Barbara N. Timmermann

Inhibitory effect of six green tea catechins and caffeine on the growth of four selected human tumor cell lines.

Green tea is an aqueous infusion of dried unfermented leaves of Camellia sinensis (family Theaceae) from which numerous biological activities have been reported including antimutagenic, antibacterial, hypocholesterolemic, antioxidant, antitumor and cancer preventive activities. From the aqueous-alcoholic extract of green tea leaves, six compounds (+)-gallocatechin (GC), (−)-epicatechin (EC), (−)-epigallocatechin (EGC), (−)-epicatechin gallate (ECG), (−)-epigallocatechin gallate (EGCG) and caffeine, were isolated and purified. Together with (+)-catechin, these compounds were tested against each of four human tumor cell lines (MCF-7 breast carcinoma, HT-29 colon carcinoma, A-427 lung carcinoma and UACC-375 melanoma). The three most potent green tea components against all four tumor cell lines were EGCG, GC and EGC. EGCG was the most potent of the seven green tea components against three out of the four cell lines (i.e. MCF-7 breast cancer, HT-29 colon cancer and UACC-375 melanoma). On the basis of these extensive in vitro studies, it would be of considerable interest to evaluate all three of these components in comparative preclinical in vivo animal tumor model systems before final decisions are made concerning which of these potential chemopreventive drugs should be taken into broad clinical trials.

Inhibition of ultraviolet B-induced c-fos gene expression and p38 mitogen-activated protein kinase activation by (-)-epigallocatechin gallate in a human keratinocyte cell line

(‐)‐Epigallocatechin gallate (EGCG), the major polyphenol isolated from green tea, is an active chemoprevention agent against cancer. However, the molecular mechanisms that underlie the inhibitory effects of EGCG are not well understood. In this study, we tested the effects of EGCG on ultraviolet (UV) B radiation–induced c‐fos gene expression in a human keratinocyte cell line, HaCaT. EGCG inhibited UVB‐induced steady‐state message and transcriptional activation of the c‐fos gene in a dose‐dependent manner. Western analyses further indicated that EGCG had an inhibitory effect on UVB‐induced accumulation of the c‐fos protein within the same dose range. To further examine the mechanism by which EGCG inhibits UVB‐induced c‐fos expression, we tested the effect of EGCG on upstream activators of the c‐fos gene. We found that EGCG significantly inhibited activation of p38 mitogen‐activated protein kinase but not c‐jun NH2‐terminal kinase or extracellular signal‐regulated protein kinase activation. Our previous studies have indicated that UVB‐induced c‐fos expression may play a key role in UVB‐induced activation of the activator protein‐1 transcription factor and EGCG‐inhibited, UVB‐induced activation of AP‐1 in HaCaT cells. Because AP‐1 is important for tumor promotion and c‐fos is a major component of AP‐1, the inhibitory effects of EGCG on c‐fos expression may further explain the anti–tumor‐promoting effects of EGCG. Mol. Carcinog. 24:79–84, 1999.

Formulation Design and Photochemical Studies on Nanocrystal Solid Dispersion of Curcumin with Improved Oral Bioavailability

Considerable interest has been focused on curcumin due to its use to treat a wide variety of disorders, however, the therapeutic potential of curcumin could often be limited by its poor solubility, bioavailability, and photostability. To overcome these drawbacks, efficacious formulations of curcumin, including nanocrystal solid dispersion (CSD-Cur), amorphous solid dispersion (ASD-Cur), and nanoemulsion (NE-Cur), were designed with the aim of improving physicochemical and pharmacokinetic properties. Physicochemical properties of the prepared formulations were characterized by scanning/transmission electron microscope for morphological analysis, laser diffraction, and dynamic light scattering for particle size analysis, and polarized light microscope, powder X-ray diffraction and differential scanning calorimetry for crystallinity assessment. In dissolution tests, all curcumin formulations exhibited marked improvement in the dissolution behavior when compared with crystalline curcumin. Significant improvement in pharmacokinetic behavior was observed in the newly developed formulations, as evidenced by 12- (ASD-Cur), 16- (CSD-Cur), and 9-fold (NE-Cur) increase of oral bioavailability. Upon photochemical characterization, curcumin was found to be photoreactive and photodegradable in the solution state, possibly via type 2 photochemical reaction, whereas high photochemical stability was seen in the solid formulations, especially CSD-Cur. On the basis of these observations, taken together with dissolution and pharmacokinetic behaviors, CSD strategy would be efficacious to enhance bioavailability of curcumin with high photochemical stability.

Prevention of photocarcinogenesis by topical administration of pure epigallocatechin gallate isolated from green tea.

Topical application of purified (-)-epigallocatechin-3-gallate (EGCG), a polyphenolic antioxidant isolated from green tea, inhibited photocarcinogenesis in BALB/cAnNHsd mice with no visible toxicity. Mice were treated with 0, 10, or 50 mg of EGCG in 200 microliters of acetone three times weekly for three weeks before ultraviolet (UV) treatments began and throughout the experiment. UV radiation consisted of five 30-minute exposures per week to banks of six FS40 Westinghouse sunlamps for 25 weeks. In the photocarcinogenesis study, mice received a total dose of approximately 2.1 x 10(6) J/m2. Skin cancer incidence in UV-irradiated mice was 96% at 28 weeks after the first UV treatment; EGCG at 10 or 50 mg reduced this incidence to 62% and 29%, respectively. UV-induced immunosuppression, assessed by the inability of UVB-irradiated mice to reject a syngeneic antigenic tumor, was not influenced by topical EGCG. Oral administration of 0, 100, or 500 mg of pure EGCG per liter of drinking water (approximately 0, 0.56, or 2.8 mg/day, respectively) did not decrease UV-induced skin tumor incidence, rate of primary tumor growth, or inability to reject antigenic tumors. Thus induction of skin tumors by UV radiation was significantly reduced by topical, but not by oral, administration of purified EGCG through a mechanism distinct from inhibition of photoimmunosuppression.

Phytochemical Adaptations to Stress

1. Introductory Chapter.- 2. Putrescine and Inorganic Ions.- 3. Phytochemical Aspects of Osmotic Adaptation.- 4. Thigmomorphogenesis: Callose and Ethylene in the Hardening of Mechanically Stressed Plants.- 5. Stress and Secondary Metabolism in Cultured Plant Cells.- 6. Temperature Stress and Lipid Modification.- 7. Morphology, Chemistry, and Genetics of Gossypium Adaptations to Pests.- 8. Bioregulation of Plant Constituents.- 9. Production of Resins by Arid-Adapted Astereae.- 10. Changes in the Levels of Plant Secondary Metabolite Production Under Water and Nutrient Stress.- Species Index.

Pharmacokinetics of the green tea derivative, EGCG, by the topical route of administration in mouse and human skin

Purpose: (−)-Epigallocatechin gallate (EGCG), the main physiologically active polyphenol of green tea, is associated with antitumor and antimutagenic activities. The goal of this study was to determine the stability and pharmacokinetic parameters of pure EGCG administered topically to human and mouse skin. Methods: EGCG was investigated by measuring drug levels of a 10% ointment formulation stored under different conditions over a period of 6 months. To determine pharmacokinetic parameters of EGCG following topical application, EGCG was applied as 10% EGCG in hydrophilic ointment USP to full-thickness mouse or human skin in vitro. The transdermal and intradermal. Penetration of EGCG was measured by reverse phase HPLC assays at different time-points. Results: The stability of EGCG in hydrophilic ointment USP was dependent on time, temperature and the degree of oxidation. For example, 10% EGCG was lost after 2 days at 37 °C, but the same formulation supplemented with 0.1% butylated hydroxytoluene (BHT) had significantly longer stability with ≥90% EGCG remaining after 130 days at 37 °C. Topical application of EGCG in hydrophilic ointment USP to human or mouse skin resulted in substantial intradermal uptake of up to 1–20% of the applied dose. However, transdermal penetration was observed only in mouse skin. Conclusion: The present study showed that topical application of EGCG in hydrophilic ointment USP achieved high concentrations in skin but negligible systemic availability. The drug was susceptible to oxidation, but if supplemented with BHT, the hydrophilic ointment formulation could potentially be used in clinical trials of skin cancer prevention.

Nordihydroguaiaretic acid: hepatotoxicity and detoxification in the mouse

Larrea tridentata (Moc & Sess) Cov. (Zygophyllaceae) is an ethnobotanically important plant found in the American Southwest and northern Mexico. Although numerous beneficial effects have been attributed to this plant, several case reports have demonstrated high doses of Larrea-containing herbals induce hepatotoxicity and nephrotoxicity in humans. Nordihydriguaiaretic acid (NDGA) is a lignan found in high amounts (up to 10% by dry weight) in the leaves and twigs of L. tridentata. Previously, NDGA has been shown to induce cystic nephropathy in the rat, however, no reports have been made concerning this compounds hepatotoxic potential. Here, we report that intraperitoneal administration of NDGA is lethal in the mouse (LD(50)=75 mg/kg). Administration is associated with a time and dose-dependent increase in serum alanine aminotransferase levels, which suggest liver damage. Indeed, freshly isolated mouse hepatocytes are more sensitive to NDGA than human melanoma cells. Furthermore, we have identified glucuronidation as a potential detoxification mechanism for NDGA. Both mono and diglucuronide conjugates of NDGA are formed after intravenous dosing. The monoglucuronide is also formed after incubation of NDGA with human hepatic microsomes; suggesting that glucuronide conjugation is important in the metabolism of NDGA by humans. In summary, this report indicates that NDGA may contribute to the hepatotoxicity of L. tridentata and provides preliminary information on NDGA metabolism.

Anti-Arthritic Effects and Toxicity of the Essential Oils of Turmeric (Curcuma longa L.)

Turmeric (Curcuma longa L., Zingiberaceae) rhizomes contain two classes of secondary metabolites, curcuminoids and the less well-studied essential oils. Having previously identified potent anti-arthritic effects of the curcuminoids in turmeric extracts in an animal model of rheumatoid arthritis (RA), studies were undertaken to determine whether the turmeric essential oils (TEO) were also joint protective using the same experimental model. Crude or refined TEO extracts dramatically inhibited joint swelling (90-100% inhibition) in female rats with streptococcal cell wall (SCW)-induced arthritis when extracts were administered via intraperitoneal injection to maximize uniform delivery. However, this anti-arthritic effect was accompanied by significant morbidity and mortality. Oral administration of a 20-fold higher dose TEO was nontoxic, but only mildly joint-protective (20% inhibition). These results do not support the isolated use of TEO for arthritis treatment but, instead, identify potential safety concerns in vertebrates exposed to TEO.

Withaferin A, a cytotoxic steroid from Vassobia breviflora, induces apoptosis in human head and neck squamous cell carcinoma.

As part of a program to discover drug leads from plant biodiversity, the present investigation was undertaken to explore the anticancer potential of compounds derived from selected Latin American plants. Bioassay-guided fractionation of a crude extract of the aerial parts of Vassobia breviflora led to the isolation of the withanolide-type steroidal lactone withaferin A (1). This compound was tested for antiproliferative activity against the head and neck squamous cell carcinoma (HNSCC) cell lines, MDA1986, JMAR, UM-SCC-2, and JHU011. The inhibitory concentrations to reduce cell viability to 50% (IC(50)) were determined by the MTS cytotoxicity assay, and 1 reduced cell viability with IC(50) values in the range 0.5-2.2 μM. A mechanistic study showed that 1 induces apoptosis and cell death in HNSCC cells as well as a cell-cycle shift from G(0)/G(1) to G(2)/M. Cells treated with 1 exhibited inactivation of Akt and a reduction in total Akt concentration. This investigation constitutes the first report of the antiproliferative activity of withaferin A (1) against head and neck squamous carcinoma.

Comparative effects of two gingerol-containing Zingiber officinale extracts on experimental rheumatoid arthritis.

Ginger (Zingiber officinale) supplements are being promoted for arthritis treatment in western societies on the basis of gingers traditional use as an anti-inflammatory in Chinese and Ayurvedic medicine. However, scientific evidence of gingers antiarthritic effects is sparse, and its bioactive joint-protective components have not been identified. Therefore, the ability of a well-characterized crude ginger extract to inhibit joint swelling in an animal model of rheumatoid arthritis, streptococcal cell wall-induced arthritis, was compared to that of a fraction containing only gingerols and their derivatives. Both extracts were efficacious in preventing joint inflammation. However, the crude dichloromethane extract, which also contained essential oils and more polar compounds, was more efficacious (when normalized to gingerol content) in preventing both joint inflammation and destruction. In conclusion, these data document a very significant joint-protective effect of these ginger samples and suggest that nongingerol components are bioactive and can enhance the antiarthritic effects of the more widely studied gingerols.