Fitoterapia 77 (2006) 561–567www.elsevier.com/locate/fitote

Immunomodulatory effects of the methanolic extract ofEpimedium alpinum in vitro

Nada Kovačević a,⁎, Miodrag Čolić b, Aleksandar Backović b,Zvezdana Došlov-Kokoruš a

a Department of Pharmacognosy, Faculty of Pharmacy, Vojvode Stepe 450, 11000 Belgrade, Serbia and Montenegrob Institute for Medical Research, MMA, Crnotravska 17, 11002 Belgrade, Serbia and Montenegro

Received 27 July 2005; accepted 6 September 2006Available online 22 September 2006

Abstract

The effect of the methanolic extract of root and rhizome of Epimedium alpinum (MEEA) on phenotype and functions of ratlymphocytes in vitro was studied. It has been found that MEEA at lower concentrations (0.1 μg/ml and 1 μg/ml) significantly enhancedproliferation of splenocytes and thymocytes triggered by concanavalin A (Con A), whereas higher concentrations of the extract (50 μg/ml–500 μg/ml) were inhibitory. The stimulatory effect of MEEA on Con A-induced proliferation of splenocytes correlated with the up-regulation of interleukin-2 receptor α (IL-2Rα) expression. In addition, increased production of IL-2 was observed when a blocking IL-2Rαmonoclonal antibody (mAb) was added to cell cultures.MEEA-suppressed proliferation of splenocytes was due to the inhibition ofIL-2 production, the down-regulation of IL-2Rα expression and the induction of apoptosis. Cellular proliferation in the presence ofinhibitory concentrations of MEEA higher than 50 μg/ml could not be restored by the addition of exogenous IL-2.© 2006 Elsevier B.V. All rights reserved.

Keywords: Epimedium alpinum; Rat lymphocytes; IL-2 production; IL-2Rα expression; Apoptosis

1. Introduction

Fifty-four Epimedium species, are mainly distributed in Far Eastern Asia (China 43 spp, Japan, Korea, Manchuria,Far Eastern Russia 6 spp) [1]. Aerial parts of several Epimedium spp. are known as traditional herbal medicine inChina, Korea and Japan, used in the treatment of infertility, chronic nephritis, osteoporosis, rheuma, asthma,cardiovascular problems, hepatitis and leukopenia [2,3].

Underground parts are used in the treatment of rheuma, asthma and menstrual irregularity [3,4]. It is believed andconfirmed through some pharmacological assays that these therapeutic activities are based on the presence of flavonoidglycosides; other active constituents of Epimedium are lignans, terpenoids, polysaccharides [4] and the alkaloidmagnoflorine [5]. Epimedium spp. contains very complex mixture of flavonoid glycosides, mostly derivatives of 8-C-

⁎ Corresponding author. Tel.: +381 11 3970379; fax: +381 11 3972840.E-mail address: nadak@pharmacy.bg.ac.yu (N. Kovačević).

0367-326X/$ - see front matter © 2006 Elsevier B.V. All rights reserved.doi:10.1016/j.fitote.2006.09.008

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prenylkaempferol [4]. Certain flavonoids, such as epimedin C, icariin or baohuoside I, isolated from Epimedium,possess immunomodulatory (enhancing or suppressive) activities [2,6,7].

Epimedium alpinum is the only representative of Epimedium in Serbia. Aerial parts of this species are used for thetreatment of lungs illness, breast tumour and as diaphoretic [8]. The presence of prenylated flavonoid glycosides [9]and magnoflorine [10] has been confirmed. In this work we studied the effect of methanolic extract of root and rhizomeof E. alpinum (MEEA) on the proliferation of rat thymocytes and splenocytes to concanavalin A (Con A) in vitro andthe mechanisms involved in the modulatory activity of the extract.

2. Experimental

2.1. Plant material

Underground parts of E. alpinum L.(Berberidaceae), collected in August 1999 at mountain Maljen (altitude 1080 m),Serbia were identified by Dr. Dmitar Lakušić (Institute for Botany, Botanical Garden “Jevremovac”, Faculty of Biology,University of Belgrade). A voucher sample was deposited in the Department of Pharmacognosy, Faculty of Pharmacy,Belgrade.

2.2. Extraction

The air-dried powdered plant material, defatted with benzine and macerated inMeOH gave a solid residue (MEEA, yield12%). The content of total flavonoids, 7.65% calculated as hyperoside, was determined by spectrophotometry [11]. Thecontent of magnoflorine (9.17%) was determinated by HPLC [10]. The content of tertiary alkaloids, ca 0.1%, wasdeterminated by gravimetric method.

2.3. Animals

Male albino Oxford rats, 9–13 weeks old, bred at the vivarium of the Institute of Medical Research, MMA, Belgrade,under conventional laboratory conditions were used. The research was conducted in accordance with the internationallyaccepted principles for laboratory animal use and care that are approved by the Ethical Committee of MMA, Belgrade.

2.4. Preparation of cells

Thymocytes and splenocytes from rats were aseptically removed, pressed through stainless meshes placed in Petridishes with the addition of phosphate buffered saline (PBS) +5% fetal calf serum (FCS), filtered through nylon gauzeto remove large debris and clumps and then washed twice by centrifugation (580 rpm) with RPMI medium (Sigma,Munich, Germany) +5% FCS. Cells were counted and viability was determined by trypan blue dye exclusion.

2.5. Cell proliferation assay

Thymocytes and splenocytes were resuspended in complete RPMI medium and adjusted at concentrations 5×106

cells/ml (thymocytes) and 2×106 cells/ml (splenocytes), respectively. Cells were cultivated for 72 h in 96-well plates(200 μl/well), in an incubator with 5% CO2, at 37 °C in the presence of a suboptimal (1 μg/ml) concentration ofconcanavalin A (Con A) with the addition of different dilutions of MEEA. Cellular proliferation was measured after18 h pulse with 1 μCi [3H]-thymidine (5 Ci/mM, Amersham, Bucks, UK). Cells were harvested on glass fiber filtersand radioactivity was measured by standard scintillation techniques. The results were expressed as mean count×min(cpm) of triplicate samples. In certain experiments rat recombinant IL-2 (20 U/ml) (Genzyme, Boston, MA) or an IL-2Rα monoclonal antibody (mAb) (5 μg/ml) (Serotec, Oxford, UK) was added to cell cultures.

2.6. Detection of IL-2 in culture supernatants

Splenocytes were cultivated in 96-well flat bottom plates (3×105/well; 200 μl) under different conditions as previouslydescribed. After 24 h, cell-free supernatants were collected and stored at −70 °C until assayed. For detection of IL-2 in the

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supernatants, the IL-2 bioassay was performed on the basis of the IL-2 concentration-dependent proliferation of the clonedmurine cytotoxic T-cell line (CTLL). CTLL were maintained continuously proliferating in complete RPMI medium withthe addition of 5×10−5 M 2-mercaptoethanol (2-ME) and recombinant human IL-2 (2U/ml). Confluent cells werewashedtwice in serum-freemedium prior to the assay to remove any remaining IL-2.Washed cells were plated in 96-well plates intriplicates (5×103 cells/well) in 100 μl RPMI medium containing 5% FCS and 5×10−5 2-ME. After that, 100 μl oflymphocyte supernatants diluted 1:10–1:50 or different dilutions of recombinant human IL-2 were added. Cells wereincubated for 18 h followed by a 6-h pulse of [3H]-thymidine as previously described. Concentrations of IL-2 insupernatants were calculated using a curve derived from IL-2 standards. The specificity of the assay was checked using ablocking anti-rat IL-2 antibody (AF-502 NA; R and D; Oxon, UK) (dilution 1:200) which inhibited CTLL proliferation inthe presence of splenocyte supernatants. The concentrations of Con A andMEEA present in diluted samples used for IL-2testing did not significantly modulate the proliferation of CTLL.

2.7. Apoptosis assays

For apoptosis, two methods were used. Splenocytes (3×105 cells/well) were cultivated in 96-well flat bottom plates for24 h under different conditions as described above. For morphological evaluation cells were stained with Türk reagent usedfor leukocyte counting, as we previously published [12]. The reagent fixes and labels nuclei, enabling clear distinctionbetween normal chromatin organization in viable cells and chromatin condensation and kariorexis in apoptotic cells. At least

Fig. 1. Effect of MEEA on in vitro proliferation of rat splenocytes (A) and thymocytes (B) triggered by Con A (1 μg/ml). Values are given as cpm±SDof triplicates. *Pb0.05; ***Pb0.005 compared to corresponding controls (cells cultivated only with Con A).

Fig. 2. Effect of MEEA on IL-2 production in vitro on rat splenocytes stimulated with Con A. Values are given as mean±SD of triplicates.***Pb0.005 compared to control (splenocytes cultivated only with Con A).

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500 cellswere calculated in eachwell. Results of quadruplicateswere given in percentages. Apoptosiswas also detected usingpropidium iodide (PI) staining as described [12]. Cells were analyzed using a flow cytometer (Coulter XL-MCL, Krefeld,Germany). The percentages of apoptotic cells with hypodiploid nuclei were determined.

2.8. Cell fluorescence

Con A-stimulated splenocytes, cultivated for 2 days with or without MEEA, were resuspended in PBSsupplemented with 2% FCS and 0.1% sodium azide (1×106 cells/tube) and incubated for 45 min at 4 °C with OX-39(anti-CD25) mAb. Control cells were incubated with an irrelevant mouse mAb non-reactive with rat antigens. Afterwashing, in PBS/FCS/sodium azide the cells were incubated for 30 min at 4 °C with polyclonal goat anti-mouse Igconjugated with FITC (Amersham) (dilution 1:50) with addition of 3% normal rat serum. After two washings in PBS,cells were resuspended in PBS and analyzed on the EPICS-XL MCL flow cytometer. The percentages of positive cellswere determined by analyzing at least 5000 events.

3. Results

The results presented in Fig. 1 show that the methanolic extract of root and rhizome of E. alpinum (MEEA) at lowerconcentrations (0.1 μg/ml and 1 μg/ml) enhanced proliferation of splenocytes triggered by Con A (1 μg/ml). Theconcentration of 0.1 μg/ml of MEEA-stimulated proliferation of thymocytes, also.

Fig. 3. Effect of MEEA on the IL-2Rα expression on Con A-stimulated splenocytes. Expression of IL-2Rα by splenocytes in cultures (48 h) wasdetermined by flow cytometry as described in the Experimental section. Values are given as mean±SD (from three different experiments). *Pb0.05;***Pb0.005 compared to control (splenocytes cultivated only with Con A).

Table 1Effect of exogenous IL-2 on MEEA-suppressed proliferation of splenocytes in the presence of Con A

MEEA (μg/ml) Proliferation (cpm)

IL-2 (−) IL-2 (+)

0 98,360±12,950 129,500±14,300*50 36,240±4880 57,390±7920*100 21,305±3960 24,807±4200250 9040±2510 8208±1250

Splenocytes were cultivated with Con A (1 μg/ml) with different concentrations of MEEA in the presence or absence of exogenous recombinant ratIL-2 (20 U/ml). Values are expressed as mean cpm±SD. *Pb0.05 compared to values in cultures without IL-2.

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In contrast, concentrations of MEEA ranging between 50 μg/ml and 500 μg/ml significantly inhibitedproliferation of splenocytes and thymocytes. Both stimulatory and inhibitory effects of MEEA were higher usingsplenocytes as responding cells. MEEA alone did not modulate spontaneous proliferation of examined cells (datanot shown).

To explore the mechanisms involved in MEEA-modulated proliferation of splenocytes, production of IL-2 in culturesupernatants and expression of IL-2Rα by Con A-stimulated splenocytes was examined. As it can be seen in Fig. 2, theconcentrations of MEEA (0.1 μg/ml and 1 μg/ml) which augmented proliferation of Con A-stimulated splenocytes didnot significantly modulate production of IL-2.

Fig. 4. Effect of MEEA on apoptosis of splenocytes. Splenocytes were cultivated in medium with or without MEEA and apoptosis was measured 24 hlater by light microscopy (A) or by flow cytometry (PI staining) (B) as described in the Experimental section. Results presented in A are given as %apoptosis±SD (sexaplicates) from one representative experiment. Results presented in B are given as histograms of PI-stained cells in control (a) andMEEA (100 μg/ml)-treated cultures (b). The percentages of cells with hypodiploid nuclei (one representative experiment) are marked by bars.(*Pb0.05); (**Pb0.01)

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However, the concentration of 0.1 μg/ml of MEEA up-regulated the IL-2Rα expression on these cells (Fig. 3). Tocheck whether utilization of IL-2 was higher in MEEA-treated cells, an IL-2Rα mAb had been added to splenocytecultures and IL-2 production was measured. The levels of IL-2 (221±45 ng/ml) in culture supernatants with MEEA(0.1 μg/ml) were statistically higher, compared to values of IL-2 in cultures without MEEA (133±40 ng/ml) (N=3;Pb0.05).

Production of IL-2 by Con A-stimulated splenocytes in the presence of higher concentrations of MEEA (50 μg/ml–250 μg/ml) and expression of IL-2Rα were significantly decreased (Fig. 2). Since exogenous addition of IL-2 onlyslightly restored proliferation of splenocytes using 50 μg/ml of MEEA and did not significantly change cellularproliferation after addition of higher concentrations of MEEA (Table 1), we asked whether the observed inhibitoryeffect of MEEA on splenocyte activation correlated with increased apoptosis. Fig. 4 shows that MEEA atconcentrations of 100 μg/ml and 250 μg/ml induced apoptosis of splenocytes.

4. Discussion

For the first time it was shown that the extract of EuropeanE. alpinum, possesses immunodulatory activity in vitro. Similaractivity has been confirmedwith extracts ofEpimedium spp. fromAsian countries, using different in vivo and in vitromodels.

It was demonstrated that investigated extract of E. alpinum, at lower concentrations, stimulated in vitro proliferationof rat lymphocytes from thymus and spleen in the presence of Con A. The responding cells were, probably, Tlymphocytes, since Con A is a well-known T-cell mitogen. In contrast, MEEA at concentrations higher than 50 μg/mlsuppressed T-cell proliferation in vitro.

It has been reported that extracts of different Epimedium spp. or pure components isolated from those extracts, showeither stimulatory [2,13] or inhibitory activity [7,14] on the immune system. However, this work provided evidencethat the same extract exerted both effects, depending on applied concentration.

There are many examples of dose-dependent effects of different plant extracts on the immune system [15–18]. Wepreviously demonstrated stimulatory and inhibitory effects of garlic extracts on T-cell proliferation in vitro [18]. Garlicextracts, at lower concentrations, stimulated production of IL-2 and expression of IL-2Rα by T cells. In contrast,MEEA at lower concentrations, up-regulated IL-2Rα, but increased production of IL-2 was observed when an anti-IL-2Rα mAb was added to the culture to prevent utilization of IL-2.

Inhibited proliferation of lymphocytes by garlic extracts was followed by lower production of IL-2 that could berestored by exogenous IL-2 [18]. In contrast, MEEA decreased IL-2 production, that could not be restored by theaddition of IL-2. Lower production of IL-2 in MEEA-treated cultures is probably a consequence of lower numbersof IL-2 secreting cells due to induction of apoptosis. Induction of apoptosis by Epimedium extracts has not beenpublished so far. However, this phenomenon might be in accordance with previous publications that certain plantalkaloids possess cytotoxic and cytostatic activities [19,20]. The best characterized alkaloid with such activities ismagnoflorine. This quarternary base alkaloid is the main alkaloid present in Epimedium spp. Magnoflorine has beenshown to suppress local graft versus host reaction in mice by interfering with the induction phase, but not with theeffector phase of the cellular immune response [19]. In addition, the alkaloid did not affect humoral immuneresponse [19]. The MEEA extract used for the experiments presented in this paper contained magnoflorine as adominant alkaloid (9.17%).

Up to now, different components with immunoenhancing activity have been isolated from Epimedium spp.Maybe the most important are flavonoids, from which epimedin C and icariin have been best characterized [6,13].Epimedin C, a triglycoside, might exert immunostimulatory activity of its own, or acting through icariin, its adiglycoside metabolite [2]. In vitro icariin increased the relative proportion of human CD8+ T cells, stimulatedNK cellular cytotoxicity and production of TNF-α by monocytes [21]. The best characterized immunosuppressiveflavonoid is baohuoside-1 [7,14]. It could also be a metabolite of icariin; it is formed by its decomposition in thepresence of enzymes [2,7]. Baohuoside-1, isolated from E. davidii, suppressed antibody and delayed-typehypersensitivity responses in mice in a dose-dependent fashion [7]. In contrast, this flavonoid did not significantlyprolong survival of cardiac grafts, suggesting its predominant immunosuppressive activity on the humoral immuneresponse. Preliminary chemical analysis of methanolic extract of underground parts of E. alpinum showed thepresence of flavonoids, too. Besides the flavonoids, it has been suggested that other components from Epimediumspp., such as certain polysaccharides, might be stimulatory on lymphocyte functions, based on observations thatthey exerted mitogenic activity on bone marrow cells [22].

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5. Conclusion

In conclusion, the obtained results clearly showed the presence of immunomodulatory components in themethanolic extract of E. alpinum underground parts. At the moment, it is not clear whether the observed stimulatoryand inhibitory effects are related to hormesis, a phenomenon observed with many pharmacologically active substances.Hormesis is characterized by the dose–response which is stimulatory at low doses and inhibitory at high doses, leadingto the biphasic, hormestic dose–response curve [23]. In contrast, different components in the extract may exert differenteffects on the immune system acting either independently, antagonistically or synergistically. In this context, previousactivation state of target cells is also important. Therefore, isolation and better characterization of biological activecomponents from plant extracts, including MEEA, are needed to resolve many of these problems.

Acknowledgements

The research has been partly supported by the Ministry of Science and Technology of Serbia, Project no. 1568.

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