Bush tea (Athrixia phylicoides): A review of the traditional uses, bioactivity and phytochemistry

SAJB-01537; No of Pages 14 South African Journal of Botany xxx (2016) xxx–xxx

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South African Journal of Botany journal homepage: www.elsevier.com/locate/sajb

Review

Bush tea (Athrixia phylicoides): A review of the traditional uses, bioactivity and phytochemistry L. Lerotholi a, S.K. Chaudhary a, S. Combrinck a,b, A. Viljoen a,b,⁎ a b

Department of Pharmaceutical Sciences, Faculty of Science, Tshwane University of Technology, Private Bag X680, Pretoria 0001, South Africa SAMRC Herbal Drugs Research Unit, Faculty of Science, Tshwane University of Technology, Private Bag X680, Pretoria 0001, South Africa

a r t i c l e Available online xxxx Edited by J Van Staden Keywords: Athrixia phylicoides Bush tea Indigenous tea Phytochemistry Antidiabetic Anti-inflammatory Antimicrobial

i n f o

a b s t r a c t Consumption of a refreshing beverage prepared from the dried leaves and twigs of Athrixia phylicoides, commonly referred to as bush tea, is widespread in South Africa. The tea has an illustrious history of use by the indigenous people of southern Africa and has the potential for commercialisation. Several ethnic groups use decoctions and pastes prepared from the plant to treat a multitude of unrelated conditions. This review is aimed at providing a comprehensive overview of the ethnopharmacology, phytochemistry, biological activities, toxicity and commercial aspects of A. phylicoides. Books, containing botanical and ethnopharmacological information were extensively consulted, and electronic databases were searched to acquire relevant articles, with no specific time frame set. Although the majority of the traditional uses indicate that A. phylicoides has strong antimicrobial properties, this is not well supported by scientific data, suggesting that more research is required involving more pathogens and the investigation of a potential alternative mode of action. The secondary metabolites of the plant have been extensively explored and several sesquiterpenes, coumarins, flavonoids and phenol carboxylic acids have been identified. However, there is a paucity of information regarding the chemical variation within the species. A multidisciplinary metabolomic approach involving sophisticated and information-rich technologies, in combination with multivariate analysis, should be undertaken to record chemotypic variation and to identify potential biomarker compounds for quality control. Some of the ethnobotanical uses have been supported through scientific studies, but a window of opportunity exists to contribute data on the pharmacological activities. Despite efforts to facilitate the commercialisation of bush tea in South Africa in an effort to grow the bio-economy of the region, more fundamental research is required to ensure the sustainability and expansion of such an industry. The commercialisation of rooibos and honeybush tea in South Africa is underpinned by basic and applied research and serves as appropriate models for the development of the bush tea industry. © 2016 SAAB. Published by Elsevier B.V. All rights reserved.

Contents 1. 2. 3.

4. 5. 6.

Introduction . . . . . . . . . . . . . . . Methods . . . . . . . . . . . . . . . . Botanical aspects . . . . . . . . . . . . . 3.1. Description and classification . . . . 3.2. Geographical distribution and habitat Phytochemistry . . . . . . . . . . . . . Ethnobotany . . . . . . . . . . . . . . . Biological activities . . . . . . . . . . . . 6.1. Antidiabetic activity . . . . . . . . 6.2. Anti-inflammatory activity . . . . . 6.3. Antimalarial activity . . . . . . . . 6.4. Antimicrobial activity . . . . . . . 6.5. Anti-oxidant activity . . . . . . . .

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⁎ Corresponding author at: Department of Pharmaceutical Sciences, Faculty of Science, Tshwane University of Technology, Private Bag X680, Pretoria 0001, South Africa. Tel.: +27 12 382 6373; fax: +27 12 382 6243. E-mail address: [email protected] (A. Viljoen).

http://dx.doi.org/10.1016/j.sajb.2016.06.005 0254-6299/© 2016 SAAB. Published by Elsevier B.V. All rights reserved.

Please cite this article as: Lerotholi, L., et al., Bush tea (Athrixia phylicoides): A review of the traditional uses, bioactivity and phytochemistry, South African Journal of Botany (2016), http://dx.doi.org/10.1016/j.sajb.2016.06.005

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L. Lerotholi et al. / South African Journal of Botany xxx (2016) xxx–xxx

7. Toxicity studies . . . . . . . . . . . . . . . 8. Factors affecting the phytochemistry of bush tea 9. Commercial aspects . . . . . . . . . . . . . 10. Conclusions . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . .

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1. Introduction A variety of plants that are indigenous to South Africa have been used traditionally as infusions or “teas” (Reichelt et al., 2012). The combination of this rich ethnobotanical heritage with a solid research foundation led to the sustainable commercialisation of indigenous herbal teas, with rooibos (Aspalathus linearis) being the most renowned (Joubert et al., 2008). Honeybush tea (Cyclopia species) has also gained popularity in recent years (Joubert et al., 2011). A third tea, Athrixia phylicoides, known as bush tea, boasts an equally impressive history of use by indigenous people of South Africa. It is most often prepared as an herbal infusion by Zulu, Sotho, Venda and Xhosa communities (Van Wyk and Gericke, 2000; Mbambezeli, 2005). However, the commercialization of bush tea is still in its infancy. The medicinal uses of A. phylicoides among these ethnic groups include the treatment of a wide variety of ailments and conditions, such as sores, boils, acne, infected wounds (Hutchings et al., 1996), hypertension, heart problems, diabetes, diarrhoea, vomiting and some skin conditions (Rampedi and Olivier, 2005). Its use as an anthelmintic, cough remedy and a purgative has also been documented (Watt and Breyer-Brandwijk, 1962). Recent field studies have further revealed that the plant is used to detoxify the body, to relieve headaches, stomach-ache, influenza and to treat leg wounds (Rakuombo, 2007). Although Rakuombo (2007) also reports on the sexual stimulant and aphrodisiac properties, the most popular use remains the use of twigs and leaves as a refreshing caffeine-free tea (Rampedi and Olivier, 2005; McGaw et al., 2013). This practice provides support and impetus for commercialisation (Araya, 2005; Rampedi and Olivier, 2005; Chellan et al., 2008; McGaw et al., 2013). However, the available scientific research is far from adequate to promote commercialization initiatives. This review aims to coherently collate the fragmented information on botanical aspects, ethnopharmacology, phytochemistry and biological properties of A. phylicoides and to further encourage research on this neglected herbal tea from South Africa. 2. Methods An extensive review of the literature, with no specific time frame set to limit the search, was carried out. The keywords “bush tea” and “A. phylicoides” were used to search electronic databases including Scopus®, ScienceDirect®, SciFinder®, Pubmed®, Google® and Google Scholar® for information. All relevant abstracts, full-text articles and regional books written in English, Afrikaans and German were studied and included where appropriate. 3. Botanical aspects 3.1. Description and classification A. phylicoides DC. belongs to the family Asteraceae (daisy family), tribe Inucleae and subtribe Athrixiinae. The Greek word thrix, meaning hair, gave rise to the genus name Athrixia, as a description of the leaves, while the specific epithet phylicoides reflects its similarity to the genus Phylica, a member of the Rhamnaceae (Mbambezeli, 2005). It is commonly known as Mohlahlaishi (Pedi), Mutshatshaila (Venda), bush tea, Bushman's tea, Zulu tea (English), Boesmans tee (Afrikaans),

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Luphephetse (Swati), Sephomolo (Sotho), Umtshanelo, Icholocholo and Itshelo (Zulu). The plant is an aromatic, perennial, leafy shrub that reaches a height of up to 1 m (Fox and Young, 1982). The stems have a white, fluffy appearance, while the leaves are alternate, sometimes radical and sessile (Fig. 1). They are described as fine, linear, 30 × 10 mm, dark green and shiny above, and grey-white and smooth below (Leistner, 2000). The leaf bases are hairy, often decurrent, lanceolate, sometimes needle-like and frequently displaying revolute margins. Flower heads are sessile, terminal and axillary (Fig. 2). The petals are a distinctive mauve colour, with contrasting bright yellow disk florets. Although the most prolific flowering time is from March to May, flowers may be present throughout the year (Mbambezeli, 2005).

3.2. Geographical distribution and habitat In South Africa, A. phylicoides is well adapted to locations of various altitudes, spanning 600 m to 2000 m above sea level (Germishuizen et al., 2006; Lehlohonolo et al., 2012). It is distributed from the northern parts of the Limpopo Province, throughout the Mpumalanga Province, to large areas of the eastern parts of South Africa including the KwaZulu–Natal and Eastern Cape Provinces, Swaziland and Lesotho (Germishuizen et al., 2006). Bush tea is widespread in coastal regions, but also grows abundantly in the Drakensberg (Fig. 3) (Fox and Young, 1982). It can be found in grasslands, forests, bushveld, and in rocky, sloping habitats (Mbambezeli, 2005). A. phylicoides is popular with gardeners that have a preference for indigenous species, since it can be used to fill open spaces in flowerbeds or be grouped together to form a purple mass when flowering (Mbambezeli, 2005).

4. Phytochemistry Flavonoids and tannins are abundant in bush tea and these phenolic compounds have potential as quality indicators for herbal teas. Hlahla et al. (2010) investigated the effects of fermentation temperature and duration on the chemical composition of A. phylicoides and the results of this study indicated that high fermentation temperature significantly increased the total polyphenol content, while the corresponding tannin content was significantly reduced. Longer fermentation resulted in a significant increase in both total polyphenols and tannins. Since tannins affect the taste of tea, sensory evaluation of the tea would assist in establishing a balance between the fermentation period and the tannin content. Tshivhandekano et al. (2014) determined the total phenolic and tannin content of leaves of A. phylicoides and reported values of 6.41 mg/100 g and 0.34 mg/100 mg, respectively. Olivier and Rampedi (2008) conducted a study on the nutrient content of A. phylicoides and reported the presence of approximately 13% non-structural carbohydrates, 8% proteins, 2.5% fatty acids, minerals (calcium, magnesium, phosphorus, potassium, sodium, iron, manganese, zinc, copper, aluminium, sulphur and fluoride), as well as traces of tannins and vitamins B1, B2, C and E. The same group (Olivier et al., 2012) compared the mineral contents of selected commercial teas and indigenous herbal teas. Rooibos and honeybush were found to contain substantially lower levels of most minerals than the other teas, which included bush tea.

Please cite this article as: Lerotholi, L., et al., Bush tea (Athrixia phylicoides): A review of the traditional uses, bioactivity and phytochemistry, South African Journal of Botany (2016), http://dx.doi.org/10.1016/j.sajb.2016.06.005

L. Lerotholi et al. / South African Journal of Botany xxx (2016) xxx–xxx

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Fig. 1. Botanical illustration of the aerial parts of Athrixia phylicoides.

The secondary metabolites produced by A. phylicoides have been extensively investigated and several essential oil constituents and phenolic compounds have been identified (Fig. 4 and Table 1). Bohlmann and Zdero (1977) isolated and identified germacrene D (1), linoleic acid (2) and p-hydroxyphenylpropan-3-ol-coumarate (3) from the leaves of A. phylicoides using silica gel column chromatography (CC). Mashimbye et al. (2006) employed CC to isolate the flavonoid 5-hydroxy-6,7,8,3′,4′,5′-hexamethoxyflavon-3-ol (4) from an acetone extract of the green leaves of A. phylicoides. A few years later, 5,6,7,8,3′,4′-hexamethoxyflavone-3-ol (4) was isolated by Mavundza et al. (2010), together with other flavonoids, 3-O-dimethyldigicitrin (5) and quercetin (6), from the ethanol extract of aerial plant parts. De Beer et al. (2011) isolated one of the major anti-oxidant compounds (6-hydroxyluteolin-7-O-β-glucoside) from an aqueous extract of the twigs and leaves using high performance countercurrent chromatography (HPCCC) combined with semi-preparative high performance liquid

chromatography (HPLC). Chlorogenic acid (7), 1,3-dicaffeoylquinic acid (8), some hydroxycinnamic acid derivatives, such as dicaffeoylquinic acids (3,4-dicaffeoylquinic acid (9) and 3,5-dicaffeoylquinic acid (10), as well as 6-hydroxyluteolin-7-O-β-glucoside (11) and quercetagetin7-O-β-glucoside (12) were also identified, using mass spectral (MS) and ultraviolet–visible (UV–vis) spectroscopy data. The presence of 5hydroxy-6,7,8,3′,4′,5′-hexamethoxyflavon-3-ol (4), which was previously identified as a novel flavonol derivative from the leaves of A. phylicoides by Mashimbye et al. (2006), was also reported by De Beer et al. (2011). Padayachee (2011) identified germacrene D (1) using gas chromatography–mass spectrometry (GC–MS) analysis, as a major compound in the essential oils isolated through hydrodistillation (0.47% yield) from seven samples of A. phylicoides. This compound was previously reported by Bohlmann and Zdero (1977). Other major compounds (N10%) identified in the volatile oil were α-pinene (13),

Please cite this article as: Lerotholi, L., et al., Bush tea (Athrixia phylicoides): A review of the traditional uses, bioactivity and phytochemistry, South African Journal of Botany (2016), http://dx.doi.org/10.1016/j.sajb.2016.06.005

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Fig. 2. Athrixia phylicoides flower (photograph: Alvaro Viljoen).

β-pinene (14), myrcene (15), β-caryophyllene (16), spathulenol (17) and caryophyllene oxide (18). Chellan et al. (2012) used high performance liquid chromatography (HPLC) with diode array detection (DAD) to quantify phenolic compounds in a hot aqueous extract of A. phylicoides. The identities of the compounds were confirmed by ultraperformance liquid

chromatography mass spectroscopy (UPLC-MS). Qualitative and quantitative differences were evident when the phenolic profile was compared to data reported by De Beer et al. (2011) for aqueous and 50% ethanolic extracts. Chlorogenic acid (7), 6-hydroxyluteolin7-O-β-glucoside (11), and quercetagetin-7-O-glucoside (12) were identified by both research groups. 5-Hydroxy-6,7,8,3′,4′,5′hexamethoxyflavon-3-ol (4) was also identified in the aqueous extract (Chellan et al., 2012), as well as hydroxybenzoic acid (19), protocatechuic acid (20), hydroxycinnamic acid (21), caffeic acid (22) and two isomers of chlorogenic acid (neochlorogenic (23) and cryptochlorogenic (24) acids). Two dicaffeoyl quinic acids (3,4-dicaffeoyl quinic (9) and 3,5dicaffeoyl quinic acids (10)) 3′-O-methylcalycopterin (25), one coumaric acid ester (p-coumaric acid 2,6-dimethyl-6-hydroxy-oct-7-enylester (26)), and four polymethoxylated flavones (5,7,3′-trihydroxy-3,6,8,4′,5′pentamethoxyflavone (27), 5,7,4′-trihydroxy-3,6,3′-trimethoxyflavone (28), 5,7-dihydroxy-3,6,8,3′,4′,5′-hexamethoxyflavone (29) and 5,4′-dihydroxy-3,6,7,8,3′-pentamethoxyflavone (30)), quercetin3′-O-glucoside (31), together with a methoxylated derivative (6methoxyquercetin-3′-O-glucoside (32), were isolated and identified by Reichelt et al. (2012) in the methanolic extract of bush tea, using high temperature liquid chromatography (HTLC) and fast centrifugal partition chromatography (FCPC). McGaw et al. (2013) used HPLC and thin layer chromatography (TLC) bioautography to screen diethyl ether, dichloromethane/ methanol, ethyl acetate, ethanol and aqueous extracts of bush tea. They identified several compounds with antimicrobial activity, including quercetin (6), caffeic acid (22), inositol (33), kaempferol (34), apigenin (35), hymenoxin (36) and oleanolic acid (37). Although many studies have been conducted to elucidate the phytochemistry of A. phylicoides, there is little information regarding the

Fig. 3. Distribution map for Athrixia phylicoides in South Africa.

Please cite this article as: Lerotholi, L., et al., Bush tea (Athrixia phylicoides): A review of the traditional uses, bioactivity and phytochemistry, South African Journal of Botany (2016), http://dx.doi.org/10.1016/j.sajb.2016.06.005

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extent of chemical variations within and between natural populations of the species. The available literature is focussed on the variation within classes of compounds (hydrolysable or condensed tannins, or phenolic compounds), rather than on single compounds or complete chemical profiles (Mudau et al., 2006, 2007a,b; Lehlohonolo et al., 2012). There is a lack of specific marker compounds despite evidence that the chemical composition of bush tea is influenced by season, altitude and edaphic factors (Mudau et al., 2006, 2007a,b, 2008; Chabeli et al., 2008; Lehlohonolo et al., 2012). 5. Ethnobotany Different parts of A. phylicoides have been traditionally used by different ethnic groups in South Africa for medicinal and other domestic purposes (Van Wyk and Gericke, 2000). For use as a tea, the aerial

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parts of the plant are collected in often mountainous areas (Fig. 5). Large bundles of material are tied together and taken home, where they are placed in a dry place or hung from the roof-beams. A decoction is made by boiling a scoop of leaves and twigs in water for about 5 min. After straining, the pleasant-tasting beverage is consumed, usually without the addition of milk or sugar (Rampedi and Olivier, 2005). The Sotho and Xhosa chew the leaves to treat sore throats, colds and coughs (Mbambezeli, 2005; Roberts, 1990). The Venda use extracts from soaked roots and leaves as an aphrodisiac (Mabogo, 1990; Van Wyk and Gericke, 2000) and as an anthelmintic (Mabogo, 1990; Mbambezeli, 2005). Venda bachelors are discouraged from drinking these root infusions (Hutchings et al., 1996) that supposedly stimulate sexual desires. The detoxifying properties of Athrixia are portrayed through its traditional use to cleanse the womb, kidneys and veins and to purify blood. Infusions are taken to treat stomach aches,

Fig. 4. Chemical structures of selected compounds isolated from Athrixia phylicoides.

Please cite this article as: Lerotholi, L., et al., Bush tea (Athrixia phylicoides): A review of the traditional uses, bioactivity and phytochemistry, South African Journal of Botany (2016), http://dx.doi.org/10.1016/j.sajb.2016.06.005

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influenza and leg wounds (Rakuombo, 2007), as well as for treating boils, headaches, infected wounds and cuts (Roberts, 1990; Joubert et al., 2008). It is also applied as a lotion to reduce acne and relieve skin rashes (Joubert et al., 2008) and used for skin cleansing. Decoctions are gargled to treat loss of voice and to counteract throat infections (Roberts, 1990). Decoctions and infusions prepared from leaves and twigs are widely used by rural people to treat hypertension, circulation and heart problems, diabetes, diarrhoea and vomiting (Rampedi and Olivier, 2005), while root decoctions are used as a purgative (Watt and Breyer-Brandwijk, 1962). One of the most common uses of A. phylicoides is for making brooms. Long branches are harvested, stripped of their leaves, and the ends (stems) are bound together to make a comfortable handle for the broom (Van Wyk and Gericke, 2000; Rampedi and Olivier, 2005).

6. Biological activities A wide range of biological activities have been reported for extracts and isolated molecules obtained from A. phylicoides, including antibacterial, antifungal, antidiabetic, antimalarial, anti-inflammatory and anti-oxidant activities (Table 2).

6.1. Antidiabetic activity In most developing and developed countries, Type 2 diabetes mellitus (T2D) is on the increase (WHO, 2015), taking its toll on national healthcare systems, in terms of costs and infrastructure. The use of indigenous herbs, including infusions and decoctions of A. phylicoides, to treat obesity and chronic diseases, such as Type 2 diabetes, is widespread in South Africa (Van Wyk and Gericke, 2000). A lower incidence of chronic diseases, particularly cardiovascular diseases and Type 2 diabetes, has been associated with a high intake of phenolic compounds (Knekt et al., 2002; Arts and Hollman, 2005). Chlorogenic acid, among other phenolic compounds, was found to upregulate glucose transporter 4 (GLUT4) and increase the expression of the peroxisome proliferator-activated receptor gamma (PPARᵧ) gene indicating that it may have antidiabetic potential (Prabhakar and Doble, 2009). Using aerial plant parts, Chellan et al. (2012) prepared three concentrations of a hot water extract (0.025, 0.050, 0.10 μg/μL) of A. phylicoides to determine the in vitro effect on cellular glucose utilization in three cell lines C2C12 (ATCC CRL-1772), Chang (ATCC CCL-13) and 3T3-L1 (ATCC CL-173). An increase in glucose uptake and metabolism (Table 2) was induced by the extract in all three cell-types. Although the oxidation of 14C-glucose to 14CO2 by C2C12 and Chang myocytes was significantly increased, no effect was observed on the conversion within 3T3-L1 cells. After exposure to the aqueous extract, the amount of glycogen stored in Chang cells was found to be significantly higher at all three concentrations tested, but there was no effect on the glycogen content in C2C12 myocytes. The assay and protocol used did not permit the quantitative determination of glycogen stored by the 3T3-L1 cells. These findings (Chellan et al., 2012) suggest that hot aqueous extracts of A. phylicoides could potentially mitigate metabolic abnormalities linked to Type 2 diabetes and obesity, by enhancing the utilisation of glucose in tissues that are responsive to insulin. Studies suggest that phenolic acids play an important role in lessening effects of diabetes and obesity (Pinent et al., 2008; Thielecke and Boschmann, 2009). Phenolic acids, 1,3-di-caffeoylquinic acid (8), chlorogenic acid (7), and other hydroxycinnamic acid derivatives found in A. phylicoides are believed to be responsible for modulating the effects of diabetes and obesity (Joubert et al., 2012). However, studies investigating glucose utilisation and storage in cancer cell lines such as Chang cells could provide erroneous outcomes, since many of the underlying metabolic processes in carcinoma cells are distinct and responses may not accurately reflect normal conditions.

6.2. Anti-inflammatory activity Decoctions prepared from the leaves of A. phylicoides are used traditionally by the Sotho to treat painful feet. The leaves are also masticated by the Sotho and Xhosa people to soothe sore throats, possibly related to inflammation of the pharynx (Watt and Breyer-Brandwijk, 1962; Roberts, 1990). These traditional uses suggest that the plant may possess anti-inflammatory activity. Padayachee (2011) applied the in vitro 5-lipoxygenase (5-LOX) assay to determine the anti-inflammatory activity of the essential oil and methanol extract of A. phylicoides (Table 2). Although the IC50 value of 25.68 μg/mL obtained for the essential oil was substantially higher than that obtained for the positive control, nordihydroguaiaretic acid (IC50 5.0 μg/mL), values in the 5-LOX assay of between 10 and 30 μg/mL can be regarded as good (Baylac and Racine, 2003). The methanol extract was found to be inactive against the enzyme at the starting concentration of 100 μg/mL (IC50 ˃ 100 μg/mL) suggesting that polar extracts and therefore “teas”, would not display anti-inflammatory activity along this pathway. However, the 5-LOX enzyme is only one of several enzymes involved in the complex process of inflammation, suggesting that the antiinflammatory activity of the plant has by no means been properly investigated and warrants further attention. 6.3. Antimalarial activity Padayachee (2011) also evaluated the essential oil and methanol extract of A. phylicoides for their potential in vitro antiplasmodial activity against a chloroquine-resistant Plasmodium falciparum (FCR-3) strain, using the titrated hypoxanthine-incorporated assay (Table 2). It was concluded that the methanol extract of A. phylicoides displayed poor activity against the parasite (IC50 83.49 ± 5.48 μg/mL). The essential oil strongly inhibited the chloroquine-resistant strain (IC50 = 1.006 ± 0.060 μg/mL), but the activity was substantially poorer than that of the positive control, quinine (0.034 ± 0.002 μg/mL). 6.4. Antimicrobial activity The many uses of bush tea that allude to infection indicate that it may display antimicrobial activity (Roberts, 1990). However, an investigation of the literature did not yield any compelling evidence to substantiate this assumption. In most of the reported studies (Table 2), polar extracts were tested against micro-organisms, probably to mimic the traditional method of preparation. Minimum inhibitory concentrations (MICs) were determined against a variety of human pathogens. McGaw et al. (2013) did not determine MICs, but instead, identified specific compounds with antimicrobial action using bioautography. Tshivhandekano et al. (2014) evaluated the in vitro antimicrobial activity of ethanol extracts of A. phylicoides against a panel of diverse human pathogens (Table 2). The microdilution technique was used over the concentration range 12.50 to 0.196 mg/mL to determine MICs and minimum microbicidal concentrations (MMC), which ranged from 1.56 to 12.50 mg/mL and from 0.78 to 12.50 mg/mL, respectively. The MIC values obtained are above 1 mg/mL and therefore do not reflect noteworthy activity as defined by Van Vuuren (2008). Similar results were obtained by Padayachee (2011) when the microbial inhibition of the methanol extract and essential oil were evaluated against a host of human pathogens (Table 2). However, a bioactive compound identified as (4-hydroxyphenyl)propyl coumaroate, also known as p-hydroxyphenylpropan-3-ol-coumarate (3) and previously isolated by Bohlmann and Zdero (1977) from A. phylicoides, was found to be highly active against Staphylococcus aureus (MIC 19.5 μg/mL). The relatively low value obtained when compared to that for the crude extract, suggests that the compound may be present in low concentrations in the extract.

Please cite this article as: Lerotholi, L., et al., Bush tea (Athrixia phylicoides): A review of the traditional uses, bioactivity and phytochemistry, South African Journal of Botany (2016), http://dx.doi.org/10.1016/j.sajb.2016.06.005

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Table 1 Chemical data and biological activities reported for selected constituents reported from Athrixia phylicoides. Compound, molecular formula, molecular weight

Type of extraction

Isolation and identification method(s)a

Biological/pharmacological References properties

Germacrene D (1) C15H24 204.35 Linoleic acid (2) C18H32O2 280.45 p-Hydroxyphenylpropan-3-ol-coumarate (3) C18H18O4 298.00 5-Hydroxy-6, 7, 8, 3′, 4′, 5′-hexamethoxyflavon-3-ol (4) C21H22O10 434.00 3-O-Dimethyldigicitrin (5) C20H20O10 420.00 Quercetin (6) C15H10O7 302.236 Chlorogenic acid (7) C16H18O9 354.31 1,3-Dicaffeoylquinic acid (8) C25H24O12 516.45 3,4-Dicaffeoylquinic acid (9) C25H24O12 516.45 3,5-Dicaffeoylquinic acid (10) C25H24O12 516.45 6-Hydroxyluteolin-7-O-β-glucoside (11) C21H20O12 464.10 Quercetagetin-7-O-β-glucoside (12) C21H20O13 480.09 α-Pinene (13) C10H16 136.24 β-Pinene(14) C10H16 136.24 Myrcene (15) C10H16 136.24 β-Caryophyllene (16) C15H24 204.351 Spathulenol (17) C15H24O 220.35 Caryophyllene oxide (18) C15H24O 220.35 Hydroxybenzoic acid (19) C7H6O3 138.12 Protocatechuic acid (20) C7H6O4 154.12 Hydroxycinnamic acid (21) C9H8O3 164.16 Caffeic acid (22) C9H8O4 180.16 Neochlorogenic acid (23) C16H18O9 354.31 Cryptochlorogenic acid (24) C16H18O9 354.31

Diethyl ether:petroleum ether (1:2) extract of the aerial parts

Silica gel CC, NMR, IR spectroscopy, UV–vis spectroscopy

Not reported

Bohlmann and Zdero (1977)

Acetone, ethanol extract of the leaves/aerial parts

Silica gel CC, Sephadex CC, NMR

Anti-oxidant

Mashimbye et al. (2006) Mavundza et al. (2010)

Ethanol extract of the aerial parts

Mavundza et al. (2010)

De Beer et al. (2011)

50% Ethanol extract of the aerial parts

CCC combined with liquid–liquid partitioning and semi-preparative HPLC, LC high resolution ESI-MS, 1H, 13C and 2D NMR spectroscopy

Essential oil from the aerial parts

Hydrodistillation, GC–MS, NMR, UV–vis spectroscopy

Anti-inflammatory, antimicrobial, antimalarial

Padayachee (2011)

Aqueous extract of the aerial parts

HPLC-DAD for quantification; UPLC-MS2 and UV–vis spectroscopy for identification

Not reported

Chellan et al. (2012)

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Table 1 (continued) Compound, molecular formula, molecular weight

Type of extraction

Isolation and identification method(s)a

Biological/pharmacological References properties

3′-O-Methylcalycopterin (25) C20H20O8 388.68 p-Coumaric acid 2,6-dimethyl-6-hydroxyoct-7-enylester (26) C19H26O4 318.175 5,7,3′-Trihydrox3,6,8,4′,5′-pentamethoxyflavone (27) C20H20O8 568.00 5,7,4′-Trihydroxy-3,6,3′-trimethoxyflavone (28) C18H16O8 360.07 5,7-Dihydroxy-3,6,8,3′,4′,5′-hexamethoxyflavone (29) C21H22O10 434.00 5,4′-Dihydroxy-3,6,7,8,3′-pentamethoxyflavone (30) C20H20O9 404.00 Quercetin-3′-O-glucoside (31) C21H20O12 464.38 6-Methoxyquercetin-3′-O-glucoside (32) C22H22O13 494.09 Inositol (33) C6H12O6 180.16 Kaempferol (34) C15H10O6 286.23 Apigenin (35) C15H10O5 270.24 Hymenoxin (36) C19H18O8 374.34 Oleanolic acid (37) C30H48O3 456.71

Methanol extract of the aerial parts

HTLC-coupled sensory-guided analysis, FCPC, preparative HPLC, NMR, LC NMR

Not reported

Reichelt et al. (2012)

Diethyl ether, dichloromethane/methanol, ethyl acetate, ethanol and aqueous extracts of the aerial parts

HPLC, TLC,

Not reported

McGaw et al. (2013)

a Isolation methods: CC = column chromatography, CCC = counter-current chromatography, DAD = diode array detection, HPLC = high performance liquid chromatography, LC–MS = liquid chromatography–mass spectrometry, HTLC = high temperature liquid chromatography, FCPC = fast centrifugal partition chromatography, TLC = thin layer chromatography, LC = liquid chromatography, ESI = electrospray ionisation, MS = mass spectrometry, NMR = nuclear magnetic resonance spectroscopy, UV–vis = ultraviolet- visible, MS2 = tandem mass spectrometry, GC–MS = gas chromatography–mass spectrometry, IR = infrared, UPLC = ultraperformance liquid chromatography.

Fig. 5. Collecting of Athrixia phylicoides in the Limpopo province of South Africa (photograph: Alvaro Viljoen).

Mabona et al. (2013) screened extracts of a variety of southern African medicinal plants, including A. phylicoides, with a history of use against dermal pathogens for their antimicrobial activity. Neither the dichloromethane:methanol or the aqueous extract displayed noteworthy activity against any of the pathogens investigated, since all the MIC values were ≥1 mg/mL. A study was conducted by Mudau and Ngezimana (2014) to evaluate the effect of different drying methods (sun, freeze, shade and oven drying) on the antimicrobial activity of A. phylicoides samples. The method of drying had no effect on the activity obtained. In fact, the overall antibacterial activity was poor, since MIC values of 6.3 mg/mL for all the Gram-negative organisms and 3.1 mg/mL was determined for the Gram-positive bacteria, with the exception of Bacillus cereus, which was even less susceptible (MIC value 6.3 mg/mL). McGaw et al. (2013) prepared extracts from dried, ground, aerial parts of the plant using water and organic solvents of various polarities. The antibacterial and antifungal activities of these extracts, at a concentration of 10 mg/mL, were determined using bioautography analysis. All the extracts were active to some extent against the Gram-negative Escherichia coli, but no activity was recorded against Pseudomonas

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Table 2 Summary of the biological activities of specific compounds or extracts of A. phylicoides. Plant parts, extract/compound Antidiabetic activity Aqueous extract of aerial parts

Assay

Organism/Cell line

Positive control(s)

Activity

Reference

In vitro glucose uptake and metabolism

Cell lines: C2C12 (CRL-1772), Chang (CCL-13), 3T3-L1 (CL-173)

1.0 μM insulin and 1.0 μM 1,1- dimethylbiguanide hydrochloride (metformin), Vehicle control (water)

For concentrations: 0.025, 0.05 and 0.1 μg/mL, respectively, amount of glucose uptake in:

Chellan et al. (2012)

C2C12 (CRL-1772) cells: 183.4 ± 32.6%, 228.3 ± 66.2%, 161.7 ± 8.5% Chang (CCL-13) cells: NNW, 134.5 ± 2.5%, 130.9 ± 5.8%

Aqueous extract of aerial parts

14 14

3T3-L1 (CL-173 cells): 143.5 ± 10.3%, 134.7 ± 18.8%, NNW For concentrations: Chellan et al. 0.025, 0.05 and 0.1 μg/mL, respectively, the 14C-glucose (2012) oxidation to 14CO2 measured as radioactivity (in fmol normalised to 1 × 106 cells) were: C2C12 (CRL-1772) cells: NNW, 2806.3 ± 751 and 2919.3 ± 428 compared to 1992.00 ± 315.95 for vehicle control;

C glucose oxidation to CO2

Chang (CCL-13) cells: NNW, 3115.7 ± 743.6 and 4476.7 ± 1620, compared to 2072.92 ± 435.52 for vehicle control;

Aqueous extract of twigs and leaves

3T3-L1 (CL-173) cells: No oxidation For concentrations: 0.025, 0.05 and 0.1 μg/mL respectively, the glycogen storage (in μg normalised to 1 × 106 cells) were:

Glycogen storage

Chellan et al. (2012)

C2C12 (CRL-1772) cells: Not affected Chang (CCL-13) cells: 13.6 ± 0.7 μg, 12.7 ± 1.8 μg and 12.7 ± 1.6 μg compared to 8.5 ± 2.88 μg for vehicle control (water) 3T3-L1 (CL-173) cells: Not detectable Antimicrobial activity Bioautography Diethyl ether, dichloromethane/methanol, ethyl acetate, ethanol and aqueous extracts of dried ground aerial parts

Ethanol extract of the leaves

Dilution method

Escherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 27853), Staphylococcus aureus (ATCC 29213), Enterococcus faecalis (ATCC 29212), Candida albicans (from a Gouldian finch) and Cryptococcus neoformans (isolated from a cheetah). Escherichia coli, Klebsiella oxytoca, Proteus vulgaris, Serratia marcescens, Salmonella typhi, Klebsiella pneumonia, Bacillus cereus, Staphylococcus aureus, Candida albicans

Not provided

Clear inhibitory zones were obtained when organic extracts were analysed by TLC bioautography. Aqueous extract inactive.

McGaw et al. (2013)

Not reported

(MIC 1.56 to 12.50 mg/mL and MMC 0.78 to 12.50 mg/mL)

Tshivhandekano et al. (2014)

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Table 2 (continued) Plant parts, extract/compound

Assay

Essential oil from the leaves Microdilution method Methanol extract of the leaves Bioactive compound (4-hydroxyphenyl propyl coumarate (38) from methanol extract of the leaves

Dichloromethane:methanol (1:1) extract of the leaves Aqueous extract of the leaves

Dilution method

Extract prepared not specified, but from the leaves

Dilution method

Organism/Cell line

Positive control(s)

Activity

Reference

Staphylococcus aureus (ATCC 12600), Enterococcus faecalis (ATCC 29212), Bacillus cereus (ATCC 11778), Staphylococcus epidermidis (ATCC 2223), Bacillus subtilis (ATCC 6051), Klebsiella pneumoniae (NCTC 9633), Escherichia coli (ATCC 8739), Yersinia entercolitica (ATCC 23715), Salmonella typhimurium (ATCC 14028), Candida albicans (ATCC 10231), Candida tropicalis (ATCC 750) Staphylococcus aureus (ATCC 25923), methicillin-resistant Staphylococcus aureus (MRSA) (ATCC 43300), gentamycin–methicillin-resistant Staphylococcus aureus (GMRSA) (ATCC 33592), Staphylococcus epidermidis (ATCC 2223), Brevibacillus agri (ATCC 51663), Propionibacterium acnes (ATCC 11827), Pseudomonas aeruginosa (ATCC 27858), Trichophyton mentagrophytes (ATCC 9533), Microsporum canis (ATCC 36299), Candida albicans (ATCC 10231) Staphylococcus aureus (ATCC 12600), Bacillus cereus (ATCC 11778), B. subtilis, B. pumilis (ATCC 21356), Enterococcus faecalis (ATCC 29212), Pseudomonas aeruginosa (ATCC 25922), Escherichia coli (ATCC 11775), Klebsiella pneumonia (ATCC 27736)

For bacteria: Ciprofloxacin

MIC 3 to N32 mg/mL MIC 1 to 6 mg/mL Active against: Staphylococcus aureus (MIC 19.5 μg/mL) Escherichia coli (MIC not determined) Candida albicans (MIC not determined)

Padayachee (2011) Padayachee (2011) Padayachee (2011)

MIC ranged from 1.0 to 3.0 mg/mL MIC ranged from 2.0 to N16.0 mg/mL

Mabona et al. (2013) Mabona et al. (2013)

Not reported

MIC range from 3.1 to 6.3 mg/mL

Mudau and Ngezimana (2014)

McGaw et al. Infusions (0.248 ± 0.012), decoctions (0.269 ± 0.015), (2007) aqueous extract (0.278 ± 0.022) and ethanol (0.174 ± 0.007) as a % of gallic acid 10.64 ± 0.08 μg/mL Mavundza et al. (2010) 2.74 ± 0.10 μg/mL Mavundza et al. (2010)

For yeast: Amphotericin B

For bacteria: Ciprofloxacin For yeast: Amphotericin B

Anti-oxidant activity Infusions, decoctions and extracts (aqueous and ethanol) of the leaves

Trolox-Equivalent Antioxidant Capacity (TEAC) and TLC-DPPH

In vitro

Gallic acid

Ethanol extract of the leaves

DPPH-spectrophotometric

In vitro

Ascorbic acid (vitamin C)

5-Hydroxy-6,7,8,3′,4′,5′ hexa-methoxyflavon-3-ol (from the leaves} 3-O-Demethyldigicitrin (from the leaves) Quercetin (from the leaves)

DPPH-spectrophotometric

In vitro

Ascorbic acid (vitamin C)

DPPH-spectrophotometric

In vitro

Ascorbic acid (vitamin C)

DPPH-spectrophotometric

In vitro

Ascorbic acid (vitamin C)

Aqueous extract of the aerial parts

ABTS, DPPH, peroxyl radical (ORAC), FRAP, iron chelating activity and inhibition of lipid peroxidation and linoleic acid emulsion oxidation

In vitro

Caffeic acid reference

Titrated hypoxanthine incorporated assay

Chloroquine-resistant Plasmodium falciparum (FCR-3) Chloroquine-resistant Plasmodium falciparum (FCR-3)

Quinine (0.034 ± 0.002 μg/mL)

1.006 ± 0.06 μg/mL

Padayachee (2011)

Quinine (0.034 ± 0.002 μg/mL)

83.489 ± 5.482 μg/mL

Padayachee (2011)

Antimalarial activity Essential oil from the leaves

Methanol extract of the leaves Titrated hypoxanthine incorporated assay

3.41 ± 0.09 μg/mL

Mavundza et al. (2010) 1.27 ± 0.25 μg/mL Mavundza et al. (2010) ABTS = 1267, DPPH = 1437, De Beer et al. (2011) ORAC = 7237 FRAP = 873 μmol Trolox equiv./g, Iron chelating activity 21.7%, inhibition of lipid peroxidation 14% and linoleic acid emulsion oxidation 86.3%.

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Table 2 (continued) Plant parts, extract/compound

Assay

Anti-inflammatory activity Essential oil from the leaves 5-Lipoxygenase assay Methanol extract of the leaves 5-Lipoxygenase assay

Organism/Cell line

Positive control(s)

Activity

Reference

In vitro In vitro

Nordihydroguaiaretic acid Nordihydroguaiaretic acid

25.68 μg/mL ˃ 100 μg/mL

Padayachee (2011) Padayachee (2011)

MIC = minimum inhibitory concentration, MMC = minimum microbial concentration, TLC = thin layer chromatography, NNW = not noteworthy, DPPH = 2,2-diphenyl-1picrylhydrazyl, ABTS = 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid, ORAC = oxygen radical absorbance capacity, FRAP = ferric reducing ability of plasma, TEAC = Troloxequivalent antioxidant capacity.

aeruginosa. The diethyl ether, dichloromethane/methanol, ethyl acetate and ethanol extracts inhibited the growth of Cryptococcus neoformans and that of the Gram-positive Enterococcus faecalis and S. aureus, as well as the fungus Candida albicans. Five antibacterial and two antifungal compounds, most of a phenolic nature, were detected, but not identified, in the organic extracts. These extracts contained more compounds with antimicrobial activity than the aqueous extract. The antimicrobial activity A. phylicoides still warrants further investigation, since plant extracts only displayed limited antimicrobial activities in the assays used. However, it is possible that individual plants from different origins may exert differential activity due to having different chemical compositions. It is also possible that the mode of microbial inhibition takes place via inhibition of quorum sensing, rather than through direct damage to the cell membranes of micro-organisms. Although the activity of bush tea extracts has been determined against a number of diverse human pathogens, in-depth research aimed at identifying compounds with antimicrobial activity must be undertaken. Such research could contribute to the establishment of quality control parameters for commercial products. 6.5. Anti-oxidant activity Herbal teas are popularly regarded as useful for the prevention and cure of diseases. During the past decade, there has been an increasing interest in the commercial development of plants, rich in antioxidants, as foods or beverages (Ivanova et al., 2005), since such compounds are perceived to contribute to the general maintenance of health. McGaw et al. (2007) screened an infusion, a decoction and extracts (aqueous and ethanol) prepared from A. phylicoides at concentrations of 0.5, 1 and 2 mg/mL, to determine their anti-oxidant activity. The A. phylicoides infusion, decoction and cold water extracts displayed good free radical scavenging ability in the Trolox-Equivalent Antioxidant Capacity (TEAC) assay, as reflected by the values obtained (Table 2). Values obtained for the decoction were higher than those reported for rooibos tea, which was explained by the higher concentrations of phenolic compounds in the bush tea extract when compared to rooibos (McGaw et al., 2007). Specific compounds with antioxidant activity were detected, but not identified, on the TLC plate sprayed with DPPH reagent. In a study conducted by Mavundza et al. (2010), the anti-oxidant activity of a crude ethanol extract of A. phylicoides, together with three isolated compounds i.e. 5-hydroxy-6,7,8,3′,4′,5′-hexamethoxyflavon3-ol (4), 3-O-demethyldigicitrin (5), and quercetin (6), were evaluated using the DPPH-spectrophotometric assay. The ethanol crude extract (3.9 to 500 μg/mL) exhibited a concentration-dependent radical scavenging activity with an EC50 value (maximal effective concentration) of 10.64 ± 0.08 μg/mL. Of the three compounds, quercetin displayed the best radical scavenging ability, while 3-O-demethyldigicitrin was the least potent (Table 2). De Beer et al. (2011) determined the antioxidant activity of an aqueous extract of A. phylicoides as reflected by its potency to scavenge free radicals using the ABTS•+, DPPH• and peroxyl radical (ORAC) scavenging assays. The reducing potential (FRAP), iron chelating activity, inhibition of lipid peroxidation and

linoleic acid emulsion oxidation assays were also used (Table 2). The excellent anti-oxidant activities of polar and aqueous extracts of A. phylicoides, as demonstrated in these assays, indicates that the tea can be used as a reliable source of anti-oxidants. A hot aqueous extract displayed better anti-oxidant activity comparable to that of fermented honeybush extracts. However, the extract exhibited lower DPPH• scavenging activity and reducing potential, than an unfermented rooibos extract. The ability of Athrixia to inhibit microsomal peroxidation was better than that of unfermented rooibos. It was found that the ORAC and ABTS•+ scavenging activities were similar to those of fermented rooibos extracts. The iron chelation activity of A. phylicoides was better than those of the honeybush and rooibos extracts. The studies of De Beer et al. (2011) and McGaw et al. (2007) were limited to only a handful of samples from each herbal tea and larger sample sets may have revealed greater variation in activity due to chemical differences between batches. However, despite these shortcomings, the studies clearly indicate that Athrixia has anti-oxidant activity that can compete with other indigenous herbal teas. Negukhula et al. (2011) conducted a study to relate the soaking (fermentation) conditions with the anti-oxidant activity of bush tea. They found that the anti-oxidant content was significantly reduced when bush tea was combined with black tea. However, when the fermentation temperature was increased to 90 °C for 3 min, there was an increase in the anti-oxidant content of the combined teas. Although the contribution of three compounds to the anti-oxidant potential was revealed by Mavundza et al. (2010), work by McGaw et al. (2007) suggests that there may be many more compounds with anti-oxidant activity present. Some of these compounds could potentially be utilised as chemical markers to determine the quality of raw materials for tea production.

7. Toxicity studies Although perceived by consumers to be safe, medicinal plants must be used with caution until the safety has been confirmed by scientific data. Toxicology studies integrate knowledge of the phytochemistry, plant part used, preparation methods and mode of administration (Mounanga et al., 2015). McGaw et al. (2007) conducted preliminary toxicological assays of A. phylicoides. The cytotoxicity of plant extracts (decoction, infusion, water and ethanol), over the concentration range 1–1000 μg/mL was evaluated in the Vero monkey kidney cell line. The LD50 values for the aqueous and ethanol extracts were above 1000 μg/mL, and 252 μg/mL, respectively, indicating the absence of toxicity. These results were confirmed on ethanol and aqueous extracts (decoction, infusion, and water) at concentrations of 0.1, 0.2, 0.5, 1, 2 and 5 mg/mL using a brine shrimp larval mortality assay. The reported LC50 values were above 1000 μg/mL for the aqueous extracts and 394 μg/mL for the ethanol extract (McGaw et al., 2007), affirming the absence of toxicity. Two A. phylicoides samples were also screened by these researchers for the presence of the notoriously toxic pyrrolizidine alkaloids, known to be produced by some members of the Asteraceae family (Wiedenfeld, 2011). Fortunately none was detected, since these

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compounds have been found to be hepatotoxic, genotoxic and carcinogenic (Fu et al., 2004). Chellan et al. (2012) exposed Chang cells to hot water extracts of fine aerial parts of A. phylicoides at concentrations of 0.025, 0.05, 0.1 μg/μL. The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) cytotoxicity assay revealed no inhibition of mitochondrial activity or reduction in Chang cell viability, implying that the extracts are not cytotoxic. The cytotoxicity of the ethanol extract, at concentrations ranging from 3.13 to 400 μg/mL, and pure isolated compounds 5-hydroxy6,7,8,3′,4′,5′-hexamethoxyflavon-3-ol (4), 3-O-demethyldigicitrin (5) and quercetin (6) from A. phylicoides, at concentrations of 1.56 to 200 μg/mL, was investigated by Mavundza et al. (2010) using the XTT (sodium 3′-[1-(phenyl amino-carbonyl)-3,4-tetrazolium]-bis-[4methoxy-6-nitro] benzene sulfonic acid hydrate) colorimetric assay. The concentration of the extract or pure compounds corresponding to 50% of the Vero cells, still alive four days after exposure and regarded as the highest non-toxic concentration to the cells was found to be 400 μg/mL. The ethanol extract displayed no toxicity (IC50 107.8 ± 0.13 μg/mL), while quercetin was only slightly toxic (IC50 value of 81.38 ± 0.33 μg/mL). The highest toxicity was ascribed to 3-Odemethyldigicitrin (5) (IC50, 28.92 ± 0.12 μg/mL) and 5-hydroxy6,7,8,3′,4′,5′-hexamethoxyflavon-3-ol (4) (IC50, 27.91 ± 0.18 μg/mL). Scrutiny of the available literature yielded only one in vivo study to assess the toxicity of an aqueous extract of A. phylicoides (Chellan et al., 2008). Potential toxicity was evaluated in mature Wistar rats by dosing them daily with the extract. Three experimental groups received 30, 90 or 180 mg dried aqueous A. phylicoides extract/kg body mass/day for 90 days. No morbidity or mortality was recorded throughout the study. Food and water intake, as well as body mass and stool production, were found to be unaffected, when compared to the control group. The extract was mildly diuretic as reflected by an increase in urine production in individuals exposed to 90 and 180 mg/kg extract. Serum alkaline phosphatase, creatinine, and urea levels were normal for all groups. Histopathology did not reveal any signs of toxicity to the liver, kidney, gastrointestinal tract and other tissues studied. None of the scientific studies aimed at investigating the toxicity of A. phylicoides has revealed detrimental effects towards cells or experimental animals. This evidence, combined with a rich history of use, in which no adverse consequences arising from the consumption of the tea have ever been documented, suggest that aqueous extracts of A. phylicoides have low toxicity, if any. 8. Factors affecting the phytochemistry of bush tea Various groups, Mudau and co-workers in particular, have investigated the effects of various environmental factors and nutrition on total phenolic compounds and tannins, as well as on the anti-oxidant activity on bush tea. A study was undertaken by Lehlohonolo et al. (2012) to determine the link between environmental factors and the phenolic composition of wild bush tea. They harvested specimens from localities differing in altitude, climate and edaphic factors. Significantly higher concentrations of soluble phenolic compounds were present in samples growing at high altitudes (944–1410 m ASL). However, there was little or no correlation between altitude and tannin content, and no significant differences in the total anti-oxidant content of samples from different altitudes. According to the study, the total polyphenol, total tannin and total anti-oxidant contents were unaffected by rainfall, temperature, soil macro elements and soil pH. Mudau et al. (2007b) investigated seasonal variations in the tannin content of wild-harvested A. phylicoides leaves. They found that the concentrations of condensed tannins were considerably higher in autumn, compared to the other seasons. Low levels of tannins are desirable in a tea, since tannins impart a bitter taste and astringency (tannin distinctiveness) to the tea (Maudu et al., 2012). The same group (Mudau et al., 2008) reported significant seasonal variation in the total anti-

oxidant content of wild-harvested bush tea, with the highest content present during winter and summer. It was proven that soil enrichment with nitrogen, phosphorous and potassium results in an increase in total phenolic compounds (Mudau et al., 2007a) and hydrolysable and condensed tannins (Chabeli et al., 2008) in bush tea. Although much research has been directed towards the effects of various factors on the production of phenolic compounds and tannins by A. phylicoides, very little has been reported on the sensory characteristics and consumer preferences linked to these compound classes. 9. Commercial aspects A. phylicoides is a multi-purpose plant that is valued as an everyday beverage and medicine (Van Wyk and Gericke, 2000). It has been harvested for generations by rural communities. Currently the main use of the plant is for the making of brooms, resulting in overexploitation, of which the signs have become evident. Rampedi and Olivier (2005) found that it has become common practice for traders from Johannesburg and Pretoria, who have realised the market potential of the brooms, to hire casual labourers to collect large quantities of the plant from the Wolkberg, a mountainous area in the Limpopo Province. Trucks, filled to overflowing with A. phylicoides, are a common sight on the highway leading to Gauteng during autumn. Brooms made from this material are sold by hawkers on the city streets. Unsustainable harvesting has led to declining populations along the lower reaches of the Wolkberg. The local people are mindful to pick only some of the branches, whereas pickers from elsewhere frequently uproot the plant, which is often unable to recover. According to the villagers, bush tea is becoming scarcer and distances of up to 12 km have to be travelled on foot to reach populations (Rampedi and Olivier, 2005). Brooms are a low-cost item and involves little value-adding, whereas research has indicated that South African indigenous plants have untapped market potential for the beverage industry. These industries, if developed sustainably, could contribute extensively to economic growth of urban and rural regions of South Africa (Rampedi and Olivier, 2005; Joubert et al., 2008). Several groups (Araya, 2005; Rampedi and Olivier, 2005; Mudau et al., 2007a, 2007b; Chellan et al., 2008) have been involved directly or indirectly in research that contributes towards commercialisation. Lehlohonolo et al. (2013) are of the opinion that bush tea must either outperform rooibos and honeybush teas in terms of its medicinal properties, or complement them, if it is to be commercially viable. Unlike rooibos and honeybush tea that are restricted to very specific habitats (Joubert et al., 2008), bush tea thrives in diverse climates and grows on a variety of soils. Propagation and mass cultivation, which are easily achievable, will counteract the increasing scarcity of the species due to unsustainable harvesting techniques. A number of studies conducted on various aspects of bush tea prepared from A. phylicoides have indicated its potential as an herbal beverage (Joubert et al., 2008; McGaw et al., 2013). The successful development of the rooibos industry in South Africa relied on fundamental research as a driver for quality control and product development, and provides a good model for the establishment of a bush tea industry. Some work has been done regarding propagation techniques as a forerunner to commercialisation. Maudu et al. (2012) compared bush tea quality of both cultivated and wild bush tea when prepared from new growth, whole plant or older plant parts. In cultivated bush tea, new growth or whole plant had better tea quality, in terms of polyphenol, tannin and anti-oxidant content, than the older leaves. However, in wild bush tea, both new and older growths were found to have comparative total polyphenol and anti-oxidant contents (Maudu et al., 2012). Araya (2005) studied the effects of applying different techniques on propagation success using cuttings and seed. Cutting position (apical vs basal), growth medium (pine bark vs sand), season (summer, autumn, winter or spring), transplanting survival of rooted apical and basal cuttings, response of basal cuttings to three hormone concentration levels (Seradix Nos. 1, 2 and 3) and light and temperature

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requirements for seed germination, were studied. The most successful vegetative propagation and best survival rate of bush tea proved to be from apical cuttings exposed to Seradix No. 2, although Seradix No. 1 provided better results with basal cuttings in pine bark. A higher percentage of cuttings formed roots in autumn and spring than in the other seasons. However, the number of roots formed was higher in spring, whereas in autumn, longer roots were obtained. The highest percentage of seeds germinated at 20 °C when exposed to continuous light (Araya, 2005).

sensing is thought to be an alternative target for antipathogenic drugs, particularly for combating bacterial infections (Vattem et al., 2007). The antidiabetic, anti-oxidant, antimalarial and anti-inflammatory have been studied but further research into the activities is needed, particularly by utilising appropriate in vivo models and addressing the mechanisms of action involved. The potential of bush tea to increase sexual desire and performance remains scientifically unexplored. Further investigations of the cardiovascular, anthelmintic and analgesic activities need to be undertaken to confirm the ethnobotanical claims matching these activities.

10. Conclusions

Acknowledgments

A lively informal trade in bush tea exists, particularly in the Gauteng region (Rampedi and Olivier, 2005), but it is focussed on a low value commodity, namely the making of brooms. Bush tea has been enjoyed by mainly rural South Africans until now as a refreshing beverage. The promotion of the plant as a caffeine-free and healthy alternative drink, competing with and perhaps even combined with rooibos and honeybush tea, has economic potential. This potential can be easily exploited, particularly since South African indigenous teas have built up a good reputation internationally. Although some detailed studies have focussed on the phytochemistry of A. phylicoides, there is little data on the chemical variation within the species, other than work done on specific groups of compounds. Unlike other indigenous South African teas that have been commercialised, A. phylicoides flourishes in a range of diverse habitats and climates, since it occurs in six of the nine provinces of South Africa (Germishuizen et al., 2006). These environmental factors tend to affect the secondary metabolites produced by plants. It is also possible that a great deal of genetic diversity exists within the species. Chemical variation should therefore be addressed prior to commercialisation of bush tea. Some studies have revealed that the chemical composition of bush tea is indeed affected by nutrients, such as the available concentrations of nitrogen, phosphorous and potassium (Mudau et al., 2006, 2007a; Chabeli et al., 2008). The volatile compounds in some cases impart many of the flavour characteristics to tea and these profiles should be more closely investigated as an indicator of sensory attributes. The non-volatile fingerprints, on the other hand, may be closely linked to the medicinal properties. Knowledge of the chemical variations that exist will assist in identifying appropriate biomarker compounds that can be used for quality control. Rooibos, for instance, is one of the herbal teas that have been successfully commercialized and chemical profiles have been studied. Aspalathin, nothofagin, orientin, iso-orientin, vitexin, isovitexin, isoquercitrin, luteolin, quercetin and chrysoeriol have been identified as marker compounds for good quality of rooibos (Joubert et al., 2008). Obtained chemical profiles can be compared with those of extracts of related species, for example A. phylicoides with Athrixia elata, which is also used for preparing beverages, to determine whether adulterated samples can be identified as part of quality control. In a review on South African herbal teas (Joubert et al., 2008), it was concluded that future research on tea species including A. phylicoides should be directed towards more comprehensive chemical characterisation of extracts, and identification of marker compounds for extract standardisation and quality control. However, little has transpired during the past seven years to fill these voids. Although the majority of traditional uses of A. phylicoides indicate that bush tea has antimicrobial activity (McGaw et al., 2013; Tshivhandekano et al., 2014), the existing data indicates only moderate activity against the organisms tested (Table 2). It is possible that certain chemotypes may be more active against micro-organisms than others, providing impetus for chemical variation studies to be undertaken and for these to be coupled to efficacy studies. No published data on the anti-quorum sensing (AQS) activity of the plant could be found. It is possible that the antimicrobial activity of bush tea, as reflected by the traditional uses, may be explained by AQS activity. Inhibition of quorum

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Please cite this article as: Lerotholi, L., et al., Bush tea (Athrixia phylicoides): A review of the traditional uses, bioactivity and phytochemistry, South African Journal of Botany (2016), http://dx.doi.org/10.1016/j.sajb.2016.06.005