Chemical and Biochemical Aspects of Dammarane Triterpene Glycosides of Korean Ginseng

Jo u rn a l o f the Pharm aceutical Society o f Korea 19, 144-158 C1975)

Chemical and Biochemical Aspects of Dammarane Triterpene Glycosides of Korean Ginseng*

B y u n g H o o n H a n an d L in K e u n W o o

N a t u r a l P r o d u c t s R e s e a r c h I n s t i t u t e , S e o u l N a t i o n a l U n i v e r s i t y , S e o u l 1 1 0

The Korean ginseng (JP a n a x g i n s e n g C .A . M e y e r) , w hich has been know n more than

*2000 years, occupies the most important place among the tonic remedies for the Far Eastern Asian peoples. According to the folkloric experiences, its long life of tonic nature is due to the innocuousness, even w ith medication of unusual high dosage. Thus, a number of scientific reports appeared in order to establish the scientific supports for the efficacy of the Korean ginseng as a drug. U n til recent decades, ginseng researches have found no definite guideline o f study, because no reproducibilities and dose dependent response were obtained in every pharmacological screening test.

A few years ago, Brekhman e t a L노그 reviewed the divergent articles on the ginsengstudies and proposed a hypothesis on the pharmacology of P a n a x g i n s e n g . It describes that dammarane glycoside, one main component of P a n a x g i n s e n g may exhibit an adaptogenic activity, Adaptogenic activity was defined essentially as the nonspecific normalizing activity irrespective of the direction of foregoing pathological shift. A lthough the validity of the hypothesis is still not completely recognized on the basis of modern experimental pharmacology, it is so much suggestive that it triggered intensive research on the chemical, pharmacological, and biochemical problems of the components. M any scientists have involved in these fields of research since then. In Japan remarkable achievements were made by Shibata on the phytochemical problems of dammarane glycosides, by Takagi e t a l, on the pharmacological problems, by Oura e t a L o n the physiological activities, in the U .S .S .R . by Elyakov on phytochemistry, and in Bulgaria by Petkov on pharmacology of the glycosides.

O n the other hand, the authors have also participated in the studies of the chemical and biochemical problems of ginseng saponins in order to elucidate the mechanism of ginseng pharmacology which is said to be a panacea.

Comprehensive reviews on the chemistry그그, pharmacology^^ and biological activities걸^®크 of various components of ginseng have already appeared elsewhere. Present review concerns with the further summary of the chemical, pharmacological, and biological studies on the ginseng saponins and a possible mechanism of action of ginseng components.

C hem istry of D am m arane T riterpene Glycosides— Elyakov e t a l P found six glycoside

♦Presented on Oct. 5, 1974 at the symposium on ^'Terpenoids'" organized by Natural Products Research Institute, Seoul National University.

144

m

nated them as panaxoside A , B, C, D, E, and F in the order of decreasing R / values. Each panaxosides were further isolated by silicagel colum n chromatography and their partial struc­

tures were reported긴. The panaxosides were divided into two groups: Panaxoside A , B and C belong to protopanaxatriol group and panaxoside D , E and F to protopanaxadiol group. The former group is less polar glycosides and has less number of sugar moiety than the latter s.

The subsequent w ork w ith the same glycoside fraction by Shibata e t o l P showed the presence of thirteen glycoside spots on the two dimensional th in layer chromatogram, and designated them as ginsenoside R x ( x； o, a, bi, t>2, c, d, e, f, gi, g2, g3, hi, and I12) in the increasing order of R/-values. He isolated them by using preparative th in layer chromatography, silica-gel colum n chromatography, and droplet counter current distribution process. T h e ir complete structures elucidated are shown in Table

Table I— C onfigurations o f dam m arane glycosides o f Panax ginseng.

G insenoside R x Aglycone Cs-Sugars Ce-Sugars Czo-Sugars

Ro O leanolic acid /S-Glucose-^-1—>2-Glucose ^-Glu*

R b i P rotopanaxadiol /S-Glucose-/S-l—»2-Glucose jS-Glu-jQ-l—»6~G1^u

Rbz P ro to panaxadio l ^-Glucose-jS-1—»2-Glucose /3-Arap-^-l 나 6-G1u

R c Protopanaxadiol ^-Glucose-/6-l—>2-Glucose ^-A ra£-^-l—♦G-GIu

R d P rotopanaxadiol j6-Glucose-/3-i—> 2-Glucose /3-Glucose

Re P rotopanaxatriol jS-Rham-jS—1—>2~Glu ^-Glu

R f P rotopanaxatriol

R g i P rotopanaxatriol /3-Glu /3-Glu

Rg2 P rotopanaxatriol /3-Rham-jS-l—>2-G1u

* T h is is linked to the carboxyl radical at C28.

Ginsenoside R bi, b2, c, and d belong to protopanaxadiol glycosides consisting of comm on prosapogenin, protopanaxadiol-35-diglucoside용\ A n d ginsenoside Re, f, gi, and g2 belong to protopanaxatriol glycosides도®^ The chemical structure of ginsenoside Ra, gs, hi, and I₁₂ were not elucidated as yet.

W h ile the authors attempted to isolate the substances having a property of stabilizing serum protein on heat denaturation, two dammarane triperpene glycosides were obtained in fine crystalline form and they were designated as panax sponin A (m p 208-^10) and C (m p 196 시 202°) in the decreasing order of R/-values. The partial structure of these saponins were determined to be protopanaxatriol diglucoside for panax saponin A고고그 and protopanaxatriol- rhamno-diglucoside for panax saponin jThe ultimate identifications of these saponins were conducted by direct comparison w ith the authentic sample of ginsenoside R g i and R e w hich were k indly supplied by Shibata. Some controversies on the structure of ginsenoside R e remain. As shown in Table I, Shibata proposed an oligoside structure효®크, protopanaxatriol - 20S-5-glucosyl-6y3-rhamno-^-l^2-gIucoside, for the structure o£ ginsenoside Re. O n the other

hand, we reported previously that panax saponin C consumes 6 moles of periodate. This sug­

gested either the monoside structure or 1 >6 type oligoside structure. The gas-chromatogram, of methylated sugars obtained by permethylation of panax saponin C followed by methanolysis.

showed only the peaks of methyl-a-2, 3, 4, 6-tetra-methoxy-glucopyranose and methyl-2, 3,4- trimethoxy-rhamnopyranoside w ith 2 ： 1 ratio. Based on these results, the authors concluded that panax saponin C possessed the monoside as its partial structure. Further confirmation o a the complete structure of the saponin is imder progress.

146 H an • W oo : Dammarane Triterpene Glycoside Vol. 19, No. 3

Dammarane g ly c o sid e s

#

F ：otoponaxadiol [I) ： R =H panaxadiol (IE} ; r = h

profopanaxatriol(H) ■ R =0H panaxatriol (EZ]; R ^zT^{o h}

Scheme 1

Chemical studies on the dammarane triterpene sapogenins were conducted by Shibata포단"나®^

and Elyakoy고®\ independently. In the early days of ginseng chemistry, G .B . Elyakov and Shibata proposed a conflicting theory on the structures of panaxatrial and protopanaxatriol, however lately G.B. Elyakov withdrew his theory on the structures. The genuine sapo­

genins are protopanaxadiol and protophanaxatriol. These can be obtained by the Smith's degradation process호*규그, namely, periodate oxidation of sugar moiety followed by alkali treat­

ment. The common acid catalyzed hydrolysis may produce artifact sapogenins, panaxadiol and panaxatriol by concurrent cyclization of side chain and by the epimerization of 20S to 20R configuration고용\ The chemical structures of these sapogenins were determined by usual spectral analysis and chemical analysis and finally by X-ray diffractometry.

Dammarane triterpenes are distributed in the plants of P a n a x genus포®\ Betulaceaa^^'^ and D ip t e r o c a r p a c e a e genus. The dammarane triterpene glycoside and their genuine aglycones are found only in the plants o£ Panax genus. The authors noticed once the structure of betulafol- ienetriol isolated from the unsaponificable fraction of fresh leaves of B e t u l a a lb a by Fisher e t because it was found to be Cs-epimer of protopanaxadiol. The authors isolated the compound from the leaves of B e t u l a l a t i f o l i a Ko m a r o v고오그 and transformed it to protopana- xadiol오®그 by follow ing simple chemical process.

Due to 3a-axial and 12/3-equatorial hydroxyl configuration of betulafolienetriol, C단-partial keto-derivative could be produced, by controlling oxydation w ith limited amount of Sarret reagent. Reduction of the 3-keto-compound w ith metalic sodium in iso-propanol produces, protopanaxadiol with good yield, A lthough the above process has no significance for indust­

rial purpose at present, may warrant further study in order to exploit the natural resources

#

September, 1975 J. Pharm. Soc. Korea 147

B e t u l a f o i i e n e t r i o l

m p 2 I O — 4®

5 - K e t o — de r i v .

Scheme 2

m p I 86 ®

p r o t o p a n a x a d i o l

for the chemical synthesis of ginseng component.

Pharm acological Significance of Damm arane Aglycone Composition—— N otew orthy p oin t in the concepts of adaptogenic activity is its normalizing property being always beneficial to host irrespective of foregoing pathological shift. The hypothesis of adaptogenic activity of ginseng glycosides was formulated by Brekhman^\ based on the experimental results conducted m ainly by using the mixture of various glycosides. Therefore, the controversy on whether the dualistic pharmacological activities were originated from single dammarane glycoside molecule or shared of different molecule, should be answered. Brekhm an suggests the former m echan­

ism. O n the other hand T a k a g i 'r e p o r t s indicate apparently the latter by show ing that glycosides of panaxadiol series display central nervous system (C N S ) depressant activity and that glycosides of panaxatriol series give rise C N S stimulant activity. In general the structural analogues display their biological activities in a way of either antagonistic or analogic. It m ay be considered from the Takagi's results that the structural congeners of ginseng glycosides, namely, glycosides of protopanaxadiol series and protopanaxatriol series are antagonistic each other as far as CN S actions concerned. O n the other hand, the authors reported that both series samples of dammarane glycosides had stimulant activity in protein synthesis. For the conveni­

ence of the argument, the pharmacological or biological activities o£ dammarane glycosides can be divided into two main categories; one is pan-cellular activity and the other is organ or tissue specific activity. Based on the various pharmacological and biochemical data concerning the dammarane glycosides of ginseng, the follow ing hypothesis is speculated; pharmacological activities of both series of glycosides, protopanaxadiol series and protopanaxatriol series m ay be either antagonistic on their tissue specific activities and analogical on their pan-cellular activities.

Thus the mixture of these two series of glycosides in an appropriate ratio, as appeared in crude extract of Korean ginseng, may be beneficial to the host by increasing the synthesis of some functional proteins due to the additive augmentation of pan-cellular activities, and w ith the disappearance of any significant behavioral symptoms due to the antagonism of tissue specific activities. Probably such two different activities may be one reason w hy so m any conflicting data came out from classical behevioral tests w ith Korean ginseng. W ith such possibility.

148 H a n • W o o ： D a m m a ra n e Triterpene Glycoside V o l. 19, N o. 3

Table I I — A pparent content* of dam m arane triterpene aglycones of K orean ginseng.

M e d ic in a l part and age

Sapogenin content

P anaxadiol P anaxatriol

R a tio f P D / P T )

M a in root:**

6-years 0.124 0.130 0.955

4-years 0.155 0.155 1.012

S ide root:**

6-years 0.556 0.380 1.465

4-years 0.474 0.316 1.500

T errestrial part;***

F o liu m :

3-years 0.0188 0.187 0.100

4-years 0.017 0.167 0.102

6-years 0.016 0.157 0.102

Stem:

3-years 0. 0022 0.0222 0.100

4-years 0.0048 0. 0476 0.101

6-years 0.0055 0.055 0.100

* T he sapogenin content in this Table do n o t denote the actual sapogenin content of Korean ginseng, since the degradation of sapogenins d u rin g acid hydrolysis o f glycosides was not considered in the calculation o f the sapogenin contents,

** Sapogenin contents were assayed by com bination of preparative th in layer chrom atographic p u r­

ific a tio n and vaniIlin- H2S0 4 c o lo u r reaction.

*** Sapogenin contents were assayed by T L C densitom etry.

the authors2®그 examined the ratio of panaxatriol contents in the main root, fibrous side root, and terrestrial herbs of Korean ginseng.

As shown in Table II, the ratio of aglycone compositions shows significantly different values in the various part of ginseng root. In the m ain root portion of Korean ginseng, the ratio of panaxatriol content approaches nearly unit value, however, in the terrestrial part pan­

axatriol content is greater than the content of panaxadiol and in the fibrous side root pana­

xadiol content is predominant. Total glycoside content of the terrestrial part is not less than that of m ain root and that of fibrous side root is more than that of m ain root. In spite of higher glycoside content, the terrestrial part and the fibrous side root of Korean ginseng have not been utilized as the substitute of the main root in practice of the oriental medicine. If we make an assumption that the terrestrial part and the fibrous side root of ginseng has not been utilized in the oriental medicine ow ing to its side action originating from the unbalanced agly­

cone ratio, the real aspect of ginseng pharmacology w hich is beneficial to host m ight be understood through the pan-cellular activity, but not through the tissue specific activity. C on­

sidering the current trends of pharmacologist's view on the ideal drug w hich is now chang­

in g from the potency-first to the safety-first, the reason why the m ain root of Korean gin-

September, 1975 J. Pharm. Soc. Korea 149

■Seng has been widely used as an miraculous drug may be understood through its properly balanced aglycone ratio. Assuming the aglycone ratio of ginseng as the decisive criterion for the indication of quality of various ginseng products, the ratio of panaxadiol to panaxatriol

T able I H - T h e ratio o f panaxadiol and panaxatriol contents in the dam m arane glycosides o f \arious ginseng species.

Samples P anaxadiol* P an ax atrio l* R a tio (p a n a x a d io l/

p a n a x a trio l)

Korean ginseng 0.65% 0.77% 0. 844

Japanese ginseng 0.179 0.35 0.511

C hikusetzu ginseng 0.739 0.313 2. 36

A m erican ginseng 0.896 1.254 0. 714

C anadian ginseng 0.896 0.739 1.212

* T he aglycone contents were assayed by the c o m b in ation of preparative th in layer c hro m a to g ra phic process a n d vanillin-HzSO# color reaction. C a lib ratio n o f the technique was checked b y standard solution o f authentic panaxadiol and panaxatriol samples.

Table -Time interval effect o f PS-A on edema suppression.

C r o u p N o.

Doses m g /k g

T im e interval* 그 ( m in )

A n im a ls w eightCgm )

E dem a at the tim e after carrageenin a d m in istra tio n

1 2 3 4 5 (lirs O

■•Control C M C 133 e 2.58 4.67 6.15 5. 33 5.15

solution s.e. i l 2 r — — — — —

S .E . 0. 555 1.03 0. 600 0. 795 0. 633

e 1.7 5.1 5. 43 4.45 4 .0

I 100 0 137 r 34.3 — 11 16.5 22.5

s.e. ± 8 .8 S .E . 0.26 ^{1.09 1.17 1.05 0.9}

e 2.11 3.47 4. 23 4.45 4. 28

n 100 60 124 r 18.1 25.7 31.3 16.5 16.8

s.e. ±10. 8 S .E . 0.83 0. 545 0. 90 1. 06 0.835

e 1.45 3. 35 3.05 3. 95 3. 67

I I I 100 120 129 r 43.7 28.3 50. 5* 26.0 28.7

s.e. ±10. 2 S.E. 0. 51 0. 76 0. 76 0. 92 0. 575

e 2.41 3. 50 3.43 2. 66 3.93

I V 100 240 136 r 6 .6 25.1 44.5* 50.1* 23.7

s.e.+9. 85 S.E. 0.64 0.286 0.71 0. 59 0. 62

e 1.37 2. 55 2.58 3. 23 2. 53

V 200 30 127 r 47.0 45.3 58. 0* 39. 5* 5 0 .7*

s.e. +8. 75 S.E. 0.328 0. 59 0. 525 0. 38 0.515

A bbreviation: C M C ; carboxym ethyl cellulose; edema v o lu m e X 10, r; percent of edema red uction . S .E .; standard error of edema volum e, s.e.; standard error o f body w eight o f anim als.

* Edem a suppression is h ig h ly significant statistically. 01}

•a) The length of tim e intervals between the m edication o f P S*A and carrageenin edema in d u c tio n .

150 H an • W oo : Dammarane Triterpene Glycoside Vcl. 19, No. 3 #

c o n te n t i n the v a rio u s g in s e n g p ro d u c ts c u ltiv a te d b y v a rio u s c o u n trie s w as e x a m in e d고®^ A s：, s h o w n in T a b le I I I , the q u a lity o f o th e r g in s e n g are n o t c o m p a ra b le w it h K o r e a n g in s e n g in respect o f the r a tio o f p a n a x a d io l to p a n a x a trio l co nten ts o f d a m m a ra n e glycosides.

Some Probelem s of B iolo gical A ctivities of D am m arane Glyccsides— W e rep orte d p r e v i­

o u s ly th e iso latio n o f a n ti- in f lam rra to ry glycosides fr o m the extract o f Panax ginseng^^^ ^ ^{a n d}

th e data are s h o w n in T a b le I V .

A s s h o w n in T a b le I V , the a n ti- in fla m m a to ry a c tiv ity o f p a n a x s a p o n in A s h o w e d the de­

layed a n d lo n g la s tin g a c tio n . T h e lo n g la s tin g a c tiv ity w as also checked i n a n o th e r experiT m e n t i n w h ic h c a rra g e e n in edem a in d u c tio n w as co n d u c te d one w e e k a fte r the m e d ic a tio n o f p a n a x s a p o n in A IC C m g /k g , p.o. A s o ne o f possible m e c h a n is m s it m a y be considered th a t th e a n ti- in fla m m a to ry a c tiv ity o f the s a p o n in m ig h t re s u lt fr o m the extended effe ct o f som e o th e r p r im a r y a c tio n , such as th e de n o v o synthesis o f c e rta in fu n c tio n a l p ro te in s. I n th is c o n n e c tio n , the a u th o rs e x a m in e d the s tim u la tin g effect o f p a n a x s a p o n in A o n the in c o r ­ p o r a tio n rate o f ^^C-leucine in to se ru m a n d liv e r p r o te in o f rat.

T able V —Effect of panax saponin A on the incorporation o f ^^C-leucine in to liver and serum protein.

E x p. N o. M aterials

Dose N o. of Radio-activity (c p m /m g protein) (m g ) mouse

Serum ( ^ ) Liver (% 0

1 C o n tro l (saline) — 3 642(100) —

P S A ( tr io l) 1 3 828(129) —

Prostisol C<^iol) 1 3 942(147) —

2 C ontrol — 3 629(100) 581(100)

P S A 1 3 1313(208) 924(159)

P S A 2 3 1515(241) 1043(179)

3 C o n tro l (saline) — 3 1058C100) 830(100)

P S A 1 3 1317(124) 1028(133)

4 C o n tro l (saline) — 3 — 529(100)

P S A 2 3 — 702(133)

M edication: Intraperitoneal injection.

^^C-leucine ： 1 /iCi/m ouse (w t.: ca. 20gm ) 2hrs. before decapitation.

Saponins: 0 .5 ^ 1 . saline soln. 6hrs before decapitation.

Three mouse livers were pooled fo r each experimental or control group.

A s s h o w n in T a b le V , p a n a x s a p o n in A a n d P ro s tis o l e n han ced the in c o r p o r a tio n rate o f C효걸-le u c in e in to s e ru m p ro te in . S u c h effect o f p a n a x s a p o n in A w as c o n firm e d b y repeated e x p e rim e n ts. T h e same e ffect b y p ro stis o l w as f u l l y in ve s tig a te d b y O u r a2 *^그 p re v io u s ly , th e r­

e fore, th e c o m p o u n d w as a d op te d as the reference fo r the b io lo g ic a l a c tiv ity . I n F i g . l , the tim e course effect o f P a n a x s a p o n in A o n the in c o ra tio n rate o f 흐분C-leucine in to liv e r p ro te in , is illu s tra te d피 .

A s s h o w n in F ig . 1, b o th th e tim e course g ra p h s o f g raded doses sho w e d m a x im u m s tim u ­ la tio n in the 4 h o u rs g r o u p a n d the e n h a n c e m e n t p r o lo n g e d fo r a lo n g tim e . T h e delayed a n d.

lo n g la s tin g te n de n c y o f the s tim u la tio n o f p r o te in synthesis s h o w n in F ig . 1 is s im ila r w i t h th a t o f a n ti- in fla m m a to ry a c tiv ity in its lo n g la s tin g a n d delayed a c tion s. Based o n th e results o f the tim e course e x p e rim e n t, it m a y be in fe r re d th a t the s tim u la tin g a c tiv ity o f p a n a x s a p o n in A o n p r o te in synthesis m a y be closely c o rre la te d w it h th e a n ti- in fla m m a to r y a c t iv it y o f the s a p o n in . T h e s tim u la tin g a c tiv ity o f g in s e n g s a p o n in o n p r o te in synthesis w as a ls o d e sc rib e d b y O u r a . H e separated a n a lc h o lic substance f r o m th e g in s e n g extract, w h ic h s h o w e d th e s tim u la tin g a c tiv ity o n the p r o te in synthesis, a n d d esign ate d th e substance as prostisoF^\

L a te r studies o n th e p ro stiso l d e m o n s tra te d th a t it w as the m ix tu r e o f s a p o n in s o f p r o to p a ­ n a x a d io l series. P ro stis o l e x h ib ite d the s t im u la tin g a c tiv itie s o n th e synthesis o f b io - m a c r o m ­ o lecules such as m essenger으®팡 N A a n d D N A - d e p e n d e n t- R N A p o lym erase 2®^ T h e e arlie st p r im a r y a c tio n o f *Trostisor* w as s h o w n to be th e s tim u la tin g a c tiv ity o n the D N A - d e p e n d e n t R N A - p o ly m e ra s e , n a m e ly , s tim u la tio n o f p r o te in synthesis a t tr a n s c r ip tio n a l leveP®\ Based o n these o bse rv atio ns, O u r a suggested the m e c h a n is m o f h o rm o n e - lik e a c tiv ity f o r th e m o d e o f a c tio n o f g in s e n g sa p o n in s. H o w e v e r , w h e n w e c o m p a re th e tim e course data o f th e a u th o r s 오분그 w ith th a t o£ O u r a 2구\ it c an be n o te d th a t th e onset o f m a x im u m s tim u la tio n o n p r o te in synthesis is s ig n ific a n tly d iffe re n t i n th e ir le n g th o f la te n t tim e . P a n a x s a p o n in A has s m a lle r m o le c u la r w e ig h t ( m o l. w t. 800) th a n th a t o f p ro stis o l ( m o l. w t . ; a p p ro x . 1, 100) a n d it sh o w s th e m a x im u m s tim u la tio n a t 4 h o u rs a fte r a d m in is tr a tio n o f the s a p o n in , h o w e v e r, i t w as re p o rte d th a t th e m a x im u m s tim u la tio n o f p r o te in synthesis w as attain e d a t 8 ^ 1 2 h r s a fte r th e a d m in is tr a tio n o f p ro stis o l. T h e a p p a re n t d e p e n d e n c y o f the la te n t tim e o n th e m o le c u la r w i g h t o f the s a p o n in m a y suggest ta c itly som e in f o r m a t io n e ith e r o n the m o le c u la r aspects o f a c tiv e fo rm s o f th e s a p o n in a t the p r o x im ity o f its rece pto r site a n d / o r u p ta k e a n d tr a n s p o r t o f these c o m p o u n d s b y cells. T h u s the m e c h a n is m o f g in s e n g p h a r m a c o lo g y is n o w b e in g u n d e rs to o d

■at the le vel o f m o le c u la r in te r a c tio n o f g in s e n g c o m p o n e n ts w it h the c e llu la r c o m p o n e n ts . I n s h o rt, the b io c h e m ic a l studies o n g in s e n g s a p o n in s p u b lis h e d h ith e r to has been c o n c e rn e d o n ly w it h the p ro b le m s o f the b io lo g ic a l responses w h ic h are m a n ife s te d b y the a d m in is t r a tio n o f g in s e n g s a p o n in . R e p o r ts c o nc e rn e d w it h th e tr a c in g th e s a p o n in i n the b io lo g ic a l system are n o t a v a ila b le a t present. T h e re fo re , the d ire c t e vidence f o r the presence o f g in s e n g s a p o n in at sub - c e llu la r a c tiv e site a t the tim e o f its m a x im u m a c tiv ity is desireable at present.

The In testin al Absorption and the Route of E xcretio n —Although the in te s tin a l a b s o rp ­ tio n o f th e s a p o n in s is p r im a r y req uisite fo r th e u n d e r s ta n d in g o f the v a r io u s p h a r m a c o lo g ic a l responses o f th e s a p o n in , fe w is a v a ila b le w i t h in c lu d in g o u r recent data단®\ R e c e n tly , th e a u th o rs e x a m in e d th e in te s tin a l a b s o rp tio n a n d th e r e n a l e x c re tio n o f g in s e n g s a p o n in . A s s h o w n in F ig . 2, p a n a x s a p o n in A appears r a p id ly i n th e bloo d, liv e r , a n d in th e u r in e as u n c h a n g e d fo r m a fte r the in tra v e n o u s a d m in is tr a tio n o f the s a p o n in . I n a n o th e r e x p e r im e n t, the u n c h a n g e d p a n a x s a p o n in A w as detected i n th e u r in e a n d b lo o d o f the r a b b it 4 h r s a fte r th e o ra l a d m in is tr a tio n o f the sa p o nin . T h e re fo re , easy a b s o rp tio n a n d easy e x c r e tio n o f p a n a x s a p o n in are observed.

T h e u n c h a n g e d s a p o n in w as also detected i n th e in te s tin a l lu m e n o f th e a n im a ls to w h ic h th e

^saponin w as a d m in is tre d b y in tra v e n o u s in je c tio n . I n o rd er to f in d o u t the e x c re tio n ro u te o f

September, 1975 J- Pharm . Soc. Korea 151

I Uf. %4

6>. i t- '고용^ <

n i

Figr. 2^ D is tr ib u tio n of panax saponin A in various extra-cellular specimens. Panax saponin A was administered to the ra ­ b bit b y intravenous injection, 200m g / 2. 0kg body w t. Panax saponin C(prot- opanaxatriol-rhamno-diglucoside) was added to every specimens as an internal references fo r the quantitative assay of panax saponin A . T L C , Solvent, C H CI3； M e O H ( 3 : 1) ； colour reaction, H2S O4 spray and heat. C, control urine;.

1, U rine 0 ^ 1 h r; 2, U rin e 1 ^ 2 hr; 3, U rine 2시 3hr; 4, U rine 3^ 4h r； 5, U rine 4 ^ 5 h r ; 6, U rine 5-*^6hr; 7, G a ll b lad ­ der; 8, S m all intestine; 9, Large inte­

stine； 10, Blood; 11, C o n tro l blood.

100 -0 C-

10 2 4 hrs

Fiff. 1— T im e course effect o f P S A on the incorporation of 고관C-Ieucine in to liver protein.

P S A Cl or 2m g/m ouse) was adm inistered intraperitoneally to mice at each designated tim e p r io r to k illin g and ^^C-leucine (1/^Ci/mouse) was injected intraperitoneally 2 hrs prio r to k illin g the anim als by decapitation. T he specific radioactivity (c p m /m g pro tein) of liver protein was assayed as described in methods. 2분그 T he control value was 529 c p m /m g protein. Three mouse livers were： pooled fo r each experimental o r control group.

A — ：2mg, ~ O ™ ：lm g

#

I

ir lU tiK > Ur

C i 2

panax saponin A in the intestinal lumen, rabbit was cannulated at bile duct and the intestinal content was analyzed to detect the presence of unchanged panax saponin A by thin layer chr­

omatography. As shown in F ig. 3, the unchanged panax sapOnin A was detected only in the bile secretion w ith one unidentified spot w hich was considered probably as the metabolite of the saponin. Therefore, we concluded that panax saponin A in the intestinal lum en seems to appear through the excretion of bile duct, but not through the direct excretion of intestinal mucosa.

152 H an • W oo : Dammarane Triterpene Glycoside Vol. 19. No.

I

!

A .

14

121

서onlloo버o

;u909J9dlIOP2CKi-I8c어SJOSq.o그

September, 1975 J. Pharm, Soc. Korea 153

A

B • «

C 00 ^• * 0 O o o

D 0 0 ^{■ v ；v d > 0}

Fig. 3—— T h in layer chrom atogram of bile. Detection o f panax saponin A in the of bile duct cannulated rabbit. Panax saponin A was adm inistered 200 m g /2kg

injection to the rabbit cannulated at bile duct. Intestinal content was collected Shours after th e ad m inistratio n o f panaxsaponin A by intravenous injection. T L C samples o f each specimen w e re prepared by o rd in ary butanol extraction. T L C ; solvent; C H C I3： M e O H C3

H2S O4 spray and heat. A , P S A ; B, collected fro m bile juice; C, collected D, collected fro m large intestine.

intestinal lu m e n by in tra v e n o u s

: 1). c o lo u r reaction ; fr o m sm all in te s tin e ;

#

•5 I8(hr.)

Fiff. 4— U rin a ry excretion - curve o£ unchanged panax saponin A . T he saponin was a d m in iste re d 175 m g / 1 .75kg. to the rabbit by intravenous injection U nch ang e d panax saponin A was assayed by preparative th in layer chrom ato graphy and vanillin-HaSO^ colour reaction®®^ P an ax s a p o n in C was added quantitatively to each specimen as the internal references fo r the assay o f panax^

saponin A .

The time course curve of urinary excretion of panax saponin A shows two phase, it constit­

utes rapidly decreasing curve from zero to six hour and slowly decreasing curve from six hour to the end of the experiment, as illustrated in Fig. 4.

Biphasic curve of urinary excretion of panax saponin A implies that this compound appear to exist both extra-cellularly and intra-cellularly i n v iv o . A t present it is uncertain whether panax saponin A itself or its metabolites exhibits biological activities.

However, as shown in Fig. 2, an unidentified spot appeared on thin layer chromatogram from samples of urine and bile of the rabbit after the administration of panax saponin A . This unidentified spot is considered to be the metabolite of panx saponin A . In addition, this unidentified substance was excreted long time. Such long lasting excretion rate of this unidentified substance coincide w ith long lasting feature of pharmacolgical activities like the anti-inflammatory and stim ulating activity of protein synthesis, so if we can put some co-relation between excretion rate and pharmacological activities of panax saponin A , the unidentified substance as metabolite of panax saponin A may play a important role. The excretion of unchanged panax saponin A was quantitatively estimated in the various extra­

cellular specimens, and illustrated in Table V I.

Table V IE x t r a - c e llu la r distribution of panax saponin A .

R o ute A m o u n t (m g ) Percent/total P S A

154 H an • W oo ： Dammarane Triterpene Glycoside Vol. 19, No. 3

U rin e 128.5 64.25

S m a ll intestine 2.34 1.17

Large intestine 12.01 6.01

B lood 12.7 ? 6.35

Gall bladder Detected ~

T o ta l 155.55 77.78

Difference 44.45 22.23

T he content of unchanged panan saponix A in the various extra-cellular specimens were analyzed six hours after the intravenous adm inistration of panax saponin A , 200mg/2kg of experimental rabbit.

As shown in Table V I, unchanged panax saponin A distributed in the extra-cellular phase reached approximately 1 1 % at six hours after the intravenous administration of the saponin. The difference between the total excretion of unchanged p a n a x saponin A and total amount administered w ill probably correspond to the amount of metabolite and or the amount of slowly excreting cellular component of panax sapinon A . Therefore, researches concerned w ith the chemical nature of the metabolite and w ith the biological activity of the substance w ill have the greatest importance in study of ginseng for future.

Labelling of Radio-isotope into P an ax Saponin A— Tracer compound labelled w ith radio­

isotope is indispensable for the studies of metabolism of ginseng saponins. One of the authors야^

established a new radio-labelling synthetic procedure by which ^^C-isopropyl fragment can be

September, 1975 J. Pharm. Soc. Korea 155

#

C H O

"""G1u( Ac) 4

A c O c 5 - G lu t A c ) 4 0 - G »o( Ac) 4

m p263° rrip23i*

HL 1모

Scheme 3 a ~ P re p a ra tio n of trisnoraldehyde derivative of panax saponin A (ginsenoside R g . )

#

/H

C ■+■ P{C6Hs)3 ► (CsH5)3 = P ~c ^

C'^Hj ^ \l I I ^C'^Hs

^ H

CH3,

S = 0 H- NaH ^{N a C H}2

S = 0

> {C6H5)3SP—C ^ C'^H3

CH5^ CH3"^

Schem e 3b— Preparation of 호센C-isopropyl-W itting reagent.

introduced to the side chain of ginseng saponin.

As shown in Scheme, the method constitutes three steps; the first step is the breakdown of side chain by osmium tetroxide oxidation fallowed by periodate oxidation, the second step is the synthesis of 고관 C-carrying W ittig reagent, and the last step re-generation of the original saponin molecule labelled w ith 호스C-isopropyl fragment by condensation of the W ittig reagent w ith the trisnor-aldehyde derivative of the saponin. This experiment was conducted w ith panax saponin A w ith overall yield more than 4 1 % , T his method may be applicable to the every dammarane glycosides of panax ginseng. Radio-labelling on the side chain w ill have

some danger of metabolic breakdown of labelled fragment during tracer studies i n v i v o .

Therefore, labelling the saponin on polycyclic skeleton is more desirable. Recently th e authors^2그 established a new synthetic procedure labelling dn polycyclic skeletdn by tritum (® H ).

A s shown in Scheme, the method constitutes the selective acetylation of hydroxyl fu n c tio n leaving one hydroxyl function in sapogenin unacetylated, oxidation of the unacetylated hydroxyl function by Sarett reagent, tritation of the resulting ketonic compound by keto-enol tautomerism and regeneration of labelled original saponin molecule by stereo-specific reduction.

These two labelling procedures w ill offer a great help in the studies of metabolism bf saponins.

of panax ginseng.

R E F E R E N C E S

1. 1.1. B rekh m an and L L D avydov, A n n . R e v , PharmacoL,^9, 419 (1969).

2. O. T anaka, D a is a Cmetabolism), 10, 86(1973),

3. K . T akagi, P r o c e e d , o f I n t e r n , G in s e n g S y m p . (T h e C entral Res. In s., Office o f M o nopoly^.

R epublic of K orea), 1974, p-119.

4. H . O ura and S. H ia i, D a is a , 10, 102 (1973).

5. M . Y a m a m o to , ib id ,, 10, 119 (1973).

6. G .B . E lyako v, L .I. Strigina, N .I. U varova, V .B . V askovsky, A .K . Dsisenke and N .K . K o che tko v,.

T e tr a h e d r o n L e tt., 1964, 3591.

7. G .B . E lyako v, and L .I. S trigina, T e tr a h e d r o n , 24, 5483 (1968).

8. S. Shibata, O. Tanaka, T . A n d o , M . Sado, S. T sushim a and T . Ohsawa, C h e m . P h a r m . B u l l . , 14, 595 (1966).

9. Y . N agai, O. T anaka and S. Shibata, T e tr a h e d r o n , 27, 881 0 9 7 1 ).

10. S. Sanada, O. Tanaka, J . S hoji, O. T anaka, and S, Shibata, C h e m . P h a r m . B u ll.y22, 421 C1974X 11. B .H . H an, Y .N . H a n and L .K . W o o , T h is J o u r n a l, 16, 129 C1972).

12. B .H . H a n and Y .N . H an, K o r . J . P h a r m a c o g ., 3, 211 (1972).

13. M . F u jita , H . Ito kaw a and S. Shibata, Y a k u g a k u Z a ss h i, 82, 1634 (1962) ； C h e m . P h a r m . B u l l . , 11. 756 C1963).

14. M . N agai, O. T anaka and S. Shibata, T e tr a h e d r o n L e tt., 1966, p-4797.

15. S. Shibata, O. T anaka, K . S o m a,Y . lid a, T . A n d o and H . N ak am ura, T e tr a h e d r o n L e t t ., 1965^

207.

16. G .B . E lyakov, A .K . Daisenko and Y .N . K lk in , T e tr a h e d r o n Lett.y 1966, 141.

17. F . S m ith , G .W , H ay and B .A . Lewis, M e th o d s in C a r b o h y d r . C h e m .,5, 361.

18. O . T anaka, M . N agai and S. Shibata, T e tr a h e d r o n L e tt*, 1967, 391.

19. S. Shibata, T . A nd o, O. Tanaka, Y . M eguro, K . S om a a n d Y . lid a, Y a k u g a k u Z a s s h i, 85, 75크 C1965).

20. F .G . Fisher and N . Seiler, Ann.^^644, 146 (I9 6 0 ).

21. Y , H irose, T . Yanagaw a, Y . Sayama, T . Igarashi and T . N akatsuka, N ip p o n M o k i z a i G a k k a is h i^

14. 36 C1968).

22. B .H . H a n . H J . C h i and Y .N . H a n , Kor. J. Pharmacog.,^4, 167 (1973).

23. B .H . H an, U npublished.

September, 1975 J. Pharm. Soc. Korea 157

H an • W oo ： Dammarane Triterpene Glycoside Vol. 19, No. 3

24. B .H . H an, C .H . K im and Y .N . H an, K o re a n B io c h e m ., J . 6, 63 (1973).

25. B .H . H an , K o r e a n J . P h a rm a c o g ,^ 3, 151 (1972).

26. L .K . W o o , B .H . Han, D .S. P ark and U .Y . R a , K o r . J . P h a r m a c o g ,, 4, 181 (1973), 27. H . O ura, S. N akashim a, K . Tsukado and Y . O hta, C h e m . P h a r m . B u ll,, 20, 980 (1972).

28. H . O ura, S. H ia i and H . Seno, C h e m . P h a r m . B u ll., 19, 1598 (1971).

29. S. H ia i, H . Oura, K . Tsugada and Y . H irai, C h e m . P k a r m . B u ll., 19, 1656 (1971).

30. B .H . H an, E.B . Lee, U .C , Y o o n and L .K . W o o , in press.

31. L .K . W o o , T h is J o u r n a l, 17, 123 (1973).

32. B .H . H a n , B .J. Song and L .K . W o o , U npublished.

33. L .K . W o o , B .H . H an, D .W . B aik and D .S . Park, T h is J o u r n a l, 17, 129 (1973).