The Anti-stress Effects of Sarcandra glabra Extract on Restraint

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din, 4-O-b-D-glycopyranosyl rosmarinic acid, rosmarinic acid, which are good antioxidants ... Natl. Acad. Sci. U.S.A., 103, 12831—12836 (2006). February 2009.
Biol. Pharm. Bull. 32(2) 247—252 (2009)

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The Anti-stress Effects of Sarcandra glabra Extract on Restraint-Evoked Immunocompromise Rong Rong HE,a Xin Sheng YAO,*,a,b Hui Ying LI,b Yi DAI,b Ying Hui DUAN,a Yi Fang LI,b and Hiroshi KURIHARA*,b a

College of Traditional Chinese Materia Medica, Shengyang Pharmaceutical University; Shengyang 110016, China: and Institute of Traditional Chinese Medicine & Natural Products, Jinan University; Guangzhou 510632, China. Received September 16, 2008; accepted November 12, 2008; published online November 19, 2008 b

Sarcandra glabra was a renowned herb traditionally used as herbal tea or food supplement to enhance mental efficiency and to recover from stress or fatigue in China. We investigated the effects of Sarcandra glabra extract (SGE), with chemical composition clearly showed by HPLC fingerprint as quality control, on immunologic response including natural killer (NK) cell activity and its antioxidative capacity in splenocytes obtained from restraint mice. Our results found that daily oral administration of SGE (125, 500 mg/kg/d) for 5 consecutive days to restrained mice alleviated the stress-induced reduction of the number of lymphocytes, the balance of CD4 T/CD8 T and NK cell activity per spleen. SGE also significantly decreased the level of lipid peroxidation and increased oxygen radical absorbance capacity (ORAC) in splenocytes. These results indicated that SGE modulate stress-attenuated immunologic response, at least, partially explained by improving antioxidative capacity in immunocytes. Key words

Sarcandra glabra; restraint stress; natural killer cell activity; oxygen radical absorbance capacity; antioxidation

Stress and frustration can trigger problems in mental and physical health, which in turn can cause many life-style diseases over a prolonged period of time. Herbs, as a good source of vitamins, minerals, and antioxidants, have played an important role in stress management. Sarcandra glabra (THUNB.) NAKAI (Chloranthaceae) was a renowned herb traditionally used as herbal tea or food supplement to remedy ailments, to enhance mental efficiency and to recover from stress or fatigue in China.1) It grows in the southern parts of China, Japan and southeastern Asia. The chemical compositions of Sarcandra glabra are volatile oil, phenolic acids, polysaccharide, etc.2) Isofraxidin, fumaric acid, terpenoid saponins and other flavonoids were isolated from the ethanolic extract of Sarcandra glabra.3) Its volatile oil is widely used in tooth-paste and chewing gum to inhibit oral bacteria.4) The whole plant is also used as herbal tea or supplementary food for its anti-infectious and anti-inflammatory effects, which are closely related to immunity.5) For these reasons, the research and development on Sarcandra glabra have attracted great attention in the international scientific community. Some studies have demonstrated that Sarcandra glabra exhibited immunological activities, which appeared to induce an increased protection against microbial and viral infections,6) cytoprotective activities,2) and anticancer activities.7) However, these data were obtained by experiments in vitro and little is known about whether Sarcandra glabra has any effects on animals. To investigate the effects of Sarcandra glabra extract (SGE) on immunological response in vivo, we used restraint loaded mice as an animal model. Restraint is a common stress-causing factor that exerts great impacts on immunological system.8) It was reported that restraint stress in mice changed the number of lymphocyte cells, altered antibodies production, and suppressed cytotoxicity of natural killer (NK) and T cells.9—12) Fukui demonstrated that restraint stress-induced change in lymphocyte cell number closely correlated with the altered antibody and cytokine levels.11) ∗ To whom correspondence should be addressed.

Reports from Sheridan’s lab have shown that restraint stress suppressed T-cell cytokine production and cytolytic T cell activity in virus-infected animals.13) They also found that restraint stress significantly modulated NK cell trafficking and cytolytic activity and contributed to elevated virus replication.14) But the molecular mechanisms underlying the link between restraint stress and these immune dysfunctions remain elusive. It was reported that NK cells and T cells were susceptible to reactive oxygen species (ROS), and would lose their activities under oxidative stress.15,16) The critical parameters of oxidation are lipid peroxidation and its production of malondialdehyde (MDA).17) To estimate the ability of protection against oxygen free radicals, a sensitive and reliable method called oxygen radical absorbent capacity (ORAC) assay was used.18) Based on this knowledge, we detected the anti-stress effects of SGE on restraint-evoked immunocompromise in mice, by evaluating the effects of SGE on recovering the number of lymphocyte cells in spleen, the balance of T lymphocyte subpopulation (CD4 T/CD8 T) and NK cell activity in splenocytes of mice. We further hypothesized the mechanisms might be associated with its anti-oxidative effects in immune cells, by detecting the production of MDA and the level of ORAC of immune cells. On the other hand, we carried out HPLC fingerprint analysis to control the quality of SGE, and we also determined compound structures of the seven main chromatographic peaks in the fingerprint. MATERIALS AND METHODS Materials and Preparation of SGE 5-Caffeoylquinic acid, 3-caffeoylquinic acid, 4-caffeoylquinic acid, caffeic acid, isofraxidin, 4-O-b -D-glycopyranosyl rosmarinic acid, and rosmarinic acid used in this study had previously been isolated from Sarcandra glabra. Mouse immnoglobulin (Ig) G1-fluorescein-isothiocyanate (FITC) or phycoerythrin (PE), anti-CD3 (FITC), anti-CD4 (PE), and anti-CD8 (PE) were all purchased from Beckman (U.S.A.). Phenylhydrazine and

e-mail: [email protected]; [email protected]

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thiobarbituric acid were purchased from Nanjing Jiancheng Bioengineering Institute (Nianjing, China). Sodium fluorescein (FL), 2,2-azobis(2-amidinopropane)-dihydrochloride (AAPH), and trolox (6-hydroxy-2,5,7,8-tetramethylchroman2-carboxylic acid) were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Sarcandra glabra was generously provided by Guangzhou Jingxiutang Pharmaceutical Co., Ltd. (Guangzhou, China), and authenticated by Professor Yao Xinsheng (College of Materia Medica, Jinan University). A voucher specimen (Z03107) was maintained in Institute of Traditional Chinese Medicine & Natural Products, Jinan University, Guangzhou 510632, China. The SGE was prepared as follows: 500 g crude dry plant was decocted with 5000 ml of boiling water for 40 min to half volume. The extracted solution was centrifuged at 3500 rpm for 30 min, and the supernatant was filtered and lyophilized. Reverse-Phase HPLC Analysis of SGE’s Chemical Composition For the quality control of the oral administration to mice, SGE chemical pattern was obtained by the PDA HPLC analysis and indicated in Fig. 1. Compared with the standard compounds, seven main chromatographic peaks were identified as follows: 5-caffeoylquinic acid (1, tR 9.0 min), 3-caffeoylquinic acid (2, tR 14.9 min), 4-caffeoylquinic acid (3, tR 16.6 min), caffeic acid (4, tR 19.2 min), isofraxidin (5, tR 27.5 min), 4-O-b -D-glycopyranosyl rosmarinic acid (6, tR 35.3 min), rosmarinic acid (7, tR 39.3 min). Animals and Treatment Seven week-old male C57BL/ 6J mice purchased from the Center of Laboratory Animal Science Research of Southern Medical University (Guangdong, China), were kept in a specific pathogen-free animal room at 231 °C with a 12-h dark–light cycle and were fed with standard laboratory diet and tap water. The animals were allowed to acclimatize to the environment for 1 week before the experiment. The experimental procedure was shown as Fig. 2. Experimental groups received oral administration of SGE dissolved in drinking water at a final concentration of 12.5 and 50 mg/ml, while the normal control group and stress control group received water only. The intakes of SGE water solution were 0.1 ml per 10 g body weight for 5 d. On the second day of administration, mice were physically restrained in a 50 ml polypropylene centrifuge tube with holes for 12 h, and then placed in the home cage with food and water before the assay. After 3 d of recovery, all mice were sacrificed and the spleens were removed. The Institutional Laboratory Animal Care approved all animal procedures. All studies were conducted in accordance with the guidelines set forth by the National Institutes of Health and the U.S. Department of Agriculture. Tumor Cell Line YAC-1 tumor cell line, a moloney virus induced mouse T cell lymphoma of A/SN origin, was obtained from Institute of Health Care Science (Suntory Ltd., Japan). The YAC-1 cell was used to test the NK cytotoxic activity for its noted sensitivity to NK cells. Cell line was cultured in RPMI-1640 containing 10% fetal bovine serum (FBS) at 37 °C in a humidified 5% CO2 atmosphere before tested. Splenocyte Preparation The spleens were collected and splenocytes were prepared by disrupting the spleen with a grinder in phosphate-buffered saline (PBS, pH 7.4). The total splenocyte number was determined with a blood–cell count-

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ing chamber (Erma, Japan). After a 10 min centrifugation at 1500 rpm to separate debris, erythrocytes were lysed using ammonium chloride reagent. The cells were washed twice with PBS and suspended in 1 ml of cold RPMI-1640 medium with 10% FBS. The splenic cell viability was determined by trypan blue exclusion. Determination of T Lymphocyte Subsets Samples containing 1106 splenocytes in RPMI-1640 medium were treated with selected monoclonal antibodies conjugated with FITC or PE (Beckman, U.S.A.). We used the following double-staining combinations: anti-CD3 (FITC)/anti-CD4 (PE), and anti-CD3 (FITC)/anti-CD8 (PE). Mouse IgG1-FITC and -PE were used as control staining. After 15 min incubation at room temperature in the dark, the cells were washed with PBS and resuspended in 0.5 ml of cold PBS and analyzed using a FACS Epics XL (Beckman, U.S.A.). Usually, 10000 cells were scanned for each sample, and the results were expressed as the percentage of cells yielding a specific fluorescence in a gated lymphocyte region. NK Cell Activity Assay The experiments employed two fluorescent stains as Piriou19) reported, 3,3-dioctadecyloxacarbocyanine perchlorate (DiO, from Sigma) which is stably integrated into the cell membrane and stains the intended cell population homogeneously, and propidium iodide (PI, from Sigma) which passes rapidly through damaged cell membranes and binds with very high affinity to the DNA. NK cell activity was detected with the freshly isolated splenic mononuclear cells. Target cells for detection of NK cell cytotoxicity were YAC-1 cell line. The YAC-1 cells were maintained in continuous suspension culture in the complete culture medium at a concentration of about 1106 cells/ml at 37 °C in a humidified 5% CO2 incubator. All cultures were split 24 h before use to ensure that the YAC-1 cells were in an exponential growth phase during the assays. DiO was firstly added to the YAC-1 cells at a concentration of 30 m mol/l, which was incubated for 15 min at 37 °C in 5% CO2. After labeling the YAC-1 cells were washed twice, counted and adjusted to 1105 cells/ml. Mixtures of the stained YAC-1 cells (0.1 ml of 1105 cells/ml) and the NK cells (0.1 ml of 1107, 5106, and 1.25106 cells/ml) were incubated at 37 °C in a humidified 5% CO2 incubator for 4 h. Another mixture of the stained YAC-1 cells (0.1 ml) and the complete culture medium (0.1 ml) were performed in parallel to serve as control for assessment of spontaneously dead YAC-1 cells. Then PI was added to the mixtures at a concentration of 5 m g/ml for 15 min at room temperature. Four groups of cells were thus identified. Intact NK cells were non-fluorescent, dead NK cells emitted red fluorescence, living YAC-1 cells exhibited green fluorescence and dead YAC1 cells were characterized by double (green–red) fluorescence. Flow cytometry was performed with a FACS Epics XL (Beckman, U.S.A.) equipped with an argon laser operating at 488 nm. Two parameter dot plots were obtained with CellQuest software (Beckman, U.S.A.). In these plots the abscissa was log scale green fluorescence and the ordinate log scale red fluorescence. In the each tested sample, the spontaneously dead YAC-1 cells were subtracted from the total dead YAC-1 cells and the NK cell cytotoxicity was expressed as the percentage of the specifically dead YAC-1 cells relative to the total YAC-1 cells. Results were plotted, and the number of cells required to produce 10% specific cytotoxic-

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Fig. 1.

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Chemical Fingerprint of SGE Was Analyzed by HPLC

Each peak of SGE in the HPLC fingerprint was identified by comparison of the retention times and UV spectra of chemically defined standard compounds. Several batches of SGE were analyzed, and similar profiles were observed. HPLC condition was as follows: Agilent series 1100 HPLC; column, Welch material XB-C18, 4.6250 mm, particle size 5 m m; mobile phase A, H2O with 0.2% HAc; mobile phase B, MeOH with 0.2% HAc; elution program: 20% B in 5 min, linear gradient from 20% B to 62% B in 55 min, 100% B in 12 min; flow rate at 0.80 ml/min; detection wavelength at 330 nm; injection volume in 10 m l and oven temperature at 35 °C.

Fig. 2.

Experimental Procedure for Mice Exposed to Restraint Stress

C57BL/6J mice were fixed in the restraint cage for 12 h and recovered for 3 d before the measurement of biochemical parameters.

ity (one lytic unit, 1 LU10) was established from the best fit of the curve. The number of LU10 per spleen (LU10/spleen) was thus calculated and used to express final results. Assays for each effector/target cell ratio were performed in triplicate. After incubation for 4 h at 37 °C under a 5% CO2 atmosphere, lysis of target cells was calculated using Eq. 1. lysis (%) 

experimental release  spontaneous release maximum release  spontaneous release

100%

(1)

The maximum release referred to the lysis obtained after adding Triton X-100 (final concentration 1%). Spontaneous release was determined by the incubation of labeled target cells in the absence of effector cells. Thiobarbituric Acid-Reactive Substances (TBARS) Assay The level of lipid peroxide in the splenocytes was determined by measuring TBARS. Splenocytes were homogenated in PBS at a concentration of about 2107 cells/ml. The splenocytes lipid peroxide content was measured using a modified version of the method of Fraga.20) Briefly, 200 m l of 0.15 mM NaOH was added and 20 m l of this diluted splenocyte homogenate was incubated at 37 °C in a water bath with or without 10 m l of 0.3 mM phenylhydrazine (in 35% methanol). After 45 min incubation, 10 m l of 1%

thiobarbituric acid in 1% acetic acid (pH 3.5) was added. The mixture was shaken vigorously and then heated at 95 °C for 45 min. After cooling, the samples were centrifuged at 3000 rpm for 10 min at 20 °C and the pink coloration of the supernatant was measured at 532 nm. ORAC Assay The procedures for the ORAC assay on SGE in vitro and splenocytes were modified from the previously described method of Kurihara.21) This assay measures the effectiveness of antioxidant components in splenocytes and SGE, by inhibiting the decline of FL fluorescence induced by a peroxyl radical generator, AAPH. Automated ORAC assay was carried out on a Labsystems Fluoroskan Ascent plate reader (Helsinki, Finland) with fluorescent filters (InfiniteTM F200, excitation wavelength, 485 nm; emission wavelength, 527 nm). Fluorescein was used as a target for free radical to attack and the reaction was initiated with AAPH, and Trolox was used as a control standard. Final results were calculated based on the difference in the area under the fluorescein decay curve between the blank and each sample. Statistical Evaluation One-way analysis of variance (ANOVA) was applied to analyze the different groups, followed by Dunnett’s post-hoc test for pair-wise multiple comparisons. Differences were considered as statistically significant at p0.05. RESULTS Effects of SGE on Splenocyte Counts and Lymphocyte Subsets in Restraint-Stressed Mice As shown in Fig. 3A, restraint stress reduced the spleen cell number significantly (p0.01), and oral administration of SGE (125, 500 mg/kg/d, 5 d) significantly improved the spleen cell number near to normal. In lymphocyte subpopulations, as shown in Fig. 3B, the ratios of CD4 T to CD8 T cells of the normal control

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Fig. 3A. Effects of SGE on the Suppression of Total Number of Spleen Cells Were Induced by Restraint Stress in Mice

Fig. 5. Effects of SGE on the Suppression of NK Cell Activity Induced by Restraint Stress in Mice

C57BL/6J mice were fixed in the restraint cage for 12 h and recovered for 3 d. The results represent the meanS.E. obtained from 7 animals, and significantly different from stress control at ∗∗ p0.05.

C57BL/6 mice were fixed in the restraint cage for 12 h and recovered for 3 d. The results represent the meanS.E. obtained from 7 animals. One LU10 is defined as the number of spleen cells necessary for a 10% specific lysis of 105 target cells. The results represent the meanS.E. obtained from animals, and significantly different from stress control at ∗∗ p0.05.

Fig. 3B.

Effects of SGE on the Mice Spleen Lymphocyte Subsets

Proportions of the lymphocyte populations in mice spleen were determined using a flow cytometer after staining with selected monoclonal antibodies conjugated with fluorescein isothiocyanate (FITC) or phycoerythrin (PE). The results represent the meanS.E. obtained from 7 animals, and significantly different from stress control at ∗∗ p0.05.

Fig. 6. Effects of SGE on the Change of TBARS in Mice Splenocytes Loaded with Restraint Stress C57BL/6 mice were fixed in the restraint cage for 12 h and recovered for 3 d. The results represent the meanS.E. for 7 animals, and significantly different from stress control at ∗∗ p0.05.

Fig. 4. Cytometry Plots in the NK Cytotoxicity Assay Detected by DiO, PI Dying Analysis (A) and (B) are FS/FL1 plot and FL1/FL3 plot in stress control group at effector : target12.5 : 1. Gate H1: lysed effector cell; Gate H2: lysed YAC-1 cells; Gate H3, live effector cell; Gate H4: live YAC-1 cells.

group and stress control group were 2.280.26 and 1.620.14, and those of oral administration of SGE (125, 500 mg/kg/d, 5 d) were 1.800.19 and 1.950.2, respectively. Effects of SGE on NK Cell Activity in RestraintStressed Mice The effects of SGE on the NK cytotoxic activity in spleen was assessed by a flow cytometer assay method using NK-cell-sensitive YAC-1 target cells (as illustrated in Fig. 4). We investigated the effects of SGE on the activity of NK cells 3 d after the stress loading, and results showed that the NK cell activity per spleen was suppressed (p0.01). However, oral administration of SGE (125, 500 mg/kg/d, 5 d) improved the suppressed NK cell activity per spleen significantly (as shown in Fig. 5). Inhibitory Effects of SGE on Splenocyte TBARS in Restraint-Stressed Mice The extent of lipid peroxidation in splenocytes was evaluated by the TBARS method. The basal value of lipid peroxide was 1.70.2 nM in splenocytes at a

Fig. 7. Curves of Fluorescence Decay Induced by AAPH in the Presence of SGE at Different Concentrations Trolox, a water-soluble vitamin E analogue, was used as a control standard. The antioxidative activity of a sample was expressed as the net area under the curve. Data were expressed as means of three experiments.

concentration of about 2107 cells. It markedly increased to 2.90.2 nM/2107 cells after restraint stress. Administration of SGE (125, 500 mg/kg/d) reduced the stress-induced increases in lipid peroxide to 2.40.1 and 2.10.3 nM/2107 cells, respectively (Fig. 6). Antioxidative Capacity of SGE in Vitro and in Vivo The ORAC value was calculated as the ratio of the area under the fluorescence decay curve for 500 m g/ml trolox as a standard. Figure 7 shows the working curves of fluorescein oxidation used as an index of resistance time for the oxidative reaction. The linear relationship between the net area and different concentrations of SGE was evaluated as shown in Fig. 7. In normal mice, the splenocytes ORAC level was

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Fig. 8. Effects of SGE on the Change in Splenocytes ORAC after Restraint Stress The ORAC unit is calculated as UK(SSampleSAAPH)/(STroloxSAAPH), K: samples multiple of dilution, S: area below the fluorescence decay curve. C57BL/6 mice were fixed in the restraint cage for 12 h and recovered for 3 d. The results represent the meanS.E. for 7 animals, and significantly different from stress control at ∗∗ p0.05.

743.041.0 U/2107 cells, while the ORAC value observed 12 h after stress was remarkably decreased (395.135.2 U/2107 cells, p0.05). Compared with the stress control group, the total antioxidative capacity of splenocytes in experimental groups significantly increased. As shown in Fig. 8, average values for ORAC by administration of SGE 125 and 500 mg/kg/d were 546.138.5 and 689.045.2 U/2107 cells, respectively. DISCUSSION In present studies, we found that restraint stress markedly reduced the total number of spleen cells and NK cell activity, and also altered the balance of CD4 T/CD8 T cells by decreasing the ratio of CD4 helper cells. These results are identical with our previous reports, in which we demonstrated that restraint stress suppressed immune responses with a nadir on day 3 after the stress loading.12) Therefore we investigated the effects of SGE on stress-induced immune dysfunction on day 3. Results indicated that the administration of SGE to stressed mice could protect against the reduction of spleen cells number and the disorder of T lymphocyte subsets. Previously, studies suggested that the maintenance of lymphocyte cell number in spleen,10) and keeping the lymphocyte subsets under homeostatic control were both important for maintaining immune functions.22) Accordingly, these results initially indicated that SGE had protective effects on immunity in mice loaded with restraint stress. Furthermore, we found SGE markedly increased in NK cells activity and ORAC level in splenocytes when compared with the restraint control. Our results suggested a correlation between immunologic response and antioxidative capacity in immunocytes. It is generally recognized that ORAC level reflects the antioxidative capacity of water soluble low molecular antioxidants in cells.23) In addition, we measured the contents of MDA to investigate the oxidative situation in spleen. The promotion of lipid peroxidation in restrained mice suggested that the stress caused oxidative stress in the splenocytes. These results indicated that stress can impair immunocompetence via the oxidation. It has been reported that restraint stress was an effective psychological stress model,24) and psychological stress co-occurs with oxidative injury in immune cells,25) which are susceptible to lose their activity due to the effects of ROS.11) It is well-known that NK cells play an important role in immunological response.26) Previous studies have been reported that restraint stress sup-

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pressed function of NK cells, leading to an increased vulnerability to infections or to the occurrence of malignant tumors.12) These reports support our results, by suggesting that SGE markedly improved immunologic response was possibly due to its antioxidative capacity in immune cells. According to traditional Chinese beliefs, Sarcandra glabra is a renowned herb as anti-infectious and anti-inflammatory supplement, which is closely related to immunologic response. Although no references described the mechanism, our results showed that SGE attenuates stress by increasing the number and activity of splenocytes in mice subjected to stress and improving antioxidative processes, through exerting antioxidative activity directly or indirectly. Because the major constituents of SGE are 5-caffeoylquinic acid, 3-caffeoylquinic acid, 4-caffeoylquinic acid, caffeic acid, isofraxidin, 4-O-b -D-glycopyranosyl rosmarinic acid, rosmarinic acid, which are good antioxidants in vitro, and these constituents may mediate its biological effects in vivo.27—30) In present studies, the ORAC value of SGE in vitro also indicates that it is a potent antioxidant. Administration of SGE significantly inhibited the production of stress-induced lipid peroxide and augmented the ORAC activity. These results suggested that SGE improved stress-induced impairment of immunity via its antioxidative property which results in activated effects on anti-infection and anti-inflammation. Considered that the quality of SGE was controlled by HPLC fingerprint, and the previous acute toxicity and mutagenicity studies approved the dietary safety of SGE.31) Therefore we concluded that the water extract of Sarcandra glabra possesses a good efficacy and safety in anti-stress treatment. Acknowledgement This work was supported by the fund of Significant Technology Project (NO.2004Z1-E5011) (Guangdong, China). REFERENCES 1) Ying G. Q., Lu H. Y., Wang H., Shanghai J. Tradit. Chin. Med., 41, 85—87 (2007). 2) Li Y., Zhang D. M., Yu S. S., Li Y., Luo Y. M., J. Nat. Prod., 69, 616— 620 (2006). 3) Zheng W. J., Wang S. F., Chen X. G., Hu Z. D., Talanta, 60, 955—960 (2003). 4) Li M. Y., Zhu C. L., Liu Z., J. Dent. Prev. Treat., 12, 24—26 (2004). 5) Takeda Y., Yamashita H., Matsumoto T., Terao H., Phytochemistry, 33, 713—715 (1993). 6) Ma S. C., Du J., Paul P. H., Deng X. L., Zhang Y. W., J. Ethnopharmacol., 79, 205—211 (2002). 7) Jiang W. Z., Kong X. L., Liang G., J. Guangxi Med. Univ., 18, 39—41 (2001). 8) Aarastad H. J., Thiele D., Seljelid R., Scand. J. Immunol., 33, 461— 472 (1991). 9) Kandil O., Borysenko M., Fed. Proc., 43, 1753—1763 (1984). 10) Bonneau R. H., Sheridan J. F., Feng N. G., Glaser R., Brain Behav. Immun., 5, 170—192 (1991). 11) Fukui Y., Sudo N., Yu X. N., J. Neuroimmunol., 79, 211—217 (1997). 12) Kurihara H., Koda H., Asami S., Kiso Y., Tanaka T., Life Sci., 70, 2509—2520 (2002). 13) Dobbs C. M., Feng N., Beck F. M., Sheridan J. F., J. Immunol., 157, 1870—1877 (1996). 14) Hunzeker J., Padgett D. A., Sheridan P. A., Dhabhar F. S., Sheridana J. F., Brain Behav. Immun., 18, 526—535 (2004). 15) Nakamura K., Matsunaga K., Cancer Biother. Radiopharm., 13, 275— 290 (1998). 16) Gelderman K. A., Hultqvist M., Holmberg J., Olofsson P., Holmdahl R., Proc. Natl. Acad. Sci. U.S.A., 103, 12831—12836 (2006).

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