Naturally Occurring Dehydrocostus Lactone Covalently Binds to KARRIKIN INSENSITIVE 2 by Dual Serine Modifications in Orobanche cumana and Arabidopsis.

Parasitic weeds, such as Orobanche and Striga, threaten crops globally. Contiguous efforts on the discovery and development of structurally novel seed germination stimulants targeting HYPOSENSITIVE TO LIGHT/KARRIKIN INSENSITIVE 2 (HTL/KAI2) have been made with the goal of weed control. Here, we demonstrate that a natural compound dehydrocostus lactone (DCL) exhibits effective "suicide germination" activity against Orobanche cumana and covalently binds to OcKAI2d2 on two catalytic serine sites with the second modification dependent on the first one. The same interactions and covalent modifications of DCL are also confirmed in AtKAI2. Further in-depth evolution analysis indicates that the proposed two catalytic sites are present throughout the streptophyte algae, hornworts, lycophytes, and seed plants. This discovery is particularly noteworthy as it signifies the first confirmation of a plant endogenous molecule directly binding to KAI2, which is valuable for unraveling the elusive identity of the KAI2 ligand and for targeting KAI2 paralogues for the development of novel germination stimulants.

The Evaluation of Debranone Series Strigolactone Agonists for Germination Stimulants in Orobanche Species.

Strigolactones (SLs) are plant hormones that regulate shoot branching. In addition, SLs act as compounds that stimulate the germination of root parasitic weeds, such as Striga spp. and Orobanche spp., which cause significant damage to agriculture worldwide. Thus, SL agonists have the potential to induce suicidal germination, thereby reducing the seed banks of root parasitic weeds in the soil. Particularly, phenoxyfuranone-type SL agonists, known as debranones, exhibit SL-like activity in rice and Striga hermonthica. However, little is known about their effects on Orobanche spp. In this study, we evaluated the germination-inducing activity of debranones against Orobanche minor. Analysis of structure-activity relationships revealed that debranones with electron-withdrawing substituents at the 2,4- or 2,6-position strongly induced the germination of Orobanche minor. Lastly, biological assays indicated that 5-(2-fluoro-4-nitrophenoxy)-3-methylfuran-2(5H)-one (test compound 61) induced germination to a comparable or even stronger extent than GR24, a well-known synthetic SL. Altogether, our data allowed us to infer that this enhanced activity was due to the recognition of compound 61 by the SLs receptor, KAI 2d, in Orobanche minor.

Effects of phenylethanol glycosides from Orobanche cernua Loefling on UVB-Induced skin photodamage: a comparative study.

Previous study has found that Orobanche cernua Loefling(OC) and its main ingredient, acteoside, possess excellently anti-photo-aging effect. In addition to acteoside, crenatoside, isoacteoside and 2'-acetylacteoside were also identified as the main phenylethanol glycosides (PhGs) in OC. To screen optimum effective substance and further clarify the photoprotective ingredients of OC, the effects of four major PhGs in OC were compared using UVB-irradiated HaCaT cells. Results indicated that acteoside, isoacteoside and 2'-acetylacteoside effectively decreased UVB-induced MMP-1 expression and stimulated type I procollagen synthesis through inhibition of MAPK/AP-1 and activation of TGF-ß/Smad pathway. Moreover, acteoside and 2'-acetylacteoside significantly reduced UVB-induced ROS and TARC secretion, which is involved in the inhibition of NF-κß/Iκßα and stimulation of Nrf2 antioxidant defense system. However, crenatoside did not show any effect on the regulation of signal cascades mentioned above. Together, our results suggested that 2'-acetylacteoside and isoacteoside also served as efficient agents against UV radiation-induced skin damage. Among them, acteoside and 2'-acetylacteoside showed a higher efficiency than that of isoacteoside, which possessed great potential in treating skin photo-damage.

Antioxidant and anticoagulant effects of phenylpropanoid glycosides isolated from broomrapes (Orobanche caryophyllacea, Phelipanche arenaria, and P. ramosa).

Holoparasitic plants of the Orobanchaceae, including Cistanche, Orobanche, and Phelipanche spp, are known for their richness of phenylpropanoid glycosides (PPGs). Many PPG compounds have been found to possess a wide spectrum of activities, such as antimicrobial, anti-inflammatory, antioxidant, and memory-enhancing. To better explore the bioactivity potential of European broomrapes (O. caryophyllacea - OC, P. arenaria - PA, P. ramosa - PR) and ten single isolated phenylpropanoid constituents, we investigated their antiradical action, protective effect against oxidation in plasma in vitro system, and influence on coagulation parameters. The tested extracts showed a scavenging activity of 50-70% of Trolox's power. The OC extract, rich in acteoside, had over 20% better antiradical potential than PR extract which was the only one containing PPGs lacking a B-ring catechol moiety in the acyl unit. Moreover, it was found that only eight tested PPGs demonstrated antioxidant potential in human plasma treated with H2O2/Fe; however, the three tested PPGs possessed anticoagulant potential in addition to antioxidant properties. It appears that the structure of PPGs, especially the presence of acyl and catechol moieties, is mainly related to their antioxidant properties. The anticoagulant potential of these compounds is also related to their chemical structure. Selected PPGs exhibit the potential for treating cardiovascular diseases associated with oxidative stress.

Evaluation of cytotoxic and anticancer effect of Orobanche crenata methanolic extract on cancer cell lines.

We aimed to assess the antitumor activity of Orobanche crenata methanolic extract and evaluate its cytotoxic effect on different cancer cell lines to develop an effective natural anticancer drug. Components of O. crenata methanolic extract were analyzed using gas chromatography-mass spectrometry. The extract's antioxidant activity was assessed by 2,2-diphenyl-1-picrylhydrazyl and ferric reducing antioxidant power procedures and cytotoxicity of the extract was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and lactate dehydrogenase assays. Caspase-3 activity was also estimated. O. crenata methanolic extract shows powerful antioxidant activity. The extract inhibited the propagation of human hepatocellular carcinoma (HepG2), human prostate cancer (PC3), human breast adenocarcinoma (MCF-7), and human colon carcinoma (HCT-116) in a dose-dependent manner. O. crenata-treated cells displayed obvious morphological structures distinctive of apoptosis. MTT assay exposed that the extract presented prevention of cell persistence in a dose-dependent means and revealed extremely cytotoxic activity against HepG2, PC3, MCF-7, and HCT-116 with 50% inhibitory concentration values 30.3, 111, 89.6, and 28.6 µg/mL, respectively, after 24 h of incubation. In addition, treatment of HCT-116 with various concentrations of the extract caused the release of lactate dehydrogenase and induction of caspase-3 activity in a dose-dependent way. In conclusion, our findings suggested that the O. crenata extract possesses potent antioxidant, cytotoxic activity, and anticancer properties which are possibly due to the principal bioactive phytochemical composites existing in this plant. These results can be used to develop new drugs for cancer treatment.

In vitro evaluation of biological activities of Orobanche crenata Forssk. leaves extract.

Orobanche crenata Forssk. (Orobanchaceae) is a holoparasitic plant noxious to legumes. Orobanchaceae are well known in traditional medicine for their hypotensive, antihyperglycemic, antispasmodic, analgesic and antimicrobial effects. In this work, the biological activities of the acetonic leaves extract were evaluated. Qualitative chemical analysis was determined through thin layer chromatography. Total phenols and flavonoids content was obtained by the Folin-Ciocalteu and the Aluminium chloride colorimetric methods, respectively. The antimicrobial activity was performed by the broth microdilution method. The extract showed antibacterial activity, particularly against Gram-positive strains, with MIC ranging from 376.00 to 3011.00 µg/ml. Besides, it exhibited antifungal effects on Candida spp. with MIC ranging from 94.10 to 3011.00 µg/ml. In addition, O. crenata inhibited superoxide anion formation and showed a DPPH quenching capacity, particularly at 80 µg/ml. Interestingly, our results are promising thus indicating the importance of O. crenata as a significant source of biologically active natural compounds.

Identification and occurrence of phenylethanoid and iridoid glycosides in six Polish broomrapes (Orobanche spp. and Phelipanche spp., Orobanchaceae).

There are about 200 holoparasitic broomrapes (Orobanchaceae) known worldwide, however, only several species have been so far investigated phytochemically. Among Orobanche s.l. are both rare and endangered species, as well as onerous crop pests. This study aims to give a phytochemical description, both qualitative and quantitative, of six broomrape species (Orobanche and Phelipanche taxa) growing in Poland, including species that have not been tested in detail (O. caryophyllacea, O. lutea, O. picridis, and P. arenaria). Sixteen metabolites, including 14 phenylethanoid glycosides (PhGs) and 2 iridoid glycosides (IrGs), were isolated and identified using NMR spectroscopy and hydrolysis, revealing the presence of two previously undescribed PhGs in P. ramosa, named ramoside A and 2'-acetylramoside A. In addition, in the example of O. caryophyllacea, we have reported as the first occurrence of IrGs in broomrapes. Concentrations of phenylethanoids, the main constituents of broomrapes, in the studied plant material (flowering shoots with haustoria) were determined using the UHPLC-PDA method. It was found that P. ramosa has been the richest source of PhGs. In addition, the differences between broomrapes have been visualized using principal component and cluster analysis. The results of the antiradical DPPH test of 13 PhGs confirmed previous findings on the relation of the antioxidant potential with the structure of phenolic moieties - phenolic acid and phenylethanoid unit.

Easy Access to Alkoxy, Amino, Carbamoyl, Hydroxy, and Thiol Derivatives of Sesquiterpene Lactones and Evaluation of Their Bioactivity on Parasitic Weeds.

It has been hypothesized that the α-methylene-γ-lactone moiety of sesquiterpene lactones is a key unit for their bioactivity. As a consequence, modifications of these compounds have been focused on this fragment. In the work reported here, two sesquiterpene lactones, namely, dehydrocostuslactone and ß-cyclocostunolide, a eudesmanolide obtained by controlled cyclization of costunolide, were chosen for modification by Michael addition at C-13. On applying this reaction to both compounds, it was possible to introduce the functional groups alkoxy, amino, carbamoyl, hydroxy, and thiol to give products in good to high yields, depending on the base and solvent employed. In particular, the introduction of a thiol group at C-13 in both compounds was achieved with outstanding yields (>90%) and this is unprecedented for these sesquiterpene lactones. The bioactivities of the products were evaluated on etiolated wheat coleoptile elongation and germination of seeds of parasitic weeds, with significant activity observed on Orobanche cumana and Phelipanche ramosa. The structure-activity relationships are discussed.

Development of phloem connection between the parasitic plant Orobanche cumana and its host sunflower.

Orobanche cumana is a root parasitic plant causing considerable yield losses in sunflower cultivation. The holoparasite fulfills its entire demand for water, minerals, and organic nutrients from the host's vascular system. In this study, the ultrastructure of the phloem connection between the haustorium of young O. cumana tubercles and the sunflower root has been examined for the first time. Parasite and host tissues were intermingled at the contact site and difficult to distinguish, but sieve-tube elements of O. cumana and sunflower could be differentiated according to their plastid ultrastructure. While O. cumana sieve-element plastids were larger, often irregular in shape and contained few, small starch inclusions, sieve-element plastids of the host were significantly smaller, always roundish with more and larger starch inclusions. This made it possible to trace the exact contact site of host and parasite sieve elements to show a direct symplastic phloem connection between the two species. The interspecific sieve plate showed more callose on the host side. This allowed detection of newly formed plasmodesmata between host sieve-tube elements and parenchymatic parasite cells, thus showing that undifferentiated cells of the parasite could connect to fully differentiated sieve elements of sunflower. Furthermore, the arrangement of phloem within the O. cumana tubercle as well as differences in sieve-element plastid ultrastructure during shoot development in O. cumana were investigated and are discussed in this paper.

From stem cells protection to skin microbiota balance: Orobanche rapum extract, a new natural strategy.

BACKGROUND: Healthy skin is a delicate balance between skin renewal and microbiota homeostasis, and its imbalance promotes premature aging and dermatological disorders. Skin stem cells are key actors in this process but their sensitivity to aging and external stressors such as UV reduces the skin renewal power. The skin microbiota has been recently described as active in the healthy skin, and its imbalance could trigger some disorders. AIMS: We hypothesized that reactivation of stem cells and maintenance of microbiota could be a disruptive strategy for younger and healthier skin. We thus developed a new plant extract that restores the entire skin renewal process by sequential activation from stem cells stimulation to microbiota protection. METHODS: We studied stem cells comportment in the presence of Orobanche rapum extract by survivin immunocytochemistry and caspases 3 and 9 dosages. We also analyzed epidermal differentiation markers by immunohistochemistry and lipids organization by GC/MS At the clinical level, we investigated the impact of O. rapum extract on microbiota and on skin aspect. RESULTS: We demonstrated an active protection of skin stem cells through the maintenance of their clone-forming capacity and resistance to UV through the overexpression of survivin coupled to caspases inhibition. Furthermore, we showed the restoration of epidermal differentiation markers and ceramide biosynthesis favorable to orthorhombic organization. Clinical studies, including microbiota analysis, showed an active skin surface renewal coupled with microbiota protection. CONCLUSION: We evidenced that our active ingredient is able to stimulate skin rejuvenation while protecting the cutaneous microbiota, creating healthier skin and thereby beauty.

Genuine and Sequestered Natural Products from the Genus Orobanche (Orobanchaceae, Lamiales).

The present review gives an overview about natural products from the holoparasitic genus Orobanche (Orobanchaceae). We cover both genuine natural products as well as compounds sequestered by Orobanche taxa from their host plants. However, the distinction between these two categories is not always easy. In cases where the respective authors had not indicated the opposite, all compounds detected in Orobanche taxa were regarded as genuine Orobanche natural products. From the about 200 species of Orobanche s.l. (i.e., including Phelipanche) known worldwide, only 26 species have so far been investigated phytochemically (22 Orobanche and four Phelipanche species), from 17 Orobanche and three Phelipanche species defined natural products (and not only natural product classes) have been reported. For two species of Orobanche and one of Phelipanche dedicated studies have been performed to analyze the phenomenon of natural product sequestration by parasitic plants from their host plants. In total, 70 presumably genuine natural products and 19 sequestered natural products have been described from Orobanche s.l.; these form the basis of 140 chemosystematic records (natural product reports per taxon). Bioactivities described for Orobanche s.l. extracts and natural products isolated from Orobanche species include in addition to antioxidative and anti-inflammatory effects, e.g., analgesic, antifungal and antibacterial activities, inhibition of amyloid ß aggregation, memory enhancing effects as well as anti-hypertensive effects, inhibition of blood platelet aggregation, and diuretic effects. Moreover, muscle relaxant and anti-spasmodic effects as well as anti-photoaging effects have been described.

Stereospecific reduction of the butenolide in strigolactones in plants.

Reductive metabolism of strigolactones (SLs) in several plants was investigated. Analysis of aquaculture filtrates of cowpea and sorghum each fed with four stereoisomers of GR24, the most widely used synthetic SL, revealed stereospecific reduction of the double bond at C-3' and C-4' in the butenolide D-ring with preference for an unnatural 2'S configuration. The cowpea metabolite converted from 2'-epi-GR24 and the sorghum metabolite converted from ent-GR24 had the methyl group at C-4' in the trans configuration with the substituent at C-2', different from the cis configuration of the synthetic H2-GR24 reduced with Pd/C catalyst. The plants also reduced the double bond in the D-ring of 5-deoxystrigol isomers with a similar preference. The metabolites and synthetic H2-GR24 stereoisomers were much less active than were the GR24 stereoisomers in inducing seed germination of the root parasitic weeds Striga hermonthica, Orobanche crenata, and O. minor. These results provide additional evidence of the importance of the D-ring for bioactivity of SLs.

Structure-activity relationships of strigolactones via a novel, quantitative in planta bioassay.

Strigolactones (SLs) are plant hormones with various functions in development, responses to stress, and interactions with (micro)organisms in the rhizosphere, including with seeds of parasitic plants. Their perception for hormonal functions requires an α,ß-hydrolase belonging to the D14 clade in higher plants; perception of host-produced SLs by parasitic seeds relies on similar but phylogenetically distinct proteins (D14-like). D14 and D14-like proteins are peculiar receptors, because they cleave SLs before undergoing a conformational change that elicits downstream events. Structure-activity relationship data show that the butenolide D-ring is crucial for bioactivity. We applied a bioisosteric approach to the structure of SLs by synthetizing analogues and mimics of natural SLs in which the D-ring was changed from a butenolide to a lactam and then evaluating their bioactivity. This was done by using a novel bioassay based on Arabidopsis transgenic lines expressing AtD14 fused to firefly luciferase, in parallel with the quantification of germination-inducing activity on parasitic seeds. The results obtained showed that the in planta bioassay is robust and quantitative, and thus can be confidently added to the SL-survey toolbox. The results also showed that modification of the butenolide ring into a lactam one significantly hampers the biological activity exhibited by SLs possessing a canonical lactonic D-ring.

Orobanche cernua Loefling Attenuates Ultraviolet B-mediated Photoaging in Human Dermal Fibroblasts.

UV radiation is the primary cause of skin photoaging, which results in an increase in matrix metalloproteinases and degradation of collagen. Developing new natural antioxidant as photoprotective agents has become a popular area of research. Orobanche cernua Loefling is a parasitic plant that is rich in phenylethanoid glycosides (PhGs). This study investigated the photoprotective effects of the ethanolic extract of Orobanche cernua Loefling (OC) and its principal component acteoside on UVB-induced photoaging as well as their underlying molecular mechanisms in normal human dermal fibroblasts (NHDFs). Biological testing demonstrated that OC and acteoside possessed significant photoprotective activities, reducing MMP and IL-6 levels while improving type-I procollagen synthesis in UVB-irradiated NHDFs. Further study showed that the protective mechanisms were the improvement of transcription factor Nrf2-mediated antioxidant defensive system, suppression of MAPK/AP-1 and activation of the TGF-ß/Smad pathway. Together, our results suggested that OC might be a promising antiphotoaging agent against UV radiation-induced skin damage.

[A new phenethyl alcohol glycoside from Orobanche coerulescens].

The constituents of the whole plant of Orobanche coerulescens were isolated and purified by using various column chromatographic techniques including D101, silica gel and ODS. The structures were identified by spectroscopic analyses including NMR and MS. A new phenylethanol glycoside was isolated from the whole plant of O. coerulescens, and was identified as 2-(3-methoxy-4-hydroxyphenyl)-ethanol-1-O- [(1→3)-O-α-L-rhamnopyranosyl-4, 6-O-di-feruloyl]-ß-D-glucopyranoside, named as orobancheoside B. Through the antibacterial activity test, orobancheoside B was proved to have certain antibacterial activity, and be one of the main active components of O. coerulescens. The research result will laid a foundation for the medicinal materials and quality control research. Activity screening, broomrape orobancheoside B has certain antibacterial activity, as one of the main active components of O. coerulescens, and to constantly improve the quality of the medicinal materials laid a foundation.

A new phenylethanoid glycoside from Orobanche cernua Loefling.

A novel phenylethanoid glycoside, 3'-O-methyl isocrenatoside (1), along with two known compounds, methyl caffeate (2) and protocatechuic aldehyde (3), were isolated from the fresh whole plant of Orobanche cernua Loefling. All the isolated compounds (1-3) were elucidated on the basis of spectroscopic analysis including IR, MS and NMR data. The cytotoxic activities of these compounds were evaluated. Results showed that 3'-O-methyl isocrenatoside (1) and methyl caffeate (2) exhibited significant cytotoxicity, with IC50 values of 71.89, 36.97 µg/mL and 32.32, 34.58 µg/mL against the B16F10 murine melanoma and Lewis lung carcinoma cell lines, respectively.

Strigolactam: New potent strigolactone analogues for the germination of Orobanche cumana.

Very recently, strigolactones have been conclusively identified as phytohormones. The progresses achieved in this field are culminating in the identification of the molecular receptors involved in the signal transduction mechanism. The exact mechanism of the mode of action of strigolactones still remains to be fully elucidated and we were interested to gain some insight into the mechanism of action of strigolactones by selectively modifying the reactivity of the lactone C-ring. Therefore, we report here the synthesis of strigolactams 1 and 16 and their surprisingly good activity on the germination of Orobanche cumana parasitic weed seeds.

Ethanol production from rape straw by a two-stage pretreatment under mild conditions.

The growing interest on rape oil as raw material for biodiesel production has resulted in an increasing availability of rape straw, an agricultural residue that is an attractive renewable source for the production of second-generation bioethanol. Pretreatment is one of the key steps in such a conversion process. In this work, a sequential two-stage pretreatment with dilute sulfuric acid (130 °C, 60 min, 2% w/v H2SO4) followed by H2O2 (1-5% w/v) in alkaline medium (NaOH) at low temperature (60, 90 °C) and at different pretreatment times (30-90 min) was investigated. The first-acid stage allows the solubilisation of hemicellulose fraction into fermentable sugars. The second-alkaline peroxide stage allows the delignification of the solid material whilst the cellulose remaining in rape straw turned highly digestible by cellulases. Simultaneous saccharification and fermentation with 15% (w/v) delignified substrate at 90 °C, 5% H2O2 for 60 min, led to a maximum ethanol production of 53 g/L and a yield of 85% of the theoretical.

[Determination of phenylethanoid glycosides in Orobanche coerulescens].

Orobanche caerulescens is an important medicinal resource in Orobanchaceae. The present study aims to establish methods for determination of acteoside, crenatoside, and total phenylethanoid glycosides in O. caerulescens, and determine the content in 15 samples to evaluate the resource utilization of this medicinal plant. The content of acteoside and crenatoside were quantitatively determined by HPLC, while total phenylpropanoid glycosides was estimated by UV-VIS spectrophotometry. According to the results, the content of acteoside was the highest in O. caerulescens, followed by crenatoside. The contents of acteoside, crenatoside, and total phenylethanoid glycosides were between 1.15% - 15.60%, 0.83% - 4.47%, and 6.78% - 27.43%, respectively, which had significant differences. The acquisition time has great influence on the content of main components of O. caerulescens. The content of phenylethanoid glycosides is higher in the samples which were collected at the flowering stage. The two determination methods were proved to be simple, accurate and reliable, and can be used to evaluate the quality and resource utilization of O. caerulescens.

Robust method for investigating nitrogen metabolism of 15N labeled amino acids using AccQ•Tag ultra performance liquid chromatography-photodiode array-electrospray ionization-mass spectrometry: application to a parasitic plant-plant interaction.

An AccQ•Tag ultra performance liquid chromatography-photodiode array-electrospray ionization-mass spectrometry (AccQ•Tag-UPLC-PDA-ESI-MS) method is presented here for the fast, robust, and sensitive quantification of (15)N isotopologue enrichment of amino acids in biological samples, as for example in the special biotic interaction between the cultivated specie Brassica napus (rapeseed) and the parasitic weed Phelipanche ramosa (broomrape). This method was developed and validated using amino acid standard solutions containing (15)N amino acid isotopologues and/or biological unlabeled extracts. Apparatus optimization, limits of detection and quantification, quantification reproducibility, and calculation method of (15)N isotopologue enrichment are presented. Using this method, we could demonstrate that young parasite tubercles assimilate inorganic nitrogen as (15)N-ammonium when supplied directly through batch incubation but not when supplied by translocation from host root phloem, contrary to (15)N2-glutamine. (15)N2-glutamine mobility from host roots to parasite tubercles followed by its low metabolism in tubercles suggests that the host-derived glutamine acts as an important nitrogen containing storage compound in the young tubercle of Phelipanche ramosa.