Biosynthesis of diosgenin in Dioscorea tubers

.AR(‘III\‘ES

OF

BIOCHEJIISTRY

ASD

Biosynthesis

BIOPHYSI(‘S

of

92,

13-16 (1961)

Diosgenin

in Dioscorea

Tubers

The inwrporation of rntlioacet,ate into diosgenin by homogen:ltcs of Ilioscorea has been demonstrated by rigorous purification of the isolated sapogenin. MewIonic acid is not incorporated into diosgenin to any significant extent under t hrse conditions. The diosgenin cont!ent, rind biosynthetic :lctivit,y of v:rriow parts of :L tjubt:r show :tppreciable ditierenres.

joribrtnda

\I’hilc much information has now ac’cumulated concerning the biosynt,hesis of steroids in animals (I), prwtically nothing is known about, the biogenesis of typical plant steroids. The format ion of rubber (‘) , carotenoids co, and other iaoprene polymers from mevalonic avid has been demonstrnt,ed in and it, seems reasonable to assume plmts, that plants synthesize steroids also from :wct:tte \.in me\-ulonic~ avid. Indeed, Sander and (irisebwh (4) have shown that tomato seedlings, grown on a nutrient rolutio1i contailiing radiowt ive wetate, incorporat,e CL4 into tomatin and, more recent#ly, Ramstad and Beal (5) have isolated labeled digitosigenin from Digitalis injec%ed with mdiowtive mevaloni~ Ad. L)ioscorca tubers art ail import,ant, source of steroidal sapogenins, and WC were therefore interested in studying the biosynthesis of steroids in this genus. Dioscorca jloribun.da tubers,’ which are rich in diosgenin, but’ also contain some yamogenin (G), were used in our experiments. Tuber homogenates were inwbatcd with radioactive acetate or merand the sapogenills then were alollntc, extra&d by the method of Morris et al. (7). 1 (ienrrollsly supplied I)y the Plant Industry Stxt ion of the U. S. Ikpartment~ of A4gricwltllre, 13rlt svillc, AI:tryl:tnd. t hrorlgh the courtesy of .Jowl)h R. H:~un, Srw Crops Research Branch.

Thr: extracts were frwtionated by adsorption chromatography on alumina columns (8), and the radioacti\Gty in aliquots of the fractions VYWdetermined.

ISKBATIOT

OF WHOLE

TT:BEKS

~1 tuber Lveighing 1,260 g. was homogenized in :t Waring blendor with 900 ml. of cracltetl ire and then transferred to :I 6-l. flask with 600 ml. water. A solution of 50 pc. sodinm ncctatem2-CL1 in 5 nil. water was :tddcd, :tnd the homogen:~te. exposed 10 the air, was stirred in the dark for 16 hr. I\ftcl :tdtlitioii of 500 ml. of ~onceritrutcd hydrochloric :icitl, the mixture was then rcflnscd for 3 hr., c~ooletl, diluted with 1 1. water, and filtcrrd by suction. TOP residue \VRS \vashed \vit h folbr l-l. port,iow of watrr and dried at 110°C’. for 2 hr. The dry mntcrinl \v:ts cst,r:rc*tcd in :I Hoxhlrt ap pur:~tjlls with 5 I. of petroleum ether (t)oililig range 30-60”) for 16 hr. and then \vith :i mixture of 6 1. of petroleum rt her xnd 600 ml. dichloromrt hune for 8X hr. The c~ombinctl extracts were cvapor:itf91, leaving 16.6 g. of seniicrystnlline niaterinl. This was dissolved in 150 nil. Iwnzene ant1 apl~lirtl to :t c~hromatogr:i~)~~ic rolumn of 500 I[. of grndc III :tl~m~ina.2 r\fttr washing with benzene. :t diosgrninwntnining fraction, weighing 13.0 g., was cluted with ether+enzene (1: 1). This fraction was rem chrorn:ttogr:1I)lle(l on :L similar col1mln. :in(l the diosgenin was elated \vith two 2-l. portions of 5% ether in benzene. Two i-mg. :lliquots of each fr:ic>-

14

HEFTMANN, TA4BLE

PURIFIUTION

ASI)

R.~DIOA~TIVITY

I OF

I~IOSCENIN Countsimin./ mmole”

hf.p.”

Compound

BENSETT

‘C

Diosgenin Diosgenin acetate Diosgenin acetate Diosgenin acet.ate” Diosgenin Diosgenin Diosgenin

,4

198-202

B

187-198

282 It 51 219 f 68

C

192.5-194

145 zt 20

1)

192-196

- 121”

115 zt 20

I<

F c:

203.5-206 203-205 229-232

- 120”

156 f 137 f

H

207-20s

15 15

henzoate

Diosgenin

111 Zt 15

a See text. b Kofler block, corrected. c In chloroform. d zko.9 error. e Found: C 76.01, H 9.76; cnlcd. C 76.27, H 9.71. tion were plated on copper planchet,s, untl t.heir radioactivity was determined under a micromil window tube in an atmosphere of Q gas. The first fraction, which weighed 3.1 g., had :I specific activity of 16.3 counts/mirl./nig.; the second one, weighing 7.5 g., had a specific :tct,ivity of 10.6 corlnt,s/min./mg. When each of the two fractions was crystallized from bellzerle-petrole~Im ether, the specific activities became idcnt,ical, 5.0 counts/ min./mg. The second diosgcnirl-containing fraction was then subjected to a series of operations and :tft,er each treatment 7-mg. aliyuot,s xere counted in duplicate to a 0.9 level of confidence. The results are summarized in Table I. Treatment A consisted of recrystallization from ethanol. One gram of this product UPS then refluxed in 15 ml. acetic anhydride for 15 min. The acetate w:~ cryst)allized from ethyl acetate (B) and then twice from glacial acetic acid (C and D). Hydrolysis of diosgenin acetate hy refusing in an escess of 5% potassium h,vdroside in methanol, followed bp recrystallieation from ethanol gave the product listed under I’. After recr>-stallization from acetone (F), diosgenin was benzoylated by the method of Barua el al. (9,. The benzoatc ~-as crystallized from ethanol ((:I and then h>-drolyzed like the :tcet.ate. The product listed under H 1~:~s crystallized from hexane. Infrared spectra of the samples listed under 1) and F were found to be identical with those of authentic diosgenin xcetut.e and diosgenin, respectively, and their melt.ing points, optical rotations, and

.ZSI) BoSSEtl

C and I un:tl,vses (cf. T:tl)le I) are likewise in agreement with the expected values. In another experiment, a tuber weighing 805 g. was incllbat,ed with 50 PC. mcvnlonic acid-2-C’“. of The s:tnl~ procedure was used for the isolation diosgenin as in the preceding experiment,. The first diosgcnin fraction from the chromntogram had a specific activity of 78 courlts/n~in./mn~ole. Ho\vever, the radioactivity of the second diesgeriill-ront:iining fraction did not differ signifirantly from the background. In order to eliminate the possibility of differences in biosynthetic :tctivitJ- between individllal tubers, the homogenate from a single tuber, xeighing 387 g., was divided in half. One half was iI)cubatcd with 50 PC. of radioactive acetate and the other one 1vit.h 50 pr. of radioact,ive mevalonate. Again, radioactive diosgenin could be isolated from the former. Its specific act,ivity, in counts,’ min./mg., on crystallization from benzene-hcsnne from et,hnnol 1.07 & was 5.69, on rerrystallization rec*r~stallizatiori from acetone 0.09, subsequent 0.99 + 0.08, and final recrystalliznt~ion from hexane 0.88 + 0.09. The diosgenin sample isolated after incubnt,ion with r:tdio:tct,ive mevalonic arid had an initial sperifir activit>- of 0.26 couIits/mill./mg., \vhich upon rerryst,allization from benzene-hexane dropped to 0.04 counts/min./mg. INCVBATION

OF PARTS

OF A TXJBER

A tuber weighing 266 g. was divided into four parts including, as shown di:rgrammatically in Fig. I : Part 1, the new growth of t,he tuber; Part, 2, the bulk of the upper (dorsal) portion of the older growth; Part 3, the lower (ventral) portion; and Part -l, a shoot, together with a small port,ion of t,uber. 1’:ach part was homogenized in a Waring blendor with an equal weight of cracked ire for containing 6 PC. 10 min., an aqueous solution sodim acetate-l-CL4 was added, and the mixture was again homogenized briefly. After being transferred to :L 2-l. flask with 250-500 nil. water, the homogenate was kept in the dark for 18 hr. At the end of this prriod, a suflicient quantity of concentrated hydrochloric acid was added to make a 3 3’ solution, and the mixture was refluxed for 3 hr. After cooling and diluting it wit,h 25OG500 ml.

FIG. 1. Dissection

of L)iosco,ea

tuber

RIOSYBTHESIS

15

OF DIOSGEMN

e-a-0-0

C. P.M. /ma.

o--0--0-0

Wt; mg.

3000-

i d ci >- zoooc 2 G s E h IOOO-

FRACTION

FIG. 2. I>istribution

of radioartivit,y

water, t.he snspension was filtered by suction, and the residue was washed free of acid with water and dried at 105” for 1 hr. The dry material was estrarted in a Soshlet, apparatus with 300 ml. of petroleum ether for 18 hr. The estrnct was evaporated to dryness and weighed. Grade III alumina (30 times the weight, of the estr:uA) was packed in a chromatographic column. The est,r:wt, was dissolved in a minimum amount of prt,rolcum ether and applied to the column. Frartions of 53 ml. each were collected, using the following ehmnt,s: Fractions No. l-2 petroleum ether, ii-4 l%, 5-6 2.5%, 7-8 5%, 9-10 lo%, 11-12 %5%, 1% 16 500/, benzene in pet.rolewn ether, 17-18 hcnzene, 19-22 5%, 23-21 lo%, 25-26 25’%, 27-28 50% ether in benzene. 29-30 ether, 31 0.50/0, 32 l%, 33 2.5’%, 34 5%, 35 lo%, and 36 20’yY0methanol in ether. Two-milligram aliquots of each fraction or, in the case of frartions weighing less t,han 2 mg., lhc t.otal fractions, were plated and counted as dcwribetl abow. Figure 2 shows graphically the (list ributiou of radioactivity in thr elllate fractions from Part 1 of the tuber. ‘I’hr clut ion 1):ttterns wcrc qlmlitatively similar

NO.

in chromatographic

Y. I

2 3 1

42 57 120 47

fractions

<..

‘-,I

0.227 0.28-l 0.212 0.519

5.9 6 .93 3.44 6.20

for all four parts of the trlbrr, k)rlt there \verp so1ue differences in the diosgenin content and in the biosynthetic activity of the different parts. The data arc summarized in Table II. The per cent of crude diosgenin is based on t hc weight of the diosger~in-collt,:riniIlg fract.ion anti the per cent incorporation on the weight of the t,ot.al lipid cstract. In each case the radioactivity of the tliosgeniwcontaining frartion \v:w relativelyIo\v, and upon rccr?st,nlliz:ttior1 and conversion to the ux-

16

HEFTMAPiN,

SENh-ETT

tate the specific activity dropped continuously. Insufficient diosgenin was obtained in this experiment to permit purification to constant specific activity.

The incorporation of radiowet& into diosgenin has been est~ablished by ext,eusive purification of the isolated sapogenin. However, calculated on the basis of the constant specific activity of 141 counts/min. /mmolc, only 0.013 % of the administered acetate was utilized for the synthesis of diosgenin, indicating t,hat acetate is largely convert,ed to ot,her products by Dioscorea tubers. Up to about 7 % of the administ)ered radioactivity can be accounted for in the lipid cxkact (cf. Table II). Chromatographic evidence (Fig. 2) suggests that, at. least six major products of biosynthesis may eventually be isolated from tubers. Whet,her any of these compounds represents a precursor in t.he biogenesis of diosgenin remains to be estahlished. Preliminary experiments, in which lkwtions l-15 from the adsorption chromataogram were incubated with tuber homogenate, indicate that they are met~abolized, but, not to diosgenin. The lower incorporation of mevalonic acid into the lipid fraction of Dioscorca tubers and the failure of this very etlicient, precursor of sterols t)o yield diosgenin deserves further investigation. Dissection of a Dioscorea tuber revealed significant differences in the diosgenin content and biosynthetic activity of morphologically different parts of the tuber. The upper port,ion near the shoot is tht

ANT)

l30XhXH

richest in the crude diosgenin fraction, while t,he incorporation of acetate by the lolver portion of the t’uber is less than in the case of the other portions. In preliminary experiments with tuber slices, we have also observed low rates of acetak incorporation, presumably because t’he tissues exposed by slicing belonged to the lower portion.

The authors are indebted to A. E. Purcell for an introdttction to some of the techniques tised and t,o ISrich Mosettig for advice and encouragement We also wish to thank H. I<. Miller for infrared analyses. and H. (;. htcCann for the determina tion of the C :~nd H and rotation values. REFEREKCES 1. HEFTMANS, I;., .4NI) ~IosETTIG, E., “Biochenistry of Steroids.” Reinhold Puhl. Corp., New York, 1960. 2. P.~Rs, It. B.. ASD I~ONXER, J., d. Biol. (‘hew. 233, 340 11958). 3. PURCELL. A. b:., THOMPSOX, G. A., JR., .4x1) ROXKER. J., J. Kiol. (‘hen!. 234, 1081 (1959). 1. SAXDER, H., ANI) (;RISEB,4VH, H., %. .\-utrcrjorwh. 13b, 755 (1958). 5. RAMSTAD. I<.. .4s1) BEAL, .J. I,., (‘Herr. & Ind. (1,ondon) 1960, 177. 6. WALL, XI. I<., Wrr~4~ar, J. J., PERLSTEIX, T., CORRELL, 1). S., ANI) C;ENTRY, H. S., J. .-la!. Pharw. :tssor. 46, 155 (195i). i.

AIORRIS, hl. P., I~OARK, U. A., hsD C.4x(3Eid, I<., J. -lqr. f’ood Chm. 6, 856 (1958). 8. REICHSTEIN, 'l'., .\ND SHOPPEE, C. W., Discrts-

sions Faraday

90~~. so. 7, 305 (1949).

9. Bh~~r.4, A. Ii., CHhKRAVARTI, R. I., .I. Indian V.IRTI.

i1954).

I)., AND CHAKR.4. (‘hem. fk)c. 31, 177