Green tannins /Avocado oil composites; suncare and skincare materials

2.1 Materials

Avocado fruits were collected from local vegetable market; the green-tea was purchased from local hypermarket. Lanolin, stearic acid and Triethanolamine (TEA) were procured from Riedel-de Haen AG (Germany), glycerin was supplied by Fluka AG, Calcium Chloride anhydrous (98%) purchased from Fizmerk chemicals (India), Ethanol (99%) purchased from Carlo Erba (France), Carbopol® 940 polymer (ULTREZ 20) was purchased from Lubrizol (USA), Deionized water was used in all experiments.

2.2 Spectroscopic means

X-ray diffractometer (XRD) (Ultima IV (185 mm)) used to investigate crystal structure of samples. Thermogravimetric analyzer (TGA) (NETZSCH TG 209 F1Iris) used to analyze thermal stability of samples in 25–800 °C range under N₂ atmosphere using a heating rate of 10 °C/min. Fourier transform infrared (FTIR) in the range of 4000–400 cm⁻¹ (Shimadzu IRAffinity^-1) supplied with attenuated total reflectance (ATR) unit for best deconvoluted functional peaks and chemical structure. 400 MHz Nuclear Magnetic Resonance (¹H NMR Bruker biospin) used to investigate chemical structure using deuterated methanol (CD₃CN). Vibro Viscometer: (SV-A Series Sine-wave Vibro Viscometer) wide range measurement (0.3–10,000 mPa.s). Laser-Diffraction Particle-size Analysis (Microtrac S3500) used for particle size distribution.

2.3 Green-tea tannins

Green tea was grounded to 1.0 mm powder size using commercial grinder. 5.0 g of grounded green tea were dissolved in 50 mL of 1:1 ratio of ethanol/water mixture. The solution was heated under 500 rpm stirring at 60 °C for 1 h. After heating, the solution was ultrasonicated for 10 min until tannins orange color appeared. The tannins extract was filtered, and filtrate was dried in the oven at 50 °C for 24 h. The brownish orange green-tea tannins powder was collected and restored in dried place until used (Das et al., 2020).

2.4 Avocado oil

Green Avocado was peeled off from avocado fruit, and the avocado skin was washed with deionized water, cut into small pieces, and left to dry overnight in an oven at 40 °C The dried pieces were grounded to 1.0 mm powder size and stored in dry place at ambient temperature. 10 g of avocado skin powder were dissolved in 60 mL ethanol and sonicated with ultrasonication device for 15 min at ambient temperature. The solution was decanted and placed in the oven at 40 °C overnight to evaporate leftover solvent and the green avocado oil was collected. The avocado oil was saved at room temperature until used. The avocado oil is known for its stability over a wide range of temperature (Espinosa-Solis et al., 2022; Chin Xuan Tan, 2019).

2.5 Four in one antiaging materials

Oil phase: In a 100 mL beaker, 4.0 g lanolin were mixed with 5.0 g stearic acid and 5.0 mL of avocado oil under vigorous stirring. The temperature was raised to 70 °C to enhance intermixing and homogeneity. The mixture was continuously stirred until homogenous oily phase was obtained. Aqueous phase: In another 100 mL beaker, 0.15% tannins (w/w) were added to 25 mL deionized water in presence of 1.0 mL triethanolamine and heated at 70 °C until homogenous aqueous phase obtained. Later on, the oil phase was slowly added to the aqueous phase, and the mixture was vigorously stirred at 1000 rpm for 10 min until the desired creamy texture was obtained. Afterwards, 5% CaCl₂ with respect to the total weight of avocado oil and tannins powder (w/w) was added to crosslink avocado oil with green-tea tannins. Finally, 10% glycerin or Carbopol® 940 moisturizer was added to give silky texture to the cream.

2.6 In-vitro antimicrobial activity

The silky material was exposed to in-vitro antimicrobial activity test against common infection bacteria, and consequently determine the optimum effective concentration of antiaging cream required for well antimicrobial activity as follows; Four different concentrations (12.5%, 25%, 50%, 100% (w/w)) of original concentration of the cream were prepared. For measurement of antibacterial activity, Agar well diffusion method was used to assess the cream antibacterial effectiveness against standard strains of bacteria; S. aureus, (ATCC 25923), Escherichia coli (E. coli) (ATCC 23452) as follows; the surface of agar plate is inoculated by distributing a the microbial inoculum over the entire agar surface. consequently, a hole with a diameter of 6–8 mm is made in sterile manner with a sterile cork borer, and a volume of 20 to 100 µL of certain concertation of antiaging cream is introduced into the well. Then, agar plates were incubated at 37 °C for 24 h. The antiaging cream diffuses in the agar medium and inhibits the growth of the microbial strain tested. The antimicrobial activity was expressed as the diameter of the inhibition zone (in mm) produced by the cream’s active ingredients. For Fungal culture test, few amount of cream was cultured and incubated in humidified Sabouraud Dextrose Agar (SDA) medium at 22 °C for 21 days to enhance the fungi growth. SDA medium is a non-selective medium used for isolation, cultivation, and maintenance of pathogenic and non-pathogenic species of fungi and yeasts at adjusted pH of 5.6 suitable enough to enhance the growth of fungi especially dermatophytes (Monwar et al., 2017).

2.7 In-vitro sun care

Sun Protection Factor (SPF) is the worldwide factor that expresses the level of protection of sunscreens against harmful solar ultraviolet rays (UVA and UVB). SPF can be measured using in-vivo or in-vitro methods. In in-vivo method, the minimal erythemal dose (MED) of UV radiation to make skin redness is measured, in presence and absence of sunscreen (Kiriiri Geoffrey et al., 2019), In in-vitro method, reliable UV spectrophotometric method is used to measure SPF value using the following relation (Santos et al., 1999);

The cream sample was synthesized as follows; in 100 mL volumetric flask 1.0 g of the antiaging cream was incubated and 100 mL ethanol were added. The flask was ultrasonicated in water bath for 15 min. In addition, different concentrations of the cream active ingredient (green tea tannins) were prepared (0.15%, 0.25%, 0.30% (w/w%)).

2.8 Viscosity

Tuning-fork vibration method that can determine wide range of viscosity measurements (0.3–10,000 mPa.s) with high accuracy technique. The sine-wave Vibro viscometer (SV-10) is able to detect the growing electromagnetic force between two thin sensor plates at fixed 30 Hz frequency and amplitude less than 1 mm. The viscosity of the antiaging cream in absence and presence of Carbopol® 940 moisturizer (5%, 10%, 15%, 20% (w/w)), which showed values in the range of 0.3–10,000 mPa.s at 22 °C.

2.9 Spreadability

For spreadability test, the successful cream is assumed to spread smoothly like silk on human skin without dragging, and should not make ascending friction during the rubbing process (Sabale et al., 2011).

2.10 Particle size distribution

Microtrac laser-diffraction (LD) particle size analyzer was used with specifications of tri–laser, multi-detector, multi-angle optical system. The particle’s measurement ranges from 0.02 to 2800 µm with accurate, reliable and reproducible particle size analysis. In order to obtain the statistical distribution of the particle size of cream, different cream portions were added in a dropwise manner to deionized water in the dispersion cell of the laser diffraction unit. The selected optical parameters were as follows: continuous phase refractive index of 1.33, and a relative refractive index of 1.59. Each sample was analyzed in triplicate at 25 °C and the data were presented as mean ± SD values.

3.1 Characterization of active ingredients

3.1.1

3.1.1 Chemical configuration of active ingredients

Polyphenolic tannins extracted from green-tea were exposed to ¹H NMR investigation using 400 MHz ¹H NMR spectrometer (Fig. 1A). Obviously, several chemical shifts exhibited the chemical structure of polyphenolic tannins as follows; CH₂ peak next to heterocyclic ring at δ = 4.09 ppm (peak 1), CH peaks inside the heterocyclic ring at δ = 5.01 and δ = 5.35 ppm, respectively (peaks 2 and 3), aliphatic OH peak at δ = 5.61 ppm (peak 4), CH peak inside the heterocyclic ring at δ = 5.96 ppm (peak 5), CH peak in aromatic benzene ring at δ = 6.64 ppm (peak 6), CH peaks in aromatic benzene ring meta position at δ = 7.01 and 7.15 ppm (peaks 7 and 8), CH peak inside the heterocyclic ring surrounded by two oxygen atoms at δ = 7.34 ppm (peak 9), and aromatic hydroxyl groups (OH groups) at δ = 7.89 ppm (peak 10). The peak at 2.0 ppm corresponds to deuterated acetonitrile (CD₃CN) solvent used. All ¹H NMR peaks provided evidence of successful extraction of polyphenolic tannins from green-tea. On the other hand, Fig. 1B shows the ATR-FTIR spectra of polyphenolic tannins and Avocado oil, the two active ingredients of antiaging cream. Table 2 illustrate the vibrational modes and wavenumber (in cm⁻¹) of polyphenolic tannins and Avocado oil. Clearly, the functional groups of tannins show distinct peaks as follows; hydroxyl group at 3232 cm⁻¹, aromatic C–H stretching at 3036, aliphatic C–H stretching at 2925 and 2888 cm⁻¹, sharp carbonyl group at 1692 cm⁻¹, conjugated aromatic C = C stretching at 1603 cm⁻¹, and C–O–C stretching at 1140 cm⁻¹, which came in accordance with NMR peak values, and confirm the chemical structure of polyphenolic tannins.

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Fig. 1

(A) ¹H NMR spectrum of green-tea tannins, (B) ATR-FTIR spectra of green-tea tannins and avocado oil.

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Table 2 Vibrational modes of polyphenolic tannins and Avocado oil, the active ingredient.ts of antiaging cream.

Active Ingredient	Wavenumber (cm−1)	Vibrational Stretching modes
Tannins Polyphenol	3232 3036 2925 and 2888 1692 1603 1140	O–H stretching Aromatic C–H stretching Aliphatic C–H stretchings C = O Stretching Conjugated C = C stretching C-O-C stretching
Avocado Oil	3382 3001 2914 and 2845 1723 1646 1622	O–H stretching of fatty acids Aromatic C–H stretching of polyunsaturated Linoleic acid Aliphatic C–H stretchings of fatty acids C = O Stretching of fatty acids C = C stretching of monounsaturated Oleic and Palmitoleic acids Conjugated C = C stretching of polyunsaturated Linoleic acid

On the other hand, Avocado oil show characteristic peaks of hydroxyl groups of fatty acids through hydroxyl groups (O–H) at 3382 cm⁻¹ and carbonyl groups (C = O) at 1723 cm⁻¹, aromatic C–H stretching of Linoleic acid at 3001 cm⁻¹, Long aliphatic CH₃ and CH₂ groups through C–H stretchings at 2914 and 2845 cm⁻¹, respectively. Moreover, characteristic C = C stretching at 1646 cm⁻¹ that corresponds to double bond of Oleic and Palmitoleic acids moieties, and conjugated C = C stretching at 1622 cm⁻¹ that corresponds to conjugated double bond of Linoleic acid moiety are other evidences of accurate chemical structure of Avocado oil.

3.1.2

3.1.2 Thermal stability

Thermal stability of active ingredients is an extremely significant step toward the applicability, transportation, short and long-run storage, and erosion factors of the so-called antiaging cream. Each sample is exposed to increasing temperature from 25 to 1000 °C at constant heating rate of 10 °C/min. The decomposition temperature is a direct evidence of how feasible the cream can resist storage and erosion factors. Fig. 2 displays the thermogravimetric analysis (TGA) and differential thermogravimetry (DTG) of the active ingredients of avocado oil, green-tea tannins and the mixture of both in the cream. The decomposition of Avocado oil (Fig. 2A) with broad band begins at 200 °C (onset) and ends at 500 °C (endset) and show multiple decomposition temperature (mid-set) at 300, 370, 410, 430, and 480 °C. The decomposition stage is referred to the onset of degradation of polyunsaturated fatty acids (Linoleic acid), followed by monounsaturated fatty acids (Oleic and Palmitoleic acids) and eventually saturated fatty acids (Palmitic and Stearic acids) (Santos et al., 2002). Whereas, the decomposition of green-tea tannins (Fig. 2B) begins at 120 °C (onset) and ends at 700 °C (endset) and show multiple decomposition temperatures (mid-set) at 200, 300, and 550 °C. The 200 °C decomposition peak corresponds to low molecular weight volatile compounds (H, CO, and CO₂), the 300 °C peak corresponds to the degradation of the laterals chains of the green-tea tannins (Zhang et al., 2011), and the 700 °C corresponds to the decomposition of flavonoid rings to form carbon skeleton (Konai et al., 2016). Fig. 2C show that tannins can bind avocado oil and form stable ingredients in presence of Ca²⁺ crosslinker, all decomposition peaks of avocado oil and green-tea tannins were present.

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Fig. 2

Thermogravimetric analysis (TGA) and Differential thermogravimetry (DTG) thermograms of (A) Avocado oil, (B) Greem-tea tannins, (C) Green-tea tannins/Avocado oil formulation in presence of Ca⁺² crosslinker.

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3.1.3

3.1.3 Crystal structure

Crystal structure of green-tea tannins was identified by X-ray diffraction pattern (XRD) using Bragg's equation as follows;

(2)

$$n\lambda =2dsin\theta$$ where n is the diffraction order (n = 1), λ is the radiation wavelength (λ = 1.5406 Å), θ is the glancing angle, and copper source of radiation (CuKα) used. The interlayer spacing (d) between tannins layers can be calculated from equation (1). Clearly, green-tea tannins displayed one broad band at 2θ = 20.6° (Fig. 3) as depicted in the literature (Zhang et al., 2015). This indicate characteristic amorphous nature of tannins. Moreover, two small intensity crystalline peaks at 2θ = 28.5° and 40.6° correspond to 3.1 and 2.2 Å interlayer spacings (d), respectively. The appearance of such small intensity crystalline peaks can be attributed to the processing technique of the gentle solvent evaporation process during the extraction of green-tea tannins.

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Fig. 3

X-ray diffraction pattern (XRD) of tannins polyphenol.

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3.2 Sun care materials; mechanism of action

Solar ultraviolet radiation covers wide range of wavelength (400–100 nm). It is divided into three wavelength regions; UVA (400–315 nm), UVB (315–280 nm) and UVC (280–100 nm) (Baker et al., 2016). UVC rays are dispersed by the ozone layer in the stratosphere layer and its penetration to earth may lead to catastrophic global warming, while the remaining rays (UVA and UVB) rays can reach the earth’s surface (Pillai et al., 2005). The chronic exposure of skin to UV rays (especially UVB rays) can induce the formation of reactive oxygen species (ROS), which cause cellular, tissue, and skin damage. They also may lead to DNA, RNA, lipid, protein damage, which eventually result with photoaging and wrinkle formation of human skin (Rittié and Fisher, 2015). When skin absorbs photon energy, excited botanic species can form by translocation of electrons from occupied orbitals (ground state) to the unoccupied orbitals (the excited state) (Ichihashi et al., 2003; Onoue et al., 2017). Jablonski suggested this mechanism of energy transfer from an excited triplet photosensitizer to an oxygen atom, and obtain an excited singlet oxygen that might play major role in the oxidization of membrane lipids and proteins, or in the oxidative damage of DNA (Hall et al., 1996; Davies, 2003). Reactive oxygen species (ROS) including hydroxyl and/or peroxyl radicals can be formed from electron or hydrogen transfer (Krumova and Cosa, 2016). At the same time, the overproduction of ROS leads to consumption of ROS-scavenging enzyme levels (such as superoxide dismutase (SOD) and catalase), which result in DNA, proteins and lipids damage, and prepare for photoaging and wrinkles formation (Kong et al., 2018). Green-tea tannins have a unique set of polyphenolic groups namely, chatechin (C), epicatechin (EC), epigallocatechin (EGC), and their gallate esters: epicatechin gallate (ECG), and epigallocatechin gallate (EGCG), are natural antioxidants and radical scavengers (Guo et al., 1999). Such structural features of green-tea tannins enable stabilization of radicals via electron delocalization and/or termination, which in turn can enhance radical scavenging and antioxidant activity of flavonoids (Quideau et al., 2011). Additionally, it has also been reported that epigallocatechin gallate (EGCG) can enhance the ROS-scavenging enzyme activity (Ji Hoon Jeong et al., 2004). As a result, green-tea tannins with their biologically active flavan-3-ols groups can be involved as radical scavenging antioxidant in fighting against photoaging and thus as natural sunscreens against sunburns and wrinkles formation (Fig. 4A) (Zillich et al., 2015). Sun protection factor (SPF) is a factor that measures how efficient sunscreens can protect human skin against UVB sunburns. Higher SPF value indicates higher capability of efficient protection against sunburns (González et al., 2008). SPF values of the green-tea tannins/Avocado oil antiaging cream were calculated using equation (1) and described in Fig. 4B. Clearly, 0.15% tannin solution showed SPF value of 6.2, when concentration of tannin increased to 0.25%, the SPF value reached 8.1. Continuous increase of tannin solution to 0.3%, led to maximum value of SPF = 15.7. This clearly confirm the role of green-tea tannins in increasing protection against sunburns and wrinkle formation. On the other hand, the SPF value of our green-tea tannin/Avocado oil antiaging cream was demonstrated to be 10.3. Although the antiaging cream contain 0.15% tannins, it showed higher SPF value than 0.15% tannin solution (SPF = 6.2), which clearly indicate that avocado oil has a significant contribution in sun protection besides its nourishing effect and antimicrobial activity. Fig. 4A illustrate the significance of sunscreen in obtaining healthy skin and the effect of UVB in obtaining damaged skin.

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Fig. 4

(A) Schematic illustration for the effect of solar UVB irradiation in presence or absence of topical antiaging cream. (B) Change of SPF value using various green-tea tannins and green-tea tannins/Avocado oil antiaging crea.

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3.3 Antimicrobial activity

Contaminated cosmetics by microorganisms is an emerging catastrophic issue to be fully discussed. In Europe from 2005 to 2018, more than 100 reports on microbiologically contaminated cosmetics were recognized. Twenty of them were children-oriented products (Michalek et al., 2019). The growth of pathogenic microorganisms is directly affected by richness of nutrients in cosmetic environment. In cross-sectional study (Dadashi and Dehghanzadeh, 2016), 52 of daily-used skin and eye cosmetics were investigated for cosmetic contamination, all cosmetics used were polluted with bacteria (95% CI = 93.1%-100.0%) and 19.2% by fungi and yeast (95% CI = 10.8%-31.9%). Contamination of cosmetics can infect the skin and cause skin acne or irritation (Maria João Carvalho et al., 2021). The above terrifying statistics rings the bell on the emerging and upcoming cosmetic contamination lesion. Two different antibacterial and antifungal activity tests were performed for our green-tea tannins/Avocado oil antiaging cream.

3.3.1

3.3.1 Antibacterial activity

Different concentrations of the green-tea tannins/Avocado oil antiaging creams (25%, 50% and 100% (w/w)) were tested. The results of the well diffusion antibacterial test (Fig. 5A) showed that the diameter of the inhibition zone was 19 mm against standard strain of E. coli using the original concentration of the sample (100% (w/w)). On the other hand, no inhibition zones for the lower concentrations (25% or 50%) were found.

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Fig. 5

(A) Antibacterial activity test, (B) Antifungal activity test, (C) Change of viscosity with Carbopol® 940 (%), (D) spreadability of green-tea tannins/Avocado oil antiaging cream., (E) Particle size distribution of the antiaging cream.

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3.4 Physicochemical properties

Carbopol® moisturizer is a synthetic high molecular weight polyacrylic acid (PAA) used in skincare and personalcare products as texture enhancers and thickening agents (Berardi et al., 2022); and also to boost collagen production and help enhance skincare characteristics of human body. In our green-tea tannins/Avocado oil antiaging cream, we added different Carbopol moisturizer to enhance viscosity and texture of the cream, the viscosity of the cream in the absence of Carbopol moisturizer was 8.8 Pa.s., whereas in the presence of ascending moisturizer concentration, the viscosity reached 10.8 Pa.s. (Fig. 5C). The perfect viscosity of cream depends on final applicability of the cream, for example the use of higher viscosity cream is favored for burned skin because it shields against infections, whereas lower viscosity cream is favored to easily spread and penetrate inside the skin for better nourishing and smoothness (Fig. 5D). Our antiaging cream show better spreadability and silky touch depending on the different viscosity values obtained. However, better penetration and skin uptake is directly correlated with smaller particle size, the use of smaller particle size can lead to easy penetration, higher skin uptake and lower retention time (Yokota and Kyotani, 2018). Fig. 5E show the particle size distribution of our antiaging creams, the average particle size of the cream was in the microscale with 1–100 μm particle size range, and average particle size of 18.5 µm. Such particle size is quite suitable for antiaging creams that properly fit to the human skin.