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INVESTIGATOR: Siddiqui, R. A.

PERFORMING INSTITUTION:
VIRGINIA STATE UNIVERSITY
PETERSBURG, VIRGINIA 23803

GREEN PAPAYA AS A POTENTIAL SOURCE FOR ANTIDIABETIC AND DIABETIC-WOUND HEALING THERAPY.

NON-TECHNICAL SUMMARY: Being overweight or obesity increases the chances of developing the type 2 diabetes. Diabetes and diabetes-related complications are serious problems not only in Virginia, but it is also a regional and national issue in America. The total cost in 2013 for diabetes management was about $245 billion. It is important to reverse/decrease the occurrence of prediabetes and to treat diabetes and its complications with less expensive and less toxic therapies. The goal of this proposal is to identify and characterize active components for anti-diabetic and wound healing properties from green papaya. Papaya has documented anti-obesity and anti-diabetic activity; however, little is known about its cellular and molecular mechanism of actions. The proposed studies are innovative because we will use various cellmodels and examine genesto characterize theeffects of papaya in regulating obesity, diabetes, and wound healing. This will help us understand how components present in papaya reducefat deposition, decrease blood glucose levels, and heal diabetic-associated wounds. These studies will provide us an opportunity for capacity building at VSU to conduct state-of-the-art research in food and nutrition science and to trainundergraduate and graduate students in nutrition science research, preparing them to serve as leaders in food and nutrition science research in the future. This study will provide scientific knowledge to be disseminated to the general public and to farmers about the potential antidiabetic benefits of papaya. This will help encourage the public to consume papaya on a regular basis to reduce or slow down the progression of diabetes in Virginia. The increased use of papaya will encourage farmers to grow this niche crop for additional income. This study will be helpful in developing products to help manage and treat diabetic complications, leading to the reduction of costs associated with diabetes management. This study, therefore, has the potential to contribute to the economies of VSU and the Commonwealth of Virginia in the future.

OBJECTIVES: The objective of the present proposal is to address a local, regional, and national emerging issue of obesity-linked diabetes and diabetes-related complication. The proposal is indirectly related to NIFA priority area dealing with obesity in general and childhood obesity in particular. The diabetes is a common metabolic complication in majority of obese people. To identify and characterize active components for reducing diabetes and diabetic-wound healing properties from green papaya (Carica papaya) will also advance our knowledge for dealing with obesity-related complications in both children and adults. It will help us reduce the clinical, and economical burden-associated with obesity. The goals of this proposal will be achieved using the following objectives:Objective 1: To perform phytochemical analysis and antimicrobial activities of papaya extracts from leaves, seeds, pulp and skin peel fractions.Objective 2: To determine antidiabetic activity of papaya extracts.Objective 3: To determine wound healing properties of papaya extracts

APPROACH: Objective 1: To perform Phytochemical analysis and antimicrobial activities of papaya extracts from leaves, seeds, pulp, and skin peel fractions.I. Characterization of Polyphenolic profile (Team: Dr. Anwar Hamama, Dr. Siddiqui, Dr. Li):We will analyze papaya extracts for their phenolic compositions using a Waters Acquity UPLC H-Class coupled with a UV-Vis, PDA, ELS and QTof MS/MS Mass detectors (Waters Corporation, Milford, MA, USA).II. Determination of antimicrobial activity (Team: Dr. Chyer Kim, Dr. Siddiqui):Antibacterial activity will be tested in both Gram-negative (Escherichia coli and Salmonella enteritidis) and Gram-positive bacteria (Bacillus subtilis and Staphylococcus aureus).Objective 2: To determine antidiabetic activity of papaya extracts.I. Induction of glucose uptake in adipose, muscle, and liver cells: (Team: Dr. Siddiqui, Dr. Li and post-doc fellow): The effect of papaya extracts for insulin mimetic or insulin sensitization on glucose uptake in adipose cells (differentiated 3T3L1 cells) will be determined using a fluorometric glucose uptake assay kit (Abcam, Cambridge, MA). .II. Modulation of insulin secretion/resistance: (Team: Dr. Robert Diegelmann, Dr. Siddiqui, Dr. Li, and post-doc fellow)We will determine if components in papaya extracts preserve beta Islet cells by measuring insulin section in an in vitro assay. The insulin secretion will be determined in a cell-free culture supernatant by using a rat Insulin ELISA kit (R & D system). For in vitro Insulin-Resistance assay, the insulin-resistant cells will be incubated with insulin or insulin + papaya extract, and glucose uptake will be quantified as described above.III. Changes at cellular and molecular levels: (Team: Dr. Siddiqui, Dr. Li, and post-doc fellow): For gene expression analysis, the highly sensitive and reliable real-time RT-PCR will be performed. The gene expression will be analyzed by real-time qRT-PCR using the Bio-Rad CFX-96 Real-Time PCR System and using RT2 SYBR Green Master mix (Bio-Rad Laboratories, Hercules, CA).Objective 3: To determine wound healing properties of papaya extracts.I. Wound scratch assay test: (Team: Dr. Robert Diegelmann, Dr. Siddiqui, Dr. Li, and post-doc fellow): This experiment will be performed using human dermal fibroblasts (HDF). Injured fibroblasts will be incubated with varying concentrations of papaya extract, and the plates will be incubated at 37°C and 5% CO2 in a microscope stage top incubator (Tpkai Hit, Japan).The scratched area covered by cell migration and proliferation will be quantified.II. Sircol collagen assay: (Team: Dr. Siddiqui, Dr. Li, and post-doc fellow):We will determine if papaya extracts stimulate collagen synthesis in fibroblast.The collagen content will be determined using the Sircol dye reagent.III. Changes at cellular and molecular levels (Team: Dr. Siddiqui, Dr. Li, and post-doc fellow):The changes in gene expression of the key mediators (Collagen, Cadherin, MMPs, VEGF, VEGFR, EGF, EGFR) will be analysed by RT-PCR and validated by quantifying the target protein using Western blot analysis as described previously (46).Statistical Analysis of Data: Data will be reported as mean ± SD for at least 5 papaya samples in each group. Comparison between treated and control groups will be made by one way ANOVA with Tukey's HSD test using SPSS Statistics 20 software. All significant values will be reported at P <0.05.

PROGRESS: 2020/03 TO 2021/03
Target Audience:Consumers, farmers, students, and scientists have an interest in the development of crops that can improve heath and prevent chronic diseases. Consumers and Farmers: The Virginia Cooperative Extenson Newsletter has published an article about papaya research at VSU to disseminate the scientific knowledge to the general public and to farmers about the potential antidiabetic benefits of papaya. Students: The dietitics interns at Virginia State University were targeted to deliver formal classroom instructions and laboratory demonstration for the health benefits of papaya. In addition, oneundergraduate students was trained in nutrition research during this period. Scienists: We have publishedonescientific research papers and presented our finding in the American Nutrition Society scientific meetings to target the scientific community. Changes/Problems:We found one unexpected observation of green papaya on lipid accumulation in liver cells. We will, therefore, further investigate antiobesity properties of green papaya. Working during 2020 was challanging as Virginia State University operation was shut down from March, 2020 to June 2020. Thereafter, we restarted working in two groups on alternate weeks. This has slowed down our progressto complete some experiments under objective 3. We have requested an extension of six months to complete the overall study. What opportunities for training and professional development has the project provided?This study has provided an excellent opportunity to train graduate and undergraduate students at VSU in Food Chemistry and Nutrition Science. In 2017- 2018, we have three graduate biology-major students for laboratory research rotations (3 weeks duration), five graduate students from biology enrolled for M.S. thesis andthree undergraduate students are worked as interns. In 2018 - 2019,three undergraduate student andtwo dietetics interns worked in research project related to papaya, ginger and hemp extracts. During 2019-2020, we were only able to train one undergraduate student as the students were not allowed to be present in our building since March, 2020. This grant has provided opportunities for students training in Food Chemistry and Nutrition Science Laboratory. Students performed research on the health benefits of papaya, plums, ginger, tomatoes, green beans, and sweet potatoes. The experience from these project will help us to explore other important agriculture produce from Virginia for health and economical benefits. How have the results been disseminated to communities of interest?1. The details of recent research is posted at PI website. 2. The data was presented at the 1890 ARD Conferences in Atlanta (April 1- 5, 2017) and in Jacksonville (March 30 - April 3, 2019). One graduate student was awarded 2nd placein poster presentation. The data was also presented to the American Nutrition Society Meeting (virtual) in May, 2020. 3. Information of current research is presented in VSU-Agriculture Research newsletter that is widely distributed to farmers, researchers, and food businesses. 4. To date (2017-2021), we have published sevenmanuscripts in peer-reviewed scientific journals and presented 15abstracts to the national conferences. What do you plan to do during the next reporting period to accomplish the goals?We plan to perform further investigation on the molecular mechanism of the antidiabetic activity of green and yellow papayas. We will also carry out further studies for objective 3 to determine wound healing properties of green and yellow papayas.

IMPACT: 2020/03 TO 2021/03
What was accomplished under these goals? We have made progress in accomplishing our goals during 2019-2020. We have performed an additional investigation to include yellow ripped papaya (not proposed initially) and compared its phytochemical analysis with green papaya (proposed initially). We have completed the following task towards objective 1-3: Objective 1: To perform phytochemical analysis and antimicrobial activities of papaya extracts from leaves, seeds, pulp, and skin peel fractions. We have completed the experiments under this objective as repored in our last report (2019). Objective 2: To determine the antidiabetic activity of papaya extracts. We performed the effect of green papaya extract on alpha-glucosidase and alpha-amylase activity. We found that none of the papaya fractions has significant activity for inhibiting alpha-glucosidase or alpha-amylase. Papaya may regulate blood glucose through other mechanisms involving glucose uptake, glucose metabolism, and stimulation of insulin secretion. We, therefore, investigated the effect of yellow and green papaya fractions on glucose uptake in liver HepG2 cells. As mentioned above, yellow papaya fractions contained higher TPC and antioxidation activity. However, green papaya extracts significantly stimulated glucose uptake in HepG2 liver cells compared to that of yellow papaya extracts. Both water and methanol extract of pulp from green papaya showed the significant stimulation of glucose uptake followed by leaves and skin. Furthermore, methanol extract of green papaya also showed inhibition of lipid droplet accumulation in liver cells. Our recent investigation indicated that green and yellow papaya pulp stimlated expression of Glut-2 receptors on liver cells. In conclusion, our data suggest that papaya pulp stimulated glucose uptake in HepG2 liver cells by inducing expression of Glut-2, a glucose transport protein. Objective 3: To determine the wound-healing activity of papaya extracts. We created an in vitro wound by scratching a monolayer of human dermal fibroblasts and monitored the movement of fibroblasts in the wounded area to assay wound healing activity of papaya extracts. Data presented show that the aqueous seeds extract from green papaya closed the wounded area by 70% (P<0.05), whereas control and other extracts (pulp, skin, and leaves) were only able to close 25-45% of the wounded area. The data indicate that seeds from green papaya have the potential for its use in diabetic wound healing. At present, we are investigating fractions from yellow papaya for their wound healing activities. Not much progress is made under this objective as the university was closed from March - June, 2020 and then we experienced several intermittent closures due to exposure of few people in our building. We have requested a six month extension to complete the proposed experiments to investigate the underlying molecular mechanism for papaya extracts on wound healing activity. Discussion and Conclusions: Our data suggest that leaves and seeds from papaya contained higher amounts of phenolic and flavonoids and possess greater antioxidation activity than those of pulp and skin fractions. However, these fractions appear to have almost no inhibitory activity on carbohydrate digestion in the intestine. In contrast, pulp from green papaya exhibited strong glucose uptake activity in liver cells, whereas skin fraction from green papaya inhibited lipid droplet formation in liver cells. The data indicated that pulp from green papaya has insulinogenic effect and can effectively lower blood glucose concentration by stimulating glucose uptake in liver. In addition, green papaya skin fraction inhibited lipid deposition in liver cells. This indicates that green papaya skin can potentially prevent diabetes associated obesity. Further experiments to accomplish goals of objective 3 to determine green papaya effect on wound healing (Objective 3) are in progress, which are to be completed in the extension period of the grant. Implications: These results will potentially have a positive financial impact on producers of green papaya and may provide an excellent source of income for growers in Virginia. The regular use of papaya may also provide an inexpensive preventive/therapeutic treatment for populations who are at a greater risk of developing diabetes and cancer, particularly older people and African Americans.

PUBLICATIONS (not previously reported): 2020/03 TO 2021/03
1. Type: Journal Articles Status: Published Year Published: 2020 Citation: Allqmani Z, Greene A, Li H, Rafie R, Beyer, AR, Witiak SM, Kaseloo PA, Siddiqui RA. (2020). Antiproliferative Effect of Green Papaya on Lymphocytic Leukemic Cells. Nutri Food Sci Int J 10(2)
2. Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Obaid Beg, Rafat Siddiqui, Haiwen Li, Reza Rafie. Characterization of Green and Yellow Papaya for Antioxidation and Glucose Uptake Stimulatory Activity in HepG2 Liver Cells. Current Developments in Nutrition, Volume 4, Issue Supplement 2, June 2020, Page 739,