Enzyme Myrosinase Research Articles

The Potential of Glucosinolates and Their Hydrolysis Products as Inhibitors of Cytokine Storms

A cytokine storm is an intense inflammatory response characterized by the overproduction of proinflammatory cytokines, resulting in tissue damage, and organ dysfunction. Cytokines play a crucial role in various conditions, such as coronavirus disease, in which the immune system becomes overactive and releases excessive levels of cytokines, including interleukins, tumor necrosis factor-alpha (TNF-α), and interferon-gamma (IFN-γ). This anomalous response often leads to acute respiratory distress syndrome (ARDS), disseminated intravascular coagulation (DIC), and multiple organ injury (MOI). Glucosinolates are plant secondary metabolites predominantly found in Brassica vegetables, but are also present in other species, such as Moringa Adens and Carica papaya L. When catalyzed by the enzyme myrosinase, glucosinolates produce valuable products, including sulforaphane, phenethyl isothiocyanate, 6-(methylsulfinyl) hexyl isothiocyanate, erucin, goitrin, and moringin. These hydrolyzed products regulate proinflammatory cytokine production by inhibiting the nuclear factor kappa-light-chain-enhancer of activated B-cell (NF-κB) signaling pathway and stimulating the nuclear factor erythroid 2-related factor 2 (Nrf2) signaling pathway. This action can alleviate hyperinflammation in infected cells and modulate cytokine storms. In this review, we aimed to examine the potential role of glucosinolates in modulating cytokine storms and reducing inflammation in various conditions, such as coronavirus disease. Overall, we found that glucosinolates and their hydrolysis products can potentially attenuate cytokine production and the onset of cytokine storms in diseased cells. In summary, glucosinolates could be beneficial in regulating cytokine production and preventing complications related to cytokine storms.

Myrosin Cells and Myrosinase Expression Pattern in Nasturtium (Tropaeolum majus L.)

Plants from the Brassicales order are known for the presence of a glucosinolate–myrosinase link, which is an important protection strategy against multiple stressors. The main goal of this study was to investigate the presence of the myrosinase enzyme and reveal the myrosin cell ultrastructure in the vegetative organs of nasturtium. The presence, localisation and expression of the enzyme myrosinase type 1 (TGG1) at different developmental stages of Tropaeolum majus L. (nasturtium) were investigated using immunohistochemical and immunofluorescent techniques. The expression of myrosinase was detected in the vegetative organs of T. majus. During plant development, within four consecutive weeks, a decrease in myrosinase expression was noticed in all studied plant organs. The location of greater myrosinase accumulation and activity is shown to be the root, contrary to the nasturtium stem and leaf, where we found the lowest myrosinase expression. Transmission electron microscopy was used to reveal the ultrastructural features of the myrosin cells of nasturtium. Myrosin cells are usually scattered between parenchyma cells and S-cells. Mostly, they are rectangular or slightly elongated in shape and can be recognised by an electron-dense large central vacuole and an expanded rough endoplasmic reticulum. The results of this study provide new data on myrosin cell morphology and the expression pattern of myrosinase in T. majus.

Exploring the therapeutic effects of sulforaphane: an in-depth review on endoplasmic reticulum stress modulation across different disease contexts.

The endoplasmic reticulum (ER) is an intracellular organelle that contributes to the folding of proteins and calcium homeostasis. Numerous elements can disrupt its function, leading to the accumulation of proteins that are unfolded or misfolded in the lumen of the ER, a condition that is known as ER stress. This phenomenon can trigger cell death through the activation of apoptosis and inflammation. Glucoraphanin (GRA) is the predominant glucosinolate found in cruciferous vegetables. Various mechanical and biochemical processes activate the enzyme myrosinase, leading to the hydrolysis of glucoraphanin into the bioactive compound sulforaphane. Sulforaphane is an organosulfur compound that belongs to the isothiocyanate group. It possesses a wide range of activities and has shown remarkable potential as an anti-inflammatory, antioxidant, antitumor, and anti-angiogenic substance. Additionally, sulforaphane is resistant to oxidation, has been demonstrated to have low toxicity, and is considered well-tolerable in individuals. These properties make it a valuable natural dietary supplement for research purposes. Sulforaphane has been demonstrated as a potential candidate drug molecule for managing a range of diseases, primarily because of its potent antioxidant, anti-inflammatory, and anti-apoptotic properties, which can be mediated by modulation of ER stress pathways. This review seeks to cover a wealth of data supporting the broad range of protective functions of sulforaphane, improving various diseases, such as cardiovascular, central nervous system, liver, eye, and reproductive diseases, as well as diabetes, cancer, gastroenteritis, and osteoarthritis, through the amelioration of ER stress in both in vivo and in vitro studies.

Bio-efficacy of mustard seed extracts against Bihar hairy caterpillar Spilosoma obliqua (Erebidae: Lepidoptera) & assessment of mustard allelo-chemicals in response to mustard aphid Lipaphis erysimi (Aphididae: Hemiptera) infestation

Mustard seed contains glucosinolate-myrosinase system of substrate-enzyme metabolism, accompanied with compartmentalized cells. Upon rupture, enzyme myrosinase leads to breakdown of glucosinolates and its metabolized products like allyl, benzyl, phenyl type of isothiocyantes and many other types like nitriles are released. The myrosinase activity is tested in three globally utilized different seed coat color mustards viz., brown mustard (Brassica juncea), white mustard (Sinapis alba) and black mustard (Brassica nigra) to demonstrate the release of glucosinolates which are known to deter insect pests. Aqueous seed extracts of these mustard types were subjected to anti-feedant activity analysis and oil extracted from these mustard seeds was subjected to insecticidal activity analysis against Spilosoma obliqua 4th instars on castor leaves. Meanwhile, the mustard leaves in open field were allowed for natural aphid infestation during early months from January to March. Thereafter, the leaf twigs of white, black and brown mustard, infested and uninfested by aphids, were collected and myrosinase activity of plants was analyzed with varying levels of important pest inflicted biochemicals in plant, viz., Total phenols, ortho-dihydric phenols and flavonols. Gas Chromatography–Mass Spectrometry analysis of mustard oil used in insecticidal activity was performed to elucidate the role of biochemicals involved in action. These compounds along with fatty acids and sterols compounds are known to have anti-feedant & insecticidal activities. Aqueous extract components were also subjected to total glucosinolate analysis, spectrophotometrically for knowing their concentration, with highest in brown (85.81 µmol/g) followed by black (59.32 µmol/g) and lowest in white mustard (47.63 µmol/g). The amount of glucosinolates, free fatty acids & sterol compounds present in different coat color mustard seed types are being correlated here with their potential bioactivities against biological plant pests. Bio-control of potentially harmful insect pests is possible by utilizing anti-feeding & insecticidal potential of mustard seed extracts.

Sulforaphane and ophthalmic diseases.

Sulforaphane (SFN) is an organosulfur compound categorized as an isothiocyanate (ITC), primarily extracted from cruciferous vegetables like broccoli and cabbage. The molecular formula of sulforaphane (SFN) is C6H11NOS2. SFN is generated by the hydrolysis of glucoraphanin (GRP) through the enzyme myrosinase, showing notable properties including anti-diabetic, anti-inflammatory, antimicrobial, anti-angiogenic, and anticancer attributes. Ongoing clinical trials are investigating its potential in diseases such as cancer, neurodegenerative diseases, diabetes-related complications, chronic kidney disease, cardiovascular disease, and liver diseases. Several animal carcinogenesis models and cell culture models have shown it to be a very effective chemopreventive agent, and the protective effects of SFN in ophthalmic diseases have been linked to multiple mechanisms. In murine models of diabetic retinopathy and age-related macular degeneration, SFN delays retinal photoreceptor cell degeneration through the Nrf2 antioxidative pathway, NF-κB pathway, AMPK pathway, and Txnip/mTOR pathway. In rabbit models of keratoconus and cataract, SFN has been shown to protect corneal and lens epithelial cells from oxidative stress injury by activating the Keap1-Nrf2-ARE pathway and the Nrf-2/HO-1 antioxidant pathway. Oral delivery or intraperitoneal injection at varying concentrations are the primary strategies for SFN intake in current preclinical studies. Challenges remain in the application of SFN in eye disorders due to its weak solubility in water and limited bioavailability because of the presence of blood-ocular barrier systems. This review comprehensively outlines recent research on SFN, elucidates its mechanisms of action, and discusses potential therapeutic benefits for eye disorders such as age-related macular degeneration (AMD), diabetic retinopathy (DR), cataracts, and other ophthalmic diseases, while also indicating directions for future clinical research to achieve efficient SFN treatment for ophthalmic diseases.

QM/MM study of the catalytic reaction of aphid myrosinase

Brevicoryne brassicae, an aphid species, exclusively consumes plants from the Brassicaceae family and employs a sophisticated defense mechanism involving a myrosinase enzyme that breaks down glucosinolates obtained from its host plants. In this work, we employed combined quantum mechanical and molecular mechanical (QM/MM) calculations and molecular dynamics (MD) simulations to study the catalytic reaction of aphid myrosinase. A proper QM region to study the myrosinase reaction should contain the whole substrate, models of Gln-19, His-122, Asp-124, Asn-166, Glu-167, Lys-173, Tyr-180, Val-228, Tyr-309, Tyr-346, Ile-347, Glu-374, Glu-423, Trp-424, and a water molecule. The calculations show that Asp-124 and Glu-423 must be charged, His-122 must be protonated on NE2, and Glu-167 must be protonated on OE2. Our model reproduces the anomeric retaining characteristic of myrosinase and indicates that the deglycosylation reaction is the rate-determining step of the reaction. Based on the calculations, we propose a reaction mechanism for aphid myrosinase-mediated hydrolysis of glucosinolates with an overall barrier of 15.2 kcal/mol. According to the results, removing a proton from Arg-312 or altering it to valine or methionine increases glycosylation barriers but decreases the deglycosylation barrier.

In silico studies on characterization of glucosinolate derivatives for inhibition ofcell cycle regulation protein BCL-2

Glucosinolates (GSL) are well-known secondary metabolites present in plants belonging to the Brassicaceae family (Cabbage, broccoli, cauliflower and brussels sprouts) and have been studied for their pharmacological properties. Glucosinolate is a sulfur-rich anionic hydrolyzed product which is good for the human diet. Through the enriched diet, the myrosinase enzyme hydrolyzes glucosinolates to form isothiocyanates against bacterial and fungal infections. Glucosinolates also show antimicrobial, anti-inflammatory, anticancer and cholinesterase inhibitor activity. The present study is to evaluate the glucosinolate substructures retrieved from the chemical database and to evaluate to understand that the BCL-2 inhibition showed the tumor growth and induced apoptosis. We retrieved 117 glucosinolate compounds and in silico druglike prediction screened the compounds and molecular docking against the BCL-2 protein structure. Based on the druglikeness analysis, we identified 39 compounds and these compounds were used for the inhibitory study with BCL2 protein. The results show that 2-Methyl-2-propenyl glucosinolate, 4-Methylpentyl glucosinolate, 4-Mercaptobutyl glucosinolate, Glucocapparin, Glucoerucin, Glucolepidiin, Glucoputranjivin, Glucoviorylin, n-Butyl glucosinolate compounds are strongly binding to BCL-2 protein structure by forming three hydrogen bonds and the resultant compounds can be used as a potential lead molecule for BCL-2 inhibitors.

Mechanical Digestion of Broccoli through Chewing and its Impact on Myrosinase Activity

Myrosinase activity on broccoli glucosinolates has been widely and extensively discussed. Purpose: Studies on cruciferous vegetables, especially broccoli, have gained significance in the fight against cancer. Glucosinolates in broccoli transformation into sulforaphane occur after its exposure through chewing. However, the relationship between individual chewing patterns and denture morphology has not been studied extensively. Research on human digestion has demonstrated how the mechanical breakdown of larger food particles into smaller ones is a crucial precursor to chemical food breakdown. This study is a comparative analysis of how chewing on broccoli tissues by different individuals enables the enzyme myrosinase to break down broccoli glucosinolates chemically. We investigate the individual chewing patterns linked to the surface anatomy of the pre-molars and molars and the myrosinase activity on broccoli glucosinolates. Methods: Three individuals chewed a 4-millimeter broccoli floret four times sequentially (2 grams), and we measured the floret length at each bite until the sample was ground. Then, we combined the chewed broccoli with distilled water, filtered it, and myrosinase activity was measured using photo spectrometric measurements and an agar diffusion test. Individual pre-molars and molars samples were measured in millimetres. Results: Data from the three human chewing mechanisms compared to the mechanical breakdown performed by an automatic mixer shows different and individually specific values. Conclusion: Individual chewing patterns link to the unique surface anatomy of the pre-molars and molars, subsequently impacting the myrosinase activity on broccoli glucosinolates’ breakdown individually

Comparing two strategies of counter-defense against plant toxins: a modeling study on plant–herbivore interactions

IntroductionVarious herbivorous insects prefer toxic plants as their hosts, although this may appear paradoxical. They have evolved specific adaptations (called counter-defenses) against the toxins. For example, the two-component chemical defense system of plants of the Brassicaceae family against herbivores consists of glucosinolates (GLSs) and the activating enzyme myrosinase. GLS hydrolysis by myrosinase leads to isothiocyanates (ITCs), which are toxic and deterrent to many insect herbivores. Two different types of counter-defenses can be distinguished: a preemptive counter-defense that prevents the GLSs from being hydrolyzed to ITCs due to metabolic redirection and direct counter-defense, where the ITCs are formed but then metabolized to non-toxic conjugates. In general, preemptive counter-defense is only a possibility if the plant stores a precursor of the toxin, which is activated upon attack. Preemptive counter-defense is believed to be more efficient due to the lower exposure to ITCs, but this has not been satisfactorily demonstrated experimentally.MethodsHere, we derive on theoretical grounds the conditions under which preemptive counter-defense reduces exposure to ITCs compared to direct counter-defense by studying the dynamics of GLS defense and counter-defense with two separate ordinary differential equation models. We model how herbivory transfers the GLSs to the insect gut with the leaf material. Thereafter, we describe the insects’ exposure to toxins by deriving the dynamics of ITCs in the gut during feeding with the two types of counter-defenses.Results and discussionBy calculating the area under the curve (AUC) of the ITC concentrations, we show, based on empirical data, that herbivores with a preemptive detoxification system are usually less exposed to ITCs. In addition, our models explain how the decline in the level of ITCs is achieved by both counter-defenses, which helps to understand the overall mechanisms and benefits of these techniques. Our results may also apply to plant defenses by inactive toxin precursors other than GLSs as well as to insects that sequester such precursors for their own defense.

Glucosinolate Accumulation and Hydrolysis in Leafy Brassica Vegetables Are Influenced by Leaf Age.

The health-beneficial effects of Brassica vegetables are mainly attributed to their high contents of glucosinolates and the products of their hydrolysis, especially isothiocyanates. Distribution of glucosinolates across plant organs can strongly vary. Here, we investigated the effect of leaf age on glucosinolate accumulation and hydrolysis in two leafy Brassica vegetables, pak choi and giant red mustard. We also evaluated the activity of the hydrolyzing enzyme myrosinase across the leaves. Finally, we assessed whether glucosinolates are transported from older leaves to younger leaves. Young leaves of both species contained more than 3-fold more glucosinolates than older ones. Accordingly, more isothiocyanates were released in the young leaves. Myrosinases fully hydrolyzed all of the amounts of glucosinolates regardless of the leaf age. Moreover, older leaves were observed to supply younger leaves with glucosinolates. Thus, this study suggests that consumers can improve the nutritional value of food by incorporating young leaves of leafy Brassicas in their diet.

Perubahan Fıtokımıa Kecambah Kembang Kol (Brassica oleraceae L., var. botrytis) sebagai Respons terhadap Cahaya Biru

The aim of this study was to observe the use of blue light on phytochemical changes during cauliflower (Brassica oleraceae L., var. botrytis) germination and the correlation between the parameters measured. The treatment was in the form of giving blue light and dark conditions as a control. Measurement of phytochemical compounds including ascorbic acid, glucosinolates, isothiocyanates and myrosinase enzymes was carried out every day for 5 days of germination. This research method is experimental research. Data obtained from measurements for all parameters are presented in the form of mean ± standard error. Furthermore, all data were analyzed using analysis of variance (ANOVA). To determine differences between groups, Duncan's multiple range test (DMRT) was used at the 95% confidence level. All data analysis used SPSS software version 24.0 (IBM, Chicago, IL, USA). In addition, the measurement of the correlation between parameters is carried out using the Pearson rank correlation coefficient. The results showed that blue light treatment significantly increased all measured phytochemical parameters. A significant positive correlation occurred between myrosinase activity and isothiocyanate formation. The glucosinolate content of cauliflower sprouts remained high during observations in the blue light treatment compared to the dark conditions. Blue light induces isothiocyanate formation and higher myrosinase activity when compared to dark conditions. The conclusion from this study is that blue light significantly increases the phytochemical changes of cauliflower sprouts.
 
 Keywords: Ascorbic Acid, Glucosinolate, Isothiocyanate, Correlation, Myrosinase

Anticancer properties of sulforaphane: current insights at the molecular level.

Sulforaphane (SFN) is an isothiocyanate with multiple biomedical applications. Sulforaphane can be extracted from the plants of the genus Brassica. However, broccoli sprouts are the chief source of sulforaphane and are 20 to 50 times richer than mature broccoli as they contain 1,153 mg/100g. SFN is a secondary metabolite that is produced as a result of the hydrolysis of glucoraphanin (a glucosinolate) by the enzyme myrosinase. This review paper aims to summarize and understand the mechanisms behind the anticancer potential of sulforaphane. The data was collected by searching PubMed/MedLine, Scopus, Web of Science, and Google Scholar. This paper concludes that sulforaphane provides cancer protection through the alteration of various epigenetic and non-epigenetic pathways. It is a potent anticancer phytochemical that is safe to consume with minimal side effects. However, there is still a need for further research regarding SFN and the development of a standard dose.

Glucosinolates: Structure, classification, biosynthesis and functions in higher plants

ABSTRACT
 Objective: To analyze concepts, structure, classification, biosynthesis and functions of glucosinolates (GSLs) in higher plants.
 Design/methodology/approach: A search was performed into recent high-impact literature related to glucosinolates (GSLs).
 Results: GSLs are secondary metabolites rich in N and S. They are divided into aliphatic, aromatic, and indole GSLs depending on the amino acid from which they arise. The products of their hydrolysis, mediated by thioglucoside glucohydrolase, thioglucosidase or myrosinase enzymes (EC 3.2.1.147), play a role in increasing tolerance to biotic and abiotic stress factors. Furthermore, given their composition, they can serve as a nutrient reservoir under nutrient deficiency conditions.
 Limitations on study/implications: GSLs are synthesized only in species of the Capparidaceae, Brassicaceae, Resedaceae, and Moringaceae families.
 Findings/conclusions: GSLs are sulfur compounds that can serve as defense mechanisms against biotic and abiotic stress factors and as sources of nutrients in plants, and molecules with important nutraceutical properties in food and human health.

Glucosinolate-extracts from residues of conventional and organic cultivated broccoli leaves (Brassica oleracea var. italica) as potential industrially-scalable efficient biopesticides against fungi, oomycetes and plant parasitic nematodes

This study aimed to standarize a protocol for obtaining a bioactive extract from broccoli (Brassica oleracea var. italica) crop residues, that is suitable for application on an industrial scale and effective in reducing plant disease incidence. For this purpose, the influence of several extraction factors in the glucosinolate (GSL) content was studied with leaves collected from two conventional broccoli fields and two organic broccoli fields. The analysis showed that lyophilization had no influence on the GSL content. Storage of plant material under two different temperatures (− 20ºC and − 80ºC) had no influence on the GSLs content of the extracts. Phytotoxicity of the extracts was studied with six different plant seeds, and also cytotoxicity was determined with human liver cells in vitro. The extracts were phytotoxic at dilutions above 10%, while cell toxicity was low. Extracts concentrations of 0.1%, 1% and 2% were tested in vitro against eight plant pathogenic fungi and two oomycetes in solid and in liquid media. The extracts reduced the growth of several plant pathogenic fungi at 2% dilution by up to 38.37% against Alternaria alternata and up to 46.55% against Sclerotinia sclerotiorum. When combined with myrosinase enzyme the effect of the extracts was enhanced, reaching inhibition values of 67.06% against A. alternata in solid medium and 68.52% against Rhizoctonia solani in liquid medium. In contrast, the same extracts increased the growth of the plant pathogenic oomycetes Pythium ultimum and Phytophthora cactorum. The effect of the same extracts in the free leaving larvae, J2s, of the plant-parasitic nematode (PPN) Meloidogyne javanica was not obvious. Minor significant differences were obtained but with no clear dose-response in nematode mortality, and no inhibition of eggs hatching was observed. These results show the industrial potential of using broccoli residues to obtain extracts with biopesticide activity against plant pathogenic fungi.

Bacterial detoxification of plant defence secondary metabolites mediates the interaction between a shrub and frugivorous birds

Many plants produce fleshy fruits, attracting fruit-eating animals that disperse the seeds in their droppings. Such seed dispersal results in a conflict between the plant and the animal, as digestion of seeds can be highly beneficial to the animal but reduces plant fitness. The plant Ochradenus baccatus uses the myrosinase-glucosinolates system to protect its seeds. We show that hydrolysis of the O. baccatus fruit glucosinolates by the myrosinase enzyme inhibited digestive enzymes and hampered digestion in naïve individuals of the bird Pycnonotus xanthopygos. However, digestion in birds regularly feeding on O. baccatus fruits was unaffected. We find that Pantoea bacteria, dominating the gut of these experienced birds as well as the fruits, thrive on glucosinolates hydrolysis products in culture. Augmentation of Pantoea protects both naïve birds and plant seedlings from the effects of glucosinolates hydrolysis products. Our findings demonstrate a tripartite interaction, where the plant-bird mutually beneficial interactions are mediated by a communal bacterial tenant.

Salad Days: Pairwise Gene Edits Food to Topple Nutrition Barriers

Salad Days: Pairwise Gene Edits Food to Topple Nutrition Barriers

Impact of Disruption and Drying Conditions on Physicochemical, Functional and Antioxidant Properties of Powdered Ingredients Obtained from Brassica Vegetable By-Products.

Reintroducing waste products into the food chain, thus contributing to circular economy, is a key goal towards sustainable food systems. Fruit and vegetable processing generates large amounts of residual organic matter, rich in bioactive compounds. In Brassicaceae, glucosinolates are present as secondary metabolites involved in the biotic stress response. They are hydrolysed by the enzyme myrosinase when plant tissue is damaged, releasing new products (isothiocyanates) of great interest to human health. In this work, the process for obtaining powdered products from broccoli and white cabbage by-products, to be used as food ingredients, was developed. Residues produced during primary processing of these vegetables were transformed into powders by a process consisting of disruption (chopping or grinding), drying (hot-air drying at 50, 60 or 70 °C, or freeze drying) and final milling. The impact of processing on powders' physicochemical and functional properties was assessed in terms of their physicochemical, technological and antioxidant properties. The matrix response to drying conditions (drying kinetics), as well as the isothiocyanate (sulforaphane) content of the powders obtained were also evaluated. The different combinations applied produced powdered products, the properties of which were determined by the techniques and conditions used. Freeze drying better preserved the characteristics of the raw materials; nevertheless, antioxidant characteristics were favoured by air drying at higher temperatures and by applying a lower intensity of disruption prior to drying. Sulforaphane was identified in all samples, although processing implied a reduction in this bioactive compound. The results of the present work suggest Brassica residues may be transformed into powdered ingredients that might be used to provide additional nutritional value while contributing to sustainable development.

A steamed broccoli sprout diet preparation that reduces colitis via the gut microbiota

Sulforaphane is a bioactive metabolite with anti-inflammatory activity and is derived from the glucosinolate glucoraphanin, which is highly abundant in broccoli sprouts. However, due to its inherent instability its use as a therapeutic against inflammatory diseases has been limited. There are few studies to investigate a whole food approach to increase sulforaphane levels with therapeutic effect and reduce inflammation. In the current study, using a mouse model of inflammatory bowel disease, we investigated the ability of steamed broccoli sprouts to ameliorate colitis and the role of the gut microbiota in mediating any effects. We observed that despite inactivation of the plant myrosinase enzyme responsible for the generation of sulforaphane via steaming, measurable levels of sulforaphane were detectable in the colon tissue and feces of mice after ingestion of steamed broccoli sprouts. In addition, this preparation of broccoli sprouts was also capable of reducing chemically-induced colitis. This protective effect was dependent on the presence of an intact microbiota, highlighting an important role for the gut microbiota in the metabolism of cruciferous vegetables to generate bioactive metabolites and promote their anti-inflammatory effects.

Anti-adipogenic Effects of Sulforaphane-rich Ingredient with Broccoli Sprout and Mustard Seed in 3T3-L1 Preadipocytes.

Glucoraphanin (GRA) is a precursor of sulforaphane (SFN), which can be synthesized by the enzyme myrosinase. In this study, we developed and validated HPLC analytical methods for the determination of GRA and SFN in mustard seed powder (MSP), broccoli sprout powder (BSP), and the MSP-BSP mixture powder (MBP), and evaluated their anti-adipogenic effects in 3T3-L1 adipocytes. We found that the analysis methods were suitable for the determination of GRA and SFN in MSP, BSP, and MBP. The content of GRA in BSP was 131.11 ± 1.84 µmol/g, and the content of SFN in MBP was 162.29 ± 1.24 µmol/g. In addition, BSP and MBP effectively decreased lipid accumulation content without any cytotoxicity. Both BSP and MBP significantly inhibited the expression of adipogenic proteins and increased the expression of proteins related to lipolysis and lipid metabolism. BSP and MBP inhibited the expression of adipocyte protein 2 (aP2), CCAAT/enhancer-binding protein-α (C/EBP-α), and peroxisome proliferator-activated receptor-γ (PPAR-γ) in 3T3-L1 adipocytes, and inhibited the expression of fatty acid synthase (FAS) through AMP-activated protein kinase (AMPK). Meanwhile, BSP and MBP also increased the expression of the lipolysis-related proteins, uncoupling protein-1 (UCP-1) and carnitine palmitoyltransferase-1 (CPT-1). Moreover, MBP exerted anti-adipogenic to a greater extent than BSP in 3T3-L1 preadipocytes.

Anti-Obesogenic Effects of Sulforaphane-Rich Broccoli (Brassica oleracea var. italica) Sprouts and Myrosinase-Rich Mustard (Sinapis alba L.) Seeds In Vitro and In Vivo.

Glucoraphanin (GRA), a glucosinolate particularly abundant in broccoli (Brassica oleracea var. italica) sprouts, can be converted to sulforaphane (SFN) by the enzyme myrosinase. Herein, we investigated the anti-obesogenic effects of broccoli sprout powder (BSP), mustard (Sinapis alba L.) seed powder (MSP), and sulforaphane-rich MSP-BSP mixture powder (MBP) in bisphenol A (BPA)-induced 3T3-L1 cells and obese C57BL/6J mice. In vitro experiments showed that MBP, BSP, and MSP have no cytotoxic effects. Moreover, MBP and BSP inhibited the lipid accumulation in BPA-induced 3T3-L1 cells. In BPA-induced obese mice, BSP and MBP treatment inhibited body weight gain and ameliorated dyslipidemia. Furthermore, our results showed that BSP and MBP could activate AMPK, which increases ACC phosphorylation, accompanied by the upregulation of lipolysis-associated proteins (UCP-1 and CPT-1) and downregulation of adipogenesis-related proteins (C/EBP-α, FAS, aP2, PPAR-γ, and SREBP-1c), both in vitro and in vivo. Interestingly, MBP exerted a greater anti-obesogenic effect than BSP. Taken together, these findings indicate that BSP and MBP could inhibit BPA-induced adipocyte differentiation and adipogenesis by increasing the expression of the proteins related to lipid metabolism and lipolysis, effectively treating BPA-induced obesity. Thus, BSP and MBP can be developed as effective anti-obesogenic drugs.