Editorial Article

Differential Spectrophotometry: Application for Quantification of Flavonoids in Herbal Drugs and Nutraceuticals

Roman Lysiuk*, Nataliia Hudz
Danylo Halytsky Lviv National Medical University, Ukraine
*Corresponding author:

Roman Lysiuk, Danylo Halytsky Lviv National Medical University, Ukraine, Tel: +38-093-65-888-53, Email: pharmacognosy.org.ua@ukr.net

The study of flavonoids is a very popular area of research on natural pharmaceutical materials. These valuable individual active principles of phenolic nature deserve a particular attention due to various pharmacological effects and are of significant importance for standardization of herbal drugs and nutraceuticals [1-6].

Flavonoids constitute one of the most distributed groups of all plant phenolics. Over 8,000 varieties of flavonoids have been identified [5]. A common part in the chemical structure of all flavonoids is carbon skeleton based on flavan system (C6-C3-C6) [2]. Flavonoids are mainly accumulated in the edible parts of plants, particularly in fruits and vegetables [2] and are ubiquitous in plant foods and drinks and, therefore, a significant quantity is consumed in daily diet [5].

Flavonoids are the major active nutraceutical ingredients in plants [5]. Nutritional randomized trials have elucidated many interesting activities of flavonoids. Attempts to chemically modify flavonoids are in progress but the food remains, obviously, the best source, although industrial approaches are welcome to reduce human dietary requests worldwide [7].

Honeybee-collected pollen and beebread are recognised as a well-balanced food. In pollen grains, most of flavonoids exist as glycosides, known as aglycones, being quercetin the major compound. Flavonoids are the secondary components of most importance in bee-bread [8]. The potential application of bee bread as a food and as a nutraceutical supplement depends in large part on its chemical composition which varies directly with the flora of the region and the time of collection by the bees [9]. Flavonoids act as prebiotics and support the growth of bifidum and lactobacilli bacteria. The nutritional value of flavonoids is probably related to their anti-inflammatory activity and through this mechanism responsible for prevention of neoplasia [10].

Natural flavonoids and their analogues have long been recognized to possess valuable properties, namely antioxidant, anti-inflammatory, hepatoprotective, antithrombotic, anti-allergic, antiviral, anticarcinogenic, and cytotoxic ones [2,3,5]. Antioxidants such as phenols, flavonoids and tannins are increasingly attracting because they are natural disease preventing, health promoting and anti-ageing substances [4]. Flavonoids possess the ability to capture superoxide, hydroxyl and lipid radicals [2]; they can act as potent antioxidants and metal chelators [5].

The fundamental role of plant-derived polyphenols contained in the human diet is to elicit a mild redox response, to activate the oxidative stress response, particularly at the mitochondria and endoplasmic reticulum levels. Flavonoids act through cell targets, involved in energy homeostasis, cell survival, and stress response. These active principles promote the cell regulatory machinery by activating those pathways leading to an optimal stress response. The dose-related effect of flavonoids might be a fundamental key to comprehending their bimodal or biphasic effect on the many aspects of cell function leading to the “proinflammatory phenotype” [7].

The analysis of the content of main active components in raw materials and phytomedicines is an essential step to evaluate the quality of products and validate the efficacy and safety of their therapeutic use [11].

Unlike synthetic drugs, the pharmacological activity from herbal drugs or phytopharmaceuticals is a result of joint action of a group of substances. Several scientific studies make clear that many natural products exhibit higher biological activity than their isolated active components. Flavonoids are chemical and pharmacological markers of great importance for the quality control of medicinal plants and phytopharmaceuticals. A variety of analytical methods can be used to quantify these compounds, however UV/Vis spectroscopy and high performance liquid chromatography (HPLC) are the most prominent ones [6]. Widely applied HPLC techniques do not allow quantifying a totality of any group of biologically active substances in plant materials.

With the information from the UV spectrum, it may be possible to identify the compound subclass or perhaps even the compound itself. Knowledge of UV spectral data of flavonoids in several solvents allow carrying out of quantification since detection can be performed at the wavelength maximum of the compound in question. These are typically to be found at 270 and 330 to 365 nm for flavones and flavonols, at 290 nm for flavanones, at 236 or 260 nm for isoflavones, at 340 to 360 nm for chalcones, at 280 nm for dihydrochalcones, at 502 or 520 nm for anthocyanins, and at 210 or 280 nm for catechins [1].

Basic physic principles concerning application of differential spectrophotometry, as an analytical method, are well characterized in [12,13]. The UV/Vis spectrophotometric determination became one of the most widely used methods for quantification of total flavonoids in raw plant materials due to its simplicity, low cost of implementation and wide availability in laboratories for quality control. This approach becomes more critical due to the high cost or absence of reference substance needed for determination of individual flavonoids [6].

Differential spectroscopy is an elegant and powerful analytical method, based on the relatively simple principles of classical absorption spectroscopy, that provides much better fingerprints than traditional absorbance spectra. The method has been applied successfuly in food analysis, clinical analysis, and environmental analysis as well, which indicates its relaibility in analytical chemistry [12].

Differential spectrophotometry is based on the formation of a complex between aluminum cation and the carbonyl and hydroxyl groups of the flavonoid with the further Uv-Vis spectrophotometric (absorption spectroscopy) assay of the formed stable complex, that provides a bathochromic displacement and the hyperchromic effect thus avoiding interference from other phenolic compounds [6,11]. To suppress self-dissociation of flavonoids acetic or hydrochloric acid are added [14].

The principle of aluminum chloride colorimetric method is that aluminum chloride forms acid stable complexes with the C-4 keto group and either the C-3 or C-5 hydroxyl group of flavones and flavonols. In addition, aluminum chloride forms acid labile complexes with the orthodihydroxyl groups in the A- or B-ring of flavonoids. The convenient colorimetric method utilizing aluminum chloride reaction to determine flavonoid contents was proved to be specific only for flavones and flavonols [14,15].

The main character of difference spectrophotometric assay is that the measured value is the difference in absorbance of two equimolar solutions of the analyte in different chemical forms which exhibit various spectral characteristics. This method for altering spectral properties of analyte due to adjustment of pH by means of acid, alkali and buffer.

Differential spectrophotometric analyses include preparation of solutions, adjustment of the spectrophotometer, checking of cuvettes, temperature control, carrying out the measurement, and calculating the result [12], therefore, preliminary purification, fractionation etc. are not required. Summarization of scientific data, related to total flavonoid determination techniques, based on the method of differential spectrophotometry after complexation with aluminium chloride, are presented in the Table 1 and might become a useful tool for choice of particular technique, considering occurrence of reference compound in the subjected plant materials or nutraceuticals.

Reference (marker) Compound

Type of flavonoid


Plant material / nutraceutical

Author, year of publication



390 nm

Daucus carota fruits

Chubka M.B., 2013 [16]



510 nm

Morus spp. leaves

Zhishen, et al., 1999 [17]


Flavone Glycoside

395 nm

403 nm

Leontodon autumnalis herb

Agrimonia eupatoria herb

Bubenchikov R.A. et al., 2016 [18]

Kurkina A.V., 2011 [19]


Favonol Glycoside

412 nm

Crataegus sanguinea fruits

Shmygareva A.A., 2017 [20]




395±3 nm

400 nm

Daucus carota fruits

Agrimonia eupatoria herb

Smalyuh O.G. et al., 2013 [21]

Kozak I.V. et al., 2017 [22]



330±2 nm

Sophora flavescens roots

Samoryadova A.B., 2015 [23]



408 nm

415 nm

415 nm

415 nm

425 nm

430 nm

510 nm

Bauhinia forficata leaves


bee bread


Agrimonia eupatoria herb

Ocimum basilicum leaves

bee bread

Marques G.S. et al., 2013 [6]

Chang Chia-Chia et al., 2002 [15]

Ivanišová E. et al., 2015 [9]

Meda A. et al., 2005 [24]

Kubinova R. et al., 2012 [25]

Silva L. et al., 2015 [11]

Zuluaga C. et al., 2015 [8]


Flavonol Glycoside

420 nm

Vaccinium myrtillus shoots

Kurkin V.A. et al., 2013 [26]


Flavone Triglycoside

397 nm

Astragalus falcatus herb

Sereda O.V. et al., 2006 [27]



Flavonol Glycoside

410 nm


410 nm


415 nm

Salix triandra leaves and branches

Centaurea scabiosa aerial part

Salvia farinacea herb

Sannikova E.G. et al., 2016 [28]


Larkina M.S. et al., 2009 [29]


Popova O.I. et al., 2016 [30]


To avoid errors in the photometric analysis of flavonoids in herbal drugs, there should be obtained in the wavelength range of 250-600 nm the absorption spectra of the test solution, the test solution with the aluminum chloride and the differential spectrum with aluminum chloride. Such approach helps to identify the analytical wavelength, to choose the standard sample and note the influence of concomitant compounds. Differential spectrophotometry has advantages over the direct method especially for medicinal plant materials containing simultaneously flavonoids and significant amounts of hydroxycinnamic acids [14].

The technique of differential spectroscopy for quantification of flavonoids’ totality, expressed as robinin, from cultivated Astragalus falcatus herb samples was developed [27], applying aluminium complex formation with the further spectra measurements. Both alcoholic solutions of extracted flavonoids and the herb extract have maximum absorption in the UV spectrum at 266 nm and 355 nm, and absorption minimum at 280-300 nm. Some difference in spectra of the plant extract and totality of flavonoids (an absorption shoulder at 325 nm) could be explained by occurrence of hydroxycinnamic acids in the total herb extract; to avoid such impact, as the measurement option, there was proposed to analyze it at the analytical wavelength 397 nm at the presence of aluminium chloride.

Among the most widely applied techniques, based on the aluminum chloride colorimetric assay, is the one, proposed by Zhishen J. et al. [17], that allows quantifying flavonoids, expressed as catechin, at the analytical wavelength 510 nm [4,31,32]. The contents of total flavonoids in 10 medicinal plant materials, originating from nine different botanical species, harvested in Poland, Lithuania and Ukraine, was determined by this UV/Vis spectrophotometric technique [31]. Also we determined the content of total flavonoids in 65 commercially available samples of herbal drugs, rich in flavonoids, originating from Ukraine, Romania, and Belarus in order to detect similarities and differences in the contents of total flavonoids and of selected essential elements [32].

Analysis of data, presented in reviewed publications, revealed that a content of total flavonoids rarely exceeds 4% (referred as a dried plant material) in herbal drugs. Amongst the prominent exceptions should be mentioned official drugs sophora flower (Sophorae japonicae flos), which in accordance with the European Pharmacopoeia (EP) [33] requirements, contains minimum 8% of total flavonoids, expressed as rutin, and minimum 6% of rutin, and sophora flower-bud (Sophorae japonicae flos immaturus) with the yield minimum 20% of total flavonoids, expressed as rutin, and minimum 15% of rutin (both dried drugs).

The EP [33] applies the method of differential spectrophotometry with aluminium chloride for determination of the percentage content of flavonoids, expressed as hyperoside, measuring absorbance at 425 nm, for the following herbal drugs: birch leaf, calendula flower, goldenrod, European goldenrod, knotgrass, motherwort. The aforementioned pharmacopoeial method comprises measurements of the absorbance of the test solution (stock solution+ aluminium chloride reagent + glacial acetic acid in methanol) at 425 nm after 30 min by comparison with the compensation solution (stock solution+ glacial acetic acid in methanol). The same principle is used for quantification of flavonoids in safflower flower (hyperoside, 420 nm), elder flower and equisetum stem (isoquercitroside, 425 nm), sophora flower and sophora flower-bud (rutin, 425 nm).

With various biological activities, flavonoids are as key candidate compounds for evaluating the quality of propolis products. The aluminum chloride complexes of propolis compounds with more functional groups absorbed stronger at 415 nm and showed the absorption maximum at longer wavelength [15]. The wavelength scans of the complexes of 15 standards with AlCl3 showed that the complexes formed by flavonols with C-3 and C-5 hydroxyl groups, such as galangin, morin and kaempferol, as well as those with extra ortho-dihydroxyl groups, such as rutin, quercetin, quercitrin and myricetin, had maximum absorbance at 415-440 nm. However, the λmax of the complexes formed by chrysin and apigenin which have only the C-5 hydroxyl and C-4 keto groups were at 395 and 385 nm, respectively. A flavone luteolin, which has the C-5 hydroxyl group and the ortho-dihydroxyl groups in B ring formed a complex that showed a strong absorption at 415 nm. Therefore, the wavelength 415 nm was chosen for absorbance measurement of propolis [15]. Differential spectroscopy assay, applying as the chromogenic agent 2% ZrOCl2 8H2O methanol solution at wavelength 412 nm, for quantification of flavonoids, expressed as rutin, in Psidium guajava leaf was developed [34].

Therefore, differential spectrophotometry in quantitative analysis of various classes of natural compounds, especially flavonoids, has become and remains a simple, rapid, valid, accurate and accessible method for determination of totality of particular group of active principles (expressed as a standard substance) in herbal materials.

Considering, that differential spectrophotometry method allows determination of flavonoid contents, expressed as a leading individual substance, the presented data might be useful for development and further standardization of herbal drugs, e.g. of nephroprotective (hypoazotemic) activity, since the flavonoids cynaroside, hyperoside, quercitin and robinin in individual state exhibit among others the aforementioned and related pharmacological effects.

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Citation: Lysiuk R, Hudz N (2017) Differential Spectrophotometry: Application for Quantification of Flavonoids in Herbal Drugs and Nutraceuticals. Int J Trends Food Nutr 1: e102.

Published: 20 September 2017


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