Comparative variability of nutrients, minerals, phenolics and anthocyanins with antioxidant potentials during fruit development stages in �ve Mulberry (Morus) cultivars

BACKGROUND: The versatility of mulberry from the Morus genus is growing among various suppliers and users globally. OBJECTIVES: To assess the variations in total sugar, acidity, minerals, phenolics and anthocyanins and antioxidant dynamics of three black mulberry ( Morrus serrata, M. nigra and M. macroura black) and two white mulberry cultivars ( M. alba and M. macroura white) during development stages. METHODS: Total sugars and titratable acidity were evaluated with AOAC methods and TPC was estimated with folin-ciocalteau method. The antioxidant activity was determined with DPPH assay, minerals were quantified with atomic absorption spectroscopy and anthocyanins were assessed with pH differential assay and identified with HPLC and LC-MS. RESULTS: Over all, M. alba (white cultivar) displayed maximum sugar (1.35%) at development stage 3 (FDS-3) followed by M. serrata (black cultivar) at FDS-5 (1.1%) and M. macroura white (white cultivar) at FDS-4 (0.9%). A decreasing trends in acidity in M. alba (0.8-0.09%), M. nigra (0.8-0.03%) and M. macroura white (0.6-0.04%) were observed during development. TPC was maximum (590 GAE/100g) in M. nigra at FDS-6 and M. serrata (450 GAE/100g) at FDS-7. Two black cultivars ( M. serrata and M. nigra


Introduction
Mulberry is the member of the Morus genus from Moraceae family [1,2].It is distributed widely from tropical to temperate climatic conditions.Morus is derived from "mora" a Latin word which means delay, possibly due to the slower growth of the mulberry buds.It is a woody plant with huge economic and medicinal significance because of numerous exceptional characteristics.M. nigra (black mulberry), Morus alba (white mulberry) and M. rubra (red mulberry) are all universally recognized species of Morus worldwide, but M. alba due to its versatility, is a prevailing species [1].The derivation the mostly cultivated mulberries are linked to some areas of China, Japan and the Himalayas foothills.According to an estimate, currently China is the leading country having largest mulberry production area (> 626, 000 ha), followed by India with nearly 280, 000 ha area [3,4].
Mulberry is manifold fruit having several small round shapes.The raw mulberry fruits have sour flavor with green color, while the ripe mulberry have anthocyanin pigments present giving it a distinctive sweet flavor, with purplish red to blackish color fruit [5].Mulberry fruit contains fatty acids, vitamins, amino acids, minerals and significant bioactive constituents like anthocyanins, chlorogenic acid, polysaccharides, quercetin and rutin [4] with greater levels of polyphenols such as carotenoids and flavonoids [6] that differs broadly among species [7,8].The contents of carbohydrates in mulberry fruit reported are from 3 to 30% with fructose and glucose as the major sugars [9].
In a lot of mulberry growing countries, the fruit is generally consumed both fresh and dried.In some occasions, the fruit is processed into juice, wine and jam as it gives very delightful taste, attractive color, high nutrients and low calories [10].In Pakistan, mulberries are native to the mountains of Himalaya and are also grown in the northern areas [1,11].The white (M.alba), red (M.rubra), and black mulberries (M.nigra) are broadly distributed in the north Pakistan, Iran and India where it is locally called toot or shahtoot (mulberry or King mulberry) which are Persian derived.In Pakistan, shahtoot principally referred to the white mulberry which is a hybrid species is appreciated for its delicious taste and consumed fresh and dried and employed for the preparation of juices, marmalades, natural dyes, liquors, and cosmetic products [12].
It is well-established that the fruits maturity stage of mulberry is very crucial indicator to portray its quality and nutritional attributes [13].For example, the fully ripened mulberry at final maturity stage have sweeter flavor as it possess five times greater sugars contrary to the fruit at semi-ripened stage [14].However, the semi to the fully ripened stages of mulberry are linked with vital biological activities like antioxidant, anti-aging, anti-atherosclerosis, antihyperglycemin, antiobesity, anti-tumor activities, immunomodulation attributes and neuro and cardio protective activities [15,16].Due to lower acidic content, the fully ripened mulberries are sweeter than the semi-ripened mulberries [17].The moisture, total crude protein, carbohydrate, ash, crude fiber, anthocyanins and phenolic contents are also different at the fully ripened stage of mulberry [16] associated to some important physiological, structural and biochemical alterations affecting principally the phytochemical and nutritional profiles of the fruit [18].It could also be due to some genetic factors, plants pre and postharvest and storage settings [19].
Although, mulberry fruits have been evaluated with attention only on the total nutrient contents yet, the assessment of periodical variations in minerals, nutrients, phenols, anthocyanins, and antioxidant activities in mulberry during development stages has not been recounted specifically from Pakistan.Consequently, here we trace the comparative variations in qualitative and quantitative aspects of macro and micro minerals, nutrients, phenolics and anthocyanins with antioxidant activities in five mulberry cultivars grown in Pakistan at different fruit development stages.

Mulberry fruits collection
Five cultivars representing four identified species of white and black Morus (Mulberry) viz., Morus nigra (black), M. alba (white), M. macroura (one black and one white) and M. serrata (black) were collected from Changa Manga and Gillian of Qasoor district, Punjab Pakistan.The fruits were harvested over the dry ice and brought to the Bioresource Utilization lab at the FBRC, PCSIR labs complex Lahore Pakistan and were sorted into different fruit development stages on the basis of their size and color (Fig. 1) and kept at -80 o C. The final fruit development stages of the 5 mulberry cultivars are shown in Figure 2 and the variations in physico-chemical characteristics during fruit development stages of 5 mulberry cultivars are provided in Table 1.

Total sugars and total titratable acidity (%)
Sugars were evaluated following the standard AOAC method no 943.03.For total sugars, 100 mg ground fruit was hydrolyzed in a boiling water bath with 2.5 N HCl and 4 ml anthrone reagent.The total sugar content was estimated at 620 nm (Shimadzu UV-1800, Japan) as glucose equivalent using standard glucose solution.
For total titratable acidity, titration of the fruit extract (10 ml) was done against 0.1 N NaOH and phenolphthalein was used as indicator.Acidity was estimated as mallic acid equivalent (0.0067g) using the following expression: (Eq.1)

Macro and micro minerals in mulberry cultivars
The content of macro and micro minerals in mulberry were calculated in accordance with the method described in AOAC method no 999.11.Briefly, 2 g mulberry fruit was ignited and transferred to muffle furnace at 550 ± 20 o C till white ash is obtained.Mineral contents of the fruit were determined by dissolving the obtained ash in 6 N HCl.The macro (Ca, Mg, Na, K and P) and micro minerals (Fe, Zn, Cu and Ni) were quantified with atomic absorption spectrophotometer (AAS) (Unicam 969, UK) [20,21].

Solvent extraction for phytochemical analysis
Solvent extraction was performed by contacting 10 g of ground mulberry fruit with methanol (10 ml) and centrifuged at 5, 000 rpm for 20 minutes.The obtained supernatant was shifted to the volumetric flask of 50 ml and using methanol the residual pellet was extracted three times and final volume was brought to 50 ml.

Total anthocyanin contents (TAC)
The quantification of total anthocyanins was done using the pH differential assay [24] and estimated on the basis of the following expression: The amount of anthocyanins was calculated from the molecular weight (MW=449.2g/mol) and molar extinction coefficient (Ɛ=26,900 M -1 cm -1 ) of cyanidin-3-glucoside and taken as mg of cyanidin-3-glucoside equivalents per 100 gram fresh weight.

Antioxidant activity of mulberry
Free radical scavenging capacity of fruit extracts of 5 mulberry cultivars was determined with DPPH • assay [25] and calculated using the following expression: ( ) (Eq. 4) where A s and A c are the sample absorbance and standard absorbance respectively.

Identification of anthocyanins in mulberry cultivars with HPLC
About 5.0 ± 0.9g of ground mulberry fruit extraction was done with 8mL solvent mixture of HPLC i. e. acetonemethanol-water-formic acid (40:40:20:0.1 v/v/v/v), followed by the addition of 200 mg Celite 545.The extract was vortexed for 2 minutes and filtered with Whatman No 4. Washing of the residue was done with 12 mL of extraction solvent followed by evaporation to dryness under vacuum using concentrator plus (Eppendorf, Germany).The residue was solubilized again in deionized water (10 mL). 1 mL portion of the extract was loaded onto Sep-Pak C18 column which was beforehand preconditioned with 2 mL methanol (100%) and deionized water (5 mL).Washing of column was done with deionized water (5 mL) to remove organic acids and sugars.The elution of anthocyanins was ensured from the column with 10 mL formic acid (0.1%) in methanol (v/v).Under vacuum, the eluent was evaporated to dry with concentrator plus (Eppendorf, Germany) and reconstituted in aqueous methanol (50%) for HLPC and LC-MS procedures.Anthocyanin identification in mulberry was performed with HPLC (PerkinElmer series 200) equipped with UV/VIS detector (PerkinElmer series 200) on Zorbax SB-C18 (4.6 x 250 mm) column with a flow rate of 0.5ml/min, at wavelength of 520 nm [20,21].

Identification of compounds with LC-MS
Molecular masses of identified mulberry peaks in HPLC were further evaluated using MS technique in a mass spectrophotometer LTQ XL (Thermo Fisher Scientific, USA) activated on +ve and -ve modes with atmospheric pressure electron spray ionization.1ml extract was examined at 10µl/sec flow rate with a mass scan range of 150 to 2000 amu.The equipment was calibrated with standard mass of cyaniding-3-Oglucoside for molecular characterization [20,21].

Statistical analysis
Data was analyzed statistically and standard deviation (SD±) was estimated in the Microsoft excel program.The data was further analyzed with one-way analysis of variance (ANOVA) to compare mean values.Differences at p < 0.05 were considered significant.
Overall, 2 white mulberry cultivars (M.alba and M. macroura white) and 1 black mulberry cultivar (M.serrate) displayed maximum sugar contents while 2 black mulberry cultivars (M.macroura black and M. nigra) showed lower sugar contents during fruit ripening stages.
The increased sugar contents at different development stages among white and black mulberry cultivars observed here could be correlated to the variations in sugar contents previously reported in other fruits like grape [26], date palm cultivars [27], guavava cultivars [28], peach and tomato [29], citrus fruit [30], sweet cherry and mulberry [31], strawberry cultivars [31,32], sunberry [33], peach and tomato [34], cherry tomato and peach [35], jamun [20], bilberry [38], litchi fruit [39], Kiwi fruit [40] and phalsa fruit [21].The total sugars content usually alter with stages of fruit development that reaches to maximum during ripening [20,21,35,36].Conversely, the accumulation patterns of sugars and levels vary between species [26, 27, 32, 35 37].For an instance, Gasim [27] validated a gradual accumulation of sugar content with variation among date palm cultivars during development stages.El Bulk et al. [28] found increase in contents of total sugar in the guava fruits during various fruit developmental stages.The maximum contents differ from 13.7 to 30.6 mg/100 ml of juice but the individual sugar contents were increased gradually with fruit maturation.The maximum variation in sugar level was 6.20 to 7.78, 1.90 to 8.00, and 5.64 to 7.67 mg/100 ml of juice for sucrose, glucose and fructose.
Kim et al. [32] reported increased sugars contents in 3 strawberry cultivars than 2 other cultivars during development stages.Dai et al. [35] also found an exponential uninterrupted increase in soluble sugar content in cherry tomato till maturity, but the concentration of soluble sugar in peach varied lower and decreased during development of fruit.In grape, also elevated soluble sugar levels of 1.2 mmol/g FM were reported during maturity [26], whereas peach and tomato have moderate (0.5 mmol/g FM) [29] and low (0.1 mmol/g FM) sugar levels [34].
In jamun and phalsa fruits, it was found that more sugars are synthesized at the final ripening stage that makes the fruit less sour and sweeter [20,21].Samkumar et al. [38] noticed elevated levels of sugars in ripe berries, with glucose and fructose dominating with low sucrose during fruit development.Trong et al. [39] found that the reducing sugars in initial stages of litchi fruit were comparatively low which reached to 2.138% in two weeks.In 8 to 10 weeks fruiting period, reducing sugars increased quickly and reached to 14.237%.Tao et al. [40] noticed that the content of sugar in Kiwi fruit altered to some extent during development of fruit before 160 days after full bloom, however the general trend of sugar was increasing that started growing fast after 160 days after full bloom, and reached to maximum during the stage of soft ripening.The variations in sugar contents in fruits could be due to the major hexoses like fructose and glucose originated from the degradation of sucrose, that are vital in the regulation and signaling during the fruit development [41].

Variation in total acidity (%) in mulberry cultivars
A decreasing trend was observed in total acidity over different stages of fruit ripening (FDS 1-7) in five mulberry cultivars (C1-C5) as shown in Figure 3B.Maximum decline was observed in M. alba (C4) from 0.8% at FDS-1 to 0.09% at FDS-4, followed by M. nigra (C2) with a decline in acidity from 0.8% at FDS-1 to 0.03% at FDS-7 and M. macroura white (C5) with a decline in acidity from 0.6% at FDS-2 to 0.04% at FDS-4.Minimum acidity of 0.05% was observed in M. serrata (C1) at FDS-2 with decline up to 0.01% at FDS-6 followed by M. macroura black (C3) with maximum acidity of 0.05% at FDS-2 and minimum 0.02% acidity at FDS-5.In general, two white mulberry cultivar (M.alba and M. macroura white) and 1 black mulberry cultivar (M.nigra) displayed maximum decline in acidity during fruit development stages.
The decreasing trend observed in total acidity in mulberry cultivars is in line with the outcomes of previous studies [20,21,[42][43][44][45][46][47][48][49][50][51][52][53] considering acidity as a crucial indicator for the determination of fruit quality [54].The levels of organic acids are usually inversely related to sugar levels during fruit development where sugars accumulate due to sugar import or starch degradation, while organic acids strongly decrease which accumulated in the young fruits [55].
Spayed and Morris [42] found that acidity in strawberry fruit increases generally in mature fruit before decreasing quickly in later ripening stages.Glew et al. [43] found that in medlar fruit, acidity first decreases in initial stages and increases in mid-ripe stages and then decreases in fully ripe stages.In peaches, Wu et al. [44] observed that organic acid increases at the intermediate maturity.In mango fruit, citric acid increase in the initial stages and then decrease in final stages [45].In the ripe wolfberry fruit, Zao et al. [48] found an initial rise and then decline in acidity in later stages.Acidity increases with fruit development in strawberry and then gradually decreases with maturity stages [47,50] and the same trend was reported in jamun cultivars [20] and phalsa [21].A decrease in contents of organic acid and acidity was also perceived during ripening of fruit in pears, peaches, apples [49], jujube cultivars [52] and ubajay fruit [53].Correspondingly during ripening of the non-climacteric lemon and orange fruits also showed a decrease in acidity that could be due to the citrate catabolism [46,51].In reality, the accumulation and metabolism of organic acids in fruits are under environmental as well as genetic control [56].

TPC variation in mulberry cultivars
Composition of TPC showed a significant increase with ripening in black mulberries whereas a very little increase was observed in white mulberry cultivars at different development stages (Fig. 3C).Maximum increase in TPC of 590 GAE/100g was observed in M. nigra (C2) at FDS-6 and 450 GAE/100g in M. serrata (C1) at FDS-7.Other cultivars including M. macroura black (C3), M. alba (C4) and M. macroura white (C5) showed minimum TPC (<200 GAE/100g) at all the developmental stages.These trends of increase in TPC recorded here in mulberry cultivars are in accordance with previous studies where increase in TPC was reported during various fruit development stages in Capsicum frutescens fruits [57], Rhus tripartitum (urcia) fruits [58], jamun cultivars [20], phalsa [21], Jujube fruit [59], Ribes stenocarpum [60], grape fruit varieties [61], navel orange [62], mulberry [63] and apple [64,65].The increase in phenols could be associated with low polyphenol oxidase content during fruit maturity [66] and also the fruit phenolic contents can be affected by many factors such as the variety, cultivation, area, ripeness, harvesting time, climatic conditions, storage time, and environment factors.However, in date palm [67], Ribes stenocarpum [60], apple [64], Prunus humilis [68] and plum [65], the total phenols trended down during development because of reduced synthesis of phenolic, or the continuous transformation of phenols into other constituents during development [69].Also the reduction of phenolic amounts may occur due to isomerization of certain compounds caused by high temperature [70].

Variation in total anthocyanin content in mulberry cultivars
Similarly, the anthocyanins content was increased with fruit ripening in M. serrata (C1), M. nigra (C2) and M. alba (C4).M. serrata and M. nigra showed significant increase in total anthocyanin contents with 14.57 to 100.06 CGE/100 g fresh weight (FW) and 14.13 to 201.87 CGE/100 g FW values from FDS-1 to FDS-7 respectively.
Whereas M. alba showed little increase from 6.51 to 6.90 CGE/100 g FW.However, a decrease in anthocyanin contents was observed in both M. macroura black (C3) and M. macroura white (C5) with 17.07 to 11.79 CGE/100 g FW and 9.03 to 2.68 CGE/100 g FW values from FDS-1 to FDS-7 respectively (Fig 3D).An increase in anthocyanin content shown here with increasing ripening stages is in agreement with the increase of anthocyanin contents in jamun [20], phalsa [21], mulberry [63], Berberis [71], blackberry [72], strawberry cultivars [32], jucara fruis [73], cheeries [74] and blueberry cultivars [75].It clearly indicates that the amount of anthocyanin content depends on the development of the mulberry fruit and the increasing concentration of anthocyanins in ripened fruits might be due to the upregulation of phenylpropanoid pathway and chalcone synthase enzyme, which are involved in anthocyanin biosynthesis [74].Also the anthocyanin accumulation is strongly regulated by development and genotype, and the environmental factors, associated to the altitude gradient, exert in the trial conditions only a finetuning influence [75].

Antioxidant activity of mulberry cultivars
DPPH radicals scavenging potentials of mulberry fruits represented as percentage inhibition are shown in Figure 3E.
Black mulberries like M. serrata (C1), M. nigra (C2) and M. macroura black (C3) showed increased antioxidant activity with almost 96% inhibition for M. macroura black (C3) at FDS-4, 80% for M. nigra (C2) at FDS-7 and 70% for M. serrata (C1) at FDS-6.Whereas the white mulberries M. alba (C4) and M. macroura white (C5) showed decreased antioxidant activity with inhibition of <20 % for both M. alba (C4) and M. macroura white (C5) at FDS-4.Over all among the mulberry cultivars, the radical scavenging abilities of three black mulberry cultivars (M. serrata, M. nigra and M. macroura black) were significantly higher whereas, antioxidant activities of white mulberry cultivars (M.macroura white and M. Alba) displayed lower antioxidant activities at all development stages.This increased antioxidant activity of black mulberry cultivars could be associated due to the increased TFC and anthocyanin contents in black cultivars as compared to the white ones.These results supports the outcomes of Saeed et al. [20,21], Yongram et al. [63] and Arfan et al. [76] where the jamun, phalsa and black/purple mulberry fruits during maturation also exhibited the strongest antioxidant activity in DPPH assay while the outcomes are not in agreement with the findings of Yan et al. [52] where the antioxidant activity in jujube fruit was found to decrease with increasing maturity.

Variation in mineral contents in mulberry cultivars
The data of nutritionally important macro (Ca, Mg, Na, K and P) and micro (Ni, Fe, Cu and Zn) minerals calculated in fruit developmental stages of all the mulberry cultivars is given in exhibited lower Ca during maturity (Fig. 4A).
Ca is a vital as well as critical component for human health as it prevents osteoporosis, which is a serious public health problem around the world [77].For older individuals, retaining Ca intake of at least 1000 to 1200 mg per day has been recommended to keep osteoporosis away [78].For this reason, Ca perceived here in mulberry cultivars is important and comparable to the Ca contents recorded for mulberry in previous inquiries [79][80][81] black and M. nigra) and 1 white cultivar (M.alba) displayed maximum Mg contents at maturity (Fig. 4B).
Mg is a significant mineral for the body to perform normal functions.Problems in the intracellular homeostasis of Mg may increase oxidative stress, alter the differentiation and proliferation mechanisms, affect the cell membrane modification and even cause cell apoptosis.Its insufficiency often causes inflammation that activates inflammation pathways and a rapid increase of pro inflammatory cytokines by the immune cells [83].The Mg contents observed here in five mulberry cultivars are comparable to the Mg contents previously reported [80,81] Liang et al. [80] reported relatively high amounts of Mg in mulberry with 202.22 mg/100g DW content.Sun et al.
[81] found the range of Mg among different varieties of mulberry fruits around 13.96 to 34.04 μg per gram respectively.

Sodium
A significant amount of Na with variations in levels were observed in all cultivars of mulberry where M. serreta (C1) at FDS-6 contained the highest 74.4 mg/100g DW Na content followed by M. nigra (C2) with Na content of 74.2 mg/100g DW at FDS-7.The minimum Na content displayed was 24.1 mg/100g DW for M. macroura black (C3) at FDS-5, 24.8 mg/100g DW for M. macroura white (C5) and 35.3 mg/100g DW for M. alba (C4) at FDS-4 respectively.Overall, the black mulberry cultivars (M.serrata and M. nigra) displayed maximum Na+ contents as compared to the white cultivars (M.alba and M. macroura white) during maturity (Fig. 4C).
Na is a most studied ion with potential repute due to its toxicity attributes [83] Na ions in the human body performs function in cellular transport of amino acids and glucose and energy production [84].It is also responsible for the maintenance of normal homeostasis of cell, regulation of electrolyte, solute balance and blood pressure.It is also vital for muscle and nerve cells and transport of substrates and nutrients across the plasma membrane [85].The values of Na perceived here are comparable to the Na content recorded for mulberry in the former studies [80,86].
Liang et al. [80] reported relatively high amounts of Na in mulberries with 41.65 to 143.36 mg/100g DW content and Koyuncu et al. [86] reported Na content in black mulberry with mean value of 277 mg/100g.
K ions are present in the body as abundant cations [87] that trigger many physiological functions.In plants, K is important for roots and leaves with multiple functions, for example; it activates certain enzymes, provides resistance against drought stress, controls turgor, water loss and wilting of leaves and triggers the opening of stomata.It increases the content of protein content and is vital during photosynthesis because it reduces respiration rate, maintains starch translocation and sugars [88].The results regarding K content obtained here in mulberry could be paralleled to the K contents recorded for mulberry in previous studies [79,80,86,89].Abd El-Malak et al. [79] found that K is the most abundant element in black, red and white mulberries with 1523.45, 1956.85 and 1979.35mg/kg contents.Liang et al. [80] 4E).As per the outcomes, the contents of P observed were higher in 1 black mulberry cultivar (M.serrata) whereas two black cultivars (M.nigra and M. macroura black) and two white cultivars (M.alba and M. macroura white) exhibited lower P contents at maturity.P is the necessary ubiquitous mineral act as a critical component of ATP production in the body [90] and compounds containing P such as IP3, cAMP and cGMP controls a number of intracellular signaling [91].The outcomes regarding the P contents shown in mulberry cultivars here could be correlated to the P contents previously reported in mulberry [80,89].Liang et al. [80] reported relatively high amounts of P in mulberry with 133.97 to 294.95 mg/100g DW content.Sánchez-Salcedo et al. [89] documented the range of P from 0.21 g/100 g in black mulberry to 0.31 g/100 g in white mulberry.

Iron
A nutritionally important amount of Fe was present in all the cultivars of mulberry where M. serrata (C1) displayed highest Fe value of 2183.35 µg/100g DW at FDS-6.M. nigra (C2) at FDS-7 and M. macroura black (C3) at FDS-5 showed average Fe contents of 1763.36 and 1615.95µg/100g DW respectively.Whereas M. alba (C4) and M. macroura white (C5) at FDS-4 displayed minimum Fe contents of 1473.43 and 1487.41 µg/100g DW.Over all, the black mulberry cultivars (M.serrata, M. nigra and M. macroura black) displayed maximum Fe contents as compared to the white mulberry cultivars (M.alba and M. macroura white) during maturity (Fig. 5A).
Fe is important for the functioning of enzymes and myoglobin [92].In the body, almost 30% Fe is stored as ferritin and hemosiderin in the bone marrow, spleen and liver.It is an important element for almost all living organisms as it triggers various metabolic processes like DNA synthesis, oxygen transport and electron transport [93].The Fe contents observed here in white and black mulberry cultivars could be paralleled to the Fe contents recorded in previous studies [79,80,81,89,94].Abd El-Malak et al. [79]  µg/100g DW at FDS-4.Over all, the Cu content was higher in black mulberry cultivars (M.serrata, M. nigra and M. macroura black) as compared to the white ones (M.alba and M. macroura white) during maturity (Fig. 5B).
Cu is a crucial micro-nutrient for plants and up to 5 to 30 mg kg −1 Cu is safe for normal functioning in plant tissues [96].The shortage of Cu causes neutropenia and anaemia, whereas the elevated level may cause liver and brain problems such as Alzheimer"s with nervous failure [96].
The results perceived here for Cu in white and black mulberry cultivars could be interrelated to the Cu contents recorded in previous studies on mulberry [79,80,81,94,97].Abd El-Malak et al. [79]  macroura white) during maturity (Fig. 5C).
Zn is the 2 nd abundant mineral in the human body with up to 2000 to 3000 milligrams that is about 90% in bones and muscles.Other body organs like the skin, brain, gastrointestinal tract, heart, lungs, the prostate; kidney, eyes, pancreas and adrenals also contain Zn in different amount.Zn proved its versatile functions in the immune system, keeping a healthy prostate, supporting healthy growth of cell, maintenance of levels of testosterone hormone, energy production, reproductive functions and protein synthesis enzymes [90,98].The Zn contents observed here in mulberry cultivars could be correlated to the Zn contents reported in the earlier investigations of several authors [79,80,81,94,97,99].Abd El-Malak et al. [79]  Such variations in the mineral contents in plants may be associated with fruit maturity and genotypes as well as some agricultural practices and ecological conditions like altitude, soil and climatic factors [100].

HPLC and LC/MS of anthocyanins in mulberry cultivars
The direct HPLC chromatograms of all mulberry cultivar extracts at their final stages are represented in Figure 6 (left) and up to five peaks (P1-P5) were detected by UV-Vis detector.Table 3 shows the MS data and possible inferences of mulberry extracts of each cultivar.According to the elution pattern of anthocyanins, P1 of each mulberry cultivar contained di glucosides and P2 and P3 contained mono glucosides of cyanidin in abundance while traces of cyanidin rutinoside and mono glucosides of malvidin, pelargonidin and delphinidin were also present (peaks P4 and P5).The right panel in Figure 4 shows the chromatograms of acid hydrolyzed extract.The chromatograms of mature mulberry fruit were compared with that of standards (cyanidin chloride, S1; pelargonidin chloride.S2; malvidin chloride, S3) where peaks 2, 3 and 4 were identified to be cyanidin, pelargonidin and malvidin, respectively while the peak 1(relatively less intense peak) was characterized as delphinidin according to the elution pattern and MS data (Figure 7).After acid hydrolysis, the anthocyanin profile had fewer peaks.
The chromatogram of C1 showed the presence of all four anthocyanins but the chromatograms of C2 generated three peaks with the absence of pelargonidin.Similarly, chromatogram of C3 also showed three peaks but peak 4 which confirms the absence of malvidin.The chromatograms of C3 and C4 produced two peaks (cyanidin and delphinidin) and one peak (cyanidin) respectively.Chromatograms of all cultivars confirmed cyanidin as the most abundant anthocyanidin in all mulberry cultivars extracts.Mass spectrum of each cultivar extract also confirmed the presence of anthocyanins in similar pattern (Table 4).Under acid hydrolysis conditions, anthocyanins are converted to corresponding anthocyanidins justifying lesser number of peaks in chromatograms of acid hydrolyzed extracts.
Delphinidin was an exception with decreased content suggesting its instability under acid hydrolysis conditions.
The presence and variations of anthocyanins like cyanidin, cyanidin rutinoside, mono glucosides of malvidin, pelargonidin and delphinidin shown here are in accordance with inquiries on mulberry [

Conclusions
The fruits of three black mulberries (M.serrata, M. nigra and M. macroura black) and two white mulberries (M. alba and M. macroura white) displayed varying quantitative nutrients, minerals and phytochemicals with antioxidant activities during fruit development.It was found that sugars in the mulberry cultivars continued to increase from the initial to final development stages where white mulberries displayed maximum increase in sugar as compared to the black ones.Total acidity was also decreased in all cultivars and TPC was higher in black mulberries while white mulberries had minimum TPC during development stages.Two black mulberries (M.serrata and M. nigra) showed significant increase in total anthocyanin but decreased in one black and one white mulberry.
An enhanced antioxidant activity of black mulberries was evident with elevated levels of Fe, Cu, Zn, Ca, Mg, Na, P and K as compared to the white mulberries during maturity.Cyanidin, mono glucosides of malvidin, cyanidin rutinoside, delphinidin and pelargonidin were the essential anthocyanins verified with HPLC and MS/MS procedures in mulberries during maturity that may be potential phyto-therapeutic agents for the prevention of diabetes and other metabolic syndromes.Convincingly, the selection of black and/or white mulberries with improved nutrition, acceptable range of metals, bioactive compounds and higher antioxidant potentials could be a significant natural cure against health related complications with lower risk of heavy metal toxicity.

Fig. 6 .Fig. 7 .
Fig. 6.HPLC analysis of five mulberry cultivars showing chromatograms of fully mature fruits with major peaks at 520nm

Table 2 and
Figures 4 & 5.The presence of micro and macro minerals was confirmed with significant variations in contents where only, Ni was not detected in any of the cultivars during maturity stage.
(C5) and M. alba (C4) at FDS-4 and M. serrata (C1) at FDS-6 presented lowest Ca contents of 18.5, 24.8, 70.6 and 93.0 mg/100g DW respectively.In general, 1 black cultivar (M.macroura black) displayed maximum Ca whereas 2 black cultivars (M.serrata and M. nigra) and the remaining 2 white cultivars (M.alba and M. macroura white) [81]d El-Malak et al.[79]found greater levels of Ca around 702.26 to 866.70 mg/kg in both red and black mulberries.Liang et al.[80]reported Ca in mulberry with 296.24 mg/100g DW content.Sun et al.[81]implemented a comparative study, where the range of Ca among different varieties of mulberry fruits recorded was 180.61 to 423.30 μg per gram respectively.
3.6.2.2.CopperAmount ofCu observed was varied among the cultivars where maximum Cu of 90.00 µg/100g DW was observed in M. macroura black (C3) at FDS-5 and minimum 72.38 µg/100g DW was noticed in M. macroura white (C5) at FDS-4.M. nigra (C2) at FDS-7 and M. serrata (C1) at FDS-6 showed intermediate Cu contents of 81.47 and 79.09 µg/100g DW.Two cultivars like M. alba (C4) and M. macroura white showed minimum Cu contents of 72.38 [94]rted 23.32mg/kg content of Cu in white mulberry.Liang et al.[80]observed Cu content from 0.162 to 0.680 mg/100g DW in mulberry cultivars with some exception.Randjelović et al.[97]reported Cu range from 1, 06 to 2, 586 ppm in mulberry fruit.Sun et al.[81]perceived 0.05 to 0.50 μg/g Cu contents in 13 different mulberry varieties and confirmed that the chemical and mineral profiling of mulberry are mutually the reason of variations in these constituents.Kalkisim et al.[94]documented the range of Cu in ripe mulberry from 2.05-109.25 µg g-1 in university campus and Siran regions of Turkey.Over all, the contents of Zn observed were also higher in black mulberry cultivars (M.serrata, M. nigra and M. macroura black) as compared to the white cultivars (M.alba and M. [94]rted highest content of Zn (47.42 mg/kg) in red mulberry as compared to the black mulberry.Liang et al.[80]observed Zn contents among different mulberry cultivars were in the range of 1.108 to 4.066 mg/100g DW.Pehluvan et al.[99]found the range of Zn in mulberry from 5.97 to 8.74 mg kg-1 from different distances of road side.Zn contents reported by Randjelović et al.[97]were in range from 18.65 to 55.37 ppm in leaves and 1.36 to 7.18 ppm in mulberry fruit.Sun et al.[81]documented the range of Zn in 13 mulberry varieties from 4.06 to 10.58 μg/g and Kalkisim et al.[94]recorded Zn contents in ripe mulberry from 24.71 to 730.94 in the industrial zone and Kelkit region of Turkey.

Table 3
Molecular mass characterization and identification anthocyanins present in mulberry cultivars