In Vitro Antimicrobial Activity of Piper retrofractum Fruit Extracts against Microbial Pathogens Causing Infections in Human and Animals

Long pepper (Piper retrofractum Vahl) is a Thai medicinal herb which has been used as one of the common ingredients in variety of Thai foods. Here, we investigated antimicrobial activities of crude bioactive metabolites extracted from fruits of P. retrofractum against 10 pathogenic organisms (bacteria and yeast) causing opportunistic infections in human or animals including Bacillus subtilis ATCC6633, Staphylococcus aureus ATCC25923, Enterococcus faecalis ATCC2921, Escherichia coli ATCC25922, Klebsiella pneumonia TISTR1843, Pseudomonas aeruginosa ATCC741, Salmonella typhi (clinical isolate), Vibrio parahaemolyticus (XN98 and 5HP), and Candida albicans ATCC90020. The results of disk diffusion test showed that the extract from methanol solvent exhibited greater antibacterial activity than other solvents with inhibition zones ranging from 0.5 to 8.0 mm, respectively. Subsequently, minimal inhibition concentration (MIC) determined by the colorimetric assay confirmed that methanol extracts showed consistent results with disk diffusion method. In summary, in vitro assays suggest that methanol is the best solvent for extraction of bioactive metabolites from P. retrofractum fruits. This crude extract can inhibit the majority of human and animal pathogens. This opens up a potential use of pepper fruits in prevention of food-contaminating microorganisms.


Introduction
Drug resistance of infectious microorganisms has been reported worldwide [1]. e most prevalent resistant species are the methicillin-resistant Staphylococcus aureus (MRSA), the extended-spectrum β-lactamase (ESBL)-producing Escherichia coli, Klebsiella pneumoniae, and multi-drug resistant Pseudomonas aeruginosa [2]. In addition, the consumption of food-contaminating microorganisms, such as Enterococcus faecalis, Escherichia coli, Salmonella typhi, and Vibrio parahaemolyticus, can pose a serious threat to human health. e existence of these microorganisms causes spoilage and often a food-borne disease [3]. Candidiasis is a fungal infection caused by Candida albicans that can affect areas such as skin, genitals, throat, mouth, and blood circulation system. It is caused by the overgrowth of C. albicans [4]. Candida infections can be difficult to treat and can reoccur after treatment. In people with weakened immune systems, candidiasis can be life threatening if it passes into the blood and spreads to vital organs [5]. Infection of V. parahaemolyticus causes acute hepatopancreatic necrosis disease (AHPND) in cultivated shrimp farm [6]. e disease spread rapidly and caused significant losses in Southeast Asian shrimp farms since 2009 [7].
Long pepper, or Piper retrofractum Vahl, is a flowering vine in the family Piperaceae, cultivated for its fruit. e dried fruit has long been used as a spice and seasoning.
ailand is located in tropical humidity zone which provides the best condition for growing P. retrofractum, especially in the central part of Kanchanaburi, Ratchaburi, Phetchaburi, and Chanthaburi provinces. Locals use P. retrofractum fruit as a key ingredient in various recipes of their traditional medicine and foods. ere has been no report of toxicity from long pepper. Fruit of long pepper was analyzed for its chemical profile. Extracted bioactive metabolites from P. retrofractum were found to contain alkaloid component, amide derivative with antiflatulent, expectorant, antitussive, antifungal, and appetizing properties. In addition, it was reported to possess gastroprotective and cholesterol-lowering properties which could be useful for application in traditional medicine [8][9][10]. e aim of this research is to extract metabolites from long pepper using different solvents and to perform in vitro antimicrobial tests against various pathogenic bacteria and yeast.

Plant Extract Sample. Fresh fruits of Piper retrofractum
Vahl (long pepper) were purchased from the botanical garden in Kanchanaburi, ailand ( Figure 1). e samples were ground in a blender. Each 250 g of the sample was then extracted with 500 mL of individual solvents including hexane, isopropanol, acetonitrile, dichloromethane, and methanol. e extraction was performed in a one-litter screw cap bottle and orbit-shaked at 150 rpm for 48 hrs at room temperature. en, the samples were filtrated by passing through Whatman No. 1 and the residue was used for cascade extraction with respective organic solvent. e filtration of each solvent was evaporated by rotary evaporator (Rotavapor ® R-300, BUCHI) to remove the solvent and dried in desiccator. e crudes were weighed and dissolved in DMSO (dimethyl sulfoxide) for the next experiments.

Antimicrobial Susceptibility Testing by the Disk Diffusion
Method. e crude extracts of P. retrofractum fruits were tested against the above pathogens by the disk agar diffusion method. e method for antibacterial disk diffusion susceptibility follows manual of antimicrobial susceptibility testing guidelines [11], and antifungal C. albicans disk diffusion susceptibility testing follows manual for antifungal disk diffusion susceptibility testing of yeasts in NCCLS guideline in 2004 [12]. All crude extracts were dissolved in dimethyl sulfoxide (DMSO). For initial screening, 40 µl of the extract was loaded onto each Whatman No. 1 filter paper disk (Ø, 6 mm) and air-dried for 20 min. ese bacteria were grown on Mueller-Hinton agar (MHA) medium (pH 7.3) except Vibrio spp. which were supplemented with 1.5% w/v NaCl and yeast using Sabouraud dextrose agar (SDA). Agar media were poured into the plates to uniform depth of 5 mm and allowed to solidify. e microbial suspensions were prepared by spectrophotometer using culture broth with adding sufficient sterile medium to adjust the transmittance to that produced by a 0.5 McFarland standard match to an optical density (OD) 0.1 at 625 nm wavelength. is procedure will yield bacterial stock suspension 1 × 10 8 cfu/ml and yeast stock suspension of 1 × 10 6 to 5 × 10 6 cfu/ml. e microbial suspension was streaked over the surface of media using a sterile cotton swab to ensure the confluent growth of the organism. e disks used were Whatman ® No. 1 papers, 6 mm in diameter, which were then aseptically applied to the surface of the agar plates at wellspaced intervals. e plates were incubated at 37°C for 24 h and observed growth inhibition zones, including the diameter of the disks, were measured. Control disks were impregnated with 10 µl of the solvent DMSO.

Determination of Minimum Inhibitory Concentration (MIC).
e crude extract has shown significant antimicrobial activities in disk diffusion method which were 2 International Journal of Microbiology selected for determination of MIC using the broth microdilution assay following NCCLS M07-A09 guideline [13] with minor modification. e microbial suspension was prepared by spectrophotometer OD 0.1 at 625 nm wavelength (0.5 McFarland standard) and contained approximately 1 × 10 8 cfu/mL and yeast suspension of 1 × 10 6 to 5 × 10 6 cfu/mL as starting inoculums. e crude extract of serial two-fold dilutions with 100 µl medium in 96-well plates was used, and then the inoculums were applied in each well with a final concentration 1 × 10 5 cfu/ml for bacteria and 0.5-2.5 × 10 3 cfu/mL for C. albicans. e microplate was incubated at 37°C for 24 hr (for bacteria) or 48 hr (for C. albicans). After incubation, 30 µL tetrazolium salt (2,3,5triphenyltetrazolium chloride, Merck), 0.02 µg/mL, was added into each well. Colorimetric interpretation was performed after 1 hr incubation; the MIC was read as the lowest concentration of antimicrobial agent at which no color change occurred [14,15].

Medicinal Plant Piper retrofractum Vahl Morphology. P. retrofractum
Vahl is a dicotyledon flower plant belonging to the Piperaceae family. It has a radical root when growing from the seed but in agriculture aerial stem transplantation is used which develops fibrous root from the pericycle layer. P. retrofractum had an adventitious root as climbing root at the node of aerial stem that was the spot-characteristic of climber herbaceous plant ( Figure 1). Simple leaf is composed of lamina as lanceolate type with 10 × 5 cm area, and the leaf base is obtuse having the entire of margin. Leaf texture was glabrous indumentum, chartaceous, succulent phyllotaxy, and small rod petiole 5-7 cm long without stipule (Figure 1(a)). IT was a perfect flower, apetalous, tubular with androecium, and gynoecium syncarpous carpel axial placentation. Fruits of P. retrofractum Vahl are finger shaped with a length of 4.5-5.2 cm and a 0.3-0.5 cm diameter. It is a multiple fruit development and has multiple seed orientation surrounded on the same receptacle. e young fruit is of green color and a small size and the ripe fruits become of a red-orange color and consist of many minuscule seeds embedded in the same receptacle as shown in Figure 2 is is the main source of herbal material for solvent extraction for future steps.

Highest Yield of Crude Extract Found in Isopropanol
Solvent. Using the same extraction protocol with equal amounts of solvents and fresh pepper fruits, five different solvents yielded dried crude production from 5.47 to 12.38% w/w (Table 1). Isopropanol solvent gave a highest yield of 12.38% (w/w), followed up by dichloromethane 11.15% (w/ w) and acetonitrile 11.15% (w/w). Lower yields 5.475% (w/ w) was produced in methanol solvents, respectively (Table 1). e column chromatography was performed after crude extraction using silica gel 60 (0.015-0.040 mm, Merck). Dichloromethane extraction and isopropanol extraction were giving pale yellow needle-like crystal 3-12 mm and unique aroma of alkaloid, shown in Figure 2(b). e result of liquid chromatography mass spectrophotometry (LCMS, micrOTOF, Bruker) was analyzed and a pure crystal with 308.1297 g/mol molecular weight was produced, and this  substance was shown to be a dimer substance form with 593.2979 g/mol molecular weight.

Solvent Extract Inhibited the Majorities of Tested Pathogens by Agar Disk Diffusion Test.
Antimicrobial susceptibility testing results of the five different solvent extracts and a control antibiotic (ampicillin) against 10 pathogenic microorganisms were presented in Table 2. Among the five solvents used, methanol gave the best inhibition result. Methanol extract inhibited 9/10 of the tested pathogens with inhibition zone in the range from 0.5 to 8 mm. Inhibition zones for C. albicans ATCC90020, S. aureus ATCC25923, P. aeruginosa ATCC741, and E. faecalis ATCC2921 were 8.0, 6.0, 4.0, and 3.5 mm, respectively. No inhibition was observed in the K. pneumonia TISTR1843 isolate and it was very weak (0.5 mm) in B. subtilis ATCC6633 and V. parahaemolyticus 5HP. Remaining isolates (E. coli ATCC25922, S. typhi (clinical isolate) and V. parahaemolyticus XN89) exhibited inhibition zone from 2 to 3 mm (Table 2). Poor inhibition results (0-2 mm) were observed in other extract solvents, except for isopropanol extract that showed 4.0 and 3.5 mm inhibition zone for S. aureus ATCC25923 and E.

Methanol Extract Minimum Inhibitory Concentration (MIC).
Since the methanol extract showed the best inhibition result by disk diffusion test, minimum inhibitory concentration (MIC) was then performed for only the methanol extract. Details of MIC testing result are shown in Table 3. Overall, the results of the two methods were consistent. Lowest MIC values were 0.5 and 5.5 and 11 µg/µL in C. albicans ATCC90020, B. subtilis ATCC6633, and S. typhi (clinical isolate), respectively. In contrast, highest MIC values were 179.5 and 90 µg/µL in P. aeruginosa ATCC741 and K. pneumonia TISTR1843, respectively. MIC ranged from 22.5 to 45 µg/µL in the remaining isolates (Table 3). e colorimetric for bacterial detection technique was incubated at 37°C for 24 hr after inoculation, and infectious yeast C. albicans was at 37°C for 24 hr incubation. en, 30 µL tetrazolium salt (0.02 µg/mL) was added into each of well. e   testing was a microdilution assay using flat shape 96-well plate. is is an example of crude metabolite extract P. retrofractum Vahl against E. coli ATCC25922. e MIC was read after tetrazolium was added which has shown the lowest concentration of antimicrobial agent at which it was colorless or changed to pale pink. e wells F1 to F6 were negative control, and the wells F7 to F12 were positive control.
colorimetric interpretation was displayed after the tetrazolium reaction with or without microorganisms within 1 hr after incubation. e color displayer was colorless with the microbial inhibition growth as a negative control in the wells F1 to F6 whereas microbial growth changed to a red color as a positive control in the well F7 to F12. e MIC was observed and read as the lowest concentration of antimicrobial agent at which no color changed or a bit color changing occurred starting from the well number 1 to the well number 12, respectively. e concentration of the row A to the row E (except row F1-6 was the negative control and F7-12 positive control) was using the two-fold dilution of various bioactive metabolite starting from the well number 1 to the well number 12, respectively. e row G tested for ampicillin and row H tested for DMSO ( Figure 3). All of the wells from H1 to H 12 were of red colors; meanwhile, the amount of DMSO used for dissolubility of testing metabolite had no effect on microbial inhibition (Figure 3).

Discussion
Health problems have recently led to the development of natural antimicrobials to control microbial diseases. Medicinal plants and spices are one of the most commonly used natural antimicrobial agents in foods and have been used traditionally for thousands of years by many cultures for controlling common health complications. e WHO reported (2017, 2018) a list of bacteria for which new antibiotics are urgently needed for antibiotic-resistant "priority pathogens" of 12 families of bacteria that pose the greatest threat to human health. ere were three families of critical priority 1 ranking: Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacteriaceae include many harmless symbionts, and many of the more familiar pathogens, such as Salmonella, Escherichia coli, Klebsiella, and Shigella, six families of high risk priority 2 ranking which are Enterococcus faecium, Staphylococcus aureus (methicillin-resistant, vancomycin-intermediate and resistant, MRSA), Helicobacter pylori, Campylobacter spp., Salmonellae and Neisseria gonorrhoeae, and three families of medium risk priority 3 ranking which are Streptococcus pneumoniae, Haemophilus influenza, and Shigella spp., respectively [16].
Natural plant product-based antimicrobials drug discovery attained paramount importance as newly discovered drugs are likely to be effective against multidrug-resistant microbes. Bacteria such as B. subtilis, S. aureus, E. faecalis, E. coli, K. pneumoniae, P. aeruginosa, and S. typhi are multidrug resistant. Huge amount of antibiotics used for treating the diseases in human and animals result in this resistance. Multidrug-resistant bacteria are generated by accumulating the multiple genes encoding resistance to a single antibiotic in one cell. Besides that, overexpression of multidrug efflux pumps genes in bacteria could generate more resistant bacteria [17]. ere have been many serious problems for treatment of these diseases in ailand. Some of these microorganisms showed severe threatening for aquatic culture, too. For example, V. parahaemolyticus is a pathogenic microorganism in aquatic animals and is associated with contaminated food problem and causes food-borne disease. V. parahaemolyticus is an aquatic zoonotic agent. Humans are infected by consuming the contaminated seafood specially shrimp. is pathogen is orally transmitted in humans and causes gastroenteritis. In shrimp culture this pathogen causes acute hepatopancreatic necrosis disease (AHPND) and a very high mortality rate and economic losses [6]. Several toxins were reported to be produced by V. parahaemolyticus such as thermostable direct hemolysin (TDH), TDH-related hemolysin (TRH), and thermolabile hemolysin (TLH) [6,7,18]. e treatment of this severe human and aquatic pathogen is done by using the antibiotic. However, there were isolates from aquaculture which were resistant to antibiotics. erefore, finding the alternative way for controlling this pathogen is necessary for human food and safety.
C. albicans was found as an infection on the mucous membranes of the intestinal tract and skin.
is study showed that crude extract from the fruit of P. retrofractum Vahl using acetonitrile or methanol has a better potential to inhibit bacterial pathogens and C. albicans. e tested organisms, particularly Gram-negative organisms, showed high resistance towards different antibiotics generally. However, this study showed that they were inhibited by crude P. retrofractum Vahl extract even at a lower concentration.
Extraction of the bioactive compounds was reported by many researchers. ese bioactive compounds belong to various groups such as tannins, alkaloids, glycosides, lignans, and terpinoids. e bioactive compounds are usually extracted by different solvents. e chemical properties and characterizations of these solvents could play a vital role in crude extraction property. Some factors should be considered when choosing the appropriate solvent in the study including solvent power (selectivity), boiling temperature, latent heat of vaporization, reactivity, viscosity, stability to heat, oxygen and light, and the last but not the least safety. In this study methanol showed better results as a solvent used in extraction.
is might be related to the polarity index of methanol (5.1) [19]. Mostly, methanol is used to extract various polar compounds; however, certain non-polar groups are fairly soluble in this solvent. Furthermore, methanol has the lowest boiling point among all the alcohols. erefore, the extraction and concentration of bioactive compounds can be more easily done by this solvent. In this research, the results showed that the amount of P. retrofractum Vahl crude extract by methanol was less than other solvents, and it might be illustrated that the concentration of active biocompounds in methanol extract might be more than others. In other words, the more bioactive compounds with higher concentrations were extracted by methanol. Further experiments to identify the components quantity of each solvent can help us for better understanding of P. retrofractum Vahl bioactive compounds against microbial pathogens.
In conclusion, P. retrofractum Vahl bioactive compounds extracted by methanol solvent have a better potential against pathogenic microorganisms. Our study showed that this crude extraction can be used as prebiotics in aquatic culture. Further study for identifying the pure bioactive International Journal of Microbiology compounds can lead to finding new antimicrobial agents for human and even aquatic culture. is research could open a new horizon and show promising results towards finding a new natural antibiotic for multidrug resistant-pathogens control with using endogenic ai food.

Data Availability
e data used to support the findings of this study are available from the corresponding author upon request.

Ethical Approval
Ethical clearance was obtained from Faculty of Science and Technology, Suan Sunandha Rajabhat University, Bangkok, ailand, with the reference number ECSSRU 2018-213.

Conflicts of Interest
e authors declare that there are no conflicts of interest regarding the publication of this paper.