Evaluation Of Antioxidant Property Of Three Herbal Formulations - An In Vitro Study

Keywords

Amla; Rosemary; Antioxidant; DPPH Assay; Endodontics.

Introduction

Oxygen is an element that is invaluable to all living organisms. Although it is an indispensable part of life, it can be both beneficial as well as poisonous to the health of a body. The free radical theory of oxygen toxicity proposed by Gerschman in 1954 states that the toxicity of oxygen is due to its partially reduced forms [1]. Free radicals are chemically active atoms that have a charge due to an excess or deficient number of electrons. They can be reactive oxygen species (ROS) or reactive nitrogen species (RNS) [2]. They are cytotoxic and are considered as the etiology for various diseases [3]. They may be beneficial in killing bacteria but may also damage the adjacent infected host tissues.

Free radicals are produced by various materials in dentistry including bleaching agents, composite resins, dental cements, ceramic restorations, metal restorations, dental implants and intracanal medicaments [4]. Hence the application of antioxidants in these fields is beneficial.

Among various antioxidants, 10% sodium ascorbate is the most widely used antioxidant in all investigations and laboratory studies [5]. Chemically, it is the sodium salt of ascorbic acid, and is also known as vitamin C. Sodium ascorbate has been proven to have strong free radical scavenging or antioxidant activity on oxidized enamel and dentin [6-8]. But, the major disadvantage of using it is that it is highly sensitive to ambient conditions such as heat, light, oxygen, humidity, pH and that loses its stability and effectiveness rapidly [9].

In the recent past, research is being focused towards use of natural herbal or plant extracts in order to develop a non-toxic, biocompatible, and effective material which can be applied safely to oxidized dental structures. Flavonoids, phenolic compounds or their derivatives in the composition of the natural plant extracts can prevent auto-oxidation via different mechanisms such as reducing or inhibiting oxygen formation, free radical scavenging, or metal ion chelating [10].

Amla (Emblicaofficinalis) is a well known antioxidant and has been used in medicine since ages. It is known to possess antioxidant, antimicrobial, anti-inflammatory properties. The presence of tannins, polyphenols and antioxidant acids are responsible for this property. In medicine, this herb has been used for the treatment of several conditions including diabetes, gastrointestinal disorders, high cholesterol, ophthalmic disorders, jaundice, diarrhoea, inflammations, etc [11, 12]. In endodontics it has been used as a root canal irrigant, for smear layer removal, as an intracanal medicament, as a guttapercha disinfectant, etc [13-15]. Rosemary (Rosmarinusofficinalis) possesses antimicrobial, antifungal, antiinflammatory and antioxidant properties. In the field of endodontics, it has been used as an irrigant, intracanal medicament [16], post-bleaching antioxidant, etc [17].

Previously our team has a rich experience in working on various research projects across multiple disciplines [18-32] Now the growing trend in this area motivated us to pursue this project. Since no previous study has combined these herbs, the aim of our study was to evaluate the antioxidant property of Amla, Rosemary, and their combination as well as compare it with that of ascorbic acid.

Materials And Methods

Preparation of the extract

Fresh amla and rosemary leaves were collected from a garden and were thoroughly double washed with running water. They were then dried under the shade for 5 days. The dried leaves were eventually crushed and powdered separately. 2 g each of Amla powder and Rosemary powder were added to 100ml of distilled water in two separate beakers and boiled at 70°C for 30mins. This allows the phytochemicals present in the powder to get activated. The extract was collected from Whatman filter paper 1, and the filtrate was further boiled at 70°C for 15 mins. 2ml of each solution was measured with a micropipette and transferred to another test tube to make the Amla-Rosemary combination. The three solutions were stored for further use at low temperature.

Evaluation of Antioxidant Property

Hydrogen donating capacity or free radical scavenging by the nanoparticles was evaluated by the DPPH measure, which depends on the reduction of the methanoliccoloured radical type of the DPPH to the non-coloured solution. A measured quantity of 0.2mM of DPPH was added to methanol solution, with the concentration of herbal solutions in a range of 10-50 µg/ml. Ascorbic acid was used as the standard. The solution was vortexed and incubated for 30 min in dark conditions. The absorbances of both test and standard were analyzed at 517 nm after the incubation period. The antioxidant activity was calculated by the equation:

% Radical scavenging activity = (Absorbance of the control - Absorbance of the Test sample )/(Absorbance of the control)×100 (Percentage Inhibition)

Statistical Analysis

All the experimental results were performed in triplicate. The data was analysed using SPSS Software (Version 23.0). One way ANOVA was used to assess the statistical significance between the groups. A p-value > 0.05 was considered statistically significant.

Results And Discussion

The absorbance values of the various samples at different concentrations is shown in Table 1.

The percentage radical scavenging activity of ascorbic acid at the different concentrations was considered as 100%. In comparison, the % radical scavenging activity of amla at 10, 20, 30, 40 and 50 µl concentrations was 75.81%, 73.32%, 90.26%, 75.85% and 68.41%. That for rosemary was 56.59%, 58.46%, 89.48%, 81.92% and 77.45%. While for the combination formulation, it was 88.56%, 84.12%, 96.02%, 88.8% and 97.56%. (Figure 3). Ascorbic acid showed the highest absorbance and antioxidant activity. The antioxidant property of the combination formulation at 50 µl conc was significantly higher than the individual extracts. The amla-rosemary combination showed results comparable to ascorbic acid.

Amla contains a wide range of tannins, alkaloids, and phenolic compounds, which make it highly antioxidant. In one previous study, extracts of a thousand different herbs were screened using sophisticated electron spin technology and Amla was one of only four chosen for both its superoxide radical scavenging activity [33]. Another study of three fruit extracts analysed their freeradical scavenging activity of hydroxyl, superoxide, nitric oxide, peroxynitrite, hypochlorous acid, etc. The authors concluded that Amla showed good efficacy in its antioxidant and radical scavenging abilities [34]. Yet another study of 30 different Thai medicinal plants traditionally used for diabetes found that five had strong antioxidant properties, of which Amla had the strongest antioxidant activity and the highest total polyphenol and tannin content [35].

For many years, it was thought that high amounts of ascorbic acid were a keystone for Amla’s antioxidant power. Now it has been proven that amla also contains high amounts of gallic and ellagic acid other than ascorbic acid which are responsible for its antioxidant property [36-38]. It may be due to this reason that some authors believe that amla is a stronger antioxidant compared to ascorbic acid [39]. This is in contradiction to the results of our study which concluded that ascorbic acid had the highest antioxi dant property. Among the most effective antioxidant constituents of rosemary, the cyclic diterpenediphenols, carnosolic acid and carnosol, carnosic acid, epirosmanol, rosmanol, methylcarnosate and isorosmanol are the most important. The extract of rosemary also contains other caffeic acid derivatives. They react with metal ions, forming chelates; which react with peroxide free radicals stabilizing them. Höulihan et al. [40] and Wu et al. [41] determined that the antioxidant properties of rosemary are attributed to its richness in isoprenoidquinones, which act as chain terminators of free radicals, and as chelators of reactive oxygen species (ROS). In addition, Gordon [42] indicated that the phenolic compounds react with the lipid and hydroxyl radicals to turn them into stable products. According to Löliger [43], carnosic acid and carnosol act as potent scavengers of peroxyl radicals.

In our study, the combination formulation showed a synergistic effect and resulted in antioxidant property comparable to that of ascorbic acid. This may be due to the increased amount of flavonoids, phenolic compounds etc in the combination formulation. A previous study by Jain et al stated that the amla and green tea showed synergistic effect when combined with other herbs such as grapeseed, anar, cinnamon etc. [44].

DPPH assay is considered as a rapid, economic and widely used method for evaluation of antioxidant activity of products using different solvents including methanol, ethanol, water, alcohol, ethyl acetate, benzene. DPPH is a coloured and stable free radical, which gets reduced in the presence of an antioxidant compound to give yellow colour [45]. DPPH test is generally conducted for the evaluation of antioxidant activity of plant extracts and hence was the method of choice for our study.

Although amla and rosemary have shown good antioxidant properties, the future scope of our study is to extend its application as a post-bleaching antioxidant to replace sodium ascorbate as a natural and less toxic alternative. This involves performing many more in vitro studies to assess the shear bond strength of composite restorations, effect on bleaching, stability of the formulation, etc before clinical trials can be performed. The ultimate decision must be made only after long term randomized clinical trials. Our institution is passionate about high quality evidence based research and has excelled in various fields [22, 46-55].

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Table 1:

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Table 1. Representing the absorbance values of the various formulations and ascorbic acid at different concentrations.

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Figure 1:

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Figure 1. Demonstrating preparation of the herbal extracts.

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Figure 2:

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Figure 2. Showing the microtiter plate containing different concentrations of the herbal formulations as well as ascorbic acid.

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Figure 3:

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Figure 3. Representing a bar graph representing the radical scavenging activity or % inhibition of the different herbal formulations at various concentrations.

Conclusion

Within the limitations of this study it can be concluded that the herbal formulations showed good antioxidant properties. The amla-rosemary combination formulation showed a synergistic effect and its antioxidant properties were comparable to that of ascorbic acid. The amla-rosemary extract has good potential to be used as a potent antioxidant in endodontics.

Acknowledgement And Declarations

The authors would like to acknowledge the institution and all the staff members of the Department of Conservative Dentistry and Endodontics for their support towards completion of this research. The authors deny any conflicts of interest associated with this paper.