DOI 10.1007/s11094-017-1618-z Pharmaceutical Chemistry Journal, Vol. 51, No. 5, August, 2017 (Russian Original Vol. 51, No. 5, May, 2017)
MEDICINAL PLANTS IMMUNOMODULATING ACTIVITY OF EXTRACT OF GENTIANA ALGIDA PALL. V. B. Khobrakova,1,3 E. R. Budaeva,1 D. N. Olennikov,1 and I. N. Zilfikarov2 Translated from Khimiko-Farmatsevticheskii Zhurnal, Vol. 51, No. 6, pp. 40 – 43, June, 2017.
Original article submitted April 30, 2015. Dry extract of Gentiana algida Pall. displayed immunomodulating activity in experiments on mouse line F1 (CBA ´ C57B1/6) with experimental azathioprine-induced immunosuppression of the cellular, humoral, and macrophage components of the immune response. It was found that the plant extract reduced the suppressive effect of the cytostatic azathioprine on the antibody response, cell-mediated immune reaction, and macrophage phagocytosis. This resulted in increases as compared with the suppressed levels of the absolute and relative amounts of antibody-forming cells, index of delayed-type hypersensitivity, phagocytic index, and phagocyte number. Dry extract of G. algida was characterized chemically by high contents of iridoids (82.07 mg/g), flavonoids (86.91 mg/g), and triterpenes (23.98 mg/g), which caused the observed immunomodulating activity. Keywords: Gentiana algida Pall., immunomodulating activity, iridoids, flavonoids, triterpenes, azathioprine, immunosuppression.
The number of human diseases caused by a suppressed immune response, among other factors, is currently growing. Therefore, the search for new efficacious immunomodulators affecting only altered segments of the immune system is critical. Many researchers focused on the immunotropic properties of plant-based preparations. Plant immunomodulating preparations are a complex of biologically active compounds in their natural combinations that are efficacious in the human body [1 – 3]. Dry extract from the aerial part of Gentiana algida Pall. [Pneumonanthe algida (Pall.) F. W. Schmidt; whitish gentian, Gentianaceae] was used in the present study. This medicinal plant is widely used in traditional Tibetan medicine to treat intoxications and for therapy of contagious diseases and lung inflammations [4]. Chemical investigations of G. algida 1
2 3
herb found flavonoids (isoorientin, isoorientin-4¢-O-glucoside [5], orientin [6]), aromatic acids (anofinic, fomannoxic) [6], iridoids (gentiopicroside [5], sweroside, trifloroside, rindoside, amplexin-1-O-Glc, 2¢-O-2,3-dihydroxybenzoylsweroside [6], 6¢-O-2,3-dihydroxybenzoylsweroside, 6¢-O-2,3-dihydroxybenzoylswertiamarin [7]), triterpenes (oleanolic acid), and sterols (sitosterol, daucosterol, stigmasterol) [6]. Biological activities of the total preparations and pure compounds from G. algida showed antibacterial [8], hemostatic [9], antitrichomonal [10], antifungal [6], and anti-inflammatory activity [11]. The high efficacy of dry extract of G. algida (EGA) for inflammatory diseases, in the pathogenesis of which immune-system disturbances play a leading role, suggested that the extract had immunomodulating properties. The goal of the present research was to determine the immunomodulating activity of dry EGA on the parameters of cellular, humoral, and macrophagic components of the immune response under experimental suppression.
Institute of General and Experimental Biology, Siberian Branch, Russian Academy of Sciences, Ulan-Ude, Buryat Republic, 670047 Russia; e-mail: evd
[email protected]. Scientific Center of Biomedical Technologies, State Research Institute of Medicinal and Aromatic Plants (VILAR), Moscow, 117216 Russia. Buryat State University, Ulan-Ude, Buryat Republic, 670000 Russia.
384 0091-150X/17/5105-0384 © 2017 Springer Science+Business Media New York
Immunomodulating Activity of Extract of Gentiana Algida Pall.
Preliminary research investigated the chemical composition of EGA using several chromatographic methods to identify seven dominant compounds belonging to the classes triterpene (oleanolic acid), iridoids (gentiopicroside, sweroside, loganic acid), and flavonoids (orientin, isoorientin, isoorientin-4¢-O-glucoside). Loganic acid was observed for the first time in this plant species according to the literature on the constituents of G. algida. The contents of isolated compounds were quantified using MC-HPLC-UV (flavonoids, iridoids) and HPTLC-densitometry (oleanolic acid). Figure 1 shows a chromatogram (MC-HPLC-UV) of EGA. The studies found that the dominant compounds in EGA were gentiopicroside (75.50 mg/g), orientin (65.23 mg/g), and oleanolic acid (23.98 mg/g). Their total concentration was 16% of the extract mass (Table 1). The total contents of iridoids, flavonoids, and triterpenes in EGA were 82.07, 86.91, and 23.98 mg/g, respectively. EXPERIMENTAL CHEMICAL PART Spectrophotometric studies used an SF-2000 spectrophotometer (Specter, Russia). Mass spectral analysis used an MAT 8200 high-resolution mass spectrometer (Finnigan). IR spectra were recorded from KBr pellets (1:100) on an FT-801 FTIR spectrometer (Simex, Russia). NMR spectra were taken on a VXR 500S NMR spectrometer (Varian). MeCN (NPK Kriokhrom, Russia, type 0), LiClO4, HClO4, H2O for HPLC CHROMASOLV® (Sigma, Switzerland); loganic acid, gentiopicroside, sweroside, orientin, isoorientin (Extrasynthese, France); and isoorientin-4¢-O-glucoside isolated earlier from Anagallidium dichotomum [12] were used in the work. Other reagents were analytically pure. Column chromatography used silica gel, reversed-phase sil-
TABLE 1. Contents of Pure Compounds in Extract of G. algida Content, mg/g
Compound
Total content, mg/g
Iridoids Loganic acid Gentiopicroside Sweroside
385 5
2 3 4
1 0
2
4
6
6 8
10
12
min
Fig. 1. Chromatogram (MC-HPLC-UV) of extract of G. algida at 254 nm: 1 ) loganic acid; 2 ) isoorientin-4¢-O-glucoside; 3 ) gentiopicroside; 4 ) sweroside; 5 ) orientin; 6 ) isoorientin.
ica gel (Sigma, Switzerland), Sephadex LH-20 (Pharmacia, Sweden), and polyamide (Woelm, Germany). Extract preparation. Air-dried raw material (aerial part) of G. algida was acquired at ZAO Etnoshop (Irkutsk, Russia). Dry extract was produced by extracting (2´) milled raw material with EtOH (60%) in a raw-material—extractant ratio of 1:15 on a water bath (50°C) with constant stirring for 60 min. The combined extracts were concentrated in vacuo (45°C) to an aqueous residue that was lyophilized. The yield of EGA was 37-38% of the raw-material mass. Fractionation of EGA. EGA (100 g) was suspended in distilled H2O (500 mL) and worked up sequentially with CHCl3, EtOAc, and BuOH. The organic extracts were concentrated in vacuo until the solvents were completely removed to produce CHCl3 (9.5 mass% of the air-dried raw material), EtOAc (18.2), and BuOH fractions (36.9). Then, the fractions were chromatographed over a column of reversed-phase silica gel (3 ´ 100 cm, H2O—MeCN, 100:0 ® 0:100). The resulting subfractions were rechromatographed over polyamide (2 ´ 60 cm, H2O—EtOH, 100:0 ® 96:4), silica gel (3 ´ 50 cm, C6H14—EtOAc, 100:0 ® 70:30; EtOAc—EtOH, 100:0 ® 70:30), Sephadex LH-20 (3 ´ 50 cm, EtOH—H2O, 96:4 ® 0:100). As a result, seven compounds were isolated and identified using UV, IR, NMR, and mass spectroscopy as oleanolic acid (1.54 g) [13], loganic acid (12 mg), gentiopicroside (264 mg), sweroside (24 mg) [14], orientin (3.47 g), isoorientin (252 mg), and isoorientin-4¢-O-glucoside (728 mg) [15].
5.63 ± 0.12 75.50 ± 1.59
82.07
TABLE 2. Effect of Extract of G. algida (EGA) on Strength of DTH
0.94 ± 0.03 Flavonoids
Orientin
65.23 ± 1.37
Isoorientin
8.89 ± 0.17
Isoorientin-4¢-O-glucoside
Animal group
86.91
12.79 ± 0.21 Triterpenes
Oleanolic acid
23.98 ± 0.59
Total contents of identified constituents
23.98 192.96
RI DTH, %
Intact, n = 10
32.42 ± 3.18
Control (azathioprine), n = 10
19.77 ± 1.40
Test (azathioprine + EGA), n = 10
30.56 ± 1.18*
Here and henceforth: *differences statistically significant for p £ 0.05 vs. the control, each group included 10 animals (n = 10).
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V. B. Khobrakova et al.
TABLE 3. Effect of Extract of G. algida (EGA) on Antibody Formation Animal group
Intact, n = 10 Control (azathioprine), n = 10 Test (azathioprine + EGA), n = 10
Absolution number Number of AFCs of AFCs in spleen per 106 splenocytes
66591 ± 4997 40717 ± 3387 67125 ± 4845*
166.1 ± 12.9 99.8 ± 8.8 185.7 ± 14.5*
Quantitative analysis of flavonoids and iridoids used microcolumn HPLC-UV (MC-HPLC-UV) on a Milichrom A-02 liquid chromatograph (ZAO EcoNova, Russia) equipped with a two-phase gradient pump, autosampler, spectrophotometric detector, and thermostatted steel column (2 ´ 75 mm) packed with ProntoSIL-120-5-C18 absorbent (Metrohm AG, Switzerland; Æ5 mm). The mobile phase was a gradient of LiClO4 (0.2 M) in HClO4 (0.006 M) (eluent A) and MeCN (eluent B) covering 5 – 80% of eluent B in 0 – 26 min. The column temperature was 35°C, mobilephase flow rate 150 mL/min, detector wavelength 254 nm. The injected sample volume was 1 mL. Contents of pure constituents were calculated from calibration curves constructed using standard samples of the compounds of purity ³ 95%. Quantitative analysis of oleanolic acid used HPTLCdensitometry as described earlier [16]. EXPERIMENTAL BIOLOGICAL PART Male mice of line F1 (CBA ´ C57B1/6) (18 – 20 g) were used in the experiments. The effects of EGA on parameters of cellular, humoral, and macrophagic components of immunity were studied using animals that were immunosuppressed by oral administration of azathioprine (Semashko OAO Moskhimfarmpreparaty, tablets) at a dose of 50 mg/kg once per day for 5 d. EGA was administered orally to immunosuppressed animals in the test group at an experimental-therapeutic dose of 200 mg/kg once per day for 14 d. Intact and control animals received purified H2O according to an analogous regime. The effect of EGA on the cellular component of the immune response was assessed from delayed-type hypersensitivity (DTH) reaction according to the standard local DTH method [17]. The condition of humoral immunity was evaluated from the number of antibody-forming cells (AFCs) as determined by the local hemolysis method [18]. The state of the macrophagic component of the immune response was determined by phagocytosis by peritoneal macrophages of Staphylococcus aureus using the Freidlin method and evaluating the phagocytic index and phagocyte number [19]. Results were processed statistically using standard statistical variance methods and the parametric Student t-criterion [20].
TABLE 4. Effect of Extract of G. algida (EGA) on Phagocytic Activity of Peritoneal Macrophages Phagocytic index, %
Phagocytic number
Intact, n = 10
81.8 ± 2.4
7.8 ± 0.6
Control (azathioprine), n = 10
42.4 ± 1.5
3.9 ± 0.3
Test (azathioprine) + EGA, n = 10
85.8 ± 4.8*
6.6 ± 0.5*
Animal group
RESULTS AND DISCUSSION EGA at a dose of 200 mg/kg affected the cell-mediated DTH reaction by restoring its index with azathioprine-induced immunosuppression. This parameter increased by 1.6 times as compared with the control (Table 2). Antibody-formation processes were affected by EGA. The drug restored the indices of the humoral immune response with azathioprine-induced immunosuppression. The absolute number of antibody-forming cells (AFCs) increased statistically significantly if EGA was administered to immunosuppressed animals, i.e., by 1.7 and 1.9 times, respectively, calculated per 106 splenocytes (Table 3). EGA affected the phagocytic activity of mouse peritoneal macrophages against Staphylococcus aureus by increasing the phagocytic index and phagocyte number by 2.0 and 1.7 times, respectively, as compared with the control data (Table 4). The results led to the conclusion that EGA was capable of weakening the suppressant activity of azathioprine on the parameters of the cellular, humoral, and macrophagic components of the immune response. Apparently, EGA is efficacious because it contains flavonoids (orientin, isoorientin, isoorientin-4¢-O-glucoside) [21], triterpenes (oleanolic acid) [22, 23], and iridoids (loganic acid, gentiopicroside, sweroside) [24], which possess immunomodulating properties. Thus, EGA is an efficacious immunomodulating agent that can be recommended for further studies intended to create novel immunomodulating plant preparations. REFERENCES 1. D. N. Lazareva, V. V. Plechev, T. V. Morugova, and L. I. Samigullina, Immunostimulatory Plants [in Russian], Ufa, Bashkortostan (2005). 2. S. N. Lebedeva and M. A. Khrebtovskii, Sib. Med. Zh., 63, 69 – 72 (2006). 3. A. A. Churin, N. V. Masnaya, E. Yu. Sherstoboev, and I. V. Shilova, Eksp. Klin. Farmakol., 71(5), 32 – 36 (2008). 4. S. M. Batorova, G. P. Yakovlev, and T. A. Aseeva, Guide to Drugs of Traditional Tibetan Medicine [in Russian], Nauka, Novosibirsk (2013).
Immunomodulating Activity of Extract of Gentiana Algida Pall.
5. M. Hostettmann-Kaldas, K. Hostettmann, and O. Sticher, Phytochemistry, 20, 443 – 446 (1981). 6. R. X. Tan, J.-L. Wolfender, W. G. Ma, et al., Phytochemistry, 41, 111 – 116 (1996). 7. R. X. Tan, J. Hu, L. D. Kong, et al., Planta Med., 63, 567 – 569 (1997). 8. T. V. Kornopol’tseva, Zh. Ts. Khotsaev, T. A. Aseeva, and L. M. Tankhaeva, Sib. Med. Zh., 73, 82 – 85 (2007). 9. E. S. Shishkina, Yu. P. Nikitin, K. A. Sobolevskaya, et al., Natural and Synthetic Drug Research [in Russian], Tomsk (1975). 10. M. A. Rubinchik, Trichomonostatic Properties of Higher Plants [in Russian], Fitontsidy, Kiev (1972). 11. Zh. Ts. Khotsaev, Development and Implementation of New Methods and Drugs of Traditional Medicine [in Russian], Moscow (2001). 12. D. N. Olennikov, Chem. Nat. Compd., 49, 1137 – 1139 (2013). 13. D. N. Olennikov and L. M. Tankhaeva, Chem. Nat. Compd., 41, 600 – 601 (2005). 14. L. J. El-Naggar and J. L. Beal, J. Nat. Prod., 43, 649 – 707 (1980). 15. C. A. Williams and B. G. Murray, Phytochemistry, 11, 2507 (1972).
387
16. D. N. Olennikov, L. M. Tankhaeva, V. V. Partilkhaev, and A. V. Rokhin, Braz. J. Pharmacogn., 22, 490 – 496 (2012). 17. A. N. Mironov (ed.), Handbook for Preclinical Drug Trials [in Russian], Part 1, Ministry of Health and Social Development of the RF, FGBU Scientific Center for Expert Evaluation of Medicinal Products, Moscow (2012). 18. A. J. Cunningham, Nature, 207, 1106 – 1107 (1965). 19. I. S. Freidlin, Use of Murine Peritoneal Macrophage Culture as a Model for Studying in vivo Cellular Mononuclear Phagocytic Systems and Their Changes Under the Influence of Biologically Active Substances: Methodical Recommendations [in Russian], Leningrad (1976). 20. G. F. Lakin, Biotherapy [in Russian], Vysshaya Shkola, Moscow (1990). 21. S. Sahreen, M. R. Khan, R. A. Khan, and N. A. Shah, BMC Complementary Altern. Med., 13, 372 (2013). 22. C. Sanchez-Quesada, A. Lopez-Biedma, and J. J. Gaforio, J. Evidence-Based Complementary Altern. Med., Art. ID 654721 (2015). 23. P. Bhandari, N. K. Patel, R. P. Gangwal, et al., Bioorg. Med. Chem. Lett., 24, 4114 – 4119 (2014). 24. E. L. Ghisalberti, Phytomedicine, 5, 147 – 163 (1998)
