Caucasian Gentiana Species: Untargeted LC-MS Metabolic Profiling, Antioxidant and Digestive Enzyme Inhibiting Activity of Six Plants

The members of Gentiana genus are widely distributed in the Caucasus region where they are used as phytoremedies, but they still have not been studied for their chemical composition and bioactivity. High-performance liquid chromatography with diode array and electrospray triple quadrupole mass detection (HPLC-DAD-ESI-QQQ-MS) was used to investigate metabolites of herb and roots of six gentians (Gentiana asclepiadea, G. cruciata, G. gelida, G. paradoxa, G. pneumonanthe, G. septemfida) grown in the Caucasus. In total, 137 compounds were found including three carbohydrates, 71 iridoid glycosides (mostly loganic acid), loganin, swertiamarin, gentiopicroside and sweroside derivatives, 40 flavones C-, O-, C,O-glycosides (such as luteolin, apigenin, chrysoeriol, and acacetin derivatives), two phenolic O-glycosides, five hydroxycinnamates, eight xanthones, and seven triterpene glycosides. Most of these compounds were identified in gentian samples for the first time. Quantitative differences were found in levels of seven iridoid glycosides, nine glycosylflavones, and two xanthones obtained by HPLC-DAD assay. The gentian extracts were evaluated for their radical-scavenging properties against DPPH and superoxide anion radicals, lipid peroxidation inhibition, and α-amylase/α-glycosidase inhibition. The herb extracts showed higher activity than root extracts. Positive correlations were found between the content of quantified phenolics and antioxidant and digestive enzymes inhibiting activity. The findings presented in our work suggest that the Caucasian gentians are a good source of bioactive phytocompounds with antioxidant and antidiabetic potential.

It is particularly significant that high-performance liquid chromatography (HPLC) profiling with diode array detection and/or mass detection was performed only for two species, (G. asclepiadea [37], G. cruciata [22]), although in limited manner. Therefore, a comprehensive and comparative study is needed for a clear understanding of the chemo-diversity of Caucasian gentians. The antioxidant studies of gentians are for the same reason and also essential to identify antioxidant principles of plants. The information about antidiabetic activity of selected gentians is still unknown; so, it would be useful to know their potential against key enzymes of carbohydrate metabolism such as α-amylase and α-glycosidase [40]. The aim of present paper was to profile soluble metabolites of six gentian herbs and roots using high-performance liquid chromatography with diode array and electrospray triple quadrupole mass detection (HPLC-DAD-ESI-QQQ-MS) techniques and to quantify selected flavonoids, xanthones and iridoid glycosides in gentian plants. In this paper, we also make a comparative study of antioxidant activity and the digestive enzyme inhibition potential of gentian extracts, and we found active compounds which were the bioactive principles of the gentians.

Liquid Chromatography Mass Spectrometric (LC-MS) Metabolite Profiling of Six Caucasian Gentians: Chemodiversity of Herbs and Roots
An assay based on high-performance liquid chromatography with diode array and electrospray triple quadrupole mass detection (HPLC-DAD-ESI-QQQ-MS) was used to profile soluble metabolites of the herbs and roots of Gentiana asclepiadea, G. cruciata, G. gelida, G. paradoxa, G. pneumonanthe, and G. septemfida collected in Caucasus. A comparison of the ultraviolet (UV) spectra, mass spectral with daughter fragmentation (MS n ) data, and retention times ( Figure S1) with reference compounds ( Figure S2) and literature data were used for identification of compounds (Table S3). The chromatograms demonstrated the presence of 137 compounds in six gentian herbs and roots (Table 1) . Typical monosaccharides of gentians are glucose and fructose, disaccharides -gentiobiose and saccharose, and trisaccharidesgentianose and gentiotriose [41]. The limitation of the RP-HPLC assay used is poor separation of isomeric carbohydrates. Therefore, an additional study is needed to clarify the carbohydrate profile of the gentians.

Iridoid Glycosides
Iridoid glycosides were the most diverse group of metabolites and it includes 71 compounds. The following types of iridoid glycosides were detected in studied gentians.
Iridoid glycosides with 2,3-dihydroxybenzoyl and hexose fragments were characterized by the primary loss of a hexosyl fragment with m/z 162 followed by the expected elimination of a The rare, for the gentians, morroniside (23) was also compared with a standard and found in the G. septemfida herb only. In addition to the mentioned iridoid glycosides, similar mass spectrometric patterns were found for twenty-one compounds. There were compounds isomeric to swertiamarin (41), gentiopicroside (56), and sweroside (44) and eighteen compounds with less obvious structures. Compound 14 gave a deprotonated ion with m/z 389 and was tentatively identified as eustomoside, an iridoid glycoside first found in Eustoma russellianum [43] and later in G. septemfida herb [28]. Seven compounds (3, 5, 6, 10, 13, 17, 19) have the molecular weight 408 and these could be isomers of eustomorusside detected in the G. septemfida herb [28]. A tentative identification was made for compound 12, which gave a deprotonated ion with m/z 471 that is usual for eustoside, which was also discovered in the G. septemfida herb [28].  (20), and sweroside-6 -O-glucoside (28).
Iridoid glycosides with 2,3-dihydroxybenzoyl, acetyl and hexose fragments were members of the largest group of iridoids found in Caucasian gentians. Their mass spectra contain a sequence of signals caused by the serial elimination of hexose (or hexoses), a 2,3-dihydroxybenzoyl fragment, an acetyl group (or groups), and final loss of glucose and liberation of an iridoid aglycone ( Figure 3). Twenty-one compounds gave an analogous fragmentation pattern. Among these were derivatives of loganic acid (82,   Three monoacetylated (93, 102, 108) and two diacetylated (113, 117) analogues of gelidoside were found in G. gelida, G. cruciata, G. pneumonanthe and G. septemfida as well as two hexosylated derivatives of gelidoside (120, 128).
One compound, amarogentin (105), was not among the mentioned types of gentian iridoid glycosides and was identified using a standard compound. Amarogentin is a common bitter component of G. lutea roots and was also found to be a trace compound in the roots of G. asclepiadea and G. pneumonanthe [27]. In our study it was detected only in G. gelida roots.
One compound, amarogentin (105), was not among the mentioned types of gentian iridoid glycosides and was identified using a standard compound. Amarogentin is a common bitter component of G. lutea roots and was also found to be a trace compound in the roots of G. asclepiadea and G. pneumonanthe [27]. In our study it was detected only in G. gelida roots.
Fourteen apigenin glycosides were the components of gentian herbs and roots. Apigenin   (Figure 7). Because the molecular weight of the loss group was 162 a.m.u., it was identified as caffeoyl. To date, only one compound satisfies these criteria, the isovitexin-7-O-(6"-caffeoyl)glucoside isolated from Bryonia herbs [61]. The discovery of five isomeric compounds illustrates the necessity for additional study to find other new isomers.

Triterpene Glycosides
Seven compounds (75, 84, 94, 95, 99, 112, 123 [42]. Triterpene glycosides are rare components of gentians and compounds with the described structural features are still unknown. In general, the chemical profiles of the studied gentian species are similar: flavonoids and iridoids accumulate predominately in all types of herbs, and iridoids are the most diverse class of compounds in the roots. Of the 137 compounds detected, 71 compounds (52% of the total) are iridoids and 40 compounds are flavonoids (29% of the total). In total, these account for more than 80% of the total variety of compounds. In gentian herb samples, 44 (G. pneumonanthe) to 58 compounds (G. paradoxa) were detected and in roots 21 (G. pneumonanthe) to 32 compounds (G. septemfida). The largest number of compounds known for the species (12) was found in the herb of G. asclepiadea, which is considered to be the most studied of the species studied. Most of the compounds mentioned in this study were found in gentian species for the first time (Table 1). Four samples have never been studied before; these include the herb and roots of G. paradoxa, and the roots of G. gelida and G. septemfida.  In general, the chemical profiles of the studied gentian species are similar: flavonoids and iridoids accumulate predominately in all types of herbs, and iridoids are the most diverse class of compounds in the roots. Of the 137 compounds detected, 71 compounds (52% of the total) are iridoids and 40 compounds are flavonoids (29% of the total). In total, these account for more than 80% of the total variety of compounds. In gentian herb samples, 44 (G. pneumonanthe) to 58 compounds (G. paradoxa) were detected and in roots 21 (G. pneumonanthe) to 32 compounds (G. septemfida). The largest number of compounds known for the species (12) was found in the herb of G. asclepiadea, which is considered to be the most studied of the species studied. Most of the compounds mentioned in this study were found in gentian species for the first time (Table 1). Four samples have never been studied before; these include the herb and roots of G. paradoxa, and the roots of G. gelida and G. septemfida.
Is it possible to say that any components are characteristic for a particular type of gentian? The most suitable example is the detection of gentisin (137) and gentioside (109) in the roots of G. asclepiadea. This occasion is really rare, because for gentians of the Pneumonanthe section, the presence of xanthones (except mangiferin) is uncharacteristic. The possible reason for this phenomenon may be the least evolutionary advancement of G. asclepiadea, which makes it close to the species of the Coelanthe section, which contains such xanthon-containing species as G. lutea and G. punctata [42].
Is it possible to say that any components are characteristic for a particular type of gentian? The most suitable example is the detection of gentisin (137) and gentioside (109) in the roots of G. asclepiadea. This occasion is really rare, because for gentians of the Pneumonanthe section, the presence of xanthones (except mangiferin) is uncharacteristic. The possible reason for this phenomenon may be the least evolutionary advancement of G. asclepiadea, which makes it close to the species of the Coelanthe section, which contains such xanthon-containing species as G. lutea and G. punctata [42].
A notable fact is the single occurrence of some hydroxycinnamates such as caffeoyl-glucose 21 and 45 in G. pneumonanthe herb or caffeoyl-glycerins 79 and 90 in G. septemfida herb. Commonly, hydroxycinnamates of various structures accumulate in the green parts of plants in the form of esters with quinic acid, shikimic acid, or glucose [65]. For plants of the genus Gentiana, such a phenomenon is extremely rare [42].
The presence of flavonoids in the form of aglycones was detected only in two species (G. asclepiadea, G. paradoxa). This is typical for gentian sections of Pneumonanthe and Aptera produced flavonoids in the form of O-, C-, C, O-glycosides and most often derivatives of apigenin (or isovitexin) and/or luteolin (or isoorientin) [26,66]. The distribution of xanthone C-glycoside mangiferin (49) within the genus Gentiana is irregular, and its presence was previously observed only in some types of sections of Pneumonanthe, Frigida, Aptera, and Chondrophyllae [67]. Mangiferin is a useful therapeutic molecule with various bioactivities [68]. New plant sources are needed and G. paradoxa herb was shown to be a mangiferin source for the first time.

HPLC-DAD Quantification of Selected Compounds in Six Caucasian Gentians: Comparison of Herb and Roots
Continuing the metabolomic study of six Caucasian gentians, we determined the quantitative content of selected compounds by high performance liquid chromatography with diode array detection (HPLC-DAD) technique [12]. Six iridoids, nine flavones and mangiferin were chosen as quantitative markers of the herb samples, and roots were analysed using seven iridoids, isoorientin-2"-O-glucoside and gentioside (Tables S4, S5, Figure S2). Comparative analysis of quantitative data showed a strong variation of iridoid, flavonoid, and xanthone content in herbs and roots (Table 2). Gentiopicroside was the predominant iridoid glycoside in herb samples of G. asclepiadea, G. cruciata, and G. pneumonanthe, and swertiamarin had maximal content in other herbs of G. gelida, G. paradoxa, and G. septemfida. The loganic acid level in gentian herb was also high and sweroside was a trace compound. Gelidoside was quantified in two herbs, G. gelida and G. septemfida, and trifloroside content was determined in G. gelida, G. paradoxa and G. septemfida. The main iridoid of roots was gentiopicroside for all samples and its derivative gentiopicroside-6"-O-glucoside was quantified only in roots. The earlier information showed a high content of gentiopicroside in G. asclepiadea, G. cruciata, and G. pneumonanthe roots from Hungary (50-60 mg/g) [27], G. cruciata herb/roots from East Serbia (10.67/19.57 mg/g; as extract) [22], and G. pneumonanthe roots from Serbia (40.02-56.68 mg/g) [18]. In this regard, the herbs and roots of six Caucasian gentians are a good source of gentiopicroside.
The following iridoids were present in much lower amounts: swertiamarin and sweroside in G. asclepiadea, G. cruciata, and G. pneumonanthe roots [27], swertiamarin and sweroside in G. cruciata herb/roots extracts [22], swertiamarin, and sweroside in G. pneumonanthe herb/roots [18]. By this data, we see a remarkable similarity between known information about iridoid content in gentian parts and the data obtained in the present study.
Flavonoids were quantifiable in all gentian herbs and G. asclepiadea roots. The basic flavonoids of herb samples were isoorientin and isoorientin-2"-O-glucoside. The content of isovitexin and its O-glycosides was less but appropriate for analysis. Isoscoparin was quantifiable in three herb samples such as G. septemfida, G. gelida, and G. pneumonanthe. Only isoorientin-2"-O-glucoside was found in G. asclepiadea roots. As previously shown, the isoorientin and isovitexin content in G. pneumonanthe herb from Serbia was 0.27-2.67 and 0.12-0.88 mg/g, respectively [18]. Isoorientin was mentioned as the dominant flavonoid of some Turkish gentian herbs such as G. asclepiadea (1.00-30.72 mg/g), G. cruciata (2.41-22.78 mg/g), G. gelida (2.64-35.16 mg/g), and G. septemfida (1.17-15.19 mg/g) [24]. By contrast to apigenin glycosides, luteolin derivatives were the principal flavonoids with high content in gentian herbs of Caucasus origin and this was also reported in early research of European and Turkish populations of various gentians.
Xanthone content in gentian herbs was formed by their mangiferin value. The herbs of G. gelida and G. septemfida were free of mangiferin and other species showed high mangiferin levels. The roots of G. asclepiadea accumulate xanthone-O-glycoside gentioside at a low level. Mangiferin content was also determined previously in G. pneumonanthe herb (0.44-5.81 mg/g) [18] and in G. asclepiadea flowers and stem extracts (0.26-1.48 mM) [37]. In view of mangiferin's importance as a bioactive compound, the herbs of four gentian species could be concluded to be a rich source of this xanthone, especially G. asclepiadea herb.
By comparing results of the quantitative profile of herbs and roots of six Caucasian gentians, it can be concluded that the herbs are a good source of iridoids, flavonoids, and xanthones, and the roots can concentrate mainly iridoids.

Bioactivity of Gentian Extracts as a Function of Phenolic Compounds Content
Antioxidant activity is one of the basic bioactivity properties of plant extracts due to the presence of the various groups of antioxidants. The gentian extracts are no exceptions, and they were previously found to be good antioxidant sources [13,22,39]. In the present work, we studied the antioxidant properties of herb and root extracts of six Caucasian gentians by three methods: 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay, superoxide anion-radical scavenging assay, and lipid peroxidation inhibition assay.  Scavenging activity against DPPH radicals of gentian herb extracts were medium to high and varied from 147.57 mg trolox/g for G. paradoxa to 580.71 mg trolox/g for G. asclepiadea (Table 3). Extracts of gentian roots were characterized by mostly low activity, with activity values <10 mg trolox/g. Using the HPLC-DAD assay coupled with pre-chromatographic reaction with DPPH radicals, the most active compounds were characterized [69,70]. Figure 9a,b show chromatograms of G. asclepiadea herb extract before and after reaction with DPPH radical. The peak areas of the most active scavengers isoorientin-2"-O-glucoside (zone iii), mangiferin (zone v), and isoorientin (zone vii) were reduced by 72-98% compared with the initial value, but iridoid glycosides such as loganic acid (zone i) and gentiopicroside (zone iv) gave week peak reduction (3-5%). The chromatograms of root extracts appeared relatively unchanged after reaction with DPPH radicals, demonstrating a low reduction of all peaks (Figure 9c,d). Analysing the scavenging activity of the selected compounds, we found that iridoid glycosides such as loganic acid and gentiopicroside have low activity (<10 mg trolox/g). Additional phenolic groups such as 2,3-dihydroxybenzoyl in iridoid structures of gelidoside (rindoside) and trifloroside can increase the radical scavenging potential, but not much (20.82-25.14 mg trolox/g). Isoorientin (2523.27 mg trolox/g) and mangiferin (3824.20 mg trolox/g) were the most active compounds found in gentian extracts. Values with different letters (i-xxxvii) indicate statistically significant differences among groups at p < 0.05 by one-way ANOVA. c as mg of trolox per g of dry extract weight; d as mg of quercetin per g of dry extract weight; e as mg of caffeic acid per g of dry extract weight; f as mg of acarbose per g of dry extract weight. DPPH-2,2-diphenyl-1-picrylhydrazyl radical scavenging activity; O 2 --superoxide anion-radical scavenging activity; LPA-lipid peroxidation inhibition activity; αAIP-α-amylase inhibitory potential; αGIP-α-glycosidase inhibitory potential.
The scavenging activity of gentian herb extracts against superoxide anion-radicals was medium to high with values of potential from 52.18 mg quercetin/g for G. paradoxa to 235.54 mg quercetin/g for G. septemfida, and root extracts had low activity (<10 mg quercetin/g). The lipid peroxidation inhibition values of gentian herb extracts were also high with activities ranging from 63.23-242.08 mg caffeic acid/g. The root extracts had low activity from <10 mg caffeic acid/g to 17.68 mg caffeic acid/g (G. asclepiadea roots). The main reason for the high activity of the herb extracts was the high phenolic content (flavonoids, xanthones), which showed the maximum intensity of antioxidant protection.
Isoorientin and mangiferin demonstrated superoxide anion-radical scavenging activity at 863.15 and 927.07 mg quercetin/g, respectively, and in the lipid peroxidation inhibition assay the parameters of activity for the same compounds were 486.56 and 522.14 mg caffeic acid/g, respectively. It was thus evident that the high content of phenolic compounds such as flavonoids and xanthones in gentian extracts gave them high antioxidant activity. This was clearly confirmed by the results of regression analysis between phenolic compound content in gentian extracts (Table S6) and their antioxidant activity values (Figure 10a). Good linearity was shown for three equations that gave high r (correlation coefficient) values from 0.9232 to 0.9752. The fact of the positive direct relationships between phenolic content and antioxidant activity of plant extracts was postulated elsewhere [71], but the compliance of this rule in case of Caucasian gentians was observed for the first time.
HPLC-DAD chromatograms (210 nm) of G. asclepiadea herb extract before (a) and after (b) pre-chromatographic reaction with DPPH radicals and G. gelida roots extract before (c) and after (d) pre-chromatographic reaction with DPPH radicals.
The inhibitory potential of gentian extracts against α-amylase and α-glycosidase as basic digestion enzymes was studied using known spectrophotometric microplate assays [57]. All herb extracts showed high activity in the range 108.85-530.11 mg acarbose/g for α-amylase inhibition and 144.77-418.80 mg acarbose/g for α-glycosidase inhibition.
The root extracts demonstrated low activity (<10-38.87 mg acarbose/g) for α-amylase inhibition and <10-25.64 mg acarbose/g for α-glycosidase inhibition. The reasons for the varied activity of gentian extracts are in the various chemical profiles and quantitative composition of the extracts. The values of inhibitory potential of pure compounds were low for iridoid glycosides (<10 mg acarbose/g), medium for isovitexin, and high for isoorientin and mangiferin. The latter two compounds were the most active inhibitors, whereas isoorientin was the most potent inhibitor of α-amylase (1242.03 mg acarbose/g in α-amylase inhibition vs. 811.10 mg acarbose/g in α-glycosidase inhibition), and mangiferin was the most potent inhibitor of α-glycosidase (296.14 mg acarbose/g in α-amylase inhibition vs. 1562.84 mg acarbose/g in α-glycosidase inhibition). It is reasonable to expect that the high phenolic content resulted in the high inhibitory activity of gentian extracts against α-amylase and α-glycosidase. Regression equation data showed good linearity for correlation graphs between phenolic compound content in gentian extracts and α-amylase/α-glycosidase inhibitory activity (Figure 10b). In view of all this, there is a need to characterise gentian herbs as potent plant sources of anti-α-amylase and anti-α-glycosidase phenolics.

Conclusions
The data obtained are in compliance with known facts about high antioxidant and antidiabetic activity of flavones with luteolin skeleton [40,71,72] as well as the xanthone mangiferin [40,68]. Both of these have two phenolic ortho-hydroxyl groups. Although Caucasian gentians contain a great number of various phytochemicals, the most abundant active compounds were flavones and mangiferin which resulted in the high efficiency of derived plant extracts as antioxidants and digestive enzymes inhibitors. To our knowledge, this is the first paper combining detailed metabolite profiling by the HPLC-DAD-ESI-QQQ-MS technique and HPLC-DAD quantification of the main compounds with an antioxidant, anti-α-amylase, and anti-α-glycosidase study of the six gentian species (herb, roots) widely distributed in the Caucasus and used as phytopharmaceuticals.

Total Extract Preparation
To prepare the total extract of gentian herb and roots the powdered sample (100 g) was triple extracted in a conical glass flask (2 L) with 60% methanol (2 L) with stirring and sonication for 60 min at 40 • C with ultrasound power of 100 W and the frequency 35 kHz. The final extracts were filtered through a cellulose filter, combined, evaporated in vacuo until dryness, and stored at 4 • C until further chemical composition analysis and bioactivity assays. The yields of total extracts of gentian herbs and roots listed in Table 4.

High-Performance Liquid Chromatography with Diode Array Detection and Electrospray Ionization Triple Quadrupole Mass Spectrometric Detection (HPLC-DAD-ESI-QQQ-MS)
Reversed-phase high-performance liquid chromatography with diode array detection and electrospray ionization triple quadrupole mass spectrometric detection (HPLC-DAD-ESI-QQQ-MS) procedure was used for phenolic compounds profiling. Experiments were performed on an LCMS 8050 liquid chromatograph coupled with diode-array-detector and triple-quadrupole electrospray ionization detector (Shimadzu, Columbia, MD, USA), using a GLC Mastro C18 column (150 × 2.1 mm, Ø 3 µm; Shimadzu, Kyoto, Japan), column temperature was 35 • C. Eluent A was 0.5% formic acid in water and eluent B was 0.5% formic acid in acetonitrile. The injection volume was 1 µL, and elution flow was 100 µL/min.
For preparation of sample solution, an accurately weighted powdered plant (100 mg) was placed in an Eppendorf tube, 2 mL of 60% methanol was added. Then the sample was extracted twice in an ultrasonic bath for 30 min at 40 • C and centrifuged (3000× g, 15 min). Combined supernatants were transferred to volumetric flask (5 mL) and the final volume was reduced to 5 mL. The resultant extract was filtered through a 0.22-µm PTFE syringe filter before injection into the HPLC system for analysis.

Validation Analysis
The linearity of HPLC-DAD quantification method was studied by injecting six concentrations (1.75-900.00 µg/mL) of the 18 reference standards (loganic acid, swertiamarin, gelidoside, gentiopicroside, gentiopicroside-6"-O-glucoside, sweroside, trifloroside, isovitexin, isovitexin-2"-O-glucoside, saponarin, apigenin-7-O-glucoside, isoorientin, isoorientin-2"-O-glucoside, isoorientin-6"-O-glucoside, luteolin-7-O-glucoside, isoscoparin, mangiferin, gentioside). Results from each analysis were averaged and subjected to regression analysis. Limits of detection (LOD) and quantification (LOQ) were determined using the following equations: LOD = (3.3 × S YX )/a; LOQ = (10 × S YX )/a, where S YX is a standard deviation of the response (Y intercept) and a is a slope of calibration curve. Intra-and inter-day variations, which are presented in terms of percent relative standard deviation (%RSD) of the analyte's peak area and variability assessed the precision of the HPLC-DAD quantification. For the intra-day variability test, the mixture solution containing 18 reference standards was analysed for five replicates within one day (56.25 µg/mL), while inter-day assay was analysed using the same concentration for intra-day precision on four different days (interval of 1 day). The repeatability test of the sample was performed on 7-fold experiments of the mixture solution contain 18 reference standards (225 µg/mL). The stability test was performed with one sample solution, which was stored at room temperature and analysed at regular intervals (0, 2, 4, 8, 12, 24 and 48 h.). For analysis of recovery data, the appropriate amounts of the powdered sample of 18 reference standards were weighted and spiked with a known amount of reference compound and then analysed five times.

Antioxidant Activity Analysis
The 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity was performed as described earlier [83]. The trolox was used as a positive control (IC 50 11.62 ± 0.23 µg/mL), and water was used as a negative control. The IC 50 value was found as the effective concentration at which DPPH radicals were scavenged by 50%. The value of antioxidant activity (A DPPH ) against DPPH radicals was found as a ratio of trolox IC 50 to sample IC 50 [A DPPH = (IC 50 Trolox / IC 50 Sample ) × 1000] and expressed as mg of trolox in 1 g of sample. Values are expressed as mean obtained from five independent experiments. The known assay [84] was used to determine superoxide anion radical scavenging activity. Quercetin was used as a positive control (IC 50 21.74 ± 0.42 µg/mL), and water was used as a negative control. The IC 50 value was found as the effective concentration at which superoxide anion radicals were scavenged by 50%. The value of antioxidant activity (A O2• ) against superoxide anion radicals was found as a ratio of quercetin IC 50 to sample IC 50 [A O2• = (IC 50 Quercetin / IC 50 Sample ) × 1000] and expressed as mg of quercetin in 1 g of sample. Values are expressed as mean obtained from five independent experiments. The previously described method [85] was used to investigate the lipid peroxidation inhibition potency. Caffeic acid was used as a positive control (IC 50 58.96 ± 1.14 µg/mL), and water was used as a negative control. The IC 50 value was found as the effective sample concentration gave 50% reduction of optical density of initial solution. The value of lipid peroxidation inhibition (I) was found as a ratio of caffeic acid IC 50 to sample IC 50 [I = (IC 50 Caffeic acid / IC 50 Sample ) × 1000] and expressed as mg of caffeic acid in 1 g of sample. Values are expressed as mean obtained from five independent experiments.

Digestive Enzymes Inhibiting Potential
The α-amylase inhibiting potential and α-glucosidase inhibiting potential were performed using spectrophotometric assays [86,87]. The α-amylase from Aspergillus oryzae (3 U/mL) and α-glucosidase from Saccharomyces cerevisae (0.5 U/mL) were used as substrates. Acarbose was used as a positive control (IC 50 311.14 ± 7.79 µg/mL for α-amylase inhibiting potential; IC 50 1282.64 ± 38.46 µg/mL for α-glucosidase inhibiting potential), and water was used as a negative control. The IC 50 value was found as the effective sample concentration gave 50% inhibition of digestive enzyme. The values of α-amylase/α-glucosidase inhibiting potential (P) was found as a ratio of acarbose IC 50 to sample IC 50 [I = (IC 50 Acarbose / IC 50 Sample ) × 1000] and expressed as mg of acarbose in 1 g of sample. Values are expressed as mean obtained from six independent experiments.

Statistical and Multivariative Analysis
Statistical analyses were performed using a one-way analysis of variance (ANOVA), and the significance of the mean difference was determined by Duncan's multiple range test. Differences at p < 0.05 were considered statistically significant. The results are presented as mean values ± SD (standard deviations) of the three replicates. Advanced Grapher 2.2 (Alentum Software Inc., Ramat-Gan, Israel) was used to perform linear regression analysis and to generate graphs.
Supplementary Materials: The following are available online at http://www.mdpi.com/2218-1989/9/11/271/s1, Table S1: Ethnopharmacological use of Gentiana species by the various Caucasus people, Table S2: Known compounds found in Gentiana species mentioned in present study (literature data), Table S3: Retention times and mass spectrometric data of compounds 1-137 found in herb and roots of six Caucasian Gentiana species , Table S4: Regression equations, correlation coefficients, standard deviation, limits of detection, limits of quantification and linear ranges for 18 compounds, Table S5: Intra-and inter-day precision, repeatability, stability and recovery for 18 compounds, Table S6: Content of selected phenolic compounds in dry extracts of gentian herbs and roots, Figure S1: High-Performance Liquid Chromatography with Electrospray Ionization Triple Quadrupole Mass Spectrometric Detection chromatogram in base peak chromatogram mode of six Gentiana herb and root extracts, Figure S2: Structures of reference compounds, Figure S3