Int J Pharm Pharm Sci, Vol 8, Issue 8, 132-141Original Article


FORMULATION DEVELOPMENT AND IN VITRO EVALUATION OF DENTAL GEL CONTAINING ETHANOL EXTRACT OF TEPHROSIA PURPUREA LINN.

NIYOGI PARTHA, PATTNAIK SNIGDHA*, MAHARANA LAXMIDHAR

School of Pharmaceutical Sciences, Siksha ‘O’ Anusandhan University, Kalinga Nagar, Ghatikia, Bhubaneswar 751003, Odisha, India
Email: pattnaiksnigdha@yahoo.com

Received: 31 Mar 2016 Revised and Accepted: 20 June 2016

ABSTRACT

Objective: Dental infections are mainly caused by the colonization of anaerobic gram-negative pathogens and a challenge was taken to formulate a dental gel containing ethanol extract of the whole plant of Tephrosia purpurea Linn. (EETP) against dental diseases that are mainly caused by Porphyromonas gingivalis.

Methods: The defatted and soxhalated ethanol extract of the whole plant of Tephrosia purpurea Linn. was estimated for its flavonoid content and the antimicrobial study was performed on clinically isolated Porphyromonas gingivalis to determine the dose with a comparison to pure flavonoid to establish the presence of bioactive components. An attempt was made to prepare an herbal bio-compatible dental gel using cold mechanical method containing EETP with varied concentrations of carbopol-934P, HPMC-K4M and chitosan and various characterization parameters were carried out.

Results: Based on flavonoid content and antimicrobial activity 2%w/v of the extract was used as the dose in all the EETP containing formulations. The pH, viscosity, gelling temperature, spreadability and swelling index of all formulations showed the suitable result. Mucoadhesive strength was found highest for the formulation with 4.0% HPMC-K4M, which also resulted in the best flavonoid diffusion with highest permeation flux. All the EETP loaded dental gel formulations extruded satisfactorily through needle no-22. The antimicrobial study on clinically isolated Porphyromonas gingivalis showed higher antimicrobial inhibition as compared to the marketed metronidazole (1% w/v) gel. Stability study confirmed all the EETP loaded dental gel formulations to be stable after one month.

Conclusion: Therefore, Basing on all aspects of the study, The dental gel containing 4.0%w/w, HPMC-K4M, 20%w/w PG with 1.0% w/w glycerol was found to be the best.

Keywords: Tephrosia purpurea Linn., Dental gel, Carbopol 934 P, HPMC-K4M, Chitosan

© 2016 The Authors. Published by Innovare Academic Sciences Pvt Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/)

INTRODUCTION

Human kind’s effort to comfort disease dates back to early civilization. From the beginning of advancement of life all cultures used natural sources like herbs as the primary health care management. There has been increasing attention paid in recent years to the oral infectious diseases and oral inflammations, in particular, infectious dental diseases. Dental infections, other wisely known as periodontitis, are the group of infections, predominantly caused by colonization of gram-negative, anaerobic pathogens on sub-gingival areas. Periodontitis can be characterized by; gum inflammation (with redness and bleeding), the formation of deep pocket (in between the teeth and gum) and loosen teeth (erosion of structure of connective tissue and bone).

Periodontitis found to eventuate due to contemporaneous gingivitis that results in connective tissue loss from the teeth [1]. Though the lesions found in both the periodontitis and gingival infections were histopathologically matched but these diseases are clinically different [2, 3]. Clinical symptoms in chronic gingivitis can be distinguished by redness and oedema formation at affected area [4], 1965, bleeding and contour change[5], an increase in gingival crevicular fluid and loosening of connective tissue [6]. According to folklore data, Tephrosia purpurea (Linn.) also known as “Sarwa wran vishapaka” has been found with all types of wound healing properties [7]. The ethanol extracts of Tephrosia purpurea Linn. was also reported to possess potential antibacterial activity [8, 9].

Basing on the above investigations, the investigation has been taken to prepare dental herbal gel formulations containing ethanol extract of Tephrosia purpurea Linn. and to evaluate them in terms of their physicochemical characteristics and in vitro drug release study.

As per U. S. P. gel is a semisolid or suspension containing small inorganic or large organic particles interpenetrated with liquid [10], pharmaceutically act as carriers for other drugs for their localized percutaneous absorption [11] and having a good visco-elastic property due to the entrapment of solvent in the network like three-dimensional colloidal structure formed by gelling agent and solvent interaction [12].

Crude drug characteristics differ dramatically to induce the activity of drug at the site of action at sufficient concentration. A drug delivery system (DDS) allied of the carrier and the drug is elevated as a revolutionary to deliver the drug at the preferable site of action, at an accurate concentration at the accurate period of time [13]. Furthermore, obstacles arising from various side effects or toxicity of synthetic drugs can be overcome by using a potent herbal drug in the form of suitable drug delivery systems that is better patient-compatible with least side effect [12]. Substantially, the challenge to formulate a delivery system with increased therapeutic activity and least side effects may be overcome by perfect fabrication of novel drug delivery systems with a proper dose of the herbal extract [14].

MATERIALS AND METHODS

Materials

Plants of Tephrosia purpurea Linn. were collected and authenticated by Regional Plant Resource Centre, Bhubaneswar, Odisha. HPMC-K4M and carbopol-934P was a gift sample from Wockhardt Ltd., Aurangabad, India. Chitosan and quercetin were purchased from Sigma, Poole, UK. Sheep Blood Agar Media was purchased from Hi-Media Laboratories Pvt. Ltd., Mumbai, India.

Methods

Preparation of plant extracts

500g of the drug powder was extracted with 3000 ml of ethanol (90%) for 24 h in soxhlet assembly. The filtered extract was dried by evaporating the solvent till a thick mass was obtained [15].

Analytical method for quantification of flavonoid

An aliquot of given concentrations of stock solution (quercetin) was scanned from 350-400 nm wavelength range in a UV-Visible spectrophotometer (Jasco V-630 Spectrophotometer, Japan, Serial No-A1259611487). From the scanning report, it was evident that the wavelength of maximum absorbance (λmax) of Flavonoid was found at 421 nm (fig. 1).

Aliquots of 0.1, 0.2, 0.3, 0.4, and 0.5 ml from the above stock solution were taken in 5 different 10 ml volumetric flasks. To each flask, 1.5 ml of methanol, 0.1 ml of 10% aluminum chloride, 0.1 ml of 1M potassium acetate and 2.8 ml of distilled water was added. The reaction mixture was kept aside at room temperature for 30 min and the volume was made up with water. The absorbance of the resulting different concentration of solutions was measured against reagent blank. The calibration curve was prepared by plotting absorbance vs concentration, and it was found to be linear over this concentration range of 10-50μg/ml (fig. 2).

0.1 g of extract was dissolved in 100 ml of ethanol to get 1 mg/ml solution. Required volume of the above solution (1 ml) was transferred into a 10 ml standard flask and colour development was carried out as that for the standard by adding 1.5 ml of methanol, 0.1 ml of 10% aluminium chloride, 0.1 ml of 1M potassium acetate and 2.8 ml of distilled water. From this 1 ml solution was taken and diluted up to 5.5 ml with distilled water. The absorbance of the test solution was measured at 421 nm against blank. Total flavonoid content was calculated in terms of percentage of quercetin equivalent per 100g [16].

Anti-gingival activity study and dose determination of EETP in comparison with pure flavonoid

The link between oral diseases and the activities of microbial species that form part of the microbiology of the oral cavity is well established and over 750 species of bacteria inhabit the oral cavity and a number of these are implicated in oral disease [17]. Out of various oral micro floras, clinically isolated Porphyromonas gingivalis were collected from Institute of Dental Sciences, Siksha‘O’ Anusandhan University, Bhubaneswar and used for the antimicrobial study. The stains were streak on sheep blood agar media in which columbia agar base (4.25%) was supplemented with sheep blood (5%), bacitracin (1%), colistin (1.537%), nalidixic acid (1.5%), hemin (0.5% of 0.1% solution) and vitamin K1 (0.1% of 0.1% solution) [18] and kept in an anaerobic condition of 10% hydrogen, 10% carbon dioxide and 80% nitrogen at 37 oC for 48hrs [18]. After 48 h the colony of the both pathogens was formed on the sheep blood agar plates and showed haemolysis effect on sheep blood showed in fig. (fig. 3). To determine the antimicrobial activity and the dose with a comparison to standard 1% metronidazole, the brain heart infusion medium was prepared by dissolving the same in distilled water. Then sample from columbia agar media was taken in brain–heart infusion broth (Oxoid) supplemented with 5 mg/l haemin (Sigma, Poole, UK), and incubated for 24 h. Ten percent inoculum was transferred to 9 ml of the same medium and incubated for 3 d. All cultures were allowed to grow at 37 oC in the above mentioned anaerobic condition.

Then 1 ml from the inoculums was added to 8 ml of the same concentration of brain–heart infusion broth (Oxoid) in six separate test tubes and numbering was done like PF2, PF3, PF4, PF5, PF6, PF7. And in one test tube, 1 ml of inoculum in 9 ml medium was taken and called as a positive control (PF1). In PF2 1 ml of 1% w/v concentration of standard metronidazole is added and taken as standard. In other five test tubes 1 ml of aqueous solution of extract in concentration of 0.010 gm/ml, 0.015 gm/ml, 0.020 gm/ml, 0.025 gm/ml, and 0.030 gm/ml was added accordingly and again kept overnight at 37oC in incubator maintaining anaerobic condition in the similar manner described above. Then optical density was measured. Results were tabulated in fig. (fig. 4) showing the minimum concentration of extract that shows the satisfactory antimicrobial activity.

To establish the bio-active compounds with a comparison to standard quercetin (PF8) of the same concentration as that of the optimized concentration of extract was tested against P. gingivalis by following the above-mentioned method (fig. 4).

Formulation of various EETP loaded dental gel formulations

Different concentration of carbopol-934P and HPMC K4M containing gel was formulated by modified cold mechanical process [19, 20]. Finally, 0.2%w/w ethanol extract of T. purpurea Linn. was added with continuous stirring.

Chitosan gel was prepared by following dispersion technique; glycerol (5%w/w) and glacial acetic acid (3%w/w) was added with continuous mixing using a mortar and pestle followed by addition of the 0.2%w/w ethanol extract of T. purpurea Linn.

All the formulations were stored in a screw capped wide mouthed beaker covered with aluminium foil in a cool and dark place [22, 23].

Characterizations of various EETP loaded dental gel formulations

pH determination

Digital pH meter was used to determine the pH of the formulations by dipping the electrode in 25 ml of distilled and deionized water containing 2g of formulated EETP loaded dental gel. The pH measurements were performed three times, and the average was noted as the result depicted for each formulation [24, 25].

Viscosity determination

Viscosity determination was carried out using Brooke-field viscometer (TV-10) connected with a thermally-controlled water bath to maintain the temperature at 37 oC. Prior to each experiment, the viscometer was equilibrated with the sample for 10 min and at a shear rate of 20rpm. The viscosity measurements obtained was the average of three readings for each formulation (n = 3).

Gelation temperature measurement

Gelation temperature was determined by following tube tilting method [26]. In this method 2g of aliquots of formulated gel was transferred to a test tube covered with aluminium foil and immersed in a temperature controlled water bath at 4oC. The samples were heated by increasing the temperature of water bath by 1oC till there was stable meniscus on 90otilting of the test tubes after equilibration for 5 min at every 10oC temperature increased. The data depicted was the average of three readings for each formulation (n = 3).

Swelling index studies

Swelling index studies were performed at 37oC using a thermo jacketed cell containing phosphate buffer saline pH 6.8 as swelling medium. About 2g of EETP loaded dental gel was taken and the study was carried out for 6 h. After 6 h, the gels were scrapped from aluminium foil and the weight was determined by removing the adhered water from the surface by blotting immediately. The swelling Index of the dental gel forming polymer was determined using the relationship [27];

$$\% St = \frac{\left( Wt - Wo \right)X100}{\text{Wo}}$$

Where ‘St’ represents the swelling of the dental gel at time‘t’.

W0 and Wt represent the initial and final weight of gelling solution and gel respectively.

All measurements were performed in triplicate (n=3) and the data noted is the average of the three readings.

Determination of spreadability

Spreadability study was performed by determining the “Slip” and “Drag” characteristics of EETP loaded dental gel [28] using a wooden block consisting of a pulley at one end. The formulations were kept on one slide and sandwiched by another slide across a length of 6 cm among the two slides. A uniform thin layer was formed by placing a 100 gm weight upon the upper slide. Then the weight was removed, and the adhered excess formulation to the slide was scraped off and a 20 gm loaded pulley was used to slip the upper slide to 6 cm distance and to separate away to the direction of applied force from the slide fixed on the board of the apparatus at the bottom. The time taken for complete separation of the two slides was recorded. The experiment was repeated for three times and the average was noted for each formulation. A shorter interval indicates better spreadability [29].

Spreadability  = m x l/t

Where, ‘m’ represents the weight tied to the upper slide (20 gm)

‘l’ represents the length of glass slide (6 cm)

‘t’ represents the time taken to separate the upper slide from the lower slide

Mucoadhesive strength determination

Mucoadhesive strength determination was done to study the mucoadhesive characteristics of prepared EETP loaded dental gel by following the method developed by Choi et al. and Yong et al. [30, 31]. The collected sheep cheek mucosal tissue was cleaned with distilled water followed by phosphate buffer saline pH 6.8 which is isotonic to the tissues and allowed to stand for sometimes at the same pH to remove all soluble components. After confirmation of integrity of the treated sheep cheek mucosal tissues by microscopic investigation [32], 2g EETP loaded dental gel was made sandwiched in between the two slides using cyanoacrylate adhesive and the mucoadhesive force of the formulated EETP loaded dental gel was determined in terms of the detaching force in the weight of water required to separate the glass plates.

In vitro release study

In vitro release studies of EETP loaded dental gels were carried out by using sigma dialysis membrane (MWCO-3500) bag. In this study, the membrane to be used was soaked overnight in phosphate buffer saline solution of pH 6.8 to allow the pores to open. Then the soaked membrane was filled with 2 gms of the EETP loaded dental gels and kept submerged in a beaker containing 60 ml of phosphate buffer saline pH 6.8 at 37±1oC temperature on a magnetic stirrer with continuous stirring at 100rpm to maintain the sink condition of the receptor medium. The samples were withdrawn at specific time intervals from the aliquot reservoir and the same volume was replaced with similar pre-warmed aliquot. The samples were assayed spectrophotometrically to quantify the amount of flavonoid at 421 nm released through the membrane.

Drug release kinetic study

The release kinetics of the flavonoid as active constituents from EETP loaded dental gel was resolved on the graphical plotting of in vitro release data in different models of drug release kinetics; Zero order kinetic model (Equation 1) as percentage of drug (cumulative amount) released in time, First order kinetic model (Equation 2) as log cumulative percentage of drug released in time and Higuchi kinetic model (cumulative percentage of drug released in square root of time) (Equation 3).

C = k0 t-------------------------- (equation 1)

Where k0 represented zero order rate constant in a unit of concentration/time,

t represented as time in hrs.

Zero order graph yield a straight line with a slope of k0 and intercept the origin of the axis [33].

log C = log C0–kt/2.303----(equation 2)

Where, C0 represents the initial concentration of drug,

k represented first order rate constant and t represents time in hrs [34].

Q = kt1/2-------------------------- (equation 3)

Where K represents the constant for the design variables of the system,

t represents time in hrs.

Hence the rate of drug release was found to be proportional to the reciprocal of the square root of time [35].

Hixon-Crowell cube root law (equation 4) data plot used to describe the drug released with surface area changed in terms of the diameter of the particle and plotted as the cube root of the percentage of drug remaining in the matrix in time [36].

3√Q0-3√Qt= KHCt-------------- (equation 4)

Where, Q0 represented as the initial amount of the drug in formulation,

Qt represented as the amount of drug released in time t in hr,

KHC represented as Hixon-Crowell rate constant [36].

Exponential drug release to the elapsed time was simply explained by Korsmeyer et al. [37] to describe the drug release by plotting as the log cumulative percentage drug release in log time (equation 5) and the exponent ‘n’ was calculated from the slope of the linear graph [37].

Q/Q0 = Ktn------------------ (equation 5)

Where, Q/Q0 represents the fraction of drug released at time t,

K represents the constant comprising the geometrical structural characteristics of drug/polymer system n represents the exponent of the drug released [37].

The diffusion exponent describe the diffusion model in a semi-empirical way as n ≤ 0.5, the Fickian diffusion model describe the release mechanism and the non-Fickian model of diffusion described the drug release mechanism as the release of the drug follow both the diffusion and the polymer dissolution mechanism where release rate is inversely proportional to polymer concentration when 0.5<n<1.0. The decrease in the release constant allows long time hydration resulting stretching of the polymer matrix which causes a decrease in rate of drug release. If n was found to be 1, the release kinetic would have been following the zero-order models and if n was found to be greater than 1, then the release mechanism would have been super case-II transport, the model that is considered when unknown release mechanism or different mechanisms control the drug release [37].

The different kinetic model describes drug release from a controlled release preparation by using different mathematical equations like; zero order diffusion model (describe the concentration independent drug release) [33] and first order diffusion model (describe the concentration dependent drug release) [34].

In accordance with Higuchi model of drug release from the polymer bed is directly proportional to the square root of time and hinge on Fickian type of diffusion [35]. But according to Hixon-Crowell cube root law, the diameter of the particle changes with time which dissolute or erode over time [36]. The values of the release exponent (n), and correlation coefficient (R2) can be calculated from all kinetic models.

In vitro permeation study

Preparation of mucosal tissue

Sheep cheek mucosa was collected from a nearby local slaughter house. A section of mucosal tissue was cut from the sheep cheek mucous membrane and was thoroughly washed with distilled water followed by isotonic phosphate buffer pH 6.8 then soaked in phosphate buffer saline pH 6.8 to remove all the soluble components [32].

Measurement of thickness of sheep cheek mucosal membrane

The sheep cheek mucus membrane was stained with haematoxylin-eosin and thickness measurement was done microscopically. Initially, the paraffin was melted at 65-70 oC to fill into the steel mould. Then at the bottom of the steel mould, the sheep cheek mucosa was placed to cut the section to a thickness of 5-7μm by placing the wax block in the microtome holder after detaching the steel mould by gentle heating followed by cooling. Then the cut section was transferred to a glass slide and stained with haematoxylin-eosin for 30 min followed by washing with isopropyl alcohol and finally with running water. Then air dried stained mucosa was placed under a microscope to measure the average thickness of the sheep cheek mucosa which was found to be 1.4x106 cm.

In vitro permeation determination

In vitro permeation of active constituent of EETP loaded dental gel was determined by Keshary-Chien (KC) diffusion cell containing 25 ml of pH 6.8 phosphate buffer [38, 39] using 2.0 ml of EETP loaded dental gel sheep cheek mucosal membrane. The samples were assayed spectrophotometrically to quantify the amount of flavonoid at 421 nm released through the membrane. The released drug percentage (cumulative) at every interval of time was calculated from the average of three determinations [40, 41] and plotted as cumulative amount drug release (Q) in time (t).

Data analysis of permeation studies of EETP loaded dental gel

The permeation flux at steady state was made out from the linear portion of the curve of the cumulative amount active medicament permeating from unit skin surface area (Q) in time (t) and the curve was extrapolated to abscissa to determine the lag time (Lt). The partition coefficient of flavonoid content in the formulation contained extract of T. purpurea Linn. was calculated as described by following equation [42].

$$\text{Partition\ coefficient} = \frac{Cs - Ceg}{\text{Ceg}}X\frac{1000}{\text{We}}$$

Where, Cs signified as the initial flavonoid concentration in aliquot, Ceg as equilibrium flavonoid concentration and We represented as the mucous membrane weight taken for study.

Reckoning of Permeability coefficient (P) was assessed from derived relation from the first law of diffusion by Fick and expressed by the following equation [43].

$$P = \frac{\text{steady\ state\ flux\ }\left( J \right)\text{Xthickness}\ of\ skin(h)}{\text{concentration}\text{\ of\ drug}\ (C)}$$

Reckoning of Diffusion Coefficient (D) was assessed from derived relation from Fick’s second law using equation [44]:

D = h2/lt

Where, h and Lt signified as the thickness of skin and lag time respectively.

Extrudability study

Extrudability study of the formulated EETP loaded dental gels were performed basing upon the weight required to extrude minimum of 0.5 cm ribbon of gel in 10 sec in the percentage of weight from the lacquered aluminum collapsible tube. More the quantities of the weight of gel extrude, the better will be the extrudability. The extrudability of each formulation was measured in triplicate and average values were calculated from the following formula; [45]:

$$Extrudability = \frac{weight\ applied\ to\ extrude\ gel\ (gm)}{area\ of\ applied\ force\ (\text{cm}^{2})}$$

Syringeability

Syringeability test was tested with the help of no-22 by inserting one gram of formulation in the syringe and it was pressed to check whether it is passing through it or not.

In vitro antimicrobial activity

In vitro antimicrobial study of the EETP loaded dental gel on clinically isolated Porphyromonas gingivalis and comparison of antimicrobial efficacy with marketed 1% metronidazole gel was carried out. Ten percent pathogenic inoculum was transferred to 9 ml of the same medium and incubated for 3 d. All cultures were grown at 37 oC in above mentioned anaerobic condition. Then 1 ml inoculum from that liquid media was added to 8 ml of the same concentration of Brain–Heart infusion broth (Oxoid) in seven separate test tubes for each pathogen and numbered as PGF1X1, PGF1X2, PGF2X1, PGF2X2, PGF3X1, PGF3X2 and PGM as a negative control. The test tube containing 1 ml inoculum in 9 ml medium for each pathogen is called as a positive control and named as PGB. In PGB 1g of marketed metronidazole, gel solution was added and taken as standard. On the other hand 1g of 2.5%w/v solution of formulated EETP loaded dental gels i.e. DF1X1, DF1X2, DF2X1, DF2X2, DF3X1, DF3X2 were added respectively to the test tubes namely PGDF1X1, PGDF1X2, PGDF2X1, PGDF2X2, PGDF3X1, PGDF3X2 and again kept overnight in 37oC in incubator maintaining anaerobic condition in an above-mentioned manner. Then optical density was measured and noted (fig. 10).

Stability study

All the formulated EETP loaded dental gels were tested for stability by storing at 40oC/75% RH for one month and observed for any change in pH, viscosity, swelling index, mucoadhesive strength, spreadability, drug content and drug release after one month. During storage, the proper storage condition was maintained by storing in clean, dry, airtight containers and keeping away from light [46].

RESULTS AND DISCUSSION

Quantification of flavonoid

From scanning graph, the flavonoid absorbance was found to be 0.182 at 421 nm (fig. 1) and a standard curve was shown in fig. 2. The total quantification count was calculated as 51.17 mg QE/gm.

Fig. 1: UV Scanning of flavonoids


Fig. 2: Standard curve of quercetin

Anti-gingival activity study and dose determination of EETP

From the fig. (fig. 3) the presence of black colour colony and haemolysis on sheep blood agar medium confirms the presence of P. gingivalis [47]. From the optical density study, it is found that+ve control (PF1) showed the optical density of 0.416±0.02, standard (PF2) showed OD of 0.095±0.04 and the 0.020 gm/ml i.e. PF5 shows the activity nearer to the standard and OD was found 0.091±0.02. So this concentration (0.020 gm/ml) was optimized to be used as the concentration of extract to be used in all the EETP dental gel formulations (fig. 4.).

Fig. 3: Black colony and haemolysis effect of P. gingivalis

Formulation of various EETP loaded dental gel formulations

Basing on the quantification data, 0.2%w/v ethanol extract of Tephrosia purpurea Linn. was used for the development of dental gel formulations using varying concentrations of carbopol 934P [0.35% (DF1X1) and 0.40% (DF1X2)], HPMC-K4M [3.5% (DF2X1) and 4.0% (DF2X2)] and Chitosan [4.0% (DF3X1) and 4.5% (DF3X2)](table 1).

Characterizations of various EETP loaded dental gel formulations

DF1X1, DF1X2, DF2X1, DF2X2, DF3X1 and DF3X2 all the formulations showed there was no alteration in pH i.e. pH 6.8.

The viscosity of various placebo dental gel formulations is directly proportional to the concentration of polymer but inversely proportional with the concentration of propylene glycol. Gelation temperature decreased with the increasing concentration of polymer but slightly increased with increased propylene glycol concentration.

Fig. 4: Antibacterial activity of different plant extracts on P. gingivalis
Values are mean±SEM of 3 replications, PF1-positive control, PF2-standard control, PF3-0.010 gm/ml of extract, PF4-0.015 gm/ml of extract, PF5-0.020 gm/ml of extract, PF6-0.025 gm/ml of extract, and, PF7-0.030 gm/ml of extract

For DF1X1, DF1X2, DF2X1, DF2X2, DF3X1 and DF3X2 viscosity was found 3718±2.09mpa s, 4242±2.33mpa s, 3641±1.99mpa s, 4024±2.05mpa s, 3425±2.31mpa s, and 3991±2.09mpa s (fig. 5). This may be due to the change of mechanical property of polymer [48].

Table 1: Composition of various optimized EETP loaded dental gel formulations

S. No.Composition (% W/W)DF1X1DF1X2DF2X1DF2X2DF3X1DF3X2
1 Extract 0.2 0.2 0.2 0.2 0.2 0.2
2 Carbopol-934P 0.35 0.40 -- -- -- --
3 HPMC-K4M -- -- 3.5 4.0 -- --
4 Chitosan -- -- -- -- 4.0 4.5
5 Acetic acid -- -- -- -- 4.5 4.5

Fig. 5: Viscosity of various EETP loaded dental gel formulations
Values are mean±SEM of 3 replications, DF1X1 and DF1X2–formulation contain Carbopol-934P 0.35% and 0.40%, DF2X1 and DF2X2-formulation contain HPMC-K4M 3.5% and 4.0%, DF3X1 and DF3X2-formulation contain Chitosan 4.0% and 4.5%

At higher gelling temperature the dental gel formulations may be in sol form within the dental cavity and at lower gelling temperature the dental gel formulations will be in gel form. The dental gel formulations in sol form possess the property of flowing out or wash out easily. So when the dental gel formulations are in sol form the residence time within the dental cavity is less and release will be less, so bioavailability is less as at 37 oC, the behaviour of the formulations changed, depending on the polymer concentration. The mono-molecular micelle forms at a lower concentration of polymer and poly-molecular micelle forms at high polymer concentration and micelle come closer to minimize their interaction with water cause the mechanical and molecular arrangement change [47]. For DF1X1, DF1X2, DF2X1, DF2X2, DF3X1 and DF3X2 gelling temperature was found to be 37.04±0.51oC, 36.55±0.51oC, 37.03±0.34 oC, 36.46±0.76 oC, 37.05±0.42 oC, 36.61±0.61oC (fig. 6).

Swelling index of DF1X2 i.e. 45.06±0.41% was found more than the Swelling index of DF1X1 i.e. 42.42±0.65%. Swelling index of DF2X2 i.e. 41.58±0.58% was found more than swelling index of DF2X1 i.e. 40.65±0.14%. Swelling index of D3X2 i.e. 43.32±0.54% was found more than swelling index of DF3X1 i.e. 39.86±0.54 % (fig. 7).

Fig. 6: Gelation temperature of various EETP loaded dental gel formulations
Values are mean±SEM of 3 replications, DF1X1 and DF1X2–formulation contain Carbopol-934P 0.35% and 0.40%, DF2X1 and DF2X2-formulation contain HPMC-K4M 3.5% and 4.0%, DF3X1 and DF3X2-formulation contain Chitosan 4.0% and 4.5%

Spreadability of DF1X2 i.e. 25.21±0.58 gm. cm/sec, was found more than the spreadability of DF1X1 i.e. 23.92±0.18 gm. cm/sec. spreadability of DF2X2i.e. 26.12±0.67 gm. cm/sec was found more than spreadability of DF2X1i.e. 24.47±0.95 gm. cm/sec. Spreadability of DF3X2 i.e. 24.85±0.35 gm. cm/sec, was found more than spreadability of F3X1i.e. 24.05±0.92 gm. cm/sec. (fig. 8).

Fig. 7: Swelling index of various EETP loaded dental gel formulations
Values are mean±SEM of 3 replications, DF1X1 and DF1X2–formulation contain Carbopol-934P 0.35% and 0.40%, DF2X1 and DF2X2-formulation contain HPMC-K4M 3.5% and 4.0%, DF3X1 and DF3X2-formulation contain Chitosan 4.0% and 4.5%

The mucoadhesive strength of DF1X2 i.e. 28±0.32 gm was found more than mucoadhesive strength of DF1X1i.e. 25±0.21 gm. Mucoadhesive strength of DF2X2 i.e. 29±0.43 gm was found more than that of DF2X1 i.e. 26±0.33 gm. Mucoadhesive strength of DF3X2 i.e. 24±0.21 gm was found more than F3X1 i.e. 21±0.24 gm (fig. 9). This can lead to a strong adhesion which may lead to the prolong drug release as more the mucoadhesive strength less would be the washout effect of the formulation, hence of active pharmaceutical ingredient.

Fig. 8: Spreadability of various EETP loaded dental gel formulations
Values are mean±SEM of 3 replications, DF1X1 and DF1X2–formulation contain Carbopol-934P 0.35% and 0.40%, DF2X1 and DF2X2-formulation contain HPMC-K4M 3.5% and 4.0%, DF3X1 and DF3X2-formulation contain Chitosan 4.0% and 4.5%

Fig. 9: Mucoadhesive strength of various EETP loaded dental gel formulations
Values are mean±SEM of 3 replications, DF1X1 andDF1X2–formulation contain Carbopol-934P0.35% and 0.40%, DF2X1 andDF2X2-formulation containHPMC-K4M 3.5% and 4.0%, DF3X1 and DF3X2-formulation contain Chitosan4.0% and 4.5%

In vitro release study of various EETP loaded dental gel formulations

The cumulative percentage drug release in the formulation DF1X1 andDF1X2 was found to be 94.043±0.379 and, 94.514±0.211 respectively after 8 hr. The drug release from gel has sustained the drug more than 7 hr. Formulation DF1X1 liberated 63.559±0.369% of its drug content within 2.0 hr. formulation DF1X2 liberated 63.090±0.131of its drug content in 2.0 hr. [fig. 10 (a)]

The cumulative percentage drug release in the formulation DF2X1and DF2X2 was found to be 97.276±0.326 and, 97.435±0.078 respectively after 8hr. The drug release from gel has sustained the drug more than 7 hr. Formulation DF2X1 liberated 65.886±0.662% of its drug content within 2.00 hr; formulation DF2X2 liberated 62.595±0.317of its drug content in 2.0hr. [fig. 10 (a)]

The cumulative percentage drug release in the formulation DF3X1andDF3X2 was found to be 93.697±0.090 and 93.719±0.130 respectively after 8hr. The drug release from gel was sustained the drug more than 7hr. Formulation DF3X1 liberated 63.051±0.111% of its drug content within 2.0 hr; formulation DF3X2 liberated 63.666±0.185% of its drug content within 2.0hr. [fig. 10 (a)]

From in vitro drug release study it was obtained that the drug release of DF1X1,DF1X2, DF2X1, DF2X2, DF3X1andDF3X2 dental gel were found to be the best which can be explained by First order release kinetic as the plot produced the highest linearity (R2= 0.9811,0.9832, 0.9762, 0.9749, 0.9749 and 0.9784 respectively) [Table 2, fig. 10 (b)].

Fig. 10 (a): Zero order plot of various EETP loaded dental gel formulations, (b): First order plot of various EETP loaded dental gel formulations
Values are mean±SEM of 3 replications, DF1X1 and DF1X2–formulation contain Carbopol-934P 0.35% and 0.40%, DF2X1 and DF2X2-formulation contain HPMC-K4M 3.5% and 4.0%, DF3X1 and DF3X2-formulation contain Chitosan 4.0% and 4.5%

The result evidenced that the rate of drug release was concentration dependent. However, the drug release kinetic was found well matched with Higuchi Model release kinetic from the curve; indicating the release of the drug increased with the square root of time [35,36]. The log cumulative percentage drug released in log time plotted for Korsmeyer-Peppas equation also demonstrate a good linearity with anomalous kinetic (R2= 0.9436, 0.9490, 0.8348, 0.8343, 0.8280 and 0.8252 respectively). The ‘n’ value was found greater than 0.5 confirmed the release of flavonoid from all EETP loaded dental gels followed non-Fickian Diffusion Kinetic [table 2, fig. 10 (e)]. Hence, the process of drug release includes two steps, first, step supposed to be the swelling of gel and second step may include the overall diffusion of the active component [48, 49]. However, the data obtained from drug diffusion study confirmed that DF2X1 and DF2X2 dental gel formulations are the best amongst the rest.

Table 2: Drug release kinetics of various EETP loaded dental gel formulations

FormulationKinetic model
Zero order
R2
DF1X1 0.7593
DF1X2 0.7612
DF2X1 0.7712
DF2X2 0.7886
DF3X1 0.7886
DF3X2 0.7511

Values are mean±SD of 3 replications


Fig. 10 (c): Higuchi plot of various EETP loaded dental gel formulations, (d): Hixson-Crowell cube root plot of various EETP loaded dental gel formulations
Values are mean±SEM of 3 replications, DF1X1 and DF1X2–formulation contain Carbopol-934P 0.35% and 0.40%, DF2X1 and DF2X2-formulation contain HPMC-K4M 3.5% and 4.0%, DF3X1 and DF3X2-formulation contain Chitosan 4.0% and 4.5%

Fig. 10 (e): Korsmeyer-Peppas plot of EETP loaded dental gel formulations, (f): Cumulative amount drug release in unit surface area of EETP loaded dental gel formulations
Values are mean±SEM of 3 replications, DF1X1 and DF1X2–formulation contain Carbopol-934P 0.35% and 0.40%, DF2X1 and DF2X2-formulation contain HPMC-K4M 3.5% and 4.0%, DF3X1 and DF3X2-formulation contain Chitosan s 4.0% and 4.5%

In vitro permeation study of various EETP loaded dental gel formulations

However, from the permeation study, it was seen that a descending order of cumulative amount permeation in the unit surface area for all the dental gel formulations was found. (DF2X2, DF2X1, DF3X2, DF3X1, DF1X2, DF1X1) [Table 3, fig. 10(f)]. Permeation flux is also high for DF2X2 and DF2X1 i.e. 0.1426±0.0031µg. cm-2. min-1 and 0.1426±0.0028µg. cm-2. min-1 and lowest flux was found in DF1X1 dental gel (0.1263± 0.0034µg. cm-2. min-1).

Extrudability study of various EETP loaded dental gel formulations

Extrudability of all the EETP loaded dental gels decreased. However, the rate of decrease in extrudability is inversely proportional to the concentration of propylene glycol. Moreover, the increase in the concentration of propylene glycol increases the force required for extruding the dental gel from the collapsible tubes. Hence, this was confirmed that all the dental gel formulations extrude satisfactorily [table 4].

Table 3: In vitro permeation parameters of various EETP loaded dental gel formulations

S. No.FormulationPermeation Flux(J) (µg. cm-2. min-1)Lag time (tL MIN)
1 DF1X1 0.1263±0.0034 15±0.5
2 DF1X2 0.1309±0.0037 15±0.5
3 DF2X1 0.1426±0.0028 15±0.5
4 DF2X2 0.1426±0.0031 15±0.5
5 DF3X1 0.1357±0.0027 15±0.5
6 DF3X2 0.1372±0.0033 15±0.5

Values are mean±SD of 3 replications.

Table 4: Extrudability of various EETP loaded dental gel formulations

S. No.FormulationExtrudability(gm/cm2)
1 DF1X1 16.05±0.032
2 DF1X2 15.65±0.030
3 DF2X1 16.67±0.010
4 DF2X2 14.12±0.010
5 DF3X1 15.85±0.050
6 DF3X2 14.67±0.020

Values are mean±SD of 3 replications

Syringeability study of various EETP loaded dental gel formulations

All the formulation easily and uniformly passed through needle no-22.

In vitro antimicrobial activity of various EETP loaded dental gel formulations

The antimicrobial study of all the EETP loaded dental gels resulted in high level of antimicrobial property than that of the standard marketed metronidazole (1% w/v) gel. DF2X2 was found to have the highest antimicrobial property [fig. 11].

Stability study

All the EETP loaded dental gel formulations were subjected for stability study for one month. After one month, all the evaluation parameters of dental gel formulation was found same or no change (fig. 5-9) as compared with that of the previous data taken at the time of preparation of the formulation thereby confirming a stable product after one month.

The in vitro drug release study data after one month is given in fig. (fig. 12).

Fig. 11: Optical density of different formulations on P. gingivalis culture
Values are mean±SD of 3 replications, PGB-positive control, PGM-negative control, PGF1X1-DF1X1 solution, PGF1X2-DF1X2 solution, PGF2X1-DF2X1 solution, PGF2X2-DF2X2 solution, PGF3X1-DF3X1 solution, PGF3X2-DF3X2 solution

Fig. 12: In vitro release study to determine Stability study of various EETP loaded dental gel formulations
Values are mean±SEM of 3 replications, DF1X1 and DF1X2–formulation contain Carbopol-934P 0.35% and 0.40%, DF2X1 and DF2X2-formulation contain HPMC-K4M 3.5% and 4.0%, DF3X1 and DF3X2-formulation contain chitosan 4.0% and 4.5%

CONCLUSION

Among all the dental gel formulations containing ethanol extract of Tephrosia purpurea Linn., DF2X2 (HPMC-K4M-3.5%w/w, PG-20%w/w, NaOH-1.0%w/w, glycerol-1.0%w/w) showed the highest drug release i.e.97.435±0.078% in 8hr. resulting in first order release kinetics (R2-0.9749).

All the EETP loaded dental gels were found to follow non-Fickian drug diffusion model which was further confirmed by permeation study; the maximum drug permeation was found to be 91.220±0.065% in 8hr.

The antimicrobial study revealed that DF2X2 was having the highest antimicrobial activity as compared to the marketed formulation. The extrudability and syringe ability data showed the satisfactory result for DF2X2.

The stability study after one month showed a little or no change of characters as that of the initial study, hence, stable. However, the reported data reveals the absence of detailed structured study of EETP contained in dental gel development in literature, and this assay seemed EETP as a component and safe dental gel that would be more acceptable and beneficial to assess its usefulness accurately.

ACKNOWLEDGMENT

The authors are thankful to Siksha ‘O’ Anusandhan University, Bhubaneswar, Odisha, India for constant support, encouragement for our research work and also to the Institute of Dental Sciences, Siksha ‘O’ Anusandhan University, Bhubaneswar for allowing us to perform a part of research work in their laboratory.

CONFLICT OF INTERESTS

Declared none

REFERENCES

  1. Listgarten MA. Pathogenesis of periodontitis. J Clin Periodontol 1986;13:418-30.
  2. Cimasoni G. Crevicular fluid updated. Monogr Oral Sci1983;12:61-152.
  3. Greenstein G. The role of bleeding upon probing in the diagnosis of dental disease. A literature review. J Periodontol 1984;55:684-8.
  4. Löe H, Theilade E, Jensen SB. Experimental gingivitis in man. J Periodontol 1965;36:177-87.
  5. Theilade E, Wright WH, Jensen SB, Löe H. Experimental gingivitis in man. II. A longitudinal clinical and bacteriological investigation. J Dent Res 1966;1:1-13.
  6. Page RC. Gingivitis. J Clin Periodontol 1986;13:345-59.
  7. Despande SS, Shah GB, Parmar NS. Antiulcer activity of Tephrosiapurpurea in rats. Indian J Pharmacol 2003;35:168-72.
  8. Kirtikar KR, Basu BD. Glossary of Indian Medicinal Plants; 1956. p. 723.
  9. Gokhale AB, Dikshit VJ, Damre AS, Kulkarni KR, Saraf MN. Influence of ethanol extract of Tephrosia purpurea Linn. on mast cells and erythrocytes membrane integrity. Indian J Exp Biol 2000;38:837-40.
  10. Honrao MS, Pabari R. Gels. Indian Pharm 2004;16-21.
  11. Nishihata T, Kamada A, Sakai K, Takahashi K, Matsumoto K, Shinozaki K, et al. Percutaneous absorption of diclofenac in rats and humans: aqueous gel formulation. Int J Pharm 1988;46:1-10.
  12. Temu MJ, Damian F, Kinget R, Mooter GVD. Intra-vaginal gels as drug delivery systems. J Women Health 2004;13:834-44.
  13. Allen MG, Prausnitz MR, McAllister DV, Cross FPM. Microneedle devices and methods of manufacture and use thereof. US Pat; 2002;6:856.
  14. Kostarelos K. Rational design and engineering of delivery systems for therapeutics: biomedical exercises in colloid and surface science. Adv Colloid Interface Sci 2003;106:147-68.
  15. The Ayurvedic Pharmacopoeia of India. 1st ed. The government of India, Delhi; 1990. p. 233-40.
  16. Patel A, Patel A, Patel A, Patel NM. Estimation of flavonoid, polyphenolic content and in vitro antioxidant capacity of leaves of Tephrosia purpurea Linn. (Leguminosae). Int J Pharm Sci Res 2010;1:66-77.
  17. Jenkinson and HF, Lamont RJ. Oral microbial communities in sickness and in health. Trends Microbiol 2005;13:589–95.
  18. Jia H, Hiroshi M, Cecilia A, Fan Y, Andrew W, Yeudall, et al. Intercellular spreading of Porphyromonas gingivalis infection in primary gingival epithelial cells. Infect Immun 2006;74:4214-23.
  19. Nglan L, Daniel G, Morris G, Denis M. Effects of hydrogen peroxide on growth and selected properties of Porphyromonas gingivalis. FEMS Microbiol Lett 1999;174:347-53.
  20. Schmolka IR. Preparation and properties of Pluronic PF‐127 gels for the treatment of burns. J Biomed Mater Res 1972;6:571‐82.
  21. Chaudhari P, Ajab A, Malpure P, Kolsure P, Sanap D. Development and invitro evaluation of thermoreversible nasal gel formulations of Rizatriptan benzoate. Indian J Pharma Edu Res 2009;43:55‐62.
  22. Saleem MT, Sanaullah, Faizan S. Formulation and evaluation of gatifloxacin topical gels. Indian Pharm 2006;7:88-92.
  23. Gupta GD, Gaud RS. The release rate of Nimesulide from different gallants. Indian J Pharm Sci 1999;61:227‐30.
  24. Rasool BKA, Abu-Gharbieh EF, Awni RA, Rasool AAA. In vitro release study of nystatin from chitosan buccal gel. Jordan J Pharm Sci 2010;3:44-55.
  25. Gupta GD, Gaud RS. Release rate of Tenoxicam from acrypol gels. Indian Pharm 2005;30:69-76.
  26. Mundada AS, Shrikhande BK. Controlled release gel of ciprofloxacin HCl for ophthalmic administration. Indian Drugs 2006;43:9-12.
  27. Pisal SS, Paradkar AR, Mahadik KR, Kadam SS. Pluronic gels for nasal delivery of vitamin B12. Part I: Preformulation study. Int J Pharm 2004;270:37-45.
  28. Tho I, Kjoniksen AL, Nystrom B, Roots J. Characterization of association and gelation of pectin in methanol-water mixtures. Biomacromolecule 2003;4:1623-9.
  29. Sanjay J, Padsalg A, Patel K, Mokale V. Formulation, development and evaluation of fluconazole gel in various polymer bases. Asian J Pharm 2007;1:63–8.
  30. Choi HG, Oh YK, Kim CK. In-situ gelling and mucoadhesive liquid suppository containing acetaminophen: enhanced bioavailability. Int J Pharm 1988;165:23–32.
  31. Yong CS, Choi JS, Quan QZ, Rhee JD, Kim CK, Lim SJ. Effect of sodium chloride on the gelation temperature, gel strength and bioadhesive force of poloxomer gels containing diclofenac sodium. Int J Pharm 2001;226:195–205.
  32. Raykar PV, Fung MC, Anderson BD. The role of protein and lipid domains in the uptake of solutes by human stratum corneum. Pharm Res 1998;5:140-9.
  33. Hadjiioannou TP, Christian GD, Koupparis MA. Quantitative Calculations in Pharmaceutical Practice and Research. New York, NY: VCH Publishers Inc; 1993. p. 345-8.
  34. Bourne DW. Pharmacokinetics. In: Banker GS, Rhodes CT. eds. Modern Pharmaceutics. 4th ed. Marcel Dekker, New York, NY; 2002;4:67-92.
  35. Higuchi T. Mechanism of sustained action medication. Theoretical analysis of the rate of release of solid drugs dispersed in solid matrices. J Pharm Sci 1963;52:1145-9.
  36. Hixson AW, Crowell JH. Dependence of reaction velocity upon surface and agitation: I-theoretical consideration. Ind Eng Chem 1931;23:923-31.
  37. Korsmeyer RW, Gurny R, Doelker E, BrP, Peppas NA. Mechanisms of solute release from porous hydrophilic polymers. Int J Pharm 1983;15:25-35.
  38. Peppas NA. Analysis of fickian and non-fickian drug release from polymers. Pharm Acta Helv 1985;60:110-1.
  39. Rajni V, Verma PRP. Diffusion study of ibuprofen from ointment base. Indian J Parm Sci 1995;57:1-6.
  40. Mohammed A, Yasmin S, Asgar A. Matrix-type transdermal drug delivery systems of metoprolol tartrate. Acta Pharm 2003;53:119-25.
  41. Ezzedeen FW, Shihab FA, Husain EJ. Percutanous diffusion of cefalexin, sulfamethoxazole and diphenhydramine from ointments. Pharmazie 1990;45:512–4.
  42. Saket MM, James KC, Kellaway IW. Partitioning of some 21 alkyl esters of hydrocortisone and cortisone. Int J Pharm 1984;21:155–66.
  43. Aslani P, Kennedy RA. Studies on diffusion in alginate gels I. Effect of cross-linking with calcium or zinc ion diffusion of acetaminophen. J Controlled Release 1996;42:75-82.
  44. Pefile SC, Smith EW, Albrecht CF, Kruger PB. The release of rooperol tetra-acetate from topical bases: in vitro studies using silicon membrane. J Controlled Release 1998; 161:237-43.
  45. Rathore RPS, Nema RK. Formulation and evaluation of topical gels of Ketoprofen. Asian J Pharm Clin Res 2008;1:12–6.
  46. Keny RV, Lourenco F. Formulation, and evaluation of thermo-reversible in situ gelling and mucoadhesive Diltiazem hydrochloride liquid suppository. Int J Pharm Biol Sci 2010;1:1-17.
  47. Nakeynma K. Porphyromonou sgingivalis and related bacteria: from colonial pigmentation to the type IX secretion system and gligingmotility. J Periodontal Res 2005;50:1-8.
  48. Korsmeyer RW, Von Meerwall E, Peppas NA. Solute and penetrant diffusion in swellable polymers. II. Verification of theoretical models. J Polym Sci Part B: Polym Phys 1986; 24:409-34.
  49. Kumar P, Mittal KL. Handbook of Microemulsion Science and Technology, Marcel Dekker, New York; 1999. p. 139-85.

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