Studies on olive-and silicone-oils-based Janus macroemulsions containing ginger to manage primary dysmenorrheal pain

Materials Science & Engineering C 100 (2019) 276–285

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Studies on olive-and silicone-oils-based Janus macroemulsions containing ginger to manage primary dysmenorrheal pain

T

Diksha Puria, Gopal Lal Khatika, Tamilvanan Shunmugaperumala,b,

⁎

a

Lovely School of Pharmaceutical Sciences, Lovely Professional University, Jalandhar-Delhi, G.T.Road (NH-1), Phagwara, Jalandhar, Punjab 144411, India National Institute of Pharmaceutical Education and Research (NIPER)-Guwahati, C/O NETES Institute of Technology & Science, NH-37, Santipur, Parli Part, Mirza, Assam 781125, India

b

ARTICLE INFO

ABSTRACT

Keywords: Janus macroemulsion Ginger Marker compound Central composite design Primary dysmenorrhea mice model Pain

Ginger (GIN) powder-loaded oil-in-water (o/w) macroemulsions were prepared based on olive-and silicone-oils. The dispersed oil droplets with paired-beans structure were evident and thus the final emulsion can be termed as Janus macroemulsions. The objectives of the present study are (1) to identify the marker compound present in GIN powder via HPLC analysis, (2) to process the GIN powder via anti-solvent precipitation technique, (3) to see the solubility of GIN powder in various single oils or oil combination, (4) to optimize the GIN-loaded o/w macroemulsions using the central composite design (CCD) with respect to mean particle size of dispersed oil droplets and highest percentage drug entrapment efficiency values (DEE) and (5) to evaluate the pain reducing activity of optimized GIN-loaded macroemulsion via in vivo primary dysmenorrhea (PD) mice model. Both predicted and obtained values of percentage DEE (76.29 Vs.76.09) and mean particle size (245.99 Vs. 272.51 μm) were almost the same indicating the CCD statistical design applicability. The optimized Janus macroemulsion was stable at 4 °C for over a period of 90 days. Using the PD mice model, the counting of writhing reaction produced by the tested GIN-loaded macroemulsions at low and high doses did not reveal significant difference in comparison to the positive control (aspirin treated). Only the high dose of GIN-loaded macroemulsion was able to restore the uterine tissue's normal histomorphological structure after the H & E staining. Nevertheless, the paired beans structure should be tested for entrapping the plant-derived drugs having dissimilar physicochemical characteristics but similar therapeutic activity.

1. Introduction Unfortunately, in conjunction with most of the synthetic medicinal principles, the plant-derived medicinally active principles have also posed the problems of poor aqueous solubility, inadequate molecular size or both, resulting in failure of absorption and thus poor systemic bioavailability [1,2]. Therefore, most of the plant-derived medicinally active principles are simply grouped under low solubility/permeability case to easily fit into a Biopharmaceutics Classification System (BCS) class IV molecule. Either in aqueous extract or dry powder, the ginger (GIN) rhizome (botanical name: Zingiber officinale, family: Zingiberaceae, order: Zingiberales, super order: Lilianae, subclass: Magnoliidae, class: Equisetopsida) is widely used as a house-hold medicine to manage the pain-associated with primary dysmenorrheal condition. The GIN contains a thermo-stable pungent principle called as 6-gingerols along with its thermo-labile analogues termed as 6-shogaols. The

gingerols can easily be converted into shogaols by degradation reaction. Indeed, the shogaols is main ingredient in dry GIN and thus it is recognized as chief biomarkers for the quality control of processed GIN commercial products [3]. In addition, exposing the aqueous extract and dry powder to different environmental condition may compromise/diminish the therapeutic potential of both gingerols and shogaols. To minimize or prevent the environment-induced therapeutic compromise of gingerols and shogaols and to solve their possible low aqueous solubility problem, it would be better idea to incorporate the GIN in a drug delivery carrier. Among the gamut of so far designed colloidal dispersion system, such as liposome, nanospheres, nanocapsules, niosomes, transferosomes, ethosomes, cubosomes, etc., another heterogeneous dispersion system called ‘oil-in-water (o/w) emulsion’ receives a considerable interest to make the successful commercial pharmaceutical formulations for hydrophobic drug moiety [4]. However, it could be of interest to

⁎ Corresponding author at: National Institute of Pharmaceutical Education and Research (NIPER)-Guwahati, C/O NETES Institute of Technology & Science, NH-37, Santipur, Parli Part, Mirza, Assam 781125, India. E-mail address: [email protected] (T. Shunmugaperumal).

https://doi.org/10.1016/j.msec.2019.01.137 Received 21 September 2018; Received in revised form 11 January 2019; Accepted 16 January 2019 Available online 24 February 2019 0928-4931/ © 2019 Elsevier B.V. All rights reserved.

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find a way for incorporating both the hydrophilic and hydrophobic drug molecules together in a single emulsion dispersion system. One of the ways which was found during the year 2000 is to develop positively-charged submicron emulsion prepared based on medium chain triglyceride (MCT) stearylamine, Lipoid-E80, glycerin, poloxamer-188, vitamin E and double distilled water [5]. This emulsion dispersion system, when stearylamine molecule was incorporated, it shows an unexpected bi-compartmental structure, termed as ‘handbag’. The handbag structure formation is believed due to the association of lipoidE80, stearylamine and poloxamer-188 at the oil water interface to prevent the random collision of dispersed oil droplets, coalescence and Ostwald ripening. While performing this activity by covering the dispersed oil droplets, with mono- or multi-molecular emulsifiers' film, the presence of cationic lipidic stearylamine promotes the elevation of polyoxyethylene structure of the hydrophilic poloxamer-188 towards the water dispersion medium of o/w emulsion system. These two competing driving forces exerted by stearylamine and poloxamer-188 ultimately lead to the formation of unexpected handbag structure or two compartmental structures within the emulsion dispersion system. The presently developed emulsions however differ significantly from the work reported by Teixeira et al. [5] in many ways. Firstly, two different non-volatile oils (silicone and olive oils), one-single emulsifier molecule (Tween 80) and double distilled water were used to make o/w emulsions. Secondly, the emulsions were produced without utilizing the so-called particle size reduction machineries (homogenizers, microfluidizers and ultrasonicator). Rather, simple magnetic and electric stirrers were enough to produce the emulsions. The emulsions thus produced having the mean particle size values in micron levels and therefore could be termed as coarse or macroemulsions [6]. The unique feature of this presently developed olive-and silicone-oils-based macroemulsions was the presence of the double-faced head structure like the one, which was shown after incorporation of cationic lipidic stearylamine emulsifier moiety into the nanosized emulsion developed by Teixeira et al. [5]. In the literature, the presence of double-faced oily head structure is simply called as ‘Janus particles’ honouring the roman God ‘Janus’. Again, from the literature, it is envisaged that the presence of double-faced head structure within the emulsion dispersion system is capable of entrapping both hydrophilic and hydrophobic drug molecules together in a confined manner. Nowadays, the term, anisotropic, is ostensibly being used to describe the Janus structure which has different surface features on the two sides. However, in the present study, the term “paired beans (Janus) structure” was analogously being used to denote the “double-faced oily head structure” and/or “unexpected bicompartmental handbag structure”. It needs to be mentioned that the selected hydrophilic drug moieties should have also been contained a measurable solubility in the surrounding continuous water medium of the o/w coarse (or macro) emulsion. Since the o/w macroemulsions are prepared based on oil, water and emulgent, it could be possible that the formulation and process (independent) variables such as GIN amounts, oil compositions, etc. can influence the dependent variables such as dispersed oil droplet size, the paired beans structure of dispersed oil droplets and the drug entrapment efficiency values in different manners when interactions occur among these different variables. Therefore, it is pertinent to apply and/ or use the response surface methodology (RSM) for the development and optimization of GIN-loaded o/w macroemulsions. The RSM is a powerful and effective statistical tool not only for analyzing the multivariate relationships between independent and response variables but also for identifying the interactions by means of a mathematical model with linear or square polynomial functions along with representing the relationships between the variables in 3-Dimentional graphical form [7,8]. Moreover, the RSM is less laborious and time consuming in comparison to the optimization by studying of one-variable at a time [9]. Among the different RSM methods which have been applied for the optimization of various drug delivery systems, the central composite design (CCD) was successfully applied previously to optimize the o/w

nanosized emulsion systems by Su et al. [10] and Zainol et al. [11]. The objectives of the present study are therefore (1) to identify the marker compound present in GIN powder via HPLC analysis, (2) to process the GIN powder via anti-solvent precipitation technique, (3) to see the solubility of GIN powder in various single oils or oil combination, (4) to optimize the GIN-loaded o/w macroemulsions using the CCD with respect to mean particle size of dispersed oil droplets and highest percentage drug entrapment efficiency values and (5) to evaluate the pain reducing activity of optimized GIN-loaded macroemulsion via in vivo primary dysmenorrhea (PD) mice model. Thus the current investigation majorly focused on the systematic optimization followed by pain-reduction capability checking in a PD mice model and verification of the histomorphological changes after the hematoxylin and eosin (H & E) staining of uterine tissue sections collected from the mice. 2. Materials and methods 2.1. Materials The GIN utilized in the present study was in fact in semi-dried rhizome form and it was obtained between July-2017 and September2017 from farmers' market, Jalandhar, Punjab, India. The castor oil, coconut oil and olive oil were purchased from Khurana oils, Ludhiana, Punjab, India. The silicone oil, ethanol, methanol and Tween 80 were procured from LOBA Chemie, Colaba, Mumbai, Maharashtra, India. Oestradiol benzoate injection was procured from Macmillon Pharmaceuticals Limited, Amritsar, Punjab, India. Oxytocin injection was obtained from Novartis India Limited, Mumbai, Maharashtra, India. All other chemicals were of analytical grade and were used as received. 2.2. Methods 2.2.1. Authentication of ginger rhizome A voucher specimen of GIN rhizome was deposited in the herbarium of the Department of Botanical and Environmental Sciences at Guru Nanak Dev University (Amritsar, Punjab, India) and after registered in the herbarium, an identification number (# 1973) was received. Following authentication, the GIN sample was stored in a desiccator until its further use. 2.2.2. Processing of ginger rhizome 2.2.2.1. Anti-solvent precipitation technique. The technique adopted by Aditya et al. [12] was followed to process the GIN powder. In brief, 500 mg GIN coarse powder (made from dried rhizome by simple grinding using the mortar and pestle) was dissolved in 10 ml of ethanol (volatile solvent). The ethanolic GIN solution was mixed with 50 ml of double distilled water (non-volatile anti-solvent). Then, the ethanol-water mixture containing the GIN was kept in a rotary evaporator (Model RV8, IKA, Germany) under the following experimental conditions: vacuum condition: −90 kPa; rotating speed; 150 rpm and temperature 45 °C. Due to the evaporation of volatile solvent, there was initially an attainment of supersaturation state for GIN particles in non-volatile anti-solvent and then followed by GIN precipitation (nucleation) state which eventually allowed the formation of GIN in fine powder form. 2.2.3. Quantitative drug analysis Since GIN rhizome consists of 6-gingerol and 6-shogaol, the drug content analysis in the macroemulsion should be aimed/based on selecting any one of the constituents of GIN as marker compound. In the present study, the already reported HPLC method [13] was fully validated and was adapted to meet the requirements for the GIN stability indicating test. In brief, a reversed-phase 250 × 4.6 mm, Agilent XDBC18 (5 μm) column furnished by Agilent Technologies (California, USA) and kept at 28 ± 0.05 °C. The mobile phase, at 1 ml/min flow rate and 277

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ran at isocratic mode with the run time of 20 min., consisted of a mixture (90:10, v/v) of methanol and water. Two milliliters of GINloaded macroemulsion was diluted with 20 ml of 90% methanol. From this, 20 μl was injected into the HPLC system. The 6-gingerol and 6shogaol were monitored with a Photodiode Array detector (PDA) at 282 nm (Agilent 1260 HPLC system, Agilent Technologies, California, USA). Furthermore, five standard solutions of 6-gingerol in 90% methanol were first prepared and appropriately diluted with 90% methanol to final 6-gingerol concentrations ranging from 2 μg/ml (minimum detectable concentration) to 10 μg/ml. All the experiments were triplicated, and the deviation ranged from 0 to 3%, indicating that the various experimental conditions were well controlled.

Table 1 Dependent and independent variables along with their goal or levels used for making central composite design (CCD) during the initial pilot screening study to generate ginger (GIN)-loaded o/w macroemulsions with the paired-beans dispersed oil droplet structure. Selected independent variables

Quantity

Levels Low High

X1: GIN fine powder produced by anti-solvent precipitation processing technique (mg) X2: Silicone oil (ml) X3: Olive oil (ml) Tween 80 (ml) Double distilled water

2.2.4. Phase solubility study The solubility of processed GIN fine powder and unprocessed GIN coarse powder was determined in triplicate using the procedure shown initially by Higuchi and Connors [14] and adopted recently by Affandi et al. [15] with slight modifications. In the present study, the single oils and oil combination selected to determine the solubility of GIN were castor oil, coconut oil, olive oil, silicone oil and silicone oil-olive oil combination (1:1 ratio). The solubility of GIN in double distilled water and double distilled water containing tween 80 was also determined. In brief, an excess amount (1–2 g) of processed GIN fine powder or unprocessed GIN coarse powder was added to 7 × 3 = 21 number of 10 ml volumetric flasks containing each one of the selected single oils (5 ml), oil combination (at the ratio of 4:1) and 9.4 ml of double distilled water with or without 0.5 ml of tween 80. All the volumetric flasks were underwent on orbital shaking in an orbital shaker (Thermo Fisher Scientific, Mumbai, India) in a temperature controlled room maintained at 25 ± 0.2 °C for 24 h. The samples were collected from each one of the volumetric flasks following filtration through 0.45 μm membrane filter (Nylon Acro Disc, Gelmen, USA). All the filtrates after suitable dilution with mobile phase solvent system were measured for GIN (6-gingerol) concentration using the validated HPLC method.

50, 100, 150, 200, 250 2, 3, 4 1, 1.5, 2 0.5 9.4

50

250

1 1

4 2

Dependent variables

Goal

Y1: Drug entrapment efficiency Y2: Particle size

Maximize Maximize

Numbers in bold indicate that the quantity of two independent variables (Tween 80 and double distilled water) were kept constant, while the quantity of other independent variables were varied.

2.2.8. Drug entrapment efficiency (DEE) An ultracentrifugation method was utilized to find out the GIN entrapment efficiency in the aqueous phase of the o/w macroemulsions [16]. The polyallomer tubes were used to hold the GIN loaded macroemulsions for centrifuging the samples in a HITACHI ultracentrifugation apparatus, operated at 50,000 rpm (̴ 162,000 ×g) at 4 °C for 2 h. After centrifugation, the bottom of the polyallomer tubes was pricked with a syringe needle to collect the aqueous phase of the o/w macroemulsion. The concentrations of GIN (6-gingerol) present both in the aqueous phase and the whole emulsion were determined by the validated HPLC method. The DEE was calculated according to the following equation [17]:

2.2.5. Preparation of oil-in-water macroemulsions The oil phase consisted of combination of silicon and olive oils mixed at a ratio of 4:1. The anti-solvent precipitation technique-processed GIN fine powder was dissolved or dispersed in the oil phase. The water phase contained the Tween 80 dissolved in double distilled water. Both oil and water phases were mixed by means of low-shear size reduction machineries [initially with magnetic stirrer (IKA® C-MAG HS7 digital S022, Werke GmbH & Co.KG, Staufen, Germany) for 5 min followed by electric stirrer (IKA® EUROSTAR 20 digital Euro-ST 20DS000, Werke GmbH & Co.KG, Staufen, Germany) at 700 rpm for 15–20 min]. The macroemulsion thus obtained was stored in well closed glass bottles at 25 °C until its further use. It should be added that an initial pilot screening study was performed with the independent variables as shown in Table 1 to confirm the formation of paired-beans dispersed oil droplets structure inside the macroemulsion.

EE (%) =

{(Ctotal × Vtotal) ( C water × Vwater )} × 100 Ctotal × Vtotal

(1)

where Ctotal is GIN amount added in whole emulsion, Vtotal is volume of emulsion prepared, Cwater is GIN (6-gingerol) detected in the water/ aqueous phase and Vwater is volume of water phase collected after centrifugation. 2.2.9. Experimental design A three-factor central composite design (CCD) was utilized to study the effect of independent variables [drug composition (50–250 mg, A) silicone oil composition (1–4 ml, B) and olive oil composition (1–2 ml, C)] on the two dependent or response variables [percentage drug entrapment efficiency (R1) and mean particle size (R2) of the Janus macroemulsions]. Hence, based on the CCD, a total of 20 experiments were run using Design Expert software (version 10.0.0.6, Stat-Ease Inc., Minneapolis, MN, USA). The independent variables and their coded levels and scheme matrix of the CCD are represented in Tables 1 and 2, respectively. The experimental runs involved 8 factorial points, 6 axial points and 6 replicates of centre points (Table 2) [18]. An appropriate polynomial model was chosen based on the significant terms (p < 0.005), the least significant lack of fit, coefficient of variance, the multiple correlation coefficient, and adjusted multiple correlation coefficient provided by Design-Expert software. To determine the repeatability of the method, the center point was repeated six times.

2.2.6. Observation with compound microscope About 50 μl of the GIN-loaded o/w macroemulsion was kept in a glass slide and covered with the cover slip. An utmost care was taken not to allow the air bubble occlusion between the slide and slip. The slide containing the macroemulsion sample was visualized under high power in a compound microscope fitted with 16 MP camera. 2.2.7. Particle size determination The average size of the dispersed oil droplets was determined by means of a Malvern Mastersizer (Malvern Instrument Ltd., Malvern, UK) at 25 °C. A laser beam of He-Ne light source at 633 nm wavelength was used. The sensitivity range was 0.02–2000 μm. About 100–200 μl of macroemulsion was mixed with 150 ml of dispersing water (Hydro S) before making the measurement. Values reported were the average oil droplet diameter of triplicate macroemulsion samples.

2.2.9.1. Generalized response surface model and statistical significance. The optimal composition and experimental conditions for the preparation of GIN-loaded Janus macroemulsions were chosen based on the situation of attaining maximum percentage drug entrapment efficiency (R1) and maximum mean particle size (R2) of 278

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temperature, 25 °C. The oil phase separation at the surface of emulsion and subsequent non-dispersion of oil droplets into the emulsion were considered as the indicator for instability problem.

Table 2 Scheme of central composite design (CCD) produced as per the Design Expert software showing the run, type, independent and response variables. Run

Type

Independent variables X1: Drug (mg)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

Axial Factorial Centre Axial Centre Factorial Factorial Factorial Center Axial Center Factorial Axial Axial Axial Center Factorial Center Factorial Factorial

200 250 150 150 150 250 50 250 150 150 150 50 75 150 150 150 50 150 50 250

X2: Silicone oil (ml) 3.5 4 3.5 2.659104 3.5 3 3 3 3.5 3.5 3.5 4 3.5 4.340896 3.5 3.5 3 3.5 4 4

Response variables X3: Olive oil (ml)

1.5 2 1.5 1.5 1.5 2 1 1 1.5 2.340896 1.5 1 1.5 1.5 0.659104 1.5 2 1.5 1 1

Drug entrapment efficiency (%) (R1) 57.86 57.87 57.87 49.37 61.74 57.88 55.8 63.85 57.18 62.09 57.29 78.76 70.08 54.89 55.97 57.86 72.38 59.03 76.09 64.78

2.2.12. In vivo study 2.2.12.1. Primary dysmenorrhea (PD) mice model. An in vivo PD mice model reported by Huang et al. [24] and Yang et al. [25] was followed with slight modification in the present study. Female BALB/c mice (6–8 week old) having 18–26 g were purchased from National Institute of Pharmaceutical Education and Research (NIPER), Mohali, Chandigarh, Punjab, India. Mice were housed under specific pathogen-free conditions in the animal house which was maintained at the temperature condition of 25–28 °C along with 12-hour light/dark cycles. All the experiments involving the mice were approved by the institutional animal ethical committee (IAEC) of School of Pharmaceutical Sciences, Lovely Professional University, Phagwara, Punjab, India. Thirty-six mice were randomly divided into 6 different groups having 6 mice each. All the mice were freely allowed to take standard diet and water ad libitum throughout this study. With the lone exception of the first group (containing 6 mice), all the other groups (from 2 to 6) were allocated oestradiol benzoate (1 mg/kg/day intraperitoneal injection) for three days to establish the animal model of primary dysmenorrhea. To confirm whether or not the mice had the building of dysmenorrhea model on the third day, an assessment was made via vaginal smear test to determine the oestrus stage of the mice and then the dysmenorrhea mice were modeled [26,27]. Six hours after the intraperitoneal injection of oestradiol benzoate on the third day, the mice belong to group 1 (normal control group) did not receive any test material but the mice belong to group 2 (negative control group) were allowed to drink saline solution freely for over the period of 7 more days. The mice belong to remaining groups (from 3 to 6) were received aspirin (positive control, 0.5 g/kg/day, per oral), placebo (0.2 ml macroemulsion without GIN per day per oral), low dose [0.2 ml (equal to 0.51 mg GIN) of processed GIN (50 mg)-loaded macroemulsion equivalent to the dose of 50 mg/kg/day, per oral] and high dose [0.4 ml (equal to 1.01 mg GIN) of processed GIN (50 mg)loaded macroemulsion equivalent to the dose of 50 mg/kg/day, per oral], respectively, for over the period of 7 more days. Thirty minutes after the corresponding test material per oral administration on the 10th day, the mice belong to all groups (from 1 to 6) were treated with the single intraperitoneal injection of oxytocin (33 U/kg) to induce the writhing response. Then, all mice were placed in a box and after 5 min, the writhing reaction times were recorded for the next 30 min. According to the Schmauss and Yaksh's standard, the writhing reaction was characterized by abdominal contraction, stretching or bending of the body, trunk and/or pelvis ending with limbs extension [28]. The analgesia effect produced in the mice due to the administration of positive control, placebo and tested macroemulsions at low and high doses was calculated by following the Eq. (3) [24].

Particle size (μm) (R2) 210.87 215.65 254.76 251.45 207.67 214.76 218.85 216.46 234.54 204.36 235.43 244.87 167.56 206.43 254.98 236.43 126.98 235.76 272.51 224.3

the dispersed oil droplets of the Janus macroemulsions, By using the polynomial regression equation, the response surface behavior was explored for the response function (Yi). The generalized response surface model is as shown below:

Yi = a 0 + a1X1 + a2X2 + a3X3 + a12X1X2 + a13X1X2 + a23X1X2a11X12 + a22X2 2 + a33X3 2

(2)

where Yi is the predicted response, Xi the independent variables, a0 is a constant, ai, aii, and aij are the linear, quadratic, and interactive coefficients, respectively [19]. An appropriate polynomial model was chosen, based on the statistical significance of the model (P,0.05) and the lack-of-fit value of the model provided by Design-Expert software not being significant [20]. The goodness of fit of the model was evaluated by coefficient determination (R2) and analysis of variance (ANOVA) [21]. Response surfaces and 3D contour plots of the fitted polynomial-regression equations were generated to visualize the interaction effect of the independent variables on response variables better [22]. 2.2.9.2. Verification of models. Quantitative comparison between the theoretical prediction and obtained experimental values was made to validate the models. The predicted error is the difference between the experimental value and the predicted value divided by predicted value [23].

Analgesia percentage =

writhing number of model group writhing number of medicated groups writhing number of model group × 100

(3)

where, model group is the mice belong to group 2 (negative control) and medicated groups are the mice belong to group from 3 to 6. All the mice were then sacrificed by cervical dislocation and the uterine tissues were harvested for histopathological examination.

2.2.10. Transmission electron microscopy A negative staining technique using 1% solution of phosphotungstic acid (PTA) sodium salt at pH 7.4 was employed. A drop of diluted emulsion [1:5 dilution with double distilled water (DDW)] was placed on a carbon‑copper grid before keeping the grid for negative staining. The excess of emulsion was wiped off by the filter paper and then air dried at room temperature before being observed under transmission electron microscopy (TEM, CM-12, Philips, Eindhoven, The Netherlands).

2.2.13. Histological analyses of uterine tissue sections by hematoxylin and eosin staining Mice uterine tissues were fixed in 10% buffered formalin for 24 h or longer at room temperature, then embedded in paraffin. Tissues were sectioned at 5 μm thickness and stained with hematoxylin and eosin (H & E) using standard protocols described by Zhang et al. [29]. Images were acquired using on Olympus microscope equipped with a DP-26 digital camera.

2.2.11. Stability study The optimized GIN-loaded Janus macroemulsion was observed over a period of 6 months at 4 °C or until instability was observed at room 279

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Fig. 1. (A) HPLC chromatogram observed with GIN (50 mg)-loaded oil-in-water macroemulsion. Insert image shows the calibration curve made by HPLC method for 6-gingerol in 90% methanol, (B) Distribution of (dispersed oil) droplet size of ginger (GIN)-loaded oil-in-water macroemulsion analyzed utilizing a Malvern Mastersizer, (C) Image of olive and silicone oils-based macroemulsion depicting a clear paired-beans dispersed oil droplets structure in photomicrography and (D) Distorted paired-beans structure in transmision eletron miroscopy.

2.2.14. Statistical management Results were reported as mean ± SEM. The obtained data were analyzed by one-way analysis of variance (ANOVA) followed by Tukey's test. Statistical significance was determined at p value < 0.05 and 0.01.

Nikam et al. [30] states that the peak of 6-shogaol possessed the retention time value of 4.442 min in the HPLC chromatogram. In the current experimental condition, the peak obtained at the retention time value of 4.48 could be related to the presence of 6-shogaol in the GIN rhizome. Although both 6-gingerol and 6-shogaol possessed multiple molecular targets, including inflammatory mediators [31], the amount of 6-gingerol present in GIN rhizome is always higher than the amount of 6-shogaol (5.68 μg/mg vs. 2.95 μg/mg, respectively) according to the report of Zhang et al. [29]. Moreover, the HPLC chromatogram shown in Fig. 1A also indicated a higher peak height for 6-gingerol in comparison to peak height produced by 6-shogaol. Hence 6-gingerol was selected as a marker compound to indicate the various characterization works (drug solubility and percentage drug entrapment efficiency) of GIN-loaded Janus macroemulsion. Furthermore, the calibration curve

3. Results and discussion 3.1. Marker compound selection Fig. 1A shows the HPLC chromatogram obtained with GIN-loaded Janus macroemulsion. Two peaks observed in the HPLC chromatogram, one at 2.880 min and another at 4.487 min, indicated the presence of 6gingerol and 6-shogaol, respectively [13,30]. In fact, the reference of 280

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Fig. 2. Response surface plots showing the interaction effects of (A) drug composition and silicone oil composition on R1, drug entrapment efficiency and (B) drug composition and silicone oil composition on R2, particle size. Response surface plot showing the interaction effects of olive oil composition and drug composition on response R1, drug entrapment efficiency (C) and response R2, particle size (D).

for 6-gingerol was also made using the HPLC method and it is shown as insert image in Fig. 1A.

3.3. Mechanism of Janus macroemulsion formation Preliminary screening experiments indicate that using the single oils (castor, coconut, olive and silicone) always showed a tendency to form the o/w macroemulsions but without the paired-beans dispersed oil droplets structure. Similarly, using the oil combination with the exclusion of silicone oil as one of the components also showed the same tendency of forming macroemulsion without the paired-bean dispersed oil droplets structure. However, the olive and silicone oils combination were able to form the o/w macroemulsions having paired-beans dispersed oil droplets structure (Fig. 1C). Accordingly, it was decided to work with olive and silicone oils combination at varying ratios for producing paired-beans dispersed oil droplets structure within the final o/w macroemulsions. However, the question of evidence to show that the macroemulsion formed indeed have Janus structure, i.e. different

3.2. Processed and unprocessed GIN solubility A pilot GIN (processed and unprocessed) solubility study was carried out for the identification of lipophilic vehicle to formulate the o/w macroemulsions having paired-beans dispersed oil droplets structure. The solubility of GIN (i.e., 6-gingerol) was tested in the selected single oils or oil mixture, water with Tween 80 and water alone (see in supplementary Table 1). It appears that the drug solubility did not vary significantly between processed and unprocessed GIN at all the solvents tested. Irrespective of the tested solvents, the solubility of GIN was ranged from 14.23 ± 0.2 to 20.8 ± 0.3 mg/ml only.

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Fig. 3. (A) Image of mice typical vaginal smear following the intraperitoneal injection of oestradiol benzoate to establish the animal model of primary dysmenorrhea (PD). Insert image shows the vaginal smear of mice at unestablished PD condition. Histomorphological changes observed after the H & E staining of uterine tissue sections collected from the mice following the treatment of normal control (B), negative control (C), positive control (D), placebo (E) and tested GIN-loaded macroemulsions at low (F) and high (G) doses under light microscope at × 200 magnification.

composition in the two parts, remains unclear. But it can be deduced from the report shown by Teixeira et al. [5] wherein the presence of cationic lipidic stearylamine promotes the elevation of polyoxyethylene structure of the hydrophilic poloxamer-188 towards the water dispersion medium of o/w emulsion system. Instead of the competitive forces generation by these two combined emulsifier molecules (stearylamine and poloxamer-188) to form unexpected handbag structure or two compartmental structures within the emulsion dispersion system, the

use of two different non-volatile oils (silicone and olive oils) and onesingle emulsifier molecule (Tween 80) might have lead to the formation of paired beans (Janus) structure in the current macroemulsion system. This could further be corroborated or substantiated with the experimental works performed by the research group of Prof. Stig E. Friberg who linked the importance of thermodynamics, interfacial tension equilibrium and interfacial free energy on the Janus and/or separate drop topology when the emulsification was performed using a 282

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Table 3 Writhing reaction and analgesia percentage values obtained for normal, negative and positive controls, placebo macroemulsion and ginger (GIN)-loaded macroemulsions at low and high doses in primary dysmenorrhea (PD) mice model. Group

Count of writhing reaction for 30 min

Analgesia (%)

0 41.67 ± 5.04 9.83 ± 1.60 37.83 ± 1.94 14.33 ± 1.97 4.67 ± 1.63

100.0 0.0 76.4a 9.2 65.6a 88.8a

Normal control (healthy mice) Negative control (PD model mice without treatment) Positive control (aspirin treated) Placebo (macroemulsion without GIN) GIN-loaded macroemulsion at low dose GIN-loaded macroemulsion at high dose a

Significant difference at p value of 0.05 (one way ANOVA).

combination olive/silicone oil with an aqueous solution of Tween 80 [32]. Additional experimental works are currently undergoing in our laboratory to see the feasibility of entrapping both hydrophobic and hydrophilic drug molecules by utilizing the paired beans (Janus) structure which was currently observed in macroemulsion system. However, the formulation parameters need to be systematically optimized firstly before exploiting the therapeutic utility of the observed paired beans structure of the macroemulsion. Thus the current investigation majorly focused on the systematic optimization followed by pain-reduction capability checking in a PD mice model and verification of the histomorphological changes after the H & E staining of uterine tissue sections collected from the mice.

exhibited a positive effect on the response of DEE values (R1) while for mean particle size values (R2), all independent variables presented negative effects except for one independent variable (ginger composition, A). The interaction coefficients with more than one factor or higher order terms in the regression equation of CCD effects represent, respectively, the interaction between terms or the quadratic relationship and thus suggesting a non-linear relationship between independent factors and response variables [34]. In this condition, the independent factors can produce a different degree of response than is predicted by the regression equation if they are varied at different levels or more than one factor is changed simultaneously [33]. In the current CCD modeling, both the DEE and mean particle size responses (R1 and R2, respectively) were affected by the interaction of independent variables and thus presenting a quadratic relationship. The interaction effect between A and C was favorable only for the mean particle size response variable R2 (positive regression coefficient value of 28.56937). However, the other two interaction effects, between A and B and between B and C, produced an inverse effect (negative regression coefficient values) against both of these two responses variables studied. Similarly, a negative regression coefficient value (−4.66184) was also observed for the interaction between A and C against the DEE response variable R1. Interestingly, quadratic effects were also noticed between all three independent variables and both of the studied two response variables (R1 and R2). The highest and positive quadratic effect for all three independent variables was noted for the DEE response variable R1 (positive regression coefficient value of 10.82848) while the highest and negative quadratic effect was seen for the mean particle size variable R2 (negative regression coefficient value of −32.2542). The coefficient significance of the quadratic polynomial models was evaluated by using Analysis of Variance (ANOVA). For any of the terms in the models, a large F-value and a small p-value indicated a more significant effect on the respective response variables [35]. The independent variables that most affect the DEE values (R1) of the macroemulsions for the linear term (main CCD effect) were drug composition (A), followed by the linear term of silicone oil composition (B) (see supplementary Table 2). However, the other one linear term (olive oil composition, C) did not indicate any significant effect (p > 0.05). The quadratic CCD effect of drug composition and silicone oil (A2 and B2) also had a significant effect (p < 0.05) on the DEE values of macroemulsions. Conversely, the effect of the other one quadratic term (olive oil composition, C2) was insignificant (p > 0.05). Furthermore, the interaction CCD effect between drug and olive or silicone oil composition (AB and/or AC) showed a significant effect (p < 0.05) on the DEE values of macroemulsions while the interaction CCD effect between silicone oil and olive oil composition (BC) showed insignificant effect (p > 0.05) on the DEE values of macroemulsions. The independent variable which exhibited the largest effect on the mean particle size value (R2) of the macroemulsions for the linear CCD effect was olive oil composition (C). The other two independent variables [drug composition (A) and silicone oil composition (B)] showed insignificant effects. The quadratic CCD effect of drug composition (A2) showed significant effect while the silicone oil (B2) and olive oil (C2)

3.4. Particle size analysis, photomicrography and transmission electron microscope Fig. 1B presents a normal droplet size distribution curve of olive and silicone oils-based macroemulsion made with 50 mg processed GIN. The Fig. 1B also shows the mean droplet diameter value [d(0.5)] of 272.51 μm. Fig. 1C depicts the microscopic image of olive and silicone oils-based macroemulsion. The photomicrography indicated the presence of dispersed oil droplets with paired-beans structure (Fig. 1C). However, the TEM image depicts the presence of distorted paired-beans oil droplets structure (Fig. 1D). 3.5. Formulation optimization by quality by design (QbD) 3.5.1. Fitting the response surface models To determine the best fitted quadratic equation in CCD model, the parameters of independent variables (such as normal and adjusted regression coefficients, regression probability and lack of fit values) along with ANOVA of regression coefficient values for CCD effects (main, quadratic and interaction) were statistically analyzed against the dependent variables (see supplementary Table 2). A positive value in the regression equation signifies an effect that favors optimization due to a synergistic effect, while a negative value indicates an inverse relationship or antagonistic effect between the factor and the response [33]. It should be mentioned that non-significant (p < 0.005) linear terms (main CCD effect) were included in the final reduced model if quadratic or interaction terms containing these variables were found to be significant (p < 0.05). In the present study, the RSM demonstrated that the second-order polynomial used for DEE values has a higher coefficient of determination (R2 = 0.9482) compared to the mean particle size value (R2 = 0.7347). The obtained coefficient of determination showed that > 90% of the response variation of the mean particle size and DEE values could be described by response surface models as the function of the main macroemulsion and preparation variables. It was observed that the lack of fit had no indication of significant (p < 0.05) for the final reduced model, therefore proving the satisfactory fitness of the RSM to the significant (p < 0.05) independent factors (variables) effect. It was also observed that only two independent variables (B and C) 283

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exhibited insignificant effects on the mean particle size (R2). The interaction CCD effect between drug and olive oil compositions (AC) showed a significant effect on the mean particle size (R2) compared to the other two interaction CCD effects (AB and BC). The final polynomial equations for the response variables studied according to the tested CCD model are shown below.

Drug entrapment efficiency (R1) = 58.56 + 1.59 × C

surface of the macroemulsion, the separated oil droplets were re-dispersible on mild shaking. But the Janus macroemulsion stored at 25 °C did show thick oil phase layer floating on the surface of the emulsion after 3 weeks storage time period indicating a possible emulsion instability problem. 3.7. Primary dysmenorrhea mice model assessment

6.19 × A + 3.44 × B 4.48 × AB

4.66 × AC

1.21 × BC + 10.83 × A2

2.29 × B2

+ 0.14 × C2

Mean particle size (R2) = 227.45 + 17.78 × A

1.25 × B

5.15 × AB + 28.57 × AC

Fig. 3A depicts the vaginal smear taken from the mice after the 3 days intraperitoneal injection of oestradiol benzoate to establish PD. Presence of characteristics features of estrus cycle such as nucleated epithelial cells, white blood cells and few keratinocytes are evident in Fig. 3A. The insert image of Fig. 3A shows the vaginal smear of mice at unestablished PD condition containing the normal cell structure.

(4)

24.48 × C

7.76 × BC

32.35 × A2 + 1.79 × B2 + 2.05 × C2

(5)

3.7.1. Writhing reaction and analgesia percentage Table 3 shows the oxytocin-induced writhing reaction observed after the administration of negative control, positive control, placebo and tested GIN-loaded macroemulsions at low and high doses in PD mice model along with the calculated analgesia percentage. Irrespective of low and high doses of GIN-loaded macroemulsion tested for oxytocin-induced writhing reaction, the obtained counting of writhing reaction did show significant difference in comparison to counting of writhing reaction observed with negative control samples. However, the counting of writhing reaction produced by the tested GIN-loaded macroemulsions at low and high doses did not reveal significant difference in comparison to the counting of writhing reaction shown by the positive control (aspirin treated). On the other hand, the analgesia percentage calculated using the Eq. (3) indicated a significant difference (one-way ANOVA) between positive control and tested GIN-loaded macroemulsions at low and high doses.

3.5.2. Response surface analysis The response surface plots for drug entrapment efficiency and particle size are presented in Fig. 2A,B,C & D. Fig. 2A demonstrates that the DEE increases with increasing silicone oil composition and decreases with increasing the composition of drug. A few explanations need to be considered to describe the observed results of DEE. First, with the rise in the oil content, the droplet disruption process becomes more difficult which is due to an increase in the flow resistance and hence the droplet break-up rate becomes severely restricted [36,37]. Second, part of the effect can be attributed to the increased rates of collision frequency, particularly at lower concentration, between the emulsion droplets followed by an ultimate increase of coalescence frequency which subsequently lead to a higher probability of coalescence of the droplets [19]. As shown in Fig. 2B, the increase in the drug amount led to decrease in particle size. The increase in particle size may result from an impoverishment of the surfactant at the interface with increasing surface of the dispersed oil phase [37]. Decrease of particle size by further increase in Tween 80 is due to the fact that the emulsifier plays a vital role in the formation of emulsion as it lowers the interfacial tension, thereby the Laplace pressure, p is reduced, and the stress required for droplet deformation is reduced [19]. Fig. 2C demonstrates that the DEE decreases with increasing olive oil composition. As shown in Fig. 2D, the increasing the drug content led to decrease in particle size as usual.

3.8. Uterinehistomorphology Fig. 3 shows also the observed histomorphological changes after the H & E staining of uterine tissue sections collected from the mice following the treatment of normal control (B), negative control (C), positive control (D), placebo (E) and tested GIN-loaded macroemulsions at low (F) and high (G) doses under light microscope at ×200 magnification. While the negative control (Fig. 3C)- and placebo (Fig. 3E)treated mice possessed discernible morphological changes in the tissue sections indicating the development of PD mice model. An almost similar histomorphological structure was visible between Fig. 3B and G. This indicates that treating the PD mice model with high dose of GINloaded macroemulsion resulted in the restoration of the normal histomorphological structure of uterine tissue sections in comparison to the normal control tissue structure. However, there was a considerable delay in the restoration of normal histomorphological structure of uterine tissue sections when the PD mice model was treated with positive control (Fig. 3D) and low dose of GIN-loaded macroemulsion (Fig. 3F).

3.5.3. Optimization of responses for formulating GIN-loaded macroemulsion An optimum GIN-loaded macroemulsion should contain the largest particle size and highest drug entrapment efficiency. By investigating the interaction CCD effect between the independent variables and evaluating the optimization constraints, the optimum GIN-loaded macroemulsion was found to have a composition of 50 mg drug, 4 ml silicone oil and 1 ml olive oil. Based on the optimum formulation, the theoretically predicted values of DEE and particle size are 76.29% and 245.99 μm, respectively. 3.5.4. Verification of the reduced models Through experimental work, it was found that the optimized formulation of GIN-loaded macroemulsion possessed a DEE value of 76.09% and particle size value of 272.51 μm. No significant difference was noted between the experimentally determined and theoretically predicted values of DEE (76.09 vs. 76.29%) and particle size values (272.51 vs. 245.99 μm) indicating suitability of CCD model tested for optimization of the Janus macroemulsions.

4. Conclusion Occasionally, few unusual liquid interfacial structures are seen in emulsion. The Janus structure reported in the present study is one of those unusual interfacial arrangements of dispersed oil droplets of the macroemulsions. With the lone exception of surface topology observed under optical microscopy, no other direct evidence was shown in the current investigation for Janus structure i.e. different composition in the two parts. Since the Janus macroemulsion was optimized in a systematic manner, the potential of the paired beans (Janus) structured of oil droplets of the o/w macroemulsions should further be exploited towards entrapping the plant-derived drugs having two dissimilar physicochemical characteristics but similar therapeutic activity.

3.6. Stability study The optimized Janus macroemulsion was stable at 4 °C for over a period of 90 days. Although slight oil phase separation was found at the 284

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Acknowledgement

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