SUPPLEMENTARY MATERIAL
Variation in phytochemicals and bioactivity of the fruits of Eleutherococcus species
cultivated in Poland.
Daniel Załuskia Zbigniew Janeczko
Department of Pharmacognosy, Collegium Medicum, Jagiellonian University, 9 Medyczna Street, 30 –
688 Cracow, Poland
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Corresponding Author:
Daniel Załuski E-mail: daniel_zaluski@onet.eu Phone: +48 12 620-55-60.
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Abstract: Neither the chemical profile and bioactivity of the fruits of Eleutherococcus species cultivated
in Poland nor the influence of long-term domestic storage of the fruits are known. No significant changes
in the phenols and flavonoids content in the freshly-dried and one-year storage fruits of E. senticosus and
E. henryi were found. The one-year storage fruits of both species exhibited a very high concentration of
Mn, Zn, Cu [mg/kg] compared to the fresh-drying ones. Spathulenol, β-longipinene and iso-longifolol
were predominant essential oil compounds. It was observed a high capacity of the freshly-dried E.
senticosus fruits to scavenge DPPH* and inhibit Hyal (EC50 0.1 mg/mL; IC50 0.58 mg/mL). Due to their
health and pharmacological benefits, a better understanding of the impact of the climate change is
important to increase pharmacological utility of the extracts.
Keywords: Eleutherococcus fruits, Polyphenols, Anti-DPPH*, Anti-hyaluronidase, Nutripharm products,
Minerals
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Experimental
Standards and Reagents. Folin-Ciocalteau reagent, 1,1-diphenyl-2-picryl-hydrazyl (DPPH), ascorbic
acid, DMSO, bovine albumin, hyaluronidase from bovine testes type I-S, Streptococcus equi hyaluronic
acid, methyl indole-3-carboxylate, sodium phosphate buffer pH 7.0 were obtained from Sigma-Aldrich.
2,4-DNPH and FeCl3, ethanol were obtained from POCH (Lublin, Poland). The acetate buffer, pH 4.5
was purchased from J.T. Baker, USA. All others reagents were of analytical grade.
Plant material. The fruits of E. senticosus (Rupr. et Maxim.) Maxim. and E. henryi Oliv. were collected
at the arboretum in Rogów (Poland) in October 2012 (voucher specimen number: SF234 and HF235). All
plant samples were deposited at the Department of Pharmacognosy, Collegium Medicum, Jagiellonian
University, Cracow, Poland. All analysis were performed using the fruits immediately after drying, and
after an one-year storage at room temperature. All samples were marked as: 1E-the ethanol extract from
the freshly-dried fruits of E. senticosus; 2E-the ethanol extract from the fruits of E. senticosus after an
one-year storage; 3E-the ethanol extract from the freshly-dried fruits of E. henryi; 4E-the ethanol extract
from the fruits of E. henryi after an one-year storage. The fruits were storaged under domestic conditions
(room temperature, paper bag, dark place).
Dried material extraction with 75% ethanol. The air-dried fruits (5 g each) from E. senticosus and E.
henryi were soaked in 50 mL 75% ethanol for 24 h. Next, the samples were subjected to triple UAE type
extraction (ultrasonic bath -Polsonic, Warsaw, Poland) using 50, 2 x 25 mL of 75% ethanol. The
extraction was performed at room temperature for 15 min for each cycle. Finally, 100 mL of each extract
was obtained. The solvents were dried with an evaporator under vacuum conditions at 45 °C and
subjected to lyophilisation. The extraction yield was calculated based on the dry weight of the extract
[%].
Total phenolic content (TPC). The total phenolic content of extracts was determined using the method
of Singleton and Rossi (Singleton, & Rossi, 1965). Gallic acid was used to calculate the calibration curve
(20-100 µg/mL; y=0.0026x + 0.044; r2=0.999), and TPC was expressed as gallic acid equivalents
(GAE/100 g dry extract). Every assay was performed in triplicate.
Total flavonoid content (TFC). The TFC in investigated samples was determined using aluminium
chloride and 2,4-dinitrophrnylhydrazine colorimetric methods (Chang, Yang, Wen, & Chern, 2002). TFC
were expressed as means (±S.E.) mg of quercetin equivalent (QEs/100 g dry extract for FeCl 3 method;
20-100 µg/mL; y=0.0041x + 0.236; r2=0.999) and as means (±S.E.) mg of hesperetin equivalent
(QEs/100 g dry extract for 2,4-DNPH method; 250-1000 µg/mL; y=6.374x - 0.098; r2=0.988). Every
assay was performed in triplicate.
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ASA of minerals. Minerals content was assayed according to requirements of AOAC 985.01. (Metals
and other elements in plants and pet foods). 0.5 g of the dried and ground fruits was put into a burning
cup, and 2 mL of pure HNO3 was added. The samples were incinerated in a MARS 5 microwave oven
(Manufactured by, CEM corporation, USA) at a temperature of 90 °C for 15 min, next 120 °C for 10 min,
and 210 °C for 30 min, and the solution was diluted to 100 ml with water. Concentrations were
determined with an Varian SpektrAA 280FS + Autosampler SPS 3 Spectrometer. Minerals and trace
elements were determined using the instrumental conditions recommended for each mineral and were
calculated based on the respective standard curve.
GC/MS/MS of essential oil. The extraction of volatile oil was carried out according to the Polish
Pharmacopoeia IX. The quantity of volatile in sample calculated in percentage (v/d.w.). The oil was
stored at 4 oC in a brown vial. A highly sensitive and accurate multiplex gas chromatography-linear ion
trap technique was used to identify components of the volatiles. GC/MS/MS was performed using Varian
4000 GC/MS/MS chromatograph equipped with Flame Ionization Detector (FID). The GC conditions
were as follows: VF-5 ms fused silica capillary column (30 m x 0.25 mm, film thickness 0.25 µm), the
oven temperature was programmed at a rate of 1 oC from 50 (held for 5 min.) to 250 oC, injector and
detector kept at 250 oC, split ratio 1 : 100. Helium was used as the carrier gas with a constant flow rate of
0.5 mL/min. The volume of injection was 1 µL of a pentane-volatiles solution (1:1). Mass spectra were
recorded at 70 eV. Mass range was from m/z 40 to 1000. The identification of individual compounds
resulted on the basis of Kovats linear retention index (LRI) determination relative to (C6–C40) n–alkanes
and retention time (RT). Compounds were further identified using their MS data compared to homemade
library mass spectra built up from pure substances (Main Lab Library).
Anti-hyaluronidase activity. The ability of the extracts to inhibit Hyal was determined by the
spectrophotometric method of Yus et al. (Yus, & Mashitah, 2012). The extract concentrations were
following 0.1; 0.5; 1.0; 1.5 mg/mL in 10% water ethanol solution. Methyl indole-3-carboxylate was used
as the positive control. Every assay was performed in triplicate.
DPPH assay. The ability of the extracts to scavenge DPPH* free radicals was determined by the
modified method of Brand-Williams et al. (Brand-Williams, Cuvelier, & Berset 1995). The methanol
solvents of the extracts at the following concentrations: 0.1; 0.5; 1.0; 1.5 mg/mL were used. Ascorbic
acid was used as a positive control (12.5; 25; 50; 75 μg/mL). Every assay was performed in triplicate.
Statistical analysis. Determinations were performed by triplicate. The obtained data were subjected to
statistical analysis using Statistica 7.0. (StatSoft, Cracow). The evaluations were analyzed for one-factor
variance analysis. Statistical differences between the treatment groups were estimated by Spearman’s (R)
and Person’s (r) test. All statistical tests were carried out at significance level of α = 0.05.
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References
Brand-Williams, W., Cuvelier, M.E., & Berset, C., 1995. Use of a free radical method to evaluate
antioxidant activity. LWT-Food Sci. Technol. 28, 25-30.
Chang, C.C., Yang, M.H., Wen, H.M., & Chern, J.C., 2002. Estimation of total
flavonoid content in
propolis by two complementary colorimetric methods. J. Food and Drug Analys. 10, 178-182.
Polish Pharmacopoeia IX. The Office for Registration of Medicinal Products, Medical Devices and
Biological Products, Warsaw, Poland. 2010.
Singleton, V.L., & Rossi, J.A., 1965. Colometry of total phenolics with phosphomolybdicphospotungstic acid reagents. AJEV. 16, 144-158.
Yus, A.Y., & Mashitah, M.D., 2012. Evaluation of Trametes lactinea extracts on the inhibition of
hyaluronidase, lipoxygenase and xanthine oxidase activities in viro. J. Phys. Sci. 23, 1-15.
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