INTRODUCTION

Currently, there is growing public concern about the treatment of captive and domesticated animals, including wild, laboratory, farm, work, zoo and companion animals (^{Webb et al., 2019}). Animal welfare is a complex and multifaceted issue with scientific, ethical, social, religious, cultural, political and economic dimensions. According to the American College of Animal Welfare: “Animal welfare refers to the state of the animal. Assessment of welfare includes consideration of the animal’s health, behavior, and biological function;” therefore, anything that contributes to or demerits the normal state of an animal affects its well-being (^{Castle et al., 2016}). On the other hand, health refers to the state of the body's systems that combat pathogens, tissue damage or physiological disorders; in other words, it is the state of an animal in relation to its attempts to confront pathology. As such, it is an important component of animal welfare (^{Broom, 2011}).

The inflammatory process is a common syndrome to numerous pathological situations of mammals. It results in the local or systemic response of the organism against harmful external stimuli of a microbial, chemical or physical type. The purpose being to eliminate the causative agent, as well as, repair damaged tissue and maintain homeostasis. This process can be divided into two phases: acute, characterized by local vasodilation and increased capillary permeability; and chronic, characterized by a greater immune response, tissue degeneration and fibrosis (^{Kumawat et al., 2012}). During the inflammatory response, pro and anti-inflammatory mediators are synthesized and secreted. These are substances that have a direct effect on inflammatory cells and blood vessels or that intervene in reactions that generate compounds that act on these cells. Among the inflammatory mediators and cell pathways are the cytokines (e.g., interferons, interleukins and tumor necrosis factor α), chemokines (e.g., monocyte chemo-attractant protein 1), eicosanoids (e.g., prostaglandins and leukotrienes) and the potent inflammation-modulating transcription factor nuclear factor κB (^{Azab et al., 2016}). Most of these molecules are derived from the phospholipid components of cell membranes, which are released as a result of their destruction (^{Kumawat et al., 2012}).

Throughout the millennia, medicinal plants have been used for the care of human and animal health, because they have a great diversity of secondary metabolites such as terpenes, alkaloids and flavonoids that have several biological properties (^{Mayer et al., 2014}; ^{Miara et al., 2019}; ^{Starlin et al., 2019}). In addition, the preference for the use of medicinal plants or phytopharmaceuticals obtained from them, continues to increase because of they lack side effects or these are minimal (^{Mayer et al., 2014}; ^{Miara et al., 2014}; ^{Starlin et al., 2019}; ^{Laudato & Capasso, 2013}).

The genus Salix, belonging to the Salicaceae family, has been used since ancient times for the treatment of fever, pain and inflammation. For these purposes, the bark of Salix alba and Salix nigra; flowers of Salix caprea and aerial parts of Salix canariensis, demonstrate a counter inflammatory response (^{Drummond et al., 2013}; ^{Gutiérrez et al., 2017}; ^{Sharma et al., 2011}; ^{Gyawal et al., 2013}; ^{Ahmed et al., 2011}).

Salix babylonica, commonly known as the weeping willow tree, is the most acknowledged species of the willows, distributed in some areas of Asia, Europe, and America. It has been used as an ornamental and medicinal plant (^{González-Alamilla et al., 2019}). In traditional medicine, the intake of an infusion of weeping willow leaves allows pain relief, whether rheumatic, muscular, head, ears, or toothache, among others (^{Waizel-Bucay, 2011}). Thus, the objective of the present work was to evaluate the anti-inflammatory activity in-vivo of a hydro-alcoholic extract of Salix babylonica leaves and identify the compounds responsible for this activity.

MATERIALS AND METHODS

RESULTS

Histological analysis

Topical application of TPA for 6 hours, induced an acute inflammatory response at the level of the dermis of the mice ear and a marked increase in the neutrophil population was noted (Figure 1). No changes in the epidermis were observed. Furthermore, when only acetone (TPA vehicle) was applied, this reaction was not observed, but rather sustained the normal appearance of the dermis particularly among the collagen fibers and fibrocytes/fibroblasts. Similarly, the application of indomethacin, hydroalcoholic extract of Salix babylonica (HESB) and ethyl acetate fraction (EAFSB) obtained from HESB, inhibited an acute inflammatory response, evidenced by the poor population of inflammatory cells, particularly neutrophils, associated with the dermis of the skin of the ear (Figure 2).

Figure 1 Mouse ear skin. H-E. 100x (a) and 400x (b). Effect of TPA, without the addition of antiinflammatory. There is a marked increase in neutrophils in the dermis (arrows, representative of at least 40 cells in the field)  

Figure 2 Mouse ear skin. H-E. 400x. Application of the organic fraction of the hydroalcoholic extract of S. babylonica. As can be seen, the presence of inflammatory cells in the dermis is minimal (arrow) 

DISCUSSION

According to the World Organization for Animal Health (OIE) Terrestrial Code, animal welfare means the physical and mental state of an animal in relation to the conditions in which it lives and dies. The guiding principles which inform the OIE’s work on the welfare of terrestrial animals include the ‘Five Freedoms’; which describe society’s expectations for the conditions animals should experience when under human control, namely: freedom from hunger, malnutrition and thirst; freedom from fear and distress; freedom from heat stress or physical discomfort; freedom from pain, injury and disease; and freedom to express normal patterns of behavior (^{OIE, 2019}).

Inflammation, an organism’s response to irritation or injury, is characterized by redness, heat, swelling and pain. Non-steroidal anti-inflammatory drugs (NSAIDs) are used for the treatment of inflammatory diseases. These compounds affect the function or production of prostaglandins. In veterinary medicine, NSAIDs are used to control, among other ailments, pain and inflammation associated with osteoarthritis in dogs and horses; as well as, for postoperative pain in dogs and cats. However, there are potential risks associated with the use of NSAIDs such as gastrointestinal ulcers/perforations and liver and kidney toxicity (^{Mathews et al., 2014}). Therefore, the search of new compounds with a counter inflammatory reaction, without the side effects of NSAIDs, is of vital importance for the maintenance of animal welfare. The use of medicinal plants is an alternative for the treatment of pain and inflammation in animals.

Plants have been proven very rich sources of structurally and biologically interesting metabolites. Less than 10% of the world’s biodiversity has been evaluated for potential biological activity, many more useful natural leader compounds await discovery (^{Gyawali et al., 2013}). In addition, the world-wide interest in herbal products has grown significantly. Cattle, horses, sheep, goats and pigs represent about 70% of the animals treated with herbal remedies, followed by poultry (9.1%), dogs (5.3%) and rabbits (4.3%) (^{Laudato & Capasso, 2013}).

Regarding the use of plants for the treatment of the inflammatory process in animals, Geranium robertianum, Artemisia abrotanum, Brassica oleracea, Avena sativa, Anagallis arvensis, Linum usitatissimum, Scrophularia canina, Hypericum perforatum, Achillea millefolium and Buxus sempervirus are used to treat or prevent mastitis in cattle because of their anti-inflammatory effect (^{Mayer et al., 2014}; ^{Laudato & Capasso, 2013}). What’s more, Aloe spp. has been used for treatment of inflammation, pains and itching in several animals; while dandelion and linseed have been used for perianal gland inflammation treatment in dogs (^{Laudato & Capasso, 2013}). Rosa canina, Matricaria chamomilla, Glycyrrhiza glabra, Aegle marmelos, Asparagus racemosus Wild, Argemone mexicana Linn, Datura metal Linn, Eclipta prostrata Linn, Moringa oleifera Lamk, Syzygium cumini, Allium cepa, Nerium oleander L, Anayclus davatus, Mentha pulegium L, Lawsonia inemis L, Olea europaea L, Ziziphus lotus L, Ruta graveolens and Ziziphus jujuba Linn, are used for the treatment of different inflammatory diseases in sheep, cattle, goats, rabbits and poultry (^{Miara et al., 2019}; ^{Laudato & Capasso, 2013}; ^{Verma, 2014}).

The Salix genus has been used in the treatment of arthritis; menstrual, dental and back pain; fevers and inflammation. Willow trees belong to the Salicaceae family and records of its medicinal use go back about 6,000 years (^{Drummond et al., 2013}; ^{Gutiérrez et al., 2017}; ^{Gyawali et al., 2013}). Salix babylonica has demonstrated antihelmintic, antiseptic, analgesic, antipyretic, antimalarial, antioxidant, anticarcinogenic, astringent, antifungal and antibacterial activity (^{González-Alamilla et al., 2019}; ^{Abdel Wahab et al., 2018}). In this work, the anti-inflammatory activity of the hydroalcoholic extract of S. babylonica leaves was evaluated and we found that it inhibited inflammation by 66.92 ± 3.20%, without significant statistical differences with respect to the reference drug, indomethacin (table 1).

There are some reports regarding the anti-inflammatory effect of extracts obtained from other species of Salix; for example, ^{Gyawali et al. (2013)}, evaluated the anti-inflammatory capacity of an methanolic extract from Salix alba bark using the formalin induced paw oedema model in rats, and found an inhibition of inflammation of 74% at a dose of 93.5 mg/Kg. Likewise, an aqueous extract from aerial parts of S. canarensis, obtained by infusion, at a dose of 105 mg/Kg, caused an 78% inhibition of the inflammation in the same model (^{Gutiérrez et al., 2017}). On the other hand, ^{Ahmed et al., (2011)} evaluated the anti-inflammatory effects of methanolic and hydroalcoholic extracts of flowers of S. caprea at 400 µg/mL, using the Human Red Blood Cell (HRCB) membrane stabilization method, and found that this extract showed 66.78 and 60.49 % of membrane protection, respectively. However, these results cannot be directly compared with that obtained in this work for the hydroalcoholic extract of S. babylonica (HESB), because the extracts evaluated are of different polarity, obtained by different methods and evaluated in different pharmacological models.

In order to identify the chemical nature of the active compounds in the hydroalcoholic extract from S. babylonica leaves, a bipartition procedure with water an ethyl acetate solvents was performed. The aqueous fraction from SBHE (AFSB) and organic fraction from SBHE (EAFSB) were evaluated in their capacity to inhibit inflammation in-vivo. As shown in table 1, EAFSB (66.92 ± 3.20 % inhibition of inflammation (% II)) was most active than AFSB (30.64 ± 3.03 %II), without significant statistical difference with respect to the extract SBHE, and the effective dose was three times lower. In addition, EAFSB had no significant statistical differences with the reference drug Indomethacin. These results suggested to us that the anti-inflammatory compounds from Salix babylonica were in the organic fraction.

Previously, we identified the flavones luteolin (Figure 3a) and luteoloside (luteolin 7-Oglucoside) (Figure 3b) as the major components of ethyl acetate fraction obtained by bipartition from hydroalcoholic extract of Salix babylonica leaves (^{González-Alamilla et al., 2019}).

Figure 3 Chemical structure of flavonoids identified as major components in EAFSB. (a) luteolin (3′,4′,5,7-tetrahydroxyflavone) and (b) luteoloside (3′,4′,5,7tetrahydroxyflavone-7-O-glucoside)  

Luteolin is a 3’,4’,5,7-tetrahydroxyflavone, a kind of flavonoid with a double bond between C2 and C3, and a carboxyl group in position 4 of ring C. Luteolin has been isolated from vegetables, fruits and medicinal plants. There are reports that evidenced its antioxidant, antimicrobial, antidiabetic, chemoprotective, chemotherapeutic, neuroprotective, antiallergic and anti-inflammatory activity. Additionally, this molecule is considered non-toxic (^{González-Alamilla et al., 2019}; ^{Aziz et al., 2018}; ^{Zhang et al., 2018}). Regarding antiinflammatory responses, luteolin suppresses pro-inflammatory cytokines expression including interleukin (IL)-6, IL-1β, IL-2, IL-8, IL-12, IL17, TNF-α, interferon (INF)-β, granulocyte-macrophage colony-stimulating factor and increase the level of antiinflammatory cytokine IL-10. It also inhibits the induction of nitric oxygen synthase (iNOS) and it’s active expression, as well as the production of NO, ROS and chemokines. These aid in controlling the migration and positioning of immunity cells, such as chemokine (CX-C motif) ligand 2 (CXCL2), chemokine (C-Cmotif) ligand 2 (CCL2), CXCL9 and CXCL8 (IL-8). Further, it also inhibits the production and release of eicosanoids, prostaglandins, and leukotrienes, as well as, pro-inflammatory adhesion molecules such as monocyte chemo-attractant protein-1 (MCP-1), intercellular adhesion molecule-1 (ICAM), and vascular cell adhesion molecular-1 (VCAM). Similarly, it prevents the NF-B pathway, protein kinase B (AKT), and the mitogen-activated protein kinase (MAPK) pathway; hyaluronidase and elastase activity; stabilization of mast cell; reduction of vascular permeability; modulation of cell membrane fluidity, active antioxidants enzymes and prevents adenosine triphosphate (ATP) from binding to spleen tyrosine kinase (Syk) or proto-oncogene tyrosine-protein kinase (^{Aziz et al., 2018}; ^{Zhang et al., 2018}; ^{Li et al., 2019}). Moreover, luteolin 7-O-glucoside (luteoloside) possesses anti-inflammatory activity too, this flavonoid inhibits the release of β-hexosaminidase (a marker of degranulation in mast cells), leukotriene C4 (LTC4), NO and PGE2 production; as well as, COX-2 and iNOS enzymatic activity. Furthermore, luteoloside is a potent inhibitor of JNK3, a protein kinase of the MAPK family that is potently activated by a variety of environmental stress and pro-inflammatory cytokines (^{Aziz et al., 2018}).

On the other hand, topical application of TPA in mice ears produced oedema with its typical symptoms: swollen, increased thickness and infiltration of pro-inflammatory cells such as neutrophils, leukocytes and lymphocytes. These cells are the key components that infiltrate the site of inflammation first and promote host defense functions and are aggregated and bonded to the walls of blood vessels on the range of 4 to 6 h after application of TPA (^{Hernandez-Valle et al., 2014}; ^{Mendes et al., 2016}; ^{Silva et al., 2017}). In the present study, samples for histological analysis were collected 6 h after stimulating with TPA, as shown in figure 1. TPA generated oedema, extravasation of fluids and increase of neutrophils in the dermis. When Indomethacin, a steroidal anti-inflammatory drug widely used as the validation for the pharmacological mode, was applied, local inflammation decreased; a similar result was observed in animals receiving treatment with hydroalcoholic extract SBHE or EAFSB (fig 2). In other words, SBHE and EAFSB induced the same response compared to the control drug. In conclusion, the histological analysis of H-E-stained mouse ears show that Salix babylonica suppresses the cells infiltration into the inflammation site induced by TPA.

CONCLUSIONS

The hydroalcoholic extract SBHE from leaves of Salix babylonica and its fractions AFSB and EAFSB display anti-inflammatory activity, EAFSB being the most active fraction. Previously, we reported that the chemical analysis of EAFSB fraction by HPLC revealed the presence of flavonoids luteolin and luteoloside (luteolin-7-O-glucoside) as major compounds. As a consequence, the anti-inflammatory effect shown by S. babylonica leaves could be attributed to the presence of those anti-inflammatory compounds. The histological analysis of the ears treated with S. babylonica suggest that these downregulate the migration of pathogenic neutrophils into the site of inflammation decreasing the production of inflammatory cytokines. Finally, our results show that S. babylonica are able to decrease inflammation and could serve as a valuable alternative for the treatment of inflammatory processes in animals.

Conflicts of Interest: The authors declare no conflict of interest.