*2.3.3 Vitamins*

#### *2.3.3.1 Vitamin A*

Berry samples were ground for vitamin A (Retinol). Berry samples were extracted with a mixture of n-hexane and ethanol. 1% BHT was added and kept in the dark environment for 1 day. At the end of this period, centrifugation was conducted at 4000 rpm (+4°C) for 10 min. The obtained supernatant was filtered with the help of Whatman filter paper and added 0.5 mL of n-hexane. Drying was then performed using nitrogen gas. The residue in the tubes was dissolved in a methanol + tetrahydrofuran mixture. Analyses were carried out in Thermo Scientific Finnigan Surveyor

model high-performance liquid chromatography (HPLC) and in amber glass vials on Tray, and autosampler using PDA array detector [55, 56].

#### *2.3.3.2 Vitamin B*

A total of 10 g of samples were weighed and homogenized. The samples were then transferred to a conical flask with 25 mL of extraction solution. A shaking water bath at an ambient temperature of 70°C was used to sonicate the solution for 40 minutes. Following sonication, the sample was cooled and filtered to make a volume of 50 mL with extraction solution. The extraction solution was again filtered with filter trips (0.45 μm), and 20 μl aliquots solution was injected into the HPLC by using an auto-sampler. A reversed-phase C-18 analytical column (STR ODS-M, 150 mm 4.6 mm ID, 5 m, Shimadzu Corporation, Japan) separated the B complex vitamins. At 40°C, the mobile phase consists of a 9:1 (v/v) combination of 100 mM sodium phosphate buffer (pH: 2.2) containing 0.8 mM sodium-1-octane sulfonate and acetonitrile. The flow rate was constant at 0.8 mL/min using a PDA detector with a 270 nm absorption rate. The peak area of the corresponding chromatogram was used to calculate B vitamins using the following equation [57]:

> ( ) ( ) <sup>1</sup> B vitamins mg100 g Concentration of standard x Area of sample / Area of Standard x Dilution factor <sup>−</sup> =

#### *2.3.3.3 Vitamin C*

Plants were sliced, frozen in liquid nitrogen, and kept at a temperature of −80°C until the analyses were completed. The extraction solution was combined with 2.5 ml of frozen crushed plant material (3% MPA and 8% acetic acid for MPA-acetic acid extraction and 0.1% oxalic acid for oxalic acid extraction). The mixture was titrated with indophenol solution (25% DCIP and 21% NaHCO3 in water) until light, but the distinct rose-pink color appeared and persisted for more than 5 seconds [58].

#### **2.4 Experimental design and statistical analysis**

The study was designed according to the "Randomized Complete Blocks" with three replicates in 12 treatments. For each application and replicate, approximately 500 g of the berry samples were taken and analyzed for the compounds to be studied. Data obtained from the study were subjected to variance analysis using the SAS-based JMP statistical package programmer. The least significant difference (LSD) test was used to separate different groups at a 5% significance level.

#### **3. Results and discussions**

Besides bodywork, vitamins, and minerals, protection of the body from diseases, blood formation, bone, dental health, etc., are required for functions. Each food contains different amounts of various vitamins and minerals. Its richest sources are fresh vegetables and fruits [59].

As shown in **Table 3**, there were significant differences among the substrates related to macro- and microelements of berries except for boron. Considering, P, K, Ca, Mg, Mn, and Cu concentrations of berries were higher in Z + C (1:1) than the
