Centella asiatica extract‐SiO2 nanocomposite: More than a drug‐delivery system for skin protection from oxidative damage

Abstract An innovative nanotechnology‐based approach was used for the preparation of Centella asiatica (C. asiatica) extract‐SiO2 nanocomposites, specifically tailored for skin protection from oxidative damage. Different amounts of C. asiatica glycolic extract (1.0, 3.0, 5.0, and 10.0 wt %) and fumed silica were used to prepare the nanocomposites by means of ball milling method. The influence of both composition of the starting mixture and milling time on the final products was evaluated by different techniques such as X‐ray powder diffraction, scanning electron microscopy, infrared spectroscopy, thermogravimetric analysis, and nitrogen sorption analysis. Results confirmed the integrity of the natural extract after the milling process, and its successful loading in the silica matrix. No cytotoxicity was observed for the obtained nanocomposites, which showed high in‐vitro ability to scavenge 2,2‐diphenyl‐1‐picrylhydrazyl and to protect human keratinocytes from damages induced with hydrogen peroxide.


| INTRODUCTION
Recently, great attention has been devoted to the use of medicinal plants in the therapeutic treatment of skin diseases, mainly because of the antioxidant and anti-inflammatory properties of their bioactives.
On this regard, Centella asiatica (C. asiatica) is of particular interest. [1][2][3][4][5][6][7] It commonly grows in many parts of the world, including Asia where has long been used in traditional medicine due to its therapeutic properties for tissue regeneration, 8 cell migration, 9 and wound repairing. 10,11 The latter is related to its ability to promote fibroblast proliferation and collagen synthesis mainly due to saponin-containing triterpene acids and their sugar esters such as asiaticoside, asiatic and madecassic acid, as well as other phytochemical constituents such as flavonoids, sesquiterpenes, plant sterols, eugenol derivatives and caffeoylquinic acids. [12][13][14] Unfortunately, the in-vivo efficacy of its main components after skin application is limited by their low bioavailability especially when formulated in topical ointments. 15 We recently developed a one-pot-production method loading silica matrix with active substances using ball milling, which allows the retention of the properties of the bioactive after the milling process along with their metabolic composition. [16][17][18] In a previous study, Vitis Vinifera ethanolic extract was delivered in the silica matrix and the obtained nanocomposite was able to improve its antioxidant activity. 17 According to these promising results, in this paper, we present a novel nanocomposite based on C. asiatica glycolic extract and fumed SiO 2 , which was prepared as new strategy to treat skin damages linked to oxidative stress. In this new biomaterial concept, C. asiatica can scavenge free radicals at the skin lesion site and can block an abnormal growth of collagen-producing keloid cells, together with antimicrobial and anti-inflammatory effects. 19,20 On the other side, the silica matrix can act as both drug delivery system and promoter of wound healing. Indeed, our previous studies already showed the controlled drug release capabilities of the silica matrix 18 while several reports suggest that silica can affect tissue repair by promoting cytokine generation for collagen synthesis (scarring), and the generation of new blood vessels (angiogenesis). 21,22 Moreover, our new strategy benefits of the high availability of silica in nature, cost-effective and easy to scale-up production of the nanocomposites. 23,24 Along with the advantages described before, the incorporation of natural products in the silica-based delivery system can protect the natural extract from physical and chemical degradation, therefore enhancing its bioavailability and pharmacological activity. 17,18 2 | MATERIALS AND METHODS 2.1 | Nanocomposite preparation C. asiatica glycolic extract (2:1 E/D ratio) was purchased from Galeno srl (Comeana, Italy). Fumed Silica (99.8%) was purchased from Sigma Aldrich. All the reagents were used as received without further purification.
In 1 g of fumed silica and variable amounts of C. asiatica glycolic extract were sealed in a 60 ml agate vial with 22.68 g of agate balls. Different compositions of the starting mixture were selected to obtain final nanocomposites with a dry extract content of C. asiatica equal to 1.0, 3.0, 5.0, and 10.0 wt % (Table 1). Ball milling was performed in a planetary mill apparatus (Fritsch GmbH, Pulverisette 5) at 100 rpm, alternating milling and rest periods at 5 min intervals to prevent an excessive overheating of the vial. All the samples were dried at room temperature for 48 h after the milling process.

| Nanocomposite characterization
X-ray powder diffraction (XRPD) data were collected using CuKα radiation on a Seifert XRD 3000 TT diffractometer in the Bragg-Brentano geometry with a step size of 0.05 2ϑ degrees, in the angle range 5 < 2ϑ < 80 . An appropriate number of counts for each step was collected to improve the signal/noise ratio. F I G U R E 1 X-ray powder diffraction patterns of the milled fumed silica and of the most representative nanocomposites (S1, S2, S6, and S8 samples) coater for 2 min) and observed under an excitation voltage of 10 kV using a SEM S-4100, HITACHI.
Fourier-transform infrared spectroscopy (FTIR) analysis of the starting mixture and of the nanocomposites was carried out with a Bruker Tensor 27 spectrophotometer, equipped with a diamond-ATR accessory and a DTGS detector. The 128 scans at a resolution of 2 cm À1 , were performed at a wave number ranging from 4000 to 400 cm À1 .
Nitrogen sorption isotherms were obtained on an ASAP2020 apparatus operating at 77 K. In order to avoid the decomposition of the organic components in the extract during the outgassing protocol, 80 C was selected as the temperature of the outgassing step. It was chosen considering the thermal stability of the natural extract, which was observed by thermogravimetric (TG) and FTIR analysis (data not reported). The specific surface area (SSA) was calculated by Brunauer-Emmett-Teller (BET) equation. 25,26 For the total pore volume (Vp), the single point adsorption at p/p 0 = 0.99 was considered.
TGA was carried out at atmospheric pressure using a Perkin Elmer instrument model TGA7, working under Ar flow (40 ml min À1 ), in the temperature range of 30-800 C with a heating rate of 10 C min À1 .
The instrument was calibrated with Curie points of Alumel, Nickel, Perkalloy and Iron standard samples (accuracy: ±2 C). The ability of samples to scavenge DPPH radicals was measured adding 40 μl of the C. asiatica extract contained in each nanocomposite to 960 μl of DPPH methanolic solutions. DPPH methanolic solution at the same dilution was also used as control.
Absorbance was measured at 517 nm after storage at room temperature for 30 min in the dark, using a UV instrument Perkin Elmer mod. Lambda 25 (Monza, Italy). Experiments were performed in triplicate. The free radical scavenging activity, expressed as percentage of antioxidant activity (AA%), was calculated according to the following formula 27 : where A is the absorbance.
The obtained results were compared with the activity of the pure C. asiatica glycolic extract at the same concentration.

| Evaluation of the nanocomposite protective effect against cell damages
HaCaT were seeded into 96-well plates and incubated at 37 C in 5% F I G U R E 3 Fourier-transform infrared spectroscopy (FTIR) spectra of the Centella asiatica (C. asiatica) glycolic extract (labeled as Ca) and of the C. asiatica-SiO 2 nanocomposites F I G U R E 4 Nitrogen adsorption/desorption isotherms for the Centella asiatica-SiO 2 nanocomposites and the fumed silica at 77 K. Red and green curves refer to adsorption and desorption, respectively Figure 1), due to the increase of the system disorder related to an increment of the milling time. 18,19,24 SEM analysis revealed the presence of particle agglomeration, typical of the milled samples (Figure 2A-C). With an increment of the milling time, a decrease in the particle size and agglomeration are observed, simultaneously with the increase in the system disorder also observed in the XRPD patterns. The agglomeration is also favored by increasing the amount of natural extract probably due to the concurrent increase of the amount of propylene glycol contained in the extract.  F I G U R E 5 Thermogravimetric (TG, 5a) and derivative (dTG, 5b) curves of the Centella asiatica-SiO 2 nanocomposites incubated with the formulations for 48 h and the cell viability was measured (Figure 7). Any cytotoxicity has been observed for the four samples, demonstrating the high biocompatibility of the formulations ( Figure 7). In particular, S2 and S6 samples are able to promote keratinocyte proliferation, as the viability was always higher than 110% irrespective of the concentration used. The biocompatibility of S7 and S9 samples is also high (around 100%), but the promotion of the cell proliferation is evident only after 48 h of treatment and is slightly lower than that observed using S2 and S6 samples. activity. [32][33][34][35][36] According to this purpose, the prepared novel silica-based biomaterials containing C. asiatica glycolic extract, totally accomplish the extract protection, and controlled release as previously found in our studies. 18 Ball milling is revealed a feasible technique for the prepara- [Correction added on November 25, 2022, after first online publication: CRUI-CARE funding statement has been added.]

CONFLICT OF INTEREST
There are no conflicts to declare.

DATA AVAILABILITY STATEMENT
The data that supports the findings of this study are available in the article ORCID Alessandra