Haruan Extract ( Channa striatus ) as an Effective Mediator in Promoting Wound Healing

Wound healing remains a major issue in surgery. None of the existing treatment modalities in caring for wounds can yet claim to be the holy grail of wound management. Channa striatus , locally known in Malaysia as Haruan , is a freshwater air-breathing carnivorous fish that is proven to influence the different phases of wound healing. As a medicinal fish, not only does Haruan have a high content of amino and fatty acids, which are essential in collagen fibre synthesis during wound recovery, it also abounds in arachidonic acid and polyunsaturated fatty acids that promote prostaglandin synthesis, a vital component of the healing process. Moreover, its antinociceptive effects could potentially reduce wound pain, an important factor in wound healing. Proteomic studies show that a quarter of the total protein detected in freeze- and spray-dried C. striatus extract are actin, myosin and tropomyosin – all molecules that play a role in the wound healing process. Proteomic profiling also reveals that Haruan possesses two types of collagen namely collagen type-I and type-II that confer tensile strength during the healing process. It is proven that collagen along with other components of the extracellular matrix form the granulation tissue which, when contracted, closes the wound and concomitantly aligns the collagen fibres in the extracellular matrix. Hence, it is inferred that Haruan promotes the maturation of granulation tissue, thereby expediting the wound healing process itself. Consequently, it could mediate a faster recovery from surgical wound coupled with a lower incidence of wound infection due to an improved and accelerated wound healing process. Additionally, Haruan has demonstrated its ability in promoting angiogenesis and cell proliferation in wound bed preparation for skin grafting. Furthermore, a Haruan aerosol concentrate can act as a wound dressing at the donor site thereby enhancing the healing process while simultane-ously exhibiting some antinociceptive properties. Haruan ’s exceptional ability in promoting wound healing together with its potential use in skin grafting would be instrumental in the field of surgery. In essence, the cumulated benefits from all the processes involved would translate into a significant reduction of hospitalisation cost; that would immensely benefit not only the patient, but also the government.


Introduction
A wound is a mechanical injury to the body leading to disruption of the normal anatomical structure and function. It can be classified into acute and chronic wounds. Acute wounds normally proceed through the reparative process in an orderly and timely manner to restore anatomical and functional integrity. Conversely, wounds that demonstrate signs of delayed and interrupted healing and fail to go through the normal healing process are termed chronic wounds [1][2][3].
Wound healing reflects a cascade of complex, highly regulated biological events to restore the body's anatomical function back to its pre-injured state. Unlike acute wounds that heal by primary intention where the edges of the wound are apposed and held together with minimal scarring, chronic wounds heal by secondary intention [4,5]; they form granulation tissue which fills the wound defects.

History of wound care and wound dressing
Wound management involves providing an optimum environment to promote healing, control bleeding and prevent infection. The history of wound care traces its origin to the Sumerians, a civilisation believed to be older than 2,000 BC [6]. In their manuscript, three healing gestures -cleansing the wounds, making the plasters and bandaging the wounds -were identified [7].
The ancient Egyptians and Greeks also contributed to the evolution of wound management. The Egyptian medical papyri documented the principle of wound closure to aid healing and the utilisation of honey, grease and lint as the main constituents of the most common plaster. It was believed that lint, a derivative of vegetable fibre, serves an absorbent role; grease or animal fat forms a barrier against bacteria; and honey, the most frequently cited ingredient in multiple topical wound preparations, possesses various healing and antibacterial properties favourable for wound healing [8,9].
Interestingly, the Greeks were the first to recognise the difference between infected and uninfected wounds, using terms such as "fresh" or "non-healing" to describe wounds [10,11]. Galen of Pergamum (120-201 AD) is a Greek surgeon who made remarkable contributions to wound and haemorrhage management. He emphasised the maintenance of wound moisture and the application of styptics consisting of basic elements with antibiotic properties for optimum wound healing [12,13]. Despite advances in modern technology, Galen's basic principles are still incorporated into the development of current wound dressings.
Additionally, the Hippocratic collection discussed the addition of wine to obstinate ulcer for maximal wound healing [13]. Indeed, in ancient times, a number of magical and mythological agents were utilised as wound dressings; they include honey, plaster, wine and milk. While some of them demonstrate significant pharmacological roles, others merely have ritualistic meanings [13].
In the modern era, a wide array of dressings and wound care products with their properties tailored to special wound care needs were invented. In fact, Winter's study [10][11][12][13], which concluded that moisturised wounds heal quicker than dry wounds, sparked an explosive burst in the evolution of wound

Stages of wound healing
The phases of wound healing is a continuum that encompasses homeostasis, inflammatory, proliferative and maturation phases under stringent regulation of growth factors, cytokines, and chemokines [38]. Admittedly, the various phases of the wound healing process can overlap and go awry anytime. The inflammatory phase is the shortest of all phases and, if arrested, wound healing will be delayed and fibrosed tissue may be formed. Meanwhile, in the proliferative phase, the wound is shrunken in size until the maturation phase. Despite the surface of the wounds being closed completely, full tensile strength might take up to twelve months to develop [3,4,[38][39][40]. Table 1 below describes the different stages, mechanisms and molecules at interplay during the wound healing process.
Unfortunately, despite the enormous efforts made in skin repair, a wound can never achieve the maximum tensile strength of a normal skin. Additionally, owing to its tight regulation by a multitude of factors, proper wound healing can be easily impeded. Indeed, chronic non-healing wounds are a common phenomenon.   sides, and mottled with a combination of black and ochre; it also has a white belly, a large head resembling a snake and a fully-toothed mouth with large scales. It preys on smaller fishes and frogs for survival. Since C. striatus is an obligate air-breather, it survives by burrowing in muds of lakes, rivers or canals, keeping its skin and air-breathing apparatus moist while surviving on stored fats [42,43]. Wild Haruan lives a solitary life except in the spawning season. Figure 2 shows a solitary Haruan in an aquarium with a glass reflection showing its white belly.
Pairs breed during most months of the year, laying hundreds of ambercoloured eggs. The eggs, guarded by both parents, are non-adhesive and they hatch within one to three days. The adults have compact muscles and a less bony structure which give them the desirable characteristics of a predatory fish [43]. Besides, they are highly aggressive predators with the ability to travel overland to exploit new bodies of water [42].    Haruan fish can be farmed though it is considered a pest or predatory species in Europe, North America and Australia, being a voracious predator and a competitor of native fish species [44,45]. When it is reared in a controlled habitat, the parameters of the aquatic environment such as the pH, temperature and water depth should be kept as close to its natural habitat as possible. For its diet, Haruan fish can be fed with a wide range of food products including formulated food [46][47][48].
However, compared to other species such as Keli or Tilapia fish, Haruan fish farming is not popular for two reasons: firstly, being a predator, it can easily eat up the surrounding aquatic animals and small terrestrial rodents; secondly, its commercial benefits have not been extensively publicised to receive enough attention [49,50].
In Malaysia, Haruan fish is cherished as a wholesome delicacy; it is served in a multitude of preparations ranging from steamed, grilled, spiced, fried, roasted, in the form of soup to even raw [51]. According to a study [52] conducted by Haemamalar and his team in Krau Wildlife Reserve, Haruan fish was reported to be one of the sources of freshwater fish among the Orang Asli (aboriginal people) tribunates.
Additionally, Haruan serves as a natural remedy for the local population. The National Health and Morbidity Survey carried out in 2014 [53], which looked at the prevalence of food supplements and the reasons for their intake, demonstrated that of the 0.68% of Malaysians consuming Haruan as a dietary supplement, 90.82% did so based on its alleged health benefits.
Thanks to the Chinese and Malay communities, Haruan has acquired a reputation for wound healing for the past several decades [54]. Poh et al did a research [55] involving a total of 134 Chinese mothers during the months of childbirth; they found out that Haruan fish was reported by a quarter of the participating women as either a necessary or a recommended food owing to its wound healing property [56]. Nonetheless, the wound healing effect of Haruan was merely anecdotal until two recently published clinical trials [57, 58] scientifically confirmed this common belief.

Cooking
Different cooking methods of Haruan fish can generate different outcomes. For instance, Haruan fish fillets preserve their nutritional value when grilled but absorb too much oil when fried, which can be detrimental to health [59]. Meanwhile, when prepared in soup, the time and heat utilised have to be properly adjusted for the snakehead fish to retain its nutritional value [60].

Topical agent
Haruan fish can also be converted into a topical agent in the form of spray or cream. This preparation involves the addition of a propellant (spray) or aqua cream (cream) to the Haruan extract [61, 62]. When Haruan is formulated into aerosol concentrate and sprayed on a wound, it will form a thin layer of dressing that acts as a protective barrier against the outside environment [63]. This minimises the physical pain as well as the mental suffering associated with dressing application and removal [64].

Haruan capsules
The principal author of this chapter worked collaboratively with the School of Pharmacy, Universiti Sains Malaysia, to process Haruan capsules for his research work together with fellow surgeons at the National Heart Institute, Kuala Lumpur, several years ago. Admittedly, oral Haruan supplement has a higher amount of concentrate which is believed to yield more merits compared to eating the flesh itself. Besides, for surgical or major traumatic wounds that involve multiple tissue layers, oral administration of Haruan extract is deemed superior to topical application [65,66]. The detailed steps in the preparation of Haruan capsules are described in

Antinociceptive properties
Pain can have a deleterious impact on wound healing [108, 109]. Coupled with chronic inflammation, prolonged pain can trigger a vicious cycle that hinders wound healing [110]. Fortunately, appropriate wound dressings with sufficient pain control can enormously improve wound healing outcomes, with accelerated wound healing and, consequently, a shorter hospital stay [111].
The antinociceptive effects of Haruan in postoperative and traumatic patients have long been discussed and reported. For instance, an earlier Haruan study [112] conducted abdominal constriction and tail flick test on mice and found that Haruan not only possesses peripherally-acting antinociceptive activity, but  its extracts could also act synergistically with other painkillers such as morphine to relieve postoperative pain and discomfort. These findings are supported by another study conducted by Solihah et al [113] who presented a similar positive result. The underlying mechanism is thought to be attributed to the presence of fatty acids and amino acids, particularly arginine, glycine and arachidonic acid, in addition to the involvement of the L-arginine-nitric oxide-cGMP pathway [42,114]. In fact, the antinociceptive effects Haruan remain relatively stable in a wide range of temperature and pH; this allows the essence to be extracted and processed safely for future use [115].

Wound healing capabilities
Freshwater fish constitute 60-70% of the animal protein intake in Malaysia [116]. Previous studies demonstrated that Haruan contains a high content of albumin which promotes the formation of collagen [117][118][119]. Moreover, it has a considerable amount of copper and zinc that can help accelerate wound healing by maintaining cell stability, besides promoting wound remodelling and the formation of blood vessels and fibrosis or scar [119][120][121][122][123][124]. Additionally, the presence of elements such as hydroxyproline, glycine, arachidonic acid and arginine in Haruan is another essential source of collagen [124][125][126].
It is therefore no surprise that the collagen content in Haruan is relatively high. Indeed, collagen plays a determining role in expediting wound healing via various mechanisms. According to Kwan et al who performed proteometric profiling of Haruan, two types of collagen, namely type I and type II collagen, were detected [127,128]. Both of them increase the tensile strength [129]. In fact, when the increased tensile strength and glycosaminoglycan of Haruan was examined and compared to cetrimide, it was reported that Haruan is superior to cetrimide in improving wound contraction and the fibroblastic phase of wound healing [61, 130,131]. Furthermore, it was found that the collagen in Haruan can help promote the maturation of granulation tissue which accelerates wound healing. Haruan can act in synchrony with the other components of the extracellular matrix to form a granulation tissue which subsequently contracts and seals the wound. Concurrently, it aligns the collagen fibres in the extracellular matrix. If the area of involvement is wide enough, the granulation tissue could be reserved for split skin grafting. The various steps of collagen involvement in wound healing are described in Figure 6. Wound healing effects of collagen [132][133][134][135].
According to the researchers [128] who performed proteomic profiling of Haruan extract at the Analytical Biochemistry Research Centre, Universiti Sains Malaysia, the proportion of actin, myosin and tropomyosin to the total protein in freeze-dried and spray-dried Haruan water extract are 25% and 26% respectively. While it is known that these three structures play a significant role in muscle contraction in the sliding filament theory, many were unaware that that they also work hand-in-hand in the wound healing process. Tropomyosin can help control cell functioning of actin while actin can regulate vital cellular functions during re-epithelisation involving cell division, cytokinesis and cell signalling, seal the embryonic wound as well as interact with myosin in the regulation of cell motility [132][133][134]. The functions of actin, myosin, and tropomyosin during the wound healing process are diagrammatically summarised in Figure 7.

Clinical trials on Haruan
To the best of our knowledge, only two clinical trials have reported the effects of Haruan in wound healing to date.
One study [57] was conducted by the principal author of this chapter. The researchers performed a double-blinded, randomised, controlled trial in 2018 at the National Heart Institute to look at the effects of Haruan fish extracts on the chest and leg surgical wounds of 253 patients after they have undergone coronary artery bypass surgery (CABG). Noting the detrimental impact of wound pain on wound healing and the interplay between wound pain, morbidity, quality of life and hospital stay [135][136][137], parameters such as wound pain and healing, mobilisation and quality of life were specifically evaluated. It was found that the wound scoring system favoured those patients who received Haruan capsules instead of placebo at day-6, six-weeks and three months postoperatively. They also discovered that Haruan extracts could alleviate wound pain, and improve quality of life with respect to energy, pain, emotion, sleep and physical level except social level based on the Nottingham Health Profile questionnaire that assesses the quality of life [57]. Therefore, Haruan extract is considered a cost-effective solution in wound healing because it decreases the percentage of wound infection thereby reducing the cost of hospitalisation. Moreover, with the patient's surgical recovery hastened, it can tremendously reduce the economic burden not only to the hospital but to the country as well.   [55,56]. The study concluded that Haruan could improve the wound's cosmetic appearance and, accordingly, patient's satisfaction. These findings are in line with a randomised controlled trial [138] which reported that Haruan extract consumers harboured a remarkably higher rate of uterine involution than the placebo group. Conversely, in the same study, the outcomes of wound healing and pain were noted to be comparable; this can be attributed to the interval used for the pain score assessment and the concomitant use of analgesics.

Potential role of Haruan in skin grafting
Skin grafting is the transfer of cutaneous tissue to cover large wounds. It can take two forms: split skin grafting, which involves the epidermis and a portion of the dermis, and full-thickness skin grafting which involves the epidermis and the entire dermis [139]. While deliberating on the pros and cons of split skin grafting as compared to full thickness skin grafting is beyond the scope of this paper, it is noteworthy that a split skin graft does not have its own blood supply; it relies on the wound bed. This is where Haruan might play an important role. In their seminal work on the bioactive proteins in C. striatus, Kwan et al [140] have shown that the fish proteins promote angiogenesis and cell proliferation. The stable, healthy and well-vascularised wound bed potentiated by Haruan action allows for skin grafts to be well taken.
The main challenge to ensure that Haruan play its magical role in promoting angiogenesis and cell proliferation lies in the wound bed preparation before skin grafting. To ensure that the wound bed is healthy, wound debridement is of utmost importance. This could be done in several ways: via a scalpel, a dermatome, or even by using a hydro-surgery device until the wound bed is really clean and healthy with some bleeding at the wound bed [141]. It has to be stressed here that without a clean and healthy wound bed, and wound edges cleared from any necrotic or purulent tissue, the added value of Haruan would be lost and the skin graft will not have a proper healing.
When the wound bed is well prepared, skin grafts will normally go through three different stages as follows: a. Imbibition: Oxygen and nutrients from the wound bed are passively absorbed by the skin graft [142].
b. Inosculation: A vascular connection is established between the cut vessels on the underside of the skin graft and the wound bed [143].
c. Revascularisation: Neoangiogenesis or new blood vessels grow into the graft from the wound bed [144].
After undergoing these stages, the skin grafts will usually need another five to seven days to adhere to the wound bed followed by the process of maturation that could last from several months to years; this includes pigmentation changes, softening and flattening [139]. As described earlier, Haruan abounds in amino acids in such as glycine, lysine and arginine, and fatty acids such as arachidonic acid, palmitic acid and docosahexaenoic acid -they all help to enhance wound healing through the initiation of several pathways including the remodelling of collagen that gives the strength to the wound, besides stimulating wound contraction [145].
Having discussed at length regarding the recipient site, we should also look at whether Haruan plays any role in wound healing at the donor site. Theoretically, the donor site requires wound care only in terms of wound dressing. Since the wound edges are not approximated at the donor site and are left to heal via secondary intention, they will be filled by granulation tissue matrix [146] and can be covered by a simple dressing only. Presumably this is another area in skin grafting where Haruan dressing might play a role. An aerosol concentrate containing Haruan water extract was formulated in an aerosol system to produce a thin film over the wound bed and serve as a dressing at the donor site [147]. The aerosol concentrate that would form a thin layer of dressing over the wound could enhance the healing process at the donor site as proven in an animal model [148], besides showing pronounced antinociceptive properties [149].

Other usages of Haruan
Apart from its aforementioned desirable features, Haruan has also been reported to confer feasible outcomes in a myriad of diseases.
Osteoarthritis is a degenerative joint disease characterised by synovial inflammation and articular cartilage degradation that leads to chronic pain and inflammation [150,151]. In osteoarthritis, a wide variety of inflammatory mediators are secreted and activated [140]. After several previous studies which demonstrated the antiinflammatory capabilities of Haruan, the role of Haruan in osteoarthritis has been extensively explored [152][153][154]. Few scientific reports on the efficacy of Haruan in osteoarthritic patients revealed promising outcomes where Haruan was shown to be superior in reducing inflammatory changes in the synovial membrane, improving the pain, symptoms and quality of life of osteoarthritic sufferers while maintaining the structure of the cartilage of the control group [155][156][157][158][159]. As osteoarthritis is a common complication of major traumatic wound injury, which necessitates skin grafting owing to the disfigurement and disabling condition, oral administration of Haruan can exert a double action, improving both wound healing and osteoarthritis.
Other functions of Haruan mentioned in the medical literature include its usage in allergic rhinitis [160,161], dermatitis [162,163], gastric ulcer [164,165], cancer [166,167], hypertension [168,169] and depression [170,171]. Unfortunately, due to the paucity of studies to date, further high-powered studies are warranted to clarify and define the role of Haruan in these diseases.

Discussion
Hong et al [65] did a scoping review on the effectiveness of Haruan extracts on wound healing; they concluded that current evidence favours the use of Haruan extracts to expedite wound healing. Indeed, optimal wound bed preparation and proper wound closure are the two fundamental goals of skin grafting regardless of the graft type [172][173][174]. With its extraordinary antimicrobial, antinociceptive and anti-inflammatory properties, Haruan is undeniably a handy tool for skin grafting. From a psychological perspective, Haruan can minimise post-operative pain and discomfort, achieve satisfactory aesthetic wound effect and improve patient postoperative quality of life. For skin grafting that covers a wound area only partially, Haruan can promote wound closure since it encourages the epithelialisation of wound. When a wound recovery is sped up with less wound infection, the duration of hospital stay will also be shortened. Consequently, expenses related to skin grafting will be cut down.
It is still inconclusive which particular biomolecules play a role in the wound healing property. However, with the advancement of technology especially in the field of proteomics, we have managed to conduct a more comprehensive protein profiling [175,176]. Although proteomics helps us to understand the interactions between the proteins in the fish and the wound, the previous protein profiling [177] were not as accurate as the new one due to the lower sensitivity of the old equipment. Conversely, the current work using Gel Elution Liquid Fractionation Entrapment Electrophoresis (GELFREE) system can maximise protein profiling [127]. The researchers at the Analytical Biochemistry Research Centre of the Universiti Sains Malaysia [127,128] also looked at the post-translational modifications (PTMs) of proteins which might be involved in the wound healing process to complement the protein profiling results. PTMs, as the name suggests, occurs following the translation of amino acids in the later part of the protein biosynthesis. They play an important role in protein regulation and are also involved in the regulation of a number of physiological functions. This helps us to appreciate how the consumption of Haruan contributes to the wound healing mechanism.
It is a known fact that structural proteins such as actin, myosin and tropomyosin are vital in the formation of muscle tissue within an organism. From the protein profiling, it was shown that 37% of all the proteins detected in the fish meat are structural proteins which play a specific role in enhancing wound healing. For example, actin gives rise to the formation of myofibroblasts which differentiated from fibroblasts containing bundles of actin microfilaments with contractile proteins such as non-muscle myosin [178][179][180]. On the one hand, both fibroblasts and myofibroblasts regulate traction force and coordinate contraction during wound closure [181]. On the other hand, tropomyosin, has been reported to regulate cell migration, particularly fibroblast and myofibroblasts [182]. This results in the promotion of rapid wound healing whenever tropomyosin is manipulated in the wound area [183,184]. Hence, the abundant presence of structural proteins in the fish meat could be a key reason why it helps in the wound healing process.
Apart from structural proteins, Haruan meat also possesses numerous enzymes including trypsin. Trypsin has been shown to enhance the healing process by potentiating fibrocyte differentiation [185]. Trypsin has also been used as a biomedicine for treating wound [186]. A clinical study conducted by Gudmunssdsdóttir et al [175] showed that native-proteins were digested by cold-adapted cod trypsin and produced an encouraging effect on the wound. These findings supported the idea that the abundant level of trypsin in Haruan meat helps in facilitating the wound healing process.
Collagen, which is essential for wound healing, is also present in the Channa striatus meat with Collagen Type-I being the most abundant [127]. Collagen is required in the different stages of wound healing including the binding process to fibronectin that helps in platelet aggregation [187], triggering angiogenesis by transforming myocytes into macrophages [188], in addition to giving support to budding capillaries [189]. A recent study by Helary et al [190] has also shown that apoptosis was prevented during chronic wound treatment by the use of concentrated collagen hydrogel that promotes cell proliferation and protects fibroblasts. Recently, mammalian collagen has been replaced by fish collagen [191] which is considered a regenerative medicine [192], a sign that the abundant collagen found in fish meat does help to advance the wound healing process.
Results from the proteomic study [127] also show that C. striatus meat is rich in calcium related proteins such as calmodulin and parvalbumin. We are aware that calcium (Ca 2+ ) plays a major role in maintaining homeostasis of the skin and is considered a key signalling molecule during wound healing [193,194]. Ca 2+ binding proteins are also known to assist in Ca 2+ signalling and skin intracellular trafficking, which includes calmodulin and calmodulin-like proteins [195]. It is also known that Calmodulin assists in keratinocytes maturation [196], proving its significant role in the wound healing process. The important role played by both calmodulin and parvalbumin in the wound healing cascade deserves to be highlighted. Expression of parvalbumin in ependymal cells has been shown to assist in tissue remodelling and wound closure [197]. Hence, it is clear that both parvalbumin and calmodulin help to transfer Ca2+ to the affected area, thereby promoting wound healing.
Proteomic profiling also revealed that more than 50% of the total proteins detected in C. striatus are uncharacterised proteins [128]. The functions of these proteins are still unknown due to the paucity of research. These proteins have been labelled as such due to the absence of any detectable homology to those proteins of known functions at both the sequence and structural level [198]. However, it is possible that one or more of the uncharacterised proteins found in Haruan play a role in the wound healing process. Indeed, the high quantity of uncharacterised proteins detected via proteomics, that is, the proteome database for C. striatus, is far from complete. At this point in time, we can safely say that while existing data have given us an insight into the proteins of Haruan, more rigorous effort must be made into the research of the uncharacterised proteins that might be involved in accelerating the wound healing process -the indisputable characteristic of C. striatus or Haruan.

Conclusion
As a wound cosmetic enhancer as well as an antimicrobial, anti-inflammatory and antinociceptive agent, Haruan fish is a promising medicinal food product for wound healing. Current evidence has illustrated the effectiveness of Haruan in wound healing, particularly in postoperative patients. This book chapter has highlighted the wonders of Haruan in wound healing associated with skin grafting. Unfortunately, in spite of the emerging role and increasing popularity of Haruan in wound healing, the use of Haruan extracts in skin grafting remains insufficient. When its merits have been fully explored, Haruan extracts could become a viable alternative to the current wound dressing regimen in skin grafting in the near future. DOI: http://dx.doi.org/10.5772/intechopen.99207