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ORIGINAL ARTICLE
Year : 2015  |  Volume : 6  |  Issue : 6  |  Page : 248-252  

Evaluation of anti-microbial activity of spore powder of Ganoderma lucidum on clinical isolates of Prevotella intermedia: A pilot study


Department of Periodontology, Maratha Mandal's Nathajirao G. Halgekar Institute of Dental Sciences and Research Center, Belgaum, Karnataka, India

Date of Web Publication8-Oct-2015

Correspondence Address:
P T Dixitraj
Deepam, TC 6/897, VARA.696, Arappura Gardens, Vattiyoorkavu P.O, Trivandrum - 695 013, Kerala
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0976-237X.166834

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   Abstract 

Aim: This study aimed at evaluating the anti-microbial activity of spore powder of Ganoderma lucidum on Prevotella intermedia isolated from subgingival plaque from chronic periodontitis patients. Settings and Design: Written informed consent was obtained from each subject enrolled in the study. The Institutional Ethics Committee granted the ethical clearance for the study.
Materials and Methods: This study included 20 patients diagnosed with chronic periodontitis. Pooled subgingival plaque samples were collected using sterile curettes from the deepest sites of periodontal pockets. The collected samples were then transported in 1 mL of reduced transport fluid. The organisms were cultured and confirmed. These organisms were then used for minimum inhibitory concentration (MIC) procedure. Statistical Analysis: Mean of the MIC value obtained was calculated.
Results: Thirteen out of the 20 clinical samples were tested that showed sensitivity at various concentrations. Five samples showed sensitivity at all concentrations. Twelve samples showed sensitivity at 8 mcg/ml. Eleven samples showed sensitivity at 4 mcg/ml, 8 samples showed sensitivity at 2 mcg/ml, and 5 samples showed sensitivity even at 1 mcg/ml. Mean MIC value of G. lucidum spore powder for P. intermedia obtained was 3.62 mcg/ml.
Conclusion: G. lucidum with its multipotential bioactivity could be used as an anti-microbial, in conjunction with conventional therapy in periodontal disease.

Keywords: Anaerobic bacteria, anti-microbial agents, Ganoderma lucidum, Gram-negative bacteria, periodontitis, Prevotella intermedia


How to cite this article:
Nayak RN, Dixitraj P T, Nayak A, Bhat K. Evaluation of anti-microbial activity of spore powder of Ganoderma lucidum on clinical isolates of Prevotella intermedia: A pilot study. Contemp Clin Dent 2015;6, Suppl S2:248-52

How to cite this URL:
Nayak RN, Dixitraj P T, Nayak A, Bhat K. Evaluation of anti-microbial activity of spore powder of Ganoderma lucidum on clinical isolates of Prevotella intermedia: A pilot study. Contemp Clin Dent [serial online] 2015 [cited 2019 Jul 19];6, Suppl S2:248-52. Available from: http://www.contempclindent.org/text.asp?2015/6/6/248/166834


   Introduction Top


Periodontitis is a disease that affects the tooth supporting tissues and is characterized by a loss of periodontal attachment including the alveolar bone.[1] The etiology of the disease is multifactorial and bacterial deposits play an essential role in the pathogenesis. The bacteria comprises predominantly of Gram-negative anaerobic rods.[1] Among them, Porphyromonas gingivalis, Prevotella intermedia, Fusobacterium nucleatum, Bacteroides spp., and Selenomonas spp. have been associated with chronic periodontitis. Black-pigmented anaerobes have been considered as the key pathogens in mixed anaerobic infections.[2] In the oral cavity, these bacteria, particularly P. intermedia, play an important role in the onset and subsequent development of the polymicrobial periodontal diseases.[3] Despite this, there are only a few published studies on this organism.

Ganoderma lucidum, a large, red mushroom, belonging to the class Basidiomycetes, is unique in its range of pharmacogenic components. Various parts of this mushroom, namely the mycelia, spores, and fruit body possess medicinal properties. The major physiologically active constituents in G. lucidum are polysaccharides, peptidoglycans, and triterpenes.[4],[5]

Spore powder of G. lucidum is widely used in Traditional Chinese Medicine.[6] Scientific research has proved that spore powder of G. lucidum has demonstrated multiple functions such as blockade of release of histamine, inhibition of an overstimulated immune system, and a regulatory effect on cellular and humoral immunity.[7],[8]

Most of the antibiotics and antivirals tend to exhibit undesired side effects and drug resistance. The mutation of the strains also complicates the issue. This makes the development of new agents an urgent necessity. Research today focuses at discovering agents that specifically inhibit viral and bacterial multiplication without affecting the normal cells. Researchers today are fast moving to tap the potential of medicinal plants and fungi with their antibacterial and antiviral activity.[9],[10]

The objective of this study was to evaluate whether or not spore powder of G. lucidum has anti-microbial activity on P. intermedia.


   Materials and Methods Top


This study included 20 patients diagnosed with chronic periodontitis. The criteria of the patients selected, included presence of bleeding on probing, probing depth ≥5 mm, and clinical attachment loss ≥3 mm. Patients on any antibiotic therapy and periodontal treatment up to 3 months prior to this study were excluded. Patients with any systemic diseases or conditions, pregnant, lactating women, and smokers were excluded from the study. Written informed consent was obtained from each subject enrolled in the study. The ethical clearance for the study was obtained from the Institutional Ethics Committee.

Pooled subgingival plaque samples were collected using sterile curettes from the deepest sites of the periodontal pockets. The samples were transported in 1 mL of reduced transport fluid. Culture procedures were carried out in the Laboratory of Molecular Biology and Immunology at our institute.

Methodology

Isolation of the organism

The plaque sample was vortexed to break down the plaque and then the sample was inoculated in blood agar with incorporated kanamycin. Then, the plates were kept in anaerobic jar for 48–72 h at 37°C. P. intermedia showed black-reddish minute colony morphology and so the plates incubated were then removed and examined for black-reddish minute colonies. Gram staining and sugar fermentation tests were performed to confirm the organism. Confirmed colonies were subcultured in thioglycollate broth to grow the organisms.[11] These organisms were then used for minimum inhibitory concentration (MIC) procedure.

Preparation of the stock solution

The stock solution was prepared by taking 1 ml of sterile saline in a sterile vial in which 10 mg of the G. lucidum spore powder was added. From this, 100 μl stock solution was used.

Minimum inhibitory concentration procedure

Ten tubes, each having 100 μl thioglycollate broth were used for the MIC procedure. One hundred μl stock solution was added in the first MIC tube containing 100 μl broth. After mixing well, 100 μl solution from this tube was transferred to the second MIC tube. This process was continued till the 10th tube. From the 10th tube which was the last tube, 100 μl final solution was discarded. The concentrations of the aqueous extract achieved by this serial dilution method were as following - 500, 250, 125, 62.5, 31.25, 16, 8, 4, 2, and 1 mcg/ml.[12] One hundred μl isolated strain of P. intermedia was added to each of the 10 such prepared MIC tubes with varying concentrations such that the final volume per tube was 200 μl. The tubes were then incubated at 37°C for 48–72 h. After the incubation period, by visual inspection of the tubes, the MIC values were determined. With the test group, positive and negative controls were put up. The positive control showing turbidity constituted broth and the bacterial strain [Figure 1]. The negative control containing broth and extract appeared clear [Figure 2]. The MIC value was obtained by visualizing each series of tubes, and the last tube with clear supernatant was taken as the MIC value. The clear supernatant was considered to be without any growth. Turbidity in the MIC tube indicated growth of the bacteria implying that the bacteria were resistant to the aqueous extract of the powdered spores of G. lucidum.
Figure 1: Turbidity implying the presence of bacterial growth

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Figure 2: Clear solution implying the absence of any growth of bacteria

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   Results Top


[Table 1] shows that 13 out of the 20 clinical samples were tested that showed sensitivity at various concentrations ranging from 500 to 1 mcg/ml.
Table 1: MIC of aqueous extract of the powdered spores of Ganoderma lucidum for Prevotella intermedia

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In our study, aqueous extract of the powdered spores of G. lucidum demonstrated anti-microbial activity on P. intermedia. Thirteen out of the 20 clinical samples were tested that showed sensitivity at various concentrations. Sensitivity was tested from 500 to 1 mcg/ml. Thirteen samples were sensitive at 16 mcg/ml. Twelve samples demonstrated sensitivity at 8 mcg/ml, 11 samples at 4 mcg/ml, and 8 samples at 2 mcg/ml. Five samples exhibited sensitivity even at 1 mcg/ml. The mean MIC value of an aqueous extract of the powdered spores of G. lucidum for P. intermedia obtained was 3.62 mcg/ml.


   Discussion Top


P. intermedia has been implicated in extra-oral and intra-oral infections. Extra-oral infections caused by P. intermedia are mainly nasopharyngeal infection and intra-abdominal infection.[13]P. intermedia belonging to the orange complex of secondary colonizers is an important pathogen involved in the pathogenesis of periodontal disease and the organism contributes for the disease progression along with other organisms. In our study, we tested the anti-microbial activity of an aqueous extract of the spores of G. lucidum against the clinical isolates of P. intermedia. Out of the 20 clinical samples tested, 13 exhibited sensitivity at various concentrations. Mean MIC value of the aqueous extract of the powdered spores of G. lucidum for P. intermedia obtained was 3.62 mcg/ml [Graph 1]. Activity of the aqueous extract of the spores of G. lucidum at 500 mcg/ml showed that 65% of organisms were sensitive and 35% of organisms were resistant [Graph 2]. Activity of the aqueous extract of the spores of G. lucidum at 16 mcg/ml showed that 65% of organisms were sensitive and 35% of organisms were resistant [Graph 3].







Yoon et al.[14] studied the antibacterial activity of the spores of G. lucidum against Gram-positive and Gram-negative bacteria by serial broth dilution method. Against standard strains and among five species of Gram-positive bacteria tested, the most prominent anti-microbial activity of G. lucidum was shown in Micrococcus luteus at a MIC of 0.75 mg/ml. Anti-microbial activity of G. lucidum was tested against the 10 species of Gram-negative bacteria and the strongest antibacterial activity was shown against Proteus vulgaris and Escherichia coli at MIC values of 1.25 mg/ml and 1.75 mg/ml, respectively. In this study, the anti-microbial activity was tested against the standard strains of various Gram-positive and Gram-negative organisms, but in our current study, clinical isolates of P. intermedia were tested. In another study by Nayak et al.[15] against the standard strains of Staphylococcus aureus, E. coli, Enterococcus faecalis and Klebsiella pneumoniae, and antibacterial effect of the aqueous extract of the spore powder of G. lucidum were studied. The results suggested that the above-mentioned microorganisms were sensitive and the MIC value for S. aureus was 125 mcg/ml, E. coli was 125 mcg/ml, E. faecalis was <2 mcg/ml, and for K. pneumoniae the MIC was 62.5 mcg/ml. In our current study, mean MIC value obtained was 3.62 mcg/ml indicating that P. intermedia was sensitive even at this low concentration. Studies demonstrated that antibacterial components present in G. lucidum such as peptidoglycans were able to inhibit Gram-positive and Gram-negative bacteria.[16],[17],[18] Some other investigators through their research suggested that tissue and cellular damage following infections may be decreased by the immunosuppressive activity of G. lucidum.[16],[17],[18]

According to Gao et al. G. lucidum and other Ganoderma species often in combination with chemotherapeutic agents have been used to treat various bacterial diseases and found that polysaccharide components were the principal bioactive components which play an important role in the antibacterial activity.[16] Smania et al. observed maximum antibacterial activity of methyl australate, a derivative from G. lucidum against E. coli and Pseudomonas aeruginosa followed by S. aureus and the least zone of inhibition was recorded for Bacillus species.[19] In our current study, property of individual components of G. lucidum were not studied, but the anti-microbial property of spore powder as a whole was assessed. Klaus and Miomir have studied the influence of various extracts isolated from G. lucidum on E. coli, Bacillus species, S. aureus, and Salmonella species. The aqueous fruiting body extract showed the maximum zone of inhibition against Bacillus species while least zone of inhibition was reported for E. coli and Salmonella species.[20] In our study, the anti-microbial activity of only the spore powder was tested against P. intermedia. Cowan reported that the most active components present in the mushroom are generally water insoluble, expecting that low polarity organic solvents would yield a more active extract,[21] In our study, an aqueous extract was used. Yet without an organic solvent, excellent MIC activity up to 1 mcg/ml against P. intermedia was demonstrated. This opens up avenues for more studies on the anti-microbial effect of organic solvent extract of the spore powder of G. lucidum on P. intermedia which could possibly show even more enhanced MIC activity.

Our study demonstrates anti-microbial activity in vitro only. However, since G. lucidum is known to have immune modulating activity,[4],[5] its effectiveness clinically could be much better and in vivo studies would probably demonstrate better control of infections due to synergistic actions. Many active substances are present in mushrooms and these individually contribute to the bioactivity observed in vitro and in vivo. Some roles of individual constituents are known whereas some still unknown. In summary, G. lucidum with its multipotential constituents may play an important role as an adjunct in the management of infectious and inflammatory diseases, periodontal disease being one. To the best of our knowledge, this is probably the first study to assess the anti-microbial activity of G. lucidum against clinical isolates of P. intermedia isolated from chronic periodontitis patients.


   Conclusion Top


G. lucidum, with its multipotential bioactivity, can be used as an anti-microbial, as an adjunct to conventional therapy in periodontal disease. Our study is probably the first of its kind to demonstrate antibacterial activity of the spore powder of G. lucidum against P. intermedia. Therefore, topical application of spore powder or aqueous or organic solvent extract directly on the infected tissues could be an efficient drug delivery system for the control of P. intermedia associated periodontitis. Systemic administration of the spore powder of G. lucidum with its proven immunomodulatory [4],[5] activity could possibly enhance the response to local drug delivery.

Source of Support:

Nil.

Conflict of Interest:

None declared.

 
   References Top

1.
Van Winkelhoff AJ, Loos BG, van der Reijden WA, van der Velden U. Porphyromonas gingivalis, Bacteroides forsythus and other putative periodontal pathogens in subjects with and without periodontal destruction. J Clin Periodontol 2002;29:1023-8.  Back to cited text no. 1
    
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Socransky SS, Haffajee AD. Periodontal microbial ecology. Periodontol 2000 2005;38:135-87.  Back to cited text no. 3
    
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Boh B, Berovic M, Zhang J, Zhi-Bin L. Ganoderma lucidum and its pharmaceutically active compounds. Biotechnol Annu Rev 2007;13:265-301.  Back to cited text no. 4
    
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Zhou X, Lin J, Yin Y, Zhao J, Sun X, Tang K. Ganodermataceae: Natural products and their related pharmacological functions. Am J Chin Med 2007;35:559-74.  Back to cited text no. 5
    
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Huie CW, Di X. Chromatographic and electrophoretic methods for Lingzhi pharmacologically active components. J Chromatogr B Analyt Technol Biomed Life Sci 2004;812:241-57.  Back to cited text no. 6
    
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Jiang ZY, Lin C, Liu XC, et al. Effects of Ganoderma lucidium polysaccharide on humoral immune function in mice. J Jinan Univ (Health Sci) 2003;24:51-3.  Back to cited text no. 7
    
8.
Ho YW, Yeung JS, Chiu PK, Tang WM, Lin ZB, Man RY, et al. Ganoderma lucidum polysaccharide peptide reduced the production of proinflammatory cytokines in activated rheumatoid synovial fibroblast. Mol Cell Biochem 2007;301:173-9.  Back to cited text no. 8
    
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Wasser SP, Weis AL. Medicinal properties of substances occurring in higher basidiomycetes mushrooms: Current perspectives. Int J Med Mushrooms 1999;1:31-62.  Back to cited text no. 9
    
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Zhong JJ, Xiao JH. Secondary metabolites from higher fungi: Discovery, bioactivity, and bioproduction. Adv Biochem Eng Biotechnol 2009;113:79-150.  Back to cited text no. 10
    
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Citron DM, Poxton IR, Baron EJ. Bacteriodes, Porphyromonas, Prevotella, Fusobacterium and other Gram-negative rods. In: Murray PR, Baron EJ, Jorgensen JH, Landry ML, Pfaller MA, editors. Manual of Clinical Microbiology. 9th ed., Ch. 58. Washington, D.C.: ASM Press; 2007. p. 911-32.  Back to cited text no. 11
    
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Schwalbe R, Steele-Moore L, Goodwin AC. Antimicrobial Susceptibility Testing Protocols. New York: CRC Press; 2007.  Back to cited text no. 12
    
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Mättö J, Asikainen S, Väisänen ML, Rautio M, Saarela M, Summanen P, et al. Role of Porphyromonas gingivalis, Prevotella intermedia, and Prevotella nigrescens in extraoral and some odontogenic infections. Clin Infect Dis 1997;25 Suppl 2:S194-8.  Back to cited text no. 13
    
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Yoon SY, Eo SK, Kim YS, Lee CK, Han SS. Antimicrobial activity of Ganoderma lucidum extract alone and in combination with some antibiotics. Arch Pharm Res 1994;17:438-42.  Back to cited text no. 14
    
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Nayak RN, Nayak A, Bhat K. Antimicrobial activity of aqueous extract of spore powder of Ganoderma lucidum – An in vitro study. J Int Oral Health 2010;2:68-74.  Back to cited text no. 15
    
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Gao Y, Zhou SH, Huang M, Xu A. Antibacterial and antiviral value of the genus Ganoderma P. Karst. Species (Aphyllophoromycetideae): A review. Int J Med 2003;5:235-46.  Back to cited text no. 16
    
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Smith J, Rowan N, Sullivan R. Medicinal Mushrooms: Their Therapeutic Properties and Current Medical Usage with Special Emphasis on Cancer Treatment; Special Report Commissioned by Cancer Research UK. Vol. 5. The University of Strathclyde in Glasgow; 2003. p. 235-46.  Back to cited text no. 17
    
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Suay I, Arenal F, Asensio FJ, Basilio A, Cabello MA, Díez MT, et al. Screening of basidiomycetes for antimicrobial activities. Antonie Van Leeuwenhoek 2000;78:129-39.  Back to cited text no. 18
    
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Smania A Jr, Monache FD, Smania EF, Cuneo RS. Antibacterial activity of steroidal compounds isolated from Ganoderma applanatum (Pers.) Pat. (Aphyllophoromycetideae) fruit body. Int J Med Mushrooms 1999;1:325-30.  Back to cited text no. 19
    
20.
Klaus A, Miomir N. Influence of the extracts isolated from Ganoderma lucidum mushroom on some microorganisms. Proc Natl Sci Matica Srpska Novi Sad 2007;113:219-26.  Back to cited text no. 20
    
21.
Cowan MM. Plant products as antimicrobial agents. Clin Microbiol Rev 1999;12:564-82.  Back to cited text no. 21
    


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