Cryptogamie,Bryologie, 2006, 27 (3): 343-348 © 2006 Adac.Tous droits réservés Antibacterialactivity in extracts of Cylindrocolea recurvifolia (Cephaloziellaceae, Marchantiophyta)and Pleurozia subinflata (Pleuroziaceae,Marchantiophyta) DanWANG, Rui-Liang ZHU* &LiQU Department of Biology,School of Life Science,East ChinaNormalUniversity, 3663 Zhong ShanNorthRoad,Shanghai 200062,China (Received 2 March 2005,accepted 20 June 2005) Abstract –Discdiffusion assay was used to screen for antibacterialactivity of aqueous, alcoholicand ether extracts of twoliverworts withdifferent habitats: Cylindrocolea recurvi- folia (Cephaloziellaceae) on moist rocks in shaded forest,and Pleurozia subinflata (Pleuro- ziaceae) on trunks or branches in forest canopy.The ether extracts of C. recurvifolia were activeagainst all seven selected bacterial species,alcoholicextracts possess antibacterial activity against six of the seven,but aqueous extracts exhibit very weakantibacterialactivity only against one of the seven bacterial species.The ether and alcoholicextracts of Pleurozia subinflata demonstrated antimicrobialactivity against six of the seven,but aqueous extracts exhibit weakantibacterialactivity only against twoof the seven bacterial species.Antibac- terialactivity of the ether extracts,expressed as MICs and MBCs, was compared with three referenceantibioticdrugs.This is the first report of antibacterialactivity tested on plants of Cephaloziellaceaeand Pleuroziaceae. The genus Cylindrocolea is newly reported for GuangxiProvinceofChina. alcoholicextract / antibacterialactivity / China / Cylindrocolea recurvifolia / ether extract / Guangxi / liverwort / MBC / MIC / oil body / Pleurozia subinflata INTRODUCTION Bryophytes areone of the important sources of antibiotics and biologically active,naturally occurring compounds (McCleary et al.,1960; Zinmeister et al.,1991). Their economic uses havebeen documented recently by Frahm (2004). Numerous recent studies also showed that bryophytes,particularly liverworts,contain anumber of antibacterialcompounds (Asakawa, 1998; Basile et al.,1998a). Although Chinahas aparticularly richliverwort flora, antibacterial activity of their extracts has not been screened extensively in mainland China (Zhu et al., 2002)owing todifficulties in the collection of pure samples from the wild and lackofexpertisein species identification. Cylindrocolea R.M.Schust.,a small mainly Gondwanalandicgenus, contains ca 12 species (Schuster, 2002). The East Asiatic taxon, C. recurvifolia *Correspondenceand reprints:[email protected] 344 D.Wang,R.-L.Zhu &L.Qu (Steph.) Inoue , is the most common member of Cylindrocolea in Chinaand Japan and it usually forms large and luxuriant populations on very wet rocks along river banks,as showninIwatsuki (2001). The large quantity of wild populations of this species in Taiwanand JiangxiofChinahas led to the finding of anew gymnomi- trane-type sesquiterpenoid (Wu &Kao, 2002)and anaturalledol (Wu et al.,1996, species reported as Cephaloziella recurvifolia ). Thus, Cylindrocolea recurvifolia became the first species of Cephaloziellaceaechemically investigated. Although C. recurvifolia has been well studied taxonomically in Japan(Inoue,1974),its dis- tribution in Chinais still poorly known. Pleurozia Dumort., the only representativeofPleuroziaceae,is ahighly specialized genus with11 species (Thiers,1993). Its diagnosticcharacters, which include apicalcell withonly twocutting faces, smaller saccatedorsallobeof the leaf withanaperturecomplex,and dimorphicperianths in sterile and fertile condition,are uniqueamong liverworts.Owing to the large plant size,many uniquemorphologicalcharacters,and interesting habitats, Pleurozia has attracted the attention of anumber of chemists toinvestigate the chemicalnatureof the genus.Flavonoids in 10 species (Mues et al.,1991) and terpenoids in two species [ P.acinosa (Mitt.) Trevis.and P.gigantea (Web.) Lindb.]havebeen reported (Wu &Asakawa, 1988; Asakawa, 2004). Pleurozia subinflata (Austin) Austin is known from China, Hawaii,Japan,SriLanka, Thailand,and Vietnam(Thiers,1993). It was listed as one of endangered bryophytes in China(Chen,1993; Wu et al.,1997, as P.giganteoides (Horik.) Inoue). During anexpedition tonortheast GuangxiProvinceinSept. 2004, we found that C. recurvifolia and P. subinflata arenot rarelocally,forming large populations.The aim of our study is toinvestigate the possible inhibitory activity of C. recurvifolia and P. subinflata against bacterial species.The present paper not only reports the first discovery of Cylindrocolea in Guangxi,China, it also shows the antibacterialactivity of cephaloziellaceous and pleuroziaceous plants for the first time. MATERIAL AND METHODS Liverwort collections InSeptember 2004, samples of Cylindrocolea recurvifolia werecollected from a single sterile population growing on moist rocks at 550 matLongtangjiang, Huajiang,XinanCo.,GuangxiProvince. Collections of severalfertile populations of Pleurozia subinflata werealsomade from forest canopy at 2000 mat Maoershan,XinanCo.,Guangxi,in September 2004. The voucher specimens, R.- L.Zhu 20040908h for C. recurvifolia, and R.-L.Zhu 20040909-63 for P. subinflata, aredeposited in the herbariumofEast ChinaNormalUniversity (HSNU). Testing microorganisms Seven bacterial species were selected for the antibacterial tests.They are Grampositive(G+): Bacillus megaterium,Bacillus subtilis,Bacillus thuringiensis, Staphylococcus aureus,and Gramnegative(G-): Escherichiacoli,Pseudomonas aeruginosa ,and Pseudomonas putida . Antibacterialactivity in extracts of twoMarchantiophyta345 Extraction and initial screening The liverwort materials werebrought back to the laboratory in plastic bags.They were washed in distilled water,dried in anoven at 40 °C, and ground intopowders.Ten grams of eachliverwort species were separately shaken in 100 ml 95% ethanol,distilled water,and ether overnight.Extracts were then filtered and stored at 4°C.For screening, the method of discdiffusion assay was used. The six mm sterilized filter paper discs that were soaked in the extracts overnight, wereplaced,after drying,ontoLB agar plates grown with the testing bacteria.The inhibitory activity was indicated by aclear zone around the disc after incubation at 37 °Cfor 24h,and the widthof the clear inhibitory zone was measured. Cultures prepared in the same way but without liverworts were used as controls.Three antibioticdrugs,Amoxicillin,Cefradine,and Ciprofloxacin Hydrochloride,dissolved in distilled water (10 µ g/ml) were used as references. The results of initial screening are showninTables 1and 2. Table 1. Antibacterialactivities of Cylindrocolea recurvifolia shownindiscdiffusion assays.The strengthof the activity is represented by the widthofinhibition zone expressed in mm. Widthofinhibition zone (mm) Bacteria Water Alcohol Ether Ciprofloxacin Cefradine Amoxicillin extract extract extract Hydrochloride Bacillus megaterium 00.9 0.4 9.07.9 7.4 B. subtilis 0.20.70.69.27.29.0 B. thuringiensis 00.5 0.8 8.06.8 9.0 Escherichiacoli 000.610.09.5 10.4 Pseudomonas aeruginosa 00.5 0.65.25.06.9 P.putida 00.4 0.5 5.8 4.4 7.2 Staphylococcus aureus 00.4 0.210.39.4 10.0 Table 2.Antibacterialactivities of Pleurozia subinflata shownindiscdiffusion assays.The strengthof the activity is represented by the widthofinhibition zone expressed in mm. Widthofinhibition zone (mm) Bacteria Water Alcohol Ether Ciprofloxacin Cefradine Amoxicillin extract extract extract Hydrochloride Bacillus megaterium 00.30.5 9.07.9 7.4 B. subtilis 00.8 1.09.27.29.0 B. thuringiensis 0.20.8 0.8 8.06.8 9.0 Escherichiacoli 0.1 0.30.5 10.09.5 10.4 Pseudomonas aeruginosa 0005.25.06.9 P.putida 00.5 0.25.8 4.4 7.2 Staphylococcus aureus 00.5 0.5 10.39.4 10.0 346D.Wang,R.-L.Zhu &L.Qu Determination of MIC (minimalinhibitory concentration) and MBC (minimal bactericidalconcentration) 50gpowders of liverwort samples were shaken overnight with500 ml ether.Extracts werefiltered,evaporated toa solid residueina rotary evaporator. 100 mg of the dry residue were then diluted in 10 ml of sterile physiologicalTris buffer (pH 7.4) solution. A serial 2-fold dilution of the plant extracts was made in sterile physiologicalTris buffer (pH 7.4) and added to the LB agar plates togive afinalconcentration range of 4-1000 µ g/ml. EachLB agar plate was incubated at 37 °Cfor 24h.After incubation the lowest concentration of eachextract showing no visible bacterialgrowth was recorded as MIC.Cultureplates containing only sterile physiologicalTris buffer (pH 7.4) were used as control. The MBC determination was performed by transferring the extracts with concentrations higher than the MIC tofreshLB agar plates,following Basile et al. (1998b). The finalconcentrations of the extracts ranged from MIC to 2000 µ g/ml. After incubation at 37 °Cfor 48 h, the MBC was reported as the lowest concentration of the extracts without visible bacterialgrowth. The MIC and MBC values werealsodetermined for the three referenceantibioticdrugs,Amoxicillin, Cefradine,and Ciprofloxacin Hydrochloride, using the aforementioned method. All the assays werecarried out in triplicates.The MIC and MBC results are shown in Tables 3 and 4. Table 3.Antibacterialactivity of ether extract of Cylindrocolea recurvifolia and Pleurozia subinflata expressed as MIC (unit: µ g/ml). Ciprofloxacin BacteriaC. recurvifoliaP. subinflataCefradine Amoxicillin Hydrochloride Bacillus megaterium 500 1254168 B. subtilis 1251258 416 B. thuringiensis 1251258 84 Escherichiacoli 250250840.25 Pseudomonas aeruginosa 500 500 32 1632 P.putida 500 500 161632 Staphylococcus