STUDIES ON THE CHEMICAL COMPOSITION OF FRUITS AND SEEDS OF PSEUDOCYDONIA SINENSIS (THOUIN) C.K. SCHNEID

Pseudocydonia sinensis (Thouin) C. K. Schneid. less known plant species in the Ukraine conditions, but the fruits were widely used in traditional Chinese medicine for the treatment of asthma, colds, sore throat, mastitis, rheumatoid arthritis, and tuberculosis. The content of protein, ash, and lipids in the seeds was found to be greater than in the pulp and peel. Monosaccharide analysis of neutral carbohydrate part showed the presence of two main sugars fructose and sucrose in the seeds, pulp, and peel. There is a higher beta-carotene content in the rind of the fruit than in the seeds and pulp. The total amount of fatty acids varied from sample to sample and contained mainly oleic acid, palmitic acid, linolenic acid, and linoleic acid. Linoleic acid in the seeds was 48.02% of total fatty acids, slightly less in the rind 42.70%. Palmitic acid, oleic acid, and linoleic acid in the pulp samples were 45.38, 21.32, and 14.93%, respectively. The total amount of amino acids found in the seeds was 105.0 g.kg-1 DM, including total essential amino acids (32.70 g.kg-1 DM). Glutamic acid was found in seeds to be the dominant free amino acid followed by aspartic acid and arginine in the seed. In our study, the antioxidant activity carried out by the DPPH method and measured by molybdenum reducing antioxidant power of peel, pulp and seeds were 9.41, 7.08, 6.21, and 158.81, 92.83, 78.58 mg TEAC.g-1 DM, respectively. Micro and macronutrients and amino acids predominated in the seeds, total fatty acids predominated in the pulp. The highest content of bioactive compounds (total polyphenols, flavonoid, and phenolic acid) and antioxidant activity was found in the peel. P. sinensis can be considered as a nourishing fruit with a copious potential with health-promoting roles and medicinal properties.


INTRODUCTION
Increasing interest in less know, non-traditional, neglected, and underutilized plant species, which could serve as a valuable source of natural bioactive compounds, has been emerged worldwide and they play important role in procuring food security to improve health and nutrition, ecological sustainability, and livelihoods. These species of rich in valuable biologically active compounds include Aronia mitschurinii A. K. Skvortsov & Maitul., Cornus mas L., Chaenomeles spp., Diospyros virginiana L., Lycium spp., Lonicera spp., Morus nigra L., Ziziphus jujuba Mill., Vaccinium spp., Sambucus nigra L.  Vinogradova et al., 2020). The direction adopted by the European Community towards sustainable crop production intensification involves "growing a wider range of plant species and varieties using combinations, sequences and rotations" (Save and Grow, 2011). It is important to cultivate little-known edible plants more widely, as they are a potential source of new biologically active substances needed for the functioning of the human body.
Pseudocydonia sinensis (Thouin) C. K. Schneid. (Chinese quince) is a less known species of the family Rosaceae Juss. and the only species in the genus Pseudocydonia C. K. Schneid., native to eastern Asia in China. This species is closely related to the East Asian genus, Chaenomeles Lindl., and to the European genus, Cydonia Mill. (Suzuki, 1994). Sometimes it is called Chaenomeles sinensis. They are distinguished by the absence of thorns and single, not clustered flowers. Cydonia oblonga differs from the Pseudocydonia sinensis plant in the presence of toothed leaves and the absence of pubescence on the fruits (Klymenko, .
In Europe, Pseudocydonia sinensis is only grown in botanical gardens and arboretums but has already proved to be an annually fruiting crop that is resistant to the climatic conditions of the continent. A more detailed study of the biology of this species will help to introduce it into widespread cultivation more quickly.
Prevail Carbohydrates ES column (250/4.6 mm) was used as a stationary phase and acetonitrile (VWR) mixed with water in a 75:25 volume ratio was used as the mobile phase.

Determination of carotenoid
Total carotenoid content expressed as beta-carotene was analyzed at a wavelength of 445 nm spectrophotometrically (VIS spectrophotometer UV Jenway Model 6405 UV/VIS). Sample (1 g) was disrupted with sea sand and extracted with acetone until complete discoloration. Petroleum-ether was added and then water, in purpose to the separation of phases. After the separation, the petroleum ether-carotenoid phase was obtained and the absorbance was measured (ČSN 560053, 1986).

Determination of mineral contents
Sample for elemental analysis was prepared using the wet ashing method in a microwave oven (Milestone 1200, Milestone, Italy). A total of 0.25 g sample matrix was decomposed in a mixture of nitric acid (6 mL) (Analytika Praha Ltd, Czech Republic) and hydrochloric acid (2 mL) (Analytika Praha Ltd, Czech Republic). After the decomposition sample was filtered using a filter with 0.45 µm pore size and filled up to 25 mL in a volumetric flask with ultrapure water. Elemental analysis was performed using ICP-OES (Ultima 2, Horiba Scientific, France) according to the procedure described by Divis et al. (2015).

Determination of amino acids
Amino acids were determined by ion-exchange liquid chromatography (Model AAA-400 amino acid analyzer, Ingos, Czech Republic) using post-column derivatization with ninhydrin and a VIS detector. A glass column (inner diameter 3.7 mm, length 350 mm) was filled manually with a strong cation exchanger in the LG ANB sodium cycle (Laboratory of Spolchemie) with average particles size 12 µM and 8% porosity. The column was tempered within the range of 35 to 95 °C. The elution of the studied amino acids took place at a column temperature set to 74 °C. A double-channel VIS detector with the inner cell volume of 5 µL was set to two wavelengths: 440 and 570 nm. A solution of ninhydrin (Ingos, Czech Republic) was prepared in 75% v/v methyl cellosolve (Ingos, Czech Republic) and in 2% v/v 4 M acetic buffer (pH 5.5). Tin chloride (SnC l2 ) was used as a reducing agent. The prepared solution of ninhydrin was stored in an inert atmosphere (N 2 ) in darkness at 4 °C. The flow rate was 0.25 (mL.min -1 ) and the reactor temperature was 120 °C.

Determination of total polyphenol, flavonoid, and phenolic acid content
The total polyphenol content (TPC) was measured by the method of Singleton and Rossi (1965) using the Folin-Ciocalteu reagent. A quantity of 0.1 mL of each sample was mixed with 0.1 mL of the Folin-Ciocalteu reagent, 1 mL of 20% (w/v) sodium carbonate, and 8.8 mL of distilled water. After 30 min in darkness, the absorbance at 700 nm was measured with the spectrophotometer Jenway (6405 UV/Vis, England). Gallic acid (25 -300 mg.L -1 ; R 2 = 0.998) was used as the standard. The results were expressed in mg.g -1 DM gallic acid equivalent.
The total flavonoid content (TFC) was determined by the modified method described by Shafii et al. (2017). An aliquot of 0.5 mL of the sample was mixed with 0.1 mL of 10% (w/v) ethanolic solution of aluminum chloride, 0.1 mL of 1 M potassium acetate, and 4.3 mL of distilled water. After 30 min in darkness, the absorbance at 415 nm was measured using the spectrophotometer Jenway (6405 UV/VIS, England). Quercetin (1 -400 mg.L -1 ; R 2 = 0.9977) was used as the standard. The results were expressed in mg.g -1 DM quercetin equivalent.
Total phenolic acid (TPA) content was determined using the method of Farmakopea Polska (1999). A 0.5 mL of sample extract was mixed with 0.5 mL of 0.5 M hydrochloric acid, 0.5 mL Arnova reagent (10% NaNO2+10% Na2MoO4), 0.5 mL of 1 M sodium hydroxide (w/v) and 0.5 mL of water. Absorbance at 490 nm was measured using the spectrophotometer Jenway (6405 UV/Vis, England). Caffeic acid (1 -200 mg.L -1 , R 2 = 0.999) was used as a standard and the results were expressed in mg.g -1 DM caffeic acid equivalents. Number of samples analyzed: 15. Number of repeated analyses: 3. Number of experiment replication: 1.

Statistical Analysis
Basic statistical analyses were performed using PAST 2.17. Data were analyzed with ANOVA test and differences between means compared through the Tukey-Kramer test (p <0.05). The variability of all these parameters was evaluated using descriptive statistics.

RESULTS AND DISCUSSION
Determining the chemical composition of Pseudocydonia sinensis is of great importance in studies into its nutritional aspects and use as raw material for industry. Proteins are macromolecules, structural units of which are called amino acids and play numerous functions that allow an organism to function and reproduce (Day, 1996). The protein content in seeds, pulp, and peel was 13.20, 1.26, and 2.32%, respectively (Table 1) After the combustion process of the plant sample at high temperatures, the plant raw transforms into a mineral residue that consists of macro-and microelements. The results obtained show the differences between the parts of the plant and are 4.33, 2.42, and 2.46% in the seeds, pulp, and peel. According to Leonel et al. ( Monosaccharide analysis of neutral carbohydrate part showed the presence of two main sugars -fructose (3.40, 34.46, and 26.00 g.kg -1 , respectively) and sucrose (9.65, 7.97, and 17.43 g.kg -1 , respectively) in the seeds, pulp, and peel, while other saccharides, such as maltose and lactose were found in low amounts only (<0.5 g.kg -1 ).

Rodríguez-Guisado et al. (2009)
analyzing the profile of sugars in Cydonia oblonga found levels of 5.31 to 10.89% for fructose, 4.08 to 5.44% for glucose, 1.51 to 2.41 of sucrose, and 0.31 to 0.42% for maltose totaling between 11.67 and 16.08% total sugars. Leonel et al. (2016) established the amount of total zinc in the range from 9.5 to 11.1 g.100g -1 . Behind the results of Rasheed et al. (2018), the amount of reducing sugar was 5.15 g.100g -1 , and the amount of non-reducing sugar was 4.61 g.100g -1 .
In this study, total fatty acids varied in different parts of Pseudocydonia sinensis and contained oleic acid, palmitic acid, linolenic acid, and linoleic acid. Linoleic acid in seeds accounted for 48.02% of total fatty acids, followed by oleic acid, accounting for 32.12% of total fatty acids ( Figure 3). Palmitic acid was the minor fatty acid in leaves,
Palmitic acid, oleic acid, and linoleic acid in the pulp samples were 45.38, 21.32, and 14.93%, respectively. Stearic acid was the minor fatty acid in leaves, accounting for 8.68% of the total fatty acids. Saturated fatty acids were the predominant fatty acids in Pseudocydonia sinensis pulp, accounting for 61.16% of the total fatty acids, while unsaturated fatty acids accounted for only 38.84%.
In peel, linoleic acid, palmitic acid, and palmitoleic acid accounted for 42.70, 28.64, and 13.91% of total fatty acids, respectively. Oleic acid was the minor fatty acid in the peel, accounting for 5.88% of the total fatty acids. Unsaturated fatty acids were also predominant in the peel, which accounted for 66.30% of total fatty acid while saturated fatty acids accounted for 33.70%. Amino acid analysis has shown that the studied Pseudocydonia sinensis seeds, pulp, and peel contained 18 amino acids (9 essential and 9 non-essential) (Figure 4).

Essential amino acids
Non-essential amino acids

Peel
The total amount of amino acids found in the seeds was 105.0 g.kg -1 DM, including total essential amino acids (32.70 g.kg -1 DM) and percentage of total essential amino acids (31.14%). Glutamic acid was found to be the dominant free amino acid (28.8 g.kg -1 ) in seeds followed by aspartic acid (10.7 g.kg -1 ) and arginine (9.8 g.kg -1 ). In the peel and pulp, the total amino acid content is found much than in the seeds, amounting to 21.4 and 13.7 g.kg -1 DM, respectively. The total non-essential amino acids in peel and pulp amounting to 11.7 and 7.2 g.kg -1 DM, respectively, and the percentage of total essential amino acids amounting to 9.7 and 6.5%, respectively).
The Cydonia oblonga fruits with 21 free amino acids identified. The sum of the 21 free amino acids ranged from approximately 316 to 1357 mg.kg -1 for Cydonia oblonga pulps and from 512 to 1820 mg.kg -1 for Cydonia oblonga peels. In what concerns the quince pulps, generally, the three most abundant free amino acids were aspartic acid, hydroxyproline, and asparagine. As a general rule, the three most abundant compounds present in quince peels were glycine, aspartic acid, and asparagine (Silva et al., 2004).
At present, little is known about the levels of trace elements in Pseudocydonia sinensis fruits and their parts such as seeds, peel, or pulp. The average contents of the elements in the different parts of Pseudocydonia sinensis are shown in Table 2.
Macroelement and trace element concentrations in the seeds samples revealing the following trend: K > P > Mg > Ca > S > Zn > Fe > Cu > Mn > Na > Al > Ni > As > Cr > Se > Pb > Cd > Hg. These elements were also detected in pulp samples according to the following order: K > Ca > P > Mg > S > Fe > Na > Zn > Cu > Al > Mn > Se > Ni > As > Cr > Pb > Cd > Hg. In the peel samples, the following concentrations were observed: K > Ca > P > Mg > S > Fe > Zn > Na > Cu > Al > Mn > Se > Ni > As > Cr > Pb > Cd > Hg.
Among biological activities inherent in plant raw material can be highlighted an antioxidant activity that had widely studied last time. A plant raw is a valuable source of antioxidants with different nature that has a therapeutic value for human health. The study of antioxidant capacity was carried out by different methods (Gupta, 2015).
In our study, the antioxidant activity carried out by the DPPH method of Pseudocydonia sinensis peel, pulp and seeds were 9.41, 7.08, and 6.21 mg TEAC.g -1 DM, respectively ( Figure 5).   Baroni et al. (2018), the antioxidant activity of Cydonia oblonga Mill. pulp, peel, seed, and jam extracts evaluated by the DPPH assay identified that methanolic peel extracts demonstrated the strongest activity, followed by pulp and seed extracts. A study by Silva et al. (2004) showed that the phenolic fraction of the seed extracts have stronger antioxidant activity than the peel and pulp extracts. Also, the different studies determined that the antioxidant activity by the DPPH method of methanol extracts of Malus domestica Borkh. cultivars were higher in peel extracts (71.7 -84.9%) than in pulp ones (43.9 -52.8%) (Manzoor et al., 2012).
Polyphenols are a large group of organic compounds with antioxidant and anti-inflammatory properties that may play a vital role in metabolic processes in the human body Flavonoids are a group of natural substances that play variable biological activities as well as other polyphenol compounds such as anti-inflammatory, antimutagenic, anticancer, antioxidative, etc. (Panche, Diwan and Chandra, 2016).
The total flavonoid content in the peel, pulp, and seeds was 18.39, 0.80, and 0.75 mg QE.g -1 DM, respectively. The total flavonoid content in the peel and pulp of Pseudocydonia sinensis fruits of different genotypes growing in Slovakia was 11.00 to 26.72 and 0.59 to 1.07 mg QE.g -1 DM, respectively (Grygorieva et al., 2020). It was previously reported (Amirahmadi, Abdollahi and Ayyari, 2017) that the total flavonoid content in fruits of closely related Cydonia oblonga species was 6.2 mg QE.g -1 .
Phenolic acids are a large group of phenolic compounds that possess numerous biological activities, among which antioxidant action (Kumar and Goel, 2019).
It was found that the total phenolic acid content varies significantly between samples ( Figure 6). The content of phenolic acids in the peel, pulp, and seeds was 5.68, 2.08, and 1.23 mg CAE.g -1 DM, respectively.
The content of phenolic compounds in the fruit of Pseudocydonia sinensis was in agreement with previous research (Hamauzu et al., 2006;Grygorieva et al., 2020). According to our previous studies (Grygorieva et al., 2020) of fruits from Slovakia, the total phenolic content in peel and pulp was 4.20 -8.39 and 1.12 -3.97 mg CAE.g -1 DM. According to Hamauzu et al. (2005), the total content of phenols in fruits of Pseudocydonia sinensis was 1280 mg.100g -1 FW. This was four and twenty times higher than in Cydonia oblonga and Malus domestica, respectively. Differences between present and previously conducted studies in the chemical composition of fruit of Pseudocydonia sinensis could be attributed to the geographical plant origin and different methods of extraction. This has been previously reported in other plant species, namely Diospyros virginiana

CONCLUSION
The chemical composition, antioxidant activity, total polyphenol, flavonoid, and phenolic acid content of peel, pulp, and seeds extracts from Pseudocydonia sinensis were studied. The content of micro and macronutrients was found in the seeds much higher than in the pulp and peel. It should also be noted that all Pseudocydonia sinensis samples are a very valuable source of potassium, which is necessary for the water and electrolyte balance. Total fatty acids predominate in the pulp than in the peel and seeds. Total amino acids predominate in the seeds than in the peel and pulp. The results revealed the highest content of bioactive compounds (total polyphenols, flavonoid, and phenolic acid) and antioxidant activity in peel compared with pulp and seeds. This study demonstrates the potential application of P. sinensis as a valuable source of natural phenolic antioxidants and can be used as raw material to elaborate diverse food products, providing important functional properties. When introduced in Ukraine and Slovakia, the amount of beneficial substances in fruits and seeds is not reduced, which makes it possible to recommend this species for wide cultivation.