The Skin Plays A Vital Role in Modulating Body Temperature, Perceiving Pain And Pressure

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ABSTRACT

Background: Green beans of Coffee arabica L. Family: Rubiaceae are a rich source of polyphenols that prevents oxidative damage to the skin. In this study, Liposomes were formulated as drug carriers to enhance the skin permeability of polyphenols for topical administration. Materials and Methods: Extracts of green coffee beans were prepared by varying the ratios of methanol and water. Phytochemical analysis and in vitro antioxidant evaluation were conducted on all the extracts to choose the best extract for further studies. The cytotoxicity potential (MTT assay) and anti-elastase activity of the chosen extract were evaluated on L929 cell lines using a flow cytometer. The liposomes of the extract were prepared by the thin film hydration method and optimized by varying the phosphatidylcholine to cholesterol ratio and gradually increasing the amount of extract. The liposomal formulation was converted into the gel using carbopol"974P as a gelling agent. The prepared liposomal suspension and the liposomal gel were evaluated for various formulation parameters. Results: The results of phytochemical analysis, in vitro antioxidant assays, and in vitro evaluation on L929 cell lines concluded that 25% methanolic extract was nontoxic with the highest phenolic content and exhibited good antielastase activity. The encapsulation efficiency of chlorogenic acid in the optimized liposomal formulation was 75%. The liposomal gel showed sustained release of the drug for up to 12 hr compared to 6-7 hr of the conventional formulation. Conclusion: The formulated green coffee bean extract-loaded liposomal-based gel may serve as a potential carrier for antiaging effects.

Keywords: Green coffee beans (GCB), Chlorogenic acid, Extract, Liposomes, Antioxidant, Antiaging.

INTRODUCTION

The skin plays a vital role in modulating body temperature, perceiving pain and pressure, and acts as a crucial barrier insults against environmental pollution making skin aging very evident.1 Long-term skin exposure to ultraviolet radiation produces free radicals which induce oxidative stress on the skin leading to the development of photoaging, sunburn, melanogenesis, immunosuppression, and photocarcinogenesis.² Free radicals are extremely reactive oxygen molecules that are engaged in forming cross-linkages with collagen molecules leading to loss of skin elasticity.3 Aging manifests itself with sagging, thinning, dryness, and spotting on the skin. Thus, with a desire of looking young, antiaging products are in great demand. Antioxidants of herbal origin have gained importance in the cosmetic industry. Antioxidants help in repairing and preventing oxidative damage caused by free radicals. The antioxidant activity of herbs is majorly due to the redox properties of phenolic compounds.4 Thus, antioxidants are found to be promising in delaying the signs of aging such as wrinkles, age spots, and fine lines.

The green coffee (Coffea arabica L, Rubiaceae)beans are a rich source of polyphenols such as chlorogenic acid and its related compounds such as caffeic acid, coumaric acid, and ferulic acid that prevent the skin from oxidative damage. Studies have revealed C. arabica extracts are found to possess antibacterial,5 antiviral,6 anti-inflammatories,7 skin wound healing, and' reduction of oxidative damage to macromolecules? and suppressive activity of metalloproteinase expression.10

Chlorogenic acid, the major polyphenolic compound of green coffee beans has antioxidant and depigmentation properties and it inhibits UV-induced skin damage. I2 According to scientific studies, green coffee beans contain a higher amount of chlorogenic acid than roasted coffee beans and other plants. "3

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The delivery of polyphenols through the skin is ineffective due to its limited penetration. Thus the formulation base or penetration enhancers that enhance its penetration is required." So liposomes were selected as drug carriers as they are amphiphilic in nature and hydrophilic molecules can easily be embedded in the concentric bilayers. Liposomes being physiologically similar to the cell membrane are non-toxic in nature. The presence of phospholipids in the liposomes improves topical absorption of the drug from the epidermis into the lower layers due to increased adhesion of the drug phospholipid complex on the skin.'5 The aim of the present study is to develop a gel containing green coffee bean extract-loaded liposomes to be used as an antioxidant cosmetic formulation for antiaging effects.

MATERIALS AND METHODS Materials

Green coffee arabica L beans were obtained from the local market of Mumbai in August 2019 and were authenticated by Dr. Harshad M. Pandit. Ex-Head and Associate Professor of Botany, Guru Nanak Khalsa College, Mumbai, India. Phosphatidylcholine was purchased from HiMedia Laboratories Pvt Ltd, Cholesterol 99% was procured from Loba Chemie Pvt Ltd, 2,2-diphenyl-1-picrylhydrazyl (DPPH), and Chlorogenic acid was purchased from Otto Chemie Pvt Ltd, Mumbai. Gallic acid and Ascorbic acid were procured from SD Fine Chemicals Ltd, Mumbai. All chemicals used in this study were of A.R. grade. Liposomes were produced using a Rotary evaporator, Rotaeva, Equitron, and UV-Visible Spectrophotometer, Shimadzu 1800 was used for evaluation of liposomes.

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Methods

Extraction Methodology

The powdered green coffee beans were defatted using petroleum ether 40-60℃(1:6 % w/v) for 3hr in a soxhlet apparatus. The defatted green coffee powder was extracted with different solvents such as water,25%methanol, 50% methanol,75% methanol, and 100%methanol for 2hr using a soxhlet system. The resulting extracts were concentrated in a porcelain dish on an electric water bath at 80℃ to remove the solvent. The prepared extracts were used for qualitative and quantitative analysis.

Total Phenolic Content

Total phenolic content was determined by the Folin-Ciocalteu reagent method using standard procedure. Gallic acid was used as a standard and total polyphenolic content was expressed as mg/g gallic acid equivalent (GAE) from the calibration curve of gallic acid.16

Spectral Overlay

UV spectrometric method was used to determine the presence of chlorogenic acid in the extract. For the preparation of standard solutions, Img/ml of standard chlorogenic acid was dissolved in phosphate buffer and further dilution was prepared to get 10 μg/ml of standard solution. For the sample solution,25% of 1 mg/ml methanolic extract was dissolved in phosphate buffer and further dilution was prepared to get 10 ug/ml of standard solution. An overlay spectrum of both the solution was obtained using UV-Vis spectrophotometry.

Quantification of Chlorogenic acid in each extract Standard solution preparation

The standard chlorogenic acid was dissolved in the solvents such as water,25% methanol,50% methanol, 75% methanol, and 100% methanol. In order to reduce the error in the preparation procedure, the chlorogenic acid 10 ppm solution was prepared in each above solvent, and then corresponding dilution ranging from concentration 2 to 10 ppm was prepared. The linearity plot was made by recording the absorbance at 324 nm using a UV-Vis spectrophotometer.

Sample solution preparation

For Sample solution preparation, the extracts were dissolved in their respective solvents and a 10 ppm solution was made. The absorbance for the sample solution was recorded at 324 nm using a UV-Vis spectrophotometer. The obtained absorbance was extrapolated in the linearity plot to find the concentration of chlorogenic acid in each extract.

Determination of Antioxidant Activities

PDF (2,2-diphenyl -1-picrylhydrazyl) antioxidant assay Radical scavenging activity of the extracts on DPPH radicals was investigated by following standard method." Ascorbic acid was used as standard. The experiment was performed in triplicate. The percentage of DPPH radical scavenging activity was plotted against each extract concentration and IC was determined. Nitric Oxide antioxidant assay

Nitric oxide radical scavenging activity was investigated by the use of the Griess Illosvoy reaction. lost empire cistanche l8 Ascorbic acid was used as standard. The experiment was performed in triplicate. The percentage of nitric oxide radical scavenging activity was plotted against each extract concentration and IC Percentage Inhibition for both the antioxidant studies was calculated using below-mentioned formula Scavenging activity was expressed as the inhibition percentage calculated using the following formula:

Determination of cell viability by MTT Assay Green coffee beans extract viz 25% methanolic extract was screened for cytotoxicity study against the L929 cell lines.200 ul of cell suspension was seeded in a 96-well plate with cell density (20,000 cells per well), without the test agent. The cells were allowed to grow for about 24 hr. An appropriate concentration of the test agent (25% methanolic extract) was added, the plate was incubated for 24 hr at 37°C in a 5% CO, atmosphere, and the spent media was removed. MTT reagent was added to a final concentration of (0.5mg/mL) of total volume and the plates were incubated for 2-3 hr in dark conditions. 100 ul of DMSO was added after removal of the MIT reagent and shaken gently. The absorbance was measured at 570nm and 630nm using a spectrophotometer on an ELISA reader. The IC.o value was determined by using a linear regression equation. Formulae used for the study:

Determination of Anti Elastase activity

The cells were cultured at the density of 3×105 cells/2 ml and incubated in a CO, incubator for 24 hr at 37°C. 1 For the anti-elastase study, the cells treated with 250uM epigallocatechin gallate (EGCG) were used as a positive control, the cells treated with 200ug/ml of 25% of methanolic extract was used as test sample(Table 6), and the cells only with culture media were used as negative control and were incubated in 2ml of culture medium for 24 hr. The tubes were centrifuged for five minutes at 300x g at 25°C and the cells were washed with PBS. 0.5 ml of BD Cytofix/Cytoperm solution was added after removal of PBS and tubes were kept undisturbed for 10 min.0.5% of bovine serum albumin (BSA) in 'S[PO OU] USEM Ol pes sem op upon %I'O pue SEd 20 μL of Mouse Anti-Human Neutrophil Elastase/ELA2 Alexa Fluor® 647-conjugated monoclonal antibody was added, mixed and incubated for 30 min at room temperature under dark condition. The cells were treated with 0.1% sodium azide and 0.5 ml of PBS and analyzed by Flow Cytometry with the excitation and emission of 650nm and 668nm respectively. In this study, test compounds namely Green coffee bean extract (25% methanolic extract) and standard Epigallocatechin gallate (EGCG) were used to evaluate their effect on ELA-2 (conjugated mouse anti-human elastase antibody) expression in L929 cell lines.

Preparation of green coffee beans extract loaded liposomes

The thin film hydration method as described by Bangham et al.1° was used to formulate liposomes. The lipid mixture was prepared by dissolving phosphatidylcholine and cholesterol in chloroform and methanol (2:1) in the round bottom flask(RBF)and glass beads were added for homogeneous film formation. The RBF was attached to a rotary flash evaporator (Roteva, equation) and allowed to rotate at 80 rpm/min in a thermostated water bath at 40°C. The organic solvents were removed by slow application of vacuum leading to homogenous lipid film formation 100% on the walls of the flask. The film was allowed to dry for LH for the complete removal of organic solvents. The dried lipid film was hydrated with phosphate buffer (pH: 5.4) containing dissolved extract (25% of methanolic extract) and then the RBF was rotated at 150 rpm/min in a thermostated water bath maintained at 46°C that is above the transition temperature of lipid. Rotation was continued for 60 min until the homogeneous suspension was obtained. The liposomes produced using this method are large and heterogeneous in size, thus bath sonication method was used for 30 min to downsize the liposomes. The green coffee beans extract-based liposomal suspension was allowed to stand for 2 hr at room temperature to ensure complete hydration. The liposomal suspension was stored in an amber-colored glass bottle in the refrigerator until further use.

Characterization of liposomes

The following are the parameters for which the prepared liposomes were characterized

Determination of Encapsulation Efficiency UV-Vis spectrophotometer was used to estimate the encapsulation efficiency of chlorogenic acid in green coffee bean extract-loaded liposomes. The absorbance of chlorogenic acid remaining in the supernatant after centrifugation was measured at 324 nm using a UV-Vis spectrophotometer. micronized purified flavonoid fraction 1000 mg uses Then, the concentration was calculated from the calibration plot obtained for standard chlorogenic acid.

The total amount of drug added to Optical Microscopy

A drop of the liposomal suspension was placed on a clean glass slide and observed under the power of 45X and 100X of the optical microscope(Motic microscope). Determination of Vesicle size, Polydispersibility Index(PDI), and Zeta Potential

Vesicle size and Polydispersibility Index were analyzed by using a dynamic light scattering instrument with a computerized inspection system (Malvern particle size analyzer). Freshly prepared liposomal batches were diluted in the ratio of 1:100 with deionized water. All measurements were done in triplicate at 25±0.5°C. The liposomal suspension was diluted with the deionized water and zeta potential was determined using Malvern Zetasizer.

Transmission Electron Microscopy(TEM)TEM analysis was carried out for optimized drug-loaded suspension by transmission electron microscopy (Tecnai T20,200Kev, FEI). A drop of the suspension was placed with the aid of a micropipette on the grid. The grid was dried well. The dried grid containing the sample was bombarded with electrons accelerated at 200 VK. Then vesicle size and liposomal morphology were visualized by TEM under vacuum (Figure 1). Preparation of Liposomal Gel

Green coffee beans extract loaded liposomal gel was prepared by using a carbopol@974P gel base. Accurately 0.5% of carbopol@974P was weighed and kept for hydration in double distilled water containing preservatives overnight. Liposomal pellets obtained after centrifugation was dispersed in distilled water by sonication and added to hydrated carbopol solution with stirring. Gelling was induced by neutralization using triethanolamine (TEA). The gel was evaluated for color, texture (smoothness and greasiness), pH, viscosity, drug content, spreadability, and in to release drug study.

In vitro drug release study

The Franz diffusion cells apparatus having a receptor compartment capacity of 22 ml and surface area of 3.91 cm2 was used to estimate in vitro drug release dialysis membrane (molecular weight cut-off 12,000-14,000)which was pre-hydrated by soaking in buffer overnight were mounted on the cells. Phosphate buffer pH 5.4 (37°C±0.5°C) was used as the receptor fluid. The reservoir solution was stirred at 100 rpm/min using a magnetic bead. 0.5 g of gel was placed in the donor compartment on the membrane. Aliquots were collected at 1,2, 3, 4, 5,6,7,8,12, and 24 hr intervals and analyzed by UV-Vis spectrophotometer at 324nm.20

RESULTS AND DISCUSSION Extraction Methodology

The percentage yields of green coffee arabica L. beans extracts are presented in Table 3. The physical appearance of all the extracts was dark yellow and dark brown in color. The 25% of the methanolic extract showed the highest percentage yield at 26.54±0.13 compared to all other extracts.

Total Phenolic Content

The Folin-Ciocalteu method was used to estimate the total phenolic content of all the extracts. The TPC of all the extracts is expressed in terms of Gallic acid equivalent (The standard curve equation: y= 0.0031 +0.3477,r2=0.9953)mentioned in Table 1. Amongst all the extracts major phenolic content is estimated in 25% of methanolic extract. Spectral Overlay Quantification of Chlorogenic acid in each extract The amount of chlorogenic acid content present in each extract is mentioned in Table 3 and Figure 2. oteflavonoid As per the results, the highest amount of chlorogenic acid was found to be present in 25% of methanolic extract.

Based on the results obtained from total phenolic content, quantification of chlorogenic acid, and antioxidant assay 25% methanolic extract was found to contain the highest phenolic content, a greater quantity of chlorogenic acid, and maximal radical scavenging activity compared to other extracts.

Determination of cell viability by MTT Assay Viability assay is an assay that determines the ability of tissues, cells, or organs to maintain or recover the state of survival. Cytotoxicity study of the test compound, green coffee bean extract against L929 cell lines in statistical data of cell cytotoxicity study by ELISA reader suggested that green coffee bean extract showed moderate cytotoxicity potential properties with Inhibitory concentration （IC） at 306.38 μg/ml compared to standard drug Camptothecin with 23ug/ml Table 5. The observation strongly suggests that the green coffee bean extract do not have significant cytotoxicity potential with concentration ranging from 12.5-200 μg/ml respectively with an incubation period of 24 hrs at 37°C(Graph 1).

Determination of Anti Elastase activity

The enzyme elastase is capable of breaking down elastin, an insoluble elastic fibrous protein that together with collagen contributes to the mechanical strength of the connective tissue. Several studies have suggested that both skin aging and anti-wrinkle effects significantly correspond to reduced elastase activity. Thus antielastase studies were carried out to confirm the utility of green coffee bean extract as an anti-aging agent. In this study, test compound namely Green coffee bean extract and Std, EGCG was used to evaluate their effect on ELA-2 (conjugated mouse anti-human Elastase antibody) expression in the L929 cell line. The concentrations of the compounds used in the experiment were as follows:

The given test compound GC bean extract suppressed the ELA-2 expression in the L929 cell line. The relative mean fluorescence intensity values of ELA-2 were almost similarly decreased in std, Epigallocatechin gallate, and test compound green coffee bean extract. (Table 7; Figure 3 and Histogram 1). Hence, GC bean can be considered a good anti-elastase agent. Preparation of green coffee beans extract loaded liposomes

In this study thin film hydration method was used to formulate liposomes. puritans vitamin c The reason for selecting this

the method is as it is the simplest method for preparing multilamellar vesicles and is capable of loading a higher mass of hydrophilic molecules such as chlorogenic acid compared to unilamellar vesicles. The thin film hydration method was found to give 75% of encapsulation efficiency for chlorogenic acid present in the extract Table 2. Thus, this method was used for the preparation of the liposomes.

Characterization of liposomes Optical Microscopy

The multilamellar vesicle was clearly observed (Figure 4). Microscopic observations were used to evaluate the shape and size of the vesicles noted for all the prepared batches.

Determination of Polydispersibility Index (PDI), Zeta Potential, and Vesicle size

PDI basically appears for the size distribution of the vesicles in a given sample. sistanche The numerical value of PDI starts from 0.0 (perfectly uniform particle size)to 1.1 (highly polydisperse particle size).In the case of lipid-based carrier systems such as liposomes and nanoliposomes a size of 0.3 and below are acceptable as it indicates a homogeneous population of phospholipid vesicles.21 Zeta potential is a significant characterization technique that is employed to understand the physical stability of the particles. A value from -30 mV to +30mV is generally considered to have sufficient repulsive force to attain better physical colloidal stability.

The thin film hydration method gives large and heterogenous multilamellar vesicles. Size analysis of the produced liposomes revealed a z- average (d.nm)size of 864.2 nm, PDI of 0.375, and Zeta Potential of -12.4 with good entrapment efficiency.

Transmission Electron Microscopy(TEM)Morphological analysis of these liposomes by TEM showed spherical-shaped liposomes. Preparation of Liposomal Gel

Liposomal dispersion was simply mixed with the polymer to give corresponding gels. The liposomal gel formulation is advantageous as the fusion of the vesicles can effectively be avoided or minimized as the polymer serves as a spacer between the liposomes. Thereby, the vesicle size is not affected because it is a controlled polymer/liposome interaction process. The liposomal-based cream formulation was not chosen as stability issues are reported in emulsion-based products due to the method as it is the simplest method for preparing multilamellar vesicles and is capable of loading a higher mass of hydrophilic molecules such as chlorogenic acid compared to unilamellar vesicles. The thin film hydration method was found to give 75% of encapsulation efficiency for chlorogenic acid present in the extract Table 2. Thus, this method was used for the preparation of the liposomes.

Characterization of liposomes Optical Microscopy

The multilamellar vesicle was clearly observed (Figure 4). Microscopic observations were used to evaluate the shape and size of the vesicles noted for all the prepared batches.

Determination of Polydispersibility Index (PDI), Zeta Potential, and Vesicle size

PDI basically appears for the size distribution of the vesicles in a given sample. The numerical value of PDI starts from 0.0 (perfectly uniform particle size)to 1.1 (highly polydisperse particle size).In the case of lipid-based carrier systems such as liposomes and nanoliposomes a size of 0.3 and below is acceptable as it indicates a homogeneous population of phospholipid vesicles.21 Zeta potential is a significant characterization technique that is employed to understand the physical stability of the particles. A value from -30 mV to +30mV is generally considered to have sufficient repulsive force to attain better physical colloidal stability. 2

The thin film hydration method gives large and heterogenous multilamellar vesicles. Size analysis of the produced liposomes revealed a z- average (d.nm) size of 864.2 nm, PDI of 0.375, and Zeta Potential of -12.4 with good entrapment efficiency.

Transmission Electron Microscopy(TEM)Morphological analysis of these liposomes by TEM showed spherical-shaped liposomes.

Preparation of Liposomal Gel

Liposomal dispersion was simply mixed with the polymer to give corresponding gels. The liposomal gel formulation is advantageous as the fusion of the vesicles can effectively be avoided or minimized as the polymer serves as a spacer between the liposomes. Thereby, the vesicle size is not affected because it is a controlled polymer/liposome interaction process. The liposomal-based cream formulation was not chosen as stability issues are reported in emulsion-based products due to

the presence of surfactant and excess oil which may interact with vesicles. Various properties such as viscosity can easily be controlled by varying the concentration of the polymer or changing the polymer type. Thus liposomal gels were prepared as they circumvent the stability issue, provide controlled release, easy to prepare, and have aesthetic appeal as skin care cosmetics.23 The prepared lipogels were pale yellow in color and opaque. 0.5% w/w of carbopol@974P was found to have good consistency and smooth appearance devoid of any aggregation. The drug content for the liposomal gel was found to be 94.16±1.3 with a viscosity of gel in the range of 13,500 cps to 16,500 cps and spreadability around 7-8 g.cm/s.

In vitro drug release profile

The outermost layer of the skin is the stratum corneum and is mainly composed of protein keratin and lipids. The intercellular lipids play a vital role in controlling percutaneous absorption. The intercellular lipids may interact with the phospholipids present in the liposomes and thereby cause swelling of the lipids without altering the multiple bilayer structure of the stratum corneum. These swollen lipids cause the drug to accumulate and form an intracutaneous depot. Although the mechanism of topically applied liposomes is not fully understood, but lipid composition, surface charge, and liposomal lamellarity primarily play an important role in drug disposition. Studies have also revealed topical delivery is also influenced by the size of liposomes.24 The in vitro drug release was performed using a dialysis membrane. The in vitro =release study of the liposomal gel has shown a sustained release effect up to 12 hr compared to 7-8 hr of conventional gel Graph 2.

CONCLUSION

Green Coffee Arabica beans extracts were screened for total phenolic content, quantification of chlorogenic acid, and antioxidant assay. Based on the results obtained from these studies 25% of methanolic extract was chosen to be incorporated into liposomes as it had shown the highest phenolic content, a greater quantity of chlorogenic acid, and better antioxidant activity. Before incorporating the extract into the liposomes it was screened for MTT and antielastase assay. Based on the MT assay the extract had no cell cytotoxicity potential and as per antielastase assay, the extract had antielastase activity. Thus the extract can be utilized to formulate antiaging liposomes. The multilamellar vesicles were prepared using the thin film hydration method. The prepared liposomes had good encapsulation efficiency and better physical stability. The extract-based liposomal suspension was formulated into a liposomal gel. As per in vitro drug release study the liposomal gel has shown a prolonged release effect of up to 12 hr compared to the conventional gel. Hence green coffee bean extract-loaded liposomes can be considered to be an effective carrier for topical delivery with antiaging potential.

ACKNOWLEDGEMENT

The authors are thankful to Stellixir Biotechechnology, Bengaluru, and Karnataka for their help in completing the MTT assay and Anti-elastase assay studies, we also thank Sprint testing solutions, Mumbai for TEM imaging and our college SVKM's Dr.Bhanuben Nanavati College of Pharmacy for providing the best facilities to conduct this study.

CONFLICT OF INTEREST

The authors declare no conflict of interest.

ABBREVIATIONS:

TPC:Total phenolic content; DPPH:2,2-diphenyl -1-picrylhydrazyl; GAE: Gallic acid equivalent; GC:Green Coffee; GCB: Green coffee beans; ELA-2 (Conjugated mouse anti-human Elastase antibody):Neutrophil Elastase; EGCG: Epigallocatechin gallate;PC:Phosphatidylcholine;CH:Cholesterol;RBF:Round Bottom Flask; TEM: Transmission Electron Microscopy; MTT: 3-(4,5-diemthylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; PDI: Polydispersibility Index;IC":Half-Maximal Inhibitory Concentration.

This article is extracted from Desai and Mallya.: Development of Green Coffee Beans Extract loaded Anti-aging Liposomal Gel