Gallic acid Substance Appraisal in Daucus carota sp. sativus and genus Vitis by Conventional Withdrawal and High Performance Liquid Chromatography

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INTRODUCTION
Gallic acid (GA) is also known as (3, 4, 5-tri hydroxy benzoic acid) the formula is C7H605. GA is naturally occurring polyphenolic compound present in fruits, vegetables and edible parts of plants. GA is a white solid, soluble in water, alcohol and melt at 235-240 o C. GA is a secondary polyphenolic metabolite in various vegetables and fruits and is a bioactive component of natural antioxidants. GA bio efficacy impact has been the subject of many research papers and reviews to date. GA has many biological properties 1 which includes antioxidant 2-3 , anticancer property [4][5] , anti-inflammatory 6 , antidiabetic 7-8 , anti-ulcerogenic 9 . GA is also safe against disorders like radical oxygen species (ROS), hyper production, oxidative stress (OS) 10 and bacterial infections 11 . GA and its derivatives have the potential to be innovative therapeutic and preventative medicines for gastrointestinal illnesses 12 . GA is phenolic acid which is derived from shikimic acid, it is basically a secondary polyphenolic metabolite. Phenolic compound plays a crucial role in maintaining a healthy society. Many bad dietary habits nowadays lower food nutritional contents 13 , and an insufficient consumption of nutritious food causes a large dietary imbalance, which is a leading cause of chronic diseases. The synthesis and concentrations of GA were determined using HPLC [14][15][16] . Fig. 1 shows the chemical structure of GA. The interaction of GA with polysaccharide can boost its value as an antioxidant in the human being [17][18][19] . Keeping in view of a breeding interest in food based approaches for chronic disease prevention so the author reported the extraction and characterization of GA were determined in carrot and grape using HPLC-UV Method.
30 min in small portions of (1:20) Methanol and 1:1 aqueous Hydrochloric acid solution in an orbital shaking incubator for one hour.
The components were cooled, screened, and the solution was filtered had been dried in a rotary vacuum evaporator at 40 o C to obtain a dense concentrate sample, which was then stored for analysis.

Standard solution and test solution preparation
10mg of GA standard was taken in a 50.0 mL volumetric flask and 5 mL of methanol was added to it followed by sonication.

Column chromatography conditions
HPLC (Agilent Technologies, 1260) with Zodiac C18 column, 250mmx4.6mm, 5micrometer was used. The chromatographic conditions applied are Detector-UV, diluent-mobile phase, 10micro liter-injection volume & wavelength-280nm, Chromatogram run time:20 min, Column and temperature cooling 35 o C and 10 o C, Isocratic pump mode and 1.0 mL/min flow rate , Methanol and water in a 700:300v/v ratio with 1.0 mL Formic acid was selected as mobile phase.

RESULTS AND DISCUSSIONS
The secondary polyphenolic metabolite GA is used to reduce ulcer, inflammatory disease, blood glucose levels, viral infections, and so on. Fruits and vegetables have a high GA content which is essential to cure diseases. Selected mobile phase with 1.0 milliliters of 1% formic acid, mix 700:300v/v methanol and water were used. A perfect baseline analyte resolution is shown in Fig. 2-5 show chromatograms of GA. Table 1 shows the GA content of selected grape and carrot samples.

Plant materials
Cultivators bought nutritious fresh fruits and vegetables (grape and carrot) from the local market in Andhra Pradesh, India. For storage of all the specified fruits and vegetables, clean and dried glass containers were utilized, and they were left at room temperature. Each kilogram of chosen fruit and vegetable substance acquired from cultivators was left to dry and scrubbed.

Preparation of sample and standard solution
The preselected materials had been slashed into small pieces or bits and positioned on a spotless filter paper. A potable grinding machine is used to finely ground the plant material. After drying, 100 g of each sample was soaked for

Method validation
The method validation study was performed by the guidelines from ICH and Association of Official Analytical Chemists.Validation parameters like LOD and LOQ, Precision at LOQ level, System suitability, Specificity, Accuracy and Linearity were studied. The standard solution of 2.5 mL is poured into a volumetric flask of 10 mL and filled it up to the mark with methanol for determining LOQ and for LOD determination 3.3 mL solution into 10 mL volumetric flasks of 10 mL and dilute with methanol to the volume. The areas of LOQ and LOD standards were presented in Table 2 and six replicates were injected to study the system suitability. In the given Table 3, %RSD value of the area (peak) of all analytes were obtained less than 2.0%. All analytes' area (peak) %RSD values were less than 2.0 percent, as shown in Table 3.

Specificity
Specificity is an important validation parameter and it discloses that the strategy is capable for tenacity of the analyte(s).
Procedure: 10 mg of GA standard was accurately weighed and diluted to the 100 mL volume with diluent. 1.0 mL of prepared solution was transferred into a volumetric flask of 50.0 mL and Sonicate to dissolve 1.0 mL of the prepared solution after being added approximately 5 mL of diluent and diluted to the volume with diluent in a 10.0 mL volumetric flask, labeled as stock Standard. Further 1.0 mL of this solution was taken into 20.0 mL volumetric flask and diluted until the desired volume was reached. The volume with diluent, labeled as standard solution.

Linearity
A linear response was observed from mode of detection and with reference to concentrations over the range of concentrations of the standard material(10ppm) uncovers linearity by planning in the reach 25%-200% concentration of Impurities. Injected all five dilutions of linearity solution (25%,50%,100%,150% and 200%) followed by blank. Record the peak area of product. Tables 4 provided linearity areas, and Fig. 6 displays a linearity graph.

Accuracy
To demonstrate the accuracy for GA impurities, recovery is performed by taking solutions(in the 50 percent, 100 percent, and 200 percent) of the proposed active concentration final product. The GA mean value was calculated and reported after each level was performed in triplicate. The LOQ level, RSD percentage, was less than 15%. In this parameter, the acceptance criteria for GA impurities recovery rates are between 80.0% and 120.0% for each concentration level. Accuracy and recovery results were and shown in Table 5 & 6.

CONCLUSION
The GA substance in fruits and vegetables varies according to cultivators, plant parts, and geographical regions.The GA substance in fruits and vegetables was studied by conventional extraction using methanol as solvent and later by using HPLC technique. Chromatographic conditions such as C18 column (250mmx4.6mm, 5µm), detection at 280nm, flow rate 1mL/min etc were applied for the determination of GA. The LOD and LOQ parameters were in the ranges of 0.11-0.9 and 0.9-3.0 µg/mL, respectively. The reported method had advantages like simple, fast, and accurate.