CA1288421C - Fertility drug and method of producing the same - Google Patents
Fertility drug and method of producing the sameInfo
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- CA1288421C CA1288421C CA000610993A CA610993A CA1288421C CA 1288421 C CA1288421 C CA 1288421C CA 000610993 A CA000610993 A CA 000610993A CA 610993 A CA610993 A CA 610993A CA 1288421 C CA1288421 C CA 1288421C
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- phytostanol
- acid
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Abstract
ABSTRACT OF THE DISCLOSURE
FERTILITY DRUG AND METHOD OF PRODUCING THE SAME
A synthetic method of producing ferulyl phyto-stanol derivative of formula I
(R : --CH3 or --C2H5) is disclosed. The method comprises acetylating ferulic acid with a mixture of pyridine and acetic anhydride, treating the product with thionyl chloride to form an acid chloride, reacting the acid chloride and phytostanol in the presence of pyridine to prepare phytostanol compound and deacetylating this compound with sodium boron hydride.
The derivative has ovulation-inducing effect and is useful in preparing fertility drugs.
FERTILITY DRUG AND METHOD OF PRODUCING THE SAME
A synthetic method of producing ferulyl phyto-stanol derivative of formula I
(R : --CH3 or --C2H5) is disclosed. The method comprises acetylating ferulic acid with a mixture of pyridine and acetic anhydride, treating the product with thionyl chloride to form an acid chloride, reacting the acid chloride and phytostanol in the presence of pyridine to prepare phytostanol compound and deacetylating this compound with sodium boron hydride.
The derivative has ovulation-inducing effect and is useful in preparing fertility drugs.
Description
27628-lD
SPECIFICATION
FERTILITY DRUG AND METHOD OF PRODUCING THE SAME
This is a divisional application of the application Serial No. 503,235 filed March 4, 1986. It should be noted that the term "invention" appearing in the specification applies to the subject matter of both the parent and the divisional applications.
Background of the Invention 1. Field oE the Invention The present invention relates to a fertility drug or an ovulation-inducing drug and a method of producing the same, and more particularly -to a fertility drug having an eEfective componen-t comprising a composition or compositions involved in Coix lacryma-jobi var. mayuem Stapf L. (herein-later described simply as "Job's tears"). The parent applica-tion relates to such a drug which is extracted from Job's tears. This applica-tion relates to components of the drug which are synthetically produced in organic reactions.
2. Prior Art Description Typically known to the world as fertility drugs are those comprising clomiphene and cyclohexil. These con-ventional fertility drugs have been used for more than 20 years and their pharmacological effects are recognized in clinical trials. However, it has been found in practice that they will often cause extraordinary sex periods, re-sulting in such troubles as multiple pregnancy and failure 8~Z~
27628-lD
of pregnancy. They often produce side effects. Under the circumstances, nevertheless, no other fertility drugs have yet become available for practical purposes.
Examination and study on novel fertility drugs have been attempted.
~ ~fi42~
lor exumple, it has been known that leaves of eorn, rye and wheat conluiil a mQterial whicll will indnce ovulalioll of domestic rabbits (Nii~ata Medical Society Bulletin; vol.78, puge 305; in 1964; Japull).
Up to the present, however, its ovulation-inducin~ effect on humall-kind hus noL yet been proved and therefore such u muterial is still not apl)licable to practical use.
Meanwllile, it llas become apparent Lhat some pharmaeoloKical effects ~ill inllere in extractunts from Job's tears alld co;x seed, u fruit of Job's tears prepared by rernoving hulls and peels froln Job's leurs sced. "Seiyuku~aku" by Ina~uki et ul; publisllcd by Nunkodo in 1975 in Japan; pa~e 162; writes as follows:
1) Tll(! o~xtrucLullt~. of .lob's t~ars ~nd eoix seed lluve d;urel.ic effeclsand tllerefore may be used for the rernedy of tumor, beriberi, nephrolith and cystolith and hurum~
2) l`hey may be used as puillkillers und crampkillers.
:1) 'I`hey ure 800d for wurts und rou~llncss.
Moreover, jL has now been eonfirmed Lllat proteins exLIueted flom unLllreshed ~owder of Job's teurs seed will spur securetioll Or humall milk (Masahiko Shi~ernitsu; Bulletin of Kumarnoto l,ocal Deprlrtment of Japan Womun Science Society; vol.~, pu~e 191; 19~). An anticullcer rnaterial can be isolùted from eoix seed (Chemieal and PllurmaeeuLical Bullctill, .Jul)ull; vol.9, pa~e ~,; 196t). Ilowever, Lhe ovuluLioll- inducin~ effecL
of .lob's tears and/or tlle extructants thereof has not yet been knowll in lhc prior alt.
Summary of the Invention It is therefore an object of the invention to provide a novel fertility dru~ of a typc diffcrent from the conventional one typicslly comprisin~ clomiphcne and cyclohexyl, capnble of inducing ovulation in physiologically nutural rnanncr, without causinK ubnormal scx pcriods.
Another object of the invention is to provide a novel metllod of sylllllelically producillK un ovulution-inducin~ maLeriul, idclllicul to that contained in Job's tears, with high efficiency, to thereby make it possible to manufacture on commercial basis a fertility drug having said ovulution-inducillg material as an effective component.
According to an uspect of the invention there is provided a fertility drug havin~ an effective component comprisin~ oil-soluble frùction of a whole or u part of Job's tears sced.
According to another aspect of the invention there is provided a t5 fertility dru8 baving an effective component comprising ferulyl stanol derivative and/or phytosterol fatty acid ester, contained in Job's tears.
'I'hc said fertilily dru8 i3 prcpurcd by incorpor~ cthyl uceLa~e into Job's tears bran to extract an oil-and-fat fraction from the Job's tears bran and collectin~ the effective component contailled in the oil-and-fat fractioll.
Accordin~ to still another aspect of the invention there is providcd a synthetic method of producin8 ferlllyl phytostanol dcrivative coml)risin~ the steps of acetylatin8 ferulic acid with a Inixture Or pyridinc alld acetic anllydride, trcatill~ the acctylaled feIulic ucid will t.hionyl chloride to prepare an ac;d clllorid(, reactillg in thc presellce of ~yridinc lhe ucid chil)lidc willl pllyl03lullol Lo form a pllylo~Lullol compoulld, dissolving the phytostanol compound into a mixture of mcthanol and chloroform, and adding sodium borohydridc to a rcsultin8 solution, with stirrin8, to thereby deacetylate the phytostanol compound.
Brief Description of Drawin~s Further objects and advantn~es of the present invention can be S fully understood from the following detailed description when resd in conjunction with the accompanying drawin8s, in which;
Figure 1 diagrammutically illustrates tlle steps of the test mcthod in Test 1 for extracting components froln a whole seed of Job's tears with extractin~ reaKents of n-hexane, ethullol, 50~ ethullol solution and water;
~ ;gure 2 is u 8raPh showin8 relationship between dosage of a purified extructant obtained from Job's teurs seed with n-hexane and the number of nuLurally produced ova alld the sl,ute of ovaria;
~ ures 3 and ~ are charts showing infra-red spectrum und nuclear 1~ maKIletic resonance spectrum of a fertility drug accordinK to one aspect ol' Llle inventioll; and Fi~ures 5 and 6 are charts showin~ infra-red spectrum and nuclear mabnetic resonunce spectrum of a fertility drug uccording to another uspect of the invention.
Detailed Description of The invention 'I'llc ~res-~ invclllioll uccordin~ lo onc u;~ccL lllcrcor is conccllcd with a fertility dru~ or an ovulatioll-inducillK drug havin~ un effective component comprising un oil-solubie fraction of a whole or u part of Job's tears seed. The oil-soluble fractio:l may be extracted from powder whicll is prepared by grindinK the whole of thc Job's tears seed.
~2 ~3~3 4 2 ~L
Alternativeiy, tbe Job's tears seed is threshed and purified in a known mullllcr to be clu~,iried inlo coix sced, bruu und hulls, floln any Or wllicll may bc extructed tlle oil-soluble fruction. In view of extractability, however, the use of bran itself or the whole seed, inclusive of the bran, is preferred.
In extraction of the oil-soluble fraction from Job's tears s~?ed, an extractin~ solvent may be n-hexane, ethyl acetate ester or any other suitable one. In the cùse of n-hexane being used, yield of the oil-soluble fraction would be decreased but it is easier to be evaporated after extraction, so that n-hexane is a preferred solvent in a practical sencc .
The fertility dru~ thus prepared has an effective eomponent of the oil-soluble fruclion extruclcd from .lob's tcurs sced or u purificd onc tllercor. A vehiclc, muLcllill~ agcnt, diluent and uny otllcr kind of udditi VCS muy bc incorl~oruled ulone or in combinulion, ul)on demund.
1'he fcrtility dru~ rnay be in the form of pellets, powder medicil)es, cul)sules, cyrup or iniection.s, and upplied orully or externally.
I:or better underslulldin~ of the invention some exemplifyin~ tcsts will l)c Nivcn llercunder.
Test l In tlli~ tesL, a water-soluble fraction und an oil-soluble fructio werc cxtructcd rc;l)(?ctively from tlle wholc s(?(?d of .lob s tCUl'S and tlle ovulution-induce effects of tlle respectivc fractions werc mcusllred and compul cd l(> cucll otll(!r .
Ille wllole sced of .lo~ s tears w~s Kroulld into powd(?r to plel)ure 5008 s&mple. This sample powder was subjected to extraction w;tll an _~_ 3 2 ~8 4 2 3L
extractill~ reagent of 1.5~ n-hexane at u lemperaturc ranKc of 15~-20~C.
After the extruction the extracting reagent was distilled under pressure reduccd condil.ions to obtain about 45~ of yellow, oily substallcc (cxtractabiliLy of about 9~ by weight - the percenta~e i3 giYen by weiglll throu~llout the Specification unlcss oLllerwise specified). This yellow, oily extructant wus deterlnined as colnponent A. Then tlle residue a~Lcl exlractioll of coMponent A was furtller sul)jected to extraction Witl 1.5Q ethanol hut only a ne~ligible ainount of an extractant was obtained.
Thc residuc was d;pped into a Icaching linuid of 50X ethanol solution.
and the leachillg liquid was concelltrated aL a temlerature below ~0C
undcr prcssurc redut:cd conditions, to thcrc~y obtaill a plccipitatt~ and about 2~ e~tractant. This cxtractant wus deterlnined as colnpon~llt B.
Thcl-, from tlle residuc ufter extractioll of colnponent ~ wa9 CXll~aclCd witll 1.5Q water, a slnall alnount of allotl~er cXtractant, WhiCIl was dclcrmined a9 COII)I)OllCllt C. In 5u~nmury, colnpollcnts A, B and C wcrc extracted in such Inunller as diagralnlnatically illustrated in PiKure 1.
I IIIU';~' COMI)Ol~ ulld C W~'I'C IIIIU I i l.U 1. i V(! I y UllU I i '~.(.'~i i 11 U kllow nallner to find that cornponent A contains ~Iyceride and such eslels as ~crulic acid cstcr, cotnpollcnL ~ conlaill~i polyulnido alld colnpollcllt C
contains arnino acid and peptidc.
Next, the ovulation-inducing effect or actiYity of the respective coMponellLs A ,B and C wcle lcstcd in Lhc following Inanller~ Morc l~articularly, 70 ~olden halnsters in thc a~e of 5 - ~ weeks W(!l'(' dcvidcd into 7 ~roups euch having 10 golden hamsters. Tlle foddcl werc I)rel)aled by addin~ to tllc basic fodder lZ of coml~ollellls A, h and C, and KiVèn orally to thc ~roup Nos. 1, 2 and 3, respectively. As thc avera~e intake of the fodder of a ~olden halnster is ~9~ a day, t90~y of Ihe ~1~2~342~
r(!3pcctivc componellts werc given to Llle respectiYc groul)s. Thcy wcre fed with such fodder for 3 weeks and then slaughtered. The 8roul~ Nos. 4, 5 and 6 were fcd in the same manner, but after 3 wecks' fecd of lhe foddcr containillg the coml)onents only the basic fodder werc ~ivcn Lhcrclo for 5 w(!elts, alld tllcn slaughtcrcd. Whereas, tbc groull No. 7 wus initi~lly given only the basic fodder for ~ weeks and tllen slaughtered.
As lo ttlt! ~('SI)(!Cl.iV(' KrOUpS, tllC S('X l)('riods WCTC olls(!rv(~l ulld the number of naturally produced ova was counted beforc they wele sllu~htcred, and after slau~htered the weight and thc state of tllc ovary were observed hy mealls of dissection. The results of thcse tests are shown in the following Table I .
38~2 T A B L E
. _ ._. . . ._ test sample comparison of nlllnber Or¦ dissection test _ _ ______ ~
~roul~ coml30nell1 scx period natural weight of state of No. udded sex pcrioc rcmurkc ovulutiol ovary(~) ovary . ,, .,. . ._ ___ ___ __ .,.,, __ ._, _ 1 A ~ days regular 17 1 2 ~.~ ~9.48 + 2.35 (~) 2 B 4 duys regular 12 + 2 1~.01 + 0.3~ (-I) C 4 days regul~r 12 + 2 17.93 + 0.23 (+) ~ A ~ d~ys regular 17 ~ 3 ¦ 19.59 + 0.2~ (++) B 4 days regular 12 -t 2 18.20 + 0.2~ (+) 6 C ~ days re~ular 12 l 2 t7.8~ + 0.56 (t) 7 4 days re~ular 12 + 2 18.03 t 0 15 (~) _ . . . , ,___ Notes: ~9 ~i~urc~ in the column of Scx Peliod show the averu8 e.
G~ tll(- collllnll of State of Ovury Ihe murk~ ) und (l~) meall rcluLive comprison in the number of corpora lutea found in cut-outs of the ovary.
~ The murk ~ identified in Lllc columll of Natural Ovulatioll mcalls lhuL Lherc could bc found a ;i~niricullce witll rc.pccl lo other 8roul~s ut u sigllificallce Icvel Or 1%.
As iL al~eurs floln Lhc re3ulLs ;howll in `lablc I Lllc compollellt A
extructcd from the whole seed of .loh s teurs with n-llcxal)c will promoLe natural ovulation witllout disturbing the ;cx periods. As a resull: of orKanic dissection Llle effective componellL contained in tllc corn!)onent A
wu-; l~roved to uct on tllc sex center so us to promotc formation of corpora lutea.
Meanwhile T~ble I AISO shows that this eftective compollellt is colltuilled only in the oil-solu5~1c fruclioll Or tlle .lob -. Lcar; sccd and is ~.carcely presellt in Lhc extractullts ohtaincd with etllullol 50 eLtlanol solulion und wùter.
~ ~ ~842~ 27628-lD
Test 2 ~ This test was carried out to find the optimal dosage of the ovulation-inducing effective component contained in the component A extracted in Test l.
First, the component A was dipped into leaching liquids of ethanol, 50~ ethanol solution and water, step by step, and then the leaching liquids were removed. Then the residue was purified by column chromatography with n-hexane. Thus, a purified extractant was obtained in a yield of 90% of the component A.
This extractant was dissolved in 0.2mQ soybean oil in quantities varied between 95~J380mg to prepare three fodder containing different quantities of the extractant, which were orally applied by injector to three groups each having lO golden hamsters, once a day, for 3 weeks. Thereafter, the test animals were slaughtered. These groups were put into the tests for the natural ovulation and the ovary weight in the same manner as in Test l, upon which matural relation of the dosage of the extractant per body weight of a golden hamster was determined with these parameters. The test results are shown in Figure 2.
As shown, the optimal dosage per body weight of a golden hamster was found to be in a range of 0.76~1.4mg/g a day.
Accordingly, provided the body weight of a grownup is 60kg, 4.6rJ8.4g/day of the purified n-hexane extractant was determined to b~ an optimal range when applied to an adult. The medication term will be dependent upon the condition of a patient and varied case by case.
Test 3 In this test, extractability of the effective component ;~ _g_ ~ 27628-lD
obtained respectively from the whole seed, coix seed, bran and hull of Job's tears was measured and compared to each other.
Respective 500g powder were prepared by grinding the coix seed, bran and hull of Job's tears seed, and extractants corresponding to component A were extracted in the same manner as in Test 1. Quantities of the extractants thus obtained and extractabilitv in the respective cases are shown below in Table II. Data for the whole seed in Table II are the reproduction of those in Table I. It will be obvious from Table II that the greatest extractability was gained in the case o~ bran, leading to the fact that the effective component is in substance contained in the bran of Job's tears seed. Thus, the effective component can be extracted with great efficiency from the bran alone or the whole seed including the bran.
TABI.E II
. _ , test sample amount of extractant.(.g). .extractability (%) _ whole seedA5.0 9.0 coix seed27.0 5.4 bran 80.0 16.0 hull 0.6 0.12 Note: The extractability is calculated by the equation of amount of extractant (g)/ sample powder (500g) X 100 The inventors have made thorough investigation of the effective ovulation-inducing material contained in Job's tears seed and found in the end that ferulyl stanol derivatives expressed by the following formula (I) and/or phytosterol fatty acid ~ster ~ 421 27628-lD
expressed by the following formula (II) has significant effect and activity on induction -lOa 3l~3~34~L
of nuturul ovulution. 13ascd on this fucl thc inventors have comc to a ~uccessful end that a novel fertility dru~ having Ull effcctivc coml)ollellt coml)rising ferulyl stnllol derivutivcs und/or l)llytostcrol fatly acid cster may ~e prel~ared.
~ormula (I) R ~ <
O
Il ,/ C ~ O ~\\~"' C = C 11 .. ..
~ ,J
Cl130 ~ (R: CI13 or '-C21k~) I:ormula (Il) ~ R, R,COO
(R~: remaining radicai of palmitic or stearic acid R2: - Cll~ or -C211s~
~ 3 8 4~3L
Stanol ferulic ~cid derivatives representcd by the formulù ( I) nay be, for exu~nple, truns-ferulyl stigmastallol, trans-ferlllyl cuml)estanol or a mixture thereof.
Phytosterol fatty acid ester represented by the forlnula ( ~) may bt~, for exu~nl)le, ~ -sito3terol palmitic acid cster, ~ -silosterol slearic ucid estcr, carnlleslelol pallnitic ucid ester, campeslcrol <;tearic uci(3 ~Islcl O~ y 011(! of lllc colnbillul.ioll~ cof.
A process for producing the above fertility drug is desclibcd in detaiI hereundt,?r.
First, ù Job's tears seed is threshed and purified il?~ a known mallncr to be scl~arated into coix seed, brall and hull. To I part of the 6run is incorl-olutcd ~ ~ 5 purts (l~y wei~lll; Ihc pall i; ~iV~'II by wei~lll. thlougholll lhe Specification unless olllerwise sl~ecified) of ethyl acetate, and thc? resullallt is agitated at 15 ~ 20C for 5 -~ 10 hours, lh~.?reby extracting ùn oil-and-fat fraction. The extract is Lhe filLered to relnove nn insolllble fraction.
To 1 purt of tlle residue is incorl)()l~Lcd 3 ~ 5 purts of elhan and lhc solulioll is u~ilatcd at 15 ~ 20(~ for 5 ~ 10 hours. lhe rcsulLulll cxtract is lllell filtcred Lo rclnovc un insolublc fracLioll.
1'he elhullol-sol~ lt? frucLioll thlls obluillcd is disLilled lo rclnove etllullol, therel3y obtuinillR all elllallol extracLulll. To 1 pUlt of Llle~
ethanol extractant is added 3 ~- 5 partx of clhyl acetate, and lhe solulioll is a~ilutcd uL 15 -` 20~C for 5 -` 10 hours, to obLuill un cLhyl acetate-soluble fraction.
The oil-and-fut fraction and the ethyi acetate-solu~le fraclioll are Inixcd to~ethcr and cthyl acetate is separatcd froM ihc InixLure. Tht?
resulLunt is supplied to a column char~ed with silicu ~el to effect ~ . .
, Kradient elution with a mixed eluent of n-bexulle and ethyl acetate whereby fructions eluted with the Inixed elucl1ts of n-llexalle and ethyl UC(?lUi(! lluvillg Mixillg lulio~ Or 3~: 1 ~ 10: 1 u~ /ol 100: 0.5 ~ 100: 1 are collected. Thc fractions thus collected are Inixed together and tht cluents are reinoved from the Inixture Lllcrcby obtainin8 ovulution-induceill~ Inaterials. The fractions eluted with lhe eluellt of ~0: 1 ^-10: 1 Inixture of n-llexulle und ethyl ucetatc will contnill ferulyl stunol derivatives and tl~e fractions eluted with the eluent huvin8 tlle tnixin~
ratio of 100: 0.5 ~ 100: ~ will COlltUill phytosterol fatty acid ester.
Ifrective Inaterials will be contailled in the nespeclive eluates obtailled with thc cluents havillg different ranges of mi~ing ratio and may be used alone or in a Inixcd state to Inanufacture llle fertility dru8 according to thc invelltion. The drug muy be a pellet a powder Incdicine n capsulc an inièction or in uny other forln and applied orally or externally.
Tllc dosaK(? of the dru6 will vury dcp-!lldillg ulll)ll tllc collditioll of u paiient an(l the way of upplicutioll but in Inost cases ~0 -~ 80~ once a ~ay Wi ~ (? cf ~CC Li VC Wll(!ll 01'~ 1 I y a~ 1.O ~11 adull.
The fertility drug of the invention having an effective componellt comprising ferulyl stallol derivativc ulld/or pllytosterol futty acid ester will be furtllcr dcscribcd l~y waY of exelnplifying tesis.
., ~ . . __ This test was carried out to observe physical and chemical characteristics of ferulyl stanol derivative. The test sample was X colnl)onent prepared in tlle saMe Inanner as in Example 3 des(:ribed later.
Illi; tcst <;ulnplc was allali~.ed by tllin luycr chrolnutoKIalllly Lo ~ 42~ 27628-lD
detect a single spot (hereinlater defined as material X) which manifested a blue-green color with sulfuric acid and was distinguishable in ultraviolet rays.
The material X was purified by alumina column chroma-tography and isolated as colorless, needle-like crystals having melting point of 156C. The material X showed positive adsorption in the Gibbs' adsorption test and had a M' peak at m/e 592 in the mass spectrometry. In the infrared spectrophotometry, as shown in Figure 3j it showed adsorption signals based on hydroxyl group at 3120r~3500cm 1, conjugated carboxyl group at 1710cm 1, double-bond of ~,~-unsaturated carbonyl group at 1640cm 1, C-C stretching ~ibration of benzene ring at 1600 and 1510cm 1, and out-of-plane deformation vibration of benzene ring at 840cm 1, respectively.
In the nuclear magnetic resonance spectrometry (CDCQ3), as shown by the chart of Figure 4, it manifested a signal pattern peculiar to phytosterol at 0.62~ 2.Oppm. Further, signals were found based on methoxy group at 3.88ppm, and the AB type spin-spincoupling (J - 16Hz) of hydrogen in the double-bond of ~,~-unsaturated carbonyl group of cinnamic acid derivative and the ABM type spin-spincoupling of hydrogen in the tri-substi-tuted benzene ring thereof was found at 6.22ppm and 7.55ppm, respectively. The singlet of lH at 5.95ppm was disappeared by adding D20. According to these results, the material X was determined to have hydroxyl group of a phenol.
The material X was subjected to alkalic hydrolysis to be separated into an acid and a neutral fractions, to both of which were applied chemical analysis to pro~e that only ferulic acid was isolated from the acid fraction (which was identified in - .
~ 8'~2~ 27628-lD
comparison with specimen). The neutral fraction was silylated in a known manner and then quantitatively analyzed by gas chromato-graphy, whereby it was identified as a mixture of stigmastanol and campestanol in a ratio of 9 : 1.
Further, the material X was acetylated with a`mixture of pyridine and acetic acid to form mono acetal in the form of colorless, platy crystals, having melting point of 155-V156C.
According to nuclear magnetic resonance spectrometry (CDCQ3), there could be seen signals based on methyl and methylene radicals of phytosterol at O.6-V2.Oppm, and a signal based on acetyl group at 2.32ppm. At 3.84ppm, a signal of singlet of 3H was found based on methyl of methoxy group. The AB type spin-spincoupling of olefin hydrogen in a,~-unsaturated carboxyl group was noted at 6.32ppm and 7.60ppm, and the ABM type spin-spincoupling of hydrogen on the tri-substituted benzene ring appeared at 7.07ppm.
Consequently, the material X was identified as a mixture of trans-ferulyl stigmastanol and trans-ferulyl campestanol in a ratio of 9 : 1. By the results of analyses including its melting point, the trans-ferulyl stigmastanol was found to be the same material as dihydro-~-sitosterol ferulic acid ester which is isolated from a corn embryo bud oil (Tamura et al; "Nippon Kagaku Zasshi (Japan Chemical Magazine)"; vol. 79, page 1011; in 1958).
Test 5 This test was carried out to reveal physical and chemical characteristics of phytosterol fatty acid ester. Y component, prepared in the same manner as in Example 3, described later, was used as a test sample.
~ 2 ~ 27628-lD
This sample was analyzed by thin-layer chromatography in a known manner to isolate a material of colorless, needle-like crystals, having a melting point of 64~v65C, which showed a spot manifesting a blue-green color with sulfuric acid and being distinguishable in ultraviolet rays. According to mass spectro-metry, a peak based on M~ was found at m/e 680. A peak based on carbonyl group was observed at 1740cm 1 in infrared spectrophoto-metry, as shown in Figure 5. In nuclear magnetic resonance spectrometry (CDCQ3), as shown by the chart in Figure 6, signals were noted based on methyl and methylene protons of phytosterol at 0.6~v2.0ppm, and -(CH2)- of long chain fatty acid residue at 1.28ppm. A signal of triplet based on methylene group adjacent carbonyl groups, a signal of multiplet (12 Hz in half width) based on hydrogen of C3 phase and a signal of multiplet based on binyl hydrogen were found at 2.25ppm, 4.6ppm and 5.35ppm, respectively. Then, the material was subjected to alkalic hydrolysis to be classified into an acid and a neutral fractions.
The neutral fraction was proved to contain sterol, which comprised ~-sitosterol and campesterol. The acid fraction was a mixture of higher fatty acid comprising in substance stearic acid and palmitic acid, which was determined by means of methylation and gas chromatography.
In conclusion, the said Y component was identified as a mixture of long chain fatty acid esters of phytosterol. A part of this material was identical to ~-sitosterol derivative mentioned by Kuksis et al in "Journal of Organic Chemistry", vol. 25, page 1209; in 1960.
~ 84~ 27628-lD
Test 6 In this test yield of stanol ferulic acid derivative and phytosterol fatty acid ester from the whole seed, coix seed, bran and hulls of Job's tears was compared to each other.
The whole seed of Job's tears was ground in a known manner, threshed and purified, and thus separated into hulls, bran and coix seed. Among 100 parts of the whole seed, 33 parts of the hulls, 15 parts of the bran and 52 parts of the coix seed were obtained. ~o these four star~ing materials were respectively added n-hexane and ethyl acetate each in a triple quantity thereof, and the solution was agitated at 15 ~20C for 5 hours, to thereby effect extraction. After the respective extracting solvents had been removed, oily components were obtained. The weight ratios of the respective oilycomponents with the starting materials are shown in the following Table III, which shows the fact that the effective components comprising ferulyl stanol derivative and phytosterol fatty acid ester are both included in the bran of Job's tears seed.
9.28842~L 27628-lD
_ _ .. _.___ .
U~
a)o ~ ~ a~
S:: h ~1 O ~ r ~1 oa) v ~ ~ ,~ ~
~d o ~ o O O ~ o ~1 0 4~
O _ _ _, , ~0 S~ ~
P~ ~ a u~
,~ O ~ o ~1 ~1 ~ o ~ O
a) ~ ~ rl ~ rl rl ~1 ~ O ~1 0 ~1 h :
a) c) 4~
H
H
H
~-~ ~
o ~ :
~ a) 0~
O
t) t) ~ ~1 ~ Ou~
O ~a) .. _ ~ rl o ~;
S~ ~ ~`1 ~ ~ ~ ~ ~1 0 ~
0~ ~ . . . .
C) ~ ~ O ~9Ln X
s~ a~
~1 ,~ I
O o ~
_ ~ _ __ ~ a) ~ ~ a) a) X
O ~1 ~ ~rl o ~; 3 ~ Q o . . . _. , ~ 421 27628-lD
Test 7 This test was carried out to determine fractions contain-ing the effective components comprising ferulyl stanol derivative and phytosterol fatty acid ester, by means of silical gel chroma-tography.
1) Preparation of Test Samples According to the method described later in Example 3, 1!250g of an oily fraction was extracted from 5kg of Job's tears seed bran with ethyl acetate and ethanol.
300g of oily fraction was subjected to column chromato-graphy using 5kg of silica gel, wherein the eluent was at first n-hexane and then mixtures of n-hexane and ethyl acetate in mixing ratios being varied to gradually increase the proportion of ethyl acetate. The eluates were 4.352g of F-I component eluted with a mixed eluent of n-hexane and ethyl acetate in a mixing ratio of 100: 1, 283g of F-II component with an eluent of a 20: 1 mixture and 8.756g of F-III component with ethyl acetate alone. F-I
component was purified by column chromatography using 250g silica gel and a mixed eluent of n-hexane and ethyl acetate in a ratio of 100: 1. F-II component was further subjected to alumina column chromatography and eluted with a 20: 1 mixture of n-hexane and ethyl acetate to obtain 128mg of F-II-l component, 1.284g of F-II-2 component, 210mg of F-II-3 component, 210mg of F-II-4 component and 55mg of F-II-5 component. Thus, seven specimens were prepared.
2) Test Method for Physiological Activities These seven specimens were dissolved into 0.2mQ of a soy bean oil to prepare various oil solutions, which were orally ~" ' .
34~:~
27628-lD
given once a day to respective groups each having 10 golden hamsters in the age of 5~ 8 weeks. The amounts of the components respectively contained in such oil solutions were made different to be 0.2mg and 0.5mg. During the administration for 3 weeks with such oil solutions, the sex periods and the number of naturally produced ova were observed, the results of which were compared with those of reference group which had been treated with 0.2mg of soy bean oil containing no sample component.
3) Results The results with respect to the natural ovulation are shown in Table IV.
TABLE IV
component amount of component addëd added 0.2mg 0.5mg . .__ ~ . . . _ _.
(reference) 12 12 F-II-l 12 10 The sex periods were 4 days for all groups including the reference and there could be found no disturbing effect on the sex period. It will be quite obvious that F-I and F-II-4 components have significant ovulation induce effect. These components were identified in a known manner to determine that F-I component was phytosterol fatty acid ester and F-II-4 component was ferulyl stanol derivative.
~ 84~ 27628-lD
Test 3 This test was performed to determine effective dosage of stanol ferulic acid derivative, an effective ovulation-inducing component of the fertility drug according to the invention.
l) Preparation of Test Sample X component was prepared in the same manner as in E~ample 3, described later, and used as a test sample. X
component was a 9 : l mixture of trans-ferulylstigmastanol and trans-ferulylcampestanol.
2) Test Method The test was carried out in the same manner as in Test 7, except the amounts of the component added in the soy bean oil were O.lmg, 0.2mg and l Omg once a day, respectively.
3) Results 3-1) Sex Period TABLE V
. . . .
amount of component added sex period (day) ~reference) 4 O~lmg 4 0.2mg 4 l.Omg 4 As shown from Table V, each group showed regular sex period.
3-2) Number of Natural Ovulation 3~2~84z~ 27628-lD
TABLE VI
amount of component number of natural added ovulation . .. ___ . ._ _ (reference) 11 + 1 O~lmg 16 + 1 0.2mg 18 + 2 l.Omg 13 + 2 Note: The mark ~ shows a significance at a significance level of 1%.
As shown from Table VI, the groups to which O.lmg and 0.2mg of the effective components were given show significance with P~O.Ol with respect to the reference group.
As the average bodv weight of a hamster used in this test was 150g and the body weight of an adult is supposed to be 60kg, the effective dosage to an adult is proved to be 40~'80mg once a day.
Test 9 This test was performed to determine effective dosage of phytosterol fatty acid ester, another effective ovulation-inducing component of the fertility drug according to the invention.
1) Preparation of Test Sample Y component was prepared in the same manner as in Example 3, described later, and used as a test sample.
2) Test ~ethod The test was carried out in the same manner as in , : `
~ Z ~84~ 27628-lD
Test 7, except the amounts of the component added in the soy bean oil were 0.lmg, 0.2mg and l.Omg once a day, respectively.
3) Results 3-1) Sex Period TABLE VI I
... . __ __ _ . _ .,_ _ amount of component added sex period ~day) (reference) 4 0.lmg 4 0.2mg 4 1.0mg _ _ It will be quite obvious from Table VII that each group showed regular sex period.
3-2)Number of Natural Ovulation TABLE VI I I
, amount of component number of natural added ovulation (reference) 12 -~ 2 0.1mg 16 + 1 0.2mg 18 ~ 2 1.0mg 15 + 1 Note: The mark ~ shows a significance at a - significance level of 1%.
As shown from Table VIII, the groups to which 0.lmg and 0.2mg of the effective components were given show significance with P<0.01 with respect to the reference group.
~ ' 27628-lD
842~L
As the average body weight of a hamster used in this test was 150g and the body weight of an adult is supposed to be 60kg, the effective dosage to an adult is proved to be 40 ~ 80mg once a day.
Ferulyl phytostanol derivative, one of the effective components having ovulation-inducing effect, may be prepared not only by extraction from Job's tears seed as described before, but also by synthetic method. Such a synthetic method of production of ferulyl phytostanol derivative is characterized by successive steps of acetylating ferulic acid with a mixture of pyridine and acetic anhydride, treating the acetylated ferulic acid with thionyl chloride to form an acid chloride, reacting in the presence of pyridine the acid chloride and phytostanol to prepare phytostanol compound, dissolving the phytostanol compound into a mixture of methanol and chloroform, and adding sodium boron hydride to a resulting solution, with stirring, to thereby deacetylate the phytostanol compound.
The respective steps will be described in detail as follows.
A) Acetylation Ferulic acid (4-hydroxy-3-methoxycinnamic acid) is dissolved into a liquid mixture of pyridine and acetic anhydride (mixing ratio of 3 : 1) in a concentration of 10 ~15 wt.%. After reflux for 4~5 hours the solvent is distilled to prepare acetylated ferulic acid.
~' ` .
27628-lD
CH=CHCOOH CH=CHCOOH
pyridine acetic anhydride OH OH
B) Formation of Acid Chloride The acetylated ferulic acid is dissolved into chloroform anhydride in a concentration of 10 ~15 wt.%. Thionyl chloride (SOCQ2) is added to the solution in a proportion of 1.5 moles per lg acetylated ferulic acid -to effect the reaction thereof at 20 ~30C for 5~-6 hours. Then, the solvent is distilled in pressure reduced conditions to prepare acid chloride.
CH=CHCOOH CH=CHCOCQ
thlonyl chloride \ OCH3 ~ OCH3 C) Reaction with Phytostanol The acid chloride thus prepared is dissolved into pyridine in a concentration of 10~-15 wt.% to prepare a first solution, and an equivalent of phytostanol is dissolved into pyridine to prepare a second solution. The first solution is dropped into the second solution in an ice-cooled condition to effect the reaction at 20--30C for 2 ~ 3 hours, and the solvent is distilled and removed.
27628-lD
34~1 CH=CHCOCQ ~ ~
~1 Hov~--pyridine r \ C =C /
¦ H
~ (R : -CH3 or -C2~5) D) Deacetylation The reactant thus obtained in the step C) is dissolved into a liquld mixture of chloroform and methyl alcohol (mixing ratio of 1 : 1~ in a concentration of 3 ~ 5 wt.%. While this solution is being agitated in an ice-cooled condition, sodium borohydride in a quantity of double equivalent is added thereto by degree. After the foaming has ceased the solution is allowed .
. ~
~ 84~ 27628-lD
to stand at 20 ~25C for 1~ 1.5 hours, a small quantity of water is added and then the solvent is evaporated in pressure reduced conditions. Thus, ferulyl phytostanol derivative is prepared.
H C--o t7 C - C H
H
~ NaBH4 CH30 1' CH30H + CHCQ3 Il-o ~ ~
\ / H
C--C
\ H
~ tR : -CEI3 or -C2H5) OH
~r ~ .
.. .
~ 42~ 27628-lD
Ferulyl phytostanol derivative thus prepared by the successive steps A) to D) may be further purified by silica gel column chromatography and then recr~stalized with methyl alcohol, thereby producing the same in a pure state. Alternatively, the purification may be carried out each time the intermediate product is formed in each step.
The followings are exemplifying tests for the method of producing ferulyl phytostanol derivative according to the invention.
Test 10 This test was carried out to examine physical and chemical characteristics of the intermediate product formed by the acetylation step A).
1) Preparation of Test Sample Test sample was prepared in the same manner as the acetylation step in Example 6, described later. The acetylated product was sub~ected to silica gel column chromatography whereby a fraction eluted with acetone was obtained, from which acetone was evaporated to prepare a purified test sample.
23 Test Method The test sample was put into tests for melting point, mass spectrometry, infrared spectrophotometry (KBr method) and nuclear magnetic resonance spectrometry (CD30D * CDCQ3) in known manner.
3) Results The test results were as follows.
mp . 195-~196 C , FD-MS m/e; 236 (M ), IR~ (KBr) Cm 3500 (COOH), 1760,1690 (~C=O), 1630 (-COOH=CH-), NMR (CD30D +
:.
~ 2~8421 27628-lD
CDCQ3), ppm: 2.32 (3H, S, COCH3), 3.82 (3H, S, OCH3), 6.32 (lH, d, J=16Hz, ~COCH=CH-), 7.07 (3H, m, aromatic ring), 7.60 (lH, d, J=16Hz, -COCH=CH-).
Conse~uently, the test sample was identified as 4-acetylferulic acid (4-acetoxy-3-methoxycinnamic acid).
Test 11 This test was carried out to observe physical and chemical characteristics of the intermediate product formed by the reaction of acetylated ferulic acid chloride and stigmastanol.
1) Preparation of Test Sample Test samp]e was prepared in the same manner as the step of formation of acid chloride in Example 6, described later. The acid chloride compound thus prepared was sub~ected to silica gel column chromato~raphy with a mixed eluent of n-hexane and ethyl acetate ester (mixing ratio of 20: 1). The eluate was recrystalized with methyl alcohol to prepare a purified test sample.
2) Test Method The tests applied to the sample were the same as in Test 10. The elementary analysis was carried out in a known manner.
3) Results A melting point was 156C. The results of the elementary analysis were C: 77.80 and ~I: 9.89~ which are in conformity to the calculated values of C: 77.60 and H: 9.78 according to the molecular formula of C41H62O5 of 4-acetylferulylstigmastanol.
The results of mass spectrometry, infrared spectro-photometry and nuclear magnetic resonance spectrometry were as ~r ~ 27628-lD
follows.
FD-MS m/e ; 634 IM ), IRv (KBr) : 1770,1710 (>C=O), 1640 (-COOH=CH-), 1600,1510 (benzene ring?, 1180,1080 (-C-O-C-), NMR
(CDCQ3), ppm : 0.6rJ2.0 (-CH3~ -CH2-), 2.32 (3H, S, COCH3), 3.84 (3H, S, OCH3), 4.70 (lH, m, -O-CH<), 6.32 (lH, d, J=16Hz, -COCH=CH-), 7.07 (3H, m, aromatic ring), 7.60 (lH, d, J=16Hz, -COCH=CH-).
In accordance with these results the test sample was identified as ~-acetylferulylstigmastanol.
Test 12 _ This test was performed to test physical and chemical characteristics of the final product prepared by the method of the invention.
1) Preparation of Test Sample Test sample was prepared and purified by the same method as in Example 6.
2) Test Method The same tests were applied as in Test 11.
3) Results A melting point was 152C. The results of the elementary analysis were C: 79.17 and H: 9.86, which are in good agreement with those of C: 79.05 and H: 10.14 calculated according to the molecular formula of C39H60O4.
The results of mass spectrometry, infrared spectrophoto-metry and nuclear magnetic resonance spectrometry were as follows.
FD-MS m/e; 592 (M ), IRv (KBr) ; Cm : 3200 - 3500 (-OH), 1710 (-CO), 1640 (-COOH=CH-), 1600,1520 (benzene ring3, ~ ' 42~
27628-lD
1180,1080 (-C-O-C-)I NMR (CDCQ3), ppm : 0.6r~2.0 (-CH3, CH2 of sterol<), 3.88 (3H, S, -OCH3), 4.72 (lH, m, -O-CH<), 5.96 (lH, S, -OH), 6.22 (lH, d, J-16Hz, -COCH=CH-), 6.98 (3H, m, aromatic ring), 7.55 (lH, d, J=16Hz, -COCH=CH-).
In conclusion, the final product was identified as trans-ferulyl stigmastanol.
For better understanding of the invention some preferred examples thereof will be given here~nder.
Example 1 5kg powder of Job's tears prepared by grinding the whole seed thereof was subjected to extraction with 15Q n-hexane at a temperature of 20C and the solvent was evaporated at a tempera-ture below 40C under reduced pressure, thereby obtaining about 450g of a yellow, oily material at extractability of about 9%.
This extractant was then purified by silica gel chromatography with n-hexane. The purified extractant was added to basic feed to prepare fodder which was given to a group having 10 golden hamsters in the age of 5~ 8 weeks. The quantities of the fodder given to a golden hamster and the purified n hexane extractant contained therein were determined l9g and 171mg a day, respect-ively. While the test animals were fed for 3 weeks the sex periods were observed, and thereafter they were slaughtered to measure the number of naturally produced ova and observe the state of the ovaria. For reference, an equivalent quantity of the basic fodder not containing the purified n-hexane extractant, was given to another group also having ten golden hamsters, to which the same tests were applied.
~ 3842~ 27628-lD
The test results are shown in Table IX, from which it is confirmed that the oil-soluble fraction of the whole seed of Job's tears has a favourable effect on natural ovulation and formation of corpora lutea, without disturbing sex periods.
3421 27628-lD
_ _ ~ ~1 +~+1 ~ .
__ , .
O
~ 4~ ~ ~ ~ O
u o ~ +l +l ~ ri 0 ~ OD ~
3:
_ ~ r~ ~ r~
R ~ ~ ~ ~ s~
~ O ~ ~1 ~
H ~. . .
u~ h ~
~H ~ ~ ~ O
E~ O ~a s~ _ .
u~ a~ ~) x h ~0 u~
Id X ~ ~ td P~ a) a) ~ ~ 4 O u, ~ u~ O a) ~ X ~r ~ O
~q E~
- ~ ~
a~ u~
a ~ p~ ~
~ o ~, ~
so~ ~ c~ ~ ~
.
u~ u~ c) rl ~ ~
~ ~ ~ o ~ ~ z ~ ~ --~
~ .
27628-lD
4Z~
Example 2 In Example l was used the whole seed of Job's tears but in this example 500g bran of Job's tears was used, and by extraction with l.S~ n-hexane at 20C was obtained about 80 g of a yellow oily material at extractability of about 16%. This material was purified by silica gel chromatography with n-hexane and the solvent was evaporated at 40C under reduced pressure, thus preparing about 72g of a purified extractant. This purified extractant was given to a group of ten golden hamsters in a proportion of 1.5mg a day per lg of the body weight of a golden hamster, and the same tests were put into practice as in Example 1, results of which are shown in Table X. As shown, the oil-soluble fraction of the bran of Job's tears seed will promote natural ovulation without disturbing the sex period and bring a significant effect on formation of corpora lutea.
~ .
, , .
~ 2 ~38421 2 7 6 2 8 -lD
~= .~ .... _ ~o _ O ~ +l o ... _ ~ ' ~ 4~ ~ ~ ~
~, o ~ .~ ~ ~
u~ ~ _ ,~ o Q
,~ ~ + I + I
3 ~ 0!:~ ~ ~:1 O ~ 1~ 4-( -~` U~
O ~ O
~ S-l ~ N ~ ~
~1 ~ ~ ~ oo ~`
~ O ~ ~1 m . . _ ,o ~ ~ 1 ~ H
o ~ ~ ~ a) ~ a~ a~ a) d ~: ,0 _~ _~ _ O ~
'S~ ~ ~ _ O '~
~ ~ U~ U~ rl tn )~ X ~1 ~ ~ ~ tl~
a~ ~ ~ (d ~O~ U~
a) ~r ~r U~
__ _ S~
. ~ O ~ ~
O ~. ~' E~ ~n 5~
a) D~ ~ O
,1 S~ Z
_ , .. . .~.. . ~, . .
, ~ 4~ 27628-lD
Example 3 50kg of Job's tears seed was threshed and purified in known manner to prepare about 6.5kg bran, among which 5kg bran was subjected to extraction with 5kg ethyl acetate at 20C for 5 hours while being agitated. This extraction procedure was repeated three times to obtain a first extractant. To 4kg of the residue was added lOkg ethanol, and the reaction solution was agitated at 20C for 5 hours. This second extraction procedure was repeated twice and then the collected extracts were filtered to remove insoluble fractions. From the ethanol-soluble fraction thus obtained was evaporated ethanol in a known manner. To 120g of the resulting component was added 360g ethyl acetate and the solution was agitated at 20C for 5 hours, to extract an ethyl acetate soluble fraction, a second extractant.
The first and second extractants were mixed together.
Ethyl acetate was evaporated and removed from the extractant mixture to obtain 1,310g of an oil-and-fat fraction.
A first portion of 500g of the oil-and-fat fraction was subjected to column chromatography charged with 8kg silica gel, whereby 450g eluate was obtained witha mixed eluent of n-hexane and ethyl acetate (mixing ratio of 20: 1). This eluate was further introduced to alumina column chromatography (800g) whereby elution was effected with a mixed eluent of n-hexane and ethyl acetate (mixing ratio of 20: 1~ and about 540mg of a fraction showing a spot in ultraviolet rays was obtained by a fraction collector. This component was subjected to reverse phase high performance liquid chromatography utilizing Rp-18 ~ Z~384~1 27628-lD
with a mixed eluent of ethyl acetate and methanol (mixing ratio of 5 : 3), to obtain about 450mg of a component having a U V
absorption. This component was further subjected to column chromatography using 50g of silica gel with a mixed eluent of n-hexane and ethyl acetate (mixing ratio of 20: 1), to elute about 330mg of a component (X component) having a U V absorption.
X component was put into the same tests as in Test 7 so that it was proved to have ovulation-inducing effect.
On the other hand, another portion of 500g of the oil-and-fat fraction was subjected to column chromatography using 8kg silica gel, with a mixed eluent of n-hexane and ethyl acetate (mixing ratio of 100:0.5), then an eluate (Y component) was obtained in a quantity of about 6.87g. Y component was also submitted to the same tests as in Test 7 and its ovulation-inducing efect was observed.
Example 4 To 3kg o Job's tears bran was added 3kg ethyl acetate and the solution was agitated at 17C for 8 hours for effecting extraction. This extraction procedure was repeated four times and extractants obtained in respective extraction were combined.
Thus, an ethyl acetate extractant was prepared. To 2.2kg of the residue was added 8kg ethanol and the mixture was agitated at 17C for 8 hours. The extract thus obtained was filtered to remove insoluble components. By evaporating ethanol from the ethanol-soluble fraction in a known manner, an extractant with ethanol was obtained in a quantity of 65g. To this extractant was added 260g ethyl acetate, and the solution was agitated at 17C
for 8 hours, to extract an ethyl acetate soluble extractant.
27628-lD
~1~8~
These two ethyl acetate extractants were combined, from which ethyl acetate was evaporated to obtain 790g of an oil-and-fat fraction.
A first portion of 300g of the oil-and-fat fraction was subjected to column chromatography using 5kg silica gel, with a mixed eluent of n-hexane and ethyl acetate (a mixing ratio of 30: 1), to obtain an eluate in a quantity of 269g. This eluate was subjected to alumina column chromatography(500g), thereby obtaining 330mg of a fraction having a spot in ultraviolet rays This fraction was subjected to reverse phase high performance li~uid chromatography utilizing Rp-18, thereby eluting with a mixed eluent of ethyl acetate and methanol (a mixing ratio of 5 : 3) to obtain about 248mg of a component having U V
absorption. This component was further subjected to column chromatography using 30g silica gel with a mixed eluent of n-hexane and ethyl acetate (a mixing ratio of 30: 1) to obtain about 190mg of a fraction having a U V absorption. This fraction was submitted to tests in the same manner as in Test 7 so that it was proved to have ovulation-inducing effect. On the other hand, the other portion of 300g of the oil-and-fat fraction was subjected to column chromatography using 5kg silica gel with a mixed eluent of n-hexane and ethyl acetate (a mixing ratio of 100:1) to obtain 4.352g of an eluate. This eluate was tested in the same manner as in Test 7 to find its significant ovulation-inducing effect.
Example 5 The procedure in Example 1 was repeated several times to obtain 40g of X and Y components, respectively, to which 150g of - 37a -~;
, ~ ~84~ 27628-lD
middle-chain fatty acid triglycerol on the market was added to make 1,000 soft capsule medicines therefrom, respectively. A
soft capsule medicine made from X component contained 40mg of the effective component (that is, a 9 : 1 mixture of trans-ferulylstigmastanol and trans-ferulylcampestanol) and a soft capsule medicine made from Y component contained 80mg of the effective component (that is, phytosterol long-chain fatty acid esters in a mixed state).
Example 6 According to the following steps trans-ferulyl-stigmastanol was synthetically manufactured.
1) Acetylation l.Og of ferulic acid (Tokyo Kasei Kougyo K.K., corres-ponding to 5.15m moles) was dissolved into 8m of a liquid mixture of pyridine and acetic anhydride in a mixing ratio of 3 : 1, and after reflux for 4 hours the solvent was evaporated.
2) Formation of Acid Chloride l.Og of the acetylated ferulic acid (4.24m mol) was dissolved into lOmQ chloroform anhydride and 0.46 mQ (6.36m moles) of thionyl chloride (Wako Jun'yaku K.K.) was added to the solution, to effect the reaction thereof at 25C for 5 hours, and then the solvent was evaporated under reduced pressure.
3) Reaction with Phytostanol The acid chloride compound thus obtained was dissolved into lOmQ pyridine anhydride and the resultant was dropped in an ice-cooled condition into lOmQ of another pyridine solution prepared by dissolving thereto 1.77g (4.24m moles) of stigmastanol (Aldrich Chemical Co.). After the solution had been allowed to -37b-~ 84X~ 27628-lD
stand at room temperature for 2 hours, the solvent was evaporated.
The remaining residue was subjected to a silica gel column, and the column was eluted with a mixed eluent of n-hexane and ethyl acetate (a mixing ratio of 20: 1). Thus, a reactant was obtained in a quantity of 1.65g.
4) Deacetylation and Purification 1.6g of the reactant was dissolved into 48mQ of a liquid mixture of chloroform and methyl alcohol ~a mixing ratio of 1 :
1), and 480mg (12.7m moles) of sodium borohydride (Wako Junlyaku K.K.) was added thereto little by little. After the foaming had ceased the reaction mixture was allowed to stand for 1 hour, and then lmQ water was added thereto. The solvent was then removed under reduced pressure.
The reaction product was dissolved into 5.OmQ of chloro-form and the solution was led to a column charged with silica gel, which was eluted with a mixture of n-hexane and ethyl acetate (a mixing ratio of 20: 1). The eluate was recrystalized with methyl alcohol to obtain 1.23g (2.08m moles) of colorless, needle-like crystals, that is trans-ferulylstigmastanol, in 83% yield.
Example 7 1) Acetylation Acetylation was carried out in the same manner as in Example 6.
2) Formation of Acid Chloride Acid chloride was formed in the same manner as in Example 6.
3) Reaction with Phytostanol The acid chloride was dissolved into lOmQ pyridine -37c-X
~ 2~842~ 27628-lD
anhydride and the resulting solution was dropped into lOmQ of a separately prepared pyridine solution containing 1.72g (4.2m moles) campestanol, in an ice-cooled condition. The solution was concentrated and then led to a silica gel column which was eluted with a mixture of n-hexane and ethyl acetate (a mixing ratio of 20: 1), thus obtaining 1.45g of a reactant.
4) Deacetylation and Purification 1.4g (2.25m moles) of the reactant was dissolved into 42mQ of a liquid mixture of chloroform and methyl alcohol (a ratio of 1 : 1), and to the solution was added little by little sodium borohydride (Wako Jan'yaku K.K.) in a total quantity of 478mg (12.6m moles). The reaction mixture was allowed to stand at room temperature for 1.5 hours after the foaming had ceased. The excessive sodium borohydride was inactivated by adding water and then the solvent was evaporated under reduced pressure.
The resulting residue was dissolved into 4.5mQ chloroform and the solution was subjected to a silica gel column which was eluted with a mixture of n-hexane and ethyl acetate (a mixing ratio of 20: 1) and recrystalized with methyl alcohol. Thus, trans-ferulylcampestanol in a quantity of 1.15g (1.73m moles) was obtained in 77~ yield.
Although this invention has been described in conjunction with specific exemplifying tests and preferred embodiments thereof, it is to be understood that many variations may be made without departing from the spirits and scopes thereof as defined in the appended claims.
-37d-~, , .
SPECIFICATION
FERTILITY DRUG AND METHOD OF PRODUCING THE SAME
This is a divisional application of the application Serial No. 503,235 filed March 4, 1986. It should be noted that the term "invention" appearing in the specification applies to the subject matter of both the parent and the divisional applications.
Background of the Invention 1. Field oE the Invention The present invention relates to a fertility drug or an ovulation-inducing drug and a method of producing the same, and more particularly -to a fertility drug having an eEfective componen-t comprising a composition or compositions involved in Coix lacryma-jobi var. mayuem Stapf L. (herein-later described simply as "Job's tears"). The parent applica-tion relates to such a drug which is extracted from Job's tears. This applica-tion relates to components of the drug which are synthetically produced in organic reactions.
2. Prior Art Description Typically known to the world as fertility drugs are those comprising clomiphene and cyclohexil. These con-ventional fertility drugs have been used for more than 20 years and their pharmacological effects are recognized in clinical trials. However, it has been found in practice that they will often cause extraordinary sex periods, re-sulting in such troubles as multiple pregnancy and failure 8~Z~
27628-lD
of pregnancy. They often produce side effects. Under the circumstances, nevertheless, no other fertility drugs have yet become available for practical purposes.
Examination and study on novel fertility drugs have been attempted.
~ ~fi42~
lor exumple, it has been known that leaves of eorn, rye and wheat conluiil a mQterial whicll will indnce ovulalioll of domestic rabbits (Nii~ata Medical Society Bulletin; vol.78, puge 305; in 1964; Japull).
Up to the present, however, its ovulation-inducin~ effect on humall-kind hus noL yet been proved and therefore such u muterial is still not apl)licable to practical use.
Meanwllile, it llas become apparent Lhat some pharmaeoloKical effects ~ill inllere in extractunts from Job's tears alld co;x seed, u fruit of Job's tears prepared by rernoving hulls and peels froln Job's leurs sced. "Seiyuku~aku" by Ina~uki et ul; publisllcd by Nunkodo in 1975 in Japan; pa~e 162; writes as follows:
1) Tll(! o~xtrucLullt~. of .lob's t~ars ~nd eoix seed lluve d;urel.ic effeclsand tllerefore may be used for the rernedy of tumor, beriberi, nephrolith and cystolith and hurum~
2) l`hey may be used as puillkillers und crampkillers.
:1) 'I`hey ure 800d for wurts und rou~llncss.
Moreover, jL has now been eonfirmed Lllat proteins exLIueted flom unLllreshed ~owder of Job's teurs seed will spur securetioll Or humall milk (Masahiko Shi~ernitsu; Bulletin of Kumarnoto l,ocal Deprlrtment of Japan Womun Science Society; vol.~, pu~e 191; 19~). An anticullcer rnaterial can be isolùted from eoix seed (Chemieal and PllurmaeeuLical Bullctill, .Jul)ull; vol.9, pa~e ~,; 196t). Ilowever, Lhe ovuluLioll- inducin~ effecL
of .lob's tears and/or tlle extructants thereof has not yet been knowll in lhc prior alt.
Summary of the Invention It is therefore an object of the invention to provide a novel fertility dru~ of a typc diffcrent from the conventional one typicslly comprisin~ clomiphcne and cyclohexyl, capnble of inducing ovulation in physiologically nutural rnanncr, without causinK ubnormal scx pcriods.
Another object of the invention is to provide a novel metllod of sylllllelically producillK un ovulution-inducin~ maLeriul, idclllicul to that contained in Job's tears, with high efficiency, to thereby make it possible to manufacture on commercial basis a fertility drug having said ovulution-inducillg material as an effective component.
According to an uspect of the invention there is provided a fertility drug havin~ an effective component comprisin~ oil-soluble frùction of a whole or u part of Job's tears sced.
According to another aspect of the invention there is provided a t5 fertility dru8 baving an effective component comprising ferulyl stanol derivative and/or phytosterol fatty acid ester, contained in Job's tears.
'I'hc said fertilily dru8 i3 prcpurcd by incorpor~ cthyl uceLa~e into Job's tears bran to extract an oil-and-fat fraction from the Job's tears bran and collectin~ the effective component contailled in the oil-and-fat fractioll.
Accordin~ to still another aspect of the invention there is providcd a synthetic method of producin8 ferlllyl phytostanol dcrivative coml)risin~ the steps of acetylatin8 ferulic acid with a Inixture Or pyridinc alld acetic anllydride, trcatill~ the acctylaled feIulic ucid will t.hionyl chloride to prepare an ac;d clllorid(, reactillg in thc presellce of ~yridinc lhe ucid chil)lidc willl pllyl03lullol Lo form a pllylo~Lullol compoulld, dissolving the phytostanol compound into a mixture of mcthanol and chloroform, and adding sodium borohydridc to a rcsultin8 solution, with stirrin8, to thereby deacetylate the phytostanol compound.
Brief Description of Drawin~s Further objects and advantn~es of the present invention can be S fully understood from the following detailed description when resd in conjunction with the accompanying drawin8s, in which;
Figure 1 diagrammutically illustrates tlle steps of the test mcthod in Test 1 for extracting components froln a whole seed of Job's tears with extractin~ reaKents of n-hexane, ethullol, 50~ ethullol solution and water;
~ ;gure 2 is u 8raPh showin8 relationship between dosage of a purified extructant obtained from Job's teurs seed with n-hexane and the number of nuLurally produced ova alld the sl,ute of ovaria;
~ ures 3 and ~ are charts showing infra-red spectrum und nuclear 1~ maKIletic resonance spectrum of a fertility drug accordinK to one aspect ol' Llle inventioll; and Fi~ures 5 and 6 are charts showin~ infra-red spectrum and nuclear mabnetic resonunce spectrum of a fertility drug uccording to another uspect of the invention.
Detailed Description of The invention 'I'llc ~res-~ invclllioll uccordin~ lo onc u;~ccL lllcrcor is conccllcd with a fertility dru~ or an ovulatioll-inducillK drug havin~ un effective component comprising un oil-solubie fraction of a whole or u part of Job's tears seed. The oil-soluble fractio:l may be extracted from powder whicll is prepared by grindinK the whole of thc Job's tears seed.
~2 ~3~3 4 2 ~L
Alternativeiy, tbe Job's tears seed is threshed and purified in a known mullllcr to be clu~,iried inlo coix sced, bruu und hulls, floln any Or wllicll may bc extructed tlle oil-soluble fruction. In view of extractability, however, the use of bran itself or the whole seed, inclusive of the bran, is preferred.
In extraction of the oil-soluble fraction from Job's tears s~?ed, an extractin~ solvent may be n-hexane, ethyl acetate ester or any other suitable one. In the cùse of n-hexane being used, yield of the oil-soluble fraction would be decreased but it is easier to be evaporated after extraction, so that n-hexane is a preferred solvent in a practical sencc .
The fertility dru~ thus prepared has an effective eomponent of the oil-soluble fruclion extruclcd from .lob's tcurs sced or u purificd onc tllercor. A vehiclc, muLcllill~ agcnt, diluent and uny otllcr kind of udditi VCS muy bc incorl~oruled ulone or in combinulion, ul)on demund.
1'he fcrtility dru~ rnay be in the form of pellets, powder medicil)es, cul)sules, cyrup or iniection.s, and upplied orully or externally.
I:or better underslulldin~ of the invention some exemplifyin~ tcsts will l)c Nivcn llercunder.
Test l In tlli~ tesL, a water-soluble fraction und an oil-soluble fructio werc cxtructcd rc;l)(?ctively from tlle wholc s(?(?d of .lob s tCUl'S and tlle ovulution-induce effects of tlle respectivc fractions werc mcusllred and compul cd l(> cucll otll(!r .
Ille wllole sced of .lo~ s tears w~s Kroulld into powd(?r to plel)ure 5008 s&mple. This sample powder was subjected to extraction w;tll an _~_ 3 2 ~8 4 2 3L
extractill~ reagent of 1.5~ n-hexane at u lemperaturc ranKc of 15~-20~C.
After the extruction the extracting reagent was distilled under pressure reduccd condil.ions to obtain about 45~ of yellow, oily substallcc (cxtractabiliLy of about 9~ by weight - the percenta~e i3 giYen by weiglll throu~llout the Specification unlcss oLllerwise specified). This yellow, oily extructant wus deterlnined as colnponent A. Then tlle residue a~Lcl exlractioll of coMponent A was furtller sul)jected to extraction Witl 1.5Q ethanol hut only a ne~ligible ainount of an extractant was obtained.
Thc residuc was d;pped into a Icaching linuid of 50X ethanol solution.
and the leachillg liquid was concelltrated aL a temlerature below ~0C
undcr prcssurc redut:cd conditions, to thcrc~y obtaill a plccipitatt~ and about 2~ e~tractant. This cxtractant wus deterlnined as colnpon~llt B.
Thcl-, from tlle residuc ufter extractioll of colnponent ~ wa9 CXll~aclCd witll 1.5Q water, a slnall alnount of allotl~er cXtractant, WhiCIl was dclcrmined a9 COII)I)OllCllt C. In 5u~nmury, colnpollcnts A, B and C wcrc extracted in such Inunller as diagralnlnatically illustrated in PiKure 1.
I IIIU';~' COMI)Ol~ ulld C W~'I'C IIIIU I i l.U 1. i V(! I y UllU I i '~.(.'~i i 11 U kllow nallner to find that cornponent A contains ~Iyceride and such eslels as ~crulic acid cstcr, cotnpollcnL ~ conlaill~i polyulnido alld colnpollcllt C
contains arnino acid and peptidc.
Next, the ovulation-inducing effect or actiYity of the respective coMponellLs A ,B and C wcle lcstcd in Lhc following Inanller~ Morc l~articularly, 70 ~olden halnsters in thc a~e of 5 - ~ weeks W(!l'(' dcvidcd into 7 ~roups euch having 10 golden hamsters. Tlle foddcl werc I)rel)aled by addin~ to tllc basic fodder lZ of coml~ollellls A, h and C, and KiVèn orally to thc ~roup Nos. 1, 2 and 3, respectively. As thc avera~e intake of the fodder of a ~olden halnster is ~9~ a day, t90~y of Ihe ~1~2~342~
r(!3pcctivc componellts werc given to Llle respectiYc groul)s. Thcy wcre fed with such fodder for 3 weeks and then slaughtered. The 8roul~ Nos. 4, 5 and 6 were fcd in the same manner, but after 3 wecks' fecd of lhe foddcr containillg the coml)onents only the basic fodder werc ~ivcn Lhcrclo for 5 w(!elts, alld tllcn slaughtcrcd. Whereas, tbc groull No. 7 wus initi~lly given only the basic fodder for ~ weeks and tllen slaughtered.
As lo ttlt! ~('SI)(!Cl.iV(' KrOUpS, tllC S('X l)('riods WCTC olls(!rv(~l ulld the number of naturally produced ova was counted beforc they wele sllu~htcred, and after slau~htered the weight and thc state of tllc ovary were observed hy mealls of dissection. The results of thcse tests are shown in the following Table I .
38~2 T A B L E
. _ ._. . . ._ test sample comparison of nlllnber Or¦ dissection test _ _ ______ ~
~roul~ coml30nell1 scx period natural weight of state of No. udded sex pcrioc rcmurkc ovulutiol ovary(~) ovary . ,, .,. . ._ ___ ___ __ .,.,, __ ._, _ 1 A ~ days regular 17 1 2 ~.~ ~9.48 + 2.35 (~) 2 B 4 duys regular 12 + 2 1~.01 + 0.3~ (-I) C 4 days regul~r 12 + 2 17.93 + 0.23 (+) ~ A ~ d~ys regular 17 ~ 3 ¦ 19.59 + 0.2~ (++) B 4 days regular 12 -t 2 18.20 + 0.2~ (+) 6 C ~ days re~ular 12 l 2 t7.8~ + 0.56 (t) 7 4 days re~ular 12 + 2 18.03 t 0 15 (~) _ . . . , ,___ Notes: ~9 ~i~urc~ in the column of Scx Peliod show the averu8 e.
G~ tll(- collllnll of State of Ovury Ihe murk~ ) und (l~) meall rcluLive comprison in the number of corpora lutea found in cut-outs of the ovary.
~ The murk ~ identified in Lllc columll of Natural Ovulatioll mcalls lhuL Lherc could bc found a ;i~niricullce witll rc.pccl lo other 8roul~s ut u sigllificallce Icvel Or 1%.
As iL al~eurs floln Lhc re3ulLs ;howll in `lablc I Lllc compollellt A
extructcd from the whole seed of .loh s teurs with n-llcxal)c will promoLe natural ovulation witllout disturbing the ;cx periods. As a resull: of orKanic dissection Llle effective componellL contained in tllc corn!)onent A
wu-; l~roved to uct on tllc sex center so us to promotc formation of corpora lutea.
Meanwhile T~ble I AISO shows that this eftective compollellt is colltuilled only in the oil-solu5~1c fruclioll Or tlle .lob -. Lcar; sccd and is ~.carcely presellt in Lhc extractullts ohtaincd with etllullol 50 eLtlanol solulion und wùter.
~ ~ ~842~ 27628-lD
Test 2 ~ This test was carried out to find the optimal dosage of the ovulation-inducing effective component contained in the component A extracted in Test l.
First, the component A was dipped into leaching liquids of ethanol, 50~ ethanol solution and water, step by step, and then the leaching liquids were removed. Then the residue was purified by column chromatography with n-hexane. Thus, a purified extractant was obtained in a yield of 90% of the component A.
This extractant was dissolved in 0.2mQ soybean oil in quantities varied between 95~J380mg to prepare three fodder containing different quantities of the extractant, which were orally applied by injector to three groups each having lO golden hamsters, once a day, for 3 weeks. Thereafter, the test animals were slaughtered. These groups were put into the tests for the natural ovulation and the ovary weight in the same manner as in Test l, upon which matural relation of the dosage of the extractant per body weight of a golden hamster was determined with these parameters. The test results are shown in Figure 2.
As shown, the optimal dosage per body weight of a golden hamster was found to be in a range of 0.76~1.4mg/g a day.
Accordingly, provided the body weight of a grownup is 60kg, 4.6rJ8.4g/day of the purified n-hexane extractant was determined to b~ an optimal range when applied to an adult. The medication term will be dependent upon the condition of a patient and varied case by case.
Test 3 In this test, extractability of the effective component ;~ _g_ ~ 27628-lD
obtained respectively from the whole seed, coix seed, bran and hull of Job's tears was measured and compared to each other.
Respective 500g powder were prepared by grinding the coix seed, bran and hull of Job's tears seed, and extractants corresponding to component A were extracted in the same manner as in Test 1. Quantities of the extractants thus obtained and extractabilitv in the respective cases are shown below in Table II. Data for the whole seed in Table II are the reproduction of those in Table I. It will be obvious from Table II that the greatest extractability was gained in the case o~ bran, leading to the fact that the effective component is in substance contained in the bran of Job's tears seed. Thus, the effective component can be extracted with great efficiency from the bran alone or the whole seed including the bran.
TABI.E II
. _ , test sample amount of extractant.(.g). .extractability (%) _ whole seedA5.0 9.0 coix seed27.0 5.4 bran 80.0 16.0 hull 0.6 0.12 Note: The extractability is calculated by the equation of amount of extractant (g)/ sample powder (500g) X 100 The inventors have made thorough investigation of the effective ovulation-inducing material contained in Job's tears seed and found in the end that ferulyl stanol derivatives expressed by the following formula (I) and/or phytosterol fatty acid ~ster ~ 421 27628-lD
expressed by the following formula (II) has significant effect and activity on induction -lOa 3l~3~34~L
of nuturul ovulution. 13ascd on this fucl thc inventors have comc to a ~uccessful end that a novel fertility dru~ having Ull effcctivc coml)ollellt coml)rising ferulyl stnllol derivutivcs und/or l)llytostcrol fatly acid cster may ~e prel~ared.
~ormula (I) R ~ <
O
Il ,/ C ~ O ~\\~"' C = C 11 .. ..
~ ,J
Cl130 ~ (R: CI13 or '-C21k~) I:ormula (Il) ~ R, R,COO
(R~: remaining radicai of palmitic or stearic acid R2: - Cll~ or -C211s~
~ 3 8 4~3L
Stanol ferulic ~cid derivatives representcd by the formulù ( I) nay be, for exu~nple, truns-ferulyl stigmastallol, trans-ferlllyl cuml)estanol or a mixture thereof.
Phytosterol fatty acid ester represented by the forlnula ( ~) may bt~, for exu~nl)le, ~ -sito3terol palmitic acid cster, ~ -silosterol slearic ucid estcr, carnlleslelol pallnitic ucid ester, campeslcrol <;tearic uci(3 ~Islcl O~ y 011(! of lllc colnbillul.ioll~ cof.
A process for producing the above fertility drug is desclibcd in detaiI hereundt,?r.
First, ù Job's tears seed is threshed and purified il?~ a known mallncr to be scl~arated into coix seed, brall and hull. To I part of the 6run is incorl-olutcd ~ ~ 5 purts (l~y wei~lll; Ihc pall i; ~iV~'II by wei~lll. thlougholll lhe Specification unless olllerwise sl~ecified) of ethyl acetate, and thc? resullallt is agitated at 15 ~ 20C for 5 -~ 10 hours, lh~.?reby extracting ùn oil-and-fat fraction. The extract is Lhe filLered to relnove nn insolllble fraction.
To 1 purt of tlle residue is incorl)()l~Lcd 3 ~ 5 purts of elhan and lhc solulioll is u~ilatcd at 15 ~ 20(~ for 5 ~ 10 hours. lhe rcsulLulll cxtract is lllell filtcred Lo rclnovc un insolublc fracLioll.
1'he elhullol-sol~ lt? frucLioll thlls obluillcd is disLilled lo rclnove etllullol, therel3y obtuinillR all elllallol extracLulll. To 1 pUlt of Llle~
ethanol extractant is added 3 ~- 5 partx of clhyl acetate, and lhe solulioll is a~ilutcd uL 15 -` 20~C for 5 -` 10 hours, to obLuill un cLhyl acetate-soluble fraction.
The oil-and-fut fraction and the ethyi acetate-solu~le fraclioll are Inixcd to~ethcr and cthyl acetate is separatcd froM ihc InixLure. Tht?
resulLunt is supplied to a column char~ed with silicu ~el to effect ~ . .
, Kradient elution with a mixed eluent of n-bexulle and ethyl acetate whereby fructions eluted with the Inixed elucl1ts of n-llexalle and ethyl UC(?lUi(! lluvillg Mixillg lulio~ Or 3~: 1 ~ 10: 1 u~ /ol 100: 0.5 ~ 100: 1 are collected. Thc fractions thus collected are Inixed together and tht cluents are reinoved from the Inixture Lllcrcby obtainin8 ovulution-induceill~ Inaterials. The fractions eluted with lhe eluellt of ~0: 1 ^-10: 1 Inixture of n-llexulle und ethyl ucetatc will contnill ferulyl stunol derivatives and tl~e fractions eluted with the eluent huvin8 tlle tnixin~
ratio of 100: 0.5 ~ 100: ~ will COlltUill phytosterol fatty acid ester.
Ifrective Inaterials will be contailled in the nespeclive eluates obtailled with thc cluents havillg different ranges of mi~ing ratio and may be used alone or in a Inixcd state to Inanufacture llle fertility dru8 according to thc invelltion. The drug muy be a pellet a powder Incdicine n capsulc an inièction or in uny other forln and applied orally or externally.
Tllc dosaK(? of the dru6 will vury dcp-!lldillg ulll)ll tllc collditioll of u paiient an(l the way of upplicutioll but in Inost cases ~0 -~ 80~ once a ~ay Wi ~ (? cf ~CC Li VC Wll(!ll 01'~ 1 I y a~ 1.O ~11 adull.
The fertility drug of the invention having an effective componellt comprising ferulyl stallol derivativc ulld/or pllytosterol futty acid ester will be furtllcr dcscribcd l~y waY of exelnplifying tesis.
., ~ . . __ This test was carried out to observe physical and chemical characteristics of ferulyl stanol derivative. The test sample was X colnl)onent prepared in tlle saMe Inanner as in Example 3 des(:ribed later.
Illi; tcst <;ulnplc was allali~.ed by tllin luycr chrolnutoKIalllly Lo ~ 42~ 27628-lD
detect a single spot (hereinlater defined as material X) which manifested a blue-green color with sulfuric acid and was distinguishable in ultraviolet rays.
The material X was purified by alumina column chroma-tography and isolated as colorless, needle-like crystals having melting point of 156C. The material X showed positive adsorption in the Gibbs' adsorption test and had a M' peak at m/e 592 in the mass spectrometry. In the infrared spectrophotometry, as shown in Figure 3j it showed adsorption signals based on hydroxyl group at 3120r~3500cm 1, conjugated carboxyl group at 1710cm 1, double-bond of ~,~-unsaturated carbonyl group at 1640cm 1, C-C stretching ~ibration of benzene ring at 1600 and 1510cm 1, and out-of-plane deformation vibration of benzene ring at 840cm 1, respectively.
In the nuclear magnetic resonance spectrometry (CDCQ3), as shown by the chart of Figure 4, it manifested a signal pattern peculiar to phytosterol at 0.62~ 2.Oppm. Further, signals were found based on methoxy group at 3.88ppm, and the AB type spin-spincoupling (J - 16Hz) of hydrogen in the double-bond of ~,~-unsaturated carbonyl group of cinnamic acid derivative and the ABM type spin-spincoupling of hydrogen in the tri-substi-tuted benzene ring thereof was found at 6.22ppm and 7.55ppm, respectively. The singlet of lH at 5.95ppm was disappeared by adding D20. According to these results, the material X was determined to have hydroxyl group of a phenol.
The material X was subjected to alkalic hydrolysis to be separated into an acid and a neutral fractions, to both of which were applied chemical analysis to pro~e that only ferulic acid was isolated from the acid fraction (which was identified in - .
~ 8'~2~ 27628-lD
comparison with specimen). The neutral fraction was silylated in a known manner and then quantitatively analyzed by gas chromato-graphy, whereby it was identified as a mixture of stigmastanol and campestanol in a ratio of 9 : 1.
Further, the material X was acetylated with a`mixture of pyridine and acetic acid to form mono acetal in the form of colorless, platy crystals, having melting point of 155-V156C.
According to nuclear magnetic resonance spectrometry (CDCQ3), there could be seen signals based on methyl and methylene radicals of phytosterol at O.6-V2.Oppm, and a signal based on acetyl group at 2.32ppm. At 3.84ppm, a signal of singlet of 3H was found based on methyl of methoxy group. The AB type spin-spincoupling of olefin hydrogen in a,~-unsaturated carboxyl group was noted at 6.32ppm and 7.60ppm, and the ABM type spin-spincoupling of hydrogen on the tri-substituted benzene ring appeared at 7.07ppm.
Consequently, the material X was identified as a mixture of trans-ferulyl stigmastanol and trans-ferulyl campestanol in a ratio of 9 : 1. By the results of analyses including its melting point, the trans-ferulyl stigmastanol was found to be the same material as dihydro-~-sitosterol ferulic acid ester which is isolated from a corn embryo bud oil (Tamura et al; "Nippon Kagaku Zasshi (Japan Chemical Magazine)"; vol. 79, page 1011; in 1958).
Test 5 This test was carried out to reveal physical and chemical characteristics of phytosterol fatty acid ester. Y component, prepared in the same manner as in Example 3, described later, was used as a test sample.
~ 2 ~ 27628-lD
This sample was analyzed by thin-layer chromatography in a known manner to isolate a material of colorless, needle-like crystals, having a melting point of 64~v65C, which showed a spot manifesting a blue-green color with sulfuric acid and being distinguishable in ultraviolet rays. According to mass spectro-metry, a peak based on M~ was found at m/e 680. A peak based on carbonyl group was observed at 1740cm 1 in infrared spectrophoto-metry, as shown in Figure 5. In nuclear magnetic resonance spectrometry (CDCQ3), as shown by the chart in Figure 6, signals were noted based on methyl and methylene protons of phytosterol at 0.6~v2.0ppm, and -(CH2)- of long chain fatty acid residue at 1.28ppm. A signal of triplet based on methylene group adjacent carbonyl groups, a signal of multiplet (12 Hz in half width) based on hydrogen of C3 phase and a signal of multiplet based on binyl hydrogen were found at 2.25ppm, 4.6ppm and 5.35ppm, respectively. Then, the material was subjected to alkalic hydrolysis to be classified into an acid and a neutral fractions.
The neutral fraction was proved to contain sterol, which comprised ~-sitosterol and campesterol. The acid fraction was a mixture of higher fatty acid comprising in substance stearic acid and palmitic acid, which was determined by means of methylation and gas chromatography.
In conclusion, the said Y component was identified as a mixture of long chain fatty acid esters of phytosterol. A part of this material was identical to ~-sitosterol derivative mentioned by Kuksis et al in "Journal of Organic Chemistry", vol. 25, page 1209; in 1960.
~ 84~ 27628-lD
Test 6 In this test yield of stanol ferulic acid derivative and phytosterol fatty acid ester from the whole seed, coix seed, bran and hulls of Job's tears was compared to each other.
The whole seed of Job's tears was ground in a known manner, threshed and purified, and thus separated into hulls, bran and coix seed. Among 100 parts of the whole seed, 33 parts of the hulls, 15 parts of the bran and 52 parts of the coix seed were obtained. ~o these four star~ing materials were respectively added n-hexane and ethyl acetate each in a triple quantity thereof, and the solution was agitated at 15 ~20C for 5 hours, to thereby effect extraction. After the respective extracting solvents had been removed, oily components were obtained. The weight ratios of the respective oilycomponents with the starting materials are shown in the following Table III, which shows the fact that the effective components comprising ferulyl stanol derivative and phytosterol fatty acid ester are both included in the bran of Job's tears seed.
9.28842~L 27628-lD
_ _ .. _.___ .
U~
a)o ~ ~ a~
S:: h ~1 O ~ r ~1 oa) v ~ ~ ,~ ~
~d o ~ o O O ~ o ~1 0 4~
O _ _ _, , ~0 S~ ~
P~ ~ a u~
,~ O ~ o ~1 ~1 ~ o ~ O
a) ~ ~ rl ~ rl rl ~1 ~ O ~1 0 ~1 h :
a) c) 4~
H
H
H
~-~ ~
o ~ :
~ a) 0~
O
t) t) ~ ~1 ~ Ou~
O ~a) .. _ ~ rl o ~;
S~ ~ ~`1 ~ ~ ~ ~ ~1 0 ~
0~ ~ . . . .
C) ~ ~ O ~9Ln X
s~ a~
~1 ,~ I
O o ~
_ ~ _ __ ~ a) ~ ~ a) a) X
O ~1 ~ ~rl o ~; 3 ~ Q o . . . _. , ~ 421 27628-lD
Test 7 This test was carried out to determine fractions contain-ing the effective components comprising ferulyl stanol derivative and phytosterol fatty acid ester, by means of silical gel chroma-tography.
1) Preparation of Test Samples According to the method described later in Example 3, 1!250g of an oily fraction was extracted from 5kg of Job's tears seed bran with ethyl acetate and ethanol.
300g of oily fraction was subjected to column chromato-graphy using 5kg of silica gel, wherein the eluent was at first n-hexane and then mixtures of n-hexane and ethyl acetate in mixing ratios being varied to gradually increase the proportion of ethyl acetate. The eluates were 4.352g of F-I component eluted with a mixed eluent of n-hexane and ethyl acetate in a mixing ratio of 100: 1, 283g of F-II component with an eluent of a 20: 1 mixture and 8.756g of F-III component with ethyl acetate alone. F-I
component was purified by column chromatography using 250g silica gel and a mixed eluent of n-hexane and ethyl acetate in a ratio of 100: 1. F-II component was further subjected to alumina column chromatography and eluted with a 20: 1 mixture of n-hexane and ethyl acetate to obtain 128mg of F-II-l component, 1.284g of F-II-2 component, 210mg of F-II-3 component, 210mg of F-II-4 component and 55mg of F-II-5 component. Thus, seven specimens were prepared.
2) Test Method for Physiological Activities These seven specimens were dissolved into 0.2mQ of a soy bean oil to prepare various oil solutions, which were orally ~" ' .
34~:~
27628-lD
given once a day to respective groups each having 10 golden hamsters in the age of 5~ 8 weeks. The amounts of the components respectively contained in such oil solutions were made different to be 0.2mg and 0.5mg. During the administration for 3 weeks with such oil solutions, the sex periods and the number of naturally produced ova were observed, the results of which were compared with those of reference group which had been treated with 0.2mg of soy bean oil containing no sample component.
3) Results The results with respect to the natural ovulation are shown in Table IV.
TABLE IV
component amount of component addëd added 0.2mg 0.5mg . .__ ~ . . . _ _.
(reference) 12 12 F-II-l 12 10 The sex periods were 4 days for all groups including the reference and there could be found no disturbing effect on the sex period. It will be quite obvious that F-I and F-II-4 components have significant ovulation induce effect. These components were identified in a known manner to determine that F-I component was phytosterol fatty acid ester and F-II-4 component was ferulyl stanol derivative.
~ 84~ 27628-lD
Test 3 This test was performed to determine effective dosage of stanol ferulic acid derivative, an effective ovulation-inducing component of the fertility drug according to the invention.
l) Preparation of Test Sample X component was prepared in the same manner as in E~ample 3, described later, and used as a test sample. X
component was a 9 : l mixture of trans-ferulylstigmastanol and trans-ferulylcampestanol.
2) Test Method The test was carried out in the same manner as in Test 7, except the amounts of the component added in the soy bean oil were O.lmg, 0.2mg and l Omg once a day, respectively.
3) Results 3-1) Sex Period TABLE V
. . . .
amount of component added sex period (day) ~reference) 4 O~lmg 4 0.2mg 4 l.Omg 4 As shown from Table V, each group showed regular sex period.
3-2) Number of Natural Ovulation 3~2~84z~ 27628-lD
TABLE VI
amount of component number of natural added ovulation . .. ___ . ._ _ (reference) 11 + 1 O~lmg 16 + 1 0.2mg 18 + 2 l.Omg 13 + 2 Note: The mark ~ shows a significance at a significance level of 1%.
As shown from Table VI, the groups to which O.lmg and 0.2mg of the effective components were given show significance with P~O.Ol with respect to the reference group.
As the average bodv weight of a hamster used in this test was 150g and the body weight of an adult is supposed to be 60kg, the effective dosage to an adult is proved to be 40~'80mg once a day.
Test 9 This test was performed to determine effective dosage of phytosterol fatty acid ester, another effective ovulation-inducing component of the fertility drug according to the invention.
1) Preparation of Test Sample Y component was prepared in the same manner as in Example 3, described later, and used as a test sample.
2) Test ~ethod The test was carried out in the same manner as in , : `
~ Z ~84~ 27628-lD
Test 7, except the amounts of the component added in the soy bean oil were 0.lmg, 0.2mg and l.Omg once a day, respectively.
3) Results 3-1) Sex Period TABLE VI I
... . __ __ _ . _ .,_ _ amount of component added sex period ~day) (reference) 4 0.lmg 4 0.2mg 4 1.0mg _ _ It will be quite obvious from Table VII that each group showed regular sex period.
3-2)Number of Natural Ovulation TABLE VI I I
, amount of component number of natural added ovulation (reference) 12 -~ 2 0.1mg 16 + 1 0.2mg 18 ~ 2 1.0mg 15 + 1 Note: The mark ~ shows a significance at a - significance level of 1%.
As shown from Table VIII, the groups to which 0.lmg and 0.2mg of the effective components were given show significance with P<0.01 with respect to the reference group.
~ ' 27628-lD
842~L
As the average body weight of a hamster used in this test was 150g and the body weight of an adult is supposed to be 60kg, the effective dosage to an adult is proved to be 40 ~ 80mg once a day.
Ferulyl phytostanol derivative, one of the effective components having ovulation-inducing effect, may be prepared not only by extraction from Job's tears seed as described before, but also by synthetic method. Such a synthetic method of production of ferulyl phytostanol derivative is characterized by successive steps of acetylating ferulic acid with a mixture of pyridine and acetic anhydride, treating the acetylated ferulic acid with thionyl chloride to form an acid chloride, reacting in the presence of pyridine the acid chloride and phytostanol to prepare phytostanol compound, dissolving the phytostanol compound into a mixture of methanol and chloroform, and adding sodium boron hydride to a resulting solution, with stirring, to thereby deacetylate the phytostanol compound.
The respective steps will be described in detail as follows.
A) Acetylation Ferulic acid (4-hydroxy-3-methoxycinnamic acid) is dissolved into a liquid mixture of pyridine and acetic anhydride (mixing ratio of 3 : 1) in a concentration of 10 ~15 wt.%. After reflux for 4~5 hours the solvent is distilled to prepare acetylated ferulic acid.
~' ` .
27628-lD
CH=CHCOOH CH=CHCOOH
pyridine acetic anhydride OH OH
B) Formation of Acid Chloride The acetylated ferulic acid is dissolved into chloroform anhydride in a concentration of 10 ~15 wt.%. Thionyl chloride (SOCQ2) is added to the solution in a proportion of 1.5 moles per lg acetylated ferulic acid -to effect the reaction thereof at 20 ~30C for 5~-6 hours. Then, the solvent is distilled in pressure reduced conditions to prepare acid chloride.
CH=CHCOOH CH=CHCOCQ
thlonyl chloride \ OCH3 ~ OCH3 C) Reaction with Phytostanol The acid chloride thus prepared is dissolved into pyridine in a concentration of 10~-15 wt.% to prepare a first solution, and an equivalent of phytostanol is dissolved into pyridine to prepare a second solution. The first solution is dropped into the second solution in an ice-cooled condition to effect the reaction at 20--30C for 2 ~ 3 hours, and the solvent is distilled and removed.
27628-lD
34~1 CH=CHCOCQ ~ ~
~1 Hov~--pyridine r \ C =C /
¦ H
~ (R : -CH3 or -C2~5) D) Deacetylation The reactant thus obtained in the step C) is dissolved into a liquld mixture of chloroform and methyl alcohol (mixing ratio of 1 : 1~ in a concentration of 3 ~ 5 wt.%. While this solution is being agitated in an ice-cooled condition, sodium borohydride in a quantity of double equivalent is added thereto by degree. After the foaming has ceased the solution is allowed .
. ~
~ 84~ 27628-lD
to stand at 20 ~25C for 1~ 1.5 hours, a small quantity of water is added and then the solvent is evaporated in pressure reduced conditions. Thus, ferulyl phytostanol derivative is prepared.
H C--o t7 C - C H
H
~ NaBH4 CH30 1' CH30H + CHCQ3 Il-o ~ ~
\ / H
C--C
\ H
~ tR : -CEI3 or -C2H5) OH
~r ~ .
.. .
~ 42~ 27628-lD
Ferulyl phytostanol derivative thus prepared by the successive steps A) to D) may be further purified by silica gel column chromatography and then recr~stalized with methyl alcohol, thereby producing the same in a pure state. Alternatively, the purification may be carried out each time the intermediate product is formed in each step.
The followings are exemplifying tests for the method of producing ferulyl phytostanol derivative according to the invention.
Test 10 This test was carried out to examine physical and chemical characteristics of the intermediate product formed by the acetylation step A).
1) Preparation of Test Sample Test sample was prepared in the same manner as the acetylation step in Example 6, described later. The acetylated product was sub~ected to silica gel column chromatography whereby a fraction eluted with acetone was obtained, from which acetone was evaporated to prepare a purified test sample.
23 Test Method The test sample was put into tests for melting point, mass spectrometry, infrared spectrophotometry (KBr method) and nuclear magnetic resonance spectrometry (CD30D * CDCQ3) in known manner.
3) Results The test results were as follows.
mp . 195-~196 C , FD-MS m/e; 236 (M ), IR~ (KBr) Cm 3500 (COOH), 1760,1690 (~C=O), 1630 (-COOH=CH-), NMR (CD30D +
:.
~ 2~8421 27628-lD
CDCQ3), ppm: 2.32 (3H, S, COCH3), 3.82 (3H, S, OCH3), 6.32 (lH, d, J=16Hz, ~COCH=CH-), 7.07 (3H, m, aromatic ring), 7.60 (lH, d, J=16Hz, -COCH=CH-).
Conse~uently, the test sample was identified as 4-acetylferulic acid (4-acetoxy-3-methoxycinnamic acid).
Test 11 This test was carried out to observe physical and chemical characteristics of the intermediate product formed by the reaction of acetylated ferulic acid chloride and stigmastanol.
1) Preparation of Test Sample Test samp]e was prepared in the same manner as the step of formation of acid chloride in Example 6, described later. The acid chloride compound thus prepared was sub~ected to silica gel column chromato~raphy with a mixed eluent of n-hexane and ethyl acetate ester (mixing ratio of 20: 1). The eluate was recrystalized with methyl alcohol to prepare a purified test sample.
2) Test Method The tests applied to the sample were the same as in Test 10. The elementary analysis was carried out in a known manner.
3) Results A melting point was 156C. The results of the elementary analysis were C: 77.80 and ~I: 9.89~ which are in conformity to the calculated values of C: 77.60 and H: 9.78 according to the molecular formula of C41H62O5 of 4-acetylferulylstigmastanol.
The results of mass spectrometry, infrared spectro-photometry and nuclear magnetic resonance spectrometry were as ~r ~ 27628-lD
follows.
FD-MS m/e ; 634 IM ), IRv (KBr) : 1770,1710 (>C=O), 1640 (-COOH=CH-), 1600,1510 (benzene ring?, 1180,1080 (-C-O-C-), NMR
(CDCQ3), ppm : 0.6rJ2.0 (-CH3~ -CH2-), 2.32 (3H, S, COCH3), 3.84 (3H, S, OCH3), 4.70 (lH, m, -O-CH<), 6.32 (lH, d, J=16Hz, -COCH=CH-), 7.07 (3H, m, aromatic ring), 7.60 (lH, d, J=16Hz, -COCH=CH-).
In accordance with these results the test sample was identified as ~-acetylferulylstigmastanol.
Test 12 _ This test was performed to test physical and chemical characteristics of the final product prepared by the method of the invention.
1) Preparation of Test Sample Test sample was prepared and purified by the same method as in Example 6.
2) Test Method The same tests were applied as in Test 11.
3) Results A melting point was 152C. The results of the elementary analysis were C: 79.17 and H: 9.86, which are in good agreement with those of C: 79.05 and H: 10.14 calculated according to the molecular formula of C39H60O4.
The results of mass spectrometry, infrared spectrophoto-metry and nuclear magnetic resonance spectrometry were as follows.
FD-MS m/e; 592 (M ), IRv (KBr) ; Cm : 3200 - 3500 (-OH), 1710 (-CO), 1640 (-COOH=CH-), 1600,1520 (benzene ring3, ~ ' 42~
27628-lD
1180,1080 (-C-O-C-)I NMR (CDCQ3), ppm : 0.6r~2.0 (-CH3, CH2 of sterol<), 3.88 (3H, S, -OCH3), 4.72 (lH, m, -O-CH<), 5.96 (lH, S, -OH), 6.22 (lH, d, J-16Hz, -COCH=CH-), 6.98 (3H, m, aromatic ring), 7.55 (lH, d, J=16Hz, -COCH=CH-).
In conclusion, the final product was identified as trans-ferulyl stigmastanol.
For better understanding of the invention some preferred examples thereof will be given here~nder.
Example 1 5kg powder of Job's tears prepared by grinding the whole seed thereof was subjected to extraction with 15Q n-hexane at a temperature of 20C and the solvent was evaporated at a tempera-ture below 40C under reduced pressure, thereby obtaining about 450g of a yellow, oily material at extractability of about 9%.
This extractant was then purified by silica gel chromatography with n-hexane. The purified extractant was added to basic feed to prepare fodder which was given to a group having 10 golden hamsters in the age of 5~ 8 weeks. The quantities of the fodder given to a golden hamster and the purified n hexane extractant contained therein were determined l9g and 171mg a day, respect-ively. While the test animals were fed for 3 weeks the sex periods were observed, and thereafter they were slaughtered to measure the number of naturally produced ova and observe the state of the ovaria. For reference, an equivalent quantity of the basic fodder not containing the purified n-hexane extractant, was given to another group also having ten golden hamsters, to which the same tests were applied.
~ 3842~ 27628-lD
The test results are shown in Table IX, from which it is confirmed that the oil-soluble fraction of the whole seed of Job's tears has a favourable effect on natural ovulation and formation of corpora lutea, without disturbing sex periods.
3421 27628-lD
_ _ ~ ~1 +~+1 ~ .
__ , .
O
~ 4~ ~ ~ ~ O
u o ~ +l +l ~ ri 0 ~ OD ~
3:
_ ~ r~ ~ r~
R ~ ~ ~ ~ s~
~ O ~ ~1 ~
H ~. . .
u~ h ~
~H ~ ~ ~ O
E~ O ~a s~ _ .
u~ a~ ~) x h ~0 u~
Id X ~ ~ td P~ a) a) ~ ~ 4 O u, ~ u~ O a) ~ X ~r ~ O
~q E~
- ~ ~
a~ u~
a ~ p~ ~
~ o ~, ~
so~ ~ c~ ~ ~
.
u~ u~ c) rl ~ ~
~ ~ ~ o ~ ~ z ~ ~ --~
~ .
27628-lD
4Z~
Example 2 In Example l was used the whole seed of Job's tears but in this example 500g bran of Job's tears was used, and by extraction with l.S~ n-hexane at 20C was obtained about 80 g of a yellow oily material at extractability of about 16%. This material was purified by silica gel chromatography with n-hexane and the solvent was evaporated at 40C under reduced pressure, thus preparing about 72g of a purified extractant. This purified extractant was given to a group of ten golden hamsters in a proportion of 1.5mg a day per lg of the body weight of a golden hamster, and the same tests were put into practice as in Example 1, results of which are shown in Table X. As shown, the oil-soluble fraction of the bran of Job's tears seed will promote natural ovulation without disturbing the sex period and bring a significant effect on formation of corpora lutea.
~ .
, , .
~ 2 ~38421 2 7 6 2 8 -lD
~= .~ .... _ ~o _ O ~ +l o ... _ ~ ' ~ 4~ ~ ~ ~
~, o ~ .~ ~ ~
u~ ~ _ ,~ o Q
,~ ~ + I + I
3 ~ 0!:~ ~ ~:1 O ~ 1~ 4-( -~` U~
O ~ O
~ S-l ~ N ~ ~
~1 ~ ~ ~ oo ~`
~ O ~ ~1 m . . _ ,o ~ ~ 1 ~ H
o ~ ~ ~ a) ~ a~ a~ a) d ~: ,0 _~ _~ _ O ~
'S~ ~ ~ _ O '~
~ ~ U~ U~ rl tn )~ X ~1 ~ ~ ~ tl~
a~ ~ ~ (d ~O~ U~
a) ~r ~r U~
__ _ S~
. ~ O ~ ~
O ~. ~' E~ ~n 5~
a) D~ ~ O
,1 S~ Z
_ , .. . .~.. . ~, . .
, ~ 4~ 27628-lD
Example 3 50kg of Job's tears seed was threshed and purified in known manner to prepare about 6.5kg bran, among which 5kg bran was subjected to extraction with 5kg ethyl acetate at 20C for 5 hours while being agitated. This extraction procedure was repeated three times to obtain a first extractant. To 4kg of the residue was added lOkg ethanol, and the reaction solution was agitated at 20C for 5 hours. This second extraction procedure was repeated twice and then the collected extracts were filtered to remove insoluble fractions. From the ethanol-soluble fraction thus obtained was evaporated ethanol in a known manner. To 120g of the resulting component was added 360g ethyl acetate and the solution was agitated at 20C for 5 hours, to extract an ethyl acetate soluble fraction, a second extractant.
The first and second extractants were mixed together.
Ethyl acetate was evaporated and removed from the extractant mixture to obtain 1,310g of an oil-and-fat fraction.
A first portion of 500g of the oil-and-fat fraction was subjected to column chromatography charged with 8kg silica gel, whereby 450g eluate was obtained witha mixed eluent of n-hexane and ethyl acetate (mixing ratio of 20: 1). This eluate was further introduced to alumina column chromatography (800g) whereby elution was effected with a mixed eluent of n-hexane and ethyl acetate (mixing ratio of 20: 1~ and about 540mg of a fraction showing a spot in ultraviolet rays was obtained by a fraction collector. This component was subjected to reverse phase high performance liquid chromatography utilizing Rp-18 ~ Z~384~1 27628-lD
with a mixed eluent of ethyl acetate and methanol (mixing ratio of 5 : 3), to obtain about 450mg of a component having a U V
absorption. This component was further subjected to column chromatography using 50g of silica gel with a mixed eluent of n-hexane and ethyl acetate (mixing ratio of 20: 1), to elute about 330mg of a component (X component) having a U V absorption.
X component was put into the same tests as in Test 7 so that it was proved to have ovulation-inducing effect.
On the other hand, another portion of 500g of the oil-and-fat fraction was subjected to column chromatography using 8kg silica gel, with a mixed eluent of n-hexane and ethyl acetate (mixing ratio of 100:0.5), then an eluate (Y component) was obtained in a quantity of about 6.87g. Y component was also submitted to the same tests as in Test 7 and its ovulation-inducing efect was observed.
Example 4 To 3kg o Job's tears bran was added 3kg ethyl acetate and the solution was agitated at 17C for 8 hours for effecting extraction. This extraction procedure was repeated four times and extractants obtained in respective extraction were combined.
Thus, an ethyl acetate extractant was prepared. To 2.2kg of the residue was added 8kg ethanol and the mixture was agitated at 17C for 8 hours. The extract thus obtained was filtered to remove insoluble components. By evaporating ethanol from the ethanol-soluble fraction in a known manner, an extractant with ethanol was obtained in a quantity of 65g. To this extractant was added 260g ethyl acetate, and the solution was agitated at 17C
for 8 hours, to extract an ethyl acetate soluble extractant.
27628-lD
~1~8~
These two ethyl acetate extractants were combined, from which ethyl acetate was evaporated to obtain 790g of an oil-and-fat fraction.
A first portion of 300g of the oil-and-fat fraction was subjected to column chromatography using 5kg silica gel, with a mixed eluent of n-hexane and ethyl acetate (a mixing ratio of 30: 1), to obtain an eluate in a quantity of 269g. This eluate was subjected to alumina column chromatography(500g), thereby obtaining 330mg of a fraction having a spot in ultraviolet rays This fraction was subjected to reverse phase high performance li~uid chromatography utilizing Rp-18, thereby eluting with a mixed eluent of ethyl acetate and methanol (a mixing ratio of 5 : 3) to obtain about 248mg of a component having U V
absorption. This component was further subjected to column chromatography using 30g silica gel with a mixed eluent of n-hexane and ethyl acetate (a mixing ratio of 30: 1) to obtain about 190mg of a fraction having a U V absorption. This fraction was submitted to tests in the same manner as in Test 7 so that it was proved to have ovulation-inducing effect. On the other hand, the other portion of 300g of the oil-and-fat fraction was subjected to column chromatography using 5kg silica gel with a mixed eluent of n-hexane and ethyl acetate (a mixing ratio of 100:1) to obtain 4.352g of an eluate. This eluate was tested in the same manner as in Test 7 to find its significant ovulation-inducing effect.
Example 5 The procedure in Example 1 was repeated several times to obtain 40g of X and Y components, respectively, to which 150g of - 37a -~;
, ~ ~84~ 27628-lD
middle-chain fatty acid triglycerol on the market was added to make 1,000 soft capsule medicines therefrom, respectively. A
soft capsule medicine made from X component contained 40mg of the effective component (that is, a 9 : 1 mixture of trans-ferulylstigmastanol and trans-ferulylcampestanol) and a soft capsule medicine made from Y component contained 80mg of the effective component (that is, phytosterol long-chain fatty acid esters in a mixed state).
Example 6 According to the following steps trans-ferulyl-stigmastanol was synthetically manufactured.
1) Acetylation l.Og of ferulic acid (Tokyo Kasei Kougyo K.K., corres-ponding to 5.15m moles) was dissolved into 8m of a liquid mixture of pyridine and acetic anhydride in a mixing ratio of 3 : 1, and after reflux for 4 hours the solvent was evaporated.
2) Formation of Acid Chloride l.Og of the acetylated ferulic acid (4.24m mol) was dissolved into lOmQ chloroform anhydride and 0.46 mQ (6.36m moles) of thionyl chloride (Wako Jun'yaku K.K.) was added to the solution, to effect the reaction thereof at 25C for 5 hours, and then the solvent was evaporated under reduced pressure.
3) Reaction with Phytostanol The acid chloride compound thus obtained was dissolved into lOmQ pyridine anhydride and the resultant was dropped in an ice-cooled condition into lOmQ of another pyridine solution prepared by dissolving thereto 1.77g (4.24m moles) of stigmastanol (Aldrich Chemical Co.). After the solution had been allowed to -37b-~ 84X~ 27628-lD
stand at room temperature for 2 hours, the solvent was evaporated.
The remaining residue was subjected to a silica gel column, and the column was eluted with a mixed eluent of n-hexane and ethyl acetate (a mixing ratio of 20: 1). Thus, a reactant was obtained in a quantity of 1.65g.
4) Deacetylation and Purification 1.6g of the reactant was dissolved into 48mQ of a liquid mixture of chloroform and methyl alcohol ~a mixing ratio of 1 :
1), and 480mg (12.7m moles) of sodium borohydride (Wako Junlyaku K.K.) was added thereto little by little. After the foaming had ceased the reaction mixture was allowed to stand for 1 hour, and then lmQ water was added thereto. The solvent was then removed under reduced pressure.
The reaction product was dissolved into 5.OmQ of chloro-form and the solution was led to a column charged with silica gel, which was eluted with a mixture of n-hexane and ethyl acetate (a mixing ratio of 20: 1). The eluate was recrystalized with methyl alcohol to obtain 1.23g (2.08m moles) of colorless, needle-like crystals, that is trans-ferulylstigmastanol, in 83% yield.
Example 7 1) Acetylation Acetylation was carried out in the same manner as in Example 6.
2) Formation of Acid Chloride Acid chloride was formed in the same manner as in Example 6.
3) Reaction with Phytostanol The acid chloride was dissolved into lOmQ pyridine -37c-X
~ 2~842~ 27628-lD
anhydride and the resulting solution was dropped into lOmQ of a separately prepared pyridine solution containing 1.72g (4.2m moles) campestanol, in an ice-cooled condition. The solution was concentrated and then led to a silica gel column which was eluted with a mixture of n-hexane and ethyl acetate (a mixing ratio of 20: 1), thus obtaining 1.45g of a reactant.
4) Deacetylation and Purification 1.4g (2.25m moles) of the reactant was dissolved into 42mQ of a liquid mixture of chloroform and methyl alcohol (a ratio of 1 : 1), and to the solution was added little by little sodium borohydride (Wako Jan'yaku K.K.) in a total quantity of 478mg (12.6m moles). The reaction mixture was allowed to stand at room temperature for 1.5 hours after the foaming had ceased. The excessive sodium borohydride was inactivated by adding water and then the solvent was evaporated under reduced pressure.
The resulting residue was dissolved into 4.5mQ chloroform and the solution was subjected to a silica gel column which was eluted with a mixture of n-hexane and ethyl acetate (a mixing ratio of 20: 1) and recrystalized with methyl alcohol. Thus, trans-ferulylcampestanol in a quantity of 1.15g (1.73m moles) was obtained in 77~ yield.
Although this invention has been described in conjunction with specific exemplifying tests and preferred embodiments thereof, it is to be understood that many variations may be made without departing from the spirits and scopes thereof as defined in the appended claims.
-37d-~, , .
Claims
THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:
1. A method for producing ferulyl phytostanol deriva-tive comprising the steps of acetylating ferulic acid with a mixture of pyridine and acetic anhydride to prepare an acetylated ferulic acid; treating said acetylated ferulic acid with thionyl chloride to form an acid chloride; re-acting said acid chloride with phytostanol in the presence of pyridine to form a phytostanol compound; dissolving said phytosterol compound into a liquid mixture of methanol and chloroform to prepare a solution; and adding to said solution sodium borohydride, with stirring, to thereby deacetylate said phytosterol compound.
2. The method according to Claim 1 wherein said phytostanol comprises stigmastanol, campestanol or a mix-ture thereof.
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:
1. A method for producing ferulyl phytostanol deriva-tive comprising the steps of acetylating ferulic acid with a mixture of pyridine and acetic anhydride to prepare an acetylated ferulic acid; treating said acetylated ferulic acid with thionyl chloride to form an acid chloride; re-acting said acid chloride with phytostanol in the presence of pyridine to form a phytostanol compound; dissolving said phytosterol compound into a liquid mixture of methanol and chloroform to prepare a solution; and adding to said solution sodium borohydride, with stirring, to thereby deacetylate said phytosterol compound.
2. The method according to Claim 1 wherein said phytostanol comprises stigmastanol, campestanol or a mix-ture thereof.
Applications Claiming Priority (9)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP60-41932 | 1985-03-05 | ||
| JP4193285A JPS61204126A (en) | 1985-03-05 | 1985-03-05 | Ovulatory agent and production thereof |
| JP60-43725 | 1985-03-07 | ||
| JP60-43726 | 1985-03-07 | ||
| JP60043725A JPS61204131A (en) | 1985-03-07 | 1985-03-07 | Ovulatory agent |
| JP4372685A JPS61204196A (en) | 1985-03-07 | 1985-03-07 | Production of phytostanyl ferulate derivative |
| JP60-45732 | 1985-03-09 | ||
| JP4573285A JPS61205212A (en) | 1985-03-09 | 1985-03-09 | Ovaluratory agent and its preparation |
| CA000503235A CA1271139A (en) | 1985-03-05 | 1986-03-04 | Fertility drug and method of producing the same |
Related Parent Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000503235A Division CA1271139A (en) | 1985-03-05 | 1986-03-04 | Fertility drug and method of producing the same |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1288421C true CA1288421C (en) | 1991-09-03 |
Family
ID=27508246
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000610993A Expired - Fee Related CA1288421C (en) | 1985-03-05 | 1989-09-11 | Fertility drug and method of producing the same |
Country Status (1)
| Country | Link |
|---|---|
| CA (1) | CA1288421C (en) |
-
1989
- 1989-09-11 CA CA000610993A patent/CA1288421C/en not_active Expired - Fee Related
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