Development and evaluation of topical microemulsion of dibenzoylmethane for treatment of UV induced photoaging

Accepted Manuscript Development and evaluation of topical microemulsion of dibenzoylmethane for treatment of UV induced photoaging Ashana Puri, Amanpreet Kaur, Kaisar Raza, Shishu Goindi, Om Prakash Katare PII:

S1773-2247(16)30235-0

DOI:

10.1016/j.jddst.2016.09.010

Reference:

JDDST 248

To appear in:

Journal of Drug Delivery Science and Technology

Received Date: 9 June 2016 Revised Date:

1 September 2016

Accepted Date: 29 September 2016

Please cite this article as: A. Puri, A. Kaur, K. Raza, S. Goindi, O.P. Katare, Development and evaluation of topical microemulsion of dibenzoylmethane for treatment of UV induced photoaging, Journal of Drug Delivery Science and Technology (2016), doi: 10.1016/j.jddst.2016.09.010. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Development and evaluation of topical microemulsion of dibenzoylmethane for

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treatment of UV induced photoaging

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Ashana Puri1#, Amanpreet Kaur1#, Kaisar Raza2, Shishu Goindi1, Om Prakash Katare1*

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Affiliations: 1

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University Institute of Pharmaceutical Sciences, Panjab University, Chandigarh, India. 2

School of Chemical Sciences and Pharmacy, Central University of Rajasthan, Ajmer, Rajasthan, India

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# First and second author contributed equally in this manuscript

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*Corresponding Author:

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Prof. Om Prakash Katare

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Professor (Pharmaceutics),

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University Institute of Pharmaceutical Sciences,

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Panjab University, Chandigarh–160014, INDIA

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Telephone:, 8054240830; 0172–2534281 (O)

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E–Mail: [email protected]

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ABSTRACT

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Dibenzoylmethane (DBM), a phytochemical agent, occurring as a minor constituent in the

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root extracts of liquorice has a potential for anti-photoaging effects. It acts as a UVA absorber

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that filters out and prevents the penetration of UV radiations into various cell components. In

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this study, DBM loaded o/w microemulsion (ME) was developed and evaluated for anti-

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photoaging effect in mice. The ME was evaluated for various physicochemical

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characteristics, stability, ex-vivo skin permeation studies and in vivo evaluation in mice model

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of UV-radiation induced photodamage. The mean globule size of DBM loaded ME was

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found to be 35.550 ± 4.879 nm. The mean cumulative amount permeated/area and skin

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retention of DBM in 24h from the ME was 6.81 and 5.16 folds higher, respectively as

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compared to the conventional cream of DBM. In vivo anti-photoaging effect on mice skin

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was evaluated in terms of visual scoring, pinch test, biochemical estimations and

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histopathological studies. Results clearly demonstrated the promising efficacy of ME

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formulation in preventing wrinkles, lesions and other macroscopic and microscopic changes

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associated with chronic UV exposure. Hence, it can be concluded that DBM ME can be used

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effectively used as sunscreen agent to protect against the damaging effect of UV rays.

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Keywords: o/w microemulsion, dibenzoylmethane, pinch test, photoaging, histopathology

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1. INTRODUCTION

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Photoaging is defined as the gross and microscopic changes due to persistent sun exposure

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[1]. It is the superposition of photodamage on the aging process and is characterized by an

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exaggeration of changes associated with chronological aging as well as qualitatively different

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changes induced by sun exposure [2]. Face, neck, ears, and dorsal aspects of the hands,

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exterior surface of forearms and the lower legs are the majorly affected areas. Clinical

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features of photodamaged skin include mottled hyperpigmentation, heliodermatitis and

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hyperplasia of elastic fibres, dryness, purpura, comedones, atrophy, telangiectasia and a

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variety of skin neoplasms (sebaceous hyperplasia, seborrheic keratosis, and actinic keratosis)

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[3-5]. The intrinsic factors like the slow and irreversible tissue degeneration which is

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inevitable and the extrinsic factors like the direct result of exposure to external elements of

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the environment, most harmful being the ultraviolet (UV) radiations simultaneously

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contribute to skin ageing[6-8].

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UVB (280-320 nm) causes intense erythema and DNA damage due to pyrimidine dimer

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formation, whereas UVA (320-400 nm) is associated with tanning and photoaging [9-11].

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UVA generates reactive oxygen species which indirectly damage DNA [12, 13]. UVA

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exposure also results in an increase in dermal inflammatory cells and decrease in human

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epidermal antigen presenting cell activity and Langerhans cell numbers [14, 15]. This UV-

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induced immunosuppression indirectly leads to the development of photocarcinogenesis and

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non-melanoma skin cancer [16, 17]. DBM is a natural phytochemical found as a minor

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constituent in the root extracts of Liquorice (Glycyrrhiza glabra in the family Leguminosae)

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[18-20]. It is a structural analogue of curcumin (diferuloylmethane) which is important

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chemical constituent of turmeric (Curcuma longa), being used as spice and coloring agent in

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food preparations. DBM has been reported to be used as a sunscreen agent to prevent the

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harmful effects of UV rays. It acts as a UVA absorber that filters out and prevents the

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penetration of the UV radiations to the vital cell components and blocks the over production

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of oxygen derived free radicals [21, 22].

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In general, majority of sunscreen products available in the market are emulsion or cream

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based. However, the nano-scaled formulations as carriers for the sunscreen agents are

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advantageous so far due to better skin retention. Moreover, UV rays penetration is reduced by

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absorption and scattering phenomena [23].

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Microemulsions (MEs) are defined as optically isotropic and thermodynamically stable

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systems of water, oil and an amphiphile and is usually with a droplet diameter in submicron

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range [24]. ME based formulations improves the solubilization of drugs, provides appreciable

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thermodynamic stability, high drug loading with low skin irritation and are easy to

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formulate[25]. MEs disperse the drug into fine oil droplets to enhance the solubility of poorly

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water-soluble drugs. These MEs can be used to deliver drugs via several routes. The topical

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route has find widespread research focus these days. DBM possesses favourable molecular

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weight (224.25 Da) and lipophilicity (log P = 3.1) for good permeation however the in vivo

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efficacy may be compromised because of poor aqueous solubility.

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Therefore, with an aim to enhance the solubility and eventually the dermal efficacy of DBM,

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ME based colloidal carrier system was designed to increase its penetration and permeability.

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It was then characterised and evaluated for in vivo anti-photoaging effect against UV induced

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photodamage.

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2. MATERIALS AND METHODS

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2.1 Chemicals

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DBM was obtained from Sigma Aldrich Co. St. Louis, USA. Captex 300 was received as a

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gift sample from Abitec Corporation, Columbus, US. Nitroblue tetrazolium and 5-5’-

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dithiobis-2-nitrobenzoic acid were obtained from Hi Media Laboratories Pvt. Ltd. Mumbai,

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India. Ultra Vitalux 300W ES (Osram, Germany), simulating the full solar spectrum (260–

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400 nm) was used as the UV source for anti photoaging evaluation in animals. All other

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chemicals and reagents were of analytical grade.

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2.2 Animals

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Female Laca mice, 6-8 weeks old and weighing 20-25 g were obtained from Central Animal

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House, Panjab University, Chandigarh, India. All the animals were housed in polypropylene

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cages at ambient temperature, with a 12 h night/day cycle, supplied with a standard pellet diet

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and water ad libitum. The animals were acclimatized for two weeks before initiating the

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experiment. Ethical approval to perform the animal protocols was obtained from Institutional

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Animal Ethics Committee, Panjab University, Chandigarh, India (IAEC/282 dated

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30.08.2012).

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2.3 Solubility studies

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Solubility of DBM in different oils (oleic acid, Captex 300, Captex 200, Captex 355 and

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castor oil) and surfactants (Tween 20, Tween 40, Tween 60 and Tween 80) was determined.

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An excess of drug was added to 2mL of each solvent taken in a screw-capped vials and kept

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at 25±2°C in a thermostat water bath shaker for 72 h. After 72 h, solutions were centrifuged

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at 12,000 rpm for 10 min [26]. The supernatant were then filtered through a membrane filter

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(0.45µm Nylon, Millipore Millex-GN) and analysed spectrophotometrically at 344nm after

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appropriate dilution with ethanol.

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2.4 Construction of pseudo-ternary phase diagrams

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Pseudo-ternary phase diagram were constructed using Captex 300 (oil), Tween 80

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(surfactant) and n-butanol (co-surfactant) using titration method to find out the range of ME

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existence region [27, 28]. Phase diagrams were prepared by titration of varied concentrations

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of oil, surfactant and co-surfactant with water. Tween 80/n-butanol was used as surfactant

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mixture (Smix) in weight ratios of 2:1. At specific surfactant/co-surfactant weight ratio, the

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ratios of oil to the Smix were varied as 1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2, 9:1. While stirring

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moderately, water was added slowly to these mixtures. After being equilibrated, the mixtures

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were assessed visually and determined as being MEs, microgel, crude emulsions or emulgel

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[29].

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2.5 Preparation of o/w MEs

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DBM (0.2%) loaded MEs with varied composition were prepared after identification of o/w

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ME region from the phase diagram (Table 1). DBM was dissolved in Captex 300 in a beaker,

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followed by addition of premix of Tween 80, n-butanol and menthol. Then, water was added

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drop wise into the above solution under constant stirring to form clear o/w ME

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spontaneously. Similarly, blank MEs were also prepared.

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Also, Conventional cream (comprising of 15% liquid paraffin, 6% sorbitan monooleate, 3%

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white bees wax, 36% white soft paraffin, and 39.8% distilled water) and aqueous suspension

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(0.5% w/w carboxymethylcellulose dispersion) containing DBM (0.2%) were also prepared

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for conducting comparative ex vivo permeation studies.

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2.6 Characterisation of the DBM loaded MEs

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2.6.1 Micromeritics and zeta potential

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Malvern’s ZetasizerTM (Malvern Instruments Ltd. UK) was used to determine the globule

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size, polydispersity index and zeta potential of selected ME. Sample (5mL) was placed in

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the cuvette and instrument recorded the intensity of fluctuation of laser beam, correlated with

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the particle size of ME droplet. Zeta potential was measured at 25°C and the electric field

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strength was around 23.2 V/cm[30].

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2.6.2 Morphology and structure

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The analysis by Transmission electron microscopy (H-7500, Hitachi, Japan) was performed

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after depositing the selected MEs on a film-coated 200-mesh gold specimen grid. Photo-

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micrographs at suitable magnifications were obtained using negative staining with 1%

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phosphotungstic acid under an electron microscope [31].

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2.6.3 Drug content and pH determination

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The percent drug content of selected ME was determined by diluting it 100 times with

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ethanol and analysing spectrophotometrically at λmax 344nm. The pH of ME was determined

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at room temperature with a glass electrode pH meter.

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2.6.4 Rheological behaviour and viscosity measurements

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The viscosity of the selected ME was measured at different angular velocities at a

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temperature of 32.0 ± 0.1ºC with a rotating-spindle Brookfield DV-II+ pro viscometer (Paar

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Physica MC1, Brookfield DV-II, UK) using spindle number 21. Shear stress was measured

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by changing the shear rate (0-100 s-1) [32].

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2.7 Thermodynamic stability studies

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Thermodynamic stability was assessed by the three step procedure as reported by Shafiq et al

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with slight modification [33].

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2.7.1 Heating cooling cycle

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Six cycles between refrigerator temperature (4°C) and 45°C with storage at each temperature

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for 48 h were studied.

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2.7.2 Centrifugation

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The formulations passed in previous step were centrifuged at 3500 rpm for 30 min.

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2.7.3 Freeze- thaw cycle.

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The formulations were subjected to freeze-thaw cycles between -21°C and 25 °C for NLT 48

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h.

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2.7.4 ME stability studies

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The developed MEs were subjected to different storage conditions i.e. 4ºC, 25ºC and 40ºC for

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3 months and periodically examined for any physical change (clarity, phase separation,

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precipitation of drug, colour change, pH) and drug content.

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2.8 Ex–vivo drug permeation and skin retention studies

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The studies were performed in triplicate using preshaved excised dorsal skin of female Laca

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mice employing vertical Franz diffusion cell assembly (PermeGear, Inc. PA, USA) with

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slight modifications. Phosphate buffer saline (PBS) pH 7.4 containing 30% isopropyl alcohol

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(v/v) and 3% Tween 80 (w/v) was used as receptor media and the cell contents were

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maintained at temperature of 32±1ºC. The ME formulations (ME 3; ME 6; ME 7), aqueous

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suspension (C1) and conventional cream (C2) containing equivalent amount (200µg) of DBM

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were applied uniformly to the skin in the donor compartment. The samples (2mL) were

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periodically withdrawn at suitable time intervals and were analyzed by a validated

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spectrophotometric method at λmax 346 nm.

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After 24 h, the skin surface in the donor compartment was rinsed with ethanol to remove

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excess drug from the surface. The receptor medium was then replaced with 50% (v/v) ethanol

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to extract drug deposited in the skin, to determine the amount of skin retention within the skin

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[34].

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The cumulative amount permeated/ area, permeation flux (µg/h/cm2) and skin retention

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(µg/cm2) were calculated. The data was statistically analyzed by one way analysis of variance

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(ANOVA) followed by Tukey’s method. Results were quoted as significant at p<0.05.

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2.9 Skin sensitivity and histopathological studies

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The electric clipper was used to remove the hair on the dorsal side of mice. Test formulations

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(0.5g) were applied on the dorsal region by uniform spreading within the area of 4 cm2.

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Observations were made for any visible changes after 4 h application of formulations for any

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signs of erythema and compared to the control untreated group [35]. The mean erythemal

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scores were recorded (ranging from 0 to 4) depending on the extent of erythema. The animals

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were then sacrificed to expose the dorsal surface. Each specimen was fixed in 10% buffered

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formalin, and then it was embedded in paraffin followed by microtomy. The staining of the

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sections was done with hematoxylin and eosin. Then the specimens were observed under a

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high power light microscope.

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2.10 Anti–Photoaging studies

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Animals were grouped into six groups; G1 (naïve control); G2 (Sham control); G3 (positive

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control); G4 and G5 treated with DBM loaded o/w ME and conventional cream respectively.

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2.10.1 UV exposure

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An area of 2 × 2 cm2 was marked on the dorsal surface of all the mice except for G1, and

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made hair free using a mild depilatory. The mice were observed for 48 h to exclude mice

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showing any reaction to the depilatory. The trauma of the shaving blade can lead to free

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radical production, hence a depilatory was preferred.

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An in–house built UV simulator was used in the study [36]. To restrict the movement of

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animals during exposure and to ensure that the radiation is homogenous, mice were

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anaesthetized using ether. Application of a dose of 167 J/cm2 (obtained by exposure for

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approximately 1.25 s from a distance of approximately 35 cm) produced a minimally

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perceptible erythema; this dose was taken as minimally erythemogenic dose (MED). UV

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exposure in the protocol was 835 J/cm2 which was 4.17 times MED and this was established

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earlier in the laboratory. At a dose of 1.5 × 105 J/cm2 (900 s), fiery red erythema with edema

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and blistering was observed, and this was denoted as the “phototoxic dose”. The distance

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from the lamp to the animals back was kept constant at 35 cm and the animals were exposed

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for 5 s, 5 times a week for 6 weeks [2].

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The animals were observed periodically for visual/morphological changes and

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subsequently these were sacrificed by cervical dislocation at the end of the study. Afterwards

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their skin was used for histological studies, protein estimation, catalase level and lipid

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peroxidation assay.

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2.10.2 Visual score

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The skin of treated mice was examined for any photodamage at 6th week. Under anaesthesia,

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the UV exposed dorsal skin of each mouse was photographed. The grade of photodamage

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was determined using a grading scale ranging from 0 for normal skin to 5 for severely

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photodamaged skin (0: no wrinkles or laxity; 1: fine striations; 2: shallow wrinkles; 3: a few

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deep wrinkles and laxity; 4: increased deep wrinkles; 5: severe wrinkles).

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2.10.3 Pinch test

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Pinch test was performed on the dorsal region of same group of animals to evaluate the

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degree of recovery after forced stretching of skin [37]. Briefly, the dorsal skin at the midline

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of mice was picked up with the thumb and fore–finger (without lifting the animal into air) for

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1 s, and it was released subsequently. The time taken until the skin recovered to the original

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state was measured.

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2.11 Biochemical estimations

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The dorsal skin of mice (all groups) was incised and a 10% w/v homogenate was prepared

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using 1.15% w/v potassium chloride in an ice bath to prevent free radical generation. The

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homogenate was centrifuged at 4000 g for 10 min and supernatants were separated for

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various estimations.

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2.11.1 Protein estimation (Biuret test)

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Accurately 0.1 mL of skin homogenate, 2.9 mL of sodium chloride (0.9% w/v) and 3 mL of

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Biuret reagent [38] were mixed together and equilibrated for 10 min at room temperature.

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The absorbance of the contents was measured at 540 nm using blank (prepared without skin

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homogenate).

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2.11.2 Catalase activity

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Although numerous procedures have been reported for measuring catalase activity; the

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method reported by Thiele, especially designed for skin was adopted. 50 µL of the

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supernatant of tissue homogenate (10%, w/v) was mixed with 3 mL of H2O2 (30 mM/L) in 50

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mM phosphate buffer (freshly prepared) in a cuvette. The change in the absorbance was

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measured at 240 nm for 2 min at an interval of 30 s [39]. The calculations were performed

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using the following equation:

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 ( / ) =

2.3   ∆ 

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Where, A1 and A2 are the initial and the final absorbance and when the time interval ∆t is 1

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min. The results were expressed as µmole H2O2 decomposed/mg of protein/min.

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2.11.3 Lipid peroxidation

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Malondialdehyde (MDA), is an indirect index of lipid peroxidation. It was assayed as

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thiobarbituric acid reacting substances (TBARS) [40]. To 0.2 mL of homogenate was added

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0.2 mL of 8.1% sodium lauryl sulphate, 1.5 mL of 20% acetic acid solution (pH adjusted to

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3.5 with 1 N sodium hydroxide) followed by addition of 1.5 mL of 0.8% freshly prepared

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thiobarbituric acid. The volume of solution was made to 4.0 mL with TDW. Subsequently the

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samples were incubated at 95ºC for 1 h, followed by cooling to room temperature and

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addition of 1.0 mL of TDW and 4.0 mL of n–butanol and pyridine (15:1). The contents were

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mixed using cylomixer, followed by centrifugation at 3500-4000 rpm for 10 min; the organic

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layer was separated and analyzed at 532 nm. The levels of lipid peroxides were expressed as

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nanomoles MDA/mg protein using the following equation:

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 ⁄ %!  # #ℎ%#  =  ' Where,  %! of malondialdehyde=1.56 x105/min/cm

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2.11.4 Superoxide dismutase (SOD) activity

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SOD activity was determined by the inhibition of nicotinamide adenine dinucleotide reduced

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nitro–blue tetrazolium (NBT) reaction system [41]. Briefly, for total SOD activity, to 100 µL

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of tissue homogenate, 2 mL of freshly prepared NBT (96 mM) and 0.5 mL of hydroxylamine

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hydrochloride (20 mM) was added and change in absorbance was measured at 560 nm for 2

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min at 30 s intervals.

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2.11.5 Glutathione (reduced) estimation

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Glutathione (reduced) was estimated by previous reported method. The sulfosalicylic acid

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(4%; 1 mL) was added to skin homogenate (1mL) and the samples were incubated at 4ºC for

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at least 1 h and then subjected to centrifugation at 1200g for 15min at 4ºC. To 0.1mL of

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supernatant, 2.7mL of phosphate buffer (0.1M), and 0.2mL of 5-5’-dithiobis-2-nitrobenzoic

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acid (0.01M) was added. The absorbance was read immediately at 412nm on a

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spectrophotometer.

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The GSH concentration was calculated as nmol/g tissue. Calculations were done using the

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formula: ()* =

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(3 ∗   ∗ 0.25) (13.6 ∗      )

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3.1 Solubility studies

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Solubility data of DBM in various oils and surfactants is depicted by Table 1. It was found to

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be freely soluble in Captex 200, Captex 300, Captex 355, and oleic acid and soluble in castor

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oil. The DBM demonstrated highest solubility in Captex 300 (217.72 ± 3.99 mg/mL), it was

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selected as the oil phase for the ME formulation. DBM was also found to be freely soluble in

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the Tween series of surfactants and Tween 80 was selected as the surfactant due to its

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maximum solubilization (222.19± 2.99 mg/mL) of DBM.

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3.2 Pseudo ternary plots and preparation of MEs

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Pseudo-ternary phase diagrams were used to determine the o/w ME region and hence, the

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actual composition of the aqueous phase, oil phase, surfactant, and co-surfactant from which

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the transparent and homogenous MEs were formed. The pseudo-ternary phase diagram with

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various weight ratios of Captex 300, Tween 80, n-butanol and water is described by Fig.1. As

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shown in the figure, stable ME was formed when the content of surfactant and co-surfactant

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mixture was more than 45 %.

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MEs employing minimum amount of surfactant and co-surfactant were selected from phase

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diagram for the preparation of ME. Composition of the different MEs (ME-1-7) prepared is

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depicted in Table 2. From the different ME compositions, ME 1 and ME 2 were turbid in

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appearance. As the surfactant and co-surfactant content was increased from 48% in ME 3 to

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50 % in ME 4 and further to 55% in ME 5, a clear, transparent and stable formulations were

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formed with no signs of precipitation of drug. Among the clear and stable ME batches, the

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ME 3 batch was selected further owing to minimum surfactant and co-surfactant content, to

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study the effect of penetration enhancer i.e. menthol during the ex-vivo permeation studies

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across mice skin. In ME 6 and ME 7 batches at constant surfactant and co-surfactant content,

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the amount of menthol was increased from 0.1% w/w to 0.5% w/w.

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The optimized batch ME 7 was subjected to further physicochemical characterization studies

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and ME 3, ME 6, ME 7 were selected for ex vivo permeation studies across mice skin.

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3.3 Characterization of prepared formulations

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The selected ME formulation (ME 7) was characterized for various parameters.

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3.3.1 Micromeritics and zeta potential

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The mean globule size and polydispersity index of optimized ME 7 batch was found to be

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35.550 ± 4.879 nm and 0.275 ± 0.003 respectively (Fig. 2). The small average diameter can

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be explained due to the co-surfactant molecules that penetrate the surfactant film, lowering

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the fluidity and surface viscosity of the interfacial film, thus decreasing the radius of

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curvature of the nanodroplets and forming transparent systems [42]. Tween 80, due to

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effective interfacial activity also has significant influence on the droplet size. Polydispersity

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index value was less than 0.5 which described the homogeneity of the optimised ME.

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Zeta potential of the formulation was found to be 0.39 mV that was near to neutral (Fig. 3).

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3.3.2 Morphology and structure

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The TEM photomicrograph of the optimzed ME 7 batch revealed the formation of spherical

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droplets. These droplets were uniform in shape and size as depicted by Fig. 4.

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3.3.3 Drug content and pH

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The drug content and pH was found to be 98.92±0.25% and 6.81±0.01respectively.This near

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neutral pH close to skin pH, allows safe and non irritating use of ME as dermal formulation.

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3.3.4 Rheological behaviour

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The optimised formulation (ME 7) exhibited Newtonian flow behaviour with viscosity of

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45.91cP. The uniformity of the nano-globules, with respect to size and shape provided this

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Newtonian flow [43].

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3.4 Thermodynamic Stability Studies

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The developed ME formulation when centrifuged at 3500 rpm for 30 min and subjected to

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freeze thaw cycles, showed no phase separation or precipitation of drug, indicating that the

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ME formulation was physically stable.

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3.4.1 ME stability studies

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The selected ME 7 remained clear even after a period of 3 months at 4ºC, 25°C and 40ºC

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temperature and were found to be consistent with respect to their drug content, pH, viscosity

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and transparency during the stability study.

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3.5 Ex-vivo permeation studies

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The mean cumulative amount permeated/area of DBM from MEs (ME 3, 6 and 7), aqueous

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suspension (C1) and conventional cream base (C2) was investigated for a period of 24 h. As

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shown in Fig. 5, the cumulative amount of DBM permeated from C1 and C2 was 2.265±

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0.177µg/cm2 and 6.578± 0.852µg/cm2 respectively. The conventional cream of DBM

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demonstrated 2.90 time increase in permeation as compared to aqueous suspension. This may

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be due to increased solubilisation of DBM when it was added into the inner phase of

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emulsion system. The cumulative amount permeated/area from the ME (ME 3) was found to

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be 22.146±0.770 µg/cm2 which is 3.366 and 9.777 time higher than conventional cream and

344

aqueous suspension of DBM. Enhanced permeability of DBM when formulated as ME can be

345

explained due to various reasons. Firstly, MEs, in particular, are known to enhance

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penetration rates in deep skin layers and decrease lag time compared to conventional

347

formulations [44-46] as they alter both the lipophilic and the polar pathway by collaborative

348

interactions of vehicle components with the stratum corneum [47]. Secondly, due to the small

349

droplet size, droplets settle down in close contact with the skin and the drug entrapped in ME

350

globules interacts more favourably with the skin cells [48]. MEs reduce the interfacial tension

351

between vehicle and skin because of their contact with the skin lipids, which results in faster

352

permeation [26]. Thirdly, enhanced drug permeation depends on the possibility of the ME

353

component entering into the skin as globules which resulted in enhanced drug accumulation

354

in the skin. This further increases the partitioning of the drug in the skin, leading to increased

355

drug concentration in the upper layers of the skin. This in turn results in higher concentration

356

gradients which act as a driving force for topical/transdermal drug delivery.

357

Further the effect of menthol was studied on the permeation profile of DBM. Menthol is

358

saturated terpene and has been widely used as a penetration enhancer. Menthol is considered

359

as a safe and effective topical OTC product according to FDA. The cumulative amount

360

permeated/area from ME 6 and ME 7 batch comprising of ME having 0.1% and 0.5%

361

menthol was 33.058 ±1.015 µg/cm2 and 44.843±0.246 µg/cm2 respectively . This 1.49 and

362

2.02 time increase in permeation as compared to ME 3 was ascribed to the altered barrier

363

properties of the stratum corneum due to menthol [49, 50]. In this study, butanol used as a co

364

surfactant also have permeation enhancing effect, and it greatly enhances the solubility of

365

DBM and might have affected the barrier properties of stratum corneum. Significant

366

enhancement in the overall skin permeation of drug was observed with ME 7 compared to C1

367

(20 times) as well as C 2 (7 times). Thus, ME 7 was selected for characterisation and in vivo

368

evaluation.

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Similarly rate of permeation flux was observed to be highest from ME 7(3.29±0.042

370

µg/cm2/h) followed by ME 6(1.610±0.014), ME 3 (1.460±0.098), C 2 (0.267±0.021µg/h/cm2)

371

and C1 (0.123±0.010µg/cm2/h).

372

The skin retention of DBM using ME 7 was 8.179±0.742 µg/cm² which was enhanced 30.29

373

times, when compared to aqueous suspension i.e. C1 (0.270±0.021 µg/cm²) and 5.16 times in

374

comparison to conventional cream i.e. C2 (1.583±0.328 µg/cm²). This effect may be due to

375

formation of depot of DBM by ME leading to improved skin retention. Thus, it can be

376

inferred that the prepared ME formulations could effectively make the drug molecules

377

accessible within skin layers, retaining them within close vicinity of the target site.

378

3.6 Skin sensitivity and histopathological studies

379

The aim of the histopathological studies was to establish the dermal tolerance of the

380

selected ME formulation (ME 7). The skin sections appear to be normal without any

381

anatomical and pathological changes after treatment as represented by Fig. 6(a) & (b).

382

Thus, the results established the safety of selected formulations on mice skin.

383

3.7 Anti-photoaging studies

384

After six weeks of UV exposure the skin of mice was evaluated visually for wrinkles, surface

385

texture, erythema, inflammation and other tests like pinch test, histopathology and

386

biochemical estimation.

387

3.7.1 Macroscopic effect of UV exposure

388

The macroscopic influence of UV irradiation on mice skin is shown in Fig. 7. The animals in

389

G1 (naïve control) showed no signs of erythema, dryness and wrinkles (Fig. 7a). Also, the

390

animals belonging to G2 (Sham control) exhibited no visual macroscopic changes at the end

391

of 6 weeks (Fig. 7b). As compared to animals in G1, UV irradiated mice (G3) demonstrated

392

development of epidermal thickening and deep/ severe wrinkles (Fig. 7c). No signs of laxity,

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lesions, deep and or severe wrinkles were observed in the mice treated with DBM ME (G4;

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Fig. 7d). Mice in G5 treated with DBM cream showed striations, laxity, shallow wrinkles and

395

erythema (Fig 7e).

396

3.7.2 Visual score

397

In the group treated with cream before UV irradiation (G5), 66.7% mice showed the

398

appearance of shallow wrinkles and 33.3% showed the appearance of deep wrinkles.

399

However, no signs of lesions or deep wrinkles were observed in ME (G4) treated group. The

400

visual scores in UV irradiated group (G3; 3.67±0.57) were significantly higher than naïve

401

(G1; zero), sham control (G2; zero) and DBM in ME (G4;0.67±0.57). There was no

402

statistically significant difference (p<0.05) observed between the visual scores of ME treated

403

groups (G4) as compared to G1 and G2 (control groups), establishing the effectiveness of the

404

developed novel ME formulation of DBM in the prevention of UV induced wrinkle

405

formation and thus, photoaging. However, the visual scores of groups G4 were significantly

406

lower as compared to cream treated group (G5; 2.33±0.57), which showed that the ME of

407

DBM was more effective than the cream in preventing photodamage.

408

3.7.3 Pinch test

409

Fig.8 depicts the time in seconds taken by the pinched skin to recover and return to normal at

410

different week intervals of 6 week study period of UV exposure. As shown in Fig. 8, time

411

taken to recover in pinch test at the end of 6 weeks by UV irradiated group (G3; 9.65±1.86s)

412

and DBM cream treated group (G5; 5.49±0.43s) was significantly higher (p<0.05) than the

413

naïve (G1; 1.54 ± 0.12s) and sham control (G2; 1.42±0.16s). However, the difference in the

414

results shown by ME (G4; 3.24±0.72s) was statistically insignificant (p<0.05) as compared to

415

the naïve and sham control. Also, the time taken to recover in pinch test by group G4 (ME)

416

was significantly (p<0.05) less than the cream-treated group (G5). This showed that pre-

417

exposure treatment with the novel ME formulation was more effective than the cream in

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preventing skin damage by UV radiation and maintaining normal skin physiology as naïve

419

control. Macroscopic changes in the skin of mice during pinch test in various treatment

420

groups at the end of experimental period of 6 weeks have been shown in Fig. 9.

421

3.7.4 Histopathological studies

422

Photoaged skin is characterised by hyperplasia of epidermis, matrix protein degradation,

423

presence of elastotic structures in the dermis along with perivenular lymphohistocytic dermal

424

infiltrates [51-53]. The microscopic effects of UV irradiation on mice skin are depicted in

425

Fig. 10. In the naïve control group (G1), epidermis had thin layer of keratin and basement

426

membrane was seen beneath the basal layer. Superficial dermis showed intact collagen and

427

elastic tissue fibres. Sweat glands were also observed in the superficial dermis. Abundant fat

428

with regular distribution of hair follicles was observed in the deeper dermal layers. Clusters

429

of sebaceous glands were attached to the hair follicles (Fig. 10a). The Sham control (G2)

430

group also showed normal fibroblasts but slight edema was observed in deep layers. The

431

basement membrane was not distinct and no signs of inflammation were seen (Fig. 10b). As

432

shown in the Fig. 10c, in the UV irradiated group, hyperplastic epidermis observed was

433

characterised by excess granuloma in stratum granulosum. Keratinocytes proliferated and the

434

cells showed focal irregularity of nuclei and some foci of acute inflammation and ulceration

435

indicating damage. Excess of keratin, inflammation and fibrous scarring in the dermis was

436

seen. Dermis showed a disarray of fibroblasts and an excess of neutrophils indicating

437

inflammation. Focal hyaline change was seen in the elastic tissue. All these observations

438

were indicative of hyperplasia and rapid growth which can be either precancerous stage or

439

cells would soon enter a cancerous stage. In the mice treated with optimised ME (G4; Fig

440

10d), skin sections showed normal morphology. Epidermis was intact, consisting of three-

441

four layers of squamous cells, thin stratum corneum and stratum granulosum. The dermis

442

showed normal collagen and fibroblasts. All the dermal appendages (sebaceous glands, sweat

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glands and hair follicles) were found to be normal. Fat and vascular capillaries were also

444

normal. Lymphocytes were absent. All these results confirmed the effectiveness of the ME

445

based formulations of DBM as sunscreen and thus preventing the cutaneous tissue from any

446

kind of damage by UV exposure. Fig.10e depicts the histopathology of cream treated skin

447

prior UV exposure. Epidermal hyperplasia with increased number of squamous cells was

448

observed. Coagulation of collagen and elastin was seen. Considerable inflammation was

449

evident by the presence of necrotic leukocytes. Neutrophils, lymphocytes and plasma cells

450

were also seen. Dense connective tissue was present and subcutaneous muscle fibres showed

451

minimal change. These histopathological markers were similar to those observed in the

452

positive control group (G3) which confirmed that the DBM cream was ineffective in

453

preventing UV induced damage as compared to the ME.

454

3.8 Biochemical estimations

455

The biochemical estimations were done to measure the level of oxidative stress which was

456

prominently due to the reactive oxygen species (ROS), skin being its major targeting site..

457

Exposure to UV radiation in photodamaged skin generated ROS. Overproduction or

458

inadequate removal of ROS from the body results in development of oxidative stress, which

459

ultimately leads to various abnormal functions like lipid peroxidation, damage of DNA,

460

protein and production of various inflammatory cytokines[54, 55].

461

3.8.1 Catalase activity

462

Catalase (CAT) is an endogenous enzyme that scavenges H2O2 in the skin by its conversion

463

to oxygen and water and thus reduces the level of oxidative stress. Catalases neutralized the

464

effect of free radicals generated as a result of UV exposure [2, 56]. It has been documented

465

that CAT activity in the skin is strongly reduced after exposure to UVA [57] and UVB [58,

466

59] irradiation. A significantly (p<0.05) decreased catalase activity (2.74 times; Table 3) was

467

observed in the UV treated group (G3) as compared to the naive control group (G1). This

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468

confirmed the UV radiations induced oxidative stress in mice (G3). Results of G2 (sham

469

control group) were not significantly different (p< 0.05) from naive control group (G1) which

470

showed that depilatory used in this experiment had no effect on activity of CAT. Significant

471

increase (p<0.05) in catalase level in groups treated with DBM loaded MEs (G4)

472

compared to the positive control (G3) was observed. The difference in catalase activity of G4

473

(ME) and G1 (naïve control) was statistically insignificant (p<0.05) whereas CAT activity

474

was significantly reduced in cream treated group (G5) as compared to the naïve control (G1).

475

These results confirmed the inability of DBM in conventional cream base in preventing UV

476

induced skin damage or restoring the normal CAT levels in comparison to the ME

477

formulation.

478

3.8.2 Lipid peroxidation

479

Free radicals generated in the body upon exposure to UV radiations oxidise cellular lipids,

480

proteins and nucleic acids, leading to local injury, genetic alterations and eventual organ

481

dysfunction. Polyunsaturated fatty acids (PUFAs), major constituents of cell membranes are

482

readily attacked by oxidizing agents and this process of lipid peroxidation is self-perpetuating

483

and highly damaging. Repetitive UV exposure leads to the formation of peroxyl free radicals,

484

which break down to form malondialdehyde (MDA). MDA further cross-links and

485

polymerizes collagen, leading to loss of skin elasticity and finally, formation of wrinkles [36,

486

60, 61]. In order to verify the induction of lipid peroxidation by UV treatment, thiobarbituric

487

acid reactive substances (TBARS) were measured in skin homogenates. The results of MDA

488

levels generated upon UV exposure for 6 weeks for various treatment and control groups

489

have been shown in Table 3. The MDA levels for the UV irradiated group were

490

approximately 4.06 times the control group (G1) indicating an increase in the MDA levels

491

caused by exposure to UV radiation. Groups treated with DBM loaded MEs (G4) showed a

492

significant decrease (p<0.05; 2.97 times) in MDA level in skin homogenates as compared to

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the positive control (G3). However, MDA levels observed in DBM cream treated group G5

494

were found to be 1.89 times less than the UV irradiated mice (G3). The difference in MDA

495

levels of G4 (ME) groups and G1 (naïve control) was statistically insignificant (p<0.05)

496

whereas the levels were significantly increased in cream treated group (G5) as compared to

497

the naïve control (G1). These results indicated the inability of conventional cream in

498

preventing UV induced skin damage and oxidative stress, subsequently.

499

3.8.3 Superoxide dismutase activity

500

Superoxide dismutase (SOD), another antioxidant enzyme in organisms, acts as a scavenger

501

of superoxide radicals and plays a major role in the cellular defense system against oxidative

502

stress and cytotoxicity [62]. A significantly (p<0.05) decreased SOD activity (2.03 times)

503

was observed in the UV treated group (G3) as compared to the naive control group (G1) that

504

confirmed the UV radiations induced skin damage and oxidative stress in mice (G3). As

505

shown in Table 3, treatment with DBM loaded ME (G4) caused a significant increase

506

(p<0.05) in SOD level in skin homogenates as compared to the positive control (G3).

507

However, SOD activity observed in cream treated group G5 was significantly (p<0.05) less

508

than that of the ME treated group. The difference in SOD activity of G4 (ME) group and G1

509

(naïve control) was statistically insignificant (p<0.05) whereas SOD activity was significantly

510

reduced in G5 as compared to the naïve control (G1). These results confirmed that DBM in

511

conventional cream base was less effective in preventing UV induced skin damage and

512

oxidative stress, subsequently as compared to the ME.

513

3.8.4 Glutathione (reduced GSH) estimation

514

Glutathione (L-g-glutamyl-L-cysteinylglycine) is chemically a nonprotein thiol involved in

515

the antioxidant cellular defense. Free glutathione is mainly present in its reduced form (GSH)

516

which gets converted to the oxidized form (GSSG) during oxidative stress. Enzyme GSH

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reductase reverts the oxidized form to the reduced form. The GSH couple (2GSH=GSSG)

518

acts as a cellular redox buffer and represents the redox environment of the cell [63, 64].

519

Decrease in the intracellular GSH has been reported in the aged tissues.

520

Significantly (p<0.05) decreased GSH (4.21 times; Table 3) was observed in the UV treated

521

group (G3) as compared to the naive control group (G1). This confirmed that GSH levels

522

decrease in photo aged skin (G3). There was significant increase in the GSH levels of ME

523

(G4) and cream treated group (G5) when compared to the UV-irradiated group (G3). Further,

524

no statistically significant difference between the ME (G4) as compared to the naïve and

525

sham control groups (G1 and G2) were observed, indicating the restoration of normal GSH

526

levels. However, the GSH levels in the cream treated group (G5) were significantly less than

527

the control groups (G1 and G2), indicating the ineffectiveness of DBM in the conventional

528

cream in preventing the photodamage and restoring the normal GSH levels as compared to

529

the ME formulations.

530

4. CONCLUSIONS

531

The results of our investigations using ME of DBM highlight the anti-photoaging efficacy of

532

this molecule, which positively reflects its utility in cosmaceuticals as a sunscreen. The

533

present investigation resulted in successful formulation of ME of DBM with desirable

534

characteristics. The results of ex vivo permeation and in vivo studies revealed that the DBM

535

loaded ME penetrated the skin with appreciable skin retention and was effective in

536

prevention of photodamage. The experimental animals treated with the prepared ME showed

537

no signs of dermatoheliosis and the results were further confirmed from histopathological and

538

biochemical studies. These significant anti-photoaging effects of DBM do warrant further

539

clinical investigations to exploit the potential benefits of ME based formulation of DBM as a

540

sunscreen in the prevention of photoaging and skin damage.

541

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FIGURE LEGENDS:

543

Fig.1: Pseudo ternary phase diagrams of Tween 80: n-butanol (2:1) using water as the

544

hydrophilic phase and Captex 300 as the hydrophobic phase

545

Fig. 2: Globule size distribution of ME 7 formulation

546

Fig 3: Zeta potential measurement of ME 7 formulation

547

Fig 4: Transmission electron micrograph of ME 7 formulation

548

Fig 5: Comparison of ex–vivo permeation profiles of different compositions of DBM through

549

mice skin (n = 3)

550

Fig 6: Histopathological evaluation of mice skin: (A) Control skin (untreated) for

551

comparison with test formulations (B) Formulation ME 7 treated group

552

Fig 7: Macroscopic changes in the mice skin after different treatments at the end of 6 weeks:

553

(A) Naïve control;G1 (B) Sham Control;G2 (C). UV–irradiated; G3 (D) ME treated; G4

554

(E). DBM cream treated; G5

555

Fig. 8: Comparative graph of pinch test for different experimental groups

556

Fig 9: Macroscopic changes in the skin of mice after pinch test upon various treatments at the

557

end of experimental period of 6 weeks. (A) Naïve control; G1 (B) Sham Control; G2 (C).

558

UV–irradiated; G3 (D) ME treated; G4

559

Fig. 10: Histopathological features of mouse skin of various experimental groups: (A) Naïve

560

control; G1 (B) Sham Control; G2 (C). UV–irradiated; G3 (D) ME treated; G4

561

cream treated; G5

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(E). DBM cream treated; G5

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TABLE LEGENDS:

564

Table 1: Solubility of DBM in different oils and surfactants

565

Table 2: Composition of different ME formulations

566

Table 3: Comparison of biochemical estimation for different experimental groups

567

Conflict of Interest

568

The authors report no conflict of interest

569

(E). DBM

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[36] S. Sharma, I.P. Kaur, Development and evaluation of sesamol as an antiaging agent, Int. J. Dermatol. 245 (2006) 200-208. [37] K. Tushkara, S. Moriwaki, M. Hotta, T. Fujimura, Y. Sugiyama-nakagiri, S. Sugawara, T. Kitahara, Y. Takema, The effect of sunscreen on skin elastase activity induced by ultraviolet A irradiation, Biol. Pharm. Bull. 28 (2005) 2302-2307.

AC C

669 670 671 672 673 674 675 676 677 678 679 680 681 682 683 684 685 686 687 688 689 690 691 692 693 694 695 696 697 698 699 700 701 702 703 704 705 706 707 708 709 710 711 712 713 714 715 716

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[41] T.V. Sirota, Use of nitro blue tetrazolium in the reaction of adrenaline autooxidation for the determination of superoxide dismutase activity, Biochemistry (Moscow) Supplement Series B: Biomedical Chemistry, 6 (2012) 254-260. [42] S. Tenjarla, Microemulsions: an overview and pharmaceutical applications, Crit. Rev. Ther. Drug Carrier Syst. 16 (1999) 461-521.

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[44] M. Kreilgaard, M.J. Kemme, J. Burggraaf, R.C. Schoemaker, A.F. Cohen, Influence of a microemulsion vehicle on cutaneous bioequivalence of a lipophilic model drug assessed by microdialysis and pharmacodynamics, Pharm. Res. 18 (2001) 593-599. [45] K. Kriwet, C. Goymann, Diclofenac release from phospholipid drug systems and permeation through excised human stratum corneum, Int. J. Pharm. 125 (1995) 231-242.

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[46] I. Sarigullu Ozguney, H. Yesim Karasulu, G. Kantarci, S. Sozer, T. Guneri, G. Ertan, Transdermal delivery of diclofenac sodium through rat skin from various formulations, AAPS PharmSciTech. 7 (2006) 88. [47] D. Kaushik, P. Batheja, B. Kilfoyle, V. Rai, B. Michiak-Kohn, Percutaneous permeation modifiers: enhancement versus retardation, Expert Opin. Drug Deliv. 5 (2008) 517-529.

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[48] S. Peltola, P. Saarinen-Savolainen, J. Kiesvaara, T.M. Suhonen, A. Urtti, Microemulsions for topical delivery of estradiol, Int. J. Pharm., 254 (2003) 99-107. [49] S. Gao, J. Singh, Int. J. Pharm. 165 (1998) 45-55. [50] M. Aqil, A. Aghad, Y. Sultana, A. Ali, Status of terpenes as skin penetration enhancers, Drug Discov. Today. 12 (2007) 1061-1067.

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AC C

717 718 719 720 721 722 723 724 725 726 727 728 729 730 731 732 733 734 735 736 737 738 739 740 741 742 743 744 745 746 747 748 749 750 751 752 753 754 755 756 757 758 759 760 761 762 763 764 765

[53] J.J. Leyden, G.L. Grove, M.J. Grove, E.G. Thorne, L. Lufrano, Treatment of photodamaged facial skin with topical tretinoin, J. Am. Acad. Dermatol. 21 (1989) 638-644. [54] M. Rahman, S. Akhter, J. Ahmad, M.Z. Ahmad, S. Beg, F.J. Ahmad, Nanomedicinebased drug targeting for psoriasis: potentials and emerging trends in nanoscale pharmacotherapy, Expert Opin. Drug Deliv. 12 (2015) 635-652. [55] M. Rahman, M. Zaki Ahmad, I. Kazmi, S. Akhter, S. Beg, G. Gupta, M. Afzal, S. Saleem, I. Ahmad, M. Adil Shaharyar, F. Jalees Ahmed, F. Anwar, Insight into the biomarkers as the novel anti-psoriatic drug discovery tool: a contemporary viewpoint, Curr. Drug Discov. Technol. 9 (2012) 48-62.

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TE D

797

[57] Y. Shindo, T. Hashimoto, Time course of changes in antioxidant enzymes in human skin fibroblasts after UVA irradiation, J. Dermatol. Sci. 14 (1997) 225-232.

EP

796

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AC C

766 767 768 769 770 771 772 773 774 775 776 777 778 779 780 781 782 783 784 785 786 787 788 789 790 791 792 793 794 795

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Table 1. Solubility of DBM in oils and surfactants Oils/ surfactants

Solubility(mg/ml) 139.96 ±1.99

Captex 300

217.72 ± 3.99

Captex 355

170.35 ± 5.66

Oleic acid

140.43 ± 2.66

Castor oil

77.05 ± 1.67

Tween 20

118.52 ± 2.33

Tween 40

123.93 ± 3.99

Tween 60

146.08 ± 5.33

Tween 80

222.19± 2.99

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Captex 200

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Table 2. Composition of different ME formulations DBM (% w/w)

Captex300 (% w/w)

ME 1

0.2

9.8

Tween80 : n-butanol (2:1) (%w/w) 40

ME 2

0.2

9.8

45

ME 3

0.2

9.8

48

ME 4

0.2

9.8

50

ME 5

0.2

9.8

55

ME 6

0.2

9.8

48

ME 7

0.2

9.8

48

menthol (% w/w)

Water (% w/w)

-

50

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Batch code

45

-

42

-

40

-

35

0.1

42

0.5

42

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Table 3. Comparison of biochemical estimation for different experimental groups GROUP

Catalase Activity ( µmoles of H2O2 consumed/min/mg protein)

Nanomoles MDA/ mg protein

1.97± 0.03

79.69±3.56

2.29±0.06

89.59±2.24

0.72±0.20

323.96±9.92

1.96±0.04

108.78±1.67

1.47±0.002

0.0109±0.0004

1.23±0.05

171.29±4.98

1.16±0.036

0.0097±0.0004

SOD units /mg protein

µmol GSH/mg protein

Sham control (G2) UV irradiated (G3)

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DBM cream treated (G5)

1.69±0.021

0.0139±0.0001

1.70±0.042

0.0137±0.0007

0.83±0.004

0.0033±0.0011

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ME treated (G4)

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Naïve control (G1)

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