A study on the effect of Haruan fish extract (Channa striatus) on wound healing and quality of life of coronary artery bypass grafting (CABG) patients: A prospective, double-blind, randomized, controlled trial [version 1; peer review: 1 approved, 1 not approved]

Background: Wound healing remains a primary problem in all surgical cases especially so when the length of incision is very significant as with cardiac bypass patients. The main objective of this study is therefore to assess the effect of Haruan fish extract (Channa striatus) on chest and leg wounds post-coronary artery bypass grafting (CABG) surgery with the optimum and standard patient care in two groups of randomized patients. Methods: This is a randomized, double blind clinical trial being conducted at the National Heart Institute, Kuala Lumpur. Two randomized groups of similar demographic and co-morbid histories planned for CABG were enrolled into the study. Both groups were blinded to the capsules being given to them preand post-operatively. Assessments were also made on wound pain, mobilization and on the health-related quality of life (HRQOL) of patients using the Nottingham Health Profile (NHP). Results: The group that received Haruan capsule showed better would healing objectively. They had better pain scores, though there was no significant difference in terms of mobilization. Overall, the HRQOL in the study group showed improved quality of life. Conclusion: Our study shows the superior effect of using Channa striatus, a local Haruan fish which is easily processed into capsules in promoting wound healing, reducing pain via its anti-nociceptive effect Open Peer Review


Introduction
Wound healing involves complex interaction between cells and mediators, which starts once the wound is inflicted, and continues for weeks. Healing is still a major problem in any kinds of surgery, particularly in cardiothoracic surgery. This is a surgery that involves a long incision over the sternum where delayed sternal wound healing is not an uncommon sight. Sternal wound infections are a known cause of morbidity and might require intervention from plastic surgeons. In severe cases, it could even lead to mortality.
Sternal wound infection is fairly uncommon as compared to leg wounds, with a frequency between 0.7% 1 and 3.3% 2 , but reports based on extended surveillance on post-discharge cases showed incidences up to 9.7% 3 . As expected, the end-result of sternal wound infection include wound dehiscence, mediastinitis, pericarditis, osteomyelitis and endocarditis 4 with associated mortality between 14% and 47% 5-9 .
Leg wound infection, though not life-threatening as chestwound, increases the overall morbidity of the patients. It is noted that there is a threefold increase in cost of managing patients with leg wound as compared to those with an uncomplicated postoperative course 7 . Complications such as wound breakdown, cellulitis, lymphangitis, fat necrosis, and delayed healing are known sequelae of leg wound infection. It is not surprising that the incidence has been reported to be as high as 24% 10-13 .
We have noticed that despite the different types of dressings being used, the rate of infection did not vary that much [14][15][16][17] . The reason for this seemingly indifferent incidence of wound infection is due to the fact that the healing was by primary intention 17 . The cause for such an infection is primarily deepseated problem of the pre-operative and intra-operative rather than merely the superficial issue of wound dressing 18 .
With that understanding, perhaps a better way in promoting wound healing would be a systemic approach rather than topical; with all other factors remaining constant. Extracts of Channa striatus, locally known as Haruan (snakehead), a fresh water, air-breathing, carnivorous fish, has been proven to influence the different phases of the wound healing process 19,20 . Laboratory studies conducted on Sprague Dawley rats have shown that Channa striatus has antinociceptive effects that could reduce postoperative pain 21-23 . Channa striatus has been shown to contain all essential amino acids for wound healing, particularly glycine. Glycine is a major component of human skin collagen that acts synergistically with other essential amino acids that facilitate in tissue healing process. It also has a high content of arachidonic acid and polyunsaturated fatty acids that can promote prostaglandin synthesis, which is a very important compound that creates and removes inflammation, which is part of the healing process 19 . It has also been shown that Channa striatus promotes remodelling of collagen by the synthesis of inter, and intra molecular protein cross-linking, producing a marked increase in the tensile strength that accentuates wound healing 24 . Furthermore, the fish also has certain fatty acids that have been reported to possess anti-inflammatory activity such as stearic acid and oleic acid 25 . Up to this point, all the above studies that looked into the healing and nociceptive properties of Channa striatus have been on animal models. This is the first study that is conducted on human subjects. We therefore conducted a prospective randomized control trial (RCT) and assess not only wound healing and wound infection after CABG, but also the health related QOL of such patients, considering the anti-htinflammatory and antinociceptive effect of Channa striatus. The potential value of patenting such natural product and it's usage on other types of surgical wounds, including laparotomy and Caesarean section; compounded with the commercial value associated, makes it even more attractive.

Study design
Laboratory. The Haruan used in this study was locally reared in a special pond for Haruan; the sex of the fish w as not determined. The depth of the pond should not be more than 4 feet for the fish to surf ace and breath, since Haruan is air breathing and carnivorous. Temperature is maintained around 25°C and pH must be maintained between 5.0 -5.5 and salinity between 0 -10 ppm. The Haruan capsules produced were derived from the flesh of the fi sh and were manufactured using strict good manufacturing practice (GMP) Guidelines by Major Interest Sdn. Bhd.
Haruan (whole fish) are procured fresh to the laboratory. These fishes are kept at -18°C until preparation. The fishes are thawed prior to gutting and cleaning. The cleaned fishes are weighed and placed into the autoclave bin. The water mixture for autoclaving is prepared in relative to fish weight with respect to the volume of the water used. Therefore, a 1:0.5 mixture would contain 1kg of fish with 0.5 kg (500ml) of water. . No preservatives were used in the preparation of Haruan capsules.
Sterilization is carried out using Hiclave HVE-50 autoclave (Hirayama, Tokyo, Japan) with temperature setting of 110°C for 15 minutes. Upon completion of the sterilization, the fish are mixed and meshed thoroughly using a clean ladle, which has been wiped with 70% alcohol. This mixture is then transferred into a large lined aluminium pan with a dimension of 24 × 18 × 1 inches and dried using a 12 tray industrial oven at 60°C for 48 hours continuously.
Upon completion, the sheets of crispy flakes are grinded using a comminuting mill and a hand grinder, Kimah Grinder Machine (CSJ-300, Penang, Malaysia) to ensure thin bone fragments are properly powdered. The resulting powder is mixed homogenously in a clear large plastic bag. Upon homogenous mixing, the powder is sieved using Retsch AS200 analytical sieve shaker (Haan, Germany), with woven wire mesh sieves of 850 micron aperture.

Production of Haruan capsules
The refined powder was filled into empty hard gelatin capsules of pharmaceutical grade which is 1005 TSE/BSE produced by Nasmir Hard Gelatin Capsules Sdn. Bhd. (Catalogue number 1NH001, Pulau Pinang, Malaysia) based on bench scale filling method using Kimah electronic capsule arranging machine, Model 400-F1 (Penai, Malaysia). The empty hard gelatin capsule shells used are of size 0 and made of halal gelatin each weighing 0.08mg. The capsules were visually inspected for defect and are weighed for standard distribution of 250 mg using Sartorious analytical digital balance (Göttingen, Germany). The standardized capsules are packed into sterile bottles using a Kimah electronic counting machine, Model JB-2B (Penai, Malaysia). The bottles are inserted with pre-packed silica beads for moisture absorbance and cotton balls to prevent spillage.

Clinical study
A randomized controlled trial (RCT) was completed with patients with coronary artery disease requiring coronary artery bypass grafting surgery were prospectively and randomly divided into two parallel groups. One group received Channa striatus capsule formulation derived from the fillet of Channa striatus and the control group received placebo (maltodextrine). The trial was conducted from January 2012 until August 2014 and the trial number as registered with the National Medical Research Registry is NMRR-17-360-34772 (Registered 13/03/2017).

Sample size calculation
We used the PS Software version 3.1.2 for power and sample size calculation. We have planned a study of a continuous response variable from independent control and experimental subjects with 1 control per experimental subject. Based on a previous study 26 we planned a study of independent cases and controls with 1 control per case. Prior data indicated that the failure rate among controls was 0.027. If the true relative risk of failure for experimental subjects relative to controls is 6.9, then we needed to study 58 experimental subjects and 58 control subjects to be able to reject the null hypothesis that this relative risk equals 1 with probability (power) 0.8. The Type I error probability associated with this test of this null hypothesis is 0.05. We used an uncorrected chi-squared statistic to evaluate this null hypothesis. Assuming a drop-out rate of 20%, the minimum sample size required was about 70 (58 + 12) subjects per group. In this study, we managed to recruit 253 patients, and 183 completed the three months follow-up. We therefore surpassed the minimum requirement of sample size of 140 subjects, allowing us reach a power of study of 0.8.

Sampling
Inclusion criteria: 1. Male or female 2. More than 18 years of age 3. Elective, urgent, or emergency coronary artery revascularization

Pre-operative
The two randomized groups based in the National Heart Institute (IJN) were matched according to sex, age, New York Heart Association (NYHA) criteria (indicates patients' hearts condition and the severity of their symptoms. Class I to Class IV), ejection fraction, and diabetic status. Randomization was done via simple computer-generated randomization with odd or even RN numbers in each group. Operative and perioperative conditions were also similar for both groups. All subjects received an identical prophylactic antibiotic regime consisting of Cefazolin 2gm at induction and 1 gm 12 hourly for 48 hours. Gentamicin 2mg/kg was also given at induction.

Intra-operative
A uniform method of wound closure and disinfection protocol was followed. Skin was disinfected with Betadine followed by povidone iodine 10%. The method for wound closure was similar for both groups, namely double layer sutures up to the intra-cutaneous skin. Going from deep up to the surface, the presternal fascia was closed with 1-0 Vicryl suture in a continuous type followed by closure of the subcutaneous tissue. Skin was closed with Monocryl 3/0 in subcuticular manner. For the leg wound, the subcutaneous tissue was closed with Vicryl 0, followed by continuous skin suture with Maxon 3/0.
Titanium clips were used to secure branches of the internal mammary artery on the chest wall and branches of the saphenous vein in the leg. Some saphenous veins branches were ligated with Silk 3/0 instead. When closing the sternum, steel wire sutures were used.

Post-operative
Both groups took either, 2 capsules (500mg) of Channa striatus, or 2 capsules of placebo (maltodextrine) daily for a minimum of six weeks, starting from day one post-operatively. If the patient was still ventilated, the capsules were broken, and the content administered via a Ryle's tube. Investigators and healthcare workers, as well as participants, were blinded to the capsules received. Only the manufacturers who supplied the drug were aware of the meaning of the label on the capsule, letter E or O.

Consent and ethics
Prior to randomization of participants, the study was explained, including risk and benefits to suitable candidates, and written consent was obtained. Consenting participants were then randomized into the trial. The study was conducted with ethical approval from the National Heart Institute Malaysia (IJNEC/04/2011) and National Medical Research Register (NMRR-17-360-34772).

Measurement
Wound Infection. Both sternal and leg wounds were inspected and assessed daily until subjects were discharged. Information at 6 weeks and 3 months postoperatively was collected from patients in the clinic or over the telephone. Each wound was scored using the ASEPSIS system (Refer to Supplementary Material 1). We realized that this is a more objective method in assessing wound infection rather than being subjective and open to biases. The end result from this scoring system will determine whether the wound is considered significant or not by obtaining a score of 21-30. If the wound is more serious with a deep seated wound infection, it will have a score of 21-30, and should there be evidence of bone infection, then the score would be >41.
Wound pain. A Visual Analogue Scale (VAS) of 0 to 10 cm was used to assess the degree of wound pain. A score of 0 cm represents "no pain" and a score of 10 represents "severe pain". Pain assessment will be recorded from day 1 and will continue daily until the subject is discharged. It will be further assessed during the clinic visit at 6 weeks and 3 months postoperatively.

Mobilization.
Similarly, a VAS of 0 to 10 cm was used to assess the degree of mobilization. A score of 0 cm represents "inability to walk" and 10 cm represents "excellent mobilization." Assessment of mobilization was made on post-operative days 3 and 4 since there was restrictions in mobility on days 1 and 2 after surgery. Similarly, it was assessed again during the clinic visit at 6 weeks and 3 months postoperatively.

Health related quality of life.
In this study, we decided to assess the health-related quality of life (HRQOL) using the Nottingham Health Profile (NHP) -Part 1. The NHP is known to be very efficient in clinical studies. It looks into parameters such as discomfort and pain apart from the general well-being of a patient. And it is found to be more objective in assessing the health status of the patients. There were 38 subjective statements which were divided into six sections on NHP Part 1 namely physical mobility, social isolation, emotional reaction, energy, pain, and sleep. Each section will have a score range from between 0-100 by adding the item weight to each answer that is deemed positive.
The questionnaire was distributed to both groups of patients before CABG and at six weeks and three months during clinic visit postoperatively. The questionnaires were administered using the questionnaire-interview approach.

Statistical analysis
Data was analysed with IBM SPSS Statistics version 24.0 and examined for normality using the Kolmogorov-Smirnoff test and stem-and-leaf plot. When normal, we proceeded with parametric tests; however, if not normal, we proceeded with the non-parametric tests.
The results were presented as means ± standard deviations for scaled measurements; with numbers and percentages for categorical measurements. The unpaired t-test was used to examine mean differences for wound infection, wound healing, pain, and mobilization across the two groups. Differences in proportions was examined using the Chi-square test.
In examining the mean QOL score, differences between the two groups (pre-operative, six weeks, and three months), the mixed mode two-way repeated measure ANOVA with post-hoc multiple comparison test (between and within subjects) of the two groups was performed.
To determine the factors influencing the change of QOL after CABG, with the dependent variable being binary (improved or worsened), we performed an initial simple logistic regression (SLogReg), and examined the statistical significance of each independent variable such as the amount of revascularization and duration of surgery on the outcome. We then performed a multiple logistic regression (MLogReg) including variables with a level of significance ≤ 0.20 in the multivariate logistic regression and controlling for the effects of possible confounding variables (sex, age, NYHA Criteria, ejection fraction, and diabetic status). A p value of 0.05 was taken as the level of significance.
Refer to Figure 1 for the study flow chart.

Results
253 patients were recruited from January 2012 till August 2014.
Only 183 patients (72.3%) completed the study up to three months follow-up. A total of 40 patients (15.8%) were lost during follow-up and 5.5% discontinued their enrolment in the study. The main reason why these patients were lost during follow-up was for a simple reason that they did not turn-up for the scheduled follow-up. This is due to the fact that IJN is the largest heart centre in the Malaysia and the patients were from all over the country. And since many were former government servants and stay in the rural areas all over the country, travelling to the city centre was not only time-consuming but expensive. Seven patients (2.8%) had their operations cancelled.
The patients were predominantly male (86.2%), the majority being Malay (86.7%), followed by Indians (15.4%) and Chinese (7.9%).  Only 10 patients (4.07%) had their chest re-opened. Known complications such as renal failure occurred in only 2 patients (0.81%) and stroke in another 2 patients (0.81%) as well. Overall, the mortality rate was 3.6% with nine patients dead. Refer to Figure 2 for study results flow chart.

Limitations
There were a few limitations to this study. The first is in getting the patients' consent in enrolling into the study. A few patients had this misconception that Haruan would cause hypertrophic wound or sometimes even keloid. This was the misperception that they had and no amount of talking or persuasion would convince them. And of course, considering that their enrolment is voluntary, their withdrawal from the study is unavoidable. Hence there were almost 30% of patients who did not manage to complete the study. The second limitation is regarding the compliance of the patients to take the capsules provided to them. While they were on the ward, this issue of compliance did not arise, since the Haruan capsules or the placebo were served by the attending staff nurses. However, compliance was not ensured when the patients were discharged from the wards. The third is regarding how the Nottingham Health Profile was administered. We decided for the questionnaire to be conducted by the researcher and not independently answered by the patients without the presence of the researcher. This could have resulted in some bias when answering the question on NHP by the patients. While we understand that this interview mode is not free from bias, it is still the least burdensome method, which only requires the respondent to speak the same language as the interviewer, and to have a basic verbal and listening skills. Considering that almost all Malaysians speak Malay and a majority of them speak English as well, this is probably a more effective way. We believe that the interview method would allow us to clarify ambigu ous questions, and maintain the motivation.

Discussion
Wound healing has always been an obsession among surgeons. And despite the advance in surgical dressings over the years, surgeons have always been trying to find novel ways in promoting wound healing. Haruan or Channa striatus has been known amongst the locals since ancient times to promote healing, and has been used as a form of nutritional supplement. Normally the fish is consumed after it is fried, grilled or boiled; as a traditional remedy for wound healing especially after delivery of caesarean section. There, however, are no clinically randomized trials so far that have been conducted to prove or disprove this belief.
Our study -the first as far as the authors are concerned -has shown the clinical benefit of Channa striatus in promoting wound healing among CABG patients. The study was conducted in a double-blind manner, and only after the results were analyzed, were the researchers informed of the content of Capsule E and Capsule O. There was no way for the researchers to know before hand of the content of the capsules.
The results scientifically confirm the long-held belief of the benefit of Haruan fish. The properties of Haruan to promote wound healing have been postulated. Perhaps it was the way in which Haruan had positive influence on the immune system in regulating the prostaglandin synthesis via the possession of ω-3 polyunsaturated fatty acids has made it effective in stimulating the healing process 20 .
The healing process requires both amino acids and fatty acids. And these have been found in abundance in Haruan fish. Compounds such as glycine, alanine, proline, arginine, serine, leucine, isoleucine, and phenylalanine are known to combine with aspartic and glutamic acid to form polypeptides. These form the basic foundation for healing to take place. Studies have demonstrated that these amino acids and fatty acids are present in Haruan 20 . Similarly, Haruan also contains a high content of arachidonic acid, 20:4ω6. This arachidonic acid is thought to be related to the wound healing property of Haruan 20 . The platelet aggregation and adhesion to the endothelial tissue that initiates blood clotting is thought to be induced by arachidonic acid.
Researchers at the Analytical Biochemistry Research Centre at the Universiti Sains Malaysia have done proteomic profiling on Haruan extract and found out that 25% and 26% of the total protein detected in freeze-and spray-dried C. striatus extract respectively are actin, myosin, and tropomyosin 27 . A recent study has also shown that actins are essential for a range of cellular functions including; the maintenance of cell shape, polarity, cell division, cytokinesis, vesicle and organelle movement, cell signaling, establishing and maintaining cell junctions, and the regulation of transcription 28 . And this process is postulated to take effect during the process of re-epithelialisation, which is part of proliferative phase during a healing process 28 .
It is also known that actin acts in synchrony with myosin in promoting wound healing. This biomechanical process in driving cell motility has previously been shown 29 .
Tropomyosin, which controls the isoform-specific regulation of diverse actin filaments, is also suggested to play a role during wound healing 30 . This finding is also confirmed by Lees et al 31 . in their report on the role of tropomyosin as a regulator of actin functioning during the process of wound healing.
So it is possible that actin, myosin and tropomyosin, which can be found in Haruan, contributed to the expedition of the wound healing process.
Two types of collagen, namely type I and type II, have also been detected in Haruan through proteomic profiling 27,32 . Collagen is a well-known substance that gives tensile strength during wound healing. In fact, it has been shown that collagen and other components of the extra-cellular matrix mainly formed the granulation tissue 33 . It is said that the granulation tissue then contracted, slowly closing the wound, and at the same time aligning the collagen fibres in the extracellular matrix 33 . This leads us to believe that the collagen found in Haruan promotes the maturation of granulation tissue, and hence, expedite wound healing.
Apart from the healing properties, Haruan has also been proven to promote the quality of life of patients' post-CABG. The HRQOL study shows that there was significant change in the energy, pain, emotion and physical level.
These findings demonstrate the much-acclaimed "rejuvenating effect" of Haruan. It is not an exaggeration to propose that the high content of fatty acids and certain amino acids coupled with the anti-nociceptive effects 34,35 might have indicated that they somehow contributed to the natural well-being of patients' post-CABG.

Conclusion
Our clinical study, though laborious, has demonstrated the effects of using Channa striatus, a local fish known as Haruan, which is easily processed into capsules in promoting wound healing, reducing pain via its anti-nociceptive effect and improving quality of life of patients after coronary artery bypass grafting surgery. This is the first randomized, double blind clinical trial being conducted on this matter although there have been anecdotal evidence on the efficacy of Haruan. It is inferred that a faster recovery from surgery confers an advantage in terms of resources to an overall economic benefit. Reduction in the percentage of wound infection also resulted in reduced hospitalization cost. We believe this will lead to the successful commercialization of Haruan, which could be reared commercially as a nutraceutical product.

Competing interests
No competing interests were disclosed.

Grant information
This study was funded by Monash University Malaysia under Major Grant, CRM279.
The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Supplementary Material 1 -definition of ASEPSIS
ASEPSIS is an acronym for Additional treatment, the presence of Serous discharge, Erythema, Purulent discharge and Separation of the deep tissue, the Isolation of bacteria and the duration of inpatient Stay. Points were allotted to the ASEPSIS system for the extent of wound disturbance showing serous (0-5) or purulent (0-10) exudate, erythema (0-5), and separation of the deep tissues (0-10). Points were also added up for criteria up to three months following surgery: antibiotic treatment, 10 points; drainage under local anaesthetic, 5 points; debridement under general anaesthetic, 10 points; positive microbiology isolate, 10 points; extended hospital stay beyond ten days due to wound infection, 5 points. The total score will then be used to reflect the severity of infection as shown: The reviewer congratulates the investigators for undertaking a challenging study that considers wound healing in patients undergoing coronary artery bypass graft surgery. The authors delineate the methodology for performing this double-blind randomized placebo-controlled trial that compares use of the Haruan fish extract to a placebo to evaluate wound healing and quality-of-life in patients undergoing coronary artery bypass graft surgery. The authors state that wound healing is a primary problem in surgical cases and, based on preliminary information, they study the Haruan fish extract. It would be good to have a standardized extract to test and to determine which may be the active components of this extract. In this study, the design seems reasonable and the outcome measures of wound pain mobilization and health-related quality-of-life using the Nottingham Health Profile is appropriate. In the abstract, the authors state that those who received the Haruan capsule showed better wound healing and had better pain scores as well as improvement in quality-of-life. The analyses seem appropriate as does the determination of the size of the study. Unfortunately only 72.3% completed the study in up to three months of followup. Additionally, the study applies mainly to males of the Malay background. Unfortunately, in the results section, there is very little evidence that the Haruan capsule actually had any benefit. It would be good to have some tables and charts demonstrating the efficacy, or not, with Haruan capsule. With regard to mean hospital stay, the results were not significant. Perhaps the study was underpowered to determine significance but, nevertheless, the results were not significant. In terms of the ASEPSIS score, there were no significant differences throughout the follow-up. Additionally, there were no significant differences in mean pain score using the visual analog scale and no difference in mobilization. With regard to the health related quality-of-life, there were no significant differences at discharge and in follow-up. Therefore, this reviewer is puzzled that the abstract states benefit when the results do not show this. Much of the issue may be related to limitations but this manuscript does not confer evidence that the Haruan capsule actually has benefit. Further, any hypothesis that wound healing would be improved by the Haruan extract is not supported here. In the discussion there is enthusiasm about using this Haruan extract with statements that this extract actually improves wound healing but it did not. This reviewer does not understand how the authors can implicate benefit or even discuss mechanisms when there is no evidence based on the results that this actually works. One other thing relates to the costs of hospitalization mentioned in the conclusion but not discussed in any detail in the manuscript.

Is the work clearly and accurately presented and does it cite the current literature? Partly
Is the study design appropriate and is the work technically sound? Yes

Are sufficient details of methods and analysis provided to allow replication by others? Yes
If applicable, is the statistical analysis and its interpretation appropriate? No wounds post-coronary artery bypass grafting (CABG) surgery. Dr Musa and his co-authors have discussed appropriately in the context of the current literature. Interestingly, the authors have used suitable methods and in my opinion, they have provided sufficient information and source data to allow other researchers to repeat different steps of wound healings in other organs of the body. I believe, no additional changes are required. The design, including sample size, wound infection, mobilization, health related quality of life, production of Haruan capsules, specifically the flow chart, and the methods, are perfectly prepared. Results are presented accurately and the conclusions are justified and supported by the data.

Is the work clearly and accurately presented and does it cite the current literature? Yes
Is the study design appropriate and is the work technically sound? Yes

If applicable, is the statistical analysis and its interpretation appropriate? Yes
Are all the source data underlying the results available to ensure full reproducibility? Yes

Are the conclusions drawn adequately supported by the results? Yes
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