Quality Management of Nutraceuticals - American Chemical Society

Chapter 3

Analysis of Flavonoids in Botanicals: A Review 1,2

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Downloaded by UNIV OF CALIFORNIA SAN DIEGO on October 18, 2013 | http://pubs.acs.org Publication Date: December 17, 2001 | doi: 10.1021/bk-2002-0803.ch003

Howard M. Merken and Gary R. Beecher 1

Food Composition Laboratory, Beltsville Human Nutrition Research Center, Agricultural Research Service, U.S. Department of Agriculture, Beltsville, MD 20705 Current address: Chemistry Department, 2339 Sciences Building, Southern Illinois University at Edwardsville, Edwardsville, I L 62026-1652 2

Flavonoids, known for their antioxidant activities i n foods, are found i n some botanicals. High-performance liquid chromatography (HPLC) has become the method of choice for flavonoid analysis in foods and botanicals. Other chromatographic methods, such as countercurrent chromatography (CCC), high-performance thin-layer chromatography (HPTLC), micellar electrokinetic capillary chromatography (MECC), and thermospray-mass spectrometry (TSP-MS), have also been used. The flavonoids found have been chiefly the glycosides of flavones and flavonols, with some coumarins and flavan-3-ols. Three flavanones were identified i n Limonium sinense, two flavanones were found after hydrolysis of Mentha piperita, and the flavanonol glycoside astilbin was found i n St. John's wort. Isoflavones have been found i n Ononis spinosa and Sophora japonica.

U.S. government work. Published 2002 American Chemical Society

In Quality Management of Nutraceuticals; Ho, C., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2001.

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Flavonoids, diphenylpropanoids of low molecular weight found i n vascular plants, are consumed by humans in the west at 100-1000 mg/day (1, 2). Most importantly, as antioxidants, they scavenge radicals by donating hydrogen, helping to prevent low density lipoproteins (LDL) from becoming oxidized, which could lead to coronary heart disease (3). Their beneficial health effects also include their ability to complex transition metal cations such as C u and F e (3, 4). Other activities include anti-AIDS, anti-arthritic, anticancer, anti hypertensive, anti-inflammatory, and antiviral activity (2). Flavonoids regenerate α-tocopherol by reducing the α-tocopherol radical (3). Flavonoids are also believed to regenerate ascorbate, which regenerates Vitamin Ε (5). Mabry and colleagues (6) published nmr (nuclear magnetic resonance) spectra of 128 flavonoids, and the ultraviolet-visible (UV-vis) spectra of 175 flavonoids, molecular extinction coefficients, and U V spectral data i n different solvents. Daigle and Conkerton (7, 8) reviewed the analysis of flavonoids by H P L C . A review by Robards and Antolovich (9) concentrated on the analytical chemistry of fruit flavonoids. The H P L C systems of food flavonoids published from 1988 to early 1999 was reviewed by Merken and Beecher (10). Middleton (2) reviewed the biological properties of flavonoids. Rice-Evans and colleagues (3) explored the mechanistic reasoning behind the antioxidant activities of flavonoids. A wealth of information about the health affects of flavonoids i n both foods and botanicals is found in a book edited by Rice-Evans and Packer (11). Flavonoids are also found in various botanicals. Flavonoid analysis is thereby necessary for an understanding as to the safety and efficacy of botanical preparations (12). This review concentrates on analysis of plants and plant extracts, as opposed to analysis of commercial preparations of botanicals or analysis of flavonoids in human tissue, blood, urine, or feces. The literature often neglects the common names of botanicals, which i n this paper were often gleaned from various websites. The literature also varies in the nomenclature of relevant flavonoid glycosides and the coumaric esters of flavonol glycosides. 2 +


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Botanical Analysis

Sample Preparation Botanical samples were generally first treated by liquid-liquid extraction (LLE). A solid phase extraction (SPE), using for example a Sep-pak Q column (73) or a Bond Elut C cartridge (14), sometimes followed. 8

1 8


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Leaves of Apocynum venetum were dried, roasted twice, and extracted and partitioned i n liquid phases (LLE) (15). The portion soluble i n ethyl acetate (EtOAc) was put through a Sephadex LH-20 column. Fifteen compounds were isolated through reversed-phase preparative thin-layer chromatography (TLC). Researchers often deglycosylated the flavonoids via acidic hydrolysis. In separate tests, Vilegas and colleagues (16) used 6% HC1 and MeOH/2% HC1 to deglycosylate two new flavonoid glycosides from Maytenus aquifolium. The monosaccharides from the methanolysis were later analyzed by G C - M S .

Analysis of Botanicals H P L C conditions were similar to those used for analysis of flavonoids in foods (10). Reversed-phase (RP) columns were usually used, although a normal phase silica gel column was used for the proanthocyanidin-rich Pycnogenol (17). Flow rates ranged from 0.8-2.0 ml/min. Detection was generally done using a diode-array detector (DAD), which can collect data over the interval of several hundred nm, and can produce chromatograms over several preset wavelengths. Detection was carried out from 254-370 nm. Anthocyanidins were generally not found i n botanical leaves or flowers, obviating analysis at 465-560 nm (10). In analyzing procyanidins in flowers and leaves of Crataegus spp., Rohr and colleagues (14) used 220 nm D A D . In comparing electrochemical detection (ECD) to diodearray detection for procyanidins, they suggested D A D , which they found more selective and easier to handle, despite the greater sensitivity of E C D .

Other Analytical Methods Non-volatile compounds can be analyzed quickly with liquid chromatography-mass spectrometry (LC-MS) (18). Thermospray-mass spectrometry (TSP-MS), using a buffer of ammonium acetate, was used to identify flavonol di- and triglycosides from the flowers of Calendula officinalis and the leaves of Ginkgo biloba and Tilia cordata. This "soft-ionization" technique limits the fragment ions so that the sugar and the aglycone can be determined. Hahn-Deinstrop and Koch (19) used thin-layer chromatography (TLC) in the identification of terpene lactones and flavone glycosides i n Ginkgo biloba. Evaluating high-performance thin-layer chromatography (HPTLC) plates densitometrically, Jamshidi and colleagues (20) claimed that H P T L C is as good as H P L C for analysis of pharmaceuticals. Maisenbacher and Kovar (21) used displacement chromatography to prepare St. John's wort oil. Their analytical methods included H P L C , H P T L C , and photometry.


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Shi and N i k i (26) list several reports of the health effects of Ginkgo biloba extract, which include the scavenging of hydroxyl and peroxyl radicals, the interaction with biologically occurring nitric oxide, and prevention of hydroxyl radical-induced cerebellar neuron death i n rats. Akiba and colleagues (35) showed that G B E can suppress aggregation of platelets exposed to a combination of F e with either ferf-butyl hydroperoxide or hydrogen peroxide, while oxidative stress induced platelet aggregation was not affected by ginkgolides A , B , and C. Downloaded by UNIV OF CALIFORNIA SAN DIEGO on October 18, 2013 | http://pubs.acs.org Publication Date: December 17, 2001 | doi: 10.1021/bk-2002-0803.ch003

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Vanhaelen and Vanhaelen-Fastre (22) combined gradient elution countercurrent chromatography (CCC) and preparative H P L C to isolate and purify seven flavonol glycosides from the leaves of Ginkgo biloba. Yang and colleagues (23) used multidimensional counter-current chromatography ( M D C C C ) to separate flavone aglycones found i n crude mixtures' of Ginkgo biloba and Hippophae rhamnoides. Their system included two high-speed countercurrent chromatography (HSCCC) systems. Micellar electrokinetic capillary chromatography ( M E C C ) has higher column efficiencies than does H P L C (24). On-line U V detection can be used for detection i n capillary electrophoresis (CE) as well as i n H P L C (25). Analyzing the results at 260 nm in both cases, Pietta and colleagues (25) used M E C C to separate several flavonol 3-O-glycosides found in Ginkgo biloba extract (GBE), and compared the results to those obtained by H P L C .

Flavonoids in Selected Botanicals

Ginkgo Biloba Extracts of Ginkgo biloba leaves have been used since ancient times i n China and since the 1960s in France and Germany to treat certain atherosclerotic diseases (26). G biloba is one of the most highly characterized botanicals available today. It contains ginkgolides and bilobalides, terpene lactones (26, 27). Also present are benzoic acid derivatives, di-trans-poly-cisoctadecaprenol, ginkgolic acids, 2-hexenal, kynurenic acids (iV-containing acids), proanthocyanidins, steroids, sterols, sugars, and waxes (28). Flavonol glycosides (Figures 1-3) are present, as are flavone glycosides (Figure 4), coumaric esters of kaempferol and quercetin (Figure 5), biflavones (Figure 6), and flavonol triglycosides (Figure 7) (13, 18, 19, 22, 25, 26, 28-34).



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OH Ο Compound astragalin * astragalin-6 -0-acetate kaempferol** kaempferol-3 -0-glucoside* kaempferol-3 -O-glucoside-7-0rhamnoside* kaempferol 3-6)-0-[y8-D-glucopyranosyl ( 1 -> 3 )-0-a-L -rhamnopyranosyl( 1 -> 2)] -0-/?-D-galactopyranoside quercetin 3-0-a-L -rhamnopyranosyl( 1 -> 6)-0-[/?-D-glucopyranosyl

H OH

Figure 15. Flavonol tetraglycosides from Maytenus aquifolium (16). Pietta and colleagues (78) found isoquercitrin, kaempferol3,7-0-dirhamnoside, kaempferol-3-0-glucoside-7-0-rhamnoside, quercetin3,7-0-dirhamnoside, quercetin-3-O-glucoside-7-rhamnoside, and tiliroside (Figures 1,3) i n the leaves of Tilia cordata, or littleleaf linden. Andrade and colleagues (50) chromatographed Centaurea erythraea, Cynara cardunculus (Cardoon aster), Hypericum androsaemum (Tutsan), Lavandula officinalis (lavender), Lippia citriodora (herb Louisa), Mentha piperita (peppermint), and Salvia officinalis (garden sage). The flavanones eriodictyol and hesperetin (Figure 8) were found in M. piperita after hydrolysis. The flavanones eriodictyol, homoeriodictyol, and naringenin (Figure 8) were among 20 flavonoids (Figures 2,3,4,16,17) identified by L i n and Chou (57) i n Limonium sinense.



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OH OH OH

OFL compound (-)-epigallocatechin-3-0-gallate H-epigallocatechin-S-O-iS'-O-methyO-gallate H-epigallocatecfon-3-Q-(3\5^

.

R l R 2 H H Me H Me Me

Figure 16. Flavan-3-ols in Limonium sinense (51).



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Compound myricetin-3 -0-/ï-galactopyranoside myricetin-3-0-(6"-0-galloyl)-/?galactopyranoside myricetin-3-0-(2"-0-galloyl)-ûirhamnopyranoside myricetin-3-0-(3"-0-galloyl)-û'rhamnopyranoside myricetin-3-0-(4"-0-galloyl)-ûrrhamnopyranoside myricetin-3-0-(2"-0-/7hydroxybenzoyl)-ar-rhamnopyranoside myricetin-S-O-ar-rhamnopyranoside quercetin-3-0-ûr-rhamnopyranoside quercetin-3-0-(2"-0-galloyl)-ar-

R3 ...

R4 ...

—

—

R5 H I

H

H

I

—

OH

I

H

H

—

OH

H

I

H

—

OH

H

H

II

—

OH H H

H H H

H H H

H H I

— — —

Rl

R2

...

— —

OH

—

Figure 17. Flavonol glycosides in Limonium sinense (51). Myricetin3-0-fi-arabinoside also found. Isoflavones are also found in a limited number of botanicals. For example, Sophora japonica, the Chinese scholar tree or Japanese pagoda tree, contains


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the isoflavone genistein (Figure 18), the flavonoids quercetin and rutin (Figure 3), and sophorabioside and sophoricioside (52). Ononis spinosa, or spiny rest harrow, contains the isoflavones biochanin A , formononetin, and genistein (Figure 18) and the flavonols kaempferol and rutin (Figures 1,3) (53).

compound biochanin A formononetin gemstein

RI OH H OH

R2 Me Me H

Figure 18. Isoflavones (structures from 6) in Ononis spinosa (53) and Sophora japonica (genistein only) (52).

Conclusion Botanical preparations are generally taken from the leaves or flowers. Botanical flavonoids are usually glycosides of flavones or flavonols, with some catechins and proanthocyanidins found i n Pycnogenol, which comes from tree bark. Catechins and flavanone glycosides are less common. H P L C is the method of choice for identification and quantitation of flavonoid glycosides and, after hydrolysis, aglycones. Other methods such as M E C C have been successfully used, but are not as prevalent i n the literature.

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