Online Copy. Extrication process of chlorogenic acid in Crofton weed and antibacterial mechanism of chlorogenic acid on Escherichia coli

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1 Extricatin prcess f chlrgenic acid in Crftn weed and antibacterial mechanism f chlrgenic acid n Escherichia cli Publicatin Inf Paper received: 21 February 2016 Revised received: 27 April 2016 Re-revised received: 5 May 2016 Accepted: 23 June Y. Zheng *, J. Liu, M.L. Ca, J.M. Deng and J. Ku Schl f Bilgical and Chemical Engineering, Panzhihua University, Panzhihua , China 2 Panzhihua Xiyu Bitechnlgy C. Ltd, Panzhihua , China *Crrespnding Authr zheng_yi_2016@163.cm 1 Intrductin Originated in Mexic, Crftn weed was intrduced th int suthern Yunnan in the middle f 20 century. It quickly spread in suthern and suthwestern China and affected the eclgical balance and threatened the grwth f crps, causing serius eclgical pressure and ecnmic lss. It is the mst infectius and harmful extic weed (Cha et al., 2007). In recent years, strengthening the cntrl n spread f Crftn weed, the research f cmprehensive utilizatin f Crftn weed has been launched t get treasure ut f the harm. As Crftn weed cntains a chemical substance, chlrgenic acid (Fu et al., 1999) that belngs t phenlic cmpunds. It is a kind f phenylprpanids frmed by the interactin between cinnamic acid and quinic acid via the shikimic acid pathway in the prcess f aerbic respiratin f plants. Chlrgenic acids are mainly effective cmpnents in Abstract Crftn weed is a perennial herb and a bilgical intruding species. The present study firstly used the rthgnal test t cmpare the differences in extractin f chlrgenic acid in leaves and stems f Crftn weed by using three kinds f slvents, namely water, ethanl and ethyl acetate. The best effect was fund by using ethnlic extractin f chlrgenic acid in Crftn weed. Further, by chsing Escherichia cli as test bject, in-vitr antibacterial test was cnducted t study the antimicrbial activities f chlrgenic acid by testing a series f indexes befre and after the interactin between chlrgenic acid and Escherichia cli, t clarify the antibacterial mechanism f chlrgenic acid n Escherichia cli. Finally, by cmparing the antibacterial activities f ischlrgenic acid A n Escherichia cli, it was cncluded that bth chlrgenic acid and ischlrgenic acid A shwed antibacterial activities against Escherichia cli, wherein chlrgenic acid had a better antibacterial effect n Escherichia cli than ischlrgenic acid A. Key wrds Antibacterial activity, Chlrgenic acid, Crftn weed, Extractin, Orthgnal test many medicines ( such as Fls Lnicerae, Herba Artemisiae, Eucmmia Bark) and in traditinal Chinese medicines (such as Fu Gan Ning, Shuang Hua injectin, acne ral liquid) which has significant pharmaclgical activities viz., antibacterial, antiviral, antixidant, anti mutagenesis, antitumr and chlaggic, as well as in antihypertensive, increasing white bld cell, stimulating central nervus system. They are widely used as additives in fds, beverages, etc., and are in great demand (Huang et al., 2012; Ca et al., 1999). Althugh chlrgenic acid exists widely in plants, but nly few kinds f plants have higher cntent. It is mainly extracted frm cca beans, hneysuckle flwer and eucmmia bark leaf which are all active pharmaceutical ingredients f traditinal Chinese medicine. Earlier research have fund that chlrgenic acid is relatively abundant in Triveni Enterprises, Lucknw (India) Jurnal f Envirnmental Bilgy, Vl. 37 (Special Issue), , September 2016

2 1050 Y. Zheng et al. Crftn weed stems and leaves, therefre, here we intend t use abundant resurce and harmful weeds-crftn weed as raw material. Hwever, the antibacterial effect and antibacterial mechanism f chlrgenic acid mnmer is nt yet fully understd and there is n available systematical research reprt. Accrdingly, in the present study, Escherichia cli, a cmmn pathgenic bacteria, was selected as test bject t test the antibacterial activity f chlrgenic acid, and thus clarifying the inhibitin mechanism f chlrgenic acid n Escherichia cli, a theretical basis fr the develpment and utilizatin f chlrgenic acids has been prvided (Da et al., 2003). Materials and Methds Experiment materials : Crftn weed leaves were cllected frm XiChang, Sichuan Prvince in Standard sample f Chlrgenic acid (brught frm Beijing XianTng Times Pharmaceutical Technlgy And Develpment c., Ltd.). High perfrmance liquid chrmatgraph (HPLC) (Agilent 1100 Series). Rtary evapratin apparatus (Shanghai YaRng Bichemical Instruments c., ltd.). Temperature cntrller heater and reflux cndensa-tin device (selfmade). All reagents are analytic pure (Beijing ChngXi Technlgy C. Ltd.) Visible ultravilet detectr, clumn temperature bx, chrmatgraphy wrkstatin. Chrmatgraphy Cnditins : Using 150 mm 4.60 mm LUNA SuC 13 (2) clumn, chsing V (methanl): V (methanl) : V (methanl) = 19:81:1.5 as mbile phase, testing with wavelength f 325 nm, at velcity f 0.5 ml min, and under 25.0 C (Li Y.S., 2013). Extractin f chlrgenic acid : Ten grams f Crftn weed 4 leaves were weighed and then grinded. Accrding t L 9(3 ) successively perfrmed the extractin 3 times, blending the filtrate and abandning the residue, then decmpressing and rtary distilling the filtrate, deslventizing and cncentrating, using HPLC t determine the cntent f chlrgenic acid in the extract, calculating extractin rati f chlrgenic acid and cntent f chlrgenic acid in extract. The experiment factr level f water extractin is shwn in Table 1. Table 2 shws, chsing water as slvent, the extractin technlgy is A 282ClD3, the extractin duratin is 2 hurs, 10 times f water, at temperature f 40 C with the PH value f 7. The rder f the factrs which affect the extractin rati f chlrgenic acid is B>A>D>C, the slid-liquid rati>extractin time>ph>extractin temperature. In ethanl extractin methd, the slid-liquid rati has the largest influence n the extractin rati f chlrgenic acid and n the cntent f chlrgenic acid in extract, while the temperature has the least influence. Ethanl Extractin : The 10.00g f Crftn weed leaves 5 were weighted and grinded. Accrding t L 16 (4 ) design methd, we successively perfrmed the extractin 3 times, blending the filtrate and abandning the residue, then decmpressing and rtary distilling the filtrate, deslventizing and cncentrating, using HPLC t determine the cntent f chlrgenic acid in the extract, calculating extractin rati f chlrgenic acid and cntent f chlrgenic acid in extract. The experiment factr level f ethanl extractin is shwn in Table 3. Table 4 shws that after chsing ethanl as slvent, the extractin technlgy is A 2B 3C 3D 4E 2, here the ethanl cncentratin is 40% and the extractin duratin is 4 hurs, i.e. 10 times f ethanl, at temperature f 80 C with the ph value f 3. The rder f the factrs which affect the extractin rati f chlrgenic acid is B>E>C>D>A, the extractin time > ph > slid-liquid rati > extractin temperature > ethanl cncentratin. Having an analysis frm the angle f cntent f chlrgenic acid in extract (mass fractin), the rder f the factrs is B>C>E>D>A, the extractin time > slid-liquid rati > ph > extractin temperature > ethanl cncentratin. In ethanl extractin methd, the extractin time has the largest impact n the extractin rati f chlrgenic acid and n the cntent f chlrgenic acid in extract, while the ethanl cncentratin has the least impact. Ethyl Acetate Extractin : The 10.00g f Crftn weed 4 leaves were weighted and grinded. Accrding t L 9(3 ) design methd, we successively perfrmed the extractin 3 times, blending the filtrate and abandning the residue, then decmpressing and rtary distilling the filtrate, deslventizing and cncentrating, using HPLC t determine the cntent f chlrgenic acid in extract, calculating extractin rati f chlrgenic acid and the cntent f chlrgenic acid in the extract. Table 6 shws, if ethyl acetate is chsen as slvent, n matter having cnsideratin frm extractin rati f Table 1 : Experiment factr level f water extractin Level Extractin Rati Temperature ph (D) Duratin h (A) f Slid t ( C) Liquid(B) : : : Jurnal f Envirnmental Bilgy, Special issue September 2016

3 Extricatin prcess f chlrgenic acid in Crftn weed 1051 Table 2 : Orthgnal test table and results f water extractin Number A B C D Extracti- Cntent f Extractin veyield chlrgenic rati f rati (%) acid in chlrgenic extract (%) acid (%) Extractiveyield rati K R Cntent f chlrgenic acid K R Extractin rati f chlrgenic acid K R Table 3 : Experiment factr level f ethanl extractin Level Ethanl Extractin Rati f Tempera ph(e) Cncen- Duratin Slid t ture ( C) tratin h (B) Liquid (C) ( D) % (A) : : : : chlrgenic acid r frm cntent f chlrgenic acid in the extract (mass fractin), the ptimum extractin technlgy is A 2B 2C 1D1, namely the extractin temperature at 50 C, ethyl acetate cncentratin is 80%, the extractin duratin is 2 hurs, and the PH value f 2. The rder f the factrs which affect the extractin rati f chlrgenic acid is A>D>C>B, the extractin temperature > ph > extractin time > ethyl acetate cncentratin. In the usage f ethyl acetate extractin, the extractin temperature has maximum effect n extractin rati f chlrgenic acid and cntent f chlrgenic acid in the extract, while the cncentratin f ethyl acetate has the least effect. Chlrgenic Acid Purificatin : NKA-9 macrprus resin 9.63g packing clumn, 5 times the vlume f distilled water, and ethanl slutin ( = 40%) which serve as eluent were adpted fr purificatin, and the purificatin results are shwn in Table 7. Chlrgenic acid and ischlrgenic acid A were purchased frm Natinal Institute fr the Cntrl f Pharmaceutical and Bilgical Prducts; 1-N-phenyl naphthylamine (NPN) was prepared frm Sigma Cmpany; Escherichia cli was cllected by Jiangxi Agricultural University micrbilgical labratry; beef extract, peptne and ther bilgical reagents were purchased frm Beijing AOBOX Bitechnlgy C., Ltd; alkaline phsphatase assay kit was cllected Nanjing Jiancheng Biengineering Institute. UV type ultravilet-visible spectrphtmeter was bught frm Japanese SHIMADZU Cmpany; 970 the CRT flurescence spectrphtmeter was bught frm Shanghai Precisin and Scientific Instrument Crpratin; DDS a cnductivity meter was btained frm Shenzhen KaDiYa Technlgy C. Ltd. Determinatin f antibacterial activity: chlrgenic acid was disslved in ischlrgenic acid A standard sample in distilled water t make 0.1 g/ml slutin as the sample f antibacterial teat. Ptassium srbate was mixed with sdium benzate t make g/ml slutin, which was used fr the cmparisn f antibacterial activity. The size f antibacterial circle was measured after the culture, and results shwed that the bigger the antibacterial circle was, the better was the antibacterial effect. Jurnal f Envirnmental Bilgy, Special issue September 2016

4 1052 Y. Zheng et al. Table 4 : Orthgnal test table and results f ethanl extractin Number A B C D E Extracti- Cntent f Extractin veyield chlrgenic rati f rati (%) acid in chlrgenic extract (%) acid (%) Extractiveyield rati K K R Cntent f chlrgenic acid K K R Extractin rati f chlrgenic acid K K R Determinatin f the cnductivity f bacterium slutin: A 0.9% NaCl slutin was used t wash the E.cli at expnential stage, s as t btain the bacterial suspensin, wherein it shuld meet the cnditin f OD 600nm= ml prepared E.cli bacterial suspensin was taken t react with 5ml chlrgenic acid material at cncentratin f 2 and 0.2 mg ml respectively fr a perid f time. After that centrifugatin (10000 r/min, 5 min) was perfrmed, and then the cnductivity f the supernatant liqur was determined. Regarding the cntrl grup where the chlrgenic acid slutin was replaced with distilled water, it was tested 3 times (nce fr every 10 mins), s as t finally determine the change in tendency f the metal in exudatin. Measurement f bsrbancy f bacterium slutin 260nm: the cllected E. cli was washed with 0.9% NaCl fr three times befre being re-suspending (OD = 0.670). By 420nm mixing 2mg/ml antibacterial activity materiel slutin with bacterial suspensin 1:1 (v/v), then values f OD260nm at different times were determined. Determinatin f micrbial slutin APK activity: 0.5 ml f 108 cfu ml E. cli bacterial suspensin was mixed with equivalent vlume f 2 mg ml, the same vlume f chlrgenic acid and ischlrgenic acid A, where sterile water was adpted as blank cntrl. Thrugh time spliced Table 5 : Experiment factr level f ethyl acetate extractin Level Temperature Ethyl Extractin ph (D) C (A) Acetate Time h (C) Cncentratin % (B) Jurnal f Envirnmental Bilgy, Special issue September 2016

5 Extricatin prcess f chlrgenic acid in Crftn weed 1053 Table 6 : Orthgnal test table and results f ethyl acetate extractin Number A B C D Extracti- Cntent f Extractin veyield chlrgenic rati f rati (%) acid in chlrgenic extract (%) acid (%) Extractiveyield rati K R Cntent f chlrgenic acid K r Extractin rati f chlrgenic acid K R Table 7 : Chlrgenic acid purificatin results = 40% Upper clumn Cllected Purified Chlrgenic Chlrgenic ethanl NKA-9 chlrgenic chlrgenic dry extract acid purity/% acid cllectin acid mass/g acid mass /g mass/g rati/% Macrprus resin Table 8 : Antibacterial activity f chlrgenic acids and cmpund preservative n E.cli Antibacterial Circle Bacterial Chlrgenic Ischlrgenic Sdium Ptassium Type acid acid A benzate srbate E.cli 23.4± ± ± ±0.2 timing sampling, it can btain the supernatant liqur f the sample interactin, and the AKP cntent was determined in accrdance with requirements n the spectrphtmeter kit. NPN t E. cli cell wall permeability test: First the test bacteria was cultivated at OD 630nm = 0.5, and bacteria was btained thrugh centrifugatin in 1000 x g at rm temperature. Then half vlume f HEPES buffer slutin f ph = 5.3 was added in it. Pipette was used t take 100 ul f bacterial suspensin int flurescence black slt which cntained 2mg ml chlrgenic acid slutin with 10mml l NPN buffer slutin. 10mml l NPN buffer slutin was regarded as a cmparisn grup fr making the ttal vlume t 200 u L. Flurescence phtmeter was used t determine the relative flurescence (excitatin wavelength=355nm, emissin wavelength=405nm). Results and Discussin Chlrgenic acid and ischlrgenic acid A shwed antibacterial activity n E.cli, and was cmpared with the cmmnly used chemical preservative sdium benzate and ptassium srbate. Table 8 shws that chlrgenic acid and ischlrgenic acid A perfrmed well n antibacterial activity, while chlrgenic acid had better effect than ischlrgenic acid A. Chlrgenic acid and ischlrgenic acid A shwed better antibacterial activity n E. cli as cmpared t chemical synthetic preservatives such as srbic acid ptassium and sdium benzate (Xing et al., 2012). Jurnal f Envirnmental Bilgy, Special issue September 2016

1054 Y. Zheng et al. The absrptin value f chlrgenic acid material acted n E.cli bacteria slutin 260nm is shwn in Fig. 1. After interactin f chlrgenic acid with E.

6 1054 Y. Zheng et al. The absrptin value f chlrgenic acid material acted n E.cli bacteria slutin 260nm is shwn in Fig. 1. After interactin f chlrgenic acid with E.cli, OD f bacterial slutin increased alng with the time and reached maximum at 60 min, where chlrgenic acid shwed maximum antibacterial activity. Cell membrane acts as a cntact with prtective barrier in bacteria. When bacteria cmes in bacteristatic agent, the cell membrane is destryed and the prtective barrier f bacteria is brken and thus, its internal electrlyte leaks int the culture slutin, leading t increased cnductivity f culture slutin. Therefre, the cnductivity change f culture slutin reflects the permeability change in bacterial cell membrane. As shwn in Fig. 2 when the cncentratin f chlrgenic acid was 2 mg ml, the cnductivity f bacterial slutin after treatment fr any times was higher than that f cntrl grup, which increased with duratin. This indicates that chlrgenic acid can increase the cell membrane permeability and result in intracellular leakage f electrlytes. Chlrgenic acid has a strnger destructive effect n cell membrane between the tw reagents. Fig. 3 shws the change in activity f E.cli alkaline phsphatase befre and after adding chlrgenic acid. Fig. 3 shws that after adding chlrgenic acid slutin int E.cli bacterial slutin, the AKP cntent f bacterial slutin becme significantly higher than that f the cntrl grup, and shwed increasing tendency. During the early interactin phase, the rising speed was slw, while the rising speed was faster after 150 min and then fllwed a steady trend; while the AKP f cntrl grup did nt change. This suggests that chlrgenic acid can destry the cmpleteness f bacterial cell wall in a shrter time and with a mre significant destrying effect as cmpared with ischlrgenic acid A (Sheng et al., 2008). NPN absrptin methd was adpted t study the effect f chlrgenic acids n E.cli cell wall permeability. If the bacterial cell wall is damaged r dysfunctinal, NPN will permeate int the cell wall t increase the flurescence intensity f the system (Run et al., 2006). As shwn in Fig. 4, after adding NPN int the E. cli bacterium suspensin with chlrgenic acids, the flurescence intensity increased with lnger duratin f actin, and the flurescence intensity reached its maximum value after 14 mins f interactin. Chlrgenic Ischlrgenic 0.37 acid acid A Time/min Fig. 1 : Change f absrbancy f nutrient slutin Cnductivity (mg/cm) OD 260nm AKP (U/L) Flurescence intensity Time/min Fig. 2 : Change f cnductivity f E.cli bacterial slutin Ntes : (a) 2 mg ml f chlrgenic acid; (b) 0.2 mg ml chlrgenic acid; (c) cntrl grup; (d) 0.2mg ml ischlrgenic acid A; e: 2mg ml ischlrgenic acid A. Chlrgenic acid Ischlrgenic acid A Time/min Fig. 3 : Change f AKP activity 120 Chlrgenic acid 80 Ischlrgenic acid A 40 Rise Fig. 4 : Change f NPN flurescence intensity after the actin f chlrgenic acid materials n E.cli a b c d e Jurnal f Envirnmental Bilgy, Special issue September 2016

7 Extricatin prcess f chlrgenic acid in Crftn weed 1055 Thrugh cmprehensive cmparisn and cnsidering extractin rate f chlrgenic acid, chlrgenic acid cntent in extract and cst issue, ethanl extractin has many mre advantages as cmpared t water and ethyl acetate extract. Water extract has many impurities, which is difficult t purify, easy t breed bacteria, but hard t be preserved. In additin, water is nt a preferred material fr being slvent, because it has disadvantages such as high biling pint and deslventizing difficulty. On the ther hand, ethyl acetate is mre cstly than ethanl, which is nt suitable fr industrial prductin. Overall ethanl extractin can vercme the drawbacks in water extractin, such as easy t mildew and als enjy price advantage as cmpared t ethyl acetate, with higher slvent yield easiness in perfrming later peratins such as filtratin, slvent recllectin and drying. The ptimum extractin parameters are as fllws: 40% cncentratin f ethanl, ethanl ph value is 3, slid t liquid mass rati is 1:10, the extractin duratin is 4 hurs, and extractin temperature is 80 C. The purity f chlrgenic acid after the purificatin by NKA-9 macrprus resin is 24.98%. Bth Chlrgenic acid and ischlrgenic acid A shwed gd antibacterial activity against E.cli. Results prve that chlrgenic acids can destry the structure f E.cli cell wall and cell membrane within a shrt perid f time, and thus increase the permeability f cell wall. On this basis, cell electrlytes, enzymes, DNA, RNA and NPN can mre easily leak int the cell wall, thus affecting the stability f cell structure and causing death f cell. Cmpared with ischlrgenic acid A, Chlrgenic acid shwed better antibacterial effect and strnger actin intensity t cell wall and membrane, s as t achieve a better antibacterial effect. Acknwledgments The authrs acknwledge the Sichuan Prvince Science and Technlgy supprt prgram- system engineering cnstructin f cmprehensive management and resurce utilizatin (N. 2014SZ0162). References Ca, H., J.T. Zhai and Y. Liu: Determinatin f chlrgenic acid cnstitute in cmpund flricme prescriptin pellet by HPLC. Mdern Instrument, 6, 107 (1999). Cha, M.M., K. Maureen and J.K. Khan : Shrt f chlrgenic acid derivatives withprmising antifungal activity. J. Birg. Medi. Chem., 15, (2007). Da, P.F. and Y.Q. Hng : Damage characteristics f Imperata cylindrical and its recent develpment f research and utilizatin. Frest Inventry and Planning, 28, (2003). Fu, Y., Q.S. Sng and Q.J. Fang : Advances in the studies n the chemical cmpnents f Eupatrium denphrum and their bilgical activities. J. Yunnan Agricul. Univ., 14, (1999). Huang, L., J.L. Tang and J. Huang: Extractin f chlrgenic acid and advances n the applied research. GaXia ShiYanShi GngZu YanJiu, 113, (2012). Li, Y.S.: Advances in the research n the distributin and extractin purificatin technlgies f chlrgenic acid in natural plants. Pharmac. J. Chinese Peple's Liberatin Army, 138, (2013). Run, Q.S., J. Yang, A.C. Ca and L. He : Advances in the studies n the chemical cmpnents and biactivity f Crftn weed as a intruding species. J. Beijing Nrmal Univ. (Natural Science), 42, (2006). Sheng, X.H., W.Q. Liu and X. Xue: Anti-herpes simplex virus effect f chlrgenic acid in-vitr. Chinese J. Nat. Medic., 6, (2008). Wang, T.Z. and Y.M. Li : The research prgress f Fls Lnicerae. West China J. Pharmac. Sci., 15, (2000). Xing, J.H., S.C. Li, W.J. Wang : The screening and identificatin f the antibacterial biactivecmpunds frm Lnicera japnica Thunb., leaves. J. Fd Chem., 28, (2012). Jurnal f Envirnmental Bilgy, Special issue September 2016

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