Influence of human gut microbiota on the metabolic fate of glucosinolates

Glucosinolates (GSLs) are secondary metabolites predominantly found in cruciferous vegetables such as broccoli, brussel sprout, cabbage and cauliflower which upon chopping and chewing will release the indigenous plant myrosinase enzyme that catalyzes the hydrolysis of GSLs. This hydrolysis releases a range of breakdown products including isothiocyanates (ITCs), which have been implicated in the cancer-protective effects of cruciferous vegetables. Certain human gut bacteria are able to metabolize GSLs and produce ITCs for human health benefits. In this work, six GSL-metabolizing bacterial strains were isolated from human faecal sample and identified. Most bacteria were capable of producing both nitriles (NITs) and ITCs from the metabolism of GSLs however Enterococcus sp. C213 and Enterococcus faecium KT4S13 produced only NITs. Enterococcus casseliflavus NCCP-53, Escherichia coli O83:H1 NRG 857C and Lactobacillus agilis R16 were able to metabolize different types (allyl, aromatic, methylthioalkyl, methylsulfinylalkyl and indolyl) of GSLs differently over 24 h of in vitro anaerobic fermentations. For all GSLs, ITC production seemed to peak between 4 and 8 h of incubation and then declined due to the inherent instability of ITCs in culture broths and buffers. In contrast, NIT productions gradually increased over time and remained relatively constant. The total percentage products from each GSL metabolism in all three bacteria never reached 100%. Interestingly, E. coli O83:H1 NRG 857C produced methylthioalkyl ITCs and NITs from methylsulfinylalkyl GSLs while E. casseliflavus NCCP-53 produced only methylsulfinylalkyl ITCs from the same GSLs. This difference was due to reductase activity in E. coli O83:H1 NRG 857C intact cells and cell-free extracts that biotransforms the sulfoxide groups of methylsulfinylalkyl GSLs to the sulfide groups. The reductase enzyme is yet to be identified at the gene and protein level, however it has been characterized using cell-free extracts in this work. This reductase is inducible by GSLs, oxygen-independent and requires Mg2+ ion and NADP(H) as co-factors for its activity with optimum pH and temperature at pH 7.0 and 37˚C, respectively. Arylsulfatase activity was also detected in this bacterium. The corresponding recombinant SUL2 enzyme (57 kDa) of E. coli O83:H1 NRG 857C expressed in BL21(DE3) exhibited arylsulfatase activity by desulfating synthetic p-nitrocatachol sulfate substrate with optimum pH and temperature at pH 6.0 and 30˚C, respectively. In addition, GSL-sulfatase activity was detected in crude extracts by being able to desulfate different intact GSLs to produce desulfo-glucosinolates (DS-GSLs) with less efficiency in comparison with the commercially available snail sulfatase from Helix pomatia. The catalytic efficiency of recombinant SUL2 enzyme for GSLs in descending order is as follows; sinigrin > glucoerucin > gluconasturtiin > glucoiberin. The DS-GSLs (except DS-glucoraphanin) then act as substrates for the recombinant GH3 enzyme defived from E. casseliflavus NCCP-53 to produce the corresponding NIT products in NB broths and the buffer with the presence of 5 mM Fe2+ ions. This enzyme (79 kDa) showed β-O-glucosidase activity for p-nitrophenyl β-D-glucopyranoside with optimum pH and temperature at pH 7.0 and 37˚C, respectively. NIT productions only occurred from the metabolism of intact GSLs in bacterial culture broths, but not in the buffers unless 5 mM Fe2+ ions are present as co-factors. Putative bacterial GSL-degrading enzymes responsible for ITC and NIT productions from GSL metabolisms are inducible by GSL in resting cells experiments. By using two-dimensional gel electrophoresis (2-DE) and liquid chromatography mass spectrometry (LC-MS/MS) for the comparative analysis between proteins obtained from cultures of L. agilis R16 and E. coli O83:H1 NRG 857C with and without GSL supplementation, upregulated/distinct proteins that may be involved in the metabolism of GSLs by these bacteria were identified. These proteins belong to (sugar) transport system, carbohydrate metabolism especially kinases and oxidoreduction process. To date, bacterial GSL-degrading enzyme is yet to be identified.Open Acces

Bioavailability of Glucoraphanin and Sulforaphane From High-Glucoraphanin Broccoli

Scope: Broccoli accumulates 4-methylsulphinylbutyl glucosinolate (glucoraphanin) which is hydrolyzed to the isothiocyanate sulforaphane. Through the introgression of novel alleles of the Myb28 transcription factor from Brassica villosa, broccoli genotypes have been developed that have enhanced levels of glucoraphanin. This study sought to quantify the exposure of human tissues to glucoraphanin and sulforaphane following consumption of broccoli with contrasting Myb28 genotypes. Methods and results: Ten participants were recruited into a three-phase, double-blinded, randomized crossover trial (NCT02300324), with each phase comprising consumption of 300 ml of a soup made from broccoli of one of three Myb28 genotypes (Myb28B/B, Myb28B/V, Myb28V/V). Plant myrosinases were intentionally denatured during soup manufacture. Three-fold and five-fold higher levels of sulforaphane occurred in the circulation following consumption of Myb28V/B and Myb28V/V broccoli soups, respectively. The percentage of sulforaphane excreted in 24 h relative to the amount of glucoraphanin consumed varied amongst volunteers from 2% to 15%, but did not depend on the broccoli genotype. Conclusion: This is the first study to report the bioavailability of glucoraphanin and sulforaphane from soups made with novel broccoli varieties. The presence of one or two Myb28V alleles results in enhanced delivery of sulforaphane to the systemic circulation

Application of Soil Bacteria as Bioinoculants to Promote Growth of Cowpea (Vigna unguiculata)

This work aimed to evaluate the capacity of soil bacteria as bioinoculants (biofertilizers) to promote cowpea (Vigna unguiculata) growth. Three pure bacterial cultures namely Acinetobacter pittii PT1.3.4 (AP), Achromobacter sp.C2.23 (AS), and Achromobacter xylosoxidans N3.4 (AX) were used as bioinoculants to enhance germination and development of cowpea seeds. Pre-decide formulations of single or mixed cultures were prepared, soaked with cowpea seeds, and cultivated on agar in a growth chamber for 7 days at 25°C. Shoot and root length were measured and percentage germination was determined. Similarly, bacterial formulations were prepared in talcum powder and were used as bioinoculants to adhere to cowpea seeds. The inoculated seeds were cultivated in pots for 28 days for the shoot and root length, fresh and dry weight, and percentage germination. Among the tested various formulations, treatment has A. pittii (AP) displayed the highest shoot length (14.67 cm) and fresh weight (0.58 g/plant) of cowpea under laboratory conditions after seven days of inoculation. Similarly, cowpea plants treated with A. pittii (AP) also have the tallest shoots (14.25 cm) under natural conditions after 7 days of inoculation, while the highest root length (10.5 cm) and fresh weight (1.57 g/plant) were recorded from the treatment of Achromobacter sp. (AS). Further, the results of the study also revealed that soil bacteria can survive for one month in talcum powder at 4°C and room temperature storage. These bioinoculants can be used for agricultural application by local farmers to mitigate the cost of chemicals that cause environmental concerns to promote sustainable agriculture in Thailand

Antioxidant properties and cytotoxic effects of Alternanthera sissoo and Alternanthera bettzickiana extracts against cancer cells

This work aimed to evaluate the antioxidant capacities and cytotoxic attributes of Alternanthera sissoo (AS) and Alternanthera bettzickiana Green (AB) which are commonly grown in Thailand, yet not extensively-studied. Dried leaves were extracted by maceration using ethanol for 48 h and ethanolic extract solution was used. Cytotoxic, anti-proliferative, anti-migratory capacities and apoptosis-related gene expressions of AS and AB extracts on MCF-7, HepG2, and HeLa cell lines were investigated using 3-(4,5-dimethylthiazolyl-2)-2, 5-diphenyltetra zolium bromide (MTT) assay, anti-colony formation and wound healing test, respectively. The antioxidant activities by DPPH assay and FRAP assay of AS (130.62±1.61 µg TE/g extract and 24.76 ± 0.54 mg Fe(II)/g extract, respectively) were significantly higher than those of AB (104.34±3.68 µg TE/g extract and 23.13± 0.90 mg Fe(II)/g extract, respectively). Likewise, total phenolic content and the total flavonoid content of AS (7.43±0.39 mg GAE/g and 184.22±3.20 mg RE/g extract, respectively) were significantly higher than AB (6.10±0.29 mg GAE/g extract and 168.07±7.90 mg RE/g extract, respectively). HPLC analysis showed the predominance of myricetin, rutin and ferulic acid. For MCF-7, HepG2 and HeLa, the lowest IC50 values of 48.53 µg/mL, 69.94 µg/mL and 43.98 µg/mL, respectively were found in AS extract at 72 h exposure using MTT assay. Apoptotic bodies, gene expression and protein expressions related to apoptosis were more pronounced in AS treatment. Both AS and AB can be served as local foods with chemopreventive effects against breast, liver and cervical cancers. This work provided a foundation for future pharmacological research on Alternanthera plant extracts

Cytotoxicity and antiproliferative activity of essential oils from lemon, wild orange and petitgrain against MCF-7, HepG2 and HeLa cancer cells

The purpose of this study was to determine the chemical composition and biological properties of the citrus essential oils (EOs) derived from orange rinds (peels) of lemon (Citrus limon), wild orange (Citrus sinensis) from Brazil extracted by the cold pressed/expressed method, and leaves and twigs of petitgrain (Citrus aurantium) from Paraguay extracted by steam distillation. These food grade EOs were evaluated for their cytotoxic activity in breast, liver, and cervical cancer cells (MCF-7, HepG2 and HeLa) via MTT assay, antiproliferative activity via colony formation assay, and antimigratory activity via wound healing assay, and apoptosis via DNA fragmentation and morphology assessment. The major compounds found in lemon EO were D-limonene (66.75%), beta-pinene (12.82%), and gamma-terpinene (11.57%), totaling over 90% of the identified compounds. For wild orange, the only predominant compound was limonene (96.60%), and the rest, found in minor amounts, included alpha-pinene, bicyclohexane, beta-pinene, beta-myrcene, 3-carene, and o-cymene. For petitgrain EO, linalyl isobutyrate (51.76%) and linalool (26.86%) were mainly detected. Based on the MTT assay, petitgrain EO was the most effective against MCF-7, HepG2 and HeLa. However, wild orange EO was the most antiproliferative and antimigratory against all three cells using the anticolony formation assay and wound healing assay, respectively. The results showed that cell death is associated with the apoptotic process, with morphological hallmarks of apoptosis including membrane blebbing and DNA fragmentation. These findings imply that the three citrus EOs might be used as active components in functional food products for chemopreventive benefits

Anticancer effects of Rhinacanthus nasutus and Acanthus ebracteatus extracts against human cervical cancer cells

Cervical cancer is second only to breast cancer in terms of incidence; however, it is the most lethal form of cancer among Thai women due to the asymptomatic nature of its early stages. This work aimed to examine cytotoxic and antiproliferative capacities of Rhinacanthus nasutus (RN) and Acanthus ebracteatus (AE) extracts against human cervical cancer cells (HeLa). Plant leaves were used for ethyl acetate extraction. The antioxidant assays, HPLC analysis, a cytotoxic MTT assay, a clonogenic assay and real-time PCR were conducted. Both RN and AE displayed similar DPPH scavenging activity (3.97 and 4.05 mg TE/g DW) and ferric reducing antioxidant power (4.79 and 4.35 mg Fe2+/g DW). However, AE was richer in total phenolic content than RN (13.30 and 10.84 mg GAE/g DW, respectively). Rutin, catechin, chlorogenic acid, and cinnamic acid were found in AE, whilst only cinnamic acid with much higher content was found in RN. Higher cytotoxicity of 91.73% against HeLa cells was found in RN (IC50 value of 62.06 µg/mL). RN showed higher antiproliferative effect (IC50 of 25.24 µg/mL) than AE (34.35 µg/mL). Genes (Bcl-2, Bax, MMP-2, MMP-9, caspase-3, p21, and cyclin D1) and proteins (cytochrome c, caspase-3 and p21) linked to apoptosis and migration were substantially more affected by RN. To conclude, both RN and AE hold promise as anticancer herbal plants against human cervical cancer; however, RN was more cytotoxic and antiproliferative in HeLa cells. RN offers a better alternative herbal medicine or complementary remedy to the standard drug for human cervical cancer treatment

Cytotoxic effects of Saccharomyces cerevisiae TC6 and Lactobacillus brevis TBRC 3003 isolated from Thai fermented foods

Purpose: To determine the cytotoxic effect, anti-colony formation effect and antimigratory effect of Saccharomyces cerevisiae TC6 isolated from Thai water kefir, and Lactobacillus brevis TBRC 3003 isolated from picked cabbage. Methods: Crude microbial extracts were obtained from whole cultures (cells and broths) using ethyl acetate as extracting solvent, and the dried extracts were redissolved in ethanol before use. Cytotoxic, antiproliferative and antimigratory effects of the two microbial extracts on MCF-7, HepG2, and HeLa were tested using 3-(4,5-dimethylthiazolyl-2)-2, 5-diphenyltetra zolium bromide (MTT), clonogenic formation and wound healing assays. Results: Lb. brevis TBRC 3003 showed the highest cytotoxicity toward HepG2 cells (IC50 of 669.72 µg/mL), while S. cerevisiae TC6 showed the highest cytotoxicity against MCF-7 (IC50 of 691.49 µg/mL) and HeLa (IC50 of 379.16 µg/mL) based on MTT assay. Anti-colony formation test showed that S. cerevisiae TC6 was most the most effective in inhibiting colony formation of HepG2 (IC50 of 311.12 µg/mL) and HeLa (IC50 of 494.64 µg/mL), while Lb. brevis TBRC 3003 was the most potent in inhibiting colony formation of MCF-7 (IC50 of 267.88 µg/mL). Conclusion: Both microbes can potentially be implemented in functional foods as bio-therapeutics with chemopreventive properties against breast, liver and cervical cancers

Cytotoxic and antiproliferative effects of Streblus asper from northeastern Thailand on A549 lung cancer cells

Thailand's second-leading cause of death is lung cancer. Thai indigenous herbal plants are sought after as an alternative treatment against lung cancer. This work aimed to examine cytotoxic, antiproliferative and antimigratory capacities of different parts of Streblus asper (SA) from northeastern Thailand on A549 lung cancer cells. Plant leaves, twigs, bark and wood were used for ethanolic extraction by maceration. The highest cytotoxicity of 85.46% was found in twig extract (IC50 57.46 µg/mL) assessed by an MTT assay. The lowest IC50 (18.42 µg/mL) was also found in twig extract using a clonogenic assay indicating its most antiproliferative activity in a long-term therapy. In addition, all SA extracts displayed antimigratory activity against A549 cells in a dose-dependent fashion, especially twig extract. Apoptotic characteristics were noticeable in SA extract treated cells. The maximum DPPH-scavenging activity, FRAP value, total phenolic and flavonoid content were found in twig extract. GC-MS analysis revealed that twig extract contained four prominent components namely ethyl-α-D-glucopyranoside, hexadecanoic acid, ethyl ester, lupeol and γ-sitosterol. Real-time PCR results showed that genes (Bcl-2, Bax, p21, and cytochrome c) linked to apoptosis were significantly affected by all SA extracts. The various SA components' ethanolic extracts exhibited moderate-to-high cytotoxic action towards A549 cells. This work will significantly advance the utilization of the plant as an alternative source of medicine for rural Thais, and it paves the way for future research to determine the active compound(s) and anticipate new drug candidates

Impact of non-thermal plasma seed priming and early development stages of two local Thai Cruciferous plants mustard green and rat-tailed radish on glucosinolates, isothiocyanates, minerals, antioxidant and anticancer activities

Over the past decade, non-thermal plasma (NTP) technology has emerged as a promising tool in the food sector. Cruciferous microgreens are known for their anticancer properties, yet the potential of certain varieties at early developmental stages remains underexplored. This study investigated the effects of NTP treatment on seed priming and plant development at 14, 21, and 28 days in mustard green (MG) (Brassica juncea (L.) Czern) and rat-tailed radish (RTR) (Raphanus sativus var. caudatus), focusing on their bioactive compounds and bioactivities. NTP treatment significantly affects stem length, fresh weight, or dry weight compared to untreated seeds. It also enhanced the production of glucosinolates, isothiocyanates (ITCs), specific minerals, total phenolics, total flavonoids, and biological activities in both plants. MG was found to contain sinigrin, gluconapin, allyl ITC, and 3-butenyl ITC, while RTR contained glucoraphasatin and raphasatin. MG exhibited IC50 values ranging from 16–78 µg/mL in cytotoxicity tests against four cancer cell lines HeLa, HepG2, MCF-7 and HT-29 with enhanced activity from NTP. RTR demonstrated greater effectiveness with IC50 values of 12–60 µg/mL with higher activity from NTP. Both plant extracts especially NTP-treated samples reduced cancer cell survival and proliferation by upregulating pro-apoptotic genes (Bax, caspase-3, and p21) and proteins while downregulating anti-apoptotic and metastatic markers (Bcl-2, MMP-9, MMP-2, and cyclin D1). NTP can enhance the therapeutic bioactivity of young plants, with both MG and RTR at 14 and 21 days of growth, showing the higher potential for anticancer applications

Omics technologies used in pesticide residue detection and mitigation in crop

In agriculture, the convenience and efficacy of chemical pesticides have become inevitable to manage cultivated crop production. Here, we review the worldwide use of pesticides based on their categories, mode of actions and toxicity. Excessive use of pesticides may lead to hazardous pesticide residues in crops, causing adverse effects on human health and the environment. A wide range of high-tech-analytical methods are available to analyze pesticide residues. However, they are mostly time-consuming and inconvenient for on-site detection, calling for the development of biosensors that detect cellular changes in crops. Such new detection methods that combine biological and physicochemical knowledge may overcome the shortage in current farming to develop sustainable systems that support environmental and human health. This review also comprehensively compiles domestic pesticide residues removal tips from vegetables and fruits. Synthetic pesticide alternatives such as biopesticide and Nano pesticide are greener to the environment. However, its safety assessment for large-scale application needs careful evaluation. Lastly, we strongly call for reversions of pesticide application trends based on the changing climate, which is lacking in the current scenario