INTRODUCTION:

Wound healing process is crucial, incorporating several stages, such as hemostasis, inflammation, proliferation, and remodeling¹^,². In diabetes, this process is disrupted due to hyperglycemia³, which induces pro-inflammatory cytokines, leading to prolonged inflammation in healing phase, impaired granulation tissue development, and the formation of chronic wound⁴.

Pro-inflammatory cytokines, including TNF-α, are significant due to their role in wound healing process⁵. Recently, several studies have investigated the inflammatory transition to the wound repair proliferative stage⁶.

Contemporary wound treatments include applying topical Purilon gel, Stimulen, Elase, and Regranex⁷^,⁸. However, topical plant products is limited despite their effectiveness in promoting healing due to composed of various antioxidant and anti-inflammatory principles ⁹. This phenomenon has led to the use of alternative therapies and natural remedies in a quick healing process ¹⁰. Plant derivative products for the healing process are Tridax procumbens linn, bark extract, Cleome viscosa extract, Psidium guajava¹¹^,¹²^,¹³^,¹⁴^.

Several plant-derived products, including Coconut Oil (VCO), have been proven to offer biological functions including anti-inflammatory, antioxidant, and other health effects based on medium-chain fatty acids (MCFAs) like lauric acid ¹⁵. VCO is pure coconut oil obtained by processing mature coconut meat naturally. In addition to the medium-chain fatty acids, it has polyphenol content with anti-inflammatory and oxidative stress activity ¹⁶. The main polyphenol identified in VCO is ferulic acid is a potent molecule with antioxidant and anti-inflammatory activities ¹⁷. A previous study that topical use of VCO indicated a quick healing than the control group ¹⁸, with potential as an anti-inflammatory and skin-protective agent ¹⁹. Previous analysis showed VCO is essential in accelerating wound healing process²⁰. Other studies have also indicated that VCO can expedite the wound healing process ¹⁸, but thus far, there has been a dearth of research investigating wound healing in diabetic conditions.

An experimental study has shown that unsaturated fatty acids (linoleic, oleic, and olenat acid) including volatile oil particularly thymoquinone, serving as the most important chemical compositions of black cumin oil, has various pharmacological effects similar to antipyretic, analgesic, anti-inflammatory, and antimicrobial drugs ^{21, 22}. The source of black cumin oil, has been long time used for various skin conditions, dermatological disorders, and in cosmeceutical formulations, for treating acne vulgaris, burns, wounds, and injuries ^{23, 24} through anti-inflammatory processes ²⁴. Furthermore, black cumin oil induces angiogenesis, fibroblast proliferation, and collagen synthesis during healing process, significantly increasing collagen formation and rate of epithelialization. ²⁵. Other studies report that black cumin oil offers valuable benefits ²⁶, showing its excellent effect on wound healing process and moisturizing ²⁷.Other research has elucidated that black cumin oil imparts an acceleration effect on wound healing (Elghory), yet scant literature delves into its utilization for wound healing processes within diabetic conditions. To date, there remains an absence of discourse surrounding the combination of VCO and black cumin oil in wound healing within diabetic contexts.

Based on the background above, this study analyzes VCO, Black cumin oil, and their combination effect on promoting wound healing in DM.

MATERIALS AND METHOD:

Materials:

Research materials were sourced from various suppliers: Sigma (USA) for Streptozotocin and RNase-free water, Thermo Fisher (CA, USA) for Triazole, and Thermo Fisher (Vilnius, Lithuania) for the cDNA Kit, GAPDH, and TNF-α gene. Additionally, VCO was acquired from PT. Indo Fureco pratman (Indonesia) and black cumin oil from PT.Habbatusauda (Indonesia).

Study design:

All experimental procedures comprising animals complied with ethical guidelines and were approved by the appropriate ethics committee with number 42/UN.16.10.D.KEPK-FF/2023. Three days after receiving an STZ injection, the study rat's FBG levels exceeded 300mg/dl, signifying diabetes. Furthermore, diabetic samples were randomly taken 6 groups (n = 5 per group). VCO, black cumin oil, and combination were applied topically with a volume of 1 ml ¹⁸ over wound area for 7 and 14 ²⁸ consecutive days. The group consists of :

Group 1: Diabetic control (DC) with no intervention;

Group 2: VCO;

Group 3: black cumin oil;

Group 4: C1 received 50% VCO and 50% black cumin oil;

Group 5: C2 received 70% VCO and 30% black cumin oil;

Group 6: C3 received 30% VCO and 70% black cumin oil.

Preparation of rat with diabetic ulcer:

The study was conducted on 30 male Wistar rats (220 and 250 g body weight (BW), placed at a controlled temperature (23 ±2^oC) and 12-h dark/12-h light cycle. Furthermore, In addition, rats are given plenty of food and water. Intraperitoneal STZ 55 mg/kg BW induces diabetes in rats housed for one week ^29,30,31. To confirm the diabetic status, fasting, and random blood glucose assays were conducted from tail vein blood once a week for four weeks. Rats identified with diabetes were anesthetized using xylazine, followed by fur removal. A 10 mm-diameter punch biopsies were used to create the incision, and each rat was placed in its separate           cage³²^,³³.

Wound tissue collection:

Skin tissue were extracted for sample on day-7 and 14. In the first stage, the rat was positioned dexter-lying, and surgery was performed to isolate the skin by excision to the subcutis depth. Subsequently, the epidermis was dissected with tweezers before being cut with scissors. After the collection of tissue samples, rats were euthanized by neck dislocation ³⁴.

TNF alfa gene expression analysis:

RNA isolation:

All tissues from experimental groups were isolated using reagents TRIzolⓇ (Thermo Fisher Scientific, CA, USA) and homogenized into a sample with a homogenizer 1 ml Reagen TRIzol™. The process was continued with the addition of 200 μl of chloroform, incubation at room temperature, and centrifugation at 12,000 x g and 4°C for 15 min. Subsequently, the top layer was added with 2x isopropanol, set for 10 min at room temperature, and centrifuged for 10 min to achieve a white pallet formation. The pellets obtained were washed with 350 μl of 70% ethanol, vortexed, and centrifuged again for 5 min at 7500 x g at 4°C. The supernatant was removed, rested for 10 min, and resuspended in 25–40 μl of RNase-free water based on the number of shells. The RNA was calculated and adjusted to a concentration of 1000 ng ³⁵^,³⁶^,³⁷.

cDNA synthesis:

The synthesis of cDNA was carried out using a synthesis kit (Thermo Fisher Scientific, Vilnius, Lithuania). ThecDNA synthesis composition was 5 μg total RNA, 1x RT buffer, 20 pmol oligodT, 4 mM dNTP, 10 mM DTT, 40 U enzyme SuperScript TMII RTase, and Nuclease Free Water with a20 μl reaction volume. Subsequently, total cDNA synthesis was performed at 52°C for 50 min with a working protocol according to the kit manual (iScript cDNA synthesis, Biorad) ³⁵^,³⁶^,³⁷.

PCR gradient amplification:

The PCR process was carried out in the amplification range for 40 amplification cycles, consisting of predenaturation at 95.0°C for 3 min. Furthermore, the initial denaturation was 94°C for 5 min, core cycle consisting of 94°C for 45 sec, 55°C for 30 sec, 72°C for 45 sec, and extension at 72°C for 7 min ³⁵^,³⁶^,³⁷.

Realtime PCR (RT-PCR):

RT-PCR used gene primers following the design and temperature optimization. The primary sequence of the alpha TNF-α was as follows ³⁸^,³⁹: 5’TGTGCCGCCGCTGTCTGCTTCACGCT-3’ 5’GATGAGGAAAGACACCTGGCTGTAGA-3’This gene concentration measurement used the relative quantification method ⁴⁰. ΔCT experiment = CT experiment target – CT experiment housekeeping. ΔCT control = CT control target – CT control housekeeping. ΔΔCT experiment = ΔCT experiment – ΔCT control. The gene expression level comparison = 2^ΔΔCT.

Histopathological analysis on animal skin tissue:

The tissue was initially fixed using a 4% phosphate-buffered formalin solution. Subsequently, the tissue was processed into paraffin blocks, which were sectioned with a microtome to 4mm thickness. These sections were stained applying Hematoxylin and Eosin for observation. Microscopic examination applies CX 33 light microscope, and photomicrographs were captured using a 3.1MP Sony Exmor CMOS camera, followed by analysis using the Betaview software. Quantitative measurements were carried out to determine the thickness of the epidermis and dermis at a 40x magnification. The measurement of the epidermal thickness was carried out by making a straight line from the basal epidermis to the upper limit of the stratum granulosum beneath the stratum corneum at 10 different points. Furthermore, dermal thickness was measured by making a straight line from the basal epidermis to the lower limit of the dermis at 10 different points. Both measurements were presented as mean values in micrometers (µm). Other histological parameters, such as edema, leucocytes, granulation, fibroblasts, collagen, and epithelization were semi-quantitatively evaluated based on criteria stated by McMinn ⁴¹^,⁴².

Data analysis:

The data analysis applies grapd prismversi 9 with ANOVA as mean ± standard deviation with a confidence interval of 95%. A p-value less than 0.05 was determined as significant.

RESULT:

Effect of VCO and Black Cumin Oil Combination on Wound Closure in Diabetic Rat:

Figure 1, 2. shows the diameter of wound closure in DC rat on days 7 and 14. The values obtained were lower compared to diabetic rat treated with topical VCO, black cumin oil, and C1, C2, and C3, where wound closure occurred perfectly.

Effect of VCO and Black Cumin Oil Combination on TNF-α Expression:

A significant decrease (p < 0.01) in TNF-α gene expression was observed in diabetic rat wound tissues topically treated with C2 and C3 compared to the control, as shown in Figure 3. Meanwhile, on day 14, a significant decrease (p < 0.01) in TNF-α gene expression was observed in diabetic rat wound tissues topically treated with C1, C2, and C3 than the control described in Figure 4.

Effect of VCO and Black Cumin Oil Combination on Wound Histology

VCO, black cumin oil, and their combination effect on the histology of diabetic rat wound, Figure 5. showed in wound treated with combination, a thicker epidermis was observed compared to another groups on day 7. Meanwhile, Figure 6. Showed on day 14, the C3 group have a thicker epidermis than the control group. Figure 7,8 showed that treatment with C1 had a thicker dermis compared to Control groups. Figure 9,10 showed Control animals showed wound characterized by incomplete epithelialization, scab-covered wound surfaces (S), granulation with loose connective tissue, several inflammatory cells, low fibroblasts, and reduced collagen. Treatment with VCO, Black Cumin Oil, or their combination showed better wound healing with complete epithelialization, lower inflammatory cell count, as well as higher fibroblast and collagen density. The combination of VCO and black cumin oil provided an enhanced histological overview compared to single treatments, with C1 yielding optimal results.

Figure 2: Wound diameter on days 1, 7, and 14 in the control group, VCO, Black cumin oil, C1= 50% VCO: 50% Black cumin oil, C2= 70% VCO: 30% Black cumin oil, C3= 30% VCO: 70% Black cumin oil

Figure 3: TNF-α expression after intervention on day 7. C1= 50% VCO: 50% Black cumin oil, C2= 70% VCO: 30% Black cumin oil, C3=30%VCO: 70 % Black Cumin oil. NS: not significant, ***p<0.001, ****p<0.0001

Figure 4: TNF-α expression after intervention on day 14. C1= 50% VCO: 50% Black cumin oil, C2= 70% VCO: 30% Black cumin oil, 70%, C3= 30% VCO : 70% Black Cumin oil. **p<0.01,***p<0.001, ****p,0,0001

Figure 5: Epidermis thickness after intervention on day 7. C1= 50% VCO: 50% Black cumin oil, C2= 70% VCO: 30% Black cumin oil, C3: 30%VCO: 70% Black Cumin oil. *p<0.05,**p<0.01

Figure 6: Epidermis thickness after intervention on day 14. Thickness. C1= 50% VCO: 50% Black cumin oil, C2= 70% VCO: 30% Black cumin oil, C3= 30%VCO: 70% Black Cumin oil. Ns: non-significant, **p<0.01

Figure 7: Dermis thickness after intervention on day 7. C1= 50% VCO: 50% Black cumin oil, C2= 70% VCO: 30% Black cumin oil, C3= 30%VCO: 70 Black cumin oil. *p<0.05 ***p<0.001, ****p<0,0001

Figure 8: Dermis thickness after intervention on day 7. Thickness. C1= 50% VCO: 50% Black cumin oil, C2= 70% VCO: 30% Black cumin oil, C3= 30%VCO: 70% Black Cumin oil. ***p<0.01, ****p<0.0001

Figure 9: Histology of skin tissue on day 7. Control group (a,m), VCO group (b,n), Black cumin oil group (c,o), Black cumin oil= 50% VCO: 50% (d,p), 70% VCO: 30% Black cumin oil (e,q), 70%Black cumin oil: 30%VCO (f,r). Showing epidermis (E) and dermis (D). granulation tissue (G) in the dermis contains collagen matrix (↓) with inflammatory cells (▼).

Figure 10: Histology of skin tissue on day 14. Control group (a,m), VCO group (b,n), Black cumin oil group (c,o), Black cumin oil= 50% VCO: 50% (d,p), 70% VCO: 30% Black cumin oil (e,q), 70%Black cumin oil: 30%VCO (f,r). Showing epidermis (E) and dermis (D). granulation tissue (G) in the dermis contains collagen matrix (↓) with inflammatory cells (▼)

DISCUSSION:

After the diabetic induction, a wound is made on the back of the neck, which healing will be delayed, according to reports on diabetic impairs wound healing⁴³. In this analysis, VCO and Black Cumin Oil, as well as their combination, accelerated wound healing process on days 7 and 14 in diabetic rat wound. Furthermore, the combination of VCO and black cumin oil in the C2 and C3 groups significantly reduced TNF-α expression. In diabetes, inflammatory cytokines and chemokines increase, such as TNF-α, leading to a prolonged healing. The TNF-α role in diabetic patients is highly significant, and its inhibition is considered a potential therapeutic study to improve healing outcomes ⁵. Moreover, an essential healing component is the inflammatory transition from proliferation stage to initiate wound healing and remodeling. During this process, prolonged inflammation is indicated by increased inflammatory cytokines, TNF-α ⁴⁴. This has led to the intensive study of TNF-α as an inflammatory cytokine is crucial in the pathogenesis of several inflammatory diseases ⁴⁵. As an important therapeutic target ⁴⁶, the RT-PCR method was applied to measure mRNA levels of inflammation-related genes such as TNF-α ⁴⁷. The decrease observed in TNF-α gene expression directly contributed to the acceleration of wound healing process⁴⁸.

A previous study has established that VCO has anti-inflammatory activity ⁴⁹^,⁵⁰. In vitro studies have also shown that VCO topical application offers anti-inflammatory activity by inhibiting cytokine levels (TNF-α, IFN-g, IL-6, IL-5, and IL-8). This plant-derived product increases skin barrier by upregulating AQP-3, filaggrin, and involucrin mRNA expression. Additionally, by protecting against UVB irradiation, VCO can treat skin disorders with permeability barrier dysfunction, particularly followed by reduced epidermal protein expression, such as atopic dermatitis and eczema¹⁹. Another study showed that VCO gel could affect TNF-α and TGF-β1 expression in periodontal tissue regeneration process in rat induced with periodontitis⁵¹. The inflammatory process was significantly increasd in diabetic wound, followed with high pro-inflammatory cytokines, and TNF ⁵². In diabetic wound, TNF-α levels are usually elevated, prolonging the inflammatory processes ⁵³. Similarly, high TNF-α can inhibit TGF-b and collagen gene expression, decreasing cell proliferation ⁵⁴.

Another report explained that black cumin has a therapeutic effect on skin healing through its anti-inflammatory, tissue growth stimulation, and antioxidative properties ⁵⁵. Black cumin oil accelerates the wound healing process⁵⁶. The IL-6, TNF-α, and NO secretion including pro-inflammatory mediators, by primary macrophages, is suppressed by black cuminaqueous extract, exerting anti-inflammatory effect in vitro⁵⁷. Furthermore, black cumin oil has been shown to accelerate wound healing, which is highly effective with honey ⁵⁸. Oil gel also has a negligible effect⁵⁹, ²⁶, capable of accelerating healing in streptozotocin-induced diabetic ras⁶⁰. The anti-inflammatory and antimicrobial properties of black cumin are included⁶¹. It was reported that NSO improved the antioxidant status associated with healing process in diabetic wounded rat by raising GPx, SOD, CAT, and GSH levels⁶². This study showed black cumin extract accelerated wound healing, leading to greater epidermal thickness, collagen fibers, and fibroblast infiltration in the treatment group compared to the control ⁶⁰. A greater granulation tissue formation, angiogenesis, fibroblast proliferation, and collagen synthesis was observed in the normal saline extract oil group than the control⁶³.

In this study, it was discovered that the histological representation of VCO treatment showed improved tissue conditions, characterized by less granulation, lower epithelium, and reduced inflammation. This suggested stimulation of wound healing with inflammation suppression and faster collagen formation, resulting in less granulation tissue width and fewer scars post-wound. Similarly, previous study stated that granulation tissues treated with VCO indicated a greater fibroblast proliferation towards the periphery and total collagen content compared to the control ¹⁸.

Treatment with black cumin oil showed a different histological pattern, characterized by thicker epithelization, higher inflammatory cell distribution, and a wider but looser granulation compared to VCO group on the same day. This histological representation showed a different wound healing pattern. Black cumin oil treatment tended to stimulate more dominant epithelium growth and did not suppress inflammatory cell infiltration compared to VCO. Another study explained that black cumin extract oil promoted wound healing by inducing angiogenesis, fibroblast proliferation, and collagen synthesis during healing ⁶⁰, resulting in wider granulation tissue. In some conditions, including type II DM, epithelization tended to be disturbed, accompanied by suppressed leukocyte function, leading to impaired wound healing, such as microcirculation disorders. Diabetic wound often depend more on granulation and reepithelialization to heal, causing poor tolerance of DM scars due to inadequate wound contraction⁶⁴. Wound healing pattern in black cumin oil treatment may be useful in conditions where wound epithelization and granulation are inappropriate, such as in type II DM, which requires further study.

The integration of Virgin Coconut Oil (VCO) and Black Cumin Oil into wound care practices holds promise for enhancing patient treatment outcomes, particularly in complex wound conditions such as those seen in diabetic patients. However, further research is warranted to validate the clinical effectiveness and safety of utilizing both oils, as well as to develop more standardized dosing guidelines and formulations. With a deeper understanding of their potential benefits and limitations, the utilization of VCO and Black Cumin Oil could serve as valuable components in the endeavor to improve wound care holistically.

The results showed that the topical application of a combination of VCO and black cumin oil on diabetic wound reduced expression of TNF-α. This showed synergistic effect of the combination in accelerating the inflammatory phase in healing of diabetic wound, where the treatment pattern combined with VCO and black cumin oil showed an intermediate effect between the histological patterns. The pattern observed was suitable for wound healing, such as diabetic ulcers, where epithelization, granulation, and leukocyte function are disrupted. However, the results are expected to yield final wound outcomes with less extensive granulation.

CONCLUSION:

This study showed that theVCO topical application and black cumin oil combination could suppress the prolonged inflammatory phase in diabetic rat wound by inhibiting TNF-α. The histological representation of wound healing in diabetic rat showed positive effect with accelerated healing in all treatments. Moreover, treatment with VCO and black cumin oil showed different wound healing pattern

CONFLICT OF INTERESTS:

The authors declare no conflict of interest.

ACKNOWLEDGMENTS:

Gratitude is expressed to andalas university for their support all lab studies and Stikes Syedza Saintika for Funding studies.

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Received on 25.12.2023 Revised on 21.03.2024

Accepted on 04.05.2024 Published on 24.12.2024

Available online from December 27, 2024

Research J. Pharmacy and Technology. 2024;17(12):6076-6083.

DOI: 10.52711/0974-360X.2024.00921

Control	VCO	BCM	C1	C2	C3
						Day 1
						Day 7
						Day 14