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ORIGINAL ARTICLE
Year : 2018  |  Volume : 16  |  Issue : 3  |  Page : 251-255

In vitro antimicrobial activity of Spinacia Oleracea against Streptococcus mutans and Lactobacillus acidophilus


Department of Public Health Dentistry, College of Dental Sciences, Davangere, Karnataka, India

Date of Submission07-Jan-2018
Date of Acceptance18-May-2018
Date of Web Publication6-Aug-2018

Correspondence Address:
Dr. Suma Bindu Adapa
Department of Public Health Dentistry, College of Dental Sciences, Davangere - 577 004, Karnataka
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jiaphd.jiaphd_6_18

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  Abstract 

Introduction: The use of traditional medicine is well known in India. The high number of bacterial resistance to antibiotics drives much of the current interest in natural antimicrobial molecules. Spinacia oleracea (spinach) possesses medicinal properties. Dental caries is prevalent in about 89% of the Indian population. Streptococcus mutans is the primary causative organism for dental caries and Lactobacillus for further development of caries. Aim: The aim of this study is to determine minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of aqueous and ethanolic extracts of spinach against S. mutans and Lactobacillus. Materials and Methods: About 10 g of freshly harvested spinach was extracted with 95% ethanol and distilled water. The extract was converted into dried powder in desiccator and was dissolved to get 20 mg/ml concentration. The MIC was determined by nine dilutions of each aqueous and ethanolic extracts made with brain–heart infusion. To determine the MBC, the MIC dilution tubes, with no visible growth, and the control tube were subcultured onto the broth media and incubated for 24 h anaerobically and the colonies were counted on the next day. Results: The ethanolic extract exhibited MIC at 12.5 μg/ml against S. mutans and at 25 μg/ml against Lactobacillus. In MBC, the ethanolic extract showed greater bacteriostatic activity against S. mutans compared to Lactobacillus acidophilus. Conclusion: There is preliminary evidence for the antimicrobial activity of spinach extracts against S. mutans and this raises the possibility that spinach may have preventive effect on dental caries.

Keywords: Dental caries, Lactobacillus acidophilus, Spinacia oleracea, Streptococcus mutans


How to cite this article:
Adapa SB, Sushanth V H, Prashant G M, Mohamed I. In vitro antimicrobial activity of Spinacia Oleracea against Streptococcus mutans and Lactobacillus acidophilus. J Indian Assoc Public Health Dent 2018;16:251-5

How to cite this URL:
Adapa SB, Sushanth V H, Prashant G M, Mohamed I. In vitro antimicrobial activity of Spinacia Oleracea against Streptococcus mutans and Lactobacillus acidophilus. J Indian Assoc Public Health Dent [serial online] 2018 [cited 2018 Nov 17];16:251-5. Available from: http://www.jiaphd.org/text.asp?2018/16/3/251/238589


  Introduction Top


Oral health is vital for overall health. Dental diseases are recognized as a disease of modern civilization and major public health problem throughout the world. Despite the advent of latest aids for oral hygiene and advancement in dental science, majority of the population suffer from dental caries and periodontal problems among which dental caries remains the most important dental health problem in the developing countries.[1] Dental caries is prevalent in about 89% of the Indian population of which 72% are residing in the rural areas whose economic values are low to afford the treatment of dental caries. Hence, there is a need to promote preventive measures that are acceptable, easily available, and cost-effective.[1],[2] Bacterium, Streptococcus mutans, is a facultative, Gram-positive anaerobic organism and is more prevalent on pit and fissure and buccal surfaces of tooth and it is the primary causative organism for the initiation of dental caries.[1],[2] The bacteria of the genus Lactobacillus is a Gram-positive, facultative anaerobe associated with progression of carious lesions, especially those in the coronal areas,[1] and it is important in further caries development, especially in the dentin.[3] As these micro-organisms play a significant role in dental caries, control of their activities prevents dental caries.

During the last two decades, there has been an increasing trend in search for new plant-derived drugs containing the medically useful alkaloids, glycosides, polyphenolics, steroids, and terpenoid derivatives.[4] About 80% of the world population mainly rely on these traditional medicines for their primary health-care needs;[5] several antimicrobial agents were isolated from plants; Spinacia oleracea Linn. (family – Chenopodiaceae) is commonly known as “spinach.” It is an erect herb with about 30–60 cm height. It is cultivated throughout the world as a leafy vegetable. Several parts of this plant are used in traditional Indian medicine for numerous therapeutic effects.[6],[7],[8] Spinach is richly packed with Vitamin A, Vitamin C, Vitamin E, Vitamin K, magnesium, manganese, folate, betaine, iron, Vitamin B2, calcium, vitamin B6, folic acid, copper, protein, phosphorus, zinc, niacin, selenium, and omega-3 fatty acids.[9] Spinach is also packed with a number of antioxidant components such as polyphenols, flavonoids, and carotenoids which are shown to possess anti-inflammatory effects, antimutagenic potential, antineoplastic effects, as well as chemopreventive activities.[10],[11] Although many studies have tested the antibacterial effect of the spinach on various organisms, there is a dearth of literature pertaining to their effect against organisms causing dental caries. In an effort to expand the spectrum of antibacterial activity of spinach, in the current study, an attempt is made to assess the antimicrobial activity of aqueous and ethanolic extracts of Spinacia oleracea leaves against S. mutans and Lactobacillus acidophilus.


  Materials and Methods Top


This is an experimental, in vitro study conducted to assess the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of spinach aqueous and ethanolic extract against S. mutans and L. acidophilus.

Samples

Raw materials used in this work were freshly harvested spinach (Spinacia oleracea), from a local farm in Davangere.

Collection of micro-organisms

Unmixed strains of S. mutans and L. acidophilus were procured from microbial type culture collection and gene bank, Chandigarh.

Culture of test organisms

Brain–heart infusion (BHI) broth was used to culture the organism. The organisms were inoculated (using inoculating loop) on the sterilized media and incubated at 37°C for 24 h.

Preparation of extract

About 10 g of the spinach was macerated or extracted with 95% ethanol and distilled water in a conical flask at wrist action rotary shaker for 48 h. The whole extract was then filtered with the help of Whatman No. 4 filter paper. The collected solvent was then evaporated to dryness with rotary vacuum evaporator at 40°C to afford a thick residue. The thick residue was then converted into dried powder in a desiccator. This dried mass was dissolved to get 20 mg/ml concentration of extract.

Minimum inhibitory concentration procedure[12]

Minimum Inhibitory Concentration (MIC) can be defined as the minimal concentration of the antimicrobial agent that will inhibit the visible growth of a microorganism after overnight incubation (Moore and Goodwin, CRC Press 2007).[12] In the present study, nine dilutions of each aqueous and ethanolic extract of spinach were made with BHI for MIC. In the initial tube, 20 μL of test material was added into 380 μL of BHI broth. For dilutions, 200 μL of BHI broth was added into the next 9 tubes separately. Then, from the initial tube, 200 μL was transferred to the first tube containing 200 μL of BHI broth. This was considered as 10–1 dilution. From 10– 1 diluted tube, 200 μL was transferred to the second tube to make 10–2 dilution. The serial dilution was repeated up to 10– 9 dilution for each extract. From the maintained stock cultures of S. mutans and L. acidophilus organisms, 5 μL was taken and added into 2 ml of BHI broth. In each serially diluted tube, 200 μL of above culture suspension was added. The tubes were incubated for 24 h and observed for turbidity [Figure 1].
Figure 1: Serial dilution of Aqueous and Ethanolic extracts of Spinach to determine MIC

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The lowest concentration of spinach extract that completely inhibited the growth of the organisms was considered as MIC.

Minimum bactericidal concentration procedure[12]

To determine the Minimum Bactericidal Concentration (MBC), the MIC dilution tubes, with no visible growth, and the control tube were (various concentrations of doxycycline and doxycycline-loaded nanoparticles ranging from 0.1 to 8 μg/ml) subcultured and incubated for 24 h anaerobically at 37°C and the colonies were counted on the next day. The organisms grown from the control tube were then compared with the organism grown from the MIC test tubes.

The test was read as follows:

  1. Similar number of colonies – indicating bacteriostatic activity only
  2. Reduced number of the colonies – indicating a partial or slow bactericidal activity
  3. No growth – if the whole inoculum has been killed – indicating bactericidal activity,


MBC was carried out to observe the bactericidal/bacteriostatic effect of the spinach extract against the organism.


  Results Top


Inhibitory effect of spinach extracts

The antibacterial activity testing of the aqueous and ethanolic extract of spinach by macrobroth dilution revealed MIC and MBC at different concentrations of the spinach extract.

Minimum inhibitory concentration and minimum bactericidal concentration of spinach extracts on Streptococcus mutans

The ethanolic spinach extract exhibited MIC at 25 μg/ml. The MIC was higher at 50 μg/ml for aqueous extract [Table 1].
Table 1: Minimum inhibitory concentration and minimum bactericidal concentration of spinach extracts on Streptococcus mutans

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In MBC, the ethanolic extract showed greater bacteriostatic activity against the S. mutans compared to the aqueous extract of spinach [Table 1].

Minimum inhibitory concentration and minimum bactericidal concentration of spinach extracts on Lactobacillus acidophilus

The MIC for the ethanolic was determined at 50 μg/ml, whereas aqueous extract exhibited MIC at 100 μg/ml [Table 2]. In MBC, the ethanolic showed greater bacteriostatic activity than aqueous extract of spinach.
Table 2: Minimum inhibitory concentration and minimum bactericidal concentration of spinach extracts on Lactobacillus acidophilus

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In comparison of L. acidophilus, more susceptibility was shown by S. mutans to the spinach extracts, i.e. lesser number of colonies compared to L. acidophilus. The spinach extract exhibited bacteriostatic activity against both the organisms but not bactericidal activity.


  Discussion Top


Dental caries is a major public health issue.[13] Natural products have recently been demonstrated as an alternative to synthetic substances for prevention of tooth decay.[1] The successful treatment of dental caries requires suppression or elimination of the oral pathogens which causes caries. S. mutans are the major cariogenic pathogens as they are vastly acidogenic, yielding short-chain acids which dissolve hard tissues of teeth.[3]L. acidophilus are pioneering microorganisms in the caries progress, especially in dentin. Studies have shown that lactobacilli are a ruling part of the flora inhabiting the extensive cavities, and their number correlates with the amount of carbohydrates.[14],[15],[16]

Although advances have been made in pharmacology and synthetic organic chemistry, the reliance on natural products, particularly on plants, remains largely unchanged (Trevor, 2001). It is well established that some plants contain compounds that are able to inhibit microbial growth (Evarando et al., 2005). Spinach (Spinacia oleracea) is an edible flowering plant in the family of Amaranthaceae. It is an annual plant (rarely biennial), which grows to a height of up to 30 cm.[17] It is regarded as a valuable dietary source of Vitamin A, nonheme iron, folate, and lutein. It has also been used medicinally in treating anemia, night blindness, tooth disorder, urinary disorder, cancer, and respiratory disorder.[18] It is considered as antioxidant, antiaging, sun protective, antipyretic, anti-inflammatory, and antibacterial agent.[19],[20] Thus, this in vitro study was planned to explore its effect on S. mutans and L. acidophilus to know if it can be used as a major agent in common risk approach against S. mutans and L. acidophilus.

The results of the present study showed that aqueous and ethanolic extract of spinach has an antibacterial effect against S. mutans and L. acidophilus. The antibacterial effect can be attributed to its silver nanoparticles (silver is a natural antimicrobial agent. Therefore, silver nanoparticles [AgNPs] exhibit great potential as novel antimicrobial agents. Besides their antimicrobial properties, AgNPs are also reported to exhibit antiangiogenesis, anti-inflammatory, and antiplatelet activities. Hence, AgNPs have diverse medical applications), total phenolics, and flavonoids.[21],[22],[23] The results could not be compared with any other study as this is the first of its kind. However, a good antibacterial activity against S. aureus,  Salmonella More Details typhimurium,  Escherichia More Details coli, Pasteurella multocida, Lactobacillus bulgaricus, Micrococcus luteus, Klebsiella pneumoniae, Proteus vulgaris, Staphylococcus epidermidis, and methicillin-resistant S. aureus was shown by the ethanol extract of Spinacia oleracea in a study by Nasim et al.[24] and Mohammed et al.[20] The aqueous extracts of spinach have an effective antimicrobial activity against severe gastrointestinal tract pathogens such as  Salmonella typhi Scientific Name Search ing to a study by Jyoti et al.[17]Spinacia oleracea showed better antibacterial activity against these bacterial strains Pseudomonas aeruginosa, Bacillus subtilis, M. luteus, and E. coli.[25] A study by Das and Chatterjee showed that the polar solvent extract of spinach plant can be used as natural antibacterial agent to cure urinary tract infection.[8]

Extract of spinach (methanolic) was effective against bacterial strains (Bacillus cereus, B. subtilis, E. coli, Enterobacter aerogenes, Enterobacter agglomerans, S. aureus, P. aeruginosa, Candida albicans, Penicillium chrysogenum, Enterococcus faecalis, K. pneumoniae, Bacillus sphaericus, Bacillus thuringiensis, and Cryptococcal meningitis) compared to other aqueous and methanolic extracts of pumpkin, suran, and ghuiya in a study by Dubey et al.[26]

Strength and limitations

In vitro studies permit a species-specific more convenient and more detailed analysis than can be done with the whole micro-organism. However, they fail to replicate the precise cellular conditions of an organism.


  Conclusion Top


From the current study, it is concluded that there is preliminary evidence for the antimicrobial activity of spinach extracts against S. mutans and L. acidophilus and this raises the possibility that spinach may have preventive use for dental caries and possibly other oral infections such as gingival and periodontal problems.

Future perspectives

Aqueous extracts of spinach have an evidence of antimicrobial activity against S. mutans and L. acidophilus. Hence, further studies can be performed on these properties to enhance the knowledge of their benefits and to check the stability of active ingredients in commercial production condition and to prove the potential of these plant extracts to be formulated in dental care products.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

 
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