Gallic Acid Substance Appraisal in Daucus Carota Sp. Sativus and Genus Vitis by Conventional Withdrawal and High Performance Liquid Chromatography

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^OC.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

Figure 1: Structure of GA Click here to View figure

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 40^oC 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

Figure 2: Blank chromatogram. Click here to View figure

Figure 3: GA Standard chromatogram. Click here to View figure

Figure 4: Carrot sample Chromatogram. Click here to View figure

Figure 5: Grape Sample chromatogram. Click here to View figure

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

Figure 6: Linearity graph. Click here to View figure

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

1. 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.
   CrossRef
2. Iqbal,Z.;Saeed ,A.;Sabri,M.;Khan,M.A.; Zafar,A.;Naeem,S.^”Extraction of volatile components from the leaves of red carrot and its free radical scavenging activity^”, World Journal  of Pharmaceutical Research ,2021,10(11),10-25.
3. Cory,H.; Passarelli ,S.;Szeto,J.;Tamez , M.;Mattei,J. ^”The role of polyphenols in human health and food systems: A mini-review^”. Frontiers in nutrition^‘,2018,5,.87.
4. Jiang,Y.;Pei,J.; Zheng,Y.  ^”Gallic Acid:^‘A Potential Anti-Cancer Agent^”. Chin. J. Integr.Med. 2022,.28,661-671.
   CrossRef
5. Ashrafizadeh, M.;Zarrabi, A.; Mirzaei ,S.;Hashemi, F.;Samarghandian, S.;Zabolian,A.; Varma,R.S.^”Gallic acid for cancer therapy: Molecular mechanisms and boosting efficacy by nanoscopical delivery^”, Food and Chemical Toxicology,2021,157,112576.
   CrossRef
6. Toyama,M.H.;Rogero,A.;de Moraes,L.L.F.;Fernandes, G.A.;da Cruz Costa,C.R.; Belchor, M.N.; De Carli,A.M.;  de Oliveira,M.A. ^”Gallic Acid as a Non-Selective Inhibitor of α/β-Hydrolase Fold Enzymes Involved in the Inflammatory Process: The Two Sides of the Same Coin^”, Pharmaceutics,2022,14,368.
   CrossRef
7. Xu, Y.;Tang,G.;Zhang,C.;Wang,N.;Feng,Y. ^”Gallic acid and diabetes mellitus: its association with oxidative Stress^”, Molecules,  2021,26, .7115.
   CrossRef
8. Chen,D.;Bai,R.;Yong ,H.;Zong,S.;Jin,C.;Liu,J.^”Improving the digestive stability and prebiotic   effect of carboxymethyl chitosan by grafting with gallic acid: In vitro gastrointestinal digestion  and colonic fermentation evaluation^”,International Journal of Biological Macromolecules,2022,214,685-696.
9. Uddin,S.J.;Afroz,M.;.Zihad,S.N.;Rahman,M.S.;Akter,S.;Khan,I.N.;Sarker,S.D.^”A^‘systematic review on anti-diabetic and cardioprotective potential of gallic acid: a widespread dietary phytoconstituent^”, Food Reviews International, 2022, 38(4),420-439.
   CrossRef
10. Alfei,S.; Marengo, B.; Zuccari,G.^”Oxidative stress, antioxidant capabilities, and  bioavailability:ellagicacidorurolithins^”, Antioxidants,2020,1 9(8),707.
    CrossRef
11. Alfei ,F.;Kanev,K.;Hofmann,M.;Wu,H.E.;Ghoneim,P.;Roelli,D. ;Zehn.^”TOX reinforces the phenotype and longevity of exhausted T cells in chronic viral infection^”, Nature, 2019,571,265-269.
    CrossRef
12. Yang,K.;Zhang,L.; Liao ,P.; Xiao,Z.;Zhang,F.; Sindaye,D.;Xin,Z.;Tan ,C.;Deng,J.;Yin,Y.; Deng,B.^” Impact of Gallic Acid on Gut Health: Focus on the Gut Microbiome, Immune Response, and Mechanisms of Action^”, Front Immunol,2020,33042163.
    CrossRef
13. Sadef ,Y.;Javed ,T.;Javed,R.;Mahmood ,A.;Alwahibi ,M.S.;Elshikh ,M.S.; Rasheed,R.A. ^” Nutritional status, antioxidant activity and total phenolic content of different fruits and vegetables’ peels^”, Plos one,2022,17(5),1-5.
14. Bagheri,A.R.;M.Ghaedi,M.^”Application of Cu-based metal-organic framework (Cu-BDC) as a sorbent for dispersive solid-phase extraction of gallic acid from orange juice samples using HPLC-UV method^”,Arabian Journal of Chemistry,2020,13(5),5218-5228.
    CrossRef
15. Singh,M.P.;Gupta,A.;Sisodia,S.S. ^”Qualitative Analysis of Gallic Acid by HPLC Method In Different Extracts of Terminalia Bellerica Roxb. Fruit^”. FABAD Journal of Pharmaceutical Sciences,2019,44(2),101-106.
16. Ciniviz,M.;Yildiz,H.^”Determination of phenolic acid profiles by HPLC in lacto‐fermented fruits and vegetables (pickle): Effect of pulp and juice portions^”. Journal of Food Processing and Preservation,2020, 44(7),1.
17. Zang,Z.;Tang,S.;Li,Z.;Chou,S.;Shu,C.;Chen,Y.;Li,B. ^”An updated review on the stability of  anthocyanins regarding the interaction with food proteins and polysaccharides^”. Comprehensive Reviews in Food Science and Food Safety,2022,1,1.
18. Queiroz,M.F.;Sabry, D.A.;Sassaki,G.L.;Rocha,H.A.O.;Costa,L.S. ^”Gallic Acid-Dextran Conjugate: Green Synthesis of a Novel Antioxidant Molecule^”. Antioxidants (Basel).2019,8(10),478.
    CrossRef

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