Identification of a regiospecific S-oxygenase for the production of marasmin in traditional medicinal plant Tulbaghia violacea.

Marasmin [S-(methylthiomethyl)-L-cysteine-4-oxide] is a pharmaceutically valuable sulfur-containing compound produced by the traditional medicinal plant, Tulbaghia violacea. Here, we report the identification of an S-oxygenase, TvMAS1, that produces marasmin from its corresponding sulfide, S-(methylthiomethyl)-L-cysteine. The amino acid sequence of TvMAS1 showed high sequence similarity to known flavin-containing S-oxygenating monooxygenases in plants. Recombinant TvMAS1 catalyzed regiospecific S-oxygenation at S4 of S-(methylthiomethyl)-L-cysteine to yield marasmin, with an apparent K m value of 0.55 mM. TvMAS1 mRNA accumulated with S-(methylthiomethyl)-L-cysteine and marasmin in various organs of T. violacea. Our findings suggest that TvMAS1 catalyzes the S-oxygenation reaction during the last step of marasmin biosynthesis in T. violacea.

Natural Products in Cardiovascular Diseases: The Potential of Plants from the Allioideae Subfamily (Ex-Alliaceae Family) and Their Sulphur-Containing Compounds.

Cardiovascular diseases (CVDs) are the leading cause of mortality worldwide and, together with associated risk factors such as diabetes, hypertension, and dyslipidaemia, greatly impact patients' quality of life and health care systems. This burden can be alleviated by fomenting lifestyle modifications and/or resorting to pharmacological approaches. However, due to several side effects, current therapies show low patient compliance, thus compromising their efficacy and enforcing the need to develop more amenable preventive/therapeutic strategies. In this scenario, medicinal and aromatic plants are a potential source of new effective agents. Specifically, plants from the Allioideae subfamily (formerly Alliaceae family), particularly those from the genus Allium and Tulbaghia, have been extensively used in traditional medicine for the management of several CVDs and associated risk factors, mainly due to the presence of sulphur-containing compounds. Bearing in mind this potential, the present review aims to gather information on traditional uses ascribed to these genera and provide an updated compilation of in vitro and in vivo studies validating these claims as well as clinical trials carried out in the context of CVDs. Furthermore, the effect of isolated sulphur-containing compounds is presented, and whenever possible, the relation between composition and activity and the mechanisms underlying the beneficial effects are pointed out.

The Phytochemistry and Pharmacology of Tulbaghia, Allium, Crinum and Cyrtanthus: 'Talented' Taxa from the Amaryllidaceae.

Amaryllidaceae is a significant source of bioactive phytochemicals with a strong propensity to develop new drugs. The genera Allium, Tulbaghia, Cyrtanthus and Crinum biosynthesize novel alkaloids and other phytochemicals with traditional and pharmacological uses. Amaryllidaceae biomolecules exhibit multiple pharmacological activities such as antioxidant, antimicrobial, and immunomodulatory effects. Traditionally, natural products from Amaryllidaceae are utilized to treat non-communicable and infectious human diseases. Galanthamine, a drug from this family, is clinically relevant in treating the neurocognitive disorder, Alzheimer's disease, which underscores the importance of the Amaryllidaceae alkaloids. Although Amaryllidaceae provide a plethora of biologically active compounds, there is tardiness in their development into clinically pliable medicines. Other genera, including Cyrtanthus and Tulbaghia, have received little attention as potential sources of promising drug candidates. Given the reciprocal relationship of the increasing burden of human diseases and limited availability of medicinal therapies, more rapid drug discovery and development are desirable. To expedite clinically relevant drug development, we present here evidence on bioactive compounds from the genera Allium, Tulgbaghia, Cyrtanthus and Crinum and describe their traditional and pharmacological applications.

Effects of light intensities and varying watering intervals on growth, tissue nutrient content and antifungal activity of hydroponic cultivated Tulbaghia violacea L. under greenhouse conditions.

Optimization of the quality and quantity of medicinal materials during cultivation could improve the value of medicinal plants. Light intensity and water availability affect physiological processes and growth of plants. Tulbaghia violacea L. (Alliaceae) bulbs and leaves are widely used traditionally in southern Africa for treatments of many ailments. The interactive effects of light intensity and watering regime on plant growth, nutrient uptake and antifungal activity of T. violacea were evaluated in the current study. Seedlings of T. violacea were grown hydroponically under two shading levels (0% and 40%) while being exposed simultaneously to one of three watering intervals: 5-day, 14-day and 21-day. Different growth parameters (plant height, plant fresh and dry weights) and tissue nutrient contents were evaluated at the end of the experiment. The antifungal activity of acetone extracts on Fusarium oxysporum were evaluated in a microdilution bioassay. Generally, significantly higher concentrations of macronutrients were recorded in the tissue of plants exposed to shorter watering interval. The results showed that different watering frequencies and light intensities significantly (p < 0.05) influenced plant growth parameters (height, and dry and fresh weights). Moreover, there were strong interactive effects of watering frequency and light intensity on most of the plant growth parameters. Remarkably, plants that were simultaneously exposed to the extended watering interval (21-day) and low light intensity showed the best anti-F. oxysporum activity. Key findings of this study revealed that shading alleviated the negative effects of water-deficit stress on plant growth and improved antifungal activity.

Protective role of wild garlic on isoproterenol-induced myocardial necrosis in wistar rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Tulbaghia violacea Harv. (TVL) is a folk medicine, native to South Africa which has previously shown antioxidant, anti-hypertensive and anti-diabetic effects. THE AIM OF THE STUDY: The aim of the current study was to investigate the protective role of wild garlic or TVL on isoproterenol (ISO)-induced myocardial necrosis in rats. MATERIALS AND METHODS: Animal (n = 6 each group) were pre and co-treated with TVL (60 mg/kg body weight) daily for 30 days. Myocardial necrosis was administrated by subcutaneous injection of ISO (85 mg/kg body weight) into rats on 29th and 30th day. On the 31st day, rats were anaesthetized and blood, heart samples were obtained for the biochemical, histopathological and molecular study. The specific protein target analysis from TVL was done by reverse docking study (reverse pharmacophore mapping) using PharmMapper. RESULTS: The levels of cardiac markers, lipid peroxidation products, and heart rate were considerably increased in ISO-induced myocardial necrosis in rats whilst plasma enzymatic antioxidants were significantly decreased. Myocardial necrotic mRNA genes were increased in ISO-induced myocardial necrosis in rats compared to controls. Pre and co-treatment with TVL and ramipril of myocardial necrosis in rats showed significant effects on all the biochemical and molecular studies evaluated. TVL reduced heart rate, prevented oxidative stress and downregulated the Fas-receptor and caspase-mediated apoptosis-signaling pathway, and heart muscle damage in myocardial necrosis in rats. The specific target protein [disulfide, bis (2-sulfhydrylethyl] from TVL mediates the protective effects. CONCLUSION: Wild garlic or TVL extract has shown a protective effect on ISO-induced myocardial necrosis in rats by increasing antioxidant production confirmed with docking studies.

Plant growth regulator interactions in physiological processes for controlling plant regeneration and in vitro development of Tulbaghia simmleri.

The endogenous auxin and cytokinin contents of in vitro regenerated Tulbaghia simmleri maintained on applied plant growth regulators in Murashige and Skoog (MS) medium were investigated using UHPLC-MS analysis. The highest number of shoots (27.6 per leaf) were produced in MS medium supplemented with 2.5 µM thidiazuron. A higher number of these shoots were rooted with 10 µM 6-(2-hydroxy-3-methylbenzylamino) purine (PI-55, cytokinin antagonist). Production of somatic embryos (SEs: 16.4-4.6, globular to cotyledonary stages) improved significantly with liquid MS medium containing 2.5 µM picloram, 2.5 µM phloroglucinol (PG) and 1.5 µM gibberellic acid or 1.5 µM PI-55 and 1.0 µM trans-zeatin. SEs (torpedo and cotyledonary stages) germinated (100%) in plant growth regulator free MS medium. The plantlets were acclimatized and all survived in the greenhouse. Higher levels of endogenous auxin, 2-oxindole-3-acetic acid (oxIAA, 371.52 pmol/g DW) and indole-3-acetylaspartate (IAAsp, 141.56 pmol/g DW) were detected in shoots from PG treatments. The roots of garden-grown mother plants possessed the highest level of indole-3-acetic acid (IAA, 630.54 pmol/g DW) and oxIAA (515.26 pmol/g DW). Cytokinins [CKs: trans-zeatin-O-glucoside (tZOG), cis-zeatin (cZ) and N6-isopentenyladenosine-5'-monophosphate (iPRMP)] levels were relatively high in shoots and roots of plantlets in vitro. However, PI-55 treatments influenced the development of plantlets promoting a higher biosynthesis level of iPRMP (418.06 pmol/g DW in root) and cZRMP (904.61 pmol/g DW in roots and 1427.83 pmol/g DW in shoots). The presented protocols offer organogenesis and somatic organogenesis systems for rapid plant regeneration of T. simmleri. In addition, the importance of exogenous and endogenous hormonal effects on in vitro plant growth and development as well as endogenous hormone metabolism signalling and transport related to the physiological processes of CK metabolism and transport are illustrated for in vitro development of T. simmleri.

Tulbaghia violacea and Allium ursinum Extracts Exhibit Anti-Parasitic and Antimicrobial Activities.

Garlic has played an important role in culinary arts and remedies in the traditional medicine throughout human history. Parasitic infections represent a burden in the society of especially poor countries, causing more than 1 billion infections every year and leading to around one million deaths. In this study, we investigated the mode of anti-parasitic activity of "wild garlics" Tulbaghia violacea and Allium ursinum dichloromethane extracts against parasites Trypanosoma brucei brucei and Leishmania tarentolae with regard to their already known antimicrobial activity. We also evaluated their cytotoxic potential against human cells. Both extracts showed a relevant trypanocidal and leishmanicidal activity, although L. tarentolae was less sensitive. We determined that the probable mode of action of both extracts is the irreversible inhibition of the activity of Trypanosoma brucei trypanothione reductase enzyme. The extracts showed a mild cytotoxic activity against human keratinocytes. They also exhibited weak-in most cases comparable-antibacterial and antifungal activity. HPLC-MS/MS analysis showed that both extracts are abundant in sulfur compounds. Thus, for the first time, the ability of Allium ursinum and Tulbaghia violacea to kill Trypanosoma sp. and Leishmania sp. parasites, probably by binding to and inactivating sulfur-containing compounds essential for the survival of the parasite, is shown.

Analysis of the phytochemical contents and antioxidant activities of crude extracts from Tulbaghia species.

OBJECTIVE: To assess the phytochemical contents and antioxidant activities of crude extracts from selected Tulbaghia species. METHODS: Standard methods were used for preliminary phytochemical analysis. The total phenolic acid contents of the plant extracts were determined using the Folin-Ciocalteu method, and the total flavonoid contents were determined using the aluminum chloride colorimetric method. 1,1-Diphenyl-2-picrylhydrazyl and 2,2'-Azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) assays were used to evaluate the antioxidant activities. RESULTS: Phytochemical screening showed flavonoids, glycosides, tannins, terpenoids, saponins, and steroids were present in the Tulbaghia species. The total phenolic acid and flavonoid contents varied in the different plant extracts, ranging from 4.50 to 11.10 mg of gallic acid equivalents per gram of fresh material and 3.04 to 9.65 mg of quercetin equivalents per gram, respectively. The IC50 values determined for Tulbaghia alliacea and Tulbaghia violacea based on 1,1-diphenyl-2-picrylhydrazyl (0.06 and 0.08 mg/mL, respectively) and 2,2'-Azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) (0.06 and 0.03 mg/mL, respectively) were low and showed they had potential antioxidant activities. CONCLUSION: Our results suggest that individual compounds from Tulbaghia species should be isolated for analysis of their antioxidant activity because some compounds may work best when pure.

Phytochemical and biological study of callus cultures of Tulbaghia violacea Harv. Cultivated in Egypt.

As in vitro plant cultures are used extensively to produce bioactive metabolites, our goal was to establish calli from Tulbaghia violacea Harv. flowers and assess the tissue phytochemically and biologically. Murashige & Skoog medium(MS) + 22.6 µM 2,4-dichlorophenoxyacetic acid +2.2 µM benzylaminopurine induced callus from flowers. Gas chromatography/mass spectrometry(GC/MS) analyses of n-hexane extracts of calli(HC) and flowers(HF) revealed 33 and 32 components(92.6 and 98.5%, respectively). Hydrocarbons were predominant in HC (55.0%), whereas a higher percentage of oxygenated compounds was found in HF(74.6%). Trans(E)-anethole(39.1%) and 16-hentriacontanone (30.3%) dominated in HF and HC, respectively. However, sulphur compounds were only detected in HF. Quantitative estimation of thiosulphinates, phenolics, flavonoids and saponins in ethanolic extracts of calli(EC) and flowers(EF) showed much higher contents in EF. Antioxidant, antimicrobial and cytotoxic screening of extracts demonstrated that EF was the most potent, followed by HF and EC; conversely, HC was inactive. Although HC and EC were less biologically active, these calli could be an alternative source of bioactive metabolites.

Tulbaghia violacea Harv. plant extract affects cell wall synthesis in Aspergillus flavus.

AIMS: This study investigates the effect that aqueous extracts of Tulbaghia violacea Harv. harbouring plant saponins, phenolics and tannins have on Aspergillus flavus ß-(1,3) glucan and chitin synthesis. METHODS AND RESULTS: Aspergillus flavus was treated with various subinhibitory concentrations of an aqueous T. violacea plant extract and the ß-(1,3) glucan and chitin content was determined together with glucan synthase and chitin synthase production respectively. CONCLUSIONS: The aqueous extract caused a significant decline (P < 0·05) in ß-glucan production in A. flavus in a dose-dependent manner when compared to the untreated sample. Further investigations showed a decrease in ß-glucan synthase production as the concentration of the plant extract was increased. A significant reduction in total chitin content corresponding to a decrease in chitin synthase production in the presence of the plant extract was also found. SIGNIFICANCE AND IMPACT OF THE STUDY: The broad spectrum activity and the efficacy of aqueous T. violacea plant extract on both ß-glucan and chitin synthesis may limit the potential of the fungus developing resistance towards it and therefore the extract is an ideal candidate for use as a potential antifungal agent.

METABOLIC EFFECTS OF TULBAGHIA VIOLACEA HARV. IN A DIABETIC MODEL.

BACKGROUND: Diabetes mellitus (DM) is a cluster of metabolic diseases with chronic hyperglycemia as a defining feature, associated with long-term organ damage and dysfunction. In this study we examined the effect of Tulbaghia violacea rhizome methanolic extract on blood glucose and lipids in normal and streptozotocin-induced diabetic rats. MATERIALS AND METHODS: Male Wistar rats (250-300g) were injected intraperitoneally (i.p.) with streptozotocin (60mg/kg body weight) to induce diabetes; or provided with distilled water for the control (CON) (3 ml/kg/b.w.) animals and treatment with TVL (60mg/kg.b.w). The rats were divided into 5 groups of 6 animals per group as follows: Non-diabetic control (NDC)-received distilled water (3ml/kg.b.w), Non-diabetic TVL (ND+TVL)-received TVL (60mh/kg b.w), Diabetic control (DC)-received distilled water (3ml/kg.b.w), Diabetic experimental (D+TVL)-received TVL (60mg/kg.b.w), Diabetic experimental (D+M)-received Metformin (250 mg/kg.b.w). All doses were administered daily via oral gavage. RESULTS: TVL-treated animals showed reduced blood glucose, increased plasma insulin, reduced plasma TG, TC, VLDL and increased HDL. Furthermore we found decreased thiobarbituric acid reactive substances (TBARS) and increased superoxide dismutase (SOD) activity and nitric oxide significantly. Regarding renal parameters we found increased blood urea nitrogen (BUN), and improved renal morphology in TVL-treated animals. CONCLUSION: Tulbaghia violacea has a hypoglycaemic effect which could due to its effects on multiple pathways of the hyperglycemic process. Further work is needed to identify the mechanism of its antidiabetic effect.

In vitro cytotoxic and pro-apoptotic effects of water extracts of Tulbaghia violacea leaves and bulbs.

ETHNOPHARMACOLOGICAL RELEVANCE: Infusions of Tulbaghia violacea (wild garlic) in water are used in traditional medicine in Southern Africa to treat numerous diseases, including cancer. Several studies have previously demonstrated the cytotoxic activities of extracts of T. violacea in cultured cancer cells. Their findings support the potential anti-cancer properties of this plant. However, these studies made use of organic solvent extraction methods, while the traditional use of the plant involves the preparation of infusions in water. MATERIALS AND METHODS: In the current study, we investigated the potential anti-cancer properties of infusions of T. violacea. We also performed a comparative study investigating the cytotoxic activities of T. violacea bulbs and leaves. A panel of four cancer cell lines (HepG2, MCF7, H157, and HT29) and one non-cancerous cell line (KMST6) was treated with the two extracts and the effects of the extracts on the growth of the cells were evaluated. We also investigated whether the growth inhibitory effects were associated with the induction of apoptosis and whether the mechanism of cell death is the result of oxidative stress and the activation of caspase-3. RESULT: We found that extracts of the leaves and not the bulbs have growth inhibitory effects and that this is the result of the induction of apoptosis, which is associated with the production of Reactive Oxygen Species (ROS) and the activation of caspase-3. The leaf extract demonstrated variable selective toxicity towards the cancer lines. Although the extract also induced cell death in the non-cancerous cell line (KMST6), we found that the levels of toxicity were lower in this cell line. CONCLUSION: this study confirms that infusions of T. violacea have potential anti-cancer activity and that this bioactivity is contained in the leaf extract. This study lends support to claims that this plant can be used to treat cancer.

Effects of Tulbaghia violacea Harv. (Alliaceae) rhizome methanolic extract on kidney function and morphology in Dahl salt-sensitive rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Tulbaghia violacea has been used traditionally for the treatment of several ailments, including hypertension. The herb has been shown to have antihypertensive properties which have been attributed to its angiotensin-converting enzymeinhibitory (ACEI) activity. It could, therefore, prove beneficial in ameliorating renal pathology associated with hypertension. To evaluate the effects of long-term administration of Tulbaghia violacea on renal function and morphology in the Dahl salt-sensitive (DSS) rat model. MATERIALS AND METHODS: Male DSS rats were treated intra-peritoneally (i.p.) as follows: methanolic extract of Tulbaghia violacea: (TVL) (50 mg/kg/b.w.), captopril: (CAP) (25 mg/kg/b.w.), or distilled water, control: (CON) (3 ml/kg/b.w.). Blood pressure (BP) was measured bi-weekly, whilst 24-h urine volumes and electrolyte concentrations were assessed weekly. Animals were sacrificed on day 49 by halothane overdose. Blood was removed for determination of plasma and serum electrolytes. Left kidney tissues were harvested for the determination of nuclear factor-kappaß (NF-kß) and transforming growth factor-ß (TGF-ß) gene expressions. RESULTS: TVL significantly reduced mean arterial pressure (MAP) and diastolic blood pressure (DBP). TVL showed reduced blood urea nitrogen, serum creatinine, total protein in urine as well as increased serum total protein. TVL decreased thiobarbituric acid reactive substances (TBARS) and increased glutathione peroxidase (GPx) and superoxide dismutase (SOD) activity and nitric oxide significantly. NF-kß and TGF-ß) gene expressions were significantly reduced in TVL and CAP treated rats. Moreover, renal morphology improved significantly in TVL and CAP treated animals. CONCLUSION: TVL and CAP demonstrated marked improvement in renal function and morphology.