Antimicrobial Efficacy of Alpinia officinale extracts against Oral Microorganism - An In-vitro Study

Priyanga Chandrasekaran¹, Shivashankar Kengadaran²

¹Department of Public Health Dentistry,

Indira Gandhi Institute of Dental Sciences, Sri Balaji Vidyapeeth, Pondicherry.

²Reader, Department of Public Health Dentistry,

Indira Gandhi Institute of Dental Sciences, Sri Balaji Vidyapeeth, Pondicherry.

*Corresponding Author E-mail: shivashankar@igids.ac.in

ABSTRACT:

Aim: To assess the antimicrobial activity of Alpinia officinale extract against S. mutans, lactobacillus, and C. albicans. Methodology: The rhizome of Alpinia officinale were identified, collected, washed, shade dried, and then ground to prepare a fine powder. 50gm of the prepared powder was dissolved in 250ml of the following solvents ethanol, acetone, ethyl acetate, chloroform and methanol at 40-50℃ for 6h. The bacterial and fungal isolates of S. mutans, Lactobacillus and C. albicans were collected and prepared. Antimicrobial and antifungal activity of extracts against S. mutans, lactobacillus and C. albicans were determined by the Disc diffusion method. Microdilution of extracts were prepared with 2X stock concentration to obtain 100 to 1.5 microgram per ml of nutrient broth to determine the minimum inhibitory concentration. Results: The most effective extract was ethanol extract followed by ethyl acetate and methanol extracts. There was substantial activity by methanol and ethyl acetate extracts. The MICs of different concentrations of ethanol extracts were found to be 1.5,3.1 and 12.5 µg/mL for S.mutans, L.acidophilus, and C.albicans respectively. Conclusion: The present study concluded that A. officinale has potential antimicrobial activities for cariogenic microorganisms.

KEYWORDS: Herbal, dental caries, periodontal disease, fungi, bacteria.

INTRODUCTION:

The WHO bulletin from 2005 claimed that "dental caries among adults was highly prevalent globally, and was affecting about 100% of the population in the majority of nations." It was one of the main causes of oral pain and the main reason individuals attend dental offices or hospitals.¹In India,dental caries prevalence increased with age, reaching a maximum of 85% in adults aged 65–74 years, up from 51.9% in 5-year-old children. Three key variables that cause dental caries include oral bacteria, the presence of fermentable carbohydrates, and the availability of tooth surfaces.²

The oral cavity contains a vast diverse category of microorganisms that are responsible for several oral and systemic infections in the human body including dental caries.³

Streptococcus mutans and Lactobacillus were the most commonly found bacteria in the oral cavity, these gram-positive cocci cause tooth decay by rapidly colonizing and altering the environmental conditions of the oral flora.³Similarly, Candida albicans found in the normal flora was one of the important causes of fungal infections in the oral cavity.⁴

Disinfectant solutions such as mouthwashes are effective ways to reduce the number of microorganisms in the oral cavity.⁵ Regular usage of mouthwash along with tooth brushing and flossing effectively reduces the bacterial population in the oral cavity.

Chlorhexidine, nystatin, and fluoride are the most commonly used mouthwashes in dentistry.Besides their benefits, they do have side effects like discoloration of teeth, sensitivity, burning of the oral mucosa, taste perturbation, and rarely swelling of parotid glands.³Considering these, herbal mouthwashes would be a better alternative to these chemical mouthwashes due to their natural ingredients for biocompatibility and lower toxicity, they can also reduce the side effects.⁶

Alpinia officinarum (Zingiberaceae family) is an aromatic plant used as medicine for colds, stomach aches, abdominal swelling, bronchitis, ulcers, diabetes, neupathia, diarrhea, vertigo, rheumatoid arthritis, inflammatory bowel diseases, etc. mostly in Far East countries.⁷Although Alpinia officinale has been used in various areas, its use in dentistry has not been explored. Hence the current study was conducted to assess the antimicrobial activity and Minimum inhibitory concentration of Alpinia officinale extract against selected cariogenic oral microorganisms.

METHODOLOGY:

Plantextraction:

The Rhizome of Alpinia officinale wasidentified and collected from the near by stores. They were washed, dried and then powdered to prepare a fine powder of50gm. The powder was dissolved in 250ml of the following solvents ethanol, acetone, ethyl acetate, chloroform, and methanol at 40-50℃ for 6h. Successive concentration, filtration, and extraction of this filtrate were done with help of the Soxhlet apparatus. The concentrated extract (10mg/mL) was stored at 4°C inairtightbottles for furtheruse (Figure 1).

Fig 1: Extraction of Alpinia officinarumextract

Identification of bacteria:

The bacterial and fungal isolates of S.mutans, Lactobacillus and C. albicans were collected and suspended in peptone broth and incubated at 37 °C for 3–4h, and used as inocula. One loopfull colony of S.mutans was picked up using inoculating loupes and streaked over the surface of cooled Mitis Salivarius Bacitracin (MSB) plates and then plates were placed in the anaerobic gas jar using anaerobic gas packs, for 24h. Similarly, colonies of C. albicans were picked up using sterile gauze sticks and spread over the slants of Saboraud's Dextrose agar and incubated Aerobically for 48h in anincubator. The same was done for lactobacillus using lactobacillus MRS agar. They were then confirmed based on morphological and colonial characteristics.

Determination of antimicrobial activity:

Antimicrobial and antifungal activity of extracts against S. mutans, lactobacillus, and C.albicans were determined by the Disc diffusion method at 100µg/disc. A total of 3 MH agar plates were prepared to assess the mean zone of inhibition. A sterile cotton swab was used to prepare lawn culture by the swab method. Discs preloaded with samples were placed over the agar surface. 0.2% chlorhexidine was used as a positive control. The agar plates were kept under incubation for 37℃ overnight incubators. The antibacterial activity was recorded with a zone scale. A zone of inhibition was recorded after 24hours. The diameter of the zone of inhibition was measured, and the antimicrobial activity of the extract was reported accordingly.

Minimum inhibitory concentration (MIC):

Microdilution of extracts were prepared with 2X stock concentration to obtain 100 to 1.5 micrograms per ml of nutrient broth. About 50µL of test pathogen at 10⁶ CFU/ml from growth on the plates were made using McFarland' sturbidity standards and introduced into all tubes and incubated for 24hours after incubation 25µL of resazurin indicator was added and incubated for 1hr. The viability of cells was noted by oxidation of indicator dye. The highest dilution failed to oxidize the dye and the medium that remains blue was considered MIC.

RESULTS:

The antimicrobial activity of Alpinia officinale was studied against bacterial and fungal isolates of S.mutans, Lactobacillus, and C.albicans were collected. Of the 5 Alpinia officinale extracts tested for antimicrobial activity, ethanol, methanol, and ethyl acetate extracts exhibited zone of growth inhibition (Figure 2). The highest zone of growth inhibition was exhibited by Ethanol extract. Ethanol and Ethyl acetate exhibited a zone of growth inhibition against all three organisms at 12mm,20mm and 14mm, and <5mm,16mm, and 18mm for L.acidophilus, S.mutans, and C.albicans respectively. Methanol extract exhibited growth inhibition only against S.mutans and C.albicans at 14mm and < 5mm respectively. There was no growth inhibition against L.acidophilus (Table 1).

Fig 2: Zone of inhibition of different extracts against oral pathogens

Table 1: Antibacterial activity of extract at 100 µg

	Acetone	Ethanol	Methanol	Ethylacetate	Chloroform	Cl.hex
L.acidophilus	0	12 mm	0	≤5 mm	0	8 mm
S.mutans	0	20 mm	14 mm	16 mm	0	≤5 mm
C.albicans	0	14 mm	≤5 mm	8 mm	0	≤5 mm

Table 2: Minimum inhibitory concentration of compound

ORGANISM	100µg	50 µg	25 µg	12.5 µg	6.25 µg	3.1 µg	1.5 µg
S.mutans	+	+	+	+	+	-	+
L.acidophilus	+	+	+	+	-	+	+
C.albicans	+	+	+	-	+	+	-
Organism				MIC µg/mL
S.mutans				3.1
L.acidophilus				6.25
C.albicans				1.5

Different concentrations (100, 50, 25, 12.5, 6.25, 3.1 and 1.5µg/ml) of ethanolic extracts were investigated to determine their MICs against test microorganisms (S mutans, lactobacillus, Candida albicans). After the addition of the Resazurin color indicator in each of the concentrations, the test tubes were noted for color change (Figure 3). The viability of cells was noted by oxidation of the indicator dye. The highest dilution that failed to oxidize the dye leaving the medium blue was considered MIC. Ethanol extracts exhibited no oxidation of the dye at 3.1µg for S mutans, 6.25 µg for L.acidophilus, and 12.5µg and 1.5µg for C.albicans (Table 2). The Minimum Inhibitory concentration for S mutans, L.acidophilus, and C.albicans were 3.1µg, 6.25 µg, and 1.5µg respectively (Table 3).

Fig 3: MIC of Alpinia officinale compound against various oral microorganisms.

DISCUSSION:

The present study was conducted to assess the antimicrobial activity and Minimum Inhibitory concentration of Alpinia officinale extracts. Five extracts of Alpinia officinale namely Ethanol, Methanol, Ethyl acetate, acetone, and chloroform were prepared and its antibacterial activity was observed using the Disc diffusion method against three organisms namely S mutans, Lactobacillus, and C. albicans. The highest antibacterial activity was exhibited by ethanol extract. Ethanol extract exhibited a zone of growth inhibition against all three organisms at 12mm, 20mm and 14mm for L.acidophilus, S.mutans, and C.albicans respectively.

Kiranmayee Rao et. al⁸in their study using Alpinia galanga extracts, a rhizome similar to Alpinia officinale, methanol extract showed the highest antimicrobial activity. It has been shown to have antibacterial properties against bacteria, fungi, yeast, and parasites. It also contains aldehydes, esters, phenols, etc. making it a choice for developingnew drug candidates.

The primary benefit of using these natural agents is that their crude extracts contain a variety of chemicals, such as phenols, acids, esters, and aldehydes, which make it more difficult for bacteria to build resistance to them than it is for synthetic antibiotics, which only contain one molecule.⁸

Gulcin Alp Avci et al in their study to determine the antimicrobial and antioxidant properties of Alpinia officinale and Zingiber officinale⁹ have found that both extracts showed potent antibacterial and antioxidant abilities and their ingestion may slow oxidation, stop or postpone the development of degenerative disorders, and their extracts may act as antibacterial agents. These results were similar to studies done by Sri Divya et al⁷where Alpinia officinarum exhibited significant antimicrobial and antioxidant properties. There are several other invitro and in-vivo studies using curcumin,^10,11Aloevera,¹² neem,¹³ cardamon,¹⁴ cumin,¹⁵ acacia,¹⁶miswak,⁵showing the antimicrobial effect of herbal medicine on oral conditions.^17-20

Since A. officinale has been widely used in culinary in the Asian continent and listed by FDA as "generally regarded as safe" may replace the hazardous and undesirable synthetic antioxidants currently employed in the industry because of its promising scavenging activity.^10,21-22

CONCLUSIONS:

The present study concluded that A. officinale has potential antimicrobial activities for both bacterial and fungal species when subjected to different concentrations of different extracts (ethanol, methanol, and ethyl acetate). The most effective extract was ethanol extract followed by ethyl acetate and methanol extracts. The MICs of different concentrations of ethanol extracts were found to be 1.5,3.1 and 12.5 µg/mL for S.mutans, L.acidophilus, and C.albicans respectively.

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