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www.gbtrp.com International Journal of Biological Technology (2010) 1(3):5-9 ISSN: 0976 - 4313

Original Article

Comparison of In vitro susceptibility of melanised and non – melanised Cryptococcus neoformans to Cinnamomum zeylanicum and Murraya paniculata S. Gouse Basha1, S. Dawood Sharief1, and S. Gokul Shankar2 1 School of Environmental Sciences, P.G. & Research Department of Zoology, The New College, Peters Road, Royapettah, Chennai - 600014, India, 2Faculty of Medicine, AIMST University, Kedah, Malaysia. *Corresponding Author: E-mail: gous79@yahoo.co.in. Received:14.07.2010;Revised:25.10.2010;Accepted:29.11.2010;Published:01.12.2010 Abstract In the present study the inhibitory activity of methanolic extracts from the bark of Cinnamomum zeylanicum (family Lauraceae) and leaves of Murraya paniculata (family: Rutaciae) were tested against 10 environmental isolates of 8 days old melanised and non–melanised Cryptococcus neoformans keeping Amphotericin B as control. Two methods namely, Disc diffusion assay and Minimum inhibitory concentration (MIC) were used according to the guidelines of the Clinical and Laboratory Standard Institute (CLSI). Methanolic extracts of both Cinnamomum zeylanicum and Murraya paniculata used against melanised and non-melanised C. neoformans isolates showed anticryptococcal activity. Keywords: Cryptococcus neoformans, Melanised, Non-melanised, 4-dihydroxy phenylalanine.

Introduction Cryptococcal infections have increased dramatically over the last years. This high incidence can be due to the explosion of acquired immune deficiency syndrome (AIDS) epidemic around the world and the use of more potent immunosuppressive agents by increasing numbers of solid organ transplant recipients (Mitchell and Perfect, 1995; Buchanan et al., 1998;Arora et al., 2001; Khan et al., 2003). Cryptococcal meningitis, the most common infection of cryptococcosis is usually chronic and uniformly fatal if untreated. Antifungal drugs, such as polyene macrolides (Amphotericin B) and azoles (itraconazole and fluconazole) are currently used in antifungal therapies with certain limitations due to side effects as toxicity and emergence of resistant strains (Saag et al., 2000). Melanin has been called an “an antifungal resistance factor,” given its ability to reduce the susceptibilities of melanised cells to antifungal drugs (Ikeda et al., 2003). The studies with amphotericin and caspofungin revealed that the activities of these drugs against melanised cells were reduced by 55 and 7%, respectively, relative to their activities against non - melanised cells (Van duin et al., 2002). Melanised C. neoformans strains exhibited reduced susceptibilities to higher concentrations of fluconazole (Ikeda et al., 2003). All these indicate the need for a © Gayathri Teknological Publication

permanent search and development of new drugs. Plants that are traditionally used in the treatment of fungal infections or related ailments could be a good source for new, safe, biodegradable and renewable antifungal drugs. Cinnamon oil is most effective against all the yeast species studied by Abdel-Mallek et al. (1994). Cinnamon bark oil is rich in cinnamaldehyde, caryophyllene, linalool and other terpenes (Paranagama, 1991). Many researchers have reported cinnamon and clove oils as a good source of antifungal compounds (Karig, 1975; Ross, 1976; Bullerman et al., 1977). The oils extracted from the leaves and fruits of Murraya paniculata exhibited strong antifungal activity against Candida albicans, and showed a modest antibacterial activity against Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus and Sarcina lutea. El-Sakhawy et al. (1998) found that the extracts of M. paniculata found active against Candida albicans ,C. tropicalis and C. luteolus.

Materials and Methods Fresh bark of the Cinnamomum zeylanicum and Murraya paniculata leaves were purchased commercially in Chennai and the taxonomic identities of these plants were confirmed at Research and Development Center, Cholayil Private Limited, Chennai. Ten isolates of C. neoformans obtained from 5


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pigeon droppings collected in Chennai which were confirmed as C. neoformans var neoformans by Canavanine glycinebromothymol blue medium (CGB) (KwonChung et al., 1982) were used. Melanisation of C. neoformans isolates Ten isolates of C. neoformans were grown in 2 sets of 10 conical flasks containing Defined Minimal Medium (DMM) (15mM glucose, 29.4mM KH2PO4, 10mM MgSO4, 13mM glycine and 3.0 µM vitaminB1, pH 5.5). To one set of conical flasks containing DMM, 1.0mM L-3, 4-dihydroxy phenylalanine (LDopa) is added to obtain melanised cells and another set of conical flasks without L-Dopa or obtaining non-melanised cells of C. neoformans. The cultures were incubated at 30oC in a rotary shaker (approx150rpm) for 8 days to obtain melanised and non – melanised cells. In vitro susceptibility test Activated cultures of 10 isolates of 8 days old melanised and non-melanised C. neoformans in Minimal media were adjusted to 1x 108 CFU/ml as per McFarland standard. 0.1 ml of both the inoculums were spread onto separate Sabouraud dextrose agar in the sterile petriplates using sterile swab. Sterile filter paper discs (7.0 mm diameter) were impregnated with 100 µg/disc, 200µg/disc and 300µg/disc of the plant extracts dissolved in 100% DMSO (dimethylsulphoxide) and dried. The discs were placed on fungal seeded plates and incubated at 370C for 48 h. Discs impregnated with only 100% DMSO served as the negative control. As a positive control, Amphotericin B (100 units/disc) was used. Following an incubation period of 48 h, plates were removed from the incubator and antifungal activity was evaluated by measuring zones of inhibition of fungal growth. The whole experiment was performed four times to minimize error. MIC determination Testing of the plant extracts for MIC was done by the broth macrodilution method. 10 isolates of 8 days old melanised and nonmelanised C. neoformans were diluted to a concentration of approximately 1x 108 CFU/ml (verified by colony counts in SDA) in separate test tubes. For susceptibility experiments, both melanised and non-melanised cells were

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suspended in minimal media were exposed to various concentrations of plant extracts. A constant volume of the melanised and nonmelanised cells in minimal media was added to each tube containing the serial dilution of the methanol extract to reach final concentrations of 16, 8, 4, 2, 1, 0.5, 0.25 and 0.125mg/ml respectively. The tubes were incubated at 350C for 72 h. Amphotericin B was used as control against tested fungi. After incubation, C. neoformans cells were plated on Sabouraud dextrose agar plates to determine their viabilities, as measured by the number of colonies. MIC was defined as the lowest concentration that did not yield visual growth. Percent survival was obtained by comparing the number of colonies relative to the number of cells not exposed to plant extracts and Amphotericin B. All experiments were repeated four times. Statistical analysis Data from the plant extracts inhibiting melanised and non-melanised C. neoformans were analyzed by Student t-test using SPSS 17 Version. P values were calculated. P values < 0.05 were considered significant. Results were reported as means ± standard deviations. In each experiment, the number of colonies in the parent suspension of control cells was considered to represent 100% survival.

Results and Discussion Bioassay results for disc diffusion and MIC of plant extracts used against 10 isolates of 8 days old melanised and non-melanised C. neoformans are listed in tables’ 1 and 2. Disc diffusion assay The mean ZOI was 15mm, 13mm and 11mm at concentrations of 300, 200 and 100 µg/disc respectively for methanolic extract of C.zeylanicum for non - melanised C. neoformans but it was 12, 11 and 10mm for methanolic extract of C. zeylanicum for melanised C. neoformans. The mean ZOI was 13mm, 11mm and 10mm at concentrations of 300, 200 and 100 µg/disc respectively for methanolic extract of M. paniculata for non melanised C. neoformans but it was 12, 10 and 9 mm for methanolic extract of M. paniculata for melanised C. neoformans. The ZOI for Amphotericin B Control was 15mm.

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Table -1. In vitro susceptibility of 10 isolates of C. neoformans to plant extracts (Disc diffusion test) Amphotericin B Methanol extract used used (µg/disc) Plants used (units/disc) Isolate Part C. neoformans Nos. used 300 200 100 100 Average ZOI (mm) of 10 isolates 1- 10

Cinnamomum zeylanicum

Non-Melanised Melanised

Bark Bark

15 12

13 11

11 10

15 15

Murraya paniculata

Non-Melanised Melanised

Leaf Leaf

13 12

11 10

10 9

15 15

Table- 2. In vitro susceptibility of 10 isolates of C. neoformans to plant extracts (MIC test)

Isolate N0s.

1- 10

Plants used

C. neoformans

Part used

Cinnamomum zeylanicum Murraya paniculata

Non-Melanised Melanised Non-Melanised Melanised

Bark Bark Leaf Leaf

Methanol extract Amphotericin B used used (mg / ml) (mg / ml) (0.125 – 16) (0. 00025) MIC range (mg/ml) of 10 isolates 1 0. 00025 1– 2 0.00025 4 0. 00025 4- 8 0.00025

Table-3. Percentage of survival of melanised and non – melanised C. neoformans at different concentrations of Cinnamomum zeylanicum Conc. (mg/ml)

Cn

C. neoformans isolate no. C

2

3

4

5

6

7

8

9

10

M

100

55 ± 2.14 NS

46 ± 5.00 NS

33 ± 2.00 NS

50 ± 3.00 NS

30 ± 2.00 NS

43 ± 1.00 NS

44 ± 2.00 NS

38 ± 5.00 NS

52 ± 2.00 NS

36 ± 4.00 NS

NM

100

49 ± 5.88

41 ± 3.00

30 ± 4.00

46 ± 2.00

28 ± 3.00

40 ± 5.00

41 ± 5.00

32 ± 2.00

48 ± 5.00

32 ± 3.00

M

100

30 ± 1.16 *

24 ± 2.00 NS

17 ± 4.00 *

34 ± 1.00 NS

22 ± 1.00 ***

33 ± 1.00 ***

26 ± 1.00 **

20 ± 1.00 **

44 ± 2.00 ***

26 ± 1.00 ***

0.125

0.25 NM

100

23 ± 5.26

19 ± 6.00

10 ± 2.00

27 ± 6.00

12 ± 1.00

23 ± 2.00

23 ± 1.00

12 ± 2.00

25 ± 1.00

17 ± 1.00

M

100

13 ± 2.93**

14 ± 3.00*

10 ± 3.00**

23 ± 1.00***

14 ± 2.00**

24 ± 1.00***

14 ± 1.00 NS

8± 2.00*

23 ± 2.00***

13 ± 1.00***

NM

100

6± 1.69

5± 2.00

2± 0.89

13 ± 1.00

6± 1.00

13 ± 1.00

12 ± 2.00

3± 1.00

7± 1.00

3± 1.00

M

100

0

0

0

13 ± 1.00***

7± 1.00***

10± 2.00***

11± 2.00***

4± 1.00**

8± 1.00***

0

NM M NM

100

0

0

0

0

0

0

0

0

0

0

100 100

0 0

0 0

0 0

0 0

0 0

0 0

0 0

0 0

0 0

0 0

0.5

1

2

1

Values represent mean ± SDM of 4 measurements *P < 0.05, ** P < 0.01, *** P < 0.001 when Melanised cells are compared with Non-melanised cells NS = Non –significant; Cn – Cryptococcus neoformans; M – Melanised; MN – Non- melanised; C Control

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Table-4: Percentage of survival of melanised and non – melanised C. neoformans at different concentrations of Murraya paniculata Conc. (mg/ml)

C. neoformans isolate no. Cn M

0.125

0.25

C 100

NM

100

M

100

NM

100

M

100

1

2

3

4

5

6

7

8

9

10

62 ± 3.24

55 ± 2.00

56 ± 2.00 NS

61 ± 3.00 NS

59 ± 4.00 NS

48 ± 4.00 NS

47 ± 1.00

66 ± 2.00

NS

NS

58 ± 7.00 NS

46 ± 4.00 NS

42 ± 3.00

43 ± 1.00

60 ± 6.00

49 ± 5.00

39 ± 3..00*

34 ± 6..00 *

55 ± 2..00**

41 ± 4..00**

43 ± 2.00 35 ± 4..00

46 ± 2..00 37 ± 7.00

24 ± 2..00

31 ± 2..00

28 ±2.00***

28 ±3.00**

27 ± 4.00

17 ± 1.00

23 ± 2.00

25 ± 2.00***

20 ± 5.00*

19 ± 2.00**

NS

NS

57 ± 3.93 51 ± 3..93

50 ± 5.00 44 ± 2..00

53 ± 3.00 49 ± 3..00

57 ± 0.00 46 ± 7..00

53 ± 3.00 43 ± 2..00

NS

NS

NS

NS

NS

46 ± 2..43 34 ± 2.20

42 ± 5..00

46 ± 8..00

38 ± 3..00

40 ± 2..00

34 ± 2..00

24 ± 2..00

36 ± 2.00***

39 ± 6.00*

34 ± 2.00**

36 ± 4.00**

24 ± 2.00***

23 ± 2.00*

**

0.5

1

2

NM

100

24 ± 1.77

23 ± 2.00

27 ± 6.46

23 ± 3.00

25± 2.00

14 ± 1.00

M

100

28 ± 3.42**

22 ± 1.00***

21 ± 4.00 NS

18 ± 4.00*

21 ± 3.00*

14 ± 1.00 NS

17 ± 3.00 17 ± 3.00

NM

100

18 ± 3.15

14 ± 1.51

16 ± 1.20

11 ± 1.51

15 ± 1.00

11 ± 1.91

15 ± 2.00

15 ± 1.16

12 ± 1.00

14 ± 0.76

M

100

14 ± 2..20*

13 ± 2..00**

14 ± 1..00***

12 ± 1..00***

13 ± 1..00***

8± 1..00***

10 ± 1..00**

16 ± 1..00*

15 ± 1..00***

13 ± 3..00*

NM

100

9± 1.16

5± 1..00

3± 1..00

4± 1..00

4± 1..00

3± 1..00

6± 2..00

11 ± 3..00

3± 1.39

7± 1..00

M

100

6± 1.77**

0

0

0

6± 3.96*

0

0

0

8± 0..89***

0 0

4

8

NS

NS

NS

NM

100

0

0

0

0

0

0

0

0

0

M

100

0

0

0

0

0

0

0

0

0

0

NM

100

0

0

0

0

0

0

0

0

0

0

Values represent mean ± SDM of 4 measurements *P < 0.05, ** P < 0.01, *** P < 0.001 when melanised cells are compared with Non-melanised cells NS = Non –significant; Cn -Cryptococcus neoformans; M - Melanised; MN - Non- melanised; C - Control Minimum inhibitory concentration (MIC) determination The MIC for methanolic extract of C. zeylanicum for non - melanised C. neoformans was 1 mg/ml whereas it was in the range of 1 to 2 mg/ml for melanised C. neoformans. The MIC for methanolic extract of M. paniculata for non - melanised C. neoformans was 4 mg/ml whereas it was in the range of 4 8mg/ml for melanised C. neoformans. The MIC for Amphotericin B Control was 0.00025 mg/ml. Percentage of survival of melanised and non – melanised C. neoformans at different concentrations of plant extracts The recovery rate of 8 days old melanised and non-melanised C. neoformans after incubation with extracts of Cinnamomum © Gayathri Teknological Publication

zeylanicum and Murraya paniculata was measured. The number of colonies of melanised and non-melanised C. neoformans decreased based upon the concentrations of plant extracts used when compared to the control (unexposed). But the isolates of nonmelanised cells were more susceptible to both the plant extracts when compared with the melanised cells. At 1mg/ml concentration of Cinnamomum zeylanicum, 6 isolates of melanised C. neoformans can be recovered but all the 10 isolates of non-melanised C. neoformans disappeared. At 4mg/ml concentration of Murraya paniculata, 3 isolates of melanised C. neoformans can be recovered but all the 10 isolates of nonmelanised C. neoformans disappeared. 8


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Decreased susceptibility of melanised C. neoformans to Cinnamomum zeylanicum and Murraya paniculata than non-melanised cells was statistically significant. The results are represented in table’s 3 and 4. Conclusion The results show that isolates of nonmelanised cells are more susceptible to Cinnamomum zeylanicum and Murraya paniculata than melanised cells. However the methanolic extract from the bark of Cinnamomum zeylanicum was found to be inhibitorier than the methanolic extract from the leaf of Murraya paniculata. In summary, accumulating evidence indicates that melanin production is an effective survival (virulence) factor of C. neoformans, and melanin may serve multiple roles in not only protecting this medically important fungus from host defenses but also provide a survival advantage in the environment. Also, the methanolic extracts from the barks of Cinnamomum zeylanicum and Murraya paniculata which showed inhibitory action against C. neoformans can be tested further to see whether it can be used as a supplement during the treatment of Cryptococcosis.

Acknowledgement The authors are thankful to the Management, Principal and Head of the Post Graduate and Research Department of Zoology, The New College, Chennai, India for providing laboratory facilities.

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and antimicrobial activity of essential oil of Murraya exotica. L. Flavour and frangrance. 13:59-62. Ikeda, R., T. Sugita, E. S. Jacobson, and Shinoda. T. 2003. Effects of melanin upon susceptibility of Cryptococcus to antifungals. Microbiol. Immunol. 47:271-277. Karig, F. 1975. Thin layer chromatography of cinnamon bark. Dtsch Apoth Ztg, 115:17811784. Khan, Z. U., Al-Anezi, A. A., Chandy, R. and Xu. J. 2003. Disseminated cryptococcosis in an AIDS patient caused by a canavanine-resistant strain of Cryptococcus neoformans var. grubii. Journal of Medical Microbiology. 52, 271–275 Kwon-Chung, K. J., Polachek, I. and Bennett, J. E. 1982. Improved diagnostic medium for separation of Cryptococcus neoformans var. neoformans (serotypes A and D)and Cryptococcus neoformans var. gattii (serotypes B and C). J. clin. Microbiol.,15: 535-537. Mitchell, T. G., Perfect, J.R. 1995. Cryptococcosis in the era of AIDS—100 years after the discovery of Cryptococcus neoformans. Clin Microbiol Rev; 8:515-48. Paranagama, P. 1991. Analysis of Sri Lankan Essential oils by gas chromatography and mass spectroscopy Colombo, Sri Lanka: ITI. Ross, F. M. 1976. Analysis of cinnamon oil by high pressure liquid chromatography. J Chromatogr, 118:273-275. Saag, M. S., Graybill, R. J., and Larsen, R. A. et al. 2000. Practice guidelines for the management of cryptococcal disease. Infectious Diseases Society of America. Clin. infect. Dis., 30: 710-718 Van Duin, D., A. Casadevall, and Nosanchuk,J. D.2002. Melanization of Cryptococcus neoformans and Histoplasma capsulatum reduces their susceptibility to amphotericin B and caspofungin. Antimicrob. Agents Chemother.46:3394–3400.

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