Secondary metabolites

Secondary metabolites (MS) are compounds derived from the biosynthesis routes of primary carbon metabolism in plants, which appear in the cytoplasm of most plant cells (^{Augustin et al., 2011}).

These compounds do not have an apparent importance however; they play an important ecological role since many of the compounds serve as defense mechanism against herbivores, viruses, bacteria and fungi (^{Pérez and Jiménez, 2011}).

Other MSs have physiological functions, such as alkaloids and pectins, which can serve to transport toxic nitrogen and other storage compounds, while phenolic compounds such as flavonoids play a role as ultraviolet ray protectors (^{Pérez and Jiménez, 2011}). MSs as opposed to primary metabolites have a restricted distribution in the plant kingdom as they are synthesized in small quantities and in a specific way determined to the genus, family or plant species (^{Ávalos and Pérez-Urria, 2009}).

Up to 2007, about 8000 polyphenols, 270 non-protein amino acids, 32 cyanogens, 10,000 alkaloids, various saponins and steroids had been reported (^{Domingo and López Brea, 2003}; Duramandiyan et al., 2006; ^{Carmona, 2007}).

MSs are grouped into four main groups: Terpenes, phenolic compounds, glycosides and alkaloids, all with different pharmacological properties as shown in Table 1.

Use of secondary metabolites as antibacterials

Plants have been considered as a sustainable option against pathogens and an important source of natural diversity due to the large number of compounds they synthesize; more than 100,000 MS have been reported in the nature of which there are some that have been shown to have an antibacterial effect (^{Montes-Belmont, 2009}, ^{Cruz-Carrillo et al., 2010}, ^{Borroto et al., 2011}, ^{Avello Lorca et al., 2012}, ^{Soto Vázquez et al., 2014}).

It is clear that in veterinary practice there are a large number of pathologies associated with the presence of bacterial pathogens which cause significant economic losses in livestock production (^{Pugh, 2002}), however, resistance to conventional antimicrobials is a factor that has limited the treatment and control of the infections caused by these pathogens (^{Pellegrino et al., 2011}), that is why antimicrobial alternatives extracted from plants have been used.

The bacterial resistance generated is due to the increase in the use of antibiotics and the respective selective pressure exerted, has caused bacteria with multiple mechanisms such as biochemical, genetic and cellular, to develop strategies to evade the action of these compounds (^{Cabrera et al. 2007}), for this reason antimicrobial alternatives extracted from plants have been used, some examples are described in Table 2.

Antibiotics have been used in veterinary medicine for three purposes: therapeutic, prophylactic and as growth promoters, the latter being a practice known since 1950, to discover that small doses of tetracycline improved the development of animals and shortly after this discovery were used other drugs such as penicillin and chloramphenicol (^{Puig et al., 2011}), it is also known that veterinary hospitals play a role in the transmission of multiresistant organisms and not only by the continuous administration of antibiotics but also by the exchange of bacteria between people and animals (^{Ríos et al., 2015}).

Resistance is a natural property of an organism (intrinsic) or achieved by mutation or acquisition of plasmids (self-replication, extrachromosomal DNA) or transposons (chromosome integrated into plasmids, transmissible DNA cassettes) (^{Cabrera et al., 2007}). Plasmids and transposons are mobile genetic elements where resistance genes are transported (^{Pérez and Robles, 2013}).

Antibiotic resistance may be the result of chromosomal mutations or the interaction of genetic material through the transport of resistance genes by means of transduction, conjugation or transposition mechanisms. There are 5 mechanisms of acquired resistance, of which the bacteria can use more than two: enzymatic modification or destruction of the antibiotic, impermeability to the antibiotic, alteration or production of new target sites, the presence of efflux pumps that expel the antibiotic and over expression of the target site (^{Cabrera et al., 2007}), although there may also be alterations in the composition and content of bacterial wall glycoproteins (^{Puig et al., 2011}).

Bacteria that are resistant to more than one antibiotic are called multiresistant bacteria, which present an even more serious problem because they have developed different resistance mechanisms (^{Pérez and Robles, 2013}).

Similarly the mechanisms by which the active components of the extracts can inhibit microbial growth are known and already mentioned: inhibition of cell wall synthesis and activation of enzymes that destroy that wall, increased permeability of the cell membrane, interference with protein synthesis and alteration of nucleic acid metabolism among others (^{Ordaz et al., 2010}).

In the case of Staphylococcus aureus, the antimicrobial effect of phenols has been associated with the ability of these compounds to produce alterations in the cytoplasmic membrane of the bacterium (^{Domingo and López Brea, 2003}).

Within the genus of importance in Veterinary Medicine are Staphylococcus, Escherichia, Salmonella, Pseudomona and Bacillus among others. Within the genus Staphylococcus the species of veterinary importance are aureus, which produces great variety of suppurative infections in wounds, mastitis, endometritis, cystitis, osteomyelitis, pyoderma in most domestic species as well as companion animals; epidermidis, which generates mastitis in ruminants; hyicus, species associated with exudative epidermis in pigs, and intermedius, which causes pyoderma, otitis, conjunctivitis, osteomyelitis in dogs (^{Velasco and Yamasaki, 2002}). In addition, this genus presents high morbidity and mortality in domestic and companion animals (^{Ríos et al., 2015}).

Some pathogenic Escherichia serotypes are associated with diarrhea in pigs, cattle, sheep, goats and horses. This genus is considered an opportunistic pathogen in urinary and respiratory tract infections, mastitis, omphalitis and different infectious processes (^{Velasco and Yamasaki, 2002}). The subspecies coli is a pathogen associated with the presence of diarrhea in small ruminants of less than 2 weeks of age, which present fever and diarrhea linked to severe damage in the intestinal epithelium altering the transport of electrolytes and water, causing a rapid dehydration to cause of hypersecretion of liquids, the morbidity rate is between 20 and 25 % and the mortality is usually greater than 50 %. On the other hand, E. coli has also been reported as a causal agent of clinical mastitis in cattle, sheep and goats producing milk, causing considerable losses due to the decrease in milk production, causing anorexia, fever, and even loss of the fourth or middle affected in extreme cases (^{Pugh, 2002}).

For its part, Bacillus anthracis, belonging to the genus Bacillus, causes sudden deaths with haemorrhages in natural holes in cattle and sheep. Some other species cause enteritis in pigs and dogs, mastitis, abortions, food poisoning, corneal ulcers, among others (^{Velasco and Yamasaki, 2002}).

Bacteria belonging to the genus Pseudomonas generate abscesses and purulent infections in different species, bovine mastitis, enteritis, pneumonia and myeloidosis in pigs as wel as in different animal species (^{Velasco and Yamasaki, 2002}).

The serotypes abortus, of the genus Salmonella, cause abortions in bovines, horses and sheep, besides generating diarreas and septicemias in different species of animals; and in the case of S. cholerasuis it generates enteritis, septicemia and pneumonia in pigs (^{Velasco and Yamasaki, 2002}).

Extracts of different species of plants with different solvents have been analyzed, while the identification of the compounds or compound groups present in these plants has been carried out, it has even been reported that these compounds tend to have a greater effect on bacterial genera which present multidrug resistance.

As a result, phenols exert an antimicrobial action against S. aureus, Salmonella thyphimurium, tannins on various bacterial genera and some viruses, flavones on Shigella and Vibrio, alkaloids against Gram positive cocci, Lactobacillus and fungi, the saponins obtained from Panax ginsen have an important effect inhibiting the growth of E. coli and S. aureus (^{Domingo and López Brea, 2003}, ^{Moreno et al., 2010}, ^{Pereira et al., 2013}).

Saponins have an antibacterial activity and can modify ruminal fermentation by suppressing protozoa and selectively inhibiting ruminal and intestinal bacteria (Lactobacillus plantarum and Enterococcus faecium in lambs) (^{Salem et al., 2010}).

In an experiment carried out in 2013 with hexanic extract of cedar, it was observed that this extract had a potent antimicrobial activity against Staphylococcus aureus; with inhibition halos of 12 mm that were responsible for this activity were the alkaloids, triterpenes or sterols, and quinones present in this extract (^{Pereira et al., 2013}).

MS anthelmintic activity

The parasitic diseases caused by gastrointestinal nematodes in ruminants represent a serious problem worldwide as they affect host productivity causing reductions in growth rates in young animals, low body conditions, reduced fertility, increased susceptibility to diseases of different origins, and increased mortality, leading to significant economic losses in livestock production (^{Moreno et al., 2010}, ^{Felice, 2015})

Drug-based anthelmintic treatment is limited due to the development of resistance of some gastrointestinal nematode populations to most commercial anthelmintics (McKellar and Jackson, 2004, ^{Epe and Kaminsky, 2013}).

This problem is common in gastrointestinal nematodes of sheep, goats, horses (^{Gonzalez et al., 2012}) and cattle (^{Encalada et al., 2008}), however this situation has been studied extensively in sheep because of the low effectiveness of the main chemicals such as benzimidazoles, imidazothiazoles and macrocyclic lactones, a study carried out in 2011 confirmed anthelmintic resistance to the main anthelmintics (Closantel, Albendazole, Ivermectin and Nitroxinil) used in the Sierra de Tabasco and North Chiapas region in sheep (^{González et al., 2012}).

One of the alternatives proposed for the control of gastrointestinal nematodes is the use of plants used in traditional herbal medicine with anthelmintic effect. The main compounds of these plants are terpenes, alkaloids, saponins, anthraquinones and tannins. Although, according to the analysis of the compounds in different plants, tannins are involved in vital functions of nematodes affecting mobility, nutrition and possibly reproduction (^{Medina et al., 2014}).

The use of plants rich in bioactive secondary metabolites and especially those containing tannins have received great attention and have been proposed as a method of control of gastrointestinal nematodes in sheep and goats, some examples are shown in Table 3 (^{Durmic and Blache, 2012}, ^{Hernández et al., 2014}).

On the other hand, plants rich in tannins have attracted the greatest attention for their effect on the gastrointestinal nematodes of ruminants. Tannin plants may have direct antiparasitic activity but may also have an indirect effect through enhancing the animals' immune responses against gastrointestinal nematodes (^{Hoste et al., 2005}). There are reports that tannins can improve resilience (fewer clinical signs, better growth and wool production) and resistance (fewer nematode eggs in feces, lower parasite load and lower fertility of parasitic females) of infected goats and sheep with gastrointestinal nematodes (^{Torres-Acosta et al., 2008}). The antiparasitic property of the secondary metabolites depends on the structure of the secondary compound, the level of ingestion and their availability in the gastrointestinal tract of the animals (^{Athanasiadou and Kyriazakis, 2004}).

Apparently the action mechanism of tannins on the infective larvae (L3) of Haemonchus contortus and Trichostrongylus colubriformis is to prevent these parasites from drawing, this prevents the gastrointestinal nematodes can be established in their site of action and can continue with their evolutionary cycle, while in adult parasites the tannins apparently attach to the mouth and possibly to the reproductive apparatus of the parasites, by the affinity of the tannins to the proline-rich proteins of the nematode cuticle (^{Torres-Acosta et al., 2008}).

The nematode Haemonchus contortus has been considered the one with the highest prevalence in small ruminants (^{Encalada et al., 2008}) and one of the main causes of economic losses in sheep production, which is why most of the in vitro studies already performed have been with Haemonchus contortus (^{López-Ruvalcaba et al., 2013}).