RESEARCH
Evaluation of cost versus antioxidant determinants in green tea dietary supplements Cindy Leslie A. Roberson, Ehab A. Abourashed, and Nancy Elsharkawy
Abstract Objective: To investigate the association between cost and (a) chemical constituents and (b) antioxidant activity as quality determinants of select green tea supplements available in the United States. Design/setting: Laboratory analysis of green tea using HPLC and antioxidant assay methods. Main outcome measures: Correlation between selected quality parameters and daily cost based on the serving size as stated in the label. Quality was defined in terms of (a) catechin levels (validated high-performance liquid chromatography method), (b) total phenolic content (Folin–Ciocalteu method), and (c) antioxidant activity (total antioxidant capacity and diphenylpicryl hydrazyl free-radical scavenging). Results: A wide range of variation in marker levels and antioxidant activity was observed in the evaluated products. Catechin levels correlated well with the total phenolic content in each product while antioxidant activities were not as consistent when correlated with catechin/polyphenol levels. There was also a low correlation between product cost and quality. Conclusion: Our results indicate that product cost does not always reflect quality, at least within the selected range of products. Thus, for a pharmacist to be able to recommend quality green tea dietary supplements, factors other than cost should be considered. J Am Pharm Assoc. 2015;55:381–389. doi: 10.1331/JAPhA.2015.14210
Cindy Leslie A. Roberson, PharmD, BCACP, Clinical Assistant Professor, Department of Pharmacy Practice, College of Pharmacy, Chicago State University, Chicago, IL Ehab A. Abourashed, MS, PhD, Associate Professor, Department of Pharmaceutical Sciences, College of Pharmacy, Chicago State University, Chicago, IL Nancy Elsharkawy, PharmD, College of Pharmacy, Chicago State University, Chicago, IL Correspondence: Cindy Leslie A. Roberson, PharmD, BCACP, Department of Pharmacy Practice, College of Pharmacy, Chicago State University, 9501 South King Drive, DH206, Chicago, IL 60628; [email protected] Funding: This work was funded by a grant from the Center for Teaching and Research Excellence, Chicago State University, and was conducted as part of the senior year capstone project for N. Elsharkawy. Previous presentations: 11th Annual Natural Supplements: An Evidenced-Based Update Conference, San Diego, California, January 29 to February 1, 2014, where the paper won first place in the basic science category of the research competition, and American Association of Colleges of Pharmacy, Grapevine, TX, July 26–30, 2014. Disclosure: The authors declare no relevant conflicts of interest or financial relationships. Acknowledgments: Kumar Mukherjee, PhD, for statistical advice. Received September 18, 2014. Accepted for publication February 23, 2015. Published online in advance of print June 12, 2015.
Journal of the American Pharmacists Association
j apha.org
JU L/A U G 2015 | 55:4 |
JAPhA 381
RESEARCH
GREEN TEA COST AND ANTIOXIDANT DETERMINANTS
D
ietary, herbal, and botanical supplementations, which belong to the biologically based therapies of complementary and alternative medicine (CAM), are increasing in popularity as a result of the publics’ desire to attain health promotion and disease prevention.1–3 Findings from the 2007 National Health Interview Survey revealed that about 4 in 10 (38%) adults aged 18 years and older and about 1 in 9 (12%) children use CAM.4 Increased demand has also been heightened by endorsements from the media, which especially targets the more information driven and information seeking “new consumers.”5 Unfortunately, the appeal of a new fad or a new dietary supplement may not necessarily translate to valid scientific findings and can lead consumers to a trail of misleading information. Since the Dietary Supplement Health and Education Act of 1994 (DSHEA) considers dietary supplements to be food, it does not follow the same regulation that prescription and over-the-counter medications have to go through for approval.6 Thus, considerable information regarding the safety, effectiveness, standardization of preparation, and product details may be lacking.6 To make matters worse, new herbal entities continue to be introduced on a regular basis in different forms and from various suppliers. This results in confusion both from the consumer’s perspective and of the health care provider’s professional role in the management and education of patients who con-
Key Points Background: ❚❚
❚❚
❚❚
Use of complementary and alternative medicine and herbal dietary supplements by a considerable section of the population has been on the rise over the last decade. Reliable quality, safety, and efficacy information for many products is still elusive. Green tea dietary supplements are an example of these products. Because of this, a driver for the recommendation of a specific product is often cost. A majority of pharmacists are uncertain about disseminating the existing and incongruent information as well as recommending specific products.
Findings: ❚❚ ❚❚ ❚❚
In the selected range of green tea products, cost does not always reflect quality. Thus, pharmacists should consider other factors when recommending these supplements. Correlation of cost versus quality in other classes of dietary supplements should be further investigated.
382 JAPhA | 5 5:4 | JUL /AUG 2 0 1 5
ja p h a.org
sume these products. One of the most accessible health care providers, pharmacists are considered experts in pharmacology. Although most pharmacists agree that they should provide recommendations with regard to supplementation use, many are uncertain about disseminating the existing and incongruent information.2 Surveys of pharmacists have shown that unfamiliarity with existing references, lack of education, and dissatisfaction with available resources can cause barriers in appropriately recommending dietary supplements.2,7,8 Patients are also less likely to disclose the use of CAM to their health care providers.9 However, biologically based supplements, while “natural,” are not necessarily safe and can be dangerous when used simultaneously with other prescribed medications without the knowledge of health care providers. Thus, with the increased demand for supplementation use, pharmacists must take on the responsibility of educating the public to avoid or minimize harm.
Objectives The goal of this study was to challenge the validity of the statement ‘you get what you pay for’ in herbal dietary supplements. We hypothesized that a direct correlation between cost and quality of antioxidant botanical supplements may not be valid at present. Using validated evaluation methods, we investigated the relationship between cost and quality by targeting products with antioxidant properties. The work presented here investigates the association between cost and (a) chemical constituents and (b) antioxidant activity as quality determinants of select green tea supplements available in the United States.
Methods The major chemical markers of green tea extract are the catechin polyphenols epigallocatechin gallate (EGCG), epicatechin gallate (ECG), epigallocatechin (EGC), and epicatechin (EC).10 These constituents are thought to be responsible for the proposed activities of green tea extracts. Quality was assessed in terms of (a) catechin levels using a validated high-performance liquid chromatography (HPLC) method; (b) total phenolic content using the Folin–Ciocalteu (FC) method; and (c) antioxidant activity using total antioxidant capacity (TAC) and diphenylpicryl hydrazyl (DPPH) free-radical scavenging assays. Product selection A total of 26 green tea (Camellia sinensis, family Theaceae) products were purchased from online vendors and various local and specialty health stores and community pharmacies in Chicago, IL. Various parameters, such as formulation, price range, vendor, and ingredients, were included in the selection process. Each product was alJournal of the American Pharmacists Association
GREEN TEA COST AND ANTIOXIDANT DETERMINANTS
lotted a unique identifier (GT1 through GT26) and the manufacturer’s information was deidentified. Instruments, chemicals, and reagents An LC-2010 HPLC system (Shimadzu, Kyoto, Japan) with a ultraviolet detector, pump, and an autoinjector, equipped with a Hypersil Gold column (C8, 3 μ, 150 mm × 4.6 mm, Thermo Scientific, Waltham, MA) and a Securityguard pre-column (C8, 4.0 mm × 3.0 mm, Phenomenex, Torrance, CA), was used to measure the levels of EC, EGC, ECG, and EGCG in all purchased products. A Synergy 2 plate reader (BioTek, Winooski, VT) and Gen5 software running under Windows XP were used to acquire and process data, respectively, for the total phenolic and antioxidant assays. All standard solutions, calibrators, quality controls, and samples were prepared using HPLC quality solvents (Fisher Scientific, Pittsburgh, PA). Reference catechins, FC, and DPPH reagents assay were obtained from Sigma Aldrich (St. Louis, MO). TAC assay kits were obtained from Zen-Bio (Research Triangle Park, NC). Standard solutions Standard stock solution. EC, EGC, ECG, and EGCG (5.0 mg each) were transferred to a 5-mL volumetric flask. Methanol (ca. 4 mL) was added and the solution was ultrasonicated for 5 minutes and then brought to a final volume of 5 mL with methanol (concentration of each standard: 1.0 mg/mL). Sample preparation. Each product was extracted by mixing the contents of 6 units (capsules, tablets, tea bags) to obtain a 100 mg sample that was transferred into a 15 mL falcon tube containing 3 mL of methanol. For tea bag products, 1000 mg was used instead of 100 mg to compensate for the nature of their content (coarse bulk green tea leaves versus dry extract in capsules and tablets). Each tube was ultrasonicated (FS30D bath, Fisher Scientific, Pittsburgh, PA) for 15 minutes followed by centrifugation (A5810 R centrifuge, Eppendorf, Hamburg, Germany) for 5 minutes. The supernatant solution was transferred to a 10 mL volumetric flask. The procedure was run in triplicate and the decanted solution in each volumetric flask was brought to volume by adding a sufficient amount of methanol (ca. 1 mL) to have sample stock solutions. The concentrations of the sample stock solutions were adjusted according to the assays performed. Determination of green tea catechins Details of the HPLC method have been published elsewhere.11 In brief, the method used a mobile phase of 0.1% formic acid in water (solvent A) and acetonitrile (solvent B) at a flow rate of 1.0 mL/min with a linear gradient programmed as follows for solvent B: 0–0.5 min 5%; 0.5– 8.0 min 25%; 8.01–9.0 min 100%; and 9.01–11.0 min 5%. Peak detection was performed at 280 nm. Chromatographic data were processed with LCSolutions software Journal of the American Pharmacists Association
RESEARCH
(Shimadzu, Kyoto, Japan) running under Windows XP. An aliquot of each sample (10 μL) was transferred to an HPLC vial and further diluted with 990 μL of methanol. The concentration of each sample was calculated as the mean of three injections (5 μL per injection). The levels of catechins (EC, EGC, ECG, and EGCG) were calculated against a 6-point calibration curve. Total phenolic content and antioxidant activity Samples for these assays were prepared from sample stock solutions at dilution factors of 10–250 depending on each product’s catechin content as determined by HPLC. The phenolic content was determined using the FC method and the antioxidant activity was measured using the TAC and DPPH free-radical scavenging assay. All analyses were done in 96–well plates. Procedures for these methods were performed according to manufacturer instructions and are summarized in Table 1. A 5-point calibration curve was generated on the same plate, using gallic acid and trolox as reference compounds for FC and antioxidant assays, respectively. The phenolic content was calculated as percent gallic acid equivalent. The antioxidant activity was similarly calculated as percent trolox equivalent. Each sample was analyzed in triplicate.
Results
Product selection Table 2 provides a summary of the products in the study including formulation, daily cost, quantity per container, and recommended serving per day. A random selection of 26 green tea supplements representing diverse market products was obtained to allow for comparison of such variables as formulation (capsules, tablets, powder, tea bags), strength (regular, double, and triple strengths), lot number (different lots from the same manufacturer), ingredients (green tea only versus multiherb), and marker levels, as stated in the label. The total number of products selected was limited by our research budget. Of all the products included, 19 were capsules, 5 were tablets, and 2 were tea bags. With the exception of two products that are mainly green tea leaves (crude bulk powder), the majority of the supplements were standardized extracts, of which five were composed of multiple herbs and six were combined with green tea crude bulk powder. The retail cost of each product and the manufacturer’s suggested serving size per day were recorded to calculate the cost of supplementation per day, based on manufacturer recommendation. Most products were recommended at one to four daily servings with a daily cost of supplementation ranging from $0.06 to $1.33. Method correlation Figure 1 describes the correlation between methods used in determining quality markers in green tea. A j apha.org
JU L/A U G 2015 | 55:4 |
JAPhA 383
RESEARCH
GREEN TEA COST AND ANTIOXIDANT DETERMINANTS
Table 1. Summary of FC, TAC, and DPPH methods Assay
Calibrator
Calibrator range (µg/mL)
Reagents
Wait time (min)
Detection (nm)
FC
Gallic acid
10–160
Sample (50 µL) + 0.2N FC reagent (µL) + 7.5% Na2CO3 (100uL)
10
650
TAC
Trolox
5–50
Sample (50 µL) + Cu2+ reagent (100 µL)
30
570
DPPH free-radical scavenging
Trolox
1.13–18
Sample (100 µL) + DPPH reagent (100 µL)
30
515
Abbreviations used: FC, Folin-Ciocalteu; TAC, total antioxidant capacity; DPPH, diphenylpicryl hydrazyl.
Table 2. Summary of green tea products used in study Daily costs ($)a
No. capsules per container
No. capsules per day
Total catechins (%)b
Total phenolics (%)b
TAC (%)b
DPPH (%)b
5.8
5.0
23.5
8.0
44.1
60.8
65.6
43.9
Samples
Formulations
GT1c
C, SE
0.11
100
4
GT2
T, SE, M
0.60
40
2
GT3
T, SE, M
0.75
60
3
42.9
57.5
59.9
45.0
GT4
T, SE, M
1.00
80
4
55.9
69.3
598.0
114.5
GT5
C, SE/P
0.24
50
4
20.3
26.7
137.5
48.6
GT6
C, SE
0.26
120
2
51.6
80.0
342.0
71.4
GT7
C, SE
1.33
30
2
70.2
85.7
366.5
78.3
GT8
C, SE
0.20
60
1
27.4
35.3
174.5
59.5
GT9
C, SE
0.06
100
1
32.3
60.0
59.7
37.0
GT10
C, P
0.21
100
3
6.4
4.0
27.0
12.7
GT11
C, SE
0.28
100
3
6.1
4.6
23.3
11.4
GT12
C, SE, M
0.38
60
3
14.5
7.8
27.9
8.0
GT13
C, P
0.08
100
1
17.6
27.0
139.4
40.0
GT14
C, SE
0.62
30
2
67.8
95.8
170.8
43.8
GT15
C, SE/P
0.49
60
2
41.2
59.7
235.5
69.1
GT16
C, SE
0.29
90
2
20.2
32.3
154.4
51.8
GT17
C, SE/P
0.10
90
1
59.3
77.5
187.3
86.8
GT18
C, SE
0.08
120
2
21.7
26.0
128.6
53.9
GT19
C, SE/P
0.25
50
2
21.2
25.3
117.6
48.1
GT20
SC, SE
0.62
60
2
22.9
24.3
110.6
37.0
GT21c
C, SE
0.33
100
4
10.5
7.2
38.7
13.9
GT22
T, SE/P
0.07
60
1
29.3
32.7
164.0
58.5
GT23
T, SE/P
0.59
90
3
39.0
48.0
214.6
64.4
GT26
SC, SE, M
0.82
20
2
24.2
27.0
132.6
52.6
GT24
TB
0.62
16
2
3.8
3.7
25.1
8.3
GT25
TB
0.31
200
4
3.8
3.6
16.5
6.6
Abbreviations used: GT, green tea; C, hard gelatin capsule; SC, soft gelatin capsule; T, tablet; TB, tea bag; P, crude bulk powder; SE, standardized extract (specific catechin and/or polyphenol concentration); M, multiherbal extracts; TAC, total antioxidant capacity; DPPH, diphenylpicryl hydrazyl. Cost per manufacturer’s suggested serving size.
a
Amount per capsule.
b
Different lot same manufacturer.
c
good correlation was obtained between catechins and polyphenols via HPLC and FC methods (Figure 1A, R2 = 0.956). A moderate correlation was obtained between the two antioxidant activity assays as measured via TAC and DPPH, (Figure 1B, R2 = 0.790). In contrast, there 384 JAPhA | 5 5:4 | JUL /AUG 2 0 1 5
ja p h a.org
were low correlations between the amount of catechins and antioxidant activity as obtained via TAC (Figure 1C, R2 = 0.494) and DPPH (Figure 1D, R2 = 0.620) assays. P
Evaluation of cost versus antioxidant determinants in green tea dietary supplements Cindy Leslie A. Roberson, Ehab A. Abourashed, and Nancy Elsharkawy
Abstract Objective: To investigate the association between cost and (a) chemical constituents and (b) antioxidant activity as quality determinants of select green tea supplements available in the United States. Design/setting: Laboratory analysis of green tea using HPLC and antioxidant assay methods. Main outcome measures: Correlation between selected quality parameters and daily cost based on the serving size as stated in the label. Quality was defined in terms of (a) catechin levels (validated high-performance liquid chromatography method), (b) total phenolic content (Folin–Ciocalteu method), and (c) antioxidant activity (total antioxidant capacity and diphenylpicryl hydrazyl free-radical scavenging). Results: A wide range of variation in marker levels and antioxidant activity was observed in the evaluated products. Catechin levels correlated well with the total phenolic content in each product while antioxidant activities were not as consistent when correlated with catechin/polyphenol levels. There was also a low correlation between product cost and quality. Conclusion: Our results indicate that product cost does not always reflect quality, at least within the selected range of products. Thus, for a pharmacist to be able to recommend quality green tea dietary supplements, factors other than cost should be considered. J Am Pharm Assoc. 2015;55:381–389. doi: 10.1331/JAPhA.2015.14210
Cindy Leslie A. Roberson, PharmD, BCACP, Clinical Assistant Professor, Department of Pharmacy Practice, College of Pharmacy, Chicago State University, Chicago, IL Ehab A. Abourashed, MS, PhD, Associate Professor, Department of Pharmaceutical Sciences, College of Pharmacy, Chicago State University, Chicago, IL Nancy Elsharkawy, PharmD, College of Pharmacy, Chicago State University, Chicago, IL Correspondence: Cindy Leslie A. Roberson, PharmD, BCACP, Department of Pharmacy Practice, College of Pharmacy, Chicago State University, 9501 South King Drive, DH206, Chicago, IL 60628; [email protected] Funding: This work was funded by a grant from the Center for Teaching and Research Excellence, Chicago State University, and was conducted as part of the senior year capstone project for N. Elsharkawy. Previous presentations: 11th Annual Natural Supplements: An Evidenced-Based Update Conference, San Diego, California, January 29 to February 1, 2014, where the paper won first place in the basic science category of the research competition, and American Association of Colleges of Pharmacy, Grapevine, TX, July 26–30, 2014. Disclosure: The authors declare no relevant conflicts of interest or financial relationships. Acknowledgments: Kumar Mukherjee, PhD, for statistical advice. Received September 18, 2014. Accepted for publication February 23, 2015. Published online in advance of print June 12, 2015.
Journal of the American Pharmacists Association
j apha.org
JU L/A U G 2015 | 55:4 |
JAPhA 381
RESEARCH
GREEN TEA COST AND ANTIOXIDANT DETERMINANTS
D
ietary, herbal, and botanical supplementations, which belong to the biologically based therapies of complementary and alternative medicine (CAM), are increasing in popularity as a result of the publics’ desire to attain health promotion and disease prevention.1–3 Findings from the 2007 National Health Interview Survey revealed that about 4 in 10 (38%) adults aged 18 years and older and about 1 in 9 (12%) children use CAM.4 Increased demand has also been heightened by endorsements from the media, which especially targets the more information driven and information seeking “new consumers.”5 Unfortunately, the appeal of a new fad or a new dietary supplement may not necessarily translate to valid scientific findings and can lead consumers to a trail of misleading information. Since the Dietary Supplement Health and Education Act of 1994 (DSHEA) considers dietary supplements to be food, it does not follow the same regulation that prescription and over-the-counter medications have to go through for approval.6 Thus, considerable information regarding the safety, effectiveness, standardization of preparation, and product details may be lacking.6 To make matters worse, new herbal entities continue to be introduced on a regular basis in different forms and from various suppliers. This results in confusion both from the consumer’s perspective and of the health care provider’s professional role in the management and education of patients who con-
Key Points Background: ❚❚
❚❚
❚❚
Use of complementary and alternative medicine and herbal dietary supplements by a considerable section of the population has been on the rise over the last decade. Reliable quality, safety, and efficacy information for many products is still elusive. Green tea dietary supplements are an example of these products. Because of this, a driver for the recommendation of a specific product is often cost. A majority of pharmacists are uncertain about disseminating the existing and incongruent information as well as recommending specific products.
Findings: ❚❚ ❚❚ ❚❚
In the selected range of green tea products, cost does not always reflect quality. Thus, pharmacists should consider other factors when recommending these supplements. Correlation of cost versus quality in other classes of dietary supplements should be further investigated.
382 JAPhA | 5 5:4 | JUL /AUG 2 0 1 5
ja p h a.org
sume these products. One of the most accessible health care providers, pharmacists are considered experts in pharmacology. Although most pharmacists agree that they should provide recommendations with regard to supplementation use, many are uncertain about disseminating the existing and incongruent information.2 Surveys of pharmacists have shown that unfamiliarity with existing references, lack of education, and dissatisfaction with available resources can cause barriers in appropriately recommending dietary supplements.2,7,8 Patients are also less likely to disclose the use of CAM to their health care providers.9 However, biologically based supplements, while “natural,” are not necessarily safe and can be dangerous when used simultaneously with other prescribed medications without the knowledge of health care providers. Thus, with the increased demand for supplementation use, pharmacists must take on the responsibility of educating the public to avoid or minimize harm.
Objectives The goal of this study was to challenge the validity of the statement ‘you get what you pay for’ in herbal dietary supplements. We hypothesized that a direct correlation between cost and quality of antioxidant botanical supplements may not be valid at present. Using validated evaluation methods, we investigated the relationship between cost and quality by targeting products with antioxidant properties. The work presented here investigates the association between cost and (a) chemical constituents and (b) antioxidant activity as quality determinants of select green tea supplements available in the United States.
Methods The major chemical markers of green tea extract are the catechin polyphenols epigallocatechin gallate (EGCG), epicatechin gallate (ECG), epigallocatechin (EGC), and epicatechin (EC).10 These constituents are thought to be responsible for the proposed activities of green tea extracts. Quality was assessed in terms of (a) catechin levels using a validated high-performance liquid chromatography (HPLC) method; (b) total phenolic content using the Folin–Ciocalteu (FC) method; and (c) antioxidant activity using total antioxidant capacity (TAC) and diphenylpicryl hydrazyl (DPPH) free-radical scavenging assays. Product selection A total of 26 green tea (Camellia sinensis, family Theaceae) products were purchased from online vendors and various local and specialty health stores and community pharmacies in Chicago, IL. Various parameters, such as formulation, price range, vendor, and ingredients, were included in the selection process. Each product was alJournal of the American Pharmacists Association
GREEN TEA COST AND ANTIOXIDANT DETERMINANTS
lotted a unique identifier (GT1 through GT26) and the manufacturer’s information was deidentified. Instruments, chemicals, and reagents An LC-2010 HPLC system (Shimadzu, Kyoto, Japan) with a ultraviolet detector, pump, and an autoinjector, equipped with a Hypersil Gold column (C8, 3 μ, 150 mm × 4.6 mm, Thermo Scientific, Waltham, MA) and a Securityguard pre-column (C8, 4.0 mm × 3.0 mm, Phenomenex, Torrance, CA), was used to measure the levels of EC, EGC, ECG, and EGCG in all purchased products. A Synergy 2 plate reader (BioTek, Winooski, VT) and Gen5 software running under Windows XP were used to acquire and process data, respectively, for the total phenolic and antioxidant assays. All standard solutions, calibrators, quality controls, and samples were prepared using HPLC quality solvents (Fisher Scientific, Pittsburgh, PA). Reference catechins, FC, and DPPH reagents assay were obtained from Sigma Aldrich (St. Louis, MO). TAC assay kits were obtained from Zen-Bio (Research Triangle Park, NC). Standard solutions Standard stock solution. EC, EGC, ECG, and EGCG (5.0 mg each) were transferred to a 5-mL volumetric flask. Methanol (ca. 4 mL) was added and the solution was ultrasonicated for 5 minutes and then brought to a final volume of 5 mL with methanol (concentration of each standard: 1.0 mg/mL). Sample preparation. Each product was extracted by mixing the contents of 6 units (capsules, tablets, tea bags) to obtain a 100 mg sample that was transferred into a 15 mL falcon tube containing 3 mL of methanol. For tea bag products, 1000 mg was used instead of 100 mg to compensate for the nature of their content (coarse bulk green tea leaves versus dry extract in capsules and tablets). Each tube was ultrasonicated (FS30D bath, Fisher Scientific, Pittsburgh, PA) for 15 minutes followed by centrifugation (A5810 R centrifuge, Eppendorf, Hamburg, Germany) for 5 minutes. The supernatant solution was transferred to a 10 mL volumetric flask. The procedure was run in triplicate and the decanted solution in each volumetric flask was brought to volume by adding a sufficient amount of methanol (ca. 1 mL) to have sample stock solutions. The concentrations of the sample stock solutions were adjusted according to the assays performed. Determination of green tea catechins Details of the HPLC method have been published elsewhere.11 In brief, the method used a mobile phase of 0.1% formic acid in water (solvent A) and acetonitrile (solvent B) at a flow rate of 1.0 mL/min with a linear gradient programmed as follows for solvent B: 0–0.5 min 5%; 0.5– 8.0 min 25%; 8.01–9.0 min 100%; and 9.01–11.0 min 5%. Peak detection was performed at 280 nm. Chromatographic data were processed with LCSolutions software Journal of the American Pharmacists Association
RESEARCH
(Shimadzu, Kyoto, Japan) running under Windows XP. An aliquot of each sample (10 μL) was transferred to an HPLC vial and further diluted with 990 μL of methanol. The concentration of each sample was calculated as the mean of three injections (5 μL per injection). The levels of catechins (EC, EGC, ECG, and EGCG) were calculated against a 6-point calibration curve. Total phenolic content and antioxidant activity Samples for these assays were prepared from sample stock solutions at dilution factors of 10–250 depending on each product’s catechin content as determined by HPLC. The phenolic content was determined using the FC method and the antioxidant activity was measured using the TAC and DPPH free-radical scavenging assay. All analyses were done in 96–well plates. Procedures for these methods were performed according to manufacturer instructions and are summarized in Table 1. A 5-point calibration curve was generated on the same plate, using gallic acid and trolox as reference compounds for FC and antioxidant assays, respectively. The phenolic content was calculated as percent gallic acid equivalent. The antioxidant activity was similarly calculated as percent trolox equivalent. Each sample was analyzed in triplicate.
Results
Product selection Table 2 provides a summary of the products in the study including formulation, daily cost, quantity per container, and recommended serving per day. A random selection of 26 green tea supplements representing diverse market products was obtained to allow for comparison of such variables as formulation (capsules, tablets, powder, tea bags), strength (regular, double, and triple strengths), lot number (different lots from the same manufacturer), ingredients (green tea only versus multiherb), and marker levels, as stated in the label. The total number of products selected was limited by our research budget. Of all the products included, 19 were capsules, 5 were tablets, and 2 were tea bags. With the exception of two products that are mainly green tea leaves (crude bulk powder), the majority of the supplements were standardized extracts, of which five were composed of multiple herbs and six were combined with green tea crude bulk powder. The retail cost of each product and the manufacturer’s suggested serving size per day were recorded to calculate the cost of supplementation per day, based on manufacturer recommendation. Most products were recommended at one to four daily servings with a daily cost of supplementation ranging from $0.06 to $1.33. Method correlation Figure 1 describes the correlation between methods used in determining quality markers in green tea. A j apha.org
JU L/A U G 2015 | 55:4 |
JAPhA 383
RESEARCH
GREEN TEA COST AND ANTIOXIDANT DETERMINANTS
Table 1. Summary of FC, TAC, and DPPH methods Assay
Calibrator
Calibrator range (µg/mL)
Reagents
Wait time (min)
Detection (nm)
FC
Gallic acid
10–160
Sample (50 µL) + 0.2N FC reagent (µL) + 7.5% Na2CO3 (100uL)
10
650
TAC
Trolox
5–50
Sample (50 µL) + Cu2+ reagent (100 µL)
30
570
DPPH free-radical scavenging
Trolox
1.13–18
Sample (100 µL) + DPPH reagent (100 µL)
30
515
Abbreviations used: FC, Folin-Ciocalteu; TAC, total antioxidant capacity; DPPH, diphenylpicryl hydrazyl.
Table 2. Summary of green tea products used in study Daily costs ($)a
No. capsules per container
No. capsules per day
Total catechins (%)b
Total phenolics (%)b
TAC (%)b
DPPH (%)b
5.8
5.0
23.5
8.0
44.1
60.8
65.6
43.9
Samples
Formulations
GT1c
C, SE
0.11
100
4
GT2
T, SE, M
0.60
40
2
GT3
T, SE, M
0.75
60
3
42.9
57.5
59.9
45.0
GT4
T, SE, M
1.00
80
4
55.9
69.3
598.0
114.5
GT5
C, SE/P
0.24
50
4
20.3
26.7
137.5
48.6
GT6
C, SE
0.26
120
2
51.6
80.0
342.0
71.4
GT7
C, SE
1.33
30
2
70.2
85.7
366.5
78.3
GT8
C, SE
0.20
60
1
27.4
35.3
174.5
59.5
GT9
C, SE
0.06
100
1
32.3
60.0
59.7
37.0
GT10
C, P
0.21
100
3
6.4
4.0
27.0
12.7
GT11
C, SE
0.28
100
3
6.1
4.6
23.3
11.4
GT12
C, SE, M
0.38
60
3
14.5
7.8
27.9
8.0
GT13
C, P
0.08
100
1
17.6
27.0
139.4
40.0
GT14
C, SE
0.62
30
2
67.8
95.8
170.8
43.8
GT15
C, SE/P
0.49
60
2
41.2
59.7
235.5
69.1
GT16
C, SE
0.29
90
2
20.2
32.3
154.4
51.8
GT17
C, SE/P
0.10
90
1
59.3
77.5
187.3
86.8
GT18
C, SE
0.08
120
2
21.7
26.0
128.6
53.9
GT19
C, SE/P
0.25
50
2
21.2
25.3
117.6
48.1
GT20
SC, SE
0.62
60
2
22.9
24.3
110.6
37.0
GT21c
C, SE
0.33
100
4
10.5
7.2
38.7
13.9
GT22
T, SE/P
0.07
60
1
29.3
32.7
164.0
58.5
GT23
T, SE/P
0.59
90
3
39.0
48.0
214.6
64.4
GT26
SC, SE, M
0.82
20
2
24.2
27.0
132.6
52.6
GT24
TB
0.62
16
2
3.8
3.7
25.1
8.3
GT25
TB
0.31
200
4
3.8
3.6
16.5
6.6
Abbreviations used: GT, green tea; C, hard gelatin capsule; SC, soft gelatin capsule; T, tablet; TB, tea bag; P, crude bulk powder; SE, standardized extract (specific catechin and/or polyphenol concentration); M, multiherbal extracts; TAC, total antioxidant capacity; DPPH, diphenylpicryl hydrazyl. Cost per manufacturer’s suggested serving size.
a
Amount per capsule.
b
Different lot same manufacturer.
c
good correlation was obtained between catechins and polyphenols via HPLC and FC methods (Figure 1A, R2 = 0.956). A moderate correlation was obtained between the two antioxidant activity assays as measured via TAC and DPPH, (Figure 1B, R2 = 0.790). In contrast, there 384 JAPhA | 5 5:4 | JUL /AUG 2 0 1 5
ja p h a.org
were low correlations between the amount of catechins and antioxidant activity as obtained via TAC (Figure 1C, R2 = 0.494) and DPPH (Figure 1D, R2 = 0.620) assays. P
