INTRODUCTION
Natural plant compounds that can substitute the use of antibiotics have been emerged from health threat of residual antibiotics and outcome of multi-drug resistant pathogenic bacteria in food including animal products. Various plant products in different area were tested and reported as antimicrobial agents and bioactive natural products (Cowan, 1999; Gautam et al., 2007; Magadula and Erasto, 2009). The plants showing antioxidant activity were also known in many reports (Wolfe et al., 2003; Kaur et al., 2006; Ordon et al., 2006; Alviano et al., 2008; Doddola et al., 2008).
The regulation has been reinforced and use of antibiotics has no scope for promotion of livestock growth. From 2006, in-feed antibiotics used in animal production have been banned by the European Union. Recently, South Korea has also banned them from the second half of 2011. For rearing of livestock without chemical antibiotics, feeding of natural products with the function such as antimicrobial agents, antioxidants and immune stimulants should be helpful for animal health. Therefore, alternative antibiotics have been regarded as most important feed additives in animal farming. Specially, plant extracts has received much attention in the research for the development of alternative antibiotics (Esquenazi et al., 2002; Wannissorn et al., 2005). The production amounts of plants are much more abundant in subtropical and tropical countries including Thailand compared with northern hemisphere including South Korea with typical four seasons.
The purpose of this study was to examine antibacterial and antioxidant activities and total contents of phenolics and flavonoids of some edible plants of Thailand and to develop natural feed additive for animal health. The plants, viz. Citrus aurantifolia Swingle (Lime) fruit and leaves, Morinda citrifolia (Beach mulberry) leaves, Sesbania grandiflora L. (Agasti sesbania) leaves, Piper sarmentosum (Wild betel) leaves, Curcuma domestica Valeton (Turmeric) roots, Cassia siamea leaves, and Cocos nucifera L. (Coconut) fruits. Tested pathogens using the plant extracts are related to wasting diseases of livestock and causative to execute great economical loss in animal production. Haemopilus parasuis, Haemopilus somnus, and Burkholderia sp. are known to induce pneumonia (Palzer et al., 2008), respiratory disease (Norcia et al., 2004), and meliodiosis (Sprague and Neubauer, 2004; Millan et al., 2007), respectively. Escherichia coli and Clostridium perfringens are causative agents of diarrhoea in piglet (Hendriksen et al., 2006).
MATERIALS AND METHODS
Plants and preparation of crude extracts
The plants studied were C. aurantifolia Swingle (Lime) fruits and leaves, M. citrifolia (Beach mulberry) leaves, S. grandiflora L. (Agasti sesbania) leaves, P. sarmentosum (Wild betel) leaves, C. domestica Valeton (Turmeric) roots, C. siamea leaves, and C. nucifera L. (Coconut) peels. The plants were cut into smaller species and air-dried in the shade for a week and finely ground. The materials were supplied by K.-C Shin (Bangkok, Thailand) and authenticated by Chang-Won Choi (Department of Biology and Medicinal Science, Paichai University, South Korea). Voucher specimens were deposited at Animal Resources and Research Center, Konkuk University. A sample of 3 g was suspended in 27 mL of methanol and the mixture was agitated in 150 rpm for 6 h. The extract was filtered and evaporated. The remained extract was resuspended with 2 mL of methanol. Methanol extract (25 μL) was used for antibacterial activity. One mL of methanol extract was mixed with 5 mL of distilled water and 5 mL of n-hexane. The mixture was vigorously mixed and then phase separation was developed by standing. Upper phase of n-hexane layer was evaporated and further solvents extractions were done as same manner of previous step. Used solvents were chloroform, ethyl acetate, and butanol, subsequently as described in Figure 1. All of prepared extracts was stored at 4°C until assay was performed. Dry matter content was analyzed by estimation of weight loss during heat dry.
Bacterial strains
Animal pathogenic bacteria used in this study, H. somnus, H. parasuis, Burkholderia sp., E. coli, and C. perfringens, are related to respiratory disease and diarrhea of piglet and other animals. All of them were collected from stock of National Veterinary Research Quarantine Service in South Korea.
Paper disc diffusion method
Antibacterial activities of each six fractions from 7 plants were determined using paper disc diffusion method, described elsewhere (Valsaraj et al., 1997). Each bacterial culture (0.1 mL) grown in Luria-Bertani broth (LB, Difco) at 37°C for 18 h was suspended in 10 mL of sterilized 0.8% agar solution. To prevent solidification of agar and heat damage of bacterial cells, 0.8% agar solution was maintained at 50°C until use. After suspension of test strain, agar solution was poured onto LB agar plate and then allowed to be solidified. Plant extracts (25 μL) was put on 6 mm sterilized paper disk and it was dried for 30 min to evaporate solvent before bacterial cells placed on suspended agar plate. Prepared test plates were incubated at 37°C for 18 h and then appeared diameters of clear zone were measured.
Determination of minimum inhibitory concentration
According to the results of screening of antibacterial activities, the minimum inhibitory concentration (MIC) of selected ten plant extracts were determined by serial dilution method on 96 well plates with turbidity measurement (Alviano et al., 2008). Plant extract was diluted with sterilized LB broth, ranged from 0.01 to 10 mg/mL, and dispensed into 96 well plate. Test strains were inoculated in prepared each well with 1:100 ratio and incubated at 37°C for 18 h. Bacterial growth was determined by measuring of turbidity at 600 nm. The MIC was determined as the lowest concentration of extracts which showed no visible growth of the test strains.
Antioxidant activity assay
The free radical scavenging activities were determined in vitro using 2-diphenyl-2-picryl hydrazyl (DPPH) photometric assay (Tadic et al., 2008) with some modification. Each extract (1 to 10 μL) was mixed into its solvent to make a total volume of 100 μL. It was mixed with 900 μL of 0.04 mg/mL DPPH in methanol solution and the mixture was reacted for 30 min with gentle agitation. Reduction of violet color induced by free radical scavenging activity was estimated spectrophotometrically at 517 nm. Each solvent used in extraction was applied as negative control and tert-butyl hydroxytoluene was used as positive control. The inhibition percentage was calculated by comparing absorbance with negative control and it was plotted against the concentration of the samples. The extract concentration that represented 50% of activity was calculated by linear regression analysis and expressed as EC50.
Determination of total polyphenols
Total polyphenol contents in the extracts were determined by the modified Folin-Ciocalteu method (Wolfe et al., 2003). Each sample (0.1 mL) was added into 2 mL of 2% Na2CO3. After the tubes were vortexed for 10 min at room temperature, 0.1 mL of 50% Folin-Ciocalteu reagent was added. The tubes were vortexed for 15 s and allowed to stand for 30 min at 40°C for color development. Absorbance was then measured at 700 nm using the Shimadzu UV-1601 spectrophotometer. Samples of extracts were evaluated at a final concentration of 1 mg/mL. Total polyphenol contents were calculated as quercetin (25 to 500 ppm) using the following equation based on the calibration curve: y = 0.0033x–0.0111, R2 = 0.9979, where x was the absorbance and y was the quercetin equivalent (μg/mL) (Kiselova et al., 2006).
Determination of total flavonoids
Total flavonoids were estimated using the method (Ordon et al., 2006). Each sample (0.5 mL) was added and mixed into 1.5 mL of 95% ethanol. To it, 0.1 mL of 10% aluminum chloride, 0.1 mL of 1 M potassium acetate and 2.8 mL of distilled water were added. After the tubes were mixed for 30 min at room temperature, the absorbance was measured at 415 nm. A yellow color indicated the presence of flavonoids. Extracts were evaluated at a final concentration of 1 mg/mL. Total flavonoid content was calculated as quercetin (5 to 100 ppm) using the following equation based on the calibration curve: y = 0.0068x–0.0165, R2 = 0.9974, where x was the absorbance and y was the quercetin equivalent (μg/mL).
RESULTS
Antibacterial activity assay
Methanol extracts of C. aurantifolia Swingle fruits showed the most broad spectrum antibacterial activity compared to other solvent extracts, except for C. perfringens (Table 1). Butanol and water fractions of it also showed very high antibacterial activities against H. somnus, related to porcine respiratory disease causative to wasting syndrome in young pigs. Antibacterial activities of C. aurantifolia Swingle leaves appeared different spectrum compared to its fruits. Methanol extract of leaves showed the antibacterial activities against both Burkholderia sp. and H. somnus, but not against E. coli, H. parsuis, and C. perfringens. However, the n-hexane and chloroform extracts from leaves showed antibacterial activities for Burkholderia sp. only whereas its fruits did not. Only butanol fraction of S. grandiflora leaves showed antibacterial activity against Burkholderia sp. related to melioidosis in pig, even in human. Only methanol extracts of P. sarmentosum leaves showed very mild antibacterial activities against both E. coli and Burkholderia sp. and moderate activity against H. parasuis. Antibacterial spectrum of ethyl acetate and water extracts from C. domestica Valeton (Turmeric) roots showed moderate to strong against C. perfringens, only. M. citrifolia L. leaves, C. siamea britt leaves, and C. nucifera L. fruits did not show any antibacterial activity against the tested pathogens in this study.
The results of the MIC were well agreed with the antibacterial patterns shown by paper disc diffusion method (Table 2). The strong activity (MIC 495 μg/mL) was shown by methanol extracts of C. aurantifolia Swingle fruits against H. somnus. Its leaves showed much lower MIC μg/mL) by chloroform extracts than methanol (281 μg/mL) and n-hexane (810 μg/mL) extracts against Burkholdeira sp. Butanol extracts of S. grandiflora L. showed antibacterial specificity to Burkholdeira sp. with MIC of 135 μg/mL. Methanol extracts of Piper sarmentosum Roxb had MICs, 502 μg/mL, 1,005 μg/mL, and 251 μg/mL against E. coli, Burkholdeira sp., and H. parasuis, respectively. The ethyl acetate and water extracts from C. domesitca Valeton roots have high specificities against only C. perfringens μg/mL and 183 μg/mL MICs, respectively.
Antioxidant activity assay
C. aurantifolia Swingle fruit showed relatively high antioxidant activity (Table 3), specifically in methanol extract (EC50, 3.46 mg/mL). Antioxidant activity of chloroform extract of C. aurantifolia Swingle leaves was higher than that (EC50, 10.27 vs 17.68 mg/mL) of methanol extract. However, no activity was detected by n-hexane extract although it showed antibacterial activity (Tables 1 and 2). Butanol extract of Sesbania grandiflora L. leaves showed no antioxidant activity. On the other hand, P. sarmentosum Roxb showed the most strong antioxidant activity with EC50, 2.70 mg/mL. Water extracts of C. domestica Valeton roots showed the moderate antioxidant activity (EC50, 18.18±0.8 mg/mL) whereas ethyl acetate fraction did not.
Total polyphenolic compounds and flavonoids
Among the plant extracts tested in the study, methanol extracts of S. grandiflora L. and chloroform extracts of C. domestica Valeton were found to have the highest amount of total polyphenol, 41.7 and 47.8 μg/mL, respectively (Table 4). The highest amount of total flavonoid recorded among all plant extracts tested was 22.5 μg/mL, which was also from methanol extract of S. grandiflora L. Methanol extracts of P. sarmentosum Roxb showed high content (19.4±0.008 μg/mL) of total flavonoid. Most of the methanol extracts of tested plants showed high amounts of total polyphenol and total flavonoid compared to other solvent extracts except for Curcuma domestica, where chloroform extract showed highest (Table 4).
DISCUSSION
In this study, 6 kinds of solvent extraction were applied to 7 edible plants of Thailand and their antibacterial activities were determined by the spectrum against 5 animal pathogenic bacteria. Some bioactive components of these plants may differ in their solubility depending on the extractive solvents used. With these results, finally 10 extracts from 4 plants were selected and their MICs and antioxidant activities were determined. As a result, C. aurantifolia Swingle fruits known as lime showed the most broad antibacterial spectrum with the strong antioxidant activity specially in methanol extracts. It has been traditionally regarded as an effective carminative fruit in Thailand and the antibacterial activity of its essential oil has been reported against Salmonella typhimurium, S. enteritidis, E. coli, C. perfringens, and Campylobacter jejuni (Wannissorn et al., 2005). In this study, we newly found that the fruit extracts of C. aurantifolia Swingle also had the antibacterial activities against Burkholderia sp., H. somnus, and H. parasuis including E. coli. However, we did not find the antibacterial activity from the fruit extracts of C. aurantifolia Swingle against C. perfringens. There may be a difference in concentration of effective compounds between essential oil and our solvent extraction. C. aurantifolia Swingle leaves contained more strong antimicrobial activity against Burkholderia sp. (Tables 1 and 2) compared to its fruits. Antibacterial properties of different parts in C. aurantifolia Swingle were reported against Gram-positive bacteria such as Staphylococcus aureus, Gram-negative bacteria including E. coli, S. paratyphi, Pseudomonas aeruginosa, and Candida albicans (Aibinu et al., 2007). Patil et al. (2009) reported that the chloroform extracts of C. aurantifolia Swingle fruit had much higher free radical-scavenging activity than acetone and methanol extracts. Immunomodulatory effect of concentrated lime juice extract was also observed on activated human mononuclear cells (Gharagozloo et al., 2001).
Although a few suggestions about antibacterial activity against skin and gastrointestinal microbes have been reported (Denyer, 1990; Doddola et al., 2008), there is little information about antimicrobial activities of S. grandiflora L. leaves. Antibacterial activity against Burkholderia sp. was found (Tables 1 and 2) in butanol fraction of S. grandiflora L. leaves. Zaidan et al. (2005) reported the antibacterial activities of methanol extracts of P. sarmentosum leaves against S. aureus and P. aeruginosa. In this study, P. sarmentosum Roxb also showed the antibacterial activities and the highest antioxidant activity, especially, its methanol extracts showed the antibacterial activities against E. coli, Burkholderia sp. and H. parasuis. C. domestica Valeton roots have been widely cultivated in Southeast Asia for the use of an additive in cooking and a traditional medicine to treat diarrhea (Ahmad-Raus et al., 2001). Interestingly, antibacterial spectrum of ethyl acetate and water extracts from C. domestica Valeton roots showed specificities against C. perfringens (Tables 1 and 2), in contrary to the report of Wannissorn et al. (2005).
M. citrifolia L. is widely grown in the Hawaii and Asia and it has been commonly called as “Noni”. It has been used as an herbal plant with effectiveness on diabetes, hypertension and immunomodulation (Takashima et al., 2007). Schäfer et al. (2008) showed the bactericidal effect against Escherichia coli in calves fed M. citrifolia (Noni) puree. Blood samples from Noni puree-fed calves displayed significantly more bacterial killing against E. coli than did controls on day 14. C. siamea britt is commonly found tree in Southeast Asia and its bark, root and flowers have been known to have medicinal values in diabetes, hypertension and insomnia (Kaur et al., 2006). Methanol extracts of C. siamea exhibited antibacterial activities against both Bacillus cereus and Listeria monocytogenes whereas M. citrifolia L. had no antibacterial activities for all tested microorganisms (Nanasombat and Teckchuen, 2009). In this study, the antibacterial activities of M. citrifolia L., C. siamea britt and C. nuricifera L. were not detected against tested pathogens. Husk of C. nuricifera L. fruits have various phenolic compounds that have the physiological effects including antileishmanial, antibacterial, antiviral and anti-neoplasic (Alviano et al., 2008). In addition, Esquenazi et al. (2002) reported that extracts of C. nuricifera showed the antibacterial activity against S. aureus. In an earlier study (Wannissorn et al., 2005), the antibacterial activity against C. perfringens was reported as very high, however, no activity was found at all of solvent fractions in this study.
Biological activities related to antibacterial and antioxidant activities may be correlated with total polyphenol and flavonoid (Rice-Evans et al., 1997; Cushnie and Lamb, 2005; Daglia, 2011). Even though the correlation was not perfectly matched in this study, methanol extracts of both Citrus aurantifolia Swingle and Piper sarmentosum Roxb possessed high antibacterial and antioxidant activities in proportion to high amounts of total polyphenol and flavonoid. In conclusion, the extracts from Citrus aurantifolia Swingle (Lime), Sesbania grandiflora L. (Agati sesbania), Piper sarmentosum Roxb (Wild betal) and Curcuma domestica Valeton (Turmeric) showed high antibacterial and antioxidant activities. They should be further tested in vivo animal experiments as alternative antibiotics in potential feed additives for the inhibition of respiratory disease and meliodiosis with diarrhea in livestock.
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