CA3066446A1 - Anti-obesity potential of garcinol - Google Patents
Anti-obesity potential of garcinol Download PDFInfo
- Publication number
- CA3066446A1 CA3066446A1 CA3066446A CA3066446A CA3066446A1 CA 3066446 A1 CA3066446 A1 CA 3066446A1 CA 3066446 A CA3066446 A CA 3066446A CA 3066446 A CA3066446 A CA 3066446A CA 3066446 A1 CA3066446 A1 CA 3066446A1
- Authority
- CA
- Canada
- Prior art keywords
- garcinol
- fat
- mammals
- obesity
- hfd
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- DTTONLKLWRTCAB-UDFURZHRSA-N (1s,3e,5r,7r)-3-[(3,4-dihydroxyphenyl)-hydroxymethylidene]-6,6-dimethyl-5,7-bis(3-methylbut-2-enyl)-1-[(2s)-5-methyl-2-prop-1-en-2-ylhex-4-enyl]bicyclo[3.3.1]nonane-2,4,9-trione Chemical compound O=C([C@@]1(C(C)(C)[C@H](CC=C(C)C)C[C@](C2=O)(C1=O)C[C@H](CC=C(C)C)C(C)=C)CC=C(C)C)\C2=C(\O)C1=CC=C(O)C(O)=C1 DTTONLKLWRTCAB-UDFURZHRSA-N 0.000 title claims abstract description 148
- QDKLRKZQSOQWJQ-JGWHSXGBSA-N Garcinol Natural products O=C([C@@]1(C(C)(C)[C@@H](CC=C(C)C)C[C@](C=2O)(C1=O)C[C@H](CC=C(C)C)C(C)=C)CC=C(C)C)C=2C(=O)C1=CC=C(O)C(O)=C1 QDKLRKZQSOQWJQ-JGWHSXGBSA-N 0.000 title claims abstract description 147
- LMFLOMBYUXRHIL-UHFFFAOYSA-N garcifuran-A Natural products COC1=C(O)C(OC)=CC(C=2C(=C3C=COC3=CC=2)O)=C1 LMFLOMBYUXRHIL-UHFFFAOYSA-N 0.000 title claims abstract description 147
- GRBCIRZXESZBGJ-UHFFFAOYSA-N guttiferone F Natural products CC(=CCCC(C(=C)C)C12CC(CC=C(C)C)C(C)(C)C(CC=C(C)C)(C(=O)C(=C1O)C(=O)c3ccc(O)c(O)c3)C2=O)C GRBCIRZXESZBGJ-UHFFFAOYSA-N 0.000 title claims abstract description 147
- 230000003579 anti-obesity Effects 0.000 title description 15
- 239000000203 mixture Substances 0.000 claims abstract description 43
- 208000008589 Obesity Diseases 0.000 claims abstract description 32
- 244000005709 gut microbiome Species 0.000 claims abstract description 31
- 235000020824 obesity Nutrition 0.000 claims abstract description 31
- 241000124008 Mammalia Species 0.000 claims abstract description 23
- 241000702462 Akkermansia muciniphila Species 0.000 claims abstract description 11
- 230000001225 therapeutic effect Effects 0.000 claims abstract description 8
- 230000004048 modification Effects 0.000 claims abstract description 5
- 238000012986 modification Methods 0.000 claims abstract description 5
- 230000001413 cellular effect Effects 0.000 claims abstract description 4
- 238000000034 method Methods 0.000 claims description 36
- 108090000623 proteins and genes Proteins 0.000 claims description 36
- 230000001965 increasing effect Effects 0.000 claims description 32
- 210000001789 adipocyte Anatomy 0.000 claims description 29
- 230000000694 effects Effects 0.000 claims description 29
- 230000014509 gene expression Effects 0.000 claims description 24
- 230000011759 adipose tissue development Effects 0.000 claims description 23
- 230000037396 body weight Effects 0.000 claims description 20
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 claims description 19
- 108010010234 HDL Lipoproteins Proteins 0.000 claims description 16
- 102000015779 HDL Lipoproteins Human genes 0.000 claims description 16
- 108010007622 LDL Lipoproteins Proteins 0.000 claims description 16
- 102000007330 LDL Lipoproteins Human genes 0.000 claims description 16
- 230000005764 inhibitory process Effects 0.000 claims description 15
- 210000002966 serum Anatomy 0.000 claims description 13
- 230000008859 change Effects 0.000 claims description 12
- 210000001596 intra-abdominal fat Anatomy 0.000 claims description 10
- 108010062497 VLDL Lipoproteins Proteins 0.000 claims description 8
- 150000003626 triacylglycerols Chemical class 0.000 claims description 8
- 101710119301 Protein delta homolog 1 Proteins 0.000 claims description 7
- 241001202853 Blautia Species 0.000 claims description 6
- 102100036467 Protein delta homolog 1 Human genes 0.000 claims description 6
- 239000002671 adjuvant Substances 0.000 claims description 6
- 210000004369 blood Anatomy 0.000 claims description 6
- 239000008280 blood Substances 0.000 claims description 6
- 239000002775 capsule Substances 0.000 claims description 6
- 239000000969 carrier Substances 0.000 claims description 6
- 235000009508 confectionery Nutrition 0.000 claims description 6
- 230000007423 decrease Effects 0.000 claims description 6
- 239000003085 diluting agent Substances 0.000 claims description 6
- 239000000839 emulsion Substances 0.000 claims description 6
- -1 gummies Substances 0.000 claims description 6
- 239000000546 pharmaceutical excipient Substances 0.000 claims description 6
- 239000000843 powder Substances 0.000 claims description 6
- 210000000229 preadipocyte Anatomy 0.000 claims description 6
- 239000000725 suspension Substances 0.000 claims description 6
- 235000020357 syrup Nutrition 0.000 claims description 6
- 239000006188 syrup Substances 0.000 claims description 6
- 239000003826 tablet Substances 0.000 claims description 6
- 241000894006 Bacteria Species 0.000 claims description 5
- 241000606125 Bacteroides Species 0.000 claims description 5
- 241000337504 Bacteroides stercorirosoris Species 0.000 claims description 5
- 241000115153 Bacteroides xylanisolvens Species 0.000 claims description 5
- 241000186560 Blautia coccoides Species 0.000 claims description 5
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims description 5
- 235000013406 prebiotics Nutrition 0.000 claims description 5
- 241000144235 Anaerobranca Species 0.000 claims description 4
- 102100024594 Histone-lysine N-methyltransferase PRDM16 Human genes 0.000 claims description 4
- 208000031226 Hyperlipidaemia Diseases 0.000 claims description 4
- 208000001145 Metabolic Syndrome Diseases 0.000 claims description 4
- 150000001200 N-acyl ethanolamides Chemical class 0.000 claims description 4
- 241000266824 Oscillospira Species 0.000 claims description 4
- 239000002621 endocannabinoid Substances 0.000 claims description 4
- 230000003827 upregulation Effects 0.000 claims description 4
- 241000605059 Bacteroidetes Species 0.000 claims description 3
- 241000192125 Firmicutes Species 0.000 claims description 3
- 208000022559 Inflammatory bowel disease Diseases 0.000 claims description 3
- 241000186660 Lactobacillus Species 0.000 claims description 3
- 102000016267 Leptin Human genes 0.000 claims description 3
- 108010092277 Leptin Proteins 0.000 claims description 3
- 102000003728 Peroxisome Proliferator-Activated Receptors Human genes 0.000 claims description 3
- 108090000029 Peroxisome Proliferator-Activated Receptors Proteins 0.000 claims description 3
- 102000007156 Resistin Human genes 0.000 claims description 3
- 108010047909 Resistin Proteins 0.000 claims description 3
- 241001261005 Verrucomicrobia Species 0.000 claims description 3
- 201000000690 abdominal obesity-metabolic syndrome Diseases 0.000 claims description 3
- 210000001593 brown adipocyte Anatomy 0.000 claims description 3
- 229940039696 lactobacillus Drugs 0.000 claims description 3
- NRYBAZVQPHGZNS-ZSOCWYAHSA-N leptin Chemical compound O=C([C@H](CO)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CC=1C2=CC=CC=C2NC=1)NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CO)NC(=O)CNC(=O)[C@H](CCC(N)=O)NC(=O)[C@@H](N)CC(C)C)CCSC)N1CCC[C@H]1C(=O)NCC(=O)N[C@@H](CS)C(O)=O NRYBAZVQPHGZNS-ZSOCWYAHSA-N 0.000 claims description 3
- 229940039781 leptin Drugs 0.000 claims description 3
- 230000019491 signal transduction Effects 0.000 claims description 3
- 241000428313 Anaerotruncus colihominis Species 0.000 claims description 2
- 241000217846 Bacteroides caccae Species 0.000 claims description 2
- 241001038648 Blautia wexlerae Species 0.000 claims description 2
- 241000605902 Butyrivibrio Species 0.000 claims description 2
- 241000193403 Clostridium Species 0.000 claims description 2
- 208000015943 Coeliac disease Diseases 0.000 claims description 2
- 208000011231 Crohn disease Diseases 0.000 claims description 2
- 241001240316 Emticicia Species 0.000 claims description 2
- 241000159554 Johnsonella ignava Species 0.000 claims description 2
- 208000012902 Nervous system disease Diseases 0.000 claims description 2
- 208000025966 Neurological disease Diseases 0.000 claims description 2
- 241001660097 Pedobacter Species 0.000 claims description 2
- 241000192142 Proteobacteria Species 0.000 claims description 2
- 241001604192 [Clostridium] alkalicellulosi Species 0.000 claims description 2
- 241001147719 [Clostridium] termitidis Species 0.000 claims description 2
- 239000003795 chemical substances by application Substances 0.000 claims description 2
- 201000010099 disease Diseases 0.000 claims description 2
- 230000003828 downregulation Effects 0.000 claims description 2
- 230000002526 effect on cardiovascular system Effects 0.000 claims description 2
- 208000019423 liver disease Diseases 0.000 claims description 2
- 102100036009 5'-AMP-activated protein kinase catalytic subunit alpha-2 Human genes 0.000 claims 2
- 101000783681 Homo sapiens 5'-AMP-activated protein kinase catalytic subunit alpha-2 Proteins 0.000 claims 2
- 230000003248 secreting effect Effects 0.000 claims 2
- 241001145875 Actinomyces naturae Species 0.000 claims 1
- 102100022089 Acyl-[acyl-carrier-protein] hydrolase Human genes 0.000 claims 1
- 241001101538 Alkaliphilus crotonatoxidans Species 0.000 claims 1
- 102100022544 Bone morphogenetic protein 7 Human genes 0.000 claims 1
- 101000964894 Bos taurus 14-3-3 protein zeta/delta Proteins 0.000 claims 1
- 241001143296 Deferribacteres <phylum> Species 0.000 claims 1
- 241001276248 Dolichospermum curvum Species 0.000 claims 1
- 241000024397 Dysgonomonas Species 0.000 claims 1
- 101000824278 Homo sapiens Acyl-[acyl-carrier-protein] hydrolase Proteins 0.000 claims 1
- 101000899361 Homo sapiens Bone morphogenetic protein 7 Proteins 0.000 claims 1
- 101000686942 Homo sapiens Histone-lysine N-methyltransferase PRDM16 Proteins 0.000 claims 1
- 101000837829 Homo sapiens Transcription factor IIIA Proteins 0.000 claims 1
- 101000611023 Homo sapiens Tumor necrosis factor receptor superfamily member 6 Proteins 0.000 claims 1
- 241000159562 Johnsonella Species 0.000 claims 1
- 241000186869 Lactobacillus salivarius Species 0.000 claims 1
- 102100029820 Mitochondrial brown fat uncoupling protein 1 Human genes 0.000 claims 1
- 108050002686 Mitochondrial brown fat uncoupling protein 1 Proteins 0.000 claims 1
- 101100439101 Mus musculus Cebpa gene Proteins 0.000 claims 1
- 102000017946 PGC-1 Human genes 0.000 claims 1
- 108700038399 PGC-1 Proteins 0.000 claims 1
- 241000030714 Parabacteroides goldsteinii Species 0.000 claims 1
- 241001607074 Peptoniphilus coxii Species 0.000 claims 1
- 241000156172 Rhodothermus clarus Species 0.000 claims 1
- 241000192031 Ruminococcus Species 0.000 claims 1
- 101001062859 Sus scrofa Fatty acid-binding protein, adipocyte Proteins 0.000 claims 1
- 102100028509 Transcription factor IIIA Human genes 0.000 claims 1
- 241001464867 [Ruminococcus] gnavus Species 0.000 claims 1
- 230000002438 mitochondrial effect Effects 0.000 claims 1
- 230000009286 beneficial effect Effects 0.000 abstract description 8
- 235000019786 weight gain Nutrition 0.000 abstract description 8
- 230000004584 weight gain Effects 0.000 abstract description 6
- 208000035150 Hypercholesterolemia Diseases 0.000 abstract description 3
- 235000009200 high fat diet Nutrition 0.000 description 75
- 241001465754 Metazoa Species 0.000 description 42
- 241000699670 Mus sp. Species 0.000 description 42
- 235000019197 fats Nutrition 0.000 description 30
- 210000000577 adipose tissue Anatomy 0.000 description 27
- 238000011282 treatment Methods 0.000 description 17
- 210000003486 adipose tissue brown Anatomy 0.000 description 15
- 210000001035 gastrointestinal tract Anatomy 0.000 description 13
- 210000001519 tissue Anatomy 0.000 description 13
- 102000014156 AMP-Activated Protein Kinases Human genes 0.000 description 12
- 108010011376 AMP-Activated Protein Kinases Proteins 0.000 description 12
- 102000004169 proteins and genes Human genes 0.000 description 12
- 235000005911 diet Nutrition 0.000 description 11
- 230000037213 diet Effects 0.000 description 10
- 230000009467 reduction Effects 0.000 description 10
- 230000004580 weight loss Effects 0.000 description 10
- 208000016261 weight loss Diseases 0.000 description 10
- 210000004027 cell Anatomy 0.000 description 9
- 231100000673 dose–response relationship Toxicity 0.000 description 9
- 230000000813 microbial effect Effects 0.000 description 9
- 241000702460 Akkermansia Species 0.000 description 7
- 235000012000 cholesterol Nutrition 0.000 description 7
- 150000002632 lipids Chemical class 0.000 description 7
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 6
- ZMJBYMUCKBYSCP-UHFFFAOYSA-N Hydroxycitric acid Chemical compound OC(=O)C(O)C(O)(C(O)=O)CC(O)=O ZMJBYMUCKBYSCP-UHFFFAOYSA-N 0.000 description 6
- 210000000593 adipose tissue white Anatomy 0.000 description 6
- NOESYZHRGYRDHS-UHFFFAOYSA-N insulin Chemical compound N1C(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(NC(=O)CN)C(C)CC)CSSCC(C(NC(CO)C(=O)NC(CC(C)C)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CCC(N)=O)C(=O)NC(CC(C)C)C(=O)NC(CCC(O)=O)C(=O)NC(CC(N)=O)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CSSCC(NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2C=CC(O)=CC=2)NC(=O)C(CC(C)C)NC(=O)C(C)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2NC=NC=2)NC(=O)C(CO)NC(=O)CNC2=O)C(=O)NCC(=O)NC(CCC(O)=O)C(=O)NC(CCCNC(N)=N)C(=O)NCC(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC(O)=CC=3)C(=O)NC(C(C)O)C(=O)N3C(CCC3)C(=O)NC(CCCCN)C(=O)NC(C)C(O)=O)C(=O)NC(CC(N)=O)C(O)=O)=O)NC(=O)C(C(C)CC)NC(=O)C(CO)NC(=O)C(C(C)O)NC(=O)C1CSSCC2NC(=O)C(CC(C)C)NC(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(N)CC=1C=CC=CC=1)C(C)C)CC1=CN=CN1 NOESYZHRGYRDHS-UHFFFAOYSA-N 0.000 description 6
- 210000004185 liver Anatomy 0.000 description 6
- 210000000056 organ Anatomy 0.000 description 6
- 238000012360 testing method Methods 0.000 description 6
- 108020004465 16S ribosomal RNA Proteins 0.000 description 5
- 238000011740 C57BL/6 mouse Methods 0.000 description 5
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 5
- 241000699666 Mus <mouse, genus> Species 0.000 description 5
- 229930002877 anthocyanin Natural products 0.000 description 5
- 235000010208 anthocyanin Nutrition 0.000 description 5
- 239000004410 anthocyanin Substances 0.000 description 5
- 150000004636 anthocyanins Chemical class 0.000 description 5
- 238000006243 chemical reaction Methods 0.000 description 5
- 206010012601 diabetes mellitus Diseases 0.000 description 5
- 235000021590 normal diet Nutrition 0.000 description 5
- 238000001543 one-way ANOVA Methods 0.000 description 5
- 238000000513 principal component analysis Methods 0.000 description 5
- 239000006041 probiotic Substances 0.000 description 5
- 235000018291 probiotics Nutrition 0.000 description 5
- 108020004414 DNA Proteins 0.000 description 4
- 230000035508 accumulation Effects 0.000 description 4
- 238000009825 accumulation Methods 0.000 description 4
- 230000001580 bacterial effect Effects 0.000 description 4
- 230000003247 decreasing effect Effects 0.000 description 4
- 238000002474 experimental method Methods 0.000 description 4
- 235000013305 food Nutrition 0.000 description 4
- 230000036541 health Effects 0.000 description 4
- 229940089491 hydroxycitric acid Drugs 0.000 description 4
- 210000003734 kidney Anatomy 0.000 description 4
- 230000000529 probiotic effect Effects 0.000 description 4
- VLEUZFDZJKSGMX-ONEGZZNKSA-N pterostilbene Chemical compound COC1=CC(OC)=CC(\C=C\C=2C=CC(O)=CC=2)=C1 VLEUZFDZJKSGMX-ONEGZZNKSA-N 0.000 description 4
- VLEUZFDZJKSGMX-UHFFFAOYSA-N pterostilbene Natural products COC1=CC(OC)=CC(C=CC=2C=CC(O)=CC=2)=C1 VLEUZFDZJKSGMX-UHFFFAOYSA-N 0.000 description 4
- 210000000952 spleen Anatomy 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- 241000087349 Anaerobranca zavarzinii Species 0.000 description 3
- 241000168061 Butyrivibrio proteoclasticus Species 0.000 description 3
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 3
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- 101710122674 Histone-lysine N-methyltransferase PRDM16 Proteins 0.000 description 3
- 206010061218 Inflammation Diseases 0.000 description 3
- 102000004877 Insulin Human genes 0.000 description 3
- 108090001061 Insulin Proteins 0.000 description 3
- 102000015494 Mitochondrial Uncoupling Proteins Human genes 0.000 description 3
- 108010050258 Mitochondrial Uncoupling Proteins Proteins 0.000 description 3
- 108010074436 Sterol Regulatory Element Binding Protein 1 Proteins 0.000 description 3
- 102100026839 Sterol regulatory element-binding protein 1 Human genes 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 230000003466 anti-cipated effect Effects 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 230000004069 differentiation Effects 0.000 description 3
- 208000035475 disorder Diseases 0.000 description 3
- 230000037149 energy metabolism Effects 0.000 description 3
- 238000013401 experimental design Methods 0.000 description 3
- 230000006870 function Effects 0.000 description 3
- 230000004054 inflammatory process Effects 0.000 description 3
- 229940125396 insulin Drugs 0.000 description 3
- 230000006372 lipid accumulation Effects 0.000 description 3
- 230000004130 lipolysis Effects 0.000 description 3
- 210000000496 pancreas Anatomy 0.000 description 3
- 238000002360 preparation method Methods 0.000 description 3
- 238000012163 sequencing technique Methods 0.000 description 3
- 238000010186 staining Methods 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 208000001072 type 2 diabetes mellitus Diseases 0.000 description 3
- 108010085238 Actins Proteins 0.000 description 2
- 102100036475 Alanine aminotransferase 1 Human genes 0.000 description 2
- 108010082126 Alanine transaminase Proteins 0.000 description 2
- 108091093088 Amplicon Proteins 0.000 description 2
- DGAKHGXRMXWHBX-ONEGZZNKSA-N Azoxymethane Chemical compound C\N=[N+](/C)[O-] DGAKHGXRMXWHBX-ONEGZZNKSA-N 0.000 description 2
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 2
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 2
- 208000027244 Dysbiosis Diseases 0.000 description 2
- 208000037487 Endotoxemia Diseases 0.000 description 2
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 2
- WZUVPPKBWHMQCE-UHFFFAOYSA-N Haematoxylin Chemical compound C12=CC(O)=C(O)C=C2CC2(O)C1C1=CC=C(O)C(O)=C1OC2 WZUVPPKBWHMQCE-UHFFFAOYSA-N 0.000 description 2
- 108010028554 LDL Cholesterol Proteins 0.000 description 2
- 108010016731 PPAR gamma Proteins 0.000 description 2
- 241000160321 Parabacteroides Species 0.000 description 2
- 102000012132 Peroxisome proliferator-activated receptor gamma Human genes 0.000 description 2
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 2
- 230000002411 adverse Effects 0.000 description 2
- 230000004075 alteration Effects 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 238000013461 design Methods 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 230000018109 developmental process Effects 0.000 description 2
- UREBDLICKHMUKA-CXSFZGCWSA-N dexamethasone Chemical compound C1CC2=CC(=O)C=C[C@]2(C)[C@]2(F)[C@@H]1[C@@H]1C[C@@H](C)[C@@](C(=O)CO)(O)[C@@]1(C)C[C@@H]2O UREBDLICKHMUKA-CXSFZGCWSA-N 0.000 description 2
- 229960003957 dexamethasone Drugs 0.000 description 2
- 229920003045 dextran sodium sulfate Polymers 0.000 description 2
- 230000000378 dietary effect Effects 0.000 description 2
- 230000003292 diminished effect Effects 0.000 description 2
- 238000009826 distribution Methods 0.000 description 2
- 230000007140 dysbiosis Effects 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 230000002708 enhancing effect Effects 0.000 description 2
- 239000000284 extract Substances 0.000 description 2
- 230000004136 fatty acid synthesis Effects 0.000 description 2
- 230000002550 fecal effect Effects 0.000 description 2
- 239000012091 fetal bovine serum Substances 0.000 description 2
- 238000007490 hematoxylin and eosin (H&E) staining Methods 0.000 description 2
- 238000001727 in vivo Methods 0.000 description 2
- 230000006698 induction Effects 0.000 description 2
- PHTQWCKDNZKARW-UHFFFAOYSA-N isoamylol Chemical compound CC(C)CCO PHTQWCKDNZKARW-UHFFFAOYSA-N 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 208000030159 metabolic disease Diseases 0.000 description 2
- 230000002503 metabolic effect Effects 0.000 description 2
- 230000009456 molecular mechanism Effects 0.000 description 2
- 238000010172 mouse model Methods 0.000 description 2
- 238000013116 obese mouse model Methods 0.000 description 2
- 239000012188 paraffin wax Substances 0.000 description 2
- 150000003904 phospholipids Chemical class 0.000 description 2
- 150000008442 polyphenolic compounds Chemical class 0.000 description 2
- 235000013824 polyphenols Nutrition 0.000 description 2
- 238000002731 protein assay Methods 0.000 description 2
- 230000011664 signaling Effects 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- DCXXMTOCNZCJGO-UHFFFAOYSA-N tristearoylglycerol Chemical compound CCCCCCCCCCCCCCCCCC(=O)OCC(OC(=O)CCCCCCCCCCCCCCCCC)COC(=O)CCCCCCCCCCCCCCCCC DCXXMTOCNZCJGO-UHFFFAOYSA-N 0.000 description 2
- 230000003442 weekly effect Effects 0.000 description 2
- 238000001262 western blot Methods 0.000 description 2
- 241000186046 Actinomyces Species 0.000 description 1
- 102000007469 Actins Human genes 0.000 description 1
- 206010002091 Anaesthesia Diseases 0.000 description 1
- 206010003497 Asphyxia Diseases 0.000 description 1
- 241000237519 Bivalvia Species 0.000 description 1
- 240000007551 Boswellia serrata Species 0.000 description 1
- 235000012035 Boswellia serrata Nutrition 0.000 description 1
- 101710168309 CCAAT/enhancer-binding protein alpha Proteins 0.000 description 1
- 102100034808 CCAAT/enhancer-binding protein alpha Human genes 0.000 description 1
- 208000005623 Carcinogenesis Diseases 0.000 description 1
- 101150061453 Cebpa gene Proteins 0.000 description 1
- 241001256780 Dolichospermum Species 0.000 description 1
- 241000827171 Dysgonomonas wimpennyi Species 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- 241000588914 Enterobacter Species 0.000 description 1
- 108700039887 Essential Genes Proteins 0.000 description 1
- 241000593508 Garcinia Species 0.000 description 1
- 235000000885 Garcinia xanthochymus Nutrition 0.000 description 1
- 102100030875 Gastricsin Human genes 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 241000282412 Homo Species 0.000 description 1
- 206010020772 Hypertension Diseases 0.000 description 1
- 206010022489 Insulin Resistance Diseases 0.000 description 1
- 108010013563 Lipoprotein Lipase Proteins 0.000 description 1
- 102100022119 Lipoprotein lipase Human genes 0.000 description 1
- 101150021539 MT-CO2 gene Proteins 0.000 description 1
- 241000736262 Microbiota Species 0.000 description 1
- 241001050508 Mucispirillum schaedleri Species 0.000 description 1
- 229910020700 Na3VO4 Inorganic materials 0.000 description 1
- CTQNGGLPUBDAKN-UHFFFAOYSA-N O-Xylene Chemical compound CC1=CC=CC=C1C CTQNGGLPUBDAKN-UHFFFAOYSA-N 0.000 description 1
- 241000566145 Otus Species 0.000 description 1
- 240000002834 Paulownia tomentosa Species 0.000 description 1
- 235000010678 Paulownia tomentosa Nutrition 0.000 description 1
- 108090000310 Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha Proteins 0.000 description 1
- 102100028960 Peroxisome proliferator-activated receptor gamma coactivator 1-alpha Human genes 0.000 description 1
- IIXHQGSINFQLRR-UHFFFAOYSA-N Piceatannol Natural products Oc1ccc(C=Cc2c(O)c(O)c3CCCCc3c2O)cc1O IIXHQGSINFQLRR-UHFFFAOYSA-N 0.000 description 1
- 101710191567 Probable endopolygalacturonase C Proteins 0.000 description 1
- 229940124158 Protease/peptidase inhibitor Drugs 0.000 description 1
- 102000001253 Protein Kinase Human genes 0.000 description 1
- 238000002123 RNA extraction Methods 0.000 description 1
- 238000011529 RT qPCR Methods 0.000 description 1
- 238000010240 RT-PCR analysis Methods 0.000 description 1
- 241001148569 Rhodothermus Species 0.000 description 1
- 241000220317 Rosa Species 0.000 description 1
- 108091006629 SLC13A2 Proteins 0.000 description 1
- 102000040739 Secretory proteins Human genes 0.000 description 1
- 108091058545 Secretory proteins Proteins 0.000 description 1
- 238000012300 Sequence Analysis Methods 0.000 description 1
- 208000006011 Stroke Diseases 0.000 description 1
- 238000000692 Student's t-test Methods 0.000 description 1
- 239000007983 Tris buffer Substances 0.000 description 1
- 235000009754 Vitis X bourquina Nutrition 0.000 description 1
- 235000012333 Vitis X labruscana Nutrition 0.000 description 1
- 240000006365 Vitis vinifera Species 0.000 description 1
- 235000014787 Vitis vinifera Nutrition 0.000 description 1
- AEOBEOJCBAYXBA-KQYNXXCUSA-N [(2r,3r,4r,5r)-2-(6-aminopurin-9-yl)-4-hydroxy-5-(phosphonooxymethyl)oxolan-3-yl] dihydrogen phosphate Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](COP(O)(O)=O)[C@@H](O)[C@H]1OP(O)(O)=O AEOBEOJCBAYXBA-KQYNXXCUSA-N 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 230000002293 adipogenic effect Effects 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 230000037005 anaesthesia Effects 0.000 description 1
- 238000000540 analysis of variance Methods 0.000 description 1
- 239000006053 animal diet Substances 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 230000003178 anti-diabetic effect Effects 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- 239000003472 antidiabetic agent Substances 0.000 description 1
- 230000001640 apoptogenic effect Effects 0.000 description 1
- YZXBAPSDXZZRGB-DOFZRALJSA-N arachidonic acid Chemical group CCCCC\C=C/C\C=C/C\C=C/C\C=C/CCCC(O)=O YZXBAPSDXZZRGB-DOFZRALJSA-N 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 238000012742 biochemical analysis Methods 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 230000006583 body weight regulation Effects 0.000 description 1
- 210000000988 bone and bone Anatomy 0.000 description 1
- 238000010804 cDNA synthesis Methods 0.000 description 1
- 230000036952 cancer formation Effects 0.000 description 1
- 231100000504 carcinogenesis Toxicity 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 230000005754 cellular signaling Effects 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 235000020639 clam Nutrition 0.000 description 1
- 238000011278 co-treatment Methods 0.000 description 1
- 210000001072 colon Anatomy 0.000 description 1
- 230000000112 colonic effect Effects 0.000 description 1
- 238000010835 comparative analysis Methods 0.000 description 1
- 238000009833 condensation Methods 0.000 description 1
- 230000005494 condensation Effects 0.000 description 1
- 238000004320 controlled atmosphere Methods 0.000 description 1
- 230000001276 controlling effect Effects 0.000 description 1
- 230000002596 correlated effect Effects 0.000 description 1
- 230000002354 daily effect Effects 0.000 description 1
- 235000014113 dietary fatty acids Nutrition 0.000 description 1
- 235000015872 dietary supplement Nutrition 0.000 description 1
- 230000003467 diminishing effect Effects 0.000 description 1
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 230000003028 elevating effect Effects 0.000 description 1
- YQGOJNYOYNNSMM-UHFFFAOYSA-N eosin Chemical compound [Na+].OC(=O)C1=CC=CC=C1C1=C2C=C(Br)C(=O)C(Br)=C2OC2=C(Br)C(O)=C(Br)C=C21 YQGOJNYOYNNSMM-UHFFFAOYSA-N 0.000 description 1
- 201000010063 epididymitis Diseases 0.000 description 1
- DEFVIWRASFVYLL-UHFFFAOYSA-N ethylene glycol bis(2-aminoethyl)tetraacetic acid Chemical compound OC(=O)CN(CC(O)=O)CCOCCOCCN(CC(O)=O)CC(O)=O DEFVIWRASFVYLL-UHFFFAOYSA-N 0.000 description 1
- 230000003203 everyday effect Effects 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 229930195729 fatty acid Natural products 0.000 description 1
- 239000000194 fatty acid Substances 0.000 description 1
- 150000004665 fatty acids Chemical class 0.000 description 1
- 239000012894 fetal calf serum Substances 0.000 description 1
- 230000037406 food intake Effects 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- BTCSSZJGUNDROE-UHFFFAOYSA-N gamma-aminobutyric acid Chemical compound NCCCC(O)=O BTCSSZJGUNDROE-UHFFFAOYSA-N 0.000 description 1
- 230000002496 gastric effect Effects 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 230000004190 glucose uptake Effects 0.000 description 1
- 210000002175 goblet cell Anatomy 0.000 description 1
- 230000012010 growth Effects 0.000 description 1
- 208000019622 heart disease Diseases 0.000 description 1
- 229920000669 heparin Polymers 0.000 description 1
- ZFGMDIBRIDKWMY-PASTXAENSA-N heparin Chemical compound CC(O)=N[C@@H]1[C@@H](O)[C@H](O)[C@@H](COS(O)(=O)=O)O[C@@H]1O[C@@H]1[C@@H](C(O)=O)O[C@@H](O[C@H]2[C@@H]([C@@H](OS(O)(=O)=O)[C@@H](O[C@@H]3[C@@H](OC(O)[C@H](OS(O)(=O)=O)[C@H]3O)C(O)=O)O[C@@H]2O)CS(O)(=O)=O)[C@H](O)[C@H]1O ZFGMDIBRIDKWMY-PASTXAENSA-N 0.000 description 1
- 229960001008 heparin sodium Drugs 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 230000013632 homeostatic process Effects 0.000 description 1
- 230000000055 hyoplipidemic effect Effects 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 239000003112 inhibitor Substances 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 230000000968 intestinal effect Effects 0.000 description 1
- 210000004347 intestinal mucosa Anatomy 0.000 description 1
- 230000003870 intestinal permeability Effects 0.000 description 1
- 208000002551 irritable bowel syndrome Diseases 0.000 description 1
- 210000005240 left ventricle Anatomy 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- 239000012139 lysis buffer Substances 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 238000002705 metabolomic analysis Methods 0.000 description 1
- 230000001431 metabolomic effect Effects 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 230000000921 morphogenic effect Effects 0.000 description 1
- 210000004877 mucosa Anatomy 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 208000008338 non-alcoholic fatty liver disease Diseases 0.000 description 1
- 206010053219 non-alcoholic steatohepatitis Diseases 0.000 description 1
- 230000035764 nutrition Effects 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 244000039328 opportunistic pathogen Species 0.000 description 1
- 201000008482 osteoarthritis Diseases 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 230000008506 pathogenesis Effects 0.000 description 1
- 230000001575 pathological effect Effects 0.000 description 1
- 239000001814 pectin Substances 0.000 description 1
- 235000010987 pectin Nutrition 0.000 description 1
- 229920001277 pectin Polymers 0.000 description 1
- 239000000137 peptide hydrolase inhibitor Substances 0.000 description 1
- 239000002953 phosphate buffered saline Substances 0.000 description 1
- CDRPUGZCRXZLFL-OWOJBTEDSA-N piceatannol Chemical compound OC1=CC(O)=CC(\C=C\C=2C=C(O)C(O)=CC=2)=C1 CDRPUGZCRXZLFL-OWOJBTEDSA-N 0.000 description 1
- 239000013641 positive control Substances 0.000 description 1
- 238000010149 post-hoc-test Methods 0.000 description 1
- 108060006633 protein kinase Proteins 0.000 description 1
- 208000020016 psychiatric disease Diseases 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 238000011552 rat model Methods 0.000 description 1
- 231100000272 reduced body weight Toxicity 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 239000011347 resin Substances 0.000 description 1
- 229920005989 resin Polymers 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 229910001220 stainless steel Inorganic materials 0.000 description 1
- 239000010935 stainless steel Substances 0.000 description 1
- 238000010972 statistical evaluation Methods 0.000 description 1
- 238000007619 statistical method Methods 0.000 description 1
- 230000004936 stimulating effect Effects 0.000 description 1
- 230000000638 stimulation Effects 0.000 description 1
- 239000011550 stock solution Substances 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 230000009469 supplementation Effects 0.000 description 1
- 230000001502 supplementing effect Effects 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 230000009885 systemic effect Effects 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 238000013518 transcription Methods 0.000 description 1
- 230000035897 transcription Effects 0.000 description 1
- 108091005703 transmembrane proteins Proteins 0.000 description 1
- 102000035160 transmembrane proteins Human genes 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
- IHIXIJGXTJIKRB-UHFFFAOYSA-N trisodium vanadate Chemical compound [Na+].[Na+].[Na+].[O-][V]([O-])([O-])=O IHIXIJGXTJIKRB-UHFFFAOYSA-N 0.000 description 1
- 235000021122 unsaturated fatty acids Nutrition 0.000 description 1
- 150000004670 unsaturated fatty acids Chemical class 0.000 description 1
- 210000001835 viscera Anatomy 0.000 description 1
- 230000037221 weight management Effects 0.000 description 1
- 210000000636 white adipocyte Anatomy 0.000 description 1
- 239000008096 xylene Substances 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
- A61P3/04—Anorexiants; Antiobesity agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/12—Ketones
- A61K31/122—Ketones having the oxygen directly attached to a ring, e.g. quinones, vitamin K1, anthralin
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
- A61P3/06—Antihyperlipidemics
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- General Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Organic Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Engineering & Computer Science (AREA)
- Obesity (AREA)
- Hematology (AREA)
- Diabetes (AREA)
- Child & Adolescent Psychology (AREA)
- Epidemiology (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Coloring Foods And Improving Nutritive Qualities (AREA)
- Medicinal Preparation (AREA)
- Acyclic And Carbocyclic Compounds In Medicinal Compositions (AREA)
- Confectionery (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
Disclosed are compositions containing garcinol for the therapeutic management of obesity. More specifically, the invention relates to the use of garcinol for a) maintaining energy balance in mammalian adipose cellular systems b) management of hypercholesterolemia and c) reducing weight gain in mammals. The modification of gut microbiota and the increase of beneficial microbe, Akkermansia muciniphila by garcinol are also disclosed.
Description
ANTI-OBESITY POTENTIAL OF GARCINOL
CROSS-REFERENCE TO RELATED PATENT APPLICATION
The present invention is a PCT application claiming priority of US provisional patent application nos. 62519949 filed on 15 June 2017 and 62523611 filed on 22 June 2017.
BACKGROUND OF THE INVENTION
Field of the invention [Paraffin] The invention in general relates to compositions for weight management.
Specifically the invention relates to compositions containing garcinol for the management of obesity, hypercholesterolemia and modification of gut microbiota.
Description of prior art [Para002] Obesity is considered to be the leading health risk for the development of various disorders like hypertension, type 2 diabetes, heart disease, stroke, osteoarthritis, and mental illness. Globally, more than 1 in 10 individuals are obese and about 36% of American adults are obese (https://www.medicalnewstoday.coln/articles/319902.php, accessed on 10 May 2018).
Obesity results due to imbalance between the energy content of food eaten and energy expended by the body to maintain life and to perform physical work. Such an energy balance framework is a potentially powerful tool for investigating the regulation of body weight.
[Para003] Conversion of white adipose tissue to brown or beige/brite is reported as an effective mechanism to utilize the undue energy abundance and increasing the energy expenditure. The role of brown adipose tissue (BAT) is well described in the following prior arts:
1. Elattar.S and Satyanarayana, "Can Brown Fat Win the Battle against White Fat?", J Cell Physiol. 2015 Oct; 230910):2311-7
CROSS-REFERENCE TO RELATED PATENT APPLICATION
The present invention is a PCT application claiming priority of US provisional patent application nos. 62519949 filed on 15 June 2017 and 62523611 filed on 22 June 2017.
BACKGROUND OF THE INVENTION
Field of the invention [Paraffin] The invention in general relates to compositions for weight management.
Specifically the invention relates to compositions containing garcinol for the management of obesity, hypercholesterolemia and modification of gut microbiota.
Description of prior art [Para002] Obesity is considered to be the leading health risk for the development of various disorders like hypertension, type 2 diabetes, heart disease, stroke, osteoarthritis, and mental illness. Globally, more than 1 in 10 individuals are obese and about 36% of American adults are obese (https://www.medicalnewstoday.coln/articles/319902.php, accessed on 10 May 2018).
Obesity results due to imbalance between the energy content of food eaten and energy expended by the body to maintain life and to perform physical work. Such an energy balance framework is a potentially powerful tool for investigating the regulation of body weight.
[Para003] Conversion of white adipose tissue to brown or beige/brite is reported as an effective mechanism to utilize the undue energy abundance and increasing the energy expenditure. The role of brown adipose tissue (BAT) is well described in the following prior arts:
1. Elattar.S and Satyanarayana, "Can Brown Fat Win the Battle against White Fat?", J Cell Physiol. 2015 Oct; 230910):2311-7
2. Zafrir B, "Brown adipose tissue: research milestones of a potential player in human energy balance and obesity-, Horm Metab Res. 2013 Oct;45( 1 0:774-85).
3. Giralt M, Villarrova F "White, brown, beige/brite: different adipose cells for different functions?" Endocrinology. 2013 Sep; 154(9):2992-3000 [Para0041 Drugs and/or natural molecules that facilitate the conversion of white to brown adipocytes are effective in the treatment/management of obesity related conditions. However, we need a better understanding of the components involved in energy expenditure and their interactions over various time scales to explain the natural history of conditions such as obesity and to estimate the magnitude and potential success of therapeutic interventions. (Kevin D. Hall, Steven B. Heymsfield, Joseph W. Kemnitz, Samuel Klein, Dale A. Schoeller, and John R.
Speakman, Energy balance and its components: implications for body weight regulation, Am J
Clin Nutr. 2012 Apr; 95(4): 989-994).
[Para005] Recently, it was observed that the gut microbiota is altered in conditions like obesity and type II diabetes. Administration of probiotics to obese individuals resulted in an effective weight loss. One particular gut microbe, Akkermansia Inucimphila was inversely associated with obesity, diabetes, cardio metabolic diseases and low-grade inflammation (Cani et al., Next-Generation Beneficial Microbes: The Case of Akkennansia mucimphila, Front.
Microbiol., 22 September 2017, https://doi. org/10.3389/fmicb.2017 .01765). Evidence show that the relative abundance of A. mucimphila increased more than 100-fold following the ingestion of prebiotics (Everard et al., 2014 Microbiome of prebiotic-treated mice reveals novel targets involved in host response during obesity. ISME J. 8, 2116-2130. doi: 10.1038/ismej2014.45).
Studies also indicated that the number of A. mucimphila was found to be lower in obese and type 2 diabetic mice and increased with antidiabetic treatments (Cani et al., Next-Generation Beneficial Microbes: The Case of Akkennansia mucimphila, Front. Microbiol., 22 September 2017, https://doi.org/10 .3389/fmicb .2017.01765). Another study observed that A.
mucimphila treatment reversed high-fat diet-induced metabolic disorders, including fat-mass gain, metabolic endotoxemia, adipose tissue inflammation, and insulin resistance. (Amandine Everard, Clara Belzer, Lucie Geurts, Janneke P. Ouwerkerk, Celine Druart, Laure B. Bindels, Yves Guiot, Muriel Derrien, Giulio G. Muccioli, Nathalie M. Delzenne, Willem M. de Vos and Patrice D. Cani, Cross-talk between Akkermansia mucimphila and intestinal epithelium controls diet-induced obesity, PNAS May 28, 2013. 110 (22) 9066-9071). Hence, increasing the viable counts of Akkerrnansia inucimphila can be an effective therapy for the management of obesity, diabetes and other metabolic disorders. The probiotic and beneficial effects of .Akkermansia muciniphila are well described in the following prior art documents.
1. Cani et al., Next-Generation Beneficial Microbes: The Case of Akkerinansia muciniphila, Front. Microbiol. , 22 September 2017, https://doi.org/10.3389/fmicb.2017.01765 2. Gomez-Gallego et al., ilkkermansict muciniphila: a novel functional microbe with probiotic properties, Beneficial Microbes, 2016; 7(4): 571-584 [Para006] Natural molecules are currently evaluated extensively for the management of obesity and related disorders. Extracts of Garcinia cambogia, have been reported to have a weight loss potential. US 7063861, discloses a weight loss composition containing garcinol and hydroxycitric acid (HCA) and optionally with anthocyanins. US8329743 also discloses a weight loss formulation containing garcinol, pterostilbene and anthocyanin. US7063861 indicates that garcinol and HCA combination increases the bioavailability of HCA bringing about an anti-obesity effect. Hence, the anti-obesity effect of garcinol per se is not reported and also cannot be anticipated from the prior art documents. Moreover, Heo et al., (Gut microbiota Modulated by Probiotics and Garcinia cambogia Extract Correlate with Weight Gain and Adipocyte Sizes in High Fat-Fed Mice Sci Rep. 2016;6:33566), reports the modulation of gut microbiota and increase in A. muciniphila by Garcinia cambogia extract without specific reference to garcinol.
The present invention solves the above problem by disclosing the anti-obesity effect and the ability of modulating the gut microbiome by garcinol.
[Para007] The principle objective of the invention is to disclose the anti-obesity effect of garcinol by bringing about weight loss and energy balance.
[Para0081 It is another objective of invention to disclose the ability of garcinol to modify the gut microbiome and increasing the viable counts of probiotic bacteria ilkkermansia muciniphila.
[Para009] It is yet another objective of invention to disclose the hypolipidemic effects of garcinol.
[Para010] The present invention fulfils the above such objectives and provides further related advantages.
SUMMARY OF THE INVENTION
[Para011]The present invention pertains to garcinol compositions for obesity management.
More specifically, the invention relates to the use of garcinol for a) the maintaining energy balance in mammalian adipose cellular systems b) management of hypercholesterolemia and c) reducing weight gain in mammals. The modification of gut microbiota and the increase of beneficial microbe. Akkermansia muciniphila by garcinol is also disclosed.
BRIEF DESCRIPTION OF THE DRAWINGS
[Para0012] Fig.la is the oil-O-red staining of adipocytes indicating a dose dependent reduction in lipid accumulation in adipocytes by garcinol.
[Para0131 Fig. lb is the graphical representation of the percentage inhibition of adipogenesis by garcinol.
[Para0141 Fig. 2 is a graphical representation showing the decrease in expression of genes related to adipogenesis in garcinol treated groups.
[Para015] Fig. 3 is a graphical representation showing the increase in expression of genes related to brown fat conversion and fat utilization in garcinol treated groups.
[Para0161 Fig. 4a is a graphical representation showing the change in weight of animals administered with different concentrations of garcinol over a period of 4 months.
[Para0171 Fig. 4b is graphical representation showing the final weight of animals administered with different concentrations of garcinol for a period of 120 days.
[Para018] Fig. 5 is a representative image showing the different fat pad regions in the mice body.
[Para019] Fig. 6 represents the change in the weight of peritoneal, mesenteric and perigonadal fat tissues treated with different concentrations of garcinol.
[Para020] Fig. 7 is a graphical representation showing the percentage reduction in visceral fat in animals administered with different concentrations of garcinol in a dose dependent manner.
[Para021] Fig. Sa and 8b are graphical representations showing the decrease in expression of genes related to adipogenesis in adipose tissues of animals administered with different concentrations of garcinol.
[Para0221 Fig. 9 is a graphical representation showing the increase in expression of genes related to brown fat conversion and fat utilization in adipose tissues of animals administered with different concentrations of garcinol.
Speakman, Energy balance and its components: implications for body weight regulation, Am J
Clin Nutr. 2012 Apr; 95(4): 989-994).
[Para005] Recently, it was observed that the gut microbiota is altered in conditions like obesity and type II diabetes. Administration of probiotics to obese individuals resulted in an effective weight loss. One particular gut microbe, Akkermansia Inucimphila was inversely associated with obesity, diabetes, cardio metabolic diseases and low-grade inflammation (Cani et al., Next-Generation Beneficial Microbes: The Case of Akkennansia mucimphila, Front.
Microbiol., 22 September 2017, https://doi. org/10.3389/fmicb.2017 .01765). Evidence show that the relative abundance of A. mucimphila increased more than 100-fold following the ingestion of prebiotics (Everard et al., 2014 Microbiome of prebiotic-treated mice reveals novel targets involved in host response during obesity. ISME J. 8, 2116-2130. doi: 10.1038/ismej2014.45).
Studies also indicated that the number of A. mucimphila was found to be lower in obese and type 2 diabetic mice and increased with antidiabetic treatments (Cani et al., Next-Generation Beneficial Microbes: The Case of Akkennansia mucimphila, Front. Microbiol., 22 September 2017, https://doi.org/10 .3389/fmicb .2017.01765). Another study observed that A.
mucimphila treatment reversed high-fat diet-induced metabolic disorders, including fat-mass gain, metabolic endotoxemia, adipose tissue inflammation, and insulin resistance. (Amandine Everard, Clara Belzer, Lucie Geurts, Janneke P. Ouwerkerk, Celine Druart, Laure B. Bindels, Yves Guiot, Muriel Derrien, Giulio G. Muccioli, Nathalie M. Delzenne, Willem M. de Vos and Patrice D. Cani, Cross-talk between Akkermansia mucimphila and intestinal epithelium controls diet-induced obesity, PNAS May 28, 2013. 110 (22) 9066-9071). Hence, increasing the viable counts of Akkerrnansia inucimphila can be an effective therapy for the management of obesity, diabetes and other metabolic disorders. The probiotic and beneficial effects of .Akkermansia muciniphila are well described in the following prior art documents.
1. Cani et al., Next-Generation Beneficial Microbes: The Case of Akkerinansia muciniphila, Front. Microbiol. , 22 September 2017, https://doi.org/10.3389/fmicb.2017.01765 2. Gomez-Gallego et al., ilkkermansict muciniphila: a novel functional microbe with probiotic properties, Beneficial Microbes, 2016; 7(4): 571-584 [Para006] Natural molecules are currently evaluated extensively for the management of obesity and related disorders. Extracts of Garcinia cambogia, have been reported to have a weight loss potential. US 7063861, discloses a weight loss composition containing garcinol and hydroxycitric acid (HCA) and optionally with anthocyanins. US8329743 also discloses a weight loss formulation containing garcinol, pterostilbene and anthocyanin. US7063861 indicates that garcinol and HCA combination increases the bioavailability of HCA bringing about an anti-obesity effect. Hence, the anti-obesity effect of garcinol per se is not reported and also cannot be anticipated from the prior art documents. Moreover, Heo et al., (Gut microbiota Modulated by Probiotics and Garcinia cambogia Extract Correlate with Weight Gain and Adipocyte Sizes in High Fat-Fed Mice Sci Rep. 2016;6:33566), reports the modulation of gut microbiota and increase in A. muciniphila by Garcinia cambogia extract without specific reference to garcinol.
The present invention solves the above problem by disclosing the anti-obesity effect and the ability of modulating the gut microbiome by garcinol.
[Para007] The principle objective of the invention is to disclose the anti-obesity effect of garcinol by bringing about weight loss and energy balance.
[Para0081 It is another objective of invention to disclose the ability of garcinol to modify the gut microbiome and increasing the viable counts of probiotic bacteria ilkkermansia muciniphila.
[Para009] It is yet another objective of invention to disclose the hypolipidemic effects of garcinol.
[Para010] The present invention fulfils the above such objectives and provides further related advantages.
SUMMARY OF THE INVENTION
[Para011]The present invention pertains to garcinol compositions for obesity management.
More specifically, the invention relates to the use of garcinol for a) the maintaining energy balance in mammalian adipose cellular systems b) management of hypercholesterolemia and c) reducing weight gain in mammals. The modification of gut microbiota and the increase of beneficial microbe. Akkermansia muciniphila by garcinol is also disclosed.
BRIEF DESCRIPTION OF THE DRAWINGS
[Para0012] Fig.la is the oil-O-red staining of adipocytes indicating a dose dependent reduction in lipid accumulation in adipocytes by garcinol.
[Para0131 Fig. lb is the graphical representation of the percentage inhibition of adipogenesis by garcinol.
[Para0141 Fig. 2 is a graphical representation showing the decrease in expression of genes related to adipogenesis in garcinol treated groups.
[Para015] Fig. 3 is a graphical representation showing the increase in expression of genes related to brown fat conversion and fat utilization in garcinol treated groups.
[Para0161 Fig. 4a is a graphical representation showing the change in weight of animals administered with different concentrations of garcinol over a period of 4 months.
[Para0171 Fig. 4b is graphical representation showing the final weight of animals administered with different concentrations of garcinol for a period of 120 days.
[Para018] Fig. 5 is a representative image showing the different fat pad regions in the mice body.
[Para019] Fig. 6 represents the change in the weight of peritoneal, mesenteric and perigonadal fat tissues treated with different concentrations of garcinol.
[Para020] Fig. 7 is a graphical representation showing the percentage reduction in visceral fat in animals administered with different concentrations of garcinol in a dose dependent manner.
[Para021] Fig. Sa and 8b are graphical representations showing the decrease in expression of genes related to adipogenesis in adipose tissues of animals administered with different concentrations of garcinol.
[Para0221 Fig. 9 is a graphical representation showing the increase in expression of genes related to brown fat conversion and fat utilization in adipose tissues of animals administered with different concentrations of garcinol.
4 [Para0231 Fig. 10a is a graphical representation showing the levels of total cholesterol and triglycerides in serum of animals administered with different concentrations of garcinol.
[Para0241 Fig. lob is a graphical representation showing the levels of LDL and VLDL in serum of animals administered with different concentrations of garcinol.
[Para025] Fig. 10c is a graphical representation showing the levels of HDL in serum of animals administered with different concentrations of garcinol.
[Para0261 Fig. 11 shows the experimental design for anti obesity study with Garcinol in HFD-induced Obesity Mice.
[Para027] Fig. 12 is a representative image showing the effect of Garcinol on HFD-induced Obesity in C57BL/6 mice. Image A is the representative photographs of each group of mice at the end of week 13. Image B shows the Photographs of perigonadal adipose tissues and image C
shows photographs of the liver.
[Para0281 Fig. 13 is a graphical representation of body weight of animal administered with various concentrations of garcinol. Body weight was monitored weekly and the average body weight of each group was expressed as the means SE, p< 0.05; a, b, c, and d significantly differed between each group.
[Para0291 Fig. 14a shows the photographs of perigonadal, retroperitoneal, and mesenteric adipose tissues of animals administered with garcinol.
[Para030] Fig. 14h is the graphical representation of adipose tissue weights of animals administered with garcinol.
[Para0311 Fig. 15a shows the representative images of each study group for the pathological assessment by H&E staining in perigonadal adipose tissue.
[Para0321 Fig. 15b is graphical representation showing the percentage frequency of adipocyte size on animals treated with garcinol. Adipocyte size was quantified under the microscope from representative sections.
[Para033] Figs. 16a and 16b show the change in the taxonomic composition of colonic bacterial communities in animals administered with garcinol. Fig 16a shows the change in the phylum and Fig. 16b represents the genus relative abundance of fecal microbiota.
[Para0341 Fig. 17a and 17b represents Principal Coordinate Analysis (PCoA) plots showing the normalized relative abundance of all samples (A) Phylum. (B) Genus [Para035] Fig. 17c represents the Heatmap showing the abundance of 50 operational taxonomic units (OTlis) significantly altered by garcinol in HFD-fed mice.
[Para036] Fig. 18a shows the effects of garcinol on protein expression of adipocyte specific factors and AMPK signaling in HFD-fed C57BL/6 Mice Perigonadal Adipose Tissue.
The protein levels of p-AMPK (Thr172), AMPK, Pref-1, SREBP-1 and PP ARy were detelmined by Western blot analysis with specific antibodies. 13-actin was used as a loading control.
[Para037] Fig. 18b is the graphical representation of the level of protein expression of adipocyte specific factors and AMPK signaling in HFD-fed C57BL/6 Mice Perigonadal Adipose Tissue.
The values indicate the relative density of the protein band normalized to 13-actin.*p<0.05;**p<0.005; compared with the HFD treatment only.
[Para0381 Fig. 19a is a graphical representation of body weight of animal administered with various concentrations of garcinol and garcinol blend.
[Para039] Fig. 19b is the representative photographs of each group of mice at the end of the study period.
[Para040] Fig. 20a is a graphical representation of perigonadal fat weights of animals administered with different concentrations of garcinol and garcinol blend.
[Para0411 Fig. 20b is a graphical representation of retroperitoneal fat weights of animals administered with different concentrations of garcinol and garcinol blend.
[Para042] Fig. 20c is a graphical representation of mesentric fat weights of animals administered with different concentrations of garcinol and garcinol blend.
DESCRIPTION OF THE MOST PREFERRED EMBODIMENTS
[Para0431 In the most preferred embodiment, the present invention discloses a method for therapeutic management of obesity in mammals, said method comprising steps of administering effective concentration of a composition containing garcinol to said mammals to bring about a) inhibition of adipogenesis b) decrease in body weight and visceral fat in said mammals. In a related embodiment, inhibition of adipogenesis in brought about by down regulation of genes selected from the group consisting of, but not limited to, peroxisome proliferator-activated receptor gamma (PPAR7), CCAAT/enhancer binding protein alpha (cEBPu), first apoptotic signal (FAS), adipocyte protein 2 (AP2), resistin and leptin. In a related embodiment, inhibition of adipogenesis is brought about by up regulation of genes selected from the group consisting of, but not limited to, phospho-adenosine monophosphate-activated protein kinase (p-AMPK), AMP-activated protein kinase (AMPK) and Preadipocyte factor I (PREF-1). In another related embodiment, the visceral fat is selected from the group consisting of mesenteric fat, peritoneal fat and perigonadal fat. In a related embodiment, the composition is formulated with pharmaceuticallylnutraceutically acceptable excipients, adjuvants, diluents or carriers and administered orally in the form of tablets, capsules, syrups, gummies, powders, suspensions, emulsions, chewable, candies and eatables.
[Para0441 In another preferred embodiment, the invention discloses a method of achieving energy balance in mammalian adipose cellular systems, said method comprising step of administering composition containing garcinol in effective amounts targeted towards mammalian pre-adipocytes and/or adipocytes to achieve effects of (a) increased inhibition of adipogenesis and (b) increased expression of factors that function individually or in combination to specifically recruit brown adipocytes or brown like (beige or brite) adipocytes, c) induce brown like phenotype (beige or brite adipocytes) in white adipocyte depots, to bring about the effect fat utilization and energy balance in said mammals. In related embodiments, the factors include the transmembrane protein mitochondrial uncoupling protein (UCP-I), the transcription co-regulators PR domain containing protein 16 (PRDM16) and Peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1a) which regulate the genes involved in energy metabolism and bone morphogenic protein 7 (BMP7), secretory protein controlling energy expenditure. In a related embodiment, the composition is formulated with pharmaceutically/nutraceutically acceptable excipients, adjuvants, diluents or carriers and administered orally in the form of tablets, capsules, syrups, gummies, powders, suspensions, emulsions, chewables, candies and eatables.
[Para0451 In another preferred embodiment, the present invention discloses a method of modifying the gut microbiota in mammals, said method comprising step of administering effective amounts of a composition containing garcinol to said mammals to brim., about change in the gut microbiota. In a related embodiment, the gut microbiota is selected from the Phylum Delerribacteres, Proteobacteria, Bacteroidetes, Verrucomicrobla and Firmicutes. In another related embodiment, the gut microbiota is selected from the genus Lactobacillus, Butyrivibrio, Clostridium, Anaerobranca, Dysgonontonas, Johnsonellaõ Rwninococcus, Bacteroides, Oscillospini, Parabacterroides, Akkermanisa, and Blautia. More specifically, the gut microbiota is selected from the group consisting of Pctrabacteroides goldsteinii, Bacteroides caccae, Johnsonella ignava, Blautia wexlerae, Dysgonomonas wimpennyi, Blautia hansenni, Anaerobranca zavarzinni, Oscillospira. eae, Mucispirillus schaedleri, Blautia coccoides, Anaerotruncus colihominis, Butyrivibro proteoclasticus, Akkermansia Lachnospora pectinoschiza, Pedobacter kwangvangensis, Alkaltphilus crotonatoxidans, lactobacillus salivaritts, Anaerivibria fipolyticus, Rhodothermus clams, Bacteroides stercorirosoris, Ruminocococcus flavefaciens, Bacteroides xylanisolvens, Rurninococcus gnavus, Clostridium termitidis, Clostridium alkalicellulosi, Emticicia oligoraphica, Pseudobutyrivibro xylanivorans, Actinomyces naturaeõ Pep/on/ph//us coxii, and Dolichospermum curvuin. In a related embodiment, modification of gut microbiota is effective in therapeutic management of diseases selected from the group consisting of obesity, cardiovascular complications, inflammatory bowel disease, Crohn's disease, Celiac disease, metabolic syndrome, liver diseases and neurological disorders. In a related embodiment, the composition is formulated with pharmaceuticallylnutraceutically acceptable excipients, adjuvants, diluents or carriers and administered orally in the form of tablets, capsules, syrups, gummies, powders, suspensions, emulsions, chewables, candies and eatables.
[Para046] In another preferred embodiment, the invention discloses a method for increasing the viable counts of Akkermansia muciniphila in the gut of mammals, said method comprising steps of administering effective amounts of a composition containing garcinol to mammals to bring about an increase in the colonies of said bacteria. In a related embodiment, the increase in the colony counts of Akkermansia mucimphila reduces body weight through the AMPK
signaling pathway by causing endocannabinoid release. In a related embodiment, the composition is foimulated with phannaceutically/nutraceutically acceptable excipients, adjuvants, diluents or carriers and administered orally in the form of tablets, capsules, syrups, gummies, powders, suspensions, emulsions, chewables, candies and eatables.
[Para0471 In another preferred embodiment, the invention discloses a method of therapeutic management of hyperlipidemia in mammals, said method comprising step of administering an effective concentration of a composition containing garcinol to brim., about the effects of (i) reducing the amount of total blood cholesterol levels; (ii) reducing the concentrations of low density lipoproteins (LDL) and very low density lipoproteins (VLDL); (iii) increasing the concentrations of high density lipoproteins (HDL) and (iv) reducing concentrations of serum triglycerides, in the blood of said mammals. In a related embodiment, the medical cause of hyperlipidemia is obesity. In a related embodiment, the composition is foimulated with pharmaceutically/nutraceutically acceptable excipients, adjuvants, diluents or carriers and administered orally in the form of tablets, capsules, syrups, gummies, powders, suspensions, emulsions, chewables, candies and eatables.
[Para0481 In another preferred embodiment, the invention discloses a composition containing garcinol for use as a prebiotic agent.
[Para049] Specific illustrative examples enunciating the most preferred embodiments are included herein below [Para050] EXAMPLE 1: Anti-obesity effects of Garcinol ¨ Study done at Sami Labs Limited, Bangalore, India and Srimad Andavan Arts & Science College, Tiruchirapalli, India [Para0511 ,A.dipogenesis inhibition and Brown fat specific gene expression by garcinol in cultured 3T3L1 and Animal tissues [Para0521 Methodology [Para0531 Preparation of Stock solutions [Para0541 Garcinol (20%) stock of 10 mg/nil was prepared in DMSO and filtered through 0.2 micron syringe filter. Stock was diluted 1000 times in DMEM to get 10 uglml final concentration and serially diluted. Insulin (Hi Media) was bought as a solution at a concentration of 20 mg/ml. This was diluted to 1 uglml in DMEM. IBMX- (Sigma) ¨Stock of 5m1V1 was prepared in DI\i1EM, and diluted 10 times to be used at a final concentration of 0.5mM.
Dexamethasone (Sigma)- A stock of 10 uM was prepared in DMEM and diluted 40 times to get a final concentration of 0.25 1.014 [Para055] Cell culture [Para0561 Mouse 3T3-L1 pre-adipocytes were cultured in DMEM containing 25 mM
glucose with 10% heat-inactivated fetal calf serum with antibiotics at 37 C and 5%
CO2. When the cells were 70-80% confluent, they were trypsinized, washed and seeded in 6 well plates at a density of 2x106 cells per well. Cells were induced to differentiate 2 d after reaching confluence (day 0), by supplementing DMEM media containing 10% Fetal Bovine Serum(FBS) along with lug/mL
insulin, 0.2511M dexamethasone, 0.5mM 1-methy1-3-isob-utyl-xanthine (IBIVLX) and different concentrations of Garcinol (20%) . From day 3 until day 7, cells were maintained in progression media supplemented with 1 ug/mL insulin and different concentrations of Garcinol (20%).
Untreated cells and undifferentiated cells grown in FCS media were taken as Adipogenesis positive and negative controls for the experiment. Quantification for amount of triglycerides accumulated in adipocytes was done by Oil red 0 staining.
[Para0571 RNA extraction [Para0581 Cells were harvested after second progression on day 7 and total RNA
was extracted using the Trizol method. Extracted RNA was treated with DNAse Ito remove any contaminating DNA and again extracted using phenol: chloroform: isoamyl alcohol extraction (24:25:1).
Quality of RNA was determined by checking the absorbance at 260/280 nm using a Nanodrop (Thermo) [Para0591 Gene expression studies in mouse fat pad [Para060] The frozen fat pads from treated and untreated animals were collected in RNA later and frozen. Approximately 100mg of the tissue was homogenized in ice and extracted with 1 ml Trizol as described earlier.
[Para061] Quantitative Real Time PCR
[Para062] 21.tg of total RNA was taken for cDNA synthesis using SuperScript III First-Strand Synthesis System (Life Technologies). Quantitative RT-PCR analysis was performed to determine the expression of brown fat specific genes in Roche Light cycler 96 using SYBR
Green master mix (Thermo Scientific). 13 actin was used as a house keeping gene The relative RNA abundance of BAT genes was normalized to the housekeeping 13 actin gene and expressed as delta delta CT (equivalent to fold change transformed by Log2).
[Para0631 Primer sequence: The primers used for the determining the expression of brown fat specific genes and genes related to adipoaenesis is given in table 1 [Para0641 Table 1: Primers used for analyzing the expression of BAT and adipogenesis specific genes Name Primer sequence BAT specific Genes m Ucpl F AGGCTTCCAGTACCATTAGGT
m Ucpl R CTGAGTGAGGCAAAGCTGATTT
mpgc 1 ctF CCC TGC CAT TGT TAA GAC C
mpgc 1 aR TGC TGC TGT TCC TGT TTT C
mprdm16 F TCCCACCAGACTTCGAGCTA
mprclm16 R ATCTCCC,_kTCCAAAGTCGGC
mBMP7 F GAGGGCTGGTTGGTGTTTGAT
mBMP7 R GTTGCTTGTTCTGGGGTCCAT
m Pectin F GAAGTCCCTC,_kCCCTCCCAA
m Pactin R GGCATGGACGCGACCA
Adipogenesis m PPAR g F TCGCTGATGCACTGCCTATG
m PPAR g R GAGAGGTCCACAGAGCTGATT
m c/EBP a F CAAGAACAGCAACGAGTACCG
m c/EBP a R GTCACTGGTCAACTCC,_kGCAC
m FAS F CIGAGATCCCAGCACITCTTGA
in FAS R GCCTCCGAAGCCAAATGAG
in AP2 F CATGGCCAAGCCCAACAT
m AP2 R CGCCCAGTTTGAAGGAAATC
[Para0651Results [Para0661 The lipids accumulated in adipocytes were quantified by Oil red 0 staining. Garcinol showed a dose dependent reduction in lipid accumulation in adipocytes (Fig. I) with 5 and 10 jig/ml showing the highest inhibition of lipid accumulation by 47.8% and 47.2%(Fig. lb).
[Para067] With respect to the genes involved in adipogenesis, PPARy is considered to be the master regulator of adipogenesis. Decrease in PPARy Expression will reduce the expression of other adipogenesis specific genes. In the present study, garcinol exhibited a dose expended reduction in the PPARy Expression and the expression genes related to adipogenesis and fatty acid synthesis like cEBPa, FAS and AP2 (Fig.2), indicating that garcinol inhibits adipogenesis in a dose dependent manner.
[Para0681 Garcinol also significantly increased the brown adipose tissue specific genes. The expression of UCP1, PRDM16, PGC la and BMP7 was increased by garcinol in a dose dependent manner (Fig. 3) suggesting that garcinol is effective in converting the white adipose tissue depots to brown or brite/beige adipose tissue thereby increasing energy expenditure by fat utilisation and lipolysis.
[Para0691 Effect of Garcinol on High Fat Diet Induced Obesity in C57 Mice [Para070] Methods [Para0711 Animals ¨ C57/BL6 mice, 6-8 weeks of age and 8 animals/Group (4Male and 4 Female) were used for the study. Animals were housed under standard laboratory conditions, air-conditioned with adequate fresh air supply (12 ¨ 15 Air changes per hour), room temperature 20.2 - 23.5 C and relative humidity 58-64% with 12 hours fluorescent light and 12 hours dark cycle. The temperature and relative humidity was recorded once daily.
[Para0721 Feed [Para0731 The animals were fed with Normal diet (9kca1 / day) and High fat diet (50kcal day) throughout the acclimatization and experimental period.
[Para074] Water was provided along with High Fat Diet to the animals throughout the acclimatization and experimental period. Water from water filter cum purifier was provided in animal feeding bottle with stainless steel sipper tubes.
[Para0751 All the studies were conducted according to the ethical guidelines of CPCSEA after obtaining necessary clearance from the committee (Approval No:
790/03/ac/CPCSEA).
a. In accordance with the recommendation of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA) guidelines for laboratory animal facility published in the gazette of India, December 15th 1998.
b. The CPCSEA approval number for the present study (Anti-obesity activity) is SAC/LkEC/BC/2017/1P.-001.
[Para076] The design of the study groups is depicted in table 2.
[Para0771 Table 2: Study design (16 weeks) Groups Treatment Diet G1 None Normal diet (9kca1 ,/0 fat) G2 None high fat diet (50kcal% Fat) G3 Garcinol (5mg/kgbw) high fat diet (50kca1 ./O Fat) G4 Garcinol (10mg/kgbw 16 weeks. high fat diet (50kca1% Fat) Garcinol (20mg/kgbw) for 16 weeks. high fat diet (50kca1% Fat) G6 Garcinol (40mg/kgbw) high fat diet (50kca1% Fat) [Para078] Body weight of the animals was recorded in all the days of experimental period. At the end of the experimental period, the animals were sacrificed by cervical dislocation. Blood was collected and Serum was separated by centrifugation and used for the analysis of biochemical parameters. The organs such as Liver Kidney, Spleen and Pancreas and Fat Tissues (Retroperitoneal, Peri-gonadal and Mesenteric) were dissected out and washed in phosphate buffered saline.
[Para079] Efficacy measurement [Para080] The following parameters were measured in the above groups:
Measurement of Body weight `k- Determination of Organ Weight = Estimation of Cholesterol (Zak et al., (2009) A new method for the determination of serum cholesterol. J Clin Endocrinoll\iletab., 94(7), 2215-2220) = Estimation of Triglycerides (Foster L.B and Dunn R.T. (1973) Stable reagents for determination of serum triglycerides by a colorimetric Hantzsch condensation method.
Clin Chem, 196, 338-340).
= Estimation of HDL Cholesterol (Burstein et al., (1970). Deteunination of HDL
cholesterol. J.lipid Res., 11, 583).
Determination of LDL and VLDL (Friedewald et al., (1972) Estimation of the concentration of Low Density Lipoprotein cholesterol in plasma without use of preparative centrifuge. J.Clin Chem.; 18:499).
[Para0811 Results [Para082] Body weight [Para083] The results indicated that garcinol inhibited weight gain in a dose dependant manner in the animals fed with high fat diet (Fig. 4a and 4b) for a period of 120 days. The percentage change in weight is depicted in the below table.
[Para0841 Table 3: Change in weight of the study animals Control IIFD G5 G10 G20 Initial 18.50 19.63 18.00 19.75 19.25 19.13 0.91 weight (g) 0.92 0.94 0.92 0.88 0.79 Final 33.75 29.33 28.25 27.37 26.00 27.5 1.37 Weight (g) 1.60 1.47 1.39 1.88 1.33 Change in 10.03 10.25 8.37 1.41 15.25 1.92 7.62 2.18 6.28 1.03 weight (g) 2.25 1.39 [Para085] Reduction in fat deposits [Para0861 The fat reduction in the different fat pad regions of mice (Fig. 5) was also evaluated.
The weights of Retroperitoneal. Peri-gonadal and Mesenteric Fat deposits after the 120 day administration of garcinol is tabulated as below [Para087] Table 4: Effect of Garcinol on Fat weight of HFD induced Mice Retroperitoneal Peri-gonadal Fat Mesenteric Fat Groups Fat (g wet tissue) (g wet tissue) (g wet tissue) 0.41 0.03 1.14 0.19 0.55 0.03 II 0.55 0.06 2.01 0.22 0.69 0.08 III 0.45 0.06 1.33 0.32 0.63 0.07 IV 0.43 0.05 1.17 0.22 0.56 0.05 0.46 0.06 1.35 0.21 0.601 0.08 VI 0.46 0.04 1.41 0.14 0.59 0.05 [Para088] Garcinol treatment significantly reduced fat accumulation in the different fat pad regions (Fig. 6). Percentage of Visceral fat was reduced by garcinol treatment (Fig. 7) with the dose of 10 mg/kg body weight showing the maximum effect.
[Para0891 Organ weights [Para090] Garcinol administration did not adversely affect the weight of the organs, suggesting that garcinol does not induce any adverse effects in critical organs. (Table
[Para0241 Fig. lob is a graphical representation showing the levels of LDL and VLDL in serum of animals administered with different concentrations of garcinol.
[Para025] Fig. 10c is a graphical representation showing the levels of HDL in serum of animals administered with different concentrations of garcinol.
[Para0261 Fig. 11 shows the experimental design for anti obesity study with Garcinol in HFD-induced Obesity Mice.
[Para027] Fig. 12 is a representative image showing the effect of Garcinol on HFD-induced Obesity in C57BL/6 mice. Image A is the representative photographs of each group of mice at the end of week 13. Image B shows the Photographs of perigonadal adipose tissues and image C
shows photographs of the liver.
[Para0281 Fig. 13 is a graphical representation of body weight of animal administered with various concentrations of garcinol. Body weight was monitored weekly and the average body weight of each group was expressed as the means SE, p< 0.05; a, b, c, and d significantly differed between each group.
[Para0291 Fig. 14a shows the photographs of perigonadal, retroperitoneal, and mesenteric adipose tissues of animals administered with garcinol.
[Para030] Fig. 14h is the graphical representation of adipose tissue weights of animals administered with garcinol.
[Para0311 Fig. 15a shows the representative images of each study group for the pathological assessment by H&E staining in perigonadal adipose tissue.
[Para0321 Fig. 15b is graphical representation showing the percentage frequency of adipocyte size on animals treated with garcinol. Adipocyte size was quantified under the microscope from representative sections.
[Para033] Figs. 16a and 16b show the change in the taxonomic composition of colonic bacterial communities in animals administered with garcinol. Fig 16a shows the change in the phylum and Fig. 16b represents the genus relative abundance of fecal microbiota.
[Para0341 Fig. 17a and 17b represents Principal Coordinate Analysis (PCoA) plots showing the normalized relative abundance of all samples (A) Phylum. (B) Genus [Para035] Fig. 17c represents the Heatmap showing the abundance of 50 operational taxonomic units (OTlis) significantly altered by garcinol in HFD-fed mice.
[Para036] Fig. 18a shows the effects of garcinol on protein expression of adipocyte specific factors and AMPK signaling in HFD-fed C57BL/6 Mice Perigonadal Adipose Tissue.
The protein levels of p-AMPK (Thr172), AMPK, Pref-1, SREBP-1 and PP ARy were detelmined by Western blot analysis with specific antibodies. 13-actin was used as a loading control.
[Para037] Fig. 18b is the graphical representation of the level of protein expression of adipocyte specific factors and AMPK signaling in HFD-fed C57BL/6 Mice Perigonadal Adipose Tissue.
The values indicate the relative density of the protein band normalized to 13-actin.*p<0.05;**p<0.005; compared with the HFD treatment only.
[Para0381 Fig. 19a is a graphical representation of body weight of animal administered with various concentrations of garcinol and garcinol blend.
[Para039] Fig. 19b is the representative photographs of each group of mice at the end of the study period.
[Para040] Fig. 20a is a graphical representation of perigonadal fat weights of animals administered with different concentrations of garcinol and garcinol blend.
[Para0411 Fig. 20b is a graphical representation of retroperitoneal fat weights of animals administered with different concentrations of garcinol and garcinol blend.
[Para042] Fig. 20c is a graphical representation of mesentric fat weights of animals administered with different concentrations of garcinol and garcinol blend.
DESCRIPTION OF THE MOST PREFERRED EMBODIMENTS
[Para0431 In the most preferred embodiment, the present invention discloses a method for therapeutic management of obesity in mammals, said method comprising steps of administering effective concentration of a composition containing garcinol to said mammals to bring about a) inhibition of adipogenesis b) decrease in body weight and visceral fat in said mammals. In a related embodiment, inhibition of adipogenesis in brought about by down regulation of genes selected from the group consisting of, but not limited to, peroxisome proliferator-activated receptor gamma (PPAR7), CCAAT/enhancer binding protein alpha (cEBPu), first apoptotic signal (FAS), adipocyte protein 2 (AP2), resistin and leptin. In a related embodiment, inhibition of adipogenesis is brought about by up regulation of genes selected from the group consisting of, but not limited to, phospho-adenosine monophosphate-activated protein kinase (p-AMPK), AMP-activated protein kinase (AMPK) and Preadipocyte factor I (PREF-1). In another related embodiment, the visceral fat is selected from the group consisting of mesenteric fat, peritoneal fat and perigonadal fat. In a related embodiment, the composition is formulated with pharmaceuticallylnutraceutically acceptable excipients, adjuvants, diluents or carriers and administered orally in the form of tablets, capsules, syrups, gummies, powders, suspensions, emulsions, chewable, candies and eatables.
[Para0441 In another preferred embodiment, the invention discloses a method of achieving energy balance in mammalian adipose cellular systems, said method comprising step of administering composition containing garcinol in effective amounts targeted towards mammalian pre-adipocytes and/or adipocytes to achieve effects of (a) increased inhibition of adipogenesis and (b) increased expression of factors that function individually or in combination to specifically recruit brown adipocytes or brown like (beige or brite) adipocytes, c) induce brown like phenotype (beige or brite adipocytes) in white adipocyte depots, to bring about the effect fat utilization and energy balance in said mammals. In related embodiments, the factors include the transmembrane protein mitochondrial uncoupling protein (UCP-I), the transcription co-regulators PR domain containing protein 16 (PRDM16) and Peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1a) which regulate the genes involved in energy metabolism and bone morphogenic protein 7 (BMP7), secretory protein controlling energy expenditure. In a related embodiment, the composition is formulated with pharmaceutically/nutraceutically acceptable excipients, adjuvants, diluents or carriers and administered orally in the form of tablets, capsules, syrups, gummies, powders, suspensions, emulsions, chewables, candies and eatables.
[Para0451 In another preferred embodiment, the present invention discloses a method of modifying the gut microbiota in mammals, said method comprising step of administering effective amounts of a composition containing garcinol to said mammals to brim., about change in the gut microbiota. In a related embodiment, the gut microbiota is selected from the Phylum Delerribacteres, Proteobacteria, Bacteroidetes, Verrucomicrobla and Firmicutes. In another related embodiment, the gut microbiota is selected from the genus Lactobacillus, Butyrivibrio, Clostridium, Anaerobranca, Dysgonontonas, Johnsonellaõ Rwninococcus, Bacteroides, Oscillospini, Parabacterroides, Akkermanisa, and Blautia. More specifically, the gut microbiota is selected from the group consisting of Pctrabacteroides goldsteinii, Bacteroides caccae, Johnsonella ignava, Blautia wexlerae, Dysgonomonas wimpennyi, Blautia hansenni, Anaerobranca zavarzinni, Oscillospira. eae, Mucispirillus schaedleri, Blautia coccoides, Anaerotruncus colihominis, Butyrivibro proteoclasticus, Akkermansia Lachnospora pectinoschiza, Pedobacter kwangvangensis, Alkaltphilus crotonatoxidans, lactobacillus salivaritts, Anaerivibria fipolyticus, Rhodothermus clams, Bacteroides stercorirosoris, Ruminocococcus flavefaciens, Bacteroides xylanisolvens, Rurninococcus gnavus, Clostridium termitidis, Clostridium alkalicellulosi, Emticicia oligoraphica, Pseudobutyrivibro xylanivorans, Actinomyces naturaeõ Pep/on/ph//us coxii, and Dolichospermum curvuin. In a related embodiment, modification of gut microbiota is effective in therapeutic management of diseases selected from the group consisting of obesity, cardiovascular complications, inflammatory bowel disease, Crohn's disease, Celiac disease, metabolic syndrome, liver diseases and neurological disorders. In a related embodiment, the composition is formulated with pharmaceuticallylnutraceutically acceptable excipients, adjuvants, diluents or carriers and administered orally in the form of tablets, capsules, syrups, gummies, powders, suspensions, emulsions, chewables, candies and eatables.
[Para046] In another preferred embodiment, the invention discloses a method for increasing the viable counts of Akkermansia muciniphila in the gut of mammals, said method comprising steps of administering effective amounts of a composition containing garcinol to mammals to bring about an increase in the colonies of said bacteria. In a related embodiment, the increase in the colony counts of Akkermansia mucimphila reduces body weight through the AMPK
signaling pathway by causing endocannabinoid release. In a related embodiment, the composition is foimulated with phannaceutically/nutraceutically acceptable excipients, adjuvants, diluents or carriers and administered orally in the form of tablets, capsules, syrups, gummies, powders, suspensions, emulsions, chewables, candies and eatables.
[Para0471 In another preferred embodiment, the invention discloses a method of therapeutic management of hyperlipidemia in mammals, said method comprising step of administering an effective concentration of a composition containing garcinol to brim., about the effects of (i) reducing the amount of total blood cholesterol levels; (ii) reducing the concentrations of low density lipoproteins (LDL) and very low density lipoproteins (VLDL); (iii) increasing the concentrations of high density lipoproteins (HDL) and (iv) reducing concentrations of serum triglycerides, in the blood of said mammals. In a related embodiment, the medical cause of hyperlipidemia is obesity. In a related embodiment, the composition is foimulated with pharmaceutically/nutraceutically acceptable excipients, adjuvants, diluents or carriers and administered orally in the form of tablets, capsules, syrups, gummies, powders, suspensions, emulsions, chewables, candies and eatables.
[Para0481 In another preferred embodiment, the invention discloses a composition containing garcinol for use as a prebiotic agent.
[Para049] Specific illustrative examples enunciating the most preferred embodiments are included herein below [Para050] EXAMPLE 1: Anti-obesity effects of Garcinol ¨ Study done at Sami Labs Limited, Bangalore, India and Srimad Andavan Arts & Science College, Tiruchirapalli, India [Para0511 ,A.dipogenesis inhibition and Brown fat specific gene expression by garcinol in cultured 3T3L1 and Animal tissues [Para0521 Methodology [Para0531 Preparation of Stock solutions [Para0541 Garcinol (20%) stock of 10 mg/nil was prepared in DMSO and filtered through 0.2 micron syringe filter. Stock was diluted 1000 times in DMEM to get 10 uglml final concentration and serially diluted. Insulin (Hi Media) was bought as a solution at a concentration of 20 mg/ml. This was diluted to 1 uglml in DMEM. IBMX- (Sigma) ¨Stock of 5m1V1 was prepared in DI\i1EM, and diluted 10 times to be used at a final concentration of 0.5mM.
Dexamethasone (Sigma)- A stock of 10 uM was prepared in DMEM and diluted 40 times to get a final concentration of 0.25 1.014 [Para055] Cell culture [Para0561 Mouse 3T3-L1 pre-adipocytes were cultured in DMEM containing 25 mM
glucose with 10% heat-inactivated fetal calf serum with antibiotics at 37 C and 5%
CO2. When the cells were 70-80% confluent, they were trypsinized, washed and seeded in 6 well plates at a density of 2x106 cells per well. Cells were induced to differentiate 2 d after reaching confluence (day 0), by supplementing DMEM media containing 10% Fetal Bovine Serum(FBS) along with lug/mL
insulin, 0.2511M dexamethasone, 0.5mM 1-methy1-3-isob-utyl-xanthine (IBIVLX) and different concentrations of Garcinol (20%) . From day 3 until day 7, cells were maintained in progression media supplemented with 1 ug/mL insulin and different concentrations of Garcinol (20%).
Untreated cells and undifferentiated cells grown in FCS media were taken as Adipogenesis positive and negative controls for the experiment. Quantification for amount of triglycerides accumulated in adipocytes was done by Oil red 0 staining.
[Para0571 RNA extraction [Para0581 Cells were harvested after second progression on day 7 and total RNA
was extracted using the Trizol method. Extracted RNA was treated with DNAse Ito remove any contaminating DNA and again extracted using phenol: chloroform: isoamyl alcohol extraction (24:25:1).
Quality of RNA was determined by checking the absorbance at 260/280 nm using a Nanodrop (Thermo) [Para0591 Gene expression studies in mouse fat pad [Para060] The frozen fat pads from treated and untreated animals were collected in RNA later and frozen. Approximately 100mg of the tissue was homogenized in ice and extracted with 1 ml Trizol as described earlier.
[Para061] Quantitative Real Time PCR
[Para062] 21.tg of total RNA was taken for cDNA synthesis using SuperScript III First-Strand Synthesis System (Life Technologies). Quantitative RT-PCR analysis was performed to determine the expression of brown fat specific genes in Roche Light cycler 96 using SYBR
Green master mix (Thermo Scientific). 13 actin was used as a house keeping gene The relative RNA abundance of BAT genes was normalized to the housekeeping 13 actin gene and expressed as delta delta CT (equivalent to fold change transformed by Log2).
[Para0631 Primer sequence: The primers used for the determining the expression of brown fat specific genes and genes related to adipoaenesis is given in table 1 [Para0641 Table 1: Primers used for analyzing the expression of BAT and adipogenesis specific genes Name Primer sequence BAT specific Genes m Ucpl F AGGCTTCCAGTACCATTAGGT
m Ucpl R CTGAGTGAGGCAAAGCTGATTT
mpgc 1 ctF CCC TGC CAT TGT TAA GAC C
mpgc 1 aR TGC TGC TGT TCC TGT TTT C
mprdm16 F TCCCACCAGACTTCGAGCTA
mprclm16 R ATCTCCC,_kTCCAAAGTCGGC
mBMP7 F GAGGGCTGGTTGGTGTTTGAT
mBMP7 R GTTGCTTGTTCTGGGGTCCAT
m Pectin F GAAGTCCCTC,_kCCCTCCCAA
m Pactin R GGCATGGACGCGACCA
Adipogenesis m PPAR g F TCGCTGATGCACTGCCTATG
m PPAR g R GAGAGGTCCACAGAGCTGATT
m c/EBP a F CAAGAACAGCAACGAGTACCG
m c/EBP a R GTCACTGGTCAACTCC,_kGCAC
m FAS F CIGAGATCCCAGCACITCTTGA
in FAS R GCCTCCGAAGCCAAATGAG
in AP2 F CATGGCCAAGCCCAACAT
m AP2 R CGCCCAGTTTGAAGGAAATC
[Para0651Results [Para0661 The lipids accumulated in adipocytes were quantified by Oil red 0 staining. Garcinol showed a dose dependent reduction in lipid accumulation in adipocytes (Fig. I) with 5 and 10 jig/ml showing the highest inhibition of lipid accumulation by 47.8% and 47.2%(Fig. lb).
[Para067] With respect to the genes involved in adipogenesis, PPARy is considered to be the master regulator of adipogenesis. Decrease in PPARy Expression will reduce the expression of other adipogenesis specific genes. In the present study, garcinol exhibited a dose expended reduction in the PPARy Expression and the expression genes related to adipogenesis and fatty acid synthesis like cEBPa, FAS and AP2 (Fig.2), indicating that garcinol inhibits adipogenesis in a dose dependent manner.
[Para0681 Garcinol also significantly increased the brown adipose tissue specific genes. The expression of UCP1, PRDM16, PGC la and BMP7 was increased by garcinol in a dose dependent manner (Fig. 3) suggesting that garcinol is effective in converting the white adipose tissue depots to brown or brite/beige adipose tissue thereby increasing energy expenditure by fat utilisation and lipolysis.
[Para0691 Effect of Garcinol on High Fat Diet Induced Obesity in C57 Mice [Para070] Methods [Para0711 Animals ¨ C57/BL6 mice, 6-8 weeks of age and 8 animals/Group (4Male and 4 Female) were used for the study. Animals were housed under standard laboratory conditions, air-conditioned with adequate fresh air supply (12 ¨ 15 Air changes per hour), room temperature 20.2 - 23.5 C and relative humidity 58-64% with 12 hours fluorescent light and 12 hours dark cycle. The temperature and relative humidity was recorded once daily.
[Para0721 Feed [Para0731 The animals were fed with Normal diet (9kca1 / day) and High fat diet (50kcal day) throughout the acclimatization and experimental period.
[Para074] Water was provided along with High Fat Diet to the animals throughout the acclimatization and experimental period. Water from water filter cum purifier was provided in animal feeding bottle with stainless steel sipper tubes.
[Para0751 All the studies were conducted according to the ethical guidelines of CPCSEA after obtaining necessary clearance from the committee (Approval No:
790/03/ac/CPCSEA).
a. In accordance with the recommendation of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA) guidelines for laboratory animal facility published in the gazette of India, December 15th 1998.
b. The CPCSEA approval number for the present study (Anti-obesity activity) is SAC/LkEC/BC/2017/1P.-001.
[Para076] The design of the study groups is depicted in table 2.
[Para0771 Table 2: Study design (16 weeks) Groups Treatment Diet G1 None Normal diet (9kca1 ,/0 fat) G2 None high fat diet (50kcal% Fat) G3 Garcinol (5mg/kgbw) high fat diet (50kca1 ./O Fat) G4 Garcinol (10mg/kgbw 16 weeks. high fat diet (50kca1% Fat) Garcinol (20mg/kgbw) for 16 weeks. high fat diet (50kca1% Fat) G6 Garcinol (40mg/kgbw) high fat diet (50kca1% Fat) [Para078] Body weight of the animals was recorded in all the days of experimental period. At the end of the experimental period, the animals were sacrificed by cervical dislocation. Blood was collected and Serum was separated by centrifugation and used for the analysis of biochemical parameters. The organs such as Liver Kidney, Spleen and Pancreas and Fat Tissues (Retroperitoneal, Peri-gonadal and Mesenteric) were dissected out and washed in phosphate buffered saline.
[Para079] Efficacy measurement [Para080] The following parameters were measured in the above groups:
Measurement of Body weight `k- Determination of Organ Weight = Estimation of Cholesterol (Zak et al., (2009) A new method for the determination of serum cholesterol. J Clin Endocrinoll\iletab., 94(7), 2215-2220) = Estimation of Triglycerides (Foster L.B and Dunn R.T. (1973) Stable reagents for determination of serum triglycerides by a colorimetric Hantzsch condensation method.
Clin Chem, 196, 338-340).
= Estimation of HDL Cholesterol (Burstein et al., (1970). Deteunination of HDL
cholesterol. J.lipid Res., 11, 583).
Determination of LDL and VLDL (Friedewald et al., (1972) Estimation of the concentration of Low Density Lipoprotein cholesterol in plasma without use of preparative centrifuge. J.Clin Chem.; 18:499).
[Para0811 Results [Para082] Body weight [Para083] The results indicated that garcinol inhibited weight gain in a dose dependant manner in the animals fed with high fat diet (Fig. 4a and 4b) for a period of 120 days. The percentage change in weight is depicted in the below table.
[Para0841 Table 3: Change in weight of the study animals Control IIFD G5 G10 G20 Initial 18.50 19.63 18.00 19.75 19.25 19.13 0.91 weight (g) 0.92 0.94 0.92 0.88 0.79 Final 33.75 29.33 28.25 27.37 26.00 27.5 1.37 Weight (g) 1.60 1.47 1.39 1.88 1.33 Change in 10.03 10.25 8.37 1.41 15.25 1.92 7.62 2.18 6.28 1.03 weight (g) 2.25 1.39 [Para085] Reduction in fat deposits [Para0861 The fat reduction in the different fat pad regions of mice (Fig. 5) was also evaluated.
The weights of Retroperitoneal. Peri-gonadal and Mesenteric Fat deposits after the 120 day administration of garcinol is tabulated as below [Para087] Table 4: Effect of Garcinol on Fat weight of HFD induced Mice Retroperitoneal Peri-gonadal Fat Mesenteric Fat Groups Fat (g wet tissue) (g wet tissue) (g wet tissue) 0.41 0.03 1.14 0.19 0.55 0.03 II 0.55 0.06 2.01 0.22 0.69 0.08 III 0.45 0.06 1.33 0.32 0.63 0.07 IV 0.43 0.05 1.17 0.22 0.56 0.05 0.46 0.06 1.35 0.21 0.601 0.08 VI 0.46 0.04 1.41 0.14 0.59 0.05 [Para088] Garcinol treatment significantly reduced fat accumulation in the different fat pad regions (Fig. 6). Percentage of Visceral fat was reduced by garcinol treatment (Fig. 7) with the dose of 10 mg/kg body weight showing the maximum effect.
[Para0891 Organ weights [Para090] Garcinol administration did not adversely affect the weight of the organs, suggesting that garcinol does not induce any adverse effects in critical organs. (Table
5).
[Para091] Table 5: Weights of kidney, spleen and pancreas in garcinol treated animals Kidney weight Spleen Weight Pancreas Weight Groups (g wet tissue) (g wet tissue) (g wet tissue) 0.42 0.02 0.19 0.01 0.15 0.01 II 0.553 0.06 0.26 0.03 0.24 0.02 III 0.49 0.03 0.25 0.02 0.23 0.01 IV 0.42 0.03 0.20 0.01 0.17 0.01 V 0.45 0.06 0.22 0.03 0.21 0.02 VI 0.42 0.04 0.23 0.02 0.22 0.02 [Para092] Gene expression: Reduction in the expression of genes related to adipogenesis was observed in fat pad of animals treated with Garcinol. Similar to Mouse 3T3-L1 cell lines, garcinol administration significantly reduced the expression of PPARy, AP2, FAS, RESISTIN
and LEPTIN in the fat pad regions (Fig. 8a and 8b). Similarly, garcinol administration effectively increased the expression of Brown fat specific genes in the mice fat pad regions (Fig.
9).
[Para093] Lipid profile: The high fat diet increased the levels of total cholesterol, LDL. VLDL
and triglycerides in the serum of study animals. High fat diet, co administered with garcinol, significantly reduced the total cholesterol and triglycerides (Fig. 10a), LDL
and VLDL (Fig.
10b) and increased the HDL levels (Fig. 10c) in the serum.
[Para0941 Conclusion:
[Para095] Garcinol treatment showed a dose dependent inhibition of adipogenesis in vitro and induced the conversion of white adipose tissue to brown or britelbeige thereby increasing fat utilisation and energy metabolism. The in vivo results indicated that Garcinol was effective in significantly reducing body weight and visceral fat accumulation at 10 mg/kg and reduced adipogenesis specific gene expression and increased brown adipose tissue specific genes in fat pad in mouse fat pads. Garcinol administration also resulted in the reduction of visceral fat and organ weights indicating that garcinol promotes lipolysis and energy metabolism. Over all, garcinol induces weight loss, reduces visceral fat and maintains health of key organs.
[Para0961 EXAMPLE 2: Anti-obesity effects of Garcinol ¨ Study done at National Taiwan University, Taipei, Taiwan [Para097] Methodology [Para098] Reagents and antibodies [Para0991 AMPK and p-AMPK (Thr172) antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA). The SREBP-1 antibody was procured from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The PPARy and Pref-1 antibodies were purchased from abeam (Cambridge, England). The mouse 13-actin monoclonal antibody was obtained from Sigma Chemical Co (St. Louis, MO, USA). The Bio-Rad protein assay dye reagent was purchased from Bio-Rad Laboratories (Munich, Germany). Xylene and hematoxylin and eosin (H&E) stain were acquired from Surgipath (Peterborough, UK). Cholesterol used as part of the animal diet was obtained from Acros Organics (Bridgewater, NJ, USA). Garcinol was procured from Sabinsa Corp. (East Windsor, NJ, USA). The purity of garcinol was determined by high-performance liquid chromatography (HPLC) to be higher than 99%.
[Para1001 Animal care and experimental design [Parana] Five-week-old male C57BLI6 mice were purchased from the BioLASCO
Experimental Animal Center (Taiwan Co., Ltd, Taipei, Taiwan) and housed in a controlled atmosphere (25 1 C at 50% relative humidity) with a 12-h light/dark cycle.
After one week of acclimation, animals were randomly distributed into normal diet (ND, 15%
energy as fat), HFD
(50% energy as fat), and HFD with 0.1% or 0.5% garcinol groups of eight mice in each group for 13 weeks. The experimental design is summarized in Fig. 11. The experimental diets were modified from the Purina 5001 diet (LabDiet, PIVII Nutrition International, St. Louis, MO, USA).
The animals had ad libitum access to food and water. Food cups were replenished with a fresh diet every day. All animal experimental protocols employed in this study were approved by the Institutional Animal Care and Use Committee of the National Taiwan University (IACUC, NTU). Upon termination of the study, the animals were sacrificed by CO, asphyxiation and dissected, and the weights of their whole bodies and selected tissues, including the liver, kidney, spleen, adipose tissues (perigonadal, retroperitoneal, and mesenteric fat) and serum were immediately collected, weighed, and photographed.
[Para1021 Histopathological examination [Para103] A portion of perigonadal fat and the median lobe of the liver were dissected and fixed in 10% buffered formalin, dehydrated with a sequence of ethanol solutions, and processed for embedding in paraffin. Sections of 3-5 1.1.m in thickness were cut, deparaffinized, rehydrated, stained with H&E, and subjected to photomicroscopic assessment. Adipocyte size was determined using Image J software (Rasband, W.S., ImageJ, U. S. National Institutes of Health, Bethesda, MD, USA).
[Para1041 Biochemical analysis [Para105] Blood samples were collected from the left ventricle under anesthesia. The samples were mixed in 10 [11_, of heparin sodium and centrifuged at 3500 rpm and 4 'V
for 10 min. The plasma was then stored at ¨80 C until use. Glutamic-pyruvic transaminase (GPT), total cholesterol (TC), TG, high-density lipoprotein (HDL), and low-density lipoprotein (LDL) levels were analyzed at the National Laboratory Animal Center, NLAC (Taipei, Taiwan) on a 7080 Biochemical Analyzer (Hitachi, Tokyo, Japan) according to the manufacturer's instructions.
[Para106] 16S rDNA gene sequencing and analysis [Para1071 Total DNA was extracted from fresh fecal samples. The purified DNA
was eluted using the innuSPEED Stool DNA kit (Analytik Jena AG, Jena, Germany) according to the manufacturer's protocol. The PCR primer sequences from Caporaso et al.,(Caporaso, J. G., Lauber, C. L., Walters, W. A., Berg-Lyons, D. et at., Global patterns of 16S
rRNA diversity at a depth of millions of sequences per sample. Proc.Natl.Acad.Sci U.S A 2011, 108 ,S'uppl I, 4516-4522) were used to amplify the 16S rRNA variable region, and the PCR
conditions were performed as mentioned in Tung el al., (Tung, Y. C., Lin, Y. H., Chen, H. J., Chou, S. C. et at., Piceatannol Exerts Anti-Obesity Effects in C57BL/6 Mice through Modulating Adipogenic Proteins and Gut Microbiota. Molecules. 2016, 21) and Chou et al., (Chou, Y.
C., Suh, J. H., Wang, Y., Pahwa, M. et at., Boswellia serrata resin extract alleviates azoxymethane (A0M)/dextran sodium sulfate (DSS)-induced colon tumorigenesis. Mol.Nutr Food Res. 2017, 61) Then, the amplicons were used to construct index-labeled libraries with the Illumina DNA
Library Preparation kit (Illumina, San Diego, CA, USA). The Illumina MiniSeq NGS System (Illumina) was employed to analyze more than 100,000 reads with paired-end sequencing (2 x 150 bp), and the metagenomics workflow classified organisms from the amplicon using a database of 16S rRNA data. The classification was based on the Greermenes database (https://greengenes.lbl.gov/). The output of the workflow was a classification of reads at several taxonomic levels: kingdom, phylum, class, order, family, genus, and species.
[Para108] Protein preparation and western blot [Para1091 Tissues were homogenized in ice-cold lysis buffer (10% glycerol, 1%
TritonX-100, 1 mi\it Na3VO4, 1 mM EGTA, 10 mi\it NaF, 1 mM Na4P207, 20 inIVI Tris buffer (pH7.9), 100 p.M
P-glycerophosphate, 137 mM NaC1, and 5 mM EDTA) containing 1 Protease Inhibitor Cocktail Tablet (Roche, Indianapolis, IN, USA) on ice for 1 h, followed by centrifugation at 17,500g for 30 min at 4 'C. The protein concentration was measured with the Bio-Rad protein assay (Bio-Rad Laboratories, Inc., Hercules, CA, USA).
[Para1101 Statistical analysis [Para111] Statistical evaluation of the significance of the differences between the groups of mice was assessed using the Student t-test. For experiments comparing multiple groups, the differences were analyzed by carrying out one-way analysis of variance (ANOVA) and Duncan's post-hoc test. Data are presented as the means E for the indicated number of independently performed experiments, and p values <0.05 were considered statistically significant. Principal component analysis (PCA) was conducted to visualize the differences between samples.
[Para1121 Results [Para1131 Body weight gain [Para114] The results indicated that that HFD feeding for 13 weeks led to significant increase in body and liver weight along with perigonadal, retroperitoneal, and mesenteric fat accumulation.
Diet supplemented with 0.1 and 0.5% aarcinol reduced body weight in a dose-dependent manner.
Co-treatment with high doses of garcinol (0.5%) and a HFD diet inhibited body weight gain, and there is no difference between HFD + 0.5% aarcinol and the ND group (Fig.12 and Fig.13).
[Para1151 Effect on White adipose tissue adipocyte size and liver homeostasis [Para116] Garcinol at concentrations of 0.5% dramatically decreased all three white adipose fat weights, compared to the HFD group, by 85.1% in terms of perigonadal weight, 92.4%
retroperitoneal weight, and 77.7% mesenteric weight (Fig. 14a and 14b).
[Para117] The average adipocyte size in perigonadal adipose tissue was evaluated by HE
staining, and the results revealed that adipocytes were enlarged in HFD-fed mice compared to those of ND mice. The increased adipocyte size was significantly reduced in the garcinol-treated mice (Fig. 15). Garcinol (0.5%) could prevent the enlargement of adipocytes induced by HFD, which made adipocyte distribution at a size of 2000 1.1m2. Importantly, adipocyte size can be prevented or inhibited by garcinol in a dose-dependent fashion (Table 6).
[Para1181 Table 6: Effect of garcinol on adipocyte size Adipocye size area ND HFD HFD +0.1% HFD +0.5%
(pm) aarcinol garcinol 2000 14.4 2.6ab 9.84 4.5b 8.38 5.3b 19.2 3.9a 15000 0.92 0.4 b 6.06 2.5 a 4.03 1.0a 1.36 0.9b >350000 0.00 0.0 c 5.10 0.9 a 3.32 1.5b The significance of the difference among the four groups was analyzed by one-way ANOVA and Duncan's multiple-range tests. The values with different letters are significantly different (p<0.05) between each group.
[Para1191 Lipid Profile: The plasma lipid profiles were also analyzed and are presented in Table 7.
[Para1201 Table 7: Lipid profile in mice administered with garcinol Ni) HFD HFD + HFD +
0.1% Gar 0.5% Gar GPT (U/L) 27.2 7.04ab 32.1 6.42a 20.5 7.26b 27.6 3.72ab T-CHO (mg/dL) 69.6 7.31 d 200.3 11.30a 179.7 11.85b 137.9 11.78' TO (mg/dL) 83.7 14.56a 85.2 13.09a 69.6 19.90ab 55.6 4.95b LDL (mg/dL) 2.4 0.41d 40.0 2.89a 33.3 0.72b 24.9 4.47' HDL (mg/dL) 57.8 6.01' 155.6 5.97a 152.6 9.73a 118.7 13.22b LDL/HDL 0.04 0.01` 0.25 0.01a 0.22 0.02b 0.21 0.03b Data are presented as the mean SE. The significance of the differences among the four groups was analyzed by one-way ANOVA and Duncan's multiple-range tests. Values not sharing the same superscript letters in the same row are significantly different among groups. p< 0.05; a, b, c, and d are significantly different between each group.
[Para121] Mice administered with garcinol at 0.1% and 0.5% had significantly diminished serum levels of both TC and TG. With respect to LDL and HDL, garcinol (0.1 and 0.5%) could reduce LDL levels, induced by HFD, in a dose-dependent manner. As the TC
decrease was brought about by HFD, the HFD group increases not only the LDL levels, but also HDL levels.
Hence, we used the LDL/HDL ratio to express this change. High and low dosages of garcinol can significantly diminish the LDL/HDL ratio compared to the HFD group.
[Para1221 Garcinol reversed HFD-induced gut dysbiosis [Para1231 The overall composition of the bacterial community in the different groups was assessed by analyzing the degree of bacterial taxonomic similarity between metagenomic samples at the genus level. Bacterial communities were clustered using PCA, which distinguished microbial communities based on HFD diet/ garcinol treatment. The gut microbiota of obese humans and HFD-fed mice is characterized by an increased Firmicutes-to-Bacteroidetes ratio (F/B ratio) (Brun, P., Castagliuolo, I., Di, L., V, Buda, A. et aL, Increased intestinal permeability in obese mice: new evidence in the pathogenesis of nonalcoholic steatohepatitis.
Am.JPhysiol GastrointesLLiver Physiol 2007, 292, G518-G525). The results indicated that the phylum level of HFD group has the higher F/B ratio compared with ND group (Fig. 16a).
Interestingly, the Garcinol treatment reduced the F/B ratio by highly elevating Bacteroides communities. In addition, garcinol treatment made the Verrucomicrobia communities rise in number (Fig. 16b). PCA of UniFrac-based pair wise comparisons of community structures disclosed a distribution of the microbial community among the four groups of mice. The main finding of PCA was that different diets promoted the development of various gut microbial communities. HFD-fed mice formed a cluster that was distinct from ND group mice, and the HFD-fed mice were also distinct from garcinol treatment mice (Fig 17a, b and c). However, high doses of garcinol (0.5%) treated mice's microbial communities were closely clustered to that of ND mice, this indicates that garcinol has a marked effect on gut microbial community composition and also reversed HFD-induced gut dysbiosis.
[Para1241 Effects of garcinol administration on the composition of gut microbial communities [Para1251 To further investigate whether the changes in the gut microbiota were induced by garcinol supplementation, we next determined the genus level of gut microbiota and used a heatmap to express the abundance of 50 OTUs significantly altered by garcinol in HFD-fed mice (Fig. 18). The results demonstrated that HFD-fed mice increased Blautia communities, which dramatically decreased in both high- and low-dose garcinol treatment groups.
The studies pointed that Blautia spp. and Enterobacter spp. were related to a HFD causing obesity in a mouse model (Becker, N., Kunath, J., Loh, G., and Blaut, M. Human intestinal microbiota:
characterization of a simplified and stable gnotobiotic rat model. Gut Microbes. 2011, 2, 25-33;
Fei, N. and Zhao, L. An opportunistic pathogen isolated from the gut of an obese human causes obesity in germfree mice. ISME.J 2013, 7, 880-884). Interestingly, the Parabacteroides, Bacteroides, and Akkertnansia genus also dramatically rose in number in the garcinol-fed mice.
Parabacteroides and Bacteroides belong to the Ba.cteroidetes phylum, and Akkertnansia belong to the Vernicomicrobia phylum; this explains why the F/B ratio behaved as it did following induction by garcinol treatment. In the heatmap, we observed that Anaerobranca zavarzinii, Blautia coccoides, and Butyrivibrio proteoclasticus communities rose in number after HFD
feeding, however garcinol administration not only lower those bacteria, but also Oscillospira eae, Mucispirillum schaedleri, Ancterotruncus collhotninis, and Lachnospirct pectinoschim. In addition, garcinol increased the numbers of Akkertnansia Bacteroides stercorirosoris, and Bacteroides xylanisolvens, which was diminished in the ND
and HFD
groups.
[Para126] Anaerobranca zavarzinii, Blautia coccoides, and Butyrivibrio proteoclasticus belong to the Firmicutes phylum; Anaerobranca zavarzinii is positively correlated with IBD patients, and Blautia coccoides was increased in HFD-induced mice model. Butyrivibrio proteoclasticus is extremely sensitive to the toxic effects of unsaturated fatty acids and associated with obesity.
On the other hand, Bacteroides stercorirosoris and Bacteroides xylanisolvens belong to the Bacteroidetes phylum, and Akk:ertnansia niuciniphila to the Verrucomicrobia phylum. Andoh et al. (Andoh, A., Nishida, A., Takahashi, K., Inatomi, 0. et al., Comparison of the gut microbial community between obese and lean peoples using 16S gene sequencing in a Japanese population.
Clin.Biochem.Nutr 2016, 59, 65-70) performed 16S rRNA sequence analysis of the gut microbiota profiles of obese and lean Japanese populations, and they found that Bacteroides stercorirosoris exists in lean Japanese people. Liu et al. (Liu, R., Hong, J., Xu, X., Feng, Q. et al., Gut microbiome and serum metabolome alterations in obesity and after weight-loss intervention. Nat. Med 2017, 23, 859-868) performed a metagenome-wide association study and serum metabolomics profiling in lean and obese, young, Chinese individuals.
They linked intestinal microbiota alterations with circulating amino acids and obesity, and indicated that Bacteroides xylanisolvens was significantly enriched in lean controls.
[Para127] Several studies have highlighted the effects of the mucin-degrading bacterium, Akkermansia inuciiiiphila, which is more abundant in the mucosa of healthy subjects than in that of diabetic patients or animals (Lion, A. P., Paziuk, M., Luevano, J. M., Jr., 1\ilachineni, S. et al., Conserved shifts in the gut microbiota due to gastric bypass reduce host weight and adiposity. Sci Transl.Med 2013, 5, 178ra41). Many studies have demonstrated the dietary effect of Akkermaiisia inticimphila and how it also inhibits obesity. Dietary supplementation of an HFD
with grape polyphenols resulted in dramatic changes in the gut microbial community structure, including a reduction in the F/B ratio and a bloom of Akkermansia muciniphila (Roopchand, D.
E., Carmody, R. N., Kuhn, P., Moskal, K. et al., Dietary Polyphenols Promote Growth of the Gut Bacterium Akkermansia muciniphila and Attenuate High-Fat Diet-Induced Metabolic Syndrome.
Diabetes 2015, 64, 2847-2858). All these studies support the suggestion that Akkertnatisia rmiciniphihi has a potential role as a probiotic with anti-obesity effects, therefore we suggest that garcinol exhibits the prebiotic role.
[Para1281 Garcinol treatment increased the number of Akkermansia spp. and affected AMPK signaling pathway by inducing endocannabinoid expression [Para129] We further investigated the molecular mechanisms by which garcinol exerts anti-obesity effects. The protein levels of AMPK, p-AMPK, PPARy, preadipocyte factor 1 (Pref-1), and SREBP-1 in RFD-fed C57BL/6 mice are shown in Fig. 19. HFD feeding resulted in decreased AMPK compared to that of the ND group, but it was increased by administration of low doses of garcinol (0.1%) in white adipose tissue. Interestingly, high dosages of garcinol (0.5%) did not elevate AMPK protein or p-AMPK protein levels. We estimated this might be associated with Akkermansia spp. Administration of A. muciniphila to HFD-induced obese mice for four weeks improved endocannabinoid content (Everard, A., Belzer, C., Geurts, L., Ouwerkerk, J. P. et al., Cross-talk between Akkerinansia ttniciniphila and intestinal epithelium controls diet-induced obesity. Proc.Natl.Acad.S'ci U.S A 2013, 110, 9066-9071) including 2-AG, 2-PG, and 2-0G. Within intestinal tissue, the increase of 2-AG reduces metabolic endotoxemia and systemic inflammation by increasing goblet cell and Treg populations.
However, in perigonadal adipose tissue, the increase of 2-AG also enhanced the storing capacity of adipose tissue by stimulating preadipocyte differentiation (via upregulation of adipocyte PPARy levels), and enhancing de novo fatty acid synthesis (via stimulation of lipoprotein lipase and upregulation of FAS levels and glucose uptake), diminishing fatty acid oxidation (via inhibition of AMPK), and enhancing triacylglycerol accumulation (via inhibition of lipolysis). 2-AG
is a phospholipid-derived lipid containing an arachidonic acid chain within its chemical structure. 2-AG is also an intermediate in triacylglycerol and phospholipid metabolism, so mice treated with HFD can readily supply the substrate for 2-AG production. Pref-1 is identified as an inhibitor of adipocyte differentiation that is highly expressed in preadipocytes and that disappears during differentiation. Garcinol treatment caused an increased protein level of Pref-1 in epididymal adipose tissue which suggests garcinol may function in the maintenance of the preadipose state in HFD-fed mice.
[Para130] Conclusion The results revealed that garcinol treatment brought about an unexpected change in the composition of the gut microbiota in mice receiving a HFD, which may affect the underlying molecular mechanisms. Moreover, these findings reinforce the concept that changes in the gut microbial community; with the goal of increasing the Akkermansia population, can prevent obesity induced by HFD.
[Para131] Example 3: Comparative evaluation of garcinol and composition containing garcinol, pterostilbene and anthocyanin for weight loss [Para1321 The present invention studied the anti-obesity effects of garcinol compared to a composition comprising garcinol, pterostilbene and anthocyanin ( garcinol blend (GB) in mammals. The study was conducted in vivo on 5 weeks old C57BLI6 male mice. A
total of 42 mice were involved in this study with 6 groups of 7 mice each. The groups were divided as in table 8.
[Para133] The high fat diet (HFD) groups were fed 45% high fat diet for 16 weeks for the induction of obesity and concurrently administered the test substance as indicated in the aforesaid table. The normal group was fed with normal diet for 16 weeks.
[Para1341 Table 8: Study Groups Group Diet Test 1 Normal Diet None 2 High Fat Diet (45%) None 3 High Fat Diet (45%) 0.1% GB
4 High Fat Diet (45%) 0.5% GB
High Fat Diet (45%) 0.1% Gar
[Para091] Table 5: Weights of kidney, spleen and pancreas in garcinol treated animals Kidney weight Spleen Weight Pancreas Weight Groups (g wet tissue) (g wet tissue) (g wet tissue) 0.42 0.02 0.19 0.01 0.15 0.01 II 0.553 0.06 0.26 0.03 0.24 0.02 III 0.49 0.03 0.25 0.02 0.23 0.01 IV 0.42 0.03 0.20 0.01 0.17 0.01 V 0.45 0.06 0.22 0.03 0.21 0.02 VI 0.42 0.04 0.23 0.02 0.22 0.02 [Para092] Gene expression: Reduction in the expression of genes related to adipogenesis was observed in fat pad of animals treated with Garcinol. Similar to Mouse 3T3-L1 cell lines, garcinol administration significantly reduced the expression of PPARy, AP2, FAS, RESISTIN
and LEPTIN in the fat pad regions (Fig. 8a and 8b). Similarly, garcinol administration effectively increased the expression of Brown fat specific genes in the mice fat pad regions (Fig.
9).
[Para093] Lipid profile: The high fat diet increased the levels of total cholesterol, LDL. VLDL
and triglycerides in the serum of study animals. High fat diet, co administered with garcinol, significantly reduced the total cholesterol and triglycerides (Fig. 10a), LDL
and VLDL (Fig.
10b) and increased the HDL levels (Fig. 10c) in the serum.
[Para0941 Conclusion:
[Para095] Garcinol treatment showed a dose dependent inhibition of adipogenesis in vitro and induced the conversion of white adipose tissue to brown or britelbeige thereby increasing fat utilisation and energy metabolism. The in vivo results indicated that Garcinol was effective in significantly reducing body weight and visceral fat accumulation at 10 mg/kg and reduced adipogenesis specific gene expression and increased brown adipose tissue specific genes in fat pad in mouse fat pads. Garcinol administration also resulted in the reduction of visceral fat and organ weights indicating that garcinol promotes lipolysis and energy metabolism. Over all, garcinol induces weight loss, reduces visceral fat and maintains health of key organs.
[Para0961 EXAMPLE 2: Anti-obesity effects of Garcinol ¨ Study done at National Taiwan University, Taipei, Taiwan [Para097] Methodology [Para098] Reagents and antibodies [Para0991 AMPK and p-AMPK (Thr172) antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA). The SREBP-1 antibody was procured from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The PPARy and Pref-1 antibodies were purchased from abeam (Cambridge, England). The mouse 13-actin monoclonal antibody was obtained from Sigma Chemical Co (St. Louis, MO, USA). The Bio-Rad protein assay dye reagent was purchased from Bio-Rad Laboratories (Munich, Germany). Xylene and hematoxylin and eosin (H&E) stain were acquired from Surgipath (Peterborough, UK). Cholesterol used as part of the animal diet was obtained from Acros Organics (Bridgewater, NJ, USA). Garcinol was procured from Sabinsa Corp. (East Windsor, NJ, USA). The purity of garcinol was determined by high-performance liquid chromatography (HPLC) to be higher than 99%.
[Para1001 Animal care and experimental design [Parana] Five-week-old male C57BLI6 mice were purchased from the BioLASCO
Experimental Animal Center (Taiwan Co., Ltd, Taipei, Taiwan) and housed in a controlled atmosphere (25 1 C at 50% relative humidity) with a 12-h light/dark cycle.
After one week of acclimation, animals were randomly distributed into normal diet (ND, 15%
energy as fat), HFD
(50% energy as fat), and HFD with 0.1% or 0.5% garcinol groups of eight mice in each group for 13 weeks. The experimental design is summarized in Fig. 11. The experimental diets were modified from the Purina 5001 diet (LabDiet, PIVII Nutrition International, St. Louis, MO, USA).
The animals had ad libitum access to food and water. Food cups were replenished with a fresh diet every day. All animal experimental protocols employed in this study were approved by the Institutional Animal Care and Use Committee of the National Taiwan University (IACUC, NTU). Upon termination of the study, the animals were sacrificed by CO, asphyxiation and dissected, and the weights of their whole bodies and selected tissues, including the liver, kidney, spleen, adipose tissues (perigonadal, retroperitoneal, and mesenteric fat) and serum were immediately collected, weighed, and photographed.
[Para1021 Histopathological examination [Para103] A portion of perigonadal fat and the median lobe of the liver were dissected and fixed in 10% buffered formalin, dehydrated with a sequence of ethanol solutions, and processed for embedding in paraffin. Sections of 3-5 1.1.m in thickness were cut, deparaffinized, rehydrated, stained with H&E, and subjected to photomicroscopic assessment. Adipocyte size was determined using Image J software (Rasband, W.S., ImageJ, U. S. National Institutes of Health, Bethesda, MD, USA).
[Para1041 Biochemical analysis [Para105] Blood samples were collected from the left ventricle under anesthesia. The samples were mixed in 10 [11_, of heparin sodium and centrifuged at 3500 rpm and 4 'V
for 10 min. The plasma was then stored at ¨80 C until use. Glutamic-pyruvic transaminase (GPT), total cholesterol (TC), TG, high-density lipoprotein (HDL), and low-density lipoprotein (LDL) levels were analyzed at the National Laboratory Animal Center, NLAC (Taipei, Taiwan) on a 7080 Biochemical Analyzer (Hitachi, Tokyo, Japan) according to the manufacturer's instructions.
[Para106] 16S rDNA gene sequencing and analysis [Para1071 Total DNA was extracted from fresh fecal samples. The purified DNA
was eluted using the innuSPEED Stool DNA kit (Analytik Jena AG, Jena, Germany) according to the manufacturer's protocol. The PCR primer sequences from Caporaso et al.,(Caporaso, J. G., Lauber, C. L., Walters, W. A., Berg-Lyons, D. et at., Global patterns of 16S
rRNA diversity at a depth of millions of sequences per sample. Proc.Natl.Acad.Sci U.S A 2011, 108 ,S'uppl I, 4516-4522) were used to amplify the 16S rRNA variable region, and the PCR
conditions were performed as mentioned in Tung el al., (Tung, Y. C., Lin, Y. H., Chen, H. J., Chou, S. C. et at., Piceatannol Exerts Anti-Obesity Effects in C57BL/6 Mice through Modulating Adipogenic Proteins and Gut Microbiota. Molecules. 2016, 21) and Chou et al., (Chou, Y.
C., Suh, J. H., Wang, Y., Pahwa, M. et at., Boswellia serrata resin extract alleviates azoxymethane (A0M)/dextran sodium sulfate (DSS)-induced colon tumorigenesis. Mol.Nutr Food Res. 2017, 61) Then, the amplicons were used to construct index-labeled libraries with the Illumina DNA
Library Preparation kit (Illumina, San Diego, CA, USA). The Illumina MiniSeq NGS System (Illumina) was employed to analyze more than 100,000 reads with paired-end sequencing (2 x 150 bp), and the metagenomics workflow classified organisms from the amplicon using a database of 16S rRNA data. The classification was based on the Greermenes database (https://greengenes.lbl.gov/). The output of the workflow was a classification of reads at several taxonomic levels: kingdom, phylum, class, order, family, genus, and species.
[Para108] Protein preparation and western blot [Para1091 Tissues were homogenized in ice-cold lysis buffer (10% glycerol, 1%
TritonX-100, 1 mi\it Na3VO4, 1 mM EGTA, 10 mi\it NaF, 1 mM Na4P207, 20 inIVI Tris buffer (pH7.9), 100 p.M
P-glycerophosphate, 137 mM NaC1, and 5 mM EDTA) containing 1 Protease Inhibitor Cocktail Tablet (Roche, Indianapolis, IN, USA) on ice for 1 h, followed by centrifugation at 17,500g for 30 min at 4 'C. The protein concentration was measured with the Bio-Rad protein assay (Bio-Rad Laboratories, Inc., Hercules, CA, USA).
[Para1101 Statistical analysis [Para111] Statistical evaluation of the significance of the differences between the groups of mice was assessed using the Student t-test. For experiments comparing multiple groups, the differences were analyzed by carrying out one-way analysis of variance (ANOVA) and Duncan's post-hoc test. Data are presented as the means E for the indicated number of independently performed experiments, and p values <0.05 were considered statistically significant. Principal component analysis (PCA) was conducted to visualize the differences between samples.
[Para1121 Results [Para1131 Body weight gain [Para114] The results indicated that that HFD feeding for 13 weeks led to significant increase in body and liver weight along with perigonadal, retroperitoneal, and mesenteric fat accumulation.
Diet supplemented with 0.1 and 0.5% aarcinol reduced body weight in a dose-dependent manner.
Co-treatment with high doses of garcinol (0.5%) and a HFD diet inhibited body weight gain, and there is no difference between HFD + 0.5% aarcinol and the ND group (Fig.12 and Fig.13).
[Para1151 Effect on White adipose tissue adipocyte size and liver homeostasis [Para116] Garcinol at concentrations of 0.5% dramatically decreased all three white adipose fat weights, compared to the HFD group, by 85.1% in terms of perigonadal weight, 92.4%
retroperitoneal weight, and 77.7% mesenteric weight (Fig. 14a and 14b).
[Para117] The average adipocyte size in perigonadal adipose tissue was evaluated by HE
staining, and the results revealed that adipocytes were enlarged in HFD-fed mice compared to those of ND mice. The increased adipocyte size was significantly reduced in the garcinol-treated mice (Fig. 15). Garcinol (0.5%) could prevent the enlargement of adipocytes induced by HFD, which made adipocyte distribution at a size of 2000 1.1m2. Importantly, adipocyte size can be prevented or inhibited by garcinol in a dose-dependent fashion (Table 6).
[Para1181 Table 6: Effect of garcinol on adipocyte size Adipocye size area ND HFD HFD +0.1% HFD +0.5%
(pm) aarcinol garcinol 2000 14.4 2.6ab 9.84 4.5b 8.38 5.3b 19.2 3.9a 15000 0.92 0.4 b 6.06 2.5 a 4.03 1.0a 1.36 0.9b >350000 0.00 0.0 c 5.10 0.9 a 3.32 1.5b The significance of the difference among the four groups was analyzed by one-way ANOVA and Duncan's multiple-range tests. The values with different letters are significantly different (p<0.05) between each group.
[Para1191 Lipid Profile: The plasma lipid profiles were also analyzed and are presented in Table 7.
[Para1201 Table 7: Lipid profile in mice administered with garcinol Ni) HFD HFD + HFD +
0.1% Gar 0.5% Gar GPT (U/L) 27.2 7.04ab 32.1 6.42a 20.5 7.26b 27.6 3.72ab T-CHO (mg/dL) 69.6 7.31 d 200.3 11.30a 179.7 11.85b 137.9 11.78' TO (mg/dL) 83.7 14.56a 85.2 13.09a 69.6 19.90ab 55.6 4.95b LDL (mg/dL) 2.4 0.41d 40.0 2.89a 33.3 0.72b 24.9 4.47' HDL (mg/dL) 57.8 6.01' 155.6 5.97a 152.6 9.73a 118.7 13.22b LDL/HDL 0.04 0.01` 0.25 0.01a 0.22 0.02b 0.21 0.03b Data are presented as the mean SE. The significance of the differences among the four groups was analyzed by one-way ANOVA and Duncan's multiple-range tests. Values not sharing the same superscript letters in the same row are significantly different among groups. p< 0.05; a, b, c, and d are significantly different between each group.
[Para121] Mice administered with garcinol at 0.1% and 0.5% had significantly diminished serum levels of both TC and TG. With respect to LDL and HDL, garcinol (0.1 and 0.5%) could reduce LDL levels, induced by HFD, in a dose-dependent manner. As the TC
decrease was brought about by HFD, the HFD group increases not only the LDL levels, but also HDL levels.
Hence, we used the LDL/HDL ratio to express this change. High and low dosages of garcinol can significantly diminish the LDL/HDL ratio compared to the HFD group.
[Para1221 Garcinol reversed HFD-induced gut dysbiosis [Para1231 The overall composition of the bacterial community in the different groups was assessed by analyzing the degree of bacterial taxonomic similarity between metagenomic samples at the genus level. Bacterial communities were clustered using PCA, which distinguished microbial communities based on HFD diet/ garcinol treatment. The gut microbiota of obese humans and HFD-fed mice is characterized by an increased Firmicutes-to-Bacteroidetes ratio (F/B ratio) (Brun, P., Castagliuolo, I., Di, L., V, Buda, A. et aL, Increased intestinal permeability in obese mice: new evidence in the pathogenesis of nonalcoholic steatohepatitis.
Am.JPhysiol GastrointesLLiver Physiol 2007, 292, G518-G525). The results indicated that the phylum level of HFD group has the higher F/B ratio compared with ND group (Fig. 16a).
Interestingly, the Garcinol treatment reduced the F/B ratio by highly elevating Bacteroides communities. In addition, garcinol treatment made the Verrucomicrobia communities rise in number (Fig. 16b). PCA of UniFrac-based pair wise comparisons of community structures disclosed a distribution of the microbial community among the four groups of mice. The main finding of PCA was that different diets promoted the development of various gut microbial communities. HFD-fed mice formed a cluster that was distinct from ND group mice, and the HFD-fed mice were also distinct from garcinol treatment mice (Fig 17a, b and c). However, high doses of garcinol (0.5%) treated mice's microbial communities were closely clustered to that of ND mice, this indicates that garcinol has a marked effect on gut microbial community composition and also reversed HFD-induced gut dysbiosis.
[Para1241 Effects of garcinol administration on the composition of gut microbial communities [Para1251 To further investigate whether the changes in the gut microbiota were induced by garcinol supplementation, we next determined the genus level of gut microbiota and used a heatmap to express the abundance of 50 OTUs significantly altered by garcinol in HFD-fed mice (Fig. 18). The results demonstrated that HFD-fed mice increased Blautia communities, which dramatically decreased in both high- and low-dose garcinol treatment groups.
The studies pointed that Blautia spp. and Enterobacter spp. were related to a HFD causing obesity in a mouse model (Becker, N., Kunath, J., Loh, G., and Blaut, M. Human intestinal microbiota:
characterization of a simplified and stable gnotobiotic rat model. Gut Microbes. 2011, 2, 25-33;
Fei, N. and Zhao, L. An opportunistic pathogen isolated from the gut of an obese human causes obesity in germfree mice. ISME.J 2013, 7, 880-884). Interestingly, the Parabacteroides, Bacteroides, and Akkertnansia genus also dramatically rose in number in the garcinol-fed mice.
Parabacteroides and Bacteroides belong to the Ba.cteroidetes phylum, and Akkertnansia belong to the Vernicomicrobia phylum; this explains why the F/B ratio behaved as it did following induction by garcinol treatment. In the heatmap, we observed that Anaerobranca zavarzinii, Blautia coccoides, and Butyrivibrio proteoclasticus communities rose in number after HFD
feeding, however garcinol administration not only lower those bacteria, but also Oscillospira eae, Mucispirillum schaedleri, Ancterotruncus collhotninis, and Lachnospirct pectinoschim. In addition, garcinol increased the numbers of Akkertnansia Bacteroides stercorirosoris, and Bacteroides xylanisolvens, which was diminished in the ND
and HFD
groups.
[Para126] Anaerobranca zavarzinii, Blautia coccoides, and Butyrivibrio proteoclasticus belong to the Firmicutes phylum; Anaerobranca zavarzinii is positively correlated with IBD patients, and Blautia coccoides was increased in HFD-induced mice model. Butyrivibrio proteoclasticus is extremely sensitive to the toxic effects of unsaturated fatty acids and associated with obesity.
On the other hand, Bacteroides stercorirosoris and Bacteroides xylanisolvens belong to the Bacteroidetes phylum, and Akk:ertnansia niuciniphila to the Verrucomicrobia phylum. Andoh et al. (Andoh, A., Nishida, A., Takahashi, K., Inatomi, 0. et al., Comparison of the gut microbial community between obese and lean peoples using 16S gene sequencing in a Japanese population.
Clin.Biochem.Nutr 2016, 59, 65-70) performed 16S rRNA sequence analysis of the gut microbiota profiles of obese and lean Japanese populations, and they found that Bacteroides stercorirosoris exists in lean Japanese people. Liu et al. (Liu, R., Hong, J., Xu, X., Feng, Q. et al., Gut microbiome and serum metabolome alterations in obesity and after weight-loss intervention. Nat. Med 2017, 23, 859-868) performed a metagenome-wide association study and serum metabolomics profiling in lean and obese, young, Chinese individuals.
They linked intestinal microbiota alterations with circulating amino acids and obesity, and indicated that Bacteroides xylanisolvens was significantly enriched in lean controls.
[Para127] Several studies have highlighted the effects of the mucin-degrading bacterium, Akkermansia inuciiiiphila, which is more abundant in the mucosa of healthy subjects than in that of diabetic patients or animals (Lion, A. P., Paziuk, M., Luevano, J. M., Jr., 1\ilachineni, S. et al., Conserved shifts in the gut microbiota due to gastric bypass reduce host weight and adiposity. Sci Transl.Med 2013, 5, 178ra41). Many studies have demonstrated the dietary effect of Akkermaiisia inticimphila and how it also inhibits obesity. Dietary supplementation of an HFD
with grape polyphenols resulted in dramatic changes in the gut microbial community structure, including a reduction in the F/B ratio and a bloom of Akkermansia muciniphila (Roopchand, D.
E., Carmody, R. N., Kuhn, P., Moskal, K. et al., Dietary Polyphenols Promote Growth of the Gut Bacterium Akkermansia muciniphila and Attenuate High-Fat Diet-Induced Metabolic Syndrome.
Diabetes 2015, 64, 2847-2858). All these studies support the suggestion that Akkertnatisia rmiciniphihi has a potential role as a probiotic with anti-obesity effects, therefore we suggest that garcinol exhibits the prebiotic role.
[Para1281 Garcinol treatment increased the number of Akkermansia spp. and affected AMPK signaling pathway by inducing endocannabinoid expression [Para129] We further investigated the molecular mechanisms by which garcinol exerts anti-obesity effects. The protein levels of AMPK, p-AMPK, PPARy, preadipocyte factor 1 (Pref-1), and SREBP-1 in RFD-fed C57BL/6 mice are shown in Fig. 19. HFD feeding resulted in decreased AMPK compared to that of the ND group, but it was increased by administration of low doses of garcinol (0.1%) in white adipose tissue. Interestingly, high dosages of garcinol (0.5%) did not elevate AMPK protein or p-AMPK protein levels. We estimated this might be associated with Akkermansia spp. Administration of A. muciniphila to HFD-induced obese mice for four weeks improved endocannabinoid content (Everard, A., Belzer, C., Geurts, L., Ouwerkerk, J. P. et al., Cross-talk between Akkerinansia ttniciniphila and intestinal epithelium controls diet-induced obesity. Proc.Natl.Acad.S'ci U.S A 2013, 110, 9066-9071) including 2-AG, 2-PG, and 2-0G. Within intestinal tissue, the increase of 2-AG reduces metabolic endotoxemia and systemic inflammation by increasing goblet cell and Treg populations.
However, in perigonadal adipose tissue, the increase of 2-AG also enhanced the storing capacity of adipose tissue by stimulating preadipocyte differentiation (via upregulation of adipocyte PPARy levels), and enhancing de novo fatty acid synthesis (via stimulation of lipoprotein lipase and upregulation of FAS levels and glucose uptake), diminishing fatty acid oxidation (via inhibition of AMPK), and enhancing triacylglycerol accumulation (via inhibition of lipolysis). 2-AG
is a phospholipid-derived lipid containing an arachidonic acid chain within its chemical structure. 2-AG is also an intermediate in triacylglycerol and phospholipid metabolism, so mice treated with HFD can readily supply the substrate for 2-AG production. Pref-1 is identified as an inhibitor of adipocyte differentiation that is highly expressed in preadipocytes and that disappears during differentiation. Garcinol treatment caused an increased protein level of Pref-1 in epididymal adipose tissue which suggests garcinol may function in the maintenance of the preadipose state in HFD-fed mice.
[Para130] Conclusion The results revealed that garcinol treatment brought about an unexpected change in the composition of the gut microbiota in mice receiving a HFD, which may affect the underlying molecular mechanisms. Moreover, these findings reinforce the concept that changes in the gut microbial community; with the goal of increasing the Akkermansia population, can prevent obesity induced by HFD.
[Para131] Example 3: Comparative evaluation of garcinol and composition containing garcinol, pterostilbene and anthocyanin for weight loss [Para1321 The present invention studied the anti-obesity effects of garcinol compared to a composition comprising garcinol, pterostilbene and anthocyanin ( garcinol blend (GB) in mammals. The study was conducted in vivo on 5 weeks old C57BLI6 male mice. A
total of 42 mice were involved in this study with 6 groups of 7 mice each. The groups were divided as in table 8.
[Para133] The high fat diet (HFD) groups were fed 45% high fat diet for 16 weeks for the induction of obesity and concurrently administered the test substance as indicated in the aforesaid table. The normal group was fed with normal diet for 16 weeks.
[Para1341 Table 8: Study Groups Group Diet Test 1 Normal Diet None 2 High Fat Diet (45%) None 3 High Fat Diet (45%) 0.1% GB
4 High Fat Diet (45%) 0.5% GB
High Fat Diet (45%) 0.1% Gar
6 High Fat Diet (45%) 0.5% Gar [Para135] Body weight was monitored weekly, and the average body weight of each group (n=7) was expressed as the means SE. The significance of difference among the six groups was analyzed by one way ANOVA and Duncan's multiple range tests. p <0.05, a, b, and c significantly different between each group.
[Para136] The results indicated that Mice fed with HFD + 0.5 % Gar groups showed the most significantly decreased body weight and prevented weight gain compared to the HFD fed group and HFD + GB group (Fig. 19a and 19b). Mice administered with HFD+ 0.5 % Gar showed the least weight gain compared to the other groups (Table 9) which is an unexpected finding and cannot be anticipated by a person skilled in the art.
[Para137] The effect of garcinol and garcinol blend on reducing the weight of perigonadal, retroperitoneal and mesenteric adipose tissues was also evaluated. The results indicated that 0.5% garcinol significantly reduced the weights of perigonadal, retroperitoneal and mesenteric adipose tissues compared to the garcinol blend (Fig. 20 a,b,c).
[Para1381 Table 9: Body weight of study animals administered with garcinol and garcinol blend ND HFD HFD+ HFD+ HFD+ HFD+
0.1%GB 0.5%GB 0.1% gar 0.5% gar Initial 21.5 1.1 21.6 + 1.1a 21.9 1.0 22.1 1.0a 2.1.5 0.7a 21.5 0.7a weight (g) Final 27.7 2.7c 38.1 3.0a 33.5 3.1b 34.0 3.3b 32.1 -1 2.6b 25.4 0.8b weight (g) Weight 6.1 1.8' 16.5 2.7a 11.6 2.8bb 10.5 2.1b 3.9 0.6b gain (g) The average body weight of each group (n=7) is expressed as the mean SE. The significance of difference among the six groups was analyzed by one way ANOVA and Duncan's multiple range tests. Value not sharing the same superscript letters in the same row are significantly different among group. p <0.05, a, b, and c significantly different between each group [Para1391 Conclusion [Para1401 Mice fed with HFD 0.5% garcinol showed significant reduction in weight compared to the garcinol blend. This is an unexpected finding and cannot be anticipated by a person skilled in the art.
[Para1411 From the abovementioned examples, it is evident that garcinol brings about inhibition of adipogenesis and promotes weight loss in a dose dependant manner compared to the garcinol blend containing pterostilbene and anthocyanin. Garcinol also modifies the gut microbiota and increases the viable colonies of beneficial microbe - Akkerinansia inuciniphila thereby maintain and improving general health and well being. The present invention confirms that garcinol is an effective anti-obesity molecule and can be effective administered as a stand alone or in combination with other weight loss ingredients for the management of obesity and related disorders.
[Para1421 While the invention has been described with reference to a preferred embodiment, it is to be clearly understood by those skilled in the art that the invention is not limited thereto.
Rather, the scope of the invention is to be interpreted only in conjunction with the appended claims.
[Para136] The results indicated that Mice fed with HFD + 0.5 % Gar groups showed the most significantly decreased body weight and prevented weight gain compared to the HFD fed group and HFD + GB group (Fig. 19a and 19b). Mice administered with HFD+ 0.5 % Gar showed the least weight gain compared to the other groups (Table 9) which is an unexpected finding and cannot be anticipated by a person skilled in the art.
[Para137] The effect of garcinol and garcinol blend on reducing the weight of perigonadal, retroperitoneal and mesenteric adipose tissues was also evaluated. The results indicated that 0.5% garcinol significantly reduced the weights of perigonadal, retroperitoneal and mesenteric adipose tissues compared to the garcinol blend (Fig. 20 a,b,c).
[Para1381 Table 9: Body weight of study animals administered with garcinol and garcinol blend ND HFD HFD+ HFD+ HFD+ HFD+
0.1%GB 0.5%GB 0.1% gar 0.5% gar Initial 21.5 1.1 21.6 + 1.1a 21.9 1.0 22.1 1.0a 2.1.5 0.7a 21.5 0.7a weight (g) Final 27.7 2.7c 38.1 3.0a 33.5 3.1b 34.0 3.3b 32.1 -1 2.6b 25.4 0.8b weight (g) Weight 6.1 1.8' 16.5 2.7a 11.6 2.8bb 10.5 2.1b 3.9 0.6b gain (g) The average body weight of each group (n=7) is expressed as the mean SE. The significance of difference among the six groups was analyzed by one way ANOVA and Duncan's multiple range tests. Value not sharing the same superscript letters in the same row are significantly different among group. p <0.05, a, b, and c significantly different between each group [Para1391 Conclusion [Para1401 Mice fed with HFD 0.5% garcinol showed significant reduction in weight compared to the garcinol blend. This is an unexpected finding and cannot be anticipated by a person skilled in the art.
[Para1411 From the abovementioned examples, it is evident that garcinol brings about inhibition of adipogenesis and promotes weight loss in a dose dependant manner compared to the garcinol blend containing pterostilbene and anthocyanin. Garcinol also modifies the gut microbiota and increases the viable colonies of beneficial microbe - Akkerinansia inuciniphila thereby maintain and improving general health and well being. The present invention confirms that garcinol is an effective anti-obesity molecule and can be effective administered as a stand alone or in combination with other weight loss ingredients for the management of obesity and related disorders.
[Para1421 While the invention has been described with reference to a preferred embodiment, it is to be clearly understood by those skilled in the art that the invention is not limited thereto.
Rather, the scope of the invention is to be interpreted only in conjunction with the appended claims.
Claims (17)
1. A method for therapeutic management of obesity in mammals, said method comprising steps of administering effective concentration of a composition containing garcinol to said mammals to bring about a) inhibition of adipogenesis b) decrease in body weight, and visceral fat in said mammals.
2. The method as in claim 1, wherein the inhibition of adipogenesis is brought about by down regulation of genes selected from the group consisting of PPAR.gamma., cEBP.alpha., FAS, AP2, resistin and leptin.
3. The method as in claim 1, wherein inhibition of adipogenesis is brought about by up regulation of genes selected from the group consisting of p-AMPK, AMPK and PREF-1.
4. The method as in claim 1, wherein the visceral fat is selected from the group consisting of mesenteric fat, peritoneal fat and perigonadal fat.
5. A method of achieving energy balance in mammalian adipose cellular systems, said method comprising step of administering composition containing garcinol in effective amounts targeted towards mammalian pre-adipocytes and/or adipocytes to achieve effects of (a) increased inhibition of adipogenesis and (b) increased expression of secretory factors that function individually or in combination to specifically recruit brown adipocytes or brown like (beige or brite) adipocytes, c) induce brown like phenotype (beige or brite adipocytes) in white alipocyte depots, to bring about the effect of fat utilization and energy balance in said mammals.
6. The method as in claim 5, wherein the secretory factors include mitochondrial UCP-1, PRDM16, PGC-1.alpha. and BMP7.
7. A method of modifying the gut microbiota in mammals, said method comprising step of administering effective amounts of a composition containing garcinol to said mammals to bring about change in the gut microbiota.
8. The method as in claim 7, wherein the gut microbiota is selected from the Phylum Deferribacteres, Proteobacteria, Bacteroidetes, Verrucomicrobia and Firmicutes.
9. The method as in claim 7, wherein the gut microbiota is selected from the genus Lactobacillus, Butyrivibrio, Clostridium, Anaerobranca, Dysgonomonas, Johnsonella, Ruminococcus, Bacteroides, Oscillospira, Parabacterroides, Akkermanisa, and Blautia.
10. The method as in claim 7, wherein the gut microbiota is selected from the group consisting of Parabacteroides goldsteinii, Bacteroides caccae, Johnsonella ignava, Blautia wexlerae, Dvsgonomonas wimpennyi, Blautia hansenni, Anaerobranca favarzinni, Oscillospira eae, Mucispirillus schaedleri, Blautia coccoides, Anaerotruncus colihominis, Butyrivibro proteoclasticus, Akkermansia muciniphila, Lachnospora pectinoschiza, Pedobacter kwangvangensis, Alkaliphilus crotonatoxidans, lactobacillus salivarius, Anaerivibria lipolyticus, Rhodothermus clarus, Bacteroides stercorirosoris, Ruminocococcus flavefaciens, Bacteroides xylanisolvens, Ruminococcus gnavus, Clostridium termitidis, Clostridium alkalicellulosi, Emticicia oligoraphica, Pseudobutyrivibro xylanivorans, Actinomyces naturae, Peptoniphilus coxii, and Dolichospermum curvum.
11. The method as in claim 7, wherein the modification of gut microbiota is effective in therapeutic management of diseases selected from the group consisting of obesity, cardiovascular complications, Inflammatory bowel disease, Crohn's disease, Celiac disease, metabolic syndrome, liver diseases and neurological disorders.
12. A method for increasing the viable counts of Akkermansia muciniphila in the gut of mammals, said method comprising steps of administering effective amounts of a composition containing garcinol to mammals to bring about an increase in the colonies of said bacteria.
13. The method as in claim 12, wherein the increase in the colony counts of Akkermansia muciniphila reduces body weight through the AMPK signaling pathway by causing endocannabinoid release.
14. A method of therapeutic management of hyperlipidemia in mammals, said method comprising step of administering an effective concentration of a composition containing garcinol to bring about the effects of (i) reducing the amount of total blood cholesterol levels; (ii) reducing the concentrations of low density lipoproteins (LDL) and very low density lipoproteins (VLDL); (iii) increasing the concentrations of high density lipoproteins (HDL) and (iv) reducing concentrations of serum triglycerides, in the blood of said mammals.
15. The method as in claim 14, the medical cause of hyperlipidemia is obesity.
16. A composition containing garcinol for use as a prebiotic agent.
17. The composition as in claim 16, wherein the composition is formulated with pharmaceutically/nutraceutically acceptable excipients, adjuvants, diluents or carriers and administered orally in the form of tablets, capsules, syrups, gummies, powders, suspensions, emulsions, chewables, candies and eatables.
Applications Claiming Priority (5)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US201762519949P | 2017-06-15 | 2017-06-15 | |
US62/519,949 | 2017-06-15 | ||
US201762523611P | 2017-06-22 | 2017-06-22 | |
US62/523,611 | 2017-06-22 | ||
PCT/US2018/037242 WO2018231923A1 (en) | 2017-06-15 | 2018-06-13 | Anti-obesity potential of garcinol |
Publications (1)
Publication Number | Publication Date |
---|---|
CA3066446A1 true CA3066446A1 (en) | 2018-12-20 |
Family
ID=64659462
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CA3066446A Pending CA3066446A1 (en) | 2017-06-15 | 2018-06-13 | Anti-obesity potential of garcinol |
Country Status (8)
Country | Link |
---|---|
EP (1) | EP3638226A4 (en) |
JP (3) | JP2020523373A (en) |
KR (1) | KR102289324B1 (en) |
AU (1) | AU2018284354A1 (en) |
BR (1) | BR112019026824A2 (en) |
CA (1) | CA3066446A1 (en) |
MX (1) | MX2019014841A (en) |
WO (1) | WO2018231923A1 (en) |
Families Citing this family (11)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
KR102136335B1 (en) * | 2020-03-24 | 2020-07-22 | (주)메디톡스 | Microorganism capable of improving liver function or inhibiting fat accumulation and use thereof |
KR102363094B1 (en) * | 2020-06-19 | 2022-02-16 | 한국식품연구원 | Predicting or Diagnosing Composition for Risk of Liver Diseases Using Human Intestinal Microbiome, Diagnosing Kit, Method For Providing Information, And Screening Method For Drugs For Preventing Or Treating Liver Diseases Using The Same |
KR102191487B1 (en) * | 2020-06-19 | 2020-12-15 | (주)메디톡스 | Microorganism capable of improving liver function or inhibiting fat accumulation and use thereof |
KR102363092B1 (en) * | 2020-06-19 | 2022-02-16 | 한국식품연구원 | Predicting or Diagnosing Composition for Risk of Obesity Using Human Intestinal Microbiome, Diagnosing Kit, Method For Providing Information, And Screening Method For Drugs For Preventing Or Treating Obesity Using The Same |
KR102363098B1 (en) * | 2020-06-19 | 2022-02-16 | 한국식품연구원 | Predicting or Diagnosing Composition for Risk of Renal Diseases Using Human Intestinal Microbiome, Diagnosing Kit, Method For Providing Information, And Screening Method For Drugs For Preventing Or Treating Renal Diseases Using The Same |
KR102363088B1 (en) * | 2020-06-19 | 2022-02-16 | 한국식품연구원 | Predicting or Diagnosing Composition for Risk of Diabetic Disease Using Human Intestinal Microbiome, Diagnosing Kit, Method For Providing Information, And Screening Method For Drugs For Preventing Or Treating Diabetes Using The Same |
KR102331485B1 (en) | 2021-08-02 | 2021-12-01 | 주식회사 바이오뱅크힐링 | Blautia wexlerae strain, and vesicles from thereof and anti-inflammation and anti-bacteria uses of thereof |
CN114605242A (en) * | 2022-04-11 | 2022-06-10 | 澳门大学 | Preparation method of phloroglucinol compound, medicine, health-care product and food |
CN114847475B (en) * | 2022-07-05 | 2022-11-29 | 广东金骏康生物技术有限公司 | Application of fermented sophora japonica rice flour for promoting growth of AKK (alkyl ketene dimer) bacteria |
CN115637242A (en) * | 2022-10-20 | 2023-01-24 | 中南大学 | Microbial composite microbial inoculum, preparation method thereof and red mud in-situ biological dealkalization method |
CN116903760A (en) * | 2023-05-25 | 2023-10-20 | 浙江大学 | Auricularia auricula polysaccharide, preparation method thereof and application thereof in inhibiting obesity |
Family Cites Families (8)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2000044468A (en) * | 1998-07-29 | 2000-02-15 | Kikkoman Corp | Lipase inhibitor and antiobestic medicine or hyperlipidemia inhibitor |
US7063861B2 (en) * | 2000-08-17 | 2006-06-20 | Sabinsa Corporation | Bioavailable composition of natural and synthetic HCA |
AU2011352073B2 (en) | 2010-12-30 | 2015-12-24 | Sami Labs Limited | Hepatoprotectant activity of garcinol |
US8329743B2 (en) * | 2011-01-10 | 2012-12-11 | Sami Labs Limited | Compositions and its use in treating obesity or inducing weight loss |
CA3092318A1 (en) * | 2012-09-20 | 2014-03-27 | Prothera, Inc. | Probiotic compositions and methods for the treatment of obesity and obesity-related conditions |
US9872840B2 (en) * | 2013-10-03 | 2018-01-23 | Delhi Institute Of Pharmaceutical Sciences And Research | Effect of garcinol in delaying the progression of diabetic nephropathy |
US10300085B2 (en) * | 2014-03-24 | 2019-05-28 | Daicel Corporation | Nutritional composition |
CA2934156A1 (en) * | 2015-11-10 | 2017-05-10 | Muhammed Majeed | Use of forskolin in the treatment of obesity |
-
2018
- 2018-06-13 BR BR112019026824-3A patent/BR112019026824A2/en unknown
- 2018-06-13 AU AU2018284354A patent/AU2018284354A1/en active Pending
- 2018-06-13 MX MX2019014841A patent/MX2019014841A/en unknown
- 2018-06-13 CA CA3066446A patent/CA3066446A1/en active Pending
- 2018-06-13 EP EP18817304.1A patent/EP3638226A4/en active Pending
- 2018-06-13 WO PCT/US2018/037242 patent/WO2018231923A1/en unknown
- 2018-06-13 KR KR1020197038979A patent/KR102289324B1/en active IP Right Grant
- 2018-06-13 JP JP2019569248A patent/JP2020523373A/en active Pending
-
2021
- 2021-08-10 JP JP2021130804A patent/JP2021185156A/en active Pending
-
2022
- 2022-04-15 JP JP2022067326A patent/JP2022097521A/en active Pending
Also Published As
Publication number | Publication date |
---|---|
JP2020523373A (en) | 2020-08-06 |
BR112019026824A2 (en) | 2020-06-30 |
JP2022097521A (en) | 2022-06-30 |
KR20200012978A (en) | 2020-02-05 |
KR102289324B1 (en) | 2021-08-12 |
AU2018284354A1 (en) | 2020-01-16 |
WO2018231923A1 (en) | 2018-12-20 |
JP2021185156A (en) | 2021-12-09 |
EP3638226A4 (en) | 2021-04-07 |
MX2019014841A (en) | 2020-02-17 |
EP3638226A1 (en) | 2020-04-22 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CA3066446A1 (en) | Anti-obesity potential of garcinol | |
He et al. | Rhizoma Coptidis alkaloids alleviate hyperlipidemia in B6 mice by modulating gut microbiota and bile acid pathways | |
Lee et al. | Blueberry supplementation influences the gut microbiota, inflammation, and insulin resistance in high-fat-diet–fed rats | |
Liu et al. | Grape seed proanthocyanidin extract ameliorates inflammation and adiposity by modulating gut microbiota in high‐fat diet mice | |
JP6954563B2 (en) | Use of pasteurized Akkermansia to treat metabolic disorders | |
Wang et al. | Targeting the gut microbiota with resveratrol: a demonstration of novel evidence for the management of hepatic steatosis | |
Lee et al. | Garcinol reduces obesity in high‐fat‐diet‐fed mice by modulating gut microbiota composition | |
Tang et al. | A synbiotic consisting of Lactobacillus plantarum S58 and hull-less barley β-glucan ameliorates lipid accumulation in mice fed with a high-fat diet by activating AMPK signaling and modulating the gut microbiota | |
Karimi et al. | Single-species versus dual-species probiotic supplementation as an emerging therapeutic strategy for obesity | |
Wu et al. | Black garlic melanoidins prevent obesity, reduce serum LPS levels and modulate the gut microbiota composition in high-fat diet-induced obese C57BL/6J mice | |
US20200030388A1 (en) | Compositions for modifying gut microbiota | |
Ge et al. | Luteolin cooperated with metformin hydrochloride alleviates lipid metabolism disorders and optimizes intestinal flora compositions of high-fat diet mice | |
US20180360776A1 (en) | Anti-obesity potential of garcinol | |
Yan et al. | Oryzanol alleviates high fat and cholesterol diet-induced hypercholesterolemia associated with the modulation of the gut microbiota in hamsters | |
Sun et al. | Tea polyphenols protect mice from acute ethanol-Induced liver injury by modulating the gut microbiota and short-chain fatty acids | |
Albuquerque et al. | Saccharomyces boulardii Tht 500101 changes gut microbiota and ameliorates hyperglycaemia, dyslipidaemia, and liver inflammation in streptozotocin-diabetic mice | |
Kou et al. | Nobiletin activates thermogenesis of brown and white adipose tissue in high‐fat diet‐fed C57BL/6 mice by shaping the gut microbiota | |
Sheykhsaran et al. | Gut microbiota and obesity: an overview of microbiota to microbial-based therapies | |
Lu et al. | Prevention of high-fat diet-induced hypercholesterolemia by Lactobacillus reuteri Fn041 through promoting cholesterol and bile salt excretion and intestinal mucosal barrier functions | |
Keshavarz Azizi Raftar et al. | The anti-fibrotic effects of heat-killed Akkermansia muciniphila MucT on liver fibrosis markers and activation of hepatic stellate cells | |
Seo et al. | Effects of kefir lactic acid bacteria-derived postbiotic components on high fat diet-induced gut microbiota and obesity | |
Yu et al. | Casein-fed mice showed faster recovery from DSS-induced colitis than chicken-protein-fed mice | |
Yu et al. | Secoisolariciresinol diglucoside-derived metabolite, enterolactone, attenuates atopic dermatitis by suppressing Th2 immune response | |
Miao et al. | Long-term use of Lacticaseibacillus paracasei N1115 from early life alleviates high-fat-diet-induced obesity and dysmetabolism in mice | |
Zhang et al. | Taraxacum officinale-derived exosome-like nanovesicles modulate gut metabolites to prevent intermittent hypoxia-induced hypertension |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
EEER | Examination request |
Effective date: 20220421 |
|
EEER | Examination request |
Effective date: 20220421 |
|
EEER | Examination request |
Effective date: 20220421 |
|
EEER | Examination request |
Effective date: 20220421 |
|
EEER | Examination request |
Effective date: 20220421 |
|
EEER | Examination request |
Effective date: 20220421 |