INTRODUCTION:

Herbal medicine is considered in folk medicine practice which depend on the usage of plant and plant products derived from plant parts like leaves, stem, bark, seed fruit, flower and root. Herbal medicines are widely used for the treatment and prevention of various diseases due to the presence of highly active pharmacological compounds. Synthetic drugs are increasing the concern about safety, toxicity and adverse effects. However, the herbal remedies believed to be harmless. We use today plant based medicine for various ailments [1]. The medicinal values of these plants were considered based on the presence of bioactive secondary metabolites like alkaloids, flavonoids and phenolic compounds. These compounds widely screened and used in discovery of new drugs from plants [2, 3]. The pure compounds have been isolated through the process of preliminary phytochemical screening. The screened compounds were characterized for their antimicrobial activity against human pathogenic microorganisms such as bacteria and fungi [4].

Azolla pinnata is otherwise known as water fern or floating fern, mostly grown in ponds and backwaters of rivers and its family is Salviniaceae. Azolla pinnata plant is used as a green manure in paddy fields and it adds nitrogen and organic matter i.e. humus to soil [5]. A. pinnata is a small, evergreen forest and it is symbiotic nitrogen fixing plant [6]. Azolla contains the flavonoids 3-Deoxyanthocyanins [7, 8]. The crude extract of A. pinnata contains phyto-constituents such as tannins, phenolic contents and flavonoids which are responsible for antioxidant activity [9]. The aquatic fern Azolla has wide applications such as fertilizer, animal feed, human feed, anticarries, antioxidant, antiviral and antimicrobial activity etc.

Tooth decay is one of the most important infections of the people of all age groups and remains a major problem worldwide [10]. Among cariogenic in particularly Streptococcus mutans is a primary cause of dental carries, it has adhere to tooth surfaces and forms biofilm. S. mutans cause dental caries gradually leads to tooth loss [11]. So many antibiotics were available for treatment of tooth loss, but they have some drawbacks such side effects and high cost. To overcome these drawbacks plant medicinal system was utilized for treatment of dental caries [12, 13]. The present study was conducted to determine antidental carries activity of aqueous, ethanol and chloroform extract of Azolla pinnata.

MATERIALS AND METHODS:

Collection and cultivation of Azolla pinnata

The Azolla pinnata fern samples were collected from Vandhavasi. Azolla was grown outdoor in cemented tanks at the Department of Microbiology, Sri Akilandeswari women’s college, Vandavasi.

Preparation of extract:

The fresh Azolla pinnata plant was collected from the tanks and were brought to the laboratory and cleaned of all the debris. The Azolla ferns were washed using double distilled water and were air dried in shade for three weeks. The dried material was then grinded and made into a powder. The dried and powdered (100 g) material was extracted with water (aqueous), ethanol (100 ml) and chloroform (100 ml) for 24 h at a temperature not exceeding the boiling point of the solvent. The obtained extracts was centrifuged at 10,000 rpm for 15 min and filtered through whatman No.1 filter paper. The supernatant was evaporated and concentrated extracts was collected and stored for further studies.

Phytochemical screening:

The phytochemical constituents of aqueous, ethanol and chloroform was characterized by following analysis methods such as steroids, alkaloids, flavonoids, terpenoids, tannins, saponins, quinines, glycosides, protein, xanthoproteins, sugars and carbohydrates [14].

Antimicrobial activity assay:

Isolation of pathogenic bacteria from clinical sample:

Pus samples were used to isolate the bacterium. The pus samples were collected aseptically and then streaked on Nutrient agar and sub cultured maintained in agar slant.

Morphological Characterization:

Morphological characterizations were done by microscopic method. The observed structure was compared with Bergey’s Manual of Determinative Bacteriology, Ninth edition [15] and the organism was identified based on standard microbiological methods. Identification of isolates were done based on colony morphology, motility, catalase test, oxidase test, coagulase test and biochemical tests like Tripal sugar iron agar, Hydrogen sulfide test, Carbohydrate fermentation test, Phenylalanine deaminase test, Methyl red test, Nitrate reduction test, Urease test, Voges proskauer, Citrate utilization test, Indole test and growth on Blood agar.

Antibacterial activity by using Agar well diffusion method:

Qualitative assay of antibacterial activity of plant extracts was performed by standard methodology i.e. agar well diffusion method. In this analysis, fresh 24 h isolated dental pathogenic organisms were spread on Muller Hinton agar. About 5 wells with 6 mm diameter were made in agar plates using gel puncture. Different concentrations of three diluted extracts (25, 50, 75 and 100 µg) were added into the well and the plates were incubated at 37°C for 24 hr. The bactericidal activity of aqueous, chloroform and ethanol was assayed by measuring the diameter of inhibition zone formed around the well.

RESULTS AND DISCUSSION:

Phytochemical screening of A. pinnata:

The crude extracts of A. pinnata showed diverse phytoprofiles with reference to solvents of the plant extracts. The aqueous extracts of A. pinnata demonstrated maximum occurrence of phytoconstituents, followed by chloroform and ethanol (Table 1). The phenol is present in all the tested extracts of A. pinnata. The tannin showed its presence in the aqueous, and ethanol extracts and saponin in chloroform extracts of A. pinnata. The carboxylic acid is present in ethanol extracts of A. pinnata. The flavonoid is present only in aqueous extracts of A. pinnata. The carbohydrate exists in aqueous and chloroform extracts of A. pinnata (Table 1). Chloroform extracts showed the occurrence of carbohydrates in the crude extracts of the A. pinnata. Steroids are present in chloroform extracts which responsible for antioxidant activities [16]. Several reports show that tannins may have potential value as cytotoxic and antineoplastic agents [17]. Tannin is almost present in aqueous extracts of A. pinnata.

FT-IR analysis:

The ethanol extract of A. pinnata showed a strong characteristic absorption bands at 3417 cm-1 for phenols and 2849 cm-1 corresponding to C-H Stretch of C=O aldehydes. Weak bands were observed at 1384 and 1321 cm-1 indicates presence of N=O Bend nitro and C–N stretch aromatic amines respectively (Figure 1 and Table 2)

Isolation and identification of dental pathogens:

Cultural and biochemical characteristics:

The isolated microorganisms were tested by various cultural andbiochemical test for identification (Table 3). Based on the morphological and cultural and biochemical characters the isolated organisms were identified as Streptococcus mutans 1, Streptococcus mutans 2 Streptococcus mutans 3 and Streptococcus mutans 4.

Antimicrobial activity of A. pinnata extracts:

In the present investigation, the inhibitory effect of different extracts viz. Aqueous, Ethanol, Chloroform from A. pinnata were evaluated against dental decaying bacterial strains. The antimicrobial activity was determined using agar well diffusion standard method. The activity was quantitatively assessed on the basis of formation of inhibition zone around the well.

Measurement of antimicrobial activity using Agar well diffusion Method:

The antimicrobial potential of the three experimental extracts was evaluated according to their zone of inhibition against dental pathogens and the results (zone of inhibition) were compared with the activity of the standard Ampicillin (1.0 mg/disc). The results revealed that all the extracts are potent antimicrobial activity against all the isolated dental microorganisms studied. Among the different solvents extracts studied ethanol and aqueous showed high degree of inhibition followed by chloroform extract.

For all the tested microorganisms Ethanol and aqueous showed maximum antibacterial activity. In Ethanol extract maximum inhibition zone diameter was obtained in Streptococcus. mutans 2 and in S. mutans 3 with diameter 24.3±1.40 mm 22.5±1.56 mm, respectively. Similarly, aqueous extract showed maximum inhibition zone with diameter of 20.5±1.02 mm in S. mutans 1 and 17.6±0.93 mm for S. mutans 4. The chloroform extract (12±0.54 – 22.6±0.56 mm) showed restrained and minimum activity, respectively. More specifically, aqueous extract represented higher susceptibility to all bacterial strains (Table 4; Figure 2-4). These results indicate better activity of ethanol extract of A.pinnata on treatment of dental diseases and followed chloroform extract than aqueous extract.

Table 1: Preliminary phytochemical evaluation of different solvent extract of A. pinnata

Phytoconstituents	Aqueous	Ethanol	Chloroform
Alkaloids	_	_	_
Phenols	₊₊	₊₊₊	₊₊
Flavonoids	₊	_	_
Saponins	_	_	₊
Proteins	_	_	_
Quinones	_	_	_
Steroids	_	_	_
Tannins	₊₊	₊	_
Xanthoproteins	_	_	_
Carboxylic acids	_	₊₊	_
Carbohydrates	₊	_	++

Table 2: Functional groups of ethanol extract of A. pinnata

Sl. No	Functional groups	cm^-1
1	C-H,C=O aldehyde , ketones	3828.34
2	C-H,C=O aldehyde , ketones	3786.81
3	O–H stretch, H–bonded alcohols, phenols	3417.03
4	C–H stretch alkenes	2917.77
5	C-H Stretch of C=O aldehydes	2849.85
6	N–H bend primary amines	1636.86
7	C–C stretch (in–ring) aromatics	1437.86
8	N=O Bend nitro	1384.27
9	C–N stretch aromatic amines	1321.08

Figure 2: Antimicrobial activity of ethanol extract of A. pinnata against dental pathogens

Figure 3: Antimicrobial activity of aqueous extract of A. pinnata against dental pathogens

Figure 4: Antimicrobial activity of chloroform extract of A. pinnata against dental pathogens

Table 3: Biochemical characters of the isolates

S. No	Tests	*Streptococcus mutans 1*		*Streptococcus mutans 2*		*Streptococcus mutans 3*	*Streptococcus mutans 4*
1	Gram staining	(+) ve cocci chain forms		(+) ve cocci chain forms		(+) ve cocci chain forms	(+) ve cocci chain forms
2	Motility	Non motile		Non- Motile		Non motile	Non motile
3	Spore	_		_		_	_
4	Catalase	_		_		_	_
5	Oxidase	_		_		_	_
6	Indole	_		_		_	_
7	Methyl red	_		_		_	_
8	Voges proskauer	_		_		_	_
9	Citrate utilization	+		+		+	+
10	Gelatin hydrolysis	_		_		_	_
11	Urease	_		_		_	_
12	TSI	A/A,G-		A/A,G-		A/A,G-	A/A,G-
13	Glucose	A+		A+		A+	A+
14	Sucrose	A+		A+		A+	A+
15	Lactose	A+		A+		A+	A+
16	Maltose	A+		A+		A+	A+
17	Coagulase test	Variable		_		_	variable
Cultural character on media
18	Nutrient agar	Whitish colonies	Whitish colonies		Whitish colonies			Whitish colonies
19	Blood agar	Alpha hemolysis	Alpha hemolysis		Alpha hemolysis			Alpha hemolysis

Table 4: Antimicrobial activity of ethanol extract of A. pinnata against dental pathogens

Conc. Ethanol extract	Zone of inhibition (mm in diameter)
Conc. Ethanol extract	*Streptococcus mutans 1*	*Streptococcus nutans 2*	*Streptococcus mutans3*	*Streptococcus mutans 4*
25µg/mL	12.7±0.52	11.4±1.12	15.2±1.62	12.1±1.52
50µg	16.7±1.33	13.6±0.33	17.5±1.53	15.0±0.72
75µg	19.5±1.02	17.5±1.33	20.8±1.23	17.5±0.92
100µg	24.3±1.40	21.0±1.32	22.5±1.56	19.0±0.56
Amphicillin	17.8±1.24	20.0±1.02	17.0±1.33	18.5±1.43

Table 5: Antimicrobial activity of aqueous extract of A. pinnata against dental pathogens

Conc aqueous extract	Zone of inhibition (mm in diameter)
Conc aqueous extract	*Streptococcus mutans 1*	*Streptococcus nutans 2*	*Streptococcus mutans3*	*Streptococcus mutans 4*
25µg	13.0±1.33	11.0±0.52	11.5±1.06	10.5±0.54
50µg	15.0±1.02	12.9±0.56	13.5±0.54	12.3±0.52
75µg	17.5±0.33	14.5±0.23	14.2±1.23	14.7±0.63
100µg	20.5±1.02	16.0±1.32	15.5±1.33	17.6±0.93
Amphicillin	12.9±0.82	20.0±1.33	17.0±1.10	17.8±1.12

Table 6: Antimicrobial activity of chloroform extract of A. pinnata against dental pathogens

Conc. Chloroform extract	Zone of inhibition (mm in diameter)
Conc. Chloroform extract	*Streptococcus mutans 1*	*Streptococcus nutans 2*	*Streptococcus mutans 3*	*Streptococcus mutans 4*
25µg	14.5±0.56	12.0±0.54	12.6±0.86	11.9±0.42
50µg	16.7±0.33	13.5±0.56	15.5±0.45	12.5±0.52
75µg	19.0±0.13	15.5±0.23	18.8±0.76	15.6±0.64
100µg	22.6±0.56	17.6±0.33	21.0±0.98	17.0±0.53
Amphicillin	17.8±0.54	20.0±0.74	17.0±0.24	18.5±0.33

Plant derived natural products such as flavonoids, terpenoids and steroids, etc have received considerable attention due to their different pharmacological properties including antimicrobial, antioxidant and antitumor activity [18, 19]. In the present study all the three plants and all the tested extracts showed the presence of phenolics in high concentrations. It is suggested that the selected three plants can be used to treat dental cavity diseases. Moreover, flavonoids present in plants possessing antifungal, antiviral antibacterial and antioxidant, anti-inflammatory and anticancer activity [20-25].

CONCLUSION:

All the extracts showed significant activity against all pathogens, but the alcoholic extract of A. pinnata showed maximum zone of inhibition against all the microorganisms. The maximum zone of inhibition was determined in ethanol and aqueous extract of A. pinnata showing great antimicrobial activity against all the experimental strains. The present investigation Azolla pinnata contain potential antimicrobial components that may be of great use for the development of pharmaceutical industries as a therapy against various diseases. The ethanol, chloroform and aqueous extracts of A. pinnata possess significant inhibitory effect against dental pathogens. The results of the study support the folkflore claim along with the development of new antimicrobial drugs from both the plants.

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Received on 13.04.2017 Modified on 17.05.2017

Research J. Pharm. and Tech. 2017; 10(6): 1891-1896.

DOI: 10.5958/0974-360X.2017.00332.8