and cytokine-modulating potential of medicinal oil formulation comprising leaf extract of Murraya koenigii and olive oil

The study investigated the wound healing effect of medicinal oil (MO) formulation prepared from Murraya koenig ii leaves extract (methanolic) incorporated in olive oil. The MO was visually transparent, homogenous, smooth in texture, the viscosity grade was observed as 140 cP and easily spreadable. Pro-inflammatory cytokines IL-1 β , IL-6, and TNF- α were significantly reduced to 82.3 ± 3.5, 156 ± 6.2, 137.3. ± 5.5 pg/ml, respectively after treatment with MO when compared to disease control animals that showed IL-1 β , IL-6, and TNF- α levels of 170 ± 6, 265 ± 7, and 288.6 ± 11, pg/ml respectively. The level of pro-inflammatory cytokine in povidone iodine solution (PIS) group was 95.3 ± 3, 162 ± 6, 177.6 ± 8.9 pg/ml of IL-1 β , IL-6, and TNF- α respectively. Interestingly, the wound-healing efficacy of MO was found better as compared to povidone iodine treated standard group and concluded that MO has excellent wound healing effect.


Introduction
Wound healing is a series of complex processes that comprise various phases such as reduction of inflammation, epithelialization, angiogenesis, matrix deposition, and remodeling (Moni et al., 2018). Cytokines are involved in the recruitment of fibroblasts and epithelial cells because of inflammatory leukocyte stimulation, which leads to the development of granulation tissue. On the other hand, persistent microbial burden, accumulation of excess inflammatory proteins and biofilm formation are also the limitations associated with drug resistance in treatment of wounds. In addition, the generation of reactive oxygen species (ROS) aggravates

Abstract
The study investigated the wound healing effect of medicinal oil (MO) formulation prepared from Murraya koenigii leaves extract (methanolic) incorporated in olive oil. The MO was visually transparent, homogenous, smooth in texture, the viscosity grade was observed as 140 cP and easily spreadable. Pro-inflammatory cytokines IL-1β, IL-6, and TNF-α were significantly reduced to 82.3 ± 3.5, 156 ± 6.2, 137.3. ± 5.5 pg/ml, respectively after treatment with MO when compared to disease control animals that showed IL-1β, IL-6, and TNF-α levels of 170 ± 6, 265 ± 7, and 288.6 ± 11, pg/ml respectively. The level of pro-inflammatory cytokine in povidone iodine solution (PIS) group was 95.3 ± 3, 162 ± 6, 177.6 ± 8.9 pg/ml of IL-1β, IL-6, and TNF-α respectively. Interestingly, the wound-healing efficacy of MO was found better as compared to povidone iodine treated standard group and concluded that MO has excellent wound healing effect.

Solvent extraction
200 g powdered sample was packed in Soxhlet apparatus and subjected to methanolic extraction through hot continuous percolation technique at 60 °C for 3 h. Finally obtained solvent containing sample was transferred into glass beaker and evaporated at room temperature (Moni et al., 2021b).

Formulation of medicinal oil (MO)
Commercially available virgin olive oil (OLIO SASSO, Italy) was purchased from a local market. The clear viscous MO was prepared by constantly agitating the mixture of olive oil and dried extract (50% w/v) at 60 °C using glass stirrer. The oil was stored in a clean, sterilized glass bottle with screw cap at room temperature for further use.

Determination of viscosity of MO
The viscosity of MO was determined using Brookfield digital viscometer (Model LVDV-E, USA) with spindle S63. A 50 ml MO was transferred in to a 50 ml beaker and then allowed to settle for 5 min. The viscosity was measured at a rotating speed of 30 rpm at room temperature.

Selection, acclimatization and grouping of animals
Healthy male Wistar rats weighing 170 -200 g were purchased from the Central Animal facility of Jazan University, Jazan. They rats were acclimatized at standard laboratory conditions (22 ± 08 °C, and relative humidity of 56 ± 6%) in institutional animal house of College of Pharmacy, Jazan University. Animals were kept free access to a standard autoclaved laboratory diet and water. Prior to commencement of experimentation, the entire study protocol was approved from Institutional Research Review and Ethics Committee (IRREC-905/1012/1441).

Experimental design
Animal were divided in to four groups of six animals in each. The group distribution is as follows: Group 1: Normal control group: The animals of the group were without wounds and therefore did not receive oil treatment. Group 2: Disease control group: The excision wounds were created on Wistar rats according to the procedure established by Moni et al. (2018). Excision wound model was adapted for the measurement of wound contraction and epithelization in rats. Animals were given light ether anesthesia (diethyl ether) and their dorsal skin was shaved using electrical shaver. A circular piece (10 mm) of full thickened skin was cut off from the pre marked area using sterile biopsy punch. Wound areas were measured and recorded on day 3, 5, 8 and 12 for all groups on a graph paper. Meanwhile animals were also inspected for sign of infection, and infected animals were excluded from the study and replaced. In this group, the animals did not receive any oil treatment.
inflammatory response and induces impairment of healing of cutaneous wounds. The development of novel pharmaceutical formulations with immunomodulatory and antibacterial properties for wound healing has a major effect on bacterial colonization of wounds. Traditionally, medicinal plants are vital sources of therapeutic principles for many diseases globally (Moni et al., 2021a;Lin et al., 2020;Rakotoarivelo et al., 2015). Murraya koenigii (M. koenigii) is an aromatic tree which belongs to the family Rutaceae, popularly known as curry leaf. It is grown in tropical and sub-tropical regions for its high medicinal value and characteristic aroma (Noolu et al., 2013;Vandana et al., 2012). M. koenigii is widely used in traditional medicine and home remedies in India, Pakistan, Sri Lanka, China and Africa (Balakrishnan et al., 2020). The leaves of M. koenigii have been used medicinally for their anthelminthic, antiemetic, analgesic, digestive, appetite-stimulating, anti-dysentery, anti-pile, anti-inflammatory, and anti-itch properties, and used for healing of cuts, bruises, and oedema (Bhandari, 2012). Globally, chronic skin wounds represent a common health problem because such injuries are related to cut, scrape or scratch the skin (Desai et al., 2012). Chronic skin wounds are a common health problem worldwide.
According to Costa et al. (2016) Olive oil improves the healing of cutaneous wounds in chronically stressed mice due to its anti-inflammatory and antioxidant properties. In our previous study, it has been reported that methanolic extract of the leaves of M. koenigii contained steroids, phytosterols, terpenes, fatty acids and furofuran lignan (Moni et al., 2021b) which indicated the presence of immunomodulatory principles as major bioactive components. In continuation of previous research, the current study sought to determine the synergistic woundhealing efficacy of a methanolic extract of M. koenigii leaves and commercially available olive oil in a MO formulation through its effect on the cytokine network.

Plant collection and processing
M. koenigii stems with leaves were collected from trees in Jazan, the capital city of Jazan province, Kingdom of Saudi Arabia. The stem with leaves was washed twice in tap water at the point of collection and kept in slanting position for a few minutes to drain out the water. The washed specimens were identified in the herbarium of Jazan University (JAZUH), with the reference number 1215 (JAZUH). A voucher specimen of the plant was also deposited at the herbarium of JAZUH for future reference. The samples were packed in polythene bags (biohazard yellow bags), tied, and transported to the laboratory. The leaves were plucked out from the stems of the plant and thoroughly washed with Millipore water to remove any impurities present. The washed leaves were dried under shade for one week. The air-dried samples were cut into small pieces and powdered using a grinder. The finely powdered leaf samples obtained were pooled and stored in an air-tight container prior to use.
Group 3: Standard drug treatment group: The animals treated with 100 µl of 10 % w/v povidone iodine solution PIS on excision wound daily twice morning and evening. Group 4: Treatment with MO: The animals treated with 100 µl of MO on excision wound daily twice morning and evening.

Measurement of wound contraction
Wound size of was measured with a transparent ruler at the outset, and subsequently at 2-day intervals up to day 12. The percentage (%) healing of wound was calculated using the following Formula 1 (Nagar et al., 2016): Initial wound area wound area on a specific day Wound healing Initial wound area = × (1)

Collection of blood specimens
On sixth day of initiation of wound, animals were anesthetized under light ether anesthesia and blood samples were collected via ocular puncture. Serum samples were separated by centrifuging at 3000 g for 10 min. Then the serum samples were stored at -20 °C for the measurement of cytokine. Sandwich enzyme linked immunosorbent assay (ELISA) kits (Abcam, USA) were used to determine/estimate the serum levels of the proinflammatory cytokines IL-1β, IL-6, and TNF-α as described below.

Measurement of pro-inflammatory cytokines
Serum levels of IL-1β, IL-6 and TNF-α were measured quantitatively using their respective rat ELISA kits (ABCAM, USA). The assays employed a simple step sandwich ELISA to determine the serum level of each cytokine. Standards and samples were simultaneously pipetted and dispensed into their respective ELISA wells and incubated at room temperature for 2.5 h. During the incubation, the cytokine present in the sample was bound to the wells coated with immobilized specific antibodies. After incubation, the wells were washed thoroughly with 1× wash solution using Biotek ELISA washer elx50, USA. Then, the specific 1× biotinylated anti-rat antibody was added and incubated at room temperature for 1 h, with mild shaking. Thereafter, the plates were washed as previously described, to remove unbound biotinylated antibody. Then, HRP-conjugated streptavidin was pipetted out and added to the wells. The wells were washed again using the same washing parameters as described earlier. Then a TMB substrate solution was added to these wells and incubated for 30 min at room temperature in dark with mild shaking. Following this, stopping solution was added, and the intensity of the colour developed was measured by determining its absorbance at 450 nm using a BioTek ELx 800 ELISA reader. The absorbance was directly proportional to the amount of each cytokine bound to its specific antibody.

Statistical analysis
Statistical analysis was performed by using Graph pad Prism software (Version 8.3.1), USA through one-way analysis of variance (ANOVA), followed by Tukey Kramer analysis as a post-hoc test.

Results and Discussion
Wound healing is a complex sequential process involving haemostasis, proliferation, vascularization, matrix production and remodeling (Cogo et al., 2021;Reinke and Sorg, 2012). Many types of cells are involved in the wound healing processes such as immune cells, endothelial cells, keratinocytes, and fibroblasts (Valluru et al., 2011;Singer and Clark, 1999). Herbs have significant role in treating various diseases and the leaves of M. koenigii have been reported for numerous medicinal properties. Earlier studies have demonstrated that aqueous extract of M. koenigii accelerated the wound healing process (Shukla and Kashaw, 2019). It has been reported that methanol extract of the leaves of M. koenigii contained bioactive compounds such as epiyangambin, stigmasterol, eucalyptol, ethyl cinnamate, a-terpineol, fatty acids, and steroids (Moni et al., 2021b). The present study was aimed at investigating the wound healing efficacy of medicinal oil (MO) prepared by mixing the methanolic extract of leaves of M. koenigii with olive oil, with respect to its effect on levels of proinflammatory cytokines which are critical in the wound healing progress.
In the present study, MO was successfully prepared using a simple triturating method. The viscosity grade of MO was 140 cP, implying that it was visually transparent, homogenous, and smooth-textured. Table 1 depicts the percentages of wound healing in the various treatment groups. During treatment, the effect of MO on wound healing was manifested from the 3 rd day onwards. The results showed that MO exhibited excellent wound healing properties as the rats fully recovered before 12 days of the study period. The wound recovery by MO was significantly higher than PIS. This may be due to the synergistic combination of methanolic extract of the leaves of M. koenigii and olive oil. In an earlier study, it was reported that olive oil promoted wound healing on excisional wound (Schanuel et al., 2019;Donato-Trancoso et al., 2016). Low kinematic viscosity of oil is essential for the bioavailability of drugs at the tissue level (Rahman et al., 2012). The MO used in this study was viscous but easily spreadable at the site of injury. Therefore, the bioactive molecules in MO penetrated the wound tissue, resulting in healing effect. The synergistic healing effect of olive oil and honey on diabetic foot ulcers has been reported in a study by Karimi et al (2019). Olive oil is composed of hydroxytyrosol, oleuropein and many phenolic compounds with antibacterial and antioxidant properties (Moustafa and Atiba 2015;Al-Waili, 2003). Moreover, olive oil contains monounsaturated fatty acids which enhance the immune function (Yaqoob et al., 1998). An earlier study suggested that a methanolic extract of M. koenigii dried leaves had a substantial anti-inflammatory and analgesic effect when compared to the conventional medication diclofenac sodium (Gupta et al., 2010). According to Ani et al. (2016) the methanolic extract of M. koenigii leaves displayed antinociceptive and analgesic effect. Hydroalcoholic extract of M. koenigii fruit possesses substantial anti-inflammatory and wound healing effects (Gummalla et al., 2016). The levels of pro-inflammatory cytokines in the treatment groups are presented in Table 2. In this study, the levels of pro-inflammatory cytokines IL-1β, IL-6, and TNF-α were increased significantly in disease control animals (Group 2) following creation of the wound. It has been reported that proinflammatory cytokines were elevated by wound creation in streptozotocin-induced diabetes (Moni et al., 2018). The levels of pro-inflammatory cytokines were decreased when the wound treated with MO, when compared with the group treated with PIS. Figure 1 shows the levels of IL-1β in the various treatment groups. Group 2 represents the disease control group in which the IL-1β level was increased 136.4% after creating the wound, when compared to normal healthy rats (normal control, i.e., Group 1). Interestingly, treatment of the wounds with MO (Group 4) markedly reduced the IL-1β level by 51.58%, a level of reduction which was significant, when compared to PIS treatment group (p < 0.05). The level of IL-6 ( Figure 2), a potent proinflammatory cytokine, was reduced significantly by about 48% by MO treatment in group 4, while IL-1β reduction level was significant, relative to that of PIS treatment in group 3 (p < 0.05). Moreover, TNF-α, a pro-inflammatory marker was reduced when the wounds were treated with MO (Group 4), when compared to PIS treatment group (Group 3; p < 0.01). Figure 3 shows the serum levels of TNF-α in the various treatment groups. Serum TNF-α was reduced by about 50%, when compared to the disease control group (Group 2). The percentage reductions in the levels of pro-inflammatory cytokines were in the order: IL-1β > TNF-α > IL-6, although the reduction levels were more or less equal. Figure 4 shows stepwise in vivo wound-healing effects of MO and PIS. It was observed that MO had better wound-healing property than PIS.
Wound-infiltrating macrophages, dendritic cells, keratinocytes, fibroblasts, and mast cells release IL-1β, a pro-inflammatory cytokine which exerts pleiotropic mode of action. IL-6 is also a pleiotropic cytokine involved in tissue injuries, chronic inflammation, autoimmunity, and bacterial infection of wounds. It is released from tissue-resident macrophages, keratinocytes, endothelial cells, and stromal cells (Li et al., 2020;Rose-John et al., 2017). IL-6 is also associated with induction of chemotaxis of leukocytes in the wound and modulates immune response during the healing process by regulating leukocyte infiltration, angiogenesis, and collagen accumulation (Ambrosch et al., 2008).

Conclusion
The results of present study indicated that MO has effective in vivo wound-healing property. MO also effectively modulated the immune system through downregulating the expressions of pro-inflammatory cytokines and may be the contributing factor in wound healing. However, further investigation is required to get more insight.