Chlorogenic Acid

Calluna vulgaris (L.) Hull (heather) is used in folk medicine for treating urinary tract disturbances and against the common cold and rheumatoid arthritis. The aim of this study was to evaluate the level of 5-caffeoylquinic (5-CQA), the most individual chlorogenic acid with the wide spectrum of pharmacological properties. Water and ethanol-water mixture were used for extraction from wild and greenhouse cultivated heather flowers. The extracts from each plant were also evaluated for the determination of total phenolic content using Folin- Ciocalteu assay and DPPH radical-scavenging activity. The 5-CQA content was higher using water-ethanol mixture in comparison to water infusion and increased in the order: wild heather < garden-violet < garden-white. Elevated temperature promotes only the water extraction yields. The extracts of C. vulgaris with high antioxidant properties may be considered as a good source of 5-CQA to be explored in pharmaceuticals, foodstuffs, feed additives and cosmetics.

Wild type E. coli expressing NtHQT and Os4CL did not produce detectable amounts of chlorogenic acid or caffeoyl-dehydroquinate after CA supplementation. In the wild type, dehydroquinate may have been rapidly converted into another downstream compound of the shikimate pathway instead of accumulating. Moreover, the expression level of ydiB , which converts dehydroqui- nate into quinate, was low. Only a small amount of quinate or dehydroquinate will be expected to be present in the cell in the absence of overexpression of ydiB [32]. ydiB and aroE mediate the conversion reaction not only from dehydroquinate to quinate but also from dehy- droshikimate to shikimate. However, aroE prefers dehy- droshikimate to dehydroquinate [22], whereas ydiB has nearly the same catalytic efficiency for dehydroshikimate and dehydroquinate [27,33]. Recent studies have shown that overexpression of ydiB does not increase shikimate,

genic acid was determined through dilution meth- od. Briefly, serial dilutions, ranging from 20.313 to 968.125 µg/ml of the chlorogenic acid were prepared volume being 30 µl. These dilute on were then applied on disc (6 mm in diameter) placed on tryptic soy agar. All the plates contain lawn of each bacterial species when A 600 reaches to 0.45 mid of log. Plates were in- cubated on above mentioned time and temperature. The ethanol used for control. Gentamycin and oxacil- lin was used as a reference compound for antibacterial activity. Antibiotics were reconstituted according to the manufacturer’s direction, filtered through a sterile 0.45 mm pore size polysulfone membrane and used on the same day.

effect of ethanol concentration on extraction yield of chlorogenic acid is shown in Figure 2(a). In the initial stage, along with the ethanol concentration increased from 50% to 70%, the extraction yield of chlorogenic acid increased rapidly; while ethanol concentration greater than 70% chlorogenic acid extraction yield was showing slow decreasing trend, and peak at 70% ethanol concentration. This is because the increase of ethanol concentra- tion leads to enhanced mass transfer dynamics, solvents and honeysuckle getting full access, and then the con- tents of chlorogenic acid dissolved increased [11]. When the ethanol concentration reached a certain level, some of chlorogenic acid was difficult to be dissolved by high concentration of ethanol, and also lead to the increase of the alcohol-soluble impurity content, resulting in a loss of chlorogenic acid content [12]. Moreover, the great- er of ethanol concentration, the more difficult to refine chlorogenic acid and it will cause wasted and the cost of production increased. Therefore, the ethanol concentration of 70% is good for the chlorogenic acid extraction. 3.2.2. Effect of Extraction Temperature on Extraction Yield of Chlorogenic Acid

The multidrug resistant E. coli strains were isolated from Urinary Tract Infected (UTI) patients and ESBL (Extended Spectrum β- Lactamase) expressing isolates were identified. The β-lactamase (βL) activity was investigated and compared for their extracellular & intracellular activity. These isolates were also been checked for the presence of different βL by PCR and found OXA, TEM and AmpC. The sequencing and mutational analysis were done for these βL. We have also projected the use of phytochemicals identified as potential antibacterials in our lab to see whether they can inhibit βL by nitrocefin assay. Automated molecular docking with all molecules was performed by using the advanced docking program AutoDock whereas the inhibitor-enzyme interactions were estimated by the Lamarckian genetic algorithm. Both, the biological and docking data suggests the potential β-lactamase inhibitory activity of phytochemicals, specifically chlorogenic acid as it is able to bind the active site pocket of OXA, TEM & AmpC and inhibit the total βL activity as performed by nitrocefin assay.

effect of chlorogenic acid isomers. The presence of seven isomers of chlorogenic acids was confirmed by LCMS (Data not shown). Earlier reports suggest that 5-Caffeoylquinic acid (5-CQA), one of the isomers of CGA, influence the glucose transport in skeletal muscle through the activation of AMPK and down-regulation of ACC [11]. 5-CQA also improves the lipid metabolism through the regulation of AMPK [12]. Our data for the first time demonstrated that CGA7 complex of chlorogenic acids exert anti-hyperlipidemic effect by promoting FFA catabolism.

YZ, a visiting staff of anesthesiology departmentt at the second affiliated hospital of Xi ’ an Jiaotong University, Shannxi Province, PR China. YZ graduated from Xi ’ an Jiaotong University and got his Ph.D degree in internal medicine. Since that time, He have been working for anesthesiology departmentt at the second affiliated hospital. His current research focuses on (1) Endoplasmic reticulum stress and Lipotoxicity (supported by the basic research plan of natural science in Shaanxi province 2014JM2 – 3034); (2) Chlorogenic Acid and hepatic protection (project supported by the supported by “ the Fundamental Research Funds for the Central Universities ” , Granted No. 08143052).; (3) Endoplasmic reticulum stress and hepatic protection (supported by the funds of our hospital. YJQN.201301).

620.7 ng/ml respectively (Table 2). Control and furosem- ide treated excretion of hippuric acid perhaps due to ben- zoic acid conjugated hippuric acid excretion [22], because generally hippuric acid is the glycine conjugate of benzoic acid. Because, hippuric acid synthesized in the liver and its production is greatly increased from benzoic acid based food substance or substances which generate benzoic acid during intermediate metabolism (e.g. polyphenols such as chlorogenic acid, quinic acid and caffeic acid) [20, 21]. Perhaps, based on the polyphenolic concentration, the hippuric acid elution was increased in the MEAE treated group urine sample, the hippuric acid excretion in the urine got increased with increase in MEAE dose in a dose depended manner. The commercial drug furosemide chemical name is (4-Chloro-N-furfuryl-5-sulfamoylan- thranilic acid 5-(Aminosulfonyl)-4-chloro-2-([2 furanyl- methyl] amino) benzoic acid) and the furosemide functional group benzoic acid conjugated with glycine may induce diuretics through hippuric acid formation, but that mechanism is still unclear.

actant cytokine involved in inflamma- tory responses, with elevated plasma levels documented in numerous condi- tions such as shock, trauma and liver al- lograft rejection (22). However, IL-6 was found to have potent antiinflammatory properties, particularly in preventing in- juries related to endotoxemia (23). Fur- thermore, IL-6 is produced by various types of lymphoid and nonlymphoid cells (24). Chlorogenic acid significantly reduced IL-6 level in septic mice, but not in a peritoneal macrophage treated by LPS. It is now evident that specifically targeting proinflammatory cytokines, such as TNF-α, IL-1β and IL-6, is not an effective treatment for sepsis because these cytokines are released early in the development of the inflammatory re- sponse, even before a patient is diag- nosed with sepsis. Recently, HMGB1 was identified as a late mediator of lethal systemic inflammation in sepsis (25). HMGB1 is released from endotoxin- stimulated macrophages in the late phase of sepsis, approximately 10 h after TNF and IL-1β have peaked. A similar delay in HMGB1 accumulation is ob- served in the serum of animals exposed to endotoxin (2). Consistent with these findings, anti-HMGB1 therapies have shown the ability to protect against sep- sis-induced mortality (26). The delayed kinetics and protective role of anti- HMGB1 therapies in vivo indicate that HMGB1 is an important therapeutic tar- get for the treatment of sepsis (13). In this study, chlorogenic acid suppressed the accumulation of serum HMGB1 in septic mice and inhibited HMGB1 re- lease from macrophages. Our results in- dicate that chlorogenic acid inhibits HMGB1 release from macrophages, at- tenuates circulating levels and improves survival in septic mice. The sources of circulating HMGB1 are secretion from monocytes and macrophages in response to LPS, TNF-α or IL-1β and release from damaged or necrotic cells (27).

The pH value of the mobile phase has an important influence on the current peaks and retention time of the analytes[35,36]. Different pH values of the mobile phase ranging from 3.4 to 5.8 were investigated in this work. As shown in Fig. 4, the adsorbed PA, CLA and CAA molecules showed different electrochemical behavior and overall retention times at different pH values. Although well- separated peaks could be obtained within the pH range tested, the mobile phase at pH 4.0 exhibited the strongest current signal and the optimal retention time. Therefore, the mobile phase consisting of methanol - 4 % acetic acid (pH 4.0) was chosen for separating the three compounds.

The aim of the present study was to characterise the effect of 5-CQA on PPAA activity 93 in vitro using potato starch as a substrate and to determine the in vitro digestibility of steam [r]

Type II diabetes (T2DM) is a metabolic disorder arising from impaired insulin action and insulin secretion, which leads to hyperglycemia and also the decimation of the body system. Acarbose, an anti-diabetic drug decreases the postprandial hyperglycemia, by slowing down the absorption of glucose through the inhibition of the carbohydrate- hydrolyzing enzyme ( α –amylase), howbeit with serious side effects. In the present study, with the aid of in silico experiment we have identified isorhamnetin from Corchorus olitorius plant to have a higher inhibitory potential on alpha-amylase (-8.5Kcal/mol) as compared with acarbose (-7.1Kcal/mol) and other compounds (caffeic acid, -6.5 Kcal/mol and chlorogenic acid, -7.3 Kcal/mol) characterized from the same plant (Corchorus olitorius). This study however corroborates the earlier wet experiment on the inhibitory properties of isorhamnetin.

It was estimated that metabolic pathway of salicylic acid rather than of chlorogenic acid is involved in the stress-induced flowering of Pharbitis nil (Japanese morning glory) plants [34]. The metabolic pathway from t-cinnamic acid to salycilic acid via benzoic acid is involved in the stress-induced flowering which can confirm also signifi- cant increasing of salicylic acid content in the stems of buckwheat plants in the phase II (beginning of flowering) compared to the phase I (formation of buds). At the same time significant increasing content of salycilic acid in the leaves and inflorescences in variant with CCC treatment in the phase II (beginning of flowering) is evidence about role of salycilic acid under plant stress conditions which could be occurred CCC treatment. Salycilic acid is an en- dogenous regulator of growth involved in a broad range of physiologic, metabolic and stress responses in plants [35]. Table 2 Content of phenolic acids in the leaves and inflorescences of buckwheat cultivar Rubra after treatment with 2% CCC

The results obtained in the present study reports the antioxidant and antimicrobial activity of A. wightii extract. Antibacterial activity of both the extract (leaf and fruit) has the significant activity against the human pathogenic organism S. aureus. The examined antimicrobial activity confirms the valuable traditional use of this herbal drug against the microbes. RP- HPLC analysis has detected four major phenolic compounds (gallic acid, ascorbic acid, chlorogenic acid, and caffeine). Both HPLC and GC–MS analysis of methanolic extract of A. wightii reveals the presence of antioxidant and antimicrobial phytochemicals. Further investigation is under conduction to explore the other poly phenolic compounds through LC-MS/MS analysis from the extract of A. wightii. Hence, we suggest that the leaves and fruits of A. wightii will be a source of natural products with potential use against pathogenic microbes in the pharmaceutical industry. Acknowledgement: The authors are grateful to UGC - Non SAP and DST-FIST, Govt of India for providing instrumental facilities. Also, we thank Dr. Ajay Kumar, Senior Technical Assistant, AIRF, JNU, New Delhi, for excellent technical GC-MS support in these studies. REFERENCES

0.8 ml/min and the absorbance of detection was 268 nm. Phenolic compound standards were: chlorogenic acid, rutin, gallic acid, caffeic acid, vanillic acid, vanillin, p-coumaric acid, and naringin dissolved in methanol, were used to identify polyphenols existing in the two extracts of Tamarix Gallica. In order to determine the peaks in HPLC, the retention times of reference standards were compared. The peak areas of the reference compounds were considered to determine the concentration of individual phenolic compounds in the extracts, and they were reported as mg/g of the extract.

Legumes are high-protein, medium-energy and micronutrient-rich food consumed in many parts of the world including Africa. This study evaluated the levels of specific phenolic compounds in three legumes. Two varieties of the common bean, (Phaseolus vulgaris L.) soybeans (Glycine max L.), and peas (Pisum sativum L.) from Rwanda were analyzed using high performance liquid chro- matography with diode array detection. The phenolic compounds were identified by comparison to the chromatographic retention times and UV spectra of known reference compounds. This study results clearly shows the presence of 11 different phenolic compounds in common beans: gallic acid, (+)-catechin, (–)-epicatechin, caffeic acid, o-coumaric acid, chlorogenic acid, quercetin, 4-hydrobenzoic acid, syringic acid, ferulic acid and vanillic acid. The concentration ranged from 0.59 to 2.27 mg/kg for epicatechin. High levels of catechin (13.5 to 57.9 mg/kg) ferulic acid (26.1 to 47.6 mg/kg) were also observed. Therefore, the results of this study show that Rwandan com- mon beans are a good source of phenolic acids in particular catechins and ferulic acid.

Docking studies revealed the interaction of the protein with the ligands, w.r.t binding energy, type of interaction and amino acids involved in interactions. Binding energy should be ideally negative. More negative the binding energy, better the binding affinity of ligand and protein (16). Table 1 & 2 give the binding energy of ligands with DHFR & DHPS proteins respectively with inhibitors viz., Chlorogenic acid, Ellagic acid, Gallic Acid, Hippuric acid and Quercetin and Standard Antibiotics viz., Clavulanic acid, Cephalosporin, Cephalosporin C, Penicillin, Sulfamethoxazole and Trimethoprim.

The following reagents were obtained from Panreac Química SL (Barcelona, Spain): gallic acid (GA), p-coumaric acid, caffeic acid, vanillic acid, ferulic acid, sinapic acid, chlorogenic acid, ellagic acid, p-hydroxybenzoic acid, protocate- chuic acid, pyrogallic acid, 3,5-dimethoxybenzoic acid, quercetin, naringenin, diethyl ether, acetic acid (glacial), anhydrous sodium sulphate and methanol. Sigma-Aldrich (Steinheim, Germany) supplied gallic acid, Folin-Ciocalteu re- agent, sodium carbonate, 2,4,6-tri (2-pyridyl)-s-triazine (TPTZ), ferric chloride hexahydrate, sodium acetate, 6-hydroxy-7,8-tetramethylchroman-2-car-carboxylic acid (Trolox), chlorogenic acid, hydrochloric acid, 2,20-Azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) diammonium salt (ABTS), sodium persulphate, sodium phosphate monobasic, sodium nitrite and aluminium chlo- ride. All reagents were of analytical grade.

The results obtained in this study demonstrate that methanol and aqueous methanol extracts of peanut leaves and roots have the ability to quench free radicals and prevent peroxides from oxidizing fluorescein. The chromatography results confirm peanut leaves and roots contain phenolic compounds. Extractions utilizing aqueous methanol more efficiently extracted phenolic compounds, which may also be correlated to an increase in antioxidant activity with the addition of water. Chlorophyll does not contribute considerably to the antioxidant activity of peanut leaves. Peanut roots grown in various regions of the country (North Carolina versus Texas) have different antioxidant activities and total phenolic content. Growing conditions such as soil type and climate are attributed to varying levels of specific antioxidant compounds in North Carolina roots as compared to Texas roots. Specific phenolic compounds do not account for all of the antioxidant activity in peanut leaves and roots, they include: caffeic acid, chlorogenic acid, ferulic acid, p-hydroxybenzoic acid, catechin, epicatechin, epicatechin gallate, gallocatechin gallate, and resveratrol. Phenolic compounds have different antioxidant abilities based on the antioxidant assay used. Reduction and quenching of free radicals by phenolic compounds is structure dependent. The total antioxidant activity attributed to the specific phenolic compounds does not equal the sum of the individual compounds due to interactions between phenolic compounds when mixed.

Time of explants collection is one of the major factors influencing the relative con- centration of the various phenolic compounds (Roussos & Pontikis, 2001a). In this study, phenolic compounds quantified by HPLC presented significant differences de- pending on the cultivars and the months. While some of the phenolic compounds in- cluding gallic acid, +(−) catechin, catechol and chlorogenic acid showed highly positive correlations with explants browning, rutin exhibited more slightly correlation with the browning. On the other hand, there were not found any correlation between browning and some phenolics such as o-coumeric acid and quercetin. Different quinones pro- duced by different phenolic compounds contribute to a different degree to the forma- tion of the brown color (Richard-Forget et al., 1992). In Olive explants, Roussos and Pontikis, 2001a found significantly positive correlations between some phenolic com- pounds (luteolin-7-glucoside, quercetin and luteolin) and the browning while they did not found any correlation between browning and some phenolics including rutin, chlorogenic acid and oleuropein.