Kinetic study of possible intermediates in the biosynthesis of chlorogenic acid in Cestrum poeppigii

p-Coumaric and 3-O-p-coumarylquinic acid seem to be important precursors of chlorogenic acid in the leaves of Cestrum poeppigii. 3-O-Cinnamylquinic acid, which has a very small metabolic activity, is of little importance in this respect. The kinetics of incorporation of radioactivity from t-cinnamic acid-3-[¹⁴C] into p-coumaric, 3-O-p-coumarylquinic, chlorogenic and 3-O-cinnamylquinic acid showed that the biosynthetic rates for these products decrease in the order shown. For p-coumaric acid, which has a markedly high metabolic activity, a turnover rate of 28 μg/hr and per gram fresh plant leaf, was calculated. Some trapping experiments with caffeic acid, and the acids mentioned above and using either t-cinnamic acid-3-[¹⁴C] or p-coumaric acid-2-[¹⁴C] as precursor, are discussed. A HPLC method for the rapid determination of phenolic acids in plant extracts, is described.     

Studies on chlorogenic acid biosynthesis in sweet potato root tissue using trans-cinnamic acid-2-14C and quinic acid-G-3H

When either trans-cinnamic acid-2-¹⁴C or quinic acid-G-³H wasadministered to sweet potato root discs, each compound was incorporatedinto chlorogenic acid. Hydrolysis analysis revealed that trans-cinnamicacid-2-¹⁴C and quinic acid-G-³H were selectively incorporatedinto the aromatic and non-aromatic moieties of chlorogenic acid,respectively. Quinic acid-G-³H was considered a more efficient precursor thantrans-cinnamic acid-2-¹⁴C, based on data of dilution values,incorporation percents and pool sizes in the tissue. No conjugatesof trans-cinnamic acid and quinic acid were detected in discsadministered trans-cinnamic acid-2-¹⁴C or quinic acid-G-³H.From these experimental results, a possible biosynthetic pathwayfor chlorogenic acid has been proposed. ¹ This paper constitutes Part 98 of the Phytopathological Chemistryof Sweet Potato with Black Rot or Injury. (Received November 2, 1971; )     

Chlorogenic acid biosynthesis in Cestrum poeppigii

The chlorogenic acid content of Cestrum poeppigii, and its ability to form the acid from labelled t-cinnamic acid, was determined at different stages of growth. In contrast to mature plants, young plants showed great seasonal variation in their chlorogenic acid content. The incorporation of radioactivity from t-cinnamic into chlorogenic acid also differed greatly during the growth period. Trapping experiments with caffeic and p-coumaric acids were performed to study the effect of large pools of these acids on the incorporation of t-cinnamic acid-3-[¹⁴C] into chlorogenic acid. The kinetics of incorporation exclude a major role for caffeic acid in the biosynthesis of chlorogenic acid.     

Isolation of 1-O-trans-p-Coumaroyl-r{beta}-D-glucopyranose from Sweet Potato Roots and Examination of Its Role in Chlorogenic Acid Biosynthesis

1-O-trans-p-Coumaroyl-rß-D-glucopyranose (p-coumaroyl-D-glucose)was isolated from slices of sweet potato root which had beenincubated with trans-cinnamic acid. Pre-loaded trans-cinnamicacid efficiently trapped the radioactivity from L-[U-¹⁴C]-phenylalanineand reduced its incorporation into chlorogenic acid by 75% ofcontrol values in disks of sweet potato root. In the root diskssupplied with trans-[3-¹⁴C]-cinnamic acid, the radioactivitywas transferred first to trans-cinnamoyl- D-glucose, then top-coumaroyl-D-glucose, and subsequently to chlorogenic acidand isochlorogenic acid. These results support our earlier propositionthat p-coumaroyl-D-glucose is involved in the biosynthesis ofchlorogenic acid as an intermediate adjacent in the pathwayto trans-cinnamoyl-D-glucose in sweet potato roots. (Received April 11, 1988; Accepted August 9, 1988)     

Turnover and metabolism of chlorogenic Acid in xanthium leaves and potato tubers

The active turnover of chlorogenic acid (3-caffeoylquinic acid(3)), a major phenolic component of Xanthium leaves and potato tuber disks, has been demonstrated in these tissues. Pulse-labelling experiments with radioactive l-phenylalanine and trans-cinnamic acid as well as direct feeding experiments with chlorogenic acid-(14)C labelled in the caffeoyl moiety have been employed in the turnover studies. The rate of turnover is calculated to be on the order of 50 to 100 mmumoles per hour per gram fresh weight of tissue.In Xanthium leaves chlorogenic acid is in part converted to an isochlorogenic acid identified by silica gel chromatography as 3,5-dicaffeoylquinic acid. Radioactivity of the caffeoyl moiety of chlorogenic acid is also incorporated into lignin-like insoluble polymers in the leaf. Turnover of chlorogenic acid in tuber tissue is largely accounted for by the incorporation of the caffeoyl moiety into insoluble polymers in the tissue.The significance of chlorogenic acid turnover is discussed in relation to the perception of the photoperiodic stimulus by leaves and to the possible role of chlorogenic acid in lignin synthesis.     

Biosynthesis of p-hydroxybenzoic acid in poplar lignin

Biosynthetic pathways to p-hydroxybenzoic acid in polar lignin were examined by tracer experiments. High incorporation of radioactivity to the acid was observed when shikimic acid-[1-¹⁴C], phenylalanine-[3-¹⁴C], trans-cinnamic acid-[3-¹⁴C], p-coumaric acid-[3-¹⁴C] and p-hydroxybenzoic acid-[COOH-¹⁴C] were administered, while incorporation was low from shikimic acid-[COOH-¹⁴C], phenylalanine-[1-¹⁴C], phenylalanine-[2-¹⁴C], tyrosine-[3-¹⁴C], benzoic acid-[COOH-¹⁴C], sodium acetate-[1-¹⁴C] and d-glucose-[U-¹⁴C]. Thus p-hydroxybenzoic acid in poplar lignin is formed mainly via the pathway: shikimic acid → phenylalanine → trans-cinnamic acid → p-coumaric acid → p-hydroxybenzoic acid.     

不同甜叶菊品种叶中绿原酸类成分的比较研究

该文以14个扦插培育的甜叶菊品种叶为材料,从8种不同型号的树脂中筛选出一种合适的大孔吸附树脂对甜叶菊叶中绿原酸类成分进行纯化前处理,采用HPLC法对不同甜叶菊品种叶中所含绿原酸类成分进行比较分析。结果表明:(1)在8种不同型号的树脂中,XAD~(-1)6对甜叶菊叶中绿原酸类成分吸附-解析性能最佳。(2)经优化,上样液浓度1.20 mg·mL~(-1)、样品溶液pH 3、解析液乙醇体积分数70%时XAD~(-1)6树脂对甜叶菊叶中绿原酸类成分具有较好的纯化效果。(3) HPLC检测分析表明,在14个品种中共检测出新绿原酸、绿原酸、隐绿原酸、异绿原酸B、异绿原酸A、异绿原酸C六种绿原酸类成分,其中主要成分均为异绿原酸A、绿原酸、异绿原酸C,而在品种3、5、13、14中没有检测出异绿原酸B。(4) 14个品种中6个绿原酸类成分的含量分别为异绿原酸A 20.55~54.3 mg·g~(-1)、绿原酸17.96~32.93 mg·g~(-1)、异绿原酸C 4.15~19.49 mg·g~(-1)、新原酸0.61~4.61 mg·g~(-1)、隐绿原酸0.52~3.11 mg·g~(-1)、异绿原酸B 0.0~3.17 mg·g~(-1),6种绿原酸类成分总量为43.9~97.8 mg·g~(-1)。可见,不同品种甜叶菊叶中绿原酸类成分含量有明显差异,富含绿原酸类成分的甜叶菊品种可用于开发获取绿原酸类物质。     

Bioavailability of chlorogenic acids following acute ingestion of coffee by humans with an ileostomy

The intestinal absorption and metabolism of 385 μmol chlorogenic acids following a single intake of 200 mL of instant coffee by human volunteers with an ileostomy was investigated. HPLC-MS³ analysis of 0-24 h post-ingestion ileal effluent revealed the presence of 274 ± 28 μmol of chlorogenic acids and their metabolites accounting for 71 ± 7% of intake. Of the compounds recovered, 78% comprised parent compounds initially present in the coffee, and 22% were metabolites including free and sulfated caffeic and ferulic acids. Over a 24 h period after ingestion of the coffee, excretion of chlorogenic acid metabolites in urine accounted for 8 ± 1% of intake, the main compounds being ferulic acid-4-O-sulfate, caffeic acid-3-O-sulfate, isoferulic acid-3-O-glucuronide and dihydrocaffeic acid-3-O-sulfate. In contrast, after drinking a similar coffee, urinary excretion by humans with an intact colon corresponded to 29 ± 4% of chlorogenic acid intake [23]. This difference was due to the excretion of higher levels of dihydroferulic acid and feruloylglycine together with sulfate and glucuronide conjugates of dihydrocaffeic and dihydroferulic acids. This highlights the importance of colonic metabolism. Comparison of the data obtained in the current study with that of Stalmach et al. [23] facilitated elucidation of the pathways involved in post-ingestion metabolism of chlorogenic acids and also helped distinguish between compounds absorbed in the small and the large intestine.     

Incorporation of some possible radioactive intermediates into chlorogenic acid in sliced sweet potato tissue

t-Cinnamic acid-2-¹⁴C, p-coumaric acid-2-¹⁴C and caffeic acid-2-¹⁴Cwere administered to discs of sweet potato roots and incorporationof each radioactive compound into chlorogenic acid was compared.The data suggest that chlorogenic acid is synthesized througheither or both of two major pathways, phenylalanine t-cinnamate t-cinnamoyl derivative p-coumaroyl derivative chlorogenicacid and phenylalanine t-cinnamate p-coumarate p-coumaroylderivative chlorogenic acid. ¹Part 75 of the phytopathological chemistry of sweet potatowith black rot and injury. ²Present address : Department of Biology, Tokyo MetropolitanUniversity, Setagaya-ku, Tokyo. (Received December 23, 1968; )     

Time-course tracer studies on the metabolism of cinnamic acid in Cestrum poeppigii

Time-course tracer studies were performed on the metabolism of trans-cinnamic acid-[3-¹⁴C] and trans-p-coumaric acid-[2-¹⁴C] in the     

Enzymic synthesis of caffeoylglucaric Acid from chlorogenic Acid and glucaric Acid by a protein preparation from tomato cotyledons

The phenylpropane metabolism of tomato (Lycopersicon esculentum Mill) cotyledons was investigated. The HPLC analysis revealed two hydroxycinnamic-acid conjugates as major components, identified as chlorogenic acid (5-O-caffeoylquinic acid) and caffeoylglucaric acid (2-O- or 5-O-caffeoyl-glucaric acid). Quantitative analyses indicated a precursor-product relationship between the chlorogenic and caffeoylglucaric acids. Protein preparations from tomato cotyledons were found to catalyze the formation of caffeoylglucaric acid with chlorogenic acid as acyl donor and free glucaric acid as acceptor molecule. This enzyme activity, possibly to be classified as hydroxycinnamoylquinic acid:glucaric acid hydroxycinnamoyltransferase, acts together with hydroxycinnamoyl-CoA: quinic acid hydroxycinnamoyltransferase.     

Labeled indole-macromolecular conjugates from growing stems supplied with labeled indoleacetic Acid : I. Fractionation

Pea (Pisum sativum var. Alaska) and bean (Phaseolus vulgaris var. Red Kidney) stem sections treated with indoleacetic acid-1-¹⁴C, indoleacetic acid-2-¹⁴C, and indoleacetic acid-5-³H were homogenized, extracted with phenol, and the water-soluble, ethanol-insoluble material subjected to further fractionation. Following an 18-hour incubation period in indoleacetic acid-1-¹⁴C, most of the label was found as nonindole-¹⁴C in high molecular weight polysaccharide, as phenol extraction is specific for both RNA and polysaccharides. With indoleacetic acid-2-¹⁴C and -5-³H, and to a lesser extent with indoleacetic acid-1-¹⁴C, radioactive indoles were obtained by hydrolysis from a heterogeneous fraction between about 500 and 30,000 molecular weight, possibly polysaccharide in nature. Indoleacetic acid accounted for 8% and indole aldehyde accounted for 21% of the total radioactivity in the extract.     

Biosynthesis of the tigloyl esters in Datura: The role of 2-methylbutyric acid

Datura innoxia plants were wick fed with (±)-2-methylbutyric acid-[1-¹⁴C] and harvested after 7 days. The root alkaloids 3α,6β-ditigloyloxytropane and 3α,6β-ditigloyloxytropan-7β-ol were isolated and degraded. In each case the radioactivity was located in the ester carbonyl group indicating that this acid is an intermediate in the biosynthesis of tiglic acid from l-isoleucine. On the other hand, (±)-2-hydroxy-2-methylbutyric acid-[1-¹⁴C], which was fed to hydroponic cultures of Datura innoxia alongside isoleucine[U-¹⁴C] positive control plants, is not an intermediate.     

Elucidation of the structure of a possible intermediate in chlorogenic acid biosynthesis in sweet potato root tissue

The structure of a possible intermediate, compound V, in chlorogenicacid biosynthesis in sweet potato root tissue was determinedas ß-1-cinnamoyl-D-glucose. The role of the compoundin chlorogenic acid biosynthesis is also discussed. ¹ This paper constitutes Part 101 of the Phytopathological Chemistryof Sweet Potato with Black Rot and Injury. (Received June 28, 1972; )     

Biosynthesis of the tigloyl esters of Datura: Cis-trans isomerism

Datura innoxia plants were wick fed with angelic acid-[1-¹⁴C] and l-isoleucine-[U-¹⁴C] to act as a positive control. After 7 days the root alkaloids 3α-tigloyloxytropane, 3α,6β-ditigloyloxytropane, and 3α,6β-ditigloyloxytropan-7β-ol were isolated and it was determined that angelic acid is not a precursor for the tigloyl moiety of these alkaloids. Tiglic acid-[1-¹⁴C] which was fed via the roots to hydroponic cultures of Datura innoxia, was incorporated to a considerable degree after 8 days.     

Metabolism of phenylpropanoids in Hydrangea serrata var. thunbergii and the biosynthesis of phyllodulcin

DL-Phenylalanine-[3-¹⁴C] and cinnamic acid-[3-¹⁴C] were fed to this plant and the label from cinnamic acid was incorporated into gallic acid, phyllodulcin and quercetin. By feeding p- coumaric acid-[U-³H], caffeic acid-[U-³H] and hydrangea glucoside A-[U-³H], it was possible to show that hydroxylation at C-3′in phyllodulcin occurs after the ring closure of dihydroisocoumarin. The biosynthetic pathway of phyllodulcin in this plant is thus: phenylalanine → cinnamic acid → p- coumaric acid → hydrangenol → phyllodulcin.     

Biosynthesis of the Actinomycin Chromophore: Incorporation of 3-Hydroxy-4-Methylanthranilic Acid into Actinomycins by Streptomyces antibioticus

Actinomycin synthesis by washed mycelia of Streptomyces antibioticus has been conducted in the presence of 3-hydroxy-4-methylanthranilate-(carboxyl-¹⁴C). Incorporation of this compound into actinomycins has been observed, which constitutes further evidence that 3-hydroxy-4-methylanthranilate is an intermediate in actinomycin biosynthesis. The position of the incorporated label has been determined to be within the actinomycin chromophore, and the label appears to be equally distributed between both halves of the chromophore. Incidental to these findings was the observation that the ¹⁴C-labeled actinomycins were subject to rapid reabsorption by the organism with actinomycin V taken up preferentially to actinomycin IV.     

Removal of the acetate-moiety of 2,4-dichlorophenoxyacetic acid in Ribes sativum

Ten minutes after uptake of 2,4-dichlorophenoxyacetic acid-1-¹⁴C(2,4-D-1-¹⁴C) by excised Ribes sativum leaves, 37·8 % of the radioactivity in water-soluble metabolites was in glyoxylic acid. When 2,4-D- 2-¹⁴C was supplied under the same conditions, 23·0 % of the radioactivity of the water-soluble rnetabolites was in glyoxylic acid. Radioactive glycine and glyoxylic acid, isolated from Ribes sativum 6 hr after uptake of 2,4-D-1-¹⁴C, contained essentially all of the ¹⁴C in the carboxyl-carbon atoms. When 2,4-D-2-¹⁴C was the precursor, the glycine isolated contained 64·8 % of its radioactivity in C₂, while 60·0 % of the radioactivity in glyoxylic acid was in C₂. The side-chain label of 2,4-D-2-¹⁴C-4-³⁶Cl was more efficiently incorporated into ethanol-insoluble plant residue than the ring-label. The metabolism of glyoxylic acid-1-¹⁴C and 2,4-D-1-¹⁴C in excised Ribes sativum leaves were compared. The data suggest a cleavage of the acetate-moiety of 2,4-D resulting in a C₂ compound, perhaps glyoxylate.     

Phenolic profiles of untransformed and hairy root cultures of Plantago lanceolata

Untransformed root cultures and Agrobacterium rhizogenes induced root cultures (hairy roots) of Plantago lanceolata were investigated for caffeic acid glycoside esters, i.e. verbascoside (V) and plantamoside (P), by HPLC. Levels of V (6–12 mg.g^–1 DW) and P (30–80 mg.g^–1 DW) from untransformed and hairy root cultures were not modified by 0.1 mM (E)-cinnamic acid addition in Murashige and Skoog's culture medium. A part of the cinnamic acid was converted into (E)-p-coumaroyl-1-O-β-D-glucopyranoside, a phenolic derivative absent from control cultures without cinnamic acid. Maximum levels of this coumaroyl ester (6–8 mg.g^–1 DW) were detected during 10 d and then slightly decreased from both root chemical profiles.     

UDPG: p-Coumarate glucosyltransferase activity in enzyme extracts from higher plants

Leaves of Coleus, Pilea, Cistus and Cestrum, and ripe tomatoes were all able to convert trans-cinnamic acid-[3-¹⁴C]into glucose esters of cinnamic acids. The pool sizes of these esters were measured by the radioisotopic dilution method, and they were found to be of the order of a few μg/g fresh plant material. 1-O-Caffeoyl-β-d-glucose in Cestrum leaves amounted to 70μg/g fresh plant material. Enzyme extracts from Cestrum leaves were able to convert trans-p-coumaric acid-[3-¹⁴C] to 1-O-p-coumaroyl-β-d- glucose, using UDPG as a source of glucose. This enzyme activity could be measured only by trapping techniques, due to the presence of considerable hydrolase activity in crude enzyme extracts.