INTRODUCTION:

More than 15,000 plants from all over the world have been studied extensively for their medicinal uses¹. According to the report of the World Health Organization (WHO), about 80% of the world population depends on herbal medicine for their primary health care². All the medical systems including Unani, Siddha, Ayurveda, and modern Allopathy systems depend on herbal-based medicines and use either adopt them or derived different methods and techniques from them³.

In the present scenario use of the plant-based drug is increasing because of no side effects, easy availability, low cost, and complete eradication of ailments and diseases. Most diseases are the result of various types of inflammatory problems and oxidative stress in the body. Inflammation in the body is the result of the interaction of living tissues and lysosomal enzymes which are released during any injury, infection, and irritation. Membrane bursting of lysozymes results in the secretion of many enzymes which catalyze or oxidize surrounding cellular proteins or other biomolecules. Presently available drugs are used to treat lysosomal constituents of activated neutrophil such as bactericidal enzymes and proteases, which cause further tissue inflammation and damage upon extracellular release or by stabilizing the lysosomal membrane⁴. The human red blood cell (HRBC) membrane is analogous to the lysosomal membrane in structure and composition⁵and its stabilization implies that the extract may as well stabilize lysosomal membranes. The stabilization of HRBC membrane by hypo tonicity induced membrane lysis can be taken as an in-vitro measure of the anti-inflammatory activity of the drugs or plant extracts⁶. Non-steroidal anti-inflammatory drugs (NSAIDs) have certain limitations and therefore cannot be used for all conditions of inflammation⁷. Inflammatory process directly or indirectly triggers the formation of reactive oxygen species (ROS) and free radicals which can establish several chronic pathological conditions or oxidative stress. Hence, it becomes important to search for anti-inflammatory agents from plant sources having fewer side effects and is able to neutralize ROS and free radicals. Antioxidants are specific biomolecules that eliminate ROS. To overcome this situation we have to take antioxidants in the form of food or medicine. After taking exogenous antioxidants, inhibition of oxidative chain reaction, and reduction of free radicals occurs which ultimately minimizes the oxidative stress induced by ROS⁸.

Ocimum basilicum L. is known as “Vantulsi” in Hindi language. Ethnobotanical study reveals that this plant is used for various ailments including headache, cough, diarrhea, constipation, warts, worms, and kidney malfunctions⁹. A recent study explored its anthelmintic, diaphoretic, antiemetic, and antidiarrhoeal potential^10-11. Another study revealed that plant contains flavonoid and phenolic compounds and their leaves are enriched with aromatic oils and vitamins¹². Fresh flowering plant of Ocimum basilicum L. shows the presence of various compounds including eucalyptol, estragole, ocimene, linalool, eugenol, acetate, 1-epibicycloses quiphellandrene, methanol, menthone, cyclohexanol, cyclohexanone, myrcenol, and nerol¹². The anti-inflammatory and antioxidant activity of Ocimum basilicum L. fruit can be investigated earlier using egg albumin assay and DPPH assay¹⁴. The present study aimed to quantify different phytochemicals for anti-inflammatory and anti-oxidative activity present in the alcoholic extract of Ocimum basilicum L.

MATERIAL AND METHODS:

Collection and Preparation Of Plant Extract:

Leaves of Ocimum basilicum were collected from the roadside vegetation at Atal Nagar, Raipur, Chhattisgarh. One specimen of plant species was submitted to the herbarium at the Department of Botany, Kalinga University, Raipur, and Chhattisgarh for future reference.

The leaves of freshly collected plants were washed under running tap water and dried under the shed. The powder (30g) of dried leaves was mixed with 300 ml ethyl alcohol for solvent extraction of phytochemicals. The alcoholic extract was kept in the desiccators for solvent evaporation, followed by storage of dry powder in air tight container and stored at room temperature until further use.

Estimation of Phytochemicals from Ethanolic Extract of Ocimum basilicum L.

Estimation of total phenolic content:

Total phenolic content was determined by Folin-Ciocalteu method^15-18. Aliquots from a stock solution of ethanolic extract (1%w/v) were mixed with 9 ml distilled water in 25 ml of volumetric flasks, followed by the addition of 1 ml of FCR. After 5 min, 10 ml of 7% sodium carbonate (Na₂CO₃) solution was added to the mixture and the final volume was made up to 25 ml. The flasks were incubated at room temperature for 90 min and the absorbance was recorded at 550 nm using a UV-Vis spectrophotometer (Labtronics - 2700). A standard plot was prepared using Gallic acid (20, 40, 60, 80, and 100 µg/ml).

Determination of tannin content:

Tannin content was estimated by Folin-Ciocalteu method^19-22. Briefly, aliquots of ethanolic extract (1% w/v) were mixed with 7.5 ml of DD/W and 0.5 ml of FCR, followed by incubation for 30 minutes at room temperature. The absorbance of the colored solution was measured at 725 nm using a UV-vis spectrophotometer (Labtronics-2700). The tannin content in alcoholic extract was determined from the standard graph of Gallic acid (y = 0.172x - 0.000, R² = 0.999) and expressed in terms of Gallic acid equivalent (µg/mg dry plant extract).

Determination of total flavonoid content:

Total flavonoid content was estimated by aluminium chloride colorimetric assay²³. Briefly, the ethanolic extract was mixed with 4ml DD/W followed by 0.3ml of 5% of sodium nitrite. After 5 min, 0.3 ml of 10% aluminium chloride was added and incubated for 5 min. After that 0.3ml of 10% aluminium chloride was added and further incubated for 5min. 2ml of 1M sodium hydroxide was added and diluted to 10ml with DD/W. Quercetin (20, 40, 60, 80,100 µg/ml) was prepared for standard plot preparation. The absorbance for test and standard solutions were determined against the reagent blank at 510 nm with a UV-Vis spectrophotometer (Labtronics-2700). The content of total flavonoids in the alcoholic extract was expressed in terms of quercetin equivalent using a standard graph (y = 0.02x + 0.007, R² = 1), (µg/mg dry plant extract).

Antioxidant Activity by Dpph Radical Scavenging Assay:

In order to evaluate the antioxidant potential of the ethanolic extract of Ocimum basilicum L., a DPPH radical scavenging assay was carried out. The color of DPPH in methanol is violet/purple. The hydrogen atom donating ability of the plant extract was determined by the decolorization of the methanolic solution of DPPH. A stock solution of ethanolic extract was prepared and different aliquots were taken in the test tubes. Freshly prepared DPPH solution(75 μl, 1.3 mg/ml) was added to all test tubes and incubate for 30 minutes. After incubation, the absorbance of the solution was recorded at 517 nm using a UV-Vis spectrophotometer (Labtronics - 2700). Ascorbic acid was used as standard and the antioxidant activity of the plant extracts was calculated by using the following formula²⁴.

AC - AS

% inhibition = ---------------- × 100

Where AC is the absorbance of DPPH alone (control), AS is the absorbance of ethanolic extract and ascorbic acid. The value of IC₅₀ was calculated from the result of the above equation and by plotting a graph of concentration versus % inhibition.

Anti-Inflammatory Activity:

Preparation of human red blood cell (HRBC) suspension:

The human blood was collected from the volunteers by standard procedure. The collected blood sample (2 ml) was mixed with an equal volume of Alsever solution (2% dextrose, 0.8% sodium citrate, 0.05 % citric acid, and 0.42% sodium chloride in water) to avoid coagulation. The content was centrifuged at 3000 rpm for 10 min and the supernatant was discarded. The cell pellet was washed with a sterile isosaline solution (0.9% NaCl (w/v)). This process was repeated until the supernatant became clear and colorless, and RBCs were packed in the bottom of the tube. The packed cell volume (PCV) was measured and reconstituted up to 40% (v/v) with 10 mM phosphate buffer saline.

Hypo tonicity induced hemolysis of HRBC:

Different concentrations of ethanolic extract of the plant were mixed with 1 ml of phosphate buffer, 2 ml of hypo saline, and 0.5 ml of HRBC suspension. The test tubes were incubated at 37^oC for 30 min. After incubation, the absorbance was recorded at 560 nm with the help of a UV-vis spectrophotometer (Labtronics-2700). Diclofenac sodium was taken as the reference standard and processed in the same manner as ethanolic extract ^25-30. The percentage of hemolysis of HRBC was calculated by the following formula:

% hemolysis = ------- × 100

Where AT is the absorbance of the test sample, AC is the absorbance of the control.

The percentage of HRBC membrane stabilization can be calculated as follows:

% protection = 100 – [------ × 100]

Data analysis:

All the experiments were performed in triplicate and the data are presented as mean± standard deviation (SD).

RESULT AND DISCUSSION:

Extraction Of Plant Material:

The soxhlet extraction of leaves of Ocimum basilicum L. resulted in powder weighing 3.01 grams. This powder was obtained from 30 grams of dry leaves of the plant. Therefore the obtained yield was 10% (w/w).

Evaluation of Phytochemicals From Ethanolic Extract Of Ocimum Basilicum L.:

The total phenolic content in the ethanolic extract of Ocimum basilicum L.was measured by Folin-Ciocalteumethod and expressed in terms of Gallic acid equivalent from the standard graph. The value obtained for the concentration of total phenols was 5.02±0.06 µg GAE/mg of ethanolic extract. The total tannin content in the alcoholic extract of the plant was found to be 7.80±0.05 µg GAE/mg from the Gallic acid standard graph. The content of total flavonoids in ethanolic extract of leaves of Ocimum basilicum L. was expressed in terms of quercetin equivalent and was 6.00±0.06µg quercetin equivalent/mg using quercetin standard graph. OcimumbasilicumL.contains a number of secondary metabolites such as tannins, phenolic, and flavonoids having a variety of functions. For instance, the number of polyphenolics and tannins was 23780.00±145.30 and 15982±341.61 µg/ml of extract respectively³¹. The quantity of such phytochemicals is highly influenced by several abiotic and biotic factors. It was observed that the Ocimum basilicum L. grown in different locations contained phenolic content in the range of 2086 to 25593mg of GAE/100-gram dry weight of plant³². In another study, the total phenolic content was estimated in the essential oil and ethanolic extract of Ocimum basilicum L.; the amount was significantly higher in the ethanolic extract (82.45 mg Pyrocatechol equivalent/g) compared to an essential oil (41.3 mg PE/g)³³. The functions of phytochemicals are to prevent pathogenic infection and provide protection to the plant. Secondary metabolites are very useful for the treatment of various ailments and human diseases.

Antioxidant Activity:

An antioxidant activity of the ethanolic extract of Ocimum basilicum L. was evaluated through DPPH radical scavenging assay and results were compared with ascorbic acid. The results shown in Figure 1 indicated that the antioxidant power of both plant extract and ascorbic acid was concentration dependent. The DPPH radical scavenging power of ascorbic acid and plant extract at 10 µg/ml concentration was 26.92±1.76 and 6.51±1.13% respectively. The percentage of DPPH radical scavenging linearly increased with the increment in the concentration. The highest values of antioxidant potential were 70.51±1.80 and 55.12±1.31% for ascorbic acid and ethanolic extract of Ocimum basilicum L. respectively at 60 µg/ml concentration. However, ascorbic acid showed comparatively higher antioxidant potential than plant extract, the results demonstrated quite a significant antioxidant power of the plant extract. The IC₅₀ value of plant extract was found to be 24.81 µg/ml. Therefore, the ethanolic extract of leaves of Ocimum basilicum L. holds considerable antioxidant potential. It was observed that the antioxidant activity of plant extract is dependent on polyphenol compounds which diminish the production of free radicals, and eliminate or neutralize the existing active species and precursors of free radicals³⁴. The phytoconstituents including polyphenols present in Ocimum basilicum L. have antioxidant capacity. Methyl eugenol is one of the important phytoconstituents present in sweet basil and contributes to the antioxidant activity of the plant. Along with methyl eugenol, alpha-pinene, cis-linaloloxide, copaene, humulene, and nerolidol present in the plant body strengthen the antioxidant potential of the plant³⁵.

Fig 1: Antioxidant activity of ascorbic acid and ethanolic extract of Ocimum basilicum L. by DPPH radical scavenging assay

Anti-Inflammatory Activity:

The comparison of the anti-inflammatory activity of ethanolic extract of Ocimum basilicum L. and diclofenac sodium is shown in Figures 2 and 3. The results indicated a significant reduction of HRBC lysis in hypotonic conditions compared to diclofenac sodium in the entire range of concentration tested (Fig. 2). The trend of hemolysis reduction was in direct relation with the concentration; higher concentration of ethanolic extract of Ocimum basilicum L. and diclofenac sodium resulted into better reduction in HRBC lysis. At 50 µg/ml concentration of diclofenac sodium, the hemolysis was 64.08±1.33% and further reduced to 39.81±2.95% at 1000 µg/ml concentration. However, the ethanolic extract of Ocimum basilicum L. displayed 27.18±2.98% lysis of HRBC at 50 µg/ml and 1.94±1.43% hemolysis at 1000 µg/ml concentration. The HRBC protection potential of ethanolic extract of Ocimum basilicum L. was quite significant compared to diclofenac sodium. At 50 µg/ml concentration of the ethanolic extract of Ocimum basilicum L., the protection against hemolysis was 2.03 times higher as compared to diclofenac sodium (Fig. 3). When the concentration of both the compounds was 1000 µg/ml, the HRBC protection potential of plant extract was 1.63 times higher compared to diclofenac sodium. The results indicated a significant membrane stabilization effect by inhibiting hypo tonicity-induced lysis of the erythrocyte membrane. The stabilization of the lysosomal membrane is important to prevent the inflammation process because the bursting or rupturing of the lysosomal membrane cause the release of lysosomal constituents of activated neutrophils responsible for tissue inflammation and damage³⁶. Hypotonicity-induced hemolysis of HRBC is associated with the shrinkage of the cell due to osmotic loss of intracellular electrolytes and fluid components. Although the exact mechanism of the membrane stabilization by the plant extract is not discovered, it can stimulate or enhances the efflux of these intracellular components^37-38. Previous studies suggested that phenolic and flavonoid compounds present in the plant extract help in the enhancement of the anti-inflammatory property. It is observed that certain flavonoids possess potent inhibitory activity against a variety of lysosomal enzymes such as protein kinase C, protein tyrosine kinases, phospholipase A2, and phosphodiesterases³⁹. The phytochemicals like flavonoids, tannins, etc. present singly or in combination and contribute to the overall anti-inflammatory activity of certain plant extracts. Various phenolic compounds are known to inhibit the synthesis of the pro-inflammatory compound during inflammation. Many flavonoids block various molecules which promote inflammation such as COX, cytokines, nuclear factor B, and matrix metalloproteinases⁴⁰. Tannin especially condensed tannin (proanthocyanidins) showed a beneficial effect on human beings as it neutralizes ROS and free radicals present in the living system⁴¹. Previous studies suggested that estragole present in this plant showed an anti-inflammatory response. But the role of the rest molecule is still unclear as extract shows a more anti-inflammatory response than estragole alone⁴² so more study is required in this field to evaluate the pharmacological property of the phytoconstituents of this plant.

Fig. 2 Hemolysis of HRBC in the presence of diclofenac sodium and ethanolic extract of Ocimum basilicum L.

Fig. 3 Protection of lysis of HRBC in the presence of diclofenac sodium and ethanolic extract of Ocimum basilicum L.

CONCLUSION:

The study portrays the efficacy of the ethanol extraction method for the recovery of different phytochemicals from Ocimum basilicum L. The ethanolic extract has good antioxidant activity. The protection against lysis of hypo tonicity-induced HRBC indicated significant anti-inflammatory potential. Therefore, the ethanolic extract of Ocimum basilicum L. can be further explored for several applications.

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

ACKNOWLEDGEMENT:

The authors are grateful to the management of Kalinga University, especially Registrar Dr. Sandeep Gandhi, Vice Chancellor Dr. Sanjeev Arora and Chairman Dr. Rajeev. Special thanks to Dr. Manoj Singh, HOD, Zoology Department, Kalinga University for his valuable help.

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Received on 13.11.2021 Modified on 27.05.2022

Research J. Pharm. and Tech 2023; 16(4):1981-1986.

DOI: 10.52711/0974-360X.2023.00325