Effect of pomegranate juice on dental plaque microorganisms (streptococci and lactobacilli).

To study the effect of pomegranate juice on dental plaque microorganisms. A clinical trial was conducted on thirty healthy volunteers aged 25-30 years who visited Out Patient Department (OPD) of Bapuji Dental College and Hospital, Davangere during the month of October 2006. Before conducting the study, thorough oral prophylaxis was done and the subjects were asked to refrain from the oral hygiene procedures for 24 hrs. Dental plaque was collected from each subject, before and after rinsing 30ml of pomegranate juice without sugar. Plaque samples were cultured using Mitis Salivarius Agar and Rogosa SL Agar media. Wilcoxon's signed rank test was used for statistical analysis. Results showed that pomegranate rinse was effective against dental plaque microorganisms. There was a significant reduction in the number of colony forming units of streptococci (23%) and lactobacilli (46%). The ruby red seeds may be a possible alternative for the treatment of dental plaque bacteria.

Introduction

‘We are what we eat’ - Some of the gravest health problems can be caused by food and yet, food can also be a cure for many as an ailment. Punica granatum L, a commonly found fruit, is found to be beneficial in maintaining oral health. The interest in plants with antibacterial and anti-inflammatory activity has increased as a consequence of current problems associated with the wide-scale misuse of antibiotics that induced microbial drug resistance. Natural products such as Astronium urundeuva, curcuma zedoaria and other herbal products have been tested with effective results. Punica granatum Linn, mostly known as “pomegranate”, is a shrub or small tree native from Asia where several of its parts have been used as an astringent, haemostatic as well as an anti-inflammatory.1 Affectionately known as the ‘jewel of winter’, the pomegranate is receiving growing acclamation for its disease-fighting abilities, largely due to its staggering antioxidant potency. The pomegranate carries with it the mystique of ancient myth and has a diverse collection of medicinal, nutritional and cosmetic uses. Over the centuries, it has been regarded as magical, mystical and healing. Traditional medicines of the Middle East and India have used the pomegranate for centuries - leaves, skin and rind included - to cure everything from conjunctivitis and sore throats to baldness and hemorrhoids.

Pomegranates contain polyphenols, tannins, ellagic acid and anthocyanins which are powerful antioxidants. The red fruits’ anthocyanidins (red pigment) contribute to the antioxidant activity, hence pomegranate juice has superior bioactivity compared to its purified polyphenols.2 Back in 1999, Israeli research on pomegranate's juice and cold pressed seed oil first showed strong antioxidant activity close to that of butylated hydroxyanisole [BHA] and green tea, and significantly greater than that of red wine. It has already been established that antioxidant activity in pomegranate juices is higher when extracted from whole pomegranates than in experimental juices obtained from the luscious red arils only. The rind of this commonly found fruit exhibits antibacterial and astringent activity. Its astringent action results in strengthened gums.3

Oral diseases start with plaque. Plaque is a sticky film of food, saliva and bacteria. Dental plaque is a biofilm which is comprised of a population of bacteria growing on the tooth surface enmeshed in a polysaccharide matrix. The researchers have shown that many of the above mentioned antioxidants can inhibit the formation of the plaque polysaccharide matrix, block the adherence of bacteria, prevent acid formation and reduce acid tolerance of cariogenic microorganisms. Recent studies demonstrate that pomegranates can support oral health and is a successful remedy for strengthening gums and fastening loose teeth. In a study by Shastravaha, treatment with the extract of Punica granatum was found to significantly improve clinical signs of chronic periodontitis.4 Earlier, when extracts of 13 Brazilian medicinal plants used in Brazilian folk medicine for the treatment of infectious diseases were screened for their antimicrobial activity against bacteria and yeasts, results found pomegranate has good activity on Staphylococcus aureus bacteria and anti-candidal activity was detected.3

The results of the above studies suggest that pomegranate is effective against oral bacteria and has an antimicrobial activity. But, much of these studies have been done using the pomegranate rind or extract of the whole pomegranate. Very few studies have been done to find the effect of experimental juices obtained from the luscious red arils only on the oral bacteria. However, the research in this area is very scarce, especially the benefits of pomegranate juice as a preventive oral health drink. Hence, an attempt is made to study the effect of pomegranate juice on selective dental plaque microorganisms (Streptococci & Lactobacilli). The null hypothesis is ‘pomegranate juice has no effect on dental plaque microorganisms’. The study hypothesis is ‘pomegranate juice has an effect on dental plaque microorganisms’.

Materials and Methods

The present study is a clinical trial conducted to find the effect of pomegranate juice on dental plaque microorganisms (Streptococci and lactobacilli). Before conducting the study, permission from the concerned authorities and ethical clearance by the ethical committee of Bapuji Dental College and Hospital was obtained. All the subjects were informed about the nature of the study and voluntary written informed consent was obtained from the participants.

Thirty healthy volunteers aged 25-30 years who visited Out Patient Department (OPD) of Bapuji Dental College and Hospital, Davangere during the month of October 2006 were included in the study. Healthy volunteers who agreed to refrain from oral hygiene procedures for 24 hours were included in the study. Subjects with medical disorders and under antimicrobial therapy or other chemotherapeutic procedures were excluded from the study. The time limit set was extended for a period of 5 days during the month of Oct 2006. Each day, around 6 subjects were given intervention and plaque samples were collected between 8.30 a.m. to 9.30 a.m.

Intervention

Thorough oral prophylaxis was done and subjects were asked to refrain from oral hygiene procedures for 24hrs. Subjects were asked to rinse 30ml of freshly prepared pomegranate juice (without sugar) for 2 minutes and the dental plaque material was collected from each subject, before and 15 minutes after rinsing.

Method of plaque collection for measuring baseline data

The subjects were asked to swish their mouth with plain water and the area of plaque collection was isolated by cotton gauze. Mesial half of buccal surfaces of the teeth 14, 16, 25, 27, 34, 36, 45 and 47 and distal half of buccal surfaces of the teeth 15, 17, 24, 26, 35, 37, 44 and 46 were used for plaque collection. Sterile stainless steel Jacquette scalers were used to collect the plaque samples. The plaque samples were collected in pre-weighed sterilized plastic 2ml test tubes & the weight of the plaque was measured using electronic balance after collecting plaque in the pre-weighed plastic tubes. Approximately 1gm of plaque was collected in the tube.

Plaque collection for measuring post exposure data

A second sample of plaque was collected from the subjects after rinsing with pomegranate juice. Distal half of buccal surfaces of the teeth 14,16, 25, 27, 34, 36, 45 & 47 and mesial half of buccal surfaces of the teeth 15, 17, 24, 26, 35, 37, 44 & 46 were used for plaque collection.

Microbiological analysis

Immediately after collection, the plaque samples were sent to the microbiological laboratory of JJM Medical College and Hospital, Davangere and transferred to the bacterial culture media by microbiological technician. 1 mg of plaque sample from each individual before and after rinsing pomegranate juice was inoculated into each of the following media with a sterilized inoculator loop - Mitis Salivarius Agar media for Streptococcal strains and Rogosa SL Agar media for Lactobacilli strains. The inoculated plates were then incubated at 37° C for 48 hours. The number of CFU (Colony Forming Units) were counted using colony counter and expressed as CFU/ mg dry weight of plaque.

Statistical analysis

The difference in the number of CFU/ mg of plaque before and after rinsing pomegranate juice was determined using Wilcoxon's signed rank test. It is the non-parametric analogue to a paired t-test which involves comparisons of differences between measurements. The significance level was set at P<0.05.

Results

The mean number of colony forming units of dental plaque microorganisms (Streptococcus mutans and lactobacilli) before and after rinsing pomegranate juice was 18.9±14.5 and 12.9±10.5 respectively. The percentage reduction in the number of colony forming units was 32%.(P<0.01). There was greater reduction in the lactobacilli strains (46%) compared to streptococci strains (23%). (Table 1)

Table 1

Number of colony forming units (CFU) of Streptococci and lactobacilli before & after pomegranate rinse

Discussion

In the present study, results showed that pomegranate juice is effective against dental plaque microorganisms decreasing the CFU by 32%. After rinsing with 30 ml of pomegranate juice, there was significant reduction in the level of dental plaque microorganisms. This is in agreement with results of Silvana et al, 2006.5

Pomegranates contain polyphenols, tannins, ellagic acid and anthocyanins. These compounds are powerful antioxidants. The red fruits’ anthocyanidins (red pigment) also contribute to the antioxidant activity. Thus pomegranate juice has superior bioactivity compared to its purified polyphenols, which illustrates the chemical synergy of the whole fruit's multiple compounds compared to single, purified, active ingredients.3 According to Ross et al (2001)6, the antiinflammatory effect of pomegranate may be attributed to its considerable immunoregulatory activity over macrophages and T and B lymphocyte subsets.

The antibacterial activity of Punica granatum Linn has been evaluated in previous studies with good results. Trivedi and Kazmi (1979)7, using extracts of fruit barks have observed an antibacterial activity of pomegranate extract against Bacillus anthracis and Vibrio cholerae while Machado et al. (2003)8 showed similar effect against Staphylococcus aureus, in agreement with Prashant and Asha (2001).9 An in vitro study10 showed that Streptococcus sanguis, a bacterial strain present in supragingival plaque was sensitive to different concentrations of pomegranate extract, which demonstrated an inhibitory action similar to that of chlorhexidine.1112 It should be highlighted, however, that in vitro studies do not reproduce exactly the oral conditions.

Due to the lack of clinical trials investigating the effect of pomegranate juice on dental plaque microorganisms, the results of the present study were evaluated by parallel inferences taken off from non specialized articles. Therefore, more controlled trials using different concentrations of pomegranate juice are necessary to verify its action upon supragingival microflora in vivo. Within the limits of the present study, it may be concluded that the pomegranate juice is effective against dental plaque microorganisms. Further research is needed to identify the real benefits of pomegranate as a therapeutic and preventive agent for dental plaque microorganisms, to identify the specific active constituents in pomegranates that could be useful as anti-caries/anti-plaque agents and the safer dose that can be ingested in humans.