Formulation, characterization of Quercus infectoria (Olivier) emulsions, and in vitro, in vivo evaluation as cosmeceutical formulation

Pharmacological properties of Quercus infectoria Olivier (galls) have been determined to be astringent, antidiabetic, antipyretic, anti‐tremor, local anesthetic, and anti‐parkinsonism. The galls of Quercus infectoria have been used for millennia in traditional oriental medicine in Asian nations to treat inflammatory illnesses.

aesthetic benefits.Topical applications employ both w/o and o/w emulsions.Without an emulsion, an emollient is frequently used to treat dry skin. 3Emulsions are the preferred delivery method in many situations, such as increasing bioavailability and masking unpleasant taste and odor.By avoiding synthetic components and adopting natural products, cosmetology is attempting to gain more recognition. 4ercus infectoria Olivier nutgall powder can be macerated in absolute methanol, filtered, and concentrated using a rotary evaporator to create Quercus infectoria Olivier methanol extract.A shrub or little tree known as Quercus infectoria Olivier (Family: Fagaceae) is primarily found in Greece, Syria, Asia Minor, and Iran.The wasp, Cypnis gallae tincotoria, attacks the small tree, causing galls to emerge on its branches.The pharmacological properties of Quercus infectoria Olivier (QI) galls have been determined to be astringent, antidiabetic, anti-parkinsonian, anti-tremor, local anesthetic, and antipyretic.The galls of QI have been used for millennia in traditional oriental formulations in Asian nations to treat inflammatory illnesses.While applying boiling and bruised galls directly to the skin efficiently treats any swelling or inflammation, gargling with hot aqueous extract of galls is highly beneficial against irritated tonsils.Powdered galls have been used to treat Hemorrhoids.5 Quercus infectoria Olivier gall extract contains quinic acid, malic acid, p-coumaric acid, gallic acid, chlorogenic acid, salicylic acid, protocatechuic acid, tannic acid, rutin, caffeic acid, rosmarinic acid, apigenin, hesperidin, coumarin, kaempferol, quercetin, naringenin and, all of which have numerous cosmetic uses.6 The purpose of this study was to create a stable emulsion with Quercus infectoria Olivier extract, in vitro and in vivo characterization by examining various factors that can have an antiaging effect.

| MATERIAL AND IN S TRUMENTS
Quercus infectoria galls were obtained from a local market and were used after identification.The materials employed in this investigation included stearic acid, cetyl alcohol, KOH, and

| Preparation of extract
To prevent contamination, the galls were washed with tap water and shade dried, crushed into powder, and stored at 25°C in an airtight sealed container.A total quantity of 100 g of galls powder were steeped in an absolute methanol, ethanol, acetone, and distilled water.The solution was filtered through a Whatman filter paper No.
1 and muslin cloth after soaking for 7 days.The solution was then concentrated using a rotary evaporator at 44°C until a gummy extract was obtained and then kept at 4°C in refrigerator.

| Antioxidant activity
Using DPPH method, the antioxidant capacity of the Quercus infectoria extracts was assessed. 7A total quantity of 95 μL of DPPH solution were combined with 5 μL of the extract to test the extract's antioxidant properties.And the mixture was incubated then in the dark for 30 min at 37°C.A 96-well microplate reader used to measure the absorbance at 517 nm after the incubation period.Ascorbic acid used as the standard.The following formula was used to compute the percentage scavenging property: A0 = Absorbance of the control.A1 = absorbance when a test or reference sample is present.

| Total phenolic contents
Folin-Ciocalteu reagent (FCR) was used in the current investigation to assess the total phenolic contents of plant extract in accordance with Kim's method with a minor modification. 8A total quantity of 1 mg of each sample was dissolved in 9 mL of water, and the mixture was vigorously blended for 5 min before being added to the Folin-Ciocalteu reagent (1.0 mL).A total quantity of 25 mL, the final volume was then obtained with water after adding 10 mL of Na2CO3 (7%) to the mixture.After 90 min of room temperature incubation, the mixture's absorbance at 750 nm was measured.The dry weight of the plant sample's total phenolic content (TPC) was calculated as grams of gallic acid equivalent (GAE)/mg.

| Total flavonoid contents
The formulation was tested for total flavonoid content (TFC) using Park's method with a few minor modifications, 9 0.1 mL from 0.3 mol/L AlCl 3 .6H 2 O was added to mixture with 0.3 mL of QI sample and 0.5 mol/L NaNO 2 .After adding 3.4 mL form 30% methanol to the mixture, absorbance was measured at 506 nm.Plant (QI) sample's total flavonoid content (TFC) was calculated as g quercetin equivalents (QE)/mg.

| Sun protection factor
Using a spectrophotometer and the previously described procedure, the formulation's in vitro sun protection factor (SPF) was determined. 10One gram of the sample was diluted with 100 mL of ethanol in a volumetric flask, and then sonicated for 5 min.A few mL of this solution were discarded after it had been sonicated and filtered through a cotton plug.Then, 5 mL of it was once more diluted with the help of ethanol to make 50.0 mL, and then 5 mL of it was diluted by using ethanol to make 25 mL.The sample's absorption was then DPPH scavenging effect ( % ) = (A0 − A1) ∕ A0 * 100 measured in the 290-320 nm region, every 5 nm, using ethanol as a blank.Using Mansur's equation, the SPF was determined. 11

| Tyrosinase enzyme inhibition activity
Tyrosinase inhibition analysis of the (QI) plant samples was carried out using Kim's method 8 with a few minor modifications, using kojic acid as a control.This procedure involved adding 60 units of enzyme in 150 mL of buffer (50 mM pH 6.8) and incubating 10 mL of the test chemical in each well for 15 min at 30°C.At 480 nm, the pre-read was recorded after incubation.Substrate was added, and each well was re-incubated for 30 min under the same conditions.After re-incubation, absorbance was once more measured on a regular basis.

| Preparation of test and control formulations
Both oil and water phases were accurately weighed in separate beakers.Prior to homogenization, aqueous phase and oil phase were both heated to reach at 75°C.While heating both phases, oily phase was introduced to aqueous phase gradually while stirring at a speed of 2000 rpm for 15 min, then 1500 revolution per minute for 10 min.
Then the speed was lowered to 1000 rpm for 10 min and 500 rpm for 5 min to finish the homogenization.Emulsion was subsequently cooled to room temperature.For the test formulation, the same process was done.Q. infectoria extract (4%) was used in the creation of the test formulation.At the same conditions used to prepare the control, the extract added to the aqueous phase, which was combined with the oily phase (stearic acid 10%, cetyl alcohol 2%, and glycerin 14%).W/W basis was used for all percentages.

| Organoleptic evaluation, spreadability, pH, and conductivity measurements
Both the Quercus infectoria loaded emulsion (QILE) and the control emulsion (CE) underwent a 3-month stability assessment.Testing for stability was done at room temperature.At predefined intervals, organoleptic criteria such as color, odor, feel, and appearance were examined.By directly dipping the probe in the emulsion, the digital pH meter, pH 197 (WTW) was used to test the pH of the QILE and the control emulsion.Three copies of each measurement were taken.Using the conductivity meter, WTW COND-197i and directly dipping the probe in emulsion, conductivity of the QILE and the control emulsion were measured in order to evaluate the stability of emulsions.Values are collected and calculated in triplicate.QILE and CE's spreadability was assessed using the glass slide method.A 1 cm circle was drawn on a glass slide and then the circle was filled with 0.5 g of emulsion.A second glass slide is then placed on top of first glass slide holding the emulsion.For 5 min, a weight of about 500 g emulsion was placed on top of the upper slide.Scale measurements were taken to identify and quantify the increase in emulsion diameter. 12

| Rheological studies
When analyzing stability and flow characteristics of derma formulations, rheology is a crucial metric.At 25°C over a period of 12 weeks, rheological characteristics including viscosity, shear rate, and shear stress of QILE and CE were measured.Utilizing a programmable rheometer (Brookfield Engineering laboratories with Spindle number CP41), QILE and CE were examined.Rheocalc version 2.5.6 was used to measure shear rate, shear stress, and viscosity.Rheological analyses were measured three times using a programmable rheometer.Each emulsion's flow behavior was assessed at 20-100 rpm using approximately 0.5-0.01g of each QILE and CE emulsions in a sample holding cup.

| Study design
Thirteen Asian origin female volunteers between the age of 22 and 35 participated in the 12-week study. 12They were chosen in accordance with the protocol's ethical requirements.After receiving the Advanced Studies and Research Board's permission, they were chosen according to ethical protocol.The Declaration of Helsinki was followed in conducting the study.Each participant who agreed to participate in this study signed a written informed consent.
Participants received thorough information regarding the goal, the procedures, and any potential side effects.All of the participants underwent a comprehensive examination to rule out any major skin conditions, particularly those affecting the face.Pregnant women, smokers, those with hypersensitivity to ingredients, and people with a history of any skin conditions were excluded from the trial, following the exclusion criteria. 13

| Patch test
Prior to in vivo trial began, all volunteers underwent a patch test on their forearms using a noninvasive procedure to determine whether they were allergic to or sensitive to the emulsions, QILE and CE.On both forearms, a 4-5 cm area chosen and marked.
Then, on the left forearm, a patch having 1 g of CE was put, and on the right forearm, a patch containing 1 g QILE.Patches had surgical dressing on them.Both patches were removed after 48 h and cleaned with sterile normal saline solution.And forearms were also examined for any signs of sensitivity, erythema and itching, or irritation to patches.After using the patch test, these values were measured.Baseline readings were also taken before applying any gel and showed 0 h time values.All subjects who had completed the patch test were handed QILE and CE jars with the coding "left" and "right" to show where the gels should be applied on cheek.To prevent the fluctuation in results caused by self-administration, each participant was told to apply a predetermined amount of emulsion, about 0.5 g on a surface measuring 100 cm 2 by them.
Subjects were told to use emulsions twice daily (in morning and in night, before bedding).Additionally, it was told to show up for readings on weeks 2, 4, 6, 8, and 12. On the day that they were supposed to read, volunteers were asked to stay for 30 min so that their bodies could adjust to the controlled laboratory temperature of 25°C and 40%RH. 12

| Panel test
Before, in vivo study began, a panel test was conducted for the QILE and CE in terms of spreadability, shine on skin, ease of application, moisturizing, irritation, and sense of feel after application.And they were asked to rub QILE and CE between their fingers and grade them on a scale of 0-5.

| Evaluation of skin mechanical properties
The skin capacitance (moisture level) was measured using a corneometer CM825 linked to probe adopter MPA5 (Courage, Khazaka electronics GmbH).This tool can measure moisture content of the epidermal skin layers as deep as 0.1 mm.The mechanical characteristics of the epidermis were measured using a cutometer fitted with an MPA5 probe (Courage + Khazaka electronics GmbH) (erythema and melanin).Elastometer linked to a probe adopter MPA5 (Courage, Khazaka electronics GmbH) with probe with 2 mm opening was used to assess skin elasticity under in vivo suction.Sebum level was measured using a sebumeter linked to a probe adopter MPA5 (Courage, Khazaka electronics GmbH).Measurements were taken in mode 1 (time/strain).A steady negative pressure of 350 milli bar was used for 18 s (the suction duration), then for 2 s (relaxation period).

| Statistical analysis
The difference in formulations (QILE, CE) and changes that occurred at various time intervals were examined using two-way ANOVA and paired sample t-test, and IBM SPSS statistic version 20.To compare the effectiveness of two formulations, QILE versus CE, a paired sample t-test was used.To examine variation across various time intervals, a two-way ANOVA was conducted.If "p" values were less than 5% (p 0.05), the results were deemed statistically significant.A mean value of the obtained values was reported.
All measurements were taken in triplicate, and the findings are shown as mean SD.GraphPad Prism 8.4.3 software was used for the statistical evaluation.

| Anti-tyrosinase inhibition activity
The Q. infectoria extract's 76% tyrosinase enzyme inhibition activity determined is shown in Figure 1A.

| Antioxidant activity
The antioxidant capacity/activity of Q. infectoria extract found 81% in comparison with the reference (ascorbic acid) in Figure 1B, indicating that it is a good candidate for derma cosmeceutical applications.

| Sun protection factor
According to this investigation, the QILE emulsion's sun protection factor (SPF) was 19.

| Total phenolic contents
A separately constructed absorbance versus concentration curve for gallic acid was used to calculate the result, which was then expressed in gallic acid equivalents (GAE).The QI extract used in this study has a total phenolic content of 56.1 mg GAE/g also confirmed from previous study. 14

| Total flavonoid contents
The dry weight of the QI (Quercus infectoria) extract sample's (TFC) total flavonoid content was reported in micrograms of quercetin equivalents (QE)/mg.A total quantity of 35.32 mg of quercetin/g of the formulation containing QI extracts and other flavonoids was found in the study.Figure 3 depicts the quercetin-total flavonoid content regression line.

| Physicochemical characterization
Controlled emulsion and QILE were placed at various temperatures (8°C, 25°C, 40°C, and 40°C 75% RH) for 3 months to conduct stability investigations.For a period of 3 months at 8°C and 25°C, the smell, color, and smoothness of emulsions did not significantly alter.
But in the latter months of the trial, there were modest variations in the color dark brown at 40°C and 40°C 75% RH.Emulsions smoothness was constant at 8°C and 25°C for the duration of the trial, but in the last months of the study, it became somewhat thinner at 40°C and 40°C 75% RH.Different physicochemical properties of the controlled emulsion and QILE optimized formulation are detailed in Table 1.

| pH, conductivity, and viscosity measurements
The viscosity, pH, and conductivity of the improved QILE formulation were examined.According to statistics acquired data, there was not difference in the formulation's viscosity, pH, and conductivity over the course of 3 months (p > 0.05).Figures 4A,B, 5A,B and 6A,B; show the viscosity, conductivity, and pH data graphically.

| Microscopy
Both the Control and QI extract-containing formulations' emulsion globules/droplets were seen to be spherical over the entire course of research.Additionally, there was a minor upward tendency in globule size over time.(Figure 7)

| Rheological behavior
The results of samples indicated that all the samples stored at different storage temperatures, over time displayed pseudo-plastic and non-Newtonian behavior.When the Ostwald power law was applied, the QILE emulsion also demonstrated shear thinning behavior and apparent viscosity of freshly prepared QILE emulsion was 436.50 cP, which suggests that cosmetology applications may be possible and decreased over time as shear rate and shear stress were raised, as seen in 8(a) and 8(b) figures.Flow index of QILE at various storage temperatures, including 8°C, 25°C, 40°C, and 40°C with 75% RH, which was less than 1, was used to confirm the pseudo-plastic and non-Newtonian behavior of QILE emulsion Table 2.

| Patch test
For the topical delivery system, notably for cosmetic and skinimproving products, this test is crucial and required.A noninvasive approach called a patch test was used to assess the likelihood of a cutaneous hypersensitivity reaction in human test volunteers.After

TA B L E 1
The stable emulsions formula selected from trials kept at 50°C for 90 days.

| Skin elasticity
In contrast to the control, QILE emulsion considerably increased volunteers' skin elasticity, as seen in this study's Figure 9B.Comparing QILE emulsion to control emulsion, the elasticity level rose by 12%.
Moreover, the improvement in skin elasticity was statistically significant at weeks 6, 8, 10, and 12 of the trial, whereas the control increased skin elasticity regularly but slightly at all study intervals.
A stable emulsion containing Quercus infectoria extract was created, and then characterized it in vitro and in vivo as a cosmetic formulation, was the aim of this work.Free radicals harm cells, but antioxidant molecules can halt and stabilize this harm. 15The DPPH method is a quick and easy testing for measuring the spectrophotometric activity of antioxidants.This study found that QILE emulsion has a significant amount of antioxidant capacity, which is compatible with the evidence already in existence and suggests they might be used in cosmetology Figure 9B.

| Skin small pore area and large pore area
Using the VisioFace® image analyzer, high-resolution pictures of the volunteer's cheeks taken at 2, 6, 8, and 12 weeks after using QILE emulsion were examined and contrasted with baseline pictures (Figure 10A,B).Software that was used in conjunction with the images was used to analyze the number of large pores, and small pores for each image.As seen in Figure 10, study showed that using QILE emulsion resulted in average reduction of 40% in small pore count and 73% in big pore count.When compared to QILE and the control emulsion at the end of the experiment, ANOVA showed significant differences in all two of the aforementioned measures.

| DISCUSS ION
Plants commonly contain phenolic compounds, also referred to as aromatic secondary metabolites.It makes likely that these compounds are what give plants their antioxidant activity as they have been linked to antioxidant characteristics. 16Phenols' hydroxyl groups have proven to be powerful free radical scavengers.Since nolic compounds found in plants. 17The Quercus infectoria extract is said to include phenolic and flavonoid components that have antioxidant properties. 6tually, sun protection factor controls the ratio of minimal erythema doses in protected and exposed skin.On both protected and unprotected skin, UV radiation during sun exposure essentially results in the least degree of erythema. 18A higher SPF number proved that the formulation offers defense against the sun's damaging UVR rays.The created emulsion offers a strong capability for sun protection, according to in vitro tests.A number of earlier investigations have shown that QILE can shield skin from photo-damage. 1 Tyrosinase enzyme mediates the rate limiting steps in the synthesis of melanin.And relative tyrosinase activity may be assessed after L-DOPA incubation.L-DOPA is converted to dopachrome by tyrosinase during incubation.A UV spectrophotometric analyzer can detect the reddish-brown pigment dopachrome.Any chemical or natural substance can test the inhibition of tyrosinase by adding that substance to the solution.By applying this idea, the QI extract's capacity to inhibit tyrosinase was identified.The polyphenolics and flavones found in QI extract may be responsible for the substantial anti-tyrosinase capability of the extract used in this investigation. 19e pH of the skin ranges from 5.0 to 7.0 because it acts as an environmental buffer.Emulgels, emulsions, and cosmetic preparations can be made with a pH range that falls within these parameters. 20e pH of the formulation decreased under various storage settings, which might be due to stearic acid oxidizing into organic acids and aldehydes.At the 0.05 level of significance, the pH shift was negligible, and these values are acceptable for topical and dermatological formulations.The formulation is stable at all temperature conditions, according to the results.Emulsion consistency and flow metrics are also used to determine emulsion stability and act as warning signs of approaching emulsion formulation destabilization.The Consistency Index (K) is used to measure emulsion consistency, whereas the Flow Index (n) is used to measure non-Newtonian flow behavior. 13e stratum corneum must be penetrated by active substances for topical delivery methods to function in vivo.In this study, it was discovered that adding herbal components to an emulsion delivery system increased skin penetration.This resulted from the action of the emulsifier and reduced size of the emulsion. 21In melanosomes of melanocytes, a chemical cascade converts L-tyrosine into melanin, which is then produced as epidermal melanin.This metabolic

Figure 2
displays the regression line for gallic acid together with the overall phenolic concentration.

F 3
I G U R E 1 (A,B) Tyrosinase enzyme inhibition and radical scavenging activity of control and QILE emulsion.F I G U R E 2 Gallic acid regression line for total phenolic contents.Quercetin regression line for total flavonoids contents.

F I G U R E 4
(A,B) Change in viscosity of control and QILE emulsions after 3 months.F I G U R E 5 (A,B) Variation in electrical conductivity of control and QILE emulsions.F I G U R E 6 (A,B) Represents the change in pH of control and QILE emulsions.

3. 10 . 5 |
Sebum levelSebumeter was used for measurement of the amount of sebum in the skin by placing a probe-equipped sebumeter tap on the cheeks of each volunteer.The results are shown in Figure9Cas a mean% change.The results are significant (p 0.01).

F
I G U R E 9 (A-C) shows variation in moisture, elasticity, and sebum level of control and QILE emulsions over the course of 12 weeks.total polyphenol content exists, it can be utilized to determine the antioxidant activity of plants.Flavonoids and their derivatives exhibit several biological actions that have an anticancer effect.Plants' ability to fight free radicals is substantially aided by the fact that flavonoids are the most common and widely distributed class of phe-

F I G U R E 1 0
(A) Fine pores and large pores at 0 days and (B) after application of QILE emulsions.F I G U R E 11 Number of fine and large pores before use (A) and after use (B) of QILE emulsion.