Study On Chemical Constituents And Anti-H₅Subtype AIV Activity Of Leaves From Lonicera Japonica Thunb.

Lonicera japonica Thunb. which belongs to the Caprifoliaceae Lonicera family withperennial evergreen habits, is common traditional Chinese medicine and widely used in food,health product, medicine field for its broad medicinal functions such as clearing heat,removing toxicity, antibacterial and antiphlogistic effects. Leaves of L. Japonica are the offalof this plant. The outputs of leaves are ten times larger than flowers. Many studies indicatedthat the active components in leaves were similar to flowers with strong pharmacologicalactivities. In this paper, the chemical components were extracted, separated, analyzed,structural identification. The anti-H₅subtype AIV activity of chemical parts was comparedleaves with flowers of L. japonica. The main ingredients and compounds separated in leaveswere measured with anti-H₅subtype AIV. The above researches provided bases for theapplication of leaves in anti-AIV instead of flowers and the main conclusions as follows:（1） The volatile components in fresh leaves and flowers of L. japonica were comparativeanalyzed with HS-SPME/GC-MS. By analyzing fresh leaves （esters53.29%, alcohols37.67%）, flower bud stage （hydrocarbons37.79%, esters30.04%, sesquiterpenehydrocarbons12.92%）, silver flowering stage （oxygenated monoterpenes28.98%,sesquiterpene hydrocarbons21.20%, esters15.91%）, gold flowering stage （alcohols25.44%,oxygenated monoterpenes23.77%, esters18.96%） in L. japonica var. chinensis wakey withthose of L. japonica in fresh leaves （alcohols83.59%）, flower bud stage （oxygenatedmonoterpenes40.47%, hydrocarbons19.72%, esters14.83%）, silver flowering stage（oxygenated monoterpenes36.61%, sesquiterpene hydrocarbons32.48%）, gold floweringstage （oxygenated monoterpenes34.07%, sesquiterpene hydrocarbons30.72%）, the mainvolatile components in leaves were alcohols and esters. The main compounds were（Z）-3-hexen-1-ol acetate and （Z）-3-hexen-1-ol. And the terpenes in leaves were very low. The65μm PDMS/DVB fiber coating was chosen for the further study considering its betterperformance in extracting more variety of compounds and larger amount. Analysis of volatilecomponents in L. japonica was suitable for its convenient and quick.（2） The volatile oil components in dry leaves and flowers of L. japonica were analyzed with SFE-CO₂/GC-MS. The optimal SFE-CO₂extraction conditions were40℃oftemperature,30MPa of pressure, and40-60mesh of sample particle size. The volatile oilcomponents in leaves were hydrocarbons （48.69%）, alcohols （28.53%）, terpenoids （10.18%）,and the main compounds were10-nonadecanol （27.14%）, hentriacontane （20.87%）,nonacosane （19.59%）. The volatile oil components in flowers were hydrocarbons （59.81%）,esters （12.12%）, and the main compounds were nonacosane （26.85%）, hentriacontane（13.28%）, heptacosane （9.10%）.（3） Thirty compounds and three proportional mixtures were isolated from leaves of L.japonica by silica gel column chromatography, sephadex LH-20, preparative HPLC. Thechemical structures of twenty eight compounds were identified by physicochemical propertiesand spectral methods, including3,4-dihydroxybenzoic acid （1）, β-sitosterol （2）, caffeic acid（3）, ρ-coumaric acid （4）, ferulic acid （5）, isoferulic acid （6）, quercetin （7）, luteolin （8）,diosmetin （9）, daucosterol （10）,3,4-O-dicaffeoylquinic acid methyl ester （11）, secoxyloganin（12）, chlorogenic acid （13）,5-O-caffeoylquinic acid （14）,4-O-caffeoylquinic acid （15）,luteoloside （16）, lonicerin （17）, isoquercitrin （18）, hyperoside （19）, demethylsecologanol（20）, secologanin （21）, rhoifolin （24）, diosmetin-7-O-β-D-glucoside （25）,3,4-O-dicaffeoylquinic acid （26）,3,5-O-dicaffeoylquinic acid （27）, isorhamnetin-3-O-glucoside（28）, rutin （29）,4,5-O-dicaffeoylquinic acid （30）, among which compound20was firstisolated from Lonicera, compound9and28were first isolated from L. japonica andcompound1,3,5,6,15,18,19,24,29were isolated from leaves of L. japonica for the fisttime. Because of the similar main compounds with flowers, leaves of L. japonica was feasiblefor further alternative flowers in poultry medicine on view of the material basis.（4） Flavonoid glycosides and caffeoylquinic acid derivatives from leaves of L. japonica byHSCCC and PRE-HPLC. Four caffeoylquinic acid derivatives including chlorogenic acid,3,4-O-dicaffeoylquinic acid,3,5-O-dicaffeoylquinic acid,4,5-O-dicaffeoylquinic acid and fiveflavonoid glycosides including lonicerin, rutin, rhoifolin, luteoloside were isolated with a twoHSCCC solvent system of methyl tert-butyl ether-n-butanol-acetonitrile-water （0.5%aceticacid）（2:2:1:5, v/v）. HSCCC is suitable for separation of active compounds from L.japonica for its fast and efficient. This study showed that an efficient method for preparativeisolation and purification high polar compounds was established combined HSCCC withPRE-HPLC.（5） Anti-H₅subtype AIV chicken embryos studies were researched comparing leaves withflowers from L. japonica using the same chemical part of volatile oils, petroleum ether,alcohol, ethyl acetate, n-butanol, aqueous extracts. The chorioallantoic cavity inoculation method was used to11-day-old SPF chicken embryos. The growth, death of chicken embryosand HA were recorded. The results showed that volatile oils, petroleum ether, alcohol, ethylacetate, n-butanol, extracts of L. japonica had the abilities of anti-AIV, while the aqueousextracts not good. The anti-AIV ability of leaves was higher than flowers excepted of volatileoils and aqueous extracts. The results showed research basis for the development of leaves. Italso showed that leaves of L. japonica was feasible for further alternative flowers in anti-AIVdrug research.（6） CPE inhibition experiment was used to evaluate the ability of crude fractions isolatedfrom leaves, flowers and compounds purified from leaves. The HA test was also be record.The preliminary results showed that the flavonoid glycosides and caffeoylquinic acidderivatives had good anti-AIV activities. When the virus inoculation amount was0.1TCID50/0.1mL, luteolin, compound23and3,4-O-dicaffeoylquinic acid methyl ester couldbe completely inhibited AIV at the minimum concentrations of94,752and752μg/mL. Thenumber of CPE is reduced and HA becoming zero. Those three compounds had the potentialin depth development value.