Fermented Honeybush Research Articles

Photoprotection and Antiaging Activity of Extracts from Honeybush (Cyclopia sp.)-In Vitro Wound Healing and Inhibition of the Skin Extracellular Matrix Enzymes: Tyrosinase, Collagenase, Elastase and Hyaluronidase.

Cyclopia sp. (honeybush) is an African shrub known as a rich source of polyphenols. The biological effects of fermented honeybush extracts were investigated. The influence of honeybush extracts on extracellular matrix (ECM) enzymes responsible for the skin malfunction and aging process-collagenase, elastase, tyrosinase and hyaluronidase-was analysed. The research also included assessment of the in vitro photoprotection efficiency of honeybush extracts and their contribution to the wound healing process. Antioxidant properties of the prepared extracts were evaluated, and quantification of the main compounds in the extracts was achieved. The research showed that the analysed extracts had a significant ability to inhibit collagenase, tyrosinase and hyaluronidase and a weak influence on elastase activity. Tyrosinase was inhibited effectively by honeybush acetone (IC50 26.18 ± 1.45 µg/mL), ethanol (IC50 45.99 ± 0.76 µg/mL) and water (IC50 67.42 ± 1.75 µg/mL) extracts. Significant hyaluronidase inhibition was observed for ethanol, acetone and water extracts (IC50 were 10.99 ± 1.56, 13.21 ± 0.39 and 14.62 ± 0.21µg/mL, respectively). Collagenase activity was inhibited effectively by honeybush acetone extract (IC50 42.5 ± 1.05 μg/mL). The wound healing properties of the honeybush extracts, estimated in vitro in human keratinocytes (HaCaTs), were indicated for water and ethanol extracts. In vitro sun protection factor (SPF in vitro) showed medium photoprotection potential for all the honeybush extracts. The quantity of polyphenolic compounds was estimated with the use of high-performance liquid chromatography equipped with diode-array detection (HPLC-DAD), indicating the highest mangiferin contents in ethanol, acetone and n-butanol extracts, while in the water extract hesperidin was the dominant compound. The antioxidant properties of the honeybush extracts were estimated with FRAP (2,4,6-Tris(2-pyridyl)-s-triazine) and DPPH (2,2-diphenyl-1-picrylhydrazyl) tests, indicating their strong antioxidant activity, similar to ascorbic acid for the acetone extract in both tests. The wound healing abilities, estimation of SPF in vitro and the direct influence on selected enzymes (elastase, tyrosinase, collagenase and hyaluronidase) of the tested honeybush extracts were analysed for the first time, indicating a high potential of these well-known herbal tea for antiaging, anti-inflammation, regeneration and protection of the skin.

Descriptive histomorphological evaluation of the testis and caudal epididymis following treatment with rooibos (Aspalathus linearis), honeybush (Cyclopia intermedia) and sutherlandia (Lessertia frutescens) in healthy and streptozotocin-induced diabetic rats

Objective: To evaluate the testis and the cauda epididymis after treating both healthy and diabetic rats with rooibos (Aspalathus linearis), honeybush (Cyclopia intermedia), and sutherlandia (Lessertia frutescens), respectively. Methods: Ninety male Wistar rats were randomly divided into nine groups (n=10 per group). Fifty rats were randomized to receive tap water, 0.1 M citrate buffer, 2.0% fermented rooibos, 4.0% fermented honeybush, or 0.2% unfermented sutherlandia. Diabetes was induced via a single intraperitoneal injection of streptozotocin (STZ) at 45 mg/kg in 40 rats. The diabetic rats were randomized to receive tap water, 2.0% fermented rooibos, 4.0% fermented honeybush, or 0.2% unfermented sutherlandia. Rats were sacrificed after seven weeks of diabetic induction. Blood was collected for hormonal assay, while the testes and caudal epididymis were retrieved for histomorphological evaluations. Results: Diabetic rats treated with 2.0% fermented rooibos exhibited a significant increase in fasting plasma glucose (FPG) versus the vehicle group (P=0.002), and a 3.6% decrease compared to the diabetic control group (P>0.05). Diabetic rats treated with honeybush or sutherlandia showed 23.6% and 15.4% decrease in FPG when compared to the diabetic control group, respectively (both P>0.05). The diabetic control rats had a significant decrease in the percentage of histologically normal seminiferous tubules compared to the control group (P=0.049). There was 21.0%, 14.0%, and 5.4% rise in the percentage of normal seminiferous tubules in diabetic rats receiving rooibos, honeybush or sutherlandia, respectively, compared to the diabetic control group. The infusion control groups (rooibos, honeybush and sutherlandia) showed normal seminiferous tubules, presence of spermatozoa in the epididymal lumen, and had normal overall architecture. Both testicular and epididymal morphology were altered in the diabetic control group, but these disruptions were mildly ameliorated by rooibos, honeybush, and sutherlandia. Conclusions: The detrimental effects of diabetes on the histomorphological architecture of the testis and epididymis are mildly ameliorated by the infusions (2.0% fermented rooibos, 4.0% fermented honeybush, and 0.2% unfermented sutherlandia).

Pharmacological Activity of Honeybush (Cyclopia intermedia) in Boar Spermatozoa during Semen Storage and under Oxidative Stress.

Simple SummaryNowadays, pig breeding is mostly conducted by artificial insemination using diluted semen stored for 1 to 5 days. During semen handling and storage, sperm quality usually declines, mainly because of oxidative stress and bacterial contamination. As cheap and natural sources of antioxidants, medicinal plants have become an alternative to the most common additives used in semen extenders. In this regard, several indigenous plants from Southern Africa have shown pharmacological activity in different animal cell types, although their effects on sperm cells have not been explored extensively. In the present study, we tested the effects of honeybush (Cyclopia intermedia) aqueous extract as a preservative of boar semen during 5 days of storage and under induced oxidative stress. Overall, this plant extract enhanced several sperm quality parameters and did not show any toxic effects. Supplementation with honeybush extract was able to improve the preservative properties of a long-term semen extender, thus confirming the beneficial use of plant extracts as natural additives for boar sperm.In recent decades, an increasing number of ethnopharmacological studies have been dedicated to medicinal plants from South African fynbos. Among these plants, honeybush (Cyclopia spp.) has become a popular tea, mainly due to its healthy properties and caffeine-free status. The antioxidant, antimutagenic, and antimicrobial properties of this plant have been reported in several cell types, but its effects on reproductive function are still unknown. Here, we assessed the effects of honeybush (Cyclopia intermedia) on boar sperm parameters under induced oxidative stress (Fe2+/ascorbate) and during five days of semen storage at 17 °C without oxidative stress. In both experiments, four concentrations (200, 50, 12.5, and 3.125 µg/mL) of fermented honeybush were tested. Our results show that honeybush enhances sperm parameters, and no toxic effects were observed at any of the tested extract concentrations. Interestingly, honeybush (12.5 µg/mL) improved the sperm motility and kinetic parameters, preserved the plasma membrane integrity, and reduced the lipid peroxidation in the samples exposed to Fe2+/ascorbate (p < 0.05). In the stored samples, positive effects of honeybush on sperm parameters (motility, kinetics, acrosome, and mitochondria) were observed from 48 h until 120 h of semen storage (p < 0.05). Our results clearly show the protective effects of honeybush on sperm samples, thus promoting its use as a natural source of antioxidants for boar semen.

Skin Hydration Effects of Scale-Up Fermented Cyclopia intermedia against Ultraviolet B-Induced Damage in Keratinocyte Cells and Hairless Mice.

Photoaging occurs by chronic skin exposure to the sun and ultraviolet irradiation and leads to skin aging accompanied by a lack of skin hydration. We previously demonstrated the photoprotective effect of fermented Cyclopia intermedia (honeybush) extract on the skin. In this study, we evaluated the skin hydration effects of scaled-up fermented honeybush extract (HU-018) against ultraviolet B (UVB) radiation in HaCaT immortalized human keratinocytes and hairless mice. Pretreating HaCaT cells with HU-018 attenuated the decreased hyaluronic acid (HA) levels and mRNA expression of genes encoding involucrin, filaggrin, and loricrin by UVB irradiation. HU-018 treatment also ameliorated the decreased stratum corneum (SC) hydration and the increased levels of transepidermal water loss (TEWL) and erythema index (EI) in hairless mice after UVB exposure. Microarray analysis revealed changes in gene expression patterns of hyaluronan synthase 2 (Has2), transforming growth factor-beta 3 (TGF-β3), and elastin induced by HU-018 in UVB-irradiated mice. Consistently, the mRNA expression of Has2, TGF-β3, and elastin was increased by HU-018 treatment. Moreover, HU-018 restored the increased epidermal thickness and collagen disorganization in skin tissue of UVB-irradiated mice. HU-018 treatment also decreased matrix metalloproteinase-1 (MMP-1) expression and increased procollagen type-1, elastin, and TGF-β1 expression. In conclusion, we found that HU-018 promoted skin hydration processes in UVB-irradiated keratinocytes and hairless mice by modulating involucrin, filaggrin, loricrin, and HA expression and ameliorating visible signs of photoaging. Thus, HU-018 may be a good skin hydration agent for skin care.

Antimicrobial and antioxidant activities of different solvent extracts from fermented and green honeybush (Cyclopia intermedia) plant material

Ethnopharmacological relevanceCyclopia intermedia is indigenous to South Africa and used to prepare honeybush herbal tea. This aromatic herbal tea has been associated with numerous health benefits, mostly based on anecdotal evidence and with very few studies reporting on the antimicrobial activities. AimThe inhibitory effect on the growth of important nosocomial microorganisms and possible association with the antioxidant capacity/content of various solvent extracts of green/unfermented and fermented honeybush plant material were determined in the current study. Materials and methodsThe agar disk diffusion assay was used as a screening assay for the antimicrobial activity of the various honeybush extracts, while the minimal inhibitory concentration (MIC) values, using the broth micro-dilution method, were determined against Streptococcus pyogenes, Staphylococcus aureus and Candida albicans. Active antimicrobial compounds were then shown using thin layer chromatography bioautography. Total antioxidant capacities and -content were also determined for each extract, while the main phenolic compounds were quantified using HPLC. ResultsSix of the eight solvent extracts of honeybush showed antimicrobial activity, with the fermented and green methanol extracts being most effective against S. aureus and C. albicans respectively, whilst the green chloroform extract was most potent against S. pyogenes. Thin layer chromatography-bioautography acknowledged the existence of active antimicrobial fractions within these different solvent honeybush extracts. Regardless of the assay, the green honeybush extracts generally exhibited the highest antioxidant capacity when compared to the fermented honeybush. The total polyphenols were also observed to be highest in green extracts when using water and methanol as solvents. In general the mangiferin and hesperidin contents were higher in the green than the fermented extracts of honeybush plant material. ConclusionsThe inhibitory activity of the various extracts against specific microorganisms was observed to be linearly proportional to the extract concentration. Although the different solvent extracts can only be considered weak antimicrobial agents, three compounds showed specific activity and should be further elucidated in future. It appears the antioxidant capacity of the various solvent herbal extracts did not relate to the antimicrobial activities; however, further work will be required to ascertain this observation. The current data also suggested that the various extracts of honeybush can be considered a good source of a unique blend of natural phytochemical antioxidants and antimicrobial compounds and should be further elucidated.

Protective effect of fermented Cyclopia intermedia against UVB-induced damage in HaCaT human keratinocytes.

BackgroundThe fermented leaves and stems of Cyclopia intermedia are used to brew honeybush tea, a herbal tea indigenous to South Africa. The aim of this study was to evaluate the protective effect of fermented honeybush extracts (FH ex) and scale-up fermented honeybush extracts (SFH ex) against ultraviolet B (UVB)-induced damage in HaCaT keratinocytes. To this end, we examined UVB-induced cell viability, antioxidant enzymes, and inflammatory mediators in HaCaT cells.MethodsUVB significantly decreased HaCaT cell viability, whereas FH ex and SFH ex did not exhibit cytotoxic effects and increased the viability of the HaCaT cells. To further investigate the protective effects of FH ex on UVB-induced oxidative stress in HaCaT cells, the activities of superoxide dismutase (SOD), catalase (CAT), matrix metalloproteinases (MMPs), pro-inflammatory cytokines and skin barrier function in terms of involucrin, filaggrin, and loricrin were analyzed.ResultsUVB-induced treatment reduced the activity of antioxidant enzymes and skin barrier function, while FH ex or SFH ex increased their activity. These results suggest that FH ex exerted cytoprotective activity against UVB-induced oxidative stress in HaCaT cells through stimulation of antioxidant enzymes activities. Furthermore, FH ex and SFH ex suppressed the UVB-induced expression of inflammatory mediators, such as IL-1β, IL-6, and IL-8, at mRNA level together with down regulation of matrix metalloproteinase (MMPs). In addition, FH ex and SFH ex reversed the phosphorylation of mitogen-activated protein kinase (MAPK) induced by UVB-irradiation. Notably, FH ex and SFH ex markedly inhibited UVB-induced activation of ERK, p38, and JNK. Thus, this agent exhibits anti-oxidative and -inflammatory effects via lowering ROS production, suppressing p38, ERK, and JNK activation, and down-regulating expression of MMPs.ConclusionsThese findings suggest that FH ex and SFH ex can be used as a skin anti-photoaging agent.

Commercial Honeybush (Cyclopia spp.) Tea Extract Inhibits Osteoclast Formation and Bone Resorption in RAW264.7 Murine Macrophages-An in vitro Study.

Honeybush tea, a sweet tasting caffeine-free tea that is indigenous to South Africa, is rich in bioactive compounds that may have beneficial health effects. Bone remodeling is a physiological process that involves the synthesis of bone matrix by osteoblasts and resorption of bone by osteoclasts. When resorption exceeds formation, bone remodeling can be disrupted resulting in bone diseases such as osteoporosis. Osteoclasts are multinucleated cells derived from hematopoietic precursors of monocytic lineage. These precursors fuse and differentiate into mature osteoclasts in the presence of receptor activator of NF-kB ligand (RANKL), produced by osteoblasts. In this study, the in vitro effects of an aqueous extract of fermented honeybush tea were examined on osteoclast formation and bone resorption in RAW264.7 murine macrophages. We found that commercial honeybush tea extract inhibited osteoclast formation and TRAP activity which was accompanied by reduced bone resorption and disruption of characteristic cytoskeletal elements of mature osteoclasts without cytotoxicity. Furthermore, honeybush tea extract decreased expression of key osteoclast specific genes, matrix metalloproteinase-9 (MMP-9), tartrate resistant acid phosphatase (TRAP) and cathepsin K. This study demonstrates for the first time that honeybush tea may have potential anti-osteoclastogenic effects and therefore should be further explored for its beneficial effects on bone.

Anti-wrinkle effects of fermented and non-fermented Cyclopia intermedia in hairless mice.

BackgroundThe fermented leaves and stems of Cyclopia intermedia are used to brew honeybush tea, an herbal tea indigenous to South Africa with reported anti-wrinkle effects. Wrinkle formation caused by photoaging clearly involves changes in extracellular matrix components and mechanical properties of the skin.MethodsThe inhibitory effects of honeybush extract and fermented honeybush on wrinkle formation were determined by analyzing skin replicas, histologically examining epidermal thickness, and identifying damage to collagen fibers.ResultsHoneybush extract and fermented honeybush reduced the length and depth of skin winkles caused by UV irradiation and inhibited thickening of the epidermal layer, in addition to suppressing collagen tissue breakdown reactions, indicating its potential use as a skin wrinkle prevention agent.ConclusionsThis in vivo study demonstrates that honeybush produces significant anti-wrinkle effects and is therefore of interest in anti-aging skin care products.

Characterization of Volatiles and Aroma-Active Compounds in Honeybush (Cyclopia subternata) by GC-MS and GC-O Analysis

Volatile organic compounds (VOCs) in fermented honeybush, Cyclopia subternata, were sampled by means of a high-capacity headspace sample enrichment probe (SEP) and analyzed by gas chromatography-mass spectrometry (GC-MS). Stereochemistry was determined by means of enantioselective GC-MS with derivatized β-cyclodextrin columns as chiral selectors. A total of 183 compounds, the majority of which are terpenoids (103; 56%), were identified by comparing their mass spectra and retention indices with those of reference compounds or tentatively identified by comparison with spectral library or literature data. Of these compounds, 37 were determined by gas chromatography-olfactometry (GC-O), using detection frequency (DF) and aroma extract dilution analysis (AEDA), to be odor-active (FD ≥ 2). (E)-β-Damascenone, (R/S)-linalool, (E)-β-damascone, geraniol, (E)-β-ionone, and (7E)-megastigma-5,7,9-trien-4-one were identified with the highest FD factors (≥512). The odors of certain compounds, that is, (6E,8Z)-megastigma-4,6,8-trien-3-one, (6E,8E)-megastigma-4,6,8-trien-3-one, (7E)-megastigma-5,7,9-trien-4-one, 10-epi-γ-eudesmol, epi-α-muurolol, and epi-α-cadinol, were perceived by GC-O assessors as typically honeybush-like.

Modulating Effects of Rooibos and Honeybush Herbal Teas on the Development of Esophageal Papillomas in Rats

Widespread consumption of herbal teas has stimulated interest in their role as cancer preventive agents. The present investigation monitored the modulation of methylbenzylnitrosamine (MBN)-induced esophageal squamous cell carcinogenesis by rooibos (Aspalathus linearis) and honeybush (Cyclopia intermedia) herbal and Camellia sinensis teas in male F344 rats. The tumor multiplicity was significantly (P < 0.05) inhibited by unfermented honeybush (45.5%), green (50%), and black (36%) teas, while the other teas exhibited weaker effects (<30% inhibition). The mean total papilloma size was reduced by unfermented rooibos (87%), unfermented honeybush (94%), and fermented honeybush (74%) due to the absence of large papillomas (>10 mm3). Reduction of the mean total papilloma number correlated with the total polyphenol (TPP) (r = 0.79; P < 0.02) and flavanol/proanthocyanidin (FLAVA) (r = 0.89; P < 0.008) intake (mg/100 g body weight) of the teas and the FLAVA (r = 0.89; P < 0.04) and flavonol/flavones/xanthones (r = 0.99; P < 0.002) intake when considering only the herbal teas. A daily TPP intake threshold of 7 mg/100 g body weight existed below where no inhibition of papilloma development was observed. Fermentation of herbal teas reduced the inhibitory effects on papilloma development associated with a reduction in the polyphenolic constituents. The inhibitory effect of herbal teas on papilloma development is associated with different flavonoid subgroups and/or combination thereof.

Photoprotection by honeybush extracts, hesperidin and mangiferin against UVB-induced skin damage in SKH-1 mice

The possible mechanism of photoprotection by polyphenolic extracts of honeybush and the two most abundant polyphenols found in honeybush, hesperidin and mangiferin were determined using a mouse model. Ethanol: acetone soluble extracts and pure honeybush compounds were applied topically to the skin of SKH-1 mice before daily exposures to ultraviolet B (UVB) (180 mJ/cm 2) for 10 days. The honeybush extracts reduced signs of sunburn, such as erythema, peeling and hardening of the skin and also significantly ( P < 0.05) reduced edema, epidermal hyperplasia and the induction of cyclooxygenase-2 (COX-2), ornithine decarboxylase (ODC), GADD45 and OGG1/2 expression. The fermented honeybush extract significantly ( P < 0.05) reduced lipid peroxidation and depletion of the antioxidant enzymes catalase and superoxide dismutase. Hesperidin and mangiferin were less effective. These results show that extracts of honeybush and to some extent, hesperidin and mangiferin, renders protection against UVB-induced skin damage. The mechanisms investigated suggest that honeybush extracts protected the skin via modulation of induced-oxidative damage, inflammation and cell proliferation. Other specific biological properties such as modulation of signaling pathways could also be involved.