INTRODUCTION:

Wound or injury is the loss or damage of tissue that can disrupt body functions that can interfere with the daily activities^1,2. The destruction of the tissue makes it easy for microorganisms to infiltrate, causing infections³. Wound healing process through some phases, namely inflammation, migration, proliferation, and maturation phase^4,5,6. Nutrients were needed to accelerate wound healing. Nutrients would play a critical role in each of the phases. The primary nutrients required for wound healing include protein, amino acids, fatty acids, and minerals (zinc, iron, and copper)^7,8,9. There are some natural product have been studied for their effect on wound healing, such as from plant Bauhinia purpurea¹⁰, Bauhinia variegate¹¹, Aerva tomentosa¹², Bridelia airyshawii¹³, Acacia catechu¹⁴, Amaranthus spinosus¹⁵, from animal such as Channa striatus¹⁶ and Monopterus albus.

Eel (Monopterus albus) is a freshwater fish that rich with nutrients needed for the acceleration of wound healing. Eels contain high protein, amino acids, and fatty acids¹⁷. Based on the previous study, the fatty acid constituent of Eel extract was dominated by oleic acid (19.7%), palmitic acid (18.7%), pentadecanoic acid (15.81%), and octadecanoic acid (4.87%)¹⁷. Nutrients content in Eels is 66.7 % protein, 10.74% fat, and pH value around 6.9. The content of arachidonic acid and docosahexaenoic in Eel are 8.25 and 6.21g/100g, respectively¹⁸. In the process of wound healing, the nutrient content is delivered to the surface of the wound to accelerate wound healing. Commonly used drug delivery systems are gel and membrane.

The gel is a semisolid dosage form that is neutral, transparent, colorless, stable, and provides good membrane strength when it dries on the skin treated to cover the wounds^19,20. Gel dosage form has several advantages compared to another semisolid dosage form including simple in the manufacture, easily applied to the skin, produce a cold feeling when applied to the skin because of the slow evaporation of water contained in the gel and easily washed after treatment and has an attractive form^21,22. The gel will dry on the surface of the skin, producing a thin layer that is strong enough to provide protection to the wound and, at the same time can absorb exudates that come out of the wound during the inflammatory phase^23,24. The gel needs time to form a membrane that functions as a wound dressing. Another dosage form that is also widely used as a wound dressing is a membrane. The ideal wound closure membrane is transparent so that the wound healing progress can be easily observed without opening the wound dressing. Other characteristics of wound dressing membranes are elastic and not sticky²⁵.

Previous studies have formulated Eel extract into gel²⁶ and membrane form²⁷. In this study, the effectiveness of these two different dosage form to accelerate the incision wound healing were evaluated. The study used four groups of animals namely negative control (untreated group), EG (a group that treats with Eel extract gel), EM (a group that treats with Eel extract membrane), and commercial preparation as a positive control. The tensile strength tests were conducted at days 3, 6, 9, and 12, in which the tensile test values indicate the wound healing ability of each sample.

MATERIALS AND METHODS:

Materials:

Materials used were polyvinyl alcohol (PVA) (VWR^® BDH Prolabo, Amerika Serikat), normal saline (Widatra Bhakti, Indonesia), glycerol (Brataco, Indonesia), propylene glycol (Brataco, Indonesia), filter paper (Whatman) ethanol (Brataco, Indonesia), methylparaben (Brataco, Indonesia), propylparaben (Brataco, Indonesia), marketed preparation as a positive control (+), distilled water. Eels were purchased from the eel farm (the same place as the source of the eels in previous studies), Bukittinggi, West Sumatera, Indonesia. Eel extract was prepared, according to Febriyenti et al.^17,27.

Preparation of Eel extracts gel and membrane:

Formulas of Eel extract gel and membrane could be seen in Table 1. Preparation and formulas of Eel extract gel and membrane refer to previous studies^26,27.

Table 1: Eel extract gel²⁶ and membrane²⁷

Ingredient	Formula (%)
Ingredient	Gel	Membrane
Eel extract	10	10
PVA	10	10
Propylen glycol	10	-
Glycerol	-	3
Methylparaben	0.1	0.1
Propylparaben	0.02	0.02
Distilled water up to	100	100

Experimental Animals:

The animal studies were conducted using the facility of Faculty Pharmacy, Universitas Andalas. Adult, healthy, male Sprague-Dawley (SD) rats, weight ranging between 200 and 250g, were selected in this study. The rats were housed in standard environmental conditions under a 12-h light-dark cycle lighting in a solid bottom cage with top-ventilated stainless steel cover. All animals were allowed to move freely and were provided access to food and water. Animal welfare guidelines were adopted from the Guide to the Care and Use of Experimental Animals^28,29,30. The handling of rats followed the protocol described by Deacon²⁸. The method’s procedures included for the animals have been approved by Animal Ethics Committee Universitas Andalas, with Ref. No. 012/KEP/FK/2015.

The effects of Eel extract gel and membrane on the healing of incision wounds:

Sprague-Dawley rats were anesthetized with inhaled ether. The animal back furs were clipped with electric clippers followed by scrubbing the skin with 70% ethanol and normal saline. Incision wounds were created by a 6 cm long full-thickness incision with a scalpel blade. Incised wounds were closed with equally spaced interrupted catguts stitch^31,32,33. After all the wounds were closed; the experimental animals were divided into the following four groups as follows:

Group 1: Wounds untreated as control negative (-)

Group 2: Wounds treated with Eel extract gel

Group 3: Wounds treated with Eel extract membrane

Group 4: Wounds treated with marketed preparation as control positive (+)

Rats were fed regular chow feed and had free access to water. Eel extract gel, membrane, or commercial preparation was applied once a day for 3, 6, 9, and 12 days. Before the preparation was applied, the surface of wound was observed visually and by used Whatman filter paper whether there are fluid or pus at the surface of wound or not.

Determination of tensile strength:

The rats were sacrificed at intervals of 3, 6, 9, and 12 days after wound creation, the stitch was removed, and tensile strength of the wounded skin was determined on the same day^34,35,36. The animals were sacrificed, and 6 cm long strips of skin having wounded area in the center were cut out. The skin was fixed in between two clamps of texture analyzer (TA.XT2 Texture Analyzer). The tensile strength was measured as the breaking load, which breaks the wound. The catguts were removed, and the cross-sectional area of the wounds was determined before measuring the breaking load of the healed wounds. The force was applied, and the force causing the wound to break was given automatically by the computer software of the texture analyzer. The tensile strength of the wounds was calculated using the following equation as provided by previous study^16,37,38:

Tensile strength = Breaking load (force)/Cross-sectional area

Where: Cross-sectional area = Thickness x Width of skin strip.

RESULTS AND DISCUSSION:

Eel extract gel²⁶ and membrane²⁷ has been formulated in a previous study. In this part, the effectiveness of the type of preparation was evaluated. Fig. 1 shows the photograph of the wound at days 3, 6, 9, and 12. There was no significant difference between the groups treated with Eel extract gel, membrane, and commercial preparation visually. As in Fig. 1, visually and by observed the filter paper, the conditions of all wounds were free of infection. There were no fluids or pus in the surface of wound. The inflammatory phase had occurred on day 0-5, which was characterized by inflammation on the wound^5,6. In the test group, the inflammatory phase did not affect increasing the exudates. Wounds remain dry, and there was not an infection. Inflammation serves to prevent bacterial invasion and prepare to continue the healing process.

Fig 1: Photograph of the incision wound healing at days 3, 6, 9 and 12

The results of the tensile strength test of wounded skin could be seen in Table 2. The tensile strength of wounded skin increased with time even though it is left untreated. Amino acids and fatty acids contain in Eel extract play an important role in accelerating the healing process³⁹. Amino acids and fatty acids important for the synthesis of collagen fibers during wound healing^40,41. Apart from nutrition like amino acid and fatty acid, wound dressings also play an important role in the healing process of wounds⁴². Wound dressing can provide a suitable environment at the surface of the wound to promote the healing process. The gel is a drug delivery system that could deliver the active ingredient to the wound. After a few minutes, the gel will dry and form the thin membrane that could cover the wound⁴³. While the drying processes, gel cause the wound too wet. The thickness of the membrane formed was not uniform. Sometime the membrane formed was too thin to cover the wound properly. In comparison, the membrane dosage form has on the ready form to apply in the surface of the wound and act as the wound dressing ⁴⁴. The membrane also could absorb the exudates and dried the wound. At the same time, membrane could prevent evaporation which could cause the wound surface become dry. Eel extract membrane provides a better result on healing the incision wound compared with Eel extract gel, commercial preparation gel (positive control) and negative control group. This result is similar to the previous study that used different active ingredient³⁸. The membrane dosage form has also given better tensile strength value compared with the gel dosage form.

Table 2: Results of the tensile strength of the rat skin with incision wound

Day-	Tensile Strength (N/cm²) at
Day-	Control (-)	Eel extract Gel (EG)	Eel extract Membrane (EM)	Control (+)
3	1.51^a ± 0.46	2.34^a ± 0.41	4.00^a ± 2.82	1.90^a ± 0.88
6	1.74^a ± 0.78	3.07^a,b ± 0.89	4.07^b ± 1.18	2.26^a ± 0.86
9	3.16^a ± 1.51	5.12^a,b ± 1.79	7.93^b ± 1.26	3.82^a ± 1.68
12	3.62^a ± 1.24	5.91^a,b ± 1.36	8.36^b ± 2.60	4.17^a ± 1.19

Means within a row with a different letter are significantly different (P<0.05)

CONCLUSION:

Eel extract formulated in the membranes form provides the highest value of the tensile strength test. Gel and membrane of eel extract have effectiveness toward the healing of incision wound in rat compared with the commercial preparation gel (positive control).

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

REFERENCES:

1. Boateng J.S, Matthews K.H, Stevens H.N.E, Eccleston G.M. Wound healing dressings and drug delivery systems: A review. J Pharm Sci. 2008; 97(8): 2892-923.

2. Cockbill S. Wounds-The Healing Process. Hospital Pharmacist. 2002; 9: 255-60.

3. Cockbill S.M.E. Dressings in Wound Management. In: Swarbrick J, editor. Encyclopedia of Pharmaceutical Technology. Third edition New York: Informa Healthcare; 2007. p. 1023 - 37.

4. Romo T, Pearson JM, Yalamanchili H, Zoumalan RA. Wound Healing, Skin2008 July 22, 2010]: Available from http://emedicine.medscape.com/article/884594-overview.

5. Molnar JA. Overview of Nutrition and Wound Healing. In: Molnar JA, editor. Nutrition and Wound Healing. Boca Raton: CRC Press; 2007. p. 1 - 14.

6. Diegelmann RF, Evans MC. Wound healing: an overview of acute, fibrotic, and delayed healing. Frontiers in Bioscience. 2004;9: 283-9.

7. NHSH Senior Management Team. 2013. Nhs Highland Wound Management Guidelines and Formulary. NHS HIGHLAND.

8. Marques SR, Peixoto CA, Messias JB, Albuquerque ARd, Junior VAdS. The effects of topical application of sunflower-seed oil on open wound healing in lambs. Acta Cir Bras. 2004;19(3): 191-203.

9. Williams JZ, Barbul A. Nutrition and wound healing. Surg Clin N Am. 2003; 83: 571-96.

10. Vijayabhaskar K, Sravanprasad M, Goli. Venkateshwarlu, Devi PS, Kumar KH, Sunil. J. Wound Healing Activity of Bauhinia purpurea in Albino Wistar Rats. Asian J Res Pharm Sci. 2011; 1(2): 47-9.

11. Sharma RK, G.P. R, Nathiya D, Sharma AK. Assessment of wound healing activity of roots of Bauhinia variegata Linn. by excision and incision model in Albino Rats. Asian J Res Pharm Sci. 2015;5(3): 145-52.

12. Kumar D, Prasad D, Dua J, Bhatnagar S. Evaluation of Wound Healing Activity of Ethanolic Extract of Aerva tomentosa Forsk. Research J Pharmacognosy and Phytochemistry. 2009; 1(3): 188-90.

13. Bagad YM, Bhurat MR, Tatiya AU, Surana SJ, Barhate SD. Evaluation of antioxidant and wound healing activity of the leaves of Bridelia airyshawii spreng. Research J Pharm and Tech. 2013; 6(4): 351-5.

14. Karwani G, Singhvi IJ, S.K. Gupta, Agarwal A. Wound Healing Activity of Hydroalcoholic Extract of Acacia catechu. Research J Pharm and Tech. 2011;4(6): 905-6.

15. Paswan SK, Srivastava S, Rao CV. Incision Wound healing, Anti-inflammatory and Analgesic activity of Amaranthus spinosus in Wistar rats. Research J Pharm and Tech. 2020; 13(5): 2439-44.

16. Laila L, Febriyenti F, Salhimi SM, Baie S. Wound healing effect of Haruan (Channa striatus) spray. International Wound Journal. 2011; 8(5): 484-91.

17. Febriyenti, Almahdy A, Mulyani D. Amino acids and fatty acids profiles of Eel (Monopterus albus) water extracts. Rasayan Journal of Chemistry. 2019; 12(3):in press.

18. Razak ZKA, Basri M, Dzulkefly K, C.N.A. Extraction and Characterization of Fish Oil from Monopterus Albus. Malaysian Journal of Analytical Sciences. 2001; 7(1): 217 - 20.

19. Carter SS. Dispensing Pharmaceutical Student. 12th ed. 1975, London: Pittman Medical.

20. Lieberman, H.A., 1989. Pharmaceutical Dosage Forms – Disperse System. Marcel Dekker Inc.

21. USP, The United States Pharmacopeia XXX - The National Formulary XXV. 2007, United States Pharmacopeial Convention, Inc.: Rockville.

22. Swarbrick J, and Boylan JC, Encyclopedia of Pharmaceutical Technology. 1992, Marcel Dekker, Inc.: New York.

23. Czaja W, Krystynowicz A, Bielecki S, Brown JRM. Microbial cellulose--the natural power to heal wounds. Biomaterials. 2006; 27(2): 145-51.

24. Stashak TS, Farstvedt E, Othic A. Update on wound dressings: Indications and best use. Clinical Techniques in Equine Practice. 2004; 3(2): 148-63.

25. Santos KSCR, Coelho JFJ, Ferreira P, Pinto I, Lorenzetti SG, Ferreira EI, et al. Synthesis and characterization of membranes obtained by graft copolymerization of 2-hydroxyethyl methacrylate and acrylic acid onto chitosan. Int J Pharm. 2006; 310(1-2): 37-45.

26. Febriyenti, Almahdy A, Dwi Mulyani, Formulation of Eel (Monopterus albus) extract gels for accelerated the wound healing, Scientia Jurnal Farmasi dan Kesehatan. 2020; 10(2): in press

27. Febriyenti, Rahmi Nofita, Khalidazia, Formulation of Eel (Monopterus albus) extract membrane for wound dressing using plasticizer, Jurnal Sains Farmasi dan Klinis. 2020; 7(1): 1-4

28. Deacon RMJ. Housing, husbandry and handling of rodents for behavioral experiments. Nat Protoc. 2006;1(2):936-46.

29. Yregard L, Lowhagen PH, Cassuto J, Nilsson U, Lindblom L, Rantfors J, et al. A new technique for the analysis of endogenous mediators released following thermal injury. Burns. 2001; 27: 9-16.

30. Olfert ED, Cross BM, McWilliam AA, editors. Guide to the care and use of experimental animals 2nd ed. Ottawa: Canadian Council on Animal Care; 1993.

31. Prasad V, Dorle AK. Evaluation of ghee based formulation for wound healing activity. J Ethnopharmacol. 2006; 107(1): 38-47.

32. Reddy JS, Rao PR, Reddy MS. Wound healing effects of Heliotropium indicum, Plumbago zeylanicum and Acalypha indica in rats. J Ethnopharmacol. 2002; 79(2): 249-51.

33. Singh M, Govindarajan R, Nath V, Rawat AKS, Mehrotra S. Antimicrobial, wound healing and antioxidant activity of Plagiochasma appendiculatum Lehm. et Lind. J Ethnopharmacol. 2006; 107(1): 67-72.

34. Akkol EK, Koca U, Pesin I, Yilmazer D, Toker G, Yesilada E. Exploring the wound healing activity of Arnebia densiflora (Nordm.) Ledeb. by in vivo models. Journal of Ethnopharmacology. 2009; 124(1): 137-41.

35. Jauregui KMG, Cabrera JCC, Ceniceros EPS, Hernandez JLM, Ilyina A. A new formulated stable Papin-pectin aerosol spray for skin wound healing. Biotechnology and Bioprocess Engineering. 2009;14: 450 - 6.

36. Gomez-Beloz A, Rucinski JC, Balick MJ, Tipton C. Double incision wound healing bioassay using Hamelia patens from El Salvador. J Ethnopharmacol. 2003; 88(2-3): 169-73.

37. Shukla A, Rasik AM, Jain GK, Shankar R, Kulshrestha DK, Dhawan BN. In vitro and in vivo wound healing activity of asiaticoside isolated from Centella asiatica. J Ethnopharmacol. 1999; 65(1): 1-11.

38. Febriyenti, Henny Lucida, Almahdy A, Istianah Alfikriyah, Muhammad Hanif, Wound Healing Effect of Honey Gel and Film, Journal of Pharmacy and Bioallied Sciences. 2019, 11(2): 176-180

39. MacKay D, Miller AL. Nutritional Support for Wound Healing. Alternative Medicine Review. 2003; 8(4): 359-77.

40. Baie SH, Sheikh KA. The wound healing properties of Channa striatus-cetrimide cream -- tensile strength measurement. J Ethnopharmacol. 2000; 71(1-2): 93-100.

41. Ghori SS, Gouse M, Panda N, Khaled S, Basheeruddin S, Baig MD, et al. Wound Healing Activity of Poly Herbal formulation. Res J Pharm Dosage Form and Tech. 2015; 7(2): 125-8.

42. Rachmasari A, Kartini RA, Alviani G, Solihah I. Healing effect of Spray gel collagen extract from Channa striata bone on burn in rats. Res J Pharma Dosage Forms and Tech. 2019; 11(4): 275-9.

43. Daphne TL, Prince V. A Study to assess the Effectiveness of Aloe Vera Gel Dressing on Wound Status among Patients with Diabetes Mellitus in Government Hospital, Dindigul. Int. J. of Advances in Nur. Management. 2019; 7(2): 159-62.

44. Saudagar RB, Gangurde. PA. Formulation, Development and Evaluation of Film-Forming Gel for Prolonged Dermal Delivery of Miconaole Nitrate. Research J Topical and Cosmetic Sci 2017; 8(1): 19-29.

Received on 16.07.2020 Modified on 02.08.2020

Research J. Pharm. and Tech. 2021; 14(6):3295-3298.

DOI: 10.52711/0974-360X.2021.00573