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Chemical and biological insights on Cotoneaster integerrimus: a new (-)- epicatechin source for food and medicinal applications.

ABSTRACT

Background: The Cotoneaster species are widely used as traditional purposes in different countries including Turkey.

Purpose: The study was performed to evaluate the biological and chemical profile of two extracts (methanol (T-Me; F-Me) and water (T-W; F-W)) from two parts (twigs and fruits) of Cotoneaster integerrimus.

Materials and methods: Antioxidant (free radical scavenging (DPPH), reducing power (CUPRAC and FRAP), phosphomolybdenum and metal chelating), enzyme inhibitory (cholinesterase, tyrosinase, [alpha]-amylase and [alpha]-glucosidase), antimicrobial (standard microorganisms and methicillin-resistant Staphylococcus aureus isolates (MRSA)) and mutagenic/antimutagenic effects (by Ames assay) were tested for biological profile. For chemical profile, total and individual phenolic components were detected for each extract.

Results: Generally, T-Me reflected the strongest biological effects with the highest level of phenolics (115. 15 mgGAEs/g extract). Also, twig extracts had more potent biological effects as compared to flower extracts. Eight-teen phenolics were identified in the extracts. (-)- epicatechin was the major constituent in all extracts and is mainly responsible for biological activities observed. Its amount present in F-W and T-W were 9.27 and 32.89 mg/g extract, respectively. Also, molecular docking was used to understand enzyme-epicatechin interactions.

Conclusion: From these results, this plant has a great potential as a health promoter for developing novel functional food ingredients and pharmaceutical preparations.

Keywords:

Cotoneaster

(-)-epicatechin

Biological effect

Chemical fingerprint

Molecular docking

Nutraceuticals

Introduction

In last decades, the uses of plant or plant products have significantly increased because chemical products are very expensive and have many unfavorable health effects. In addition, several studies have suggested that there is negative correlation between the consumption of the plant products and the incidence of some chronic diseases such as atherosclerosis, Alzheimer's diseases and diabetes mellitus (Dangles et al., 2012; Ninfani et al., 2005; Tsao et al., 2010). This phenomena lead to the emergence of the terms of "nutraceuticals" to indicate the functional food able to prevent or facilitate the treatment of such diseases. Nutraceuticals contribute to provide nutritional components (vitamins, mineral, radical scavengers, antioxidants or fiber etc.) and active molecules possessing precise pharmacological properties, able to maintain or restore a healthy metabolism (Chauha et al., 2013). From this point, nutraceuticals and their connections are one of the most interested subjects in the scientific area. In this direction, uninvestigated plants could be considered as an important pool to develop new nutraceuticals.

The genus Cotoneaster belongs to the Rosaceae family and is represented by eight species in Turkey (Davis, 1972). Plants belonging this genus commonly called as "Dag musmulasi or Tavsan elmasi" in the different region of Anatolia. In addition, the Cotoneaster species are widely used as culinary plants in different countries including Turkey (Baytop, 1999; Cakilcioglu and Turkoglu, 2010; Khan et al., 2008). Previously, some bioactive compounds were reported in the genus (El-Mousallamy et al., 2000; Khan et al., 2008; Sokkar et al., 2013; Zengin et al., 2014) but so far, there is no information about biological activities and chemical fingerprint of C. integerrimus. Therefore, the objectives of the present work were (i) to evaluate total and individual phenolic components of two extracts (methanol and water) from two parts (twigs and fruits) of C. integerrimus; (ii) to assess their biological activities including antioxidant, enzyme inhibitory, antimicrobial, mutagenic and antimutagenic effects.; (iii) to design molecular approach for enzyme inhibitory effect; (iv) to provide a starting point for new studies on other Cotoneaster species. The obtained results could contribute to the development of new functional food ingredients and drug formulations.

Materials and methods

Plant material

Cotoneaster integerrimus was collected from Kayseri-Turkey (Kayseri-Hisarcik, dry slopes) during the end of flowering season (2014). Taxonomic identification of the plant material was confirmed by the senior taxonomist Dr. Murad Aydin Sanda, from the Department of Biology, Selcuk University. The voucher specimen has been deposited at the Herbarium of the Department of Biology, Selcuk University, Konya-Turkey (Voucher number: TY7629).

Preparation of the solvent extracts

To produce methanol extracts, the air-dried samples (5g) of twigs or fruits of Cotoneaster integerrimus were macerated with 100 ml of solvent with methanol at room temperature for 24 h. For water extracts, the air-dried samples (5g) were extracted by boiling deionized water (100 ml) for 15 min. Methanol was removed with a rotary evaporator. The water extract was freeze-dried. All extracts were stored at +4[degrees]C until analyzed. The extraction yields are given in Table 1.

Total phenolics, flavonoids and phenolic composition

The total phenolics content was determined by Folin-Ciocalteu method (Slinkard and Singleton, 1977) with slight modification and expressed as gallic acid equivalents (GAEs/g extract), while total flavonoids content was determined by Al[Cl.sub.3] method (Zengin et al., 2014) with slightly modification and expressed as rutin equivalents (REs/g extract).

Phenolic compounds were evaluated by RP-HPLC (Shimadzu Scientific Instruments, Tokyo, Japan). Detection and quantification were carried out with a LC-10ADvp pump, a Diode Array Detector, a CTO-10Avp column heater, SCL-10Avp system controller, DGU-14A degasser and SIL-10ADvp auto sampler (Shimadzu Scientific Instruments, Columbia, MD). Separations were conducted at 30[degrees]C on Agilent[R] Eclipse XDB C-18 reversed-phase column (250 mm x 4.6 mm length, 5 [micro]m particle size). Phenolic compositions of the extracts were determined by a modified method of Zengin et al. (2014). Gallic acid, protocatechuic acid, (+)-catechin, p-hydroxybenzoic acid, chlorogenic acid, caffeic acid, (-)-epicatechin, syringic acid, vanillin, p-coumaric acid, ferulic acid, sinapic acid, benzoic acid, o-coumaric acid, rutin, hesperidin, rosmarinic acid, eriodictyol, trans-cinnamic acid, quercetin, luteolin, kaempferol and apigenin were used as standards. Identification and quantitative analysis were done by comparison with standards. The amount of each phenolic compound was expressed as miligram per gram of extract using external calibration curves, which were obtained for each phenolic standard.

Biological activities evaluation

Antioxidant (DPPH radical scavenging, reducing power (CUPRAC and FRAP), phosphomolybdenum and metal chelating (ferrozine method)) and enzyme inhibitory activities (cholinesterase (Elmann's method), tyrosinase (dopachrome method), [alpha]-amylase (iodine/potassium iodide method) and [alpha]-glucosidase (chromogenic PNPG method)) determined by the method described by Zengin et al. (2014). The results were expressed as [IC.sub.50] (the concentration of plant extract required to scavenge 50% of free radical or enzyme) and [EC.sub.50] (the effective concentration at which the absorbance was 0.5). Antimicrobial, mutagenic/antimutagenic properties were determined with broth-dilution and Ames assays, respectively. (Zengin et al., 2014)

Molecular docking

Ligand preparation

(-)-Epicatechin structure was downloaded from the Zinc database (Irwin and Shoichet, 2005). The molecule was minimized by AMI force field and by Amber until converged to minimum potential energy with limit of RMSD of 0.01 Kcal/A mol.

Enzymes preparation

Acetylcholinesterase (AchE), crystal structure PDB; 1B41 (Kryger, et al., 2000) [alpha]-Amylase crystal structure, PDB:4GQR (Williams et al., 2012), Mushroom Tyrosinase crystal PDB: 2Y9X (ismaya et al., 2011) and [alpha]-Glucosidase crystal structure PDB: 3W37 (Tagami et al., 2013) were downloaded from the RCSB Protein Data Bank (Berman et al., 2000). The structures were manually cleaned from crystallization water molecules, salts and other small molecules by PyMol (PyMOL Molecular Graphics System), the structures were neutralized at pH = 7, and fixed from errors by the Swiss Pdb Viewer (Guex and Peitsch, 1997). All the enzymes were processed with Gromacs 4.6 (Berndsen et al., 1995; Lindahl et al., 2001). In case of AchE, crystallized without inhibitor, the topology of the enzyme was calculated by pdb2gmx routine included in Gromacs 4.6, by using the OPLS-AA/L force field, then the enzyme was solvated into a cubic box of water, generated considering 1 nm distance from the edge of the enzyme. In case of [alpha]-Amylase, Tyrosinase and [alpha]-Glucosidase, the enzymes were processed with Gromacs 4.6 in complex with their co-crystallized inhibitors (Berndsen et al., 1995). The topology of the enzymes were calculated by pdb2gmx routine included in Gromacs 4.6, by using the GROMOS96 43al force field. The topology of the inhibitors, Myricetin (for cr-Amylase), Tropolone (for Tyrosinase) and Acarbose (for [alpha]-Glucosidase) were calculated by submitting the molecules to the PRODRG2 server and processed by Amber force field (Schiittelkopf and Van Aalten, 2014) then manually added to the topology files of the enzymes in order to generate the respective enzyme-inhibitor complexes. Then, each enzyme-inhibitor complex was solvated into a cubic box of water, as previously described for AchE. The net negative charge of the complexes was neutralized by adding [Na.sup.+] ions. Finally the systems were minimized by the energy minimization, by a steepest descent minimization, through the Gromacs 4.6 MD engine mdrun, until they reached the minimum potential energy. At this point the minimized enzymes were extracted from the water box, and used for docking experiments.

Docking experiments

The docking experiments were carried out by Vina Autodock (Trott and Olson, 2010). The grid for docking was generated by Autodock tools (ADT) (Sanner, 1999) by centring the grid on the co-crystallized inhibitor present in the crystal structures downloaded. Only in the case of acetylcholinesterase, the binding pocket was centred around the residues His447, covering all the residues reported in literature to be part of the enzymatic pocket: Ser203, Glu334, Tyrl24, Trp286, Tyr341, Asp74 (Lushington et al., 2006). The ratable bonds of (-)-epicatechin were selected by default. After the docking calculations, Vina Autodock generated nine poses for each enzyme-(-)-epicatechin complex. In Fig. 1 are reported the binding for each enzyme (-)-epicatechin complex. In Fig. 2 are reported the interactions found between the enzyme and the (-)-epicatechin, calculated by Scroedinger Maestro free tool (Schrodinger Release, 2015).

Statistical analysis

All the assays were carried out in triplicate. The results were expressed as mean values and standard deviations (mean [+ or -] SD). Statistical differences between the samples were analyzed using one-way analysis of variance (ANOVA) followed by Tukey's honestly significant difference post hoc test with a =0.05, respectively. All the analyses were carried out by using SPSS v14.0 software.

Results and discussion

Phytochemical composition

Phenolic compounds are the most important group of plant bioactive metabolites. Total phenolic and flavonoid contents of C. integerrimus extracts are shown in Table 1. The highest phenolic content was detected in T-Me with 115.15 mgGAEs/g extract, followed by T-W and F-W, respectively. The total phenolic content of T-Me was over three times higher than those in F-Me, which had the lowest total phenolic content. As for total flavonoid content, a similar trend was observed with total flavonoids and the content ranged between 2.03 and 16.29 mgREs/g extract. The flavonoid content of T-W was about 2-fold higher than that of F-W. Apparently, the twig extracts had the higher levels of bioactive compounds as compared to fruit extracts. In our previous study (Zengin et al., 2014), the water extract of C. nummularia had the highest level of phenolics (266.39 mgGAEs/g extract) as compared to ethyl acetate (81.11 mgGAEs/g extract) and methanol (251.82 mgGAEs/g extract). The results indicate that content of phenolics in C. nummularia extracts (water and methanol) is higher than that in all C. integerrimus extracts.

The C. integerrimus extracts were analyzed with a HPLC-DAD in order to identify their main components. The identified components are listed in Table 2. 23 standard phenolics were analyzed and which 18 of them were identified in the studied extracts.

(-)-epicatechin was the major phenolic in the all extracts and the content ranged from 9.27 (in F-W) to 32.89 mg/g extract (in T-W).

Other major components in the extracts were ferulic acid, chlorogenic acid, (-t-)-catechin and p-hydroxybenzoic acid. Syringic acid, vanillin, luteolin, kaempferol, sinapic acid, benzoic acid, rosmarinic acid were not detected in these extracts. The major components exhibit a broad spectrum of biological activities including antioxidant, anticancer, antimicrobial, enzyme inhibitors and antimutagenic (Cox et al., 2015; Geetha et al., 2004; Park et al., 2004; Shay et al., 2015; Yilmaz and Toledo, 2004). Thus, the bioactive components may be contributed to the biological activities of the studied extracts. Also, the major components could be considered as important indicators to evaluate biological and chemical fingerprints. In contrast to our results, ferulic acid was found to be major phenolic in C. nummularia extracts, followed by chlorogenic acid and (-)- epicatechin (Zengin et al., 2014).

Antioxidant properties

The use of only one method does not reflect the antioxidant activity of plant extracts. In this sense, the antioxidant effects of C. integerrimus were evaluated by different spectrophotometric assays including free radical scavenging (DPPH), reducing power (FRAP and CUPRAC), phosphomolybdenum and metal chelating. (For further details of the reactions on (-)-epicatechin see the Supporting Material). The results of the antioxidant assays are summarized in Table 3.

DPPH is a stable free radical and widely used to evaluate free radical scavenging abilities of plant extracts. The antioxidant compounds react with the radical and convert to picryl hydrazine from as evidenced through discoloration (from purple to yellow). The discoloration is reflected hydrogen donation ability of antioxidant compounds. As shown in Table 3, the free radical scavenging activity can be ranked as T-Me > T-W > F-W > F-Me. Also, T-Me exhibited two times more reducing ability in the assay than F-Me. None of the tested extracts exceed trolox antioxidant efficiency. [IC.sub.50] for trolox was detected at concentration of 0.19 mg/ml.

Reducing ability is considered as one of the most important antioxidant mechanisms, which can reflect electron donating ability of antioxidants. For this purpose, the reductive abilities of the extracts were tested by CUPRAC and FRAP assays. Similar to DPPH results, T-Me and T-W presented the highest cupric and ferric reducing ability, respectively. In any case, the reducing abilities of the extracts were lower than that of trolox. Phosphomolybdenum assay based on the reduction of Mo (VI) to Mo (V) by antioxidants and then green phosphate/Mo (V) complex at acidic pH. The decreasing sequence of phosphomolybdenum activities of the tested extracts was T-Me> T-W>F-Me>FW. Also, T-Me was more potent than trolox.

Transition metals (especially iron), play role as a pro-oxidant in the lipid peroxidation. From this point, antioxidant compounds, namely metal chelators, can deactivate these metal ions and prevent lipid oxidation. As shown in Table 3, the water extracts showed better activities as compared to methanol extracts. Interestingly, T-Me exhibited the weakest chelating activity, which possess the highest phenolic concentration. The metal chelating activity decreased in the following order: T-W>F-W>F-Me>T-Me. However, EDTA is excellent chelator with [IC.sub.50] of 28.90 [micro]g/ml.

At the whole, our results are displayed that phenolic contents are consistent with the assessed antioxidant test (except for metal chelating). Generally, the DPPH, CUPRAC, FRAP and phosphomolybdenum values of the extracts decreased in the same order as the total phenolic content (T-Me>T-W>F-W>F-Me). In this context, the phenolic compounds could be considered as major contributors to antioxidant effect of C. integerrimus extracts. These approaches were verified by many researchers who suggested that phenolics are major antioxidants (Carocho and Ferreira, 2013; Prochazkova et a!., 2011; Sevgi et al., 2015). Also, similar results were obtained in our previous study for C. nummularia extracts (Zengin et al., 2014). As to metal chelating activity, several researchers claimed that the metal chelation play a minor role in all antioxidant properties of phenolic compounds (Rice-Evans et al., 1996). Again, the observed metal chelating activity could be explained with the presence of some non-phenolic chelators, for example peptides and ascorbic acid (Nithiyanantham, et al., 2013).

Inhibitory potentials on pharmacological enzymes

In recent decades, Alzheimer's disease (AD) and Diabetes mellitus (DM) are considered as global health problems because the prevalence of the diseases will grow by day by. For example, someone in USA develops AD every 67 seconds in today. By 2050, one new case of AD is expected to develop every 33 seconds. (Alzheimer Association, 2014) Similar to AD, 366 million people are affected with DM and this is expected to rise to 552 million by 2030 (Whiting et al., 2011). From this point, effective approaches to prevent, delay and treat to these diseases are urgently needed. In this direction, key enzyme inhibitory theory is assumed as one of the most effective strategy for the treatment of the health problems. According to this theory, acetylcholinesterase (AChE) manages the level neurotransmitter (acetylcholine (ACh)) at synaptic area (Murray et al., 2013). Deficit in the neurotransmission are major complication in the AD and thereby AChE inhibitors are significant for the progression of AD. As for DM, the inhibition of carbohydrate metabolism enzymes (especially [alpha]-amylase and [alpha]-glucosidase) is an effective tool for the management of DM. The enzymes catalyze the digestion of carbohydrate and increase blood glucose level (Kumar et al., 2012). Again, tyrosinase is a key enzyme in the synthesis of melanin and the inhibition of tyrosinase could be considered as a major therapeutic approach for the treatment of skin disorders (SD) (Loizzo et al., 2012). At this point, several synthetic and oral inhibitors are designed to manage the mentioned diseases, but they exhibited several side effects such as gastrointestinal disorders and hepatotoxicity (Chen et al., 2012; Chopra et al., 2011; Dong et al., 2012). To this end, many researches focused on alternative inhibitors from natural sources that could manage the mentioned health problems with fewer side effects.

The enzyme inhibitory activities of the extracts were tested against AChE, BChE, tyrosinase, [alpha]-amylase and [alpha]-glucosidase using ELISA microplate reader. The results are given in Table 4. T-Me exhibited the strongest AChE inhibitory activity with [IC.sub.50] of 1.07 mg/ml, followed by F-Me, T-W and F-W. The results revealed the AChE inhibitory activity of T-W was 10-fold higher than that of F-W. Clearly, methanol extracts were more active against AChE than water extracts. However, BChE inhibitory activities of the extracts can be ranked as T-W> F-W > F-Me> T-Me. In addition, galanthamine has an excellent inhibitor effect on both AChE and BChE. The observed differences could be explained with the differences of active site in the enzymes. Similar case was reported by several researchers (Senol et al., 2010; Tundis et al., 2015). As to tyrosinase inhibitor effects, the best inhibitory effect were observed in T-Me and F-W found to be inactive against tyrosinase. Similar to tyrosinase inhibitory activity, the highest [alpha]-amylase and [alpha]-glucosidase inhibitory activity was achieved by T-Me. Again, the observed [alpha]-glucosidase inhibitory activity appears quite higher than that of the standard anti-diabetic drug, namely acarbose. T-W and F-Me had the lowest inhibitory potential against [alpha]-amylase and [alpha]-glucosidase, respectively. The strongest activity for T-Me might be related to the higher concentration of phenolics in the extract. These results are in accordance with Fatiha et al (2015) and Zhang et al (2015) who reported that there is linear correlation between total phenolics and enzyme inhibitory activity. Also, the same approaches were obtained in C. nummularia extracts (Zengin et al., 2014). Again, several phenolics were reported as strong tyrosinase, [alpha]-amylase and [alpha]-glucosidase inhibitors. To summarize, T-Me had the higher inhibitory activity on tested enzymes (except for BChE) as compared to other extracts. To the best of our knowledge, the enzyme inhibitory activity of C. integerimus is reported here for the first time.

Molecular docking

As shown in Figs. 1 and 2, (-)-epicatechin is able to penetrate in the enzymatic pocket, of all the enzymes analysed in this experiment, in the same way of the respective co-crystallized inhibitors. The most interesting aspect is that this molecule can form several hydrophobic interactions with the residues surrounding the enzymatic pockets. In case of the tyrosinase, the complex is also stabilized by one hydrogen bond between (-)-epicatechin and Met280, in the case of [alpha]-glucosidase the complex is stabilized by two hydrogen bonds with Tyr357 and His626. Tyrosinase enzyme is a metal-enzyme containing two atoms of copper, but it was found that (-)-epicatechin doesn't bind directly to these atoms but has a hydroxyl group directed toward one Cu atom, in a similar way of tropolone.

The inhibition mechanism is rather attributable to a competition with the substrate for the enzymatic pocket. The docking scores are reported in Table 5 for each enzymes. The best value was reached with the enzyme [alpha]-amylase, however the docking score of the best pose calculated on different enzymes, should not be used for a direct explanation of the activity. It looks reasonable to conclude that the range of [IC.sub.50] is the same for the four enzymes and it falls in the micromolar range, basing on the in vitro enzymatic assays (Table 4).

Antimicrobial evaluation

In this study, extracts of fruit and twigs of C. integerrimus were evaluated in terms of antimicrobial and anti-MRSA activity by broth microdilution method according to Zengin et al. (2014). Obtained results are presented in Table 6

According to the results, fruit methanol and water extracts of C. integerrimus exhibited weak antimicrobial activity with the doses ranging between 12.5-6.25 mg/ml. Except for MRSA strain 8 (ES 93) all test microorganisms were resistant to these extracts. Especially, standard bacteria strains were resistant to water extract of fruit and no MIC values were observed against those strains. T-Me extract revealed significant antimicrobial activity at doses ranging between 0.195-6.25 mg/ml. S. aureus ATCC 43300 (MRSA), MRSA 1 to 7, and 10, 11, and 14 numbered strains were affected from this extract. Also, the lowest MIC values were determined against MRSA 8, 9, 12 and 13 numbered strains (Table 6). It was seen that T-Me extract of C. integerrimus had strong anti-MRSA activity and moderate antifungal capacity. When the T-W extract was evaluated, it was determined that the MIC values were distributed ranging between 0.390-12.5 mg/ml (Table 6). Although standard bacteria affected from this extract at concentrations of 1.562-12.5 mg/ml, these MIC values can be considered as weak antibacterial activity. But water extract showed moderate antifungal activity against C. albicans. While anti-MRSA activity was also observed against MRSA isolates 6 to 9, and 11 to 13 at a concentration of 0.390, MIC values were determined as 0.781 mg/ml against MRSA 1-5,10, and 14 numbered strains.

Our results showed that T-Me and T-W extracts of C. integerrimus manifested significant antifungal and anti-MRSA activity. In our previous study, methanol, ethyl acetate, and water extracts of Cotoneaster nummularia manifested antibacterial and anti-MRSA activity at concentrations ranging between 0.039-2.5 mg/ml (Zengin et al., 2014). In general it is obviously seen that C. integerrimus twig extracts are more effective than C. nummularia extracts against standard bacteria and MRSA isolates. In that study, ferulic acid, catechin and epicatechin were major phenolic compounds in C. nummularia and significant biological activities were related to these phenolics. Also epicatechin, ferulic acid and catechin are major ingredient of the T-Me extracts, so the remarkable antimicrobial activities of C. integerrimus may be attributed to the presence of these phenolics. These approaches were confirmed by several researchers (Cetin-Karaca and Newman, 2015; Park et al., 2004).

Mutagenic/Anti-mutagenic evaluation

Table 7 shows the mean number of revertants/plate, the standard deviation and the mutagenic ratio (MR) after the treatments with the four extracts of C. integerrimus, observed in S. typhimurium strains TA98 and TA100 in the presence (+S9) and absence (-S9) of metabolic activation. The results showed that only positive control plates induced an increase in the number of revertant colonies relative to the negative control, indicating mutagenic activity reaching a mutagenic ratio of higher than 2.0 (Table 7). None of the extracts induced two fold or greater increase in the mean number of revertants relative to the negative control group, in the presence or absence of S9. This means that C. integerrimus extracts were not found to be mutagenic in Ames Assay (Table 7).

The antimutagenic effect of each extract was estimated from the mean number of revertants/plate, the standard deviation (SD) and the percent inhibition (% I) of the mutagenic activity of 4-NPDA and 2-AF for TA98 strain; SA and 2-AA for TA100 strain on treatment with the three concentrations of the plant extracts. The results obtained from the assay were presented in Table 8. Cotoneaster T-Me extract can be considered as strong antimutagenic at a dose of 5000 [micro]g/plate against 4-NPDA, while other two concentrations were moderately antimutagenic for TA 98 strain without S9. When tested against 2-AF in the presence of S9, T-Me revealed excellent antimutagenic activities with ratios of 94% and 82% at concentrations of 5000 and 1000 [micro]g/plate, respectively (Table 8). Similarly T-Me extract manifested excellent antimutagenic activity against 2-AA at doses of 5000 and 1000 [micro]g/plate, respectively in the presence of metabolic activation for TA100 strain, while these doses were strong and moderately antimutagenic against SA without S9 (Table 8). It can be stated that S9 enzymes ameliorated the action of mutagens at higher doses. While T-W extract showed moderately antimutagenic activity at 5000 and 1000 [micro]g/plate doses against 4-NPDA in the absence of S9 for TA98 strain, it exhibited more than 40% inhibition against 2-AF, and concentrations in the range of 10,000-1000 [micro]g/plate achieved 91% and 73% inhibition, respectively, making the extract a very strong antimutagenic in the presence of metabolic activation system for TA98 (Table 8). Also 500|xg/plate concentration was found to be moderate antimutagenic with S9. Associated with SA, T-W was described as strong and moderately antimutagenic at concentrations of 5000 and 1000 [micro]g/plate for TA100 strain without S9 mix. By the addition of S9 enzymes, inhibition ratio and antimutagenic activity were increased from 41% to 74% against 2-AA, making it very strong antimutagenic (Table 8).

When the F-Me extract evaluated, it was determined that only 5000 [micro]g/plate dose of extract showed strong antimutagenic activity with a rate of 46% in the absence of S9 for TA98 (Table 8). The other doses were found to be moderately antimutagenic. When the F-Me treated with 2-AF, the extract revealed only moderate antimutagenic activity at a dose of 5000 [micro]g/plate for TA98 with S9. The others were determined to be non antimutagenic (Table 8). In contrast to this situation, F-Me extract was found to be non antimutagenic against SA in the absence of S9 for TA100 strain, but 5000 [micro]g/plate dose of extract showed moderately antimutagenic activity by the addition of S9 mix against 2-AA (Table 8). When combined with 4-NPDA, F-W extracts induced the inhibition greater than 40%, reaching 60%, 75%, and 79%, respectively in the absence of S9 for TA 98 and ranking them as strongly antimutagenic (Table 8). But only 5000 [micro]g/plate doses were assigned to be moderately antimutagenic against mutagenic action of 2-AF in the presence of S9 for TA98 strain. The lower doses showed no antimutagenic activity. The same situation was observed for TA100 strain. While F-W extract showed strong antimutagenic activity against SA at a dose of 5000 [micro]g/plate for TA100, the antimutagenicity ratios of F-W against 2-AA were decreased by the addition of S9 metabolic activation enzymes and these doses were determined to be weak antimutagenic (Table 8). In our previous study, we determined that Cotoneaster nummularia water extract revealed strong antimutagenic action against 2-AF and 2-AA with inhibition rates greater than 40%, reaching 49%, 50%, 55%, and 59% in the presence of S9 both for TA98 and TA100 strains (Zengin et al., 2014). In this study methanol and water extracts of Cotoneaster twig and fruit manifested excellent antimutagenic activity against mutagenic action of 2-AA and 2-AF at the highest doses (5000 [micro]g/plate). The ratio of strong inhibitions of mutagens by C. integerrimus extracts found in this study has a strong consistency with the results of study conducted by Zengin et al. (2014).

Overall, it can be stated from the study that metabolic activation enzymes increased inhibition rates, reaching 96% at some concentrations, against mutation induced by 2-AF and 2-AA both for TA98 and TA100 strains. But in some extracts S9 decreased the antimutagenicity rates. These results suggest that extracts, with high antimutagenic activity in the presence of S9, should be suitable for evaluation concerning CyP450 modulations effects and it could be explained by finding that medicinal plants might contain compounds capable of inhibiting the CyP450 required for activating these mutagens (Del-Toro-Sanchez et al., 2014). Buening et al. (1981) determined that some plant metabolites are potent inhibitors of cytochrome c (P450) reductase. As a result, Cotoneaster extracts have significant antimutagenic capacities against well known mutagens and it can be considered as a natural antimutagenic agent source. This situation might be explained with the presence of (-)- epicatechin and similarly it was reported by Geetha et al (2004) as the most effective antimutagenic agent.

Conclusion

As far as we know, this study is the first report on biological and chemical profile of C. integerrimus extracts. The extracts have remarkable biological effects with higher level of phenolics. Generally, twig extracts exerted stronger biological activities as compared to fruit extracts, (-)-epicatechin might be major component responsible for the biological activity as detected in the study. This approach was confirmed by chemical reactions and molecular docking analysis. Finally, C. integerrimus proved to be an excellent source of natural-bioactive agents and the plants seems to serve as prospective material for the exploration of new multi-functional food and drug formulations. This study could provide new perspectives on other Cotoneaster species.

http://dx.doi.org/10.1016/j.phymed.2016.06.011

ARTICLE INFO

Article history:

Received 23 December 2015

Revised 12 April 2016

Accepted 14 June 2016

Conflict of interest

We wish to confirm that there are no known conflicts of interest associated with this publication and there has been no significant financial support for this work that could have influenced its outcome.

Supplementary materials

Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.phymed.2016.06.01l.

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Ahmet Uysal (a), Gokhan Zengin (b), *, Adriano Mollica (c), Erdogan Gunes (b), Marcello Locatelli (c,d), Turgut Yilmaz (e), Abdurrahman Aktumsek (b)

(a) Deparment of Medicinal Laboratory, Vocational School of Health Services, Selcuk University, Turkey

(b) Selcuk University, Science Faculty, Department of Biology, Konya-Turkey

(c) University "G. d'Annunzio" Chieti-Pescara, Department of Pharmacy, 66100, Chieti-Italy

(d) Interuniversity Consortium of Structural and Systems Biology, 00136, Roma- Italy

(e) Ankara University, Faculty of Pharmacy, Department of Pharmaceutical Botany, Ankara-Turkey

Abbreviations: ABTS, 2,2'-azino-bis(3-ethylbenzothiazoline)-6-sulfonic acid; AChE, acetylcholinesterase; BChE, butyrylcholinesterase; ChE, Cholinesterase; CUPRAC, Cupric reducing antioxidant capacity; DPPH, 1,1-diphenyl-2- picrylhydrazyl; FRAP, ferric reducing antioxidant power; GAE, gallic acid equivalent; PNPG, 4- Ntrophenyl-[alpha]-D-glucopyranoside; RE, rutin equivalents; RP-HPLC, reversed-phase high-performance liquid chromatography.

* Corresponding author at: Department of Biology, Selcuk University, Konya- Turkey.

E-mail address: gokhanzengin[R]selcuk.edu.tr (G. Zengin).

Table 1
Extraction yields, total phenolic and flavonoid
contents of the extracts (meaniSD) *.

Part     Solvent    Abbreviations   Yields (%)

Twig     Methanol   T-Me            15.25
         Water      T-W             14.13
Fruit    Methanol   F-Me            32.70
         Water      F-W             25.35

         Total phenolics          Total flavonoids
Part     (mg GAEs/g extract) **   (mg REs/g extract) ***

Twig     115.15 [+ or -] 1.39a    16.29 [+ or -] 0.43a
         96.98 [+ or -] 0.97b     6.02 [+ or -] 0.16b
Fruit    38.47 [+ or -] 0.57d     2.03 [+ or -] 0.20d
         42.70 [+ or -] 0.61c     3.96 [+ or -] 0.15c

* Different letters (a, b, c and d) in the extracts
indicate significant difference (p< 0.05).

** CAEs, gallic acid equivalents.

*** REs, rutin equivalents.

Table 2
Phenolic components in the solvent extracts from
Cotoneaster integerrimus (mg-g extract) (mean [+ or -] SD) *.

No   Phenolic Components    T-Me                  T-W

1    Gallic acid            0.04 [+ or -] 0.001a  nd
2    Protocatechuic acid    0.68 [+ or -] 0.04b   0.89 [+ or -] 0.04a
3    (+)- Catechin          3.95 [+ or -] 0.02a   2.27 [+ or -] 0.12b
4    p-Hydroxybenzoic acid  2.51 [+ or -] 0.03b   2.37 [+ or -] 0.03c
5    Chlorogenic acid       6.81 [+ or -] 0.08b   8.29 [+ or -] 0.08a
6    Caffeic acid           1.45 [+ or -] 0.04b   1.58 [+ or -] 0.04a
7    (-)- Epicatechin       19.05 [+ or -] 1.15b  32.89 [+ or -] 1.13a
8    Syringic acid          nd                    nd
9    Vanilin                nd                    nd
10   p- Coumaric acid       0.11 [+ or -] 0.01b   0.29 [+ or -] 0.01a
11   Ferulic acid           4.48 [+ or -] 0.02b   9.26 [+ or -] 0.26a
12   Sinapic acid           nd                    nd
13   Benzoic acid           nd                    nd
14   o-Coumaric acid        0.01 [+ or -] 0.01c   0.09 [+ or -] 0.01a
15   Rutin                  0.21 [+ or -] 0.01b   nd
16   Hesperidin             0.14 [+ or -] 0.01a   0.15 [+ or -] 0.01a
17   Rosmarinic acid        nd                    nd
18   Eriodictyol            0.61 [+ or -] 0.02a   0.10 [+ or -] 0.01b
19   trcms-Cinnamic acid    nd                    0.14 [+ or -] 0.01a
20   Quercetin              0.94 [+ or -] 0.02a   nd
21   Luteolin               nd                    nd
22   Kaempferol             nd                    nd
23   Apigenin               nd                    0.30 [+ or -] 0.01

No   F-Me                   F-W

1    0.04 [+ or -] 0.01b    nd
2    nd                     nd
3    0.06 [+ or -] 0.01c    nd
4    2.90 [+ or -] 0.03a    1.40 [+ or -] 0.02d
5    4.54 [+ or -] 0.11c    3.99 [+ or -] 0.08d
6    0.87 [+ or -] 0.02c    0.83 [+ or -] 0.02c
7    11.36 [+ or -] 0.24c   9.27 [+ or -] 1.19d
8    nd                     nd
9    nd                     nd
10   0.03 [+ or -] 0.01d    0.06 [+ or -] 0.01c
11   nd                     nd
12   nd                     nd
13   0.69 [+ or -] 0.01b    0.95 [+ or -] 0.01a
14   0.03 [+ or -] 0.01c    0.07 [+ or -] 0.01b
15   0.29 [+ or -] 0.01a    0.31 [+ or -] 0.01a
16   nd                     nd
17   nd                     nd
18   nd                     nd
19   nd                     0.05 [+ or -] 0.01b
20   nd                     0.07 [+ or -] 0.01b
21   nd                     nd
22   nd                     0.17 [+ or -] 0.02
23   nd                     nd

* Different letters (a, b, c and d) in the extracts indicate
significant difference (p < 0.05). nd, not detected.

Table 3
Total antioxidant (by phosphomolybdenum method),
radical scavenging, reducing power and metal chelating
activities of the extracts (mean [+ or -] SD) *.

           Phosphomolybdenum      CUPRAC
           (E[C.sub.50]:          (E[C.sub.50]:
Sample     [micro]g/ml)           [micro]g/ml)

T-Me       0.36 [+ or -] 0.015d   336.50 [+ or -] 12.78c
T-W        0.53 [+ or -] 0.020c   348.98 [+ or -] 4.54c
F-Me       1.24 [+ or -] 0.023b   657.42 [+ or -] 12.41a
F-W        1.38 [+ or -] 0.041a   596.21 [+ or -] 5.81b
Trolox     0.41 [+ or -] 0.004d   62.76 [+ or -] 1.03d
EDTA **    --                     --

           FRAP
           (E[C.sub.50]:            DPPH (I[C.sub.50]:
Sample     [micro]g/ml)             mg/ml)

T-Me       265.38 [+ or -] 3.20c    1.06 [+ or -] 0.001c
T-W        257.35 [+ or -] 3.09c    1.09 [+ or -] 0.001c
F-Me       498.82 [+ or -] 12.89a   2.44 [+ or -] 0.074a
F-W        376.41 [+ or -] 1.85b    1.85 [+ or -] 0.016b
Trolox     34.13 [+ or -] 0.08d     0.19 [+ or -] 0.006d
EDTA **    --                       --

           Chelating effect
Sample     (I[C.sub.50]: mg/ml)

T-Me       6.24 [+ or -] 0.087a
T-W        1.47 [+ or -] 0.024c
F-Me       6.14 [+ or -] 0.121a
F-W        2.14 [+ or -] 0.018b
Trolox     -
EDTA **    28.90 [+ or -] 0.100d

* Different letters (a, b, c and d) in the extracts indicate
significant difference (p< 0.05).

** EDTAE, disodium edetate (as [micro]g/ml).

Table 4
Enzyme inhibitory activity of the extracts
(mean [+ or -] SD) *.

Sample             Acetyl cholinesterase   Butyryl cholinesterase
                   (I[C.sub.50]: mg/ml)    (I[C.sub.50]: mg/ml)

T-Me               1.07 [+ or -] 0.001c    5.98 [+ or -] 0.373a
T-W                2.11 [+ or -] 0.022b    2.80 [+ or -] 0.022c
F-Me               1.72 [+ or -] 0.035b    3.89 [+ or -] 0.091b
F-W                12.84 [+ or -] 0.232a   2.67 [+ or -] 0.412c
Galanthamine **    2.60 [+ or -] 0.001d    5.10[+ or -]0.100d
Kojic acid         --                      --
Acarbose           --                      --

Sample             Tyrosinase             [alpha]-Amylase
                   (I[C.sub.50]: mg/ml)   (I[C.sub.50]: mg/ml)

T-Me               1.35 [+ or -] 0.002c   2.02 [+ or -] 0.078cd
T-W                4.15 [+ or -] 0.128a   11.61 [+ or -] 0.259b
F-Me               1.84 [+ or -] 0.009b   2.72 [+ or -] 0.021c
F-W                ni ***                 14.80 [+ or -] 0.931a
Galanthamine **    --                     --
Kojic acid         0.13 [+ or -] O.OOld   --
Acarbose           --                     1.30 [+ or -] 0.006d

Sample             [alpha]-Glucosidase
                   (I[C.sub.50]: mg/ml)

T-Me               1.26 [+ or -] 0.085d
T-W                5.22 [+ or -] 0.081b
F-Me               8.81 [+ or -] 0.191a
F-W                5.25 [+ or -] 0.021b
Galanthamine **    --
Kojic acid         --
Acarbose           2.19 [+ or -] 0.102c

* Different letters (a, b, c and d) in the extracts indicate
significant difference (p < 0.05).

** As [micro]g/ml.

*** ni, no inhibition.

Table 5
Docking scores of (-)-epicatechin on
acetylcholinesterase, [alpha]-amylase, tyrosinase,
[alpha]-glucosidase calculated by Vina Autodock.

(-)-epicatechin    Acetylcholinesterase   [alpha]-Amylase

                   Docking scores (Kcal / mol)

Pose 1             -8.1                   -8.7
Pose 2             -7.9                   -7.9
Pose 3             -7.8                   -7.8
Pose 4             -7.5                   -7.8
Pose 5             -6.9                   -7.8
Pose 6             -6.8                   -7.2
Pose 7             -6.7                   -7.2
Pose 8             -6.5                   -7.2
Pose 9             -6.3                   -7.1

(-)-epicatechin    Tyrosinase   [alpha]-Glucosidase

                   Docking scores (Kcal / mol)

Pose 1             -6.8         -7.7
Pose 2             -6.7         -7.5
Pose 3             -6.5         -7.0
Pose 4             -6.5         -7.0
Pose 5             -6.1         -6.8
Pose 6             -6.1         -6.8
Pose 7             -6.1         -6.5
Pose 8             -6.1         -6.3
Pose 9             -6.0         -6.3

Table 6
MIC values of Cotoneaster integerrimus extracts
against standard microorganisms and MRSA isolates.

Tested Microorganisms       MIC values of Cotoneaster
                            Extracts (mg/ml)

                            F-Me       F-W        T-Me       T-W

Escherichia coli ATCC       6.25       --         6.25       12.5
  25922
Pseudomonas aeruginosa      6.25       --         6.25       12.5
  ATCC 15442
Staphylococcus aureus       6.25       --         1.5625      0.7812
  ATCC 25923 (MSSA)
Klebsiella pneumoniae       6.25       --         6.25       12.5
  ATCC 70603
Staphylococcus aureus       6.25       12.5       0.3906      0.7812
  ATCC 43300 (MRSA)
Salmonella enteritidis      6.25       --         3.125      12.5
  ATCC 13076
Streptococcus pneumoniae    6.25       --         1.5625      3.125
  ATCC 10015
Sarcina lutea ATCC 9341     6.25       --         0.7812      1.5625
Candida albicans            6.25        6.25      0.3906      0.3906
Candida parasilopsis        6.25        6.25      3.125       0.7812
MRSA strain 1 (ES 16)       6.25        6.25      0.3906      0.7812
MRSA strain 2 (ES 25)       6.25       12.5       0.3906      0.7812

MRSA strain 3 (ES 29)       6.25        6.25      0.3906      0.7812
MRSA strain 4 (ES 67)       6.25       12.5       0.3906      0.7812
MRSA strain 5 (ES 68)       6.25       12.5       0.3906      0.7812

MRSA strain 6 (ES 69)       6.25        6.25      0.3906      0.3906

MRSA strain 7 (ES 75)       6.25       12.5       0.3906      0.3906

MRSA strain 8 (ES 93)       1.5625      6.25      0.1953      0.3906

MRSA strain 9 (ES 100)      6.25        6.25      0.1953      0.3906

MRSA strain 10 (ES 107)     6.25       12.5       0.3906      0.7812
MRSA strain 11 (ES 110)     6.25       12.5       0.3906      0.3906

MRSA strain 12 (ES 123)     6.25       12.5       0.1953      0.3906
MRSA strain 13 (ES 124)     6.25       12.5       0.1953      0.3906

MRSA strain 14 (ES 128)     6.25       12.5       0.3906      0.7812

Tested Microorganisms       MIC value of     MIC value of
                            Gentamicin       Oxacillin
                            ([micro];g/ml)   ([micro];g/ml)

Escherichia coli ATCC         2.44
  25922
Pseudomonas aeruginosa        9.76
  ATCC 15442
Staphylococcus aureus         2.44           0.25
  ATCC 25923 (MSSA)
Klebsiella pneumoniae         2.44
  ATCC 70603
Staphylococcus aureus        78.12           64
  ATCC 43300 (MRSA)
Salmonella enteritidis        4.88
  ATCC 13076
Streptococcus pneumoniae      2.44
  ATCC 10015
Sarcina lutea ATCC 9341       4.88
Candida albicans            312.5
Candida parasilopsis        312.5
MRSA strain 1 (ES 16)       156.25           16
MRSA strain 2 (ES 25)       312.5            [greater than or
                                               equal to] 128
MRSA strain 3 (ES 29)       312.5            32
MRSA strain 4 (ES 67)       156.25           32
MRSA strain 5 (ES 68)       156.25           [greater than or
                                               equal to] 128
MRSA strain 6 (ES 69)       312.5            [greater than or
                                               equal to] 128
MRSA strain 7 (ES 75)       156.25           [greater than or
                                               equal to] 128
MRSA strain 8 (ES 93)        78.12           [greater than or
                                               equal to] 128
MRSA strain 9 (ES 100)       78.12           [greater than or
                                               equal to] 128
MRSA strain 10 (ES 107)     156.25           8
MRSA strain 11 (ES 110)     156.25           [greater than or
                                               equal to] 128
MRSA strain 12 (ES 123)      78.12            16
MRSA strain 13 (ES 124)      78.12           [greater than or
                                               equal to] 128
MRSA strain 14 (ES 128)      78.12           [greater than or
                                               equal to] 128

Molecular docking

Table 7
Mutagenic activity expressed as mean number of
revertants/plate [+ or -] standard deviation and
mutagenicity ratio of water and methanol extracts of
Cotoneaster twig and fruit towards S. typhimurium TA98
and TA100 strains with Anri wifhnnf

                        Concentration   Number of [His.sup.+]
                        (mg/plaque)     Revertants/plaque

                                        Methanol Extracts

                                        TA 98

                                        S9 (-)                MR

([dagger])              100 [micro]1/   34 [+ or -] 3 (a)     1
  Negative Control        plaque
[R] Positive Control                    513 [+ or -] 81 (b)   15
                        0               37 [+ or -] 5 (a)     1.08
                        5000            49 [+ or -] 5 (a)     1.44
Cotoneaster Twig        1000            55 [+ or -] 3 (a)     1.61
                        500             41 [+ or -] 4 (a)     1.20
([dagger])              100 [micro]1/   34 [+ or -] 3 (a)     1
  Negative Control        plaque
[R] Positive Control                    513 [+ or -] 81 (b)   15
                        0               37 [+ or -] 5 (a)     1.08
                        5000            44 [+ or -] 5 (a)     1.29
Cotoneaster Fruit       1000            46 [+ or -] 3 (a)     1.35
                        500             42 [+ or -] 6 (a)     1.23

                        Concentration   Number of [His.sup.+]
                        (mg/plaque)     Revertants/plaque

                                        Methanol Extracts

                                        TA 98

                                        S9 (+)                  MR

([dagger])              100 [micro]1/   41 [+ or -] 5 (a)       1
  Negative Control        plaque
[R] Positive Control                    3100 [+ or -] 109 (b)   76
                        0               38 [+ or -] 6 (a)       0.92
                        5000            52 [+ or -] 3 (a)       1.27
Cotoneaster Twig        1000            45 [+ or -] 5 (a)       1.09
                        500             37 [+ or -] 3 (a)       0.9
([dagger])              100 [micr0]1/   41 [+ or -] 5 (a)       1
  Negative Control        plaque
[R] Positive Control                    3100 [+ or -] 109 (b)   76
                        0               38 [+ or -] 6 (a)       0.92
                        5000            50 [+ or -] 1 (a)       1.21
Cotoneaster Fruit       1000            48 [+ or -] 7 (a)       1.17
                        500             33 [+ or -] 6 (a)       0.8

                        Concentration   Number of [His.sup.+]
                        (mg/plaque)     Revertants/plaque

                                        Methanol Extracts

                                        TA 100

                                        S9 (-)                  MR

([dagger])              100 [micro]1/   143 [+ or -] 6 (a)      1
  Negative Control        plaque
[R] Positive Control                    2022 [+ or -] 82 (b)    14
                        0               154 [+ or -] 10 (a)     1.07
                        5000            155 [+ or -] 10 (a)     1.08
Cotoneaster Twig        1000            137 [+ or -] 21 (a)     0.95
                        500             146 [+ or -] 16 (a)     1.02
([dagger])              100 [micro]1/   143 [+ or -] 6 (a)      1
  Negative Control        plaque
[R] Positive Control                    2022 [+ or -] 82 (bc)   14
                        0               154 [+ or -] 10 (a)     1.07
                        5000            156 [+ or -] 8 (a)      1.09
Cotoneaster Fruit       1000            165 [+ or -] 7 (a)      1.15
                        500             208 [+ or -] 18 (b)     1.45

                        Concentration   Number of [His.sup.+]
                        (mg/plaque)     Revertants/plaque

                                        Methanol Extracts

                                        TA 100

                                        S9 (-)                    MR

([dagger])              100 [micro]1/   135 [+ or -] 11 (a)       1
  Negative Control        plaque
[R] Positive Control                    5244 [+ or -] 157 (b)     39 *
                        0               156 [+ or -] 9 (a)        1.15
                        5000            181 [+ or -] 14 (a) (b)   1.34
Cotoneaster Twig        1000            169 [+ or -] 6 (a)        1.25
                        500             155 [+ or -] 10 (a)       1.14
([dagger])              100 [micro]1/   135 [+ or -] 11 (a)       1
  Negative Control        plaque
[R] Positive Control                    5244 [+ or -] 157 (c)     39
                        0               156 [+ or -] 9 (a)        1.15
                        5000            183 [+ or -] 13 (b)       1.35
Cotoneaster Fruit       1000            194 [+ or -] 9 (b)        1.43
                        500             188 [+ or -] 14 (b)       1.40

                        Concentration   Number of [His.sup.+]
                        (mg/plaque)     Revertants/plaque

                                        Water Extracts

                                        TA 98

                                        S9 (+)                MR

([dagger])              100 [micro]1/   34 [+ or -] 3 (a)     1
  Negative Control        plaque
[R] Positive Control                    513 [+ or -] 81 (c)   15
                        0               37 [+ or -] 5 (a)     1.08
                        5000            36 [+ or -] 3 (a)     1.05
Cotoneaster Twig        1000            52 [+ or -] 1 (b)     1.52
                        500             41 [+ or -] 3 (a)     1.30
([dagger])              100 [micro]1/   34 [+ or -] 3         1
  Negative Control        plaque
[R] Positive Control                    513 [+ or -] 81       15
                        0               37 [+ or -] 5         1.08
                        5000            39 [+ or -] 7         1.14
Cotoneaster Fruit       1000            45 [+ or -] 7         1.32
                        500             40 [+ or -] 0         1.17

                        Concentration   Number of [His.sup.+]
                        (mg/plaque)     Revertants/plaque

                                        Water Extracts

                                        TA 98

                                        S9 (+)                  MR

([dagger])              100 [micro]1/   41 [+ or -] 5 (a)       1
  Negative Control        plaque
[R] Positive Control                    3100 [+ or -] 109 (c)   76
                        0               38 [+ or -] 6 (a)       0.92
                        5000            54 [+ or -] 6 (a)       1.31
Cotoneaster Twig        1000            58 [+ or -] 3 (b)       1.41
                        500             48 [+ or -] 4 (a)       1.17
([dagger])              100 [micro]1/   41 [+ or -] 5 (a)       1
  Negative Control        plaque
[R] Positive Control                    3100 [+ or -] 109 (b)   76
                        0               38 [+ or -] 6 (a)       0.92
                        5000            42 [+ or -] 0 (a)       1.02
Cotoneaster Fruit       1000            51 [+ or -] 4 (a)       1.24
                        500             39 [+ or -] 6 (a)       0.95

                        Concentration   Number of [His.sup.+]
                        (mg/plaque)     Revertants/plaque

                                        Water Extracts

                                        TA 100

                                        S9 (-)                 MR

([dagger])              100 [micro]1/   143 [+ or -] 6 (a)     1
  Negative Control        plaque
[R] Positive Control                    2022 [+ or -] 82       14
                        0               154 [+ or -] 10 (a)    1.07
                        5000            125 [+ or -] 13 (a)    0.87
Cotoneaster Twig        1000            120 [+ or -] 13 (a)    0.83
                        500             112 [+ or -] 9 (a)     0.78
([dagger])              100 [micro]1/   143 [+ or -] 6 (a)     1
  Negative Control        plaque
[R] Positive Control                    2022 [+ or -] 82 (b)   14
                        0               154 [+ or -] 10 (a)    1.07
                        5000            129 [+ or -] 11 (a)    0.90
Cotoneaster Fruit       1000            178 [+ or -] 4 (a)     1.24
                        500             174 [+ or -] 33 (a)    1.21

                        Concentration   Number of [His.sup.+]
                        (mg/plaque)     Revertants/plaque

                                        Water Extracts

                                        TA 100

                                        S9 (+)                  MR

([dagger])              100 [micro]1/   135 [+ or -] 11 (a)     1
  Negative Control        plaque
[R] Positive Control                    5244 [+ or -] 157 (b)   39
                        0               156 [+ or -] 9 (a)      1.15
                        5000            178 [+ or -] 11 (a)     1.31
Cotoneaster Twig        1000            149 [+ or -] 7 (a)      1.1
                        500             138 [+ or -] 20 (a)     1.02
([dagger])              100 [micro]1/   135 [+ or -] 11 (a)     1
  Negative Control        plaque
[R] Positive Control                    5244 [+ or -] 157 (c)   39
                        0               156 [+ or -] 9 (a)      1.15
                        5000            141 [+ or -] 10 (a)     1.04
Cotoneaster Fruit       1000            201 [+ or -] 14 (b)     1.48
                        500             169 [+ or -] 13 (a)     1.25

MR: Mutagenicity ratio

2-Aminofluorene (7.5 [micro]g/plate) was used as positive
indirect mutagen in the presence of S9 mix; 4-nitro-O-
fenilendiamine (5 [micro]g/plate) was used as positive
direct mutagen in the absence of S9 mix for S. typhimurium
TA98 strain.

2-Aminoanthracene (5 [micro]g/plate) was used as positive
indirect mutagen in the presence of S9 mix; Sodium azide
(5 ([micro]g/plate) was used as positive direct mutagen in
the absence of S9 mix for S. typhimurium TA100.
(abc) Differences between groups having the same letter in
the same column are not statistically significant (ANOVA,
Tukey, p>0.05)

([dagger]) Negative control: DMSO (100 ([micro]l/plate) was
used as negative control forS. typhimurium TA98 and TA100
both in the presence and absence of S9

[R] Positive controls:

Table 8
Antimutagenicity of Cotoneaster twig and fruit
extracts towards S. typhimurium TA98 and TA100 strains
with and without metabolic activation (S91.

Extracts        Concentration        Number of his *
                ([micro]g/plate)     revertants/plate

                                     TA 98

                                     S9 (-)                % I

* Negative      100 [micro]l/        30 [+ or -] 1 (a)
  Control         plate
[R] Positive                         530 [+ or -] 18 (d)   0
  Control
                0                    29 [+ or -] 3 (a)
                5000                 218 [+ or -] 2 (b)    62
T-Me            1000                 342 [+ or -] 2 (c)    38
                500                  366 [+ or -] 24 (c)   33
* Negative      100 [micro]l/plate   30 [+ or -] 1 (a)
  Control
[R] Positive                         530 [+ or -] 18 (c)   0
  Control
                0                    29 [+ or -] 3 (a)
                5000                 354 [+ or -] 13 (b)   35
T-W             1000                 342 [+ or -] 1 (b)    38
                500                  647 [+ or -] 18 (d)   0
* Negative      100 [micro]l/plate   30 [+ or -] 1 (a)
  Control
[R] Positive                         530 [+ or -] 18 (d)   0
  Control
                0                    29 [+ or -] 3 (a)
                5000                 301 [+ or -] 10 (b)   46
F-Me            1000                 344 [+ or -] 4 (c)    37
                500                  366 [+ or -] 24 (c)   33
* Negative      100 [micro]l/plate   30 [+ or -] 1 (a)
  Control
[R] Positive                         530 [+ or -] 18 (d)   0
  Control
                0                    29 [+ or -] 3 (a)
                5000                 132 [+ or -] 10 (b)   79
F-W             1000                 156 [+ or -] 19 (b)   75
                500                  231 [+ or -] 11 (c)   60

Extracts        Concentration        Number of his *
                ([micro]g/plate)     revertants/plate

                                     TA 98

                                     S9 (+)                  % I

* Negative      100 [micro]l/        44 [+ or -] 3 (a)
  Control         plate
[R] Positive                         3100 [+ or -] 109 (d)   0
  Control
                0                    53 [+ or -] 3 (a)
                5000                 251 [+ or -] 8 (ab)     94
T-Me            1000                 593 [+ or -] 25 (b)     82
                500                  2227 [+ or -] 74 (c)    29
* Negative      100 [micro]l/plate   44 [+ or -] 3 (a)
  Control
[R] Positive                         3100 [+ or -] 109 (e)   0
  Control
                0                    53 [+ or -] 3 (a)
                5000                 328 [+ or -] 21 (b)     91
T-W             1000                 890 [+ or -] 38 (c)     73
                500                  1908 [+ or -] 85 (d)    39
* Negative      100 [micro]l/plate   44 [+ or -] 3 (f)
  Control
[R] Positive                         3100 [+ or -] 109 (g)   0
  Control
                0                    53 [+ or -] 3 (f)
                5000                 2324 [+ or -] 61 (fg)   25
F-Me            1000                 3174 [+ or -] 42 (g)    0
                500                  3534 [+ or -] 73  (g)   0
* Negative      100 [micro]l/plate   44 [+ or -] 3 (a)
  Control
[R] Positive                         3100 [+ or -] 109 (d)   0
  Control
                0                    53 [+ or -] 3 (a)
                5000                 2251 [+ or -] 39 (b)    28
F-W             1000                 2841 [+ or -] 23 (c)    9
                500                  2854 [+ or -] 45 (c)    8

Extracts        Concentration        Number of his *
                ([micro]g/plate)     revertants/plate

                                     TA 100

                                     S9 (-)                   % I

* Negative      100 [micro]l/        139 [+ or -] 12 (a)
  Control         plate
[R] Positive                         2022 [+ or -] 82 (c)     0
  Control
                0                    163 [+ or -] 17 (a)
                5000                 1185 [+ or -] 110 (b)    45
T-Me            1000                 1548 [+ or -] 92 (b)     25
                500                  1502 [+ or -] 42 (b)     28
* Negative      100 [micro]l/plate   139 [+ or -] 12 (a)
  Control
[R] Positive                         2022 [+ or -] 82 (d)     0
  Control
                0                    163 [+ or -] 17 (a)
                5000                 1262 [+ or -] 50 (b)     41
T-W             1000                 1483 [+ or -] 105 (bc)   29
                500                  1745 [+ or -] 8 (cd)     15
* Negative      100 [micro]l/plate   139 [+ or -] 12 (a)
  Control
[R] Positive                         2022 [+ or -] 82 (c)     0
  Control
                0                    163 [+ or -] 17 (a)
                5000                 1629 [+ or -] 13 (b)     21
F-Me            1000                 1588 [+ or -] 103 (b)    23
                500                  1600 [+ or -] 76 (b)     23
* Negative      100 [micro]l/plate   139 [+ or -] 12 (a)
  Control
[R] Positive                         2022 [+ or -] 82 (c)     0
  Control
                0                    163 [+ or -] 17 (a)
                5000                 890 [+ or -] 20 (b)      61
F-W             1000                 1505 [+ or -] 93 (bc)    28
                500                  I774 [+ or -] 12 (bc)    13

Extracts        Concentration        Number of his *
                ([micro]g/plate)     revertants/plate

                                     TA 100

                                     S9 (+)                   % I

* Negative      100 [micro]l/        153 [+ or -] 10 (f)
  Control         plate
[R] Positive                         5244 [+ or -] 157 (h)    0
  Control
                0                    169 [+ or -] 14 (f)
                5000                 391 [+ or -] 8 (g)       96
T-Me            1000                 1604 [+ or -] 76 (g)     72
                500                  5195 [+ or -] 106 (h)    1
* Negative      100 [micro]l/plate   153 [+ or -] 10 (f)
  Control
[R] Positive                         5244 [+ or -] 157 (h)    0
  Control
                0                    169 [+ or -] 14 (f)
                5000                 1495 [+ or -] 14 (g)     74
T-W             1000                 3698 [+ or -] 115 (gh)   30
                500                  5932 [+ or -] 105 (h)    0
* Negative      100 [micro]l/plate   153 [+ or -] 10 (f)
  Control
[R] Positive                         5244 [+ or -] 157 (gh)   0
  Control
                0                    169 [+ or -] 14 (f)
                5000                 3197 [+ or -] 44 (g)     40
F-Me            1000                 4873 [+ or -] 98 (h)     7
                500                  4925 [+ or -] 120 (h)    6
* Negative      100 [micro]l/plate   153 [+ or -] 10 (f)
  Control
[R] Positive                         5244 [+ or -] 157 (gh)   0
  Control
                0                    169 [+ or -] 14 (f)
                5000                 4511 [+ or -] 102 (g)    14
F-W             1000                 5565 [+ or -] 139 (h)    0
                500                  5382 [+ or -] 165 (gh)   0

% I: Inhibition

2-Aminofluorene (7.5 [micro]g/plate) was used as positive
indirect mutagen in the presence of S9 mix; 4-nitro-0-
fenilendiamine (5 [micro]g/plate) was used as positive direct
mutagen in the absence of S9 mix for S. typhimurium TA98
strain.

2-Aminoanthracene (5 [micro]g/plate) was used as positive
indirect mutagen in the presence of S9 mix; Sodium azide
(5 [micro]g/plate) was used as positive direct mutagen in
the absence of S9 mix for S. typhimurium TA100.

(abcde) Differences between groups having the same letter in
the same column are not statistically significant (ANOVA,
Tukey, p>0.05)

(fgh) Differences between groups having the same letter in
the same column are not statistically significant (ANOVA,
Tamhane, p>0.05)

* Negative control: DMSO (100 [micro]l/plate) was used as
negative control for S. typhimurium TA98 and TA100 both in
the presence and absence

[R] Positive controls:


----------

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Author:Uysal, Ahmet; Zengin, Gokhan; Mollica, Adriano; Gunes, Erdogan; Locatelli, Marcello; Yilmaz, Turgut;
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Report
Geographic Code:1USA
Date:Sep 15, 2016
Words:10179
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