# Development of Niacinamide/Ferulic Acid-Loaded Multiple Emulsion and ItsIn Vitro/In Vivo Investigation as a Cosmeceutical Product

Authors: Sidra Huma; Haji Muhammad Shoaib Khan; Shakeel Ijaz; Muhammad Sarfraz; Hafiz Saqib Zaka; Aousaf Ahmad
Journal: BioMed Research International
 (2022)

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## Abstract

Objective. Multiple emulsions have the ability to incorporate both lipophilic and hydrophilic actives in the same preparation and facilitate permeation of active ingredients through skin. The current study was aimed at formulating niacinamide/ferulic acid-loaded stable multiple emulsion (MNF) and its in vitro/in vivo characterization as a cosmeceutical product. Methods. Both the compounds were evaluated for their radical scavenging potential by the DPPH method and FTIR analysis. Then, placebo and active formulations were prepared using a double emulsification method and were investigated for stability testing (changes in color, odor, and liquefaction on centrifugation, pH, and globule size) for a period of three months. Afterwards, MNF was investigated for in vitro sun protection factor, rheological studies, entrapment efficiency, zeta potential, zeta size, and ex vivo permeation. Moreover, after ensuring the hypoallergenicity and safety, it was also checked for its cosmeceutical effects on human skin using noninvasive biophysical probes in comparison with placebo. Results. Results demonstrated that MNF showed a non-Newtonian behavior rheologically and both MNF and placebo were stable at different storage conditions. Entrapment efficiency, zeta potential, and zeta size were 93.3%, -5.88 mV, and 0.173 μm, respectively. Moreover, melanin, sebum, and skin erythema were significantly reduced while skin elasticity and hydration were improved. Conclusion. It is evident that niacinamide and ferulic acid can be successfully incorporated in a stable multiple emulsion which has potent cosmeceutical effects on human skin.


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## Body

## 1. Introduction

Human skin is continuously exposed to ultraviolet radiations (UVR) and many other detrimental effects of environment that lead to photodamaging of skin manifested by inflammation, dryness, hyperpigmentation, wrinkles, and roughness [1]. Antioxidants are prime elements that protect the skin from UVR damage and cause the diminished melanin level with improved hydration and elasticity of the skin. In recent years, synthetic compounds used as antioxidants are very helpful in diminishing the excessive reactive oxygen species (ROS) production and improve the endogenous antioxidant defense system of the body [2]. For the delivery of these antioxidants for skin disorders, topical delivery systems are used most commonly [3] like emulsions which increase the flux through skin [4]. Multiple emulsions, which are also termed double emulsions or emulsions of emulsions, are novel development in the field of emulsion technology in which globules of dispersed phase contain smaller dispersed droplets. Hence, they are complex systems in which internal and external phases are alike and the intermediate phase separates these two phases. For their stabilization, both hydrophilic and hydrophobic surfactants are required. In multiple emulsions, both hydrophilic and lipophilic drugs can be incorporated. Multiple emulsions are becoming very popular and gaining attention of researchers as a topical drug delivery system owing to many advantageous features such as high encapsulating capacity, protection of entrapped drug, low viscosity due to an external water phase, smooth texture, and low chances of irritation [4, 5].Niacinamide also known as nicotinamide is an amide analogue of vitamin B3 and is a hydrophilic substance. It is a prime component of oxido-reduction coenzymes nicotinamide adenine dinucleotide (NAD) and nicotinamide adenine dinucleotide phosphate (NADP) which are involved in hydrogen transfer. In epidermal barrier functions, niacinamide has promising role in stabilizing effects by reducing the transepidermal water loss (TWEL) and hence increases the hydration level of skin [6]. After epicutaneous applications of niacinamide, it has antimicrobial, antipruritic, photoprotective, sebostatic, skin lightening, and antiaging effects. Nowadays, niacinamide has extensive usage in the treatment of melasma and hyperpigmentation. Niacinamide has sufficient bioavailability and is used in cosmetics in different concentrations [7, 8].Ferulic acid is a hydrophobic phenolic compound, also named as 4-hydroxy-3-methoxycinnamic acid (hydroxycinnamic acid). It is mostly found in plants, fruits, and vegetables. Ferulic acid is considered to be a superior antioxidant because it can stay longer in blood in comparison to other phenolic acids. As an antioxidant, ferulic acid is used to neutralize the free radicals to repair the damaged cells in muscle tissues [9]. It is commonly used with vitamins A, E, and C to enhance their activities and delay the photoaging process [10]. It has many skin benefits attributed to its strong radical scavenging potential. It has antiwrinkle, antipigmentation, photoaging (antiaging), skin lightening, and photoprotective effects owing to its ability to absorb the UVR [11].The aim of present research work was to formulate niacinamide/ferulic acid-loaded stable multiple emulsion (MNF) and itsin vitro/in vivo characterization as a cosmeceutical product.


## 2. Materials and Methods

### 2.1. Materials

ABIL-EM 90 (Franken Chemicals, Gebinde), paraffin oil (Merck KGOA, Darmstadt, Germany), magnesium sulfate (Merck KGaA, Darmstadt, Germany), 2,2-diphenyl-1-picrylhydrazyl (DPPH) (Sigma Chemical Co., USA), niacinamide (99% purity) (Tianjin Zhongrui Pharmaceutical Co., Ltd., China), and ferulic acid (Sigma-Aldrich) were used in this study. Distilled water was prepared at the Laboratory of Cosmeceuticals, Department of Pharmaceutics, IUB, Pakistan.


### 2.2. Antioxidant Activity

The antioxidant activity of both niacinamide and ferulic acid was determined by using the DPPH method. To check the antioxidant activity, 10μl of drug solution and 90 μl of DPPH solution were taken in each well of a 96-well plate. The content was mixed and incubated for 30 minutes in a dark place. After the incubation, absorbance was measured by using a spectrophotometer at wavelength 517 nm. Vitamin C (ascorbic acid) was used as the standard. Percentage scavenging effect was calculated using the following formula [12].
(1)Freeradicalscavengingactivty%=Abs.ofcontrol−Abs.ofsampleAbs.ofcontrol×100.


### 2.3. Fourier Transform Infrared Spectroscopy (FTIR) Analysis

FTIR of both drugs was conducted by using Tensor 27 FTIR, Bruker Optics GmbH (Ettlingen, Germany). The outcome of FTIR analysis was measured by % transmittance against a specific wave number for the respective functional group, conducted in 450–4000 cm–1 wave number range.


### 2.4. Trials for Stable MNF Formulation

Several trials were performed to prepare the stable multiple emulsion consisting of niacinamide and ferulic acid. Initially, primary emulsion of MNF was stabilized by modifying the concentrations of liquid paraffin and lipophilic surfactant (ABIL-EM 90) while keeping the drug constant. Afterwards, a number of multiple emulsions were formulated from already prepared stabilized primary emulsion. Several formulations were prepared for the most stable multiple emulsion by altering the content of the hydrophilic surfactant (Tween 80). A feed scheme of the combinations of ingredients for trials is presented in Table1.Table 1
Trials for stable MNF formulation.
(a)For primary emulsionFormulationLiquid paraffin (%w/w)ABIL-EM 90 (%w/w)Magnesium sulfate (%w/w)Niacinamide (%w/w)Ferulic acid (%w/w)Water (%w/w)MF1132.50.550.5100MF21330.550.5100MF31340.550.5100MF4142.50.550.5100MF51430.550.5100MF61440.550.5100MF7152.50.550.5100MF81530.550.5100MF91540.550.5100(b)FormulationFor multiple emulsionTween 80 (%w/w)Primary emulsion (%w/w)Water (%w/w)MF100.580100MF110.880100MF12180100


### 2.5.In Vitro Sun Protection Factor

The sun protection factor of both niacinamide and ferulic acid and MNF was checked by diluting it with ethanol. To calculate the data, Mansur’s equation was used and absorption was recorded every 5 nm at 290 to 320 nm [13].
(2)SPF=CF×∑290320EEλ×1λ×Absλ,where CF is the correction Factor, EE is the erythemal effect spectrum, and Abs is the absorbance of sunscreen.


### 2.6. Preparation of Placebo and MNF

For the development of MNF, a double emulsification method was adopted by taking the ingredients indicated in Table2. In the first step, primary emulsion (w/o) was prepared and both drugs were dissolved in different phases of primary emulsion according to their solubility. In the second step, a proportion of the primary emulsion was dispersed in an external (continuous) phase consisting of the secondary emulsifier. For preparation of primary emulsion, the oil phase comprised of ABIL-EM 90, liquid paraffin, and ferulic acid while the aqueous phase was consisted of distilled water, hydrated magnesium sulfate, and niacinamide. Both the oil phase and aqueous phase (without magnesium sulfate, niacinamide, and ferulic acid) were heated up to 75±5°C in a preheated water bath. After that, magnesium sulfate and niacinamide were added in the aqueous phase and ferulic acid was dissolved in the oil phase. Afterwards, dropwise addition of the aqueous phase and the oil phase was conducted with constant stirring at 2000 rpm for 20 minutes which was then lowered to 1000 rpm for 5 minutes. Next, the rotation was slowed to 500 rpm till the system was homogenized completely. At room temperature, the primary emulsion was then cooled down. Primary emulsion was then added to the external phase containing water and Tween 80, and stirring speed was 700 rpm for 20 minutes and 500 rpm for 1 hour. Then, the formation of MNF was confirmed by microscopy.Table 2
Composition of placebo and MNF (100 g).
Primary emulsionLiquid paraffin13%ABIL-EM 903%Magnesium sulfate0.5%Niacinamide5%Ferulic acid0.5%Distilled waterq.s.Multiple emulsionTween 800.8%Primary emulsion80%Distilled waterq.s.The same procedure was adapted to formulate placebo (without actives).


### 2.7. Characterization of Placebo and MNF

For stability studies, MNF and placebo were kept at several temperatures, i.e., 8°C, 25°C, 40°C, and 40° C with 75% RH, for a time span of 3 months and assessed for different parameters (color, odor, pH, microscopic evaluation, and liquefaction on centrifugation). To access the mean globule size and structure of globules, microscopic evaluation of both placebo and MNF was checked using an optical microscope equipped with a high-resolution camera and imaging software minisee®.


### 2.8. Rheology

Rheological studies of both placebo and MNF were performed by using a rotational rheometer model (DVIII ultra, Brookfield, USA). The share rate was set at 20 to 65, and the speed of the rheometer was 10 to 32 rpm and viscosities were measured at 25°C. For analysis of procured data, the software Rheocalc V. 2.6 was utilized. Rheological features and consistency of samples were calculated by using the Ostwald-de Waele power law [14].
(3)τ=Kγn,where K is the consistency index, γ is the share rate, τ is the share stress, and n is the flow index.


### 2.9. Entrapment Efficiency

Entrapment efficiency of MNF was checked by an indirect ultracentrifugation method. The freshly prepared sample was centrifuged at 120000 rpm for 45 minutes at room temperature. The supernatant was separated, and data was analyzed by using a spectrophotometer at wavelength of 232.5 nm. Free drug was calculated by using the following formula [15].
(4)EE%=TotalDrug−FreeDrugTotalDrug×100.


### 2.10. Zeta Potential, Zeta Size, and PDI

By using the laser Doppler electrophoretic mobility measurement technique, both zeta potential and zeta size were measured at 25°C.


### 2.11.Ex Vivo Permeation Studies

Ex vivo permeation of MNF (containing niacinamide and ferulic acid) was estimated in the Franz cell by the artificial cellulose acetate membrane at 37±0.5°C. Plain drug solution (niacinamide and ferulic acid) was used as the standard. The cellulose acetate membrane having an area of 1.76 cm2 was immersed in phosphate buffer of pH 5.5 and mounted in the diffusion cell. The diffusion cell having a 12 mL volume in the receiver section and the fluid in this compartment were agitated by using magnetic beads at slow speed of 30 rpm. 2 ml aliquot was removed at time intervals of 0, 0.5, 1, 2, 3, 4, 5, 6, and 8 hours, and the fresh butter was refilled in the compartment every time. The absorbance was taken by using a UV spectrophotometer at 232.5 nm, and data was analyzed by using the following formula [4].
(5)Kp=JssCv,where Kp is the permeability coefficient, Jss is the steady-state flux, and Cv is the total donor concentration of formulation.For further evaluation ofex vivo permeability studies by a model-dependent approach, the data was analyzed by fitting them in the zero-order, first-order, Higuchi, and Korsmeyer-Peppas models.


### 2.12. NoninvasiveIn Vivo Studies

#### 2.12.1. Ethical Approval and Study Design

The present research study was approved by the Board of Advanced Study and Research (BASR) and the institutional ethics committee, Faculty of Pharmacy, the Islamia University of Bahawalpur, with reference no. 128-2021-/PHEC. The study design for current research work was single-blind random study which was designed for 3 months. In that study, effects of MNF and placebo were checked on thirteen healthy insensitive female volunteers with age 20-40years. The volunteers were investigated for any skin allergy and disease by a dermatologist. A consent form with possible risk and protocols of study was signed by each volunteer, and they were asked to continue their diet but avoid using any other skin preparation during this study period.


#### 2.12.2. Inclusion Criteria for Volunteers

(1)
All volunteers participating in the study must be healthy(2)
All of the volunteers should have healthy skin and no history of any hypersensitivity(3)
No one among volunteers should be using any facial creams at least two weeks before and during the three-month study period(4)
In order to measure biophysical parameters, volunteers should be available in the laboratory on specific time(5)
Prior to measuring the readings, volunteers should wait for half an hour in order to acclimatize the laboratory conditions, i.e., room temperature


#### 2.12.3. Exclusion Criteria for Volunteers

(1)
A participant on any steroidal therapy shall be excluded from the study(2)
A person with any hypersensitivity or skin acne issue should be excluded from the study(3)
Any participant failing to comply the study guidelines shall be considered excluded(4)
A person having extraordinary hairy skin should be excluded for participating in the study(5)
A person which is already involved in a similar study should not be considered for selection


#### 2.12.4. Safety Evaluation by the Patch Test

Before the start of the study, a patch test was performed by checking melanin and erythema level on all the thirteen volunteers to check the skin sensitivity. After 24 h, the volunteers with no skin allergic reaction were provided with two creams marked as right and left which indicate the site of application. The measurements were taken at 0, 2, 4, 6, 8, 10, and 12 weeks of the study.


#### 2.12.5. Skin Erythema and Melanin Level

Skin melanin and erythema level was measured by using Mexameter® by Courage+Khazaka Electronic GmbH, Germany, which works on the principle of light absorption and emission with three specific wavelengths: 568, 660, and 880 nm. To avoid the chances of error, three consecutive readings were taken on a tested area and their mean was calculated.


#### 2.12.6. Skin Sebum Level

Sebumeter® EM 25 (Courage+Khazaka Electronic GmbH, Germany) was used to check skin sebum level. A plastic sebumeter tape is present in the probe of the sebumeter which measures the skin sebum level by following the principle of light transmission through a sebum-coated opaque plastic strip by photometric transparency. To avoid the chances of error, three consecutive readings were taken on a tested area and their mean was calculated.


#### 2.12.7. Skin Moisture Level

Corneometer® by Courage+Khazaka Electronic GmbH, Germany, was used to check the skin moisture level. The probe used for hydration level measurement works on the principle of diffusion of the electric field through the stratum corneum (epidermis), and moisture content is measured as dielectric constant-dependent capacitance changes of water. To avoid the chances of error, three consecutive readings were taken on a tested area and their mean was calculated.


#### 2.12.8. Skin Elasticity Level

Elastometer® EM 25 by Courage+Khazaka Electronic GmbH, Germany, was used to check the skin elasticity properties. The probe of the elastometer works on the principle of suction and stretching of skin and measures the elasticity on 0-99 range. To avoid the chances of error, three consecutive readings were taken on a tested area and their mean was calculated.


### 2.13. Mathematical and Statistical Analysis

Obtained data was statistically analyzed by SPSS version 23. Two-way ANOVA using LSD and paired samplet-test was applied for comparison of data with formulation and multiple comparisons of placebo and active formulations, respectively. P<0.05 was selected as the level of significant difference.


## 2.1. Materials

ABIL-EM 90 (Franken Chemicals, Gebinde), paraffin oil (Merck KGOA, Darmstadt, Germany), magnesium sulfate (Merck KGaA, Darmstadt, Germany), 2,2-diphenyl-1-picrylhydrazyl (DPPH) (Sigma Chemical Co., USA), niacinamide (99% purity) (Tianjin Zhongrui Pharmaceutical Co., Ltd., China), and ferulic acid (Sigma-Aldrich) were used in this study. Distilled water was prepared at the Laboratory of Cosmeceuticals, Department of Pharmaceutics, IUB, Pakistan.


## 2.2. Antioxidant Activity

The antioxidant activity of both niacinamide and ferulic acid was determined by using the DPPH method. To check the antioxidant activity, 10μl of drug solution and 90 μl of DPPH solution were taken in each well of a 96-well plate. The content was mixed and incubated for 30 minutes in a dark place. After the incubation, absorbance was measured by using a spectrophotometer at wavelength 517 nm. Vitamin C (ascorbic acid) was used as the standard. Percentage scavenging effect was calculated using the following formula [12].
(1)Freeradicalscavengingactivty%=Abs.ofcontrol−Abs.ofsampleAbs.ofcontrol×100.


## 2.3. Fourier Transform Infrared Spectroscopy (FTIR) Analysis

FTIR of both drugs was conducted by using Tensor 27 FTIR, Bruker Optics GmbH (Ettlingen, Germany). The outcome of FTIR analysis was measured by % transmittance against a specific wave number for the respective functional group, conducted in 450–4000 cm–1 wave number range.


## 2.4. Trials for Stable MNF Formulation

Several trials were performed to prepare the stable multiple emulsion consisting of niacinamide and ferulic acid. Initially, primary emulsion of MNF was stabilized by modifying the concentrations of liquid paraffin and lipophilic surfactant (ABIL-EM 90) while keeping the drug constant. Afterwards, a number of multiple emulsions were formulated from already prepared stabilized primary emulsion. Several formulations were prepared for the most stable multiple emulsion by altering the content of the hydrophilic surfactant (Tween 80). A feed scheme of the combinations of ingredients for trials is presented in Table1.Table 1
Trials for stable MNF formulation.
(a)For primary emulsionFormulationLiquid paraffin (%w/w)ABIL-EM 90 (%w/w)Magnesium sulfate (%w/w)Niacinamide (%w/w)Ferulic acid (%w/w)Water (%w/w)MF1132.50.550.5100MF21330.550.5100MF31340.550.5100MF4142.50.550.5100MF51430.550.5100MF61440.550.5100MF7152.50.550.5100MF81530.550.5100MF91540.550.5100(b)FormulationFor multiple emulsionTween 80 (%w/w)Primary emulsion (%w/w)Water (%w/w)MF100.580100MF110.880100MF12180100


## 2.5.In Vitro Sun Protection Factor

The sun protection factor of both niacinamide and ferulic acid and MNF was checked by diluting it with ethanol. To calculate the data, Mansur’s equation was used and absorption was recorded every 5 nm at 290 to 320 nm [13].
(2)SPF=CF×∑290320EEλ×1λ×Absλ,where CF is the correction Factor, EE is the erythemal effect spectrum, and Abs is the absorbance of sunscreen.


## 2.6. Preparation of Placebo and MNF

For the development of MNF, a double emulsification method was adopted by taking the ingredients indicated in Table2. In the first step, primary emulsion (w/o) was prepared and both drugs were dissolved in different phases of primary emulsion according to their solubility. In the second step, a proportion of the primary emulsion was dispersed in an external (continuous) phase consisting of the secondary emulsifier. For preparation of primary emulsion, the oil phase comprised of ABIL-EM 90, liquid paraffin, and ferulic acid while the aqueous phase was consisted of distilled water, hydrated magnesium sulfate, and niacinamide. Both the oil phase and aqueous phase (without magnesium sulfate, niacinamide, and ferulic acid) were heated up to 75±5°C in a preheated water bath. After that, magnesium sulfate and niacinamide were added in the aqueous phase and ferulic acid was dissolved in the oil phase. Afterwards, dropwise addition of the aqueous phase and the oil phase was conducted with constant stirring at 2000 rpm for 20 minutes which was then lowered to 1000 rpm for 5 minutes. Next, the rotation was slowed to 500 rpm till the system was homogenized completely. At room temperature, the primary emulsion was then cooled down. Primary emulsion was then added to the external phase containing water and Tween 80, and stirring speed was 700 rpm for 20 minutes and 500 rpm for 1 hour. Then, the formation of MNF was confirmed by microscopy.Table 2
Composition of placebo and MNF (100 g).
Primary emulsionLiquid paraffin13%ABIL-EM 903%Magnesium sulfate0.5%Niacinamide5%Ferulic acid0.5%Distilled waterq.s.Multiple emulsionTween 800.8%Primary emulsion80%Distilled waterq.s.The same procedure was adapted to formulate placebo (without actives).


## 2.7. Characterization of Placebo and MNF

For stability studies, MNF and placebo were kept at several temperatures, i.e., 8°C, 25°C, 40°C, and 40° C with 75% RH, for a time span of 3 months and assessed for different parameters (color, odor, pH, microscopic evaluation, and liquefaction on centrifugation). To access the mean globule size and structure of globules, microscopic evaluation of both placebo and MNF was checked using an optical microscope equipped with a high-resolution camera and imaging software minisee®.


## 2.8. Rheology

Rheological studies of both placebo and MNF were performed by using a rotational rheometer model (DVIII ultra, Brookfield, USA). The share rate was set at 20 to 65, and the speed of the rheometer was 10 to 32 rpm and viscosities were measured at 25°C. For analysis of procured data, the software Rheocalc V. 2.6 was utilized. Rheological features and consistency of samples were calculated by using the Ostwald-de Waele power law [14].
(3)τ=Kγn,where K is the consistency index, γ is the share rate, τ is the share stress, and n is the flow index.


## 2.9. Entrapment Efficiency

Entrapment efficiency of MNF was checked by an indirect ultracentrifugation method. The freshly prepared sample was centrifuged at 120000 rpm for 45 minutes at room temperature. The supernatant was separated, and data was analyzed by using a spectrophotometer at wavelength of 232.5 nm. Free drug was calculated by using the following formula [15].
(4)EE%=TotalDrug−FreeDrugTotalDrug×100.


## 2.10. Zeta Potential, Zeta Size, and PDI

By using the laser Doppler electrophoretic mobility measurement technique, both zeta potential and zeta size were measured at 25°C.


## 2.11.Ex Vivo Permeation Studies

Ex vivo permeation of MNF (containing niacinamide and ferulic acid) was estimated in the Franz cell by the artificial cellulose acetate membrane at 37±0.5°C. Plain drug solution (niacinamide and ferulic acid) was used as the standard. The cellulose acetate membrane having an area of 1.76 cm2 was immersed in phosphate buffer of pH 5.5 and mounted in the diffusion cell. The diffusion cell having a 12 mL volume in the receiver section and the fluid in this compartment were agitated by using magnetic beads at slow speed of 30 rpm. 2 ml aliquot was removed at time intervals of 0, 0.5, 1, 2, 3, 4, 5, 6, and 8 hours, and the fresh butter was refilled in the compartment every time. The absorbance was taken by using a UV spectrophotometer at 232.5 nm, and data was analyzed by using the following formula [4].
(5)Kp=JssCv,where Kp is the permeability coefficient, Jss is the steady-state flux, and Cv is the total donor concentration of formulation.For further evaluation ofex vivo permeability studies by a model-dependent approach, the data was analyzed by fitting them in the zero-order, first-order, Higuchi, and Korsmeyer-Peppas models.


## 2.12. NoninvasiveIn Vivo Studies

### 2.12.1. Ethical Approval and Study Design

The present research study was approved by the Board of Advanced Study and Research (BASR) and the institutional ethics committee, Faculty of Pharmacy, the Islamia University of Bahawalpur, with reference no. 128-2021-/PHEC. The study design for current research work was single-blind random study which was designed for 3 months. In that study, effects of MNF and placebo were checked on thirteen healthy insensitive female volunteers with age 20-40years. The volunteers were investigated for any skin allergy and disease by a dermatologist. A consent form with possible risk and protocols of study was signed by each volunteer, and they were asked to continue their diet but avoid using any other skin preparation during this study period.


### 2.12.2. Inclusion Criteria for Volunteers

(1)
All volunteers participating in the study must be healthy(2)
All of the volunteers should have healthy skin and no history of any hypersensitivity(3)
No one among volunteers should be using any facial creams at least two weeks before and during the three-month study period(4)
In order to measure biophysical parameters, volunteers should be available in the laboratory on specific time(5)
Prior to measuring the readings, volunteers should wait for half an hour in order to acclimatize the laboratory conditions, i.e., room temperature


### 2.12.3. Exclusion Criteria for Volunteers

(1)
A participant on any steroidal therapy shall be excluded from the study(2)
A person with any hypersensitivity or skin acne issue should be excluded from the study(3)
Any participant failing to comply the study guidelines shall be considered excluded(4)
A person having extraordinary hairy skin should be excluded for participating in the study(5)
A person which is already involved in a similar study should not be considered for selection


### 2.12.4. Safety Evaluation by the Patch Test

Before the start of the study, a patch test was performed by checking melanin and erythema level on all the thirteen volunteers to check the skin sensitivity. After 24 h, the volunteers with no skin allergic reaction were provided with two creams marked as right and left which indicate the site of application. The measurements were taken at 0, 2, 4, 6, 8, 10, and 12 weeks of the study.


### 2.12.5. Skin Erythema and Melanin Level

Skin melanin and erythema level was measured by using Mexameter® by Courage+Khazaka Electronic GmbH, Germany, which works on the principle of light absorption and emission with three specific wavelengths: 568, 660, and 880 nm. To avoid the chances of error, three consecutive readings were taken on a tested area and their mean was calculated.


### 2.12.6. Skin Sebum Level

Sebumeter® EM 25 (Courage+Khazaka Electronic GmbH, Germany) was used to check skin sebum level. A plastic sebumeter tape is present in the probe of the sebumeter which measures the skin sebum level by following the principle of light transmission through a sebum-coated opaque plastic strip by photometric transparency. To avoid the chances of error, three consecutive readings were taken on a tested area and their mean was calculated.


### 2.12.7. Skin Moisture Level

Corneometer® by Courage+Khazaka Electronic GmbH, Germany, was used to check the skin moisture level. The probe used for hydration level measurement works on the principle of diffusion of the electric field through the stratum corneum (epidermis), and moisture content is measured as dielectric constant-dependent capacitance changes of water. To avoid the chances of error, three consecutive readings were taken on a tested area and their mean was calculated.


### 2.12.8. Skin Elasticity Level

Elastometer® EM 25 by Courage+Khazaka Electronic GmbH, Germany, was used to check the skin elasticity properties. The probe of the elastometer works on the principle of suction and stretching of skin and measures the elasticity on 0-99 range. To avoid the chances of error, three consecutive readings were taken on a tested area and their mean was calculated.


## 2.12.1. Ethical Approval and Study Design

The present research study was approved by the Board of Advanced Study and Research (BASR) and the institutional ethics committee, Faculty of Pharmacy, the Islamia University of Bahawalpur, with reference no. 128-2021-/PHEC. The study design for current research work was single-blind random study which was designed for 3 months. In that study, effects of MNF and placebo were checked on thirteen healthy insensitive female volunteers with age 20-40years. The volunteers were investigated for any skin allergy and disease by a dermatologist. A consent form with possible risk and protocols of study was signed by each volunteer, and they were asked to continue their diet but avoid using any other skin preparation during this study period.


## 2.12.2. Inclusion Criteria for Volunteers

(1)
All volunteers participating in the study must be healthy(2)
All of the volunteers should have healthy skin and no history of any hypersensitivity(3)
No one among volunteers should be using any facial creams at least two weeks before and during the three-month study period(4)
In order to measure biophysical parameters, volunteers should be available in the laboratory on specific time(5)
Prior to measuring the readings, volunteers should wait for half an hour in order to acclimatize the laboratory conditions, i.e., room temperature


## 2.12.3. Exclusion Criteria for Volunteers

(1)
A participant on any steroidal therapy shall be excluded from the study(2)
A person with any hypersensitivity or skin acne issue should be excluded from the study(3)
Any participant failing to comply the study guidelines shall be considered excluded(4)
A person having extraordinary hairy skin should be excluded for participating in the study(5)
A person which is already involved in a similar study should not be considered for selection


## 2.12.4. Safety Evaluation by the Patch Test

Before the start of the study, a patch test was performed by checking melanin and erythema level on all the thirteen volunteers to check the skin sensitivity. After 24 h, the volunteers with no skin allergic reaction were provided with two creams marked as right and left which indicate the site of application. The measurements were taken at 0, 2, 4, 6, 8, 10, and 12 weeks of the study.


## 2.12.5. Skin Erythema and Melanin Level

Skin melanin and erythema level was measured by using Mexameter® by Courage+Khazaka Electronic GmbH, Germany, which works on the principle of light absorption and emission with three specific wavelengths: 568, 660, and 880 nm. To avoid the chances of error, three consecutive readings were taken on a tested area and their mean was calculated.


## 2.12.6. Skin Sebum Level

Sebumeter® EM 25 (Courage+Khazaka Electronic GmbH, Germany) was used to check skin sebum level. A plastic sebumeter tape is present in the probe of the sebumeter which measures the skin sebum level by following the principle of light transmission through a sebum-coated opaque plastic strip by photometric transparency. To avoid the chances of error, three consecutive readings were taken on a tested area and their mean was calculated.


## 2.12.7. Skin Moisture Level

Corneometer® by Courage+Khazaka Electronic GmbH, Germany, was used to check the skin moisture level. The probe used for hydration level measurement works on the principle of diffusion of the electric field through the stratum corneum (epidermis), and moisture content is measured as dielectric constant-dependent capacitance changes of water. To avoid the chances of error, three consecutive readings were taken on a tested area and their mean was calculated.


## 2.12.8. Skin Elasticity Level

Elastometer® EM 25 by Courage+Khazaka Electronic GmbH, Germany, was used to check the skin elasticity properties. The probe of the elastometer works on the principle of suction and stretching of skin and measures the elasticity on 0-99 range. To avoid the chances of error, three consecutive readings were taken on a tested area and their mean was calculated.


## 2.13. Mathematical and Statistical Analysis

Obtained data was statistically analyzed by SPSS version 23. Two-way ANOVA using LSD and paired samplet-test was applied for comparison of data with formulation and multiple comparisons of placebo and active formulations, respectively. P<0.05 was selected as the level of significant difference.


## 3. Results

### 3.1. Antioxidant Activity

The antioxidant activity of niacinamide and ferulic acid was 93% and 95%, respectively, when compared with the standard (ascorbic acid) as indicated in Figure1(a) which showed that both the compounds are potential candidate for topical cosmeceutical applications.Figure 1
Radical scavenging potential and sun protection factor of niacinamide, ferulic acid, and MNF.
(a)(b)


### 3.2. Fourier Transform Infrared Spectroscopy (FTIR) Analysis

Functional groups of niacinamide and ferulic acid were identified by FTIR analysis. Spectra for niacinamide and ferulic acid are presented in Table3 and Figures 2(a) and 2(b), respectively.Table 3
Spectral interpretation of FTIR spectra of drugs (niacinamide and ferulic acid).
Sr. noDrugPeak values (cm-1)Functional group1Niacinamide3352N-H3154.2N-H2924.09C-H1676C=O1389C-C1090C-H2Ferulic acid3432O-H2917C-H1662.61C=O1592C=C1511C=C1429C=CFigure 2
FTIR analyses of (a) niacinamide and (b) ferulic acid.
(a)(b)


### 3.3.In Vitro Sun Protection Factor

The SPF values of niacinamide, ferulic acid, and MNF were23±0.90, 35±1.20, and 40±1.60, respectively, as indicated in Figure 1(b). The results signify that not only active compounds but also their loaded formulation has high SPF values.


### 3.4. Organoleptic Evaluation

In organoleptic evaluation, different parameters like color, odor, and liquefaction on centrifugation were checked for both placebo and MNF that were kept at different storage conditions (8°C, 25°C, 40°C, and 40°C with 75% relative humidity) for the time period of 3 months. The color of placebo and MNF was white and off-white, respectively, which remains unchanged throughout the study period of 3 months. All the formulations that were kept at 8°C and 25°C were stable throughout the study period of 3 months, while slight liquefaction on centrifugation was observed in formulations that were kept at 40°C and 40°C with 75% relative humidity (RH) after 90 days as indicated in Table4.Table 4
Organoleptic evaluation of placebo and MNF.
T5emp. (°C)Time (days)0171530456090PMNFPMNFPMNFPMNFPMNFPMNFPMNFPMNFColor and odor8−−−−−−−−−−−−−−−−25−−−−−−−−−−−−−−−−40−−−−−−−−−−−−−−−−40+RH−−−−−−−−−−−−−−−−Liquefaction8−−−−−−−−−−−−−−−−25−−−−−−−−−−−−−−−−40−−−−−−−−−−−−−−++40+RH−−−−−−−−−−−−++++−: no change; +: slightly change; MNF: multiple emulsion loaded with niacinamide and ferulic acid; P: placebo; RH: 75% relative humidity.


### 3.5. pH

pH of both placebo and MNF was checked that were kept at different temperature conditions (8°C, 25°C, 40°C, and 40°C with 75% relative humidity) for the time period of 3 months. pH of freshly prepared placebo and MNF was6.4±0.11 and 6.2±0.12, respectively, which was reduced with passage of time as indicated in Figures 3(a) and 3(b). When two-way ANNOVA was applied by using the LSD test, there is an insignificant difference by taking 8°C as the reference, and according to the paired t-test, there was a significant difference between pH values of MNF and placebo.Figure 3
Change in pH of placebo and MNF.
(a)(b)


### 3.6. Microscopic Evaluation

Globule size determination was done for both placebo and MNF that were kept at different storage conditions (8°C, 25°C, 40°C, and 40°C with 75% relative humidity). The shape of globules for both placebo and MNF was spherical and remained the same throughout the study period. The globule size of fresh MNF and after 3 months was4.730±1.50μm and 6.49±1.20μm, respectively, which was slightly increased with time as indicated in Figures 4(a) and 4(b).When two-way ANNOVA was applied by using LSD, there is an insignificant difference by taking 8°C as the reference, and according to the paired t-test, there was a significant difference between microscopic values of MNF and placebo.Figure 4
Microscopic evaluation of placebo MNF.
(a)(b)


### 3.7. Rheological Evaluation

The results showed that all of the samples kept at several storage temperatures, i.e., 8°C, 25°C, 40°C, and 40°C with 75% RH showed pseudo-plastic non-Newtonian behavior with time, and by applying the Ostwald power law, the MNF showed the shear thinning behavior. The apparent viscosity of freshly prepared MNF was 446.99 cP which was decreased with the passage of time and by increasing the shear rate and shear stress as indicated in Figures5(a)–5(d). The flow index of MNF at diverse storage points, i.e., 8°C (refrigerator), 25°C (incubator), 40°C (incubator), and 40°C (incubator) with 75% RH, was less than 1 confirming the pseudo-plastic non-Newtonian behavior of MNF.Figure 5
Rheological analysis of MNF of (a) fresh formulation and after (b) 30 days, (c) 60 days, and (d) 90 days.
(a)(b)(c)(d)


### 3.8. Entrapment Efficiency

For calculating how much drug is entrapped in the emulsion system, entrapment efficiency is one of the best methods. The entrapment efficiency of MNF was 93.3% as indicated in Table5.Table 5
Zeta size, polydispersity index, zeta potential, and entrapment efficiency of MNF.
FormulationZeta size (nm)PDIZeta potential (mV)EE (%±SD)MNF1730.359-5.8893.3±0.325


### 3.9. Zeta Potential, Zeta Size, and PDI

Results of zeta potential, zeta size, and PDI of MNF are shown in Table5 and Figure 6. Zeta potential was found to be -5.88 mV whereas zeta size and PDI were 173 nm (0.173 μm) and 0.359, respectively.Figure 6
Zeta potential, zeta size, and PDI of MNF.
(a)(b)


### 3.10.Ex Vivo Permeation Studies

The permeation of niacinamide/ferulic acid-loaded MNF and plain drug solution (as standard) is indicated in Figure7. The graph shows that incorporation of niacinamide in combination with ferulic acid enhances the permeation in comparison with plain drug solution. The percent cumulative drug permeation of MNF and drug solution was 81.575% and 67.044%, respectively, in 8 hours of study. About 30% of drug was released in the first 4 hours and 70% of drug released in 8 hours from the drug solution. MNF manifested nonlinear biphasic drug release with an initial rapid release phase (31%) in the first 4 hours and then a slower release (81%) in the next 8 hours.Figure 7
Ex vivo permeation studies.The permeability coefficient and flux of MNF and drug solution are indicated in Table6. The data obtained from ex vivo permeability studies was analyzed by putting them in different kinetic models, and results are indicated in Table 7. According to results from all kinetic models, Korsmeyer-Peppas was found to be the best suitable model for MNF having R2 value 0.9991 at pH 5.5.Table 6
Permeation data analysis.
FormulationFluxjss (μg cm2/min)Permeability coefficientKp (cm/min)At 5.5 pHDrug solution0.09060.00165MNF0.10580.00192Table 7
Kinetic models forex vivo permeation studies.
FormulationZero-order kineticsFirst-order kineticsHiguchi modelKorsmeyer-PeppasR2KoR2K1R2kHR2NDrug solution0.95750.1400.90180.0020.74662.2860.99861.474MNF0.93810.1670.85730.0020.71472.7040.99911.620


### 3.11. NoninvasiveIn Vivo Studies

#### 3.11.1. Safety Evaluation by the Patch Test

The patch test was performed on the selected human volunteers to check their erythema level before startingin vivo studies. The results indicated in Figure 8(a) showed that no increase in erythema level was found in any of volunteers which confirmed that MNF was safe and suitable for the topical application in selected volunteers.Figure 8
Noninvasivein vivo investigations of placebo and MNF on different skin parameters for 12 weeks. (a) Skin erythema for the patch test. (b) Skin erythema, (c) skin melanin, (d) skin sebum, (e) skin moisture, and (f) skin elasticity. Values are expressed as the mean±SD and ∗P≤0.05, where n=13.
(a)(b)(c)(d)(e)(f)


#### 3.11.2. Skin Erythema Level

According to results, there was a decrease in erythema levels of volunteers manifested by both MNF and placebo. As demonstrated in Figure8(b), this decrease was marked and more pronounced by MNF as compared to placebo, i.e., 7 times more than that of zero time value. Moreover, unlike placebo, this decrease was regular and statistically significant at 2, 4, 6, 8, 10, and 12 weeks of the study. The paired t-test showed that there was a significant difference between decreased levels in erythema manifested by MNF and placebo.


#### 3.11.3. Skin Melanin Level

Figure8(c) shows that skin melanin level was decreased by MNF but increased by placebo during the study period. The percentage change in skin melanin level by MNF was very ostentatious, i.e., about 8 times the initial zero time value. It was also statistically significant at 2, 4, 6, 8, 10, and 12 weeks of the study. Moreover, the paired t-test proved that the changes in skin melanin level induced by MNF as well as placebo were statistically significant from each other.


#### 3.11.4. Skin Sebum Level

Skin sebum level of the volunteers was increased by placebo with passage of time while MNF decreased it as indicated in Figure8(d). The percentage change in sebum level by MNF was very marked and about 14 times the initial zero time value. Furthermore, it was found to be statistically significant at the 2nd, 4th, 6th, 8th, 10th, and 12th week of the study, whereas the effect of placebo on the sebum level was not regular and was significant from zero time value only at the 8th, 10th, and 12th weeks of the study. According to the paired t-test, there was a significant difference between changes in sebum level by MNF and placebo.


#### 3.11.5. Skin Moisture Level

An increase in the moisture content of skin of volunteers was observed by both MNF and placebo during the study period as indicated in Figure8(e). But the increase in moisture level by MNF was more pronounced, i.e., 7 times more than the initial zero time value. Statistical analyses (two-way ANNOVA) using the LSD test showed that there was a significant difference in moisture level by MNF at the 2nd, 4th, 6th, 8th, 10th, and 12th week while at the 12th week by placebo. The paired t-test demonstrated that there was a significant difference between moisture levels by both MNF and placebo.


#### 3.11.6. Skin Elasticity Level

In the present study, unlike placebo, MNF markedly increased the skin elasticity in volunteers as depicted in Figure8(f). The percentage increase in elasticity level by MNF was 9 times the initial zero time value. Moreover, this increase is statistically significant at 2, 4, 6, 8, 10, and 12 weeks of the study, whereas placebo decreased the skin elasticity, but this decrease was not regular as at the 10th week, there was a mild increase in the elasticity level.


## 3.1. Antioxidant Activity

The antioxidant activity of niacinamide and ferulic acid was 93% and 95%, respectively, when compared with the standard (ascorbic acid) as indicated in Figure1(a) which showed that both the compounds are potential candidate for topical cosmeceutical applications.Figure 1
Radical scavenging potential and sun protection factor of niacinamide, ferulic acid, and MNF.
(a)(b)


## 3.2. Fourier Transform Infrared Spectroscopy (FTIR) Analysis

Functional groups of niacinamide and ferulic acid were identified by FTIR analysis. Spectra for niacinamide and ferulic acid are presented in Table3 and Figures 2(a) and 2(b), respectively.Table 3
Spectral interpretation of FTIR spectra of drugs (niacinamide and ferulic acid).
Sr. noDrugPeak values (cm-1)Functional group1Niacinamide3352N-H3154.2N-H2924.09C-H1676C=O1389C-C1090C-H2Ferulic acid3432O-H2917C-H1662.61C=O1592C=C1511C=C1429C=CFigure 2
FTIR analyses of (a) niacinamide and (b) ferulic acid.
(a)(b)


## 3.3.In Vitro Sun Protection Factor

The SPF values of niacinamide, ferulic acid, and MNF were23±0.90, 35±1.20, and 40±1.60, respectively, as indicated in Figure 1(b). The results signify that not only active compounds but also their loaded formulation has high SPF values.


## 3.4. Organoleptic Evaluation

In organoleptic evaluation, different parameters like color, odor, and liquefaction on centrifugation were checked for both placebo and MNF that were kept at different storage conditions (8°C, 25°C, 40°C, and 40°C with 75% relative humidity) for the time period of 3 months. The color of placebo and MNF was white and off-white, respectively, which remains unchanged throughout the study period of 3 months. All the formulations that were kept at 8°C and 25°C were stable throughout the study period of 3 months, while slight liquefaction on centrifugation was observed in formulations that were kept at 40°C and 40°C with 75% relative humidity (RH) after 90 days as indicated in Table4.Table 4
Organoleptic evaluation of placebo and MNF.
T5emp. (°C)Time (days)0171530456090PMNFPMNFPMNFPMNFPMNFPMNFPMNFPMNFColor and odor8−−−−−−−−−−−−−−−−25−−−−−−−−−−−−−−−−40−−−−−−−−−−−−−−−−40+RH−−−−−−−−−−−−−−−−Liquefaction8−−−−−−−−−−−−−−−−25−−−−−−−−−−−−−−−−40−−−−−−−−−−−−−−++40+RH−−−−−−−−−−−−++++−: no change; +: slightly change; MNF: multiple emulsion loaded with niacinamide and ferulic acid; P: placebo; RH: 75% relative humidity.


## 3.5. pH

pH of both placebo and MNF was checked that were kept at different temperature conditions (8°C, 25°C, 40°C, and 40°C with 75% relative humidity) for the time period of 3 months. pH of freshly prepared placebo and MNF was6.4±0.11 and 6.2±0.12, respectively, which was reduced with passage of time as indicated in Figures 3(a) and 3(b). When two-way ANNOVA was applied by using the LSD test, there is an insignificant difference by taking 8°C as the reference, and according to the paired t-test, there was a significant difference between pH values of MNF and placebo.Figure 3
Change in pH of placebo and MNF.
(a)(b)


## 3.6. Microscopic Evaluation

Globule size determination was done for both placebo and MNF that were kept at different storage conditions (8°C, 25°C, 40°C, and 40°C with 75% relative humidity). The shape of globules for both placebo and MNF was spherical and remained the same throughout the study period. The globule size of fresh MNF and after 3 months was4.730±1.50μm and 6.49±1.20μm, respectively, which was slightly increased with time as indicated in Figures 4(a) and 4(b).When two-way ANNOVA was applied by using LSD, there is an insignificant difference by taking 8°C as the reference, and according to the paired t-test, there was a significant difference between microscopic values of MNF and placebo.Figure 4
Microscopic evaluation of placebo MNF.
(a)(b)


## 3.7. Rheological Evaluation

The results showed that all of the samples kept at several storage temperatures, i.e., 8°C, 25°C, 40°C, and 40°C with 75% RH showed pseudo-plastic non-Newtonian behavior with time, and by applying the Ostwald power law, the MNF showed the shear thinning behavior. The apparent viscosity of freshly prepared MNF was 446.99 cP which was decreased with the passage of time and by increasing the shear rate and shear stress as indicated in Figures5(a)–5(d). The flow index of MNF at diverse storage points, i.e., 8°C (refrigerator), 25°C (incubator), 40°C (incubator), and 40°C (incubator) with 75% RH, was less than 1 confirming the pseudo-plastic non-Newtonian behavior of MNF.Figure 5
Rheological analysis of MNF of (a) fresh formulation and after (b) 30 days, (c) 60 days, and (d) 90 days.
(a)(b)(c)(d)


## 3.8. Entrapment Efficiency

For calculating how much drug is entrapped in the emulsion system, entrapment efficiency is one of the best methods. The entrapment efficiency of MNF was 93.3% as indicated in Table5.Table 5
Zeta size, polydispersity index, zeta potential, and entrapment efficiency of MNF.
FormulationZeta size (nm)PDIZeta potential (mV)EE (%±SD)MNF1730.359-5.8893.3±0.325


## 3.9. Zeta Potential, Zeta Size, and PDI

Results of zeta potential, zeta size, and PDI of MNF are shown in Table5 and Figure 6. Zeta potential was found to be -5.88 mV whereas zeta size and PDI were 173 nm (0.173 μm) and 0.359, respectively.Figure 6
Zeta potential, zeta size, and PDI of MNF.
(a)(b)


## 3.10.Ex Vivo Permeation Studies

The permeation of niacinamide/ferulic acid-loaded MNF and plain drug solution (as standard) is indicated in Figure7. The graph shows that incorporation of niacinamide in combination with ferulic acid enhances the permeation in comparison with plain drug solution. The percent cumulative drug permeation of MNF and drug solution was 81.575% and 67.044%, respectively, in 8 hours of study. About 30% of drug was released in the first 4 hours and 70% of drug released in 8 hours from the drug solution. MNF manifested nonlinear biphasic drug release with an initial rapid release phase (31%) in the first 4 hours and then a slower release (81%) in the next 8 hours.Figure 7
Ex vivo permeation studies.The permeability coefficient and flux of MNF and drug solution are indicated in Table6. The data obtained from ex vivo permeability studies was analyzed by putting them in different kinetic models, and results are indicated in Table 7. According to results from all kinetic models, Korsmeyer-Peppas was found to be the best suitable model for MNF having R2 value 0.9991 at pH 5.5.Table 6
Permeation data analysis.
FormulationFluxjss (μg cm2/min)Permeability coefficientKp (cm/min)At 5.5 pHDrug solution0.09060.00165MNF0.10580.00192Table 7
Kinetic models forex vivo permeation studies.
FormulationZero-order kineticsFirst-order kineticsHiguchi modelKorsmeyer-PeppasR2KoR2K1R2kHR2NDrug solution0.95750.1400.90180.0020.74662.2860.99861.474MNF0.93810.1670.85730.0020.71472.7040.99911.620


## 3.11. NoninvasiveIn Vivo Studies

### 3.11.1. Safety Evaluation by the Patch Test

The patch test was performed on the selected human volunteers to check their erythema level before startingin vivo studies. The results indicated in Figure 8(a) showed that no increase in erythema level was found in any of volunteers which confirmed that MNF was safe and suitable for the topical application in selected volunteers.Figure 8
Noninvasivein vivo investigations of placebo and MNF on different skin parameters for 12 weeks. (a) Skin erythema for the patch test. (b) Skin erythema, (c) skin melanin, (d) skin sebum, (e) skin moisture, and (f) skin elasticity. Values are expressed as the mean±SD and ∗P≤0.05, where n=13.
(a)(b)(c)(d)(e)(f)


### 3.11.2. Skin Erythema Level

According to results, there was a decrease in erythema levels of volunteers manifested by both MNF and placebo. As demonstrated in Figure8(b), this decrease was marked and more pronounced by MNF as compared to placebo, i.e., 7 times more than that of zero time value. Moreover, unlike placebo, this decrease was regular and statistically significant at 2, 4, 6, 8, 10, and 12 weeks of the study. The paired t-test showed that there was a significant difference between decreased levels in erythema manifested by MNF and placebo.


### 3.11.3. Skin Melanin Level

Figure8(c) shows that skin melanin level was decreased by MNF but increased by placebo during the study period. The percentage change in skin melanin level by MNF was very ostentatious, i.e., about 8 times the initial zero time value. It was also statistically significant at 2, 4, 6, 8, 10, and 12 weeks of the study. Moreover, the paired t-test proved that the changes in skin melanin level induced by MNF as well as placebo were statistically significant from each other.


### 3.11.4. Skin Sebum Level

Skin sebum level of the volunteers was increased by placebo with passage of time while MNF decreased it as indicated in Figure8(d). The percentage change in sebum level by MNF was very marked and about 14 times the initial zero time value. Furthermore, it was found to be statistically significant at the 2nd, 4th, 6th, 8th, 10th, and 12th week of the study, whereas the effect of placebo on the sebum level was not regular and was significant from zero time value only at the 8th, 10th, and 12th weeks of the study. According to the paired t-test, there was a significant difference between changes in sebum level by MNF and placebo.


### 3.11.5. Skin Moisture Level

An increase in the moisture content of skin of volunteers was observed by both MNF and placebo during the study period as indicated in Figure8(e). But the increase in moisture level by MNF was more pronounced, i.e., 7 times more than the initial zero time value. Statistical analyses (two-way ANNOVA) using the LSD test showed that there was a significant difference in moisture level by MNF at the 2nd, 4th, 6th, 8th, 10th, and 12th week while at the 12th week by placebo. The paired t-test demonstrated that there was a significant difference between moisture levels by both MNF and placebo.


### 3.11.6. Skin Elasticity Level

In the present study, unlike placebo, MNF markedly increased the skin elasticity in volunteers as depicted in Figure8(f). The percentage increase in elasticity level by MNF was 9 times the initial zero time value. Moreover, this increase is statistically significant at 2, 4, 6, 8, 10, and 12 weeks of the study, whereas placebo decreased the skin elasticity, but this decrease was not regular as at the 10th week, there was a mild increase in the elasticity level.


## 3.11.1. Safety Evaluation by the Patch Test

The patch test was performed on the selected human volunteers to check their erythema level before startingin vivo studies. The results indicated in Figure 8(a) showed that no increase in erythema level was found in any of volunteers which confirmed that MNF was safe and suitable for the topical application in selected volunteers.Figure 8
Noninvasivein vivo investigations of placebo and MNF on different skin parameters for 12 weeks. (a) Skin erythema for the patch test. (b) Skin erythema, (c) skin melanin, (d) skin sebum, (e) skin moisture, and (f) skin elasticity. Values are expressed as the mean±SD and ∗P≤0.05, where n=13.
(a)(b)(c)(d)(e)(f)


## 3.11.2. Skin Erythema Level

According to results, there was a decrease in erythema levels of volunteers manifested by both MNF and placebo. As demonstrated in Figure8(b), this decrease was marked and more pronounced by MNF as compared to placebo, i.e., 7 times more than that of zero time value. Moreover, unlike placebo, this decrease was regular and statistically significant at 2, 4, 6, 8, 10, and 12 weeks of the study. The paired t-test showed that there was a significant difference between decreased levels in erythema manifested by MNF and placebo.


## 3.11.3. Skin Melanin Level

Figure8(c) shows that skin melanin level was decreased by MNF but increased by placebo during the study period. The percentage change in skin melanin level by MNF was very ostentatious, i.e., about 8 times the initial zero time value. It was also statistically significant at 2, 4, 6, 8, 10, and 12 weeks of the study. Moreover, the paired t-test proved that the changes in skin melanin level induced by MNF as well as placebo were statistically significant from each other.


## 3.11.4. Skin Sebum Level

Skin sebum level of the volunteers was increased by placebo with passage of time while MNF decreased it as indicated in Figure8(d). The percentage change in sebum level by MNF was very marked and about 14 times the initial zero time value. Furthermore, it was found to be statistically significant at the 2nd, 4th, 6th, 8th, 10th, and 12th week of the study, whereas the effect of placebo on the sebum level was not regular and was significant from zero time value only at the 8th, 10th, and 12th weeks of the study. According to the paired t-test, there was a significant difference between changes in sebum level by MNF and placebo.


## 3.11.5. Skin Moisture Level

An increase in the moisture content of skin of volunteers was observed by both MNF and placebo during the study period as indicated in Figure8(e). But the increase in moisture level by MNF was more pronounced, i.e., 7 times more than the initial zero time value. Statistical analyses (two-way ANNOVA) using the LSD test showed that there was a significant difference in moisture level by MNF at the 2nd, 4th, 6th, 8th, 10th, and 12th week while at the 12th week by placebo. The paired t-test demonstrated that there was a significant difference between moisture levels by both MNF and placebo.


## 3.11.6. Skin Elasticity Level

In the present study, unlike placebo, MNF markedly increased the skin elasticity in volunteers as depicted in Figure8(f). The percentage increase in elasticity level by MNF was 9 times the initial zero time value. Moreover, this increase is statistically significant at 2, 4, 6, 8, 10, and 12 weeks of the study, whereas placebo decreased the skin elasticity, but this decrease was not regular as at the 10th week, there was a mild increase in the elasticity level.


## 4. Discussion

The present study was aimed at formulating a stable niacinamide/ferulic acid-loaded multiple emulsion (MNF) and itsin vitro and in vivo characterization as a cosmeceutical product. Free radicals cause the damage to cells, and this damage is prevented and stabilized by antioxidant substances [16]. To evaluate the antioxidant activity by spectrophotometry, the DPPH method is a very rapid and easy assay. In the present study, both niacinamide and ferulic acid showed strong antioxidant potential, which is in accordance with the previously available data and suggests their potential use in cosmetology [17].The FTIR spectrum of niacinamide confers the C–H, C=O, C–C, and C–H stretching at 2924 cm–1, 1676 cm–1, 1389 cm–1, and 1090, respectively. N–H bands appeared at 3352 cm–1 and 3154 cm–1. These results are supported by the previously reported work [18]. For ferulic acid, peaks at 3432 cm–1, 2917 cm–1, and 1662 cm–1 manifested C–H stretching, C–H bond stretching, and C=O bond stretching, respectively. C=O bond stretching was pointed at 1592 cm–1, 1511 cm–1, and 1429 cm–1. Results of spectra are supported by the previously reported work [19].Sun protection actually determines the ratio between minimum erythema doses of protected skin and unprotected skin. It is basically UV energy that is required to produce minimum erythema from sun on protected skin and unprotected skin [20]. Higher value of SPF showed that the product is very effective against the deleterious effects of UVR of sunlight. Both the compounds (niacinamide and ferulic acid) individually as well as the developed formulation manifested strong capacity of sun protection in vitro. Previous researches have shown that both of these compounds have potential to prevent the skin from photodamage [21, 22].Microscopic evaluation is a useful tool for investigating the characteristics of multiple emulsions that give insight into their stability. Hence, the slower the increase in the globule size, the more the stability of the emulsions and vice versa [23]. In case of multiple emulsions, increase in globule size is an indicator of destability which may be due to many reasons like coalescence of droplets, rupturing of oil layers, shrinkage, and swelling of internal droplets [24]. The present formulation exhibited no change in the globule shape whereas there was mild increase in the globule size during the course of 90 days when kept at higher temperature and humidity. This mild increase does not affect the stability of the formulation as manifested in centrifugation testing during this period. Moreover, ABIL-EM 90 has been proven very efficient to make the topical formulations stable [25].Human skin pH ranges 4.5–6, averaging 5.5. In the case of topical cosmeceutical products, pH is of much importance as if it is not in the prescribed range, it will adversely affect the skin [26]. Decrease in pH of both placebo and MNF in the current study may be due to the decomposition of paraffin oil, but this decrease is insignificant and lies within the prescribed limits throughout the study period [27].In cosmetic preparations, rheological properties are of paramount importance as they affect the spreadability of formulation onto the skin. Formulations with poor rheological attributes do not spread evenly onto the skin and hence failed to provide required cosmeceutical outcomes [28]. In the current study, both the MNF and the placebo showed pseudo-plastic non-Newtonian behavior with time, and by applying the Ostwald power law, the MNF showed the shear thinning behavior. The non-Newtonian behavior of emulsion shows that this formulation spreads evenly on the skin and enhances the absorption of drug through skin [29], and this non-Newtonian behavior was due to the continuous deformation by applying high shear stress and then reform to its original state due to the Brownian motion after removal of share stress [27].Zeta potential is the expression of charge on the surface of the globules and an important parameter indicative of stability. The higher the zeta potential, the more the stability of the formulation and vice versa. Moreover, its negative value suggests that electrostatic repulsion between the globules will prevent their aggregation and thereby stabilize the formulation [30].The external phase in multiple emulsions provides the gradient for the solubilized drug present in the internal phase and affects the drug release in comparison to other formulations. Multiple emulsions prepared by the double emulsification method have higher entrapment efficiency and slow release form formulation making it controlled release. This release pattern may be due to more homogenous particle size that also makes it more stable [31]. Incorporation of both the lipophilic surfactant (ABIL-EM 90) and hydrophilic surfactant (Tween 80) enhances the permeation, which means that there are chances of enhancement of its absorption and bioavailability too in the form of multiple emulsions [4, 31]. Results from ex vivo permeation studies showed that MNF manifested nonlinear biphasic drug release pattern where the initial release of ferulic acid/niacinamide might be due to the release of drug from the external aqueous phase (niacinamide) whereas the second prolonged phase would be the slow release of ferulic acid from the oil phase. Similar results were obtained by Sawant et al. in their study [15]. According to results from all kinetic models, Korsmeyer-Peppas was found to be the best suitable model for MNF. Incorporation of ferulic acid/niacinamide enhances its permeation which means that there are chances of enhancement of its absorption and bioavailability too [32].The human skin is mainly damaged by high level of ROS which are produced due to photooxidative reactions. In the current study, the decrease in erythema level of volunteers’ skin was due to the presence of niacinamide/ferulic acid. Both the compounds have the capacity to inhibit the production and regulation of proinflammatory cytokines (IL-1, IL-6, IL-10, and IL-12) [8] and inflammatory mediators (COX-2 and PGE2) which are involved in the photoinduced erythema [1, 17]. The decrease in melanin level by MNF was due to the presence of these compounds which have the tendency to inhibit melanogenesis by inhibiting melanocytic proliferation and tyrosinase activity which is a key enzyme for melanin synthesis [9].The activity of the sebaceous gland was responsible for the production of sebum whose overproduction can lead to large pores and many skin diseases like acne vulgaris due to microbial infestation [33]. In the present study, significant decrease in sebum level is attributed to the activity of niacinamide and ferulic acid present in MNF [34, 35], while the slight increase in sebum level was observed in the case of placebo which can be related to the presence of the paraffin oil [36].Developing a formulation which acts on the dermis and stimulates collagen is of prime importance which relates to the hydration of the skin. Moisturizers play an important role in this regard and improve many impaired functions like atopic skin and contact dermatitis related to dry skin [37]. The increase in the moisture level is due to the moisturizing effects of both niacinamide and ferulic acid, which were used in MNF. Previous studies showed that reduction in inflammation, erythema, and profilaggrin play an important role in maintaining the skin hydration level. Profilaggrin proteins have moisturizing ability and maintain the water loss from skin (epidermis) [1, 6]. Slight increase in moisture level by placebo may be due to occlusive effect of the oil phase which reduces the water loss from the epidermis by filling the gap between stratum corneum cells [38].Skin elasticity is improved by niacinamide used in MNF that inhibits the dermal matrix glycation and increased concentration of collagen (dermal remodeling) [39]. Niacinamide and ferulic acid both increase the level of NADP which is also involved in the increased production of collagen [6, 17].


## 5. Conclusion

Current research work showed that niacinamide/ferulic acid-loaded stable multiple emulsion with its strong antioxidant and sun protective attributes has potent cosmeceutical effects on skin manifested by decreased melanin and sebum level as well as increased moisture and elasticity.



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*Source: 1725053-2022-03-17.xml*