Phenolic Compound Analysis and Pharmacological Screening of Vitex agnus-castus Functional Parts

Vitex agnus-castus is a medicinal plant of the Verbenaceae family, widely used in traditional medicine. This study is aimed at investigating the functional variability of phenolic compounds in different parts (leaves, stems, flowers, roots, and seeds) of Vitex agnus-castus methanolic extracts and at assessing their in vitro antidiabetic, antioxidant, and antibacterial activities. The results of HPLC-DAD-QTOF-MS indicated the presence of 25 phenolic compounds with a remarkable variability between plant parts; high levels were registered in chlorogenic, vanillic, 3,4-dihydroxybenzoic, and 3-hydroxybenzoic acids; hesperidin; and luteolin. V. agnus castus fruits and stems presented higher antioxidant activities. The extracts inhibited the growth of five pathogenic bacteria with MIC values documented between 7.81 and 31.25 mg/mL. In vitro antihyperglycemic effect revealed higher effect in flowers (2921.84 μg/mL) and seeds (2992.75 μg/mL) against α-glucosidase and of leaves (2156.80 μg/mL) and roots (2357.30 μg/mL) against α-amylase. The findings of this showed that V. agnus castus is a promising source for antidiabetic bioactive compounds. However, further investigations regarding the evaluation of in vivo antidiabetic effects of these compounds are needed.


Introduction
Vitex agnus-castus L, the chaste tree, is a shrub belonging to the Vitex genus of the Verbenaceae family. It is scattered on the shores and on the shores of the Mediterranean and Asia. V. agnus castus is a deciduous plant that reaches heights up to 5 m [1]. The leaves are opposite, in the form of a hand, composed of five to seven radiating leaves which are carried on a main stem. The leaves are linear, lanceo-late, toothed, dark green above, and gray under a very tight felt. From August to October, panicles of flowers with many flowers form. The flowers are fragrant and of a blue, lilac, pink, or white color. The berries resemble peppercorns, hard, violet to black, yellowish in the interior, half covered with their green calyces, and containing four seeds. The smell is aromatic and spicy, the taste warm and special. The parts used today as herbal medicine are the ripe dried berries.
Recently, essential oils and organic extracts from V. agnus castus showed promising properties including antibacterial, antioxidant, anticancer, and anticorrosion [7][8][9][10][11]. However, at our knowledge, there is no study that investigated a comparison between different plant parts of V. agnus castus. Therefore, the aims of this study are to determine the phenolic compounds of V. agnus castus methanolic extract from the leaves, stems, flowers, roots, and seeds using HPLC-DAD-QTOF-MS analysis and to investigate their functional properties (antioxidant, antidiabetic, and antibacterial).

Experimental Procedures
2.1. Collection and Identification of the Plant. Vitex agnuscastus was harvested at the edge of Oued Baht Caidat of Al Mokhtar, Sidi Al Kamel of the province of Sidi Kacem which corresponds to the following coordinates: latitude 34°13 ′ 00 ″ North, longitude 5°42 ′ 00 ″ West, altitude 194 m. The plant was collected in August 2014 at the time of their flowering stage. The samples (leaves, roots, stems, flowers, and seeds) were rinsed, dried in a dry place, aired in the absence of light, and finally crushed using a grinder. The species was identified the Prof. Zidane Lahcen (Département Biologie, Productions Végétales Animales et Agro-Industrie, Ibn Tofail University).

Extraction and Phenolic Compound Quantification.
The extraction of the various parts was carried out under cold conditions by maceration of 5 g of the vegetable powder in 40 mL of methanol in solution for 24 hours with stirring. The filtrates obtained were placed in closed bottles and then stored in the fridge (put temp 4-7°C). The extract concentration was calculated relatively to the plant material and used for all assays. The different parts of the plant were assessed for polyphenolic contents. Total polyphenol content was determined by the Folin-Ciocalteu method [12], the flavonoids by the aluminum chloride (AlCl 3 ) colorimetric method, and the tannins condensed by the sulfuric vanillin reaction. The total polyphenol content was expressed in mg gallic acid equivalent/g extract (mg GE/g extract), the total flavonoid content in quercetin equivalent mg/g extract (mg QE/g extract), and condensed tannins in mg catechin equivalent/g extract (mg CE/g extract).
2.3. Chromatographic Analysis. Phenolic compound analysis was performed using HPLC-DAD-QTOF-MS [13]. The system consisted of a binary pump (G1312A, Agilent Technologies, Inc., Wilmington, DE, USA) and an auto sampler (G1330B, Agilent Technologies, Inc. Wilmington, DE, USA) coupled to a mass spectrometer equipped with an electrospray ionizer source (MS; ESI-; Micro mass Quattro Micro; Waters, Milford, MA, USA). Reversed phase HPLC separation was carried out using a zorbax C18 column Zorbax (100 mm × 2:1 mm × 1:7 μm, Agilent Technologies, Santa Clara, CA, USA). The mass spectrometer was operated in negative ion mode with the following parameters: capillary voltage, 3.0 kV; cone voltage, 20 V; and extractor, 2 V. Source temperature was 100°C, desolvation temperature was 350°C, cone gas flow was 30 L/h, and desolvation gas flow was 350 L/h. The mobile phase components were 0.1% formic acid (A) and acetonitrile with 0.1% formic acid (B). The mobile phase gradient was as follows: 0 min, 90% A; 0-18 min, 30% A; 18-20 min, 30% A; 20-23 min, 30% A; 23-25 min, 90% A; and 25-30 min, 90% A. The injection volume was 10 μL, and the column temperature was 35°C. The flow rate of the mobile phase was 0.5 mL/min. The phenolic acids were identified on the basis of their retention times, MS spectra, and molecular-ion identification.

α-Amylase
Inhibition. In α-amylase inhibition assay, 250 μL of the sample was mixed with 250 μL of α-amylase (240 U/mL, in 0.02 M phosphate buffer, pH 6.9, with 0.006 M NaCl). After incubating at 37°C for 10 min, 250 μL of 1% (w/v) soluble starch (in 0.02 M phosphate buffer, pH 6.9) was added and the mixture was further incubated at 37°C for 30 min, followed by adding 500 μL of DNS color reagent, and stopped by heating in boiling water bath for 10 min. After cooling to room temperature, the mixture was diluted with 2 mL of buffer and the absorbance was measured at 540 nm. The inhibition percentage (%) was calculated by the following equation for both enzymatic assays: where AC is the absorbance of the control, ACb is the control blank, AS is the absorbance of the sample, and AS is the absorbance of the sample blank.

α-Glucosidase
Inhibition. The α-glucosidase enzyme (0.1 U/mL) and substrate p-nitrophenyl-α-D-glucopyranoside (p-NPG, 1 mM) were dissolved in potassium phosphate buffer (0.1 M, pH 6.7), and all samples were dissolved in distilled water. The inhibitor (150 μL) was preincubated with the enzyme (100 μL) at 37°C for 10 min, and then, the substrate (200 μL) was added to the reaction mixture. The enzymatic reaction was performed at 37°C for 30 min. The reaction was then terminated by the addition of Na 2 CO 3 (1 M, 1 mL). All samples were analyzed in triplicate with different concentrations to determine the IC 50 values, and the absorbance was recorded at 405 nm [13].
2 BioMed Research International 2.6. Free Radical Scavenging Activity. The free radical scavenging activity of the extracts was measured by 1.1-diphenyl-2-picrylhydrazil (DPPH) as described by Huang et al. [14]. Briefly, 0.2 mm solution of DPPH in methanol was prepared and 0.5 mL of this solution was added to 2.5 mL of plant extract and was allowed to stand at room temperature for 30 min, and then, absorbance was read at 517 nm against blank samples. Lower absorbance of the reaction mixture indicated higher free radical scavenging activity. The radicalscavenging activity (RSA) was calculated as a percentage of DPPH discoloration. The following equation was used: where AD is the absorbance value of the DPPH blank sample and AE is the absorbance value of the test solution. AE was evaluated as the difference between the absorbance value of the test solution and the absorbance value of its blank. Thin layer chromatography was used to identify the active compounds against DPPH radicals [15]. The chemical revelator is a methanolic solution of DPPH with a concentration of 2 mg/mL for the detection of extracts as well as compounds having antioxidant activity [16]. The frontal ratios of the spots resulting from the separation were calculated.

Ferric
Reducing/Antioxidant Power. The ferric ion (Fe 3+ ) reducing antioxidant power (FRAP) method was used to measure the reducing capacity of extracts with a slight modification, which involves the presence of extracts to reduce the ferricyanide complex to the ferrous form. The absorbance was measured at 700 nm at the reaction time of 30 min. The reducing power of the extracts was represented as ascorbic acid equivalent (mg AAE/g edw).
2.8. Trolox Equivalent Antioxidant Capacity. The Trolox equivalent antioxidant capacity (TEAC) was used. The assay is based on the inhibition of the absorbance of the radical cation 2,2 ′ -azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS+) solution when it is exposed to an antioxidant as described. The ABTS+ cation radical was produced by the reaction between 10 mL of 2 mm ABTS in H 2 O and 100 mL of 70 mm potassium persulfate, stored in the dark at room temperature for 24 h. The ABTS+ solution was then diluted with methanol to obtain an absorbance of 0.70 at K = 734 nm and adjusted the temperature to 30°C. Samples were prepared in triplicate by diluting 50 mL of extracts in 2 mL of the ABTS + solution diluted with methanol and left to react for 1 min. Absorbances were recorded at 734 nm. The antioxidant activity samples were expressed as TEAC values, defined as the concentration of standard Trolox with the same antioxidant capacity of the extract under investigation.
2.9. Antibacterial Activity. The antimicrobial activity was examined against five bacterial strains: Staphylococcus aureus CIP 483, Bacillus subtilis CIP 5262, Escherichia coli CIP 53126, Pseudomonas aeruginosa CIP 82118, and Salmonella enterica CIP 8039. For the minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) of our crude extract, a modified resazurin microtitre-plate assay was used as reported by Sarker et al. [17]. Any color changes from purple to pink or colorless were recorded as positive. The lowest concentration at which color change occurred recorded as the MIC value; the minimum bactericidal concentration (MBC) is the lowest concentration of substance that leaves at most 0.01% of surviving germs [18].
2.10. Statistical Analysis. The significance of differences between multiple averages was determined by one-way analysis of variance (ANOVA), followed by Tukey's post hoc test at a 5% significance level.   Table 1). The lowest content of polyphenols was that of the stems (4:21 ± 1:53 mg GE/g), while the highest value was that of the flower part (13:11 ± 1:28 mg GE/g). The flavonoid levels followed an inverse trend. The tannin levels ranged between 2:72 ± 0:73 mg CE/g for stems and 9:29 ± 1:47 mg CE/g for flowers. The results of HPLC-DAD-QTOF-MS are shown in Table 2. As listed, Vitex extracts showed an important diversity and variability between plant parts. The chlorogenic acid (main phenolic compound) is present in all plant parts in the following concentrations: 42019:9 ± 2269:07 μg/kg (leaves), 295098 ± 15640:2 μg/kg (roots), 213138 ± 11935:7 μg/kg (stems), 259626 ± 13500:6 μg/kg (flowers), and 122900 ± 4547:28 μg/kg (seeds). Moreover, the chemical analysis by HPLC-MS was identified 11 types of flavonoids; the most abundant is luteolin with high levels especially in the flower part with a value of 77047:5 ± 4006:47 μg/kg. Importantly, other previous studies have also revealed numerous phenolic compounds in V. agnus castus extracts [6,[19][20][21]. Indeed, the rate of phenolic compounds found in our results is not exactly similar to those identified in these reposted works. This difference is certainly due the geographical origins of the plant. However, in our study, the variability between chemical compounds in different parts can be explained by the ability of        Figure 6: Chemical structures of identified bioactive compounds. 6 BioMed Research International previous studies have indicated the antidiabetic effects of V. agnus castus [22]. The antidiabetic action of this plant is not mediated by the inhibition of enzymes involved in hydrocarbon catabolism, while it is related to its capacity to hypoglycemic state via hormonal interaction mechanisms [22]. Berrani et al. [9] showed that methanolic extracts of V. agnus castus aerial part exhibit a remarkable in vivo antidiabetic effect in diabetic rats induced by streptozotocin. Indeed, the administration of this extract at 300 mg/kg induced a decrease in blood glucose after 2 h and improved lipid profile after 21 days [9]. In addition, other plants belonging to other families have exerted important in vitro antidiabetic effects via the inhibition of α-amylase and α-glucosidase [23,24].   These plants contain phenolic compounds similar to those identified in V. agnus castus which they are certainly responsible for these effects.
Main bioactive compounds (chlorogenic acid, luteolin, vanillic, hesperidin, and luteolin are the) identified in agnus castus are involved certainly in these antidiabetic effects. In fact, literature reports showed that these compounds possess promising in vitro and in vivo antidiabetic properties with several mechanisms of action. Indeed, several studies reported the antidiabetic effects of luteolin [25][26][27]. In an in vitro study, Kyungpook et al. [27] showed that luteolin exhibits an important inhibition of α-glucosidase (36% at 0.5 mg/mL) compared with acarbose used as a standard drug, the inhibition of αamylase was less potent than that of acarbose. Yan et al. [26] reported that luteolin reduces the activity of α-glucosidase at a dose dependent-manner (IC 50 = 1:72 ± 0:05:10 −4 mol/L). Moreover, this inhibition was mediated by a noncompetitive inhibition action with a single inhibition site on α-glucosidase (K i = 1:40 ± 0:02 × 10 −4 mol/L) [26]. On the other hand, this compound inhibited the activity of several intestinal enzymes such as α-glucosidase, maltase activity, and sucrase in an animal model [25]. Chlorogenic acid showed also remarkable antidiabetic effects [28][29][30]. In vitro studies reported that chlorogenic acid inhibits α-amylase and α-glucosidase (key enzymes linked to type 2 diabetes) activities in a dosedependent manner [30]. Nicasio et al. [28] reported that chlorogenic acid decreases plasma glucose surge during the glucose tolerance test in streptozotocin-induced diabetic rats [29]. Karthikesan et al. [31] reported that chlorogenic acid increases the levels of plasma insulin and glycogen, decreases glycosylated hemoglobin, and reverses the altered activities of some enzymes implicated in glucose metabolism such as glucose-6-phosphatase, fructose-1,6-bisphosphatase, glucokinase, and hexokinase in an animal model [31]. Moreover, mechanistic studies revealed that chlorogenic acid exhibits a stimulation of glucose transport in L6 skeletal muscle cells and increases GLUT 4 translocation through AMPK activation [32]. In addition, this acid inhibited the gluconeogenesis through the downregulation of gluconeogenic enzyme G6Pase, stimulates glucose uptake in skeletal muscles by increasing GLUT-4 expression and translocation to plasma membrane, and increases AMPK phosphorylation in timeand dose-dependent manners [32].

Antioxidant Activity.
Recently, several scientific investigations revealed that oxidative stress plays an important role in the development of numerous pathologies such as cancer, diabetes, and autoimmune diseases. The antioxidants are the molecules which eliminate these unstable free radicals and therefore inhibit the rate of oxidation and protect cells from damage [33]. These bioactive molecules are mostly used for the prevention and treatment of oxidative stress-related diseases such as diabetes, Alzheimer's disease, and cancer [34]. Several tests have been developed for the analysis of in vitro and in vivo antioxidant activity [35]. Furthermore, to study the antioxidant activity of our plant extracts, the ability to scavenge the stable free radical DPPH and the cation ABTS and their ferric reducing antioxidant power (FRAP) was evaluated.
According to DPPH assay results (Table 1), the flower part exhibited a high IC 50 value = 0:199 mg/mL, followed by roots (IC 50 = 0:283 mg/mL), and then stems and leaves. The lowest activity was attributed to seeds with 0.612 mg/mL. The antioxidant effect based on thin layer chromatography revealed inhibitory compounds in the plant parts. These constituents are capable of stabilizing the DPPH radicals. The results of the antioxidant activity evaluated by FRAP assay are shown in Table 1, used as ascorbic acid equivalent reference. We observed that the flowers, roots, and stems (1:347 ± 0:08, 1:293 ± 0:06, and 0:926 ± 0:17 mg AAE/g, respectively) exhibited a high reducing power in comparison to leaves (0:488 ± 0:14 mg AAE/g) and seeds (0:615 ± 0:09 mg AAE/g). The plant part ability to inhibit the ABTS+ radical was obtained using the ABTS assay with Trolox in a calibration reference curve. The discoloration test results are reported in Table 1. As shown, we noted that the roots (27:668 ± 0:46 mg TE/g) had the most active effect compared to other parts. Several other studies have reported the antioxidant effects [36,37]. These works revealed different findings according to the used methods and type of tested extract.
3.4. Antibacterial Activity. The antibacterial activity was evaluated by the microdilution method. The results were expressed by MIC and MBC values. Methanol extracts of different parts showed remarkable antibacterial powers with some variability depending on the part of plant and the tested strain. The MIC and MBC values are listed in Table 4. As summarized, the MIC and CMB obtained ranged between 7.81 and 31.25 mg/mL. Indeed, methanolic extracts of flowers showed the lowest MIC and MBC values against tested strains especially P. aeruginosa (MIC = MBC = 7:81). The ration  Table 4. As shown, methanolic extracts of different parts revealed a bactericidal power by a ratio of CMB/MIC between 1 and 2 (less than 4). Moreover, because of the development of bacterial resistance, infectious diseases became a serious problem of human health. Different sources have been screened for identification and isolation of new bioactive molecules with antibacterial properties. This work revealed that extracts from different parts of Vitex agnus-castus inhibited bacterial growth with the lowest concentrations. Other studies have reported the antibacterial effects of Vitex agnus-castus [38][39][40]. Our results are in consonance with those obtained in these reported works with certain difference, which could be related to the difference in chemical compounds, bacterial strains, and used methods. Moreover, the antibacterial action of Vitex agnus-castus extracts is most probably due to the major phenolic compounds such as luteolin and chlorogenic acid that show remarkable antibacterial effects [41,42].

Conclusion
The present work has highlighted the richness of Vitex agnus-castus in phenolic compounds, especially flavonoids and tannins, which are most likely responsible for these biological activities. The results of the present study demonstrated that the methanolic extracts of the different parts of the Vitex plant are effective inhibitors of α-amylase and α-glucosidase, which can help be of value in lowering postprandial glucose levels. However, the major compounds responsible for the inhibitory action of α-amylase and α-glucosidase have yet to be characterized, isolated, and tested for in vitro and in vivo antidiabetic activity. This can be beneficial in the development of new antidiabetic agents from indigenous plant resources.

Data Availability
All used data in this study were cited in the manuscript.

Ethical Approval
This study did not include the in vivo experimental procedures with a mouse model.