Megha P. Raj, C. Rubina Reichal1, S. Manju2, M. Shobana2, M. Sangeetha3
1Professor, Department of Pharmaceutics, Department of Pharmaceutical Chemistry,
Cherraan’s College of Pharmacy, Coimbatore - 641039, Tamil Nadu, India.
2,3Assistant Professor, Department of Pharmaceutics, Department of Pharmaceutical Chemistry,
Cherraan’s College of Pharmacy, Coimbatore - 641039, Tamil Nadu, India.
*Corresponding Author E-mail: rubinareichel@gmail.com
ABSTRACT:
KEYWORDS: Ocimum Basilicum, Phytosomes, Topical drug delivery, antimicrobial activity.
INTRODUCTION:
The genus Ocimum belongs to the family Lamiaceae. Ocimum basilicum possesses analgesic, antiulcerogenic, antidepressant, anticonvulsant, anti-hyperlipidemic, anti-inflammatory,antioxidant,antiplateletproperty, antithrombotic,anti-microbial,insecticidal, immunomodulatory and cytotoxicity effect. It is mostly used to treat anxiety, cold, fevers, migraines, diabetes, menstrual cramps, sinusitis, cardiovascular diseases, nerve pain, insect bites, and headache1. It also has spasmolytic, carminative, hepatoprotective, diuretic and stimulating properties that lead to the production of various drugs from Ocimum in pharma industries2,3. In recent years, herbal remedies have been considered as dietary supplement for disease prevention and as alternative/complementary medicine4,5.
Phytosomes are said to be containing natural herbal formulations. Most of the Plants are having medicinal properties because to the presence of many active constituents which are mainly the secondary metabolites like flavonoids, terpinoids, tannins, glycosides, alkaloids etc6,7. The active constituents present in the plants are mostly hydrophilic in nature. The therapeutic efficacy of herbal extracts are quickly destroyed by the enzymes present in the intestinal gut. Hence, advanced researches are done for the specific site delivery of these plant derived products8. The term “phyto” means plant and “some” means cell like. It is also called as herbosomes. This is an advanced methodology, where extract of the plant or the hydrophilic phytoconstituents are mixed with phospholipids to produce more lipid stable molecular complexes, thereby it enhances the absorption and bioavailability of phytoconstituents9,10.
In the past decades considerable attention has been focused on the development of novel drug delivery systems for herbal drugs11. Herbal drugs are becoming more popular in the modern world for their ability to cure various diseases with less toxic effects and better therapeutic effects. Because of the reasons and based on the literatures the objective of the present study was to prepare and evaluate the Ocimum basilicum as phytosomal gel12,13.
Materials:
Ocimum Basilicum leaves were obtained as a gift sample from Hapamuni herbals. And all other solvents and chemicals used were of analytical grade.
The leaves were dried and powdered. After that it was sequentially extracted with ethanol using Soxhlet apparatus. The solvent was removed and concentrated using water bath. The prepared extracts were stored for further studies14.
Determination 𝝀max:
The stock solution was prepared by dissolving 10mg of powdered Ocimum Basilicum extract in the range of 20-120μg/ml and the diluted solutions absorbance were measured in UV spectrophotometer at 320nm15,16.
Preparation of Phytosomes:
Accurately weighed quantity of lecithin and cholesterol were taken in Round bottom flask (RBF) and it was dissolved in 10ml of chloroform then sonicated for 10mins. The organic layer was removed and the thin layer of phospholipids was formed. This is then mixed with ethanolic extract of Ocimum basilicum. Again these are sonicated, then it was stored at freezer (2.8℃) for further use16,17.
Table 1: Composition of Phytosome formulation of Ocimum Basilicum
|
Formulation Code |
Ocimum basilicum extract |
Cholesterol |
Lecithin |
Ethanol (ml) |
Chloroform |
Sonication Time (min) |
|
F1 |
10 |
15 |
40 |
5 |
5 |
5 |
|
F2 |
10 |
15 |
45 |
5 |
5 |
10 |
|
F3 |
10 |
15 |
50 |
5 |
5 |
15 |
|
F4 |
10 |
15 |
55 |
5 |
5 |
20 |
|
F5 |
10 |
15 |
60 |
5 |
5 |
25 |
|
F6 |
10 |
15 |
65 |
5 |
5 |
30 |
|
F7 |
10 |
15 |
70 |
5 |
5 |
35 |
Evaluation of Phytosomal complex:
The prepared phytosomal complex were studied for Microscopic view, Fourier Transformer-Infra Red Spectroscopy (FTIR) analysis, Percentage Practical Yield, Entrapment efficiency, Drug content as per the standard procedures.18,19,20.
The particle size of Ocimum basilicum phytosomes was measured by particle size analyzer.21,22,23
Zeta potential is the most important parameter for physical stability of phytosomes. 5ml of this diluted sample was transferred to a cuvette and the zeta potential was measured22.
The in-vitro diffusion study was carried out by using Franz Diffusion Cell. The egg membrane was mounted between the donor and receptor compartment. The receptor compartment contains 15ml of pH 6 phosphate buffer and the temperature was maintained at 37˚C and was constantly stirred. The samples were withdrawn at specific time intervals and they were analyzed at 320nm using UV spectrophotometer23.
The optimized formulation was tested in-vitro for its antimicrobial activities against P.aeruginosa by paper disc diffusion method known antibiotic such as Gentamycin was used as a reference (as standard drug) against bacteria. From inhibition zone, anti-microbial activity was critically examined.
Scanning electron microscopy study was done to determine the surface morphology, size and shape of prepared Ocimum basilicum phytosome formulation. 24
Formulation of Phytosomal Gel of Ocimum basilicum
Carbopol 934 is dispersing into distilled water and the gel base was prepared. 0.1% phytosomal solution of Ocimum basilicum is added to ethanol and this is mixed with the Carbopol gel base and other ingredients are mixed. The optimized formulation was prepared and the gel was stored in a suitable container.
Figure 1: Ocimum basilicum Phytosomal Gel
Table 2: Formulation of Phytosomal Gel of Ocimum basilicum
|
Ingredients |
FOR 25gm |
|
Carbopol 934 |
0.25gm |
|
Triethanolamine |
0.125ml |
|
Methyl paraben |
0.25ml |
|
Ethanol + 0.1% phytosome solution |
2.5ml |
|
Distilled water |
21.8ml |
The optimized gel was tested for homogeneity , Measurement of pH, Drug content, Rheological study, Spreadability, Extrudability and In-vitro drug release study24,25
Drug release kinetics was performed using model dependent method in which the dissolution profile of the formulation has been subjected various kinetics like zero order, first order, Higuchi’s and Korsmeyer-Peppas model24.
Stability Studies:
The stability studies were conducted for the optimized formulation as per ICH guidelines.26
Optical Microscopy:
Optical microscopy was observed that the vesicles formed and were found to be of uniform size and shape.
Figure 2: Microscopic view of Ocimum basilicum phytosomes
From the FTIR –Studies, the complex formation was indicated by the formation of strong hydrogen bonding between hydroxyl group of phospholipids and extract phytoconstituents in O. basilicum phytosome form. And there was no appearance or disappearance of peaks, and it showed that the excipients are more compatible.% Practical yield of different formulations was shown in Table no .3 have higher % Practical yield of 93.72%. The entrapment efficiency was calculated from the absorbance obtained from the supernatant solution. The formulation F6 showed highest release entrapment efficiency of 89.71%. The drug content of Ocimum basilicum extract in the complexes was found to be in the range of 79.36% - 89.86%. The formulation F6 showed the maximum drug content of 89.86%. The results were shown in Table no 3.
Table 3: Results of Percentage Practical Yield, Percentage Entrapment Efficiency & Drug Content
|
Formulations |
Percentage Practical Yield |
Percentage Entrapment Efficiency |
Drug Content (%W/W) |
|
F1 |
90.28 |
86.10 |
87.03 |
|
F2 |
88.15 |
83.94 |
86.41 |
|
F3 |
86.51 |
78.38 |
85.15 |
|
F4 |
84.56 |
74.43 |
83.51 |
|
F5 |
82.47 |
70.91 |
81.47 |
|
F6 |
93.72 |
89.71 |
89.86 |
|
F7 |
80.69 |
66.15 |
79.36 |
It was observed that the average particle size was found to be 393nm for optimized formulation (F6) Ocimum basilicum phytosomes complex.
Figure 3: Particle size of Ocimum basilicum phytosomes
The magnitude of the zeta potential is similarly charged particles of Ocimum basilicum phytosomes complex confirm the high integrity in the complex and the zeta potential value was found to be -11.3 indicating good stability of the formulation.
Figure 4: Zeta Potential Distribution of Ocimum basilicum Phytosomes
In-vitro Drug Diffusion Study of Phytosomes:
From the In-vitro Drug Diffusion Study, the Formulation 6(F6) showed the higher cumulative percentage of drug release at 24th hour .Thus formulation (F6) was selected as best formulation and it was incorporated into a gel by using Carbopol 934 as a polymer, it was shown in figure 5.
Figure 5: In-vitro Drug Diffusion Profile
Table 4: Results of Anti-microbial activity
|
Sl. No |
Test parameter |
Method |
Results (Zone of Inhibition in mm) |
|||
|
Ocimum Basilicum extract |
Gentamycin (120mcg) |
|||||
|
10 μl |
50 μl |
100 μl |
||||
|
1 |
Antimicrobial activity- P.Aeruginosa |
Lab method |
32 |
60 |
83 |
30 mm |
The results showed in Table 4 that the formulation was effective to kill bacteria.
Figure 6: SEM image of aqueous extract of Ocimum Basilicum Phytosome (F6)
Scanning electron microscopy showed that the vesicles are spherical in shape and smooth in nature shown in figure 6.
Evaluation of Gels of Phytosome Complex:
From the visual inspection of the prepared gel formulation (F6) showed good appearance and homogeneity. The pH of optimized gel formulation was 5.5. The drug content of the optimized gels was estimated spectrophotometrically at 320nm. The drug content in which the best formulation contained 91.71% showed in Table 5. The viscosity of the optimized gel formulation was found by the Brookfield’s viscometer. The viscosity of the formulation increases as concentration of polymer increases. The viscosity of the optimized gel was found to be 9641 centipoise. Spredability denotes the extent of area to which the gel readily spreads on application to skin or the affected part. The spredability of the fabricated gel formulation was carried out and it was found to be 4.5cm which denoted that it has good spredability.
The optimized gel formulation was shown optimum extrudability. Because, the extrudability was decreased within the concentration of gelling agent.
Table 5: Evaluation parameters of Gel
|
Formulation |
Homogeneity |
pH |
Drug Content (%) |
Viscosity (Centipoise) |
Spreadability (cm) |
Extrudability (gm/cm2) |
|
Optimized gel |
Good |
5.5 |
91.71 |
9641 |
4.5 |
9.1 |
The release profile obtained from the best optimized formulation was fitted to various kinetic equation to know the mechanism of drug release as indicated by the maximum r2 value.
Figure 7: Zero Order Release Kinetics
Figure 8: First Order Release Kinetics
Figure 9: Higuchi Release Kinetics
Figure 10: Korsmeyer Peppas Model
From the release kinetic data the optimized formulation was fitted with various kinetics equations. From the graphical representation it can be understood that this layer is best fit into Zero order kinetics which has shown a regression coefficient (r2) of 0.9689 and Higuchi model (r2= 0.9354). Peppas equation was used to analyze the release pattern of the formulation and the value of “n” was found to be 0.45<n<0.89, indicating the drug release follows Non- Fickian diffusion, the results are shown in Figure 7 to 10.
Stability Studies:
|
Sl. No |
Parameters |
Initial |
30th Day |
45th Day |
|
1 |
Homogeneity |
Good |
Good |
Good |
|
2 |
Drug Content (%) |
91.71 |
91.67 |
91.64 |
|
3 |
pH |
6 |
6 |
6 |
|
4 |
Spreadability |
4.5 |
4.5 |
4.4 |
|
5 |
Extrudability (gm/cm2) |
9.1 |
9.1 |
9.1 |
|
6 |
Viscosity |
9641 |
9638 |
9630 |
|
7 |
% Cumulative Release |
98.85 |
98.80 |
97.71 |
For the optimized gel formulation the stability study was carried out and there were no marked changes in the optimized gel formulation. The results were shown in Table.6 and it showed that the formulated gel was stable during the stability studies.
DISCUSSION:
Ocimum Basilicum plant is selected based on the literature survey. The results of FTIR study confirmed that the complex formulation.UV spectrophotometric study was used to determine the λmax of Ocimum basilicum. (320nm).Ocimum basilicum phytosomes were prepared by antisolvent precipitation technique. A total of 7 formulations were prepared. All the formulations were evaluated for pH, spreadability, extrudability, drug content, rheological studies and in-vitro drug diffusion study. The optimized formulation showed excellent antimicrobial activity. From this formulation (F6) was selected as best formulation based on the in-vitro release and it was incorporated into a gel by using Carbopol 934 as a polymer.The prepared phytosomal gel formulation was subjected to various evaluation studies such as percentage yield, solubility, drug content, entrapment efficiency, in-vitro drug release studies. Release kinetic data revealed that the gel formulation followed Non-Fickian diffusion mechanism. Stability studies were conducted as per ICH guidelines and it showed that no significant change in homogeneity, drug content, pH, spreadability, extrudability, viscosity, in-vitro diffusion studies.
CONCLUSION:
Ocimum basilicum plant is useful in the management of anticonvulsant, anti-hyperlipidemic, anti- inflammatory, antioxidant, antiplatelet property, anti-thrombotic, anti-microbial, insecticidal, immunomodulatory and cytotoxicity effect. Phytosome technology has proved to be better absorption and bioavailability over conventional herbal extracts. From this study, Phytosomal gel of Ocimum basilicum was successfully prepared and it was evaluated with all characterization studies. The prepared phytosomal gel was safe, convenient and efficient and also showed better penetration into the skin. Thus it can be concluded that the topical phytosomal gel may serve as promising dosage form in the treatment of skin disease.
ACKNOWLEDGEMENT:
The authors are thankful to the Chairman and the Principal, Cherraan’s College of Pharmacy, Coimbatore, India for providing valuable guidance and support to carry out this research work.
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Received on 30.10.2021 Modified on 29.11.2021
Accepted on 21.12.2021 © RJPT All right reserved
Research J. Pharm. and Tech 2022; 15(10):4649-4654.
DOI: 10.52711/0974-360X.2022.00780