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Anti-microbial properties of Scutellaria baicalensis and Coptis chinensis, two traditional Chinese medicines

Anti-microbial properties of Scutellaria baicalensis and Coptis chinensis, two traditional... Volume 4 † Number 2 † June 2011 10.1093/biohorizons/hzr014 Advance Access publication 4 May 2011 ......................................................................................................................................................................................................................................... Research article Anti-microbial properties of Scutellaria baicalensis and Coptis chinensis, two traditional Chinese medicines Francesca S. Leach* Old School Studio, Hammerwood Road, Ashurst Wood, East Grinstead, West Sussex RH19 3RX, UK. * Corresponding author: Email: francesca.leach@bathspa.org Supervisor: N. Chaffey, Bath Spa University, Bath, UK. ........................................................................................................................................................................................................................................ High incidence of resistance to pharmaceutical antibiotics among microbes in hospital environments prompts the search for alternative sources of anti-microbial chemicals. A largely underexploited resource in this regard is plants used in traditional Chinese medicine (TCM). In this investigation, anti-microbial properties of water extracts of two herbs used in TCM—Scutellaria baicalensis Georgi (Huang Qin) and Coptis chinensis Franch (Huang Lian)—against Escherichia coli B, coagulase-negative staphylococcus and Saccharomyces cerevisiae were examined using the disc diffusion method with water as a negative control and vancomycin as the positive control for coagulase-negative staphylococcus. Coptis chinensis appeared more potent than S. baicalensis against the three microbes used in the main experiments. Against E. coli B, the mean width and standard error of the kill zone was 3.9+ 0.6 and 13.3+ 0.7 mm for S. baicalensis and C. chinensis, respectively. Against coagulase-negative staphylococcus, the mean kill zone widths were 6.6+ 1.1 and 11.0+ 1.0 mm for S. baicalensis and C. chinensis, respectively. Against S. cerevisiae, the mean kill zone widths were 8.4+ 1.0 and 12.6+ 1.4 mm for S. baicalensis and C. chinensis, respectively. When compared with the positive control, C. chinensis was comparable in effect to vanco- mycin against coagulase-negative staphylococcus, whereas S. baicalensis was less effective than vancomycin. Further experiments inves- tigated the use of herbs in combination and minimum inhibitory concentration. A limited number of further tests were conducted with other bacteria; E. coli 8879 (NCIMB 8879), Staphylococcus aureus (NCIMB 9518), Micrococcus luteus and Bacillus megaterium; both herbs killed all of the other bacteria, but C. chinensis appeared more potent than S. baicalensis. Diffusion disc technique provided a useful method to evaluate the anti-microbial effects of the two herbs, both of which showed promise as new anti-microbial agents. Key words: Scutellaria baicalensis, Coptis chinensis, anti-microbial, Escherichia coli B, coagulase-negative staphylococcus, disc diffusion, traditional Chinese medicine. Submitted September 2010; accepted February 2011 ........................................................................................................................................................................................................................................ (Huang Qin) and Coptis chinensis (Huang Lian) on Introduction Escherichia coli B, coagulase-negative staphylococcus and Bacterial resistance to antibiotics is becoming an increasing Saccharomyces cerevisiae. problem the world over. In order to combat this new Both a Gram-negative and a Gram-positive bacteria problem, novel antibiotic compounds/substances need to were chosen E. coli and coagulase-negative staphylococ- be found which are both effective and safe. Traditional cus, respectively, because Gram-negative bacteria are Chinese medicine (TCM) is an emerging research area in eth- more resistant to anti-microbials than Gram-positive nobotany and may provide a useful source of novel anti- bacteria due to their additional outer membrane layer. microbial compounds. The yeast S. cerevisiae was chosen as it is much more dif- This research aims to investigate the anti-microbial prop- ficult to identify safe drugs with antifungal properties erties of two herbs used in TCM, Scutellaria baicalensis than it is to find anti-microbials, because both humans ......................................................................................................................................................................................................................................... The Author 2011. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.5), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. 119 Research article Bioscience Horizons † Volume 4 † Number 2 † June 2011 ......................................................................................................................................................................................................................................... and fungi are eukaryotic whereas bacteria are resulting in the UK Department of Health launching new prokaryotic. strategies such as the ‘UK Anti-microbial Resistance Humans have a long history of using plants. The earliest Strategy and Action Plan’ (2000) to improve the efficacy of evidence of their use specifically as medicine comes from the current practices and staff. This problem is exacerbated fossil records from around 60 000 years ago, during the by not only the bacteria showing a wide resistance to most Middle Palaeolithic age. From this point on, human of the current antibiotics but also other anti-microbial pro- history is rich with examples of human use of plants as ducts such as disinfectants and antiseptics. medicine. The occurrence of bacterial resistance has been largely Although conservative estimates suggest that there are attributed to the overuse of antibiotics and the continual 250 000 species of gymnosperms and angiosperms on the exposure of the bacteria to low levels of antibiotics. The Earth, only around 15% have been phytochemically evalu- spread in resistance among bacteria can largely be attributed 3,4 ated and 6% screened for biological activity. This leaves a to four main areas: the occurrence of new species of patho- 15 15 wealth of unexplored potential new medicines, but increas- genic soil microbes, new resistance mechanisms, new ing industrial growth and urbanization of uncultivated and mutations occurring in the chromosome or plasmid DNA agricultural land mean that many of these species could be and the transfer of already existing resistant genes to other lost in the coming years, often without their medicinal prop- species or genera of bacteria. This ability to transfer the erties being recognized. resistance information via a plasmid is more widespread Analysis of methods used in plant-derived drug discovery than once understood to be, as it is now thought to occur suggests that scientific research, based on plants commonly between Gram-negative and Gram-positive bacteria. This used in traditional medicine, was an effective approach. would account for the occurrence of efflux pumps in This use of traditional medicines as sources of new drugs is Gram-positive bacteria. supported by the World Health Organisation (WHO) who In Gram-negative and Gram-positive bacteria, efflux throughout the world has 19 collaborating centres for tra- pumps enable the bacteria to expel a vast range of anti- ditional medicine, seven of which are in China, with microbial agents from antibiotics through to dyes and 13,16 another five throughout East Asia such as in Japan and organic solvents. The pump, which straddles the phos- Korea. pholipid bi-layer and the inner membrane transporter, recog- nizes substances and ejects them via the outer membrane Anti-microbial activities of medicinal plants channel into the external medium. This is done without Many common plants and herbs are known to have anti- the need for the toxic substances to accumulate prior to microbial effects, such as Chickweed, Echinacea, garlic and expulsion. thyme. Various papers have been written on this topic, Western medicine badly needs to find new anti-microbial often focusing on herbs used in Western medicine or herbs agents. Ethnobotanical remedies offer a promising avenue used by localized or indigenous communities, where the of research. TCM has the advantage over other traditional plants and their uses are uncommon in western medicine. medicinal practices, of a well-documented history of use. Although up to 50% of currently used pharmaceuticals are The bacteria that will be tested against these agents are derived from, or based on, plant products, none of them coagulase-negative staphylococcus and E. coli B. There are used as anti-microbials. are many strains of E. coli and although many are harmless, some can cause severe illness and even death. Anti-microbial resistance in bacteria S. aureus similarly to E. coli can cause diarrhoea and vomit- Bacteria are present in practically every environment on the ing which are typical symptoms of food poisoning. Earth and we are confronted daily by thousands of bacteria Coagulase-negative staphylococcus and E. coli B are less which live both on us and in us. These bacteria both help us, harmful species of S. aureus and E. coli, respectively, and 11 12 such as bifidobacterai, and can also make us ill. so these shall be used for the majority of the experiment. Throughout the world, there is growing concern about the Summary of relevant literature increasing prevalence and damage caused by outbreaks of illness such as infections caused by methicillin-resistant The literature review showed that a number of Chinese herbs Staphylococcus aureus. show promise as anti-microbial agents. There is an urgent Many of the antibiotics in use today are based upon a need to find new agents to combat disease caused by resistant small number of original compounds, such as penicillin, bacteria. Two of the most promising agents appear to be 18,19 20 with differences that arise from substitutions of chemical C. chinensis and S. baicalensis; these will be the groups at one point of the molecule. This small difference subject of my experimental investigation. means that bacteria can quickly adapt to the change in The Chinese name for Coptis sp. Franch, from the 13 21 chemical composition and become resistant again. Ranunculeae family, is Huang lian. There are 16 species Antibiotic-resistant bacteria are a major health concern, worldwide, six of these grow in mainland China, and ......................................................................................................................................................................................................................................... 120 Bioscience Horizons † Volume 4 † Number 2 † June 2011 Research article ......................................................................................................................................................................................................................................... only Coptis quinquefolia in Taiwan. Of these, C. chinensis, weighed into a labelled sterile McCartney bottle. A bottle C. deltoidea and C. tetoides are typically found in herbal of deionized, sterilized water was then heated to the markets in China and Taiwan. The rhizome has been boiling point, cooled to 838C and 5.4 ml of this was added found to be a rich source of the alkaloid berberine. In to the McCartney bottle. The infusion was then shaken for reference books, Huang Lian is specified most often as 6 min and left upright for 6 min to allow the suspended con- C. chinensis. It is mainly used traditionally as a heat purga- tents to settle as otherwise these can block the function of the tive, anti-inflammatory, de-toxicant and to clear damp. pipette. About 0.15 ml of this infusion was then loaded onto The Chinese name for S. baicalensis Georgi, from the sterile discs (13 mm in diameter) which were then placed in a Lamiaceae family is Huang Qin and has been found to 448C incubator under sterile conditions in order to dry contain high levels of the flavones wogonoside and baica- thoroughly. lin. Both these herbs are traditionally used to treat con- ditions brought about from excess heat and damp in the Preparation of the microbial spread plates body, including bacterial gastroenteritis (H. Li, personal Nutrient broth communication). Nutrient broth was made by adding 1 g of ‘Lab-lemco’ powder, 2 g of yeast extract, 5 g of peptone and 5 g of Materials and Methods sodium chloride per litre of distilled water. This was then mixed until all the powder had dissolved. This was then dis- Standard disc diffusion methodology was used to test two tributed into McCartney bottles in 10 ml quantities and then Chinese herbs against populations of common bacterial sterilized by autoclaving at 1218C for 15 min. A 24 h bac- pathogens and yeast. terial culture was made by taking one loop of culture from a Diffusion disc technique refrigerated slope culture, and putting it into 10 ml of nutri- ent broth. This was then placed in an incubator at 308C for Disc diffusion technique involved placing the two sterile 24 h for all microbes. 13 mm diffusion discs loaded with the anti-microbial agent on to each prepared agar spread plate inoculated with the microbe under test. The plates were then inverted and Nutrient agar incubated for 24 h at 378C for coagulase-negative staphylo- Nutrient agar was then made using 1 g of ‘Lab-lemco’ coccus, E. coli 8879 (NCIMB 8879), S. aureus (NCIMB powder, 2 g of yeast extract, 5 g of peptone, 5 g of sodium 9518), Micrococcus luteus and Bacillus megaterium or chloride and 15 g of agar per litre of distilled water. This 308C for E. coli B and S. cerevisiae. was then boiled until the powder had completely dissolved, Initially, a pilot study was carried out to investigate the then sterilized by autoclaving at 1218C for 15 min. After most effective method for extraction of anti-microbial being cooled to 458C in a water bath, it was used to herbal components, appropriate infusion concentration and prepare pour plates using sterile Petri dishes. incubation time in addition to the overall feasibility of the study. Ringer’s solution Preparation of the herbs Ringer’s solution was made using 1/4 strength Ringer’s sol- Samples of the two herbs, C. chinensis Franch and ution tablets of the formula 2.25 g of sodium chloride, S. baicalensis Georgi, were bought from a reputable UK 0.105 g of potassium chloride, 0.12 of calcium chloride TCM dispensary: The Institute of Chinese Medicine in and 0.05 g of sodium bicarbonate per litre. One tablet London (The Institute of Chinese Medicine, 44-46 was dissolved in 500 ml of distilled water and distributed Chandos Place, London WC2N 4HS). in 9 ml amounts into McCartney bottles. These were then sterilized by autoclaving at 1218C for 15 min. Infusions Pilot studies showed that hot water extraction was most Spread plates effective and traditional infusion concentration was suitable. Herbal infusions were prepared fresh each day, and infusions To prepare the bacterial spread plates, a microbial solution were labelled to identify the herb and the infusion replicate was first made by adding 1 ml of the 24 h bacterial culture number. The herb infusions were prepared by first grinding to 9 ml of sterile Ringer’s solution to make a 1 in 10 dilution 1 g of one herb sample in a pestle and mortar to a fine for each bacterium. Spread plates were then prepared and powder. The preparation was based on the traditional use adding 0.5 ml of bacterial solution of E. coli B or coagulase- of the herbs, and the amounts used were based on the negative staphylococcus onto pre-prepared agar plates. Plates instructions given with tea bags of these herbs as produced were labelled to identify the bacteria species, bacterial by the suppliers. Then, exactly 0.6 g of the sample was dilution factor. ......................................................................................................................................................................................................................................... 121 Research article Bioscience Horizons † Volume 4 † Number 2 † June 2011 ......................................................................................................................................................................................................................................... Minimum inhibitory concentration Determination of minimum inhibitory concentration (MIC) was achieved provisionally by first making an infusion of both herbs at the concentration used above. This was then diluted by half four times and once using a 3:1 ratio of infu- sion to water, so that a dilution series of 75%, 50%, 25%, 12.5%, 6.25% and 3.125% of the original infusion concen- tration was made. Three discs were made at each concen- tration, for each herb. For each organism, one disc of each concentration was plated. The diameter of the kill zone was measured as above, for each disc, after 24 h incubation time. Interaction Interaction was investigated using the method as above, except that instead of loading 0.15 ml of a single herb sample onto each disc, 0.075 ml of each of the two herbs Figure 1. Scutellaria baicalensis’ antimicrobial effect on E. coli B producing was loaded onto a single disc so that the total volume of infu- both a reduction zone (Line A on the left) and a kill zone (Line B on the right). sion loaded onto each disc was still 0.15 ml. Measurement of anti-microbial effect Contact time The measure of herbal potency was the diameter of the kill The effect of very short contact times between the zone around each disc on each plate. As shown in Fig. 1, herb-loaded disc and the spread plate was investigated. The the reduction zone is the outer area where there is still discs were prepared using the method as above. Instead of some bacteria present, whereas the inner kill zone is comple- the dried discs being placed onto the Petri dishes and left tely clear and absent of microbial growth. The diameter of for 24 h, they were removed after contact times of 15, 30 s, each disc’s inhibition zone was measured using a good- 1, 3, 5 and 7 min. The kill zone diameter was measured quality metric ruler, three times in different orientations to after the standard time of 24 h. allow a mean to be taken. Measuring was done by the same person to ensure consistency of technique and to try Analysis and reduce the human error. Data were tabulated, recording The six measures of the kill zone diameter for each exper- the details of the herbal preparation, the microbe and exper- imental condition (herb, sample, infusion number, tempera- imental conditions. ture, microbe, incubation time) were used to derive the kill After measuring the diameter of the kill zone, the diameter zone width, by subtraction of the disc diameter (13 mm). of the disc (13 mm) was subtracted to obtain the kill zone The kill zone width was then used to calculate the mean width. The reduction zones were measured separately, but kill zone width, standard deviation and variance of the kill these data are not presented here. zone width. Statistical analysis to test for differences between herbs Replicates and controls, and between different experimental conditions, Replications of all variables in the experiment were con- was carried out on the data using appropriate statistical tests, ducted. The infusion of each sample of each herb in hot such as ANOVA, independent and paired t-tests using the water was replicated six times. Two samples of each herb statistical software package Minitab 13.32. were made into three individual infusions. Each infusion was loaded onto 12 sterile discs, 6 for each microbe. Each Pooling replicate data microbe was also tested against a negative deionized, steri- lized water control disc. Vancomycin was used as a positive The variation between different infusions of the same herb control disc for coagulase-negative staphylococcus. One was tested using independent t-tests, and the results were control Petri dish, with both controls on it, was used per pooled where conditions were replicated apart from the infu- three Petri dishes of herb extract tested. sion number, if there was no significant difference between Once the initial data had been collected, further studies infusions. There was no significant difference by infusion were carried out. These tested the herbs against the microbes: number, for example, for sample 1, S. baicalensis,378C, E. coli 8879, S. aureus, M. luteus, B. megaterium and infusion 2 and 3 compared (t ¼ 0.43 (10), P ¼ 0.676). S. cerevisiae. Consequently, replications of different infusion numbers ......................................................................................................................................................................................................................................... 122 Bioscience Horizons † Volume 4 † Number 2 † June 2011 Research article ......................................................................................................................................................................................................................................... but same experimental conditions were pooled where Comparison of the anti-microbial effect of C. chinensis possible. and S. baicalensis with the commercial antibiotic vancomycin As shown in Fig. 4, the action of vancomycin on coagulase- Results negative staphylococcus produced kill zone diameters of similar size to those produced by C. chinensis. S. baicalensis The experiments were conducted in the Microbial Biology produced kill zones of smaller diameter than vancomycin. Laboratories at Bath Spa University between 29 September Neither S. baicalensis nor C. chinensis had a significantly and 3 November 2009. greater anti-microbial effect than vancomycin on coagulase- negative staphylococcus (for S. baicalensis: t ¼ 0.69 (22), Measurement of anti-microbial effect of C. chinensis P ¼ 0.496) (for C. chinensis: t ¼ 0.025 (22), P ¼ 0.98). and S. baicalensis on E. coli B, coagulase-negative Results are only shown for coagulase-negative staphylo- staphylococcus and S. cerevisiae coccus as vancomycin had no anti-microbial effect on S. baicalensis had a measurable anti-microbial effect on E. coli B. Hence, both S. baicalensis and C. chinensis have coagulase-negative staphylococcus, E. coli B and a significantly greater anti-microbial effect than vancomycin S. cerevisiae. Figure 2 shows that the size of the kill zone on E. coli B (for S. baicalensis: t ¼ 4.99 (10), P ¼ 0.0005) was smallest on E. coli B and largest on S. cerevisiae. The (for C. chinensis: t ¼ 13.17 (16), P, 0.0001). statistical tests showed that effects were significantly greater than that of water, the negative control, for all the organ- The MIC of infusion of either C. chinensis or S. baicalensis isms. The least inhibitory effect of S. baicalensis was seen needed for anti-microbial action on E. coli B which produced a mean width of inhibition The reduction in anti-microbial effects with lower concen- diameter of 3.3 mm, but was still highly significant compared trations of the herbal infusion was tested. Figure 5 shows with the control (t ¼ 5.15 (10), P ¼ 0.0004). results for S. baicalensis, which produced an inhibitory C. chinensis had an anti-microbial effect which was effect (kill zone ¼ 8.3 mm) at 50% original infusion strength greater than that of S. baicalensis. Figure 3 shows that (original infusion strength was the standard strength used in C. chinensis has its greatest effect on E. coli B, being all other experiments) on coagulase-negative staphylococcus. highly significantly different from the water control (t ¼ On E. coli B, S. baicalensis produced a small inhibitory effect 13.17 (16), P, 0.0001) and producing a mean ring of inhi- at 12.5% of the original infusion strength. On S. cerevisiae, bition width of 13.26 mm. Figure 4. Antimicrobial effects on the microbe S. albus, comparing Figure 2. Antimicrobial effect of S. baicalensis on E. coli B, S. albus and S. baicalensis, C. chinensis and the antibiotic vancomycin. Error bars show S. cerevisiae; mean kill zones and error bars showing standard error. standard error. Figure 5. Comparison of the effect of diluting the S. baicalensis infusion to Figure 3. Antimicrobial effect of C. chinensis on E. coli B, S. albus and 75%, 50%, 25%, 12.5%, 6.25% and 3.125% of the original infusion strength S. cerevisiae; mean kill zones and error bars showing standard error. had on S. albus, E. coli B and S. cerevisiae on mean kill zone width. ......................................................................................................................................................................................................................................... 123 Research article Bioscience Horizons † Volume 4 † Number 2 † June 2011 ......................................................................................................................................................................................................................................... Figure 6. Comparison of the effect of diluting the C. chinensis infusion to Figure 9. Comparison of the mean kill zone width produced in S. cerevisiae 75%, 50%, 25%, 12.5%, 6.25% and 3.125% of the original infusion strength by the two herbs acting alone or in combination. had on S. albus, E. coli B and S. cerevisiae on mean kill zone width. Figure 7. Comparison of the mean kill zone width produced in S. albus by Figure 10. Comparison of the mean kill zone width on E. coli 8879, the two herbs acting in combination or alone. Staphylococcus aureus, M. luteus and B. megaterium produced by S. baicalensis and C. chinensis. E. coli B and S. cerevisiae, respectively. For both coagulase- negative staphylococcus (Fig. 7) and E. coli B (Fig. 8), the effect of the combined herbs fell between that of S. baicalensis and C. chinensis alone. The difference in effect between combined and single herb applications was significantly different for E. coli B(t ¼ 31.86 (10), P ¼ 0.0001). S. cerevisiae was different; as shown in Fig. 9, the effect of the combined herb was consistently lower than that of either of the herbs alone and statistically significantly lower than C. chinensis (t ¼ 2.6 (10), P ¼ 0.023), suggesting that the two herbs are antagonistic with S. cerevisiae. Figure 8. Comparison of the mean kill zone width produced in E. coli Bby the two herbs acting alone or in combination. Effects of S. baicalensis and C. chinensis on a range of other bacteria: pilot testing 25% strength of the original infusion was needed to produce Figure 10 shows the results using a range of other bacteria. and inhibitory effect (kill zone ¼ 1 mm). Figure 6 shows the S. baicalensis showed inhibitory effects on all other bacteria results for C. chinensis, which produced an inhibitory effect tested. C. chinensis showed greater levels of inhibition than on coagulase-negative staphylococcus at 25% strength of the S. baicalensis on all the other bacteria tested. The greatest original infusion. On E. coli B, C. chinensis produced a large levels of inhibition were seen by the inhibition of M. luteus inhibitory effect (kill zone ¼ 10.3 mm) at 12.5% of the orig- by C. chinensis (kill zone width of 36.78 mm with sample 2). inal infusion. C. chinensis produced an inhibitory effect (kill zone ¼ 4.3 mm) on S. cerevisiae at 12.5% strength of the original infusion. Effect of length of contact time between the herbal disc and the bacterial population: Pilot testing Interaction between C. chinensis and S. baicalensis The investigation was prompted by the incidental, repeated Figures 7–9 show the effects of discs containing 50% of each observation of kill zones produced on an area of the plate of the two herbs (labelled S.b þ C.c) compared with the discs where a disc had been accidentally dropped and left in of one herb only on coagulase-negative staphylococcus, contact with the prepared spread plate for a few seconds. ......................................................................................................................................................................................................................................... 124 Bioscience Horizons † Volume 4 † Number 2 † June 2011 Research article ......................................................................................................................................................................................................................................... The figures below show results after contact times ranging width in coagulase-negative staphylococcus, E. coli B and from 15 s to 7 min, instead of the standard 24 h. S. cerevisiae. Additionally, C. chinensis was active at far S. baicalensis produced measurable kill zone widths in lower concentrations than S. baicalensis, especially against coagulase-negative staphylococcus with the 15 s contact E. coli B where it was still active at 3.125% of the original time. The size of the kill zones then decreased gradually infusion strength. These results suggest that weak infusions over the 30 s and 1 min applications, before a peak at or small doses of C. chinensis may have a powerful effect 3 min (4 mm) similar to that at 7 min (4.3 mm). The kill and therefore might make it a convenient anti-microbial zone width in E. coli Bby S. baicalensis was low until the substance. 7 min contact time which increased the kill zone width The high levels of inhibition shown by E. coli Bby from 1 to 4.3 mm. C. chinensis agrees with other studies. The inhibition of C. chinensis produced little inhibition of coagulase- S. cerevisiae by S. baicalensis is consistent with by the find- negative staphylococcus until the 3 min contact time. With ings of Cole et al. that diterpenoid isolates from E. coli B, however, C. chinensis produced small but increas- S. baicalensis have antifungal activity. ing rings of inhibition from the 15 s contact time. The kill Lin et al. found Coptidis rhizoma to inhibit 100% of zone width was the greatest with the 3 min contact time. growth of both S. aureus and E. coli, along with Porphyromonas gingivialis, Streptococcus mutans and S. sanguis. Franzblau and Cross also found C. chinensis Discussion and S. baicalensis to inhibit both E. coli and S. aureus. The two herbs showed a strong anti-microbial effect on the In the experiments that investigated interaction, the range of test organisms. Against coagulase-negative staphy- two-herb combination had a measurably smaller anti- lococcus, the herbs were comparable in effect to the anti- microbial effect than C. chinensis as a single herb. The com- biotic vancomycin. The herbs therefore show promise as bination of herbs produced a significantly greater zone of new anti-microbial agents, but these in vitro results must inhibition than S. baicalensis alone on E. coli B(t ¼ 3.704 be interpreted with caution, as they need to be tested in vivo. (10), P ¼ 0.0041). With coagulase-negative staphylococcus, When compared with the negative control water, the herb combination produced a measurably greater C. chinensis had a significantly greater effect on the organ- (0.43 mm), but not significantly different kill zone width isms E. coli B, coagulase-negative staphylococcus and from the single herb application. This interaction may seem S. cerevisiae at a P . 0.0001 level. S. baicalensis also had a surprising when the fact that most Chinese herbal prescrip- significantly greater effect on the organisms E. coli B, tions/medicines consist of a combination of different herbs coagulase-negative staphylococcus and S. cerevisiae at a is taken into account. It seems that in coagulase-negative sta- P . 0.0005 level when compared with the negative control phylococcus and E. coli B rather than being synergistic, the water. effect is additive, producing a kill zone width result that Both C. chinensis (P, 0.0001) and S. baicalensis (P, falls between the results for the two herbs when acting 0.005) had significantly greater levels of inhibition on alone. This finding is reflected by other studies, which E. coli B, than the positive control vancomycin. Since vanco- showed with the addition of measurements of in vivo cyto- mycin has no inhibitory effect on E. coli B, this comparison kine modulation that although the anti-microbial effect is is slightly unrealistic and would be improved if an antibiotic additive, the overall effect, including cytokine modulation, that was effective against Gram-negative bacteria such as was synergistic. In S. cerevisiae, however, it seems the E. coli B had been used. On coagulase-negative staphylococ- herb combinations action is conflicting, producing a far cus, vancomycin showed similar levels of inhibition to lower result than the two herbs alone. C. chinensis (mean inhibition by vancomycin 10.25 mm Results from the contact time series need more investi- and by C. chinensis 10.29 mm) and greater levels of inhi- gation. Contact times of 15 and 30 s between the bition than S. baicalensis (mean inhibition 5.68 mm). S. baicalensis disc and coagulase-negative staphylococcus Incubation of E. coli B with S. baicalensis produced produced kill zones of greater width than the 1 min disc reduction zones in addition to the kill zones. The reduction application. Similarly with E. coli B, the 15 s application zone was an area that, although not entirely clear of had a greater inhibitory action than the 30 s, 1, 3 and microbial growth, was still far clearer than the areas of the 5 min applications. This pattern was not present in the plate with uninterrupted microbial growth. When consider- C. chinensis results which show a trend of increasing kill ing the results, only the kill zones were take into account zone width with increasing contact time. However, with as any possible anti-microbial substance would need to both S. baicalensis and C. chinensis, there is a notable fully kill the microbe and not just reduce its level. peak at the 3 min contact time. The results for the trials looking at MICs were interesting. The anti-microbial activity of S. baicalensis and S. baicalensis was required in higher concentrations to C. chinensis is often attributed to the action of the flavonoids C. chinensis in order to produce a kill zone of similar they contain. These claims can be corroborated by testing the ......................................................................................................................................................................................................................................... 125 Research article Bioscience Horizons † Volume 4 † Number 2 † June 2011 ......................................................................................................................................................................................................................................... 3. Fabricant D, Farnsworth N (2001) The value of plants used in traditional pure flavonoid extracts against the bacteria in question medicine for drug discovery. Environ Health Perspect 109: 69–75. directly. When tested, although flavone showed no anti- 4. Verpoorte R (2000) Pharmacognosy in the new millennium: lead-finding and microbial activity against P. aeruginosa, polyhydroxylated biotechnology. J Pharm Pharmacol 52: 253–262. related flavonoids did exhibit anti-microbial properties. 5. Rastogi S, Kaphle K (2008) Sustainable traditional medicine: taking the inspi- Kong et al. found a close correlation between the ultra rations from ancient veterinary science. Evid Based Complement Altern Med; performance liquid chromatography spectrum and the anti- doi:10.1093/ecam/nen071. bacterial activities of Rhizoma coptidis on E. coli, determin- 6. NR Farnsworth, DJ Chadwick, J Marsh, eds (1994) Ethnopharmacology and ing that the probable antibacterial components were drug development. Ethnobotany and the Search for New Drugs, Ciba Foundation Symposium, Vol. 185, pp. 2–11. Chichester: John Wiley & Sons. berberine, jatrorrhizine and palmatine. The activity of 7. WHO (2010) WHO Collaborating Centres for Traditional Medicine [Online]. these active components was found to vary with the place 27 Available from: http://www.who.int/medicines/areas/traditional/ of production and time of harvest. collabcentres/en/index.html (accessed 15 February 2010). Fan et al. also found protoberberine alkaloids (PBAs) 8. Hoffmann D. (1994) New Holistic Herbal, 3rd ed. London: Thorsons. ISBN: extracted from Coptis chinensis to inhibit the growth of 978-1852301934. S. aureus. Of these PBAs, berberine showed the highest 9. Cowan MM (1999) Plant products as anti-microbial agents. Clin Microbiol Rev levels of anti-microbial activity. 12: 564–582. 10. Horner-Devine C, Carney K, Bohannan B (2004) An ecological perspective on bacterial biodiversity. Proc R Soc Biol Soc 271: 113–122. Conclusion 11. Mitsuoka T (1990) Bifidobacteria and their role in human health. J Ind Diffusion disc technique was used to evaluate the anti- Microbiol Biotechnol 6: 263–267. microbial effects of two herbs used in TCM: S. baicalensis 12. NHS (2008) Germs: Home Truths [Online]. Available from: http://www.nhs. uk/livewell/homehygiene/pages/homehygienetrivia.aspx (accessed 3 and C. chinensis. These herbs demonstrated powerful anti- January 2010]. microbial effects in vitro, against E. coli B, coagulase- 13. Lyon BR, Skurray R (1987) Anti-microbial resistance of Staphylococcus aureus: negative staphylococcus, S. cerevisiae, E. coli 8879, genetic basis. Microbiol Rev 51: 88–134. S. aureus, M. luteus and B. megaterium compared with the 14. Neu HC (1992) The crisis in antibiotic resistance. Science 257: 1064–1073. water control. The action of C. chinensis was comparable 15. Courvalin P (1994) Transfer of antibiotic resistance genes between to the antibiotic vancomycin. The results need to be tested Gram-positive and Gram-negative bacteria. Antimicrob Agents Chemother in vivo. 38: 1447–1451. 16. Zgurskaya H, Nikaido H (2002) Multidrug resistance mechanisms: drug efflux across two membranes. Mol Microbiol 37: 219–225. Acknowledgements 17. Food Standards Agency (2009). Food Poisoning; Food Bugs [Online]. I would like to thank Dr Nigel Chaffey for his direction and Available from: http://www.eatwell.gov.uk/healthissues/foodpoisoning/ abugslife/ (accessed 22 September 2009). encouragement on this research; Jenny Beard for her skills 18. Franzblau S, Cross C (1986) Comparative in vitro anti-microbial activity of and assistance in the laboratory, Darrel Watts and Graham Chinese medicinal herbs. J Ethnopharmacol 15: 279–288. Smith for their guidance about statistics and Dr Han Li for 19. Lin SJ, Chen CS, Lin SS et al. (2006) In vitro anti-microbial and in vivo cyto- her advice about herbs. kine modulating effects of different prepared Chinese herbal medicines. Food Chem Toxicol 44: 2078–2085. Funding 20. Tan KB, Vanitha J (2004) Immunomodulatory and anti-microbial effects of some traditional Chinese medicinal herbs: a review. Curr Med Chem 11: I gratefully acknowledge the funding provided by Bath Spa 1423–1430. University. 21. Cheng K, Chang H, Su C, Hsu F (1997) Identify Coptis species using random amplified polymorphic DNA. Bot Bull Acad Sin 38: 241–244. 22. Zhu Y (1998). Chinese Materia Medica: Chemistry, Pharmacology and Author biography Applications. Amsterdam: Taylor & Francis. F.L. studies BSc Hons Biology at Bath Spa University. She has 23. Chang HM, But PP (1987) Pharmacology and Applications of Chinese Materia Medica. London: World Scientific Publishing Co. Pte. Ltd. a particular interest in health, disease and other cultures’ 24. Maidment C, Dyson A, Haysome I (2006) A study into the anti-microbial approaches to these. In the future, she hopes to do vocational effects of cloves (Syzgium aromaticum) and cinnamon (Cinnamomum zeyla- postgraduate training in the healthcare field. nicum) using disc- diffusion assay. Nutr Food Sci 36: 225–230. 25. Bridson EY (1998) The OXOID Manual, 8th ed. Basingstoke: OXOID Limited. References 26. Minitab statistical software 13.32 (2000). Available from www.minitab.com (computer software). 1. Alberts B, Johnson A, Lewis J, Raff M, Roberts K, Walter P (2002) Molecular 27. Kong W, Zhao Y, Xiao X et al. (2009) Spectrum-effect relationships between Biology of the Cell. New York: Garland Science. ultra performance liquid chromatography fingerprints and anti-bacterial 2. Solecki R (1975) Shanidar IV, a Neanderthal flower burial in northern Iraq. activities of Rhizoma coptidis. Anal Chim Acta 634: 279–285. Science 190: 880–881. ......................................................................................................................................................................................................................................... 126 Bioscience Horizons † Volume 4 † Number 2 † June 2011 Research article ......................................................................................................................................................................................................................................... 28. Cole M, Bridge P, Dellar J, Fellows L, Cornish C, Anderson J (1991) Antifungal antibiotic resistant strains of Pseudomonas aeruginosa in Taiwan. Am J activity of Neo-clerodane diterpenoids from Scutellaria. Phytochemistry 30: Chin Med 35: 1047–1060. 1125–1127. 30. Fan D, Xiao X, Ma X (2008) Calorimetric study of the effect of protoberberine 29. Liu CS, Cham MT, Yang CH, Chang HW, Chen CH, Chuang LY (2007) alkaloids in Coptis chinensis Franch on Staphylococcus aureus growth. Antibacterial properties of Chinese herbal medicines against nosocomial Thermochim Acta 480: 49–52. ........................................................................................................................................................................................................................................ ......................................................................................................................................................................................................................................... http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Bioscience Horizons Oxford University Press

Anti-microbial properties of Scutellaria baicalensis and Coptis chinensis, two traditional Chinese medicines

Leach, Francesca S.
Bioscience Horizons , Volume 4 (2) – Jun 4, 2011
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Volume 4 † Number 2 † June 2011 10.1093/biohorizons/hzr014 Advance Access publication 4 May 2011 ......................................................................................................................................................................................................................................... Research article Anti-microbial properties of Scutellaria baicalensis and Coptis chinensis, two traditional Chinese medicines Francesca S. Leach* Old School Studio, Hammerwood Road, Ashurst Wood, East Grinstead, West Sussex RH19 3RX, UK. * Corresponding author: Email: francesca.leach@bathspa.org Supervisor: N. Chaffey, Bath Spa University, Bath, UK. ........................................................................................................................................................................................................................................ High incidence of resistance to pharmaceutical antibiotics among microbes in hospital environments prompts the search for alternative sources of anti-microbial chemicals. A largely underexploited resource in this regard is plants used in traditional Chinese medicine (TCM). In this investigation, anti-microbial properties of water extracts of two herbs used in TCM—Scutellaria baicalensis Georgi (Huang Qin) and Coptis chinensis Franch (Huang Lian)—against Escherichia coli B, coagulase-negative staphylococcus and Saccharomyces cerevisiae were examined using the disc diffusion method with water as a negative control and vancomycin as the positive control for coagulase-negative staphylococcus. Coptis chinensis appeared more potent than S. baicalensis against the three microbes used in the main experiments. Against E. coli B, the mean width and standard error of the kill zone was 3.9+ 0.6 and 13.3+ 0.7 mm for S. baicalensis and C. chinensis, respectively. Against coagulase-negative staphylococcus, the mean kill zone widths were 6.6+ 1.1 and 11.0+ 1.0 mm for S. baicalensis and C. chinensis, respectively. Against S. cerevisiae, the mean kill zone widths were 8.4+ 1.0 and 12.6+ 1.4 mm for S. baicalensis and C. chinensis, respectively. When compared with the positive control, C. chinensis was comparable in effect to vanco- mycin against coagulase-negative staphylococcus, whereas S. baicalensis was less effective than vancomycin. Further experiments inves- tigated the use of herbs in combination and minimum inhibitory concentration. A limited number of further tests were conducted with other bacteria; E. coli 8879 (NCIMB 8879), Staphylococcus aureus (NCIMB 9518), Micrococcus luteus and Bacillus megaterium; both herbs killed all of the other bacteria, but C. chinensis appeared more potent than S. baicalensis. Diffusion disc technique provided a useful method to evaluate the anti-microbial effects of the two herbs, both of which showed promise as new anti-microbial agents. Key words: Scutellaria baicalensis, Coptis chinensis, anti-microbial, Escherichia coli B, coagulase-negative staphylococcus, disc diffusion, traditional Chinese medicine. Submitted September 2010; accepted February 2011 ........................................................................................................................................................................................................................................ (Huang Qin) and Coptis chinensis (Huang Lian) on Introduction Escherichia coli B, coagulase-negative staphylococcus and Bacterial resistance to antibiotics is becoming an increasing Saccharomyces cerevisiae. problem the world over. In order to combat this new Both a Gram-negative and a Gram-positive bacteria problem, novel antibiotic compounds/substances need to were chosen E. coli and coagulase-negative staphylococ- be found which are both effective and safe. Traditional cus, respectively, because Gram-negative bacteria are Chinese medicine (TCM) is an emerging research area in eth- more resistant to anti-microbials than Gram-positive nobotany and may provide a useful source of novel anti- bacteria due to their additional outer membrane layer. microbial compounds. The yeast S. cerevisiae was chosen as it is much more dif- This research aims to investigate the anti-microbial prop- ficult to identify safe drugs with antifungal properties erties of two herbs used in TCM, Scutellaria baicalensis than it is to find anti-microbials, because both humans ......................................................................................................................................................................................................................................... The Author 2011. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.5), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. 119 Research article Bioscience Horizons † Volume 4 † Number 2 † June 2011 ......................................................................................................................................................................................................................................... and fungi are eukaryotic whereas bacteria are resulting in the UK Department of Health launching new prokaryotic. strategies such as the ‘UK Anti-microbial Resistance Humans have a long history of using plants. The earliest Strategy and Action Plan’ (2000) to improve the efficacy of evidence of their use specifically as medicine comes from the current practices and staff. This problem is exacerbated fossil records from around 60 000 years ago, during the by not only the bacteria showing a wide resistance to most Middle Palaeolithic age. From this point on, human of the current antibiotics but also other anti-microbial pro- history is rich with examples of human use of plants as ducts such as disinfectants and antiseptics. medicine. The occurrence of bacterial resistance has been largely Although conservative estimates suggest that there are attributed to the overuse of antibiotics and the continual 250 000 species of gymnosperms and angiosperms on the exposure of the bacteria to low levels of antibiotics. The Earth, only around 15% have been phytochemically evalu- spread in resistance among bacteria can largely be attributed 3,4 ated and 6% screened for biological activity. This leaves a to four main areas: the occurrence of new species of patho- 15 15 wealth of unexplored potential new medicines, but increas- genic soil microbes, new resistance mechanisms, new ing industrial growth and urbanization of uncultivated and mutations occurring in the chromosome or plasmid DNA agricultural land mean that many of these species could be and the transfer of already existing resistant genes to other lost in the coming years, often without their medicinal prop- species or genera of bacteria. This ability to transfer the erties being recognized. resistance information via a plasmid is more widespread Analysis of methods used in plant-derived drug discovery than once understood to be, as it is now thought to occur suggests that scientific research, based on plants commonly between Gram-negative and Gram-positive bacteria. This used in traditional medicine, was an effective approach. would account for the occurrence of efflux pumps in This use of traditional medicines as sources of new drugs is Gram-positive bacteria. supported by the World Health Organisation (WHO) who In Gram-negative and Gram-positive bacteria, efflux throughout the world has 19 collaborating centres for tra- pumps enable the bacteria to expel a vast range of anti- ditional medicine, seven of which are in China, with microbial agents from antibiotics through to dyes and 13,16 another five throughout East Asia such as in Japan and organic solvents. The pump, which straddles the phos- Korea. pholipid bi-layer and the inner membrane transporter, recog- nizes substances and ejects them via the outer membrane Anti-microbial activities of medicinal plants channel into the external medium. This is done without Many common plants and herbs are known to have anti- the need for the toxic substances to accumulate prior to microbial effects, such as Chickweed, Echinacea, garlic and expulsion. thyme. Various papers have been written on this topic, Western medicine badly needs to find new anti-microbial often focusing on herbs used in Western medicine or herbs agents. Ethnobotanical remedies offer a promising avenue used by localized or indigenous communities, where the of research. TCM has the advantage over other traditional plants and their uses are uncommon in western medicine. medicinal practices, of a well-documented history of use. Although up to 50% of currently used pharmaceuticals are The bacteria that will be tested against these agents are derived from, or based on, plant products, none of them coagulase-negative staphylococcus and E. coli B. There are used as anti-microbials. are many strains of E. coli and although many are harmless, some can cause severe illness and even death. Anti-microbial resistance in bacteria S. aureus similarly to E. coli can cause diarrhoea and vomit- Bacteria are present in practically every environment on the ing which are typical symptoms of food poisoning. Earth and we are confronted daily by thousands of bacteria Coagulase-negative staphylococcus and E. coli B are less which live both on us and in us. These bacteria both help us, harmful species of S. aureus and E. coli, respectively, and 11 12 such as bifidobacterai, and can also make us ill. so these shall be used for the majority of the experiment. Throughout the world, there is growing concern about the Summary of relevant literature increasing prevalence and damage caused by outbreaks of illness such as infections caused by methicillin-resistant The literature review showed that a number of Chinese herbs Staphylococcus aureus. show promise as anti-microbial agents. There is an urgent Many of the antibiotics in use today are based upon a need to find new agents to combat disease caused by resistant small number of original compounds, such as penicillin, bacteria. Two of the most promising agents appear to be 18,19 20 with differences that arise from substitutions of chemical C. chinensis and S. baicalensis; these will be the groups at one point of the molecule. This small difference subject of my experimental investigation. means that bacteria can quickly adapt to the change in The Chinese name for Coptis sp. Franch, from the 13 21 chemical composition and become resistant again. Ranunculeae family, is Huang lian. There are 16 species Antibiotic-resistant bacteria are a major health concern, worldwide, six of these grow in mainland China, and ......................................................................................................................................................................................................................................... 120 Bioscience Horizons † Volume 4 † Number 2 † June 2011 Research article ......................................................................................................................................................................................................................................... only Coptis quinquefolia in Taiwan. Of these, C. chinensis, weighed into a labelled sterile McCartney bottle. A bottle C. deltoidea and C. tetoides are typically found in herbal of deionized, sterilized water was then heated to the markets in China and Taiwan. The rhizome has been boiling point, cooled to 838C and 5.4 ml of this was added found to be a rich source of the alkaloid berberine. In to the McCartney bottle. The infusion was then shaken for reference books, Huang Lian is specified most often as 6 min and left upright for 6 min to allow the suspended con- C. chinensis. It is mainly used traditionally as a heat purga- tents to settle as otherwise these can block the function of the tive, anti-inflammatory, de-toxicant and to clear damp. pipette. About 0.15 ml of this infusion was then loaded onto The Chinese name for S. baicalensis Georgi, from the sterile discs (13 mm in diameter) which were then placed in a Lamiaceae family is Huang Qin and has been found to 448C incubator under sterile conditions in order to dry contain high levels of the flavones wogonoside and baica- thoroughly. lin. Both these herbs are traditionally used to treat con- ditions brought about from excess heat and damp in the Preparation of the microbial spread plates body, including bacterial gastroenteritis (H. Li, personal Nutrient broth communication). Nutrient broth was made by adding 1 g of ‘Lab-lemco’ powder, 2 g of yeast extract, 5 g of peptone and 5 g of Materials and Methods sodium chloride per litre of distilled water. This was then mixed until all the powder had dissolved. This was then dis- Standard disc diffusion methodology was used to test two tributed into McCartney bottles in 10 ml quantities and then Chinese herbs against populations of common bacterial sterilized by autoclaving at 1218C for 15 min. A 24 h bac- pathogens and yeast. terial culture was made by taking one loop of culture from a Diffusion disc technique refrigerated slope culture, and putting it into 10 ml of nutri- ent broth. This was then placed in an incubator at 308C for Disc diffusion technique involved placing the two sterile 24 h for all microbes. 13 mm diffusion discs loaded with the anti-microbial agent on to each prepared agar spread plate inoculated with the microbe under test. The plates were then inverted and Nutrient agar incubated for 24 h at 378C for coagulase-negative staphylo- Nutrient agar was then made using 1 g of ‘Lab-lemco’ coccus, E. coli 8879 (NCIMB 8879), S. aureus (NCIMB powder, 2 g of yeast extract, 5 g of peptone, 5 g of sodium 9518), Micrococcus luteus and Bacillus megaterium or chloride and 15 g of agar per litre of distilled water. This 308C for E. coli B and S. cerevisiae. was then boiled until the powder had completely dissolved, Initially, a pilot study was carried out to investigate the then sterilized by autoclaving at 1218C for 15 min. After most effective method for extraction of anti-microbial being cooled to 458C in a water bath, it was used to herbal components, appropriate infusion concentration and prepare pour plates using sterile Petri dishes. incubation time in addition to the overall feasibility of the study. Ringer’s solution Preparation of the herbs Ringer’s solution was made using 1/4 strength Ringer’s sol- Samples of the two herbs, C. chinensis Franch and ution tablets of the formula 2.25 g of sodium chloride, S. baicalensis Georgi, were bought from a reputable UK 0.105 g of potassium chloride, 0.12 of calcium chloride TCM dispensary: The Institute of Chinese Medicine in and 0.05 g of sodium bicarbonate per litre. One tablet London (The Institute of Chinese Medicine, 44-46 was dissolved in 500 ml of distilled water and distributed Chandos Place, London WC2N 4HS). in 9 ml amounts into McCartney bottles. These were then sterilized by autoclaving at 1218C for 15 min. Infusions Pilot studies showed that hot water extraction was most Spread plates effective and traditional infusion concentration was suitable. Herbal infusions were prepared fresh each day, and infusions To prepare the bacterial spread plates, a microbial solution were labelled to identify the herb and the infusion replicate was first made by adding 1 ml of the 24 h bacterial culture number. The herb infusions were prepared by first grinding to 9 ml of sterile Ringer’s solution to make a 1 in 10 dilution 1 g of one herb sample in a pestle and mortar to a fine for each bacterium. Spread plates were then prepared and powder. The preparation was based on the traditional use adding 0.5 ml of bacterial solution of E. coli B or coagulase- of the herbs, and the amounts used were based on the negative staphylococcus onto pre-prepared agar plates. Plates instructions given with tea bags of these herbs as produced were labelled to identify the bacteria species, bacterial by the suppliers. Then, exactly 0.6 g of the sample was dilution factor. ......................................................................................................................................................................................................................................... 121 Research article Bioscience Horizons † Volume 4 † Number 2 † June 2011 ......................................................................................................................................................................................................................................... Minimum inhibitory concentration Determination of minimum inhibitory concentration (MIC) was achieved provisionally by first making an infusion of both herbs at the concentration used above. This was then diluted by half four times and once using a 3:1 ratio of infu- sion to water, so that a dilution series of 75%, 50%, 25%, 12.5%, 6.25% and 3.125% of the original infusion concen- tration was made. Three discs were made at each concen- tration, for each herb. For each organism, one disc of each concentration was plated. The diameter of the kill zone was measured as above, for each disc, after 24 h incubation time. Interaction Interaction was investigated using the method as above, except that instead of loading 0.15 ml of a single herb sample onto each disc, 0.075 ml of each of the two herbs Figure 1. Scutellaria baicalensis’ antimicrobial effect on E. coli B producing was loaded onto a single disc so that the total volume of infu- both a reduction zone (Line A on the left) and a kill zone (Line B on the right). sion loaded onto each disc was still 0.15 ml. Measurement of anti-microbial effect Contact time The measure of herbal potency was the diameter of the kill The effect of very short contact times between the zone around each disc on each plate. As shown in Fig. 1, herb-loaded disc and the spread plate was investigated. The the reduction zone is the outer area where there is still discs were prepared using the method as above. Instead of some bacteria present, whereas the inner kill zone is comple- the dried discs being placed onto the Petri dishes and left tely clear and absent of microbial growth. The diameter of for 24 h, they were removed after contact times of 15, 30 s, each disc’s inhibition zone was measured using a good- 1, 3, 5 and 7 min. The kill zone diameter was measured quality metric ruler, three times in different orientations to after the standard time of 24 h. allow a mean to be taken. Measuring was done by the same person to ensure consistency of technique and to try Analysis and reduce the human error. Data were tabulated, recording The six measures of the kill zone diameter for each exper- the details of the herbal preparation, the microbe and exper- imental condition (herb, sample, infusion number, tempera- imental conditions. ture, microbe, incubation time) were used to derive the kill After measuring the diameter of the kill zone, the diameter zone width, by subtraction of the disc diameter (13 mm). of the disc (13 mm) was subtracted to obtain the kill zone The kill zone width was then used to calculate the mean width. The reduction zones were measured separately, but kill zone width, standard deviation and variance of the kill these data are not presented here. zone width. Statistical analysis to test for differences between herbs Replicates and controls, and between different experimental conditions, Replications of all variables in the experiment were con- was carried out on the data using appropriate statistical tests, ducted. The infusion of each sample of each herb in hot such as ANOVA, independent and paired t-tests using the water was replicated six times. Two samples of each herb statistical software package Minitab 13.32. were made into three individual infusions. Each infusion was loaded onto 12 sterile discs, 6 for each microbe. Each Pooling replicate data microbe was also tested against a negative deionized, steri- lized water control disc. Vancomycin was used as a positive The variation between different infusions of the same herb control disc for coagulase-negative staphylococcus. One was tested using independent t-tests, and the results were control Petri dish, with both controls on it, was used per pooled where conditions were replicated apart from the infu- three Petri dishes of herb extract tested. sion number, if there was no significant difference between Once the initial data had been collected, further studies infusions. There was no significant difference by infusion were carried out. These tested the herbs against the microbes: number, for example, for sample 1, S. baicalensis,378C, E. coli 8879, S. aureus, M. luteus, B. megaterium and infusion 2 and 3 compared (t ¼ 0.43 (10), P ¼ 0.676). S. cerevisiae. Consequently, replications of different infusion numbers ......................................................................................................................................................................................................................................... 122 Bioscience Horizons † Volume 4 † Number 2 † June 2011 Research article ......................................................................................................................................................................................................................................... but same experimental conditions were pooled where Comparison of the anti-microbial effect of C. chinensis possible. and S. baicalensis with the commercial antibiotic vancomycin As shown in Fig. 4, the action of vancomycin on coagulase- Results negative staphylococcus produced kill zone diameters of similar size to those produced by C. chinensis. S. baicalensis The experiments were conducted in the Microbial Biology produced kill zones of smaller diameter than vancomycin. Laboratories at Bath Spa University between 29 September Neither S. baicalensis nor C. chinensis had a significantly and 3 November 2009. greater anti-microbial effect than vancomycin on coagulase- negative staphylococcus (for S. baicalensis: t ¼ 0.69 (22), Measurement of anti-microbial effect of C. chinensis P ¼ 0.496) (for C. chinensis: t ¼ 0.025 (22), P ¼ 0.98). and S. baicalensis on E. coli B, coagulase-negative Results are only shown for coagulase-negative staphylo- staphylococcus and S. cerevisiae coccus as vancomycin had no anti-microbial effect on S. baicalensis had a measurable anti-microbial effect on E. coli B. Hence, both S. baicalensis and C. chinensis have coagulase-negative staphylococcus, E. coli B and a significantly greater anti-microbial effect than vancomycin S. cerevisiae. Figure 2 shows that the size of the kill zone on E. coli B (for S. baicalensis: t ¼ 4.99 (10), P ¼ 0.0005) was smallest on E. coli B and largest on S. cerevisiae. The (for C. chinensis: t ¼ 13.17 (16), P, 0.0001). statistical tests showed that effects were significantly greater than that of water, the negative control, for all the organ- The MIC of infusion of either C. chinensis or S. baicalensis isms. The least inhibitory effect of S. baicalensis was seen needed for anti-microbial action on E. coli B which produced a mean width of inhibition The reduction in anti-microbial effects with lower concen- diameter of 3.3 mm, but was still highly significant compared trations of the herbal infusion was tested. Figure 5 shows with the control (t ¼ 5.15 (10), P ¼ 0.0004). results for S. baicalensis, which produced an inhibitory C. chinensis had an anti-microbial effect which was effect (kill zone ¼ 8.3 mm) at 50% original infusion strength greater than that of S. baicalensis. Figure 3 shows that (original infusion strength was the standard strength used in C. chinensis has its greatest effect on E. coli B, being all other experiments) on coagulase-negative staphylococcus. highly significantly different from the water control (t ¼ On E. coli B, S. baicalensis produced a small inhibitory effect 13.17 (16), P, 0.0001) and producing a mean ring of inhi- at 12.5% of the original infusion strength. On S. cerevisiae, bition width of 13.26 mm. Figure 4. Antimicrobial effects on the microbe S. albus, comparing Figure 2. Antimicrobial effect of S. baicalensis on E. coli B, S. albus and S. baicalensis, C. chinensis and the antibiotic vancomycin. Error bars show S. cerevisiae; mean kill zones and error bars showing standard error. standard error. Figure 5. Comparison of the effect of diluting the S. baicalensis infusion to Figure 3. Antimicrobial effect of C. chinensis on E. coli B, S. albus and 75%, 50%, 25%, 12.5%, 6.25% and 3.125% of the original infusion strength S. cerevisiae; mean kill zones and error bars showing standard error. had on S. albus, E. coli B and S. cerevisiae on mean kill zone width. ......................................................................................................................................................................................................................................... 123 Research article Bioscience Horizons † Volume 4 † Number 2 † June 2011 ......................................................................................................................................................................................................................................... Figure 6. Comparison of the effect of diluting the C. chinensis infusion to Figure 9. Comparison of the mean kill zone width produced in S. cerevisiae 75%, 50%, 25%, 12.5%, 6.25% and 3.125% of the original infusion strength by the two herbs acting alone or in combination. had on S. albus, E. coli B and S. cerevisiae on mean kill zone width. Figure 7. Comparison of the mean kill zone width produced in S. albus by Figure 10. Comparison of the mean kill zone width on E. coli 8879, the two herbs acting in combination or alone. Staphylococcus aureus, M. luteus and B. megaterium produced by S. baicalensis and C. chinensis. E. coli B and S. cerevisiae, respectively. For both coagulase- negative staphylococcus (Fig. 7) and E. coli B (Fig. 8), the effect of the combined herbs fell between that of S. baicalensis and C. chinensis alone. The difference in effect between combined and single herb applications was significantly different for E. coli B(t ¼ 31.86 (10), P ¼ 0.0001). S. cerevisiae was different; as shown in Fig. 9, the effect of the combined herb was consistently lower than that of either of the herbs alone and statistically significantly lower than C. chinensis (t ¼ 2.6 (10), P ¼ 0.023), suggesting that the two herbs are antagonistic with S. cerevisiae. Figure 8. Comparison of the mean kill zone width produced in E. coli Bby the two herbs acting alone or in combination. Effects of S. baicalensis and C. chinensis on a range of other bacteria: pilot testing 25% strength of the original infusion was needed to produce Figure 10 shows the results using a range of other bacteria. and inhibitory effect (kill zone ¼ 1 mm). Figure 6 shows the S. baicalensis showed inhibitory effects on all other bacteria results for C. chinensis, which produced an inhibitory effect tested. C. chinensis showed greater levels of inhibition than on coagulase-negative staphylococcus at 25% strength of the S. baicalensis on all the other bacteria tested. The greatest original infusion. On E. coli B, C. chinensis produced a large levels of inhibition were seen by the inhibition of M. luteus inhibitory effect (kill zone ¼ 10.3 mm) at 12.5% of the orig- by C. chinensis (kill zone width of 36.78 mm with sample 2). inal infusion. C. chinensis produced an inhibitory effect (kill zone ¼ 4.3 mm) on S. cerevisiae at 12.5% strength of the original infusion. Effect of length of contact time between the herbal disc and the bacterial population: Pilot testing Interaction between C. chinensis and S. baicalensis The investigation was prompted by the incidental, repeated Figures 7–9 show the effects of discs containing 50% of each observation of kill zones produced on an area of the plate of the two herbs (labelled S.b þ C.c) compared with the discs where a disc had been accidentally dropped and left in of one herb only on coagulase-negative staphylococcus, contact with the prepared spread plate for a few seconds. ......................................................................................................................................................................................................................................... 124 Bioscience Horizons † Volume 4 † Number 2 † June 2011 Research article ......................................................................................................................................................................................................................................... The figures below show results after contact times ranging width in coagulase-negative staphylococcus, E. coli B and from 15 s to 7 min, instead of the standard 24 h. S. cerevisiae. Additionally, C. chinensis was active at far S. baicalensis produced measurable kill zone widths in lower concentrations than S. baicalensis, especially against coagulase-negative staphylococcus with the 15 s contact E. coli B where it was still active at 3.125% of the original time. The size of the kill zones then decreased gradually infusion strength. These results suggest that weak infusions over the 30 s and 1 min applications, before a peak at or small doses of C. chinensis may have a powerful effect 3 min (4 mm) similar to that at 7 min (4.3 mm). The kill and therefore might make it a convenient anti-microbial zone width in E. coli Bby S. baicalensis was low until the substance. 7 min contact time which increased the kill zone width The high levels of inhibition shown by E. coli Bby from 1 to 4.3 mm. C. chinensis agrees with other studies. The inhibition of C. chinensis produced little inhibition of coagulase- S. cerevisiae by S. baicalensis is consistent with by the find- negative staphylococcus until the 3 min contact time. With ings of Cole et al. that diterpenoid isolates from E. coli B, however, C. chinensis produced small but increas- S. baicalensis have antifungal activity. ing rings of inhibition from the 15 s contact time. The kill Lin et al. found Coptidis rhizoma to inhibit 100% of zone width was the greatest with the 3 min contact time. growth of both S. aureus and E. coli, along with Porphyromonas gingivialis, Streptococcus mutans and S. sanguis. Franzblau and Cross also found C. chinensis Discussion and S. baicalensis to inhibit both E. coli and S. aureus. The two herbs showed a strong anti-microbial effect on the In the experiments that investigated interaction, the range of test organisms. Against coagulase-negative staphy- two-herb combination had a measurably smaller anti- lococcus, the herbs were comparable in effect to the anti- microbial effect than C. chinensis as a single herb. The com- biotic vancomycin. The herbs therefore show promise as bination of herbs produced a significantly greater zone of new anti-microbial agents, but these in vitro results must inhibition than S. baicalensis alone on E. coli B(t ¼ 3.704 be interpreted with caution, as they need to be tested in vivo. (10), P ¼ 0.0041). With coagulase-negative staphylococcus, When compared with the negative control water, the herb combination produced a measurably greater C. chinensis had a significantly greater effect on the organ- (0.43 mm), but not significantly different kill zone width isms E. coli B, coagulase-negative staphylococcus and from the single herb application. This interaction may seem S. cerevisiae at a P . 0.0001 level. S. baicalensis also had a surprising when the fact that most Chinese herbal prescrip- significantly greater effect on the organisms E. coli B, tions/medicines consist of a combination of different herbs coagulase-negative staphylococcus and S. cerevisiae at a is taken into account. It seems that in coagulase-negative sta- P . 0.0005 level when compared with the negative control phylococcus and E. coli B rather than being synergistic, the water. effect is additive, producing a kill zone width result that Both C. chinensis (P, 0.0001) and S. baicalensis (P, falls between the results for the two herbs when acting 0.005) had significantly greater levels of inhibition on alone. This finding is reflected by other studies, which E. coli B, than the positive control vancomycin. Since vanco- showed with the addition of measurements of in vivo cyto- mycin has no inhibitory effect on E. coli B, this comparison kine modulation that although the anti-microbial effect is is slightly unrealistic and would be improved if an antibiotic additive, the overall effect, including cytokine modulation, that was effective against Gram-negative bacteria such as was synergistic. In S. cerevisiae, however, it seems the E. coli B had been used. On coagulase-negative staphylococ- herb combinations action is conflicting, producing a far cus, vancomycin showed similar levels of inhibition to lower result than the two herbs alone. C. chinensis (mean inhibition by vancomycin 10.25 mm Results from the contact time series need more investi- and by C. chinensis 10.29 mm) and greater levels of inhi- gation. Contact times of 15 and 30 s between the bition than S. baicalensis (mean inhibition 5.68 mm). S. baicalensis disc and coagulase-negative staphylococcus Incubation of E. coli B with S. baicalensis produced produced kill zones of greater width than the 1 min disc reduction zones in addition to the kill zones. The reduction application. Similarly with E. coli B, the 15 s application zone was an area that, although not entirely clear of had a greater inhibitory action than the 30 s, 1, 3 and microbial growth, was still far clearer than the areas of the 5 min applications. This pattern was not present in the plate with uninterrupted microbial growth. When consider- C. chinensis results which show a trend of increasing kill ing the results, only the kill zones were take into account zone width with increasing contact time. However, with as any possible anti-microbial substance would need to both S. baicalensis and C. chinensis, there is a notable fully kill the microbe and not just reduce its level. peak at the 3 min contact time. The results for the trials looking at MICs were interesting. The anti-microbial activity of S. baicalensis and S. baicalensis was required in higher concentrations to C. chinensis is often attributed to the action of the flavonoids C. chinensis in order to produce a kill zone of similar they contain. These claims can be corroborated by testing the ......................................................................................................................................................................................................................................... 125 Research article Bioscience Horizons † Volume 4 † Number 2 † June 2011 ......................................................................................................................................................................................................................................... 3. Fabricant D, Farnsworth N (2001) The value of plants used in traditional pure flavonoid extracts against the bacteria in question medicine for drug discovery. Environ Health Perspect 109: 69–75. directly. When tested, although flavone showed no anti- 4. Verpoorte R (2000) Pharmacognosy in the new millennium: lead-finding and microbial activity against P. aeruginosa, polyhydroxylated biotechnology. J Pharm Pharmacol 52: 253–262. related flavonoids did exhibit anti-microbial properties. 5. Rastogi S, Kaphle K (2008) Sustainable traditional medicine: taking the inspi- Kong et al. found a close correlation between the ultra rations from ancient veterinary science. Evid Based Complement Altern Med; performance liquid chromatography spectrum and the anti- doi:10.1093/ecam/nen071. bacterial activities of Rhizoma coptidis on E. coli, determin- 6. NR Farnsworth, DJ Chadwick, J Marsh, eds (1994) Ethnopharmacology and ing that the probable antibacterial components were drug development. Ethnobotany and the Search for New Drugs, Ciba Foundation Symposium, Vol. 185, pp. 2–11. Chichester: John Wiley & Sons. berberine, jatrorrhizine and palmatine. The activity of 7. WHO (2010) WHO Collaborating Centres for Traditional Medicine [Online]. these active components was found to vary with the place 27 Available from: http://www.who.int/medicines/areas/traditional/ of production and time of harvest. collabcentres/en/index.html (accessed 15 February 2010). Fan et al. also found protoberberine alkaloids (PBAs) 8. Hoffmann D. (1994) New Holistic Herbal, 3rd ed. London: Thorsons. ISBN: extracted from Coptis chinensis to inhibit the growth of 978-1852301934. S. aureus. Of these PBAs, berberine showed the highest 9. Cowan MM (1999) Plant products as anti-microbial agents. Clin Microbiol Rev levels of anti-microbial activity. 12: 564–582. 10. Horner-Devine C, Carney K, Bohannan B (2004) An ecological perspective on bacterial biodiversity. Proc R Soc Biol Soc 271: 113–122. Conclusion 11. Mitsuoka T (1990) Bifidobacteria and their role in human health. J Ind Diffusion disc technique was used to evaluate the anti- Microbiol Biotechnol 6: 263–267. microbial effects of two herbs used in TCM: S. baicalensis 12. NHS (2008) Germs: Home Truths [Online]. Available from: http://www.nhs. uk/livewell/homehygiene/pages/homehygienetrivia.aspx (accessed 3 and C. chinensis. These herbs demonstrated powerful anti- January 2010]. microbial effects in vitro, against E. coli B, coagulase- 13. Lyon BR, Skurray R (1987) Anti-microbial resistance of Staphylococcus aureus: negative staphylococcus, S. cerevisiae, E. coli 8879, genetic basis. Microbiol Rev 51: 88–134. S. aureus, M. luteus and B. megaterium compared with the 14. Neu HC (1992) The crisis in antibiotic resistance. Science 257: 1064–1073. water control. The action of C. chinensis was comparable 15. Courvalin P (1994) Transfer of antibiotic resistance genes between to the antibiotic vancomycin. The results need to be tested Gram-positive and Gram-negative bacteria. Antimicrob Agents Chemother in vivo. 38: 1447–1451. 16. Zgurskaya H, Nikaido H (2002) Multidrug resistance mechanisms: drug efflux across two membranes. Mol Microbiol 37: 219–225. Acknowledgements 17. Food Standards Agency (2009). Food Poisoning; Food Bugs [Online]. I would like to thank Dr Nigel Chaffey for his direction and Available from: http://www.eatwell.gov.uk/healthissues/foodpoisoning/ abugslife/ (accessed 22 September 2009). encouragement on this research; Jenny Beard for her skills 18. Franzblau S, Cross C (1986) Comparative in vitro anti-microbial activity of and assistance in the laboratory, Darrel Watts and Graham Chinese medicinal herbs. J Ethnopharmacol 15: 279–288. Smith for their guidance about statistics and Dr Han Li for 19. Lin SJ, Chen CS, Lin SS et al. (2006) In vitro anti-microbial and in vivo cyto- her advice about herbs. kine modulating effects of different prepared Chinese herbal medicines. Food Chem Toxicol 44: 2078–2085. Funding 20. Tan KB, Vanitha J (2004) Immunomodulatory and anti-microbial effects of some traditional Chinese medicinal herbs: a review. Curr Med Chem 11: I gratefully acknowledge the funding provided by Bath Spa 1423–1430. University. 21. Cheng K, Chang H, Su C, Hsu F (1997) Identify Coptis species using random amplified polymorphic DNA. Bot Bull Acad Sin 38: 241–244. 22. Zhu Y (1998). Chinese Materia Medica: Chemistry, Pharmacology and Author biography Applications. Amsterdam: Taylor & Francis. F.L. studies BSc Hons Biology at Bath Spa University. She has 23. Chang HM, But PP (1987) Pharmacology and Applications of Chinese Materia Medica. London: World Scientific Publishing Co. Pte. Ltd. a particular interest in health, disease and other cultures’ 24. Maidment C, Dyson A, Haysome I (2006) A study into the anti-microbial approaches to these. In the future, she hopes to do vocational effects of cloves (Syzgium aromaticum) and cinnamon (Cinnamomum zeyla- postgraduate training in the healthcare field. nicum) using disc- diffusion assay. Nutr Food Sci 36: 225–230. 25. Bridson EY (1998) The OXOID Manual, 8th ed. Basingstoke: OXOID Limited. References 26. Minitab statistical software 13.32 (2000). Available from www.minitab.com (computer software). 1. Alberts B, Johnson A, Lewis J, Raff M, Roberts K, Walter P (2002) Molecular 27. Kong W, Zhao Y, Xiao X et al. (2009) Spectrum-effect relationships between Biology of the Cell. New York: Garland Science. ultra performance liquid chromatography fingerprints and anti-bacterial 2. Solecki R (1975) Shanidar IV, a Neanderthal flower burial in northern Iraq. activities of Rhizoma coptidis. Anal Chim Acta 634: 279–285. Science 190: 880–881. ......................................................................................................................................................................................................................................... 126 Bioscience Horizons † Volume 4 † Number 2 † June 2011 Research article ......................................................................................................................................................................................................................................... 28. Cole M, Bridge P, Dellar J, Fellows L, Cornish C, Anderson J (1991) Antifungal antibiotic resistant strains of Pseudomonas aeruginosa in Taiwan. Am J activity of Neo-clerodane diterpenoids from Scutellaria. Phytochemistry 30: Chin Med 35: 1047–1060. 1125–1127. 30. Fan D, Xiao X, Ma X (2008) Calorimetric study of the effect of protoberberine 29. Liu CS, Cham MT, Yang CH, Chang HW, Chen CH, Chuang LY (2007) alkaloids in Coptis chinensis Franch on Staphylococcus aureus growth. Antibacterial properties of Chinese herbal medicines against nosocomial Thermochim Acta 480: 49–52. ........................................................................................................................................................................................................................................ .........................................................................................................................................................................................................................................

Journal

Bioscience Horizons – Oxford University Press

Published: Jun 4, 2011

Keywords: Scutellaria baicalensis Coptis chinensis anti-microbial Escherichia coli B coagulase-negative staphylococcus disc diffusion traditional Chinese medicine

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