Studies On Chemical Constituents And Pharmacological Activity Of The Leaves Of Forsythia Suspensa(Thunb.)

Forsythia suspensa（Thunb.）Vahl is flowering deciduous shrubs belonging to the Forsythia genus of Oleaceae family.It is widely distributed in Shandong,Henan,Hebei,Shanxi,Shaanxi,Gansu,Hubei,Sichuan and other provinces of China.The fruits of F.suspensa have detoxification,and reducing swelling activity,which was used to treat ulcer,scrofula,acute mastitis,erysipelas,anemopyretic cold,febrile disease early,coma,exanthesis,fever,thirst,anuresis,odynuria,etc.Studies have found that many types of compounds were found in F.suspensa,such as triterpenoids,phenethyl alcohol glycosides,lignans,flavonoids and so on,which have a variety of activities,such as antibacterial,anti-inflammatory,anti-cancer,anti-viral and anti-oxidation.Some active ingredients in F.suspensa,for example,forsythin and forsythiaside A,exist in F.suspensa leaves with higher level than in the fruits of F.suspensa.The leaves of F.suspensa have been used as tea for local residents to keep healthy in longevity village in Wu’an of Hebei and Pingding of Shanxi for several hundreds of years.So,to figure out the chemical composition and its possible physiological activity of F.suspensa leaves,is very essential for us to provide some scientific basis for its medicinal alternative use instead of its fruits.In that case,leaves could be well developed and used,that is,turning waste into treasure.Objective: In order to enrich the chemical compounds library of Forsythia genus and to find new effective ingredients,the chemical constituents of F.suspensa leaves were studied by using a variety of methods,such as silica gel column chromatography,reverse phase silica gel column chromatography,Sephadex LH-20 column chromatography.The isolated compounds were identified by UV,¹H-NMR,¹³C-NMR,HMBC,HSQC,¹H-¹H COSY,NOESY and so on.Methods: The leaves of F.suspensa of 4.4 Kg（dry weight）were extracted with ethanol and divided into petroleum ether（60⁹0℃）,CH₂CL₂,EtOAc and n-butanol parts.The parts of Et OAc and n-butanol were isolated and purified by methods of macroporous resin chromatography,silica gel column chromatography,reversed phase column chromatography,Sephadex LH-20 column chromatography.26 compounds have been isolated,and their structures were deduced with UV,1D and 2D NMR,MS,et al.Anti-proliferation inhibitory activity to human tumor cell and anti-tyrosine phosphatase（PTPs）activity of these compounds were tested.Results: 26 compounds were isolated from the leaves of F.suspensa,including twelve triterpenoids,four lignans,three flavonoids,three phenyl glycosides,four other compounds: β-Ursolic acid（1）,Dammarolic acid（2）,Corosolic acid（3）,Suspenamide A（4）,6’’-Acetyl forsythin（5）,Hovenic acid（6）,Forsythin（7）,（+）Epipinoresinol-4’-β-d-glucoside（8）,β-D-Glucopyranose,1-[3-（4-hydroxyphenyl）-2-propenoate]（9）,Quadranoside IV（10）,Arjunglucoside II（11）,Kaempferol（12）,Quercetin（13）,Trans-caffeic acid methyl ester（14）,（+）-Phillygenin（15）,Suspensanoside A（16）,Niga-ichigoside F1（17）,Arjunglucoside I（18）,（19）Protocatechuic acid（20）,Suspensanoside C（21）,Suspensanoside B（22）,Forsythoside A（23）,Forsythoside I（24）,Glucose（25）,Rutin（26）.Among these compounds,Suspensanoside A（16）,Forsythin（7）,（+）Epipinoresinol-4’-β-D-glucoside（8）,Kaempferol（12）,（+）-Phillygenin（15）and Nigaichigoside F1（18）Exhibited significant antiproliferative activity against MCF-7 and BGC-823 cancer cells.On the other hand,Suspenamide A（4）showed strong PTPs inhibitory activity.Conclusion: Twenty-six compounds were isolated and identified from the EtOAc and n-butanol extracts of F.suspensa leaves,Among these compounds,five are new compounds,including three triterpenoids: Suspensanoside A（16）,Suspensanoside B（23）,Suspensanoside C（22）,an anthranilic acid derivative,Suspenamide A（4）,and a lignan,6’’-Acetyl forsythin（5）.Asiatic acid（2）,Corosolic acid（3）,Hovenic acid（6）,b-D-glucopyranose,1-[3-（4-hydroxyphenyl）-2-propenoate]（9）,Arjunglucoside II（10）,quadranoside IV（11）,kaempferol（12）,trans-caffeic acid methyl ester（14）,niga-ichigoside F1（17）,arjunglucosi de I（18）are isolated for the first time from Forsythia genus.This is the first report about the biphenyl-4-sulfonyl group being found as an acyl substituent in natural products（compound 4）.The new compound,Suspensanoside A（16）,showed potent anti-proliferative activity with IC₅₀ values of 12.44 ± 0.69 μM against MCF-7 and 15.44 ± 0.99 μM against BGC-823.Forsythin（7）,（+）epipinoresinol-4’-β-D-glucoside（8）,Kaempferol（12）,（+）-Phillygenin（15）and Nigaichigoside F1（17）showed strong anti-proliferative activity on dose-dependent manner to MCF-7 tumor cells.Their IC₅₀ values range from 11.22 ± 0.59 to 24.30 ± 1.01 μM.Besides,Suspenamide A（4）exhibited potent inhibition activity against different PTPs（Cdc25B,PTP-MEG2,PTP1 B,SHP-1）.