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Flavonol acyl glucosides from the aril of Schotia brachypetala Sond. and their antioxidant, antibacterial, and

antimalarial activities

Jan H van der WesthuizenDirectorate Research Development,

University of the Free State, Bloemfontein, South Africa

4

South African Biodiversity

• South Africa is one of richest centres in the world in diversity of plant species

• More than 20 000 different plant species• Particularly the seeds have not been well

studied

Seeds from South African plants

• A variety of seeds were collected and their methanol extracts screened for bioactivity in autobiographic DPPH radical scavenging and the acetylcholinesterase inhibition bioassays (TLC) and for antibacterial and antiplasmodial activity

• S. brachypetala was chosen for further investigation

Cuendent, M., Hostettmann, K., Potterat, O. (1997). Iridoid glucosides with free radical scavenging properties from Fragraea blumei. Helv. Chim. Acta 80, 1144-1151. Marston, A.; Kissling, J.; Hostettmann, K. Phytochemistry Analysis 2002, 51-54.

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Strong DPPH radical scavenging activity of the MeOH extract of S. brachypetala aril on TLC

Sample load on TLC: 10 μL of a stock solution (10 mg/mL)

Moderate Antiplasmodial activity of the MeOH extract of S. brachypetala aril IC50 ± s.d. (μg/mL) 18.95 ± 2.97

E. coli ATCC 8739

K. pneumoniae ATCC 13883

Staph. aureus ATCC 25923

E. faecalis ATCC 29212

Crude extract 0.5 8.0 16.0 8.0

Culture control >16.0 >16.0 >16.0 >16.0

Negative control >16.0 >16.0 >16.0 >16.0

Ciprofloxacin control (μg/mL)

0.63 0.12 0.30 0.63

Antibacterial activities of the crude MeOH extract of the MeOH extract of S. brachypetala aril MIC values (mg/mL).

NO Inhibition of acetylcholinesterase by the MeOH extract of S. brachypetala aril on the TLC

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Schotia brachypetala trees Mpumalanga Province

8

Flower of Schotia brachypetela

Fruit pods of Schotia brachypetala with the yellow arils

The aril is a specialised outgrowth from the attachment point of the seed

10

Some traditional uses of S. brachypetala

• Traditional healers use a decoction of the bark to treat dysentery and diarrhoea, nervous heart conditions, flu symptoms, excessive beer drinking and as an emetic and to strengthen the body. The roots are also used to treat diarrhoea and heartburn

• The seeds can be roasted and eaten and the plant was listed in a review for its potential value as a new crop

Watt J.M., Breyer-Brandwijk M.G. The Medicinal and Poisonous Plants of Southern and Eastern Africa. 2nd ed. 1962. E. & S. Livingstone (Edinburgh), Livingstone, London. A. Hutchings. Zulu Medicinal Plants. University of Natal Press, Pietermaritzburg (1996).

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“Semi-bioguided” isolation strategy

• Active fractions, not necessarily active compounds, were identified

• All the compounds, not necessarily the active ones, in the active fractions were isolated

• Isolated compounds were then evaluated for their biological activity

• Whole process was monitored by TLC• Reduce time in contact with chromatographic

materials

HPLC chromatogram of the methanol extract of Schotia brachypetala aril

(detection: 254 nm)

Column: Phenomenex C18 reverse phase column (150 x 4.6 mm, 5 micron); solvents: A: distilled H 2O + 0.1% formic acid, B: 70% MeOH/ H2O + 0.1% formic acid, gradient: 0-15 min, 10-30% B; 15-25 min, 30-70% B; 25-27 min, 70-100% B; 27-35 min, 100% B; flow rate: 1 ml/min; injection: 10 μl of a stock solution of S. brachypetala aril MeOH extract at a concentration of 10 mg/ml in MeOH.

13

Countercurrent Chromatograpy

• Countercurrent chromatography was employed as the main chromatography technique

• In this technique, the stationary phase and mobile phase are two immiscible solvents and no solid support (e.g. silica gel) is present

• Adsorption of analyses to the solid phase and decomposition is avoided and a high recovery rate is thus achieved

• By using fast spinning coils, it is possible to pump two immiscible solvents in opposite directions in the same tube

• Easy scaling up to preparative level a major advantage

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High-speed Contercurrent Chromatography (HSCCC) equipment

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Solvent selection and coil loading

• The choice of solvent systems was based on TLC analyses• Sample (5 mg) was dissolved in a mixture of 1 ml of the upper

and 1 ml of the lower phase and 10 μL of each phase was deposited on a TLC plate.

• Solvent systems which afforded an even distribution of the analytes between upper and lower phase were chosen

• The coils of the chromatograph were first filled with stationary phase and then the mobile phase was pumped into the apparatus under rotation until conditions were stable and no more stationary phase eluted. The sample was then dissolved in equal amounts of upper and lower phases and injected via a sample loop

16

Preparative isolation scheme of cCompounds 1, 2, 3 and 6

17

Preparative isolation scheme of compounds 3, 4 and 5

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Preparative isolation scheme of compound 7

19

Compounds 1 and 2

O

OOH

O

OH

OR

OCH3

OO

HO

O

OH

HO OH

1 R = H2 R = CH3

3

5

7

8

10

1'

4'

6'1''5''

6''

1'''

3'''

4'''

6'''

9'''

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Structure elucidation of 1

• Molecular ion (m/z 625.1539 [M+H]+ ) corresponds with a molecular formula of C31H28O14

• One of the six oxygenated carbon resonances in the δ 60-80 region correlates with a three proton singulet in the HSQC spectrum, suggesting a methoxy group

• The other five, and an anomeric carbon at 99.78 correlating with doublets in the 3.6 to 5.1 region, suggest a sugar.

• Acid hydrolysis gives β-glucose (TLC, ref standard)• D-glucose via GC of thiazolidine derivative• The J = 15.75 Hz suggests a trans double bond• The significant downfield shift of two H-6'' resonances (δ 4.62 and 4.29)

suggests that the coumaryl moiety is attached at 6''- OH. Corroborated by the HMBC correlation between the ester carbonyl at 167 and H-6 ''

• Basic hydrolysis give coumaric acid

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1H NMR spectrum (600 MHz, CD3OD) of compound 1

O

OOH

O

OH

OH

OCH3

OO

HO

O

OH

HO OH

22

13C NMR spectrum (150 MHz, CD3OD) of compound 1

O

OOH

O

OH

OH

OCH3

OO

HO

O

OH

HO OH

23

HSQC spectrum of compound 1

O

OOH

O

OH

OH

OCH3

OO

HO

O

OH

HO OH

24

HMBC spectrum of compound 1

O

OOH

O

OH

OH

OCH3

OO

HO

O

OH

HO OH

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MS identification of coumaric acid moiety

Product ion scan of coumaric acid reference standard samples showing the precursor ion [M - H]- at m/z 163 and the product ions produced by collision induced dissociation.

Product ion scan of m/z 163 fragment in MS of compound 1 (M+:m/z 623) (negative mode)

-MS2 (163.00) CE (-18): 1.807 to 2.101 min from Sample 1 (Sample001) of 1.wiff (Heated Nebulizer) Max. 3.3e6 cps.

30 40 50 60 70 80 90 100 110 120 130 140 150 160 170m/z, amu

5%

10%

15%

20%

25%

30%

35%

40%

45%

50%

55%

60%

65%

70%

75%

80%

85%

90%

95%

100%

Re

l. Int. (%

)

119.1

163.3

117.393.1 162.090.8 145.094.8 104.0 121.3 160.2 164.1114.965.141.1 135.362.837.235.2 155.5

-MS2 (163.00) CE (-18): 1.837 to 2.131 min from Sample 1 (Sample001) of 1.wiff (Heated Nebulizer) Max. 2.0e5 cps.

30 40 50 60 70 80 90 100 110 120 130 140 150 160 170m/z, amu

5%

10%

15%

20%

25%

30%

35%

40%

45%

50%

55%

60%

65%

70%

75%

80%

85%

90%

95%

100%

Re

l. Int. (%

)

119.1

163.3

94.7 118.093.0 162.137.035.3 145.245.2 103.8 160.2120.8

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HPLC identification of coumaric acid

HPLC chromatogram of p-coumaric acid (reference standard) at a retention time of 1.92 minutes.

HPLC chromatogram of p-coumaric acid in hydrolised sample at a retention time of 1.92 minutes

XIC of -MRM (1 pair): 163.0/119.0 amu from Sample 1 (Sample005) of 5.wiff (Heated Nebulizer), Smoothed Max. 4.1e6 cps.

0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5Time, min

0%

5%

10%

15%

20%

25%

30%

35%

40%

45%

50%

55%

60%

65%

70%

75%

80%

85%

90%

95%

100%

Re

l. Int. (%

)

1.92

XIC of -MRM (1 pair): 163.0/119.0 amu from Sample 1 (Sample005) of 7.wiff (Heated Nebulizer), Smoothed Max. 3.9e5 cps.

0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5Time, min

0%

5%

10%

15%

20%

25%

30%

35%

40%

45%

50%

55%

60%

65%

70%

75%

80%

85%

90%

95%

100%

Re

l. Int. (%

)

1.92

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Absolute configuration of hydrolised sugars

GC chromatogram of the thiazolidine derivatives of the acid hydrolyzed sugars from the crude MeOH extract

Chromatogram of the thiazolidine derivatives of the mixture of the acid hydrolyzed sugars from the crude MeOH extract spiked with the thiazolidine derivative of L-glucose

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Compounds 3 to 5

O

OOH

O

OH

OR1

OR2

OHO

OH

HO OH

3 R1 = H, R2 = CH3

4 R1 = CH3, R2 = CH3

5 R1 = H, R2 = H

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Compounds 6

O

OOH

O

OH

OH

OH

OHO

OH

HO OH

RR

6

1H NMR spectrum (600 MHz, CD3OD) of compound 6

O

OOH

O

OH

OH

OH

OHO

OH

HO OH

RR

6

J2,3 = 11.5 Hz (trans)

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210

215

220

225

230

235

240

245

250

255

260

265

270

275

280

285

290

295

300

305

310

315

320

325

330

335

340

345

350

355

360

365

370

375

380

385

390

395

400

-40000

-30000

-20000

-10000

0

10000

20000

30000

40000

ECD spectrum for compound 60.10 mM; 0.047 mg/mL 0.05 mM; 0.023 mg/mL 0.025 mM; 0.012 mg/mL

Wavelength (nm)

Mol

ar E

llip

tici

ty

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Compound 7

O

OOH

HO

OH

OH

O OO

O OH

OHOOH

OHHO

O

OH

OH

OH

Glu

Xyl

Rham

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1H NMR spectrum (600 MHz, CD3OD) of compound 7

O

OOH

HO

OH

OH

O OO

O OH

OHOOH

OHHO

O

OH

OH

OH

Glu

Xyl

Rham

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13C NMR spectrum (150 MHz, CD3OD) of compound 7

O

OOH

HO

OH

OH

O OO

O OH

OHOOH

OHHO

O

OH

OH

OH

Glu

Xyl

Rham

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Sugars via acid hydrolysis of compound 7

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Fragmentation MS of compound 7

O

OOH

HO

OH

OH

O OO

O OH

OHOOH

OHHO

O

OH

OH

OH

Glu

Xyl

Rham

Antimicrobial activities

Culture, media preparation and (MIC) assays were performed according to methodology adopted from NCCLS (2003) guidelines

Antiplasmodial and DPPH radical scavenging activities of the crude MeOH

extract and isolated compounds

The antimalarial activity of the pure compounds was determined using the tritiated hypoxanthine incorporation assay against the chloroquine-resistant FCR-3 strain of Plasmodium falciparum (Desjardins et al., 1979). A 0.022% DPPH solution (25 ml) in MeOH was

added to a solution of the compound to be tested at different concentrations in MeOH (115 mL). Absorbance at 517 nm was measured after 30 min and the percentage of activity was calculated (Cavin et al., 1998).

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Radical scavenging activity of 1 and 2

O

OOH

O

OH

OR

OCH3

OO

HO

O

OH

HO OH

1 R = H2 R = CH3

3

5

7

8

10

1'

4'

6'1''5''

6''

1'''

3'''

4'''

6'''

9'''

40

4'-OH required DPPH scavenging (quinone methide)

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CO-WORKERS

Kun Du, University of the Free State, South Africa (MSc thesis)Andrew Marston (deceased), University of the Free State, South AfricaSandy F. van Vuuren, University of the Witwatersrand, South Africa (antimicrobial bioassay) Robyn L. van Zyl, University of the Witwatersrand, South Africa (antiplasmodial bioassay)Christina Coleman, University of Mississippi, USAPieter C. Zietsman, National Museum, Bloemfontein, South Africa (plant collection)Susan L. Bonnet, University of the Free State, South AfricaDaneel Ferreira, University of Mississippi, USA (CD spectra)

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Acknowledgements

• University of the Free State for financial assistance

• Multi-Disciplinary University Traditional Health Initiative (MUTHI): Building Sustainable Research Capacity on Plants for Better Public Health in Africa European Union Seventh Framework programme. Grant number 266005

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