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Elizabeth Baldwin, Jinhe Bai, Anne Plotto, John Manthey, Smita Raithore, Sophie Deterre, Wei Zhao, Cecilia do Nascimento Nunes, Philip A. Stansly, and James A. Tansey

, thus, contributing to yield reductions. HLB symptomatic fruit that impart off-flavor to the juice have lower sugars, sometimes higher acids, higher levels of bitter limonoids and astringent flavonoids as well as an altered volatile profile ( Baldwin et

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Anne Plotto, Elizabeth Baldwin, Jinhe Bai, John Manthey, Smita Raithore, Sophie Deterre, Wei Zhao, Cecilia do Nascimento Nunes, Philip A. Stansly, and James A. Tansey

, higher acids, higher limonoids and some flavonoids, and lower top-note esters (ethyl acetate and ethyl butanoate) than juice of fruit from healthy trees or asymptomatic fruit from infected trees ( B aldwin et al., 2010 ; B assanezi et al., 2009 ; D

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Amit Vikram, G.K. Jayaprakasha, and Bhimanagouda S. Patil

Our recent studies have shown that certain citrus limonoids protect from colon cancer based on cell and animal studies. Animal studies also suggest that citrus juice protects from osteoporosis. To understand the structure–function relationship through animal studies requires a large amount of purified limonoids. Since certain limonoids are present in low concentration, it is a challenge to obtain the required quantity of different limonoids. In this context, we report the purification of limonin 17-ß-D glucopyranosides (LG), and deacetylnomilinic acid 17-ß-D glucopyranoside (DNAG). However, DNAG was isolated in relatively large amount from sour orange (Citrus aurantium L.) seeds. Defatted seed powder was extracted with methanol and purified using column chromatography to obtain multigrams of DNAG. While LG was found in lower concentration, a large concentration of hesperidin was also purified in this process. This project is based upon work supported by the USDA-CSREES under Agreement USDA IFAFS #2001 52102 02294 and USDA #2005-34402-14401 “Designing Foods for Health” through the Vegetable & Fruit Improvement Center.

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Deepak Dandekar, G. K. Jayaprakasha, and Bhimanagouda Patil

Citrus consumption has been shown to promote human health due to presence of several bioactive compounds. In the process of understanding the health benefits of citrus, we need to isolate and characterize these compounds. Limonoids are one of such prominent, but lesser-known phytonutrients that have been shown to prevent cancers of the mouth, skin, lung, breast, and colon. With the growing interest in the health-promoting properties of citrus limonoids, the demand for these bioactives has significantly increased. It has been critical to explore environment-friendly extraction methods rather than using hazardous organic solvents. A water-based hydrotropic extraction of limonoid aglycones from sour orange (Citrus aurantium L.) seeds was developed. Two hydrotropes, sodium salicylate (Na-Sal) and sodium cumene sulfonate (Na-CuS), were studied for extraction efficiency using the Box Behnken experiment design method. The extraction efficiency of prominent aglycone limonin was observed depending on hydrotrope concentration, extraction temperature, and percentage of raw material loading. Response Surface Analysis (RSA) of data predicted the optimum conditions for maximum yield. Recovery of aglycones from filtered extract is also easily achieved by mere dilution using water at pH 3 or 7 or by partitioning the extract with dichloromethane. At optimum conditions, limonin yield of 0.46 mg/g seeds in the case of Na-Sal extraction and 0.65 mg/g seeds in the case of Na-CuS extraction was achieved. The results demonstrated that the hydrotropic extraction process of limonoid aglycones has practical commercial importance. This project is based upon work supported by the USDA–CSREES IFAFS #2001 52102 02294 and USDA–CSREES #2005-34402-14401 “Designing Foods for Health” through the Vegetable and Fruit Improvement Center.

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Jun Yu*, Romeo Toledo, Rakesh Singh, Leonard Pike, and Bhimanagouda Patil

Grapefruit seeds were studied for the extraction of limonoids using supercritical CO2 extraction (SC-CO2) technique. Limonin aglycone was successfully extracted with SC-CO2 directly from grapefruit seeds; and the limonin glycoside was extracted using SC-CO2 and ethanol as co-solvent from the spent seeds after the extraction of limonin aglycone. In an effort to optimize the extraction conditions of limonin aglycone, pressure, temperature, time effects were investigated. Various times of extraction, CO2 flow rate and the feeding modes of CO2 were also employed to obtain the highest yield of limonin aglycone. Optimal conditions to achieve the highest limonin aglycone (0.63 mg/g seeds) were 48.3 MPa, 50°C and 60 min with CO2 bottom feeding, flow rate about 5 L/min. The extraction conditions for limonin glycoside to achieve highest yield were further optimized. The highest extraction yield (0.62 mg limonin glycoside/g seeds) were at 48.3 MPa, 50°C, 30% molar fraction of ethanol (XEth =0.30) and 40 min with CO2 top feeding, flow rate about 5 L/min. The results demonstrated that supercritical CO2 extraction of limonoids from grapefruit seeds, a citrus juice industry byproduct, has practical significance for commercial production.

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Clark Wilson, G.K. Jayaprakasha, and Bhimanagouda Patil

Open column chromatography is an effective and common technique for the separation and purification of chemical constituents. Limonoids are found in significant quantities in citrus fruits. Citrus limonoids have documented anti-cancer activity in several types of cancer, such as breast, colon, skin, and neuroblastoma in animal models and in vitro cell culture studies. Furthermore, limonoids have shown anti-inflammatory properties and inhibitory effects on bone resorption. In addition to many potential health benefits, limonoids have also shown antifungal and insect anti-feedant properties. To meet the large demand of limonoids for bioactivity studies, defatted grapefruit seeds were extracted using acetone and concentrated under vacuum. The dried extract was loaded onto a silica gel column and eluted with mixtures of dichloromethane and ethyl acetate with increasing polarity to obtain three compounds. The purity of the compounds (1–3) have been analyzed by HPLC and the structures have been identified by using NMR spectra and mass spectra as nomilin, limonin, and deacetylnomilin, in respective order of elution. The results will be presented in greater detail on the poster. This project is based upon work supported by the USDA-CSREES under Agreement USDA IFAFS #2001 52102 02294 and USDA #2005-34402-14401 “Designing Foods for Health” through the Vegetable & Fruit Improvement Center.

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Shibu M. Poulose, Edward D. Harris, and Bhimanagouda S. Patil

Limonoids are triterpinoids unique to citrus and neem trees with potential cancer-preventing properties in animals and human cell lines. Antioxidant activity and apoptotic induction are thought to be the principal effects of citrus limonoids in the antiproliferative properties, but this postulate lacks firm experimental evidence. In this study four highly purified 17 β-D glucopyranosides of citrus, limonin glucoside (LG), obacunone glucoside (OG), nomilinic acid glucoside (NAG), and deacetylnomilinic acid glucoside (DNAG), were tested for their effects against human SH-SY5Y neuroblastoma cells. Neuroblastomas account for 10% of childhood cancers, and in our study the cultured cells were treated with different concentrations and different time intervals. Micromolar levels of LG and OG significantly (P ≤ 0.001) stopped cell growth and induced cell death in 24 hours, but had no adverse effect over Chinese hamster ovary (CHO) cells at the highest toxic level tested. The viability studies were based on trypanblue exclusion and dimethylthiazol diphenyltetrazolium (MTT) reduction assays. The limonoids significantly increased the downstream caspases 3/7 activity (P ≤ 0.005) within 12 hours of treatment, suggesting an explicit role of apoptotic induction, which was confirmed by flow cytometry and DNA fragmentation assays. Highest S phase cell number was reduced by LG, followed by OG, NAG, and DNAG as compared to the known inhibitor camptothecin. Structural variations of limonoids could be ascribed to antioxidant activity. This study strongly supports apoptosis induction as an anticancer mechanism of citrus limonoids. Funded by USDA 2001-52102-11257 and 2004-34402-14768.

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Shibu M. Poulose*, Jennifer S. Brodbelt, Leonard M. Pike, and Bhimanagouda S. Patil

Limonoids, chemically related triterpinoids predominantly found in citrus and neem relatives, are known to play a pivotal role in the prevention of different types of cancer and cardiovascular diseases. Since the concentrations of these compounds are low in the plant tissues, the isolation of pure compounds is the limiting factor for the individual activity studies in animal models. In this study, combinations of chromatographic techniques were used to isolate limonoid aglycones and limonoid glucosides from citrus byproducts such as seeds and molasses. The compounds were initially extracted with different polar solvents and the concentrated extracts were passed through a series of adsorbent resin (SP-70) and ion-exchange resins (WA-30, Dowex-50, Q-sepharose) to remove further impurities. The use of increasing ionic strength of NaCl from 0 to 800 mm to release the exchanged compounds from the ion exchange columns further separated the limonoids from flavonoids, which was confirmed through TLC, UV, and analytical HPLC methods. Individual compounds were further purified using flash chromatography and preparative HPLC methods and identified by using LC-MS analysis. Direct crystallization of limonin resulted in a 17% increase in the yield as compared to the previously reported methods. The results suggest that application of these purification methods are useful for the bulk purification of compounds in order to further investigate their biological activity.

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Kranthi Kiran Mandadi, G. K. Jayaprakasha, Farzad Deyhim, and Bhimanagouda S. Patil

Citrusprovedbeneficial to human health in preventing cardiovascular disorders, hypertension, anemia, and several cancers, including colon, lung, skin, stomach, and breast, in animal and cell culture studies. For the first time, current study was focused to determine whether orange juice and grapefruit juice increase bone mass in an orchidectomized (ORX) rat model of osteoporosis. Thirty-six male Sprague-Dawley rats were randomly divided into four groups of 1) sham; 2) ORX; 3) ORX + orange juice (OJ); and 4) ORX + grapefruit juice (GJ). All rats were fed to the mean intake of the sham group and were provided with freshly squeezed grapefruit or orange juice for 60 days. There was a numerical improvement in femoral density with OJ and GJ compared to the ORX group. Ultimate bone strength and femoral cortical area diameter (mm) increased (P < 0.05) with OJ and GJ compared to the ORX group. Furthermore, bone fracture withstand threshold time(s) increased (P < 0.05) with OJ and GJ compared to the ORX group. Beneficial effects of citrus juices on bone strength could be related to its bioactive triterpene compounds and nutrient composition. Isolation of triterpenes using specific combinations of food-grade adsorbent and ion exchange resins yielded pure limonin-17-β-D-glucoside and limonin along with obacunone, nomilin, and deacetylnomilin. Identification and characterization of the isolated limonoids were performed by specific analytical techniques, such as HPLC, LC-MS, and NMR. Further studies are needed to determine the protective effects of limonoids on bone homeostasis. Funded by USDA 2001-52102-11257 and 2004-34402-14768.

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Jose L. Perez, G.K. Jayaprakasha, and Bhimanagouda S. Patil

Grapefruit has potential health-promoting properties due to the presence of multitude bioactive compounds. Ongoing cell culture and animal studies in our lab using limonoids and flavonoids have provided strong evidence of their protective properties for preventing chronic diseases. Studies related to D-glucarate, a natural, nontoxic bioactive compound found in grapefruit, has not been explored. One of the derivatives, such as D-glucaro-1,4-lactone, is reported to be a potent ß-glucuronidase inhibitor. With the inhibition of ß-glucuronidase enzyme, glucuronidation will be favored. Glucuronidation is a conjugation process through which potentially carcinogenic environmental toxins can be neutralized. In this context, quantification of glucarate using HPLC was developed. Samples from grapefruits were prepared by heating fruit extract with distilled water. Further, the extract was homogenized and centrifuged. The supernatant was treated with petroleum ether to remove non-polar substances. Then the extract was subject to ion exchange chromatography. Fractions were collected and analyzed by analytical HPLC for the quantification of D-glucarate content and its lactone. This project was supported by the USDA-CSREES grant for Designing Foods for Health through the Vegetable and Fruit Improvement Center.