Proanthocyanidins in Sea Buckthorn (HippophaÃ« rhamnoides L

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Proanthocyanidins in Sea Buckthorn (Hippophaë rhamnoides L.) Berries of Different Origins with Special Reference to Influence of Genetic Background and Growth Location Wei Yang, Oskar Laaksonen, Heikki Kallio, and Baoru Yang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.5b05718 • Publication Date (Web): 22 Jan 2016 Downloaded from http://pubs.acs.org on January 23, 2016

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Journal of Agricultural and Food Chemistry

Proanthocyanidins in Sea Buckthorn (Hippophaë rhamnoides L.) Berries of Different Origins with Special Reference to Influence of Genetic Background and Growth Location Wei Yang , Oskar Laaksonen , Heikki Kallio #, Baoru Yang †

†

†

†

Food Chemistry and Food Development, Department of Biochemistry, University of Turku, #

The Kevo Subarctic Research Institute, University of Turku, FI-20014 Turku, Finland

*

Author to whom correspondence should be addressed

Baoru Yang Professor of Food Chemistry and Food Development Department of Biochemistry University of Turku Tel: +358 2 333 68 44; E-mail: [email protected]

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†*

ACS Paragon Plus Environment


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ABSTRACT

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Wild sea buckthorn berries from Finland (Hippophaë rhamnoides ssp. rhamnoides) and

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China (ssp. sinensis), as well as berries of two varieties of ssp. rhamnoides cultivated in

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Finland and five of ssp. mongolica cultivated in Canada were compared based on the content

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and composition of proanthocyanidins (PAs). Among all the samples, only B-type PAs were

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found. The content of dimeric, trimeric, tetrameric and total PAs ranged between 1.4–8.9

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mg/100 g, 1.3–9.5 mg/100 g, 1.0–7.1 mg/100 g and 390 - 1940 mg/100g DW, respecvtively.

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The three subspecies were separated by three validated factors (R2 0.724, Q2 0.677). in the

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PLS-DA model. Significant differences in total PAs were found between the ssp. rhamnoides

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and ssp. mongolica samples (p < 0.05). In ssp. rhamnoides, samples grown in the north

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Finland were characterized by high amount of total PAs, typically 2-3 times higher than in

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the level found south. In ssp. sinensis altitude did not have a systematic effect on the PA

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composition, suggesting the significance of interaction between genetic background of

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growth location.

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Keywords: HILIC; Hippophaë rhamnoides; latitude; proanthocyanidins; sea buckthorn; subspecies; varieties

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INTRODUCTION

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Proanthocyanidins (PAs, also known as condensed tannins) are oligomeric or polymeric

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compounds composed of flavan-3-ol subunits. PAs are widely distributed in flowers, fruits,

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seeds, leaves, and bark of plants. PA oligomers and polymers consisting only of catechin or

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epicatechin (C or EC) subunits are called procyanidins (PC), whereas prodelphinidins (PD)

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are oligomers and polymers containing gallocatechin or epigallocatechin (GC or EGC).

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Significant progress has been achieved in understanding biosynthesis of PAs and their flavan-

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3-ol precursors1 and their transportation to vacuole.2-4 PAs accumulate in many organs and

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tissues of plants to provide protection against pathogens and herbivores, to reinforce plant

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tissues, and to maintain seed dormancy.5,6 They widely exist in foods such as cereals, fruits,

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berries, vegetables, nuts, wines, and plant-based beverages, where they have an affect on

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sensory properties.5,7 PAs play a major role in astringency and bitter taste.8 PAs with low

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degree of polymerization (DP) have been reported to be more bitter than astringent.

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Compounds with higher DP are generally more astringent than bitter, and intensity of

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astringency increases along with the increasing polymerization.8,9 Moreover, the ratio of

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PC:PD may result in the overall astringency perception.10

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Recent investigations suggest that PAs may have potential health effects due to their

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antioxidative,11 antimicrobial12 and anti-inflammatory activities13 as well as due to protection

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against cardiovascular diseases.14 In addition, PAs have also shown anticancer activities via

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induction of apoptosis and inhibition of cell proliferation in vivo and in vitro.15

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Most PAs consumed are degraded by gut microflora,16 and the degradation metabolites

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have potential health benefits.17,18 Both absorbable and nonabsorbable PAs are bioactive.18

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The absorbable PAs with DP < 4 appear in plasma, and their glucuronidated, methylated, and

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sulphated metabolites are detected in urine.19-21 The nonabsorbable PAs may have direct 3 `



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effects on the intestinal mucosa and exert potential prebiotic benefits affecting the

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composition and metabolism of gut microbiota.17,22

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Sea buckthorn (Hippophaë rhamnoides L.) has recently attracted a great deal of attention

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for its nutritional, medicinal and environmental impact.23 In recent years, many investigations

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have demonstrated the pharmacological effects of sea buckthorn berries.24-28 Sea buckthorn

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berries are known to have a distinctively sour, bitter, and astringent sensory profile.29,30

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Although PAs are found in lower contents than other groups of phenolic compounds, they

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may have an essential role in the astringent and bitter sensation. In water solution, the

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threshold of polymeric PAs for astringency, especially for puckering astringency has been

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reported as 22 mg/L, and the threshold concentrations of PA dimers for bitterness and

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astringency were 120 and 208 mg/L, respectively.31,32

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Qualitative and quantitative methods have been developed for analysis of sea buckthorn

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PAs 33-36 but the content and composition of different origins, varieties and growth conditions

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is not well known.

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The aim of the present study was to investigate the profiles and contents of PAs in different

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subspecies of sea buckthorn. Thus, wild and cultivated berries of three major subspecies of

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sea buckthorn from China, Finland, and Canada were analyzed. The results provide the new

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information supporting berry breeding and cultivation as well as industrial utilization of sea

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buckthorn.

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MATERIALS AND METHODS

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Plant Materials. Berries of wild Hippophaë rhamnoides ssp. rhamnoides (two locations in

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Finland) and of wild ssp. sinensis (ten locations in six provinces in China) as well as two

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varieties of ssp. rhamnoides (two lacations in Finland) and five varieties of ssp. mongolica

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(one location in Canada) were harvested during August–October 2008 (Table 1) .

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Berries were harvested randomly from the bushes when optimally ripe, pooled and mixed

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well for each sample. Ripeness was determined by the local sea buckthorn experts. The

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berries were frozen immediately after picking and stored at −20 °C until analyzed.

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Sample Preparation and purification. Extraction and purification of PAs were carried out

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with the method applied in our previous investigation.36 In brief, about 10 g of berries were

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weigthd accurately in duplicate, thawed at room temperature for 15 min and crushed with a

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disperser (IKA T25-Digital Ultra Turrax, Staufen, Germany) at 7000 rpm. The pulp was

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extracted three consecutive times with 30 mL of a mixture of acetone, water and acetic acid

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(80:19.5:0.5, v/v) by sonicating for 15 min during each time of extraction. After

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centrifugation and combining of the supernatants, acetone was removed by a rotary

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evaporator under reduced pressure at 40 °C. The aqueous solution was defatted with

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petroleum ether (2 × 10 mL) and the remaining aqueous extract was filtered through a 0.20

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µm regenerated cellulose (RC) filter (15 mm i.d., phenomenex, Torrance, CA). The final

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extract (c.a. 25 mL) was used for Sephadex LH-20 column chromatography (CC).

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A 120 mm × 20 mm i.d. glass column packed with five grams of Sephadex LH-20

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(Pharmacia, Uppsala, Sweden) was activated and rinsed with 100 mL water. The crude

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proanthocyanidin extract was loaded to the column. The sugars were eluted with 150 mL

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water and the flavonoids with 100 mL methanol in water (20:80, v/v). After elution of PAs

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with 150 mL (70:30, v/v, Fraction), the column was cleaned with 100 mL methanol. The flow

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rate was maintained at 1 mL/min with a Alitea-XV peristaltic pump (Bioengineering, Wald,

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Switzerland). Solvents in PA solution were removed with a vacuum rotary evaporator

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(40 °C). The PAs were re-dissolved in 1 mL methanol and filtered through a PTFE filter (13

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mm i.d., 0.22 µm, VWR International, West Chester, PA) before analyses.

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HILIC-ESI-SIR analysis. The instruments, gradient program, and ESI-MS conditions

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were the same as in our previous study.36 The total flow rate was reduced to 0.5 mL/min in

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order to have the whole flow directed to the mass spectrometer after the UV detector.

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Quantitative analysis. Quantive analysis of dimeric, trimeric and tetrameric PAs was

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carried out by HILIC-ESI-SIR method using an external standard as described previously.36

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The calibration curve was constructed by analysing standard solutions of procyanidin B2

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(Extrasynthese, Genay, France) in methanol in the concentration range of 0.01–10 mg/100

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mL. To standardize the ionization efficiency in ESI-MS, epicatechin in a prpoper molar

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concentration was used as an internal standard to correct the peak areas in the SIR mode.

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The total proanthocyanidin content was determined spectrophotometrically using BL-

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DMAC assay and an external standard.37 DMAC reagent (Sigma-Aldrich, St. Louis, MO) of

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1 mg/mL concentration in acidified ethanol (75 mL of 91% ethanol, 12.5 mL of concentrated

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hydrochloric acid, 12.5 mL of distilled water) was prepared fresh for the assay. The reaction

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was performed in a 96-well plate. 10 µL of sea buckthorn extract solution, 60 µL of

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extraction solvent and 100 µL of DMAC solution were mixed into each well. The final

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volume was 170 µL for each well. The microplate was read for absorption at 640 nm by a

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plate reader (Hidex, Finland) against a blank containing 70 µL ethanol and 100 µL DMAC

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solution. The calibration curve was constructed by reaction of DMAC with procyanidin B2 in

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acidified ethanol at final concentrations of 100–2000 mg/100 mL in the wells. The absorbance

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at 30 min was used to calculate PA concentration with the calibration curve. Content of each

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sample was expressed as mg/100 g PA in dried weight (DW) of berries.

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Statistical Analysis. Statistical analyses were carried out with SPSS 16.0.1 (SPSS, Inc.,

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Chicago, IL). Differences in the composition between samples of different varieties were

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analyzed with one-way analysis of variance (ANOVA).

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Partial least squares regression discrimination analysis (PLS-DA) was used to explain the

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difference between three subspecies according to the proanthocyanidin contents (X-data;

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n=16) in the berry samples (n= 20 × 2). Principal component analysis (PCA) was applied to

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further examine the deviations of proanthocyanidin variables within each subspecies. Full

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cross validation was used to determine the correct number of factors or components.

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Multivariate models were created using Unscrambler X, version 10.3 (CAMO Software, Oslo,

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Norway).

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RESULTS AND DISCUSSION

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Identification of proanthocyanidins. The purified PA fractions were analyzed by HILIC

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chromatography coupled with electrospray ionization mass spectrometry in negative ion

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mode. The major PA constituents of all the samples were similar to those previously found in

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wild ssp. rhamnoides berries (Pyhämaa, Finland), and PAs with DP up to eleven were

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found.36 All the compounds represented the B-type PAs. The main components as PA dimers

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(Dim-1, Dim-2, and Dim-3), trimers (Tri-1, Tri-2, Tri-3 and Tri-4) and tetramers (Tet-1, Tet-

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2, Tet-3, Tet-4 and Tet-5) were scanned in twelve channels by HILIC-ESI-MS-SIR analyses

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and displayed in Supplemental Figure 1. It is important to notice that each peak displayed

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may contain several heterogeneous PAs (e.g. C-C or EC-EC or C-EC). In addition, elution

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order did not always follow the DP values, which could not be identified.

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Compositional profile of proanthocyanidins. The total PA content in each sample was

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determined in duplicate by BL-DMAC method, and the contents of the PA oligomers with

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ESI-MS-SIR analysis. The contents of PAs in all the samples are collected in Table 2.

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The total content of PAs was the highest in the berries of variety ‘Tytti’ (H. rhamnoides ssp.

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rhamnoides) grown in Kittilä (1940 mg/100g DW) and the lowest in the berries

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‘Prozcharachnaya’ (H. rhamnoides ssp. mongolica) grown in Canada (390 mg/100g DW). 7 `



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The content of PA dimers, trimers and tetramers ranged between 1.4–8.9 mg/100 g, 1.3–

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10.7 mg/100 g, and 1.0–8.3 mg/100 g DW, respectively, covering only a small protion (0.5–

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5 %) of the total PAs in any of the samples studied (Table 2). Typically the Chinese berries

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were rich in PAs of higher degrees of polymerization.

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The content and composition of PAs were compared among the three subspecies. Ssp.

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rhamnoides had the highest content of total PAs (1205 ± 544 mg/100g DW), followed by ssp.

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sinensis (832 ± 292 mg/100g DW) and ssp. mongolica (644 ± 171 mg/100g DW). The high

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deviation within ssp. rhamnoides was due to the extremely high values in berries grown far

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north in Lapland, beyond 68 °N. ANOVA test was performed to determine any significant

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differences among the three subspecies. Significant differences between ssp. rhamnoides and

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ssp. mongolica samples were found for contents of total PAs, PA dimers, Dim-2, Dim-3, Tri-

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1, Tet-1 and Tet-3. Statistically, ssp. sinensis did not differ significantly from the other two

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subspecies in total PAs (Table 2).

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PLS-DA model was created in order to examine the possible classification of the sea

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buckthorn samples according to their PA contents (Figure 1). In the model, the three

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subspecies were relatively well classified with three validated factors (R2 0.724, Q2 0.677).

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The sinensis berries (R2 0.697, Q2 0.640), located mainly on the right of the scores plot along

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the Factor-1, were characterized as containing at lower levels all PAs (excluding the variable

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total PAs which was very close to the central axis) than the berries of the other two

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subspecies. The second factor separated the other subspecies; rhamnoides (R2 0.705, Q2

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0.658) contained more trimers and tetramers, whereas mongolica (already with two factors;

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R2 0.771, Q2 0.775) contained more dimers. The three subspecies were sufficiently separated

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by PLS-DA models. For further analysis, three separate PCA models were created to study

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the variations in PA contents within the subspecies. It is of special importance to notice the

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significance of growth location on composition when concluding the differences and

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similarities between the subspecies.

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Variation within ssp. rhamnoides

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Within ssp. rhamnoides, the variety ‘Tytti’ grown in Kittilä had the highest total content of

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PAs (1941 ± 92 mg/100 g DW). In contrast, the lowest value (594 ± 3 mg/100g DW) among

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this subspecies was found in ‘Terhi’ from Sammalmäki. Samples from Taapajärvi were rich

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in PA trimers and tetramers. The highest content of PA dimers was found in Pyhämaa (PHY)

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(Table 2). The variation within ssp. rhamnoides was further examined in a PCA model.

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Figure 2A shows scores and loadings plots of the PCA models with two validated

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components within the Finnish ssp. rhamnoides subspecies, which in general contained more

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PAs than the other subspecies (Table 2, Figure 1). The major variation within rhamnoides

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was detected between the wild berries on the first component. The berries from Taapajärvi on

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the right side of the plot (with 52% of the variation) contained more PD trimers and tetramers

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(Tri-4 and Tet-5), whereas wild berries from Pyhämaa (on the left) contained more PC

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trimers and tetramers (Tri-1 and Tet-1). Moreover, the PCA model of ssp. rhamnoides

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showed that the second component with 29% of the variation separated the varieties ‘Tytti’

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and ‘Terhi’ grown in Kittilä (north) from those Sammalmäki (south), with more total PAs,

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Dim-1 and Dim-2 in north (Figure 2A).

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The locations close to 60 °N were in southern Finland and test fields at 67 °N as northern

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Finland. The total PA content was significantly higher in the north in both wild and cultivated

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ssp. rhamnoides berries. In ‘Tytti’ and ‘Terhi’ the ratio was close to 3:1 whereas in the wild

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ones the ratio was around 2:1 (Table 2). Independent t-test revealed statistically significant

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differences in composition in all the three north-south comparisons. Likewise, higher

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abundances of Dim-1 and Tet-3 were found in samples from North Finland for all the three

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subspecies. Vice versa, the contents of Dim-3, Tri-1, Tet-1 and Tet-2 were higher in the

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samples from the south. For other oligomers, latitude showed impact on ‘Tytti’ and ‘Terhi’

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with typically same trends, but differing from those in the wild berries.

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Growth locations in Sammalmäki and Pyhämaa are on the shore of the Baltic Sea with

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different climate than the more continental Taapajärvi and Kittilä. This may partially explain

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the characteristic differences in the PA contents among the berries (Table 2) .

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In order to further highlight the differences between the two rhamnoides varieties ‘Tytti’

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and ‘Terhi’, and to discriminate between samples from northern and southern Finland, an

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additional PCA model was established. Within the varieties ‘Terhi’ and ‘Tytti’, the two first principal components accounted for 84%

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of total variance (Supplemental Figure 2). A clear separation between the northern and

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southern samples was observed in the first component (62%). The northern samples were

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grouped along the right side of the plot containing more total PAs and Dim-1, while the

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southern samples were grouped on the left side of the plot more abundant in trimers and

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tetramers, especially ‘Terhi’ from Sammalmäki was characterized by a high content of

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dimers and PDs (Dim-3, Tri-4 and Tet-5).

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Hellström et al. investigated PAs in a variety of fruits and berries and reported the total

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content of PAs in sea buckthorn berries of undefined subspecies/cultivar to be 1500 mg/100g

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DW.38 This was higher than the values found in common fruits such as kiwi fruit (70

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mg/100g DW), cherry (140 mg/100g DW), grape (140 mg/100g DW), banana (250 mg/100g

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DW), peach (300 mg/100g DW), apple, (600 mg/100g DW) and gooseberry (850 mg/100g

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DW). The content in lingonberry, again, was clearly above the levels of the other fruits and

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berries investigated (2350 mg/100g DW). The highest total contents of PAs found in sea

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buckthorn samples in our study were close to the values reported by Hellström et al. Teleszko

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et al reported the proanthocyanidin contents in some cultivars of Russian sea buckthorn (H.

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rhamnoides ssp. mongolica) to be close to 100 mg/100 g fresh berry flesh.39 The dry matter

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content in sea buckthorn berries varies in the range of 10-20% of fresh berries. Therefore, the

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values reported in these studies are within the reasonable range of variation caused by

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different subspecies, cultivars and growth environments.

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Temperature and radiation as climatic conditions typically correlate negatively with

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latitude. Although the day length increases even up to no sunset in summer, the growth

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season is always shorter and total irradiation less in northern Finland. The concentrations of

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anthocyanins and flavonols in bog bilberries40, anthocyanins in bilberries41 as well as soluble

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proanthocyanidins in juniper (Juniperus communis) needles42 were all increased in northern

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Finland compared with those in southern Finland. These unique factors from higher latitude

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were reported to have mainly a positive impact on the biosynthesis of flavonoids in plants.43

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A sharp increase in catechins and flavonols was observed during sweet potato leaf exposure

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to a long day photoperiod.44 Low temperature has been shown to induce anthocyanin

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synthesis in various species.45 Moreover, due to the depletion of the ozone layer, the level of

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UV radiation has increased in high latitude areas.43 UV-B irradiation enhanced the

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concentration of flavonol as protection against potential damage in a variety of plant

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species.46 All these results were in agreement with the variation of total PAs in the current

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study. However, for a better understanding of the effect of latitude with complex impact of

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climate conditions, detailed and long term studies are still needed.

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Variation within ssp. mongolica

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Five varieties of ssp. mongolica of Russian origin were cultivated in Canada. All these five

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varieties were grown in the same orchard in Québeck and were compared with each other.

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The content of total PAs in berries ranged from 390 to 880 mg/100g DW. ‘Prevoshodnaya’ 11 `



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had the highest content of total PAs, and the lowest was found in ‘Prozcharachnaya’. Also

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dimers, trimers and tetramers were present with low in ‘Prozcharachnaya’, followed by

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‘Vitaminaya’.

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As showen in Figure 2B in the PCA model the varieties ‘Chuiskaya’, ‘Oranzhevaya’ and

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‘Prevoshodnaya’ were located on the right of the plot on the first component (68%) along

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with all the PA variables in the loadings plot. These three varieties were separated by the

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second (18%) and third (9%; not shown) components. The ‘Prevoshodnaya’ samples had the

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highest total content of PAs and correlated with variables Dim-3, Tet-2 and Tet-3, whereas

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‘Chuiskaya’ samples on the other side of the second component correlated with variables

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Dim-1 and Dim-2. The ‘Prozcharachnaya’ samples correlated negatively with all PA

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variables.

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Variation within ssp. sinensis

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The wild sea buckthorn berries collected from ten different locations in China varied

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widely from each other. In Sichuan, the berries were collected at three different altitudes of

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2000 m, 2500 m and 3000 m, and in Shanxi at 1515 m and 2182 m. In Qinghai the altitude

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was 3115 m. The rest of berry samples were from locations below 1500 m (Table 1). Results

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of PA analyses were presented in Table 2. The samples from Sichuan (SC) had the highest

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contents of total PAs and lowest contents of dimers, trimers and tetramers, whereas opposite

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results were seen in the samples from Heilongjiang (HL), which had the lowest contents of

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total PAs and the highest contents of the oligomers.

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More detailed variation was show in PCA model for H. rhamnoides ssp. sinensis berry

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samples (Figure 2C). The first two components contained 91% of variance within the PA

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content data. The majority of the compound variables in the loadings plot located on right

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side of the first component along with the Hebei (HB), Heilongjiang (HL) and Shanxi (SX), 12 `


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samples whereas the Sichuan (SC) samples were on the opposite side of the component. The

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second principal component separated the SC samples (altitude 2000 m) from the other

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samples, including those SC samples grown at altitudes of 2000 and 3000 m. Interestingly,

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the compounds that correlate with SC-2000 samples along the second component were Dim-1,

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Tri-1 and Tet-1, indicating the accumulation of PC in SC samples in ratio to PD. These

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findings indicate that the SC-2000 samples may have a more intense puckering sensation in

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astringency perception compared with the other samples.

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The sinensis samples were also compared to study the effect of altitude and latitude on the

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composition of PAs. Despite some outliers, clearly decreasing trends were seen in the

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contents of PA dimers, trimers and tetramers as the altitude increased and as the latitude

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decreased (Supplementary Figure 3A-3C). In contrast, the total PAs content slightly

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increased as the altitude increased and as the latitude decreased (Supplementary Figure 3D).

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To investigate the association between altitude and latitude with the composition of PAs,

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bivariate correlation analysis (Spearman's correlation) and partial correlation analysis were

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performed with SPSS. The Spearman's correlation coefficients were presented in Table 3,

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showing significant correlation between the latitude and most of the PAs. Latitude was

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positively correlated to Dim-3, Tri-3, Tri-4, Tet-4, Tet-5, dimers, trimers and tetramers, but

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negatively correlated to total PAs (p < 0.01). Altitude was negatively correlated to all the

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compounds of PAs with few significant correlation (Tri-3, Tri-4 and Tet-5 ). It is worth noting

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that Dim-3, Tri-4, Tet-5 and the main components of Tri-3, Tet-4 were all PD, we speculated

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that PD was of the essence in oligomeric PAs positively correlated with latitude.

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In partial correlation analysis, most of the PAs correlated positively with latitude and

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negatively with altitude. The content of dimers had a significant positive correlation with the

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latitude, while total PAs correlated negatively with the latitude (p < 0.01) (Table 3).

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Furthermore, samples from SX (1512-2182) and SC (2000-3000) were each compared as 13 `



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altitude-stratified samples without interference of the varying longitudes and latitudes (Table

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2). In SX samples, the contents of dimers, trimers and tetramers increased as the altitude

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increased from 1512 m to 2182 m, whereas the total PAs decreased. In SC samples, the

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contents of dimers, trimers, tetramers and total PAs decreased as the altitude increased from

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2000 m to 2500 m, whereas the opposite trends were found in altitude increased from 2500 m

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to 3000 m.

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Compared to the altitude, latitude was found to have more significant impact on PA content

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in the sea buckrhorn. When other factors were held constant, variation in PAs content as a

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result of altitude as a single factor was not significant (Table 3). It is worth noting that the

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small number of samples in the study needs to be considered when making conclusions can

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be made on the basis of the results.

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An earlier study has reported positive correlation between latitude and the content of

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phenolic compounds in bog bilberry.40 The results were in agreement with the changing trend

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of dimers, trimers and tetramers in the current study. In contrast, the total PAs i.e. higher

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degrees of polymerization correlated negatively with latitude. On the other hand, Mateus et

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al. suggested the contents of PAs in grape skins and seeds were higher at lower altitudes.47

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Moreover, a substantial part of the observed genetic variation among different populations of

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H. rhamnoides ssp. sinensis. could be attributed to altitudinal factor.48 The result in H.

302

rhamnoides ssp. rhamnoides showed an opposite situation in the content of total PAs, latitude

303

was assumed as a major impacting factor. This indicates that genetic factor may be the prime

304

cause affecting the content of PAs in the berries of ssp. rhamnoides. Furthermore, the effects

305

of latitudes and altitudes are outcomes of complex interaction of the genetic and the

306

environmental factors such as soil conditions and climatic conditions.

307

In conclusion, proanthocyanidins in the berries of three subspecies (ssp. rhamnoides, ssp.

308

mongolica and ssp. sinensis of Hippophaë rhamnoides) were analysed by HILIC-DAD-ESI-

14 `


Page 14 of 30

Page 15 of 30


309

MS. All of them were B-type PAs. The composition and content of PAs in sea buckthorn

310

were influenced by genetic background and interaction with growth location. Among the

311

three subspecies, all of them were separated in PLS-DA models. In ssp. rhamnoides, the sea

312

buckthorn berries grown in northern Finland had higher amount of total PAs, whereas lower

313

amounts of oligomeric PAs were found in varieties ‘Tytti’ and ‘Terhi’ from northern Finland.

314

In ssp. sinensis, latitude was positively correlated to all dimers, trimers and tetramers, but

315

negatively correlated to total PAs. Compared with latitude, altitude showed less impact on

316

PAs in sea buckthorn berries. This is the first systemic comparison of the content and

317

composition of PAs in sea buckthorn of different subspecies and varieties. The study

318

provided valuable information for berry breeding cultivation, and utilization of sea buckthorn.

319

320

321

Supporting Information

322

Supplementary Figure 1 presents selected ion recording (SIR) chromatograms analyzed by

323

ESI-MS in purified fractions. Scores and loading plots for wild Finnish sea buckthorn

324

(subspecies rhamnoides) are summarized in Supplementary Figure 2. Correlations between

325

spatial parameters and contents of proanthocyanidins are presented in Supplementary

326

Figure 3. This material is available free of charge via the Internet at http://pubs.acs.org.

327

328

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329

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464 465

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Figure captions

467

Figure 1. PLS-DA model for sea buckthorn samples (n=20×2) classified according to

468

subspecies (rhamnoides, green triangle; mongolica, red dots; sinensis, blue box) with the

469

proanthocyanidin contents (variables n=16). Abbreviations of the sample names refer to

470

Table 1 and the compounds refer to Table 2.

471

Figure 2. PCA models for three subspecies of sea buckthorn as proanthocyanidin as variables

472

(n=16). A. Scores and loading plots for subspecies rhamnoides (n=6×2); B. mongolica

473

(n=5×2); C. sinensis (n=9×2). Abbreviations of the sample names refer to Table 1 and the

474

compounds refer to Table 2.

475

476

23 `



477

Page 24 of 30

Table 1. Information of Sea Buckthorn Samples

Subspecies

Growth site (abbreviation) a

longitude

latitude

Altitude (m)

Harvest Time

Sammalmäki, Finland (SAM)

22°09′ E

60°23′ N

1

Aug 28, 2008

Kittilä, Finland (KIT)

24°37′ E

68°02′ N

210

Oct 10, 2008

Sammalmäki, Finland (SAM)

22°09′ E

60°23′ N

1

Aug 28, 2008

Kittilä, Finland (KIT)

24°37′ E

68°02′ N

210

Aug 28,2008

Taapajärvi, Finland (TAA)

24°42′ E

67°07′ N

1

Oct 4, 2008

Pyhämaa, Uusikaupunki, Finland (PYH)

21°15E

60°54'N

1

Sep 29, 2008

Chuiskaya

Québec, QC, Canada

71°17′ W

46°47′ N

100

Sep 3, 2008

Oranzhevaya

Québec, QC, Canada

71°17′ W

46°47′ N

100

Sep 3, 2008

Vitaminaya

Québec, QC, Canada

71°17′ W

46°47′ N

100

Sep 3, 2008

Prevoshodnaya

Québec, QC, Canada

71°17′ W

46°47′ N

100

Sep 3, 2008

Prozcharachnaya

Québec, QC, Canada

71°17′ W

46°47′ N

100

Sep 3, 2008

Heilongjiang, China (HL)

127º06′ E

47º14′ N

210

Dec 15, 2008

Heibei, China (HB)

116º34É

41º17′ N

832, 818

Oct 16, 2008

Shanxi, China (SX)

113º52É

37º05′ N

1515, 2182

Oct 16, 2008

Sichuan, China (SC)

106º54É

31º01′ N

3000, 2500, 2000

Oct 15, 2008

Qinghai, China (QH)

101º23É

36º45′ N

3115

Oct 25, 2008

Inner Mongolia, China (IM)

109º48É

39º47′ N

1480

Oct 25, 2008

Variety Terhi

rhamnoides

Tytti

Finnish (wild)

mongolica

sisensis

Chinese (wild)

478

24 `


Page 25 of 30

479


Table 2. Proanthocyanidins Content in Sea Buckthorn Samples (mg/100g DW) Sample

Dim-1

Dim-2

Dim-3

Tri-1

Tri-2

Tri-3

Tri-4

Tet-1

Tet-2

Tet-3

Tet-4

Tet-5

* Dimers

# Trimers

† Tetramers

Total PAs

1205±544b

Comparison of subspecies rhamnoides L.

0.5±0.2b

1.3±0.5b

2.5±1.1a

0.7±0.4a

1.4±0.3b

1.8±0.5b

2.6±0.9b

0.4±0.2a

0.6±0.2b

0.6±0.1b

1.1±0.3b

2.4±0.8b

4.3±0.9b

6.5±1.5b

5.0±1.0b

mongolica L.

1.0±0.6b

0.7±0.2a

5.7±0.8b

1.9±0.5b

1.2±0.2b

1.6±0.4ab

2.8±0.9b

0.9±0.3b

0.6±0.9b

0.4±0.1a

0.8±0.3ab

2.5±0.9b

7.6±1.2c

7.5±1.7b

5.2±1.5b

644±171a

sisensis L.

0.3±0.1a

0.6±0.2a

2.1±8.0a

0.5±0.4a

0.8±0.3a

1.2±0.6a

1.7±1.0a

0.3±0.2a

0.3±0.1a

0.4±0.1a

0.7±0.3a

1.5±1.0a

3.0±1.1a

4.2±1.9a

3.2±1.5a

832±292ab

Comparison of ssp. rhamnoides L. variety ‘Terhi’ between north (KIT) and south (SAM) Finland North

0.6±0.0b

1.1±0.0a

0.9±0.1a

0.6±0.1a

1.2±0.1a

1.6±0.0a

2.0±0.0a

0.3±0.0a

0.6±0.00a

0.7±0.0b

1.1±0.1a

1.9±0.0a

2.6±0.1a

5.4±0.0a

4.5±0.1a

1515±69b

South

0.4±0.0a

1.1±0.0a

3.0±0.0b

1.3±0.0b

1.7±0.0b

1.8±0.0b

2.2±0.3b

0.7±0.0b

0.9±0.00b

0.6±0.0a

1.0±0.0a

2.1±0.0b

4.5±0.0b

7.0±0.1b

5.3±0.0b

594±3a

Comparison of ssp. rhamnoides L. variety ‘Tytti’ between north (KIT) and south (SAM) Finland North

1.0±0.0b

2.1±0.0b

1.5±0.0a

0.4±0.0a

1.1±0.2a

1.7±0.0a

2.3±0.0a

0.2±0.0a

0.4±0.01a

0.6±0.0b

1.1±0.0b

2.1±0.0a

4.6±0.0a

5.4±0.1a

4.5±0.0a

1941±92b

South

0.3±0.0a

1.1±0.0a

2.9±0.3b

0.8±0.0b

1.4±0.1b

1.7±0.0a

2.4±0.0a

0.4±0.0b

0.6±0.01b

0.5±0.0a

0.9±0.0a

2.3±0.0b

4.2±0.3a

6.2±0.0b

4.7±0.1b

642±8a

Comparison of ssp. rhamnoides L. Wild Seabuckthorn between north (PYH) and south (TAA) Finland North

0.6±0.0b

1.5±0.0b

2.5±0.1a

0.3±0.1a

1.7±0.0b

3.0±0.1b

4.6±0.0b

0.1±0.0a

0.4±0.0a

0.8±0.0b

1.8±0.0b

4.0±0.1b

4.7±0.1a

9.5±0.2b

7.1±0.1b

1643±61b

South

0.3±0.0a

0.8±0.0a

4.2±0.4b

1.1±0.0b

1.1±0.1a

1.5±0.0a

2.2±0.0a

0.5±0.1b

0.5±0.0b

0.4±0.0a

0.8±0.0a

2.0±0.0a

5.3±0.4a

5.8±0.1a

4.1±0.1a

894±6a

880±7d

Comparison of ssp. mongolica L. varieties Prevoshodnaya

0.7±0.0b

0.7±0.0b

6.7±0.0c

2.1±0.0c

1.5±0.0b

2.0±0.2c

3.5±0.0cd

1.1±0.1c

0.7±0.01d

0.4±0.0b

0.9±0.2c

3.0±0.0dc

8.1±0.0cd

9.0±0.2de

6.2±0.3d

Prozcharachnaya

0.4±0.0a

0.7±0.0b

4.3±0.1a

1.1±0.0a

1.1±0.0a

1.1±0.0a

1.4±0.0a

0.5±0.0a

0.4±0.0a

0.3±0.0a

0.4±0.0a

1.2±0.0a

5.4±0.1a

4.6±0.1a

2.8±0.0a

389±3a

Chuiskaya

2.0±0.0d

1.1±0.0d

5.8±0.1b

2.4±0.0c

1.1±0.0a

1.6±0.0b

3.2±0.0c

1.2±0.1cd

0.6±0.0c

0.3±0.0ab

0.9±0.0bc

2.7±0.0c

8.9±0.1c

8.3±0.1c

5.6±0.1c

570±2b

Oranzhevaya

1.4±0.0c

0.9±0.0c

5.6±0.0b

1.9±0.0b

1.2±0.0a

2.0±0.0c

3.7±0.0d

0.7±0.1b

0.5±0.0b

0.4±0.0ab

1.3±0.0d

3.8±0.0e

7.9±0.0bcd

8.8±0.1d

6.8±0.1e

700±8c

Vitaminaya

0.4±0.0a

0.4±0.01a

6.1±0.1bc

1.9±0.0bc

1.1±0.0a

1.4±0.0b

2.2±0.0b

0.9±0.0b

0.5±0.0b

0.3±0.1ab

0.7±0.0b

1.9±0.0b

7.0±0.1b

6.7±0.0b

4.4±0.1b

681±5c

Comparison of ssp. sisnensis L. among Heilongjiang (HL), Hebei (HB), Shanxi (SX), Sichuan (SC), Qinghai(QH) and Inner Mongolia (IM) HL

0.4±0.0

0.7±0.1ab

3.0±0.3ab

0.5±0.1

1.0±0.1b

1.8±0.1b

3.1±0.3d

0.2±0.2

0.4±0.0

0.5±0.0b

1.2±0.1c

2.9±0.2c

4.1±0.3b

6.4±0.5b

5.1±0.3b

577±5a

HB

0.4±0.1

0.7±0.2ab

2.7±0.9ab

0.6±0.2

1.0±0.3b

1.8±0.5ab

2.6±0.9cd

0.2±0.1

0.4±0.1

0.5±0.1ab

0.9±0.1c

2.3±0.9bc

3.8±1.2ab

5.9±1.9ab

4.3±1.2ab

735±55a

SX

0.2±0.1

0.5±0.3ab

2.3±0.4b

0.5±0.3

0.9±0.3b

1.5±0.4ab

2±0.6b

0.3±0.1

0.4±0.1

0.5±0.1ab

1±0.3abc

1.9±0.6b

3.1±0.8ab

4.9±1.6ab

4±1.2ab

681±20a

SC

0.2±0.1

0.4±0.2a

1.2±0.2a

0.5±0.6

0.5±0.2a

0.7±0.2a

0.7±0.1a

0.3±0.4

0.3±0.2

0.3±0.1a

0.4±0.1a

0.5±0.1a

1.8±0.4a

2.4±1a

1.7±0.7a

1173±321c

QH

0.3±0.0

0.8±0.0b

2.4±0.0b

0.4±0.0

0.7±0.0ab

1.0±0.0b

1.3±0.0a

0.2±0.0

0.3±0.0

0.3±0.0ab

0.6±0.0b

1.1±0.0a

3.5±0.1b

3.4±0.0ab

2.6±0.1ab

818±1ab

IM

0.2±0.1

0.5±0.0ab

2.5±0.1b

0.5±0.0

0.6±0.0ab

1.1±0.1b

1.3±0.1a

0.2±0.1

0.2±0.0

0.3±0.0ab

0.5±0.0a

0.9±0.1a

3.2±0.2b

3.6±0.2ab

2.1±0.2ab

574±5a

Altitude comparison of ssp. sisnensis; SiChuan (SC) SC 3000

0.3±0.0b

0.5±0.0b

1.4±0.1b

0.2±0.0a

0.5±0.0ab

0.8±0.1b

0.9±0.1b

0.1±0.0a

0.3±0.0ab

0.4±0.0c

0.4±0.1b

0.6±0.1b

2.2±0.1b

2.4±0.2ab

1.8±0.2ab

985±9a

SC 2500

0.1±0.0a

0.2±0.0a

1.1±0.1ab

0.1±0.0a

0.2±0.0a

0.5±0.1a

0.6±0.1a

0.1±0.0a

0.1±0.0a

0.2±0.0a

0.2±0.0a

0.4±0.1a

1.4±0.2a

1.3±0.2a

1.0±0.1a

949±13a

SC 2000

0.4±0.0b

0.4±0.0ab

1.0±0.0a

1.3±0.0b

0.7±0.0b

0.7±0.0b

0.8±0.0ab

0.8±0.0b

0.5±0.1b

0.3±0.0b

0.4±0.0b

0.5±0.0ab

1.8±0.0ab

3.4±0.0b

2.5±0.1b

1587±52b

Altitude comparison of ssp. sisnensis; ShanXi (SX)

480

SX 1512

0.1±0.0a

0.3±0.0a

1.9±0.0a

0.3±0.0a

0.7±0.0a

1.1±0.0a

1.5±0.0a

0.2±0.0a

0.3±0.0a

0.4±0.0a

0.8±0.0a

1.5±0.0a

2.4±0.0a

3.5±0.0a

3.0±0.1a

698±4b

SX 2182

0.4±0.0b

0.8±0.0b

2.7±0.1b

0.8±0.0b

1.1±0.1b

1.8±0.1b

2.5±0.1b

0.4±0.0b

0.5±0.0b

0.6±0.0b

1.3±0.1b

2.4±0.1b

3.8±0.1b

6.3±0.3b

5.0±0.2b

664±3a

*

#

†

Means ± standard deviation; Significant differences (p < 0.05) are marked as a-e; Dimers are sums of Dim-1, Dim-2 and Dim-3; Trimers are sums of Tri-1, Tri-2, Tri-3 and Tri-4; Tetramers

25 ` ACS Paragon Plus Environment


481

are sums of Tet-1, Tet-2, Tet-3, Tet-4 and Tet-5.

26 ` ACS Paragon Plus Environment

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Page 27 of 30

482


Table 3. Correlation coefficients between the compositional parameters of sea buckthorn berries and the latitude and altitude of growth sites. Sample

Dim-1

Dim-2

Dim-3

Tri-1

Tri-2

Tri-3

Tri-4

Tet-1

Tet-2

Tet-3

Tet-4

Tet-5

Dimers

Trimers

Tetramers

Total PAs

Spearman’s correlation coefficients Latitude

0.361

0.489

0.784**

0.335

0.687*

0.872**

0.872**

0.192

0.240

0.557*

0.789**

0.853**

0.775**

0.812**

0.749**

-0.803**

Altitude

-0.296

-0.121

-0.450

-0.395

-0.565

-0.655*

-0.652*

-0.172

-0.299

-0.371

-0.531

-0.634*

-0.441**

-0.725

-0.592

-0.595

Partial correlation coefficients Latitude

0.221

0.711*

0.794**

-0.267

0.385

0.555

0.611

-0.386

-0.114

0.323

0.604

0.580

0.763**

0.452

0.455

-0.745**

Altitude

-0.051

0.574

0.436

-0.326

-0.043

-0.019

-0.058

-0.353

-0.243

0.019

0.155

-0.048

0.442

-0.123

-0.086

-0.481

483

* p

Proanthocyanidins in Sea Buckthorn (HippophaÃ« rhamnoides L

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