Effect of pomegranate and aloe vera extract on

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ORIGINAL RESEARCH

Effect of pomegranate and aloe vera extract on streptococcus mutans: An in vitro study Priya Subramaniam, Shilpy Dwivedi, Eswara Uma, Girish Babu K. L. Department of Pedodontics, The Oxford Dental College, Hospital and Research Centre, Bommanahalli, Hosur Road, Bangalore, India

A B S T R A C T Introduction: The aim of the present in vitro study was to evaluate and compare the antibacterial effect of pomegranate and aloe vera extracts on Streptococcus mutans. Materials and Methods: Hydroalcoholic extracts of pulp from both Punica granatum (pomegranate) and Aloe barbadensis miller (aloe vera) were prepared to concentrations of 5, 25, 50 and 100%. Pure sorbitol powder dissolved in distilled water was taken as the negative control. Streptococcus mutans (S mutans) was isolated from saliva by inoculation on to Mitus Salivarius Bacitracin (MSB) agar, which was then streaked onto agar plates containing Brain Heart Infusion. In each petridish, wells were prepared and using a sterile micropipette, 125l of the specific concentration of the extract (pomegranate/ aloe vera/ sorbitol) was deposited in each well. This was done in triplicate for each concentration of the extracts. The effect of different concentrations of the extracts on S mutans was observed and the data was subjected to statistical analysis. Results: Pomegranate extract showed significantly higher inhibitory effect on S mutans at all concentrations (P≤0.05). On comparison of all three extracts at different concentrations, a significant difference (P≤0.05) was observed only at 50 and 100% concentrations. The inhibitory effect of pomegranate extract was significantly different when compared to aloe vera and sorbitol extracts. (P≤0.01). Discussion: Pomegranate extract has a significant antibacterial effect on S mutans at all concentrations. Keywords: Aloe vera, dental caries, pomegranate, streptococcus mutans

Introduction In recent times, there has been an explosion of interest shown in the use of plants for the treatment of various diseases. Research on the medicinal and therapeutic value of these natural products has also increased. People are aware of the ill-effects associated with over prescription of antibiotics. There is also concern about increasing resistance developed by micro-organisms against antimicrobials. The use of plants and their derivatives which possess preventive and therapeutic effects could contribute to oral health. Punica granatum Linn, commonly known as pomegranate, is the Access this article online Quick Response Code: Website: www.dentalhypotheses.com DOI: 10.4103/2155-8213.103920

predominant member of two species comprising the Punicaceae family. The pomegranate is a distinctive fruit borne from a small long-living tree cultivated throughout the Mediterranean region, as far north as the Himalayas, in Southeast Asia, and in California and Arizona in the United States.[1] The ripe fruit can be up to five inches wide with a deep red, leathery skin, is grenade-shaped, and crowned by the pointed calyx. The fruit contains many seeds (arils) separated by white, membranous pericarp, and each seed is surrounded by small amounts of red pulp (mesocarp). The plant is known to have both medicinal and nutritional benefits. It is used in several systems of medicine for a variety of ailments. In ayurvedic medicine, the pomegranate is considered “a pharmacy unto itself” and as a remedy for diabetes in the Unani system of medicine practised in the Middle East and India.[1,2] Various components of the plant such as the leaves, flower, roots and bark; and extracts of the fruit including the pericarp, seed oil and juice have been

Corresponding Author: Dr. Priya Subramaniam, Department of Pediatric Dentistry, The Oxford Dental College, Hospital and Research Centre, Bommanahalli, Hosur Road, Bangalore-560 068, India. E-mail: [email protected] Jul-Sep 2012 / Vol 3 | Issue 3

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used.[3] According to Lansky, the pomegranate is an extraordinary, albeit mysterious (and messy), fruit with a complete medicinal power contained within its juice, peel and seeds.[4] Pomegranate is a potent antioxidant with anticarcinogenic and anti-inflammatory properties. The potential therapeutic properties of pomegranate are wide ranging and include treatment of cancer,[5] cardiovascular disease, [6,7] diabetes, [8] and dental conditions. [1] The biochemical constituents that make it therapeutically beneficial are ellagic acid, ellagitannins (including punicalinand punicalagin), punicic acid, flavonoids, anthocyanidins, anthocyanins, estrogenic flavonols, flavonols and flavones. A low concentration of pomegranate extract was found to inhibit Staphylococcal enterotoxin A production. This implicatestheir potential antibacterial therapeutics with the added ability to inhibit enterotoxins. [9] Pomegranate byproducts and punicalagins inhibited the growth of human gut microflora like clostridia and Staphyloccocusaureus.[10] Aloe barbadensis Miller (family Lilaceae) commonly known as aloe vera is a perennial succulent xerophyte, a cactus-like plant that is easily grown in hot and dry climates and widely distributed in Asia, Africa and other tropical areas. It consists of two different parts, each of which produces substances with completely different compositions and therapeutic properties. The outer green rind includes the vascular bundles, and the inner colourless parenchyma contains the aloe gel. The pericyclic tubules, which are present just beneath the outer green rind of the leaves cells, produce a bitter yellow exudate (latex) that has laxative properties.

have been attributed to the polysaccharides (mainly acemannan) in the inner leaf parenchymatous tissue.[11,14] The chemical composition of aloe veraleaves is highly complex and includes vitamins B1, B2, B6, C and betacarotene; enzymes such as amylase, alkaline phosphatase, superoxide dismutase, lactic dehydrogenase, and lipase; saccharides (mannose, glucose);inorganic compounds (calcium, chlorine, copper, potassium, zinc, chromium) an danthraquinones such as barbaloin and emodin.[15] Yamaguchi et al, reported the presence of aluminium, boron, barium, calcium, iron, magnesium sodium, phosphorus, silicon and strontium in aloe vera gel.[16] Aloe vera has been used as a moisturizing agent and for the treatment of skin abrasions, minor burns and irritations.[17-19] Aloe vera has been included in many herbal products for oral use, including dentifrices and mouth rinses. S mutansis the chief oral pathogen in the etiology of dental caries. Due to their antimicrobial properties, pomegranate andaloe veramay be useful in preventing dental caries by acting on the dental pathogen, S mutans. Hence, this preliminary study was undertaken to assess the effect of aloe vera and pomegranate extracts against S mutans.

Materials and Methods Prior to carrying out this in vitro study, the study protocol was presented to the review committee of the institution, following which approval and permission was granted by the institutional review board.

Technically, the term “pulp” or “parenchyma tissue” refers to the intact fleshy inner part of the leaf including the cell walls and organelles; while the “gel” or “mucilage” refers to the viscous clear liquid within the parenchyma cells.[11] This gel is a clear, soft, moist, slippery and thin tasteless jelly- like material and is removed from the leaves after separation from the inner cellular debris. The gel consists of 99.5% water[12] and the remaining 0.5-1% solid material consists of a range of compounds including water soluble and fat soluble vitamins, minerals, enzymes, polysaccharides, phenolic compounds and organic acids.[13]

Pulp from both Pomegranate and Aloe verawas used. Fresh ripe pomegranate fruits were obtained from the local market in Bangalore city, India. Extracts were prepared at Bhat Biotech Private Limited, an ISO 9001 certified laboratory in Bangalore, India. After washing, the peel/ rind (pericarp) was removed and the pulp (mesocarp) of the fruit was separated from the seeds (endocarp) by hand with the help of a mortar and pestle. Leaves were taken from aloe vera plants and its pulp was extracted by hand filleting method, as described by Ramachandra and Rao.[20] Pure sorbitol powder was obtained from Gulshan Polyols Limited, Mumbai, India. It was dissolved in distilled water and was taken as the negative control.

The foliage, extract and resin of the plant are known to present antimicrobial, anti-inflammatory, anticancer and wound healing properties. Many of the medicinal benefits

The pulp of both aloe vera and pomegranate were separately mixed with 80% concentration of ethanol obtained from Microlabs Limited, Bangalore. The pulp-

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ethanol mix was then centrifuged at 3000rpm for 10 minutes and the supernatant collected was allowed to evaporate over a dry oven. The gelatinous extract thus prepared was weighed and using distilled water, serial dilutions of 5mg/95ml, 25mg/75ml, 50mg/50ml and 100mg (w/v) were made in order to obtain 5, 25, 50 and 100% concentrations, respectively. S mutans (ATCC: 25175) was first isolated from saliva by inoculation on Mitis-Salivarius-Bacitracin agar. The inoculums were adjusted to 0.5 McFarland turbidity standards, according to guidelines given by National Committee on Clinical Laboratory Standards.[21] It was then streaked onto agar plates containing Brain Heart Infusion agar. Five wells were prepared on each agar plate using a sterile stainless-steel template. Using a sterile micropipette 125l of specific concentration of each extract (pomegranate/aloe vera/ sorbitol) was dispensed into each well. This was done in triplicate for every concentration so as to overcome any inadvertent technical errors. Considering standardised difference as 1.07 with a two-sided 5% significance level and a power of 80%, a sample size of 15 measurements for each concentration was necessary. Following 48 hrs of incubation at 37°C, zones of inhibition (that is, locations where no growth of bacteria was present) were examined around the wells. These appeared as a clear, circular halo surrounding the wells. Diameters of the inhibition zones were measured using a Hi Antibiotic Zone Scale (Hi Media Laboratories, Mumbai, India). The mean diameter of the well’s measurements (in mm.) represented the inhibition value. All the measurements were carried out by a single examiner who was blinded about the various test groups and concentrations. The examiner repeated the

Figure 1: Zone of inhibition seen with pomegranate extract


measurements for five wells in each group. (15 wells in three groups) Cohen’s kappa statistic was used to measure the reproducibility by comparing with the original measurements. A kappa value of 0.88 showed that the intra-examiner agreement was almost perfect. Statistical analysis was carried out by a statistician who was blinded to the data. The statistical software namely, Statistical Package for Social Sciences (SPSS) Version 15.0 was used. The Confidence Interval was taken at CI = 95%. Student ‘t’ test was used to determine the inhibitory effect of the individual extracts. Chi square test was used to compare all three extracts and the independent t test was applied to compare pomegranate with aloe vera and sorbitol. P-values 0.05 were considered statistically significant and P-values 0.01 as highly significant.

Results Pomegranate extract showed a highly significant inhibitory effect against S mutans at all concentrations (P≤ 0.05) [Figure 1]. A significant inhibitory effect was seen with extracts of aloevera [Figure 2] and sorbitol at only 100% concentration [Table 1]. On comparison of all three extracts at different concentrations, a significant difference was observed only at 50% and 100% concentrations (P≤0.05) [Table 2]. The inhibitory effect of pomegranate extract was significantly different from that of aloe vera and sorbitol extracts (P≤0.01) [Table 3].

Discussion There has been a recent resurgence of interest in the use of natural products and their effect on oral health. Their potential antibacterial effect could be of use in the prevention and treatment of oral diseases.

Figure 2: Zone of inhibition seen with aloe vera extract

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Table 1: Zone of inhibition exhibited extracts at each concentration Extract Pomegranate

Aloe vera

Sorbitol

Concentration (%)

Number

Diameter (mm) Mean ± SD

CIat 95%

100 50 25 5 100 50 25 5 100 50 25 5

15 15 15 15 15 15 15 15 15 15 15 15

15.33 ± 0.58 11.00 ± 1.00 9.33 ± 1.15 3.00 ± 1.00 0.83 ± 0.15 No inhibition No inhibition No inhibition 0.23 ± 0.06 No inhibition No inhibition No inhibition

15.04 to 15.62 10.49 to 11.51 8.74 to 9.92 2.49 to 3.51 0.75 to 0.91

Table 2: Comparison of extracts at each concentration Extract Pomegranate Aloe vera Sorbitol Pomegranate Aloe vera Sorbitol Pomegranate Aloe vera Sorbitol Pomegranate Aloe vera Sorbitol

Concentration (%) 100

P value 0.011*

50

0.011*

25

0.061

5

0.061

*P ≤ 0.05 significant

Table 3: Comparison of pomegranate with aloe vera and sorbitol Concentration (%) 100 50 25 5

Aloe vera 0.000* 0.000* 0.000* 0.000*

Sorbitol 0.000* 0.000* 0.000* 0.007*

*P ≤ 0.01 highly significant

Dental caries continues to be the most common oral disease in infants and children, and its prevalence continues to rise in the developing countries. Two wellknown and commonly available plants; pomegranate and aloe vera were selected for their inhibitory effect on S mutans, as it is strongly associated with the initiation of dental caries. They were compared with sorbitol, a poly alcohol that is fermented by S mutans slowly and is considered to have a low cariogenic potential.[22] The pomegranate fruit has been consumed for centuries without adverse effects or toxicity. The antimicrobial activity of Punicagranatum Linn has been widely investigated. The rind or peel, juice and seedsof the fruit have been studied.[23-26] Human trials examining 102

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0.19 to 0.27

P value 0.000* 0.003* 0.005* 0.035* 0.011* 0.021* -

the antibacterial properties of pomegranate extracts have primarily focused on oral bacteria.[26-30] In comparison to petroleum ether, chloroform, methanol and water extracts of Punicagranatum, the methanolic extract was found to be most effective against all tested microorganisms.[31] In an in vivo study, rinsing with hydroalocoholic extract of pomegranate resulted in reduction of micro-organisms of dental plaque[27] Methanol extract of pomegranate peel at concentrations of 8and 12mg/ml was effective against L acidophilus, S mutans and S salivarius.[24] The use of mouth rinses that contain extracts of pomegranate have demonstrated reduction in plaque microflora. They were seen to bring about changes in salivary measures relevant to oral health. The changes were reduced total protein, reduced activities of aspartate aminotransferase (an indicator of cell injury), reduced alpha-glucosidase activity (a sucrose degrading enzyme), increased activities of the antioxidant enzyme ceruloplasmin (which could give better protection against oral oxidant stress) and increased radical scavenging.[32] Topical applications of pomegranate preparations have been found to be particularly effective in controlling oral inflammation, as well as bacterial and fungal counts in periodontal disease and Candida associated denture-stomatitis.[27,28,30] The ellagitannin, punicalagin, was thought to be responsible for pomegranate’s antibacterial activity.[27] In an in vitro study on Candida albicans using extracts of different natural products, Punicagranatum Linn showed the highest anti-fungal activity.[23] Studies have shown the specific antimicrobial action of Punicagranatum Linn on dental biofilm bacteria, i.e., disturbance of polyglycan synthesis, and thus acting on the adherence mechanisms of these organisms to the dental surface. Jul-Sep 2012 / Vol 3 | Issue 3


Streptococcus sanguis was found to be sensitive to different concentrations of pomegranate extract.[33] The synergistic action of the pomegranate constituents appears to be superior to that of a single constituent. The largest components of the fruit extract are tannins and polyphenolics.[34] There is a growing interest in the use of tannins in the prevention of dental caries. The effect of tannins on microbial metabolism can be measured by their action on membranes. They can cross the cell wall that is composed of several proteins and polysaccharides, and bind to its surface. They can precipitate proteins and may also suppress many enzymes such as glucosyl transferase. The seeds of the fruit contain a lower concentration of tannins compared to the outer carp. The antibacterial agents present in pomegranate- the hydrolysable tanninsform complexes of high molecular weight with soluble proteins, increase bacterial lysis and interfere with bacterial adherence.[35] Vasconcelos et al, suggested that pomegranate might be used in the control of adherence of different microorganisms in the oral cavity.[36] Pomegranate is a potent antioxidant. Ellagic acid has been detected in human plasma following consumption of pomegranate juice.[37] No toxic effects of the antioxidant polyphenol punicalagin, abundant in pomegranate juice, have been reported.[38] There are more than 300 varieties of the aloe plant but the aloe barbadensis variety exhibits the best medicinal properties.[39] The polysaccharides found in aloe gel are not stable, especially under stress conditions such as heat, the presence of acid and enzymatic activities. A standardized and consistent production process is vital for preserving the natural biological activity of the aloe gel.[40] Aloe gel will lose its complete potency on exposure to sunlight for more than two hours, as it is easily oxidised. There are conflicting results with regard to the therapeutic benefits of aloe vera gel. This could be due to the use of plants from different geographical locations with variations in their chemical composition and also because of different isolation techniques that were used to extract compounds from the aloe leaf pulp.[15] Previous researchers have used aloe vera based oral products;[39,41,42] whereas in the present study only the aloe vera extract was used. Unfortunately, because of improper processing procedures, many of these so-called aloe products contain, very little or virtually Jul-Sep 2012 / Vol 3 | Issue 3

no active ingredients, namely, mucopolysaccharides. In order to avoid contamination of internal fillet with the yellow sap, the traditional hand-filleting method of processing Aloe leaves was developed.[20] The hand filleting method was used to separate the parenchyma from the inner portion of the aloe leaves in order to obtain aloe vera gel. Only by hand filleting the leaf it is able to cleanly separate the gel from the rind. This method separates the gel from the bitter yellow exudate present in the outer green rind of the leaves. This is of critical concern because the highest concentration of potentially beneficial aloe constituents are found in this mucilage, as this layer represents the constituents synthesized by the vascular bundle cells empowered by energy developed in the green (chlorophyll- containing) rind cells through sun induced photosynthesis.[20] An in vitro study demonstrated that aloe vera tooth gel was as effective as commercially popular toothpastes in controlling growth of S mutans, S sanguis, Aviscosus and C albicans. Aloe vera tooth gel showed enhanced antibacterial effect against S mitis.[39] Studies have reported significant reduction of gingivitis and plaque accumulation after the use of a mouth rinse and dentifrice containing aloe vera.[41,42] The antimicrobial effects of aloe vera has been attributed to the plant’s anthraquinones: aloe emodin, aloetic acid, aloin, anthracine, anthranol, barbaloin, chrysophanic acid, ethereal oil, ester of cinnamonic acid, isobarbaloin and resistannol.[43] In a randomized controlled clinical trial, an aloe vera containing toothpaste showed significant improvement in gingival and plaque index scores as well as in microbiological counts.[44] However, the precise amount of aloe vera present in these various products has not been given. The percentage of a therapeutic agent in a dentifrice usually ranges from 0.4% to 1.0% of the total formulation.[45] So the presence of other anti-plaque agents in a dentifrice can mask the effect of aloe vera per se. Other agents such as sodium lauryl sulphate, menthol and fluoride can promote a moderate anti-plaque effect.[46,47] Hence, Villalabos et al,[42] used higher concentration of aloe vera (50%) and used a mouth rinse containing only aloe vera as the active ingredient, favouring its action without interference of other components. Fani and Kohanteb concluded that Aloe vera gel at optimum concentration could be used as an antiseptic for prevention of dental caries and periodontal diseases.[48] In our study also, aloe vera was effective only at 100% concentration, which is in accordance to an earlier report.[41]

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A gel containing 10% Punicagranatum Linn extract was not efficient in preventing supragingival dental plaque formation and gingivitis.[49] In our study pomegranate was highly effective against S mutans at all concentrations. However, an earlier study found methanolic extract of pomegranate peel to be effective against S. mutans, L. acidophilus and S. salivarius only at concentration of 8 and 12 mg/ml.[24] A randomized clinical study on adults concluded that a gel containing pomegranate extract was efficient in treating gingivitis when used along with mechanical cleaning in controlling plaque and gingivitis.[50] In the present study, pomegranate showed higher inhibitory effect against S mutans in comparison to both aloe vera and sorbitol. Although sorbitol was used as the negative control, it did not support the growth of S mutans. Rather it showed inhibition at 100% concentration, which was similar to that of aloe vera. In vitro studies do not reproduce the exact oral conditions. This was only a preliminary in vitro study that demonstrated the antimicrobial effect of pomegranate on S mutans. Therefore, more clinical trials using different concentrations of Punica granatum Linn extract are necessary to verify its action upon other oral microflora in vivo.

Conclusion Since pomegranate pulp extract showed inhibition of S mutans growth even at low concentrations, it could be incorporated in tooth wipes for infants or in an alcoholfree mouth rinse. Further research will be needed to identify the real benefits of the active ingredients of pomegranate as a therapeutic and preventive agent for dental caries and gingivitis, in addition to its common use in popular medicine. Long-term clinical trials can be done using more isolates from clinical samples. However, the potential use of pomegranate as a convenient alternative to antimicrobial products, especially for children should be considered.

Acknowledgement The authors acknowledge the laboratory services of Bhat Biotech Private Limited, Bangalore.

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Cite this article as: Subramaniam P, Dwivedi S, Uma E, Girish Babu KL. Effect of pomegranate and aloe vera extract on streptococcus mutans: An in vitro study. Dent Hypotheses 2012;3:99-105. Source of Support: Nil, Conflict of Interest: No.

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