Iridoids and Flavonoids of Four Siberian Gentians: Chemical Profile and Gastric Stimulatory Effect

Some Gentiana species have been used by the nomadic people of Siberia as bitter teas or appetizers to eliminate digestive disorders (dyspepsia, heartburn, nausea, etc.). We studied the most frequently used gentians: Gentiana algida, G. decumbens, G. macrophylla and G. triflora. The aim of the present study was to evaluate the phytochemical features and gastrostimulatnt activity of these four gentian herbs. Five iridoids, seven flavones and mangiferin were detected in gentian herbs after analysis by microcolumn-RP-HPLC-UV-ESI-MS. A componential phytochemical profile of the G. decumbens herb is presented for the first time, as well as information about distinct phytochemicals found in gentian herbs. HPLC quantification of the specific compounds of gentian herbs demonstrated the high content of iridoids (24.73–73.53 mg/g) and flavonoids (12.92–78.14 mg/g). The results of biological activity evaluation of four gentian decoctions demonstrated their good ability to stimulate acid-, enzyme- and mucin-forming functions of the stomach attributed to mostly by iridoids and flavonoids. In general, it can be claimed that the gentian decoctions can be used as effective and safe appetizers and are also a good source of biologically active agents.


Introduction
The basis of the nutritional system of Siberian people has traditionally consisted of products rich in animal protein and fat. Despite its profitable energy value, animal protein is a category of food with reduced digestibility, so excessive consumption leads to a variety of digestive disorders (dyspepsia, heartburn, nausea, etc.). To facilitate the process of digestion a variety of herbal products with a bitter taste was used [1][2][3]. The most commonly used herbal bitter species were gentianaceous plants that grow everywhere in the meadows and steppes of Siberia. These herbs are given uncomplicated names, like white or blue bitter herbs (plants with white or blue flowers), and their derivative beverages are referred to as bitter teas (гашуун ундан, Buryatian; аhыы утах, Yakutian; йдарйичу умивўн, Evenkian) [4]. Ethnopharmacological investigations allow us to identify the "bitter herbs" as various species of the genera Halenia [5][6][7][8], Gentianopsis [9], Anagallidium [10] and Gentiana. As part of an ongoing study of the chemistry and pharmacology of gentianaceous bitter teas, we present the results of analysis of the herbs of the four most frequently used gentians, including G. algida Pall. G. algida is a little gentian with white flowers which grows in crude marshy meadows of Central Siberia and the northwestern part of China. In Traditional Chinese Medicine (TCM) and Traditional Tibetan Medicine (TTM) practice this plant is used as a remedy for the treatment of pneumonia, throat diseases, infections and wounds [11,12]. Some phytochemicals have been discovered in G. algida herbs of Chinese origin, including iridoids, sterols, oleanolic acid, flavones, anofinic acid and fomannoxin [11][12][13][14][15]. The latter two compounds have demonstrated antifungal activity against Cladosporium curcumerinum [12]. G. decumbens is a typical plant of the steppefied meadows of all Siberian regions. The wide distribution of this species resulted in the most frequent application as a food plant in the form of tea, decoctions or infusions. TTM recommends G. decumbens for gastric diseases and some infections. To date there is no chemical data describing the phytochemical profile of G. decumbens. As for G. macrophylla, vegetating from Yakutian Siberia in the north to central China in the south, it is one of the most popular remedies in TCM, in which its roots are used to dispel rheumatism and ease pain [16]. Iridoids, triterpenes, sterols, and flavonoids have been isolated from roots of G. macrophylla [17]. The aerial part of this plant has been poorly investigated, although some iridoids and flavonoids from the flowers are known [18]. G. triflora is a large gentian with navy blue flowers which commonly grows in Siberian callows. In TTM the herb of G. triflora is used for the treatment of chronic stomach diseases, as it reduces gastric acidity, inflammation and cholecystitis [19]. The roots of G. triflora are used in TCM in many formulations for poor appetite and digestive problems. As in the case of G. macrophylla, the chemical compounds of G. triflora roots have been better studied than those of the herb. Only two flavones have been detected in G. triflora leaves-isoorientin and its 4′-O-glucoside [20]. To summarise known data devoted to these four gentian herbs (G. algida, G. decumbens, G. macrophylla, G. triflora), a poor general knowledge level of their chemical and pharmacological data can be confirmed. More characterisation is required of these plants that are widely used in everyday life.
In the present work, the herbs of G. algida, G. decumbens, G. macrophylla, G. triflora were submitted to analysis of specific classes of phytochemicals (iridoids, phenolic compounds). HPLC characterisation of a series of gentian herbs was conducted with the aim of quantifying phytochemicals. The influence of the decoctions of four gentian herbs on gastric secretion was also investigated.

Phytochemicals of Four Siberian Gentians Herbs
Phytochemicals of the Gentianaceae family include a wide spectrum of compounds with various chemical structures. Most common are iridoids, flavonoids, and xanthones [21]. Known data about phytochemicals of the Gentiana species included in the present investigation has indicated a low level of scrutiny of herb compounds in comparison to root constituents, e.g., about thirty compounds were isolated from G. macrophylla roots [16,17] vs. eight compounds discovered in G. macrophylla flowers [18]. Information on the chemical composition of G. decumbens is absent; therefore, in the first stage of the phytochemical study of the gentian teas we conducted an investigation of the G. decumbens herb.
Two flavones (isoorientin-4′-O-glucoside and isoorientin) had previously been described in the G. triflora herb [20]; however, lutonarin, mangiferin, saponarin, isovitexin and isoscoparin are newly identified compounds of this plant. Of particular note is the discovery of 1,3,6,7-tetrahydroxyxanthone 2-C-glucoside (mangiferin) in G. triflora. This compound occurs sporadically in the Gentianaceae family and is often found with C-glycosylflavones [22] and has previously been described in the other members of the Pneumonanthe section of Gentianaceae family which includes G. triflora; these are G. asclepiadea [29] and G. pneumonanthe [11].

HPLC Quantification of the Iridoids and Phenolic Compounds of Four Gentian Herbs
Quantification of the main phytochemicals in the gentian herbs was realised by an MC-HPLC-UV procedure previously applied for analysis of the Anagalidium dichotomum herb [10]. Of the 12 compounds including five iridoids, six flavone-C/O-glycosides and mangiferin, from eight to 11 analytes were found in detectable levels in 16 samples of the gentian herbs. All the contents are summarised in Table 3.  The results showed that the total content of quantifiable compounds in the gentian herb samples was 53.96-64.07 mg/g in G. macrophylla herb, 72.97-81.13 mg/g in G. decumbens herb, 76.18-105.18 mg/g in G. algida herb, and 122.36-151.81 mg/g in G. triflora herb. The highest iridoids content, 67.92-73.53 mg/g, was detected in G. triflora herb which dominate compound was loganic acid-6′-Oβ-D-glucoside (61.23-66.81 mg/g). The prevalence of this component observed in G. decumbens herb (51.37-61.37 mg/g) and G. macrophylla herb (15.93-24.09 mg/g). Gentiopicroside was a main iridoid of G. algida herb containing it as 27.71-45.87 mg/g. Detectable amounts of sweroside and swertiamarin were found in G. algida (2.09-7.80 mg/g and 1.95-3.19 mg/g) and G. macrophylla herbs (0.30-0.51 mg/g and 0.27-0.94 mg/g). The maximal concentration of flavonoids was in G. triflora (50.09-78.14 mg/g), an herb with a highest content of isoorientin-4′-O-glucoside (22.67-40.27 mg/g). The G. algida, unlike of other species, contained the high level of isoorientin (21.18-39.53 mg/g) which content in other herbs was 1.93-4.14 mg/g in G. decumbens, 4.30-7.11 mg/g in G. macrophylla, and 14.25-31.16 mg/g in G. triflora. The contents of isovitexin and saponarin were on the quantifiable levels in all species reaching 1.37-7.08 mg/g and 0.19-4.06 mg/g, respectively. The measurable amounts of isosaponarin were discovered only in G. triflora herb (0.57-3.87 mg/g). Contents of isoscoparin in gentian herbs were 0.92-1.53 mg/g in G. decumbens, 1.37-2.64 mg/g in G. macrophylla, 2.06-4.19 mg/g in G. triflora, and undetectable in G. algida. The presence of the discovered xanthone mangiferin was observed in G. triflora herb only (1.97-3.71 mg/g).
To summarize the results of HPLC quantification of the specific compounds of four gentians it should be concluded that the herbs of G. algida, G. decumbens, G. macrophylla and G. triflora are the good source of iridoids and flavonoids.

Influence of the Gentian Decoctions and Individual Compounds on Gastric Secretory Function
Oral intragastral administration of the gentian herb decoctions in a dose of 100 mg/kg stimulated acidand enzyme-forming functions of the stomach ( Figure 5). In all experimental groups an increase of gastric juice volume, total and free HCl concentration as well as pepsin concentration was observed. Maximal output of gastric juice volume (90.8% compared to the control group), total and free HCl concentration (25.5% and 51.1%) and pepsin concentration (57.4%) was observed in the G. algida group. G. triflora decoction was characterised by a medium increase of all the parameters of stomach secretion, while G. decumbens and G. macrophylla preparations demonstrated respectively weak activity.  Among individual compounds which were applied in a dose of 20 mg/kg, gentiopicroside had the most pronounced stimulation activity on gastric juice secretion (114.9% compared to the control group), both total and free HCl concentrations (88.7% and 92.3%) and pepsin production (90.1%). Loganic acid-6′-O-glucoside demonstrated a less pronounced influence on the secretory parameters of the stomach; however, the differences in the results obtained compared to the control group allow us to describe it as a stimulator with medium effectiveness. Flavone-C-glucoside (isoorientin) and its O-derivative (isoorientin-4′-O-glucoside) had the weakest activity, confirming the leading role of iridoids for stomach secretion stimulation.
Discussing the possible reasons for the observed differences it should be mentioned that the main chemical feature of G. algida decoction is a high gentiopicroside content. This iridoid compound is characterised as the most bitter that suppose it has the most pronounced stimulation action on digestive secretions by activating a trigger system of the digestive tract. Meanwhile, despite G. decumbens, G. macrophylla, and G. triflora decoctions were less bitter, we can describe them as gastric stimulants although with a less pronounced effect. Interestingly, although the main role of gentiopicroside and gentiopicroside-containing remedies for stimulation of gastric secretion was discussed previously [30], this ability of the loganic acid derivative is demonstrated in this paper for the first time.
The mucin concentration in stomach juice is another important indicator of stomach secretory activity. Mucin is an important protective factor for the gastric mucosa and consists of water and glycoproteins that cover the entire gastrointestinal mucosa. It is capable of acting as an antioxidant, and thus can reduce mucosal damage mediated by oxygen free radicals [31]. In this study, an increase of mucin secretion (14.2%-52.5% compared with the control) was observed in rats administered with the gentian teas, indicating the ability of the mucosal membrane to protect the mucosa from physical damage, irritant stomach secretions (HCl and pepsin) and back-diffusion of hydrogen ions.
All the phytochemicals studied influence mucin secretion positively with the highest effectiveness for gentiopicroside (137.2% compared with the control). The values of stimulation of loganic acid-6′-O-glucoside, isoorientin and isoorientin-4′-O-glucoside were close (36.6%-48.2%) and significantly higher than in the control but 2.8-3.8 times lower than the same parameter of gentiopicroside.
Generally, this study proved stimulant activity of the decoctions of four gentian herbs on the secretory functions of the stomach which is due to iridoids as well as flavonoids. The fact of simultaneous enriching of stomach juice by HCl, pepsin and mucin allows characterising of gentian decoctions as good and safe stimulators of gastric function.
Summarising the research, the striking insight of the nomadic people of Siberia in empirically identifying the positive effects of certain plant products on the human body evokes admiration. As a result of numerous investigations they managed to find the relationship between consumption of bitter herbal teas (mostly gentianaceous plants) with a consequent improvement of the digestion process. Certainly they could not explain the true nature of the impact of bitter tea on humans, but empirical knowledge gained during evolution was the basis for the introduction to modern customs the positive tradition of drinking bitter appetizers before eating. As a result of our study we are able to identify the cause of the use of bitter gentian teas as appetizers. The decoctions of the four species studied (G. algida, G. decumbens, G. macrophylla, G. triflora) are characterised by their high content of bioactive phytochemicals. In particular, the iridoids show a predominant influence on the secretion of digestive juices in the stomach and thus promote the process of digestion. Generally, G. algida, G. decumbens, G. macrophylla and G. triflora herbs could be promising candidates for further studies designed to obtain more evidence on their components with potential gastroprotective and digestive activity.

Plant Material
The samples of four gentian species were collected in the flowering period in the Siberian Regions

Polyamide Solid Phase Extraction (SPE) of Iridoids and Flavonoids
The sample of dried milled herb (1 g) was added to distilled water (100 mL), heated on heater plate and boiled 10 min. The mixture was left to stand at room temperature for 15 min, and than filtered under reduced pressure. A polyamide column (1.5 g) was prepared, primed with methanol (40 mL) followed by tridistilled water (td-water, 80 mL). An aliquot (1 mL) of gentian decoction was loaded on the column. Sequential elution was done with 30 mL of td-water (iridoids fraction) and 70% ethanol (phenolic fraction). The fractions after SPE extraction were concentrated to 1 mL, filtered through a 0.22-μm PTFE syringe filter before HPLC separation and stored at −10 °C prior analysis.
MC-HPLC-UV quantification experiments were carried out at the same chromatographic conditions with a UV-detection at 254 nm (iridoids) and 334 nm (flavonoids, mangiferin). An accurately weighted, dried, and powdered gentian herb tea samples (100 mg) was placed in a conical flask. Then 4 mL of 60% ethanol was added and the mixture was weighted. The sample was then extracted for 30 min at 40 °C in ultrasonic device UZV-2.8 (Sapfire, Moscow, Russia) with an ultrasound power of 100 W and frequency of 35 kHz, equipped with a temperature controller and a digital timer. After cooling, the flask weight was reduced to initial sign, and the resultant extract was filtered through a 0.22-μm PTFE syringe filter before injection into the HPLC system for analysis. Stock solutions of standards were made by accurately weighing 1 mg of loganic acid, loganic acid-6′-O-β-D-glucoside, swertiamarin, sweroside, gentiopicroside, isoorientin, isoorientin-4′-O-glucoside, isovitexin, saponarin, isosaponarin, isoscoparin, and mangiferin and dissolving it in 20 mL of methanol/DMSO in a volumetric flask. The appropriate amounts of stock solutions were diluted with methanol in order to obtain standard solutions containing 0.25-1.00 mg/mL. As all the compounds used for quantification were well-separated in experiment conditions mixtures of standards were analyzed. Prepared solutions were stored at 4 °C for no more than 72 h. The results are presented as mean values ± SD (standard deviations) of the three replicates.

Influence of Gentian Herbal Decoctions and Individual Compounds on Gastric Secretion
The experimental procedures relating to the animals were authorised by the Institute of General and Experimental Biology's Ethical Committee (protocol No LM-0324, 27.01.2012) before starting the study and were conducted under the internationally accepted principles for laboratory animal use and care. Male rats (Sprague Dawley; six weeks old) were purchased from the "Pushchino" Laboratory Animal Breeding House, Moscow. Rats weighing ca. 200-270 g (seven weeks old) were used for the study. The animals were kept under standard pathogen-free conditions at a constant temperature (25 °C), humidity (55%) on a 12 h light/dark cycle, and were fed food and water ad libitum. The experiments were performed using 10-12 rats per group after more than 24 h fasting. Pylorus ligation was performed by the method of Shay et al. [39] with slight modifications as recorded by Kurasawa et al. [40]. The rats were treated with solutions of gentian decoctions in 0.9% NaCl (100 mg/kg) or individual compounds in 1% Tween-80 aqueous solution (20 mg/kg), while the control group was given an equal volume of 0.9% NaCl or 1% Tween-80 aqueous solution. All the treatments were administered orally. Rats were under anaesthesia about 3 min before pylorus ligation, and awoke within 15 min after the ligation. The abdomen of the rats was incised and the stomach and duodenum were exposed. The pyloric portion of the stomach was gently taken out and occluded with a ligature. At 0-4 h after pylorus ligation, rats were sacrificed, the stomach was removed, and gastric contents were collected. The gastric contents obtained from pylorus-ligated rats were centrifuged at 3000 g for 10 min, and the volume of each sample was measured as gastric juice volume. The sample was re-centrifuged for 10 min at 3000 g, and used for measuring free [41] and total HCl concentration [42], pepsin activity [43] and mucin concentration [44].

Statistical 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.

Conclusions
The results of this study confirm the expressed potential of the four gentian plants for the production of bioactive compounds, and validate the ethnomedical use of the herb of Gentiana algida, G. decumbens, G. macrophylla and G. triflora as useful nutritional appetizers. Generally, chemical profiles of the mentioned gentian herbs as well as their gastric stimulant activity have not been previously reported. Our current findings demonstrated a scientific rationale for the use of gentian decoctions as a source of bioactive iridoids and phenolics (flavonoids, mangiferin). Also, the studied plants were proved as remedies for the treatment of gastric disorders (hypoacidity). With regard to general recommendations, it could be claimed that the gentian decoctions might be explored in the medicine industries.