Honeybush Tea (Cyclopia sp.)

CHAPTER

12

Honeybush Tea (Cyclopia sp.): A Traditional South-African Tisane Adam Kokotkiewicz, Maria Luczkiewicz Department of Pharmacognosy, Faculty of Pharmacy, Medical University of Gdansk, Gdansk, Poland

Abbreviations 2-AAF 2-acetylaminofluorene ABTS 2,2’-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) AFB1 aflatoxin B1 CFR Cape Floristic Region CHP cumyl hydroperoxide DEN diethylnitrosamine DMBA 7,12-dimethylbenz(a)anthracene DPPH 2,2-diphenyl-1-picrylhydrazyl ERa estrogen receptor alpha ERß estrogen receptor beta FB1 fumonisin B1 FRAP ferric ion reducing antioxidant power GSH glutathione GSSG glutathione disulfide GST-a glutathione S-transferase alpha HPLC high-performance liquid chromatography LC-MS liquid chromatography-mass spectrometry MBN methylbenzylnitrosamine MMS methyl methanesulfonate NIRS near infrared spectroscopy NMR nuclear magnetic resonance TP total polyphenols TPA 12-O-tetradecanoylphorbol-13-acetate UDP-GT uridine 5’-diphospho-glucuronosyltransferase UVB ultraviolet B

INTRODUCTION The Cape Floristic Region is one of the richest biodiversity hotspots in terms of species number and endemism rate (Turpie et al., 2003). The area comprises only 4% of South Africa, yet hosts over 8,600 species of vascular plants, with endemics constituting roughly 65% of its flora Tea in Health and Disease Prevention. DOI: 10.1016/B978-0-12-384937-3.00012-4 Copyright Ó 2013 Elsevier Inc. All rights reserved.

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SECTION 2 Miscellaneous Teas and Tea Types: Non-Camellia sinensis (Goldblatt, 1997). Apart from their unique character and scientific importance, many species found in the CFR also are of significant economic and medicinal value (Turpie et al., 2003; Van Wyk, 2008, 2011a, b). Among commercially-exploited South-African plants, there are shrubs of the genus Cyclopia (Fabaceae), which are used in the production of the honeybush herbal tea. Although its manufacture is known to have been practiced for at least one hundred years, it remained fairly unknown until the revival of the honeybush tea industry in the 1990s (Du Toit et al., 1998; Joubert et al., 2008a, 2011). The tea itself, prepared mainly from fermented leaves and stems of several Cyclopia spp., is characterized by a sweet, distinctive, honey-like flavor and a lack of caffeine, making it an attractive beverage for everyday use (Kokotkiewicz and Luczkiewicz, 2009). As a result of its growing popularity in overseas markets, honeybush has become an important export commodity. The development of a successful tea industry, however, required the improvement of production and the standardization of procedures. Numerous studies on pretreatment, fermentation and drying have been undertaken in order to provide plant material of the finest quality (Joubert et al., 2008a, 2011). Detailed investigations into the chemical composition and biological activity of Cyclopia extracts have also been made. It has been shown that honeybush teas are rich in polyphenols and, as such, are biologically active and possess substantial antioxidative potential (McKay and Blumberg, 2007; Joubert et al., 2008a, 2009; Kokotkiewicz and Luczkiewicz, 2009). This review comprises the most relevant data on the botanical characteristics, chemistry, application and biological activity of Cyclopia plants.

BOTANICAL CHARACTERISTICS AND CULTIVATION

142

The Cyclopia genus (Fabaceae family, Podalyrieae tribe) includes over 20 species of endemic shrubs, mainly associated with the fynbos plant formation of the CFR in South Africa (Kokotkiewicz and Luczkiewicz, 2009). Their range extends from Cederberg Mountains (Western Cape) to Cape Peninsula in the south and Port Elizabeth in Eastern Cape Province (Schutte-Vlok, 1998; Joubert et al., 2011). Cyclopia plants inhabit mostly sandy, acidic, infertile soils and exhibit nitrogen-fixing abilities (Spriggs and Dakora, 2007, 2009a, b; Sprent et al., 2010; Joubert et al., 2011). As a result of frequent fires in fynbos shrublands, two survival strategies have emerged within Cyclopia spp. Resprouters, like C. intermedia and C. genistoides, are characterized by a multi-stem habit and strongly developed underground part (lignotuber), capable of surviving fires and resprouting thereafter. Reseeders, like C. subternata, are usually destroyed by fire and subsequently recover their populations from soil-stored seeds (Sutcliffe and Whitehead, 1995; Du Toit et al., 1998; Joubert et al., 2011). Cyclopia plants are characterized by trifoliate leaves, paired, fused bracts, unifloral inflorescences and yellow, sweet-scented flowers with a thrust-in calyx base (De Nysschen et al., 1996; Van der Bank et al., 2002) (Figure 12.1). At present, the plant material for honeybush tea production is obtained mainly from just a few species, namely C. intermedia, C. subternata, C. genistoides and, to a lesser extent, C. sessiliflora, which are found in different parts of the CFR (Table 12.1). For many years, the tea was manufactured exclusively from wild-harvested plants, but as the honeybush industry expanded, Cyclopia populations faced the threat of overexploitation (Du Toit et al., 1998). In order to keep up with the growing demand, efforts were undertaken to establish commercial cultivars of Cyclopia plants. Two species, C. genistoides and C. subternata, were selected for cultivation, and the first orchards have already been established. The plants can be propagated by either seeds or cuttings (Joubert et al., 2008a, 2011).

HONEYBUSH TEA MANUFACTURE The plant material was traditionally collected during the flowering period, which falls in May or September, depending on the species used (Kokotkiewicz and Luczkiewicz, 2009). As the demand for honeybush tea increased, many producers decided to extend the harvesting period (Du Toit et al., 1998). In order not to put the plants under stress, the plant material is currently harvested mainly before the flowering, in summer to late autumn (Joubert et al., 2008a, 2011).

CHAPTER 12 Honeybush Tea (Cyclopia sp.): A Traditional South-African Tisane

FIGURE 12.1 Dried, Unfermented C. intermedia (A) and C. genistoides (B) Materials. White squares are 11cm. (Photo by Adam Kokotkiewicz, previously unpublished.)

The harvested plants are cut into small pieces with a fodder cutter and then subjected to oxidation (hereinafter referred to as ‘fermentation’) in order to develop the characteristic brown color and distinctive sweet flavor. Traditional honeybush manufacture involves the use of a curing heap and requires substantial amounts (ca. 1.5e2.5 tons) of harvested plants. For fermentation, the shredded plant material is formed into a pile (up to 5 m in diameter and 2 m high) and covered with canvas. As a result of exothermic reactions, the temperature inside the heap can reach up to 60
C, while outer parts of the pile remain unheated. As the fermentation proceeds, the heap is turned over every 12 hours to ensure proper aeration and uniform processing. After 3e5 days, the plant material is spread on canvas sheets and sun-dried (Du Toit et al., 1998). The heap fermentation, although inexpensive, has several disadvantages and as such is no longer practiced in the modern honeybush industry (Joubert et al., 2008a, 2011). Firstly, it provides little control of plant processing, which depends strongly on weather conditions. The poor heap aeration and ambient temperature in outer layers of the pile may result in incomplete fermentation of the plant material (Du Toit et al., 1998). Another problem is the extensive mold and bacterial growth, resulting from long fermentation times at relatively low temperatures. The presence of biological contaminants can pose a serious threat to honeybush tea manufacturers, as the obtained product might fail to meet the microbial standards of target countries (Du Toit et al., 1999). In fact, Salmonella contamination substantially hampered the rooibos (Aspalathus linearis) tea industry in the 1980s (Du Toit et al., 1998). In order to provide better-quality tea, controlled high-temperature fermentation has been researched. It has been shown that the curing of the plant material in an oven at 90
C for 36 h (or 70
C for 60 h) results in fine-quality tea, characterized by rich flavor, deep-brown color and the absence of microbial contaminants. The presence of flowers proved to be beneficial, but not necessary for the development of the sweet honeybush aroma (Du Toit and Joubert, 1999). Pretreatment with water results in more-uniformly fermented material and better beverage characteristics. The inactivation of peroxidase and polyphenol oxidase by hot water treatment does not impair the fermentation process, which indicates its chemical, rather than

143

SECTION 2 Miscellaneous Teas and Tea Types: Non-Camellia sinensis

TABLE 12.1 Major Commercially Exploited Cyclopia Species Species

Common Name abc

C. intermedia E. Mey

Bergtee (‘mountain tea 1’)

C. subternata Vog.

Vleitee (‘valley tea’)abc

C. genistoides (L.) Vent.

Kustee (‘coastal tea’)abc

C. sessiliflora Eckl. and Zeyh.

Heidelbergteeabc

Geographical Distribution

Source of Plant Material

From Swellendam (Western Cape) to Uitenhage (Eastern Cape), Langeberg, Swartberg and Kouga Mountainsabc From Heidelberg (Western Cape) to Uitenhage (Eastern Cape), Tsitsikamma and Outeniqua Mountains, Langkloof valleyabc Western Cape, coastal regions from Yzerfontein (north of Cape Town) to Gourits River, Overberg regionabc Langeberg mountains near Heidelberg (Western Cape)abc

Wild-harvested plantsabd

Wild-harvested and cultivated plantsab

Wild-harvested and cultivated plantsab Wild-harvested plantsab

Table by Adam Kokotkiewicz, previously unpublished a Joubert et al., 2008a b Joubert et al., 2011 c Schutte-Vlok, 1998 d Du Toit et al., 1998

144

enzymatic nature (Du Toit and Joubert, 1998a). After fermentation, the plant material is sundried, but faster, controlled drying at elevated temperature can also be applied without adverse effect on the organoleptic qualities of the tea (Du Toit and Joubert, 1998b). At present, hightemperature fermentation takes place in rotary fermenters for 18e60 h (Joubert et al., 2008a). Sun-drying is practiced thereafter, but rotary driers can also be used during adverse weather conditions (Du Toit and Joubert, 1998b; Joubert et al., 2008a, 2011). The production process is shown in Figure 12.2. Although honeybush is especially popular in its oxidized form, the unfermented (‘green’) version of the tea is gaining popularity because of its higher phenolics content and superior antioxidant potential (Joubert et al., 2008c, 2010). Unfermented honeybush can be obtained by simply hot-air drying the shredded plant material. The drawback of this method is the enzymatic degradation of chlorophyll and phenolic compounds during heating, resulting in substantial browning of the processed leaves and giving them the look of low-quality fermented tea (Joubert et al., 2010). In order to prevent detrimental color changes, the plant material should be steamed directly after comminution and subsequently dried without unnecessary delay. Low-temperature storage of the final product also favors green color retention (Joubert et al., 2010). The honeybush tea industry, with annual production of less than 300 tons, is still small in comparison to rooibos (Joubert et al., 2008a, 2011). However, it has undergone substantial modernization during the last decade and is steadily growing. Substantial amounts of the tea are exported, mainly to The Netherlands, Germany, United Kingdom and USA (Joubert et al., 2008a, 2011).

TRADITIONAL AND MODERN USE The medicinal use of Cyclopia plants dates back to a few centuries ago, and many healing effects have been attributed to the consumption of honeybush extracts, without indicating any specific species. The infusions are thought to stimulate appetite, treat colic in babies and stimulate lactation in breastfeeding women. They are also believed to alleviate arthritic pains, combat skin ailments and act as an expectorant (Du Toit et al., 1998; Joubert et al., 2008a). The reports are anecdotal, and no clinical trials have so far been undertaken to confirm them

CHAPTER 12 Honeybush Tea (Cyclopia sp.): A Traditional South-African Tisane

FIGURE 12.2 Honeybush Tea Production Scheme. (Scheme by Adam Kokotkiewicz, previously unpublished.)

(Joubert et al., 2008a). Although honeybush infusions can still be used in traditional herbal medicine, nowadays they are used mostly as a beverage for everyday use. The absence of caffeine and low tannin levels make them suitable for patients with heart and digestive disorders, as well as for children (Du Toit et al., 1998). Honeybush infusions can be served either hot, often with the addition of milk and sugar, or as iced tea blended with fruits or juices (Du Toit et al., 1998; Kokotkiewicz and Luczkiewicz, 2009). The infusion is traditionally prepared by pouring hot water over the leaves and letting it brew for 10 min. When a larger amount of coarse material is used, the same leaves can be used several times, simply by decantation and adding fresh portions of hot water. The infusion should be kept warm, as undesired flavors may form during a few days of storage at ambient temperature. Another method of honeybush tea preparation involves boiling ca. 4e6 g of material in 1 l of water for 10e15 min (Du Toit et al., 1998). At present, both fermented and unfermented honeybush teas are commonly available in tea bags, which makes their brewing less time-consuming. Blends of honeybush, rooibos and other African plants are also available (Joubert et al., 2008a).

CHEMICAL COMPOSITION AND QUALITY CONTROL The major secondary metabolites found in honeybush plants are of polyphenolic character. TP content of dried hot water extracts of unfermented material from different Cyclopia species is slightly lower in comparison to C. sinensis (green and black) and rooibos teas (Joubert et al., 2008c). Tannin level is low, as it constitutes ca. 16e30% of TP content of fermented C. maculata, depending on processing time at 70
C (Du Toit and Joubert, 1998a). The chemical composition of Cyclopia plants has been much examined since the 1990s. One multispecies survey revealed the presence of mangiferin (xanthone) and hesperetin-O-glycoside (flavanone) in the leaves of nearly all examined plants (De Nysschen et al., 1996). However, further investigations focused on major commercially exploited species, i.e. C. intermedia, C. subternata, C. genistoides and, to a lesser extent, C. sessiliflora (Joubert et al., 2008a). It has been shown that the predominant polyphenols in the above plants are xanthones, mangiferin and isomangiferin, as well as the flavanone hesperidin (Table 12.2). Several other polyphenols were also isolated from C. intermedia and C. subternata, and subsequently identified by NMR (Ferreira et al. 1998; Kamara et al., 2003, 2004). These include flavanones, flavones, flavonols, isoflavones, flavanes and coumestans (Table 12.2). Additionally, the presence of several compounds has been confirmed by LC-MS (Joubert et al., 2008c; De Beer et al., 2009;

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SECTION 2 Miscellaneous Teas and Tea Types: Non-Camellia sinensis

TABLE 12.2 Polyphenolic Derivatives Found in Cyclopia Plants Compound Type

Common Name

Xanthone

Mangiferin Isomangiferin

Flavanone

Hesperetin (3’,5,7-trihydroxy-4’methoxyflavanone) Hesperidin (hesperetin-7-O-rutinoside) Eriodictyol (3’,4’,5,7tetrahydroxyflavanone) Eriocitrin (eriodictyol-7-O-rutinoside) Eriodictyol-5-O-glucoside Eriodictyol-7-O-glucoside Naringenin (4’,5,7-trihydroxyflavanone) Prunin (naringenin-7-O-glucoside) Narirutin (naringenin-7-O-rutinoside) Naringenin-5-O-rutinoside Butin (3’,4’,7-trihydroxyflavanone)

Flavone

Isosakuranetin-7-O-glycoside (5,7dihydroxy-4’-methoxyflavanone-7-Oglycoside) Luteolin (3’,4’,5,7-tetrahydroxyflavone)

146 Scolymoside (luteolin-7-O-rutinoside) 5-Deoxyluteolin Diosmetin (3’,5,7-trihydroxy-4’methoxyflavone) Vicenin-2 (apigenin-6,8-di-C-glucoside) Flavonol

Flavan-3-ol Flavan Isoflavone

Kaempferol-5-O-glucoside Kaempferol-6-C-glucoside Kaempferol-8-C-glucoside Kaempferol-3-O,6-C-diglucoside 3-Hydroxy-6-[O-a-apiofuranosyl(1’”/6”)-ß-D-glucopyranosyloxy]-3’,4’methylenedioxyflavonol (-)-Epigallocatechin-3-O-gallate 3’,4’,5,7-Tetrahydroxyflavan-5-Oglucoside Formononetin (7-hydroxy-4’methoxyisoflavone) 7-[O-a-Apiofuranosyl-(1’”/6”)-ß-Dglucopyranosyloxy]-4’-methoxyisoflavone (formononetin-7-O-diglycoside) 3’-Hydroxydaidzein (3’,4’,7trihydroxyisoflavone) Orobol (3’,4’,5,7-tetrahydroxyisoflavone) Afrormosin (4’,6-dimethoxy-7hydroxyisoflavone) Wistin (afrormosin-7-O-glucoside)

Occurence in Major Commercially Exploited Species C. C. C. C. C.

intermediaabcdefg, C. subternataadefghij, genistoidesacdefgjklm, C. sessilifloraacdfg intermediabcef, C. subternatadefij, genistoidescdefjm, C. sessilifloracdf intermediabdg, C. genistoidesg

C. intermediabcdefg, C. subternatadefghij, C. genistoidescdefgjkm, C. sessilifloracdefg C. intermediab C. intermediadef, C. subternatadefghij, C. genistoidesgj, C. sessilifloradfg C. intermedian C. intermedian C. intermediab C. intermedian C. intermediadg, C. subternatadhg, C. genistoidesd, C. sessilifloradg C. intermedian C. intermediao, C. subternatao, C. sessiliflorao C. intermediaa, C. genistoidesa

C. C. C. C. C.

intermediab, C. subternatadhij, genistoidesdjm, C. sessiliflorad subternatahi subternatah intermedian

C. intermediao, C. subternatao, C. sessiliflorao C. intermedian C. subternatah C. intermedian C. intermedian C. intermediam C. subternatah C. subternatah C. intermediab C. intermedian

C. C. C. C.

intermediao, C. subternatao, sessiliflorao subternatah intermediab

C. intermedian

CHAPTER 12 Honeybush Tea (Cyclopia sp.): A Traditional South-African Tisane

TABLE 12.2 Compound Type

Coumestan

Polyphenolic Derivatives Found in Cyclopia Plantsdcontinued Common Name Calycosin (3’,7-dihydroxy-4’methoxyisoflavone) Pseudobaptigenin (7-hydroxy-3’,4’methylenedioxyisoflavone) Fujikinetin (7-hydroxy-6-methoxy-3’,4’methylenedioxyisoflavone) Medicagol Flemichapparin Sophoracoumestan B

Occurence in Major Commercially Exploited Species C. intermediab C. intermediab C. intermediab C. intermediab C. intermediab C. intermediab

Table by Adam Kokotkiewicz, previously unpublished a De Nysschen et al., 1996 b Ferreira et al., 1998 c Joubert et al., 2003 d Joubert et al., 2008c e Kokotkiewicz et al., 2009 f De Beer and Joubert, 2010 g Verhoog et al., 2007a h Kamara et al., 2004 i De Beer et al., 2009 j Mfenyana et al., 2008 k Joubert et al., 2006 l Joubert et al., 2008b m Verhoog et al., 2007b n Kamara et al., 2003 o De Nysschen et al., 1998

De Beer and Joubert, 2010). Substantial qualitative and quantitative differences in polyphenolic content exist between various Cyclopia species. Unfermented C. genistoides can be considered to be a rich source of xanthones, with concentrations of mangiferin and isomangifering varying from 2.9e5.9% and 0.54e1.37%, respectively. On the other hand, C. intermedia has a significantly higher hesperidin content (1.66e1.76%) than C. genistoides (ca. 1%) (Joubert et al., 2003; Kokotkiewicz et al., 2009). Among honeybush plants, C. subternata is distinguished by high levels of the flavanone eriocitrin (0.23e0.47%) and the flavone scolymoside (ca. 0.5%) (Kokotkiewicz et al., 2009; Joubert et al., 2010). Low amounts of scolymoside aglycone, luteolin, have also been found in some Cyclopia spp. (Joubert et al., 2008c). Differences in chemical composition can also be observed within the same species. It has been shown that two C. genistoides types differed significantly in terms of mangiferin and hesperidin content; the Overberg type contained more mangiferin than the West Coast Type, whereas hesperidin prevailed in the latter. Mangiferin content was also affected by harvesting time, as it significantly decreased (5.94e5.21%) during a 15-week period from the end of March to midJuly (Joubert et al., 2003). Honeybush fermentation results in a substantial decrease in the amount of polyphenols. It has been shown that 60 h-long fermentation at 70
C causes at least 13e17% and 23% loss of TP content in C. intermedia and C. maculata, respectively (Du Toit and Joubert, 1998a; Du Toit and Joubert, 1999). In a comparative study, it has been shown that C. genistoides is least affected by the oxidation process, retaining 77% of its original TP content. C. subternata, C. intermedia and C. sessiliflora were more susceptible, losing over 40% of their polyphenols (Joubert et al., 2008c). In the case of green honeybush, which does not undergo fermentation, enzymatic degradation of polyphenolic compounds is triggered by shredding and subsequent drying of the harvested plants. However, this phenomenon can be effectively inhibited when the material is steamed directly after comminution. Interestingly, the concentration of

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SECTION 2 Miscellaneous Teas and Tea Types: Non-Camellia sinensis scolymoside in unfermented C. subternata increases during the drying process, presumably as a result of eriocitrin oxidation (Joubert et al., 2010). Cyclopia plants contain no quinolizidine alkaloids and are caffeine-free (De Nysschen et al., 1998; Joubert et al., 2008a). Among non-polyphenolic compounds, tyrosol and benzaldehyde derivatives, as well as pinitol and some organic acids have been identified in C. intermedia and C. subternata (Table 12.3). Moreover, a number of volatile compounds have been found in C. genistoides. The predominant compound found in the volatile fraction of unfermented material is 6-methyl-5-hepten-2-one, whereas linalool prevails in fermented honeybush tea (Le Roux et al., 2008). Honeybush tea contains a certain amount of macro- and micro-elements, although it cannot be regarded as a particularly rich source of minerals. In comparison to green and black teas, honeybush tea infusions are generally characterized by low mineral content, except for Ca (8.5e14.8 mg/l), Fe (0.06e0.09 mg/l) and Mg (10.8e15.8 mg/l), which were present in similar (Fe, Mg) or higher (Ca) amounts. Higher concentrations of macro- and micro-elements were recorded in unfermented C. intermedia (Malik et al., 2008). Fluoride content is low (0.03e0.09 mg/l depending on steeping time), but amounts as high as 0.59 mg/l were also reported (Joubert et al., 2008a; Malinowska et al., 2008). Apart from phytochemical surveys focused on the identification of secondary metabolites, research has been undertaken to develop fast and reliable analytical methods to be used for quality control of honeybush teas. It has been shown that an aluminium chloride colorimetric method can be used for mangiferin determination in unfermented C. genistoides (Joubert et al., 2008b). Among other spectroscopic methods, NIRS was applied to identify mangiferin and hesperidin in green C. genistoides plant material. This method seems to be appropriate for screening purposes, especially when samples of high mangiferin/hesperidin content are to be analyzed. NIRS also has several advantages, as there is no need for extract preparation and it enables the use of fine-ground plant material (Joubert et al., 2006, 2008a). Intra- and interspecies variation in phenolic composition of Cyclopia plants poses a challenge for quality control units. Although spectroscopic methods can be applied to perform quick, screening

148

TABLE 12.3 Non-Polyphenolic Secondary Metabolites Found in Cyclopia Plants Compound Type

Common Name

Phenolic acid Organic acid Benzaldehyde derivative

p-Coumaric acid (4-hydroxycinnamic acid) ()-Shikimic acid 4-[O-a-Apiofuranosyl-(1”/2’)-ß-Dglucopyranosyloxy]benzaldehyde Tyrosol (p-hydroxyphenethyl alcohol) 2-{4-[O-a-Apiofuranosyl-(1”/6’)-ß-Dglucopyranosyloxy]phenyl}ethanol (tyrosol diglycoside) 4-Glucosyltyrosol 6-Methyl-5-hepten-2-one Linalool a-Terpineol Geraniol Nerol Limonene Hexanal (þ)-Pinitol

Phenethyl alcohol derivative

Unsaturated ketone Monoterpene alcohol

Cyclic terpene Alkyl aldehyde Cyclitol

(Table by Adam Kokotkiewicz, previously unpublished.) a Ferreira et al., 1998 b Kamara et al., 2004 c Kamara et al., 2003 d Le Roux et al., 2008

Occurence in Major Commercially Exploited Species C. intermediaa, C. subternatab C. subternatab C. intermediac C. intermediac C. intermediac

C. C. C. C. C. C. C. C. C.

subternatab genistoidesd genistoidesd genistoidesd genistoidesd genistoidesd genistoidesd genistoidesd intermediaa, C. subternatab

CHAPTER 12 Honeybush Tea (Cyclopia sp.): A Traditional South-African Tisane

analyses, HPLC is the method of choice when unambiguous identification of individual polyphenols is needed (Joubert et al., 2008a; De Beer and Joubert, 2010).

ANTIOXIDANT PROPERTIES AND BIOLOGICAL EFFECTS Honeybush extracts are rich in polyphenols, and as such exhibit substantial antioxidant properties, which were demonstrated in several comparative studies (Table 12.4). In most in vitro studies, Cyclopia extracts proved to exhibit lower antioxidant activity in comparison to green, black and rooibos teas. A wide variety of experimental models have been used, including ABTS, DPPH, superoxide ion and hydroxyl radicals scavenging, as well as linoleic acid oxidation assays (Du Toit et al., 2001; Lindsey et al., 2002; Steenkamp et al., 2004; Ivanova et al., 2005). However, the results of the experiments are inconclusive, as in many cases no information on the processing state, or even species used, is given. The most comprehensive study on antioxidant in vitro activity of honeybush involved the use of both fermented and unfermented materials from several commercially important Cyclopia species, as well as rooibos and C. sinensis (Joubert et al., 2008c). The antioxidant potential of unfermented rooibos and green tea proved to be superior in all assays. However, the antioxidant activity of some honeybush samples was comparable to fermented rooibos and C. sinensis teas. In a FRAP assay, unfermented C. intermedia was more active than fermented rooibos, black and oolong tea, while C. genistoides was comparable to those teas in terms of antioxidant activity. In the same assay, the activity of unfermented C. subternata and C. sessiliflora was similar to that of black tea. Green C. sessiliflora inhibited microsomal lipid peroxidation to the same degree as fermented rooibos. Fermentation substantially lowered antioxidant activity of the analyzed plants, which was reflected in most assays. Considering intra- and interspecies variation in phenolic composition and the influence of fermentation parameters on TP content, none of the examined Cyclopia species can be considered as the most valuable in terms of antioxidant activity (Joubert et al., 2008a, c). Antioxidant activity of honeybush extracts is related to high mangiferin content, which proved to be one of the most potent Cyclopia antioxidants. However, the results of in vitro tests may not correspond to antioxidant activity in vivo (Joubert et al., 2008a, 2009). In fact, mangiferin is weakly absorbed and it undergoes bacterial hydrolysis to its aglycone, norathyriol (Bock et al., 2008; Joubert et al., 2009, Liu et al., 2011). Research with the use of a pig model also revealed that norathyriol may undergo further degradation to phenolic acids (Bock and Ternes, 2010). No free mangiferin, but several of its matabolites, including norathyriol were detected in blood plasma of pigs fed with Cyclopia extracts (Bock et al., 2008). These results should be taken into consideration in further studies concerning antioxidant and biological effects of honeybush extracts in animal and human models. Antimutagenic activity of honeybush extracts was demonstrated in in vitro, ex vivo and in vivo models. In a comparative study using a Salmonella typhimurium mutagenicity assay, C. intermedia extracts exhibited stronger activity against the mutagens requiring metabolic activation, i.e. 2-AAF and AFB1, than against directly acting mutagens (MMS, H2O2, and CHP). The activity against 2-AAF was weaker in comparison to rooibos, whereas the activity against AFB1 was similar in both honeybush and rooibos extracts (Marnewick et al., 2000). In another study, the activity of fermented rooibos against AFB1 was comparable to unfermented C. intermedia and C. subternata. On the other hand, unfermented C. intermedia and C. sessiliflora, in comparison to fermented rooibos, provided almost the same activity against 2-AAF. The lowest activity, both against AFB1 and 2-AAF, was recorded for fermented C. intermedia (Van der Merwe et al., 2006). Antimutagenic activity of Cyclopia extracts was also tested ex vivo, with the use of cytosolic and microsomal liver fractions of rats fed with fermented and unfermented honeybush. Protection against AFB1 and 2-AAF was provided by cytosolic fractions obtained from animals fed with unfermented honeybush, while the fermented plant material was only effective in the case of AFB1. Microsomal liver fractions of rats fed with both types of honeybush tea were active only against AFB1 (Marnewick et al., 2004). In another study, livers of rats

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SECTION 2 Miscellaneous Teas and Tea Types: Non-Camellia sinensis

TABLE 12.4 Studies on Biological, Antimicrobial and Antioxidant Activity of Cyclopia Extracts, and the Metabolism of the Relevant Polyphenols Observed Phenomena

Examined Speciesa

Plant Materialb

Type of Extractc

Experimental Modelc

Modulation of chemicallyinduced mutagenesis

C. int.

U; F

Water

In vitro; Ames test with Salmonella typhimurium TA98, TA100 and TA102 strains

C. C. C. C.

U; F

int. sub. sess. gen.

150 C. int.

U; F

C. int.

U; F

C. int.

U; F

Major Findingsc

Inhibitory effect against 2-AAF and AFB1 in the presence of metabolic activation (rat liver S9 homogenate); weak inhibition of mutagenesis induced by directly acting mutagens MMS, CHP and H2O2 Water In vitro; Ames Inhibitory effect test with of C. intermedia, C. subternata and Salmonella typhimurium C. sessiliflora extracts TA98, TA100 and against 2-AAF and TA102 strains AFB1 in the presence of metabolic activation (rat liver S9 homogenate); enhanced mutagenic activity of 2-AAF in the presence of unfermented C. genistoides extract Inhibitory effect of Water Ex vivo; Ames cytosolic fractions test performed against 2-AAF and with the use of subcellular liver AFB1; inhibitory effect fractions of male against AFB1 and Fischer rats fed enhancement of with Cyclopia 2-AAF-induced extracts for mutagenesis by 10 weeks microsomal fractions Suppressed tumor EtOH/DMK In vivo; female development resulting soluble ICR mice from the application of fractions of subjected to Cyclopia extract onto MeOH DMBAthe skin after DMBA extracts treatment followed by TPA initiation and before TPA promotion (90% application in order to induce and 84% inhibition for unfermented and skin tumors fermented teas, respectively) Water In vivo; male Significantly reduced Fischer rats amount of induced subjected to tumors (45.5% MBN-treatment inhibition) and the mean total papilloma in order to size (94% inhibition) in induce animals fed with

Literature Marnewick et al., 2000

Van der Merwe et al., 2006

Marnewick et al., 2004

Marnewick et al., 2005

Sissing et al., 2011

CHAPTER 12 Honeybush Tea (Cyclopia sp.): A Traditional South-African Tisane

TABLE 12.4

Studies on Biological, Antimicrobial and Antioxidant Activity of Cyclopia Extracts, and the Metabolism of the Relevant Polyphenolsdcontinued

Observed Phenomena

Examined Speciesa

Modulation of C. int. chemicallyinduced mutagenesis and liver oxidative status

Modulation of hepatic phase II drug metabolizing enzymes and liver oxidative status

C. int.

Plant Materialb

U; F

U; F

Type of Extractc

Water

Water

Experimental Modelc

Major Findingsc

esophageal papillomas

unfermented honeybush extract for 25 weeks (starting 7 days after MBNtreatment); weaker effects observed for fermented plant material; clear correlation between increased polyphenol content and reduced number of papillomas Marginally diminished number of DENinitiated liver lesions in animals fed with Cyclopia extracts (starting 1 week after initiation and continued until the end of the experiment); DEN-FB1-induced reduction of CAT activity and increase of GSH:GSSG ratio counteracted by the extract; liver lipid peroxidation (measured as TBARS levels) alleviated by honeybush extract Decreased concentration of GSSH and increased level of GSH in rat liver; increased GSH:GSSH ratio; increased activity of cytoplasmatic GST-a (unfermented and oxidized material); increased activity of UDP-GT (unfermented tea); unaltered serum levels of alanine and aspartate aminotransferases and alkaline phosphatase; unaltered serum levels of bilirubin (total and conjugated),

In vivo; male Fischer rats subjected to DEN-treatment (initiation), followed by FB1feeding (21 days, starting 3 weeks after DEN delivery) in order to induce liver damage

In vivo; male Fischer rats fed with Cyclopia extracts for 10 weeks

Literature

Marnewick et al., 2009

151

Marnewick et al., 2003

Continued

SECTION 2 Miscellaneous Teas and Tea Types: Non-Camellia sinensis

TABLE 12.4

Studies on Biological, Antimicrobial and Antioxidant Activity of Cyclopia Extracts, and the Metabolism of the Relevant Polyphenolsdcontinued

Observed Phenomena

Examined Speciesa

Plant Materialb

Type of Extractc

Experimental Modelc

Photoprotective activity

C. int.

U; F

EtOH/DMK soluble fractions of EtOH extracts

In vivo; female SKH-1 mice subjected to UVB irradiation

Phytoestrogenic activity

C. C. C. C.

U; F

Water; MeOH

In vitro; whole cell receptor binding assay (COS-1 cells transiently transfected with either ERa or ERß receptors)

U

MeOH

In vitro; whole cell receptor binding and ERE-containing

152 int. sub. sess. gen.

C. gen.

Major Findingsc creatinine, total cholesterol, total protein and iron Reduced erythema, edema, epidermal hyperplasia and lipid peroxidation in animals subjected to topical application of Cyclopia extracts prior to daily UVBirradiations (10 subsequent days); induction of COX-2 and ODC and depletion of CAT and SOD in irradiated animals counteracted by honeybush extracts; weaker protection observed for individual honeybush components hesperidin and mangiferin Significant estrogenic activity (particularly towards ERß receptors) demonstrated for C. genistoides and C. subternata; stronger effects recorded for methanol extracts from unfermented material; substantial diversity of estrogenic effects within C. genistoides; weak estrogenic activity of C. intermedia and C. sessiliflora extracts; no phytoestrogenic activity demonstrated for mangiferin and hesperidin Transactivation of promoter sections of genes containing estrogen response

Literature

Petrova et al., 2011

Verhoog et al., 2007a

Verhoog et al., 2007b

CHAPTER 12 Honeybush Tea (Cyclopia sp.): A Traditional South-African Tisane

TABLE 12.4 Observed Phenomena

Studies on Biological, Antimicrobial and Antioxidant Activity of Cyclopia Extracts, and the Metabolism of the Relevant Polyphenolsdcontinued Examined Speciesa

Plant Materialb

Type of Extractc

Experimental Modelc

Major Findingsc

elements only via ERß; proliferation of breast cancer cells of estrogen-sensitive MCF-7-BUS cell line induced by extracts and polyphenols found in Cyclopia; proliferation of estrogen-insensitive MDA-MB-231 cell line induced only by honeybush extracts; inhibition of E2induced MCF-7-BUS cells proliferation by Cyclopia extracts; substantial diversity of estrogenic effects within C. genistoides; binding to SHBG demonstrated for all extracts Substantial estrogenic In vitro; whole Water; activity demonstrated cell receptor MeOH; sequential binding (MCF-7- for one C. genistoides and one C. subternata BUS cells) and nonmethanol extract (all sequential promoter assays); substantial reporter (EREextracts diversity of estrogenic (EA, EtOH, containing effects within promoter MeOH, C. subternata; highest reporter 50% transfected into potency and efficacy MeOH, (comparable to that of T47D-KBluc water) cells); E-screen commercial phytoestrogen (MCF-7-BUS preparations) cells) and demonstrated for alkaline sequential methanol phosphatase and ethyl acetate (Ishikawa Var-I C. subternata extracts, cells) assays respectively; considerable estrogenic activity recorded for hot water infusions (E-screen and alkaline phosphatase assays) EtOH/ In vitro; Bacteriostatic effect Water Escherichia coli on E. coli, diminishing (DH5a strain) after 48 h and Botrytis (C. subternata and

Literature

promoter reporter assays (COS-1 cells transiently transfected with either ERa or ERß receptors); MTT cell proliferation assay (MCF-7BUS and MDAMB-231 cells); competitive SHBG binding assay

Antimicrobial activity

C. gen. C. sub.

U

C. sub. C. gen.

U

Mfenyana et al., 2008

Coetzee et al., 2008

Continued

153

SECTION 2 Miscellaneous Teas and Tea Types: Non-Camellia sinensis

TABLE 12.4 Observed Phenomena

Studies on Biological, Antimicrobial and Antioxidant Activity of Cyclopia Extracts, and the Metabolism of the Relevant Polyphenolsdcontinued Examined Speciesa

Plant Materialb

Type of Extractc

Experimental Modelc

Major Findingsc

cinerea (STEU 6253 strain)

Antioxidant activity

C. C. C. C.

int. sub. sess. gen.

U; F

Water

C. int.

F

Water

C. int.

NS

Water

NS

NS

Water

NS

NS

Water; EtOH; hexane

154

C. genistoides); stimulation of B. cinerea growth by C. subternata extract (10 mg/ml); reduced B. cinerea spore germination in the presence of C. genistoides extract (100 mg/ml) ABTS radical Inhibition of Fe2þscavenging induced lipid assay; FRAP; rat peroxidation by liver microsomal 24-42%, depending lipid on species and peroxidation fermentation stage; assay lower antioxidant potential recorded for unfermented teas (except C. genistoides in microsomal lipid peroxidation test); antioxidant effects of honeybush extracts similar or lower than those observed for Aspalathus linearis and Camellia sinensis teas Honeybush Superoxide antioxidant potential anion and hydroxyl radical demonstrated to be higher than for roselle scavenging assay (Hibiscus sabdariffa), but lower than for rooibos (Aspalathus linearis) ABTS radical Cyclopia antioxidant scavenging activity proved to be assay slightly lower than for rooibos (Aspalathus linearis) DPPH radical Cyclopia antioxidant scavenging activity demonstrated assay to be remarkably lower than for rooibos (Aspalathus linearis) Linoleic acid Antioxidant activity of oxidation assay aqueous extract demonstrated to be lower than for rooibos, hibiscus and green tea; antioxidant

Literature

Joubert et al., 2008c

Steenkamp et al., 2004

Ivanova et al., 2005

Du Toit et al., 2001

Lindsey et al., 2002

CHAPTER 12 Honeybush Tea (Cyclopia sp.): A Traditional South-African Tisane

TABLE 12.4 Observed Phenomena

Metabolic fate of mangiferin and hesperidin

Metabolic fate of mangiferin

Studies on Biological, Antimicrobial and Antioxidant Activity of Cyclopia Extracts, and the Metabolism of the Relevant Polyphenolsdcontinued Examined Speciesa

C. gen.

C. gen.

Plant Materialb

U

U

Type of Extractc

Water/ EtOH (80/20)

Water/ EtOH (80/20)

Experimental Modelc

In vivo; female pigs fed with Cyclopia extract for 11 days

In vivo; female pigs fed with Cyclopia extract for 11 days

Major Findingsc potential of ethanolic and hexane extracts proved to be higher than for rooibos but lower than for hibiscus flowers Norathyriol (mangiferin aglycone), but no free mangiferin found in the blood plasma; six mangiferin and hesperidin metabolites detected in the urine (norathyriol, norathyriol monoglucuronide, methyl mangiferin, hesperetin, hesperetin monoglucuronide and eriodictyol monoglucuronide); mangiferin and norathyriol found in the feces; no hesperidin or metabolites ascribed to its intake detected in pig feces Several phenolic acids (4-hydroxybenzoic acid, 3,4dihydroxybenzoic acid, 2,4,5trixydroxybenzoic acid, 2,4,6trixydroxybenzoic acid, 3hydroxyphenylacetic acid, 3,4dihydroxyphenylacetic acid) detected in the feces

Literature

Bock et al., 2008

155 Bock and Ternes, 2010

(Table by Adam Kokotkiewicz, previously unpublished.) a C. int., C. intermedia; C. sub., C. subternata; C. sess., C. sessiliflora; C. gen., C. genistoides; NS, not specified b U, unfermented; F, fermented; NS, not specified c Abbreviations: 2-AAF, 2-acetylaminofluorene; ABTS, 2,2’-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid); AFB1, aflatoxin B1; CAT, catalase; CHP, cumyl hydroperoxide; COX-2, cyclooxygenase-2; DEN, diethylnitrosamine; DMBA, 7,12-dimethylbenz(a)anthracene; DMK, acetone; DPPH, 2,2-diphenyl-1picrylhydrazyl; E2, 17-ß-estradiol; EA, ethyl acetate; ERa, estrogen receptor alpha; ERß, estrogen receptor beta; ERE, estrogen response element; EtOH, ethanol; FB1, fumonisin B1, FRAP, ferric ion reducing antioxidant power; GSH, glutathione; GSSG, glutathione disulfide; GST-a, glutathione S-transferase alpha; MeOH, methanol; MBN, methylbenzylnitrosamine; MMS, methyl methanesulfonate; ODC, ornithine decarboxylase; SHBG, sex hormone-binding globulin; SOD, superoxide dismutase; TBARS, thiobarbituric acid reactive substances; TPA, 12-O-tetradecanoylphorbol-13-acetate; UDP-GT, uridine 5’-diphospho-glucuronosyltransferase; UVB, ultraviolet B

SECTION 2 Miscellaneous Teas and Tea Types: Non-Camellia sinensis fed with honeybush extracts showed elevated antioxidant status, represented by higher levels of GSH and increased GSH:GSSG ratio. Moreover, increased activities of microsomal GST-a and microsomal UDP-GT were recorded in rat livers. The observed effects may contribute to antimutagenic activity of Cyclopia previously observed ex vivo. However, it is also possible that honeybush tea components directly interact with the applied mutagens (Marnewick et al., 2003). In another in vivo experiment, liver lipid peroxidation in rats treated with DEN followed by FB1 was significantly decreased when C. intermedia extracts were included in the diet. The number of liver lesions was also diminished in honeybush-fed animals (Marnewick et al., 2009). Honeybush extracts were also shown to exhibit protective effects when applied externally. In an experiment with the use of hairless SKH-1 mice, topical application of Cyclopia extracts significantly reduced sunburn damage induced by UVB irradiation. The above effect is most probably multifactorial and results from UV-absorbing properties of the preparation, modulation of enzyme activities and the reduction of oxidative stress (Petrova et al., 2011). Apart from the previously described activities, Cyclopia extracts substantially inhibited the development of DMBA-induced skin tumors in ICR mice. In the presented model, antimutagenic activity of both fermented and unfermented honeybush proved to be stronger than rooibos but significantly weaker in comparison to green tea (Marnewick et al., 2005). Anticancer activity of Cyclopia was also observed in rats subjected to MBN treatment in order to induce esophageal papillomas. The experiment showed that both the number and size of the tumors were significantly reduced in the case of animals fed with honeybush extracts (Sissing et al., 2011).

156

In recent years, the phytoestrogenic potential of extracts obtained from Cyclopia plants has been examined, however, no in vivo study has been conducted so far (Table 12.4). In in vitro models, methanolic extracts from unfermented C. subternata and C. genistoides were shown to exhibit highest estrogenic activity, mainly towards ERb (Verhoog et al., 2007a). Substantial differences in estrogenic activity were observed within C. genistoides. Extracts from different harvestings acted via ERa, ERb, or two types of receptors simultaneously (Verhoog et al., 2007b). Interestingly, mangiferin and hesperidin, which are predominant compounds in the polyphenolic fraction in Cyclopia plants, provided no estrogenic activity. On the other hand, significant phytoestrogenic effects were observed in the case of luteolin, formononetin and naringenin which were either absent or present in low quantities in the analyzed extracts. The data suggests that the recorded activity may be attributable to honeybush components which have not yet been identified (Verhoog et al., 2007a, b). In another experiment, it was also demonstrated that noticeable phytoestrogenic activity can be provided by water extracts from unfermented C. genistoides. Moreover, some of the sequential extracts obtained from green honeybush were shown to possess phytoestrogenic activity comparable to that of commercial preparations (Mfenyana et al., 2008). Due to substantial phytoestrogenic activity, green honeybush seems to be especially suitable for the preparation of commercial nutraceuticals. However, care should be taken to select proper material, as plants from various harvestings differ greatly in terms of estrogenic effects (Verhoog et al., 2007b). The antifungal and antibacterial activity of honeybush extract has not been extensively investigated so far. However, the growth of Escherichia coli and spore germination of Botrytis cinerea were significantly inhibited by Cyclopia extracts at certain concentration ranges. Considering the above, Cyclopia plants can be further evaluated for the presence of natural antimicrobial and antifungal agents (Coetzee et al., 2008).

SUMMARY POINTS l l l l

Indigenous South-African herbal tea characterized by sweet, honey-like flavor manufactured from leaves and stems of several Cyclopia spp. both fermented (oxidized, ‘red’) and unfermented (‘green’) teas available lack of caffeine and low tannin content

CHAPTER 12 Honeybush Tea (Cyclopia sp.): A Traditional South-African Tisane

l l

l

l l

l

high polyphenol content, including xanthones, flavanones and flavones antioxidant properties lower or similar to those of Camellia sinensis and rooibos (Aspalathus linearis) teas substantial antimutagenic activities comparable to those of green, black and rooibos teas, demonstrated in in vitro, ex vivo and in vivo models noticeable phytoestrogenic activity demonstrated in vitro substantial inter- and intra-species differences in chemical composition, antoxidant activity and biological effects health benefits in humans yet to be examined.

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SECTION 2 Miscellaneous Teas and Tea Types: Non-Camellia sinensis Joubert, E., Manley, M., Maicu, C., De Beer, D., 2010. Effect of pre-drying treatments and storage on color and phenolic composition of green honeybush (Cyclopia subternata) herbal tea. J. Agric. Food Chem. 58, 338e344. Joubert, E., Otto, F., Gru¨ner, S., Weinreich, B., 2003. Reversed-phase HPLC determination of mangiferin, isomangiferin and hesperidin in Cyclopia and the effect of harvesting date on the phenolic composition of C. genistoides. Eur. Food Res. Technol. 216, 270e273. Joubert, E., Richards, E.S., Van der Merwe, J.D., et al., 2008c. Effect of species variation and processing on phenolic composition and in vitro antioxidant activity of aqueous extracts of Cyclopia spp. (honeybush tea). J. Agric. Food Chem. 56, 954e963. Kamara, B.I., Brand, D.J., Brandt, E.V., Joubert, E., 2004. Phenolic metabolites from honeybush tea (Cyclopia subternata). J. Agric. Food Chem. 52, 5391e5395. Kamara, B.I., Brandt, E.V., Ferreira, D., Joubert, E., 2003. Polyphenols from honeybush tea (Cyclopia intermedia). J. Agric. Food Chem. 51, 3874e3879. Kokotkiewicz, A., Luczkiewicz, M., 2009. Honeybush (Cyclopia sp.) e A rich source of compounds with high antimutagenic properties. Fitoterapia 80, 3e11. Kokotkiewicz, A., Wnuk, M., Bucinski, A., Luczkiewicz, M., 2009. In vitro cultures of Cyclopia plants (honeybush) as a source of bioactive xanthones and flavanones. Z. Naturforsch C 64, 533e540. Le Roux, M., Cronje, J.C., Joubert, E., Burger, B.V., 2008. Chemical characterization of the constituents of the aroma of honeybush, Cyclopia genistoides. S. Afr. J. Bot. 74, 139e143. Lindsey, K.L., Motsei, M.L., Ja¨ger, A.K., 2002. Screening of South African food plants for antioxidant activity. J. Food Sci. 67, 2129e2131. Liu, H., Wang, K., Tang, Y., et al., 2011. Structure elucidation of in vivo and in vitro metabolites of mangiferin. J. Pharm. Biomed. Anal. 55, 1075e1082. McKay, D.L., Blumberg, J.B., 2007. A review of the bioactivity of South African herbal teas: Rooibos (Aspalathus linearis) and honeybush (Cyclopia intermedia). Phytother. Res. 21, 1e16. Malik, J., Szakova, J., Drabek, O., et al., 2008. Determination of certain micro and macroelements in plant stimulants and their infusions. Food Chem. 111, 520e525. Malinowska, E., Inkielewicz, I., Czarnowski, W., Szefer, P., 2008. Assessment of fluoride concentration and daily intake by human from tea and herbal infusions. Food Chem. Toxicol. 46, 1055e1061.

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Marnewick, J.L., Batenburg, W., Swart, P., et al., 2004. Ex vivo modulation of chemical-induced mutagenesis by subcellular liver fractions of rats treated with rooibos (Aspalathus linearis) tea, honeybush (Cyclopia intermedia) tea, as well as green and black (Camellia sinensis) teas. Mutat. Res. 558, 145e154. Marnewick, J.L., Gelderblom, W.C.A., Joubert, E., 2000. An investigation on the antimutagenic properties of South African herbal teas. Mutat. Res. 471, 157e166. Marnewick, J., Joubert, E., Joseph, S., et al., 2005. Inhibition of tumour promotion in mouse skin by extracts of rooibos (Aspalathus linearis) and honeybush (Cyclopia intermedia), unique South African herbal teas. Cancer Lett. 224, 193e202. Marnewick, J.L., Joubert, E., Swart, P., et al., 2003. Modulation of hepatic drug metabolizing enzymes and oxidative status by rooibos (Aspalathus linearis) and honeybush (Cyclopia intermedia), green and black (Camellia sinensis) teas in rats. J. Agric. Food Chem. 51, 8113e8119. Marnewick, J.L., Van der Westhuizen, F.H., Joubert, E., et al., 2009. Chemoprotective properties of rooibos (Aspalathus linearis), honeybush (Cyclopia intermedia) herbal and green and black (Camellia sinensis) teas against cancer promotion induced by fumonisin B1 in rat liver. Food Chem. Toxicol. 47, 220e229. Mfenyana, C., De Beer, D., Joubert, E., Louw, A., 2008. Selective extraction of Cyclopia for enhanced in vitro phytoestrogenicity and benchmarking against commercial phytoestrogen extracts. J. Steroid Biochem. Mol. Biol. 112, 74e86. Petrova, A., Davids, L.M., Rautenbach, F., Marnewick, J., 2011. Photoprotection by honeybush extracts, hesperidin and mangiferin against UVB-induced skin damage in SKH-1 mice. J. Photochem. Photobiol. B. 103, 126e139. Schutte-Vlok, A.L., 1998. Not all milk and honeybush tea. One of the fynbos’ characteristic genera, Cyclopia, faces several threats. Veld & Flora 84, 90e91. Sissing, L., Marnewick, J., de Kock, M., et al., 2011. Modulating effects of rooibos and honeybush herbal teas on the development of esophageal papillomas in rats. Nutr. Cancer 63, 600e610. Sprent, J.I., Odee, D.W., Dakora, F.D., 2010. African legumes: a vital but under-utilized resource. J. Exp. Bot. 61, 1257e1265. Spriggs, A.C., Dakora, F.D., 2007. Competitive ability of selected Cyclopia Vent. rhizobia under glasshouse and field conditions. Soil Biol. Biochem. 39, 58e67. Spriggs, A.C., Dakora, F.D., 2009a. Field assessment of symbiotic N2 fixation in wild and cultivated Cyclopia species in the South African fynbos by 15N natural abundance. Tree Physiol. 29, 239e247.

CHAPTER 12 Honeybush Tea (Cyclopia sp.): A Traditional South-African Tisane

Spriggs, A.C., Dakora, F.D., 2009b. Symbiotic performance of selected Cyclopia Vent. (honeybush) rhizobia under nursery and field conditions. Symbiosis 48, 143e153. Steenkamp, V., Fernandes, A.C., Van Rensburg, C.E.J., 2004. Antioxidant scavenging potential of South African export herbal teas. S. Afr. J. Bot. 70, 660e663. Sutcliffe, M.A., Whitehead, C.S., 1995. Role of ethylene and short-chain saturated fatty acids in the smokestimulated germination of Cyclopia seed. J. Plant Physiol. 145, 271e276. Turpie, J.K., Heydenrych, B.J., Lamberth, S.J., 2003. Economic value of terrestrial and marine biodiversity in the Cape Floristic Region: implications for defining effective and socially optimal conservation strategies. Biol. Conserv. 112, 233e251. Van der Bank, M., Chase, M.W., Van Wyk, B.-E., et al., 2002. Systematics of the tribe Podalyrieae (Fabaceae) based on DNA, morphological and chemical data. Bot. J. Linn. Soc. 139, 159e170. Van der Merwe, J.D., Joubert, E., Richards, E.S., et al., 2006. A comparative study on the antimutagenic properties of aqueous extracts of Aspalathus linearis (rooibos), different Cyclopia spp. (honeybush) and Camelia sinensis teas. Mutat. Res. 611, 42e53. Van Wyk, B.E., 2008. A broad review of commercially important southern African medicinal plants. J. Ethnopharmacol. 119, 342e355. Van Wyk, B.E., 2011a. The potential of South-African plants in the development of new food and beverage products. S. Afr. J. Bot. 77, 857e868. Van Wyk, B.E., 2011b. The potential of South-African plants in the development of new medicinal products. S. Afr. J. Bot. 77, 812e829. Verhoog, N.J.D., Joubert, E., Louw, A., 2007a. Screening of four Cyclopia (honeybush) species for putative phytooestrogenic activity by oestrogen receptor binding assays. S. Afr. J. Sci. 103, 13e21. Verhoog, N.J.D., Joubert, E., Louw, A., 2007b. Evaluation of the phytoestrogenic activity of Cyclopia genistoides (honeybush) methanol extracts and relevant polyphenols. J. Agric. Food Chem. 55, 4371e4381.

159