Ind 43834967

8/2/2019 Ind 43834967

http://slidepdf.com/reader/full/ind-43834967 1/10

JFS C: Food Chemistry and Toxicology

HPLC Analysis of Catechins, Theaflavins, andAlkaloids in Commercial Teas and Green TeaDietary Supplements: Comparison ofWaterand 80% Ethanol/Water ExtractsMENDEL FRIEDMAN, C AROL E. LEVIN, SUK -H YUN CHOI, ETSUKO K OZUKUE, AND NOBUYUKI K OZUKUE

ABSTRACT: To help meet the needs of consumers, producers of dietary tea supplements, and researchers for in-formation on health-promoting tea compounds, we compared the following conditions for the extraction of tea leaves and green tea-containing dietary supplements: 80% ethanol/water at 60 ◦C for 15 min and boiled water for5 min. Thefollowing 7 catechins, 4 theaflavins, and3 alkaloidswere separatedin a 70-min single HPLC analysis: (−)-epigallocatechin, (−)-catechin, (+)-epicatechin, (−)-epigallocatechin-3-gallate, (−)–gallocatechin-3-gallate, (−)-epicatechin-3-gallate, (−)-catechin-3-gallate, theaflavin, theaflavin-3-gallate, theaflavin-3-gallate, theaflavin-3,3-digallate, caffeine, theobromine, and theophylline. The following ranges of concentrations of flavonoids (catechins

plustheaflavins) in the tealeaves extracted with 80% ethanol were observed (in mg/g): in 32 black teas, 19.8 to 115.1;in 24 green teas, 12.3 to 136.3; in 14 specialty teas, 4.9 to 118.5; in 7 herbal teas, 0 to 46.0. Total alkaloids in all teasrangedfrom 0 to 32.6 mg/g. Significantly greater amountsof flavonoids were extractedfrom thetea leaveswith aque-ous ethanol than with boiled water. Levels of tea catechins in 10 capsules sold as dietary supplements were about50 to 75% lower than the amounts listed on the labels. Catechin content of 4 commercial green tea extracts rangedfrom 96 to 696 mg/g. The results make it possible to maximize the extraction of tea compounds to better relate theflavonoid and alkaloid content of teas and dietary tea supplements to their health-promoting effects.

Keywords: alkaloids, dietary supplements, flavonoids, HPLC, tea leaves

Introduction

Interest in teas, tea extracts used as dietary supplements, and tea

compoundsarisesfromthefactthatteaflavonoids(catechinsandtheaflavins) arereported to havebeneficialprotective effects against

cholesterol (Maron and others 2003); diabetes (Fukino and others

2005); chronic liver disease (Ruhl and Everhart 2005); breast (Seeley

andothers2005),lung(Yangandothers2005),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-

scribethe useof caffeineto enhancemental activity (Rao andothers

2005) and running performance (McLellan and others 2005) and to

treat apnea (absence of breathing) (Schmidt 2005) and migraine

MS 20060096 Submitted 2/13/2006, Accepted 4/29/2006. Authors Friedmanand Levinare with Western Regional Research Center, Agricultural ResearchService, 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: [email protected]).

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

supplementsand cosmetics toselectteas,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, wehad 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 homeuse (Friedmanand others 2005). Other investigatorsused

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(DelRioand others2004); (b)boilingwaterfor 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(Linandothers2003);(h)80%

C328 JOURNAL OF FOOD SCIENCE—Vol. 71, Nr. 6, 2006 C 2006 Institute of Food Technologistsdoi: 10.1111/j.1750-3841.2006.00090.xFurther reproduction without permission is prohibited

8/2/2019 Ind 43834967


C : F o o d C h e m i s t r y & T o x i c o l o g y

Efficient extraction of tea compounds . . .

methanol for3 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

thataqueousethanolefficientlyextracted flavonoids fromtea 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 andalkaloids.We therefore comparedlevelsof flavonoidsandalkaloidsextractedwithaqueousethanoltolevelsextractedwith

boiled water.

Themainobjectivesofthisstudywereto(a)simultaneouslydeter-

mine, by a previously validatedHPLC method (Friedmanand 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 andalkaloidsof 15greentea extracts (10capsules,1 gum,

and4 powders)sold at retail to theamountslisted on thelabels.The

results demonstrate the value of aqueous ethanol for the efficient

extraction of tea compounds, and the lack of correlation between

observedflavonoidcontentof thedietarysupplements andamountslisted on the labels.

Materials and Methods

MaterialsTeas 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-3-gallate,

and theaflavin-3, 3-digallate were obtained from Wako ChemicalCo. (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.

Extraction and analysis of commercial tea leaves andgreen tea dietary supplements

For extraction with 80% ethanol, each sample (approximately

1.5 g) was placed into a 250-mL flask equipped with a reflux con-

denser. This was followed by addition of 50 mL of a mixture of

ethanol: water (80:20, v/v). Each sample was then heated in a water

bath for 15 min at 60 ◦C, sonicated for 5 min, and then cooled. The

residue was extracted twice more with the same solvent. The com-

bined supernatants were then filtered through a 0.45-μm Milliporenylon filter.

For extraction with water (in this study, only theophylline in all

teas and in green tea products and catechins and alkaloids in green

tea dietary supplements were analyzed), each tea sample (approx-

imately 1.5 g) was placed into a 250-mL flask to which was added

250mL of water previously brought to theboiling point.The sample

was then stirred slowly with a magnetic stirrer for 5 min. The av-

erage temperature during the extraction, determined by measuring

the temperature of the water at 0, 1, 2, 3, 4, and 5 min intervals after

boiling,was approximately 90 ◦C. The cooledsample wasfiltered on

a 0.45 μm Millipore nylon filter before analysis.

The effect of the following variations to the cited conditions on

the efficiency of the extractions were also evaluated: (a) ethanol in

the aqueous-ethanol solvent: 10, 20, 40, 60, 80, or 95%; 60 ◦C for

15 min; and (b) time: 3, 5, 10, 15, or 20 min; 80% ethanol at 60 ◦C.

HPLC was carried out on a Hitachi liquid chromatograph model

665-II equipped with an Autosampler (model 655A-40, Hitachi Ltd.,

Tokyo,Japan).Thestainlesssteelcolumn(250mm×4.0mmi.d.)was

packed with Inertsil ODS-3v (5 μm particle diameter, GL Sciences,

Tokyo, Japan). The column temperature of 30 ◦C was maintained

witha Shimadzucolumn oven CTO-10vp(Shimadzu, Kyoto, Japan).

The gradient system consisted of a mixture of acetonitrile and 20

mMKH2PO4. The flow rate was1 mL/min. A Shimadzuphoto diodearrayUV-VISdetector(modelSPD-10Avp,Kyoto,Japan)was setfrom

200 to700 nm. The tea extract (10μL) wasinjected into thecolumn.

Separateanalyses,each intriplicate,werecarriedout with3 separate

extracts prepared from 3 different tea bags or green tea extracts for

each sample.

Theinitialcompositionofthemobilephase,consistingof7%(v/v)

solvent A (100%acetonitrile)and 93%of solvent B (20mM KH2PO4),

was maintained for 7 min. Solvent A was then increased linearly to

10% at 20 min, 15% at 25 min, 20% at 30 min, and 25% at 45 min to

70 min. Programming was then continued in the isocratic mode as

follows: 40% A at 70.1 to 75.0 min and 7% A at 75.1 to 90.1 min.

Spiking-extraction, linearity of concentration plots, and lim-

its of detection (LOD) were determined as described previously (Friedmanand others 2005). Thisreferencecontains values for these

parameters for 13 tea compounds, but not for theophylline. These

values for theophylline were determined in this study. Theophylline

eluted at approximately13 min, the concentration-response plot

was linear between 0 and 800 ng, LOD was 1.9 ng, and recovery

after spiking ranged from 95 and 107% of added sample. Recoveries

of catechins and alkaloidsfrom spiked green tea powders (capsules)

determined in this study ranged from 85% to 115%.

Statistical analysisThe square root or log transformation was used to stabilize the

variance prior to one-way analyses of variance (ANOVA) between

the 77 teas among the respective sums of 7 catechins, 4 theaflavins,

and 11 catechinsand theaflavins. Zeros were deleted from one-way

analyses (ANOVA) since they would result in a negative bias of the

error variance estimates. Dunnet’s one-tailed test was used to test

for decreases from the 10th highest ranked tea (SAS 2000).

Results and Discussion

Analytical aspectsThe previously validated HPLC-UV method for the analysis of the

compositionofaqueousteaextracts(Friedmanandothers2005)was

alsofoundto be effectivefor 80%ethanol/water extracts. Themobile

phase, consisting of mixturesof acetonitrileand 20 mM KH2PO4 and

programmed in linear/isocratic modes, separated, in a single run,

all 14 catechins, theaflavins, and alkaloids. The elution positions of a mixture of the 14 standardsand chromatograms of representative

black, green, pouching (oolong), and herbal teas as well as 2 green

tea extracts are shown in Figure 1. To our knowledge, this is the first

report on the simultaneous determination of these tea compounds

by HPLC.

We determined the effect of varying the ethanol concentration

from 10% to 95% during aqueous ethanol extraction at 60 ◦C on

the composition of a representative black and green teas. The data

(not shown) indicate that ethanol concentrations up to 80%did not

significantly affect the levels of extracted tea compounds. We also

noted that 80% ethanol/water evaporated at a faster rate than 20%

ethanol/water and that the amounts extracted with 95% ethanol

were lower than with the other ethanol/water mixtures.

URLs and E-mail addresses are active links at www.ift.org Vol. 71, Nr. 6, 2006— JOURNAL OF FOOD SCIENCE C329

8/2/2019 Ind 43834967



Black Tea80% ethanol extract

Green Tea80% ethanol extract

0 20 40 60

Pouchong Tea80% ethanol extract

Herbal Tea80% ethanol extract

1

1

1

1 2

2

2

2

3

3

3

4

4

4

6

6

6

7

7

7

7

8

8

8

9

9

9

10

10

10

11 121314

5

5

A b s o r b a n c e , 2 8 0 n m

1

2

3

4

5

6

78

9

10 11

12

1314

Standards

0 20 40 60

Tea Capsule Hwater extract

1 2 3

4

65

7

8

9

10

Tea Capsule H80% ethanol extract

1 2 3

4

65

7

8

9

10

Tea Powder M

water extract

1 3

4

6

7

89

10

Tea Powder M80% ethanol extract

13

4

6

7

8

9

10

Retention time (min)

Figure 1 --- HPLCchromatograms of amixture of 7 catechins, 4theaflavins, and 3 alkaloidstandards, selected teas,and green tea extracts.Peak 1, theobromine; 2,theophylline; 3,(−)-epigallocatechin; 4,caffeine; 5, catechin; 6,(+)-epicatechin; 7,(−)-epigallocatechingallate; 8, (−)-gallocatechingallate; 9, (−)-epicatechingallate; 10, (−)-catechingallate; 11, theaflavin; 12,theaflavin 3-gallate; 13,theaflavin 3-gallate; 14,theaflavin 3,3-digallate.

Additional studies also showed that varying the extraction time

from3to20minfor80%ethanolat60◦Calsodidnotsignificantlyin-

fluence the levelsof all tea compounds, with the possible exception

of epicatechin-3-gallate. Based on these observations and to avoid

evaporation of ethanol at higher temperaturesduring the workup of

the extracts, we selected 60 ◦C and 15 min as convenient standard

conditions for extractions with the 80% ethanol.

Composition of tea leavesLevels of individual catechins in teas extracted with 80%

ethanol or water. For the 77 teas evaluated in this study we use

samplenumbersbutnotteabrandnames.Thebrandnamesofthese

teas are listed in Friedman and others (2005). Table 1 compares the

composition of these teas after extraction with 80%ethanol at 65 ◦C

for 15 min to amounts extracted with boiled water for 5 min.

Forthe7individualteacatechins,theamountsextractedwith80%

ethanol are in most cases greater than the corresponding levels ex-

tracted with water. The differential trends in the amounts extracted

with the 2 solvents become more apparent for individual and to-

tal theaflavin and total flavonoid (catechin plus theaflavin) levels

outlined below.Levels of individual theaflavins in black teas. For the

theaflavins in black teas extracted with 80% ethanol, the levels (in

mg/g tea) for theaflavin (TF) ranged from 0.4 (#16) to 9.1 (#15) and

for the water extracts from 0 to 5.4 (#15). For theaflavin-3-gallate

(TF3G), the corresponding values are from 0 to 5.9 (#3) and 0 to 2.1(#23); for theaflavin-3-gallate (TF3G), from 0 to 3.8 (#18) and 0 to

0.8 (#21 and #30); and for theaflavin-3,3 -digallate (TF33G), from

1.8 (#28) to 10.9 (#30) and 0 to 6.4 (#31). Green teas contained no

theaflavins. Only 2 of the 14 specialty teas (#60 and #67) and one

of the 7 herbal teas (#74) contained theaflavins. The data show that

significantlygreateramounts of theaflavins were extractedwith 80%

ethanol than with water.Totallevelsofcatechinsandtheaflavins. Itisalsoinstructiveto

compare the trends in the sums of the concentrations of the 7 cate-

chins and4 theaflavins (seetotal FLAV columnin Table1), calculated

from theindividualvalues to thecorresponding sums present in the

water extracts of the same teas. For 32 black teas, the sum of the 11

catechins and theaflavins (in mg/g tea) extracted with 80% ethanol

C330 JOURNAL OF FOOD SCIENCE—Vol. 71, Nr. 6, 2006 URLs and E-mail addresses are active links at www.ift.org

8/2/2019 Ind 43834967


8/2/2019 Ind 43834967



ranged from 19.8 (#4) to 115.9 (#31) and for the water extracts, from

6.5 (#1) to 72.5 (#32). Most black teas contained both catechins and

theaflavins.

The sum of 7 catechins (total CATS column in Table 1) for the 32

black teas (in mg/g tea) extracted with 80%ethanol rangedfrom 9.6

(#3) to 111.0 (#29) and for water extracts, from 5.4 (#1) to 69.5 (#32).

For the sum of the 4 theaflavins (total TFS column in Table 1), the

valuesfor the80% ethanol extracts rangedfrom 3.7(#27) to 20.7 (#3)

and for the water extracts, from 0 to 8.8 (#18). Such high catechin

levels insomeof the black teas appear not to have been reported inthe published studies mentioned above.

The sum of 7 catechins for the 24 green teas (in mg/g dry tea)

extracted with 80%ethanol ranged from 12.3 (#1) to 136.3 (#56) and

for water extracts, from 4.4 (#33) to 100.0 (#56). The green teas con-

tained no theaflavins.

The sum of 7 catechins for the 14 specialty teas (in mg/g tea)

extracted with 80%ethanol ranged from 4.9 (#59) to 118.5 (#70) and

for the water extracts, from 3.4 (#57) to 83.1 (#70). The sum of 7

catechins for the 7 herbal teas (in mg/g tea) extracted with 80%

ethanol ranged from 0 to 46.0 (#77) and for the water extracts, from

1.2 (#71) to 26.4 (#77). The data show that greater total amounts of

flavonoids are extracted by 80% ethanol than by water.

A statistical profile using one-way analyses of variance (ANOVA,Dunnet’s one-tailed test) between 77 teas among the respective

sums of 7 catechins, 4 theaflavins, and 11 catechins and theaflavins

wasusedtodefineasubsetof10teascontainingthehighestamounts

of catechins and theaflavins extracted with both solvents. See foot-

note b to Table 1.

Factors that may affect extraction of tea compounds from

leaves. Extraction of the 7 catechins and 4 theaflavins with 80%

ethanol at 60 ◦C for 15 min was more effective than extracting with

water for 5 min at an average temperature of approximately 90 ◦C,

conditions used worldwide to prepare tea infusions. Both individ-

ual and total levels of catechins and theaflavins extracted with 80%

ethanol differed significantly from amounts extracted with water.

Possible reasons for these differences are not readily apparent, ex-

cept to suggest that geographical origin, soil composition, differ-

ences inthe compositionof different leaves,postharvest treatments,

and physical structure of thedifferent leavesmay influence thesus-

ceptibilities of dry tea leaves to extraction (Wang and others 2000;

Astill and others 2001; Lin and others 2003). It is also likely that

positively charged metal ions (minerals) also present in tea leaves

(Ferraraand others 2001)chemically chelate to phenolicOH groups

of flavonoids and that thefiber matrix of the leaves preferentially or

selectively binds or physically occludes some of the catechins and

theaflavins. Such physicochemical forces may make it more diffi-

cult to extract tea compounds from leaves. Still another possibility

is that levels of high-molecular weight theaflavins exceed solubili-

ties in water but not in theaqueous ethanol. Similar considerations

may govern the extraction of potato glycoalkaloids from leaves of the potato plant (Brown and others 1999).

As mentioned in the Introduction, to extract biologically active

compounds from tea leaves, other investigators used a wide range

of solvents and conditions. The previously reported levels of these

compounds also vary widely. The values we obtained with 80%

ethanol compare favorably with the highest levels reported in the

published studies. In addition,this is thefirst report on theanalysis

of all 14 tea compounds in a single HPLC run.

Purine (methylxanthine) tea alkaloids. Teas also contain 3

purine alkaloids: theobromine, theophylline, and caffeine. Table 1

compares the levels of these alkaloids after extraction with either

80% ethanol at 65 ◦C for 15 min to amounts extracted with boiled

waterfor 5 min. Belowwe compare thetrends in thelevels extracted

with the 2 solvents.

Thecaffeinecontent (inmg/g of tea) forblackteas extractedwith

80% ethanol ranged from 12.9 (#15) to 30.3 (#30) and the amounts

extracted with water, from 3.5 (#1) to 26.7 (#30). For green teas, the

corresponding ranges are from 0.5 (#33) to 26.8 (#51) and from 0 to

23.9 (#41); and for specialty teas, from 4.3 (#58) to 31.9 (#68) and 2.1

(#58) to29.7 (#67).Herbal teas contained noor low levelsof caffeine.

The theobromine content (in mg/g of tea) of all 77 teas extracted

with 80% ethanol ranged from 0.5 (#33) to 2.5 (#71) and with water,from 0 to 3.2 (#30). Theophylline levels ranged from 0 to 1.6 (#17)

for both extracts. Total alkaloid levels for all teas ranged from 0 to

32.7mg/gtea(#68).Mostpreviousstudiesdonotreporttheophylline

levelsof teas. Thestatisticalanalysismentioned earlier was also ap-

plied to caffeine and theobromine levels of the teas. See footnote

b in Table 1. The data show smaller differences in the amounts of

alkaloids extracted by the 2 solvents compared to much larger dif-

ferences for the flavonoids described earlier.

Composition of dietary green tea supplementsGeneral aspects. Although green tea extracts are widely used in

biomedicalstudies,therearefewpublishedstudiesontheactuallev-

els of tea flavonoids and alkaloids in green tea extracts sold at retailas capsules or powders as health-promoting dietary supplements

(neutraceuticals) (Laiand Roy 2004). Onesuch study (Manningand

Roberts 2003) of 7 commercial green tea products determined by

HPLCrevealed widevariability and muchloweramountsin thecon-

tent of 5 catechins (extracted with 80 ◦C water and allowed to steep

for 8 h) compared to claims on the labels. The authors recommend

thatthe phytochemicalcontent of suchdietary supplements should

be standardized so that consumers can make a rational choice.

To contribute to this effort, we determined the flavonoid and al-

kaloid content of 15 commercial green tea extracts sold as capsules

or powders. Table 2 lists these products and the compositional data

as listed on the labels. Table 3 compares the levels of 7 individual

catechins, their sums, levelsof the3 alkaloids, andtheir sums deter-

minedby HPLC with wateror 80%ethanolextracts.Forcapsules, we

also calculated the amounts per capsule listed in the last column of

the table. None of the samples contained theaflavins. What follows

is an attempt to relate our findings to the compositional data listed

on the labels.

Green tea gum and capsules. We determined the content of 7

catechins and 3 alkaloids in 1 product sold at retail as a gum and 10

products sold as capsules. The data in Table 3 show that (a) unlike

the results from tea leaves described earlier, the values determined

with the ethanolic extracts weregenerally only approximately 5% to

10% higher than the corresponding values for both individual and

total catechin levels obtained with the water extracts; (b) the total

catechin content of the gum (A in Table 2 and 3) of 10 mg/piece

is much lower than the 120 mg/piece listed on the label; (c) thetotal catechin content (in mg/g) of the capsules ranged from 41.6

(E in Table 3) to 379.9 (D); the corresponding range per capsule is

from 21.5 (C) to 176.1 (D); and(d) total catechin (polyphenol) levels

obtained in the present study are approximately 50% to 75% lower

than the values listed on the labels of these products.

All capsules also contained caffeine, theobromine, and theo-

phylline. Thedata show that (a) theamounts of individual andtotal

alkaloidsextracted withboth solvents did not differ significantly; (b)

values (in mg/g) for caffeine ranged from 16.7 (C) to 78.5 (F); and

(c) corresponding theobromine levels ranged from approximately

0.4 (C) to approximately 2.5 (G, K) and of theophylline levels, from

approximately 0.1 (C) to 2.2 (D). There is a 3-fold variation in the

C334 JOURNAL OF FOOD SCIENCE—Vol. 71, Nr. 6, 2006 URLs and E-mail addresses are active links at www.ift.org

8/2/2019 Ind 43834967




Table 2 --- Commercial green tea extracts sold as capsules, gums, and powders

Product Name of product Label Size Comments from labels

A Mega-T dietary supplement,green tea chewing gum

120 mg 24 pieces Blend of green tea (leaf) powder, white tea (leaf) powder, andEGCG

B Green tea capsules 100 mg 60 capsules Each capsule contains green tea extract (leaf); 50%catechins (polyphenols)

C Green tea capsules 315 mg 90 capsules Green tea extract (leaf) = 14% polyphenols = 44 mgD White tea capsules 315 mg 90 capsules White leaf and flower extract. 90% polyphenols = 283.5 mgE Green tea capsules 100 mg 120 capsules Green tea extract, 100 mg; catechins (polyphenols), 50 mgF Green and white tea

capsules, organic

500 mg 60 Vcaps White peony and green sencha tea (leaf). Minimum 40%

polyphenols (200 mg), 3 to 6% theanine (15 to 30 mg), and5 to 9% caffeine (25 to 45 mg)

G Green extract capsules 315 mg 100 capsules Natural whole herb, Camellia sinensis ; each capsulecontains the polyphenol catechins of 3 cups of green teawith less than 1/5 caffeine of 1 cup (5 oz.)

H Green tea complex capsules 1000 mg 50 caplets Standardized green tea extract (leaves): 790 mg leaf extract(25% polyphenols, 8.5% EGCG, 5% caffeine) plus greentea powder (leaf), 210 mg

I Mega-T dietary supplementgreen tea

600 mg 30 caplets Metabolic support blend provides 90 mg EGCG and 50 mgcaffeine/capsule; contains green tea and several herbalextracts (fo ti root, gacinia fruit, guarana seed,bladderwrack kelp, and gotu kola whole plant)

J Green tea complex 1000 mg 60 caplets 790 mg green tea extract (Camellia sinensis ) (25%polyphenols, 8.5% EGCG, 5% caffeine); 210 mg green teapowder (Camellia sinensis) (leaf)

K Green tea extract 315 mg 100 capsules Natural whole herb; 2 capsules contain 630 mg green tea

extract (Camellia sinensis ) (aerial) (standardized to contain15% polyphenols, 95 mg)L Instant green tea beverage

mix2.2 g 32 tubes 300 mg of antioxidants per serving from fresh green tea

leavesM Green tea leaf-BRMa 5 g Powder Store at room temperatureN Green tea leaf-XRMa 5 g Powder Store at room temperatureO Green tea polyphenol 10 g Powder Storage: +4 ◦C

aThe characterization data of BRMsTM (Botanical reference material) from a specific genus and species of plant. XRMTM reference material comes with determinedvalues for their chemical composition and physical properties along with the methods used to obtain these values.

alkaloid levels among the capsules, with caffeine approximately 20

times greater than levels of theobromine and theophylline.

A possible reason why the amounts extracted with the 2 solvents

from both capsules and powders do not differ significantly may be

that we are dealing with catechins and alkaloids that have already

been extracted from tea leaves. Strictly speaking, the solvents do

not extract but rather dissolve the catechins from the matrices of

the commercial products. We do not know why catechin levels show

such wide variation among the capsules and why these levels differ

significantly from the amounts listed on the labels. Are catechins

degradedduringstorageby interactingchemically orphysically with

other capsuleconstituentssuch ascellulose,starch, andlactoseused

as fillers (excipients)? This aspect merits study.

Green tea powders. We also determined the catechin and alka-

loid levels of 4 commercial green tea powders. As is the case with

capsules, the amounts extracted with the 2 solvents tended to be

generally similar. Table 3 shows that the catechin levels (in mg/g)

for the 4 products ranged from 96.0 (L) to 696.9 (O), a 7.3-fold vari-

ation from highest to lowest value. Levels of alkaloids ranged fromapproximately 24 (L) to approximately 85 (N), a 3.5-fold variation.

The label on the tea beverage mix (L in Table 2) states that it pro-

vides 300 mg of antioxidants per serving of 2.2 g. The determined

total catechin amount of 206.5 mg per serving corresponds to 69%

of the amount shown on the label. The analytical data provided by

the supplier of the ChromaDex TM XRM powder (M) agree well with

total catechins we are reporting (52.5% for the 80% ethanol extract

and 49.4% for the water extract compared with 52.2% reported by

ChromaDex TM). However, individual catechin in the extracts deter-

mined in this study differed from the data on the label. We found

morethan twice the amount of (−)-epigallocatechin and about half

theamountof(+)-epicatechincompared to their analysis.Our anal-

ysis also revealed the presence of small amounts of gallocatechin-

3-gallate and catechin-3-gallate.

Conclusions

The results obtained in this study indicate that aqueous ethanol

is an excellent solvent for theextractionand analysis in a single

determination by HPLC of 7 tea catechins, 4 theaflavins, and3 alka-

loids from tea leaves. Extraction of tea compounds from the leaves

with80% ethanol at 60 ◦C was generally moreefficient thanreported

extraction of the same teas with hot water alone. The greater ex-

traction efficiency with aqueous ethanol seems to be influenced by

both thetypeof teaas wellas thechemicalstructure ofthe individual

flavonoids.Becausebiologicalpotenciesof individual teaflavonoids

vary widely (Cabrera and others 2003; Xu and others 2004; Cooper

and others 2005a, 2005b; Cushnie and Lamb 2005; Friedman and

others 2006), studies on the relationship between composition and

healtheffects of teas shouldevaluateboth water and ethanol/water

extracts. Thepossible variability in the composition of the same tea

brands originating fromdifferent retailers or tea batches also meritsfurther study.

Because theamountsof teacompounds in greentea dietarysup-

plements extractedwith the 2 solvents were significantly lower than

the amounts listed on the labels, our data suggest that consumers

could benefit by more accurate labeling of dietary supplements for

flavonoid(polyphenol) content.Moreover,because noneof theeval-

uated supplements contained beneficial theaflavins, there is a need

for new supplements containing both catechinsderived from green

teas and theaflavins derived from black teas.

Acknowledgment We thank Dr. Bruce E. Mackey for statistical analysis.


8/2/2019 Ind 43834967


8/2/2019 Ind 43834967




References Astill R, Birch MR, Dacombe C, Humphrey PG, Martin PT. 2001. Factors affecting the

caffeine and polyphenol contents of black and green tea infusions. J Agric FoodChem 49:5340–7.

Aucamp JP, Hara Y, Apostolides Z. 2000. Simultaneous analysis of tea catechins, caf-feine, gallic acid, theanine and ascorbic acid by micellar electrokinetic capillary chromatography. J Chromatogr A 876:235–42.

Barnes PJ. 2006. Drugs for asthma. Br J Pharmacol 147(Suppl 1):S297–303.Berube-Parent S, Pelletier C, Dore J, Tremblay A. 2005. Effects of encapsulated green

tea and Guarana extracts containing a mixture of epigallocatechin-3-gallate andcaffeine on 24 h energy expenditure and fat oxidation in men. Br J Nutr 94:432–6.

Bettuzzi S, Brausi M, Rizzi F, Castagnetti G, Peracchia G, Corti A. 2006. Chemopre-vention of human prostate cancer by oral administration of green tea catechins in

volunteers with high-grade prostate intraepithelial neoplasia: a preliminary reportfrom a one-year proof-of-principle study. Cancer Res 66:1234–40.

BonoliM, ColabufaloP, PelilloM, GallinaToschiT, LerckerG. 2003. Fast determinationof catechins and xanthines in tea beverages by micellar electrokinetic chromatog-raphy. J Agric Food Chem 51:1141–7.

Brown MS, McDonald GM, Friedman M. 1999. Sampling leaves of young potato(Solanum tuberosum) plants for glycoalkaloid analysis. J AgricFood Chem47:2331–4.

Cabrera C, Gimenez R, Lopez MC. 2003. Determination of tea components with an-tioxidant activity. J Agric Food Chem 51:4427–35.

Camouse MM, Hanneman KK, Conrad EP, Baron ED. 2005. Protective effects of teapolyphenols and caffeine. Expert Rev Anticancer Ther 5:1061–8.

Choe E, Min DB. 2006. Chemistry and reactions of reactive oxygen species in foods. JFood Sci 70:R142–59.

Cooper R, Morre DJ, Morre DM. 2005a. Medicinal benefits of green tea: part I. Review of noncancer health benefits. J Altern Complement Med 11:521–8.

Cooper R, Morre DJ,MorreDM. 2005b. Medicinal benefits of green tea:part II.Review of anticancer properties. J Altern Complement Med 11:639–52.

CushnieTP,LambAJ.2005.Antimicrobialactivityofflavonoids.Int JAntimicrobAgents

26:343–56.Del RioD, StewartAJ, Mullen W,Burns J,LeanME, BrighentiF, CrozierA. 2004.HPLC-MSn analysis of phenolic compounds and purine alkaloids in green and black tea.J Agric Food Chem 52:2807–15.

Fernandez PL,PablosF, MartinMJ, GonzalezAG. 2002. Study of catechinand xanthinetea profiles as geographical tracers. J Agric Food Chem 50:1833–9.

Ferrara L, Montesno D, Senatore A. 2001. The distribution of minerals and flavonoidsin the tea plant (Camellia sinensis). Farmaco 56:397–401.

Friedman M. 1997. Chemistry, biochemistry, and dietary role of potato polyphenols. A review. J Agric Food Chem 45:1523–40.

FriedmanM,KimS-Y,LeeS-J,HanG-P,HanJ-S,LeeR-K,KozukueN.2005.Distributionof catechins, theaflavins, caffeine, and theobromine in 77 teas consumed in theUnited States. J Food Sci 70:C550–9.

FriedmanM, HenikaPR, Levin CE,Mandrell RE,Kozukue N.2006. Antimicrobialactiv-ities of tea catechins and t heaflavins and tea extracts against Bacillus cereus . J FoodProt 69:354–61.

Fukino Y, Shimbo M,AokiN, OkuboT, Iso H. 2005.Randomizedcontrolledtrial foraneffect of green tea consumption on insulin resistance and inflammation markers. JNutr Sci Vitaminol ( Tokyo) 51:335–42.

Goldstein J, Silberstein SD, Saper JR, Elkind AH, Smith TR, Gallagher RM, Battikha JP,

Hoffman H, Baggish J. 2005. Acetaminophen, aspirin, and caffeine versus suma-triptan succinate in the early treatment of migraine: results from the ASSET trial.Headache 45:973–82.

Kao YH, Chang HH, Lee MJ, Chen CL. 2006. Tea, obesity, and diabetes. Mol Nutr FoodRes 50:188–210.

Khokhar S, Magnusdottir SG. 2002. Total phenol, catechin, and caffeine contents of teas commonly consumed in the United Kingdom. J Agric Food Chem 50:565–70.

LaiPK, Roy J.2004. Antimicrobialandchemopreventivepropertiesof herbsandspices.Curr Med Chem 11:1451–60.

Lee BL, Ong CN. 2000. Comparative analysis of tea catechins and theaflavins by high-performance liquid chromatography and capillaryelectrophoresis. J Chromatogr A 881:439–47.

LinYS, TsaiYJ,Tsay JS,Lin JK.2003. Factorsaffecting thelevelsof teapolyphenolsandcaffeine in tea leaves. J Agric Food Chem 51:1864–73.

Manning J, Roberts JC. 2003. Analysis of catechin content of commercial green teaproducts. J Herb Pharmacother 3:19–32.

Maron DJ, Lu GP, Cai NS, Wu ZG, Li YH, Chen H, Zhu JQ, Jin XJ, Wouters BC, Zhao J.2003. Cholesterol-lowering effect of a theaflavin-enriched green tea extract: a ran-domized controlled trial. Arch Intern Med 163:1448–53.

McLellan TM, Kamimori GH, Voss DM, Bell DG, Cole KG, Johnson D. 2005. Caffeine

maintains vigilance and improves run times during night operations for SpecialForces. Aviat Space Environ Med 76:647–54.

Menet MC, Sang S, Yang CS, Ho CT, Rosen RT. 2004. Analysis of theaflavins andthearubiginsfrom black tea extract byMALDI-TOF massspectrometry. J AgricFoodChem 52:2455–61.

Murase T, Haramizu S, Shimotoyodome A, Tokimitsu I. 2006. Reduction of diet-induced obesity by a combination of tea-catechin intake and regular swimming.Int J Obes (Lond) 30:561–8.

Nam SH, Choi SP, Kang MY, Koh HJ, Kozukue N, Friedman M. 2005a. Bran extractsfrom pigmented rice seeds inhibit tumor promotion in lymphoblastoid B cells by phorbol ester. Food Chem Toxicol 43:741–5.

Nam SH, Choi SP, Kang MY, Kozukue N, Friedman M. 2005b. Antioxidative, antimu-tagenic, and anticarcinogenic activities of rice bran extracts in chemical and cellassays. J Agric Food Chem 53:816–22.

Nam SH, Choi SP, Kang MY, Koh HJ, Kozukue N, Friedman M. 2006. Antioxidativeactivities of bran extracts from twenty one pigmented rice cultivars. Food Chem94:613–20.

PineiroZ, PalmaM, BarrosoCG. 2004. Determinationof catechinsby means of extrac-tion with pressurized liquids. J Chromatogr A 1026:19–23.

Rao A, Hu H, Nobre AC. 2005. The effects of combined caffeine and glucose drinks on

attention in the human brain. Nutr Neurosci 8:141–53.Ruhl CE, Everhart JE. 2005. Coffee and tea consumption are associated with a lower

incidence of chronic liver disease in the United States. Gastroenterology 129:1928–36.

Sakakibara H, Honda Y, Nakagawa S, Ashida H, Kanazawa K. 2003. Simultaneous de-termination of all polyphenols in vegetables, fruits, and teas. J Agric Food Chem51:571–81.

SAS. 2000. Release 9.1.3. Cary, NC: SAS Institute.SchmidtB. 2005. Methylxanthinetherapyfor apneaof prematurity:evaluationof treat-

ment benefits and risks at age 5 years in the international Caffeine for Apnea of Prematurity (CAP) t rial. Biol Neonate 88:208–13.

Schulz-Stubner S. 2005. Theuse of small-dose theophyllinefor thetreatmentof brady-cardia in patients with spinal cord injury. Anesth Analg 101:1809–11.

SeeleyD,MillsEJ,WuP,VermaS,GuyattGH.2005.Theeffectsofgreenteaconsumptionon incidence of breast cancer and recurrence of breast cancer: a systematic review and meta-analysis. Integr Cancer Ther 4:144–55.

ThornfeldtC. 2005. Cosmeceuticals containingherbs:fact,fiction,and future.Derma-tol Surg 31:873–80.

Wang LF, Kim DM, Lee CY. 2000. Effect of heat processing and storage on flavanols

and sensory qualities of green tea beverage. J Agric Food Chem 48:4227–32. Xu JZ, Yeung SY, Chang Q, Huang Y, Chen ZY. 2004. Comparison of antioxidant ac-tivity and bioavailability of tea epicatechins with their epimers. Br J Nutr 91:873–81.

YangCS,LiaoJ, YangGY,Lu G.2005.Inhibition oflungtumorigenesisby tea. Exp Lung Res 31:135–44.


Ind 43834967

Documents

Transcript of Ind 43834967