Studies On Isodon Serra Of The Diterpenoids And Its Quality Evaluation

ObjectiveIsodon serra(Maxim.)Hara,namely "xihuangcao",,"xiongdancao" in Chinese.It belongs to Labiatae.Biological activity studies performed on the spieces have shown that it has heat clearing and detoxifying,promoting blood circulation by removing blood stasis,antibacterial and anti-inflammatory treatment of various hepatitis,antitumor etc.After a long period of research on I.serra,we have found that diterpenoids gathered in ethyl acetate parts.Previous chemical investigations on Isodon serra have isolated and identified three 6,7-seco-ent-kauranes compounds,namely nodosin,isodocarpin and sculponeatinA,which have been found strong activity in vitro.Firstly,this paper focus on the three diterpenoids to seperation and enrichment.Furthermore,enrichment the extactions of the diterpenoids.Secondly,establishing the quality standard of the extactions of diterpenoids and I.serra.In addition,to develop a method about quantitative analysis of multi-components by single-marker to determination the five diterpenoids components in I.serra simultaneously.Methods1.To seperate the three 6,7-seco-ent-kauranes compounds,we utilization of silica gel,Sephadex LH-20,MCI and ODS column chromatography,and determine the structure by using IR,UV and nuclear magnetic resonance(NMR)technology in combination with related literature data.2.Compare the different separation processes,Sephadex LH-20 and MCI column chromatography and determine the better one.3.Establish a RP-HPLC method to determination the contents of enmein,sculponeatinA,nodosin,isodocarpin and lasiokaurin in I.serra materials and diterpenoids extactions.The conditions of the quantitative annalysis:Kromasil C18(4.6 mm×250 mm,5?m)analytical column,mobile phase consisted of solvent A(methanol)and solvent B(0.1%formic acid),elution profile:0-38 min,40%A;38-42 min,40-55%A;42-63 min,55-60%A.Column temperature:30?,DAD wavelength:254 nm,flow rate:0.7 ml/min.4.Establish a relative correction factor method regarding nodosin as internal standards,we have investigated relative correction factors on four kinds of chromate-graphic column,Kromasil-C18(4.6 mm×250 mm,5 ?m),Diamond C18(4.6 mm×250 mm,5?m),Phenomenex-C18(4.6 mm×250 mm,5 ?m),Hypersil-Ci8(4.6 mm×250 mm,5 ?mn)and three kinds of high performance liquid chromatographs such as Waters 2695,Agilent 1260,LC-20AT.Finally compare the differences between the calculated values and measured values.Results1.In total,we obtain nodosin 7.2 g,isodocarpin7.4 g,sculponeatinA 101 mg.2.Diterpenoids extactions weighing 88.7g after freeze drying.3.The contents range of enmein,sculponeatinnA,nodosin,isodocarpin,lasiokaurin in I.serra are:1.85 mg·g-1,2.54 mg·g-1,6.34mg·g-1,1.34 mg·g-1 ? 1.27 mg·g-1;The average contents of the five diterpenoids in leaves are 1.89 mg·g-1,3.84 mg·g-1,8.84 mg·g-1,1.81 mg·g-1,1.52 mg·g-1;And the average contents in the stem of I.serra are 1.17 mg·g-1,0.62 mg·g-1,1.47mg·g-1,0.31mg·g-1,0.36mg·g-1.The results show that the content of 5 components is different in different batches,the contents of five diterpenoids in leaves is significantly higher than in stems.Futhermore,the contents of sculponeatinA,nodosin,isodocarpin and lasiokaurin in the leaves is about 5 times of the stems.The contents of 5 components in the extactions are:53.89 mg·g-1,27.24 mg·g1,100.31 mg·g-1,112.61 mg·g-1,7.18 mg·g-1.Studies have shown that isodocarpin,lasiokau-rin are concentrated in 30:1 elution parts of sillica column;enmein,sculponeatin A,nodosin are concentrated in 20:1 elution parts of sillica column.4.There is no difference of QAMS and external determination method.ConcludesDiterpenoids are rich in EtOAc part and the diterpenoids components are all 6,7-seco-ent kauranes.All these results indicat that the method is simple accurate and repeatable,and can be used for the quality assessment of Isodon serra.