The in vitro antimicrobial and antioxidant activity of leaves of medicinal plants with phytobiotic potential in animal production

Little is known about which secondary metabolites are responsible for inhibiting pathogenic bacteria and reducing the pro-oxidant effect on the leaves of four medicinal plants used as phytobiotic in animal production. The aim of this study was to evaluate the antimicrobial and antioxidant activity of four medicinal plants (Anacardium occidentale, Psidium guajava, Morinda citrifolia and Moringa oleifera.) in vitro. A total of six bacterial strains were inoculated, then minimum bactericidal concentration (MBC) was evaluated in ne powder and minimum inhibitory concentration (MIC) and MBC were determined on the aqueous extract. Also, the in vitro antioxidant activity was evaluated through 1,1-diphenyl-2-picryl-hydrazyl, as well as the main secondary metabolites were identied and quantied by chromatographic analysis. The results showed that Anacardium occidentale and Psidium guajava leaves had higher antimicrobial activity against all bacterial strains. In addition, Morinda citrifolia inhibited S. aureus in the aqueous extract, although without in vitro bactericidal effect, while Moringa oleifera leaf did not show antimicrobial effect. All plants showed antioxidant capacity, standing out Anacardium occidentale and Psidium guajava. Mainly the leaves of Anacardium occidentale showed high concentrations of quercetin 3-O-glucoside-7-O-rhamnoside, kaempeferol-7-O-glucoside, quercetin, caffeic acid, and cinnamic acid. Apparently, the antimicrobial and antioxidant activity are due to the main polyphenolic compounds identied in medicinal plants (mainly Anacardium occidentale and Psidium guajava); however, further studies are necessary to elucidate the exact mechanism. myricetin 3-O-galactoside, myricetin 3-O-glucoside, and myricetin 3-O-arabinoside, where mainly present in A. occidentale and P. guajava leaves. This is consistent with the results obtained in the antimicrobial experiment of staphylococcal strains, in both forms, ne powder and as aqueous extract, any antimicrobial with the other strains. These results al. several studies that probe the antimicrobial and antioxidant properties of M. citrifolia based on its chemical compounds in the plant parts. Also, antibacterial activity found by al. et al. (2012) specically with Staphylococcus aureus. The difference in terms of the least antimicrobial effect in this study could be due to the use of methanolic extract. The antioxidant activity of the leaves of M. citrifolia was the lowest of among the four plants. Very little literature has been published the antioxidant capacity of the leaves of this plant. Besides, there are several investigations that show this quality in its fruits al. Thorat Sunder et al. (2016) demonstrated the multiple uses of M. citrifolia in livestock and poultry as a natural growth promoter due to its immunomodulatory, antioxidant, and hypocholesterolemic properties.


Introduction
The European Union totally eliminated the use of growth-promoting antibiotics in animal production on January 1st, 2006, prompting many countries to reduce or eliminate these synthetic products. Subtherapeutic antibiotics are known to cause microbial resistance and cross-resistance with other microorganisms that inhabit animals and humans. Therefore, there is a growing interest in research to nd natural alternatives to antibiotics; especially medicinal plants with bene cial phytochemical compounds and with antimicrobial, anti-in ammatory and antioxidant properties .
In that context, phytogenic additives have proven to be an important alternative to enhance the genetic expression of farm animals without the use of dietary antibiotics. Thus, phytogenic feed additives are included among supplements in order to positively affect feed quality, animal health and animal products caused by their speci cally effective substances (Karásková et al. 2015). Likewise, approximately 80% of the population in developing countries use medicinal plants systematically for humans and animals, in Cuba 1,170 species of these plants are reported, where 56% are known for their curative and preventive properties (Ramírez et al. 2020). Plants for antibacterial purposes are used to heal wounds, relieve digestive and oral discomfort, and additives in the farm animal diet, among others (Martínez et al. 2013;Más et al. 2016;Aroche Ginarte et al. 2017;Salazar Bell et al. 2017;Aroche et al. 2018). Thus, plants such as Anacardium occidentale (A. occidentale), Psidium guajava (P. guajava), Morinda citrifolia (M. citrifolia), and Moringa oleifera (M. oleifera) are some of the most used as adjuvants to therapeutic treatment in different diseases in humans and animals.
Anacardium occidentale, (family Anacardiaceae) is commonly used in the infection treatment, hemorrhages, diarrheal processes, and diabetes in animals, and also have shown that small concentrations could increase the egg production and egg quality and could decrease pig's diarrheal syndrome (Sunderam et al. 2019;Khatib et al. 2020;Siracusa et al. 2020). Its antimicrobial activity has also been veri ed in the ethanolic extract of owers, bark, and leaves, relating it to the alkaloid, saponin, phenolic acid and tannin contents (da Silva et al. 2016).
Several investigations have shown that leaves, fruits, bark and roots of P. guajava have been used to alleviate several illnesses, such as gastrointestinal diseases, diarrheal syndrome, stomach pain, diabetes mellitus, hypertension, wound healing, in ammations and obesity. Also, in animal production, P. guajava has been shown to promote egg production, eggshell thickness and reduce the liquid feces of pigs after weaning (Gupta et al. 2019;Salihu Abdallah et al. 2019;Weli et al. 2019;Ceballos-Francisco et al. 2020).
Morinda citrifolia are also popular for their variety of bene ts in human health and animal production, such as antimicrobial, anticancer, antioxidant, antiin ammatory, analgesic, cardiovascular, among others (Senthilkumar et al. 2016;Sunder et al. 2016;Thorat et al. 2017). Those properties made possible to include its leaves and fruits in poultry and pig diets with positive effects on egg production and body weight in order to increase the animal performance (Sunder et al. 2016;Aroche Ginarte et al. 2017;Salazar Bell et al. 2017;Aroche et al. 2018).
Moringa oleifera as a functional food in human health and animal production is very popular, especially for its high nutritional content of protein, minerals and vitamins Wang et al. 2018;Su and Chen, 2020). Likewise, bene cial effects of M. oleifera have been found due to its anticancer, antiin ammatory, antidiabetic, antioxidant and antimicrobial activity (Siddhuraju and Becker, 2003;Dhakad et al. 2019). Regarding to animal use of M. oleifera, several authors recommend it as a feasible source of nutrients for ruminant and non-ruminant animals (Mahfuz and Piao, 2019;Su and Chen, 2020;Valdivie et al. 2020).
These four plants have a marked global interest for animal production, due to their nutraceutical properties to improving productive indicators, intestinal health, and quality of the nal product (e.g., egg, meat and milk) in animals (Martínez et al. 2012(Martínez et al. , 2013Más et al. 2015Más et al. , 2016Aroche Ginarte et al. 2017;Cañete Sera et al. 2017;Salazar Bell et al. 2017;Aroche et al. 2018;Ramírez et al. 2020). However, little is known about which of these plants has the highest antibacterial and antioxidant potential related to animal production; which would allow to elucidate the medicinal bene ts reported animals of zootechnical interest; therefore, the objective of this investigation was to determine the antimicrobial and antioxidant effect of A. occidentale, P. guajava, M. citrifolia y M. oleifera leaves and aqueous extract in vitro.

Plant Material
Leaves of A. occidentale, P. guajava, M. oleifera and M. citrifolia were collected in Granma province, Cuba, in February/2019, during the low rainy season; this zone is characterized by a at topography and charcoal brown soil, authenticated by specialists from the Faculty of Agricultural Sciences of the University of Granma. The plants were more than one year old and without any sign of pathology. The leaves were dried in the shade, with free air circulation to constant weight and then dried in a stove (WSU 400, German) with air recirculation for 1 hour at 60°C. Subsequently, the leaves were crushed in a hammer mill with parallel blades, at 1 mm of size. The samples were stored at room temperature (26°C) in fully airtight plastic bags until further use.
In vitro experiments were performed at the Feed Research Institute of the Chinese Academy of Agricultural Sciences to determine the antibacterial and antioxidant activity of the leaves and their aqueous extracts.

Preparation of the Fine Powder and Aqueous Extract
To obtain the ne powder, the leaves were ground in a commercial grain crushing machine (Zhejiang Horus Industry and Trade Co., Ltd., Zhejiang, China) through a 40 mesh (0.45 mm) sieve (Yoston, China) and stored in completely airtight bags until use for microbiological tests.
Also, 16.67 g of the leaves of each plant were weighed and mixed with 500 mL of water (30:1, v/w) for aqueous extraction. The aqueous extract was obtained by the sonication method, using an ultrasonic extractor (model SY-1000E, China) for 50 minutes at 50°C, allowed to stand for 1 hour, and ltered through Whatman lter paper. No. 1. It was subsequently condensed through a rotary evaporator (model RE-2000, China), under reduced pressure at 45°C at 60 rpm.
The extract was frozen at -80°C for at least 4 hours, and nally dried in a lyophilizing machine (model LGJ-18, China).

Minimum Bactericidal Concentration of Fine Powder
The MBC of the ne powder from leaves of the four plants was determined for triplicated. Bacterial culture was inoculated and incubated for 12 hours, later, 90 mm diameter petri dishes were prepared with Mueller-Hinton Agar (MHA) at different concentrations of the ne powder. Each bacterial culture of 100 µL of was inoculated, which consisted in strains of enterotoxigenic Escherichia coli (ETEC) K88 + , Escherichia coli ATCC 1515, Staphylococcus aureus (S. aureus): ATCC 43300 and ATCC 25923, Salmonella enteritidis (S. enteritidis): ATCC 3377, and Salmonella typhimurium (S. typhimurium): ATCC 14028. In the rst period, concentrations of 5, 15 and 30 mg/mL of ne powder in the culture medium were tested to identify the minimum concentration of each plant in that range. Then, concentrations less than 5 mg/mL were used for leaves of A. occidentale and P. guajava; 5 to 15 mg/mL of ne powder in culture medium in the case of M. citrifolia and 15 to 30 mg/mL of ne powder of culture medium with M. oleifera.

Minimum Inhibitory Concentration and Minimum Bactericidal of Aqueous Extract from the Plants
For this study, aqueous extract of the four plants was used, thus a stock solution of 13 mg/mL was prepared, which was used to prepare in serial dilutions of 13, 6.5, 3.25, 1.63, 0.81, 0.41, 0.2, 0.1, 0.05, 0.03, and 0.01 mg/mL. The inoculum of E. coli (ETEC K88 + ), S. aureus (ATCC 43300), and S. typhimurium (ATCC 14028) were prepared in culture medium at a concentration of approximately 15 x 10 7 CFU/mL compared to theoretical optical density (550 nm absorbance) that de nes the level of 0.50 in the McFarland turbidimetric scale. Then, 200 µL/well of each dilution was placed in 96-well microplates and 2 µL of each bacterial culture was inoculated for triplicates, incubated for 12 hours at 37°C to determine its absorbance in a plate reader (ELISA, BIO-TEK, Synergy HT).
Determination of the MBC was carried out for triplicated, 100 µL of supernatant from those wells where bacterial growth was inhibited and seeded with a sterile glass triangular spatula in 90 mm diameter petri dishes with Mueller-Hinton Agar (MHA), and incubated for 12 hours at 37°C.

Antioxidant Activity
Antioxidant activity of aqueous extract of leaves from the four plants was evaluated with DPPH− (Shen et al. 2010) where a solution of 0.1 mM of DPPH− in methanol was prepared. Later, 1 mL of this solution was taken and vigorously mixed in a vortex with 3 mL of the different concentrations (10, 5, 2.5, 1.25, 0.625, 0.313, 1,156, 0.078, 0.039, 0.020 and 0.010 mg/mL) of the extract, and 200 µL of each concentration were placed in a 96-well microplate. The solutions were left to stand at room temperature in the dark for 30 min and then, the absorbance at 517 nm was measured with the use of a plate reader (ELISA brand, BIO-TEK, Synergy HT). BHT was used as reference. Low absorbance values indicate high free radical scavenging capacity, or high antioxidant capacity, which was calculated using the following formula: where A0 is the absorbance of the control reaction, and A1 is the absorbance in the presence of the extracts and the reference. All samples were evaluated in triplicate and the results were averaged and shown as IC 50 values (mg/mL).

Pretreatment Method
The sample leaves (40 mg) from A. occidentale, P. guajava, M. citrifolia and M. oleifera were added to 4 mL of extractant and were shaken under ultrasonic for 30 min. Then, centrifugation for 5 min to take the supernatant and the membrane was done. EDTA buffer solution: weigh 7.10 g of anhydrous sodium hydrogen phosphate, 1.95 g of disodium edetate, 8.40 g of citric acid, and dissolve in 650 mL of water.

Mass spectrometry (MS)
Electrospray ionization (ESI) in positive/negative mode with a MS2 Scan was used in MS. The drying gas temperature was 250°C and dry gas ow rate of 7 L/min, with an atomizing gas pressure of 35 psi. Sheath gas temperature of 325°C and sheath gas ow of 11 L/min was used, and a fragmentor of 80 V; 100 V; 120 V with a cell accelerator voltage of 5 V was used.

Description
The pretreatment was separately extracted with methanol and acetonitrile. The results showed that the extraction with methanol was better. In the full scan of the parent ion, the positive and negative ion modes are used for simultaneous scanning, and the results can be mutually veri ed. The addition of formic acid to the mobile phase increased the sensitivity of the compound both in positive ions and in negative ion mode. The addition of ammonium acetate improved

Qualitative Method
Forty mg sample of leaves from A. occidentale and P. guajava were added to 4 mL of extractant and were shaken under ultrasonic for 30 min. Then, centrifugation for 5 min to take the supernatant and the membrane was done.
EDTA buffer solution: weight 7.1 g of anhydrous sodium hydrogen phosphate, 1.95 g of disodium edetate, 8.4 g of citric acid, and dissolve in 650 mL of water.
MS was performed on a Sciex Triple Quad 4500 MS/MS, and electrospray ionization coupled with multiple reaction monitoring (MRM) model. The resulting optimized values were as follows: source temperature 450°C; ion spray voltage 4500 V; collision gas: 9 psi; curtain gas 10 psi; ion source gas (GS 1) 18 psi; and ion source gas (GS 2) 0 psi.

Statistical Analysis
Data were processed by simple classi cation ANOVA in a completely randomized design. Before this, the normality of the data was veri ed using the Kolmogorov-Smirnov test and for uniformity of variance, the Bartlett test. When the effects were signi cant, the means were separated using Duncan's test at the signi cance level of P ≤ 0.05. All analyzes were carried out in accordance with the SPSS statistical software, version 21.0 (SPSS Inc., Chicago, IL, USA).

Results
The MBC of the leaves of A. occidentale; P. guajava; M. citrifolia and M. oleifera against six strains of pathogenic bacteria is showed in Table 1. Leaves of A. occidentale showed the greatest bactericidal effect in the study, mainly against Escherichia coli K88 and Staphylococcus aureus (ATCC 25923) with concentrations of 4 and 1 mg/mL, respectively. Likewise, the leaves of P. guajava showed a bactericidal effect by reducing the growth of Gram negative and Gram-positive bacteria with a concentration of 11 mg/mL for E. coli K88 + . Also, the leaves of M. citrifolia and M. oleifera only showed bactericidal activity against the strains of S. aureus (ATCC 43300; ATCC 25923) although with higher doses (8-16 mg/ml) than the inhibitory effects of the leaves of A. occidentale and P. guajava.  Likewise, a higher concentration of the aqueous extract of P. guajava (compared to the aqueous extract of A. occidentale leaves) is necessary to inhibit and eliminate S. typhimurium (ATCC 14028), similar occurred for the bactericidal effect of this product against S. aureus (ATCC 43300). The aqueous extract of M. citrifolia only inhibited the growth of S. aureus (ATCC 43300) at doses of 6.5 mg/mL, however, it did not show bactericidal activity at the concentrations studied (maximum concentration of 13 mg/mL). Also, the M. oleifera extract did not show inhibitory or bactericidal activity. Table 3 shows the IC 50 of the aqueous extract of the leaves of the four plants. A. occidentale plant with the highest free radical trapping activity compared to the other three plants, as it re ects the lower IC 50 , being even lower (P<0.001) than the positive control butylated hydroxytoluene (BHT). Furthermore, P.
guajava did not show (P>0.05) statistical differences with A. occidentale and BHT. However, M. oleifera and M. citrifolia had the lowest results in antioxidant activity, as they require the highest concentration to inhibit the 1,1-diphenyl-2-picryl-hydrazyl (DPPH−) reaction. The phytochemical compounds identi ed in the four plants and compared with those reported in the literature are shown in Table 4, where the information on antibacterial and antioxidant activity was compared based on the scienti c literature. Is remarkable the presence of powerful antibacterial compounds (Table 4) Table 1 and 2.
Also, the content of principal compounds from A. occidentale and P. guajava leaves are shown on Table 5, where it is observed the higher concentration of quercetin 3-O-glucoside-7-O-rhamnoside, kaempferol-7-O-glucoside, quercetin, caffeic acid and cinnamic acid from A. occidentale leaves compared to P. guajava.

Discussion
A. occidentale is known for its antibacterial properties, mainly in its owers, bark and leaves (da Silva et al. 2016). In addition, it has been used in the prevention and treatment of oral diseases (being the rst contact of the digestive system with the food) by inhibiting the bacteria in this cavity and therefore the formation of bio lm (Anand et al. 2015). Also, Melo Menezes et al. (2014) found that both crude extract and isolated tannins of A. occidentale have inhibitory activity against microorganisms that are part of the composition of oral bio lm. Therefore, they hypothesized that the mechanisms of the antimicrobial action of tannins, the enzymatic inhibition, the modi cation of cellular metabolism by its action on the membranes and binding with metal ions, decrease the access to metabolism to the microorganisms that are outside the bio lm. The present study results showed a potent antimicrobial and antioxidant activity, which is related to the high content of polyphenols and avonoids contained in its leaves, in addition to other medicinal compounds. Souza et al. (2017) observed the antioxidant and anti-in ammatory activity in vitro in A. occidentale leaves extract when used in RAW 264.7 macrophage cells due to the lower oxidative damage of these cells and the decrease in in ammatory parameters induced by LPS stimulation. Additionally, Brito et al. (2020), pointed out that pentagalloil hexoside, a precursor to the formation of hydrolyzed tannins such as ellagitannins and gallotanins, was found in all the organs of A. occidentale, these chemical compounds are responsible for several functional properties, with higher emphasis on the antimicrobial activity. Thus, the present study showed that A. occidentale was the plant with the highest antimicrobial and antioxidant capacity compared to the other three plants studied.
Regarding the effect of A. occidentale in animal production, speci cally in poultry and pig production,  found that dietary supplementation with 1.0% of a mixed powder made from 40% of A. occidentale leaves powder increased growth performance and decreased the diarrhea incidence in weaned piglets. Furthermore, Más et al. (2016) showed that the dietary inclusion in low concentrations of A. occidentale and P. guajava leaves powder promoted growth and reduced dehydration in pigs before and after weaning. In this sense, Aroche et al. (2018) showed positive results in feed e ciency and IgG production when they added 0.5% of a mixture of plants representing 60% of A. occidentale in broiler diets.
P. guajava has also shown strong bactericidal activity on its leaves and aqueous extract, as it requires a small amount to eliminate bacteria such as E. coli, S. aureus, and Salmonella. Similarly, Salihu Abdallah et al. (2019) veri ed that the aqueous and methanolic extracts of P. guajava leaves have antimicrobial activity against S. aureus and S. typhi. The aqueous extract was effective with MIC of 12.5 mg/mL for both bacteria and MBC between 25 and 50 mg/mL for S. aureus and S. typhi respectively. In this study, the concentrations of aqueous extract were necessary to obtain the MIC and MBC against these bacteria, and were lower than those aforementioned, which may be due to the variety of the plant used, the origin, the extraction methods, among other factors. Also, Chero Nepo and Ruiz Barrueto (2016) determined that the alcoholic extract of P. guajava inhibits the growth of Streptococcus mutans due to its bactericidal power.
Regarding the antioxidant activity, Flores et al. (2015) identi ed the chemical composition of seven cultivars of P. guajava and founded a high content of avonoids, in addition of anthocyanins, proanthocyanins, triterpenes and other compounds. Likewise, Feng et al. (2015) and Flores et al. (2015) showed that there is high correlation between avonoid content and the antioxidant capacity of the plant, which agree with our ndings, where P. guajava was the second plant to show a high antioxidant power.
On the other hand, M. oleifera is a multipurpose plant with multiple nutritional bene ts, but also has been studied for its antimicrobial and antioxidant effects, since its use in human and animal nutrition is increasingly popular (Wang et al. 2018). Likewise, M. citrifolia has innumerable health bene ts, however, when these two plants are compared with A. occidentale and P. guajava, they may be at a disadvantage due to the lower content of secondary metabolites responsible for the aforementioned activity. This research demonstrated the marked difference for antimicrobial and antioxidant effect of both the leaves and the aqueous extract of A. occidentale and P. guajava compared to M. citrifolia and M. oleifera.
However, in the case of M. oleifera, researchers such as Siddhuraju and Becker (2003), determined that this plant presents high antioxidant power in its ethanolic and methanolic extracts, which was related to abundant avonoid content, especially quercetin and kaempferol. Shih et al. (2011) found high antioxidant activity in the ethanolic extract of various parts of this plant, where the leaves showed the highest activity, with an IC 50 of 0.287 mg/mL, which is less than that found in this study (0.603 mg/mL). This difference could be due to the difference on the extraction (aqueous) method used this study. In relation to animal production, authors such as Zhang et al. (2018) found positive effects of M. oleifera on performance of fattening pigs, with a marked effect due to increased activity of the enzyme superoxide dismutase and decreased serum malondialdehyde concretion.
M. citrifolia only inhibited the growth of staphylococcal strains, in both forms, as ne powder and as aqueous extract, however, did not show any antimicrobial effect with the other bacterial strains. These results agree with Almeida et al. (2019), whom reported several studies that probe the antimicrobial and antioxidant properties of M. citrifolia based on its chemical compounds in the plant parts. Also, antibacterial activity was found by Pandiselvi et al. (2019) and Sunder et al. (2012) speci cally with Staphylococcus aureus. The difference in terms of the least antimicrobial effect in this study could be due to the use of methanolic extract. The antioxidant activity of the leaves of M. citrifolia was the lowest of among the four plants. Very little literature has been published about the antioxidant capacity of the leaves of this plant. Besides, there are several investigations that show this quality in its fruits (Senthilkumar et al. 2016;Sunder et al. 2016;Thorat et al. 2017). Sunder et al. (2016) demonstrated the multiple uses of M. citrifolia in livestock and poultry as a natural growth promoter due to its immunomodulatory, antioxidant, and hypocholesterolemic properties.
Polyphenols are the major secondary metabolites distributed in all plants, with higher emphasis on iso avonoids, anthocyanins, avonols, and avones in A. occidentale and P. guajava. The quanti cation of the main secondary metabolites in these two plants (A. occidentale and P. guajava) such as quercetin 3-Oglucoside-7-O-rhamnoside, chicoric acid, kaempeferol-7-O-glucoside, caffeic acid, and cinnamic acid could support the antibacterial and antioxidant effects found in this study.
Theoretically, authors such as Sharaf et al. (2000) and Roepke and Bozzo (2013) have mentioned that 3-O-glucoside-7-O-rhamnoside is a rare secondary metabolite in plants with proven antioxidant and antimicrobial properties against E. coli. Furthermore, caffeic and chicoric acids have potential as antidiabetic agents (Tousch et al. 2008), already demonstrated by Kamtchouing et al. (1998) and Mukhtar et al. (2004) who found a reduction in glucose concentration in laboratory mice when they used extracts of A. occidentale and P. guajava, respectively. In addition, the avonoid kaempferol-7-O-glucoside was identi ed and quanti ed in the leaves of A. occidentale, which is a phytochemical widely studied for its antimicrobial properties (Singh et al. 2011). Moreover, cinnamic acid is an organic acid that occurs naturally in many medicinal plants and quanti ed in both medicinal plants, it has low toxicity and a wide spectrum of functional activities, this secondary metabolite has antibacterial, antiviral and antifungal properties (Sova, 2012), which supports the effect antimicrobial found in the leaves of the plant in study (Tables 1 and 2

Conclusions
It is concluded that A. occidentale and P. guajava are the plants with the highest antimicrobial and antioxidant activity in their leaves and aqueous extract. M. oleifera has good antioxidant in vitro activity, although it does not have high antimicrobial power; and M. citrifolia is the plant that has the least antioxidant activity in its aqueous extract.