JPS6234734B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a novel therapeutic agent for nephritis. Therefore, the present invention is directed to a compound that is represented by the molecular formula C ₂₃ H ₃₀ O ₆ and contains a hydroxyl group, an α/β-unsaturated carbonyl group, and a carbon-carbon double bond of an aromatic ring as an active ingredient. The present invention relates to a therapeutic agent for nephritis characterized by: The novel compound (substance of the present invention) contained as an active ingredient in the therapeutic agent for nephritis of the present invention has an anti-complement effect, has a therapeutic effect on nephritis, and has the following physical and chemical properties. have (1) Molecular formula ₂₃ H ₃₀ O ₆ (2) Molecular weight 402 (3) Solubility Soluble in methanol, ethanol, acetone, acetic acid ethyl ester, dimethyl sulfoxide, pyridine, acetic acid, ether, and chloroform.
Slightly soluble in benzene, insoluble in water, hexane, and petroleum ether. (4) Constituent elements The constituent elements are carbon, hydrogen, and oxygen, and do not include nitrogen, sulfur, halogen, etc. (5) Elemental analysis values C: 68.58% H: 7.55% Calculated values as C ₂₃ H ₃₀ O ₆ C: 68.64% H: 7.51% (6) Infrared absorption spectrum (IR) The IR analysis chart for KBr tablets is shown in As shown in Figure 1. For example, the main absorption peak is 3400
(S), 2920 (M), 4670 (S), 1610 (S),
1585(S), 1430(M), 1390(S), 1310
(S), 1240 (S), 1195 (M), 1115 (M),
1040(M), 1005(M), 990(M), 960(M),
940 (M), 880 (M), 815 (W), 765 (W),
There are absorptions at 550 (W), 515 (W) cm ^-1, etc., especially at 3400 cm ^-1 (hydroxyl group), 1670 cm ^-1 (α/β-unsaturated carbonyl group), 1610, 1585 cm ^-1 (aromatic ring carbon The presence of a hydroxyl group, an α/β-unsaturated carbonyl group, and an aromatic ring is recognized through the intervening double bonds). (7) Ultraviolet absorption spectrum (UV) The results of UV analysis using ethanol (EtOH) as the solvent, sample concentration of 11.9 mg/640 ml EtOH, and cell length of 1 cm are shown in Figure 2. 246 (ε
= 16474), 307 (ε = 6659) and 350 (ε =
5427) nm, maximum absorption is observed. (8) Color reaction (i) 2,4-dinitrophenylhydrazine reagent, phosphomolybdenum reagent, Torrence reagent,
Shows a positive reaction. Shows negative in ferric chloride reaction. (9) Thin layer chromatography (TLC) Using “Silica Gel 60F ₂₅₄ ” manufactured by Merck & Co.
As a result of TLC analysis, the Rf values were as follows. (i) Ethyl acetate-chloroform-acetic acid (volume ratio 50:50:2) Rf value = 0.34 (ii) Benzene-butanol-acetic acid (volume ratio 60:
15:5) Rf value = 0.58 (iii) Benzene-acetic acid ethyl ester-acetic acid (volume ratio 40:50:2) Rf value = 0.42 Physicochemical properties and other instrumental analysis data of (1) to (9) above, and From the results of the physical and chemical properties of various substances derived from the substance of the present invention, it is estimated that the substance of the present invention is a terpene-based substance having a hydroxyl group, an α/β-unsaturated carbonyl group, and an aromatic ring. In any case, the compound having the above molecular formula is a novel compound that has not been described in any literature, and is identified as a novel terpene derivative. The substance of the present invention can be produced using, for example, a microorganism belonging to the genus Stachybotrys. In the above, the microorganism is not limited as long as it belongs to the genus Stachybotrys, and all known microorganisms and microorganisms newly isolated and obtained by the present inventors can be used. Examples of typical known microorganisms belonging to the genus Stachybotrys include the following. Stachybotrys alternans IFO 9355 Stachybotrys alternans IFO 9355 Stachybotrys chartarum IFO 5369 Stachybotrys chartarum IFO 7222 Stachybotrys cylindrospora 8858 Stachybotrys cylindrospora 8858) Γ Stachybotrys echinata 7525 (Stachybotrys echinata 7525) Γ Stachybotrys reniformis 7067 ( Stachybotrys reniformis 7067) Furthermore, in the present invention, bacteria belonging to the genus Stachybotrys that were newly isolated and obtained by the present inventors as described below can also be advantageously used. The isolated and obtained bacteria are new bacterial species as described below, and the present inventors have identified them as Stachybotrys sp. K-76, T
-789 and T-791. Collection site 1 Stachybotrys sp. K-76 Isolated from soil in Ishitan City, Okinawa Prefecture. 2 Stachybotrys sp. T-789 Isolated from soil in Naruto City, Tokushima Prefecture. 3 Stachybotrys sp. T-791 Isolated from soil in Tokushima City, Tokushima Prefecture. Properties on various culture media 1 Stachybotrys sp. K-76 Culture characteristics of this strain on various media based on macroscopic and microscopic observations (shown in Figures 3 and 4) are as described below. A. Macroscopic Observation It shows extremely good growth on various media commonly used for culturing filamentous fungi, but the settlement of mychospores is not always good, except on oatmeal agar media. The growth conditions in typical media are described below. (1) Malt extract agar medium Growth is relatively slow, and large colonies of 30 to 35 mm in size are formed after 30 days at 27°C. The village grows in a circular shape, with a large jagged edge around the periphery. The surface of the colony is flat, and in the center there are thick, circular pine-like aerial filaments that grow densely, and the color gradually changes from white at the initial stage of cultivation to light ivory (Lt-Ivory, 2ca). From around the second week of culture, the mycospores begin to grow.
Although a small amount is formed, it is hardly visible to the naked eye. No sclerotia or other sexual spore organ tubes are formed. The reverse side is colorless to copper tan (5ie). The diffusible dye is Copper Tan,
5ie). (2) Potato Glucose Agar Medium Growth is good and 45% after culturing at 27℃ for 30 days.
Reaching ~47mm. The village grows in a convex shape at the center, with jagged edges and concentric folds extending from the center of the village. From the early stage of cultivation, it is immediately covered with a thick layer of white to pearl pink (4ca) mycelia, and as the cultivation progresses, the color of the mycelium becomes grayish-black (Gray.i). A large number of mycospore masses are formed. A large amount of droplets can also be seen. The colors on the back are Rust Tan (5le) to Light Rose Brown (6 1/2 lg),
The diffusible dye is Butterscotch.
Scotch, 3ne), but only slightly. (3) Siapetsk agar medium: Good growth, 40~ after 30 days of culture at 27℃
Reaches 45mm. It is folded in concentric circles, and the village has a convex central ridge. The periphery is wavy. The color on the back is Luggage
Tan, 4ne). On the surface of Toast Tan (4lg), pale colorless to white mycelia grow concentrically from the center which is not covered by aerial mycelia. No droplets are observed, but the center is very wet. Almost no inchospores are formed. The diffusible pigment is weak and gold (Gold,
2le). (4) Oatmilk agar medium Growth is extremely rapid, reaching 60-70 mm after 30 days of culture at 27°C. The colony is thin and flat, and colorless to white mycelia are formed on the entire surface from the early stage of culture. Upright mycelia of 3 to 4 mm in size occur only near the center of the colony,
It's not secret. In the second week of culture, epiphytes of mycospores cover the entire village and the color changes to light olive drab (Lt Olive Drab, 1li). The back side is Beige Brown (3ig) to Tan (Tan,
3ie). The diffusible pigment Colonial Yellow (2ca) is faintly produced. 2 Stachybotrys sp. T-789 The characteristics of culturing the T-789 strain on various media based on macroscopic and microscopic observations (shown in Figure 5) are as described below. The morphological characteristics observed are Stachybotrys sp. K
- Exactly matches strain 76. However, there are differences in their properties on the culture medium. The following two types of media, which are the most different, will be described. A. Macroscopic observation (1) Malt extract agar medium Growth is extremely slow after 30 days of culture at 27°C.
17~20mm. The area around the village grows in waves and is localized. The village has a convex bulge in the center and radial folds in the periphery. Aerial hyphae are thin and epiphytic over the entire surface, and spore formation gradually occurs from the white color at the initial stage of culture, taking on a mole (MOle, l) color in the center and growing densely, but spore formation can be seen in a ring toward the periphery. The back side is Cork Tan (4ie). Diffusible pigments are abundant and amber (Amber, 3le). (2) Tuapetsk agar medium Growth is good, 45mm after 30 days of culture at 27℃
Grows in. The village forms radial folds with a convex center, and the periphery shows wave-like growth.
Thin, white aerial mycelium grows concentrically outward from the central convex part, and spore formation occurs concentrically only at the periphery, and the convex part is extremely moist with dissolved hyphae. The back side is mustard brown (Mustard Brown, 2pi). Does not produce diffusible pigments. 3 Stachybotrys sp. T-791 Deuteromystobacterium The characteristics of culturing the T-791 strain on various media based on macroscopic and microscopic observations (shown in FIG. 6) are as follows. A. Macroscopic observation (1) Malt extract agar medium Growth is rapid and irregular, and the color of the underside is tan (3ie) to dark crown (Dk brown, 3pn). The village is flat and sporulation is good. The hyphae exhibit a biscuit (2ec) to tan (3ie) color, and exudate droplets are observed. Does not produce diffusible pigments. (2) Potato Glucose Medium Growth is extremely rapid and cultured at 27℃.
It reaches 70mm in 30 days. At the periphery, thin white mycelia are observed spreading in a tree-like manner, and droplets are also noticeable. There is almost no sporulation. The back side is light amber (Lt
Amber, 3ie). No diffusible dye. (3) Tuapetsk agar medium: Poor growth (4) Synthetic Mucor agar medium: Poor growth (5) Oatmilk agar medium: Very good growth, covering the shear in two weeks. The colony is thin and flat, and hyphae are abundantly formed from the early stage of culture, and spore formation is rapid. The bacterial flora color changes from white to dark brown (Dk Brown, 3pn) as the culture progresses. A large amount of droplets are produced on the hyphae. Does not produce diffusible pigments. Physiological properties K-76, T-789, T-791, etc. are all aerobic bacteria and have the following physiological properties. 1 Stachybotrys sp. K-76 PH Temperature Conditions for growth 3.5-11.5 15-35°C Optimal growth conditions 6.0-9.5 20-32°C 2 Stachybotrys sp. T-789 PH Temperature growth conditions 3.5-11.5 15-38℃ Optimal growth conditions 4.5-10.5 20-32℃ 3 Stachybotrys sp. T-791 PH Temperature growth conditions 3.5-11.5 15-38℃ Optimal growth conditions 4.5-9.5 20-30℃ Morphological properties 1 Stachybotrys sp. K-76 The following is clear from Figures 3 and 4. That is, sclerotia and other sexual organs were not observed on the various media, and asexual sporulation of the aschospore type was observed. Hyphae are formed on various types of media, are intricately branched, and have hyphae widths of 2 to 4 microns that extend vertically and horizontally. The pedicel has simple branches from the hyphae (no branching from one pedicle to other pedicels), with 3 to 4 septa from the podocyte at the base, and is either erect or gently curved. and expand.
The hyphae width of the pedicel is 4.3-4.7μ at the base and 3.6-4.5μ at the center. The tip of the pedicel swells slightly and measures 7.9-9.3 x 3.6-4.7μ from the apex.
Three to seven oval to bottle-shaped stalks are formed upright. The stalks are smooth and colorless to pale yellowish brown. The mycospores are produced by basophilically connecting from the tips of the sycamores and do not form straight chains, but instead form semi-moon-shaped clusters of 7 to 26 pieces. The size of the pedicel is 4.9-8.0
×3.3-4.7μ, subglobular to ovoid, and its surface is rough to warty. Its color tone ranges from dark eye buoy (Dk Lvy 24 po) to gray-black. No coating of viscous substance was observed on the mycospore mass. The taxonomic position of this bacterium, which has the above mycological properties, is explained in the inspection handbook “The Genera of Hyphomycetes from Soil”.
Genera Of Hyphomycetes From Soil,
The Williams & Wilkins Company, G.L.
Barron, 1968)” and “A Manual of Soil Fundy” (A.
MANUAL OF SOIL FUNGI, The Iowa
State University Press Ames Iowa
USA, J.C. Gilman, 1971) and The Genera of Hundhyay Sporrating in Piyur Kayalchayar (The
Genera of Fungi Sporolating in Pure
Cultre, VERAG VON J, CREMER 3301
LEHRE, JAVON ARX, 1970), etc. According to Saccardo's classification system, this bacterium belongs to the genus Stachybotrys in the family Hyphomycetes Demathyaceae. That is, the characteristics of this strain have the property that it does not have asci or other sexual reproductive organs, produces dark brown inchospores from the inflorescence, and that the produced inchospores gather in a half-moon shape at the apex of the inflorescence. The characteristics match those of the genus Stachybotrys. The properties of this bacterium can be found in the above search guide and in Bisbee GR.
(1943); Trans.Brit.Mycol.soc., 26:133
−143, Zutsuk RK (1946): Mycologia,
38:69–76, Baron GL (1961), Can.J.Bot.
, 39: 153-157 and IFO archived standard strains, the inchospores of this strain K-76 have rough and wart-like protrusions, and are ovoid to ovoid.
Stachybotrys lobulata (IFO) in that it has an oval shape.
5369) was determined to be the most similar. However, the pedicel of Stachybotrys robulata is 1 mm.
It also differs from the K-76 strain, which forms simple branches, in that it has the property of branching and elongating the stalks from one stalk. The K-76 strain produces a large amount of diffusible pigment and its sporulation is extremely poor on other agar media except potato glucose agar and oatmeal agar, compared to Stachybotrys lobulata. grows very quickly on various media and forms flat colonies without folds. In addition, spore formation is so good that it represents the color of the bacterial flora in most media except for Czapetzk agar, and no diffusible pigment is produced, or even if it is produced, it is very weak. Based on the above-mentioned differences, we concluded that this strain K-76 is a new bacterial species different from Stachybotrys lobrata, and that it is a new strain different from Stachybotrys lobrata. It was named strain K-76. 2 Stachybotrys sp. T-789 The following can be seen from Figure 5. That is, Stachybotrys sp. There is a slight difference in size from K-76, but they are completely identical morphologically. The above mycological properties were compared to the aforementioned Stachybotrys sp. Stachybotrys lobrata was identified by referring to the search guide and literature as well as the K-76 strain and by comparing it with the IFO standard strain.
IFO 5369 and Stachybotrys sp. Most similar to K-76. This strain is observed to form good inchospores on Czapetsk agar medium, but the former two strains have a cultural difference in that almost no inchospore formation is observed on Czapetsk agar medium. Also, the aforementioned Stachybotrys sp. K-
As mentioned in the section on strain 76, there is a clear difference in morphological characteristics from Stachybotrys lobrata, and the optimal growth range is wide between PH4.5 and 10.5, so this strain is the K-76 strain. It is different from Therefore, for the above reasons, this bacterial strain is recognized as a new bacterial species, Stachybotrys sp. It was named T-789. 3 Stachybotrys sp. T-791 The following can be seen from Figure 6. That is, the inflorescence pedicel forms simple branches and stands upright. It swells slightly into a club shape at its tip. However, the standard strain Stachybotrys echinata IFO 7525 and
There is no significant bulk as observed in 8856. The mycelium shows a width of 4.0-4.5μ, slightly larger than the inflorescence. The pedicel, which has podocytes and branches upright from the vegetative or aerial hyphae, has 2 to 3 septa and has a size of 40 to 80 x 3.0 to 3.5 μm. No thorn-like projections are observed on the cell wall of the pedicel, which is smooth. 3 to 6 sycamores are produced from the apical ampulla of the sycamore stalk, and 4.3 to 5.2 x 3.0 to 4.3 to 5.2
Continuously produces spherical to subglobular inchospores with a size of 4.2μ and one cell with spiny projections24~
Forms a chain of 70 spores. The sycamore exhibits a sake bottle-shaped shape and measures 6.9-10.7 x 3.5-4.7μ. The stalk and stalk are colorless. The stalks are coffee (coffee, 3pn) to black in color. According to the taxonomic position of this bacterium having the above-mentioned mycological properties, it belongs to the genus Stachybotrys (genus Menmoniella) when searched using the search guide given in the section on the K-76 strain mentioned above. In other words, this strain T- does not have asci or other sexual reproductive organs and continuously produces dark brown inchospores from the inflorescence, and the produced spores are long chains.
The characteristics of strain 791 are consistent with those of the genus Stachybotrys (genus Menmoniera). The properties of this bacterium can be found in the above search guide and in Zutsuk RK (1946, Mycologia, 38:69-76) and Smith G. (1962, Trans, Brit. Mycol.
Soc., 45: 387-394) and other documents and IFO preserved standard strain, and the present strain T-791 was identified.
The strain's ability to continuously produce chocospores from the chotes in a basal-like manner determined that the strain belongs to the genus Menmoniella named by Hohnel (Menmoniella echinata). However, based on the report by Zutsuk and Smith mentioned above, this bacterial strain T-791 was considered to be a species related to Stachybotrys echinata, and thus Stachybotrys echinata IFO 7525
and 8856. Morphologically, the cell wall of this strain T-791 does not have the thorn-like protrusions that are specific to both standard strains, and the tip of the pedicel is 2 to 3 times the width of the hyphae of the pedicle in the standard strain. Compared to this strain, no significant expansion was observed in this strain. Furthermore, the two standard strains showed good growth on medium, especially on potato glucose agar medium, forming slightly raised circular colonies, with abundant hyphae, and significant spore formation. In contrast, this strain exhibits tree-like, irregular growth as described above, and has poor hyphal formation and spore formation. Based on the above differences in mycological properties, this bacterial strain was recognized as a new bacterial species, Stachybotrys sp. It was named T-791. In addition, the color display description is from "Color Harmony Manual, 1958,
Container Corporation of America) labeling laws. The new strain mentioned above, Stachybotrys sp.
K-76, Stachybotrys sp. T-789 and Stachybotrys sp. T-791 has microbial accession numbers to FERM-P No. 3801 (FERM-P No. 3801), FERM-P No. 3802 (FERM-P No. 3802), and FERM-P No. 3802, respectively. Bacteria No. 3803
(FERM-P No. 3803). The production of the substance of the present invention using the microorganism belonging to the genus Stachybotrys is specifically carried out as follows. That is, the microorganism is first cultured in a medium containing conventional nutrients and additives. Examples of nitrogen sources commonly used as a culture medium include soybean flour, soybean oil, corn steep liquor, yeast extract, dried yeast, oatmeal, meat extract, casein hydrolyzate, ammonium salts, nitrates, etc. Carbon sources include: Examples include glucose, glycerin, maltose, starch, lactose, sucrose, and molasses. Examples of additives added to the medium include inorganic salts such as calcium carbonate, sodium chloride, magnesium sulfate, and phosphoric acid. may contain a trace amount of salt. Cultivation can be carried out in a normal aqueous medium containing the above-mentioned culture medium, by surface culture or deep aeration agitation culture.
It is preferable to perform deep aeration agitation culture. The culture conditions are normally aerated, pH is 3.5 to 11.5, preferably 4.5 to 9.5, and the culture temperature is 15 to 35°C, preferably 20 to 32°C, and the culture can be advantageously carried out usually for 3 to 7 days. In the method of the present invention, the substance grown in the culture solution is then collected after the above-mentioned culture. The collection method is not particularly limited, and any known method that utilizes the physical and chemical properties of the produced substance can be employed. For example, differences in solubility with impurities, differences in adsorption affinity for ordinary adsorbents such as activated carbon, XAD-2, silica gel, ion exchange resins, Cephadex, etc.
This can be carried out by a method that utilizes the difference in distribution ratio between two liquid phases, or by a combination of these methods. More specifically, the culture solution is filtered or centrifuged according to a conventional method to remove bacterial cells in advance. Next, methanol is added to the supernatant, stirred, and left to stand for 2 to 3 hours, and the precipitate is removed by centrifugation again. After further extraction with the same amount of ethyl acetate, the solvent was distilled off. After pouring the extract into methanol and passing the methanol solution through an activated carbon column, the solvent of the eluate is distilled off. The residue is gel-filtered with Cephadex LH-20, each fraction obtained is subjected to the anti-complement activity test described above, the active portion is collected, and the substance of the present invention is purified and isolated by distilling off the solvent. . When used as a therapeutic agent for nephritis, the substance of the present invention is usually administered in the form of a pharmaceutical composition together with its pharmacological carrier. As carriers, diluents or excipients such as fillers, fillers, binders, wetting agents, disintegrants, surfactants, lubricants, etc., which are commonly used to prepare drugs according to the usage form, can be used. These can be appropriately selected depending on the dosage unit form of the nephritis therapeutic agent. As the dosage unit form of the nephritis therapeutic agent, various forms can be selected depending on the therapeutic purpose, and typical examples include tablets, pills, powders, liquids, suspensions, emulsions,
Examples include granules, capsules, suppositories, injections (solutions, emulsions, suspensions), and the like. When forming tablets, a wide variety of carriers conventionally known in this field can be used, such as lactose, sucrose, sodium chloride, glucose, urea, starch, calcium carbonate, kaolin, crystalline cellulose, silicic acid, etc. Excipients, water, ethanol, propanol, simple syrup, glucose solution, starch solution, gelatin solution, carboxymethylcellulose, shellac, methylcellulose, potassium phosphate, binders such as polyvinylpyrrolidone, dry starch, sodium alginate, agar powder, laminaria powder , disintegrants such as sodium bicarbonate, calcium carbonate, Twin, sodium lauryl sulfate, stearic acid monoglyceride, starch, lactose, disintegration inhibitors such as sucrose, stearin, cocoa butter, hydrogenated oil, etc.
Absorption enhancers such as quaternary ammonium bases and sodium lauryl sulfate, humectants such as glycerin and starch, adsorbents such as starch, lactose, kaolin, bentonite, and colloidal silicic acid, purified talc,
Examples include lubricants such as stearate, boric acid powder, macrogol, and solid polyethylene glycol. When forming into a pill form, a wide variety of carriers conventionally known in this field can be used, such as excipients such as glucose, lactose, starch, cocoa butter, hydrogenated vegetable oil, kaolin, and talc, powdered gum albia, and tragacanth. Examples include binders such as gelatin and ethanol, and disintegrants such as laminaria and agar. Furthermore, tablets may be coated with conventional coatings as required, such as sugar-coated tablets, gelatin-coated tablets, enteric-coated tablets, film-coated tablets, or double-coated tablets.
It can be a multi-layered tablet. When forming into a suppository, a wide variety of conventionally known carriers can be used, such as polyethylene glycol, cacao butter, higher alcohols, esters of higher alcohols, gelatin, semi-synthetic glycerides, and the like. When prepared as injections, solutions, emulsions and suspensions are preferably sterilized and isotonic with blood, and when formed into the form of solutions, emulsions and suspensions, diluents are used. All those commonly used in this field can be used, such as water, ethyl alcohol, propylene glycol, ethoxylated isostearyl alcohol, polyoxylated isostearyl alcohol,
Examples include polyoxyethylene sorbitate and sorbitan ester. In this case, the nephritis therapeutic agent may contain a sufficient amount of salt, glucose, or glycerin to prepare an isotonic solution, and may also contain conventional solubilizing agents, buffers, soothing agents, preservatives, etc. Further, coloring agents, preservatives, perfumes, flavoring agents, sweeteners, etc. and other pharmaceutical agents may be included in the therapeutic agent, if necessary. The amount of the substance of the present invention to be contained in the therapeutic agent for nephritis of the present invention is not particularly limited and can be appropriately selected within a wide range, but is usually 1 to 70% by weight, preferably 5% by weight of the composition.
~50% by weight. There are no particular limitations on the use of the therapeutic agent for nephritis of the present invention, and it can be administered in a manner appropriate for various forms. For example, tablets, pills, solutions, suspensions, emulsions, granules, and capsules are administered orally. In the case of an injection, it is administered intravenously alone or mixed with a normal replacement fluid such as glucose or amino acids, and furthermore, if necessary, it is administered alone intramuscularly, intradermally, subcutaneously, or intraperitoneally. Suppositories are administered rectally. The dosage of the therapeutic agent for nephritis of the present invention is appropriately selected depending on the purpose of use, symptoms, etc., but the amount of the substance of the present invention is usually used in the range of about 0.5 to 20 mg/Kg per day. The therapeutic agent of the present invention has anti-complement activity and is also useful as a therapeutic agent for autoimmune diseases, collagen diseases and rheumatism. In order to further clarify the present invention, synthesis examples of the substances of the present invention are listed below as reference examples, and pharmacological test results and manufacturing examples of the therapeutic agents of the present invention are also listed. Reference example 1 Stachybotrys sp. Pour K-76 into a 500ml Sakaguchi flask and add 100ml of a medium with the following composition.
Culture was performed at 28°C and PH=6 for 4 days with reciprocal shaking. Glycerin 0.5% Starch 1.0〃 Lactose 0.2〃 Soybean flour 0.5〃 Yeast extract 0.1〃 Malt extract 0.2〃 CaCO ₃ 0.3〃 MgSO ₄ 0.05〃 30 volumes of one seed culture obtained above, 20 volumes in a jar fermenter A medium having the above composition was added and cultured for 5 days at a culture temperature of 28° C., an aeration rate of 1/medium·min, and a stirring rate of 300 revolutions/min. The obtained culture solution was centrifuged at 8,000 rpm to remove bacterial cells, and methanol from step 5 was added to this supernatant, stirred and left for 3 hours, then centrifuged to remove the precipitate, etc. of ethyl acetate. After the solvent of the acetic acid ethyl ester layer was distilled off under reduced pressure, the residue was dissolved in methanol and passed through an activated carbon column. The eluate was concentrated to dryness under reduced pressure, and then chloroform:
It has anti-complement activity by dissolving it in ethyl acetate = 1:1 (V/V), gel-filtering it with a Sephadex LH-20 column, taking the anti-complement activity peak, and distilling off the solvent. 2.0 g of a pale yellow weakly acidic substance was obtained (the physical and chemical color properties of this substance are as described above). Reference example 2 Known Stachybotrys cartarum IFO
5369 was placed in a 500 ml Sakaguchi flask with 100 ml of a medium with the following composition, and cultured with reciprocating shaking at 28°C and pH = 6 for 4 days.
I did it for days. Glycerin 0.5% Starch 1.0〃 Lactose 0.2〃 Soybean flour 0.5〃 Yeast extract 0.1〃 Malt extract 0.2〃 CaCO ₃ 0.3〃 MgSO ₄ 0.05〃 30 volumes of 2 bottles of the seed culture obtained above, 20 volumes of the above Put the culture in the composition and culture temperature
Culture was carried out for 5 days at 28°C, aeration rate of 1/min of medium, and stirring rate of 300 revolutions/min. The obtained culture solution was centrifuged at 8,000 rpm to remove the bacterial cells, and methanol from step 5 was added to this supernatant, stirred and left for 3 hours, then centrifuged to remove the precipitate, and an equal amount Extract with ethyl acetate. After the solvent in the ethyl acetate layer was concentrated to dryness under reduced pressure, the residue was dissolved in methanol and passed through an activated carbon column. The eluate was concentrated to dryness under reduced pressure, and then chloroform:ethyl acetate = 1:1 (V/ V), gel filtration was performed on a Sephadex LH-20 column, the active peak was taken, and the solvent was distilled off to obtain 2.2 g of a pale yellow weakly acidic substance with anti-complement activity. The physical and chemical properties were consistent with those obtained in Reference Example 1.) Reference example 3 Stachybotrys sp. By culturing and purifying T-789 in a medium with the following composition adjusted to PH = 7.5 at 32°C in the same manner as in Reference Example 1, a pale yellow, weakly acidic property with anti-complement activity was obtained. The physical and chemical properties were consistent with those obtained in Reference Example 1.) Glycerin 0.5% Glucose 1.2〃 Corn steep liquor 0.5〃 Dried yeast 0.1〃 Malt extract 0.2〃 MgSO ₄ 0.05〃 NaCl 0.3〃 Reference example 4 Stachybotrys sp. T-791 was cultured and purified in a medium with the following composition adjusted to PH = 5.5 at 25°C in the same manner as in Reference Example 1 to obtain a pale yellow weakly acidic substance with anti-complement activity. The physical and chemical properties were consistent with those obtained in Reference Example 1.) Glycerin 0.5% Starch 1.0〃 Sugar 0.2〃 Soybean flour 0.5〃 Peptone 0.1〃 Malt extract 0.2〃 MgSO ₄ 0.3〃 HCl 0.05〃 Pharmacological test results Anti-complement activity effect Anti-complement activity is described in "Immunochemistry" No. 830-834 It is measured and confirmed in accordance with the test method described in Page (published by Asakura Shoten, edited by Yuichi Yamamura et al., 1973). That is, in a test tube, 0.5 ml of an aqueous dispersion of the substance of the present invention, 0.5 ml of sensitized blood cells (EA) at 1×10 ⁸ cells/ml, 1 ml of veronal buffered saline (GVB ⁺⁺ ) containing isotonic gelatin, and GVB ⁺⁺ Collect 0.5 ml of complement serum (GPC) diluted 150 times with liquid, moisturize at 37°C for 60 minutes, add 5 ml of ice-cold physiological saline, centrifuge, and then measure the absorbance of the separated supernatant. is measured using an absorbance meter OD ₄₁₃ to determine how much hemolysis of sensitized blood cells is suppressed by the substance of the present invention. The absorbance and anti-complement activity values measured according to the above method are
Shown in the table.

[Table] The blank in Table 1 above is a sample in which only GVB ⁺⁺ , gpc, and EA were collected without collecting the inventive substance, and the control* is a sample in which 0.5 ml of EA and 2.0 ml of water were collected. Sample showing % hemolysis. The anti-complement activity value was determined for sample No. 1 from which the substance of the present invention was collected.
From the OD ₄₁₃ value of the blank (sample No. 2 and 3)
Compare the value (OD′) after subtracting the OD ₄₁₃ average value*
It is determined by the value obtained by dividing the OD ₄₁₃ average value of (sample Nos. 4 and 5) by the value (OD') obtained by subtracting the blank OD ₄₁₃ average value, ie, the hemolysis ratio (y). Therefore, if the hemolytic ratio (y) is not less than 1, it can be considered to have anti-complement activity. It can be said that the smaller the value of the hemolysis ratio (y) is, the higher the anti-complement activity is, and the substance of the present invention is found to have superior anti-complement activity from this test result. Therapeutic effect on nephrotoxin-type nephritis Rat nephrotoxin (hereinafter abbreviated as "NT")
can be obtained as follows. Mouse kidney cortex was homogenized with an equal volume of saline, and the homogenate was mixed with Freund´s
complate adjuvant (manufactured by Difco) in a 1:1 ratio, and 2 ml of the resulting mixture was added to a rabbit (weight 3100).
g) to be immunized by intramuscular injection. After 1.5 months, blood was collected from the rabbit's heart to obtain serum. The obtained serum is inactivated at 56°C for 30 minutes and then fractionated by salting out with 40% saturated ammonium sulfate. iminoglobulin-γ-
Collect globulin (IgG) fraction and NT
get. Male Wistar rats weighing 150-160 g are used for the test. The substance of the present invention is
Based on the time of administration 1 hour before NT administration, 24
Administer intravenously at a dose of 3 mg per animal once a day every hour from 3 days before to 4 days afterward. In addition, 1 ml of NT was intravenously injected into the tail vein of the mouse. Chlorophyllin (CP) was used as a comparative compound (dose of 10 mg per animal once a day), and physiological saline was used as a control. Proteinuria (the total amount excreted in the urine in 24 hours) is determined by Bovine Serim by sulfosalicylic acid.
Measured by turbidity method using albumin) as a control. The results obtained are shown in Table 2.

[Table] The number of days is the number of days that have passed since administration was administered 1 hour before NT administration. The amount of proteinuria in Table 2 is expressed in mg/day. The protein uria in mice under normal conditions is 0.5 to 5 mg/day, but if it exceeds this value, especially if it exceeds 10 mg/day, it can be said that nephritis has developed. As is clear from Table 2, in the case of the control group, from the time of administration to 7
The daily amount of proteinuria was the same as that of mice under normal conditions, indicating that the primary reaction was suppressed. Furthermore, the substance of the present invention exhibits an inhibitory effect about three times that of CP. Therapeutic effect on Heymann type nephritis Wistar female mice weighing 180 to 200 g are used in the test. Remove the mouse kidney cortex and homogenize it with an equal volume of physiological saline. The homogenate was centrifuged at 1500G for 1 hour, and the supernatant was collected using the method of Edginton et al. [TS
Edgington at al, Journal of Experimental
Madicinl, 127 , 555] and Friend Complate Adjuvant 37Ra.
(manufactured by Difco) at a ratio of 0.4:1 as an adjuvant, and 0.5 ml is injected into the abdominal cavity of the mouse. After that, proteinuria 100mg/every two weeks
Administer the same amount of this adjuvant until the end of the day. (The period is approximately 6 to 8 weeks.) The mouse with Heyman type nephritis (weight
The substance of the present invention is intraperitoneally administered to animals (300 to 350 g) at a dose of 3 mg per animal every 24 hours for 7 days, and the amount of proteinuria (mg/day) is measured in the same manner as above. In addition, CP was used as a comparison compound (once per day per animal).
A similar test was conducted using physiological saline as a control group. The results obtained are shown in Table 3.

[Table] The weight of the mouse 2-3 weeks after the start of the experiment
The amount of protein uria is 400-500g, and the normal amount of proteinuria is 5-500g.
It is thought to be 15mg/day. As is clear from Table 3, the substance of the present invention can cure Heymann type nephritis.
The effect of the substance of the present invention is about three times that of CP. Acute Toxicity An acute toxicity test of the substance of the present invention was conducted in mice by intraperitoneal administration. The resulting LD ₅₀ value is 40
It was mg/Kg. Production Example 1 Sodium salt of the substance of the present invention 500 mg Glucose 250 mg Distilled water for injection Appropriate amount Total volume 5 ml Dissolve the sodium salt of the substance of the present invention and glucose in distilled water for injection, and then inject into a 5 ml ampoule. After purging with nitrogen, sterilize under pressure at 121°C for 15 minutes to obtain an injection. Production Example 2 Sodium salt of the substance of the present invention 500mg Sodium sulfite 5mg Distilled water for injection Appropriate amount Total amount 5ml An injection is obtained according to Production Example 1. Production Example 3 Substance of the present invention 750mg Semi-synthetic glyceride base Appropriate amount Total amount 2000ml Add the substance of the present invention to the semi-synthetic glyceride base,
Mix and suspend at 50℃, then pour into a mold.
After cooling naturally, take it out to obtain a suppository. Production Example 4 Substance of the present invention 750mg Vitamin E 90mg Semi-synthetic glyceride base Appropriate amount Total volume 2000ml A suppository is obtained according to Production Example 3. Production Example 5 Substance of the present invention 150g Abysiel (trade name, manufactured by Asahi Kasei Corporation) 40g Cornstarch 30g Magnesium stearate 2g TC-5 (trade name, hydroxypropylmethyl)
Cellulose 10g Polyethylene glycol-600 3g Castor oil 40g Methanol 40g The substances of the present invention, Abysiel, cornstarch and magnesium stearate were mixed, polished and sugar-coated.
Compress the tablets with an R10mm kine. The obtained tablet is TC-
5. Coat with a film coating agent consisting of polyethylene glycol-6000, castor oil and methanol to produce film coated tablets. Production Example 6 The substance of the present invention 100g Abysiel 40g Cornstarch 30g Magnesium stearate 2g Methyl acrylate-methacrylic acid copolymer 5.7g Triacetin 0.6g Ethanol 50.4g The substance of the present invention, Abysiel, cornstarch and magnesium stearate were mixed, polished, and coated with sugar.
Compress the tablets with an R10mm kine. The resulting tablets are coated with a film coating agent consisting of methyl acrylate-methacrylic acid copolymer, triacetin, and ethanol to produce enteric-coated tablets. Production Example 7 Substance of the present invention 150.0g Citric acid 1.0g Lactose 33.5g Dicalcium phosphate 70.0g Pluronic F-68 30.0g Sodium lauryl sulfate 15.0g Polyvinylpyrrolidone 15.0g Polyethylene glycol (Carbowax)
4.5g polyethylene glycol (Calbox 600)
45.0g Cornstarch 30.0g Dry sodium lauryl sulfate 3.0g Dry magnesium stearate 3.0g Ethanol Appropriate amounts of the present invention substance, citric acid, lactose, dicalcium phosphate, Purone F-68 and sodium lauryl sulfate are mixed. The above mixture is sieved through a No. 60 screen and wet granulated with an alcoholic solution consisting of polyvinylpyrrolidone, Carbowax 1500 and 6000. Add alcohol if necessary to make the powder into a pasty mass. Add cornstarch and continue mixing until uniform particles are formed. Pass it through a No. 10 screen, put it in a tray and put it in an oven at 100℃ for 12~
Dry for 14 hours. The dry particles are sieved through a No. 16 screen, dried sodium lauryl sulfate and dried magnesium stearate are added and mixed, and compressed into the desired shape using a tablet machine. The core is treated with varnish and sprinkled with talc to prevent moisture absorption. A subbing layer is applied around the core. Apply varnish a sufficient number of times for internal use. Further subbing layers and smooth coatings are applied to make the tablet perfectly round and smooth. Pigmented coatings are applied until the desired shade is obtained. After drying, the coated tablets are polished to a uniform gloss.

[Brief explanation of the drawing]

FIG. 1 is an infrared absorption spectrum analysis diagram of the substance of the present invention, FIG. 2 is an ultraviolet absorption spectrum analysis diagram of the substance of the invention, and FIGS. 3 and 4 are the infrared absorption spectrum analysis diagram of the substance of the present invention. S.P. Micrograph of K-76, Figure 5 is Stachybotrys. S.P. T-
The micrograph of 789 and Figure 6 are Stachybotrys.
S.P. A micrograph of T-791 is shown.

Claims (1)

[Claims] 1 Nephritis characterized by containing as an active ingredient a compound represented by the molecular formula C ₂₃ H ₃₀ O ₆ and having a hydroxyl group, an α/β-unsaturated carbonyl group, and a carbon-carbon double bond of an aromatic ring in the molecule therapeutic agent.