Benzoates and in situ formed benzene in food supplements and risk assessment.

Upward trend in the use of food supplements urged the assessment of their safety. Eighty-eight liquid herbal supplements collected in Novi Sad (Serbia) in 2018 (36 samples) and 2021 (52 samples) were analysed for the presence of benzoates and sorbates (HPLC-UV) and benzene (HS-GC/MS). Benzoic acid varied from 599 to 9253 mg/kg and sorbic acid between 185 and 1658 mg/kg. The acceptable daily intake of sorbic acid was not reached, but in case of benzoic acid, it was exceeded by 5.3% of the samples. The presence of benzene was confirmed in 41.2% of benzoate preserved supplements (0.9-51.7 µg/kg). Benzene exposure revealed no health concern: maximum hazard quotients ranged from 0.39% (toddlers) to 0.84% (adolescents); minimum margins of exposure were between 35,680 (adolescents) and 77,419 (toddlers); estimates of lifetime cancer risk did not reach one extra cancer case per 100 000 persons. However, measures to mitigate benzene presence in food should be considered.

Larvicidal, antioxidant and biotoxicity assessment of (2-(((2-ethyl-2 methylhexyl)oxy)carbonyl)benzoic acid isolated from Bacillus pumilus against Aedes aegypti, Anopheles stephensi and Culex quinquefasciatus.

Mosquitoes are a vector for many dreadful diseases known for their public health concern. The continued use of synthetic insecticides against vector control has led to serious environmental impacts, human health problems, and the development of insect resistance. Hence, alternative mosquito control methods are needed to protect the environment and human health. In the present study, the bioefficacy of (2-(((2-ethyl-2 methylhexyl)oxy)carbonyl) benzoic acid isolated from Bacillus pumilus were tested against Aedes aegypti, Culex quinquefasciatus and Anopheles stephensi. The isolated bioactive compound was characterized through thin layer chromatography (TLC), UV-visible spectroscopy (UV), Fourier-transform infrared spectroscopy, nuclear magnetic resonance spectroscopy, and gas chromatography-mass spectrometry analysis. The pure compound caused a high percent mortality rate in a dose-dependent manner, the obtained values were 96, 82, 69, 50 and 34%; 86, 72, 56, 43, and 44%; 100, 90, 83, 70 and 56% against Ae. aegypti, Cx. quinquefasciatus, and An. stephensi respectively. The effective lethal concentration values (LC50) were 13.65, 14.90 and 9.64 ppm against Ae. aegypti, Cx. quinquefasciatus, An. Stephensi, respectively. The effect of (2-(((2-ethyl-2 methylhexyl)oxy)carbonyl) benzoic acid significantly increased the superoxide dismutase, catalase, α, ß esterase and Glutathione-S-transferase level after 24 h of the treatment period. The comet assay confirmed that isolated compound causes DNA damage in all tested insects. Histopathological examinations of treated larvae showed shrunken body posture, damaged epithelial cells and microvillus as compared to control organisms. The biosafety of the isolated compound was assessed against G. affinis and did not produce mortality which confirmed that the activity of the isolated compound is species specific. The current study concludes that the critical success factors of new insecticidal agent development are based on the eco-compatibility and alternative tools for the pesticide producing industry.

Chemical profiling and cytotoxic activity of 150-year old original sample of Jerusalem Balsam.

Herbal formulations have been used in ethnomedicine and pharmacy around the world for thousands of years. One of them is Jerusalem Balsam (JB), which came into use in the seventeenth century. Today, people still produce and use it regularly as prophylactic supplement. JB has been widely used in Europe since the nineteenth century, as a remedy possessing antibacterial, antifungal and anti-inflammatory activities. The composition of the product was not known, although possible formulations were reported. In this study the original sample, which dated back to 1870, was investigated for chemical composition and cytotoxic activity. The obtained results were compared with results from more recently produced samples. Several tests were carried out, namely GC-MS, UPLC-PDA-Q-TOF-MS and MTT. Only the 150-year old sample showed a significant cytotoxic activity on cancer cell lines. At a concentration of 125 µg/mL after 72 h of incubation, the original sample inhibited almost 90% of cell metabolic activity, while contemporary samples showed none or little activity. None of the tested samples showed a significant impact on normal cells. These results may be attributed to the activities of benzoic acid and its derivatives, cinnamic acid derivatives, vanillin, group of sesquiterpenes and cembrene.

Mineral Elements and Active Ingredients in Root of Wild Paeonia lactiflora Growing at Duolun County, Inner Mongolia.

Roots of wild Paeonia lactiflora are often used as herbs in traditional Chinese medicine. In this study, the contents of potassium (K), calcium (Ca), phosphorus (P), magnesium (Mg), iron (Fe), manganese (Mn), copper (Cu), and zinc (Zn) and the concentrations of three active ingredients such as paeoniflorin (PF), catechin (CA) and benzoic acid (BA) in roots of wild P. lactiflora collected from Duolun County of Inner Mongolia in China were evaluated. The results showed that the mean contents of eight elements followed the following order: Ca > K > P > Mg > Fe > Zn > Mn > Cu, and the concentrations of three active ingredients decreased in the order: PF > CA > BA. It was found that PF concentration was positively correlated with the contents of Fe and Mn. However, the concentration of CA was linearly decreased with Mg content. Moreover, BA concentration showed positive linear dependence upon the contents of P and Mn. Results of stepwise regression analyses showed that 39.2% of the variance in PF concentration could be explained by Fe content, whereas 28.1% of the CA concentration changes could be explained by Mg content; moreover, 42.5% of the variance in BA concentration could be accounted for by the combination of Mn and P contents. In a word, the concentrations of active ingredients in roots of P. lactiflora can be changed by adjusting mineral elements levels in roots to meet the need of appropriate quality control of P. lactiflora.

Quantitative analysis of different batches of raw, wine-processed, and vinegar-processed Paeoniae Alba Radix using ultra-performance convergence chromatography coupled with photo diode array detection.

Supercritical fluid chromatography is a safe and ecofriendly analytical technique that has not been fully applied to the analysis of traditional Chinese medicine. This is the first study on the separation of six quality markers-paeoniflorin, albiflorin, benzoyl paeoniflorin, oxypaeoniflorin, gallic acid and benzoic acid-from raw, wine-baked and vinegar-baked Paeoniae Alba Radix (PAR) by Supercritical fluid chromatography. Optimum separation was achieved on an HSS C18 SB column (100 × 3.0 mm, 1.8 µm particles) with a gradient elution of high-purity carbon dioxide as mobile phase A and methanol-acetonitrile (70:30, v/v) with 0.10% phosphoric acid as mobile phase B. The flow rate was set at 0.7 mL/min for 15.0 min. The method was validated in terms of the overall intraday and interday precision, with relative standard deviations (RSDs) of 0.87-2.87 and 1.47-3.63%, respectively. The recoveries were 98.10-103.60% with an RSD of 1.00-3.40%. The stability of the RSD values was in the range 1.10-3.78%. The developed approach was successfully applied and provides a valuable reference for the quality assessment of PAR and processed PAR. The results also revealed that the standardization of processing technology is of great significance to the fluctuations in quality before and after the processing of traditional Chinese medicine.

Phenolic profile and antioxidant activity of extracts prepared from fermented heat-stabilized defatted rice bran.

Heat-stabilized, defatted rice bran (HDRB) serves as a potential source of phenolic compounds which have numerous purported health benefits. An estimated 70% of phenolics present in rice bran are esterified to the arabinoxylan residues of the cell walls. Release of such compounds could provide a value-added application for HDRB. The objective of this study was to extract and quantify phenolics from HDRB using fermentation technology. Out of 8 organisms selected for rice bran fermentation, Bacillus subtilis subspecies subtilis had the maximum phenolic release of 26.8 mg ferulic acid equivalents (FAE) per gram HDRB. Response surface methodology was used to further optimize the release of rice bran phenolics. An optimum of 28.6 mg FAE/g rice bran was predicted at 168 h, 0.01% inoculation level, and 100 mg HDRB/mL. Fermentation of HDRB for 96 h with B. subtilis subspecies subtilis resulted in a significant increase in phenolic yield, phenolic concentration, and radical scavenging capacity. Fermented rice bran had 4.86 mg gentistic acid, 1.38 mg caffeic acid, 6.03 mg syringic acid, 19.02 mg (-)-epicatechin, 4.08 mg p-courmaric acid, 4.64 mg ferulic acid, 10.04 mg sinapic acid, and 17.59 mg benzoic acid per 100 g fermented extract compared to 0.65 mg p-courmaric acid and 0.36 mg ferulic acid per 100 g nonfermented extract. The high phenolic content and antioxidant activity of fermented HDRB extract indicates that rice bran fermentation under optimized condition is a potential means of meeting the demand for an effective and affordable antioxidant.

Microbial catabolism of procyanidins by human gut microbiota.

SCOPE: A major portion of ingested procyanidins is degraded by human microbiota in the colon into various phenolic compounds. These microbial metabolites are thought to contribute to the health benefits of procyanidins in vivo. The objective of this study was to identify and quantify the microbial metabolites of procyanidins after anaerobic fermentation with human microbiota. METHODS AND RESULTS: (-)-Epicatechin, (+)-catechin, procyanidin B2, procyanidin A2, partially purified apple and cranberry procyanidins were incubated with human microbiota at a concentration equivalent to 0.5 mM epicatechin. GC-MS analysis showed that common metabolites of all six substrates were benzoic acid, 2-phenylacetic acid, 3-phenylpropionic acid, 2-(3'-hydroxyphenyl)acetic acid, 2-(4'-hydroxyphenyl)acetic acid, 3-(3'-hydroxyphenyl)propionic acid, and hydroxyphenylvaleric acid. 5-(3',4'-Dihydroxyphenyl)-γ-valerolactones and 5-(3'-hydroxyphenyl)-γ-valerolactones were identified as the microbial metabolites of epicatechin, catechin, procyanidin B2, and apple procyanidins but not from the procyanidin A2 or cranberry procyanidin ferments. 2-(3',4'-Dihydroxyphenyl)acetic acid was only found in the fermented broth of procyanidin B2, A2, apple, and cranberry procyanidins. The mass recoveries of microbial metabolites range from 20.0 to 56.9% for the six substrates after 24 h of fermentation. CONCLUSION: Procyanidins, both B-type and A-type can be degraded by human gut microbiota. The microbial metabolites may contribute to the bioactivities of procyanidins.

Comparative analysis of the main bioactive components of San-ao decoction and its series of formulations.

A high performance liquid chromatographic (HPLC) method with diode array detection (DAD) was established for simultaneous determination of seven main bioactive components in San-ao decoction and its series of formulae (San-ao decoction, Wu-ao decoction, Qi-ao decoction and Jia-wei San-ao decoction). Seven compounds were analyzed simultaneously with a XTerra C(18) column (4.6 mm × 250 mm, 5 µm) using a linear gradient elution of a mobile phase containing acetonitrile (A) and a buffer solution (0.02 mol/L potassium dihydrogen phosphate and adjusted to pH 3 using phosphoric acid) (B); the flow rate was 1.0 mL/min. The sample was detected with DAD at 210, 254 and 360 nm and the column was maintained at 30 °C. All the compounds showed good linearity (r2 > 0.9984) in the tested concentration range. The precisions were evaluated by intra-day and inter-day tests, and relative standard deviation (R.S.D.) values within the range of 0.83%–2.53% and 0.64%–2.77% were reported, respectively. The recoveries of the quantified compounds were observed to cover a range from 95.34% and 104.82% with R.S.D. values less than 2.72%. The validated method was successfully applied for the simultaneous determination of seven main bioactive components including ephedrine (1), amygdalin (2), liquiritin (3), benzoic acid (4), isoliquiritin (5), formononetin (6) and glycyrrhizic acid (7) in San-ao decoction and its series of formulae. The results also showed a wide variation in the content of the identified active compounds in these samples, which could also be helpful to illustrate the drug interactions after some herbs combined in different formulations.

Antioxidant capacity of teas and herbal infusions: polarographic assessment.

Hydrogen peroxide scavenging (HPS) activity of unfermented (green, yellow, and white), partially fermented (oolong), and completely fermented (black) tea ( Camellia sinensis ), maté ( Ilex paraguariensis ), and various herbal infusions, as well as individual compounds (flavan-3-ols, flavonols, cinnamic and benzoic acids, and methylxanthines), was assessed by recently developed direct current (DC) polarographic assay. Correlations of tea and herbal infusion HPS activity with total phenolic content determined using the Folin-Ciocalteu assay (FC-GAE) (0.81 and 0.93), ferric reducing/antioxidant power (FRAP) (0.97 and 0.92), 1,1-diphenyl-2-picrylhydrazyl (DPPH) (0.77 and 0.80), and 2,2'-azinobis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) scavenging (0.86 and 0.86) were statistically significant. Correlations between relative antioxidant capacity index (RACI), calculated by assigning all applied assays equal weight, and HPS (0.98), FRAP (0.97), ABTS (0.89), and DPPH (0.89) confirmed DC polarographic assay reliability when applied individually. Correlation analysis, ANOVA, and Levene and Tukey's HSD tests unequivocally confirmed this reliable, rapid, and low-cost assay validity, clearly demonstrating its advantages over spectrophotometric assays applied.

Quantitation of flavanols, proanthocyanidins, isoflavones, flavanones, dihydrochalcones, stilbenes, benzoic acid derivatives using ultraviolet absorbance after identification by liquid chromatography-mass spectrometry.

A general method was developed for the systematic quantitation of flavanols, proanthocyanidins, isoflavones, flavanones, dihydrochalcones, stilbenes, and hydroxybenzoic acid derivatives (mainly hydrolyzable tannins) based on UV band II absorbance arising from the benzoyl structure. The compound structures and the wavelength maximum were well correlated and were divided into four groups: the flavanols and proanthocyanidins at 278 nm, hydrolyzable tannins at 274 nm, flavanones at 288 nm, and isoflavones at 260 nm. Within each group, molar relative response factors (MRRFs) were computed for each compound based on the absorbance ratio of the compound and the group reference standard. Response factors were computed for the compounds as purchased (MRRF), after drying (MRRFD), and as the best predicted value (MRRFP). Concentrations for each compound were computed based on calibration with the group reference standard and the MRRFP. The quantitation of catechins, proanthocyanidins, and gallic acid derivatives in white tea was used as an example.

Ultra high performance liquid chromatography-time of flight mass spectrometry for analysis of avocado fruit metabolites: method evaluation and applicability to the analysis of ripening degrees.

We have developed an analytical method using UHPLC-UV/ESI-TOF MS for the comprehensive profiling of the metabolites found in the methanolic extracts of 13 different varieties of avocado at two different ripening degrees. Both chromatographic and detection parameters were optimized in order to maximize the number of compounds detected and the sensitivity. After achieving the optimum conditions, we performed a complete analytical validation of the method with respect to its linearity, sensitivity, precision, accuracy and possible matrix effects. The LODs ranged from 1.64 to 730.54 ppb (in negative polarity) for benzoic acid and chrysin, respectively, whilst they were found within the range from 0.51 to 310.23 ppb in positive polarity. The RSDs for repeatability test did not exceed 7.01% and the accuracy ranged from 97.2% to 102.0%. Our method was then applied to the analysis of real avocado samples and advanced data processing and multivariate statistical analysis (PCA, PLS-DA) were carried out to discriminate/classify the examined avocado varieties. About 200 compounds belonging to various structural classes were tentatively identified; we are certain about the identity of around 60 compounds, 20 of which have been quantified in terms of their own commercially available standard.

Evaluation of the biochemical components and chromatic properties of the juice of Vaccinium macrocarpon Aiton and Vaccinium oxycoccos L.

Benzoic acid, total anthocyanins, soluble solids, titratable acidity, and colour properties in juice of the American cranberry Vaccinium macrocarpon and the European cranberry Vaccinium oxycoccos were investigated. Berry juices of V. macrocarpon cultivars were distinguished by their higher total anthocyanin and benzoic acid amounts. These cultivars accumulated on average 43.11 mg/l of benzoic acid and 92.45 mg/l of total anthocyanins. The levels of benzoic acid and total anthocyanins in V. oxycoccos cultivars were 17.52 mg/l and 42.54 mg/l, respectively. The V. macrocarpon cultivars 'Franklin', 'Le Munyon', 'Searles', and 'Early Richard' were selected as the best according to the enhanced total anthocyanins and benzoic acid amounts. The separation of anthocyanins by HPLC-UV-VIS revealed the presence of six anthocyanins, with peonidin-3-galactoside being the most prevalent. Galactoside together with glucoside conjugates comprised the largest percentage of total anthocyanins in the juices of V. macrocarpon and V. oxycoccos cultivars.

[RP-HPLC determination of main chemical components in different parts and different harvest periods of Paeonia lactiflora].

The contents of gallic acid, catechin, albiflorin, paeoniflorin, benzoic acid and paeonol extracted in different growth years, collecting season and of different parts of Paeonia lactiflora were determined. The results showed that the contents of catechin and paeoniflorin in Paeonia lactiflora collected in autumn are the highest, and the contents of benzoic acid was lower than that of those collected at other time. The longer is the age of Paeonia lactiflora, the higher is the contents of catechin and paeoniflorin. The contents of catechin and paeoniflorin in the root of Paeonia lactiflora were higher than those in other parts of the plant. There is a certain content of paeoniflorin in the leaves of Paeonia lactiflora. Judging from the result, paeoniflorin is synthesized in the leaf and then transported to the root. Catechin is not synthesized in the leaf, but mainly in the root. Paeonia lactiflora should be collected in autumn, and immature plant should not be collected.

[Determination of salylic acid, syringic acid, benzoic acid and anthranilic acid in Radix Isatidis by HPCE].

OBJECTIVE: To develop a simple and rapid capillary electrophoresis (CE) method for the separation and determination of four active organic acids including salicylic acid, syringic acid, benzoic acid, and anthranilic acid in Radix Isatidis. METHOD: The HPCE system consisted of a fused-silica capillary column of 47.3 cm (38.3 cm to the detector) x50 microm i.d. and a mixture ofacetonitrile-borate buffer (15% acetonitrile, 25 mmol L(-1) borate, 15 mmol L(-1) beta-CD, pH 9.10) solution as the operating buffer. The applied voltage was 11.5 kV and the UV detection was set at 220 nm. The effects of the applied voltage, detection wavelength, and the pH of buffer, the concentration of buffer, acetonitrile and beta-CD were investigated. RESULT: The linear calibration rang was 3.0-90 mg L(-1) (r=0.9994) for salylic acid, 4.0-120 mg L(-1) (r=0.9995) for syringic acid, 2.0-60 mg L(-1) (r=0.9998) for benzoic acid and 5.0-100 mg L(-1) (r=0.9992) for anthranilic acid. The recoveries of salylic acid, syringic acid, benzoic acid and anthranilic acid were 95.9%-102.6%, 98.6%-103.4%, 98.7%-104.1%, 96.1%-104.3% respectively. The detection limits of salylic acid, syringic acid, benzoic acid and anthranilic acid were 0.7, 1.1, 1.2 and 1.5 mg L(-1), respectively.

[Analysis of benzoic acid content in Radix Paeoniae Alba treated by different process].

OBJECTIVE: To analyze the benzoic acid content in the Radix Paeoniae Alba treated by three kinds of process. METHODS: After harvesting, the fresh roots were processed as follows: stored in low temperature, dried after boiling a few minutes and dried immediately. Then the content of benzoic acid was determined by UV spectrophotometry. RESULTS: The content of benzoic acid in the above material without outer part of the cortex was at the higher level than those with outer part of the cortex. In material without outer part of the cortex, the content of benzoic acid was 0.0635% in stored sample, 0.0248% in poaching sample, 0.0639% in dried sample, 0.6635% in part of the cortex. CONCLUSION: Benzoic acid was mostly distributed in the root cortex, Radix Paeoniae Alba treated by boiling can reduce the benzoic acid content. Fresh roots without cortex are comparatively economical and reasonable in distilling Paeoniflorin.

Development of HPLC and NACE methods for the simultaneous determination of benzoic and sorbic acids in sour snap beans containing oil.

The practical methods were developed for the simultaneous determination of benzoic acid (BA) and sorbic acid (SA) in sour snap bean samples containing oil. BA and SA in the samples were extracted by ultrasonication with water, followed by cleanup procedures with precipitation for removing the potential proteins and with petroleum ether liquid-liquid extraction for removing the edible oil contained in the samples. The HPLC method was developed using Supelco C18 (250 mm x 4.6 mm id, 5 microm) as column, MeOH-20 mM NH(4)Ac (25:75 v/v) at 1.0 mL/min as the mobile phase and 230 nm as the detection wavelength. The optimal NACE method was established with a running buffer of 20.0 mM NH(4)Ac in 95% MeOH (pH* 10.6), and an applied voltage of -30 kV over a capillary of 50 microm id x 48.5 cm (40 cm to the detector window), which gave a baseline separation of BA and SA, and as well as of the blank matrix within ca. 10 min. Both HPLC and NACE methods gave the relatively lower limits of quantification at about 0.01-0.02 and 0.04-0.05 mg/kg, respectively, whereas the overall recoveries were larger than 85.0%. The proposed methods have been successfully applied to measure 15 real sour bean samples and the content profile of BA and SA in sour bean samples was obtained and evaluated.

[Identification of flavonoids in phytoextracts by spectral methods].

The multicomponent phyto-extracts made of the wild herbs and berries were investigated by spectral methods. The biologically active substances--flavonol quercetin and oxybenzoic acids were identified in them. The content of the flavonoids in the above-mentioned phyto-extracts upon freezing and subsequent storage was determined.

[Determination of adulteration musk by GC-MS, SPME and HS2000].

Musk is one of the rare traditional Chinese medicinal materials and is usually sophisticated with different adulterants. Some new adulterants have been detected by GC-MS, SPME and HS2000.

Production and analysis of organic acids in hairy-root cultures of Isatis indigotica Fort. (indigo woad).

Hairy roots were induced from both cotyledon and hypocotyl explants of Isatis indigotica Fort. (indigo woad) through transformation with Agrobaterium rhizogenes strain A4, R1601 and ATCC15834. The results showed that the cotyledons were the preferred explants to hypocotyls and A4 was the most suitable A. rhizogenes strain for the transformation and induction of hairy roots of I. indigotica. High-voltage paper electrophoresis (HVPE) analysis demonstrated the production of mannopine in hairy roots and confirmed the successful transfer of Ri T-DNA (root-inducing transferred DNA) of A. rhizogenes into the I. indigotica genome. Five organic acids, namely CPQ [3-(2-carboxyphenol)-4(3 H )-quinazolinone], syringic acid, salicylic acid, benzoic acid and 2-aminobenzoic acid, which were considered as main antiviral components of I. indigotica, were detected in natural roots, hairy roots and liquid media with high-performance capillary electrophoresis. The results showed CPQ production in hairy roots was significantly higher than that in natural roots. Our results also revealed that all the five organic acids could be excreted from hairy roots into liquid media, and the concentrations of organic acids in the liquid media paralleled those in hairy roots. The hairy roots of I. indigotica grew fast and showed an S-shaped growth curve that reached its apex on the day 24 of culture with a 20-fold increase in fresh weight compared with the starting inoculums. The accumulation of the two organic acids CPQ and syringic acid in liquid media paralleled the growth of hairy roots. MS [Murashige, T. and Skoog, F. (1962) Physiol. Plant. 15, 473-497] medium or half-strength MS medium supplemented with 30 g/l maltose was found to be best for hairy-root culture and accumulation of CPQ.

Isolation and quantitative analysis of phenolic antioxidants, free sugars, and polyols from mango (Mangifera indica L.) stem bark aqueous decoction used in Cuba as a nutritional supplement.

An aqueous decoction of mango (Mangifera indica L.) stem bark has been developed in Cuba on an industrial scale to be used as a nutritional supplement, cosmetic, and phytomedicine. Previously we reported its antioxidant activity, and we concluded that the product could be useful to prevent the production of reactive oxygen species and oxidative tissue damage in vivo. A phytochemical investigation of mango stem bark extract has led to the isolation of seven phenolic constituents: gallic acid, 3,4-dihydroxy benzoic acid, gallic acid methyl ester, gallic acid propyl ester, mangiferin, (+)-catechin, (-)-epicatechin, and benzoic acid and benzoic acid propyl ester. All structures were elucidated by ES-MS and NMR spectroscopic methods. Quantitative analysis of the compounds has been performed by HPLC, and mangiferin was found to be the predominant component. Total polyphenols were assayed also by the Folin-Ciocalteu method. The free sugars and polyols content was also determined by GC-MS.