Ribes himalense as potential source of natural bioactive compounds: Nutritional, phytochemical, and antioxidant properties

Abstract Ribes himalense Royle ex Decne. (family Saxifraaceae, subfamily Grossulariaceae, genus Ribes) is a wild berry fruit with illustrated health‐promoting features, which widely distributed in Northwest China are deficiently exploited. This study aimed to assess the potential of a Ribes himalense as a source of natural bioactive compounds through characterizing its nutraceutical characteristics, phytochemicals properties, and antioxidant ability. Fresh berries were quantitatively analyzed for proximate composition, minerals, vitamins, amino acids, total polyphenols, total flavonoids, anthocyanins, procyanidin, and polysaccharides contents through China National Food Safety Standard; the characterization and identification of extracts of wild berries obtained with ethanol 30%, ethanol 50%, and ethanol 95% were firstly performed by UPLC‐Triple‐TOF‐MS2. Furthermore, antioxidant activity of the ethanol extract was evaluated via different assay methods such as DPPH, ABTS, and FRAP. The results indicated that the most important bioactive composition was procyanidin (0.72%), polyphenols (0.49%), total flavonoids (0.38%), vitamin C (64.6 mg/100g FW), and K (218.44 mg/100 g FW), and a total of 95 compounds were detected with polyphenols, flavonoids, and proanthocyanidins as the dominant, and also ethanol extract possessed stronger antioxidant activity. These results suggested that Ribes himalense fruit has great potential in protecting human health, with the focus on the development of functional products.

and a VWD detector. The separation was carried out at 37°C using a Phenomenex Gemini-NX column (250 × 4.6 mm, 5 μm). Mobile phase A was acetonitrile/H 2 O (50:50, v/v), and mobile phase B was 0.05 mol/L sodium acetate solution (6.80 g sodium acetate trihydrate was dissolved in 900 ml water, adjust the pH to 4.0-5.0 with glacial acetic acid, and dilute to 1,000 ml with water), gradient elution for 45 min. The flow rate of 1 ml/min, the injection volume was 10 μl, and the detection wavelength was 360 nm.

| Mineral content determination
2.5 g samples were weighed into PTFE inner tank and added with 10 ml nitric acid, cover the safety valve and soak overnight, and digested in microwave digestion instrument. When the digestive liquid was colorless and transparent or slightly yellow, cooled, constant volume with ultrapure water to 25 ml, mix well for later use. The minerals were determined according to the standard method of China National Food Safety Standard (GB 5009.268-2017). Potassium (K), calcium (Ca), magnesium (Mg), iron (Fe), zinc (Zn), manganese (Mn), and copper (Cu) contents were determined by using an inductively coupled plasma spectrometer (ICPS, OES-725, USA).

| Vitamin B1
The vitamin B1 (VB1) was analyzed using China National Food Safety Standard method (GB 5009.84-2016). VB1 was extracted with 60 ml 0.1 mol/L hydrochloric acid and 2.0 ml mixed enzyme solution (1.76 g papain and 1.27 g amylase, with water constant volume to 50 ml). VB1 was determined by a high-performance liquid chromatography (HPLC) method. A Waters 1525 Series HPLC system equipped with a fluorescence detector and a Dikma Spursil C 18 -EP column (250 × 4.6 mm, 5 μm). Mobile phase was 0.05 mol/L sodium acetate solution and methanol (65:35, v/v) with gradient elution for 12 min, excitation wavelength 375 nm, emission wavelength 435 nm, flow rate of 0.8 ml/min, and injection volume of 10 μl.

| Vitamin B2
The vitamin B2 (VB2) was determined by a HPLC method (GB 5009.85-2016). Vitamin B 2 was extracted using 60 ml 0.1 mol/L hydrochloric acid and 2 ml mixed enzyme solution (2.345 g papain and 1.175 g taka-diastase, with water constant volume to 50 ml) and was determined by a Waters 1525 Series HPLC system equipped with a fluorescence detector and a Dikma Spursil C 18 -EP column (250 × 4.6 mm, 5 μm). Mobile phase was 0.05 mol/L sodium acetate solution and methanol (65:35, v/v) with gradient elution for 14 min, excitation wavelength 462 nm, emission wavelength 522 nm, flow rate of 1 ml/min, and injection volume of 10 μl.

| Vitamin C
The vitamin C (ascorbic acid) was analyzed by a HPLC method (GB 5009.86-2016), which a Shimadzu LC-2030C 3D Series HPLC system equipped with a PDA detector and a Shim-pack GIST C 18 column (250 × 4.6 mm, 5 μm). After the sample was dissolved in 20 g/L metaphosphoric acid, the absorbance was measured at 245 nm. Mobile phase was 20 mM metaphosphoric acid solution and methanol (98:2, v/v), gradient elution for 20 min, flow rate of 0.7 ml/min, and injection volume of 10 μl. The content of vitamin C was calculated on the basis of the calibration curve of L (+) ascorbic acid that acted as the standard reference.

| Vitamin E
The vitamin E (tocopherol) was extracted with 50 ml mixture of pe- The residues in the evaporating flask were dissolved in methanol by stages and transferred to a 10-ml volumetric flask. The solution was set to scale and passed through an organic mesofiltration membrane 0.22 μm for HPLC determination (GB 5009.82-2016), which Agilent 1260 Series HPLC system equipped with a UV detector and a Dikma Spursil C 18 column (250 × 4.6 mm, 5 μm). Mobile phase was methanol/H2O (98:2, v/v), gradient elution for 40 min, flow rate of 1 ml/ min, and injection volume of 10 μl. The measurements at 294 nm against a blank sample, standard curves made with pure tocopherol were used for this purpose.

| Determination of total phenolic content
The total polyphenol contents were determined according to spectrophotometric method of China Food Safety Standard (T/ AHFIA005-2018) which involved the reduction in Folin-Ciocalteu reagent by phenolic compounds. 1 ml of extract sample and standard (3,4,5-trihydroxybenzoic acid) was incubated with 2.5 ml Folin-Ciocalteu's reagent, shaken, and then 2.5 ml of 15% (w/v) Na 2 CO 3 was added and the solution hatched at 40°C water bath for 60 min, respectively. Absorbances of samples were measured at 778 nm using Cari-300 Ultraviolet-visible spectrophotometer (Varian Corp., USA). Gallic acid was used as the standard and formulated into standard series with concentrations of 0, 4, 8, 12, 20, and 30 mg/L, draw a standard curve, and the content of total polyphenols in the solution to be measured was calculated according to the standard curve. The results were indicated as gallic acid equivalent mg GAE/g fresh weight.

| Determination of total flavonoids content
The total flavonoids contents were investigated according to spectrophotometric method. Add 25 ml of 75% ethanol to 1 g of sample, sonicate for 30 min, filter into a 50-ml volumetric flask, continue to add 75% ethanol to dilute to the mark, shake well, draw 2 ml of filtrate into a 50-ml volumetric flask, and process according to the above steps, to get the sample solution which measures the absorbance at 510 nm using Cari-300 Ultravioletvisible spectrophotometer, prepare a standard curve with rutin (0.2094 mg/ml) as a standard, and calculate the total flavonoid content. The total flavonoid content values were represented as rutin equivalents (RTE), that is, mg RTE/g fresh weight, and determined via a calibration curve.

| Determination of polysaccharide content
Weigh 0.5 g of the sample into a 150-ml conical flask, add 50 ml of 75% ethanol and ultrasonic for 30 min, filter, add about 100 ml of water to the filter residue, heat, and boil on a heating plate for 30 min. Cool to room temperature, transfer the contents to a 250ml volumetric flask, add distilled water to wash the conical flask 3 times and transfer them into the volumetric flask together, add water to dilute to the mark, and shake well. Precisely pipet 3 ml, add 3 ml of Sevag reagent (chloroform: n-butanol, 4:3, v/v), centrifuge at 9,000 rpm for 5 min, discard the middle denatured protein layer and the lower organic layer, and the aqueous phase continued to repeat the above operation until no denatured protein appears between the water phase and the organic layer. Dilute the solution 10 times, accurately pipette 0.5 ml of the solution into a test tube with stopper, add distilled water to make up to 2 ml, add 1 ml of 5% phenol solution blending, slowly add 5 ml of concentrated sulfuric acid, color reactions can be carried out after the shake, draw the standard curve with D-anhydrous glucose as the standard, and use the ultraviolet spectrophotometer to determine the polysaccharide content of the sample at 490 nm.

| Determination of anthocyanin content
The content of anthocyanins was determined by Varian Cari-300 Bio spectrophotometry using the pH differential method as described (Llivisaca et al., 2018). Put 75-100 mg of the sample in a 50-ml brown volumetric flask, 25 ml extract (concentrated HCl: methanol, 4:96, v/v) was added, sealed, ultrasonic at 40°C for 30 min, and cooled to room temperature to obtain the test solution. According to the test solution: buffer solution (1:5, v/v), prepare two dilutions of the test solution, one of which is diluted with potassium chloride buffer (0.025 M, pH 1.0), and the other is used sodium acetate buffer (0.4 M, pH 4.5). Using distilled water as a blank control, equilibrate the two diluted solutions prepared above for 20 min, then measure the absorbance was then measured at λ max and 700 nm in each solution, and the anthocyanin content was estimated using the following formula (Jiang, Yang, & Shi, 2017): (Molecular Weight) = molecular weight of cyanidin 3-glucoside is 449.2 g/mol; DF = dilution factor; ɛ = molar extinction coefficient of cyanidin 3-glucoside is 29,600 L/mol·cm; l = path length, cm. All samples were analyzed in triplicate.

| Determination of proanthocyanidins content
Procyanidin and vanillin were purchased from Shanghai Chunyou Biotechnology Co. (Shanghai, China). The content of procyanidin in the extract solution was determined using the standard vanillin-Hydrochloric acid method (Fang et al., 2020). Vanillin/methanol solution (4 g/100 ml) of 3 ml and concentrated HCl of 1.5 ml were added to 1 ml of the extract solution in a 25 ml tube with plug (out of light), mixed well. Color development at room temperature for 15 min, with methanol as the control. The absorbance was measured at 500 nm using a Cari-300 Bio Ultraviolet-visible spectrophotometer, and the content of procyanidins was calculated by standard curve method.

| Extract preparation
Fresh fruit was pressed to produce fruit juice, which was eluted with 30% ethanol with AB-8 macroporous resin to obtain component 1 (Fr 1). Component 2 (Fr 2) was obtained by 60% ethanol elution.
Component 3 (Fr 3) was obtained by eluting with 95% ethanol. The extracts were stored at 4°C prior to further assay. Three components were concentrated by rotary evaporation, and 4-5 ml of which were absorbed, respectively, into the EP tube to obtain three samples. The samples were treated as follows: Fr1 samples were concentrated to dry, followed by 10 ml 50% acetonitrile-aqueous solution, ultrasonic for 2 min, centrifugation at 10,000 rpm for 20 min, and the supernatant was taken for testing; Fr2 samples were ultrasonic for 5min, centrifuged at 10,000 rpm for 30 min, and the supernatant was taken for testing; the Fr3 sample was concentrated to dry, then 2ml 50% acetonitrile-aqueous solution was added, followed by ultrasonic for 2 min, centrifugation at 10,000 rpm for 20 min, and the supernatant was taken for testing.   the accurate relative molecular mass of the primary mass spectrum, and the molecular formula is fitted within the mass deviation range of 5 × 10 −6 through the peakview 1.2 software, and compare with the literature database which makes a preliminary guess for each chromatographic peak. Secondary mass spectrometry with good signal-tonoise ratio was screened to obtain its information of chromatographic peaks, and corresponding fragment ions of compounds were obtained. Then the chemical composition was further predicted according to the fragmentation of ions and combined with literature data.

| Assay of antioxidant activity in vitro
The antioxidant activities of the 30%, 60%, and 95% ethanol extracts which is µmol Fe (II)/g fresh weight, and determined via a standard curve (y = 1.2416x + 0.0134, R 2 = 0.9996). The analyses were carried out in triplicate for each concentration.

| Statistical analysis
The experimental data used statistical software SPSS 20.0 for variance analysis, Duncan's multiple range test is used to separate the means, and the results are reported as mean ± SD. A p value of .05 is considered a statistically significant difference. Origin 8.5 software was used to draw graphs.

| Proximate analysis and bioactive compounds contents
Proximate composition and phytochemical component of R.
These compounds are found in abundance in Ribes berries and contain active molecules that have health benefits. Furthermore, phenolics, flavonoids, polysaccharide, anthocyanin, and procyanidine have been reported to possess antioxidant, anti-inflammatory, anticancer, and antihyperglycemia properties (Braga et al., 2018;Ieri et al., 2015). These data have proved that R. himalense fruits contain both nutrient and bioactive substances, it will satisfy in which natural and functional.  (Plessi et al., 2007;Sánchez-Castillo et al., 1998).

| Vitamin content
The vitamin content of R. himalense fresh fruit from wild is presented in  (Donno et al., 2015(Donno et al., , 2018. Furthermore, content of vitamin C reached 64.6 mg/100g, in fruit market; vitamin C has been corresponding to more than 65% of antioxidant and antiviral activity in many fruits and their beverages (Mditshwa et al., 2017;Padayatty et al., 2003). Thus, the high content of vitamin C in this fruit makes it suitable for further development and application in commercial market.

| Amino acid profiles
Amino acids (AA), the most important chemical elements in the world, are divided into essential amino acids and nonessential amino acids. The essential amino acids, defined as one that the body cannot make in sufficient amounts to maintain growth or nitrogen balance, must be absorbed from food and supply human body (Rose et al., 1948;Rose & Smith, 1949). AAs are important fundamental units of vital tissues, proteins and peptides (including enzymes and hormones), neurotransmitters, nourishment, and transporters. Thus, they are arousing great scientific interest for the researchers (Wahl & Holzgrabe, 2016). Table 2 shown the amino acids profile of Ribes himalense fruit.
Tryptophan with 0.08 ± 0.01 g/100 g and cysteine with 0.01 g/100 g were the amino acids with lower presence.
This is the first study analyzing the nutritional profile of R. himalense samples from undeveloped wild area of Qinghai Plateau.
Although we could observe differences among all samples, in general, the variance found between the nutritional compositions of the
The MS spectrometry results and fragmentation characteristics of the components of Fr1 are shown in Table 3. were numbered by their elution order and summarized in Table 4.

| Flavan-3-ols
Compound 17  stereoisomer is always higher than that of (+)-catechin on C18 inverse-phase column, which means that catechin is eluted earlier than epicatechin (Mena et al., 2012 (Chai et al., 2020;Laczkó-Zöld et al., 2018;Lin et al., 2016). Moreover, previous findings on Ribes species fruits, in- Berl., indicated that these berries are the richest in polyphenols, such as anthocyanins, flavonoids, and phenolic acids, which play a vital role in the prevention and control of various illnesses through equilibriuming the oxidative and antioxidation factors in the human body (Carole et al., 2019;Delazar et al., 2010;Hurst et al., 2020;Laczkó-Zöld et al., 2018). Therefore, phenolic compounds in the R. himalense fruits could act as a main contributor to their antioxidant activity, which was consistent with the result of total phenolic content measurements.

| CON CLUS IONS
In summary, the structure of the detected compounds was identified by the following methods: (a) unambiguously recognized by comparing with reference standard compounds, such as analysis of nutrients content (including minerals, amino acids, vitamins); (b) characterized by cleavage pathways and typical fragment ions according to the relevant references; (c) preliminarily identified by searching Scifinder and Reaxy databases, like phenolic acids, flavonoids, proanthocyanidins, anthocyanins, and organic acids.
Berry is a fruit loved by consumers and is recognized as a food with multiple health benefits. In this work, we have carried out nutritional properties, chemical characterization, and antioxidant activities of the small berries of R. himalense picked from the plateau to clarify whether they is still a good source of this health showed that R. himalense berries are an excellent source of bioactive compounds with prospects of various health-promoting functions, particularly phytochemicals performing considerable antioxidant capacity and conducing to the prophylaxis of some diseases caused by oxidative stress. As far as we know, this was the first report of systematic analysis of the chemical constituents and nutrients of R. himalense fruit. Based on the results obtained, because of its nutrition and chemical composition, the R. himalense proved to be a good choice for enriching the daily diet and could also be regarded as a rich natural source of nutrients with high antioxidant potential. Therefore, it should be widely used in modern nutritious food, cosmetics, and pharmaceutical industries to study functional products with potential health benefits.

CO N FLI C T S O F I NTE R E S T
The authors have declared no conflicts of interest for this article.

E TH I C A L S TATEM ENT
This study does not involve any human or animal testing.