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In this paper we show that metabolic engineering in Cucurbita pepo hairy roots can be used to both effectively increase and modify cucurbitacins. Cucurbitacins are highly-oxygenated triterpenoids originally described in the Cucurbitaceae family, but have since been found in 15 taxonomically distant plant families. Cucurbitacin B, D, E and I are the most widespread amongst the Cucurbitaceae and they have both important biological and pharmacological activities. In this study C. pepo hairy roots were used as a platform to boost production and alter the structures of the afore mentioned cucurbitacins by metabolic engineering to potentially provide new or more desirable bioactivities. We report that the ability to induce cucurbitacin biosynthesis by basic Helix-Loop-Helix transcription factors is partially conserved within the Cucurbitaceae and therefore can potentially be used as a biotechnological tool to increase cucurbitacins in several genera of this family. Additionally, overexpression of a novel acyltransferase from cucurbitacin producing Iberis amara generates a hitherto undescribed acetylation at the C3-hydroxyl group of the cucurbitadienol backbone. While overexpression of the cytochromes P450 CsCYP88L2 and McCYP88L7 from Cucumis sativus and Momordica charantia (respectively), results in accumulation of new spectral feature as revealed by High resolution liquid chromatography mass spectroscopy analysis; the m/z of the new peak supports it might be a cucurbitacin hydroxylated at the C19 position in C. pepo hairy roots. Finally, this paper is a case study of how hairy roots can be used to metabolically engineer and introduce novel modifications in metabolic pathways that have not been fully elucidated. [ABSTRACT FROM AUTHOR]

In this paper we show that metabolic engineering in Cucurbita pepo hairy roots can be used to both effectively increase and modify cucurbitacins. Cucurbitacins are highly-oxygenated triterpenoids originally described in the Cucurbitaceae family, but have since been found in 15 taxonomically distant plant families. Cucurbitacin B, D, E and I are the most widespread amongst the Cucurbitaceae and they have both important biological and pharmacological activities. In this study C. pepo hairy roots were used as a platform to boost production and alter the structures of the afore mentioned cucurbitacins by metabolic engineering to potentially provide new or more desirable bioactivities. We report that the ability to induce cucurbitacin biosynthesis by basic Helix-Loop-Helix transcription factors is partially conserved within the Cucurbitaceae and therefore can potentially be used as a biotechnological tool to increase cucurbitacins in several genera of this family. Additionally, overexpression of a novel acyltransferase from cucurbitacin producing Iberis amara generates a hitherto undescribed acetylation at the C3-hydroxyl group of the cucurbitadienol backbone. While overexpression of the cytochromes P450 CsCYP88L2 and McCYP88L7 from Cucumis sativus and Momordica charantia (respectively), results in accumulation of new spectral feature as revealed by High resolution liquid chromatography mass spectroscopy analysis; the m/z of the new peak supports it might be a cucurbitacin hydroxylated at the C19 position in C. pepo hairy roots. Finally, this paper is a case study of how hairy roots can be used to metabolically engineer and introduce novel modifications in metabolic pathways that have not been fully elucidated.

STW 5 (Iberogast®; Steigerwald Arzneimittelwerk GmbH, Darmstadt, Germany) contains nine plant extracts and possesses well-documented overall efficacy in functional gastrointestinal disorders (FGID). Little is known about the onset of symptom relief. Twenty-nine centers in Germany recruited 272 patients with established FGID. These patients were treated with STW 5 for approximately 3 weeks in this noninterventional study. Patients assessed the severity of their gastrointestinal complaints before and at defined times after the intake of STW 5 (10 cm visual analogue scale; VAS). Fifteen minutes after the first dose, the severity of gastrointestinal complaints had decreased by 1.4 cm (mean; initial mean: 5.2 of 10 cm). After 1 h, more than 90% of the maximum effect of 3.2 cm on the 10 cm VAS had been reached. Most patients with symptoms experienced a marked improvement within 5, 15 or 30 min of taking STW 5. Absolute improvements were larger in patients with more pronounced baseline complaints. Subgroups with upper (80% of the study population) and lower FGID (20%) did not present major differences. Neither did subgroups by age and duration of complaints. Treatment with STW 5 resulted in rapid improvement of symptoms. [ABSTRACT FROM AUTHOR]

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Iberis amara is used in herbal medicine where it has been shown to have heterogeneous pharmacological effects. In this study, ultrasound-assisted supercritical CO 2 extraction (UASCE) of total flavonoids from Iberis amara was optimized. The maximal total flavonoids content (TFC) was achieved at 25 MPa pressure, 46 °C temperature, 0.34 W/mL ultrasonic energy density, which was 18% higher than that obtained with traditional supercritical CO 2 extraction. Furthermore, kinetics study showed that ultrasound primarily improves the extraction rate during constant extraction rate period. High correlation (r = 0.78) exists between TFC and antioxidant activity of extracts. Moreover, HPLC analysis revealed that ultrasound introduction can effectively increase the individual concentrations of interested flavonoids (rutin, kaempferol, quercetin, catechin) in extracts in the range from 16.8% to 45.1%. In conclusion, this study suggests that UASCE is a feasible combined technique to produce flavonoids from Iberis amara on an industrial level as well as for analytical purposes. [Display omitted] • Flavonoids was acquired by ultrasound assisted supercritical CO 2. • Extraction parameters were optimized by response surface methodology. • The optimal combined process was superior to conventional techniques. • Iberis amara is a good source for natural flavonoids. [ABSTRACT FROM AUTHOR]

Abstract: Inflammation is a common mechanism of many gastrointestinal diseases. Therefore, it is interesting to know, whether complex phytopharmaceuticals known to modulate gastrointestinal motor function reveal also anti-inflammatory properties. We tested the fixed herbal combination product STW 5 (Iberogast®) and its main component Iberis amara fresh plant extract (STW 6) to characterize their protective potential in an experimental inflammation model in vitro. The test system consisted of ileum/jejunum segments from male Wistar rats. Inflammation was evoked by intraluminal instillation of 2,4,6-trinitrobenzenesulfonic acid (TNBS) for 30min. Preincubation of TNBS together with STW 5 and STW 6 prevented the TNBS-induced inhibition of ACh-induced contractions. No differences were found between water-dissolved and ethanol-dissolved extracts. STW 5 and STW 6 reduced morphological changes induced by TNBS in mucosal and muscle layers. The IL-10 mRNA measured by qRT-PCR was not influenced by TNBS but increased by STW 5 and STW 6. The TNBS-induced increase in the TNFα-mRNA expression was suppressed by STW 5 but not by STW 6. Additionally, STW 5 decreased TNFα release in LPS-stimulated human monocytes. STW 6 influenced neither the TNFα-mRNA nor the TNFα release. These findings demonstrate that STW 5 reduced inflammation-induced alterations in ileum/jejunum segments. The effects were associated with a restoration of the disturbed ACh-induced contraction, pathohistological protection and inhibition of TNFα. STW 6 may contribute to the protective effect of STW 5 mainly by increasing IL-10 pathway but not by influencing TNFα. [Copyright &y& Elsevier]

Abstract: Functional dyspepsia is a common chronic disorder with non-specific upper abdominal symptoms which can not be explained by organic or biochemical abnormalities. The dyspeptic symptoms are very compromising and bothersome and result in a substantial reduction of quality of life. The substantial direct and indirect medical and economical costs induce a high socioeconomic interest in the pathogenesis and the treatment options of this disease. Over the past 30 years several theories about the etiology of the symptoms in functional dyspepsia patients have been put forward. These include disorders of gastrointestinal motility, acid secretion, visceral hypersensitivity, adaptation and accommodation, Hp-infection, mucosal inflammation and finally genetic predisposition. There is increasing evidence that functional dyspepsia is a multi-causal disorder, which leads to altered processing of afferent information from the gastrointestinal tract to the CNS. Autonomic hypersensitivity and altered central processing could be a common phenomenon whereas motility changes, inflammation or altered secretion could increase neural afferent inputs. Treatment of this complex disorder could and should involve these different levels of symptom generation. Thus different approaches with anti-secretory, spasmolytic, prokinetic and anti-inflammatory effects and most preferably reduction of visceral hypersensitivity seem logical. This could explain the variety of drugs which show a positive symptomatic response. This could also offer the conclusion, whether a combination of these drugs could be clinically superior which remains to be proven. And this could offer a logical approach for the use of substances with a multi-target action, e.g. STW 5. [Copyright &y& Elsevier]

Abstract: To correlate the pharmacological effects of the fixed herbal combination STW 5 (Iberogast®) containing nine extract components with its confirmed clinical efficacy, ex vivo/in vitro absorption tests were performed. For the investigation, the everted gut sac technique and, in a pilot study, the Caco-2-cell model were used. The absorption rate of the extracts was determined by measuring characteristic marker substances of each of the individual extracts using HPLC or GC techniques. The results allow us to conclude that the investigated substances from STW 5 possess a good bioavailability, which is in accordance with the rapid onset of the therapeutic efficacy and explains its known pharmacological effects and clinical efficacy in terms of multiple drug action and multi-target therapy, respectively. [Copyright &y& Elsevier]

Abstract: Introduction: A limited number of drugs are available for the treatment of functional dyspepsia and irritable bowel syndrome. The efficacy of STW 5 (Iberogast®) was previously shown in clinical trials. Since visceral hypersensitivity seems to be the prime pathomechanism of functional gastro-intestinal disorders, the aim of this study was to explore whether STW 5 reduces intestinal afferent sensitivity in the upper gastrointestinal tract. Methods: Two groups of male Wistar rats were pretreated with either the herbal preparation STW 5 or its vehicle (30.8% ethanol). Then, after 2h, general anesthesia was induced by pentobarbitone (60mgkg−1 i.p.) and extracellular multi-unit afferent recordings were obtained from mesenteric afferents innervating the proximal jejunum. The intestinal afferent nerve response to increasing doses of 5-HT and bradykinin were quantified as well as afferent discharge following a ramp distension of the adjacent intestinal loop from 0 to 60cm H2O. Results: Afferent discharge to 5-HT and bradykinin increased dose-dependently. Following the different doses of 5-HT, the peak in afferent nerve discharge was always reduced after pretreatment with STW 5 compared to controls with a response of 110±5imps−1 after STW 5 and 128±3 in vehicle controls at the maximum dose (40μgkg−1; ; mean±SEM). For bradykinin, afferent responses were reduced following STW 5 at the 20 and 40μgkg−1 dose but not at 10μgkg−1 (40μgkg−1: 176±7imps−1 following STW 5 versus 200±6imps−1 in controls; ). The ramp distension of the intestinal loop stimulated a rise in intestinal afferent nerve discharge that was always lower in the STW 5 pretreated group compared to vehicle controls with the exception of the discharge rate at the pressure level of 0 and 20cm H2O (all other pressures up to 60cm H2O ). Conclusions: Sensitivity of intestinal afferents to mechanical and chemical stimuli is reduced following treatment with the herbal preparation STW 5. This mechanism may help to explain why STW 5 relieves dyspeptic and bowel symptoms in patients. [Copyright &y& Elsevier]

Abstract: Functional dyspepsia (FD) is a disorder that involves impaired gastric accommodation, antral hypomotility, and upper abdominal pain. The herbal drug STW 5 (Iberogast®) is used to successfully treat FD patients. Here, we report in vitro data revealing the mode of action of STW 5 and its individual herbal extracts on gastric motility. STW 5 evoked a relaxation of the proximal stomach but increased antral motility. Both effects are myogenic. The extracts of Angelica root, chamomile flower and liquorice root mimicked the inhibitory effects in the proximal stomach whereas the extracts of greater celandine herb, Melissa leaf, caraway fruit and bitter candy tuft increased motility of the proximal stomach. All extracts increased motility in the antrum comparable to the effects of STW 5. We conclude that the differential effects of STW 5 on proximal and distal stomach motor activity are not caused by solely spasmolytic or anti-spasmolytic effects of the individual components. It is suggested that the individual extracts target transduction mechanisms that are specifically expressed in the proximal vs. distal stomach. We present a rationale for the differential effect of STW 5 which is a result of the combined actions of its individual components and reason that the inhibitory effects in the proximal and the excitatory effects in the distal stomach may contribute to symptom relief in FD patients treated with STW 5 (Iberogast®). [Copyright &y& Elsevier]

Abstract: STW 5 (Iberogast®), a phytomedicine agent consisting of a fixed combination of nine individual plant extracts, is widely used in the treatment of dyspepsia and motility related disorders. Little if anything is known on the possible influence on electrophysiological properties of intestinal smooth muscle by which STW 5 causes its beneficial effects. The aim of the present study was to investigate whether herbal extracts influence electrophysiological parameters of large and small intestine. For this purpose intracellular recordings of smooth muscle cell (SMC) of the circular muscle layer of different parts of mouse intestine were performed using standard microelectrode techniques. The resting membrane potential (RMP), excitatory and inhibitory neurotransmission in proximal colon, the frequency and the amplitude of slow waves in small intestine were investigated. The RMP of SMC was −46.4±3.8mV, in the colon and −59±1.3mV, in small intestine. STW 5 significantly depolarized the RMP of colonic (16.6±2.2mV, , ) and jejunal (9.6±1.6mV, , ) SMC. This depolarizing effect can be mainly attributed to the constituents of chamomile flower, Angelica root and greater celadine herb. Following the electrical field stimulations (EFSs), junction potentials are influenced in a distinct manner. Excitatory junctions potentials (EJPs) of the colon were not significantly reduced (13.1±4.8 vs. 10.1±2.8 n.s., ) but fast (fIJP) and slow (sIJP) inhibitory junction potentials of the murine colon are reduced significantly by STW 5 (fIJP: 21.6±8.1 vs. 11.6±2.1 and sIJP: 12.1±3.3 vs. 6.1±1.3 , ). The basal frequency of small intestinal slow waves was 39.5±1.4min−1 and the amplitude was 23.1±0.9mV, . STW 5 significantly reduced amplitude and frequency of the slow waves (11.7±0.8mV; 33.5±3.4min−1, , ). This effect on slow waves represents the summation of effects of the nine individual phytoextracts. Whereas Angelica root and chamomile flower completely blocked the slow wave activity, bitter candy tuft increased the frequency and amplitude, greater celadine herb reduced frequency and amplitude of the slow wave, peppermint leaf reduced frequency and left amplitude unchanged and liquorice root, caraway fruit and lemon balm leaf had no effects in basic electrophysiological properties of SMC. This study demonstrates that STW 5 causes changes in SMC RMP, excitatory and inhibitory neurotransmission and slow wave rhythmicity. These effects represent a summation effect of different constituents of this phytotherapeuticum and prove that STW 5 has characteristic effects on intestinal electrophysiology. [Copyright &y& Elsevier]

Abstract: Functional gastro-intestinal diseases as the irritable bowel syndrome are very common in the population and are characterized by a broad spectrum of symptoms which mostly are related to spastic or paralytic intestinal function without defined histopathological changes of the tissue. Due to the multifactorial pathogenesis a multifactorial therapy with multi-target action seems to be reasonable. STW 5 (Iberogast®), its constituent herbal extracts and some isolated compounds were used in an in vitro model provided by intestinal samples from guinea pig in order to test their activity on histamine-induced contractions and spontaneaous motility, respectively. For comparison the known spasmolyticum papaverine was used. The results show that the lytic effect of the phytotherapeuticum on histamine-induced contraction represents additively the actions of the different components and corresponds to approx. 10μM of papaverine. Spontaneous peristaltic motion was differently modulated by the various constituent extracts. The experiments with silibinin, glycyrrhicine, chelidonine, and protopine showed that the effects of the extracts were not comparable to those of the respective chemical constituents. [Copyright &y& Elsevier]

Abstract: The protective effect of a commercial preparation (STW 5, Iberogast®), containing the extracts of bitter candy tuft, lemon balm leaf, chamomile flower, caraway fruit, peppermint leaf, liquorice root, Angelica root, milk thistle fruit and greater celandine herb, against the development of gastric ulcers was previously reported in an earlier publication (Khayyal et al., 2001). All extracts produced a dose dependent anti-ulcerogenic effect associated with a reduced acid output, an increased mucin secretion, an increase in prostaglandin E2 release and a decrease in leukotrienes. The effect on pepsin content was not uniform and did not seem to bear a relationship with the anti-ulcerogenic activity. The best effects were observed with the combined formulation, STW 5. Furthermore, the effect of the latter in protecting against the development of rebound gastric acidity was examined experimentally in rats and compared with the effect of some commercial antacid preparations (Rennie®, Talcid® and Maaloxan®). A model of testing rebound acidity was developed by inducing a marginal increase in gastric acidity through the administration of indomethacin, in such a way that it could be easily neutralized, allowing any eventual secondary increase in acidity to be measured within a few hours of administration. In addition, the serum gastrin level was measured after drug treatment to establish any correlation between it and any rebound acidity. The results obtained demonstrated that STW 5 did not only lower the gastric acidity as effectively as the commercial antacid, but it was more effective in inhibiting the secondary hyperacidity. Moreover, STW 5 was capable of inhibiting the serum gastrin level in rats, an effect which ran parallel to its lowering effect on gastric acid production. [Copyright &y& Elsevier]

Abstract: Clinical studies with the fixed herbal combination product STW 5 (Iberogast®) have indicated an efficiacy comparable to metoclopramide (5-HT3 antagonist) and cisapride (5-HT4 agonist) in functional gastro-intestinal diseases like functional dyspepsia (FD) and irritable bowel syndrome (IBS). Since serotonin (5-HT3 and 5-HT4) and muscarinic M3 receptors are known to play a central role in the etiology of FD and IBS, the extracts contained in STW 5 and several of their phytochemical components were studied in vitro for binding affinities to these receptors of the intestine. STW 5 inhibited the binding of 3H-GR113808 and 3H-4-DAMP to 5-HT4 and M3 receptors, respectively, about 10 times more potently than the binding of 3H-GR65630 to 5-HT3 receptors. IC50 values for STW 5 did correspond to extract dilutions of 1:1000 (M3 binding) and 1:2000 (5-HT4 binding). In addition, STW 5 also potently inhibited the binding to opioid receptors with an IC50 value of 1:2000. Of the nine herbal extracts contained in STW 5, the fresh plant extract of bitter candy tuft (Iberis amara) selectively inhibited binding to M3 receptors, while ethanolic extracts of celandine herb and chamomile flower were selective to 5-HT4, and liquorice root to 5-HT3 receptors. Binding affinities to human recombinant 5-HT3, 5-HT4 and M3 receptors were qualitatively similar to those of the corresponding intestinal receptors. The benzylisoquinoline alkaloid berberine had significant inhibitory action on 5-HT4 and M3 binding, showing IC50 values of 40ng/ml (100nM) and 200ng/ml (500nM), respectively, but is present in the extract of celandine herb only in traces, so that also for the celandine extract a cooperative effect of several phytochemical constituents can be assumed. These in vitro data indicate that 5-HT4 (to a lesser degree 5-HT3), muscarinic M3, and opioid receptors represent target sites for the treatment of FD and IBS with STW 5 (Iberogast®). [Copyright &y& Elsevier]

Abstract: Since inflammation is a common mechanism of many gastrointestinal diseases, reactive oxygen metabolites may play an important role in their pathophysiology. Therefore it is interesting to know, whether phytopharmaceuticals known to modulate gastroinstinal motor function reveal also antioxdiative properties. We tested STW 5 (Iberogast®), its constituent nine different plant extracts, and some isolated compounds which are present in STW 5 for characterizing their antioxidative and radical quenching activities. The test assays consisted in pure chemical and complex celluar systems in which different types of reactive species were produced. Quantification of the effects was based on chemiluminescence reactions. The results show that all extracts contribute to the effect of the complete remedy STW 5, in the chemical systems in a strongly additve manner, in the cellular systems in a supraadditve manner. The largest contributions resulted from the extracts from peppermint and melissa leafs. Comparison of effects from isolated phytochemical compounds from the extracts with that of the extracts itself shows that usually the extract is more effective than the monosubstance which indicates also the synergism within the whole plant extracts. This means that the plant extracts present in STW 5 provide strong radical quenching activities that could also be involved in the therapeutic gastrointestinal actions. [Copyright &y& Elsevier]

Abstract: A combination of ethanolic extracts from nine medicinal plants is successfully used in STW 5 (Iberogast®) for treatment of gastrointestinal disorders. To elucidate possible modes of action, the focus of this study is on antioxidant properties of the phytomedicine STW 5. In fact, functional gastrointestinal diseases, such as non-ulcer dyspepsia (NUD) and irritable bowel syndrome, are often initiated by or correlated to inflammatory processes, where oxidants such as reactive oxygen species (ROS) play a crucial role. Prominent in vivo sources of ROS generation are represented by the enzymes xanthine oxidase (XOD) or myeloperoxidase (MPO). Applying these enzymes in models in vitro, we show that STW 5 and its components possess strong antioxidant activities. Depending on the model investigated, even pro-oxidant activites of single components of STW 5 could be observed. Interestingly, these effects were absent in STW 5, indicating cooperation between the components. Moreover, if one of the component extracts of STW 5 is omitted, the antioxidant activity is reduced. Thus we conclude that all the single extracts combined in STW 5 are of importance for the therapeutic effect, working in concert. The component of STW 5 performing best in vitro differed with the model investigated, respectively, with ROS and ROS generators. In the XOD system, the extracts of lemon balm leaf and peppermint leaf showed the best antioxidant result, whereas concerning MPO driven chlorination reactions, bitter candy tuft extract was the most efficient antioxidant. Best protection against peroxynitrite induced oxidation of methionine like sulfur-compounds exhibited the STW 5 components lemon balm leaf, Matricaria flower and peppermint leaf. [Copyright &y& Elsevier]

Abstract: The quality of a phytomedicine is defined by the quality of the herbal drug, the manufacturing of the drug preparations and the properties of the finished product, taking into account the special requirements of the individual herbal species in accordance with Good Manufacturing Practice (GMP) standards [2003. Medicinal Products for Human and Veterinary Use. Eudralex, vol. 4 (2003/94/EC)]. The quality control of the complete process is based on pharmacognostic methods, characteristic fingerprint chromatograms, defined amounts of marker substances, physicochemical characteristics and microbiological monitoring. For a herbal multi-component preparation used in multi-target therapy, these pharmaceutical prerequisites have to be ensured for all components and for their combination, as is exemplified by Iberogast® (STW 5) a fixed combination of hydroethanolic extracts of bitter candytuft (Iberis amara), angelica root (Angelicae radix), milk thistle fruit (Silybi mariani fructus), celandine herb (Chelidonii herba), caraway fruit (Carvi fructus), liquorice root (Liquiritiae radix), peppermint herb (Menthae piperitae folium), balm leaf (Melissae folium) and chamomile flower (Matricariae flos) using in the therapy of gastrointestinal compliants (). The prerequisites for the quality of each of its components according to actual standards are at first the cultivation of the plant material according to the Guidelines for Good Agricultural Practice (GAP) conditions of Medicinal and Aromatic Plants [1998. Z. Arzn. Gew. Pfl. 3, 166–178] to yield a defined raw material of high quality. Characteristic compounds of the extracts had to be identified and different analytical methods such as HPLC, with low coefficients of variation had to be developed to analyze each of the standardized ethanolic extracts and the finished product. At the example of the extract of I. amara these necessary investigations are described. The variability of the plant material in its natural habitats, the identification of characteristic compounds and exemplary chromatograms for fingerprint evaluation and quantification are shown. These data are required for characterization of the profile of the active substances in the finished product. [Copyright &y& Elsevier]

A systematic review referring to efficacy and tolerability of the herbal combination Iberogast® (iberis amara planta totalis, Chelidonii herba, Cardui matriae fructus, Melissae folium, Carvi fructus, Liquiritiae radix, Angelicae radix, Matricariae flos, Menthae piperitae folium) was performed in patients with functional dyspepsia. Three placebo-controlled trials and a reference-controlled trial showed a statistical significant and therapeutical relevant reduction of the gastrointestinal symptom-scores in 595 patients. The therapeutic efficacy was also found in one observational study (2267 patients). In accordance with the available evidence Iberogast® seems to be an effective phytotherapeutic preparation to reduce the symptoms of dyspepsia yet, without central nervous side effects. [ABSTRACT FROM AUTHOR]

The S-locus glycoprotein gene, SLG, which participates in the pollen-stigma interaction of self-incompatibility, and its unlinked homologue, SLR1, were analyzed in Raphanus sativus and three self-incompatible ornamental plants in the Brassicaceae. Among twenty-nine inbred lines of R. sativus, eighteen S haplotypes were identified on the basis of DNA polymorphisms detected by genomic Southern analysis using Brassica SLG probes. DNA fragments of SLG alleles specifically amplified from eight S haplotypes by PCR with class I SLG-specific primers showed different profiles following polyacrylamide gel electrophoresis, after digestion with a restriction endonuclease. The nucleotide sequences of the DNA fragments of these eight R. sativus SLG alleles were determined. Degrees of similarity of the nucleotide sequences to a Brassica SLG ( S 6 SLG) ranged from 85.6% to 91.9%. Amino acid sequences deduced from these had the twelve conserved cysteine residues and the three hypervariable regions characteristic of Brassica SLGs. Phylogenetic analysis of the SLG sequences from Raphanus and Brassica revealed that the Raphanus SLGs did not form an independent cluster, but were dispersed in the tree, clustering together with Brassica SLGs. These results suggest that diversification of the SLG alleles of Raphanus and Brassica occurred before differentiation of these genera. Although SLR1 sequences from Orychophragmus violaceus were shown to be relatively closely related to Brassica and Raphanus SLR1 sequences, DNA fragments that are highly homologous to the Brassica SLG were not detected in this species. Two other ornamental plants in the Brassicaceae, which are related more distantly to Brassica than Orychophragmus, also lacked sequences highly homologous to Brassica SLG genes. The evolution of self-incompatibility in the Brassicaceae is discussed. [ABSTRACT FROM AUTHOR]

• Ultrasound-supercritical carbon dioxide is efficient for cucurbitacin E extraction. • The optimal hybrid process is superior to traditional techniques. • Calculated Sovová model fitted well with the extraction curve. • Iberis amara can be considered as a suitable source for cucurbitacin E production. Cucurbitacin E (CuE) is an active compound with heterogeneous biological activity and is abundant in Iberis amara seeds. To extract CuE from the seeds, a novel procedure using ultrasound-assisted supercritical CO 2 extraction (UASCE) was exploited in this study. The Taguchi design method was adopted for optimizing the experimental conditions of UASCE using L 25 (56) orthogonal array. The highest CuE yield (8.61 ± 0.06 mg/g, dry mass) for UASCE was achieved at 60 min dynamic extraction time, 25 MPa pressure, 50 °C, 2 mL/min CO 2 flow rate, 12% co-solvent concentration, and 200 W ultrasonic power. The obtained results revealed that UASCE improved the CuE yield by 26.1% and decreased extraction duration by 34.8% as well as reduced the usage amount of CO 2 by 52% compared with orthodox supercritical CO 2 extraction. Furthermore, the kinetics study demonstrated that the adopted Sovová models fitted well with the experimental extraction curves. The convective mechanism plays a prominent role in UASCE process and mass transfer coefficients increased with the amplification of ultrasonic power. This hybrid technique can be considered as an efficient and promising extraction procedure to obtain CuE from Iberis amara seeds in industrial scale. [ABSTRACT FROM AUTHOR]