Authentification of crude sample
Crude samples of garlic and ginger were sent for authentification to CSIR- National Institute of science communication and information resources.
The crude sample of Aceclofenac was received as a gift sample from East African Company, Dehradun.
Extraction of essential oil
Steam distillation was employed for extraction of essential oil from buds of
Allium sativum. 100 gm of buds were crushed placed in conical flask which was connected to the Clevenger apparatus. Appropriate amount of distilled water was filled in the flask and heated up to boiling point. Steam triggered the release of the aromatic molecules present in the garlic buds. The combination of steam and essential oils was present and collected into measuring cylinder and allowed to stand for about 5 hr after which oil layer got separated and was collected.
(Bagudo et al., 2014).
Same procedure was adopted for crushed rhizomes of
Zingiber officinale.
Phytochemical screening og isolated material
Various tests were performed on isolated essential oils from
Allium sativum and
Zingiber officinale and extract from
Sapindus mukorossi. Various tests are explained as follows:
Specific gravity of oil
(Bagudo et al., 2014).
Take around 10 ml of oil in a pre-weighed weighing bottle. The specific gravity of oil can be calculated as.
Where:
W1= Weight of empty weighing bottle +oil.
W0 = Weight of weighing bottle.
V0= Volume of oil used.
Saponification value determination
Add 30 ml of ethanolic KOH to a flask containing around 2 g of the precisely weighed extracted oil. For an additional 30 minutes, it was connected to a condenser to ensure that the sample was completely dissolved. After cooling add 1 ml of phenolphthalein titrate with 0.5 M HCl until a pink endpoint.
Where:
S= Implies sample titre value.
B= Blank titre value.
M= Molarity of the HCl.
56.1 represents molecular weight of KOH.
TLC of ginger essential oil
Essential oil of
Zingiber officinale was also analyzed by Thin layer chromatographic analysis method.
Mobile phase
Combination of Toluene: Ethyl Acetate: Formic Acid in the ration of 9:1:2.
TLC plates: Percolated silica gel 60F254.
visualizing agent: Anisaldehyde- sulfuric acid.
Plates were visualized both in day light and UV short and long wavelength. The
Rf value of spots was calculated (
Syed Shariq Mian, 2019).
Emulgel formulation
For the formulation take mentioned quantity of methanol and dissolve drug and essential with continuous stirring. Dissolve carbopol-934 in water into another flask. Continue stirring till carbopol is completely dissolved. To neutralize carbopol-934 add triethanolamine. Add drug solution to oily phase with little amount of Tween 80 with continuous stirring.
Finally solution was continuously stirred for upto 2 hours. Lastly add benzyl alcohol and ethylene glycol with continuous stirring.(Singh V.K 2013) Similarly nine different batches were formulated. For
Allium sativum formulations were coded as FA1-FA9 (Table 1) and in case of
Zingiber officinale coding were FZ1-FZ9 (Table 2).
Evaluation of emulgels
All the formulated emulgel loaded with natural oils were further evaluated for various parameters. All formulation were inspected for color and homogeneity.
For pH determination disperse 1.0 gm of formulation in 20 ml of distilled water and note pH using calibrated PH meter (
Patel, 2011).
To measure spreading coefficient a circle with 1 cm diameter is marked in glass plate and spread 0.5 gm of formulated gel within circle. Place a second glass plate above it. Place around 500 gm of weight above the first glass plate for around 5 min. It cause an increase in the diameter of circle due to the spreading of the emulgel which can be calculated (
Nanda, 2014). Brookfield Viscometer was used for Viscosity Measurement in cps.
For extrudability test aluminum collapsible tubes were filled with 20 g emulgel and exrtrudability was observed (
Kumar, 2010).
To measure Drug content 100 mg of emulgel was dissolved in 100 ml of mixture of phosphate buffer solution pH 6.8 and methanol (60:40) solution and stirred for about 2 hrs. This solution was filtered and the absorbance was measured at 273 nm (
Trivedi, 2013).
Franz diffusion cell was used to study
in vitro drug release and artificial transdermal membrane was employed to mimic the skin membrane (
Shivhare, 2009).
In the receiver compartment phosphate buffer (pH 7.4) was placed and in the donor compartment about 1 gm of Emulgel formulation was placed. The receptor phase was continuously stirred at temperature 37±0.5°C maintained during the process. Between regular time intervals 1 ml of the sample was taken and replaced with fresh buffer solution in receiver compartment. Withdrawn samples were analyzed at 273 nm.
Graphical study was done after plotting release order curves in Excel sheet to further study the type of drug release.
Based on all evaluation parameters best formulation were selected and further subjected to
in-vivo studies.
In vivo studies
For
in-vivo studies authors took permission from IAEC with protocol ID: SIP/IAEC/PCOL/01/2022. Best formulations selected from evaluation studies were used for
in-vivo studies employing albino Wistar rats (200–220 g) to access both analgesic as well as anti - inflammatory activity of best selected emulgel formulation. All the animals were kept under standard laboratory conditions.
All the rats were divided into different groups (n=6):
Group 1- untreated group (Control).
Group 2- received Drug (Standard).
Group 3- treatment with FAB.
Group 4- treatment with FZB.
For analgesic activity method described by Khan
et al was used and around 300 mg of formulation was gently rubbed on dorsal surface of the rat’s right hind paw. After about 30 min subcutaneously 5% formalin (20 μl) was injected over plantar aponeurosis of the right hind paw of rat. The time taken by rats for responses like licking and biting was an indicator of pain response was recorded in seconds for 5 min after injection
(Khan et al., 2015).
Carrageenan-Induced Paw Edema Model was used to evaluate the anti-inflammatory effect of emulgel. 0.1 ml of 1% of carrageenan was administered subcutaneously to right hind paw of rat in the sub-plantar surface. All the formulations were rubbed around half an hour before the carrageenan administration. The paw volume was measured at regular time intervals with the help of a digital Vernier caliper
(Panthong et al., 2007).
SEM analysis
After the in-vivo studies the best formulation was applied to excised rat skin and sent for SEM analysis.
