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Article

The Potential of Tecoma stans (Linn.) Flower Extract as a Natural Antioxidant and Anti-Aging Agent for Skin Care Products

by
Panikchar Wichayapreechar
1,*,
Anchalee Prasansuklab
2,3,
Pimchanok Charoongchit
4 and
Ranit Charoenjittichai
1,*
1
Department of Cosmetic Sciences, School of Pharmaceutical Sciences, University of Phayao, Phayao 56000, Thailand
2
College of Public Health Sciences, Chulalongkorn University, Bangkok 10330, Thailand
3
Center of Excellence on Natural Products for Neuroprotection and Anti-Ageing, Chulalongkorn University, Bangkok 10330, Thailand
4
Department of Pharmaceutical Care, School of Pharmaceutical Sciences, University of Phayao, Phayao 56000, Thailand
*
Authors to whom correspondence should be addressed.
Cosmetics 2024, 11(6), 214; https://doi.org/10.3390/cosmetics11060214
Submission received: 22 October 2024 / Revised: 23 November 2024 / Accepted: 6 December 2024 / Published: 8 December 2024
(This article belongs to the Special Issue Current and Future Trends in Cosmetics Research: The 10th Anniversary of Cosmetics)
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Versions Notes

Abstract

:
Tecoma stans belongs to the Bignoniaceae family and possesses various pharmacological activities, including antimicrobial, anti-inflammatory, antifungal, antioxidant, and wound-healing activities. Although numerous studies have highlighted the beneficial effects of T. stans extracts, the impacts of different solvents on its biological activities, particularly its inhibitory effect on skin degradation enzymes (collagenase, elastase, and hyaluronidase assay), have not been reported. This study aims to explore the effects of different solvent extractions on the total phenolic and total flavonoid contents, antioxidant and anti-aging activities, and cytotoxicity. The most suitable extract was selected for incorporation into an anti-aging product. T. stans flowers were extracted using hexane, ethyl acetate, absolute ethanol, and deionized water through maceration. The aqueous extract yielded the highest extraction efficiency (40.73%), followed by absolute ethanol, ethyl acetate, and hexane. The phytochemical screening results revealed that all T. stans flower extracts contained phenolics, flavonoids, terpenoids, and alkaloids. Among the various solvents tested for T. stans flower extraction, absolute ethanol demonstrated the highest total phenolic content (24.10 ± 2.07 mg gallic acid equivalents (GAE)/g extract), followed by deionized water (20.83 ± 1.28 mg GAE/g extract). The highest total flavonoid content was observed in the ethyl acetate extract (205.11 ± 7.83 mg catechin equivalents (CE)/g extract), with ethanol showing a significantly lower concentration (140.67 ± 1.92 mg CE/g extract). In terms of antioxidant activity, the aqueous extract exhibited the most potent effects, with IC50 values of 0.600 ± 0.005 mg/mL for the DPPH• assay and 0.207 ± 0.001 mg/mL for the ABTS•+ assay. For anti-aging assays, the absolute ethanolic extract demonstrated the highest enzyme inhibition activity at 1 mg/mL, with collagenase, elastase, and hyaluronidase inhibition rates of 89.49% ± 2.96%, 94.61% ± 2.33%, and 82.56% ± 2.27%, respectively. Moreover, at a concentration of 50 µg/mL, the absolute ethanolic extract exhibited lower cytotoxicity, with human keratinocyte (HaCaT) cell viability of 78% ± 8.47%, which was significantly higher than that of the other extracts. An anti-aging gel containing 0.05% w/w of the ethanolic T. stans extract demonstrated physical and physicochemical stability during three months of storage at ambient temperatures, 4 °C, 45 °C, as well as after six cycles of heating/cooling tests. These findings suggest that the ethanolic extract of T. stans flower has potential as a safe and effective anti-aging agent for cosmeceutical products.

1. Introduction

As the largest organ in the human body, the skin plays a vital role in safeguarding internal organs from the external environment. It serves several essential functions, such as regulating body temperature, maintaining water content, and protecting the body from sunlight or ultraviolet light [1,2]. The skin is frequently exposed to direct UV radiation. Chronic or unprotected exposure to UV radiation is the primary external factor that significantly contributes to skin damage, particularly premature skin aging [3]. UV radiation from sunlight initiates a cascade of physical changes within the skin through intricate pathways. Upon exposure to UV irradiation, skin cells activate components that generate reactive oxygen species (ROS), triggering downstream signaling cascades. These include the phosphorylation of mitogen-activated protein kinases (MAPKs) and the activation of the activator protein-1 (AP-1) transcription factor in the epidermis and upper dermis. AP-1 significantly impacts collagen homeostasis by stimulating the transcription of several collagen-degrading enzymes, such as MMP-1, MMP-3, and MMP-9, while inhibiting the production of procollagens [2,4]. Moreover, excessive ROS levels can indirectly activate hyaluronidase, elastase, and collagenase, which are responsible for degrading the main components of the extracellular matrix: hyaluronic acid, elastin, and collagen, respectively. The presence of these enzymes in the skin contributes to visible signs of aging, such as wrinkles and skin laxity [5,6]; therefore, retarding and inhibiting oxidative stress, collagenase, elastase, and hyaluronidase may be effective strategies for preventing skin damage.
Topical anti-aging products, such as gel or cream, often contain antioxidants and anti-aging agents that can maintain cellular metabolism, stimulate the production of the extracellular matrix components, and delay the aging process [7,8,9]. Antioxidants are compounds that inhibit autoxidation by either preventing the formation of free radicals or disrupting their propagation [10]. Additionally, anti-aging agents encompass substances that enhance cellular repair through diverse mechanisms, including anti-glycation, anti-tyrosinase, anti-collagenase, anti-inflammatory, anti-elastase, anti-hyaluronidase, and moisturizing properties. Additionally, these agents stimulate cell proliferation, exhibit anti-cell senescence effects, and inhibit the production of matrix metalloproteinases [11,12]. Natural compounds derived from various sources are recognized for their roles as antioxidants and anti-aging agents. Notable examples include astaxanthin, sourced from the freshwater microalgae Haematococcus pluvialis; resveratrol, found in grape seeds; epigallocatechin gallate, abundant in green tea; and curcumin, derived from Curcuma longa [10,13]. Phenolic compounds, including polyphenols, flavonoids, and polyphenolic acids, are naturally occurring constituents found in various plant organs, including vegetables, fruits, seeds, flowers, leaves, rhizomes, and bark [10,14]. The mechanisms of phenolic compounds may be either electron transfer, hydrogen atom transfer, sequential proton transfer, single-electron transfer, or transition metal chelation. These mechanisms collectively contribute to the inhibition of free radical formation [15]. Hence, our study focuses on seeking natural antioxidants and anti-aging properties that are both effective and safe for use in anti-aging products.
Tecoma stans (Linn.) Juss. ex Kunth (Bignoniaceae), commonly known as yellow bells, is a prominent ornamental shrub extensively distributed in the plains of the Indian subcontinent. It is recognized as an invasive species in South Africa, Australia, Argentina, tropical regions of Asia, and the Pacific Islands [16]. Traditionally, T. stans has been employed in primary care medicine, especially for managing hyperglycemia, providing cardioprotection, and alleviating diarrhea [17]. It is a source of bioactive compounds that can be extracted from its flowers, including alkaloids, flavonoids, phenols, saponins, and quinones [18]. A variety of biologically active components have been extracted from the flowers of T. stans, including tecostamine, tecomine, coumaroyl-spermidine analogs, O-glucuronyl O-methyl quercetin, hydroxyphenyl ethyl O-coumaroyl O-deoxyhexosyl hexoside, and dihydroxyphenyl ethyl O-caffeoyl O-deoxyhexosyl hexoside [19]. T. stans flowers exhibit antioxidative activity, wound-healing properties, anti-proliferative effects, anti-cancer, anti-diabetes, and antimicrobial activities [20,21,22,23]. However, the potential of T. stans flower extract to inhibit skin aging, particularly through the inhibition of collagenase, elastase, and hyaluronidase, has not been previously explored. This study presents, for the first time, an investigation into the anti-aging properties of various solvent extracts derived from T. stans flowers.
In this study, we aim to evaluate natural anti-aging agents derived from the T. stans flowers. Furthermore, we explored the effect of solvent polarity on extraction yield and evaluated a variety of bioactivities, including antioxidant, anti-collagenase, anti-elastase, and anti-hyaluronidase properties, as well as skin toxicity. Additionally, anti-aging skincare products containing the potential of T. stans flower extracts were developed, and their stability was evaluated.

2. Materials and Methods

2.1. Chemicals and Reagents

All standards, reagents, and chemicals were of analytical grade. Absolute ethanol, 95% ethanol, chloroform, ethyl acetate, dimethyl sulfoxide, glacial acetic acid, hexane, hydrochloric acid, and sulfuric acid (H2SO4) were sourced from RCI Labscan (Bangkok, Thailand). Catechin, gallic acid, epigallocatechin (EGCG), Trolox, quercetin, acetic anhydride, aluminum chloride, calcium chloride, ferric chloride (FeCl3), potassium persulphate, sodium acetate, sodium carbonate, sodium chloride, sodium hydroxide, sodium nitrite, ABTS•+ (2,2′-Azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt), DPPH• (2,2-Di(4-tert-octylphenyl)-1-picrylhydrazyl), Folin–Ciocalteu’s phenol reagent, elastase from porcine pancreas (E.C.3.4.21.36), Tris-HCl, N-[3-(2-furyl) acryloyl]-Leu-Gly-Pro-Ala (FALGPA), N-Succinyl-Ala-Ala-Ala-p-nitroanilide (AAAPVN), collagenase from Clostridium histolyticum (EC.3.4.23.3), hyaluronidase from bovine testes (E.C.3.2.1.35), hyaluronic acid sodium salt from rooster comb, p-dimethyl-amino benzaldehyde, tricine, fetal bovine serum (FBS), and Dulbecco’s Modified Eagle Medium (DMEM: high glucose) were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) was purchased from a supplier in Canada (Bio Basic, Inc., Markham, ON, Canada).

2.2. Preparation and Extraction of Tecoma stans (Linn.)

The fresh flowers of T. stans were collected in May 2022 from Phayao Province, Thailand (19°02′03.8″ N, 99°53′00.9″ E), a region characterized by mountainous terrain, a tropical savanna climate, and an elevation of approximately 380 m above sea level (Figure 1A,B). To identify the species of T. stans, the plant was classified by an expert from the School of Pharmaceutical Science, University of Phayao, Phayao, Thailand, and a voucher specimen was preserved. The flowers were carefully cleaned, washed with water, and then dehydrated in a hot-air oven at 50 °C for a few days. Afterward, the dried flowers were finely ground (Figure 1C) using a cutting mill (A11 basic, IKA, Staufen, Germany). The extraction of T. stans flowers was performed through maceration with different solvents, including hexane, ethyl acetate, absolute ethanol, and deionized water. Briefly, the T. stans powder (250 g) was immersed in each solvent (750 mL) and shaken for 48 h at ambient temperature in the dark (repeated 3 times extraction), following the methods of Wichayapreechar [24], with minor modifications. After that, each extraction was filtered through Whatman™ filter paper No. 1 using a Buchner funnel (Cytiva, MA, USA). The filtered organic solvent was concentrated to dryness using a rotary evaporator under reduced pressure and at a controlled temperature of 50 °C to achieve the crude extracts. The deionized water was lyophilized, and all crude extracts were stored in a freezer at −20 °C for further analysis. The extraction yield (%) was calculated using the following equation:
Extraction yield (%) = (W1/W2) × 100
where W1 is the weight of the T. stans extract, and W2 is the weight of the T. stans powder.

2.3. Preliminary Phytochemical Screening of T. stans Extracts

Phytochemical screening is a qualitative method used to identify chemical compounds in plants, particularly secondary metabolites, through simple and inexpensive tests. A small sample (5 mg/mL in absolute ethanol) from each T. stans flower extract obtained through different solvent extractions was evaluated for its phytochemical constituents, including flavonoids, phenolic, terpenoids, steroids, and alkaloids, following the methods of Abioye [25], Harbourne [26], and Shaikh [27], with minor modifications. The presence of the main secondary metabolites in T. stans was observed via color change.

2.3.1. Determination of Flavonoid Compounds

The Shinoda test is used to detect the presence of flavone structures, which are an important subgroup of flavonoid compounds. Briefly, a few pieces of magnesium ribbon were added to the test solution, followed by 2–3 drops of concentrated hydrochloric acid [25,26]. The appearance of an orange, magenta, or red color confirms the presence of flavonoids.

2.3.2. Determination of Phenolic Compounds

The ferric chloride test is employed to identify the presence of phenolic compounds. Briefly, 2–3 drops of 10% w/v FeCl3 solution were added to the test solution [25,26]. The appearance of blue, violet, or dark green coloration confirms the presence of phenolic compounds.

2.3.3. Determination of Terpenoid Compounds

Salkowski’s test is a qualitative method used to screen for terpenoid compounds. In this procedure, 2 mL of chloroform was added to the test solution, followed by the careful addition of 2 mL of concentrated H2SO4 to create a distinct layer [26,27]. The appearance of a reddish-brown color in the core layer indicates the presence of a steroidal ring, suggesting the presence of terpenoids.

2.3.4. Determination of Steroid Compounds

The Liebermann–Burchard test is a colorimetric assay used for the detection of steroids. It involves the reaction of the steroid with acetic anhydride and sulfuric acid, producing a color change indicating the presence of the steroid. In brief, 2 mL of chloroform was added to the test solution, followed by the gradual addition of 1 mL of acetic anhydride and 2 mL of concentrated H2SO4 [26,27]. The development of a greenish-blue color confirms the presence of steroidal structures, suggesting the presence of steroids.

2.3.5. Determination of Alkaloid Compounds

Dragendorff’s test is a rapid phytochemical screening method used to detect alkaloids in the sample. Briefly, the test sample was combined with 2 mL of Dragendorff’s reagent [26,27]. The presence of an orange-red, orange, or reddish-brown precipitate in the test solution confirms the presence of alkaloids.

2.4. Total Phenolic Content of T. stans Extracts

The total phenolic content (TPC) of various solvent extracts was measured using the Folin–Ciocalteu colorimetric method based on a previously described procedure [24], with slight modifications. In a 96-well microplate, 20 µL of the T. stans extract (1 mg/mL) solution was combined with 100 µL of 10% v/v Folin–Ciocalteu’s phenol reagent. After incubating for 5 min, 80 µL of 20% w/v sodium carbonate solution was added to each well. The reaction mixture was then incubated in the dark for 30 min at ambient temperature. Absorbance was subsequently measured at 760 nm using a microplate reader (BioTek Instruments, Winooski, VT, USA). A calibration curve was generated by diluting gallic acid in absolute ethanol to final concentrations of 5–30 µg/mL (R2 = 0.9998; Figure S1). The TPC was expressed as mg of GAE/g dry extract.

2.5. Total Flavonoid Content of T. stans Extracts

The total flavonoid content (TFC) of various solvent extracts was assessed using the colorimetric method for aluminum chloride complexation following a previously established procedure [24], with minor modifications. In brief, 50 µL of T. stans extract (1 mg/mL) was combined with 30 µL of a 5% w/v sodium nitrite solution in a 96-well microplate and allowed to mix for five minutes. Then, 30 µL of 10% v/v aluminum chloride solution was added, and the mixture was stored in the dark for six minutes. After that, 90 µL of 1 M sodium hydroxide was added, and the reaction mixture was incubated for an additional 20 min at ambient temperature. The microplate reader (BioTek Instruments, Winooski, VT, USA) was used to measure the absorbance at 500 nm for this assay. A calibration curve was generated by diluting catechin in absolute ethanol to final concentrations of 10–200 µg/mL (R2 = 0.9993; Figure S2). The amount of TFC was expressed as mg of CE/g dry extract.

2.6. Antioxidant Activities of T. stans Extracts

The antioxidant activities of T. stans extracts obtained using various solvents to scavenge free radicals were evaluated using the DPPH• and ABTS•+ assays. These spectrophotometric methods are widely recognized for assessing the antioxidant potential of natural plants and products.

2.6.1. DPPH• Radical Scavenging Activity

The DPPH• radical scavenging assay relies on the reduction of the 1,1-diphenyl-2-picrylhydrazyl (which has a deep purple color) to 1,1-diphenyl-2-picryl hydrazine (which is either a pale-yellow color or colorless). In this assay, an antioxidant reacts with DPPH• radical through both single-electron transfer and hydrogen atom transfer mechanisms, leading to the formation of a new radical. The reaction mechanism is determined using a spectrophotometric method. The antioxidant capacities of various T. stans extracts were determined via the DPPH• assay, as described in a prior study [24], with slight modifications. In brief, 70 µL of 0.2 mM DPPH solution was combined with 70 µL of each T. stans extract solution at final concentrations ranging from 100 to 2000 µg/mL. The mixture was incubated in the dark, covered with aluminum foil, for 20 min. The absorbance at 517 nm was monitored using a microplate reader (BioTek Instruments). Trolox served as a positive control, with final concentrations ranging from 0.5 to 15 µg/mL. All experiments were performed in triplicate (Table S1). The following equation was used to calculate the percentage inhibition for DPPH• radical scavenging activity:
% Scavenging activity = [(Acontrol − Asample)/Acontrol] × 100
where Acontrol represents the absorbance of the blank, and Asample represents the absorbance of the sample. The antioxidant capacities of all T. stans extracts were expressed as IC50 values (DPPH•: Table S1 and ABTS•+: Table S2).

2.6.2. ABTS•+ Radical Scavenging Activity

The ABTS•+ radical scavenging activity is a spectrophotometric method used to evaluate the total antioxidant capacity of various compounds. This method quantifies the ability of these compounds to reduce the dark blue ABTS cation radical to its colorless form, ABTS. The antioxidant activities of all T. Stan extracts were assessed using the ABTS•+ assay [24], with minor modifications. Briefly, a 7 mM ABTS•+ solution in deionized water was prepared and combined with 140 mM potassium persulfate at a 1:1 ratio and then incubated in the dark at room temperature for 16 h. Subsequently, the mixture was diluted with distilled water at a 1:49 ratio to achieve an optimized absorbance of 0.80 ± 0.20 at 734 nm. T. stans extracts were prepared in 95% ethanol to a final concentration between 50 and 1000 µg/mL. A 210 µL reaction mixture, consisting of 70 µL of each sample concentration and 140 µL of ABTS•+ reagent, was protected from light at ambient temperature for 10 min. The absorbance at 734 nm was measured using a microplate reader (BioTek Instruments). A positive control in this assay was Trolox at final concentrations ranging from 1.25 to 30 µg/mL. All experiments were performed in triplicate (Table S2). The percentage inhibition of ABTS•+ radical scavenging activity was determined using Equation (2).

2.7. Inhibition of Skin Aging-Related Enzymes

Collagen, elastin, and hyaluronic acid are crucial components in the dermis layer that maintain skin firmness, elasticity, and flexibility. Collagenase, elastase, and hyaluronidase are enzymes responsible for the degradation of collagen, elastin, and hyaluronic acid, respectively. Therefore, compounds that inhibit these enzymes may help delay skin aging, including the formation of wrinkles. The most common in vitro methods for assessing anti-aging efficacy are based on spectrophotometric methods to measure anti-collagenase, anti-elastase, and anti-hyaluronidase activities [12].

2.7.1. Inhibition of Collagenase Activity

An evaluation of the anti-collagenase activity of all T. stans extracts was conducted according to a previously described method by Wichayapreechar [24], with some modifications. Firstly, 0.8 unit/mL collagenase solution was prepared by dissolving collagenase from Clostridium histolyticum (ChC–EC.3.4.23.3) in 50 mM Tricine buffer (pH 7.5) with 400 mM sodium chloride and 10 mM calcium chloride. In brief, 30 μL of each extract solution (at concentrations of 0.5 and 1 mg/mL) was combined with 50 mM Tricine buffer (60 μL) and collagenase solution (30 μL) at ambient temperature, followed by a 10 min incubation. Subsequently, 2 mM FALGPA substrate (60 μL) in 50 mM Tricine buffer (pH 7.5) was added to the mixture to initiate the reaction, which was then incubated continuously for 20 min. The absorbance at 335 nm was measured using a microplate reader (BioTek Instruments). EGCG (100 μg/mL) served as a positive control. The control test consisted of Tricine buffer mixed with collagenase enzyme and the FALGPA substrate. All experiments were performed in triplicate. The percentage of anti-collagenase activity was calculated according to the following equation:
% Anti-collagenase activity = [(Abs control − Abs sample)/Abs control] × 100
where Abs control denotes the absorbance of the control, and Abs sample denotes the absorbance of each sample.

2.7.2. Inhibition of Elastase Activity

An evaluation of the anti-elastase activity of all T. stans extracts was conducted according to a previously described method by Wichayapreechar [24]. Porcine pancreatic elastase (PE–E.C.3.4.21.36) was prepared at a concentration of 1 mM, along with 2.0 mM N-Succinyl-Ala-Ala-Ala-p-nitroanilide (AAAPVN) in 0.2 mM Tris-HCl buffer (pH 8.0). A mixture consisting of 20 μL of T. stans extract solution (at concentrations of 0.5 and 1 mg/mL), 90 μL of Tris-HCl buffer, and 10 μL of elastase enzyme was pre-incubated at ambient temperature for 5 min. The reaction was initiated by the addition of 80 μL of AAAPVN, and the mixture was subsequently incubated at ambient temperature for 15 min. The absorbance at 410 nm was measured using a microplate reader (BioTek Instruments). EGCG (100 μg/mL) served as a positive control. The control consisted of a mixture of Tris-HCl buffer, elastase enzyme, and the AAAPVN substrate. All experiments were performed in triplicate. The percentage of anti-elastase activity was calculated according to the following equation:
% Anti-elastase activity = [(Abs control − Abs sample)/Abs control] × 100
where Abs control denotes the absorbance of the control, and Abs sample denotes the absorbance of each sample.

2.7.3. Inhibition of Hyaluronidase Activity

An evaluation of the anti-hyaluronidase activity of all T. stans extracts was conducted according to Lee et al. [28], with minor modifications. In brief, 3000 units/mL of hyaluronidase from bovine testes (EC. 3.2.1.35) and the sodium salt of hyaluronic acid from a rooster comb (5 mg/mL) were prepared in 0.1 M acetate buffer (pH 3.5). The reaction mixture included 100 µL of hyaluronidase enzyme, 100 µL of each T. stans extract (0.5 and 1 mg/mL), and 500 µL of hyaluronic acid solution. This mixture was incubated at 37 °C for 20 min. After incubation, 2 mL of p-dimethyl-amino benzaldehyde solution (prepared by dissolving 4 g of p-dimethyl-amino benzaldehyde in 350 mL of 100% acetic acid and 50 mL of 10 mol/L HCl) was added to the mixture. Subsequently, 200 µL of the reaction mixture was measured at 570 nm using a microplate reader (BioTek Instruments). Quercetin (100 μg/mL) served as a positive control. The control experiment consisted of an acetate buffer mixed with hyaluronidase enzyme and the hyaluronic acid solution. All experiments were performed in triplicate. The percentage of anti-hyaluronidase activity was calculated according to the following equation:
% Anti-hyaluronidase activity = [(Abs control − Abs sample)/Abs control] × 100
where Abs control denotes the absorbance of the control, and Abs sample denotes the absorbance of each sample.

2.8. Cytotoxicity of T. stans Extracts

2.8.1. Cells and Cell Culture Environment

HaCaT cells, immortalized human keratinocytes (CLS Cell Lines Service, Heidelberg, Germany) were cultured in high-glucose Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) in a humidified environment with 5% CO2 at 37 °C.

2.8.2. Determination of Cell Viability

HaCaT cells are widely utilized for studying epidermal pathophysiology and serve as an excellent model for assessing anti-aging effects [29]. The cellular toxicity of T. stans extracts was determined using an MTT colorimetric assay [24], with minor modifications. Briefly, HaCaT cells (5.0 × 103 cells/well) were seeded into 96-well plates and incubated overnight with 5% CO2 at 37 °C. The cells were subsequently treated with T. stans extracts at concentrations ranging from 1 to 100 μg/mL and incubated under the same conditions for 24 h. After the incubation period, 20 μL of MTT reagent (5 mg/mL) was added to each well, and the plates were further incubated at 37 °C with 5% CO2 for 4 h. The medium was then removed, and 150 μL of DMSO was used to dissolve the formazan crystals and the absorbance was monitored at 570 nm using a microplate reader (Enspire®, PerkinElmer, Waltham, MA, USA). The control group was untreated cells cultured in a complete medium containing 0.1% DMSO. Cell viability greater than 90% was considered non-cytotoxic. Cell viability (%) was calculated using the following equation:
% Cell viability = (Abs treated cells/Abs untreated cells) × 100
where Abs untreated cells is the absorbance of the control, and Abs treated cells is the absorbance of the treated cells.

2.9. Anti-Aging Skincare Formulation

The T. stans extract in the skincare product was formulated as a gel. The composition of the product (Table 1.) was prepared using a cold process. First, the weighed amount of the gelling agent was slowly dispersed into 40 mL of deionized water in a beaker, with continuous stirring using an overhead stirrer (RW 20 digital, IKA, Staufen, Germany) at a speed of 400 rpm until a homogeneous gel was formed. Humectants (glycerin, panthenol, and sodium hyaluronate) were mixed with deionized water while stirring continuously until a homogeneous was achieved. This mixture was subsequently added to the previously prepared gel. Subsequently, other components, including emollients, thickeners, antioxidants, and preservatives, were slowly added to the gel formulation and mixed at 400 rpm using an overhead stirrer. Finally, an active ingredient (0.05% T. stans extract) was slowly poured into the formulation.

2.10. Stability Test

The stability of the T. stans skincare product was conducted through long-term and accelerated studies according to Srisuksomwong et al. [30], with minor modifications. In the long-term stability assessment, the product was stored under various conditions: at ambient temperature, 4 °C in a refrigerator, and 45 °C in a hot-air oven. Evaluations were conducted monthly over three months. Additionally, accelerated stability studies were performed using a heating/cooling cycling method consisting of six cycles [31]. Each cycle involved 24 h in a hot-air oven at 45 °C, followed by 24 h in the refrigerator at 4 °C. The characteristics of the product, including color, odor, pH, homogeneity, phase separation, and viscosity, were assessed before and after the study.

2.11. Statistical Analysis

The statistical analysis was performed using SPSS Version 24 for Windows (SPSS, Chicago, IL, USA). All data are presented as the mean ± SD from three independent experiments (n = 3). One-way ANOVA was used for statistical analysis, followed by Tukey’s post hoc test. Values of p < 0.05 and p < 0.01 were considered statistically significant.

3. Results and Discussion

3.1. T. stans Extraction Yield and Phytochemical Screening of Different Solvent Extracts

Extraction is a critical initial step for isolating phytochemicals, and determining the optimal conditions for extraction is essential to enhance both the yield and content. The chemical properties of the phytochemicals, the extraction method, the amount of sample, the solvent selection, and the presence of interfering substances all influence the extraction efficiency. The extraction yield depends on various factors, such as the solvent’s polarity, the pH of the media, the extraction temperature, the extraction time, and the composition of the sample [32,33,34]. In this study, different polarities of the solvents, including hexane, ethyl acetate, absolute ethanol, and deionized water, were used for the floral T. stans extraction at an equal number of samples, solvent, extraction method, time, and temperature. The results in Table 2 show that the highest yield (40.73%) was obtained from the extraction of T. stans flowers using deionized water, followed by 28.3% from absolute ethanol. In contrast, the extractions with ethyl acetate and hexane resulted in lower yields, at 6.83% and 3.29%, respectively. This result indicates that the extraction yield increases with the polarity of the solvent used. The highest extraction yield of T. stans flowers contributed from water extraction may attribute to the higher solubility of its compounds in the aqueous medium than in ethanol, ethyl acetate, and hexane. A much higher yield was observed in the water extraction of T. stans flower, reflecting the abundance of hydrophilic compounds present. In contrast, lower-polarity solvents can only extract a smaller number of low-polarity compounds, which are less present in the T. stans flower. This corresponds to the previous study by Gonçalves et al. [19], which reported that the extract amount of T. stans flowers collected from hydroethanolic fraction was higher than that from dichloromethane and hexane. Jeyaraj et al. [35] also found that Clitoria ternatea flower extract using water as a solvent provided an extraction yield higher than that which was extracted using pure acetone but less than that using pure ethanol and methanol. Similar results from the extraction of Limnophila aromatica have shown that the yields from water extraction are higher than those from pure ethanol and pure acetone. The results are aligned with the results from Sun and Tomkinson’s study [36] for wheat straw, which indicated that the yield of its extracts from dichloromethane is lower than the mixed solvent with ethanol.
The phytochemicals extracted from plant materials are influenced by the type of solvent used [37]. This study employed preliminary phytochemical screening techniques due to their cost effectiveness, ease of implementation, and minimal resource requirements, making them useful for detecting phytoconstituents [25,27]. The qualitative phytochemical tests for flavonoids, phenolics, terpenoids, steroids, and alkaloids are summarized in Table 2. Notably, the T. stans flower extract exhibited a higher intensity of color change, indicating a greater concentration of compounds. Among the solvent extracts of T. stans flowers, the absolute ethanol extract exhibited the highest level of phytochemicals, followed by the ethyl acetate, deionized water, and hexane extracts, respectively. Flavonoids and phenolics were predominantly found in ethanolic, deionized water, and ethyl acetate extracts. In contrast, steroids were present in low concentrations across all extracts. These findings align with previous preliminary phytochemical studies. Raju et al. [38] reported the presence of flavonoids, saponins, carbohydrates, glycosides, and tannins in methanolic and ethyl acetate extracts of T. stans flowers, while steroids, alkaloids, and proteins were not detected. Similarly, Rajamurugan et al. [23] and Pulipati et al. [39] observed alkaloids, glycosides, saponins, carbohydrates, tannins, phenolics, steroids, and flavonoids in the ethanolic extract of T. stans flowers. Further supporting our screening results, Gonçalves et al. [19] conducted a comprehensive analysis of the phytochemical constituents in solvent extracts and fractions derived from T. stans flowers. Their study, utilizing gas chromatography, revealed that the hexane fraction was predominantly composed of fatty acid esters, including ethyl palmitate, ethyl linoleate, ethyl linolenate, and ethyl stearate, along with phytosterols such as β-sitosterol and campesterol. Additionally, through liquid chromatography coupled with a diode array detector and mass spectrometry, they identified O-glycosylated flavones and flavonols (e.g., O-glucuronyl O-methyl quercetin and O-hexosyl luteolin), alkaloid compounds (e.g., tecomine, tecostamine, and coumaroyl-spermidine analogs), and phenylethanoid glycosides (e.g., dihydroxyphenyl ethyl O-caffeoyl O-deoxyhexosyl hexoside and hydroxyphenyl ethyl O-coumaroyl O-deoxyhexosyl hexoside) in the ethanol, hydroethanolic, dichloromethane, and ethyl acetate extracts and fractions. Our preliminary phytochemical screening suggests that T. stans flowers, extracted using deionized water, absolute ethanol, and ethyl acetate, are abundant in bioactive compounds, particularly flavonoids and phenolics. The chemical property of the solvent significantly influences both the extraction efficiency and the phytochemical profile. Further research is necessary to isolate and characterize the specific compounds responsible for the observed bioactivities in T. stans flower extracts.

3.2. Total Phenolic Contents, Total Flavonoid Contents, and Antioxidant Activities of Different Extraction Solvents of T. stans

Regarding the total phenolic content (TPC) and total flavonoid content (TFC) in the various extraction solvents of T. stans flowers, the results presented in Table 3 indicate that the highest TPC of 24.10 ± 2.07 mg GAE/g extract was obtained from the absolute ethanol extract, significantly surpassing the TPC from deionized water and ethyl acetate. In contrast, the lowest TPC, at 6.90 ± 0.47 mg GAE/g extract, was found in the hexane extract. Notably, an increase in solvent polarity was associated with higher TPC in T. stans flower extracts. Additionally, the highest TFC was recorded in the ethyl acetate extract at 205.11 ± 7.83 mg CE/g extract, followed by ethanol, hexane, and water. Interestingly, this contrasts with the findings of Gonçalves et al. [19], who reported a higher TPC (47.30 to 190.80 mg GAE/mg extract) than TFC (6.00 to 25.20 mg quercetin equivalents (QE)/mg extract) in T. stans flower extracts. Furthermore, the ethyl acetate extract exhibited the highest levels of phenolic compounds, while the dichloromethane extract contained significantly elevated levels of flavonoids. Alonso-Castro et al. [40] revealed that the TPC and TFC obtained from the distilled water extraction of T. stans leaves were 12.0 ± 2.1 g GAE/kg extract and 1.2 ± 0.1 g QE/kg plant extract, respectively. Jeyaraj et al. [35] found no significant difference in the TPC of C. ternatea flower extracts when using pure ethanol, acetone, or distilled water as solvents. Ethanol is known to be an effective solvent for extracting polyphenols. Additionally, the presence of water in plant extraction may enhance the diffusion process, thereby facilitating the extraction of phenolic compounds from plant tissues [33,41,42,43]. The types and concentrations of polyphenols present in plants vary widely between species. The influence of solvent polarity on TPC and TFC is dependent on the specific chemical properties of the compounds being extracted [35,44,45].
Antioxidants and their properties are valuable features of plant extracts, offering potential benefits in preventing diseases and cellular aging associated with oxidative damage. Phenolic compounds and flavonoids are recognized as powerful antioxidants due to their ability to scavenge the radicals of the lipid peroxyl, hydroxyl, and superoxide anions by donating hydrogen or electrons to form stable radical intermediates [46,47]. The antioxidant activities of various plant extracts are strongly correlated with their TPC and TFC [48,49,50]. In this study, the antioxidant capacities of T. stans flower extracts were evaluated using the DPPH• and ABTS•+ assays, with the results represented by IC50 values summarized in Table 3. The deionized water extract demonstrated the highest DPPH• and ABTS•+ radical scavenging potential, with the lowest IC50 values of 0.600 ± 0.004 and 0.207 ± 0.001 mg/mL, respectively, significantly surpassing those of the ethyl acetate and absolute ethanol extracts. Conversely, the hexane extract exhibited the lowest radical scavenging activities, with IC50 values of 1.378 ± 0.057 and 0.871 ± 0.063 mg/mL for DPPH• and ABTS•+ assays, respectively. Trolox standards used in the assays had IC50 values of 3 and 9 µg/mL for DPPH• and ABTS•+ radicals, respectively. A consistent trend was observed, where the radical scavenging potential of the ABTS•+ assay was lower than that of the DPPH• assay, likely due to the aqueous nature of the ABTS•+ assay favoring hydrophilic compounds with antioxidant capacity [51,52]. Although the DPPH• and ABTS•+ scavenging abilities of T. stans flower extracts were lower than those of Trolox (p < 0.05), the results indicate that these extracts possess proton-donating capabilities and may function as free radical inhibitors or scavengers, acting as primary antioxidants. Furthermore, the antioxidant activities of T. stans flower extracts tended to correlate with extraction yield, phenolic compound concentrations, and flavonoid content. In this study, we indicated that water extracts exhibited greater antioxidant activity than solvent extracts, possibly due to lower levels of TPC and TFC in the latter. This correlation aligns with previous studies by Jeyaraj et al. [35], Gonçalves et al. [19], and Mohammed et al. [45]. Gonçalves et al. [19] demonstrated that the DPPH• radical scavenging activities of ethanolic, hydroethanolic, ethyl acetate, and dichloromethane extracts from T. stans flowers were more effective than those of hexane extracts. The ethyl acetate extract showed the lowest LC50 value at 2.99 ± 0.56 µg/mL with the highest TPC at 190.80 ± 4.09 µg/mL compared to the extracts from other solvents. The other study revealed that at a concentration of 200 mg/mL, the ethyl acetate extracts from the leaves and branches of T. stans exhibited the highest effects of the DPPH• radical scavenging activity at 83.4 ± 0.31% and 82.06 ± 0.54%, respectively, followed by chloroform and methanol extracts; in contrast, aqueous extracts showed the lowest effects [53].

3.3. Assessments of Anti-Aging Activities of Different Solvent Extracts from T. stans

This study represents the first report on the anti-aging properties of T. stans. The anti-aging activities of T. stans extract obtained from various solvent extractions were investigated regarding their capacity to inhibit the activities of collagenase, elastase, and hyaluronidase. The study revealed that the T. stans extracts obtained using all solvent polarities exhibited anti-collagenase, anti-elastase, and anti-hyaluronidase effects in a dose-dependent manner (Figure 2, Figure 3 and Figure 4). In the assessment of anti-collagenase activity, the ethanolic extract of T. stans exhibited the highest inhibitory effect, approximately 75% to 90%. In contrast, the T. stans extract obtained using hexane showed the lowest collagenase inhibitory effect, ranging from approximately 55% to 64%. Notably, the extracts derived from deionized water and ethyl acetate demonstrated comparable collagenase inhibitory effects, ranging from 66% to 76% (Figure 2). Furthermore, the ethanolic extract of T. stans exhibited considerable anti-elastase activity, approximately 83% to 95%. The anti-elastase activity of deionized water extract from the flowers of T. stans showed the lowest elastase inhibitory effect, ranging from approximately 65% to 70%, while T. stans extracts obtained using ethyl acetate and hexane demonstrated equal elastase inhibitory effects, ranging from 70% to 85% (Figure 3). The ethanolic extract of T. stans exhibited the highest anti-hyaluronidase activity, ranging from approximately 70% to 83%. This was followed by the deionized water extract, which demonstrated an activity of approximately 67% to 76%. The ethyl acetate extract showed a range of activity from approximately 38% to 70%, while the hexane extract exhibited the lowest activity, ranging from approximately 30% to 47% (see Figure 4). While all different solvent extracts from T. stans flower exhibited lower anti-aging activity compared to the positive controls—EGCG, which is known for its anti-collagenase and anti-elastase properties, and quercetin, recognized for its anti-hyaluronidase activity—these T. stans extracts nonetheless displayed promising anti-aging effects at concentrations of 0.5 and 1.0 mg/mL.
According to our data, all extracts of T. stans contain flavonoids, phenolics, terpenoids, steroids, and alkaloids. These plant secondary metabolites have diverse chemical structures that influence their antioxidant and anti-aging properties. Numerous studies show that both flavonoids and phenolic acids, found in ethanolic, methanolic, deionized water, and ethyl acetate extracts, exhibit potent antioxidant activity [54,55,56,57,58]. The inhibition of free radicals by antioxidant compounds from natural plants can prevent the elevation of collagenase, elastase, and hyaluronidase enzyme levels, which are known to accelerate the skin aging process and the formation of wrinkles. By preserving the function of collagen, elastin, and hyaluronic acid—critical for maintaining skin elasticity, flexibility, and moisture—this inhibition may help mitigate the aging process [13,24,57,59,60,61,62]. Interestingly, the T. stans extract obtained using absolute ethanol demonstrated the highest anti-aging activity based on anti-collagenase, anti-elastase, and anti-hyaluronidase assays. The remarkable anti-aging activities of the ethanolic extract may be attributed to its phytochemical compounds (flavonoid, phenolic, terpenoid, and alkaloid compounds) and antioxidant properties, which are associated with elevated total phenolic and total flavonoid contents. Furthermore, the T. stans extracts obtained using deionized water and ethyl acetate also exhibited significant anti-aging activities, as indicated by their higher antioxidant activities compared to that of absolute ethanol extract (Table 2 and Table 3). These results indicate that anti-aging activities may be associated not only with the antioxidant properties of the extracts but also with other compounds or the synergistic interactions of the phytochemical constituents present in T. stans extracts.

3.4. Effect of Solvent Polarity on the Cytotoxicities of T. stans Extracts

Generally, the active ingredients in cosmeceutical products should have the least toxicity towards normal skin cells. In addition, keratinocyte damage serves as a key player in skin aging by reducing the skin’s barrier function and repairing the ability [63]. Hence, in this study, the cytotoxic effects of all four T. stans extracts were evaluated on the human keratinocyte cell line (HaCaT) by using the MTT cell viability assay.
After 24 h treatment with various concentrations of solvent extracts (1–100 µg/mL), the HaCaT cell viability decreased in a dose-dependent manner (Figure 5). The non-cytotoxic concentrations for the deionized water and absolute ethanol extracts ranged from 1 to 25 µg/mL, whereas the ethyl acetate and hexane extracts exhibited lower non-cytotoxic ranges, between 1 and 5 µg/mL. These findings implied that the extracts of T. stans with deionized water and absolute ethanol are safer compared to the rest of the extracts, which can be considered permissible for skin applications.

3.5. Stability Test of an Anti-Aging Skincare Formulation

Signs of aging skin typically include decreased hydration, as well as the presence of dark spots and blemishes, which are often attributable to an impaired skin barrier. To address these issues, the selection of substances in skincare formulations that enhance skin moisture retention is crucial. This can be achieved through the combination of humectants, occlusive agents, and emollients [64,65,66,67]. In this study, the skincare products maintain skin integrity and appearance by incorporating humectants such as glycerin, panthenol, and sodium hyaluronate while also providing an occlusive effect via emollients (glyceryl glucoside, PEG-7 glyceryl cocoate, and coco caprylate/caprate). Additionally, Calipro Moist, a mixture of emollients, was included in the formulation to enhance the skin feel and act as a thickener, thereby stabilizing the formulation alongside SEPINOV™ EMT 10 (Table 1). The ethanolic T. stans flower extract demonstrated significantly enhanced antioxidant capacity and anti-aging potency in inhibiting collagenase, elastase, and hyaluronidase in vitro. Therefore, T. stans flower extracted using absolute ethanol is suitable for formulation as an anti-aging skincare product. The formulated skincare gel base appeared in a white, opaque, semi-solid form, while the ethanolic T. stans extract-containing gel exhibited a light yellowish color, as shown in Figure 6A and Figure 6B, respectively.
The physicochemical properties of the skincare products were evaluated after three months of long-term stability testing and after the sixth cycle of the heating/cooling cycling test. All samples of the T. stans extract-containing gel showed no significant changes (p > 0.05) in any evaluated parameters compared to the initial samples, which were light yellow, opaque gels with no separation (Table 4, Figure 7). The pH of the formulation demonstrated an acceptable value for skin application, while its viscosity facilitates prolonged retention at the application site, thereby extending the time available for the diffusion of active ingredients.

4. Conclusions

This study evaluates the anti-aging properties of Tecoma stans flower extract, focusing on its ability to inhibit in vitro skin-degradation enzymes, including collagenase, elastase, and hyaluronidase, as well as its antioxidant potential. Our results demonstrate that solvent polarity plays a crucial role in the extraction process, with polar solvents, particularly ethanol and water, yielding higher extraction efficiencies compared to non-polar solvents like hexane and ethyl acetate. Higher antioxidant activities, as measured using the DPPH• and ABTS•+ radical scavenging assays, were observed in the deionized water, ethyl acetate, and ethanol extracts, respectively, and these activities correlated with higher concentrations of total phenolic and flavonoid compounds. Both ethanolic and aqueous extracts, at concentrations of 0.5 and 1 mg/mL, showed significant inhibition of collagenase, elastase, and hyaluronidase enzyme activities, being positively correlated with their antioxidant properties. The ethanolic extract was found to be non-cytotoxic to human keratinocyte cells at concentrations up to 50 µg/mL, suggesting its safety for topical applications.
Additionally, a prototype anti-aging gel formulated with 0.05% w/w ethanolic extract demonstrated excellent physical and physicochemical stability for over three months under various storage conditions, including ambient temperature, 4 °C, 45 °C, and heating/cooling cycles. These findings highlight the potential of T. stans ethanolic extract as a safe and effective ingredient for anti-aging cosmetic formulations. However, to assess its efficacy and dermal safety, further comprehensive clinical studies on human subjects are required. Future research should also explore the long-term effects of such formulations and their impact on skin health to better understand their potential as skin anti-aging agents.

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/cosmetics11060214/s1. Figure S1: Linearity relationship between absorbance vs concentration of gallic acid, Figure S2: Linearity relationship between absorbance vs concentration of catechin, Table S1: Linearity equation of %DPPH radical scavenging activity to determine IC50 of each T. stans extracts, Table S2: Linearity equation of ABTS activity to determine IC50 of each T. stans extracts.

Author Contributions

Conceptualization, P.W.; methodology, P.W. and A.P.; validation, P.W. and R.C.; formal analysis, P.W. and R.C.; investigation, P.W., P.C., A.P. and R.C.; resources, P.W. and A.P.; data curation, P.W.; writing—original draft preparation, P.W., P.C., A.P. and R.C.; writing—review and editing, P.W., P.C., A.P. and R.C.; visualization, P.W.; supervision, P.W. and R.C.; project administration, P.W.; funding acquisition, P.W. and R.C. All authors have read and agreed to the published version of the manuscript.

Funding

This research partially received funding from Mae Fah Luang University Health and Beauty Science Park for experiments on antioxidant activities and anti-aging skincare formulation.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The original contributions presented in this study are included in the article/Supplementary Materials. Further inquiries can be directed to the corresponding author(s).

Acknowledgments

The authors are grateful to the Department of Cosmetic Sciences, School of Pharmaceutical Sciences, University of Phayao, and the Center of Excellence on Natural Products for Neuroprotection and Anti-Ageing, Chulalongkorn University, for providing instruments and their facilities. The authors would like to thank Nattathida Jiwkhuntod, Pimlaphach Suraphon, and Atchara Sintarom for their support of this project. Acknowledgement is also extended to the support and funds from Mae Fah Luang University Health and Beauty Science Park.

Conflicts of Interest

The authors declare no conflicts of interest.

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Cosmetics 11 00214 g001
Figure 1. Tecoma stans (Linn.) (T. stans). (A) The T. stans tree; (B) the T. stans flowers; and (C) the dried fine powder of T. stans.
Figure 1. Tecoma stans (Linn.) (T. stans). (A) The T. stans tree; (B) the T. stans flowers; and (C) the dried fine powder of T. stans.
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Figure 2. Collagenase inhibitory activities of T. stans extracts derived from solvents of differing polarities. Values are expressed as the mean ± SD from three independent experiments. An asterisk (*) indicates a significant difference (p < 0.05) between sample concentrations within the solvent. The different letters (a–c) denote statistically significant differences among the solvents at a sample concentration of 0.5 mg/mL (p < 0.05). The different letters (A–C) denote statistically significant differences among the solvents at a sample concentration of 1 mg/mL (p < 0.05).
Figure 2. Collagenase inhibitory activities of T. stans extracts derived from solvents of differing polarities. Values are expressed as the mean ± SD from three independent experiments. An asterisk (*) indicates a significant difference (p < 0.05) between sample concentrations within the solvent. The different letters (a–c) denote statistically significant differences among the solvents at a sample concentration of 0.5 mg/mL (p < 0.05). The different letters (A–C) denote statistically significant differences among the solvents at a sample concentration of 1 mg/mL (p < 0.05).
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Figure 3. Elastase inhibitory activities of T. stans extracts derived from solvents of differing polarities. Data are presented as the mean ± SD from three independent experiments. An asterisk (*) indicates a statistically significant difference (p < 0.05) between sample concentrations within the solvent. The different letters (a–c) denote statistically significant differences among the solvents at a sample concentration of 0.5 mg/mL (p < 0.05). The different letters (A–C) denote represent statistically significant differences among the solvents at a sample concentration of 1 mg/mL (p < 0.05).
Figure 3. Elastase inhibitory activities of T. stans extracts derived from solvents of differing polarities. Data are presented as the mean ± SD from three independent experiments. An asterisk (*) indicates a statistically significant difference (p < 0.05) between sample concentrations within the solvent. The different letters (a–c) denote statistically significant differences among the solvents at a sample concentration of 0.5 mg/mL (p < 0.05). The different letters (A–C) denote represent statistically significant differences among the solvents at a sample concentration of 1 mg/mL (p < 0.05).
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Figure 4. Hyaluronidase inhibitory activities of T. stans extracts obtained using solvents of varying polarities. Data are shown as the mean ± SD from three independent experiments. An asterisk (*) indicates a statistically significant difference (p < 0.05) between sample concentrations within the solvent. The different letters (a–c) denote statistically significant differences among the solvents at a sample concentration of 0.5 mg/mL (p < 0.05). The different letters (A–D) denote statistically significant differences among the solvents at a sample concentration of 1 mg/mL (p < 0.05).
Figure 4. Hyaluronidase inhibitory activities of T. stans extracts obtained using solvents of varying polarities. Data are shown as the mean ± SD from three independent experiments. An asterisk (*) indicates a statistically significant difference (p < 0.05) between sample concentrations within the solvent. The different letters (a–c) denote statistically significant differences among the solvents at a sample concentration of 0.5 mg/mL (p < 0.05). The different letters (A–D) denote statistically significant differences among the solvents at a sample concentration of 1 mg/mL (p < 0.05).
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Figure 5. The percentage viabilities of HaCaT cells treated with different concentrations of T. stans extracts for 24 h. The values are expressed as the mean ± SD in triplicate experiments, with * p < 0.05 and ** p < 0.01 vs. control.
Figure 5. The percentage viabilities of HaCaT cells treated with different concentrations of T. stans extracts for 24 h. The values are expressed as the mean ± SD in triplicate experiments, with * p < 0.05 and ** p < 0.01 vs. control.
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Figure 6. Appearance of skincare formulation (A) without T. stans extract and (B) with T. stans extract.
Figure 6. Appearance of skincare formulation (A) without T. stans extract and (B) with T. stans extract.
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Figure 7. Appearance of skincare formulation with T. stans extract after stability test: (A) ambient temperature; (B) 4 °C in a refrigerator; (C) 45 °C in a hot-air oven; and (D) heating/cooling cycling.
Figure 7. Appearance of skincare formulation with T. stans extract after stability test: (A) ambient temperature; (B) 4 °C in a refrigerator; (C) 45 °C in a hot-air oven; and (D) heating/cooling cycling.
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Table 1. The components of the skincare formulation.
Table 1. The components of the skincare formulation.
Ingredients (INCI Name)Trade Name%w/wFunction in Formula
AquaAquaqs. to 100Solvent
GlycerinGlycerin3.0Humectant
Glyceryl GlucosideGlyceryl Glucoside2.0Emollient
PanthenolPanthenol1.0Humectant
Sodium hyaluronate Sodium hyaluronate0.2Humectant
PEG-7 glyceryl cocoateCETIOL HE1Emollient
Pentaerythrityl Tetra-di-t-butyl HydroxyhydrocinnamateTinogard ® TT0.05Antioxidant
Coco Caprylate/caprateCETIOL C5C2Emollient
Hydroxyethyl Acrylate/Sodium Acryloyldimethyl Taurate CopolymerSEPINOV™ EMT 102.7Gelling agent
Water, Glycerin, Biosaccharide Gum-1, Hydroxypropyl Guar, Hydroxyethylcellulose, 1,2-Hexanediol, EthylhexylglycerinCalipro moist1Emollient/thickener
Ethylhexylglycerin (and) PhenoxyethanolEthylhexylglycerin (and) Phenoxyethanol0.8Preservative
Teccoma stan (Linn.) extract Teccoma stan (Linn.) extract0.05Active ingredient
Table 2. Percentage of extraction yield and preliminary phytochemical screening of T. stans flower extracted using various solvents.
Table 2. Percentage of extraction yield and preliminary phytochemical screening of T. stans flower extracted using various solvents.
SolventsExtraction Yield (%)Phytochemical Screening
Flavonoid CompoundsPhenolic Compounds Terpenoid Compounds Steroids Compounds Alkaloid Compounds
Hexane3.29+++++++
Ethyl acetate6.83+++++++++
Absolute ethanol28.30+++++++++++
Deionized water40.73+++++++
(+) denotes presence; (++) and (+++) denote stronger color intensity.
Table 3. Total phenolic contents, total flavonoid contents, and antioxidant activities of T. stans extracted using various solvents.
Table 3. Total phenolic contents, total flavonoid contents, and antioxidant activities of T. stans extracted using various solvents.
SolventsTotal Phenolic Contents
(mg GAE/g Extract)		Total Flavonoid Contents
(mg CE/g Extract)		Antioxidant Activity
DPPH• Assay
(IC50 Value, mg/mL)		ABTS•+ Assay
(IC50 Value, mg/mL)
Hexane 6.90 ± 0.47 d131.22 ± 3.74 c1.378 ± 0.057 d*0.871 ± 0.063 c*
Ethyl acetate 12.66 ± 0.11 c205.11 ± 7.83 a0.847 ± 0.011 b*0.219 ± 0.027 a*
Absolute ethanol 24.10 ± 2.07 a140.67 ± 1.92 b0.935 ± 0.003 c*0.465 ± 0.027 b*
Deionized water 20.83 ± 1.28 b112.05 ± 1.90 d0.600 ± 0.004 a*0.207 ± 0.001 a*
Trolox--0.003 ± 0.0010.009 ± 0.001
Values are expressed as the mean ± SD (n = 3); Different letters denote statistically significant differences between treatment groups at p < 0.05. An asterisk (*) indicates a significant difference (p < 0.05) between treatment groups compared with Trolox.
Table 4. Physicochemical properties of skincare products that contain the T. stans flower extract.
Table 4. Physicochemical properties of skincare products that contain the T. stans flower extract.
ParameterInitiationAmbient Temperature4 °C
in a Refrigerator		45 °C
in a Hot-Air Oven		Heating/Cooling Cycling
(6 Cycles)
Color Light yellowLight yellowLight yellowLight yellowLight yellow
pH5.53 ± 0.015.24 ± 0.015.37 ± 0.035.22 ± 0.015.34 ± 0.02
HomogeneityExcellentExcellentExcellentExcellentExcellent
Phase separationNoNoNoNoNo
Viscosity (Pa.s)17.20 ± 0.6517.09 ± 0.5716.27 ± 0.1416.36 ± 0.2216.39 ± 0.81
All values in the table are represented as mean ± SD (n = 3).
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MDPI and ACS Style

Wichayapreechar, P.; Prasansuklab, A.; Charoongchit, P.; Charoenjittichai, R. The Potential of Tecoma stans (Linn.) Flower Extract as a Natural Antioxidant and Anti-Aging Agent for Skin Care Products. Cosmetics 2024, 11, 214. https://doi.org/10.3390/cosmetics11060214

AMA Style

Wichayapreechar P, Prasansuklab A, Charoongchit P, Charoenjittichai R. The Potential of Tecoma stans (Linn.) Flower Extract as a Natural Antioxidant and Anti-Aging Agent for Skin Care Products. Cosmetics. 2024; 11(6):214. https://doi.org/10.3390/cosmetics11060214

Chicago/Turabian Style

Wichayapreechar, Panikchar, Anchalee Prasansuklab, Pimchanok Charoongchit, and Ranit Charoenjittichai. 2024. "The Potential of Tecoma stans (Linn.) Flower Extract as a Natural Antioxidant and Anti-Aging Agent for Skin Care Products" Cosmetics 11, no. 6: 214. https://doi.org/10.3390/cosmetics11060214

APA Style

Wichayapreechar, P., Prasansuklab, A., Charoongchit, P., & Charoenjittichai, R. (2024). The Potential of Tecoma stans (Linn.) Flower Extract as a Natural Antioxidant and Anti-Aging Agent for Skin Care Products. Cosmetics, 11(6), 214. https://doi.org/10.3390/cosmetics11060214

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