Pyrus communis L. (Pear) and Malus domestica Borkh. (apple) leaves lipoidal extracts as sources for beta-sitosterol rich formulae and their wound healing evaluation

Abstract Pyrus communis L. (cv. Le-Conte) (pears) and Malus domestica Borkh. (cv. Anna) (apples) are economic fruit crops cultivated in Egypt. Their leaves were assessed for their beta-sitosterol content and found to have 9.4 mg/g dried leaves wt and 5 mg/g dried leaves, respectively. So we used the lipoidal leaves extracts in the formulation of eight beta-sitosterol-rich emulgels from which the most stable formulae were tested for their antimicrobial activity. Finally, the formulae which exerted antimicrobial activity were biologically evaluated for wound healing against well-known wound healing ointment Mebo® which is composed mainly of 0.25% beta-sitosterol in a base of sesame oil and beeswax. Wound contraction was statistically different in both formulae F3 and F8 from both control and Mebo® groups which indicated better wound healing activity of these formulae ensured by further histopathological study of the healed wounds. Graphical Abstract


Introduction
Current literature review describe that Pyrus communis fruits exhibit antioxidant and anti-ulcerative activity (Hamauzu et al. 2007), Antibacterial, antiseptic, antipyretic, aphrodisiac, astringent, diuretic, laxative and, anti-hyperglycemic (Duke 2002), also Malus domestics fruits and leaves have good antioxidant activities due to their phenolics content (Wojdyło et al. 2008) moreover, they exhibit neuroprotective activities (Cheng et al. 2014, El-Hawary et al. 2021) and cancer chemopreventive activity (Jaganathan et al. 2014). The previous literature reported the presence of beta-sitosterol in P. communis L. fruits and seeds (G orna s et al. 2016) and M. domestica Borkh. fruits (Wo zniak et al. 2018) and leaves as the main reported sterol, also triterpenes were reported, for example, ursolic acid, chemical derivatives, oleanolic and masilinic acid (He andLiu 2007, J€ ager et al. 2009). Moreover, three triterpenoids were isolated previously from P. communis stem bark (Mehta et al. 2003) and beta-sitosterol in P. communis L. twigs (Liu and Zuo 1987). Beta-sitosterol is known for anti-inflammatory and angiogenic activity (Moon et al. 1999, Choi et al. 2002 which resulted in the compound's potent wound healing activity, therefore this prompted our interest to use these extracts as a cheap source of beta-sitosterol. Many topical products are formulated by incorporation of beta-sitosterol in aim to be used as wound and burn healing formulae, for example, Mebo V R which showed good wound healing activity beside the low toxicity and lower cost in comparison to silver sulphadiazines products (Jewo et al. 2009). As reported previously, beta-sitosterol is the major phytosterol in P. communis L. and M. domestica Borkh. different organs, therefore, these plants became of interest to use their leaves lipoidal extracts as cheap, available, and probably effective raw material for wound healing formulae for the first time. Furthermore, examination of their activity biologically in comparison to Mebo V R .

Results and discussion
The GC/MS analysis of pear and apple leaves unsaponifiable fractions was reported in Table 1S representing several compounds with beta-sitosterol as the main compound in both extracts. So assays of beta-sitosterol in pear and apple leaves' n-hexane extracts and dried leaves were carried out by the method (Vorobyova et al. 2014) with beta-sitosterol standard calibration curve Figure 1S and concentrations for the extracts and the dried leaves were reported in Table 2S. The estimated concentrations showed that pear and apple leaves n-hexane extracts are rich, economic, and available sources of beta-sitosterol. Therefore, pear and apple leaves' n-hexane extracts were formulated to utilize them as cheap, non-greasy and effective beta-sitosterol-rich wound healing formulae due to the reported beta-sitosterol anti-inflammatory and angiogenic activity (Moon et al. 1999, Choi et al. 2002 which resulted in its potent wound healing. Eight formulae (F1-F8) were produced and their compositions were reported in a Table 3S. They were evaluated physically for their appearance shown in Figure 2S, pH, spreadability, and their rheological properties and the results were presented in Table  4S. The apparent viscosities were determined at increasing shear rates for the determination of the rheological behavior of the formulation (Tanriverdi and Yapar 2016). After evaluation of the eight formulae physical properties, accelerated stability testing was carried out as reported in the methods to select the stable formulae to precede the biological evaluation on them. There were five formulae F2, F3, F4, F7 and F8 which passed all accelerated stability tests and reserve their physical properties as a result, they were chosen for antimicrobial evaluation and finally, the best two formulae were selected for wound healing evaluation.
Firstly the antimicrobial activity was initiated to the five stable formulae by the method (Hindler et al. 1994), the results were represented in Table 5S. From the previous results Table 4S and 5S, F3 and F8 formulae were selected for in vivo wound healing testing.
Evaluation of wound healing activity of formulae F3 and F8 was processed according to (Mukherjee andSuresh 2000, Esimone et al. 2006). Results were introduced in a Table 6S and the rate of wound healing was represented for the four groups in Figures 3S and 4S. It was noticed that almost all the wounds of the treated group (4) completely healed after 14 days. F3 and F8 (Group 3 and 4) significantly enhanced wound healing compared to Mebo V R (Group 2). Wound healing can be attributed to the high beta-sitosterol content of n-hexane fractions used in these formulations, which was known for its wound healing activity (Moon et al. 1999), Moreover may be to the synergistic effect of other present sterols. The fastest wound healing was exerted by F8 then F3 and finally Mebo V R .
The histopathological alterations severity in the skin of the different experimental groups was represented in Table 7S and presented in Figures 5S-9S.
From the previous results, it was found that formulation (8) of P. communis L. leaves n-hexane extract followed by formulation (3) of M. domestica Borkh. leaves nhexane extract exhibited rapid wound healing activities according to the pharmacological and histopathological evaluation of their wound healing activity in comparison to the wound healing well-known market formula Mebo V R .

Experimental section
Figures from 1S-9S and tables from 1S-7S were provided as Supplementary data.

Conclusion
As a result, these two formulae F8 and then F3 can be recommended as good, economic, and applicable natural wound healing formulae that can be applied in largescale industry especially they yielded competitive wound healing results with the standard market product. professor of pathology, faculty of veterinary medicine, Cairo University for his help in carrying out the histopathological part.