Phytonutrient Core

Director: Qing-Yi Lu, PhD
Co-Director: Vay Liang W. Go, MD
Research Assistant: Lifeng Zhang, PhD

• Specific Aims

  1. - To provide standardization and analysis of plants/herbal extracts being studied in individual projects using HPLC, GC, GC/MS, LC/MS, and other established analytical methods;
  2. - To develop novel methods as needed to investigate bioactive and marker compounds in plant/herbal extracts and to assess animal plasma/tissue bioavailability of the phytonutrients;
  3. - To utilize the core laboratories of the UCLA Molecular Instrumentation Center as necessary to further identify and characterize the specific structures of compounds found in the extracts.

 

The UCLA Center for Human Nutrition houses two fully equipped laboratories for chemistry and analysis:

*Pharmanex Phytochemical Laboratory;
*Mark Hughes Cellular and Molecular Nutritional Laboratory.

- 1 Thermofinnigan LCQ Classic LC-MS/MS with ESI and APCI sources coupled to an HP 1100 HPLC with a diode array-UV detector;
- 1 Finnigan Trace GC/MS;
- 1 Perkin Elmer Biosystem Mariner Time of Flight High Resolution MS coupled to an HP 1100 HPLC with photodiode array-UV detector;
- 4 Analytical HPLCs: two 2690 Waters HPLC systems with diode array-UV detector, Scanning Fluorescence Detector and Coulochem II- Electrochemical Detector;
- 2 Preparative scale LC 4000 Waters systems with UV detectors equipped with numerous semi-prep and preparative columns and 2 NovaPrep 800 HPLCs;
- 1 Semi-prep Rainin HPLC with Gilson RI (Refractive Index), Dynamax UV and ELSD (Evaporative Light Scattering Detector).

Access to Other Core Facilities at UCLA

*The UCLA Molecular Instrumentation Center
- Mass Spectrometry Laboratory
- Proteomics and Biochemistry Instrumentation Facility
- The Magnetic Resonance Facility
- Materials Laboratory
- Crystallography Laboratory
*The UCLA Pasarow Mass Spectrometry Laboratory
*Harbor-UCLA Stable Isotope Core Laboratory

 

Phytonutrient Core will provide a wide range of services in support of project and other investigators

- Isolation of bioactive phytochemicals using bioassay-guided fractionation (e.g. Glycyrrhiza uralensis L.).
- Structural determination of purified active compounds using MS, NMR, UV, IR and chemical reactions.
- Analytical methods development and method validation using HPLC, GC, LC/MS, TLC, and wet chemistry.
- Certification of standardized botanical preparations for all research studies conducted in the program project.
- Providing product quality information to the working group of the National Advisory Council for Complementary and Alternative Medicine.

 

Available HPLC and GC methods following extraction:

From plants:
- Quantitation of carotenoids, tocopherols, retinol by HPLC
- Quantitation of isoflavones (genistein and daidzein) by HPLC
- Quantitation of hypericin and other phytochemicals in Hypericum perforatu
- Quantitation of monacolins in Chinese Red Yeast Rise by HPLC
- Quantitation of ginsenosides in Panax pseudoginseng Wall.
- Quantitation of glycyrrhizic acid in Glycyrrhiza uralensis L. (Chinese licorice)
- Quantitation of green tea catechins by HPLC
- Quantitation of polyphenols of Scutellaria baicalensis by HPLC
-Quantitation of fatty acids from Saw Palmetto by GC.

From body fluids
- Quantitation of plasma carotenoids, tocopherols, retinol by HPLC
- Quantitation of urinary and plasma isoflavones (genistein and daidzein) by HPLC
- Quantitation of plasma lovastatin and lovastatin acid by HPLC and LC/MS
- Quantitation of urinary and plasma green tea catechins by HPLC
- Quantitation of plasma, urinary and tissue polyphenols of Scutellaria baicalensis by HPLC
- Quantitation of plasma fatty acids by GC

Studies and Results

The primary aim of the Phytonutrient Core laboratory continues to be the support of Center investigators with resources to perform various chemical analyses.  UCLA Center for Excellence in Pancreatic Diseases has three research projects: Project 1 – the effects of phytochemicals and metabolism in pancreatic diseases (phytochemicals include embelin (EM), ellagic acid (EA), rottlerin (RT), lycopene (Lyc) and curcumin (Cur)).  Project 2 – role of polyphenols in regulating lipid inflammatory process in pancreatic cancer (using extract of Scutellaria baicalensis Georgi and green tea or green tea polyphenols); and project 3 – the effects of flavonoids on pancreatic carcinogenesis and angiogenesis (using quercetin, apigenin and genistein).  In less than 4 years, our core laboratory has developed scores of methods for sample preparation and chemical analysis. Following is a list of all the measurements we performed for each project followed by a brief description of results:

 

In supporting of project 1, we have:

  1. examined the purity of EM, EA, Cur, RT and Lyc using HPLC and/or LC-MS;
  2. measured the long-term stability of EM, RT and Lyc in common organic solvents;
  3. examined the bioavailability of Lyc and EA in liver, pancreas and plasma of nude mice injected (ip weekly) EA (25mg/kg) and mixed Lyc-EA (1mg/kg-1mg/kg and 2mg/kg-5mg/kg) for 4 weeks (n=4 for each treated group);
    1. determined the distribution of Lyc in the liver, pancreas and plasma of xenograft mice injected (ip weekly) EA (25mg/kg) and mixed Lyc-EA (1mg/kg-1mg/kg and 2mg/kg-5mg/kg) for 5 weeks (n=5 for each treated and untreated group);
    2. examined the bioavailability of EM (70 mg/kg) and RT (20 mg/kg) in C57BL/6 mouse plasma (blood collected at 1, 3 and 24 hr after gavage, n=3 per group);
    3. measured the stability of EM, EA and RT in animal diets containing EM, EA, EM+EA or EM+RT;
    4. determined the distribution of EM and RT in plasma, pancreas, subcutaneous tumor, lung and liver of the mice fed a diet containing EM, EA, RT, EM+EA or EM+RT for 6 weeks (n=6 for 5 treatment group and a control group);
    5. examined the plasma absorption of EM in mice (orthotopic pancreatic xenograft model) fed diet containing EM, EA or EM+EA (n=6 for each treatment and a control group);
    6. examined the stability of EM in aqueous buffers (pH=5, 7 and 9) and in ex vivo mouse liver homogenates;
    7. examined the stability of RT in aqueous buffers (pH=5, 7 and 9) and in ex vivo mouse liver homogenates;
    8. prepared EA–methyl-b-cyclodextrin complex (EA-MebCD) in 1:2 mole ratio;
    9. examined the bioavailability of EA-MebCD in tissues of intestine, liver, and pancreas and in blood of Sprague-Dawley rats administered EA-MebCD (equivalent to 0.4 g EA/kg) by gavage, in comparison to the rats administered EA (same dose) only. Rats were sacrificed 30 min or 1 hr after the administration (n=3 per group);
    10. prepared Cur-hydroxypropyl-g-cyclodextrin complex in various ratio and measured the solubility of Cur.

Lyc was detected in plasma, lung, liver and pancreas of mice injected with higher dose, whereas in lower dose lycopene was found in the tissues of liver and pancreas only.  EA was not found tissue or plasma of nude mouse. EM was found in plasma but not in the tissues sampled. RT was found in tissues of pancreas, pancreatic tumor, liver and lung collected and in plasma of mice after feed with diet containing RT.

Ellagic acid is known to be insoluble in many common solvents. In order to increase solubility and bioavailability, we prepared and administered rats with EA-MebCD complex. We found levels of EA were higher in both plasma and pancreas tissue after administering with EA-MebCD, in comparison to the rats administered with EA only.

 

In supporting of project 2 we have:

  1. procured the extract of Scutellaria baicalensis Georgi (SB) from Cortex Scientific Botanicals (Ojai, CA) in compliance with NCCAM interim policy on the botanical product quality;
  2. quantified SB flavonoids baicalin, baicalein, oroxylin A, wogonin, wogonoside, oroxylin A-7-O-glucuronide in SB extract;
  3. provided the solubility information on preparing SB stock solution for in vitro studies;
  4. examined the concentration change of bioactive constituents baicalin and baicalein in cell culture medium with pancreatic cancer MiaPaca cells treated with 25 mM of baicalin or baicalein in presence and in absence of catalase, as well as in presence and in absence of serum;
  5. determined the stability of SB flavonoids in animal diet at days 0 and 5;
  6. determined the distribution of SB in tissues of liver, pancreas, subcutaneous tumor and in plasma of the mice fed diet containing 0.1 or 1% SB extract for 6 weeks in a preliminary study (n=2 for each treatment group);
  7. determined the distribution of SB in tissues of colon, small intestine, lung, liver, pancreas, kidney, prostate, subcutaneous pancreatic tumor and in plasma of the mice fed a diet containing 1% SB extract for 13 weeks (n=8 for treatment and untreated control group);
  8. determined the metabolic stability of baicalin in homogenates of colon, small intestine, lung, liver, pancreas, kidney and prostate tissue collected from control mice after addition of known concentrations of baicalin;
  9. examined the stability of baicalin in liver homogenates (pH=5 and pH=7) and in plasma. Samples were taken at 0, 30, 60, 90, and 120 min after the addition of known concentration of baicalin.
  10. examined the stability of baicalin in aqueous buffer (pH=5 and pH=7) and in plasma. Samples were taken at 0, 30, 60, 120, 210 and 1440 min after the addition of known concentration of baicalin.

Using HPLC, we confirmed baicalin (20.6%, wt/wt) is the major flavonoid component in the SB extract. We detected small amount of oroxylin A-7-O-glucuronide (1.5%), wogonoside (3.0%), baicalein (1.2%), wogonin (<1.0%), and oroxylin A (<1.0%) present in the extract (data not included in CoA from the manufacturer). To investigate the absorption of SB extract in pancreas and tumor as target tissues, we also evaluated concentrations of metabolites in lung and liver, as lung and liver are common sites of metastases.  In addition, we investigated metabolites in the tissues of small intestine, colon, kidney, and prostate. We found all tissues from mice fed a diet containing 1% of SB accumulated baicalein, wogonin, and oroxylin A (methylated baicalein), as well as their glucuronides/sulfates.  Levels of total baicalein, wogonin, and oroxylin A are highest in colon (334.6 nmol/g tissue), followed by small intestine, liver, kidney, prostate, pancreas, lung, and tumor (2.32 nmol/g). Moreover, about half of the baicalin is metabolized to the 6-O-methylated oroxylin A compound in tissues investigated with the exception of prostate.

To investigate metabolic stability of baicalin in mouse tissue homogenates we added 2.24 or 22.40 nmol/g tissue of baicalin in various control mouse tissue homogenates in pH 7 buffer and incubated at 37oC for 2 hr under anaerobic and dark conditions.  Results showed the conversion of baicalin to baicalein occurred in all tissues investigated (colon, small intestine, liver, kidney, prostate, pancreas, and lung). In liver homogenates, enzymatic hydrolysis at pH 7 occurred almost immediately after addition of baicalin (t=0 min) and reached a maximum with 60% molar conversion at 30 min.  These data suggest that baicalin undergoes ex vivo hydrolysis and phase II metabolic pathways in various tissues (Lu et al. Pancreas. in press).

 

In supporting of project 3 we have:

  1. examined the purity of quercetin, apigenin and genistein using HPLC and/or LC-MS;
  2. examined the concentration change of quercetin (30 mM) in cell culture medium RPMI and determined the uptake of quercetin in MiaPaca cells;
  3. determined the stability of quercetin in animal diets (5%) at days 0 (baseline), 1, 2, 3, 4 and 7;
  4. determined plasma absorption of quercetin in nude mice after feeding with 5, 7.5 or 10% of quercetin for 8 weeks in a preliminary study (n=4 for each treatment group, blood collected at 2, 4, 6 and 8 weeks);
  5. determined the plasma absorption of quercetin and its metabolites in the mice (orthotopic pancreatic xenograft model) fed a 5% quercetin diet for 8 weeks (n=16  for treatment and control group);
  6. determined the distribution of quercetin and its metabolites in tissues of  pancreas, tumor, lung and liver and in plasma of the mice (orthotopic pancreatic xenograft model) fed 0.2 or 1% quercetin for 6 weeks (n=12 for each treatment group and a control group);
  7. determined the stability of apigenin in animal diets (0.01 and 0.05%) at days 0, 1, 2 and 3;
  8. determined the absorption of apigenin in plasma and tumors of the orthotopic pancreatic xenograft mice fed a diet containing 0.01 or  0.05% apigenin for 6 weeks in a preliminary study (n=5 for each treatment group);
  9. determined the distribution of apigenin and its metabolites in plasma and tumors of the orthotopic xenografts mice fed a diet containing 0.2% apigenin for 6 weeks (n=8 for a treatment and a control group).

 

We reported for the first time that quercetin and its metabolites are distributed in pancreas and pancreatic xenograft tumors of mice after administering with 0.2 or 1 % quercetin diet for 6 weeks.  All investigated organs (liver, lung, pancreas and tumor) contained a considerable proportion of deconjugated quercetin and isorhamnetin (methylated quercetin), ranging from 40-100% of the total quercetin concentration.  In tumor 64% of quercetin and 75% of isorhamnetin are aglycone forms.  In blood, conjugates were found almost exclusively in plasma (>98%). We reported chemotherapeutic drug gemcitabine co-treatment with quercetin reduced absorption of quercetin in circulatory system and liver. In addition, we found quercetin was accumulated in pancreatic cancer MiaPaCa-2 cells after cells were exposed to quercetin (30 μM) in vitro.  However, quercetin is very unstable and its level declined to half of its initial concentration (30 μM) in 1 hr in cell culture medium (Zhang et al.: J. Agric. Food Chem. 2010, 58, 7252–7257).

For the nude mice receiving orthotopic injection of pancreatic cancer MiaPaCa-2 cells and fed 0.2% apigenin diet for 6 weeks, we found apigenin and its metabolite luteolin accumulated in plasma and tumor. Compared with a base diet-fed mice, apigenin-fed mice had smaller tumors (0.7±0.1 vs 0.9±0.1 g, P=0.058) and this correlated with tumor apigenin level (HPLC; Pearson correlation -0.65, P<0.05) (submitted for publication).

 

Other Studies and Results

In addition to support Center investigators with resources to perform various chemical analyses, we have conducted and completed following research projects:

 

1. Profiling of carotenoids, tocopherol and fatty acids in California Hass avocado  A total of 95% of American avocado production is located in southern California.  The California Hass avocado (Persea americana) is an example of a domesticated berry fruit that matures on the tree during its growing season but ripens only after being harvested. Avocados are typically harvested multiple times during the growing season in California. Previous research has demonstrated potential health benefits of avocados and extracts of avocado against inflammation and cancer cell growth, but seasonal variations in the phytochemical profile of the fruits being studied may affect the results obtained in future research. Therefore, in the study, avocados were harvested in January, April, July, and September, 2008, from four different growing locations in California (San Luis Obispo, Ventura, Riverside, and San Diego) and analyzed for total fat content, fatty acid profile, carotenoids, and vitamin E.  We found a significant increase in total carotenoid and fat content of avocados from all regions as the season progressed from January to September. Four carotenoids not previously described in the avocado were identified and quantified. The total content of carotenoids was found to be highly correlated with the total fat content (r = 0.99, p < 0.001) demonstrating a remarkable degree of constancy of carotenoid intake per gram of fat content in the California Hass avocado. Our study suggests that research on the potential health benefits of the avocado should consider the time of harvest, degree of ripening, growing area of the particular fruits being studied, and the total phytochemical profile. These steps will enable researchers to account for potential nutrient-nutrient interactions that might affect their research outcomes (Lu et al. J. Agric. Food Chem. 57: 10408-13, 2009).

 

2. In-vitro tracer-based investigation on the effect of loquat leaves extract toward glucose metabolism using HepG2 Sesquiterpene isolated from dried loquat leaves (Eriobotrya japonica (Thunb.) Lindl.) has been reported to have anti-hyperglycemia properties in diabetic animal model. In a joint effort with investigators of the Metabolomic Core of the Center, we have prepared loquat leave extract and obtained active ingredient sesquiterpene through our collaboration with the researchers at Institute of Botany, Jiangsu Province and the Chinese Academy of Sciences, Nanjing, China.  We have investigated the effect of loquat leaves extract on glucose metabolism in HepG2 cells using [1, 2-13C2]-glucose as the tracer and also compared the effect of the purified sesquiterpene to that of metformin, an antidiabteic drug known for its insulin sensitizing effect. Human hepatoma HepG2 cells were grown in tissue cell culture in Dulbecco’s modified Eagle’s medium containing 50% [1, 2 13C2]-glucose in the presence of metformin (1 mM) or sesquiterpene (25 mg/mL). Cells were harvested in 48 hours and mass isotopomers of ribose and deoxyribose were determined. Metformin reduced formation of m1 by G6PDH and increased formation of m2 by TK/TA in ribose/deoxyribose. Using phenotypic phase plane analysis, a significant difference was observed (P< 0.05) in the glycolysis-pentose cycle pathway between loquat extracts and metformin. Our results suggest that loquat extract acts on different metabolic pathways than those of metformin to achieve its anti-hyperglycemic effect (abstract presented in Experimental Biology 2011, Washington D.C., April 9-14, 2011).

 

3. Quantitative proteomic analysis of proteins altered by green tea treatment in pancreatic cancer HPAF-II cells  Green tea and its polyphenols have been shown to exhibit multiple antitumor activities in various cancers, but studies on the pancreatic cancer are very limited.  In order to identify the cellular targets of green tea action, we exposed a green tea extract (GTE) to human pancreatic ductal adenocarcinoma HPAF-II cells, and performed two-dimensional gel electrophoresis of the cell lysates.  We identified 32 proteins with significantly altered expression levels. These proteins are involved in drug resistance, gene regulation, motility, detoxification and metabolism of cancer cells.  In particular, we found GTE concomitantly inhibited molecular chaperones heat-shock protein 90 (Hsp90), its mitochondrial localized homologue Hsp75 (tumor necrosis factor receptor-associated protein 1, or Trap-1) and heat-shock protein 27 (Hsp27).  Western blot analysis confirmed the inhibition of Hsp90, Hsp75 and Hsp27 by GTE.  Furthermore, we showed that GTE inhibited Akt activation and the levels of mutant p53 protein, and induced apoptosis and growth suppression of the cells. Our study has identified multiple new molecular targets of GTE and provided further evidence on the anticancer activity of green tea in pancreatic cancer (manuscript submitted).

Significance 

Extended laboratory studies supported epidemiological findings and provided evidence that quercetin and other flavonoids have many biological activities, such as antitumor and antiproliferative effects on varies human cancers.  However, most studies investigated biological activities in cultured cells by using the free form of the flavonoid, e.g. quercetin or aglycone, and did not take absorption and metabolism into consideration for the interpretation of results. Dietary flavonoids were present in the human circulating system predominantly as conjugates of glucuronides and sulfates that are likely to have differential biological activities and distribution patterns in tissues and cells compared with flavonoid aglycones.  More importantly, bioactive food components must be sufficiently absorbed in the gastrointestinal tract and reach pharmacological levels in target tissues to have the potential to exert biological activity.  In the past year Phytonutrient Core has made great effort to quantify and identify flavonoids from SB extract and their metabolites including the glucuronide and sulfate metabolites in the target tissue and tumors of the animals fed with SB diet. Moreover, we have expanded the studies of chemical stability in common organic solvents to aqueous buffers at various pHs to mimic physiological conditions,  to ex vivo tissue homogenates and to the metabolic stability study.  Our data on flavonoid tissue distribution may be used to predict the organ site(s) most likely to benefit from the treatment, and to provide important information on predicting chemopreventive or therapeutic efficacy of flavonoids investigated.

Future Plans

We will continue to monitor the stability of phytonutrients of interests.  We will measure the uptake of these chemicals in the cells and culture medium and identify their metabolites using HPLC or LC/MS/MS.  We will also determine the bioavailability of these phytochemicals in animal models and identify their metabolites.  Currently we are investigating chemical properties and bioavailability of embelin and rottlerin (project 1). Literatures on these aspects are very limited.
During past few years, we have used an in-house proteomic technique and performed quantitative and qualitative proteomic analyses of proteins altered by green tea treatment in pancreatic cancer HPAF-II cells (manuscript submitted for publication). Proteomics is a powerful tool that can directly analyze protein expression at the posttranslational level and allows the qualitative and quantitative assessment of a broad-spectrum of proteins that can be related to specific cellular responses.  We plan in the future to advance our proteomic studies utilizing samples from in vivo models to aid the identification of new molecular targets and to integrate protein identification with phytonutrient metabolism.