記住我
N.Swathi
and N.V.S.Venugopal*
Department of Chemistry, GITAM School of Science, GITAM (Deemed to be University), Visakhapatnam-530045, A.P, India.
Corresponding Author E-mail: vnutulap@gitam.edu
Article Publishing History
Article Received on : 16 Dec 2022
Article Accepted on :
Article Published : 22 Mar 2023
Gallic acid(GA) contains three hydroxy groups, which are catalyzed by enzymes during the methylation or sulphation reactions. The present research portrays GA substance appraisal in a root vegetable Carrot (Daucus carota sp. sativus) and botanically a berry like grapes (genus Vitis). Conventional withdrawal process was performed by using methanol as solvent followed by maceration and distillation process. The study sought to quantify and compare the contribution of GA in Carrot and grapes for the nutritional care of society. The determination of GA content was quantified using HPLC (High performance liquid chromatography) method with. Zodiac C18(250mmx 4.6mm, 5µm) column. The other chromatographic conditions applied are detection at 280nm and flow rate 1mL/min. The detection limit (0.11–0.9 μg/mL) and quantification limit (0.9–3.0 μg/mL) were obtained. The reported method was validated and has the advantage of being fast, simple and accurate.
KEYWORDS:Characterization; Daucus carota sp. sativus; Extraction; Gallic acid; genus Vitis; High performance liquid chromatography
Download this article as:
Copy the following to cite this article:Swathi N, Venugopal N. V. S. Gallic Acid Substance Appraisal in Daucus Carota Sp. Sativus and Genus Vitis by Conventional Withdrawal and High Performance Liquid Chromatography. Orient J Chem 2023;39(2).
Swathi N, Venugopal N. V. S. Gallic Acid Substance Appraisal in Daucus Carota Sp. Sativus and Genus Vitis by Conventional Withdrawal and High Performance Liquid Chromatography. Orient J Chem 2023;39(2). Available from: https://bit.ly/3lANnzq
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 235-240OC.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 [14-16]. Figure.1 shows the chemical structure of GA. The interaction of GA with polysaccharide can boost its value as an antioxidant in the human being [17-19]. 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.
Chemicals and reagents
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, 100g of each sample was soaked for 30 minutes 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 40oC 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, 250mm x 4.6mm, 5micrometer was used. The chromatographic conditions applied are Detector- UV ,diluent- mobile phase, 10micro liter-injection volume & wavelength-280nm, Chromatogram run time:20 minutes, Column and temperature cooling 35 ºC and 10ºC, Isocratic pump mode and 1.0 mL/min flow rate , Methanol and water in a 700:300 v/v ratio with 1.0ml 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 700:300 v/v methanol and water were used. A perfect baseline analyte resolution is shown in Figures 2 – 5 show chromatograms of GA. Table 1 shows the GA content of selected grape and carrot samples.
Table 1: GA in grape and Carrot
Fruit/Vegetable
S1
S2
S3
S4
S5
Grape white(mg/100g)
0.281
0.361
0.279
0.452
0.618
Carrot root vegetable(mg/100g)
0.658
0.698
0.714
0.874
0.657
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 3.3mL solution into 10 mL volumetric flasks of 10ml 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%.
Table 2: Standard solution of LOQ and LOD (Area)
Standard solution
First
second
third
fourth
Fifth
sixth
Average
SD
RSD%
Gallic acid
232.72
232.68
232.85
233.48
233.34
240.8
234.31
3.20
1.36
Area of LOQ solution
First
second
third
Fourth
fifth
sixth
Average
SD
RSD%
Gallic acid
74.77
73.96
74.62
74.62
74.30
73.77
74.34
0.40
0.54
Area of LOD solutions
Average
SD
RSD%
First
second
third
Gallic acid
20.99
20.96
20.98
20.98
0.02
0.07
RSD: relative standard deviation
SD: Standard deviation
All analytes’ area (peak) % RSD values were less than 2.0 percent, as shown in table 3.
Table 3: Percentage of RSD
Name
Standard solution
First
second
third
fourth
Fifth
Sixth
Average
SD
RSD%
Gallic acid
242.05
241.53
239.67
239.65
237.09
238.43
239.74
1.86
0.78
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 100mLvolume 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 figure 6 displays a linearity graph.
Table 4: Linearity
S.No
Injection Id
Areas of linearity
Percentage
Gallic acid
1
S1
25
75.63
2
S2
50
114.25
3
S3
100
234.72
4
S4
150
347.02
5
S5
200
478.48
Correlation coefficient: 0.9985
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.
Table 5: Accuracy.
LOQ
First
second
third
Average
SD
%RSD
69.94
73.62
77.84
73.80
3.95
5.36
Accuracy at 50%
115.28
119.05
120.89
118.41
2.86
2.42
Accuracy at 100%
241.18
241.51
234.51
239.07
3.95
1.65
Accuracy at 200%
453.25
484.76
475.37
471.13
16.18
3.43
Table 6: Recovery Percentage
Name
LOQ
Accuracy at 50%
100%
200%
Result
Result
Result
Result
Gallic acid
107.84
103.21
107.03
100.41
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( 250mmx 4.6mm, 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.
Acknowledgement
All authors are thankful to the management, Department of chemistry, School of science, GITAM (Deemed to be University) Visakhapatnam, Andhra Pradesh, India for their support and encouragement.
Conflict of Interest
There are no conflict of interest.
References
Szczepańska ,J.; Barba,F.J.;Skąpska,S.;Marszałek, K. ”High pressure processing of carrot juice: Effect of static and multi-pulsed pressure on the polyphenolic profile, oxidoreductases activity and colour”, Food Chemistry,2020, 307, 125549.
This work is licensed under a Creative Commons Attribution 4.0 International License.
Related Posts
留言 (0)