ACTIVITY OF ANTHRAQUINONES FROM Rubia cordifolia ... anthraquinones have been identified, in which munjistin and purpurin are predominant. The cell ...
Pharmaceutical Chemistry Journal
Vol. 41, No. 11, 2007
MEDICINAL PLANTS CHEMICAL COMPOSITION AND PHARMACOLOGICAL ACTIVITY OF ANTHRAQUINONES FROM Rubia cordifolia CELL CULTURE N. P. Mishchenko,1 S. A. Fedoreev,1 V. M. Bryukhanov,2 Ya. F. Zverev,2 V. V. Lampatov,2 O. V. Azarova,2 Yu. N. Shkryl’,3 and G. K. Chernoded3 Translated from Khimiko-Farmatsevticheskii Zhurnal, Vol. 41, No. 11, pp. 38 – 41, November, 2007. Original article submitted November 20, 2006.
The results of qualitative and quantitative determination of anthraquinones in Indian madder (Rubia cordifolia L.) cell culture are presented. According to the 1H and 13C NMR, UV, IR, and mass-spectroscopic data, seven anthraquinones have been identified, in which munjistin and purpurin are predominant. The cell culture preparation exhibits antiinflammatory activity, which is manifested by an antiexudative effect and antiproliferative action during the rapid development of a model edema. A decrease in the antioxidant state without significant suppression of enzymatic activity is demonstrated.
Anthraquinones are frequently encountered in plant species of the Rubiaceae family, the best known of which is common madder (Rubia tinctorium L.). The root and rhizome of this plant contain up to 36 anthraquinone derivatives [1]. Another promising source of anthraquinones among species of the Far-Eastern flora of this family is Indian madder (Rubia cordifolia L.). Extracts of R. cordifolia (RC) rhizome are known to loosen bladder stones and possess hepatoprotective properties [2]. The relatively low content of biologically active substances in RC stimulates the development of biotechnological methods for the commercial production of RC biomass capable of synthesizing considerable amounts of secondary metabolites. This study is devoted to determining the qualitative and quantitative composition of anthraquinones in RC cell culture and evaluating the pharmacological activity of a preparation based on these anthraquinones.
1 2 3
EXPERIMENTAL CHEMICAL PART The experiments were performed with preparations made of RC root calluses. The cell culture (RC-I strain) was obtained from rhizomes of wild-growing plants collected in the Southern part of the Maritime Territory. The cultures were grown on the WB/NAA nutrient medium containing 0.5 mg/liter of 6-benzyladenine and 2.0 mg/liter of a-naphthylacetic acid as described elsewhere [3]. The UV spectra of ethanol solutions were measured using an UVmini-1240 spectrophotometer (Shimadzu, Japan). The IR absorption spectra were recorded on a Vector 22 FT-IR (Bruker, Germany) spectrophotometer using deuterated chloroform and dichloromethane as solvents. The 1H and 13 C NMR spectra were obtained on a DPX-300 (Bruker, Germany) spectrometer operating at 300 MHz (1H nuclei) and 125 MHz (13C nuclei), using DMSO-d6 and deuterated chloroform as the solvents. The mass spectra were recorded using an AMD-604S high-resolution mass-spectrometer with an electron-impact ionization source operating at an electron beam energy of 70 eV. The melting points were determined using a Koefler heating stage (Leica VMTG, Austria). Anthraquinones were identified by HPLC using an Agilent Model 1100 chromatograph equipped with a tunable-wavelength VWD G1314A detector (Agilent, Germany) and a Hypersil BDS-C18 (5 mm) 250 ´ 4.6 mm column
Pacific Institute of Bioorganic Chemistry, Far-Eastern Division, Russian Academy of Sciences, Vladivostok, 690022 Russia. Altai State Medical University, Barnaul, Altai Territory, 656038 Russia. Institute of Biology and Soil Science, Far-Eastern Division, Russian Academy of Sciences, Vladivostok, 690022 Russia.
605 0091-150X/07/4111-0605 © 2007 Springer Science+Business Media, Inc.
606
N. P. Mishchenko et al.
thermostatted at 30°C and eluted at flow rate of 1 ml/min with A (1% acetic acid solution) and B (acetonitrile containing 1% of acetic acid) solvents in a linear gradient mode: 10 – 70% B for 25 min. Sample preparation: a 100-mg sample of callus (or a pharmaceutical preparation) was wetted with 0.6 ml of 2 N HCl, and mixed with 3.0 ml of C2H5OH (with periodic shaking) for 1 h, and filtered through 0.45 mm Neilon (Dacron) filter with a diameter of 13 mm (KURABO, Japan). A 10-ml aliquot was applied on the chromatograph column. The HPLC data were processed and analyzed using ChemStation program package (Agilent Technologies GmbH, Waldbronn, Germany). The total content of anthraquinones in pharmacological preparations and callus were determined spectrophotometrically [3]. Isolation of anthraquinones from RC callus cells. Dried and comminuted RC calluses (250 g) were wetted with 2 M ethanol solution of HCl (250 ml) and allowed to stand in air for 15 h. Then, the cellular mass was dried and extracted with hot MeOH for 8 h. The extract was concentrated in vacuum to a final volume or 200 ml and redistributed between water (2 liters) and chloroform (2 liters). The chloroform phase was separated, the solvent was removed, and the residue was partitioned by repeated gel-permeation column chromatography on Sephadex LH-120 (Pharmacia Fine Chemicals, Sweden) eluted with a chloroform – methanol (10 : 1) system. This procedure gave six colored fractions, which yielded (after removal of the solvents and crystallization of the residues) 38 mg of munjistin (II), 7 mg of alizarine (III), 3 mg of munjistin methyl ether (VI), 18 mg of purpurin (V), 5 mg of xanthopurpurin (IV), and 2 mg of ethylxanthopurpurin (VII).4 The aqueous phase was partially evaporated (to a final volume of about 300 ml) and partitioned by a low-pressure reverse-phase column chromatography on a Toyopearl HW-40 (TOSOH, Japan) TSK gel column eluted with an aqueous C2H5OH solution containing 0.6% HCOOH in a gradient concentration (10 – 50%) mode. This procedure gave 243 mg of ruberitrinic acid (I). The chemical structures of all isolated compounds were established on the basis of 1H and 13C NMR spectroscopy 4
Numbers of compounds correspond to their order of yield from HPLC column.
data (COSY, HSQC, and HMBC techniques) and confirmed by the results of UV, IR, and mass-spectrometric measurements and by comparison to published data for the known substances [4, 5]. It should be noted that compound VII was isolated in this study for the first time. Xanthopurpurin ethyl ether (VII) appeared in the form of whiskers of a lemon-yellow color, with m.p., 186 – 190°C; UV spectrum in C2H5OH [lmax, nm (lg e)]: 231 (4.3), 242 (4.4), 282 (4.3), 413 (3.9); IR spectrum in CDCl3 (nmax, cm – 1): 1674, 1633, 1608, 1594; 1 H NMR spectrum in CDCl3 (d, ppm): 1.48 (t, 3H, J 7 Hz, CH3), 4.18 (q, 2H, 7 Hz, CH2), 6.69 (d, 1H, 2.5 Hz, 2-H), 7.28 (d, 1H, 2.5 Hz, 4-H), 7.79 (m, 2H, 6-H, 7-H), 8.25 – 8.37 (m, 2H, 5-H, 8-H), 12.88 (s, OH); mass spectrum [m/z (Irel)]: 268 [M]+ (100), 212 (45), 184 (22). RC cell culture preparation for biological tests. The pharmacological investigation was performed using a preparation containing the entire complex of anthraquinones extracted from RC cells. For this purpose, 30-day-grown callus cells were dried, comminuted, and exhaustively extracted in a setup for continuous hot extraction with removal of a waxy residue. The cellular mass was dried, mixed with 2 N HCl solution in C2H5OH (50 ml/100 g cells), and allowed to stand overnight. Then, the cells were sequentially extracted with hot chloroform and ethanol. After removal of the solvent, the ethanol extract yielded a syrup-like residue, which was redistributed between water and ethyl acetate. The ethyl acetate layer was separated, washed with water until neutral reaction of the wash water, combined with the chloroform extract, and evaporated to dryness. The residue (3.0 g) was dissolved on heating in 100 g of ethanol. To this solution was added 50 g lactose, the solvent was evaporated, and the residual mixture was dried to constant weight. Thus, we obtained a purified complex of RC anthraquinones supported on lactose (in what follows RC preparation). EXPERIMENTAL BIOLOGICAL PART The pharmacological investigation was performed on a group of both male and female white mongrel rats weighing 200 – 250 g, which were kept in individual cages with free access to water and meals. The tests were carried out in ac-
TABLE 1. Retention Times of Anthraquinones on Hypersil BDS-C18 Column and Their Content in RC Calluses and in the Cell Culture Preparation Anthraquinone fraction I
II
III
IV
V
VI
VII
Total content of anthraquinones, %
9.60 7.4
11.11 60.3
15.44 4.8
17.18 1.2
17.40 20.2
19.50 0.3
21.22 0.2
2.14
3.5
62.4
5.5
0.8
22.5
0.4
0.3
0.76
Parameter
Retention time*, min Content of anthraquinones in RC callus, % Content of anthraquinones in RC preparation, %
* For HPLC conditions specified in the text.
Chemical Composition and Pharmacological Activity of Anthraquinones
cordance with the rules adopted by the European Convention for Protection of Animals. The anti-inflammatory activity of RC preparation was studied on conventional models of acute and chronic inflammation against the background of 14-day administration of RC anthraquinones in a dose of 100 mg/kg (p.o.) with a starch jelly. Animals in the control group received an equivalent amount of pure starch jelly for the same period of time. The acute inflammation was induced by subplantar injection (0.1 ml) of a 1% carrageenan solution. The edema volume growth was monitored by phethismometry for 4 h after phlogogen injection. In the chronic inflammation test, the process development was monitored by determining the dry and wet weight of the granuloma formed around a sterile 20-mg cotton ball implanted under the skin [6]. The course of the free-radical oxidation (FRO) process in vivo was studied using the model of inflammatory edema induced in the hind paws of rats by subplantar injections (0.2 ml) of a 3% formalin solution, which is accompanied by a pronounced oxidative stress. This model allows the induced FRO to be studied as an interaction between the prooxidant and antioxidant factors [7]. After termination of the treatment with RC anthraquinones, the oxidative stress was induced, and the blood characteristics of rats in the test group was determined 2 – 3 days after phlogogen injection and compared to the data for the intact and untreated control groups. The general prooxidant activity (GPA) and the total concentration index of all prooxidants and free-radical metabolites were evaluated by determining the content of lipid peroxidation (LPO) products interacting with thiobarbituric acid in the blood plasma. In addition, we also determined the concentration of malonic dialdehyde (MDA) and other thiobarbiturate-sensitive products (TBSPs) formed in the course of LPO. The antioxidant activity in the erythrocyte hemolysate was evaluated in terms of a change in the integral index of general antioxidant activity (GAA) and the content of particular antioxidant enzymes catalase (CAT, [1.11.1.6]), of superoxide dismutase (SOD, [1.15.1.1]), and glutathiuone peroxidase (GPO, [1.11.1.9]). The experimental data were statistically processed by the method of variational series and expressed in terms of the Student criterion. RESULTS AND DISCUSSION Previously, we reported [3] that the RC cell culture produces munjistin (II) and purpurin (V) in an amount reaching
TABLE 2. Effect of the RC Cell Culture Preparation (100 mg/kg) on the Cotton Granuloma Development in Rats Group
n
Wet weight, mg Dry weight, mg Difference, mg
Control
28
84.6 ± 1.77
14.8 ± 0.47
69.8 ± 2.49
RC preparation
32
62.4 ± 3.64*
10.8 ± 1.04*
51.6 ± 4.88
607
90% of the total content of anthraquinones. In the present study, we have isolated some other anthraquinone fractions (in addition to II and V) from a methanol extract of RC, including the compounds known to occur in this plant – ruberitrinic acid (I), alizarine (III), xanthopurpurin (IV), munjistin methyl ether (VI) – and the previously unreported ethoxy derivative of xanthopurpurin (VII). O 8 7 6
5
8a
10a
9
10
O
OH 9a
4a
R
1
2 3
4
1
R 2
R I VII
I: R = O-b-D-Xyl-(1-6)-b-D-Gl, R1 = R2 = H II: R = COOH, R1 = OH, R2 = H III: R = OH, R1 = R2 = H IV: R = H, R1 = OH, R2 = H V: R = R2 = OH, R1 = H VI: R = COOCH3, R1 = OH, R2 = H VII: R = R2 = H, R1 = OC2H5
The isolated compounds were used as reference samples in selecting the optimum conditions for a chromatographic analysis of producers in the RC cell culture. Several techniques for the detection of anthraquinones in R. tinctorum by means of reverse-phase HPLC have been reported so far [8 – 11]. These techniques are based on the detection of aglycons formed upon the hydrolysis of extracts, during which anthraquinone glycosides such as ruberitrinic acid and lucidine primeveroside form alizarine. In the present investigation, the method originally proposed by Kuzovkina, et al. [10] was modified so as to analyze simultaneously both anthraquinones and glycosides. Table 1 gives the retention times for the identified anthraquinones and presents data on their content in the RC calluses and combined pharmacological preparation. It should be noted that the content of the ethoxy derivative of xanthopurpurin (VII), which was isolated from the
TABLE 3. Effect of the RC Cell Culture Preparation (100 mg/kg) on the Prooxidant Activity and Antioxidant Protection System State in Blood Plasma of Rats upon Formalin Injection Group Parameter Intact
Control
Test
TBSP, mmole GPA, %
2.5 ± 0.18
4.2 ± 0.18*
3.4 ± 0.12*
45.1 ± 1.06
60.1 ± 1.25*
61.4 ± 1.27*
CAT, %
12.2 ± 1.27
22.4 ± 1.02*
18.3 ± 0.43*
SOD, % GPO, % GAA, %
16.9 ± 0.81 233.0 ± 7.3 73.7 ± 0.51
28.3 ± 1.05* 243.0 ± 11.2 87.8 ± 0.86*
27.1 ± 0.82* 195.3 ± 5.0* 41.6 ± 1.0*
* Reliable differences from the intact control group; underlined figures refer to reliable differences from the untreated control group.
608
N. P. Mishchenko et al.
Edema volume growth, cm3
1.2 1
1.0
3 0.8
2
0.6 0.4 0.2 0
1
2
Time, h
3
4
Fig. 1. Effect of the RC cell culture preparation (100 mg/kg) on the dynamics of model carrageenan-induced paw edema development in rats: (1 ) control group; (2 ) test group; (3 ) asterisks show the level of reliable changes ( p < 0.001) relative to the control.
RC samples for the first time, varies within 0.1 – 0.5% of the total amount of anthraquinones both in the combined preparation and in various samples of RC callus, which confirms the biosynthetic origin of this compound. Biological tests on the model of acute inflammatory reaction induced by carrageenan in rats revealed a pronounced antiphlogistic effect of the RC preparation upon a prolonged (two-week) preliminary administration (Fig. 1). The anti-inflammatory effect of RC preparation reached maximum already 1 h after carrageenan injection and somewhat decreased by the end of the test, while still being statistically reliable. The maximum decrease in the edema volume growth reached 38.5%, which allowed this result to be considered as a pronounced antiinflammatory effect. Taking into account the efficacy of treatment in the period of fast edema growth and the fact that the initial stage of carrageenan-induced edemation is related to the effect of phlogogen on the release of certain inflammation mediators, we may suggest that the preparation under consideration affects the inflammation start mechanisms and inhibits the release of inflammation mediators, probably, due to a membrane-stabilizing action [12]. The study of antiproliferative properties showed that the wet granuloma weight in rats treated with the RC preparation was significantly smaller as compared to that in the untreated control group (Table 2). Administration of the RC preparation also led to a significant decrease in the dry weight of granulomatous tissues, which was about 27% lower as compared to the control ( p < 0.001). Therefore, the proposed preparation substantially suppresses the development of both acute and chromic inflammation. The subplantar injection of formalin led to a sharp activation of LPO processes, which was manifested by an increase in the amount of TBSPs (by a factor of about 1.7) and in the GPA index (from 45.1 to 60.1%) as compared to the control ( p < 0.001). The increase in the prooxidant system
activity was accompanied by subsequent activation of the antioxidant system enzymes that led to an increase in the integral GAA index (Table 3). Against the background of accumulation of the free-radical species generated as a result of the formalin-induced oxidative stress, the presence of RC preparation led to a decrease in the formation of MDA and other TBSPs by a factor of about 1.2 relative to the control. At the sane time, the GPA index was not changed significantly as compared to that in the control group, which is indicative of the insignificant influence of the RC preparation on the GPA. Therefore, this preparation is apparently not directly involved in the neutralization of free-radical species. The oxidative stress induction in the control group led to activation of the antioxidant mechanisms, which was manifested as a significant increase in the activity of antioxidant system enzymes (CAT and SOD) and led to the corresponding growth in the GAA index (by more than 19%). The presence of the RC preparation somewhat suppressed (by a factor of about 1.2) the increased activity of GPO and CAT in rats with formalin-induced edema, while virtually not influencing the SOD activity. At the same time, the RC preparation led to a significant decrease in the GAA index as compared to that in the intact and untreated control animals (by 52.6 and 43.6%, respectively). The results of experiments modeling the conditions of enhanced free-radical oxidation showed a predominant effect of the RC preparation on the antioxidant status. A substantial decrease in this status in the absence of significant suppression of the enzymatic activity suggests that nonenzymatic mechanisms of the antioxidant protection are probably operative. These mechanisms are related to the presence of water-soluble compounds (iron ascorbate, glutathione, uric acid, etc.) and fat-soluble components of cell membranes (a-tocopherol, retinol, etc.) in the blood plasma, which are activated under the action of the RC preparation. In conclusion, seven anthraquinones have been identified in a preparation obtained from RC cell culture, which account for about 95% of the total content of anthraquinones in this plant material. The RC preparation possesses pronounced anti-inflammatory properties, which are probably related to suppression of the general antioxidant activity. ACKNOWLEDGMENTS This study was supported in part by the Russian Foundation for Basic Research (project No. 06-04-48068) and the Presidium of the Russian Academy of Sciences (“Molecular and Cell Biology” Program). REFERENCES 1. G. C. H. Derksen and T. A. van Beek, in: Studies in Natural Product Chemistry, Rubia tinctorum L., Atta-ur-Rahman (ed.), Elsevier, Amsterdam (2002), p. 629.
Chemical Composition and Pharmacological Activity of Anthraquinones
2. A. H. Gilani and K. H. Janbaz, Phytother. Res., 9, 372 – 375 (1995). 3. N. P. Mischenko, S. A. Fedoreyev, V. P. Glazunov, et al., Fitoterapia, 70, 552 – 557 (1999). 4. H. Itokawa, K. Mihara, and K. Takeya, Chem. Pharm. Bull., 31(7), 2353 – 2358 (1983). 5. R. A. Muzychkina, Natural Anthraquinones. Biological Properties and Physicochemicals Characteristics, G. A. Tolstikov (ed.), PHASIS, Moscow (1998). 6. A Practical Guide on the Experimental (Preclinical) Investigation of New Pharmaceuticals [in Russian], Pharmacological Committee of the USSR Ministry of Public Health, Moscow (2000),
609
7. V. M. Bryukhanov, Ya. F. Zverev, Ya. S. Arbuzov, et al., Ros. Fiziol. Zh. im. I. M. Sechenova, 90(8), 35 – 36 (2004). 8. K. Sato, T. Yamazaki, E. Okuyama, et al., Phytochemistry, 30(5), 1507 – 1509 (1991). 9. G. C. H. Derksen, T. A. van Beek, A. Groot, and A. Capelle, J. Chromatogr., 816, 277 – 281 (1998). 10. I. N. Kuzovkina, O. V. Mantrova, I. E. Al’terman, et al., Fiziol. Rast., 43(2), 291 – 298 (1996). 11. P. Banyai, I. N. Kuzovkina, L. Kursinszki, and E. Szoke, Chromatographia, 63, (Supp. 13), S111 – S114 (2006). 12. G. Ya. Shvarts and R. D. Syubaev, Farmakol. Toksikol., 45(1), 46 – 49 (1982).
