POLYPHENOLS AS ADJUVANT IN TOSSING OUT ACNE VULGARIS (PUSTULES): A NOVEL FORMULATION STRATEGY

POLYPHENOLS AS ADJUVANT IN TOSSING OUT ACNE VULGARIS (PUSTULES): A NOVEL FORMULATION STRATEGY

S. K. Janani, Raman Suresh Kumar ^*, Sai Surya Nikitha Upadhyayula and Chenmala Karthika

Department of Pharmaceutics, JSS College of Pharmacy, JSS Academy of Higher Education and Research, Ooty, Nilgiris - 643001, Tamil Nadu, India.

ABSTRACT: Acne vulgaris is a skin condition in which people lose their self-esteem and ultimately lead to depression. It is more common among the adolescent age groups. According to GBD (Global burden of disease) study, about 85% of the populations are being affected. Various medications are available for the treatment, but, its uses are limited due to side effects like skin irritation. In our study, we have formulated a novel combination nanoemulsion gel containing adapalene and resveratrol for the treatment of pustules, assuming that it won’t produce much skin irritation and also expecting a synergistic activity. The spontaneous emulsification method was employed for the preparation of nanoemulsion, and it was incorporated into carbopol 934 gel. Further, various evaluation tests, including in-vitro and in-vivo studies, were performed. Cumulative percentage drug release was more for marketed formulation as compared to prepared formulation, whereas, the prepared formulation had a better efficacy as compared to marketed formulation. Thus, we can say that the formulation has exhibited its synergistic activity on combining with a polyphenol. Additionally, as an outcome of the skin irritation study, the formulation did not produce any skin irritation. Hence, we can conclude that the novel formulation containing adapalene and resveratrol served as a better candidate for the management of acne (pustules).

Acne vulgaris, Flavonoid, nanoemulsion gel, Adapalene, Synergistic activity

INTRODUCTION: Acne vulgaris is a common skin condition that affects most people with ages ranging from 12-24 ¹. Basically, acne can be categorized into blackheads, whiteheads, papules, pustules and nodules. Mechanisms involved in the evolution of acne are hyper-proliferation of follicular epithelium, excess sebum production from sebaceous gland, Propionibacterium acne (bacteria), and finally inflammatory mediators like interleukins, tumor necrosis factor, fatty acids, and other bacteria-derived mediators.

Though various treatment methods are available, the most preferred one is topical medications because of the ease of administration and their direct action on the affected area, and they can be chosen either as a monotherapy or a combination therapy. The main aim of our study is to formulate a combination therapy using a polyphenol as an adjuvant. Adapalene is used as the choice of drug.

The reason for choosing adapalene is that it is a retinoid derivative, used as the first-line therapy, and moreover, compared to other retinoid derivatives like tretinoin and isotretinoin, adapalene has less skin irritation. Resveratrol is used as an adjuvant, assuming that it will produce a synergistic effect with the drug. It is produced naturally by many plants, and it is present in the skin of red grapes, blueberries, raspberries, and mulberries ².

It has been reported that resveratrol acts against the bacteria (p.acne) causing acne ³. Because of these reasons, we have opted resveratrol as an adjuvant. Apart from adapalene and resveratrol, an additional ingredient used in the formulation can act against acne (i.e.) tea tree oil, obtained from Melaleuca alternifolia. Several studies have proved that tea tree oil can act against acne. It is the terpinen-4-ol and α-terpineol found in the oil, which is responsible for action ⁴.

We have selected nanoemulsion gel for the topical application. The reason for choosing nano-technology is that it can easily penetrate into the skin by crossing the stratum corneum, and it can produce therapeutic action. In this work, we have induced pustules (a moderate form of acne) and compared the efficacy of the prepared formulation with that of the marketed formulation (Adapalene + benzoyl peroxide).

MATERIALS AND METHODS:

Materials: Adapalene was obtained as a gift sample from Arti industries. The marketed formulation was obtained as a gift sample from the apex, Chennai, India. Capryol 90 was obtained from Gattefose India Pvt. Ltd., Mumbai. Isopropyl myristate 98% was obtained from Avra.

Methods:

Preformulation Studies: ⁵

• Compatibility Studies:
• DSC (Differential Scanning Calorimetry): ^{2, 6} To check the purity of the drug and compatibility between the drug and excipients, DSC were carried out.
• FTIR (Fourier Transformer Infrared Spectroscopy): ^{7, 8} Again, the compatibility between the drug and excipients was determined using FTIR.
• Screening of Oil, Surfactant and Co-Surfactant:
• Selection of Oil Based on the Solubility: ⁹ Solubility of drug and polyphenol was checked in different oils by shake flask method. Adapalene and resveratrol were added to different oils in a volumetric flask and kept at 37 ºC in an orbitrary shaker for 24 h to determine the solubility.
• Selection of Surfactant and Co-surfactant: Kolliphor and ethanol was selected as surfactant and co-surfactant for the preparation of nano-emulsion.
• Formulation of Nanoemulsion: ^{10, 11} Spontaneous emulsification methods were employed. We have prepared 2 formulations, namely F1 (Adapalene + 5 mg resveratrol) and F2 (Adapalene +10 mg resveratrol) to check the synergistic activity of the flavonoid.

Initially, we have dissolved adapalene and resveratrol in tea tree oil and capryol 90, respectively. After dissolving, we mixed both the oils (tea tree oil and capryol 90) as well as smix together. Later the samples were titrated with water to get a translucent to transparent liquid. Further, it was subjected to thermodynamic stability studies. Further, the stable ones have incorporated into carbopol 934 gels.

• Evaluation of Nanoemulsion:

• Thermodynamic Stability Studies: ^{12, 13} Samples were subjected to thermodynamic stability studies, including heating and cooling cycle, centrifugation, and freeze-thaw cycle. Then, the stable ones were taken further.

Characterization of Nanoemulsion: Particle size, PDI (polydispersity index), and zeta potential ¹⁴ using zeta sizer (Malvern Instruments) was determined.

Preparation of Gel and Incorporation of Nanoemulsion: ¹⁵ Carbopol 934 at different concentrations including 0.4%, 0.6%, and 0.8% was dissolved in a suitable quantity of water. 0.8% concentration of carbopol was selected since it formed a clear gel. Then, the nanoemulsion was incorporated into a gel, and it was further neutralized with triethanolamine to maintain the pH between 6.8-7.4.

Characterization of Adapalene Loaded Nano-emulsion Gel:

Determination of pH: pH of the prepared formulation was checked with the help of a digital pH meter ^{16, 17}.

Viscosity Determination: ¹⁸ Viscosity of the formulation was checked without any dilution.

It was determined by using Brookfield DV-II programmable viscometer. Spindle no 28 was allowed to rotate at 40 rpm at 19 ºC for 10 min. The initial and final values were considered.

Spreadability of the Formulation: Simple hand spreading technique was employed to determine the ease of administration.

Determination of Extrudability: ¹⁹ Formulation was filled into the lacquered aluminum collapsible tube, and weight was applied to it. The extruded quantity was weighed.

Ex-vivo Studies: ²⁰ Franz diffusion cell was used for this study. Phosphate buffer 7.4 was used as the media. The formulations were kept on the donor compartment, and later 5 ml of samples were withdrawn from the receptor compartment at different time intervals like 1, 2, 3, 4, 5, 6, 7, and 8 h and replaced with fresh buffer solution. All the samples were analyzed using UV by simultaneous determination method. Finally, the cumulative percentage of drug release was determined ²¹.

In-vivo Study: Rabbits weighing about 2.5-4 kg of either gender were taken. All the animals were kept in the laboratory 10 days prior to the initiation of the experiment in order to get adapted to the environmental surroundings by getting permission from the Institutional animal ethics committee (IAEC) (Registration no: 118/PO/ReBi/S/1999/ CPCSEA), JSS College of Pharmacy, Udhagamandalam, Tamilnadu, India.

Skin Irritation Study: ^{22, 23} Skin irritation study was carried out according to “Acute Dermal Irritation/Corrosion test” as given in the OECD (404) GUIDELINE. First patch of 2 formulations (F1 and F2) and blank formulation (without drug) was applied on the skin and removed after 3min. In the same manner, it was applied for 1 h and 4 h. Since there was no skin irritation, it was observed again for 24 h, 48 h, and 72 h. And it was also kept till 14^th day for the reversibility of the effect.

Induction of Pustules: ²⁴ Hairs on the back were shaved (30 cm²- 40 cm² area). Marking was carried out in such a way that in one area SLS (sodium lauryl sulphate) and in another area HgCl₂ (Mercuric chloride) was used for induction. HgCl₂ (10%, 15%) and SLS (5%) was used for the induction of pustules by dispersing in petrolatum. After the application of chemicals, the animals were kept for 24 h. After 24 h, the pustules got induced to those animals which was exposed to HgCl₂. SLS didn’t produce any results. Thus, HgCl₂ was considered for the induction of pustules.

Treatment of Pustules Using Marketed and Prepared Formulation: The formulations were applied once daily and observed for efficacy. Group 1 was treated with prepared formulation (F1), group 2 was treated with 2^nd prepared formulation (F2), and group 3 and 4 was treated with the marketed formulation.

RESULTS AND DISCUSSION:

Preformulation Study:

(a) Compatibility Studies:

DSC (Differential Scanning Calorimetry): Endothermic peaks were obtained for the drug as well as for the physical mixture. Since the peaks were remaining the same in all the thermograms, it indicates that there is no interaction between the drug and the excipients.

Thus, it can be concluded that drugs and excipients are compatible. DSC for adapalene, resveratrol and carbopol 934 was found to be 327.59 ºC, 268.31 ºC, and 95 ºC, respectively. While coming to the physical mixture, there was a slight peak shift for all three peaks. Thus, again the compatibility was determined using FTIR.

FTIR: There was no disappearance or appearance of additional peaks. Shifting was also not present. Thus, it indicates that the drug and excipients are compatible and doesn’t produce any interactions. FTIR data are given in Table 1.

TABLE 1: FTIR DATA

Solubility Studies: Solubility data is given in Table 2. Based on the results, tea tree oil and capryol 90 were selected as the oil phase. The solubility of adapalene was more in tea tree oil, probably due to the affinity of the drug towards the oil whereas, the solubility of resveratrol was only 10 mg/ml in tea tree oil.

Hence, it was a compulsion to opt for the other oils. 45 mg of the resveratrol solubility was achieved with 1ml of Capryol 90. The reason could be the ester group of capryol 90, which would have influenced the solubility. Therefore, tea tree oil and capryol 90 were taken as the oil phase.

TABLE 2: SOLUBILITY STUDIES

SD:  Standard deviation

Screening of Surfactant and Co-surfactant: In the present study, different surfactants and co-surfactant were tested in order to check the formation of stable nanoemulsion. But, most of them were unstable and divided into two layers. The combination of kolliphor and ethanol was selected for the formulation, and different ratios were prepared in order to pick up the stable one for the final formulation. Since, the nanoemulsion is obtained by spontaneous emulsification method the addition of water makes the difference and leads to the formation of either emulsion or nanoemulsion.

Characterization of Nanoemulsion: Out of 8 samples, only 6 samples were finally selected whereas, the other 2 samples were not stable due to phase separation. Further, the following parameters were determined.

Particle Size and PDI (Poly Dispersity Index): Particle size and PDI was expected to be <100 nm and <1. Selected sample was having low particle size of 39.99 nm. They also exhibited broad size distribution.

In addition, PDI value of 0.234 was also within the range as compared to that of other samples. The maximum percentage intensity of the particle in this formulation was found to be 25%.

Zeta Potential: The normal value of zeta potential is between -30 mV and +30 mV.

For the prepared formulation, it was found to be -0.665 mV. Thus, it is a stable formulation.

Selection of Gelling Agent: Compared to 0.4% and 0.6%, 0.8% concentration was not having a free-flowing capacity until and unless external stress was applied, and it also had good viscosity. Thus, 0.8% gel was prepared, and nanoemulsion was incorporated.

Characterization of Nanoemulgel:

Determination of pH: Formulation pH was found to be 6.4-6.5, which was equivalent to skin pH. Hence, it can be said that the formulation is compatible and may not produce skin irritation.

Viscosity Determination: The consistency of the formulations F1 and F2 were 79350 cp and 68650 cp, respectively, which are normally considered as good for gels.

Spreadability: Ease of application was taken into consideration.

Extrudability:

Ex-vivo Drug Release Study: Simultaneous determination method was employed. Release of drug from the prepared formulation as well as the marketed formulation is given in Fig. 1-3. The release of adapalene, resveratrol, and benzoyl peroxide was determined at 282 nm, 306 nm, and 236 nm, respectively. From the graph, it was clear that even though the prepared formulation was a nanoemulsion, the marketed formulation was having more drug release, which may be due to a higher dose of drug (5 mg) in marketed formulation as compared to that of the prepared formulation (1 mg).

TABLE 3: REPRESENTS THE EXTRUDABILITY OF THE FORMULATION WITH INCREASING WEIGHT

FIG. 1: CUMULATIVE DRUG AND FLAVONOID RELEASE FROM F1 FORMULATION

In-vivo studies:

Skin Irritation Study: There was no skin irritation, redness, or erythema observed during this study.

Hence, we can say that the formulation is safe for topical application. Table 4 represents the visual observation of the skin irritation study.

Treatment of Pustules and Comparison of the Efficacy: Group 1 and 2, which were treated with prepared formulation, showed better efficacy as compared to that of the marketed formulation.

Table 5 represents the animals before and after the treatment. The mechanism of action of the prepared formulation may be as given in Fig. 4 since the prepared formulation is a nanoemulsion and it had a capacity to penetrate easily into the stratum corneum as compared to the marketed formulation, thus producing a better efficacy.

FIG. 4: MECHANISM OF ACTION OF THE PREPARED FORMULATION ON THE ACNE

In addition, tea tree oil would have also exhibited its action. Moreover, resveratrol would have shown its synergistic activity by acting as an adjuvant.

From Table 5, it is clearly visible that the prepared formulation had more efficacies as compared to that of the marketed formulation.

CONCLUSION: Pustules are one of the moderate to severe forms of acne, in which the top layer of the skin will be covered with pus. The main aim of our study was to decrease the treatment time that is taken for the management of acne vulgaris. In addition, to decrease the scars as well as the skin irritation caused by other retinoid derivatives. Moreover, the addition of resveratrol has also been served as an adjuvant and helped in decreasing the treatment time. Since we have implemented nanotechnology in this work, the formulation can easily cross through the startum corneum and act on the affected area. In addition to these factors, we have also used tea tree oil in our formulation. Tea tree oil possesses different activities like anti-bacterial and anti-oxidant etc. And it has also been widely used for the treatment of acne. In our study, tea tree oil would have also exhibited its action on pustules. Thus, the preparation of nanoemulsion gel containing adapalene and resveratrol was useful in the treatment of pustules, and moreover because of this synergistic activity, we can also reduce the dose of the drug (adapalene). Even the treatment time for the prepared formulation was less as compared to that of the marketed formulation. The treatment time taken by marketed formulation was 19-26 days, whereas prepared formulation took only 11-12 days for the treatment.  Hence, it can be said that the prepared formulation had improved efficacy in the treatment of pustules. Therefore, it can be concluded that the novel combination of adapalene and resveratrol would serve as a better combination therapy in the management of acne vulgaris (pustules).

ACKNOWLEDGEMENT: The author would like to thank “Sir Dorabgi Tata Trust” for providing financial assistance for carrying out this research work.

CONFLICTS OF INTEREST: Authors declare that there no conflicts of interest.

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    How to cite this article:

    Janani SK, Kumar RS, Upadhyayula SSN and Karthika C: Polyphenols as adjuvant in tossing out acne vulgaris (pustules): a novel formulation strategy. Int J Pharm Sci & Res 2021; 12(2): 845-52. doi: 10.13040/IJPSR.0975-8232.12(2).845-52.

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