Influence Of In Vitro Human Digestion Simulation On The Phenolics Contents And Biological Activities Of The Aqueous Extracts From Turkish Cistus Species Part 1

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Abstract: Oxidative stress is one of the significant precursors of various metabolic diseases such as diabetes, Parkinson's disease, cardiovascular diseases, cancer, etc. Various scientific reports have indicated that secondary plant metabolites play an important role in preventing oxidative stress and its harmful effects. In this respect, this study was planned to investigate the phenolic profile and antioxidant and antidiabetic potentials of the aqueous extracts from Turkish Cistus species by employing in vitro methods. In vitro digestion simulation procedure was applied to all extracts to estimate the bioavailability of their phenolic contents. Total phenolic, flavonoid, phenolic acid, and proanthocyanidin contents were determined for all phases of digestion. In addition, changes in the quantity of the assigned marker flavonoids (salidroside, hyperoside, and quercitrin) were monitored by High-Performance Thin Layer Chromatography(TLC) analysis. The antioxidant activity potentials of the extracts were studied by various methods to reveal their detailed activity profiles. On the other hand, in vitro a-amylase and a-glucosidase enzymes and advanced-glycation end product(AGE)inhibitory activities of the extracts were determined to evaluate the antidiabetic potentials of extracts. The results showed that aqueous extracts obtained from the aerial parts of Turkish Cistus species have rich phenolic contents and potential antioxidant and antidiabetic activities; however, their bioactivity profiles and marker flavonoid concentrations might significantly be affected by human digestion. The results exhibited that total phenolic contents, antioxidant activities, and diabetes-related enzyme inhibitions of the bioavailable samples were lower than non-digested samples in all extracts.

Keywords: Turkish Cistus species; antioxidant activity; human digestion simulation; HPTLC; diabetes

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1. Introduction

The Cistaceae family is composed of shrubs, and annual or perennial herbaceous plants and the genus Cistus is one of the broadly distributed members of this family. More than 50 Cistus species are distributed worldwide, and they are commonly called "rockrose" [1]. Previous in vitro and in vivo investigations demonstrated that Cistus species possess antiviral, antidiabetic, antioxidant, antimicrobial, and anti-inflammatory activities [2,3]. Different phenolic compounds(phenolic acids, flavonoids, proanthocyanidins) and terpenes were isolated from Cistus species, and their therapeutic benefits are generally attributed to these components[4,5].

In Turkey, five Cistus species grow naturally, ie, C. salvifolius L., C. paroiflorus Lam, C.monspeliensis L., C.laurifolius L.and C.creticus L.[6]. In the ethnobotanical records of Turkish folk medicine, various organs of Cistus species are frequently documented as a remedy. Infusions prepared from the branches of C. laurifolius, C. salviifolius, and C. creticus are ingested orally against diabetes in Edremit (Balikesir) district [7]. Decoctions prepared from the flowers of C.creticus and C.salvifolius are used internally against peptic ulcers in Marmaris (Mugla) [8], while a decoction of the unopened flower buds of C. laurifolius is used for the same purpose. In Western Anatolia, the decoction of C. laurifolius leaves is used internally against fever and stomachache and externally, via bathing, against rheumatic pain [9].

Cistanche can improve immunity

It is a well-proven fact that the elevated aggregation of reactive oxygen species (ROS)triggers oxidative stress, which is one of the significant precursors of various metabolic disorders such as cancer, diabetes, cardiovascular problems, Alzheimer's disease, etc. [10]Therefore, antioxidant utilization has become a common holistic approach for preventing or treating such conditions in current scientific practice. The antioxidant activities of the plant extracts have been reported by a tremendous number of researchers[11-14]. As a common approach, the antioxidant potential of the plant extracts is generally devoted to their phenolic contents. A large body of evidence is available in the scientific literature that Cistus species are also rich in phenolic profiles and eventually have a considerable degree of antioxidant activity. However, the bioavailability concept of these phytochemicals in the body has not been considered in most of these studies.

It is a well-known fact that gastrointestinal tract conditions influence phenolic compounds due to different pH conditions, enzyme actions, and microbiota. On the other hand, the chemical structures of phenolic compounds and the plant matrix are also important factors affecting their bioavailability [15]. Therefore, in the present investigation, the in vitro digestion simulation method was applied to all extracts to estimate the bioavaility of phenolic contents. In order to monitor the transitions, total phenolic, flavonoid, phenolic acid, and proanthocyanidin contents were determined in all phases of digestion. Moreover, the antioxidant activities of the extracts were studied by mechanistically different spectrophotometric methods to reveal their comprehensive activity profiles. The antioxidant potentials of all samples obtained by the digestion process were investigated with DPPH and DMPD (free radical scavenging), FRAP and CUPRAC(metal-reducing potential), and TOAC(total antioxidant capacity) assays. Previously, salidroside, hyperoside, and quercitrin were determined as the marker flavonoids of Cistus species by Guzelmeric et al. [16]Therefore, the qualitative and quantitative determination of these flavonol glycosides was carried out with the High-Performance Thin Layer Chromatography system and their bioavailability indexes were estimated.

Diabetes mellitus (DM) is a common metabolic disorder and is described by decreased insulin secretion by pancreatic β-cells or a lack of responses of the body to insulin. There are two types of DM: Insulin-dependent (Type I) and non-insulin-dependent(Type II) [17]. One of the treatment strategies for Type II DM is to control postprandial hyperglycemia, which is defined as "a significant rise of blood sugar concentration in the bloodstream following a meal".Inhibition of the key digestive enzymes, including o-amylase and a-glucosidase, is essential to controlling postprandial hyperglycemia. In the gastrointestinal system, o-amylase digests the starch into reducing sugars such as maltodextrin, lactose, and maltose, and α-glucosidase breaks these sugars into glucose. Therefore, inhibition of digestive enzymes is regarded as a possible mode of action to treat postprandial hyperglycemia [18]On the other hand, elevated blood glucose levels may trigger the formation of AGEs which are defined as"compounds formed as a result of enzymatic glycation reaction (Maillard)between reducing sugars and proteins, nucleic acids and lipids".Elevated accumulation of AGEs in the body may induce many diabetic complications, including nephropathy, neuropathy, retinopathy, etc.[19]. Aminoguanidine, imagine and metformin is examples of synthetic inhibitors for AGEs, and acarbose, miglitol, and voglibose are synthetic inhibitors for digestive enzymes and have been in use for the past decades [20,21]. However, clinical trials and in vivo experiments demonstrated the side effects of these synthetic inhibitors, such as hepatotoxicity, abdominal distention, flatulence, meteorism, anemia, vomiting, heart failure, etc. [21,22]. Due to stach harmful effects, multiple studies have involved the inhibitory potentials of the plant extracts on AGEs [23-25]. It has been reported that phytochemicals, particularly phenolic compounds such as phenolic acids, flavonoids, and proanthocyanidins, significantly inhibited the formation of AGEs and related enzyme actions,i.e.,α-amylase and α-glucosidase [26-28].

Since water extraction (infusion or decoction) is the common preparation technique in traditional medicine, this study was carried out on the aqueous extracts from Turkish Cistus species before and after the in vitro gastrointestinal digestion simulation. In this respect, the phenolic profiles and antioxidant and antidiabetic potentials of the aqueous extracts and their digestion metabolites were comparatively investigated. According to the reference survey, inhibitory activities ofCistus extracts on AGEs were studied for the first time in this study. In addition, quantitative analysis of the marker flavonoids was also accomplished by HPTLC analysis. In vitro digestion simulation technique was applied to all extracts to monitor the alterations in the concentrations and the biological activity profiles of the phenolic compounds in the gastrointestinal conditions.

2. Results

2.1. Estimation of the Phenolic Contents of the Samples

According to the results shown in Table 1, aqueous extract of C. saloifolius had higher total flavonoid, phenolic and phenolic acid contents than other studied species, while ND (non-digested) samples of C.creticus and C.laurifolius possessed the highest proanthocyanidin contents. The most significant decrease was detected in the total proanthocyanidins contents of all extracts. Proanthocyanidin amounts of IN (bioavailable) samples were undetectable in all aqueous extracts. As a result, the phenolic contents of the aqueous extracts were negatively affected by the in vitro human digestion simulation procedure.

The abbreviations for samples are ND: Non-digested; PG: Postgasttic; IN: Bioavailable; BAvl: Bioavailability index; Results were stated as the mean of triplicates 士 standard deviation （S.D.） and as mg gallic acid equivalents （GAE） in 1 g sample； Results were expressed as the mean of triplicates ± standard deviation (S.D.) and as mg quercetin equivalents (QE) in 1 g sample; D Results were expressed as the mean of triplicates ± standard deviation (S.D.) and as mg caffeic acid equivalents (CAE) in 1g sample; F Results were expressed as the mean of triplicates ± standard deviation (S.D.) and as mg catechin equivalent (CE)in 1 g sample;* Abbreviations of the aqueous extracts: CCA for C. creticus, CLA for C.laurifolius, CMA for C.monspeliensis, CPA for C.parviflorus, CSA for C.salviifolius.Different letters in the same row indicate significance (p<0.05).

As presented in Table 2, salidroside and hyperoside contents in the aqueous extract of C. salviifolius were relatively higher than those of the other species, while quercitrin was not found. On the other hand, quercitrin was found in the highest concentration in all simulation samples of the aqueous extract from C.creticus, but its concentration reduced significantly in bioavailable samples. Additionally, the HPTLC chromatogram and overlay UV spectra of references and the corresponding spots in the tracks of all extracts were presented in Figure 1.

The abbreviations for samples are ND: Non-digested; PG: Postgastric; IN: Bioavailable; BAvI: Bioavailability index; BResults were given as mg/g dry extract and experiments were performed independently three different times;* Abbreviations of the aqueous extracts: CCA for CC. creticus, CLA for C. laurifolius, CMA for C.monspeliensis, CPA for C.parviflorus, CSA for C.salviifolius.Different letters in the same row indicate significance (p<0.05).

Figure 1. (A) Overlay UV spectra of salidroside and the corresponding spots in the tracks of all extracts. (B) Overlay UV spectra of hyperoside and the corresponding spots in the tracks of all extracts. (C) Overlay UV spectra of quercitrin and the corresponding spots in the tracks of all extracts. (D)HPTLC chromatograms of:1.CCA ND,2.CCA PG,3.CCA IN,4.CLA ND，5.CLA PG，6.CLA IN，7.Tiliroside （Rf ≈0.65），8.Hydroxide （Rf ≈0.35），9.Quercitrin （Rf≈0.45），10.CMA ND，11.CMAPG,12.CMAIN,13.CPAND,14.CPAPG,15.CPAIN,16.CSAND,17.CSAPG,18.CSAIN.Mobilephase:EtOAc/CHCl2/CHCOOH/HCOOH/H2O(100:25:10:10:10:10):11);Derivatization: NPR reagent. Visualization: 366 nm.

2.2.Estimation of Antioxidant Activity of the Samples

As presented in Table 3, bioavailable samples of Cistus extracts exhibited weaker radical scavenging antioxidant activity than their non-digested and post-gastric counterparts. ND and PG samples of all aqueous extracts showed significant DPPHradical scavenging activity and possessed lower ECso values than reference compound BHT (ECso value:5.83±0.2 μg/mL）. However， all extracts displayed a weaker DMPD radical scavenging activity than the reference compound Trolox （5.82±0.37μg/mL）.ND， PG, and IN samples of CPA possessed better DMPD activity compared to samples of other extracts.

The abbreviations for samples are ND: Non-digested; PG: Postgastric; IN: Bioavailable; BAvI: Bioavailability index； Results were presented as ECso in μg/mL equivalents and experiments were performed independently three different times. ECso value of the reference compound"BHT" in DPPH scavenging activity was determined as 5.83±0.2 ug/mL； C Results were presented as EC50in μg/mL equivalents and experiments were performed independently three different times. ECso value of the reference compound Trolox in DMPD scavenging activity was determined as 5.82±0.37 μg/mL； P Results were expressed as mM FeSO4 equivalents in a 1g sample and experiments were performed independently three different times. FRAP activity of the reference compound "BHT" was detected as 4.06±0.42mM FeSO4 eq. in1 g sample; F Results were given as mg ascorbic acid equivalent (AAE) in 1 g sample and experiments were performed independently three different times; results were given as mg ascorbic acid equivalent (AAE)in 1 simple and experiments were performed independently three different times;*Abbreviations of the aqueous extracts: CCA for C. creticus, CLA for C. laurifolius, CMA for C.monspeliensis, CPA for C.parviflorus, CSA for C.salviifolius. Different letters in the same row indicate significance (p<0.05).

Similar to radical scavenging activity assays, bioavailable samples of Cistus extracts also showed weaker metal-reducing and total antioxidant activities than the non-digested and post-gastric samples. All ND samples of the extracts displayed significant ferric reducing antioxidant activity, which was stronger than the reference compound BHT (4.06±0.42mM FeSO4 equivalent). Among PG samples, only CSA(44±0.16mMFeSO4 equivalent) had better activity than BHT. In the CUPRAC assay, ND and PG samples of CSA were detected as most potent among the samples of other species. Discretely, IN sample of CCA had better CUPRACactivity than bioavailable samples of other aqueous extracts.

2.3.Diabetes-Related Enzyme Inhibition Activity

As indicated in Table 4, concentration-dependent enzyme inhibitory activity was seen in all aqueous extracts. While ND samples of CPA and CSA (75.89%±0.62,80.34%±0.19, respectively) exhibited somewhat higher α-amylase inhibitory activity than acarbose (75.80% ±0.02) at the 1mg/mL concentration; only the ND sample of CSA exhibited higher α-glucosidase inhibitory activity than the reference compound quercetin in both concentrations.

The abbreviations for samples are ND: Non-digested; PG: Postgastiic; IN: Bioavailable; BAvl: Bioavailability index; Results were stated as the mean of triplicates ± standard deviation (S.D.) and acarbose 'was used as control group with75.8±0.02% inhibition at 1 mg/mL, 65.45±0.01% inhibition at0.5mg/mL; Results were expressed as the mean of triplicates ± standard deviation (S.D.) and quercetin was used as a control group with 80.4±0.03% inhibition at 1 mg/mL,69.t66±0.05% inhibition at 0.5 mg/mL; D Results were expressed as the mean of triplicates ± standard deviation (S.D.) and quercetin was used as control group with 89.33±3.47% inhibition at1mg/mL,72.03±3.04% inhibition at 0.5 mg/mL;* Abbreviations of the aqueous extracts: CCA for C.creticus,CLA for C.laurifolius,CMA for C.monspeliensis, CPA for C.parviflorus,CSA for C.salviifolius.Different letters in the same row indicate significance (p<0.05).

This article is extracted from Molecules 2021, 26, 5322. https://doi.org/10.3390/molecules26175322 https://www.mdpi.com/journal/molecules