The genus Yersinia (Y.) contains more than a dozen validly described species1 of facultative anaerobes widespread within the environment. Members of the genus are also widely-associated with animal populations. Many of these bacteria are clinically significant as they can be pathogenic to humans. The zoonotic nature of these bacteria poses a unique set of challenges in the treatment and prevention of associated disease as they can persist in non-human hosts until they opportunistically infect susceptible people. Diseases caused by Yersinia spp. vary in severity and mortality, ranging from the highly devastating bubonic and pneumonic plagues (Yersinia pestis) to the gastrointestinal-distressing yersiniosis (Yersinia enterocolitica).2
Yersinosis is an acute gastrointestinal infection responsible for over 100,000 cases of illness annually in the United States.3 Typically characterized by combinations of abdominal pain, fever or diarrhoea, infection commonly originates from the ingestion of contaminated food or water. Indeed, approximately 90% of all reported cases originate from food/water sources.4 Prevention is further complicated as Y. enterocolitica can grow at 4 °C and thus refrigeration does not provide sufficient protection from illness.5 Though rarely life-threatening, the economic drain caused through temporary incapacitation from yersiniosis means that the probing for effective treatment strategies is of particular importance. One such strategy involves utilizing natural resources such as plants with documented antibacterial capabilities. Therefore, these relatively untapped reservoirs may provide novel treatment options for yersiniosis.
Terminalia is one of the most useful genera of therapeutic plants globally. The genus consists of approximately 200-250 species of flowering trees, many of which have uses in several traditional medicinal systems.6 Whilst numerous therapeutic properties are known for Terminalia spp., the antibacterial activity has been particularly well reported. Extracts prepared from the fruit of the Australian species Terminalia ferdinandiana (Kakadu plum) have potent growth inhibitory activity against an extensive panel of pathogens including bacteria associated diarrhoea and dysentery7 as well as the bacterial triggers of rheumatoid arthritis (Proteus mirabilis)8 and multiple sclerosis (Acinetobacter baylyi and Pseudomonas aeruginosa).7 Leaf extracts from the same species have also been shown to inhibit growth of the same bacteria, as well as a microbial trigger of ankylosing spondylitis (Klebsiella pneumoniae).8 Similarly, African Terminalia spp. have also been shown to be effective bacterial growth inhibitors. Terminalia stenostachya and Terminalia spinosa have strong antibacterial activity against a broad spectrum of medicinally important bacteria including several Mycobacterium spp., Enterococcus faecalis, Staphylococcus aureus, Vibrio cholera, Bacillus anthracis, K. pneumoniae, Salmonella typhi, P. aeruginosa and Escherichia coli.9 Recent studies have demonstrated the growth inhibitory activity of Terminalia sericea and Terminalia prunioides against pathogenic10-12 and food spoilage bacteria.13
The Indian Terminalia spp. are used in several traditional medicinal systems (including, Ayurveda Siddha and Unani) and their therapeutic uses have been extensively documented. Many of the Indian Terminalia spp. are used to treat various diseases (Table 1) and numerous recent investigations have reported on their antimicrobial properties. Leaf and branch extracts of Terminalia arjuna have antibacterial activity against a wide panel of microbes.14,15 Terminalia chebula has traditional uses in Ayurveda for the treatment of numerous diseases and conditions16 and has potent antibacterial activity.14 A recent study even highlighted their potential in the prevention and treatment of the endospore forming bacterium Bacillus anthracis.27 Terminalia alata, Terminalia bellirica and Terminalia catappa also have broad spectrum antibacterial activity.15 However, despite the relative wealth of antibacterial studies for the Indian Terminalia spp., there is a lack of studies screening Terminalia spp. for the ability to inhibit Y. enterocolitica growth, and thus their therapeutic value for the prevention and treatment of yersiniosis. This study was undertaken to examine the ability of selected Indian Terminalia spp. with extensive usage in Ayurvedic medicine for the ability to inhibit Y. enterocolitica growth.
|Plant Species||Part Utilized in This Study||Common Name/s||Traditional Medicinal Uses||Known Constituents||References|
|Terminalia chebula||fruit||Chebulic Myroblan, Black Myroblan, Haritaki, Inknut||Used externally to treat fungal infections and cutaneous wounds and in the prevention of inflammation of the mucosal membrane of the mouth. Used internally as a laxative and is known for its purgative effects. Known to have uses in the treatment of asthma and coughs.||Terflavin B and chebulinic acid||6|
|Terminalia arjuna||branch||Arjuna, Koha, White Marudah||Treatment of cardiovascular disorders as well as anti-inflammatory properties. Known to aid in the elimination of cholesterol. Also an analgesic and an antioxidant.||Triterpenoids, flavonoids, tannins, gallic and ellagic acid, sitosterol, proanthocyanidins||6|
|Terminalia catappa||fruit||Indian almond, tropical almond, umbrella tree||Therapeutic effects for liver related diseases, anticancer activity as well as effective in the blocking of HIV reverse transcriptase. Additionally known to have antidiabetic benefits.||Flavonoids (including kaempferol, quercetin), tannins, saponins and phytosterols||6|
MATERIALS AND METHODS
Plant source and extraction
The Terminalia chebula Retz. (fruit), Terminalia arjuna Roxb. Wight & Arn. (branch) and Terminalia catappa L. (fruit) plant materials utilized in this study were provided by Dr. Paran Rayan, Griffith University. Voucher samples of all plant specimens are deposited at the School of Natural Sciences, Griffith University. The plant materials were thoroughly desiccated in a Sunbeam food dehydrator and the dried materials stored at -30°C. Prior to usage, the materials were thawed and ground into a coarse powder. Individual 1 g amounts of the material were then weighed into separate tubes and 50 mL of deionised water, methanol, chloroform, hexane or ethyl acetate were added. All solvents used were analyticalreagent grade and were obtained from Ajax (Australia). The ground plant materials were separately extracted in each solvent for 24 hours at 4 °C through gentle shaking. The extracts were then filtered through filter paper (Whatman No. 54) under vacuum, followed by drying by rotary evaporation in an Eppendorf concentrator 5301. The resulting extracts were weighed and suspended in 10 mL sterilized deionised water containing 1 % DMSO.
Qualitative phytochemical studies
Phytochemical analysis of the extracts was achieved as previously described [20, 21] and used to determine the presence of triterpenoids, saponins, cardiac glycosides, tannins, phytosteroids, phenolic compounds, flavonoids, anthraquinones, and alkaloids.
Clinical Yersinia enterocolitica strain
The clinical isolate strain of Yersinia enterocolitica used in this study was supplied by Ms. Jane Gifkins of the School of Natural Sciences Griffith University, Australia. All growth studies were performed using nutrient agar (Oxoid Ltd., Australia) under aerobic conditions. Incubation was at 30 °C and the stock culture was subcultured and maintained in nutrient broth at 4 °C.
Evaluation of antimicrobial activity
Antimicrobial activity of all plant extracts was determined using a modified disc diffusion assay.22, 23 Briefly, 100 μL of Y. enterocolitica was grown in 10 mL of fresh nutrient broth until they reached a count of ~108 cells/mL. Volumes of 100 μL of the bacterial suspension were spread onto nutrient agar plates and tested for antibacterial activity using 5 mm sterilised filter paper discs. Discs were impregnated with 10 μL of Terminalia spp. extracts, allowed to dry and placed onto inoculated plates. The plates were left at 4 °C for 2 h before incubation at 30 °C for 24 h. The diameters of the inhibition zones were measured to the closest whole millimetre. Each assay was performed in at least triplicate. Mean values (± SD) are reported in this study. Standard discs of chloramphenicol (10 μg) were obtained from Oxoid (Australia) and were used as positive controls to compare antibacterial activity. Filter discs impregnated with 10 μL of distilled water (containing 1% DMSO) and used as a negative control.
Minimum inhibitory concentration (MIC) determination
The minimum inhibitory concentrations (MIC) of each plant extract was determined as previously described.24,25 Briefly, each individual plant extract was diluted in deionised water and tested across a decreasing concentration gradient. Discs were impregnated with 10 μL of the extract dilutions, allowed to dry and placed onto plates inoculated with Y. enterocolitica. The assay was performed in triplicate as outlined above and graphs of the zone of inhibition versus concentration were plotted. Linear ln regression was utilized to determine MIC values.
Reference toxin for toxicity screening
Potassium dichromate (K2Cr2O7) (AR grade, Chem-Supply, Australia) was prepared in sterilized deionized water (4 mg/mL) and serially diluted in artificial seawater for use in the Artemia franciscana nauplii bioassay.
Artemia franciscana nauplii toxicity screening
Toxicity of all extracts were determined using an adapted Artemia franciscana nauplii lethality assay.26,27 Briefly, 400 μL of seawater containing ~43 (mean 43.2, n = 155, SD 14.5) A. franciscana nauplii were added to wells of a 48 well plate and immediately used in the bioassay. Volumes of 400 μL of reference toxin or the diluted plant extracts were transferred to the wells and incubated at 25 ± 1 °C under artificial light (1000 Lux). Negative controls (400 μL seawater) and all test treatments were run in triplicate for each plate. The wells were monitored at regular intervals and the number of dead were counted. The nauplii were classified as dead if no movement was detected within 10 seconds. After 24 h, all nauplii were sacrificed and counted to determine the total % mortality per well. The LC50 with 95% confidence limits for each treatment was determined using probit analysis.
+++ indicates a large response; ++ indicates a moderate response; + indicates a minor response; - indicates no response in the assay. CFW = T. chebula aqueous fruit extract; CFM = T. chebula methanolic fruit extract; CFC = T. chebula chloroform fruit extract; CFH = T. chebula hexane fruit extract; CFE = T. chebula ethyl acetate fruit extract; ABW = T. arjuna aqueous branch extract; ABM = T. arjuna methanolic branch extract; ABC = T. arjuna chloroform branch extract; ABH = T. arjuna hexane branch extract; ABE = T. arjuna ethyl acetate branch extract; PFW = T. catappa aqueous fruit extract; PFM = T. catappa methanolic fruit extract; PFC = T. catappa chloroform fruit extract; PFH = T. catappa hexane fruit extract; PFE = T. catappa ethyl acetate fruit extract.
Liquid extraction yields and qualitative phytochemical screening
Extractions of 1 g of various dried Terminalia spp. materials with various solvents resulted in dried plant extracts ranging from 22 to 144 mg (T. arjuna branch extracts), 62 to 634 mg (T. chebula fruit extracts) and 144 to 447 mg (T. catappa fruit extracts) (Table 1). Methanolic and aqueous T. chebula extracts provided considerably greater yields of extracted material relative to the ethyl acetate, chloroform and hexane counterparts. Interestingly, the opposite trend was evident for the T. catappa fruit extracts, with the lower polarity chloroform and hexane extracts yielding higher masses of extracted material than in the higher polarity extracts. Substantially lower yields were recorded in all T. arjuna branch extracts. The dried extracts were resuspended in 10 mL of deionised water (containing 1 % DMSO), resulting in the concentrations presented in Table 2.
Qualitative phytochemical studies showed that the aqueous and methanolic extracts generally had a wide range of phytochemicals (Table 2). All Terminalia spp. generally contained substantial levels of phenolics, especially water soluble phenolics. Additionally, these extracts generally resulted in high levels of tannins and flavonoids and moderate to high levels of saponins. Similarly, the ethyl acetate extracts had comparable phytochemical profiles to the methanolic and aqueous counterparts. However, most classes of compounds were present in low abundances. Conversely, the hexane and chloroform extracts of most of the Terminalia spp. typically only had low to moderate levels of phenolics, tannins and flavonoids and were mostly devoid of detectable levels of the other phytochemical classes.
To determine the ability of the Terminalia spp. fruit and branch extracts to prevent Y. enterocolitica growth, 10 μL of each extract was screened using a disc diffusion assay. Bacterial growth inhibition was evident in 9 of the 15 extracts screened (60%) (Figure 1). The T. chebula aqueous, methanolic and ethyl acetate extracts were the most potent inhibitors of growth (as judged by zone of inhibition), with inhibitory zones of 19.7 ± 0.6, 24 ± 1.0 and 21.3 ± 0.6 mm respectively. These are significantly better inhibition than the chloramphenicol control (10 μg), which had an inhibitory zone of 9 ± 0.6 mm. Similarly, all T. catappa extracts tested had comparable Y. enterocolitica growth inhibition to the chloramphenicol control. Of these, the aqueous T. catappa proved the most effective extract, with an inhibitory zone of 12 ± 1.7 mm. The antimicrobial efficacy was further quantified by determining the MIC values (Table 3). The T. chebula extracts were the most potent inhibitors of Y. enterocolitica growth, with MIC values of 653, 85 and 64 μg/mL for the aqueous, methanolic and ethyl acetate extracts respectively (~0.6-6.5 μg infused into the disc). The T. arjuna methanolic extract showed low to moderate activity (3000 μg/mL). All other extracts were inactive, with MIC > 10,000 μg/mL.
Quantification of toxicity
All extracts were initially screened in the assay at 2000 μg/mL (Figure 2). Potassium dichromate was also tested as a reference toxin in the bioassay. Potassium dichromate was rapid in its induction of mortality, with nauplii death evident within the first 3 h of exposure and 100 % mortality evident within 4-5 h (unpublished results). The methanolic and aqueous extracts of all Terminalia spp. displayed apparent toxicity in the Artemia nauplii bioassay, with ≥50% mortality rates at 24 h. The mortality for all other extracts were not significantly different to that observed in the seawater control.
|Extract||MIC (μg/mL)||LC50 (μg/mL)|
[i] Numbers indicate the mean MIC and LC50 values of triplicate determinations. -indicates no bacterial growth inhibition was evident, or that an LC50 value could not be obtained as the mortality did not reach 50% for any dose tested. CFW = T. chebula aqueous fruit extract; CFM = T. chebula methanolic fruit extract; CFC = T. chebula chloroform fruit extract; CFH = T. chebula hexane fruit extract; CFE = T. chebula ethyl acetate fruit extract; ABW = T. arjuna aqueous branch extract; ABM = T. arjuna methanolic branch extract; ABC = T. arjuna chloroform branch extract; ABH = T. arjuna hexane branch extract; ABE = T. arjuna ethyl acetate branch extract; PFW = T. catappa aqueous fruit extract; PFM = T. catappa methanolic fruit extract; PFC = T. catappa chloroform fruit extract; PFH = T. catappa hexane fruit extract; PFE = T. catappa ethyl acetate fruit extract.
To further enumerate the effect of toxin concentration on the induction of mortality, the extracts were serially diluted in artificial seawater to test across a range of concentrations in the Artemia nauplii bioassay. Table 2 shows the LC50 values of the extracts towards A. franciscana. No LC50 values are reported for the chloroform, hexane or ethyl acetate extracts of any Terminalia spp. as ≤50% mortality was seen across concentrations tested. All other extracts were deemed nontoxic as they yielded LC50 values significantly greater than 1000 μg/mL following 24 h exposure. Extracts with LC50 values of ≥1000 μg/mL towards Artemia nauplii are deemed to be nontoxic as previously reported .
Terminalia spp. have been used for a broad range of medicinal purposes by traditional healers from a wide variety of ethnic and cultural groupings. The best documented of these are the traditional Indian medicinal systems, particularly the Ayurveda. Ayurvedic practitioners utilise various Terminalia spp. for a wide variety of medicinal purposes including abdominal and back pain, coughs and colds, conjunctivitis, diarrhoea and dysentery, fever, headache, heart disorders, inflammation, leprosy, pneumoniae, sexually transmitted diseases, worms, wounds, haemorrhages, ulcers, and as a general tonic.6 Many of these diseases are caused by microbial pathogens, indicating the potential of these plants as antimicrobial agents. Indeed, recent investigations have reported on their antibacterial properties of several Indian Terminalia spp. extracts prepared from several parts of T. arjuna have antibacterial activity against a wide panel of microbes.6, 16, 17 T. chebula is also used in Ayurvedic medicine for the treatment of numerous microbial diseases and conditions6,18 and displays potent antibacterial activity against a panel of microbes.16 T. catappa has also been reported to have broad spectrum antibacterial activity.17
The growth inhibitory activity of the T. chebula extracts against Y. enterocolitica is particularly noteworthy for the development of future antibiotic chemotherapeutics against the gastrointestinal disease yersiniosis. Potent Y. enterocolitica growth inhibitory activity was evident in the T. chebula fruit extracts. The methanolic and ethyl acetate extracts were the most potent growth inhibitors, with MIC values substantially <100 μg/mL.
Whilst an examination of the phytochemistry of the Terminalia spp. was beyond the scope of our study, a commonality of this genus is their relatively high levels of a number of tannin components including exifone (4-galloylpyrogallol), ellagic acid dehydrate, trimethyl ellagic acid, chebulic acid, corilagen, castalagin and chebulagic acid.6,8-10 Gallotannins have been reported to inhibit the growth of a broad spectrum of bacterial species29 through a variety of mechanisms including binding cell surface molecules including lipoteichoic acid and proline-rich cell surface proteins,30,31 and by inhibiting glucosyltransferase enzymes.32 Ellagitannins have also been reported to exert antibacterial activity via several mechanisms including interaction with cytoplasmic oxidoreductases and by disrupting bacterial cell walls.29, 31
It is likely that other phytochemical classes also contribute to the growth inhibitory properties of these extracts. Our qualitative phytochemical screening studies indicate that polyphenolics, flavonoids, saponins, and terpenes were present in most Terminalia spp. extracts. Terpenoids have been previously reported to have potent broad spectrum antibacterial activity33 and therefore may contribute to the inhibitory activity against S. pyogenes. Many studies have also reported potent antibacterial activities for a wide variety of flavonoids.34 Further phytochemical evaluation studies and bioactivity driven isolation of active components is required to further evaluate the mechanism of Y. enterocolitica growth inhibition.
Importantly, our findings also demonstrate that the Indian Terminalia spp. extracts tested in our study were nontoxic towards Artemia franciscana nauplii, with LC50 values substantially > 1000 μg/mL. Extracts with LC50 values > 1000 μg/mL towards Artemia nauplii are defined as being nontoxic.28 Whilst our preliminary toxicity studies indicate that these extracts may be safe for use as Y. enterocolitica growth inhibitors, studies using human cell lines are required to further evaluate the efficacy of these extracts.
Aqueous, methanolic and ethyl acetate T. chebula extracts were potent inhibitors of Y. enterocolitica growth.
The T. chebula methanolic and ethyl acetate extracts were particularly potent with MIC values of 85 and 64 μg/mL respectively.
The T. arjuna branch extract was moderately active (3000 μg/mL).
All other extracts were either low efficacy, or completely devoid of growth inhibitory activity.
All Indian Terminalia spp. extracts were nontoxic in the Artemia nauplii assay.