The antimicrobial activities of extracts from Chinese herbs commonly available in the local Malaysian Chinese medicine halls against three field isolates and one reference 

strain of Brucella melitensis were evaluated. A total of ten herb extracts were obtained via ethanol extraction. Antibacterial screenings were done using disc diffusion method. Herb extracts with inhibitory zones of 10 mm or more in diameter were further subjected to the Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) determination. Of the 10 herbs, which were Lunicera japonica, Flos Lonicera, Coptis chinensis, Adrographis paniculata, Isatis indigotica, Radix paeoniae rubra, Polygonum orientale, Galla chinensis, Semen plantaginis, Fructus forsythia and Cortex phellodendrim, four were found to possess inhibitory effect against Brucella melitensis strains. The herbs are Coptis chinensis, Radix paeoniae rubra, Galla chinensis, and Cortex phellodendrin. The MIC ranged from 3.75 to 30 mg/mL. It was suggested that these four herbs are potential alternatives for the treatment or prevention of brucellosis caused by Brucella melitensis

Bejo, Siti Khairani

Khoo, Wen Wen

Universiti Putra Malaysia Institutional Repository

435th Proceedings of the Seminar in Veterinary Sciences, 5 - 8 January 2010Screening of Chinese Medicinal Herbs for the Inhibition of  Brucella melitensisKhoo Wen Wen & 1Siti Khairani Bejo1Department of Veterinary Pathology and Microbiology Faculty of Veterinary Medicine, Universiti Putra MalaysiaAbstractThe antimicrobial activities of extracts from Chinese herbs commonly available in the local Malaysian Chinese medicine halls against three field isolates and one reference strain of Brucella melitensis were evaluated.  A total of ten herb extracts were obtained via ethanol extraction.  Antibacterial screenings were done using disc diffusion method.  Herb extracts with inhibitory zones of 10 mm or more in diameter were further subjected to the Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) determination.  Of the 10 herbs, which were Lunicera japonica, Flos Lonicera, Coptis chinensis, Adrographis paniculata, Isatis indigotica, Radix paeoniae rubra, Polygonum orientale, Galla chinensis, Semen plantaginis, Fructus forsythia and Cortex phellodendrim, four were found to possess inhibitory effect against Brucella melitensis strains. The herbs are Coptis chinensis, Radix paeoniae rubra, Galla chinensis, and Cortex phellodendrin. The MIC ranged from 3.75 to 30 mg/mL. It was suggested that these four herbs are potential alternatives for the treatment or prevention of brucellosis caused by Brucella melitensis. Keywords:  Brucella melitensis, minimum inhibitory concentration (MIC), herbal extract.IntroductionBrucellosis is a zoonotic disease caused by the bacterium from the Brucella spp. This disease is endemic in many areas of the world including Malaysia. It is characterized by chronic infection in animals involving many species such as cattle, swine, sheep, goat and even horses leading to abortion, infertility, genital infection and the formation of localized lesions in many parts of the body tissues (Galloway J.H,1972).  Human beings are susceptible to several biotypes of this agent namely Brucella abortus, Brucella canis, Brucella melitensis and Brucella suis but are resistant to infection by Brucella neotomae and Brucella ovis. The organism is of public health importance as it can cause debilitating disease (Malta fever) like fever and chills with frequent relapses which can persist for months if left untreated. Current control measures in farms are disease surveillance and investigative studies, vaccination, test and slaughter programs to contain and eradicate the disease. With these measures, brucellosis is no longer a threat in Western Europe but is still prevalent in Malaysia and other parts of the world (Swabe, 1999). The wide practice of the application of B. abortus S19 vaccination of susceptible young female animals either with the full dose or the reduced dose together with the  slaughter of positive animals has resulted in the significant decrease of the 44overall rate of infection in countries like Egypt (Refai et al., 1990). For conventional human treatment, doxycycline and rifampin combination are used for six weeks to combat the infection. Streptomycin is included in severe cases. The tedious treatment regime and ease of transmission of this disease highly suggest a quest for alternatives or improvement on ways to manage this disease. Ethnopharmacology is the scientific study correlating ethnic groups, their health, and how it relates to their physical habits and methodology in creating and using medicines. Since global eradication of animal brucellosis  will not be realistic  in the near future due to socio-economic and political factors, and since the development of a satisfactory human vaccine currently is also not achieved yet, there exists a need for optimal antibiotic treatment schedules. These would ideally minimize the percentage of treatment failures and relapses whilst simultaneously being affordable for populations of low socio-economic status as well as being convenient in order to ensure adequate patient adherence. Ethnopharmacology is a very good alternative to explore in the sense that historically both human and animal medicine have relied heavily on plant materials.  Major pharmaceutical companies had started off by selling plant extracts whilst a quarter of all prescription drugs currently sold in the western world still use active ingredients derived from plants (Cox and Balick, 1997). The first stage in a drug development program using plants as the starting material is the collection and analysis of data on the uses of plants by various native cultures. Ethnobotany, ethnomedicine, folk medicine and traditional medicine can provide information that is useful as a pre-screen  to select plants for experimental pharmacological studies. There are a number of Chinese herbs available in Malaysia that has potential to treat brucellosis. However, the potential of the herbs has not been evaluated yet. In this study, 10 types of commonly available Chinese herbs were tested in bioassay systems that are believed to predict the action of these drugs in humans. The goal of this ethnopharmacology study is to identify drugs to alleviate human illness via analysis of plants assumed to be useful in Chinese culture throughout the world. The objectives of this study are to determine the antibacterial effects and minimal inhibitory concentrations of Chinese crude herbs against Brucella melitensis.Materials and MethodsDried HerbsA total of ten dried herbs were obtained from Chinese medicinal shop. The herbs were Lunicera japonica, Flos lonicera, Coptis chinensis, Adrographis paniculata, Isatis indigotica, Radix paeoniae rubra, Polygonum orientale, Galla chinensis, Semen plantaginis, Fructus forsythia and Cortex phellodendrim. The origins of these herbs are from various parts of China.45Herb Extract PreparationThirty grams of herbs were macerated with 80% ethanol (500 mL) using a blender and left for five days at room temperature. The mixture was filtered and evaporated at 60ºC to obtain the dried herb extracts. Brucella Isolates One reference strain (Brucella melitensis 16M), one isolate from goat (Brucella melitensis 293) and two isolates from sheep (Brucella melitensis 183 and Brucella melitensis 4611) were used.  Antibacterial Screening (Disc Diffusion Method)Extract concentration of 30 mg/mL was made by mixing 30 mg crude extract with one millilitre (1 mL) of 3% DMSO. Seventy microlitres (70 µL) of each extract was added to blank discs. The herb extract infused discs were dried at 60ºC in an oven overnight. Negative control were prepared by adding 70 µL of 3% DMSO to blank discs and dried at 60ºC overnight while Clavumox disc was used as positive control. Twenty mililitre Mueller Hinton Agar (MHA) plate were seeded with 20 ul suspension of test microorganism in 0.5 McFarland concentrations. One herb extract infused disc, one positive control disc and one negative control disc were put in each MHA plate. Triplicates were done. Inhibition zones diameters were read using the Aura Image (Oxoid). Extract with inhibition zone diameter of ten millimetres (10 mm) or more were subjected to MIC determination. Determination of MIC of herb extracts on Brucella melitensisTwo-fold serial dilution methods were used. Briefly, 30 mg/mL crude extracts were filtered through 0.25 µm millipore filter for sterilization. The extracts were serially diluted starting from 30 mg/mL up to 0.015 mg/mL. Dilutions were made in 96-well microplates.  Two hundred microlitres (200 µL) of 30 mg/mL extracts were dispense in the 1st column and 100 µL Brucella broth medium in the 2nd to 12th well. Two-fold serial dilution was done by pipetting 100 of extract from the 1st well into the 2nd well and so on. Twenty microliter (20 µL) of Brucella inoculum was added to every well. Streptomycin (0.05 mg/mL) was used as positive control while the negative control was made up of medium and inoculums. Triplicates were done for all four isolates. The plates were wrapped in parafilm and incubated at 37 ºC for three days. The growth of inoculum was determined by turbidity. Clear wells indicate absence of bacteria growth. The MIC of herbs was the lowest concentration in the medium that completely inhibit visible growth. Determination of MBC of herb extracts on Brucella melitensisThe MBC of herb extract was carried out by inoculating ten microlitres (10 µL) of mixture from each well on MHA plate.  Inoculated MHA plates were incubated at 37ºC for three days. The lowest concentration that yielded no growth after subculturing was taken as MBC.46Results and DiscussionThe antimicrobial screening results of the herb extracts are shown in Table 1. Four herbs showed inhibitory effects on the organism namely, Coptis chinensis, Radix paeoniae rubra, Galla chinensis and Cortex phellodendrim. Galla chinensis showed the largest inhibition zone which were, 39 mm for Brucella melitensis 16M, 36 mm for Brucella melitensis 293, 33 mm for Brucella melitensis 183 and 40 mm for Brucella melitensis 4611. Cortex phellodendrim showed the smallest inhibition zone which is, 13 mm for Brucella melitensis 16M, 12 mm for Brucella melitensis 293, 12 mm for Brucella melitensis 83 and 15 mm for Brucella melitensis 4611. The larger the inhibition zone the stronger the inhibitory effect of the tested sample. As Galla chinensis has the largest inhibitory zone, it means that it has the strongest inhibitory effect on Brucella melitensis and has high potential to be used as an antimicrobial agent against this microorganism.  In fact, the inhibitory zone of Galla chinensis is almost similar to Clavumox which has the highest zone of 45 mm thus, making it a potential alternative drug for management of infections. The other six herbs did not show any inhibitory zone thus, they do not have the potential to be used as anti Brucella agents. Among the four Brucella isolates, Brucella melitensis 293 is the most resistant to the inhibitory effect of the herbs because the inhibitory zones are the smallest which are 14 mm for Coptis chinensis, 14 mm for Radix paeoniae rubra, 32 mm for Galla chinensis and 12 mm for Cortex phellodendrin.Table 1.  Inhibition Zone Diameter in mm of Herb Extracts on Four Strains of Brucella melitensisHerbsBrucella melitensis 16MBrucella melitensis 293Brucella melitensis 183Brucella melitensis 4611Lunicera japonica 0 0 0 0Flos lonicera 0 0 0 0Coptis chinensis 22 14 29 22Andrographis paniculata 0 0 0 0Isatis indigotica 0 0 0 0Radix paeoniae rubra 18 14 18 16Galla chinensis 39 32 33 40Semen plantaginis 0 0 0 0Fructus forsythia 0 0 0 0Cortex phellodendrim 13 12 12 15Clavumox 44 42 45 453% DMSO Infused Blank Disc 0 0 0 047The antimicrobial activities of different extracts tested are shown in Tables 2 to 5. Bactericidal activity was confirmed by determining the MBC:MIC ratio. The MBC:MIC ratio describes a relationship between the minimum in vitro bactericidal concentration and the MIC of an antibiotic. If the MBC:MIC ratio of a pathogen is between 1:1 to 2:1, the drug is considered to be bactericidal against that pathogen. For Brucella melitensis 16, all herbs fulfill the ratio except for Coptis chinensis which has a ratio of 4:1 and Cortex phellodendrin which did not show any effects at the tested concentration.Table 2.  MIC and MBC of herb extracts on Brucella melitensis16HerbsBrucella melitensis  16MMIC (mg/mL) MBC(mg/mL) MIC-to-MBC ratioCoptis chinensis 7.5 30 1: 4Radix paeoniae rubia 7.5 7.5 1: 1Galla chinensis 3.75 3.75 1: 1Cortex phellodendrin nd nd ndnd - not determined because sample was not active at the highest concentrationFor Brucella melitensis 293, all herbs fulfill the ratio except for Cortex phellodendrin which did not show any inhibitory effects at the tested concentration.Table 3.   MIC and MBC of herb extracts on Brucella melitensis 293HerbsBrucella melitensis  293MIC (mg/mL) MBC(mg/mL) MIC-to-MBC ratioCoptis chinensis 3.75 7.5 1: 2Radix paeoniae rubia 7.5 7.5 1: 1Galla chinensis 3.75 3.75 1: 1Cortex phellodendrin nd nd ndnd - not determined because sample was not active at the highest concentrationFor Brucella melitensis 183, the results were the same as for Brucella melitensis 293.48Table 4.  MIC and MBC of herb extracts on Brucella melitensis 183HerbsBrucella melitensis  183MIC (mg/mL) MBC(mg/mL) MIC-to-MBC ratioCoptis chinensis 3.75 7.5 1: 2Radix paeoniae rubia 7.5 7.5 1: 1Galla chinensis 3.75 3.75 1: 1Cortex phellodendrin nd nd ndnd - not determined because sample was not active at the highest concentrationFor Brucella melitensis 4611, the MIC-to-MBC ratio was the same with the previous 2 strains.  However, MIC and MBC values were higher.Table 5.  MIC and MBC of herb extracts on Brucella melitensis 4611HerbsBrucella melitensis  4611MIC (mg/mL) MBC(mg/mL) MIC-to-MBC ratioCoptis chinensis 7.5 15 1: 2Radix paeoniae rubia 3.75 3.75 1: 1Galla chinensis 3.75 3.75 1: 1Cortex phellodendrin nd nd ndnd - not determined because sample was not active at the highest concentrationFrom the results, 2 herbs showed bactericidal activity which are, Radix paeoniae rubia and Galla chinensis. Cortex phellodendrin did not show bactericidal activity at the highest concentration tested while Coptis chinensis an MBC-to-MIC ratio that exceeded the bactericidal ratio of 1:2.  In comparison to previous work by Zhu et al., (2009) who assessed the herbs’ effects on Ureaplasma urealyticum, the findings are similar where Coptis chinensis, Radix paeoniae rubra, Galla chinensis and Cortex phellodendrim also yielded potential inhibitory effect on Brucella melitensis. However, the MICs of these herbs on the two different bacteria are different. Taking Brucella melitensis 16M as a comparison to Ureaplasma urealyticum, the MICs of Coptis chinensis on Brucella melitensis 16M is 7.5 mg/mL while on Ureaplasma is 20 mg/mL, MICs of Radix paeoniae rubia on Brucella melitensis 16M is 7.5 mg/mL while on Ureaplasma is 2.5 mg/mL, MICs of Galla chinensis on Brucella melitensis 16M is 3.75 mg/mL while on Ureaplasma is 1.25 mg/mL. Cortex phellodendrin did not yield any detectable MIC on Brucella melitensis at the highest tested concentration of 30 mg/mL while it yielded an MIC of 495 mg/mL on Ureaplasma urealyticum. From these comparisons and studies, it shows that these herbs can be developed into useful antimicrobials that can act against more than one type of bacteria. References Cox and Balick (1997). Plants, People and  Culture: The Science of Ethnobotany. 1st edition. Henry Holt & Company, USA.Galloway, J.H. (1972).  Farm Animal Health and Disease Control.  1st edition,Lea & Febiger., Philadelphia.Refai, M., El-Gibaly, S. and  Adawi, A. (1990). Initiation of a national brucellosis control program in Egypt. In: Adams, L. G. (Ed.), Advances in Brucellosis Research. Texas A&M University, Texas USA.Swabe, J. (1999).  Animals, Disease and Human Society – Human-animal relations and the rise of veterinary medicine.  1st edition, Routledge., NY.Zhu, C. T., Dong, C. L., Kong, Z. Y., Liu, L. X. and Yao, Y. Y. (2009). Mocrodilution inhabitation test of Chinese herbs to assess their effect against clinical strains of Ureaplasma urealyticum in vitro. J Nanjing Med Uni 23: 143-145.

Screening of chinese medicinal herbs for the inhibition of 

Brucella melitensis

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Screening of chinese medicinal herbs for the inhibition of Brucella melitensis

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