Processing, properties and benefits of green tea

Green tea, like the other types of tea, is an infusion of dried and processed leaves of Camellia sinensis, a member of the Theaceae family.
The processing of the leaves leading to the product ready for use is such as to minimize the oxidation of the compounds contained in them, particularly phytochemicals such as catechins, which are polyphenols belonging to the class of flavonoids and the most responsible for the health benefits of green tea.
Having undergone no significant chemical modifications, leaves retain green color, whereas the beverage, prepared with one tea bag per person, or in case of loose tea, one teaspoon per person, for an infusion time of about 3 minutes in water at 75 °C, is golden yellow in color.
Some organoleptic properties of green tea, such as the flavor, that is more delicate and lighter than that of black tea, and the health properties, which have always been recognized in East Asia cultures, depend on leaf processing.
Only recently scientists started studying the health benefits of tea consumption, highlighting its role in preventing many diseases, such as cardiovascular diseases and some types of cancer.
It has been shown that tea polyphenols, particularly catechins, are able to activate intracellular signaling pathways by binding to membrane receptors and/or entering the cell and binding to cytoplasmic, mitochondrial or nuclear receptors. Then, depending on the cell type, they activate or inhibit some cellular processes.
Given the high consumption of tea in the world, even small effects on health could have significant effects on public health.

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Camellia sinensis

Camellia sinensis is an evergreen plant native to South, East, and Southeast Asia, which is now cultivated in at least 30 countries, mostly in tropical or subtropical climates, although some varieties grow in Cornwall and Washington State.
In nature, Camellia sinensis can grow up to 15-20 meters (49-65 ft), whereas in plantations it is pruned to less than 1,5 meters to facilitate leaf harvesting.
It can grow up to altitude of 1,500-2,000 meters (4,900-6,550 ft), and many of the high-quality teas are produced from such crops, as the plant grows slowly and the leaves acquire a better flavor.
The most cultivated varieties, of the four known, are Camellia sinensis var. sinensis, native to China, and Camellia sinensis var. assamica, native to India.
The different types of tea are produced from fresh leaves. Young leaves are preferred over older leaves that are considered to be inferior in quality.
Fresh leaves are rich in water-soluble polyphenols, especially catechins and glycosylated catechins. The major catechins in green tea are epigallocatechin-3-gallate or EGCG, the most active, epigallocatechin, epicatechin 3-gallate, epicatechin. Catechin, gallocatechin, catechin gallate, and gallocatechin gallate are also present, although in lower amount.

Skeletal formula of gallocatechin gallate, one of the catechins found in green tea
Gallocatechin gallate

These polyphenols account for 30%-42% of the dry leaf weight. Caffeine accounts for 1,5-4,5% of the dry leaf weight.
In addition to leaf processing, the organoleptic properties of the beverage are influenced by cultivar, characteristics of the soil where the plant grown up, methods of cultivation, altitude, climate, and time of year in which leaf harvest occurs.

How green tea is made

The differences in leaf processing, which lead to the different types of tea ready for consumption, cause different degrees of oxidation of the compounds present in them, especially catechins.
During green tea manufacturing, oxidative processes, both enzymatic and chemical, are minimized. After harvesting, leaves are exposed to sunlight for 2-3 hours and withered/dried. Then, the processing proceeds through three steps:

  • heat treatment;
  • rolling;
  • drying.

Heat treatment, short and gentle, is crucial for the quality and properties of the beverage. It can done either with a steam, the traditional Japanese method, or by dry cooking in hot pans, that is similar to a roasting method and is the traditional Chinese method. Heat treatment inactivates enzymes and then prevents the enzymatic oxidation processes, particularly those involving polyphenols. It also removes the grassy smell, and evaporates, in the case of the traditional Chinese method, part of the water of the leaf, which constitutes about 75% of its weight, making it softer, thus facilitating the next step.
Heat treatment is followed by the rolling step, that facilitates the subsequent drying step and, destroying the leaf tissue, favors the release of aromas, thus improving the quality of the product.
The drying, the last step, improves the appearance of beverage and leads to the production of new compounds.

Health benefits of green tea

In East Asia cultures, mainly in China and Japan, tea drinking has always been associated with health benefits. Below is a brief review of the results of epidemiological and laboratory studies that have analyzed the effects that green tea consumption can play in preventing many diseases. EGCG, which is the most abundant catechin in green tea accounting for about 60% of the polyphenols present in dried leaves, seems to play the main role.
At the molecular level, the galloyl groups at positions 3 and/or 3′ appear to be essential for many of the effects exerted by catechins.

Cardiovascular disease

Cardiovascular disease is the main cause of deaths worldwide, particularly in low- and middle-income countries, with an estimate of about 17 million deaths in 2008 that could increase up to 23.3 million by 2030.
Daily tea consumption, especially green tea, seems to be associated with a reduced risk of developing cardiovascular disease, such as hypertension and stroke.
Among the proposed mechanisms, the improved bioactivity of the endothelium-derived vasodilator nitric oxide, due to the action of tea polyphenols that could enhance nitric oxide synthesis and/or decrease its breakdown by superoxide anions, seem to be important.


Cancer

Several epidemiological and laboratory studies have shown encouraging results with respect to the preventive role of tea consumption, especially green tea, against the development of some cancers such as those of the oral cavity, digestive tract, and lung among those who have never smoked.
Tea polyphenols seem to act not only as antioxidants, but also as compounds that, directly, can influence gene expression and various metabolic pathways.

Antiviral activity

Recent studies have highlighted antiviral effects of catechins, particularly EGCG of green tea and theaflavins of black tea, especially against positive single-stranded RNA viruses, to which the Coronaviridae family, and then SARS-CoV-1 and SARS-CoV-2, belongs.
The antiviral properties of EGCG appear to be due to its structural characteristics, namely, the presence of pyrogallic and galloyl groups.

Starch digestion

In vitro studies have shown that green tea and black tea polyphenols can reduce the glycemic index of starchy foods. Hence, they could be useful in controlling their glycemic index in vivo. This effect seems to be due to the inhibition of pancreatic alpha-amylase and other digestive enzymes, and to a direct interaction between starch and phytochemicals that would reduce the surface area of starch granules available for enzyme activity. Green tea appears to be equally effective against both gluten-containing foods, against which black tea appears less effective, and gluten-free foods. For more information, see the article on tea polyphenols.

Weight loss

During weight loss and weight-loss maintenance it is important to keep as constant as possible the daily energy expenditure.
Since the 90s, it has been proposed that green tea, by virtue of its content of caffeine and catechins could be of help for:

  • maintaining, or even increasing, daily energy expenditure;
  • increasing fat oxidation.

In addition to these potential lipolytic and thermogenic effects, catechins and caffeine could act on other targets, such as intestinal absorption of fat and energy intake, perhaps through their effect on gut microbiota and gene expression.
And products for weight loss and weight maintenance based on green tea extracts have been marketed. It should be noted that these products contain catechins and caffeine in much higher amount than beverage.
How much truth is there in green tea’s fat burning effect?
The issue seems to have been resolved by a meta-analysis of 15 studies on weight loss and intake of these products. The study showed that green tea-based products induces, in overweight and obese adults, a weight loss that is:

  • not statistically significant;
  • very small;
  • probably not clinically important.

Hence, on the basis of scientific evidence, green tea does not seem to be helpful in fat loss nor in weight maintenance.

Anticariogenic activity

Animal and in vitro studies have shown that tea, and particularly its polyphenols, seem to possess:

  • antibacterial activity against cariogenic bacteria, such as Streptococcus mutans, as in the case of  green tea EGCG;
  • inhibitory action on salivary and bacterial amylase, in which black tea thearubigins and theaflavins are more effective than green tea catechins;
  • inhibitory action on acid production in the oral cavity.

All these properties make green tea and black tea beverages with potential anticariogenic activity.

References

Arab L., Khan F., and Lam H. Tea consumption and cardiovascular disease risk. Am J Clin Nutr 2013;98:1651S-1659S. doi:10.3945/ajcn.113.059345

Clifford M.N., van der Hooft J.J.J., and Crozier A. Human studies on the absorption, distribution, metabolism, and excretion of tea polyphenols. Am J Clin Nutr 2013;98:1619S-1630S. doi:10.3945/ajcn.113.058958

Dwyer J.T. and Peterson J. Tea and flavonoids: where we are, where to go next. Am J Clin Nutr 2013;98:1611S-1618S. doi:10.3945/ajcn.113.059584

Goenka P., Sarawgi A., Karun V., Nigam A.G., Dutta S., Marwah N. Camellia sinensis (Tea): implications and role in preventing dental decay. Phcog Rev 2013;7:152-156. doi:10.4103/0973-7847.120515

Green R.J., Murphy A.S., Schulz B., Watkins B.A. and Ferruzzi M.G. Common tea formulations modulate in vitro digestive recovery of green tea catechins. Mol Nutr Food Res 2007;51(9):1152-1162. doi:10.1002/mnfr.200700086

Grassi D., Desideri G., Di Giosia P., De Feo M., Fellini E., Cheli P., Ferri L., and Ferri C. Tea, flavonoids, and cardiovascular health: endothelial protection. Am J Clin Nutr 2013;98:1660S-1666S. doi:10.3945/ajcn.113.058313

Huang W-Y., Lin Y-R., Ho R-F., Liu H-Y., and Lin Y-S. Effects of water solutions on extracting green tea leaves. Sci World J 2013;Article ID 368350. doi:10.1155/2013/368350

Hursel R. and Westerterp-Plantenga M.S. Catechin- and caffeine-rich teas for control of body weight in humans. Am J Clin Nutr 2013;98:1682S-1693S. doi:10.3945/ajcn.113.058396

Hursel R., Viechtbauer W. and Westerterp-Plantenga M.S. The effects of green tea on weight loss and weight maintenance: a meta-analysis. Int J Obesity 2009;33:956-961. doi:10.1038/ijo.2009.135

Jurgens T.M., Whelan A.M., Killian L., Doucette S., Kirk S., Foy E. Green tea for weight loss and weight maintenance in overweight or obese adults. Editorial group: Cochrane Metabolic and Endocrine Disorders Group. 2012:12 Art. No.: CD008650. doi:10.1002/14651858.CD008650.pub2

Lambert J.D. Does tea prevent cancer? Evidence from laboratory and human intervention studies. Am J Clin Nutr 2013;98:1667S-1675S. doi:10.3945/ajcn.113.059352

Lorenz M. Cellular targets for the beneficial actions of tea polyphenols. Am J Clin Nutr 2013;98:1642S-1650S. doi:10.3945/ajcn.113.058230

Mathur A., Gopalakrishnan D., Mehta V., Rizwan S.A., Shetiya S.H., Bagwe S. Efficacy of green tea-based mouthwashes on dental plaque and gingival inflammation: a systematic review and meta-analysis. Indian J Dent Res 2018;29(2):225-232. doi:10.4103/ijdr.IJDR_493_17

Mhatre S., Srivastava T., Naik S., Patravale V. Antiviral activity of green tea and black tea polyphenols in prophylaxis and treatment of COVID-19: a review. Phytomedicine 2020;153286. doi:10.1016/j.phymed.2020.153286

Sharma V.K., Bhattacharya A., Kumar A. and Sharma H.K. Health benefits of tea consumption. Trop J Pharm Res 2007;6(3):785-792.

Yang Y-C., Lu F-H., Wu J-S., Wu C-H., Chang C-J. The protective effect of habitual tea consumption on hypertension. Arch Intern Med 2004;164:1534-1540. doi:10.1001/archinte.164.14.1534

Yuan J-M. Cancer prevention by green tea: evidence from epidemiologic studies. Am J Clin Nutr 2013;98:1676S-1681S. doi:10.3945/ajcn.113.058271

Xu J., Xu Z., Zheng W. A review of the antiviral role of green tea catechins. Molecules 2017;22(8):1337. doi:10.3390/molecules22081337

Blood pressure, hypertension and dietary sodium

A high sodium (Na+) intake (the main source is salt or sodium chloride NaCl) contributes to blood pressure raise, and hypertension development.
Many epidemiologic studies, animal studies, migration studies, clinical trials, and meta-analyses of trials support this, with the final evidence from rigorously controlled, dose-response trials. Furthermore, in primitive society Na+ intake is very low and people experience very low hypertension, and the blood pressure increase with age does not occur.
Probably, sodium intake effect sizes are to be underestimated!

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Recommended daily intake

Sodium’s physiologic requires are very low; in fact, the minimum recommended Na+ intake for maintain life is 250 mg/day (Note: iodized salt is an important source of dietary iodine in the United States and worldwide).
An Americans consumes the mineral in great excess of physiologic requires: despite the guidelines from the Departments of Agriculture and Health and Human Services, during the period from 2005 through 2006 the average salt intake in USA is of 10.4 g/day for the average man and 7.3 for the average woman, amount in excess regarding preceding years.
A study published on February 2010 on “The New England Journal of Medicine” have shown that “A population-wide reduction in dietary salt of 3 g per day (1200 mg of Na+ per day) is projected to reduce the annual number of new cases of coronary heart disease (CHD) by 60,000 to 120,000, stroke by 32,000 to 66,000, and myocardial infarction by 54,000 to 99,000 and to reduce the annual number of deaths from any cause by 44,000 to 92,000″ (Bibbins-Domingo et all., see References). These benefits are similar in magnitude to those from:

  • a 50% reduction in tobacco use;
  • a 5% reduction in body mass index among obese adults;
  • a reduction in cholesterol levels.

These benefits regard all adult group age, black and nonblack, male and female. The benefits for black are greater than nonblack, in both sex and all age group. It’s estimated an annual savings of $10 billion to 24 $ billion in health care costs.
Clinical trials have also documented that a reduced Na+ intake can lower blood pressure in the setting of antihypertensive medication, and can facilitate hypertension control.
But, in USA dietary salt intake is on the rise!
So, it is recommended, to prevent hypertension development, a reduction in its intake and, in view of the available food supply and the currently daily Na+ intake, a reasonable recommendation is an upper limit of 2.3 g/day (5.8 g/day of salt).
How achieves this level? It can be achieved:

  • cooking with as little salt as possible;
  • refraining from adding salt at the table;
  • avoiding highly salted, processed foods.

Food sources of sodium

They include:

  • salt used at the table: up to 20% of the daily salt intake;
  • salt or sodium compounds added during preparation or processing foods: between 35 to 80% of the daily sodium intake comes from processed foods.A major source of sodium is salt, or sodium chlorideWhich foods are?
    Processed, smoked or cured meat and fish e.g. sliced salami, sausage, salt pork, tuna fish in oil etc.; meat extracts and sauce, salted snack, soy sauce, barbecue sauce, commercial salad dressing; prepackage frozen foods; canned soup, canned legumes; cheese etc.
    There are also many sodium-containing additives as disodium phosphate (e.g. in cereals, ice cream, cheese), monosodium glutamate (i.e. meat, soup, condiments), sodium alginate (e.g. in ice creams), sodium benzoate (e.g. in fruit juice), sodium hydroxide (e.g. in pretzels, cocoa product), sodium propionate (e.g. in bread), sodium sulfite (e.g. in dried fruit), sodium pectinate (e.g. syrups, ice creams, jam), sodium caseinate (e.g. ice creams and other frozen products) and sodium bicarbonate (e.g. baking powder, tomato soup, confections).
    So pay attention to ingredients!
  • Inherent sodium of foods. Generally low in fresh foods.

The blood pressure response to lower dietary Na+ intake is heterogeneous with individuals having greater or lesser degrees of blood pressure reduction. Usually the effect of reduction tend to be greater in blacks, middle-aged and older persons, and individuals with hypertension, diabetes or chronic kidney disease.
Furthermore genetic and dietary factors influence the response to sodium reduction.

Diet modifies response of blood pressure to sodium

Some components of the diet may modify response of blood pressure to sodium.

  • A high dietary intake of calcium and potassium rich foods, such as fruit, vegetable, legumes (e.g. Mediterranean diet), and low-fat dairy products (e.g. DASH diet), may prevent or attenuate the rise in blood pressure for a given increase in sodium intake.
  • Some evidences, seen primarily in animal model, suggest that high dietary intake of sucrose may potentiate salt sensitivity of blood pressure.

Note: high Na+ intake can contribute to osteoporosis: they result in an increase in renal calcium excretion, particularly if daily calcium intakes are low.

References

Appel L.J., Brands M.W., Daniels S.R., Karanja N., Elmer P.J. and Sacks F.M. Dietary approaches to prevent and treat HTN: a scientific statement from the American Heart Association. Hypertension 2006;47:296-308. doi:10.1161/01.HYP.0000202568.01167.B6

Bibbins-Domingo K., Chertow G.M., Coxson P.G., Moran A., Lightwood J.M., Pletcher M.J., and Goldman L. Projected effect of dietary salt reductions on future cardiovascular disease. N Engl J Med 2010;362:590-599. doi:10.1056/NEJMoa0907355

Cappuccio FP. Overview and evaluation of national policies, dietary recommendtions and programmes around the world aiming at reducing salt intake in the population. World Health Organization. Reducing salt intake in populations: report of a WHO forum and technical meeting. WHO Geneva 2007;1-60.

Chen J, Gu D., Jaquish C.E., Chen C., Rao D.C., Liu D., Hixson J.E., Lee Hamm L., Gu C.C., Whelton P.K. and He J. for the GenSalt Collaborative Research Group. Association Between Blood Pressure Responses to the Cold Pressor Test and Dietary Sodium Intervention in a Chinese Population. Arch Intern Med. 2008;168:1740-1746. doi:10.1001/archinte.168.16.1740

Denton D.,  Weisinger R., Mundy N.I., Wickings E.J., Dixson A., Moisson P., Pingard A.M., Shade R., Carey D., Ardaillou R., Paillard F., Chapman J., Thillet J. & Michel J.B. The effect of increased salt intake on blood pressure of chimpanzees. Nature Med 1995;10:1009-1016. doi:10.1038/nm1095-1009

Ford E.S., Ajani U.A., Croft J.B., Critchley J.A., Labarthe D.R., Kottke T.E., Giles W.H, and Capewell S. Explaining the decrease in U.S. deaths from coronary disease, 1980-2000. N Engl J Med 2007;356:2388-2398. doi:10.1056/NEJMsa053935

Geleijnse J.M., Witteman J.C., den Breeijen J.H., Hofman A., de Jong P., Pols H.A. and Grobbee D.E. Dietary electrolyte intake and blood pressure in older subjects: the Rotterdam Study. J Hyperten 1996;14:73741.

Harlan W.R. and Harlan L.C. Blood pressure and calcium and magnesium intake. In: Laragh J.H., Brenner B.M., eds. Hypertension: pathophysiology, diagnosis and management. 2end ed. New York: Raven Press 1995;1143-1154

Holmes E., Loo R.L., Stamler J., Bictash M., Yap I.K.S., Chan Q., Ebbels T., De Iorio M., Brown I.J., Veselkov K.A., Daviglus M.L., Kesteloot H., Ueshima H., Zhao L., Nicholson J.K. and Elliott P. Human metabolic phenotype diversity and its association with diet and blood pressure. Nature 2008;453:396-400. doi:10.1038/nature06882

Pickering T.G. New guidelines on diet and blood pressure. Hypertension 2006;47:135-136. doi:10.1161/01.HYP.0000202417.57909.26

Simpson F.O. Blood pressure and sodium intake. In: Laragh J.H., Brenner B.M. eds. Hypertension: pathophysiology, diagnosis and management. 2end ed. New York: Raven Press 1995;273-281

Strazzullo P., D’Elia L., Kandala N. and Cappuccio F.P. Salt intake, stroke, and cardiovascular disease: meta-analysis of prospective studies. BMJ 2009;339:b4567. doi:10.1136/bmj.b4567

Tzoulaki I., Brown I.J., Chan Q., Van Horn L., Ueshima H., Zhao L., Stamler J., Elliott P., for the International Collaborative Research Group on Macro-/Micronutrients and Blood Pressure. Relation of iron and red meat intake to blood pressure: cross sectional epidemiological study. BMJ 2008;337:a258. doi:10.1136/bmj.a258

Weinberger M.H. The effects of sodium on blood pressure in humans. In: Laragh JH, Brenner BM, eds. Hypertension: pathophysiology, diagnosis and management. 2end ed. New York: Raven Press 1995;2703-2714.

Writing Group of the PREMIER Collaborative Research Group. Effects of comprehensive lifestyle modification on blood pressure control: main results of the PREMIER Clinical Trial. JAMA 2003;289:2083-2093. doi:10.1001/jama.289.16.2083

World Health Organization, International Society of Hypertension Writing Group. 2003 World Health Organization (WHO)/International Society of Hypertension (ISH) statement on management of hypertension. Guidelines and recommendations. J Hyperten 2003;21:1983-1992.

Foods high in anthocyanins, their absorption and metabolism

Together with catechins and proanthocyanidins, anthocyanins and their oxidation products are the most abundant flavonoids in the human diet.
Examples of anthocyanin rich foods are:

  • certain varieties of grains, such as some types of pigmented rice (e.g. black rice) and maize (purple corn);
  • in certain varieties of root and leafy vegetables such as aubergine, red cabbage, red onions and radishes, beans;
  • but especially in red fruits.

Example of anthocyanin rich food
Anthocyanins are also present in red wine; as the wine ages, they are transformed into various complex molecules.
Anthocyanin content in vegetables and fruits is generally proportional to their color: it increases during maturation, and it reaches values up to 4 g/kg fresh weight (FW) in cranberries and black currants.
These polyphenols are found primarily in the skin, except for some red fruits, such as cherries and red berries (e.g. strawberries), in which they are present both in the skin and flesh.
Glycosides of cyanidin are the most common anthocyanins in foods.

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Anthocyanin rich fruits

  • Berries are the main source of anthocyanins, with values ranging between 67 and 950 mg/100 g FW.
  • Other fruits, such as red grapes, cherries and plums, have content ranging between 2 and 150 mg/100 g FW.
  • Finally, in fruits such as nectarines, peaches, and some types of apples and pears, anthocyanins are poorly present, with a content of less than 10 mg/100 g FW.

Cranberries, besides their very high content of anthocyanins, are one of the rare food that contain glycosides of the six most commonly anthocyanidins present in foods: pelargonidin, delphinidin, cyanidin, petunidin, peonidin, and malvidin. The main anthocyanins are the 3-O-arabinosides and 3-O-galactosides of peonidin and cyanidin. A total of 13 anthocyanins have been detected, mainly 3-O-monoglycosides.

Anthocyanin absorption

Until recently, it was believed that anthocyanins, together with proanthocyanidins and gallic acid ester derivatives of catechins, were the least well-absorbed polyphenols, with a time of appearance in the plasma consistent with the absorption in the stomach and small intestine. Indeed, some studies have shown that their bioavailability has been underestimated since, probably, all of their metabolites have not been yet identified.
In this regard, it should be underlined that only a small part of the food anthocyanins is absorbed in their glycated forms or as hydrolysis products in which the sugar moiety has been removed. Therefore, a large amount of these ingested polyphenols enters the colon, where they can also suffer methylation, sulphatation, glucuronidation and oxidation reactions.

Anthocyanins and colonic microbiota

Few studies have examined the metabolism of anthocyanins by the gut microbiota in the colon.
Within two hours, it seems that all the anthocyanins lose their sugar moieties, thus producing anthocyanidins.
Anthocyanidins are chemically unstable in the neutral pH of the colon. They can be metabolized by colonic microbiota or chemically degraded producing a set of new molecules that have not yet fully identified, but which include phenolic acids such as gallic acid, syringic acid, protocatechuic acid, vanillic acid and phloroglucinol (1,3,5-trihydroxybenzene). These molecules, thanks to their higher microbial and chemical stability, might be the main responsible for the antioxidant activities and the other physiological effects that have been observed in vivo and attributed to anthocyanins.

References

de la Rosa L.A., Alvarez-Parrilla E., Gonzàlez-Aguilar G.A. Fruit and vegetable phytochemicals: chemistry, nutritional value, and stability. 1th Edition. Wiley J. & Sons, Inc., Publication, 2010

de Pascual-Teresa S., Moreno D.A. and García-Viguera C. Flavanols and anthocyanins in cardiovascular health: a review of current evidence. Int J Mol Sci 2010;11:1679-1703. doi:10.3390/ijms11041679

Escribano-Bailòn M.T., Santos-Buelga C., Rivas-Gonzalo J.C. Anthocyanins in cereals. J Chromatogr A 2004:1054;129-141. doi:10.1016/j.chroma.2004.08.152

Han X., Shen T. and Lou H. Dietary polyphenols and their biological significance. Int J Mol Sci 2007;9:950-988. doi:10.3390/i8090950

Manach C., Scalbert A., Morand C., Rémésy C., and Jime´nez L. Polyphenols: food sources and bioavailability. Am J Clin Nutr 2004;79(5):727-747. doi:10.1093/ajcn/79.5.727

Tsao R. Chemistry and biochemistry of dietary polyphenols. Nutrients 2010;2:1231-1246. doi:10.3390/nu2121231