Potential of Cissampelos pareira L. Pectin as a Bioactive Compound in Moisturizing and Anti-Aging Applications

Abstract

Cissampelos pareira is a plant known for its edible and medicinal properties, with bioactive potential in various applications. This study aimed to isolate and characterize pectin from C. pareira (PCP) and assess its safety, as well as its efficacy in skin moisturizing and anti-aging effects on human volunteers. The highest yield of PCP extraction was achieved after a 45 min extraction period. Chemical composition analysis using Fourier transform infrared (FT-IR) spectroscopy confirmed the presence of a characteristic pectin structure. Optimized PCP samples revealed high methoxy pectin content, degree of esterification, and galacturonic acid concentration, along with consistent physicochemical properties. PCP exhibited antioxidant activity and showed no cytotoxicity at 1 mg/mL, and effectively reduced hydrogen peroxide-induced oxidative damage in NIH/3T3 cells. Stability assessment indicated that PCP maintains its physical appearance and total polysaccharide content after heating–cooling periods. None of the PCP formulations caused skin irritation among volunteers. In an 8-week clinical trial, subjects who used serum formulations containing 0.5% w/w and 1.0% w/w PCP showed significant improvements in skin hydration and elasticity compared to the placebo serum group. While skin smoothness, scaliness, and wrinkling also improved, these anti-aging results were not statistically significant. The results indicated PCP is a promising candidate for skin care applications due to its safety, stability, antioxidant activity, and demonstrated efficacy in enhancing skin hydration and elasticity.

1. Introduction

Increasing consumer demand for natural products has resulted in the incorporation of plant-based extracts as active components in cosmeceutical and nutricosmetic formulations [1]. Bioactive compounds present in natural ingredients provide beneficial properties, such as antioxidant, anti-aging, and anti-inflammatory activities, which support applications in skincare, hair care, and oral health [2]. For instance, Camellia sinensis extracts contain catechin derivatives (e.g., epicatechin) that contribute to sustained moisturization, reduced skin roughness, and sebum production. Additionally, Vitis vinifera extracts are rich in resveratrol, which can inhibit UV light-mediated skin aging. Carica papaya extracts contain flavonoids (e.g., kaempferol and quercetin) and phenolic acids (e.g., ferulic acid, caffeic acid) that are widely commonly incorporated in anti-aging skin care formulations [1].

Pectins are large polysaccharides present in the primary cell walls of plants, where they play an important role in regulating ionic balance, cellular growth, and cell separation processes [3]. Due to their water-absorbing ability and capacity to slow gastrointestinal transit, pectins are used in managing diarrhea, particularly in children, and have been applied to facilitate wound healing [4]. Pectin is widely used for its thickening and gelling properties, which are greatly influenced by its fine structure, determined by both the botanical source and extraction conditions [5]. Notably, low methoxyl (LM) pectin can form gels at room temperature under appropriate conditions, enhancing its utility in temperature-sensitive cosmetic and pharmaceutical applications, where product stability is essential.

Cissampelos pareira (C. pareira), commonly known as “Velvetleaf” or “Khruea ma noi” in Thailand (Figure 1), is a well-known climbing plant of the Menispermaceae family with medicinal applications [6]. It can be found in various regions including India, Asia, East Africa, and subtropical regions of the Americas [7]. Traditional medicine has utilized this plant to treat ailments such as ulcers, rheumatism, fever, asthma, cholera, diarrhea, and rabies [8]. Crude extracts of C. pareira exhibit several pharmacological effects, including anti-inflammatory, antimicrobial, and antioxidant activities [9]. Phytochemical analysis of ethanolic extract from C. pareira leaves revealed the presence of bioactive compounds, including alkaloids, saponins, glycosides, carbohydrates, tannins, phenolics, flavonoids, steroids, protein, amino acids, triterpenoids, and lignins [10]. The isolated compounds from C. pareira, obtained through column chromatography using MCI gel CHP20P, Sephadex LH 20, and silica gel, include amentoflavone, kaempferol 3-O-β-D-glucopyranoside, and kaempferol 3-O-β-D-glucuronopyranoside [11]. According to Singthong et al. [12], galacturonic acid was identified as the primary constituent of pectin extracted from C. pareira leaves, with trace amounts of neutral sugars. Structural analysis, combining carboxyl reduction and methylation studies, revealed the main structure of C. pareira pectin as a 1,4-linked D-galacturonan. Currently, there is no available information in the literature regarding the cosmetic application of pectin from C. pareira (PCP). This study aims to expand the preparation and standardization of PCP in terms of physicochemical properties and antioxidant activity. Furthermore, assessments were conducted to evaluate cytotoxicity and cellular antioxidant accumulation. Additionally, a clinical study was conducted to evaluate the safety, moisturizing effects, and anti-aging efficacy of PCP on human volunteers.

2. Materials and Methods

2.1. Chemical Materials

The chemicals used for extraction were of commercial grade, while those employed in standardization and cellular studies were of reagent grade. Those used in topical formulations were of cosmetic grade. Ethanol (95%) was purchased from Northern Chemicals and Glasswares Co., Ltd. (Chiang Mai, Thailand) and RCL Labscan (Bangkok, Thailand). DPPH (2,2′-diphenyl-1-picrylhydrazyl), L-ascorbic acid, phenol, hydrochloric acid, sodium hydroxide, and phenolphthalein were sourced from Sigma-Aldrich (Burlington, MA, USA). Sulfuric acid (98%), hydrogen peroxide, and apple pectin were obtained from Merck (Darmstadt, Germany). Glucose was purchased from Chem-Supply Pty Ltd. (Gillman, SA, Australia). Dulbecco’s Modified Eagle’s Medium (DMEM), fetal bovine serum (FBS), and penicillin/streptomycin solution were supplied from Sigma-Aldrich (St. Louis, MO, USA). Olive oil and sodium hyaluronate were purchased from Namsiang Co., Ltd. (Bangkok, Thailand). Glycerol was sourced from Emery Oleochemicals (Selangor, Malaysia), while butylene glycol was from KH Neochem (Chuo-ku, Japan). Phenoxyethanol (and) caprylyl glycol (and) sorbic acid were supplied by Ashland (Covington, KY, USA). Polyacrylate crosspolymer-11 was obtained from Clariant (Álava, Spain). Sodium lauryl sulfate was sourced from BASF (Thai) Ltd. (Bangkok, Thailand). Distilled water was acquired from the Scientific & Technological Instruments Centers, MFU (Chiang Rai, Thailand).

2.2. Plant Material

C. pareira leaves were purchased from the Surin Khruea Ma Noi farm located in Surin Province, Thailand. The leaves were washed with tap water and dried in a tray dryer (XUE343, France Etuves, Chelles, France) at 60 °C for 6 h. Subsequently, the dried leaves were pulverized into powder using an electric blender (EM-ICE, Sharp Corporation, Osaka, Japan).

2.3. PCP Preparation

The PCP was prepared according to [13] with slight modification. The dried leaf flakes of C. pareira were subjected to extraction for three different time intervals: 15 min, 30 min, and 45 min. Initially, 5 g of each sample was pre-washed with 95% ethanol in a solid-to-ethanol ratio of 1:10 (w/v). The mixture was then placed in a water bath at 75 °C under three different conditions. Afterward, the ethanol solution was separated from the sample using a filter cloth, and the dried sample was retained. The extraction process of pectin started by combining the sample with distilled water, adjusted to pH 2.0 ± 0.05 using 0.1 N HCl, in a ratio of 1:50 (w/v). The mixture was heated in a water bath at 75 °C under three different conditions. The resulting mixture was filtered through a filter cloth to obtain a concentrated sample solution. The filtrate was further concentrated using a rotary evaporator (N-1000V-WD, EYELA, Tokyo, Japan). To precipitate PCP, the concentrated solution was vigorously stirred with 95% ethanol for 5–10 min. After precipitation, the solution was refrigerated for 24 h, followed by filtering and washing the PCP with 95% ethanol three times. The obtained pectin was subsequently dried in a hot air oven (Memmert UF110, Memmert USA LLC, Eagle, WI, USA) at 45 °C for 24 h, blended into powder form using an electric blender (EM-ICE, Sharp Corporation, Osaka, Japan), and kept in an air-tight container at 4 °C until further use.

2.4. Chemical Characterization of PCP

2.4.1. FT-IR Spectroscopy

FT-IR spectra were recorded using a Golden-gate Diamond single reflectance ATR on a FT-IR spectrophotometer (Spectrum-GX FT-IR, PerkinElmer, Shelton, CT, USA) with DTGS detector (PerkinElmer, Shelton, CT, USA). The spectra were recorded in the absorbance mode from 4000 to 400 cm⁻¹ (mid-infrared region) at the resolution of 4 cm⁻¹ and with 128 scans. Spectra readings for each sample condition and apple pectin standard were performed.

2.4.2. Methoxy Content (%), Degree of Esterification (%), and Galacturonic Acid (%)

Methoxy content, degree of esterification (DE), and galacturonic acid of PCP were determined [14]. Initially, 500 mg of pectin powder was transferred into a 250 mL flask, followed by 2 mL of ethanol for moisture. Next, 100 mL of carbon dioxide-free water was added to dissolve the powder, followed by adding 5 drops of phenolphthalein. The solution was titrated with 0.5 M sodium hydroxide for the initial titer. Subsequently, 10 mL of 0.5 M sodium hydroxide was added to the initial titer, vigorously shaken, and allowed to stand for 15 min. Afterward, 0.5 M hydrochloric acid was added and shaken until the pink color completely disappeared. The resulting solution was mixed with 5 drops of phenolphthalein and titrated with 0.5 M sodium hydroxide, vigorously shaken, and terminated once a faint pink color was visible (endpoint). The endpoint represented the saponification titer (the final titer). The methoxy content, DE, and galacturonic acid were calculated from the formula described by Wisidsri and Thungmungmee [15].

2.5. Physicochemical Analysis of PCP

2.5.1. Physical Appearance, pH, and Viscosity

The test involved visual and olfactory observations to evaluate changes in color and smell, respectively. A pH meter (SevenCompact, Mettler Toledo, Greifensee, Switzerland) was used to monitor the pH value. A viscometer (RVDV2T AMETEK Brookfield, Middleborough, MA, USA) was used to measure viscosity using a selected spindle. The viscosity of 1.0% w/v pectin solution was determined using spindle no.2 (AMETEK Brookfield, Middleborough, MA, USA) at 60 rpm, following the manufacturer’s recommendations based on the texture of the solution.

2.5.2. Total Polysaccharide Content

The phenol-sulfuric acid technique with slight modifications [16] was used for the estimation of total polysaccharide content. A standard curve was prepared using a stock solution of glucose, prepared in distilled water. Aliquots were taken from this solution to obtain glucose concentrations ranging from 100 to 800 µg/mL. To each 1 mL of glucose solution, 1 mL of 5% phenol solution was added, followed by 5 mL of concentrated sulfuric acid. A blank and sample were prepared in the same manner, but instead of the standard, 1 mL of distilled water or extract solution was used, respectively. All test tubes were heated at 95 °C for 10 min. The absorbance was measured at 490 nm. The concentration of polysaccharides was expressed as mg glucose equivalent per µg of extract.

2.5.3. Solubility and Swelling Capacity

The solubility of PCP (10 mg) was determined in deionized water (10 mL). Briefly, the pectin extract was saturated in each solvent overnight, and the clear supernatants were transferred into evaporating dishes and evaporated until completely dried. Weight of the dried residue, along with the reference, was determined and expressed as the solubility (%) [17]. The swelling of pectin extract was assessed by hydrating a sample (0.1 g) in a measuring cylinder with distilled water (10 mL) for 24 h. The volume (mL) occupied by the hydrated sample was recorded and calculated as swelling capacity (%) [18].

2.5.4. In Vitro Hydration and Oil Absorption Capacity

In vitro hydration was determined by mixing the polysaccharide (100 mg) with water (6 mL) in a centrifuge tube and allowing it to hydrate for 18 h. The mixture was then subjected to centrifugation at 7000× g for 60 min. After decanting the supernatant, the weight of residue was recorded. The hydration capacity was calculated as the amount of water retained by the pellet (g of water per g of dry sample weight). In addition, the oil absorption capacity was accessed using the same procedure, but olive oil was used instead of water [19].

2.6. Antioxidant Activity by 2,2-Diphenyl-1-Picrylhydrazyl (DPPH) Assay

The antioxidant activity was determined by a DPPH scavenging assay [19]. PCP was dissolved in distilled water for variable concentrations (0.1–5.0 mg/mL). A quantity of 100 μL of the dissolved PCP was mixed with 100 μL of 6 × 10⁻⁵ M DPPH solution, which was performed in a 96-well plate and allowed to stand for 30 min without direct exposure to light. The absorbance was measured at 517 nm using a microplate reader (SPECTROstar Nano, BMG LABTECH, Ortenberg, Germany). In addition, distilled water and L-ascorbic acid were used as the negative and positive controls, respectively. The DPPH scavenging capacity of the experiment was expressed as the percentage of DPPH radical inhibition and half maximal inhibitory concentration (IC₅₀).

2.7. Cell Culture

The NIH/3T3 fibroblasts (ATCC^® CRL-1658^TM) were obtained from the American Type Culture Collection (Manassas, VA, USA). The cells were cultured at 37 °C in a humidified incubator with 5% CO₂ (Binder CB210, BINDER GmbH, Tuttlingen, Germany) using Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin solution. The cell passages ranging from 16 to 23 were utilized for the experiments.

2.7.1. Cytotoxicity Assay

A protocol stated by Myo and Khat-udomkiri in 2022 [20] was used to perform the MTT assay to assess the mitochondrial functionality of the NIH/3T3 cells. The cells were treated with either ascorbic acid (AA) or PCP, while DMEM without the FBS supplement was used as the control for 24 h. The formazan crystals were dissolved using dimethyl sulfoxide solution and quantified spectrophotometrically at 570 nm. Cell viability (%) was calculated using the following equation:

Cell viability (%) = (Absorbance sample/Absorbance control) × 100(1)

2.7.2. Cellular Antioxidant Activity

The cellular antioxidant assay was performed using the method described by Khat-udomkiri et al. [21]. NIH/3T3 cells were exposed to ascorbic acid (AA) and PCP, while cells maintained in DMEM medium without fetal bovine serum (FBS) supplementation served as the control for 24 h. Subsequently, the cells were treated with hydrogen peroxide (H₂O₂). Cell viability (%) was assessed and quantified using the MTT assay.

2.8. Stability Test in Accelerated Conditions

2.8.1. Stability of Pectin

A 1.0% w/v sample solution was prepared by dissolving PCP in distilled water for stability testing. The samples were transferred to clean screw-cap universal bottles to prevent vapor or gas leakage into the environment. They were subjected to an accelerated stability test consisting of six heating-cooling cycles, alternating between 45 and 4 °C for 24 h at each temperature. Physical appearance (color and odor), pH, viscosity, antioxidant activity (DPPH assay), and total polysaccharide content were evaluated at the initial (T0), third (T3), and sixth (T6) cycles.

2.8.2. Stability of Serum Products

Formulation of Serum Products

The serum formulations were prepared as cosmetic products. Briefly, six serum formulations were developed based on the extracts used (Table 1). FB was a base serum used as a placebo. Formulations F1 to F3 contained 0.2% w/w, 0.5% w/w, and 1.0% w/w of PCP, respectively. F4 was a serum containing 0.50% w/w pectin from apples and F5 was a serum containing 0.50% w/w sodium hyaluronate. Each moisturizer serum was formulated by pre-mixing the water phase materials, including distilled water, glycerol, and butylene glycol. Then, the thickener agent, polyacrylate crosspolymer-11, was added to modify the rheology of the serum. Finally, the extract and preservatives were added, and the mixture was stirred until homogeneous. Serum products are divided into six formulations depending on the extract. FB was a placebo.

Centrifugation Test

A centrifugation test was used to determine the resistance of the serum under centrifugal force. The assay was conducted following the requirements of the ANVISA cosmetic product [22]. Ten grams of each sample were placed in 17 mm diameter centrifuge tubes and subjected to centrifugation at 3000 rpm for 30 min.

Heating-Cooling Cycle

All samples were conducted with the same methods as previously described. The physical appearance (color and odor), pH, and viscosity changes of the samples were determined. Viscosity was measured using a viscometer with a spindle no.4 (AMETEK Brookfield, Middleborough, MA, USA) at 200 rpm.

2.9. Clinical Evaluation in Volunteers

2.9.1. Ethical Conduct

The study protocol in human volunteers was conducted according to the guidelines of the Declaration of Helsinki. Prior to human study, the protocol was screened and approved by the Mae Fah Luang University Ethics Committee, Thailand with COA number EC 22124-17.

2.9.2. Volunteer Recruitment

Thirty-five healthy Thai male and female volunteers aged between 25 and 50 years old were screened and enrolled according to inclusion and exclusion criteria. The inclusion criteria required participants to be healthy, with no lesions on the tested area, not currently involved in other clinical trials, and without a history of allergies to any ingredients in the formulation. The exclusion criteria included individuals with any skin diseases or abnormalities and those currently taking other medications. All procedures were thoroughly explained to the volunteers, and informed consent was obtained before the study was initiated.

2.9.3. Safety Test

The epicutaneous closed patch test was used to evaluate the safety profile of PCP. The patch test was performed using 8 mm Finn chambers (SmartPractice, McDowell, WV, USA). The serums FB and F1 to F5, as test samples, were filled in Finn chambers prior to the application on the anterior upper arm of volunteers. The 0.5% sodium lauryl sulfate (SLS) solution was used as a positive control, while deionized water was used as a negative control. All volunteers were instructed to avoid water, strenuous exercise, excessive sweating, and scratching. After 24 h, the patch was removed, and the patched area was evaluated for irritation and allergic reactions at 30 min, 24 h, 48 h, and 72 h after patch removal. The test results were then analyzed according to the grading criteria for skin reactions provided by the Cosmetic, Toiletry, and Fragrance Association (CTFA) Guidelines. The scores were determined by calculating the mean irritation index (M.I.I.) as described by An et al. [23].

2.9.4. Evaluation of Moisturizing and Anti-Skin Aging Efficacy

A single-blinded, placebo, simple randomized trial was conducted to assess the moisturizing and anti-skin aging efficacy. For a single-blind study, the participants did not know which side of their arm received serum containing PCP or placebo serum. Participants underwent a two-week rest period after the epicutaneous patch test and were assessed for any adverse events at the test site before. Only those without adverse events were included in the subsequent study. In the efficacy study, participants were asked to apply 0.1 mL of serum twice a day (morning and night) to a 3 × 3 cm² area on their forearm for 8 consecutive weeks. The measurements were performed under controlled conditions (temperature 20 ± 2 °C and 40–60% relative humidity). Before measurements, the subjects were instructed to properly rinse their left and right forearms with a given cleansing product and acclimate to the ambient atmosphere for at least 10–20 min. Skin parameters at the baseline (W0) and after applying the serum for 1–8 weeks (W1–W8) were measured by using the following devices:

Corneometer^® CM 825, Tewameter^® TM 300, and Cutometer^® MPA 580 (Courage Khazaka electronic GmbH, Cologne, Germany) were utilized with MPA CTplus software (first accessed in February 2023) to assess skin hydration, trans-epidermal water loss (TEWL), and skin elasticity, respectively. Measurements were performed on well-defined areas of the left and right forearm. For the evaluation of skin elasticity, parameters of R2 as the overall elasticity of the skin, including creep and creep recovery, R5 as pure elasticity ignoring viscoelastic creep, and R7 as firmness were obtained from the Cutometer^® [24].

Visioscan^® VC98 USB (Courage Khazaka electronic GmbH, Cologne, Germany) was used to undertake skin microtopography on the left and right forearm in well-defined measurement areas utilizing the SELS (Surface Evaluation of the Living Skin) software (first accessed in February 2023) equipped by the device. Parameters for smoothness (SEsm), scaliness (SEsc), and wrinkles (Sew) were recorded.

All parameters measured during the long-term studies were presented as a percentage of change from baseline (Week 0), calculated with the following formula:

(2)$\%\mathrm{Change}=\left[{\displaystyle \frac{\mathrm{Week} n - \mathrm{Week} 0}{\mathrm{Week} 0}}\right]\times 100,$

where Week n represents the value for n = 1, 2, 3 and 4.

2.10. Statistical Analysis

Statistical analysis data were presented as the means ± S.D. One-way analysis of variance (ANOVA) was performed to assess the differences between groups using SPSS version 25.0. The level of significance was set at p < 0.05. The Shapiro-Wilk test was used to determine the normality of the data distribution since the data have smaller samples (n < 50). For the clinical study, the statistical analysis data were presented as means ± SEM and Kruskal-Wallis one-way analysis of variance was used to evaluate the differences between groups, as the sample data are significantly different than a normal population.

3. Results and Discussion

3.1. Preparation of Pectin C. pareira (PCP) Extract

Ethanol was utilized for removing epicuticular wax, which acts as a natural film covering the outer surface of plant leaves [25,26], It was also used to precipitate carbohydrates including pectin. Pectins are hydrophilic polysaccharides that extensively interact with water molecules in aqueous solution. Organic precipitants may interrupt these pectin-water interactions to separate the pectins from the solution. Ethanol and isopropanol are commonly used to purify pectins from the solution [27]. Because isopropanol toxicity is well documented, ethanol was safer to use as an extraction solvent for the pectin in this study. To prove the optimal extraction conditions for maximizing pectin yield by time at 15, 30, and 45 min, Figure 2 illustrates the physical appearance of the extracted PCP solution after refrigerating for 24 h, the filtered and washed PCP, and the dried PCP. The results showed that an extraction time of 45 min yielded the highest percentage (18.07 ± 2.04%), although there is no significant difference compared to the yield obtained at 30 min (17.00 ± 1.11%) (Figure 3). This value was higher than the C. pareira pectin yield of 3% reported by Wasidsri and Thungmungmee [15]. However, it was lower than the yields reported by Singthong et al. [14] (21.5–28.9%) and Linn et al. [28] (25–35). These differences are likely due to variations in the extraction condition and the origins of the pectin, which influence both the quantity and quality of the extracted pectin [14,15,28].

3.2. Chemical Characterization of PCP

3.2.1. FT-IR Spectroscopy

The FT-IR spectra of the crude extracts were compared with those of commercial apple standard pectin to confirm the polysaccharide characteristics. It was observed that the FT-IR spectra of all crude polysaccharides exhibited similar absorption patterns to the commercial pectin, as shown in Figure 4. The analysis of the FT-IR spectra revealed distinct absorption bands in PCP. The spectrum of pectin exhibited a board band at 3500–3200 cm⁻¹, corresponding to the O–H stretching. The sharp band at 3000–2500 cm⁻¹ is attributed to the C–H stretching vibration. Additionally, strong bands were observed at 1730–1760 cm⁻¹ and 1600–1630 cm⁻¹, indicating the presence of esterified C=O groups of methylated carboxyl groups and COO-symmetric stretching, respectively [29]. Medium bands at 1310–1390 cm⁻¹ and less significant bands at 1000–1300 cm⁻¹ were assigned to O–H bending and C=O stretching, respectively. The FT-IR spectra between the wavenumbers 950–1200 cm⁻¹ are considered as the “fingerprint” region for carbohydrates, which allows the identification of major chemical groups in polysaccharides. The position and intensity of the bands are specific for each polysaccharide [30]. Furthermore, an increase in the intensity of the absorbance of ester carbonyl groups at wavenumbers 1730–1760 cm⁻¹ indicates a high degree of esterification, whereas the intensity of the free carboxyl stretching band decreased [31].

3.2.2. Methoxy Content (%), Degree of Esterification (%), and Galacturonic Acid (%)

This was evaluated in conjunction with the qualitative characteristics of the pectin, including methoxy content, degree of esterification (DE), and galacturonic acid. Methoxy content serves as a key molecular parameter for pectin classification, representing the proportion of carboxyl groups within the pectin molecules that exist as the methyl ester relative to the total esterified groups. Additionally, the methoxy content is a crucial factor in regulating the setting time of pectins and their gel-forming capacity [17]. This study (Table 2) showed that the methoxy content of PCP was 1.71%, 2.48%, and 6.67% for extraction times of 15, 30, and 45 min, respectively. An increase in methoxy content led to enhanced spreading quality and sugar-binding capacity of the pectin. Correspondingly, the degree of esterification is another molecular index for pectin classification that describes the extent to which carboxyl groups in pectin molecules exist as methyl esters [32]. The DE for PCP samples was found to be 11.92%, 19.76%, and 29.42% for extraction times of 15, 30 and 45 min, respectively. Based on the %DE presented in Table 1, the pectin in this study can be categorized as low methoxy pectin (LMP) as its value is below 50%. This result of a low degree of esterification is similar to that found in other studies [12,14,15]. The galacturonic acid of PCP was found to be 10.68%, 15.53%, and 41.74% for extraction durations of 15, 30, and 45 min, respectively. According to Willats et al. [33], pectin consists of a chain of galacturonic acid units linked by (1,4)-linked α-D-galacturonic acid units. The high galacturonic acid content in PCP may indicate the approximate value of the present pectin. The selected conditions were mainly considered from the yielded content of crude extract. All extraction times exhibited characteristics of low methoxy pectin (LMP), as indicated by methoxyl content below 8.16% and a DE below 50%, consistent with the reference criteria [34]. The 45 min extraction condition exhibited the highest yield extraction (%) and the galacturonic acid showed a significant difference (p < 0.05). Therefore, an extraction time of 45 min was selected for further study.

3.3. Physicochemical Properties of PCP

3.3.1. Physical Appearance, pH, and Viscosity

Basic standardization of PCP involved the determination of its physical appearance, pH, and viscosity, with the results presented in Table 3. The 1.0% w/v pectin solution exhibited a dark green color and green odor. The pH value of PCP was 3.74 ± 0.04, indicating its acidic nature. Pectin is primarily composed of D-galacturonic acid units connected by α-(1–4) glycosidic linkages, with several carboxyl or hydroxyl groups dispersed along the backbone, while a small percentage of neutral sugars may be present as side chains [35]. The viscosity of the 1.0% w/v PCP solution was 614.00 ± 3.53 cps. Viscosity is a rheological property that characterizes the flow qualities of a substance. The evaluation of viscosity plays a significant role in assessing consistency, fluidity, and stability over time [36].

3.3.2. Total Polysaccharide Content

The colorimetric determination of the polysaccharide content involves dehydrating polysaccharides using concentrated sulfuric acid, resulting in the production of furfural derivatives. The reaction of furfural derivatives with phenol produces a distinct color [37]. In Table 3, the PCP exhibited the total polysaccharide content of 20.44 ± 0.16 µg glucose/µg extract (equivalent to 20.44 ± 0.16 mg/g extract). Comparing our findings with other studies, noticeable differences in polysaccharide content can be observed, highlighting the variability across sources. For example, Cucumis melo polysaccharide [38] exhibited a total polysaccharide content of 0.23 ± 0.01 mg glucose/mg extract (or 230 mg/g extract), while cocoa pod husk [39] demonstrated an even higher content, ranging between 270 and 320 mg/g extract. Both are significantly higher than that of PCP. In contrast, the PCP exhibited a greater polysaccharide content than Ceylon spinach, which contained 0.37 ± 0.00 mg/g extract [40]. Pectin and other polysaccharides contain numerous hydroxyl groups in their chemical structures, which are believed to contribute to their antioxidant properties [41,42]. Additionally, the pectin demonstrated anti-inflammation and anti-aging properties [43].

3.3.3. Solubility and Swelling Capacity

The dried crude extract exhibited a high solubility in water, with a value of 99.93 ± 0.01% (Table 3). Generally, pectin is soluble in purified water. Monovalent cation salts of pectinic and pectin acids are typically soluble in water, while di- and trivalent cation salts tend to be weakly soluble or insoluble. The swelling capacity of hydrogels increases with the hydrophilicity of the hydrogel network [13,44]. In this work, the dried PCP was hydrated, and underwent swelling in deionized water, resulting in a thick, viscous solution of hydrogel. The swelling capacity was valued at 0.40 ± 0.00 (Table 2). The swelling capacity holds potential for various applications, including its utilization in cosmetic products as a thickening or suspending agent.

3.3.4. In Vitro Hydration and Oil Absorption Capacity

The water absorption or water-holding capacity (in vitro hydration efficacy) is a critical factor influencing hydrogel formation and determining its suitability for biomedical and cosmetic applications [39]. The in vitro hydration capacity of PCP is recorded as 9.79 ± 0.90 mL/g) in Table 2, which is attributed to its high polysaccharide content. The hydrophilic side chains present in the carbohydrate structure work together to regulate water absorption levels. On the other hand, the extract yielded a negative result in the oil absorption capacity assay (Table 3). The oil absorption capacities are influenced by the hydrophobic regions (backbone) of the polysaccharide structure [18].

3.4. Antioxidant Activity

DPPH^• is a stable free radical that possesses a deep purple color and a strong absorption around 517 nm. The antioxidant compounds in the medium reduce the DPPH radical by donating an electron or a hydrogen atom, thereby converting it into a more stable molecular product by donating. This reduction is accompanied by a color change, from the purple of the DPPH radical to the pale yellow of its reduced form, enabling quantification of antioxidant activity through spectrophotometric analysis [45]. The results revealed that PCP concentrations ranging from 0.1 to 5.0 mg/mL exhibited inhibitory performance ranging from 26.04 ± 1.03% to 105.82 ± 4.33%, with an IC₅₀ value of 0.72 ± 0.02 mg/mL (Table 3). The IC₅₀ is close to the value reported previously, which was 0.54 mg/mL [15]. Although the antioxidant activity of PCP is not as potent as that of standard ascorbic acid, its IC₅₀ values are within the range of other natural sources, such as mangosteen rind pectin (IC₅₀ = 0.161 ± 0.03 mg/mL) [46], Conocarpus erectus pectin (IC₅₀ = 0.189 mg/mL) [47], Cucumis melo polysaccharide extract (IC₅₀ = 0.214 ± 0.006 mg/mL) [38], and cocoa pod husk polysaccharide (IC₅₀ = 0.217 ± 0.006 mg/mL) [39] and apple pectins (IC₅₀ = 2.75 ± 0.09 to 5.24 ± 0.08 mg/mL) [48]. This consistency suggests that PCP could serve as a viable alternative in applications where natural antioxidants are preferred.

3.5. Cell Culture

3.5.1. Cytotoxicity Assay

Cytotoxicity was assessed by exposing cells to varying concentrations of AA and PCP to establish their maximum non-toxic concentrations. The cytotoxicity assay results for AA and PCP are shown in Figure 5. Cells treated with 0.001 mg/mL and 0.005 mg/mL of AA demonstrated viability rates of 94.66 ± 0.50% and 84.98 ± 1.83%, respectively, indicating an absence of cytotoxic effects at these concentrations [20]. Cell survival was significantly reduced when exposed to AA concentrations ranging from 0.01 to 0.1 mg/mL, with cell viability below 80%. PCP concentrations ranging from 5.0 to 50.0 mg/mL decreased NIH/3T3 cell viability to below 80%. In contrast, PCP concentrations of 0.5 mg/mL and 1.0 mg/mL were non-cytotoxic, as cell viability remained above 80%.

3.5.2. Cellular Antioxidant Activity

The results of the cellular antioxidant effect of PCP on NIH/3T3 fibroblasts under the oxidative stress induced by 400 μM H₂O₂ are presented in Figure 6. Compared to the control group, treatment with H₂O₂ resulted in a significant reduction in cell viability, decreasing to 53.66 ± 4.99% (p < 0.05). However, in NIH/3T3 fibroblasts pre-treated with AA at 0.005 mg/mL, cell viability increased to 59.48 ± 3.14%. Furthermore, PCP at concentrations of 0.5 and 1 mg/mL showed a statistically significant increase in cell viability (65.57 ± 3.52% and 74.51 ± 3.14%, respectively) compared to those exposed to H₂O₂ alone (p < 0.05). Consistent with previous findings indicating the antioxidant activity of PCP [15], the present study concludes that PCP exerts a protective effect against H₂O₂-induced oxidative stress in NIH/3T3 fibroblasts.

3.6. Stability Test by Accelerated Condition

3.6.1. Stability of PCP

The stability of PCP was assessed through a heating and cooling process over six cycles, as shown in Table 4. There was no change in color or odor observed after the stability test. The pH, viscosity, and antioxidant activity of the extract exhibited significant differences (p < 0.05) between T0 and T6, whereas the polysaccharide content remained consistent (p > 0.05). Consequently, 1.0% w/v pectin solution was acidic with a pH ranging from 3.74 ± 0.04 to 3.92 ± 0.06. In terms of viscosity, an increase in the temperature of liquid leads to an increase in the average kinetic energy of molecules. This effect was observed in our experiment, although the viscosity of T3 and T6 showed no significant difference (p > 0.05). The DPPH radical scavenging activity decreased as the IC₅₀ value increased. The antioxidant ability of polysaccharides is closely related to their molecular weight. Polysaccharides with low molecular weights tend to have more reductive hydroxyl group or carboxyl group terminals to accept and eliminate free radicals [49,50]. Therefore, the molecular weight of PCP may have changed after the stability test, leading to a decrease in antioxidant activity. However, there was a significant difference in antioxidant activity from T0 to T3 (p < 0.05), whereas no significant decrease was observed between T3 and T6 (p > 0.05).

3.6.2. Stability of Serum Products

All serums (FB, F1–F5) exhibited distinct characteristics including color, smell, spreadability, and texture. Serums containing PCP at concentrations of 0.2% w/w (F1), 0.5% w/w (F2), and 1.0% w/w (F3) were pale yellow, pale green, and dark green, respectively, depending on the PCP concentration. FB and F5 were colorless while F4 displayed a pale orange color. Serums containing PCP had a green odor. The odor was stronger corresponding to a higher extract percentage. FB, F4, and F5 were odorless. All serums remained homogeneous after centrifugation at 3000 rpm for 30 min. There were no changes observed in terms of color or odor. The pH of a serum product is critical as fluctuations in readings could indicate product instability or contamination during production and storage. Additionally, the changes in pH value suggest the possibility of a chemical reaction between the raw materials used in the product, which could affect its efficacy and safety [51]. After stability testing, there was no change in color or smell. The data in Table 5 showed no significant difference (p > 0.05) from the initial measurements. These results indicated that serums are physically stable and acceptable. Cosmetic preparations should maintain a viscosity within a defined range to ensure physicochemical stability throughout their shelf life and during application by the user.

3.7. Clinical Evaluation in Volunteers

3.7.1. Skin Safety

The epicutaneous patch test is accepted as a gold standard technique for evaluating skin safety. Patch test results are valuable for classifying substances as either irritants or allergens. All tested serums demonstrated an absence of irritant and allergic effects in human volunteers, with the M.I.I. score similar to deionized water (M.I.I. < 0.20), whereas SLS showed slight irritation (0.20 < M.I.I < 0.50), with an M.I.I value 0.23. Therefore, all serums (FB and F1 to F5) can be further used to study their efficacy in human volunteers.

3.7.2. Evaluation of Moisturizing and Anti-Skin Aging Efficacy

This study investigated the efficacy of PCP (F1–F3) in skin moisturizing and anti-aging effects over 8 weeks. Formulation with the same composition without PCP was used as a placebo serum (FB). Serums containing 0.5% w/w pectin from apples (F4) and 0.5% w/w sodium hyaluronate (F5) were used as positive controls. The amount of water in the stratum corneum (SC) has a positive impact on the skin’s appearance as well as its barrier function. Numerous non-invasive techniques have been developed to assess the skin moisture level and the effects of various moisturizers on skin moisture content. The Corneometer^® CM 825, Tewameter^® TM 300, and Visioscan^® VC 98 are commonly used instruments in dermatological research [52].

The Corneometer^® utilizes the capacitance method to measure the water content (skin hydration) of the SC. Since water has the highest di-electrical constant in the skin, increased capacitance values indicate an increase in the skin’s water content. The mean change in water content after measurements of the SC was converted into arbitrary units (AU) [52,53]. Comparing each serum across different time points, all formulations improved skin hydration over time, with varying patterns of significance across different week pairs. Only the serum containing 0.50% w/w sodium hyaluronate (F5) demonstrated significant improvement in hydration earlier, starting in week 2 and continuing through week 8. In contrast, the serums FB, F1, F2, F3, and F4 significantly improved skin hydration (p < 0.05) from week 1, with effects sustained through weeks 3 to 8. However, Figure 7a illustrates that serums containing PCP (F2 and F3) exhibited significant differences (p < 0.05) compared to FB, similar to F4 and F5 in week 3. Therefore, it can be concluded that the efficiency of 0.5% w/w PCP and 1.0% w/w PCP was similar to that of positive controls.

One of the most essential properties of skin barrier is trans-epidermal water loss (TEWL). Skin barrier impairments are typically associated with higher TEWL, while healthy skin is associated with lower TEWL. To measure the water evaporating from the skin, a probe with sensors that detect variations in water vapor density is placed in contact with the skin surface [54]. The result after applying the serum products for 8 weeks is shown in Figure 7b. A decrease in TEWL of all products was observed with no significant difference (p > 0.05) compared to FB in the same week. After applying each serum at different time points, serums FB, F1, and F3 showed no significant differences in TEWL across all weeks. In contrast, F2 showed a significant decrease (p < 0.05) in TEWL from week 3 and maintained its prevention of TEWL until week 8, similar to pectin from apples (F4), whereas sodium hyaluronate (F5) showed a significant decrease (p < 0.05) in TEWL in week 8. Therefore, it can be concluded that 0.5% w/w PCP showed as much efficiency as 0.5% w/w pectin from apples, which was the positive control.

Moisturizing agents retain water by forming hydrogen bonds between their polar groups and water molecules and providing film for water locking. Polyols, such as glycerol and sorbitol, are commonly used due to their hydroxyl groups. Compared with polyols, polysaccharides contain more hydroxyl, carboxyl, acetyl, and other polar groups in their chains, giving them excellent hydrophilic activity [55]. Hyaluronic acid, a widely recognized polysaccharide, has long been regarded as a benchmark for moisturization. In this study, PCP demonstrated no significant difference compared to hyaluronic acid in terms of skin hydration and TEWL, making it a promising alternative. Furthermore, enhanced stratum corneum hydration and maintenance of TEWL has been reported for polysaccharide-based formulations with both short-term and long-term applications [19,39,56].

Skin aging is a complex process characterized by a decline in elasticity and an increase in wrinkling, dryness, and roughness [57]. The Cutometer^® MPA 580 is used to measure the mechanical properties of the skin, especially its elasticity [58]. The Cutometer^® MPA 580 functions by mechanically deforming the upper layer of the skin through the application of negative pressure that pulls it into the probe opening. This allows the measurement of skin resistance and its ability to recover from such deformation (elasticity). The values obtained are then used to generate a deformation curve from which elasticity (R) parameters are calculated. Among these parameters, overall elasticity (R2), net elasticity (R5), and firmness (R7) are indicators of the influence of aging on the skin’s mechanical properties [59]. These parameters are associated with the function of the skin’s elastic fibers and can indicate the changes due to genetic, physiological, or acquired pathological skin conditions [60]. The results indicated a general improvement in skin elasticity after using all serum formulations (Figure 8). Serum containing 0.5% w/w PCP (F2) demonstrated a significant increase in overall elasticity (R2) compared to the placebo (FB) in week 2 (p < 0.05). Likewise, serum containing 1.0% w/w PCP (F3) exhibited a significant difference (p < 0.05) compared to the placebo (FB) as well as the serum containing 0.5% w/w pectin from apples (F4) in week 3. Furthermore, it displayed significantly higher overall elasticity (R2) compared to the serum containing 0.5% w/w sodium hyaluronate (F5) in week 3. Therefore, it can be concluded that increasing the concentration of PCP leads to improved skin elasticity. Moreover, 1.0% w/w PCP had a higher efficiency in enhancing skin elasticity compared to 0.5% w/w sodium hyaluronate in week 3. Figure 8b indicates a slight increase in net elasticity (R5) of the skin after using all serums over time. Furthermore, it was revealed that 1.0% w/w PCP exhibited a higher efficiency in improving net elasticity (R5) compared to 0.2% w/w PCP with a significant difference (p < 0.05) in week 3. As the results showed, skin elasticity slightly improved each week after applying the serums. However, the results in week 6 showed a reduction in skin elasticity improvement. Lastly, the insignificant change in R7 indicated no change in skin firmness (R7) by all formulations.

The Visioscan^®, which comprises a high-resolution camera and a halogenide light source, was utilized to analyze the changes in skin topography. The software employs the surface analysis of living skin (SELS) method to analyze the image data and calculate several SELS parameters, which are used to evaluate the skin surface both quantitatively and qualitatively [61]. The parameters used in this study included skin smoothness (SEsm), scaliness (SEsc), and wrinkling (SEw). Figure 9 illustrated a decrease in SEsm values in volunteers after applying serums, indicating that the skin has become smoother, although the difference was not statistically significant (p > 0.05). Skin scaliness (SEsc) decreased in all serum formulations after 6 weeks of application, as shown in Figure 10, although the difference was not statistically significant (p > 0.05). Lower SEsc values suggested that the skin became more hydrated over time. It was supported by the values obtained from Corneometer^® MPA 580 for skin hydration. A smaller SEsc value corresponds to higher skin moisture, as treatment with moisturizing or anti-aging formulations tends to reduce SEsc values [62]. Regarding the SEw result (Figure 11), it showed that all serum formulations reduced the extent of wrinkles after application compared to the placebo, although the difference was not statistically significant (p > 0.05). The results in terms of the percentage change in skin smoothness, scaliness, and wrinkles in week 6 were lower compared to other weeks.

4. Conclusions

Pectin from C. pareira leaves exhibited antioxidant activity with considerable amounts of total polysaccharides. The stability of pectin extract did not show statistical significance in terms of organoleptic characteristics and total polysaccharide content, but it showed a significant change in pH, viscosity, and antioxidant potential. At a concentration of 1 mg/mL, PCP displayed no cytotoxicity and effectively reduced H₂O₂-induced oxidative damage in NIH/3T3 cells. In the long-term clinical study, serum containing 0.5–1.0% w/w PCP showed statistically significant improvements in skin hydration and elasticity. However, changes in skin smoothness, scaliness, and wrinkle appearance were not significantly different from baseline values. The results of this study suggest that PCP is a promising, safe, and hydrating ingredient with potential applications for a variety of industries, including pharmaceuticals and cosmetics.

Author Contributions

S.C.: Methodology, Investigation, Formal analysis, Writing—Original Draft, Writing—Review and Editing. N.K.-u.: Methodology, Investigation, Formal analysis, Writing—Original Draft. T.T.-U.: Investigation, Formal analysis, Writing—Original Draft. N.D.: Methodology, Investigation, Writing—Original Draft. N.S.: Writing—Original Draft. M.P.V.: Writing—Original Draft, Writing—Review and Editing. T.S.: Conceptualization, Methodology, Investigation, Formal analysis, Writing—Original Draft, Writing—Review and Editing, Visualization, Supervision, Project administration, Funding acquisition. All authors have read and agreed to the published version of the manuscript.

Institutional Review Board Statement

The study was conducted according to the guidelines of the Declaration of Helsinki and approved by the Ethics Committee of Mae Fah Luang University. The certificate of analysis (COA) and protocol numbers are 037/2023 and EC 22124-17, respectively (Approval issued on 7 February 2023).

Informed Consent Statement

Informed consent was obtained from all the volunteers involved in the study.

Data Availability Statement

Data are contained within the article.

Acknowledgments

The authors would like to thank Mae Fah Luang University (MFU) for providing laboratory facilities and financial support. The authors also warmly thank Panita Mahawong and Thanaporn Khonpudsa, School of Cosmetic Science, MFU, for recording the clinical results.

Conflicts of Interest

The authors declare no conflicts of interest.

Footnotes

Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Figures and Tables

Figure 1. C. pareira.

Figure 2. The extracted PCP solution after refrigerating for 24 h (a), the filtered and washed PCP (b), and the dried PCP (c).

Figure 3. The percentage yield of the PCP extracted using 95% ethanol as extraction solvent, under each condition at 15, 30, and 45 min. * indicates a significant difference (p [less than] 0.05) from the percentage yield of PCP. Data are represented as the mean ± S.D. from triplicate measurements.

Figure 4. Fourier transform infrared spectra of PCP under each condition at 15, 30, and 45 min, and the apple pectin standard.

Figure 5. Cytotoxicity of ascorbic acid (AA) and PCP in different concentrations. * indicates the significant difference from the control by LSD test (p [less than] 0.05).

Figure 6. Cellular antioxidant activity of ascorbic acid (AA) and PCP. * indicates the significant difference from the control by LSD test (p [less than] 0.05). # indicates the significant difference from the H2O2 by LSD test (p [less than] 0.05).

Figure 7. Percentage change in skin hydration as measured by the Corneometer® (a) and in TEWL as measured by the Tewameter® (b). Values are given as mean ± SEM from triplicate measurements. * indicates the significant difference between FB and each formulation in the same week (p [less than] 0.05).

Figure 8. (a) Percentage change in skin elasticity (R2) as measured by the Cutometer®. Values are given as mean ± SEM from triplicate measurements. (b) Percentage change in skin elasticity (R5) as measured by the Cutometer®. Values are given as mean ± SEM from triplicate measurements. (c) Percentage change in skin firmness (R7) as measured by the Cutometer®. Values are given as mean ± SEM from triplicate measurements. * indicates the significant difference between each formulation in the same week (p-value [less than] 0.05).

Figure 8. (a) Percentage change in skin elasticity (R2) as measured by the Cutometer®. Values are given as mean ± SEM from triplicate measurements. (b) Percentage change in skin elasticity (R5) as measured by the Cutometer®. Values are given as mean ± SEM from triplicate measurements. (c) Percentage change in skin firmness (R7) as measured by the Cutometer®. Values are given as mean ± SEM from triplicate measurements. * indicates the significant difference between each formulation in the same week (p-value [less than] 0.05).

Figure 9. Percentage change in skin smoothness (SEsm), measured by Visioscan®. Values are given as mean ± SEM from triplicate measurements.

Figure 10. Percentage change in skin scaliness (SEsc), measured by Visioscan®. Values are given as mean ± SEM from triplicate measurements.

Figure 11. Percentage change in skin wrinkling (SEw), measured by Visioscan®. Values are given as mean ± SEM from triplicate measurements.

Table 1

Composition of serum containing various active ingredients.

Ingredients	Functions	Formulations (% w/w)
		FB	F1	F2	F3	F4	F5
Distilled water	Diluent	79.0	78.8	78.5	78.0	78.5	78.5
Glycerol	Humectant	10.0	10.0	10.0	10.0	10.0	10.0
Butylene glycol	Humectant	10.0	10.0	10.0	10.0	10.0	10.0
Phenoxyethanol (and) Caprylyl Glycol (and) Sorbic Acid	Preservative	0.5	0.5	0.5	0.5	0.5	0.5
Polyacrylate Crosspolymer-11	Thickener agent	0.5	0.5	0.5	0.5	0.5	0.5
Pectin from apple	Moisturizing Agents	-	-	-	-	0.5	-
Sodium Hyaluronate	Moisturizing Agents	-	-	-	-	-	0.5
C. pareira extract	Moisturizing Agents	-	0.2	0.5	1.0	-	-

Table 2

Methoxy content (%), degree of esterification (%), and galacturonic acid (%) of PCP.

Extraction Conditions			Result
Temperature(°C)	Time(min)	pH	Methoxy(%)	Degree of Esterification (%)	Galacturonic Acid (%)
75	15	2.04 ± 0.00	1.71 ± 0.16 a	11.92 ± 4.13 a	10.68 ± 0.97 a
75	30	2.03 ± 0.00	2.48 ± 0.16 b	19.76 ± 5.36 b	15.53 ± 0.97 b
75	45	2.03 ± 0.00	6.67 ± 0.27 c	29.42 ± 1.47 c	41.74 ± 1.68 c

Values are given as mean ± S.D. from triplicate measurements. Different letters in the same column indicate significant differences (p < 0.05).

Table 3

Physicochemical and antioxidant properties of PCP.

Property	Value
pH (at 1% w/v PCP)	3.74 ± 0.04
Viscosity (at 1% w/v PCP)	614.00 ± 3.53
Total polysaccharide content (µg glucose/µg extract)	20.44 ± 0.16
Solubility in water (%)	99.93 ± 0.01
Swelling capacity in water (ml/g)	0.40 ± 0.00
Water absorption (ml/mg)	39.79 ± 0.90
Oil absorption (ml/mg)	N/A
Antioxidant activity (IC50, mg/mL)	0.72 ± 0.02

Values are given as mean ± S.D. from triplicate measurements. N/A is the data is not available.

Table 4

Stability of 1.0% w/v PCP solution after heating–cooling cycles.

Parameters	Cycles
	T0	T3	T6
pH	3.74 ± 0.04 a	3.82 ± 0.03 a	3.92 ± 0.06 b
Viscosity (mPa.s)	614.00 ± 3.53 a	601.53 ± 1.85 b	598.13 ± 0.75 b
Total polysaccharide content (µg glucose/µg extract)	20.44 ± 0.16 a	20.24 ± 0.12 a	20.19 ± 0.49 a
IC50 of DPPH assay (mg/mL)	0.72 ± 0.02 a	1.34 ± 0.08 b	1.37 ± 0.08 b

Values are given as mean ± S.D. from triplicate measurements. Different letters in the different columns indicate significant differences (p < 0.05).

Table 5

Stability of serum (pH and viscosity values) after heating–cooling cycles.

Formulations	Cycles
	T0		T3		T6
	pH	Viscosity (mPa.s)	pH	Viscosity (mPa.s)	pH	Viscosity (mPa.s)
FB	5.25 ± 0.04	766.50 ± 2.61	5.32 ± 0.02	765.13 ± 3.81	5.32 ± 0.03	761.37 ± 1.02
F1	5.15 ± 0.03	744.43 ± 1.20	5.09 ± 0.04	741.70 ± 1.73	5.11 ± 0.03	739.60 ± 1.45
F2	4.99 ± 0.03	817.73 ± 1.79	5.05 ± 0.04	819.57 ± 1.32	5.01 ± 0.03	816.53 ± 0.57
F3	4.70 ± 0.02	934.83 ± 1.36	4.66 ± 0.04	938.77 ± 2.35	4.67 ± 0.06	933.50 ± 1.15
F4	5.59 ± 0.03	798.50 ± 0.85	5.62 ± 0.02	797.70 ± 2.43	5.65 ± 0.02	791.20 ± 0.82
F5	5.15 ± 0.04	870.47 ± 0.60	5.21 ± 0.02	869.13 ± 3.68	5.26 ± 0.03	864.20 ± 1.25

Values are given as mean ± S.D. from triplicate measurements. Not sharing the letters in the different columns indicates no significant differences (p > 0.05).

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