JPS591425A - Sesquiterpene lactone, extract containing same and manufacture, and sesquiterpene lactone medicine - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

Detailed Description of the Invention Technical Field The present invention relates to sesquiterpene lactones and sesquiterpene lactone-containing plant extracts and preparations for use in medicine, %1
' Extracts and formulations useful in the treatment of migraines and various joint and bronchial conditions.

Prior Art The incidence of migraine is said to be in the range of 12% of the population. Although the exact cause has not been elucidated, some factors in the causal relationship have been well reported.

neurohormonal and local hormonal (autacoid) factors,
For example, norato-renarin, 5-hydroxytryptamine (serotonin), histamine and prostaglandins are involved in migraine, probably acting on the cerebro-vascular level.

A large body of evidence suggests that variations in blood vessel diameter play a role in the course of migraine. Such reports demonstrated constriction of cerebral cortical blood vessels and subsequent dilation during a migraine attack. 6 is thought to be due to the release of amines such as noradrenaline from neurons and the release of 5-hydroxytryptamine from platelets. Mast cells also release histamine, which plays an important role in a variant of migraine known as cluster headache (migraine neuralgia). Certain agents with anti-noradrenergic, anti-5-hydroxytryptamine and antihistamine activities have all been used to alleviate migraine symptoms. Prostaglandins (PG), such as PGFJ2. PGF2α.

PGI2 (prostacyclin) or thromboxane A
Evidence that 2 is involved is due to the fact that non-steroidal anti-inflammatory drugs such as aspirin, which act by inhibiting the synthesis of glosstaglandin, are highly effective in the prevention and acute treatment of migraine.

Regarding the causes of arthritic conditions, inhibition of the synthesis of grosstaglandin by nonsteroidal anti-inflammatory drugs, such as aspirin or indomethacin, significantly implicates grosstaglandin as an inflammatory mesoietor, at least in rheumatoid arthritis. . Glonutaglandin-like substances are present in the inflammatory perfusate of experimental animals. At low concentrations, E-prostaglandins are associated with the pain-producing effects of other agonists, such as histimine, 5-hydroxytryptamine, and plasmakinin (e.g., bradykinin), as well as other features of the inflammatory response, such as increased vascular permeability, erythema, and leukocytes. It is a significant synergist of accumulation and platelet aggregation.

Patients with bronchial asthma and bronchitis experience attacks of wheezing and difficulty breathing. These symptoms are due to bronchial narrowing resulting from spasmodic or sticky secretions of bronchial smooth muscle, or more commonly from a combination of both. The exact causes of bronchoconstriction in asthma are still not fully understood, but endogenous neurohormonal and local hormonal hormones such as acetylcholine, 5-hydroxytryptamine (serotonin), histamine, some prostaglandins, and It is believed that leukotrienes are involved.

Allergic reactions to substances in inhaled dust or absorbed from food are thought to be manifested by bronchoconstriction or urticaria or circulatory collapse caused by the release of histamine or other endogenous substances.

To treat bronchospasm lff1k, drugs with antispasmodic action are used, such as aminophylline. Drugs with antihistamine activity are also used in asthma and other conditions related to allergic phenomena, such as wheezing and hay fever.

It has been known for many years that sesquiterpene lactones exist in plants, such as plants of the Asteraceae family [Yoshio et al.
shio), Mabry and Timmermann [Sesquiterpene
Lactone, Chemistry, NMlt and Plant Distribution (5eaquiterpeneLa
ctones, Chemistry + NMRa
nd Plant Diatribu On), Tokyo Oaza Publishing 1973].

Among these plants, Natsushirogi ((Tanacatu)
mparthenium: Formerly id Chrysan
themumparthenium) has been recommended in Chinese medicine as an effective treatment for migraines when ingested. Rigorous medical studies of this treatment for migraines have apparently not been published, but have not considered which components of the plant are responsible for this relieving effect.

Many years ago, it was reported that the sesquiterpene lactones, parthenolide and santamarin, could be extracted from F. occidentalis using light oil and chloroform, respectively [Sorm et al., Co. 11. C
Zech, Chem, Comm (1961),
26°803 and Romo et al., Tetrahedron, (1965), Phantom 1
．． 1741].

These documents do not indicate possible medical uses for sesquiterpene lactones.

・The cell-positive effects of ruthenolide and several other sesquiterpene lactones have also been reported [Lee et al., Cancer Research].
ch), (1971), 31.1649]. However, this document does not suggest that sesquiterpene lactones or extracts containing them are useful for treating migraines, arthritis, or bronchial diseases.

More recently, certain sesquiterpene lactones have been shown to be effective inhibitors of cara r-nan-induced edema and chronic adjuvant-induced arthritis in rodents [Hall, Starnes et al.
), Lee 4 and Wadde l 1
), J. Pharm, Set, (1979
), 68.537]. However, this document does not suggest that sesquiterpene lactones have pharmacological activity against any complaint, let alone migraine.

OBJECTS OF THE INVENTION The object of the present invention is to suitably extract a component having pharmacological action from Nasushirogiku and to provide a useful pharmaceutical component.

By repeatedly and selectively extracting the summer white material and extensively testing the extract in vitro for its biological activity, particularly its antispasmodic activity in the smooth muscle assay, we have discovered that among the vast majority of the plant's constituents, It has been found that compounds within the range of existing sesquiterpene lactones have properties that are highly effective in the treatment of migraine headaches. This means that although the presence of some such compounds in plants has been known for many years, nowhere in the literature has it been disclosed or suggested that these compounds have a useful effect on migraines. Especially since there wasn't one. Additionally, at least some of the reports regarding its use in the treatment of migraines suggest ingesting an aqueous extract of the plant as a "tea."

Does this mean that sesquitil lactone is the active ingredient, since sesquitil lactone is not expected to be extracted from plants by this method? show.

It has also been found that such compounds are effective in treating not only migraines, but also bronchial asthma and arthritic conditions such as rheumatoid arthritis, osteoarthritis and similar arthritis. This could not have been predicted from the completely unrelated cytotoxic effect reported by Lii et al. (see loc. cit.).

Structure and effect of the invention The present invention provides a sesquiterpene lactone-containing extract f for use in medicine, particularly in the treatment of migraine, asthma or arthritic conditions:
Copper is preferably used in organic solvents that are essentially non-polar, such as oil, hexane, chloroform or oils, such as saturated or unsaturated long chains (e.g. from 10 to 25 carbon atoms). ) hydrocarbons or fatty acids, such as commercially available vegetable or animal oils, such as coco oil, soybean oil, fish oil, readers of these, such as polyoxyethylated fatty acids, regenerated glycerides and esters of said compounds, such as sorbitan. A sesquiterpene lactone-containing extract recovered by extraction with esters.

The sesquiterpene lactone-containing extract can be obtained by mixing dried and pulverized feverfew leaves with a non-polar solvent and allowing the mixture to stand for extraction. Alternatively, the dry powder is thoroughly extracted with a non-polar organic solvent, for example in a Soxhlet apparatus.

Sesquiterpene lactones, including compounds that we have found to be effective against one or more of the conditions of migraine, asthma, and arthritis, include dharmafuranolide and zonoid guayanolide, especially those with lactone rings. It is a sesquiterpene lactone with an α-methylene substituent.

The extract may contain several such sesquiterpene lactones.

Dharmafuranolide, which can be extracted from Natsushirogiku, has the following formula; I found that.

Another darumafuranolide found to be effective and the extract containing it have the formula (5): a We obtained this compound by extracting it from Natsushiroki with a non-polar solvent. It is believed that it was newly isolated. We call this "chrysantemonin."

This compound does not appear to be described anywhere in the literature.

Total hydrolysis of chrysantemonine gave the compound of formula (6): As discussed below, we suspect that this may be a particularly effective compound.

Certain guayanolides and extracts containing the compounds found to be effective have formulas (3)' and (4), respectively. We obtained these compounds by extracting them from Natsushirogiku with a nonpolar solvent, and believe that we have newly isolated these compounds. We refer to these compounds as "paltolide" (3) and "chrysanthemolide" (4). These compounds do not appear to be described anywhere in the literature.

Another interesting guayanolide extracted from Nassirochicum is chrysalthymine A of the following formula (A): 20 This compound and the Nasciultimin extract containing this compound are effective against migraine, asthma and arthritic conditions. I knew it was possible.

According to another aspect, the present invention provides a pharmaceutical sesquiterpene lactone formulation, which comprises (,) having at least two kermacrane nuclei in the molecule [1i4 epoxy groups bonded to adjacent carbon atoms; or guayanolides having hydroxy, ether or ester groups, or pharmaceutically acceptable derivatives of such compounds.

The present invention further provides all such darmafuranolide (a) and guayanolide (b) as such or their pharmaceutically acceptable derivatives, including all racemic mixtures and individual isomers of such compounds.

rlumacranolide (a), e.g. dimers and trimers of 1
These compounds appear to be more effective than kermacranolides containing a single kermacrane nucleus, each of which preferably has one or more functional substituents. . Therefore, would it be possible to administer these compounds at lower dosage levels and achieve equivalent efficacy? It is believed that this can be achieved.

Similarly, polyfunctional guayanolides, such as guayanolide (b) with an enzyme function at position 10, appear to be more effective than guayanolides without such polyfunctionality.

Particularly preferred darmafuranolides are the above-mentioned trimeric chrysantemonin (5) and the dimer obtained by hydrolyzing this, as well as pharmaceutically acceptable derivatives of these compounds.

Particularly preferred guayanolides are represented by the formula (3)'
and (4) paltolide and chrysanthemolide and pharmaceutically acceptable derivatives of these compounds.

The pharmaceutical preparation according to the present invention is obtained by extraction or synthetically produced from Nasushirogiku as described above, and/or
Alternatively, it can be converted into a pharmaceutically acceptable derivative by synthesis, and such formulations also fall within the scope of the present invention.

The present invention provides pure, essentially pure sesquinalpene lactones for use in the treatment of migraine headaches.

The sesquiterpene lactone-containing extract or preparation according to the present invention V can be obtained from Natsushirogiku by the following method.

The leaves of Natsushirogiku are freeze-dried and then crushed. The dry powder is treated in a Sock-Nuret apparatus with an organic solvent, e.g.
Extract thoroughly with 0/40' petroleum ether (ie light oil). The extract is evaporated to dryness and chromatographed on a silica column. The column is eluted by increasing the polarity of the solvent, for example from 100% petroleum ether with boiling point 60/40' to 40% methanol in chloroform. Collect fractions and combine as necessary to account for their thin layer chromatography. The combined fractions are tested for antispasmodic activity as described below. Those fractions showing 1.00% inhibition of the agonist are further fractionated. These sub-fractions are then tested. Optionally, after further purification (usually by preparative thin layer chromatography), all of the active ingredient can be isolated. The pure compound can be recrystallized from a suitable solvent and analyzed spectroscopically, for example by IHNMR, 13c spectroscopy, infrared spectroscopy or ultraviolet spectroscopy.

The sesquiterpene lactone-containing extract or preparation according to the invention can also be obtained from the plant by mixing the dried and crushed leaves of the plant with a non-polar solvent to form a suspension and leaving the mixture to leave the plant. It can be obtained by extracting sesquiterpene lactones.

Next, beeswax or other mineral wax, colloidal silica,
Cellulose derivatives, such as Cal? in the presence or absence of oxymethylcellulose and/or natural gums, such as agar or xanthan, starch, starch conductors and suitable carbohydrates,
The suspension may be stabilized using suitable solid fat mixtures with or without the addition of wetting agents such as nlupitan fatty acid ester and lecithin.

Furthermore, the pure sesquiterpene lactone according to the invention
Sesquiterpene lactones can be obtained synthetically by standard methods of producing sesquiterpene lactones, for example by photolysis of genones and other monoepoxides, such as compounds of the formula:

These epoxides can then be separated from each other if desired.

Other methods that can be used to produce sesquiterpene lactones, including pharmaceutically acceptable derivatives, are disclosed by Waadel et al. [J, Pharm.

Sci, (1979), 68.715].

Pure compounds can be made into formulations according to the present invention pc for use in medicine by conventional operations.

The formulations according to the invention can be administered as such, ie in the form of plant extracts or as organically synthesized sesquiterpene lactones alone, but usually mixed with pharmaceutically acceptable excipients. The formulation may include one or more extractive and/or synthetic sesquiterpene lactones.

If the extract is obtained by making a stable suspension of dried and ground Fatsushirogi leaves in a non-polar solvent, the suspension can be filled into one-piece soft capsules or two-piece hard capsules sealed with KM. 1 unit dose, preferably about 0.2 with or without other pharmaceutically active substances.
A formulation according to the invention can be made by making into capsules containing an amount of 5 to 200 mg of Natsushirogiku leaves.

Alternatively, the dried and ground summer beetles can be left in a non-polar solvent for a sufficient period of time to extract, such as 14 days, and then squeezed to form a cold infusion and filtered to obtain the extracted solution.

The suspension, steeping liquid or extraction solution can be mixed with a surfactant or a mixture thereof, such as polyethoxy fatty acid sorbitan esters, hepnotic and diglycerides, acetylated glycerides to create a self-emulsifying system.

The formulations according to the invention can be administered as such, ie in the form of plant extracts or as organically synthesized sesquiterpene lactones alone, but usually mixed with pharmaceutically acceptable excipients. The formulation may include one or more extractive and/or synthetic sesquiterpene lactones.

For the treatment of migraine, formulations may be administered orally or parenterally and are conveniently administered in the form of tablets or liquid suspensions. In addition to the sesquiterpene lactones, the formulation also contains other known anti-migraine preparations, analgesics and anti-emetics, such as propranolol hydrochloride, ergotamine tartrate, methysergide maleate, dihydroergotamine mesylate, clonidine hydrochloride, Meteptenmucate, Buclizine dihydrochloride, Metoclopramide hydrochloride, Bizotifen hydrate malate, Aspirin and other nonsteroidal anti-inflammatory drugs, Valacetamol and other minor analgesics, Benmezocine hydrochloride, Prochlorperazine, Caffeine , mesolobamate, ethoheptanone citrate, zomevirac, mezotazinol hydrochloride.

The dosage of active ingredient may be from 0.25 to 200 ml per day. Generally, 0.25 to 20 m9 per day is sufficient;
For the treatment of acute attacks of migraine or similar disorders, the optimal dosage range would be higher, about 2In9 to 200rn9 per day.

It is best administered in oral dosage forms made as tablets, capsules, or liquid suspensions on a daily basis or on a regular basis as prescribed by a physician. A single administration is effective, but can be repeated as necessary. However, there are cases when it is best to administer by suppository, inhalation or injection.

For oral administration, the formulation may be mixed with conventional tableting or encapsulating excipients, or made as a suspension in a non-toxic liquid vehicle that is acceptable for oral administration.

If desired, the formulation can also be provided in capsule form for sustained release over a certain period of time. For parenteral administration, the formulation may be presented as a suspension in a suitable sterile injection vehicle, such as a sterile saline solution.

Suitable methods of preparing formulations suitable for oral or parenteral administration will be apparent to those skilled in the art, given the desired dosages set forth above. The same applies to rectal administration, which is also possible.

The excellent effects of the formulation according to the invention are: (a) treatment of classic migraine and common migraine; (b) treatment of cluster migraine;
and (c) approved for the treatment of migraine and other headaches before and during menstruation.

The preparation is useful in preventing the above types of headaches, reducing the frequency of attacks,
Effective in reducing severity and duration.

The formulations of the invention are also effective in reducing nausea and nausea associated with such attacks or as separate symptoms. The formulations of the invention can also be used to treat acute attacks of headaches of the type mentioned above and reduce their duration, severity and associated symptoms.

The exact reason for the effectiveness of these preparations in the treatment of migraine is not known, but they are highly effective against biogenic amines and prostaglandins released into the local or systemic circulation in migraine disorders and related conditions. Assume that there is a change in cerebrovascular reactivity. Based on this hypothesis, the efficacy of the formulations of the invention in the treatment of migraine may be either by stabilizing vascular smooth muscle cell membranes and directly suppressing or reducing cell membrane reactivity to biogenic amines and prostaglandins, or by intracranial This is thought to be the result of an indirect effect by inhibiting the effects of these substances on the nerves to blood vessels.

The preparations according to the invention contain neurohormonal transmitters and autocoids, such as acetylcholine, noradrenaline, 5
- It was found to block the effects of hydroxyletryptamine, histamine, planokinin and prostaglandin E2 on smooth muscle.

Furthermore, when the extract is habitually administered to guinea pigs, 5-
Smooth muscle reactivity to hydroxyletryptamine and histamine gradually decreases. Indeed, it has been found that such trials may be conducted on specific formulations to determine their effectiveness in treating migraine.

For the treatment of arthritic conditions, the formulations of the invention may be administered orally, rectally, parenterally or by inhalation;
Conveniently it is in the form of tablets, capsules, suppositories or liquid suspensions.

The dosage of the active ingredient may be from 0.25 to 200 mg per day. Generally, 0.25 to 20 m9 per day is sufficient, but
For the treatment of acute attacks of chronic arthritis or similar disorders, the optimal dosage range is higher, about 2 ny to 2 ny per day.
00 qytehrou.

It is best administered in oral dosage forms made as tablets or liquid suspensions on a daily basis or on a regular basis as prescribed by a physician. A single administration is effective, but can be repeated as necessary. However, there are cases when it is best to administer by suppository, inhalation or injection.

Formulations according to the invention may further contain other anti-arthritic agents, including non-steroidal anti-inflammatory agents, analgesics, skeletal muscle relaxants, steroids, gold and (nidiramine).

Such drugs are, for example: aspirin, indomethacin, piroxicam, penorilate, ibuzolofen, racetamol, salicylamine, diflunisal, etohegtason, fenoprofen (calcium phenozolonate).
, flufenamic acid, mefenamic acid, nanoloxen sodium, ketoprofen, phenylbutacin, sulindac, penicillamine, salsalate, fenclofenac, flurbiprofen, fenbufen, fueglazone, sodium aurothiomalate, naproxen, penoxaglofen, aloxypurine, hydroxychloroquinine sulfate, azazolopagne, tolmetin, choline magnesium trisalicylate, diclofenac, adrenal steroids such as grednisone or prednisolone.

Oral administration of formulations for treating arthritis has been described above.

The effects of the formulation according to the invention include (a) treatment of rheumatoid arthritis; (b) treatment of osteoarthritis; and
elty syndrome, Stll's disease, systemic lupus erythematosus, polyarteritis nodosa, scleroderma, gout, inability to relax, Crohn's disease, chronic pulcellosis, ankylosing spondylitis, It is found in the treatment of arthritis associated with sarcoidosis, soft spots and gonorrhea.

The formulation according to the invention is useful for the prevention of the above-mentioned arthritis,
It is effective in reducing the frequency, severity and duration of attacks. These compounds can also be used to treat acute attacks of the arthritis described above and reduce its duration, severity and associated symptoms.

Although the exact reason for the effectiveness of these preparations in treating arthritis is unknown, phrostaglandin and other substances such as histamine, 5-hydroxytryptamine, and It is speculated that pradikinin is released into and around the joints. Phrostaglandin potentiates the pain-inducing effects of other substances. Based on this hypothesis, the effectiveness of the formulation according to the invention in the treatment of arthritic conditions is due to its anti-f.

This is believed to be the result of staglanson action, antihistamine action, anti-5-hydroxytryptamine action, and anti-pradykinin action.

It has been found that the preparation according to the invention has a pronounced antispasmodic effect on a wide range of smooth muscle spasmorons.

The formulation according to the invention therefore blocks the action of histamine, 5-hydroxytryptamine, pradikinin and frostaglandin E2. These formulations counteract the mesoietic effects most commonly associated with chronic inflammation. We have found it useful to conduct such tests on certain formulations to determine their effectiveness in treating rheumatoid arthritis and related conditions.

For the treatment of asthma, the formulations of the invention can be administered orally, rectally, parenterally or by inhalation.

The dosage of active ingredient may be from 0.25 to 200 In9 per day. Generally, 0.25 to 201n9 per day is sufficient, but for the treatment of acute attacks of asthma or similar disorders, the optimal dosage range may be higher, about 21n9 to 200n9 per day.

It is best administered in oral dosage forms made as tablets or capsules or suspensions on a daily basis or on a regular basis as prescribed by a physician. A single administration is effective, but can be repeated as necessary. However, there are cases when it is best to administer by suppository, inhalation or injection.

In addition to the sesquiterpene lactones, the formulation may contain other ingredients, such as bronchodilators, antihistamines and anti-infectives, such drugs being, for example: isozolenaline sulfate, orcilcinarin. Epinephrine, terbutaline sulfate, theophylline, choline theophylinate, aminophylline, ephedrine hydrochloride, papaperine hydrochloride, ibratrobium promide, atropine metonitrate, peclomezone diprobionate, phenotyrol beautifying water aVS, betamethacin, inetaline Mesylate or hydrochloride, phenylephrine hydrochloride or monolithic salt, thenyldiamine hydrochloride, lenolotyrol salt modified salt, deptrobincuen mW, butetamate citrate, epinephrine, diraenylbilarine hydrochloride, sodium chromoglycate , etamiphylline musylate, theophylline monoethanolamine, ephedrine hydrochloride, puphyrine, guaifuenesin, diphenyldramine salt and other histamines, Hl-
Receptor antagonists, Duglophylline, Methoxyphenamine hydrochloride, Rimiterol prefecture salt, Hyosine hydrobromide, Salbutamol sulfate, Ketotifen hydrogen fumarate, and antifungal, bactericidal and antiviral agents.

Oral administration for the treatment of asthma is as described above.

The effects of the formulation according to the invention include (a) treatment of bronchial asthma;
(b) Treatment of bronchoconstriction associated with tidal bronchitis; (
C) Focused on the treatment of symptoms associated with histamine release in allergic hypersensitivity, such as hay fever and anaphylaxis.

This formulation can also be administered with other bronchodilators, antihistamines and antiinfectives as mentioned above.

The reason for the effectiveness of these substances in the treatment of asthma and other allergic reactions is that they contain various endogenous substances that can be released into the local or systemic circulation, such as acetylcholine, histamine, 5- and droxytriftamine, prazikinin. and its ability to prevent or suppress smooth muscle spasm caused by glostaglandin. The superior efficacy of the formulations of the present invention in the treatment of bronchoconstriction is due to the ability to stabilize smooth muscle cell membranes and directly suppress or reduce the reactivity of cell membranes to biogenic amines, glosstaglandins and poly(peptides), or to stabilize smooth muscle cell membranes and directly suppress or reduce the reactivity of cell membranes to biogenic amines, glosstaglandins and poly(peptides), or This may be the result of an indirect effect by inhibiting the action of these substances on the nerves and/or on the mucus-secreting cells.

The formulation according to the invention blocks the smooth muscle contractions caused by acetylcholine, 5-hydroxytryptamine, histamine, glosstaglandin E2 and pradikinin. In fact, we have found it useful to conduct such antispasmodic tests on certain formulations to determine their effectiveness in treating bronchial diseases.

EXAMPLES Next, the extraction of the preparation according to the present invention from Nasushirokiku and the effects of such a preparation will be explained in detail based on the drawings and examples.

The figure shows the extraction and isolation procedure starting from a light oil extract of Nashiriki.

In the drawings, l"PTLCJ stands for r preparative thin layer chromatography", numbers, e.g. 1 to 8, represent combined fractions, and new compounds or mixtures are designated by the following codes: DJ 156a - the formula ( 3chi's Paltrid, DJ
177b - chrysanthemolide of formula (4) above, DJ
140a - Chrysantemonine of formula (5) above, D
J 140a H2-Compound of formula (6) above, DJ
61m-long chain fatty acid ester, DJ 192a-/4'ruthenolide and unidentified terpene, DJ 124m-mixture of fatty acid esters, DJ
179al - Possibly a mixture of reynosine, chrysalthymine A and the new sesquiterben lactone dihydroleinosine, DJ 179cm A complex sesquiterpene lactone containing α-methylene-γ-lactone.

1. General Description A. Chromatography (,) Adsorbent Silica Dal is an adsorbent used in all chromatography fc. The silica dal used for column chromatography was Silica Dull 60 (Merck), and the silica 9 for thin layer chromatography (-(TLC) was Silica Dull (Merck) and Silica Dull GF254 (Merck). Conventional method. TLC plates for analysis and preparation were Silicadal G15g and Silicadal CF25415.
It was produced by mixing N with 60rrLt of distilled water. The slurry was applied in a 0.25 m thick layer onto a 20 x 20 crn glass plate. Plates were air dried and activated at 105°C for 1 hour. Silica Dal G3
A plate impregnated with silver nitrate was prepared by mixing 0 g with 60 g of a 10 mm solution of silver nitrate in distilled water.

The slurry was applied to the layers as described above.

(b) Solvents used for elution of solvent-based columns and development of TLC plates are described in appropriate sections.

(c) Detection of components on TLC plate Detection of components requires short-wave ultraviolet light (254 nm) and 20V in distilled water.
/v sulfuric acid followed by heating at 105°C for 10 minutes was used.

B, Infrared edge spectra obtained using a Unicam SP 200 spectrometer on solid films, Nujorumul, chloroform or KBr disks.

C1 ultraviolet spectrum
In-Elmer Lambda) 3 UV/VIS spectroscopy was taken in ethanol for spectroscopic analysis with a path length of 1 inch using a UV/VIS spectrometer 1.

D, melting point Measured with an electrothermal melting point measuring device and is not corrected.

E, H-1 nuclear magnetic resonance spectra in deuterochloroform using tetramethylsilane (TMS) as an internal standard.
r) WH400 spectrometer at 400MHz or Nl colet NT 200 spectrometer at 200MHz
Recorded in MHz.

F, C-13 nuclear magnetic resonance spectra were purified in Bruker (Bruker) in deuterochloroform using tetramethylsilane (TMS) as an internal standard.
er) using a Vl/H400 spectrometer, 100.6 M
Recorded in Hz.

1H and 15C resonances are indicated by δ (TMS δ 0.0), using the following abbreviations: =Il, -heavycycle; d, doublet: t1
Triple ring; q, quadruple ring; m, multiple ring and J1 coupling constant.

G, quality analysis spectrum 18 or 70 eV at direct temperature of 180-240℃
and recorded on an AEIMS902 high-resolution mass spectrometer or a VG Micromass 168 spectrometer.

In January 1980, 5.8 kg of Natsushirogiku leaves were transferred to the Royal Hospital Road Chelsea Physic Garden, London SW3.
Garden, Royal Ho5pital Ro
ad) and collected from Chemical Laboratory Instruments (Chemical Laboratory Instruments).
Lyophilization was performed using a Lnatruments SB4 freeze dryer. A certified specimen of the dry aerial part of the plant was placed in the museum at Chelsea University. The freeze-dried leaves lost 82.8911 when dried, resulting in a final weight of 1 kg. This was ground and thoroughly extracted in a Soxhlet apparatus with light oil having a boiling point of 40-60°C. The extract was evaporated to dryness under reduced pressure using a rotary evaporator to obtain a yellow oily residue of 44.9%.

3. Antispasmodic effect of light oil extract 50m9 of light oil extract was used as agonists such as acetylcholine (Ach) and 5-hydroxytributamine (
5-HT) and histamine were used to test the antispasmodic effect.

A guinea pig weighing 200 to 300 g was killed by hitting the head. The proximal ileum was removed, the length of 2-3 cm was cleaned, and Krebs solution (NaCt118.4, K
Cl4.7, CaCl22.5, MgSO41,2,
KHPOl, 2, NaHCOs 25 and glucose 1
IIWffi in 1.54 mm/-e), and 029 in this
A mixture of 5q6 and 5% Co2 was blown in at a constant flow rate.

The ileum was set up to record longitudinal contractions at equal lengths.

Log (dose k) one response song #'f acetylcholine, 5
- for hydroxytryptamine and histamine, from which ED5o doses were determined and repeated doses were given until a constant response was reached.

The antagonist, a light oil extract, was then dissolved in a small amount of dimethyl sulfoxide and dissolved in Krebs' solution at 10-4, j9/
It was made into W degree of ml. Add this to the bath containing the ileum and
I left it for a minute. After thorough washing with Krebs' solution to remove residual antagonist, the response of the tissue to the agonist was recorded and the rate of change was calculated as +ii1'.

A control experiment was performed in exactly the same manner except that the antagonist was omitted. (This experiment is important because leaving untreated tissue for 30 minutes often increases the sensitivity of the tissue to exogenous agonists). Light oil extract is 10 to 3 yuzu agonist
It showed 0chi suppression.

4. Crude separation of light oil extract 125 g of light oil extract was collected and subjected to a silica del column (5
The degree of separation was investigated in terms of components present and large-scale testing. The Yairot test was found to be satisfactory and 40.9 kg of the light oil extract was loaded into a column containing 1.2 kg of silica dal made into a slurry by mixing with negative oil having a boiling point of 40-60°C. 200 ml fractions were collected. The fractions were examined by TLC and the appropriate ones were combined (see drawing). The solvent elution of the column and the overlay of the combined fractions are shown in Tables 1 and 2.

Table 1 Solvent elution of column chromatography of light oil extract 1-24 Light oil with boiling point 40-60°C 25-36 Ethyl acetate in light oil 1.0 circulation v
/v37-50 25 嬬v/v51-65 5.
0%v/V66-95
7.5 superior v/v96-112
10.0 section v/V113-118
25.0% v/v119-166
” 50.0 嬬v/v16
7-174 75.0%
v/v 175-180 Ethyl Acetate 181-185 Chloroform in Ethyl Acetate 1.0% v/v 186-188
5.0 section v/v189-192
10.0 cycles v/v193
-195' 25.
0% v/v '196-201 Chloroform in ethyl acetate 50.0 v/V2O2-217 Chloroform 218-237 Methanol in chloroform
Section 10.0 v/V Table 2 From column chromatography of light oil extract 1-
8 2.995 7.59
- 26 2.692 6.
727-50 0.380 - 1.051
- 61 5.850 13
．． 7162-70 1.170 2.9
71-74 0.230 0.675-
80 0.700 1.881-84
1.800 4.585-86
0.764 1.91 87-92 2
．． 380 6.0g3-101 1.
520 3.8102-111
1.878 No. 7112-11B
1.409 3.511g-123
1,7434,4 124-1437,62519,1 144-1762,0595,2 177-2000,7962,0 201-2281,6464,1 The combined fractions from the antispasmodic light oil extract were added to the It was tested for antispasmodic activity as described in section. The results are shown in Table 3.

6. Extraction boiling point of Naushirokiku using chloroform 4
950 g of plant material previously extracted with light oil at 0-60° C. was thoroughly extracted with chloroform in a Soxhlet apparatus for 9 days. The extract was evaporated under reduced pressure using a rotary evaporator to obtain 469 as a green oily residue.

7. Antispasmodic action of chloroform extract Chloroform extract 50n+9 was tested for antispasmodic action as described above (see part 3). One of the three yuzu agonists
00 ratio suppression was obtained.

8. Crude separation of chloroform extract 4.5 minutes of chloroform extract was transferred to a silica column column (12
The degree of separation was investigated in terms of components present and large-scale testing by chromatography. 100ml fractions were collected and combined as appropriate for TLC. The solvent elution of the column and the weight of the combined fractions are shown in Tables 4 and 5.

Table 3 1-8 0 0 09-26
0 0 027-50
20 31 3351-6
1 89 100 9662-7
0 26 32 2671-74
55 87 5075-80
91 100 10081-
84 0 0 085-8
6 12 27 2687-92
5 42 3993-101
60 86 79102-111
58 62 66112-118
20 42 35119-1
23 84 97 97124-
143 91 Zoo 10
0144-176 100 100
100177-200 81 10
0 100 Table 4 Number of solvent elution fractions in column chromatography of chloroform extract Solvent 1-12
Ethyl acetate 50.0% M/V13-16 in light oil
Ethyl acetate in light oil 75.0 v/v
l 7-27 Ethyl acetate 28-32 Chloroform in ethyl acetate 1
0.0% v/v 33-36 Chloroform in ethyl acetate 20.0% v/v 37-40
Chloroform in diethyl acid 50.01v/v4
1-45 Chloroform 46-54 Methanol in chloroform 10.0 V/V 55-62 Methanol in chloroform 25.0 V/V 63-68
Less than the weight of the combined fractions of methanol in chloroform 50.0 v/v Table 5 - Margin 1-2 0.429 9.
53-4 0.417 9.
35-6 0.171 3.
87-8 0.097 2.2
'1-12 0.201 4.
513-18 0.362
8.119-22 0.273
6.123-25 0.410
9.226-39 0.264
5.940-54 0.94
0 20.94.369 97
．． The combined fractions from the chloroform extracts were tested for antispasmodic activity as described in part 3 above.

The results are shown in Table 6. All fractions showed 100% antagonism towards the agonist of 3Si. Therefore, the pharmacological effects were not investigated further. However, the extract was fractionated and new compounds were isolated in the hope of aiding in elucidating the structure of active substances from light oil extracts.

Table 6 1-2 9 0 263-4 4
6' 50 1005-6 91 9
4 937-8 60 65 7
89-12 52 54 6313-18
22 33 8019-22 0
0 023-25 0 0
026-39 43 32 1640
-54 48 47 3510, Valaxicon 144 from light oil extract to fractions 144 to 176 from light oil extract are three agonists Ach,
10-'j;l/- for 5-HT and histamine
showed 100% inhibition.

A. Separation of fractions 144-176 (A) Chromatography of these fractions (1,959,9) on a silica dull column (40 g) L7'c. 100
- fractions were collected and combined as appropriate for TLC. The solvent elution of the column and the weight of the combined fractions are shown in Tables 7 and 8.

Table 7 @ Fractions 144-176 (A) from purchased oil extract
Column chromatography solvent A l 7
Hexane A 8-25 Ethyl acetate 10% in hexane
v/vA26-50 Ethyl acetate in hexane 20% v/vA51-65 Ethyl acetate in hexane 40% v/vA66-78
Ethyl acetate 60% v/vA in hexane
79-85 Ethyl acetate A86 Chloroform Table 8 Fractions 144-176 from light oil extract (A)
Column chromatography combined A I-70,0
221, I A 8-9 0.014 0.7AIO-1
80,0170,9 A19-24 0.048 2.5A25-
29 0.096 4.9A30-34
0.211 10.8A35-36 0.
146 7.5A37-38 0.102
5.2A39-46 0.262 13
．． 4A47-52 0.177 9.0
A53-58 0・218 11.1A
59-65 0.267 13.6A6 low 67 0.104 5.3A68
-72 0.125 6.4B, Antispasmodic Effect of Horns 144-176 (A) 50 ml larger fractions were tested for antispasmodic activity as described in Part 3 above. The results are shown in Table 9.

Table 9 Fractions 144 to A25-29 from salmon oil extract
77 100 87A30-34
60 48 55A35-36
94 100 100A37-38
97 98 97A39-46
83 93 92A47~5
2 100 100 100
A53-58 100 1.00
100A59-65 100
100 100A6 low 67
90 100 100A
68-72 87 90
92A73-85 75
80 70 From Table 9, it can be seen that the extract from L. occidentalis according to the invention, obtained by extraction with a non-contact solvent, has a particularly high antispasmodic effect, which may be useful in the treatment of migraines. Therefore, the 3-glaze agonist Ac
We believe that the fraction that suppresses all of h, 5-HT and histamine by a factor of 100 would be at least stimulatory for migraine. These fractions may also be useful as anti-inflammatory agents and in the treatment of arthritis and bronchial diseases. All of these fractions contain sesquiterpene lactones, as shown below.

Further study of the fraction showing inhibition of 100q6 against the agonist of C. 3. Tested.

Fractions A59-65 were separated by preparative TLC. 204~ on a 20x20m TLC plate (
It was placed on 4 sheets (thickness: 0.25 so) and developed with chloroform:methanol (98:2).

There were 8 bread P under ultraviolet light (254nrn). The components of each band were extracted with chloroform in a conventional manner.

The main component of fractions A59-65, i.e., Chihand 6, was crystallized as fine needle-like crystals (26 ny) of DJ 140 m in chloroform and methanol.

6′α-ofctoyl”-kaα-(3a-hydroxy(l
og ε) 210 (4,01) nm SIR rainbow solid 7
471ax 3470 (hydroxyl), 1760 (ゝC=O1/r-lactone), 1715 (]c-=o, ester),
1.07 (3H, d, J=7.0 Hz, H-5a
a or 6as).

1.21 (3H, d, J=7.0 Hz, H-6
aa or 5as).

132 (3H+ s r H-151ri + 133
(3H+ a +H-7as) +1, 43 (3
B, 5lH-15'8), 1.44 (3H,s,
H-15't+) 11, 59 (3H, a, H-14s
), 1.97 (1B, dd, J = 8.0.2
．． 0 Hz, H-9'), 2.03 (IH, m, H
-9.

2, 18 (IH, d, J=10.0 Hz, H-5)
, 2.38 (IH.

d, J=9.5 Hz, H-5//), 2.42
(IH, d, J=11.5Hz, H-5')1
2.5 8 (IHlml H-31) 1 2.8
2 (IH, dd, J = 9.0, 9.0 Hz,
H-7'), 2.89 (IH, dd, J = 9
．． 5, 9.5 Hz, H-7'), 2.99 (
IH.

Snake, H-1, 3.08 (IH, Megumi, H-7)
, 3.36 (IH, see.

H-1), 3.99 (IH, m, H=1'),
4.01 (IH, dd.

J=11.5, 9.0 Hz, H-6'), 4.
09 (IH, dd.

J = 9.5, 9.5 Hz, H-6,
4.1 3 (IH, dd, J = 10.0,
10.0 Hz, H-6), 4.78 (IH, d
, J=2.0 Hz, H-14'), 4.81
(IH, d, J = 2.0 Hz.

H-14'), 5.09 (IH, d, J = 2.OH
z, H-14').

5.1 2 (IH, d, J = 2.0 Hz, H-
14, 5.19 (IH.

m, H-8') + 5.33 (IHld, J=3
．． 5 Hz, H-13 &) 15, 36 (IH, 2, H-8, 5.39 (IH, d, J=3
．． 5Hz + H-13〃a) + 5.94 (2
H + shi H-8a) + 6-05 (IH, dd, J
=5.5, 1.0 Hz, H-13'a), 6
．． 07 (IH, d, J = 3.5 Hz, H-13
b), 6.09 (IH, dd.

J = 5.5, 1. OHz, H-13'b), 6
．． 13 (IH, d, J= 3.5 Hz, H-
43'b); MS: m/z (%+ r e
l −+ n t , ) +381 (1,7), 367
(2,0), 273(2,1).

261 (1,7), 259 (1,9), 247 (2
, 4).

246 (8,1), 245 (4,7), 243 (11,
0).

229 (13,0), 228 (39,0), 226 (1
4,2), 213 (12,2), 202 (6,3).

201 (10,7), 200 (10,2), 199 (6
, 0), 198 (5, 2), 197 (5, 7).

185 (7,0), 183 (9,1), 173 (6,4
).

169 (7,1), 167 (7,9), 157 (8,6
).

156 (8,2), 97 (34,5), 95 (15,2
).

94 (100,0), 83 (33,1), 71 (19,
8), 43(11,3).

(1) Hydrolysis of DJ 140m Dissolve 52 Da of DJ 140a (combined with mother liquor) in sooxane (5 Go) and add 2CO3 (i.e. 5 Go) to the stoichiometric amount.
- 245 µt of a solution containing 500 Isulphin) was added.

The reaction was monitored by TLC. While gradually increasing the temperature,
Seven days after adding 6≦more bases, only the starting material could be detected. A 2% solution of KOH in H2O was added and the solution was refluxed for 30 minutes. After post-treatment in a conventional manner, the mixture was used as a developing solvent in CHCl3:M.0H (iL9:2).
Separated by preparative TLC using. Ultraviolet light (2
More than 20 bands appeared by irradiating the plate with 54 am), spraying the edge of the plate with 20% v/v healthy acid, and heating.

The main component present in band 2 was extracted with CHCl.

DJ 140a (ie the starting material) is present in Band 3. This was extracted, hydrolyzed again using 2% w/v KOH solution and worked up as before. Extracts from this organic solvent soluble portion and the original hydrolysis band 2 were identical on TLC. The mixtures were combined and purified by TLC using CHCL3:MeOH (98:2) as the developing solvent. Deployed Great three times. By spraying sulfuric acid on the edges of the plate as before and heating it, the main component appeared in Siband 2.

Using this component as a developing bath agent, CHCl5: MeOH (
Further purification was performed by TLC using 98:2).

After extraction in CHCl, the material was extracted with CHCl and hexane as colorless plate crystals (5 m9) of DJ 140aH2. Epoxy-4α-hydroxydarmacle-11taH melting point 159°C (min #); UV: λ (log ε); 2
09(3,99)nm; IR: v solid 74
″ax 3′ 500 (OH), 1760 (c=o, r
- Rough 1, 32 (3H1 view, H-15m), 1.
40 (3H, le H-15's).

1, 48 (IH, Megumi, H-8), 1.55 (3
H, Le H-14g).

1,77 (IH,m, H-9'), 2.06 (
IH, LeH-9').

2, 23 (IH, ShiH-8), 2.30 (IH,
d, J=14.5Hz), 2.44 (IH,m,
H-7'), 2.46 (IH, d, J = 11
．． 5 Hz, H-5'), 2.59 (IH, dd
, J=14.5.

5.0 Hz), 2.68 (IH, d, J = 9
．． 5 Hz, H-5).

3.01 (If(, shiH-8'), 3.20 (I
H, m, H-7).

3, 35 (IH, s, H-1), 3.92 (I
H, Dan, J=11.5°8, 0 Hz, H-6'),
4.03 (IH, m, H-1'), 4.1'
3 (IH, dd, J = 9.5, 9.5 Hz,
H-6), 4.91 (IH.

d, J = 1.5 Hz, H-14'), 5
．． 15 (IH, d, J=1, 5 Hz, H-14
), 5.35 (IH, d, J = 3.5 Hz
.

H-13&), 5.89 (IH, dd, J = 5
．． 5, 0.7 Hz.

H-13'a), 6.07 (IH, dd, J
=5.5, 0.7 Hz.

H-13'b), 6.09 (IH, d, J =
3.5 Hz, H-13b); 13CNMR: δ(1
00.6MHz, CDCLs), 15.10.

18.69 and 29.10 (C-14, C-15 and C
-15'), 23.40, 33.35, 34.
64.

38.09.38.77.65.04 and 66.08 (
C-2, C-2', C-3, 0-3', C-8゜0-9
and C-9'), 43.24 and 44.31 (C-7 and C-7'), 50.72 (C-8').

55.30, 61.39 and 73.27 (C-4°C-
10 and C-4'), 50.86, 62.90°65.
66 and 68.88 (C-1, C-1', C-5 and C
-5'), 75.35 and 79.95 (C-6 and C-
6'), 118.00 and 118.64 (C-13 and C-13'), 135.69 (C-14').

137.92 (C-10'), 140.98 (C-11
).

170.60 and 178.64 (C-12 and C-12
').

C-11' not detected MS: m/z (%,
relative strength).

430(1,1), 356(1,3), 342(1,3
).

281 (2,7), 281 (1,0), 280 (4
,3).

279 (12,9), 266 (1,4); 265 (7,
1).

261 (1,3), 247 (2,7), 2
28(5,0).

207 (8,9), 167 (15,4), 150 (10
, 5).

149 (100), 113 (5,3), 99 (6,1)
.

98 (5,4), 97 (6,4), 94 (9,7).

87 (5,5), 85.1 (11,1), 85.0 (3
0,6).

83.1 (7,8), 83.0 (46,0), 71 (
18,4).

70 (10,5), 69 (15,2), 60 (5,6)
.

57 (50,7), 56 (16,9), 55 (22,9
); Accurate mass measurement: Actual trace j value: 265.1449:C
45H2,04 requires 265.1440; Actual value: 279. :l 241 : C15H4,05
requires 279.1232.

(b) Fraction A33-58 and A6 low 67
Add two sets of fractions to 0.236. ＠), chromatography was performed on a silica column column (10 g).

100- fractions were collected and combined as appropriate for TLC. The 11 elutions of the column and the weights of the combined fractions are shown in Tables 10 and 11.

Table 10 Fractions A33-58 and A6 from light oil extract
Number of solvent elution fractions for row 67 column chromatography Solvent Aa 1-19 Noriform 2.5% in hexane V/VAa 20-22 Chloroform in hexane ssv/vAa 23-30 Chloroform 10% in hexane vZvAa 31-63
Glue oo form in hexane 20qbv/vAa
64-98 Chloroform 30% in hexane
v/v Table 11 Fractions A33-58 and A from light oil extract
Number of charge fractions of the combined fractions of 6-row 67 column chromatography Weight Total weight Aa 1-
46 0.007 3.0Aa47-49
0.019 8. IAa50-63
0.039 16.5Aa64-69 0
．． 048 20.3Aa70-76 0.0
30 12.7Aa77-93 0.056
23.7Aa94-98 0.028
11.90.227 96.2 Fraction Aa70-76 contains only one main component, which is crystallized from chloroform and methanol;
DJ 140m 14m9, identical to that obtained from fraction A39-65 above, was obtained.

Chloroform:methanol (98:2) as developing solvent
Further fraction Aa was obtained by preparative TLC using
50 63 (0,039,S') were isolated. Three bands appeared under UV light (254 nm) and these components were extracted with chloroform.

Band 2 fraction Aa 50-63 main component C1
81ψ available, DJ 17 from chloroform and hexane
7b crystallized as colorless needle-like crystals (8~), melting point 116-117°C (decomposition); IR rainbow solid 74''''',
3540 (OH), 176°Tn&X (/”=Ol r-lactone), C=C/X in 1665
.

1,73 (2H,m, H-9a) 11.98 (
2H1ml H-8s)+2,18 (2H, shiH-2
a), 2.31 (IH, and J=13.5, 11
．． 0 Hz, H-7), 2.81 and 3.97 (I
H each, br 11g, C-3 and C-50dama)
, 4.41 (IH, m, H-3), 4.88
(IH, s, H-15m), 5.21 (IH, a,
H-15b), 5.23 (IH, d, J=-1
1.0Hz.

H-6), 5.450H,a, J=3, s Hz,
n-x3m)+6,18 (IH, d, J = 3.
5 Hz, H-13b): MS: m/z (
91! relative intensity), 280 (2,0), 279 (2,6
), 278 (0,2), 267 (3,4).

266(5,5), 265. (16,8), 264(4
0,8) , 263 (17,9) , 262 (6
,2).

258 (4,9), 260 (8,0), 2
58(4,9).

230 (22,0), 226 (10,5), 223 (7
32), 222 (18,2), 220 (10,7).

180 (18,6), 179 (11,9), 169 (1
0,9), 166 (13,2), 165 (18,0).

164 (14,7), 163 (14,6), 161 (1
0,4), 157 (20,4), 155 (14,1).

153 (10,7), 152 (19,2), 151 (2
1,9), 150 (24,7), 143 (24,0).

137 (32,9), 135 (21,1), 129 (2
0,1), 125 (25,8), 124 (30,1)
.

123 (37,4), 111 (43,4), 110 (4
5,4), 109 (46,6), 101(5
4,0).

97 (68,1), 95 (74,3), 85 (58,9
).

84 (65,7), 83 (100,0), 81 (64,
0).

69 (68,7); Accurate mass measurement: Actual value 278,1
150: C45H1805 is 278.1154 8
Meng.

(i) Acetylation of DJ 177b DJ 177b
4ny was acetylated using birinone and acetic anhydride in a conventional manner. After working up in the usual manner, spots for the starting material and less polar products were present in the TLC test. Although the material is still a little too early to be completely purified, 'H NMR spectrum of the crude product showed the following additional signals indicative of the acetate product: δ, CDC4
3: 2.16 (3H.

, CH, Co2-) and 4.60 (IH, Megumi,
H-3).

(c) Fraction A47-52 Fractions 47-52 (0,123 g) were chromatographed on a silica column column (11'). 10
0rnt fractions were collected and combined as appropriate for TLC. The solvent elution of the column and the weight of the combined fractions are shown in Tables 12 and 13.

Chloroform:methanol (98:2) as developing solvent
Fraction Ab34- by preparative TLC using
56 was further isolated. Four bands appeared under ultraviolet light (254 nm), and the components of Nanagi et al. were extracted with chloroform. The main component, i.e. the component contained in band 3, is 201%
This was crystallized using chloroform and n-4 ester. This material was a mixture of sesquidelben lactones and survived all f# preparation procedures. Therefore, this material was not tested further.
Table 2 Solvent elution of column chromatography of fraction A47-52 from light oil extract Ab 1-8 Chloroform i in hexane 0
% V/VAb 9-20 Glue O in hexane
CI Form 2Q%v/vAb21-56 Glue in hexane oo Form 25fDv/vAb57
Chloroform Table 13 Fraction from Light Oil Extract A47-520 Column Chromatography Combined Fraction Weight Ab 1-19 0.015 12.2A
b20-23 0.004 3.3Ab
24-33 0.039 31.7Ab
34-56 0.053 43.0Ab5
7 0.007 5.70.118
95.9 The component in band 1 (11m9) was further purified with preparative Ti and C. Chloroform:methanol (
98:2) for 7 times.

Four bands appeared under ultraviolet light (254% m).

These components were extracted with chloroform. The component contained in Band 1 of the latter plate was 6■. This was crystallized from chloroform and n-pentane and DJ 17
9ml was obtained. This material is probably a mixture of sesquiterpene lactones, although it shows only one spot in the TLC test.

The signals seen in the 'HNMR spectrum indicate that it is reynosine, chrysaldemine B and possibly a dihydro derivative of reynosine.

Due to the lack of material, no further testing was done at this stage, but the possibility that the material is a trimer rather than DJ 140a cannot be ignored.

11. 7-lactones 124-143 from light oil extracts
Study (B) Fractions 124-143 from the light oil extract showed 100% inhibition against the two agonists 5-HT and histamine and 91% inhibition against Ach.

A, re-separation of fractions 124-143 (B) These fractions (7,525 g) were separated on a silica gel column (
150 g).

100 mA' fractions were collected and combined as appropriate for TLC. The column solvent elution and combined fraction weights are shown in Tables 14 and 15.

Table 14 Fractions 124-143 (B) from light oil extract
Solvent elution of column chromatography B1-27
Hexane 828-45 Chloroform i% in hexane,
7vB 46-51 Chloroform in hexane
2%φB 52-60 Chloroform in hexane 5%φB 61-67 Chloroform in hexane 10%v/vB 68-80 Chloroform in hexane 20%V/VB 81-136 Chloroform in hexane 40%v/vB137-149
Glue oo form 60% v/v B150-1 in hexane
70 Chloroform in hexane Table 13 ~ B17
l-194 Chloroform B195-203 Chloroform 9 in methanol
0% Sil Table 15 Fractions 124-143 (B) from light oil extract
Weight of the combined fractions of column chromatography Fraction number Weight (g) Total -B
1-9 0.011 0. IB 1
0-90 0.040 0.5B 91
-106 0.025 0.3B107
-111 2.484 33.0B11
2-115 1.537 20.4B1
16-122 0.490 6.5B1
23-130 0.316 4.
2B131-147 0.252
3.3B148-163 0.454
6.0B164-171 0.20
0 2.6.

B172-196 0.810 1
0.8B197-198 0.682
9.1B199 0.07
8 1.0B200
0.083 1. lB20l-204
0,0480,6 7,51099,6 B, antispasmodic action of fraction 124-143 (B) 50
~The more desired fractions were tested for antispasmodic activity as described in section 3 above. The results are shown in Table 16.

Table 16 Fractions 124-143 (B) from light oil extract
Antispasmodic effect, tested at IQ/17ful, suppression of υt margin fraction -B107
-111 100 100 100B112
-115 100 100 100B116
-122 95 100 100B123
-130 82 100 98B131
-147 78 100 100B14
8-163 51 88 97B16
4-171 22 29 9081
72-196 3 44 100B1
97-198 16 58 33B1
99 31 72 65B20
0 27 42 20C, Fraction Testing Fractions Showing 100 H Inhibition Against Three Agonists Fraction B107-111 showed 100 H inhibition against all three agonists. Fraction (2,4
34 g) was chromatographed on a silica gel column (120 g'). 100 d fractions were collected and combined as appropriate for TLC.

The solvent elution of the column and the weight of the combined fractions are
It is shown in Table 7 and Table 18.

Table 17 Fractions from Light Oil Extract B107-1110 Column Chromatography Solvent Elution Ba 1-5 Chloroform in Hexane 1
0% Ba 6-11 Chloroform in hexane 20 tB & 12-70 Chloroform in hexane 30 t Ba 7l-73 ri o o *ru A Table 18 Fractions from light oil extract B 107-1110 Column chromatography combined Weight of fraction Ba 1-20 0.112 4.6
Ba21-32 0.465, 19.
1B Maro 33 0.057
2.3Ba34-55 1.4
21 58.4Ba56-58 0.
128 5.3B 59-68 0
．． 130 5.3Ba69-70
0.094 3.9Ba71-73
0.030 1.2 Fluxi Enen B1
The main component of 07-111, that is, Sapflux carbon Ba34
The main component contained in -55 is crystallized from chloroform and hyhexane to obtain /4' ruthenolide (47B++) as colorless plate crystals. Melting point 113 ~ conjugated), 16+o (
:c=c2), 125o(-φ-〇-φ-)am-1;
'HNMR: δ (400MHz, CDCts), 1.3
2 (dH9s.

H-15m), 1.73 (3H, see, H-14g), 2
．． 81 (IHld, J=8.5Hz, H-5),
2.81 (IH, m, H-7), 3.87 (IH, ad
, J=8.5, 8.5Hz, H-6), 5.23(I
H, dd.

J-12,5,3,0Hz, H-1), 5.64 (IH
, d, J = 3.8Hz.

H-13a), 6.35 (IH, d, J = 3.8Hz,
H-13b):”CNMR:δ(100,6MHz
, CDC6ρ, 16.97, 17.24 (C-14
and C-15), 24.11, 30.64 (C-8 and C-3), 36.35, 41.19 (C-2 and C-9
), 47.65 (C-7), 61.45 (C-5)
, 66.36 (C-4), 82.38 (C-6), 12
1.08 (C-1), 125.24 (C-13).

134.51 (C-10), 139.21 (C-11)
, C-12 not detected: MS: m/z (%, r
el, int, ), 248(1,5).

233 (1,1), 230 (8,1), 190 (9,3
), 107(8,4).

105 (12,2), 95 (8,9), 91 (13,6
), 81(12,4).

79 (11,7), 77 (9,2), 67 (9,6),
58 (24,8).

55 (12,0), 53 (17,3), 43 (100,
0).

This material was identical in all respects to the authentic sample supplied by Sorm (F, Sorm).

12, fractions 75-80 from light oil extract (C
) Fraction 75-80 (C) from light oil extract was
100% against two agonists 5-HT and histamine
showed 91% inhibition against ACh.

A, reseparation of fractions 75-80 (C) 0, 659
was chromatographed on a column of silica gel (60,!i'). Collect a fraction of 100m/T
It was approved as suitable for LC.

The weight of the combined fractions and the solvent elution of the column are
It is shown in Table 9 and Table 20.

Solvent Elution of Column Chromatography of Fraction 75-80 (C) from Table 19 Light Oil Extract C1-3 Chloroform in Hexane 5CH04-12
Chloroform in hexane 10% C13-2
9 Chloroform in hexane 20 parts C50-33 Chloroform in hexane 40% bacteria 20 parts Fractions from light oil extract 75-80 Column chromatography of (C) Combined fractions 1 C16-190,0365,5 C20-240,44668,6 C25-260,0446,8 G27-39 0.103 1
5.80.649 99.8 All these subfractions are 10-'gALl
It had no antispasmodic effect at concentrations of , and was therefore not tested further.

Fraction 177-200 from the light oil extract showed 100% inhibition against the two agonists 5-HT and histamine and 81% inhibition against AchIC.

A. Re-separation of fractions 177-200 (D) ~ These fractions (0,729,!i+) were chromatographed on a silica gel column (30,9).

100 mJ fractions were collected and combined as appropriate for TLC. The column solvent elution and the weight of the combined fractions are shown in Tables 21 and 22.

Table 21 Furakujon 177-200 (D) from light oil extract
IJ) Column chromatography solvent elution D 1-12 Ethyl acetate 25%φ in hexane
013-17 Ethyl acetate 40%v in hexane
/vD18-24 Ethyl acetate 6 in hexane
0%φD25-31 Ethyl acetate 80% v/v in hexane D32-36 Ethyl acetate D37-40 Chloroform in ethyl acetate 1
0%φTable 22 Weight of combined 7 fractions of column chromatography of fraction 177-20) from light oil extract D 1-3 0.037 51D 4
-8 0.090 12.3D 9-1
0 0.049 6.7Dll-15
0,08812,1 016-200,24032,9 D21-26 0.160 21.9D
27-45 0.070 9.6 total = 0.734 100.6 B, antispasmodic action of fraction 177-200 (9) 50
The greater than mg fraction was tested for antispasmodic activity as described in section 3 above.

The results are shown in Table 23.

Table 23 Fraction 177-200 (D) from light oil extract
Antispasmodic effect of Ach 5-H
T Histamine DI-4649691 D4-8 81 92
93Dll-1597100100 D16-20 100 100
100D21-26 100 100
100D27-45 47
Test fraction D16-20 (0,175 g), a fraction showing 100% inhibition against the 52 67a0 agonist, was further separated by preparative TLC using chloroform:methanol (95:5) as developing solvent. did. The plate was developed three times. Three bands appeared under UV light (254 mm). These components were extracted with chloroborum. The main component, that is, the component contained in Band 1, is 59■, which crystallizes from chloroform and methanol as a colorless plate crystal (26m9) of DJ 154m (gamma-lactone), 16
75 (:C=C:, conjugate), 1260cmt5-0-φ
-, :r-4# し)' ) am-' : ' HN
MR: δ (400MHz, CDCl2), 1.15 (
3H,s, H=15m), 1.58(3H,s-,H-
14g), 2.87 (IH, d, J = 11.0Hz.

H-5), 3.30 (IH9m+H-7), 3
．． 32 (IH, d, J=1.3H2,H-3),
3.57 (IH, a*J=i, anz+u-zL4.
09 (IH, dd, J = 11.0, 10.0Hz, H
-6), 5.54 (IH, d, J==3.5Hz*
H-13a), 6.19 (IH, d, J=3
．． 5Hz, H-13b);MS:rrN/z(%
, relative intensity) 279 (0,7), 278 (2,1), 2
63 (2,3), 262 (4,7).

260(3,1), 249(3,1), 231(6,3
), 217(8,6).

203 (9,4), 189 (12,5), 175 (15
, 6), 165 (26, 6), 151 (40, 6), 1
49 (23,4), 147 (28,9).

123 (26,6), 121 (33,6), 112 (8
8,3), 109 (42,2), 97 (35,9),
95 (93,8), 91 (41,4).

85 (47,7), 83 (44,5), 81 (36,7
), 79(38,3).

77 (35,9), 71 (78,1), 69 (52,3
), 67(49,2).

57(93,8), 55(71,1), 53(
75,8), 43(100,0).

The melting point in the literature is 250°C.

This material was identical in all respects to the literature data for chrysalthymine A.

(b) Fraction D21-26 Chloroform:methanol (95:5) as developing solvent
Fraction D21- by preparative 'rLC using
26 (0.110 g) was further isolated. The plate was developed three times. Below the ultraviolet edge (254 mm), three /J nodes appeared by spraying the edge of the plate with H2SO4 and heating. These components were extracted with chloroform.

The main component of fraction D21-26, that is, the component contained in band 2, was crystallized from chloroform and hexane to obtain colorless needle crystals (16m9) of DJ 156a (':
::C=O, r-lacto:/), 1660 (two C=C:
, conjugate).

1640 (;C-(,: ), HNMR: δ(4
00MHz, CDC3).

1.25 (3H8s, H-14a) *1.35 (3H+
a+M-15a).

1.47 (LH, m, H-8), 1.62 (,2H, m
, H-2s).

1.67-2.04 (6H,brm+H-3+st-H
-9m+2xOHa).

2.16 (IH, i, H-8), 2.39 (LH, dd
, J=12.3°12.3Hz, H-5), 2.63(
IH, m, H-1), 2.70 (IH.

m, H-7), 4.24 (IH, dd, J=12.
3.9.5Hz, 1(-6).

5.54 (II (, d, J=3.5Hz, H-13m)
, 6.25 (IH, d.

J=3.5Hz, H-13b), CNMR:
δ (100,6MHz.

CDC63), 23.51.24.25 (C-14 and C-15).

25.01.25.33 (C-2 and C-8), 39.
34,43.77 (C-3 and C-9), 47.2
2 (C-7), 49.78 (C-1).

55.35 (C-5), 74.72 (C-4), 79.
88 (C-10).

82.74 (C-6), 120.37 (C-13), 1
38.45 (C-11), 169.37 (C-12):
MS: rrV/z (% relative intensity).

266 (0,4), 265 (0,9), 264 (1,8
), 263(18).

262 (3,6), 261 (2,7), 260 (4,5
), 249(3,0).

248 (6,0), 247 (7,2), 246 (18,
0), 245(6,3).

244 (13,5), 232 (4,5), 231 (15
,3),230(10,0),229(9,0),22
8 (19,8), 215 (12,6).

213 (18,9), 203" (22,5), 191 (
22,5), 190 (27,0), 188 (24,3)
, 175(24,3), 173(225).

159 (24,3), 105 (45,0), 95 (44
, 6), 93 (44, 1).

91 (59,5), 83 (85,6), 81 (51,4
), 79(49,5).

71 (42,3), 69 (47,7), 57 (59,5
), 55(91,9).

43 (100,0): Accurate mass measurement; Actual value: 26
4.1364; C15H2oO4 requires 264.1362.

Fractions 119-123 from the test light oil extract of 5-
Shows 97% inhibition against I (T and histamine, A
It showed inhibition of 84ch against ch.

A, re-separation of fractions 119-12. These fractions (1,5839) were transferred to a silica gel column (30
Chromatography was carried out using a filter.

100m/! 7 lactations were collected and combined as appropriate for TLC. The column solvent elution and combined fraction weights are shown in Tables 24 and 25.

Table 24 Fractions 119-123 from light oil extract (IE
) Number of solvent elution fractions in column chromatography Solvent E1-2
Hexane E3-5 Chloroform in hexane 10 φ
E 6-31 Chloroform 2 in hexane
0fDV/VE32 Chloroform Table 25 Fractions 119-123 (E) from light oil extract
Weight of combined fractions of column chromatography El-13, 0,0100,6 F14 0.007 0.4
゜15 0.058 3.7E
16-18 0.450 28.4
E19-21 0.201 12.
7E22-31 0.148 9
．． 3E32-36 0.251 1
5.91i:37-38 0.064
4.0E39
0.395 25. OB, antispasmodic action of fraction 119-123 (EE) 501n9
A number of these fractions were tested for antispasmodic activity as described in section 3 above. The results are shown in Table 26.

Fraction 119-123 (E) from the 26th light light oil extract
antispasmodic effect, xo-'i/ml test, fraction number inhibition multi-E15
67 97 91E16-18
57 96 89E19-21
85 100 100E22-31
28 100 92E32-36
85 100 98FJ37-38
56 100 100E39
The test fraction E15-21 of the fractions showing 78 100 91C0 agonist inhibition was combined and TL using chloroform:hexane (75:25 tl) as the developing solvent.
Purified with C. Extract the main components and compare them with the authentic specimen by chromatography and spectroscopic analysis! 1l-
It was identified as 4'ruthenolide.

As can be seen from the above, when Oshi and Natsushirogiku are extracted with a non-polar solvent, fractions with high antispasmodic activity are obtained, and sesquiterpene lactones can be obtained from these fractions.

Compounds 140a, 156a and 177b (which we believe are not disclosed in the literature) have the following mobilities in thin layer chromatography:

Compound Solvent Rf14
0a Chloroform/methanoflu (98/2)
0.49-1 development 156a Chloroform/methanol (9515)
0.35-3 times development 177b Chloroform/methanol (98/2)
0.34-Example 1 of manufacturing one-time expansion capsules Mix crushed Natsushirogiku leaves and soybean oil in the following proportions: Crushed Natsushirogiku leaves 10.0 kg Soybean oil 100.0 hours active The mixture was left unstirred for 14 days to extract the components, then lecithin 2.2 kg hydrogenated fat 30.0 hours
Row
It stabilized at 10.0 kg.

The mixture is filled into soft cassisels or hard capsules corresponding to the dosage of dried leaves 25111&. This fills a 7.5 oval soft capsule or a M2 hard capsule of target filling type 1380.5~.

Alternatively, the mixture is expressed and the resulting cold infusion is stabilized with one or more of the suspensions mentioned above and filled as an oily extract into soft or two-piece hard gelatin capsules.

Example 2 Mix crushed Natsushirogiku leaves and cod liver oil in the following proportion: Natsushirogiku leaves 10.0 to 9 liver oil
The mixture is left for 14 days to extract the active ingredient.

The resulting mixture is filtered and the p-liquid is filled into soft capsules or two-piece hard capsules with a fill weight of 250 m9 per unit dose, corresponding to a dry aggregate of 25 cm.

Alternatively, the suspension or cold soak liquid is mixed with a surfactant or mixture thereof, such as polyethoxy fatty acid sorbitan esters, mono- and di-glycerides, acetylibiglycerides, to create a self-emulsifying system.

The resulting formulation is filled into soft capsules or two-piece hard capsules.

It is assumed that the mixtures and formulations improve the absorption of oily active particles via the phosphorus system.

Example 3 Mix the following ingredients: Cold infusion of dried Natsushirogiku leaves (as above) 100
．． 0 to 9 polyethoxy sorbitan monooleate
37.0ki9 Mixture of mono- and di-glycerides
29.0 to 9 These ingredients are mixed and filled into soft capsules or two-piece hard capsules corresponding to dry Natsushirogiku Aggregate 251n9 at a dosage fill volume of 415 ml.

The soft or two-piece hard gelatin capsules can be used orally or rectally.

These capsules may be treated with gastric resistant coatings, such as hydroxymethylzorobylcellulose, to delay disintegration or absorption, or the contents may be mixed with polymeric matrix materials known to the pharmaceutical industry to achieve controlled rates. can release the active ingredient.

Margin below

[Brief explanation of the drawing]

The figure is a flow sheet showing the operation for extracting and isolating the active ingredient from Natsushirogiku. Patent applicant: Nidoward Stewalt Johnson (2 others) Patent application agent: Akira Aoki, patent attorney Kazuyuki Nishidate, patent attorney (1) Yukio, patent attorney Akira Yamaguchi 493104 7252-4
C493/14 7252-
4 C-493/22 72
52-4 C//CC07D 493104 303100 7043-4
C307100) 7043
-4C (C07D 493/14 303100 7043-4
C307100) 7043
-4C (C07D 493/22 303100 7043-4
C307100) 704
3-4C Priority Claim 0 September 22, 1982 ■ United Kingdom (GB) C8227062 0 Inventor Peter John Highlands Berkshire Nisel 66 Kiwell Ticket Maidranhead Staubings Nistit The Co 0 Inventor Deborah Margaret Hylands United Kingdom Berkshire Nissel 66 Kwell Ticket Maidranhead Staubings Nistit the Cottagues 1 Applicant Peter John Hylands United Kingdom Berkshire Nissel 66 Kwell Ticket・Maidranhead Stowinges Estate 1 ■Applicant Deborah Margaret Highlands United Kingdom Berkshire Nisel 616 Kiwell Ticket Maidranhead Stow 5 Bings Nistit The Co.

Claims (1)

[Claims] 1. Tanacetum parth
A sesquiterpene lactone-containing extract for pharmaceutical use, which is an extract recovered from P. enlum by extraction with a non-polar solvent. 2. The extract according to claim 1, which contains a plurality of sesquiterpene lactones. 3. The extract according to claim 1, wherein the nonpolar solvent is light oil, hexane, chloroform, or oil. 4. The extract according to claim 1, 2 or 3, wherein the sesquiterpene lactone has an α-methylene substituent on the lactone ring. 5. The extract according to any one of claims 1 to 4, wherein the sesquiterpene lactone is dharmafuranolide. 6. The extract according to claim 5, wherein darumafuranolide'' (a) is 74 ruthenolide of formula '', or (b) has formula (5) 6a. 7. Sesquiterpene lactone is The extract according to any one of claims 1 to 4, which is guayanolide. 8. Guayanolide (,) is chrysalthymine A of formula (A): or (b) 75E formula (3
)'. 9. A pharmaceutical sesquiterpene lactone preparation containing darmafuranolide containing at least two rlumacrane nuclei in the molecule. 10. A sesquiterpene lactone formulation according to claim 9, wherein the lumacranolide has the formula (5): U or is a pharmaceutically acceptable derivative of a compound of formula (5) or (6). 11. Pharmaceutical sesquiterpene lactate/preparation comprising guayanolide having an oxygen functional group at position 10 of the guayanan nucleus. 12. Guayanolide has the formula (3)':
or a compound (1) in which the α-methylene group of the compound of formula (3)' is substituted with a vicinal diol, or a compound of formula (3)' or a pharmaceutically acceptable derivative of the above compound (1). or has the guaquino-1-nodoka formula (4): or has an epoxy group and α of the compound of formula (4)
- Compound (ii) in which at least one methylene group is substituted with a vicinal diol, or a compound of formula (4) or a pharmaceutically acceptable derivative of the compound (11) The sesquiterpene lactone preparation according to item 11. 13. The preparation according to any one of claims 9 to 12, wherein the sesquitil (lactone) is recovered by extraction from Nasushirogiku. 14. An excipient that can additionally contain the pharmaceutical vcH. 15. The extract according to any one of claims 1 to 8 or the formulation according to any one of claims 9 to 13, comprising: The extract or preparation according to claim 14, which further contains one or more other known anti-arthritic agents. 16. The extract or preparation according to claim 14, which further contains one or more other known bronchodilators, antihistamines, and anti-infectives. Extract or preparation 1 according to claim 14]. 17. Claims 1 to 16 further include one or more known anti-migraine ingredients, analgesics and/or antiemetics.
Extract or preparation according to any one of paragraphs. 18. Extracts or formulations according to any one of claims 1 to 17, suitable for administration orally, rectally, parenterally or by inhalation. 19. The extract or formulation according to claim 18 in the form of a tablet, capsule or liquid suspension. 20. The extract or preparation according to claim 19, which is in the form of a capsule-filled preparation, which is made by suspending particles of the dried plant Festulae in oil obtained by extracting sesquiterpene lactones from the dried plant. 21. Extract or formulation according to any one of claims @1 to 20 for use in the treatment of migraine conditions. 22, containing chrysaldemin A or parthenolide
- The extract or preparation according to claim 21. 23. The extract or preparation according to any one of claims 1 to 20 for use in the treatment of asthma or bronchial symptoms. 24. The extract or preparation according to any one of items 1 to 20 for use in treating symptoms of arthritis. 25. Claims 1 to 25 for use as an anti-inflammatory agent
Extract or formulation according to any one of paragraph 20. 26. Sesquiterpene lactones including darumafuranolides containing at least two chelmacrane nuclei in the molecule. 27. Claim 26, wherein darmafuranolide has formula (5): or formula (6): or is a pharmaceutically acceptable derivative of said compound of formula (5) or (6).
Sesquiterpene lactones listed in Section. 28. A sesquiterpene lactone containing guayanolide having an oxygen functional group at position 10 of the guayanan nucleus. 29, guayanolide has the formula <31': the following blank space H, or a compound of the formula (3)' in which the α-methylene group is substituted with a vicinal diol, or a compound of the formula (3)
)' or a pharmaceutically acceptable derivative of said compound (i), or the guayanolide is of formula (4): or the epoxy group of the compound of formula (4) and α
- Compound (11) in which at least one methylene group is substituted with vicinal diol, or a compound of formula (4), or a pharmaceutically acceptable derivative of said compound (11), according to claim 27. sesquiterpene lactones. Formula (3)', (4), (
5) or (6) sesquiterpene lactone. 31. Substantially pure claims 26-30
The sesquiterpene lactone according to any one of paragraphs. 32. A substantially pure sesquiterpene lactone for use in the treatment of migraine. 33. Pharmaceutical sesquiterpene lactone-containing extraction comprising treating Natsushirogi with a non-polar solvent, total evaporation of the solvent and chromatography of the resulting crude extract using the non-polar solvent as at least the first eluent. How things are manufactured.