PRELIMINARY STUDIES ON THE CHEMICAL CHARACTERIZATION AND ANTIOXIDANT CAPACITY OF POLYPHENOLS FROM SAMBUCUS SP.
A.M. ANTONa, A.M. PINTEAb, D.O. RUGINĂb, Z.M.SCONŢAb, D. HANGANUa, L. VLASE c, D. BENEDEC a
aDepartment of Pharmacognosy, “Iuliu Haţieganu” University of Medicine and Pharmacy, 13, E. Isac Street, 400023 Cluj-Napoca, Romania
b University of Agricultural Sciences and Veterinary Medicine, Cluj-Napoca, 3-5 Mănăştur Street, 400372, Cluj-Napoca, Romania
cDepartment of Pharmaceutical Technology and Biopharmaceutics, “Iuliu Haţieganu” University of Medicine and Pharmacy, 13, E. Isac Street, 400023 Cluj-Napoca, Romania
The aim of this study was focused on the chemical characterization of phytochemical compounds and their antioxidant capacity of Sambucus nigra L. (black elder) and Sambucus ebulus (dwarf elder) berries. The primary sources of antioxidant capacities in the evaluated elderberry extracts were phenolic acids and flavonoids (flavonols and anthocyanins) determined by liquid chromatography-mass spectrometry (LC-PDA-MS).
The most abundant anthocyanin in elderberry fruits was cyanidin-3-O-sambubioside, which accounted for more than half of all anthocyanins identified in the berries. Quercetin- 3-O-rutinoside was the major flavonoid found in elderberry extracts, in higher quantity in Sambucus nigra. The antioxidant capacity data obtained for crude-extracts proved that the Sambucus ebulus had the highest antioxidant activity, being the richest one in anthocyanins.
(Received November 8, 2012; Accepted July 18, 2013)
Keywords: Elderberry, Flavonoids, Anthocyanins, Antioxidant capacity
1. Introduction
Sambucus nigra L. (black elder) and Sambucus ebulus L. (dwarf elder) belong to Adoxaceae family, being two widespread species which grow on sunlight-exposed locations in most parts of Europe. Sambucus nigra L. cultivars are planted for ornamental purposes and the elderflower extracts are used as beverage or food flavorings [1]. Recently, much attention has been focused on natural compounds that may be beneficial in reducing oxidative stress-induced diseases. Elderberries contain organic acids and flavonols glycosides [2] and anthocyanins [3, 4].
Nevertheless, the anthocyanins present in elderberries are important for the beneficial health effects associated with their antioxidant properties and their ability to protect against colon cancer [5], influenza A and B virus [6], respective Helicobacter pylori [7] infections has been demonstrated.
The antioxidant activity of flavonols and anthocyanins is due to the presence of ortho 3',4'- dihydroxy moiety in the B ring and meta 5,7-dihydroxy arrangements in the A ring [8, 9].
Literature data on the overall chemical composition of elderberry fruits are scarce, only few studies revealed anthocyanin and polyphenolic content levels of various Sambucus nigra L.
cultivars [2-4, 10]. Even if the content of phenolics in berries is affected by genetic differences,
__________________________ __________
* Corresponding author: [email protected]
environmental conditions, degree of maturity at harvest it is very important for pharmaceutical and alimentary industry to know the chemical composition and the antioxidant capacity of different species [10]. Therefore, the objective of our study was to characterize the phenolic acid, flavonol and anthocyanin content from Sambucus nigra L. and Sambucus ebulus L.
berry extracts and to evaluate their antioxidant capacity.
2. Material and methods 2.1. Reagents
The standard compounds, including cyanidin-3-O-glucoside, cyanidin, gallic acid (GAE), quercetin, and 2,2’-Diphenyl-1- picrylhydrazyl (DPPH) were obtained from Sigma Chemical Co.
(St. Louis, MO). Reagents such as butylhydroxytoluene (BHT), acetonitrile, formic acid, ethanol and methanol were purchased from Merck (Darmstadt, Germany). Cyanidin-3-sambubioside, cyanidin-3,5-diglucoside standard compounds were purchased from Extrasynthese (Lyon, France).
Water used for experiments was treated in a Milli-Q water purification system.
2.2. Extraction
The fruits of black elder and dwarf elder were harvested at the end of August, 2011 at their optimum fruit maturity (Hunedoara, Romania). Vouchers No. 530 and 531 were deposited in the Herbarium of the Department of Pharmaceutical Botany of the Faculty of Pharmacy, “Iuliu Haţieganu” University of Medicine and Pharmacy, Cluj-Napoca, Romania. For obtaining extracts 5g of fruits were extracted by grinding the sample at 20.000 rpm by ultraturax with acidified methanol (0.3% HCl, v/v). The extracts were collected after a centrifugation at 3500 rpm and the residues were re-extracted until the extraction solvents became colorless. The crude extracts were filtrated by multiple layers of cotton and then were concentrated by rotary evaporation at 35°C.
Prior to HPLC analysis were redisolved in acidified methanol, centrifuged at 5000 rpm and filtered through 0.45 μm nylon filter.
2.3. LC-MS analysis
Samples were analysed on an Agilent Technologies 1100 HPLC system (Chelmsford, MA) equipped with G1322A degasser, G13311A binary gradient pump, G1313A autosampler and G1316A UV detector. Volumes of 5 μl were injected on a Zorbax SB C-18 column (3.5 μm, 100 x 3.0 mm) at temperature of 48°C. Data were collected at 330 nm until 17.5 min then at 370 nm.
Mobile phase was consisted in methanol (solvent A) and acetic acid 0.1% (v/v) (solvent B). The solvent elution started with a linear gradient with 5% A and continued for 35 min up to 42% A.
The flow rate was 1 ml/min min. The HPLC system was coupled with an Agilent mass spectrometer (LC/MSD Ion Trap VL). Identification of flavanols was performed via LC-MS in negative mode ionisation on an Agilent 1100 Technologies mass spectrometer equipped with an electrospray ionisation (ESI) interface. Nitrogen was used as desolvation and collision gas.
Desolvation gas (nitrogen) was heated at 360 ºC and nebulizer pressure was set to 65 psi [11,12,13].
2.4. HPLC-PDA analysis
In order to obtain the anthocyanin-rich fraction the elderberry crude-extracts were semipurified following the procedure described previously by He et al. (2011) [14]. The aqueous extract (50 μL) was filtered through a C-18 Sep-Pak cartridge (sorbent mass = 50 mg) (Waters Corp., Milford, MA), previously activated with methanol followed by equilibration with 0.01%
aqueous HCl. Anthocyanins and other polyphenolics were adsorbed onto the Sep-Pak column while sugars, acids, and other water-soluble compounds were removed by washing the minicolumn with 2 volumes of 0.01% aqueous HCl. The second fraction, containing polyphenols
(other than anthocyanins), was subsequently eluted with 2 volumes of ethyl acetate. Anthocyanins were eluted with 4 volumes of methanol containing 0.01% HCl. The acidified methanol fraction was concentrated by using a rotaevaporator at 35˚C until the methanol was completely removed.
The anthocyanin-rich fraction obtained was then dissolved in acidified deionized and sterile water.
For the acid hydrolysis of anthocyanin-rich fraction (2 ml) was mixed with 2M HCl (2 ml) in a screw-cap test tube. The mix was capped and hydrolyzed for 1 h at 90˚C, then cooled in an ice bath.
2.5. Radical scavenging activity (DPPH)
The DPPH scavenging activity assay was done according to a method reported by Brand- Williams et al. [15]. A volume of 1ml of freshly prepared DPPH solution (0.1 g/l) solution was allowed to react with 1ml sample. The ability of the polyphenolic compounds to act as free radical scavengers against DPPH radical was tested by measuring the disappearance of the absorbance after 30 min at 40 °C from the moment when antioxidant solution was applied. The antioxidant activity was calculated as follows:
% DPPH scavenging activity = (1-[Asample/Acontrol t=o]) 100
The absorbance was read at 517 nm, against a reagent blank (JASCO V-630, Japan).
Standard curve was prepared using different concentrations of butylhydroxytoluene and the results were expressed as percent of control.
2.6. Statistical analysis
The data were expressed as mean ± standard deviation (SD) from three replicates for each sample. Significance of difference was defined at the 5% level (p<0.05).
3. Results and discussion
3.1. Polyphenol compounds composition
The profile of phytochemicals of the investigated elderberry cultivars by HPLC-MS analysis revealed the presence of four polyphenolic compounds in both Sambucus sp. fruit extracts (Fig. 1-2). One phenolic acid - p-coumaric acid, two flavonoid glycosides quercetin-3-O-glucoside (also known as isoquercetrin) and quercetin-3-O-rutinoside (also known as rutin) and the aglicon quercetin we identified in the black elderberry crude-extract. Quercetin-3-O-rutinoside was the major phenolic compound found in the fruits of Sambucus nigra (29.02 mg/100 g FW) and the results are in agreement with those obtained by other authors. Quercetin-3-O-glucoside (8.69 mg/100 g FW) was the second major polyphenol identified in our samples, according to data reported previously [16]. The aglicon quercetin in our study was approximately 1/50 of the amount of quercetin-3-O-rutinoside comparing to 1/10 of the amount reported by Veberic et al.,(2009) [2].
In the Sambucus ebulus berry extract hyperoside (1.60 mg/100 g FW) and quercetin-3-O- rutinoside (1.20 mg/100 g FW) were the major polyphenolic compounds identified. After the acidic hydrolysis of the extracts the identified compounds were kaempferol and quercetin (Table 1, Fig.1b, Fig.2b). Kaempferol was found in fewer amounts (0.23 mg/100 g FW) in Sambucus nigra crude-extract comparing to Sambucus ebulus (0.57 mg/100 g FW) sample.
DAD sambu O-gluc and s sambu shown major standar were h compa g FW) literatu author cyanid varied anthoc then fo others anthoc presen hydrol Sambu
Fig.1. In extract (b).
Fig.2. Indiv (b). Peak
3.2. Antho The anthoc analysis. E ubioside-5-gl coside) these sambubioside ubioside pure n in Table 2 a anthocyanin rds and pub higher in Sa aring to those ). The unide ure data and rs reported th din-3-O-gluco
between 27 cyanin conten
or Sambucus authors [2 cyanidins fro nce of cyanid
lization proc ucus nigra
ndividual poly Peak identific O-rutin
vidual polyph k identificatio quercetin ocyanins com
cyanins from lderberries ucoside, cya e consisted o
e. The ant e standard. T and Fig. 3, 4 ns identified blished data.
ambucus ebu e detected in entified com d it UV max he major anth oside [2, 4].
70 and 630 nt (TAC) ob s nigra (272 , 16]. The om the glyc din-3-O-gluc cedure. Anth
and Sambuc
yphenols of Sa cation: 1- hyp noside; 4- que
enols of Samb on: 2- querceti n; 5-quercetin mposition m Sambucus s
were found anidin-3,5-O of three aglyc thocyanin q The data rega 4. Cyanidin-3 based on th Cyanidin 3 ulus (244.79 Sambucus n mpound (peak ximum abso hocyanins in
Similar amo mg/ 100 g F btained this w
.87 mg/100 extracts we cosilated for coside in hy hocyanidins cus ebulus
ambucus ebulu peroside; 2- qu ercetin; 5-quer
bucus nigra cr in-3-O-glucos n, 6-kaempher
sp. berries ha d to contain O-diglucoside
cons (cyanid quantification
arding antho 3-O-sambubi their retentio 3-sambubiosi 9 mg/100 g nigra (134.94 k 3) could b orbance at 5 n Sambucus n
ounts for cya FW were re was higher fo
g FW), data ere subjected rms and the ydrolyzed ex such as c extracts (Ta
us crude-extra uercetin-3-O-g rcetin, 6-kaem
rude-extract (a side, 3- querce rol, 7- p-coum
ave been sep n five major e, cyanidin-3 din, pelargon
n was perf ocyanin ident ioside and cy on times, UV
ide and cyan FW, respec 4 mg/100 g F be a cyanidi
16 nm. Con nigra L. as c anidin-3-O-s eported prev or Sambucus a in agreeme d to acid h en analyzed xtract may b yanidin and able 1). Pel
act (a) and hy glucoside, 3- q mpherol.
a) and hydroly etin-3-O-rutin
aric acid.
parated and id r anthocyan 3-O-sambubi
idin) glycosy formed usin tification and yanidin-3-O- V-VIS spect nidin-3- O - ctively 84.53 FW, respectiv n-3-O -rutin nsistent with yanidin-3-O ambubioside iously [2]. R s ebulus (502 ent with the hydrolysis in
by HPLC-P be explained d pelargonid largonidin-3-
ydrolyzed quercetin-3-
lyzed extract noside; 4-
dentified by nins (cyanidi
ioside, cyan ylated with g ng cyanidin d quantificat -glucoside w tra comparin -glucoside c
3 mg /100 vely 44.83 m noside accor h our results O -sambubios
e from elderb Regarding th 2.73 mg/100
results obtai n order to PDA analysi d by an inco din were fo
-O -glucosi HPLC- in-3-O- nidin-3- glucose n 3-O-
tion are were the ng with contents
g FW) mg /100
ding to s, other ide and berries, he total
g FW) ined by release is. The omplete ound in
de and
pelargonidin-3-O-sambubioside were detected for the first time in elderberry extracts by Wu et al., (2004) [4].
0 2 4 6 8 10 12 14 16 18 20
9
Area (mAU)
Retention time (min)
5
6
7
A 8
0 5 10 15 20 25 30 35
Area (mAU)
Retention time (min) 10
11
12
Fig.3. Individual anthocyanins and anthocyanidins of Sambucus ebulus anthocyanin rich- fraction (A) and hydrolyzed extract (B). Peak identification 5- Cyanidin-3-O- sambubioside-5-glucoside; 6- Cyanidin-3,5-diglucoside, 7- Unknown; 8- Cyanidin-3-O-
sambubioside; 9- Cyanidin-3-O-glucoside, 10- Cyanidin-3-O -glucoside; 11- Pelargonidin; 12- Cyanidin.
0 2 4 6 8 10 12 14 16 18 20
9
Area (mAU)
Retention time (min)
5
6
7 A 8
0 5 10 15 20 25 30 35
Area (mAU)
Retention time (min) 10
11
12 B
Fig.4. Individual anthocyanins and anthocyanidins of Sambucus nigra anthocyanin rich- fraction (A) and hydrolyzed extract (B) extracts. Peak identification 5- Cyanidin-3-O- sambubioside-5-glucoside; 6- Cyanidin-3,5-diglucoside, 7- Unknown; 8- Cyanidin-3-O-
sambubioside; 9- Cyanidin-3-O-glucoside, 10- Cyanidin-3-O-glucoside; 11- Pelargonidin; 12- Cyanidin.
Table 1. The aglycons from the Sambucus sp. berries extracts after acid hydrolysis (mg /100 g FW)
Identified compound λmax Rt Peak Sambucus nigra Sambucus ebulus (mg/100 g FW)
Total flavonol aglycons 2.60 ± 0.16 1.06 ± 0.06
Quercetin 280, 365 23.0 5 2.37 ± 0.13 0.49 ± 0.08
Kaempferol 270, 370 31.5 6 0.23 ±0.18 0.57 ± 0.05
Total anthocyanindins 121.87 ± 2.05 175.03 ± 2.39
Cyanidin-3-O-glucoside 278, 516 12.7 10 19.56 ± 1.24 32.22 ± 2.21 Pelargonidin 274, 520 14.6 11 11.89 ± 1.52 19.72 ± 1.98 Cyanidin 275, 523 27.5 12 109.98 ± 3.41 155.31 ± 2.98
Table 2. Retention times and the individual polyphenols concentration in Sambucus nigra and Sambucus ebulus berry extracts
Compound λmax (nm) Peak Rt
Concentration (mg/100 g FW) Sambucus nigra Sambucus
ebulus
Phenolic acid 0.55 ± 0.01 -
p-coumaric acid 284 7 8.72 0.55 ± 0.01 -
Total flavonoids 38.26 ± 1.43 3.44 ± 0.045
Hyperoside 366 1 18.70 - 1.60 ± 0.04
Quercetin-3-O-glucoside 366 2 19.70 8.69 ± 1.23 0.44 ± 0.02 Quercetin-3-O-rutinoside 367 3 20.20 29.02 ± 2.1 1.20 ± 0.07
Quercetin 370 4 23.10 0.55 ± 0.98 0.20 ± 0.05
Total anthocyanins 272.87 ± 1.11 502.73 ± 1.83
Cyanidin-3-O-sambubioside-
5-glucoside 275, 525 5 4.7 23.34 ± 0.68 42.92 ± 1.56
Cyanidin-3,5 -diglucoside 275, 516 6 6.8 47.1 ± 0.78 91.63 ± 3.21
Unknown 271, 518 7 10.4 22.66 ± 0.21 38.86 ± 0.87
Cyanidin-3-O-sambubioside 279, 518 8 12.2 134.94 ± 2.65 244.79 ± 3.25 Cyanidin-3-O-glucoside 275, 517 9 13 44.83 ± 1.23 84.53 ± 0.26
nd- not detected
3.3. Antioxidant activity evaluation by DPPH assay
Various phytochemical components, such as phenolic acids, flavonols and anthocyanins are known to be responsible for the antioxidant capacity of fruits. The antioxidant activity of elderberry crude extracts was evaluated by DPPH assay and the data obtained are presented as percentage values in Table 3. The method used is based on the reduction of the radical DPPH in the presence of hydrogen donating antioxidant. The strongest radical scavenging activity showed Sambucus ebulus, while the activity of Sambucus nigra berry crude extract was considerably lower. The butylated hydroxytoluene (BHT), a synthetic scavenger showed a reduced antioxidant activity than those exerted by polyphenolic compounds from Sambucus ebulus extract. The antioxidant activity evaluated by oxygen radical absorbance capacity was higher for elderberries than for gooseberries, black and red currants [4]. In agreement with literature data our results showed that the antioxidant activity of elderberries is positively correlated to their anthocyanin content. Similar results were obtained for chokeberries, blackberries, red raspberries, black raspberries and strawberries extracts [17-19].
Table 3. Radical scavenging activity determined by DPPH assay
Elderberry crude-extracts DPPH radical scavenging activity (%)
Sambucus nigra 63.26 ± 1.05
Sambucus ebulus 83.17 ± 1.21
BHT 81.39 ± 0.85
4. Conclusions
Elderberries are predominately used as processed food or dietary supplements, therefore it is useful to know their content on phenolic acids, anthocyanins and flavonoids. Sambucus nigra and Sambucus ebulus both contained especially anthocyanins as cyanidin 3-O- sambubioside and cyanidin 3-glucoside. The species with the highest amounts of total analyzed anthocyanins and flavonoids was Sambucus ebulus, which also showed the higher potential to scavenge the radical DPPH.
Acknowledgement
This research was supported by UMF Cluj-Napoca Research Grant no.
22714/49/06.10.2010 and no. 27020/43/15.11.2011 References
[1] L. Christensen, K. Kaack, X. Fretté, Eur. Food Res. Technol., 227(1), 293 (2008) [2] R. Veberic, J. Jakopic, F. Stampar, V. Schmitzer, Food Chem., 114 (2) 511 (2009)
[3] J. I. Nakajima, I. Tanaka, S. Seo, M. Yamazaki, K. Saito, J Biomed Biotechnol, 5 241 (2004) [4] X. Wu, L. Gu, R. L. Prior, S. Mckay, J. Agric. Food. Chem., 52 (26), 7846 (2004)
[5] P. Jing, J. A. Bomser, S. J. Schwartz, J. He, B. A. Magnuson, M. M. Giusti, J. Agric. Food.
Chem., 56(20), 9391 (2008)
[6] Z. Zakay-Rones, E. Thom, T. Wollan, J. Wadstein, J. Int. Med. Res., 32(2), 132 (2004)
[7] S. Zafra-Stone, T. Yasmin, M. Bagchi, A. Chatterjee, J. A. Vinson, D. Bagchi, Mol Nutr Food Res, 51 (6): 675-683 (2007)
[8] C. Rice-Evans, N. Miller, G. Paganga, Trends Plant Sci., 2 (4), 152(1997)
[9] C. A. Rice-Evans, N. J. Miller, G. Paganga, Free Radical Biol. Med., 20(7), 933 (1996) [10] R. Zadernowski, M. Naczk, J. Nesterowicz, J. Agric. Food Chem., 53 (6), 2118 (2005) [11] I. Nencu, L. Vlase, V. Istudor, L.E. Dutu, C.E. Gîrd, Farmacia 60(4),493 (2012)
[12] M. Compaore, C.E. Lamien, A. Lamien-Meda, L. Vlase, M. Kiendrebeogo, C. Ionescu, O.G. Nacoulma, Nat. Prod. Res. 26(11), 1069 (2012)
[13] R.N.T. Meda, L. Vlase, A. Lamien-Meda, C. E. Lamien, D. Muntean, B. Tiperciuc, I. Oniga, O.G. Nacoulma, Nat. Prod. Res. 25(2),93 (2011)
[14] J. He and Giusti, M. M., J. Chromatogr. A, 1218 (44), 7914 (2011)
[15] W. Brand-Williams, M. E. Cuvelier, C. Berset, LWT - Food Science and Technology, 28 (1), 25 (1995)
[16] J. Lee, C. E. Finn, J. Sci. Food Agric., 87(14), 2665 (2007)
[17] A. Castañeda-Ovando, M. D. L. Pacheco-Hernández, M. E. Páez-Hernández, J. A. Rodríguez, C. A. Galán-Vidal, Food Chem., 113(4), 859 (2009)
[18] J. Dai, A. Gupte, L. Gates, R. J. Mumper, Food Chem. Toxicol., 47 (4): 837-847 (2009)
