Antibacterial evaluation of guava leaves extract and its effect on reactive oxygen species formed by calcium hydroxide and chlorhexidine mixture.

Background: Reactive oxygen species (ROS) are formed when mixing calcium hydroxide (CH) with chlorhexidine (CHX). Aim: To analyze the antibacterial efficacy of aqueous (AGL) and ethanolic (EGL) extracts of Psidium guajava leaves against root canal bacteria such as Streptococcus mutans, Staphylococcus aureus, Lactobacillus acidophilus and Enterococcus faecalis, to evaluate the antioxidant potential of the extracts and its ability to counteract the ROS. Materials and
Methods: Three different concentrations (10%, 25%, and 35%) of AGL and EGL were prepared. The antibacterial efficacy was analyzed using the Kirby-Bauer disc diffusion method. The different concentrations of both AGL and EGL were evaluated for their antioxidant property using ultraviolet spectrometer. Further, the ability of these extracts on neutralizing the ROS formation was analyzed using mass spectrometer.
Results: CH and CHX mixture exhibited maximum antibacterial activity followed by the same mixture with 35% EGL. EGL showed greater antioxidant activity than AGL extract at all concentrations. Conclusions: The addition of natural antioxidants like guava leaves extract decreases ROS formed by CH and CHX mixture. Copyright:

INTRODUCTION

Debridement and disinfection protocols are performed to eliminate persistent microorganisms on the canal surface and the dentinal tubules which can impair the healing of periapical lesions.[123] Among the multitude of microorganisms present in the infected root canal, Enterococcus faecalis, a facultative anaerobe has the ability to persist in harsh environmental conditions, which exist in endodontically treated teeth. Enterococci and Streptococci are highly prevalent in root-filled teeth with persistent periapical lesion.[4] Few bacteria of Actinomyces genera have the capacity to survive in the periapical region and prevent healing.[1] Various intracanal medicaments such as calcium hydroxide (CH) and chlorhexidine (CHX) are essential to control persistent microbial infections.

A mixture of CH and CHX has better antimicrobial efficacy than CH alone because of their synergic effect.[56] CHX induces reactive oxygen species (ROS) production in an alkaline environment. Studies have demonstrated that 2% CHX when associated with CH liberates parachloroaniline and ROS.[678] These are oxygen-containing chemically reactive molecules which are by-products formed during the normal metabolism of oxygen. It plays a major part in cell signalling and hemostasis. ROS levels increase intensely during oxidative stress leading to significant damage to the cellular components causing many diseases in humans.[9] Hence, there is a need for adding an agent to remove the excess ROS formed by CH and CHX mixture which does not reduce the antibacterial efficacy since it is used as intracanal medicament.

Antioxidants or ROS scavengers are essential in preventing and controlling human diseases. Many of the antioxidant compounds are derived from plant sources. Studies demonstrated the antioxidant property of the different parts of the guava plant. Guava is rich in Vitamin C and A.[1011] Conventionally, it has been used in the management of toothache, gingivitis, wounds, ulcers, coughs, sore throat, vomiting, diarrhea, dysentery, gastroenteritis, diabetes, hypertension, and obesity.[10] Guava leaf extract has antioxidant, anti-inflammatory, anti-spasmodic, and antimicrobial activity. Anthocyans, alkaloids, flavonoids, tannins and terpenoids contribute for its therapeutic effects.[12]

Hence, the study objectives were to analyze the antibacterial efficacy of AGL and EGL against Streptococcus mutans, Staphylococcus aureus, Lactobacillus acidophilus and E. faecalis, to evaluate the antioxidant potential of the extracts and its ability to counteract the reactive oxygen formation (ROS) formed when mixing CH with CHX.

MATERIALS AND METHODS

Preparation of AGL and EGL

The preparation of the extracts was adopted from Preethi et al.[13] Guava leaves were washed, shade dried, macerated, and powdered using the dry grinder and stored in an airtight bottle. 10, 25, and 35 g of guava leaves powder were dissolved in 90 ml, 75 ml, and 65 ml of sterile distilled water and heated for 2 h at 100°C. The extracts were filtered and the water filtrate was frozen and lyophilized. This was redissolved in water to obtain different concentrations of AGL. 10, 25, and 35 g of guava leaves powder were dissolved in 90 ml, 75 ml, and 65 ml of ethanol and then filtered to obtain 10%, 25%, 35% of EGL.

Antibacterial analysis

The antibacterial analysis was conducted for the extracts, CH (Prevest DenPro, india) +2% CHX (Asep-RC, Anabond Stedman Pharma Research [P] Ltd., India) (positive control [PC]), distilled water (negative control) and 10, 25, 35% of the extracts [AGL and EGL] and its combination with PC using Kirby-Bauer disc diffusion method. Microbial strains of S. mutans, E. faecalis, and S. aureus and L. acidophilus were obtained. Standard 4 mm diameter wells were punched into agar plates using blunt end of a pipette. Using a sterile applicator 0.5 ml of each bacterial suspension was swabbed over the surface of agar plates. A micropipette was used to deliver 50 μL of test solution into the wells. The agar plates were incubated at 37°C for 24 h prior measuring the zone of inhibition.

Evaluation of the anti-oxidant potential of the extracts

The antioxidant potential of 10%, 25% and 35% concentration AGL and EGL were analysed using 2, 2 diphenyl-1-picryl hydrazyl (DPPH) assay. 0.1 mM solution of DPPH radical solution in ethanol was prepared. A volume of 2 ml of the prepared solution were mixed with 1 ml of AGL solutions and incubated at room temperature in dark. The absorbance of DPPH radicals in the mixture was measured using ultraviolet spectrophotometer (SL 159-ELICO, REMI Instruments division, Mumbai) at 517 nm. L-ascorbic acid was used as a PC for both AGL and EGL under the same assay conditions.

Evaluating ROS Formation

PC and all possible combination of the extracts with PC were evaluated for ROS and parachloroaniline using mass spectrometer (LCQ ADVANTAGE MAX, United states) immediately after preparation. The samples were mixed with 500 μl of solvent methanol (99.8%) and injected into the mass spectrometer by an injection. The ion separation was achieved by using the electron impact ionization method. The ions were then detected electronically and the resulting information was analyzed.

RESULTS

Maximum antibacterial activity was noted with PC followed by PC with 35% EGL and other groups as shown in Figure 1a. No zone of inhibition was noted in the negative control. All four microorganisms showed varied sensitivity to different combination of agents.

Figure 1

(a) Antibacterial activity of different groups, (b) antioxidant activity of aqueous and ethanol guava leaves extract by 2, 2 diphenyl-1-picryl hydrazyl method, (c) analysis of parachloroaniline using mass spectrometry

Antioxidant activity

The antioxidant activity of EGL was better than AGL. 35% EGL showed the best antioxidant activity among the different concentrations analyzed [Figure 1b].

Analysis of parachloroaniline and reactive oxygen species using mass spectrometry

All experimental groups showed a peak value of ROS formation ranging from 8.73 to 8.74 m/Z [Figure 2]. The ROS was reduced by the addition of both AGL and EGL. 35% EGL was better in reducing the ROS. The maximum production and maximum reduction of para-chloroaniline (PCA) was exhibited by PC and PC +35% EGL, respectively [Figure 1c].

Figure 2

Mass spectrometric graph showing ROS peak ranging from 8.73 to 8.74 m/Z. (a) CH + CHX, (b) CH + CHX + 10% AGL, (c) CH + CHX + 25% AGL, (d) CH + CHX + 35% AGL, (e) CH + CHX + 10% EGL, (f) CH + CHX + 25% EGL, (g) CH + CHX + 35% EGL. CH: Calcium hydroxide, CHX: Chlorhexidine

DISCUSSION

Root canal instrumentation is inefficient in eliminating microorganism from inaccessible areas such as accessory and lateral canals, isthmus, and the dentinal tubules which may serve as a source of infection. Hence, the use of irrigants during treatment and the intracanal medicament in between the appointments are mandatory.[1]

CH has antimicrobial activity, tissue dissolving ability and induces hard-tissue formation.[514] The antimicrobial activity of CH is directly influenced by its high alkalinity. CHX is an effective broad-spectrum antimicrobial agent. CHX has substantive antibacterial efficacy which is effective against strains resistant to Ca (OH) 2.[15] The CHX liberates PCA along with ROS as a function of alkaline environment, heat, and time.[16]

ROS are small highly reactive short-lived molecules formed by incomplete single-electron reduction of oxygen. They are bactericidal in nature. ROS is essential for cell proliferation and signaling. However, increase in the ROS levels during oxidative stress results in disruption of the cell structures, proteins, DNA, lipid peroxidation, and oxidative inactivation of specific enzymes.[9] They are implicated in neurodegenerative diseases, diabetes and cancer.[9]

A dynamic equilibrium exists between ROS activity and counteraction by antioxidants in normal conditions.[17] Under oxidative stress, ROS production and activity increases and the defense capacity decreases.[17] ROS scavengers or antioxidant plays a significant role in the prevention of human diseases. Numerous herbs are known to have antioxidant potential. Guava leaves extract has higher antioxidant potential compared to the other parts of the tree.

Many researchers have performed antibacterial screening selectively of guava essential oils, and solvent extracts.[181920] It has been reported that the phenolic compounds penetrate the bacterial cell membrane and causes cell lysis.[21] The antibacterial analysis reveals that the PC (combination of CH and CHX) showed maximum antibacterial activity which could be due to the massive production of ROS.[6] The type of solvent and the method of extraction may play a role in the antibacterial activity of any natural extract. EGL was better than AGL when used alone or in combination with CH and CHX. Cytoplasmic membrane depolarization and inhibition of macromolecular synthesis are responsible for the antimicrobial action of polyphenolic compounds.[19] Tannins inhibit microbial growth by chelation of iron leading to iron deprivation, metabolic disturbances by inhibition of oxidative phosphorylation, deprivation of substratum, and extracellular enzyme inhibition.[19]

Ascorbic acid is present in abundance in guava leaves which may be responsible for its antioxidant potential.[10] EGL exhibited higher antioxidant activity than AGL which could be due to the actions of flavonoids, polyphenols, and tannins present in EGL.[12] The antioxidant potential of both extracts appears to be concentration dependent. Maximum production of ROS was noted in PC (CH and CHX), which could be due to the alkaline pH created by hydroxyl ions a dissociation product of CH. The addition of guava leaf extract reduced the ROS formation when CH is used along with CHX with EGL more effective than AGL.

CONCLUSIONS

The combination of CH and CHX generates greater amount of ROS and PCA which has adverse effects on the periapical tissues. The addition of higher concentrations of guava leaves extract reduces the ROS significantly. 35% EGL showed greater antioxidant activity and least generation of PCA without adversely affecting the antimicrobial activity of the CH-CHX combination.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.