The Natural Products Journal
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The Natural Products Journal, 2016, 6, 126-133 ISSN: 2210-3155 eISSN: 2210-3163
Herbal Formulation Against Dental Caries Causing Microorganisms Using Extracts of Stevia Rebaudiana Leaves (A Natural Sweetner) BENTHAM SCIENCE
Ridhi Mehtaa, Ram Kumar Pundirb, Dhanashree Sakharec and Anil K. Sharmaa,* a
Department of Biotechnology, Maharishi Markandeshwar University, Mullana, Ambala, Haryana, India; bDepartment of Biotechnology, Ambala College of Engineering, Kurukshetra University, Ambala, India; cDepartment of Orthodontics, M.M. Institute of Dental Sciences & Research, Mullana (Ambala) Haryana, India-133207 Abstract: Stevia Rebaudiana leaves have functional and sensory properties superior to those of many other high-potency sweeteners. Dental caries is an infectious microbiological disease that affects almost every human. There is a linear correlation of sugar consumption with dental caries in populations worldwide. Present invention was directed to design and develop Stevia based polyherbal formulation with enhanced performance in oral care as Stevia inhibits the growth of dental caries causing microorganisms. In the present study, twelve pure isolates grown on Snyder agar medium and five MTCC strains i.e. Streptococcus mutans, Staphylococcus aureus, Curvularia lunata, Escherichia coli and Bacillus subtilis were screened for antimicrobial activity through agar well diffusion method. Metabolites from plant leaves were extracted using six different solvents and investigated for phytochemical properties. Few cultures were found to be belonging to Staphylococcus species, Bacillus sp., Clostridium sp., Neisseria sp., and Escherichia. coli. On the basis of maximum zone of inhibition produced against cultures, diethyl ether extracts of plant were effective against bacteria and fungi, followed by petroleum ether and chloroform and finally minimum inhibitory concentration and minimum bactericidal concentration of extracts were performed. The phytochemical screening of different extracts of Stevia showed the presence of various secondary metabolites. Herbal formulation prepared in diethyl ether extract displayed significant bioactivity against all twelve isolated cultures and standard cultures. The study holds importance as one needs to be familiarized with alternatives to sugar and type of food products that are available with substitute sweetening agents, as we have the responsibility to reduce the incidence of dental caries by emphasizing the role of sugars in caries formation.
Keywords: Stevia rebaudiana, dental caries, herbal formulation, antimicrobial, phytochemical, snyder media. Received: November 25, 2015
Revised: February 04, 2016
INTRODUCTION Dental caries is a chronic, infectious, transmissible, biobehavioural disease that extends throughout the life span affecting almost every human-being, second only to common cold [1, 2]. Carbohydrates present in food are converted via oral microbial flora to acids that are able to demineralize the tooth structure. If demineralization surpasses saliva and other remineralisation events, then these tissues gradually break down, producing dental caries (cavities, holes in the teeth). Two groups of bacteria i.e. Streptococcus mutans and Lactobacillus are mainly responsible for initiating caries [3]. Acid-producing bacteria utilize fermentable carbohydrates such as sucrose, fructose and glucose resulting in the production of lactic acid [4]. The National Oral Health Survey and Fluoride mapping – studies in 2003 reported that 72.5% of 12 year old children and 75.4% of 15 year old children had dental caries [5]. There are 4 types of caries based on the location where they are seen on the tooth and the causative organisms for each type as given below:
*Address correspondence to this author at the Department of Biotechnology, MMEC, M. M. University, Mullana-Ambala-133207, India; Tel: +918059777758; Fax: +91-1731-274375; E-mail:
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2210-3163/16 $58.00+.00
Accepted: February 15, 2016
1) Pit and Fissure Caries – Streptococcus mutans, Streptococcus sanguis, other Streptococci, Lactobacillus Sp., Actinomyces Sp. 2) Smooth Surface Caries- S. mutans, S. salivarius. 3) Root Surface- filamentous rods, S. mutans, S. sanguis, Streptococcus salivarius. 4) Deep Dentinal Caries- Lactobacillus filamentous rods and S. mutans.
sp,
other
The development of dental plaque is a multistep mechanism. Initial attachment of S. mutans is followed by its gathering and proliferation, resulting in the formation of a sessile, exopolymer-shrouded society known as a biofilm [6]. Replacement of fermentable carbohydrates by xylilitol, saccharin, and aspartame has been resourcefully used for reducing caries [7]. However, long-term exploitation of artificial sweeteners is not recommended because of their apparent role as carcinogen [8, 9]. It is decisive to inspect medicinal plants which show enhanced activity and then can be standardized and formulated. This will facilitate the promotion of extracts from plants like Stevia and rational use of quality, safe and efficacious traditional remedies. The growing research in herbal treatments has led to the discovery of various phytochemicals to limit the virulence of © 2016 Bentham Science Publishers
Herbal Formulation Against Dental Caries Causing Microorganisms
dental caries causing microorganisms Table 1. Because of the difficulty to ensure adequate removal of plaque by mechanical means, there is a great interest in the use of therapeutic mouthwashes to be adjuncts to the mechanical approaches. Mouthwashes have the ability to deliver therapeutic ingredients and benefits to all accessible surfaces of mouth including interproximal surfaces. They also remain effective for extended period of time depending on their composition [10]. In humans, Chinese herbs have been used in the past practices to reduce body stress, promote intestinal health, and enhance body immunity [11]. Use of Stevia in forms like mouth washes will be effective with fewer side effects especially in diabetes patients since they have many medications for their health concern [12]. MATERIALS AND METHODS Maintenance of Clinical Isolates Seven subjects with age ranging from 18 to 53 were selected from various clinics for the study. Biofilm samples were taken from suspected carious teeth to specify the tested sample and to confine the causative cariogenic bacteria as much as possible. Sterile cotton swabs were gently pressed on the portion of teeth with carious lesions and rotated 2-3 times. Then, swab was immediately dipped in the tube with sterile distilled water. These samples were taken in tubes with Snyder test agar media to detect caries activity. Microorganisms that use the dextrose in the medium, acidify it and the pH indicator, bromocresol green, changes color from blue-green to yellow. The tubes were then transported back to laboratory and incubated at 37 oC. The readings were taken at 24, 48, 72 and 96 hrs to detect the colour change in media [13]. The isolates were identified using the standard protocols reported earlier [14-16]. Collection of The Candidate Plant The leaves of Stevia rebaudiana plants were collected from Chaudhary Devi Lal Herbal Nature Park, Chuharpur, Yamunanagar (Haryana) and brought to the laboratory for further study. Extraction Using Various Solvents Stevia plants that reached the maximum growth stage (mature stage before flowering) were harvested by cutting the plant at 5-10 cm from the ground. Leaves were air dried in shade and finely powdered.100g of powdered plant material was dissolved in enough sterilized distilled water, methanol, diethyl ether, acetone, petroleum ether and chloroform to make 500 ml of extract. The mixture was kept undisturbed at room temperature for six days in a sterile flask covered with aluminium foil to avoid evaporation and subjected to filtration through sterilized whatman no. 1 filter paper. After filtration, the extracts were concentrated. Collected residual metabolites were stored in labeled sterile screw-capped bottles at -20 °C. Extracts thus obtained were immediately evaluated for antibacterial and antifungal activities [17].
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Standardization of Test Microorganism The test microorganisms were standardized using 0.5 McFarland standards. McFarland Standards were used as reference to adjust the density of microbial suspensions so that their number would be within a given range. 0.5 McFarland gives approximate cell density of 1.5 x 108 CFU/ml, having absorbance of 0.132 at a wavelength of 600 nm [18]. Antimicrobial Activity of Different Extracts of Stevia Against Isolated Dental And Procured Pathogenic Strains (MTCC) Screening of Stevia extracts prepared using six solvents for antimicrobial activity against isolated dental caries causing microorganisms and five known cultures i.e. Staphyloccocus aureus (MTCC 737), Streptococcus mutans (MTCC 497), Bacillus subtilis (MTCC 441), E. coli (MTCC 41 ) and Curvularia lunata ( MTCC 2030) were collected from Microbial Type Culture Collection (MTCC), IMTECH Chandigarh, done by agar well diffusion method. Minimum Inhibitory Concentration (MIC) Minimum Bactericidal Concentration (MBC)
And
The minimum inhibitory concentration (MIC) is defined as the lowest concentration of the antimicrobial agent that will inhibit the visible growth of a microorganism after overnight incubation [18-20]. Various metabolites of Stevia were optimized for their activities using different concentrations (100 µg/ml, 250 µg/ml, 500 µg/ml and 750 µg/ml). Antibiotic Susceptibility The antibiotic resistance of isolated cultures was assessed using antibiotic discs (Hi media Laboratories Pvt. Ltd. Mumbai, India) on Snyder agar plates. Resistance was assessed against Tetracyclin (30μg), Co-Trimoxazole (25μg), Cloxacillin (1μg), Cefuroxime (30μg), Cephotaxime (30 μg) and Lincomycin (2μg).
Table 1.
List of natural compounds used in herbal formulation against dental caries causing bacteria.
S.No
Materials
Quantity
1
Stevia rebaudiana
40
2
Clove (Eugenia aromatic)
40
3
NaCl
40
4
Neem
40
5
Tulsi
40
6
Seesam oil
40
7
Mint
40
128 The Natural Products Journal, 2016, Vol. 6, No. 2
Mehta et al.
Phytochemical Screening The preliminary phytochemical evaluation of part of powdered leaves of S. rebaudiana was done using standard methods as described by Harbone J.B. [21]. 2.9 Herbal Formulation Using Extracts Including Stevia Stevia, Clove, NaCl, Neem, Tulsi and Mint were powdered and then mixed with sesame oil. 50 grams of this mixture was dissolved in 250 ml of diethyl ether solvent, which showed best activity against the isolates. The filtrate was collected using Whattman No. 1 filter paper. This filtrate was then used to check the antimicrobial activity against the tested cultures. RESULTS Bacteriological Analysis of Dental Caries Samples Isolation and Selection of Dental Caries Bacteria Forty seven bacterial isolates were obtained on Snyder agar plates out of which twelve were selected on the basis of colour change of the medium from green to yellow. SRD10, SRD 15, SRD 16, SRD18, SRD19, SRD 22, SRD 26, SRD28, SRD 30, SRD31 and SRD32. Characterization of Bacterial Isolates Of the twelve isolates, six isolates i.e. SRD2, SRD10, SPM15, SPM16, SPM18 and SRD22 were Gram +ve and cocci shaped except SRD 18 which was rod shaped. Other six isolates i.e. SRD19, SRD26, SRD28, SRD30, SRD31 and SRD32 were Gram –ve and cocci shaped except SRD31 and
Table 2.
SRD32 which were rod shaped Tables 2 and 3. It is speculated that Gram -ve rod shaped might be E. coli while Gram +ve cocci shaped might belong to Staphylococcus spp. Similarly Gram + rod shaped might belong to Clostridium or Bacillus spp. while gram negative cocci might belong to Neisseria spp. Antibiotic Susceptibility Test In the present study, all the isolates were reported to be sensitive to Tetracycline and Co-Trimoxazole. Isolates were found to be resistant to Cloxacillin except two isolates i.e. SRD15, SRD32, which were reported to be sensitive. Isolates namely SRD2, SRD15, SRD16, SRD18, SRD19, and SRD26 were resistant to Cefuroxime while rest of them were sensitive. Isolates such as SRD2, SRD16, SRD26 and SRD28 were resistant to Cephotaxime and rest of the isolates were found to be sensitive. Only one single isolate i.e. SRD10 was resistant to Lincomycin and rest of them were sensitive. Antimicrobial Activity of Stevia Extracts The means of diameters of zones of growth inhibition produced by extracts of Stevia rebaudiana on the isolated as well as standard strains have been shown in Tables 4 and 5 respectively. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration of various extracts of Stevia rebaudiana against all the cultures has been shown in Table 6. Phytochemical Screening The results of the phytochemical screening of the powdered S. rebaudiana have been shown in Table 7.
Biochemical characterization of selected bacterial isolates. (-) represents poor or no activity, (+) represents mild activity and (++) represents strong activity.
S. No.
Strains
Oxidase
Catalase
Urease
Indole Acetic Acid
1
SRD2
_
+
+
+
2
SRD10
_
++
+
+
3
SRD15
_
+
+
_
4
SRD16
_
_
_
+
5
SRD18
_
++
+
+
6
SRD19
_
++
+
_
7
SRD22
_
++
+
+
8
SRD26
_
++
+
+
9
SRD28
_
_
_
+
10
SRD30
_
+
+
_
11
SRD31
_
+
_
+
12
SRD32
_
+
+
+
Herbal Formulation Against Dental Caries Causing Microorganisms
Table 3.
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129
Biochemical tests of selected isolates.
S. No.
Strains
Citrate
Amylase
Fermentation of Carbohydrates Glucose
Sucrose
Lactose
1
SRD2
+
+
G
AG
AG
2
SRD10
+
_
AG
_
_
3
SRD15
+
+
G
AG
A
4
SRD16
+
_
A
A
A
5
SRD18
+
+
AG
AG
AG
6
SRD19
+
+
AG
AG
_
7
SRD22
+
+
AG
AG
AG
8
SRD26
+
+
AG
_
_
9
SRD28
_
_
_
_
_
10
SRD30
_
+
A
A
A
11
SRD31
_
+
A
_
_
12
SRD32
_
_
A
_
_
13.
Control
_
_
_
_
_
In the fermentation of carbohydrates test, gas formation is represented by (G), acid formation by (A) and gas and acid formation both by (AG).
Table 4.
Zone of inhibition results of antimicrobial activity of the extracts of Stevia rebaudiana leaves against isolated cultures.
Inhibition (in mm)
SRD 2
SRD10
SRD 15
SRD 16
SRD 18
SRD 19
SRD 22
SRD 26
SRD 28
SRD30
SRD31
SRD32
Petroleum ether
18
11
NA
20
NA
NA
23
12
NA
NA
11
NA
Methanol
13
11
11
11
17
15
13
11
14
19
18
19
Water
11
18
11
11
16
NA
11
11
18
11
16
13
Diethyl ether
12
13
11
12
20
17
21
27
26
16
20
23
Chloroform
NA
12
NA
24
NA
13
15
19
21
NA
13
11
Acetone
NA
12
11
13
12
NA
11
19
17
NA
NA
11
NA: No Activity.
Table 5.
Zone of inhibition results of antimicrobial activity of the extracts of Stevia rebaudiana leaves against standard cultures.
Zone of Inhibition (in mm)
B.subtilis
E.coli
S.mutans
S.aureus
C.lunata
Petroleum ether
12
13
19
18
NA
Methanol
NA
12
16
15
12
Water
19
19
NA
12
NA
Diethyl ether
18
18
18
21
18
Chloroform
11
16
18
15
14
Acetone
11
12
19
14
12
NA represents no activity.
Herbal Formulation Herbal formulation was prepared in diethyl ether extract which showed best activity against almost all the twelve
isolated cultures and cultures collected from MTCC i.e. four bacteria, S. mutans, S.aureus, E. coli, B.subtilis and one mold C. lunata,.
130 The Natural Products Journal, 2016, Vol. 6, No. 2
Mehta et al.
Fig. (1). Graph showing antimicrobial activity of different extracts of Stevia against dental caries causing pathogens by agar well diffusion method.
DISCUSSION A new interest has been developed in the last decade to seek phytochemicals from plants and their antimicrobial activities [22]. S. rebaudiana leaf extracts demonstrated antibacterial activity against dental caries causing microorganisms. In an experiment conducted by Ghosh et al., it was reported that petroleum ether extracts were found to have highest antimicrobial activity against S. aureus, Enterococcus faecalis and Pseudomonas aeruginosa [23]. While against other three bacterial species Escherichia coli, Proteus mirabilis, Bacillus subtilis, the best activity was displayed by water, ethanol and acetone extracts with an IZD of 11 mm, 10.6 mm and 10.3 mm respectively. Methanol extract gave the highest zone of inhibition against P. aeruginosa while minimum inhibition against S. aureus. Current study demonstrated that amongst all the extracts of Stevia, diethyl ether extract showed highest antimicrobial activity against seven bacterial isolates including SRD18, SRD22, SRD26, SRD28, SRD31 and SRD32 with S. aureus forming an IZD (Zone of diameter) of 20mm, 21mm, 27mm, 26mm, 20mm, 23mm and 21mm respectively. The petroleum ether extract of Stevia was active against SRD2, SRD16, SRD22, S. mutans and S. aureus. Methanolic extracts were active against SRD2, SRD18, SRD19, SRD22, SRD30, SRD31, SRD32, S.mutans and S.aureus and against one fungus Curvularia lunata. Chloroform extract of Stevia was active against SRD16, SRD26, SRD28, E.coli, S. mutans, S.aureus and the fungus C. lunata. Acetone extract was found to be active against SRD26, SRD28, S.mutans, S.aureus, E.coli and Curvularia lunata. Preethi et al. reported that petroleum ether extract gave lowest MIC values against all the bacterial isolates tested [24]. A lowest MIC
value of 0.390 μg/ml was recorded against Bacillus subtilis, Pseudomonas vulgaris and Streptococcus pneumoniae. Methanol extract showed the MIC values in the range of 0.781 to 6.25 μg/ml. In the present study, the MIC values of methanolic extract of Stevia against SRD2, SRD19, SRD18, E.coli and S.aureus were found to be 250 μg/ml, 500 μg/ml, 250 μg/ml, 500 μg/ml and 100 μg/ml respectively. The MIC value of diethyl ether extract of Stevia against SRD 15, SRD 18, SRD 19, SRD 26, SRD 28, SRD 2, SRD 10, S. aureus and E. coli was 100 μg/ml and 500 μg/ml for S. mutans.750 μg/ml for C. lunata, 250 μg/ml for B. subtilis and 500 μg/ml for B. subtilis. The MIC values of petroleum ether extract of Stevia against SRD 2 was 750 μg/ml, SRD 18 μg/ml and SRD 22 μg/ml was 250 μg/ml, 500 μg/ml for SRD 16 and 100 μg/ml for S. aureus. The MIC value of acetone extract of Stevia against S. aureus was reported to be 250 μg/ml. On a similar note, the MIC value of chloroform extract of Stevia against SRD 16, SRD 26 and SRD 28 were found to be 100 μg/ml, 250 μg/ml and 100 μg/ml respectively. Also MBC values of methanolic extract of Stevia against SRD2, SRD18, E.coli were found to be more than 750 μg/ml while MBC value of 100 μg/ml was observed for S. aureus. Similarly MBC values of petroleum ether extract of Stevia against SRD2 and S. aureus were found to be more than 750 μg/ml, while for SRD 18 it was found to be 500 μg/ml. On a similar note, MBC values of diethyl ether extract of Stevia against SRD28 S. aureus, C. lunata and E. coli were found to be more than 750 μg/ml followed by 750 μg/ml for SRD32, 500 μg/ml for SRD 10, SRD19, SRD18 and S. mutans. However for SRD2 and B. subtilis, the MBC value of 250 μg/ml was observed while for SRD15 it was found to be 100 μg/ml. Previous studies conducted by Preethi et al. demonstrated the
Herbal Formulation Against Dental Caries Causing Microorganisms
Table 6.
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131
Minimum inhibitory concentration and minimum bactericidal/ fungicidal concentration of Stevia metabolites obtained from different solvents.
S. No.
Cultures
Stevia Metabolites from Solvents
MIC (in µg/ml)
MBC (in µg/ml)
1
SRD15
Diethyl ether
100
100
2
SRD2
Methanol
250
>750
3
SRD19
Methanol
500
750
4
E.coli
Methanol
250
>750
5
SRD18
Methanol
500
>750
6
SRD19
Diethyl ether
100
500
7
S. aureus
Petroleum ether
100
>750
8
SRD26
Diethyl ether
100
500
9
SRD28
Diethyl ether
100
>750
10
SRD2
Petroleum ether
750
>750
11
S. aureus
Methanol
100
100
12
SRD2
Diethyl ether
100
250
13
SRD10
Diethyl ether
100
500
14
S. aureus
Acetone
250
250
15
S. aureus
Diethyl ether
100
>750
16
S. mutans
Diethyl ether
500
500
17
C. lunata
Diethyl ether
750
>750
18
SRD18
Petroleum ether
250
500
19
SRD16
Petroleum ether
500
750
20
SRD22
Petroleum ether
250
750
21
SRD18
Diethyl ether
250
500
22
SRD16
Chloroform
100
>750
23
SRD26
Chloroform
250
500
24
E. coli
Diethyl ether
100
>750
25
SRD28
Chloroform
100
750
26
SRD10
Water
100
750
27
B. subtilis
Diethyl ether
250
250
28
SRD32
Diethyl ether
500
750
132 The Natural Products Journal, 2016, Vol. 6, No. 2
Table 7.
Results of The Phytochemical Screening Of Powdered S. rebaudiana.
S.No 1
2
3
4
5
Chemical Test
8
Methanol
Diethyl ether
Petroleum ether
Chloroform
Water
a.
Molisch Test
+
+
_
+
+
+
b.
Fehilings Test
+
+
_
+
_
+
c.
Benedicts Test
+
+
_
+
+
+
a.
Biuret Test
+
+
+
+
_
+
b.
Millions Test
+
+
+
_
_
+
a.
Drogendroffs Test
_
+
+
+
_
+
b.
Mayers Test
_
+
+
+
_
+
Protein:
Alkaloid:
Flavanoids: a.
Ammonia Test
+
+
+
+
_
_
b.
Sodium Hydroxide
+
+
_
+
_
_
c.
Conc. H2SO 4
_
+
_
+
_
_
_
+
+
+
+
+
Steroids: Salkowski Test
Test for saponins: a.
7.
Acetone
Carbohydrate:
a. 6
Mehta et al.
Foam formation test
Tannins and phenolic compounds: a.
Ferric chloride Test
+
+
_
_
_
+
b.
Lead acetate test
+
+
_
_
_
+
_
+
+
+
+
+
Glucosides: a.
Killer-Killani Test
+ = present; - = absent.
preliminary phytochemical analysis of crude extracts of leaves and flowers of S. rebaudiana which exposed the presence of various phytochemicals such as high concentration of alkaloids in chloroform and hexane leaf extracts and a moderate amount of flavonoids found in methanol, chloroform and petroleum ether flower and ethanol leaf extracts [24]. Higher concentrations of phenols were also found in the ethanol leaf extract. Similarly higher concentrations of steroids were reported in ethanol and hexane leaf extracts. Ethanol leaf extract has been reported to contain high concentrations of tannins when compared with the other extracts. In the present study, carbohydrates were found to be present in all the extracts of Stevia except in the diethyl ether extract. Proteins were found to be present in all the extracts except the chloroform extract of Stevia. On the other hand, Alkaloids were present in all the extracts except acetone and chloroform extract of Stevia. Flavanoids were found to be present in all the extracts except chloroform and aqueous extract of Stevia. Steriods were only
present in petroleum ether and methanol extracts while saponins were present in all the extracts except the acetone extract of Stevia. Tannins and phenolic compounds were reportedly present in acetone, methanol and water extracts of Stevia. Glucosides were present in all the extracts except the acetone extract of Stevia. Inadequate literature is available for herbal formulation of Stevia extract against dental caries causing microorganisms. A number of toothpastes are currently being used as credible means of oral hygiene products yet none of these fulfill the complete requirements of oral healthcare. Earlier studies by Mohire et al. reported that herbal extracts possess satisfactory antimicrobial activity against most of the dental pathogens [25]. Authors in the present study tried to constitute an in vitro herbal formulation consisting of neem, clove, sodium chloride, mint, tulsi, seesam oil and Stevia extracts using various solvents which was further evaluated for the antimicrobial activity against dental caries causing microorganisms. Among the various extracts of Stevia, herbal formulation
Herbal Formulation Against Dental Caries Causing Microorganisms
using diethyl ether extract (greenish translucent in appearance) showed promising results against almost all the isolates and standard cultures comprising of B. subtilis, C. lunata, S. mutans, S.aureus and E. coli.
The Natural Products Journal, 2016, Vol. 6, No. 2 [4] [5]
[6]
CONCLUSION Out of 47 isolated bacterial cultures, twelve pure isolates grown on Snyder agar medium were found to be dental caries causing microorganisms. Isolates such as SRD2, SRD10, SRD15, SRD16, SRD18, SRD19, SRD22, SRD26, SRD28, SRD30, SRD31 and SRD32 were considered to be dental caries causing strains. Few cultures were found to be belonging to Staphylococcus species, Bacillus sp., Clostridium sp., Neisseria sp., Escherichia.coli. Stevia metabolites extracted using various solvents determined the antimicrobial activity against twelve isolates and five dental caries causing cultures, obtained from MTCC. Diethyl ether extracts were found to be most effective displaying significant activity against bacteria and fungi, followed by petroleum ether and chloroform. Phytochemical analysis further revealed that Stevia plants are a good source of carbohydrates, proteins, alkaloids, flavonoids, saponins and glucosides. The formulation prepared using diethyl ether as the solvent system was found to be most active against almost all the twelve isolates and S. mutans, S.aureus, C. lunata, E. coli and B.subtilis and hence can be recommended to be added in the mouth wash. Obviously, further research is required to establish pharmacological implications of extracts derived from this plant. Therefore, Stevia leaf extracts pose as an ideal applicant for future investigations to get insight about their uses for pharmaceutical and natural plant-based products. CONFLICT OF INTEREST The authors confirm that this article has no conflict of interest.
[7] [8]
[9] [10]
[11]
[12]
[13] [14] [15]
[16] [17]
[18] [19]
ACKNOWLEDGEMENTS Authors would like to acknowledge M.M. University Mullana (Ambala) for providing all the requisite facilities for the said work.
[20]
[21]
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