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34 1-346, 1997 © 1997 by Gustav Fisch er Verlag
Effect of Vietnamese Ginseng on the phagocytosis in vitro and in vivo
N. 1. 1. HUONG" 3, K. MATSUMOT01, N. 1. NHAM3, N. H. QUANG3, N. M. DUC 3, K. YAMASAKI2 and H. WATANABE1*
Department of Pharm acology, Research Institute for Wakan -Yaku (Oriental M edicines), To yama M edical and Phar maceutical University, Japan 2 Department of Biologica l Acti ve Subs tance s, Institute of Phar maceuti cal Sciences, Hi roshim a Un iversity Scho ol of M ed icine, J apan 3 T he Science-Pro d uctio n Cent re of Vietna mese Ginseng , Ho Chi M in h Universit y of Medicine and Pha rmacy, Vietnam
Th e effects of Vietnamese ginseng cru de extract (VG extr act ), tot al sapo nin (VG saponin ) and its major saponin co mpo nent, majonoside-R 2, on phagocytosis were exam ined in mice by bactericidal and car bo n clear an ce tests. Escherichia coli (E. coli) ATCC 25 922 was used to ind uce th e ac ute tox icity and activa te the phagocytic activi ty of ph agocytes in both in vitro and in vivo bactericidal tests. Pretreatment with VG extrac t (500 mg/kg, or al administr ati on , p.o.) and majonoside-R2 (50 mg/kg, intrape rito neal administration, i.p.) protected the animals from th e acute toxicity of E. coli AT CC 25922 and significantly increased the phagoc ytic index in bot h in vitro an d in vivo bactericidal tests. Moreover, VG extract (100- 500 mg/kg, p.o.), VG sapo nin (25 rug/k g, i.p.) and majonoside-R2 (10 mg/kg, i.p.), as well as zymosan A, a non-specific phago cytic stimulant, also incr eased the ph agocytic ind ex eva luated by th e carbon clearance test. T hese results indicate th at Vietn amese ginse ng enhances the phagocy tic activity of phagocyres, an d suggest that majonoside-R2 plays an import ant rol e in th is effecr. Key words: Vietn am ese ginseng, maj on oside-R2 , phagocytos is, Escherichia coli, ca rbon clea rance test.
. Vietn amese ginseng (VG) iPanax uietname nsis H a et Grus hv. Araliaceae ), a new Panax species, has been used as a "secret medicinal plant " of the Seda ng ethn ic minor ity in Vietn am for the tre atment of serious illness and as a potent pan acea in traditional med icine. Pharm aco logical studies on VG have sho wn th at thi s ginseng prod uces th e stimulato ry effects on th e central nervo us system, and exhibits ant ifatigue, antioxidant, antistress and antibacterial act ivities (N harn, 1989 ). Th e antibacteria l effect on pathogenic Streptococci appears to be cha rac teristic of VG, since other ginsengs such as Panax ginseng have not been reported to have such an effect. M or eover, polyacetylenic compound s of VG exhibit a pot ent suppressive action on Gra m-po sitive cocci and derrnatophytes (N ham et al., 1995 ). Sapo nins isolated from med icinal plants have been fou nd to exhibit cyto toxic, a ntitumor and imm unomodulating activities (Lacaille-Dubois and Wagner, 1996 ). Singh et al. (198 4) were the first to show ex perimenta lly that Panax ginseng enhanced th e immune respo nse in mice. Moreover, Panax ginseng and its co mpo nents reportedly exhi bit irnmun om odu lating activity (Keranova et al., 1990; M at sud a et al., 1987; Saita et al. 1993; Sun et a1. 1994), and reverse stress-induced immunosuppression (Saito and Okamoto, 1996 ). However, until now no report is available on th e ef-
1995). It is well known that immune responses are produced primarily by phagocytic leukocytes.) or in saline for intraperitoneal administration (i. the function of R. The concentration of 5xl0 7 bacteria/ml that caused the 0% of lethal level was used for the in uiuo bactericidal test. Animals Swiss albino male mice (20-25 g. respectively. into mice in a constant volume of 10 ml/kg.S. Then. Our preliminary experiment indicated that the lethal level was more than 85% at concentrations of more than 3xl0 8 bacteria/ml.p. The mixture was washed twice by Hanks solution and centrifuged at 800 rpm for 5 min to discard the non-phagocytosed bacteria.29%. T. The acute toxicity of Escherichia coli ATCC 25922 Escherichia coli ATCC 25922 (E. libitum. Huong et al.S. coli.2. and :2: 75% represents no protective effect (-). 1995.1 ml of leukocytes and 0.o.1 ml of 3 x 10 8 E.E. The animals were housed in groups of 20 per cage for at least 1 week before starting the experiments. coli by modifying the methods previously reported (Ben- .. the leukocytes remained in the supernatant fluid. coli ATCC 25922 solution was injected i. The survival ratio of < 50%. and immunostimulating effect (++).o. and settled for 20 min at room temperature. and that majonoside-RZ. 1976). twice a day for 5 days before (11 times in total) and for 3 days after (5 times in total) i. Shizuoka. By this sedimentation. Thus. coli ATCC 25922) was suspended in saline and various concentration of E. :2: 50%. respectively. and male BALB/c mice (20-25 g. Materials and Methods Materials VG extract.). in experimental animals has been evaluated by the carbon clearance test and the increased carbon clearance indicates an increased phagocytic activity.27% and 22. such as neutrophils and eosinophils. One hour after the last administration. Yields of VG extract. Japan SLC. the purpose of this study is to investigate. smeared on glass slides and then rapidly dried in air. Wagner et al. (1980). Pasteur Institute of Ho Chi Minh City. 1989). Housing conditions were thermostatically maintained at 24 ± 1 °C In vivo phagocyticactivity of leukocytes (bactericidal assay) We determined the kinetics of blood clearance of injected E. blood sample was taken aseptically from the tail vein into a tube containing heparin. These samples were stained with Giemsa stain and scored under the oil immersion objective for the percentage of leukocytes containing bacteria per 100 leukocytes (% phagocytic activity) (Williams and Chase. The number of surviving animals was measured 72 h after injection. 1976). (1994) suggested that the improvement of general immune defences is one of the index to evaluate the antistress activity of medicinal plants and their biologically active components. respectively.p. contributes to the effect of VG (Huong et al. 1996). Furthermore. Moreover. for 5 days (11 times in total) before assay.. VG saponin and majonoside-R2 were dissolved in distilled water for oral administration (p. Food and water were given ad fect of VG or its saponin components on the immune system. The upper one third supernatant layer of the erythrocyte column (sediment) that was rich in neutrophil polymorphonuclear leukocytes was collected.67% in VG extract and VG saponin. In vitro phagocytic activity of leukocytes (microscopic assay) Test drugs were administered p. We have previously demonstrated that VG attenuates pathophysiological changes caused by psychological stress and conditioned fear in mice.E. Japan) were used for the experiments. VG extract. Test drugs were administered p. 13. immunostimulating effect (+).E. VG saponin and majonoside-R2 were prepared as previously reported (Huong et al.p. whether VG and majonoside-R2 modulate the phagocytic activities of neutrophils and R. Quantitative analysis using high-performance liquid chromatography revealed that the contents of majonoside-RZ were 8.. We selected this concentration for the in vitro bactericidal test.o.342 N. VG saponin and majonoside-R2 were 41. The concentration of bacteria that produced the lethal levels of 85-90% and 0% was selected for in vitro and in vivo phagocytic experiments. T. The end point of the entire phagocytic process performed by phagocytic cells in the circulation is measurable through the bactericidal assay (Williams and Chase. and a relative humidity of 55 ± 5% with a 12 h light-dark cycle (lights on: 0730-1930). 0.p.p.. respectively.S. injection of 3xl0 8 cells/ml of E. or i. a major constituent of VG which has not been isolated from Panax ginseng (Due et al. The protective effect of the test drugs was evaluated as described by Delaveau et al. Samples of phagocytes were removed from the suspension. 1993). or i. usually plays an important role in keeping the homeostasis of the human body and in the cellular host defense mechanism (Silverstein et al. the network of phagocytic tissue macrophages which. act as a first line of defense against infection. was previously termed "the reticuloendothelial system" or R. coli suspension were mixed in test tubes and incubated at 37°C for 20 min. polymorphonuclear leukocytes. Vietnam). with the use of bactericidal and carbon clearance tests. together with endothelial cells and polymorphs. In these responses.2 and 5.
01 vs. or i... respectively. 50 100 250 500 Veh icle i. * P < 0. After incubation at 37°C for 24 h.5 .7 33 . n = 20 ). Table 1. The weig ht of these or gan s was ex pressed as mg % (organ weig ht/1 00 g body weight) .p. Zy mosan A (Sigma Chemi cal Co . injection of 5 x 10 7 Escherichia coli AT CC 25922. respectively. 4 4 870 . for 5 da ys befor e an d 3 days after i. 335. animals were co ntinuo usly administered test dru gs for 3 days after E.. Th e ca rbon suspension wa s injected i. 9 4 38. coli injecti on and th en liver an d spleen w ere remove d . Briefly. 30 2. H annover..p. Gunther Wagn er.1 .05 (Fisher's Exac t probability test) Table 2.4 " 5974 . In some experiments..0 63 . The phagoc ytic ind ex K is calculated from the formu la: K = (log C 1 -log C 2 ) / (t 2 .8.o.3 26 . Co lloidal carb on (Pelikan drawin g ink.. Th e protect ive effect of test d rugs wa s expressed as the surv ival ra tio (%) and immunostim ulating index . 10 50 111 6 2/15 4/15 5115 12/16 ': ' 1/2 0 7/1 1'" 10112 * 6. of anima ls survi ved after 72 h Sur vival ra tio (%) Immunostimulating index Vehicle p.3 13.p. coli ATCC 25 922. the num ber of residual bacterial colonies (bac teria/ml blood sample) was determined.o. Germ an y) was centrifuged at 5.6"* ## Spleen 42 7.0 5..p.3 + + ++ VG ex tra ct and maj on oside-R 2 were ad ministered p. 1959.:: 5 0%.o. vehicle-treated group (Student 's z-rest. naive group an d ## P < 0.6 83. In vivo phagocytic activity of macrophages The ph agocytic activity of macr ophages was estimated by th e carbo n clearance test (Biozzi et a!.5 % gelatin as a sta bilizer. with an 18 h suspension of 5 x 10 7 E.Effect of Vietnamese Ginseng on the phagocytosis in vitro an d in vivo acerraf et al. 'f" P < 0. Animals were sacrificed 72 h after E.. H ere: th e surv ival ratio of < 50 % .8 ': " ' ## 500 VG extract was administered daily for 5 days before and 3 days after i. 'f P <0. Effect of VG extract o n cha nges in the weight of mouse liver and spleen ca used by Escherichia coli injectio n Treatm ent Naive Vehicl e VG extra ct Doses (mg/kg.. Okirnura et a!.) Organ we ight (mg %) Liver 41 03. 59.1 . 1989).o.5 (t l ) and 10 min (t 2 ) Statistical analysis Fisher's Exact probability test was used to anal yze the survival rati o (%) following E.5 624 . coli injection and killed to remo ve liver and spleen.p. and z 75 % was exp ressed as no protective effect (-) . USA) was dissolved in saline and injected i.p. injection of 3 x 10 8 Escherichia coli ATCC 25922. 71. Effects o f Vietn a mese ginseng ex tract and majonoside-R 2 on the acu te to xicit y o f Escherichia coli AT CC 25922 in mice. Ongsakul et a!. an d irnmun ostimulati ng effect (++).6.. resp ectively. Th e blood samples were hemol yzed in 2 ml 0.05.. Venou s blood (25 ul) was taken by retro-orbiral veno us plexu s pu ncture 0.000 rpm for 15 min and diluted 1:3 in sterilized saline conta ining 1. Treatment Dos es mg/kg No. 1985 ). 341.1 % sodium ca rbonate and th e optica l density of the solutio n at 600 nm wa s measured using a Beckman DU 64 0 spectro photo meter to determine th e concent ration of carb on in th e peripheral blood at the time sta ted. VG extr act p.. 1 ml were tak en asepticall y from the tail vein 1 hand 4 h after injection and were spread on MacConkey agar follo wing appropriate dilution in trypticase soy bro th.p.t Il Here: C] and C 2 are the blood concentrations of carbon at time t) and t 2 • Test drugs were administered p.p. p. The weight of these org an s was calcul ated as the percentage of body weight (mg%) . Majonosi de-R2 i. 34 3 after injection of the ca rbon suspensio n. for 5 days as a positive control. into the tail vein thro ugh a glass syringe in a volume of 10 mllkg.v. The phagocytic dat a of tw o or more than two gro ups was ana lyzed by Student 's z-tesr or one-way an alysis of variance (AN OVA) followed by Dunn ett 's test.o.3.3 75. 1953 . Blood samples of approximatel y 0. for 5 days before the carbon clearance test. MO. coli ATCC 25922 adm inistr at ion . o r i. 55.01 vs. . immu nostim ulating effect (+). a nimals pretreated with test drugs (11 times ad ministrat ion in total ) were injected i.
x 5 days. o 100 500 VGextract (mg/kg..) and majon oside-R2 (50 mg/kg. Zymosan A (ZYM) was injected i... or i.. Each column represents the mean ± SEM (n = 15 ). Test drugs were administered p. p..) or majonosid e-R2 (l0-50 mg/k g.. x 5 days. as well as a nonspecific ph agocytic sti m ulant zymos an A (30 .05 and 'f 'f P <0. * Saline MR-2 ZYM 10 30 Saline MR-2 ZYM 50 50 (mg/kg. Effects of Vietnamese ginseng extract lve extract ... coli injection. c.p. ind icating positiv e im m unostim ula ting action..... Fu rthermore.o. Fig. (n = 7.'-----'-.L-l. (C)] on phagocytosis in the carbon clearance test._J. . H uang et a l.) or majonoside-R2 (50 mg/kg. ve extract (500 mg/kg.. 2.. Fig..o. i..) for 5 da ys dose-dependently exert ed a protective act ivity aga ins t the acute toxic ity induced by i. .. i.o. i. 2B ) and majonoside-R2 (l0 mg/kg/day.. VG ex tract (500 mg/kg.p. The surviva l ratio of VG extract (500 mg/k g )..L_. e 0 __ * 100 ""'0 1000 ~ Z e =. (A) in vitro 80 60 40 * 20 Vehicle VG extract Vehicle Majonoside-R2 o. p. x 5 da ys.p.. i. M oreo ver.. Blood samples were taken 1 h (A) or 1 h and 4 h (B) after the last administration of test drugs. VG extract (10 0-5 00 mg/kg/day. x 5 days) .. .) en ha nce d the phagocytic ac tivity of neutrophil pol ymorph onuclear leukocyte s in th e in vitro bactericidal test. VG sa ponin (25 mg/kg/day. p. lA. One a nd 4 h after bacterial injecti on .p.) 200 After 4 h ·2 ~ '1_ - ..) was administered twice a day for 5 days (11 times in tota l) before assays.o.----I. for 5 days before the colloidal carbon injection.. respectively.. total saponin IVe saponin. th e VG extract-treated gro u p sho wed a significantly sma ller number of residual bacteria in the circulation th an the vehicle control grou p. VG extract pretreatment a lso increased the kin et ics of ba ct eri al clearance after injection of 5 x 10 7 E. VG and majonoside-R2 on the phagocytosis in vitro and in vivo As sho w n in Fig. indicating th at VG extract tr eatment enhances ph ago cytosis (Fig..L. Escherichia coli ATCC 25922 was applied to activate phagocytosis in the concentrations of 3x l 08 and 5 x 107 in in vitro or in vivo test.. Effects of VG extract.... ~ e 2000 ~~ "". increased th e ph agocytic index in the carbon clearance tes t. The phagocytic activity was expressed as the percentage of leukocytes containing bacteria per 100 leukocytes (% phagocytic activity) in (A) or the number of residual bacterial colonies (bacteria/nil blood sample) in (B)...p.p.p. Fig. i..L..344 N... Each column represents the mean ± S.05 vs. T.. <. 2e).) (B) in vivo 3000 After 1 h 0.p.12). Effect of Vietnamese ginseng extract (Ve extract) and majonoside-R2 on phagocytosis in both ill vitro (A) and in vivo (B) bactericidal tests. Fig. (B)) and majonoside-R2 [MR2.. p < 0. T... p < 0. coli (Ta ble 1 ). (A)). Vehicle..... 1B). coli..--'o 25 50 VGsaponin (mg/kg. 1. for 5 days as a positive contra!' The phagocytic activity was expressed as the phagocytic index K.. vehicle groups (Student's r-test).M. vehicle groups (Student's t-test or Dunnett's test).an d rnajonosideR2 (5 0 mg/kg) -treated groups reached more tha n 75 % at 72 h after E . i..o..~. p.p. injection of 3 x 10 8 E. Lp.01 vs. ':..) Vehicle VG extract Vehicle VG extract Fig.o. p.5 0 mg/kg/day. Results The protective effect of VG extract and majonoside-R2 on the acute toxicity of Escherichia coli ATCC 25922 Pretreatment w ith VG extract (50-500 mg/kg..&J ell ell III . i.p.. 2A ).E.
Th e authors gratefully acknowledge Dr. the liver and spleen. treat ment with VG extract (500 mg/kg/day.J. .: Effects of Vietna mese gins eng o n opioid ago nist. Planta Med. 1'. R..E.: Saponins from Vietna mese ginsen g.S. 41: 229-235 . 54: 447. S.S. H .: A rev iew of the biolo gical and pharmacological act ivities of sa ponins.: Quantitative study o f th e gran ulopectic acti vity of the ret iculoendothelial system.. N . I.: Immunomod ulating act ivity of ginsenoside Rg1 from Panax ginseng. P. 70: 1-1 0. Ta ken together. A. Ito . Du e..S. M. 0 . ]pn.: Pharmacognostical and chem ical stu dies on Vietn amese ginseng. a major constituent of VG. 1980 .. N. Panax uietnam ensis H a et Grushv. Pharmacol.]. In concl usion. Client. : Impaired blood clearance of bac teria and ph agocyt ic act ivity in vitamin A-defic ient rat s. Pharmacol. K.. R. Meyer (IX) Pro tective effect of Red Gin seng on infection (2) On phagocytic activity of mouse reticu loendothelial system.E. Ara liacea ea. Furu ya. Behav. an d Halpern. Acknowledgemen ts 345 Discussion The present study clearl y demonstrat es that pretreatment with Vietna mese ginseng enha nces the phagocytic acti vity of phagocy tes in both ill vitro an d ill vivo tests. VG may enhance the phagocytosis by additive action on energy-co nsuming proces s.. D. 1985 ). activity for phago cytosis... H o Chi M inh Universit y o f Medici ne and Pharmacy fo r their genero us gifts of Escherichia coli ATCC 25922 an d cult ure med ium . . M . 1989)... Panax uietnamensis H a et Gru sh v. we fou nd that administration of VG extract significantly increase d the weigh t of the liver and spleen after bacterial injection compa red with vehicle-trea tment. H . Kitagawa.Biologica l properties. M. Du e. N. 41: 2010-2014. The exact mechanism underlying the activatio n of phagoc ytosis by VG remains to be clar ified. and Tanaka. M . Bot. 110: 2 7-48. References Bcnacerraf.. relative weight of R. G. and Watanabe.and foot shoc k stress-i nd uced a ntinccice ption in mice .: A qua ntitative study of the kine tics of blood clear ance of p H-labeled Escherichia coli and Staphylococci by the reticuloendothelial system.. N . Araliaceae. Phagocyto sis reportedly behaves much like muscle cells during exercise and is an energ y-consuming process (Weiss. Shiba ta . 1995.J. H uong. 198 9. K. 1989 ). N . O hta ni. M. Moreover.. 34 : 441 . 1'. Ip n. and Lamb. M . Benacerraf. Pharm. Due.141. Sirisinha.. 35: 31-3 5.: Stud y o n Panax oietna mensis H a er Grus hv. M . a nd Schlossman. play an importa nt ro le in phagocytic activity.1 959. A. De.: Func tio n of reticu loendothelial system on CC l4 -ind uced liver injury in mice. Neyc hev. op son ic facto rs in serum. 1993. 1'. Kenarova. and Wagn er. B... Exp.. P.o. c. 1'. A... M . Ta ni. an emeritu s professor of Tok yo Un iversit y. Lalloue tt e. Botan y . Th e ph agocytic proces s is known to be divided into tw o distinc t phases: attachment and ingestio n. Thes e inelud e bacteria or particle size. Pharmacal. 1'. M .1987. act ivation and pro liferatio n of the phagocyres an d R. Many facto rs have been repor ted to be invo lved in th is pro cess. Sho yakugal:« Z assbi 41 : 135. C . and the phagoc ytic activity of Kupffer cells in the liver is depre ssed by hepatit is or hepa tic damage (O kazaki et aI. K.acaille-Dubois.4 32. and Kasai .E. 1985. suggesting an increase in R. Proc. N. H. A.. We are also indebted to Dep artm ent s of Microbio logy in the fa cult y of M edicine and the Faculty o f Pharmacy. the pre sent results give preliminar y evidence that VG is ca pab le of mod ulating the imm une system and th at majon oside-R2. P. ]pn. Nharn. Pharmacol. Phytomedicine 3 : 33.1990.. 0. 1995... 1996. p. Ogawa. B.and co ndi tioned fear stress-ind uced antinocicept ion . Ok azaki. . H . for their encouragement.J. It has been reported th at VG increa ses the endura nce time in the experimenta l swim test throu gh the effect on the energ y metabolism (Nham.S. Yama saki . B. 52: 42 7. M atsum ot o. I. E. Br. 178: 204-208 .39. K. 40: 49-54. Aral iaceae . Matsumoto.: Cru de saponin extracted from Vietn am ese ginseng and its majo r constitu ent ma jonoside-R2 attenu at e the psychological st ress. Herba Pol. 1976 ). M . and Watan abe . N . 1'. Shoj i Shiba ta. by plant extracts.45 4. Nharn. Biozzi. 1'.E. Bioi. ] . Collec ted in central Vietn am . : Stimulat ion of the phag ocy tic activity o f R.S. bloo d flow th rou gh liver and spleen. . M at suda. Nh am . increases Kupffer cells and enhance s the acti vity of cytochrome PASO and other hepatic enzymes. Effect of restraint stress on delayed type hypersensitivity (DT H ) response. Kasai . H adjiivanova. Both neutrop hils and mac rophages contain large stores of glycogen and creatine phospha te which are required dur ing phagoc ytosis (Silverstein et aI. an d Dr.. M . 1'. Thus. (Williams and Chase. De laveau ..386 . and Mizuno. and Petkov. 1'. K..457. Biochem. ... Sebestycn. S. T. Med. On gsaku l. 1985. 1'. Due. x 8 days) significantly increased the weight of liver and spleen compared wit h th ose of vehicle control. Kubo .: Pharmaco logical study on Panax ginseng C. ]. Pathol. H uong. and Yam au chi.Chemistry .. Med. 1'.. N .]pn. Nham.. .T issue culture . Phytomedicine 2: 363.]. Okim ura. Exp.. K. The pre vious study by N ham (1989) has demonstrate d that VG extrac t exer ts a protective activity aga inst CC l4 -ind uced liver injury. and suggests th at the activation of phagocytosis is one of th e mechanisms underlying the antibacteria l activi ty of VG.. Luan. and Saka mo to. 1996. 1'... N . organs. Exp. M .: Stress and immune responses III. N . etc. A. 1972). 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