*t* Student's test; \*\*\* *p* < 0.001; \* *p* < 0.05; <sup>a</sup> difference between paw volume increase (%) seen for ZIZ-L and B-L; <sup>b</sup> difference between paw volume increase (%) seen for IND and B-L.

The results of our research indicate that the proposed topical dosage form is of appropriate quality and has demonstrated its efficacy and safety in animal models. From an organoleptic point of view, a clear difference was observed in the aspect and the texture between the base alone and the *Ziziphus jujuba* Mill. hydrophobic ointment. Still, both displayed adequate characteristics, typical for the corresponding pharmaceutical semi-solid preparations. Even if a high amount of extract was incorporated into the base, a homogenous and stable ointment was obtained. Hydrophobic ointment bases have been reported in the literature also for other healing extracts of herbal origin, such as *Urtica simensis* Hochst. ex A.Rich. [79], a mixture of herbal extracts from *Salvadora persica* L., *Azadirachta indica* A. Juss, and *Calendula officinalis* L. [80], or an extract from the *Acanthus polystachyus* Delile leaves [81].

Regarding pH, a slight decrease in the value was observed after including the extract in the base, but of little practical significance, as it is within the limits imposed by the compendial standards [52]. Taking into account the results recorded for cutaneous tolerance, the ointment pH seems proper for the recommended use. Additionally, the pH proved to be stable during the evaluated period.

In terms of spreadability (an important parameter for ointments), a minor difference in the behavior was noticed after adding the extract to the base [82]. The final spreading ability of the ointment was mainly influenced by the base and less by the extract included. The spreading properties displayed by the studied ointment reveal suitable structural and viscoelastic attributes and appropriate viscosity. The rheological behavior remains constant over time, confirming the product's stability.

Concerning the tolerance of the developed ZIZ-L ointment, the dermal irritation tests proved that it was well accepted, not inducing any reversible or non-reversible skin damage, and not affecting the somatic-motor functions. As animal skin is considered to be very sensitive to most pharmaceutical ingredients [83], the results obtained provided strong evidence for the lack of risk when applying the proposed formulation even for a prolonged time. This is in part due to the selected lipophilic base, as shown by the results, but also to the plant extract. The dried extract of *Ziziphus jujuba* leaves was included in a high amount and was incorporated by suspension, leading to no deleterious dermal responses, including erythema and edema.

This in vivo experiment confirmed that ZIZ-L ointment treatment significantly accelerated wound healing in Group 2, compared to control Groups 1 (untreated) and 3 (B-L treated). Wound healing resulted in a considerable decrease in the damaged epidermis length compared to controls. Visual examination showed that the epidermis recovery was faster in the wound treated with the ointment containing *Ziziphus jujuba* Mill. extract than those treated with the lipophilic base, suggesting that the extract owns a potent therapeutic

effect in the wound healing process. The wound closure percentage displayed by the ZIZ-L ointment application was similar to the one achieved after the reference product administration, confirming that the proposed formulation has the potential for treating skin wounds. This supports the traditional use of this species leaves for wound healing, as reported in India. Whereas previous research reported wound healing activity for extracts prepared from this species' fruits, bark or roots, our research was focused on a leaf extract prepared with 70% ethyl alcohol [84–86].

*Ziziphus jujuba* Mill leaves extract enhances skin wound healing through multiple mechanisms. The major active ingredients that were identified in the extract were chlorogenic acid, quercetin and rutin. The mechanisms of action are complex. Chlorogenic acid promoted fibroblastic and remodeling phases of wound healing. It accelerated the wound closure in the presence of keratinocyte [87]. Quercetin improves wound healing by inhibition of matrix metalloproteinases, which are normally inhibited by plasminogen activator inhibitor 1 (PAI-1) [88]. Also, it promotes a normal regeneration, not a fibrosis because it influences positive cell migration and proliferation, increases surface αV integrin and decreases β1 integrin in wounds, and increases the production of collagen fibers which are well oriented in sub-epidermal tissue [89,90]. Rutin promotes wound healing by several mechanisms: it enhances the production of antioxidant enzymes in the presence of erythroid 2-related factor 2 (NRF2), inhibits the expression of matrix metalloproteinases (MMPs) and decreases the expression of vascular endothelial growth factor (VEGF). It also induces the expression of the neurogenic-related protein (UCH-L1) [91].

Regarding the in vivo anti-inflammatory effect of *Ziziphus* ointment, the registered results show its potential properties. Even though the activity proved to be slower than the one displayed by the reference product, it was significantly higher than the lipophilic base action. The results support using the ointment as an herbal remedy for curative purposes in various topical inflammatory processes. Considering the chemical composition of *Ziziphus jujuba* Mill. leaves extract, its anti-inflammatory effect is due to its major constituents. Chlorogenic acid has antioxidant properties and because of that, it reduces the expression of inflammatory molecules. It inhibits phospholipase A2, cyclooxygenases and lipoxygenases, and reduces the concentrations of prostanoids and leukotrienes, especially PGE2 (Prostaglandin E2), IL-1β (Interleukin 1 beta), interferon-γ, monocyte chemotactic protein-1, and macrophage inflammatory protein-1α [92,93]. Quercetin inhibits cyclooxygenase (COX) and lipoxygenase (LOX) which catalyzes the production of inflammation molecules especially LTB-4 [94,95]. Also, it inhibits lipopolysaccharide (LPS)-induced tumor necrosis factor α (TNF-α) production in macrophages and LPS-induced IL-8 production [96,97]. It can also inhibit the production of tryptase and histamine and the downregulation of vascular cell adhesion molecule 1 (VCAM-1) and CD80 expression [98]. Rutin inhibits cytokines (e.g., TNF-a, IL-6) that are highly expressed and secreted by macrophages in inflammations [99]. It also activates nuclear factor-κB and extracellular regulated kinases 1/2 by HMGB1 (High mobility group box 1) [100].

We have several reasons to consider that the wound healing process from this study consists of regeneration and not fibrosis. The injuries that we treated with the *Ziziphus* ointment are mild and the epithelial tissue that are involved has an important regenerative potential [101]. The time of complete healing was short, less than a month. Furthermore, the stage of inflammation within the healing process is shortened (several days) by phenolic compounds and flavonoids from the *Ziziphus* extract that have anti-inflammatory properties. This fact may avoid a determination of fibrosis [102,103].

*Ziziphus jujuba* Mill. leaves' ointment exhibited pronounced wound-healing effects and moderate anti-inflammatory characteristics, thereby supporting its usefulness as a medicinal therapy.

#### **4. Conclusions**

The present evaluated an ointment containing 10% dried ethanolic extract from *Ziziphus* jujuba Mill., formulated in a hydrophobic base consisting of petrolatum, but also cetyl

alcohol, cholesterol, coconut oil and butylhydroxyanisole. The leaf extract used to prepare the ointment contains chiefly rutin (29.836 mg/g), quercetin (15.180 mg/g), and chlorogenic acid (350.96 μg/g); it also contains various amounts of phenolic acids, other flavonoids, fatty acids, organic acids and other compounds.

The formulated ointment has stable organoleptic properties related to those of the extract, is homogeneous, has a slightly acid pH (5.41–5.42), and appropriate rheological properties. It has demonstrated good tolerability following single and repeated administration in rat experiments. In rat wound models, the ointment resembled Cicatrizin in terms of its healing ability; both products accelerated healing, the effect being comparable.

The tested ointment showed an anti-inflammatory effect compared to the control group. In comparison to indomethacin, although the effect was slightly more pronounced for the herbal ointment, the difference was not statistically significant.

Due to its accessibility, good tolerance, and efficacy demonstrated in experimental models, the developed ointment is a promising therapy for wound healing and would be worth further exploring its benefits in a clinical setting.

**Author Contributions:** Conceptualization, M.-V.H., R.A., E.A.O., E.M. and A.I.A.; methodology, E.A.O., E.M., O.C.S, . and E.-I.G.; software, R.A.; validation, all authors.; formal analysis, R.A., O.C.S, ., E.M. and E.A.O.; investigation, E.A.O., E.M., O.C.S, ., E.O., A.I.A., C.B., E.-I.G., L.E.D., C.S.S. and E.N.; resources, M.-V.H., R.A.; data curation, R.A.; writing—original draft preparation, M.-V.H., R.A., E.O., E.A.O., E.M., O.C.S, ., C.B., C.S.S. and E.N.; writing—review and editing, all authors.; visualization, M.-V.H.; supervision, R.A., E.A.O., E.M. and L.E.D.; project administration, M.-V.H., R.A.; funding acquisition, R.A. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was supported by UMF Carol Davila—project number 33894/11.11.2014.

**Institutional Review Board Statement:** The animal study protocol was approved by the Bioethics Commission of the Faculty of Pharmacy, University of Medicine and Pharmacy Carol Davila, Bucharest (997/7 October 2016).

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data underlying this article will be shared upon reasonable request to the corresponding author.

**Conflicts of Interest:** R.A. received consultancy or speakers' fees from UCB, Sandoz, Abbvie, Zentiva, Teva, Laropharm, CEGEDIM, Angelini, Biessen Pharma, Hofigal, AstraZeneca, and Stada. All other authors report no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

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