Quantitative Extraction of p-Coumaric Acid and Ferulic Acid in Different Gramineous Materials and Structural Changes of Residual Alkali Lignin

Ferulic acid(FA)and p-coumaric acid(pCA)in bagasse,wheat straw,corn straw,and corncob were extracted by alkaline hydrolysis and characterized by gas chromatography(GC)and gas chromatography-mass spectrometry(GC-MS).It was found that the FA and most of the pCA in gramineous biomass could be dissociated and released after being treated with 1 M NaOH at 100℃for 4 h.The yields of pCA/FA in bagasse,wheat straw,corn straw,and corncob determined by GC-FID are 39.8/11.5,13.7/11.0,28.0/11.0,and 35.1/14.5 mg/g,respectively.The raw materials and the treated solid residues were characterized by gel-state 2D Heteronuclear Single Quantum Coherence Nuclear Magnetic Resonance(2D HSQC NMR).It was found that only a small amount of lignin was detected in the residue after alkali treatment,indicating that the alkali treatment conditions can effectively cleave the FA and pCA.Additionally,the lignin in the alkali solution was recovered and characterized by 2D HSQC NMR.The FA was not able to be detected by NMR,whereas a small amount of pCA remained in the alkali lignin.This study reveals the structural change of residual lignins during the quantitative isolation of FA and pCA,which is essential for the selective isolation of pCA/FA and valorization of residual alkali lignin.

Events associated with apoptotic effect of p-Coumaric acid in HCT-15 colon cancer cells

AIM:To investigate the events associated with the apoptotic effect of p-Coumaric acid,one of the phenolic components of honey,in human colorectal carcinoma(HCT-15)cells.METHODS:3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltertazolium-bromide assay was performed to determine the antiproliferative effect of p-Coumaric acid against colon cancer cells.Colony forming assay was conducted to quantify the colony inhibition in HCT15 and HT 29 colon cancer cells after p-Coumaric acid treatment.Propidium Iodide staining of the HCT15 cells using flow cytometry was done to study the changes in the cell cycle of treated cells.Identification of apoptosis was done using scanning electron microscope and photomicrograph evaluation of HCT 15cells after exposing to p-Coumaric acid.Levels of reactive oxygen species(ROS)of HCT 15 cells exposed to p-Coumaric acid was evaluated using 2’,7’-dichlorfluorescein-diacetate.Mitochondrial membrane potential of HCT-15 was assessed using rhodamine-123 with the help of flow cytometry.Lipid layer breaks associated with p-Coumaric acid treatment was quantified using the dye merocyanine 540.Apoptosis was confirmed and quantified using flow cytometric analysis of HCT15 cells subjected to p-Coumaric acid treatment after staining with YO-PRO-1.RESULTS:Antiproliferative test showed p-Coumaric acid has an inhibitory effect on HCT 15 and HT 29 cells with an IC50(concentration for 50%inhibition)value of 1400 and 1600μmol/L respectively.Colony forming assay revealed the time-dependent inhibition of HCT 15 and HT 29 cells subjected to p-Coumaric acid treatment.Propidium iodide staining of treated HCT 15cells showed increasing accumulation of apoptotic cells(37.45±1.98 vs 1.07±1.01)at sub-G1phase of the cell cycle after p-Coumaric acid treatment.HCT-15 cells observed with photomicrograph and scanning electron microscope showed the signs of apoptosis like blebbing and shrinkage after p-Coumaric acid exposure.Evaluation of the lipid layer showed increasing lipid layer breaks was associated with the growth inhibition of p-Coumaric acid.A fall in mitochondrial membrane potential and increasing ROS generation was observed in the p-Coumaric acid treated cells.Further apoptosis evaluated by YO-PRO-1 staining also showed the timedependent increase of apoptotic cells after treatment.CONCLUSION:These results depicted that p-Coumaric acid inhibited the growth of colon cancer cells by inducing apoptosis through ROS-mitochondrial pathway.

p-Coumaric acid ameliorates ethanol-induced kidney injury by inhibiting inflammatory cytokine production and NF-κB signaling in rats

Objective: To examine the effects of p-coumaric acid on ethanol-induced kidney injury in Swiss Wistar rats.Methods: Ethanol(25% v/v) was used to induce nephrotoxicity in rats. p-Coumaric acid was orally administered at 50, 100, or 200 mg/kg body weight. The levels of oxidative parameters were determined; pro-inflammatory biomarkers were analyzed by Western blotting and apoptotic protein was analyzed by immunohistochemistry. Results: Ethanol treated rats showed decreased levels of antioxidants and aberrant production of pro-inflammatory cytokines(IL-6, IL1β, TNF-α), NF-κB activation and imbalance of proand anti-apoptotic proteins(Bcl-2, Bax, caspase 3). Meanwhile, p-coumaric acid restored antioxidant levels and decreased the levels of inflammatory cytokines, NF-κB, and proapoptotic proteins and increased Bcl-2 expression. Conclusions: p-Coumaric acid ameliorates ethanol-induced kidney injury by restoring antioxidant production and suppressing cellular apoptosis and inhibiting NF-κB expression.p-Coumaric acid should be further investigated as a promising candidate for ethanol-induced kidney toxicity.

p-Coumaric acid alleviates adriamycin-induced hepatotoxicity in rats

Objective:To evaluate the effect of p-coumaric acid against adriamycin-induced hepatotoxicity in rats.Methods:The rats were divided into 4 groups.The control group received solvent;the p-coumaric acid group was treated with 100 mg/kg of p-coumaric acid orally for five consecutive days;the adriamycin group was administered with a single dose of adriamycin(15 mg/kg,i.p.),and the p-coumaric acid+adriamycin group was given p-coumaric acid five days before adriamycin administration.Twenty-four hours after the last administration,blood samples were collected for biochemical analysis,and liver tissues were removed for histopathological and immunohistochemistrical studies.Moreover,the levels of tissue lipid peroxidation and enzyme activities of glutathione peroxidase,superoxide dismutase,and catalase in liver tissue were measured.Results:Treatment with p-coumaric acid protected the liver from the toxicity of adriamycin by attenuating the increase in alkaline phosphatase,alanine transaminase,aspartate transaminase,total bilirubin,total cholesterol,triglyceride,and low-density lipoprotein cholesterol and lessening the decrease in high-density lipoprotein cholesterol and albumin.p-Coumaric acid also raised the levels of glutathione peroxidase,superoxide dismutase,and catalase,as well as decreased lipid peroxidation in liver tissue and hepatic IL-1βexpression.Additionally,histopathological study confirmed the protective effect of p-coumaric acid against liver damage.Conclusions:p-Coumaric acid can alleviate adriamycin-induced hepatotoxicity.

Simultaneous determination of gallic acid and p-coumaric acid in rat plasma by UPLC-MS/MS and its application to a comparative pharmacokinetic study after oral administration of monomer compound and red wine extract

A rapid,sensitive and selective ultra high performance liquid chromatography-tandem mass spectrometry(UPLCMS/MS)method was developed and validated for simultaneous determination of gallic acid(GA)and p-coumaric acid(CA)in rat plasma.Plasma samples were extracted by methanol and separated on an ACQUITY UPLC BEH C18 column(1.7μm,100 mm×2.1 mm)using gradient elution consisting of acetonitrile–0.2%formic acid within a runtime of 4.0 min.The detection was performed in multiple reaction monitoring(MRM)mode with negative ionization.The linear range was 20–20000 ng/mL for both GA and CA,with lower limits of quantification of 20 ng/mL.Intra-day and inter-day precisions were within 5.4%and 10.0%,respectively and the accuracy(relative error,RE,%)was less than 7.2%and–4.9%,respectively.The mean absolute extraction recoveries of both analytes and IS from rat plasma were all more than 82.6%.The validated method was successfully applied to the comparative pharmacokinetic study of GA and CA in rat plasma after oral administration of GA and CA monomers and red wine extract,respectively.It was found that both the area under the curve(AUC)and t1/2 of the two constituents were remarkably increased for red wine extract group than that in monomer group,indicating the priority of intake of red wine to active component monomer.

Impact of Expressing p-Coumaryl Transferase in Medicago sativa L. on Cell Wall Chemistry and Digestibility

The addition of p-coumaric acid (pCA) to lignin molecules is frequently found in members of the grass family. The role of this addition is not clearly understood, but is thought to potentially aid in the formation of syringyl-type lignin. This is because the incorporation is as a conjugate of pCA ester linked to sinapyl alcohol, a major component of lignin. The forage legume alfalfa (Medicago sativa L.) does not contain appreciable levels of pCA in its more heavily lignified stem tissues. The maize p-coumaryltransferase (pCAT) gene was used to transform alfalfa to determine its impact upon lignin composition and its potential to alter cell wall digestibility. A constitutive expression vector using the cassava vein mosaic virus (CsVMV) promoter was used to drive expression of maize pCAT in alfalfa. Expression of the pCAT transgene was detected in both leaves and stems. Though there was a range of pCAconcentration in transformed alfalfa stems (0.2 - 1.79 micrograms (μg)), this was a clear increase over bound pCA in control stems (0.15 - 0.2 mean = 0.17 micrograms (μg)). This did not lead to consistent responses concerning total lignin in the stem tissues. Leaf tissue, on the other hand, already has a relatively high level of pCA (0.85 - 1.2, mean = 0.99 micrograms (μg)) and those expressing pCAT gene showed on average a small increase, but there is a wide range of values among the transformants (0.38 - 1.55, mean = 1.06 micrograms (μg)). Lignin in leaves did not appear to be significantly impacted. However, incorporation of pCA into the wall appears to cause a shift in lignin composition. Testing the pCAT expressing stem cell walls for digestibility using a rumen in vitro system showed there was no change in the digestibility of the stem compared to empty vectors and control alfalfa stems. Although expression of pCAT gene in alfalfa changes the amount of wall bound pCA, it does not appear to change lignin levels or impact digestibility.

Induced Effects of Exogenous Phenolic Acids on Allelopathy of a Wild Rice Accession(Oryza longistaminata,S37)

Four exogenous phenolic acids, including salicylic acid, fumalic acid, p-coumaric acid and p-hydroxybenzonic acid, were used to investigate the regulatory effects on allelopathy of a wild rice accession of S37 （Oryza Iongistaminata）, which is a known allelopathic rice. The four exogenous phenolic acids induced the enhancement of the allelopathic potential of wild rice S37 in target weeds though the weed-suppressive activities were low, and the inducible effects were dependent on the specific phenolic acid, concentration and treatment time. After foliar application of exogenous phenolic acids, the inhibition rates for plant height, root length and fresh weight of barnyard grass （Echinochioa crus-galli） were significantly higher than those of the control. Especially at the concentration of 100 mg/L, the inhibition rates for plant height and fresh weight of barnyard grass by fumalic acid were 38.12% and 26.31% higher than those of the control, showing that fumalic acid was more effective compared with other phenolic acids in inhibiting monocotyledon weed growth. Furthermore, the weedsuppressive activity of aqueous extract from the leaves of wild rice S37 treated with exogenous phenolic acids was increased, and it peaked at 48 h after the treatment with the aqueous extract, and then gradually declined.

Comparison among Cornelian Cherry and <i>Prunus cerasus</i>According to Phenolic Content and Antioxidant Capacity by Three Various Methods of Extraction

Cornelian cherry and Prunus cerasus with red pigments possess precious source of flavonoids and phenolic acids which have various applications in treatment of various health problems. This study is conducted to compare different methods of extraction (shaking incubator, soxhelet, ultrasonic) were applied in order to identify the best method which shows the highest rate of antioxidant capacity by DPPH and ferric reducing antioxidant power (FRAP) methods and total phenolic compounds via Folin-Ciocalteu procedure, p-coumaric acid content of fruits were evaluated by high performance liquid chromatography (HPLC). As a result, cornelian cherry with 1313.13 mg/Kg average TPC score exhibits higher total phenolic content than Prunus cerasus with 1270 mg/Kg. It’s notice worthy that there was a slight difference among antioxidant activity in two fruits. Consequently, DPPH revealed nearly stronger antioxidant activity for Prunus cerasus while cornelian cherry had a little more potent antioxidant activity according to FRAP Test. p-coumaric acid content was almost twice in Prunus cerasus (10.8 mg/ml) than cornelian cherry (5.6 mg/ml). In addition, both shaking incubator and ultrasonic extraction procedures were more efficient than soxhelet in two fruits.

Changes of chlorogenic acid content and its synthesis-associated genes expression in Xuehua pear fruit during development

According to synthetic pathway of plant chlorogenic acid （CGA）, the expression patterns of genes encoding enzymes that are associated with CGA synthesis were studied in normally developed Xuehua pear fruit. The study demonstrated that CGA content in peel and flesh of Xuehua pear decreased as fruit development progressed, with a higher level in peel. The expression levels of PbPAL 1, PbPAL2, PbC3H, PbC4H, Pb4CL 1, Pb4CL2, Pb4CL6, PbHC T1 and PbHC T3 genes decreased in fruit, which was consistent with the pattern of variation in CGA content. That indicated that these genes might be key genes for influencing fruit CGA synthesis in Xuehua pear. However, Pb4CL7 gene expression profile is not consistent with variation of CGA content, hence, it may not be a key gene involved in CGA synthesis.

Mono-and dioleyl p-coumarate phenolipids and their antioxidant activity in a muscle food model system

Response surface methodology (RSM) was used to optimize the degree of esterification of p-coumaric acid to triolein via lipase-catalyzed acidolysis, and enzyme load, reaction time and mole ratio of substrates were selected as variables in the experimental design. The results showed that the model employed was highly sufficient for determining the effectiveness and interaction of three selected variables, enzyme load, reaction time and the mole ratio of substrates, on the dependent variable, the degree of esterification. Although the optimization point was not found in the selected range of the three variables, the steepest ascent analysis suggested that an increase of these three variables might lead to a stationary point. However, based on the limitations on increasing the range of tested variables, including possible oxidation of synthesized lipids and increased cost, the degree of esterification so yielded in the designed central composite design should be the one closest to the possible ideal optimized degree. The p-coumarates so produced exhibited varying antioxidant performance in the tested muscle food model, which could be explained by their different lipophilicity. Moreover, the potential health benefits of synthesized phenolic lipids have been discussed.

Molecular exposition of broad‑spectrum antibacterial efficacy by p‑coumaric acid from an edible mushroom Termitomyces heimii:in vitro and in silico approach

P-Coumaric acid was previously reported to contain antioxidant,antidiabetic,anti-inflammatory,antiplatelet,antiulcer and anticancer activities.Along with these,the present work has been conducted to study the antibacterial activity of p-coumaric acid.It could be used to control broad-spectrum microbiome-based inflammation or in cancer control.HPLC analysis of methanolic extract from the mushroom Termitomyces heimii has exhibited a rich fraction of p-coumaric acid(p-CA),which in the pure form showed significant bactericidal potentials.To predict the molecular interactions associated with the bactericidal mechanism of p-CA,the transmembrane protein sequences of Staphylococcus aureus and Escherichia coli were retrieved from the IMG–JGI database,screened and then aligned using Clustal X2 and PHYLIP 3.69 softwares.The common sequences were subjected to tertiary structure prediction using Phyre2 server,followed by quality assessment through the Ramachandran plot.Next,the 3D molecular structure of p-CA was downloaded from PubChem and docked with the selected tertiary structures using Patchdock and showed higher affinity towards 12 common transmembrane protein structures,amongst which CDP-diacylglycerol–glycerol-3-phosphate 3-phosphatidyl transferase(PgsA)exhibited best dock-ing with p-CA on the basis of ACE value(–249 kcal/mol).This fact revealed that p-CA can block the normal functioning of membrane-bound enzyme PgsA,consequently leading to the interruption in the synthesis and recycling of an essential membrane component phosphatidylglycerol(PG),resulting in membrane disruption followed by cell lysis.Here,for the first time we reported the molecular insights and fundamental biochemical events underlying the bactericidal action by p-CA,to explore new perspective to combat multidrug-resistant bacteria.

Stability and degradation of hydroxysafflor yellow A and anhydrosafflor yellow B in the Safflower injection studied by HPLC-DAD-ESI-MS^n

Safflower is a popular Chinese medicinal plant and Safflower injection is extensively used for the clinical treatment of cerebrovascular and cardiovascular diseases. In this study, HPLC-DAD-ESI-MSn was utilized to study the stability and degradation of the two major but chemically unstable bioactive compounds hydroxysaffior yellow A and anhydrosaffior yellow B, in Safflower injection. The impact of light irradiation, temperature, and pH on the stability of these two compounds were studied. The results showed that hydroxysafflor yellow A and anhydrosafflor yellow B could degrade at high temperature （〉60 ℃） or extreme pHs （pH ≤ 3.0 or 〉7.0）, but not under light irradiation. The common degradation product was p-coumaric acid. Chemical structures of the other degradation products were characterized by LC-MS. Hypothetical degradation pathways were proposed. In addition, ADP-induced platelet aggregation tests showed that the degradation of anhydrosaffior yellow B could reduce the anticoagulation activities of Safflower injection. Our results suggest that temperature and pH are critically important for the preparation and storage of Safflower injection.