorption in the gut. The structure of the plant matrix is also a prominent factor in the bioavailability of phenolics; phenolic compounds can bind to macromolecules in the plant matrix, such as fibers, proteins, and lipid molecules. Thus, only the liberated phenolic components from the matrix may become absorbable from the gastrointestinal tract. Moreover, different pH values and enzymatic actions of the intestinal microbiota are among the other crucial factors affecting the transformation in the chemical structure of phenolic compounds [36]. In light of these data, we can hypothesize that different results obtained from similar types of experimental studies may be due to the complexity of the digestion system and the composition of the plant matrix. As presented in Table 3, the bioavailable samples of Cistus extracts exhibited a weaker antioxidant activity than the non-digested and post-gastric counterparts due to their lower phenolic contents.

Moreover, both extracts of C. saluifolius displayed better antioxidant activity in DPPH, CUPRAC, FRAP, and TOAC assays when compared to other species. Besides, both extracts of C.paroiflorus showed higher DMPD radical scavenging activity than the extracts of other species. As indicated in Table 1, the total phenolics, flavonoids, and phenolic acid contents of C. salviifolius were higher than in the other samples. Thus, the higher antioxidant potential of the C. saloiifolius extracts may be related to its phenolic contents.

Moreover, the reduction in antioxidant potentials, inhibitory activities on carbohydrate-related enzymes, and AGEs of the bioavailable samples may be related to the decline in marker flavonoid glycosides. However, Cistus extracts also contained other phenolic substances, as indicated in Figure 1. Therefore, a detailed chromatographical analysis of the extracts is required to monitor the influence of digestion on biological activity.

The inhibitory potential of plant extracts on digestive enzymes has recently attracted more attention due to the safety concern of synthetic inhibitors. Therefore, the inhibitory effect of plant extracts was determined in several studies, and this effect was generally associated with phenolic substances such flavonoids, phenolic acids, proanthocyanidins, etc. Sun et al. [37] suggested that polyphenolic compounds display their inhibitory effects by binding with the enzymes mentioned above with the help of hydrophobic forces and non-covalent bonds. Therefore, inhibition of α-amylase and α-glucosidase enzyme activity by polyphenols is related to their molecular structures. While this interaction mechanism has been studied using different techniques such as inhibition kinetics, molecular docking fluorescence quenching, etc., no certain conclusion has been acquired yet [38]. However, numerous studies have reported that digestive enzyme inhibitions of the plant extracts are directly related to their phenolic contents. Similar studies on the enzyme inhibitory activities of Cistus species have also been previously reported. Sayah et al. [39] investigated the α-amylase and a-glucosidase inhibitory activities of the 80% methanolic and aqueous extracts from C. monspeliensis and C. saluifolius. Their results were in accordance with the present study, revealing that C. salvijfolius aqueous extract demonstrated higher α-glucosidase （IC50 μg/mL∶0.95±0.14） and o-amylase（IC50 μg/mL∶217.1±0.15）inhibitory activity than the C.monspeliensis aqueous extract （IC50 μg/mL∶14.58±1.26） and （ICsn ug/mL:886.10±0.10), respectively. The inhibitory rates of both aqueous extracts on these enzymes were higher than the reference compound acarbose (ICso ug/mL:18.01±2.00)Similar to our study, they found a correlation with the enzyme inhibitory rates and the total phenolic and flavonoid amounts. Both total phenolic and total flavonoid contents of the C. salvijfolius aqueous extract(408.43±1.09mg GAE and 140.00±1.15 RE, respectively) were higher than the C.monspeliensis aqueous extract (261.76 ±1.9mg GAE and 78.00±1.15 RE respectively). Orhan et al. [40] also investigated the digestive enzyme inhibitory potentials of 80% aqueous and ethanolic extracts from the leaves of C.laurifolius. According to their results,80% ethanolic extract (71.7%±0.6) displayed a strong α-amylase inhibitory activity compared to aqueous extract (39.3% ±2.2)at 1 mg/mL concentration. They suggested that phenolic compounds, especially flavonoids, directly affect insulin secretion by preventing beta-cell apoptosis and supporting anti-diabetic activity. This hypothesis, correlated with our results, marks that the ND sample of aqueous extracts of C. salviifolius exerted the highest total flavonoid and phenolic contents and the greatest o-amylase and o-glucosidase inhibitory activities. As given in Table 4, the aqueous extract of C. salviifolius exhibited better inhibitory activities on digestive enzymes than the other extracts.

Additionally, IN samples of the aqueous extracts contained lower phenolic and flavonoid amounts than ND samples and thus revealed lower enzyme inhibitory activities in the present work. While phenolic contents of the extracts were negatively affected by the digestion procedure, they still exhibited significant digestive enzyme inhibitory activity. In several studies, flavonoid glycosides were reported as the major metabolites of plant extracts with strong a-amylase and α-glucosidase inhibitory potentials [41-43]While the inhibitory mechanism of phenolic compounds on the digestive enzymes has not been revealed yet, structure-activity relationship studies have been carried out on some phenolic components such as flavonoids, phenolic acids, proanthocyanidins and tannins Structure-activity relationship studies about flavonoids showed that C2=C3 double bond of C-ring enhances the digestive enzyme inhibition potential of such compounds. Since this bond elevates the electron density, the strength of interaction between the flavonoid and enzyme is increased [44]. In addition, hydroxyl groups on C-5 and C-7 facilitate the o-amylase inhibitory potential of flavonoids. It was also suggested that hydroxylation of flavonoids skeleton positively affects their a-amylase and α-glucosidase enzyme inhibitory poten-tials[45]. As indicated earlier, all marker flavonoids in the present study were flavonol glycosides bearing these structural requirements described above. However, after in vitro digestion procedure, their related activities declined significantly in bioavailable samples of the aqueous extracts.

Generally, the inhibitory potential of the plant extracts on AGEs is related to several factors, i.e., their phenolic contents, antioxidant potentials, metal chelating capabilities, protein interactions, and AGE receptor blocking activities[13]. As presented in Table 4, bioavailable samples of the aqueous extracts displayed lower AGE inhibitory activities compared to ND samples since in vitro digestion procedure adversely affected the phenolics content of the extracts. According to the current study outcomes, the ND sample of aqueous extract of C.salvifolius possessed the highest AGE inhibitory activity as well as total phenolic and flavonoid contents. In comparison, IN sample of aqueous extract of C.monspeliensis showed the weakest AGE inhibition potential, which may be ascribed to its lowest total flavonoid and phenolic contents. Since flavonoids have a widespread distribution in plant extracts, fruits, vegetables, and beverages, several studies have intensified on the inhibition potential of flavonoids on AGE formations. The same flavonoids assigned as the marker phenolics in this study were also reported as the marker compounds in other studies[46-48].

Similar to digestive enzyme inhibition, AGE inhibitory activities of flavonoids were stimulated by the C2=C3 double bond and hydroxylation of A and C rings. However, sugar attachment to the flavonoid skeleton leads to decreased inhibitory activity [49]. On the other hand, Cervantes-Lauren et al. [50] suggested that flavon-3-of glycosides possessed a greater AGE inhibition potential than the other flavonoid glycosides. This hypothesis may be an explanation for the decreased AGE inhibition in bioavailable samples. For instance, quercitrin and hyperoside were not detected in bioavailable samples of the aqueous extract of C.monspeliensis, which is the reason for the lower activity of IN samples of C.monspeliensis compared to the extracts from other species. Even though quercitrin was not detected in the aqueous extract of C. saloifolius, significantly higher amounts of hyperoside and salidroside may be the cause of its high AGE inhibition activity. In general, a positive correlation was observed between the marker flavonoid contents of the samples and their inhibitory potentials on AGEs.

4. Material and Methods

4.1.Chemicals

All references, enzymes, and chemicals employed in the experiments were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Analytical grade materials were used in the experiments.

4.2.Plant Samples

Aerial parts of C.creticus and C.saloifolius were gathered from the campus of Yeditepe University (Kayisdagi, Istanbul) during the last week of April 2018. Aerial parts of C.mon-speliensis and C.paroiflorus were gathered from near AlacatI Kutlu Aktas Balaji, Cesme, Izmir in the second week of May 2018. Aerial parts of C. laurifolius were collected from Kemer-Doganhisar road, Konya, in the first week of June 2018. Prof. Dr. Erdem Yesilada authenticated plant materials. Voucher specimens for C.creticus (YEF18013),C.lauri-folios (YEF18017),C.monspeliensis(YEF18015),C.paroifforus(YEF18016)and C.salvifolus (YEF18014) were stored at the Herbarium of the Department of Pharmacognosy, Faculty of Pharmacy, Yeditepe University, Istanbul, Turkey.

4.3.Extraction Procedure

Aqueous extraction was chosen since it is commonly used as a preparation technique in traditional medicine. The coarsely air-dried and powdered aerial parts of Cistanche species (100 g) were extracted with hot distilled water(80℃,1.5L)by using a shaking device for 15 min. Then, aqueous extracts were filtered through a filter paper and evaporated to dryness under reduced pressure. After the lyophilisation procedure was completed, extracts were dissolved in distilled water for further processing (non-digested sample: ND) (the yield of extracts:13.04% for C.creticus,15.7% for C.laurifolius,12.8% for C.monspeliensis,14.94% for C.parviflorus,14.74% for C.salvifolius).

4.4.In Vitro Human Digestion Simulation Method

The human digestion simulation model was applied to Cistus samples in vitro, following the method detailed earlier by Celeb et all.[34].Firstly,1g NaCl and 1.6g pepsin were dissolved in 500 mL of distilled water to obtain a simulated gastric fluid solution (SGF). Later, the pH of the SGF solution was arranged to 2 with HCl(5 M);17.5 mL of this solution was mixed with 2.5 mL of plant samples, and this mixture was situated in the shaking water bath at 37℃ for 2 h to imitate the peristaltic movements of the digestion system. After 2 h, the samples were put into an ice bath to inactivate the enzymatic reactions;2 mL of the samples was set aside as a"post-gastric"(P)sample for further experiments. A cellulosic dialysis membrane loaded with a proper amount of NaHCO3(1 M, pH7) was located in the cold sample solutions; thus, gastrointestinal absorption was mimicked. Then, 4.5 mL of bile acids/pancreatin solution was combined with the solutions, and the mixture was incubated for another 2 h at 37 ℃. Finally, the fluid inside the dialysis membrane was acquired as the "bioavailable" sample (IN). After the procedure was over, all samples were preserved at -20℃ for further experiments. 4.5.In Vitro Estimation of Phenolic Profile

4.5.1.Total Phenolic Content Assay

Spectrophotometric determination of the total phenolic content of the samples was conducted in a 96-well plate template according to the method detailed earlier by Barak et al.【32】；75 uL of NagCO3（20% in H2O） was added to 20 μL of freshly prepared sample and reference solutions. Then， 100 μL of Folin-Ciocalteu reagent was combined with the mixture. After a 30 min incubation period at room temperature in the dark, the absorbance was measured at 690 nm. Gallic acid was used as a reference solution at different concentrations to establish a calibration curve and total phenolic contents were expressed as gallic acid equivalents (GAE).

4.5.2.Total Flavonoid Content Assay

Spectrophotometric determination of the total flavonoid content of the samples was managed in a 96-well plate template in accordance with the previously explained method by Bardakci et al.[51];150 uL of 75% ethanol,10 uL of aluminum chloride, and 10 uL of 1M sodium acetate trihydrate were combined with 50 μL of sample and reference solutions， separately. Then, these mixtures were incubated at darkroom temperature for 30 min. After the incubation period, absorbance was calculated at 405 nm. Quercetin was employed as a reference solution at different concentrations to establish a calibration curve and total flavonoid contents were represented as quercetin equivalents (QE). 4.5.3.Total Phenolic Acid Content Assay

The total phenolic acid content of the samples was measured spectrophotometrically following the procedure reported formerly by Barak et al. [52]. Firstly, proper amounts of sodium nitrite and sodium molybdate were dissolved in distilled water to obtain the Arnow reagent. Then, 1 ml of the samples was combined with 1 mL of Arnow reagent, 1 mL of 0.1 M HCl, and 1 mL of 1M NaOH, separately. After that, the volume of the mixture was adjusted to 10 mL with distilled water, and the absorbance was read immediately at 490 nm. Caffeic acid was employed as a reference solution at different concentrations to get a calibration curve, and total phenolic acid contents were given as caffeic acid equivalents (CAE).

4.5.4.Total Proanthocyanidin Content Assay

Spectrophotometric determination of the total proanthocyanidin content of the samples was conducted in a 96-well plate template by following the method of Barak et al. [1]Briefly，25 uL of the sample solutions was mixed with 150 μL of 4% vanillin and 75 μL of HCl solutions (32%), respectively. After 15 min incubation time at darkroom temperature, the absorbance was adjusted to 492 nm. Catechin hydrate was utilized as a reference solution at different concentrations to obtain a calibration curve. Methanol was employed as a control solution. The total proanthocyanidin content of the samples was stated as catechin equivalents (CE).

4.6.Free Radical Scavenging Activity Assays 4.6.1.DPPH Radical Scavenging Activity Assay

DPPH radical scavenging activity of the samples was determined in a 96-well plate template following the method modified by Celeb et al.[53]. At first,150 uM of DPPH solution was freshly prepared. Then，200 μL of DPPH solution was mixed with 25 μL of sample solutions. Then, this mixture was incubated at darkroom temperature for 50 min. The absorbance was calculated at 540 nm. Butylated hydroxytoluene (BHT) was employed as a reference solution at different concentrations. Methanol was used as a control solution. The activity of samples was presented as EC50, corresponding to the concentration showing 50% activity.

4.6.2.DMPD Radical Scavenging Activity Assay

DMPD+(N, N-dimethyl-p-phenylenediamine) radical scavenging activity of the samples was carried out in a 96-well plate template according to the method described earlier by Inan et al. [13]. Firstly,100 mM DMPD+ solution, 0.05 M FeCl; 6H2O solution and 0.01 M acetate buffer were freshly prepared. Then,1 mL of DMPD solution,100 mL of acetate buffer, and 0.2mL of FeCl； 6H2O solution were mixed， and later 15 μL of sample solutions were combined with 210 μL of this mixture. After the incubation period at dark-room temperature for 50 min, the absorbance was measured at 492 nm. Trolox was used as a reference solution at different concentrations to obtain a calibration curve. Concentrations of the sample solutions were 1 mg/mL. DMPD radical scavenging activities of the samples were stated as Trolox equivalents(TE). 4.7.Metal Reducing Activity Assays

4.7.1.Ferric Reducing Antioxidant Power Assay (FRAP)

Determination of FRAP activity of the samples was accomplished in a 96 well-plate template following the procedure reported earlier by Bardakci et al.[54]. At the beginning of the experiment, FRAP reagent was formed bymixing acetate buffer, ferric-tripyridyltriazine, and FeCl; 6H2O solutions. After that, the FRAP reagent was put inside the 37℃ oven for 30min. Then,10 uL of the sample solutions were combined with 30 uL of distilled water and 260 μL of FRAP reagent， respectively. After incubation time at 37℃for 30min.， the absorbance was adjusted to 593 nm. A standard curve was built by employing different molarities of ferrous sulfate (0.25-2 mM) solution to evaluate the results. BHT was used as a reference solution at different concentrations. FRAP activities of the samples were presented as mM FeSO4 in 1 g dry extract.

4.7.2.Cupric Reducing Antioxidant Capacity Assay (CUPRAC)

CUPRAC activity of the samples was determined in a 96-well plate template following the method modified by Celeb et al. 【31】； 85 μL of CuSO4 （10 mM）， neocupraine, and ammonium acetate solutions and 51 uL of distilled water were added to 43 uL of sample solutions, separately. Following an incubation period (20 min) at 50℃in a water bath, the absorbance was measured at 450 nm. Ascorbic acid was used as a reference solution at different concentrations to acquire a calibration curve. CUPRAC activities of the samples were presented as ascorbic acid equivalents (AAE). 4.8. Total Antioxidant Activity Assay(TOAC) Total antioxidant activity determination of the samples was performed in a 96-well plate template following the procedure by Celeb et al. [55]. At first, a certain amount of sodium phosphate monobasic, ammonium molybdate tetrahydrate, and sulfuric acid was mixed to acquire the TOAC solution. Then， 300 μL of TOAC solution was added to 30 μL of sample solutions. After the incubation period at 95℃ in a water bath for 90 min, the absorbance was determined at 690 nm. Ascorbic acid was used as a reference solution at different concentrations to acquire a calibration curve. TOAC activities of the samples were represented as ascorbic acid equivalents (AAE). 4.9.Estimation of Bioavailability Index

The bioavailability index (BAvI) was calculated according to the theoretical equation described by Inan et al.[13]: BAVI=CIN/CND The "bioavailability index"(BAvI) was described as the ratio of the number of phenolics in the bioavailable sample (IN) to that in the non-digested sample(ND). 4.10. Quantification of Marker Flavonoids by HPTLC

The quantitative determination of salidroside, hyperoside, and quercitrin concentrations in all simulation samples (ND, PG, IN) of the aqueous extracts from Cistus species was carried out by high-performance thin-layer chromatography (HPTLC) (CAMAG, Muttenz, Switzerland) following the method validated by Guzelmeric et al.[16]. Hyperoside, salidroside, and quercitrin were prepared in 25,50 and 100 ug/mL concentrations, and the concentrations of freshly prepared sample solutions were adjusted to 10mg/mL. These solutions were applied to normal phase glass-backed silica gel plates (20 cm × 10 cm, Merck， Darmstadt， Germany） with certain volumes （1-5 μL standard solutions and 5 uL sample solutions） by using 100 μL syringes （Hamilton， Bonaduz， Switzerland）. The application procedure was performed with Linomat 5 sample applicator. The development process was performed in Automated Development Chamber(ADC 2) and ethyl acetate: dichloromethane. acetic acid. formic acid: water (10:25:10:10:10:10me) was selected as a mobile phase. Then, the plates were derivatized with Natural Product Reagent (NPR)(1g diphenylboricacid 2-aminoethylesterin 200mL of ethyl acetate) in the immersion device (CAMAG). While hyperoside and quercitrin were analyzed spectrophotometrically at 260 nm, the amount of salidroside was measured at 330 nm by a UV scanner. Rf values of the standards were determined as hyperoside （≈0.35）， quercitrin （≈0.45）， tiliroside （≈0.65）.The correlation coefficients（r2） were found to be ×0.98 for the quantification of the marker flavonoids.

4.11.Inhibitory Activity on Diabetes-Related Enzymes

4.11.1. α-Glucosidase Inhibitory Activity

α-glucosidase inhibitory activities of the non-digested and bioavailable samples obtained from the aqueous extracts of Cistus species were examined following the method explained earlier by Balan et al. [56]. Firstly, proper amounts of monosodium phosphate and disodium phosphate were mixed with procuring 100mM phosphate buffer (pH7). o--glucosidase enzyme was dissolved in phosphate buffer to obtain the a-glucosidase solution （0.2U/mL）. Then 170 uL of phosphate buffer，20 μL of the α-glucosidase solution, and 20 uL of sample solutions were combined and incubated in a 37°Coven for 15 min. After that，20 μL of 2.5 mM p-nitrophenyl-α-D-glucopyranoside solution in 100 mM potassium phosphate buffer (pH7.0) was added to the mixture, and another incubation period was executed at 37°C for 15 min. Then， 80 μL of 0.2 M sodium carbonate solution was appended to the mixture to terminate the reaction. Absorbance was measured at 405 nm. Quercetin solution at different concentrations was used as a reference. Results were estimated as the percentage of inhibitory activity in 1 mg/mL and 0.5 mg/mL concentrations of ND and IN samples of the aqueous extracts of Cistus species. 4.11.2. α-Amylase Inhibitory Activity o-amylase inhibitory activity of non-digested and bioavailable samples obtained from the aqueous extracts of Cistus species was determined by following the procedure detailed previously by Balan et al.[56]. Spectrophotometric determination of a-amylase inhibitory activities of the samples was performed by employing DNS (3,5-dinitrosalicylic acid) reagent. As stated in the method, maltose is formed from the conversion of the starch, and the yellow color of alkaline DNS is turned into the orange-red color due to maltose produced from starch. Thus,96 mM DNS solution was prepared from the mixture of sodium potassium tartrate solution (dissolved in 2 M NaOH) and a certain amount of DNS (dissolved in distilled water). Then,20 mM sodium phosphate buffer with 6.7mM NaCl (co-factor of u-amylase enzyme) was prepared at 20°C(pH:6.9). the a-Amylase enzyme （1U/mL） and starch（10 mg/mL） were dissolved in this buffer. After that，50 μL of sodium phosphate buffer and 10 μL of a-amylase enzyme solution were mixed with 20 μL of the sample solutions. This mixture was incubated at 37°C for 45 min. After the incubation period， 20 μL of the starch solution was added to the mixture. Another incubation period was started at 37°C for 45 min. The same procedure was applied to the samples without the addition of w-amylase enzyme solution called "sample background".The control group was studied with the same procedure in the absence of sample solutions. Absorbance was measured at 540 nm. Acarbose was used as a reference solution at different concentrations. Results were presented as a percentage of inhibitory activity in 1 mg/mL and 0.5 mg/mL concentrations of ND and IN samples from aqueous extracts of Cistus species. 4.11.3.AGE Inhibitory Activity AGE inhibitory activity of non-digested and bioavailable samples from aqueous extracts of Cistus species was determined following the method described by Starow-icz et al. [57]. Before any process,1 mg/mL and 0.5 mg/mL concentrations of ND and IN samples were freshly prepared. Then,1 mL of 10 mg/mL concentration of bovine serum albumin (BSA)solution was added to 1 mL of ND and IN sample solutions. Control samples were prepared without adding ND and IN sample solutions, and the blank samples were prepared without adding 0.5 M glucose. Then, all prepared samples were incubated for 40 h in a shaking water bath at 55 °C. After the incubation period ended, Thermo ScientificTM VarioskanTMLUX multimode microplate reader was used in the 370 nm excitation/440 nm emission range to calculate fluorescence intensity. Quercetin was used as a reference solution at different concentrations.

4.12.Statistical Analysis

All experiments were performed independently three different times. The GraphPad Prism software program (6.1 version) was utilized to determine the parametric or non-parametric distribution of the data. One-way ANOVA Tukey's Multiple comparisons analysis section was used to evaluate TPC, TFC, TPAC, TSC, TPACC, FRAP, CUPRAC, TOAC, DPPH, and DMPD assays. On the other hand, the results of o-amylase, α-glucosidase, and AGE inhibition experiments were examined by two-way ANOVA Sidak's multiple comparisons analysis. Significant results were shown with p<0.05. 5. Conclusions

In the present study, the aqueous extracts of all Cistus species recorded in Turkish flora were investigated for their phenolic profiles and in vitro antioxidant and antidiabetic potentials. Since decoction or infusion is the common form in traditional medicines, using aqueous extracts for the activity assessment in experimental studies is particularly important. On the other hand, hydrophilic constituents in the aqueous extract are subjected to a series of metabolic transformations in the gastrointestinal system once ingested. Therefore for correct activity evaluation of traditional formulations, activities of the bioavailable metabolites should also be investigated.

This is the first study to examine the consequences of the in vitro human digestion simulation method on Turkish Cistus species. In addition, the inhibition potential of Turkish Cistus species on AGEs was studied in this study for the first time. Furthermore, salidroside, hyperoside, and quercitrin were assigned marker flavonoids, and alterations in their concentrations were monitored in the in vitro digestion procedure. While phenolic contents and antidiabetic and antioxidant activities of the extracts were negatively affected by gastrointestinal digestion procedures, they still exhibited significant bioactivity. According to the results, C.saloifolius extract was detected as the most potent plant in terms of phenolic content and antioxidant and anti-diabetic activities. In conclusion, aerial parts from Turkish Cistus species have rich phenolic contents and potential antioxidant and anti-diabetic activities. While the in vitro digestion simulation method was employed to evaluate bioavailability, it might not fully mimic the metabolic pathways occurring in the organism. Therefore, further in vivo and clinical studies are required to assess the bioavailability of the phenolic compounds and their contribution to the reported pharmacological effects in detail.

This article is extracted from Molecules 2021, 26, 5322