UHPLC-QTOF-MS phytochemical profiling and in vitro biological properties of Rhamnus petiolaris (Rhamnaceae)

Industrial Crops & Products 142 (2019) 111856

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UHPLC-QTOF-MS phytochemical profiling and in vitro biological properties of Rhamnus petiolaris (Rhamnaceae)

T

Gabriele Rocchettia, Maria Begoña Miras-Morenoa, Gokhan Zenginb,⁎, Ismail Senkardesc, Nabeelah Bibi Sadeerd, Mohamad Fawzi Mahomoodallyd, Luigi Lucinia,⁎ a

Department for Sustainable Food Process, Università Cattolica del Sacro Cuore, Via Emilia Parmense 84, 29122 Piacenza, Italy Department of Biology, Faculty of Science, Selcuk University, Campus, Konya, Turkey c Department of Pharmaceutical Botany, Faculty of Pharmacy, Marmara University, Istanbul, Turkey d Department of Health Sciences, Faculty of Science, University of Mauritius, 230 Réduit, Mauritius b

A R T I C LE I N FO

A B S T R A C T

Keywords: α-glucosidase Tyrosinase Bioactive compounds Metabolomics Orthogonal projection to latent structures

The genus Rhamnus has a great attention as source of bioactive compounds. So, this work aimed to investigate phytochemical profile and biological activity of water and methanolic extracts of different parts of Rhamnus petiolaris Boiss. & Balansa, namely twigs, leaves, mature and unmature fruits. The in vitro antioxidant activity, enzyme inhibitory properties, along with their polyphenol and anthraquinone profiles were determined by untargeted metabolomics. Results showed that methanolic and aqueous unmature fruit extracts were the most effective 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) scavenger (470.96 mg trolox equivalent (TE)/g and 394.96 mg TE/g, respectively). The aqueous unmature fruit extract displayed the most potent cupric and ferric reducing power and showed the highest total phenolic contents (TPC) (137.17 mg gallic acid equivalent (GAE)/g). The methanolic twig extract showed the highest enzymatic inhibitory property against of α-glucosidase, tyrosinase, acetylcholinesterase (AChE), and butyrylcholinesterase (BChE). On the basis of the correlation coefficients calculated separately for all experimental parameter pairs, flavonols and anthocyanins highly correlated with DPPH, whereas tyrosols correlated with BChE activity. Multivariate statistics following untargeted metabolomics allowed to describe the differences in polyphenols and anthraquinones, as affected by the extraction solvent used. Mature and unmature fruits were substantially comparable and were affected in a similar way by the extraction conditions, while different profiles were recorded for Rhamnus leaves and twigs. These findings indicate that the recovery efficiencies of specific subclasses of compounds (above all when considering flavonoids, phenolic acids and anthraquinones) as a function of the matrix and extraction chosen, significantly affect the phytochemical profile and biological activity of Rhamnus.

1. Introduction For centuries, human life has been revolving around plants in the attempt to maintain good health and manage common ailments (Petrovska, 2012). Although the usage of plants was based solely on the intuitive nature of humans, due to the absence of proper techniques to confirm plants’ curative properties, humankind advancing contemporary science acknowledged the usage of various medicinal plants and included them in modern pharmacotherapy (Petrovska, 2012). As an ample evidence showed that 70–80% of the world’s population still depends on herbal medicine as their primary health support (Yoo et al., 2018). Recently, Allkin Bob from the Royal Botanic Gardens, Kew,

⁎

recorded around 28,000 plant species as medicinal plants (Allkin, 2017). Medicinal plants quickly attracted the attention of scientists since the eureka moment which came along the discovery of taxol, the blockbuster anti-cancer drug from Pacific yew tree, by Stierle et al., in 1993 clearly gratifying the curative nature of plants (Stierle et al., 1993). Since then, the screening of medicinal plants quickly mushroomed and the inclusion of health-promoting plant extracts in nutrition gained popularity. In line with current global trend, this study aimed to study is Rhamnus petiolaris Boiss. & Balansa (Family: Rhamnaceae). The genus Rhamnus is a major group in Angiosperms, consisting of 113 accepted species names, 75 synonyms and 286 unassessed summing up to 474

Corresponding authors. E-mail addresses: [email protected] (G. Zengin), [email protected] (L. Lucini).

https://doi.org/10.1016/j.indcrop.2019.111856 Received 17 June 2019; Received in revised form 23 August 2019; Accepted 8 October 2019 0926-6690/ © 2019 Elsevier B.V. All rights reserved.

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2. Materials and methods

scientific species names (The Plant List, 2013). The word Rhamnus is known under the term rhamnos in both Greek and Latin languages. From the Greek definition, rhamnos means ‘a kind of prickly plant or spiny shrub’ while the Latin rhamnos means ‘buckthorn or Christ’s thorn’ (Quattrocchi, 2016). It is a global plant since it is distributed in East Asia, North and South America and in the subtropical regions of Africa (Akkemik et al., 2018; Flora of China, 2007). Morphologically, Rhamnus species are shrubs with undivided leaf blades. Most species have yellowish green flowers with four to five ovate-triangular sepals. The plant possessed a special feature which characterized itself as a natural dyeyielding plant. The berry-like fruits that they produced contain yellow dyes which are usually used as printing ink and in medical lore (Akkemik et al., 2018; Dogan et al., 2003; Flora of China, 2007; Kayabasi and Arlı, 2001). Interestingly, literature showed that dyeyielding plants possess significant antimicrobial properties. A previous study conducted by Comlekcioglu et al. (2017) on the aqueous fruit extract of R. petiolaris showed good antibacterial activities. R. petiolaris, locally known as Cehri in Turkey, is an evergreen shrub or small-sized plant endemic to Turkey (Ozturk et al., 2013). From this point, the member of the genus Rhamnus has great potential as industrial plants in pharmaceutical and medicinal applications. As in the case of many other medicinal plants, different parts of R. petiolaris are used in folklore medicine. For instance, the fruit possess laxative property similarly to other Rhamnus species (Ozturk and Altay, 2017; Ozturk et al., 2013). Phytochemical screening of R. petiolaris is rather scarce and till date only few studies have been reported on the compounds present. In 1974, Wagner et al (Wagner et al., 1974), isolated two flavonol-3-O-triosides identified as rhamnazin-3-O-[α-Lrhamnopyranosyl (1 → 4)-α-L-rhamnopyranosyl (1 → 6)]-β-D-galactopyranoside and rhamnetin-3-O-α-L-rhamnopyranosyl (1 → 2)-α-Lrhamnopyranosyl (1 → 6)]-β-D-galactopyranoside from the fruit. Later in 1994 Ozipek et al (Ozipek et al., 1994)., isolated a new acylated flavonol glycoside from the berries. Out of the 113 accepted members of the Rhamnus genus, R. petiolaris is an underexplored plant and is poorly understood. Despite a long story of use, to the best of our knowledge no studies have been reported on the comprehensive phytochemical and biological profile of this plant. In fact, this species lacks considerable scientific attention in terms of its pharmacological properties and phytochemical profiles. There has been no attempt to evaluate the inhibitory activity of various extracts of R. petiolaris on key enzymes linked to human ailments, namely diabetes and Alzheimer’s disease. Consequently, to shed more light on this plant, the present study provides a deeper and broader understanding on antioxidant capacity and possible inhibitory action against α-amylase, α-glucosidase, tyrosinase, acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). Because the choice of extraction solvents is one of most important steps in the phytochemical studies, we selected methanol and water as solvents in the study. Methanol is one of most common solvents in phytochemical literatures (Alam et al., 2013; Belwal et al., 2018) and water was selected to provide traditional way (for example tea). Besides, the development of high-resolution platforms for the analysis of bioactive compounds is allowing to gain a clear picture of the phytochemical composition of a plant of interest. In this work, the phytochemical profile of R. petiolaris was investigated using a metabolomics-based approach. Indeed, metabolic profiling or fingerprinting, using comprehensive databases for the annotation steps can improve the understating of the wide phytochemical composition of plants and foods. Taken together, obtained results could provide a significant starting point on R. petiolaris to evaluate as an industrial plant in several applications.

2.1. Plant material The studied plant materials were collected from Nevsehir (central Anatolia region, Turkey) in July-August 2018 (20 July 2018 for twigs, leaves and unmature fruit samples; 20 August 2018 for mature fruits). The scientific identification was performed by Dr. Ismail Senkardes (botanist at Marmara University, Department of Pharmaceutical Botany, Istanbul, Turkey). Then, voucher specimen was deposited in the herbarium of Marmara University (voucher number: MARE 20217). The plant parts (twigs, leaves, unmature and mature fruits) were separated and kept for 10 days at room temperature for drying. Then, these samples were powdered with a laboratory mill. 2.2. Extraction methods For methanol extracts, the samples were stirred overnight (24 h) at room temperature (5 g in 100 ml solvent). After filtration, the extracts were concentrated using a rotary evaporator under vacuum at 40 °C. For water extracts, the infusions were prepared (5 g of samples were kept in 100 ml of boiled water for 20 min) and then were lyophilized. All extracts were stored at +4 °C until analyses. 2.3. Screening of bioactive compounds The total phenolic and flavonoid contents were determined using the common assays; Folin-Ciocalteu and AlCl3 assays, respectively (Uysal et al., 2017). The assays results were spectrophotmetrically recorded (765 nm for total phenolic and 415 nm for total flavonoid). Results were expressed as gallic acid (mg GAEs/g extract) and rutin equivalents (mg REs/g extract) for respective assays. Afterwards, the untargeted phenolic and anthraquinone profile of the different Rhamnus extracts was investigated by means of ultra-highpressure liquid chromatograph coupled to quadrupole-time-of-flight mass spectrometer (both from Agilent Technologies). The experimental conditions for the analysis of plant extracts by means of untargeted metabolomics were optimized in previously works (Rocchetti et al., 2018, 2019b). Briefly, the mass spectrometer was run in the positive scan mode (50–1100 m/z) and chromatographic separation was achieved on an Agilent Zorbax eclipse plus C18 column (50 × 2.1 mm i.d., 1.8 μm dp). The LC mobile phase A consisted of water while mobile phase B was methanol (LCMS grade, VWR International Ltd). An inhouse custom database of anthraquinones coupled to the comprehensive phenolics database exported from Phenol-Explorer (http://phenolexplorer.eu/) was used for identification; with this purpose, the whole isotopic pattern (monoisotopic accurate mass, isotopic ratio and isotopic accurate spacing) was considered. The approach used allowed great confidence in the annotation. In particular, the compounds were identified according to a Level 2 of identification (i.e., putatively annotated compounds). The post-acquisition data filtering (Agilent Profinder B.06) retained only those compounds putatively annotated within 100% of replications in at least one condition. Finally, in order to provide more quantitative information, polyphenols were firstly ascribed into classes and subclasses and then quantified, as reported in a previous work (Rocchetti et al., 2019b). The phenolic standards used were: cyanidin, catechin, luteolin, ferulic acid, sesamin, 5-pentadecylresorcinol, resveratrol and tyrosol. Additionally, a calibration curve of aloin (Sigma grade, Sigma-Aldrich, S. Louis, MO, USA) was built in order to provide a total anthraquinone content for the different Rhamnus organs. Results were finally expressed as mg phenolic equivalents/g dry extract. 2.4. Determination of in vitro antioxidant and enzyme inhibitory effects The 2

metal

chelating,

phosphomolybdenum,

ferric

reducing

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abundant classes equivalents of compounds were phenolic acids (45 putatively annotated compounds), followed by flavones and tyrosols (43 compounds), flavonols (30 compounds), anthocyanins (28 compounds), lignans (12 compounds), alkylphenols and stilbenes (i.e., 7 and 3 compounds, respectively). For a more detailed description, polyphenols and anthraquinones identified are presented into supplementary material, together with annotation scores and composite mass spectra. Independently from the extraction solvent, some polyphenols were particularly abundant and characteristics of each organs (supplementary material). Rhamnus leaves were abundant in isomers of diferuloylquinic acid (phenolic acids), cyclolariciresinol (lignans), glycosidic forms of kaempferol, quercetin and patuletin (flavonols), isoflavonoids (such as glycitin and 6″-O-malonylglycitin) and cyanidin 3O-glucosyl-rutinoside. The unmature fruits were particularly abundant in glycosidic forms of anthocyanins, flavones (such as apigenin and nepetin) and flavonols (such as isorhamnetin 3-O-galactoside and isorhamnetin 7-O-rhamnoside). The twigs were characterized by high values of anthocyanins (i.e., cyanidin 3-O-glucoside and cyanidin 3-Orutinoside), flavone equivalents (such as chrysoeriol 7-O-apiosylglucoside and 6-hydroxyluteolin 7-O-rhamnoside), flavonols (namely glycosidic forms of kaempferol and quercetin) and furanocoumarins (in particular isopimpinellin and bergapten), while caffeic acid 4-O-glucoside was the most represented phenolic acid. Interestingly, mature fruits showed the highest abundances when compared with the other Rhamnus parts, being particularly rich in cyanidin 3-O-glucosyl-rutinoside, delphinidin 3-O-rutinoside and pelargonidin 3-O-(6′′-succinyl-glucoside). Furthermore, when compared with the other extracts, mature fruits were the richest source of flavonols (above all glycosidic forms of quercetin and kaempferol), while the most abundant hydroxycinnamic acid was found to be 1,2-disinapoylgentiobiose. One of the exclusive phenolic compounds characterizing R. petiolaris is rhamnazin (Wagner et al., 1974); it is a dimethoxyflavone that is quercetin in which the hydroxy groups at the 3′ and 7 positions have been replaced by methoxy groups. In our experimental conditions, we revealed a high abundance of this compound above all in water extracts of mature fruits (supplementary material). The untargeted metabolomics-based approach allowed to identify a wider set of compounds when compared with targeted approaches, thus demonstrating that this plant was particularly rich in flavonoids (i.e., anthocyanins and flavonols). Afterwards, by exploiting a semiquantitative analysis from the UHPLC-QTOF data, all phenolic compounds identified were classified and expressed according to a standard per class as mg/g dry extract. The results of the semi-quantitative analysis were finally reported in Table 1. According to this analysis, an increase in anthocyanin content (on average +30%) was observed when moving from unmature to the mature fruits (Table 1). In fact, mature fruit (both methanol and water extracts) presented the highest values of these pigments (i.e., 80.58 and 69.34 mg/g, respectively), according to its black colour. Similar results are also reported in other fruit species. For instance, significantly higher anthocyanin contents were recently reported in different wild mature fruits, compared with unmature fruits (Belwal et al., 2019). The increasing concentration of anthocyanins in mature fruits might be due to the up regulation of phenylpropanoid pathway and chalcone synthase enzyme, which are involved in anthocyanin biosynthesis. Therefore, the increase of anthocyanin content promoted both colour and health-promoting properties of these fruits (Belwal et al., 2019). In regard to the other phenolic subclasses, the most abundant values (in terms of mg/g dry extract) were phenolic acid followed by flavonol/flavanol equivalents. It was interesting to notice that both methanol and water extracts of mature fruits presented again the highest values (i.e., 28.42 and 22.10 mg/g, respectively) when compared with the R. petiolaris parts. Overall, using methanol as extraction solvent determined a higher efficiency in the recovery of phenolic acids, although the highest value (p < 0.05) for this class of compounds was recorded in aqueous extracts of Rhamnus leaves (i.e., 37.02 mg/g).

antioxidant power (FRAP), cupric reducing antioxidant capacity (CUPRAC), ABTS, and DPPH activities of the extracts were assessed following the methods described by Uysal et al. (2017). The antioxidant activities were reported as trolox equivalents (mg TE/g extract), whereas EDTA (mg EDTAE/g extract) was used for metal chelating assay. The possible inhibitory effects of the extracts against cholinesterase (by Ellman’s method), tyrosinase, α-amylase and α-glucosidase were evaluated using standard in vitro spectrophotometric bio-assays (Uysal et al., 2017). The enzyme inhibitory actions of extracts were assessed as equivalents of kojic acid (KAE) for tyrosinase, galantamine (GALAE) for acetyl and butyryl cholinesterase, and acarbose (ACAE) for α-amylase and α-glucosidase. 2.5. Statistical analysis A one-way analysis of the variance (ANOVA) was done using the software PASW Statistics 25.0 (SPSS Inc.) to investigate significant differences (p < 0.05, Duncan’s post hoc test) for each assay carried out. Besides, Pearson’s correlation coefficients (p = 0.01, two-tailed) were also calculated in PASW Statistics 25.0. Thereafter the raw metabolomic dataset exported from the Mass Profiler Professional B.12.06 (Agilent technologies) was elaborated into SIMCA 13 software (Umetrics, Malmo, Sweden). A supervised orthogonal projection to latent structures discriminant analysis (OPLS-DA) model was created to separate the variation between groups into predictive and orthogonal components. Hotelling’s T2 and model cross-validation were then carried out, using CV-ANOVA (p < 0.01) and inspecting the results of permutation testing to exclude overfitting. The goodness-of-fit (R2Y) and the goodness-of-prediction (Q2Y) were also produced. Finally, VIP selection method was used to identify the most discriminating compounds according to the extraction techniques. In particular, the compounds possessing a VIP score > 1 were retained and further elaborated. 3. Results and discussion 3.1. Untargeted screening of polyphenols and anthraquinones in different Rhamnus organs The total bioactive components of the solvent extracts of R. petiolaris were firstly investigated spectrophotometrically in terms of total phenolic content (TPC) and total flavonoid content (TFC) as presented in supplementary material. As highlighted by numerous researchers, fruits are good carriers of bioactive phytochemicals especially phenolic component which act as a protective barrier against a multitude of diseases (Carocho and Ferreira, 2013; Kähkönen et al., 1999; Martins et al., 2016). Indeed, results in this study corroborated with this fact showing that both methanolic and aqueous extracts of mature and unmature fruits possessed the highest TPC and TFC compared with other plant part extracts studied. For instance, methanolic and aqueous immature fruit extract showed the highest TPC value of 109.99 and 137.17 mg GAE/g respectively in contrast the methanolic and aqueous leaf extracts possessing the lowest TPC with 46.07 and 41.82 mg GAE/g respectively. On the other hand, flavonoids were abundantly present in mature fruits in contrast to immature fruits. The highest TFC were recorded in both methanolic and aqueous mature fruit extracts with 161.40 and 157.97 mg GAE/g respectively while the lowest TFC was observed in the aqueous twig extract (36.37 mg GAE/g). Afterwards, the untargeted phenolic and anthraquinone profiles of the different Rhamnus extracts was investigated by means of a metabolomic approach, based on UHPLC-ESI-QTOF mass spectrometry. The experimental plan designed allowed to putatively annotate 237 compounds, being characterized by 211 polyphenols and 26 anthraquinones. It is important to underline that an overall identification score higher than 75% was considered, in order to increase the confidence in annotation. With respect to the phenolic composition, the most 3

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± ± ± ± ± ± ± ± 4.05 1.85 2.42 3.58 4.08 1.80 2.36 2.74 22.89 ± 0.78 10.17 ± 4.45a 8.52 ± 1.60a 14.57 ± 1.65b 17.09 ± 4.18b 6.61 ± 0.79a 6.06 ± 0.21a 17.66 ± 1.37b 12.05 ± 0.33 11.62 ± 8.58ab 28.42 ± 5.86d 10.46 ± 1.08a 6.67 ± 0.31a 17.91 ± 1.50bc 22.10 ± 2.80cd 4.99 ± 1.60a 0.88 0.17a 0.29b 0.33ab 0.24c 0.21a 0.91b 0.19a ± ± ± ± ± ± ± ± 3.49 1.75 2.56 2.20 3.49 1.63 2.52 1.53 7.77 8.60bc 15.45c 5.00a 2.25ab 4.98a 27.47c 2.65a ± ± ± ± ± ± ± ± 37.01 62.52 80.58 35.09 45.29 40.22 69.34 24.67 9.62 38.86 59.37 15.85 13.41 31.02 44.16 32.27 Water

Twigs Unmature fruits Mature fruits Leaves Twigs Unmature fruits Mature fruits Leaves MeOH

* Results are expresses as mean values (mg/g) ± standard deviation (n = 3). Superscript letters within each column indicate homogeneous sub-classes as resulted from ANOVA (p < 0.01; Duncan’s post-hoc test).

0.00a 0.00a 0.00ab 0.01e 0.00c 0.00b 0.02c 0.00d ± ± ± ± ± ± ± ± 0.01 0.01 0.02 0.70 0.06 0.03 0.08 0.67 5.53 7.54bc 12.59bc 6.42bc 1.55a 0.58ab 4.16ab 1.14c ± ± ± ± ± ± ± ± 30.43 27.23 32.90 28.67 13.18 23.65 22.78 37.02 0.03 0.11ab 0.12bc 0.11a 0.06ab 0.06ab 0.06ab 0.06c ± ± ± ± ± ± ± ± 0.21 0.35a 0.45ab 0.45c 0.33c 0.16a 0.33ab 0.74b

0.76 0.43 0.46 0.29 0.41 0.42 0.37 0.61

Stilbenes Phenolic acids

bc d

Alkylphenols

c

Lignans

c

Tyrosols

ab

Flavonols

c

Flavones

a

Anthocyanins Extraction Yield (%) Parts Solvent

Table 1 Extraction yields (%) and quantification per classes of phenolics identified from UHPLC-ESI-QTOF-MS in the different parts of the plant (i.e., twigs, mature fruits, unmature fruits and leaves) and considering the two different extraction solvents (methanol and water)*.

G. Rocchetti, et al.

Table 2 Total anthraquinone content of the different Rhamnus organs*. Solvent

Parts

Total anthraquinone content (mg/g)

MeOH

Twigs Unmature fruits Mature fruits Leaves Twigs Unmature fruits Mature fruits Leaves

0.09 0.14 0.22 1.18 0.17 0.28 0.62 1.13

Water

± ± ± ± ± ± ± ±

0.01a 0.01ab 0.01bc 0.13f 0.03abc 0.03c 0.01d 0.10e

* Results are expressed as mean value (mg aloin/g dry matter) ± standard deviation (n = 3). Superscript letters within each column indicate homogeneous sub-classes as resulted from ANOVA (p < 0.01; Duncan’s post-hoc test).

Regarding the anthraquinone profile, the UHPLC-QTOF untargeted approach allowed us to identify also the most common anthraquinones characterizing Rhamnus species, including emodin (C15H10O5), physcion (C16H12O5) and chrysophanol (C15H10O4) (Duval et al., 2016). Anthraquinone derivatives are the largest group of natural quinones, constituting the largest group of natural pigments with about 700 compounds described. In this regard, the most frequently reported compounds are emodin, physcion, catenarin and rhein. Anthraquinones can be found in all parts of the plants, such as roots, rhizomes, fruits, flowers and leaves (Duval et al., 2016). In order to provide a total anthraquinone content in the different Rhamnus extracts, all the anthraquinones annotated were quantified according to a standard of aloin and then expressed as total content. The values obtained (mg aloin/g dry matter) are reported in Table 2. As can be observed, both leaf extracts (i.e. methanol and water) were characterized by the highest content (p < 0.05) of total anthraquinones (on average 1.16 mg/g). For the other organs, values ranged from 0.09 (recorded in twigs extracted in methanol) up to 0.62 mg/g (when considering mature fruits extracted in water). Our quantitative results were difficult to compare with literature, considering the lack of works on this Rhamnus species. However, the anthraquinone contents detected were generally comparable with others from some previous works (Locatelli et al., 2011), analyzing barks of two other Rhamnus species, such as R. catharticus and R. orbiculatus. In fact, these authors reported a total content of 1.64 and 0.75 mg/g, when considering R. catharticus and R. orbiculatus, respectively. Interestingly, the most represented anthraquinone derivative in R. catharticus and R. orbiculatus was found to be physcion (i.e. 67.8% and 81.3%, respectively). After the initial screening of polyphenols and anthraquinones in the different Rhamnus extracts by UHPLC-QTOF, multivariate statistics was used in order to group and then discriminate samples according to their phytochemical profiles. In this regard, the supervised OPLS discriminant analysis was found to be particularly effective for this purpose. The OPLS-DA score scatter plot obtained is reported in Fig. 1; as can be observed, mature and unmature fruits possessed a similar phytochemical profile when compared with twigs and leaves that were distributed differently in the hyperspace along the two latent vectors (i.e. t1 and t2). Therefore, this statistical approach confirmed that the organs analysed were characterized by distinctive phenolic and anthraquinone signatures. The OPLS model parameters were found to be excellent, recording high R2Y and Q2Y values, being 0.99 and 0.92, respectively. Besides, no outliers were outlined by Hotelling’s T2 range (supplementary material), with more than acceptable cross-validation parameters, as resulted by CV-ANOVA (p < 0.001) and permutation testing (N = 100). In order to identify the most discriminant compounds contributing to the differences outlined in the OPLS, the variable selection method VIP (variable importance in projection) was used. The polyphenols and anthraquinones allowing to discriminate the different organs are reported in supplementary material. Overall, 114

4

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Fig. 1. OPLS-DA on the phenolic and anthraquinone profile of the different Rhamnus extracts when considering the different organs (i.e., twigs, unmature fruits, mature fruits and leaves).

validation parameters (supplementary material), thus confirming the robustness of the OPLS model based on both phenolic and anthraquinone profiles for discriminating the different extraction methods. Overall, according to our data on the polyphenol composition (Table 1), methanol and water used as extraction solvents were supposed to promote a different extraction of some compounds according to their polarity. Therefore, the extraction solvent was found to play a key role in determining the discrimination of the extracts tested, even though some plant tissues were characterized by distinct profiles (such as twigs and leaves) as outlined into the OPLS score plot (Fig. 2). Afterwards, the variable selection method VIP was used to highlight the most discriminant compounds involved in the separation observed. In this sense, 94 compounds belonging to both polyphenols and anthraquinones possessed a VIP score higher than 1. The VIP markers outlined by VIP were reported in Table 3, together with VIP scores and standard errors. Looking to the VIP markers, flavonoids were the most common classes of phenolics reaching 50 compounds, followed by 22 low molecular weight phenolics (such as phenolic terpenes and tyrosol equivalents), phenolic acids (11 compounds, mainly hydroxycinnamics) and anthraquinones (7 compounds). The results obtained in this work pointed out that the phytochemical profiles of Rhamnus are heavily affected by the different extraction solvent used. In this regard, both methanol and water allowed to recover with a different efficiency some specific compounds. Overall, methanol was found more efficient in promoting differences between the different plant materials tested. In fact, the alcoholic solvent determined a distinct separation between twigs and leaves, while the aqueous-extracts of the same plant matrices showed a more similar profile (Fig. 2). Finally, in order to better understand the different incidence in recovering bioactive compounds as promoted by the different extraction solvents, a fold-change analysis on the VIP markers was provided (Table 3). In particular, mature and unmature fruits were characterized by a similar FC-distribution (i.e. 63% of the VIP markers changed with similar trends) thus justifying the clusters observed for these plant tissues into the OPLS-DA score plot (Fig. 2). Interestingly, methanol allowed to recover high contents of flavanones and anthocyanins when considering twigs. In this regard, the compound most affected by the alcoholic extraction was naringin 4′-O-glucoside, being characterized by a strong up-accumulation (FC = 20.44); regarding anthocyanins, the 80% of VIP markers was found to be up-accumulated in this tissue. Interestingly, the most characterizing Rhamnus compound, i.e. rhamnazin (3,7-

compounds possessed a VIP score higher than 1 (supplementary material), with the following distribution: 51 flavonoids, 20 phenolic acids, 20 lower-molecular-weight compounds (such as tyrosols), 18 anthraquinones, 4 lignans and 2 stilbenes. Looking to the markers outlined by VIP, 3 compounds were characterized by the highest scores, being hydroxycaffeic acid (1.58), quercetin 3-O-rutinoside (1.45) and delphinidin 3-O-rutinoside (1.44). Among the VIP markers, glycosidic forms of flavonols (i.e. quercetin), flavones and anthocyanins were found as important into the OPLS model. Interestingly, both physcion and emodin (two of the most important Rhamnus anthraquinones; Moreira et al., 2018) were included into the VIP list, recording a VIP score of 1.04 and 1.19, respectively. These latter compounds are particularly studied for their bioactive properties. In this regard, physcion is a dihydroxyanthraquinone that derives from a 2-methylanthraquinone that is widely isolated and characterized from both terrestrial and marine sources. Physcion has a role as an apoptosis inducer and it is characterized by several protective actions, such as antineoplastic, hepatoprotective anti-inflammatory, antibacterial, and antifungal (Moreira et al., 2018). Regarding emodin, it has been described as a purgative anthraquinone found in several plants, especially R. frangula and R. sphaerosperma (Moreira et al., 2018). 3.2. Effect of the extraction solvents on the phytochemical profiles In order to compare the different extractions tested, using methanol and water extraction solvents, multivariate statistics was applied to the metabolomics-based data. Multivariate analysis following metabolomic workflows is usually performed by using unsupervised (such as hierarchical clustering) and supervised statistical tools (such as OPLS discriminant analysis). Generally, the use of class membership criteria characterizing supervised methods allows a better separation between classes into the score plot space. Furthermore, the OPLS-DA is also able to effectively separate Y-predictive variation from Y-uncorrelated variation in X (data matrix). Therefore, an OPLS-DA score plot was built considering each different extraction condition, and finally provided in Fig. 2. In our experimental conditions, a complete separation between methanolic and water extracts was achieved, with clear differences related to the plant material tested. Notably, the discriminant model indicators were found to be excellent, being R2Y (the goodness-of-fit) = 0.99 and Q2Y (goodness-of-prediction) = 0.89, with more than adequate cross5

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Fig. 2. OPLS-DA on the phenolic and anthraquinone profile of the different Rhamnus extracts when considering different extraction solvents (i.e., methanol vs water).

investigated to determine the correlation with the corresponding in vitro antioxidant activities. It is important to highlight that the aqueous unmature fruit extract showing the highest TPC equally exhibited the highest antioxidant activity in most of the assays (except for DPPH) thus showing a good correlation between phenolic compounds and antioxidant potential. In a recent study, Benamar et al. (2019) evaluated antioxidant activity of different extracts from Rhamnus lycioides subsp. oleoides leaves and ethyl acetate and methanol extracts were noted as most active extracts with the high level of phenolics and anthraquinones. Similarly, the methanol extract of leaves exhibited more ability when compared with water extract in our study. Ben Ammar et al. (2019) isolated some compounds from R. alternatus leaves and barks and some of the compounds exhibited significant DPPH scavenging abilities. In a previous study, Moussi et al. (2015) tested R. alternatus leaves extracts and the total phenolic contents varied from 78.91 to 712.58 μg GAE/mL. Also, the high levels of phenolics and flavonoids in four Rhamnus extracts was reported by Kosalec et al. (2013). In accordance with our results, Boussahel et al. (2015) found higher level of flavonoids in the methanol extract of R. alternatus bark than water extract. Our finding (46.07 mg GAE/g extract) was also very close that of the methanol extract of R. intermedia leaf (46.40 mg GAE/g extract) reported by Šamec et al. (2012). In addition, the authors reported high antioxidant abilities (by DPPH, ABTS and FRAP assays) for leaf extract when compared with bark extract. This fact could evidence that phenolic compounds are main contributors to antioxidant abilities of the members of the genus Rhamnus. Stef et al. (2009) evaluated antioxidant capacity of eleven medicinal plants in Romania and they reported the highest DPPH scavenging ability of R. frangula extracts. Generally, the members of the Rhamnus genus was reported highly active antioxidant agents and this phenomena could be contributed the industrial usage of the members of the Rhamnus genus.

dimethylquercetin), was better extracted in water than methanol when considering each plant tissue tested (Table 3). Besides, methanol was found to be also the best extraction solvent for the recovery of two anthraquinones, being 8-C-glucosyl-7-O-methylaloediol (FC = 15.67) and aloenin-2′-p-coumaroyl ester (FC = 13.04), when considering Rhamnus leaves and unmature fruits, respectively. Besides, physcion was better extracted in water than methanol, recording the highest recovery in unmature fruits extracted with water (FC = 5.78). Overall, our results agreed with those reported in literature (Lou et al., 2016; Rocchetti et al., 2019a; Złotek et al., 2016) outlined different extraction efficiencies of bioactive compounds depending on both the extraction solvents and technologies used. 3.3. In vitro antioxidant activities The in vitro antioxidant activities of R. petiolaris were determined by a series of assays including radical scavenging (DPPH and ABTS), reducing power (FRAP and CUPRAC), total antioxidant capacity (phosphomolybdenum) and metal chelating ability. As displayed in Table 4, the best DPPH scavenging properties were exhibited by the mature and unmature fruit extracts. For instance, the highest DPPH activity was observed by the methanolic unmature fruit extract (470.96 mgTE/g) while the lowest activity was observed by the aqueous leaf extract (76.64 mgTE/g). In addition, the highest ABTS scavenging potential was displayed by the aqueous unmature fruit extract (394.96 mgTE/g) and the weakest ABTS scavenger was found to be the methanolic leaf extract (90.70 mg/TE/g). The total antioxidant activity of the extracts was also evaluated using phosphomolybdenum assay involving an electron-transfer reaction based on the reduction of molybdenum (VI) to molybdenum (V) by the antioxidant (Meshkini, 2016). In this study, the highest antioxidant activity was exhibited by the aqueous unmature fruit (2.96 mgTE/g) and the least active extract was aqueous leaf (1.59 mgTE/g) which was in agreement with its TPC since the extracts displayed highest and lowest TPC accordingly. The reducing powers of the different extracts were also evaluated in terms of CUPRAC and FRAP. Results showed that aqueous unmature fruit extract have the most significant reducing potential on CUPRAC and FRAP (772.73 and 1049.61 mg TE/g). Similarly, the aqueous unmature fruit extract exerted the highest metal chelating effect (40.57 mg EDTAE/g) which is in accordance with its TPC and the aqueous twig extract exerting the least effect (8.17 mg EDTAE/g) agreeing with its TFC. The TPC and TFC of the different Rhamnus extracts (supplementary Table 2) were

3.4. Inhibitory activity on key enzymes The enzyme inhibitory potential of R. petiolaris extracts were evaluated against tyrosinase, α-amylase, α-glucosidase, and two cholinesterases (AChE and BChE). The findings are represented in Table 5. The selected target enzymes play a pivotal role in maintaining and regulating bodily functions. For example, tyrosinase regulates the production of melanin pigments, by a process defined as melanogenesis, produced by the melanocytes, in order to protect the skin against 6

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Table 3 Discriminant polyphenols and anthraquinones identified by VIP (variable importance in projection) selection method following supervised OPLS-DA modelling as function of the different extraction solvent (methanol vs water). Compounds are provided together with VIP scores (measure of variables importance in the OPLS model). Class

Compound

VIP score

LogFC [Leaves MeOH vs H20]

LogFC [Unmature fruits MeOH vs H2O]

Log FC [Mature fruits MeOH vs H2O]

LogFC [Twigs MeOH vs H2O]

Anthraquinones

Chrysophanol Aloenin-2'-p-coumaroyl ester Aloesone 8-C-glucosyl-aloesol Physcion 8-C-glucosyl-7-O-methylaloediol 7-O-Methylaloeresin A Pelargonidin Delphinidin 3-O-(6”-acetyl-galactoside) Delphinidin 3-O-(6”-acetyl-glucoside) Cyanidin 3-O-(6”-acetyl-glucoside) Delphinidin 3-O-arabinoside Cyanidin Cyanidin 3-O-(6”-caffeoyl-glucoside) Delphinidin 3-O-rutinoside Malvidin 3-O-(6”-p-coumaroyl-glucoside) Cyanidin 3-O-(6”-succinyl-glucoside) Dihydroquercetin 3-O-rhamnoside Dihydromyricetin 3-O-rhamnoside Dihydroquercetin Theaflavin Prodelphinidin dimer B3 Naringin 4'-O-glucoside Naringenin 7-O-glucoside Eriodictyol Eriocitrin Apigenin 6-C-glucoside 6-Hydroxyluteolin 7,3',4'-Trihydroxyflavone Luteolin 7-O-(2-apiosyl-glucoside) Cirsimaritin Hispidulin Nepetin Geraldone Apigenin 6,8-C-galactoside-C-arabinoside Apigenin 7-O-apiosyl-glucoside Apigenin 7-O-glucoside Jaceosidin Apigenin 6,8-C-arabinoside-C-glucoside Chrysoeriol 7-O-apiosyl-glucoside Apigenin 7-O-glucuronide Morin Galangin Kaempferol 3-O-xylosyl-glucoside Quercetin 3-O-glucosyl-xyloside Kaempferol 3-O-rhamnoside Kaempferol 3-O-acetyl-glucoside Isorhamnetin 3-O-rutinoside Isorhamnetin Methylgalangin 3-Methoxynobiletin Isorhamnetin 3-O-galactoside 3,7-Dimethylquercetin Patuletin 3-O-glucosyl-(1-6)-[apiosyl(12)]-glucoside 6”-O-Acetyldaidzin Daidzin Glycitin Pinoresinol Conidendrin Todolactol A Dimethylmatairesinol 5-Heptadecylresorcinol 4-Methylcatechol 3-Methylcatechol 4-Vinylphenol Curcumin Syringaldehyde p-Anisaldehyde Sinapaldehyde

1.62 1.28 1.25 1.12 1.09 1.06 1.04 1.79 1.57 1.41 1.41 1.31 1.29 1.19 1.07 1.06 1.00 1.89 1.09 1.07 1.38 1.16 1.38 1.08 1.38 1.06 1.90 1.61 1.58 1.45 1.33 1.25 1.20 1.19 1.17 1.17 1.15 1.13 1.07 1.01 1.01 1.61 1.58 1.45 1.35 1.35 1.31 1.28 1.20 1.19 1.19 1.14 1.13 1.01

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.73 0.76 0.94 0.96 0.91 1.01 1.23 1.12 1.26 1.39 0.81 0.92 0.99 1.04 0.86 1.09 1.37 0.86 0.97 1.16 1.07 1.57 1.36 0.82 1.41 1.22 0.86 0.36 1.19 0.96 1.25 1.22 0.90 1.71 1.15 1.15 1.08 1.46 1.13 0.92 1.21 0.37 1.2 0.97 0.77 1.15 0.97 1.05 0.90 1.34 1.71 1.59 1.46 0.96

−4.52 8.62 2.69 0.45 3.53 15.67 −0.19 −5.73 −5.61 0.92 −6.29 −6.97 −2.07 1.37 1.29 1.61 −3.95 1.30 0.21 −14.71 −5.77 2.05 20.44 1.22 4.90 5.71 1.37 0.18 1.00 −7.30 −0.12 −1.88 0.73 9.62 −12.61 −12.61 3.60 −6.54 −12.61 −12.60 −0.12 0.18 1.00 −7.30 −21.25 0.99 −0.86 0.93 0.74 9.63 −0.50 −23.24 −6.54 2.63

6.77 13.04 6.77 1.01 −5.78 −8.07 0.13 1.47 −0.50 12.02 11.38 16.44 −0.18 3.28 8.96 1.34 −1.69 1.41 7.56 −13.28 −0.34 −0.83 −6.38 −6.88 −6.38 11.92 1.51 −0.55 17.87 −2.87 0.54 5.92 −2.84 −0.29 1.26 1.26 0.82 −1.23 −0.50 0.63 19.07 −0.55 17.87 −2.87 −19.26 −1.59 −12.91 1.05 −2.84 −0.29 −6.42 −0.63 −1.23 −3.76

1.85 5.17 4.90 0.02 −0.96 −1.76 −0.45 1.06 12.88 7.36 0.56 −6.12 0.79 −1.14 0.65 −0.35 1.03 1.09 1.11 0.58 0.83 −3.44 −4.83 0.64 −4.83 0.37 1.08 0.51 0.82 −0.60 −0.63 0.12 −2.27 11.63 0.69 0.69 0.16 −2.38 −0.34 −0.69 −0.35 0.51 0.82 −0.61 −11.76 −0.33 −6.63 −0.11 −2.27 11.63 0.43 −1.59 −2.38 2.06

−0.53 −0.30 −0.33 7.15 −0.23 −8.27 1.03 0.44 13.33 1.02 1.03 1.87 9.54 8.90 −0.58 4.83 −1.55 1.30 7.05 14.28 15.53 −7.14 0.59 7.26 0.59 −15.41 15.62 2.17 19.55 −9.79 7.93 1.16 1.04 −0.04 0.60 0.60 −0.62 −5.82 −1.14 −0.29 5.11 2.17 19.55 −9.79 −0.94 −7.19 1.54 0.85 1.04 −0.04 0.22 −8.01 −5.83 3.84

1.48 1.19 1.19 1.95 1.49 1.48 1.19 1.55 1.31 1.28 1.00 1.12 1.09 1.01 1.15

± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.87 0.97 1.22 0.59 1.04 0.93 1.20 0.88 0.67 0.67 1.47 1.02 1.20 0.79 0.87

1.34 −2.69 1.25 20.29 5.27 −2.25 −0.95 19.98 −1.74 −1.74 −6.94 −4.42 −0.01 −12.86 0.15

5.25 0.70 1.36 17.81 0.57 −1.29 −1.38 17.21 −1.56 −1.56 0.82 −0.46 −5.13 −0.61 1.22

6.34 1.70 0.47 20.17 0.55 −0.89 11.40 12.21 −0.65 −0.44 0.62 4.12 2.53 −19.76 1.68

8.10 −15.64 1.90 12.15 1.97 −4.40 2.13 21.58 −1.11 −1.11 −0.38 1.21 0.34 1.02 7.68

Anthocyanins

Dihydroflavonols

Flavanols Flavanones

Flavones

Flavonols

Isoflavonoids

Lignans

Alkylphenols

Curcuminoids Hydroxybenzaldehydes Hydroxycinnamaldehydes

(continued on next page) 7

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Table 3 (continued) Class

Compound

VIP score

LogFC [Leaves MeOH vs H20]

LogFC [Unmature fruits MeOH vs H2O]

Log FC [Mature fruits MeOH vs H2O]

LogFC [Twigs MeOH vs H2O]

Hydroxycoumarins Methoxyphenols Other polyphenols

Umbelliferone Guaiacol Catechol Pyrogallol Phlorin Carnosol Rosmanol Carvacrol Thymol Oleoside dimethylester Ligstroside-aglycone Hydroxytyrosol Oleoside 11-methylester p-HPEA-AC 4-Hydroxybenzoic acid 4-O-glucoside Isoferulic acid 3-Sinapoylquinic acid p-Coumaric acid 4-O-glucoside 3,5-Dicaffeoylquinic acid Ferulic acid 4-O-glucoside Feruloyl glucose Caffeic acid 4-O-glucoside p-Coumaroyl tyrosine Avenanthramide 2f Homovanillic acid

1.69 1.28 1.61 1.57 1.46 1.37 1.35 1.05 1.01 1.49 1.22 1.17 1.07 1.01 1.39 1.49 1.40 1.27 1.26 1.24 1.24 1.12 1.07 1.03 1.09

8.03 −1.74 −2.25 0.48 1.76 −0.12 −6.03 0.33 0.33 −23.96 0.68 −12.50 −13.55 −0.12 −0.22 −22.20 0.93 0.03 21.88 −1.44 −1.44 −0.87 0.82 −1.58 −0.01

−0.24 −1.56 −1.91 1.37 −0.20 −0.50 −0.50 −6.73 −18.76 −0.51 0.96 −6.10 6.94 −8.30 0.01 −0.63 −1.37 0.24 17.71 19.28 0.43 −0.20 5.56 −2.70 −5.11

8.08 −0.44 −2.23 −0.02 1.71 −0.35 −0.35 −10.65 −0.35 −2.96 −0.52 −1.66 −0.48 −0.88 6.58 −7.42 −14.89 −0.09 −12.19 −0.35 0.01 1.36 −0.77 −1.41 2-53

8.78 −1.11 −0.93 0.37 −0.06 18.98 8.09 1.00 1.00 −3.83 −6.48 1.41 0.63 −1.47 1.08 −21.01 2.29 1.33 0.10 1.49 1.49 0.30 6.71 11.15 0.33

Phenolic terpenes

Tyrosols

Hydroxybenzoics Hydroxycinnamics

Hydroxyphenylacetics

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.83 0.67 1.20 1.06 0.96 1.11 1.10 1.41 1.43 0.83 1.26 1.02 1.27 0.78 1.13 0.91 0.86 1.60 1.18 1.09 1.05 1.38 1.40 1.15 1.20

strongest inhibitory effect (0.58 mmol ACAE/g) followed by the aqueous leaf extract (0.09 mmol ACAE/g) while for the α-glucosidase activity, it is the methanolic twig extract which exerted the highest activity followed by the aqueous unmature fruit extract (0.42 mmol ACAE/g). It is worthy to point out that the methanolic twig extract showed the highest activity against four enzymes AChE, BChE, tyrosinase and αglucosidase except for α-amylase. Although, the aqueous unmature and methanolic mature fruit extracts have the highest TPC and TFC respectively, they did not possess the highest enzymatic inhibitory effects. It could be projected that there may not be any vital link between TPC or TFC and enzymatic properties but more directly linked to other bioactive compounds. In fact, different phenolic and flavonoid classes have different biological potentials (Kumar and Pandey, 2013). So far, little evidence has been found enzyme inhibitory effects of the members of the genus Rhamnus. The present findings seem to be consistent with other research (Ben et al., 2018) which found the best amylase and glucosidase inhibitory effects for methanol extract of R. frangula leaves. Also, R. lycioides subsp. oleoides anthraquinone rich extract exerted a notable anti-acetylcholinesterase activities (Benamar et al., 2019). In this context, the presented paper is the first scientific

harmful radiation (Athipornchai and Jullapo, 2018). On the other hand, the enzyme α-amylase breaks down long carbohydrate chains and αglucosidase breaks down starch and disaccharides into glucose. These enzymes serve as major digestive enzymes and help in intestinal absorption. Importantly, α-amylase and α-glucosidase are potential targets in producing lead compounds for the management of diabetes (Gulçin et al., 2018; Nair et al., 2013). AChE and its related enzyme BChE take part in the hydrolysis of acetylcholine and butyrylcholine, which are related to neurological disorders including Alzheimer’s disease (AD) (Pope and Brimijoin, 2018). It is known that BChE hydrolyses acetylcholine faster than AChE (Colović et al., 2013). Additionally, cholinesterase inhibitors also help to manage the leading cause of blindness which is glaucoma (Weinreb et al., 2014). As observed in Table 5, the most effective AChE and BChE inhibitor was found to be the methanolic twig extract (4.20 and 2.96 mg GALAE/g respectively). It is important to highlight that most of the extracts showed inactivity in terms of BChE inhibition in contrast to AChE. Moreover, the same extract, i.e. methanolic twig, showing significant cholinesterase inhibition equally exhibited the highest tyrosinase activity (137.14 mg KAE/g) and the least active is aqueous unmature fruit extract (13.03 mg KAE/ g). In terms of α-amylase, the methanolic leaf extract showed the Table 4 in vitro antioxidant activity of the different Rhamnus extracts*. Solvent

Parts

DPPH (mgTE/g)

ABTS (mgTE/g)

CUPRAC (mgTE/g)

FRAP (mg TE/g)

Phosphomolybdenum (mmol TE/ g)

Metal chelating ability (mg EDTAE/g)

MeOH

Twigs Mature fruits Unmature fruits Leaves Twigs Mature fruits Unmature fruits Leaves

165.23 ± 5.10e 378.95 ± 3.91c 470.96 ± 0.45a

252.52 ± 6.05d 186.88 ± 2.79e 268.76 ± 5.50c

401.52 ± 5.53e 518.40 ± 7.50d 660.55 ± 7.73b

517.70 ± 9.77e 569.80 ± 5.55d 761.62 ± 7.94b

2.54 ± 0.11b 1.77 ± 0.13e 2.33 ± 0.10cd

11.31 ± 1.28e 18.28 ± 1.78d 28.38 ± 1.30c

76.64 ± 0.54g 150.54 ± 3.46f 291.81 ± 8.30d 388.02 ± 2.77b

90.70 ± 0.81g 282.82 ± 8.66b 279.33 ± 1.55b 394.96 ± 10.23a

158.65 368.69 529.55 772.73

110.22 ± 2.13g 506.41 ± 6.9f 716.09 ± 2.98c 1049.61 ± 13.25a

1.91 2.42 2.23 2.96

0.07e 0.10bc 0.07f 0.04a

28.55 ± 2.82c 8.17 ± 0.62f 18.41 ± 2.40d 40.57 ± 0.03a

54.39 ± 1.29h

113.03 ± 1.09f

123.58 ± 1.42h

104.71 ± 1.97g

1.59 ± 0.12d

Water

± ± ± ±

6.64g 7.57f 5.49c 8.63a

± ± ± ±

31.70 ± 0.94b

* Values are expressed are mean ± standard deviation (n = 3). DPPH: 2,2-diphenyl-1-picrylhydrazyl; ABTS: 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid); CUPRAC: Cupric reducing antioxidant capacity; FRAP: Ferric reducing antioxidant power. TE: Trolox equivalent; EDTAE: EDTA equivalent. Different letters indicate significant differences in the extracts as resulted from ANOVA (p < 0.01; Duncan’s post-hoc test). 8

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Table 5 Enzyme inhibitory activity of the different Rhamnus extracts*. Solvent

Parts

AChE inhibition (mg GALAE/g)

MeOH

Twigs Mature fruits Unmature fruits Leaves Twigs Mature fruits Unmature fruits Leaves

4.20 2.38 2.74 3.67 1.04 na na 0.39

Water

± ± ± ± ±

0.02a 0.05d 0.20c 0.40c 0.20e

± 0.12f

BChE inhibition (mg GALAE/g)

Tyrosinase inhibition (mg KAE/g)

Amylase inhibition (mmol ACAE/g)

2.96 ± 0.28a na na 1.03 ± 0.31b na na na na

137.14 ± 0.97a 127.94 ± 2.28c 127.69 ± 1.38c 134.71 ± 0.46b 29.16 ± 1.37d 13.87 ± 1.27e 13.03 ± 0.84e 14.65 ± 2.03e

0.57 0.55 0.48 0.58 0.14 0.16 0.13 0.09

± ± ± ± ± ± ± ±

0.04ab 0.03b 0.02c 0.01a 0.01d 0.01d 0.01d 0.01e

Glucosidase inhibition (mmol ACAE/g) 1.93 1.90 1.70 1.86 1.43 0.42 1.00 0.67

± ± ± ± ± ± ± ±

0.01a 0.03a 0.03c 0.01b 0.01d 0.02g 0.01e 0.03f

* Values are expressed as mean ± standard deviation (n = 3). AChE: Acetylcholinesterase; BChE: Butyrylcholinesterase; GALAE: Galatamine equivalent; KAE: Kojic acid equivalent; ACAE: Acarbose equivalent; n.a.: not active. Different letters indicate significant differences in the extracts as resulted from ANOVA (p < 0.01; Duncan’s post-hoc test).

matrices, compared with targeted approaches. Furthermore, for the first time, a detailed description of polyphenols and anthraquinone profiles in this plant species has been achieved. Therefore, these preliminary findings could be very useful to produce active extracts warrant further attention in an endeavor to develop new phytopharmaceuticals, nutraceuticals or food additives.

evidence on enzyme inhibitory effects of R. petiolaris extracts and thus it might provide a great contribution to scientific platform. 3.5. Correlations between phytochemical profiles and bioactive properties The relationship between the quantitative profiles of polyphenols and the biological activity of R. petiolaris were statistically analyzed. With this aim, the Pearson’s correlation coefficients were established between polyphenols and their respective antioxidants and enzymes investigated to determine possible linear relationship between them. The data are presented in supplementary material. It was observed that the highest and most significant positive linear relationship exist between anthocyanins: DPPH (0.82: p < 0.01), flavonols: DPPH (0.91: p < 0.01) and TFC: DPPH (0.86: p < 0.01). Furthermore, a strong negative correlation exists between stilbenes: CUPRAC (-0.80: p < 0.01) and tyrosols: DPPH (-0.74: p < 0.05). It is important to underline that alkylphenols do not show any significant contribution to the antioxidants and enzymes except BChE showing a positive correlation of 0.67 (p < 0.01). From the data obtained by Pearson’s correlations, we established no correlation between polyphenols and amylase activity in contrary to glucosidase which showed a moderate correlation of 0.58 (p < 0.01) with flavones and 0.52 (p < 0.05) with lignans. As proposed by Ozer et al. (2018), the determination of phytochemicals responsible for amylase activity needs further biological tests to be verified, including biological activity guided chromatography techniques. Similarly, any of the polyphenols investigated correlate with tyrosinase except TPC-MS showing a weak relationship (0.41: p < 0.05). Finally, flavonols, stilbenes, anthraquinones and phenolic acids showed no significant correlation with any enzyme investigated.

Declaration of Competing Interest The authors declare no conflict of interest. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.indcrop.2019.111856. References Akkemik, U., Ok, T., Eminagaoglu, O., Fırat, M., Aksoy, N., 2018. Rhamnus L. Çehriler. In: Akkemik, U. (Ed.), Türkiye’nin Doğal-Egzotik Ağaç ve Çalıları. Orman Genel Müdürlüğü Yayınları, Ankara, pp. 248–271. Alam, M.N., Bristi, N.J., Rafiquzzaman, M., 2013. Review on in vivo and in vitro methods evaluation of antioxidant activity. Saudi Pharm. J. 21, 143–152. Allkin, B., 2017. Useful plants –medicines: at least 28, 187 plant species are currently recorded as being of medicinal use. In: Willis, K.J. (Ed.), State of the World’s Plants 2017. Royal Botanic Gardens, Kew, London (UK) Available from: https://www.ncbi. nlm.nih.gov/books/NBK464488/. Athipornchai, A., Jullapo, N., 2018. Tyrosinase inhibitory and antioxidant activities of Orchid (Dendrobium spp.). S. Afr. J. Bot. 119, 188–192. Belwal, T., Ezzat, S.M., Rastrelli, L., Bhatt, I.D., Daglia, M., Baldi, A., Devkota, H.P., Orhan, I.E., Patra, J.K., Das, G., 2018. A critical analysis of extraction techniques used for botanicals: trends, priorities, industrial uses and optimization strategies. TracTrend Anal. Chem. 100, 82–102. Belwal, T., Pandey, A., Bhatt, I.D., Rawal, R.S., Luo, Z., 2019. Trends of polyphenolics and anthocyanins accumulation along ripening stages of wild edible fruits of Indian Himalayan region. Sci. Rep. 9, 5894. Ben, A.B., Jemel, I., Bhat, R.S., Onizi, M.A., 2018. Inhibitory effects of various solvent extracts from Rhamnus frangula leaves on some inflammatory and metabolic enzymes. Cell. Mol. Biol. 64, 55–62. Ben Ammar, R., Miyamoto, T., Chekir-Ghedira, L., Ghedira, K., Lacaille-Dubois, M.-A., 2019. Isolation and identification of new anthraquinones from Rhamnus alaternus L and evaluation of their free radical scavenging activity. Nat. Prod. Res. 33, 280–286. Benamar, H., Rarivoson, E., Tomassini, L., Frezza, C., Marouf, A., Bennaceur, M., Nicoletti, M., 2019. Phytochemical profiles, antioxidant and anti-acetylcholinesterasic activities of the leaf extracts of Rhamnus lycioides subsp. Oleoides (L.) Jahand. & Maire in different solvents. Nat. Prod. Res. 33, 1456–1462. Boussahel, S., Speciale, A., Dahamna, S., Amar, Y., Bonaccorsi, I., Cacciola, F., Cimino, F., Donato, P., Ferlazzo, G., Harzallah, D., 2015. Flavonoid profile, antioxidant and cytotoxic activity of different extracts from Algerian Rhamnus alaternus L. bark. Pharmacogn. Mag. 11, S102. Carocho, M., Ferreira, I.C.F.R., 2013. A review on antioxidants, prooxidants and related controversy: natural and synthetic compounds, screening and analysis methodologies and future perspectives. Food Chem. Toxicol. 51, 15–25. Colović, M.B., Krstić, D.Z., Lazarević-Pašti, T.D., Bondžić, A.M., Vasić, V.M., 2013. Acetylcholinesterase inhibitors: pharmacology and toxicology. Curr. Neuropharmacol. 11, 315–335. Comlekcioglu, N., Aygan, A., Kutlu, M., Kocabas, Y.Z., 2017. Antimicrobial activities of some natural dyes and dyed wool yarn. Iran. J. Chem. Chem. Eng. Research Note Vol 36.

4. Conclusions This study sets out to study the biological propensities of the aqueous and methanolic extracts of Rhamnus petiolaris Boiss. & Balansa (twigs, leaves, mature and unmature fruits). The extracts showed different biological activities, according to the matrix considered. The methanolic unmature fruit was more effective as DPPH and ABTS scavenger, whereas the aqueous unmature fruit extract displayed the highest cupric and ferric reducing power. The highest phenolic contents were found in mature and unmature fruits, independently from the extraction solvent used. Correlation results showed that flavonols and anthocyanins were highly correlated with DPPH and tyrosols were in a good linear relationship with BChE activity. In conclusion, data provided from the present study tend to show that the different extractions used were able to promote the recovery of specific subclasses of compounds with different efficiencies (above all when considering flavonoids, phenolic acids and anthraquinones). This work underlines the potential of untargeted metabolomics in providing better insights and comprehensive descriptions of the phytochemical composition of plant 9

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Ozipek, M., Calis, I., Ertan, M., Ruedi, P., 1994. Rhamnetin 3-p-coumaroylrhamninoside from Rhamnus petiolaris. Phytochemistry 37, 249–253. Ozturk, M., Altay, V., 2017. An overview of the endemic medicinal and aromatic plants of Turkiye-conservation and sustainable use. I. International Congress on Medicinal and Aromatic Plants (Konya, 10-12 May 2017). pp. 11 10. Ozturk, M., Uysal, I., Gucel, S., Altundag, E., Dogan, Y., Baslar, S., 2013. Medicinal uses of natural dye-yielding plants in Turkey. Res. J. Text. App. 17, 69–80. Petrovska, B.B., 2012. Historical review of medicinal plants’ usage. Pharmacogn. Rev. 6, 1–5. Pope, C.N., Brimijoin, S., 2018. Cholinesterases and the fine line between poison and remedy. Biochem. Pharmacol. 153, 205–216. Quattrocchi, U., 2016. CRC World Dictionary of Medicinal and Poisonous Plants: Common Names, Scientific Names, Eponyms, Synonyms, and Etymology (5 Volume Set). CRC press. Rocchetti, G., Giuberti, G., Gallo, A., Bernardi, J., Marocco, A., Lucini, L., 2018. Effect of dietary polyphenols on the in vitro starch digestibility of pigmented maize varieties under cooking conditions. Food Res. Int. 108, 183–191. Rocchetti, G., Blasi, F., Montesano, D., Ghisoni, S., Marcotullio, M.C., Sabatini, S., Cossignani, L., Lucini, L., 2019a. Impact of conventional/non-conventional extraction methods on the untargeted phenolic profile of Moringa oleifera leaves. Food Res. Int. 115, 319–327. Rocchetti, G., Lucini, L., Rodriguez, J.M.L., Barba, F.J., Giuberti, G., 2019b. Gluten-free flours from cereals, pseudocereals and legumes: phenolic fingerprints and in vitro antioxidant properties. Food Chem. 271, 157–164. Šamec, D., Kremer, D., Grúz, J., Jurišić Grubešić, R., Piljac-Žegarac, J., 2012. Rhamnus intermedia Steud. Et Hochst.–A new source of bioactive phytochemicals. Croat. Chem. Acta 85, 125–129. Stef, D.S., Gergen, I., Trasca, T.I., Harmanescu, M., Lavinia, S., Ramona, B., Heghedus, M.G., 2009. Total antioxidant and radical scavenging capacities for different medicinal herbs. Rom. Biotech. Lett. 14, 4704–4709. Stierle, A., Strobel, G., Stierle, D., 1993. Taxol and taxane production by Taxomyces andreanae, an endophytic fungus of Pacific yew. Science 260, 214. The Plant List, 2013. The Plant List. (Accessed 6th July 2018). http://www.theplantlist. org/. Uysal, S., Zengin, G., Locatelli, M., Bahadori, M.B., Mocan, A., Bellagamba, G., De Luca, E., Mollica, A., Aktumsek, A.J.Fip., 2017. Cytotoxic and enzyme inhibitory potential of two Potentilla species (P.speciosa L. and P. reptans Willd.) and their chemical composition. Front. Pharmacol. 8, 290. Wagner, H., Ertan, M., Seligmann, O.J.P., 1974. Rhamnazin-und rhamnetin-3-O-trioside aus Rhamnus petiolaris. Phytochemistry 13, 857–860. Yoo, S., Nam, H., Lee, D., 2018. Phenotype-oriented network analysis for discovering pharmacological effects of natural compounds. Sci. Rep. 8, 11667. Złotek, U., Mikulska, S., Nagajek, M., Świeca, M.J., 2016. The effect of different solvents and number of extraction steps on the polyphenol content and antioxidant capacity of basil leaves (Ocimum basilicum L.) extracts. Saudi J. Biol. Sci. 23, 628–633.

Dogan, Y., Başlar, S., Mert, H.H., Güngör, A., 2003. Plants used as natural dye sources in Turkey. Econ. Bot. 57, 442–453. Duval, J., Pecher, V., Poujol, M., Lesellier, E., 2016. Research advances for the extraction, analysis and uses of anthraquinones: a review. Ind. Crop. Prod. 94, 812–833. Flora of China, 2007. Flora China 12, 139–162. http://www.efloras.org/florataxon.aspx? flora_id=2&taxon_id=128246. Gulçin, İ., Taslimi, P., Aygün, A., Sadeghian, N., Bastem, E., Kufrevioglu, O.I., Turkan, F., Şen, F., 2018. Antidiabetic and antiparasitic potentials: inhibition effects of some natural antioxidant compounds on α-glycosidase, α-amylase and human glutathione S-transferase enzymes. Int. J. Biol. Macromol. 119, 741–746. Kähkönen, M.P., Hopia, A.I., Vuorela, H.J., Rauha, J.-P., Pihlaja, K., Kujala, T.S., Heinonen, M., 1999. Antioxidant activity of plant extracts containing phenolic compounds. J. Agric. Food Chem. 47, 3954–3962. Kayabasi, N., Arlı, M., 2001. Cehri (Rhamnus petiolaris)’den Elde Edilen Renkler. Tarim Bilimleri Dergisi 7, 128–134. Kosalec, I., Kremer, D., Locatelli, M., Epifano, F., Genovese, S., Carlucci, G., Randić, M., Zovko Končić, M., 2013. Anthraquinone profile, antioxidant and antimicrobial activity of bark extracts of Rhamnus alaternus, R. fallax, R. Intermedia and R. Pumila. Food Chem. 136, 335–341. Kumar, S., Pandey, A.K., 2013. Chemistry and biological activities of flavonoids: an overview. Sci. World J. 16 2013. Locatelli, M., Epifano, F., Genovese, S., Carlucci, G., Koncić, M., Kosalec, I., Kremer, D., 2011. Anthraquinone profile, antioxidant and antimicrobial properties of bark extracts of Rhamnus catharticus and R. orbiculatus. Nat. Prod. Commun. 6, 1275–1280. Lou, S.-N., Lai, Y.-C., Hsu, Y.-S., Ho, C.-T., 2016. Phenolic content, antioxidant activity and effective compounds of kumquat extracted by different solvents. Food Chem. 197, 1–6. Martins, N., Barros, L., Ferreira, I.C.F.R., 2016. In vivo antioxidant activity of phenolic compounds: facts and gaps. Trends Food Sci. Technol. 48, 1–12. Meshkini, A., 2016. Acetone extract of almond hulls provides protection against oxidative damage and membrane protein degradation. J. Acupunct. Meridian Stud. 9, 134–142. Moreira, T.F., Sorbo, J.M., Souza, Fd.O., Fernandes, B.C., Ocampos, F.M.M., de Oliveira, D.M.S., Arcaro, C.A., Assis, R.P., Barison, A., Miguel, O.G., 2018. Emodin, physcion, and crude extract of Rhamnus sphaerosperma var. Pubescens induce mixed cell death, increase in oxidative stress, DNA damage, and inhibition of AKT in cervical and oral squamous carcinoma cell lines. Oxid. Med. Cell. Longev. 1–18 2018. Moussi, K., Nayak, B., Perkins, L.B., Dahmoune, F., Madani, K., Chibane, M., 2015. HPLCDAD profile of phenolic compounds and antioxidant activity of leaves extract of Rhamnus alaternus L. Ind. Crop. Prod. 74, 858–866. Nair, S.S., Kavrekar, V., Mishra, A., 2013. In vitro studies on alpha amylase and alpha glucosidase inhibitory activities of selected plant extracts. Euro J. Exp. Bio. 3, 128–132. Ozer, M.S., Kirkan, B., Sarikurkcu, C., Cengiz, M., Ceylan, O., Atılgan, N., Tepe, B., 2018. Onosma heterophyllum: phenolic composition, enzyme inhibitory and antioxidant activities. Ind. Crop. Prod. 111, 179–184.

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