The Structure of Phytolaccoside G

Journal of the Pharmaceutical Society of Korea 21， 159-162 C1977)

The Structure of Phytolaccoside G

W o n S i c k W o o a n d S a m S i k K a n g

N a tu ra l Products Research Institute, Seoul N ation al University, S eoul 1 1 0

(Received July 3

1977)

Abstract—The structure of phytolaccoside G (I), one of the minor saponins of the roots of Phytolacca americana, has been elucidated as 3-jQ-D-xylopyranosyljaligonic acid. And phytolaccoside B ( II) is 30- methylester of phytolaccoside G.

The roots of Phytolacca americana (Phytolaccaceae) w hich are being used as a fo lk medicine in treating edema and rheumatism, has been described to contain a large am ount of antiinflammatory saponins1). This paper describes the structural elucidation of phytolaccoside G .

Phytolaccoside G ( I ) , C

H

O

• 2H

0 , mp 276-8°, M

d+77°, on acid hydrolysis, gave jali- gonic acid2)，mp 318-9° and d-xylose. Methylation of I w ith C H

N

gave d im e th y le s te r(III), C

H

O

, mp 181-184°. Exhaustive methylation of I by H akom ori， s method3) gave the per- methylated product(V ), which showed N M R signals for five tertiary methyls(<5 0. 70

1. 22

seven O-methyls (d 3.31

3 .6 2 )， an anomeric proton (d 4.20, 1H, d, J —8 H z ) and an olefinic proton (3 5.48, m, 1H ). The mass spectrum showed the molecular ion peak at m /e 748 (C

H

O

) and two peaks at m /e 557 and 175 which corresponded to mass of genin portion minus H + formed by cleavage of C-O bond at x and that of sugar portion by cleav­

age at y, respectively. Besides these peaks, the tw o retro-Diels-Alder fragmentation ions from the ion of mass 557 appeared at m le 306 and 219 and important peaks at m je 247

246

233, 187, 173, 133 formed by further fragmentation from the R D A fragment ion of mass 306 were also observed. From the above results, it was apparent that I was composed of one molecule each of d-xylose and jaligonic acid.

Phytolaccoside G was oxidized w ith H IO

and two moles of reagent was consumed. H y d ­ rolysis of the oxidation product gave jaligonic acid. Formation of jaligonic acid indicated that xylose must have been attached to the C-2 or C-3 position rather than C-23 position and 2- mole-reagent consumption indicated that xylose was present as pyranose form rather than furanose form. Methanolysis of IV gave di-O-methyljaligonic acid dim ethylester(V) w hich showed in its N M R spectrum five tertiary methyl signals (3 0. 60

1.14)， tw o m ethyl ether signals (3 3.33, s, 6 H ), two methylester signals (3 3.59 and 3.71, 3H each), an A B quartet (3 3. 06 and 3. 32, 1 H each, *7=10 H z) and a multiplet (d 5. 38, 1 H ) due to olefinic proton.

The presence of a doublet (1 H ) centered at d 5.01 C /二-3 H z) in the N M R spectrum o£

160 Woo • Kang : The Structure of Phytolaccoside G Vol. 21, No 3

acetate of V clearly indicated that 3/3-OH of IV was not methylated. T his result suggests that xylose unit is linked to C-3 of jaligonic acid in the saponin.

/3-Orientation of glycosidic linkage was suggested not only from the coupling constant of the anomeric proton signal of permethylated product, but also by molecular rotation difference between the saponin and the sapogenin (Table I) .

Table I —Molecular rotations of phytolaccoside G and related substance.

Substance 〔 。 0

〔 m 〕d

Observed value

Phytolaccoside G +77 +500.5°

Jaligonic acid +113.7 +589°

〔M〕d contribution of xylose unit -88.5°

Literature value4)

Methyl-a-D-xylopyranoside +249°

Methyl ᄀ S-D-xylopyranoside -107°

A m e n d m e n t— — Phytolaccoside B was earlier shown to be 3--d -glucopyranosy 1 phytolacca- genin5). However, further examination showed that sugar unit of the saponin was d-xylose and not d-glucose. A n d methylphytolaccoside B was identical to dimethylphytolaccoside G (N M R , M S, mmp, co-TLC). Therefore, the constitution of phytolaccoside B ( I I ) was revised as 3- j3-D-xylopyranosylphytolaccaprenin.

I R i= R 2 = R 3 = H II Ri=M e, R

'-=R

= H I I I R i=R2=M e, R3=H IV Ri=R2=R3=M e

E X P E R IM E N T A L

M p， s are uncorrected. N M R spectra were taken in

using T M S as internal reference

at 100 M H z.

Iso lation of phytolaccoside G ( I ) — The saponin mixture was applied to a silica gel column and eluted w ith M eO H -C H C b (gradient) to give a G fraction w hich was crystallized from ethylacetate saturated w ith water, mp 276〜 278°, M ^ 5+77° (c —0.5 in M e O H ), IRJJS?

cm-1; 3410(OH), 1705(acid), 1000

llOO(glycoside).

Anal, Calcd for C35H 54O n • 2H20 : C, 61.21; H , 8.51. Found

C, 61.61; H , 8. 23.

The saponin (100 m g ) was heated w ith H Cl-M eO H under reflex for 3hr. The product was diluted w ith H

O and extracted w ith E t

. The E t

extract was crystallized from M e O H to give needles of jaligonic acid, m p 318

319°， w hich was identified by direct comparison w ith an authentic sample (mmp, co-TLC, I R and M S ). T he aqueous layer was hydrolyzed w ith HC1, neutralized w ith A g

CC

and evaporated in vacuo. The residue was found to be d-xylose by T L C (MeOH-CHCliB-Acetone-NH^OH

5 12 13 12, R f 0. 35; MeOH- -CHC13-NH4OH-H20==30 ： 60 ： 3 ： 4, R f 0.11).

D im e th y le s te r (III)ᅳ The saponin (100 m g ) in M e O H (20 m l) was treated w ith C H

N

in the usual manner. The crude product was purified by P L C (C H C b- M eO H -N H ^O H - H ^O ^^O : 4 ：1 ：3) and crystallized from M eO H to give needles of I I I ，mp 181 〜 1840，w hich is identical w ith methylphytolaccoside B.

H I O r A lk a li degradation of I

A M e O H soln of I (32. 5 m g ) was added to 20 m l of 0. 01 M H IO

, the volume made up to 35 m l w ith M e O H and the soln kept in the dark at 4° until I was completely disappeared on T L C (about 10 days). A n aliquot was removed and H IO

was estimated w ith 0 .0 1 M NasAsOs in the usual way. The consumption of oxidant was 1.99 moles. To the remainder soln, 1 ml of ethyleneglycol was added, after standing at room tem p­

erature for

hr, diluted w ith H

O and extracted w ith B u O H . The B u O H extract was heated under reflux in 10 m l of 5% N aO H -M eO H for 5 hr. The reaction m ixture was acidified and extracted w ith E t

(X The E t

(〕 extract was crystallized from M e O H to yield needles of ja li­

gonic acid, which was identical w ith an authentic sample (m m p, co-TLC).

P e rm e th y la tio n of I

According to the H akom ori， s method, N a H (200 m g ) was stirred w ith dimethylsulfoxide (5 m l) at 70ᄋ for 45 m in under nitrogen gas flow. T o this methylsul- finyl anion soln, 200 m g of I in 5 m l of dimethylsulfoxide was added and the m ixture was stirred for 30 m in at room temperature under nitrogen gas flow. C H

I (5 m l) was added and the reaction m ixture was allowed to stand at room temperature overnight w ith stirring.

After dilution w ith water, the mixture was extracted w ith C H C I

and the organic layer was washed w ith water, dried and concentrated. The residue was chromatographed over silica gel and eluted w ith C H C I

to give permethylaied product ( I V ) ，which was purified from MeOH- H 20 to give a white powder, m p 111〜 40， [세 f~82.

(c = 0 . 34 in M e O H ), IRkbJ cm -1:

O H (n il) 1730 (ester).

M ethanolysis of IV

A solution of IV (100 m g) in 20 m l of 3% H Cl-M eO H was refluxed for

hr. The reaction m ixture was concentrated, diluted w ith water and filtered. Ppt was crystallized from M e0 H - H 20 to yield a white powder of di-O-methyljaligonic acid dimethyl- ester (V ) ，mp 105〜 107o.

A nal. Calcd. for C

H

O

C, 71.05; H , 9.47. F ou nd

C, 69. 84; H , 9.87.

The filtrate was extracted w ith C H C I

. The C H C I

extract was found to be methyl-2, 3, 4-tri-O-methyl-D-xyloside by T L C (ethylacetate, R f 0 .65

0.55) and G L C (column, 5%

N P G S on Gas Chrom Q, 100

120 mesh, 2.2 m X 4 m m ; column temp., 170°; F ID temp., 200°; injector temp., 190°; N

flow, 45 m l/m in

tR(m in ), 2.2

2 .8 ).

A c e ty la tio n of V — The sample (50 m g) was heated w ith AC

O (0 .5 m l) and C

H

N (0 .5 m l) for 2 hr. Pouring onto ice gave solids which were purified from MeOH-H^O to yield a white powder, mp 85

89°; N M R :

0.73

1 .

(

X C H

)

2.12(3 H , s, C H

C O ), 3.11 and 2. 87 (1 H each, A B quartet, J —10 H z), 3.26(3 H, s, O M e), 3.30(3 H, s, O M e), 3. 60(3 H , s, C O O M e), 3.71(3 H , s, C O O M e), 5.01(1 H

d, J —3 Hz, H-3) and 5.38 (1 H, m, H-12).

R E F E R E N C E S

1. W.S. Woo, K.H. Shin and S.S. Kang, Kor. J • Pharmacog .， 7

47 C1976); W.S. Woo and K.H- Shin, J . Pharm. Soc. Korea, 20，149 (1976).

2. W.S. Woo, Lloydia, 36, 326 (1973).

3. S. Hakomori, J . Biochem.y 55

205 C1964).

4. I. Kitagawa, A. Inada and I. Yosioka, Chem. Pharm. Bull., 23，2268 C1975).

5. W.S. Woo and S.S. Kang, Phytochemistry, 15，1315 (1976