Study On The Chemical Constituents And Quality Evaluation Methods Of The Resin Of Commiphora Myrrha

The genus Commiphora(Burseraceae)with more than 150 species,is mainly distributed in the tropical and subtropical regions,especially in Northeastern Africa,Southern Arabia and India.The myrrha recorded in Chinese Pharmacopoeia 2020 edition included dry resin of Commiphora myrrha or C.molmol,which is divided into natural myrrha and gum myrrha.Myrrha is used for relieveing pain and promoting blood circulation for a long time since Tang Dynasty.The quality standard of myrrha varies in different countries and regions due to its complex sources and chemical constituents.Thus,it is urgent for the establishment of a more scientific and reasonable standard for myrrha and its products through modern analysis technology.In this paper,the chemical constituents of the resin of C.myrrha were studied systematically.As a result,20 componuds were separated and purified through chromatographic column separation and recrystallization.The structures were elucidated by MS,1D-/2D-NMR COSY,HSQC,HMBC,ROESY)as:myrrhterpenes A-B(1-2),myrrhone(3),dihydropyrocurzerenone(4),curzerenone(5),rel-1 S,2S-epoxy-4R-furanogermacr-10(15)-en-6-one(6),(1 E)-3-methoxy-8,12-epoxygermacra-1,7,10,11-tetraen-6-one(7),alismol(8),myrrhterpenoid C(9),(-)-myrrhalactone A(10),(+)-myrrhalactone A(11),commipholinone(12),dihydrocurcolone(13),curcolone(14),eudesm-4(15)-ene-1β,6α-diol(15),rel-2R-methoxy-4R-furanogermacra-1(10)E-en-6-one(16),2α-methoxy-8α-hydroxy-6-oxogermacra-1(10),7(11)-dien-8,12-olide(17),myrrhanolide B&C(18&19),and myrrhalactone C(20).Myrrhterpenes A-B were new cadinane-type sesquiterpenes,and compound 8 and 17 were isolated from C.myrrha for the first time.The anti-inflammatory activity was tested by measuring the inhibitory effects on the NO production in LPS-induced RAW 264.7 macrophage cells,and compounds 1,2,6,7,10,11-15,17 and 20 displayed potent anti-inflammatory activities with IC50 values of 0.73-18.36 μM.The quality evaluation method myrrha was studied by using 18 samples of the resin of C.myrrha,a sample of the resin of C.molmol,2 samples of vinegar-processed C.myrrha,2 samples of vinegar-processed C.molmol and 9 samples of vinegar-processed C.myrrha formula granules.The thin-layer chromatography method was established after investigating different extraction solvents,developing solvents and chromogenic agents.The samples were extracted by n-hexane under ultrasonics for 30 min,silica gel GF254 TLC plate was used,wiht a mixture of cyclohexane and diethyl ether(4:1)as developing solvent.10%sulfuric acid in ethanol was selected as chromogenic agent.The resin of C.myrrha and C.molmol can be distingluished through this method.The HPLC characteristic fingerprint spectra of myrrha and its formula granules were established.The octadecyl silane bonded silica gel was selected as the stationary phase,methanol(A)and water(B)were used as mobile phases with a gradient elutiion at a flow rate of 0.8 ml/min.The detection wavelength was 270 nm and the column temperature was 30℃.The results of precision,repeatability,stability and retardance were all good enough for the requirements.Nine common peaks of the resin of C.myrrha were determined and five of them were identified.Thirteen common peaks of the resin of myrrha formula granules were determined and seven of them were identified.The resin of C.myrrha and C.molmol can be distinguished by this method.The content determination of myrrhone was set up by HPLC.The octadecyl silane bonded silica gel was used as stationary phase,and methanol(A)and water(B)were used as mobile phases with a isocratic elution at a flow rate of 0.8 ml/min.The detection wavelength was 240 nm and the column temperature was 30℃.The linear range of myrrhone was 5.219～83.496 μg(R2=0.9999),the limit of detection was 4.4375 pg/ml and the limit of quantitation was 13.3125 pg/ml.The average recovery(n=9)of the resin of C.Myrrha was 100.3%with RSD as 1.57%.The average recovery(n=9)of myrrha formula granules was 103.1%with RSD as 1.33%.The results of precision,repeatability and stability were all good enough for the requirements.The myrrhone contents in the resin of C.myrrha and its formula granules were 0.137～1.735 mg/g and 0.197～1.218 mg/g,repectively.