Experimental immunology
The effect of Rhodiola kirilowii extracts on pigs’ blood leukocytes metabolic (RBA) and proliferative (LPS) activity, and on the bacterial infection and blood leukocytes number in mice

Introduction

As a part of our continuing searching for non-toxic natural immunostimulatory remedies, we screened a large panel of immunotropic activities of aqueous and hydro-alcoholic extracts of Rhodiola rosea (RR) and Rhodiola quadrifida (RQ). After performing experiments on mice, rats and pigs, we obtained evidence of their in vivo and in vitro immunomodulatory activity [1-10]. Rhodiola extracts have stimulated both non-specific and specific cellular immunity. In vivo, they increased number of blood leukocytes and their metabolic activity, improved the motility of splenic lymphocytes, and lowered the intensity of bacterial infection. In vitro, RR and RQ extracts potentiated respiratory burst (RBA) and potential killing (PKA) activity of blood leukocytes. As well, as specific immunity was concerned, RR and RQ extracts have stimulated in vitro and in vivo mitogen-induced lymphocyte proliferation and local graft-versus-host reaction (GVH) performed against semi-allogeneic and xenogeneic histocompatibility barrier. The anti-oxidative influence of RR extracts and their immunological action via CD4+ Tcells were established [1, 2].

We also performed some experiments with Rhodiola kirilowii (RK) extracts [9, 11-13] confirming the results obtained in vitro for RR and RQ species. In vivo, RKW and RKA extracts enhanced the ability of lymphocytes derived from parental strain mice (fed with these extracts) to induce local GVH reaction in F1 hybrids and have stimulated metabolic activity of mice blood granulocytes (chemiluminescence induced by Zymosan) collected from mice fed with RKW or RKA for 7 days [11-13]. The aim of this study was to evaluate : firstly, the in vitro effect of RKW and RKA extracts on respiratory burst activity (RBA) and LPS-induced mitogenic response of leukocytes isolated from the blood of pigs; and, secondly, the in vivo effect of these extracts on the intensity of bacterial infection and the number of blood leukocytes in mice fed with RKW or RKA extract for 7 days.

Material and methods

Preparation and chemical analysis of extracts



Rhodiola kirilowii (Crassulaceae) roots and rhizomes were cultivated, collected and identified in the Research Institute of Medicinal Plants (RIMP), presently Institute of Natural Fibres and Medicinal Plants, Poznań. The cultivation was established by vegetative propagation. The plant growth was controlled: the reaction and mineral components of the soil, the air temperature, the average sum of humidity, and rain as well as the sun periods were monitored permanently. Voucher specimen is kept in the herbarium of Department of Botany, Breeding and Agriculture in Plewiska near Poznań.

Sample extractions were prepared by the methods as described below. Aqueous extract: finely powdered roots were extracted two times with water (extraction was performed: first – 2 hour and second 1 hour long) in the ratio raw material/solvent 1/5, in the temperature 40-45°C. The supernatants were mixed together and after centrifugation at 3000 rpm for 15 min were lyophilized. Hydro-alcoholic extract: finely powdered roots were extracted with ethanol/water solution (1/1, v/v) in the ratio raw material/solvent 1/10 by the percolation method. Then the percolates were lyophilized which was preceded by the distilling off the ethanol in the temperature 40-45°C. DER values of extracts were: 5.09/1 for RKW and 3.27/1 for RKA. Extracts were stored at –70°C until used .

The content of phenolic compounds was determined by means of two methods: ultra performance liquid chromatography (UPLC) and high-performance liquid chromatography (HPLC).



Ultra performance liquid chromatography



An ultra performance liquid chromatograph connected to a tandem mass spectrometer (UPLC-MS/MS; Waters) was used for chemical analyses. The separation of analytes was performed on an Acquity UPLC BEH C18 column, 1.7 µm 2.1 × 50 mm (Waters). Mobile phase: phase A: 0.1% (v/v) HCOOH solution in water, phase B: 0.1% (v/v) HCOOH solution in acetonitrile. Mobile phase flow rate was: 0.20 ml/min. The assay was performed in gradient elution: 0.0 min – 97% of phase A, 7.5 min – 68% of phase A, 9.0 min – 97% of phase A. Column temperature was 25°C; ion source temperature: 120°C; desolvation temperature: 350°C. Gas flow rate: desolvation gas: 700 l/h; cone gas: 10 l/h. Peaks were identified by spiking the samples with standard compounds.



High-performance liquid chromatography



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 × 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 l = 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.

Spectrofotometric analysis of tannins was performed according to the methods of European Pharmacopoeia on UV-Visible Spectrometer Cintra 20 GBC 9. (Determination of tannins in herbal drugs. European Pharmacopoeia, 6 ed. 2008; 1: 255).



Animals



Mice



The study was performed on 8-10-weeks old female inbred Balb/c mice, 20–22 γ of body mass, delivered from the Polish Academy of Sciences breeding colony.



Pigs



Blood for in vitro immunological experiments was collected from the vena cava cranialis of four PWZ piglets, 4-5 month old, 40-50 kg body mass, females.



Leukocyte subpopulations



RK extracts were administered to the groups of 8 mice each, per os, in daily doses of 50, 100, 200 or 400 µg. These doses corresponded to about 25, 50, 100 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 a mean number of leukocytes in 1 mm3 of blood ± standard error of the mean (SEM).



Bacterial infection



Mice were fed RK extracts 400 µg daily, in 10 % ethyl alcohol, or 0.04 ml of 10% alcohol as a control, for 7 days. On the day 8-ght 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].



In vitro studies



Leukocytes were isolated from the blood by centrifugation at 2000xg for 30 min at 4°C on the Gradisol L or G gradient (Aqua-Medica, Poland), washed three times in PBS and resuspended in RPMI 1640 medium (Sigma) supplemented with 10% of FCS(Gibco), at a stock concentration of 2 × 106 cells/ml of medium. For RBA test, cells were preincubated for 2 hours at 37°C in the presence of RK extracts in concentrations 1, 5, 10, 20 and 50 µg per ml.



Respiratory burst activity test



The metabolic activity of blood phagocytic cells was determined based on the measurement of intracellular respiratory burst activity (RBA) after stimulation with PMA (Phorbol Myristate Acetate, Sigma [3]. The isolated cells were resuspended in RPMI-1640 medium (Sigma) at 106 cells/ml. On 96-well U-shaped microplates, 100 µl of the isolated blood leukocytes was mixed with 100 µl of a 0.2% nitro blue tetrazolium (NBT, Sigma) solution in 0.2 M phosphate buffer at pH 7.2, and 1 µl of PMA at a concentration of 1 mg/ml in ethanol was added. After 30 min of incubation at 37°C, the supernatant was removed from each well. The cell pellet was washed with absolute ethanol and, three times, with 70% ethanol and it was dried at room temperature. The amount of extracted reduced NBT after incubation with 2 M KOH and DMSO (dimethylsulfoxide, Sigma) was measured colorimetrically at 620 nm in a microplate reader (Tecan Sunrise). All samples were tested in triplicate, and the results are presented as mean values ± SD.



Proliferative response to lipopolysaccharide



Proliferative response of pigs blood leukocytes after stimulation with mitogen lipopolysaccharide (LPS), was determined by MTT spectrophotometry (OD 570 nm) using (3-[4,5- dimethylthiazol-2yl]-2,5-diphenyltetrazolium bromide – 3-[4,5-dimethyl-2-thiazol]-2,5-diphenyl-2H-tetrazolium bromide), as described previously [3].

MTT (Sigma) was dissolved in PBS at a concentration of 5 mg/ml and filtered. On 96-well culture plates (Sarstetd, USA), 100 µl of blood lymphocytes in RPMI 1640 containing 10% FCS, 2 mM L-glutamine, 0.02 mM 2-mercaptoethanol, 1% hepes buffer, penicillin/streptomycin (100 U/100 µg/ml) was mixed with 100 µl of RPMI 1640 containing lipopolysaccharide (LPS, 20 µg/ml). After 72 h incubation at 37°C in a 5% carbon dioxide atmosphere (Memmert Incubator), 50 µl of MTT solution was added into each well, and plates were incubated for 4 h at 37°C. After incubation the plates were centrifuged (1,400 g, 15°C, 5 min). Supernatants were removed and 100 µl of DMSO (Sigma) were added into each well and incubated for 15 min at room temperature. After incubation the solubilized reduced MTT was measured colorimetrically at 620 nm in a microplate reader (Tecan Sunrise). All samples were tested in triplicate, and the results are presented as mean values. Rhodiola extracts were present in culture medium during whole cultivation period (72 hours) in concentrations 1, 10, and 50 µg/ml.

For all experiments animals were handled according to the Polish law on the protection of animals and NIH (National Institutes of Health) standards. All experiments were accepted by the local Ethical Committee (nr 1/N/WDP-1/19.01.2006).

Statistical evaluation of the results

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

Results and discussion

In experiments with pigs leukocytes in cell culture (Fig.1 and Fig. 2), both extracts in concentration 5 and 10 µg/ml stimulated their metabolic (respiratory burst) and in concentration 10 µg/ml proliferative (LPS) activity (p < 0.001). In the presence of 1µg/ml only RKA extract was effective (p < 0.05 for RBA and p < 0.01 for LPS). High concentrations of RK extracts were ineffective (20 µg/ml) or inhibitory (50 µg/ml). LPS, commonly used as B-cell mitogen, can stimulate also other cells taking part in innate immunity (monocytes, macrophages, dendritic cells) by Toll-like receptors [14]. The Toll-like receptors recognize a few highly conservative structures existing in prevailing microorganisms [15]. Previous in vitro study performed on rats and pigs blood leukocytes did not revealed cytotoxic effects of RK extracts up to the 1000 µg/ml concentration. Our present results are in agreement with our previously performed studies, where similar modulatory effect on leukocytes RBA, PKA and proliferative activity was observed after leukocytes incubation in extracts of other Rhodiola species – Rhodiola rosea and Rhodiola quadrifida [3-5]. In these previous experiments RBA and PKA stimulatory activity of hydro-alcoholic extracts of RR and RQ constantly was more pronounced than the activity of corresponding water extracts. One would expect such modulatory effect, because Rhodiola extracts contain various phenolic compounds, some of them with antioxidant activity [2, 16, 17]. Anti-oxidant activity of epigallocatechin gallate (EGCG), compound abundant in green tea, but also present in RK extracts (see Table 1) is well documented (reviewed in [13]). In vivo experiments performed in mice showed beneficial effect of feeding mice with Rhodiola kirilowi aqueous (RKW) or 50% hydro-alcoholic (RKA) extracts on Pseudomonas aeruginosa infection (Fig. 3).

It was found that the infection intensity was highly significantly lower, in comparison to the control, when mice obtained daily, for 7 days before P. aeruginosa i.p. inoculation, 0.4 mg of RKA or RKW extract (p < 0.001). Accordingly, both extracts significantly increased blood leukocytes (both lymphocytes and granulocytes) number in mice, at 0.1 and 0.2 mg (RKW) and 0.1 mg (RKA) daily dose (Fig. 4a and 4b). Again, in 0.1 mg dose stimulatory effect was more pronounced in mice fed with RKA than in mice fed with RKW extract (0.001 vs.0.05), but disappeared in mice fed with 0.2 mg RKA extract daily doses.

There are some papers about direct in vitro anti-viral and anti-bacterial activity of Rhodiola extracts and their compounds [18-21]. However, we rather think that in our present in vivo study, antibacterial effect of RK extracts was indirect, mediated by activated leukocytes migrating to the peritoneal cavity .

From the results presented in this study, as well as from all our previous reports on immunotropic activity of Rhodiolas, one may conclude, that extracts of plants belonging to various Rhodiola species (R. rosea, R. quadrifida, R. kirilowii) may represent valuable source of substances with immunostimulatory potential. However, one should remember, that many herbal remedies (among them extracts of Rhodiola), being stimulatory in lower doses, in higher doses presented inhibitory effects, affecting proliferation, metabolic activity and mobility of lymphocytes. So, establishing of proper dose is extremely important [22].

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: ed. by Bajaj YPS, 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, Skopinska- 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, Skopinska-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. Skopinska-Różewska E, Wójcik R, Siwicki AK, et al. (2008): The effect of R. 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. Skopinska-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.

 8. Bany J, Zdanowska D, Skopinska-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, Skopinska-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.

10. 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: 265-72.

11. Wójcik R, Siwicki AK, Skopińska-Różewska 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.

12. 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.

13. 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.

14. Dąbrowski MP, Stankiewicz W (2011): Immunostimulatory effects of lipopolysaccharide. Centr Eur J Immunol 36: 85-86.

15. Słotwiński R, Słotwińska SM, Bałan BJ, Kędziora S (2009): Toll-like receptor signaling pathway in sepsis. Centr Eur J Immunol 34: 124-128.

16. Mielcarek S, Mscisz A, Buchwald W, et al. (2005): Phytochemical investigation of Rhodiola sp. Root extracts. Herba Polonica 51(S1): 159.

17. Wiedenfeld H, Zych M, Buchwald W, Furmanowa M (2007): New compounds from Rhodiola kirilowii. Sci Pharmac 75: 29-34.

18. 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.

19. 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.

20. Kratz JM, Andrighetti-Frohner 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.

21. 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.

22. Hartwich M (2010): The importance of immunological studies on Rhodiola rosea in the new effective and safe herbal drug discovery. Centr Eur J Immunol 35: 263-266.