INTRODUCTION:

Medicinal plants have been used as traditional treatments for numerous human diseases for thousands of years and in many parts of the world. Many plant-derived medicines used against oral microbial pathogens [1]. Over 750 species of bacteria inhabit in the oral cavity and a number of these are implicated in oral diseases [2,3]. Despite several agents being commercially available, these chemicals can alter oral microbiota and have undesirable side-effects such as vomiting, diarrhea and tooth staining [4].

Acmella oleracea has been used as a traditional medicine for toothache, rheumatism and fever[5].The most active antiseptic alkaloid extracted from this plant, is found effective at extremely low concentrations against blood parasites, and indeed is a poison to most invertebrates while remaining harmless to warm-blooded creatures [6].

Acmella oleracea extract has been tested against various yeast and bacteria was essentially inactive [7].The isolation and total synthesis of the active ingredients have been reported [8]. The leaves are used as immunomodulatory, adaptogenic, toothpaste, lithotriptic, antiscorbitic, ailagogine and digestive [9].

Clerodendrum infortunatum is one of the commonly used plants in ethnomedicine for its various medicinal properties. Different species of Clerodendrum genus have been traditionally used over centuries and their antioxidant and hepatoprotective potential have already been proved [10]. The plant was found to contain triterpenes, steroids and flavonoids[11]. The leaves and roots of C. infortunatum are used as antimicrobial activity [12]. The ethanolic extract is shown to have significant anti-microbial activity comparable to the standard drug tetracycline [13].

Young twigs is used as a tooth brush strengthen gum and teeth [14].Leaves yield quercetin-3-galactoside, rutin, friedelin, taraxosterol, lupeol, β-amyrin along with psoralen,bergapten and β-sisterol [15]. So far no more works have been done this kind of plants, hence the present works undertaken the following objectives, such as to identified the antimicrobial activity of Acmella oleracea, C. infortunatum and F. benghalensis against chosen microorganisms viz, Bacillus subtilis, Lactobacillus acidophilus, Aspergillus niger and Candida albicans.

MATERIALS AND METHODS:

Preparation of plant extracts:

Plant materials were collected from Thiruvananthapuram and identified plant from agriculture department Thiruvananthapuram. The fresh leaves were harvested and properly washed in tap water (H₂O), and then rinsed in sterile distilled H₂O. The leaves were divided into two equal parts; one part was dried in the hot air oven at 40°C for 3 days, while the second portion was blended fresh using an electric blender. The dried roots were pulverized, using sterile laboratory mortar and pestle, to obtain a powered form. These were stored in airtight glass containers protected from sunlight until required for further analysis.

Bacterial isolates:

Isolates of Lactobacillus acidophilus and Bacillus subtilis and fungi Aspergillus niger and Candida albicans, were obtained from the stock culture in Biotechnology laboratory, Malankara Catholic College, Mariagiri. Biochemical tests were performed to re-identify and confirm the identity of the isolates. Fresh plates of the test bacteria were made from the isolate cultures obtained on agar slants. Discrete colonies of fresh cultures of the different bacterial isolates were then picked and suspended in 5 ml Nutrient broth (NB, Oxoid), in well-labeled sterile Bijou bottles, and incubated for 24 h at 37ºC prior to antimicrobial susceptibility testing.

Extraction of plant material:

Cold and hot extraction with H₂O, and soxhlet extractions with methanol (99%) and Benzene [16].Twenty (20) g of each sample was weighed into 100 ml of the solvent (methanol and Benzene). For cold extraction the samples and solvent were stirred every 30 min for 3 h and allowed to stand for 24 h, while for hot extraction the samples and solvent were heated for 30 min and stirred every 30 min for 3 h and allowed to stand 24 h. After preparation of the crude extract as described, the organic extracts were diluted using 50% dimethylsulphoxide (DMSO), while the aqueous extracts were reconstituted using sterile distilled H₂O to make the final concentrations which kept in refrigerator till used.

Determination of antimicrobial activity:

Antibacterial activity of the aqueous and organic extracts of the plant sample was evaluated by the cup plate agar diffusion method [17].Bacterial cultures were adjusted to 0.5 McFarland turbidity standards and inoculated onto Mueller Hinton agar (MHA, Oxoid) plates (diameter: 15 cm). A sterile cork borer was used to make a well (6 mm in diameter) on the MHA plates. Aliquots of 100 μl of extract dilutions, reconstituted in 50% DMSO (for organic extracts) and distilled water (for aqueous extracts) at concentrations of 200, 150, 100 and 50 mg/ml, were applied in each of the wells in the culture plates previously seeded with the test organisms. The cultures were incubated at 37oC for 24 h. A well was made in each of the culture plates and filled with 20 μl of 10 mg/ml of ciprofloxacin and streptomycin as positive controls, and sterile filter paper soaked in sterile glycerol served as a negative control. Antimicrobial activity was determined by measuring the zone of inhibition around each well (excluding the diameter of the well). For each extract, three replicate trials were conducted against each organism.

Determination of Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC):

The Minimum Inhibitory Concentration (MIC) of the extracts was determined for each of the test organisms in triplicate in test tubes. To 0.5 ml of varying concentrations of the extracts (5, 25, 50, 75, 100, 125, 150, 175 and 200 mg/ml) in test tubes, Nutrient broth (2ml) was added and then a loopful of the test organism, previously diluted to 0.5 McFarland turbidity standard, was introduced. The procedure was repeated on the test organisms using the standard antibiotics (ciprofloxacin and streptomycin). A tube containing Nutrient broth only was seeded with the test organisms, as described above, to serve as controls. The culture tubes were then incubated at 37oC for 24 h. After incubation the tubes were then examined for microbial growth by observing for turbidity.

To determine the MBC, for each set of test tubes in the MIC determination, a loopful of broth was collected from those tubes that did not show any growth and inoculated onto sterile Nutrient agar by streaking. Nutrient agar plates only were also streaked with the respective test organisms to serve as controls. All the plates were then incubated at 37oC for 24 h. After incubation the concentration at which no visible growth was seen was noted as the Minimum Bactericidal Concentration (MBC).

Effect of temperature and pH on antimicrobial activity of extracts:

Five milliliters of 100 mg/ml of methanol extracts were constituted in test tubes and treated at 4oC in the refrigerator, and at 60 and 100^oC in water bath for 30 min and then tested for antibacterial activity as described earlier. To determine the effect of pH, methanol extracts in test tubes were treated at a pH of 2.5, 5 and 10, using 1 N HCl and 1 N Na- OH, respectively, for 30 min. After 30 min of treatment, the extracts were neutralized (pH 7) using 1 N HCl and 1 N NaOH, respectively, and then tested for antimicrobial activity as described earlier.

Phytochemical Screening:

Phytochemical tests were carried out on the methanolic extract of Acmella oleracea, Clerodendrum infortunatum and Ficus benghalensis [18].

Statistical analysis:

The mean values of each experimental group were calculated from 3 replicates for each sample. Then the mean values were tested for significance using statistical tests such as student’s t test and ANOVA.

RESULTS:

Phytochemical screening of the leave plant extracts revealed the presence of carbohydrates, alkaloids, tannins, saponins, Flavanoids, anthraquinone, glycosides, steroids, terpenoids and sterols. Though, Flavanoids, Anthraquinone and glycosides absence in benzene extract of Ficus bengalensis not detected in the leaves extract. results of the preliminary phytochemical showed Table 1.

Table 1: Phytochemical constituents of leaves from Acmella olerecea, Clerodendron infortunatum and Ficus bengalensis

Phytochemical constituents	Benzene			Methanol
Phytochemical constituents	A o	C i	F b	A o	C i	F b
Alkaloids	+	++	++	+	+	+
Carbohydrates	+	++	+	+	-	+
Tannins	+	-	++	+	+	+
Saponins	+	+++	+	+	+	+
Flavanoids	+	+	-	+	+	-
Anthraquinone	+	+	-	+	-	-
glycosides	+	+	-	+	+	+
Steroids	+	+	+++	+	+	+
Terpenoids	+	++	+	+	-	+
Sterols	+	+	++	-	-	-
Methylated sterols	-	-	+	-	-	-

A o - Acmella olerecea

C i - Clerodendrum infortunatum

F b - Ficus bengalensis

The plants used in the study were collected from Thiruvananthapuram district. The present study mainly focuses on the bioactive screening of the plants such as Acmella olerecea, Clerodendron infortunatum and Ficus bengalensis against some of the pathogens causing oral diseases.

Antimicrobial activity of methanolic and benzene plant extracts:

The antimicrobial activity of the methanol extracts of Acmella olerece, Clerodendron infortunatum and Ficus bengalensis were determined against bacteria (Lactobacillus acidophilus and Bacillus subtilis) and fungi (Aspergillus niger and Candida albicans) by disc diffusion method. Among the 4 species of microorganisms, Acmella olerece methanolic extract showed extremely antimicrobial activity against Aspergillus niger (17.8mm) and minimum as well as similar activity against Lactobacillus acidophilus (12.2mm) and C. albicans (12.2mm). Clerodendron infortunatum methanolic extract showed highly significant antimicrobial activity against Lactobacillus acidophilus (14.2mm) and minimum activity against A. niger (12mm). Ficus bengalensis methonolic extract showed highly significant antimicrobial activity against C. albicans (14.2mm) and minimum activity against A. niger (12mm).

Furthermore, the consistent similar activity was observed in L. acidophilus and Bacillus subtilis (14.2mm) on both plant of A. olerece and C. infortunatum. However maximum (14.8) and minimum (12) antimicrobial activity was noted on both fungi C. albicans and A. niger when treated with methanol extract of C. infortunatum and

F. bengalensis respectively (Table 2).

Table 2: Antimicrobial activity of Benzene and methanolic extracts of three medicinal plants on Bacteria and fungi

Tested organisms	Benzene			Methane
Tested organisms	A o	C i	F b	A o	C i	F b
Lactobacillus acidophilus	12.2	9.8	8	14.2	14.2	12.4
Bacillus subtilis	11	10.2	10	12.7	12.7	13.8
Aspergillus niger	17.8	8.8	9	14.2	12	12.7
Candida albicans	12.2	12.4	12	12.4	13.8	14.8

A o - Acmella olerecea

C i - Clerodendron infortunatum

F b - Ficus bengalensis

Minimum inhibitory concentration (MIC):

The 7µl dilution of crude extract of Acmella olerecea showed Minimum inhibitory concentration (in OD) against Lactobacillus acidophilus (0.07nm), and Candida albicans (0.08nm) (Fig. 1).

Fig.1: Minimum inhibitory concentration (MIC) of three medicinal plants on Bacteria and fungi

Minimum bactericidal concentration (MBC):

Result of minimum bactericidal concentrations of Acmella olerecea, Ficus bengalensis and Clerodendron infortunatum methanolic extracts against Lactobacillus acidophilus and Candida albicans were recorded Table 3.

Effect of Temperature and pH on antimicrobial activity:

The effect of temperature on the antimicrobial activity of leaf extracts of three plants (Table-4) showed that the activity of extracts increased with an increase in temperature. The activity of the extracts against S. typhi at 30ºC (untreated) (14 mm zone of inhibition) increased to 22

Table-3: Minimum bactericidal concentration (MBC) of methanolic extracts of plants.

	Medicinal Plant Leaves Extracts
Dilution(μl)	*Acmella olerecea*		*Clerodendron infortunatum*		*Ficus bengalensis*
Dilution(μl)	MIC	MBC	MIC	MBC	MIC	MBC
3	+	123	+	112	+	95
4	+	75	+	56	+	42
5	+	32	+	21	+	18
6	+	12	+	8	+	6
7	-	0	-	0	-	0

Table 4: Effect of temperature (ºC) on the antibacterial activity (mm) of leaf extracts (methanol and benzene) of three medicinal plants of the tested organisms.

Medicinal Plant Leaves Extracts		*Acmella olerecea*			*Clerodendron infortunatum*			*Ficus bengalensis*
Temperature (°C)		35	45	55	35	45	55	35	45	55
*Tested* *Microorganisms*	*L. acidophilus*	13	17	14	13	15	14	13	17	12
	*Bacillus subtilis*	13	18	16	18	24	13	12	14	10
	*Aspergillus niger*	15	22	12	13	16	18	15	13	12
	*Candida albicans*	16	15	14	11	22	20	15	10	11

Table 5: Effect of pH on the antibacterial activity of leaf extracts of three different plants (20 mg/ml) on the tested organisms.

Medicinal Plant Leaves Extracts		*Acmella olerecea*			*Clerodendron infortunatum*			*Ficus bengalensis*
P^H		5	7	8	5	7	8	5	7	8
*Tested* *Microorganisms*	*L. acidophilus*	13	15	12	11	15	13	13	14	13
	*Bacillus subtilis*	10	13	11	14	22	13	17	16	12
	*Aspergillus niger*	13	15	12	18	16	15	12	13	15
	*Candida albicans*	15	16	13	13	20	16	15	14	10

DISCUSSION:

The presence of bioactive substances have been reported to confer resistance to plants against bacteria, fungi and pests and therefore explains the demonstration of antibacterial activity by the plant extracts used in this study[19].The use of medicinal herbs in the treatment and prevention of infectious diseases has attracted the attention of scientists worldwide [21-24]. In the present study methanol and benzene extract of Acmella olerecea, Clerodendron infortunatum and Ficus bengalensis showed the activity against Lactobacillus acidophilus, Bacillus subtilis, Candida albicans and Aspergillus niger. The antimicrobial activity of the methanol extracts of Acmella olerece, Clerodendron infortunatum and Ficus bengalensis were determined against bacteria (Lactobacillus acidophilus and Bacillus subtilis) and fungi (Aspergillus niger and Candida albicans) by disc diffusion method. Among the 4 species of microorganisms, Acmella olerece methanolic extract showed highly significant antimicrobial activity against Aspergillus niger (17.8mm) and minimum activity against Lactobacillus acidophilus (12.2mm) and Candida albicans (12.2mm). Clerodendron infortunatum methanolic extract showed highly significant antimicrobial activity against Lactobacillus acidophilus (14.2mm) and minimum activity against Aspergillus niger (12mm). Ficus bengalensis methanolic extract showed highly significant antimicrobial activity against Candida albicans (14.2mm) and minimum activity against Aspergillus niger (12mm). The influence of extract dilution on the antimicrobial activity of Acmella olerece, Clerodendron infortunatum and Ficus bengalensis against various organisms is statistically significant (p<0.05).

The antimicrobial activity of the benzene extracts of Acmella olerece, Clerodendron infortunatum and Ficus bengalensis were determined against bacteria (Lactobacillus acidophilus and Bacillus subtilis) and fungi (Aspergillus niger and Candida albicans) by disc diffusion method. Among the 4 species of microorganisms, Acmella olerece benzene extract showed highly significant antimicrobial activity against Aspergillus niger (13.7mm) and minimum activity against Lactobacillus acidophilus (9.8mm) and Candida albicans (12.2mm). Clerodendron infortunatum benzene extract showed highly significant antimicrobial activity against Candida albicans (12.4mm) and minimum activity against Aspergillus niger (8.8mm). Ficus bengalensis benzene extract showed highly significant antimicrobial activity against Candida albicans (12mm) and minimum activity against Lactobacillus acidophilus (8mm).

The 7µl dilution of crude extract of Acmella olerecea showed Minimum inhibitory concentration against Lactobacillus acidophilus (0.07nm), and Candida albicans (0.08nm). The 7µl dilution of crude extract of Clerodendron infortunatum showed Minimum inhibitory concentration (in OD) against Lactobacillus acidophilus (0.07nm), and Candida albicans (0.9nm). The 7µl dilution of crude extract of Ficus bengalensis showed Minimum inhibitory concentration against Lactobacillus acidophilus (0.05nm), and Candida albicans (0.06nm). In this study it was observed that the MIC of the active plants extracts are lower than the MBC and MFC, suggesting that the plant extracts were bacteriostatic at lower concentration and bactericidal at higher concentration [25].

Phytochemical screening of the leave plant extracts revealed the presence of carbohydrates, alkaloids, tannins, saponins, Flavanoids, anthraquinone, cardiac glycosides, steroids, terpenoids and sterols. Methylated sterols were not detected in the leaves extract. The presence of these phytochemical compounds is linked to biological activity, such as protection of the plant against infections[26,27].The presence of these glycoside moieties, some of which are known to be structurally and chemically related to sex hormones (oestrogens, gestrogens and androgens known to inhibit tumor growth) and flavonoids protects against gastric infections [28,29].The presence of alkaloids in this plant is also of great importance to humans because of their medicinal values as significant quantities are used as anti-malarial, analgesic and as stimulants. This therefore gives credence to some of the ethnomedical uses of the plant [30].

Acmella olerece methanolic extract showed highly significant antimicrobial activity against Aspergillus niger (22mm) at 45°C and minimum activity against Aspergillus niger (12mm) at 55°C. Clerodendron infortunatum methanolic extract showed highly significant antimicrobial activity against Bacillus subtilis (24mm) at 45°C and minimum activity against Candida albicans (11mm) at 35°C. Ficus bengalensis methanolic extract showed highly significant antimicrobial activity against Lactobacillus acidophilus (17mm) at 45°C and minimum activity against Candida albicans (10mm) at 55°C. The influence of temperature on the antimicrobial activity of Acmella olerece, Clerodendron infortunatum and Ficus bengalensis against various organisms is statistically significant (p<0.05). In terms of the effect of temperature on activity of the crude extracts, results showed that increase in temperature of the crude extracts increased the activity of the crude extracts. This could suggest the reason why traditional healers often boil plant extract before they are taken by the sick persons.

Acmella olerece methanolic extract showed highly significant antimicrobial activity against Candida albicans (16mm) at pH 7 and minimum activity against Bacillus subtilis (10mm) at pH 5. Clerodendron infortunatum methanolic extract showed highly significant antimicrobial activity against Bacillus subtilis (22mm) at pH 7 and minimum activity against Lactobacillus acidophilus (11mm) at pH 5. Ficus bengalensis methanolic extract showed highly significant antimicrobial activity against Candida albicans (10mm) at pH 8 and minimum activity against Aspergillus niger (12mm) at pH 7. The influence of pH on the antimicrobial activity of Acmella olerece, Clerodendron infortunatum and Ficus bengalensis against various organisms is statistically significant (p<0.05). Effect of pH on activity indicated that test organisms were more susceptible to acidic pH of the crude plant extracts than to the alkaline pH of the same crude extracts. This is an indication that the crude plants can be stable under the acidic conditions of the stomach and the gastro-intestinal tract and therefore can be formulated to be taken orally.

ACKNOWLEDGEMENTS:

The authors are grateful to Dr. Rajagopal (Head of the Department) and the Staff Members of Biotechnology Department, Malankara Catholic College, Mariagiri, Kanyakumari, Tamilnadu, India for their constant encouragement and support.

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Received on 30.08.2014 Modified on 10.09.2014

Research J. Pharm. and Tech. 7(11): Nov. 2014 Page 1264-1269