Сhemical composition of peatland small cranberry ( Vaccinium oxycoccus ) for potential use as functional ingredient

Introduction. The aim of the present study was to determine, for the first time, the small cranberries ( Vaccinium oxycoccus ) chemical composition depending on depending on growing soil. Materials and methods. The analyses were made on ripe fruits of the plant, harvested from Romania, Suceava county, Coșna commune, in a peat area. The mineral content was determined using a coupled-mass spectrometer (ICP – MS). DPPH method was used for antioxidant activity determination. Folin Ciocalteu method and HPLC analysis were used for polyphenol content determination. Results and discussion. A value of 84.1% was obtained for fruits’ moisture content, close to the values found in other studies. For ash, a relatively low value of 1.38% was obtained that shows a low mineral content, corresponding to the low mineral content of the soil. However, the mineral content of the analyzed fruits is higher than that reported by other studies . For most of the elements there was a positive correlation between mineral content in fruits and soil. A significant amount of calcium was observed in the studied cranberries, close to the average means reported in other studies. Magnesium falls within the limits determined in other studies, and the copper content as well. The contents of manganese, iron and zinc were lower than in other studies. The contents of arsenic, mercury and stibium were below the maximum approved limits and led is under the detection limit. Regarding the antioxidant capacity, the inhibiting percent of free radicals and total content of polyphenols is higher in aqueous extract than in the ethanolic extract and medium content in the studied fruits, in comparison with other reported data: 340.78 mg/100g in aqueous extract and 254.20 mg/100g in ethanolic extract.


Introduction
Peatland small cranberry (Vaccinium oxycoccus) is a plant from Vaccinium genus from Ericaceae family, which includes over 450 species from Europe, North America, Central America, Japan and Asia, Central and South-Eastern Africa, Madagascar (Mabberley et al., 1997).Different blueberries species (Vaccinium myrtillus, V. ashei, V. angustifolium, V. corymbosum), cranberries (V.macrocarpon, V. oxycoccos), huckleberry (V.ovatum, V. parvifolium), lingonberry (V.vitis-idaea) are the most known berries of this genus (Jurikova et al., 2018).The most known cranberry, the big cranberry, called American cranberry (V.macrocarpon), originates from the eastern and central North America, including Canadian Eastern territory and found in the highlands moors from North East and Great Lakes of United States (Česonienė et al., 2016).Its fruits Its fruits have been used in local medicine from the XVI century or even earlier (Narwojsz et al., 2019).The European relative of the big cranberry less known and studied is small cranberry, V. oxycoccus, with a larger geographical distribution.
The small cranberries (Vaccinium oxycoccus) were gathered on peatlands from the North of Carpathian Mountains.The aim of the present study was to determine the small cranberries (V.oxycoccus) chemical composition, particularly the content of moisture, ash, trace elements, nitrites, polyphenols, and vitamin C, as well as their antioxidant activity, and to compare the present paperwork results with the those from other studies on cranberries grown on different areas.
Ripe berries used for the study were harvested during September -October.Soil samples were taken from the same area to determine ash and minerals contents.

Determination of moisture content
The moisture content was determined by weighing berries sample, dried in oven till the mass remained constant.The moisture content was calculated by the following formula: C, % = (mS -mDS)•100/mS, were mS is sample mass before drying, g; mDS is sample mass after drying, g.

Determination of ash
To determine ash content, fruit and soil samples were calcinated at 800 ºC using a Nabertherm LE 2/11/R6 Muffle Furnace ( Arvinte et al., 2019).The ash percentage was calculated by the following formula: C, % = mCS•100/ms where mS is sample mass before calcination, g; mCS is sample mass after calcination, g.

Determination of mineral content
The mineral content was determined by using ICP -MS Agilent Technologies 7500 series spectrometer; the samples were digested with concentrated nitric acid and hydrochloric acid (Arvinte et al., 2019).The metal concentration (C) was expressed in mg/100 g of sample and was calculated by formula: where a is standard concentration, mg/L; V is a volume of the acid that dissolves the sample, L; m is mineralized sample mass, g.
The mineral content was determined in Vaccinium oxycoccus fruits and in soil, taken from the area where the fruits were harvested.

Determination of nitrite content
The determination of nitrite content in small cranberries was made using Griess method (Neacsu and Chirigiu, 2006).In slightly acidic medium (acetic acid) the nitric acid (nitrite) mixed with sulfanilic acid and α-naphthylamine form a red nitric acid.The intensity of pink color of the nitric compound formed as a diazotizing reaction between sulfanilic acid and nitrites from deproteinized watery extract and the subsequent coupling with α-naphthylamine was measured.Also, the substance absorbance was measured at 520 nm wavelength versus a blank.

Determination of the antioxidant capacity
The determination of the antioxidant capacity was made through DPPH (2-diphenyl-1picrilhidrazil), which is one of the most stable nitrogen alkyls and commercially available, with a maximum absorption UV-VIS at 517 nm.DPPH methanol solution has a maximum absorption at 517 nm wavelength, due to the unpaired electron.In the presence of an antioxidant, the electron pairs, resulting the simple form of DPPH-H, discolored, and the absorption disappears.The result of the decolorating is directly proportional with the number of the captured electrons, so inversely proportional with the absorbance.Determinations were made for watery extract: 5 g sample in 200 mL of water were boiled for 30 minutes and alcoholic extract: 1 g sample in 50 mL of ethanol, obtained on an ultrasound tub, at 40 ºC and 25 KHz frequency from the plant berries.5 mL of extract were mixed with 2.5 mL solution DPPH, 6•10 -5 M. Absorbance was determined on a fiber optic spectrophotometer from Ocean Optics, 5 min read time.
The inhibit percent of the free radicals, by comparison with DPPH was calculated with the formula: where A0 is standard substance absorbance and AP is analyzed sample absorbance (Paduret at al., 2016).

Determination of the total polyphenol content
The determination of the total content of polyphenols was made through Folin-Ciocâlteu method, on the same extracts of 5 g samples in 200 mL water.Polyphenols are substances with antioxidant specificity.They are aromatic chemical compounds with more hydroxyl groups inserted on aromatic ring.Due to this structure, they can be oxidized by Folin Ciocâlteu reagent (Fc), with the formation of blue coloration with maximum of absorption at 700 nm.Folin -Ciocâlteu indicator is specific only to the phenol compounds with reducing properties.The calibration curve was made using gallic acid solution (Su et al., 2007).The sample for analyses was made from 2 mL extract, 1 mL Folin Ciocâlteu reagent and 8 mL of 7.5% Na2CO3 solution, mixed for 5 minutes, left to still for 30 minutes in the dark and the reading of the sample absorbance was done at 750 nm (Sripakdee et al., 2015).
The result is expressed as Fc indicator, achieved by calculus formula: where A750 is absorbance read at a 750 nm; d is sample dilution.

Identification and determination of the polyphenol content
Samples of 10 g of berries stepped into 100 mL of solvent (ethanol, ether and benzene) were analyzed.Phenolic extracts were analyzed using a liquid chromatography High Performance System (Shimadzu, Kyoto, Japan) equipped with liquid chromatograph LC-20 AD, auto sampler SIL-20A, column oven CTO-20AC and a detector with diode array SPD-M-20A.The separation was made on a Phenomenex Kinetex® 2,6 μm Bifenil 150 × 4,6 mm, column with 0,5 mL/min flow, and temperature controlled at 25°C.The sample injection volume was of 10 mL.A solvent system consisting of 0.1% acetic acid in water (A solvent) and acetonitrile (B solvent) was used with the following gradient: starting with 100% A and installing a gradient to obtain 5% B at 6.66 min, 40% B at 66.6 min and 80% B at 74 min, according to the method previously described with some modifications (Palacios et al., 2011).Solvent flow was of 1 mL/min.Phenol compounds were identified based on standard materials retention times and the quantification was made through chromatogram recording absorbance as oppose to external standards, at 280 nm for gallic acid, protocatechuic acid, vanillic acid, p-hydroxibenzoic acid and 320 nm for chlorogenic acid, caffeic acid, pcoumaric acid, rosmarinic acid, myricetin, quercetin, luteolin and kaempferol.All the standard calibration curves showed high linearity levels.
The analysis was done through infrared spectroscopy with Fourier transform (FT-IR) using a Nicolet i-20 spectrophotometer (ThermoScientific, SUA).The spectra were recorded in transmission mode using ATR system in the wave number rage of 4000-400 cm −1 at 4 cm −1 resolution.SpectraGryph-spectroscopy software was used to display spectra.The samples (peatland cranberries extract in ethanol, ether and benzene) were placed on the ATR crystal and the spectra were recorded in triplicates.

Determination of vitamin C content
The samples were obtained through mixing 5 g grounded berries with 12 mL acid solution (10% perchloric acid and 1% o-phosphoric), completed with mono-potassium phosphate 0.02 mol/L.The samples were analyzed with HPLC Agilent Zorbax Extend-C18 columns, 5 μm particle size, I.D. × L 150 cm × 4.6 mm, flow rate was 1.0 mL/min.

Chemical composition of cranberries
The moisture content of peatland cranberries was 84.1% (Table 1).This explains the fact that these plants grow only in swamp areas, with high humidity, as compared with blueberries (Vaccinium myrtillus) and lingonberries (Vaccinium vitis-idea), which grow in swamps along with peatland small cranberries, but also on alpine meadows and even in alpine forests.The results corroborate with other studies (Table 2), which identified moisture content from 84.2% up to 92% for small cranberries, the highest moisture content was detected in berries collected in spring, after the snow has melted et al., 2016).One can remark a strong correlation between the low minerals content in small cranberries and that of soil.However, the mineral content of small cranberries is much higher than the figures obtained in other studies (0.20-0.28%) (Česonienė et al., 2016).
Soil samples were taken from two distinct areas, as peat soil is heterogeneous.Regarding the ash content, the highest values were found in the soil, not in the fruit.The difference were not significant at p<0.05%.On the other hand, the moisture content was lower in the soil, differences being not significant at 95% Most of the mineral elements found in the soil are also found in the cranberries fruit, but in smaller amounts (Table 3).There is an important calcium quantity in the studied peatland small cranberries, 11.04 mg/100 g, close to the average 13.0 mg/100 g related to other studies and significantly higher than the average calcium quantity from Vaccinium macrocarpon, 7.8 mg/100 g (Česonienė et al., 2016).Also, magnesium with 5.19 mg/100 g is situated between established limits by other studies, 2.9-9.1 mg/100 g.Manganese content of 0.05 mg/100 g is lower as opposed to other results 0.9-4.00mg/100 g, although the manganese content in the soil is relatively high.Iron quantity of 0.23 mg/100 g is significantly lower than the one reported, 0.11-0.42mg/100g (Česonienė et al., 2016), but the majority is represented by iron divalent ion.Zinc quantity of 0.03 mg/100 g has lower values than in other determinations, 0.1 -0,19 mg/100 g, as the coppery quantity of 0.10 mg/100 g fits in other determinations limits, reaching the higher limit, 0.04 -0.19 mg/100 g, and it is higher than in Vaccinium macrocarpon, 0.04 -0.06 mg/100 g (Česonienė et al., 2016).Arsenic, mercury and antimony are below the approved limits and lead is under detection limits (Amariei et al., 2017).The difference in elements composition was significant (at 95% confidence level) for all the samples studied.(a,b and c) refer to the comparison of the same element between the different samples; results followed by superscript letters are significantly different (p < 0.05%) according to Turkey's post hoc test.
Comparison of the mineral content, mg/100 g, in peatland small cranberries with mineral content of the soil on which these berries grow is shown in Table 4. Antioxidant capacity and the polyphenol contents in peatland small cranberries are shown in Table 5.It could be observed that the free radicals inhibition percentage was higher in aqueous extract.Wild cranberries antioxidant properties were compared with the grown cultivars, and a significantly higher antioxidant activity in wild cranberries was noticed (Borowska et al., 2009).Likewise, cranberries had a higher antioxidant potential than lingonberry, the adjacent plant from the peatland (Brown et al., 2012).One can observe that watery extract is richer in polyphenols than the alcoholic extract, the difference was significant p<0.05%.
By comparison (Table 6), other studies presented a polyphenol total content of 226.5 mg/100 g of fresh weigh (Narwojsz et al., 2019).Other authors determined a polyphenol total content, which varies from 288.5 to 456.3 mg/100 g (Borowska et al., 2009), up to 705 mg/100 g (Denev et al., 2013).One can notice a medium content in the studied berries.According to the results obtained by different authors, polyphenol total content is significantly higher in wild cranberries as opposed to grown varieties, 100.4 -154.8 mg/100 g (Borowska et al., 2009)

Identification of polyphenols and determination of their content
Polyphenol content in extracts in ethanol, ether and benzene is shown in Table 7.  (Kähkönen et al., 1999(Kähkönen et al., , 2001) ) 59-64% (Wang et al., 2000) 69.6% (Vattem et al., 2005) Total polyphenols, mg/100 g 254.20 340.78 705 (Denev et al., 2013) 288.5-456.3 (Borowska et al., 2009) 226.5 (Narwojsz et al. 2019) 347-389 (Česonienė et al., 2009)  One can see that the ethanolic extract is much richer in polyphenols than the ether and benzene extracts, excepting the P-coumaric acid, which appears in benzene extract (Table 8).It is obvious the p-hydroxibenzoic acid presence, as it appears in other research studies, its quantity value being very close to that found in large cranberry (Vaccinium macrocarpon) (Zuo et al., 2002).Likewise, one can observe the presence of the chlorogenic acid, pcoumaric acid (Jurikova et al., 2018, Zuo et al., 2002) as other researchers report, but also the rosmarinic acid which was not identified in other studies.All the difference found were not significant at 95% confidence level.As other authors show, the major flavonoids which appear in cranberries are quercetin and myricetin (Chen et al., 2001).The high content of quercetin, 5.15 mg/100 g of frozen weight, was reported (Ehala et al., 2005).Although the content found in the studied cranberries was lower, 0.39 mg/100 g, it is nevertheless a significant quantity.This substance contributes mainly in the antioxidant activity of the fruit.As for the quercetin content, the peatland cranberries are in the third place among the berries that contain this polyphenol, after elder and blueberries, and among vegetables only French and red onion have more quercetin than peatland cranberries (Kiviranta et al., 1988).There are multiple studies regarding this bitter-taste flavonoid, which has unique biological properties than can improve mental and physical performance and can reduce infection hazards.Quercetin stands out through anti-carcinogenic, anti-inflammatory, antiviral, antioxidant and psycho-stimulant activities and also through the capacity of lipid per-oxidation inhibition, platelet aggregation and mitochondrial biogenesis simulation (Li et al., 2016;MLcek et al., 2016).Quercetin may play an important role in treating several degenerative diseases (Palle et al., 2017;Pandey et al., 2012;Suganthy et al., 2016) without side effects or insignificant side effects.It was shown that quercetin and other flavonoids prevent the formation of atherosclerotic plaque, aggregation platelets (antithrombin effects) and favors the cardiovascular smooth muscles relaxation (Formica and Regelson, 1995).The quercetin antivirals' properties were investigated and proved on a large number of studies (Noor et al., 2021).As for myricetin content, cranberries are among the richest berries in this element (USDA, 2011).From the structural point of view, myricetin is similar to quercetin and it might have many of the latest functions (Ross et al., 2002).Studies show that myricetin has a therapeutic effect on many diseases (Song et al., 2021).

Vitamin C content
Vitamin C content in the studied berries (Table 8) was 5.98 mg/100 g, lower than the reported quantity in other studies, 15.3-30 mg/100 g (Česonienė et al., 2016), although the obtained results of vitamin C quantities in cranberries are quite different (Jurikova et al., 2018;Tikuma et al., 2014).It was shown that too ripened berries have a much lower vitamin C content, average 1.03 mg/100 g (Jurikova et al., 2018;Viskelis et al., 2009).Likewise, during storage a part of ascorbic acid transforms into its active biologic isomer, dehyidroascorbic acid.Vitamin C content is positively correlated with antioxidant activity of these berries (Jurikova et al., 2018).

Conclusion
The obtained results show the antioxidant potential of peatland small cranberries and their possible usage in maintaining health, ageing and oxidative stress diminishing.It is known that quercetin and other polyphenols identified in peatland small cranberry have antioxidant, anti-inflammatory, vasodilatory, antithrombotic, antihypertensive, anticancer and probiotic properties (Lu et al., 2017), and this allows to recommend these cranberries for consumption as functional food.

Table 1 Content of moisture and ash in peatland small cranberries and the soil they grow on
bFor difference assessment was performed Student t-test.Values followed by a,b are statistically different at 95% confidence level.

Table 8 Comparison of the polyphenols and vitamin C content, mg/100 g, in small cranberries with those available in the literature Polyphenol compound
Note: *N/A, not available.