Phytochemicals and antioxidant capacity of some tropical edible plants

Objective To find biological functions such as antibacterial and antioxidant activities in several tropical plants and to investigate the possibility of antibiotic substitute agents to prevent and treat diseases caused by pathogenic bacteria. Methods Plants such as Poncirus trifoliata fruit (Makrut), Zingiber officinale Rosc (Khing), Areca catechu L. (Mak), Solanum melongena L. I (Makkhuayao), and Solanum melongena L. II (Makhurapro) were extracted by methanol, n-hexane, chloroform, ethyl acetate, butanol and water. The free radical scavenging activities were measured using 2-diphenyl-2-picryl hydrazyl photometric assay. Antibacterial activities with a minimum inhibitory concentration (MIC) were observed by agar diffusion assay against pathogenic strains of Escherichia coli, Burkholderia sp., Haemopilus somnus, Haemopilus parasuis, Clostridium perfringens, and Pantoea agglomerans. Results Poncirus trifoliata fruit methanol extract showed antibacterial activities against gram-negative and gram-positive pathogens. Additionally, this showed the strongest antibacterial activity against Burkholderia sp. and Haemopilus somnus with MIC 131 μg/mL, respectively. Areca catechu L. water extract showed antibacterial activities against Burkholderia sp., Haemopilus somnus, and Haemopilus parasuis. The MIC value for Haemopilus parasuis was 105 μg/mL in this. Antioxidant activity of Zingiber officinale Rosc n-hexane extract showed 2.23 mg/mL effective concentration 50% (EC50) value was the highest activity among tropical plants extracts. Total polyphenol content in Zingiber officinale Rosc methanol extract was 48.4 μg/mL and flavonoid content was 22.1 μg/mL showed the highest values among tested plants extracts. Conclusion Taken together, these results suggest that tropical plants used in this study may have a potential benefit as an alternative antibiotics agent through their antibacterial and antioxidant activities.


INTRODUCTION
It is important to prevent and treat livestock diseases for improving productivity of livestock. Although antibiotics have played a key role for those, they have caused various side effects such as the generation of antibiotics-resistant bacteria [1]. As a result, consumers' demand for eco-friendly agricultural products has been gradually increasing. Therefore, many countries are prohibiting the use of antibiotics.
Today, there has been an increasing concern to find alternative antibiotics substitutes in livestock diseases treatment particularly those of various natural products sources including plants to replace synthetic antibiotics used in livestock that are easy to obtain and have considerably few side effects [2,3]. Antibiotics substitutes derived from plants sources can pro mote the growth of livestock by preventing livestock diseases without causing tolerance to livestock and human, and they can reduce environmental pollution [4].
There are approximately 250,000 to 500,000 species of plants with pharmacological activity on this planet, but some plants have been investigated and reported for their antimicrobial activity and physiological activity [5][6][7]. Therefore, the screening of more plant and the research of antibacterial activity may play a critical role in the development of antibiotic substitute agents. Meanwhile, studies on plant extracts, such as subtropical and tropical plants have been actively conducted for the development of alternative agents [8,9]. Southeast Asian countries have an abundance of plant species because of their tropical climate that supports plants' growth [10]. In our previous study, we observed the bioactivities of several tropical plants as follows. Citrus aurantifolia Swingle fruits and leaves showed the strongest antibacterial activity against Haemopilus somnus and Burkholderia sp., respectively. Piper sarmentosum Roxb leaves showed antibacterial activities against Escherichia coli (E. coli), Burkholderia sp. and Haemopillus parsuis. In addition, Citrus aurantifolia Swingle, Piper sarmentosum Roxb, Curcuma domestica Valeton roots, and Sesbania grandifloraL. have antioxidant effects [10].
Following the previous study, this experiment was conducted to screen other tropical plants such as Poncirus trifoliata fruit (Makrut), Zingiber officinale Rosc (Khing), Areca catechu L. Poncirus trifoliata fruit belonging to the Rutaceae family in the Citrus genus has been widely used in folk medicine as a remedy for inflammation, gastric and dysentery [11]. Zingiber officinale Rosc belonging to Zingiberaceae family is a medicinal plant widely used in treatment of bacterial infections because of their antimicrobial activity [12]. Areca catechu L. is one of the fruit plants of Palmaceae family and has been used to treat dysentery in East and North Asian countries [13]. Additionally, Solanum melongena L. cultivated worldwide throughout the tropics and subtropics belongs to the Leptostemonum Clade of the spiny solanums and is one of the most highly antioxidant vegetables, known as "eggplant" [14].
To date, there has been scant information on studies of alternative agents for the promotion of livestock health related to the bioactivity of these tropical plants.
Therefore, this study was conducted to investigate biological functions such as antibacterial and antioxidant activities and measure the content of polyphenol and flavonoid in other tropical plants to provide fundamental data for the antibiotics substitute agents that prevent and treat diseases caused by pathogenic bacteria.

Preparation of plant extracts
Poncirus trifoliata fruit (Makrut), Zingiber officinale Rosc (Khing), Areca catechu L. (Mak), Solanum melongena L. I (Makkhuayao), and Solanum melongena L. II (Makhurapro) were selected for this study. They purchased from an authorized market (AOR TOR KOR) in Bangkok, Thailand. They were cut into smaller species and dried by using the dryer (Eyela WFO-600SD, Tokyo Rikakikai Co. Ltd, Tokyo, Japan) for three days and then ground finely with the grinder (DA-505, DAESUNG ARTLON, Paju, Korea). Those plants were extracted with water, methanol, n-hexane, chloroform, ethyl acetate, and butanol using the same method as in the previous study [10]. In briefly, a sample of 3 g was suspended in 27 mL methanol and was agitated with 150 rpm using the stirrer (KMC-130SH, Vision Scientific Co, Daejeon, Korea) for six hours, and then filtered and evaporated. The extract was resuspended with 2 mL methanol (methanol extract). Methanol extract 1 mL was mixed with n-hexane: H 2 O (1:1) and mixed vigorously and then separated by standing. Upper phase was collected (n-hexane extract) and same manner of previous step was processed to collect extracts of chloroform, ethyl acetate, and butanol in order.

Antibacterial activity assay
Antibacterial activities of plant extracts were measured by the paper disk diffusion method, as described elsewhere [15]. Aliquots (25 μL) of each extract were dropped on sterilized paper with 6 mm of diameter and placed on suspended agar plate with pathogenic bacteria. And prepared plates were incubated at 37°C for 18 hours and appeared diameters of clear zone were measured. The pathogenic bacterial strains selected for this study were E. coli, Burkholderia sp., Haemopillus somnus, Haemopillus parsuis, Clostridium perfringens, and Pantoea agglomerans. They were obtained from the National Veterinary Research Quarantine Service in Korea.

Determination of minimum inhibitory concentration
The minimum inhibitory concentration (MIC) values are used to determine susceptibilities of bacteria to drugs and evaluate the activity of new antibacterial agents [16]. According to the evaluation of antibacterial activities, MIC values of plant extracts were observed by broth micro-dilution method [17]. Plant extracts were diluted with sterilized Luria Bertani broth and dispensed in 96-well microtiter plates. Each tested strain was adjusted to 10 7 to 10 8 CFU/mL and diluted 1:100 ratio. And then they were added to the prepared each well and incubated at 37°C for 18 hours. The absorbance values were measured at 600 nm using microplate reader. The MIC value was determined as the lowest concentration of plant extracts showed no visible turbidity of the test strains.

Antioxidant activity assay
The free radical scavenging activities of plant extracts were determined in vitro by 2-diphenyl-2-picryl hydrazyl (DPPH) photometric assay [18] with some modification. Briefly, 1 to 10 μL of each extract added to its solvent to make total volume of 100 μL and mixed with 900 μL of 0.04 mg/mL DPPH methanol solution and then stirred gently to react for 30 minutes at room temperature. The absorbance values were measured at 517 nm. Each solvent used in extraction was used as a negative control and tert-butyl hydroxytoluene was used as a positive control. The percentage antioxidant activity was calculated by comparing sample absorbance with negative controls and was plotted against sample concentrations. The antioxidant activities were expressed as EC 50 value means the concentration at 50% of antioxidant activity.

Total polyphenol contents of tropical plants
Total polyphenol contents of plant extracts were evaluated by the modified Folin-Ciocalteu method [19]. Na 2 CO 3 solution (2 mL, 2%) was added to 0.1 mL extract and mixed for 10 minutes at room temperature. After the vortex, 0.1 mL of 50% Folin-Ciocalteu reagent was added and mixed again for 15 seconds, and then incubated for 30 minutes at 40°C to develop color. The absorbance was measured at 700 nm using UV-1601 spectrometer (Shimadzu, Kyoto, Japan). Plant extracts were evaluated at a final concentration of 1 mg/mL. Total polyphenol content of plant extracts was calculated as quercetin (25 to 500 ppm) using the following equation based on the calibration curve: y = 0.0033x-0.0111, R 2 = 0.9979, where x was the absorbance and y was the quercetin equivalent (μg/mL) [20].

Total flavonoid contents of tropical plants
Total flavonoids were estimated using the aluminum chloride colorimetric method [21]. Plant extract 0.5 mL was added to 1.5 mL of 95% ethanol. After mixture, 0.1 mL of 10% aluminum chloride, 0.1 mL of 1 M potassium acetate and 2.8 mL distilled water were added, respectively. They were mixed for 30 minutes at room temperature, the absorbance was measured at 415 nm using UV-1601 spectrometer (Shimazu, Japan). Extracts were evaluated at a final concentration of 1 mg/mL. Total flavonoid content of plant extracts was calculated as quercetin (5 to 100 ppm) using the following equation based on the calibration curve: y = 0.0068x-0.0165, R 2 = 0.9974, where x was the absorbance and y was the quercetin equivalent (μg/mL).

RESULTS AND DISCUSSION
In vitro Antibacterial activity of tropical plants As shown in Table 1, methanol extracts of Poncirus trifoliata fruits showed to have antibacterial activities against six animal pathogens such as E. coli, Haemopillus somnus, Burkholderia sp., Haemopillus parsuis, Clostridium perfringens, and Pantoea agglomerans. Especially, it showed the strongest antibacterial activity against Haemopilus somnus, a common disease-causing bacterium of boveine including respiratory disease, conjunctivitis, mastitis, and polyarthritis as a gram-negative pleomorphic bacterium worldwide [22,23]. Whereas other solvent extracts did not show antibacterial activities. Zingiber officinale Rosc, known as the medicinal plant [12], had moderate antibacterial activity (11.9 to 13.0 mm) against Clostridium perfringens in n-hexane, Chloroform, ethyl acetate, and butanol extract fractions. Clostridium perfringens as a gram-positive bacterium cause various forms of acute enteritis and fatal enterotoxemias in animals [24]. Ethyl acetate and butanol fractions of it appeared antibacterial activities (9.9 to 11.5 mm) against Pantoea agglomerans that may cause severe and occasionally fatal infections in animals and humans when introduced into the organs [25]. Water extract of Areca catechu L. had inhibitory effect on Burkholderia sp. (12.5 mm), Haemopillus somnus (12.3 mm), and Haemopillus parsuis (12.8 mm) and methanol of it showed antibacterial activity (12.5 mm) against Burkholde ria sp. related to melioidosis in animals and humans [10]. It was reported that Areca catechu leaf extract had the inhibitory effects against inflammation in vivo and vitro [13]. According to Al-Bayati, Areca catechu nut water extract showed antibacterial activity against E. coli [26]. However, all solvent extracts of this showed no antibacterial activity against E. coli in this study.
Solanum melongena L. I (Makkhuayao) extract did not exhibit antibacterial activity against the tested pathogens in most solvent extracts but showed antibacterial activity (10.1 mm) against Pantoea agglomerans in the butanol fraction. And butanol extract of Solanum melongena L. II (Makhuapro) showed antibacterial activities against Burkholderia sp. (12.4 mm) and Pantoea agglomerans (9.7 mm), respectively.

Minimum inhibitory concentration against animal pathogenic bacteria
MIC were tested against six animal pathogenic bacteria using broth micro-dilution method for plants extracts with antibacterial activities ( Table 2). These results were like the antibacterial patterns shown by paper disc diffusion assay. Methanol extract of Poncirus trifoliata fruits appeared displayed MIC values with a range of 131 to 262 μg/mL against six pathogenic bacteria. Only its methanol extract showed MIC value 262 μg/mL and exhibited antibacterial activity against E. coli, a pathogen that infects cattle and swine colonies [27]. Zingiber officinale Rosc displayed the activity with MIC value of 240 μg/mL for the chloroform fraction and 690 μg/mL for n-hexane against Clostridium perfringens, a pathogen causing enterotoxenia of livestock [24]. Ethyl acetate extract of it showed MIC value of 247 μg/mL against Haemopillus parsuis and 1,980 μg/mL against Pantoea agglomerans. Butanol extracts of it had MIC values of 720 μg/mL, 1,140 μg/mL, and 1,140 μg/mL against Burkholderia sp., Haemopillus parsuis, and Pantoea agglom  2) Mean ± standard error (n = 3).

Total polyphenol content and flavonoid content
Total polyphenol content and flavonoid content were rep-resented in Table 3. Total polyphenol amount of Zingiber officinale Rosc (Khing) methanol extract exhibited the highest vale as 48.4±0.00 μg/mL among all plant extracts, also the highest among them. This value was like that of Curcuma domestica Valeton (47.8 μg/mL) belonging to the same ginger family [10]. Conversely, Danciu et al [29] reported that total polyphenol content of this ethanol extract showed 16 mg GAE/g. Ghasemzadeh et al [30] recorded that polyphenol content in Malaysian Zingiber officinale root extract had 10.22 to 13.5 mg gallic acid/g. Thus, the polyphenol contents of Zingiber officinale in several studies represented various levels.
The content of active phytochemicals is known to be highly influenced by extraction conditions such as solvent, temperature, and extraction time [31]. Most ginger plants probably contain a considerable number of polyphenols. Total flavonoid content of Zingiber officinale Rosc (Khing) methanol extract was 22.1±0.01 μg/mL, the largest among tested plants extracts. Those of the rest plants extracts showed 6.2 to 19.8 μg/mL and total flavonoid con- tent in the methanol extracts of tested plants showed higher compared to those of other solvent extracts. Those of methanol extracts in Poncirus trifoliata fruit and Areca catechu L. were 37.4±0.02 μg/mL and 38.7±0.01 μg/mL, respectively. Total polyphenol content of Solanum melongena L. I (Makkhuayao) and Solanum melongena L. II (Makhuapro) were 30.2±0.01 mg/mL and 34.8±0.01 mg/mL in butanol extracts, higher than those of other solvent extracts. In Sola num melongena L. II, the total polyphenol content of most solvent extracts were above 20 μg/mL and the total flavonoid contents were 10 μg/mL, and they showed to be slightly different among extracts except for Hexane extract (polyphenols 22.7 to 34.7 μg/mL vs flavonoids 10.9 to 15.9 μg/mL). Poncirus trifoliata fruit plant has been widely used in folk medicine as a remedy for inflammation, gastric and dysentery [11]. Additionally, several studies suggested that this had the ability of herbal medicine for anti-helicobacter pylori activity, anticancer, and anti-anaphylactic activities [11,32]. In this study, Poncirus trifoliata fruit in methanol extract contained 37.4 μg/mL of total polyphenol and 18.2 μg/mL of flavonoid, and thus showed antioxidant activity and above moderate antimicrobial effect with relative low MIC value. This extract may be beneficial to livestock health.
Although content of polyphenol and flavonoid in Areca catechu L. water extract were relatively lower compared those of other solvent extracts, antioxidant and antibacterial activities including MIC value of it were favorable. With these results, these bioactive components of it probably have different solubility depending on the extractive solvents used.

Antioxidant activity of tropical plants
As shown Table 4, Zingiber officinale Rosc (Khing) exhibited antioxidant activities with the range of 1.10 to 2.23 mg/mL in n-hexane, chloroform, and butanol extracts.
Zingiber officinale Rosc, known as ginger, is the nutraceutical aromatic plant used as the spice and medicine worldwide [29]. This root extract has been used in Chinese and Indian traditional herbal medicine to treat indigestion, vomiting, arthritis, rheumatism, pains, cramps, fever, and infection [33]. It has been reported that this extract possesses valuable pharmacological phytocompounds attributed to antioxidant, antiinflammatory, immunomodulatory, antihyperglycemic, hypolipidemic, analgesic, and cardioprotective properties [33,34]. Tohma et al. 's results showed that ethanol extract of Zingiber officinale Rosc had better antioxidant activity than water extract of it. However, methanol and water extracts of this did not have antioxidant activities in this study [35]. Conversely, the antioxidant activity of n-hexane extract was the highest in this study, consistent with Račková 's results that n-hexane extract showed better DPPH radical-scavenging activity than other solvent extracts [36]. It was demonstrated that hydroethanolic extract of Zingiber officinale Rosc had strong free-radical reducing activity [33].
Antioxidant activity of Poncirus trifoliata fruit was 1.47±0.42 mg/mL in methanol extract but other extracts including water extract did not have antioxidant activities. Yu et al [37] reported that 70% ethanol extract of this showed more efficacy than water extract in the coronary from the pig. Areca catechu L grows throughout South, Southeast Asia and several Pacific Ocean islands [38]. This plant is the most commonly used as herbal medicine to treat dysentery and dysuria in Indonesia [13] and as an anthelmintic in humans and animals for a lengthy time in India and China [38]. Areca catechu L. showed antioxidant activity with 2.11±0.49 mg/mL in water extract with moderate inhibition (12.3 to 12.8 mm) on Burkholderia sp., Haemopillus somnus, and Haemopillus parsuis. Also, it was 1.06±0.11 mg/mL in methanol extract that appeared antibacterial activity against Burkholderia sp. These results were like Chavan' s results that in methanol and water extracts of it, total polyphenol content of Areca catechu L methanol extract showed higher than that of water extract and anti-oxidant activities showed relatively higher than those of other solvent extracts except acetone extract of it [39]. However, our result did not match the Hamsar et al. ' s study result that Areca catechu methanol extract exhibited higher scavenging effect than water extract [38]. Plants have potential as antioxidants because they are rich sources of polyphenols including flavonoids. Although total polyphenol and flavonoids contents of methanol extract were higher than those of water extract, the EC 50 value of water extract was higher than that of methanol extract in this study.
Solanum melongena L cultivated from tropical to temperate is one of the most consumed vegetables worldwide and one www.ajas.info of the most highly antioxidant vegetables [14]. Antioxidant activities of Solanum melongena L. I (Makkhuayao) and So lanum melongena L. II (Makhuapro) were 1.85±0.27 mg/mL and 1.66±0.14 mg/mL in butanol extracts, respectively. Namely, it seems that high antioxidant activities are correlated with the higher content of polyphenol (30.2 and 34.8 μg/mL) and flavonoid (13.9 and 15.9 μg/mL) compared to those of other extracts. Polyphenols have been active antioxidants as plant secondary metabolites [29]. According several studies, the antioxidant activity of it was correlated with the abundance of polyphenols and flavonoids [40,41]. Specifically, there is a result that Solanum melongena peel extract has been significantly higher in free radical scavenging activity [14]. Taken together, the correlation between antibacterial and antioxidant activities and amounts of total polyphenol and flavonoid was not perfectly matched in this study. Nevertheless, methanol extract of Poncirus trifoliata fruit (Makrut) possessed antibacterial activities against all pathogens selected for this study. Especially, this extract possessed strong antibacterial activity against Haemopillus somnus. This extract also contained total polyphenol and flavonoid higher amounts than the other solvents extracts and showed the antioxidant activity in proportion to these contents. In addition, hexane, chloroform, and butanol extracs of Zingiber officinale Rosc (Khing), water extract of Areca catechu L. (Mak), and butanol extract of Solanum melongena L. II (Makhuapro) showed moderate antibacterial and antioxidant activities.
The objective of this study was to identify suitable antibiotics using several tropical plants to improve productivity owing to disease prevention and treatment in livestock. Based on these results in this study, tropical plants have contained abundantly antioxidants such as polyphenol and flavonoids and biological functions such as antimicrobial and antioxidant activities have been effective. Tropical plants extract used in this study are possible substitute antibiotics. Therefore, further research on more tropical plants are needed to screen for their bioactivities and enable development of better antibiotic substitutes for animal health. The probable reason for this could be the fact that they may be used as a resource to improve livestock health and can be easily used in such circumstances where they are widely available and can be harvested in large quantities throughout the year.

CONFLICT OF INTEREST
We certify that there is no conflict of interest with any financial organization regarding the material discussed in the manuscript.