Formulation and Biomedical Activity of Oil-in-Water Nanoemulsion Combining Tinospora smilacina Water Extract and Calophyllum inophyllum Seeds Oil

Abstract

Introduction: Tinospora smilacina is a native plant used in traditional medicine by First Nations peoples in Australia to treat inflammation. In our previous study, an optimised Calophyllum inophyllum seed oil (CSO) nanoemulsion (NE) showed improved biomedical activities such as antimicrobial, antioxidant activity, cell viability and in vitro wound healing efficacy compared to CSO.

Methods: In this study, a stable NE formulation combining T. smilacina water extract (TSWE) and CSO in a nanoemulsion (CTNE) was prepared to integrate the bioactive compounds in both native plants and improve wound healing efficacy. D-optimal mixture design was used to optimise the physicochemical characteristics of the CTNE, including droplet size and polydispersity index (PDI). Cell viability and in vitro wound healing studies were done in the presence of CTNE, TSWE and CSO against a clone of baby hamster kidney fibroblasts (BHK-21 cell clone BSR-T7/5).

Results: The optimised CTNE had a 24 ± 5 nm particle size and 0.21± 0.02 PDI value and was stable after four weeks each at 4 °C and room temperature. According to the results, incorporating TSWE into CTNE improved its antioxidant activity, cell viability, and ability to promote wound healing. The study also revealed that TSWE has >6% higher antioxidant activity than CSO. While CTNE did not significantly impact mammalian cell viability, it exhibited wound-healing properties in the BSR cell line during in vitro testing. These findings suggest that adding TSWE may enhance CTNE's potential as a wound-healing treatment.

Conclusion: This is the first study demonstrating NE formulation in which two different plant extracts were used in the aqueous and oil phases with improved biomedical activities.

Keywords: RSM; in vitro; medicinal plants; nanoemulsion; wound healing.

Figure 1

Liquid chromatography–mass spectrometry (LC-MS) chromatogram of Tinospora smilacina leaf water extract.

Figure 2

Three-dimensional response surface plot representing the effect of (A) Calophyllum inophyllum seed oil (CSO) (%), (B) Tween 80 (%) and (C) Tinospora smilacina water extract: high pure water (TSWE: HPW) (%) on (a) droplet size (nm) and (b) polydispersity index.

Figure 3

(a) Illustration of Tinospora smilacina water extract and Calophyllum inophyllum seed oil nanoemulsion (CTNE), (b and c) transmission electron microscopy of droplets in the CTNE. Scale bar (b) 500 nm and (c) 100 nm.

Figure 4

Cell Viability profile by MTT assay of Calophyllum inophyllum seed oil (CSO), Tinospora smilacina water extract (TSWE) and CSO nanoemulsion (CTNE) and TSWE at 24 h on hamster kidney (BSR) cell line.The x-axis indicates the NE concentration matched with the equivalent TSWE and CSO concentration content available in NE. The untreated control contained cells with media only. Each value is the mean of three replicates with standard deviation (±2 SD) and all conditions were significantly different to the untreated control analysed by two-way-ANOVA (n = 3; p < 0.05).

Figure 5

DPPH radical scavenging of Calophyllum inophyllum seed oil (CSO), Tinospora smilacina water extract (TSWE) and CSO nanoemulsion (CTNE) and TSWE. The DPPH results are the mean of three independent experiments with error bars ±2SD. The x-axis indicates the NE concentration matched with the equivalent TSWE and CSO concentrations available in NE.

Figure 6

(a) Percentage wound closure observed for Calophyllum inophyllum seed oil (CSO), Tinospora smilacina water extract (TSWE) and CSO nanoemulsion (CTNE) and TSWE at 24 h and 48 h in hamster kidney (BSR) cells. Blank indicates untreated cells (negative control), FGF indicates Fibroblast Growth Factor (positive control) and cells with media only negative control. Each value is the mean of three individual experiments with standard deviation (±2 SD) and analysed by two-way analysis of variance (ANOVA) (n = 3; p < 0.0001). (b) Relative concentration of components available in CTNE.

Figure 7

Wound closure photomicrograph of Calophyllum inophyllum seed oil (CSO), Tinospora smilacina water extract (TSWE) and CSO nanoemulsion (CTNE) and TSWE at 24 h and 48 h in hamster kidney (BSR) cells. (a) Untreated control. (b) Fibroblast growth factors (FGF; positive control). (c) CTNE:62.5 µg/mL. (d) TSWE:22.5 µg/mL. (e) CSO: 4.3 µg/mL. Black solid lines represent the wound size (μm) of the BSR cell line monolayer.