INTRODUCTION:
The use of plants containing bioactive flavonoids and vitamins as therapeutic agents has been published. Plants produce flavonoids as a result of interactions with their environment, increasing their resistance to predation, pest attacks, and bacterial infections1,2-4. Moreover, there are studies on the potential of Quercetin lipoidal nano systems for treating dermatitis, and on the chemopreventive potential of Quercetin for breast cancer by targeting signaling pathways5,6.
Also, concerning the effectiveness of Quercetin on Polymicrobial Biofilms on Catheters, and indications of quercetin's benefits for diseases associated with aging7,8. Quercetin is also anti-oxidative, anti-oxidative, anti-inflammatory, anti-proliferative, anti-carcinogenic, anti-diabetic, anti-viral, and is a natural chemotherapy agent9,10. Quercetin was found to be a key compound against inflammatory and apoptotic proteins in an in silico study. Researchers suggest investigating chemical entities for anti-inflammatory and anti-tethering properties based on the in silico testing of Quercetin against inflammatory and apoptotic proteins11.
Researchers have studied quercetin combined with vitamin C to be an anti-oxidant for various types of treatments, including anti-inflammatory, antiviral, and anti-cancer, as well as a supporting supplement for Covid-1912,13. As is known, these two components are found in many plants, including tomato plants. Tomatoes (Solanum lycopersicum L.) are rich in carotenoid compounds, polyphenols, and vitamin C, serving as antioxidants. As well as phenolic compounds (phenolic acids and flavonoids), carotenoids (lycopene, coenzyme Q-10, and beta-carotene), vitamins (ascorbic acid, vitamin A), and glycoalkaloids (tomatin). The most abundant carotenoid in tomatoes is lycopene, while the most abundant polyphenol is flavonoids. Tomatoes contain phenolic acids as well as flavonoids such as quercetin, routine, kaempferol, and naringenin. Tomatoes also grow in Tanah Karo, North Sumatra, Indonesia, and are an important functional food. Traditional uses of tomatoes include spices and juice14,15.
Research on determining the content of quercetin mixtures spectrophotometrically has been carried out on the Simultaneous Quantification of Quercetin and Gallic Acid in Neolamarckia cadamba16. In addition, spectrophotometric methods are often used to determine mixture levels after mathematical software has been developed for computers. Spectrophotometric methods have evolved rapidly for quantifying drug mixture concentrations containing chromophores. The integration of MATLAB software, along with computer technology and mathematical calculations, has improved the analysis of spectrophotometric data by enhancing efficiency, simplicity, speed, and cost-effectiveness. Multivariate calibration methods in MATLAB accurately determine drug mixture levels in pharmaceutical formulations. The UV spectrophotometric method using mean centering ratio spectra has been developed to eliminate the absorption contribution of reagents and matrices in mixtures. The Mean Centering Ratio Spectra (MCR) method was developed for the direct determination of two or three components in mixtures without prior separation steps or derivatization in drug analysis17,18.
Nowadays, An RP-HPLC method was employed to concurrently quantify rutin and quercetin in Morus alba L19. HPLC-Tandem mass is utilized to measure the levels of quercetin and vitamin C compounds individually20. Quercetin and its mixtures in Arjunarishta prepared by HPTLC Densitometry21, Beside it, HPLC with ultraviolet detection has been developed to determine ascorbic acid and rutin in pure form and pharmaceutical dosage forms simultaneously22. and quercetin and its mixtures are analyzed using capillary zone electrophoresis, and UHPLC-Tandem mass23. Currently, HPTLC technique is employed to detection and quantification of anti-oxidant marker24, Quercetin and its metabolites were determined in rat plasma using ultra-high performance liquid chromatography with tandem mass spectrometry detection25., and simultaneous quantitative analysis of quercetin and gallic acid in Leea indica by HPTLC26.
A spectrophotometer's applications in pharmaceutical analysis are frequent. Degradation products and other co-occurring components may interfere with direct UV absorption measurements. As a result, the concentration of the analyte and chemicals that are unrelated to the analyte are often represented by the measured absorption. Therefore, mean centering ratio spectra, or MCR, an application method of UV spectrophotometry, is used to carry it out27. Moreover, other publications suggest that UV spectrophotometry with mean centering of ratio spectra can reduce or eliminate interference, and other unnecessary absorptions28.
The application methods have principles and base ways of working in the use of spectrophotometric methods, as well as objectives validating these methods on the effect of matrix components found in tomatoes other than quercetin and ascorbic acid. The goal of this study is to evaluate quercetin and ascorbic acid simultaneously without any treatment in the preparation of the dried extract separation method by UV spectrophotometry using mean centering ratio spectra methods.
MATERIAL AND METHODS:
Instrument:
Quercetin and ascorbic acid were measured using a Shimadzu 1800 brand UV-visible spectrophotometer, UV-Probe 2.42 (Shimadzu) and MATLAB software (The Mathworks).
Materials:
Pharmaceutical grade of Ascorbic Acid p.a.(E.Merck Ltd) ,Quercetin (Sigma-Aldrich ) and Methanol p.a, Ethanol absolute, n Hexane p.a (E.Merck).
Tomato Extract Preparation:
The dried and ground tomatoes were weighed, macerated in 70% methanol for 72 hours, stirred occasionally, filtered, and the dregs re-macerated for 48 hours. After collecting the filtrate, it was evaporated at 40oC with a rotary evaporator (Buchi) until a concentrated extract was obtained. Various solvents with different polarities were used for the liquid extraction method to fractionate the extract. A mixture of equal amounts of methanol and n hexane was used to dissolve one gram of thick extract using a 100ml separating funnel, shaken until completely extracted so that a layer of methanol and a layer of n-hexane were formed. As these two compounds are soluble in methanol, the methanol layer was used as a sample1,28,
UV spectrophotometric using mean centering ratio spectra methods.
Experiment Procedure:
A simultaneous determination of quercetin and ascorbic acid in a tomato extract was conducted using MCR methods with a solvent methanol pro analysis at the maximum wavelength of 244nm for quercetin (concentration of 6.6mg/ml) and 222nm for ascorbic acid (concentration of 8.0mg/ml). As part of the MCR process, MATLAB software is used to calculate the mean centering value, and the measurement value is obtained using that value. To match the quercetin content ratio in the measurement, standard solutions of ascorbic acid are added to the assay preparation for tomato extract. Using ascorbic acid as a divisor would absorb the concentrations of the Lamber-Beer law in the area of 0.2 to 0.617,18.
Standard solution preparation:
A 100-ml volumetric flask was used to contain the standard mixture, consisting of 50 mg quercetin and 50 mg ascorbic acid powder. and dissolved in methanol, and the volume was adjusted to the marked line with a concentration of 500µg/mL respectively. To make a working solution with a concentration of 25µg/mL, 5 mL of each solution was transferred to a separate 100 mL volumetric flask and diluted in methanol to the mark line29.
Selection of wavelengths based on spectral characteristics:
Quercetin and ascorbic acid using ultraviolet spectrophotometry were determined within the 200-320 nm wavelength range for MCR measurements. The maximum absorption wavelengths for quercetin and ascorbic acid were subsequently determined through calculation29.
Process of constructing spectrum ratios.
Construction of the spectrum ratio: with a system of divisors, the spectrum (3-11ug/ml) of quercetin is divided by the spectrum of 9ug/mL ascorbic acid to make the first series of ratio spectra. Similarly, the spectrum of ascorbic acid (5 -13ug/mL) was divided by the spectrum of 8ug/mL quercetin to produce a second set of ratio spectra. MATLAB software (Mathworks) was used to generate ratio spectra for ascorbic acid and quercetin17,18.
The construction of calibration graphs for MCR
There were five concentrations of quercetin working solution pipetted each time: 3ug/mL, 5ug/mL, 6ug/mL, 8 ug/mL, and 9ug/mL. There were also five concentrations of ascorbic acid working olution pipetted each time: 5ug/mL, 7ug/ml, 9ug/mL, 11ug/mL, and 13 ug/ml.
The equation for the regression line was obtained by computing the mean centering value and amplitude from the first ratio of quercetin to ascorbic acid, measured at 244nm and 221nm, respectively. This calculation was performed using concentrations of quercetin and ascorbic acid30,31
Measurements of Validation parameter:
Validation measurements of the MCR caculation in UV spectrophotometry were done on the following parameters32,33
Linearity:
Ascorbic acid and quercetin working solution absorption spectra were measured at 221 and 244nm, respectively. Regression equations are used for each component to calculate amplitude values. As a general rule, the regression equation obtained has the following formula32,33.
Y= aX + b [1]
Notes: Y = Amplitude a = Slope X = Concentration (μg/mL) b = Constant.
Accuracy
Three repetitions of 80%, 100%, and 120% were used to measure accuracy. The analyte component of each range is 70%, and the standard component is 30%. The difference in results was compared with the theoretical concentration32,33. Then, substance levels are obtained as a comparison of the results obtained with the theoretical results:
Ca- Cb
% R=------------------- X 100 [2]
CC
Note: %R = Percentage of recovery, Ca = Concentration after addition, CC = Theoretical concentration before addition, and Cb = Concentration of the standard substances addition
Calculation of the relative standard deviation (RSD).
The RSD calculation is done using the regression equation using the following formula30,31.
SD
RSD %= ------------------ x 100 [3]
Where RSD = Relative standard deviation, SD = Standard deviation,
and = Average data.
Determination of Limit of detection (LOD) and limit of quantification (LOQ)
LOD and LOQ are calculated as follows31.
3.3 x SD 10 x SD
LOD = ---------------- LOQ =-----------
Slope Slope
Note: SD = Standard deviation, Slope = a (y = ax+b)
Preparation of quercetin and ascorbic acid mixture:
At measuring 50ml, enter 0.7ml of quercetin working solution and 1.1ml of ascorbic acid working solution, dilute with methanol until the line marks, and shake until homogeneous. So a solution was obtained containing 7.0µg/mL quercetin and 9.0µg/mL ascorbic acid. The absorbance was measured at a wavelength of 200–400nm29. The same method is used to create the ratio spectrum and MCR.
Simultaneously determining quercetin and ascorbic acid mixture in dried extract:
The MCR application was used to determine the levels of quercetin and vitamin C in tomato extract.
Weighed one gram of the thick tomato extract sample and put it in a 100ml volumetric flask, dissolved it with methanol, and homogenized it with a sonicator for 15 minutes. Each mL of solution contains 10mg of tomato extract. After the solution is filtered, approximately 10 ml of the first filtrate is discarded, and the next filtrate is collected. Take 1.7ml of the filtrate and pipette it into a volumetric flask of 50ml. Afterward, 200-400nm wavelengths were used to measure the absorption. To determine the absorption values of vitamin C and quercetin, a ratio spectrum was created, then mean-centered as in the working solution procedure, and the mean centering value obtained was entered into the regression equation obtained for the quercetin and vitamin C working solution17,18.
RESULTS AND DISCUSSION:
Extraction of quercetin and vitamin C from tomatoes:
The results of tomato maceration with methanol solvent for 120 hours were then evaporated at 40şC using a rotary evaporator (Buchi). Then it was dissolved in 100 mL of methanol, and N-hexane was added in the same volume ratio, and the resulting mixture was separated into two layers. The methanol layer was chosen as the sample. This methanol layer was evaporated at a temperature of 60şC until completely dry, so that 10.98 grams of dry extract were obtained1,28.
In the next stage, 50mg of the extract was dissolved in a 50-ml methanol solution. The 5 ml extract solution was pipetted and diluted into a 10 ml flask. then the absorption is measured at wavelengths ranging from 200 to 400 nm. The recorded results are illustrated in Figure 1 below.
Fig.1. Spectrum absorption a) Tomatoes extract 5 ug/ml and
b) Ascorbic acid an Quercetin Mixture
Figure 1 shows that the spectral profile of dried tomato extract is the same as that of the standard solution mixture of quercetin and ascorbic acid. However, the concentrations of these components in the spectrum are more pronounced in the tomato extract. Consequently, the tomato extract is confirmed to contain both quercetin and ascorbic acid15.
To quantify the levels of quercetin and ascorbic acid in the extract, the MCR method is recommended. The development of MCR from quercetin and ascorbic acid is based on mathematical equations and facilitates the determination of mixtures of two components simultaneously without requiring any isolation17,18.
Development of an absorption spectrum system for the MCR method:
The technique of utilizing the absorption of molecules in UV spectrophotometry has been applied to measure drug concentrations in analytical chemistry. This is particularly significant in the quest to develop more straightforward, rapid, and dependable analytical methods17,18. In the analysis of herbal extracts, this method also encounters a fundamental challenge due to the spectral proximity and overlap of quercetin and ascorbic acid in the UV range, posing difficulties in simultaneously determining the levels of both components17,18. Because the overlapping spectra of quercetin and ascorbic acid lead to complications, as illustrated in Figure 2.
Fig.2. Overlapping absorption spectrum of quercetin and ascorbic acid
Based on the information presented in Figure 2, the maximum absorption spectrum results for quercetin in the methanol solvent were identified as 244nm, while for ascorbic acid, they were identified as 222nm29. Figure 2 as created with a combination of the spectra of 6..6ug/ml quercetin and 8.0ug/ml ascorbic acid.
The illustration reveals that a significant portion of the absorption spectrum of ascorbic acid overlaps with quercetin. Consequently, the direct spectrophotometric method proves inadequate for determining a mixture of quercetin and ascorbic acid. In contrast to the direct spectrophotometric method, the MCR method proves effective in determining the mixture of quercetin and ascorbic acid17,18.
Construction of quercetin and ascorbic acid at absorption in spectrum:
Assessment of the absorption condition of working solutions containing quercetin and ascorbic acid in methanol solvent was done by measuring the absorption of working solutions at varying concentrations (3-9) ug/mL for quercetin and (5 – 13)ug/mL for ascorbic acid. Figure 3 displays the absorption spectra for various concentrations29.
Fig.3. Absorption spectra a) quercetin at various concentrations
b) Ascorbic acid at various concentrations
Referring to Figure 3, it is evident that the spectra of quercetin and ascorbic acid in Methanol solvent remain consistent despite variations in concentration. This indicates the stability of the solvent mixture when applied to quercetin and ascorbic acid solutions. Therefore, the measurements align with the acceptable use of solvents in spectrophotometric methods, as long as the solvent's impact is limited to altering the maximum wavelength of the drug component by no more than 3% of the literature-stated wavelength2.
A range of wavelengths between 200–300nm was used to measure the absorption spectra of quercetin working solutions. Divided by the normal spectrum of ascorbic acid, these spectra are referred to as the ratio spectrum, or MC. The absorption spectra of working solutions with different concentrations were recorded in the 200–280 nm wavelength range for the quantification of ascorbic acid. A MRC was constructed by dividing these spectra by the normal spectrum of quercetin, as shown in Figure 3. In Figure 4, the maximum wavelength is 244.0nm, and the amplitude of the signal at that wavelength is used to determine the concentration of ascorbic acid. SMCR spectrophotometry begins by constructing ratio spectra and determining divisor concentration. For Lambert Beer law compliance, the divisor concentration was selected from the concentration range17,18.
Ratio spectrum construction from MCR:
The MCR method was created taking into account previous research, using the central average of the obtained spectrum ratios. By adopting this approach, derivative steps can be eliminated, resulting in a better signal-to-noise ratio. Various wavelength ranges were experimented, and it was found that the most favorable results for quercetin and ascorbic acid were obtained, with using wavelength ranges of 200 - 300nm and 200 - 280nm, respectively. In addition, this study tested the effect of divider concentration on method selectivity. Various concentrations of both quercetin and ascorbic acid were experimented. That the utilization of different concentration ratios does not exert a noteworthy impact on the maximum wavelength specificity for identifying quercetin and ascorbic acid. As a result, the standard spectrum for each quercetin and ascorbic acid is employed as a reference point instead of concentration-based dividers17,18.
The tomato extract samples that will be determined are the respective contents of quercetin and ascorbic acid. In the compilation, spectrophotometric determination requires a dividing factor for quercetin of 75μg/mL, and for ascorbic acid of 40μg/mL. However, because the concentration of ascorbic acid measured was relatively small and was below the minimum absorption limit of 0.2, an additional amount of working solution was required so that the measurement met the requirements of Lambert Beer's law. Spectrum ratio analysis is carried out to reduce the influence of other substances17,18. Figure 4 illustrates the results obtained.
Fig.4. Ratio Absorption spectrum ratio a.) the ascorbic acid
with 75ug/ml quercetin as divisor b) quercetin with
40µg/mL ascorbic acid as divisor
Referring to Figure 4, it is evident that the spectra of quercetin and ascorbic acid in the methanol solvent remain consistent despite variations in concentration. This indicates the stability of the solvent mixture when applied to quercetin and ascorbic acid solutions. Therefore, the measurements align with the acceptable use of solvents in spectrophotometric methods, as long as the solvent's impact is limited to altering the maximum wavelength of the drug component by no more than 3% of the literature-stated wavelength17.
A range of wavelengths between 200–300nm was used to measure the absorption spectra of quercetin working solutions. Divided by the normal spectrum of ascorbic acid, these spectra are referred to as the ratio spectrum, or MC. The absorption spectra of working solutions with different concentrations were recorded in the 200–280 nm wavelength range for the quantification of ascorbic acid. A MRC was constructed by dividing these spectra by the normal spectrum of quercetin17, as shown in Figure 3.
In Figure 4, the maximum wavelength is 244.0nm, and the amplitude of the signal at that wavelength is used to determine the concentration of ascorbic acid. MCR spectrophotometry begins by constructing ratio spectra and determining the divisor concentration. For Lambert - Beer law compliance, the divisor concentration was selected from the concentration range17.
Fig.5: Spectrum MC a) Queretin b) Ascorbic Acid
According to figure 5, the amplitude spectrum for quercetin can be obtained at 206nm based on amplitude vs. concentration (3-11) ug/ml. Similarly, (5-13)g/ml of ascorbic acid has a wavelength of 240nm and a spectrum shown in figure 5b. A mean centering value and calibration curve can then be calculated.
Quercetin and ascorbic acid standard solution calibration curve30,31
The calibration curve in the MCR method is established by plotting a graph of concentrations against the MC value in Table 1, specifically the results obtained from MCR.
Table 1: MC values of the standard solution oquercetin at 206 nm
and ascorbic acid at 240 nm
|
No |
Concentration (ug/mL) |
MC Value |
No |
Concentration (ug/ml) |
MC Value |
|
1 |
0 |
0 |
1 |
0 |
0 |
|
2 |
3 |
0.7828 |
2 |
5 |
0.1457 |
|
3 |
5 |
1.0270 |
3 |
7 |
0.1943 |
|
4 |
7 |
1.4307 |
4 |
9 |
0.2428 |
|
5 |
9 |
1.7596 |
5 |
11 |
0.2991 |
|
6 |
11 |
2.0457 |
6 |
13 |
0.3738 |
|
a. Quercetin |
b.Ascorbic acid |
||||
The regression equations derived through the MCR method for quercetin and ascorbic acid are provided in Figure 5 below:
Fig. 6. Calibration curve of a) Quercetin and b) Ascorbic acid
The calibration of MC quercetin standard and ascorbic acid gives a linear regression equation shown figure 6a for the quercetin regression equation Y = 0.01787 X + 0.1402 with a R2 value of 0.9905. In addition figure 6b for ascorbic acid at Y = 0.028X + 0.9965 with R2 = 0.9965. The selection of these regression equations was based on their respective r values22,23 The calculated r value is compared with the table r value at the 99% confidence level with the degrees of freedom set at four. It is clear that the calculated r values for ascorbic acid and quercetin exceed the minimum limits of the corresponding table values. This suggests that the equations exhibit strong linearity as they approach a value R2 <-1 30. Therefore, the MCR method can be applied to determine the levels of quercetin and ascorbic acid in a mixture of quercetin and ascorbic acid.
Validation criteria test for MCR:
A validity test is carried out on the criteria of accuracy, linearity, continuance, LOD, and LOQ32,33, with the results shown below in Table 1.
Table 2. Validation test of MCR method from tomatoes fruit
|
Parameter |
Quercetin |
Vitamin C |
|
Accuracy (%) |
100.01 |
100.07 |
|
Precision(%) |
0.70 |
0.49 |
|
Linearity |
0.9905 |
0.9965 |
|
LOD (ug/ml) |
4.3508 |
1.0132 |
|
LOQ (ug/ml) |
13.1844 |
3.0702 |
The results indicate that quercetin and ascorbic acid achieved accuracy levels of 100.01% and 100.07%, respectively, meeting the specified accuracy criteria of 98-102% 20. The precision requirements, with RSD values of 0.70% for quercetin and 0.49% for ascorbic acid, were also satisfied, as they were below the threshold of <2%. Quercetin demonstrated LOD and LOQ values of 4.3508µg/mL and 13.1844µg/mL, while ascorbic acid exhibited values of 1.0132µg/mL and 3.0702µg/mL, indicating the method's capability for sensitive detection and quantification. In summary, the MCR method is affirmed to possess excellent exactness and correctness, as well as low detection and quantitation limits32,33.
Therefore, it can be affirmed that the MCR method has fulfilled the validation criteria for determining quercetin and ascorbic acid levels, considering parameters such as exactness, correctness, continuance, LOD, and LOQ20,21.
Determination of quercetin and ascorbic acid in tomatoes extract sample
The process begins by preparing a solution of tomato extract in methanol solvent and analyzing the absorption spectrum within the wavelength range of 200 to 400 nm. Next, this spectrum is divided by a dividing factor and produces a ratio spectrum for each component. The MATLAB application was used on the spectrum of the ratio of quercetin and ascorbic acid to obtain the MC value17,18. then calculated by the equation of regression30,31.
To establish the rate of sample, inputting the amplitude value at the highest peak into the regression equation is undertaken. The MC values of quercetin and ascorbic acid in tomato extract are presented in Table 2, while the statistical results for determining these compounds are detailed in Table 3.
Table 3. MC values of quercetin and ascorbic acid on tomatoes extract
|
Repetition |
MC values (Y) |
|
|
Quercetin |
Ascorbic Acid |
|
|
1. |
0.3579 |
0.0941 |
|
2. |
0.3594 |
0.0944 |
|
3. |
0.3525 |
0.0910 |
|
4. |
0.3722 |
0.0935 |
|
5. |
0.3626 |
0.0958 |
|
6. |
0.3614 |
0.0955 |
Table 4: Quercetin and Ascorbic acid content in 100 grams of fresh tomatoes
|
No |
Percentage content(%) |
|
|
Quercetin |
Ascorbic acid |
|
|
|
(0.47 ±0.03) % |
(1,31 ± 0.07) % |
Based on the results from the extraction of tomatoes (Figure 1) to the validation testing of the MCR method and determination of levels regarding the content of quercetin and ascorbic acid in tomatoes and Table 2 regarding the validation of the MCR method and the results of testing quercetin and ascorbic acid in tomatoes), it can be stated that the MCR method can be used for simultaneous determination of quercetin and ascorbic acid levels in tomatoes.
CONCLUSION:
The spectra of the mean centering ratio prove it is a rapid, reliable, and consistent quantitative spectrophotometric method. Its application in the analysis of quercetin and ascorbic acid in laboratory and pharmaceutical tomato extracts has yielded promising results. The determined levels of quercetin and ascorbic acid in the tomato extract were 2.0%/kg and 4.0%/kg, respectively. The exactness and correctness of the method were verified, demonstrating an average exactness of 100.01% for quercetin and 100.31% for ascorbic acid. Additionally, the relative standard deviation (RSD) values for quercetin and ascorbic acid were 0.03% and 0.06%, respectively.
ACKNOWLEDGEMENT:
The author expresses gratitude to the Chancellor of USU and the USU Research Institute for granting permission, providing financial support, and offering access to laboratory facilities for the 2023 talent scheme reputation research focused on the analysis of quercetin and ascorbic acid content in tomatoes.
The author affirms that there are no instances of conflicting interests.
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Received on 20.02.2024 Revised on 14.07.2024 Accepted on 16.10.2024 Published on 28.01.2025 Available online from February 27, 2025 Research J. Pharmacy and Technology. 2025;18(2):823-830. DOI: 10.52711/0974-360X.2025.00122 © RJPT All right reserved
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