Supplementary material for: [https://doi.org/10.1016/j.jep.2017.11.013]Related to published version: [http://cherry.chem.bg.ac.rs/handle/123456789/2068]Related to accepted version: [http://cherry.chem.bg.ac.rs/handle/123456789/3153

Arsenijević, Jelena

Božić, Dragana

Maksimović, Zoran

Milenković, Marina

Samardžić, Stevan

Tešević, Vele

Journal of Ethnopharmacology

English

Cherry - Repository of the Faculty of Chemistry;  University of Belgrade

  
 
 
 
 
 
 
 
 
 
 
 
Supplementary data for the article: 
 
Samardžić, S.; Arsenijević, J.; Božić, D.; Milenković, M.; Tešević, V.; Maksimović, Z. 
Antioxidant, Anti-Inflammatory and Gastroprotective Activity of Filipendula Ulmaria (L.) 
Maxim. and Filipendula Vulgaris Moench. J. Ethnopharmacol. 2018, 213, 132–137. 
https://doi.org/10.1016/j.jep.2017.11.013  
 
 
Supplementary material 
 1 
Antioxidant, anti-inflammatory and gastroprotective activity of Filipendula 
ulmaria (L.) Maxim. and Filipendula vulgaris Moench 
Stevan Samardžić*, Jelena Arsenijević, Dragana Božić, Marina Milenković, Vele 
Tešević, Zoran Maksimović 
 
 Corresponding author. Tel: +381 11 3951 321; E-mail: stevans@pharmacy.bg.ac.rs 
 
A. Isolation and structure elucidation of constituents of  
Filipendula vulgaris Moench flowers. 
  
 
1. General 
Silica gel (0.063–0.200 mm, Merck, 
Germany), Sephadex LH-20 (GE 
Healthcare Bio-Science, Sweden) and 
octadecyl silica gel (Sigma-Aldrich, USA) 
were employed for column 
chromatography. Final purification was 
performed using TLC cellulose plates 
(Merck, Germany) or Agilent 1260 LC 
System equipped with an automated 
fraction collector 1364C and 
semi-preparative Zorbax SB-C18 column 
(9.4×250 mm, 5 μm). 
NMR spectra (1H, 400 MHz; 13C, 100 
MHz) were recorded on Bruker AscendTM 
400 instrument. Prior to analysis, all 
samples were dissolved in deuterated 
methanol (MeOH-d4). TMS was used as 
the internal standard. 
MS and UV spectral data of the isolated 
compounds were obtained by LC-PDA-MS 
analysis using Agilent 1260 liquid 
chromatograph. Mass spectral data were 
acquired with Agilent 6130 single 
quadrupole mass detector (negative ion 
mode, mass range 100–1000 m/z). Ion            
source (electrospray ionization) operating 
parameters were set as follows: drying gas 
flow 9 L/min (N2), nebulizer pressure  
40 psi, drying gas temperature 350 °C, 
capillary voltage 3500 V and fragmentor 
voltages 100 and 250 V. UV spectra were 
obtained with a G4212B PDA detector. 
 
2. Isolation of constituents 
 
2.1. Preparation of fractions F1 and F2 
Dried and ground flowers of F. vulgaris 
(178 g) were successively bi-macerated 
with organic solvents of increasing 
polarity: n-hexane, dichloromethane and 
methanol (flowers : solvent ratio, 1:10, 
w/v). The resulting extracts were dried 
under reduced pressure. A portion (47 g) of 
the obtained methanol extract was 
suspended in water and partitioned with 
ethyl acetate. In the next step, the organic 
layer was separated and concentrated in 
vacuum to give 4.7 g of fraction (F1) 
further used in the isolation process. 
The ethyl acetate insoluble fraction (8 
g) of methanol extract of defatted F. 
vulgaris flowers, prepared according to 
procedure described by Ćebović and 
Maksimović (2012), was subjected to silica 
gel column chromatography and eluted 
with solvents of increasing polarity 
 
   
Supplementary material                              Samardžić et al. 
 2 
(CH2Cl2/MeOH/H2O/CH3COOH, 
90/10/0.4/0.1→70/30/1.2/0.3 v/v/v) to 
provide 40 subfractions (100 mL). Based 
on chemical composition similarity, 
subfractions 21–36 were combined to yield 
2.6 g of the fraction here designated as F2. 
 
2.2. Isolation procedure 
Mixture of F1 (0.7 g) and F2 (0.65 g) 
was partitioned on Sephadex LH-20 
column using ethanol as the mobile phase. 
Sixteen subfractions were collected (A1–
A16, 50 mL each). Subfraction A10 was 
subsequently purified by semi-preparative 
RP-HPLC (30% acetonitrile, flow rate 3.3 
mL/min) to give 5.3 mg of 1. Compound 2 
(150.1 mg) precipitated from combined 
subfractions A12–A14. Residue obtained 
after removal of 2 was submitted to 
preparative TLC (cellulose, 30% 
CH3COOH) to yield 4.2 mg of 3. 
Mixture of F1 (1.6 g) and F2 (0.65 g) 
was partitioned on Sephadex LH-20. This 
time the elution was performed with 
mixtures of ethanol and methanol 
(80/20→0/100 v/v), mixture of methanol, 
acetone and water (80/14/6 v/v/v) and 
finally 70% acetone. Forty subfractions 
were obtained (B1–B40, 50 ml each). 
Subfractions B14–B15 were separated 
using preparative TLC (cellulose, 30% 
CH3COOH) and 17.8 mg of mixture of 4 
and 5 was isolated. Semi-preparative 
RP-HPLC of combined fractions B31–B32 
(20% acetonitrile, flow rate 3.3 mL/min) 
yielded 6.1 mg of 6.  
For conduction of in vivo 
pharmacological experiments, additional 
amount of 2 was obtained by repeating the 
aforementioned procedure with minor 
adjustments, whereas new quantities of 6 
were isolated by vacuum liquid 
chromatography separation of combined 
subfractions (B29–B30 and B33–B34, 
262.9 mg) over octadecyl silica gel eluted 
with 20% acetonitrile. 
 
3. Structure elucidation of the isolated 
constituents 
UV and NMR (1H, 13C) spectra of the 
isolated compounds were in agreement 
with the previous reports. Their molecular 
masses corresponded to the determined 
structures. Based on literature data, 
compound 1 was identified as kaempferol 
4′-O-β-D-glucoside (Lim et al., 2004; 
Scheer and Wichtl, 1987); 2 was 
determined to be spiraeoside (quercetin 
4′-O-β-D-glucoside) (Fossen et al., 1998; 
Lim et al., 2004; Pavlović, 2008); 3 
presented kaempferol 3-O-(2″-O-galloyl)-
β-D-glucoside (astragalin 2″-O-gallate) 
(Isobe et al., 1980; Ochir et al., 2013); 
mixture of 4 and 5 was identified as 
mixture of quercetin 3-O-(2″-O-galloyl)-β-
D-glucoside and quercetin 3-O-(2″-O-
galloyl)-β-D-galactoside (2:1 w/w, based 
on HPLC-UV chromatograms and NMR 
spectra integration), respectively (Isobe et 
al., 1979; Kawakami et al., 2011; Kim et 
al., 1998; Ochir et al., 2013; Pakulski and 
Budzianowski, 1996; Yazaki et al., 1989); 
and finally, the structure of 6 was 
established as tellimagrandin II (Chen et 
al., 2014; Feldman and Sahasrabudhe, 
1999; Wilkins and Bohm, 1976). 
 
 
   
Supplementary material                              Samardžić et al. 
 3 
B. HPLC analysis of F. vulgaris and F. ulmaria lyophilized flower infusions 
HPLC analysis of the lyophilized flower 
infusions of F. ulmaria and F. vulgaris was 
performed on an Agilent 1100 system 
coupled to a DAD detector and equipped 
with Zorbax Eclipse XDB-C18 analytical 
column (4.6×250 mm, 5 μm, Agilent). 
Quantification was done by the external 
standard method. Detailed description of 
the used procedures is given in the 
manuscript (section 2.3.). Mobile phase 
consisting of solvent A (H3PO4 in H2O, pH 
2.75) and solvent B (solvent A : 
acetonitrile, 10 : 90 v/v) was used for 
gradient elution as presented in the Table 
1S. Program 1 was used for the 
quantification of astragalin, spiraeoside 
and salicylic acid, whereas the contents of 
gallic acid, ellagic acid, hyperoside and 
isoquercitrin were determined using 
program 2. 
 
Table 1S. Elution programs. 
Elution program 1  Elution program 2 
time  
(min) 
solvent B 
(%v/v) 
flow rate  
(mL/min) 
 time 
(min) 
solvent B 
(%v/v) 
flow rate  
(mL/min) 
0 
5 
15 
20 
25 
30 
35 
38 
10 
25 
25 
30 
50 
70 
10 
10 
0.8 
0.8 
0.8 
0.8 
0.8 
0.8 
0.8 
0.8 
 0 
5 
15 
19 
25 
28 
30 
32 
10 
15 
20 
24 
54 
70 
10 
10 
0.8 
0.8 
1.7 
1.2 
0.8 
0.8 
0.8 
0.8 
 
 
4. References 
Ćebović, T., Maksimović, Z., 2012. 
Hepatoprotective Effect of Filipendula 
hexapetala Gilib. (Rosaceae) in Carbon 
Tetrachloride-induced Hepatotoxicity in Rats. 
Phytother. Res. 26, 1088–1091. 
Chen, Y., Wang, J., Ou, Y., Chen, H., Xiao, S., Liu, 
G., Cao, Y., Huang, Q., 2014. Cellular 
antioxidant activities of polyphenols isolated 
from Eucalyptus leaves (Eucalyptus grandis × 
Eucalyptus urophylla GL9). J. Funct. Foods 7, 
737–745. 
Feldman, K.S., Sahasrabudhe, K., 1999. 
Ellagitannin Chemistry. Syntheses of 
Tellimagrandin II and a Dehydrodigalloyl 
Ether-Containing Dimeric Gallotannin 
Analogue of Coriariin A. J. Org. Chem. 64, 
209–216. 
Fossen, T., Pedersen, A.T., Andersen, Ø.M., 1998. 
Flavonoids from red onion (Allium cepa). 
Phytochemistry 47, 281–285. 
Isobe, T., Fukushige, T., Noda, Y., 1979. A new 
flavonoid glycoside from Polygonum nodosum. 
Chem. Lett. 8, 27–30. 
Isobe, T., Ito, N., Noda, Y., 1980. Minor flavonoids 
of Polygonum nodosum. Phytochemistry 19, 
1877. 
Kawakami, K., Shibukura, Y., Kanno, T., Furuki, 
T., Aketa, S., Hirayama, M., 2011. 
Identification of 2″-Galloylated Flavonol 
3-O-Glycosides Accumulating in Developing 
Leaves of Persimmon. Phytochem. Anal. 22, 
403–410. 
Kim, H.J., Woo, E.-R., Shin, C.-G., Park, H., 1998. 
A New Flavonol Glycoside Gallate Ester from 
Acer okamotoanum and Its Inhibitory Activity 
 
   
Supplementary material                              Samardžić et al. 
 4 
against Human Immunodeficiency Virus-1 
(HIV-1) Integrase. J. Nat. Prod. 61, 145–148. 
Lim, E.-K., Ashford, D.A., Hou, B., Jackson, R.G., 
Bowles, D.J., 2004. Arabidopsis 
glycosyltransferases as biocatalysts in 
fermentation for regioselective synthesis of 
diverse quercetin glucosides. Biotechnol. 
Bioeng. 87, 623–631. 
Ochir, S., Yuki, T., Kanazawa, T., Nishizawa, M., 
Yamagishi, T., 2013. Two Galloylated 
Flavonoids as Antioxidants in Rosa gallica 
Petals. Chem. Nat. Compd. 49, 940–942. 
Pakulski, G., Budzianowski, J., 1996. Quercetin 
and Kaempferol Glycosides of Dionaea 
muscipula from in vitro Cultures. Planta Med. 
62, 95–96. 
Pavlović, M., 2008. Doctoral dissertation: 
Proučavanje sastojaka Anthemis triumfetti 
(Asteraceae) i poređenje sa drugim vrstama 
podroda Cota. University of Belgrade – Faculty 
of Pharmacy, Belgrade. 
Scheer, T., Wichtl, M., 1987. On the Occurrence of 
Kaempferol-4′-O-β-D-glucopyranoside in 
Filipendula ulmaria and Allium cepa. Planta 
Med. 53, 573–574. 
Wilkins, C.K., Bohm, B.A., 1976. Ellagitannins 
from Tellima grandiflora. Phytochemistry 15, 
211–214. 
Yazaki, K., Shida, S., Okuda, T., 1989. 
Galloylhomoarbutin and related polyphenols 
from Pyrola incarnata. Phytochemistry 28, 
607–609. 
 


Supplementary material for the article: Samardžić, S.; Arsenijević, J.; Božić, D.; Milenković, M.; Tešević, V.; Maksimović, Z. Antioxidant, Anti-Inflammatory and Gastroprotective Activity of Filipendula Ulmaria (L.) Maxim. and Filipendula Vulgaris Moench. Journal of Ethnopharmacology 2018, 213, 132–137. https://doi.org/10.1016/j.jep.2017.11.013

Faculty of Chemistry Repository - Cherry

             Supplementary data for the article:  Samardžić, S.; Arsenijević, J.; Božić, D.; Milenković, M.; Tešević, V.; Maksimović, Z. Antioxidant, Anti-Inflammatory and Gastroprotective Activity of Filipendula Ulmaria (L.) Maxim. and Filipendula Vulgaris Moench. J. Ethnopharmacol. 2018, 213, 132–137. https://doi.org/10.1016/j.jep.2017.11.013    Supplementary material  1 Antioxidant, anti-inflammatory and gastroprotective activity of Filipendula ulmaria (L.) Maxim. and Filipendula vulgaris Moench Stevan Samardžić*, Jelena Arsenijević, Dragana Božić, Marina Milenković, Vele Tešević, Zoran Maksimović   Corresponding author. Tel: +381 11 3951 321; E-mail: stevans@pharmacy.bg.ac.rs  A. Isolation and structure elucidation of constituents of  Filipendula vulgaris Moench flowers.    1. General Silica gel (0.063–0.200 mm, Merck, Germany), Sephadex LH-20 (GE Healthcare Bio-Science, Sweden) and octadecyl silica gel (Sigma-Aldrich, USA) were employed for column chromatography. Final purification was performed using TLC cellulose plates (Merck, Germany) or Agilent 1260 LC System equipped with an automated fraction collector 1364C and semi-preparative Zorbax SB-C18 column (9.4×250 mm, 5 μm). NMR spectra (1H, 400 MHz; 13C, 100 MHz) were recorded on Bruker AscendTM 400 instrument. Prior to analysis, all samples were dissolved in deuterated methanol (MeOH-d4). TMS was used as the internal standard. MS and UV spectral data of the isolated compounds were obtained by LC-PDA-MS analysis using Agilent 1260 liquid chromatograph. Mass spectral data were acquired with Agilent 6130 single quadrupole mass detector (negative ion mode, mass range 100–1000 m/z). Ion            source (electrospray ionization) operating parameters were set as follows: drying gas flow 9 L/min (N2), nebulizer pressure  40 psi, drying gas temperature 350 °C, capillary voltage 3500 V and fragmentor voltages 100 and 250 V. UV spectra were obtained with a G4212B PDA detector.  2. Isolation of constituents  2.1. Preparation of fractions F1 and F2 Dried and ground flowers of F. vulgaris (178 g) were successively bi-macerated with organic solvents of increasing polarity: n-hexane, dichloromethane and methanol (flowers : solvent ratio, 1:10, w/v). The resulting extracts were dried under reduced pressure. A portion (47 g) of the obtained methanol extract was suspended in water and partitioned with ethyl acetate. In the next step, the organic layer was separated and concentrated in vacuum to give 4.7 g of fraction (F1) further used in the isolation process. The ethyl acetate insoluble fraction (8 g) of methanol extract of defatted F. vulgaris flowers, prepared according to procedure described by Ćebović and Maksimović (2012), was subjected to silica gel column chromatography and eluted with solvents of increasing polarity     Supplementary material                              Samardžić et al.  2 (CH2Cl2/MeOH/H2O/CH3COOH, 90/10/0.4/0.1→70/30/1.2/0.3 v/v/v) to provide 40 subfractions (100 mL). Based on chemical composition similarity, subfractions 21–36 were combined to yield 2.6 g of the fraction here designated as F2.  2.2. Isolation procedure Mixture of F1 (0.7 g) and F2 (0.65 g) was partitioned on Sephadex LH-20 column using ethanol as the mobile phase. Sixteen subfractions were collected (A1–A16, 50 mL each). Subfraction A10 was subsequently purified by semi-preparative RP-HPLC (30% acetonitrile, flow rate 3.3 mL/min) to give 5.3 mg of 1. Compound 2 (150.1 mg) precipitated from combined subfractions A12–A14. Residue obtained after removal of 2 was submitted to preparative TLC (cellulose, 30% CH3COOH) to yield 4.2 mg of 3. Mixture of F1 (1.6 g) and F2 (0.65 g) was partitioned on Sephadex LH-20. This time the elution was performed with mixtures of ethanol and methanol (80/20→0/100 v/v), mixture of methanol, acetone and water (80/14/6 v/v/v) and finally 70% acetone. Forty subfractions were obtained (B1–B40, 50 ml each). Subfractions B14–B15 were separated using preparative TLC (cellulose, 30% CH3COOH) and 17.8 mg of mixture of 4 and 5 was isolated. Semi-preparative RP-HPLC of combined fractions B31–B32 (20% acetonitrile, flow rate 3.3 mL/min) yielded 6.1 mg of 6.  For conduction of in vivo pharmacological experiments, additional amount of 2 was obtained by repeating the aforementioned procedure with minor adjustments, whereas new quantities of 6 were isolated by vacuum liquid chromatography separation of combined subfractions (B29–B30 and B33–B34, 262.9 mg) over octadecyl silica gel eluted with 20% acetonitrile.  3. Structure elucidation of the isolated constituents UV and NMR (1H, 13C) spectra of the isolated compounds were in agreement with the previous reports. Their molecular masses corresponded to the determined structures. Based on literature data, compound 1 was identified as kaempferol 4′-O-β-D-glucoside (Lim et al., 2004; Scheer and Wichtl, 1987); 2 was determined to be spiraeoside (quercetin 4′-O-β-D-glucoside) (Fossen et al., 1998; Lim et al., 2004; Pavlović, 2008); 3 presented kaempferol 3-O-(2″-O-galloyl)-β-D-glucoside (astragalin 2″-O-gallate) (Isobe et al., 1980; Ochir et al., 2013); mixture of 4 and 5 was identified as mixture of quercetin 3-O-(2″-O-galloyl)-β-D-glucoside and quercetin 3-O-(2″-O-galloyl)-β-D-galactoside (2:1 w/w, based on HPLC-UV chromatograms and NMR spectra integration), respectively (Isobe et al., 1979; Kawakami et al., 2011; Kim et al., 1998; Ochir et al., 2013; Pakulski and Budzianowski, 1996; Yazaki et al., 1989); and finally, the structure of 6 was established as tellimagrandin II (Chen et al., 2014; Feldman and Sahasrabudhe, 1999; Wilkins and Bohm, 1976).      Supplementary material                              Samardžić et al.  3 B. HPLC analysis of F. vulgaris and F. ulmaria lyophilized flower infusions HPLC analysis of the lyophilized flower infusions of F. ulmaria and F. vulgaris was performed on an Agilent 1100 system coupled to a DAD detector and equipped with Zorbax Eclipse XDB-C18 analytical column (4.6×250 mm, 5 μm, Agilent). Quantification was done by the external standard method. Detailed description of the used procedures is given in the manuscript (section 2.3.). Mobile phase consisting of solvent A (H3PO4 in H2O, pH 2.75) and solvent B (solvent A : acetonitrile, 10 : 90 v/v) was used for gradient elution as presented in the Table 1S. Program 1 was used for the quantification of astragalin, spiraeoside and salicylic acid, whereas the contents of gallic acid, ellagic acid, hyperoside and isoquercitrin were determined using program 2.  Table 1S. Elution programs. Elution program 1  Elution program 2 time  (min) solvent B (%v/v) flow rate  (mL/min)  time (min) solvent B (%v/v) flow rate  (mL/min) 0 5 15 20 25 30 35 38 10 25 25 30 50 70 10 10 0.8 0.8 0.8 0.8 0.8 0.8 0.8 0.8  0 5 15 19 25 28 30 32 10 15 20 24 54 70 10 10 0.8 0.8 1.7 1.2 0.8 0.8 0.8 0.8   4. References Ćebović, T., Maksimović, Z., 2012. Hepatoprotective Effect of Filipendula hexapetala Gilib. (Rosaceae) in Carbon Tetrachloride-induced Hepatotoxicity in Rats. Phytother. Res. 26, 1088–1091. Chen, Y., Wang, J., Ou, Y., Chen, H., Xiao, S., Liu, G., Cao, Y., Huang, Q., 2014. Cellular antioxidant activities of polyphenols isolated from Eucalyptus leaves (Eucalyptus grandis × Eucalyptus urophylla GL9). J. Funct. Foods 7, 737–745. Feldman, K.S., Sahasrabudhe, K., 1999. Ellagitannin Chemistry. Syntheses of Tellimagrandin II and a Dehydrodigalloyl Ether-Containing Dimeric Gallotannin Analogue of Coriariin A. J. Org. Chem. 64, 209–216. Fossen, T., Pedersen, A.T., Andersen, Ø.M., 1998. Flavonoids from red onion (Allium cepa). Phytochemistry 47, 281–285. Isobe, T., Fukushige, T., Noda, Y., 1979. A new flavonoid glycoside from Polygonum nodosum. Chem. Lett. 8, 27–30. Isobe, T., Ito, N., Noda, Y., 1980. Minor flavonoids of Polygonum nodosum. Phytochemistry 19, 1877. Kawakami, K., Shibukura, Y., Kanno, T., Furuki, T., Aketa, S., Hirayama, M., 2011. Identification of 2″-Galloylated Flavonol 3-O-Glycosides Accumulating in Developing Leaves of Persimmon. Phytochem. Anal. 22, 403–410. Kim, H.J., Woo, E.-R., Shin, C.-G., Park, H., 1998. A New Flavonol Glycoside Gallate Ester from Acer okamotoanum and Its Inhibitory Activity     Supplementary material                              Samardžić et al.  4 against Human Immunodeficiency Virus-1 (HIV-1) Integrase. J. Nat. Prod. 61, 145–148. Lim, E.-K., Ashford, D.A., Hou, B., Jackson, R.G., Bowles, D.J., 2004. Arabidopsis glycosyltransferases as biocatalysts in fermentation for regioselective synthesis of diverse quercetin glucosides. Biotechnol. Bioeng. 87, 623–631. Ochir, S., Yuki, T., Kanazawa, T., Nishizawa, M., Yamagishi, T., 2013. Two Galloylated Flavonoids as Antioxidants in Rosa gallica Petals. Chem. Nat. Compd. 49, 940–942. Pakulski, G., Budzianowski, J., 1996. Quercetin and Kaempferol Glycosides of Dionaea muscipula from in vitro Cultures. Planta Med. 62, 95–96. Pavlović, M., 2008. Doctoral dissertation: Proučavanje sastojaka Anthemis triumfetti (Asteraceae) i poređenje sa drugim vrstama podroda Cota. University of Belgrade – Faculty of Pharmacy, Belgrade. Scheer, T., Wichtl, M., 1987. On the Occurrence of Kaempferol-4′-O-β-D-glucopyranoside in Filipendula ulmaria and Allium cepa. Planta Med. 53, 573–574. Wilkins, C.K., Bohm, B.A., 1976. Ellagitannins from Tellima grandiflora. Phytochemistry 15, 211–214. Yazaki, K., Shida, S., Okuda, T., 1989. Galloylhomoarbutin and related polyphenols from Pyrola incarnata. Phytochemistry 28, 607–609.  

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