Studies On The Chemical Constituents And Pharmacological Activity Of Rhododendron Micranthum Turcz

Rhododendron micranthum is a species of Rhododendron,Ericaceae,with nicknames of zhaobai DuJuan,xiaohua DuJuan.It is mianly distributed in Henan,Hubei,Hunan,Sichuan,northeast,north and northwest of China,also existed in Korea.The branches and leaves of R.micranthum as a traditional Chinese medicine is recorded in the Dictionary of the Medicinal Plant.R.micranthum has been used clinically to relieve pain for thousands of years,and was used to treat chronic bronchitis,arthralgia due to wind and dampness,backache,dysmenorrheal,postpartum arthritis pain,dysentery,and fracture.Up to now,some kinds of chemical constituents,including grayanane diterpenoids,flavonoids,terpenoids,phenols and volatile oils have been reported from this plant.In order to study the chemical composition of R.micranthum and to elucidate the chemical basis of its biological activity,a systematically chemical investigation was carried out on the ethyl acetate fraction of the ethnol extract.The structures of the isolated compounds were identified on the basis of 1D-and 2D-NMR and their pharmacological activities were screened on analgesic models.Objective:The aim is to study the chemical constituents of the branches and leaves of Rhododendron micranthum by using various techniques such as silica gel column chromatography,Sephadex LH-20,preparative high performance liquid chromatography and recrystallization purification.Their structures were identified on basis of spectral data such as UV,IR,HRMS,1D-NMR,2D-NMR spectroscopic analyse.And the activeties of the isolated compounds were evaluated on analgesic models.Methods:The air-dried branches and leaves of Rhododendron micranthum?110 kg?were crushed and extracted with 95%ethanol by reflux for three times.The ethanol extract was evaporated under reduced pressure to yield a residue?12 kg?.The residue was scattered in water,partitioned with petroleum ether,dichloromethane,ethyl acetate and n-butanol,respectively.After evaporated the solvent,we got four portions of extracts.Isolation and purification were carried out through various chromatographic techniques such as silica gel column chromatography,sephadex LH-20,medium pressure,column chromatography,preparative HPLC.Refractive index?RI?detector was used to isolate those non-UV absorbing compounds.Purity of compounds was tested by HPLC-DAD-ELSD.Structures of compounds were elucidated by the spectral data of UV,IR,HRMS,¹H-NMR,¹³C-NMR,DEPT,¹H-¹H COSY,HMQC,HMBC,NOESY and CD.The analgesic effect of diterpenoids was evaluated in writhing test on mice.Results:Fifty-one compounds were isolated from the ethyl acetate fraction of ethanol extract of Rhododendron micranthum.These compounds include 18 diterpenoids?1^*-3^*,4-18?,12 lignans?19-30?,4 sesquiterpenoids?31-34?,3 phenols?35-47?and 4 other compounds?75-78?.The structures of these compounds were identified as follows:6-deoxy-grayanotoxin XVIII 3-O-?-D-glucoside?1^*?,6-deoxy-1-epi-graynoto-xin XVIII 3-O-?-D-glucoside?2^*?,grayanotoxin XVIII 6-O-?-D-glucoside?3^*?,grayanotoxin I?4?,grayanotoxin III?5?,grayanotoxin II?6?,grayan otoxin IV?7?,grayanotoxin VII?8?,grayanotoxin IX?9?,grayanotoxin VIII?10?,grayanotoxin XVIII?11?,pierisformosin A?12?,6-deoxy-1-epi-grayanotoxin XVII?13?,kalmanol?14?,grayanoside B?15?,pierisformos ide B?16?,pierisformoside F?17?,pieroside B?18?,?7S,8S?-4-hydroxy-9'-O-??-L-rhamnopyranosyl?-3,3',5,-trimethoxy-8-O-4'-neoligan?19?,icarisideE₁?erythro??20?,icariside E₂?threo??21?,?2S,3R?-2,3-dihydro-3-hydroxy methyl-7-methoxy-2-?4'-hydroxy-3'-methoxyphenyl?-5-benzofuranpr-opanol3a-O-?-L-rhamnopy-ranoside?22?,?8R,7'S,8'S?-5,5'-dimethoxy-7-oxolaricir esinol 9'-O-?-D-xylopyranoside?23?,schizandriside?24?,?-?-lyoniresinol3?-O-?-D-xylopyranosi-de?25?,?-?-secoisolariciresinol-O-?-L-rhamnopyranosi de?26?,ssioriside?27?,?-?-secoisolariciresinol-9-O-?-D-xylopyranoside?28?,prupaside?29?,alutaceuol?30?,3?-[?-?-D-glucpyrnosyl?o-xy]eudesma-4?14?.11?13?-dien-12-ol?31?,zedoarolide B?32?,10,11-dihydro-10,11-dihyd roxynerolidol?33?,schensianol A?34?,esculetin?35?,sco-polin?36?,2-[4-?3-Hydroxyproxypropyl?-2-methoxphenoxy]propane-1,3-di-ol?37?,threo-3-?4-hydroxy-3,5-dimethoxyphenyl?-3-ethoxypropane-1,2-diol-?38?,3-?4-hyd roxy-3,5-dimethoxyphenyl?propane-1,2-diol?39?,pisoninol?40?,2-phenyle thyl-?-D-glucopyranoside?41?,4-O-?-D-glucopyranosyl-2-O-methylphloroac etophenone?42?,4-ally-2,6-dimethoxyphenyl 1-O-?-D-apiofuranosyl-?1''-6'?-O-?-D-glucopyranoside?43?,eugeny l-O-?-D-apiofuranosyl-?1''-6'?-O-?-D-g lucopyranoside?44?,4-allyl-2,6-dimethoxyphenolglucoside?45?,benzyl2-h ydroxy-4-O-?-D-glucopyranosyl-benzoate?46?,amarusine A?47?,9?-O-?-D-glucopyranosyloxy-5-megastigmen-4-one?48?,?-?-3-oxo-?-ionyl-O-?-D-glu copyranoside?49?,?Z?-3-Hexenyl?-D-glucopyranoside?50?,oct-1-en-3-yl?-glucopyranoside?51??The analgesic activities of compounds 1,2,4-18 were evaluated in the writhing test.At a dose of 0.1 mg/kg,compounds 4 showed significant analgesic activity?*P<0.01?.Compounds 5,9,14 reduced the writhes of mice by 70%,44.3%and 42.9%,respectively,at the dose of 0.1mg/kg.Compounds6,7,8,10,11,13 inhibited the writhes of mice by 54.3%,42.9%,27.1%,12.1%,30.0%,and 38.6%,respectively at the dose of 1.0 mg/kg.At a dose of10 mg/kg,compounds 1,12 showed analgesic activity?*P<0.05?.Conclusions:Three new compounds,together with 48 known compounds were isolated from the ethyl acetate portions of the ethanol extract of Rhododendron micranthum Turcz.The compounds covered 8 diterpenoids,12 lignans,4 sesquiterpenoids,13 phenols and 4 others.Among these compounds,there are 3 new compounds?1*-3*?.Among others which were known,27 compounds?12,16,17,19-24,26,28,29,31-34,37-40,43-49?were isolated for the first time from the geus Rhododendron;5 compounds?15,18,25,27,30?were isolated from Rhododendron micranthum Turcz for the first time.All the 3 new compounds separated from Rhododendron micranthum were diterpenoid glycosides.Among three new compounds,compound 3 is the first 6-OH grayanane diterpenoid glycoside.All the diterpenoids were evaluated on the writhing test.Based on the results of the writhingtest,structure-activityrelationships?SARs?ofgrayanane diterpenoids were summarized:?1?grayanane diterpenoid glycosides were less active and less toxic comparing with corresponding aglycones,?2?3?-OH,5?-OH,10?-CH₃ substitutions were necessary for the activity,?3?activity decreased when there was double bond between C-10 and C-20,?4?the activity of grayananes with 1?-OH were more potent than grayananes with1?-OH,?5?the activity of grayananes with 14-OAc were more potent than grayananes with 14-OH,?6?C-15,16 become double bond had no influence on activity.