Progress 04/15/20 to 04/14/24
Outputs
Target Audience:This research aimed to develop knowledge, technology, and nutraceutical products to benefit individuals with obesity and related ailments. The information and products developed stand to greatly benefit these individuals. In addition to obesity victims, the effort benefits grape growers, customers, and the industry as a whole by increasing sales, market value, and income. FAMU expanded its nutrigenomics research capabilities to improve phytochemical constituent content in fruit and vegetable crops for medicinal use, meet consumer/stakeholder/clientele needs, and train current and future students in cutting-edge technologies. Project achievements were shared with consumers, grape producers, industry, academia, USDA, collaborators, 1890 and 1862 land-grant universities, and outreach workers through various educational and outreach programs. Faculty and students from biology, chemistry, pharmacy, and community groups received training in cell biology, nutraceutical screening, and interactive courses to improve their skills and knowledge. Materials such as posters, and research articles were created with the assistance of extension specialists to promote project outcomes. Changes/Problems:We faced several challenges in executing the project to meet the proposed objectives. Due to pandemic, we were unable to bring undergraduate and graduate students to the lab for providing appropriate in person training in various experimental techniques and data collection and analysis protocols to accelerate the experiments. In addition, due to social distancing and density limitation guidelines access to the lab was also restricted to the researchers. The Research Associate who was originally hired to conduct the obesity project research has left the university for a faculty position necessitating finding another qualified individual. A new candidate (Dr. Meenakshi Agarwal) was hired, and she worked hard to complete this project, overcome all obstacles, and finish it on time. We also worked with scientists from the College of Pharmacy on campus to complete the prescribed experiments on time and provide students with hands-on experience to complete project duties and satisfy project objectives. With the challenges, we were able to meet the project objectives, draft a manuscript, which is currently under review. In addition, outcomes were delivered in the form of oral and poster presentations to the stakeholders. What opportunities for training and professional development has the project provided?1. One undergraduate student was trained in berry sample collection and processing, phytochemical extraction and quantification, adipose cell culturing methods, RNA isolation and characterization techniques, and other related biochemical techniques. The project participants were also provided training in experimental design and a wide range of biochemical and analytical techniques, data collection and analysis, and report writing. 2.Two post-doctoral research associates were trained in berry developmental stage assessment, biochemical evaluation, sample processing, RNA extraction and quantification, electrophoretic techniques, Primer design, PCR technique, bioinformatics, related molecular and cellular techniques, cell culture, isolation and characterization of bioactive molecules, instrumental analysis, and nutraceutical product development and testing. The researchers were also trained in experimental design, troubleshooting, data collection and processing, scientific proposal writing, project execution and management, report writing and manuscript preparation.3. Collaborations were made with other FAMU partners and faculties were trained in the cell culture concepts and techniques. How have the results been disseminated to communities of interest?The overall goal of this research is to determine the usefulness muscadine grape in controlling adipose cell growth, multiplication and fat accumulation for controlling obesity, and provide aplatform to train students and faculty for building expertise in nutraceutics, workforce development as well asbenefit theconsumersbymitigating the obesity and fat accumulation, add valueand sustain the muscadine grape industry. Accordingly, the research outcomeswere shared with the concerned groups and stakeholders through various outreach activities and other electronic and print media. Some of thedessemination means and mediaare identified below. 1. Conference presentations 2. Workshops 3. Florida Legislature Appreciation Day 4. Professional meetings 5. Social Media pages, etc. 6. Guest lectures, and 7. Research article publishing What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Ripe berries (EL-38 stage) from ~40 different muscadine grape cultivars were collected, stilbenoids extracted, vacuum-dried, dissolved in methanol, and subjected to HPLC. The HPLC analysis revealed that muscadine berry extracts contained four different stilbenes namely, t-piceid, t-resveratrol, ε-viniferin and t-pterostilbene with distinct retention times where t-piceid and t-resveratrol are the major constituents of muscadine berry. Based on the highest stilbenes content, two muscadine cultivars, Sothern Home and Pineapple were chosen for the obesity study. Since berry metabolites are an important factor that impacts the nutritional value of grapes, the Total phenolic content (TPC), Total flavonoid content (TFC), and antioxidant activity of whole muscadine berry extracts were determined. The results showed a higher level of TPC and antioxidant activity in cv. Southern Home as compared to the cv.Pineapple. Whereas the TFC was found to be higher in cv. Pineapple. The methanolic extracts of berry were vacuum-dried and re-dissolved in DMSO at a concentration of 100 mg/ml and used in the bioactivity studies using Pre-adipocyte cells (3T3-L1) as described below. Pre-adipocyte cells (3T3-L1) were purchased from ATCC and used to assess lipid accumulation in the cells. The cells were cultured in Dulbecco's Modified Eagle's medium containing 10% fetal bovine serum at 37°C in a 95% humidified incubator under 5% CO2 atmosphere. However, this culture media failed to support the pre-adipocyte cell growth. Extensive studies on four media sources showed that the most effective media to support pre-adipocyte cell growth is Corning DMEM media with a >90% growth and increased cell adherence. Using the Corning DMEM basal media, we were able to culture the pre-adipocytes cells in the pre-adipocyte media and used in bioactivity evaluation studies as below. The pre-adipose cells were seeded into plates, incubated overnight, and exposed to muscadine grape berry extracts (cvs. Southern Home and Pineapple) in DMSO at a concentration of 0 to 75 mg/ml. The results showed a ~10% reduction in cell viability at the berry extract concentration of 0.6 mg/ml, ~15% reduction at a berry extract concentration of 1.2 mg/ml to 4.7 mg/ml, ~30% reduction at 9.4 to 18.8 mg/ml, ~40 % reduction at 37.5 mg/ml and ~50 % reduction at a concentration of 75 mg/ml. Berry extracts from both Southern Home and Pineapple cultivars were found to show a similar effect on 3T3-L1 pre-adipose cell viability. A concentration of 2.3 mg/mL was chosen for both the extracts and used in subsequent studies. At this concentration, more than 85% of the cells were found to be alive as compared to the untreated control. To determine the impact of berry extract treatment on pre-adipose cell growth at the molecular level, total RNA was isolated from control and treated cells, its purity measured, and used for C-DNA synthesis and qPCR-based gene expression analyses. The resulting gene expression data was examined to determine the response of genes associated with obesity and measure differences in gene expression between control and muscadine berry extract treated cells. The results revealed that treatment of pre-adipose cells with berry extract from muscadine grape cv. Pineapple showed upregulation of Atrn and Ramp3 genes by a factor of 2 and 150, respectively while the expression of genes Nr3c1, Npy1r, Agrp, B2m, Bdnf, Cpd, Cpe,Ghr, Hprt, and Ppargc1a was downregulated in the range of 0.1 and 0.5 in the treated cells compared to untreated cells. Further, the gene expression studies with berry extracts from cv. Southern Home showed that expression of Agrp, Atrn,Npy1r, and Sigmar1 genes was elevated by 1.5-fold in treated cells while expression of Ramp3 gene increased by150-fold compared to the control cells which is known to encode 'Receptor Activity Modifying Protein 3'. This result is consistent with the report of Liu et al. 2018 (Peptides, 110, pp.10-18) suggesting that mice lacking Ramp3 exhibited increased obesity and fat tissue accumulation. In addition, Liu et al., (2018) inferred that Ramp3 reduces adipocyte cell growth and weight gain in mice lacking estrogen. Our result also showed downregulation of genes B2m, Bdnf, Cpd, Cpe, Ghr, Gusb, Hprt, Hsp90ab1, II1r1, Nr3c1, Sort1, and Zfp91. However, it is of particular interest to note that the genes B2m and Zfp91 were profoundly downregulated compared to other genes. The B2m gene is known to code for Beta-2 Microglobulin. In this regard, Nadler et al., 2000 (Proceedings of the National Academy of Sciences, 97(21), pp.11371-11376) discovered that b2m with reduced expression is associated with obesity in mice. The gene, Zfp91 which encodes zinc finger protein promotes tumor growth in patients with acute myeloid leukemia (Unoki et al., 2003, International Journal of oncology, 22(6), pp.1217-1223). Our results showed decreased expression of the Zfp91 gene following treatment with muscadine berry extract suggesting that it may be also involved in adipose cell growth/ multiplication and is associated with obesity. Further, to examine the impact of MGEs on adipose cells, the 3T3-L1 preadipocytes were cultured in DMEM containing FBS. Two days after confluence (day 0), the cells were stimulated to differentiate with DMEM containing 10% FBS, 1.0 μM of dexamethasone, 0.5 mM of isobutylmethylxanthine, and 1.0 μg/mL of insulin for two days. From day 4 onwards, the differentiation media were replaced with 10% FBS/DMEM media containing 1.0 μg/mL of insulin. The media was changed every two days until the cells were harvested. Several trial experiments were performed to optimize the differentiation process for preadipocytes into adipocytes cells. The cell differentiation was monitored under the microscope. Further, the lipid accumulation was observed using an optimized protocol for lipid O staining. The cells were fixed with 10% formalin for 40 minutes, rinsed twice with water, followed by the addition of 60% isopropanol and incubation for 5 minutes. Afterwards, isopropanol was discarded and replaced with Oil Red O solution to cover the cell surface and incubated for 10 minutes. The cells were rinsed four times with sterile water and visualized under the bright filled and fluorescence microscopy. The differentiated cells were treated with both the MGEs and examined for lipid accumulation until 15 days of incubation. Samples were harvested and stained. The results showed that administration of both MGEs resulted in a considerable decrease in lipid accumulation in 3T3-L1 adipocytes while maintaining the cell viability as observed by Oil red staining. Placing emphasis on the molecular underpinnings of obesity is a crucial component in the development of potentially effective therapeutic agents targeting weight gain. As a result, the current study was aimed to profile the mRNA expression of numerous genes involved in obesity in 3T3-L1 mouse cells that had been treated with the investigated berry extracts at safe concentrations. mRNA expression studies were performed following the above-described protocol. The treatment of 3T3-L1 cells at various phases resulted in the upregulation and downregulation of numerous genes associated with the obesity pathway. The cells treated with cv. Pineapple exhibited a decrease in the expression of genes Adipor1, Hsp90ab1, and Ptpn1, while the expression of genes Cntfr, Hrh1, and Zfp91 increased. In contrast, the genes Ptpn1, IL6ra, Insr, Atrn, Nr3c1, and Adipor1 were downregulated in cells treated with muscadine grape cv. Southern Home berry extract. In summary, this investigation revealed that the muscadine berry extract induced notable modifications in gene expression and a substantial decrease in lipid accumulation, thereby indicating its potential to regulate the activity of hormones associated with obesity.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2024
Citation:
Muscadine Grape Bio-Prospecting: A Path to Adiposity Control
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2024
Citation:
Exploring Muscadine Grape Phytochemicals to Support Healthy Aging.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2024
Citation:
Effective Phytochemicals Content of Muscadine Grape for Promoting Apoptosis and Abrogation of Cancer Cell Growth to Prevent Disease Progression
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2024
Citation:
Safeguarding the Phytochemicals Content and Composition and Health Value of Muscadine Grape Products to Preserve Health Value and Bioactivity.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2024
Citation:
Phytochemicals Content and Composition, and Nutraceutical Value of Muscadine Grape Products.
|
Progress 04/15/23 to 04/14/24
Outputs
Target Audience:This research aimed to develop knowledge, technology, and nutraceutical products to benefit individuals with obesity and related ailments. The information and products developed stand to greatly benefit these individuals. In addition to obesity victims, the effort benefits grape growers, customers, and the industry as a whole by increasing sales, market value, and income. FAMU expanded its nutrigenomics research capabilities to improve phytochemical constituent content in fruit and vegetable crops for medicinal use, meet consumer/stakeholder/clientele needs, and train current and future students in cutting-edge technologies. Project achievements were shared with consumers, grape producers, industry, academia, USDA, collaborators, 1890 and 1862 land-grant universities, and outreach workers through various educational and outreach programs. Faculty and students from biology, chemistry, pharmacy, and community groups received training in cell biology, nutraceutical screening, and interactive courses to improve their skills and knowledge. Materials such as posters, and research articles were created with the assistance of extension specialists to promote project outcomes. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?1. One undergraduate student was trained in berry sample collection and processing, phytochemical extraction and quantification, adipose cell culturing methods, RNA isolation and characterization techniques, and other related biochemical techniques. The project participants were also provided training in experimental design and a wide range of biochemical and analytical techniques, data collection and analysis, and report writing. 2. Two post-doctoral research associates were trained in berry developmental stage assessment, biochemical evaluation, sample processing, RNA extraction and quantification, electrophoretic techniques, Primer design, PCR technique, bioinformatics, related molecular and cellular techniques, cell culture, isolation and characterization of bioactive molecules, instrumental analysis, and nutraceutical product development and testing. The researchers were also trained in experimental design, troubleshooting, data collection and processing, scientific proposal writing, project execution and management, report writing and manuscript preparation.3. Collaborations were made with other FAMU partners and faculties were trained in the cell culture concepts and techniques. How have the results been disseminated to communities of interest?The overall goal of this research is to determine the usefulness muscadine grape in controlling adipose cell growth, multiplication and fat accumulation for controlling obesity, and provide a platform to train students and faculty for building expertise in nutraceutics, workforce development as well as benefit the consumers by mitigating the obesity and fat accumulation, add value and sustain the muscadine grape industry. Accordingly, the research outcomes were shared with the concerned groups and stakeholders through various outreach activities and other electronic and print media. Some of the dessemination means and media are identified below. 1. Conference presentations 2. Workshops 3. Florida Legislature Appreciation Day 4. Professional meetings 5. Social Media pages, etc. 6. Guest lectures, and 7. Research article publishing What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
The objective of this study was to assess the potential of phytochemicals-rich whole muscadine grape berry extract in regulating the activity of obesity through the utilization of an in vitro cell culture model. We employed two muscadine cultivars: Pineapple, and Southern Home. Since metabolites are important for the nutritional value of grapes, we measured the Total phenolic content (TPC), Total flavonoid content (TFC), and antioxidant activity of whole muscadine berry extracts. We found a higher level of TPC and antioxidants in cv. Southern Home as compared to the Pineapple. Whereas the TFC was found higher in cv. Pineapple. This is in line with past findings that different muscadine cultivars had varying metabolite contents. Our study revealed that the administration of muscadine grape extract resulted in a considerable decrease in lipid accumulation in 3T3-L1 adipocytes while maintaining the cell viability as observed by Oil red staining. Placing emphasis on the molecular underpinnings of obesity is a crucial component in the development of potentially effective therapeutic agents targeting weight gain. As a result, the current study aimed to profile the mRNA expression of numerous genes involved in obesity in 3T3-L1 mouse cells that had been treated with the investigated extracts at safe concentrations. The treatment of 3T3-L1 cells at various phases resulted in the upregulation and downregulation of numerous genes associated with the obesity pathway. The cells that were subjected to treatment with cv. Pineapple exhibited a decrease in the expression of Adipor1, Hsp90ab1, and Ptpn1, while the expression of Cntfr, Hrh1, and Zfp91 increased. In contrast, the following genes were downregulated in cells treated with cv. Southern Home: Ptpn1, IL6ra, Insr, Atrn, Nr3c1, and Adipor1. In its entirety, this research investigation revealed that the berry extract induced notable modifications in gene expression and a substantial decrease in lipid accumulation, thereby indicating its potential to regulate the activity of hormones associated with obesity. This study emphasizes the encouraging prospects of whole muscadine grape berry extract, which is abundant in phytochemicals, as an innovative therapeutic substance to address complications associated with obesity. Students and faculty members received training in cell culture, as well as biochemical and molecular techniques. The findings of the research were disseminated through poster and oral presentations at both the departmental and conference levels. A research article has been written and subsequently submitted to a peer-reviewed journal for review
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2024
Citation:
Muscadine Grape Bio-Prospecting: A Path to Adiposity Control.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2024
Citation:
Exploring Muscadine Grape Phytochemicals to Support Healthy Aging.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2024
Citation:
Effective Phytochemicals Content of Muscadine Grape for Promoting Apoptosis and Abrogation of Cancer Cell Growth to Prevent Disease Progression.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2024
Citation:
Safeguarding the Phytochemicals Content and Composition and Health Value of Muscadine Grape Products to Preserve Health Value and Bioactivity.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2024
Citation:
Phytochemicals Content and Composition, and Nutraceutical Value of Muscadine Grape Products.
|
Progress 04/15/22 to 04/14/23
Outputs
Target Audience:The focus of this research is to develop knowledge, technology and nutraceutic products for the benefit the individuals suffering from obesity and other adiposity related ailments who stand to greatly benefit from the information generated and nutraceutic products developed to remedy the adiposity associated ailments. Besides the obesity victims, the other interested groups and audience will be the grape growers, consumers, and the industry by and large who stand to greatly benefit from the project outcome through increased sales, market value and income. This project will also help establish cutting edge nutraceutics research and development programs at the public and private sectors with focus on using muscadine grape for developing novel nutraceutic products and agro-pharmaceuticals to prevent/remedy various ailments. The ongoing research activities will help establish and strengthen nutrigenomics research capability at FAMU for enhancing or altering phytochemical constituent content of fruit and vegetable crops for medicinal use to meet the consumer/stakeholder/clientele needs and demands as well as help provide training ground for current and future students in cutting edge technologies. The project accomplishments will be communicated to the consumers, grape growers, industry, academic community, USDA, collaborators and other 1890 and 1862 land-grant Universities, and outreach personnel using diverse educational and outreach activities. The research outcome will be communicated to the stakeholders through field demonstrations and workshops to train farmers, grape growers on varietal selection, application of appropriate cultural practices for high quality grape production to safeguard health value and marketing strategies. The results obtained in the experimental studies will be published in peer reviewed scientific journals. The summary of interesting findings will be also published as popular articles in leading newspapers. The literature and educational materials will be made available to potential beneficiaries, consumers, growers, and industry at CVSF/FAMU website as documents, provide hands on training to students in molecular and cellular biology, proteomics, and transcriptomics. The interested faculty and students from biology, chemistry and pharmacy, and community groups will be trained in cell biology involving nutraceutical screening and interactive courses focused on enhancing their skills and knowledge. Suitable propagation materials like posters, brochures and publications will be made with the help of extension specialist and used in dissemination of project outcome. Changes/Problems:We encountered some difficulties in observing the transition of pre-adipose to adipocyte cell morphology. Only 20% of the cells with different morphologies representing adipocyte cells were observed. As reported by previous studies, the Oil red O staining of adipocytes reveals the correlation between the staining intensity and the degree of cell differentiation. Using this concept, we have optimized the staining technique to differentiate the preadipocyte and adipocyte cells. We have also successfully observed the neutral lipid's oil red O staining and the nuclei's blue staining pattern. However, we noticed only a few differentiated adipocytes cells following red O staining of adipocyte cells. These issues have slowed the progress and required extensive evaluation and modification of protocol to be able to monitor cell differentiation. What opportunities for training and professional development has the project provided?1. One undergraduate student was trained in berry sample collection and processing, phytochemicals extraction and quantification, adipose cell culturing methods, protein and RNA isolation and characterization techniques and other related biochemical techniques. The project participants were also provided training in experimental design and a wide range of biochemical and analytical techniques, data collection and analysis, and report writing. 2. Two graduate students were provided training in experimental design, berry maturity determination, phytochemicals isolation, purification and quantification, RNA and protein extraction and other related molecular and cellular techniques, data collection and processing to obtain training in cutting edge gene and protein techniques as well as data collection, processing and analysis tools for use in their thesis research. 3. Two post-doctoral research associates were trained in berry developmental stage assessment, biochemical evaluation, sample processing, RNA and protein extraction and quantification, electrophoretic techniques, Primer design, PCR technique, bioinformatics, related molecular and cellular techniques, cell culture, isolation and characterization of bioactive molecules, instrumental analysis, and nutraceutic product development and testing. The researchers were also trained in experimental design, troubleshooting, data collection and processing, scientific proposal writing, project execution and management, report writing and manuscript preparation. How have the results been disseminated to communities of interest?The overall goal of this research is to determine the usefulness muscadine grape to control adipose cell growth, multiplication and fat accumulation for controlling obesity, and intended to benefit the growers, students, consumers, individuals with related ailments and the grape industry. Accordingly, the research results were shared with the concerned groups through various outreach activities and other means. Some of these means and media are identified below. 1. Conference presentations 2. Workshops 3. Florida Legislature Appreciation Day 4. Professional meetings 5. Social Media pages etc. 6. Guest lectures What do you plan to do during the next reporting period to accomplish the goals?The differentiated adipose cells will be employed in the next step, which will be to monitor the expression of genes that are linked with obesity and result will be compared with the pre-adipocyte cells. The differentiation protocol will be optimized to convert the pre-adipose cells to adipose cells. It is known that throughout the process of cells differentiating into adipocytes, lipid droplets accumulate in the cytoplasm. To differentiate preadipocytes from adipocytes, the oil red O staining technique will be used. A culture will be generated using the adipose cell line and treated with varying concentrations of grape berry extract prepared from different muscadine grape cultivars. The gene expression studies will be carried out in three replications using the total RNA extracts followed by their conversion to cDNA. Following the above format, real-time quantitative PCR (RT-qPCR) will be conducted to examine differential gene expression between untreated control and berry extract treated cell lines. This study will focus on investigating the effect of berry extracts on expression of leptin, ghrelin, and others obesity-related genes. In addition, we will investigate the effect of muscadine berry extract treatment on expression of various obesity risk factors including proteins and metabolites content and composition of pre-adipose and adipose cells. Proteins and metabolites will be extracted from both control and berry extract treated cells and their profiles will be analyzed using 2-D gel electrophoresis and HPLC.
Impacts
What was accomplished under these goals?
The primary goal of this project is to determine the efficacy of muscadine grape berry extract to limit the adipose cell growth and multiplication while sparing non-adipose/fat cells for use in controlling obesity. Further, this study includes monitoring changes in gene and protein expression to better understand the pathways leading to adipose cell growth and multiplication at the molecular and cellular level. For this purpose, pre-adipose cells, 3T3-L1 MBX cells isolated from mouse embryo were purchased from ATCC (Manassas, VA, USA). Initially, we had difficulty in culturing the pre-adipose 3T3-L1 MBX cells despite following recommended protocol. This required extensive revision of culture protocol to define the effective culture media. After extensive revisions to the published culture protocol and modifying the media components we were able to successfully culture the pre-adipose cells and used in the following experiments. The pre-adipose cells were grown as a mono layer under a controlled environment in T75 flasks using revised Dulbecco's Modified Eagle Medium (DMEM), supplemented with 4 mM L-glutamine, 10% heat-inactivated fetal bovine serum (FBS), 100 U/mL penicillin and 0.1 mg/mL streptomycin (1% P-S). The optimized culture protocol was then used to measure the effect of muscadine grape berry extracts on viability of 3T3-L1 MBX cells at the pre-adipose phase. Ripe berries were harvested, powdered and extracted with methanol. The extract was vacuum-dried and re-dissolved in DMSO at a concentration of 150 mg/ml and used in the bioactivity studies. The pre-adipose cells were seeded into plates, incubated overnight and exposed to muscadine grape berry extracts (cvs. Southern Home and Pineapple) in DMSO at a concentration of 0 to 75 mg/mL. Cell viability analysis was established using the average of three independent experiments. The results showed a ~10% reduction in cell viability at the berry extract concentration of 0.6 mg/ml, ~15% reduction at a berry extract concentration of 1.2 mg/ml to 4.7 mg/ml, 30% reduction at 9.4 to 18.8 mg/ml, ~40 % reduction at 37.5 mg/ml and ~50 % reduction at a concentration of 75 mg/ml. Berry extracts from both Southern Home and Pineapple cultivars found to show similar effect on 3T3-L1 pre-adipose cell viability. A concentration of 2.3 mg/mL was chosen for both the extracts and used in subsequent studies. At this concentration more than 85% of the cells were found to be alive as compared to the untreated control. To determine the impact of berry extract treatment on pre-adipose cell growth at the molecular level, total RNA was isolated from control and treated cells, its purity measured, and used for C-DNA synthesis and qPCR-based gene expression analyses. The resulting gene expression data was examined to determine the response of genes associated with obesity and measure differences in gene expression between control and muscadine berry extract treated cells. The results revealed that treatment of pre-adipose cells with berry extract from muscadine grape cv. Pineapple showed upregulation of Atrn and Ramp3 genes by a factor of 2 and 150, respectively while the expression of genes Nr3c1, Npy1r, Agrp, B2m, Bdnf, Cpd, Cpe, Ghr, Hprt, and Ppargc1a was downregulated in the range of 0.1 and 0.5 in the treated cells compared to untreated cells. Further, the gene expression studies with berry extracts from cv. Southern Home showed that expression of Agrp, Atrn, Npy1r, and Sigmar1 genes was elevated by 1.5-fold in treated cells while expression of Ramp3 gene increased by150-fold compared to the control cells which is known to encode 'Receptor Activity Modifying Protein 3'. This result is consistent with the report of Liu et al. 2018 (Peptides,110, pp.10-18) suggesting that mice lacking Ramp3 exhibited increased obesity and fat tissue accumulation. In addition, Liu et al., (2018) inferred that Ramp3 reduces adipocyte cell growth and weight gain in mice lacking estrogen. Our result also showed downregulation of genes B2m, Bdnf, Cpd, Cpe, Ghr, Gusb, Hprt, Hsp90ab1, II1r1, Nr3c1, Sort1, and Zfp91. However, it is of particular interest to note that the genes B2m and Zfp91 were profoundly downregulated compared to other genes. The B2m gene is known to code for Beta-2 Microglobulin. In this regard Nadler et al.,2000 (Proceedings of the National Academy of Sciences,97(21), pp.11371-11376) discovered that b2m with reduced expression is associated with obesity in mice. The gene, Zfp91 which encodes zinc finger protein promotes tumor growth in patients with acute myeloid leukemia Unoki et al., 2003 (International journal of oncology,22(6), pp.1217-1223). Our result showing decreased expression of Zfp91 gene following treatment with muscadine berry extract suggest that it may be also involved in adipose cell growth/multiplication and associated with obesity. Further investigation on expression of genes associated with obesity using differentiated adipose cells and comparison with undifferentiated pre-adipose cells will shed light on involvement of these genes in regulating the adipose cell growth and multiplication and adiposity. Further, evaluating diverse muscadine grape genotypes with varying phytochemicals content and composition for their effect on expression of obesity related genes will help determine the efficacy of muscadine grape to mitigate obesity development.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2022
Citation:
Identification and characterization of molecular and cellular components to augument sugar metabolism for increasing sugar content of muscadine grape
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2022
Citation:
Synergistic action of stilbenes has potent cytotoxic activity resveratrol alone
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2022
Citation:
Efficacy of muscadine grape phytochemicals to modulate prostate cancer progression
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2022
Citation:
Exploiting grape phytochemicals to promote healthy ageing
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2022
Citation:
Muscadine grape as a potential prebiotic and probiotic source to promote gut health
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2022
Citation:
Identifying and characterizing parthenocarpic gene in muscadine grape
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2022
Citation:
Development of value added functional foods from muscadine grapes
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2022
Citation:
Towards developing non-leathry muscadine table grape:identifying and characterizing gene associated with leathery skin in muscadine grape
|
Progress 04/15/21 to 04/14/22
Outputs
Target Audience:The audience for the outcome of this research are health-conscious consumers, obese individuals, grape growers, wineries and grape industry by and large. This project has helped establish a cutting edge agro-nutraceutical research program with a focus on using muscadine grape juice and whole berry for developing novel products as supplements to help prevent obesity and/or inhibit lipid accumulation. The research will determine the efficacy of muscadine grape whole berry phytochemicals extract to control lipid accumulation in adipocytes and help identify the molecular and cellular mechanisms involved and establish obesity research at FAMU in agro-nutraceuticals. The newly built research capacity will help enhance or alter nutraceutical/nutritional properties of muscadine grapes, other small fruit crops and medicinal plants of commercial importance. The project has invocated faculty innovation, productivity, and outreach activities. The value-added nutraceutical products that will be developed based on final project outcome help promote muscadine grape marketability and justify establishment of new vineyards using nutraceutically-important grape cultivars. Outcome of this research has been shared with the Florida Grape Grower Association, Viticulture Advisory Council of Florida as well as with other grape growers and consumer groups. In addition, the research outcome was also communicated to the community, consumers and growers at the Association of Research Directors of I890 Institutions Meeting and several other community events to appraise and educate the interested groups. The project has initiated discussions and potential for collaborations with peers for furthering the studies. Besides, the project has also instilled interest in students and scientists interested in learning about molecular and cellular basis of adiposity and fat accumulation, and application of agro-nutraceuticals to prevent/manage obesity. During this period the project related findings have been propagated to various individuals including community groups, public, students, women, children, grape growers, grape industry personnel, biomedical scientists, etc., through various outreach activities and scientific publications. Changes/Problems:The pandemic and prescribed guidelineslimited options for in person training of undergraduate and graduate students in the lab. Hence, it was difficult to provideappropriate in person training in various sample collection and processing protocols, analytical techniques and data collection and analysis protocols normally employed in the laboratory basedexperiments. Further, social distancing,density limitation andaccess to the lab was limited which curtailed training activities. The Research Associate whowas hired to conductthe obesity projectresearch has left the university for a faculty position necessitating finding another qualified individual. Because of the requiredexpertise in the area and lack ofsuitable candidates it was difficult to findand recruit a qualified individual who is experienced in this area and capable oftacklingtheexperiments identified in the project. However, the project personnel collaborated with scientists from College of Pharmacy on campus and workingto optimize the cell culture protocols to execute the prescribed experiments in a timely manner,andprovidestudent experiential learningto accomplish the project tasks formeeting project objectives. Now that most restrictions are lifted, we hopeto follow the experimental workplanand complete the tasksin a timely manner. We believe with the new collaborative arrangements and support from other researcherswe will be able to conduct the proposed experiments to successfully complete the project. What opportunities for training and professional development has the project provided?Because of the pandemic, limited opportunitiesexisted for in person training of students. However, as the guidelines were relaxed towards the later part of the year limited access was provided for in person training of students. Some of these activities are described below: 1. One undergraduate student was trained in berry sample collection and processing, phytochemicals extraction and quantification, adipose cell culturing methods, protein and RNA isolation and characterization techniques and other related biochemical techniques. The project participants were also provided training in experimental design and a wide range of biochemical and analytical techniques, data collection and analysis, and report writing. 2. Two graduate students were provided training in experimental design, berry maturity determination, phytochemicals isolation, purification and quantification, RNA and protein extraction and other related molecular and cellular techniques, data collection and processing to obtain training in cutting edge gene and protein techniques as well asdata collection, processing and analysis tools for use in their thesis research. 3. Two post-doctoral research associates were trained in berry developmental stage assessment, biochemical evaluation, sample processing, RNA and protein extraction and quantification, electrophoretic techniques, Primer design, PCR technique, bioinformatics, related molecular and cellular techniques, cell culture, isolation and characterization of bioactive molecules, instrumental analysis, and nutraceutic product development and testing. The researchers were also trained in experimental design, troubleshooting, data collection and processing, scientific proposal writing, project execution and management, report writing and manuscript preparation. How have the results been disseminated to communities of interest?The overall goal of this research is to determine the usefulness muscadine grape to control adipose cell growth,multiplication and fat accumulation for controlling obesity, and intended to benefit the growers, students, consumers, individuals with related ailments and the grape industry. Accordingly, the research results were shared with the concerned groups through various outreach activities and other means. Some of these means and media are identified below. 1. Journal articles 2. Conference presentations 3. Florida Grape Grower Association meeting 4. Workshops 5. Florida Legislature Appreciation Day 6. Professional meetings 7. Social Media pages etc. 8. Guest lectures What do you plan to do during the next reporting period to accomplish the goals?This project is intended to evaluate the efficacy of muscadine grape extracts to control obesity by regulating the expression of obesity hormones, leptin and ghrelin using adipocytes. This requiredfirst establishing the pre-adipocyte cell culture and then their transformation to adipocytes for using in the bioactivity evaluation studies. Hence, the pre-adipocyte cell culture has to be established first and then the pre-adipocytes must be transformed to adipocyte cells using transformation media. However, we had encountered a problemin successfully establishing and growing the pre-adipocytes. After numerous attempts and trials we have now optimized and established the pre-adipocyte cell culture system. They will be transformed to adipocytes and used to evaluate the efficacy of selected muscadine grape genotypes differing in phytochemicals content and composition to regulateadipocyte cell multiplication and fat accumulation. The adipocyte cell culture will be used to determine the effect of muscadine berry extract on fat absorption and accumulation where the adipocyte cells will be treated with Nile red/BODIPY stain for determining fat content of cells.Subsequently, additional muscadine genotypes differing in phytochemicals content and composition will be screened to determine the extent of genetic variation in bioactivity level among muscadine genotypes and increase the portfolio of genotypes to obtain a comprehensive idea on their bioactivity level. We will also apply a variety of post-harvest stilbene induction techniques to increase resveratrol content of the berry for enhancing the bioactivity levelof non-performing muscadine genotypes. We will further explore the observed anti-obesity effects at the molecular and cellular levels by monitoring muscadine berry extract induced changes at the gene and protein levels in adipocyte cells. For determining the transcriptomics profile, RNA will be isolated from the muscadine berry extract treated and control adipocytes and sent for RNA sequencing to determine the treatment effect on leptin and ghrelin gene expression.For determining the effect of muscadine berry extracttreatment on expression/suppression of fat absorption and fat accumulation pathway genes, protein will be isolated from treated adipocyte cells and sent for protein sequencing. These studieswill help identify the genes and pathways responding to the treatment of 3T3-L1 cells with muscadine grape berry extract. The quantitative genomics and proteomics studies will help better understand the global genome and proteome expression changes in adipocytestreated with muscadine berry extract. Outcome of this research has the potential to identify the elite genotypes and therapeutic targets for developing novel supplements from muscadine grape berry. Further, the data will help discover molecular pathway by which muscadine berry extracts induce anti-obesity effects. The transcriptome and epigenetic studies can shed light on the molecular pathways involved in anti-obesity effect of muscadine berry extracts in obese cells as well as leptin and ghrelin protein and their gene expression and regulation.
Impacts
What was accomplished under these goals?
Muscadine grape is a rich source of polyphenolics including secondary metabolites like stilbenes, anthocyanins, flavonoids, etc. Plant secondary metabolites (SMs) are derivatives of primary metabolites produced by plants due to variousphysiological changes. The SMs significantly improve plant healthand survival under various environmental stresses and thereby act as important primary metabolites. Stilbenes are defense substances produced as phytoalexins by many plants including grapes. Besides supporting plant defense they also have a wide range of health benefits. Among the stilbenes, resveratrol has been credited as being potentially responsible for preventing various ailments. In view of the documented health benefits of stilbenes this study is designed to evaluate the efficacy of muscadine grape in wholesome to mitigate the obesity hormones (leptin and ghrelin) expression to regulate adiposity. Fully ripened muscadine grape berries (EL-38 stage) with diverse berry characteristics, skin color and phytochemicals composition (cvs. Jane bell, Pineapple, and Southern Home) were collected from FAMU research vineyard and transported to the lab. The berries were powdered using liquid nitrogen and extracted with various aqueous and non-aqueous solvents, and their combination using a homogenizer and stirred overnight. The homogenate was centrifuged, and a portion of the supernatant used for quantifying phenolics, flavonoids and stilbenes content and composition by HPLC. The HPLC analysis revealed that muscadine berry extracts contained four different stilbenes namely, t-piceid, t-resveratrol, ε-viniferin and t-pterostilbene with distinct retention times where t-piceid and t-resveratrol are the major constituents of muscadine berry. The total phenolics and flavonoids content of the berry extracts was determined by the Folin-Ciocalteu colorimetric method while the total flavonoids content (TFC) estimated according to the previously reported method with some modifications. The antioxidant activity was measured using the DPPH radical-scavenging method and data expressed in µM Trolox equivalents/g fresh weight. The phenolics content and composition of muscadine berry extracts was determined by HPLC and calibrated using the phenolics standards. These data was derived to reveal the phytochemicals status of the selected genotypes. For testing the biological activity of muscadine berry extracts on adipocyte phenotype, first pre-adipocytes must be cultured and transformed to adipocyteswhich takes two to three weeks after seeding the cells into the experimental plates. The pre-adipocytes have a fibroblast-like appearance. The pre-adipocyte cells (3T3-L1) were purchased from ATCC (ATCC CL-173) and cultured on ATCC 3T3-L1 culture media (DMEM with 10% FBS, 1% nonessential amino acids, 2 mM L-glutamine, 100 units/ml of penicillin, and 100 μg/ml of Streptomycin and 250 mg/mL amphotericin-B) at +37C. However, this culture media failed to support the pre-adipocyte cell growth and establish a viable pre-adipocyte cell culture. Repeated attempts to culture the pre-adipocyte cells using modified media failed to support their growth. Subsequently, a new batch of pre-adipose cell lines were purchased, and attempts made to establish a viable pre-adipocyte cell culture using culture media from four different vendors. The media tested were 1) ATCC 3T3-L1 media - DMEM (ATCC - Cat. No. 30-2002), 10% fetal bovine serum (ATCC- Cat. No.30-2020); 2) 3T3-L1 Sigma media (Cat. No. DIF001-1KT); 3) Gibco MesenPRO RS Medium (reduced-serum medium), CTS StemPro MSC SFM (serum-free medium); and 4) 3T3-L1 Corning media (Cat. No. 10-013-CV).Comparative analysis of performance of pre-adipocyte cells on these four media sources showed that the most effective media to support pre-adipocyte cell growth is Corning DMEM (Dulbecco's Modified Eagle's Medium) media (Cat. No. 10-013-CV) which gives the cell viability more than 90% as well as increasedcell adherence.Using the Corning DMEM basal media, we were able to successfully rejuvenate the pre-adipocyte cells from frozen stock, and effectively maintain the pre-adipocytes cell growth in the pre-adipocyte media. As a result of above experimentation we were able to define the culture media suitable for successfully culturing pre-adipocytes and differentiate them into adipocytes for use in the bioactivity evaluationstudy. The media identifiedfor successfully establishing the pre-adipocytes is described below: Basal medium: 90% DMEM, HEPES pH 7.4, Biotin. Pre-adipocytes medium: 90% DMEM- high glucose, 10% Fetal Bovine Serum (FBS), Penicillin, Streptomycin. Differentiating media: 90% DMEM- high glucose, 10% Fetal Bovine Serum (FBS), Penicillin, Streptomycin, Biotin, 1 µg/ml Insulin, 1µm Dexamethasone, 0.5 mM 3-Isobutyl-1-methylxanthine (IBMX). We have also found that at 10% CO2 concentration the 3T3-L1 cells grew faster, and stacking more than 3 flaks reduced cell growth. The pre-adipocyte cells have a fibroblast-like appearance. Thepre-adipocyte cells are being subjected to differentiation to adipocytes using the transformation media to define the optimum transformation culture conditions. The pre-adipocyte cells began to resemble adipocytes two to three days after differentiation, rounding up, producing and storing fat. The cells seem post-confluent, balled up, and "star-shaped" once the differentiation phase has begun. We believe that we have optimized and defined the pre-adipocyte cell culture and their transformation media. The 3T3-L1pre-adipocytes are now being grown in themore suitable media and in its 5th passage. They will be cultured until the 8thpassagefor better reproducibility and then transferred to thetransformation media for transforming the pre-adipocyte cells to adipocyte cells. These adipocyte cells will be used for determining the efficacy of muscadine berry extract to regulate adipocyte cell multiplication. The muscadine berry extracts prepared above will be dissolved in DMSO and different amounts (1 to 1000µg) added to the adipocyte cells cultured above. The multiplication and fat accumulation of adipose cellswill be monitored for 24, 48 and 72 hrs after the addition of muscadine berry extract to determine their growth and effectiveness of muscadine berry extract on limiting adipose cell line multiplication and fat accumulation.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
Rahman MA, Balasubramani SP and Sheikh MB. 2021. Molecular characterization and phylogenetic analysis of MADS-box gene AGL11 associated with stenospermocarpic seedlessness in muscadine grapes. Genes 12(2): 232. doi: 10.3390/genes12020232
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
Darwish AG, Das PR, Ismail A, Gajjar P, Balasubramani SP, Sheikh MB, Tsolova V, Sherif SM and El-Sharkawy I. 2021. Untargeted Metabolomics and Antioxidant Capacities of Muscadine Grape Genotypes during Berry Development. Antioxidants 2021, 10, 914. https://doi.org/10.3390/ antiox10060914.
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Progress 04/15/20 to 04/14/21
Outputs
Target Audience:The target audience for the outcome of this research is general health conscious public, obeseindividuals, health conscious individuals, grape growers, wineries and grape industry by and large. This project has helped establish a cutting edge agro-nutraceutical research program with a focus on use of muscadine grape berry extracts for developing functional foods to help prevent obesity and/or inhibit lipid accumulation. The project has enabled establishing molecular and cellular studies for understanding the effects of select muscadine grape whole berry phytochemical extracts on molecular mechanisms involved in lipid accumulation in adipocytes and establish capacity at FAMU in nutraceuticals research for enhancing or altering nutraceutical/nutritional properties of muscadine grapes, other small fruit crops and medicinal plants of commercial importance. The project has invocated faculty innovation, productivity, and outreach activities. The value-added nutraceutical products that will be developed based on final project outcome will help promote muscadine grape marketability and justify establishment of new vineyards using nutraceutically-important grape cultivars. Outcome of this research has been shared with the Florida Grape Grower Association, Viticulture Advisory Council of Florida as well as with other grape growers. In addition, the research outcome was also communicated to the community, consumers and growers at the Biannual Grape Harvest Festival, Association of Research Directors of I890 Institutions Meeting and several other community events to appraise and educate the interested groups. The projecthas initiated discussions and potential for collaborations with peers for furthering the studies. Besides, the project has also instilled interest in students and scientists interested in learning about molecular and cellular basis of adiposity development, cancer progression and application of agro-nutraceuticals to prevent/manage obesity. During this period the project related findings have been propagated to various individuals including community groups, general public, students, women, children, grape growers, grape industry personnel, biomedical scientists, etc. Changes/Problems:Due to pandemic, we were unable to bring undergraduate and graduate students to the lab for providing appropriate in person training in various experimental techniques and data collection and anlaysis protocols to accelerate the experiments. In addition, due to social distancing and density limitation guidelines access to the lab was also restricted to the researchers. However, the researchers strived to accomplish maximum output under the circumstances and complete the experiments in a timely manner to accomplish various project tasks. We hope this year will be different and enable us to execute the workplan and complete the proposed experiments as scheduled in a timely manner. What opportunities for training and professional development has the project provided?Due to pandemic, students were not allowed to be present in the lab for in person training. Hence, limited training was provided to undergraduate students using in-person and online sources where and when possible. The training included literature collection, experimental design, experimental methodology, data collection and analysis protocols, viewing and developing standard operation protocol. The students were enabled to make presentation of their learnings in the under-graduate seminar organized by FAMU. The researchers and post-doc working in the project were trained in berry collection and processing, preparation of stilbene extracts, HPLC analysis of stilbenes, animalcell culture and in vitro digestion methods for natural products. Furthermore, opportunities were also provided to the post-doc for developing individual research proposals to seek funding for enhancing their career and professional development. How have the results been disseminated to communities of interest?Due to the pandemic situation, Grape Harvest Festival, anannual event of FAMU Viticulture Center could not be conducted. This could have facilitated the propogation of results to thousands of general public who usually attend this festival. With social gathering restrictions in place, we could do limited dissemination efforts. However, we are currently in the process of finalizing atleast two manuscripts which will be communicated shortly. We are also considering other options to publicize the health benefits of Muscadine grape through popular writings in local news papers, brochures, pamphlets and FAMU extension magazine. What do you plan to do during the next reporting period to accomplish the goals?In the year 2021 2022, we intend to complete the following tasks: 1) Collect fresh berries during the vintage (July to September 2021) 2) Prepare fresh stilbene and total phenolics extracts from frsh berries and estimate the quantities of stilbenes and total phenolics to determine the seasonal effect on stilbenes content and composition 3) Complete the in vitro digestion studies using various muscadine grape varieties and assess the effect on stilbenes and total phenolics content and composition on digestive process 4) Treat the 3T3-L1 cells with muscadine grape extracts to assess their modulatory effect on lipid accumulation 5) Develop primers for the leptin and ghrelin genes identification and establish the protocol for evaluating their expression in 3T3-L1 cells
Impacts
What was accomplished under these goals?
Pre-adipocyte cells (3T3-L1) werepurchased from ATCC. Cell culture was established in the lab by performingthe following protocol. The 3T3-L1 preadipocytes were cultured in Dulbecco's Modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS). At two days after confluence (day 0), the cells were stimulated to differentiate with DMEM containing 10% FBS, 1.0 μM of dexamethasone, 0.5 mM of isobutylmethylxanthine, and 1.0 μg/mLof insulin for two days. From day 4 onwards, the differentiation media were replaced with 10% FBS/DMEM media containing 1.0 μg/mLof insulin. Themedia waschanged every two days until the cells were harvested. All media contained 100 μg/mLof streptomycin and 100 U/mLof penicillin. Cells were maintained at 37°C in a 95% humidified incubator with 5% of CO2 atmosphere. These cells are being used for muscadine berry extract treatment to assess lipid accumulation, leptin and ghrelin gene expression.Ripe berries (EL-38 stage) from ~40 different cultivars of muscadine grapeswere collected from the vineyard of the Center for Viticulture and Small Fruit Research, Florida Agriculture and Mechanical University, Tallahassee, FL, USA (30.47 latitude and 84.17 longitude) during vintage of 2019and 2020. The stilbenoids were extracted according to the protocol established in-house (Balasubramani et al., 2019). Ten grams (10 g) of berry was ground with liquid nitrogen and the ground powder was mixed with 7 mL of methanol, and then homogenized for 10 min on a vortex and further extracted for 24 h under darkness. The suspension was centrifuged at 14,000 rpm for 15 min. The supernatant was removed carefully, and the resulting residue was extracted a second time with 3 mL of methanol and ethyl acetate (1:1,v/v), as described above. The pooled organic solvent extracts were vacuum-dried in a concentratorat room temperature in the dark. A portion of the vacuum concentrated extract of the berries was dissolved in 1 mL HPLC grade methanol and filtered through a 0.22 µm nylon membrane filter and subjected to HPLC. The analysis of stilbenoids was carried out using an HPLC system equipped with Waters 2487 dual UV detector and 1525 gradient pump. The HPLC pumps, autosampler, and detectors were controlled via Waters Empower software (Empower 3 service pack 2) supplied by Waters Corporation. The analytical column Luna RP C18 (4.6 × 250 mm; particle size, 5 μm) and guard cartridge (C18 4 × 3.0 mm) were used for this study. The column temperature was maintained at 25°C. The gradient elution profile was as follows: 90% solvent water (B), 10% solvent acetonitrile (A) (0-18 min); 85% A, 15% B (18-23 min); 85% A 15% B (23-30 min); 10% A, 90% B (30-35 min). The flow rate was set at 0.4 mL/min. A volume of 2 µL of each sample was injected to resolve and quantify individual stilbenoids. Three injections were performed in sequence for each biological replicate. UV absorbance detection was recorded using dual wavelengths at 285 and 305 nm. A mixture of the standards was prepared using 1, 2, 3, 4, 5, and 6 ng of each of the four stilbenoids and used for calibration and quantification of stilbenoids. The standards, t-piceid, t-resveratrol, ε-viniferin, and t-pterostilbenewere prepared and used as described above. Samples and calibration standards were run in triplicates. Chromatograms were acquired with different retention times for each of the stilbenes and the area under the curve (AUC) was calculated using Empower III software. The linearity ranges of the calibration curves were R2= 0.9906. Quantification of stilbenoids from thecultivars was based on the calibration curves obtained from the respective standards. A standardized static in vitro gastrointestinal digestion method was adapted from Xionget al. (2020). Simulated salivary fluid (SSF), simulated gastric fluid (SGF), and simulated intestinal fluid (SIF) electrolyte stock solutions were prepared with the corresponding electrolytes according to the referenced parameters. In the oral phase, 0.5 g of Muscadine grape berrysuspended in 0.5 mL watermixed with 0.35 mL of SSF and minced together, followed by addition of 0.05 mL of 1500 U/mL porcine pancreas α-amylase solution, and 2.5 μL of 0.3 M CaCl2, and 97.5 μL of water (pH 7.0), in sequence, and thoroughly mixed for 2 min. In the gastric phase, the 1 mL oral bolus was mixed with 0.75 mL of SGF electrolyte stock solutions, 1.6 mL porcine pepsin stock solution of 25,000 U/mL, 5 μL of 0.03 M CaCl2, 0.02 mL of 1 M HCl, and 65 μL of water (pH 3.0). The reaction vessel was placed into a shaking incubator at 37°Cfor 2 h. In the intestinal phase, 2 mL of gastric chyme was mixed with 1.1 mL of SIF electrolyte stock solution, 0.5 mL of a pancreatin solution 800 U/mL, 0.25 mL fresh bile (160 mM in fresh bile), 4 μL of 0.3 M CaCl2, 15 μL of 1 M NaOH and 131 μL of water (pH 7.0), and was shaken for 2 h at 37°C. The digested material from each sample (digests) was freeze-dried for extraction and analysis. The recovery index (RI) of stilbenes and total phenols will be estimated by HPLC and biochemical methods following the standard protocol. We have also established a method for 3T3-L1 cell culture and preliminary trials were performed using the berry extracts. HPLC analysis of the extracts has revealed that the stilbenes content of muscadine grape varieties and the extent of genetic variation during different vintage seasons. Based on the HPLC results we have generated a catelogue of high-stilbene containing and low-stilbene containing muscadine grape varieties. We have also developed a post harvest stilbene induction method in house to enhance the stilbenes content of freshly harvested muscadine berries. We hope to use this method for enhancing the stilbenes content of commercial muscadine grape genotypes and evaluate them for bioactivity potential. We have been also working on a in vitro digestion protocol as described above. The optimized protocol will help simulate the in vivo conditions to learn the details of muscadine grape digestion in human intestine. The preliminary observations have indicated that the recovery of total polyphenolsafter in vitro digestion is about 50%. We are in the process of analyzing the extract of digestate for calculating the recovery percentage of different stilbenes. We have also developed some muscadine grape berry and juice based nutraceutical products such as breakfast bars, syrups, resins, etc., for testing their potential in controling apetite by modulating leptin and ghrelin expression.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2020
Citation:
Molecular assessment of genes linked to immune response traits of honey bees in conventional and organically managed apiaries,
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