Patent Application: US-201615368655-A

Abstract:
phytoecdysones for use in improving muscle quality in obese and / or sarcopenic mammals , preferably , obese mammals subjected to a low - calorie diet . the phytoecdysones are advantageously incorporated into a food composition . the phytoecdysones can be derived from plants , such as quinoa .

Description:
in the invention , it is proposed to provide a concentrated dose of pure phytoecdysones or using a phytoecdysone - rich plant extract to improve the muscle condition of individuals suffering from obesity , sarcopenia or sarcopenic obesity . according to the invention , it is possible to provide this dose of phytoecdysones in the form of an extract from a plant such as quinoa , incorporated for example into a food forming part of the normal diet of an individual . specifically , 4 grams of quinoa extract enriched with 0 . 5 % of phytoecdysones by weight contain 20 milligrams of phytoecdysones . in order to obtain the same quantity of phytoecdysones from quinoa seeds it would be necessary to consume 50 to 100 grams of untreated seeds ( dini et al ., 2005 ). the quinoa extract according to the invention may contain up to 50 times more phytoecdysones than the quinoa seeds from which the extract is derived . the method involves a sequential extraction with water , adding 500 g of quinoa seeds to 2 litres of boiling water , the whole being maintained for 5 minutes at 80 ° c . the water is eliminated and a second extraction is performed with 2 litres of an ethanol - water mix ( 1 : 1 ) applying constant agitation for 20 minutes at 80 ° c . sequential extraction of this kind eliminates saponins from the extract , these being abundant in quinoa seeds ( muir et al ., 2002 ), and which would give a bitter taste to said extract . the ethanol extract is filtered through miracloth ™, evaporated to dryness and taken up in 400 ml of absolute ethanol , leaving an abundant insoluble residue . the ethanol fraction is filtered or centrifuged and then dried . chromatographic analysis ( hplc ) shows that this extract contains 2 ± 0 . 2 % by weight of 20 - hydroxyecdysone ( 20e ). a quantity of between 150 and 200 milligrams of phytoecdysones is obtained per kilogram of treated quinoa seeds , of which 85 - 90 % is 20 - hydroxyecdysone and the remainder ecdysteroids with very similar structures such as makisterone a , 24 - epi - makisterone a , 24 ( 28 )- dehydro - makisterone a or 20 , 26 - dihydroxyecdysone . the structures of these compounds are illustrated in fig9 . most notably , an extract analogous with extract a , suitable for use in connection with the invention , is sold under the name quinolia ®, a registered u . s . trademark owned by an applicant ( biophytis sa ). ii . experimental study of the effect of 20 - hydroxyecdysone and extract a on the muscle composition of mice subjected to a high - fat diet the effect of phytoecdysones is observed on mice subjected to a high - fat diet during 3 weeks . the hf high - fat diet involved the intake of large amounts of fat in the form of lard . the mice selected for the study were male c57bl / 6j mice , 6 weeks old at the start of the experiment . mice not subjected to a high fat diet , forming a normal diet control group , were tested in parallel . the mice in the study were grouped according to the dietary regimes and treatments to which they were subjected : normal or control diet ( lf ), high - fat diet ( hf ), high - fat diet supplemented with quinoa extract ( hfq ) and high - fat diet supplemented with pure 20 - hydroxyecdysone ( hf20e ). the mice were subjected to the dietary regimes detailed in table 1 below for three weeks and the mice fed a high - fat diet were treated in parallel with pure 20e or extract a ( 2 % 20e ). the concentration of 20e was adjusted to equal 40 mg per kg of food . in light of the average food intake of the mice , the dose of 20e administered corresponded in the two treatments to 5 mg of 20e per kg of body weight per day . the food was supplied in excess every day for both dietary regimes and all three treatments . on average , 40 g of food was provided per cage per day or 6 . 5 g of food per mouse per day . table 1 below sets out in greater detail the composition of the diets to which mice were subjected : fig1 shows the lean body mass ( carcass defatted ) of the animals at the end of the experiment . administration of a high - fat diet has reduced carcass weight by 5 % compared to the control group . this result is consistent with the reduction of muscle protein synthesis resulting from such a diet ( anderson et al ., 2008 ). supplementation with extract a has not led to a significant increase , but the 20 - hydroxyecdysone has produced an increase that has allowed the mice to return virtually to the same level as the normal diet . following sacrifice , aliquots of muscle ( quadriceps ) were collected for analysis . fig2 contains a graph plotting muscle triglyceride content against diet and associated treatment . as expected , a trend was observed towards a higher increase in triglyceride content for the muscle in mice fed a high - fat diet compared to the control group of mice fed with the control diet ( 30 % increase ). in the mice that had received a treatment in association with the high - fat diet , administration of pure 20e or extract a shows a trend towards lower muscle triglyceride content of 26 % and 6 % respectively . fig3 contains a graph plotting muscle protein content against diet and associated treatment . the high - fat diet shows a trend towards lower (− 5 %) muscle protein content compared to mice fed the control diet . in the mice that had received a treatment in association with the high - fat diet , treatment with pure 20e or extract a shows a trend towards higher muscle protein content by 5 % and 13 %, respectively , compared to hf treatment alone . fig4 contains a graph plotting quantities of gene transcripts ( mrna ), as measured in the muscle , against diet and associated treatment . the quantities have been normalized with respect to the quantities measured in the muscles of mice fed the control diet . the high fat diet produced a sharp decrease in the quantity of gene transcripts coding for uncoupling proteins ucp2 and ucp3 compared to the quantities measured in mice fed the control diet . in the mice that had received a treatment in association with the high - fat diet , administration of pure 20e resulted in an increase in the quantity of ucp3 gene transcripts and a tendency to increased quantities of ucp2 gene transcripts . in mice that had received a treatment in association with the high - fat diet , administration of extract a led to an increase in the quantity of ucp2 and ucp3 gene transcripts . the high - fat diet led to a decrease in the quantity of gene transcripts coding for cpt - 1 intracellular fatty acid transporter relative to the amount measured in mice fed the control diet . treatment with pure 20e and extract a therefore tends to restore transcript levels to those seen for the control diet . these changes are consistent with an improvement in muscle oxidative capacity due to treatment with pure 20e and extract a . the animals fed the high - fat diet consumed a quantity of food providing them with the same amount of energy ( kcal ) as animals fed the standard diet ( fig5 ). this is also true for the animals receiving extract a or pure 20e . conversely , the energy expenditure of the latter was higher ( 9 %) than that of the animals fed the high - fat diet alone ( fig6 ). this difference , although small , has important implications , because its effect was cumulative over the duration of the experiment . the administration of pure 20e , like that of extract a , prevents the lipid deposition and protein loss in muscle induced by a high - fat , lard - based diet . both treatments promote the metabolism of fatty acids taken up in excessive amounts in muscle due to the administration of the high - fat diet . the increased energy expenditure combined with constant food intake may explain the observed differences in the accumulation of fat . this increased energy expenditure was not due to increased locomotor activity ( as measured in metabolic cages ); it appears therefore to be due to increased thermogenesis . iii . clinical double - blind study of the effects of extract a on obese individuals subjected to a low - calorie diet for 6 weeks the effect of extract a was studied on protection of lean mass during a low - calorie diet . the effect of extract a was studied in a double - blind clinical study involving obese subjects following a low - calorie diet for 6 weeks . protection of lean mass was assessed by measuring muscle strength using a “ grip test ” and by estimating lean body mass in a dxa scan analysis of body composition . the muscle strength and lean mass data are estimated values . to take into account differences in the duration of the low - calorie diet phase , the grip test and lean body mass data were initially calculated per day actually completed before being multiplied by the 42 days corresponding to the average duration of the low - calorie diet phase undergone by the volunteers . measurement of the loss of lean body mass during the low - calorie diet phase the effect of extract a on protection of lean body mass was studied during a low - calorie diet period . the product leads to a slight tendency to greater protection of lean body mass compared with the placebo ( fig7 ). it is likely that the metabolic constraints of a stringent diet outweigh all other considerations and indeed , in studies conducted outside any such diet period , the “ anabolic ” effects of 20 - hydroxyecdysone were significantly enhanced by supplementary intake of proteins ( simakin et al ., 1988 ). the effect of administration of extract a on muscle quality in obese subjects subjected to a low - calorie diet was studied . the measured changes in grip test results after 6 weeks of diet ( fig8 ) evidence greater protection of muscle strength in subjects ingesting extract a supplements (− 0 . 55 kg ) than in those who received a placebo (− 1 , 70 kg ). administration of extract a provides obese subjects with enhanced protection of lean body mass as is shown by the dxa scan analysis , with a trend towards lower loss in the case of extract a compared to the placebo . muscle quality is also better protected by administration of extract a , the loss being smaller compared to the group receiving the placebo . anderson s r , gilge d a , steiber a l , previs s f . 2008 . diet - 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