|Year : 2015 | Volume
| Issue : 3 | Page : 331-336
Formulation and evaluation of antimicrobial activity of Morus alba sol-gel against periodontal pathogens
Shilpa Gunjal1, Anil V Ankola1, Udaykumar Bolmal2, Kirankumar Hullatti3
1 Department of Public Health Dentistry, KLE VK Institute of Dental Sciences, KLE University, Belgaum, Karnataka, India
2 Department of Pharmaceutics, KLE College of Pharmacy, KLE University, Belgaum, Karnataka, India
3 Department of Pharmacognosy, KLE College of Pharmacy, KLE University, Belgaum, Karnataka, India
|Date of Web Publication||14-Sep-2015|
Department of Public Health Dentistry, KLE VK Institute of Dental Sciences, KLE University, Belgaum - 590 010, Karnataka
Source of Support: None, Conflict of Interest: None
Background: Periodontitis has a multifactorial etiology, with primary etiologic agents being pathogenic bacteria that reside in the subgingival area. Recent advances in the field of alternative medicine introduced various herbal products for the treatment of periodontitis. Aim: To assess and compare the antimicrobial activity of Morus alba sol-gel with chlorhexidine sol-gel against ATCC standard strains of Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, and Tannerella forsythia. Materials and Methods: Crude extract of Morus alba leaves was prepared by Soxhlet method by using ethanol as a solvent. Phytochemical screening of the crude extract of M. alba was performed to check the various chemical constituents. M. alba sol-gel and chlorhexidine sol-gel were formulated using Pluronic f127 and Pluronic f108 and compared for their antimicrobial activity. The minimum inhibitory concentration of both the gels was performed using agar well diffusion technique. Results: The minimum inhibitory concentration of M. alba sol-gel and chlorhexidine sol-gel against A. actinomycetemcomitans is 19 and 17 mm, T. forsythia is 12 and 21 mm, and P. gingivalis is 16 and 18 mm, respectively. Conclusion: Both M. alba and chlorhexidine sol-gel exhibited potent antimicrobial activity against periodontal pathogens.
Keywords: Antibacterial activity, Morus alba, periodontal microorganisms, thermoreversible sol-gel
|How to cite this article:|
Gunjal S, Ankola AV, Bolmal U, Hullatti K. Formulation and evaluation of antimicrobial activity of Morus alba sol-gel against periodontal pathogens. J Indian Assoc Public Health Dent 2015;13:331-6
|How to cite this URL:|
Gunjal S, Ankola AV, Bolmal U, Hullatti K. Formulation and evaluation of antimicrobial activity of Morus alba sol-gel against periodontal pathogens. J Indian Assoc Public Health Dent [serial online] 2015 [cited 2020 Jan 18];13:331-6. Available from: http://www.jiaphd.org/text.asp?2015/13/3/331/165299
| Introduction|| |
Chronic periodontitis is an infectious disease resulting in inflammation within the supporting tissues of the teeth, progressive attachment loss, and bone loss and is characterized by periodontal pocket formation and/or recession of the gingiva.  Periodontitis has a multifactorial etiology, with the primary etiologic agents being pathogenic bacteria residing in the subgingival area. More than 700 different bacterial species colonize the oral cavity, but only a few of these are thought to be potential periodontal pathogens.  Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, and Tannerella forsythia are known as the main pathogens of periodontal disease, and treatment of periodontal disease is associated with success in removing and reducing these microorganisms. ,, Treatment of periodontal pocket is based on mechanical debridement of the tooth surface followed by proper maintenance of oral hygiene. Limitations of mechanical debridement are inability to access the periodontal pockets which leads to early microbial re-colonization and recurrence of the periodontal pocket. , Thus, local drug delivery systems (gels, microchip, fiber, etc.,) emerged into the market.
Local delivery devices are system designed to deliver agents into the periodontal pocket to retain therapeutic levels for a prolonged period of time. One such local drug delivery system which has controlled release property, less time consumption, and noninvasive is a gel. Gels are formulated using poloxamers (polyoxyethylene and polyoxypropylene units). These high-molecular-weight poloxamers have the ability to form a thermoreversible gel.  The advantage of thermoreversible sol-gel/hydrogel system is that they are shear thinning systems which show the temperature-dependent gelation. The addition of poloxamers results in the formation of gel system. Poloxamers exhibit temperature-dependent sol to gel phase transition. The aqueous solutions of these polymers are liquid at room temperature but gel at body temperature. Once injected, it is retained in the periodontal pocket for a long time, thus reducing the frequency of administration. 
For centuries, plants have been used throughout the world as drugs and remedies for various diseases, including infectious diseases. , These drugs serve as prototypes to develop the more effective and less toxic medicines. , According to WHO, medicinal plants would be the best source for obtaining a large variety of drugs. , Many plants have been used as remedies for diseases and offer biologically active compounds that possess antimicrobial properties. ,
Of the many herbal derivatives, Morus alba is one such plant which has garnered great attention because of its antioxidative, antidiabetic, antibacterial, antiviral, and anti-inflammatory properties. ,, M. alba also known as white mulberry is cultivated throughout the world, wherever silkworms are raised. The leaves of the white mulberry are the main food source for the silkworms. It is a popular medicinal plant that belongs to family Moraceae, and has long been used commonly in Ayurvedic and many of traditional systems of medicine. To the best of our knowledge, M. alba has been investigated for its use in dentistry only against pathogens causing dental caries. Hence, through this in vitro study, an attempt is made to explore the use of crude extract of M. alba in a sol-gel formulation against periodontal pathogens. Thus, the aim of the present study is to formulate and compare the thermosensitive sol-gel of M. alba with chlorhexidine against the periodontal microorganisms.
| Materials and methods|| |
Prior to the start of the study, ethical approval was obtained from recognized KLE University (Ref. no. KLEU/Ethic/14-15/D-79). This study is a part of the ongoing clinical trial.
The chemicals used in the gel preparation are Pluronic f127, Pluronic f108, chlorhexidine gluconate solution, and methyl paraben. The entire chemicals were of IP grade.
Plant collection and crude extraction
Leaves of M. alba were collected from Belgaum, Karnataka, India. The plant was authenticated from the taxonomist, Regional Medical Research Centre, ICMR (Voucher specimen is RMRC-988). The leaves were washed thoroughly in water and shade-dried. The dried leaves were coarsely ground. The coarse powder was extracted with solvent 99.9% of ethanol at room temperature using Soxhlet method of extraction.  The extract was stored at 4°C until further use.
Freshly prepared crude extract of M. alba leaves using ethanol as a solvent was qualitatively tested for the presence of chemical constituents. Phytochemical screening of the crude extract was performed using the following chemicals and reagents.
A fraction of the extract was treated with 3-5 drops of Wagner's reagent (1.27 g of iodine and 2 g of potassium iodide in 100 ml of water) and observed for the formation of reddish-brown precipitate (or coloration).
Ferric chloride test
A fraction of the extract was treated with aqueous 5% ferric chloride and observed for the formation of deep blue or black color.
2 ml of the extract was treated with 10% alcoholic ferric chloride solution and observed for the formation of blue or greenish color solution.
Alkaline reagent test
To the test solution (0.5-1 ml), few drops of sodium hydroxide solution (10%) were added. Formation of an intense yellow color, which turns colorless on the addition of few drops of dilute sulfuric acid, indicates the presence of flavonoids.
Proteins and amino acids
About 0.5-1 ml of the sample was taken in a test tube and it was boiled with 0.2% solution of ninhydrin. Appearance of violet color confirms the presence of proteins in the sample.
Sterols and triterpenoids
About 5 ml of test solution was boiled with few drops of acetic anhydride, cooled, and then concentrated sulfuric acid was added along the sides of the test tube. Appearance of red color in the lower layer indicates the presence of sterols, whereas yellow color in the lower layer indicates the presence of triterpenoids.
Few drops of Molisch's reagent were added to 2 ml of the various extracts. This was followed by a slow addition of 0.2 ml of concentrated sulfuric acid along the sides of the test tube. The mixture was then allowed to stand for 2-3 min. Appearance of a red or a dull violet color at the interphase of the two layers indicated a positive test.
1 g of the extract was dissolved in 10 ml of distilled water in a test tube and vigorously shaken for 1-2 min. Formation of honeycomb froth 1cm in height and lasting for a minimum of 30 minutes indicates the presence of saponins.
5 ml of each extract was treated with 2 ml of glacial acetic acid in a test tube and a drop of ferric chloride solution and 1 ml of concentrated sulfuric acid were added along the sides of the test tube. A brown ring at the interface indicated the presence of deoxysugar characteristic of cardiac glycosides. A violet ring may appear below the ring, while in the acetic acid layer, a greenish ring may form.
Fixed oil and fats
Filter paper test
A small quantity of extract was pressed between the filter paper. Appearance of oil stain on the paper indicated the presence of fixed oils.
The chemical constituents were identified by the characteristic color changes and precipitation reactions using standard protocol. ,
The standard strains of A. actinomycetemcomitans
(ATCC 29523), P. gingivalis (ATCC 33277), and T. forsythia (ATCC 43037) were procured from Promochem, Bengaluru.
The chemicals used for gel preparation were of IP grade. Both the gels were prepared using the same chemicals except for the active ingredients. Initially, 18 g of Pluronic F 127 (18%, w/v) was slowly dissolved in 100 ml of distilled water (maintaining ice bath at 4°C) with continuous stirring using a magnetic stirrer. It is followed by the addition of 6 g of Pluronic F 108 (6%, w/v) to the above solution and stirred at a low speed (300-400 rpm) in order to avoid frothing. The above solution was stored in a container, sealed with aluminum foil, and refrigerated overnight at 4°C until clear solution was obtained. The above steps are common for both the gels. For M. alba gel, 16 g of M. alba extract (16%, w/v), and for chlorhexidine gel 1 ml of chlorhexidine gluconate (1%, w/v) were added slowly to the above solutions with continuous stirring using a magnetic stirrer followed by the addition of a preservative, 100 mg of (0.1% w/v) of methyl paraben. The final volume of the gel was 100 ml. The chlorhexidine and M. alba sol-gel were stored in a refrigerator at 4°C until further investigations are carried out. ,
The antimicrobial assay was determined by the agar well diffusion method. Antibacterial activity of the gels was determined on brucella agar medium. In a conical flask, 4.3 g of brucella agar base was suspended in 100 ml of distilled water. The conical flask was heated to boil to dissolve the medium completely. Later it was sterilized by autoclaving at 15 lbs pressure (121°C) for 15 min. It was cooled to 50°C (for T. forsythia, 100 μl of 0.001% of n-acetyl muramic acid was added) and then aseptically added 5% (v/v) sterile defibrinated sheep blood. It was mixed well before pouring into sterile Petri plates. The medium was poured into sterile disposable Petri plates. After the medium was solidified, plates were kept in a refrigerator until further use. The Petri plates were removed from the refrigerator and dried in an incubator at 37°C. A. actinomycetemcomitans, P. gingivalis, and T. forsythia were grown in thioglycollate broth mixed with horse serum, incubated for 48 h under anaerobic conditions. With the help of a micropipette, 50 μl of bacteria as per 0.5 McFarland standards were taken, spread on the agar plate, and lawn cultured with the help of a sterile swab. Two wells were made in the nutrient agar plate using a sterile cork borer measuring 4 mm depth and 6 mm diameter. In one well, 70 μl of chlorhexidine gel and in another well 70 μl of M. alba gel were filled with the help of micropipette. The plates were then incubated for 48 h in an anaerobic jar within 20-30 min of agar well diffusion. After incubation, the plates were observed for the zone of inhibition around the well and the diameters of the inhibition zones were measured in mm using a Hi antibiotic zone scale.  The assay was carried out in triplicates and the result thus obtained is taken as the mean of the three readings.
| Results|| |
[Table 1] shows the qualitative phytochemical investigation of M. alba that showed the presence of alkaloids, carbohydrates, glycosides, saponins, sterols, triterpenes, fats and oils, phenols, tannins, flavonoids, proteins, and amino acids in ethanolic extract.
|Table 1: Phytochemical analysis of crude extract of M. alba using ethanol solvent |
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[Table 2] shows the antimicrobial activity of sol-gels determined by agar well diffusion method. The sol-gel prepared by using M. alba and chlorhexidine exhibited potent antimicrobial activity toward all the periodontal pathogens. M. alba gel showed wider zone of inhibition (19 mm) against A. actinomycetemcomitans when compared to chlorhexidine gel (17 mm). Whereas chlorhexidine gel had a wider zone of inhibition (21 mm) compared to M. alba gel (12 mm) against T. forsythia.
|Table 2: Antimicrobial activity of M. alba and chlorhexidine gel against periodontal microorganisms (zone of inhibition) by Agar well diffusion method* |
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| Discussion|| |
In the present in vitro study, thermosensitive/thermoreversible gels were formulated and antimicrobial activities of gels have been assessed for periodontal pathogens using agar well diffusion method.
Gels are the polymeric network which increases the contact time of the drug at the administration sites, thereby increasing the absorption of the drug due to its mucoadhesive property. However, due to its semisolid nature, there is difficulty in application. To ease the application procedure, the sol-gel has been formulated in such a way that it is liquid during application, but turns out into a gel when it comes into contact with the oral mucosa.  The ideal gel formulation for the treatment of periodontitis should exhibit a high value of mucoadhesion and retention within the pocket for the desired period of time, show controlled release of the drug, and be easily delivered into the periodontal pocket preferably using a syringe. 
In the present study, the antibacterial activity of M. alba gel was found to be 19 mm, 16 mm, and 12 mm against standard strains of A. actinomycetemcomitans, P. gingivalis, and T. forsythia, respectively. Chlorhexidine gel was used as a positive control which is accepted as gold standard in dentistry. It has a broad spectrum of antimicrobial activity against a wide variety of bacteria (both Gram-positive and Gram-negative) and fungi. Its mechanism of action being decreasing the pellicle formation, alteration of bacterial adhesion to the tooth surface, and alteration of bacterial cell wall, ultimately leading to cell death.  The most widely used concentration of chlorhexidine gel formulations available in the market is 1%. Hence, in the present study, the same concentration was used in the formulation of the gel.
Antimicrobial activity revealed that a zone of inhibition of both the gels against A. actinomycetemcomitans and P. gingivalis was almost similar. Whereas the diameter of the inhibition zone was greater for chlorhexidine gel than M. alba gel against T. forsythia. Even though the M. alba gel demonstrates less antimicrobial activity against T. forsythia compared to chlorhexidine gel, it has an added advantage over adverse effects. 
Antimicrobial activity of mucoadhesive gel prepared from the extract of Quercus brantii and Coriandrum sativum showed 17.23 ± 0.2 mm zone of inhibition against P. gingivalis, while M. alba sol-gel showed 16 mm.  Literature reveals few studies of in vitro antibacterial activity of gel, as most of the studies of the gels have been in vivo. The antimicrobial activity of M. alba sol-gel could not be compared with other sol-gel as most of the gels have not been assessed for their antimicrobial activity against periodontal pathogens.
In the present study, leaves of the M. alba plant have been assessed for its antibacterial activity and thus found to be effective against periodontal microorganisms. In another study, root bark of M. alba was found to possess antibacterial activity against periodontal pathogens like Actinobacillus actinomycetemcomitans and P. gingivalis with minimum inhibitory concentration of 1000 μg/ml and 8 μg/ml, respectively.  Kuwanon C, Mulberrofuran G, and Albanol B present in the M. alba leaves showed strong antibacterial activity with minimum inhibitory concentrations ranging from 5 to 30 mg/ml.  Therefore, all the parts of the M. alba plant were found to possess antibacterial activity.
Phytochemical analysis of M. alba extract revealed the presence of all major bioactive compounds like tannins, flavonoids, saponins, and alkaloids. These bioactive compounds are known to act by a different mechanism and exert antimicrobial action. Tannins, belonging to polyphenol family, have been reported to possess the antibacterial activity against periodontal pathogens.  Tannins precipitate microbial proteins and prevent the development of microorganisms.  Polyphenols increase the antioxidant ability of oral fluids and prevent periodontal disease.  Therefore, tannins present in M. alba extract might be responsible for exerting antimicrobial activity against periodontal pathogens. This promising gel might lead to a new avenue in the prevention and treatment of periodontitis.
Although the antibacterial activity of sol-gel was assessed against periodontal pathogens, the concentration of each phytoconstituent and the chemical constituent that might be responsible for the activity was not determined. Further, in vivo studies should be conducted to know the effectiveness of M. alba sol-gel.
| Conclusion|| |
The present study throws light on the formulation of thermoreversible gel using M. alba and assessing its antimicrobial activity against the most common periodontal pathogens A. actinomycetemcomitans, P. gingivalis, and T. forsythia. The study also concludes that M. alba leaves contain pharmaceutically important phytochemicals like tannins, flavonoids, triterpenes, alkaloids, saponins, phytosterols, carbohydrates, fats, and oils.
We would like to thank Venus Ethoxyethers Pvt Ltd., Biocholim Ind. Estate, Goa, for providing Pluronic 127 and 108, Unilab Chemicals and Pharmaceuticals Pvt Ltd., Mumbai, for providing chlorhexidine gluconate solution, and Gujarat Organics Ltd., for providing Methyl paraben as gift samples.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Table 1], [Table 2]