Are polyphenols the life extending structures in humic substances?

Are polyphenols the life extending structures in humic substances? From: J. Berger & R. Sommer, Max-Planck-Institut für Entwicklungsbiologie, Tübingen Nadine Saul 1, Kerstin Pietsch 1, Ralph Menzel 1, Stephen Stürzenbaum 2, and Christian Steinberg 1 1 Humboldt-Universität zu Berlin, Laboratory of Freshwater & Stress Ecology, 12437 Berlin, Germany 2 King s College London, Analytical and Environmental Science Division, London SE1 9NH, United Kingdom

Longevity

Longevity with HS? female + Humic Substance male?

survivors (%) The effects of humic substances on C. elegans as exemplified by HuminFeed 100 50 Lifespan during HuminFeed exposure 0 mg/l 2.5 mg/l HF (0.2 mm DOC) 5 mg/l HF (0.4 mm DOC) 25 mg/l HF (2 mm DOC) 0 1 3 5 7 9 11 13 15 17 19 21 23 25 days

Tannins: Contrasty polyphenols a) Tannic acid, b) Gallic acid, c) Ellagic acid, d) Catechin

Tannins: Contrasty polyphenols Plants synthesize polyphenols (PP) for their defense against fungi, bacteria and parasites the creation of colors to attract insects PP have antioxidative, anti-inflammatory, and antimicrobial capacities PP protect against Cardiovascular and neurodegenerative diseases Cancer Oxidative stress Ageing

Tannins: Contrasty polyphenols Tannins showed health-promoting and toxic properties have protein-binding & precipitating capacities are anti-nutritional substances due to precipitation of digestive enzymes binding of nutritional proteins astringent taste

Individuals alive (%) Individuals alive (%) Longevity via tannin exposure 100 0 µm TS 100 µm 100 0 µm GS 300 µm 50 50 0 1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 0 1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 100 0 µm ES 50 µm 100 0 µm CT 200 µm 50 50 0 1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 0 1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 Days Days

3 proven mechanisms for life extension

3 proven mechanisms for life extension Hormesis

3 proven mechanisms for life extension Hormesis Calorie Restriction

3 proven mechanisms for life extension Hormesis Calorie Restriction Targeted molecular modulation

3 proven mechanisms for life extension Hormesis Calorie Restriction Targeted molecular modulation

Hormesis = mild stress (e.g. a toxin in low concentrations) with beneficial effects on organisms Beneficial effects = e.g. longer life or enhanced stress resistance Hormesis in C. elegans: Radiation, inorganic & organic substances and heat stress

Hormesis

0 50 100 150 200 250 300 350 400 0 100 200 300 400 500 600 700 800 0 50 100 150 200 250 300 0 100 200 300 400 500 600 700 800 Survivors after oxidative stress (%) Survivors after heat stress (%) Mean lifespan (%) Are tannins hormetic? 130 120 110 100 90 TA * * GA * EA * CT * * * 80 180 160 140 120 100 80 60 180 160 140 120 100 80 60 40 20 * * * * Concentration (µm)

survivors after heat stress (%) And what about HuminFeed? 80 Thermal Stress Resistance 70 60 50 40 30 Control HuminFeed 20 10 0 0.2 mm DOC 0.4 mm DOC 4 mm DOC

Mean lifespan in days survivors (%) And what about HuminFeed? 100 50 0 mg/l 2.5 mg/l HF (0.2 mm DOC) 5 mg/l HF (0.4 mm DOC) 25 mg/l HF (2 mm DOC) 0 1 3 5 7 9 11 13 15 17 19 21 23 25 days 15,5 15 14,5 14 13,5 13 12,5 Control 0.2 mm DOC 0.4 mm DOC 2 mm DOC

Body length (µm) Offspring per nematode Are there side effects or is it real hormesis? 1600 1550 1500 1450 * 320 300 280 1400 1350 1300 1250 260 240 220 1200 200

Offspring after 85h Are there side effects or is it real hormesis? 18 16 14 12 10 8 6 4 2 0

Length alteration (%) Reproduction alteration (%) Are there side effects or is it real hormesis? 103 102 101 100 115 110 99 98 105 97 96 95 A1 A6 A1 A6 A1 A6 0.2 mm DOC 0.4 mm DOC 2 mm DOC 100 95 0.2 mm DOC 0.4 mm DOC 2 mm DOC

3 proven mechanisms for life extension Hormesis Calorie Restriction Targeted molecular modulation

3 proven mechanisms for life extension Hormesis Calorie Restriction Targeted molecular modulation

Calorie Restriction Calorie Restriction= Longevity and ageing deceleration by reducing the amount of food to about 30-50 % of the ad libitum amount Tannins possess antinutritional capacities

Calorie Restriction 1989: Start of a Calorie Restriction rhesus monkey experiment They get only 30% of the ad libitum food amount So far: Less age related diseases like cancer, heart disease or diabetes Better brain function Extension of the median lifespan

Calorie Restriction 1989: Start of a Calorie Restriction rhesus monkey experiment They get only 30% of the ad libitum food amount Healthy, but hungry ad libitum Calorie Restriction

Individuals alive (%) Calorie Restriction in C. elegans 100 no food (A6) 50 ad libitum 0 1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39 41 43 45 Days

Quantity of offspring related to control (%) Attraction Assay ~ ~ ~ 6 alternating drops of food bacteria (with and without test substance) 1 L1-Larvae per drop ~ ~ ~ Waiting for 96h Counting the offspring per drop 180 160 TA 100 µm GA 300 µm EA 50 µm CT 200 µm 140 * 120 100 80 60 *

Change of pharyngeal pumping (%) Pharyngeal Pumping Frequency 120 115 * 110 105 100 95 90 85 80 3. Day 6. Day 9. Day 0 0.2 0.4 4 mmmmmmmm0

Mean lifespan variation (%) Lifespan of the eat-2 mutant 125 120 115 110 N2 eat-2 105 100 95 TA 100 µm GA 300 µm EA 50 µm CT 200 µm HF 0.4 mm

3 proven mechanisms for life extension Hormesis Calorie Restriction Targeted molecular modulation

3 proven mechanisms for life extension Hormesis Calorie Restriction Targeted molecular modulation

Insulin / IGF-1 (insulin/insulinlike growth factor) pathway Insulin/DAF-28 DAF-2/insulin receptor AGE-1 DAF-18 PDK-1 AKT-1, AKT-2 DAF-16 Translocation in the nucleus and regulation of diverse genes Enhanced stress response & Longevity

p38 MAPK pathway

TGF-beta pathway TGF-β = Transforming growth factor beta Controls proliferation, cellular differentiation, immunity, apoptosis, lifespan, etc

Microarray

Microarray & Mutant Strains Results Both, Tannic acid & Huminfeed, modulate 3 known longevity pathways: The TGF-beta signaling The p38 MAPK signaling The Insulin like signaling They seem to modulate the same molecular mechanisms of longevity

Do the properties of HF changed after enrichment with hydroxybenzene? Chemical enrichment of HuminFeed by a polycondensation reaction with hydroquinone or benzoquinone: lifespan extension not changed, but increased tolerance to thermal stress delayed onset of reproduction even more growth reduction stronger pumping activity of the pharynx

Conclusion Longevity possible via HF & tannins Hormetic action of TA & EA, tendency for HF No clear hints for Calorie Restriction Molecular background: TGF-beta p38 MAPK insulin like signaling Enrichment with hydroxybenzene molecules boost HF action

Conclusion Due to numerous overlapping actions, tannins might be the trigger of longevity in humic substances

Thank You! Menzel et al. Enrichment of Humic Material with Hydroxybenzene Moieties Intensifies Its Physiological Effects on the Nematode Caenorhabditis elegans. Environ. Sci. Technol. 2011, 45, 8707 8715 Saul et al. Diversity of Polyphenol Action in Caenorhabditis elegans: Between Toxicity and Longevity. J. Nat. Prod. 2011, 74, 1713-1720

HuminFeed HuminFeed (Humintech GmbH, Düsseldorf, Germany) was made by an alkaline extraction process of highly oxidized lignite Prior to extraction, the raw lignite material was milled and homogenized The humate solution was separated from the humin via sedimentation of the latter

Chemical Enrichment with Hydroquinone HuminFeed (1 g) was dissolved in 2.5 ml of distilled water and further diluted in water to a volume of 50 ml the ph was adjusted to ph 7 addition of 500 mg of the monomer hydroquinone in the presence of oxalic acid and formaldehyde stirring for 1 h at 100 C precipitation by the addition of 6 M HCl at ph 1, filtration, and freeze-drying