Daily consumption of a chocolate rich in flavonoids 2 decreases cellular genotoxicity and improves 3 biochemical parameters of lipid and glucose 4 metabolism . 5

In recent years, Atherosclerotic Cardiovascular Disease (ACVD), Obesity and Diabetes, 14 have increase exponentially worldwide. In the present work, we evaluate the genoprotective effect 15 of consuming a flavonoids-rich chocolate and the improvement in the biochemical parameters 16 related to the prevention and treatment of cardiovascular risk and metabolic syndrome in young 17 Mexican adults. A randomized, placebo-controlled, double-blind study was undertaken in the 18 Autonomous University of Baja California. The treatments were a daily intake of 2 grams of dark 19 chocolate containing 70% cooca or milk chocolate. Total phenolic compounds and flavonoids were 20 evaluated in both chocolates. Anthropometrical and Biochemical parameters were measured in the 21 84 participants before and after the study. Buccal epithelial genotoxicity was also evaluated from 22 the beginning to the end of the experiment in the participants. Result suggested that flavonoids of 23 cocoa intake have protective effects against DNA damage, and Biochemical parameters (total 24 cholesterol, triglycerides, and LDL-cholesterol level in blood) and anthropometrical parameters 25 (waist circumference) were also improved after six months of daily intake of 2 grams of dark 26 chocolate with a 70% of cocoa. 27


Introduction
Atherosclerotic Cardiovascular Disease (ACVD) is the leading cause of death around the world [1].
Obesity, high levels of LDL, triglycerides and low levels of HDL are associated with the development of ACVD [2].Other affections such as Diabetes, insulin resistance and hypertension are also risk factors to develop cardiovascular diseases [3]- [5].The incidence of all these pathologies are increasing worldwide and they and they are becoming a public health problem.
Diet is one of the major lifestyle factors that can significantly influence the incidence and progression of chronic diseases such as cardiovascular disease, diabetes and cancer.Cocoa has been consumed by humans since at least as early as 460 A.D [6].Increasing amount of reports have shown that the consumption of cocoa and dark chocolate exerts several beneficial effects on cardiovascular health and endothelial dysfunction [7]; Observations are consistent with reports that dark chocolate lower blood pressure [7] and improve endothelium-dependent vasodilatador responses [8].Cocoa, especially dark chocolate, contains high level of flavanols such as epicatechin and catechin, and procyanidins.Flavanols in cocoa are present as either the monomers (-) epicatechin and (+) catechin or oligomers of epicatechin and/or catechin, called proanthocyanidins or condensed tannins.
Flavonoids have been widely reported by their many benefical effects in health.For example, some reports showed that epicatechins improved vascular function, reduce BP, improve insulin sensitivity, and reduce platelet activity [9].
In recent years, increasing attention has been given to compounds that induce genetic damage by various mechanisms.There is evidence that the mutagenic events and genotoxic agents may play an important role in the cause and/or progression of human diseases other than cancer [10].Recently, cells from the oral epithelium have been used for the evaluation of exposure to various genotoxic agents, associated with its recognized sensitivity for the assessment of DNA damage.The toxicological relevance of the micronucleus test is well defined: it is a multi-target genotoxic endpoint, assessing not only clastogenic and aneugenic events but also some epigenetic effects, which is applicable in different cell types.Micronuclei in exfoliated epithelial cells are widely used as a noninvasive biomonitoring process suitable for the detection genotoxicity.
In the present work, we evaluate the genoprotective effect of consuming a flavonoids-rich chocolate and the improvement in the biochemical parameters related to cardiovascular risk and metabolic syndrome in young Mexican adults.

Study population
Table 1 also describes important participant characteristics such as sex, education level, physical activity and Cardiometabolic co-morbidities (diabetes and hypertension).The physical activity was classified as inactive, moderate active and active (Table 1), and it was assessed via scoring criteria of the International Physical Activity Questionnaire of World Health Organization [11].The majority of the studied population was university students, with normal blood pressure but high triglyceride and glucose levels (Table 2).Also they presented an average body mass index (BMI) of 32.1, with an incidence of 68% (Table 1).According to the World Health Organization, for adults over 20 years old, a BMI between 30.00 and 34.9 falls into one of the category of Obesity Class I [12].

Frequency in nuclear abnormalities in buccal epithelial cells
The abnormalities of the nuclei in the buccal epithelial cells were 14.4% at the beginning, participants of both experimental groups showed abnormalities of broken egg nucleus, micronucleus and binucleus (Figure 1).

Determination of total phenolic and flavonoids content
Results showed that 54.6% of phenolic content in the used dark chocolate were from the flavonoids group; this percentage of flavonoids was significant higher compared with the 18% of flavonoids contained in milk chocolate.The relation flavonoids/polyphenols showed that the used commercial dark chocolate with 70% cocoa was significant better source of flavonoids compared with the used milk chocolate.Moreover, taking into account that 2 grams of dark chocolate were provided to each participant in the study; approximately 69.6µmol of (-)-catechin equivalents of flavonoids were provided to each participant daily in the treatment with commercial dark chocolate with 70% cocoa (FRC), and 20.8 µmol of (-)-catechin equivalents of flavonoids were provided to participant in milk chocolate treatment (MC) .

Characteristics of study population
84 participants were recruited for the present study most of them were university students (94%), of which 38 % were current smokers, and 65 % of them reported having a habit of moderate physical activity.67% presented a BMI in a range of 30-34.9ldue to they were classified with Obesity class I. half of them were randomly included to the treatment where commercial dark chocolate with 70% cocoa was provided and the other half were treated with milk chocolate.The principal reported risk factor by participants was dyslipidemia, as 61.90 % of participants presented at least one of the following anomalies: total cholesterol ≥190 mg/dl (≥4.9 mmol/l), TAG ≥150 mg/dl (≥1.7 mmol/l), LDL-cholesterol ≥115 mg/dl (≥3.0 mmol/l), HDL-cholesterol <40 mg/dl for men and <46 mg/dl for women and/or taking hypolipid medications.

Determination of total phenolic and flavonoids content
According to results, the content of phenolics was slightly less in milk chocolate compared with dark chocolate, however it has been reported that sugar content could affect the measurement of phenolic content by Folin-Ciocalteu method.Interestingly, the flavonoid content was 3 fold higher in the dark chocolate compared with the milk chocolate used for the present study.Since the percentage of flavonoids/phenols (%) was significantly higher in the dark chocolate than in the milk chocolate, the used dark chocolate was a better source of flavonoids compared with the commercial milk chocolate used in the present study.
According to the flavonoid content (Table 2) in the daily chocolate intake and to that there were no significant difference in the intake of fruits and vegetables reported by the participants (Table 3), it was assumed that the flavonoid intake of participants treated with the commercial dark chocolate with 70% cocoa was significantly higher than the flavonoid intake of participants treated with the commercial milk chocolate.

Changes in measured parameters by daily dark chocolate consumption
Dietary variables did not significant change by the consumption of flavonoid-rich chocolate.
However, certain anthropometric and biochemical variables varied after the flavonoid-rich-chocolate consumption (Table 2).The waist circumference of participants was significant lower (P>0.05)after de study, change could not be attributed to changes in dietary since Total energy intake, and the proportion of carbohydrates and lipids were similar before and after the study.Interestingly, total blood cholesterol, triglycerides and LDL-cholesterol significantly decreased after six months of daily intake of dark chocolate with high content of flavonoids (P<0.05).
There has been previously reported that dark chocolate intake could improve LDL levels in blood [13].Moreover, it was also reported that the consumption of flavonoids could significantly increase lipid oxidation [14], [15], thus flavonoids intake might improve lipid biochemical parameters by the modulation of reverse cholesterol transport in gut and liver [16].Regarding to HOMA-IR and fasting plasma glucose both parameters were significantly decreased after 6 months of daily intake of the flavonoid-rich chocolate.HOMA-IR is a homeostatic model assessment (HOMA) to determine insulin resistance (IR) in β-cells.It is calculated with the proportion of plasma glucose and insulin levels.According to the results of the present study, and in accordance with previous reports [17], daily flavonoid-rich chocolate intake was significantly associated with a lower HOMA-IR (P<0.05).
Some flavonoids, such as genistein and epicatechin has been previously associated with a lowering blood glucose concentration [14], [18].Finally, and not less important blood pressure was significantly improved by the consumption of flavonoid-rich chocolate compared with milk questions on habitual daily consumption of chocolate during the previous week.The participants reported their frequency of consumption ranging from 1 to 7 days of consumption.They also selected the serving size based on how many packaged portions take every day.

Determination of total phenolic content
Total phenolic content determination was made using the Folin-Ciocalteu method [22], Briefly, Extraction was performed with 80% methanol in water (v/v), then total polyphenols content was determined by using Coulter DU 520 Spectrophotometer at 750 nm.A standard curve was developed using Gallic acid (Sigma-Aldrich, Michigan, USA).

Determination of flavonoids content
Flavonoids were evaluated in the 80% methanol in water (v/v) extracts with a colorimetric method according to previous reports [22], using a Coulter DU 520 Spectrophotometer at 510 nm.A standard curve was developed with (-)-catechin (Sigma-Aldich, Michigan, USA).

Epithelial genotoxicity, biochemical and anthropometric parameters measure (dependent variables)
After 4 weeks of daily consumption of flavonoid-rich chocolate a blood sample was obtained for each participant in order to measure biochemical parameters.Standard laboratory assays were used to measure Biochemical parameters with IDEXX Catalyst Dx® equipment.Anthropometric parameters were also measure every 4 weeks with a digital weighing scale and a measuring tape.
Genotoxicity was measured in each participant by micronuclei assay.Briefly, exfoliated cells were collected by a non-invasive sampling method from oral mucosa.Buccal cavity cells are obtained by scraping the cheeks with a tongue depressor.The samples are transferred dropwise to pre-cleaned slides.Then, the slides are air-dried and fixed with 80% methanol.After that slides were stained with hematoxylin for 3-5 minutes and then with eosin for 5-8 minutes.Genotoxic damage was determined by the observation of slides in the microscope; the cellular malformations were observed and counted.

Statistical analyses
Data were analyzed by using MiniTab and GraphPad Prism (GraphPad Software Inc., CA, USA) software.Results are reported as mean ± SEM.Statistical differences for mean data obtained from present study were analyzed by two way ANOVA.Correlation was made using MiniTab MiniTab General Linear Model.

Conclusions
In conclusion, flavonoids of cocoa have protective effects against DNA damage, it is suggested that the reducing genotoxic stress effect is related with the antioxidant activity of flavonoids and the modulation of CYP450.However, further in vivo studies are needed to determine their mechanism of action of antigentotoxic effect.Biochemical parameters (total cholesterol, triglycerides, and LDL-cholesterol level in blood) and anthropometrical parameters (waist circumference) were also improved after six months of dark chocolate with a 70% of cocoa intake.Interestingly daily flavonoid-rich chocolate intake also improve fasting plasma glucose levels and insulin resistance parameter (HOMA-IR).These effects were attributed to the proportion of flavonoids in the chocolate

Table 1 .
Characteristics of study population at recruitment.

Table 2 .
Phenolic and Flavonoid content in used dark and milk chocolates for the study.

22 July 2018 doi:10.20944/preprints201807.0399.v1
Changes in measured parameters by daily chocolate consumptionDietary variables did not significant change by the consumption of flavonoid-rich chocolate.Certain anthropometric and biochemical variables varied after the flavonoid-rich-chocolate consumption.Dietary, anthropometric and biochemical variables measured in the present studied are reported in

Table 2 .
Every variable was measured in each participant before and after the study of milk or dark chocolate daily consumption for 6 months.

Table 3 .
Dietary, anthropometric and biochemical variables before and after the study of flavonoid-rich chocolate consumption for 6 months.