Effect of ultrasound-enhanced Nigella sativa seeds oil on wound healing: An animal model.

OBJECTIVES: Nigella sativa oil, ultrasound, and moist-exposed burn ointment (MEBO) have been suggested as noninvasive treatments for a number of inflammatory conditions and to accelerate wound healing. The aim of this study was to evaluate the efficiency of pulsed and continuous modes of ultrasound either alone or through phonophoresis, with N. sativa oil, or MEBO ointment in the treatment of chemical burns.
METHODS: Thirty-five local rabbits were randomly divided into seven equal groups: pulsed ultrasound, continuous ultrasound, topical N. sativa oil, pulsed phonophoresis, continuous phonophoresis, topical MEBO ointment, and control group. Wound surface area was measured on days 0, 7, 14, and 21 using metric graph paper and photographs.
RESULTS: Significant differences were found between pre- and post-treatment wounds in all groups, except for the control group, in favor of the pulsed phonophoresis. In contrast, low results were found in favor of continuous ultrasound.
CONCLUSION: Ultrasound, topical application of N. sativa oil, phonophoresis, and MEBO ointment have the potential to accelerate wound healing induced by chemical burns. Such treatment modalities may be used to treat wounds.

Introduction

Wound healing represents a clinical dilemma whenever exact and suitable wound management is necessary. Many studies have investigated wound supervision, with more attention paid to distinct therapeutic procedures and the improvement of strategies for all types of wound management. Wound healing involves differential cell proliferation, extracellular components, and the effects of soluble chemical mediators, including growth factors and cytokines. Although the healing process is ongoing, it can be classified into four major steps: coagulation, the inflammatory phase, the reproductive phase, and the wound reconstructive phase, including scar tissue production. A suitable strategy to treat wounds can affect clinical outcomes.

Because of the dynamic structure of wounds and the high number of possible treatment modalities, it is not feasible to state which product is preferred for a specific wound, despite recommendations being made for various wound dressings. Nigella sativa (NS) has been used as a natural source of medicinal treatment for thousands of years by different societies and cultures throughout the world, as well as a food supplement to promote good health. It is very popular for the principle of respecting the holy prophet Muhammad P.B.U.H; “Hold on to use the Black seed, for it has a remedy for every illness except death.” Here, the term “hold on to” indicates the prolonged use.

NS seeds have been regularly and broadly used in herbal medicine to treat and prevent various skin injuries and diseases. NS oil (NSO) contains many types of proteins, saponins, and alkaloids. Most of the physiological effects of NS seeds are due to the presence of thymoquinone, which produces both essential and volatile oils, These have been reported to protect the body, especially the skin, against neurological disorders and hepatotoxicity caused by chemicals or a disease.11, 12 The beneficial effects associated with the use of NS seeds and thymoquinone may be due to their cell protection activities and antioxidant responses, and their influence on inflammatory mediators. Studies into major types of burns have gained the attention of inspectors, in terms of the value of the skin's biological role. Despite this, few studies have reported results of traditional low-intensity ultrasound on the healing of full-thickness burns. Accordingly, further studies investigating the value of ultrasound in the management of burn wounds are needed. Hence, the present study determined the effectiveness of different modes of ultrasound, phonophoresis, NSO, and MEBO on wound healing and tissue regeneration of full-thickness chemical burns. Phonophoresis is the transdermal administration of a medication with the aid of ultrasound. The transmission force of ultrasound emission pushes the medication distant from the transducer approaching the target area; the heat generated by the ultrasound intensifies the possibility of dissipation to the tissue leading to dilatation of the blood vessels, and the streaming induced by the ultrasound modifies cell permeability and improves tissue dispersion. Furthermore, sonic waves enhance the active potential of the drug, leading to more efficient distribution within the target tissue. MEBO is a local ointment produced to manage burns. It has a slight yellow-brown appearance, comprising wax originating from bees, sesame seed oil, proteins, fats, and carbohydrates. The principal constituent is beta-sitosterol. Additionally, MEBO comprises Chinese herbal constituents, such as Radix Scutellaria, Cortex Phellodendri, and Rhizoma Coptidis, which are thought to alleviate pain and improve tissue regeneration. Application of MEBO is an accessible approach to maintain moistened background for proper restoration, and for the formation of new epithelial tissue with no need of further spreading dressing. It was supposed to administer pain-free treatment and diminish the number of solutions needed for resuscitation.

Materials and Methods

Material and instruments

NSO was extracted by staff members from the College of Pharmacy, Aljouf University. MEBO ointment (0.25% beta-sitosterol) was from Gulf Pharmaceutical Industries, UAE. The ultrasonic device (model Sonopuls 490u) was from ENRAF NONIUS B.V (Netherlands). The measuring instrument included a transparent plastic film, metric graph paper (1 mm2), and a digital camera (NIKON D7500; Malaysia).

Animals

Thirty-five albino rabbits, approximately 20-weeks-old at the beginning of the study, and weighing 1000–1250 g, were obtained from the animal house, College of Pharmacy, Aljouf University. The rabbits were housed individually under hygienic conditions with determined environmental parameters that provided sufficient air passage, food complements, and lighting. Rabbits had unrestricted access to water and food (standard food as a grain). The enclosures containing each group were cleaned regularly. Anesthesia was provided by chloroform (bioethics agreement according to the Ethical Committee, Aljouf University, KSA).

Procedures

The dorsal surface of each rabbit was stripped mechanically and left to rest for day. On the following day, each rabbit was anesthetized with chloroform, as described by Divekar and Naik before induction of the burn wound. Concentrated HCl (38%) was applied topically to the bare skin (three drops) to provoke burn. The area of the burn was bathed with normal saline and then debrided to eliminate the residual dead tissue.

Experimental design: this was a double blind randomized comparative study conducted in an animal model.

After burn induction, rabbits were randomly divided into seven equal groups; A, B, C, D, E, F, and G. (Five rabbits in each group):

Group (A): Five rats received pulsed ultrasound.

Group (B): Five rats received continuous ultrasound.

Group (C): Five rats received topical application of NSO.

Group (D): Five rats received pulsed phonophoresis using NSO.

Group (E): Five rats received continuous phonophoresis through the use of NSO.

Group (F): Five rats received topical application of MEBO ointment.

Group (G): Five rats received local wound care.

Interventions

Treatment protocol

Treatment was initiated in each group the day following burn induction. Group A (PUS) received pulsed ultrasound, in which a sterile plastic bag filled with aquasonic gel was applied to all wounds and surrounding tissues to prevent any cross-contamination of the device. Pulsed ultrasonic (applicator 1.9 cm2) was applied over the gel pad while the ultrasound head was moved in a circular motion over the wound cavity. Holding the rabbits securely was essential to ensure the wound area was covered by the ultrasound applicator with the following parameters; pulsed duty cycle 20% (2 ms on, 8 ms off) and power density of 0.8 W/cm2, for 6 min, five times a week, over 3 consecutive weeks.

Group B (CUS) received continuous ultrasound with the following parameters: pulsed duty cycle 100% and power density 0.5 W/cm2, 6 min, five times a week, for 3 consecutive weeks.

Group C (NS) received topical application of extracted NSO, daily, with a 3 mL volume that dogged by a disinfected syringe then emptied to the burn area and set aside by non-adherent sterile gauze for three consecutive weeks.

Group D (PPH) received pulsed phonophoresis of NSO, in which a 3 mL volume of oil were dogged by a disinfected syringe then emptied onto the burn area. A sterile plastic drape was applied to all wounds and surrounding tissues to prevent any cross-contamination of the device. The plastic drape should overlap the wound margins by at least 3 cm and its surrounding ends fixed on to rabbit skin by adhesive tape. A pulsed ultrasound (applicator 1.9 cm2) was applied over a plastic drape and movement was initiated over the wound cavity or wound scab, then over the wound margins in all groups. Ultrasound therapy was in the ‘on’ mode only in group (A) with the following parameters; pulsed duty cycle 40% (4 ms on, 6 ms off) and power density 0.5 W/cm2, 5 min each session, five times a week, for 3 consecutive weeks.

Group E (CPH) received continuous phonophoresis of NSO through the aforementioned technique.

Group F (MEBO) received topical application of MEBO ointment to the wound bed twice daily for 3 consecutive weeks.

Group G (Control) received daily local wound care with normal saline.

Measurement protocols

All measurements were made 24-h post-burn, and at the end of each successive week over a 3-week period. Wound surface area was measured by the tracing method as described by Bohannon and Pfaller. A clean transparent sheet was placed on the wound. The wound margins were determined with a fine-tipped marker. The area of each wound was outlined three times to ascertain the reliability of the procedure. After outlining, the transparent sheet surface facing the burn was cleaned with sterilized cotton and ethanol. Carbon paper was placed over the metric graph paper (1 mm2). The transparent sheet was fixed over the carbon paper with white paper in between, and the outline was copied by tracing on the graph paper. The wound surface area was determined in millimeters-squared using metric graph paper. The surface area was transformed into centimeters-squared. The average of the three examinations was determined and used to denote the total burn surface area.

Photographs, used to determine changes in the healing process at the beginning of the study, and after 7, 14, and 21 days, were obtained with a digital camera with a span of 1.5 ft between the wound bed and the camera. at 90°.

Data analysis

Data were examined by SPSS v.20 using two-way ANOVA and Tukey's tests, and the results are presented as the mean ± standard deviation (mean ± SD) with a confidence interval of 95%. A p-value less than 0.05 was recognized as significant.

Results

One-way ANOVA revealed significant differences in the WSA values of all groups (A, B, C, D, E, and F) after 21 days of treatment, except for the control group (Group G), with p-values of <0.001, <0.001, <0.001, <0.001, <0.001, <0.001, and 0.214, respectively (Table 1).

Table 1

Mean and standard deviation (confidence interval 95%) for WSA before and after treatment in all groups.

Mean, standard deviation, and p-value for WSA before and after the treatment (cm2)
	0 days	7 days	14 days	21 days
Group A	14.26 ± 0.24	13.72 ± 0.28a	12.84 ± 0.57a	11.7 ± 0.54a
Group B	14.14 ± 0.34	13.64 ± 0.46a	13.04 ± 0.59a	11.96 ± 0.74a
Group C	14.4 ± 0.26	13.2 ± 0.11a	11.68 ± 0.19a	10.52 ± 0.28a
Group D	14.08 ± 0.53	13.36 ± 0.26a	11.36 ± 0.48a	9.52 ± 0.38a
Group E	14.14 ± 0.28	13.14 ± 0.94a	12.2 ± 0.91a	11.1 ± 1.13a
Group F	14.28 ± 0.54	13.88 ± 0.61a	13.42 ± 0.93a	10.18 ± 0.26a
Group G	14.18 ± 0.28	14.1 ± 0.20	14 ± 0.16	13.9 ± 0.19

WSA: wound surface area.

Statistically significant.

When comparing the results of WSA before treatment, there was a significant difference after 7, 14, and 21 days with p values of 0.029, <0.001, and <0.001, respectively; the p-value at the beginning of the treatment was 0.882, as displayed in Table 1.

Two-way ANOVA showed that there was a significant effect of treatment type, time, and the interaction of treatment type and time, with p-values of <0.001, <0.001, and <0.001, respectively, as presented in Table 2.

Table 2

Mean difference (confidence interval 95%) between all groups in the study after 7, 14, and 21 days.

Groups and time			Mean difference (confidence interval 95%)
Group A	0 days	7 days	0.54000
		14 days	1.42000a
		21 days	2.56000a
	7 days	14 days	0.88000a
		21 days	2.02000a
	14 days	21 days	1.14000a
Group B	0 days	7 days	0.50000
		14 days	1.10000a
		21 days	2.18000a
	7 days	14 days	0.60000
		21 days	1.68000a
	14 days	21 days	1.08000a
Group C	0 days	7 days	1.22000a
		14 days	2.70000a
		21 days	3.86000a
	7 days	14 days	1.48000a
		21 days	2.64000a
	14 days	21 days	1.16000a
Group D	0 days	7 days	0.72000
		14 days	2.72000a
		21 days	4.56000a
	7 days	14 days	2.00000a
		21 days	3.84000a
	14 days	21 days	1.84000a
Group E	0 days	7 days	1.00000
		14 days	1.94000a
		21 days	3.04000a
	7 days	14 days	0.94000
		21 days	2.04000a
	14 days	21 days	1.10000
Group F	0 days	7 days	0.40000
		14 days	0.86000
		21 days	4.10000a
	7 days	14 days	0.46000
		21 days	3.70000a
	14 days	21 days	3.24000a
Group G	0 days	7 days	0.08000
		14 days	0.18000
		21 days	0.28000
	7 days	14 days	0.10000
		21 days	0.20000
	14 days	21 days	0.10000

Statistically significant.

In terms of the efficacy of each treatment, the results favored the pulsed phonophoresis group (Group D) with high percentage, while the least effect was in the control group (Group G) with very low percentage as revealed in Table 3.

Table 3

Percentage improvement in wound surface area in all groups.

Percentage improvement in all groups (%)
	7 days	14 days	21 days
Group A	3.8	6.4	8.9
Group B	3.5	4.4	8.3
Group C	8.5	11.3	9.9
Group D	5.1	14.9	16.2
Group E	7.1	7.2	9
Group F	2.8	3.3	14.2
Group G	0.6	0.7	0.7

Discussion

Wound healing is the process through which broken tissue is repaired as similarly as it reaches to its healthy status, while wound contraction is way of diminishing the surface area of the wound. This principle depends on the capability of the tissue to adjust, the nature and degree of impairment, and the overall status and vitality of the tissue. The repair of skin injury is characterized by an established and definite array of physiological issues that are associated with wound contraction and advancing the regeneration and reconstruction of the spoiled tissue. Recent studies have found that wound healing sequelae might be hastened and improved with the aid of herbal or synthetic composites. Plant ingredients could enhance the property of wound restoration.

In the current study, a chemical wound was induced with concentrated HCL in rabbits to investigate the wound healing capacity as well as the antimicrobial load of pulsed ultrasound, continuous ultrasound, topical application of NSO, transdermal delivery of NSO through both pulsed and continuous modes of ultrasound, and the topical application of MEBO ointment. The outcomes of our study provide strong evidence for the ability of these treatment modalities to accelerate wound closure and healing of burns in an animal model after 21 days of treatment; the pulsed phonophoresis group was favored. The better results of group D and the superiority to other groups (pulsed phonophoresis) may be attributed to the use of NSO, which was augmented by pulsed ultrasound; NSO enhanced wound healing in farm animals. Furthermore, ether extracts of NSO applied topically to infected skin wounds in mice promote healing by diminishing both total and absolute differential white blood cells, cutaneous germs, inflammation, bacterial augmentation, and tissue damage. Aqueous extracts of NS presented weak free radical scavenging activity, induced gingival fibroblast reproduction, and enhanced wound contraction properties, although it had a non-significant impact on collagen construction. It also resulted in increased levels of growth factors. NSO exerts a range of treatment effects, which reduce inflammation, decrease blood glucose level, bactericidal action, remove free radicals, potentiate the immune system, are antiallergic, and lower blood pressure. The anti-inflammatory and antioxidant activities of NSO may be due to the presence of fixed and essential oils. The anti-inflammatory and analgesic effects of NSO may be due to the high amount of thymoquinone, which is an effective component of oil.

Fatty acids are essential compounds of the cell membrane, and are required to assure epidermal well-being and to maintain the water barrier of the skin. Fatty acids, such as oleic and linoleic acid, might promote wound healing through improvement of the entire quantity of cells transferring over the wound track during the compensation process. Furthermore, fatty acids also activate neutrophils, which play an important role in wound healing through the release of cytokines and growth factors, enhancement of antibacterial activity, and the removal of destroyed tissue. Ultrasound at lower intensities, similar to those utilized in the current study to enhance the transdermal delivery of oils, diminish cellular destruction through the use of pulsed acoustic power, which accelerates fibroblast reproduction and improves tissue restoration in different kinds of wounds. In contrast, Cambier and Vanderstraeten assessed the impact of pulsed LITUS with a duty cycle of 20%. They used LITUS on 20 female rats with full-thickness burns for 5 min over 6 weeks, and found non-significant histological data.

The results of the current study showed that the pulsed ultrasound led to better results than continuous ultrasound when applied alone or through the phonophoresis technique. This is because low-intensity ultrasound and the pulsed mode initiate the mechanical effects of ultrasound, which allow drugs to be transferred and to be deposited in target tissues the power which thermal influence does not admit and this harmonious with Loyd et al., who declared that phonophoresis promotes the transdermal penetration of a drug by mechanical impacts such as cavitation property and acoustic power. The drugs are incorporated within the intracellular space, between cells and in the appendages. While appropriating ultrasound for delivering drugs through the skin, blood flow increases in target tissues and the drug crosses the skin stratum corneum, transferring immediately to the blood circulation before reaching the target tissues where it induces a rapid effect. Numerous laboratory studies have attempted to clarify the impacts of ultrasound on the wound healing process. To date, ultrasound has been shown to induce cellular enhancements, the formation of new collagen, improved tensile strength of collagen fibers, formation of new blood vessels, wound approximation, stimulation of both fibroblasts and macrophages, breakdown of fibrin, modification of inflammation, and advancement of the proliferative process of wound.

MEBO treats wounds with moisture, which originates from the oil, and berberine. Additionally, it inhibits microbial growth and encourages the rapid formation of epithelial tissue. This is thought to be supported by beta-sitosterol, a corticosteroid located in different plants, mainly soya. MEBO diminishes the vulnerability of the burn, which, as noted by Smahel et al., restricts tissue destruction and promotes restorative effects. Although the in vitro antibacterial effects of MEBO have not been explained, studies have shown that it limits wound infection, probably by hindering invasion of eschar formation via injurious microbes. Maintenance of organic serum on the wound surface inhibits the desiccation of cells and improves the movement of keratinocytes over the wound. It also enhances the capacity of common cytokines and growth agents to heal wounds in ways that are yet to be described. This study was limited to a small sample size, with the hope that future studies using large sample sizes will gain more evidence-based results through the use of different plant sources that can be delivered to the body via phonophoresis.

Conclusion

Wound healing can be accelerated by various mechanisms that promote wound contraction and inhibit inflammation by means of pulsed phonophoresis using NSO as an adjunct treatment with limited side effects.

Conflict of interest

The authors have no conflict of interest to declare.

Ethical approval

All the animals which had been included in this study were evaluated and treated according to the principles of the guide for the care and use of laboratory animals (DHHS Publ. No. (NIH) 85-23, Revised 1985) and conformed to the approval of the ethical committee of Aljouf University, KSA.

Authors' contribution

HE designed the study, conducted phonophoresis studies, wrote the article, and carried out careful revision of this article. SS conducted the statistical analysis interpretation. SA supervised the work. All authors have critically reviewed and approved the final draft and are responsible for the content and similarity index of the manuscript.