SUPPLEMENTARY MATERIALS
New chalcanonol glycoside from the seeds of saw palmetto;
antiproliferative and antioxidant effects
Fatma M. Abdel Bar1
1
Pharmacognosy department, Faculty of pharmacy, Mansoura University, Mansoura
35516, Egypt
*To whom correspondence should be addressed. Email: fatma_maar@yahoo.com
Abstract
A new chalcanonol glycoside dimer ;bis-O-[(I-4`) → (II-6`)]--hydroxyphloretin-2`O--glucoside (1), in addition to six known compounds viz. (-)-epicatechin (2) and ()-epiafzelechin (3), 4-hydroxybenzoic acid (4), protocatechuic acid (5),methylgallate
(6), β- sitosterol (7) and -sitosterol-3-O-glucoside (8) were isolated from the seeds of
saw palmetto. The structures of the isolated compounds were established from
analysis of their MS and 1D and 2DNMR spectroscopic data. The antiproliferative
activities of the isolated compounds towards PC3, the human prostate cancer cells
were investigated. Amongst the isolated compounds, the new compound and the
sterolic derivatives showed antiproliferative effects. Screening of the antioxidant
effects of the isolated compounds by ABTS+ radical assay revealed that the isolated
phenolics were active free radical scavengers.
Key words:Saw palmetto seeds,Serenoarepens,chalcanonol glycoside, (-)-epicatechin,
(-)-epiafzelechin, methylgallate.
Contents
1
H and 13C-NMR spectral data of compounds 2-6.
Page
3
Table S1. Antioxidant assay results of compounds 1-8 by ABTS method.
4
Isolation procedure of compounds7and8.
4
Isolation procedure of compounds 1-6.
4
Identification of compounds 7 and 8.
5
Figure S1. Summary of the isolation procedure of compounds 1-6 from the
6
ethyl acetate fraction of saw palmetto seeds.
Figure S2: 1H-NMR spectrum (500 MHz, CD3OD) of compound 1.
7
Figure S3: 13C-NMR spectrum (125 MHz, CD3OD) of compound 1.
8
Figure S4: HSQC spectrum (CD3OD) of compound 1.
9
Figure S5: HMBC spectrum (CD3OD) of compound 1.
10
Figure S6: HMBC correlations of compound 1.
11
1
H and 13C-NMR spectral data of compounds 2-6:
Compound 2:13C-NMR (125 MHz, CD3OD): δC79.9 (C-2), 67.5 (C-3), 29.3 (C-4),
157.6 (C-5), 96.5 (C-6), 158.0 (C-7), 96.0 (C-8), 157.4 (C-9), 100.1 (C-10), 132.3 (C1`), 115.4 (C-2`), 145.8 (C-3`), 145.9 (C-4`), 116.0 (C-5`) and 119.5 (C-6`).1H-NMR
(500 MHz, CD3OD): δH4.82 [1H, brs, H-2], 4.18 [1H, brs, H-3], 2.75 [1H, dd, J= 16.8,
2.3 Hz, H-4a], 2.86 [1H, dd, J= 16.7, 4.4 Hz, H-4b], 5.96 [1H, d, J= 2.1 Hz, H-6], 5.98
[1H, d, J= 2.0 Hz, H-8], 7.01 [1H, brs, H-2`], 6.76 [1H, d, J= 8.2 Hz] and 6.81 [1H,
dd, J= 8.4, 1.2 Hz].
Compound 3:13C-NMR (125 MHz, CD3OD): δC79.9 (C-2), 67.5 (C-3), 29.4 (C-4),
157.3 (C-5), 95.9 (C-6), 158.0 (C-7), 96.2 (C-8), 157.5 (C-9), 100.0 (C-10), 131.6 (C1`), 115.7 (C-2`/6`), 129.2 (C-3`/5`) and 157.0 (C-4`).1H-NMR (500 MHz, CD3OD):
δH4.76 [1H, brs, H-2], 4.07 [1H, brs (triplet-like), H-3], 2.62 [1H, dd, J= 16.8, 2.4 Hz,
H-4a], 2.75 [1H, dd, J= 16.8, 4.5 Hz, H-4b], 5.81 [1H, d, J= 2.0 Hz, H-6], 5.84 [1H, d,
J= 2.1 Hz, H-8], 7.20 [2H, J= 8.5 Hz, H-2`/6`] and 6.67 [2H, d, J= 8.6 Hz, H-3`/5`].
Compound 4, 4-hydroxybenzoic acid; 1H-NMR [CD3OD] proton signals at 6.83
(2H, d, J= 8.6, H-4/6) and 7.89 (2H, d, J= 8.6, H-3/7);
13
C-NMR [CD3OD] carbon
signals at c 170.3 (C-1), 163.3 (C-5), 132.8 (C-3/7), 122.8 (C-2) and 116.1 (C-4/6).
Compound 5, protocatechuic acid; 1H-NMR [CD3OD] proton signals at 7.48 (brs,
H-3), 6.83 (d, J= 8.2, H-6) and 7.46 (dd, J= 8.3, 1.8, H-7); 13C-NMR [CD3OD] carbon
signals at c 170.6 (C-1), 151.6 (C-5), 146.0 (C-4), 124.1 (C-7), 123.1 (C-2), 117.8 (C3) and 115.9 (C-6).
Compound 6, methylgallate; 1H-NMR [CD3OD] proton signals at 7.09 (2H, s, H3/7) and 3.79 (3H, s, 6-OCH3); 13C-NMR [CD3OD] carbon signals at c 169.3 (C-1),
146.4 (C-4/6), 139.8 (C-5), 121.5 (C-2), 110.3 (C-3/7) and 52.6 (6-OCH3).
Table S1. Antioxidant assay results of compounds 1-8 by ABTS method.
Compounds Absorbance (A) % inhibition
0.069
85.31
1
0.063
86.59
2
0.075
84.04
3
0.064
86.38
4
0.074
84.25
5
0.067
85.74
6
0.407
13.40
7
0.305
35.01
8
Ascorbic acid
0.062
86.80
Acontrol
0.470
0
Isolation procedure of compounds 7 and 8:
The n-hexane fraction (20 g) was chromatographed over silica gel column (70 x 4 cm
i.d.) and eluted with n-hexane-EtOAc mixtures of increased polarity and 100 mL
fractions were collected, crystallization from fraction (60-63), eluted with n-hexaneEtOAc (85:15 v/v) afforded compound 7, 20 mg.
Similarly, the chloroform fraction (1.5 g) was chromatographed over silica gel column
(50 x 3 cm i.d.) and eluted with n-hexane-EtOAc mixtures of increased polarity and 20
mL fractions were collected, fractions (65-66) eluted with n-hexane- EtOAc (60:40
v/v) afforded compound 8, 12 mg.
Isolation procedure of compounds 1-6:
The EtOAc fraction (9.0 g) was chromatographed on silica gel column (50 x 4 cm i.d.)
and eluted with CH2Cl2- MeOH mixtures of increased polarity and 100 mL fractions
were collected. Fraction (37-44) eluted with CH2Cl2- MeOH (9:1 v/v), was separated
on Sephadex LH-20 using medium pressure column, MPLC (35 x 3 cm i.d.) packed in
CH2Cl2- MeOH (9.5: 0.5 v/v), eluted with CH2Cl2- MeOH (9:1 v/v) and 10 mL
fractions were collected to afford compound 4, 8 mg. Fraction (53-68) eluted with
CH2Cl2- MeOH (9:1 v/v), was separated on Sephadex LH-20 using MPLC (35 x 3 cm
i.d.) packed in CH2Cl2- MeOH (9.5: 0.5 v/v), eluted with mixtures of CH2Cl2- MeOH
with increased polarity (5%, 10% and 15%, respectively) and 10 mL fractions were
collected, subfraction (25-28), eluted with CH2Cl2- MeOH (10%) afforded compound
5, 7 mg, subfraction (30-32) eluted with the same polarity afforded compound 3, 8 mg
and subfraction (39-46) eluted with CH2Cl2- MeOH (15%) afforded compound 2, 15
mg. Fraction (82-90), 500 mg, eluted with CH2Cl2- MeOH (8:2 v/v) was separated by
Sephadex LH-20 using MPLC (35 x 3 cm i.d.), eluted with CH2Cl2- MeOH (8:2 v/v)
and
10
mL
fractions
were
collected,
subfraction
(16-20),
50
mg
was
rechromatographed on Sephadex LH-20 MPLC, eluted with CH2Cl2- MeOH (93:7
v/v) and 10 mL fractions were collected to give compound 1(9 mg), subfraction (2125) was rechromatographed on silica gel column and eluted with n-hexane-CH2Cl2
(5:5 v/v) and 10 mL fractions were collected to give compound 6,12 mg.
Identification of compounds 7 and 8:
Compound 7 was obtained as colourless needles, m.p.138 - 140 oC, soluble in
chloroform. TLC of 7 showed Rf value of 0.25 using silica gel GF254 and petroleum
ether- EtOAc (9: 1, v/v), and acquired pink colour when sprayed with vanillin/ sulfuric
acid spray reagent and heated at 110 ˚C. It has no behavior under UV 254 and UV366
light.Compound 7 was identified as -sitosterol by co-chromatography against
authentic sample.
Compound 8 was obtained as white amorphous powder. It is soluble in methylene
chloride-methanol. TLC of 8 showed Rf value of 0.30 using EtOAc-methanol (98:2,
v/v). It gave positive Molish’s test indicating its glycosidal nature. It has no behavior
under UV254 and UV366 light, acquires a pink colour after spraying with vanillin/
sulfuric acid spray reagent and heating at 110 ˚C. Compound 8 was identified as
sitosterol-3-O-β-glucoside by co-chromatography against authentic sample.
Figure S1. Summary of the isolation procedure of compounds 1-6 from the ethyl acetate fraction of saw palmetto seeds.
Figure S2: 1H-NMR spectrum (500 MHz, CD3OD) of compound 1.
Figure S3: 13C-NMR spectrum (125 MHz, CD3OD) of compound 1.
Figure S4: HSQC spectrum (CD3OD) of compound 1.
Figure S5: HMBC spectrum (CD3OD) of compound 1.
HO
4``
6``
5`` O
1``
HO
2`` OH
HO 3``
3`
O
O
2`
II-B
HO
4`
5`
1`


`
2
1
3
II-A
OH
6
6`
4
5
OH
O
5`
4`
4``
HO
HO
6``5`` O
3``
2`
1`` O
2
OH
I-B
3`
HO
3
6` OH
`
1`

I-A

1
4
5
6
O
2`` OH
HMBC correlations
Figure S6: HMBC correlations of compound 1.
OH