Periodontitis is a disease, inflammatory in nature, which affects the supporting structures of the teeth and is characterized by periodontal pocket formation, progressive attachment loss, and bone loss. Dental plaque and its micro-organisms are considered the primary etiologic factor of periodontitis. Around 700 bacterial species of diverse nature colonize the oral cavity, but only a few of these are potential periodontal pathogens. Some of the primary pathogens of periodontal disease are Porphyromonas gingivalis, Tannerella forsythia, Treponema denticola, and Aggregatibacter actinomycetemcomitans.[2,3]
The critical objectives of periodontal therapy include the elimination of dental plaque and the factors that aid in plaque accumulation and retention. Scaling and root planing (SRP) includes eliminating plaque and calculus which helps in re-establishing the periodontal tissues to a healthy condition. However, several studies have reported that SRP alone will not be able to efficiently remove most of the causal bacteria, and bacteria may persist in the treated sites. Hence, systemic and locally delivered pharmacologic substances are used as an adjunct to SRP.
Local drug delivery (LDD) devices are the systems designed to deliver a drug directly into the periodontal pocket resulting in the penetration of the drug into the periodontal tissues and retaining the therapeutic level within the periodontal pocket for an extended time frame. This dual effect on pocket microflora and the pathogens invading tissue can improve clinical results without any undesired systemic effects. Several antibiotics have been used in LDD to eliminate resident periodontal pathogens and thus provide an adjunctive therapy to the mechanical treatment. Undesirable side effects such as drug resistance, toxicity, drug hypersensitivity, development of opportunistic infections, and drug interactions are often encountered.
For centuries, plants, plant products, and herbs are being used globally as remedial agents against several diseases, including infectious diseases. Plant extracts are used in dentistry for their anti-inflammatory, antimicrobial properties and are also used as antiseptic, antioxidant, antifungal, antiviral, and analgesic agents.[9–12] The development of a herbal extract-based LDD agent with controlled release can give better results, lesser side effects and could act as a therapeutic adjunct to mechanical debridement in the treatment of periodontal disease.[13,14]
Moringa oleifera (MO), commonly known as the drumstick tree, is an important plant of medicinal value which is found in several tropical regions and used to treat various diseases.[14,15] Most of its parts, such as leaves, seeds, roots, flowers, and fruits, can provide antitumor, anti-inflammatory, and antibacterial effects, which can be attributed to the chemical constituents such as flavonoids, phytosterols, glycosides, tannins, and amino acids present in these. To date, no studies have been done to evaluate the role of M. oleifera as an LDD agent for periodontal treatment.
Hence, this in vitro experimental study aimed to design, formulate, and evaluate the antibacterial efficacy of M. oleifera extract for periodontal drug delivery and conduct in vitro characterization of the LDD gel developed.
MATERIALS AND METHODS
The study was designed as an in vitro experimental study. Ethical clearance for the study was obtained from the Institutional Ethical Committee.
The source ingredients used for the study were dried, and cold-milled M. oleifera leaves powder (Madurai, India), 70% ethanol, 2% v/v acetic acid, propylene glycol triethanolamine (Bengaluru, India), and chitosan (85% deacylated) of medium molecular weight (Cochin, India) were procured. Polyethylene glycol 6000 (PEG 6000), Carbopol 940 was obtained commercially (Bengaluru, India).
The leaves of M. oleifera were used for the preparation of the extract as they are a richer source of phenolics than seed and fruit extracts. When different extraction methods for M. oleifera leaves were compared, maceration of MO dried leaves powder with 70% ethanol was recommended regarding simplicity, convenience, and economic feasibility of the extract with highest contents of flavonoids, phenolics, and antioxidant activity. The same method was followed in the present study [Figure 1]. Ten gram of dried and powdered MO leaves was soaked with 400 ml of 70% ethanol (1:40 w/v) at the room temperature (28°C ± 2°C) for 72 h with intermittent stirring. The extract was filtered and procured through Whatman no. 1 filter paper and was re-extracted again until the extraction was depleted. It was then evaporated in a hot air oven at 90°C.
Assessment of the minimum inhibitory concentration (MIC) of Moringa leaves extract against P. gingivalis was done as follows [Figure 2]: nine dilutions of extract were obtained using thioglycollate broth. In the primary tube, 20 μl of the extract was combined with 380 μl of thioglycollate broth. For further dilutions, 200 μl thioglycollate broth was put in to the following nine tubes individually. 200 μl was then transferred from the primary tube to the first tube with 200 μl of thioglycollate broth to obtain 10−1 dilution. From this tube with 10−1 dilution, 200 μl was transferred to the second tube for a 10−2 dilution. The series of dilutions were repeated till a 10−9 dilution of the extract. From the maintained stock cultures of standard strains of P. gingivalis ATCC 33277, 5 μl were added to 2 ml thioglycollate broth. 200 μl of the culture suspension was added to each serially diluted tube. The tubes were then incubated in a temperature-controlled anaerobic jar at 37°C for 48–72 h and analyzed for turbidity.
For the preparation of M. oleifera gel, the method of preparation by Aslani et al. was followed with slight modifications [Figure 3]. Carbopol 940 has excellent cross-linking and swelling properties. This increases the viscosity of the formulation and results in controlled drug release. Chitosan exhibits favorable properties such as nontoxicity, biocompatibility, and biodegradability. Herbal extracts are poorly soluble compounds and require a carrier to be soluble in the gel base. In the present study, PEG was used to solubilize the MO extract into the gel base and it also shows high thermal stability.
To formulate the chitosan gel, 0.5 g Chitosan (85% deacylated) was dissolved in 10 ml of 2% (v/v) acetic acid solution with continuous stirring with the help of a mechanical stirrer at 3600 rpm for half an hour.
To formulate the carbopol 940 gel, carbopol 940 (0.5 g) was added to water (10 ml) and dissolved and kept aside to swell for 30 min. It was then constantly stirred by a mechanical stirrer at 3000 rpm for 30 min.
To formulate the PEG gel, 0.5 g PEG 6000 (PEG 6000) was added to 10 ml of water and dissolved with frequent mixing with the help of a mechanical stirrer for 20 min at 2000 rpm. 1 mg of M. oleifera leaves extract was then added and stirred until completely dissolved. Further, PEG gel containing the MO extract was added to Carbopol gel and combined well. This mixture was then added to Chitosan gel and was combined well together using the mechanical stirrer at ~5000 rpm for half an hour. The pH was then adjusted to neutral using triethanolamine. 2 ml of propylene glycol was added as the plasticizer and stirred slowly. The stirring was carried out for 1 h till a clear gel was obtained.
Probe sonication was performed after mixing the solution to obtain a homogenous dispersion (QSONICA SONICATORS, MODEL CL-334, Newtown, CT) at 20 s on cycle and then 5 s off-cycle for 10 min.
Assessment of the MIC of drug-loaded gel against P. gingivalis was done as follows [Figure 4]: The MIC of drug-loaded gel against P. gingivalis was assessed following the same procedure mentioned earlier for the MO leaves extract.
The gel formulation was characterized in vitro for clearness, color, homogeneity, consistency, and existence of particles [Figure 5]. The consistency of the formulation was assessed by squeezing a little amount of the gel in between the index finger and thumb.
The determination of pH in gel formulations was done as follows: The pH meter was calibrated before each use using a standard buffer solution at neutral pH 7. 1 g of the gel formulation added to 10 ml purified water. The electrode was introduced into this sample at room temperature for 10 min, and the reading was noted. The mean pH was evaluated after noting the pH values thrice [Figure 6].[24,25]
Tests for thermodynamic stability included centrifugal test, freeze and thaw test, heating − cooling cycle, syringeability study, viscosity determination, and ex vivo mucoadhesive strength.
For the centrifugal test, 48 h after preparation, the gel formulation was transported into tubes and centrifuged using a centrifugal device at 2000 rpm for 60 min (Centrifuge 5430). The stability of the formulated gel against centrifugal force was evaluated at 5, 15, 30, and 60 min.
The heating − cooling cycle of the formulation was assessed by keeping it at 0°C and 45°C alternately for 48 h and checked for any changes in its physical properties.
For the freeze and thaw test, the formulated gel was subjected to −20°C and 20°C for 24 h each for three consecutive cycles and checked for any physical changes.
Syringeability of the formulation was assessed by passing the gel formulation through a 26 G needle, which is commonly used for periodontal drug delivery.
The viscosity of the formulation, in centipoise, was determined using Brookfield DV-II + viscometer (Brookfield Engineering Labs Inc., USA). After mixing, the formulated MO extract gel was added to a test tube, and its viscosity was determined using spindle no. 4. The measurements were made over a range of speed settings from 10 rpm to 100 rpm at 25°C. Table 1 shows the corresponding readings.
Ex vivo mucoadhesive strength was assessed using a fabricated mucoadhesive strength test apparatus [Figure 7] and freshly collected porcine buccal mucosa. The apparatus consisted of a vertical stand for support and a metallic base. The stand included a fixed and a movable platform. The movable platform was balanced with the apparatus using a shaft to keep the apparatus steady. A pan was put on the apparatus to hold the weight and to measure the bio-adhesive strength. The porcine cheek mucosa was procured from a slaughterhouse, and it was used in 2 h of slaughtering the animal. Adipose and loose tissue was removed and the mucosa was rinsed in the distilled water and phosphate buffer (pH 6.8) at 37°C. After preparing the tissue, a 3 cm2 sized mucosal layer was fixed on the immovable platform using cyanoacrylate gum [Figure 8].
The sample film was attached over the movable platform similarly using cyanoacrylate gum. It was hydrated with 15 µL of phosphate buffer for 30s for swelling and initial hydration. The movable platform was shifted toward the fixed platform horizontally to bring it into contact with the surface of the mucosa. A 20 g preload was kept over the movable platform as the initial pressure for 3 min to establish good attachment of the mucosal surface to the film. Weights were proportionately added to the attached pan with the movable platform at a particular time intervals. The total weight required for complete detachment of the film was observed, and the following formula was used to calculate the mucoadhesive strength:
Force of adhesion = (Bioadhesive strength × “‘g”) ÷ 1000
Bond strength = Force of adhesion ÷ Area of film surface
(“g” = 9.81 m/s2) is referred to as the acceleration due to gravity. The weight in grams that is required to separate the film and the mucosal surface is the bioadhesive strength. The energy needed for detaching the two systems is called the bond strength.
Evaporation and solvent removal after the maceration process of M. Oleifera leaves powder in 70% ethanol yielded a dark green semi-solid concentrated extract. The MIC of Moringa extract against P. gingivalis was assessed to be 0.8 μg/ml. P. gingivalis was sensitive to any concentrations above 0.8 μg/ml of the prepared extract [Table 2].
A combination of carbopol and Chitosan polymers with a well-dispersed and stable system in an aqueous solution was used as the gel base, which is considered an excellent candidate for controlled drug delivery.[30,31] 1 mg of the ethanolic extract of M. oleifera was used to formulate the mucoadhesive gel; which was more than the MIC against P. gingivalis. The gel obtained after probe sonication was homogenous, semi-solid, and light yellowish-green in color.
The MIC of drug-containing gel against P. gingivalis was evaluated as 25 μg/ml. This revealed that the prepared formulation of M. oleifera extract gel showed bactericidal properties against P. gingivalis [Table 2].
The last objective of the present study was to evaluate the physicochemical properties of the formulated MO extract gel by its in vitro characterization. The formulated gel was light yellowish green in color, opaque, and had a semi-solid consistency [Table 3]. The gel had flexibility and a smooth and uniform surface. The homogeneity, physical appearance, and consistency of the gel were good.
Periodontal pockets have been shown to have a mean pH of 7.09 ± 0.07 in periodontal disease, with no large variations in relation to the pocket depth. In the present study, the average pH of the drug-containing gel was 7.13, which could neither interfere with the drug release nor cause any harmful effect or irritation to the patient.
Tests for thermodynamic stability performed were the centrifugal test, freeze and thaw test, and heating and cooling test. The MO extract gel exhibited good stability in all the experimental conditions.
Gel formulations, as LDD agents in periodontal therapy, are gently delivered into subgingival pockets with syringes, ensuring an equal distribution of the drug. The formulated M. oleifera extract gel was syringeable through a 26 G needle. The results of the Syringeability study indicated that the formulated M. oleifera extract gel was syringeable through a 26 G needle, which is commonly used for LDD in periodontal therapy.
MO extract gel showed suitable viscosity, resulting in the controlled release of drug in the periodontal pocket [Table 1]. The mucoadhesive strength of the drug-containing gel was observed as 29.5 g. Force of adhesion was calculated as 0.289 N, whereas the bond strength was 0.096 Nm−2.
Periodontal disease occurs due to a series of complex, multifactorial, and polymicrobial infections that result in damage to the tooth-supporting structures. Conventionally, mechanical therapy (SRP) and systemic and local anti-infective therapy are used to treat periodontal pockets. Various medicinal plants and their derivatives have been used to prevent and treat oral infections in recent times. Recent studies have investigated the benefits of using plant-based products on various periodontal clinical parameters such as gingival inflammation and gingival bleeding.
Many studies have been done to assess the phytochemistry of M. oleifera and its pharmacological uses.[16,37] The antioxidant polyphenolic investigation done by high-performance liquid chromatography in ethanolic extract of M. oleifera leaves showed the presence of phenolic acids (gallic, chlorogenic, ellagic, and ferulic acid) and flavonoids (kaempferol, quercetin, vanillin, and rutin). Polyphenols are promising antimicrobial and immunomodulatory agents in controlling periodontal diseases. Implementing LDD devices containing high concentrations of polyphenols, especially flavonoids with their anti-inflammatory effects, could provide a possible role in managing or preventing periodontitis.[40,41]
The antimicrobial effectiveness, explained in terms of MIC, is the least amount of the compound that can of prevent the growth of the offending microorganism. To date, many studies have been done to assess the antimicrobial effectiveness of M. oleifera. The evaluated results showed that the M. oleifera leaves extract showed anti-bacterial properties against both Gram-positive and Gram-negative bacteria, however, it exhibited more potent antimicrobial activity against Gram-negative bacteria than Gram-positive strains.
Although subgingival plaque contains over 500 bacterial species, several studies have reported that P. gingivalis is one of the chief etiological agents in the pathogenesis and development of periodontal disease and is appropriately termed as keystone pathogen. The initial objective of the current study was to evaluate the MIC against P. gingivalis, which is considered a keystone pathogen in periodontitis. This study is the first to evaluate the antibacterial effectiveness of MO leaves extract toward P. gingivalis, and the results showed that M. oleifera leaves ethanol extract possessed bactericidal properties against P. gingivalis even at a lower amount of 0.8 μg/ml.
Pterygospermin, benzyl glucosinolate, and benzyl isothiocyanate isolated from M. oleifera leaves reportedly have bactericidal activities against several bacterial species. Furthermore, the leaves of M. oleifera contain flavonoids, tannins, saponins, phytochemicals, and other phenols, that exhibit antimicrobial properties via cell membrane perturbations.[31,45] This would suggest that further studies can isolate the specific compounds from the MO extract and evaluate their antimicrobial activity against P. gingivalis. In periodontal disease, the innate immune response is the initial line of defence. The ability of MO extract to inhibit bacterial growth could enable the host immune response to destroy the invading pathogens. When incorporated into a controlled delivery device, MO extract could enhance clinical performance and furthermore provide a cost-effiective local therapy. An ideal formulation of a LDD agent for the treatment of periodontitis should have adequate retention and mucoadhesion within the periodontal pocket for the required duration of time, exhibit controlled drug release properties, and the mode of delivery into the periodontal pocket should be easy.
Our study’s second objective was to formulate a mucoadhesive gel using M. oleifera leaves extract at its MIC, against P. gingivalis for periodontal drug delivery.
Chitosan, Carbopol 940, and PEG 6000 were used in combination as the gel base in the present study. The molecular weight of Carbopol is high and it swells in water up to 1000 times its original volume to create a sizeable adhesive surface with mucin and offer adequate mucoadhesiveness. Carbopols with mucin results in a secondary bio-adhesive bond‚ where as with other polymers it forms a superficial bio-adhesive bond. Carbopols, added as mucoadhesive polymers within the formulations allow enhanced contact within the mucosa and longer retention. This allows sustained drug delivery and reduces the frequency of administration. Adding Carbopol 940 also increases the viscosity of the formulation and decreases its rate of release. Carbopol 940 has excellent cross-linking and swelling properties. The gel thickens with the swelling of the polymer, and penetration of water is reduced which leads to controlled release of the drug. Considering these properties, Carbopol 940 was the drug carrier used in the present study.
Chitosan is a hydrophilic biopolymer procured by the deacetylation of chitin, which has both bio-adhesive and permeabilizer properties. Chitosan is also nontoxic, bio-compatible, and bio-degradable. Chitosan also exhibits antimicrobial, wound healing, haemostatic, and tissue regenerative properties, which makes it an agent of interest in the dental field.[48,49]
The antimicrobial action of chitosan can be classified as extracellular effects, intracellular effects, or both. Anionic interaction between the anionic regions in the cell wall of bacteria such as carboxylic acids and phospholipids, and cations from the amino groups of chitosan has been shown as the mechanism behind its antibacterial effects. Gel formulations of chitosan show good bioadhesive properties, significantly contributing to drug retention within the periodontal pocket and also to the drug release in an extended manner. It has been proposed that this is brought about by the positive charge on chitosan, that also aids in its prolonged effect on the epithelium after physically being removed from the surface. Our results showed that M. oleifera leaves extract possessed bactericidal properties against P. gingivalis even at a lower concentration. Hence, the use of chitosan along with Moringa oliefera extract provides an additive antimicrobial effect and also aids in bioadhesion within the periodontal pocket.
Advantages of PEG are high thermal stability and the ability to solubilize poorly soluble compounds like herbal extracts into gel base.
The MO extract gel formulated as a periodontal drug delivery agent showed stable physicochemical properties during the in-vitro characterization. Viscosity determines the amount of drug that is released from the gel. Viscosity of the gel and the drug release rate are inversely proportional to each other. MO extract gel showed adequate viscosity, which can be linked to the combination of polymers used for the formulation. The formulation showed excellent mucoadhesive strength, this characteristic can result in drug retention within the periodontal pocket for the desired time period and results in sustained release of the drug. This can reduce the frequency of administration, which would help in achieving good patient compliance and better clinical efficacy.
The current study has some limitations. One of the limitations is that we could not attribute the bactericidal property to an isolated compound in the MO extract. Another limitation of our study was that it was an in-vitro study, and only one combination of concentrations of polymers was used. In the present study, evaluation of drug content uniformity and drug release studies, as it was a whole leaf extract, was a challenge. Further research can be done on various concentrations of bio-adhesive polymers within the gel to find out the ideal combination of polymers for the gel formulation that would give the ideal mucoadhesive properties. Drug release tests can be performed to evaluate the release kinetics of the drug. Furthermore, clinical trials must be carried out to evaluate the effectiveness of the formulated gel clinically as an adjunct periodontal drug delivery agent.
The study with respect to the formulation of moringa gel is the first of its kind to formulate a LDD gel with M. oleifera extract. The current study showed that M. oleifera leaves extract possesses a bactericidal effect against P. gingivalis and is a potent botanical extract for periodontal drug delivery formulation against periodontitis, as P. gingivalis is suggested to be the keystone pathogen in the etiopathogenesis of periodontitits. Incorporating the extract into the gel base, which contains a combination of polymers, improves the drug’s stability, muco-adhesiveness, and controlled release. Hence, it can be concluded that M. oleifera leaves extract can be used to treat periodontal diseases as a LDD agent.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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