Determination of Heraclenin and Heraclenol in Heracleum candicans D.C. by TLC

A simple TLC method has been developed for the simultaneous determination of heraclenin and heraclenol in the roots of Heracleum candicans D.C. The analytes were separated on silica gel F₂₅₄ plates with toluene:ethyl acetate (7:3) and scanned using densitometry at 366 nm. The method was validated in terms of precision, repeatability and accuracy. The linear range for heraclenin was found to be 4 - 10 μg per spot with correlation coefficient of 0.997 while for heraclenol it was 1–5 μg per spot with a correlation coefficient of 0.985. The two compounds were quantified in different samples of H. candicans and were found to be present in the range of 1.02 – 1.36% and 0.29 – 0.43% w/w. The method was found to be very simple, accurate, precise and economical and can be used for routine quality control.     

Quantitative determination of furocoumarins in samples of “Carapiá” by capillary gas chromatography

Summary The occurrence of furocoumarins in the Moraceae has already been demostrated. We present here the results concerning the chemical composition and quantification of furocoumarins from “carapiá” (Dorstenia species, Moraceae) employed in Brazil because of its medicinal properties against skin diseases. A capillary gas chromatographic procedure is described for the simultaneous determination of the furocoumarins (psoralen, bergapten, pimpinellin, and isopimpinellin) in rhizomes and aerial parts ofDorstenia tubicina, Dorstenia asaroides andDorstenia vitifolia and in commercial samples. The method is shown to be sensitive and reproducible, and may have application in the analysis of “carapia” crude drugs.     

Two nematicidal furocoumarins from Ficus carica L. leaves and their physiological effects on pine wood nematode (Bursaphelenchus xylophilus)

The ethanol extract of the Ficus carica L. leaves was tested to show strong nematicidal activity against pine wood nematode (PWN), Bursaphelenchus xylophilus, causing 90.93% corrected mortality within 72 h at 1.0 mg/mL. From the ethyl acetate soluble fraction of the F. carica L. leaves extract, the main nematicidal constituents were obtained by bioassay-guided isolation and identified as linear furocoumarins bergapten (1) and psoralen (2) by mass and NMR spectral data analysis. Bergapten and psoralen had significant nematicidal activity against PWN with the LC₅₀ values of 97.08 aKSnd 115.03 μg/mL within 72 h, respectively. The two furocoumarins could inhibit the activities of amylase, cellulase and acetylcholinesterase (AchE) from PWN. The morphologies of PWNs changed much after they were treated by bergapten and psoralen. The physiological effects of bergapten and psoralen on PWN might provide helpful clues to elucidate their nematicidal mechanisms.     

Overpressured Layer Chromatographic (OPLC) Separation of Closely Related Furocoumarins

Abstract

In this paper, an OPLC method for the analytical separation of eight furocoumarins is described. Of the eight compounds investigated, four are linear furocoumarins (psoralen, bergapten, 8-methoxypsoralen, iso-pimpinellin) and four are angular furocoumarins (angelicin, sphondin, iso-bergapten, pimpinellin). In preliminary experiments, optimization of the mobile phase was made using the “PRISMA” model on TLC plates in unsaturated chambers. Evaluation of the final optimization steps for OPLC separations on HPTLC plates was done densitometrically. With the elaborated OPLC system, seven compounds could be baseline separated, and the resolution between iso-bergapten and angelicin was better than 1.3. Application of the method is demonstrated with the analysis of furocoumarin-containing root extracts from Heracleum sphondylium, H. mantegazzianum and Pastinaca sativa.     

STUDIES ON THE PHOTO-C4-CYCLO-ADDITION REACTIONS BETWEEN SKIN-PHOTOSENSITIZING FUROCOUMARINS AND NUCLEIC ACIDS*

Abstract— Among the natural or synthetic furocoumarins (psoralens) a group exists which has interesting biological properties, the best known of which is skin-photosensitization. The mechanism of action has remained unclarified for a long time. Furocoumarins lack photooxidative properties; they act by a mechanism that does not require oxygen and are therefore different from photodynamic substances. Photosensitizing furocoumarins when irradiated at 365 nm react with nucleic acids to give a C₄-cyclo-addition to the 5,6-double bond of the pyrimidine bases engaging their 3,4- or 4‵,5‵-double bond. Differences exist in the behaviour of the various furocoumarins; psoralen reacts equally well with native DNA, with denatured DNA and with RNA, whereas bergapten, xanthotoxin and 8-methylpsoralen at room temperature react to a much greater extent with native DNA than with denatured DNA and with RNA. A temperature effect has also been observed. In the case of native DNA an intercalation, occurring in the dark, of furocoumarins between two adjacent base pairs of the double helix is suggested as the first step in the reaction. The photoreaction is not accompanied by breaks in the polynucleotide chain or by conformational modifications of the macromolecule. A parallelism was observed between the order of activity of the substances of this group for photoreaction with native DNA and for skin-photosensitization. Ehrlich ascites tumor cells lose completely their capacity of transmitting the tumor after irradiation in the presence of psoralen, bergapten and xanthotoxin. By hydrolysis of DNA extracted from Ehrlich ascites tumor cells irradiated in the presence of psoralen a photoadduct between psoralen and thymine was isolated.     

Candibirin A,a furano­coumarin dimer isolated from Heracleum candicans Wall.

Candibirin A [systematic name: 9,9′‐(1,4‐dioxane‐2,5‐diyldi­methyl­ene­dioxy)­di(7H‐furo­[3,2‐g]­chromen‐7‐one)], a new furano­coumarin dimer, was isolated from Heracleum candicans Wall . ¹H NMR and MS spectra had indicated that the title compound was a dimer of heraclenin or heraclenol, but the linkage structure and its chirality were undetermined. The dioxane linkage, having the R,R configuration, has now been elucidated from di­methyl sulfoxide‐solvated crystals, C₃₂H₂₈O₁₀·2C₂H₆OS. Candibirin A is thus a dimerization product from heraclenin formed by reaction at the epoxy group. Di­methyl­form­amide‐solvated crystals, C₃₂H₂₈O₁₀·C₃H₇NO, adopt a different conformation, with a folded structure that differs from the extended structure in the dimethyl sulfoxide solvate. However, the puckering of the dioxane linker unit is very similar in the two conformers. This result shows that the rotation of the ether bonds, in the linker between the furano­coumarin and dioxane moieties, causes the conformational flexibility of (I).     

High-performance thin layer chromatographic quantification of bioactive psoralen and daidzein in leaves of Ficus carica L

This study was undertaken to quantify psoralen and daidzein by high-performance thin layer chromatography (HPTLC). The methanolic extract of 10 mg mL(-1) concentration solution was prepared for HPTLC quantification of psoralen and daidzein. HPTLC aluminium-backed plates coated with 0.2 mm layers of silica gel 60 F(254) were used as the stationary phase. The working standard solution of psoralen and daidzein was applied along with the test sample solution by means of Camag Linomat IV sample applicator. R (f) values of psoralen and daidzein were found to be 0.60 and 0.88, whilst as their percentage values in methanolic extract were found to be 3.02% and 5.64% (w/w), respectively. A simple quantitative estimation method of psoralen and daidzein by HPTLC is reported that can be used for the quality control of marketed preparations containing Ficus carica. However, further study is warranted to isolate and quantify active constituents present in the leaves of F. carica by sophisticated techniques.     

In vitro and In vivo Antiproliferative Effect of a Combination of Ultraviolet‐A and Alkoxy Furocoumarins Isolated from Umbelliferae Medicinal Plants,in Melanoma Cells

We examined the effects of six furocoumarins with alkoxy groups at the C‐5 or C‐8 position isolated from Umbelliferae medicinal plants on cell proliferation, and their mechanisms of action against B16F10 melanoma cells or in melanin‐possessing hairless mice implanted with B16F10 cells, under UVA irradiation. Three furocoumarins with an alkoxy group at C‐5, isoimperatorin (1), oxypeucedanin (2) and oxypeucedanin hydrate (3), showed antiproliferative activity and caused G₂/M arrest at concentrations of 0.1–10.0 μm . Furthermore, three furocoumarins with an alkoxy group at C‐8, imperatorin (4), heraclenin (5) and heraclenol (6), inhibited the proliferation of melanoma cells and cell cycle at G₂/M at concentrations of 0.1–1.0 μm . UVA plus 1, 2, 3, 4 and 6 reduced tumor growth and final tumor weight in B16F10‐bearing mice at a dose of 0.3, 0.5 or 1.0 mg kg^?1 (intraperitoneal injection). UVA plus 1, 3 and 6 increased Chk1 phosphorylation and reduced cdc2 (Thr 161) phosphorylation in melanoma cells. We suggest that the antitumor actions of UVA plus furocoumarins with an alkoxy group at C‐5 or C‐8 were due to G₂/M arrest of the cell cycle by an increase in phosphor‐Chk1 and decrease in phospho‐cdc2.     

Simultaneous determination of urinary metabolites of methoxypsoralens in human and Umbelliferae medicines by high-performance liquid chromatography

A high-performance liquid chromatography method has been developed for the determination of coumarins and furocoumarins (psoralens). Nine coumarins and furocoumarins are separated simultaneously on a Hypersil C(8) (25 cm x 4.6-mm i.d.) column with a gradient of methanol and acetonitrile aqueous solution as mobile phase at 1.0 mL/min with two-channel UV-vis absorbance detection. The limits of detection are 0.366, 0.219, 0.317, 0.440, 0.536, 0.300, 0.531, 0.531, 0.237, and 0.280 ng/mL for coumarin, 7-hydroxycoumarin, 7-methoxycoumarin, citropten (5,7-dimethoxycoumarin), 7-ethoxy-4-methylcoumarin, psoralen, xanthotoxin (8-methoxypsoralen), bergapten (5-methoxypsoralen), isopimpinellin (5,8-dimethoxypsoralen), and imperatorin (9-isopenteneoxypsoralen), respectively. Human urine is analyzed 1-6 days after ingestion of the oral Chinese medicines. This lead to the conclusion that the concentration of coumarins and furocoumarins is higher than that of the control urine. The coumarins and furocoumarins are detected at 312 and 249 nm, respectively.     

PHOTOREACTIONS AT 3655Å BETWEEN PYRIMIDINE BASES AND SKIN-PHOTOSENSITIZING FUROCOUMARINS

Abstract After irradiation at 3655 Å of an aqueous frozen solution containing thymine and psoralen, a new photocompound was isolated by column chromatography. It contains a furocoumarin and a pyrimidine-moiety linked together by the formation of a cyclobutane ring (see formulas II and III). By irradiation at 2537 Å in acetic acid solution, the photocompound breaks up again yielding psoralen and thymine. From an aqueous frozen solution containing cytosine and psoralen irradiated at 3655 Å, an analogous photocompound was obtained, which, however, consists of the addition to psoralen of a uracil molecule, instead of a cytosine one (IV, V). It has been stated that the hydrolytic deamination of the cytosine moiety to the uracil one takes place during the working up of the photocompound in aqueous solution after irradiation. Substances with properties similar to those above were obtained from bergapten (5-methoxy-psoralen) and thymine, from psoralen and thymidine or thymidylic acid, irradiated at 3655 Å.
The new substances may be considered as model compounds in explaining the photoreactions which take place between the skin-photosensitizing furocoumarins and DNA upon irradiation at 3655 Å.     

无花果叶中有效成分鉴别及含量测定

近年来,无花果叶片中所含成分分析主要集中在同类型物质或整类物质,而对多种类型单一有效物质的定性定量研究较少。 本文采用溶剂浸提-超声波辅助法对无花果叶片中的有效成分进行了提取,利用气相色谱-质谱和超高效液相色谱-离子阱质谱对有效成分进行了鉴定。 结果表明,无花果叶中主要成分为绿原酸、芦丁、补骨脂素和佛手苷内酯。 采用单因素实验法,确定了4种成分的最佳提取工艺。 利用高效液相色谱建立了绿原酸、芦丁、补骨脂素和佛手苷内酯的定量分析方法。 绿原酸在1.84~9.2 μg范围内有良好的线性关系(R²=0.9991),回收率为95.2%;芦丁在3.4~17 μg范围内有良好的线性关系(R²=0.9981),回收率为96.8%;佛手内脂在4~20 μg范围内有良好的线性关系(R²=0.9998),回收率为89.5%;补骨脂素在4~20 μg范围内有良好的线性关系(R²=0.9963),回收率为87.1%。     

New coumarin from the roots of Prangos pabularia

The new coumarin 1, yuganin A (7-methoxy-8-((1S,2S)-1,2,3-trihydroxy-3-methylbutyl)-2H-chromen-2-one) along with nine known coumarins, heraclenol 3′-O-β-D-glucopyranoside (2), oxypeucedanin hydrate 3′-O-β-D-glucopyranoside (3), heraclenol (4), oxypeucedanin hydrate (5), osthole (6), oxypeucedanin (7), heraclenin (8), isoimperatorin (9), imperatorin (10) and the disaccharide sucrose (11), have been isolated from the roots of Prangos pabularia, and the structures of these isolated compounds were elucidated by spectroscopic means, especially, UV, HR-ESIMS, and 1D and 2D NMR spectroscopy. Furthermore, the anti-melanogenic effect of yuganin A and its inhibitory effect on B16 cells were evaluated. Yuganin A may be useful in the treatment of hyperpigmentation and as a skin-whitening agent in the cosmetics industry.     

Isopimpinellin Is Not Phototoxic in a Chick Skin Assay

Abstract— Synthetic isopimpinellin (5,8-dimethoxypsoralen), confirmed to contain as impurities only trace quantities at most of psoralen, bergapten (5-methoxypsoralen) and xanthotoxin (8-methoxypsoralen), is not phototoxic when tested in a chick skin bioassay system. These findings are at variance with earlier studies showing isopimpinellin to be phototoxic against chick skin and support the conclusion that isopimpinellin is photobiologically inactive. As recently proposed by others, the several reports of isopimpinellin photoactivity are most likely attributable to contamination by small amounts of highly active psoralens such as bergapten or xanthotoxin.     

A Reverse Phase Technique for Separating the Linearc Furano Coumarins in Celery

Abstract

A reverse phase method for separating the four linear furanocoumarins psoralen, xanthotoxin, bergapten, and isopimpinellin in celery is described. The photosensitizing compounds, psoralen, xanthotoxin, and bergapten were also quantified in celery blades and leaves. The method utilizes a phenyl-bonded HPLC column with a methanol-water solvent system. This system allows the use of a detection wavelength (225 nm) more appropriate for these linear furanocoumarins than is currently used with normal phase systems.     

Elemental analysis of brahmi (<Emphasis Type="Italic">Bacopa</Emphasis> <Emphasis Type="Italic">monnieri</Emphasis>) extracts by neutron activation and its bioassay for antioxidant,radio protective and anti-lipid peroxidation activity

Brahmi (Bacopa monnieri) leaves, known as nervine tonic in Ayurveda, and its aqueous (BA), methanolic (BM) and aqueous–methanolic (BAM) extracts were analyzed for 7 minor (Al, Fe, Na, K, Ca, P, Cl) and 18 trace (As, Au, Ba, Br, Co, Cr, Cu, Hf, Hg, La, Mn, Rb, Se, Sm, Sr, Th, V, Zn) elements by INAA. BAM extract showed maximum contents of Na, K, Cl and significant amounts of Mn, Co, Zn. It was also found as effective scavenger of DPPH radicals with 33.5% total phenolic content, highest γ-ray radioprotective effect and higher anti lipid peroxidation activity.     

FUROCOUMARIN SENSITIZATION INDUCES DNA-PROTEIN CROSS-LINKS

The capacity of some linear and angular furocoumarins to induce DNA-protein cross-links by UVA (320–400 nm) irradiation has been evaluated in Chinese hamster ovary cells. Two linear furocoumarins, psoralen and 8-methoxypsoralen appeared to be capable of inducing DNA-protein cross-links to a noticeable extent. 4'-Methylangelicin and 4,4'-dimethylangelicin formed only reduced amounts of DNA-protein cross-links, while angelicin and 4,6,4'-trimethylangelicin seemed to be unable to induce significant levels of this lesion. The biological significance of this damage remains to be elucidated, but it might have an important role in furocoumarin sensitization. In the examined compounds, the capacity for inducing DNA-protein cross-links appears to be a property of the skin phototoxic furocoumarins. This result suggests the hypothesis of a connection between this damage and the formation of skin erythemas.     

Molecular mechanics and dynamics of DNA-furocoumarin complexes: effect of the aromatization of the pyrone ring on the intercalation geometry

Results of molecular mechanics and dynamics calculations on intercalation complexes of DNA with various furocoumarins (psoralen, angelicin, 7-methylpyrido[3,4-c]psoralen and 7-methylpyrido[4,3-c]psoralen) and their corresponding aromatized derivatives are presented. These calculations were undertaken with the aim to elucidate the roles of the pyrone and pyridine moieties in the interactions which tend to orient the furocoumarins and pyridopsoralens between DNA base pairs. It appears that the intercalation geometries are very similar for the furocoumarins and related aromatized compounds. Therefore the oxygen and nitrogen atoms of the pyrone and pyridine moieties are not important in the orientation of the drug within the oligonucleotide.     

PHOTOINACTIVATION OF ENZYMES BY LINEAR AND ANGULAR FUROCOUMARINS

Abstract Furocoumarins with linear (psoralen, 8-methylpsoralen, 8-methoxypsoralen and 3-carbethox-ypsoralen) and angular molecular structures (angelicin and 4,5'-dimethylangelicin) were found to inactivate enzymes to different extents through UV-A irradiation. Moreover, enzymes with different structures (glutamate dehydrogenase, lysozyme, 6-phosphogluconate dehydrogenase, enolase, thermoly-sine and ribonuclease) are inactivated to different extents by the same furocoumarin. UV-A irradiation produces both covalent incorporation of the furocoumarins into the protein molecule and photodegra-dation of amino acid residues; the latter phenomenon seems to be mainly responsible for the photoinactivation process. A close correlation was found between the capacity of the furocoumarins to photoinactivate enzymes and their capacity to modify free amino acids.
A study of the effects of quenchers of various forms of activated oxygen on the photoinactivation of glutamate dehydrogenase, used as a model enzyme, and psoralen and 8-methylpsoralen as a reference for furocoumarins, showed that singlet oxygen is the species most involved in the photoinactivation process.     

Furocoumarins photolysis products induce differentiation of human erythroid cells

Psoralens, also known as furocoumarins, are a well-known class of photosensitizers largely used in the therapy of various skin disease. In this study we have evaluated the effects of crude pre-irradiated solutions of furocoumarins derivatives on (a) erythroid differentiation and apoptosis of human leukemic K562 cells and (b) hemoglobin synthesis in cultures of human erythroid progenitors derived from the peripheral blood. To prove the activity of a mixture of photoproducts generated by UVA irradiation of the three psoralen derivatives 5-methoxypsoralen (5-MOP) 8-methoxypsoralen (8-MOP), and angelicin (ANG), we employed the human leukemic K562 cell line and the two-phase liquid culture procedure for growing erythroid progenitors. The results obtained demonstrate that pre-irradiated solutions of psoralen derivatives significantly induce erythroid differentiation of K562 cells irrespective of the type of derivative used, suggesting that the active photoproduct(s) share a common structure. Interestingly, solutions of psoralens irradiated in anaerobic conditions do not exhibits erythroid inducing ability, indicating that the effect is mostly due to photooxidized psoralen products. In erythroid precursor cells, psoralens photolysis products stimulates at low concentrations an increase of hemoglobin A and hemoglobin F. Altogether, these data suggest that photoproducts of psoralen warrant further evaluation as potential therapeutic drugs in beta-thalassaemia and sickle cell anaemia.     

SKIN-PHOTOSENSITIZING FUROCOUMARINS: PHOTOCHEMICAL INTERACTION BETWEEN DNA AND —O14CH3 BERGAPTEN (5-METHOXY-PSORALEN)

Abstract— For some years the mechanism of the photosensitizing effects displayed by some furocoumarins on various biological substrates (human and guinea-pig skin, bacteria cultures, mammalian cells adapted to in vitro growth, viruses) have been studied. Recently it has been pointed out that a photoreaction occurs between the photosensitizing furocoumarins and DNA after irradiation at 3655 Å. By use of a labeled furocoumarin, i.e.—O¹⁴CH₃ bergapten or 5-methoxy-psoralen, this has been confirmed and more extensively studied. During the irradiation a stable combination of the furocoumarin with native DNA takes place with a quantum yield of 5·2 × 10^-3. It is probable that the reactive sites of DNA are the pyrimidine bases. Yeast-RNA and the same DNA after heat-denaturation or in the presence of high NaCl concentration photoreact at a much reduced rate. This photoreaction may explain some various biological photosensitizing effects produced by furocoumarins.