Chemically characterised Pimenta dioica (L.) Merr. essential oil as a novel plant based antimicrobial against fungal and aflatoxin B1 contamination of stored maize and its possible mode of action

Abstract

The chemical characterisation of Pimenta dioica essential oil (PDEO) revealed the presence of 50 components, amongst which α-Terpineol (30.31%) was the major component followed by β-Linalool (6.75%) and γ-Terpinene (4.64%). The oil completely inhibited the growth of aflatoxin B₁ secreting strain Aspergillus flavus LHP-VS-8 and aflatoxin B₁ production at 2.5?µL/mL and 1.5?µL/mL, respectively. The oil caused dose dependent reduction of methylglyoxal (an AFB₁ inducer), enhanced leakage of Ca²⁺, Mg²⁺ and K⁺ ions and significantly reduced ergosterol content of fungal plasma membrane. During in situ experiments, PDEO exhibited complete protection of fumigated maize cob slices from fungal infestation without affecting seed germination. The chemically characterised PDEO is recommended as a plant based preservative and shelf life enhancer of food commodities by preventing fungal growth, AFB₁ production and lipid peroxidation. This is the first report on PDEO as inhibitor of AFB₁ secretion and methylglyoxal biosynthesis.     

First report of toxicity of Xylopiaparviflora (A. Rich.) Benth (Annonaceae) root bark's essential oil against cowpea seed bruchid,Callososbruchus maculatus Fabricius (Coleoptera: Chrysomelidae: Bruchinae)

The fumigant toxicity of Xylopia parviflora (A. Rich.) Benth (Annonaceae) root bark's essential oil (EO) against cowpea seed bruchid, Callosobruchus maculatus, was investigated in the laboratory. Dose had significant (P < 0.0001) effect on mortality at 6 hours after treatment (HAT) at a concentration of 6.25 μL/mL air which exerted 81.70% mortality, while there was no mortality in all other lower doses. At 12 HAT, 75.05% and 90.00% mortality were observed at doses of 3.15 and 6.25 μL/mL air, respectively. It was significantly (P < 0.05) higher than the mortality (50.58%) observed when 0.78 μL/mL air was applied. The lethal time for 50% of assayed adults (LT₅₀) obtained when the bruchid was exposed to X. parviflora EO at a dose of 6.25 μL/mL air (2.71 h) was significantly lower than LT₅₀ obtained at exposure of bruchid to other lower doses of 0.78–3.15 μL/mL air.     

Use of MALDI-TOF MS to Discriminate between Aflatoxin B1-Producing and Non-Producing Strains of Aspergillus flavus

Aflatoxin B₁ (AFB₁) is one of the most toxic mycotoxins. One of the producers of AFB₁ is Aspergillus flavus. Therefore, its rapid identification plays a key role in various sectors of the food and feed industry. MALDI-TOF mass spectrometry is one of the fastest and most accurate methods today. Therefore, the aim of this research was to develop the rapid identification of producing and non-producing strains of A. flavus based on the entire mass spectrum. To accomplish the main goal a different confirmatory MALDI-TOF MS and TLC procedures such as direct AFB₁ identification by scraping from TLC plates, A. flavus mycelium, nutrient media around A. flavus growth, and finally direct AFB₁ identification from infected wheat and barley grains had to be conducted. In this experiment, MALDI-TOF mass spectrometry with various modifications was the main supporting technology. All confirmatory methods confirmed the presence of AFB₁ in the samples of aflatoxin-producing strains of A. flavus and vice versa; AFB₁ was not detected in the case of non-producing strains. Entire mass spectra (from 2 to 20 kDa) of aflatoxin-producing and non-producing A. flavus strains were collected, statistically analyzed and clustered. An in-depth analysis of the obtained entire mass spectra showed differences between AFB₁-producing and non-producing strains of A. flavus. Statistical and cluster analysis divided AFB₁-producing and non-producing strains of A. flavus into two monasteries. The results indicate that it is possible to distinguish between AFB₁ producers and non-producers by comparing the entire mass spectra using MALDI-TOF MS. Finally, we demonstrated that if there are established local AFB₁-producing and non-producing strains of A. flavus, the entire mass spectrum database identification of aflatoxigenic A. flavus strains can be even faster and cheaper, without the need to identify the toxin itself.     

Antioxidant,cholinesterase inhibition activities and essential oil analysis of Nelumbo nucifera seeds

Nelumbo nucifera seeds’ essential oil (EO), crude extract and subsequent fractions were evaluated for their DPPH, ABTS and superoxide anion-free radical scavenging and cholinesterase inhibitory activities. The ethyl acetate fraction and EO showed outstanding antioxidant activities with IC₅₀ values of 191, 450 μg/mL (DPPH), 123, 221 μg/mL (ABTS) and 69, 370 μg/mL (superoxide anion). The ethyl acetate fraction and EO also caused significant inhibition of acetylcholinesterase and butyrylcholinesterase with IC₅₀ values of 70 ± 0.6, 64 ± 0.8 and 75 ± 0.3, 58 ± 0.2, in dose-dependent manner. The first ever gas chromatography–mass spectrometry analysis of the EO obtained from N. nucifera seeds resulted in identification of 19 constituents, mainly comprised of oxygenated sesquiterpenes responsible for their promising bioactivity. The crude and fractions revealed the presence of saponins, flavonoids, steroids, alkaloids, terpenoids and cardiac glycosides in phytochemical investigation.     

Amino acid profiling and antimicrobial activity of Cucurbita moschata and Lagenaria siceraria seed protein hydrolysates

Cucurbita moschata and Lagenaria siceraria seed proteins were extracted and hydrolysed with trypsin in order to recover antibacterial peptides. Amino acid content and molecular weight distribution were estimated to justify their co-relationship with antimicrobial activity. Antimicrobial activities of C. moschata and L. siceraria seed protein hydrolysates against three Gram-negative bacteria and two Gram-positive bacteria were evaluated. Seed protein hydrolysates of both of these plants have significantly higher activity against Acinetobacter baumannii (p < 0.05). The lethal concentration (LC₅₀) values of L. siceraria hydrolysates (LSH) and C. moschata hydrolysates (CMH) were 70 ± 6.2 and 135.6 ± 4.5 μg/mL in viable count method and 73.2 ± 2.9 and 122.9 ± 3.2 μg/mL in turbidity method, respectively, against A. baumannii. Based on the above findings, seed protein hydrolysates of these plants may be considered as nutritional food and functional antimicrobial agents in food system.     

Cumin scented leaf essential oil of Cinnamomum chemungianum: compositions and their in vitro antioxidant, α-amylase, α-glucosidase and lipase inhibitory activities

As part of our work on prospecting of Cinnamomum of the Western Ghats, chemical compositions, antioxidant, α-amylase, α-glucosidase and lipase inhibitory activities of leaf essential oil (EO) of Cinnamomum chemungianum were evaluated. Chemical characterisation of the cumin scented leaf EO from five locations in the southern Western Ghats revealed that they were highly varied. EO from Kannikatti (CC²) exhibited good α-amylase inhibitory activity with IC₅₀ value of 5.97 μg/mL, whereas the EOs from Chemungi (CC¹) and Athirumala (CC⁵) showed better α-glucosidase inhibition with IC₅₀ of 56.65 and 62.12 μg/mL, respectively. The EOs from Chemungi, Kannikatti and Athirumala were found to inhibit lipase with IC₅₀ of 919.75, 923.17 and 838.46 μg/mL, respectively. The EO of C. chemungianum may be used in food products as it has cumin scent and mild inhibitory activities.     

Activity-guided investigation of Carissa carandas (L.) roots for anti-inflammatory constituents

The present study was structured to investigate the anti-inflammatory potential of the extracts, fractions and compounds isolated from Carissa carandas (L.) roots. Bioassay guided fractionation of methanol extract based on inhibitory potential towards proinflammatory mediators (TNF-α, IL-1β and nitric oxide (NO)) led to the identification of stigmasterol (1), lupeol (2), oleanolic acid (3), carissone (4) and scopoletin (5) as potential anti-inflammatory agents. Carissone (4) (IC₅₀ = 20.1 ± 2.69 μg/mL) and scopoletin (5) (IC₅₀ = 24.6 ± 1.36 μg/mL) exhibited significant inhibition of NO production comparable to specific NO inhibitor (L-NAME; IC₅₀ = 19.82 ± 1.64 μg/mL) without affecting the cell viability. Also, 4 and 5 at a concentration of 30 μM were found to inhibit 41.88–53.44% of TNF-α and IL-1β. To the best of our knowledge, this is the first report displaying the anti-inflammatory effects of C. carandas (L.) roots, partially mediated by inhibition of TNF-α, IL-1β and NO.     

Development of a Lateral Flow Strip with a Positive Readout for the On-Site Detection of Aflatoxin B1

Aflatoxin B₁ is one of the contamination indicators for food safety monitoring. The rapid and effective assessment and determination of AFB₁ in food is of great importance to dietary safety. The lateral flow assay shows advantages in its simplicity, and rapidity, and provides a visual readout, while the available lateral flow assay for AFB₁ requires a competitive format that produces readings inversely proportional to the AFB₁ concentration, which is counterintuitive and may lead to a potential misinterpretation of the results. Herein, we developed a positive readout aptamer-based lateral flow strip (Apt-strip) for the detection of AFB₁. This Apt-strip relies on the competition between AFB₁ and fluorescein-labeled complementary DNA strands (FAM-cDNA) for affinity binding to limited aptamers against AFB₁ (AFB₁-Apt). In the absence of AFB₁, AFB₁-Apt hybridizes with FAM-cDNA. No signal at the T-line of the Apt-strip was observed. In contrast, AFB₁-Apt binds to AFB₁ in the sample, and then a part of the FAM-cDNA is hybridized with the free AFB₁-Apt, at which time the other unreacted FAM-cDNA is captured by A35-Apt on the T-line. The signal was observed. This method achieved fast detection of AFB₁ with a detection limit (DL) of 0.1 ng/mL, positive readout, and increased sensitivity.     

AFB1–AP Conjugate for Enzyme Immunoassay of Aflatoxin B1 in Corn Samples

Abstract

This article describes the conjugation between aflatoxin B₁ (AFB₁), one of the major mycotoxins, and alkaline phosphatase (AP), one of the most used enzymes for immunoassays. In addition, an application of the ELISA method for aflatoxin B₁ determination in corn is presented. Three AFB₁–AP conjugates in different toxin–enzyme ratios were prepared and tested. The ELISA results, developed with the most effective conjugate obtained, showed a satisfactory working range between 2.4 and 4000 ng of toxin/g of corn. The detection limit was 2 ng/g in corn samples, and recoveries ranged from 105 to 120%.     

Urease inhibitory profile of extracts and chemical constituents of Pistacia atlantica ssp. cabulica Stocks

The current study was designed to evaluate the urease inhibitory profile of extract and fractions of Pistacia atlantica ssp. cabulica Stocks followed by bioactivity-guided isolated compounds. The crude extract was found significantly active with urease inhibitor (95.40% at 0.2 mg/mL) with IC₅₀ values of 32.0 ± 0.28 μg/mL. Upon fractionation, ethyl acetate fraction displayed 100% urease inhibition with IC₅₀ values of 19.9 ± 0.51 μg/mL at 0.2 mg/mL. However, n-hexane and chloroform fractions exhibited insignificant urease inhibition. Similarly, the isolated compound, transilitin (1) and dihydro luteolin (2) demonstrated marked urease attenuation with 95 and 98% respectively, at 0.15 mg/mL. Both the isolated compounds showed marked potency with IC₅₀ values of 8.54 ± 0.54 and 9.58 ± 2.22 μg/mL, respectively. In short, both the extract and fractions and isolated compounds showed marked urease inhibition and thus a useful natural source of urease inhibition.     

Antileishmanial diterpenoid alkaloids from Aconitum spicatum (Bruhl) Stapf

The crude extracts of tubers of Aconitum spicatum (Bruhl) Stapf were investigated for in vitro antileishmanial activity against Leishmania major. The dichloromethane extract at pH 2.5 showed antileishmanial activity with IC₅₀ value of 27.10 ± 0.0 μg/mL. Chromatographic purification of the dichloromethane extract led to isolation of three C-19 norditerpenoid alkaloids indaconitine (1), chasmaconitine (2) and ludaconitine (3). Compounds 3 and 2 showed antileishmanial activity with IC₅₀ = 36.10 ± 3.4 and 56.30 ± 2.1 μg/mL, respectively. Compound 1 was less effective (IC₅₀ > 100 μg/mL). The cytotoxicity of compounds 1, 2 and 3 studied against MCF7, HeLa and PC3 cancer cell lines and 3T3 normal fibroblast cell line did not show cytotoxicity at 30 μM.     

Biological activity and LC-MS profiling of ethyl acetate extracts from Nitraria sibirica (Pall.) fruits

Nitraria sibirica is a traditional Uighur medicine. This study was undertaken to investigate the bioactivity of N. sibirica fruit extract and to evaluate their chemical compositions. The ethyl acetate extract from N. sibirica fruits exhibited the potential antioxidant activity (SC₅₀ = 30.17 ± 0.06 μg/mL) and protein tyrosine phosphatase 1B inhibitory activity (IC₅₀ = 7.15 ± 0.03 μg/mL) in vitro. In order to investigate the active constituents in this extract, a LC-QTOF-MS/MS method was developed and established. A total of 28 compounds including seven cinnamic acids, nine benzoic acids and 12 flavonoids were identified or partially characterised according to the accurate mass and the characteristic fragment ions at low and high collision energy. Most of them were reported for the first time in this plant. Phytochemical profiles of the active extract will help the development and utilisation of N. sibirica in food and medicine.     

Chemical constituents of essential oil of endemic Rhanterium suaveolens Desf. growing in Algerian Sahara with antibiofilm,antioxidant and anticholinesterase activities

Twenty compounds were detected in the essential oil of Rhanterium suaveolens representing 98.01% of the total oil content. Perillaldehyde (45.79%), caryophyllene oxide (24.82%) and β-cadinol (5.61%) were identified as the main constituents. In β-carotene–linoleic acid assay, both the oil and the methanol extract exhibited good lipid peroxidation inhibition activity, with IC₅₀ values of 17.97 ± 5.40 and 11.55 ± 3.39 μg/mL, respectively. In DPPH and CUPRAC assays, however, the methanol extract exhibited a good antioxidant activity. The highest antibiofilm activity has been found 50.30% against Staphylococcus epidermidis (MU 30) at 20 μg/mL for essential oil and 58.34% against Micrococcus luteus (NRRL B-4375) at 25 mg/mL concentration for methanol extract. The in vitro anticholinesterase activity of methanol extract showed a moderate acetylcholinesterase inhibitory (IC₅₀ = 168.76 ± 0.62 μg/mL) and good butyrylcholinesterase inhibitory (IC₅₀ = 54.79 ± 1.89 μg/mL) activities. The essential oil was inactive against both enzymes.     

GC×GC/qMS analyses of Campomanesia guazumifolia (Cambess.) O. Berg essential oils and their antioxidant and antimicrobial activity

The present study investigated the essential oil obtained from Campomanesia guazumifolia (Cambess.) O. Berg, an aromatic plant used in Brazilian folk medicine. The chemical composition was performed by GC×GC/qMS. The antioxidant and antimicrobial activities were evaluated by DPPH and BCB and, MIC assays, respectively. Sixty-eight compounds were identified in the oil, where the major compounds were bicyclogermacrene (15%), globulol (5%) and spathulenol (5%). Sesquiterpene hydrocarbons (29 compounds) and oxygenated sesquiterpenes (20 compounds) were the most representative classes of terpenes. DPPH (IC₅₀ value 26.1 ± 0.5 μg/mL) and BCB (68.3 ± 1.5%) values indicated a significant antioxidant activity. The essential oil strongly inhibited Staphylococcus aureus (MIC 15 ± 0.1 μg/mL), Escherichia coli (MIC 25 ± 0.2 μg/mL) and Candida albicans (MIC 5 ± 0.1 μg/mL). The results give a deeper understanding of the chemical composition and report for the first time the antioxidant and antimicrobial potential of the C. guazumifolia essential oil.     

Chemical composition and in vitro antibacterial and antiproliferative activities of the essential oil from the leaves of Psidium myrtoides O. Berg (Myrtaceae)

In this study, the chemical composition and antibacterial and antiproliferative potential of the essential oil obtained from fresh leaves of Psidium myrtoides (PM-EO) against oral pathogens and human tumour cell lines were investigated for the first time. GC-FID and GC-MS analyses showed that trans-β-caryophyllene (30.9%), α-humulene (15.9%), α-copaene (7.8%), caryophyllene oxide (7.3%) and α-bisabolol (5.3%) are the major constituents of PM-EO. The antibacterial activity of PM-EO against a panel of oral pathogens was investigated in terms of their minimal inhibitory concentrations (MIC) using the broth microdilution method. PM-EO displayed moderate activity against Streptococcus mitis (MIC = 100 μg/mL), S. sanguinis (MIC = 100 μg/mL), S. sobrinus (MIC = 250 μg/mL), and S. salivarius (MIC = 250 μg/mL), and strong activity against S. mutans (MIC = 62.5 μg/mL). The antiproliferative activity in normal (GM07492A, lung fibroblasts) and tumour cell lines (MCF-7, HeLa, and M059 J) was performed using the XTT assay. PM-EO showed 50% inhibition of normal cell growth at 359.8 ± 6.3 μg/mL. Antiproliferative activity was observed against human tumour cell lines, with IC₅₀ values significantly lower than that obtained for the normal cell line, demonstrating IC₅₀ values for MCF-7 cells (254.5 ± 1.6 μg/mL), HeLa cells (324.2 ± 41.4 μg/mL) and M059 J cells (289.3 ± 10.9 μg/mL). Therefore, the cytotoxicity of PM-EO had little influence on the antibacterial effect, since it showed antibacterial activity at lower concentrations. Our results suggest that PM-EO is a promising source of new antibacterial and antitumour agents.     

LC Simultaneous Determination of the Free Forms of B Group Vitamins and Vitamin C in Various Fortified Food Products

An LC–UV screening method for simultaneous determination of ascorbic acid (C), and the free forms of thiamine (B₁) riboflavin (B₂), niacin (B₃), pyridoxine (B₆) in enriched food products was developed and validated. The chromatographic separation was accomplished within 18 min using a gradient of water with 0.1% formic acid (pH 2.5) and methanol with 0.1% formic acid on a C₁₈ reverse phase column (5 μm, 150 × 3.2 mm) while detection was performed at two wavelengths (266 and 290 nm). Sample preparation was based on an extraction method originally developed for vitamin C. This procedure besides extracting vitamin C was extended to the extraction of the free forms of vitamins B₁, B_2, B_3, B₆ and B₉. The developed analytical method was successfully applied for the simultaneous determination of the vitamin C content along with the free vitamin B forms of three different enriched food products.

     

LC Simultaneous Determination of the Free Forms of B Group Vitamins and Vitamin C in Various Fortified Food Products

An LC–UV screening method for simultaneous determination of ascorbic acid (C), and the free forms of thiamine (B₁) riboflavin (B₂), niacin (B₃), pyridoxine (B₆) in enriched food products was developed and validated. The chromatographic separation was accomplished within 18 min using a gradient of water with 0.1% formic acid (pH 2.5) and methanol with 0.1% formic acid on a C₁₈ reverse phase column (5 μm, 150 × 3.2 mm) while detection was performed at two wavelengths (266 and 290 nm). Sample preparation was based on an extraction method originally developed for vitamin C. This procedure besides extracting vitamin C was extended to the extraction of the free forms of vitamins B₁, B_2, B_3, B₆ and B₉. The developed analytical method was successfully applied for the simultaneous determination of the vitamin C content along with the free vitamin B forms of three different enriched food products.     

Rapid detection of aflatoxin B1 on membrane by dot-immunogold filtration assay

Immunofiltration assay for mycotoxins in which nitrocellulose membrane (NCM) was used as a support and enzyme was used as the label has been developed since the late 1980s. As colloidal gold is a good labeling substance that can accelerate antibody-antigen reaction which result can be read directly by naked eyes, the colloidal gold particles could replace the enzyme to be labeled to antibody in aflatoxin B₁ (AFB₁) immunoassay. Dot-immunogold filtration assay (DIGFA) of AFB₁ on NCM was developed in this study. At first, the colloidal gold was synthesized and colloidal gold-monoclonal antibody (McAb) conjugates against AFB₁ were prepared at pH 7.0 of colloidal gold solution, 0.018 mg/mL of McAb. Then the colloidal gold-McAb conjugates were used to develop AFB₁ DIGFA, which detection time was only 15 min, six times less than that of ELISA. With this method to determine the standard AFB₁ solution, the results demonstrated a visual detection limit of approximately 2 ng/mL of AFB₁, which was similar to that of ELISA. This method had good specificities for AFG₁, AFG₂ and AFM₁ and a little cross-reactivity with AFB₂. 45 food samples collected from the markets were subjected to DIGFA and the results showed that one corn sample was positive and in agreement that of HPLC. It is suggested that DIGFA developed in current study has a potential use as a rapid and cost-effective screening tool for the determination of AFB₁ in foods in the field within 15 min without complicated steps.     

Bioactive DOPA melanin isolated and characterised from a marine actinobacterium Streptomyces sp. MVCS6 from Versova coast

The melanin pigment produced from Streptomyces sp., MVCS6 was isolated and dihydroxyphenyalanine (DOPA) melanin compound was biochemically identified and spectroscopically characterised (ultraviolet and FT-IR). DOPA melanin showed a promising activity as an antibacterial natural product against 12 pathogenic bacteria from hospital isolations, particularly, against Pseudomonas aeruginosa RMMH7 (inhibition zone of 18 ± 0.02 at 30 μg/disc, and MIC of 10 ± 0.02 μg/mL) and Vibrio parahaemolytics RMMH12 (inhibition zone of 15 mm ± 0.03 at 30 μg/disc, and MIC of 14 ± 0.02 μg/mL). Moreover, in vitro evaluation of reducing power (Ascorbic Acid Equivalent (160 μg/mL)), DPPH radical-scavenging (89%), NO-scavenging (72%) and lipid peroxidation activities (89.6%) were determined. Cytotoxicity of DOPA melanin against cervical cancer cell line showed a dose–response activity, and IC₅₀ value was found to be 300 μg/mL. These results would open the way to propose Streptomyces sp. MVCS6 as a promising source of bioactive eumelanin with therapeutic potential in medicine.     

Polyphenolic profiling of Ipomoea carnea Jacq. by HPLC-DAD and its implications in oxidative stress and cancer

Ipomoea carnea Jacq. is an important folklore medicinal plant, assessed for its underexplored biological potential. Antioxidant, cytotoxic, antiproliferative and polyphenolic profile of whole plant was evaluated using various techniques. Maximum extract recovery (29% w/w), phenolic [13.54 ± 0.27 μg GAE/mg dry weight (DW)] and flavonoid (2.11 ± 0.10 μg QE /mg DW) content were recorded in methanol-distilled water (1:1) flower extract. HPLC-DAD analysis quantified substantial amount of six different polyphenols ranging from 0.081 to 37.95 μg/mg extract. Maximum total antioxidant and reducing potential were documented in methanol-distilled water and acetone-distilled water flower extracts (42.62 ± 0.47 and 24.38 ± 0.39 μg AAE/mg DW) respectively. Ethanol-chloroform root extract manifested highest free radical scavenging (IC₅₀ of 61.22 μg/mL) while 94.64% of the extracts showed cytotoxicity against brine shrimps. Ethanol leaf extract exhibited remarkable activity against THP-1 cell line (IC₅₀ = 8 ± 0.05 μg/mL) and protein kinases (31 mm phenotype bald zone).