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Rooibos Tea: Research into Antioxidant and Antimutagenic Properties

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HerbalGram. 2003;59:34-45 ?American Botanical Council (Buy This Issue)

by Laurie Erickson

Antioxidants are hot topics in the health news these days, and an herbal tea called rooibos (pronounced ROY-boss) is becoming popular partly because it is being marketed as a healthy beverage with high levels of antioxidants. The rooibos plant (Aspalathus linearis (Burm. f.) Dahlgren, Fabaceae) is a South African flowering shrub used to make a mild-tasting tea that has no caffeine, very little tannin, and significant amounts of polyphenol antioxidants. Although the tea is new to many Americans, it has been made in the Cedarberg mountain region of South Africa for generations. Distributors are promoting the tea for numerous health benefits, citing recent studies that show some antioxidants found in rooibos tea may protect against cancer, heart disease, and stroke. What the evidence for these claims?

A Note on Tea Terminology
In the strict sense, the word tea has been reserved for infusions made from leaves of the evergreen shrub Camellia sinensis (L.) Kuntze, Theaceae, while infusions made from herbs such as rooibos have been called tisanes. Over time, however, the common use of the word tea has been extended to include herbal infusions, and this relaxed usage is followed here. Rooibos is often referred to as red tea because it makes a vibrant red-colored tea, which can be confusing because black tea and hibiscus herbal tea are also sometimes called red tea.

Botanical Description
Rooibos is a shrubby legume that is indigenous to the mountains of South Africa Western Cape.^1-3 The genus Aspalathus includes more than 200 species native to South Africa.^{2 -5 A} . linearis is a polymorphic species; various wild forms have been described, each with characteristic morphology and geographical distribution.^1-3 Some forms are prostrate and remain less than 30 cm (1 foot) tall, while other forms grow erect and may reach up to 2 m (about 6 feet) in height.^1-3,6 The types of wild rooibos that have been used to make tea are sometimes referred to as the Red, Black, Grey, and Red-Brown types.^1,2

The type of A. linearis that is cultivated commercially for tea is the Red type, also known as the Rocklands type;^1,6 it is native to the Pakhuis Pass area in the northern Cedarberg region.⁶ The Rocklands type grows erect, up to 1.5 m (about 5 feet) in height. It has a single basal stem that divides just above the ground surface into multiple thin branches that carry bright green, needle-like leaves of about 10?0 mm (0.4?.6 inches) in length.⁷ The plant produces small yellow flowers in spring through early summer,⁶ and each flower generates a one-seeded leguminous fruit.^4,5

Rooibos has adapted to coarse, nutrient-poor, acidic soil and hot, dry summers.^4,5,8 In addition to a network of roots just below the soil surface, the plant has a long tap root that reaches as deep as 2 m (about 6 feet) and helps the plant find moisture during summer drought.⁵ As a legume, rooibos contains nodules of nitrogen-fixing bacteria on its roots; this characteristic helps the plant survive in the poor Cedarberg soils and minimizes the need for fertilizing commercial crops with nitrogen.⁸ The bacteria convert nitrogen dioxide to biologically useful ammonia in a process known as nitrogen fixation. The plant absorbs the nitrogen and benefits from it in exchange for providing the bacteria with food sources created from photosynthesis.

One study found genetic variations between four morphologically different populations of A. linearis.¹ The authors suggest that the wild forms of A. linearis might be used to improve characteristics, such as yield and disease resistance, of the cultivated form. They also observe that because the cultivated Rocklands form is being grown outside of its original Pakhuis Pass location, this introduction of the cultivated form into new areas could threaten the genetic integrity of the wild forms in these areas.

A later study⁷ showed genetic differences between populations of A. linearis that are sprouters (plants that can resprout from a deep rootstock to regenerate after a fire) and populations that are seeders (plants that rely on producing plentiful seeds to reproduce). The authors suggest that reseeding is the primitive character state in A. linearis and resprouting is a derived state that evolved to help the plant survive in a region prone to wildfires. The rooibos plant that is commercially grown for tea is the seeder type.⁷

In addition to differences in morphology and genetics, researchers have found differences in chemistry between various populations of A. linearis.^6,9 Van Wyk, of the Department of Botany at Rand Afrikaans University , presented results of his tests on the different wild populations of rooibos, showing significant variations in the polyphenol profile by population.⁹

Historical Background

More than 300 years ago, indigenous inhabitants of the mountainous regions of South Africa Western Cape were the first to collect wild rooibos and use it to make tea.¹⁰ These people discovered that they could brew a sweet, tasty tea from rooibos leaves and stems that they cut, bruised with wooden hammers, fermented in heaps, and then sun-dried. Botanists first recorded rooibos plants in 1772 when they were introduced to the tea by the Khoi people.¹⁰

Rooibos became a cultivated crop by the early 1930s, has been grown commercially since World War II, and now is exported to countries worldwide, including Germany, Japan, the Netherlands, England, Malaysia, South Korea, Poland, China, and the United States .¹⁰ In 1999, about 29 percent of South Africa total rooibos sales were exported to 31 countries.¹⁰ The quantity of rooibos exported in 2000 was two and a half times greater than the quantity exported in 1999, and exports continue to grow.¹⁰ The small towns of Clanwilliam and Wupperthal, north of Cape Town in the Cedarberg region, have a long history of rooibos cultivation; these towns are popular tourist stops because of their beautiful rural scenery and their role in the rooibos industry.

Roughly 70 percent of the bulk rooibos that is exported goes through Clanwilliam-based Rooibos Ltd. <www.rooibosltd.co.za>, a partnership of private growers/processors and a cooperative of large and small farmers in the area. The rooibos is sold in a variety of products in Europe, Asia, and, increasingly, America . Other South African companies that market rooibos tea products include Khoisan, Cape Natural Tea Products, and Coetzee & Coetzee. International demand for rooibos has been increasing since trade sanctions against South Africa were lifted following the demise of apartheid in the 1990s. Since 1999, the nonprofit organization Agribusiness in Sustainable Natural African Plant Products (ASNAPP, <www.asnapp.org>) has helped small farmers in and around Wupperthal to introduce sustainable methods of rooibos cultivation that allow them to compete in the world market. ASNAPP is sponsored by the U.S. Agency for International Development, Rutgers University , and Stellenbosch University . Through Stellenbosch University , ASNAPP also helped the farmers at Wupperthal fund construction of a tea court to process rooibos.

Rutgers University provides a quality control program for ASNAPP Wupperthal tea program, evaluating parameters such as color, taste, aroma, pH, moisture content, cleanliness, total phenol content, and antioxidant capability for tea samples collected from the industry in general and from all the growers in the Wupperthal tea program.¹¹ Data from their analyses are made available to the farmers and also to prospective buyers via product specification sheets.

The Perishable Products Export Control Board (PPECB) of South Africa ensures that all exported rooibos products pass a phytosanitary inspection and are certified to be free of bacteria and impurities.^4,10 In order to pass these health and safety tests, rooibos producers steam pasteurize the tea as the final step before packing. Organic rooibos is also monitored by various international organizations that provide organic certification, such as the German firms Ecocert and Lacon.

Harvesting and Processing: Fermented and Unfermented Rooibos

When rooibos is cultivated commercially, the needle-like leaves and stems are usually harvested in the summer, which corresponds to January through March in South Africa.⁴ The plants are cut to about 30 cm (1 foot) from the ground at harvest time and begin another major growth cycle the following spring. The harvested rooibos is processed two different ways, producing two types of tea. The green leaves and stems are either bruised and fermented or immediately dried to prevent oxidation. The traditional fermented tea is processed today in much the same way as the indigenous people processed it hundreds of years ago, including the sun-drying step, but the tools are more sophisticated now.

The fermented type is called red tea because fermentation turns the leaves and the resulting tea a rich orange/red color; this distinctive color led to the Afrikaans name rooibos, which means "red bush." The unfermented type, often called green rooibos, contains higher levels of polyphenol antioxidants because fermented rooibos loses some antioxidants during the fermentation process. The unfermented type was developed to maximize antioxidant levels in response to recent interest in the health benefits associated with the antioxidants found in C. sinensis teas. Unfermented rooibos tea is a tan/yellow color rather than the rich reddish color of fermented rooibos.

Both types of rooibos tea are available plain or flavored, loose or in tea bags, organic or conventionally grown. Rooibos is graded according to color, flavor, and cut length, with the highest grade labeled "supergrade." The tea has a smooth, non-bitter flavor that is pleasant hot or chilled. The unfermented variety has a very mild "green" taste reminiscent of green tea but without the astringency; the fermented type is quite different, with a stronger sweet and fruity taste. The mild flavor of rooibos has made it popular in multi-ingredient herbal tea blends.

Antioxidants in Rooibos
Free radicals (unstable molecules that have lost an electron) can damage the DNA in cells, leading to cancer, and they can oxidize cholesterol, leading to clogged blood vessels, heart attack, and stroke. Antioxidants can bind to free radicals before the free radicals cause harm. Some antioxidants are called polyphenols because these substances contain a phenolic ring in their chemical structure. Polyphenols are common in plants; they act as pigments and sunscreens, as insect attractants and repellants, and as antimicrobials and antioxidants.^12,13 The polyphenol group is further divided into subgroups such as flavonoids and phenolic acids. Polyphenols can also be classified as monomeric (molecules containing a single unit) or polymeric (larger molecules containing more than one unit). As described in this section, laboratory studies have found that rooibos tea contains polyphenol antioxidants, including flavonoids and phenolic acids, that are potent free radical scavengers.

Flavonoids: The polyphenol antioxidants identified in rooibos tea include the monomeric flavonoids aspalathin, nothofagin, quercetin, rutin, isoquercitrin, orientin, isoorientin, luteolin, vitexin, isovitexin, and chrysoeriol.^14-19 Currently, rooibos is the only known natural source of aspalathin.¹⁵ Nothofagin is similar in structure to aspalathin and has only been identified in one other natural source besides rooibos: the heartwood of the red beech tree (Nothofagus fusca (Hook F.) Oerst, Nothofagaceae), which is native to New Zealand.²⁰

A recent analysis of fermented rooibos measured the levels of all the flavonoids listed above except nothofagin (see Table 1).¹⁹ Of the 10 flavonoids measured, the three that occurred in largest amounts were aspalathin, rutin, and orientin, followed by isoorientin and isoquercitrin. Nothofagin was identified by mass spectrometry but was not quantified because a standard was not available. The amount of nothofagin in fermented and unfermented rooibos was estimated to be about three times less than aspalathin in one study.²⁰ Aspalathin and nothofagin arepresent in relatively large amounts in unfermented rooibos tea,^19,20 but some of the aspalathin and nothofagin oxidizes to other substancesduring fermentation; thus, fermented rooibos contains less aspalathin and nothofagin than unfermented rooibos.²⁰ The change in polyphenol composition is the reason the tea changes color with fermentation.²⁰

Phenolic Acids: In addition to flavonoid antioxidants, rooibos also contains phenolic acids that have been shown to have antioxidant activity.^14,18,21 Like flavonoids, phenolic acids are polyphenol substances that are found in fruits, vegetables, and whole grains. The phenolic acids identified in rooibos tea, in decreasing order of antioxidant activity as measured in one study²¹ with the commonly used 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging assay, include caffeic acid, protocatechuic acid, syringic acid, ferulic acid, vanillic acid, p-hydroxybenzoic acid, and p-coumaric acid.^14,18 Using the DPPH assay, caffeic acid was just as active an antioxidant as the most potent flavonoids tested (quercetin, isoquercitrin, and aspalathin).²¹

Total Polyphenol Content: Despite some promotional claims, a serving of rooibos tea has less total polyphenols than the same size serving of green or black tea. Serving size varies, but for comparison purposes a 150 to 200 ml serving is often used (about 3/4 of a standard baking measuring cup). Elizabeth Joubert, Ph.D., specialist researcher at South Africa ARC Infruitec-Nietvoorbij and a rooibos expert, says that the total polyphenol content of an average 150 to 200 ml serving of rooibos tea can be as much as 60 to 80 mg, depending on factors such as the brewing time and amount of leaves used.²² For comparison, one study found that brewing black tea leaves for 1 to 3 minutes at a concentration of 1 g leaves per 100 ml water resulted in black tea that contains 128 to 199 mg of polyphenols per 200 ml serving of tea.²³ The types of polyphenols in rooibos tea are different than those in green and black teas, so the potential health benefits of the teas cannot be compared solely on their total polyphenol content. Rooibos tea does not contain epigallocatechin gallate (EGCG), which is a polyphenol in green tea that has shown anticarcinogenic and antioxidant capabilities, but many of the polyphenols in rooibos tea are also strong antioxidants.

Quercetin and Luteolin: Two of the flavonoids in rooibos tea, quercetin and luteolin, are potent antioxidants found in many fruits and vegetables. Studies in vitro (in the test tube) have shown that these antioxidants can cause cancer cells to "commit suicide," referred to as apoptosis.^24-27 Quercetin decreased primary tumor growth and prevented metastasis in a model of pancreatic cancer.²⁵ Luteolin and quercetin inhibited proliferation of thyroid²⁸ and colon²⁹ cancer cells, respectively, in vitro. Quercetin inhibited cyclooxygenase-2 (COX-2) expression in colon cancer cells, which may help prevent colon cancer.^30,31 Both luteolin and quercetin can block the formation of lipid peroxides.^32-34

Although studies like these show quercetin and luteolin are strong antioxidants, researchers haven yet determined whether enough of either of these two flavonoids are present in rooibos tea and absorbed by the body to have beneficial effects. As shown in Table 1, recent analysis of fermented rooibos found considerably more quercetin than luteolin,¹⁹ but even quercetin was present in much lower amounts than aspalathin, orientin, and rutin.

Based on the data in Table 1, a 150 ml serving of fermented rooibos tea made with 2.5 g of tea leaves has about 0.27 mg of quercetin; for comparison, one study found that C. sinensis contains 1.5 to 3.75 mg of quercetin per 150 ml serving of tea.³⁵ A previous study³⁶ found 1.5 mg of quercetin per 150 ml serving of fermented rooibos, but that may be an upper limit. Joubert says that the 1.5 mg estimate is probably high,²² but emphasizes that these estimates will vary with parameters such as the brewing time and the amount of water and tea leaves used. At any rate, the amount of quercetin per serving of rooibos is a small percentage of the total polyphenol content per serving of rooibos.

Aspalathin and Nothofagin: A unique polyphenol that is one of the most abundant monomeric flavonoids in rooibos tea,^19,20 aspalathin seems to contribute to the antioxidant capabilities of rooibos,²¹ but aspalathin is not as well studied as quercetin and luteolin. Nothofagin is similar in structure to aspalathin and may have similar antioxidant capabilities.

Joubert says that chief research technologist Petra Snijman of the Program on Mycotoxins and Experimental Carcinogenesis (PROMEC) at the Medical Research Council of South Africa recently developed a way to isolate pure aspalathin and nothofagin from rooibos. Joubert says, "According to unpublished in vitro studies done at ARC Infruitec-Nietvoorbij, aspalathin compared well with quercetin in terms of antioxidant activity, except in a fat medium where quercetin demonstrated much higher potency than aspalathin. What is important in these comparative studies is the test environment. Relative efficacy will depend on the test system used (the polarity of the medium, the type of free radical that needs to be scavenged, etc.)."²²

Joubert co-authored a study²¹ that found aspalathin compared well to other antioxidants with the DPPH radical scavenging assay. The study measured the antioxidant capability of many of the flavonoids and phenolic acids found in rooibos tea and compared them to several reference standards such as alpha-tocopherol (vitamin E). The percent inhibition of the DPPH radical by quercetin, isoquercitrin, aspalathin, rutin, luteolin, and alpha-tocopherol was 98.27, 91.99, 91.74, 91.18, 90.85, and 75.10, respectively (using a 0.25 mole ratio of antioxidant to DPPH). All of the flavonoids tested showed potent hydrogen donating abilities with DPPH except for vitexin, which only had a 7.26 percent inhibition even at a 0.5 mole ratio to DPPH.

According to the data in Table 1, a 150 ml serving of fermented rooibos made with 2.5 g of tea leaves has about 3 mg of aspalathin; since the amount of nothofagin was measured to be three times less than aspalathin in one study,²⁰ a 150 ml serving of fermented rooibos has on the order of 1 mg of nothofagin. A serving of unfermented rooibos has considerably more aspalathin and nothofagin than an equal serving of fermented rooibos because a portion of these flavonoids oxidizes to other substances during fermentation.²⁰

Orientin and Rutin: Orientin and rutin are two of the other most abundant monomeric flavonoids in rooibos,¹⁹ and both have been associated with health benefits. Orientin is a potent free radical scavenger. It reduced by half the number of cancer-associated changes in cells of human blood exposed to radiation.³⁸ When mice were exposed to radiation, orientin protected against lipid peroxidation in the liver and also reduced damage to the bone marrow and gastrointestinal tract.^39,40 Rutin, a flavonoid found in buckwheat (Fagopyrum esculentum Moench, Polygonaceae) and some fruits and vegetables, seems to help maintain the strength of capillary walls; oral rutin as well as oral and topical o-(beta-Hydroxylethyl)-rutoside (HR) have been used to treat hemorrhoids, varicose veins, and the lower leg edema associated with venous insufficiency and venous hypertension.^41-46 According to the data in Table 1, a 150 ml serving of fermented rooibos tea made with 2.5 g of tea leaves has about 2.5 mg of orientin and 3.2 mg of rutin.

Total Antioxidant Capability: Although the 10 flavonoids in Table 1 are important because they are known to have antioxidant properties, they only represent a small percentage of the total polyphenol content of a serving of fermented rooibos tea. A 150 to 200 ml serving of rooibos can have up to 60 to 80 mg of total polyphenols,²² and Table 1 shows that a 150 ml serving of fermented rooibos made with 2.5 g of leaves has about 14 mg of the 10 flavonoids in the table. Many other polyphenols are present, but they have not all been identified or quantified.

To assess the antioxidant capability of rooibos tea as a whole, researchers compared the antioxidant activity of rooibos tea extracts to that of green and black tea extracts with the DPPH radical scavenging assay as well as the beta-carotene bleaching method.⁴⁷ All the teas showed strong antioxidant activity with both methods. Using the DPPH method, the ranking from highest to lowest antioxidant activity was green tea (90.8 percent inhibition), unfermented rooibos (86.6 percent), fermented rooibos (83.4 percent), and black tea (81.7 percent). Green tea was significantly higher than the others (P < 0.05), but the other three teas did not differ from each other significantly with respect to DPPH inhibition. Using the beta-carotene bleaching method, the ranking was green tea, black tea, fermented rooibos, and unfermented rooibos. The relative ranking varies with the type of test because the substance to be tested will have different reactivity to the different oxidizing agents used. These tests only measure the antioxidant capability of substances outside of the body and done provide data on whether the antioxidants are absorbed by the body and effective after the food is consumed.

In this study, all the tea extracts were diluted to the same amount of soluble solids rather than to the amounts of solids found in the teas.⁴⁷ This method allows a comparison of antioxidant capability on a mass equivalent basis, but does not reflect a comparison of the antioxidant strength of equal volume servings of the teas. Although the soluble solid content varies with the method of tea preparation, it usually decreases in the order green tea, black tea, unfermented rooibos, fermented rooibos.⁴⁷ The percent of soluble solids represented by polyphenols is similar for the four teas and the DPPH antioxidant activity is similar on a mass equivalent basis, so the DPPH antioxidant capability of equal-sized servings will decrease in the order of the soluble solid content.⁴⁷ Black and green teas have over twice as much soluble solids as rooibos tea when prepared conventionally, so over two 200 ml servings of rooibos tea would need to be consumed to receive the same antioxidant benefit (as measured by DPPH) as one 200 ml serving of black or green tea (or the rooibos would need to be brewed to twice the standard concentration).⁴⁷ This result agrees with the data given previously for 60 to 80 mg polyphenols for a 150 to 200 ml serving of rooibos tea²² as compared to 128 to 199 mg polyphenols for a 200 ml serving of black tea.²³

The studies referenced above show that rooibos tea contains antioxidants that have positive effects when tested as isolated substances and that the tea as a whole has good antioxidant activity in vitro. So, do all these antioxidants in rooibos tea lead to health benefits for tea drinkers?

Rooibos Research in Live Animals and Animal Cells

Laboratory studies have demonstrated potential health benefits of rooibos in vitro (in test tubes) and in vivo (in live animals), but human studies have not been conducted. Much more research is needed, but the studies so far look intriguing.

Fermented Rooibos against Mutagens: Researchers found that fermented rooibos tea reduced cancer-associated changes in animal cells induced by the mutagens benzo[a]pyrene (B(a)P) and mitomycin C (MMC) both in vitro and in vivo.⁴⁸ The in vitro part of the study measured chromosomal aberrations in animal cells caused by exposure to the mutagens. The cells were treated with tea extract either at the same time as the mutagen or after the mutagen. Some of the tests used rat liver microsomal enzyme, called S9, to provide metabolic activation of the mutagen; B(a)P requires metabolic activation, but MMC can act with or without it.

Both green tea and rooibos tea suppressed aberrant cells caused by B(a)P and MMC in the presence of S9, but rooibos showed a greater suppression of aberrant cells than did green tea (see Table 2). In fact, when the cells were exposed to B(a)P and S9 simultaneously with rooibos tea, the highest concentration of rooibos tea (1000 microgram/ml) completely inhibited the aberrant cells, bringing their percentage down to the level of the controls that were not exposed to any mutagen. Also, rooibos tea suppressed aberrant cells caused by MMC both with and without the presence of S9, but green tea showed no suppression without S9. Treating the cells simultaneously with the mutagen and tea extract caused a greater protective effect than treating the cells with tea extract following exposure to the mutagen (compare Tables 2 and 3).