Supporting information to:

Anti candidiasis agents from a Tanzanian plant, Combretum zeyheri

Deborah KB Runyoro1@; Santosh K Srivastava*1 Mahendra P. Darokar2

Ngassapa D Olipa3, Cosam C Joseph4, Mecky IN Matee5

Affiliation:

1Medicinal Chemistry Division; 2Molecular and Bioprospection Division, Central Institute of Medicinal and Aromatic Plants, Lucknow, Utter Pradesh, India

3School of Pharmacy, Muhimbili University of Health and Allied Sciences, Dar es Salaam; Tanzania

4 Chemistry Department, University of Dar es Salaam, Dar es Salaam; Tanzania

5School of Medicine, Muhimbili University of Health and Allied Sciences, Dar es Salaam; Tanzania

Correspondence

Santosh K. Srivastava, Central Institute of Medicinal and Aromatic Plants, P.O.CIMAP, Lucknow–226015, India

Phone: +91-522-2718581; Fax: +91-522-2342666; E-mail:

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Extraction and isolation of Anti candidiasis constituents

The shade dried leaves (10 kg) of C. zeyheri were powdered and extracted with methanol: water (80: 20) using repeated maceration for one week at room temperature. The solvent removed under vacuum from the pooled MeOH extracts at 40-50o C. The resultant MeOH extract in water so obtained was successively partitioned with n- hexane and ethyl acetate respectively. The n-hexane and ethyl acetate fractions were dried in vacuo at 40-50o C whereas the aqueous fraction was freeze-dried using Edwards freeze drier.

The bioactive ethyl acetate fraction (50 g) was subjected to flash chromatographic (5cm x 28cm, Silica gel H) separation. Step gradient elution with each fraction of 60 ml was carried out from CH2Cl2 to EtOAc: CH2Cl2 (3:1). Fractions were pooled on the basis of their TLC profile. The Flash fractions 5-10 (1.8g) eluted with EtOAc: CH2Cl2 (1:3) were further purified over VLC-I (2.7cm x 8cm). Step gradient elution (as above) of VLC with each fraction of 100 ml was carried out. The VLC-I fractions 23-24 eluted with CH2Cl2: CHCl3 (1:1) afforded isomeric mixture of 1(185 mg). During elution of fraction 38 with EtOAc: CH2Cl2 (3:1), the Flash got choked, hence the flash column was eluted back with MeOH and the recovered mixture (24g) was further resolved over VLC-II (9.5cm x 6cm). Step gradient elution of VLC-II with CH2Cl2, EtOAc and MeOH in different proportions in increasing order was carried out. Each fraction of 200 ml was collected. Fractions 22-32 (3g) eluted with EtOAc: CH2Cl2 (3:1) were mixture, hence further subjected to VLC-III (2.7cm x 8cm) and gradient eluted with CH2Cl2, CHCl3 and MeOH in increasing order. Fractions of 100ml each were collected. Fractions 47- 53, eluted with CHCl3: CH2Cl2 (3:1) yielded a white powder of isomeric mixture 2 (34 mg), while fractions 77-125 (175mg) eluted with CHCl3: MeOH (98.5: 1.5) were again purified over VLC-IV (2.7cm x 8cm) and gradient elution with fractions of 100ml each was carried out. The fractions 56-80 eluted with CHCl3: MeOH (99: 1) afforded compound 3 (40mg), while fractions 274-304 eluted with CH2Cl3: MeOH (97.5: 2.5) afforded compound 4 (629 mg).

Anticandida activity assay

Disk diffusion assay:

The antimicrobial activity was determined according to the methods of Bauer et al. and Wannisorn et al. All strains were sub cultured from -80oC stock culture into 5 mL Sabouraud dextrose broth (SDB, Hi-Media), and incubated for 24 h at the desired temperatures. The inoculums of the test microbes were prepared equivalent to the 0.5 McFarland Standards, as described in NCCLS protocols. Uniform lawns of each of the selected microbes were made using 100 μL inoculums on nutrient SDA plate. Then filter paper (Whatman) discs (5 mm) containing test compounds (5 μL) were placed over the seeded plates. The plates were incubated at 28oC for 48 h. The activity was measured in terms of zone of microbial growth inhibition. First, the diameter of the total zone was measured (including disc). Then the diameter of the disc was subtracted from the total zone to obtain the net zone of growth inhibition. The tests were performed in triplicate to confirm the findings. The net zone of growth inhibition above 10 mm was considered as highly active, 4-10 mm moderately active, and less than 4 mm either weakly active or inactive.

Broth dilution assay:

The minimum inhibitory concentration (MIC) was determined using the two fold serial micro broth dilution technique using a 96 well micro plate. The test compounds (1-6) were added to sterile Sabouraud dextrose broth (SDB) in microtiter plates before the diluted microbial suspension (final inoculum of 104 cfu/mL) was added. The samples were assayed in triplicate. The MIC values were taken as the lowest concentration of the test compound in the wells of the microtiter plate that showed no turbidity after 24 h of incubation at 28oC for 48 h. The turbidity of the wells in the microtiter plate was interpreted as visible growth of the microorganism.

1H and 13C-NMR spectroscopic data of 1-4

The 1H and 13C-NMR spectroscopic data of 1-4 have been presented in table (S1 and S2)

Table S1: 1HNMR chemical shifts (in δ) for selected peaks of the isolated compounds

H / Compounds
1.1 and 1.2 / 2.1 and 2.2 / 3 / 4
2β / - / 4.08m / 4.26dt / 4.32d (J10.5 Hz)
3a / 3.45t (J 8.28, 7.51 Hz) / 3.38 d (J 9.22) / 3.41d (J 9.3 Hz) / 4.16d (J 9.3Hz)
6a / - / 4.84s / 5.02s
12 / 5.48 / 5.44s br / 3.32dd / 5.50
18 / 2.62d (J 11.1 Hz); (3.27dd [J 3.6Hz, 13.5 Hz]) / 3.29dd, (2.60d [J 11.1Hz]) / 5.55s / 3.25 dd (J 3.00, 13.2Hz)
23a / - / - / 4.32 (J 10.5 Hz)
23 β / - / - / 3.98 (J 10.39Hz)
H-23, 24, 25, 26, 27, 29, 30 / 0.89, 0.96, 1.00, 1.02,1.04 (3H each, 1.23 (6H) / 0.93, 0.96, 0.98, 0.99, 1.05, 1.24, 1.25 / 1.44,1.75, 1.68,1.55, 1.26, 0.94, 1.00 / 1.65, 1.70, 1.55, 1.18, 0.89,0.96

Table S2: 13CMR chemical shifts (in δ) for the isolated compounds

C / Compounds
1.1 and 1.2 / 2.1 and 2.2 / 3 / 4
1 / 39.4t / 47.9t (48.1t) / 50.5 t / 50.1t
2 / 28.3t (28.6t) / 68.8d / 69.2 d / 69.2d
3 / 78.4d / 84.1d / 84.6 d / 78.8d
4 / 39.5s / 39.9s / 41.0s / 44.6s
5 / 56.1d / 56.1d / 57.1d / 49.3d
6 / 19.0t / 19.1t / 68.1 d / 67.9d
7 / 33.8t (33.5 t) / 33.7t / 41.7 t / 41.2
8 / 40.3s (40.0s) / 40.1s (40.3s) / 39.9 s / 39.6s
9 / 48.3d / 48.4d / 49.2 d / 48.9d
10 / 37.5s / 38.8s / 38.9 s / 38.4s
11 / 23.9t / 24t / 24.4 t / 24.2t
12 / 125.8d (122.8d) / 122.9d (125.7d) / 123.2 d / 123.0d
13 / 139.5s (145s) / 145.1s (139s) / 144.6 s / 144.4s
14 / 42.8s (42.4s) / 42.5s (42.8s) / 43.3 s / 43.0s
15 / 28.9t (28.6t) / 28.5t (28.9t) / 28.7 t / 28.5t
16 / 25.1t (24.0t) / 24.1t (25.1t) / 24.4 t / 24.0t
17 / 48.3s / 46.9s / 47.0 s / 46.9s
18 / 53.8d (42.2d) / 42.3d (53.7d) / 42.6 d / 42.3d
19 / 39.6 d (46.7t) / 46.7t (39.6d) / 47.2 t / 46.8t
20 / 39.7 d (31.1s) / 31.1s (39.6d) / 31.3 s / 31.1s
21 / 31.3t (34.5t) / 34.5t (31.3t) / 34.8 t / 34.6t
22 / 37.6t (33.5t) / 33.4t (37.5t) / 33.6 t / 33.4t
23 / 29.0q / 29.5q / 29.5 q / 66.8t
24 / 16.7q / 16.9q / 19.5 q / 15.8q
25 / 15.9q (15.7q) / 17.6q (17.1q) / 18.9 q / 19.1q
26 / 17.7q / 17.7q / 19.0 q / 18.8q
27 / 24.1q (26.3q) / 26.3q (23.9) / 26.7 q / 26.5q
28 / 179.9s (180.2s) / 180.2s (179.9 s) / 180.4 s / 180.3s
29 / 17.7q (33.5q) / 33.4q (21.5q) / 33.6 q / 33.4q
30 / 21.6q (24.0q) / 24.1q (17.6q) / 24.2 q / 24.0q

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