Prospects of Proteolytic Enzymes & Bioflavonoids in Management of Tissue Repair & Wound Healing in Animals
Ramesh Jagtap, D J Kalita, Sujit Kumar
Introduction:
In livestock, several infectious diseases like HS, FMD, Black quarter, Lumpy skin disease, PPR, Blue tongue, infectious mastitis, frequently led to huge economic loss to dairy owner. Apart from physical injuries and surgical wounds, these infections are associated with tissue and organ damage, often directly or indirectly by invading pathogens. Though, few pathogens may not directly damage tissue, it is often a natural consequence of their virulence factors or toxins, produced for survival, proliferation in tissues and for transmission to another animal. The tissue and organ damage often manifests as symptoms of disease which can be useful for diagnosis and further for treatment. Tissue damage activates a tissue healing response with acute inflammation followed by either complete repair or replacement fibrosis, depending on regenerative capacity of tissues and extent or type of damage. Repeated or persistent damage often led to continuous activation of repair pathways, consequent chronic inflammation, tissue fibrosis and sclerosis. Many times, indirectly, animals’ own immune response in the form of inflammation or immunopathology can led to tissue damage. Although, several medicines are useful for the treatment of tissue and organ damage, proteolytic enzymes like papain, bromelain or bacterial, fungal proteases and also flavonoids namely rutin have shown to possess many beneficial properties that help in tissue healing process.Proteolytic enzymes that are produced endogenously often as precursor proteins, serve many functions in normal as well as pathological situations. They are involved in the regulation of cell maturation and multiplication; collagen synthesis; and the removal of dead tissues following inflammation. In human medicine, proteolytic enzymes or proteases, from many sources like bacteria, fungi, or bromelain from pineapple plant, papain from papaya plant and flavonoids like rutin from certain fruits and vegetables; evidenced therapeutic properties and demonstrated promising clinical applications. Despite several potential therapeutic and health benefits, till date there are very few scientific publications on the effect of proteolytic enzymes and flavonoids preparations in farm animals. There are other kind of proteases mostly derived from microbes and are used in livestock and poultry feed. These proteases possess broad substrate specificity, can efficiently hydrolyze soluble proteins and thus used for breakdown of various vegetable proteins in livestock and poultry feeds. Scientific community has recognized the diverse therapeutic effects of proteolytic enzymes of plant origin and few phytochemicals like flavonoids used for therapeutic purpose or as nutraceuticals.
Proteolytic Enzymes & Bioflavonoids:
Proteolytic enzymes namely bromelain, papain etc. & bioflavonoids like Rutin, are frequently seen in medical practice due to their wide repertoire of properties and beneficial effects. The key properties of such preparations are strong antioxidant effect, antimicrobial properties and proteolytic activities. These preparations contain various phytochemicals or metabolites, mixture of proteinases or proteolytic enzymes and various bioflavonoids.
The efficacy of these preparations as Neutraceutical or for therapeutic use is well demonstrated in medical practice. Although there are limited evidences in farm animals, cellular effects at protein or DNA level could be similar both in human and animals. Various beneficial properties of few such enzymes, which are well demonstrated in various in-vitro model studies as well as in human subjects are given below.
Bromelain:
Bromelain is a complex mixture of proteolytic enzymes derived from stem and fruit of pineapple plant (Ananas cosmosus) and possesses notable nutraceutical and therapeutic properties. It is used since ancient times in folk medicine and still interest is continued. It contains, proteolytic enzymes, minerals, protease inhibitors, organic acids, colored pigments, and organic solvents. Several proteolytically active components isolated from bromelain, most active being Proteinases, which comprise ~2% of the total proteins. Commercial bromelain is isolated and extracted by various methods. The variation in composition noted with method of purification and the source; from stem with high protease content, compared to bromelain derived from the fruit. Stem bromelain is a mixture of different thiol endopeptidases and also contain metabolites like phosphatase, glucosidase, peroxidase, cellulase, escharase, and several protease inhibitors.
The potential therapeutic value of bromelain attributed mostly to multiple proteolytic fractions; other components or factors also exert potential beneficial effects. In medical practice, bromelain has not only been used to treat various health problems, it is also popular as a nutritional supplement to promote health. Bromelain is absorbed into intestines, and remains biologically active with a half-life of ~6–9 h (Mialovyts’k, 2003).
Important pharmacological properties and potential benefits of Bromelain:
Anti-inflammatory and immuno-modulatory activity:-
It has been proved as potential anti-inflammatory; suppress chronic inflammation in several in-vitro studies, through modulation of several inflammatory mediators including interleukin (IL)-1β, IL-6, interferon (INF)-γ and tumor necrosis factor (TNF)-α. It regulates lymphocyte homing and migration to the sites of inflammation. Bromelain exerts potential immuno-modulatory effect through activation of natural killer cells and enhance production of granulocyte-macrophage-colony stimulating factor, IL-2, IL-6 and decreases the activation of T-helper cells. Bromelain mediates anti-inflammatory action (as shown in Fig.1) by reducing pro-inflammatory prostaglandins (Graf 2000), reduces the level of bradykinin, reduces plasma fibrinogen as it enhances serum fibrinolytic activity, thus reduce distention and permeability of capillaries.
Fig.1 Effect of Bromelain on mediators of acute Inflammation
Antimicrobial activity:
Bromelain supplementation protects animals against diarrhea caused by bacterial enterotoxins from E coli and Vibrio cholerae (Mynott et al., 1997).
It lowers the pathogen attachment to mucosa by modifying the receptor attachment sites and influences the intestinal secretory signaling pathways (Chandler and Mynott et al., 1998).
Its synergistic activity with antibiotics is well proven, as it is reported to increases blood and urine levels of certain antibiotics in human. Bromelain increases bioavailability and reduces the side effects that are associated with various antibiotics. Combined bromelain and antibiotic therapy were shown to be more effective than antibiotics alone in pneumonia, bronchitis, cutaneous Staphylococcus infection, thrombophlebitis, cellulitis, pyelonephritis, and in perirectal and rectal abscesses, sinusitis and urinary tract infections.
Debridement of wounds, acceleration of tissue healing and anti-edematous properties:
In medical practice, there are substantial evidences of its tissue or wound healing potential. It is demonstrated that within 4 days of bromelain oral administration, completely heal the bruises or wounds on the face and associated haematomas. In rat model of traumatically-induced hind leg edema, bromelain has demonstrated significant reduction of the edema.
Bromelain has been used successfully to debride the dead and necrotic tissues from third-degree burn injuries in porcine models. The removal of damaged tissue from wounds or second/third degree burns is termed as debridement. Bromelain can be beneficial for debridement of necrotic tissue and acceleration of healing. Bromelain contains escharase which is nonproteolytic, selectively act on dead and necrotic tissues sparing normal protein substrate or various glycosaminoglycan substrates and thus responsible for this effect. In wound tissues, it also accelerates the recovery of blood perfusion and partial pressure of oxygen.
It notably accelerates surgical wound healing by reducing pain and inflammation if administered before surgery. Bromelain is found to be effective in reducing swelling, bruising, and pain in most post-surgery cases.
Fibrinolytic activity, effect on blood coagulation and anti-hypertensive activities:
Multiple studies also demonstrated the fibrinolytic activity, reduction of thrombus formation and reduction of platelet aggregation. It is demonstrated in in-vitro and in-vivo studies that bromelain is an effective fibrinolytic agent as it stimulates the conversion of plasminogen to plasmin, resulting in increased fibrinolysis by degrading fibrin. Bromelain influences blood coagulation by increasing the serum fibrinolytic ability and by inhibiting the synthesis of fibrin, a protein involved in blood clotting. It notably prolongs prothrombin time and activated partial thromboplastin time when used at higher concentration.
Papain:
Papain, the plant enzyme extracted from papaya catalyzes the breakdown of proteins by hydrolysis. It has been shown to be as effective as aspirin in several studies. It is a natural treatment for wounds after surgical procedures, as well as burns, bedsores and skin ulcers. Papain aids in elimination of fibrin layer of wound or tumors or fibrous tissue or breaking up deadly blood clots and thereby allowing the body access to the softer, more vulnerable tissues. Studies have shown besides as an aid to digestion, papain possesses diverse beneficial properties. It is used as an ingredient in various enzymatic debriding preparations, in the care of some chronic wounds to clean up dead tissue.
Rutin:
Flavonoids are one of the important phytochemicals and possess several beneficial properties. One of the key properties is its antioxidant effect. One such bioflavonoid is Rutin, which is a commonly called as Rutoside or Vitamin P. Chemically, it is a glycoside comprising of flavonolic aglycone quercetin along with disaccharide rutinose. It has demonstrated a number of pharmacological activities, including antioxidant, cytoprotective, vasoprotective, anticarcinogenic, neuroprotective and cardioprotective activities.
Important pharmacological properties and potential benefits of Rutin:
Various effects on Central Nervous system (CNS): Rutin has demonstrated the neuroprotective effect on brain ischemia and helps in reduction of neuro-inflammation. It has shown to be effective in neurodegenerative disorders as it suppresses pro-inflammatory cytokines by reducing TNF-α and IL-1β production in microglia.
Analgesic and Anti-arthritic:
Rutin interacted strongly with cyclooxygenase, forming a number of specific hydrogen bonds and possess both peripheral and central anti-nociceptive activity (Selvaraj et al., 2014). The anti-arthritic properties exhibited by inhibiting both acute and chronic inflammation and found to be very active in the chronic stage of inflammation. Due to its antimicrobial properties, it can be effective in septic arthritis cases (Han, 2009) and also effective in osteoarthritis cases.
Anti-oxidative:
Rutin prevents oxidative damage in endothelial cells, improve endothelial functions by augmenting their NO production (Ugusman et al., 2014).
Anti-ulcer effects:
Exhibits anti-ulcer effects through concentration-dependent inhibition of gastric ATPase or proton pump, which is demonstrated in Goat.
Anti-osteoporotic and anti-osteopenic effect:
Rutin also act as an “osteoblast stimulant’ and thus help in prevention of bone fractures.
Anticataract and ophthalmic effect:
It helps to prevent cataract by inhibition of glycation and thus reduction of formation of advanced glycation end products (AGE), which are associated with cataract.
Diuretic effect:
It exerts diuretic effect thorough its metabolite, Quercetin. It induces nitric oxide release, leading to increasing renal vaso-relaxation by increasing kidney filtration.
Antimicrobial activities:
It showed in-vitro antibacterial activities against E. coli Proteus, Shigella spp. and Klebsiella sp. Pseudomonas, Bacillus spp. and Salmonella spp.
Rutin shows antiviral activity against retroviruses, orthomyxoviruses, herpes viruses, hepatitis B virus and hepatitis C virus. Also showed antifungal activity against certain Candida spp. and can be used in treatment of septic arthritis caused by C. albicans.
Due to its varied properties rutin help to protect various vital organs or shows activities like neuroprotective, hepatoprotective, cardio-protective, spleeno-protective, nephro-protective, protection of lung tissue, etc.
Proteolytic enzymes and bioflavonoids on Veterinary perspective:
Considering the wide repertoire of biochemical, pharmacological properties and well proven benefits in medical practice; the proteolytic enzymes and bioflavonoids, would certainly benefit livestock or farm animals when administered as Neutracutical or as therapeutic agents against specific disease conditions or illness. There are reports of use of proteolytic enzymes and bioflavonoids, with better tolerance and comparable effects with non-steroidal anti-inflammatory drugs (Diclofenac sodium), proving its strong analgesic and anti-inflammatory effects in human patient (Ueberall et al., 2016). Similar effects are also expected in animal patients for the treatment of various inflammatory conditions like pneumonia, arthritis or osteoarthritis, mastitis, etc.
Moreover, wounds are very common in farm animals as animals frequently encounter physical injuries, bruising, muscle soreness, fracture, maggotted wounds and sometime burns. The post-surgical wounds also require proper follow up supportive therapy with analgesics, anti-inflammatory and nutritional supplements for few days, depending on the surgical procedures carried out. The management of such wounds and associated inflammation is imperative as it affects the production and health of the animals. Due to multiple properties like debridement, anti-thrombotic, anti-inflammatory, antioxidant activity, immunomodulatory, etc., these preparations can be very helpful in management of most types of wounds including surgical wounds.
Bioflavonoids, proteolytic enzymes for Mastitis management:
Bovine mastitis, usually caused by microbial infection is one of the costliest and prevalent diseases affecting dairy cows worldwide. Mammary tissue damage reduces the number and activity of epithelial cells and consequently contributes to decreased milk production. The death of mammary tissue induced by either apoptosis or necrosis. Strong immune reactions, microbes and their products contribute to epithelial tissue damage.
Moreover, Mastitis also considered as immunity associated disease due to strong immune response and characterized by strong influx of somatic cells, primarily polymorphonuclear neutrophils (PMN), into the mammary gland. The blood-milk barrier is compromised by migration of more and more immune cells, which worsen the damage to mammary tissues. Meanwhile, the reactive oxygen intermediates produced by PMN can also damage the mammary tissue.
In vitro and in vivo studies suggest that the use of antioxidants and other protective compounds can be beneficial in mastitis control program. Flavonoids like Rutin having strong antioxidant capacity can be helpful in mastitis control program.
The other protective compounds in case of mastitis can be proteolytic enzymes. Due to activities like prevention of apoptosis, anti-inflammatory, analgesic, fibrinolytic activity, debridement of dead and necrotic tissues, bromelain and papain can be beneficial in treatment and control of mastitis.
At cell and molecular level, nuclear factor- κB (NF-κB) signaling has critical role in immunity and inflammation. The NF-κB has an essential role in many biological processes, such as cell survival, immune response, inflammation and development. It further, regulates the transcriptional expression of specific inflammatory mediators to initiate inflammation of the mammary epithelial cells.
In the pathogenesis of mastitis of bacterial etiology (E. Coli, Staphylococcus spp.), lipopolysaccharides (LPS) of bacteria, bind to Toll-like receptors (TLRs) on mammary epithelial cells and elicits NF-κB signaling pathway to accelerate its pathogenesis. There are several reports show that the blocking of NF-κB signaling could be a useful strategy for mastitis control. It is well demonstrated that bromelain inhibit expression or block activation of NF-κB, thus prevents further signaling pathway.
Further, it is well proven that the preparations containing antioxidants, especially flavonoids and also the proteolytic enzymes inhibit or reduce the expression of various inflammatory mediators like INF-γ and TNF-α, also activates natural killer cells and augments the production of granulocyte-macrophage-colony stimulating factor, IL-2, IL-6 and decreases the activation of T-helper cells in several inflammatory conditions. Proteolytic enzymes, as bromelain affect expression of Transforming growth factor (TGF)-β, one of the major regulators of inflammation in patients affected by osteomyelofibrosis and rheumatoid arthritis. Thus, these preparations decrease majority of inflammatory mediators and has demonstrated a significant role as an anti‑inflammatory agent in various conditions. Oral administration of such proteolytic enzymes and flavonoids, can be helpful for faster recovery from mastitis.
Conclusion:
Farm animals often suffer tissue and organ damage, whether due to physical trauma, surgical procedures or due to many infections directly or indirectly. Cell damage activates healing response, initiated mostly as acute inflammation. The preparations containing Proteolytic enzymes & bioflavonoids possess wide repertoire of pharmacological properties namely analgesic, anti-inflammatory, anti-microbial, anti-thrombotic or fibrinolytic, debridement of necrotic tissues, anti-arthritic, anti-oxidant, etc. Due to their many beneficial properties and proven therapeutic benefits, these preparations certainly can be beneficial in the management of acute and chronic inflammation, pain, associated with tissue or organ damage and thus for acceleration of tissue healing process, in farm animals, though there are limited published reports of use in farm animals. Mastitis is highly prevalent disease of dairy animals worldwide with severe economic impact leading to high loss to dairy farmers. As like many infectious diseases, severe cell and tissue damage of mammary gland tissues occur in mastitis due to pathogen invasion, multiplication and further due to immunopathology. Owing to many pharmacological benefits, proteolytic enzymes & bioflavonoids preparations would certainly be helpful as a supportive therapy in controlling inflammation, discomfort and thus for faster recovery from mastitis.
- Note: References can be made available on request
