Phenolic acid derivatives with potential anticancer properties--a structure-activity relationship study. Part 1: methyl, propyl and octyl esters of caffeic and gallic acids

The antiproliferative and cytotoxic properties of polyphenolic acid derivatives, structurally related with the natural models caffeic and gallic acids, have been tested in human cervix adenocarcinoma cells (HeLa). Simultaneous structural information was obtained for these compounds through theoretical ab initio methods. This study was conducted for the following esters: methyl caffeate (MC, 1), propyl caffeate (PC, 2), octyl caffeate (OC, 3), methyl gallate (MG, 4), propyl gallate (PG, 5) and octyl gallate (OG, 6). A significant growth-inhibition effect was assessed for some of these compounds, clearly dependent on their structural characteristics. Marked structure-activity relationships (SARs)--namely the number of hydroxyl ring substituents--were found to rule the biological effect of such systems.     

Histological study of cell death in digital malformations induced by 5-azacytidine: suppressive effect of caffeine

The present study investigated microscopically the process of 5-azacytidine (5-AC)-induced digital teratogenesis and caffeine's suppressive effect on this process. Three distinct zones of programmed cell death were observed in control and caffeine-treated embryos 3 hours after 5-AC injection: the preaxial and postaxial ectodermal regions and the central part of the mesodermal regions. 5-AC temporarily suppressed programmed cell death in the ectoderm and mesoderm 3 hours after it was injected. However, caffeine promoted programmed cell death; normal programmed cell death was observed in the limb buds of embryos whose dams were treated with 5-AC and caffeine. The percentage of total cell death in hindlimb buds of embryos treated with 5-AC and caffeine was higher than that from embryos treated with 5-AC, whereas 5-AC-induced digital malformations were reduced by post-treatment with caffeine. Cell death reached a maximum 12 hours after the injection in limb buds from 5-AC and caffeine-treated embryos and at 24 hours in the 5-AC treated embryos. Furthermore, in the 5-AC and caffeine-treated embryos, the frequency of cell deaths at 12 hours increased almost linearly with the doses of caffeine in parallel with the reduction of 5-AC-induced malformation frequency by caffeine. These results suggest that although induced cell death may be one of the factors leading to digital malformations produced by 5-AC, it is not essential, and the existence of other factors affecting the pattern formation of the limb bud is proposed.     

Changes in biological production of the cyanobacterium, Nostoc sp. strain MAC, under subinhibitory concentrations of tannic acid and related compounds

The effects of gallic acid, methyl gallate, propyl gallate and tannic acid on cell growth, protein synthesis, photosynthesis, membrane function and metabolic activity of Nostoc sp. strain MAC were quantitatively investigated. Treatment of MAC with 1/2 inhibitory concentrations of tannic acid and related compounds resulted in a severe decline in biological production. Chlorophyll a and c-phycocyanin syntheses were inhibited by over 90%. Glutamine synthetase and nitrate reductase activities were suppressed by at least 45% and 56%, respectively. The percentage inhibition of total cell yield was around 40%, whereas that of total protein was around 80%. In addition, cellular potassium loss was 2–5 times that of control cultures and was accompanied by a loss in phosphate of about 1.2 times that of control cultures. However, gallic acid did not inhibit c-phycocyanin synthesis, nor did tannic acid or propyl gallate inhibit the activity of glutamine synthetase. Methyl gallate had no effect on electrolyte efflux. The control of biomass accumulation in relation to the production of off-flavor compounds in cyanobacteria by natural tannin compounds may have important aquacultural implications. This revised version was published online in June 2006 with corrections to the Cover Date.     

Modification of X-Ray-Induced Killing of HeLa S3 Cells by Inhibitors of DNA Synthesis

After irradiation of HeLa S3 cells with 220 kv x-rays during G1, treatment with any of six inhibitors of DNA synthesis results in the progressive enhancement of cell killing (loss of colony-forming ability). Incubation with hydroxyurea, cytosine arabinoside, or hydroxylamine reduces survival five- to twentyfold in about 8 hr, following an x-ray dose of 400 rads. In contrast, treatment with 5-fluorodeoxyuridine, deoxyadenosine, or thymidine after this same dose reduces survival less than twofold during a comparable time interval. These differences occur at drug concentrations which reduce the rate of DNA synthesis by at least 95% (except in the case of hydroxylamine, which inhibits DNA synthesis to a smaller extent), but which kill no unirradiated cells during the treatment periods. When inhibition of DNA synthesis with either hydroxyurea or cytosine arabinoside is reversed by addition of appropriate precursors of DNA, the enhancement is abolished. With hydroxyurea, the rate of cell killing is dependent on the dose of x-rays previously administered, and the extent of enhancement seems to be related to the drug concentration. Imposition of a delay between irradiation and addition of hydroxyurea does not abolish the enhancement effect, but instead causes a proportional lag in its inception. Postirradiation treatment of S phase cells with either hydroxyurea or cytosine arabinoside also enhances killing. Furthermore, unlike early G1 cells, S cells (and, as shown previously, cells blocked at the G1-S transition) are sensitized by preirradiation exposure to hydroxyurea.     

The distal boundary of myogenic primordia in chimeric avian limb buds and its relation to an accessible population of cartilage progenitor cells

Using chimeras consisting of chick embryos that had received substitution grafts of quail somites, we have determined the distalmost extension of the myogenic primordia in the outgrowing wing bud at 5 days of incubation. At Hamburger-Hamilton stage 25 the most distal premuscle cell is consistently 300 mum or more from the apex of the wing mesoblast. The stage 25 wing tip resembles very early whole limb buds in not having proceeded beyond the mesenchymal state or having expressed markers of terminal differentiation. However, unlike early whole limb buds it is free of a myogenic subpopulation. We therefore propose that the stage 25 wing tip is the appropriate system for in vitro and molecular studies of cartilage differentiation.     

没食子酸丙酯和没食子酸异丁酯对人红细胞膜结构与功能的影响

本文应用荧光探剂ANS(1—苯胺—8萘磺酸)、NPN(N—苯基—1—萘胺)和DPH(1.6—二苯基—1.3.5—已三烯)观察没食子酸丙醋和没食子酸异丁酯对人红细胞膜流动性和相变温度以及Na~ -K~ ATP酶活性的影响.实验结果指出该两种化合物均能:(1)降低与膜结合的荧光探剂强度但不改变探剂在水相与膜相的分配比例:(2)降低膜脂的相变温度,增加膜的流动性;(3)抑制红细胞膜Na~ -K~ ATP酶活性;(4)标记红细胞膜的DPH偏振度随化合物浓度的增加而降低,膜的流动性增加.在给定的浓度范围内,两种化合物的效应表现为明显的量效关系与构效关系.从上述结果推测该两种化合物可能是通过改变膜脂结构、膜蛋白的脂类环境而调节膜的功能,成为其治疗疾病的机理之一.     

Overexpression of S-adenosylmethionine decarboxylase.（SAMDC） in Xeno-pus embryos activates maternal program of apoptosm as a “fail-safe”mechanism of early embryogenesis

In Xenopus, injection of S-adenosylmethionine decarboxylase (SAMDC) mRNA into fertilized eggs or 2-cell stage embryos induces massive cell dissociation and embryo-lysis at the early gastrula stage due toactivation of the maternal program of apoptosis. We injected SAMDC mRNA into only one of the animalside blastomeres of embryos at different stages of cleavage, and examined the timing of the onset of theapoptotic reaction. In the injection at 4-and 8-cell stages, a considerable number of embryos developed intotadpoles and in the injection at 16-and 32-cell stages, all the embryos became tadpoles, although tadpolesobtained were sometimes abnormal. However, using GFP as a lineage tracer, we found that descendant cellsof the blastomere injected with SAMDC mRNA at 8-to 32-cell stages are confined within the blastocoel atthe early gastrula stage and undergo apoptotic cell death within the blastocoel, in spite of the continued development of the injected embryos. These results indicate that cells overexpressed with SAMDC undergo apoptotic cell death consistently at the early gastrula stage, irrespective of the timing of the mRNA injection.We assume that apoptosis is executed in Xenopus early gastrulae as a “fall-safe“ mechanism to eliminate physiologically-severely damaged cells to save the rest of the embryo.     

Overexpression of S-adenosylmethionine decarboxylase (SAMDC) in Xeno-pus embryos activates maternal program of apoptosis as a "fail-safe" mechanism of early embryogenesis

In Xenopus, injection of S-adenosylmethionine decarboxylase (SAMDC) mRNA into fertilized eggs or2-cell stage embryos induces massive cell dissociation and embryo-lysis at the early gastrula stage due toactivation of the maternal program of apoptosis. We injected SAMDC mRNA into only one of the animal side blastomeres of embryos at different stages of cleavage, and examined the timing of the onset of theapoptotic reaction. In the injection at 4- and 8-cell stages, a considerable number of embryos developed intotadpoles and in the injection at 16- and 32-cell stages, all the embryos became tadpoles, although tadpolesobtained were sometimes abnormal. However, using GFP as a lineage tracer, we found that descendant cellsof the blastomere injected with SAMDC mRNA at 8- to 32-cell stages are confined within the blastocoel atthe early gastrula stage and undergo apoptotic cell death within the blastocoel, in spite of the continueddevelopment of the injected embryos. These results indicate that cells overexpress     

"Chemical surgery" as an approach to study morphogenetic events in embryonic mouse limb

Cytosine arabinoside (Ara-C) or retinoic acid (RA) was injected into pregnant mice in doses which induce a high incidence of limb defects. Within 4 hr of the treatment, extensive cell death was observed in the embryonic limb buds. However, the location of necrotic cells and the eventual limb defects were different for the two chemicals. Ara-C killed cells in those regions of the limb which were undergoing active proliferation. RA, on the other hand, had no effect on actively dividing cells but was lethal to cells of chondrogenic lineage at stages when their proliferation rate had fallen 7- to 10-fold below the original rate. In all cases, an excellent correlation between the location of dead cells (as seen 4 hr after drug treatment) and the eventual bony defects (as seen in the term fetuses) was observed. The unique properties of Ara-C and RA have been exploited in determining the relative levels of cytodifferentiation in the embryonic mouse limb buds. It is concluded that in the limbs of early 11th day mouse embryos (comparable to chick stage 19–20), differentiation of future skeletal elements has not yet begun. However, by the 12th day (comparable to chick stage 23), cell populations destined to form most of the future cartilages (except for digits) have already been established.     

Effects of cadmium on formation of the ventral body wall in chick embryos and their prevention by zinc pretreatment.

BACKGROUND: Cadmium (Cd) is an established experimental teratogen whose effects can be reversed by pretreatment with zinc. Mesodermal development is a frequently reported target for Cd teratogenicity. The aim of this study was to examine the mechanisms of Cd induced body wall defects in chick embryos. METHODS: Chick embryos in shell-less culture were treated with 50 microl of cadmium acetate (8.9 x 10(-5) M Cd(2+)) at 60-hr incubation (H.-H. stages 16-17). Controls received equimolar sodium acetate. Other embryos were treated with various concentrations of zinc acetate and then with Cd or NaAc 1 hrs later. Development was evaluated 48 hrs later. Resin-embedded 1-microm sections were examined at earlier stages. RESULTS: Cd caused embryolethality (35%), ventral body wall defect with malpositioned lower limbs (40%), and weight reduction in survivors. After 4-hr treatment with Cd, breakdown of junctions between peridermal cells with rounding up and desquamation occurred. Shape changes were also seen in the basal layer of the ectoderm. At 4 hr, cell death was evident in lateral plate mesoderm, somites, and neuroepithelium; the lateral plate mesoderm began to grow dorsally, carrying the attached limb buds with it. Zn pretreatment protected against the lethal, teratogenic, and growth-retarding effects of Cd, as well as ectodermal changes and cell death. CONCLUSIONS: Cd disrupts peridermal cell adhesion and induces cell death in the mesoderm. This may result in abnormal growth of lateral plate mesoderm and in a body wall defect. Zn pretreatment prevents both the gross teratogenic effects and the cellular changes, most likely by competition with Cd.     

Cytotoxic effects of ethylene glycol monomethyl ether in the forelimb bud of the mouse embryo

The role of cytotoxicity in digital maldevelopment in CD-1 mouse embryos was examined following dosage with ethylene glycol monomethyl ether (EGME) on gestation day (gd) 11. Patterns of cell necrosis in the forelimb buds of embryos collected from dams given EGME orally at doses of 100, 250 or 350 mg/kg were characterized by staining with Nile blue A. Cell death was induced in the mesenchymal tissue and to some extent in the limb bud ectoderm, including the apical ectodermal ridge in a dose-related manner. The area of preaxial physiological cell necrosis was enlarged by EGME, and the shape of the limb buds was altered 24 hr after treatment. Preaxial tissue and the predigital chondrocyte condensations were reduced or missing following 250 and 350 mg EGME per 1 kg. Light and electron microscope evaluations of forelimb buds revealed the presence of phagocytic vacuoles and condensed, fragmented cytoplasm, which indicate cytotoxicity, as early as 2 hr following EGME, a maximum effect being observed 6 hr after the dose was administered. Although the severity of the cytotoxic response appeared to be dose-related, comparison with the incidence of digital malformations in near-term fetuses indicates that the loss of mesenchymal tissue is partially compensated for as formation of the limb progresses.     

Hydroxyurea and p-aminophenol are the suicide inhibitors of ascorbate peroxidase

Guaiacol peroxidase from spinach catalyzes the oxidation of p-aminophenol to produce the aminophenoxy radical as the primary product which is converted further into a stable oxidation product with an absorption peak at 470 nm. The p-aminophenol radicals oxidize ascorbate (AsA) to produce monodehydroascorbate radicals. Kinetic analysis indicates that p-aminophenol radicals also oxidize monodehydroascorbate to dehydroascorbate. Incubation of AsA peroxidase from tea leaves and hydrogen peroxide with p-aminophenol, p-cresol, hydroxyurea, or hydroxylamine results in the inactivation of the enzyme. No inactivation of the enzyme was found upon incubation of the enzyme with these compounds either in the absence of hydrogen peroxide or with the stable oxidized products of these compounds. The enzyme was protected from inactivation by the inclusion of AsA in the incubation mixture. The radicals of p-aminophenol and hydroxyurea were produced by AsA peroxidase as detected by their ESR signals. These signals disappeared upon the addition of AsA, and the signal characteristic of monodehydroascorbate was found. Thus, AsA peroxidase is inactivated by the radicals of p-aminophenol, p-cresol, hydroxyurea, and hydroxylamine which are produced by the peroxidase reaction, and it is protected from inactivation by AsA via the scavenging of the radicals. Thus, these compounds are the suicide inhibitors for AsA peroxidase. Isozyme II of AsA peroxidase, which is localized in chloroplasts, is more sensitive to these compounds than isozyme I. In contrast to AsA peroxidase, guaiacol peroxidase was not affected by these various compounds, even though each was oxidized by it and the corresponding radicals were produced.     

Structural Features Required for Inhibition of Soybean Lipoxygenase-2 by Propyl Gallate : Evidence that Lipoxygenase Activity Is Distinct from the Alternative Pathway

The ability of 19 structural analogs of propyl gallate to inhibit purified soybean seed (Glycine max [L.] Merr. var. Ransom) lipoxygenase-2 (EC 1.13.11.12) was determined. The results indicate that the o-dihydroxy and not the ester function of propyl gallate is essential for inhibition of lipoxygenase. Catechol thus represents the minimum inhibitory structure. Among those compounds possessing an o-dihydroxy function, the K_i′ for inhibition of lipoxygenase is directly related to the lipophilicity of the inhibitor as measured by the octanol-water partition coefficient. The structural features of propyl gallate necessary for inhibition of lipoxygenase were found to differ from those required for inhibition of the plant mitochondrial alternative pathway. This further supports the concept that the alternative oxidase and lipoxygenase are functionally distinct species.     

Enhanced porphyrin accumulation using dendritic derivatives of 5-aminolaevulinic acid for photodynamic therapy: an in vitro study

Intracellular porphyrin generation following administration of 5-aminolaevulinic acid has been widely used in photodynamic therapy for a range of malignant and certain non-malignant lesions. However, cellular uptake of 5-aminolaevulinic acid is limited by its hydrophilic nature and improved means of delivery are therefore being sought. Highly branched polymeric drug carriers known as dendrimers are a promising new approach to drug delivery. The aim of this study was to investigate the efficacy of dendrimers conjugated with 5-aminolaevulinic acid for porphyrin production in the transformed PAM 212 keratinocyte cell line and skin explants. Each dendritic derivative incorporated three 5-aminolaevulinic acid residues which were conjugated as esters via methyl or propyl linkers to a central tertiary carbon whose remaining terminal bore an amino, aminobenzyloxycarbonyl or nitro group. In the cell line, all compounds were more efficient at low concentrations compared to equimolar 5-aminolaevulinic acid for porphyrin production, with the most efficient incorporating the longer propyl linker. This compound was also the most lipophilic according to partition coefficient measurements. The intracellular porphyrin fluorescence levels showed good correlation with cellular phototoxicity following light exposure for all the compounds, together with minimal dark toxicity. Our findings indicate that the key factors influencing the efficacy of the dendritic derivatives are lipophilicity and steric hindrance within the dendritic structure which could restrict access to intracellular esterases for liberation of 5-aminolaevulinic acid. These findings should be taken into account in the design of larger dendrimers of 5-aminolaevulinic acid.     

Amelioration by leucovorin of methotrexate developmental toxicity in rabbits

Methotrexate (MTX) is lethal or teratogenic to embryos of all species tested. New Zealand white rabbit embryos are relatively resistant to the embryolethal effects of MTX. However, when pregnant does were injected iv with 19.2 mg MTX/kg on gestational day 12, virtually all surviving fetuses exhibited multiple malformations of the head, limbs, and trunk. MTX is a structural analogue of folic acid that competitively inhibits dihydrofolate reductase, thereby preventing formation of folinic acid and essentially stopping one carbon metabolism. One carbon metabolism is important in the synthesis of methionine, histidine, glycine, and purine bases that are required for the de novo synthesis of DNA. Presumably these metabolic effects of MTX relate directly to its mechanism of developmental toxicity. An ameliorative treatment has been tested utilizing i.v. injection of pregnant rabbits with leucovorin (LV), a close structural analogue of folinic acid (the product of the inhibited enzyme), at various times after MTX exposure. When LV was injected at times up to 24 hours after MTX fewer malformed fetuses resulted and the incidence of specific malformations was reduced. When given at times up to 20 hours after MTX administration, LV virtually eliminated the grossly apparent effects of MTX at term. In the forelimb bud, MTX increased the extracellular space surrounding limb bud mesenchymal cells within 8-10 hours; this process continued through 16 hours and remained unabated by 24 hours. Mesenchymal cell nuclei became hyperchromatic and pyknotic during this time period. By 24 hours, a moderate amount of cellular debris was observed in the mesenchymal compartment of limb buds from approximately one-third of the embryos examined. Endothelial cell nuclei of the limb bud vasculature did not exhibit the histopathological alterations observed in the mesenchymal cells. Limb buds from embryos injected with LV at times up to 6 hours after MTX were histologically normal. When LV treatment was delayed until 16 or 20 hours after MTX, mesenchymal nuclei regained normal appearance within 2 hours of treatment; further, the abnormally large intracellular space began to decrease during the next 4 hours. Cellular debris was not a prominent feature of limb buds from LV-treated embryos examined at any time. Embryos from rabbits injected with LV at 24 hours after MTX exhibited either typical MTX-induced lesions or a sequence of reparative events similar to those described for the 16 and 20 hour LV-treated embryos.(ABSTRACT TRUNCATED AT 400 WORDS)     

Tissue browning of in vitro cultures of Scots pine: Role of peroxidase and polyphenol oxidase

Callus cultures from shoot tips of mature Scots pine ( Pinus sylvestris L.) were characterized by rapid browning and an inability to regenerate. The peroxidase (POD) and polyphenol oxidase (PPO) activities and relationship to browning in such cultures were compared with embryogenic and non-embryogenic cultures of Scots pine, started from immature embryos of three different pine clones. The browning in callus cultures derived from pine buds was visible approximately after 2 weeks of culture, and continued thereafter until the callus was dark brown and poorly growing. The non-embryogenic cultures induced from immature embryos showed either light yellow coloring or browning, whereas the embryogenic cultures showed browning. POD activity increased during the first 4 weeks in callus tissue initiated from pine buds, and was significantly higher than in pine buds or cultures derived from immature embryos. The ability of cultures initiated from pine buds to oxidize catechol was notably high compared with cultures initiated from immature embryos, regardless of the time of measurement. Addition of catalase revealed that both POD and PPO were able to use catechol as substrate. An antibody raised against broad bean ( Vicia faba ) chloroplast PPO was used to recognize PPO. One polypeptide with a molecular mass of 50 kDa was detected in all pine samples on SDS-PAGE and non-denaturing PAGE. Another polypeptide with a molecular mass of 70 kDa was shown exclusively in the light-yellow non-embryogenic cultures. The results suggest that especially the high POD activities in callus tissues started from mature trees cause rapid and early browning and possibly subsequent cell death.     

Development and apoptosis of pre-implantation porcine nuclear transfer embryos activated with different combination of chemicals

Artificial activation of oocytes is a pre-requisite for successful cloning by nuclear transfer (NT). This study investigated effect of different combination of activation chemicals such as electric pulse (E), thimerosal (Thi) + dithiothreitol (DTT), 6-dimethylaminopurine (6-DMAP), or cycloheximide (CH) on the developmental ability and the frequency of apoptosis of porcine NT embryos during the culture in vitro. NT embryos activated with chemicals showed significantly higher developmental rate to blastocyst stage compared to embryos activated with E alone (21.5%-26.6% vs. 15.7%, respectively). Of chemicals, Thi + DTT supported higher development to blastocyst stage as compared to 6-DMAP or CH (26.6% vs. 21.5%-23.4%, respectively). Apoptosis of NT embryos were analyzed by using a terminal deoxynucleatidyl transferase-mediated deoxyuridine 5-triphosphate nick-end labeling (TUNEL) assay. The onset of apoptosis of embryos activated E alone was on Day 4, whereas embryos activated with chemicals showed apoptosis on Day 3 post-activation NT embryos exposed to chemicals for activation had higher frequency of apoptosis compared to that of embryos exposed to E alone from Day 3 to Day 7 during the culture. In conclusion, this study shows that chemical activation after fusion could increase not only the developmental ability of porcine NT embryos but also the mean cell number with an increased ratio of inner cell mass (ICM) to trophectoderm (TE) cells. However, the chemical activation also could increase the frequency of apoptosis and induced apoptosis earlier in porcine NT embryos.     

n‐Octyl Gallate as Inhibitor of Pyruvate Carboxylation and Lactate Gluconeogenesis

The alkyl gallates are found in several natural and industrial products. In the latter products, these compounds are added mainly for preventing oxidation. In the present work, the potencies of methyl gallate, n‐propyl gallate, n‐pentyl gallate, and n‐octyl gallate as inhibitors of pyruvate carboxylation and lactate gluconeogenesis were evaluated. Experiments were done with isolated mitochondria and the isolated perfused rat liver. The potency of the gallic acid esters as inhibitors of pyruvate carboxylation in isolated mitochondria obeyed the following decreasing sequence: n‐octyl gallate > n‐pentyl gallate > n‐propyl gallate > methyl gallate. A similar sequence of decreasing potency for lactate gluconeogenesis inhibition in the perfused liver was found in terms of the portal venous concentration. Both actions correlate with the lipophilicity of the compounds. The effects are harmful at high concentrations. At appropriate concentrations, however, octyl gallate should act therapeutically because its inhibitory action on gluconeogenesis will contribute further to its proposed antihyperglycemic effects.     

Analysis of phenolic compounds in health care products by low‐pressure liquid‐chromatography with monolithic column and chemiluminescent detection

This paper presents a new application for monolithic columns with low‐pressure chromatographic separation using an flow injection analysis configuration with chemiluminescent detection for the determination of a mixture of phenolic compounds: phloroglucinol, 2,4‐dihydroxybenzoic acid, salicylic acid, methyl paraben and n‐propyl gallate. The procedure consists of the separation of these compounds on a reverse‐phase ultra‐short monolithic column with pH 3.0 acetate buffer and 5% acetonitrile as carrier phase. The detection is based on a chemiluminescence measurement coming from Ce(IV)–Rhodamine 6G chemistry with the incorporation of two different chemiluminescent chemical conditions in the chromatographic setup in order to enhance the sensitivity for the different phenolic compounds. All separation and detection variables were optimized to propose a determination method. The analysis is performed in 280?s, with the sampling frequency being some 13 h^?1. The calibration function is a double reciprocal function obtaining good results within two orders of magnitude. The limits of detection were 8.8 × 10 ^?8 m (phloroglucinol), 2.7 × 10 ^?8 m (2,4‐dihydroxybenzoic acid); 2.3 × 10 ^?8 m (salicylic acid); 5.2 × 10 ^?8 m (methyl paraben) and 4.1 × 10 ^?6 m (n‐propyl gallate), and the relative standard deviations at a medium level of the linear range were 4.4% (phloroglucinol), 2.8% (2,4‐dihydroxybenzoic acid), 5.2% (salicylic acid), 3.6% (methyl paraben) and 6.8% (n‐propyl gallate). The method was applied and validated satisfactorily for the determination of these compounds in healthcare products, comparing the results against an HPLC reference method. Copyright © 2009 John Wiley & Sons, Ltd.