Experimental immunology
The in vivo effect of Rhodiola quadrifida extracts on the antibody production, on the blood leukocytes subpopulations and on the bacterial infection in mice

Introduction

The genus Rhodiola (Crassulaceae) consists of many species. These plants originate from arctic regions of Asia, Europe and North America, and are used in traditional medicine of many countries, mainly as adaptogens, anti-depressants, and anti-inflammatory drugs. The most known is Rhodiola rosea. Extracts of Rhodiola rosea are present on the European market as dietary supplements. Our investigations on the immunotropic effects of extracts obtained from three Rhodiola species – rosea, quadrifida and kirilowii, began more than fifteen years ago [1, 2]. Since this time, we published the results of many in vitro and in vivo experimental studies on the immunotropic and anti-angiogenic effects of extracts prepared from underground parts of Rhodiola rosea, Rhodiola quadrifida and Rhodiola kirilowii. We presented for the first time stimulatory effect of extracts prepared from these three Rhodiola species on some parameters of specific and non-specific cellular immunity in mice, rats and pigs [3-13]. We also reported anti-angiogenic activity of these extracts on tumor-induced cutaneous angiogenesis [14-17]. The aim of the present study was to investigate the influence of feeding mice for 7 days R. quadrifida aqueous (RQW) or 50% hydro-alcoholic (RQA) extracts on the subsequently induced bacterial infection (Pseudomonas aeruginosa) in mice, on the leukocyte blood subpopulations in their blood and on the anti-sheep red blood cells (SRBC) antibody production. Pseudomonas aeruginosa is one of the most dangerous pathogens for patients during cancer therapy, subjected to immunosuppressive treatment, or people immunocompromised from the other reasons.

Material and methods

Preparation and analysis of extracts



Rhizomes of R. quadrifida were collected in Altai mountain in Mongolia, thanks to Dr H. Wiedenfeld. The Mongolian plant material was identified; voucher specimen was deposited at the herbarium of the Institute of Botany of Mongolian Academy of Science in Ulaanbaatar. Sample extractions were performed as described before [3]. Briefly, aqueous extracts: finely powdered roots were extracted with water two times in the temperature 40-45°C. The supernatants were combined and after centrifugation at 3000 rpm for 15 min, were lyophilized. Hydro alcoholic extracts: finely powdered roots were extracted with ethanol/water solution (1/1, V/V) by the percolation method. The percolates were lyophilized which was preceded by the distilling off the ethanol in the temperature 40-45°C. Extracts were dissolved in 10% ethyl alcohol before administration to the animals.

High-performance liquid chromatography (HPLC) analysis was performed (with the samples diluted with methanol) on Agilent 1100 HPLC system, equipped with photodiode array detector. For all separations a Lichrospher 100 RP18 column (250.0 mm × 4.0 mm, 5 µm) from Merck was used. The mobile phase consisted of 0.05% phosphoric acid in water (A) and acetonitrile (B), applied in the following gradient elution: from 95A/5B to 80A/20B for 30 min then from 80A/20B to 20A/80B for 5 min and an isocratic elution for 15 min. Each run was followed by an equilibration period of 10 min. The flow rate was adjusted to 1 ml/min, the detection wavelength set to DAD at  = 205 nm, 220 nm, 254 nm, 330 nm and 20 µl of samples was injected. All separations were performed at a temperature of 25°C. Peaks were identified by spiking the samples with standard compounds and comparison of the UV-spectra and retention times.



Animal



Studies were performed on B6C3F1 hybrid mice, males, at the age of 7-9 weeks, 22-27 γ of body mass, delivered from own breeding colony (leukocytes and bacterial infection study) and on 7-9 weeks old female Balb/c mice, 20-25 γ of body mass, delivered from the Polish Academy of Sciences breeding colony (antibody study).



Study of antibody production



Mice were fed Rhodiola extract or 10% alcohol (controls) for 7 days, before intraperitoneal injection of 0.2 ml 10% SRBC suspension. Animals received daily 40 or 200 µg of the extract (feeding with use of Eppendorf pipette). These doses corresponded to about 20 and 100 mg given to 70 kg person (applying the coefficient equal 7 for adjusting differences between mouse and human in relation of the surface to body mass). Mice received drugs by Eppendorf pipette, in 0.04 ml of 10% ethyl alcohol. Control mice were fed 0.04 ml of 10% ethyl alcohol. Each experimental or control group consisted of ten animals. Mice were bled in anesthesia from retro-orbital plexus 7 days after immunization.

The antibody level was evaluated with haemagglutination assay in heat inactivated (56°C, 30 min) sera. After performing a series of sera dilutions, 0.5% SRBC were added and the mixture was incubated for 60 min at room temperature, then centrifuged (10’, 150 g) and shaken. The hemagglutination titer was evaluated in a light microscope – as the last dilution in which at least 3 cell conglomerates were present in at least 3 consecutive fields at objective magnification 20×.



Leukocyte subpopulations



Rhodiola quadrifida extracts were administered to the groups of 8 mice each, per os, in daily doses of 50 or 400 µg. These doses corresponded to about 25 or 200 mg given to 70 kg person. Mice were bled in anesthesia from retro-orbital plexus 7 days after immunization and sacrificed with Morbital. Counting of leukocytes and blood smears examination were performed by routine methods. The results are presented as inhibitory or stimulatory indices, calculated by dividing number of cells (lymphocytes or granulocytes) in 1 ml of blood of each experimental animal, by mean number of cells/ml of blood of animals belonging to the corresponding control groups.



Bacterial infection



Mice were fed R. quadrifida extracts 400 µg daily, in 10% ethyl alcohol, or 0.04 ml of 10% alcohol as a control, for 7 days. On the day eighth mice were infected intraperitoneally (i.p.) with Pseudomonas aeruginosa strain ATCC (27853). Four hours after administration of 0.1 ml of bacteria suspension (3 × 107 CFU) the mice were anaesthetized with barbiturates and killed by spinal dislocation after which the livers were isolated. The livers were homogenized and the number of viable bacteria were estimated by plating after 24 hours growth on Cetrymide agar (Merck) [8].

All experiments were approved by Local Ethical Committee.



Statistical evaluation of the results



The results were verified statistically (GraphPad Prism software package) by two-way ANOVA and Bonferroni post-test (leukocytes and antibody production) and by one-way ANOVA and Dunnett’s multiple comparison test (bacterial infection).

Results

The results of the estimation of blood leukocytes number are presented on the Fig. 1. Both lymphocytes and granulocytes numbers in the blood collected from mice fed R. quadrifida water extract were significantly lower than in the respective controls. In the case of R. quadrifida hydro-alcoholic extract, however, no such differences were observed. Moreover, mice fed higher RQA dose presented more lymphocytes in their blood than their corresponding controls.

The results of the experiments with bacterial infection are presented on the Fig. 2. RQA extract significantly diminished number of bacteria in livers of infected mice. Mice fed aqueous RQW extract presented no differences from mice belonging to the control group.

There were no effect of both extracts on anti-SRBC antibody production in lower 0.04 mg dose, in higher dose (0.2 mg) aqueous (RQW) extract diminished anti-SRBC antibody response (p < 0.01).

Discussion

In the previous studies, performed in vitro, we observed enhancement of intracellular respiratory burst and potential bactericidal activity in rat blood leukocyte cultures in the presence of R. rosea, R. kirilowii and R. quadrifida extracts [3, 5, 9, 10]. We also reported inhibitory effect of R. rosea extracts on bacterial infection in mice [8]. In the present paper we confirmed this last effect for the hydro-alcoholic extract obtained from R. quadrifida. Aqueous extract from R. quadrifida, however, have not presented inhibitory activity. The reason for this difference is not clear. One might hypothesize that it is connected with different pattern of leukocytosis in mice pretreated with these two types of R. quadrifida extracts. In mice fed hydro-alcoholic extract number of blood leukocytes significantly increased, in mice fed aqueous extract number of blood leukocytes significantly decreased (both lymphocytes and granulocytes). Accordingly, antibody production was diminished in mice fed 0.2 mg of aqueous extract. We have not found reports on the influence of R. quadrifida on humoral immunity. In our previous study on the influence of R. rosea roots on the antibody production we obtained stimulation of this parameter of humoral immunity [1]. Also Guan et al. and Mishra et al. reported adjuvant effects of extracts and compounds isolated from R. rosea and Rhodiola imbricata [18, 19]. It remains to elucidate, whether the lack of anti-bacterial effect presented by water extract of R. quadrifida might be connected with the presence of factors impairing lympho- and granulopoiesis, or factors toxic for mature leukocytes. Previous in vitro study performed on rats and pigs blood leukocytes did not reveal cytotoxic effects up to the 1000 µg/ml concentration. However, concentrations higher than 10 µg/ml diminished respiratory burst and potential killing activity, but without difference between both types of extracts [4, 11]. In other in vivo study, we observed again stimulatory effect of R. quadrifida hydro-alcoholic extract on cellular immunity in mice (graft-versus-host reaction) and no effect of R. quadrifida aqueous extract on this parameter [5]. Mielcarek et al. [20] performed phytochemical investigation of Rhodiola extracts (prepared from underground parts of R. rosea and R. quadrifida) using high performance thin layer chromatography (HPTLC), HPLC and spectrophotometric methods. Substantial differences were found in the qualitative and quantitative composition of the extracts. Generally, hydro-alcoholic extracts contained more glycosides and phenolic acids than aqueous extracts, with one exception – the highest concentration of gallic acid was observed in aqueous extract of R. quadrifida.

There are some papers about direct in vitro anti-viral and anti-bacterial activity of Rhodiola extracts and gallic acid [21-24]. We could not exclude such possibility, but we rather think that in our present in vivo study, antibacterial effect of Rhodiola hydro-alcoholic extract was indirect, mediated by activated leukocytes migrating to the peritoneal cavity from the blood. Lack of effect of aqueous extract may be connected with high content of gallic acid and its negative influence on the level and activity of white blood cells (mononuclears and granulocytes). It was reported, that gallic acid inhibits cell viability and induces apoptosis in human monocytic cell line U937 [25], and induces neuronal cell death through activation of c-Jun N-terminal kinase and downregulation of Bcl-2 [26]. Gallic acid derivative, propyl gallate, inhibits the growth of endothelial cells via caspase-independent apoptosis [27]. It was also reported that gallic acid inhibits the growth of HeLa cervical cancer cells via apoptosis and/or necrosis [28]. Derivatives of gallic acid induce apoptosis in tumoral cell lines and inhibit lymphocyte proliferation [29].

References

 1. Furmanowa M, Olędzka H, Michalska M, et al. (1995): Rhodiola rosea L. in vitro regeneration and the biological activity of roots. In: Biotechnology in Agriculture and Forestry 33: Medicinal and Aromatic Plants VIII. Bajaj YP (ed.). Springer-Verlag, Berlin, Heidelberg; 412-426.

 2. Furmanowa M, Skopińska-Różewska E, Rogala E, Hartwich M (1998): Rhodiola rosea in vitro culture phytochemical analysis and antioxidant action. Acta Societat Botan Pol 67: 69-73.

 3. Siwicki AK, Skopińska-Różewska E, Hartwich M, et al. (2007): The influence of Rhodiola rosea extracts on non-specific and specific cellular immunity in pigs, rats and mice. Centr Eur J Immunol 32: 84-91.

 4. Wójcik R, Siwicki AK, Skopińska-Różewska E, et al. (2008): The in vitro influence of Rhodiola quadrifida extracts on non-specific cellular immunity in pigs. Centr Eur J Immunol 33: 193-196.

 5. Skopińska-Różewska E, Wójcik R, Siwicki AK, et al. (2008): The effect of Rhodiola quadrifida extracts on cellular immunity in mice and rats. Pol J Vet Sci 11: 105-111.

 6. Skopińska-Różewska E, Bychawska M, Sommer E, Siwicki AK (2008): The in vivo effect of Rhodiola quadrifida extracts on the metabolic activity of blood granulocytes in mice. Centr Eur J Immunol 33: 179-181.

 7. Wójcik R, Siwicki AK, Skopińska-Różewska E, et al. (2009): The in vitro effect of R. quadrifida and R. kirilowii extracts on pigs blood lymphocyte response to mitogen ConA. Centr Eur J Immunol 34: 166-170.

 8. Bany J, Zdanowska D, Skopińska-Różewska E, et al. (2009): The effect of Rhodiola rosea extracts on the bacterial infection in mice. Centr Eur J Immunol 34: 35-37.

 9. Wójcik R, Siwicki AK, Skopińska-Rózewska E, et al. (2009): The effect of Chinese medicinal herb Rhodiola kirilowii extracts on cellular immunity in mice and rats. Pol J Vet Sci 12: 399-405.

10. Skopińska-Różewska E, Bychawska M, Białas-Chromiec B, Sommer E (2009): The in vivo effect of R. rosea and R. quadrifida hydro-alcoholic extracts on chemokinetic activity of spleen lymphocytes in mice. Centr Eur J Immunol 34: 42-45.

11. Wójcik R, Siwicki AK, Skopińska-Różewska E, et al. (2009): The in vitro influence of R. kirilowii extracts on blood granulocytes potential killing activity (PKA) in pigs. Centr Eur J Immunol 34: 158-161.

12. Skopińska-Różewska E, Bychawska M, Białas-Chromiec B, et al. (2010): The in vivo effect of Rhodiola kirilowii extracts on blood granulocytes metabolic activity in mice. Centr Eur J Immunol 35: 20-24.

13. Skopińska-Różewska E, Sokolnicka I, Siwicki AK, et al. (2011): Dose-dependent in vivo effect of Rhodiola and Echinacea on the mitogen-induced lymphocyte proliferation in mice. Pol J Vet Sci 14: 265-272.

14. Skopińska-Różewska E, Mścisz A, Sommer E, et al. (2005): The influence of different Rhodiola extracts on murine tumor cells angiogenic activity. Herba Polonica 51: 172.

15. Skopińska-Różewska E, Malinowski M, Wasiutyński A, et al. (2008): The influence of R. quadrifida 50% hydro-alcoholic extract and salidroside on tumor-induced angiogenesis in mice. Pol J Vet Sci 11: 97-104.

16. Skopińska-Różewska E, Wasiutyński A, Sommer E, et al. (2008): The influence of Rhodiola rosea, R. kirilowii, and R. quadrifida extracts on cutaneous angiogenesis induced in mice after grafting of human kidney cancer tissue. Centr Eur J Immunol 33: 185-189.

17. Skopińska-Różewska E, Hartwich M, Siwicki AK, et al. (2008): The influence of R. rosea extracts and rosavin on cutaneois angiogenesis induced in mice after grafting of syngeneic tumor cells. Centr Eur J Immunol 33: 102-107.

18. Guan S, He J, Guo W, et al. (2011): Adjuvant effects of salidroside from Rhodiola rosea L. on the immune responses to ovalbumin in mice. Immunopharmacol Immunotoxicol 33: 738-743.

19. Mishra KP, Chanda S, Shukla K, Ganju L (2010): Adjuvant effect of aqueous extract of Rhodiola imbricate rhizome on the immune responses to tetanus toxoid and ovalbumin in rats. Immunopharmacol Immunotoxicol 32: 141-146.

20. Mielcarek S, Mscisz A, Buchwald W, et al. (2005): Phytochemical investigation of Rhodiola sp. Root extracts. Herba Polonica 51: 159.

21. Jeong HJ, Ryu YB, Park SJ, et al. (2009): Neuraminidase inhibitory activities of flavonols isolated from Rhodiola rosea roots and their in vitro anti-influenza viral activities. Bioorg Med Chem 17: 6816-6823.

22. Wong YC, Zhao M, Zong YY, et al. (2008): Chemical constituents and anti-tuberculosis activity of root of Rhodiola kirilowii. Zhongguo Zhong Yao Za Zhi 33: 1561-1565.

23. Kratz JM, Andrighetti-Fröhner CR, Kolling DJ, et al. (2008): Anti-HSV-1 and anti- HIV-1 activity of gallic acid and pentyl gallate. Mem Inst Oswaldo Cruz 103: 437-442.

24. Zuo G, Li Z, Chen L, Xu X (2007): Activity of compounds from Chinese herbal medicine Rhodiola kirilowii (Regel) Maxim against HCV NS3 serine protease. Antiviral Res 76: 86-92.

25. Kim NS, Jeong SI, Hwang BS, et al. (2011): Gallic acid inhibits cell viability and induces apoptosis in human monocytic cell line U937. J Med Food 14: 240-246.

26. Kang MK, Kang NJ, Jang YJ, et al. (2009): Gallic acid induces neuronal cell death through activation of c-Jun N-terminal kinase and downregulation of Bcl-2. Ann N Y Acad Sci 1171: 514-520.

27. Han YH, Moon HJ, You BR, et al. (2010): Propyl gallate inhibits the growth of endothelial cells, especially calf pulmonary arterial endothelial cells via caspase-independent apoptosis. Int J Mol Med 25: 937-944.

28. You BR, Moon HJ, Han YH, Park WH (2010): Gallic acid inhibits the growth of HeLa cervical cancer cells via apoptosis and/or necrosis. Food Chem Toxicol 48: 1334-1340.

29. Serrano A, Palacios C, Roy G, et al. (1998): Derivatives of gallic acid induce apoptosis in tumoral cell lines and inhibit lymphocyte proliferation. Arch Biochem Biophys 350: 49-54.