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Volatile oils of prickly ash (Zanthoxylum) pericarps have various potential biological functions with considerable relevance to food, pharmacological, and industrial applications. The volatile profile of oils extracted from prickly ash pericarps obtained from 72 plantations in China was determined by gas chromatography and mass spectrometry. Several chemometric analyses were used to better understand the volatile oil profile differences among different pericarps and to determine the key factors that affected geographical variations in the main volatile constituents of oils. A total of 47 constituents were detected with D-limonene, alfa-myrcene, and linalool as the most abundant. The volatile profile of pericarp oils was significantly affected by prickly ash species and some environmental factors, and the key factors that affected volatile profile variations for different prickly ash species were diverse. Chemometric analyses based on the volatile oil profile could properly distinguish Z. armatum pericarps from other pericarps. This study provides comprehensive information on the volatile oil profile of pericarps from different prickly ash species and different plantations, and it can be beneficial to a system for evaluating of pericarp quality. Moreover, this study speculates on the key environmental factors that cause volatile oil variations for each species, and can help to obtain better prickly ash pericarp volatile oils by improving the cultivated environments.

Zanthoxylum bungeanum (Rutaceae), a popular food flavoring and traditional Chinese medicine ingredient, is an important cash crop. Its leaves are rich in flavonoids with multiple bioactivities. How...

Green prickly ash (Zanthoxylum armatum) and red prickly ash (Zanthoxylum bungeanum) fruit have unique flavor and aroma characteristics that affect consumers' purchasing preferences. However, differences in aroma components and relevant biosynthesis genes have not been systematically investigated in green and red prickly ash. Here, through the analysis of differentially expressed genes (DEGs), differentially abundant metabolites, and terpenoid biosynthetic pathways, we characterize the different aroma components of green and red prickly ash fruits and identify key genes in the terpenoid biosynthetic pathway. Gas chromatography-mass spectrometry (GC-MS) was used to identify 41 terpenoids from green prickly ash and 61 terpenoids from red prickly ash. Piperitone was the most abundant terpenoid in green prickly ash fruit, whereas limonene was most abundant in red prickly ash. Intergroup correlation analysis and redundancy analysis showed that HDS2, MVK2, and MVD are key genes for terpenoid synthesis in green prickly ash, whereas FDPS2 and FDPS3 play an important role in the terpenoid synthesis of red prickly ash. In summary, differences in the composition and content of terpenoids are the main factors that cause differences in the aromas of green and red prickly ash, and these differences reflect contrasting expression patterns of terpenoid synthesis genes.

Zanthoxylum plants, important aromatic plants, have attracted considerable attention in the food, pharmacological, and industrial fields because of their potential health benefits, and they are easily accessible because of the wild distribution in most parts of China. The chemical components vary with inter and intraspecific variations, ontogenic variations, and climate and soil conditions in compositions and contents. To classify the relationships between different Zanthoxylum species and to determine the key factors that influence geographical variations in the main components of the plant, the fatty acid composition and content of 72 pericarp samples from 12 cultivation regions were measured and evaluated. Four fatty acids, palmitic acid (21.33&ndash, 125.03 mg/g), oleic acid (10.66&ndash, 181.37 mg/g), linoleic acid (21.98&ndash, 305.32 mg/g), and linolenic acid (0.06&ndash, 218.84 mg/g), were the most common fatty acid components in the Zanthoxylum pericarps. Fatty acid profiling of Zanthoxylum pericarps was significantly affected by Zanthoxylum species and geographical variations. Stearic acid and oleic acid in pericarps were typical fatty acids that distinguished Zanthoxylum species based on the result of DA. Palmitic acid, palmitoleic acid, trans-13-oleic acid, and linoleic acid were important differential indicators in distinguishing given Zanthoxylum pericarps based on the result of OPLS-DA. In different Zanthoxylum species, the geographical influence on fatty acid variations was diverse. This study provides information on how to classify the Zanthoxylum species based on pericarp fatty acid compositions and determines the key fatty acids used to classify the Zanthoxylum species.

Zanthoxylum bungeanum is an economically important tree species of edible, medicinal, and ornamental value. The trees grow in varied climates from tropical to subtropical regions owing to their high resistance to adverse environment. The unstable environmental temperature is one of the important factors affecting the yield and quality of Z. bungeanum. To investigate complex responses to cold stress in Z. bungeanum, we compared cold-tolerant “Fuguhuajiao” (FG) and cold-sensitive “Fengxiandahongpao” (FX) varieties. Physiological measurements and transcriptome sequencing were performed using plant leaves collected at six different timepoints (0, 1, 3, 6, 12, and 24 h) of 4 °C treatment. Chemometrics analysis showed that the physiological cold response in FX at late stage was similar to that in FG at early stage. Based on orthogonal partial least squares discriminant analysis (OPLS-DA), the variable important in projection (VIP) values of peroxidase (POD) and soluble protein were 1.11 and 1.41, which were identified as important physiological indices in the two varieties. Under low temperature stress, there were 3513 differentially expressed genes in FG and 25,157 in FX. Weighted gene co-expression network analysis (WGCNA) revealed that many cold-response genes and pathways were enriched in the MEfirebrick4 module which had a high correlation with POD. Hub genes identified in this module indicated that Z. bungeanum can respond and adapt to cold stress by altering signal transduction, plant hormones, transcription factors, protein modification, functional proteins, metabolism, cell structure, light response, and the circadian clock. Endocytosis-related gene ERD7, abscisic acid pathway-related gene PP2C, late embryogenesis abundant gene LEA D-29, and two plasma membrane/lipid raft-associated remorin genes ZB01477 and pp34 are located at the core of the network, and all play pivotal roles in cold responses. These results of this study highlight cold response mechanisms at the physiological and molecular levels in Z. bungeanum and will contribute to a better understanding of low temperature resistance in related plants.

Melanin is a broad term for a family of complex organic macromolecules that contain phenol or indole groups. Melanins are found in animals, plants, and microorganisms and are widely used in medicine, food, and chemical industries. However, at present, the synthesis of plant melanin is still unclear and hinders the development and utilization of melanin. To clarify the composition of plant melanin and the key synthetic genes, we observed the cytological observation of the melanin-forming tissue of ZB seeds and analyzed the transcriptome and metabolite profiles of different developmental stages. We measured seed melanin content and gene expression levels and used redundancy analysis (RDA) to identify key genes that affected melanin synthesis. The seed melanin content was 3.85 % at the green fruit stage (S2) and reached a peak of 4.33 % at the fruit maturity stage (S4). Tyrosine and hydroquinone contents were highest at S2: 830 mg/kg and 77.23 mg/kg, respectively. Catechin, L-epicatechin, procyanidin B1, and p-coumaric acid were the main flavonoids in ZB seeds and reached their highest levels at S2. RDA analysis confirmed that S2 was the key period of melanin synthesis and that ZbPPO, ZbLAR, ZbTYRP1, and ZbTYR had crucial roles in the synthesis of ZB melanin-related substances.

The WRKY family of transcription factors (TFs) includes a number of transcription-specific groupings that play important roles in plant growth and development and in plant responses to various stresses. To screen for WRKY transcription factors associated with drought stress in Zanthoxylum bungeanum, a total of 38 ZbWRKY were identified and these were then classified and identified with Arabidopsis WRKY. Using bioinformatics analyses based on the structural characteristics of the conservative domain, 38 WRKY transcription factors were identified and categorized into three groups: Groups I, II, and III. Of these, Group II can be divided into four subgroups: subgroups IIb, IIc, IId, and IIe. No ZbWRKY members of subgroup IIa were found in the sequencing data. In addition, 38 ZbWRKY were identified by real-time PCR to determine the behavior of this family of genes under drought stress. Twelve ZbWRKY transcription factors were found to be significantly upregulated under drought stress and these were identified by relative quantification. As predicted by the STRING website, the results show that the WRKYs are involved in four signaling pathways&mdash, the jasmonic acid (JA), the salicylic acid (SA), the mitogen-activated protein kinase (MAPK), and the ethylene signaling pathways. ZbWRKY33 is the most intense transcription factor in response to drought stress. We predict that WRKY33 binds directly to the ethylene synthesis precursor gene ACS6, to promote ethylene synthesis. Ethylene then binds to the ethylene activator release signal to activate a series of downstream genes for cold stress and osmotic responses. The roles of ZbWRKY transcription factors in drought stress rely on a regulatory network center on the JA signaling pathway.

Fresh Zanthoxylum bungeanum Maxim. (Z. bungeanum) easily lose its color attributes and nutritional properties during drying. This study aimed to evaluate the influences of four different drying techniques, including shade drying (SD), hot air drying (HAD), far−infrared drying (FID), and freeze drying (FD) on the quality maintenance of ‘Fengjiao’ Z. bungeanum. The color attributes, essential oil content and compositions, total alkylamides content (TALC), total anthocyanin content (TAC), total phenol content (TPC), total flavone content (TFC), and antioxidant activity of Z. bungeanum were determined. Results found that HAD caused the most reduction in its nutritional components; SD maintained higher retention of essential oil compositions, but resulted in the lower TAC, TPC, TFC, and antioxidant activity compared with FID and FD treatments. However, FD maintained the highest nutrients content and antioxidant activity of Z. bungeanum. Thus, FD is an efficient drying method that shows potential applications in maintaining the quality of Z. bungeanum.

Selective tissue factor-factor VIIa complex (TF-FVIIa) inhibitors are viewed as promising compounds for treating thrombotic disease. In this contribution, we describe multifaceted exploratory SAR studies of S1′-binding moieties within a macrocyclic chemotype aimed at replacing cyclopropyl sulfone P1′ group. Over the course of the optimization efforts, the 1-(1H-tetrazol-5-yl)cyclopropane P1′ substituent emerged as an improved alternative, offering increased metabolic stability and lower clearance, while maintaining excellent potency and selectivity.

Incorporation of a methyl group onto a macrocyclic FVIIa inhibitor improves potency 10-fold but is accompanied by atropisomerism due to restricted bond rotation in the macrocyclic structure, as demonstrated by NMR studies. We designed a conformational constraint favoring the desired atropisomer in which this methyl group interacts with the S2 pocket of FVIIa. A macrocyclic inhibitor incorporating this constraint was prepared and demonstrated by NMR to reside predominantly in the desired conformation. This modification improved potency 180-fold relative to the unsubstituted, racemic macrocycle and improved selectivity. An X-ray crystal structure of a closely related analogue in the FVIIa active site was obtained and matches the NMR and modeled conformations, confirming that this conformational constraint does indeed direct the methyl group into the S2 pocket as designed. The resulting rationally designed, conformationally stable template enables further optimization of these macrocyclic inhibitors.

Eight isobutylhydroxyamides, including three new (1-3), qinbunamides A-C, and five known sanshools (4-8), ZP-amide A (4), ZP-amide B (5), ZP-amide E (6), ZP-amide C (7), and ZP-amide D (8), were isolated from the pericarps of cultivated Zanthoxylum bungeanum Maxim, cultivated in Qinling mountain area, Shaanxi, China. The structures of all compounds were determined on the basis of spectroscopic techniques, including 1D and 2D NMR analysis and comparison with previously reported data. Compounds 1 and 2 are the first example of isobutylhydroxyamides containing an ethoxy group, and compound 3 is a rare C11 fatty acid-containing sanshool existing in genus Zanthoxylum. The tested compounds enhanced nerve growth factor (NGF)-mediated neurite outgrowth (neurotrophic activity) in rat pheochromocytoma (PC12) cells, but were inactive in the inhibitory effects on the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical and growth of HCT116 cells at concentrations of 50μM.

Zanthoxylum bungeanum is an important commercial crop of the Rutaceae family, its peel is used as a condiment and also in traditional Chinese medicine. Seeds are rich in fatty acids but are usually rejected. To investigate the feasibility of using Z. bungeanum seeds to make biodiesel, techniques including GC MS, transcriptome sequencing and RT-qPCR were used to analyse the fatty acids content and gene expression levels in seeds of five developmental stages. The results show that nine fatty acids are produced and that these accumulate during seed development, to reach the highest levels at maturity. At maturity, the levels of elaidic acid and palmitic acid are highest, each accounting for 22 % of the total. These are followed by linoleic acid, linolenic acid, oleic acid and palmitoleic acid, which account for 18, 15, 7 and 12 % of the total, respectively. The contents of saturated and unsaturated fatty acids continued to accumulate during seed development. At maturity, the content of saturated fatty acids was 46.48 mg/g and the content of unsaturated fatty acids was 142.05 mg/g. Using transcriptome sequencing, the expression levels of 20 genes related to fatty-acid synthesis were determined. A fatty acids synthesis pathway model was established based on gene function annotation information. The results of inter-group correlation analysis and RDA analysis show that ENR, ECR and SAD1 play active roles in fatty acids synthesis in Z. bungeanum and were identified as key genes for fatty acids synthesis.

Zanthoxylum bungeanum Maxim. (Z. bungeanum) is well known for its pharmaceutical properties and pungent taste due to its alkylamide compounds, among which hydroxyl-α-sanshool has the highest content. The differences in the hydroxyl-α-sanshool profiles among 12 varieties of Z. bungeanum collected from a common garden were evaluated by RP-HPLC (reversed-phase high-performance liquid chromatography), HPLC fingerprint and chemometric analyses. The results showed that the peel of You Huajiao (S12) exhibited the highest content (46.936 ± 0.122 mg/g) and Fugu Huajiao (S2) exhibited the lowest content (2.235 ± 0.077 mg/g). The twelve varieties were divided into four groups, and their scores of similarity were between 0.424 and 1. Moreover, hydroxyl-α-sanshool was further proven to be the key compound in Z. bungeanum. To explore the molecular mechanisms of hydroxyl-α-sanshool causing these differences, 6656 differentially expressed genes (DEGs) were identified from three varieties(S2, S3 Fengxian Dahongpao and S12)and showed a significant difference in hydroxyl-α-sanshool expression, as determined by transcriptome analysis, including 10 DEGs related to the biosynthesis of unsaturated fatty acids and 9 DEGs related to valine biosynthesis, which were probably involved in hydroxyl-α-sanshool biosynthesis. The results of qRT–PCR (quantitative real-time–polymerase chain reaction) analysis for 6 DEGs selected after functional annotation were equivalent to those detected by transcriptome sequencing, implying that these DEGs may be the key genes involved in hydroxyl-α-sanshool biosynthesis. The present comprehensive analysis provides, for the first time, insight into the directional biosynthesis of alkylamide compounds, the selection of key functional genes and quality breeding in Z. bungeanum for further study.

On the basis of a crystal structure of a phenylpyrrolidine lead and subsequent molecular modeling results, we designed and synthesized a novel series of macrocyclic FVIIa inhibitors. The optimal 16-membered macrocycle was 60-fold more potent than an acyclic analog. Further potency optimization by incorporation of P1' alkyl sulfone and P2 methyl groups provided a macrocycle with TF/FVIIa Ki = 1.6 nM, excellent selectivity against a panel of seven serine proteases, and FVII-deficient prothrombin time EC2x = 1.2 μM. Discovery of this potent, selective macrocyclic scaffold opens new possibilities for the development of orally bioavailable FVIIa inhibitors.

The structure-activity relationships (SAR) of six-membered ring replacements for the imidazole ring scaffold is described. This work led to the discovery of the potent and selective pyridine (S)-23 and pyridinone (±)-24 factor XIa inhibitors. SAR and X-ray crystal structure data highlight the key differences between imidazole and six-membered ring analogs.

Inhibitors of the Tissue Factor/Factor VIIa (TF-FVIIa) complex are promising novel anticoagulants that show excellent efficacy and minimal bleeding in preclinical models. On the basis of a zwitterionic phenylglycine acylsulfonamide 1, a phenylglycine benzylamide 2 was shown to possess improved permeability and oral bioavailability. Optimization of the benzylamide, guided by X-ray crystallography, led to a potent TF-FVIIa inhibitor 18i with promising oral bioavailability, but promiscuous activity in an in vitro safety panel of receptors and enzymes. Introducing an acid on the pyrrolidine ring, guided by molecular modeling, resulted in highly potent, selective, and efficacious TF-FVIIa inhibitors with clean in vitro safety profile. The pyrrolidine acid 20 showed a moderate clearance, low volume of distribution, and a short t 1/2 in dog PK studies.

Aminoisoquinoline and isoquinoline groups have successfully replaced the more basic P1 benzamidine group of an acylsulfonamide factor VIIa inhibitor. Inhibitory activity was optimized by the identification of additional hydrophobic and hydrophilic P′ binding interactions. The molecular details of these interactions were elucidated by X-ray crystallography and molecular modeling. We also show that decreasing the basicity of the P1 group results in improved oral bioavailability in this chemotype.

A 6-amidinotetrahydroquinoline screening hit was driven to a structurally novel, potent, and selective FVIIa inhibitor through a combination of library synthesis and rational design. An efficient gram-scale synthesis of the active enantiomer BMS-593214 was developed, which required significant optimization of the key Povarov annulation. Importantly, BMS-593214 showed antithrombotic efficacy in a rabbit arterial thrombosis model. A crystal structure of BMS-593214 bound to FVIIa highlights key contacts with Asp 189, Lys 192, and the S2 pocket.

Pyridine-based Factor XIa (FXIa) inhibitor (S)-2 was optimized by modifying the P2 prime, P1, and scaffold regions. This work resulted in the discovery of the methyl N-phenyl carbamate P2 prime group which maintained FXIa activity, reduced the number of H-bond donors, and improved the physicochemical properties compared to the amino indazole P2 prime moiety. Compound (S)-17 was identified as a potent and selective FXIa inhibitor that was orally bioavailable. Replacement of the basic cyclohexyl methyl amine P1 in (S)-17 with the neutral p-chlorophenyltetrazole P1 resulted in the discovery of (S)-24 which showed a significant improvement in oral bioavailability compared to the previously reported imidazole (S)-23. Additional improvements in FXIa binding affinity, while maintaining oral bioavailability, was achieved by replacing the pyridine scaffold with either a regioisomeric pyridine or pyrimidine ring system.

A multidisciplinary, fragment-based screening approach involving protein ensemble docking and biochemical and NMR assays is described. This approach led to the discovery of several structurally diverse, neutral surrogates for cationic factor VIIa P1 groups, which are generally associated with poor pharmacokinetic (PK) properties. Among the novel factor VIIa inhibitory fragments identified were aryl halides, lactams, and heterocycles. Crystallographic structures for several bound fragments were obtained, leading to the successful design of a potent factor VIIa inhibitor with a neutral lactam P1 and improved permeability.

Heterocyclic amide isosteres were incorporated into a phenylglycine-based tissue factor/factor VIIa (TF–FVIIa) inhibitor chemotype, providing potent inhibitors. An X-ray co-crystal structure of phenylimidazole 19 suggested that an imidazole nitrogen atom effectively mimics an amide carbonyl, while the phenyl ring forms key hydrophobic interactions with the S1′ pocket. Exploration of phenylimidazole substitution led to the discovery of potent, selective and efficacious inhibitors of TF–FVIIa.

The structure of recombinant tick anticoagulant peptide (rTAP) complexed to bovine factor Xa at 3.0 A resolution reveals the structural basis for the specificity and the high affinity of rTAP. Three N-terminal residues, Tyr501, Asn502 and Arg503, play a critical role in the complex formation as suggested by earlier mutagenic studies and the ornithodorin-thrombin complex. Unexpectedly, the side-chain of Tyr501 is located in the S1 pocket, although factor Xa favors arginine as a P1 residue. Arg503 is located at the aryl binding pocket and forms a salt-bridge with Glu97 of factor Xa. The autolysis loop, which is disordered in the uninhibited factor Xa structure, is involved in the formation of the complex as a part of the secondary binding site. The C-terminal helix of rTAP interacts with factor Xa as a secondary binding determinant. The N-terminal residues of rTAP reorganize during the formation of the factor Xa-rTAP complex from the conformation found in the solution into an extended conformation. The presence of the secondary binding site confirms the proposed two-step kinetic mechanism based on the results of a mutagenesis study.

In this Letter, we describe the synthesis of several nonamidine analogs of biaryl acid factor VIIa inhibitor 1 containing weakly basic or nonbasic P1 groups. 2-Aminoisoquinoline was found to be an excellent surrogate for the benzamidine group (compound 2) wherein potent inhibition of factor VIIa is maintained relative to most other related serine proteases. In an unanticipated result, the m-benzamide P1 (compounds 21a and 21b) proved to be a viable benzamidine replacement, albeit with a 20–40 fold loss in potency against factor VIIa.

Crystals of two recombinant antichymotrypsin (rACT) variants have been prepared: variant rACT-T345R crystallizes in space group P2(1) (a = 109.2 A, b = 79.4 A, c = 111.9 A, beta = 116.3 degrees, with 2 molecules in the asymmetric unit), and variant ACT' crystallizes in space group P2(1)22(1) (a = 69.7 A, b = 77.2 A, c = 83.8 A, with one molecule in the asymmetric unit). The latter variant is an engineered dimer having the P3-P3' hexapeptide sequence of the related serpin, alpha 1-proteinase inhibitor, substituted for the corresponding wild-type sequence. Crystals of each variant diffract to a limiting resolution of 2.5 A, which represents the best diffraction yet achieved for a crystalline, inhibitory serpin. The exceptional quality of ACT' crystals probably arises from favorable protein-protein interactions as well as a stabilizing disulfide crosslink engineered between the monomers.

The complex of bovine factor Xa and recombinant tick anticoagulant peptide (rTAP) was crystallized in two different crystal forms using polyethylene glycol as a precipitant. Form I belongs to space group P42212 with unit-cell dimensions a = b = 133.1, c = 68.8 Å. It contains one complex per asymmetric unit and diffracts to 3.0 Å resolution. Form II belongs to P41212 (or P43212) with dimensions a = b = 126.5, c = 146.7 Å; it contains two complexes per asymmetric unit and diffracts to 2.5 Å. The crystals of both forms consist of factor Xa (MW = 45.3 kDa) and rTAP (MW = 6.7 kDa).

The peptidyl trifluoromethyl ketones Ac-Phe-CF3 (1) and Ac-Leu-Phe-CF3 (2) are inhibitors of chymotrypsin. They differ in Ki (20 and 2 microM, respectively) as well as in their kinetics of association with chymotrypsin in that 1 is rapidly equilibrating, with an association rate too fast to be observed by steady-state techniques, while 2 is "slow binding", as defined by Morrison and Walsh [Morrison, J. F., & Walsh, C. T. (1988) Adv. Enzymol. Relat. Areas Mol. Biol. 61, 202], with a second-order association rate constant of 750 M-1 s-1 at pH 7.0 [Imperiali, B., & Abeles, R. (1986) Biochemistry 25, 3760]. The crystallographic structures of the complexes of gamma-chymotrypsin with inhibitors 1 and 2 have been determined in order to establish whether structural or conformational differences can be found which account for different kinetic and thermodynamic properties of the two inhibitors. In both complexes, the active-site Ser 195 hydroxyl forms a covalent hemiketal adduct with the trifluoromethyl ketone moiety of the inhibitor. In both complexes, the trifluoromethyl group is partially immobilized, but differences are observed in the degree of interaction of fluorine atoms with the active-site His 57 imidazole ring, with amide nitrogen NH 193, and with other portions of the inhibitor molecule. The enhanced potency of Ac-Leu-Phe-CF3 relative to Ac-Phe-CF3 is accounted for by van der Waals interactions of the leucine side chain of the inhibitor with His 57 and Ile 99 side chains and by a hydrogen bond of the acetyl terminus with amide NH 216 of the enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)