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A natural product library screen identified a synergistic combination of curcumin and ursolic acid as a nutraceutical option for treating prostate cancer. Subsequent in vitro and in vivo testing showed substantial promise in the combination which deemed it ready for human translation. However, poor bioavailability has prevented this combination from impacting cancer therapy. A novel formulation for ursolic acid and curcumin was developed by (1) attempting to increase dissolution rates with a reduced particle size and (2) enhancing solubility with lipid-based formulations. A hot-melt rotating disk process prepared the active compounds in the lipid-based microsphere formulations. The formulations were then optimized based on both solubility and process yield with available excipients. Our target was 1200mg/day of curcumin and 300mg/day of ursolic acid. A phase 1 clinical trial design was performed by sequentially enrolling three groups (curcumin, ursolic acid, and combination). Healthy patients were recruited without a history of prostate cancer. Blood was collected for bioavailability measurements and rectal swabs for gut microbiome analysis at baseline, 6 hours, 24 hours, and two weeks post supplementation intake. The primary outcome was safety using Common Terminology Criteria for Adverse Events (CTCAE v.4.03). Mass spectroscopy measured the bioavailability of the active parent compounds in blood. Lastly, we used 16s V1-2/3-4 RNA sequencing to measure the effects on the human gut microbiome. The results indicate that lipid-enhanced ursolic acid and curcumin are safe and well-tolerated. However, renal function was identified as a laboratory value that should be monitored with future studies evaluating this combination. Curcumin improved the bioavailability of ursolic acid when given in combination. The presence of ursolic acid did not appear to alter blood levels of curcumin or its metabolites. Thus, a potential mechanism for synergistic effects of the combination may involve increased bioavailability of ursolic acid. The combination also altered the microbiome favoring the production of the B vitamin biotin, a cofactor in immune regulation and a feature associated with lower prostate cancer risk. In conclusion, a combination of ursolic acid and curcumin is safe and should be evaluated in Phase II clinical trials for possible treatment of prostate cancer.

Food contamination from hepatitis A virus (HAV) is a great concern to food producers worldwide. Finding an innovative approach to inactivate HAV on food contact surfaces and on different produce remains a challenge. Using chemical disinfectants (e.g. chlorine) is an effective way to inactivate HAV on fomites, but it maybe unfavorable for food products. While heat inactivation of HAV remains the most efficient way to inactivate HAV when present in foods, most foodborne outbreaks of HAV are related to ready-to-eat (RTE) foods including produce which do not undergo further heating. Therefore, finding compounds with effective anti-HAV activities will be of great benefit to the food sector. In our study, oleanolic acid (OA) and ursolic acid (UA) have been investigated for their anti-HAV properties. OA at 600 μg/ml and UA at 360 μg/ml showed 2.27±0.67 and 1.33±0.35 log PFU/ml reduction after a 1 h treatment, respectively. Furthermore, to increase virus inactivation, photodynamic inactivation (PDI) was applied, which uses oxygen, light and a photosensitizer to produce reactive oxygen species (ROS). Grape seed extract (GSE) and oleanolic acid with known antiviral properties were tested as photosensitizers. Conditions using UV light at 254 nm with a distance of 72 cm and doses (energy density) of 0.012±0.000, 0.020±0.001, 0.040±0.001, 0.061±0.002, 0.081±0.002 and 0.121±0.003 J/cm² for 3, 5, 10, 15, 20 and 30 min exposure times, respectively were applied for the PDI experiments. However, the acquired viral reductions by GSE and OA mediated PDI were attributed to UV light more than ROS production. Future work may include the use of different light sources for illumination, and the use of UA as a potential photosensitizer compound.

Currently, breast cancer becomes the most frequently diagnosed and the second deadly cancer among women in United States. American Cancer Society reported that an estimated 234,190 new cases of diagnosed and 40,730 new cases of death would occur in United States in 2015. Despite of sex, age, race, pregnancy, breastfeeding and other factors, current therapies, such as breast-conserving surgery (BCS), radiation therapy and systemic therapy (hormone and targeted therapy) seem no longer effectively to control the growth of human breast cancer. Thus, there is an increasing demand to develop effective and alternative cancer treatments to improve the current breast cancer therapies and optimize the recovery procedure. Phytochemicals from fruits and vegetables are suggested to be associated with reducing the risk of chronic diseases, such as cancer. The advantages of choosing phytochemical as anticancer agents such as easily approachable, large diversity make them great potential to be developed to a new treatment for cancer. For example, EGCG and quercetin, widely distributed in fruits and vegetables, have been reported to exhibit high antioxidant, anti-inflammatory and anti-cancer effects. However, the molecular mechanism of actions of phytochemicals in the prevention of cancer is not fully understood yet. In my Ph.D. project, ursolic acid (UA), a triterpenoid, has been used for anti- proliferation and cytotoxicity studies towards MDA-MB-231 human breast cancer cells by methylene blue assay. Results determined that cell proliferation of MDA-MB231 cells can be effectively inhibited by ursolic acid in a non-cytotoxic concentration range. Thus, UA was chosen as candidate phytochemical in the study of signaling pathways in MDA-MB-231 cells, specifically focused on anti-proliferation and apoptosis pathways. On the anti-proliferation signaling pathway, the expressions of a serious of key proteins, including PCNA, CDK-4, cyclinD1, p21, p-p53, p-p38, pASK 1 and TRAF-2, were all measured by immunoblotting assay. Results demonstrated that there was a dose-dependent relationship between UA concentrations and expression of all these proteins. In addition, p38 inhibitor study showed the reversing effect on the expression of PCNA and CyclinD1 inhibited by UA. In the apoptosis signaling pathway, the expressions of Bax, Bxl-2 and cleaved caspase-9 and 3 were also measured by immunoblotting assay. Data suggested that UA induce mitochondria apoptotic pathway in MDA-MB-231 in a dose-dependent manner. Moreover, apoptosis effect induced by UA was also confirmed by TUNEL assay. At last, two-way combination studies on UA and other phytochemicals (EGCG, quercetin, and resveratrol) were performed to test the additive and synergistic effect in MDA-MB-231 cells. Results revealed that combinations of these phytochemicals exhibited the additive and potential synergistic effect in MDA-MB-231 cells. Future work on three- or more way of combination is necessary to elucidate the mechanism of synergy among phytochemicals in fruits and vegetables In summary, ursolic acid had anti-proliferative effect in MDA-MB-231 human breast cancer cells through p38/MAP kinase pathway by regulating cell cycle cyclinD1/CDK family and PCNA proteins. Meanwhile, ursolic acid induced apoptosis through mitochondria death pathway by regulating the expression of Bax/Bcl-2 family proteins and caspase family proteins through p38/MAP kinase pathway. Ursolic acid had additive and potential synergistic effect with other phytochemicals. These data provide insights in developing new cancer therapy using ursolic acid and other phytochemicals.

A series of pentacyclic triterpenes found in P. frutescens, including ursolic acid (UA), oleanolic acid (OA), augustic acid (AA), corosolic acid (CA), 3-epi-corosolic acid (3-epiCA), maslinic acid (MA), and 3-epi-maslinic acid (3-epiMA) were evaluated for their effects on skin tumor promotion by 12-O-tetradecanoylphorbol-13-acetate (TPA). UA was also evaluated in a combination with resveratrol (Res) for possible combinatorial chemopreventive activity. All triterpene compounds significantly inhibited skin tumor promotion by TPA. MA and 3-epiCA, were significantly more effective than UA at inhibiting tumor development. Topical pretreatment with all of these compounds significantly inhibited epidermal proliferation induced by TPA, however, CA, 3-epiCA and MA were more effective than UA. All compounds reduced skin inflammation and inflammatory gene expression induced by TPA, however, 3-epiCA and MA were identified to be more effective than UA. Finally, the ability of these compounds to alter epidermal signaling pathways associated with skin tumor promotion by TPA was also evaluated. The greater ability of 3-epiCA and MA to inhibit skin tumor promotion was associated with greater reduction of Cox-2 and Twist1 proteins and inhibition of activation of IGF-1R, Stat3 and Src. The effect of combining UA + Res for combinatorial inhibitory effects on skin tumor promotion were also examined. The combination of UA + Res produced a greater inhibition of TPA-induced epidermal hyperproliferation, epidermal inflammatory signaling, and inflammatory gene expression when compared to UA or Res alone. Furthermore, NF-kB, Egr-1, and AP-1 DNA binding activities following TPA treatment were dramatically decreased by the combination of UA + Res. Treatment with UA + Res during skin tumor promotion by TPA produced greater inhibition of tumor multiplicity and tumor size than with either agent alone. Collectively, the current data demonstrate that UA and related triterpenes as well as the combination of UA + Res inhibited skin tumor promotion by TPA via effects on multiple cellular and biochemical/molecular mechanisms associated with this process similar to calorie restriction. Of the tritperpenes tested, 3-epiCA and MA were the most active. Furthermore, the favorable anti-tumor promoting effects of combining UA + Res suggest that phytochemical combination therapy may be a more efficacious strategy for cancer chemoprevention.