In vitro Antifungal Effects of Hesperetin and Silibinin

Background: Increasing resistance of microorganisms against available antimicrobial agents is of major concern among scientists and clinicians worldwide. To overcome the resistance and other drawbacks of the current antimicrobial drugs and to obtain more efficacious drugs, an antimicrobial drug having a novel mode of action should be developed. The flavonoid compounds have demonstrated for their therapeutic potential as antimicrobial agents previously. Aim: To evaluate the antifungal efficacy of plant derived compounds such as hesperetin (HSP) and silibinin (SBN) compounds in vitro. Materials and Methods: In this study, we evaluated the antifungal effect of two plant derived flavonoid compounds i.e., SBN and HSP against Candida albicans, Candida tropicalis, Malassezia furfur, Aspergillus niger, Penicillium oxalicum. The disk diffusion test was performed to test the antifungal effect of HSP and SBN with three different concentrations i.e., 15, 20, 25 mg/ml of HSP and SBN. The antifungal efficacy was studied for 72 h. Results: Our results indicate that SBN has significant antifungal effect than that of HSP. The high sensitivity of these compounds was found against C. albicans, C. tropicalis. Conclusion: These drugs could be good candidate to combine with regular antimicrobial agents.


INTRODUCTION
A new spectrum of human fungal infections is increasing due to increased cancer, AIDS, and immunocompromised patients.The increased use of antifungal agents also resulted in the development of resistance and it makes necessary to discover new classes of antifungal compounds to cure fungal infections. 1 Candida albicans is a polymorphic fungus which is the predominant cause of invasive candidiasis, superficial and deep tissue fungal infections.In particular, the invasive candidiasis than on scalps without dandruff. 9Penicillium oxalicum, a plant pathogen and it has been recently found that causes an opportunistic fungal infection in patients with acute myeloid leukemia, diabetes mellitus, and chronic obstructive pulmonary disease. 10ncreasing resistance of microorganisms against available antimicrobial agents is of major concern among scientists and clinicians worldwide.It is reported that pathogenic viruses, bacteria, fungi, and protozoa are more and more difficult to treat with the existing drugs due to development of resistant. 11To overcome the drawbacks of the current antimicrobial drugs and to obtain more efficacious drugs, an antimicrobial drug having a novel mode of action should be developed.Plant-derived flavonoids are a large group of naturally occurring phenylchromones found in fruits, vegetables, tea, and wine.Drugs derived from natural sources play a significant role in the prevention and treatment of human diseases. 12Hence, in this study, we tested the antifungal effect of hesperetin and silibinin against different fungal species.Silibinin (SBN) is a flavonolignan extracted from the seeds of a medicinal plant Silybum marianum (Asteraceae), commonly called "milk thistle". 13esperetin (HSP) is isolated from citrus fruits that have previously been found to exert anticancer activity through a variety of mechanisms. 14Both HSP and SBN (Figure 1) have been reported to have wide spectrum of biological activities.5][16][17] However, studies regarding their antifungal effect are scanty.Hence, in the present study we evaluated the antifungal efficacy of hesperetin and silibinin against different fungal species.

Experimental design
The fungal cultures in Sabouraud's Dextrose Agar plate were filled with three different concentrations i.e., 15, 20, 25 mg/ml of HSP and SBN.The antifungal efficacy was studied for 72 h.

Disk diffusion method
The disk diffusion test was performed to test the antifungal effect of HSP and SBN . 18On sterile plates containing Sabouraud's Dextrose Agar, the fungal cultures were swabbed.Then, sterile discs (made from Whatman filter paper) each about 5 mm diameter impregnated with the HSP and SBN separately was placed on the inoculated plates.Similarly, each plate was placed with a sterile disc.The plates were incubated at 28°C for 72 h for evaluation.The diameter of inhibition zones formed around the disk was measured in millimeter.The test was carried out in triplicate in each run of the experiments.

Antifungal activity of HSP
The results from this study clearly show that HSP has antifungal efficacy only against C. albicans, C. tropicalis, Malassezia furfur species.Hesperetin has produced dose-dependent inhibition of C. tropicalis and Malassezia furfur fungal growth, whereas, in low concentrations hesperetin did not show any effect in the growth of C. albicans.The maximum inhibitory effect was seen at maximum dose tested in this study i.e., 25 mg/ml.This flavonoid exhibits higher efficacy against C. tropicalis and Malassezia furfur when compared to C. albicans (Figure 2).Hesperetin did show any significant change in the growth of Aspergillum niger and Penicillium oxalicum.

Antifungal activity of SBN
We also tested the antifungal efficacy of the SBN against the C. albicans, C. tropicalis, Malassezia furfur, Aspergillum niger and Penicillium oxalicum.The results obtained from present study shows that SBN has antifungal efficacy only against C. albicans, C. tropicalis, Malassezia furfur among fungal organism tested.Silibinin shows high sensitivity against Candida species i.e., C. albicans, C. tropicalis when compared to other species (Figure 3).In fact, SBN did show any significant change in the growth of Aspergillum niger and Penicillium oxalicum.The maximum effect was observed with higher concentration of SBN used in this study.

DISCUSSION
Human fungal pathogens cause invasive infections are hidden killers, taking lives of 1.5 million people every year. 19Therapeutic applications of immunosuppressive drugs, the use of broad spectrum antibiotics against various clinical settings are responsible for an increasing number of immunocompromised patients and consequent opportunistic fungal infections worldwide.An impaired immune system provides favorable conditions for pathogenic and non-pathogenic micro-organisms. 3esperetin, a flavanone glycoside predominantly found in citrus fruits, exhibits a wide array of biological properties. 20Studies have shown the antimicrobial effect of HSP especially against bacterial strains and came out with promising results. 21In the present study, HSP shows antifungal activity against Candida species i.e., C. albicans (only in higher concentration) and C. tropicalis.The HSP treatment also shows inhibitory effect against Malassezia furfur.On the other hand antimicrobial effect of SBN has also been concretely proved against wide variety of bacterial  strains. 17,22The antifungal effect of SBN found in this study is marginally greater than HSP.Moreover, SBN shows higher efficacy against C. albicans and C. tropicalis.Flavonoids are hydroxylated phenolic substances synthesized by plants in response to microbial infection.Flavones are phenolic structures containing one carbonyl group and the addition of a 3-hydroxyl group yields a flavonol.Flavonoids have been found to be effective antimicrobial substances against a wide array of microorganisms.Their activity is probably was suggested due to their ability to complex with extracellular and soluble proteins and to complex with fungal cell wall.It also been said that high lipophilic nature of flavonoids also disrupts fungal membranes. 23In light of the above reports it is suggested that the profound antifungal effect of HSP and SBN found in this study could be due to their action on fungal membrane disruption and subsequent inhibition of its growth.Collectively, this study clearly shows that SBN possess significant antifungal efficacy than HSP.It should be brought to light that both these flavonoids show efficacy only against C. albicans, C. tropicalis and Malassezia furfur and did not show any efficacy against the Aspergillum niger and Penicillium oxalicum.Furter, it has to emphasize that in previous studies flavonoid compounds have demonstrated for their therapeutic potential as antimicrobial agents.The synergistic effect of flavonoids combined with commonly utilized antibiotics is well supported in the literature and this strategy is emerging as an important complementary treatment modality in research. 17Accordingly, the present study would be of immensely useful to identify some of the efficacious plant derived flavonoid compounds which possess antifungal effect.

CONCLUSION
In conclusion, this study clearly indicates the fact that SBN has the significant antifungal effect against Candida species when compared to HSP.These drugs could be good candidate to combine with regular antimicrobial agents.However, their exact role behind the antifungal effect is further warranted.
Hesperetin (C16H14O6; CAS No. 69097-99-0) was purchased from Sigma Chemicals, India.Silibinin (C 25 H 22 O 10 ; CAS No. 22888-70-6) was purchased from Sigma Chemicals, India.It is a mixture of two diastereomers (silybin A and silybin B).All other chemical used in this study purchased locally and were of analytical grade.