| ObjectiveTo find the anti-inflammatory effective substances of Rubiae radix et rhizoma,spectrum-effect relationships were applied in this paper.HPLC/UV technology was also used to establish chemical fingerprints.The overall chemical information and pharmacodynamic data were acquired from the chemical fingerprints and the tests of suppressing inflammation and alleviating pains of various samples originated from different sources.Coupled with chemometric and mathematical statistical methods,the spectrum-effect relationships of Rubiae radix et rhizoma between chemical fingerprints and anti-inflammatory effects were developed,and then the characteristics peaks associated with pharmacodynamic effects in the fingerprints were located,and thus the active ingredients were speculated.Methods1.The establishment of Rubiae radix et rhizoma fingerprintHPLC-DAD method was introduced to analyze Rubiae radix et rhizoma samples from different sources.Mobilephase,elution gradient chromatographic column and detected wavelength etc.were optimized to complete the systemic methodology and establish the HPLC fingerprint of Rubiae radix et rhizoma.What’s more,the similarity evaluation of chromatograms was proeessed by computer aided interpretation.The quantitative fingerprint charaeteristic was treated with cluster analysis.Based on the retention time and the ultraviolet(UV)-absorption(190 to 800 nm)of the known standard compounds,the main ingredients were identified.2.Preliminary evaluation of analgesic property of Rubiae radix et rhizomaKM mice was pretreatmented with water extract and alcohol extract of Rubiae radix et rhizoma(30,15,7.5,4.5g,1.5g/kg,p.o.)once.After 1 h,the acetic acid-induced writhing in mouse pain model was established by injecting acetic acid(0.6%,0.2mL per mice).The number of writhing times of mouse was observed within 20 minutes.The pharmacological effects between water extract and alcohol extract of Rubiae radix et rhizoma were compared by the efficacy evaluation of analgesic rate.3.Preliminary evaluation of anti-inflammatory property of Rubiae radix et rhizome3.1 Inhibiting effect of primary and secondary inflammation of adjuvant-induced arthritisTo evaluate inhibiting effect of secondary inflammation,the freund’s complete adjuvant-induced arthritis model in rats was duplicated.To study the inhibiting effect of primary inflammation,wistar rat was with pretreatment with alcohol extract of Rubiae radix et rhizoma(10、5g/kg,p.o.)1 h before the model was duplicated and last for 14 days.The volume difference of rat paw was measured and the inhibition rate were evaluated.To study the inhibiting effect of secondary inflammation,wistar rat was treatment with alcohol extract of Rubiae radix et rhizoma(10、5、2.5g/kg,p.o.)for 14 days(q.d.),since 11 days after induced inflammatory.The volume difference of rat paw was measured and the inhibition rate were evaluated as well.After the end of the experiment,the paw was collected for the histopathological evaluation.3.2 The effect on carrageenan-induced rat paw edema modelSD rat was with pretreatment with alcohol extract of Rubiae radix et rhizoma(10、5、2.5g/kg,p.o.)once.1 h after the administration,the carrageenan-induced rat paw edema model was duplicated and the volume difference of rat paw as well as the inhibition rate were evaluated.4 h after the carrageenan challenge,rat was sacrificed.The paw was collected for the analysis of IL-6 and TNF-α.4.Study on the correlation between characteristic of Rubiae radix et rhizoma fingerprint and pharmaeodynamicsThe established fingerprint method was applied to analyze 10 batches of Rubiae radix et rhizoma from different origin to gain the quantifiable fingerprint characteristics.The analgesic effect of 10 batches of Rubiae radix et rhizoma in mice was evaluated by writhing test.And the anti-inflammatory effect on paw edema induced by freund’s complete adjuvant was investigated in rats.At the Premise of the acquired charaeteristic of fingerprint and pharmacodynamiedata,grey relation alanalysis was selected to show what the constituents represented by fingerprint contributed to pharmaeodynamies.Results1.The establishment of Rubiae radix et rhizoma fingerprintThe Rubiae radix et rhizome fingerprint was established.Nine common peaks were calibrated.The similarities of 23 batches of Rubiae radix et rhizoma samples were 0.851～0.990,while the 5 batches of adulterants were 0.051～0.757.28 batches of samples were divided into two groups:genuine and adulterants by using cluster analysis.Data of HPLC-PAD analysis profile indicated that 5 ingredients,rubimaillin,6-hydroxyrubiadin,purpurin,alizarin,rubiadine,were indentified in Rubiae radix et rhizome.2.Preliminary evaluation of analgesic property of Rubiae radix et rhizomaBoth water extract and ethanol extract could decrease the numbers of times of mice’s writhing,which showed an analgesic effect.Comparison between water extract and ethanol extract,ethanol extract have an better analgesic effect.3.Preliminary evaluation of anti-inflammatory property of Rubiae radix et rhizome3.1 Inhibiting effect of primary and secondary inflammation of adjuvant-induced arthritisEthanol extract of Rubiae radix et rhizoma have an inhibiting effect on both primary inflammation and secondary inflammation.It showed significant inhibition of adjuvant-induced arthritis in rats paw edema,abating inflammatory responses in histopathologic examination.3.2 The effect on carrageenan-induced rat paw edema modelData of anti-inflammatory property evaluation indicated that oral administration of alcohol extract of Rubiae radix et rhizoma(10、5、2.5g/kg)significantly reduced carrageenan-induced paw edema and attenuated the levels of IL-6 and TNF-a,in a dose-dependent manner.4.Study on the correlation between characteristic of Rubiae radix et rhizoma fingerprint and pharmacodynamicsAccording to the relationa lgarde,the sequence of conrtibution to analgesic effect is,peak 7>peak 5>peak 6>peak 18>peak 3>peak 8>peak 11>peak 9>peak 17>peak 13>peak 15>peak 16>peak 4>peak 20>peak 10>peak 19>peak 12>peak 1>peak 2>peak 14.While to anti-inflammatory effect is,peak 11>peak 8>peak 18>peak 5>peak 3>peak 7>peak 17>peak 10>peak 13>peak 6>peak 4>peak 9>peak 20>peak 16>peak 19>peak 15>peak 12>peak 1>peak 2>peak 14.It can be concluded that analgesic and anti-inflammatory effects are the total results of chemical constituents group in Rubiae radix et rhizome.The four identified components which were B,E,were highly correlated with the effect of analgesic and anti-inflammatory.And the molecular weight of peak 3、peak 5、peak 6、peak 7、peak 8、peak 9、peak 11、peak 17、peak 18 was as below:457.97、578.02、299.89、283.91、620.04、756.17、269.95、410.02、283.98ConclusionThis study established the fingerprint of Rubiae radix et rhizome,identified the effective component group represented by fingerprint contributed to pharmacodynamic,and provide a reference for establishing fingerprint of TCM’s spectrum-effect relationship and screening the active ingredient. |
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