the active ingredient of the chondroprotective agent according to the present invention is a flavonoid , which is widely present in the vegetable kingdom . typical flavonoid compounds include flavones , flavonols , flavanones , and flavanonols . flavanones contain an asymmetric carbon atom at the 2 - position , and flavanonols contain asymmetric carbon atoms at the 2 - and 3 - positions , and such compounds may be present as the stereoisomers . these stereoisomers can also be used in the present invention . further , the saccharides present in the above naturally occurring glycosides are not particularly limited . as examples of the naturally occurring glycosides , there may be mentioned glucoside , galactoside , fructoside , rhamnoside , rutinoside ( that is , rhamnoglucoside ), arabinoside , xyloside , apioglucoside , and robinobioside . in the present invention , any naturally occurring flavonoids may be used as the above present substance . the flavonoid compounds and naturally occurring glycosides thereof shown in the following table 1 are preferable . the compounds nos . 34 to 51 in table 1 include a single bond as x . thus , the carbon atom in the 2 - position or the carbon atoms in the 2 - and 3 - positions are asymmetrical , and there exist stereoisomers . it is known that pinocembrin includes (±) and ( s ) isomers ; naringenin , sakuranetin , hesperetin and eriodictyol include (±), ( r ), and ( s ) isomers ; pinobanksin includes ( 2r - trans ) and ( 2s - trans ) isomers ; aromadendrin and fustin include trans -(±), ( 2r - trans ), and ( 2s - trans ) isomers ; taxifolin and ampelopsin include trans -(±), ( 2r - trans ), ( 2s - trans ), and ( 2r - cis ) isomers . it is possible to use , as the flavonoids , compounds isolated and purified from naturally occurring plants or chemically synthesized . many compounds described in table 1 are commercially available . for example , it is possible to obtain flavone , apigenin , luteolin , acacetin , linarin , diosmetin , baicalein , fisetin , kaempferol , quercetin , hesperetin , and hesperidin from funakoshi co ., ltd ., tokyo . examples of the acute toxicity of the present substance are as follows : mouse ld 50 of quercetin ( oral administration ): 160 mg / kg and mouse ld 50 of fisetin ( intravenous injection ): 180 mg / kg . further , no abnormalities were observed for a week after hesperetin was administered orally to balb / c mice ( female , seven weeks old ) at the dose of 100 mg / kg . the same results were obtained where hesperidin , acacetin , diosmetin , apigenin , luteolin , or kaempferol was administered . as a pharmacological effect , the present substance exhibits the function to inhibit destruction of chondrocyte matrix in chondrocyte culture ( derived from cartilage of rabbit shoulder and knee joints ) ( see example 1 as below ). accordingly , the present substance is useful as a chondroprotective agent for treating various types of arthropathy accompanying the cartilage destruction of joints . examples of such arthropathy are rheumatoid arthritis , osteoarthritis , periarthritis humeroscapularis , shoulder - arm - neck syndrome , lumbago , etc . the chondroprotective agent containing the present substance as an active ingredient may be in the form of any conventional formulation . the chondroprotective agent may contain the present substance alone , or a mixture of the present substance with any pharmaceutically acceptable carrier or diluent . the chondroprotective agent may contain the active ingredient in an amount of 0 . 01 to 100 percent by weight , preferably 0 . 1 to 70 percent by weight . the chondroprotective agent of the present invention may be administered orally or by some other routes . the dose of the chondroprotective agent according to the present invention varies with the patient ( animal or human ), age , individual differences , state of illness , and the like . generally speaking , however , when a human is treated , the dose of oral administration of the present substance is in the range of 0 . 1 to 500 mg / kg ( body weight ) per day , preferably 0 . 5 to 200 mg / kg ( body weight ), which is usually divided into 1 to 4 dosages in a day , although the dose outside the above range may sometimes be administered . the present invention now will be further illustrated by , but is by no means limited to , the following examples . the cartilages were sterilely extracted from the shoulder and knee joints of rabbits ( new zealand white rabbits ) ( body weight of 1 to 1 . 5 kg ). the cartilages were thoroughly washed with pbs (−) ( free of ca 2 + , mg 2 + ), hanks &# 39 ; solution and 0 . 1 % edta - pbs (−), and then cut into small segments ( 1 mm × 1 mm × 1 mm ). after pbs (−) containing 0 . 1 % edta was added , the segments were allowed to stand in an incubator of 37 ° c . for 30 minutes . then , the segments were treated with a trypsin solution ( 0 . 25 %) at 37 ° c . for one hour to remove the connective tissue adhered to the cartilage . after the supernatant had been removed , the cartilages were treated for about 2 to 2 . 5 hours in a ham f - 12 medium containing 10 % fetal bovine serum ( fbs ) and 0 . 2 % collagenase . then , the collagenase solution was centrifuged ( 1500 r . p . m . ), and the residual chondrocytes were washed twice with a ham f - 12 medium ( chondrocyte culture medium ) containing 10 % fbs . finally , the resulting suspension was adjusted so that the chondrocytes were suspended in the concentration of 3 × 10 5 cells / ml in the chondrocyte culture medium . the chondrocytes were seeded in an amount of 1 ml / well on 24 - well plates . the chondrocytes became confluent after 4 days . the experiment were performed within two weeks after reaching the confluent stage . ( b ) addition of compounds to be tested and proteoglycan depleting agents the chondrocyte culture medium which had been used for cultivating the chondrocytes was removed from each well and 800 μl of fresh serum - free s - clone medium containing 0 . 1 % human serum albumin was added . further , 100 μl of s - clone medium containing the compounds to be tested ( containing the compound in the concentration of 10 fold the final concentration ; dmso concentration = 2 . 5 %) was added . the chondrocytes were cultured in the presence of carbon dioxide ( 5 %) and air ( 95 %) for 2 hours . then , the proteoglycan depleting agent , pma ( phorbol myristate acetate ) ( final concentration = 0 . 1 μg / ml ) was added into the culture medium of the chondrocytes . compounds of present invention : apigenin ( present substance no . 5 ), luteolin ( present substance no . 8 ), acacetin ( present substance no . 11 ), linarin ( present substance no . 12 ), diosmetin ( present substance no . 13 ), baicalein ( present substance no . 15 ), fisetin ( present substance no . 16 ), kaempferol ( present substance no . 17 ), quercetin ( present substance no . 22 ), hesperetin ( present . substance no . 41 , ( s ) isomer ), and hesperidin ( present substance no . 42 , ( s ) isomer ) ( all from funakoshi co .) proteoglycan depletion was determined by the measurement of the glycosaminoglycan ( major constituent of proteoglycan , hereinafter referred to as gag ) content following digestion of the chondrocyte matrix with papain . after 2 days , the supernatant of the chondrocyte culture was removed . then , 1 ml of 0 . 03 & amp ; papaine solution was added to the remaining chondrocyte matrix layer and a reaction was performed at 65 ° c . for 1 hour to liberate the gag from the matrix layer . the content of the gag in the treated papaine solution was determined by the 1 , 9 - dimethylmethylene blue method ( refer to r . w . farndale , biochim . biophys . acta ., vol . 883 , pp . 173 to 177 , 1986 ). the gag content in the chondrocyte matrix of the control test wherein the proteoglycan depleting agent was not added was shown as “ 100 ”, and the relative amount of the gag of each experiment except the control test was calculated by by following formula : wherein a represents the gag content of the control tests wherein the proteoglycan depleting agent was not added , and b represents the gag content wherein the proteoglycan depleting agents were added alone or the gag content wherein the proteoglycan depleting agents and the compounds to be tested were added . the gag contents of the control tests varied in a range of 11 . 23 to 59 . 0 μg / ml , depending on the period from the time when the chondrocytes became confluent until the time when the chondrocytes were used in the above experiment . the results are shown in table 2 . the gag content is the value of the mean value ± standard error ( n = 3 to 6 ). for each of the compounds to be tested , the control test and the proteoglycan depleting test wherein the proteoglycan depleting agent was added were carried out and the results thereof are also shown . the significance was determined by student &# 39 ; s t - test with respect to the proteoglycan depleting test wherein the proteoglycan depleting agent was added . the results of the determination are shown as follows : in comparison with the gag content in the control tests wherein the proteoglycan depleting agent was not added , the addition of the proteoglycan depleting agents , pma , induced a loss of gag content . under these conditions , the present compound significantly inhibited or reduced the loss of gag content , and showed a function to inhibit or suppress the proteoglycan depletion . on the other hand , indomethacin , a conventional analgesic and anti - inflammatory agent , did not show the function to inhibit or suppress the proteoglycan depletion , but caused a significant exacerbation on the proteoglycan depletion . the mixture was kneaded using 32 parts by weight of a wetting agent , ethanol . then , the kneaded mixture was glanulated by wet granulation and dried to obtain the granule . as explained above , the present substance strongly inhibits proteoglycan depletion from the chondrocyte matrix and exhibits a function to protect cartilage . further , the present substance has low toxicity . accordingly , the present substance is very useful for the treatment of arthropathy , such as rheumatoid arthritis , osteoarthritis , periarthritis humeroscapularis , shoulder - arm - neck syndrome , lumbago , and so on . although the present invention has been described with reference to specific embodiments , various changes and modifications obvious to those skilled in the art are deemed to be within the spirit , scope , and concept of the invention .
a chondroprotective agent comprising a flavonoid compound of the general formula : wherein r 1 to r 9 are , independently , a hydrogen atom , hydroxyl group , or methoxyl group and x is a single bond or a double bond , or a stereoisomer thereof , or a naturally occurring glycoside thereof is disclosed . the above compound strongly inhibits proteoglycan depletion from the chondrocyte matrix and exhibits a function to protect cartilage , and thus , is extremely effective for the treatment of arthropathy .