Results of a preliminary study on the effects of a compound on telomeres length, biological and physiological parameters

Telomeres are ribonucleoprotein structures forming a protective buffer at the ends of chromosomes [1] Photo 1. They preserve the integrity of the genetic material during the cell cycle and correspond to tandem repetitions of nucleotide sequences (TTAGGG) associated with several dozens of regulatory proteins including telomerase, which is the only enzyme capable of replicating telomeres. In the absence of telomerase activity, which depends on the catalytic subunit Telomerase Reverse Transcriptase (TERT), the end of the DNA molecule is truncated by about 50 to 200 nucleotides at each S phase of the cell cycle. The TERT gene is suppressed in almost all postnatal somatic cells, with the exception of stem cells and lymphoid cells. The telomere length is therefore truncated in older organisms [2].


Introduction
Telomeres are ribonucleoprotein structures forming a protective buffer at the ends of chromosomes [1] Photo 1.
They preserve the integrity of the genetic material during the cell cycle and correspond to tandem repetitions of nucleotide sequences (TTAGGG) associated with several dozens of regulatory proteins including telomerase, which is the only enzyme capable of replicating telomeres. In the absence of telomerase activity, which depends on the catalytic subunit Telomerase Reverse Transcriptase (TERT), the end of the DNA molecule is truncated by about 50 to 200 nucleotides at each S phase of the cell cycle. The TERT gene is suppressed in almost all postnatal somatic cells, with the exception of stem cells and lymphoid cells. The telomere length is therefore truncated in older organisms [2]. This shortening of aging telomeres is directly related to cell division. Indeed, DNA polymerases cannot replicate the ends of linear chromosomes. Thus, each cycle of DNA replication has a loss of genetic material [2]. Since the telomere does not contain coding sequences, there is no loss of genomic information. In addition, telomeres prevent chromosome ends from being recognized as DNA damage. Telomeres are therefore involved in the process of preserving the integrity of the genome and are essential for proper cell function.
In those cases where no mechanism comes into play to regenerate the telomeres, if telomere shortening occurs at each cell replication cycle, this indicates that the cell cannot live indefi nitely.
The division of cells in the absence of an effective telomere maintenance mechanism, will in fact lead to the formation of short telomeres which will in turn lose their protective function and be reported to the cell as damaged DNA. An activation of the cellular senescence pathways called p53 and pRb / p16 [3] can then be observed, which induces an interruption of cellular proliferation, an entry into senescence or death by apoptosis, according to cellular type.
The Hayfl ick limit is the maximum number of cell divisions that a cell can undergo [4]. It provides a link between the length of the telomere and the lifespan of the cell.
Replicative telomere erosion can be compensated by the action of an enzyme, telomerase, a specialized reverse transcriptase that uses a specifi c template RNA to extend the 3 'strand of the chromosome ends. There are also telomeraseindependent telomerase elongation mechanisms based on homologous recombination events called ALT (alternative lengthening of telomeres) [5].
In addition to cellular proliferation, chronic oxidative stress has been shown to be a major causal factor in telomere shortening and cellular senescence [6].
Measured by various techniques in peripheral blood leukocytes, the average size of telomeres is a marker of the "biological age" of individuals and their exposure to chronic stress under various physiological and pathological conditions [7]. In humans, the telomere size varies on average from 10 to 4 kb from birth to 80 years of age and undergoes erosion of a few dozen bases per year. The length of the telomeric sequence, shorter in men than in women, is linear and inversely correlated with age. It is a genetically determined individual characteristic (heritability of 70-80%), which is characterized by a large variability. At a given age, the average size of leukocyte telomeres is therefore the result of three variables: inherited length, the proliferation rate of immune cells, and the level of exposure to chronic oxidative stress.
Hematopoietic cells and endothelial cells share the same embryogenic origins [11], which could be one of the essential elements of the link between telomere size, atherosclerosis and cardiovascular pathologies. Thus, regardless of age, telomeres could be involved in the initiation and /or progression of cardiovascular disease.
Overall, for the same chronological age, the mortality rate from infections or cardiovascular disease in subjects over 60 years of age with the shortest telomeres is three to eight times higher than those with the longest telomeres [12]. This data accounts for the critical role of telomeres at the interface of the molecular systems involved in aging, cell proliferation, tissue renewal, oxidative stress, infl ammation, and carcinogenesis [13].
Several molecules have appeared on the market in recent years, due to their supposed action on telomeres. Astragalus, a plant used in traditional Chinese medicine, could act as an activator of telomerases. Astragalus and one of its derivatives (Astragalosides) appear to be metabolized to Cycloastragenol (CA), a telomerase activator [14]. Some products from Astragalus have shown their value in vitro and in animal experiments [15][16][17] and have been evaluated in humans with encouraging fi rst results. In a randomized, double-blind versus placebocontrolled study of 117 subjects aged 53 to 87 years, Salvador et al. showed that Astragalus derivatives signifi cantly increased telomerase size over a one-year follow-up period compared to the placebo [18].
We are reporting the fi ndings below of an open prospective pre-study in healthy volunteers, the interest of a new ASFC molecule on telomere elongation and its cardiovascular action.

Population
Ten healthy volunteers (6 men, 4 women), average age 59.4 ± 8.19 years old (average age females: 55.2 ± 9.14 years old, average age males: 63.6 ± 4.87 years), with no specifi c history included between November 2017 and February 2018. All subjects signed informed consent before inclusion in the protocol. The volunteer subjects came all from our medical and paramedical team. No subject had active cardiovascular disease at the time of inclusion. All changes in physical activity or drug intake during the study period, as well as the occurrence of any negative side effects, were reported.

ASTCOQ02
The telomerase activator tested ASTCOQ02, combining Astragalus extracts (including astragaloside IV and cycloastragenol, olive fruit (including hydroxytyrosol), zinc oxide and seed extract). The product was in the form of an oral capsule, used by this clinical pre-study in healthy volunteers, with a dosage of 2 capsules (one in the morning and one in the evening) per day. The daily dose (2 capsules per day) included in total: It is a product marketed as a food supplement with no recognized toxicity.

Cardiovascular evaluation and other measures
The medical and socio-economic characteristics of the subjects were collected by a standardized questionnaire, including the medical-surgical antecedents and cardiovascular co-morbidities. All subjects benefi ted at TO, M1 (1 month) and -and the length of the PQ interval at the ECG.
The evoked potentials P300 constitute late cognitive evoked potentials demonstrating the quality of the cortical activities.
The P300 is very clearly correlated with cerebral aging and provides a distinction between neurodegenerative diseases such as Alzheimer's disease and thymic disorders [20][21][22].

Statistical analysis
Mean values and standard deviations of the overall population of 10 healthy volunteers and 2 independent groups: men and women were gathered according to clinical and biological parameters, telomere size, arterial compliance results, P300. The average of the different groups were evaluated by a parametric statistical test: student's t-test, with a signifi cance level p set at 5%.

/ On the length of telomeres
The "long-life" telomere activator requires a few weeks' time for incorporation and action. This is observed in our pre-study between T0 to M1, where one simply observes the physiological shortening of telomeres before the "long-life" action.

/ On arterial rigidity
The evaluation of arterial stiffness shows a signifi cant improvement at 6 months of total arterial compliance (

/ On the p300
The measurement of the P300 cognitive potentials evoked also shows an improvement, but insignifi cant, at 6 months of P300 for all 10 healthy volunteers (298.10 ± 19.51 msec at T0 for the 10 subjects, versus 282.40 ± 29.94 msec to M6).

Discussion
This pre-study evaluated the compliance and the good tolerance of the "long-life" product at 6 months.
All healthy volunteers continued the study to term and no side effects were reported during the entire six-month follow-up period. Biological assessments have not revealed any biological alteration. Analysis of the T0, M1 and M6 electrocardiogram showed no abnormality in independent analysis by a cardiologist through a single-blind study. The "long-life" telomere activator requires a few weeks' time for incorporation and effects. This is observed in our pre-study between T0 to M1, in which one can simply observe the physiological shortening of telomeres before "long-life" effects.
Our study revealed, after the lag phase of the product from In fact, the telomere point measurement has no value as such. It is the measure of the variation in the size of our telomeres, in one way or another, which constitutes a major piece of information on the future quality of longevity.
It is also possible that the "long-life" telomerase activator has a more targeted and rapid effects on short telomeres and is the most likely to bring the cell into senescence and apoptosis.
New studies on the action mechanism of the "long-life" molecule, at different dosages, during a longer study period, a larger number of subjects included, and with the presence of a control group, are in progress.
Our pre-study has also shown interesting cardiovascular results. The increase in arterial stiffness has been validated as a strong independent marker of cardiovascular mortality in both hypertensive and normotensive patients [23].  Several studies have demonstrated a formal link between telomere length and senescence, including endothelial cells [24], but also with numerous cardiovascular atherosclerotic pathologies [25][26][27]. The respective roles of chronic infl ammation, oxidative stress, and telomere shortening on the occurrence of atherosclerosis remain debatable [28,29].
In many cases, coronary diseases appear within the context of atherothrombosis and cellular senescence, which have been the focus of attention for many years [37][38][39] [43].
In 2011, Depinho et al reported an improvement in tissue degeneration in elderly mice by activation of telomerases [44].
This work has been confi rmed on many animal models.
However, there is currently little data on the possible therapeutic strategies that can be used to protect telomere length or elongation, particularly in the context of cardiovascular disease. Physical exercise appears to positively infl uence telomere length [45].
The WOSCOPS study showed that statins could have a benefi cial effect on telomere length, thus contributing, in addition to their lipid-lowering effect, to preserving the integrity of the vessel wall of patients at risk, by decreasing the senescence of endothelial cells [46,47]. Oral antidiabetic drugs such as Pioglitazone, belonging to another pharmacological class, appear to have some benefi cial properties on telomere biology by increasing telomerase activity [48,49]