Results of quality control of tannins, flavonoids and coumarins contained in products based on
The quality control of raw materials and products from plants is one of the topics most discussed by universities and health surveillance agencies. One of the primary tools used to ensure the reliability of production processes is the use of Standard Operating Procedures (SOPs). SOPs sequentially describe the steps of a particular methodology so that it can be reproduced by different analysts, which minimises variations in their implementation and improves the standardisation of the final product.
Several techniques, such as high performance liquid chromatography, gas chromatography and mass spectrometry, can be used in SOPs to control the quality of plant phenolic compounds [1,2]. However, these compounds have a characteristic spectrum produced by the double bonds in the aromatic rings and substituent positions that facilitates their identification and the development of spectrometric analytical techniques is easily accomplished. In this sense, spectrophotometric methods are more practical, reproducible and inexpensive than other techniques and are therefore favoured for the development of analytical methodologies for such determinations.
In addition to producing compounds such as carbohydrates, lipids, proteins and nucleic acids directly involved in their essential growth functions, plants have an arsenal of enzymes capable of producing, processing and accumulating several other substances not necessarily related to the maintenance of their life . All of these reactions can be defined as secondary metabolism, the products of which provide advantages for both survival and species perpetuation in the plant’s ecosystem . However, this protection has a cost for the plant because metabolic resources that could increase its biomass are used to produce these compounds. In addition to protection, secondary metabolites perform important ecological functions such as inhibiting the germination and growth of other plants, attracting both pollinators and seed-dispersing animals and providing chemical defences against microorganisms .
Phenolic compounds, which have one or more hydroxyl groups linked to an aromatic ring, stand out from other classes of plant secondary metabolites because they are widely distributed and have various ecological functions that are scientifically proven to have numerous pharmacological activities and are well represented by tannins, flavonoids and coumarins.
Tannins are water soluble phenolic compounds with a molecular weight between 500 and 3000 Daltons and may be chemically classified into two groups: hydrolysable tannins and condensed tannins [6,7]. Hydrolysable tannins are connected by ester-carboxyl linkages, which undergo hydrolysis under acidic and basic conditions . Figure 1 presents an example of hydrolysable tannin (gallotannin), connected through a polyol (usually β-D-glucose) with the hydroxyl group esterified by gallic acid. Polyphenols connected with ellagic acid are called ellagitannins .
Condensed tannins, also known as proanthocyanidins (Figure 2), can contain dozens of units of flavan-3-ols (catechin) or flavan-3,4-diols (leucoanthocyanidins). These units have a complex structure and are resistant to hydrolysis; however, they can be soluble in aqueous organic solvents because of their structure .
Hydrolysable and condensed tannins may occur in the same plant simultaneously. However, the hydrolysable tannins are characteristic of Magnoliopsida herbaceous and woody plants and are restricted to certain taxonomic families. Ellagitannins have been used as taxonomic markers, particularly for Hemamelidaceae, Dilenidaceaa and Rosaceae. Condensed tannins have been identified in all plant groups, including Gymnosperms and Pteridophytes .
These secondary metabolites were initially identified by their astringent taste and capacity to bind proteins, which allows for the precipitation and formation of complexes with collagen skin fibres to increase their resistance to water and heat. Chemically speaking, hydrophobic interactions and hydrogen bonds between the phenolic groups in tannins and some macromolecules explain these features. However, the stability of the formed complexes only results after the formation of covalent bonds via the oxidation of tannins by quinones .
Since antiquity, plants containing tannins have been used medicinally as anti-inflammatory, antimicrobial, antitumor and antiviral agents and to treat both wound sand burns . Tannins are also used to manufacture beverages and process animal skin into leather. Some researchers have shown that tannins protect plants against attack by herbivores and pathogens .
Although the use of tannin in the tanning industry has become restricted, interest in studying the ingestion of foods containing tannins to prevent diseases such as atherosclerosis or certain types of cancer has increased because of various epidemiological studies. Some studies report that the complexation of tannins with proteins gives them an important role in controlling bacteria, fungi and insects [13-15]. Other studies examined the inhibitory action of the enzyme reverse transcriptase  and the anticarcinogenic activity associated with green tea and diets rich in fruits containing tannins . It is generally believed that the pharmacological activity of tannins occurs via their complexation with metal ions, antioxidant activity or the ability to complex with macromolecules.
Flavonoids comprise a group of natural substances with great structural diversity and there are currently more than nine thousand known flavonoids that do not occur in humans but can be found in various plant parts such as the leaves, fruits, bark, roots, stems and flowers [22,23]. Flavonoids (Figure 3) are composed of a simple skeleton containing two phenol rings connected by a propionic chain; where ring A is the acetate derivative and both ring B and the three-carbon bridge are derived via a shikimate pathway, which may be associated with carbohydrates (heterosides), un associated (aglycones) or polymerised further (anthocyanins) .
This class of metabolites has several biological functions, such as defence against both herbivores and pathogens, the perpetuation of the species by attracting seed dispersing animals, protection from ultraviolet rays and allelopathy [24,25]. Flavonoids also possess important pharmacological properties, such as antioxidant, antiinflammatory, anti-thrombogenic, antimicrobial, anticancer, antidiabetic and hypocholesterolemic activities [23,26].
Studies show that flavonoids are chemical markers responsible for various pharmacological activities performed by the genus
Coumarins are lactones of
Coumarins are used as antioxidants, anti-HIV drugs, antispasmodics, spasmolytics, hypolipidemics, hypotensives and vasodilating agents ; however, they are also used in food flavouring, perfumes, tobacco and cosmetic products . It is estimated that the daily human exposure to coumarins from cosmetics and perfumes is 0.04 mg/kg/day .
Coumarins are a class of secondary metabolites widely distributed throughout the Plantae kingdom and found in Fungi and Bacteria as well . In plants, coumarins are found frequently in the families of Apiaceae, Rutaceae and Asteraceae, and less frequently in the families of Fabaceae, Oleaceae, Moraceae and Thymeleaceae .
Coumarins have a characteristic UV spectrum due to the nature and position of their substituents, which facilitates both their identification and the development of analytical spectrophotometric techniques . For these reasons,
2. Problem statement
Despite extensive literature presenting various analytical methods, the development of an SOP is often difficult for three reasons: 1) the work does not detail the difficulties and adjustments required to implement the methodology, 2) the steps are not clearly presented for reproduction and 3) the limits of interpretation are not discussed. One criterion recommended by the National Sanitary Surveillance Agency (Agência Nacional de Vigilância Sanitária - ANVISA) in Brazil for the standardisation of herbal drugs is the active compound content or chemical class, which is the total concentration of tannins for products based on
Thus, this paper presents research protocols adopted by our research group to study the levels of tannins, flavonoids and coumarins from plant extracts and the experimental application of these SOPs to analyse products sold in markets (pharmacies and natural product stores) as phytomedicines "All medicine is obtained using solely active raw vegetables. It is characterised by knowledge of the effectiveness and risks of their use, as well as the reproducibility and consistency of its quality. Its efficacy and safety are validated through ethnopharmacological surveys of use, documentation, technical and scientific publications or clinical trial phase 3". "Medicinal plant or their parts, after collection processes, stabilisation and drying and can be full, erasures, crushed or powdered" .
"All medicine is obtained using solely active raw vegetables. It is characterised by knowledge of the effectiveness and risks of their use, as well as the reproducibility and consistency of its quality. Its efficacy and safety are validated through ethnopharmacological surveys of use, documentation, technical and scientific publications or clinical trial phase 3".
"Medicinal plant or their parts, after collection processes, stabilisation and drying and can be full, erasures, crushed or powdered" .
3. Standard operating procedures (POP)
The following SOPs describe the chemical classes to be analysed and the chemical basis of the methods. They provide a detailed list of all the reagents required for the preparation and describe the experimental procedure to be followed. Finally, there is a list of references used in the development of the SOP.
3.1. Standard operating procedure for the quantification of tannins
(1) Tannic acid (0.1 mg/mL, w/v): Dissolve 10 mg of tannic acid in 100 ml of distilled water.
(2) Folin-Ciocalteu reagent (10%, v/v): Dilute 5 ml of Folin-Ciocalteu reagent with 45 mL of distilled water.
(3) Sodium carbonate Na2CO3 (7.5%, w/v): Dissolve 7.5 g of Na2CO3 in 100 ml of distilled water. If necessary, solubilising the solution on a heating plate and magnetic stirrer.
(4) Methanol (80%, v/v): Dilute 800 ml of methanol with 200 ml of distilled water.
The reagent volume is sufficient to examine a maximum of 100 analyses.
Amorim E. L. C, Nascimento J. E., Monteiro J. M., Peixoto Sobrinho T. J. S, Araújo T. A. S., Albuquerque U. P. A simple and accurate procedure for the determination of tannin and flavonoid levels and some applications in ethnobotany and ethnopharmacology. Functional Ecosystems and Communities 2008; 2(1) 88-94 .
Santos S. C., Mello J. C. P. Taninos. In: Simões C. M. O., Schenkel E. P., Gosmanm, G., Mello J. C. P., Mentz L. A., Petrovick P. R. (ed.) Farmacognosia: da planta ao medicamento. Porto Alegre: Universidade Federal do Rio Grande do Sul; 2004. p.615-656 .
3.2. Standard operating procedure for the quantification of flavonoids
(1) Rutin (0.1 mg/mL, w/v): Dissolve 10 mg of rutin in 100 ml of methanol.
(2) Acetic acid solution (60%, v/v): Dilute 30 ml of acetic acid with 20 ml of methanol.
(3) Pyridine Solution (20%, v/v): Dilute 40 ml of pyridine with 160 ml of methanol.
(4) Aluminium chloride solution AlCl3 (5%, w/v): Dissolve 5 g AlCl3 in 100 mL of methanol. If necessary, complete dissolution via magnetic stirring.
(5) Methanol (80%, v/v). Dilute 80 ml of methanol with 20 ml of distilled water.
The reagent volume is sufficient to examine a maximum of 100 analyses.
Peixoto Sobrinho T. J. S, Silva C. H. T. P., Nascimento J. E., Monteiro J. M., Albuquerque U. P., Amorim E. L. C. Validação de metodologia espectrofotométrica para quantificação dos flavonóides de
Zuanazzi, J. A. S.; Montanha, J. A. Flavonóides. In: Simões C. M. O., Schenkel E. P., Gosmanm, G, Mello J. C. P., Mentz L. A., Petrovick P. R. (ed.) Farmacognosia: da planta ao medicamento. Porto Alegre: Universidade Federal do Rio Grande do Sul; 2004, p.577-614 .
3.3. Standard operating procedure for the quantification of coumarins
(1) 1,2-benzopyrone (1 mg/mL, w/v): Dissolve 10 mg of coumarin in 10 ml of distilled water.
(2) Lead acetate (5%, w/v): Dissolve 2,5 g of lead acetate in 50 ml of distilled water.
(3) Hydrochloric acid solution, HCl (0.1 M, v/v): Dilute 10 ml of concentrated hydrochloric acid with 1000 ml of distilled water.
(4) Methanol (80%, v/v): Dilute 80 ml of methanol with 20 ml of distilled water.
The reagent volume is sufficient to examine a maximum of 100 analyses.
Kuster R. A. M., Rocha L. A. M. A. Cumarinas, coronas e cantinas. In: Simões CMO, Schenkel EP, Gosmanm, G, Mello JCP, Mentz LA, Petrovick PR. (ed.) Farmacognosia: da planta ao medicamento. Porto Alegre: Universidade Federal do Rio Grande do Sul; 2004, p.537-556 .
Osório O. K., Martins J. L. S. Determinação de cumarina em extrato fluido e tintura de guaco por espectrofotometria derivada de primeira ordem. Brazilian Journal of Pharmaceutical Sciences 2004; 40 (4) 481-486 .
Analysis of the active component levels in raw plant materials and phytomedicines is essential for the safety and efficacy of pharmaceutical products . The quantification of active compounds in herbals is still only incidentally performed due to the presence of active phytocomplexes plants and their extracts , which complicates their analysis. Through this framework, the use of standardised extracts focusing on specific groups of active components ensures the chemical homogeneity of the product, which improves product quality . The compounds selected for this quality adjustment process should be the same as the assets in the product .
In this way, five products containing
4.1. Calibration curves
To quantify the active components, calibration curves with increasing concentrations proportional to their absorbance were constructed. A correlation equation was obtained from these curves (generally linear) of the type
The calibration curve constructed for tannic acid and tannins and used to quantify
A calibration curve was constructed from rutin to quantify flavonoids in products from
The correlation equation and coefficient obtained from the calibration curve used to analyse coumarins in products containing
4.2. Content of active principles
The results of quality control of tannins of
|MI1||12.57 ± 2.15a||17.12%|
|MI2||4.04 ± 0.23b||5.75%|
|MI3||5.61 ± 0.55bc||9.76%|
|MI4||7.72 ± 0.84c||10.84%|
|MI5||11.81 ± 1.00a||8.44%|
|BF1||4.89 ± 0.11a||2.33%|
|BF2||7.27 ± 0.39a||5.41%|
|BF3||50.38 ± 5.36b||10.64%|
|BF4||65.98 ± 3.62c||5.49%|
|MG1||3.06 ± 0.20a||6.67%|
|MG2||5.17 ± 0.59b||11.40%|
|MG3||6.80 ± 0.24c||3.46%|
|MG4||1.63 ± 0.20d||12.50%|
|MG5||4.49 ± 0.20b||4.55%|
Values are mean ± standard deviation. Values followed by the same letter in column are not statistically different (n = 6, p<0.05).
Analysis of variance (ANOVA) is one way to indicate significant differences (p<0.01) for the drugs of
Analysis of four products containing
Of the five products from
The quantitative analysis of raw vegetables and phytomedicines is a fundamental quality control process that leads to security, stability, consistency and effectiveness in the produced phytomedicines . It is important to emphasise the need for standardisation in analysing herbal medicines to determine the concentration of their active components in raw vegetable materials as well as for species identification.
This chapter provides easily reproducible standard operating procedures (SOPs) for the quality control of raw materials and herbal plants to ensure a minimal standard of quality in products sold. The implementation of these SOPs allows for the analysis of samples sold in establishments in Recife/PE and reveals an inconsistency in the concentration of tannins, flavonoids and coumarins within these products.
The low level of these metabolites may alter their effectiveness and more rigorous quality control and standardisation of these products is required to prevent compromising their therapeutic activity.
- "All medicine is obtained using solely active raw vegetables. It is characterised by knowledge of the effectiveness and risks of their use, as well as the reproducibility and consistency of its quality. Its efficacy and safety are validated through ethnopharmacological surveys of use, documentation, technical and scientific publications or clinical trial phase 3".
- "Medicinal plant or their parts, after collection processes, stabilisation and drying and can be full, erasures, crushed or powdered" .