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DOI: 10.1055/s-2000-9774
Smooth Muscle Relaxing Activity of Gentiopicroside Isolated from Gentiana spathacea
Dr. M. Alejandra Rojas
Molina
Departamento de Investigación Química y Farmacológica de Productos Naturales Facultad
de Química Universidad Autónoma de Querétaro
Cerro de las Campanas 76010
Querétaro
Mexico
Email: rojasa@sunserver.uaq.mx
Phone: +52 42 163730
Publication History
Publication Date:
31 December 2000 (online)
Abstract
Bioassay directed fractionation of the (1 : 1) chloroform-methanol extract of Gentiana spathacea H.B.K (Gentianaceae) led to the isolation of gentiopicroside (gentiopricrin) (1), the major spasmolytic component of the plant. Gentiopicroside inhibited, 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 significantly blocked by this monoterpene glucoside, which suggests that this compound might be interfering with calcium influx into the smooth muscle cells.
Gentiana spathacea H.B.K. (Gentianaceae), commonly known as “Juana Mi Pila”, is a plant highly valued by the Otomi Indians of the State of Querétaro (México) for treating gastrointestinal ailments. This species is also employed by mestizo populations who have access to the same vegetal resources and share ethnobotanical information with the Otomies [1]. G. spathacea has not been previously subjected to any chemical or pharmacological study. However, phytochemical investigations of related species have allowed the isolation or identification of various secoiridoid glycosides [2], [3], [4], [5], [6], [7], some flavonoids [8], [9], [10], phenolic compounds [11] and alkaloids [12].
Recently, we have established a multidisciplinary research program to obtain smooth muscle relaxing agents potentially valuable for the development of drugs useful in the therapy of gastrointestional and/or cardiovascular diseases. As a part of this investigation, it was found that the chloroform-methanol (1 : 1) extract of G. spathacea displayed a significant concentration-dependent inhibition of the spontaneous contractions of isolated guinea-pig ileum (Fig. [1]). The extract of G. spathacea (IC50 = 4.6 μg/ml) was 2.3 times more potent than the extract of Datura lanosa (IC50 = 10.6 μg/ml) (Table [1]), which was used as a positive control due to its high contents of scopolamine and atropine [13].
Column chromatography on silica gel of the extract of G. spathacea afforded seven primary fractions. F5 significantly inhibited the spontaneous contractions of the ileum (67 % of inhibition) when tested at the IC50 of the original extract. F5 was extensively chromatographed to yield gentiopicroside (1). Figure [1] shows the concentration-response curve for gentiopicroside (IC50 = 2.8 μg/ml), which was nearly two times more active than the original extract. The potency of 1 was also compared with that of nifedipine, a calcium entry blocker. In this case, 1 turned out to be 15 times less potent than the positive control (Table [1]).[]
In an initial attempt to characterize the mechanism of action of 1, its effect on cholinergic, histaminergic and calcium-induced contractions (BaCl2 and KCl) was assessed. Gentiopicroside significantly inhibited the contractions induced by KCl (32.2 % of inhibition) and BaCl2 (inhibition = 52.4 %) and also produced a moderate decrease on cholinergic (19 % of inhibition) and histaminergic (22.6 % of inhibition) contractions (Fig. [2]). Since the tested spasmogens induce ileum contractions by different modes of action, the antagonism elicited by 1 suggested that it might be acting at a common step in the contraction mechanism induced by these agonists. Furthermore, the results obtained in the experiments with KCl and BaCl2, which produce contractions mainly due to an increased Ca2+ influx into the smooth muscle cell, supported the hypothesis that gentiopricroside might be blocking the entry of extracellular Ca2+. However, this behavior does not rule out the possibility that 1 competes with Ca2+ binding proteins such as calmodulin.
Test substance | Emax | IC50 (μg/ml) | Potency |
Nifedipine | 85.14 ± 1.74 | 0.188 ± 0.03 | 1.000 |
D. lanosa extract | 85.64 ± 8.10 | 10.59 ± 3.20 | 0.018 |
G. spathacea extract | 71.86 ± 1.98 | 4.58 ± 1.10 | 0.041 |
Gentiopicroside | 95.87 ± 3.66 | 2.80 ± 0.71 | 0.067 |
Values are means ± s.d.; n = 6 (95 % confidence intervals). | |||
Potency was obtained by the formula: IC50 nifedipine/IC50 test substance, assuming a value of 1.00 for Nifedipine. |


Fig. 1Concentration-response curves showing the relaxatory effects of the extract of Gentiana spathacea and gentiopicroside on isolated guinea-pig ileum. Values are expressed as the percentages of inhibition of contractile responses calculated as the mean from six data ± s.d., p < 0.05.

Fig. 2Effect of gentiopicroside on the contractions induced by BaCl2 (3 × 10-4 M), KCl (60 mM), acetylcholine (1 × 10-4 M) and histamine (1 × 10-3 M) on isolated guinea-pig ileum. p < 0.05 (ANOVA). Gentiopicroside was tested at its IC50 for the inhibition of spontaneous contractions of the ileum (2.80 μg/ml).
Materials and Methods
The aerial parts of G. spathacea were collected in the locality of Amealco, State of Querétaro (México) in November 1997. A voucher specimen (Serrano No. 72) has been deposited in the Ethnobotanical Collection of the Herbarium of Querétaro “Dr. Jerzy Rzedowski” (QMEX) located at the Faculty of Natural Sciences, University of Querétaro. Air-dried, ground plant material (305 g) was macerated three times with CHCl3-MeOH (1 : 1). The extract was evaporated to dryness in vacuo to yield 92.5 g of a dark green sticky residue, which was subjected to open column chromatography on silica gel (600 g). Elution was accomplished with a gradient of hexane/CHCl3/acetone/MeOH [hexane (3 l), hexane-CHCl3, 88 : 2 (1.2 l); hexane-CHCl3, 9 : 1 (2 l); hexane-CHCl3, 1 : 1 (12 l); CHCl3 (9 l), CHCl3-acetone, 8 : 2 (8 l); acetone (8 l); acetone-MeOH, 9 : 1 (5.1 l); acetone-MeOH, 6 : 4 (2.2 l) and MeOH (2 l)]. A total of 210 fractions (250 ml each) were collected and pooled on the basis of their TLC profile to render seven primary fractions (F1 - F7). These fractions decreased the spontaneous contractions of isolated guinea-pig ileum, displaying the following percentages of inhibition: F1 (20.46 ± 5.3), F2 (29.80 ± 10.0), F3 (30.43 ± 5.9), F4 (20.83 ± 6.0), F5 (67.21 ± 8.0), F6 (41.12 ± 6.8) and F7 (39.06 ± 4.0). The most active fraction F5 (24.11 g) eluted with CHCl3-acetone (8 : 2) and acetone was further chromatographed over a silica gel (480 g) column eluted with CHCl3, acetone and methanol. Secondary fractions 86 - 112 eluted with acetone-MeOH (1 : 1) yielded 4.6 g of impure 1, which was purified by preparative HPLC (pump: Waters 600; detector: Waters 486 tunable absorbance set at λmax 254 nm; column: Waters Prep Nova Pak HR C18, 300 × 19 mm i.d., 6 μm, 60 Å; mobile phase: MeOH-H2O, 22 : 78; flow rate: 8 ml/min) to afford 1.063 g of 1 (amorphous white powder, tR 234 - 280 ml, 0.35 % dry weight). Physical constants (m.p., αD) and spectral data (UV, MS, NMR) of compound 1 were consistent with reported values [2], [14]. Copies of the original spectra are obtainable from the author of correspondence.
Guinea-pig ileum segments (1 cm) were isolated and prepared for recording the contractions using force transducers (Grass FT03) connected to a polygraph (Grass 7D) as previously described [15]. After a stabilization time of 30 min, a 10-min control period was recorded. Then, the extracts and pure compounds were added to the bath at different concentrations and the responses were recorded during 10 min. Concentration-response curves for the test substances were plotted. Fractions derived from G. spathacea extract were tested only at the IC50 of the original extract. The effect of the extracts, fractions and pure compounds was determined by comparing the areas under the curve inscribed by the frequency and the amplitude of the ileum contractions before and after the application of the test materials. Areas were calculated from the polygraph tracing using an analog-digital tablet (CPLAB-10) and specially-designed software.
The effect of gentiopicroside on the contractions induced by Ach (1 × 10-4 M), histamine (1 × 10-3 M), BaCl2 (3 × 10-4 M) and KCl (60 mM) was studied. In these experiments, 1 was administered to the ileum, previously stimulated by the agonists, at a concentration corresponding to its IC50 for the inhibition of ileum spontaneous contractions and the responses were recorded during 10 min. The effect of 1 was determined by comparing the average amplitude of the ileum contractions with the amplitude of the contractions obtained when the agonists alone were added to the bath.
All the results are expressed as the mean of 6 experiments ± sd. Concentration-response curves (CRC) for the extracts, gentiopicroside and nifedipine were plotted and the experimental data from the CRC were adjusted by the non-linear, curve-fitting program ORIGIN 4.0. The statistical significance (p < 0.05) of differences between means was assessed by an analysis of variance (ANOVA) followed by a Dunnet's test [16].
#Acknowledgements
This research was partially financed by Consejo Nacional de Ciencia y Tecnología (CONACYT, Proyecto No. 25762-N), Secretaría de Educación Pública (FOMES 98-23-04) and Consejo de Ciencia y Tecnología del Estado de Querétaro (CONCYTEQ). We also thank M. Sc. V. Serrano and M. Sc. Ricardo Pelz for collection and identification of plant material and Angélica Feregrino for technical assistance. The authors acknowledge the facilities provided by Dr. Rachel Mata, Universidad Nacional Autónoma de México for registration of spectra. Special thanks are due to M. Sc. José Alfredo Zepeda Garrido (Rector of the University of Querétaro) and Q. M. J. Merced Esparza García (Director of the Faculty of Chemistry, University of Querétaro) for facilities provided during the development of this research.
#References
- 1 Rojas A,, Bah M,, Rojas J I,, Serrano V,, Pacheco S.. Spasmolytic activity of some plants used by the Otomi Indians of Querétaro (México) for the treatment of gastrointestinal disorders. Phytomedicine. 1999;; 6 367-71
- 2 Mpondo E M,, Chulia A J.. 6′-O-β-D-Glucosylgentiopricroside: A new secoiridoid from Gentiana asclepiadea. Planta Medica. 1988;; 54 185-6
- 3 Ma W G,, Fuzzati N,, Wolfender J L,, Hostettman K,, Yang C R.. Rhodenthoside A, a new type of acylated secoiridoid glyocside from Gentiana rodhentha. . Helvetica Chimica Acta. 1994;; 77 1660-71
- 4 Tan R X,, Wolfender J L,, Ma W G,, Zhang L X,, Hostettmann K.. Secoiridoids and antifungal aromatic acids from Gentiana algida. Phytochemistry. 1996;; 41 111-6
- 5 Tan R X,, Wolfender J L,, Zhang L X,, Ma W G,, Fuzzati N,, Marston A,, Hostettmann K.. Acyl secoiridoids and antifungal constituents from Gentiana macrophylla. . Phytochemistry. 1996;; 42 1305-13
- 6 Tan R X,, Kong L D.. Secoiridoids from Gentiana siphonantha. . Phytochemistry. 1997;; 46 1035-8
- 7 Tan R X,, Kong L D,, Wei H X.. Secoiridoid glycosides and an antifungal anthranilate derivative from Gentiana tibetica. . Phytochemistry. 1998;; 47 1223-6
- 8 Wolfender J L,, Maillard M,, Hostettmann K.. Liquid chromatographic-thermospray mass spectrometric analysis of crude plant extracts containing phenolic and terpene glycosides. Journal of Chromatography. 1993;; 647 183-90
- 9 Ling C N,, Kuo S H,, Chung M I,, Ko F N,, Teng C M.. A new flavone C-glycoside and antiplatelet and vasorelaxing flavones from Gentiana arisanensis. . Journal of Natural Products. 1997;; 60 851-3
- 10 Ko F N,, Chu C C,, Lin C N,, Chang C C,, Teng C M.. Isoorientin-6″-O-glucoside, a water-soluble antioxidant isolated from Gentiana arisanensis. . Biochimica et Biophysica Acta. 1998;; 1389 81-90
- 11 Atkinson J E,, Gupta P,, Lewis J R.. Some phenolic constituents of Gentiana lutea. . Tetrahedron. 1969;; 24 1507-11
- 12 Marekov N L,, Popov S S.. The structure of gentioflavine, a new alkaloid of some Gentiana species. Tetrahedron. 1968;; 24 1323-6
- 13 Bye R,, Mata R,, Pimentel J.. Botany, ethnobotany and chemistry of Datura lanosa (Solanaceae) in Mexico. Anales del Instituto de Biología. Universidad Nacional Autónoma de México, Serie Botánica 1991;; 61 21 - 42.
- 14 van der Sluis W G,, van der Nat J M,, Spek A L,, Ikeshiro Y,, Labedie R P.. Gentiogenal, a conversion product of gentiopicrin (gentiopicroside). Planta Medica. 1983;; 49 211-5
- 15 Rojas A,, Cruz S,, Rauch V,, Bye R,, Linares E,, Mata R.. Spasmolytic potential of some plants used in Mexican traditional medicine for the treatment of gastrointestinal disorders. Phytomedicine. 1995;; 2 51-5
- 16 Bailey N T..
Simple experimental design and the analysis of variance. In: Statistical Methods in Biology. Cambridge:; Cambridge University Press, 1995: 234 - 6
Dr. M. Alejandra Rojas
Molina
Departamento de Investigación Química y Farmacológica de Productos Naturales Facultad
de Química Universidad Autónoma de Querétaro
Cerro de las Campanas 76010
Querétaro
Mexico
Email: rojasa@sunserver.uaq.mx
Phone: +52 42 163730
References
- 1 Rojas A,, Bah M,, Rojas J I,, Serrano V,, Pacheco S.. Spasmolytic activity of some plants used by the Otomi Indians of Querétaro (México) for the treatment of gastrointestinal disorders. Phytomedicine. 1999;; 6 367-71
- 2 Mpondo E M,, Chulia A J.. 6′-O-β-D-Glucosylgentiopricroside: A new secoiridoid from Gentiana asclepiadea. Planta Medica. 1988;; 54 185-6
- 3 Ma W G,, Fuzzati N,, Wolfender J L,, Hostettman K,, Yang C R.. Rhodenthoside A, a new type of acylated secoiridoid glyocside from Gentiana rodhentha. . Helvetica Chimica Acta. 1994;; 77 1660-71
- 4 Tan R X,, Wolfender J L,, Ma W G,, Zhang L X,, Hostettmann K.. Secoiridoids and antifungal aromatic acids from Gentiana algida. Phytochemistry. 1996;; 41 111-6
- 5 Tan R X,, Wolfender J L,, Zhang L X,, Ma W G,, Fuzzati N,, Marston A,, Hostettmann K.. Acyl secoiridoids and antifungal constituents from Gentiana macrophylla. . Phytochemistry. 1996;; 42 1305-13
- 6 Tan R X,, Kong L D.. Secoiridoids from Gentiana siphonantha. . Phytochemistry. 1997;; 46 1035-8
- 7 Tan R X,, Kong L D,, Wei H X.. Secoiridoid glycosides and an antifungal anthranilate derivative from Gentiana tibetica. . Phytochemistry. 1998;; 47 1223-6
- 8 Wolfender J L,, Maillard M,, Hostettmann K.. Liquid chromatographic-thermospray mass spectrometric analysis of crude plant extracts containing phenolic and terpene glycosides. Journal of Chromatography. 1993;; 647 183-90
- 9 Ling C N,, Kuo S H,, Chung M I,, Ko F N,, Teng C M.. A new flavone C-glycoside and antiplatelet and vasorelaxing flavones from Gentiana arisanensis. . Journal of Natural Products. 1997;; 60 851-3
- 10 Ko F N,, Chu C C,, Lin C N,, Chang C C,, Teng C M.. Isoorientin-6″-O-glucoside, a water-soluble antioxidant isolated from Gentiana arisanensis. . Biochimica et Biophysica Acta. 1998;; 1389 81-90
- 11 Atkinson J E,, Gupta P,, Lewis J R.. Some phenolic constituents of Gentiana lutea. . Tetrahedron. 1969;; 24 1507-11
- 12 Marekov N L,, Popov S S.. The structure of gentioflavine, a new alkaloid of some Gentiana species. Tetrahedron. 1968;; 24 1323-6
- 13 Bye R,, Mata R,, Pimentel J.. Botany, ethnobotany and chemistry of Datura lanosa (Solanaceae) in Mexico. Anales del Instituto de Biología. Universidad Nacional Autónoma de México, Serie Botánica 1991;; 61 21 - 42.
- 14 van der Sluis W G,, van der Nat J M,, Spek A L,, Ikeshiro Y,, Labedie R P.. Gentiogenal, a conversion product of gentiopicrin (gentiopicroside). Planta Medica. 1983;; 49 211-5
- 15 Rojas A,, Cruz S,, Rauch V,, Bye R,, Linares E,, Mata R.. Spasmolytic potential of some plants used in Mexican traditional medicine for the treatment of gastrointestinal disorders. Phytomedicine. 1995;; 2 51-5
- 16 Bailey N T..
Simple experimental design and the analysis of variance. In: Statistical Methods in Biology. Cambridge:; Cambridge University Press, 1995: 234 - 6
Dr. M. Alejandra Rojas
Molina
Departamento de Investigación Química y Farmacológica de Productos Naturales Facultad
de Química Universidad Autónoma de Querétaro
Cerro de las Campanas 76010
Querétaro
Mexico
Email: rojasa@sunserver.uaq.mx
Phone: +52 42 163730


Fig. 1Concentration-response curves showing the relaxatory effects of the extract of Gentiana spathacea and gentiopicroside on isolated guinea-pig ileum. Values are expressed as the percentages of inhibition of contractile responses calculated as the mean from six data ± s.d., p < 0.05.

Fig. 2Effect of gentiopicroside on the contractions induced by BaCl2 (3 × 10-4 M), KCl (60 mM), acetylcholine (1 × 10-4 M) and histamine (1 × 10-3 M) on isolated guinea-pig ileum. p < 0.05 (ANOVA). Gentiopicroside was tested at its IC50 for the inhibition of spontaneous contractions of the ileum (2.80 μg/ml).