TITLE: 

  ABSTRACT.OBJECTIVE:
The current study was intended to evaluate and compare the in vivo efficacy of Calotropis gigantea mouth rinse as anticariogenic agents with 0.2% chlorhexidine and Listerine mouth rinse.

ABSTRACT.MATERIALS AND METHODS:
An experimental crossover study was conducted on sixty school children aged 14–15 years. Participants were divided into three Groups A, B, and C. In all children, baseline unstimulated saliva samples were collected and assessed for Streptococcus mutans counts. The study was conducted in three phases; each phase lasted for 8 days separated by a washout period of 15 days in between them. Groups A, B, and C were treated with 0.2% chlorhexidine, Listerine and C. gigantea extract mouth rinses, respectively, in the Phase I and assessed for salivary S. mutans counts. Subsequently, mouth rinses were crossed over as dictated by the Latin square design in Phase II and III.

ABSTRACT.RESULTS AND CONCLUSION:
All the three mouth rinses have individually shown a statistically significant reduction in the salivary S. mutans counts.

BODY.INTRODUCTION:
In the present scenario of medical and pharmaceutical advancement, microbes have evolved to change their metabolism and genetic structure to gain resistant against the drugs used in the treatment of various infectious diseases.[1] Global antibiotic resistance by bacteria is becoming an amounting public health concern. To prevail over microbial drug resistant, researchers are looking forward for the development of alternative and novel drugs in pursuit to discover new antibacterial agents.[2]  Plants are in use as medicinal resource since prehistoric age. Plants contain loads of biologically active molecules with diverse medicinal properties. The traditional and folk medicinal system uses the plant products for the treatment of various infectious diseases.[1] Plant-produced compounds are of more interest in recent past as they are a source of safe and more effective substitutes against the synthetically produced antimicrobial agents.[3] The World Health Organization has estimated that about 80% of people living in various Asian and African countries still rely on medicines derived from plant products for some part of their major health care needs. Utilizing the healing powers of plants is an ancient idea, more so in India. At present, 25%–50% of all pharmaceuticals dispensed around the world are of plant origin, and only few among them have been used as antimicrobials.[4]  Dental caries is a familiar microbial disease affecting humans. It is an infectious, chronic disease of multifaceted etiology resulting in destruction of tooth structure by acid produced by microorganisms found in dental plaque, leading to early loss of tooth structure in children and young adult.[56] It has been established that cariogenic microorganisms, especially Streptococcus mutans, play a decisive role in the pathogenesis of dental caries as it is involved in the initiation of almost all carious lesions of enamel.[7] Research in the field of caries prevention has been focusing on ways of reducing or totally eliminating cariogenic microflora from the oral cavity. Numerous studies have shown that caries can be prevented by regular toothbrushing and flossing. Nevertheless, most of the studies have revealed that it is difficult to eliminate S. mutans from pits, fissures, and proximal surfaces by mechanical means alone. Consequently for effective caries control, these methods should be combined with the chemotherapeutic agents.[8] The use of broad-spectrum antimicrobial mouth rinses as an additive to patient's oral hygiene and in delivery of active agents to teeth and gums has assumed greater importance with the recognition that most individuals are unable to maintain sufficient oral hygiene with mechanical means alone.[289]  The bisbiguanide chlorhexidine is considered as the "gold standard" in anticariogenicity, and has been researched at wide span. It is currently the most potent chemotherapeutic agent in use against S. mutans. It has bactericidal activity against both Gram-positive and Gram-negative bacteria.[510] Another mouth rinse Listerine is a product which is about 115 years old and is also a broad-spectrum antiseptic mouth rinse, whose active ingredients are the four essential oils, namely, thymol, menthol, eucalyptol, and methyl salicylate. Listerine is also extremely effective against a wide range of Gram-negative and Gram-positive microorganisms, commonly found in dental caries.[58] These two mouth rinses chlorhexidine and Listerine are widely used as chemotherapeutic agents in plaque control; even then, the incidence of dental care is high. Furthermore, these being modern medicines, it has its own side effects such as altered taste perception, metallic taste, alcohol content, staining of teeth, and many others. All of which is leading to its nonfavorable among many individuals.[5810]  Consequently, the onus is on researchers to come up with robust, novel, effective, feasible, and newer strategies to manage this disease.[5] One such strategy would be to verify the colossal wealth of medicinal plants. Calotropis gigantea is a traditional medicinal plant belonging to the family of "Asclepiadaceous," habitat of Asian countries that includes India, Indonesia, Malaysia, Thailand, Sri Lanka, China, and others, and it is bestowed with enormous medicinal property.[3] There are reports of tribal people using this plant parts to cure numerous illnesses such as toothache, earache, sprain, anxiety, pain, epilepsy, diarrhea, and mental disorders.[1] The root bark of this plant is used as medicine in the treatment of leprosy, piles, wounds, tumors, parasitic infections, and dysentery. In literature, it was also reported that alcoholic root extract of C. gigantea showed analgesic, anticonvulsant, anxiolytic, and sedative effect in albino rats.[11] Literature review reveals that no studies have been conducted to assess the antimicrobial activity of C. gigantea against S. mutans. As researches related to herbal mouth rinses are scarce and as clinicians, we are at cross road when patient seek our opinion about such herbal mouth rinses. There is a need for research in this area to generate the requisite evidence to proclaim the helpfulness of these herbal products.  Hence, the current study was conducted with an aim to assess and compare the effect of C. gigantea extract mouth rinse, 0.2% chlorhexidine mouth rinse and Listerine mouth rinse individually on salivary S. mutans level on 14–15 years schoolchildren of Jaipur.

BODY.MATERIALS AND METHODS.STUDY DESIGN AND POPULATION:
The present in vivo, experimental, crossover study (within participants utilizing Latin square design) was conducted among selected school children aged between 14 and 15 years. The study protocol was approved by the Ethical Review Panel. Permission to carry out the study was obtained from the school authorities. Only those going children who had at least one cavitated active carious lesion (clinical selection criteria) and a salivary S. mutans count of equal to or more than 105 CFU per ml of saliva (microbiological selection criteria) were included in the study.  Participants with a known history of hypersensitivity to any product used in the study, suffering from illness which might influence the salivary flow, unable to fulfill with the study appointment schedules, having a record of antibiotic therapy in the previous 1 month till the start of the study, having any systemic illness, and who were already using any mouth rinse were excluded from the study.

BODY.MATERIALS AND METHODS.SAMPLING:
Sixty school going children were selected from the three different schools. From each school, twenty children were selected randomly who were fulfilling the proposed criteria. Written informed consent was obtained from selected children and from their parents before the beginning of examination by discussing with them the need of the study.

BODY.MATERIALS AND METHODS.PREPARATION OF ETHANOLIC EXTRACT:
Fresh leafs of C. gigantea plant were collected, they were thoroughly washed in running water, dried in natural light for 48 h, and powdered. Twenty grams of this powder was soaked in 200 ml of ethanol for about 24 h. The supernatant that formed was filtered through Whatman No. 1 filter paper. This filtrate was evaporated in sunlight and dissolved in distilled water to get the required concentration of 2.5% C. gigantea.

BODY.MATERIALS AND METHODS.BASELINE SALIVA COLLECTION AND MICROBIAL ANALYSIS:
All the children selected for investigation were given clear instructions to refrain from eating for 1 h before collection of saliva. From each child, 1 ml of unstimulated whole saliva was collected by asking them to let saliva get collected in the floor of the mouth without swallowing it for at least 1 min and then to spit out into the sterile container. The containers were then directly transported to the oral pathology laboratory, where it was processed straight away.  Using a stirrer, the collected saliva sample was manually stirred and homogenized. Hundred microliter of this saliva sample was diluted with 1 ml of normal saline (1:10 dilution). Using an inoculation loop (2 mm inner diameter), 5 μl of the 1:10 diluted saliva sample was streaked on a mitis salivarius-bacitracin agar (MSB) which is the selective medium for S. mutans. The MSB agar plates were then incubated at 37°C for 48 h in an atmosphere consisting of 95% nitrogen and 5% carbon dioxide. After the incubation period of 48 h, S. mutans appeared on the culture plate as small, rough, raised, and adherent colonies. Those colonies which were atypical were further confirmed by mannitol and sorbitol test. Electronic colony counter was used for counting of colonies. The obtained data were the baseline S. mutans level.

BODY.MATERIALS AND METHODS.TEST PROCEDURE:
Three mouth rinses 0.2% chlorhexidine mouth rinse (Hexidine, ICPA Health Products Ltd., 233-A, Adarsh Industrial Estate, Sahar Road, Andheri East, Mumbai 400099, India), Listerine mouth rinse (Listerine Smart Rinse, Johnson and Johnson Private Ltd., 501 Arena Space, Jogeshwari East, Mumbai 400060, India), and 2.5% C. gigantea extract mouth rinse (self-prepared) were tested for their anticariogenic properties. The present study was a crossover study utilizing the Latin square design, so sixty schoolchildren were divided into three Groups A, B, and C. Every child in each group was subjected to all three mouth rinses in a phased manner as shown in the diagram. In Phase I, Groups A, B, and C were assigned 0.2% chlorhexidine, Listerine and 2.5% calotropis mouth rinses, respectively. Every child was instructed to rinse twice daily, once in morning after breakfast and once in night after dinner before going to bed for seven continuous days with 10 ml (undiluted) of the given mouth rinse for 1 min and then spit out the rinse. A measuring cup was provided to the children to dispense 10 ml of the given mouth rinse. Children were instructed not to eat or drink anything for a minimum of half an hour after rinsing.  On the 8th day, children were instructed to use mouth rinse once in the morning after breakfast. One hour after completing the mouth rinse from all children, 1 ml of unstimulated saliva samples was collected. The collected saliva sample was subjected to microbiological procedure as done earlier during baseline salivary S. mutans CFU/ml count assessment. Phase I of mouth rinse was followed by a washout period of 15 days during which children were asked to stop using the assigned mouth rinse. After the washout period of 15 days, the selected children were assigned with the next mouth rinse in a phased manner. After each phase, unstimulated saliva sample was again collected and was subjected to microbiological procedure. During the entire course of the study, the children were instructed to stick to their routine oral hygiene habits.

BODY.MATERIALS AND METHODS.STATISTICAL ANALYSIS:
For intragroup and intergroup comparison of salivary S. mutans counts at baseline and on day 8, paired t-test (GraphPad software, California) and one-way ANOVA (XLSTAT software, Addinsoft) were used, respectively.

BODY.RESULTS:
In this crossover trial, analysis of the data is done by two methods. One by comparing the observations on each group separately at different phases of the trial, and another with respect to each mouth rinse. In the current study, both the analyses have been done, Phases I, II, and III of Group A are compared and similarly Phases I, II, and III of Group B and Group C are compared [Table 1 and Graph 1]. The overall results for 0.2% chlorhexidine, Listerine and 2.5% calotropis extract mouth rinses for the total sample are also compared [Tables 2–4 and Graph 2].   Table 1 Mean reduction in Streptococcus Mutans count in Group A   Graph 1Intergroup comparison of Streptococcus mutans reduction  Table 2 Mean reduction in Streptococcus Mutans count in Group B   Table 3 Mean reduction in Streptococcus Mutans count in Group C   Table 4 Inter group comparison in Mean reduction in Streptococcus Mutans count   Graph 2Mean reduction in Streptococcus mutans count  Individually, each of the mouth rinse significantly reduced the salivary S. mutans count which was reflected in the difference between pre- and post-test counts. When all the three mouth rinses were compared for their ability to reduce salivary S. mutans count although there was a numerical difference statistically, it was found to be not significant.

BODY.DISCUSSION:
From the ancient era to the current times, in India and other parts of the world, plants, animals, and other natural things have shown a considerable influence on culture, tradition, and civilization of man. From the beginning of civilization, human beings have worshiped plants and such plants are conserved as inherited resources and were used as food, fodder, fiber, fertilizer, fuel, and febrifuge and in every other way possible.[12] In modern days, medicinal plants are being probed and explored as an alternate resource to get therapeutic compounds based on their medicinal properties.[1] C. Gigantea is one such plant with many therapeutic principles and other economic values.[12]  In ancient ayurvedic medicine, the plant C. gigantea is known as "Sweet Arka."[12] C. gigantea is a traditional medicinal plant; it belongs to the family of Asclepiadaceous habitat of Asian countries. The plant grows up to 2–4.5 m long. It has light green leaves and milky stem. The leaves are very much tender in nature.[3] C. gigantea is scientifically acknowledged for its cytotoxic, antipyretic, antimicrobial, and wound healing properties. C. gigantea has been medicinally used to treat ailment such as fevers, cough, cold, nausea, vomiting, rheumatism, indigestion, eczema, asthma, elephantiasis, leprosy, and diarrhea.[313]  Microorganisms are the foremost etiologic agents of numerous medically important infectious diseases occurring in man and animals. Dental caries is one such dreaded microbial infectious disease occurring in oral cavity on the hard surfaces of teeth and predominantly affecting all the populations of the world.[7] Researchers have confirmed that teeth are demineralized when they are exposed to salivary bacteria and carbohydrates in vivo. S. mutans constitutes a majority of the total bacteria present in dental caries samples.[71014] Reducing their levels in the oral cavity would provide a rationale for the prevention of dental caries. Mouth rinses obtained from artificial products are extensively used as adjuncts to oral hygiene and in the delivery of active agents to the teeth and gums. The ability of these rinses to influence plaque formation and to alter the course of gingival inflammation has been studied at length.[15]  Natural products of plant origins have shown to possess abundant pharmacological properties such as antimicrobial, anti-inflammatory, and cytostatic effects, and they are been used as folk medicine throughout the world since ancient times. The presence of bioactive compounds comprising saponins, tannins, flavonoids, alkaloids, and many others is attributed for their effectiveness against microorganisms.[12] C. gigantea is one such plant; it is drought resistant, saline tolerant to a fairly high degree, grows natural all over the world. Various parts of this plant encompass an immense potential ability in treating a wide range of diseases and disorders. Its phytochemical components constitute alkaloids, tannins, flavonoids, terpenoids, amino acids, saponins, steroids, cardiac glycosides, and many more.[12] Tannins form irreversible complexes with proline-rich protein 33 resulting in the inhibition of cell protein synthesis. Alkaloids are one of the largest groups of phytochemicals in plants; they have remarkable effects on decreasing pain perception in humans; this has led to the development of powerful painkiller medications. Flavonoids, another constituent, exhibits a wide range of biological activities such as antimicrobial, anti-inflammatory, analgesic, antiallergic, cytostatic, and antioxidant properties.[2411]  The present study was designed to evaluate the anticariogenic efficacy of natural product of plant C. gigantea extract in mouth rinse form against salivary S. mutans and compare with two other commonly use artificial mouth rinses 0.2% chlorhexidine and Listerine. Search of previous researches did reveal that no studies have been conducted to evaluate the effect of C. gigantea on S. mutans.  The C. gigantea extract was prepared at 2.5% concentration as this particular concentration exhibited the maximum zone of inhibition against S. mutans among the different concentrations that were investigated in the pilot study. The other two mouth rinses selected were 0.2% chlorhexidine mouth rinse (ICPA Health Products Ltd.) and Listerine mouth rinse (Johnson and Johnson Private Ltd.). In the study, a standardized volume of 10 ml and rinsing time of 1 min was instructed to each child. The washout period of 15 days between the two phases was used to trim down the carry-over effect of the intervention as the salivary S. mutans counts returned to baseline levels within 15 days.[8]  0.2% chlorhexidine mouth rinse effectively reduced the salivary S. mutans counts to a statistically significant level. Listerine mouth rinse also significantly reduced the salivary S. mutans counts. Similar results for 0.2% chlorhexidine and Listerine were seen in other earlier studies.[58] This shows a clear evidence to suggest that 0.2% chlorhexidine and Listerine is a potent antibacterial agent against S. mutans. The bactericidal efficacy of these essential oil-containing mouth rinses used in this study has been long recognized and represents the primary mechanism by which it exerts its clinical effects.[7]  Results from this in vivo study of 2.5% C. gigantea mouth rinse could not be compared with other studies as no other in vivo studies have assessed the effect of Calotropis on S. mutans. However, an in vitro study of C. gigantea has shown antimicrobial activity against S. mutans and lactobacilli. It was found to be effective at as low as 1.25% concentration also.[16]  Furthermore, earlier studies on the antimicrobial activity of C. gigantea root bark extracts have revealed its antibacterial potentials against Sarcina lutea, Bacillus megaterium, Bacillus subtilis, Shigella sonnei, Escherichia coli, and Pseudomonas aeruginosa. Studies on the aqueous extract of C. gigantea leaves have exhibited the antibacterial activity against six clinical isolates of bacteria Staphylococcus aureus, Klebsiella pneumoniae, Bacillus cereus, Pseudomonas aeruginosa, Micrococcus luteus, and E. coli. Anti-Candida activity of the C. gigantea leave extracts was reported against clinical isolates of Candida albicans, Candida tropicalis, Candida krusei, and Candida parapsilosis.[12413]  In the present study, we made an attempt to assess the antimicrobial effect of 2.5% C. gigantea extract against S. mutans. C. gigantea showed a significant reduction in salivary S. mutans between baseline and posttest values in each group and also in total sample. These can be attributed to various phytochemical components present in C. gigantea. Thus, this study is distinctive as until now no similar studies have been reported in literature.

BODY.CONCLUSION:
C. gigantea represents a rich source of valuable medicinal compounds and contains high antibacterial property and can be further explored for the isolation of its bioactive compound. This is an encouraging result which clearly favors promotion of C. gigantea as a mouth rinse. However, this is an initial attempt in assessing the effect of S. mutans; further, long-term studies need to be performed to investigate the effect of C. gigantea on dental caries before it can be confidently recommended. Furthermore, researches should be directed on various other herbs that can possess significant antimicrobial properties which can be developed as effective cariostatic agents.

BODY.CONCLUSION.FINANCIAL SUPPORT AND SPONSORSHIP:
Nil.

BODY.CONCLUSION.CONFLICTS OF INTEREST:
There are no conflicts of interest.

Question: Compared to Control what was the result of C. gigantea extract mouth rinse on Streptococcus mutans count?
Evidence: All the three mouth rinses have individually shown a statistically significant reduction in the salivary S. mutans counts.
Answer: significantly decreased