Patent Application
20140024706
1. Field of the Invention
The invention in general pertains to compositions for adipogenesis inhibition. More specifically, the present invention discloses a composition comprising scirpusin A and scirpusin B and anti-adipogenesis/anti-obesity potential thereof.
2. Description of Prior Art
Scirpusin A as a hydroxystilbene dimer from Xinjiang wine grape has been previously reported by Kong Q et al in J Sci Food Agric. 2010 Apr. 15; 90(5):823-8. Scirpusin A has been noted for its amyloid-beta-peptide aggregation inhibitory activity (Rivière C et al (2010)), singlet oxygen quenching and DNA protective activity (Kong Q et al (2010)) and beta-secretase inhibitory activity (Jeon SY et al (2007)).
Scirpusin B is a well established vaso-relaxing dimer of piceatannol and has been obtained in large amounts from passion fruit (Sano S et al, “Identification of the strong vaso-relaxing substance scirpusin B, a dimer of piceatannol, from passion fruit (Passiflora edulis) seeds, J Agric Food Chem. 2011 Jun. 8; 59(11):6209-13. Scirpusin B is also noted for its mild GSH activity (Maruki-Uchida H et al (2013)) and anti-HIV properties (Yang G X et al (2005)).
It has been previously reported that hexane extract of Cyperus rotundus tuber extracts exhibit anti-obesity properties. (Administration of Cyperus rotundus rhizomes extract prevents Weight Gain in Obese Zucker rats. Lemaure et al. 2007. Phytother Res. 21: 724-730.). The hexane fraction has been characterized to contain α-Cypernone, Rotundene, β-selinene, Calamenene, Cyperene, d-cadinene, Cyperotundone, Cadalene, Patchoulenone, Nootkatene, Sugeonol, g-calacorene, Kobusone, Cyperol, Isokobusone and Epi-a-selinene (Yadav et al. International Journal of Pharmaceutical and Clinical Research 2010; 2(1): 20-22). But the present invention discloses anti-obesity activity in ethyl acetate fraction of Cyperus rotundus This ethyl acetate fraction does not contain any of the many constituents of the hexane fraction. The present ethyl acetate fraction contains stilbenoid derived compounds, a class of compounds not reported to be occurring Cyperus rotundus by any investigator thus far. Hence it is the unique combination of the unexpected discovery of the occurrence of stilbenoid derived compounds and further their anti-obesity action. It is also a surprising finding that following the bioactivity guided fractionation of the rhizomes from Cyperus rotundus, a subfraction of ethyl acetate layer was characterized by the concentrated presence of two piceatannol dimers scirpusin A and scirpusin B which showed excellent anti-adipogenic effect in comparison to another subtraction that was concentrated with piceatannol along with dimers scirpusin A and scirpusin B. Thus the inventors of the present invention demonstrate for the first time the presence of scirpusin A and scirpusin B in the ethyl acetate fraction of the rhizomes Cyperus rotundus and anti-adipogenesis/anti-obesity potential thereof. comprising
It is thus the principle objective of the present invention to disclose a composition scirpusin A and scirpusin B and anti-adipogenesis/anti-obesity potential thereof.
It is another objective of the present invention to disclose a method of inhibiting adipogenesis in mammalian cells using a composition comprising scirpusin A and scirpusin B.
It is yet another objective of the present invention to disclose a method of managing obesity in mammals using a composition comprising scirpusin A and scirpusin B.
It is a further objective of the present invention to disclose a method of obtaining compositions comprising A. scirpusin A and scirpusin B and B. piceatannol and its dimers scirpusin A and scirpusin B through bioactivity guided fractionation of the rhizomes of Cyperus rotundus.
The present invention fulfills the aforesaid objectives and provides further related advantages.
The present invention discloses compositions comprising scirpusin A and scirpusin B and anti-adipogenesis/anti-obesity potential thereof. The invention also discloses a method of managing obesity in mammals using a composition comprising scirpusin A and scirpusin B. The present invention further discloses a method of obtaining compositions comprising A. scirpusin A and scirpusin B and B. piceatannol and its dimers scirpusin A and scirpusin B through bioactivity guided fractionation of the rhizomes of Cyperus rotundus. Other features and advantages of the present invention will become apparent from the following more detailed description, taken in conjunction with the accompanying images, which illustrate, by way of example, the principle of the invention.
In the most preferred embodiment the present invention relates to anti-adipogenic/anti-obesity composition comprising scirpusin A and scirpusin B represented by STR#1 and STR#2 respectively.
In another most preferred embodiment, the present invention relates to a method of inhibiting adipogenesis in mammalian cells, said method comprising step of bringing to contact adipogenic mammalian cells with a composition comprising scirpusin A and scirpusin B represented by STR#1 and STR#2 respectively.
In another most preferred embodiment, the present invention relates to the method of therapeutically inhibiting obesity caused by adipogenesis in mammals, said method comprising step of dietary supplementation of a composition comprising scirpusin A and scirpusin B represented by STR#1 and STR#2 respectively to a mammal in need of said therapeutic inhibition.
In another most preferred embodiment, the present invention relates to the use of a composition comprising scirpusin A and scirpusin B represented by STR#1 and STR#2 for inhibiting adipogenesis in mammalian cells.
In an alternate embodiment, the present invention also relates to a process for the bioactivity guided fractionation of the rhizomes of Cyperus rotundus to obtain anti-adipogenic/anti-obesity compositions comprising A. scirpusin A and scirpusin B represented by STR#1 and STR#2 and B. piceatannol and its dimers scirpusin A and scirpusin B represented by STR#1 and STR#2 respectively, said process comprising the steps of:
The present inventors investigated the hexane extract referred in step 2 preceding and found that Scirpusin A & Scirpusin B were not present. Hence hexane extract in step 1 is constitutionally different from ethyl acetate fraction detailed in step 7. Thus the ethyl acetate extract of Cyperus rotundus is quite different from the hexane extract that was the subject of investigation in Lemaure et al. 2007. Phytother Res. 21: 724-730
The following sections of this specification consist of illustrative examples of the most preferred embodiments of the present invention.
Methodology:
Dried rhizomes of Cyperus rotundus were pulverized to form a coarse powder. The pulverized powder was then extracted with 3 volumes of hexane followed by heating, reflux for 3 hours and filtering to obtain the hexane soluble fraction and spent material. The spent material is further extracted with 3 volumes of methanol followed by heating, reflux for 3 hours and filtering to obtain the methanol soluble active fraction and spent material. The methanol soluble fraction is solubilized in aqueous methanol and successively partitioned with chloroform (CHCl3), Ethyl acetate (EtOAc) and methanol to obtain the chloroform layer, ethyl acetate layer and the aqueous methanol layer respectively. The chloroform layer, ethyl acetate layer and the aqueous methanol layer are subjected to further bioactivity guided fractionation, wherein the bioactivity parameter was the ability of the chloroform layer, ethyl acetate layer and the aqueous methanol layer to inhibit adipogenesis in 3T3-L1 mouse adipocytes (mammalian adipocytes). The steps of the Oil Red O staining technique as adapted from Salazar Olivo et al (1995), Wu Z et al (1998), Fu M et al (2005) to study extent of adipogenesis inhibition is explained in EXAMPLE 1A herein below. The results are mentioned in Table A.
Terminal differentiation of adipocytes is accompanied by the accumulation of great amounts of lipids in large cytoplasmic vesicles. A common assay to measure adipocyte differentiation in cell culture is with the dye Oil Red-O (ORO). ORO is a lipid-soluble bright red dye which is a reliable indicator of adipocyte differentiation.
Principle: Oil Red O (Solvent Red 27, Sudan Red 5B, C.I. 26125, and C26H24N4O) is a lysochrome (fat-soluble dye) diazo dye used for staining of neutral triglycerides and lipids on frozen sections and some lipoproteins on paraffin sections. It has the appearance of a red powder with maximum absorption at 518(359) nm. Oil Red O is one of the dyes used for Sudan staining. Similar dyes include Sudan III, Sudan IV, and Sudan Black B. The staining has to be performed on fresh samples, as alcohol fixation removes the lipids. Oil Red O largely replaced Sudan III and Sudan IV, as it provides much deeper red color and the stains are therefore much easier to see. Oil red 0 is an oil soluble dye. Oil soluble dyes exhibit greater solubility of the dye in lipoid substances in the tissues/cells, than in the usual hydro alcoholic dye solvents. Hence, it will deeply stain the cells.
3T3-L1 cells approximately 60×104 cells are seeded for 48-72 hrs to get 70-80% confluence. After 48 hrs 200 μl of AIM (Adipogenesis induction medium) freshly prepared is added. 72 hrs later 200 μl APM (Adipogenesis progression medium) with the test compounds in different concentrations is added to the wells. The cells are incubated for 48 hrs in a humidified atmosphere (370 C) of 5% CO2 and 95% air. The supernatant is collected and stored for the estimation of leptin, adiponectin, IL-6 and TNF-alpha. Cells are fixed by adding 100 μl of 10% formalin and ORO staining is done. OD is read at 492 nm in microplate reader.
The results are expressed as IC50 values using Graphpad prism software. The percentage of inhibition of adipogenesis is calculated as follows,
% Inhibition=C−T/T*100
Where C-absorbance of Oil red O in differentiating/undifferentiated cells
T-absorbance of Oil red O in sample treated differentiating/undifferentiated cells.
The ethyl acetate layer exemplified the best bioactivity in terms of adipogenesis inhibition with an IC50 (μg/ml) value of 9.39. This fraction was then subjected to column fractionation to identify the bioactivity (adipogenesis inhibition) biomarker. Column fractionation involved the step of eluting sub fractions of the ethyl acetate layer with increasing polarity of methanol: chloroform mixture. The sub fractions of ethyl acetate layer are labeled as I, II, III and IV are subjected to bioactivity (anti-adipogenesis) evaluation. The essential steps of anti-adipogenic activity evaluation involves the procedure outlined herein above EXAMPLE 1A. The results are summarized herein below in Table B.
Sub fractions III and IV were then subjected to LC-MS with both fractions being enriched in piceatannol dimers scirpusin A and scirpusin B (
Ionization parameters: APCI positive mode, Source voltage—4.50 kV, Capillary temperature—225 degrees, Capillary voltage—43.00 V.
Data interpretation: Mass of Scirpusin A is reported to be 470.13. The mass [M+H] observed at 18.77 min in positive ionization mode using the above protocol is 471.08. Mass of Scirpusin B is reported to be 486. The mass [M+H] observed at 17.94 min in positive ionization mode is 487.05.
The first level of confirmation of the presence of dimers of Piceatannol in the Cyperus extract was based on this preliminary information on mass. Scirpusin A was directly confirmed by direct comparison with an authentic sample of Scirpusin A.
Subtractions were then subjected through the preparative HPLC to obtain purified dimer scirpusin B which was then studied using the analytical tools High Resolution Mass Spectroscopy (FIRMS), liquid chromatography-mass spectrometry (LC-MS/MS) and Nuclear Magnetic Resonance Spectroscopy (NMR) to be confirmed as scirpusin B. Data from the HRMS indicated [M+H]=487.138 which matched very well with the structure of the dimer and reported data (Sano et al., 2011) on the same (
EFFICACY EVALUATION FOR ANTI-OBESITY EFFECT OF A CYPRO-AF (active ethyl acetate fraction) AND CYPRO-D1 (ethyl acetate subfraction naturally enriched in piceatannol dimers scirpusin A and scirpusin B) EXTRACTS IN MICE
Objective of the test: The objective of the study was to evaluate the efficacy of Cypro-AF and Cypro-D1 extracts for anti-obesity effect in C57 mice.
Test System details:
Test Performance details
Husbandry
Grouping: Grouping of animals was done on the last day of acclimatization by body weight randomization and stratification method. Grouping of animals was done such that body weight variation of animals used does not exceed ±20% of the mean body weight of each group.
Study Design: The animals were divided into 6 groups viz., Group 1, 2, 3, 4, 5 and 6 consisting of 10 animals (5 male and 5 female) each. The group details, doses and number/sex of animals per group are presented in the following table:
Formulation Details and Dosage
The test items Cypro-AF and Cypro-D1 were dissolved in distilled water for formulating different doses. Freshly formulated test items were administered through oral route by gavage. The volume of dosage per animal was maintained at 10 ml/kg body weight for all the animals throughout the study period. The following table provided details of the test formulation.
Obesity induction: The G1 Control group animals were fed with normal control diet feed D12450B containing 10 kcal % fat and the G2 to G6 group animals were fed with high fat diet feed D12492 containing 60 kcal % fat during the induction of obesity and during main study.
Main Study:
Main study was started after the induction of obesity. The 3 doses of Cypro-AF and 1 dose of Cypro-D1 were administered to animals from Day 28 daily consecutively for a period of 27 days. The feeding of the diets continued in the main study was done in induction of obesity. The G1 Control and G2 High fat diet control group animals administered with distilled water while other groups animals received test items from Day 28 to Day 54 of the study period. The dose volume of administration was maintained according to the weekly body weight of individual animals. The total duration of the study was 61 days (7 days Acclimatization period+27 days Induction of obesity+27 days Main study).
Observations
The following observations were made for during the study period.
Feed Consumption
Individual animal feed consumption were recorded. Weekly average feed consumption was calculated and recorded.
Body Weight
Individual animal body weights were recorded on the day of receipt on Day 1 and weekly (±1 day) thereafter during the study period.
Clinical observations
All the animals were clinically observed twice daily during the study period.
Clinical Pathology
At the completion of the study period, blood samples were collected from the animals in tubes containing potassium ethylene di-amide tetra acetic acid (K2-EDTA) anticoagulant for hematology and without anticoagulant for clinical chemistry. The blood samples collected in tubes without anticoagulant were centrifuged at 3000 rpm for 10 minutes to obtain serum. Blood samples were collected humanely from retro-orbital plexus puncture method under mild ether anesthesia with the help of a fine capillary tube. The following hematology and clinical chemistry parameters were analyzed.
Hematology
The following hematology parameters were estimated using Sysmex, KX-21 (Transasia Bio-Medicals Ltd., India):
Clinical Chemistry
The following clinical chemistry parameters were analyzed using the “Erba Mannheim Chem Touch analyzer” (Transasia Bio-Medicals Ltd., India) from serum samples.
Pathology
After the completion of the study period, on Day 55, all the animals were humanely sacrificed by exposing them to excess carbon-di-oxide in gas chamber and subjected to following external and internal gross necropsy.
Gross Necropsy
The animals were subjected to external and internal gross pathological examinations.
Organ Weights
The following organs from all animals was trimmed of any adherent tissue, as appropriate and weighed wet as soon as possible to avoid drying: Brain, Thymus, Liver, Adrenals, Kidneys (paired organs), spleen, Heart, Ovaries/Testes (paired organs).
Fat Deposits Weights
The following fat deposits from all the animals was collected and weighed.
1. Epididymal Fat
2. Brown Fat
3. Ovarian Fat
Statistical Analysis and Report Preparation
The raw data obtained from the present study were subjected to computer statistical processing. The computer printout of the data (in the form of appendix) was verified with the original raw data. After verification, the data was subjected to One-way ANOVA (Analysis of Variance) with Dunnett's post test for the data on body weights, hematology and clinical chemistry parameters, organ weights using GraphPad Prism version 5.01, GraphPad Software. All analyses and comparisons will be evaluated at the 95% level of confidence (P<0.05), indicated by the designated by the superscripts of a where G1 is compared to G3, G4, G5, and G6 and b where G2 is compared to G3, G4, G5, and G6 throughout the report as stated below: *: Statistically significant (P<0.05) wherever applicable.
The data were subjected to One way—ANOVA statistical analysis by comparing the following:
G1 group {Control group (with 10 kcal % Fat)} to G3 group {CYPRO-AF 50 mg/kg+High fat diet (with 60 kcal % Fat)}, G4 group {CYPRO-AF −100 mg/kg+High fat diet (with 60 kcal % Fat)}, G5 group {CYPRO-AF 200 mg/kg+High fat diet (with 60 kcal % Fat)} and G6 group {CYPRO-D1 10 mg/kg+High fat diet (with 60 kcal % Fat)} as represented below:
G2—High fat diet Control (with 60 kcal % Fat) to G3 group {CYPRO-AF 50 mg/kg+High fat diet (with 60 kcal % Fat)}, G4 group {CYPRO-AF −100 mg/kg+High fat diet (with 60 kcal % Fat)}, G5 group {CYPRO-AF 200 mg/kg+High fat diet (with 60 kcal % Fat)} and G6 group {CYPRO-D1 10 mg/kg+High fat diet (with 60 kcal % Fat)} as represented shown below:
Results
Feed Consumption
The summary of weekly average feed consumption of male and female animals is presented in Table-1 and Table-2 respectively. There were no statistical significant differences in the feed consumption of animals during the study period.
Body Weight
The summary of weekly body weight of male and female animals is presented in Table-3 and Table-4 respectively.
In male animals, there was statistical significant increase in mean weekly body weight values of on Day 21 in G3 group {CYPRO-AF −50 mg/kg+High fat diet (with 60 kcal % Fat)}, G4 group {CYPRO-AF −100 mg/kg+High fat diet (with 60 kcal % Fat)}, G5 group {CYPRO-AF −200 mg/kg+High fat diet (with 60 kcal % Fat)} and G6 group {CYPRO-D1 −10 mg/kg+High fat diet (with 60 kcal % Fat)} compared to G1 group {Control group (with 10 kcal % Fat)}. These changes were considered to be due to difference in fat content of the feed.
In male animals, there was statistical significant increase in mean weekly body weight values of on Day 28 in G4 group {CYPRO-AF −100 mg/kg+High fat diet (with 60 kcal % Fat)}, G5 group {CYPRO-AF −200 mg/kg+High fat diet (with 60 kcal % Fat)} and G6 group {CYPRO-D1 −10 mg/kg+High fat diet (with 60 kcal % Fat)} compared to G1 group {Control group (with 10 kcal % Fat)}. These changes were considered to be due to difference in fat content of the feed.
In male animals, there was decrease in mean weekly body weight values of on Day 35, 42, 49 and 55 in G3 group {CYPRO-AF −50 mg/kg+High fat diet (with 60 kcal % Fat)}, G4 group {CYPRO-AF −100 mg/kg+High fat diet (with 60 kcal % Fat)}, G5 group {CYPRO-AF −200 mg/kg+High fat diet (with 60 kcal % Fat)} and G6 group {CYPRO-D1 −10 mg/kg+High fat diet (with 60 kcal % Fat)} compared to G1 group {Control group (with 10 kcal % Fat)}. These changes were considered to be due to administration of test items.
In female animals, there was statistical significant increase in mean weekly body weight values of on Day 21 in G4 group {CYPRO-AF −100 mg/kg+High fat diet (with 60 kcal % Fat)}, G5 group {CYPRO-AF −200 mg/kg+High fat diet (with 60 kcal % Fat)} and G6 group {CYPRO-D1 −10 mg/kg+High fat diet (with 60 kcal % Fat)} compared to G1 group {Control group (with 10 kcal % Fat)}. These changes were considered to be due to difference in fat content of the feed.
In female animals, there was statistical significant increase in mean weekly body weight values of on Day 28 in G3 group {CYPRO-AF −50 mg/kg+High fat diet (with 60 kcal % Fat)}, G4 group {CYPRO-AF −100 mg/kg+High fat diet (with 60 kcal % Fat)}, G5 group {CYPRO-AF −200 mg/kg+High fat diet (with 60 kcal % Fat)} and G6 group {CYPRO-D1 −10 mg/kg+High fat diet (with 60 kcal % Fat)} compared to G1 group {Control group (with 10 kcal % Fat)}. These change were considered to be due to difference in fat content of the feed.
In female animals, there was decrease in mean weekly body weight values of on Day 35, 42, 49 and 55 in G3 group {CYPRO-AF −50 mg/kg+High fat diet (with 60 kcal % Fat)}, G4 group {CYPRO-AF −100 mg/kg+High fat diet (with 60 kcal % Fat)}, G5 group {CYPRO-AF −200 mg/kg+High fat diet (with 60 kcal % Fat)} and G6 group {CYPRO-D1 −10 mg/kg+High fat diet (with 60 kcal % Fat)} compared to G1 group {Control group (with 10 kcal % Fat)}. These change were considered to be due to administration of test items.
Clinical Observations
The summary of clinical signs of male and female animals is presented in Table-5 and Table-6 respectively. The animals were found to healthy and normal in health status during the clinical observations during the study period.
Hematology
The summary of hematological parameters estimations of male and female animals is presented in Table-7 and Table-8 respectively.
Hematology parameters statistical analysis comparison between G1 to G3, G4, G5, and G6
Mean Corpuscular Hemoglobin Concentration (MCHC)
In male animals, there was statistical significant increase in mean MCHC value of G4 group {CYPRO-AF −100 mg/kg+High fat diet (with 60 kcal % Fat)} compared to G1 group {Control group (with 10 kcal % Fat)}. These changes can be considered as incidental as there was no dose dependent response.
Mean Corpuscular Volume and Mean Corpuscular Hemoglobin
In female animals, there was statistical significant increase in mean MCV and MCH values of G3 group {CYPRO-AF −50 mg/kg+High fat diet (with 60 kcal % Fat)} compared to G1 group {Control group (with 10 kcal % Fat)}. These changes can be considered as incidental as there was no dose dependent response.
Mean Corpuscular Hemoglobin and Mean Corpuscular Hemoglobin Concentration
In female animals, there was statistical significant increase in mean MCH and MCHC values of G5 group {CYPRO-AF −200 mg/kg+High fat diet (with 60 kcal % Fat)} compared to G2 group {High fat diet control group (with 60 kcal % Fat)}. This change can be considered as incidental as there was no dose dependent response.
Clinical Chemistry
The summary of clinical chemistry parameters estimations of male and female animals is presented in Table-9 and Table-10 respectively.
20.09**ab ± 7.51
Clinical chemistry parameters statistical analysis comparison between G1 to G3, G4, G5, and G6
Total Proteins
In female animals, there was statistical significant decrease in mean Total protein values of G3 group {CYPRO-AF −50 mg/kg+High fat diet (with 60 kcal % Fat)} and G4 group {CYPRO-AF −100 mg/kg+High fat diet (with 60 kcal % Fat)} compared to G1 group {Control group (with 10 kcal % Fat)}. These changes were considered to be due to difference in fat content of the feed.
Triglycerides
In female animals, there was statistical significant decrease in mean Triglyceride values G3 group {CYPRO-AF −50 mg/kg+High fat diet (with 60 kcal % Fat)}, G4 group {CYPRO-AF −100 mg/kg+High fat diet (with 60 kcal % Fat)}, G5 group {CYPRO-AF −200 mg/kg+High fat diet (with 60 kcal % Fat)} and G6 group {CYPRO-D1 −10 mg/kg+High fat diet (with 60 kcal % Fat)}compared to G1 group {Control group (with 10 kcal % Fat)}. These change were considered to be due to difference in fat content of the feed.
Total Cholesterol
In male animals, there was statistical significant increase in mean Total Cholesterol value of G3 group {CYPRO-AF −50 mg/kg+High fat diet (with 60 kcal % Fat)} and G6 group {CYPRO-D1 10 mg/kg+High fat diet (with 60 kcal % Fat)} compared to G1 group {Control group (with 10 kcal % Fat)}. These change were considered to be due to difference in fat content of the feed.
In female animals, there was statistical significant increase in mean Total Cholesterol values of G5 group {CYPRO-AF−200 mg/kg+High fat diet (with 60 kcal % Fat)} compared to G1 group {Control group (with 10 kcal % Fat)}. This change can be considered due to difference in fat content of the feed.
High Density Lipids
In male animals, there was statistical significant decrease in mean High density lipids value of G5 group {CYPRO-AF−200 mg/kg+High fat diet (with 60 kcal % Fat)} compared to G1 group {Control group (with 10 kcal % Fat)}. This change can be considered due to difference in fat content of the feed.
Low Density Lipids
In male animals, there was statistical significant increase in mean Low density lipids value of G3 group {CYPRO-AF−50 mg/kg+High fat diet (with 60 kcal % Fat)}, G4 group {CYPRO-AF−100 mg/kg+High fat diet (with 60 kcal % Fat)} and G6 group {CYPRO-D1 −10 mg/kg+High fat diet (with 60 kcal % Fat)} compared to G1 group {Control group (with 10 kcal % Fat)}. These change were considered to be due to difference in fat content of the feed.
Very Low Density Lipids
In female animals, there was statistical significant increase in mean Very low density lipids value of G5 group {CYPRO-AF−200 mg/kg+High fat diet (with 60 kcal % Fat)} compared to G1 group {Control group (with 10 kcal % Fat)}. This change can be considered due to difference in fat content of the feed.
Clinical Chemistry Parameters Statistical Analysis Comparison Between G2 to G3, G4, G5, and G6
Triglycerides
In male animals, there was decrease in mean Triglycerides values of G3 group {CYPRO-AF 50 mg/kg+High fat diet (with 60 kcal % Fat)}, G4 group {CYPRO-AF−100 mg/kg+High fat diet (with 60 kcal % Fat)}, G5 group {CYPRO-AF 200 mg/kg+High fat diet (with 60 kcal % Fat)} and G6 group {CYPRO-D1 10 mg/kg+High fat diet (with 60 kcal % Fat)} compared to G2 group High fat diet Control (with 60 kcal % Fat). This decrease in mean Triglycerides values changes could be due the effect of the test items.
In female animals, there was statistical significant decrease in mean Triglycerides values of G6 group {CYPRO-D1 10 mg/kg+High fat diet (with 60 kcal % Fat)} compared to G2 group High fat diet Control (with 60 kcal % Fat). This decrease in mean Triglyceride values changes could be due the effect of the test items.
There was decrease in mean Triglyceride values of G3 group {CYPRO-AF 50 mg/kg+High fat diet (with 60 kcal % Fat)}, G4 group {CYPRO-AF 100 mg/kg+High fat diet (with 60 kcal % Fat)} and G5 group {CYPRO-AF 200 mg/kg+High fat diet (with 60 kcal % Fat)} compared to G2 group High fat diet Control (with 60 kcal % Fat). These decrease in mean Triglyceride values changes could be due the effect of the test items.
Total Cholesterol
In male animals, there was decrease in mean Total Cholesterol values of G3 group {CYPRO-AF 50 mg/kg+High fat diet (with 60 kcal % Fat)}, G4 group {CYPRO-AF−100 mg/kg+High fat diet (with 60 kcal % Fat)} and G5 group {CYPRO-AF 200 mg/kg+High fat diet (with 60 kcal % Fat)} compared to G2 group High fat diet Control (with 60 kcal % Fat). This decrease in mean Total Cholesterol values changes could be due the effect of the test items.
In female animals, there was decrease in mean Total Cholesterol values of G3 group {CYPRO-AF 50 mg/kg+High fat diet (with 60 kcal % Fat)}, G4 group {CYPRO-AF 100 mg/kg+High fat diet (with 60 kcal % Fat)}, and G6 group {CYPRO-D1 10 mg/kg+High fat diet (with 60 kcal % Fat)} compared to G2 group High fat diet Control (with 60 kcal % Fat). These decrease in mean Total Cholesterol values changes could be due the effect of the test items.
High Density Lipids
In male animals, there was statistical significant decrease in mean High density lipids values of G3 group {CYPRO-AF 50 mg/kg+High fat diet (with 60 kcal % Fat)}, G4 group {CYPRO-AF−100 mg/kg+High fat diet (with 60 kcal % Fat)}, G5 group {CYPRO-AF 200 mg/kg+High fat diet (with 60 kcal % Fat)} and G6 group {CYPRO-D1 10 mg/kg+High fat diet (with 60 kcal % Fat)} compared to G2 group High fat diet Control (with 60 kcal % Fat).
The statistical significant decrease in mean High density lipid values changes could be due the effect of the test items.
In female animals, there was statistical significant decrease in mean High density lipids values of G3 group {CYPRO-AF 50 mg/kg+High fat diet (with 60 kcal % Fat)}, G4 group {CYPRO-AF 100 mg/kg+High fat diet (with 60 kcal % Fat)}, G5 group {CYPRO-AF 200 mg/kg+High fat diet (with 60 kcal % Fat)} and G6 group {CYPRO-D1 10 mg/kg+High fat diet (with 60 kcal % Fat)} compared to G2 group High fat diet Control (with 60 kcal % Fat). These decreases in mean High density lipid values changes could be due the effect of the test items.
Low Density Lipids
In male animals, there was decrease in mean Low density lipids values of G5 group {CYPRO-AF 200 mg/kg+High fat diet (with 60 kcal % Fat)} compared to G2 group High fat diet Control (with 60 kcal % Fat). This decrease in mean Low density lipid values changes could be due the effect of the test items.
Very Low Density Lipids Values
In male animals, there was decrease in mean Very low density lipids values of G3 group {CYPRO-AF 50 mg/kg+High fat diet (with 60 kcal % Fat)}, G4 group {CYPRO-AF−100 mg/kg+High fat diet (with 60 kcal % Fat)}, 05 group {CYPRO-AF 200 mg/kg+High fat diet (with 60 kcal % Fat)} and G6 group {CYPRO-D1 10 mg/kg+High fat diet (with 60 kcal % Fat)} compared to G2 group High fat diet Control (with 60 kcal % Fat). This decrease in mean Very low density lipid values changes could be due the effect of the test items.
In female animals, there was marginal decrease in mean Very low density lipids values of G3 group {CYPRO-AF 50 mg/kg+High fat diet (with 60 kcal % Fat)}, G4 group {CYPRO-AF 100 mg/kg+High fat diet (with 60 kcal % Fat)}, and G6 group {CYPRO-D1 10 mg/kg+High fat diet (with 60 kcal % Fat)} compared to G2 group High fat diet Control (with 60 kcal % Fat). These decreases in mean Very low density lipid values changes could be due the effect of the test items.
Conclusion: From the present study, it can be concluded that the test items Cypro-AF and Cypro-D1 had an effect on decreasing parameters such as HDL, Triglycerides, Cholesterol, LDL and VLDL concentrations in high fat diet induced obese male and female C57 animals at 50, 100 and 200 mg/kg Bwt of Cypro-AF and 10 mg/kg Bwt of Cypro-D1. No significant statistical changes were observed in the organ weights and fat deposits upon necropsy of animals.
While the invention has been described with respect to a preferred embodiment it is to be clearly understood by those skilled in the art that the invention is not limited thereto. Rather, scope of the invention is to be interpreted only in conjunction with the appended claims.
This application is non-provisional filing of provisional application No. 61/672,849 filed on 18 Jul. 2012, the contents of which are incorporated herein by reference.
| Number | Date | Country | |
|---|---|---|---|
| 61672849 | Jul 2012 | US |
