INTRODUCTION:

Stroke is a neurological deficit caused by an acute focal injury of the central nervous system due to cerebral infarction, intracerebral haemorrhage and subarachnoid haemorrhage. It is one of the leading causes of disability and death¹. Among all strokes ischemic stroke (IS) accounts for 70% in India².

Thrombolysis is the only effective treatment for acute ischemic stroke patients coming within 4.5 hrs of symptoms of onset with improved functional outcomes at 3 and 6 months³. An untreated IS patient loses 1.9 million neurons in each minute and every 15minute reduction of the door to needle time there is a 5% lower odds of in-hospital mortality⁴. Various oral antiplatelet agents including aspirin, clopidogrel and dipyridamole are being used routinely for treatment of acute ischemic stroke. It was reported that early and prompt use of aspirin in patients with suspected acute ischemic stroke reduces the recurrent stroke, death in hospital and improves functional recovery. Cerebral edema occurs in most cerebral infracts ad caused early death. To minimize edema formation before clinically significant increase in intracranial pressure osmotherapy which includes IV mannitol or glycerol is given⁵. Glycerol may act as a free radical scavenger, antioxidant, and activator of plasma prostaglandin (PGI2), resulting in vasodilation. An improvement in ischemic brain energy metabolism after glycerol administration has also been postulated. In the present investigation an attempt was made to develop a novel formulation containing aspirin and glycerol. The main objective is to develop an emulsion composition with glycerol as continuous phase using tween 80 as emulsifying agent and to evaluate for physical and chemical stability.

MATERIALS AND METHODS:

Materials:

Aspirin, glycerol, methanol, acetonitrile, Tween 80, span 80 were all purchased from SD fine chemicals, Hyderabad and olive oil was purchased from the local market of Mahabubnagar.

METHODS:

Establishment of analytical method Several analytical methods are reported^6,7. Here, in this investigation acetonitrile was used as the spectrophotometric solvent to dissolve aspirin. Working standard solutions were prepared in the concentration range 2 to 10µg/ml and a spectrum was recorded for median concentration using UV Visible spectrophotometer (Lab India, UV 3000) and λmax was noted. The absorbances were determined at the λmax. A standard graph was plotted for concentration vs absorbance.

Determination of partition coefficient:

A 5ml of oil (coconut oil or olive oil) and 5ml of Glycerol were taken in a separating funnel. Accurately 10mg of Aspirin was added to the oil glycerol mixture vigorously shaken for 30 minutes. After shaking the mixture was set aside by keep on a tripod stand over night to equilibrate the aspirin in both the phases. Then the oil phase and Glycerol phase were separated and estimated the drug concentration in each phase. 1ml of oil/ glycerol phase was taken and dissolved in few ml of acetonitrile and made up to 10ml with the same solvent. The solution is centrifuged in order to separate the oil droplets measured the absorbance in UV- visible spectrophotometer and partition coefficient and log p value were calculated using standard graph.

Formulation Development and preparation of oil/Glycerol emulsion:

Based on partition coefficient studies olive oil is selected as oil phase. The continuous phase is Glycerol or a mixture of Glycerol and water in different ratios. The emulsion compositions are given in table 1. A quantity of oil was taken in a beaker and accurately weighed quantity of aspirin was added to the oil and dissolved in by keeping the mixture on a water bath at 55⁰C. Continuous phase is prepared by dissolving tween 80 in Glycerol or Glycerol water mixture. The continuous phase is added to the oil phase slowly dropwise while mixing using a high shear homogenizer at 5000rpm until a clear milky emulsion is formed and mixing is continued for 15 minutes. Then emulsion is transferred in to a clean dried bottle and kept aside for 24 hrs then evaluated.

Evaluation of prepared formulation

Prepared emulsions has been evaluated by

i) Visual observation

ii) Determination of drug content

iii) Particle size analysis

iv) In vitro diffusion studies

v) Stability

i) Visual observation:

The prepared formulations were observed visually for phase separation.

ii) Determination of drug content:

A quantity of emulsion equivalent to 1mg of drug was transferred in to a 10ml volumetric flask and few ml of acetonitrile was added to the volume and sonicated for 5minutes to extract the drug into solvent. Then the solution is centrifuged to separate the oil globules. From the supernatant 0.1ml was transferred into another volumetric flask and made up to 10 ml with acetonitrile to produce 10µg/ml of aspirin and absorbance was measured at λmax. Then drug content and %drug content was calculated using the standard graph.

Table 1 Compositions of emulsions

Code/Ingredient	F1	F2	F3	F4	F5	F6	F7	F8	F9	F10
Aspirin(mg)	100	50	50	50	50	50	0	50	50	50
Olive oil (ml)	3	2.5	2.5	2.5	2.5	2.5	1.5	1.5	1.5	1.5
Glycerol (ml)	7	6.5	6.5	6.5	3.25	6.5	4.25	4.25	6.37	8.5
Span 80 (gms)	0.22	0.74	1.11	1.48	3.25	2.2	0	0	0	0
Tween 80 (gms)	0.078	0.26	0.39	0.52	1.11	0.78	1.65	1.65	1.65	1.65
Water	-	-	-	-		--	4.25	4.25	2.13	0

iii) Particle size analysis:

Calibration of ocular/eyepiece micrometer

● In Stage micrometer, the linear scale of 1.0mm length is engraved. The 1mm is divided into 100 equal parts.

● One division of standard stage micrometers is equal to 10 μm or 0.01mm.

● Here, one division is equal to the space between any two divisions.

● We have calibrated the eyepiece micrometer by superimposing a stage micrometer with an eyepiece micrometer.

● In this method, we observed that the two lines completely overlapped the ocular micrometer scale with the stage micrometer scale.

● According to the ocular micrometer, the two lines are superimposed from 20 to 55 divisions i.e 35 divisions.

● Whereas in stage micrometers, it is from 0 to 5 divisions.

1stage division = 10μm

5 stage division = 50μm

35 divisions on ocular micrometer = 50μm

1 ocular division = 1 x 50/35 = 1.42μm.

For measuring particle size one drop of emulsion was placed on a glass slide and covered with a coverslip and 100 particles were taken into account in different microscopic fields. The diameters of the particles were noted.

iv) In Vitro diffusion studies:

● Egg shell was dissolved in 0.1N HCl for 1hour. Semipermeable membrane was obtained by washing with water in a petri dish.

● 100ml of buffer solution was prepared by dissolving 0.23g of Disodium hydrogen phosphate, 0.019g of Potassium dihydrogen phosphate and 0.8g of Sodium chloride in sufficient water to make up to 100ml.

● 100ml beaker was taken and filled with 80 ml of buffer solution.

● A glass tube with two sides open was taken and semipermeable membrane was tied with thread on one end and was dipped in to the beaker containing buffer solution with the help of a burette stand.

● A one ml of emulsion was placed in the tube with semipermeable membrane and the entire assembly was placed on the magnetic stirrer.

● Magnetic stirrer was switched on and operated at 50 rpm. After every 15 minutes 1ml of sample solution was taken out in to the test tube separately and marked as 15 mins, 30 mins, 45mins and 60 mins respectively by replacing fresh buffer.

● In each test tube 0.1ml was pipetted out into a volumetric flask and made up to 10ml with buffer solution.

● The absorbances were observed with the wavelength 282nm and noted to calculate the cumulative percent drug released.

Fig 1 In Vitro diffusion assembly

v) Stability studies:

● The emulsions were centrifuged at 2000 rpm for 1 hr and visually observed for phase separation.

● The emulsions were kept at room temperature protecting from the light and moisture and observed for phase separation and drug content periodically i.e., every week for a period of three months.

RESULT:

Construction of calibration curve

The median standard concentration was scanned in UV Visible spectrophotometer between the wavelengths 200 to 400nm and the λmax was determined as 282nm. Then absorbance was measured for all working standards at λmax. The procedure was repeated in triplicate and the results were given in table 1. The curve was linear in the concentration range 2 to 10µg/ml, correlation coefficient was 0.9735 and the regression equation was y = 0.0077x.

Fig 1 Calibration curve of aspirin in acetonitrile.

Table 2. Partition coefficient of aspirin

Oil	Log P
Olive oil	0.4899
Coconut oil	0.3647

The oil phase was selected based on oil /glycerol partition coefficient of aspirin. The log p value of aspirin was more in olive oil compared to coconut oil.

Formulation development of emulsions:

Various oral antiplatelet agents including aspirin are being used routinely for treatment of acute ischemic stroke and the recommended loading dose is 300mg thereafter 75mg and various aspirin formulations are developed^8,9,10,11,12. Such a large dose of oral aspirin may cause gastric bleeding and ulceration. Cerebral edema which occurs in ischemic stroke can be minimized by giving 20% intravenous glycerol. Aspirin oil in water or oil in glycerol emulsions are not reported yet. Therefore the present investigation is aimed to prepare oil/glycerol emulsion for intravenous administration in ischemic patients^13,14. At the beginning a combination of emulsifying agents that is span 80 and tween 80 were chosen based on HLB values because the required HLB for olive oil is 7. Various concentrations of emulsifying agents from 3% to 15% and in various ratios were tried from F1 to F6, the emulsion was formed but phase separation was observed. From F1 to F6 glycerol was used as continuous phase. Later glycerol and water in 50:50 ratio in F7 and F8 75:25 ratio in F9 was and 100:0 in F10 were used as continuous phase and span 80 is omitted, only tween 80 was used as emulsifying agent. The emulsion compositions F7 to F10 were stable and no phase separation was observed.

Evaluation of emulsions

1 Visual observation

Table 3

Code	Observation
F1	Phase separation observed
F2	Phase separation observed
F3	Phase separation observed
F4	Phase separation observed
F5	Phase separation observed
F6	Phase separation observed
F7	No phase separation
F8	No phase separation
F9	No phase separation
F10	No phase separation

Fig 2 Formulations F1-F10

2. Drug content

Drug content was determined and the results were depicted in table 4

Table 4 percentage drug content

Formulation	% drug content			Avg	Stdv
F8	68.6	72.5	76.5	72.53	3.95
F9	108.67	99.65	97.8	102	5.816
F10	103.46	107.98	98.76	103.4	4.61

3. Particle size:

The particle size was determined by optical microscopy and the average particle size for F8, F9 and F10 were 6.04, 3.38 and 7.8 µm respectively.

Fig 3 Particle size and size distribution of composition F8

Fig 4 Particle size and size distribution of composition F9

Fig 5 Particle size and size distribution of composition F10

4. In vitro diffusion studies:

Rate of drug release followed first order kinetics and an immediate burst release effect was observed within 15 minutes.

Fig 6 Cumulative percent drug released vs time

5. Stability studies:

After centrifugation at 2000rpm for 1hr, there was no phase separation indicating that the formulations F8 to F10 were physically stable. Stability studies were carried out for a period of 3 months at room temperature there was no phase separation and drug content was found to be greater than 95%. Hence it is concluded that the formulations F8 to F10 were physically and chemically stable.

DISCUSSION:

According to the Indian guidelines for stroke management 2011, aspirin (an antiplatelet agent) is recommended in acute ischemic stroke immediately within 24 to 48 hours as part of overall management of stroke. Clinical deterioration can occur in 25% of patients after initial stroke assessment, possibly because of stroke progression, brain edema, haemorrhage, and recurrent ischemia. Brain edema is a major cause of early death after stroke. Therefore multidisciplinary care is required for management of the potential complications due to complexity of stroke. To minimize the edema formation before clinically significant increase in intracranial pressure, osmotherapy which includes Intravenous mannitol (0.25 to 0.5g/kg) and/or glycerol or 3% normal saline is also recommended by Indian stroke association. Hence in this investigation an attempt was made to prepare oil/glycerol emulsion loaded with aspirin to treat ischemic stroke.

The investigation was carried out systematically. Initially aspirin calibration curve is constructed in acetonitrile as it was a stable media for aspirin according to the literature. The curve was linear in the concentration range 2 to 10µg/ml. The regression coefficient was found to be 0.9735 and the equation was y=0.0077x. Based on oil/glycerol partition coefficient studies olive oil was selected as oil phase.

The emulsions are prepared by high shear homogenization using tween 80 and span 80 as emulsifying agents. The emulsifying agents were selected based on hydrophilic lypophilic balance (HLB). Total 10 formulations were prepared. F1 to F7 were prepared with glycerol as continuous phase¹⁵ and varying concentrations of emulsifying agents but we observed phase separation. Composition F8 was prepared with 15 % tween 80 as emulsifying agent and 50:50 glycerol water mixture as continuous phase and F9 glycerol: water in 75:25 ratio. The composition F10 was prepared with 15% tween 80 and 100% glycerol as continuous phase. The compositions F8 to F10 were found to be stable physically because there was no phase separation. Drug content was determined as 68±3.95%, 108±5.816%, 103±4.61% respectively in compositions F8, F9 and F10. Rate of drug release followed first order kinetics and an immediate burst release effect was observed within 15 minutes. The particle size was determined by optical microscopy and the average particle size for F8, F9 and F10 were 6.04, 3.38 and 7.8 µm respectively. After centrifugation at 2000rpm for 1hr, there was no phase separation indicating that the formulations F8 to F10 were physically stable. Stability studies were carried out for a period of 3 months at room temperature there was no phase separation but creaming was observed and drug content was found to be greater than 95%. Hence it is concluded that the formulations F8 to F10 were physically and chemically stable.

CONCLUSION:

An oil/glycerol emulsion was prepared successfully with narrow particle size, high drug loading efficiency using high shear homogenization. Based on particle size, drug content and drug release properties the best formulation was F9. The physical and chemical stability studies indicate that the formulations are stable and animal studies are yet to be done.

CONFLICT OF INEREST:

The authors have no conflicts of interest regarding this investigation

ACKNOWLEDGEMENTS:

We acknowledge the chairman and correspondent of Smt Sarojini Ramulamma College of Pharmacy for providing all the facilities to carry out this investigation.

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Received on 17.05.2022 Modified on 14.06.2022

Asian J. Res. Pharm. Sci. 2022; 12(3):177-182.

DOI: 10.52711/2231-5659.2022.00030