Studies On The Chemical Constituents And Bioactivities From The Leaves Of Rhododendron Micranthum And Rhododendron Auriculatum (Ericaceae)

Grayanane diterpenoids are the unique metabolic constituents of Ericaceae plants,especially in the genera of Rhododendron and Pieris.In recent years,grayanane and related diterpenoids isolated from Ericaceae plants showed good analgesic activities.Therefore,this stimulates our interest in discovering new diterpenoids with analgesic activities from Ericaceae plants.We studied on the chemical constituents and biological activities from the leaves of the Ericaceae plants Rhododendron micranthum Turcz.and Rhododendron auriculatum Hemsl..A total of 128 compounds were isolated and identified by various separation methods such as spectroscopy,chemical methods,and single crystal X-ray diffraction analysis.Among them,51 new compounds were identified,including 3 new skeleton compounds.The isolated compounds involved diterpenoids,triterpenoids,sesquiterpenoids,monoterpenes,lignans,and flavonoids.In addition,most of the isolates were evaluated for the anti-inflammatory,anti-tumor,PTP1 B inhibition,immune regulation,as well as analgesic activities.And some of them showed significant analgesic activities.This study is of great significance for the development of new natural lead compounds with analgesic activity in the later clinical stage.Chapter 1.Study on the chemical constituents and bioactivities of n-butanol fractions from the leaves of R.micranthum.Rhododendron micranthum,a semi-green or evergreen shrub,mainly grows in North and Northeast China,and Shandong,Henan,Sichuan,Hunan,and Hubei provinces.The branches and leaves of this plant have been used as traditional medicine to treat rheumatoid arthritis,puerperal arthrodynia,menoxenia,hypertension,fracture,dysmenorrhea,and chronic tracheitis,in China.Previous phytochemical investigations revealed the presence of micranthane,grayanane and leucothane type diterpenoids,and flavonoids in R.micranthum.However,there is no report of diterpenoid glucosides from R.micranthum.In order to exploring novel and active diterpenoid glycosides from this plant,the n-Bu OH fraction of the leaves of R.micranthum was investigated,leading to the isolation of 56 compounds including 26 new glucosides(1–23,37–38,and 45–46).Compound 1 represents the first example of a 3?-oxygrayanane diterpenoid glucoside,compounds 2–5 are the first examples of 5?-hydroxy-1-?H-grayanane diterpenoids,compounds 4-6,8,and 9 represent the first examples of grayanane glucosides with the glucosylation at C-16.Compounds 14–16 represent the first examples of 15?-hydroxy-leucothane diterpenoids,leucothane diterpene diglucosides,and 9?-hydroxy-leucothane diterpenoids,respectively.Compounds 17 and 18 are the second and third examples of 4,5-seco-ent-kaurane diterpenoids,and this is the first time to report 4,5-seco-ent-kaurane-type diterpenoids from the genus of Rhododendron.Compounds 18 and 19 are the first examples of 5?-H-grayan-1(10),9(11)-diene-6-one diterpenoids.In addition,compounds 1–26,28–31,37–40,42,45–48,and 51–56 were isolated from this plant for the first.The absolute configuration of the glucose was determined by chemical methods,the absolute configuration of 18 was determined by Mo2(OAc)4-induced ECD,and the absolute configurations of compounds 1,2,4,14,and 30 were assigned by single crystal X-ray diffraction analysis.This is the first report of the crystal structures of grayanane and leucothane diterpenoid glucosides.In addition,the diterpenoid,sesquiterpenoid,and phenylpropanoid glucosides were evaluated for their anti-tumor and anti-inflammatory.Compound 46 showed good anti-tumor activities against HL-6,SMMC-7721,MCF-7,and SW480 cells.The analgesic activities of the isolated diterpenoids 1–22,24–28,30,and 32–36 were evaluated by acetic acid-induced writhing test in mice.Compounds 2,3,7–10,12–16,17–19,21,26,30,and 32–36 showed significant analgesic activities,with the inhibition rates in excess of 50%.In particular,compounds 35 and 36 exhibited more potent analgesic activity than morphine at a dose of 0.04 mg/kg,with the inhibition rates of 71.5% and 69.3%,respectively.Chapter 2.Study on the chemical constituents and bioactivities from the leaves of R.auriculatumRhododendron auriculatum,belong to the genus of Rhododendron,an evergreen shrub or small tree,is native to China,and widely distributes in northern Guizhou,western Hubei,southern Shanxi,and eastern Sichuan of China.In mainland china,the bark and roots have been used as a folk medicine to treat cough.To date,there is no report of any diterpenoids from R.auriculatum,although seven known compounds including two triterpenoids and five phenolic compounds have been reported from the branches of R.auriculatum.Therefore,in a course of search for novel diterpenoids from the Ericaceae plants,the leaves of R.auriculatum were investigated.In this study,77 compounds including 24 new ones(57–80,and 92)were isolated and identified from the leaves of R.auriculatum.Compound 57 represents the first example of a 5,20-epoxy-grayanane diterpenoid.Compound 58 is the first example of a 3?,5?-dihydroxy-1-?H-grayanane diterpenoid.Compounds 62 and 63 represent the first example of 19-hydroxygrayanane and grayan-5(6)-ene diterpenoids,respectively.Compounds 70–72 represent the first examples of 1,10-seco-grayanane diterpenoids.And all the isolated compounds were obtained from this plant for the first time.The absolute configurations of compounds 58–60,66–68,70,74,76,77,82,and 84 were determined by single crystal X-ray diffraction analysis for the first time.And all of the isolated diterpenoids(57-91,2,24,28,30,33–35,and 73a)were evaluated for their analgesic activities.Compounds 58,60,62,63,66–68,71,74–77,79 and 81 showed significant analgesic effects at the dose of 5 mg/kg.In addition,compounds 33 and 35 exhibited more potent analgesic activity than morphine at a dose of 0.04 mg/kg,with the inhibition rates of 58.5% and 54.1%,respectively.