2.1 Materials
BM seed oil was obtained from Suyash Ayurveda, Gujarat. Lipoid, GmbH provided a complimentary sample of Phospholipon 90G (PL90G) from Germany. Hexane, ethanol, dimethyl sulphoxide (DMSO), triethanolamine (TEA), potassium hydroxide (KOH), sodium hydrogen sulphate (NaHSO4) were purchased from Mumbai-based Loba Chemie Pvt. Ltd. India. Sodium sulphate anhydrous was procured from Thomas Baker Pvt. Ltd. Mumbai, India. Carbopol 934, Yeast Extract-Peptone-Dextrose, Roswell Park Memorial Institute- 1640 medium, Mueller Hinton Broth, Mueller Hinton Agar media were procured from Himedia Laboratories, Pvt, Ltd. Mumbai, India. Formaldehyde solution was procured from CDH Pvt. Ltd., New Delhi, India. Clotrimazole cream, used in the in vivo studies, was procured from local pharmacy. Other chemicals were of analytical grading, and glasswares were borosilicate.
2.2 Methods
2.2.1 Analysis of BM seed oil by Gas Chromatography (GC)
Derivatization of fatty acids present in BM seed oil
The fatty acids in seed oil of BM (20 µL) were transformed to fatty acid methyl esters (FAME) for evaluation using gas chromatography/mass spectroscopy. Fatty acids in 20 µL of oil were transesterified at 55°C for 1.5 hours in 0.7 mL of 10 N KOH, and 5.3 mL of methanol in a water bath. This was allowed to cool before adding 0.58 ml of 24 N H2SO4 and reversing the process. After another 1.5 hours of incubation at 55°C, the sample was cooled down. 3ml hexane was added and then vortexed for 5 minutes followed by centrifugation at 5000 rpm for 5 minutes. The esterified compound obtained in the hexane layer was further tested by GC for estimation of alkyl derivatives that is methyl esters formation of fatty acids (FAME) ( Ito et al., 2014; European Pharmacopeia, 5th ed).
Chromatographic system
On the gas chromatograph (GC-2014), a flame ionization detector and a 0.25 mm 30 m fused silica capillary column encapsulated with a 0.25 µm FAMEWAX™ column (cat # 12497) were installed (GC-2014). The injection port and detector temperatures were kept at 225°C and 230°C, respectively. The column temperature process was initially set to 165°C for the first 30 minutes, then increased at a rate of 1.5°c/min to 220°C, where it remained stable for the final 15 minutes. The carrier gas, helium, was partitioned with a flow ratio of 1:40 at an initial temperature of 40 cm/sec and a flow rate of about 0.5 mL/min.
2.2.2 Physical characterization of oils
BM seed oil was evaluated for physical characteristics which included color, odor and clarity.
2.2.3 Development of BM seed oil-loaded EVs
In the present research work, EVs were formulated using BM seed oil for treating VVC using the ethanol injection-sonication method.
BM seed oil and PL90G were dissolved in ethanol (organic phase) and then the organic phase was firmly injected dropwise into the aqueous phase with a syringe for half an hour with constant stirring at 1000 rpm and sonicated for 20 minutes. The prepared BM oil loaded EVs were refrigerated for further characterization and evaluation. The preparation process is exhibited in the Fig. 1.
2.2.4 Selection of Phospholipid
The ability of various phospholipids to form ethanolic vesicles (EVs) with desired properties was tested. PL 90G, Phospholipon 85G (PL 85G), and liquid lecithin were among the phospholipids studied. Phospholipids were used without further purification.
2.2.5. Selection of concentration of BM seed oil, ethanol, and phospholipid
A total of 6 formulations were synthesized with BM seed oil, ethanol and phospholipid ranging in concentration from 5–15%w/w, 10–25%w/w, and 5–10% w/w respectively during preliminary studies (Touitou et al. 2000), displayed in Table 1.
Table 1
BM seed oil, phospholipid and ethanol concentrations in various preliminary EV formulations
Formulations
|
BM seed oil (g) (F1-F6)
|
Ethanol (g)
|
PL 90G (g)
|
F1
|
0.5
|
1
|
1
|
F2
|
0.5
|
2
|
1
|
F3
|
0.5
|
2.5
|
1
|
F4
|
1.5
|
1
|
1
|
F5
|
1.5
|
2
|
1
|
F6
|
1.5
|
2.5
|
1
|
2.2.6 In-process Vesicle Monitoring
Throughout the studies, prepared EVs were monitored for structural integrity and micromeritics using a high accuracy optical microscope (magnification: 40X, 100X) equipped with a camera. During the studies, various vesicular properties such as shape, vesicle count, percent light transmittance, disruption, lamellarity, vesicle coalescence, aggregation, and physical stability of the drug for 15 days in the vesicles were supervised.
2.2.7 Systematic optimization of BM seed oil loaded EVs as per the experimental design
QbD is a systematic, organised process concerned with pharmaceutical product quality. It then runs specific key factors representing the independent variables and examines their impact on the dependent observed responses. In other words, QbD provides and implements a composition — for the model to be designed and meet the specified standards. Response surface methodology (RSM) is a tool that generates a large amount of data with minimal input. (Ismail et al., 2021).
A 3-factor, 3-level BBD was used to optimise BM seed oil-loaded EVs (BBD). The chosen dependent variables were the concentration of BM seed oil (X1), PL 90G (X2), and ethanol (X3), applied at 3 distinct levels of each variable, viz. low (5%), medium (17.5%), and high (25%). The response variables viz. vesicle size, PDI and entrapment efficiency (%EE) were evaluated using numerous ethanolic vesicular formulations prepared according to the design. Table 2 outlines the 17 experimental runs analysed, as well as the coded values for the studied factors. Following the evaluation of prepared EVs for the response variables, optimization statistics analysis was accomplished. Design Expert® ver.10.0.1 was used to produce 3-D response surface plots and 2-D contour plots for the response surface evaluation (MS Stat-Ease Inc., Minnepolis, MN) (Negi et al., 2015).
Table 2
Factor combinations as per the chosen experimental design (i.e., BBD) for BM seed oil-loaded EVs
Codes of EVs
|
Trial No.
|
Coded Factor levels
|
X1
|
X2
|
X3
|
F1
|
1
|
-1
|
0
|
1
|
F2
|
2
|
-1
|
0
|
-1
|
F3
|
3
|
1
|
0
|
-1
|
F4
|
4
|
-1
|
-1
|
0
|
F5
|
5
|
0
|
0
|
0
|
F6
|
6
|
0
|
1
|
-1
|
F7
|
7
|
0
|
0
|
0
|
F8
|
8
|
0
|
0
|
0
|
F9
|
9
|
0
|
-1
|
1
|
F10
|
10
|
0
|
0
|
0
|
F11
|
11
|
0
|
0
|
0
|
F12
|
12
|
1
|
-1
|
0
|
F13
|
13
|
1
|
0
|
1
|
F14
|
14
|
-1
|
1
|
0
|
F15
|
15
|
1
|
1
|
0
|
F16
|
16
|
0
|
-1
|
-1
|
F17
|
17
|
0
|
1
|
1
|
Actual unit translation of coded levels
|
|
(BM-EVs)
|
Coded level (EV1 to ETHO17)
|
-1
|
0
|
1
|
X1: BM seed oil (%)
|
5
|
10
|
15
|
X2: PL (%)
|
5
|
7.5
|
10
|
X3: Ethanol (%)
|
10
|
17.5
|
25
|
2.2.8 Characterization of prepared EVs
Particle size of BM seed oil loaded EVs
Particle size and ethanol concentrations have an impact on skin permeability and are considered as vital parameters. The small vesicle size allows for good penetration through the skin. Zetasizer from Malvern, UK was used to assess the average diameter and PDI of the prepared BM seed oil-loaded EVs (Malvern, UK). The samples (1ml) were analysed 24 hours after they were prepared. The samples were inserted in a polystyrene sample cell, and the results were kept track of. To circumvent multi-scattering phenomena, EV dispersions were diluted 1/4th with distilled water prior to measuring of particle size, and each sample test was performed three times. A photomultiplier was used to quantify the intensity of the laser light dispersed by the samples at a 90º angle. Because of random collisions with solvent molecules, particles suspended in fluids were in Brownian motion. The diffusion coefficient is inversely proportional to particle size, according to the Stokes-Einstein equation. The width of the size distribution was measured using PDI. A PDI of less than 0.4 indicates that the vesicles are homogeneous and monodisperse (Pathan et al., 2016).
High resolution Transmission Electron Microscopy (HR-TEM) BM seed oil-loaded EVs
The samples' surface morphology was evaluated using HR-TEM. A drop of prepared EVs was put on a carbon film-covered copper grid prior to the observation, and the specimen was negatively stained with a single drop of 1% aqueous sodium phosphotungstate solution. After removing excess liquid, the specimen was allowed to air dry prior to TEM evaluation (Safwat et al., 2017).
Field emission scanning electron microscope (FESEM) of the BM seed oil-loaded EVs
FESEM also assessed the shape and size of the EVs. On a clear glass stub, a single drop of EVs was mounted. They were then air-dried and encapsulated with a very thin gold coating to allow for FESEM visualisation.
FESEM has been developed as a modern version of SEM with a magnification of approximately 300,000x and an increasing voltage range of about 0.1 to 30 kV is used. Despite having the same instrument configuration, FESEM is preferred over SEM due to higher image resolution. The cold cathode field emitter of a field emission gun (FEG) heats up the tungsten filament by immersing it in a large power potential gradient, which distinguishes the FESEM from the SEM. The electric gun creates a strong vacuum in the cabinet, outperforming the traditional tungsten filament and enabling for faster scanning than SEM. Furthermore, using metal apertures and magnetic lenses, electron beams in FESEM are constrained and concentrated into a thin monochromatic beam (Mahmood et al., 2017).
Percent entrapment efficiency (%EE)
The GC FAME method was used to calculate the %EE of BM oil-loaded EV. Using the centrifugation method, the % EE was measured in triplicate. The EVs were centrifuged at 10000 rpm (M/s REMI CPR 24) for 10 minutes at 4o C. After lysing with hexane, the drug content was identified in both the vesicular sediment and clear supernatant.
The following equation was used to calculate the drug's % EE:
where, T denotes the total amount of drug found in both the sediment and the filtrates, and C denotes the amount of drug found only in the supernatant (Negi et al., 2017).
2.2.9 Preparation of ethanolic vesicular hydrogel
Carbopol 934 tends to form a very good consistency and clear gel at low concentrations. A small amount of distilled water was used to soak Carbopol 934 for an hour. Triethanolamine was then used to adjust the pH to neutral. Under continuous stirring, the required amount of BM seed oil-loaded EVs were added to the swollen polymer until homogeneous, clear, and transparent gel was achieved (Shukla et al., 2020; Indora and Kaushik, 2015).
2.2.10 Characterization of ethanolic vesicular hydrogel
Rheological studies
Rheological analysis is a crucial component of quality control. Rheological parameters such as yield point, flow behaviour, thixotropy, and apparent viscosity can all help predict how a product will react during preparatory work, handling, and finally skin application. Rheological measurements were conducted within 24 hours of preparation (Wojciechowska et al., 2021).
Rheological studies on the optimised ethanolic vesicular hydrogel were carried out using a rotary type rheometer (Rheolab QC, M/s Anton-Paar GmbH, Vienna, Austria) equipped with DG26 spindle geometry for assessing the sample's viscosity and torque and a water jacket (C-LTD80/QC) to keep a constant temperature of 25°c. The optimised formulation was subjected to individual shear rate and shear stress conditions, and data were analysed using Rheoplus/32 ver 3.40. Shear stress was gradually increased from 0.1 to 100 s− 1. For all samples evaluated in triplicate, the mean value was calculated using the Power-law and Herschel-Bulkey models (Garg et al., 2017).
2.2.11 Minimum Inhibitory Concentration (MIC) determination of BM seed oil loaded EVs and ethanolic vesicular hydrogel in fungal strains
Fungal strain of Candida (ATCC 90028) was used for this study. At 4°c, the fungal strains were maintained in YPD broth and agar media.
MIC is referred as the extract’s minimum concentration needed to prevent/arrest the development of all microorganisms in the culture (Soumya and Nair, 2012).
Heat maps were designed to assist in the visualisation of antifungal susceptibility data, with optical density values represented quantitatively with colour. In flat-bottom 96-well microtiter plates containing RPMI 1640, the MIC of BM seed oil-loaded EVs and ethanolic vesicular hydrogel against the test fungus was determined.
The BM seed oil-loaded EVs and ethanolic vesicular hydrogel were selected from the 10% x-axis range. Each well received 50 µl of working inoculum suspension (1×108 cfu/ml). A number of wells in each plate were set aside for sterility (no inoculum), inoculum survival (no sample solution), and inhibitory effect control with dimethyl sulfoxide (DMSO) (no sample added). For 48 hours, the plate was incubated at 30°C. The colour change was then visually observed after the dye resazurin was added. Colour change from purple to pink or colourless indicated growth. MIC values were determined by the lowest concentrations at which the colour changed from purple to pink.
2.2.12 Anticandidal activity by agar well diffusion
The agar well diffusion assay is commonly used to test the antifungal potential of BM seed oil loaded EVs and ethanolic vesicular hydrogels. Agar well diffusion was carried out with the help of agar extract and yeast extract peptone dextrose broth (YPD). The media was autoclaved for 15–20 minutes at 121° cointegrated at 15psi. Following that, the media was transferred into Petri plates within the laminar airhood and agar was allowed to solidify. After the medium solidified, four 4 mm diameter wells were snipped out of the agar and each well received 10 µl of the antifungal agent (fluconazole, positive control), BM seed oil loaded EVs, ethanolic vesicular hydrogel, and DMSO (negative control). The plates were kept for 42–72 hours at 35ºc. The diameter of a clear zone of inhibition for fungal growth was later determined.
2.2.13 Vaginal candidiasis mice model for in vivo studies
For the current study, pathogen-free BALB/c female mice (20–25 gm) were divided into four groups. The animals were kept in the in vivo testing facility at 25 ± 3ºC with a 12-hour light/dark cycle, and were fed a nutritious diet and pure water. The CPCSEA approved all animal experiments and the study through the Institutional Animal Ethics Committee of Shoolini University (Protocol no. IAEC/SU/21/01).
C. albicans was cultured on Sabouraud's dextrose agar. Prior to vaginal C. albicans infection, mice were given a single cyclophosphamide dose, intraperitoneally (200 mg/kg body weight). Estradiol valerate in the dose of 0.5 mg/mice was subcutaneously given from day − 3 to day + 4 (8 days) to stimulate the condition of pseudo estrous. For three days, vagina of mice was inoculated with a clean and fresh suspension of C. albicans (50 µl) comprising 1.2 × 109 colony forming unit/ml (cfu/ml) (day 0, 1, 2).
Itching, vaginal discharge, redness, and high swelling appeared three days later. The test substances were administered intravaginally to mice (50 mg each of BM seed oil, BM seed oil-loaded EVs, and ethanolic vesicular hydrogel) after infection was confirmed. Infection control was presented by a group that was infected but not treated, while negative control was presented by a group that was neither infected nor treated. The standard group received marketed cream (clotrimazole, 50 mg) once daily for three days (Srivastava et al., 2018).
2.2.14 Histopathological analysis of vaginal tissue
Following the completion of the in vivo study, the animal was killed and the medicated skin area was removed. Each sample was fixed in 10% formalin solution before being cut into transverse sections for histological evaluation. Before being examined microscopically for changes in vaginal tissue, samples were properly analysed and stained with haematoxylin-eosin (Kirici et al., 2021).