Assessment of the effect of drying on Brassica greens via a multiplex approach based on LC-QTOF-MS/MS, molecular networking, and chemometrics along with their antioxidant and anticancer activities.

Turnip (Brassica rapa var rapa L.) leaves are a rich source of versatile bioactive phytochemicals with great potential in the food and herbal industries. However, the effect of drying on its constituents has never been studied before. Hereto, three drying techniques were compared, namely, lyophilization (LY), vacuum oven (VO), and shade drying (SD). Chemical profiling utilizing liquid chromatography-quadrupole time-of-flight tandem mass spectrometry (LC-QTOF-MS/MS) combined with chemometrics showed the different impacts of the drying methods on the phytochemical composition of the alcoholic leaf extracts. Unsupervised principal component analysis (PCA) and supervised partial least squares-discriminant analysis (PLS-DA) of the LC-QTOF-MS/MS data showed distinct distant clustering across the three drying techniques. Loading plots and VIP scores demonstrated that sinapic acid, isorhamnetin glycosides, and sinapoyl malate were key markers for LY samples. Meanwhile, oxygenated and polyunsaturated fatty acids were characteristic for SD samples and oxygenated polyunsaturated fatty acids and verbascoside were characteristic for VO samples. LY resulted in the highest total phenolics (TP) and total flavonoid (TF) contents followed by SD and VO. LY and SD samples had much higher antioxidant activity than VO measured by 2,2-diphenyl-1-picrylhydrazyl (DPPH), oxygen radical absorbance capacity (ORAC), and iron metal chelation assays. According to the anticancer activity, the drying methods were ranked in descending order as SD > LY â‰« VO when tested against colon, breast, liver, and lung cancer cell lines. Among the identified compounds, flavonoids and omega-3 fatty acids were key metabolites responsible for the anticancer activity as revealed by partial least squares (PLS) regression and correlation analyses. In conclusion, compared to LY, SD projected out as a cost-effective drying method without compromising the phytochemical and biological activities of Brassica greens. The current findings lay the foundation for further studies concerned with the valorization of Brassica greens.

Vetiver aerial parts and roots ameliorate rheumatoid arthritis in complete Freund's adjuvant rat model, a phytochemical profiling and mechanistic study.

ETHNOPHARMACOLOGICAL RELEVANCE: Vetiver (Chrysopogon zizanioides) is indigenous to India where it is traditionally used to relief rheumatisms, lumbagos and sprains. Vetiver anti-inflammatory activity has not been previously investigated, and its specific interactions with body inflammation cascade remain largely unknown. AIM OF THE STUDY: The present work was performed to validate the ethnobotanical use of the plant and compare the anti-inflammatory activities of the ethanolic extracts of the most traditionally used part (aerial part) to that of the root. Furthermore, we attempt to reveal the molecular mechanism of this anti-inflammatory activity in correlation to the chemical composition of C. zizanioides aerial (CA) and root parts (CR). MATERIALS AND METHODS: Ultraperformance liquid chromatography coupled to high resolution mass spectrometry (UHPLC/HRMS) was used for comprehensive analysis of both CA and CR. The anti-inflammatory effect of both extracts was evaluated in complete Freund's adjuvant (CFA)-induced RA model in Wistar rats. RESULTS: Phenolic metabolites were predominant in CA and 42 were identified for the first time, while only 13 were identified in CR. Meanwhile, triterpenes and sesquiterpenes were confined to the root extract. In CFA arthritis model, CA showed better anti-inflammatory activity than CR marked by an increase in serum level of IL-10 with simultaneous decrease in pro-inflammatory markers; IL-6, ACPA and TNF-α and was evident in histopathological examination. This anti-inflammatory effect was accompanied by down-regulation of JAK2/STAT3/SOCs3, ERK1/ERK2, TRAF6/c-FOS/NFATC1, TRAF6/NF-κB/NFATC1 and RANKL pathways which were all upregulated after CFA injection. These pathways were modulated to larger extent by CA, except for ERK1/ERK2 which was downregulated more effectively by CR. This differential effect between CA and CR can be explained by the variability in their phytoconstituents profile. CONCLUSION: In agreement with the ethnobotanical preference, CA extract was more effective than CR extract in reducing the symptoms of RA probably due to its enrichment with flavonoids, lignans, and flavolignans. Both CA and CR reduced the production of inflammatory cytokines through modulating various biological signaling pathways. These findings support the traditional use of vetiver leaves as a remedy for RA and suggest that the use of the whole plant may offer advantage by synergistically affecting more inflammatory pathways.

Preparation and optimization of a molecularly imprinted polymers - solid phase extraction system for the extraction of bioactive sesquiterpene lactones of Ambrosia maritima plant.

A solid phase extraction (SPE) system for sesquiterpene lactones of damsissa was developed utilising molecularly imprinted polymers (MIPs). The prepared MIPs had a mesoporous structure and particle size of ≈2.65 µm with 3.99 nm pore size. Additionally, MIPs exhibited high thermal stability with degradation temperature between 209 and 459 °C. Optimized MIP-SPE protocol conditions were set at loading step: 1 mL ethanol; washing step: 1 mL water; eluting step: 4 mL methanol. Developed MIP-SPE system showed a binding capacity of 66.66 mg/g based on Langmuir isotherm which was selected as the best fitting model isotherm. Good selectivity coefficients were observed for neoambrosin of 2.37, 1.31 and 1.14 against umbelliferone, quercetin glucoside and p-coumaric acid, respectively. Furthermore, the proposed MIP-SPE protocol displayed some potential in the isolation of sesquiterpene lactones from damsissa plant extract and laid a foundation for the development of more selective MIPs to nonpolar natural products.

Outgoing and potential trends of composition, health benefits, juice production and waste management of the multi-faceted Grapefruit Citrus Χ paradisi: A comprehensive review for maximizing its value.

Grapefruit (GF) Citrus Χ paradisi Macfad (F. Rutaceae) is one of the major citrus fruits that encompass a myriad of bioactive chemicals and most unique among citrus fruits. Nevertheless, no study has yet to assess comprehensively its multitudinous constituents, health benefits, and valuable waste products. Hereto, the present review provides an updated comprehensive review on the different aspects of GF, its juice production, waste valorization, enhancement of its byproducts quality, and compared to other citrus fruits. Grapefruit uniqueness among other citrus fruits stands from its unique taste, flavor, and underlying complex chemical composition. Despite limonene abundance in peel oil and grapefruit juice (GFJ) aroma, nootkatone and sulfur compounds are the key determinants of its flavor, whereas flavanones contribute to its bitter taste and in conjunction with limonoids. Different postharvest treatments and juice processing are reviewed and in context to its influence on final product quality and or biological effects. Flavanones, furanocoumarins, and limonoids appear as the most prominent in GF drug interactions affecting its metabolism and or excretion. Valorization of GF peel is overviewed for its utilization as biosrobent, its oil in aromatherapy, limonene as antimicrobial or in cosmetics, fruit pectin for bioethanol production, or as biosorbent, and peel phenolics biotransformation. The present review capitalizes on all of the aforementioned aspects in GF and further explore novel aspects of its juice quality presenting the full potential of this valued multi-faceted citrus fruit.

Sesquiterpene lactones; Damsin and neoambrosin suppress cytokine-mediated inflammation in complete Freund's adjuvant rat model via shutting Akt/ERK1/2/STAT3 signaling.

ETHNOPHARMACOLOGIAL RELEVANCE: Although Damsissa (Ambrosia maritima) is traditionally used as anti-inflammatory and diuretic, the biological activity and mechanism of action of its major constituents are to be elucidated. AIM: to decipher the anti-arthritic potential of damsin (DMS) and neoambrosin (NMS) and to unfold their molecular signaling in complete Freund's adjuvant (CFA)-induced arthritis model. MATERIALS AND METHODS: the right hind paw was inoculated with CFA (0.1 ml) at day 0 and 7 while treatments were started from the 14th day and continued for 2 weeks. Rats were randomly assigned into 4 groups; normal group (NRML), CFA-induced arthritis group, CFA-induced arthritis treated with DMS and NMS (10 mg/kg/day) as 3rd and 4th group; respectively. RESULTS: Throughout experimental period, treatments ameliorated the increase of paw volume, knee joint diameter and nociception tests as reflected in open field arena. Also, DSM and NMS suppressed phosphorylation of Akt, STAT-3, ERK1/2 which was further mirrored by inactivation of GSK3ß and downregulation of MCP-1 together with CCN1 and NF-kß in hind paw tissue. Concomitantly, inflammation markers; TNF-α, IL-6, -12 were lowered as confirmed microscopically during examination of hind paw tissue. CONCLUSION: DSM and NMS-induced suppression of NF-kß subdues clinical features of RA most probably through repression of Akt/ERK1/2/STAT3 pathway. Therefore, DMS and NMS can serve as safe and effective treatment for rheumatoid arthritis, one of the most disabling chronic, inflammatory and painful autoimmune disease.

Ambrosin, a potent NF-κß inhibitor, ameliorates lipopolysaccharide induced memory impairment, comparison to curcumin.

Despite its poor bioavailability, curcumin is a promising natural polyphenol targeting NF-κß. NF-κß is a target for new therapeutics because it plays a pivotal role in the pathophysiology of Alzheimer disease (AD). In contrast, ambrsoin, a sesquiterpene lactone which is a potent NF-κß inhibitor, is scarcely studied in AD models. The current work aims to assess the efficacy of ambrosin as a possible remedy for AD. In silico studies showed that bioavailability and BBB permeability could be favorable for ambrosin over curcumin. Memory impairment was induced in mice by single intraperitoneal injection of LPS (0.4 mg/kg). Treated groups received curcumin (100 mg/kg) or ambrosin at doses (5 or 10 mg/kg) for 7 days. Mice in treated groups showed a significant improvement in memory functions during Morris water maze and object recognition tests. Curcumin and ambrosin (10 mg/kg) inhibited the upsurge of NF-κßp65 transcript and protein levels. Consequently, downstream pro-inflammatory and nitrosative mediators were inhibited, namely, TNF-α, IL-1ß, COX-2 and iNOS. BACE1 was inhibited, thereby reducing amyloid plaques (Aß) deposition and eventually reducing inflammation and apoptosis of neurons as revealed by immunohistopathological examination. In conclusion, ambrosin can be repurposed as AD remedy after further pharmacokinetic/pharamacodynamic assessments. It could serve as an additional lead drug for AD therapeutics.

Synthesis and 2D-QSAR study of dispiropyrrolodinyl-oxindole based alkaloids as cholinesterase inhibitors.

In this work, we describe the regioselective synthesis of some new dispiro[indene-2,3'-pyrrolidine-2',3″-indoline]-1,2″(3H)-dione 4-29 attributable to the previously described methods. All the new chemical entities were assessed in-vitro as inhibitors of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) enzymes; while no significant inhibitory activity for the tested compounds were assigned on AChE, compounds 4, 27, 29, 28 and 15 were the most active against BChE enzyme with IC50 = 13.7 µM, 21.8 µM, 22.1 µM, 22.9 µM and 24.9 µM respectively compared to Donepezil (IC50 = 0.72 µM). Compound 4 was found to have a mixed type mode of inhibition, the bioactivity of the new chemical entities (N = 26, n = 5, R2 = 0.893, R2 cvOO = 0.831, R2 cvMO = 0.838, F = 33.32, s2 = 0.003) was elucidated via a statistically significant QSAR model utilizing CODESSA-Pro software that validated the observed results.

Protective action of Crateva nurvala Buch. Ham extracts against renal ischaemia reperfusion injury in rats via antioxidant and anti-inflammatory activities.

ETHNOPHARMACOLOGICAL RELEVANCE: Crateva nurvala stem bark is commonly used in Ayruveda in treatment of many renal injuries, e.g., urinary lithiasis, diuretic and nephroprotective. However, its protective effect against renal ischaemia/reperfusion, the major cause of acute kidney injury, has never been studied. Moreover, no comprehensive chemical profiling of its extracts was recorded. AIM OF THE STUDY: Assessment of the protective effect of the plant extracts against renal ischaemia/reperfusion and elucidation of the possible mechanism of action. Then, to determine its bioactive constituents using modern UPLC-HRMS technique. MATERIAL AND METHODS: Unilateral ischaemia was induced by clamping the left renal artery for 1h then reperfusion for 24h. Rats were divided in 4 groups: i) sham-operated group, ii) ischaemia/reperfusion, I/R group, iii) I/R protected by previous administration of Crateva leaves extract, CLE group and iv) I/R protected by previous administration of Crateva bark extract, CBE group. At the end of reperfusion, blood samples were analyzed for renal function biomarkers. Kidneys were examined histopathologically and their homogenates were used in determining the intracellular levels of oxidative stress, inflammatory, and apoptosis markers. RESULTS: Leaves and bark extracts attenuated the deleterious effects of I/R apparent in reducing LDH, creatinine and blood urea nitrogen levels. The extracts reduced the oxidative stress by replenishing the glutathione levels and Nrf2 factor levels. Moreover, extracts decreased levels of pro-inflammatory TNF-α, NF-κß and IL-6; which ultimately resulted in reducing the pro-apoptotic caspase-3. Bark and leave extracts have quite similar chemical profile where 42 compounds of various chemical classes were identified. Flavonoids are the major class of the bioactive phytochemicals CONCLUSION: C. nurvala extracts had effectively ameliorated the deleterious effects of renal I/R by mainly counteracting oxidative stress and presumably inflammation. Consequently, it can be used as a complementary treatment with other agents. In this aspect, leaves stand as a sustainable alternative to bark. The presented chemical profiling can be used in future standardization and quality control of the drug.

Metabolome based volatiles profiling in 13 date palm fruit varieties from Egypt via SPME GC-MS and chemometrics.

Dates (Phoenix dactylifera L.) are distributed worldwide as major food complement providing a source of sugars and dietary fiber as well as macro- and micronutrients. Although phytochemical analyses of date fruit non-volatile metabolites have been reported, much less is known about the aroma given off by the fruit, which is critical for dissecting sensory properties and quality traits. Volatile constituents from 13 date varieties grown in Egypt were profiled using SPME-GCMS coupled to multivariate data analysis to explore date fruit aroma composition and investigate potential future uses by food industry. A total of 89 volatiles were identified where lipid-derived volatiles and phenylpropanoid derivatives were the major components of date fruit aroma. Multivariate data analyses revealed that 2,3-butanediol, hexanal, hexanol and cinnamaldehyde contributed the most to classification of different varieties. This study provides the most complete map of volatiles in Egyptian date fruit, with Siwi and Sheshi varieties exhibiting the most distinct aroma among studied date varieties.

O-Methyltransferases involved in biphenyl and dibenzofuran biosynthesis.

Biphenyls and dibenzofurans are the phytoalexins of the Malinae involving apple and pear. Biosynthesis of the defence compounds includes two O-methylation reactions. cDNAs encoding the O-methyltransferase (OMT) enzymes were isolated from rowan (Sorbus aucuparia) cell cultures after treatment with an elicitor preparation from the scab-causing fungus, Venturia inaequalis. The preferred substrate for SaOMT1 was 3,5-dihydroxybiphenyl, supplied by the first pathway-specific enzyme, biphenyl synthase (BIS). 3,5-Dihydroxybiphenyl underwent a single methylation reaction in the presence of S-adenosyl-l-methionine (SAM). The second enzyme, SaOMT2, exhibited its highest affinity for noraucuparin, however the turnover rate was greater with 5-hydroxyferulic acid. Both substrates were only methylated at the meta-positioned hydroxyl group. The substrate specificities of the OMTs and the regiospecificities of their reactions were rationalized by homology modeling and substrate docking. Interaction of the substrates with SAM also took place at a position other than the sulfur group. Expression of SaOMT1, SaOMT2 and SaBIS3 was transiently induced in rowan cell cultures by the addition of the fungal elicitor. While the immediate SaOMT1 products were not detectable in elicitor-treated cell cultures, noraucuparin and noreriobofuran accumulated transiently, followed by increasing levels of the SaOMT2 products aucuparin and eriobofuran. SaOMT1, SaOMT2 and SaBIS3 were N- and C-terminally fused with the super cyan fluorescent protein and a modified yellow fluorescent protein, respectively. All the fluorescent reporter fusions were localized to the cytoplasm of Nicotiana benthamiana leaf epidermis cells. A revised biosynthetic pathway of biphenyls and dibenzofurans in the Malinae is presented.

Control of plant defense mechanisms and fire blight pathogenesis through the regulation of 6-thioguanine biosynthesis in Erwinia amylovora.

Fire blight is a devastating disease of Rosaceae plants, such as apple and pear trees. It is characterized by necrosis of plant tissue, caused by the phytopathogenic bacterium Erwinia amylovora. The plant pathogen produces the well-known antimetabolite 6-thioguanine (6TG), which plays a key role in fire blight pathogenesis. Here we report that YcfR, a member of the LTTR family, is a major regulator of 6TG biosynthesis in E. amylovora. Inactivation of the regulator gene (ycfR) led to dramatically decreased 6TG production. Infection assays with apple plants (Malus domestica cultivar Holsteiner Cox) and cell cultures of Sorbus aucuparia (mountain ash, rowan) revealed abortive fire blight pathogenesis and reduced plant response (biphenyl and dibenzofuran phytoalexin production). In the presence of the ΔycfR mutant, apple trees were capable of activating the abscission machinery to remove infected tissue. In addition to unveiling the regulation of 6TG biosynthesis in a major plant pathogen, we demonstrate for the first time that this antimetabolite plays a pivotal role in dysregulating the plant response to infection.

Biosynthesis of the biphenyl phytoalexin aucuparin in Sorbus aucuparia cell cultures treated with Venturia inaequalis.

Aucuparin is the most widely distributed biphenyl phytoalexin in the rosaceous subtribe Pyrinae, which includes the economically important fruit trees apple and pear. The biphenyl scaffold is formed by biphenyl synthase, which catalyzes biosynthesis of 3,5-dihydroxybiphenyl. Conversion of this precursor to aucuparin (3,5-dimethoxy-4-hydroxybiphenyl) was studied in cell cultures of Sorbus aucuparia after treatment with an elicitor preparation from the scab-causing fungus Venturia inaequalis. The sequence of the biosynthetic steps detected was O-methylation - 4-hydroxylation - O-methylation. The two alkylation reactions were catalyzed by distinct methyltransferases, which differed in pH and temperature optima as well as stability. Biphenyl 4-hydroxylase was a microsomal cytochrome P450 monooxygenase, whose activity was appreciably decreased by the addition of established P450 inhibitors. When fed to V. inaequalis-treated S. aucuparia cell cultures, radioactively labeled 3,5-dihydroxybiphenyl was not only incorporated into aucuparin but also into the dibenzofuran eriobofuran, the accumulation of which paralleled that of aucuparin. However, biphenyl 2'-hydroxylase activity proposed to be involved in dibenzofuran formation was detected in neither microsomes nor cell-free extracts in the presence of NADPH and 2-oxoglutarate, respectively. Nevertheless, a basis for studying biphenyl biosynthesis at the gene level is provided.

Biosynthesis of the antimetabolite 6-thioguanine in Erwinia amylovora plays a key role in fire blight pathogenesis.

Sulfur for fire: The molecular basis for the biosynthesis of the antimetabolite 6-thioguanine (6TG) was unveiled in Erwinia amylovora, the causative agent of fire blight. Bioinformatics, heterologous pathway reconstitution in E. coli, and mutational analyses indicate that the protein YcfA mediates guanine thionation in analogy to 2-thiouridylase. Assays in planta and in cell cultures reveal for the first time a crucial role of 6TG in fire blight pathogenesis.

Formation of biphenyl and dibenzofuran phytoalexins in the transition zones of fire blight-infected stems of Malus domestica cv. 'Holsteiner Cox' and Pyrus communis cv. 'Conference'.

In the rosaceous subtribe Pyrinae (formerly subfamily Maloideae), pathogen attack leads to formation of biphenyls and dibenzofurans. Accumulation of these phytoalexins was studied in greenhouse-grown grafted shoots of Malus domestica cv. 'Holsteiner Cox' and Pyrus communis cv. 'Conference' after inoculation with the fire blight bacterium, Erwinia amylovora. No phytoalexins were found in leaves. However, both classes of defence compounds were detected in the transition zone of stems. The flanking stem segments above and below this zone, which were necrotic and healthy, respectively, were devoid of detectable phytoalexins. The transition zone of apple stems contained the biphenyls 3-hydroxy-5-methoxyaucuparin, aucuparin, noraucuparin and 2'-hydroxyaucuparin and the dibenzofurans eriobofuran and noreriobofuran. In pear, aucuparin, 2'-hydroxyaucuparin, noreriobofuran and in addition 3,4,5-trimethoxybiphenyl were detected. The total phytoalexin content in the transition zone of pear was 25 times lower than that in apple. Leaves and stems of mock-inoculated apple and pear shoots lacked phytoalexins. A number of biphenyls and dibenzofurans were tested for their in vitro antibacterial activity against some Erwinia amylovora strains. The most efficient compound was 3,5-dihydroxybiphenyl (MIC=115 µg/ml), the immediate product of biphenyl synthase which initiates phytoalexin biosynthesis.