2. Digitoxin, a “cardiac glycoside”, is used to treat congestive heart failure and cardiac arrhythmia Derived from cholesterol, with shortening and lactonization of side chain, plus several sugar units attached at C3-OH Digitoxin competes with K + for a binding site on enzyme potassium-ATPase (potassium blocker/antagonist) inhibiting Na-K ATPase pump By blocking this activity, it causes Ca 2+ to be transported out more slowly; so heart muscle is exposed to Ca 2+ for a longer period of time As a result, the heart contracts more forcefully (“inotropic effect”) It also increases cholinergic stimulation to the heart, which slows it down (steadier, stronger heartbeat with more rest between beats) Too high a dose of Digitalis can be toxic, but the therapeutic dose is about 50% of the toxic dose so it must be taken with care. Similar compounds (saponins) have been used as arrow poisons!

3. Structural variation among the cardiac glycosides (cardenolides) gitoxigenins are lower in activity than the digitoxigenins gitaloxigenins are unstable and usually lose the R=formyl group

4. Tetraterpene precursor phytoene is made in the same way by joining 2 GGPPs Triterpenes: Precursor squalene is made by tail-to-tail joining of two FPPs

5. precursor to cholesterol & other animal steroids precursor to many plant sterols Folding and cyclization of squalene oxide leads to sterol/steroid precursors more details next slide

6. How folding and cyclizations lead to sterols & triterpenes Folding pattern leading to protosteryl cation, precursor of cycloartenol & lanosterol

7. Folding pattern leading to most pentacyclic triterpenoids in plants Chair-chair-chair-boat configuration leads to dammarenyl cation

8. Precursor to ginsenosides found in Panax ginseng (p.224) Common pentacyclic triterpenes Folding pattern leading to most pentacyclic triterpenoids in plants, continued

9. Skeletal numbering system of pentacyclic triterpenoids Some bioactive pentacyclic triterpenoids Triterpenoid saponins have sugars attached to 3-OH and soapy properties. Some triterps are esterified at 3-OH based on lupeol Betulinic acid Ursolic acid based on  -amyrin Oleanolic acid based on  -amyrin R = H = Ursolic acid R = OH = Pomolic acid R Anti-proliferative activity Anti-inflammatory activity Ursolic acid derivatives inhibit tumor cell proliferation @ <10 uM Pomolic > ursolic Anti-viral activity (HIV) reduced production of p24 viral protein in infected T cells at 1.4 uM Synthetic esters of BA were effective at nM conc. (Kashiwada, et al, 2000) Replace COOH with CH 3 : lupeol

10. Cancer fighter from Cranberry Waste??? Ursolic acid in cranberries first reported 1934 Waxy component of peel 1950’s: triterpenoids isolated from cranberry pulp (Laakso) Considered “wastes” from the processing of berries to make juice What do we know about ursolic acid today?

11. Ursolic acid affects numerous pathways associated with inflammation and carcinogenesis in vitro  Inhibits proliferation of many tumor cell lines  Induces apoptosis in liver, leukemia, human prostate cancer models, arrests cells in G 1 phase; caspase activation, c-IAP down-regulation observed  Decreases MMP-9 expression in human fibrosarcoma (may decrease tumor metastasis)  Decreases COX-2 expression (anti-inflammation)  Inhibits activation of NF-kB (anti-inflammation) in vivo  Decreases inflammation in mouse-paw edema model  Decreases FSaII murine fibrosarcoma growth in mouse model (IV administration)  Diet containing 0.10% UA delayed breast tumor appearance, significantly decreased tumor volume in mice (DeAngel, et al, 2010)

12. Ursolic acid (UA) Ursolic acid & its hydroxycinnamoyl esters are plentiful in cranberry peels cis-3-O-p-hydroxycinnamoyl ursolic acid (cis-HC-UA) trans-3-O-p-hydroxycinnamoyl ursolic acid (trans-HC-UA) Content in cranberry products, other Vaccinium berries, inhibition of matrix metalloproteinases Kondo, et al, JFSA, 2011 Cytotoxicity in tumor cells and structure elucidation by 1 H & 13 C NMR; Murphy, et al, JAFC, 2003

15. NCI screening in tumor cell lines shows HC-UA esters antiproliferative at  M concentrations Cell lineTumor type GI 50 (  M) cis HC-UAtrans HC-UA MCF-7Breast1.41.9 MDA-MB-435Breast4.38 DU-145Prostate4.15.3 NCI-H460Lung2.22.6 NCI-H322MLung1.210 HT-29Colon519 HCT-116Colon1.53.5 RPMI-8226Leukemia1.86 SF-295CNS3.02.7 RXF 393Renal1.51.8 TK-10Renal1.211 SN12CRenal1.72.4 * M. Kondo, M.S. Thesis, UMass Dartmouth, 2006

16. Ursolic acid dramatically reduced formation of colon tumor colonies in a 14-day soft agar colony formation bioassay Liberty, et al, Acta Horticulturae, 2009

17. Ursolic acid also increased apoptosis in colon tumor cells Liberty, et al, Acta Horticulturae, 2009

18. “Saponins” have a triterpenoid aglycone and several sugars attached “soapy” properties also make some saponins toxic, because they can lyse red blood cells Glycyrrhizin also inhibits ROS production by neutrophils, modulates interferon, interleukins what type of skeleton?

19. Steroids: numbering system and possible ring conformations Properties depend on Ring fusion stereochemistry Functional group positions estradiol sitosterolprogesteroneergosterol rare! cholesterol

20. Ginseng (Panax ginseng) Traditional Asian medicinal herb Roots are dried & peeled (white) or steamed (red) Contains triterpenoid saponins (“ginsenosides”) with multiple sugars attached Taken as a general restorative, “adaptogen” to help body cope with stress Traditional uses: anemia, diabetes, gastrointestinal, insomnia, impotence Effects on CNS—stimulate or relax? Better concentration? Studies indicate ginseng may modulate blood pressure, enhance cerebral blood flow and increase mitochondrial energy metabolism.

21. Japanese ginseng dammaraneoleanane Panax japonicus is used to treat diabetes in traditional Chinese medicine Other properties: expectorant, antitussive, sedative, analgesic Previous study isolated polyacetylenes with  -glucosidase inhibitory activity

22. Summary 37 compounds isolated (2 new ones) LC-MS/MS patterns of isolated compounds matched those in the plant extracts 3 bioassays were conducted: Cytotoxicity to lung, colon, prostate (DU145) and nasopharyngeal (KB) cells: Compound 1 was cytotoxic to DU145, KB cells (6 – 7  g/mL) but not to lung or colon cells. Inhibition of superoxide anion generation and elastase release by neutrophils (antioxidant/antiinflammatory effects) was determined by measuring ferricytochrome c reduction normally inhibited by SOD: Several oleanane glycosides inhibited superoxide and elastase production. Inhibition of  -glucosidase, an enzyme used to metabolize carbohydrates (a measure of anti-diabetic activity) was determined by measuring enzyme activity on a substrate: Compounds 14 & 15 had moderate anti-diabetic activity.

23. Cholesterol Main functions and properties Cell membrane structure & fluidity Precursor of steroid hormones and bile acids Carried by lipoproteins in esterified form Found in eggs, dairy, meat Molecule is flat due to the trans-fused configuration of the B, C & D rings 8 chiral carbons = 256 possible stereoisomers (only one occurs in nature!) Diet, medicine and cholesterol levels Most cholesterol in the body does not come directly from food sources but is biosynthesized in response to body’s needs However, excess dietary cholesterol can inhibit uptake by cells and dietary fats affect cholesterol transport & metabolism High HDL levels reduce cholesterol level in bloodstream Many cholesterol-lowering drugs (statins) work by interfering with cholesterol biosynthesis Statin structure mimics that of HMG Co-A - competitively inhibits HMG Co-A reductase, a key enzyme in IPP biosynthesis

24. Formation of isoprene units by the mevalonate pathway The only pathway to isoprenoids in animals Pathway to steroids in animals; sterols and triterpenoids in plants Rxns occur in the cytosol from 6 C to 5 C inhibited by statins

25. Sterol skeletons: variation is mainly in the side chain on the D ring, additional methylation on other rings

26. Some common plant and fungal sterols Soybean-derived sterols were found to lower LDL cholesterol levels by 10-15% Soybeans contain high concentrations of stigmasterol, sitosterol & campesterol -may be esterified with fatty acids or glycosylated. Ergosterol is commonly produced by fungi, synthesis is blocked by miconazole and other azole drugs, weakening fungal membranes. Fucosterol is found in brown algae stanols have reduced C=C campestanol, esterified has dramatic LDL lowering H 2, Pt

27. Medicinal applications of sterols (Dewick, Ch. 5)  Dietary plant sterols have been known to reduce cholesterol levels in laboratory animals for many years, which has led to the introduction of plant sterol esters as food additives, particularly in margarines, as an aid to reducing blood levels of low density lipoprotein (LDL) cholesterol (risk factor for heart disease)  Plant sterol esters are usually obtained by esterifying sitosterol with fatty acids to produce a fat-soluble product. Regular consumption of this material (1.3 g per day) is shown to reduce blood LDL cholesterol levels by 10–15%. The average diet will normally include small amounts of plant sterol esters.  How it works: The plant sterols are more hydrophobic than cholesterol and have a higher affinity for micelles involved in fat digestion, effectively decreasing intestinal cholesterol absorption. Plant sterols themselves are not absorbed from the GI tract.  Related materials used in a similar way are plant stanol esters. Stanols are obtained by hydrogenation of plant sterols: mainly sitostanol and campestanol. These are then esterified with fatty acids.

28. More on sterols and stanols  Regular consumption of plant stanol esters (3.4 g per day) is shown to reduce blood LDL cholesterol levels by an average of 14%. Stanols are usually transesterified with rapeseed oil, which is rich in unsaturated fatty acids.  Clinical trials report that daily consumption of 1.5 – 3 g of phytosterols & stanols reduce total cholesterol by 8 – 17% (Nutraingredients.com)  Patients with metabolic syndrome who consumed 4 g/day of plant sterols in yogurt had a 20% drop in LDL and triglyceride levels  Foods fortified with plant sterols include spreads (such as Benecol and Take Control), beverages (such as Minute Maid Heart Wise) and snacks (such as Nature Valley Healthy Heart Chewy Granola Bars).  Rye bread: consumption of high fiber rye bread enriched with plant sterols reduced total cholesterol by 5%, LDLs by 8%  Cranberries contain  -sitosterol

29. YAMS! YUM!

30. Biosynthetic route to an animal steroid: progesterone biosynthesis Some Dioscorea species (yams) esp. those grown in Mexico have high content of saponins 10,000 tons of tubers/year  steroid drugs The sapogenin aglycone (typicaly diosgenin) is obtained by fermentation of the yams, followed by heating with aqueous acid to fully hydrolyze sugars, then the diosgenin is extracted. Diosgenin is also marketed to treat menopause symptoms. Diosgenin: a natural precursor to synthetic hormones Prepares uterus for pregnancy Released on ovulation Intermediate in corticosteroid biosynthesis

31. A semi-synthetic route to progesterone used in production of birth control pills From soybeans Key step since most steroid hormones have small substituent on D ring

32. Some key steroids in human physiology Animal steroids – Derived from cholesterol, most animal steroids have the substituent on the D ring shortened or removed completely. Corticosteroids and progesterones have a short chain, estrogens and androgens typically have no chain but may be oxygenated on the D ring Glucocorticoids are produced by the adrenal cortex & have many functions - Glucose metabolism - Involved in carbohydrate synthesis and storage in liver - Play a role in inflammatory processes Aldosterone is involved in balancing electrolytes Promotes Na + retention and K + excretion

33. Estrogens Most phytoestrogens are products of the shikimate pathway, not isoprenoids! Suppress ovulation and androgen formation Control menstrual cycle Aromatic ring introduces greater planarity Some cancer cells are estrogen or androgen-sensitive due to cellular receptors that bind steroids. Examples: MCF-7 breast cancer cells (estrogen) LNCaP prostate cancer cells (androgen)

34. Testosterone and estrogens produced through same pathway Cholesterol Natural and synthetic “anabolic” steroids Development and maintenance of male sex characteristics Promote muscle growth but side effects include reduced fertility, increased aggression

35. Vitamin D Cholecalciferol is a sterol (isoprenoid) formed photochemically in animal skin from 7-dehydrocholesterol by sun’s irradiation Also found in yeasts and some plants as D2 (ergocalciferol) which only differs in side chain structure Dietary sources include liver and dairy products, fish liver oils Vitamin D3 is hydroxylated in the body to its active forms, first calcidiol and then to calcitriol (Figure 5.106, p58 Dewick). Key role: Ca metabolism Calcitriol stimulates the absorption of calcium and phosphate in the intestine and the mobilization of calcium from bone Other physiological functions include a role in immune modulation, hormone secretion, and cell differentiation Vitamin D deficiency is common in Northern latitudes due to reduced exposure to sunlight Rickets = inability to calcify the collagen matrix of growing bone, causing lack of rigidity in the bones (children) or osteoporosis In most countries, milk and cereals are fortified with vitamin D3

36. Vitamin D is a product of the mevalonate pathway, made from a cholesterol derivative. B ring opens up allowing for rotation and isomerization Ergocalciferol (Vit D 2 ) is prepared from ergosterol made by some yeasts Saccharomyces cerevisiae

37. Studies in human populations Epidemiological studies follow a selected population over a defined period of time, monitor intake of the test substance as well as other health factors, habits, lifestyle and assess the effect of these parameters on the number and type of disease-related incidents. Clinical trials usually use a randomized, placebo-controlled, double-blind design to administer treatment over a defined period, then assess the difference in specific measurable endpoints between the populations

38. Recent findings on Vitamin D and health Cancer: Vitamin D thought to improve cancer survival – high levels associated with 40% less colon cancer mortality (Dana Farber study) Incidence of certain cancers higher in Northern latitudes CVD: patients age 50+ with Vit. D deficiency were 45- 78% more likely to develop cardiovascular diseases & mortality (Utah study) Diabetes: Vitamin D supplementation reduced insulin resistance in a New Zealand study Crohn’s disease: Vit.D induced expression of NOD2 gene for defense against inflammatory bacteria Bone health: a study of n = 70,000 showed Vit D + Ca supplement reduced fractures by 16% (BMJ study); Vit.D (600 IU) + Ca (1200 mg) with weight-bearing exercise recommended to prevent osteoporosis.

39. Vitamin D and immunity An 2009 study published by researchers at Oregon State University reveals that Vitamin D is so crucial to the functioning of your immune system that the ability of vitamin D to boost immune function and destroy invading microorganisms has been conserved in the genome for over 60 million years. In primates, this action of "turning on" an optimal response to microbial attack only works properly in the presence of adequate vitamin D, which is produced as a result of sun exposure, and is available in smaller amounts from dietary sources.sun exposure Vitamin D prevents the "adaptive" immune response from over- reacting, thereby reducing inflammation, and appears to suppress that immune response. At the same time vitamin D boosts the innate immune response by turning on production of antimicrobial peptides. The overall effect may help to prevent the immune system from overreacting (may reduce risk of autoimmune conditions).inflammationimmune system http://www.naturalnews.com/027231_Vitamin_D_immune_system_v accines.htmlhttp://www.naturalnews.com/027231_Vitamin_D_immune_system_v accines.html Also check out “The miracle of Vitamin D” at http://www.westonaprice.org/The-Miracle-of-Vitamin-D.html http://www.westonaprice.org/The-Miracle-of-Vitamin-D.html

40. in vitro study: Vitamin D 2 induces apoptosis in human leukemia cells Chen, W-J et al, Induction of Apoptosis by Vitamin D2, Ergocalciferol, via Reactive Oxygen Species Generation, Glutathione Depletion, and Caspase Activation in Human Leukemia Cells. J. Agric. Food Chem. 2008, 56, 2996–3005 Investigated the ability of D2 to induce apoptosis (programmed cell death) in HL-60 leukemia cells and the mechanistic pathways involved Found that D2 induced apoptosis primarily through an oxidative pathway involving reactions in the mitochondria (caspase activation and modulation of regulatory proteins)