Experimental immunology
The in vitro influence of Rhodiola quadrifida extracts on non-specific cellular immunity in pigs

Introduction
In the previous studies performed in rats we have found that extracts from R. rosea and R. quadrifida (traditional medicines from Asia, known for adaptogenic and antioxidant properties), when present in culture medium in concentration up to 10 µg/ml, enhanced blood leukocytes non-specific and specific immunologic activity in vitro [1, 2]. Chemical composition of extracts prepared from these two Rhodiola species are different. Compounds common for them are gallic and chlorogenic acids, tyrosol, and salidroside [3, 4]. The aim of this work was to study the in vitro nonspecific immunomodulatory activity of aquaeous and hydro-alcoholic extracts of under-ground parts of Rhodiola quadrifida in leukocyte cultures established from pig’s blood.


Material and Methods
Rhizomes and roots of R. quadrifida were collected in Altai mountain in Mongolia, thanks to dr H. Wiedenfeld, and further processed in Research Institute of Medicinal Plants (RIMP) in Poznań. The raw material was washed, cut into thick slices and dried in natural conditions.

Preparation and chemical analysis of Rhodiola extracts
Sample extractions were prepared by the methods as described below. Aqueous extracts: 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 lyophylized. Hydroalcoholic extracts: 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 lyophylized which was preceded by the distilling off the ethanol in the temperature 40-45°C. Dry extracts were stored under silica gel in the exsiccator in the room temperature.
All the samples were diluted in methanol. HPLC analysis was performed on Agilent 1100 HPLC system, equipped with photodiode array detector. For all separation a Lichrospher 100 RP18 column (250.0 × 4.0 mm, 5 µm) from Merck was used. The mobile phase consisted 0.05% phosphoric acid in water (A) and acetonitrile (B), applied in the following gradient elution: from 95A/5B in 30 min to 80A/20B then from 80A/20B in 5 min to 20A/80B and an isocratic elution in 15 min to the end. Each run was followed by an equilibration period for 10 min. The flow rate was adjusted to 1 ml/min, the detection wavelength set to DAD at λ=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 assigned by spiking the samples with standard compounds and comparison of the UV-spectra and retention times.

Animals
Blood for immunological experiments was collected from the vena cava cranialis of four PWZ piglets, 4-5 month old, 40-50 kg body mass, females. Experiments were approved by Local Ethical Committee.

Immunological studies
Leucocytes were isolated from blood by centrifugation at 2000g for 30 min at 4°C on the Gradisol G gradient (Aqua-Medica, Poland), washed three time in PBS and resuspended in RPMI 1640 medium (Sigma) supplemented with 10% of FCS (Foetal Calf Serum, Gibco-BRL) at a stock concentration of 2 × 106 cells/ml of medium. Viability of cells was checked by supravital staining with 0.1% w/v trypan blue. For RBA and PKA tests cells were preincubated for 2 hours at 37°C in the presence of R. quadrifida extracts, in concentrations 1, 5, 10, 20 and 50 µg/ml of culture medium.

RBA test
The metabolic activity of blood phagocyting cells (mostly granulocytes) was determined based on the measurement of intracellular respiratory burst after stimulation by PMA (phorbol myristate acetate, Sigma), as described by Chung and Secombes and adapted by Siwicki et al. [5]. The isolated cells were resuspended in RPMI-1640 medium (Sigma) at 106 cells/ml. On 96-well U-shaped microplates 100 µl of isolated blood leukocytes were mixed with of 100 µl of 0.2% nitro blue tetrazolium (NBT, Sigma) solution in 0.2 M phosphate buffer at pH 7.2 and added 1 µl of PMA at concentration 1 mg/ml in ethanol. After 30 min of incubation at 37°C, the supernatant was removed from each well. The cells pellet was washed with absolute ethanol and than three times in 70% ethanol and 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 plate microreader (MRX 3 Dynatech). All samples were tested in triplicate.

PKA test
Potential bactericidal activity of phagocyting cells was determined in isolated blood leukocytes stimulated with killed microorganisms, according to the method presented by Rook et al. [6]. On 96-well U-shaped microplates 100 µl of leucocytes were mixed with 100 µl of 0.2% NBT in phosphate buffer at pH 7.2 and 10 µl of killed Staphylococcus aureus strain 209P (containing 106 bacteria). The mixture was incubated 1h at 37°C and the supermatant was removed. The cell pellet was washed with absolute ethanol and three times with 70% ethanol and dried at room temperature. This was followed by the addition of 2M KOH and DMSO to each well. The amount of extracted reduced NBT was measured at 620 nm in a plate microreader (MRX 3 Dynatech). All samples were tested in triplicate.

Statistical analysis
The results of experiments performed on the leukocytes collected from the blood of 4 pigs were analysed statistically by a one-way ANOVA and the significance of differences between groups was verified with a Tukey’s Multiple Comparisons Test (GraphPadPrism software package).

Results
In in vitro studies both extracts were non-toxic at concentrations 50, 100, 200, 400, 800, and 1000 µg/ml after 24, 48, 72 and 94 hours of cells cultures.
The results of RBA test are presented on the Fig. 1. Significant, dose-dependent increase of activity was observed up to the concentration of 10 µg/ml of culture medium, and in higher concentrations of both types of extracts, significant, dose-dependent decrease of activity was seen. Similar results were obtained in PKA test (Fig. 2).

Discussion
The production of free oxygen radicals is a critical component of the killing process of bacteria by granulocytes. Present findings obtained in vitro in pigs confirmed our earlier results obtained in vitro in rats for Rhodiola quadrifida, and in rats and pigs for Rhodiola Rosea
[1, 7]. Both extracts stimulated granulocytes activity in lower doses, and were inhibitory in the highest (50 µg/ml) dose applied. As we have not observed cytotoxic effects up to the high 1000 µg/ml concentration, this inhibition might be connected with anti-oxidant activity of higher than 10 µg/ml concentrations [8].
Information about immunotropic activity of Rhodiolas is almost absent. Rhodiolas are known as adaptogens and antidepressants, increasing resistance to the harmfull effects of various stressors [9, 10]. The beneficial effects of adaptogens are associated with the hypothalamic-pituitary-adrenal (HPA) axis and the sympatho-adrenal-system (SAS), both of them may influence immunological reactions. The immunomodulatory effects of antidepressants were also described [11]. More experimental work is needed for elucidate the mechanism of immunomodulatory effect of Rhodiolas on granulocytes in vitro and in vivo activity.

References
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