**2. Scientific history**

562 Selected Topics in DNA Repair

Thus, in a particular sense, this dietary supplement combination contemplates the provision of a method of treating a human subject consisting of selectively administering to the subject resveratrol material, carotenoid material, nicotinamide material, DMAE material, zinc source and quinic acid material in daily dosage amounts effective, in combination, to improve resistance to DNA damage, enhance DNA repair capacity, and stimulate immune function. In a specific example of currently preferred dosage range for humans, about 100- 500 mg resveratol material, about 100 mg of carotenoid material, about 100 mg of nicotinamide material, about 100-200 mg DMAE material, about 10 mg of zinc source material and between 250-700 mg quinic acid source material are administered daily in this method (Pero and Garret 2010). So far as the author is aware, this particular combination of ingredients devoid of other bioactives has heretofore already been recognized as being

The discovery that natural products should not be combined into a natural medicine unless one tests whether each ingredient is additive to the overall desired biological effect, and that one way to accomplish this endpoint is to not combine natural products that have similar modes of action and thus competitive routes of absorption and excretion without first testing the combination for additive effects. That is to say, the present invention avoids inhibited uptake and absorption of natural products, thereby obtaining additive biological effects, by combining only natural products having well defined different, and thus potentially non-competitive modes of action which is, for example, the case with the exclusive combination of carotenoids + nicotinamide + zinc (Pero 2000,

Diet supplementation of humans or animals for example, by the oral, intraperitoneal, intravenous, subcutaneous or intramuscular routes of administration with the combination of carotenoids + nicotinamide/niacin + an appropriate zinc salt at a dose of this combination that exceeds a normal dietary levels was effective. This practice showed that dietary supplementation containing this combination together with simultaneous supplementation of other nutrients and/or natural products cannot enhance immune function (Payette, H. et al 1990; Zhang et al 1995), but when daily doses of carotenoids (as lycopene at 20 mg and Vitamin E 36 IU), niacin (120 mg) zinc salt (12 mg), reservatol (300 mg) and a quinic acid material (400 mg) were administered in the absence of other known DNA repair enhancers, and above dietary levels, the resistance to oxidative cellular DNA damage, and enhancement of DNA repair and immune function were observed in the clinic (Pero and Lund 2011). These data were taken as proof of concept for this dietary supplement

A clinical evaluation already published (Pero 2000, Sheng et al 1998) was also determined by comparing each individual's biological response before and after supplementation. In such a manner, each individual became his own control; e.g. the male subjects were given baseline measurements of resistance to cellular DNA damage, enhancement of DNA repair and stimulation of immune function once a week for 4 weeks, and then they were supplemented daily and the same measurements repeated once a week for the last 5 weeks of a 7 week intervention period. The before measurements (i.e. n = 4) were the baseline biological response parameters to be compared to the after measurements (i.e. n = 5). One individual was not supplemented to provide a control for the supplemented individuals. The data from this experimental design has taught that resistance to cellular DNA damage, enhancement of DNA repair and stimulation of immune function were all significantly modulated by a combination of carotenoids + nicotinamide + zinc when administered as an exclusive drug

synergistic or even effective (Pero 2000, Sheng et al 1998).

to avoid metabolic competition and synergize DNA repair.

Sheng et al 1998).

In 2010 there was an extensive review article published entitled "Historic development of Uncaria preparations and their related bioactive components" (Pero 2010b). This overview has specific relevance to the current review because it was the first recognition of the concept that ingredients could in fact convey properties of enhancement of DNA repair. Before this time, there were no clear cut examples where the process of DNA repair could be shown to be stimulated to higher levels of activity by exogenous nutrients or supplements in our environment. Previously it was believed that DNA repair which regulated our genetic integrity could not afford to be anything less than perfect to satisfy the requirements of orderly evolutionary change. Now it is quite accepted that even genes need to have nutritive treatment, functionality repaired and developed to maturity during life.

Uncaria sp. is a well known herbal medicine used for generations by the Ashinka Indians native to the Amazon basin. There have been two sets of bioactive ingredients for which Uncaria extracts have been developed and standardized. The first are oxindole alkaloids, initially studied and described in 1967; the second are a set of molecules know as Carboxy Alkyl Esters (CAEs ™) first identified and described in 1997 as the bioactive ingredients in AC-11® (Reviewed in Pero 2010b). More recently in 2005 (Sheng et al 2005) it was shown that one of the acid moieties of CAEs is quinic acid, and it is now documented to be one of the more effective DNA repair ingredients found in Uncaria spp, or brightly-colored berry extracts that also contain quinic acid (Stoner et al 2008, Pero 2006), and in turn can enhance DNA repair. It is important to remember that quinic acid is a natural-occurring alpha hydroxy organic acid quite ubiquitous in berries, and also the metabolic source of all other aromatic compound production in plants via the shikimate pathway (Pero 2010a); e.g. the bioactive agents in berries such as hydroxy organic acids (hydroxy benzoic, hydroxy cinnamonic and caffeic acids), flavinoids, and ellagic acid (Stoner et al 2008).

The progression of events that established Cat's Claw extracts as the most consistent and potent DNA repair enhancer of anti-aging effects has become obvious, and signaled why some elements are built of the knowledge of the later events that have happened, to paint a more complete picture of how DNA repair regulates aging. A chronology of events that remain unbroken and additive of each other, that Pero and colleagues have in turn built and learned from are as follows:

Enhancing DNA Repair by Combining only Dietary Supplement

biologic testimony to the impotance of this pathway to human health.

**DNA repair and anti-aging effects** 

**3.1 The reservatrol material** 

mechanism to dietary restriction.

which results in an improved biological cellular function.

**3.2 The carotenoid material** 

**3.3 The nicotinamide material** 

Ingredients that do not Metabolically Compete in Order to Achieve Synergism 565

increase as the causative importance of this research area to human disease development becomes better known (Wood et al 2001). So far nearly all areas of DNA repair are represented by defective metabolism such as: base excision repair (BER) (glycosylases, endonucleases), PARP (poly ADP ribose polyemerase), direct reversal of damage, repair of DNA protiein crosslinks, mis- matched excision repair (MMR), nucleotide excision repair (NER), homologous recombination, non-homlogous end-joining, modulation of nucleotide pools, DNA polymerase, editing and processing nucleases, Rad 6 pathway, chromatin structure, and genes defective in disease that modulate DNA damage. The mere fact there are so many variant ways to become diseased by defective DNA repair mechanisms is a

**3. A dietary supplement composition that induces rehydration and enhances** 

The known molecular mechanisms of resveratrol are described herein. The main effects of resveratrol are to regulate cell cycle events that favor growth arrest allowing DNA repair enhancement before cells die from DNA damage blockage of cell replication (Valenzano et al 2006; Gatz et al 2008; Feng et al 2002; Whyte et al 2007). There are changes in both gene and protein expression, such as the up-regulation of p53 and p21 and the down-regulation of cyclin A, chk1, CDC27, and Eg5 (a mitotic motor protein). Resveratrol also alters the intracellular Smad signaling of the TGF-β pathway. Finally, dietary restriction, the beststudied life-extension treatment, causes overexpression of SIRT 1 (Cohen et al. Science 2004), and since these effects are not additive to resveratrol, they suggest that a similar molecular

The known molecular mechanisms of carotenoids are described herein. The exact mechanism of action of carotenoids such as beta carotene is not fully understood but it is commonly accepted scientifically that one primary mechanism is to directly scavenge oxygen derived free radicals produced either as by-products of metabolism or from exogenous environmental exposures (Lieber 1993; Bohm et al 1993; Regnault et al 1993; Riso 1999). As a free radical scavenger, carotenoids can be expected to reduce or protect against the chemical damage induced in DNA, RNA and protein of cells by toxic environmental exposures or endogenous cellular metabolic errors that ultimately can result in a disease state. On the other hand, nicotinamide and zinc salts do not possess this chemical property

The known molecular mechanisms of nicotinamide are described herein. Nicotinamide and its metabolic equivalent nicotinic acid (niacin, vitamin B) or even tryptophan which is the synthetic precursor to niacin is the main precursor for the formation and maintenance of the cellular pool of NAD (Bernofsky 1980; Olsson et al 1993). NAD is essential for cellular ATP production and maintenance of the cell's redox potential, and it is also the substrate for the DNA repair enzyme, poly ADP-ribosyl transferase (ADPRT). Niacin deprivation decreases the NAD pools significantly both in tissue culture cells (Jacobson, E et al 1992) and animal


**Prevalence of DNA repair deficiencies in the general population.** There are now more than 130 DNA repair - regulated genes identified that also can influence individual susceptibility to DNA damage, and as a consequence, the incidence of human diseases. The number has increased dramatically in the last 20 years, and no doubt will continue to increase as the causative importance of this research area to human disease development becomes better known (Wood et al 2001). So far nearly all areas of DNA repair are represented by defective metabolism such as: base excision repair (BER) (glycosylases, endonucleases), PARP (poly ADP ribose polyemerase), direct reversal of damage, repair of DNA protiein crosslinks, mis- matched excision repair (MMR), nucleotide excision repair (NER), homologous recombination, non-homlogous end-joining, modulation of nucleotide pools, DNA polymerase, editing and processing nucleases, Rad 6 pathway, chromatin structure, and genes defective in disease that modulate DNA damage. The mere fact there are so many variant ways to become diseased by defective DNA repair mechanisms is a biologic testimony to the impotance of this pathway to human health.
