Phytornedicine Vol (4), pp 34 1-346, 1997 © 1997 by Gustav Fisch er Verlag Effect of Vietnamese Ginseng on the phagocytosis in vitro and in vivo N 1 HUONG" 3, K MATSUMOT01, N 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 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 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 Summary 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 Introduction 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- 342 N T T Huong et al fect of VG or its saponin components on the immune system It is well known that immune responses are produced primarily by phagocytic leukocytes In these responses, polymorphonuclear leukocytes, such as neutrophils and eosinophils, act as a first line of defense against infection Furthermore, the network of phagocytic tissue macrophages which, together with endothelial cells and polymorphs, was previously termed "the reticuloendothelial system" or R.E.S usually plays an important role in keeping the homeostasis of the human body and in the cellular host defense mechanism (Silverstein et al., 1989) The end point of the entire phagocytic process performed by phagocytic cells in the circulation is measurable through the bactericidal assay (Williams and Chase, 1976) Moreover, the function of R.E.S in experimental animals has been evaluated by the carbon clearance test and the increased carbon clearance indicates an increased phagocytic activity We have previously demonstrated that VG attenuates pathophysiological changes caused by psychological stress and conditioned fear in mice, and that majonoside-RZ, a major constituent of VG which has not been isolated from Panax ginseng (Due et al., 1993), contributes to the effect of VG (Huong et al., 1995, 1996) Wagner et al (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 Thus, the purpose of this study is to investigate, with the use of bactericidal and carbon clearance tests, whether VG and majonoside-R2 modulate the phagocytic activities of neutrophils and R.E.S Materials and Methods Materials VG extract, VG saponin and majonoside-R2 were prepared as previously reported (Huong et al., 1995) Yields of VG extract, VG saponin and majonoside-R2 were 41.2, 13.2 and 5.29%, respectively Quantitative analysis using high-performance liquid chromatography revealed that the contents of majonoside-RZ were 8.27% and 22.67% in VG extract and VG saponin, respectively VG extract, VG saponin and majonoside-R2 were dissolved in distilled water for oral administration (p.o.) or in saline for intraperitoneal administration (i.p.) Animals Swiss albino male mice (20-25 g, Pasteur Institute of Ho Chi Minh City, Vietnam), and male BALB/c mice (20-25 g, Japan SLC, Shizuoka, Japan) were used for the experiments The animals were housed in groups of 20 per cage for at least week before starting the experiments Housing conditions were thermostatically maintained at 24 ± °C and a relative humidity of 55 ± 5% with a 12 h light-dark cycle (lights on: 0730-1930) Food and water were given ad libitum The acute toxicity of Escherichia coli ATCC 25922 Escherichia coli ATCC 25922 (E coli ATCC 25922) was suspended in saline and various concentration of E coli ATCC 25922 solution was injected i.p into mice in a constant volume of 10 ml/kg The number of surviving animals was measured 72 h after injection The concentration of bacteria that produced the lethal levels of 85-90% and 0% was selected for in vitro and in vivo phagocytic experiments, respectively Our preliminary experiment indicated that the lethal level was more than 85% at concentrations of more than 3xl0 bacteria/ml We selected this concentration for the in vitro bactericidal test The concentration of 5xl0 bacteria/ml that caused the 0% of lethal level was used for the in uiuo bactericidal test Test drugs were administered p.o or i.p twice a day for days before (11 times in total) and for days after (5 times in total) i.p injection of 3xl0 cells/ml of E coli The protective effect of the test drugs was evaluated as described by Delaveau et al (1980) The survival ratio of < 50%, :2: 50%, and :2: 75% represents no protective effect (-), immunostimulating effect (+), and immunostimulating effect (++), respectively In vitro phagocytic activity of leukocytes (microscopic assay) Test drugs were administered p.o or i.p for days (11 times in total) before assay One hour after the last administration, blood sample was taken aseptically from the tail vein into a tube containing heparin, and settled for 20 at room temperature By this sedimentation, the leukocytes remained in the supernatant fluid The upper one third supernatant layer of the erythrocyte column (sediment) that was rich in neutrophil polymorphonuclear leukocytes was collected Then, 0.1 ml of leukocytes and 0.1 ml of x 10 E coli suspension were mixed in test tubes and incubated at 37°C for 20 The mixture was washed twice by Hanks solution and centrifuged at 800 rpm for to discard the non-phagocytosed bacteria Samples of phagocytes were removed from the suspension, smeared on glass slides and then rapidly dried in air 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, 1976) In vivo phagocyticactivity of leukocytes (bactericidal assay) We determined the kinetics of blood clearance of injected E coli by modifying the methods previously reported (Ben- Effect of Vietnamese Ginseng on the phagocytosis in vitro an d in vivo acerraf et al., 1959; Ongsakul et a! 1985 ) Briefly, a nimals pretreated with test drugs (11 times ad ministrat ion in total ) were injected i.p with an 18 h suspension of x 10 E coli ATCC 25 922 Blood samples of approximatel y ml were tak en asepticall y from the tail vein hand h after injection and were spread on MacConkey agar follo wing appropriate dilution in trypticase soy bro th After incubation at 37°C for 24 h, the num ber of residual bacterial colonies (bac teria/ml blood sample) was determined In some experiments, animals were co ntinuo usly administered test dru gs for days after E coli injection and killed to remo ve liver and spleen The weight of these org an s was calcul ated as the percentage of body weight (mg%) 34 after injection of the ca rbon suspensio n Th e blood samples were hemol yzed in ml 0.1 % sodium ca rbonate and th e optica l density of the solutio n at 600 nm wa s measured using a Beckman DU 64 spectro photo meter to determine th e concent ration of carb on in th e peripheral blood at the time sta ted The phagoc ytic ind ex K is calculated from the formu la: K = (log C -log C ) / (t - t Il Here: C] and C are the blood concentrations of carbon at time t) and t • Test drugs were administered p.o or i.p for days before the carbon clearance test Zy mosan A (Sigma Chemi cal Co , MO, USA) was dissolved in saline and injected i.p for days as a positive control 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!., 1953 ; Okirnura et a!., 1989) Co lloidal carb on (Pelikan drawin g ink, Gunther Wagn er, H annover, Germ an y) was centrifuged at 5,000 rpm for 15 and diluted 1:3 in sterilized saline conta ining 1.5 % gelatin as a sta bilizer Th e ca rbon suspension wa s injected i.v into the tail vein thro ugh a glass syringe in a volume of 10 mllkg Venou s blood (25 ul) was taken by retro-orbiral veno us plexu s pu ncture 0.5 (t l ) and 10 (t ) Statistical analysis Fisher's Exact probability test was used to anal yze the survival rati o (%) following E coli ATCC 25922 adm inistr at ion 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, respectively Table Effects o f Vietn a mese ginseng ex tract and majonoside-R on the acu te to xicit y o f Escherichia coli AT CC 25922 in mice Treatment Vehicle p.o VG extr act p.o Veh icle i.p Majonosi de-R2 i.p Dos es mg/kg 50 100 250 500 10 50 No of anima ls survi ved after 72 h Sur vival tio 111 2/15 4/15 5115 12/16 ':' 1/2 7/1 1'" 10112 * 6.3 13.3 26 33 75.0 5.0 63 83.3 (%) Immunostimulating index + + ++ VG ex tra ct and maj on oside-R were ad ministered p.o o r i.p., respectively, for da ys befor e an d days after i.p injection of x 10 Escherichia coli ATCC 25922 Th e protect ive effect of test d rugs wa s expressed as the surv ival tio (%) and immunostim ulating index H ere: th e surv ival ratio of < 50 % ,;:: 0%, and z 75 % was exp ressed as no protective effect (-) , immu nostim ulating effect (+), an d irnmun ostimulati ng effect (++), resp ectively * P < 0.05 (Fisher's Exac t probability test) Table 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, p.o.) Organ we ight (mg %) Liver Spleen 500 41 03.8;; 341 4 870 6;; 30 2.4 " 5974 ;; 335.6"* ## 42 7.1 ;; 59 38.3;; 55.5 624 ;; 71.8 ':" ' ## VG extract was administered daily for days before and days after i.p injection of x 10 Escherichia coli AT CC 25922 Animals were sacrificed 72 h after E coli injecti on and th en liver an d spleen w ere remove d The weig ht of these or gan s was ex pressed as mg % (organ weig ht/1 00 g body weight) 'f P