Effect of vietnamese ginseng on the phag

WATANABE1* 1Department of Pharm acology, Research Institute for Wa kan -Yaku Oriental M ed icines, To yama M edical and Phar maceu tical University, Japan 2Department of Biologica l Acti

Phytornedicine Vol 4 (4), pp 34 1-346, 1997

©1997by Gustav Fisch er Verlag

Effect of Vietnamese Ginseng on the phagocytosis

N M DUC3, K YAMASAKI2 and H WATANABE1*

1Department of Pharm acology, Research Institute for Wa kan -Yaku (Oriental M ed icines), To yama M edical

and Phar maceu tical University, Japan

2Department of Biologica l Acti ve Subs tance s, Institute of Phar maceuti ca l Sciences, Hi roshim a Un iversity

Scho ol of M ed icine, J apan

3T he Science-Pro d uctio n Cent re o f Vietna mese Ginseng , Ho Chi M in h Universit y of Medicine a nd Pha

r-macy, Vietnam

Summary

Th e effects o f Vietnamese ginseng cru de extract (VG extr act ), tot al sa po nin (VG saponin ) and its major sa po n in co mpo nent, majonoside-R 2, on phagocytosis were ex am ined in mice by

bacte-ricidal 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 vitroandin vivo

bac-tericidal tests Pretreatment with VG extrac t (500 mg/kg, or al admin istr ati on ,p.o.)and

majono-side-R2 (50 mg/kg, intrape rito nea l administration, i.p.) protected the an imals from th e acute

tox-icity of E.coliAT CC 25922 and significantly increased the phagoc ytic index in bot hin vitroan d

in vivobactericidal tests Moreover, VG extract (100 - 500 mg/kg,p.o ),VG sa po 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

stimu-lant, also incr eased the ph agocytic ind ex eva luated by th e carbon clea rance test T hese results

in-dicate th at Vietn amese ginse ng enhances the phagocy tic activity of ph agocyres, an d suggest that

majonoside-R2 plays an imp ort ant rol e in th is effecr

Key words: Vietn am ese ginseng, maj on oside-R2 , phagocytos is,Escherichia coli,ca rbon clea r-ance test

Introduction

Vietn amese ginseng (VG) iPanax uietname nsis H a et

Grus hv Araliaceae ), a newPanax species, has been used as

a "s ecret medicinal plant " of the Seda ng ethn ic minor ity in

Vietn am for the tre atment of seri ous 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

stimula-to ry effects on th e central nervo us system, and exhibits

ant ifatigue, antioxidant, antistress and antibacterial act

iv-ities (N harn, 1989 ) Th e antibacteria l effect on pathogenic

Streptococciappears to be cha rac teristic of VG, since other

ginsengs such as Panax ginsenghave not been reported to

ha ve such an effect M or eover, polyacetylenic compou nd s

of VG exhibit a pot ent suppressive action on Gra m-po sitive

cocciand derrnatophyte s (N ham et al., 1995 )

Sapo nins isolated from med icinal plants have been fou nd

to exhibit cyto toxic, a ntitumor and imm unomod ulating ac-tivities (Lacaille-D ubois and Wagner, 1996 ) Singh et al (198 4) were the first to show ex perimenta lly that Panax ginsengenha nced th e immune respo nse in mice Moreover,

Panax ginsengand its co mpo nents reportedly exhi bit irn-mun 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

pri-marily by phagocytic leukocytes In these responses,

poly-morphonuclear leukocytes, such as neutrophils and

eosino-phils, act as a first line of defense against infection

Further-more, 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

ho-meostasis of the human body and in the cellular host

de-fense 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

as-say (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

de-fences is one of the index to evaluate the antistress activity

of medicinal plants and their biologically active

compo-nents 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

pre-pared 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

sap-onin 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

experi-ments The animals were housed in groups of 20 per cage

for at least 1 week before starting the experiments Housing

conditions were thermostatically maintained at 24 ± 1 °C

and a relative humidity of 55± 5% with a 12 h light-dark cycle (lights on: 0730-1930) Food and water were givenad libitum.

The acute toxicity of Escherichia coli ATCC 25922 Escherichia coliATCC 25922(E coliATCC 25922) was suspended in saline and various concentration of E coli

ATCC 25922 solution was injected i.p.into mice in a con-stant 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 experi-ments, respectively Our preliminary experiment indicated that the lethal level was more than 85% at concentrations

of more than 3xl08bacteria/ml We selected this concentra-tion for theinvitro bactericidal test The concentration of 5xl07 bacteria/ml that caused the 0% of lethal level was used for theinuiuo bactericidal test

Test drugs were administeredp.o.ori.p.twice a day for

5 days before (11 times in total) and for 3 days after (5 times in total) i.p.injection of 3xl08cells/ml of E.coli.The protective effect of the test drugs was evaluated as de-scribed 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

assay)

Test drugs were administered p.o.or i.p.for 5 days (11 times in total) before assay One hour after the last admin-istration, blood sample was taken aseptically from the tail vein into a tube containing heparin, and settled for 20 min

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 3 x 108

E colisuspension were mixed in test tubes and incubated

at 37°C for 20 min The mixture was washed twice by Hanks solution and centrifuged at 800 rpm for 5 min to discard the non-phagocytosed bacteria Samples of phagoc-ytes 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 immer-sion objective for the percentage of leukocytes containing bacteria per 100 leukocytes (% phagocytic activity) (Wil-liams and Chase, 1976)

In vivo phagocyticactivity of leukocytes (bactericidal assay)

We determined the kinetics of blood clearance of injected

E.coliby modifying the methods previously reported

(Ben-Effect of VietnameseGinsengon the phagocytosis in vitro an d in vivo 34 3 acerraf et al., 1959; On gsakul et a! 1985 ) Briefly, a nimals

pretreated with test drugs (11 times ad ministrat ion in total )

we re injectedi.p.with an 18 h suspension of 5 x 107E

co-li ATCC 25 922 Blood samples of approximatel y 0 1 ml

were tak en asepticall y from th e tail vein 1 hand 4 h after

injection and were spread on MacConkey agar follo win g

appro pr iate dilution in trypticase soy bro th After

incuba-tion at 37°C for 24 h, the num ber of residual bacterial

col-onies (bac teria/ml blood sample) was determined In some

experiments, animals were co ntinuo usly administered test

dru gs for 3 da ys after E.coliin jection and killed to remo ve

liver and spleen The weight of these org an s was calcul ated

as th e percentage of body weight (mg%)

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 min and diluted 1:3 in sterilized saline co nta

in-ing 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 o f 10 mllkg Venou s blood (25ul)was taken by

ret-ro-orbiral veno us plexu s pu ncture 0.5 (tl )and 10 min (t2)

after injection of the ca rbon suspensio n Th e blood samples were hemol yzed in 2 ml 0.1 % sodium ca rbonate and th e optica l density of the solutio n at 600 nm wa s measured us-ing a Beckman DU 64 0 spectro photo meter to determine th e concent ration of carb on in th e periphera l blood at the time sta ted The ph agoc ytic ind ex K is ca lculated from the

for-mu la:

K = (log C1-log C2 ) / (t2 - t Il Here: C] and C2are th e blood co ncentrations of carbon

at time t) and t2 • Test drugs were administeredp.o. or i.p.for 5 da ys befor e the carbon clearance test Zy mosan A (Sigma Chemi -cal Co , MO, USA) was dissolved in saline and injected i.p.

for 5 days as a positive control

Statistical analysis

Fisher's Exact probability test was used to anal yze the survival rati o (%) following E.coliATCC 25922 adm

inis-tr at ion T he phagocytic dat a o f tw o or more than two gro ups was ana lyzed by Student 'sz-tesror one-way an alysis

of variance (AN OVA) followe d by Dunn ett 's test, respec-tively

Table 1 Effects o f Vietn a mese ginseng ex tract a nd majonoside-R 2 on the acu te to xicit y o fEscherichia coliAT CC 25 922 in mice Treatment

Vehiclep.o.

VG extr actp.o.

Veh iclei.p.

Majonosi de-R2i.p.

Dos es No of anima ls Sur vival ra tio

Immuno-stimulating index

+ + ++

VG ex tra ct and maj on oside-R 2 were ad ministeredp.o.o ri.p.,respective ly, for 5 da ys befor e an d 3 d ays afteri.p.injecti on of 3 x 108

Escherichia coliATCC 25922 Th e protect ive effect of test d rugs wa s expressed as the surv ival ra tio(%) a nd immunostim ulating index

H ere: th e surv iva l ratio of < 50 % ,;:: 5 0 %, and z75 % was exp ressed as no protective effect (-) , immu nostim ulating effect(+) ,an d irn-mun ostimu lati ng effect(++),resp ectively * P < 0.05 (Fisher's Exac t probability test)

Tab le 2 Effect of VG extract o n cha nges in the weight of mouse liver and spleen ca used byEscherichia coliinjectio n

VG ex tra ct 500 5974 5 ;; 335.6"* ## 62 4 1 ;; 71 8 ':"' # #

VG extract was administered daily for 5 d ays before and 3 days afteri.p.injection of 5 x 107Escherichia coliAT CC 25922 Animals w ere sacri ficed 72 h afterE coliinjecti on a nd 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) P <0.05 , P < 0.01 vs naive group an d ## P < 0.01 vs vehicle-tre ated group (Student 's z-rest, n 20 )

344 N T T.H uang et a l.

o 25 50 VGsaponin (mg/kg, Lp.)

Saline MR-2 ZYM

50 50 (mg/kg, i.p.)

0 ' -'- -

-'-Saline MR-2 ZYM

10 30

o 100 500 VGextract (mg/kg, p.o.)

o L-l I L_ L L _J

Effects ofVGextract,VGand majonoside-R2 on the phagocytosis in vitro and in vivo

As sho w n in Fig lA, VG ex tract (50 0 mg/kg, p.o )and maj on oside-R2 (50 mg/kg, i.p.)en ha nce d the phagocytic

ac t ivity of neutrophil pol ymorph onuclear leukocyte s in th e

in vitrobactericidal test M oreo ver, VG extract pretreat-ment a lso increased the kin et ics o f ba ct eri al clearance after injection o f 5 x 107E coli.One a nd 4 h after bacterial in-jecti on , th e VG extract-treated gro u p sho wed a

significant-ly sma ller number of residual bacteria in the circulation

th an the veh icle control grou p, indicating th at VG extract

tr eatment enh ances ph ago cytosis (Fig 1B)

Fu rthermore, VG extract (10 0-5 0 0 mg/kg/day,p.o.x 5

d ays, Fig 2A ), VG sa poni n (25 mg/kg/day, i.p.x 5 da ys, Fig 2B ) and majonoside-R2 (l0 mg/kg/day, i.p.x 5 days, Fig 2 e), as well as a nonspecific ph agocytic sti m ulant zy-mos an A (30 - 5 0 mg/k g/day, i.p. x 5 days) , increased th e

ph agocytic index in the carbon clearance tes t

Fig 2 Effects of Vietnamese ginseng extract lve extract , (A)), to-tal saponin IVe saponin, (B)) and majonoside-R2 [MR2,(C)]on phagocytosis in the carbon clearance test Test drugs were

adminis-tered p.o or i.p for 5 days before the colloidal carbon injection Zymosan A (ZYM) was injected i.p for 5 days as a positive

con-tra!' The phagocytic activity was expressed as the phagocytic index

K Each column represents the mean±S.E.M (n = 7- 12) ': p < 0.05 and 'f 'f P <0.01 vs vehicle groups (Student'st-test or Dunnett's test)

After 4 h

Vehicle VG extract

200

*

After 1 h Vehicle VG extract Vehicle Majonoside-R2

Vehicle VG extract

40

20

60

·2 ~' 1 _

c

~ e

.&J ell 2000

- III

ell

*

""'0

~e

~

=

Z

(B) in vivo

3000

(A) in vitro

80

The protective effect ofVGextract and majonoside-R2

on the acute toxicity of Escherichia coli ATCC 25922

Pretreatment w ith VG extract (50-50 0 mg/kg, p.o )or

maj ono sid e-R2 (l0-50 mg/k g, i.p.)for 5 da ys

dosedependentl y ex ert ed a protective act ivity aga ins t the acute toxic

-ity in duced byi.p.injection of 3 x 108E coli(Ta ble 1 ) The

su rviva l ratio of VG extract (500 mg/k g ) an d rnajonoside

-R2 (5 0 mg/kg) -treated groups r eached more tha n 75 % at

72 h afte r E coli inject ion, ind icating positiv e im m un

o-st im ula ting action

Results

Fig 1 Effect of Vietnamese ginseng extract (Ve extract) and

ma-jon oside-R2 on phagocytosis in both illvitro (A) andin vivo (B)

bactericidal tests Vehicle, ve extract (500 mg/kg, p.o.) or

majon-oside-R2 (50 mg/kg, i.p.) was administered twice a day for 5 days

(11 times in tota l) before assays Escherichia coliATCC 25922

was applied to activate phagocytosis in the concentrations of

3x l 08and 5 x 107inin vitro orin vivo test, respectively Blood

samples were taken 1 h (A) or 1 h and 4 h (B) after the last

admin-istra tion of test drugs The phagocytic activity was expressed as

the percentage of leukocytes containing bacteria per 100

leuko-cytes(%phagocytic activity) in (A) or the number of residual

bac-terial colonies (bacteria/nil blood sample) in (B) Each column

represents the mean±SEM (n=15 ).<.p < 0.05 vs vehicle groups

(Student's r-test)

Effect of Vietna meseGinsengon the ph agocytosis in vitro and in vivo 345

As summarized in Table 2, treat ment with VG extract

(500 mg/kg/da y,p.o. x 8 days) significantly increased the

weight of liver and spleen compared wit h th ose of vehicle

control

Discussion

The present study clearl y demonstrat es th at pretreatment

with Vietna mese ginseng enha nces the phagocytic acti vity

of phagocy tes in both ill vitroan d ill vivo tests, and

sug-gests th at the activation of phagocytosis is one of th e

mech-an isms underlying the mech-antibacteria l activi ty of VG

Th e ph agocytic proces s is known tobe divided into tw o

distinc t phases: attachment and ingestio n Many facto rs

have been repor ted to be invo lved in th is pro cess Thes e

inelud e bacteria or particle size, receptor attach ment, op son

-ic facto rs in serum, relative weight o f R.E.S organs, act

iva-tion and pro lifera tio n of th e phagocyres an d R.E.S., bloo d

flow th rou gh liver and spleen, etc (Williams and Chase ,

1976 ) Moreover, the liver and spleen, the organs rich in

mononuclea r ph agocytes (macro phages), play an

impor-ta nt ro le in phagocytic activity, and th e phagoc ytic activity

of Kupffer cells in the liver is depre ssed by hepatit is or

he-pa tic damage (O kazaki et aI., 1985 ) The pre vious study by

N ham (1989) has demonst rate d that VG extrac t exer ts a

protective activity aga inst CC l4-ind uced liver injury,

in-creases Kupffer cells and en hance s the acti vity of

cyto-chrome PASOand other hepatic enzymes In th e prese nt

study, we fou nd th at ad ministration of VG extract

significantly increase d th e weigh t of the liver and spleen after bac

-terial injection compa red with vehicle-trea tment,

suggest-ing an increase in R.E.S activity for phago cytosis Thus, it

wo uld be expected that such p rot ective effects of VG on th e

liver injury contribute to th e enhancement of phagocytic

activity by VG extract

Phagocyto sis reportedly behaves much like muscle cells

during exercise and is an energ y-consuming process (Weiss,

1972) Both neutrop hils and mac rophages co ntain large

stores of glycogen and creatine phospha te which are

re-quired dur ing phagoc ytosi s (Silverstein et aI., 1989 ) It has

been reporte d th at VG increa ses th e endura nce time in the

experimenta l swim test throu gh th e effect on th e energ y

metabolism (N ham, 1989) Ta ken together, VG may

ehance the phagocytosis by additive action on energy-co

n-suming proces s The exact mechanism underlying the

acti-vatio n of phagoc ytosis by VG remains to be clar ified

In concl usion, th e pre sent result s give preli minar y

evi-dence that VG is ca pab le of mod ulating the imm une system

and th at majon oside-R2, a major constituent of VG, pla ys

an important ro le in th e stimulatory effect of VG on

phag-ocytic activity

Acknowledgemen ts

Th e a uthors gra tefully acknowledge Dr Shoj i Shiba ta, a n emer-itu s profe ssor of Tok yo Un iversit y, an d Dr Osa m u Tanaka, an emer itus pro fessor of H iro shim a Un iversit y, for their encourage-ment We are also indebted to Dep artm ent s o f Microbio log y in the

fa cult y of M edicine and the Faculty o f Pharmacy, H o Chi M inh Universit y o f Medici ne and Ph a rm acy fo r their genero us gifts o f

Escherichia coliATCC 25922 an d cult ure med ium

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Address

H Wata na be, Department of Pharm acology, Research In-stitute for Wakan-Yaku (Oriental Medicines), Toyama Medical and Pharmaceutical University, 2630 Sugitani , Toyama 930-01, Japan

Tel.: +81-764-34-2281; Fax: +81-764-34-5056; e-mail: hwatanab@ms.toyama-mpu.ac jp