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Planta Med 2000; 66(2): 178-180
DOI: 10.1055/s-0029-1243126
Letter

© Georg Thieme Verlag Stuttgart · New York

Chemical Composition and Seasonal Variations in the Amount of Secondary Compounds in Gentiana lutea Leaves and Flowers

Nebojša Menković1*, Katarina Šavikin-Fodulović1 , Katica Savin2
  • 1Institute for Medicinal Plants Research, Belgrade, Yugoslavia
  • 2Faculty of Pharmacy, Department of Pharmacognosy, Belgrade, Yugoslavia
Further Information

Dr. Nebojša Menkovič

Institute for Medicinal Plants Research

Tadeuša Koščuška 1

11000 Belgrade

Yugoslavia

Email: katarinasf@hotmail.com

Email: ksf@eunet.yu

Fax: +38-111182072

Publication History

Received: April 9, 1999

Accepted: July 25, 1999

Publication Date:
24 November 2009 (online)

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Table of Contents #

Abstract

The chemical investigation of MeOH extracts of Gentiana lutea leaves and flowers showed that xanthones were one of the dominant class of compounds. Secoiridoids and flavonoids were also recorded. The amount of secondary metabolites varied depending on development stage. In the phase of flowering, leaves are rich with compounds possessing C-glycoside structures while O-glycoside structures accumulate mainly before flowering.

In both folk and modern medicine, the roots of Gentiana lutea L. (Gentianaceae) are very popular as a stomachic as well as a component in preparations showing beneficial effects in gall and liver diseases [1]. Recent investigations pointed out an interesting chemical composition of the aerial parts of G. lutea. The presence of isogentisin and two flavonic heterosides was reported in leaves of G.lutea by Hostettmann et al. [2]. Isogentisin revealed potent monoamine oxidase (MAO) inhibition [3]. Bakuridze et al., (1991) reported the presence of mangiferin in the aerial parts of G.lutea [4], a xanthone compound showing antioxidative [5] [6], antidepressant and some other pharmacological activities [7] [8]. The present paper deals with the isolation and quantification of the secondary metabolites in G.lutea leaves and flowers.

As the aerial parts of G.lutea contain secoiridoid, flavonoid and xanthone compounds which possess characteristic optical activity in UV, high performance liquid chromatography with DAD detection represented a method of choice for the analysis of this complex mixture. Two procedures previously reported [9] [10] were tested. Both procedures showed relatively good separation according to the literature, but mangiferin was weakly separated from gentiopicrin and isoorientin. For this reason, a new gradient mode (described in Materials and Methods) was tested and a better separation between the three compounds was managed. Corresponding compounds from chromatograms are described in [Table 1]. Identification was made by comparison with autentic standards.

Table 1 Compound from leaves and flowers of G.lutea.
Common name Rt (min) UVλmax (nm) Spectral analysisb
1 Swertiamarina L 17.5 238 a
2 GentiopicrinL, F 22.1 242, 272 a + b
3 MangiferinL, F 25.5 240, 259, 316, 364 a + b
4 Isoorientina L, F 30.4 254, 268, 349 a
5 Isovitexin1L, F 31.9 268, 336 a
6 Isogentisin-3-O-β-D-primeverosideL, F 34.5 236, 258, 302, 368 a + b
7 IsogentisinL, F 41.5 236, 259, 306, 372 a + b
a Substances compared with reference samples
b a = HPLC-UV; b = MS, IR, 1H-NMR;
L – compound detected in leaves; F – compound detected in flowers

The aerial parts of G.lutea were harvested at four natural habitats during the phase of flowering. The amount of secondary compounds varied in the samples, but it appeared not to be correlated with altitude ([Table 2]).

Table 2 The amount of secondary metabolites in leaves and flowers of G.lutea harvested on different natural habitats during three seasons (1993, 1995, 1997), (results represent the mean of the three seasons).
Compound Suvobor Stara Planina Koritnik Ošljak
L F L F L F L F
mangiferin 3.98 ± 0.11 1.14 ± 0.04 3.01 ± 0.11 0.91 ± 0.05 2.79 ± 0.12 0.77 ± 0.04 2.66 ± 0.11 0.79 ± 0.05
isoorientin 0.93 ± 0.05 0.41 ± 0.02 0.76 ± 0.04 0.43 ± 0.03 0.80 ± 0.04 0.36 ± 0.03 0.51 ± 0.04 0.32 ± 0.03
isogentisin-primeveroside 0.19 ± 0.02 0.09 ± 0.01 0.21 ± 0.02 0.07 ± 0.01 0.18 ± 0.02 0.09 ± 0.02 0.15 ± 0.02 0.07 ± 0.01
isogentisin 0.11 ± 0.01 0.31 ± 0.02 0.10 ± 0.01 0.34 ± 0.03 0.15 ± 0.02 0.28 ± 0.03 0.12 ± 0.02 0.34 ± 0.03
gentiopicrin 0.82 ± 0.06 3.29 ± 0.12 0.85 ± 0.04 3.02 ± 0.13 0.72 ± 0.05 2.11 ± 0.10 0.59 ± 0.03 2.34 ± 0.09
swertiamarin 0.65 ± 0.04 – 0.65 ± 0.03 – 0.39 ± 0.03 – 0.38 ± 0.02 –
Legend: L – leaves; F – flowers.
Results were obtained by HPLC and are presented as % of dry weight.

The amounts of secondary compounds were recorded in wild plants at various stages of development every month during the period from March till October during three seasons. The seasonal variation in the content is shown graphically ([Fig. 1] and [Fig. 2]). Mangiferin and isoorientin reached their maximum between June and July which coincides with the period of flowering. In the case of mangiferin a rapid fall in the amount was recorded in May and August, but only slight changes in the isoorientin level. In contrast, the maximum of the accumulation of isogentisin-primeveroside was recorded in April, and isogentisin in May, while a large decrease in their amounts was noticed during the flowering phase ([Fig. 1]). The amount of isogentisin-primeveroside was again increased in October.It could be concluded that during phase of flowering, leaves are rich with compounds possessing C-glycoside structures while O-glycoside structures accumulate mainly before flowering. The accumulation of secoiridoids ([Fig. 2]) showed more or less constant increase during the vegetation period reaching the maximum in October.Seasonal variations in the amount of secondary metabolites were also recorded in the roots and rhizomes of G.lutea. The greatest content of gentisin-primeveroside was detected in April while gentisin reached its maximum in October. The dynamics of the accumulation of secoiridoids in the roots was completely different in comparison to leaves. Two maximums of accumulation of gentiopicrin were recorded (April and October) while the amounts of swertiamarin were relatively constant during the whole vegetation period.

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Fig. 1 Variation in the concentrations of γ-pyrones during the development of G.lutea leaves (results were obtained by HPLC and are presented as percentage of dry weight).

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Fig. 2 Variation in the concentrations of secoiridoids during the development of G.lutea leaves (results were obtained by HPLC and are presented as percentage of dry weight).

In the samples examined in the present study the determination of the general chromatographic pattern was a particularly simple and quick analysis method, and permitted identification and evaluation of the characteristic components in the crude drug, that is the secoiridoids and γ-pyrones. Also, the suggestion of Nikolaev et al. [11] to use not only the root but also the aerial parts of G.lutea as a medicinal material seems to be justified.

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Materials and Methods

Plant material was harvested at four natural localities: Suvobor (800m), Stara Planina (1800m), Koritnik (2000m) and Ošljak (1850m) during 1990–1997.A voucher specimen (No. 15 795) has been deposited in the herbarium at the Institute of Botany and Botanical Garden ”Jevremovac”, University of Belgrade.

Air-dried samples of leaves and flowers were extracted with MeOH under reflux for 30 minutes in a water bath (50 °C). The ratio between sample and solvent was 1 : 10. Extracts were filtered, supplemented to 10 ml and were used for TLC and HPLC analyses. TLC. a) Conditions for γ-pyrones – Layer: TLC Cards Cellulose F254, 0.1 mm (Fluka); Solvent system: 15 % HOAc in water (v/v); Detection: The TLC was observed in day light and UV254/366 before and after spraying with 5 % AlCl3 in EtOH b). Conditions for secoiridoids – Layer: Silica gel 60 F254, 0.2 mm (Merck); Solvent system: CHCl3 – EtOH (75 + 25 v/v); Detection: chromatogram was observed on UV254/366 after successive spraying with 0.2 % Fast Red Salt B in water and 20 % K2CO3 in water. HPLC. Instrument: Hewlett Packard HPLC model 1090, DAD detection (HP 1040A); Column: Lichrospher RP-18 (5µ) 250 × 4 mm I. D. (Merck); Mobile phase A: acetonitrile; Mobile phase B: HPLC water with 1 % 0.1N H3PO4; Flow rate: 0.8 ml/min; Elution by linear gradient according to the following scheme: start: phase B 98 %; phase B 90 % 5–18min; phase B 85 % 20–25min; phase B 70 % 30min; phase B 30 % 40min; Phase B 0 % 50min. UV spectra of MeOH solutions were measured using a G113AA HP 8543 advanced UV-VIS spectrometer. IR spectra were measured using Perkin Elmer FT-IR 1725X (KBr pellets) spectrometer. MS data were obtained on a Finnigan MAT 8230 (DCI isobutane) mass spectrometer at 150eV. The 1H- NMR spectra were recorded in CDCl3 at the room temperature on a Varian Gemini 2000 operating at 200 MHz.

Mangiferin (3) was isolated from the leaves of G.lutea collected in the phase of flowering according to Glizin et al., [12]. Isogentisin-3-O-β-D-primeveroside (6) was isolated from the aerial parts of 6–7 months old seedlings collected from the experimental field on Suvobor, where (6) was the most abundant compound [13]. Isogentisin (7) was obtained from flowers of G.lutea [13].

The compounds were identified by their physical and spectroscopic data. Standards: Swertiamarin (2), isoorientin (5), and isovitexin (6) were purchased from Roth (Germany). Gentiopicrin (3), gentisin, and gentisin-7-O-β-D-primeveroside were previously isolated at the Institute for Medicinal Plants Research from roots of G.lutea [14]. Their structures were confirmed using UV, IR and 1H-NMR techniques. Quantification was performed using HPLC. Quantities of secondary metabolites were calculated from calibration curves (5 points for each standard supstances were used for the establishment of calibration curves). Defined concentrations of standard supstances were added to analysed samples and proportional increase of absorptions were obtained. The results are presented as percentage of dry weight ± standard deviations. Standard deviations were calculated using arc-sin transformed data.

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References

Dr. Nebojša Menkovič

Institute for Medicinal Plants Research

Tadeuša Koščuška 1

11000 Belgrade

Yugoslavia

Email: katarinasf@hotmail.com

Email: ksf@eunet.yu

Fax: +38-111182072

#

References

Dr. Nebojša Menkovič

Institute for Medicinal Plants Research

Tadeuša Koščuška 1

11000 Belgrade

Yugoslavia

Email: katarinasf@hotmail.com

Email: ksf@eunet.yu

Fax: +38-111182072

   Permissions and Reprints
Zoom Image

Fig. 1 Variation in the concentrations of γ-pyrones during the development of G.lutea leaves (results were obtained by HPLC and are presented as percentage of dry weight).

Zoom Image

Fig. 2 Variation in the concentrations of secoiridoids during the development of G.lutea leaves (results were obtained by HPLC and are presented as percentage of dry weight).