Moringa oleifera leaf extract–loaded phytophospholipid complex for potential application as wound dressing

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Abstract

Research has indicated that Moringa oleifera (MO) leaves aqueous extract possesses wound healing activity. However, the wound delivery of a plant aqueous extracts is often poor due to the large molecular sizes and poor lipid solubility of the polyphenols. In this study, a phyto-phospholipid complex (phytosome) containing MO aqueous extract was developed to maximize topical wound delivery of MO. Phytosomes containing MO (MOPCT) were formulated and characterised in terms of morphology, particle size, polydispersity, zeta potential, and encapsulation efficiency. In vitro normal human dermal fibroblast cells (NHDF) cytotoxicity and cell migration assay was also carried out on filtered MOPCT and controls. Under TEM, the MOPCT appeared as multilamellar vesicles with an average size of 198 ± 21 nm and a zeta potential −28.30 ± 1.31 mV. The encapsulation efficiency showed that quercetin (82.8%) kaempferol (52.2%), chlorogenic acid (15.6%), rosmarinic acid (8.44%) were present in the filtered MOPCT. The filtered MOPCT exhibited the highest NHDF cell migration and proliferation rate compared to the controls. The MOPCT also did not cause cytotoxicity at concentrations <1.5 mg/mL. In conclusion, MO phytosomes were successfully developed and exhibited a significant potential to be formulated into a functional wound dressing.

Introduction

Moringa oleifera (MO), commonly known as Merunggai among countries in Southeast Asia, is a highly valued medicinal plant found in the tropics and the subtropics. The leaves of MO have been traditionally used in the treatment of sores; alleviating fever, sore throat, bronchitis, and eye and ear infections; and to reduce glandular swelling [1]. Over the years, both in vitro and in vivo models have well documented the wound healing property of MO leaves [2,3]. Oral administration of the aqueous extract of MO leaves to male Swiss albino mice demonstrated a faster healing time in resutured incisions and dead space wounds [4]. Another study showed the highest potency of aqueous extract in stimulating cell proliferation and normal human dermal fibroblast (NHDF) cell migration with lowest toxicity compared with methanol and ethanol extracts. In addition, the aqueous extract of MO leaves exhibited potent anti-inflammatory effects demonstrated by the inhibition of carrageenan-Choose an item. induced edema in albino rats [5]. The aqueous extract was also found to reduce the levels of inflammatory markers in macrophages, including induced nitric oxide synthase (iNOS), tumor necrosis factor-α (TNF-α), and interleukin-1β (IL-1β) [6]. These properties of the MO leaf extract encourage its development into a commercially available wound healing aid.

Several bioactive phytochemicals such as polyphenols are water-soluble molecules. However, the large molecular sizes and the high water solubility hinder the topical absorption of polyphenols, thereby limiting their ability to pass across lipid-rich biological membranes [7]. In recent years, phytochemicals have been complexed with phospholipids, primarily phosphatidylcholine to form phytophospholipid complexes (or phytosomes), a type of liposomal formulation that can encapsulate plant bioactive compounds to facilitate drug penetration into cell membranes, thus increasing topical therapeutic effectiveness and reducing the risk of systemic effects [8,9]. It has been reported that liposomal formulations provide a prolonged local therapeutic action [10]. In addition, liposomal formulations are known to increase the permeability of active compounds across the stratum corneum [11]. Phytosomes have emerged as a promising delivery system as the phospholipid structure mimics the structural component of the mammalian cell membrane [8], thereby enabling the active compounds to passively penetrate the lipoidal membrane without damaging the cells [12]. Therefore, phytophospholipid complex formulations are increasingly being explored to formulate plant extracts.

To our knowledge, there are yet no commercially available wound healing agents developed using the MO leaf extract; moreover, the potential of the leaf extract to be developed into liposomal formulations for wound healing applications has not yet been widely explored. Therefore, the objectives of this study were to formulate phospholipid complexes loaded with the MO aqueous leaf extract and to characterize the physicochemical properties of the phospholipid complexes as well as the cytotoxicity and in vitro wound healing effects of the MO phytosome formulations.

Section snippets

Chemicals and reagents

The chemicals soybean-derived l-alpha-lecithin (purity ≥97%) (Acros Organics, USA), cholesterol (cholesterin) (Nacalai Tesque, Kyoto, Japan), vicenin-2 (HPLC grade, purity ≥99%) and Tween® 80 (Sigma-Aldrich, Germany), dichloromethane (Ajax Finechem, Australia), N-hexane (95% purity) and UHPLC grade acetonitrile (J.T. Baker, USA), ammonium formate (purity >98%) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), and formic acid (purity >98%) (Tokyo Chemical Industry, Japan) were used as

Extraction and phytochemical screening of MO aqueous leaf extract

The MO aqueous leaf extract was extracted by macerating ground MO leaves in deionized water. A clear, dark brown color extract was obtained after the filtration. The average yield of the extract was calculated using its dry weight after the extraction and freeze-drying process. Ground MO leaves weighing 35 g resulted in a yield of 14.3 ± 0.5 g of dried MO aqueous leaf extract. The yield of the MO aqueous leaf extract was 40.8% ± 1.3%.

Phytochemical screening of MO aqueous leaf extract

Chlorogenic acid, kaempferol, quercetin, rosmarinic acid,

Conclusions

Phospholipid complexes containing MO leaf extract are spherical in shape and exist in the form of multilamellar vesicles. Chlorogenic acid, kaempferol, quercetin, rosmarinic acid, rutin, and vicenin-2 are detected in the MO aqueous leaf extract. In addition to rutin and vicenin-2, four other compounds have were encapsulated into MOPCT with efficiency as high as 82.8%. Through MTT assay, MOPCT was found to be nontoxic to NHDF cells at doses 1.5 mg/mL and below. Based on the cell migration assay,

Declaration of competing interest

The authors have no conflicts of interest in this study.

Acknowledgement

This research is financially supported by the NRGS grant (NH1014 S016), Ministry of Agriculture, Goverment of Malaysia. Research facilities support was provided by the Universiti Kebangsaan Malaysia. Fresh MO leaves were collected by Pn Hazni Mohammad, a senior science officer from the Faculty of Pharmacy, University Kebangsaan Malaysia. The leaves were authenticated by Dr Shamsul Bin Khamis from Herbarium University Kebangsaan Malaysia, Bangi.

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