A poly-herbal formulation from traditionally used medicinal plants as a remedy for oral hygiene

Microorganisms play an important role in the development of periodontal diseases in human that lead to serious complications. Since ages, medicinal plants, singly or in combinations are used for the treatment of various dental disorders. However, their scientific validation and characterization is scarce. Therefore, the goal of this study was to develop knowledge based poly-herbal formulation as a remedy for oral hygiene. After validations of antimicrobial potential, extracts of 11 plants were combined in different ratios, and finally one poly-herbal formulation was selected for further characterization. Antimicrobial activity was determined through disc and well diffusion assays. Further level of tumour necrosis factor (TNF-α) for assessing anti-inflammatory activity and 3-(4,5Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay for safety assessment was determined. High performance thin layer chromatography (HPTLC) fingerprint profile was developed using thin layer chromatography (TLC) scanner and densitometer. The poly-herbal formulation, PHF 1205 showed antimicrobial activity against both pathogenic bacteria and fungi with zone of inhibition ranging from 7 to 13 mm. PHF 12-05 was found to possess anti-inflammatory activity by significantly (P<0.05) reducing the level of tumour necrosis factor (TNF-α). HPTLC characterized PHF 12-05 was found to be quite stable and safe. The observations clearly indicate that PHF 12-05 holds promise in improving oral hygiene and hence help in preventing oral problems.


INTRODUCTION
Oral infections are one of the most common diseases worldwide leading to dental caries and periodontal disease (Petersen et al., 2005).Dental caries is a dynamic process causing progressive destruction of hard tooth substance involving demineralisation of the inorganic part and dissolution of the organic portion (Winston and Bhaskar, 1998).Periodontitis, a destructive gum disease, may progress irreversibly in breaking down supporting periodontal structures which results in loss of *Corresponding author.E-mail: mp.darokar@cimap.res.in.tooth and about 20% population of the world is affected by these diseases (Petersen, 2005(Petersen, , 2008)).The tooth decay which occurs due to oral infections highly affects the adults and aged people and is sometimes responsible for early death (Marsh and Martin, 1992;Petersen, 2005).These diseases are mainly caused by plaque forming bacteria and yeast that reside in the oral cavity which metabolize sucrose to organic acids dissolving calcium phosphate in teeth, causing decalcification and eventually the decay (Loesche, 2007).Dental treatment is usually a high expense remedy and it mainly utilizes some antiseptics as well as antibacterial agents like triclosan, chlorhexidine, and amine based fluorides.But the major drawback of these products is that they possess significant toxicity and are also responsible for the staining of teeth and burning sensation on tongue (Grundermann et al., 2000;Chung et al., 2006).Further, these products are beyond the reach of common people in developing countries (Akpata and Akinrimisi, 1977;Homer et al., 1990).Most of the plant species are traditionally used for thousands of years to cure or prevent oral diseases in various parts of world (Tichy and Novak, 1998).For example, use of chewing sticks (miswak) is very common in India and Africa, because of its proven antibacterial activity against microorganisms infecting oral cavity (Lewis, 1980).Plants like Achyranthes aspera, Azadirachta indica and Curcuma longa have been used long ago to cure pyorrhoea, tooth ache, and gum infections.Syzygium aromaticum is used for years to relieve tooth ache (Ambasta, 2000).Therefore, rationale of this study was to scientifically validate the claims of traditionally used medicinal plants, and based on them, develop a poly herbal formulation which can be used for the cure or prevention of oral/dental problems.

Preparation of plant extracts
Plant materials, A. aspera- Bark (Voucher Specimen No. 13651), S. aromaticum-Fruit (Voucher Specimen No. 13656) mucorosai-Seed (Voucher Specimen No. 1223) were obtained in the month of September 2011 from National Gene Bank of Medicinal and Aromatic plants of CSIR-CIMAP, Lucknow, India.The plant material was identified by Dr. S. C. Singh (Botanist, Pharmacognosy Department) and Dr. A. K. Gupta (Scientist, Genetic Resource Management).Plant materials were shade dried, pulverized and 500 g of the powdered material was soaked in 1000 ml of methanol, water, and water: alcohol (80:20), respectively for overnight and were refluxed using Soxhlet apparatus.The extracts were concentrated under reduced pressure using rotary evaporator (Bucchi) and were stored at 4°C for further use.The extracts were solubilised in dimethyl sulfoxide (DMSO, Sigma, USA) for in-vitro assays.

Preparation of poly-herbal formulation
Since hydro-alcoholic extracts of A. aspera, S. aromaticum, P. nigrum, M. elengi, A. indica, C. longa, Z. officinale, S. persica, A. nilotica, Z. armatum and S. mucorosai exhibited better antimicrobial activity, they were used for the development of the formulation.The bioactive hydro-alcoholic extracts were suspended all together in distilled water as per the ratio given in Table 1.The solution was heated at 60°C for 5 min, cooled to room temperature, and sodium chloride (common salt) was added, stirred thoroughly, and volume was made up with distilled water and filtered.The formulation was stored in sterile glass bottle (Schott Duran, Australia) for further studies at 4°C.

Disc/Well diffusion assay
The microbial growth inhibitory potential of the selected plant extracts was determined by using the agar disc diffusion method (Clinical and Laboratory Standards Institute (CLSI), 2006a).Inoculums, for all test microorganisms were prepared by mixing a few microbial colonies with sterile normal saline solution and comparing the turbidity with that of the standard 0.5 McFarland solution which is equivalent to 10 6 to 10 8 CFU/ml.Out of this, 100 µl were inoculated on agar medium (MHA for bacteria and SDA for fungi).Plant extracts were dissolved in DMSO (Merck, India) to a final concentration of 100 mg/ml and 5 µl from this stock was transferred onto sterile filter paper disc (Whattman-1, 5 mm diameter).Disc prepared with only DMSO served as negative control and standard clinically used antibiotics served as positive control.
The plates were then incubated at 37°C for 24 h for bacteria and 28°C for 24 h for fungal strains.For evaluating antimicrobial property of formulation, 50 µl of the formulation was added into each well prepared in agar plate with a sterile 10 mm cork-borer at equidistance.All tests were performed in triplicate and zones of inhibition were measured from the edge of each disc/well after the incubation period.

Minimum inhibitory concentration (MIC)
The antimicrobial activity of the plant extracts was determined by micro dilution broth assay using 96 'U' bottom micro-titre plates (Greiner, Germany) as per CLSI guidelines (CLSI, 2006b).Extracts were serially diluted by two folds (starting from 2000 to 3.9 µg/ml) in MHB for bacteria and in SDB for fungi.The broth was inoculated with 10.0 µl of diluted 24 h grown culture of test organism with a titre equivalent to 0.5 McFarland standards.The inoculated plates were then incubated at 37°C and 28°C, respectively for bacteria and fungi.
The growth was recorded spectrophotometrically at 600 nm using spectramax 190-microplate reader (Molecular Devices, Sunnyvale, CA, USA).The MIC value was determined from the turbidometric data as the lowest concentration of the extracts showing growth inhibition equal to or greater than 80% as compared to the control.All experiments were performed in triplicate to rule out any error during the procedure.

Isolation of peritoneal macrophages
Peritoneal macrophages were isolated using the method reported Distilled Water --q.s.
-by Bawankule et al. (2008).All procedures were conducted under aseptic conditions.Mice were euthanized by cervical dislocation under ether anesthesia and peritoneal exudates cells (PEC) were obtained by intra-peritoneal injection of phosphate buffer saline, pH 7.4 (Sigma Chemicals Co. USA).Macrophages in the PEC suspension were isolated by the cell adhesion method.The PEC were suspended in RPMI 1640 medium (Sigma Chemicals Co. USA) containing 10% fetal calf serum (Gibco, USA) and incubated for 4 h at 37°C with 5% CO2 in an incubator using 96 well plates (Nunc, Germany).After removing non adherent cells along with medium, the adherent cells were resuspended in RPMI 1640 medium.The cells were used for experiments as resident peritoneal macrophages at a concentration of 2 × 10 10 viable cells/ml.

Expression of TNF-α from lipo-polysaccharide (LPS) stimulated peritoneal macrophages
Peritoneal macrophages were cultured at 37°C with 5% CO2 in humidified air for 48 h with LPS (E. coli 050: B5; Sigma Chemical Co., USA) at the concentration of 1 µg/ml.TNF-α activity in culture supernatants was estimated by Enzyme Immune Assay (EIA) using commercial kit for mouse TNF-α (Pierce Endogen, Rockford, USA) in culture supernatant and measured using Griess reagent (Sigma Chemical Co.USA) (Bawankule et al., 2008).The samples were frozen and stored at -80°C for further use.

Safety assessment
Safety evaluation of PHF was determined by using the aforementioned isolated peritoneal macrophages and culture method.3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) stock solution at 0.1 mg/ml was added to each well having peritoneal microphages and incubated at 37°C for 2 h.Dark blue formazan crystals formed were solubilised by adding 100 µl DMSO to each well and the absorbance was measured at 540 nm (Eun et al., 2004;Inaba et al., 1996).

Standardization parameters
Various standardization parameters were studied including organoleptic properties, pH, heavy metal content, stability, microbial load and viscosity as per the methods described earlier by Pandey et al. (2012).Different organoleptic characters such as colour, odour, taste, etc., of the samples were evaluated following the methods described by Siddiqui and Hakim (1995).The viscosity of the developed formulation was determined using Brookfield Viscometer (Model no.DV-11+Pro) at various RPM's at room temperature.The sample volume used for the study was 7.2 ml.The spindle no.18 with small sample adopter was used for the determination of viscosity.Microbial analysis was carried out for determination of microbial contamination as per procedures reported earlier (Ministry of Health and Family Welfare, 2010; WHO Guideline, 2007).This test included total bacterial, total yeast and mould count.

Preparation of samples by acid digestion method
Dried and processed sample (0.5 to 1.0 g) was taken in 150 ml conical flask.Acid mixture of HNO3:HClO4 (4:1) (v/v) ml was added in the flask and heated continuously till the solution is colourless and reduced the contents of acid mixture of HNO3:HClO4 (4:1) (v/v) to about 2 to 3 ml by continuous heating.After cooling to room temperature, 30 ml of distilled water was added and filtered through Whatman No. 1 filter paper into a 100 ml volumetric flask and the volume was made-up to 100 ml with distilled water and finally the sample was processed through Induced Coupled Plasma (Unichem Model 969) following the conditions described earlier (Lohar, 2007) for the presence of heavy metals like lead (Pb), cadmium (Cd), nickel (Ni), chromium (Cr) and cobalt (Co).The presence of micronutrients such as calcium (Ca), cupper (Cu), iron (Fe), magnesium (Mg), manganese (Mn), and zinc (Zn) were also determined.

Stability studies
The PHF was stored in a glass reagent bottles at different temperatures of 0, 37, 42°C and at room temperature (20°C minimum and 35°C maximum) for three months, and finally, the samples were checked for their antimicrobial activity, clarity of solution, pH and microbial contamination and organoleptic properties.

High performance thin layer chromatography (HPTLC) fingerprinting
After optimization using Vario System of mobile phase, a well resolved separation was achieved by using the mobile solvent chloroform:methanol (85:15, v/v) and 60F TLC plate (Merck Cat # 1.05729.001).Spots were applied using micropipette in the ratio 2:5:10 µl.The plates were run in ascending mode for a distance of 9.0 cm in a vertical twin trough chamber that was previously saturated for 2.0 min with the mobile solvent.After TLC run, the air dried plates were immersed in freshly prepared vanillin-sulphuric acid derivatizing reagent (vanillin:ethanol:H2SO4, 1 g:95 ml:5 ml) followed by heating at 110°C for 15 min.The densitometry scanning (CAMAG, Muttnez, Switzerland) was performed in the reflectance/ absorbance mode, slit width 6.00 to 0.40 mm, scanning speed 20 mm/sec and data resolution 10 mm/step, detection wave length 610 nm.By following the method described earlier (Verma et al., 2009).

RESULTS
The hydro-alcoholic extract of all the selected plant species was observed to exhibit antimicrobial activity against one or the other pathogen used for testing.The hydro-alcoholic extract of plant species C. longa, A. nilotica, and S. aromaticum was active against the majority of the tested microorganisms, followed by A. indica, P. nigrum and others.The minimum inhibitory concentration of hydro-alcoholic extract was found to be in the range of 62.5 to 1000 µg/ml (Table 2).Several formulations containing bioactive hydro-alcoholic extracts of 11 plant species in various ratios were prepared.Finally, a poly-herbal formulation (PHF12-05) showing best antimicrobial activity and optimal representation of all the plants was selected for further studies.PHF12-05 exhibited strong antimicrobial activity against all pathogenic strains with zone of microbial growth inhibition ranging from 7 to 13 mm (  3017).Although, activity of PHF12-05 did not match with the standard antibiotics; however, it was comparable with respect to zone of growth inhibition.Similarly, PHF 12-05 exhibited anti-inflammatory activity in terms of expression of TNF-α that was increased in LPS stimulated peritoneal macrophages and was observed to be decreased significantly (P<0.05) after treatment with PHF12-05 in dose dependent manner (Figure 1).Further, PHF12-05 was found to be safe against peritoneal microphages up to 100 µg/ml concentration.The HPTLC fingerprint of the PHF12-05 at three different concentrations: 2, 5 and 10 µl loaded on TLC plates showed same pattern of resolution.However, the numbers of peaks obtained were more in higher concentration (10 µl) as compared to lower concentrations.At 620 nm, 2, 5 and 10 µl track showed seven, eight and nine peaks, respectively with R f values ranging from 0.01 to 0.95 and peak area ranging from 10261.0 to 27505.8 (Figure 2).Separation of peaks was observed to be better at 254 nm.
The formulation (PHF 12-05) was dark brown in colour, with a pungent odour and bitter taste.It was quite stable, since no significant changes were observed in pH, antimicrobial activity, consistency/clarity and microbial load when stored at temperatures/humidity of 4°C/28, 28°C/40, 37°C/25 and 42°C/30 (Table 5).Heavy metals contamination was observed to be much below the maximum permissible limits in such preparations (Table 4).

DISCUSSION
Microorganisms such as bacteria and yeast that reside in oral cavity are responsible for oral infections leading to plaque formation, tooth decay and periodontal disease (Marsh and Martin, 1992;Moynihan and Petersen, 2004;Petersen, 2005;Loesche, 2007;Khan, 2009;Deshpande et al., 2010;Gur et al., 2006).In underdeveloped and developing countries, majority of the populations rely on the traditional means of preventions and cure.Large numbers of plant species are known for thousands of years to prevent or cure oral diseases (Tichy and Novak, 1998).The observations of this study are in accordance with traditional use of the selected plant species taken up in this study for the oral hygiene (Table 1).For example, use of chewing sticks (miswak) is very common in India and Africa, because of its proven antibacterial activity against microorganisms infecting oral cavity (Lewis, 1980).The medicinal plants used in the formulation of PHF 12-05 were active against the tested microorganism with MIC ranging from 62.5 to 1000 µg/ml, which correlates well with the earlier reports of antimicrobial activity of these plants.For example, S. aromaticum is reported to be active against bacterial strains (Pandey and Singh, 2011) and C. albicans (Nzeako et al., 2006) are known to cause oral infections.P. nigrum (Black pepper) inhibited both Gram positive and Gram negative
When the extracts of the selected plants were combined together in form (formulation PHF 12-05), it was observed that the antimicrobial effect was enhanced significantly.In traditional systems, it is also well known that the medicinal plants exhibit better activity in combinations than when used individually (Khan, 2009;Pandey et al., 2012).
Plants probably have least side effects as they are widely used as dietary intake as well as in form of various traditional remedies including various combinations and formulas since ancient time.Microbial infections are also known to evoke inflammatory reactions leading to severe pain and wound that is difficult to manage (Bawankule et al., 2008).The formulation (PHF 12-05) was found to be anti-inflammatory and safe.HPTLC fingerprint of the extracts and herbal formulations are considered to be quality control parameters (Panda et al., 2012).The HPTLC fingerprint of PHF 12-05 was found to be quite distinct at three different wavelengths and can be used as quality control purpose.Heavy metal contamination, microbial load and stability of the extracts and herbal formulations are important for their commercial utilization (Meena et al., 2012).PHF 12-05 was found to be containing heavy metal and microbial contaminations less than the permissible limits and was stable at various temperatures and humidity conditions.

Conclusion
The observations of this study clearly suggest the suitability of PHF 12-05 as herbal remedy for maintaining oral hygiene, since it possesses potent antimicrobial activity against bacterial and yeast strains which are major cause of dental infections.Besides, this formulation also possesses anti-inflammatory activity, especially those caused by the microorganisms.The quality control parameters, stability and less heavy metal and microbial contamination make this formulation suitable for human use.Further studies may be required for the isolation and chemical characterization of the active ingredients in plant extract for the development of a novel agent(s) against dental caries.

Table 1 .
Composition of poly herbal formulation PHF12-05 and traditional importance of plant species.

Table 2 .
Minimum inhibitory concentrations of plant extracts against human pathogenic microorganisms through broth dilution assay.

Table 3 .
Antimicrobial activity of poly herbal formulation PHF 12-05 through well diffusion assay.

Table 5 .
Parameters showing stability of poly-herbal formulation PHF12-05 after storage for two months.