Study On The Chemical Constituents And Bioactivities For The Leaves Of Rhododendron Molle (Ericaceae)

Rhododendron molle(Blume)G.Don.is widely distributed in Zejiang,Jiangsu,Jiangxi,Anhui,Fujian,Hunan,Hubei,Henan,Guangxi,Guangdong,Guizhou,Sichuan and Yunnan provinces of China and grown on mountain slopes,grassy hillsides,and ridge woods at an altitude of about 1000 meters.The whole plant of R.molle could cure diarrhea,vomiting or cramps.In clinic,this plant was always used to treat rheumatoid arthritis and injuries.In pharmaceutical industry,this species was commonly applied as anesthetics,analgesics,and pesticide.Its root extract can significantly improve the deterioration of chronic nephritis.Previous chemical studies mainly fouced on the flowers,roots,and fruits of R.molle,and the leaves of R.molle have never been reported.Considering the flowers and fruits of this plant are seasonal,the roots are not renewable.By contrast,the regeneration ability of the leaves is strong,the source of the leaves is rich,and the research of the leaves is green.Thus,we studied on the chemical constituents in leaves of R.molle for the first time.A total of 132 compounds were isolated by silica gel columns,RP C18 columns,Sephadex LH-20 columns,and the semipreparative RP C18 HPLC methods.These compounds were identified by spectroscopic analysis(UV,IR,NMR,MS,CD),quantum chemical calculations,and single crystal X-ray diffraction analysis.And 92 new compounds(1–39,41,42,45–71,76–79,82–89,103–108,110–112,and 117–119)(including 27 new skeleton diterpenoids,1–27)and 33 single crystals(including a derivative 11a),have been assigned.The chemical types of these obtained components involved nor-sesquiterpenoid,diterpenoid,triterpenoid,lignin as well as flavonoid and so on.These findings enriched our knowledge about the chemical diversity of R.molle.Compound 1–8 are the first example of diterpenoids yielded by twice enzymatic Wagner-Meerwein rearrangement through the grayanane diterpenoid,possessing tetracyclo [10.3.1.01,10.04,8]hexadecane scaffold.Compound 9 is the first example of new skeleton diterpenoid obtained by the grayanane diterpenoid through once enzymatic Wagner-Meerwein remote rearrangement,which bears a rare 7-oxabicyclo[4.2.1]nonane core fused with three cyclopentanes.Compound 10 is so similar with the grayanane diterpenoid except that the 20-CH3 is located at C-1.Compound 11 and 12 are highly mutated new scaffold diterpenoids with a free benzene ring.Compound 13 is the first 5,6-seco-14-nor-grayanane diterpenoid with nineteen carbons.Compound 14 and 15 are new scaffold diterpenoids originated from the mollane type diterpenoid by once enzymatic Wagner-Meerwein rearrangement,possessing a new 16-oxo-pentacyclo [7.5.3.0.01,9.03,7.012,15]heptadecane skeleton.Compound 16 is obtained by thrice enzymatic Wagner-Meerwein rearrangement originated from the grayanane type diterpenoid,bearing a tetracyclo[10.3.1.01,10.03,7]hexadecane scaffold.Compound 17 is the first example that 20-CH3 participated in the formation of the benzene ring.Compound 18–32 are all seco-grayanane type diterpenoids afforded by oxidative cleavages of carbon-carbon bonds.Mollane type diterpenoids 33–39 possess long conjugate structures,which are very rare for its highly oxidation degrees.And compounds 41 and 42 are highly oxygenated kalmane type diterpenoids.Compounds 45 is a grayanane dimer,C-2 and C-14' is connected by an oxygen bridge.Other new diterpenoids(46–71,76–79,82–89,103–108,110–112,117–119)include 2,3-epoxygrayanane,5,9-epoxygrayanane,and 5,6-acetonylgrayanane and so on.In addition,the anti-inflammatory,immunomodulatory,and PTP1 B inhibitory activities of obtained diterpenoids were investigated.Results suggested compounds 72,73,75,79,84,95–99,and 101 exhibit anti-inflammatory bioactivity,the IC50 value of them range from 2.8 to 35.4 ?M.And rhodojaponin II(73)shows very potent anti-inflammatory bioactivity,the IC50 value is 2.8 ?M.New skeleton rhodochinane type diterpenoids 1 and 3 could stimulate the proliferation of CD8 cells and inhibit the proliferation of CD4,Tregs cells as well as lymphocyte B cells(enhancement rates ranging from-6.23% to-15.00%),leading to the proliferation of lymphocyte T cells to improve immunity(enhancement rates range from 25.82% to 125.38%).Moreover,compounds 7–9,11–17,21–30,35–39,41,42,47,50,55,62,66,67,85,118,and 119 exhibit PTP1 B inhibitory bioactivity,the IC50 value of them range from 5.25 to 58.94 ?M.And mollanol E(36)shows very potent anti-inflammatory bioactivity,the IC50 value is 5.25 ± 0.17 ?M.The further study suggested the mollactolides A–C(28–30)are competitive type inhibitors,the Ki value of them range from 12.39 to 17.99 ?M.In order to improve the PTP1 B inhibitory activity of 28,the phenylsulfonic acid was treated with 28.The results indicated that the IC50 and Ki of obtained mollactone B 3-O-sulfate(28a)are 0.22 ± 0.05 and 7.73 ?M,respectively.For the purpose of illustrating the mode of action between the natural compounds and PTP1 B enemzy,a molecular docking study was performed.Rusulted indicated that large numbers of inhibitors could interact with the catalytic site Cys215 of PTP1 B enemzy.In this paper,a comprehensive study suggested that the leaves of R.molle possess diverse chemical components and extensive bioactivities.Thus,the leaves should be regarded as a new officinal part of R.molle.These results provide clues to design narural non-steroidal anti-in-flammatory drugs,immunoenhancers as well as novel PTP1 B inhibitors from the leaves of R.molle.