Mana L. Tereschuk, Mario D. Baigon, Lucia I. C. de Figueroa, and Lidia R. Abdala
The flavonoids, constituting one of the most numerous and widespread groups of natural plant constituents, are important to humans not only because they contribute to plant colors but also because many members are physiologically active. These low-molecular-weight substances, found in all vascular plants, are phenylbenzopyrones. Over 4000 structures have been identified in plant sources, and they are categorized into several groups (Fig. 1). Primarily recognized as pigments responsible for the autumnal burst of hues and the many shades of yellow, orange, and red in flowers and food, the flavonoids are found in fruits, vegetables, nuts, seeds, stems, flowers, and leaves as well as tea and wine and are important constituents of the human diet (1). They are prominent components of citrus fruits and other food sources. Flavonols (quercetin, myricetin, and kaempferol) and flavones (apigenin and luteolin) are the most common phenolics in plant-based foods. Quercetin is also a predominant component of onions, apples, and berries. Such flavanones as naringin are typically present in citrus fruit, and flavanols, particularly catechin, are present as catechin gallate in such beverages as green or black tea and wine. Some major sources of flavonoids are outlined in Table 1 (2). The daily intake of flavonoids in humans has been estimated to be approx 25 mg/d, a quantity that could provide pharmacologically significant concentrations in body fluids and tissues, assuming good absorption from the gastrointestinal tract (3).
Biological activity of flavonoids was first suggested by Szent-Gyorgyi 1938 (4), who reported that citrus peel flavonoids were effective in preventing the capillary bleeding and fragility associated with scurvy. The broad spectrum of biological activity within the group and the multiplicity of actions displayed by a certain individual members make the flavonoids one of the most promising classes of biologically active compounds (5).
From: Methods in Molecular Biology, vol. 268: Public Health Microbiology: Methods and Protocols Edited by: J. F. T. Spencer and A. L. Ragout de Spencer © Humana Press Inc., Totowa, NJ
Flavonoids possess important effects in plant biochemistry and physiology, acting as antioxidants, enzyme inhibitors, precursors of toxic substances, pigments, and light screens. In addition, these compounds are involved in photosensitization and energy transfer, actions of plant growth hormones and growth regulators, control of respiration and photosynthesis, morphogenesis and sex determination, and defence against infection (6). Several reports (7-9) indicate that plant flavonoids activate bacterial nodulation genes involved in control of nitrogen fixation, suggesting important relationships between particular flavonoids and activation and expression of genes.
Dietary Sources of Flavonoids and Phenolic Acids
Catechins Tea, red wine
Flavanones Citrus fruits
Flavonols (e.g., quercetin) Onions, olives, tea, wine, apples Anthocyanidins Cherries, strawberries, grapes, colored fruits
Caffeic acid Grapes, wine, olives, coffee, apples, tomatoes, plums, cherries
Flavonoids have been recognized to possess antiallergic, anti-inflammatory, antiviral, antiproliferative, and anticarcinogenic activities; they also affect some aspects of mammalian metabolism (6). Epidemiological studies have indicated that high fla-vonoid consumption is associated with reduced risk of chronic diseases such as cardiovascular diseases (1,10).
The resurgence of interest in traditional medicine during the past two decades, together with efforts in expanded pharmacognosy has rekindled an interest in flavonoids and the need to understand their interaction with mammalian cells and tissues (6).
Plants have been used by humans since the beginning of human culture for various purposes, connected to survival, including medicine. Such folk or ethnomedical uses represent leads that may shortcut the discovery of modern therapeutic drugs, either directly from the plants or from their synthetic analogs. In fact, 74% of the 121 biologically active plant-derived compounds currently in use worldwide have been discovered through follow-up research to verify the authenticity of folk or ethnomedical uses of plants (11).
The World Health Organization (WHO) estimates that 80% of people living in developing countries use traditional medicine almost exclusively. Medicinal plants form the principle component of traditional medicine. This means that some 3,300 million people use medicinal plants on a regular basis; they should therefore be studied in terms of safety and efficacy (12).
Medicinal components from plants also play an important role in conventional western medicine. In 1984, Farnsworth et al. (13) identified 119 secondary plant metabolites that are used globally as drugs. It has been estimated that 14-28% of higher plant species are used medicinally, but only 15% of all angiosperms have been investigated chemically, and 74% of pharmacologically active plant-derived components were discovered after following up on ethnomedicinal use of the plant (14). Because the numbers of resistant strains of microbial pathogens have been growing since methycillin-resistant Staphylococcus aureus appeared, it is critically important to develop new antimicrobial compounds for these and other microorganisms (15).
In Argentina, native people have been using plants as remedies for infectious diseases for centuries. Many plant preparations have been used in the treatment of diarrhea and respiratory diseases, and these are still being used by rural populations (16).
Species of the family Asteraceae are some of the most useful, and some South American members were reported previously to show pharmacological activities (17-19). Leaf infusions from members of Tagetes (Asteraceae) have been used in folk medicine to treat stomach and intestinal diseases (20), and several Tagetes species have been found to possess biological activity (16,21-23).
The genus Tagetes (family: Asteraceae; tribe: Tageteae; subtribe: Tagetinae, sensu Strother ) comprises 56 strongly aromatic species, some of them known as marigolds (25). In Argentina, the genus Tagetes is composed of 12 species, some of which are used in popular medicine; their use has been supported by several studies on their biological activity (16,23).
Nine species of Tagetes are widely distributed in the northwest Argentina. Three members of this group are used frequently in popular medicine in Tucuman: T. minuta, T. pusilla, and T. terniflora. T. minuta is a native plant known as suico or chinchilla that has been used as an antimicrobial, an antihelminthic, a diuretic, and an antispas-modic, and against intestinal diseases (20,26). T. pusilla, known as ams del cerro, is used as a condiment and its infusion as an antisyphilitic, carminative, diuretic, and tonic (20). T. terniflora is also a native plant known as suico-suico or quichia. It is used by people in Argentina and Ecuador as a condiment in soups (27). Other studies on the biological activity of the genus Tagetes showed active thiophenes with larvi-cidal activity (28) and 3-OCH3 flavone derivatives as inhibitor agents of picornavirus and vesicular stomatitis virus (29).
Experiments that functioned on the antimicrobial activity of methanolic extracts and fractions (ethyl acetate and aqueous) from T. minuta, T. pusilla, and T. terniflora have been carried out against Gram-positive and Gram-negative bacteria, with interesting results. The same fractions were inactive against Lactobacillus, Zymomonas, and Saccharomyces species. An absence of antimicrobial activity against nonpatho-genic human bacteria could be beneficial for intestinal disease treatments, in which the intestinal flora must be preserved.
6-OH and 6-CH3O flavonoid derivatives (quercetagetin and patuletin) from leaves and flowers of T. minuta, T. pusilla, and T. terniflora has been identified (30). T. minuta and T. pusilla contain the following major flavonoid compounds: quercetagetin, quercetagetin-7-O-arabinosyl-galactoside, quercetagetin-3-O-arabinosyl-galactoside, quercetagetin-7-O-glucoside, patuletin, patuletin-7-O-glucoside, and isorhamnetin. However T. terniflora has a different flavonoid pattern: quercetagetin-7-O-arabinosyl-galactoside, quercetin, quercetin-3-O-arabinoside, quercetin-3-O-galactoside, isorhamnetin-3,7-diglucoside and isorhamnetin.
The major component of the extract isolated from leaves of T. minuta, the flavonol quercetagetin-7-O-arabinosyl-galactoside, showed significant antimicrobial activity on the pathogenic microorganism tested (16). The recently isolated flavonol quercetagetin-7-O-glucoside from T. pusilla and T. minuta exhibited better antimicrobial activity than the compound cited above (31). Although T. pusilla had an identical flavonoid compound profile compared with T. minuta, antimicrobial activity against almost all microorganisms was lower than that of T. minuta (23).
Recent studies with flavonoid glycosylated derivatives show that monohydroxy-lated flavonoids in the B-ring are absorbed without cleavage of the |3-glycosidic bond because of the resistance of this bond to the stomach HCl and the action of pancreatic enzymes action, whereas diglycoside derivatives are absorbed in the colon after deglycosylation by microorganisms. The authors also noted the low absorption efficiency of the related aglycone (quercetin) (32). These studies and the antimicrobial activity found in quercetagetin 7-0-glucoside allow us to consider this flavonoid a promising antibacterial agent with good bioavailability in humans (31).
The object of this chapter is to help readers in the beginning of studies of antimicrobial activity of flavonoids isolated from three native Tagetes species from Argentina. The separation procedure is described but identification is not included. The methodology to determine antimicrobial activity against Gram-positive, Gram-negative bacteria as well as yeasts is also described.
Leaves (50 g) of Tagetes minuta (HBK), Tagetes terniflora (HBK), and Tagetes pusilla (HBK) were collected from different areas of Tucuman, northwest Argentina. Sample specimens were deposited in the Teodoro Meyer Herbarium (LIL).
2.2. Extraction of Flavonoids (see Note 1)
2.3. Separation of Flavonoids
1. TBA: reagent-grade tertiary butanol/glacial acetic acid/water (3:1:1). TBA is used for both paper chromatography (PC) and thin-layer chromatography (TLC; cellulose).
2. 15% and 30% Acetic acid, for PC and TLC (cellulose).
3. Acetic acid/chloride acid/water (30:3:10), for PC and TLC (cellulose).
7. Ethyl acetate/glacial acetic acid/formic acid/water (100:11:11:26), for TLC (silica).
8. Visualization by spraying with natural product reagent (1% methanolic solution of diphenylboric acid ethylamino ester) followed by 5% ethanolic polyethylene glycol 4000 (33).
10. Ethyl acetate.
11. Trifluoracetic acid (TFA; solution A) for high-performance liquid chromatography (HPLC) analysis.
12. Acetonitrile (solution B) for HPLC analysis.
13. Separatation funnels (125 and 250 mL) with glass stopcock.
14. Rotary evaporator with temperature-controlled water bath, attached to a water pump (Buchi Rotavapor R 110).
15. Whatman 3MM paper (46 x 57-cm sheets).
16. Chromatographic cabinet (Chromatocab).
17. Chromatography column, glass with solvent reservoir, and glass stopcock (1 x 50 cm).
18. Sephadex LH-20 (Pharmacia-Biotech 17-0090-10).
19. Fraction collector (Gilson FC203B).
20. TLC aluminum sheets (20 x 20 cm), silica gel 60 (without fluorescent indicator) with concentrating zone (Merck 5582).
21. TLC aluminium sheets (20 x 20 cm) cellulose F, thickness 0.1 mm (Merck 5574).
22. Glass tank with glass cover (Desaga).
23. HPLC equipment.
24. Analytical C18 column (Phenomenex).
25. Ultraviolet viewing lamp (366 nm).
26. Recording UV-VIS spectrophotometer (Beckman DU 640).
28. Ungraduated pipet.
The microorganisms to be used are Gram-positive and Gram-negative bacteria as well as yeasts (see Note 2).
2.5. Antimicrobial Assays
2.5.1. Culture Media and Devices for Microorganisms Growth Media to be used must correspond to the specific microorganism.
1. Commercial Mueller-Hinton broth (Biokar), 23 g/L, is used for almost all Gram-positive and Gram-negative bacteria as a suitable medium for antimicrobial screening.
2. de Man-Rogosa-Sharpe (MRS) broth: 10g/L polypeptone, 10 g/L meat extract, 5 g/L yeast extract, 20 g/L glucose, 5 g/L sodium acetate, 2 g/L ammonium citrate, 2 g/L KH2PO4, 0.25 g/L MgSO4 x 7H2O, 0.058 g/L MnSO4 x 4H2O, and 1.08 mL/L Tween-80 at a final pH 6.4. Sterilize at 121°C for 20 min. This medium is used for Lactobacillus sp.
3. Zym broth containing: 50 g/L glucose, 10 g/L yeast extract, 1 g/L KH2PO4, 1 g/L MgSO4 x 7H2O, 1 g/L (NH4)2SO4, final pH 5.3. Sterilize at 121°C for 20 min. This medium is used for Zymomonas mobilis.
4. YEPD: 10 g/L peptone, 20 g/L glucose, and 10 g/L yeast extract, final pH 4.5. Sterilize at 121°C for 20 min. This medium is used for Saccharomyces cerevisiae.
5. Incubator shaker (rotary) with temperature and speed control.
6. Recording UV-VIS spectrophotometer (Beckman DU 640).
2.5.2. Antimicrobial Assays in Solid Medium
1. 2 and 4% Commercial Mueller-Hinton agar (Biokar) for Proteus vulgaris.
2. 0.6% Commercial Mueller-Hinton agar (Biokar).
6. Chloramphenicol as standard antibiotic.
7. Magnetic stirrer.
8. Teflon-coated magnetic stirring bars.
9. Petri dishes: glass (100 x 15 mm) or plastic (90 x 15 mm).
11. Sterile tips.
2.5.3. Antimicrobial Assays in Liquid Medium
1. Commercial Mueller-Hinton broth (Biokar).
2. Magnetic stirrer.
3. Teflon-coated magnetic stirring bars.
4. Vortex mixer.
6. Sterile tips.
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