Arch Pharm Res Vol 31, No 9, 1153-1159, 2008 DOI 10.1007/s12272-001-1282-6 http://apr.psk.or.kr
Synergistic Immunostimulating Activity of Pidotimod and Red Ginseng Acidic Polysaccharide against Cyclophosphamide-induced Immunosuppression
Xiao Fei Du, Cheng Zhe Jiang1, Chun Fu Wu, Eun Kyung Won1, and Se Young Choung1 Department of Pharmacology, Shenyang Pharmaceutical University, Shenyang, China and 1Department of Hygienic Chemis- try and Kyung Hee East-West Pharmaceutical Research Institute, College of Pharmacy, Kyung Hee University, Seoul, Korea
(Received May 6, 2008/Revised July 22, 2008/Accepted September 5, 2008)
We investigated the synergistic effect of combined treatment with red ginseng acidic polysaccharide (RGAP) from Panax ginseng C.A. Meyer and pidotimod in cyclophosphamide-treated mice. The combination of pidotimod and RGAP restored concanavalin A-induced splenic T cell proliferation and LPS-stimulated B cell proliferation significantly. The production of nitric oxide from peritoneal macrophages was increased by the combinations. NK cell activity was increased by RGAP alone or in combination with pidotimod. A synergistic increase in the level of serum IL-12 and interferon- gamm was observed when the combination of the two was used. RGAP alone or in combination with pidotimod modulated the level of serum C-reactive protein to a near-normal level. These results indi- cate that combinations of pidotimod and RGAP are synergistic and suggest that combination therapy using pidotimod and RGAP for improving immune activity may provide an additional benefit over the use of the two drugs by themselves. Key words: Panax ginseng C.A. Meyer, Red ginseng acidic polysaccharide, Pidotimod, Cyclophos- phamide, Immunostimulation, Combination
INTRODUCTION et al., 1989), hypoglycemic activities (Konno et al., 1985; Konno et al., 1983), and antitumor activities (Lee et al., Panax ginseng C.A. Meyer (Korean ginseng, mainly 1997; Moon et al., 1983). Li et al. reported that coarse produced in Korea and northeast China), is a herbal root polysaccharide from Panax quinquefolium could improve and has been extensively used as a traditional medicine lymphocyte transformation in immunosuppressed mice and functional food in the Orient for more than 3,000 (Li, 1996). Among the polysaccharides which are isolated years. The known biochemical and pharmacological acti- from ginseng root, it is considered that acidic polysac- vities of ginseng include antiaging, antidiabetic, anticar- charides were more effective than neutral polysaccharides cinogenic, analgesic, antipyretic, antistress, antifatigue, in their immunostimulating activities. They were found to and tranquilizing activities, as well as the promotion of activate macrophage and NK cells, induce the production DNA, RNA, and protein synthesis activities (Abe et al., of nitric oxide synthase (NOS), and increase the number 1979; Huang, 1989; Sun et al., 1991). We reviewed the of macrophages (Kim et al., 1990; Lim et al., 2004). In biochemical and pharmacological studies of Korean addition, polysaccharides isolated from Panax ginseng ginseng and found them to be mainly concentrated on showed strong stimulation of inducible NOS (iNOS) in ginseng saponins (ginsenosides) as active components. RAW 264.7 cells (Friedl et al., 2001). However, non-saponin components have also received a Pidotimod ((R)-3-[(S)-(5-oxo-2-pyrrolidinyl)carbonyl] great deal of attention for their mitogenic activities (Eun thiazolidine-4-carboxylic acid) is a biological immuno- regulatory modifier synthesized by Poli Industria Chimica, Correspondence to: Se Young Choung, Department of Hygienic Chem- Italy (Magni et al., 1994). It is the first compound of a new istry and Kyung Hee East-West Pharmaceutical Research Institute, Col- class of immunity modifiers with peptide-like structures, lege of Pharmacy, Kyung Hee University, Seoul 130-701, Korea Tel: 82-2-961-0372, Fax: 82-2-961-0372 and has been well characterized and is known to be E-mail: [email protected] chemically pure (Crimella et al., 1994). Treatment with
1153 1154 X. F. Du et al. pidotimod increased macrophage activity and humoral water ad libitum. The experimental protocol was approv- immune functions (Coppi and Manzardo, 1994). Fur- ed by our institutional animal care and use committee. thermore, pidotimod is able to activate NK cells, a front The mice were divided into seven groups of twelve. All line of defense that is capable of quickly expressing itself animals received cyclophosphamide (Choongwre Pharma towards aggressors (Migliorati et al., 1994). Company, Korea) intraperitoneally once per three days to An essential facet of traditional oriental medicine is establish the immunosuppressive animal model, except “combination”. The modern concept of “combination” for control group which received normal saline. The dif- relates to molecules possessing the same or similar func- ferent groups were treated with the following: pidotimod tions, which are combined as a group and which show (Yungjin Pharm. Company, Korea) at a dose of 200 mg/ particular activities (Wu and Wu, 2002). The advantage kg given orally; RGAP at a dose of 300 mg/kg given to the use of the combination would be to reduce drug orally; and the combinations of pidotimod (200 mg/kg) dosages, thereby lowering the potential risk of toxicity, and RGAP at different doses of 33, 100, and 300 mg/kg. and also to reduce the development of drug resistance. Mice were sacrificed after three weeks of administration. Many chemicals including many clinically used drugs Bodyweight gain (percentage) and relative weight of the when exposed under certain doses and durations suppress spleen (organ weight/100 g of bodyweight) were determin- the immune response and may cause increased incidences ed for each animal. of infectious diseases and cancers (Barron et al., 2003). Therefore, cyclophosphamide was used as an immuno- Peritoneal macrophage culture suppressant in this study and the synergistic effect of the Murine peritoneal cells were harvested by peritoneal combined treatment of red ginseng acidic polysaccharide lavage with 5 mL of PBS, according to a procedure re- (RGAP) from Panax ginseng C.A. Meyer and pidotimod ported previously (Komutarin et al., 2004). The cells was investigated in BALB/c mice. The purpose of this were kept cold on ice until transferred and plated in a study has been to confirm the synergistic effect of the complete medium consisting of RPMI-1640 with 10% combination to provide a reference for the combinational FBS, 50 µM 2-mercaptoethanol, 100 U/mL penicillin, and application of western and oriental medicines. 100 mg/ml streptomycin. After incubating at 37oC in 5%
CO2 for 2 h, the non-adherent cells were removed by MATERIALS AND METHODS washing twice with cold PBS. The adherent cells were used to perform the following experiment. Materials RGAP was provided by Korean Tobacco and Ginseng Assay of NO production and cell viability company (Korea). It was isolated from Panax ginseng Peritoneal macrophages (2 × 105 cells per well) from C.A. Meyer as described previously (Kyeong, 2000). BALB/c mice were cultured in the absence or presence of RGAP was confirmed to be the acidic polysaccharide LPS (20 µg/mL). NO synthesis was examined by assay- fraction composed of about 56.9% uronic acid, 28.3% ing the culture supernatants for nitrite, the stable reaction neutral sugar and less than 1% protein. The analysis of product of NO with molecular oxygen, using the Griess component sugars in RGAP by GC revealed that the reagent (An et al., 2006). Briefly, 50 µL of supernatant polysaccharides in RGAP contained about 51.8 mole % was removed from the conditioned medium and incubat- glucuronic acid, 26.1 mole % glucose, and 5.1 mole % ed with the same volume of modified Griess reagent at galacturonic acid as major components, and arabinose, room temperature for 15 min. The absorbance at 540 nm rhamnose, and galactose as minor components. One milli- was measured by means of an automatic microplate gram of RGAP contained less than 0.006 EU (endotoxin reader (JASCO, V-530). units) of endotoxin, which did not affect the experimental Cell viability was evaluated by an MTT reduction assay result obtained by RGAP. (Byun et al., 2006). After various treatments, the cells All the reagents were purchased from Sigma-Aldrich were co-incubated with MTT (0.25 mg/mL) for 4 h at (USA) except for those as indicated. 37oC. The formazan crystals in the cells were solubilized with DMSO. The level of MTT formazan was determined Animals and treatments by measuring the optical density at a wavelength of Specific pathogen-free male BALB/c mice (six-week- 540 nm. old) were purchased from Koatech Company, Korea. They were acclimatized for at least three days before the Assay of splenocyte proliferation experiment. Mice were housed in an air-conditioned SPF BALB/c mice were sacrificed and their spleens were room with a 12-h light/dark cycle, and provided with removed aseptically. A single cell suspension was pre- Certified Rodent Diet (Koatech Company, Korea) and tap pared after cell debris and clumps had been removed. Pidotimod and Red Ginseng Acidic Polysaccharide 1155
Splenocytes were washed three times with PBS and then Table I. Effects of pidotimod and RGAP alone or by com- resuspended in RPMI-1640 medium containing 5% FBS. bination against cyclophosphamide-induced immunosuppres- They were seeded in 96-well plates at 2 × 105 cells per sion on bodyweight gain (percentage) and relative organ weight (organ weight/100 g of bodyweight) of spleen well and incubated in the absence or presence of either bodyweight/ Spleen/bodyweight LPS (1 µg/mL) or Concanavalin A (Con A, 0.25 µg/mL). Group ∆ After the cells had been cultured for 72 h at 37oC in a bodyweight (%) (%) a a humidified 5% CO2 incubator, the number of proliferating control 17.31±1.19 4.07±0.14 cells was determined by the MTT assay at a wavelength Cy 07.51±0.51 b 2.89±0.04 b of 540 nm. Pi+Cy 15.63±2.47 a 3.32±0.08 c R(300) +Cy 10.92±1.12 b 3.42±0.19 c Assay of splenic NK cell activity Pi+R(33)+Cy 14.26±1.91 a 2.98±0.08 b After the preparation of a single splenocyte suspension, Pi+R(100)+Cy 17.05±2.40 a 3.38±0.15 c equal volume of Histopaque® was added and centrifuged Pi+R(300)+Cy 14.42±1.25 a 3.40±0.14 c at 400 g for 30 min at room temperature to separate Cy: cyclophosphamide; Pi: pidotimod; R: RGAP. The results are mononuclear cells. The upper layer to within 0.5 cm of presented as the mean±S.E.M. of 12 animals per group. Values the opaque interface was transferred and seeded at 2 × with different superscripts are significantly different at P<0.05. 105 cells/mL followed by washing with PBS. YAC-1 cells were used as target cells. The effecter-to-target cell ratio rate of increase of bodyweight to just 7.51% compared to was 50:1. After the cells had been co-cultured for 72 h at the control value of 17.31%. Bodyweight was significantly o 37 C in humidified 5% CO2, the number of proliferating restored when mice were treated with pidotimod alone or cells was determined by the MTT assay. Splenic NK cell in combination with RGAP. activity was calculated as follows: Relative spleen weight was extremely reduced to 2.89% by cyclophosphamide. Treatments with pidotimod and NK cell activity RGAP individually increased the relative spleen weight
=[(ODY +ODNK − OD(Y+NK))/(ODY +ODNK)] × 100% to 3.32% and 3.42%, respectively. When pidotimod was combined with RGAP, the spleen index was also signifi- where ODY = optical density of wells containing YAC-1 cantly restored. cells only; ODNK = optical density of wells containing
NK cells only; and OD(Y+NK) = optical density of wells Effect of pidotimod and RGAP on NO production by containing both YAC-1 cells and NK cells. LPS-stimulated mouse peritoneal macrophages To determine the effect of pidotimod and RGAP on the Assay of cytokines and CRP in serum production of NO of mouse peritoneal macrophages, the Blood was obtained from the posterior vena cava and cells were stimulated by LPS (20 µg/mL) for 24 h. The centrifuged at 3,000 rpm and 4oC for 15 min. The amounts results indicated that LPS and/or samples did not affect of IL-12, IFN-γ, and CRP in the supernatant were analyzed the viability of macrophages in the present study (data by an enzyme-linked immunosorbent assay using com- not shown). Fig. 1 shows that the stimulation of perito- mercial ELISA system assay kits (Pierce Biotechnology neal macrophages with LPS resulted in a manifold in- Inc., USA). crease in NO production compared to the control group in the absence of LPS. Cyclophosphamide significantly Statistical analysis inhibited the LPS-stimulated NO production by 35.9%, The data were expressed as mean±S.E.M. The groups compared to the LPS-stimulated control group. The mean were analyzed using an analysis of variance (ANOVA) values for the pidotimod- or RGAP-treated groups were followed by the Dunnett’s test for multiple comparisons not significantly different from the mean value of the (SPSS version 13.0). A p value of less than 0.05 was cyclophosphamide-treated group. However, NO production considered significant. was significantly increased by combinations of pidotimod (200 mg/kg) and RGAP at doses of 100 and 300 mg/kg RESULTS to 86.1% and 87.1%, respectively, compared to the single treatment. Effect of pidotimod and RGAP on bodyweights and spleen weights of mice Effect of pidotimod and RGAP on splenocyte prolif- The effects of pidotimod and RGAP on the bodyweights eration and spleen weights of mice are presented in Table I. The effects of pidotimod and RGAP on mitogen-stimu- Cyclophosphamide elicited a significant reduction in the lated murine spleen lymphocyte proliferation are pre- 1156 X. F. Du et al.
sented in Fig. 2(a) and (b). It is apparent that treatment with cyclophosphamide significantly reduced Con A- or LPS-stimulated cell proliferation to 47.7% and 40.8% compared to the control group in the absence of Con A or LPS, respectively. The results also show that the strongly diminished Con A-induced proliferative response in cyclo- phosphamide-treated mice was greatly recovered by pido- timod, RGAP, and the combinations of the two. However, the decrease in B cell proliferation was only significantly restored by the combination of pidotimod and RGAP (300 mg/kg).
Fig. 1. Effects of pidotimod and RGAP alone or by com- Effect of pidotimod and RGAP on splenic NK cell bination against cyclophosphamide-induced immunosuppres- activity sion on NO production by LPS-stimulated mouse peritoneal The splenic NK cell activity was determined by examin- macrophage. The results are presented as the mean ± S.E.M. ing cytotoxicity towards YAC-1 cells by means of an MTT of 12 animals per group and expressed as percentage values, assay. As shown in Fig. 3, cyclophosphamide significantly taking the LPS-treated control group as 100%. Values with inhibited the NK cell activity to 59.4%. When the im- different superscripts are significantly different at P<0.05. munosuppressed mice were treated with pidotimod and RGAP individually or simultaneously, the decline in the NK activity was significantly reversed compared to the cyclophosphamide-treated mice.
Effect of pidotimod and RGAP on cytokines and CRP Mouse serum IL-12, IFN-γ, and CPR were evaluated. As shown in Table II, cyclophosphamide injection caused significant reductions in the levels of IL-12, IFN-γ, and CRP to 67.0%, 82.8%, and 50.0%, respectively. In con- trast to the individual treatments, combined treatment of
Fig. 2. Effects of pidotimod and RGAP alone or by com- Fig. 3. Effects of pidotimod and RGAP alone or by com- bination against cyclophosphamide-induced immunosuppres- bination against cyclophosphamide-induced immunosuppres- sion on mice splenic T cell (a) and B cell (b) proliferations. sion on mice splenic NK cell activity. The results are The results are presented as the mean ± S.E.M. of 12 animals presented as the mean±S.E.M. of 12 animals per group and per group and expressed as percentage values taking control expressed as percentage values taking control group as 100%. group with Con A or LPS as 100%. Values with different Values with different superscripts are significantly different at superscripts are significantly different at P<0.05. P < 0.05. Pidotimod and Red Ginseng Acidic Polysaccharide 1157
Table II. Effects of pidotimod and RGAP alone or by com- against cyclophosphamide. In addition, the combinations bination against cyclophosphamide-induced immunosuppres- are suggested to have greater sensitivity for the T cells of sion on mice serum IL-12, IFN-γ, and CRP levels splenocytes than the B cells, which was manifested in a Group IL-12 IFN-γ CRP better T-cell-mediated immunostimulating effect. NK cell control 100.0±12.6 a 100.0±5.3 a 100.0±14.5 a activity of splenocytes was determined to assess the effects Cy 067.0±05.5 b 082.8±1.9 b 050.0±10.6 b of samples on innate immune responses. Shin reported Pi+Cy 061.0±04.0 b 088.4±1.9 b 052.4±10.9 b that RGAP restored NK cell activity suppressed by R(300) +Cy 082.3±07.1 ab 096.9±3.5 a 083.4±09.8 ac paclitaxel (Shin et al., 2004). Our results have further Pi+R(33)+Cy 073.7±07.3 b 099.8±3.8 a 042.5±06.7 b shown that treatments with pidotimod and RGAP, both Pi+R(100)+Cy 127.9±08.3 c 100.5±4.7 a 059.4±12.1 b individually and in combination, can enhance NK cell Pi+R(300)+Cy 079.8±04.7 ab 102.1±4.9 a 086.2±11.5 ac activity. Macrophages are an integral part of the immune Cy: cyclophosphamide; Pi: pidotimod; R: RGAP. The results are presented as the mean±S.E.M. of 12 animals per group. Values system, playing an important role in the initiation and with different superscripts are significantly different at P<0.05. regulation of immune response (Gordon, 1998). Macro- phage activation by LPS, the major component of the cell walls of gram-negative bacteria, results in the release of pidotimod and RGAP (100 mg/kg) acted synergistically several inflammatory mediators such as NO and the pro- in enhancing the IL-12 level that had been decreased by inflammatory cytokines (An et al., 2006). NO is a highly cyclophosphamide. A significant increase in levels of reactive molecule produced from a guanidine nitrogen of serum IFN-γ was observed when mice were treated with NOS., The iNOS, the high-output isoform, is highly ex- RGAP. Pidotimod showed no effect like this until it was pressed in LPS-activated macrophage of the three NOSs combined with RGAP. Similarly, RGAP alone or in com- (Petros et al., 1994), which is mediated by cytokines, bination with pidotimod induced elevations in serum CRP INF-γ, and tumor necrosis factor alpha (TNF-α) (Kroncke against cyclophosphamide and restored CRP to near- et al., 1998). Low concentrations of NO from activated normal levels. macrophages are beneficial, and along with other reactive nitrogen intermediates, are responsible for cytostatic and DISCUSSION cytotoxic activity against infectious organisms and tumor cells. In addition, NO plays a regulatory role in the func- Integrative medicine, combining the theories and treat- tion of natural killer cells and in the expression of cytokines ments of both western medicine and traditional medicine, such as IFN-γ and transforming growth factor-β (Bogdan has become the developing trend of medicine along with et al., 2000). A combination of pidotimod and RGAP has the social development since 1970’s (Feng, 1977; Liu, been shown to significantly restore NO production sup- 2003). Previous reports have indicated some progress in pressed by cyclophosphamide, which supports the results pre-clinical and clinical research (Chen et al., 2003; Wang that they also increased INF-γ production in a dose- and Zhou, 2003). In this study, we have demonstrated the dependent manner in this study. synergistic effect of a combination of RGAP and pido- IL-12 is known as a T cell stimulating factor, which can timod in immunosuppressant models induced by cyclo- stimulate the growth and function of T cells. It is involv- phosphamide. The action of cyclophosphamide is primarily ed in the differentiation of naïve T cells into Th1 cells directed towards the depletion of B lymphocytes. Cyclo- and stimulates the production of IFN-γ and TNF-α from phosphamide has also been reported to induce the de- T and NK cells. Pidotimod and RGAP acted synergisti- pletion of T cells (Mahiou et al., 2001; Rollinghoff et al., cally in enhancing the IL-12 level, which was decreased 1977) and the deficiency of macrophages (Santosuosso et by cyclophosphamide, whereas individual treatments al., 2002). Accordingly, cyclophosphamide impaired all showed no enhancing effect. Moreover, when combined of the immune parameters examined in this study. with RGAP, pidotimod was able to fully restore the Lymphocyte proliferation induced by Con A is commonly decrease of IFN-γ induced by cyclophosphamide. IFN-γ used as a method of detection for T lymphocyte im- is involved in the regulation of immune and inflammatory munity in vitro, while lymphocyte proliferation induced responses. It is produced in activated T cells and NK by LPS is often used to detect B lymphocyte immunity cells. Thus, our findings suggest that RGAP may include (Cerqueira et al., 2004). Cyclophosphamide-induced sup- some factors capable of inducing T helper activity and pression of ConA-induced T-lymphocyte proliferation and up-regulating NK cell activity. CRP is a plasma protein, LPS-induced B-lymphocyte proliferation was identified in an acute phase protein produced by the liver (Lind, this study. Combined treatment with pidotimod and RGAP 2003). It is also believed to play an important role in in- significantly enhanced the proliferation of splenocytes nate immunity as an early defense system against infec- 1158 X. F. Du et al.
tions. It was found that RGAP is capable of preventing Y., Efficacy of traditional Chinese medicine and Western the decrease of CRP caused by cyclophosphamide and medicine in the treatment of Ureaplasma urealyticun and modulating it to a near-normal level, which suggests that Chlamydia trachomatis infectious chronic prostatitis (report it does not cause any inflammatory reaction. of 48 cases). Zhonghua Nan Ke Xue, 9, 202-206 (2003). In summary, the present study has demonstrated that Coppi, G. and Manzardo, S., Experimental immunological the combination of pidotimod and RGAP acts synergisti- screening tests on pidotimod. Arzneimittelforschung, 44, cally in enhancing the immunostimulating effect in cyclo- 1411-1416 (1994). phosphamide-induced immunosuppressed mice. Pidotimod Crimella, T., Orlandi, R., Bocchiola, G., Anders, U., and and RGAP used in combination significantly restored the Stradi, R., Analytical and chemical profile of pidotimod. spleen index decreased by cyclophosphamide, and also Arzneimittelforschung, 44, 1405-1410 (1994). increased Con A- and LPS-induced splenocyte prolifera- Eun, S. M., Hung, N. K., Nam, L. K., and Cheung, K. Y., tion. The cotreatments also enhanced NK cell activity and Growth promoting activities of a macromolecular fraction NO production by peritoneal macrophages. IL-12 and from fresh ginseng. Korean J. Ginseng Sci., 13, 215-221 IFN-γ in serum were restored by combined treatments. In (1989). view of these data the finding that combined therapy with Feng, T. Y., Taking the road of combining traditional Chinese pidotimod and RGAP in our immunosuppressed model is and western medicine. Chin Med J (Engl), 3, 8-12 (1977). of particular importance. The effect of combination of Friedl, R., Moeslinger, T., Kopp, B., and Spieckermann P. G., pidotimod and RGAP on the humoral immune system is Stimulation of nitric oxide synthesis by the aqueous extract already being addressed and our findings will follow in of Panax ginseng root in RAW 264.7 cells. Br. J. Pharmacol., future communications. 134, 1663-1670 (2001). Gordon, S., The role of the macrophage in immune regulation. ACKNOWLEDGEMENTS Res. Immunol., 149, 685-688 (1998). Huang, Y. S., Effect of ginsenosides Rb1 and Rg1 on lipid This research was supported by Science Research peroxidation of rat in vitro. Zhongguo Yi Xue Ke Xue Yuan Foundation for International Special Project, Kyung Hee Xue Bao, 11, 460-462 (1989). University, (KHU-20071063). Kim, Y. S., Kang, K. S., and Kim, S. I., Study on antitumor and immunomodulating activities of polysaccharide frac- REFERENCES tions from Panax ginseng: Comparison of effects of meutral and acidic polysaccharide fraction. Arch. Plarm. Res., 13, Abe, H., Arichi, S., Hayashi, T., and Odashima, S., Ultrastruc- 330-336 (1990). tural studies of Morris hepatoma cells reversely transform- Komutarin, T., Azadi, S., Butterworth, L., Keil, D., ed by ginsenosides. Experientia, 35, 1647-1649 (1979). Chitsomboon, B., Suttajit, M., and Meade, B. J., Extract of An, H. J., Jeong H. J., Um J. Y., Kim H. M., and Hong S. H., the seed coat of Tamarindus indica inhibits nitric oxide Glechoma hederacea inhibits inflammatory mediator release production by murine macrophages in vitro and in vivo. in IFN-gamma and LPS-stimulated mouse peritoneal macro- Food Chem. Toxicol., 42, 649-658 (2004). phages. J. Ethnopharmacol, 106, 418-424 (2006). Konno, C., Murakami, M., Oshima, Y., and Hikino, H., Isola- Barron, M. G., Heintz, R., and Krahn, M. M., Contaminant tion and hypoglysemic activity of panaxans Q,R,S,T and exposure and effects in pinnipeds: implications for Steller U,glycans of Panax ginseng roots. J. Ethnopharmacol, 69- sea lion declines in Alaska. Sci. Total Environ., 311, 111- 74 (1985). 133 (2003). Konno, C., Sugiyama, K., Kano, M., Takahashi, M., and Bogdan, C, Rollinghoff, M., and Diefenbach, A., The role of Hikino, H., Isolation and hypoglycemic activity of panaxans nitric oxide in innate immunity. Immunol. Rev., 173, 17-26 A,B,C,D and E,glycans of Panax ginseng roots. Planta (2000). Medica, 434-436 (1983). Byun, J. A., Ryu, M. H., and Lee, J. K., The immunomodula- Kroncke, K. D., Fehsel, K., and Kolb-Bachofen, V., Inducible tory effects of 3-monochloro-1,2-propanediol on murine nitric oxide synthase in human diseases. Clin. Exp. Immunol., splenocyte and peritoneal macrophage function in vitro. 113, 147-156 (1998). Toxicol. In Vitro, 20, 272-278 (2006). Kyeong, M. P., Tae, C. J., Young, S. K., Han, J. S., Ki, Y. N., Cerqueira, F., Cordeiro-Da-Silva, A., Gaspar-Marques, C., and Jong, D. P., Immunomodulatory effect of acidic poly- Simoes, F., Pinto M. M., and Nascimento M. S., Effect of saccharide fraction from Korean red ginseng (Panax ginseng). abietane diterpenes from Plectranthus grandidentatus on T- Natural Product Sciences, 6, 31-35 (2000). and B-lymphocyte proliferation. Bioorg. Med. Chem., 12, Lee, Y. S., Chung, I. S., Lee, I. R., Kim, K. H., Hong, W. S., 217-223 (2004). and Yun, Y. S., Activation of multiple effector pathways of Chen, Z. Q., Shang, X. J., Ye, Z. Q., Lu, F. E., and Huang, G. immune system by the antineoplastic immunostimulator Pidotimod and Red Ginseng Acidic Polysaccharide 1159
acidic polysaccharide ginsan isolated from Panax ginseng. function. Arch. Pharm. Res., 123-129 (1983). Anticancer Res., 323-331 (1997). Petros, A., Lamb, G., Leone, A., Moncada, S., Bennett, D., Li, Y. Ma, X., Qu, S., Wang, L., Du, B., and Wei, Z., Effect of and Vallance, P., Effects of a nitric oxide synthase inhibitor CPPQ (coarse polysaccharide from Panax quinquefolium) in humans with septic shock. Cardiovasc. Res., 28, 34-39 on immunologic function of immunosuppressive mice (1994). induced with cyclophosphamide. Baiqiuen Yike Daxue Rollinghoff, M., Starzinski-Powitz, A., Pfizenmaier, K., and Xuebao, 137-139 (1996). Wagner, H., Cyclophosphamide-sensitive T lymphocytes Lim, T. S., Na, K., Choi, E. M., Chung, J. Y., and Hwang, J. suppress the in vivo generation of antigen-specific cyto- K., Immunomodulating activities of polysaccharides isolated toxic T lymphocytes. J. Exp. Med., 145, 455-459 (1977). from Panax ginseng. J. Med. Food, 7, 1-6 (2004). Santosuosso, M., Divangahi, M., Zganiacz, A., and Xing, Z., Lind, L., Circulating markers of inflammation and atheroscle- Reduced tissue macrophage population in the lung by rosis. Atherosclerosis, 169, 203-214 (2003). anticancer agent cyclophosphamide: restoration by local Liu, L. M., Establishment and development of clinical theory granulocyte macrophage-colony-stimulating factor gene for integration of traditional Chinese and western medicine. transfer. Blood, 99, 1246-1252 (2002). Zhong Xi Yi Jie He Xue Bao, 1, 244-246 (2003). Shin, H. J., Kim, Y. S., Kwak, Y. S., Song, Y. B., Kim, Y. S., Magni, A., Signorelli, G., and Bocchiola, G., Synthesis and and Park, J. D., Enhancement of antitumor effects of preliminary pharmacological evaluation of pidotimod, its paclitaxel (taxol) in combination with red ginseng acidic enantiomer, diastereomers and carboxamido derivatives. polysaccharide (RGAP). Planta Med, 70, 1033-1038 (2004). Arzneimittelforschung, 44, 1402-1404 (1994). Sun, X. B., Matsumoto, T., Kiyohara, H., Hirano, M., and Mahiou, J., Walter, U., Lepault, F., Godeau, F., Bach, J. F., and Yamada, H., Cytoprotective activity of pectic polysacchari- Chatenoud, L., In vivo blockade of the Fas-Fas ligand des from the root of panax ginseng. J. Ethnopharmacol., pathway inhibits cyclophosphamide-induced diabetes in 31, 101-107 (1991). NOD mice. J. Autoimmun., 16, 431-440 (2001). Wang, X. L. and Zhou, Y. H., Comments on treatment of Migliorati, G., Nicoletti, I., and Riccardi, C., Immunomo- severe acute respiratory syndrome by integrated traditional dulating activity of pidotimod. Arzneimittelforschung, 44, Chinese and western medicine. Zhong Xi Yi Jie He Xue 1421-1424 (1994). Bao, 1, 155-157 (2003). Moon, C. K., Sim, K. S., Lee, S. H., Park, K. S., Yun, Y. P., Wu, F. E. and Wu, W., Development of new Chinese medicine Ha, B. J., and Lee, C. C., Antitumor activity of some phy- with