HPLC Analysis of Catechins, Theaflavins, and Alkaloids in Commercial Teas and Green Tea Dietary Supplements: Comparison of Water and 80% Ethanol/Water Extracts

JFS C: Food Chemistry and Toxicology

HPLC Analysis of Catechins, Theaflavins, and Alkaloids in Commercial Teas and Green Tea Dietary Supplements: Comparison of Water and 80% Ethanol/Water Extracts

Mendel Friedman, Carol E. Levin, Suk-Hyun Choi, Etsuko Kozukue, and Nobuyuki Kozukue

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Introduction[pic 2]

nterest in teas, tea extracts used as dietary supplements, and tea compoundsarisesfromthefactthatteaflavonoids(catechinsand theaflavins) are reported to have beneficial protective effects against cholesterol (Maron and others 2003); diabetes (Fukino and others 2005); chronic liver disease (Ruhl and Everhart 2005); breast (Seeley andothers 2005), lung(Yangandothers 2005), prostate(Bettuzziand others 2006), and skin cancers (Camouse and others 2005); obesity (Berube-Parent and others 2005; Murase and others 2006; Kao and others 2006); and pathogenic microorganisms (Cushnie and Lamb 2005; Friedman and others 2006). Flavonoid-containing cosmetics (cosmeceuticals) are also of interest (Thornfeldt 2005). For discus- sions of possible antioxidative and reactive oxygen species (ROS)- suppressing mechanisms of the beneficial effects of tea flavonoids and related dietary ingredients see Friedman (1997), Nam and

others (2005a, 2005b), and Choe and Min 2006.

Teas also contain 3 pharmacologically active purine (methylx- anthine) alkaloids: caffeine, theobromine, and theophylline. These are also reported to exhibit beneficial effects. Selected studies de- scribe the use of caffeine to enhance mental activity (Rao and others 2005) and running performance (McLellan and others 2005) and to treat apnea (absence of breathing) (Schmidt 2005) and migraine

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MS 20060096 Submitted 2/13/2006, Accepted 4/29/2006. Authors Friedman and Levin are with Western Regional Research Center, Agricultural Research Service, US Dept. of Agriculture, Albany, CA 94710. Authors Choi, Kozukue, and Kozukue are with Uiduk Univ., Dept. of Food Service Industry, San 50 Yugeom, Gangdong, Gyeongju, Gyongbuk 780-713, Korea. Direct inquiries to author Friedman (E-mail: mfried@pw.usda.gov).

headaches (Goldstein and others 2005) and of theophylline and theobromine to treat asthma (Barnes 2006) and bradycardia (slow- ness of heartbeat) (Schulz-Stubner 2005).

The cited studies suggest the need to define the composition of teas sold commercially as tea bags and of green tea extracts sold as dietary supplements in the form of capsules, gums, powders, and tea drinks. Although tea is generally consumed after water infusion, isolated tea extracts and individual tea compounds are widely used to prepare dietary supplements and tea beverages sold at retail and in biomedical studies. Detailed knowledge of the composition will allow consumers, researchers, and producers and distributors of supplementsandcosmeticstoselectteas, extracts, andsupplements with the highest content of beneficial compounds.

All analytical methods for quantifying biologically active com- pounds present in tea leaves involve extraction, separation, and analysis. To help define the composition of commercial teas, we had previously validated an HPLC method to analyze, in a single run, levels of 7 catechins, 4 theaflavins, and 2 purine alkaloids in a large number of commercial teas extracted with hot water to simu- late home use (Friedman and others 2005). Other investigators used a wide variety of extraction conditions, resulting in a wide variety of measured concentrations of flavonoids and alkaloids from dry tea leaves. Previous studies include the use of (a) boiling water for 3 min (Del Rio and others 2004); (b) boiling water for 5 min (Khokhar and Magnusdottir 2002; Bonoli and others 2003); (c) boiling water for 10 min (Aucamp and others 2000); (d) water at 80 ◦C for 30 min (Sakakibara and others 2003); (e) water at 90 ◦C for 30 min (Lee and Ong 2000); (f ) acetonitrile/water for 1 h (Fernandez and others

          2002);(g) ethanolatroomtemperature(Linandothers 2003);(h) 80%

[pic 4][pic 5]C328  JOURNAL OF FOOD SCIENCE—Vol. 71, Nr. 6, 2006

⃝C 2006 Institute of Food Technologists doi: 10.1111/j.1750-3841.2006.00090.x

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Efficient extraction of tea compounds . . .

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methanol for 3 h, then with 80% methanol containing 0.15% HCl for 3 h (Cabrera and others 2003); (i) 80% acetone for 2 wk (Menet and others 2004); and (j) pressurized liquids (Pineiro and others 2004).

In the course of studies designed to relate tea and rice bran flavonoids to anticarcinogenic and antimicrobial effects, we noted that aqueous ethanol efficiently extracted flavonoids from tea leaves and dark rice brans (Friedman and others 2006; Nam and oth- ers 2006). These observations suggested the need to better define the potential of aqueous ethanol as an extraction solvent for tea flavonoids and alkaloids. We therefore compared levels of flavonoids and alkaloids extracted with aqueous ethanol to levels extracted with boiled water.

Themainobjectivesofthisstudywereto(a) simultaneouslydeter- mine, by a previously validated HPLC method (Friedman and others 2005), levels of 14 tea catechins, theaflavins, and alkaloids in a sin- gle analysis; (b) compare levels of tea constituents extracted with ethanol/water to previously reported levels (except theophylline) of water extracts of the same 77 teas; and (c) compare levels of flavonoids and alkaloids of 15 green tea extracts (10 capsules, 1 gum, and 4 powders) sold at retail to the amounts listed on the labels. The results demonstrate the value of aqueous ethanol for the efficient extraction of tea compounds, and the lack of correlation between observed flavonoid content of the dietary supplements and amounts listed on the labels.

Materials and Methods

Materials

Teas were purchased as tea bags in local markets and restau- rants and from the Stash Tea Co. (Portland, Oreg., U.S.A.). Di- etary green tea supplements were obtained from local stores and commercial companies. (−)-Epigallocatechin, (+)-catechin, (−)- epicatechin, (−)-epigallocatechin gallate, (−)-gallocatechin gallate, (−)-epicatechingallate,(−)-catechingallate, caffeine, theobromine, and theophylline were obtained from Sigma-Aldlrich (St. Louis, Mo., U.S.A.). Theaflavin, theaflavin-3-gallate, theaflavin-3r-gallate, and theaflavin-3, 3r-digallate were obtained from Wako Chemical Co. (Osaka, Japan). HPLC grade solvents were filtered through a 0.45-μM membrane (Millipore, Bedford, Mass., U.S.A.) and de- gassed in an ultrasonic bath before use.