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14.1 Introduction

The process that leads from plants to bioactive pure constituents requires multidisciplinary collaboration. This review summarizes the last two decades of results on Gentianaceae species of the rich Serbian and Montenegrin flora. In the search for biologically active and new compounds, targeted collection of wild-growing plants with special emphasis on endemics was based on chemotaxonomic and ethnomedicinal information of the corresponding genera. Combined efforts led to the isolation and structure elucidation of biologically active secondary metabolites, mostly belonging to three types of chemical structures, typical of the Gentianaceae, i.e., xanthones, flavone C-glycosides and secoiridoids (Šavikin-Fodulovic et al. 2002).

14.2 Genus Gentiana

Gentiana L. is a large cosmopolitan genus with about 400 species, occurring in alpine habitats of temperate regions of Asia, Europe, and the Americas. In the central regions of the Balkan Peninsula can be found 11 species of the genus Gentiana , such as G. lutea , G. punctata , G. cruciata , G. asclepiadea , G. pneumonanthe , G. kochiana , G. dinarica , G. verna , G. tergestina, G. utriculosa , and G. nivalis (Jovanović-Dunjić 1973). The best known is yellow gentian, G. lutea L., distributed in both Serbia and Montenegro.

14.2.1 Gentiana lutea L. (Yellow Gentian)

Gentiana lutea (Fig. 14.13a) is very popular as a stomachic as well as a component in preparations showing beneficial effects in gall bladder and liver diseases (Blumenthal 1998; Tasic et al. 2004). The roots and rhizome of G. lutea (Gentianae radix) are described in European Pharmacopoeia 7.0 (Ph. Eur. 7.0), (2011) as well as in many national pharmacopoeias (Pharmacopoea Yugoslavica 1984). The material is stabilized (unfermented), yellow colored, and very bitter. It is also used in traditional medicine, and in the course of his ethnomedicinal studies , the Serbian pharmacognosist Tucakov (1996) concluded that the yellow gentian root is among the most important remedies of the mountain inhabitants in Serbia. The fermented root, with a maroon-colored cross section, exhibiting a smell and taste like dried figs, is used for preparing alcoholic beverages (Tasic et al. 2004). Some studies showed that the root possesses cholagogue, anthelmintic , anti-inflammatory, and antimicrobial activity (Öztütk et al. 1998; Pontus et al. 2006; Menković et al. 1999; Šavikin et al. 2007). Underground organs of yellow gentian are characterized as having a diverse chemical composition, with secoiridoids as the main constituents. LC-ESI TOF MS chromatogram of G. lutea underground organs collected on Mountain Suvobor, Serbia, is presented in Fig. 14.1 with a tentative identification of the compounds in Table 14.1.

Fig. 14.1
figure 1

LC-ESI TOF MS chromatogram of G. lutea underground organs

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Table 14.1 Tentatively identified components in the extract using LC-ESI TOF MS and UV data of G. lutea underground organs
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The bitter taste of the roots originates from secoiridoids . Among them, the most abundant are 1–3.

In addition, the roots contain biphenyl derivatives such amarogentin (5) located in bark of the roots, as well as the closely related amaropanin (4) and amaroswerin (6). Amarogentin is one of the most bitter natural compounds known (Wagner et al. 1983).

Another group of pharmacologically active constituents are xanthones 7–9.

The aerial parts of yellow gentian showed themselves to be very interesting and promising plant material (Menković et al. 2000). LC-ESI TOF MS chromatogram of G. lutea leaves collected from plants on the Mount Suvobor, Serbia, is presented in Fig. 14.2 with details of the compounds in Table 14.2.

Fig. 14.2
figure 2

LC-ESI TOF MS chromatogram of G. lutea leaves

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Fig. 14.3
figure 3

LC-ESI TOF MS chromatogram of a methanolic extract of Gentiana dinarica leaves

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Fig. 14.4
figure 4

LC-ESI TOF MS chromatogram of a methanolic extract of G. kochiana leaves

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Fig. 14.5
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LC-ESI TOF MS chromatogram of a methanolic extract of G. kochiana roots

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Fig. 14.6
figure 6

LC-ESI TOF MS chromatogram of roots methanolic extract of G. asclepiadea

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Fig. 14.7
figure 7

LC-ESI TOF MS chromatogram of a methanolic extract of G. asclepiadea aerial parts

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Fig. 14.8
figure 8

LC-ESI TOF MS chromatogram of a methanolic extract of G. utriculosa aerial parts

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Fig. 14.9
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LC-ESI TOF MS chromatogram of a methanolic extract of G. punctata roots

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Fig. 14.10
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LC-ESI TOF MS chromatogram of a methanolic extract of G. punctata aerial parts

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Fig. 14.11
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LC-ESI TOF MS chromatogram of a methanolic extract of Gentianella austriaca aerial parts

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Fig. 14.12
figure 12

LC-ESI TOF MS chromatogram of S. punctata leaves

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Table 14.2 Tentatively identified components in the extract using LC-ESI TOF MS and UV data of G. lutea leaves
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The presence of 8 (Menković et al. 2000) and two flavonoid heterosides, 10 and 11, was reported in the leaves of G. lutea by Hostettmann et al. (1973).

From the aerial parts of G. lutea , three types of secondary metabolites, including xanthones, flavonoids, and secoiridoids were isolated in the laboratories of the authors of this chapter.

Xanthones

C-Glucoxanthone mangiferin (12) was accompanied by gentioside (9) and isogentisin (8).

C-Glucoflavones

The aerial parts of G. lutea contained C-glucoflavones 13–15:

Secoiridoids

The aerial parts of G. lutea yielded seven secoiridoids, namely 1–3 and 16–19:

14.2.2 Gentiana dinarica Beck

Gentiana dinarica (Fig. 14.13b) is a rare, endemic, perennial species distributed in the Apennine and Balkan Peninsula. It grows on carbonate soils in subalpine and alpine regions at an altitude of 800–2300 m. According to authors’ knowledge, it is not used in traditional medicine. In their investigations, plant materials (aerial parts and roots) were collected on the Mount Tara (1300 m), Serbia. A LC-ESI TOF MS chromatogram of G. dinarica leaves is presented in Fig. 14.3 with compounds identified in Table 14.3. The aerial parts of G. dinarica contained secoiridoids and C-glucoflavones, but xanthones were absent. Three C-glucoflavones, 13, 15, and 10, were isolated from aerial parts.

Fig. 14.13
figure 13

Flowers of some species of the family Gentianaceae (pictures taken from nature). a Gentiana lutea , b G. dinarica , c G. kochiana , d G. asclepiadea , e G. urticulosa, f G. punctata , g Swertia perennis, and h S. punctata

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Table 14.3 Tentatively identified components in the extract using LC-ESI TOF MS and UV data of G. dinarica leaves
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A methanolic root extract contained three xanthones, namely 20–22 and two C-glucoflavones 13 and 10. Xanthone aglycones were absent. Secoiridoid components isolated both from roots and from aerial parts were 1–3 and 5. Considerable amount of compound 5 was found in the roots, which could be of interest.

14.2.3 Gentiana kochiana E.P. Perrier and Songeon (Syn. G. acaulis L.)

Gentiana kochina (Fig. 14.13c) is a small gentian native to Central and Southern Europe, from Spain east to the Balkans, growing especially in the mountainous regions, such as the Alps, Cevennes, and Pyrenees, at an elevation of 800–3000 m. It grows in the mountain meadows with acidic silicate soil, inhabiting mostly south aspects. Extracts of this plant are used in traditional medicine in Tuscany (Italy) as an antihypertensive remedy (Manganelli et al. 2000).

The aerial parts and/or roots collected on the Mount Komovi, Montenegro, contained exclusively 1,3,7,8-tetraoxygenated xanthones, such as 23, its 1-O-primeveroside (24), 25 and gentiacaulenin 26, and their heterosides (27–29). Roots contain secoiridoids 1–3 and 18. LC-ESI TOF MS chromatograms of G. kochiana leaves and roots are given in Figs. 14.4 and 14.5 and in Tables 14.4 and 14.5.

Table 14.4 Tentatively identified components in the extract using LC-ESI TOF MS and UV data of G. kochiana leaves
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Table 14.5 Tentatively identified components in the extract using LC-ESI TOF MS and UV data of G. kochiana roots
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Xanthones

14.2.4 Gentiana asclepiadea L. (Willow Gentian)

Gentiana asclepiadea (Fig. 14.13d) is distributed in Central and Southern Europe from Schwarzwald to Olimp Mountain. It grows in high-mountain pastures and at the edges of forests to the subalpine tops of the mountains. It is also found in beech and spruce forests.

As the roots contain a similar chemical composition to yellow gentian, it is also used in traditional medicine as a remedy for poor appetite, for digestive problems, and for hepatitis A virus infections (Saric 1989; Mihailović et al. 2011). Although G. asclepiadea is very abundant in the flora of Serbia, comprehensive phytochemical and pharmacognostic investigations of plant material have not been carried out. From roots and rhizomes of G. asclepiadea, secoiridoid 1 was isolated together with its 6′-O-β-d-glucoside (30). Underground organs also contained sugars with gentianose being dominant. Unlike yellow gentian, the roots of willow gentian did not contain xanthone compounds. LC-ESI TOF MS chromatogram of G. asclepiadea roots is shown in Fig. 14.6 and in Table 14.6.

Table 14.6 Tentatively identified components in the extract using LC-ESI TOF MS and UV data of G. asclepiadea roots
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Secoiridoids

Aerial parts also contain sugars (fructose , glucose, gentiobiose, gentianose) similar to those in roots. The polyphenolic complex was diverse, being composed of flavonoids and xanthones . LC-ESI TOF MS chromatogram of G. asclepiadea aerial parts is depicted in Fig. 14.7 and in Table 14.7.

Table 14.7 Tentatively identified components in the extract using LC-ESI TOF MS and UV data of G. asclepiadea aerial parts
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C-Glucoflavones such as 11 and 13 and their glycosides 11, 31, 33, and 10 were detected. The second group of γ-pyrone constituents were xanthone C-glucosides, with mangiferin (12) and its two O-glucosides 34 and 35 as the main constituents.

Flavonoids

Xanthones

14.2.5 Gentiana utriculosa L. (Bladder Gentian)

Gentiana utriculosa (Fig. 14.13e) is an annual plant species found in Central Europe, mainly in the mountains of Italy and the Balkan Peninsula (Tutin 1972). According to our knowledge, it is not used in traditional medicine in the central regions of the Balkan Peninsula.

Analysis of the methanolic extract of the aerial parts showed the presence of secoiridoids, flavonoids, and xanthones. LC-ESI TOF MS chromatogram of G. utriculosa aerial parts is presented in Fig. 14.8 and in Table 14.8.

Table 14.8 Tentatively identified components in the extract using LC-ESI TOF MS and UV data of G. utriculosa aerial parts
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Previous phytochemical investigation of the G. utriculosa led to the isolation of xanthone aglycones 23 and 26, as well as C-glycosides 12, 13, and 15 (Hostettmann and Jacot-Guillarmod 1977). Examination by the authors of G. utriculosa originating from the Mount Tara, West Serbia, revealed xanthone O-glycosides such as 24, 36, and 37, and 4-C-glucoxanthones 12 and 38, the latter being detected for the first time in the genus Gentiana (Janković et al. 2009).

In addition, C-glucoflavones, 11 and 13 and 39, were isolated from the same extracts.

14.2.6 Gentiana punctata L. (Spotted Gentian)

Gentiana punctata (Fig. 14.13f) is a subalpine species of Southeast and Central Europe. The roots of spotted gentian are the officinal substitute for G. lutea , according to Yugoslav Pharmacopoeia IV (Ph. Yug. IV). As the roots and rhizomes contain bitter secoiridoids, they are also used in traditional medicine for the treatment of gastrointestinal tract diseases, as well as in the production of aperitifs (Tasić et al. 2004). G. punctata is an endangered species due to destructive harvesting (Šavikin-Fodulović et al. 2003).

Phytochemical investigations of G. punctata revealed significant differences in the chromatographic profiles of methanolic extracts of roots and aerial parts (Menković et al. 1998). In roots, a secoiridoid complex was dominant among secondary metabolites. Among them, 1 was the most abundant compound. However, flavonoids were not detected, but xanthone aglycon 7 was recorded. LC-ESI TOF MS chromatogram of G. punctata roots is presented in Fig. 14.9 and in Table 14.9.

Table 14.9 Tentatively identified components in the extract using LC-ESI TOF MS and UV data of G. punctata roots
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The most abundant secoiridoid in the aerial parts was 17, while 1 and 2 were also detected together with numerous flavonoid compounds. Xanthones were absent. LC-ESI TOF MS chromatogram of G.  punctata aerial parts is presented in Fig. 14.10 and in Table 14.10.

Table 14.10 Tentatively identified components in the extract using LC-ESI TOF MS and UV data of G. punctata aerial parts
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14.3 Genus Gentianella Moench.

The genus contains about 250 species distributed over all continents. Six Gentianella species can be found in Serbia (Jovanović-Dunjić 1973). The main secondary metabolites of the genus are xanthones, flavone C-glucosides, and secoiridoids (Šavikin et al. 2010, Janković 2005). Gentianella species are not used in traditional medicine in the central regions of the Balkan Peninsula .

14.3.1 Gentianella austriaca (A & J Kerner) Holub

Gentianella austriaca is distributed in Southeast Europe and the Central Balkans. It grows on different bedrocks at altitudes of 950–2400 m in mountain and subalpine meadows and pastures, and rocky regions, even in the vegetation of glaciers (Jovanović-Dunjić 1973). The species, collected during flowering at the Mount Kopaonik in Serbia (at ca. 1750 m), was characterized by the presence of three classes of compounds typical for the Gentianaceae, such as secoiridoids, C-glucoflavones , and xanthones. The isolated and identified compounds in the aerial parts of G. austriaca were bellidifolin (40), demethylbellidifolin (41), corymbiferin (42), demethylbellidifolin-8-O-glucoside (43), bellidifolin-8-O-glucoside (swertianolin) (44), corymbiferin-1-O-glucoside (45), veratriloside (46), lanceoside (47), swertisin (48), campestroside (49), together with 1, 12 and 13. Compounds 46 and 47 have been found for the first time in the genus. The dominant compound isolated from the aerial parts was 40 and its 8-O-glycosyl derivatives. The roots also contained all the above-mentioned xanthones , except 49 and the flavonoids. LC-ESI TOF MS chromatogram of G. austriaca aerial parts is presented in Fig. 14.11 and in Table 14.11.

Table 14.11 Tentatively identified components in the extract using LC-ESI TOF MS and UV data of Gentianella austriaca aerial parts
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Xanthones

In the course of chemosystematic studies of the family Gentianaceae from Serbia and Montenegro, three additional Gentianella species were also collected at the time of flowering, namely G. albanica (Jav.) Holub and G. crispata (Vis.) Holub at the Mount Hajla, at ca. 1900 m, situated between Montenegro and Serbia (Kosovo), and G. bulgarica (Velen.) Holub on the slopes of the same mountain (at ca. 800 m). The HPLC/DAD (high-performance liquid chromatography with diode array detection) chromatograms of the MeOH (methanol) extracts of the aerial parts of these species indicated considerable similarity with G. austriaca , revealing almost the same constituents (Janković et al. 2005).

14.4 Genus Swertia

Due to the numerous pharmacological properties of its constituents, the genus Swertia has received considerable attention (Šavikin-Fodulovic et al. 2002; Šavikin et al. 2010). Some members of the genus, such as S. japonica , S. chirata, S. hookeri, S. macrosperma , S. petiolata, and S. calycina, have been used in traditional medicine in the Far East for many years. Among their active principles , xanthones (mostly 1,3,7,8- and 1,3,5,8-tetraoxygenated) have special significance because of their various biological activities, e.g., antidepressant , antileukemic, antitumor, antitubercular, choleretic, diuretic, antimicrobial, antifungal, anti-inflammatory, antiviral, cardiotonic, and hypoglycemic activities (Peres et al. 2000; Neerja et al. 2000).

Among the European Swertia species, only S. perennis (Fig. 14.13g) is recognized officially according to Flora Europaea, whereas a provisional status has been assigned to S. punctata Baumg (Tutin 1972). Tan and Vladimirov (2001) claimed that S. punctata is the precisely defined species. S. punctata is also described in flora of Serbia (Jovanović-Dunjić 1973), as the only species of the genus occurring in Serbia.

14.4.1 Swertia Punctata Baumg.

Swertia punctata Baumg. (Fig. 14.13h) is distributed in the Central Balkans. It grows in a zone of mountain wetland pastures, meadows, and turfs, mostly on silicate and serpentine substrates at altitude, between 1500 and 2000 m. Its population in Serbia is scarce and endangered, as reported recently in the Red Book of Serbian Flora (Jovanović 1999). Although several Swertia species are used extensively in traditional medicine, S. punctata is not mentioned in traditional medicine until the present times.

Methanol extracts have been analyzed of the aerial parts and roots of the S. punctata originating from Stara Planina, East Serbia (Menković et al. 2002). LC-ESI TOF MS chromatogram of S. punctata leaves is presented in Fig. 14.12 and in Table 14.12.

Table 14.12 Tentatively identified components in the extract using LC-ESI TOF MS and UV data of S. punctata leaves
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Apart from 12 and 13, the compounds isolated from S. punctata are listed below:

1,3,5,8-Tetrasubstituted xanthones

In addition to 40 and 44, the following 1,3,5,8-tetrasubstituted xanthones were identified:

1,3,7,8-Tetrasubstituted xanthones

The roots of S. punctata afforded norswertianin-1-O-glucoside (20), as well as the following 1,3,7,8-tetrasubstituted xanthones (Menković et al. 2002):

14.5 Biological Activity

Due to the diverse chemical composition and the presence of secoiridoids, xanthones, and C-glucoflavones , the numerous pharmacological activities have been demonstrated of Gentianaceae species (Jensen and Schripsema 2002; Neerja et al. 2000; Pinto et al. 2005; Šavikin et al. 2010).

The bitter principles (secoiridoids), the usual constituents of the genus, stimulate secretion of gastric juices and bile, thus aiding appetites and digestion. G. lutea , as the officinal medicinal plant , was investigated extensively and activities, such as increasing salivation, appetite stimulation, and choleretic and immune-stimulating activity were reported (Öztütk et al. 1998, 2006). Gentiopicrin , a dominant compound in the secoiridoid complex, was indicated as the main active compound. Secoiridoids also exhibit several other biological activities. Swertiamarin and sweroside inhibited the growth of Bacillus cereus , B. subtilis , Citrobacter freundii and Escherichia coli , swertiamarin was active against Proteus mirabilis and Serratia marcescens , while sweroside inhibited the growth of Staphylococcus epidermidis (Kumarasamy et al. 2003).

Methanolic extracts of flowers and leaves of Gentiana lutea , together with the isolated compounds mangiferin (12), isogentisin (8), and gentiopicrin (1), were used to investigate the antimicrobial activity. Both extracts and isolated compounds showed antimicrobial activity on a range of Gram-positive and Gram-negative bacteria as well as the yeast Candida albicans  (Šavikin et al. 2009). The synergistic activity of the pure compounds may be responsible for the excellent antimicrobial effect of the extracts.

Gentiopicrin (1) has shown spasmolytic activity inhibiting, in a concentration-dependent manner, the spontaneous contractions of isolated guinea pig ileum. Contractions induced by histamine, acetylcholine, BaCL2, and KCL on the ileum were also blocked significantly by this monoterpene glucoside, which suggests that this compound might be interfering with calcium influx into the smooth muscle cells (Rojas et al. 2000).

In a previous study, in vitro experiments showed that mangiferin (12) inhibited the cytotoxic action of ionizing irradiation (doses of 6 and 8 μGy) only on normal resting human PBMC (peripheral blood mononuclear cell), not stimulated for proliferation. Orally consumed G. lutea extract showed the potential to reduce the cytotoxic effect of irradiation on normal human immunocompetent cells PBMC of some healthy people, without changing the susceptibility of malignant cells to be destroyed by irradiation (Menković et al. 2010).

As reviewed by Neerja et al. (2000), bellidifolin (40), methylbellidifolin (51), methylswertianin (54), and mangiferin (12) isolated from the Swertia species exhibited various biological effects such as hypoglycemic, hepatoprotective, antituberculous, antioxidant, antimalarial, and anti-inflammatory activities. The xanthones, gentiacaulenin (26) and gentiakochianin (25), are responsible for antihypertensive activity, exerting a vasodilator action on in vitro aortic rings, probably linked to the blocking of the ryanodine-sensitive Ca++ channels (Chericioni et al. 2003).

Inhibition has been observed of type A and type B monoamine oxidases by a number of xanthones (Suzuki et al. 1980, 1981). Diethyl ether extracts of G. kochiana as well as gentiacaulenin (26) and gentiakochianin (25) were tested for CNS (central nervous system) pharmacological activity in rodents (Tomić et al. 2005). Extracts and 26 strongly inhibited rat microsomal MAO (monoamine oxidase) A. Examinations of behavior on mice showed that 10-day s.c. administration of the extract (20 mg/kg) decreased significantly immobility score in a forced swimming test and strongly inhibited ambulation and stereotypy in an open-field test. Studies suggest some antidepressant therapeutic potential of G. kochiana that is presumably connected to the action of 26.

Xanthones 25 and 26 were also identified as the active principles responsible for in vitro antiglioma action of ether and methanolic extracts of G. kochiana (Isakovic et al. 2008). These compounds induced cell cycle arrest in G2/M and G0/G1 phases, respectively, in both C6 rat glioma and U251 human glioma cell lines. Both xanthones reduced mitochondrial membrane potential and increased the production of reactive oxygen species in glioma cells, but only the effects of 25 were pronounced enough to cause caspase activation and subsequent apoptotic cell death. The assessment of structure-activity relationship showed that dihydroxylation at positions 7, 8 of the xanthonic nucleus is the key structural feature responsible for the ability of gentiakochianin to induce microtubule-associated G2/M cell blockage and apoptotic cell death in glioma cells.

Hudecová et al. (2012) showed that G. asclepiadea exerts antioxidant activity and enhances DNA repair of hydrogen peroxide- and silver nanoparticle-induced DNA damage. The same study also showed antioxidant, antigenotoxic, and biomodulatory effects of G. asclepiadea extracts on various cells (including lymphocytes and HEK 293 cells) exposed to different agents such as H2O2, Zeocin, and AgNPs (silver nanoparticles).

The aerial parts of Gentianella austriaca were evaluated for their antioxidative activity and protective properties on irradiated human peripheral blood lymphocytes in vitro (Leskovac et al. 2007). Aqueous ethanolic extracts showed protective effects, decreasing the incidence of radiation-induced micronuclei by 35.56 %, and significantly reduced lipid peroxidation for 30.88 %. The radioprotective effects of the water-soluble xanthones demethylbellidifolin (41), demethylbellidifolin-8-O-glucoside (43), bellidifolin-8-O-glucoside (44), and the flavonoid swertisin (48) were also reported (Jankovic et al. 2008). Among the compounds examined, the highest reduction by 27.92 % in the incidence of micronuclei was observed in irradiated lymphocytes treated with swertisin (48).

In addition, isovitexin (15), the co-occurring constituent of G. lutea leaves, exhibits considerable antioxidative and hypoglycemic effects. The potential antioxidative effect of the extract of the leaves of G. lutea could be assigned to xanthones and flavonoids, presumably mangiferin (12) and isoorientin (13), respectively. It is well known (Rice-Evans et al. 1995) that the potential of flavonoids and xanthones to exert antioxidative effects is strongly dependent on their structure and the substituents of the heterocyclic and B rings. As far as the antiradical activity is concerned, the following structural features are essential:

  1. (a)

    3′,4′-Dihydroxy pattern (ring B)

  2. (b)

    2,3-double bond conjugated with keto group (ring A)

  3. (c)

    3,5-Dihydroxy substitution (ring A)

  4. (d)

    Free 7-OH; it was established that glycosylation in positions 3, 5, and 7 reduces the activity.

The structure of isoorientin (13) fulfills most of the above conditions with ortho-3′,4′-OH groups, 5-OH and 7-OH, as well as a 2,3-double bond conjugated with the keto group. This could explain considerable antioxidative activity of 13. 3′ and 4′-O-glucosides of 13, lacking free ortho-3′,4′-OH groups that exhibit rather small inhibitory capacity (<1 %), (Burda and Oleszek 2001). In swertisin (48), free OH groups are only at C-4′ i C-5, whereas 7-OH is methylated, causing much lower activity in 48 (3 %) in comparison with that in 13 (66 %). At the same time, the considerable antioxidative activity of mangiferin (12) could be explained by the structural features similar to those in isoorientin (Krstić-Milošević 2008).

Based on the comparison of pharmacologic effects of the main components in the extracts of G. lutea leaves, one could expect antidiabetic, hepatoprotective, and anti-inflammatory effects. Thus, the continuation would be justified of the chemical investigation of this extract, aimed to discover a new source of drugs for use in phytotherapy. The use of yellow gentian leaves in the therapy instead of roots could have a protective effect on this species endangered by overexploitation. In addition, in the ethnomedicine of Turkey, as well as in the Indian medicine Ayurveda, Gentiana olivieri , possessing a similar chemical profile to G. lutea, is used as tonic and bitter as well as for its antidiabetic, appetizer, and antipyretic effects (Sezik et al. 2005, Chopra et al. 2006, Mansoor and Malghani 1999).

Recently, Turkish authors have proved the hypoglycemic activity of the extracts of this species in which 13 was a dominant constituent, and at the same time, its hepatoprotective effect was evaluated (Orhan et al. 2003).

Similarly, in the Chinese province of Mongolia, the people use a plant “guixincao” in the form of a tea to cure colds, to clean blood by removing toxins and pathogens, as well as a diuretic  (Min-Hui et al. 2010). This plant, identified as Gentianella acuta (Michaux.) Hulten, exhibits the chemical profile rather similar to that of G. austriaca with demethylbellidifolin , bellidifolin , and the corresponding heterosides being dominant.

Moreover, according to the chemical composition, S. punctata could be attractive as a source of medicinal raw material, but since its population is small, efforts are now concentrated on determining alternative ways for biomass production.

14.6 Experimental Design

14.6.1 Plant Material

For analysis, aerial parts and roots were collected from all species investigated. G. lutea was collected on the Mount Suvobor 750 m, Serbia; G. dinarica on the Mount Tara 1300 m, Serbia; G. kochiana on the Mount Komovi 2100 m, Montenegro; G. asclepiadea on the Mount Hajla 1900 m, Montenegro; and G. utriculosa on the Mount Tara (1100 m), Serbia. Four species of genus Gentianella were examined in the present studies. G. albanica and G. crispata were collected on the Mount Hajla at ca. 1900 m situated between Montenegro and Serbia (Kosovo), G. bulgarica on the slopes of the same mountain at ca. 800 m, and G. austriaca at Mount Kopaonik in Serbia at ca. 1750 m. S. punctata was collected on Stara mountain (1900 m), Serbia.

14.6.2 Chromatographic Techniques

A combination of different preparative chromatographic techniques was applied to isolate pure compounds. The analogous basic separation scheme, shown in Fig. 14.14 for Swertia punctata , was applied to isolate pure compounds from all extracts examined.

Fig. 14.14
figure 14

Isolation of some compounds from Swertia punctata leaves

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The spectra were recorded with the following instruments: IR, Perkin-Elmer FT-IR spectrometer 1725 X; 1H and 13C NMR 1D and 2D NMR, Varian Gemini 2000 (200 MHz for 1H), and Bruker AMX 500 (500 MHz for 1H); UV, G113AA HP 8543 advanced UV–Vis spectrometer; DCIMS (150 eV, isobutane), Finnigan MAT mass spectrometer 8230, double focusing (BE geometry); ESIMS (a sample, dissolved in MeOH–H2O, 1:1) Finnigan MAT 900, double focusing (EB geometry) equipped with a Finnigan MAT electrospray interface; optical rotations, Perkin-Elmer 141 MC polarimeter; melting points (not corrected), Boetius PHMK apparatus; analytical HPLC, Hewlett Packard HPLC model 1090, DAD detector (HP 1040), column, Lichrospher RP-18 (5 ı̀), 250 × 4 mm I.D. (Merck); preparative medium pressure chromatography (MPLC), Lobar column (silica gel Si 60, size A or B); dry-column flash chromatography (DCFC), silica gel Si 60; MN polyamide DC 6; TLC, 0.2 mm, silica gel 60 F 254 Merck, detection under UV or by heating after spraying with 50 % H2SO4; column chromatography (CC), polyamide-6-powder (polycaprolactam); Sephadex LH-20; elemental C,H-analysis, combustion method (Fig. 14.14).

14.6.2.1 LC-ESI TOF MS Analysis of MeOH Extracts

Air-dried plant material (0.5 g) was extracted with MeOH (10 ml) at room temperature for 48 h. The solvent was removed under vacuum at room temperature. Before the analysis, the extract was dissolved in MeOH and the concentration was adjusted to ca. 10 mg/ml, followed by filtration through a Teflon Millipore filter HV, with a pore size 0.45 µm.

  1. 1.

    Injection volume: 5 μl.

  2. 2.

    Instrument: Agilent MSD ESI TOF coupled with Agilent 1200 series RR, liquid chromatography.

  3. 3.

    Column: LiChrospher 100 RP 18e, 150 × 4.0 mm i.d. 5.0 μm.

  4. 4.

    Mobile phase: A (water + 0.2 % HCOOH) + B (MeCN).

  5. 5.

    Elution: combination of gradient and isocratic modes according to following scheme:

    Time (min)

    Phase A (%)

    Phase B (%)

    0

    98

    2

    5

    90

    10

    18

    90

    10

    20

    85

    15

    25

    85

    15

    30

    70

    30

    40

    30

    70

    50

    30

    70

    51

    98

    2

    55

    98

    2

  6. 6.

    Flow rate: 0.995 ml/min.

  7. 7.

    MSD conditions: drying gas (N2) flow 12 l/min; nebulizer pressure 45 psig; drying gas temperature 350o; capillary voltage, 4000 V; fragmentor voltage, 140 V; skimmer, 60 V; Oct RF voltage 250 V; positive mode, mass range m/z 100–2500; 10,000 transients/scan.

14.7 Conclusions

The limited selection of bioactive plant constituents of some Gentiana, Gentianella, and Swertia species and the therapeutic applications described here give a general idea of the progress achieved over the last two decades. These achievements have been possible, in part, thanks to the development of techniques in separation and spectroscopy. LC DAD-ESI TOF MS chromatograms of plant extracts are presented and discussed.

Among numerous bioactive extracts, those of G. lutea leaves emerge as very promising since one could expect antidiabetic, hepatoprotective, and anti-inflammatory effects. Thus, the continuation is justified of the chemical investigation of this extract, aimed to discover a new source of drugs used in phytotherapy. The use of yellow gentian leaves instead of roots in therapy could have a protective effect on this species endangered by overexploitation. Moreover, according to its chemical composition, Swertia punctata could be attractive as a source of medicinal raw material, but since its population is small, efforts should be focused in finding alternative ways for biomass production.