Cyclopia Maculata Research Articles

Honeybush (Cyclopia spp.) phenology and associated arthropod diversity in the Overberg region, South Africa

Background: Cyclopia is endemic to regions of the Cape Floristic Region across the Eastern and Western Cape provinces of South Africa and is commonly known as honeybush. Honeybush has historically been used as an herbal tea, and has proven medicinal properties. Honeybush biomass and extracts are used in the functional foods and cosmetics sectors, both locally and overseas. The growing demand for honeybush calls for increased agricultural production and a shift away from the predominantly wild harvested supply.Objectives: The current study aimed to address the lack of baseline knowledge on honeybush phenology and its associated arthropod community to advance sustainable production of commercially valued plants in the genus.Method: The study was conducted on wild and cultivated Cyclopia species (Cyclopia maculata and Cyclopia genistoides) at respective sites in the Overberg region. Sampling took place from April 2014 to April 2015 using qualitative methods for recording seasonal honeybush phenology and suction sampling for aboveground arthropods. Focal insect taxa (Coleoptera, Hemiptera, Thysanoptera, Lepidoptera and Hymenoptera) were sorted and identified to family level and classified into functional feeding guilds.Results: Qualitative phenology observations of wild C. maculata and cultivated C. genistoides indicated a high level of congruency in seasonality of phenophase stages. Associated arthropod assemblages contained a diversity of families per functional feeding group, namely phytophagous, zoophagous and omnivorous taxa, with high seasonal variability.Conclusion: Findings highlight the complexity of ecological elements to be taken into consideration for ecologically sound honeybush cultivation. Outcomes can be applied to land management practices and governance policies promoting sustainable agroecosystems in honeybush production areas.

Cyclopia Extracts Enhance Th1-, Th2-, and Th17-type T Cell Responses and Induce Foxp3+ Cells in Murine Cell Culture.

Cyclopia genistoides, one of the traditional South African medicinal plants, and other species of the same genus offer noteworthy phenolic profiles, in particular high levels of the anti-allergic xanthone mangiferin. Hot water and 40% ethanol-water (v/v) extracts, prepared from C. genistoides, Cyclopia subternata, and Cyclopia maculata, were tested for immune-regulating activity in vitro using murine splenocytes and mesenteric lymph node cells. The 40% ethanol-water extracts of C. genistoides and C. subternata significantly enhanced production of several types of cytokines, including IL-4, IL-17, and IFN-γ, by antigen-stimulated splenocytes. A concentration-dependent response was observed, noticeably for IFN-γ production. The activity of the extracts did not correlate with the content of any of the major phenolic compounds, indicative that other extract constituents also play a role in immunomodulation. Additionally, the increased ratio of CD4+CD25+Foxp3+ Treg cells to total CD4+ cells indicated induction of Foxp3+ cells when mesenteric lymph node cells were cultured in the presence of these two extracts. This study is the first reporting immunostimulatory activity for Cyclopia, which are widely consumed as the herbal tea known as honeybush, underpinning further investigations into the potential use of its extracts as adjuvants for mucosal immunotherapy.

Analysis of honeybush tea (Cyclopia spp.) volatiles by comprehensive two-dimensional gas chromatography using a single-stage thermal modulator.

The applicability of comprehensive two-dimensional gas chromatography (GC×GC) using a single-stage thermal modulator was explored for the analysis of honeybush tea (Cyclopia spp.) volatile compounds. Headspace solid phase micro-extraction (HS-SPME) was used in combination with GC×GC separation on a non-polar × polar column set with flame ionisation (FID) detection for the analysis of fermented Cyclopia maculata, Cyclopia subternata and Cyclopia genistoides tea infusions of a single harvest season. Method optimisation entailed evaluation of the effects of several experimental parameters on the performance of the modulator, the choice of columns in both dimensions, as well as the HS-SPME extraction fibre. Eighty-four volatile compounds were identified by co-injection of reference standards. Principal component analysis (PCA) showed clear differentiation between the species based on their volatile profiles. Due to the highly reproducible separations obtained using the single-stage thermal modulator, multivariate data analysis was simplified. The results demonstrate both the complexity of honeybush volatile profiles and the potential of GC×GC separation in combination with suitable data analysis techniques for the investigation of the relationship between sensory properties and volatile composition of these products. The developed method therefore offers a fast and inexpensive methodology for the profiling of honeybush tea volatiles. Graphical abstract Surface plot obtained for the GC×GC-FID analysis of honeybush tea volatiles.

Modifying the sensory profile of green honeybush (Cyclopia maculata) herbal tea through steam treatment

Green honeybush (Cyclopia spp.), superior to the traditional “fermented” product in bioactive phenolic content, is not well accepted by consumers due to its predominant vegetal aroma. Steam treatment of fresh, shredded plant material (0, 30, 60, 90 and 120 s) and dried, shredded plant material (0, 1, 2, 3 and 4 min) of Cyclopia maculata was investigated to improve the aroma of the infusion, but without compromising green colour and phenolic content of the product. Steam treatment increased and decreased fruity and vegetal aroma attribute intensities, respectively. The dried plant material was less susceptible than the fresh plant material to the impact of steam. The fresh and dried plant material should be steam-treated for at least 60 s and 2 min, respectively. Steam treatment had little impact on leaf colour, although the total chlorophyll content was decreased. The individual polyphenol content of the plant material also remained largely unaffected, with reductions in the content of some compounds only when the fresh plant material was steam-treated for 30 s (mangiferin, isomangiferin, 3-β-d-glucopyranosyliriflophenone and hesperidin) and dried plant material steam-treated for 4 min (eriodictyol-O-glucopyranoside). Except for hesperidin, longer steam treatment of the fresh plant material did not affect its individual phenolic content.

By-product of honeybush (Cyclopia maculata) tea processing as source of hesperidin-enriched nutraceutical extract

Cyclopia maculata (honeybush) plant material was evaluated to determine its potential as renewable source material for hesperidin-enriched extract preparation. Stems contained significantly higher hesperidin levels than leaves, while “fermentation” (high-temperature oxidation process), an essential processing step of the herbal tea product, honeybush, had no effect (P>0.05) on hesperidin content. Extraction of hesperidin from the by-product of honeybush tea processing, containing mostly stems, was optimised using response surface methodology (RSM). Four extraction parameters, namely ethanol concentration (% v/v), time (min), temperature (°C), and solvent:solid ratio (mL/g), were evaluated. Second order polynomial mathematical models for hesperidin yield and hesperidin content of the extract were constructed from RSM results and verification experiments indicated good accuracy and predictive ability. Practical process restrictions limited global optimisation and only an optimum for ethanol concentration could be predicted. The choice of process parameter values, feasible for industrial application, will depend on the product application and a cost-benefit analysis of the unit operations of the process.

Germination characteristics of dimorphic honeybush (Cyclopia spp.) seed

Cyclopia is a genus of leguminous shrubs endemic to the fynbos biome of South Africa. Cyclopia spp. are used to make honeybush tea for which a high market demand has led to the overharvesting of Cyclopia species in the wild. Consequently, it has become important to study the cultivation requirements of honeybush in order to support farmers with the cultivation of this crop. Honeybush seeds are known to have either physical or combinational dormancy upon maturity, depending on the species. The plants also produce colour-dimorphic (green and brown), mature seeds in the same pod. A study of honeybush (Cyclopia maculata, Cyclopia genistoides and Cyclopia subternata) seed anatomy was carried out using light and dissection microscopy. It is hypothesized that seed physical dormancy is attributable to a relatively thick (3.5μm) cuticular layer of the integument, a layer of macrosclereid cells, and the presence of a hygroscopically activated hilar valve. There is an absence of other structural openings by which moisture may enter the seed. No consistent structural differences were found amongst the three species or their colour-dimorphic seed. Seeds of the same three honeybush species were divided by colour and their germination response to selected variables (scarification, stratification, seed age and germination temperature) was evaluated. Three dormancy-breaking treatments (wet heat, dry heat and microwave energy) were identified as potential alternatives to conventional sulphuric acid scarification. Highest germination percentage of scarified seed for the three species tested was obtained after 3weeks of cold stratification at 2°C and incubated at 15°C. Brown C. subternata seeds stored for three years gave a significantly higher germination percentage than seed stored for one year only. In non-treated seed, brown seeds had a higher germination percentage than green seeds. In treated seed (i.e. scarified and/or stratified), green seeds had a better germination percentage than brown seeds.

Stability, clinical efficacy, and antioxidant properties of Honeybush extracts in semi-solid formulations.

Background:Honeybush extracts (Cyclopia spp.) can be incorporated into skin care products to treat conditions such as skin dryness and can function as an anti-oxidant.Objective:To formulate Honeybush formulations and test it for antioxidant activity, skin penetration, and skin hydrating effects.Materials and Methods:Semi-solid formulations containing either Cyclopia maculata (2%) or Cyclopia genistoides (2%) underwent accelerated stability studies. Membrane release studies, Franz cell skin diffusion and tape stripping studies were performed. Antioxidant potential was determined with the 2-thiobarbituric acid-assay and clinical efficacy studies were performed to determine the formulations' effect on skin hydration, scaliness, and smoothness after 2 weeks of treatment on the volar forearm.Results:The formulations were unstable over 3 months. Membrane release, skin diffusion studies, and tape stripping showed that both formulations had inconclusive results due to extremely low concentrations mangiferin and hesperidin present in the Franz cell receptor compartments, stratum corneum-epidermis, and epidermis-dermis layers of the skin. Honeybush extracts showed antioxidant activity with concentrations above 0.6250 mg/ml when compared to the toxin; whereas mangiferin and hesperidin did not show any antioxidant activity on their own. The semisolid formulations showed the potential to emit their own antioxidant activity. Both formulations improved skin smoothness, although they did not improve skin hydration compared to the placebos. C. maculata reduced the skin scaliness to a larger extent than the placebos and C. genistoides.Conclusion:Honeybush formulations did not penetrate the skin but did, however, show antioxidant activity and the potential to be used to improve skin scaliness and smoothness.

Sensory profiling of honeybush tea (Cyclopia species) and the development of a honeybush sensory wheel

Several Cyclopia species (Cyclopia sessiliflora, Cyclopia longifolia, Cyclopia genistoides, Cyclopia intermedia, Cyclopia subternata and Cyclopia maculata), used as honeybush herbal tea, were analyzed using descriptive sensory analysis in order to develop a generic honeybush sensory wheel. It was found that the “characteristic” sensory profile of honeybush could be described as “floral”, “sweet-associated”, “fruity”, “plant-like” and “woody” with a sweet taste and a slightly astringent mouthfeel, whereas other attributes defined differences in the sensory characteristics between the Cyclopia species. The species could be divided into three distinct groups: group A (C. sessiliflora, C. intermedia and C. genistoides) associated with “fynbos-floral”, “fynbos-sweet” and “plant-like” attributes, group B (C. longifolia and C. subternata) with “rose geranium” and “fruity-sweet” and group C (C. maculata) with “woody”, “boiled syrup” and “cassia/cinnamon”. The large sample set enabled the development of a generic honeybush sensory wheel, comprising of 30 attributes, organized into positive and negative attributes in primary and secondary tiers. Gas chromatographic-olfactometry of the aroma fraction of a sample of C. maculata with a prominent spicy aroma indicated a high level of eugenol, the only aroma-active compound that associated with a spicy aroma.

Aqueous extract of unfermented honeybush (Cyclopia maculata) attenuates STZ-induced diabetes and β-cell cytotoxicity.

New strategies, which include β-cell protection, are required in the treatment of T2D, as current drugs demonstrate little or no capacity to directly protect the vulnerable β-cell against diabetes-induced cytotoxicity. In this study we investigated the ameliorative effect of pre-treatment with an aqueous extract of unfermented Cyclopia maculata (honeybush) on STZ-induced diabetes and pancreatic β-cell cytotoxicity in Wistar rats after demonstrating a protective effect in vitro in RIN-5F cells. The amelioration of STZ-induced diabetes was seen in the reduction of the area under the curve, determined by the oral glucose tolerance test, as well as fasting glucose levels in extract-treated rats. Pre-treatment with extract also improved serum triglyceride levels and the glucose-to-insulin ratio. Pre-treatment with the extract or the drug, metformin, increased the β-cell area in islets, with a concomitant increase in β-cell proliferation at the higher extract dose (300 mg/kg/d), but not the lower dose (30 mg/kg/d). Subsequently, the in vitro tritiated thymidine incorporation assay showed that the extract was not mitogenic in RIN-5F cells. STZ-induced elevation of plasma nitrite levels was reduced in extract-treated rats, but no changes were observed in their serum catalase, serum glutathione, liver lipid peroxidation and liver nitrotyrosine levels. Pre-treating the rats with extract ameliorated the diabetic effect of STZ in Wistar rats, with evidence of pancreatic β-cells protection, attributed to the presence of high levels of antioxidants such as the xanthones, mangiferin and isomangiferin.

Cyclopia maculata (honeybush tea) stimulates lipolysis in 3T3-L1 adipocytes

We have previously, for the first time, demonstrated that hot water extracts of Cyclopia maculata and Cyclopia subternata, endemic South African plants that are consumed as herbal teas, inhibit adipogenesis in 3T3-L1 adipocytes. The aim of this study was to extend the anti-obesity investigations of these plants by quantifying lipolysis in mature 3T3-L1 adipocytes. Glycerol concentration in culture supernatants was used as a marker of adipocyte lipolysis. Isoproterenol, a β-adrenergic agonist and a known lipolytic agent, was used as a positive control in our assays. Lipolysis was stimulated by all extracts, although statistical significance was noted for fermented (oxidised) C. maculata only. A concentration of 80μg/ml of C. maculata extract induced maximal lipolysis (1.8-fold, p<0.001). The increased lipolysis was accompanied by an increase in the expression of hormone sensitive lipase (1.6-fold, p<0.05) and perilipin (1.6-fold, p<0.05). The plant extracts, at the concentration range assayed (0–100μg/ml), were not cytotoxic in terms of mitochondrial dehydrogenase and adenosine-5′-triphosphate activity. These results showed that C. maculata stimulates lipolysis in mature 3T3-L1 adipocytes, providing further support for the anti-obesity effects of Cyclopia spp.

Cyclopia maculata and Cyclopia subternata (honeybush tea) inhibits adipogenesis in 3T3-L1 pre-adipocytes

The stems, leaves and flowers of Cyclopia have been consumed as a herbal tea ‘honeybush tea’ to treat various medical ailments since the 19th century. Plant polyphenols are reported to inhibit adipogenesis in cell and animal models of obesity. The aim of this study was to assess the effect of hot water extracts of two Cyclopia species, C. maculata and C. subternata on obesity in an in vitro model. The total polyphenol content of unfermented C. subternata, unfermented C. maculata and fermented C. maculata extracts was 25.6, 22.4 and 10.8g GAE/100g, respectively. The major compounds present in the extracts were: the flavonoid, phloretin-3′,5′-di-C-glucoside in C. subternata, the xanthone, mangiferin in unfermented C. maculata and the flavanone, hesperidin in fermented C. maculata. All of the plant extracts inhibited intracellular triglyceride and fat accumulation, and decreased PPARγ2 expression. The higher concentrations of unfermented C. maculata (800 and 1600μg/ml) and C. subternata (1600μg/ml) were cytotoxic in terms of decreased mitochondrial dehydrogenase activity. Both fermented and unfermented C. maculata, at concentrations greater than 100μg/ml, decreased cellular ATP content. Cyclopia maculata and C. subternata inhibit adipogenesis in vitro, suggesting their potential as anti-obesity agents.

Leaf and stem anatomy of honeybush tea (Cyclopia species, Fabaceae)

The leaf and stem microstructure of commercialised Cyclopia species (Cyclopia intermedia, Cyclopia subternata, Cyclopia maculata, Cyclopia genistoides) is described and illustrated for the first time. Leaflet characters of diagnostic value include the shape, margin (flat or revolute), bundle sheath extensions, crystals (aggregates, solitary crystals and crystal sand), relative size of the upper and lower epidermal cells and thickness of the cuticle. The study revealed an abundance of phenolic compounds, not only in the leaf epidermal cells and leaf bundle sheaths, but also in the secondary phloem, xylem rays, xylem parenchyma and pith, showing that the inclusion of older stems may be acceptable and does not necessarily lower the quality of the product.

ALTERATION IN THE MINERAL NUTRITION OF PURELY SYMBIOTIC AND NITRATE-FED NODULATED LEGUMES EXPOSED TO ELEVATED UV-B RADIATION

The effects of elevated UV-B radiation and two nitrogen (N) sources on mineral nutrition of Cyclopia maculata, a UV-B-sensitive species, was assessed together with UV-B-tolerant symbiotic legumes Vigna unguiculata, Glycine max, Lupinus luteus, Vicia atropurpurea, Podalyria calyptrata and Virgilia oroboides. Plants were grown outdoors on tables under moderately or highly elevated UV-B exposures which simulated 15% and 25% ozone depletion above Cape Town in South Africa. Exposing purely symbiotic plants of V. unguiculata to elevated UV-B132 radiation decreased the tissue concentration of calcium (Ca) in leaves, phosphorus (P) in stems and roots, potassium (K) in roots, and boron (B) in roots and nodules relative to UV-B162 or ambient control. In contrast, seedlings of C. maculata, V. oroboides, L. luteus, and V. atropurpurea plants exposed to elevated UV-B radiation increased the concentration of several nutrients in leaves, stems and roots whereas P. calyptrata showed no changes in the mineral nutrition. The reported reduction in plant dry matter yield in C. maculata was accompanied with increased concentrations of P, K, Ca, magnesium (Mg), copper (Cu), and manganese (Mn) in photosynthetic leaves and stems, suggesting that the reported decrease in biomass yield could not be attributed to limitations associated with nutrient uptake. Our results suggest that factors other than limitations of nutrient uptake contributed to the reduced biomass accumulation of purely symbiotic and nitrate (NO3 −)-fed nodulated seedlings of C. maculata.

Impact of increased ultraviolet-B radiation stress due to stratospheric ozone depletion on N2 fixation in traditional African commercial legumes

Reports of diminished nodule formation and nitroge-nase activity in some Asian tropical legumes exposed to above-ambient levels of ultraviolet-B (UV-B: 280-315nm) radiation have raised concerns as to the impact of stratospheric ozone depletion on generally poorly developed traditional African farming systems confronted by the high cost and limited availability of chemical fertilisers. These rely on N 2 -fixing legumes as the cheapest source of N for maintaining soil fertility and sustainable yields in the intrinsically infertile and heterogeneous African soils. In view of this, we examined the effects of supplemental UV-B radiation approximating 15% and 25% depletions in the total ozone column on N 2 fixation in eight traditional African commercial legume species representing crop, forest, medicinal, ornamental and pasture categories. In all categories examined, except medicinal, supplemental UV-B had no effect on root non-structural carbohydrates, antho-cyanins and flavonoids, known to signal Rhizobiaceae micro-symbionts and promote nodule formation, or on nodule mass, activity and quantities of N fixed in different plant organs and whole plants. In contrast, in the medicinal category Cyclopia maculata (Honeybush) a slow growing commercially important herbal beverage with naturally high flavonoid concentrations, displayed decreased nodule activity and quantities of N fixed in different plant organs and whole plants with increased UV-B. This study's findings conclude negligible impacts of ozone depletion on nitrogen fixation and soil fertility in most traditional African farming systems, these limited to occasional inhibition of nodule induction in some crops.

Responses to ultraviolet-B radiation by purely symbiotic and NO3-fed nodulated tree and shrub legumes indigenous to southern Africa.

Purely symbiotic and NO3-fed nodulated seedlings of Virgilia oroboides (Bergius) T.M. Salter, Cyclopia maculata (L.) Vent and Podalyria calyptrata Willd. were exposed to biologically effective ultraviolet-B radiation (UV-B) to assess the effects of above- and below-ambient UV-B on growth, symbiotic function and metabolite concentrations. Seedlings were grown outdoors either on tables under ambient or 34 or 66% above-ambient UV-B conditions (UV-B100 control, UV-B134 and UV-B166, respectively), or in chambers providing below-ambient (22% of ambient) UV-B (UV-B22) along with a UV-A control and a photosynthetically active radiation (PAR) control. Exposure of seedlings to UV-B166 radiation reduced (P < or = 0.05) leaf and stem dry mass by 34 and 39%, respectively, in C. maculata, and reduced leaf nitrogen concentration (%N) by 12% in V. oroboides. Nodule %N in C. maculata and stem %N in P. calyptrata also decreased (P < or = 0.05) in response to UV-B22 radiation compared with the UV-A control, but not compared with the PAR control. Concentrations of flavonoids, soluble sugars and starch were unaltered by the UV-B treatments. Application of 1 mM NO3 to UV-B166-treated seedlings increased whole-plant dry mass of V. oroboides and P. calyptrata by 47 and 52%, respectively. Dry mass of organs, nodule %N and total N concentration of these species also increased with NO3 application. However, NO3 supply decreased (P < or = 0.05) nodule dry mass, stem %N and leaf %N as well as root and leaf anthocyanin concentrations in C. maculata. In terms of UV-B x N interactions, dry mass of stems, roots, nodules and total biomass of NO3-fed C. maculata seedlings were reduced, and nodule %N, total N and leaf anthocyanins were depressed by the UV-B134 and UV-B166 treatments relative to UV-B100-treated seedlings. Although we found that above-ambient UV-B had no effects on growth and symbiotic function of V. oroboides and P. calyptrata seedlings, feeding NO3 to these species increased (P < or = 0.05) seedling growth. In contrast, purely symbiotic C. maculata seedlings were sensitive to the UV-B166 radiation treatment, and adding NO3 further increased their sensitivity to both the UV-B134 and UV-B166 treatments.

The effect of pretreatment on the fermentation of honeybush tea (Cyclopia maculata)

Different pretreatments (bruising, hot and cold water) were used to study their effect on fermentation as measured by the quality parameters of honeybush tea (Cyclopia maculata). Bruising and pretreatment with water (hot and cold) were investigated as means to increase the fermentation rate and to improve product quality. The development of the desired dark-brown colour and honey-like flavour during fermentation was faster in hot and cold water treated material and gave a more uniform coloured product than with the bruising pretreatment. Liquor characteristics also improved with water pretreatment. A red–brown extract, compared with the light yellow–brown of untreated samples, was obtained with the water pretreatment. Hot water pretreatment inactivated the enzymes polyphenol oxidase (PPO) and peroxidase (POD), but did not terminate or impair fermentation. The inactivation of the enzymes, together with high temperature (>60°C) fermentation, indicated a chemical oxidation process rather than an enzymatic reaction in honeybush tea fermentation. © 1998 SCI.

The effect of pretreatment on the fermentation of honeybush tea (Cyclopia maculata)

Different pretreatments (bruising, hot and cold water) were used to study their effect on fermentation as measured by the quality parameters of honeybush tea (Cyclopia maculata). Bruising and pretreatment with water (hot and cold) were investigated as means to increase the fermentation rate and to improve product quality. The development of the desired dark-brown colour and honey-like flavour during fermentation was faster in hot and cold water treated material and gave a more uniform coloured product than with the bruising pretreatment. Liquor characteristics also improved with water pretreatment. A red–brown extract, compared with the light yellow–brown of untreated samples, was obtained with the water pretreatment. Hot water pretreatment inactivated the enzymes polyphenol oxidase (PPO) and peroxidase (POD), but did not terminate or impair fermentation. The inactivation of the enzymes, together with high temperature (>60°C) fermentation, indicated a chemical oxidation process rather than an enzymatic reaction in honeybush tea fermentation. © 1998 SCI.

The effect of pretreatment on the fermentation of honeybush tea (Cyclopia maculata)

The effect of pretreatment on the fermentation of honeybush tea (Cyclopia maculata)