Physicochemical Characteristics, Chemical composition and In vitro Antioxidant activities of Essential oil from Ocimum basilicum L. Leaves
Asegele Desta1, Krishna Chaithanya K1, Naveen Kumar A.D2,
Prasanthi Cheekurumelli3, John Dogulas Palleti4, Sudhish Rai5. Zenebe Hagos1*
1Department of Chemistry, College of Natural and Computational Sciences,
Aksum University, Axum, Ethiopia.
2Department of Medicine, Texila American University, Lilayi, Lusaka, Zambia.
3Department of Microbiology, St. Ann's College for Women, Malkapuram, Visakhapatnam, A.P. India.
4Research and Development, Centre for Computational and Biological Sciences (CCBS),
48-12-17, Srinagar, Near RTC Complex, Visakhapatnam - 530016, Andhra Pradesh, India.
5Department of Pharmacognosy, Jagrani Devi Pharmacy College, Baradwar, Shakti, Chhattisgarh, India.
*Corresponding Author E-mail: zenebehagos.2003@gmail.com
ABSTRACT:
Plant-derived medicines offer a safer alternative to synthetic options, providing significant therapeutic benefits and more economical treatment options. This study aimed to assess the physicochemical characteristics, chemical composition, and antioxidant properties of the essential oil extracted from Ocimum basilicum L. leaves harvested in Wukro. The oil was obtained using the hydrodistillation method, and its chemical composition was analyzed using GC-MS. various parameters including density, specific gravity, pH, boiling point, refractive index, acid value, saponification value, and ester value were considered. Antioxidant activities were evaluated using DPPH, ABTS, FRAP, NO inhibition, and OH free radical assays. The yield of oil was determined to be 0.14±0.016%, with GC-MS identifying 30 compounds. Major components included eugenol (32.69%), Octadecatrienoic acid (12.00%), and ethylisoallcholate (9.25%). Antioxidant activity increased with higher concentrations of the essential oil, suggesting synergistic effects among its components. These findings support the potential use of the essential oil as a natural source of antioxidants.
KEYWORDS: Essential oil, Ocimum basilicum L., Chemical composition, GC-MS, Antioxidant.
INTRODUCTION:
Aromatic plants and essential oils have been used since ancient times and are still today for the treatment of various infectious and non-infectious diseases1. Essential oils are a mixture of various volatile compounds such as terpenoids and phenolic compounds showed significant therapeutic importance against chronic obstructive pulmonary disease, lower respiratory infection, and tuberculosis2. Since 2017, our research group has already reported that the phytochemical analysis and in vitro antioxidant activities of different parts of the selected medicinal plants such as Moringa stenopetal3, Cordia africana L4 and Anogeissus leiocarpa5.
The oxygen and nitrogen free radicals are synthesized during aerobic respiration and metabolic pathways play a key role in signal transduction pathways and cell-cell communication6. Antioxidants are a group of chemical substances capable of inhibiting the oxidation reactions by donating electrons to pro-oxidant substances without becoming radical itself7. Oxidative stress is an imbalance between both pro-and antioxidant systems, which leads to damage to cellular constituents lead to cell injury leading to cell death and pathogenesis of various chronic diseases like carcinomas, coronary heart disease, and many other health problems related to advancing age8.
Many plants have been used because of their antioxidants, which are due to compounds synthesized in the secondary metabolism of the plant9. These products are bioactive phytochemical constituents that produce definite physiological actions in the human body. Phenolic compounds are some of the bioactive phytochemicals which are part of the essential oils10. In recent years, the antioxidant potential of plants has attracted the attention of the scientific community and also protects the living cells from oxidative damage that occur due to the formation of free radicals and reactive oxygen species during metabolic activity11.
Ocimum basilicum L. plant belongs to the Lamiaceae family and is commonly referred to as sweet basil, which is commonly cultivated in many tropical and temperate countries in Asia, Africa, Central, and South America12 and it is a good source of antioxidants and antibacterial agents. O. basilicum L. oil is composed of many chemicals, a few of which are found at relatively high concentrations including methyl cinnamate, linalool, eugenol, citral, 1,8-cineole, estragole, methyl eugenol13. Different works of literature reported that the O.basilicum L. contains monoterpene hydrocarbons, oxygenated monoterpene, sesquiterpene hydrocarbons, oxygenated sesquiterpene, triterpene, flavonoids, and aromatic compounds14.
In Ethiopia, O.basilicum L. has been used for different purposes traditionally without scientific knowledge considering the advantages of the plant and tendency of the community in using it scientific investigation of the chemical composition, physicochemical characteristics, and in vitro antioxidant activity of O.basilicum L. plant is needed and there are no previously reported researches that show the chemical composition of the plant in the study area. Therefore, the present study was to investigate the physicochemical characteristics, chemical composition, and in vitro antioxidant activity of the essential oil of O. basilicum L. leaves.
MATERIALS AND METHODS:
A fresh healthy green leaves of O.basilicum L. was collected from Wukro town, Eastern Zone of Tigray which is 42km far from Mekelle in the month of December 2018. The plant material was authenticated by Department of Biology. Mekelle University, Mekelle, Ethiopia.
The collected fresh leaves of O. basilicum L. was washed by tap water followed by distilled water in order to remove unwanted substances from the leaves. Then, it was dried under shade at a room temperature for about nine days in a neat material because if it is exposed to direct drying by sunlight or oven the volatile components of the leaves can evaporate and it was grinded in to powder by grinding machine.
Two hundred fifty grams (250g) of powdered O. basilicum L. leaves was taken and subjected to hydrodistillation for four hours. During this process, the essential oil or volatile oil came with the water vapor. Since water is denser than the essential oil, it came out before the oil and stored in the beaker and the oil was kept until all the volatile oil were evaporated from the plant. Then, the oil was kept with sealed vials. The water left during extraction was dried over anhydrous sodium sulphate and stored in sealed vials at 40C until analysis 15.
Physicochemical characteristics provide a base line for suitability of oils16. The physicochemical characteristics of the essential oil such as color, odor, density, solubility, optical activity, refractive index, specific gravity, total acid number, iodine value and saponification value were determined.
The yield of the oil from O. basilicum L. leaves was calculated based on dried weight of plant sample which is given as follows17
Weight of oil
% Yield = -------------------------- ×100
Weight of sample
To determine the density of the essential oil first the weighted dried beaker and labeled as wo. 1ml of the oil was taken and poured to the beaker carefully and weighed again and labeled as w1. The weight of the essential oil was then calculated by subtracting w0 from w1. finally taking the known volume of the oil the density was determined using the following formula18.
Density = (Mass of oil)/ (Volume oil)
Specific gravity is the ratio of the density of respective substance to the density of water at 4°C 19. The specific gravity of the oil was obtained by dividing the density of the oil to the density of the water.
Density of oil
Specific gravity = -------------------------------
Density of Water
Digital polar meter was used to determine the optical rotation of the essential oil of O.basilicum L. The oil was filled to the sample cell and the optical rotation was recorded from the polarimeter20. Then, the specific rotation was determined by the formula;
α
[α] = -----------
l x p
Where α = optical rotation, l = length of sample cell,
p =densty of the oil p = densty of the oil.
The refractive index of the oil sample was determined with the help of Abbe refract meter model (Adt44D+). Two drops of oil were placed on the prism with the help of syringe and the prism was firmly closed by tightening the screw head. The apparatus was allowed to stand for 5min, then the reading was recorded from the display screen21.
Total acid number (TAN):
A one gram of oil was poured to the conical flask and mixed with few drops of phenolphthalein as indicator. Then titration was carried out with 0.1N KOH until the end point was obtained with constant stirring. Dark pink color was observed and the volume of 0.1NKOH was noted and finally the acid value was calculated by the following formula22.
V X N x 56.1
A. V. = ----------------------------------
W
Where V = volume of potassium hydroxide used N = normality of Potassium hydroxide W = weight in g of the sample.
Two grams of oil was weighed into a conical flask. Next 10ml of carbon tetrachloride and 20ml of the Iodine bromide solution were added to the flask and the solution was kept in dark for 30min at room temperature. Then 15ml of 10% potassium iodide solution with 100ml of distilled water was added to the flask. Finally, the resulting solution was titrated against 0.1M sodium thiosulphate (Na2S2O3), using starch as indicator. The end point where the blue-black coloration becomes colorless. A blank titration was carried out at the same time starting with 10 ml carbon tetrachloride. Iodine value was then calculated by the following formula19.
(vb- Vs) X N X 12.69
Iodine value = ----------------------------
Weight of sample
Where vb = 0.1 N sodium thiosulfate required (ml) by blank vs = 0.1 N sodium thiosulfate required (ml) by sample N = Normality of sodium thiosulfate solution.
Two grams of oil sample was weighted into a clean dried conical flask and 25ml of alcoholic potassium hydroxide (KOH) was added. A reflux condenser was attached to the flask and heated for an hour with periodic shaking. The appearance of clear solution indicated the completion of saponification. Then 1ml of 1% phenolphthalein indicator was added and the hot excess alkali was titrated with 0.5M hydrochloric acid (HCl) until it reached the end point where it turned colorless. A blank titration was carried out at the same time and under the same condition. The Saponification value was calculated as follows23
(Vo - V1 ) X N
Saponification value =------------------------- X 56.1
m
Where vo = 0.5 N HCl required (ml) by the blank V1= 0.5 N HCl required (ml) by the sample m = weight in g of the sample.
To determine solubility, different solvents were taken (water, methanol, acetic acid, diethyl ether, and benzene). The above-mentioned solvents were taken from their stock solutions in different bottles. To determine the solubility of the essential oil, 1 ml from each solvent and 1ml from the sample were taken by using pipette24.
A portion of the essential oil was poured to test tube and a thermometer was inserted and placed on a heating mantle, it was observed that oil in the beaker started circulating leading to boiling of oil and the temperature was read from the thermometer. This temperature was taken as boiling temperature of the oil25.
A portion of oil was poured in to a clean dry beaker. The pH electrode was standardized with buffer solution and then immersed to the oil sample. The pH value was then read and recorded from the electrode26
Determination of ester value:
The ester value is the number of mg of KOH required to saponify the esters in 1 gm of a sample. In this study, it was determined from the saponification value and the acid value, and was calculated by using the following formula27.
Ester value = Saponification value – Acid value
To conduct the GC-MS analysis 1μl of the essential oil was mixed with 1μl of Methanol and injected to the GC-MS with the following instrumental conditions. The analysis of the sample was performed using gas chromatograph (GC, Model CP-3800, coupled with a mass spectrometer (MS, Model Saturn 2200,) system in India. The separation was done using a VF-5ms fused silica capillary column (5% phenyl- dimethyl polysiloxane, 30m × 0.25mm, film thickness 0.25µm). For MS detector, electron impact (EI) ionization system with ionization energy of 70 eV was used. Helium gas was used as a carrier gas at a constant low rate of 1ml. Injector and mass transfer line temperature were set at 250oC. The optimization condition for oven temperature was programmed for 1 min at 500C, 50 to 2400C at 50C min-1 hold for 5 minutes at 2400C. The injection of the samples was carried out with the auto-sampler for 1µl with a split ratio 1:20. The conditions of analysis and specification of the instrument were optimized for a better separation and resolution. Identification of components was based on matching with standard NIST electronic library.
In vitro antioxidant assays:
Table 1: The yield of O.basilicum L. leaves
|
Amount of sample used for extraction (g) |
Time used for extraction (hr.) |
Amount of oil obtained (g) |
Yield (%) (w/w) |
Previous reports |
|
|
|
|
||||
|
250 |
4 |
0.35± 0.04 |
0.14± 0.016 |
0.5± 0.03 |
0.37% |
W/W = weight by weight
The color of the essential oil was isolated from O.basilicum L.was pale yellow liquid and characteristic odour. The obtained yield was very low 0.14±0.016% which was collected in the month of December, 2018, when compared with previous studies, 0.5±0.03 which was collected in the month of October, 200933. Another researcher reported that the O. basilicum L. grown in Serbia and Montenegro had an essential oil yield of 0.37%34. Great variations in the essential oil yield of O.basilicum L. across geographic regions might be attributed to variable agroclimatic conditions and different agronomic techniques for cultivating, and harvesting time35
Table 2: Physicochemical properties of O. basilicum L. essential oil
|
S. No |
Parameters |
Values and results |
|
|
1 |
State at room temperature |
Liquid |
|
|
2 |
Color |
Pale yellow |
|
|
3 |
Odor |
Spicy |
|
|
4 |
Solubility |
Water |
Slightly soluble |
|
Methanol |
Slightly soluble |
||
|
Chloroform |
Soluble |
||
|
Diethyl ether |
Soluble |
||
|
Benzene |
Soluble |
||
|
5 |
Specific gravity |
0.85 |
|
|
6 |
Boiling temperature (oC) |
76.5 |
|
|
7 |
Refractive index |
1.34520 |
|
|
8 |
Optical rotation(ml/gxdm) |
-0.70588 |
|
|
9 |
pH |
7.73 |
|
|
10 |
Saponification value |
43.7580 |
|
|
11 |
Acid value |
2.805 |
|
|
12 |
Ester value |
40.953 |
|
|
13 |
Density |
0.85 |
|
|
14 |
Iodine value |
28.224 |
|
As showed in table 2, the essential oil of O. basilicum L. leaves was slightly soluble in water and methanol while it was soluble in organic solvents such as chloroform, diethyl ether and benzene. This difference in solubility is due to the different polarity of the solvents and nature of the components of the essential oil, indicating the components of the essential oil contains polar and none polar components. The polar components such as phenols are soluble in polar solvents (water, methanol) and the nonpolar components like terpenes are soluble in nonpolar solvents (diethyl ether, benzene)36. The values reported previously for these parameters were 0.980, 54oC, 1.50, 1.520, 4.2, -5.99, 189.8, 11.1, and 178.8. The variation of these values might be geographical location, climatic condition or seasonal variation37. Refractive index describes how fast light travels through the material. As depicted from table 2, the essential oil different to the standard refractive index of essential oils (1.47950-1.48950)38. Acid value is an indirect method for the determination of free fatty acid in oil sample and its edibility. Oil with low free fatty acids has more significant usage and is edible. The total acid number (TAN) values recorded in this study was 2.805 this value is found in the permissible limits i.e. 10 mg KOH/g of oil and found to be suitable for dietary purposes, as it contains lower fatty acid contents39.
Saponification values determined was found to be 43.7580 it is useful in perfumery industry40. The oil is unsaturated because the oil has lower iodine value. The iodine value observed for the oil sample was 28.224, this value was found below the permissible range for semi-drying of oil (100-300)41.
The essential oil of the leaves of O.basilicum L. was subjected to GC–MS analysis. The identified components of the essential oil of O.basilicum L. leaves were shown figure-1
Figure 1: GC-MS Analysis of Essential oil of O.basilicum L. leaves
DPPH radical scavenging activity:
The DPPH radical scavenging activity of the essential oil from O. basilicum L. and the standard ascorbic acid at the concentrations range from 100-500μg/ml were shown in the figure-2a, both essential oil and standard ascorbic acid significantly reduced the DPPH radical with increasing concentrations. Essential oils and ascorbic acid have exhibited promising in vitro DPPH radical scavenging activity ranges from 11.17%-57.95% and 23.1%-76.54% at the concentrations rages from 100-500μg/ml with the IC50 values 474.±0.91 and 299 ±1.5μg/ml respectively. Pripdeevech et.al.42 is also reported that the essential oil from O. basilicum L from Thailand, the IC50 of the essential oil was also higher than that of the standard ascorbic acid this reveals lower scavenging activity.
ABTS radical scavenging activity:
In the figure-2b the ABTS free radical scavenging activity of the essential oil of O.basilicum L. and Ascorbic acid at 100-500µg/ml were showed range of 21.7-56.13%, and 28.5-852% with IC50 values of 311.57µg/ml and 241.18µg/ml respectively. Our results are correlated with highest antioxidant capacity was reported by43
Hydroxyl radical scavenging assay:
As shown in figure -2c the hydroxyl radical scavenging activity of the essential oil of O. basilicum L. leaves and the standard ascorbic acid was increased with increased concentration of the essential oil and ascorbic acid. The percentage of hydroxyl radical scavenging activity of essential oil from O. basilicum L. and ascorbic caid at concentration of at100-500µg/ml was found to be 17.2-50.7% and 23.56-69.25% with IC50 values of 398.17 μg/ml and 231.19μg/ml respectively. The previous reports also suggested that the antioxidant capacity of the oil was correlated with the major proportion of volatile components in the oil and to compounds possessing a phenolic ring44.
Nitric Oxide radical scavenging assay:
Figure 2: 2a, 2b, 2c, 2d; 2,2-diphynyl-1-pycryl hydrazyl (DPPH), 2,2-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid (ABTS), Hydroxyl radical (OH), Nitric Oxide radical scavenging assay (NO) assays of essential oil from O. basilicum L and ascorbic acid standard
The antioxidant activity of the essential oil was determined by measuring its ability to transform Fe3+ to Fe2+. The reducing power was confirmed by the changes of yellow color of the test solution to various shades of green and blue depending on the concentration of the essential oil and the result was presented in figure-2e. the reducing power of the essential oil and the standard ascorbic acid increased with an increase in concentration and the absorbance was measured at 700 nm, as showed in figure-2e essential oil of O.basilicum L. and ascorbic acid showed 0.37-0.97 and 0.43-1.18 O.D in the 100-500µg/ml respectively.
Both the essential oil and ascorbic acid caused a color change from yellow to blue to reveal that the essential oil has a reducing power towards free radical. Previous reports shown that, the essential oil was with lower antioxidant activity than BHT 46 and the present study exhibited lower activity than the standard.
Figure 2: 2e Reducing power activity of essential oil from O. basilicum L and ascorbic acid standard
The essential oil extracted from O. basilicum L. leaves was found to contain significant compounds, including eugenol (32%), ethyl iso-allocholate (19.00%), 9,12,15-octadecatrienoic acid, 2,3-bis[(trimethylsilyl)oxy]propyl ester (12.00%), phenol, 2-methoxy-5-(1-propenyl) (4.70%), silanediol, dimethyl (4.27%), eucalyptol (3.37%), D-fructose, diethyl mercaptal, pentaacetate (2.99%), and α-terpineol (1.98%), as identified through GC-MS analysis. The physicochemical characteristics of the essential oil were found to meet acceptable quality standards. Moreover, the essential oil exhibited potent free radical scavenging activity against DPPH, ABTS, and OH free radicals, attributed to the synergistic effects of its phyto-constituents. This suggests that the essential oil from O. basilicum L. leaves could serve as a natural source of antioxidants, potentially useful in the treatment of various diseases caused by free radicals.
ACKNOWLEDGMENTS:
The authors truthfully thank to the Department of Chemistry, Aksum University, Axum, Ethiopia for providing laboratory facilities, this project work was funded by Research and Postgraduate studies directorate, Aksum University, Axum, Tigray region, Ethiopia in the form Student Project work.
CONFLICT OF INTEREST:
The authors confirm that this article content has no conflict of interest.
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Received on 15.09.2020 Modified on 14.03.2023
Accepted on 29.04.2024 © RJPT All right reserved
Research J. Pharm. and Tech 2024; 17(5):2352-2358.
DOI: 10.52711/0974-360X.2024.00368