Supplementary contentto:
Antimalarial potential of extracts and isolated compounds from four species of genus Ammannia
Harish C. Upadhyaya,#
Brijesh S. Sisodiab,c,#
Jyoti Agrawalb
Anirban Palb
Mahendra P. Darokarb
Santosh K. Srivastavaa
Affiliation
aMedicinal Chemistry Department, b Molecular Bioprospection Department; Central Institute of Medicinal and Aromatic Plants (CSIR-CIMAP), P.O.- CIMAP, Lucknow–226015, India
Correspondence:
Santosh K. Srivastava, Central Institute of Medicinal and Aromatic Plants (CSIR-CIMAP), P.O.- CIMAP, Lucknow–226015, India
Phone: +91-522-2359623; Fax: +91-522-2342666; E-mail:
cPresent address:
Dept. of Clinical Biochemistry, RRA Podar Ayurveda Cancer Research Institute(CCRAS, Dept. of AYUSH), Dr. Annie Besant Road, Worli,Mumbai-400 018,India
#Both authors contributed equally
Extraction and fractionation of plant material for antimalarial activity
Shade dried and finally milled samples of A. multiflora (whole plant, 100 g), roots and aerial parts of A. baccifera(50 g each), roots and aerial partsof A. verticillata(100 g each) and A. coccinea (whole plant, 100 g)were separately extracted at room temperature (28±20C) with methanol (100 ml x 3, 24 hour each), filtered through Whatman filter paper No. 1 and the combined extracts were separately dried under vacuum at 40°C until dry. In this way the respective crude methanolic extracts: A. multiflora (AM, 10.5 g); roots (BR, 6.5 g) and aerial parts (BA, 7.1 g) of A. baccifera; roots (VR, 7.0 g) and aerial parts (VL, 13.0 g) of A. verticillata; and crude methanolic extract of A. coccinea (AC, 9.5 g) were obtained. A portion (~0.5 g) of all the crude methanolic extracts were kept for bioactivity evaluation and the rest amounts were separately dissolved in distilled water and successively partitioned with n-hexane, chloroform, and n-butanol(each 3 x 100 ml). The combined extracts were separately evaporated under reduced pressure to afford n-hexane, chloroform and n-butanolfractions respectively.On fractionation, AM (10.0 g) afforded n-hexane (AM1, 2.4 g), chloroform (AM2, 0.3 g) and n-butanol (AM3, 3.2 g) fractions.The methanolic extracts of roots of A. baccifera(BR, 6.0 g) afforded n-hexane (BR1, 1.8 g), chloroform (BR2, 90 mg) and n-butanol(BR3, 0.5 g) fractions, while the methanolic extract of aerial parts of A. baccifera(BA, 6.5 g) afforded n-hexane (BA1, 2.4 g), chloroform (BA2, 0.15 g) and n-butanol(BA3, 0.7 g) fractions. The methanolic extract of roots of A. verticillata (VR, 6.5 g) afforded n-hexane (VR1, 0.64 g), chloroform (VR2, 0.50 g) and n-butanol(VR3, 1.80 g) fractions while methanolic extract of aerial parts of A. verticillata (VL, 12.5 g) afforded n-hexane (VL1, 1.68 g), chloroform (VL2, 1.10 g) and n-butanol(VL3, 4.82 g) fractions. Similarly, methanolic extract of A. coccinea(AC, 9.0 g) afforded n-hexane (AC1, 1.20 g), chloroform (AC2, 0.85 g) and n-butanol(AC3, 2.05 g) fractions. The extract/fraction samples were kept in an air tight sample bottle and stored at −20°C until used for antimalarial activities.
Isolation of Compounds fromA. multiflora
Extraction and fractionation
The authenticated samples of A. multiflora (whole plant) were shade dried, pulverized and extracted with methanol. The dried methanolic extract was dissolved in distilled water and successively partitioned with n-hexane, chloroform, and n-butanol. The details of the extraction and fractionation procedures are being presented in following flowchart.
Chromatographic separation of Chloroform fraction
TLC profile of chloroform extract showed presence of a major compound, hence a portion (0.6g) of this was purified over silica gel column (CC, 60-120 mesh, 12 g). Gradient elution of the column was carried out with solvents of increasing polarity using n-hexane and chloroform in various proportions. A total of 58 fractions of 50 ml each were collected. The CC fraction 25-51 eluted with n-hexane-chloroform (2.5:7.5 to 1:9) afforded a pure compound M-1(412 mg).
Chromatographic separation of n-butanolfraction
The n-butanol fraction was a very complex mixture; hence a portion (12 g) of this was subjected to vacuum liquid chromatographic (VLC-1, 7.5 cm x 7.5 cm) separation over Silica gel H (125 g). Stepwise gradient elution of VLC-1 column was carried out with solvents of increasing polarity viz. n-hexane, chloroform, methanol in various proportions. Fractions of 50 ml each were collected. A total of 426 fractions were collected and pooled on the basis of their TLC profile. The VLC-1 fraction 64-73 eluted with chloroform-methanol (99.5:0.5) afforded a compound, M-2 (35.0 mg). The VLC-1 fraction 135-152 eluted with chloroform-methanol (93:7), afforded the compound M-3(10.5 mg). The VLC-1 fraction 184-197 eluted with chloroform-methanol (92.5:7.5), furnished a compound M-4 (11 mg), while VLC-1 fraction 203-219 eluted with chloroform-methanol (92.5:7.5) furnished another compound, M-5 (14 mg).
The VLC-1 fraction 257-324 (940 mg) eluted with chloroform-methanol (91.5:8.5 to 9:1) was a complex mixture hence further purified over VLC-2 (4.5x 3.5 cm, 35 ml) using silica gel-H (14 g). Gradient elution of VLC-2 was carried out using chloroform, methanol and water in various proportions. The stepwise gradient elution of VLC-2 was carried out with lower layer of chloroform-methanol-water, monitoring polarity of the eluent by methanol and a total of 53 fractions were collected and pooled on the basis of their TLC profile. The VLC-2 fractions 35-37 eluted with chloroform-methanol-water (65:15:10), furnished a compound, M-6 (37 mg).
Further, VLC-1 fraction 335-352 (435 mg) eluted with chloroform-methanol (88:12) were re-purified over flash chromatograph (FC-2) using silica gel-H (14 g). The stepwise gradient elution of FC-2 was carried out with lower layer of chloroform-methanol-water, monitoring polarity of the eluent by methanol and a total of 103 fractions were collected and pooled on the basis of their TLC profile. The FC-2 fraction 48-51 (37 mg) eluted with chloroform-methanol-water (65:14:10) were mixture, hence subjected for purification over preparative TLC (eluted with lower layer of chloroform-methanol-water, 65:30:10) which furnished a yellow compound, M-7 (11 mg).
Isolation of Compounds fromA. baccifera
Extraction and fractionation
A comparative TLC profile of the methanolic extracts of roots and aerial parts of A. baccifera and there similar fractions (i.e. fractions partitioned with same solvent) were developed and it was found that the TLC profiles were more or less similar. Therefore, for the isolation of the compounds, the whole plants of A. baccifera were taken. The dried and pulverized 700 g sample of A. baccifera (whole plant) was extracted with methanol (3 x24 h). The extract was dried under reduced pressure. The dried methanolic extract was fractionated according to the following procedures.
Chromatographic separation of Chloroform fraction
The TLC profile of chloroform fraction showed presence of a major compound. In order to purify, the major portion (1.0 g) of chloroform extract was subjected to column chromatography (CC, silica gel 60-120 mesh, 18 g, column diameter 1x 31 cm). The step-wise gradient elution of CC was carried out with n-hexane, chloroform and methanol in various proportions. A total of 104 fractions of 50 ml each were collected and pooled according to their similarity in behaviour on TLC plate. The fraction 58-66, eluted with n-hexane-chloroform (12:88) afforded a pure compound, B-1 (22 mg) while the fraction 70-92, eluted with n-hexane-chloroform (10:90 to 2:98) afforded the compound B-2 (380 mg).
Chromatographic separation of n-butanolfraction
The major portion (7.0 g) of n-butanol fraction was subjected to vacuum liquid chromatographic (VLC, 7.5 cm x 7.5 cm) separation over Silica gel H (100 g), and the stepwise gradient elution of the VLC was carried out with chloroform and methanol. A total of 325 fractions of 50 ml each were collected and pooled on the basis of their TLC profile. Fraction 114-123, eluted with chloroform-methanol (93:7) afforded the compound B-3(13.0 mg). The VLC fraction 140- 230, eluted with chloroform-methanol (93:7 to 70:30) was mixture of compounds (V-1, 950 mg), while the VLC fraction 315-316, eluted with methanol afforded the compound B-4 (8.2 mg). In order to purify, the VLC fraction V-1 (950 mg) was subjected to flash chromatographic (FC, 2.5 x 24 cm) separation over silica gel-H (particle size 10 M, 40 g) and the step-wise gradient elution of FC columns was carried out with chloroform and methanol. A total of 170 fractions of 50 ml each were collected and pooled on the basis of their behaviour on TLC plate. The V-1 fraction 91-95, eluted with chloroform-methanol (98: 2) afforded a pure compound B-5 (11 mg), while the V-1 fraction 136-150, eluted with chloroform-methanol (96.5:3.5 to 96:4) afforded the compound B-6 (30 mg). The V-1 fraction 163-180, eluted with chloroform-methanol (92:8 to 88:12) was a mixture of compounds containing two major compounds and hence subjected for further purification by preparative TLC (chloroform-methanol-water, 65:35:10) which furnished two pure compounds, B-7 (28 mg) and B-8 (18 mg).
Spectroscopic data of compound B-5: Yellow powder; IR (KBr) ˅max: 3448, 2927, 1654, 1445 cm-1; ESI-MS (m/z):303 [M+H]+; 1H-NMR (300 MHz, MeOD): 6.48 (1H, d, J=1.8 Hz, H-6) 6.79 (1H, d, J=1.8 Hz, H-8), 7.69 (1H, d, J=2.1 Hz, H-2’), 7.00 (1H, d, J=8.4 Hz, H-5’), 7.43 (1H, dd, J=8.4 Hz, 2.1, H-6’); 13C-NMR (75 MHz, MeOD): See Table S1. The compound B-5 was characterized as ‘quercetin’ (Figure S1).
Table S1: 13C NMR data of AB-5 in MeODPosition / δC / Position / δC
2 / 147.3 / 10 / 104.5
3 / 135.5 / 1’ / 122.0
4 / 177.8 / 2’ / 115.8
5 / 160.3 / 3’ / 146.4
6 / 98.3 / 4’ / 149.2
7 / 164.0 / 5’ / 116.2
8 / 95.4 / 6’ / 121.0
9 / 156.1
Figure S1: Structure of B-5
Spectroscopic data of compound B-8:Yellow amorphous powder; IR (KBr) ˅max: 3415, 2940, 1660, 1603, 1455 cm-1;ESI-MS (m/z): 633 [M+Na]+; 1H-NMR (300 MHz, MeOD): 6.13 (1H, d, J=2.1 Hz, H-6), 6.32 (1H, d, J=2.1 Hz, H-8), 7.65 (1H, d, J=2.1 Hz, H-2’), 6.81 (1H, d, J=6.9 Hz, H-5’), 7.50 (1H, dd, J=6.9, 2.1 Hz, H-6’); sugars δH: 5.04 (1H, d, J=7.5 Hz, H-1”), 3.30 & 3.75 (H-6”), 4.45 (1H, s, H-1”’), 1.06 (3H, d,J=6.3 Hz, H-6”’) and 3.46, 3.39, 3.38, 3.37, 3.26, 3.21; 13C-NMR (75 MHz, MeOD): See Table S2. The compound B-8 was characterized as quercetin-3-O-rutinoside, commonly called as ‘Rutin’ (Figure S2).
Table S2:13C NMR data of AB-8 in MeODPosition / δC / Position / δC
2 / 158.5 / 6’ / 122.2
3 / 135.5 / 1’’ / 104.3
4 / 179.4 / 2’’ / 75.6
5 / 159.4 / 3’’ / 78.1
6 / 100.0 / 4’’ / 73.8
7 / 167.0 / 5’’ / 77.2
8 / 94.9 / 6’’ / 68.6
9 / 163.0 / 1’’’ / 102.4
10 / 105.7 / 2’’’ / 71.2
1’ / 122.7 / 3’’’ / 72.1
2’ / 116.1 / 4’’’ / 72.3
3’ / 145.9 / 5’’’ / 69.7
4’ / 149.8 / 6’’’ / 17.9
5’ / 117.6
Figure S2: Structure of B-8
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