Patent Application: US-52544705-A

Abstract:
a method of extracting phytoprogestogenic compounds from herbs and their use in preparing a medicament for treating humans requiring progesterone replacement or supplement . assay systems to quantify progesterone and progestogenic receptor activity and a method for assembling a kit for the assays are also provided .

Description:
different herbs reported to have “ uterotonic activity ” were identified and screened for progestogenic activity using a cell - based reporter gene assay and have discovered two herbs with significant progestogenic activity . extracts from the rhizoma chuanxiong , the dried rhizome of ligusticum chuanxiong hort , were able to specifically activate the progesterone receptor but not the related androgen , estrogen or glucocorticoid receptors . dose response studies in cells indicate that predicted therapeutic serum levels for rhizoma chuanxiong crude extract , prepared as by methods described are between 6 . 25 μg / ml to 100 μg / ml , and the effective oral dose would be between 31 . 25 mg to 500 mg . extracts containing these phyto - progestogens from rhizoma chuanxiong are useful to treat health problems requiring progesterone therapy , supplementation or replacement including fetal support , menstrual disorders , amenorrhoea , menorrhagia , polycystic ovarian syndrome , pregnancy complications , endometriosis , contraception , menopause , endometrial hyperplasia and endometrial cancer . these phytoprogestogens alone , or in combinations with other compounds , are also useful for oral contraception , for hormonal replacement therapy , for treating endometriosis , neuroprotective and neuroregenerative agents in stroke and traumatic brain injuries . it was also observed that compound ( s ) co - eluting with two known phthalides , 3 - butyl - 4 , 5 - dihydrophthalide and 3 - butyl - phthalide , from rhizoma chuanxiong strongly activate the progesterone receptor . these compound ( s ), which co - elute with 3 - butyl - 4 , 5 - dihydrophthalide and 3 - butyl - phthalide or herbal extracts enriched therewith , are useful for harmone replacement therapy and the treatment of health problems requiring progestogenic supplementation or therapy as described above . the present invention also provides novel methods for separating fractions of rhizoma chuanxiong , enriched for 3 - butyl - 4 , 5 - dihydrophthalide , 3 - butyl - phthalide and components and co - elutes , with strong progestogenic activity using solvent - solvent partitioning , solid - phase fractionations and high - performance liquid chromatography ( hplc ). these methods can be used to standardize and fingerprint rhizoma chuanxiong extracts using solvent - solvent partitioning solid phase fractionations , hplc , mass spectrometry ( ms ), and nuclear magnetic resonance imaging ( nmr ). since the bioactive compounds co - elutes with 3 - butyl - 4 , 5 - dihydrophthalide and 3 - butyl - phthalide , these compounds can be used for the measurement and standardization of the bioactivity rhizoma chuanxiong and its extracts . novel methods have been devised to detect summated bioavailabillty and bioequivalence of multiple steroidogenic compounds in serum , following drug administration to humans and animal models . it has also been established that rhizoma chuanxiong extract is an effective progestogenic drug when administered both subcutaneously and orally to animal models . in the progestogen receptor assay of the invention , hela cells grown in 24 - well microtiter plates were transiently co - transfected with two plasmids using lipofectamine . the first plasmid consisted of dna encoding the full length human progesterone receptor ( pr — b ), and the second a progesterone reporter gene ( pre2 - tata - luc ) comprising a luciferase reporter gene driven by two copies of the progesterone response element from the aminotransferase gene . cells were exposed to the ligands in rpmi 1640 medium , supplemented with 10 % charcoal - stripped fetal calf serum , 2 mm l - glutamine , 0 . 1 mm non - essential amino acids and 1 mm sodium pyruvate for 42 - 48 hours at 37 ° c . in a 5 % carbon dioxide incubator . replicate wells were exposed to vehicle only ( ethanol ) and used as negative controls . after the period of incubation , cells were rinsed with pbs buffer , lysis buffer ( promega corp ) was added , and cell lysates collected for measurement of luciferase activity . progestogenic activity of the ligands was expressed as fold - increase in luciferase activity compared with control cells exposed to vehicle only . all data points were in triplicate . to determine the sensitivity of progesterone receptor assay , the transfected cells were exposed to increasing doses of one natural ( progesterone ), and one synthetic ( megesterol acetate ) progestogen ( fig1 ). both progesterone and megesterol acetate were able to dose - dependently increase reporter gene activity at doses ranging from 0 . 1 nm onwards , reaching peak activity at 10 μm , wherein a 300 - fold increase in hromonal activity was observed over controls exposed to vehicle only . thus the assay system of the invention provides an effective way to screen for nano - molar quantities of bioactive progestogens . novel uses of extracts from rhizoma ligusticum chuanxiong ( l . c .) for progesterone therapy . as will be clear from example 1 below , from among 13 herbs screened , extracts derived from rhizoma ligusticum chuanxiong ( l . c .) were identified as possessing strong progestogenic activity . the most potent were l . c . extracts which displayed progestogenic activity comparable to progesterone itself ( example 1 ). since progesterone and its derivatives are commonly used for hormone replacement therapy and other progesterone deficient states ; crude herbal extracts of rhizoma ligusticum chuanxiong with defined progestogenic activity can be useful to treat health problems requiring progestogen therapy . such conditions include menstrual disorders , amenorrhoea , menorrhagia , polycystic ovarian syndrome , pregnancy complications , endometriosis , contraception , menopause , endometrial hyperplasia and endometrial cancer . the extracts in combinations with other compounds are also useful for oral contraception , for hormonal replacement therapy , for treating endometriosis , as neuroprotective and neuroregenerative agents in stroke and traumatic brain injuries . novel methods to efficiently extract compounds from rhizoma ligusticum chuanxiong that specifically activate the progesterone receptor . various combination of ethanol / water , methanol / water and water alone , were tested to determine the best solvent for extracting progestogenic compounds from l . c . extraction with 100 % ethanol proved to be the most effective method for producing a crude extract with the highest progestogenic activity , comparable to progesterone itself ( 100 nm ) ( table 2 ). in contrast , extraction with methanol resulted in activity that was only one - third of 100 % ethanol . extraction with water resulted in an inactive extract . the method of extraction comprising soaking of powdered l . c . in 100 % ethanol for 5 - 7 days at 37 ° c ., that results in l . c . crude extract with maximal progestogenic activity is a novel and non - traditional method . it was also observed that while crude l . c . alcoholic extract has strong progestogenic activity , it does not have estrogenic , glucocorticoid or androgenic activity . thus it is clear that l . c . extract contains compounds that can specifically activate the progesterone receptor but not other closely related members of the steroid receptor family . moreover it has an additive effect on progesterone action but has no antagonistic effects on the action of progestogens , estrogens , glucocorticoids or androgens . l . c . extracts prepared in the above manner are therefore particularly useful in health conditions in which pure and specific progestogenic effects are required and where cross reaction with other steroid receptor are contraindicated . it is believed that the crude l . c . extract and progesterone act through the same molecular mechanisms since their activity are blocked by the progesterone antagonist ru486 in an identical manner ( example 4 ). thus the amount of l . c . required to achieve therapeutic effects can be inferred by comparison with the biological activity of physiological levels of progesterone . the range of serum progesterone concentrations in the human luteal phase is 15 to 90 nm , and these doses of progesterone elicit 150 - to 200 - fold increases in reporter gene activity ( fig1 ). comparable 150 - to 200 - fold increases in reporter gene activity can be obtained with 6 . 25 μg / ml to 100 μg / ml of l . c . ( fig2 ), suggesting that these doses of l . c . represent therapeutic serum dose ranges . assuming 100 % absorption and distribution to the total blood volume of 5000 ml , the oral dose of l . c . crude extract required for therapeutic effect is predicted to be between 31 . 25 mg to 500 mg . these doses of l . c . crude extract can be administered orally to treat conditions requiring progesterone replacement or supplementation as discussed above . new methods to purify l . c . fractions that are highly enriched for progestogenic activity the present invention also provides a novel method to obtain l . c . fractions that are enriched for progestogenic activity . such bioactive fractions are obtained by performing solid phase extractions using reverse phase c18 or diol matrices in glass columns ( example 7 ). using reverse phase c18 matrix , progestogenic activity is mainly present in fractions eluted with 80 % to 100 % methanol . with dry - packed diol columns , maximal activity was observed with fractions eluted with 30 % etoac : 70 % dcm , and lesser degrees of activation with the 100 % dcm and 60 % etoac : 40 % dcm elutes . removal of unwanted annins can be achieved by first partitioning these into the butanol and water fractions , using solvent / solvent partitioning ( example 8 ). further purification if desired can be achieved using preparative hplc . in this method a thermoquest hypurity elite c18 column and a isocratic mobile phase of 45 % acn : 55 % water can be used . under these conditions , purified fractions can be expected to exhibit typical hplc chromatograms as demonstrated in example 10 . in one manifestation , crude extract is first put through solvent / solvent partition to remove tannins , and then eluted through diol solid phase columns using polar solvents like hexane and dcm ( example 9 ). the elute when subjected to hplc , separates into highly purified subfractions with strong progestogenic activity ( example 10 ). the present invention also provides for reproducible novel methods to obtain l . c . extracts with defined purity , consistency and biological activity . these highly purified l . c . extracts can be use to treat conditions requiring progesterone therapy , supplementation or replacement . such conditions include menstrual disorders , amenorrhoea , menorrhagia , polycystic ovarian syndrome , pregnancy complication , endometriosis , contraception , menopause , endometrial hyperplasia and endometrial cancer . the extracts in combinations with other compounds are also useful for oral contraception , for hormonal replacement therapy , for treating endometriosis , as neuroprotective and neuroregenerative agents in stroke and traumatic brain injuries . a key problem in botanical drugs is the lack of methods to standardize and quality control raw materials , herbal decoctions , the manufacturing process and the finished herbal products . discovery of progestogenic properties of l . c . herbal extract will enable a person skilled in the art to use this property in appropriate cell - based or other assays to measure potency , purity and quality of the raw herbs and other manufactured herbal products derived from it . for example , progestogenic effects of the herbs can be measured with progestogenic - reporter gene assays in cell lines , and with ligand - binding assays ; among others . it has also been observed that characteristic hplc conditions can be used to fingerprint l . c . herbal extracts . in one manifestation , l . c . extracts after preliminary solvent partition and solid phase extraction , can be subjected to hplc c18 analysis ( example 10 ) resulting in typical hplc chromatograms with distinct subfraction ( fig1 ). since the biological activity of each subfraction has been documented , these characteristic hplc patterns , especially peaks corresponding to the relative quantity and resolution of each subfraction , can be used to standardize l . c . herbal extract . if a higher level of standardization is required , these hplc fractions can be subjected to nmr analyses , whereby characteristic patterns and chemical shifts can be observed ( fig1 a , b , c , d ). those skilled in the art will realize that these methods of purification of active compounds can be adapted for standardization of the biological effect of the l . c . extracts . the methods of the present invention also enable those skilled in the art to devise methods for standardization and quality control of l . c . extracts using bioassays , solid phase fractionation , hplc and nmr fingerprinting . discovery of the progestogenic effects of senkyunolide ( a ) and / or 3 - butyl - phthalide and / or minor components co - eluting with them . two known phthalides , 3 - butyl - 4 , 5 - dihydrophthalide and 3 - butyl - phthalide were purified , isolated and identified from l . c ., along with components which co - elute with them , as potent activators of the progesterone receptor . those skilled in the art will realize that the descriptions contained herein provides novel methods for measurement and standardization of the bioactivity l . c . and its extracts based on the presence of 3 - buty - 4 , 5 - dihydrophthalide and 3 - butyl - phthalide , 3 - butyl - 4 , 5 - dihydrophthalide and 3 - butyl - phthalide , or components which co - elute with them , or herbal extracts enriched for them , are also useful for hormone replacement therapy and the treatment of human or animal health problems requiring progestogenic supplementation or therapy . products derived from 3 - buty - 4 , 5 - dihydrophthalide and 3 - butyl - phthalide , or components which co - elutes with them , are also expected to exhibit progestogenic effects and therefore can be used for progesterone therapy novel methods to detect summated activity of multiple steroidogenic compounds in serum following drug administration to animal models . novel methods to perform pharmacokinetic and / or bioequivalence studies in human or in animal models have also been provided herein . these depend on the use of simple , robust techniques to precipitate proteins and extract bioactive small molecules from serum following parenteral or oral administration to animal models . the summated bioactivity of these molecules can then be tested in the receptor - based bioassay of the invention . total biological activity of these extracted small molecules when compared to reference pharmaceutical compounds , enables the determination of bioequivalence . the assay of the present invention is useful as a biomarker of progestogenic action that can predict actual physiological changes due to progesterone in animals and in the human . the assay of the invention can measure progestogenic activity accurately and rapidly in days , compared to 4 - 8 weeks for traditional animal models of progestogenic activity , like the pregnancy maintenance and endometrial transformation models . the present invention is of use in the pharmaceutical industry where a rapid and discriminating biomarker to predict of the efficacy potential steroid - active drugs in - vivo is required . since many steroidal pharmaceutical compounds are coming off patent , generic drug manufacturers need to prove bioequivalence to regulatory authorities . the present invention has great utility in this regard as a rapid and relatively inexpensive way to determine the absorption , distribution , metabolism , bioavailability and bioequivalence of pure compounds or complex mixtures compared to reference pharmaceutical compounds . proof of principle that l . c . extract is an effective progestogenic drug when administered both subcutaneously and orally the data in examples 12 and 13 prove that l . c . ethanolic extract is efficiently absorbed when administered orally , that it remained biologically active in serum after absorption in the intestinal tract , and after the first - pass effect in the liver . because the effect of l . c . declined rapidly after 5 hours ( example 13 ), it is believed that a 6 hourly ( 4 times a day ) oral dosing regime will be required to maintain high progesterone activity throughout the day . mpa is used at a dose of 50 mg / weekly ( 7 . 1 mg / day ) for endometriosis which is roughly equivalent to the dose of mpa ( 10 mg ) in our experiments . since l . c . can achieve a therapeutic equivalence of mpa 10 mg if administered at a dose of 2 gm 6 hourly , the preferred dosage for lc crude extract for therapeutic use in humans would be about 250 mg to 5 gm every 6 hours or 1 gm to 30 gm per day . the use of purified extracts enriched for bioactive progestogens would reduce the dose further . it has also been established in animal models that the progestogenic herbal lc extract is effective when administered subcutaneously or orally . the maximum progestogenic effect of the extract of the invention is equivalent to that observed with the gold standard progestogenic drug , depo - provera . l . c . at the above doses can therefore be used for the same indications as depo - provera , and these include abnormal menstrual bleeding , endometriosis , contraception , menopausal vasomotor symptoms , endometrial and renal carcinoma , and breast cancer . the present invention will now be described with reference to the following examples , which are illustrative and should not be construed as limiting the scope of the invention in any manner . to begin the search for phyto - progestogens in traditional chinese herbal medicines , the pharmacopoeia of the people &# 39 ; s republic of china ( 1997 ) was consulted and 13 herbs reported to have “ uterotonic activity ” were selected for screening . separate portions of the different chosen herbs were extracted with respective different concentration of solvents : 70 % and 100 % ethanol and 70 % and 100 % methanol . this was carried out by first powdering the herbs , mixing with the solvent , and allowing the mixture to soak for 5 - 7 days at 37 ° c ., after which the respective extracts in the supernatant were filtered with whatman grade i ( 11 μm pore size ) filter paper . the herbal residues were re - extracted a second time with the same solvent for 2 - 3 hours . filtered extracts were combined and dried in a rotary evaporator . this process was repeated with different combinations of solvents , namely 50 %, 70 % and 100 % ethanol in water ; and 70 % and 100 % methanol in water for each of the 13 selected herbs . dried extracts were weighed and re - suspended in 100 % ethanol or methanol to a concentration 50 mg / ml . each herbal extract was screened for progestogenic activity in - vitro at a final concentration of 50 μg / ml . of the 13 herbal extracts screened , rhizoma ligusticum chuanxiong ( l . c .) displayed the greatest progestogenic activity . the herb l . c ., extracted with a 100 % ethanol , demonstrated a 323 - fold higher activity compared to controls exposed to vehicle alone as shown in table 1 . this was about 80 % of the progestogenic activity observed with progesterone ( 100 nm ). in comparison , the other 11 extracts did not activate the pr . thus it was discovered fro the first time that potent phyto - progestogens are present in extracts from the dried rhizome of l . c ., a traditional chinese herb . various combinations of ethanol / water , methanol / water and water alone were tested to determine the best solvent for extracting progestogenic compounds from l . c . under conditions described in example 1 . extraction with a 100 % of ethanol was the most effective method for producing a crude extract with the highest progestogenic activity , comparable to that of progesterone itself ( 100 nm ) ( table 2 ). in contrast , extraction with 100 % methanol resulted in activity that was only one - third of 100 % of ethanol . extraction with water resulted in an inactive extract . this established a non - traditional method for extraction using 100 % ethanol and repeated soaking for 5 - 7 days at 37 ° c . that resulted in a l . c . crude extract with maximal progestogenic activity . in all subsequent experiments , extraction was performed with this method . the same methods as in example 1 and 2 were adopted . the progestogenic activity of l . c . crude extract was compared to a physiological concentration of pg ( 100 nm ) ( fig2 ). doses of l . c . from 6 . 25 μg / ml to 100 μg / ml resulted in a dose - dependent increase in progestogenic activity . the maximal activity of l . c . crude extract , at 50 μg / ml , was equivalent to that observed for 100 nm progesterone . since the maximum serum concentration of progesterone in women is 90 nm , it is predicted that a serum l . c . level of 50 μg / ml would be sufficient to achieve maximal therapeutic progestogenic effect . the physiological range of progesterone concentrations in the human luteal phase is 15 to 90 nm , and these doses of progesterone elicit 150 - to 200 - fold increase in reporter gene activity ( fig1 ). comparable 150 - to 200 - fold increases in reporter gene activity are obtained with 6 . 25 μg / ml to 100 μg / ml of l . c . ( fig2 ), suggesting that these doses of l . c . represent therapeutic serum dose ranges . assuming 100 % absorption and serum distribution to the total blood volume of 5000 ml , the oral dose of l . c . crude extract required for therapeutic effect is predicted to be between 31 . 25 mg to 500 mg . inhibition of l . c . and progesterone action by the progesterone receptor antagonist , ru486 , and possible therapeutic uses of l . c . the same methods as in example 1 and 2 were adopted . to determine the mechanism whereby l . c . activates the progsterone receptor , the effect of ru486 ( a specific progesterone receptor antagonist ) on l . c . activity was examined . the progestogenic effect of l . c . was dose - dependently inhibited by ru486 , in a manner similar to that observed for progesterone itself ( fig3 ). thus doses of ru486 ≧ 0 . 01 nm were able to completely inhibit the activity of fixed concentrations of both l . c . crude extract and progesterone . since ru 486 acts by binding competitively to the ligand - binding pocket of the pr lbd , our data confirm that components in l . c . activate pr by specifically binding to the pr lbd in a manner similar to that for progesterone itself . given that l . c . extract and progesterone act through similar mechanisms , l . c . herbal extract can be used for health problems for which progesterone replacement or supplementation is thereapeutically indicated . such conditions include menstrual disorders , amenorrhoea , menorrhagia , polycystic ovarian syndrome , pregnancy complications , endometriosis , contraception , menopause , endometrial hyperplasia and endometrial cancer . the extracts or phyto - progestogens from l . c ., alone , or in combinations with other compounds , are also useful for oral contraception , for hormonal replacement therapy , for treating endometriosis , neuro - protective and neuro - regenerative agents in stroke and traumatic brain injuries . the same methods as in example 1 and 2 were adopted . the presence of l . c . did not block the activity of progesterone . addition of increasing doses of pg to a fixed concentration ( 50 μg / ml .) of l . c . produced an additive increase in progestogenic activity ( fig4 ) suggesting that l . c . can be used to boost the effects of endogenous progesterone . absence of agonistic and antagonistic activity on other steroid receptors , gr , ar , erα and erβ . the same methods as in example 1 and 2 were adopted . although l . c . exhibits progestogenic activity in a dose - dependent manner , doses of l . c . from 3 . 125 μg / ml to 50 μg / ml did not result in any activation of gr , ar , erα and erβ reporter gene systems ( fig5 ). similarly , the introduction of increasing concentrations of l . c crude extract did not elicit significant synergistic or antagonistic effect on the gr , ar , erα and erβ reporter gene systems in the presence of hc ( 1 nm ), dht ( 1 nm ), and e2 ( 1 nm ) ( fig6 a , b , c and d ) respectively . the absence of cross - receptor activation with closely - related steroid receptors suggests that the action of l . c . was highly specific to the progesterone receptor . solid phase fractionation of l . c . with the use of c18 and diol columns to identify the bioactive compounds in l . c . crude extract , bioassay - guided fractionation was performed . preliminary isolation was performed with solid phase c18 and diol columns . reverse phase c18 powder was packed into glass columns , after which 4 mls of l . c . ( 50 mg / ml ) was applied . elution of l . c . fractions was performed with solvents of decreasing polarity in the following order : 30 % meoh , 80 % meoh , 100 % meoh and finally dcm . the fractions were collected separately dried down , reconstituted in ethanol and their bioactivity measured . progestogenic activity was mainly present in fractions eluted with 80 to 100 % methanol in water ( fig7 a ). smaller amounts were presen tin the 30 % methanol fraction . in contrast , negligible activity was observed with 100 % dcm , the most hydrophobic solvent . fractionation was also performed with diol , a silica matrix . diol powder was added to l . c . ( 50 mg / ml ) extract and the mixture dry - packed on top of a diol column . elution was performed using a least polar solvent to the most polar solvent . the elution solvents used were , 100 % hexane ( d1 ), 50 % dcm : 50 % hexane ( d2 ), 100 % dcm ( d3 ), 10 % etoac : 70 % dcm ( d4 ), ( d5 ), 100 % etoac ( d6 ), 20 % meoh : 80 % etoac ( d7 ) and finally 100 % meoh ( d8 ). fig7 b shows the progestogenic activity of the fractions obtained with pr bioassay as described in fig1 . maximal activity was observed with d4 ( 30 % etoac : 70 % dcm ) fraction , and lesser degrees of activation with d3 ( 100 % dcm ) and d5 ( 60 % etoac : 40 % dcm ). these studies suggest that the bioactive compounds were of intermediate polarity since they co - elute with solvents of mixed polarity in both the diol and reverse phase c18 matrices . studies herein also suggest that diol and reverse phase c18 columns used with solvents of intermediate polarity can be used to obtain l . c . fractions that are enriched for progestogenic compounds . to further define the conditions under which separation of progestogenic fractions can be obtained , solvent / solvent partition of l . c . extracts was performed . crude l . c . extract in ethanol ( 360 ml ) was mixed with water ( 40 ml ) to give a 90 % ethaol solution , in which 400 ml of hexane was added . the hexane was removed and the ethanol : water ratio adjusted to 3 : 2 to obtain optimal partition of dcm / ethanol - water interface . an equi - volume of dcm was added to the mixture . after this separation , dcm layer was removed and butanol was added . the butanol layer was separated from the water layer . all 4 solvent fractions were dried down , weighed , and their constituents tested for the pr bioactivity . under these conditions , the majority of the bioactivity resided in the dcm and hexane fractions ( fig8 ). the most potent was the dcm fraction , where dose - dependent increase in pr activity was observed such that 25 μg / ml of this fraction was equivalent to 100 nm of progesterone . the hexane fraction also exhibited strong bioactivity and 50 μg / ml of this fraction was equivalent to 100 nm of progesterone . in comparison , the buanol and water fractions did not show any bioactivity . since tannins ( substances known to interfere with receptor - based assays ) partition mainly to butanol and water fractions , this method is useful for removing these unwanted tannins from bioactive fractions . the method of solvent / solvent partition can therefore be used to obtain dcm and hexane l . c . fractions , enriched for progestogenic compounds , which are free of tannins . second separation step of l . c . after solvent partition , with the use of the diol solid matrix as the dcm fraction obtained from solvent / solvent partition exhibited greatest activity , it was used for further bioassay - guided fractionation . the dcm fraction was subected to diol solid - phase extraction and eluted with solvents of increasing polarity as described in example 7 . the fractions were collected and analyzed for bioactivity . fractions d1 , d2 and d3 , eluted with 100 % hexane , hexane / dcm ( 1 : 1 ) and 100 % dcm respectively , showed the most progestogenic activity ( fig9 ). thus the combination of solvent / solvent partition ( described in example 9 above ) and diol solid phase extraction , using non - polar solvents like hexane and dcm , is useful to further purify fractions from l . c . that are enriched for progestogenic compounds . to further separate the bioactive components of l . c ., the diol fraction d1 ( obtained as in example 9 ) was subjected to preparative hplc analyses using a thermoquest hypurity elite c18 column and a isocratic mobile phase of 45 % acn : 55 % water . under these conditions , diol fraction d1 exhibited a typical hplc chromatogram from which 7 distinct subfractions can be recognized ( fig1 ). these subfractions ( with elution times as noted in fig1 ) were dried , weighed and their progestogenic activities determined . progestogenic activity was greatest in subfractions 6 and 7 , eluting from 23 . 1 min to 40 min ( fig1 ). decreasing amounts of bioactivity was observed in subfractions 5 and 1 , with elution periods of 20 . 51 to 23 min . and 2 to 12 . 5 min . respectively . in contrast minimal bioactivity was detected in subfractions 2 to 4 eluting from 12 . 51 to 20 . 5 min . our data indicate that more than one compound in l . c . is responsible for the progestogenic activity of l . c ., since bioactive subfractions 1 and 5 - 6 are separated by inactive subfractions . the hplc chromatogram as shown fig1 can be used as a standard for biological activity of l . c . under these hplc conditions , the subfractions 6 and 7 ( eluting from 23 . 1 min to 40 min ) can be expected to contain the most potent progestogenic compounds . nmr finger printing of the fraction 6 obtained from hplc fractionation to identify the bioactive compounds , the hplc fraction with the highest yield ( subfraction 6 ) was subjected to nmr analysis . a known phthalide , senkyunolide ( a ) or 3 - butyl - 4 , 5 - dihydrophthalide , was identified in subfraction 6 , using 1d - and 2d - nmr , 1 h - 1 h cosy , 1 h - 13 c ghsqc spectral analyses ( fig1 a , b , c , d ). senkyunolide ( a ) has the molecular formula c 13 h 16 o 2 corresponding to five double - bond equivalents . the nmr data for senkyunolide ( a ) revealed resonances consistent with an enter carbonyl carbon ( 13 c 165 . 0 ) and a 3 , 4 - disubstituted 1 , 3 cyclohexadiene ( 1 h δ 5 . 96 , 6 . 10 and 13 c 130 . 0 , 117 . 0 , 125 . 0 and 137 . 5 ). evident were resonances consistent with a butyl group ( 1 h δ 1 . 93 , 1 . 38 , 1 . 38 , 0 . 93 and 13 c 33 . 0 , 27 . 0 , 24 . 0 and 14 . 5 ) and an oxymethine ( 13 c 84 . 5 and 1 h δ 5 . 06 ). analysis of ghmbc showed important correlations from 1 h δ 2 . 54 to 13 c 165 . 0 , 137 . 5 , 130 . 0 , 125 . 0 and 117 . 0 ppm and 1 h δ 5 . 06 to 13 c 165 . 0 , 130 . 0 , 125 . 0 , 117 . 0 and 33 . 0 and 27 . 0 ppm . this allowed the gross structure of the compound to be proposed as shown ( fig1 a ). this data is consistent with the reported structure ( yu , 1983 ). using similar analyses a related second compound , 3 - butyl - phthalide ( fig1 b ), was characterized in subfraction 6 . analyses of 1d - nmr data show that the principal constituent of subfraction 6 is senkyunolide ( a ), constituting 95 % of the dried extract by weight . 3 - butyl - phthalide , forming 2 % of the extract is a minor constituent . thus it is very likely that senkyunolide ( a ) and / or 3 - butyl - phthalide and / or minor co - elutes , are progestogenic and can be used to treat conditions requiring progesterone replacement therapy or supplementation . furthermore , senkyunolide ( a ) and / or 3 - butyl - phthalide and / or minor co - elutes , can be used as markers for progestogenic bioactivity in l . c . extracts . detection of progestogenic activity in serum following drug administration to animal models l . c . extract was administered to rat models and its progestogenic effect in serum compared with vehicle ( negative control ) and a reference progestogenic drug , 6α - methyl - 17α - hydroxy - progesterone acetate ( depo - provera , mpa ). l . c . extract ( 1 gm / ml ), vehicle ( 1 ml ) and mpa ( 10 mg / ml ) were injected subcutaneously to male sprague - dawley rats and peripheral blood ( 150 μl ) sampled at indicated time intervals for 24 hours from tail veins . male rats were selected because of their low endogenous progesterone levels . collected blood samples were stored at 4 ° c . until processed . whole blood was centrifuged at 4 ° c . for 5 minutes and serum collected . two parts of extraction solvent , consisting of acetonitrile : methanol ( 3 : 1 ), was added to one part of serum and the mixture vortexed for 30 sec to precipitate proteins . the precipitate was then removed by centrifugation at 10 , 000g for 5 min . the clear supernatant containing bioactive small molecules was stored at − 20 c for bioassay . subsequently 6 μl of this supernatant ( equivalent to 2 μl of serum from the rat ) was used for the progestogenic bioassay . to increase the assay sensitivity , hela cells were pre - exposed to charcoal - stripped serum in phenol free rpmi for at least 7 days prior to performing the assay . as shown in fig1 , progestogenic activity in serum rose to a peak within 30 min of subcutaneous injection of the reference drug mpa . the peak progestogenic activity observed with mpa was maintained for more than 24 hours and was about 5 - fold higher than serum from rats administered vehicle only . this level of progestogenic activity is equivalent to the maximum bioactivity of mpa extract when tested in - vitro ( black bar ). serum from rats injected with lc displayed a slower but still significant rise in progestogenic activity reaching a peak after 270 min . at its peak , lc activity was comparable to that observed with saturating doses of mpa in - vitro and in - vivo . high levels of progesterone activity was observed from 120 to 330 min after injection and declined to 31 % of peak levels after 24 hours . this proves the principle that the technology described in this example can accurately quantify the summated bioactivity of progestogenic compounds in serum following administration of progestogenic drugs to animal models . progestogenic activity of lc in a rat model following oral administration to determine the pharmacokinetics and bioequivalence of lc , we measured the progestogenic effect of rat serum sampled at defined time points after oral doses of lc ( 2 g / 2 ml ), mpa ( 10 mg / 2 ml ), and vehicle only ( 2 ml 60 % ethanol ). serum samples were extracted and progesterone effect measured as in example 12 . rats fed mpa exhibited a rapid and highly significant rise in serum progestogenic action reaching a peak after 120 min ( fig1 ). high levels of progestogenic effect were maintained throughout the 24 hour of sampling . in contrast , rats administered vehicle only exhibited baseline progesterone activity that was about 5 - fold lower than that observed with mpa . rats fed lc showed a steady rise in progesterone activity reaching a peak after 210 min . this peak was maintained for an hour before dropping rapidly to baseline after 300 min . rats fed either mpa or lc displayed peak progesterone activity that was comparable to that observed with saturating doses of progesterone in - vitro . based on these pharmacokinetic / pharmacodynamic profiled , we conclude that oral doses of lc can result in maximal progesterone activity in serum . in our model , significant effects were observed after 60 min , peaks at 210 min and returns to baseline after 270 min following lc administration . these data prove that lc ethanolic extract is efficiently absorbed when administered orally and that it remains biologically active in serum after absorption in the intestinal tract and after the first - pass effect in the liver . because the effect of lc declines rapidly after 5 hours , we conclude that a 6 hourly ( 4 times a day ) oral dosing regime might be required to maintain high progesterone activity throughout the day . mpa is used at a dose of 50 mg / weekly ( 7 . 1 mg / day ) for endometriosis which is roughly equivalent to the dose of mpa ( 10 mg ) in our experiments . since lc can achieve a therapeutic equivalence of mpa 10 mg if administered at a dose of 2 gm 6 hourly , the preferred dosage for lc crude extract for therapeutic use in humans would be about 250 mg to 5 gm every 6 hours , or 1 gm to 30 gm per day . the use of purified extracts enriched for bioactive progestogens would reduce the dose further . a ) discovery of extracts from the rhizoma chuanxiong , the dried rhizome of ligusticum chuanxiong hort ., a traditional chinese herb , which specifically activates the progesterone receptor ( phyto - progestogens ). b ) novel methods to efficiently extract progestogenic compounds from rhizoma chuanxiong . c ) prediction of therapeutic serum levels for rhizoma chuanxiong crude extract , prepared as by methods described in ( b ), as being between 6 . 25 μg / ml to 100 μg / ml , and the effective oral dose being between 31 . 25 mg to 500 mg . d ) use of extracts , containing these phyto - progestogens from rhizoma chuanxiong , to treat health problems requiring progesterone therapy , supplementation or replacement including fetal support , menstrual disorders , amenorrhoea , menorrhagia , polycystic ovarian syndrome , pregnancy complications , endometriosis , contraception , menopause , endometrial hyperplasia and endometrial cancer . e ) use of extracts , containing these pyto - progentogens from rhizoma chuanxiong , alone , or in combinations with other compounds , for oral contraception , for hormonal replacement therapy , for treating endometriosis , neuroprotective and neuroregenerative agents in stroke and traumatic brain injuries . f ) discovery of compound ( s ) co - eluting with two known phthalides , 3 - butyl - 4 , 5 - dihydrophthalide and 3 - butyl - phthalide , from rhizoma chuanxiong which strongly activates the progesterone receptor . g ) use of compound ( s ), which co - elutes with 3 - butyl - 4 , 5 - dihydrophthalide and 3 - butyl - phthalideor or herbal extracts enriched for them , for hormone replacement therapy and the treatment of health problems requiring progestogenic supplementation or therapy as described in ( b ) and / or ( c ). h ) new and simple methods to separate fractions of rhizoma chuanxiong , enriched for 3 - butyl - 4 , 5 - dihydrophthalide , 3 - butyl - phthalide and components and co - eluates , with strong progestogenic activity using solvent - solvent partitioning , solid - phase fractionations and high - performance liquid chromatography ( hplc ). i ) novel methods for standardization and fingerprinting of rhizoma chuanxiong extracts using solvent - solvent partitioning solid phase fractionations , hplc , mass spectrometry ( ms ), and nuclear magnetic resonance imaging ( nmr ). j ) novel methods for measurement and standardization of the bioactivity rhizoma chuanxiong and its extracts based on the presence of 3 - 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