Study On The Extraction And Purification Process Of 3-hydroxy-4-prenyl-5-methoxystilben-2-carboxylic Acid From Pigeonpea [Cajanus Cajan(L.) Millps.] Leaves And Its Antibacterial Activity

In the present study,the microwave-assisted extraction(MAE)of 3-hydroxy-4-prenyl-5-methoxystilben -2-carboxylic acid(Abbreviation:HPMSCA)from the leaves of pigeonpea [Cajanus cajan(L.)Millsp.]was studied for the first time,and the process for separation and purification of HPMSCA by macroporous absorption resin,silica gel column chromatography, recrystallization,etc.was optimized.Meanwhile,the preliminary antimicrobial activity of HPMSCA was also investigated.The results as follow:1.An RP-HPLC-PAD method for the determination of HPMSCA was developed firstly as follows:The chromatographic column was Symmetry Shield^?RP₁₈,150 mm×3.9mm,5μm; mobile phase was methanol-water-formic acid(80:19.9:0.1,v/v/v),flow rate was 1 mL/min; PAD at 259.2 nm,injection volume 10μL,and column temperature 35℃.The determination of HPMSCA in the RP-HPLC-PAD condition showed that the R.S.D. of the precision and repeatability with this method were all less than 3%.2.The microwave-assisted extraction of HPMSCA from pigeonpea leaves was first time to optimize,and the optimum extraction parameters were as follows:The apparatus power:300 WExtraction solvent:80%EtOHRatio of liquid to solid:30:1Extration cycle:2 cyclesExtraction time:each cycle 1 minHPMSCA could be rapidly and effectively extracted under the above-mentioned conditions,the extraction yields of HPMSCA was 19.00 mg/g,while the content was 5.922% in the extractum.3.The optimum process for the purification of HPMSCA from pigeonpea leaves was obtained firstly,and the results were as follows:(1)The HPMSCA in extracts were enriched by NKA-9 resin with the 30%EtOH as feed solution,in which the content of HPMSCA was 0.039 mg/mL,and the feed process was performed with 3 BV/100g(Dry Resin)in the flow rate of 1.5 BV/h.And the desorpt was performed with 5 BV/100g(Dry Resin)as the solution of 85～90%EtOH,the flow rate was 1 mL/min.After enriched by NKA-9 resin under the above conditions,the contents of HPMSCA increased 3-fold to 18.4%,the recovery yields was 69.15%.(2)Pigeonpea leaves extracts after separated by NKA-9 resin were purified by normal- phase medium pressure silica gel column chromatography.An gradient elution was performed with CHCl₃-Petroleum Ether(10～15/1,v/v)on the column,in which filled silica gel of 300 to 400 meshes,the quantity ratio of sample/silca gel was 1:4,the flow rate was 10 mL/min.And after this process the content of HPMSCA was 52.19%,the recovery was 73.35%。(3)The further purification of HPMSCA was done with reverse-phase medium pressure ODS column chromatography combined recrystallization.The quantity ratio of sample/ODS silica gel was 1:100;the flow phase was MeOH-H₂O-CH₂O₂(76.5:23.2:0.3,v/v/v);and crystallization was done in methanol,recrystallization was finished with solution of CHCl₃-Petroleum Ether(1:1,v/v).The HPMSCA white powder with more than 93%in purity was obtained,the recovery was 62.82%.Finally,the yield of HPMSCA through the whole purification process was 31.86%.4.The preliminary antibacterial activity showed that HPMSCA has rather strong inhibition effect on Proteus vulgaris,Staphylococcus epidermidis,Staphylococcus aureus and Bacillus subtilis,among the four strains,Proteus vulgaris belongs to Gram-negative bacteria (G^-),and Staphylococcus epidermidis,Staphylococcus aureu belong to Gram-positive bacteria (G⁺).The bacterial inhibition diameters were 15 mm,18 mm,20 mm and 15.5 mm, respectively.The mean MIC of the four strains was 25μg/mL,and that of MBC was 50μg/mL. The bacterial inhibition diameters of HPMSCA to the other G^-strains including Escherichia coli and Pseudomonas aeruginosa were all 6 mm,the MIC and MBC were all higher than 200μg/mL,which demonstrated there were no the antibacterial action.While under the maximum concentration of 200μg/mL,there were also no antibacterial action observed to the fungi of Candida albicans and Aspergillus niger.In summary,the article was on the base of determinated of HPMSCA,and taken the investigation on the microwave-assisted extraction of HPMSCA,together with its separation and purification by column chromatography,while the HPMSCA with more than 93%in purity was obtained,corresponded to the material the yield was 5.79 mg/g.And end with the preliminary antibacterial activity study which showed obvirous antibacterial effect.This reseach provided significant theoretical basis for the further exploitation and utilization of pigeonpea resource,as well as the development of new drugs from HPMSCA.