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7(43), pp. 2859-2865, 29 November, 2013DOI: 10.5897/AJPP2012.1513ISSN 1996-0816 2013 Academic n Journal of Pharmacy andPharmacologyFull Length Research PaperScreening of antibacterial activity of 20 Chinese herbalmedicines in YunnanDing Yan-Jiao1,2, Zuo Guo-Ying1*, Hao Xiao-Yan2, Wang Gen-Chun1 and Han Jun31Research Center for Natural Medicines, Kunming General Hospital, PLA, Kunming 650032, China.2College of Pharmacy, Guiyang Medical College, Guiyang 550004, China.3Yunnan Traditional Chinese Medical College, Kunming 650500, China.Accepted 17 June, 2013The 80% ethanol extracts from 20 Chinese herbal medicines collected in Yunnan Province, China, werescreened for antibacterial activity against Staphylococcus aureus (SA), methicillin-resistantStaphylococcus aureus (MRSA), Escherichia coli (EC) and Pseudomonas aeruginosa (PA), includingantifugal activity against Candida albicans (CA) by the agar hole diffusion test. The extracts wereprepared through macerating at room temperature and hot refluxing procedures. 19 of the 20 extractsshowed effects with different potency and their inhibition zone diameters (IZD) ranged from 9 to 27 mm.The extracts from Psychotria henryi Levl and Marsdenia tinctoria R. Br. (maceration and refluxextraction) were the most active against both SA and MRSA, with mean IZDs 19.7 to 22.0 mm. Theanti-SA and MRSA active extracts were further determined for minimum inhibitory concentration (MIC)and minimum bactericidal concentration (MBC) by serial dilution with a standard broth microdilutionmethod. 12 extracts showed effects on both SA and MRSA strains with MICs/MBCs ranges among 32 to 1024 / 64 to 1024 mg/L, respectively. P. henryi, M. tinctoria and Sapium baccatum Roxb also showedinhibition against EC, PA and CA strains, and displayed a broad spectrum of antimicrobial activities.Key words: Chinese herbal, methicillin-resistant Staphylococcus aureus (MRSA), minimum inhibitoryconcentrations lococcusaureus(MRSA) occurred in early 1961 (Jevons, 1961), it hasbecome a troublesome pathogen responsible for seriousinfections owing to its resistance to a large group ofantibacterial agents, resulting in high morbidity andmortality. It is resistant not only to β-lactam antibiotics,but also to other antibacterial groups, such asfluoroquinolones, tetracyclines, macrolides, lincosamidesand aminoglycosides. Some strains that are partially orfully resistant to vancomycin have been found (Pantostiand Venditti, 2009). The search for novel drugs to combatMRSA infection is urgently needed.Traditional Chinese medicine (TCM) has accounted fora wealth of health needs over thousands of years inChina (The Compiler Group, 2009). A great number ofplant species have been used as healing herbs in TCMfor the treatment of infectious diseases, such as skinulcers, cold, diarrhea, pneumonitis and tuberculosis, etc(Jiangsu New Medical College, 1977; Sima et al., 2012;Jinous and Fereshteh, 2012; Muhammad et al., 2012).There is increasin interest in researching Chinese herbaldrugs for application as antibacterial agents (Zuo et al.,*Corresponding author. E-mail: [email protected] Tel: 86-871-477-4941.

2860Afr. J. Pharm. Pharmacol.Table 1. The antibacterial agent-susceptibility testing results of MRSA strains.MRSA StrainMRSA092MRSA098MRSA111Resistant EV,CLI,AZM,PEN,OXS,FUR, LEV,ERY,PIP/T, CTX VIntermediate (I)NoneGMNoneSusceptible (S)VAN,GAT,PIP/T,FOS,LNZVAN,FOS,LNZVAN,LNZAMP: ampicillin; AZM: azithromycin; CAT: cefathiamidine; CFP/SU: cefoperazone /sulbactam; CFZ: cefazolin; CLI:clindamycin; CTX: Cefotaxime; ERY: erythromycin; FOS: fosfomycin; FUR: cefuroxime; GAT: gatifloxacin; GM: gentamicin;LEV: levofloxacin; LNZ: linezolid; OXS: oxacillin; PEN: Penicillin; PIP/T: piperacillin/tazobactam; CFP/SU: cefoperazone/sulbactam; VAN: Vancomycin.2008). For the sake of finding more anti-MRSA plants, weherein report the screening of 20 southern Yunnanmedicinal plants for in vitro inhibition against clinicalMRSA isolates, including their effects on pathogens ofEscherichia coli, Pseudomonas aeruginosa and Candidaalbicans.MATERIALS AND METHODSBacterial strainsStrains of S. aureus (SA, ATCC25923), E. coli (EC, ATCC25922), P.aeruginosa (PA, ATCC27853), C. albicans (CA, ATCCY0109) andantibiotic susceptibility disks were provided by the National Instituteof Control of Pharmaceutical and Biological products (NICPBP,Beijing, China). MRSA strains were clinical isolates from infectioussamples of critically ill patients in Kunming General Hospital (KGH)(Table 1). Pathogen purification and identification (including colonialmorphology, gram staining and coagulase testing) were conductedin our clinical microbiology laboratory and further confirmed bystandard cefoxitin disk diffusion test following Clinical andLaboratory Standards Institute (CLSI, 2007) standard procedures.The tested MRSA strains were multi-drug resistant to β-lactams,aminoglycosides, fluoroquinolones and macrolides, and they weresusceptible to vancomycin (Table 2).PlantsThe selected plants were collected in Xishuangbanna of YunnanProvince in August, 2011. They were identified at the BotanyDepartment, Kunming Institute of Botany (KIB), the ChineseAcademy of Sciences. The voucher specimens were preserved atthe herbarium of KIB (Table 2).Media usedStandard Mueller-Hinton agar and broth (MH-A and MH-B), andSabouraud agar and broth (S-A and S-B) (Tianhe Microbial AgentsCo., Hangzhou, China) were used as the bacterial and fungalculture media, respectively.Extract preparationTwo types of 80% ethanol extracts were prepared from the 20Southern Yunnan plants by the macerating extraction at roomtemperature (25 C) and the hot reflux extraction (85 C),respectively as the follows: An amount (50 g, each) of powderedair-dried aerial parts of the plant material was macerated with 80%ethanol (150 ml) for one week, filtered and the residue was furthermacerated twice with the same amount of solvent overnight andfiltered after sonication for 30 min. The filtrates were combined andthe solvent was evaporated at 40 C under reduced pressure toafford each of the plant extracts. Another amount (50 g, each) of thesame plant material was refluxed with 80% ethanol for two hours,filtered and the residue was further refluxed twice with the sameamount of solvent and filtered. The filtrates were combined and thesolvent was evaporated at 40 C in vacuum to afford each of theplant extracts (Table 3).Susceptibility testThe ethanol extracts of the 20 plants were initially subjected tosusceptibility test according to the agar diffusion method on MH-A(for the bacteria) or S-A (for C. albicans) plates (Zuo et al., 2008).Briefly, crude extracts were prepared at concentrations of 50 mg/mlin dimethylsulfoxide (DMSO). The microbial suspension was firstlyplated onto the agar plates with inoculums of 1.5 10 8 CFU/ml forthe bacteria and 5 105 CFU/ml for C. albicans. Then, holes of 6.0mm diameter each in the agar plates were punched with a sterilizedhole-puncher. The samples were pipetted into the holes with nooverflowing. The plates were incubated at 35 C for 24 h andmeasured and the inhibition zone diameters (IZDs) recorded. Forevery experiment, a sterility check (DMSO and medium), negativecontrol (DMSO, medium and inoculums) and positive antibacterialcontrol (vancomycin) were included (Table 3). All experiments wereperformed in triplicate.The samples of IZDs 12 mm against standard S. aureus (SA)were subjected to assay of their IZDs against MRSA (Table 4). Thesubsequent samples with IZDs 12 mm against one of the MRSAstrain (MRSA111) were further subjected to assay of their minimuminhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) for MRSA by standardized broth microdilutiontechniques (Zuo et al., 2008). Briefly, the starting inoculums were 5 105 CFU/ml. The 96-well plates were incubated at 35 C for 24 haccording to CLSI guidelines (CLSI, 2006, 2007). All experimentswere determined in duplicate, with concentrations ranging up to2048 mg/L. For the MBC assays, 0.1 ml aliquots from drug dilutionwells with no visual growth inhibition were plated onto MH-A. Thelowest drug concentration that yielded three or fewer microorganismcolonies was recorded as the MBC NCCLS, 1999). Vancomycin (EliLilly Japan K.K., Seishin Laboratories) was also used as a positivecontrol anti-MRSA agent (Table 5).

Yan-Jiao et al.2861Table 2. Species of the 20 medicinal N48Ulcer, dysmenorrheal, rheumatism and ostalgia, injuries from falls456Aphanamixis grandifolia Bl.Blumea densiflora DC.Cinnamomum bejolghota (Buch.-Ham.)SweetCitrus medica L.Croton tiglium L.Duranta repens eaeVerbenaceaeHITBC5184KUN51KUN6627Fructus piperis NigriPiperaceaeKUN54788Hedyotis capitellata Wall. ex G. DonRubiaceaeKUN12179Heracleum candicans Wall. ex DCUmbelliferaeKUN1245110Illicium simonsii Maxim.MagnoliaceaeIBSC12511Lignum santali AlbiSantalaceaeHITBC56361213Marsdenia tinctoria R. Br.Millettia pachycarpa um indicum Mill.ApocynaceaeHITBC38315Psychotria henryi LevlRubiaceaeKUN6642161718Radix curcumae WenyujinRumulus uncariae Macrophyllae cum UncisSapium baccatum 506KUN27019Scutellaria discolor Wall. ex Benth.LamiaceaeKUN688Dyspepsia, coughExpelling phlegm, anthelminthicMalaria, subduing swelling and detoxicatingDissolving phlegm and detoxicating, dyspepsia, diarrhea,epigastralgiaCold and rheumatism, malaria, fracturesAsthma, cough and cold, chronic bronchitis, stomach ache,rheumatalgia, injury from fallUlcer,anthelminthicpneumonitis, cough and asthma, nausea and vomiting,ecchymoma, paralysisEpigastralgia, hepatomegaly, rheumatism and osteodyniaInjury from fall, fracture, acute gastroenteritisCardiotonic and dieresis, expelling phlegm, analgesia, removingblood stasis, epilepsyStrengthening spleen and dehumidify, regulating vital energy andanalgesiaMenoxenia, jaundice, hemuresis, epilepsyHeadache, serious dysentery, convulsion and hypertensionMenoxenia, dyspepsia, pa-lengTympanitis,diminish inflammation, rheum, fever, tuberculosis,injury from fall20Toona ciliata Roem.var.pubescens e antibacterial agent susceptibility spectrum ofthe tested three MRSA strains is listed in Table 1.They were used for anti-MRSA evaluation. The 20Folk useRheumatism and analgesiaRheumatism and analgesiaTinea, chronic diarrhea, chronic dysentery, uterine bleedingYunnan medicinal plants are shown in Table 2,together with their plant families, specimennumbers and folk uses. The initial antimicrobialscreening results (IZDs) of 50 mg/ml ethanolextracts of the 20 plant extracts against standardSA, that is, methicillin-susceptible Staphylococcusaureus (MSSA, ATCC25923) and other standardstrains of EC, PA and CA are shown in Table 3. 19of the 20 extracts showed effects with different potency and their IZDs ranged from 9 to 27 mm.

2862Afr. J. Pharm. Pharmacol.Table 3. Antimicrobial activities of 20 plant ethanol extracts (IZD unit: mm).SpeciesA. grandifolia (macerating extraction)A. grandifolia (reflux extraction)B. densiflora (macerating extraction)B. densiflora DC. (reflux extraction)C. bejolghota (macerating extraction)C. bejolghota (reflux extraction)C. medica (macerating extraction)C. medica (reflux extraction)C. tiglium (macerating extraction)C. tiglium (reflux extraction)D. repens (macerating extraction)D. repens (reflux extraction)DMSOF. piperis (eflux extraction)F. piperis (macerating extraction)H. candicans (macerating extraction)H. candicans (reflux extraction)H. capitellata (macerating extraction)H. capitellata (reflux extraction)I. simonsii (macerating extraction)I. simonsii (reflux extraction)L. santali (macerating extraction)L. santali (reflux extraction)M. pachycarpa (macerating extraction)M. pachycarpa (reflux extraction)M. tinctoria (macerating extraction)M. tinctoria (reflux extraction)N. indicum (macerating extraction)N. indicum (reflux extraction)P. henryi (macerating extraction)P. henryi (reflux extraction)R. curcumae (macerating extraction)R. curcumae (reflux extraction)R. uncariae (macerating extraction)R. uncariae (reflux extraction)S. baccatum (macerating extraction)S. baccatum (reflux extraction)S. discolor (macerating extraction)S. discolor (reflux extraction)T. ciliata Roem. var. pubes (reflux extraction)T. ciliata Roem.var.pubescens (Franch.) Hand.-Mazz. (macerating extraction)Extractionyield 000160130020111201500160013110000SA: Staphylococcus aureus (ATCC25923); EC: Escherichia coli (ATCC25922); PA: Pseudomonas aeruginosa (ATCC27853); CA: Candidaalbicans (ATCCY0109).The anti-MRSA activities of the selected 17 extractswhich were active with IZDs 10 mm against MSSAwere shown in Table 4. The extracts from Psychotriahenryi Levl and Marsdenia tinctoria R. Br. (macerationand reflux extraction) were the most active against bothSA and MRSA, with mean IZDs 19.7 to 22.0 mm (Table 4).

Yan-Jiao et al.2863Table 4.The testing results of the 17 extracts with IZD 10mm against SA and MRSA strains (unit: mm).SpeciesSAMRSA92MRSA98MRSA111P. henryi (reflux extraction)M. tinctoria (macerating extraction)M. tinctoria (reflux extraction)P. henryi (macerating extraction)S. baccatum (macerating extraction)S. baccatum (reflux extraction)I. simonsii (macerating extraction)R. uncariae (reflux extraction)C. bejolghota (reflux extraction)I. simonsii (reflux extraction)S. discolor (reflux extraction)C. tiglium (reflux extraction)D. repens (macerating extraction)D. repens (reflux extraction)C. tiglium (macerating extraction)R. Uncariae (macerating extraction)T. ciliata (macerating 1617171015000017212221181414141515120130121000Mean SEM(MRSA, n 3)22.0 2.521.3 0.920.7 0.919.7 1.317.7 0.918.0 3.117.7 2.317.0 1.516.3 1.315.7 0.314.7 1.511.0 5.511.0 1.09.7 4.84.0 4.03.3 3.30.0 0.00.0 0.0SA: Staphylococcus aureus (ATCC25923); MRSA: methicillin-resistant Staphylococcus aureus.The anti-SA and MRSA active extracts were furtherdetermined for minimum inhibitory concentration (MIC)and minimum bactericidal concentration (MBC) by serialdilution with a standard broth microdilution method. 12extracts showed effects on both SA and MRSA strainswith MICs/MBCs ranges between among 32 to 1024 /64 to 1024 mg/L, respectively. P. henryi, M. tinctoriaand Sapium baccatum Roxb also showed inhibitionagainst EC, PA and CA strains, and displayed a broadspectrum of antimicrobial activities.DISCUSSIONThe screening results revealed that most of the plantextracts were much more active against Gram positivepathogen (SA) than those of Gram negative pathogens(EC and PA), which is a common phenomenon of plantantimicrobial property (Tegos et al., 2002). None of theplants showed strong inhibition against PA (Table 3). The4 plant extracts from D. repens (reflux extraction), M.tinctoria (macerating extraction), S. baccatum (refluxextraction) and P. henryi (reflux extraction) showed IZDsof 9 to 12 mm against standard EC. There were 15 plantextracts active against standard CA and the IZDs were inthe ranges of 10 to 20 mm. Some plants showed a widespectrum of antimicrobial activity and were in agreementwith their folk uses (Table 3). However, several extractsshowed no antimicrobial activity at 50 mg/ml which werenot consistent with their folk uses for infectious diseases.It might be due to the antimicrobial potency which wasmuch lower in this study and the different criterion for theantimicrobial judgment. We also noted the inconsistentvalues between IZD and MIC of an extract; this might bedue to the different diffusion capacity of the activeconstituents in the culture media of agar and broth.It was found that different extraction methods produceddifferent yields of active extracts. Meanwhile, the highertemperature of extraction may cause the active ingredientchange, hence the antimicrobial activity decreases(Tables 3 to 5).In this study, the 80% ethanol extracts of 20 plant materials were screened for their antimicrobial activities forthe first time. We found that P. henryi was active againstnot only Gram-positive bacteria but also Gram-negativebacteria. The chemical composition and pharmacologicaleffects of P. henryi has not been reported yet, but therewere reports (Muhammad et al., 2003; Kerber et al.,2001) about the same genus, that is, P. sychotriabrachyceras and P. sychotria klugii which containedindole alkaloids. So, it is worthy to further investigate theanti-MRSA components. Our study suggested that M.tinctoria showed promising activity against MSSA andMRSA. The plant contained steroidal components such astinctoralactone and marsdenone (Chowdhury et al., 1993)and showed antifertility activity (Chowdhury et al., 1994).But its anti-MRSA effect has not been noted previously.Further research for the corresponding active

2864Afr. J. Pharm. Pharmacol.Table 5. MICs and MBCs of the 12 extracts with IZD 10mm against SA, MRSA and PA strains (unit: mg /L).StrainParameterMICMBCPA1024n.a.ATCC 259233264MRSA 09264128MRSA 098128512MRSA 1113264S. baccatum (macerating extraction)MICMBCn.a.n.a.32128512102412851232128R. Uncariae (reflux S. baccatum (reflux extraction)MICMBC2048n.a.64128512102425651264256P. henryi (macerating 048M. tinctoria (macerating . tinctoria (reflux 12I. simonsii (macerating 4I. simonsii (reflux 048D. repens L. (macerating 22048C. bejolghota (reflux 42048S. discolor (reflux .2440.9760.4880.976P. henryi (reflux extraction)SA: Staphylococcus aureus (ATCC25923); MRSA: methicillin-resistant Staphylococcus aureus; PA: Pseudomonas aeruginosa(ATCC27853); n.a.: not active at the concentration up to 2048 mg/L.components from these plants and their systematic antiMRSA properties are been carried out.81073126, 81173504).REFERENCESACKNOWLEDGEMENTThis research was financially supported by the grant fromthe National Natural Science Foundation of China (NSFCChowdhury AKA, Hashim MF, Sen BC, Khan OF, Ahmed M (1994).Antifertility principles from Marsdenia tinctoria: Pharmacological andphytochemical studies. Pure Appl. Chem. 66:2343-2346.Chowdhury AKA, Sen BC, Hashim MF, AHMED M (1993). Tinctoramine:

Yan-Jiao et al.a steroidal alkaloid from Marsdenia tinctoria. Phamazie 48(8):628629.Clinical and Laboratory Standards Institute. Performance standards forantimicrobial susceptibility testing. Seventeenth informationalsupplement. M100-S17.CLSI, 2007.Wayne, PA.Jevons MP (1961). "Celbenin"-resistant staphylococci. Brit. Med. J.1:124-125.Jiangsu New Medical College (1977). Dictionary of Chinese MateriaMedica. Shanghai Science and Technology Press. Shanghai, China.Kerber VA, Gregianini TS, Paranhos JT, Schwambach J, Farias F, FettJP, Fett-Neto AG, Zuanazzi JA, Quirion JC, Elizabetsky E, HenriquesAT (2001). Brachycerine, a novel monoterpene indole alkaloid fromPsychotria brachyceras. J. Nat. Prod. 64(5):677-679.Jinous A, Fereshteh R (2012). Chemistry, pharmacology and medicinalproperties of Peganum harmala L. Afr. J. Pharm. Pharmacol.6(22):1573-1580.Muhammad A, Ghiasuddin, Anwar S, Naveed M, Ashfaq AK. Bina SS(2012). Evaluation of Viburnum grandiflorum for its in-vitropharmacological screening. Afr. J. Pharm. Pharmacol. 6(22):16061610.Muhammad I, Dunbar DC, Khan SI, Tekwani BL, Bedir E, Takamatsu S,Ferreira D, Walker LA (2003). Antiparasitic alkaloids from Psychotriaklugii. J. Nat. Prod. 66(7):962-967.National Committee for Clinical Laboratory Standards ( 1999).Methods for Determining Bactericidal Activity Antimicrobial Agents.Approved Guidelines. Document M26-A. CLSI (formerly NCCLS),Wayne, PAPantosti A, Venditti M (2009). What is MRSA? Eur. Respir. J. 34(5):1190-1196.2865Sima N, Mahdieh A, Narges K (2012). Evaluation of analgesic and antiinflammatory effects of fresh onion juice in experimental animals. Afr.J. Pharm. Pharmacol. 6(23):1679-1684.Tegos G, Stermitz FR, Lomovskaya O, Lewis K (2002). Multidrug pumpinhibitors uncover remarkable activity of plant antimicrobials.Antimicrob. Agents Chemother. 46(10):3133-41.The Compiler Group (2009). Shen Nong’s Materia Medica. TianjinAncient Books Publishing House.Tianjin, China.Zuo GY, Wang GC, Zhao YB, Xu GL, Hao XY, Han J, Zhao YB (2008).Screening of Chinese medicinal plants for inhibition against clinicalisolates of methicillin-resistant Staphylococcus aureus (MRSA). J.Ethnopharmacol. 120:287–290.

7 Fructus piperis Nigri Piperaceae KUN5478 Dissolving phlegm and detoxicating, dyspepsia, diarrhea, epigastralgia 8 Hedyotis capitellata Wall. ex G. Don Rubiaceae KUN1217 Cold and rheumatism, malaria, fractures 9 Heracleum candicans Wall. ex DC Umbelliferae KUN12451 Asthma, cough and cold, chronic bronchitis, stomach ache,