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2TMR Integrative Medicine  2019, 3: e19011.       DOI: 10.12032/TMRIM201903011
    
Effect of gloriosa superba in combination with fluconazole on anti-Candida activity against biofilm development and mature biofilm growth patterns
Somwanshi Sachin M1,*(), Dhawale Shashikant C1, Zore Gajanan B2, Choudhary Harshad W1
1Department of Pharmacology, School of Pharmacy, Swami Ramanand Teerth Marathwada University, Vishnupuri, Nanded- 431606, (M.S.) India
2School of Life Sciences, Swami Ramanand Teerth Marathwada University, Vishnupuri, Nanded- 431606, (M.S.) India.
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Highlights:

Use of G. Superba L. alone or in combination with available antifungal drugs would be a novel approach against drug resistant biofilms of C. albicans. In future it will be useful to avoid side effects associated with high dosages and long term usage of the conventional antifungal drugs during anti- biofilm therapy.

Abstract

This study was to evaluate the efficacy of methanolic extract of G. superba L in combination with Fluconazole against biofilm development and mature biofilms of C. albicans. Synergism between MEGS and Fluconazole combination against biofilm formation was evident with FICI of 0.187. Combination of MEGS and Fluconazole did not have synergistic potential against mature biofilm growth, evidenced in FICI of 0.916. MIC of standard Fluconazole was found to be 0.5 and >0.5 in biofilm development and mature biofilm respectively.



Key wordsG. superba L.      FICI      MTT      Germ tube assay     
Published: 09 June 2019
Corresponding Authors: Sachin M Somwanshi   
E-mail: sachinsomwanshi007@gmail.com
Cite this article:

Somwanshi Sachin M, Dhawale Shashikant C, Zore Gajanan B, Choudhary Harshad W. Effect of gloriosa superba in combination with fluconazole on anti-Candida activity against biofilm development and mature biofilm growth patterns. 2TMR Integrative Medicine, 2019, 3: e19011.  

URL:

https://www.tmrjournals.com/im/EN/10.12032/TMRIM201903011     OR     https://www.tmrjournals.com/im/EN/Y2019/V3/IVolume 3/88888

Sr. no. Conc. of extract in mg/ml Average mean Percentage inhibition growth compared to control mean
Total cell Total germ tube
I II I II Mean ± SD
1 0 104 80 83 71.42 77.21 5.79
2 0.0025 149 29 19.13 19 19.06 0.065
3 0.05 133 15 17 7.45 12.22 4.775
4 1 82 2 4 1.11 2.55 1.445
5 2 86.5 0.5 0 0.88 0.44 0.44
6 4 73.5 0 0 0 0 0
Table 1 Effect of MEGS on germ tube growth of C. albicans
Figure 1 Germ tube cell growth of C. albicans ATCC 90028, in presence of various concentrations of MEEGS. Germ tube cell growth was analyzed as absorbance of cell density at 595 nm. Where absorbance of colored end product was measured at 450 nm and compared with that of control biofilms
Figure 2 Microphotographs showing inhibition of C. albicans Germ tube formation in presence of MEGS A) Control without MEGS; B) Treatment with 1mg.mL-1 of MEGS; C) Germ tube growth in presence 2mg.mL-1 D) Concentration 4mg/ml of MEGS. The complete inhibition of biofilm and presence of only a layer of yeast form cells in fig. C and fig. D and lowering in density of Germ tube growth in fig. B, compared to that of dense network of biofilm in control, fig. A

Sr. No
Concentration of MEGS
(In mg/ml)
Inhibition of growth (%)
I II Mean SD
1 0 100 100 100 0
2 0.0025 95 92 93.5 1.5
3 0.05 88 80 84 4
4 1 75 70 72.5 2.5
5 2 55 45 50 5
6 4 35 29 32 3
Table 02 MIC of MEGS on planktonic growth
Sr. no. Conc. of
Extract
Mg/ml
Absorbance at 450 nm indicating growth Percentage of growth compared to control
I II I II Mean ± SD
1 0 0.727 0.554 100 100 100 0
2 0.0025 0.589 0.792 81.01 142.96 111.98 30.975
3 0.05 0.556 0.655 76.47 118.23 97.35 20.88
4 1 0.358 0.45 49.24 81.22 65.23 15.99
5 2 0.212 0.205 29.16 37 33.08 3.92
6 4 0.196 0.180 26.96 32.49 29.72 2.765
Table 03 Effect of MEGS on Biofilm development of C. albicans
Figure 3 Development of biofilm growth of C. albicans ATCC 90028, in presence of various concentrations of MEEGS. Development of biofilm growth as analyzed as absorbance of cell density at 595 nm. Biofilm developments were quantified using MTT assay, where absorbance of colored end product was measured at 450 nm and compared with that of control biofilms
Figure 4 Microphotographs showing inhibition of C. albicans Biofilm development in presence of MEGS. A) Control without MEGS; B) Treatment with 1mg.mL-1 of MEGS; C) Biofilm growth in presence 2mg.mL-1 D) Concentration 4mg/ml of MEGS. The complete inhibition of biofilm and presence of only a layer of yeast form cells in fig. D and lowering in density in fig. C, compared to that of dense network of biofilm in control, fig. A
Sr. no. Conc. of
Extract
Mg/ml
Absorbance at 450 nm indicating growth Percentage of growth compared to control
I II I II Mean ± SD
1 0 0.790 0.689 100 100 100 0
2 0.5 0.705 0.675 89.24 97.96 93.6 4.36
3 1 0.691 0.635 87.35 92.16 89.75 2.40
4 2 0.557 0.545 70.50 79.10 74.8 4.3
5 4 0.430 0.405 54.43 58.78 56.60 2.17
6 8 0.350 0.314 44.30 45.57 44.93 0.63
Table 04 Effect of MEGS on Mature Biofilm of C. albicans
Figure 5 Mature of biofilm growth of C. albicans ATCC 90028, in presence of various concentrations of MEEGS. Mature biofilm growth as analyzed as absorbance of cell density at 595 nm. Mature biofilm were quantified using MTT assay, where absorbance of colored end product was measured at 450 nm and compared with that of control biofilms.
Figure 6 Microphotographs showing inhibition of C. albicans Mature Biofilm in presence of MEGS. A) Control without MEGS; B) Treatment with 1mg/ml of MEGS; C) Biofilm growth in presence 2mg/ml. D) Concentration 4mg/ml of MEGS. The complete inhibition of biofilm and presence of only a layer of yeast form cells in fig. D and lowering in density of growth in fig. C, compared to that of dense network of biofilm in control fig. A
Growth of C. albicans MIC (mg/ml) FICI Effect
Alone In combination
Fluconazole MEGS Fluconazole MEGS
Biofilm development 0.5 4 0.031 0.25 0.187 Synergistic
Mature biofilm >0.5 8 0.25 4 0.916 Indifference
Table 05 Effect of MEGS on Development and mature Biofilm Candida albicans in combination with Fluconazole
Figure 7 Microphotographs showing inhibition of C. albicans biofilm development in combination with Fluconazole. A) Control without MEGS; B) MEGS alone 4mg/ml conc. C) Fluconazole alone >0.5 mg/ml concentration D) Complete inhibition of biofilm presence of Combination (0.25Fluco. + 4 mg/ml MEGS) and the lowering in density of biofilm growth in fig. B, compared to that of dense network of biofilm in control fig. A
Figure 8 Microphotographs showing inhibition of C. albicans Mature biofilm formation in combination with Fluconazole. A) Control without MEGS; B) MEGS alone 8mg/ml conc. C) Fluconazole alone >0.5 mg/ml concentration D) Complete inhibition of biofilm presence of Combination (0.25Fluco.+ 4 mg/ml MEGS) and the lowering in density of biofilm growth in fig. B, compared to that of dense network of biofilm in control fig. A
Growth Parameter MICs of Fluconazole
Planktonic growth 0.2 mg/ml
Biofilm Formation 0.5 mg/ml
Mature biofilm > 0.5 mg/ml
Table 06 Effect of Fluconazole on C. albicans
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