Ethanolic Fenugreek Extract: Its Molecular Mechanisms Against Skin Aging And The Enhanced Functions By Nanoencapsulation

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Abstract: Fenugreek, or Trigonella foenum-graecumL.(family Leguminosae)seeds are typically used as food supplements to increase postnatal lactation. Fenugreek extract displays antioxidative and anti-inflammatory properties, but its mechanisms against skin aging have not been exploited. In this research, we are the first to define an in vitro collagenase inhibitory activity of fenugreek extract (ICso =0.57±0.02mg/mL),which is 2.6 times more potent than vitamin C(IC50=1.46mg/mL). Nanoencapsulation has been applied to improve the extract stability and subsequently enhanced its bioactivities. Liponiosome encapsulating fenugreek extract (LNF) was prepared using a high-speed homogenizer, resulting in homogeneous spherical nanoparticles with sizes in the range of 174.7±49.2 nm,0.26±0.04 in PdI,and 46.6 ±7.4% of entrapment efficiency. LNF formulation significantly facilitated a sustained release and significantly enhanced skin penetration over the extracts, suggesting potential use of LNF for transdermal delivery. The formulated LNF was highly stable, not toxic to human fibroblast, and was ableto enhance cell viability, collagen production, and inhibit MMP1,MMP9, IL-6,and IL-8 secretions compared to the extract in the co-cultured skin model. Therefore, ethanolic fenugreek extract and its developed LNF display molecular mechanisms against skin aging and could potentially be used as an innovative ingredient for the prevention of skin aging.

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Keywords: fenugreek; rutin; anti-collagenase; anti-aging; collagen production; liposomes; nanoencapsulation; lipid particles; transdermal delivery; sustained release

1. Introduction

Skin aging is a biological process induced by both intrinsic and extrinsic factors. Intrinsic aging is caused by changes in the elasticity of the skin over time, while extrinsic aging is attributed to UV radiation, pollution, and smoking. Exposure to UV radiation (photoaging)is a predominant factor [1,2]. The most important structures of the extracellular matrix (ECM) are collagen, elastin, and glycosaminoglycans (GAGs) [3]. Common features of skin aging include the generation of reactive oxygen species (ROS) and degradation of the ECM by overexpressed matrix metalloproteinases (MPs).Cumulative oxidative stress and UV irradiation have been shown to increase the activity of degradative enzymes, espe-cially collagenase and elastase [1,4,5]. This contributes to overall skin behaviors, including the loss of tensile strength and elasticity, and resultant wrinkling, and dryness. To cope with skin aging, research mainly focuses on the reduction of oxidative stress (antioxidant)and ECM degradation (anti-collagenase and anti-elastase)[6-8]. Natural products have attracted interest as a new generation of cosmeceuticals. There is numerous evidence that plants possess a variety of active compounds, for example, terpenoids, alkaloids, and phe-nolic compounds [9-11]. flavonoids Trigonella foenum-graecum L. (fenugreek), belonging to the family Leguminosae, is a herbaceous plant that typically grows in western Asia, northern India, northern Africa, and the Mediterranean [12]. Fenugreek seeds are commonly used as a food, spice, galactagogue, and traditional medicine for diabetes[13-15]. Additionally, a toxico-logical study showed the safety of fenugreek seeds when used as a dietary supplement [16]. Fenugreek seeds contain numerous phytochemicals, including alkaloids, saponins, and flavonoids (rutin,quercetin, and vitexin) [17]. Many studies reported fenugreeK's antidi-abetic, anticancer, antimicrobial, antioxidative, and anti-inflammatory activities [18-23]Rutin is a compound of fenugreek that has beeen investigated for its anti-aging and antioxi-dant activities, as well as inhibition of collagen degradation [24-26]. Although there is no report implicating fenugreek extract in cosmeceuticals, it is expected to display biological properties that show these potential functions. Nevertheless, the general issues of using nat-ural extract in cosmetic products are poor pernetration into the skin, solubility, and stability. Nanoencapsulation is a technique that has been used to(i) improve stability,(ii) increase water solubility, (iii) enhance skin permeatior and absorption, and iv) slowly release active ingredients [27,28]. There are many different types of nanoparticles, such as liposomes, niosomes, and liponiosomes (a combination of liposomes and niosomes). Liponiosomes are composed of phospholipid and non-ionic surfactants that enhance the capability and control the release of nano-carriers[29]. The morphology of liponiosomes resembles that of liposomes. However, liponiosomes are more flexible and adaptable and, importantly, function differently. The advantages of liponiosome carriers are that they are deformable to penetrate pores that are much smaller than the carrier's size and have the capability to transport both water and fat-soluble agents. Hence, liposomes are promising carriers for delivering an active ingredient into the skin [30]. In this study, a fenugreek extract using rutin as an analytical marker was validated and identified. The biological activities of fenugreek extract were elucidated. Fenugreek has shown anti-collagenase activity and increased collagen production, which can refer to the anti-aging property. Liponiosome encapsulating fenugreek extract (LNF) was developed and characterized. Cytotoxicity of LNF was observed in human dermal fibroblasts using cell-based assays. These also included an investigation of molecular mechanisms on LNF anti-aging properties. The re-sults suggested that LNF is non-toxic to dernnal fibroblast cells and can inhibit the secretion of MMP1,MMP9,IL-6 and IL-8 upon UV-induced skin aging. Thus, our findings support the implication of fenugreek extract and LNF as innovative anti-aging agents, highting their potential as active ingredients in cosmeceutical products.

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2. Results

2.1. UHPLC Validation and Identification of Rutin in Fenugreek Extract

After ethanol maceration, the oily yellowish-brown paste of fenugreek extract was obtained. The identification of rutin content as an analytical marker was investigated using UHPLC. Optimum separation of rutin from fenugreek extract was achieved with a retention time of approximately 2.458 min (Figure 1),and was confirmed by using LC-MS with the detected product ion at 301 (Supplementary Figure S1). The validation of rutin was performed using AOAC guidelines [31,32] and is shown in Table 1. The equation in the table represents good linearity with a coefficient of determination(r2)of 0.998, with accuracy measured by the percentage of the recovery. Sensitivity was indicated using Limit of Detection (LOD) and Limit of Quantitation (LOQ) at 5.17 and 15.67 ug/mL, respectively The fenugreek extracts were prepared at various concentrations and the amount of rutin in the extract was interpreted using the area under the curve, relative to the standard curve of the commercial rutin reference. Results showed that the prepared extract contains 7.73±0.40mg/gofrutin.

2.2.In Vitro Collagenase Inhibition and Collagen Production of Fenugreek Extract

An anti-aging activity of fenugreek extract was firstly identified by in vitro collagenase inhibitory assay. We observed that the IC50 ofrutin trihydrate standard, fenugreek extract, and epigallocatechin gallate (EGCG) as a positive control was 0.06±0.56±0.02,and 0.11±0.01 mg/mL, respectively (Figure 2a,b). Notably, the inhibitory concentration of fenugreek extract was significantly lower than ascorbic acid (IC50=1.46±0.02mg/mL), indicating that fenugreek extract and rutin trihydrate standard exhibit anti-collagenase activity. The ability of fenugreek extract on collagen production was next investigated using human dermal fibroblast. Cells were treated with 125 ug/mL of extract and vitamin Cas a positive control for7 and 14 days. Cells were stained with Sirius Red and stained collagens were dissolved with NaOH. hesperidin uses As shown in Figure 2b, the Sirius Red represents the collagen content. The amount of stained collagen was significantly enhanced in extract-treated cells (163% and 131%) compared to a vehicle control (225% and 187%) at day 7 and day 14, respectively. This induction was similar to the effect of vitamin C-induced collagen production and was significantly higher than rutin-induced collagen production in human fibroblast cells. This result demonstrated that fenugreek extract induces collagen production in human dermal fibroblast cells.

2.3.Physicochemical Characterizations of Formulated LNF

To improve fenugreek extract stability and improve its appearance, formulation ofLNF was performed using all ingredients demonstrated in Table 2, resulting in a yellow-gold paste substance (Figure 3a: lower left) which is easily dispersed in water to obtain a yellowish solution (Figure 3a: right).

The LNF displayed a miscellaneous and soft herbal scent. The physicochemical characterization of formulated LNF was investigated using dynamic light scattering (DLS)and is shown in Table 3. The particle size had a range of 174.7±49.2 nm, with a low polydispersity index (PdI=0.26±0.04)and negatively charged surface due to phospho-lipids [33]. The encapsulation and loading efficiencies were approximately 46.6±7.4% and 33.5±4.0%,respectively. A micrograph shows the morphology of formulated particles as a spherical particle encapsulating fenugreek extract inside Figure 3a, with diameters in the range of 50-150 nm (Figure 3b). Endothermic peaks in the DSC thermogram (Figure 3c) located at 45.8℃represent the melting temperature of LNF, which is between the melting temperatures of liposome at 44.2 ℃and niosome at 47.5 ℃, respectively. oteflavonoid The morphology and thermal behavior could confirm that LNF displays the physical characteristics of both liposomes and niosomes.

LNF was kept at 4℃,25°C, and 40°C for 3months to study its stability in terms of particle size, PdI, and Z potential using the DLS technique. The pH and viscosity of the obtained particles were also investigated1. We observed a slight increase in the particle sizes stored at 40°C compared to 4℃, but these changes were not statistically different. lost empire cistanche Additionally, there was no significant change in the other characteristics of formulated LNF, including pH, viscosity, and % encapsulation.These results all suggested that the obtained particles were physiochemically stable (Figure 4).

2.4.Releasing Profile and Skin Penetration of Formulated LNF

The releasing profile (Figure 5a) demonstrated that fenugreek extract was released simultaneously, whereas its liposomes were detected 2 h later as 6.98±0.37% and increased to 23.92±1.41% at 24 h, suggesting the sustained release behavior of LNF.In order to investigate the potency of LNF on transdermal delivery, skin penetration was performed using ex vivo porcine skin. Imaging mass microscope (IMS) analysis was employed to observe a cumulative amount of rutin that was transported across the porcine skin of rutin standard, the extract,and LNF. In Figure 5b, we observed that fenugreek extract and LNF showed a higher amount of accumulated rutin on the skin compared to rutin standard. This result indicated that the permeability of fenugreek extract and LNF was greater and deeper than rutin at 24 h. micronized purified flavonoid fraction 1000 mg uses Additionally, skin penetration of formulated LNF was noticeably enhanced compared to those of extract. According to the releasing profile and ex vivo skin permeation study, LNF possibly enhances the efficiency of the extract by increased permeation and controlled release.

This article is extracted from Pharmaceuticals 2022, 15, 254. https://doi.org/10.3390/ph15020254 https://www.mdpi.com/journal/pharmaceuticals