Phenolic profiles of cultivated, in vitro cultured and commercial samples of Melissa officinalis L. infusions

Melissa officinalis L. (lemon balm) is normally consumed as an infusion and presents therapeutic properties, such as sedative, carminative and antispasmodic, also being included in some pharmaceutical preparations. The phenolic profiles of different samples of lemon balm, prepared as infusions, were evaluated by HPLC-DAD-ESI/MS. The profiles were compared in order to understand the differences between cultivated, in vitro cultured and commercial (bags and granulated) samples. All the samples showed a similar phenolic profile, presenting differences only in the quantities found of each compound. Rosmarinic acid was the most abundant compound, being higher in commercial samples, especially in tea bag sample (55.68 mg/g of infusion) and lower in in vitro cultured sample (15.46 mg/g). Moreover, dimers, trimers and tetramers of caffeic acid were identified and quantified for the first time in lemon balm. Only one flavonoid, luteolin-3′-O-glucuronide was found in all the samples, ranging from 8.43 mg/g in commercial granulate sample to 1.22 mg/g in in vitro cultured sample. Overall, cultivated and in vitro cultured samples presented the lowest amounts of phenolic compounds (59.59 and 30.21 mg/g, respectively); otherwise, commercial samples showed the highest contents (109.24 mg/g for tea bag and 101.03 mg/g for granulate sample). The present study shows that infusion of lemon balm can be a source of phenolic compounds, known for their bioactive effects.     

Chemical composition and in vitro antioxidative activity of a lemon balm (Melissa officinalis L.) extract

The leaf material of lemon balm (Melissa officinalis L.) was extracted with 450 ml/l aqueous ethanol by medium pressure liquid-solid extraction. The total phenolic content of the extract was estimated as gallic acid equivalents by Folin-Ciocalteu reagent method and a qualitative-quantitative compositional analysis was carried out using high performance liquid chromatography coupled with photodiode array detection. The lemon balm extract contained hydroxycinnamic acid derivatives and flavonoids with caffeic acid, m-coumaric acid, eriodictyol-7-O-glucoside, naringin, hesperidin, rosmarinic acid, naringenin, hesperetin being identified based on their chromatographic behaviour and spectral characteristics. The extract was also investigated for potential in vitro antioxidant properties in iron(III) reduction, iron(II) chelation, 1,1-diphenyl-2-picrylhydrazyl, 2,2′-azinobis(3-ethylbenzothiazoline-6-sulphonate), superoxide anion and nitric oxide free-radical scavenging, and inhibition of β-carotene-linoleic acid bleaching assays. The extract demonstrated antioxidant activity in all the assays. However, it was not as potent as the positive controls except in the β-carotene-linoleic acid bleaching assay, where its activity was superior to that of gallic and caffeic acids and statistically indistinguishable from quercetin and BHA. The exceptionally high antioxidant activity and the fact that this assay is of biological relevance warrants further investigation of lemon balm extract in ex vivo and in vivo models of oxidative stress.     

Flavone-di-C-glycosides in citrus juices from Southern Italy

6,8-Di-C-glucopyranosylapigenin and 6,8-di-C-glucopyranosyldiosmetin were detected by HPLC–DAD–ESI–MS–MS in a variety of Southern Italian Citrus juices (orange, lemon, bergamot, citron, mandarin, clementine). In some juices, the amounts of C-glycosides detected were significant. 6,8-Di-C-glucopyranosylapigenin is characteristic of orange juice, while 6,8-di-C-glucopyranosyldiosmetin is the most important C-glycoside in lemon and citron juice. In bergamot juice the concentrations of 6,8-di-C-glucopyranosylapigenin and 6,8-di-C-glucopyranosyldiosmetin are similar. Clementine juice is distinctive as the amounts of both C-glycosides present are negligible.     

Chemical composition, in vitro anti-yeast activity and fruit juice preservation potential of lemon grass oil

The anti-yeast activity of lemon grass oil was evaluated against several food spoiling yeasts (Saccharomyces cerevisiae, Zygosaccharomyces bailii, Aureobasidium pullulans, Candida diversa, Pichia fermentans, Pichia kluyveri, Pichia anomala and Hansenula polymorpha) through disc diffusion and microbroth dilution method. The minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) varied from 0.28 to 1.3 mg/ml and 0.56 to 4.5 mg/ml, respectively, where highest MIC (2.25 mg/ml) was shown by A. pullulans and lowest MIC (0.28 mg/ml) was shown by C. diversa and P. anomala. Kill time assay was conducted for selected three yeast strains where S. cerevisiae showed highest reduction (3 log cfu) in viability within 24 h exposure of lemon grass oil at MFC level. Further, the anti-yeast efficacy of lemon grass oil alone and in combination with thermal treatment was evaluated in real food system i.e. mixed fruit juices. Chemical composition analysis of lemon grass oil by gas chromatography–mass spectrometry (GC–MS) revealed that the dominant compounds were geranial (40.5%), neral (30.7%), geranyl acetate (5.1%), caryophyllene (2.5%). Present results established the superior performance of integrated (thermal–lemon grass oil) treatment over the individual exposure (lemon grass oil or thermal treatment alone) for fruit juice preservation.     

Response surface optimised extraction and chromatographic purification of rosmarinic acid from Melissa officinalis leaves

The extraction of lemon balm (Melissa officinalis) leaves with aqueous methanol was optimised using response surface methodology. Fifteen runs were conducted following a Box-Behnken design (BBD) followed by ridge analysis using the concentration of methanol, the extraction temperature and time as the independent variables and taking the extraction yield of RA from lemon balm as the response variable. The optimal extraction conditions were a methanol concentration of 59.0% (v/v), a temperature of 54.8 °C and a time of 64.8 min, which gave a maximal RA yield of 46.1 mg RA/g dry materials. The RA extract was loaded onto a column packed with Sephadex LH-20 and then was eluted with 100% methanol, which resulted in RA with a purity of 38.8% and a yield of 43.8%. The purity of RA increased by 3.1-fold when compared to its initial purity in the extract obtained from extraction.     

Nutrition analysis by nanoparticle-assisted laser desorption/ionisation mass spectrometry

We analysed the bioactive compounds in Panax ginseng C.A. Meyer by using nanoparticle-assisted laser desorption/ionisation (nano-PALDI) mass spectrometry (MS). To this end, we prepared manganese oxide nanoparticles (d = 5.4 nm) and developed a nano-PALDI MS method to analyse the standard ginsenosides and identify these ginsenosides in an extract of P. ginseng. The nanoparticles served as an ionisation-assisting reagent in MS. The mass spectra did not show any background interference in the low-m/z range. Our pilot study showed that the nanoparticles could ionise the standard ginsenosides and also respective lipid and ginsenosides in the extract without the aid of chemical and liquid matrices used in conventional MS methods. Analysis of the post-source decay spectra obtained using nano-PALDI MS will yield information regarding the chemical structure of the analyte.     

Phenolic profiles of in vivo and in vitro grown Coriandrum sativum L.

Coriandrum sativum L. is a source of a variety of polyphenols and other phytochemicals, related to its high antioxidant activity and to its use for indigestion, rheumatism, and prevention of lipid peroxidation damage. Plant cell cultures are a means to study or to produce some active metabolites, such as polyphenols. This technique was applied to the investigation of coriander, and a detailed analysis of individual polyphenols in vivo and in vitro grown samples was performed. The in vivo vegetative parts showed quercetin derivatives as the main flavonoids and quercetin-3-O-rutinoside (3296 mg/kg dw) was the main polyphenol found in this part of coriander. The fruits revealed only phenolic acids and derivatives, caffeoyl N-tryptophan hexoside (45.33 mg/kg dw) being the most abundant phenolic derivative. In vitro samples also gave a high diversity of polyphenols, being C-glycosylated apigenin (2983 mg/kg dw) the main compound. Anthocyanins were only found in clone A, which was certainly related to its purple pigmentation, and peonidin-3-O-feruloylglucoside-5-O-glucoside was the major anthocyanin found (1.70 μg/kg dw). In vitro culture can be used to explore new industrial, pharmaceutical, and medicinal potentialities, such as the production of secondary metabolites like flavonoids.     

Quantification of total polysaccharides and triterpenoids in Ganoderma lucidum and Ganoderma atrum by near infrared spectroscopy and chemometrics

Two chemometrics, the partial least-squares (PLS) and radial basis function (RBF) network were performed to develop a quantification method for total polysaccharides and triterpenoids in Ganoderma lucidum and Ganoderma atrum from different origins based on near infrared reflectance spectroscopy (NIR). The influences of spectral window and spectral pre-treatments were initially studied in the construction of PLS model. The best result was obtained when the standard normal transformation (SNV) +1st derivative spectrum over 4100–7750 cm⁻¹ was used for the modelling. Then based on each principle, both of the two models were optimised respectively. The final results with high determination coefficient (R²) (higher than 0.973, 0.989 for PLS and RBF, respectively) and low root mean square errors of prediction (RMSEP) (low to 0.1109 and 0.01298 for polysaccharides and triterpenoids, respectively) confirm the good predictability of the two models. The overall results show that NIR spectroscopy combined with chemometrics can be efficiently utilised for accurate analysis of routine chemical compositions in G. lucidum and G. atrum.     

Isolation and characterization of a novel analyte from Bacillus subtilis SC-8 antagonistic to Bacillus cereus

In this study, an effective substance was isolated from Bacillus subtilis SC-8, which was obtained from traditionally fermented soybean paste, cheonggukjang. The substance was purified by HPLC, and its properties were analyzed. It had an adequate antagonistic effect on Bacilluscereus, and its spectrum of activity was narrow. When tested on several gram-negative and gram-positive foodborne pathogenic bacteria such as Salmonella enterica, Salmonella enteritidis, Staphylococcus aureus, and Listeria monocytogenes, no antagonistic effect was observed. Applying the derivative from B. subtilis SC-8 within the same genus did not inhibit the growth of major soybean-fermenting bacteria such as Bacillus subtilis, Bacillus licheniformis, and Bacillus amyloquefaciens. The range of pH stability of the purified antagonistic substance was wide (from 4.0 to >10.0), and the substance was thermally stable up to 60 °C. In the various enzyme treatments, the antagonistic activity of the purified substance was reduced with proteinase K, protease, and lipase; its activity was partially destroyed with esterase. Spores of B. cereus did not grow at all in the presence of 5 μg/mL of the purified antagonistic substance. The isolated antagonistic substance was thought to be an antibiotic-like lipopeptidal compound and was tentatively named BSAP-254 because it absorbed to UV radiation at 254 nm.     

Comparative study of the effect of the maturation process of the olive fruit on the chlorophyll and carotenoid fractions of drupes and virgin oils from Arbequina and Farga cultivars

Two varieties of olive fruit (Arbequina and Farga) have been characterized by their contents and types of chlorophyll and carotenoid pigments during the olive ripening. Independently of the initial content, the pigment concentrations decreased with fruit maturity. Chlorophyllides a and b, esterified xanthophylls and α-carotene were only detected in Arbequina fruits. Moreover, the synthesis of esterified xanthophylls, when the skin fruit colour changed from green to turning-colour only, was observed in this variety. The pigments transferred from fruit to the oil were also studied. All the pigments that were found in the fruit were transferred to the oils, in addition to derivative pigments associated with acidic medium in the oil extraction process. The destruction of the chlorophyll fraction was greater than of the yellow pigments during the olive oil extraction process.     

Antioxidant, antiacetylcholinesterase and antimicrobial activities of Cymbopogon schoenanthus L. Spreng (lemon grass) from Tunisia

Aqueous extract, proanthocyanidin rich extract, and organic extracts of Cymbopogon schoenanthus L. Spreng (lemon grass) shoots from three different locations in South Tunisia were screened for their antioxidant, acetylcholinesterase and antimicrobial activities. In addition to the evaluation of these activities, the contents of flavonoids and total phenolic compounds were determined.Antioxidant activity measured by DPPH assay showed that the proanthocyanidin extract exhibited higher antioxidant activity than the aqueous extract. Extract concentration providing 50% inhibition (IC₅₀) ranged from 16.4 ± 6.8 μg/mL to 26.4 ± 6.8 μg/mL. The antioxidant activity was also determined using the β-carotene/linoleic acid bleaching test. The best results (IC₅₀ = 0.11 ± 0.10 mg/mL) were obtained with the proanthocyanidin extract of the plants collected from the desert region (Dhibat).The greatest acetylcholinesterase inhibitory activity (IC₅₀ = 0.23 ± 0.04 mg/mL) was exhibited by the ethyl acetate and methanol extracts of the plants collected from the mountainous region. It seems that extracts obtained with more polar solvents gave better results.The proanthocyanidin extracts showed a good antimicrobial activity against Streptococcus sobrinus at low concentration (MIC = 4 mg/mL). Therefore, these extracts could be used to prevent carious lesions by inhibiting S. sobrinus growth.     

Supercritical Fluid Extraction of Essential Oil Components from Lemon-Scented Botanicals

Essential oils were extracted from four different varieties of lemon-scented plants utilizing supercritical fluid extraction. The plants used included: lemon eucalyptus (Eucalyptus citriodora), lemon balm (Melissa officinalis), lemon bergamot (Monarda citriodora), and lemongrass (Cymbopogon citratus). Extracts from three plants of each species were analysed using gas chromatography and confirmed with GC-MS. The compounds identified for each plant species were: lemon balm (citronellal, neral, geranial, neral acetate, caryophyllene, and caryophyllene oxide), lemon bergamont (thymol, thymol methylester, and α -terpine), lemon eucalyptus (citronellal, neral, caryophyllene, and caryophyllene oxide), and lemongrass (neral, geranial, and caryophyllene). Supercritical fluid extractions were performed at three pressures (13,790, 27,580, and 41,370 kPa) and two extraction temperatures (40 and 60 °C). The results indicated that the mass percent extracted increased with increasing temperature and pressure, as was the ratio of the compounds in the different samples. The effect of particle size reduction on the extraction yield was also investigated. The extraction yield did not increase due to the increase in surface area as would be expected, but decreased. This has been attributed to the fact that the compounds of interest are extremely volatile and were lost to the atmosphere during the grinding process.     

Biological activities of flavonoids from pathogenic-infected Astragalus adsurgens

The present study was aimed at the identification of biological components from pathogenic-infected Astragalus adsurgens with an activity-guided purification process. Fifteen flavonoids were obtained from the most active ethyl acetate fraction and identified as astradsurnin (1), a new chalcone derivative, together with fourteen known flavonoids (2-15). These compounds were tested for antibacterial activities against five bacteria and cytotoxic activities against two selected cancer cells. Within the series of flavonoids tested, compounds 4 and 15 were the most active against Escherichia coli, Bacillus cereus, Staphylococcus aureus, Erwinia carotovora and Bacillus subtilis with MICs ranging from 7.8 to 31.3 μg/ml. Moreover, compounds 4 and 5 exhibited moderate activity against HL-60 and SMMC-7721 cells with IC₅₀ values between 5 and 10 μg/ml. To our knowledge, this is the first time that it has been reported on the biological and chemical study of A. adsurgens infected by Embellisia astragali.     

Synthesis of cyclopropane fatty acids in Lactobacillus helveticus and Lactobacillus sanfranciscensis and their cellular fatty acids changes following short term acid and cold stresses

An implemented GC method to separate and quantify the cell cyclopropane fatty acids lactobacillic (C19cyc11) and dehydrosterculic (C19cyc9) was used to study the adaptive response to sublethal acid and cold stresses in Lactobacillus helveticus and Lactobacillus sanfranciscensis. The comparison of the composition of cellular fatty acids of the two strains and their changes after 2 h of stress exposure under micro-aerobic and anaerobic conditions indicated that the aerobic biosynthetic pathway for unsaturated fatty acids is prevalent in L. sanfranciscensis, while the anaerobic pathway is prevalent in L. helveticus. Indeed in the latter strain, in the presence of a source of oleic acid and under micro-aerobic conditions, C18:1n11 and its post-synthetic derivative C19cyc11 accounted for overall proportion ranging from 52 to 28% of the total FAs. On the other hand L. sanfranciscensis synthesizes by aerobic pathway C18:1n9 and transforms it to C19cyc9. However in this species the cumulative level of these two FAs did not exceed 30%. The relevant proportion of dodecanoic acid in the latter species suggests that carbon chain shortening is the principal strategy of L. sanfranciscensis to modulate fluidity or chemico-physical properties of the membranes.     

Characterization of a new anthocyanin in black raspberries (Rubus occidentalis) by liquid chromatography electrospray ionization tandem mass spectrometry

Anthocyanin composition of black raspberry (Rubus occidentalis) was studied using high-performance liquid chromatography coupled to photodiode array (PDA) detection and electrospray ionization mass spectrometry (LC-ESI/MS) and tandem mass spectrometry (MS/MS). Pelargonidin 3-rutinoside was isolated and identified in black raspberries using HPLC, UV–Vis spectroscopy, MS, and NMR spectroscopy. No pelargonidin derivative had been previously found in Rubus occidentalis. In addition, the presence and identities of four previously reported anthocyanins (cyanidin 3-glucoside, cyanidin 3-sambubioside, cyanidin 3-rutinoside and cyanidin 3-xylosylrutinoside) were confirmed by HPLC/MS and MS/MS analyses.     

The microbial diversity of water kefir

The microbial diversity of water kefir, made from a mixture of water, dried figs, a slice of lemon and sucrose was studied. The microbial consortia residing in the granules of three water kefirs of different origins were analyzed. A collection of 453 bacterial isolates was obtained on different selective/differential media. Bacterial isolates were grouped with randomly amplified polymorphic DNA (RAPD)-PCR analyses. One representative of each RAPD genotype was identified by comparative 16S rDNA gene sequencing. The predominant genus in water kefirs I and II was Lactobacillus, which accounted for 82.1% in water kefir I and 72.1% in water kefir II of the bacterial isolates. The most abundant species in water kefirs I and II were Lactobacillus hordei and Lb. nagelii followed by considerably lower numbers of Lb. casei. Other lactic acid bacteria (LAB) were identified as Leuconostoc mesenteroides and Lc. citreum in all three water kefirs. The most abundant species in water kefir III was Lc. mesenteroides (28%) and Lc. citreum (24.3%). A total of 57 LAB belonging to the species of Lb. casei, Lb. hordei, Lb. nagelii, Lb. hilgardii and Lc. mesenteroides were able to produce exopolysacchrides from sucrose. Non LABs were identified as Acetobacter fabarum and Ac. orientalis. The Acetobacter species were more prevalent in consortium III. Cluster analyses of RAPD-PCR patterns revealed an interspecies diversity among the Lactobacillus and Acetobacter strains. Aditionally, Saccharomyces cerevisiae, Lachancea fermentati, Hanseniaospora valbyensis and Zygotorulaspora florentina were isolated and identified by comparison of partial 26S rDNA sequences and FTIR spectroscopy.     

Coumarins of Matricaria chamomilla L.: Aglycones and glycosides

The identity and quantity of coumarin-like compounds in leaves and anthodia of Matricaria chamomilla L. were studied by LC-DAD and NMR. So far, two monosubstituted coumarins, herniarin and umbelliferone, and two herniarin precursors were identified therein. In this paper, two other coumarin glycosides and one aglycone were confirmed. Skimmin (umbelliferone-7-O-β-d-glucoside), daphnin (daphnetin-7-O-β-d-glucoside) and daphnetin (7,8-dihydroxycoumarin) were found for the first time in diploid and tetraploid leaves and anthodia of M. chamomilla L. Daphnetin is known as a strong sensitizer, so this compound and its glycosidic derivative can contribute to the allergic potential of chamomile. Commercial chamomile preparations were tested for their presence.     

Prevalence and comparison of Streptococcus infantarius subsp. infantarius and Streptococcus gallolyticus subsp. macedonicus in raw and fermented dairy products from East and West Africa

Streptococcus infantarius subsp. infantarius (Sii) and Streptococcus gallolyticus subsp. macedonicus are members of the Streptococcus bovis/Streptococcus equinus complex (SBSEC) associated with human infections. SBSEC-related endocarditis was furthermore associated with rural residency in Southern Europe. SBSEC members are increasingly isolated as predominant species from fermented dairy products in Europe, Asia and Africa. African variants of Sii displayed dairy adaptations to lactose metabolism paralleling those of Streptococcus thermophilus including genome decay. In this study, the aim was to assess the prevalence of Sii and possibly other SBSEC members in dairy products of East and West Africa in order to identify their habitat, estimate their importance in dairy fermentation processes and determine geographic areas affected by this potential health risk. Presumptive SBSEC members were isolated on semi-selective M17 and SM agar media. Subsequent genotypic identification of isolates was based on rep-PCR fingerprinting and SBSEC-specific16S rRNA gene PCR assay. Detailed identification was achieved through application of novel primers enhancing the binding stringency in partial groES/groEL gene amplification and subsequent DNA sequencing. The presence of S. thermophilus-like lacS and lacZ genes in the SBSEC isolates was determined to elucidate the prevalence of this dairy adaptation. Isolates (n = 754) were obtained from 72 raw and 95 fermented milk samples from Côte d'Ivoire and Kenya on semi-selective agar media. Colonies of Sii were not detected from raw milk despite high microbial titers of approximately 10⁶ CFU/mL on M17 agar medium. However, after spontaneous milk fermentation Sii was genotypically identified in 94.1% of Kenyan samples and 60.8% of Kenyan isolates. Sii prevalence in Côte d'Ivoire displayed seasonal variations in samples from 32.3% (June) to 40.0% (Dec/Jan) and isolates from 20.5% (June) to 27.7% (Dec/Jan) present at titers of 10⁶–10⁸ CFU/mL. lacS and lacZ genes were detected in all Kenyan and 25.8% (June) to 65.4% (Dec/Jan) of Ivorian Sii isolates. Regional differences in prevalence of Sii and dairy adaptations were observed, but no clear effect of dairy animal, fermentation procedure and climate was revealed. Conclusively, the high prevalence of Sii in Kenya, Côte d'Ivoire in addition to Somalia, Sudan and Mali strongly indicates a pivotal role of Sii in traditional African dairy fermentations potentially paralleling that of typical western dairy species S. thermophilus. Putative health risks associated with the consumption of high amounts of live Sii and potential different degrees of evolutionary adaptation or ecological colonization require further epidemiologic and genomic investigations, particularly in Africa.