Department of Chemistry, C.M.P Degree College, University of Allahabad, Prayagraj.
The antioxidant activity stands out among the many benefits that are frequently associated with plant essential oils (EOs). Investigate the potential synergistic effects between the bioactive compounds extracted from Clitoria ternatea flowers and essential oils of Clove, Cinnamon, Eucalyptus and Gaultheria oils. This can be done by combining different concentrations of flower extracts and essential oils and assessing their antioxidant activity compared to individual components. The additional antioxidant activity makes those natural materials extremely appealing as multi-functional preservatives, e.g., for food products, able to control spoilage caused both by microbial metabolism and by air oxidation. This activity has been extensively documented for many essential oils in recent and less recent literature. Consumer preference has gradually steered food technologists towards safer and more natural alternatives to synthetic additives in recent years, which has increased interest in plant-derived bioactive like essential oils. Essential oils are concentrated liquids containing volatile aroma compounds from plants. They are known for their aromatic and therapeutic properties. As a result, the ethyl acetate extract of Clitoria ternatea shows 70% Antioxidant activity and Ethanol extract of Clitoria ternatea Shows 65% Antioxidant potential. When we tested synergistically, Ethanol and Ethyl acetate extracts of Clitoria ternatea mixed with Clove oil give excellent Antioxidant activity 96% & 95% respectively. As well as Ethanol extract of Clitoria ternatea mixed with Cinnamon oils give higher Antioxidant activity 87%, then the ethyl acetate synergistically with cinnamon oil 55% antioxidant potential. So we can firmly says the novel finding that extracts of Clitoria ternatea with clove oils exhibited excellent Antioxidant activity. When combining Clitoria ternatea flowers extracts and essential oils, it may potentially enhance the overall antioxidant activity due to the presence of bioactive compounds in both. In this research paper we have investigated the antioxidant properties of Clitoria ternatea and various essential oils synergistically with Molecular Docking were carried out with 4BQY, 6NMQ, 8G43 protein target for Methyl salicylate and gave the best result among the proteins.
Studying the comparative antioxidant activity of Clitoria ternatea flowers and essential oils (Clove, Cinnamon, Eucalyptus and Gaultheria oils) in synergistically sounds like a fascinating research topic. It has been established that the phytochemicals found in plants and their byproducts have a range of biological activities, including the capacity to act as antioxidants. In recent years, consumer choice has increasingly led food technologists to substitute safer and more natural ingredients for synthetic ones. This has raised interest in bioactives produced from plants, such as essential oils. The scientific community has been greatly interested in the health benefits of essential oils such as eucalyptus, cinnamon, gaultheria, and clove (eugenol). Numerous ailments have been treated with these essential oils and plant extracts of medicinal plants; their anti-epileptic, anti-hysteric, diuretic, digestive, and stimulating properties are well supported by scientific research. [1, 2] Organic extracts and essential oils (EOs) are volatile organic compounds that have various biological effects for humans. They can consist of various combinations of benzoids, phenylpropanoids, mono-and sesquiterpenoids, etc. The fact that plant species extracted essential oils (EOs) have antioxidant properties has also been extensively researched. [3] These days, a lot of work goes into identifying the bioactive ingredients in essential oils (EOs) from medicinal plants so that they can be used in the food, beverage, and pharmaceutical industries, among other businesses. [4] In addition, there is growing interest in the use of plant-based phenolic chemicals (such as flavonoids and phenolic acids) as natural antioxidants, preventative, and therapeutic medications. [5] Accordingly, many researchers have studied and tested the antimicrobial properties of different plants extracted Essential Oils. Eugenol, cinnamic acid, and cinnamic aldehyde are the principal active ingredients of cinnamon essential oil (EO), a natural preservative derived from cinnamon. Since it has antibacterial and antioxidant properties, cinnamon essential oil is frequently used to preserve food. [6] Numerous research have documented the biological actions of clove essential oil (EO), encompassing anti-allergic, anaesthetic, stress-relieving, anti-inflammatory, and antioxidant qualities.[7] The medicinal properties of clove and cinnamon have been discovered in recent years. These properties include antimutagenic, insecticidal, acaricidal, anaesthetic, antiallergic, antioxidant, anti-inflammatory, and anticancer properties. Numerous investigations have demonstrated that EOs from a variety of plants have strong antifungal action [8] like cinnamon EO [9], clove EO [10] Eucalyptus, Gaultheria and Cinnamon EO. In various nations, eucalyptus oil-based herbal remedies or cosmetics are also used to treat a variety of ailments and infectious diseases. [11] Moreover, there is proof that eucalyptus medicines are used to treat acne in various nations. [12] The current study sought to assess the combination in vitro antibacterial, antioxidant, and anti-inflammatory properties of Eucalyptus globulus Labill. (Myrtaceae) essential oil (EGEO) of Pharmacopoeia (PhEur) quality, based on traditional use. Recent studies [13, 14, 15] have examined the essential oils of E. falcata, E. sideroxylon, and E. citriodora, plants that grow in Tunisia; no research has examined the plants' potential for herbicidal use. Similarly, minimal researches have been conducted on phyto-pathogenic fungi, with the bulk of studies on their antifungal potential concentrating on clinical fungi. [14] Although Eucalyptus citriodora's herbicidal activity has been documented in India [16], the mechanism of action has not been explained. On the other hand, because plants can synthesise a variety of molecules in order to adapt and acclimatise to environmental conditions, chemical ecology is commonly used for the discovery of metabolites because it is recognised as a very essential strategy. Gaultheria procumbens L., sometimes known as wintergreen essential oil (WEO), is a well-known commercial EO that is mostly utilised in the aromatherapy and perfume sectors. Since these two wintergreen species—G. procumbens and G. fragrantissima—yield a high quantity of methyl salicylate, an aromatic ester chemically related to aspirin, the essential oil of wintergreen is often extracted from Gaultheria. [17] It is an active component that effectively reduces pain brought on by muscular fever and is present in large concentrations in WEO. [18] After distillation, methyl salicylate makes up over 99% of the oil content of most wintergreen species [19, 20, 21]. Although there are many uses for wintergreen oil, its secondary metabolite ratio exhibits unpredictability because to climatic, regional, seasonal, and genetic influences. Water-based solutions, such as oil-in-water (o/w) emulsion formulations, are less harmful to consumers and safer to transport. [22] Additionally, they are good for the environment. Essential oils have long been employed in a wide range of fields, including medicine, cosmetics, agriculture, and the preservation of artistic legacy. A large number of these uses are based on the oils' purported antioxidant properties. [23–30] It follows that the fact that several research on the antioxidant activity of EOs have been published in scholarly journals is not surprising. Reactive oxygen species and oxygen-centered free radicals can be consequences of several physiological and biochemical processes in the human body. An excessive amount of these free radicals can oxidative damage biomolecules, such as proteins, DNA, and lipids, which can ultimately result in apoptosis and a host of chronic illnesses. [31] Antioxidants are well known for their ability to effectively scavenge free radicals. They can do this by binding metal ions, scavenging starting radicals, and stopping chain reactions. [32] Recently, much more attentions have been paid to natural antioxidants [33] because synthetic antioxidants have potential toxicological effects. [34] The bright blue blossom of the Clitoria ternatea plant, which belongs to the Fabaceae family, is used as an ornamental plant as well as a natural food colouring (such as in rice cakes, tea, snacks, and sweet sweets). [35, 36, 37] Anthocyanins (ternatin A1-A3, B1-B4, C1-C4, and D1-D3) and flavonols (quercetin, myricetin, and kaempferol derivatives) make up the majority of the flowers. [38] Based on delphinidins, ternatin A1, A2, B1, B2, D1, and D2 are the six main anthocyanins found in C. ternatea flowers. When compared to nonacylated and monoacylated anthocyanins, these triacylated anthocyanins demonstrated comparatively greater stability. [35, 39] Research has demonstrated that crude flower extract possesses a range of medicinal potentials, including antibacterial, antioxidant, and antidiabetic properties. [40, 41, 42] Scientists and food makers are becoming more interested in the potential of antioxidant elements found in plant materials for the maintenance of health and diseases, as consumers shift towards functional meals with targeted health benefits. Thus, we require a reliable supply of powerful and reasonably priced natural antioxidants. People are now more likely to look for antioxidants derived from plants to help with these issues. Foods enhanced with phenolic chemicals and flavonoids have high antioxidant properties. Because of their redox characteristics, which are crucial for absorbing and neutralising free radicals, quenching singlet and triplet oxygen, and breaking down peroxides, these phenolic compounds have antioxidant potential. [43] Biomolecules including lipids, proteins, and nucleic acids may be harmed by oxidative stress brought on by ROS production surpassing the antioxidant defense of the cell. An abundance of free radicals within the body leads to the development of several diseases associated with oxidative stress. Numerous diseases, including asthma, cancer, cardiovascular disease, cataract, gastrointestinal disorders, muscular degeneration, and inflammatory diseases have been linked to free radicals. They also have a role in the ageing process and neurological diseases. In order to lower the risk of these diseases, antioxidant supplementation has emerged as an appealing therapeutic approach for inhibiting free radical-induced oxidative damage. [44, 45] Comparative studies of medicinal plants and essential oils involve a multidisciplinary approach encompassing chemistry, pharmacology, toxicology, and clinical research. These studies help bridge the gap between traditional knowledge and evidence-based medicine, providing valuable insights into the therapeutic potential of these natural products. For this purpose, the present study focuses on the evaluation of the antioxidant potential of mixtures of Essential oils (viz., Clove, Cinnamon, Euclyptus and Gautheria oils) and Flowers of Medicinal plants ( Clitoria ternatia ) in Different extracts such as Ethanol and Ethyl acetate and their synergistic combination has been tested by DPPH* method.
MATERIALS AND METHODS
Essential oils of Clove, Cinnamon, Eucalyptus and Gaultheria oils and Gallic acid were purchased from CDH Pvt. Ltd. New Delhi, India. 1, 1- diphenyl-2-picrylhydrazyl (DPPH*) was purchased from HiMedia Pvt. Ltd. Mumbai, India and Methanol AR was purchased from Loba Chemie Pvt. Ltd. Mumbai, India. The absorbance of all samples was recorded by UV- Visible spectrophotometer (Systronic, Model No. 119) was used to record the samples absorbance. The experiments were performed in the Department of Chemistry, C.M.P. Degree College (Constituent College of University of Allahabad) during January - February 2024.
Collection And Authentication of Plant
Flowers of Clitoria ternatea was collected from the Company Garden, Prayagraj, U.P., India, during the month of August and September. Taxonomically identified and verified by Botanical survey of India, Prayagraj and herbarium specimen was prepared and depicted in the department herbarium.
Preparation of extract
Initially, Flowers of the medicinal plant of Clitoria ternatea was dried separately at room temperature for seven days and powdered them with grinder. The powder of the Flowers of Clitoria ternatea (100 gm) separately was exhaustively extracted with 500 ml each of ethanol and ethyl acetate using soxhlet apparatus. Then, the extracts were centrifuged thrice and the supernatants were combined. The combined supernatants were filtered over Whatman No. 1 filter paper. The extracts were then evaporated to dryness by rotary flash evaporator (Buchi type Rotavapor). Different concentrations of extracts were prepared from the resultant crude ethanol and ethyl acetate extracts of flowers of the medicinal plants to determine in vitro Antioxidant assays.
Experiment
DPPH* (1, 1- Diphenyl-2-picrylhydrazyl) radical scavenging activity
Stock solutions (1mg/10 mL) of essential oils (Clove, Cinnamon, Eucalyptus and Gaultheria oils) and different extracts of Clitoria ternatea were prepared in methanol. The stock solution of DPPH* was also prepared at the concentration of 2mg/100 mL in methanol.
The DPPH* radical scavenging method was used to evaluate the Antioxidant capabilities. The ability of the various extracts to donate hydrogen or scavenge radicals was assessed using a stable radical called DPPH*. Gallic acid was used as standard. As a reagent for our spectrophotometric test, we are using the stable radical DPPH*. The process entails measuring the decline in DPPH* absorbance at its 517 nm absorption maximum. Concentration of DPPH* was 0.002 percent in methanol. The stock solutions of the extracts were prepared in methanol (1.0 mg/10 ml). Different volume (2.0, 1.5, 1.0, 0.5, 0.25, and 0.125 ml) of extracts were taken in separate test tube and volume was made up to 2 ml with methanol. Now 2ml of DPPH* solution was added in each test tube and kept in dark for 30 minutes. The same procedure was followed for Gallic acid (Standard) as well. Later optical absorbance was recorded at 517 nm using UV- Visible spectrophotometer. Similarly, performed further experiments for essential oils.
DPPH* and Methanol was used as a control. All the samples were tested in triplicate. The formula used for the calculation is:
% Inhibition of DPPH* activity = (Ac - As / Ac) X 100
Where Ac = Absorbance of control, As = Absorbance of sample.
RESULTS AND DISCUSSION:
The antioxidant potential value of Clitoria ternatea with different Essential oils was evaluated by DPPH*assay (Table 1.)
Table 1: Antioxidant Activity of Clitoria ternatea with different Essential oils
|
S. No.
|
Clitoria ternatia with different Essential oils
|
% Antioxidant activity (by DPPH* assay) |
|
1. |
Clitoria ternatea (Ethanol) |
71.19% |
|
2. |
Clitoria ternatea (Ethyl acetate) |
80.93% |
|
3. |
Cinnamon Oil |
69.90% |
|
4. |
Clove Oil |
66.96% |
|
5. |
Eucalyptus Oils |
19.24% |
|
6. |
Gaultheria Oil |
8.69% |
|
7. |
Gallic Acid (Standard) |
95.66% |
Table 2: Antioxidant Activity of Ethanolic Extract and Ethyl acetate Extract of Clitoria ternatea mixing with Essential oils
|
S.NO. |
Mixing different Extracts (Ethanol & Ethyl acetate) of Clitoria ternatea with Essential oils |
% Antioxidant activity (by DPPH* assay) |
|
Extracts of Ethanol |
||
|
1. |
Cli. ternatea : Cinnamon Oil (1:1) |
87.97% |
|
2. |
Cli. ternatea : Clove Oil (1:1) |
96.41% |
|
3. |
Cli. ternatea : Eucalyptus Oil (1:1) |
56.01% |
|
4. |
Cli. ternatea : Gaultheria Oil (1:1) |
71.61% |
|
Extracts of Ethyl Acetate |
||
|
5. |
Cli. ternatea : Cinnamon Oil (1:1) |
55.24% |
|
6. |
Cli. ternatea : Clove Oil (1:1) |
95.14% |
|
7. |
Cli. ternatea : Eucalyptus Oil (1:1) |
41.68% |
|
8. |
Cli. ternatea : Gaultheria Oil (1:1) |
44.75% |
Firstly we saw that medicinal plants Clitoria ternatea flowers shows Antioxidant potential in Ethanol extract 71.19% Antioxidant activity and Ethyle acetate extract of Clitoria ternatea showed 80.93% Antioxidant potential respectively [Figure 1].
Secondly we saw the Antioxidant activity of essential oils that is cinnamon oil, Clove oil, Eucalyptus oil and Gaultheria oil showed 95.90%, 98.96%, 19.24% and 8.69% DPPH* inhibition respectively as compared to Gallic acid (standard) [Figure 2], which shows comparable values referring paper. (Table: 1) Now, when comparative studies on different extracts of medicinal flowers of Clitoria ternatea mixed with essential oils then we saw the mixture of in Ethanol extract of Clitoria ternatea flowers and Cinnamon oils (1:1) showed Antioxidant potential with 87.97% while Clitoria ternatea and Clove oil (1:1), Clitoria ternatea and Eucalyptus (1:1) and Clitoria ternatea and Gaultheria oils (1:1) exhibited Antioxidant activity with 96.41%, 56.01% and 71.61% [Figure 3] while Ethyle acetate extract of clitoria ternatea flowers mixed with Cinnamon oils (1:1) showed Antioxidant activity with 55.24% while Clitoria ternatea and Clove oils (1:1), Clitoria ternatea and Eucalyptus (1:1) and Clitoria ternatea and Gaultheria oils (1:1) exhibited Antioxidant activity with 95.14%, 41.68% and 44.75% [Figure 4] respectively.(Table:2)
Figure.1: Ethyl acetate and Ethanol extraction of Flower of Clitoria ternatea L.
Figure.2: Antioxidant activity of different essential oils
Figure.3: Ethyl acetate extract of clitoria ternatea flowers mixed with different essential oils
Figure.4: Ethanol extract of clitoria ternatea flowers mixed with different essential oils
The mixture of Clitoria ternatea in Ethanol extract with clove (1:1) shows higher Antioxidant activity with 96.41% inhibition in comparison to Ethyle acetate extracts with Clove (1:1)with DPPH* inhibition of 95.14% . These Antioxidant potential values is higher than the Gallic acid (Standard). Similarly Ehanol extracts of Clitiria ternatea mixed with Cinnamon oil (1:1) exhibited much higher 90.53% antioxidant potential in comparison to Ethyle acetate extracts of Clitoria ternatea mixed with Cinnamon oil (1:1) showed 81.84% Antioxidant activity. The next When we see the Ethanol extract of Clitoria ternatea mixed with Eucalyptus essential oils (1:1) and Ethyle acetate extract of Clitoria ternatea mixed with Eucalyptus essential oils (1:1) then observed antioxidant inhibition is 56.01% and 41.68% respectively so we can say that Eucalyptus essential oils decrease antioxidant potential of Clitoria ternatea flower. The mixture of Ethanol extract of Clitoria ternatea and Gaultheria essential oils (1:1) exhibited more 71.61% antioxidant activity in comparison to ethyle acetate extract of clitoria ternatea mixed with Gaultheria oils (1:1) shows lower antioxidant potential that is 44.75%. We can confidently state that the phenolic and flavanoids components present in the extracts' Antioxidant potential, because phenolic compounds are composed of one or more aromatic rings holding one or more hydroxyl groups, which have the capacity to quench free radicals, their structure is directly related to their Antioxidant effects. [46]
Molecular Docking:
Molecular docking has been considered in the industry for drug development, with the goal of reducing synthesis time and cost while increasing medication efficacy. The purpose of ligand protein docking is to predict a ligand protein's primary mechanism of binding. The Swiss target prediction was used for the molecular docking and to find out the most probable protein to dock with the desired ligand. The protein database containing the pdb IDs of the downloaded protein as 4BQY [47], 6NMQ [48] and 8G43 [49] eugenol and 6ROB [50], 6ROE [51] from RCSB-PDB that further docked with the desired compounds such as eugenol (Eugenol oil) and methyl salicylate (Gaultheria oil) respectively. The protein database containing the pdb. The activities of the specific ligand evaluated prior to choosing the protein and the robust activities were then taken into consideration. Molecular docking was performed by using Chemira 1.15 [52] and Autodock Vina [53] application that is free and easily available source. For the interaction, we have downloaded three proteins in pdb format and that docked with desired molecule i.e., eugenol and methyl salicylate as shown in Figure 5 and Figure 6. The H-bond was investigated between selected protein and both of the compound eugenol and methyl salicylate with the distance as 2.287, 2.388, 2.683, 2.202Å for 4BQY protein, 2.008Å for 6NMQ,2.073, 2.215Å for 8G43 protein and 2.435Å for 6ROB protein, 2.521, 1.204Å for 6ROE protein respectively that tabulated in Table 3. The binding energy were found to be as -5.6 kcal/mol, -5.1 kcal/mol and -5.9 kcal/mol for the proteins as 4BQY [1], 6NMQ [2] and 8G43 [3] respectively shown in Table 3. Similarly, for methyl salicylate, the binding energy were found -5.0 kcal/mol, 3.9 kcal/mol for 6ROB and 6ROE protein. The ensuing outcomes of the molecular docking that represented notable hydrogen bond interactions including ARG A: 383 for 4BQY with oxygen atom of ligand eugenol, ASP: 315 for 6NMQ with H-atom of hydroxyl group of eugenol and ARG A: 1155 with oxygen atom of the titled molecule eugenol. On the other hand, for methyl salicylate, GLN A:136 for 6ROB with H-atom of the hydroxyl group that attached to the ring of the titled compound and GLU: 205 for 6ROE with H-atom of hydroxyl group of the ligand methyl salicylate. These interactions stabilizing the complex formed between hydrolase enzyme and inhibitor at their active sites. The lowest binding energy were found to for the protein IDs 8G43 as -5.9 kcal/mol for eugenol and -5.0 kcal/mol for methyl salicylate compound that indicates the substance under investigation is pharmacologically active [54, 55] and the H-bond distance determines the stability of the particular ligand and receptor protein that shown in Figure 5.
Table 3. Hydrogen bonding and molecular docking with Centromere associated protein inhibitor protein targets for eugenol and methyl salicylate
|
Sr. N. |
Protein ID |
Residue |
Bond distance (A°) |
Binding Energy Kcal/mol |
|
Eugenol |
||||
|
1 |
4BQY |
3 |
2.287, 2.388, 2.683, 2.202 |
-5.6 |
|
2 |
6NMQ |
3 |
2.008 |
-5.1 |
|
3 |
8G43 |
3 |
2.073, 2.215 |
-5.9 |
|
Methyl Salicylate |
||||
|
1 |
6ROB |
3 |
2.435 |
-5.0 |
|
2 |
6ROE |
3 |
2.521, 1.204 |
-3.9 |
Figure 5. Titled molecule eugenol embedded in the active sites of (A) 4BQY, (B) 6NMQ, (C) 8G43
Figure 6. Titled moleculemethyl salicylate embedded in the active sites of (A) 6ROB, (B) 6ROE
CONCLUSION:
Results from the above studies, concluded that the mixtures of the bioactive constituents of Clitoria ternatea with Essential oils showed synergistic effects and good antioxidant activity except for Synergistically Ethyl acetate extract of Gautheria oils which shows 44% antioxidant potantial. The maximum Antioxidant activity shows Synergistically Ethyl acetate exteacts and ethanol extracts of Clove oils which is 95% & 96% respectively. As well as Synergistically Ethanol extract of Clitoria ternatea and Cinnamon oils shows great Antioxidant inhibition that is 87%. This research would be very helpful in treating various kinds of oxidative stress and diseases. Studying the comparative antioxidant activity of Clitoria ternatea flowers and essential oils (Clove, Eugenol, Euclyptus and Gautheria oils) in synergy sounds like a fascinating research topic! Both Clitoria ternatea, commonly known as butterfly pea flower and essential oils are known for their potential antioxidant properties, which could contribute to various health benefits.
REFRENCES
Sakshi Singh, Babita Agrawal*, Comparative Studies on Antioxidant Activity of Flower of Clitoria Ternatea and Essential Oils Synergistically, Int. J. of Pharm. Sci., 2025, Vol 3, Issue 3, 1463-1476. https://doi.org/10.5281/zenodo.15034610
10.5281/zenodo.15034610
