Physico-chemical properties and nutritional composition of fruits of the wild Himalayan strawberry ( Fragaria nubicola Lindle.) in different ripening stages

Abstract

BACKGROUND:

Genus Fragaria (family - Rosaceae), popular edible berry fruits with delicious flavor and considerable health benefits has many wild relatives in the Himalayan region.

OBJECTIVES:

The objective of the study was to characterize variability in morphology, physicochemical properties, phytochemicals, and antioxidant activity in wild Himalayan Strawberry, Fragaria nubicola along the ripening stages and among the genotypes.

METHOD:

Morphological and physicochemical properties, thiamine, carotenes, total phenolic content, phenolic compounds, total flavonoids, flavonols, tannins, anthocyanins, and antioxidant activity (ABTS, DPPH, and FRAP assays) were determined in four ripening stages of berry fruits.

RESULTS:

Morphological attributes (diameter, length, volume, and fresh weight of berries) have shown considerable variations among the genotypes and increased significantly (p < 0.05) with the ripening stages. The physicochemical properties such as juice content, pH and moisture content also increased with the ripening, however, the pomace content decreased with the ripening. Anthocyanin content increased significantly (p < 0.05) with ripening and reached at maximum level after full ripening. A successive decrease in free and bounded total phenolic, flavonoid, and flavonol contents was observed with the ripening except in total tannin content. However, a reverse trend of these phenolics was observed in juice along with the ripening. The antioxidant activity measured by three in vitro assays increased with the ripening. Phenolics were extracted higher in the acidified methanolic solvent (extracted free and bounded phenolics) as compared to methanolic solvent (extracted free phenolics). Phenolic compounds quantified by RP-HPLC analysis were extracted higher in acidified methanol as compared to methanol, except chlorogenic acid content.

CONCLUSION:

The results showed quantitative changes in free and bounded phenolics and morphological and functional traits along with the ripening. Also, this important genetic resource exhibited potential utility in the breeding of strawberry improvement programs and as an alternative resource of rich phytonutrients and antioxidants as a functional food.

Keywords

Antioxidant berry fruits genetic resource himalayan berries phenolics wild strawberry

1 Introduction

Himalayan region is known for rich floristic diversity including, valuable wild edible fruits dominated by various wild berries of the Rosaceae family [1]. These berry fruits have attracted global interest due to their high nutritional value, rich vitamins, beneficial flavonoids, phenolics, anthocyanins, and antioxidants. These valuable secondary metabolites of berries have health protection ability, which worked as antioxidants, prevent or delay the oxidation of lipid, DNA, or other macromolecules through inhibiting oxidative chain reactions [2, 3]. Thus, in many epidemiological studies, consumption of berry fruits has been associated with the lower risk of non-communicable diseases such as cardiovascular diseases, diabetes, cancer and neurodegenerative disorders [4], through improvement in LDL oxidation, lipid peroxidation, total plasma antioxidant capacity, dyslipidemia, and glucose metabolism [5]. However, the beneficial effect of these valuable bioactive compounds largely depends upon their extractability due to their conjugation with carbohydrate molecules. Glycosylation of these compounds leads to their altered biochemical properties, cellular transport, storage, and turnover in the plant cells [6]. It has been estimated that around 35–65%of phenolic compounds in the Rubus berries are bounded with glucose, cellulose, hemicellulose, lignin, pectin, and other structural proteins [7]. This association provides both physical and chemical barriers to plants, astringency to prevent insect attacks, protection against radiation, and antibacterial, antifungal, and antimicrobial functions [8, 9].

Fragaria genus (family Rosaceae), commonly known as strawberry, are berry fruits popularised for their unique flavor and considerable health benefits [10]. The Fragaria genus comprises twenty wild species, three naturally occurring hybrids, and two cultivated hybrids [11]. Production of cultivated species reached 9.22 million tonnes in 2017 all over the world [10], and its hybridization compatibility with other wild species has increased the possibility of expansion in other areas and its global productivity. Modern cultivated strawberry varieties are the results of breeding efforts made among F. ananassa (garden strawberry), F. vesca (wild strawberry), F. moschata (musk strawberry), and many other wild exotic species [10]. Among the wild species, Fragaria nubicola Lindle. (common name - Himalayan Strawberry) is a perennial herb, commonly grows in shady places along forest edges within the altitudinal range of 2100 to 4000 m asl [12]. This species is native to the Himalaya, distributed from Afghanistan to Myanmar, India, Bhutan, Nepal, Pakistan and South-west China including Tibet. It preferred to grow in open areas, edges of forests, disturbed areas along with roads and tracks with light shade and under moist conditions [13]. Its edible fruits are broadly ovoid or depress ovoid with 5.5–16.5 mm long and 7.0–17.5 mm wide having shiny and surface between achenes with anthocyanin-tinged monopodial stolon [13].

Generally, Fragaria species represented by low-growing perennials, having evergreen and trifoliolate leaves, insect-pollinated white actinomorphic flowers and different accessory aggregate fruits characters such as color, shape, and achene and calyx positions at maturity. Fruits of commercial Fragaria have therapeutical benefits in cardiovascular diseases and dyslipidemia. The health benefits of Fragaria fruits are associated with the presence of phenolic compounds such as phenolic acids and anthocyanins, which suggests its possible application in multiple therapeutical areas [10]. Accumulation of valuable metabolic constituents along the ripening in cultivated strawberry has been a matter of interest due to rapid changes in its quality attributes with fruit maturation [14 –22]. Fragaria nubicola fruits can not only be an alternative option for valuable nutrients but can also be utilized as a good genetic backup in narrow genetic base-bearing cultivated strawberry species [23]. The presence of the species in a wide geographical range along the heterogeneous mountainous environmental conditions of Himalaya created greater possibilities of finding a new set of health-beneficial phytochemical diversity. However, morphological variability, physicochemical properties and nutritional composition of fruits altered with ripening stages have not been evaluated in the fruits of F. nubicola. Keeping this in view, the current study attempted to determine changes in morphology, physicochemical properties, nutritive constituents, phytochemicals, and in vitro antioxidant activity during the ripening of berry fruits. Phenolic compounds were also quantified in berry fruits of the species as these compounds are the most valuable active constituents in fruits of Fragaria. Phenolic constituents were characterized in juices, methanolic and acidified methanolic extract to determine the free and bounded phenolic content in the species. This will be the first quantitative assessment of important morphological traits, nutritive and bioactive constituents in F. nubicola.

2 Materials and methods

2.1 Plant material

Fresh berry fruits of F. nubicola were collected from the wild population of Pangthang area, Gangtok, Sikkim State of India (Latitude: 27° 21’ 48”, Longitude: 88° 34’ 04”; altitude: 2150 m asl). Only one berry fruit per plant located at least 30 cm away from each other was harvested and has been considered as a separate genotype for morphological analysis. The ripening stages were determined by maturation time, size and change in color of berries. Ripening stages were considered as small and green berries (Stage-1), maturing berries with an initial color change to light red color (Stage-2), fully grown and ripened berries with red color (Stage-3), and ripened dark red berries (Stage-4). Furthermore, samples of fully matured berries were randomly collected (termed as mixture samples) for phenolic composition and nutritive analysis (riboflavin, β-carotene, and total carotene). Immediately after collection, 20 berry fruits were randomly selected from each ripening stage for measuring morphological parameters. While physicochemical characters (juice content, pH of juice, pulp pomace, and moisture content) was conducted in triplicates from ripening groups of berries. All the infected and damaged berries were removed, and only healthy berries were further processed for analysis. Each group of berries was washed with water to remove dust particles and dried properly before any measurement.

2.2 Reagents and instruments

2,2-Diphenyl-2-picrylhydrazyl (DPPH) radical, 2,2-azinobis-3-ethylbenzthiazoline-6-sulphonic acid (ABTS), 2,4,6-tri-2-pyridyl-1,3,5-triazin (TPTZ), gallic acid, ascorbic acid, tannic acid, quercetin, chlorogenic acid, caffeic acid, p-coumaric acid, 3- hydroxy-benzoic acid, catechin, valanic acid, naringin, rutin, riboflavin, thiamine and β-carotene were procured from Sigma–Aldrich (Steinheim, Germany). Sodium carbonate, potassium persulphate, ferric chloride, sodium acetate, potassium acetate, meta-phosphoric acid, aluminum chloride, glacial acetic acid, β-carotene, tetrahydrofuran, potassium dichromate, n-hexane, methanol, ethanol and hydrochloric acid were procured from Qualigens (Mumbai, India).

2.3 Morphological and physicochemical characterization

The width and length of 20 fresh berry fruits (each stage) were measured using a digital Vernier caliper with the sensitivity of 0.01 mm (Traceable Digital Caliper-6”, VWR International, Milano, Italy), and fresh weight determined using a digital weighing machine with the sensitivity of 0.1 mg (Model CY510, Citizen). The volume of berry fruits was determined by liquid displacement using water as a solvent. Berry fruits (50 g in three replicates) were squeezed by hand for obtaining the juice and juice content was recovered through passed from a cheesecloth. After recovery of juice, pulp pomace was obtained, and its content was determined using a digital weighing machine. The pH of berry juice was measured using a digital pH meter (ECPH-70042S digital pH meter, Eutech Thermo Scientific, USA). A total of 50 g berry fruits (3 replicates) were kept in a hot air oven at 65°C for drying and moisture content was calculated based on dry weight obtained.

2.4 Extraction preparation for quantification of phenolics and antioxidant activity

Fresh berry fruits (20 g) in each ripening stage were used for the preparation of extract for analysis of phenolics (total phenolic, flavonoid, flavonol, tannin contents) and antioxidant activity. Berry fruits were carefully homogenized using the grinder and kept in a flask with 50 ml aqueous methanol (80%v/v) and acidified methanol (80%methanol with 1N HCl in 4:1), thereafter placed in the water bath at 60°C for 1 hour. After cooling, the supernatant was kept for continuous shaking at room temperature for 15 hours. The extract was filtered and the filtrate was centrifuged at 8,000 rpm for 10 minutes. The supernatant was stored at 4°C and used for quantification of phenolics and antioxidant activity within two days of extraction.

2.5 Determination of total phenolic, flavonoid, flavonol and tannin content

Total phenolic content in extracts were determined by Folin-Ciocalteu’s colorimetric method [19]. Briefly, in 0.25 ml of diluted methanolic extract, 2.25 ml distilled water and 0.25 ml Folin-Ciocalteu’s reagent was added and allowed to stand for reaction up to 5 minutes. This mixture was neutralized by 2.50 ml of 7%sodium carbonate (w/v) and kept in dark at room temperature for 60 minutes. The absorbance of the resulting blue color was measured at 765 nm using a UV-VIS spectrophotometer (Hitachi U-2001). Quantification was done based on a standard curve of gallic acid prepared in 80%methanol (v/v) and results were expressed in mg gallic acid equivalent (GAE)/g of fresh weight (fw) of fruits.

Total flavonoid content in the extract of berry fruits was determined by the aluminum chloride colorimetric method [24]. Briefly, 0.50 ml of methanolic extract of the sample was diluted with 1.50 ml of distilled water followed by the addition of 0.50 ml of 10%(w/v) aluminum chloride, 0.10 ml of 1M potassium acetate and 2.80 ml of distilled water. This mixture was incubated at room temperature (∼20°C) for 30 minutes. The absorbance of the resulting reaction mixture was measured at 415 nm. Quantification of flavonoid content was done based on a standard curve of quercetin prepared in 80%methanol and results were expressed in mg quercetin equivalent (QE)/g fresh weight (fw) of fruits. Total flavonol was estimated using the method of Dhyani et al., [25]. Briefly, in 2.0 ml of extract, 2.0 ml aluminium chloride ethanolic solution (2%w/v) and 3.0 ml sodium acetate solution (5%) were added. The absorbance of the resulting solution was recorded at 440 nm after incubation (2.5 h) at room temperature (∼20°C). Quantification of total flavonol content was done based on a standard curve of catechin prepared in 80%(v/v) methanol and results were expressed in mg quercetin equivalent (QE)/g fresh weight (fw) of fruits.

Total tannin content was estimated following Bhatt et al. [26] with minor modifications. Briefly, 5 ml extract was added to 0.5 ml Folin–Dennis reagent (0.5 N) and 1 ml sodium carbonate solution (7%). The solution was diluted up to 10 ml with distilled water. After thorough mixing, the flasks were allowed to stand in a water bath at 25°C for 20 min. The absorbance of the resulting greenish-blue color was measured at 700 nm using a UV–vis spectrophotometer. The quantification of tannin was performed at 700 nm based on a standard curve of tannic acid prepared in 80%(v/v) methanol. Results were expressed in mg tannic acid equivalent (TAE)/g of fresh weight.

2.6 Quantification of phenolic compounds

Twenty microliter extract of each sample was used in triplicate for HPLC analysis (L-7100 series pump and L-7400 series UV-VIS detector, Merck-Hitachi, Japan). Phenolic compounds were separated by using 4.6×250 mm i.d.5μm, Spherisorb, C18 column. The Mobile phase used for the study included water, methanol and acetic acid in the ratio of 80:20:1 with a flow rate of 0.8 ml/min in isocratic mode. The spectra of phenolic compounds were recorded at 254 nm for gallic acid, catechin, trans-cinnamic acid, vanillic acid, and p-coumaric acid, and 280 nm for rutin, naringenin, caffeic acid, and chlorogenic acid. The identification of phenolic compounds was done based on the retention time of the corresponding external standard. UV-VIS spectra of the pure standard were used for plotting the standard calibration curve at different concentrations. The repeatability of quantitative analysis was less than 3.0 %. The mean value of content was calculated with±standard deviation (SD). The results were expressed as mg/g of fresh weight (fw) of fruits.

2.7 Extraction and quantification of total monomeric anthocyanins

For quantification of anthocyanins, 5 g of berry fruits were homogenized with 50 ml of 80%(v/v) acidified ethanol (95%ethanol + 5%1.5N HCl). After thorough mixing, the samples were allowed to stand for overnight incubation at room temperature followed by filtering the extract. Samples were stored at 4°C until analysis within 4 days.

Total anthocyanin contents were determined by the pH-differential method as described in Badhani et al. [3]. The extracts were appropriately diluted in two buffers, 0.025 M potassium chloride pH 1.0 and 0.4 M sodium acetate pH 4.5. After 15 min of incubation at room temperature, absorbance was measured at 520 nm and 700 nm with a UV-VIS spectrophotometer using distilled water as a blank. The absorbance difference between the pH-1.0 and pH-4.5 samples was calculated: $A = (A 520 nm - A 700 nm) pH 1.0 - (A 520 nm - A 700 nm) pH 4.5$

The monomeric anthocyanin pigment concentration was calculated using the following equation: $Monomeric anthocyanin pigment (mg / l) = (A \times MW \times DF \times 1000) / (ɛ \times 1);$

Where, MW = 449.2 and ɛ= 26,000, respectively, are molecular weight and molar absorptive of cyanidin-3-glucoside, which was used as a standard; DF is the dilution factor; l is the path length. The total monomeric anthocyanins were represented as mg/g fw.

2.8 Extraction and quantification of thiamine content

For quantification of thiamine content, fruits (10 g) were homogenized with 50 ml of ethanolic sodium hydroxide and filtered. In 10 ml of filtered solution, 10 ml of potassium dichromate was added for color development. The quantification of thiamine content was performed at 360 nm and a standard curve of thiamine content was prepared in 80%(v/v) ethanol for comparison [27].

2.9 Extraction and quantification of β-carotene

To measure the total carotenoid and β-carotene content, 10 g of fresh berries were homogenized in an ice bath with 5 mL acetone in a cold mortar pestle. Consequently, 1.0 g anhydrous sodium sulfate (Na₂SO₄) was added to the achieved homogenization and was elutriated using a paper filter. Filtrated solution reached a volume of 10 mL with acetone and was centrifuged for 10 min at 5000 rpm. The upper phase was collected, and the absorbance of the solution was measured at 662, 645, and 470 nm wavelengths [28]. Acetone was used as a control and the carotenoids and β- carotene of each extract were calculated using the formulas as follows. $Chlorophyll a (Ca) = 11.75 A 662 - 2.350 A 645$ $Chlorophyll b (Cb) = 18.61 A 645 - 3.960 A 662$ $Total carotenoids = 1000 A 470 - 2.270 Ca - 81.4 Cb / 227$ $β – carotene = 0.854 A 479 - 0.312 A 645 + 0.039 A 663 - 0.005 .$

2.10 Antioxidant activity

2.10.1 Free radical - scavenging ability by the use of DPPH cation (DPPH assay)

Traditional DPPH assay described by Nowicka et al. [29] was used for this study with few modifications. Cation solution of 0.1 mM DPPH prepared in 80%methanol (2.7 ml) was mixed with 0.9 ml sample extract and kept in dark at room temperature (∼20°C) for 20 minutes. A reduction in the absorbance at 520 nm was recorded. Results were expressed in millimole (mM) ascorbic acid equivalent (AAE) per 100 g fresh weight (fw) of fruits.

2.10.2 Free radical - scavenging ability by using ABTS radical cation (ABTS assay)

Total antioxidant activity was measured following the improved ABTS method described by Jugran et al. [30]. ABTS salt (7.0μM) and potassium persulphate (2.45μM) was added for the production of ABTS cation (ABTS^.+) and kept in dark for 16 hours at 23°C. ABTS^.+solution was diluted with distilled water till an absorbance of 0.700±0.005 at 734 nm was obtained. Diluted ABTS^.+solution (3.90 ml) was added in 0.10 ml of methanolic extract and allowed to stand for six minutes in dark at room temperature (∼20°C), and absorbance recorded at 734 nm using UV-VIS spectrophotometer corresponding to a blank prepared with 80%(v/v) methanol. A standard curve of various concentrations of ascorbic acid was prepared in 80%v/v methanol for the equivalent quantification of antioxidant potential. Results were expressed in millimole (mM) ascorbic acid equivalent (AAE) per 100 g fresh weight (fw) of fruits.

2.10.3 Ferric reducing antioxidant power (FRAP) activity

Ferric reducing antioxidant power (FRAP) assay was performed following Bahukhandi et al. [31] with some modifications. FRAP reagent was prepared by adding 10 volume of 300 mM acetate buffer (i.e., 3.1 gram of sodium acetate and 16 ml glacial acetic acid/l), 1 volume of 10 mM 2,4,6-tri-2-pyridyl-1,3,5-triazine (TPTZ) in 40 mM HCl and 1 volume of 20 mM ferric chloride. The mixture was pre-warmed at 37°C and 3.0 ml of the mixture was added to 0.10 ml methanolic extract and kept at 37°C for 8 minutes. Absorbance was taken at 593 nm by using a UV-VIS spectrophotometer. A blank was prepared by ascorbic acid and results were expressed in millimole (mM) of ascorbic acid equivalent (AAE) per 100 g fresh weight (fw) of fruits.

2.11 Statistical analysis

Only one berry fruit was obtained from a single plant and thus, variability in morphological characters among genotypes was calculated in terms of range, standard deviation, and coefficient of variation. Data of nutritive analysis was carried out based on five replicates from extraction. Significant differences among mean values of ripening stages were tested using Duncan’s multiple range test (P≤0.05) using SPSS software Version 17.0 (SPSS Inc., Chicago., IL). An unpaired t-test was used to compare the mean values of the phenolics extracted in the different solvent systems (methanolic and acidified methanolic).

3 Results and discussion

3.1 Morphological and physicochemical characterization

Morphological characters showed great variability among the genotypes and ripening stages (Table 1). With the ripening stages, diameter, length and fresh weight increase significantly (p < 0.05, each) with the development of berry fruits. On an average basis, the diameter of berry fruit was observed as 6.18 mm in Stage-1, 8.18 mm in Stage-2, 9.87 mm in Stage-3, and 12.64 mm in Stage-4. Similarly, the length of berry fruit was recorded as, 6.57 mm in Stage-1, 8.39 mm in Stage-2, 10.31 mm in Stage-3, and 12.94 mm in Stage-4. Fresh fruit weight was recorded as 0.13 g in Stage-1, 0.23 g in Stage-2, 0.35 g in Stage-3, and 0.69 g in Stage-4. However, among the different genotypes, a high level of variation was observed in diameter, length, and fresh weight of berry fruit. In consideration of variance among genotypes, the diameter of berry fruit exhibited coefficient of variance (CV) as 15.38%in Stage-1, 11.30%in Stage-2, 17.20%in Stage-3, 23.14%in Stage-4, and 18.89%in a mixture of all mature berries. Similarly, CV in the length of berry fruit was recorded between 7.61%to 30.01%in different ripening stages. Also, the CV in fresh berry fruit weight was between 2.01%to 12.15 %in different ripening stages.

Table 1
Genotypic variation in morphological characters in different ripening stages of Fragaria nubicola berries

Stage-1 Stage-2 Stage-3 Stage-4 All mature mixed fruits

Range (CV) Mean±SD Range (CV) Mean±SD Range (CV) Mean±SD Range (CV) Mean±SD Range (CV) Mean±SD

Diameter (mm) 3.99–9.09 (15.38) 6.18±0.97^a 6.26–10.50 (11.30) 8.18±0.96^b 7.73–13.10 (17.20) 9.87±1.31^a 10.18–17.01 (23.14) 12.64±1.71^d 7.41–13.44 (18.89) 10.69±1.42

Length (mm) 4.55–9.09 (18.77) 6.57±1.11^a 6.99–10.17 (7.61) 8.39±0.80^b 8.08–11.79 (7.61) 10.31±0.99^c 9.62–17.85 (30.01) 12.94±1.97^d 7.29–15.63 (31.63) 9.99±1.78

Fresh weight (g) 0.06–0.31 (3.00) 0.13±0.06^a 0.10–0.41 (2.01) 0.23±0.07^b 0.23–0.51 (20.0.) 0.35±0.06^c 0.40–1.50 (12.15) 0.69±0.29^d 0.28–1.24 (7.58) 0.68±0.23

	Stage-1	Stage-2	Stage-3	Stage-4	All mature mixed fruits
Diameter (mm)	3.99–9.09 (15.38)	6.18±0.97^a	6.26–10.50 (11.30)	8.18±0.96^b	7.73–13.10 (17.20)	9.87±1.31^a	10.18–17.01 (23.14)	12.64±1.71^d	7.41–13.44 (18.89)	10.69±1.42
Length (mm)	4.55–9.09 (18.77)	6.57±1.11^a	6.99–10.17 (7.61)	8.39±0.80^b	8.08–11.79 (7.61)	10.31±0.99^c	9.62–17.85 (30.01)	12.94±1.97^d	7.29–15.63 (31.63)	9.99±1.78
Fresh weight (g)	0.06–0.31 (3.00)	0.13±0.06^a	0.10–0.41 (2.01)	0.23±0.07^b	0.23–0.51 (20.0.)	0.35±0.06^c	0.40–1.50 (12.15)	0.69±0.29^d	0.28–1.24 (7.58)	0.68±0.23

The range represents the minimum and minimum values in different genotypes; CV –Coefficient of variance calculated from 20 genotypes; SD-Standard deviation; Diameter, length, and fresh weight were calculated individual berry fruit samples; Different superscript letters after mean±SD in a row are significantly (p < 0.05) different from each other in ripening stage.

Previously, Staudt [13] described the shape of mature ovoid or depresses ovoid fruit as 9.4 mm (ranged between 5.5 to 16.5 mm) long and 13.3 mm (ranged between 7.0 to 17.5 mm) wide, which is in accordance with the current study. However, a greater variability among individual berry fruits collective from heterogeneous environmental conditions. In berries, shape and size are determined by the development and enlargement of individual achene, which is individually a true fruit. Thus, the development of shape and size is controlled by genetically programmed enlargement of the ovary in the pistil and the success rate of pollination [32]. Although berry of F. nubicola was observed smaller as compared to cultivated strawberry (F. ananassa) [33, 34], it might be an important fruit species of Himalaya to cope with the nutritional security of the region. Also, a high degree of variation was recorded in fruit traits, and any successful breeding program required larger genetic variation of important traits along with the expression of these traits in variable environmental conditions [3]. Heritability of diverse quality-related traits is an important aspect of the screening for the introduction of a species into the cultivation and further breeding program. Thus, there are possibilities of varietal improvement in F. nubicola, but it is also a good genetic resource for introducing new traits in cultivated strawberries due to the high hybridization capability in the Fragaria genus.

Likewise, the volume of berry fruits also increased significantly (p > 0.05) with ripening stages and was observed as 121.33 mm³ in Stage-1, 346.67 mm³ in Stage-2, 450.00 mm³ in Stage-3, and 1170.00 mm³ in Stage-4 (Table 2). Similarly, juice content of berries also increased from 37.33%(Stage-1) to 64.0%(Stage-4) with ripening. However, juice pH increased with ripening stages and reached 3.45 (Stage-1) to 3.85 (Stage-4). The increase in pH was sharp with initial stages but slowed in later stages of ripening. Pulp pomace content decreased with the ripening stage and was observed to reduce from 48.60%(Stage-1) to 30.44%(Stage-4). In contrary, moisture content recorded among ripening stages as 83.10%in Stage-1, 87.45%in Stage-2, 88.57%in Stage-3, and 90.35%in Stage-4. The change in acidity resembles the utilization of organic acids in the enhanced metabolic process and synthesis of other building blocks during the ripening process [35].

Table 2

Physico-chemical parameters in different ripening stages of Fragaria nubicola berries

Ripening Stage	Volume (mm³)	Juice (ml/100 g)	Juice pH	Pulp pomace content (%)	Moisture (%)
Stage-1	121.33±7.51^a	37.33±2.31^a	3.45±0.00^a	48.6.±1.72^d	83.1.±0.66^a
Stage-2	346.67±92.38^b	45.33±1.15^b	3.55±0.05^a	41.08±1.47^c	87.45±0.75^b
Stage-3	450.00±30.00^c	54.00±2.00^c	3.80±0.00^c	34.24±2.06^b	88.57±0.43^c
Stage-4	1170.00±112.69^d	61.33±3.06^d	3.85±0.05^c	30.44±1.69^a	90.35±0.42^d
All mix	583.33±88.20	64.00±3.27	3.75±0.07^c	30.75±2.5	89.33±0.77

Different superscript letters after mean±SD in a row are significantly (p < 0.05) different from each other in the ripening stage.

Increased juice and moisture content with maturity have directly associated with changes in structure configuration of pectin in the cell wall, which ultimately determined the physical properties of fruits. During ripening, cell enlargement is facilitated by increased solubilization of pectin polysaccharides, loss of natural sugars, and increase in polyuronides [15, 16]. However, in greenhouse-grown strawberry fruits (F. ananassa Duch, Cv. Albion) pH increased in six maturation stages from 3.39 to 3.80, and moisture content increased from 91.9 to 92.5%, respectively [20]. Interestingly, in cultivated strawberry (Fragaria×ananassa) pH of fruit juice initially decreased with the maturation of berries but started to increase after 21 days of fruit-set till fruit maturation and full ripening [14]. In Fragaria×ananassa, malic, shikimic, and ascorbic acids have been observed as major organic acids and reached their optimum level at 28 days after fruit set and then decreased gradually. It shows an inverse relationship with titratable acidity with pH in F. ananassa during fruit ripening [14]. Also, in different fruits parts of F. ananassa, external tissue pH was slightly lower than the internal tissue pH (3.46 and 3.61, respectively) [36].

3.2 Nutritive constituents

Anthocyanins, known as one of the most popularised secondary metabolites in berries of Fragaria species. In F. nubicola, level of total monomeric anthocyanin increased significantly (p < 0.05) with fruit development and recorded as 0.38 mg/g in Stage-1, 0.55 mg/g in Stage-2, 1.36 mg/g in Stage-3, and 1.60 mg/g in Stage-4. In the mixture of all mature berries, it was observed as 1.46 mg/g (Fig. 1). Generally, the accumulation of anthocyanin pigments in berries is the most important indicator of ripeness and fruit quality of strawberries [21]. These values of anthocyanin content in the present study were recorded comparatively higher than previously reported values in cultivated strawberries [14 , 37] as well as lower than few other reports on cultivated strawberries [38]. However, comparable content of anthocyanins was found in F. nubicola as in cultivated strawberries [39]. However, the accumulation of anthocyanin in berries of F. nubicola along the different ripening stages follows the same pattern as cultivated strawberry [14, 20].

Fig. 1

Anthocyanin content in different ripening stages of Fragaria nubicola berries.

Since anthocyanin pigment accumulation and stability have been affected by the pH of juice, it has been reported that in strawberry fruit (Fragaria×ananassa Duch.) citric and malic acid concentrations increased in the external tissues and internal tissues during ripening, which acts as a buffer solution for pH in the cells. pH above the threshold level (probably above pH 4) affected the stability of red flavylium cation by nucleophilic attacks by water molecules at 2 and 4 positions and formed colorless hemiacetal, or carbinol molecule [36]. Thus, in cultivated strawberries, pH has been considered as a major contributing factor for the stability of the anthocyanin pigment, and the pH change from 3.21 to 3.81 during ripening altered the flavylium form from 37 to 13%[40].∥Among other nutritive constituents, riboflavin (0.29 mg/g fw), β-carotene (0.05 mg/g fw), and total carotene (76.18 mg/g fw) were also quantified in a total mixture of the mature berries of the species (Table 3). Earlier, β-carotene content has been reported between 5.63–10.02 mg/100 g fw and total monomeric anthocyanin between 59.9 to 143.7 mg/100 g fw in fruits of 21 cultivars of strawberry fruit (Fragaria×ananassa Duch.) from India [41]. However, β-carotene and anthocyanins were not detected in another Himalayan species F. indica collected from Uttarakhand, India [26]. Overall, species can be considered as a better source of anthocyanin, β-carotene, and other nutrients for the nutritional requirements of consumers.

Table 3

Nutritive chemical constituents (mg/g) in Fragaria nubicola berries

Ripening Stage	All mixed samples
Monomeric anthocyanins	1.46±0.04
Riboflavin	0.29±0.01
β-carotene	0.05±0.02
Total carotene	76.18±3.64

3.3 Phenolics, flavonoids, flavonols, and tannin content

Phenolic constituents (total phenolic content, total flavonoids, total flavonols, and total tannins) are the diverse group of secondary metabolites and frequently present as bounded with structural molecules (cell membrane-associated molecules) or carbohydrates molecules in storage organelles to increase their in planta turnover, solubility, inter and/or intracellular transport and stability [42]. Thus, acid hydrolysis is applied to extract bounded phenolics [43]. Keeping this in view, free phenolic present in the juice, free phenolic (methanolic extract), and free and bounded phenolic (extractable in acidified methanolic solvent) were analyzed. Results indicated that extraction of different phenolics significantly improved (p < 0.05) with acidified methanolic solvent as compared to aqueous methanolic solvent in all the ripening stages (Table 4). These results revealed that the acid hydrolysis method led to the release of higher bound phenolics from the fruits of F. nubicola. Acid hydrolysis improved extraction of total phenolic content with 3 to 10%higher yield than methanolic extraction in different samples. Similarly, an increase in extraction was improved by 60%to 110%in total flavonoids and 27%to 115%by total flavonol content using acid hydrolysis in different ripening stages. Also, extraction of tannin was improved by 33%to 115%using acid hydrolysis in different ripening stages. Meyers et al. [44] reported that bound phenolics in strawberries account for approximately 5%of the total phenolic content, which is in accordance with the current study. In F. nubicola, the extractability of phenolics has been reported to be influenced by different solvents with different polarities [31].

Table 4
Phenolics (mg/g) extracted in methanol, acidified methanol and fruit juice among the different ripening stages of Fragaria nubicola berries

Ripening Stage Total phenolics Total flavonoids Total flavonols Total tannins

Methanolic Acidified methanolic Juice Methanolic Acidified methanolic Juice Methanolic Acidified methanolic Juice Methanolic Acidified methanolic Juice

Stage-1 8.19±0.06^d 8.47±0.14^c * 1.32±0.15^a 0.83±0.01^c 1.32±0.01^c * 3.49±0.73^a 2.32±0.01^d 2.95±0.04^a * 2.74±0.04^a 1.89±1.11^a * 3.98±0.01^c 2.48±0.05^d

Stage-2 7.96±0.01^c 8.18±0.03^b * 2.21±0.10^b 0.87±0.04^c 1.11±0.01^b * 4.58±1.37^b 2.41±0.01^c 3.16±0.00^b * 2.81±0.01^b 2.41±0.01^c * 3.60±.0.01^a 2.36±0.08^c

Stage-3 7.40±0.02^a 8.10±0.05^b * 2.40±0.06^c 0.62±0.01^b 1.30±0.01^c * 8.51±0.29^c 1.91±0.01^b 4.11±0.02^d * 3.60±0.02^c 2.18±0.01^b * 3.70±0.01^b 2.15±0.03^b

Stage-4 7.52±0.05^b 7.73±0.06^a * 2.81±0.15^d 0.57±0.01^a 0.92±0.01^a * 7.75±2.22^d 1.79±0.01^a 3.36±0.05^c * 3.83±0.05^d 2.45±0.01^c * 4.14±0.01^d 1.88±0.01^a

Ripening Stage	Total phenolics	Total flavonoids	Total flavonols	Total tannins
Stage-1	8.19±0.06^d	8.47±0.14^c *	1.32±0.15^a	0.83±0.01^c	1.32±0.01^c *	3.49±0.73^a	2.32±0.01^d	2.95±0.04^a *	2.74±0.04^a	1.89±1.11^a *	3.98±0.01^c	2.48±0.05^d
Stage-2	7.96±0.01^c	8.18±0.03^b *	2.21±0.10^b	0.87±0.04^c	1.11±0.01^b *	4.58±1.37^b	2.41±0.01^c	3.16±0.00^b *	2.81±0.01^b	2.41±0.01^c *	3.60±.0.01^a	2.36±0.08^c
Stage-3	7.40±0.02^a	8.10±0.05^b *	2.40±0.06^c	0.62±0.01^b	1.30±0.01^c *	8.51±0.29^c	1.91±0.01^b	4.11±0.02^d *	3.60±0.02^c	2.18±0.01^b *	3.70±0.01^b	2.15±0.03^b
Stage-4	7.52±0.05^b	7.73±0.06^a *	2.81±0.15^d	0.57±0.01^a	0.92±0.01^a *	7.75±2.22^d	1.79±0.01^a	3.36±0.05^c *	3.83±0.05^d	2.45±0.01^c *	4.14±0.01^d	1.88±0.01^a

Different superscript letters after mean±SD in the column are significantly (p < 0.05) different from each other in the ripening stage. ^* represented that values are significantly higher (p < 0.05) than other extraction methods.

Among the ripening stages, free phenolic content, and acidified methanol extractable (free and bounded) total phenolic content decreased with ripening stages, however, these values were found as 8.19 mg and 8.47 mg/g GAE, respectively in stage-1 and gradually decreased up to 7.52 mg and 7.73 mg/g GAE, respectively in stage-4. Similarly, methanol extractable (free) and acidified methanol extractable (free and bounded) total flavonoid and total flavonol contents also decreased with the ripening stage. Nevertheless, free, and acid extractable free and bounded tannin content increased with ripening stages of berries and recorded as 1.89 mg/g and 3.98 mg/g in stage-1 and 2.45 mg/g and 4.14 mg/g TAE, respectively in stage-4. Juice obtained from the squeezing of the berry fruits was also rich in total phenolic content (1.32 to 2.81 mg/g GAE), total flavonoids (3.49 to 8.51 mg/g QE), total flavonols (1.79 to 2.41 mg/g QE), and total tannin content (1.88 to 2.48 mg/g TAE). Interestingly, the content of these all phenolics increased in juice with the ripening of berries, except total tannin content, which showed a reverse pattern than others phenolics.∥Earlier, total phenolic content has been recorded as 3.08 mg/g GAE, total flavonoids as 2.63 mg/g QE, total flavonols as 1.80 mg/g QE, tannin contents as 1.42 mg/g TAE and total proanthocyanidin as 1.01 mg/g catechin equivalent in fresh berries of F. nubicola collected from Parvati valley of Himachal Pradesh, west Himalaya [31]. In comparison to various cultivars of F. ananassa, F. nubicola comprises a relatively higher amount of total phenolics, flavonoids, and anthocyanin content [45]. In Indian cultivation conditions, total phenolic content has been reported between 59.9 to 143.7 mg/100 g fw in 21 cultivars of F. ananassa. Likewise, total phenolic content has been reported between 1.9 mg/g to 5.7 mg/g GAE fw in 18 strawberry cultivars (1 wild, 3 commercial, and 14 non-commercial cultivars) and these values changed significantly among the corresponding years of plantation [46]. However, total phenolic content was found relatively higher (3.17 mg to 4.43 mg/g GAE) in six cultivars of strawberries (Kent, Elsanta, Selva, Elkat, Dukat and Senga Sengana) grown under commercial plantation with conventional farming practice in northwest Poland [47]. In Allstar and Chandler cultivars of strawberries, total phenolic content has been reported between 0.89 mg to 2.05 mg/g GAE under different cultivation systems [48]. Also, in Korona strawberries, agricultural practices such as fertilization, mulch color, early forcing, and planting date, and environmental factors like light and growing area were found major influencing factor for the accumulation of phenolic content and different phenolic compounds, although, these values remain below than the current study [49]. Thus, F. nubicola can be claimed as a rich source of phenolics and anthocyanins. Generally, species growing in wild habitats accumulated higher phenolic content, which is considered as plant defense molecules against herbivory, pathogen attack, plant to plant communication and interaction, allelopathy, and abiotic stresses (e.g., drought, chilling, and radiation) [50]. Thus, bringing the species into cultivation, it is required to maintain such beneficial traits along with the introduction of new growth and productivity-related traits.∥Earlier, a decreasing trend of total phenolic content and increasing trend of anthocyanin was recorded in fruits of cultivated strawberry along with the maturation (F. ananassa Duch, Cv. Albion) in greenhouse conditions [20]. Besides, decreasing pattern of phenolic accumulation with maturation has also been reported in Sweet Charlie and Camarosa varieties of strawberry [51]. Also, a similar pattern of changes in total phenolic content was recorded in ‘Selva’ strawberries during ripening [10]. These results of phenolics, flavonoids and anthocyanins are in correspondence with the trend shown by other Himalayan wild edible fruits i.e., Myrica esculenta, Berberis asiatica, Rubus ellipticus, Pyracantha crenulata, and Morus alba [24]. Thus, ripen fruits are suitable for obtaining more phytonutrients from these berries.

3.4 Composition of phenolic compounds

Phenolics are a group of many active natural compounds having health-promoting biological properties due to high antioxidant capacity, thus major phenolic compounds present in the species were also qualified in mature berry fruits in methanolic and acidified methanolic extract. In the current study, 8 phenolic compounds (i.e., gallic acid, catechin, chlorogenic acid, vanillic acid, caffeic acid, trans-cinnamic acid, naringin and rutin) were detected in the F. nubicola (Table 5). Among these, most of these phenolic acids have been already detected in the different commercial Fragaria (strawberry) cultivars [52, 53]. F. nubicola berries were dominated by gallic acid (19.33 mg/100g in free form and 13.81 mg/100g fw free and bounded form). It is reported that the dominant compound in strawberry, ellagitannins consist of glucose esterified with hexahydroxy-diphenic acid and gallic acid [19, 54]. In another study, Bhatt et al. [26] detected gallic acid (7.26 mg/100 g) and catechin as major phenolic compounds in Fragaria indica.

Table 5
Phenolic compounds extracted in methanol, acidified methanol in Fragaria nubicola berries

Phenolic compounds Methanolic extractable (free compounds) Acidified methanolic extractable (free and bounded compounds)

Gallic acid 19.33±3.80^* 13.81±0.89

Catechin 16.13±0.18 22.28±0.48^*

Chlorogenic acid 36.36±1.24 ND

Vanillic acid 28.14±1.85 66.56±5.15^*

Caffeic acid ND 13.26±0.04

trans-cinnamic acid ND 37.27±3.72

Naringin ND 24.10±4.72

Rutin ND 6.52±0.42

Phenolic compounds	Methanolic extractable (free compounds)	Acidified methanolic extractable (free and bounded compounds)
Gallic acid	19.33±3.80^*	13.81±0.89
Catechin	16.13±0.18	22.28±0.48^*
Chlorogenic acid	36.36±1.24	ND
Vanillic acid	28.14±1.85	66.56±5.15^*
Caffeic acid	ND	13.26±0.04
trans-cinnamic acid	ND	37.27±3.72
Naringin	ND	24.10±4.72
Rutin	ND	6.52±0.42

^* Represented that values are significantly higher (p < 0.05) than other extraction methods. ND – Not detected.

Results revealed that acidified methanolic solvent was able to extract higher content of phenolic compounds, except chlorogenic acid, which was only detected in methanolic extract (36.36 mg/100 g fw). Velde et al., [53] found gallic acid, catechin, rutin and ellagic acid in lower quantity in methanol extraction but the concentration of these phenolic compounds increased after acid hydrolysis along with additional detection of epicatechin, ferulic acid, quercetin, and kaempferol in Camarosa and Selva cultivars of strawberry. It has been reported that phenolic compounds present in strawberry can be classified into three different classes based on structural configuration, phenolic acids (caffeic, ferulic, p-coumaric, p-hydroxybenzoic, and ellagic acid); flavonols (quercetin, kaempferol); and (c) anthocyanins (cyanidin, pelargonidin, and pelargonidin derivatives). Among these, anthocyanins have been reported as a dominant group [55] and largely contribute to antioxidant activity and related health benefits of the strawberry fruits, such as anticancer, antidiabetic and aging progression [56 –59]. During the ripening, phenylalanine ammonia-lyase, the first enzyme involved in phenolic biosynthesis, and chalcone synthase, the first committed enzyme in flavonoid biosynthesis were found to increase significantly increasing light intensities in strawberry [60]. In recent transcriptome sequencing revealed that FvMYB10 has been recognized as a regulatory transcription factor for anthocyanin biosynthesis and its expression as influenced by light [61]. Thus, light can have an influencing factor for anthocyanin accumulation in F. nubicola. Also, the transformed anthocyanin gene in strawberries can alter enhanced content and resulting antioxidant and cytotoxic properties [62] and can be used for up-scaling the quality of the strawberries. The composition of phenolic acid and anthocyanin showed qualitative and quantitative variation among cultivars of cultivated strawberries [44]; thus, compositional variability in phenolics in F. nubicola can be a new dimension in altering the commercial strawberry phenolic profile during breeding programs.

3.5 Antioxidant activity

Antioxidant activity across different ripening stages in berries of F. nubicola extracted by methanolic and acidified methanolic extracts was measured by three different in vitro assays (Table 6). Among both the solvent extractions, the ABTS assay revealed 2 to 12%higher values of antioxidants in the acidified methanolic extract as compared to methanolic extract. Similarly, in the DPPH assay, it was an increase of 7 to 8 %in the acidified methanolic extract as compared to methanolic extract, However, with FRAP assay no significant difference was obtained in antioxidant activity in both the extraction solvents. Results also revealed a slight increase in antioxidant activity with ripening stages of berries with all three in vitro assays. Antioxidant activity measured by ABTS assay showed that in methanolic extract, antioxidant activity was constant in Stage-1 to stage-3 and increased significantly in stage-4, however, in acidified methanolic extract, the increase was not significant with the ripening stage of berries. With DPPH assay, antioxidant activity increased with ripening stages in both the extracts, however, this was constant in stage-3 and stage-4. However, with the FRAP assay unclear pattern was obtained among the ripening stage in methanolic extracts but antioxidant activity was decreased with ripening stages. In juice samples, an increasing pattern was obtained with the ripening of berries with all three in vitro assays.

Table 6
Antioxidant activity (mM AAE/100g fw) in different ripening stages of Fragaria nubicola berries

Ripening Stage ABTS DPPH FRAP

Methanolic Acidified methanolic Juice Methanolic Acidified methanolic Juice Methanolic Acidified methanolic Juice

Stage-1 11.30±0.22^a 12.65±0.09^b * 11.39±0.32^a 5.19±0.09^a 5.61±0.10^a * 1.30±0.01^a 3.67±0.08^c 3.79±0.05^c * 3.08±0.22^a

Stage-2 11.29±0.08^a 12.46±0.07^a * 11.61±0.21^b 5.67±0.01^b 6.09±0.01^b * 2.70±0.03^b 3.62±0.11^c 3.54±0.12^ab 3.11±0.12^a

Stage-3 11.31±0.09^a 12.61±0.07^b * 11.68±0.14^b 5.82±0.02^c 6.22±0.03^c * 3.12±0.07^c 3.32±0.06^a 3.57±0.12^b * 3.29±0.22^b

Stage-4 12.08±1.16^b 12.38±0.07^a * 12.22±0.22^c 5.84±0.01^c 6.26±0.03^c * 4.45±0.02^d 3.52±0.01^b 3.48±0.01^a 3.43±0.10^c

Ripening Stage	ABTS	DPPH	FRAP
Stage-1	11.30±0.22^a	12.65±0.09^b *	11.39±0.32^a	5.19±0.09^a	5.61±0.10^a *	1.30±0.01^a	3.67±0.08^c	3.79±0.05^c *	3.08±0.22^a
Stage-2	11.29±0.08^a	12.46±0.07^a *	11.61±0.21^b	5.67±0.01^b	6.09±0.01^b *	2.70±0.03^b	3.62±0.11^c	3.54±0.12^ab	3.11±0.12^a
Stage-3	11.31±0.09^a	12.61±0.07^b *	11.68±0.14^b	5.82±0.02^c	6.22±0.03^c *	3.12±0.07^c	3.32±0.06^a	3.57±0.12^b *	3.29±0.22^b
Stage-4	12.08±1.16^b	12.38±0.07^a *	12.22±0.22^c	5.84±0.01^c	6.26±0.03^c *	4.45±0.02^d	3.52±0.01^b	3.48±0.01^a	3.43±0.10^c

Different superscript letters after mean±SD in columns are significantly (p < 0.05) different from each other in the ripening stage. ^* represented that values are significantly higher (p < 0.05) than other extraction methods.

These values of antioxidant activity were comparatively higher than previously reported on the species collected from Himachal Pradesh of the Indian Himalayan Region using the same in vitro assays [31]. However, antioxidant activity in F. nubicola was comparable to values of 90 cultivars of Fragaria×ananassa Duch using DPPH and ABTS assay [29]. Previously, Olennikov et al. [22] found that antioxidant activity measured by the same in vitro assays of F. viridis fruits showed an increasing trend with 3 ripening stages using ABTS and FRAP assay but the decreasing trend with DPPH assay with fruit ripening [22]. However, it has been reported that in the Green Osogrande and Green Camino Real cultivar of strawberries, antioxidant activity increased with fruit ripening [18]. F. nubicola fruits have well recognition for antioxidants and fresh fruit juice (10 ml/kg, oral administration) introduced in induced ischemia-reperfusion in Albino Wistar rats improved the neurobehavioral parameters, such as motor performance, neurological status, grasping ability, forelimb strength improvement in balance and co-ordination with antioxidant enzymes, such as catalase superoxide dismutase [63]. Thus, the potential of these wild berries can be harnessed as nutraceuticals due to their health-promoting effects.

4 Conclusion

Strawberries are considered as a model plant for the study of the ripening process in non-climacteric plants, in which the ripening process is independent of biochemical changes regulated by ethylene synthesis and a high surge of respiration. The physicochemical and nutritional composition analysis in berries of Fragaria nubicola indicates significant importance of the species for nutraceutical development as well as opens an avenue for its domestication. Present research suggested that for harnessing the maximum potential of the species, ripening stages needs to be considered. Mostly, nutritional and physicochemical properties were recorded higher with the maturity of berries. For plant breeders, it is essential to enhance the level of anthocyanins and other bioactive compounds coupled with other fruit quality parameters (i.e., self-life, flavor, texture, and other sensory characters), and agronomic parameters (yield, cropping system, pathogen resistance, etc.). Nevertheless, considering free and bounded forms the total phenolics, flavonoids, and flavonols contents were increased with successive ripening stages of berries except for total tannin however, a reverse trend was observed for juice. Antioxidant activity of free and bound forms have shown an irregular trend however juice has shown an increase in antioxidant activity with ripening stages. This investigation allowed an opportunity to conduct further research on the use of beneficial traits such as better productivity, self-life, nutritive value, synchronized harvesting time, and many other quality-related fruit traits to the target species through breeding for bringing the species into cultivation.

Footnotes

Acknowledgments

We thank Director GBPNIHE for use of the facilities and encouragement. We are very grateful to an anonymous reviewer for key suggestions and grammatical improvement in the manuscript during the review process. POB is thankful to the Himalayan Fellowship program (mountain Division, GBP-NIHE) for a partial research grant for supporting the current work.

Funding

The authors report no funding.

Conflict of interest

The authors have no conflict of interest to report.

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