|Year : 2019 | Volume
| Issue : 4 | Page : 155-162
Development and validation of spectrophotometric procedure for quantitative determination of flavonoid content used to control the quality of mixture herbal product
Olga Alekandrovna Smyslova1, Dmitry Olegovich Bokov2, Olga Georgievna Potanina3, Tatyana Mikhailovna Litvinova1, Irina Alexandrovna Samylina1
1 Institute of Pharmacy, Sechenov First Moscow State Medical University, Moscow, Russia
2 Institute of Pharmacy, Sechenov First Moscow State Medical University; Federal Research Center for Nutrition, Biotechnology and Food Safety, Moscow, Russia
3 Peoples' Friendship University of Russia (RUDN University), Moscow, Russia
|Date of Web Publication||1-Oct-2019|
Assoc. Prof. Dmitry Olegovich Bokov
Sechenov First Moscow State Medical University, 8 Trubetskaya Street, Bldg. 2, Moscow, 119991; Federal Research Center for Nutrition, Biotechnology and Food Safety, 2/14 Ustyinsky Pr., Moscow, 109240
Source of Support: None, Conflict of Interest: None
Species, known as mixture herbal products, are compositions of several types of crushed, sometimes whole, medicinal plant materials with additives; they are a widely used dosage form in the Russian Federation. A large range of species are produced at the pharmaceutical companies. In pharmacopoeial analysis, the most popular and widely used method for the determination of flavonoids, suitable for the standardization of species, is the method of differential spectrophotometry, based on the complexation of flavonoids with aluminum chloride. In accordance with modern requirements for the drugs production, the validation of analytical methods is a prerequisite for the creation of pharmacopoeial monographs projects regulating the quality of pharmaceutical substances of plant origin. Therefore, it is necessary to validate analytical methods for their intended use in evaluating the drug quality. This article discusses/presents the main stages of development and validation (by parameters: accuracy, precision, specificity, linearity) of the methodology for determining total flavonoid content using original species “Fitourol” as a model.
Keywords: Medicinal plant materials, mixture herbal products, pharmacopoeial analysis, species, spectrophotometric analysis method, total flavonoid content, validation
|How to cite this article:|
Smyslova OA, Bokov DO, Potanina OG, Litvinova TM, Samylina IA. Development and validation of spectrophotometric procedure for quantitative determination of flavonoid content used to control the quality of mixture herbal product. J Adv Pharm Technol Res 2019;10:155-62
|How to cite this URL:|
Smyslova OA, Bokov DO, Potanina OG, Litvinova TM, Samylina IA. Development and validation of spectrophotometric procedure for quantitative determination of flavonoid content used to control the quality of mixture herbal product. J Adv Pharm Technol Res [serial online] 2019 [cited 2022 Aug 15];10:155-62. Available from: https://www.japtr.org/text.asp?2019/10/4/155/268456
| Introduction|| |
Species, mixture herbal products (MHP) is one of the oldest dosage forms, which has retained its value to date due to a number of advantages. These include the presence of active ingredients in the raw material in almost native form, ease of preparation of aqueous extracts that have high affinity to the body due to the nature of the extractant and the natural origin of biologically active compounds (BAS), and low cost. Species under different names (teas, herbal mixtures, MHP, herbal drug mixtures, herbal collections, herb-herb combinations, etc.,) are included in many pharmacopoeias of the world and are still used in almost all traditional medical systems., In folk Russian medicine, MHP have always been one of the main dosage forms. MHP were included in all editions of the domestic pharmacopoeia of Russia.
According to the State Pharmacopoeia of the Russian Federation, IV edition, MHP are mixtures of two or more types of medicinal plant raw materials of various processing methods, possibly with the addition of substances of mineral, synthetic, plant, and animal origin. When standardizing one component herbal drug or multicomponent MHP, the quantitative content of one BAS group (essential oils, tannins, phenolic compounds, and flavonoids) or several BAS groups [Table 1] are assessed., Growing interest in the various biological activities of herbal flavonoids and phenolics outlines the need of determination their content in herbal products.,
|Table 1: Standardization of mixture herbal products (quantitative content of biologically active substances)|
Click here to view
The spectrophotometric method is the most popular and widely used quantitative method for the flavonoid determination in pharmacopoeial analysis; it is suitable for MHP standardization. The total flavonoid content (TFC) is calculated in terms of the prevailing component with or without the addition of (in rare cases) the complexing agent, aluminum chloride.,,, Together with chromatographic fingerprint analysis, TFC can be helpful for determining the identity, consistency, and stability of herbal drugs containing multiple botanicals. Spectrophotometry method has been widely used in the standardization of herbal substances for determination of various BAS groups; by itself, and also in conjunction with other methods, it allows to achieve acceptable results for standardization.,,,,
In accordance with modern requirements for the production of drugs, the validation of analytical methods is a prerequisite for their inclusion in the projects of pharmacopoeial monographs on pharmaceutical substances of plant origin. Therefore, it is necessary to validate analytical methods for their intended use in evaluating the quality of medicines. Evaluation of analytical methods by the validation parameters serves as the basis for the systematic approach formation to the choice of methods for assessing the content of a drug and increasing its standardization level. It cannot but affect the quality of regulatory documentation, and therefore, the quality of pharmaceutical products.,
Previously, we have developed a multicomponent plant collection “Fitourol,” consisting of medicinal plant materials: Cowberry leaves (Vaccinium vitis-idaea [L.]), horsetail herb (Equisetum arvense [L.]), burdock roots (Arctium lappa [L.]), dill fruits (Anethum graveolens [L.]), wormwood herb (Artemisia vulgaris [L.]). In the course of the conducted pharmacological studies, it was established that the water extract of the “Fitourol” collection has a diuretic, saluretic, antiinflammatory, antioxidant, nephroprotective, phosphorus-calcium normalizing metabolism, as well as a pronounced litholytic effect in experimental urolithiasis in laboratory animals caused by intragastric administration of sublethal doses of vitamin D2. The wide range of pharmacological activity of the “Fitourol” collection makes it a promising herbal remedy for use in urological practice.
The aim of this study is to consider the main development and validation stages of methods for determining the content of TFC on the example of the original collection “Fitourol” MHP. For this MHP, a technique was developed for quantifying TFC in terms of luteolin-7-glycoside by differential spectrophotometry with a complexing agent.
| Materials and Methods|| |
The development of a spectrophotometric method for the quantitative determination of TFC in MHP was carried out by Spectronic Helios Alfa ultraviolet-visible spectrophotometer (Thermo Electron Corporation, England) with 10 mm layer thickness cuvette. A standard sample (SS), luteolin-7-glycoside (CAS No. 5373-11-5, Sigma-Aldrich, USA) was used in the analysis. The MHP was prepared in laboratory conditions; its composition includes: Vaccinii vitis-idaeae folia 30%, Equiseti arvensis herba 30%, Arctii radices 15%, Anethi graveolentis fructus 15%, and A. vulgaris herba 10%.
In the present study, the information-analytical method and the system analysis method were used. In the course of the study, regulatory and technical documentation and validation guidelines for analytical methods were analyzed.
| Results and Discussion|| |
Development of method for quantitative determination of total flavonoid content in the mixture herbal products
The method of differential spectrophotometry, based on the complexation reaction with aluminum chloride (III), is used to determine the flavonoids. As a result, a bathochromic shift of the flavonoid absorption maximum occurs. This method is suitable for the analysis and standardization of MHP, because it allows to analyze the total content of glycosides and aglycones of flavonoids of various groups.
The choice of a SS for calculating TFC is based on one condition: The flavonoid standard with aluminum chloride (III) should have a similar curve and a close absorption maximum at the same wavelength, as the absorption maximum of MHP flavonoids with the same complexing agent. Luteolin-7-glycoside was chosen as SS; it has a similar curve and a close maximum absorption in the same area of 402 ± 5 nm with a maximum absorption of flavonoids in “Fitourol” MHP with aluminum chloride (III) [Figure 1].
|Figure 1: Absorption spectra of the complexes of “Fitourol” mixture herbal product flavonoids (1) and luteolin-7-glycoside standard sample solution (2) with aluminum chloride|
Click here to view
The influence of a number of parameters is studied to select the optimal conditions for the extraction of the total flavonoids from the MHP sample. These include the nature of the extractant, fineness level, the ratio of raw materials and extractant, time and number of extraction [Table 2].
|Table 2: Total flavonoid content in of the “Fitourol” mixture herbal products extracts depending on the extraction conditions|
Click here to view
Optimal extraction conditions were established experimentally. The results indicate that the optimal conditions for the analysis are: The extractant is 70% ethanol, raw materials: Extractant ratio is 1:10, particle size (fineness level) is 2 mm, double extraction for 60 min. It formed the basis for the quantitative determination method of TFC in terms of luteolin-7-glycoside in the “Fitourol” MHP. Further, the description of the method is given.
Quantitative determination of flavonoid content in terms of luteolin-7-glycoside
A sample of the raw material (analyzed MHP) is crushed to the size of particles passing through a sieve with 2 mm holes. Next, the MHP accurately weighed quantity about 10.0 g is taken, placed in a 200 ml flask, 50 ml of 70% ethanol is added. The flask with the sample in the alcohol-water mixture is heated in a boiling water bath under reflux from the moment of boiling for 1 h. After cooling, resulting extract is filtered through the filter paper into a volumetric flask (100 ml) to separate the raw material particles. The extraction is repeated with 50 ml of 70% ethanol for 1 h. After cooling, the alcohol-water extract is filtered into the same volumetric flask that was filled up to mark with 70% ethanol (solution A). 1.0 ml of solution A is placed in a 25 ml volumetric flask, 2 ml of 2% aluminum chloride (III) solution is added and flask is filled up to mark with 70% ethanol.
Preparation of the reference solution
1 ml of solution A is placed in a 25 ml volumetric flask, 0.1 ml of diluted acetic acid is added, and flask is filled up to mark with 95% ethanol (the solution should be freshly prepared).
The optical density of the obtained solutions was measured by a spectrophotometer after 40 min at a wavelength of 402 ± 5 nm in a cuvette with a layer thickness of 10 mm. The optical density of luteolin-7-glycoside SS solution is measured at the same time at a wavelength of 402 ± 5 nm.
Preparation of the luteolin-7-glycoside standard sample solution
1.0 ml of SS, luteolin-7-glycoside, is placed in a 25 ml flask, 2 ml of a 2% aluminum chloride (III) solution is added, and flask is filled up to mark with 70% ethanol. A solution containing 1.0 ml of SS luteolin-7-glycoside SS, 0.1 ml of diluted acetic acid and 95% ethyl alcohol up to 25 ml is used as a reference solution.
The TFC in the analyzed MHP in terms of luteolin-7-glycoside and absolutely dry raw materials in percent (X) is calculated by the formula:
D* – Optical density of the test solution;
D – Optical density of luteolin-7-glycoside SS solution;
M* – Mass of the MHP sample, g;
M – Mass of CO luteolin-7-glycoside SS, g;
W – Mass loss on drying of the MHP sample, %.
Preparation of luteolin-7-glycoside standard sample solution
A accurately weighed quantity of CO luteolin-7-glycoside SS (0.03 g) is placed in a 100 ml volumetric flask, dissolved in 50 ml of 70% ethanol and flask is filled up to mark with 70% ethanol, stirred.
Validation of methods for quantitative determination total flavonoid content in mixture herbal products
Validation assessment of the developed methodology is estimated by the criteria: Trueness, precision (repeatability and reproducibility), specificity, and linearity.
The trueness of the method is established by checking the method with model SS solutions. Model solutions are prepared in three concentrations with a SS content in % to the initial concentration of 80%, 100%, and 120%. The determination is carried out in triplicate for each concentration. The usability criterion is the average % recovery when checking solutions of 80%, 100%, 120% concentrations, that are corrected for 100% and its average value should be in the range of 98.0%–102.0%. Luteolin-7-glycoside SS solutions were used as model solutions. The initial concentration of luteolin-7-glycoside was 0.4 mg in 25 ml. According to our results, the % recovery ranged from 99.49% to 101.99%, and its average value was 99.93% [Table 3].
The testing of method repeatability is carried out in different days using a spectrophotometer by the same specialist on one MHP sample in six replications with 100% concentration of the active substance in the test solution. The assessment of repeatability is carried out by coefficient of variation (CV). The acceptance criterion is performed when the variation coefficient for six measurements is not more than 2%. The TFC in terms of luteolin-7-glycoside in “Fitourol” MHP was 0.43%. According to our results, CV is in the range from 1.22% to 1.26% [Table 4].
The reproducibility is established according to the results of analysis of one collection sample in two laboratories in six replications with 100% concentration of the active substance in the test solution. The acceptance criterion is the value of the variation coefficient for six measurements, which should be no more than 2%. The TFC in terms of luteolin-7-glycoside in the collection “Fitourol” was 0.43%. The variation coefficient value for six measurements was 1.35% [Table 5].
The determination of linearity is carried out at five different dilutions levels of the test solution in the range of at least 80%–100% of the analyte concentration in the test solution. The criterion for acceptability of linearity was the correlation coefficient value, it should be no <0.98, and the data set can be described by a straight line. Determination of linearity was performed at five concentrations levels of luteolin-7-glycoside SS. For the analysis, an accurately weighed quantity 0.02 g of luteolin-7-glycoside SS was weighed, placed in a 25 ml volumetric flask, 20 ml of 70% ethanol was added and flask was filled up to mark with 70% ethanol (solution A). Next, 1 ml; 2 ml; 3 ml; 4 ml; 5 ml of solution A were placed in five 25 ml volumetric flasks. To the solutions 2 ml of a 2% aluminum chloride (III) solution was added and flasks were filled up to mark with 70% ethanol, stirred. The optical density of each obtained solutions was measured by a spectrophotometer at a wavelength of 402 ± 5 nm in 10 mm layer thickness cuvette. In the experiment, the linearity of the method is observed in the concentration range of 0.32–1.6 mg%; it is confirmed by a high correlation coefficient (r = 0.999) [Table 6] and [Figure 2].
|Figure 2: Graph of optical density versus CO concentrations of luteolin-7-glycoside. OY - optical density, OX - concentration, mg%|
Click here to view
The specificity of the method is confirmed by MHP flavonoids' light-absorbance at a certain wavelength compared with SS. The acceptance criterion is the identity of the SS absorption spectrum of with aluminum chloride with the MHP absorption spectrum with the same complexing agent. According to the developed method of TFC quantitative determination, the colored complexes of “Fitourol” MHP and CO luteolin-7-glycoside SS were obtained. The absorption spectra of the obtained complexes were recorded by a spectrophotometer at the wavelength range from 350 to 460 nm. The presented absorption spectra [Figure 1] confirm the identity. Therefore, specificity is satisfactory. The method specificity is also proved by the method with additives. The test is carried out on one sample of collection in 3 concentrations with SS additives. The acceptance criterion is the value of the relative experimental error with additives, which must be within the random error of the method. The test was carried out on one sample of the “Fitourol” MHP in three concentrations with the addition of luteolin-7-glycoside SS. The relative error value of experiments with additives is within the random error of the method, which indicates the absence of a systematic error in TFC determination and the method specificity [Table 7] and [Table 8].
The quantitative determination method of the TFC in the “Fitourol” MHP in terms of luteolin-7-glycoside can be positively evaluated by parameters: Trueness, precision (repeatability and reproducibility), specificity, and linearity [Table 9].
|Table 9: The validation results of the quantitative determination method of total flavonoid content in the “Fitourol” mixture herbal products in terms of luteolin-7-glycoside|
Click here to view
| Conclusion|| |
Thus, the main stages of the development and validation of methods for the quantitative determination of the TFC in terms of luteolin-7-glycoside in the “Fitourol” MHP are considered. This algorithm can be used in the development of regulatory documentation for MHP, the main biologically active substances of which are flavonoids. The method, developed and validated according to this algorithm, can be used in setting standards and developing projects of Pharmacopoeia monographs to include the State pharmacopoeia of the Russian Federation of a subsequent edition.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
Yong EL, Wong SP, Shen P, Gong YH, Li J, Hong Y. Standardization and evaluation of botanical mixtures: Lessons from a traditional Chinese Herb, Epimedium
, with oestrogenic properties. Novartis Found Symp 2007;282:173-88.
Che CT, Wang ZJ, Chow MS, Lam CW. Herb-herb combination for therapeutic enhancement and advancement: Theory, practice and future perspectives. Molecules 2013;18:5125-41.
Kiseleva TL, Smirnova YA, Blinkov IL, Dronova MA, Tsvetaeva EV. Brief Encyclopedia of Modern Herbal Medicine with the Basics of Homeopathy: A Reference Book of a Practitioner. Moscow: Publishing House of the Professional Association of Naturotherapists; 2010.
Kaur S, Mondal P. Study of total phenolic and flavonoid content, antioxidant activity and antimicrobial properties of medicinal plants. J Microbiol Exp 2014;1:5.
Choudhary N, Sekhon BS. An overview of advances in the standardization of herbal drugs. J Pharm Educ Res 2011;2:55.
Atanassova M, Georgieva S, Ivancheva K. Total phenolic and total flavonoid contents, antioxidant capacity and biological contaminants in medicinal herbs. J Univ Chem Technol Metall 201;46:81-8.
Folashade O, Omoregie H, Ochogu P. Standardization of herbal medicines – A review. Int J Biodivers Conserv 2012;4:101-2.
Bagirova VL, Severtsev VA. Tinctures, Extracts, Elixirs and their Standardization. St. Petersburg: SpecLit; 2001.
Kurkin VA, Pravdivtseva OE. St. John's Wort: Results and Prospects of Creating Medicines. Samara: Sam SMU, Fort; 2008.
Khasanova SR, Potanin AP, Kudashkina NV. Development of methods for the quantitative determination of the amount of flavonoids in the plant collection “Cardiofit”. Bashk Chem J 2013;20:60-2.
Xie P, Chen S, Liang YZ, Wang X, Tian R, Upton R. Chromatographic fingerprint analysis – A rational approach for quality assessment of traditional Chinese herbal medicine. J Chromatogr A 2006;1112:171-80.
Galvão MA, Ferreira MR, Nunes BM, Santana AS, Randau KP, Soares LA. Validation of a spectrophotometric methodology for the quantification of polysaccharides from roots of Operculina macrocarpa
(jalapa). Rev Bras Farmacogn 2014;24:683-90.
Grubesić RJ, Vuković J, Kremer D, Vladimir-Knezević S. Spectrophotometric method for polyphenols analysis: Prevalidation and application on Plantago
L. species. J Pharm Biomed Anal 2005;39:837-42.
Aisha AF, Abu-Salah KM, Ismail Z, Majid AM. Determination of total xanthones in Garcinia mangostana
fruit rind extracts by ultraviolet (UV) spectrophotometry. J Med Plants Res 2013;7:29-35.
Bokov DO. Standardization of snowdrop (Galanthus
L.) herbal pharmaceutical substances by ultraviolet-spectrophotometry. Asian J Pharm Clin Res 2018;11:207-11.
da Silva LA, Pezzini BR, Soares L. Spectrophotometric determination of the total flavonoid content in Ocimum basilicum
L. (Lamiaceae) leaves. Pharmacogn Mag 2015;11:96-101.
Arzamastsev AP, Sadchikova NP, Kharitonov YY. Validation of analytical methods. Pharm 2006;4:3-6.
Nikam PH, Kareparamban J, Jadhav A, Kadam V. Future trends in standardization of herbal drugs. J Appl Pharm Sci 2012;2:38-44.
Smyslova OA, Markaryan AA, Evdokimova OV, Glazkova IU, Yaroshenko MA. Characteristics of the new comprehensive herbal medicine for the treatment and prevention of urolithiasis. Biol Med 2015;7:12-8.
Smyslova OA, Litvinova TM, Glazkova IU, Denisova MN, Ermakov DA, Suvorova II. Pharmacotherapeutic efficiency of the new complex urological herbal medicine in experimental urolithiasis. Int J Green Pharm 2017;11:730-6.
[Figure 1], [Figure 2]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6], [Table 7], [Table 8], [Table 9]