Research About Composition Of Lycopus Lucidus Turcz. Var. Hirtus Regel From Korean Chinese Folk Medicine Based On Antibacterial Activity

Zelan as the Korean Chinese folk medicine is the dry aerial part of Lycopus lucidus Turcz.var.hirtus Regel.It has some pharmacological effects such as anti-bacterial and anti-inflammatory effects,needs to be further investigated and explored.The aims of this study were to isolate the monomer compound with anti-bacterial activity,and establish basis for the further development of Zelan.In this study,methanol was used as extracting solvent and heat-refluxing method was used to extract Zelan,the concentrated extract was extracted with petroleum ether and ethyl acetate for three times.The ethyl acetate extract with better anti-bacterial activity was performed to separate and purify by various chromatographic methods such as Sephadex LH-20 gel chromatography,silica gel column chromatography and ODS column chromatography.A total of 22 compounds were identified by a variety of spectroscopy methods as:β-sitosterol(1),lupeol(2),betulinic aldehyde(3),betulinic acid(4),ursolic acid(5),euscaphic acid(6),oleanic acid(7),apigenin(8),luteolin(9),trans-cinnamic acid(10),4-hydroxy-3-methoxycinic acid(11),p-hydroxybenzoic acid(12),3,4-dihydroxybenzoic acid(13),3,4-dihydroxybenzaldehyde(14),(+)-(R)rosmarinic acid(15),(+)-(R)-methyl rosmarinate(16),(+)-(R)-methyl rosmarinate and(-)-(S)-methyl rosmarinate(17),quercetin-3-O-β-D-glucopyranoside(18),luteolin-7O-β-D-glucopyranoside(19),scutellarin(20),rutin(21),stearic acid(22).Among them,compounds 2,3,6,10,11,12 and 17 were separated from Zelan for the first time.All compounds were carried out anti-bacterial activity and the results showed that compounds 2～9,14～19 and 22 exhibited antibacterial activity.It was the first report for the antibacterial activity of compound 17.The results of minimum inhibitory concentration(MIC)showed that compounds 9,16 and 17 had strong inhibitory effect against Escherichia coli KCTC(1924)and Quinolone-resistant Staphylococcus aureus CCARM(3519),the MIC values were 4～16μg/mL.Compound 9 had a strong inhibitory effect on a variety of bacteria,which were 4 μg/mL of the MIC value of Methicillin-resistant Staphylococcus aureus CCARM(3167),S.aureus(503)and S.aureus KCTC(209);for Bacillus cereus(1661)inhibit the inhibition,the MIC value was 16μg/mL.Compounds 15,16 and 18 had good activity against Quinolone-resistant Staphylococcus aureus CCARM(3505)and the MIC value could reach 16 to 32 μg/mL.Compound 15 had good antibacterial activity of Bacillus subtilis(1021)and compound 17 on B.cereus(1661)with MIC values of 16 μg/mL,respectively.Compounds 9,16 and 18 showed inhibitory effects on Canidia albicans(7535),and the MIC values were 64 μg/mL.Compound 5 had same MIC value of 32 μg/mL against MRSA CCARM(3167),QRSA CCARM(3519)and E.coli KCTC(1924).The minimum bactericidal concentration(MBC)values of compounds 5,6,7,14,18 and 19 were less than 4 times MIC,then the compounds were not easily resistant to bacteria.From bacterial growth curve,it was found that compounds 9 and 16 could significantly inhibit the growth of E.coli KCTC(1924),which was enhanced with the increase of drug concentration.Nowadays,the content standard data of Zelan is not described in the Pharmacopoeia.We found that the contents of compounds 4 and 15 were larger than other compounds,so determined to analyze the content of these two compounds by HPLC simultaneously,and the results could provide a reference for the content standard of Zelan in the Pharmacopoeia.In the results,compound 4 acid has a good linear relationship at the concentration range of 0.02～1 mg/mL,Y=3609X+17374(R2=0.9997);the regression equation of compound 15 is Y=31992X-8037.8(R2=0.9996)at the concentration range of 0.001～0.03 mg/mL.The RSD of the precision,stability and repeatability test in the method validation of the two compounds were all less than 2%(n=6),and the sample recovery rate was between 98%and 102%(RSD=0.18%and 0.23%),so it can be confirmed that the HPLC method can accurately determine the content of compounds 4 and 15 in Zelan.In the results,the content of compounds 4 and 15 in Zelan were 36.8 mg/g and 11.4 mg/g,respectively.