US20030068388A1 - Compositions and methods for their preparation from lepidium - Google Patents
Compositions and methods for their preparation from lepidium Download PDFInfo
- Publication number
- US20030068388A1 US20030068388A1 US10/138,030 US13803002A US2003068388A1 US 20030068388 A1 US20030068388 A1 US 20030068388A1 US 13803002 A US13803002 A US 13803002A US 2003068388 A1 US2003068388 A1 US 2003068388A1
- Authority
- US
- United States
- Prior art keywords
- lepidium
- composition
- component
- benzyl
- column
- 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.)
- Granted
Links
- 241000801118 Lepidium Species 0.000 title abstract description 174
- 239000000203 mixture Substances 0.000 title abstract description 155
- 238000000034 method Methods 0.000 title abstract description 49
- 150000001875 compounds Chemical class 0.000 claims description 10
- 239000000126 substance Substances 0.000 claims description 6
- 239000000463 material Substances 0.000 abstract description 36
- 201000001880 Sexual dysfunction Diseases 0.000 abstract description 8
- 231100000872 sexual dysfunction Toxicity 0.000 abstract description 8
- 206010028980 Neoplasm Diseases 0.000 abstract description 4
- 201000011510 cancer Diseases 0.000 abstract description 4
- 238000002955 isolation Methods 0.000 abstract description 3
- MDKCFLQDBWCQCV-UHFFFAOYSA-N benzyl isothiocyanate Chemical compound S=C=NCC1=CC=CC=C1 MDKCFLQDBWCQCV-UHFFFAOYSA-N 0.000 description 37
- 150000004665 fatty acids Chemical class 0.000 description 35
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 34
- 239000003125 aqueous solvent Substances 0.000 description 33
- 238000010828 elution Methods 0.000 description 32
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 30
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 29
- 235000014113 dietary fatty acids Nutrition 0.000 description 29
- 239000000194 fatty acid Substances 0.000 description 29
- 229930195729 fatty acid Natural products 0.000 description 29
- 238000004366 reverse phase liquid chromatography Methods 0.000 description 25
- 229930182558 Sterol Natural products 0.000 description 22
- 150000003432 sterols Chemical class 0.000 description 22
- 235000003702 sterols Nutrition 0.000 description 22
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 21
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 21
- 150000004676 glycans Chemical class 0.000 description 21
- 229920001282 polysaccharide Polymers 0.000 description 20
- 239000005017 polysaccharide Substances 0.000 description 20
- QAADZYUXQLUXFX-UHFFFAOYSA-N N-phenylmethylthioformamide Natural products S=CNCC1=CC=CC=C1 QAADZYUXQLUXFX-UHFFFAOYSA-N 0.000 description 18
- 240000000759 Lepidium meyenii Species 0.000 description 17
- 235000000421 Lepidium meyenii Nutrition 0.000 description 17
- 241000196324 Embryophyta Species 0.000 description 16
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 16
- 235000001014 amino acid Nutrition 0.000 description 16
- 150000001413 amino acids Chemical class 0.000 description 16
- 230000005526 G1 to G0 transition Effects 0.000 description 15
- 241001465754 Metazoa Species 0.000 description 15
- 239000003480 eluent Substances 0.000 description 14
- 235000012902 lepidium meyenii Nutrition 0.000 description 14
- 238000011068 loading method Methods 0.000 description 13
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 description 13
- 239000000741 silica gel Substances 0.000 description 13
- 229910002027 silica gel Inorganic materials 0.000 description 13
- 241000700159 Rattus Species 0.000 description 11
- 238000000605 extraction Methods 0.000 description 11
- 239000012071 phase Substances 0.000 description 11
- 239000007787 solid Substances 0.000 description 10
- WGQKYBSKWIADBV-UHFFFAOYSA-N benzylamine Chemical class NCC1=CC=CC=C1 WGQKYBSKWIADBV-UHFFFAOYSA-N 0.000 description 9
- OCSIXVSXYFNTQS-UHFFFAOYSA-N n-benzyloctanamide Chemical compound CCCCCCCC(=O)NCC1=CC=CC=C1 OCSIXVSXYFNTQS-UHFFFAOYSA-N 0.000 description 9
- -1 N-substituted amides Chemical class 0.000 description 8
- 239000000546 pharmaceutical excipient Substances 0.000 description 8
- 239000000843 powder Substances 0.000 description 8
- 239000000243 solution Substances 0.000 description 8
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 7
- 241000699670 Mus sp. Species 0.000 description 7
- 125000001570 methylene group Chemical group [H]C([H])([*:1])[*:2] 0.000 description 7
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 6
- MLGPKWUKOQAAGI-UHFFFAOYSA-N N-benzylhexadecanamide Chemical compound CCCCCCCCCCCCCCCC(=O)NCC1=CC=CC=C1 MLGPKWUKOQAAGI-UHFFFAOYSA-N 0.000 description 6
- 150000001408 amides Chemical class 0.000 description 6
- 230000037396 body weight Effects 0.000 description 6
- 238000005100 correlation spectroscopy Methods 0.000 description 6
- 239000000284 extract Substances 0.000 description 6
- 150000002576 ketones Chemical class 0.000 description 6
- 239000002002 slurry Substances 0.000 description 6
- 238000004809 thin layer chromatography Methods 0.000 description 6
- 239000002253 acid Substances 0.000 description 5
- 230000008878 coupling Effects 0.000 description 5
- 238000010168 coupling process Methods 0.000 description 5
- 238000005859 coupling reaction Methods 0.000 description 5
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 5
- 238000005325 percolation Methods 0.000 description 5
- BDERNNFJNOPAEC-UHFFFAOYSA-N propan-1-ol Chemical compound CCCO BDERNNFJNOPAEC-UHFFFAOYSA-N 0.000 description 5
- 238000011282 treatment Methods 0.000 description 5
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 4
- 229920001429 chelating resin Polymers 0.000 description 4
- 239000012141 concentrate Substances 0.000 description 4
- 201000010099 disease Diseases 0.000 description 4
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 4
- 238000001704 evaporation Methods 0.000 description 4
- 150000002500 ions Chemical class 0.000 description 4
- 238000001819 mass spectrum Methods 0.000 description 4
- 239000003960 organic solvent Substances 0.000 description 4
- 230000000144 pharmacologic effect Effects 0.000 description 4
- 238000000926 separation method Methods 0.000 description 4
- 239000007858 starting material Substances 0.000 description 4
- ZWEHNKRNPOVVGH-UHFFFAOYSA-N 2-Butanone Chemical compound CCC(C)=O ZWEHNKRNPOVVGH-UHFFFAOYSA-N 0.000 description 3
- 201000009030 Carcinoma Diseases 0.000 description 3
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 description 3
- 238000010521 absorption reaction Methods 0.000 description 3
- 125000003277 amino group Chemical group 0.000 description 3
- 125000001797 benzyl group Chemical group [H]C1=C([H])C([H])=C(C([H])=C1[H])C([H])([H])* 0.000 description 3
- 125000002915 carbonyl group Chemical group [*:2]C([*:1])=O 0.000 description 3
- 239000001913 cellulose Substances 0.000 description 3
- 229920002678 cellulose Polymers 0.000 description 3
- 230000003750 conditioning effect Effects 0.000 description 3
- 235000019439 ethyl acetate Nutrition 0.000 description 3
- 230000008020 evaporation Effects 0.000 description 3
- 150000002193 fatty amides Chemical class 0.000 description 3
- 238000004108 freeze drying Methods 0.000 description 3
- 238000004128 high performance liquid chromatography Methods 0.000 description 3
- 201000001881 impotence Diseases 0.000 description 3
- 238000003305 oral gavage Methods 0.000 description 3
- 239000002904 solvent Substances 0.000 description 3
- 238000001551 total correlation spectroscopy Methods 0.000 description 3
- MYRTYDVEIRVNKP-UHFFFAOYSA-N 1,2-Divinylbenzene Chemical compound C=CC1=CC=CC=C1C=C MYRTYDVEIRVNKP-UHFFFAOYSA-N 0.000 description 2
- IMFQYAJJXFXVMM-UHFFFAOYSA-N 1-(isothiocyanatomethyl)-4-methoxybenzene Chemical compound COC1=CC=C(CN=C=S)C=C1 IMFQYAJJXFXVMM-UHFFFAOYSA-N 0.000 description 2
- 238000001644 13C nuclear magnetic resonance spectroscopy Methods 0.000 description 2
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 2
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 2
- AOJJSUZBOXZQNB-TZSSRYMLSA-N Doxorubicin Chemical compound O([C@H]1C[C@@](O)(CC=2C(O)=C3C(=O)C=4C=CC=C(C=4C(=O)C3=C(O)C=21)OC)C(=O)CO)[C@H]1C[C@H](N)[C@H](O)[C@H](C)O1 AOJJSUZBOXZQNB-TZSSRYMLSA-N 0.000 description 2
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 description 2
- 229910004373 HOAc Inorganic materials 0.000 description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
- KZSNJWFQEVHDMF-BYPYZUCNSA-N L-valine Chemical compound CC(C)[C@H](N)C(O)=O KZSNJWFQEVHDMF-BYPYZUCNSA-N 0.000 description 2
- 241000699666 Mus <mouse, genus> Species 0.000 description 2
- 206010049565 Muscle fatigue Diseases 0.000 description 2
- LRHPLDYGYMQRHN-UHFFFAOYSA-N N-Butanol Chemical compound CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 description 2
- 208000035823 Non-specific autoimmune cerebellar ataxia without characteristic antibodies Diseases 0.000 description 2
- ONIBWKKTOPOVIA-UHFFFAOYSA-N Proline Natural products OC(=O)C1CCCN1 ONIBWKKTOPOVIA-UHFFFAOYSA-N 0.000 description 2
- PPBRXRYQALVLMV-UHFFFAOYSA-N Styrene Chemical compound C=CC1=CC=CC=C1 PPBRXRYQALVLMV-UHFFFAOYSA-N 0.000 description 2
- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 description 2
- HEDRZPFGACZZDS-MICDWDOJSA-N Trichloro(2H)methane Chemical compound [2H]C(Cl)(Cl)Cl HEDRZPFGACZZDS-MICDWDOJSA-N 0.000 description 2
- KZSNJWFQEVHDMF-UHFFFAOYSA-N Valine Natural products CC(C)C(N)C(O)=O KZSNJWFQEVHDMF-UHFFFAOYSA-N 0.000 description 2
- PBCJIPOGFJYBJE-UHFFFAOYSA-N acetonitrile;hydrate Chemical compound O.CC#N PBCJIPOGFJYBJE-UHFFFAOYSA-N 0.000 description 2
- 150000007513 acids Chemical class 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 239000007900 aqueous suspension Substances 0.000 description 2
- LGJMUZUPVCAVPU-UHFFFAOYSA-N beta-Sitostanol Natural products C1CC2CC(O)CCC2(C)C2C1C1CCC(C(C)CCC(CC)C(C)C)C1(C)CC2 LGJMUZUPVCAVPU-UHFFFAOYSA-N 0.000 description 2
- 238000001460 carbon-13 nuclear magnetic resonance spectrum Methods 0.000 description 2
- BJDCWCLMFKKGEE-CMDXXVQNSA-N chembl252518 Chemical compound C([C@@](OO1)(C)O2)C[C@H]3[C@H](C)CC[C@@H]4[C@@]31[C@@H]2O[C@H](O)[C@@H]4C BJDCWCLMFKKGEE-CMDXXVQNSA-N 0.000 description 2
- 238000004587 chromatography analysis Methods 0.000 description 2
- 230000001143 conditioned effect Effects 0.000 description 2
- 238000004821 distillation Methods 0.000 description 2
- 238000000921 elemental analysis Methods 0.000 description 2
- 239000002024 ethyl acetate extract Substances 0.000 description 2
- 235000013305 food Nutrition 0.000 description 2
- 238000002290 gas chromatography-mass spectrometry Methods 0.000 description 2
- 235000013922 glutamic acid Nutrition 0.000 description 2
- 239000004220 glutamic acid Substances 0.000 description 2
- 238000003919 heteronuclear multiple bond coherence Methods 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- 208000000509 infertility Diseases 0.000 description 2
- 230000036512 infertility Effects 0.000 description 2
- 231100000535 infertility Toxicity 0.000 description 2
- 238000002329 infrared spectrum Methods 0.000 description 2
- 229960004592 isopropanol Drugs 0.000 description 2
- 229920005610 lignin Polymers 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- HQKMJHAJHXVSDF-UHFFFAOYSA-L magnesium stearate Chemical compound [Mg+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O HQKMJHAJHXVSDF-UHFFFAOYSA-L 0.000 description 2
- JGHZJRVDZXSNKQ-UHFFFAOYSA-N methyl octanoate Chemical compound CCCCCCCC(=O)OC JGHZJRVDZXSNKQ-UHFFFAOYSA-N 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 235000021313 oleic acid Nutrition 0.000 description 2
- 239000002245 particle Substances 0.000 description 2
- FDPIMTJIUBPUKL-UHFFFAOYSA-N pentan-3-one Chemical compound CCC(=O)CC FDPIMTJIUBPUKL-UHFFFAOYSA-N 0.000 description 2
- WEXRUCMBJFQVBZ-UHFFFAOYSA-N pentobarbital Chemical compound CCCC(C)C1(CC)C(=O)NC(=O)NC1=O WEXRUCMBJFQVBZ-UHFFFAOYSA-N 0.000 description 2
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 description 2
- 239000003495 polar organic solvent Substances 0.000 description 2
- 238000002953 preparative HPLC Methods 0.000 description 2
- 229920005989 resin Polymers 0.000 description 2
- 239000011347 resin Substances 0.000 description 2
- 241000894007 species Species 0.000 description 2
- 238000001228 spectrum Methods 0.000 description 2
- 238000002211 ultraviolet spectrum Methods 0.000 description 2
- 239000004474 valine Substances 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- OILXMJHPFNGGTO-UHFFFAOYSA-N (22E)-(24xi)-24-methylcholesta-5,22-dien-3beta-ol Natural products C1C=C2CC(O)CCC2(C)C2C1C1CCC(C(C)C=CC(C)C(C)C)C1(C)CC2 OILXMJHPFNGGTO-UHFFFAOYSA-N 0.000 description 1
- OYHQOLUKZRVURQ-NTGFUMLPSA-N (9Z,12Z)-9,10,12,13-tetratritiooctadeca-9,12-dienoic acid Chemical compound C(CCCCCCC\C(=C(/C\C(=C(/CCCCC)\[3H])\[3H])\[3H])\[3H])(=O)O OYHQOLUKZRVURQ-NTGFUMLPSA-N 0.000 description 1
- WRIDQFICGBMAFQ-UHFFFAOYSA-N (E)-8-Octadecenoic acid Natural products CCCCCCCCCC=CCCCCCCC(O)=O WRIDQFICGBMAFQ-UHFFFAOYSA-N 0.000 description 1
- DNIAPMSPPWPWGF-GSVOUGTGSA-N (R)-(-)-Propylene glycol Chemical compound C[C@@H](O)CO DNIAPMSPPWPWGF-GSVOUGTGSA-N 0.000 description 1
- CHRJZRDFSQHIFI-UHFFFAOYSA-N 1,2-bis(ethenyl)benzene;styrene Chemical compound C=CC1=CC=CC=C1.C=CC1=CC=CC=C1C=C CHRJZRDFSQHIFI-UHFFFAOYSA-N 0.000 description 1
- 238000005160 1H NMR spectroscopy Methods 0.000 description 1
- XILIYVSXLSWUAI-UHFFFAOYSA-N 2-(diethylamino)ethyl n'-phenylcarbamimidothioate;dihydrobromide Chemical compound Br.Br.CCN(CC)CCSC(N)=NC1=CC=CC=C1 XILIYVSXLSWUAI-UHFFFAOYSA-N 0.000 description 1
- LQJBNNIYVWPHFW-UHFFFAOYSA-N 20:1omega9c fatty acid Natural products CCCCCCCCCCC=CCCCCCCCC(O)=O LQJBNNIYVWPHFW-UHFFFAOYSA-N 0.000 description 1
- BMYNFMYTOJXKLE-UHFFFAOYSA-N 3-azaniumyl-2-hydroxypropanoate Chemical compound NCC(O)C(O)=O BMYNFMYTOJXKLE-UHFFFAOYSA-N 0.000 description 1
- OQMZNAMGEHIHNN-UHFFFAOYSA-N 7-Dehydrostigmasterol Natural products C1C(O)CCC2(C)C(CCC3(C(C(C)C=CC(CC)C(C)C)CCC33)C)C3=CC=C21 OQMZNAMGEHIHNN-UHFFFAOYSA-N 0.000 description 1
- QSBYPNXLFMSGKH-UHFFFAOYSA-N 9-Heptadecensaeure Natural products CCCCCCCC=CCCCCCCCC(O)=O QSBYPNXLFMSGKH-UHFFFAOYSA-N 0.000 description 1
- YHOWUFKFMVMIPR-OXPDKAOFSA-N CCC(=O)/C=C/C=C/C=C/C(=O)CCCCCCCC(=O)NCC1=CC=CC=C1 Chemical compound CCC(=O)/C=C/C=C/C=C/C(=O)CCCCCCCC(=O)NCC1=CC=CC=C1 YHOWUFKFMVMIPR-OXPDKAOFSA-N 0.000 description 1
- ZXVBFJHGCMSOAG-XVNWVHQASA-N CC[C@H](O)/C=C/C=C/C=C/C(=O)CCCCCCCC(=O)NCC1=CC=CC=C1 Chemical compound CC[C@H](O)/C=C/C=C/C=C/C(=O)CCCCCCCC(=O)NCC1=CC=CC=C1 ZXVBFJHGCMSOAG-XVNWVHQASA-N 0.000 description 1
- 244000003240 Caesalpinia gilliesii Species 0.000 description 1
- 235000014161 Caesalpinia gilliesii Nutrition 0.000 description 1
- SGNBVLSWZMBQTH-FGAXOLDCSA-N Campesterol Natural products O[C@@H]1CC=2[C@@](C)([C@@H]3[C@H]([C@H]4[C@@](C)([C@H]([C@H](CC[C@H](C(C)C)C)C)CC4)CC3)CC=2)CC1 SGNBVLSWZMBQTH-FGAXOLDCSA-N 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical group [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 241000714933 Chryseobacterium nakagawai Species 0.000 description 1
- BTEISVKTSQLKST-UHFFFAOYSA-N Haliclonasterol Natural products CC(C=CC(C)C(C)(C)C)C1CCC2C3=CC=C4CC(O)CCC4(C)C3CCC12C BTEISVKTSQLKST-UHFFFAOYSA-N 0.000 description 1
- 238000000134 MTT assay Methods 0.000 description 1
- 231100000002 MTT assay Toxicity 0.000 description 1
- 206010057672 Male sexual dysfunction Diseases 0.000 description 1
- 239000005913 Maltodextrin Substances 0.000 description 1
- 229920002774 Maltodextrin Polymers 0.000 description 1
- 238000005481 NMR spectroscopy Methods 0.000 description 1
- 238000011887 Necropsy Methods 0.000 description 1
- 229920001890 Novodur Polymers 0.000 description 1
- 239000005642 Oleic acid Substances 0.000 description 1
- ZQPPMHVWECSIRJ-UHFFFAOYSA-N Oleic acid Natural products CCCCCCCCC=CCCCCCCCC(O)=O ZQPPMHVWECSIRJ-UHFFFAOYSA-N 0.000 description 1
- 235000021314 Palmitic acid Nutrition 0.000 description 1
- 229920002472 Starch Polymers 0.000 description 1
- FEWJPZIEWOKRBE-UHFFFAOYSA-N Tartaric acid Natural products [H+].[H+].[O-]C(=O)C(O)C(O)C([O-])=O FEWJPZIEWOKRBE-UHFFFAOYSA-N 0.000 description 1
- HZYXFRGVBOPPNZ-UHFFFAOYSA-N UNPD88870 Natural products C1C=C2CC(O)CCC2(C)C2C1C1CCC(C(C)=CCC(CC)C(C)C)C1(C)CC2 HZYXFRGVBOPPNZ-UHFFFAOYSA-N 0.000 description 1
- 150000001242 acetic acid derivatives Chemical class 0.000 description 1
- NIXOWILDQLNWCW-UHFFFAOYSA-N acrylic acid group Chemical group C(C=C)(=O)O NIXOWILDQLNWCW-UHFFFAOYSA-N 0.000 description 1
- 125000002015 acyclic group Chemical group 0.000 description 1
- 229940009456 adriamycin Drugs 0.000 description 1
- 239000003463 adsorbent Substances 0.000 description 1
- 150000001298 alcohols Chemical class 0.000 description 1
- 125000001931 aliphatic group Chemical group 0.000 description 1
- 229930013930 alkaloid Natural products 0.000 description 1
- 150000003797 alkaloid derivatives Chemical class 0.000 description 1
- 150000001336 alkenes Chemical class 0.000 description 1
- 125000005907 alkyl ester group Chemical group 0.000 description 1
- 125000000217 alkyl group Chemical group 0.000 description 1
- DTOSIQBPPRVQHS-PDBXOOCHSA-N alpha-linolenic acid Chemical compound CC\C=C/C\C=C/C\C=C/CCCCCCCC(O)=O DTOSIQBPPRVQHS-PDBXOOCHSA-N 0.000 description 1
- 235000020661 alpha-linolenic acid Nutrition 0.000 description 1
- 125000003368 amide group Chemical group 0.000 description 1
- 230000003444 anaesthetic effect Effects 0.000 description 1
- 230000003466 anti-cipated effect Effects 0.000 description 1
- 239000008346 aqueous phase Substances 0.000 description 1
- 150000004945 aromatic hydrocarbons Chemical class 0.000 description 1
- 125000003118 aryl group Chemical group 0.000 description 1
- FCPVYOBCFFNJFS-LQDWTQKMSA-M benzylpenicillin sodium Chemical compound [Na+].N([C@H]1[C@H]2SC([C@@H](N2C1=O)C([O-])=O)(C)C)C(=O)CC1=CC=CC=C1 FCPVYOBCFFNJFS-LQDWTQKMSA-M 0.000 description 1
- 229940076810 beta sitosterol Drugs 0.000 description 1
- NJKOMDUNNDKEAI-UHFFFAOYSA-N beta-sitosterol Natural products CCC(CCC(C)C1CCC2(C)C3CC=C4CC(O)CCC4C3CCC12C)C(C)C NJKOMDUNNDKEAI-UHFFFAOYSA-N 0.000 description 1
- 238000009835 boiling Methods 0.000 description 1
- 210000000481 breast Anatomy 0.000 description 1
- XNNQFQFUQLJSQT-UHFFFAOYSA-N bromo(trichloro)methane Chemical compound ClC(Cl)(Cl)Br XNNQFQFUQLJSQT-UHFFFAOYSA-N 0.000 description 1
- 239000001506 calcium phosphate Substances 0.000 description 1
- 229910000389 calcium phosphate Inorganic materials 0.000 description 1
- 235000011010 calcium phosphates Nutrition 0.000 description 1
- SGNBVLSWZMBQTH-PODYLUTMSA-N campesterol 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)CC[C@@H](C)C(C)C)[C@@]1(C)CC2 SGNBVLSWZMBQTH-PODYLUTMSA-N 0.000 description 1
- 235000000431 campesterol Nutrition 0.000 description 1
- 239000002775 capsule Substances 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 235000014633 carbohydrates Nutrition 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 125000004432 carbon atom Chemical group C* 0.000 description 1
- 150000001732 carboxylic acid derivatives Chemical class 0.000 description 1
- 235000011472 cat’s claw Nutrition 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 150000001793 charged compounds Chemical class 0.000 description 1
- 229940112822 chewing gum Drugs 0.000 description 1
- 235000015218 chewing gum Nutrition 0.000 description 1
- 238000003776 cleavage reaction Methods 0.000 description 1
- 239000011280 coal tar Substances 0.000 description 1
- 210000001072 colon Anatomy 0.000 description 1
- 238000004440 column chromatography Methods 0.000 description 1
- 239000000470 constituent Substances 0.000 description 1
- 150000004292 cyclic ethers Chemical class 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 239000011928 denatured alcohol Substances 0.000 description 1
- VILAVOFMIJHSJA-UHFFFAOYSA-N dicarbon monoxide Chemical compound [C]=C=O VILAVOFMIJHSJA-UHFFFAOYSA-N 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 239000007884 disintegrant Substances 0.000 description 1
- 238000002518 distortionless enhancement with polarization transfer Methods 0.000 description 1
- 239000002552 dosage form Substances 0.000 description 1
- 239000003937 drug carrier Substances 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 206010016256 fatigue Diseases 0.000 description 1
- 230000035558 fertility Effects 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 239000000796 flavoring agent Substances 0.000 description 1
- 235000019634 flavors Nutrition 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 239000005350 fused silica glass Substances 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 238000001052 heteronuclear multiple bond coherence spectrum Methods 0.000 description 1
- 238000003929 heteronuclear multiple quantum coherence Methods 0.000 description 1
- 238000000990 heteronuclear single quantum coherence spectrum Methods 0.000 description 1
- ZSIAUFGUXNUGDI-UHFFFAOYSA-N hexan-1-ol Chemical compound CCCCCCO ZSIAUFGUXNUGDI-UHFFFAOYSA-N 0.000 description 1
- QNVRIHYSUZMSGM-UHFFFAOYSA-N hexan-2-ol Chemical compound CCCCC(C)O QNVRIHYSUZMSGM-UHFFFAOYSA-N 0.000 description 1
- KDCIHNCMPUBDKT-UHFFFAOYSA-N hexane;propan-2-one Chemical compound CC(C)=O.CCCCCC KDCIHNCMPUBDKT-UHFFFAOYSA-N 0.000 description 1
- 238000000265 homogenisation Methods 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- 229910052500 inorganic mineral Inorganic materials 0.000 description 1
- QXJSBBXBKPUZAA-UHFFFAOYSA-N isooleic acid Natural products CCCCCCCC=CCCCCCCCCC(O)=O QXJSBBXBKPUZAA-UHFFFAOYSA-N 0.000 description 1
- 150000002540 isothiocyanates Chemical class 0.000 description 1
- 125000000468 ketone group Chemical group 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 235000020778 linoleic acid Nutrition 0.000 description 1
- OYHQOLUKZRVURQ-HZJYTTRNSA-N linoleic acid group Chemical group C(CCCCCCC\C=C/C\C=C/CCCCC)(=O)O OYHQOLUKZRVURQ-HZJYTTRNSA-N 0.000 description 1
- 229960004488 linolenic acid Drugs 0.000 description 1
- KQQKGWQCNNTQJW-UHFFFAOYSA-N linolenic acid Natural products CC=CCCC=CCC=CCCCCCCCC(O)=O KQQKGWQCNNTQJW-UHFFFAOYSA-N 0.000 description 1
- 239000000314 lubricant Substances 0.000 description 1
- 210000004072 lung Anatomy 0.000 description 1
- 235000019359 magnesium stearate Nutrition 0.000 description 1
- 229940035034 maltodextrin Drugs 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- MYWUZJCMWCOHBA-VIFPVBQESA-N methamphetamine Chemical compound CN[C@@H](C)CC1=CC=CC=C1 MYWUZJCMWCOHBA-VIFPVBQESA-N 0.000 description 1
- 125000001434 methanylylidene group Chemical group [H]C#[*] 0.000 description 1
- 125000000956 methoxy group Chemical group [H]C([H])([H])O* 0.000 description 1
- 239000011707 mineral Substances 0.000 description 1
- 150000002826 nitrites Chemical class 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 229920001778 nylon Polymers 0.000 description 1
- 125000002801 octanoyl group Chemical group C(CCCCCCC)(=O)* 0.000 description 1
- ZQPPMHVWECSIRJ-KTKRTIGZSA-N oleic acid Chemical compound CCCCCCCC\C=C/CCCCCCCC(O)=O ZQPPMHVWECSIRJ-KTKRTIGZSA-N 0.000 description 1
- 150000002889 oleic acids Chemical class 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 239000006186 oral dosage form Substances 0.000 description 1
- 150000007524 organic acids Chemical class 0.000 description 1
- 235000005985 organic acids Nutrition 0.000 description 1
- 239000011368 organic material Substances 0.000 description 1
- 239000012074 organic phase Substances 0.000 description 1
- 238000012856 packing Methods 0.000 description 1
- 235000019629 palatability Nutrition 0.000 description 1
- 150000002943 palmitic acids Chemical class 0.000 description 1
- 210000003899 penis Anatomy 0.000 description 1
- 229960001412 pentobarbital Drugs 0.000 description 1
- 238000005191 phase separation Methods 0.000 description 1
- 229920001568 phenolic resin Polymers 0.000 description 1
- 239000005011 phenolic resin Substances 0.000 description 1
- 229920000058 polyacrylate Polymers 0.000 description 1
- 229920006122 polyamide resin Polymers 0.000 description 1
- 229920001225 polyester resin Polymers 0.000 description 1
- 239000004645 polyester resin Substances 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 150000004804 polysaccharides Polymers 0.000 description 1
- 229920002451 polyvinyl alcohol Polymers 0.000 description 1
- 235000019422 polyvinyl alcohol Nutrition 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 238000011321 prophylaxis Methods 0.000 description 1
- 210000002307 prostate Anatomy 0.000 description 1
- 230000007065 protein hydrolysis Effects 0.000 description 1
- 238000000425 proton nuclear magnetic resonance spectrum Methods 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 238000010992 reflux Methods 0.000 description 1
- 230000002441 reversible effect Effects 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 229920006395 saturated elastomer Polymers 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 239000000377 silicon dioxide Substances 0.000 description 1
- KZJWDPNRJALLNS-VJSFXXLFSA-N sitosterol 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)CC[C@@H](CC)C(C)C)[C@@]1(C)CC2 KZJWDPNRJALLNS-VJSFXXLFSA-N 0.000 description 1
- 229950005143 sitosterol Drugs 0.000 description 1
- 239000002689 soil Substances 0.000 description 1
- 239000008247 solid mixture Substances 0.000 description 1
- 238000007711 solidification Methods 0.000 description 1
- 230000008023 solidification Effects 0.000 description 1
- 238000001694 spray drying Methods 0.000 description 1
- 239000008107 starch Substances 0.000 description 1
- 235000019698 starch Nutrition 0.000 description 1
- HCXVJBMSMIARIN-PHZDYDNGSA-N stigmasterol 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)/C=C/[C@@H](CC)C(C)C)[C@@]1(C)CC2 HCXVJBMSMIARIN-PHZDYDNGSA-N 0.000 description 1
- 235000016831 stigmasterol Nutrition 0.000 description 1
- 229940032091 stigmasterol Drugs 0.000 description 1
- BFDNMXAIBMJLBB-UHFFFAOYSA-N stigmasterol Natural products CCC(C=CC(C)C1CCCC2C3CC=C4CC(O)CCC4(C)C3CCC12C)C(C)C BFDNMXAIBMJLBB-UHFFFAOYSA-N 0.000 description 1
- 125000000185 sucrose group Chemical group 0.000 description 1
- 239000002344 surface layer Substances 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 239000000454 talc Substances 0.000 description 1
- 229910052623 talc Inorganic materials 0.000 description 1
- 235000002906 tartaric acid Nutrition 0.000 description 1
- 239000011975 tartaric acid Substances 0.000 description 1
- YLQBMQCUIZJEEH-UHFFFAOYSA-N tetrahydrofuran Natural products C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- QORWJWZARLRLPR-UHFFFAOYSA-H tricalcium bis(phosphate) Chemical compound [Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O QORWJWZARLRLPR-UHFFFAOYSA-H 0.000 description 1
- 150000005671 trienes Chemical group 0.000 description 1
- 238000001291 vacuum drying Methods 0.000 description 1
- 229920002554 vinyl polymer Polymers 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/31—Brassicaceae or Cruciferae (Mustard family), e.g. broccoli, cabbage or kohlrabi
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P15/00—Drugs for genital or sexual disorders; Contraceptives
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P15/00—Drugs for genital or sexual disorders; Contraceptives
- A61P15/08—Drugs for genital or sexual disorders; Contraceptives for gonadal disorders or for enhancing fertility, e.g. inducers of ovulation or of spermatogenesis
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P15/00—Drugs for genital or sexual disorders; Contraceptives
- A61P15/10—Drugs for genital or sexual disorders; Contraceptives for impotence
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
Definitions
- the present invention relates to compositions containing particular components that can be obtained from a plant which can have pharmaceutical applications. More particularly, the plant genus is Lepidium.
- Lepidium meyenii commonly called maca or Peruvian ginseng, is a perennial plant having a fleshy, edible, tuberous root. Another species is Lepidium peruvianum . The maca root is consumed for food and is also consumed for its pharmacological properties; for example to enhance fertility. (See Leon, J., Economic Botany, 18:122-127(1964)) Maca has also been used to treat chronic fatigue. (Steinberg, P., Phil Steinberg's Cat's Claw News, Vol. 1, Issue 2, July/August (1995).
- the present invention relates to an isolated, Lepidium-derived composition that is essentially free of cellulose and lignin and that has about 40% of polysaccharides that can be isolated from Lepidium plant material (a Lepidium polysaccharide component).
- the composition can also contain an aqueous component and a component, designated a Lepidium amino acid component, having amino acids that can be isolated from Lepidium plant material and.
- the Lepidium amino acid component has about 70% or more proline, 5% or more glutamic acid, and 5% or more valine.
- the composition has about 0.3% benzyl isothiocyante and about 0.5% of a component, called a macamide component, having amides of fatty acids that can be isolated from Lepidium material.
- a component called a macamide component, having amides of fatty acids that can be isolated from Lepidium material.
- the composition has about 45% or more of a Lepidium polysaccharide component.
- the composition is combined with one or more pharmaceutically acceptable excipients.
- the present invention relates to a composition having about 0.3% or more benzyl isothiocyanate, about 0.3% or more of a macamide component, about 1% or more of fatty acids that can be isolated from Lepidium plant material (a Lepidium fatty acid component), and about 0.15% of sterols that can be isolated from Lepidium plant material (a Lepidium sterol component).
- the composition has between about 2% and about 5% benzyl isothiocyanate, about 0.2% and about 1% Lepidium sterol component, between about 10% and about 20% Lepidium fatty acid component, and about 3% to about 7% macamide component.
- the composition has between about 5% and about 9% benzyl isothiocyanate, between about 1% and about 3% of Lepidium sterol component, between about 20% and about 30% of a Lepidium fatty acid component, and about 10% or more of macamide component.
- benzyl isothiocyanate between about 1% and about 3% of Lepidium sterol component, between about 20% and about 30% of a Lepidium fatty acid component, and about 10% or more of macamide component.
- One or more of the foregoing compositions can be combined with a pharmaceutically acceptable excipient.
- the invention relates to novel fatty acid amides: N-benzyl octanamide having the chemical formula C 15 H 23 NO, N-benzyl-16(RS)-hydroxy-9-oxo-10E,12E,14E-octadecatrieneamide having chemical formula C 25 H 35 NO 3 , and N-benzyl-16(S)-hydroxy-9-oxo-10E,12E,14E-octadecatrieneamide having the chemical formula C 25 H 35 NO 3 .
- the present invention relates to a process of obtaining an isolated Lepidium-derived composition including the steps of:
- step b) concentrating the composition of step b) to isolate the composition.
- the aqueous solvent is water. More preferably, the aqueous solvent is a mixture of alcohol, preferably SDA, and still more preferably, a mixture of 75 vol-% SDA and 25 vol-% water.
- the process includes the further steps of:
- the invention process includes the further step of g), eluting the reverse phase chromatography column with a second elution volume of aqueous solvent to form a second effluent from which the composition can be isolated by, for example, the step of concentrating the second effluent.
- the second elution volume has two or more gradient volumes that are sequentially eluted and the resulting second effluent is fractionate collected as gradient fractions so that at least one gradient fraction is collected for each gradient volume eluted.
- the gradient fractions can be combined and concentrated to obtain an isolated Lepidium derived composition that has about 0.3% or more of benzyl isothiocyanate, about 0.15% or more Lepidium sterol component, about 1% or more Lepidium fatty acid component, and about 0.3% or more of macamide component.
- each of the two or more gradient volumes comprises a mixture of an alcohol and water and wherein the vol-% alcohol in the first gradient volume is about 20 vol-% or more and the vol-% alcohol in any subsequent gradient volume is equal to or greater than the vol-% alcohol in the immediately previously eluted gradient volume.
- the invention provides a process for producing the composition having between about 5% and about 9% of benzyl isothiocyanate, between about 1% and about 3% of Lepidium sterol component, between about 20% and about 30% of Lepidium fatty acid component, and d) about 10% or more of macamide component comprising the steps of:
- the preferred plant material in any embodiment is Lepidium meyenii.
- the present invention relates to a method of treating or preventing cancer in an animal, preferably a human, by administering a cancer treating or preventing effective amount of any of the hereinabove described isolated Lepidium-derived compositions.
- the present invention relates to a method for treating sexual dysfunction in an animal, preferably a human, by administering a sexual dysfunction treating or preventing amount of any of the hereinabove described isolated Lepidium-derived compositions to the animal.
- the animal is a female animal and the sexual dysfunction is infertility.
- the animal is a male animal and the sexual dysfunction is a sub-normal libido.
- the animal is a male animal and the sexual dysfunction is impotence.
- Alcohol refers to a lower aliphatic alcohol having from one to six carbon atoms.
- Aqueous component This term refers to that part or portion of a composition that is made-up of one or more aqueous solvents.
- Aqueous solvent means water or a single phase having an organic solvent that is miscible with water.
- miscible organic solvents include but are not limited to methanol, ethanol, isopropanol, n-propanol, acetone, and acetonitrile.
- Other miscible organic solvents are known to the skilled artesian.
- Benzyl isothiocyante includes benzyl isothiocynate and its methoxy derivatives.
- Column volume refers to the volume of the space defined by the inner surface of the chromatography column or chamber that surrounds the stationary phase or a reverse phase chromatography column. Column volume is abbreviated herein as CV.
- Fractionate collecting When used in connection with an effluent or a gradient effluent, or a gradient fraction, the term fractionate collecting denotes that the effluent or gradient effluent is segregated into at least two portions or aliquots.
- Lepidium amino acid component This term refers to that part or portion of a composition that is made-up of amino acids that can be isolated from Lepidium plant material.
- Lepidium composition is a composition having at least one of a Lepidium amino acid component, a Lepidium fatty acid component, a Lepidium polysaccharide component, or a macamide component.
- Lepidium fatty acid component This term refers to that part or portion of a composition that is made-up of fatty acids that can be isolated from Lepidium plant material.
- Lepidium plant material refers to plant matter from any part of a plant of the genus Lepidium. Examples of Lepidium plant matter include, but are not limited to, matter from Lepidium meyenii and Lepidium peruvianum.
- Lepidium polysaccharide component This term denotes that part or portion of a composition that is made-up of polysaccharides that can be isolated from Lepidium plant material.
- Lepidium sterol component As used herein, the term refers to that part or portion of a composition that is made-up of sterols that can be isolated from Lepidium plant material.
- Macamide Means amides and N-substituted amides of fatty acids that can be isolated from Lepidium plant material.
- Percent (%) Unless otherwise limited or modified, percents and percentages described herein are on a weight basis.
- the chemical composition of plant material from a particular plant species varies with, for example, the conditions under which the plant is grown (for example soil and climate).
- a particular compound or mixture of compounds can exhibit pharmacological efficacy over a readily ascertainable range of composition and dosage. Therefore, it is understood that the percentages recited throughout are meant to include such variations outside the stated percentage or percentage ranges as would be anticipated by the skilled artesian.
- Substantially Free of cellulose Means having 5% or less of cellulose and lignin combined.
- SDA Means special denatured alcohol, typically a mixture of 97% to 95% ethanol with 3% to 5% of methanol or coal tar.
- First Lepidium compositions of the invention can be obtained from an extraction composition in a first process.
- Starting material for a first process is Lepidium plant material, preferably Lepidium meyenii .
- Lepidium plant material for example the root of Lepidium meyenii is reduced in size to pieces having nominal dimensions between about 0.1 mm and 30 mm.
- the pieces of Lepidium plant material are contacted with aqueous solvent.
- the contacting in this or any embodiment may be by any suitable means as are known in the art; for example, percolation, vat extraction, counter current extraction, and the like.
- the contacting is for a time from about 2 hr. to about 18 hr.
- the contacting is carried out at a temperature above the solidification temperature (or where applicable the phase separation temperature) but below the boiling point of the aqueous solvent. Typically, the contacting is conducted from 20° C. to 75° C., with 40° C. to 50° C. being preferred.
- the aqueous solvent which is an extraction composition containing a first Lepidium composition
- the concentration can be by any of the means as are known in the art such as evaporation, distillation, and lyophilization, to mention a few.
- First Lepidium compositions generally have 30% or more, preferably about 40% or more, of a Lepidium polysaccharide component and about 1% or more of a macamide component. Typically, 70% or more of the polysaccharide units of a polysaccharide of a Lepidium polysaccharide component are sucrose units.
- a macamide component includes N-benzyl octanamide (macamide A), N-benzyl-16-hydroxy-9-oxo-10E,12E,14E-octatrieneamides (macamide B), and N-benzyl-9,16-dioxo-10E,12E,14E -octadecatrieneamide (macamide C).
- First Lepidium compositions further contain about 8% or more of a Lepidium amino acid component.
- the Lepidium amino acid component of a first Lepidium composition typically has 70% or more proline, 5% or more glutamic acid, and 5% or more valine.
- First Lepidium compositions also generally contain up to 1% of benzyl isothiocyanate.
- First Lepidium compositions can also contain about 0.5% to about 1.5% Lepidium fatty acid component.
- First Lepidium compositions of the present invention are useful for their nutritional value and are useful for the treatment or prophylaxis of carcinomas.
- First Lepidium compositions are also useful for treating sexual dysfunction in particular sub-normal libido and impotence in males and infertility in women.
- the composition of the extraction composition and the composition of the first Lepidium composition obtained therefrom can be changed by changing the aqueous solvent.
- the aqueous solvent is an alcohol-water mixture having 75 vol-% SDA
- the first Lepidium composition generally contains about 0.8% or more benzyl isothiocyanate and a Lepidium fatty acid component of about 1% or more.
- the aqueous solvent is 90 vol-% or more water
- e first Lepidium composition has less than about 0.1% of benzyl isothiocyanate and less than about 2% Lepidium fatty acid component.
- a first Lepidium composition having a Lepidium polysaccharide component of about 45% or more, a Lepidium fatty acid component between about 1% and about 2%, and less than about 1% each Lepidium sterol component and benzyl isothiocyanate is obtained by a suitably adapted first process that includes a reverse phase liquid chromatography process adapted to elute a first effluent containing a first Lepidium composition.
- RPLC reverse phase liquid chromatography
- the column packing stationary phase, or adsorbent
- is non-polar typically having a dipole moment of about 3 or less.
- Silica gel that has been treated to provide it with a bonded surface layer that is paraffinic in nature is an example of a useful stationary phase for reverse phase chromatography.
- Silica gels having permanently bonded C 8 to C 18 alkyl groups are commercially available as a stationary phase.
- WP-Octadecyl from J.T. Baker Corp, Phillipsburg, N.J., 08865.
- Reverse phase liquid chromatography columns are eluted with eluents of decreasing polarity which causes the more polar compounds loaded on a column to elute first.
- Reverse phase liquid chromatography stationary phases of organic material are also known.
- Polymers of vinyl aromatic compounds, for example styrene, that are crosslinked with polyvinylic aromatic hydrocarbons, for example divinyl benzene, can be used as stationary phases for reverse phase liquid chromatography.
- These organic polymeric stationary phases are made by processes that yield small, extremely rigid, macroreticular particles.
- Crosslinked acrylic polymers are also useful as stationary phases for reverse phase liquid chromatography, as are polyvinyl alcohols (alkylated or non-alkylated).
- Suitable stationary organic phases for RPLC are commercially available.
- styrenic and acrylic stationary phases are available from the Rohin and Haas Company, Philadelphia, Pa., under the trade name Amberlite®.
- Styreneic stationary phases are also available under the trade name Amberchrom® from Tossohass, Montgomeryville, Pa.
- Polyamide resins e.g. nylons
- polyester resins e.g. polyester resins
- phenolic resins are also useful stationary phases for the reverse phase chromatography processes of the present invention.
- organic solvents are suitable mobile phases, or eluents, for reverse phase liquid chromatography.
- Lower alcohols such as methanol, ethanol, and propanol, as well as nitrites such as acetonitrile, are suitable as organic eluents.
- Lower aliphatic ketones such as acetone, methyl ethyl ketone, and diethyl ketone, as well as cyclic ethers such as tetrahydrofuran, can also be used.
- Dimethyl formamide, dimethyl sulfoxide, and alkyl esters of acetic acid such as ethyl acetate can also be used.
- aqueous solvents that are mixtures of water and an alcohol, for example, methanol, ethanol, n-propanol, iso-propanol, n-butanol, and n-and sec-hexanol, are particularly useful as mobile phases or eluents for the RPLC processes of the present invention, which in certain embodiments are carried out using an eluent of variable composition.
- an elution volume which is a volume of aqueous solvent applied to the column, can be a gradient eluent having two or more gradient volumes, the composition of which can be the same or different, or the compositon of the gradient eluent can be varied continuously during elution.
- the composition of the elution volume that is a gradient eluent can vary step-wise, linearly, sigmoidally, exponentially, logarithmically, parabolically, or hypyperbolically during elution.
- concentration of gradient eluents are determined by the concentration of polar organic solvent necessary to elute products from the stationary phase and by the requirement that the polar organic solvent be miscible to form a single phase at the required concentration.
- the initial alcohol concentration in the elution volume is 10 volume percent (10 vol-%) or less and is increased as separation and purification proceeds.
- the reverse phase liquid chromatography systems used to practice the present invention may be either preparative or analytical. Preparative columns have larger loading capacity and are typically larger in size.
- the separation is typically carried out at 20° C. to 30° C. However, a temperature up to about 45° C. can be used.
- the separation may be carried out at high pressure (500-200 psi) or moderate pressures (100-500 psi) or, preferably, at lower pressures (10-100 psi).
- the reverse phase liquid chromatography column can be conditioned by eluting the column with a conditioning volume of a conditioning liquid, preferably an aqueous solvent, more preferably water.
- the conditioning volume is preferably between about 1 and about 10 column volumes.
- the material to be treated is applied to the preferably conditioned reverse phase chromatography column as a solution, a slurry, or a loading concentrate obtained by evaporating an aqueous solvent, preferably alcohol, from an extraction composition containing the product.
- aqueous solvent preferably alcohol
- the product to be treated is solid, it may be mixed with a suitable solid carrier, for example treated or untreated silica gel, and the solid mixture placed on top of the solid support.
- Loading of the column is accomplished by eluting the solution, slurry, or loading concentrate through the column; or, when the product to be treated is admixed with silica gel, by eluting the column with a loading elution volume.
- elution of the solution, slurry, loading concentrate, or loading elution volume is followed by elution with a washing elution volume comprising an aqueous solvent having the same composition as the aqueous solvent of the solution, slurry, or loading concentrate used to load the column stationary phase.
- the washing elution volume when one is used, is preferably between about 1 and about 10 column volumes.
- Starting material for this adapted first process is a first Lepidium composition made by the previously described first process in which the aqueous solvent is an alcohol-water mixture having between about 65 vol-% and about 85 vol-%, preferably about 75 vol-% of alcohol, preferably SDA.
- the stationary phase of the RPLC column is a styrenic resin, preferably a crosslinked styrene-divinylbenzene resin such as Amberlite® XAD-16HP available from Rohm and Haas.
- the first Lepidium composition from a first process, combined with water (5-7 L per kg of first composition), is eluted through an RPLC column to apply the first composition to the column.
- the column is then eluted with an elution volume effective to elute a first effluent containing a first Lepidium composition, the solids component of which includes about 45% or more of a Lepidium polysaccharide component.
- the elution volume contains aqueous solvent that is preferably water or an alcohol-water mixture having at least about 90 vol-% water and the elution volume amounts generally to 4 to 7, preferably 6, column volumes.
- the effluent is collected and concentrated to yield a first Lepidium composition having a Lepidium polysaccharide component of about 45% or more.
- the first process can be farther adapted to produce a second effluent by including, after elution of a first elution volume, the step of eluting a second elution volume that includes an aqueous solvent.
- Second Lepidium composition is obtained by concentrating the second effluent.
- the concentrating can be by any suitable means as know in the art such as evaporation, distillation, lyophilization, and the like.
- the second elution volume is typically to 4 to 10 column volumes.
- the second effluent contains a second Lepidium composition having between about 0.3% and about 12%, preferably between about 2% and about 10%, more preferably between about 5% and about 10% of benzyl isothiocyanate; between about 0.3% and about 2.7%, preferably between about 1% and about 2.5% of a Lepidium sterol fraction; and between about 10% to about 65%, preferably between about 10% and about 25% of a Lepidium fatty acid component.
- the second elution volume has at least two gradient volumes, each of which contains an aqueous solvent.
- the identity or composition of the aqueous solvent in each of the two or more gradient volumes can be the same or it can be different. Preferably it is different.
- the second effluent resulting from the elution of the second elution volume can be fractionate collected into at least as many gradient fractions as there are gradient volumes eluted.
- the fractionate collected gradient fractions can be combined, or they can be maintained separately.
- the gradient fractions can be concentrated to obtain second Lepidium compositions.
- the second elution volume is an alcohol-water mixture having 70 vol-% and preferably 80 vol-% or more alcohol, preferably SDA.
- the second Lepidium composition obtained in this process contains between about 0.2% and about 10%, preferably between about 2% and about 5%, of benzyl isothiocyanate; between about 0.15% and about 3%, preferably between about 0.2% and about 1%, of a Lepidium sterol component; between about 1% and about 65%, preferably between about 10% and about 25% of a Lepidium fatty acid component; and between about 0.3% and about 0.5% of a macamide component, wherein the macamide component comprises about 30% or more macamide B and 20% or more macamide C.
- Second Lepidium compositions typically have less than about 5%, preferably less than about 1%, of either a Lepidium polysaccharide component or a Lepidium amino acid component.
- a second Lepidium composition can also be obtained from a second extraction composition from a second process.
- residual Lepidium plant material that remains after separating the first extraction composition is contacted in a second contacting step with an aqueous solvent, preferably a mixture of an alcohol and water having about 80 vol-% or more, preferably about 90 vol-% or more alcohol, preferably SDA, to produce a second extraction composition or liquor.
- an aqueous solvent preferably a mixture of an alcohol and water having about 80 vol-% or more, preferably about 90 vol-% or more alcohol, preferably SDA
- the process of contacting in the second contacting step can be the same as that in the first contacting step of a first process or it can be different.
- the liquor is separated from residual Lepidium plant material and concentrated to obtain a second Lepidium composition.
- the separating can be by any process known in the art; for example centrifugation, filtration, or decanting.
- the second Lepidium composition obtained by a second process R-A) has between about 7% and about 9% of benzyl isothiocyanate, a Lepidium sterol component of between about 1% and about 3%/o, a Lepidium fatty acid component between about 10% and about 20%, and a macamide component of between about 0.3% and about 0.5%.
- a Lepidium sterol component includes about 50% or more ⁇ -sitosterol and also contains stigmasterol and campesterol.
- a Lepidium fatty acid component contains between about 0.5% and about 1.2% 9,16-dioxo-10E,12E,14E-octadecatrieneoic acid between about 1.5% and about 3.5% of 16-hydroxy-9-oxo-10E,12E,14E-octadecatrieneoic acid between about 30% and about 35% linoleic acid, and between about 15% and about 22% linolenic acid.
- a Lepidium fatty acid component also contains oleic acid.
- the second composition also contains about 0.2% or more of a macamide component, wherein the macamide component comprises 1% or less macamide A, about 10% or more macamide B, and about 20% or more macamide C.
- the present invention also provides a third Lepidium composition that contains a mixture of a pharmaceutically acceptable excipient with a first Lepidium composition, a second Lepidium composition, or with both a first and second Lepidium composition.
- Pharmaceutically acceptable excipients are any materials that do not interfere with the pharmacological activity of the third composition or degrade the bodily functions of the animal to which it can be administered, but facilitate fabrication of dosage forms or actual administration of the composition; for example by improving palatability of oral dosage forms.
- Examples of pharmaceutically acceptable excipient include but are not limited to maltodextrin, calcium phosphate, and fused silica.
- Pharmaceutically acceptable excipients also include flavorants.
- Third compositions of the present invention can be made, for example, by combining about 25% to about 50% (dry weight) of a first or second Lepidium composition of the present invention and about 75% to about 25% (dry weight) of one or more pharmaceutically acceptable excipients, combining this mixture with water (5 liter per kg of solids) and homogenizing the mixture.
- a Silverson Model 14 RT-A homogenizer (Silverson Corporation, East Longmeadow, Mass.) is an example of an apparatus suitable for carrying-out the homogenization.
- the homogenized composition is then dried to obtain a third Lepidium composition.
- the drying may be carried-out by any means as are known in the art; for example spray drying, oven dying, rotary vacuum drying, or lyophilization.
- the present invention provides novel amides of fatty acids, namely; N-benzyl octanamide (also called macamide A or MA-3), racemic and enantiomerically pure N-benzyl-16-hydroxy-9-oxo-10e,12e,14e-octadecatrienamide (also called macamide B or MA-S-4), and N-benzyl 9,16-dioxo-10e,12e,14e-octadecatrienamide (also called macamide C or MA-9). All of these N-substituted amides are members of the class macamides, as that term is used herein.
- N-benzyl amides of the present invention which are useful in the prevention or treatment of carcinomas, can be synthetically prepared, or obtained from Lepidium plant material, preferably Lepidium meyenii , by chromatographic processes.
- the chromatographic process is particular useful for obtaining enantiomerically pure N-benzyl-16(S)-hydroxy-9-oxo-10e,12e, 14e-octadecatrienamide.
- N-benzyl amides of the present invention can be synthetically prepared by various methods (See Barstaw, L. E. et al., J. Org. Chem., 36, 1305,(1971)).
- N-benzyl amides of the present invention can be made by refluxing the corresponding carboxylic acid and benzyl amine with triphenylphosphene and bromotrichloromethane.
- the N-benzyl amides of the present invention can be isolated from Lepidium plant material by c chromatographic process.
- Starting material for isolation of the N-benzyl amides of the present invention by chromatographic processes is a dry powder loading composition formed by mixing with silica gel (60-100 mesh) the residue from rotary vacuum concentration of an ethyl acetate extract of an aqueous suspension of a first Lepidium composition from a first process in which the aqueous solvent contains a mixture of about 75% SDA and about 25% water.
- the dry powder loading composition is applied to a chromatography column that is the eluted with a series of gradient volumes.
- the resulting gradient eluents are fractionate collected and compared by thin layer chromatography (TLC). Fractionate collected gradient eluents having similar TLC patterns are combined and combined fractions are further treated by column chromatography.
- TLC thin layer chromatography
- the methods of preventing or treating carcinomas, libido-related male sexual dysfunction, male impotence, and muscle fatigue comprise administering or dosing an effective amount of a composition, which can be a third Lepidium composition, that contains a first or second Lepidium composition, or both.
- a composition which can be a third Lepidium composition, that contains a first or second Lepidium composition, or both.
- effective amount will be recognized by clinicians but includes an amount effective to either (1) reduce the symptoms of the disease or condition sought to be treated or prevented (i.e. cancer, sexual dysfunction), (2) induce a pharmacological change relevant to treating or preventing the disease sought to be treated or prevented, or (3) prevent the occurrence of the disease or condition.
- the Lepidium compositions used in the method of the present invention can be administered by any route.
- Compositions of the present invention are administered alone, or are combined with a pharmaceutically-acceptable carrier or excipient according to standard pharmaceutical procedures.
- Preferably, Lepidium compositions are administered orally as a third Lepidium composition.
- the compositions of the present invention are used in the form of tablets, capsules, chewing gum, and the like. In the case of tablets, various disintegrants such as starch, and lubricating agents such as magnesium stearate and talc can be used.
- compositions of the invention can include pharmaceutically acceptable acid addition salts, particularly those obtained with mineral acids, for example hydrochloric or hydrobromic acid.
- mineral acids for example hydrochloric or hydrobromic acid.
- organic acids for example tartaric acid, can also be used.
- compositions are dosed at between 0.1 g and 10 g per kg of body weight per day (is this about right?).
- HPLC analysis of Maca product were performed on a Hewlett Packard Series 1100 HPLC using an phenomenex, Luna C-8 column.
- GC/MS analysis of Maca product was performed on an HP-5973 MSD using a Supelco SAC-5 capillary column.
- Roots of Lepidium meyenii (4.6 Kg) were cut to a nominal dimension of about 1 cm and contacted with an aqueous solvent (75 vol-% SDA and 25 vol-% water; 25 L per kg root) at 45° C. by percolation. Decoction was separated from the plant material and concentrated to yield a first composition (2.1 Kg), denoted NE, having a solids content of 77% (i.e. 1.6 Kg on a dry basis).
- the solids component included, based on the solids present; 0.89% benzyl isothiocyanate, 0.079% of a Lepidium sterol component, 1.46% of a Lepidium fatty acid component, 8.72% of a Lepidium amino acid component, and 41.9% of a Lepidium polysaccharide component.
- Roots of Lepidium meyenii (500 g) were reduced in size to a nominal dimension of 0.5 cm and contacted with 14 L water by percolation. The aqueous phase was separated from residual plant material and evaporated to dryness to obtain 20 g of a tacky product.
- the tacky product had a Lepidium sterol component of ⁇ 0.01%, a Lepidium fatty acid component of ⁇ 0.1%, a Lepidium amino acid component of 9%, and A Lepidium polysaccharide component of 44%.
- the residual plant material from example 2 was contacted with 15 L of 100% SDA by percolation to form a liquor.
- the liquor was separated and concentrated to yield 10 g of a second Lepidium composition having 7.8% benzyl isothiocyanate, a Lepidium sterol component of 1.8%, a Lepidium fatty acid component of 22%, and a macamide component of 12%. No Lepidium amino acid component was found in the composition.
- a first Lepidium composition (1.6 Kg) obtained according to the process of example 1 (i.e. NE), was slurried with water (8.8 L per kg of dry NE) for about 30 min. The slurry was applied to a RPLC column (15 cm. ⁇ 100 cm packed with Amberlite® XAD-16 (Rohm and Haas Co.) that had been preconditioned with 20 column volumes of water. The column was eluted with a first elution volume (6 column volumes) of 100 vol-% water.
- the resulting first effluent was collected and evaporated to dryness to yield 1.5g of a first Lepidium composition having 0.15% benzyl isothiocyanate, 0.07% of a Lepidium sterol component, 1.8% of a Lepidium fatty acid component, 10% of a Lepidium amino acid component, and 48% of a Lepidium polysaccharide component.
- the composition had less than 1% of a macamide component.
- the RPLC of example 4 was eluted with a second elution volume (6 column volumes) that was made-up of 100% SDA.
- the resulting effluent was collected and evaporated to dryness to yield 162 g of a second Lepidium composition having 4.1% benzyl isothiocyanate, 0.4% of a Lepidium sterol component, 12% of a Lepidium fatty acid component, 4.4% of a macamide component, and no Lepidium amino acid component or Lepidium polysaccharide component.
- a first Lepidium composition (1.6 Kg NE on a dry basis), obtained according to the method of example 1, was slurried with water (6.3 L per kg of first Lepidium composition) for about 30 min. The slurry was applied to a RPLC column (15 cm ⁇ 100 cm) packed with Amberlite® XAD-16 resin (Rohm and Haas Co) that had been preconditioned with about 20 column volumes of water. The column was eluted with a first elution volume of 4 column volumes of water. The column was then eluted with a second elution volume that was made-up of five gradient volumes, each having a volume equal to 4 column volumes and each made-up of a mixture of SDA and water.
- the gradient fractions had, respectively, 20 vol-%, 40 vol-%, 60 vol-%, 80 vol-%/o, and 100 vol-% SDA.
- the gradient fractions corresponding to each of the gradient volumes were fractionate collected and analyzed (HPLC and GC).
- the gradient fractions were free of both an Lepidium amino acid component and a Lepidium polysaccharide component.
- the gradient fractions contained other components as set-out below in Table 1. TABLE 1 Content of Various Lepidium Components in Gradient fractions of a RPLC Process. Fr.* Fr. Fr. Fr. Fr. Fr.
- Cells were planted in 96 well flat bottom plates with low evaporation lids. Three cell lines per plate were seeded in 0.2 ml medium per well. Each cell line was planted at the optimum concentration for its particular growth rate: HT-29 and A-549, 5000 c/ml; MCF-7, 15000 c/ml; A-498, 10,000 c/ml; PC-3, 15000 c/ml; and PACA-2, 10,000 c/ml. Products were tested at various dilutions (at least ten) to determine the ED 50 .
- mice 22 ⁇ 1.5 g were dosed for 21 days with Ig of either of two third Lepidium compositions.
- One group (group I) was dosed with a third Lepidium composition made with a first Lepidium composition obtained according to example 1.
- a second group group (II) was dosed with a third Lepidium composition made with a second Lepidium composition obtained according to example 5.
- Each mouse was individually placed in water (25 ⁇ 2° C.) and observed. The elapsed time at which a mouse remained submerged. The results are collected in Table 4.
- TABLE 4 Muscle Fatigue Results Animal Duration of Swim Time In Number Seconds Group (n) (mean ⁇ SD) p Control Group 15 110.07 ⁇ 2.58 Group I 15 124.07 ⁇ 3.30 ⁇ 0.01 Group II 15 144.13 ⁇ 3.52 ⁇ 0.01
- mice 22 ⁇ 1.5 g; 15 per group were dosed by oral gavage for 21 days with 1 g/day of either of two third Lepidium compositions.
- Male mice in one group (group-I) were dosed with a third Lepidium composition made with a first Lepidium composition obtained according to example 1.
- Male mice in a second group (group II) were dosed with a third Lepidium composition made with a second Lepidium composition obtained according to example 5.
- a control group received no third Lepidium composition.
- Mice were ear-tagged or color coded for identification. On the 21 st day, 1 ⁇ 2 hour after dosing, each male was placed in a cage with two mice and observed under darkroom conditions.
- mice were observed for three hours and the number intromissions were recorded. Intromission was indicated by a characteristic rearward lunge by the male terminating coitus. Results are summarized in table 5 below. TABLE 5 Intromission Results. Number of Intromissions Group (mean ⁇ SD) p Control 16.33 ⁇ 1.78 Group I 46.67 ⁇ 2.39 ⁇ 0.01 Group II 67.01 ⁇ 2.55 ⁇ 0.01
- Testectomies were performed on a sufficient number of male rats using pentobarbital, 45 mg/kg, as anesthetic. Rats were treated postoperatively for 3 days with sodium penicillin, 2,000 U/kg. Rats were earcoded and color-coded for identification and divided into 7 groups. Three groups of 10 rats each (Set A) were dosed by oral gavage for 21 days with a third Lepidium composition made with a first Lepidium composition obtained according to example 1. Rats in each of the three groups received a different dosage. A second set of three groups of 10 rats each (Set B) were dosed for 21 days with a third Lepidium composition made with a second Lepidium composition obtained according to example 5. Rats in each of the three groups received a different dosage. A control group of 10 rats received no third Lepidium composition.
- the N-benzyl amides of the present invention can be isolated from Lepidium plant material by chromatographic process.
- Starting material for isolation of the N-benzyl amides of the present invention by chromatographic processes is a dry powder loading composition formed by mixing with silica gel (60-100 mesh) the residue from rotary vacuum concentration of an ethyl acetate extract of an aqueous suspension of a first Lepidium composition from a first process in which the aqueous solvent contains a mixture of about 75% SDA and about 25% water.
- the dry powder loading composition is applied to a silica gel column (130-270 mesh) and the column is then eluted with, in sequence, five elution volumes, each about 8 column volumes that contain mixtures of n-hexane and acetone in the following ratios (vol-% n-hexane:vol-% acetone): 10:1, 5:1, 2:1, 1:1, and 0:1.
- the column effluent is divided into 18 fractions.
- the 25 th through 27 th liter of effluent make-up a sixth fraction.
- the 41 st through 491 ⁇ 2 th liter of effluent make-up a ninth fraction.
- the 63 rd through 70 th liter of effluent make-up a twelfth fraction.
- N-benzyl octanamide can be obtained by chromatographic treatment of the sixth fraction by treating the sixth fraction on a silica gel column using a n-hexane-acetone mobile phase (3:1). N-benzyl octanamide can be isolated from the effluent (all of it? What part?) by removing mobile phase from the effluent.
- N-benzyl octanamide can be represented, without regard to stereochemistry, as:
- N-benzyl-9,16 dioxo-10E,12E14E-octadecatriene amide (MA-9, macamide C) can be isolated from the ninth fraction by further chromatographic treatment of the fraction on a silica gel column using 30 elution volumes of hexane/acetone as mobile phase. Fractions 15-20 (i.e. effluent from elution volumes 15-20) were further treated by preparative IPLC (Dynamax-60A column) using acetonitrile-water gradient eluent (5:95 to 95:5 vol. ratio, acetonitrile:water) to obtain N-benzyl-9,16-dioxo-10E,12E,14E-octadecatrienamide.
- the structure of macamide C can be represented, without regard to stereochemistry, as:
- the structure of mamide B can be represented, without regard to stereochemistry, as:
- N-benzyl-16(S)-hydroxy-9-oxo-10E,12E,14E-octadecatrienamide (MA-S-4, macamide B) can be isolated from the twelfth fraction by preparative BPLC (Dynamax C-18 column) of the twelfth fraction using an acetonitrile-water gradient mobile phase (10:90 to 90:10 volume ratio acetonitrile:water).
- the sixth fraction (1.8 g from 25 th through 27 th liters collected) was chromatographed over silica gel (60 g) using hexane/acetone (3:1, 2 l) as the mobile phase to give 25 mg of N-benzyloctanamide (macamide A or MA-3).
- the ninth fraction (3.4 g; 41 st through 49 th liters collected) was initially separated over silica gel column (120 g, 130-270 mesh) into 30 fractions (100 nm each).
- the twelfth fraction (10 g) was chromatographed on silica gel eluting with CHCl 3 /EtOAc (2:1, 200 ml per fraction, total 5 l).
- the fractions (10-15) mainly contained N-benzyl-16(S)-hydroxy-9-oxo-10E,12E,14E-octadecatrienamide (MA-S-4 or macamide B) were combined and purified by preparative HPLC run on a Dynamax C-18 column using an acetonitrile/0.1% HOAc gradient system (0-60 min; from 10% Acetonitrile to 90% Acetonitrile) as eluent to give 33 mg of MA-S4.
- MA-3 showed the similar UV and IR spectra as those of N-benzyl-hexadecamide (MA-1) indicating that MA-3 was a fatty amide.
- the 13 C resonance of a carbonyl at ⁇ 173.2 supported the amide structure.
- the benzene ring and the amide group accounted for all the five unsaturation degrees. Therefore, the fatty acid moiety was acyclic.
- the DEPT and 13 C NMR spectra showed the signals for total eighteen carbons, including one methyl, eight methylenes, seven methines, two quaternary carbons for the fatty acid moiety. Thus, a C 18 fatty acid moiety was proposed.
- MA-S-4 was determined to be N-benzyl-16(S)-hydroxy-9-oxo-10E,12E,14E-octadecatrienamide.
- the EI mass spectrum of MA-9 showed a molecular ion peak at m/z 395 consistent with the molecular formula C 25 H 33 NO 3 .
- the 13 C resonance of the carbonyl carbon at ⁇ 173.3 favored that MA-9 was a fatty amide.
- H-3 and H-4 ( ⁇ 1.19), H-7 and H-6 ( ⁇ 1.19) were observed.
- the H-5 signals were regarded to account the left two protons.
- MA-9 was determined to be N-benzyl-9,16-dioxo-10E,12E,14E-octadecatrienamide.
- MA-9 is a new compound and named macamide C.
Landscapes
- Health & Medical Sciences (AREA)
- Natural Medicines & Medicinal Plants (AREA)
- Life Sciences & Earth Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Reproductive Health (AREA)
- Endocrinology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Epidemiology (AREA)
- Microbiology (AREA)
- Mycology (AREA)
- Medical Informatics (AREA)
- Gynecology & Obstetrics (AREA)
- Botany (AREA)
- Biotechnology (AREA)
- Alternative & Traditional Medicine (AREA)
- Pregnancy & Childbirth (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Acyclic And Carbocyclic Compounds In Medicinal Compositions (AREA)
- Medicines Containing Plant Substances (AREA)
- Medicinal Preparation (AREA)
Abstract
Description
- This application is a continuation of U.S. patent application Ser. No. 09/878,141, filed Jun. 8, 2001, which is a divisional application of U.S. patent application Ser. No. 09/261,806, filed Mar. 3, 1999, now U.S. Pat. No. 6,267,995, the disclosure of which is herein incorporated by reference in its entirety.
- The present invention relates to compositions containing particular components that can be obtained from a plant which can have pharmaceutical applications. More particularly, the plant genus is Lepidium.
-
- Johns,Ethnobiology, 1:208-212(1981), studied the biologically active aromatic glycosinolates present in the plant and also reported that benzyl isothiocyanate was the principal isothiocyanate in the plant, with p-methoxybenzyl isothiocyanate being present in relatively smaller amount. The role of these species in reproduction was discussed.
- Dini et al.,Food Chem., 49:347-349(1994) described the total content of carbohydrates and amino acids (free and from protein hydrolysis) and listed 20 saturated and unsaturated paraffinic acids (principally linoleic, palmitic, and oleic acids) present in the plant. A sterol fraction containing five sterols (identified as their acetates) was also reported. An alkaloid fraction was reported but not characterized.
- In one aspect, the present invention relates to an isolated, Lepidium-derived composition that is essentially free of cellulose and lignin and that has about 40% of polysaccharides that can be isolated from Lepidium plant material (a Lepidium polysaccharide component). The composition can also contain an aqueous component and a component, designated a Lepidium amino acid component, having amino acids that can be isolated from Lepidium plant material and. Typically, the Lepidium amino acid component has about 70% or more proline, 5% or more glutamic acid, and 5% or more valine. In certain embodiments, the composition has about 0.3% benzyl isothiocyante and about 0.5% of a component, called a macamide component, having amides of fatty acids that can be isolated from Lepidium material. In an particular embodiment, the composition has about 45% or more of a Lepidium polysaccharide component. In another embodiment, the composition is combined with one or more pharmaceutically acceptable excipients.
- In another aspect, the present invention relates to a composition having about 0.3% or more benzyl isothiocyanate, about 0.3% or more of a macamide component, about 1% or more of fatty acids that can be isolated from Lepidium plant material (a Lepidium fatty acid component), and about 0.15% of sterols that can be isolated from Lepidium plant material (a Lepidium sterol component). In a preferred embodiment, the composition has between about 2% and about 5% benzyl isothiocyanate, about 0.2% and about 1% Lepidium sterol component, between about 10% and about 20% Lepidium fatty acid component, and about 3% to about 7% macamide component. In a particularly preferred embodiment, the composition has between about 5% and about 9% benzyl isothiocyanate, between about 1% and about 3% of Lepidium sterol component, between about 20% and about 30% of a Lepidium fatty acid component, and about 10% or more of macamide component. One or more of the foregoing compositions can be combined with a pharmaceutically acceptable excipient.
- In another aspect, the invention relates to novel fatty acid amides: N-benzyl octanamide having the chemical formula C15H23NO, N-benzyl-16(RS)-hydroxy-9-oxo-10E,12E,14E-octadecatrieneamide having chemical formula C25H35NO3, and N-benzyl-16(S)-hydroxy-9-oxo-10E,12E,14E-octadecatrieneamide having the chemical formula C25H35NO3.
- In yet another embodiment, the present invention relates to a process of obtaining an isolated Lepidium-derived composition including the steps of:
- a) contacting Lepidium plant material with an aqueous solvent,
- b) separating the contacted aqueous solvent from the Lepidium plant material, and
- c) concentrating the composition of step b) to isolate the composition.
- Preferably, the aqueous solvent is water. More preferably, the aqueous solvent is a mixture of alcohol, preferably SDA, and still more preferably, a mixture of 75 vol-% SDA and 25 vol-% water.
- In yet other embodiments, the process includes the further steps of:
- d) applying the first Lepidium composition from step c) to a reverse phase liquid chromatography column,
- f) eluting the reverse phase chromatography column with a first elution volume of aqueous solvent effective to elute a first effluent from which a composition having about 45% or more Lepidium polysaccharide component is isolated by, for example, concentration.
- In yet another embodiment, the invention process includes the further step of g), eluting the reverse phase chromatography column with a second elution volume of aqueous solvent to form a second effluent from which the composition can be isolated by, for example, the step of concentrating the second effluent.
- In a preferred embodiment, the second elution volume has two or more gradient volumes that are sequentially eluted and the resulting second effluent is fractionate collected as gradient fractions so that at least one gradient fraction is collected for each gradient volume eluted. The gradient fractions can be combined and concentrated to obtain an isolated Lepidium derived composition that has about 0.3% or more of benzyl isothiocyanate, about 0.15% or more Lepidium sterol component, about 1% or more Lepidium fatty acid component, and about 0.3% or more of macamide component. Preferrably, each of the two or more gradient volumes comprises a mixture of an alcohol and water and wherein the vol-% alcohol in the first gradient volume is about 20 vol-% or more and the vol-% alcohol in any subsequent gradient volume is equal to or greater than the vol-% alcohol in the immediately previously eluted gradient volume.
- In yet another embodiment, the invention provides a process for producing the composition having between about 5% and about 9% of benzyl isothiocyanate, between about 1% and about 3% of Lepidium sterol component, between about 20% and about 30% of Lepidium fatty acid component, and d) about 10% or more of macamide component comprising the steps of:
- a) contacting Lepidium plant material with an aqueous solvent comprising about 90 vol-% or more water,
- c) concentrating the contacted aqueous solvent to make a residue of Lepidium plant material,
- d) contacting the residue of Lepidium plant material with aqueous solvent comprising a mixture of an alcohol and water having about 90 vol-% alcohol or more to form a liquor, and
- e) concentrating the liquor to obtain the composition.
- The preferred plant material in any embodiment isLepidium meyenii.
- In still another embodiment, the present invention relates to a method of treating or preventing cancer in an animal, preferably a human, by administering a cancer treating or preventing effective amount of any of the hereinabove described isolated Lepidium-derived compositions. In another embodiment, the present invention relates to a method for treating sexual dysfunction in an animal, preferably a human, by administering a sexual dysfunction treating or preventing amount of any of the hereinabove described isolated Lepidium-derived compositions to the animal. In a preferred embodiment, the animal is a female animal and the sexual dysfunction is infertility. In a more preferred embodiment, the animal is a male animal and the sexual dysfunction is a sub-normal libido. In a yet more preferred embodiment, the animal is a male animal and the sexual dysfunction is impotence.
- As used herein, the following terms have the following meanings.
- Alcohol: The term alcohol refers to a lower aliphatic alcohol having from one to six carbon atoms.
- Aqueous component: This term refers to that part or portion of a composition that is made-up of one or more aqueous solvents.
- Aqueous solvent: The term aqueous solvent means water or a single phase having an organic solvent that is miscible with water. Examples of miscible organic solvents include but are not limited to methanol, ethanol, isopropanol, n-propanol, acetone, and acetonitrile. Other miscible organic solvents are known to the skilled artesian.
- Benzyl isothiocyante: includes benzyl isothiocynate and its methoxy derivatives.
- Column volume: Column volume refers to the volume of the space defined by the inner surface of the chromatography column or chamber that surrounds the stationary phase or a reverse phase chromatography column. Column volume is abbreviated herein as CV.
- Fractionate collecting: When used in connection with an effluent or a gradient effluent, or a gradient fraction, the term fractionate collecting denotes that the effluent or gradient effluent is segregated into at least two portions or aliquots.
- Lepidium amino acid component: This term refers to that part or portion of a composition that is made-up of amino acids that can be isolated from Lepidium plant material.
- Lepidium composition: A Lepidium composition is a composition having at least one of a Lepidium amino acid component, a Lepidium fatty acid component, a Lepidium polysaccharide component, or a macamide component.
- Lepidium fatty acid component: This term refers to that part or portion of a composition that is made-up of fatty acids that can be isolated from Lepidium plant material.
- Lepidium plant material: As used herein, Lepidium plant material refers to plant matter from any part of a plant of the genus Lepidium. Examples of Lepidium plant matter include, but are not limited to, matter fromLepidium meyenii and Lepidium peruvianum.
- Lepidium polysaccharide component: This term denotes that part or portion of a composition that is made-up of polysaccharides that can be isolated from Lepidium plant material.
- Lepidium sterol component: As used herein, the term refers to that part or portion of a composition that is made-up of sterols that can be isolated from Lepidium plant material.
- Macamide: Means amides and N-substituted amides of fatty acids that can be isolated from Lepidium plant material.
- Percent (%): Unless otherwise limited or modified, percents and percentages described herein are on a weight basis. The chemical composition of plant material from a particular plant species varies with, for example, the conditions under which the plant is grown (for example soil and climate). A particular compound or mixture of compounds can exhibit pharmacological efficacy over a readily ascertainable range of composition and dosage. Therefore, it is understood that the percentages recited throughout are meant to include such variations outside the stated percentage or percentage ranges as would be anticipated by the skilled artesian.
- Substantially Free of cellulose: Means having 5% or less of cellulose and lignin combined.
- SDA: Means special denatured alcohol, typically a mixture of 97% to 95% ethanol with 3% to 5% of methanol or coal tar.
- First Lepidium compositions of the invention can be obtained from an extraction composition in a first process. Starting material for a first process is Lepidium plant material, preferablyLepidium meyenii. Lepidium plant material, for example the root of Lepidium meyenii is reduced in size to pieces having nominal dimensions between about 0.1 mm and 30 mm. The pieces of Lepidium plant material are contacted with aqueous solvent. The contacting in this or any embodiment may be by any suitable means as are known in the art; for example, percolation, vat extraction, counter current extraction, and the like. The contacting is for a time from about 2 hr. to about 18 hr. The contacting is carried out at a temperature above the solidification temperature (or where applicable the phase separation temperature) but below the boiling point of the aqueous solvent. Typically, the contacting is conducted from 20° C. to 75° C., with 40° C. to 50° C. being preferred. After the contacting, the aqueous solvent, which is an extraction composition containing a first Lepidium composition, is separated from residual plant material and the extraction composition is concentrated until the extraction composition has a solids component generally of at least about 70%, of which typically 40% is a Lepidium polysaccharide component. In this or any embodiment, the concentration can be by any of the means as are known in the art such as evaporation, distillation, and lyophilization, to mention a few.
- First Lepidium compositions generally have 30% or more, preferably about 40% or more, of a Lepidium polysaccharide component and about 1% or more of a macamide component. Typically, 70% or more of the polysaccharide units of a polysaccharide of a Lepidium polysaccharide component are sucrose units. A macamide component includes N-benzyl octanamide (macamide A), N-benzyl-16-hydroxy-9-oxo-10E,12E,14E-octatrieneamides (macamide B), and N-benzyl-9,16-dioxo-10E,12E,14E -octadecatrieneamide (macamide C). First Lepidium compositions further contain about 8% or more of a Lepidium amino acid component. The Lepidium amino acid component of a first Lepidium composition typically has 70% or more proline, 5% or more glutamic acid, and 5% or more valine. First Lepidium compositions also generally contain up to 1% of benzyl isothiocyanate. First Lepidium compositions can also contain about 0.5% to about 1.5% Lepidium fatty acid component. First Lepidium compositions of the present invention are useful for their nutritional value and are useful for the treatment or prophylaxis of carcinomas. First Lepidium compositions are also useful for treating sexual dysfunction in particular sub-normal libido and impotence in males and infertility in women.
- In a first process of the present invention, the composition of the extraction composition and the composition of the first Lepidium composition obtained therefrom can be changed by changing the aqueous solvent. When the aqueous solvent is an alcohol-water mixture having 75 vol-% SDA, the first Lepidium composition generally contains about 0.8% or more benzyl isothiocyanate and a Lepidium fatty acid component of about 1% or more. When the aqueous solvent is 90 vol-% or more water, e first Lepidium composition has less than about 0.1% of benzyl isothiocyanate and less than about 2% Lepidium fatty acid component.
- In a preferred embodiment, a first Lepidium composition having a Lepidium polysaccharide component of about 45% or more, a Lepidium fatty acid component between about 1% and about 2%, and less than about 1% each Lepidium sterol component and benzyl isothiocyanate is obtained by a suitably adapted first process that includes a reverse phase liquid chromatography process adapted to elute a first effluent containing a first Lepidium composition. In reverse phase liquid chromatography (RPLC), the column packing (stationary phase, or adsorbent) is non-polar, typically having a dipole moment of about 3 or less. Silica gel that has been treated to provide it with a bonded surface layer that is paraffinic in nature is an example of a useful stationary phase for reverse phase chromatography. Silica gels having permanently bonded C8 to C18 alkyl groups are commercially available as a stationary phase. For example, WP-Octadecyl from J.T. Baker Corp, Phillipsburg, N.J., 08865. Reverse phase liquid chromatography columns are eluted with eluents of decreasing polarity which causes the more polar compounds loaded on a column to elute first.
- Reverse phase liquid chromatography stationary phases of organic material are also known. Polymers of vinyl aromatic compounds, for example styrene, that are crosslinked with polyvinylic aromatic hydrocarbons, for example divinyl benzene, can be used as stationary phases for reverse phase liquid chromatography. These organic polymeric stationary phases are made by processes that yield small, extremely rigid, macroreticular particles. Crosslinked acrylic polymers are also useful as stationary phases for reverse phase liquid chromatography, as are polyvinyl alcohols (alkylated or non-alkylated). Suitable stationary organic phases for RPLC are commercially available. For example, styrenic and acrylic stationary phases are available from the Rohin and Haas Company, Philadelphia, Pa., under the trade name Amberlite®. Styreneic stationary phases are also available under the trade name Amberchrom® from Tossohass, Montgomeryville, Pa. Polyamide resins (e.g. nylons), polyester resins, and phenolic resins are also useful stationary phases for the reverse phase chromatography processes of the present invention.
- Many organic solvents are suitable mobile phases, or eluents, for reverse phase liquid chromatography. Lower alcohols, such as methanol, ethanol, and propanol, as well as nitrites such as acetonitrile, are suitable as organic eluents. Lower aliphatic ketones such as acetone, methyl ethyl ketone, and diethyl ketone, as well as cyclic ethers such as tetrahydrofuran, can also be used. Dimethyl formamide, dimethyl sulfoxide, and alkyl esters of acetic acid such as ethyl acetate can also be used. Mixtures of such solvents in various proportions can be used when it is desired to elute or wash the column with solvents of varying polarity. Applicants have found that aqueous solvents that are mixtures of water and an alcohol, for example, methanol, ethanol, n-propanol, iso-propanol, n-butanol, and n-and sec-hexanol, are particularly useful as mobile phases or eluents for the RPLC processes of the present invention, which in certain embodiments are carried out using an eluent of variable composition. Thus, an elution volume which is a volume of aqueous solvent applied to the column, can be a gradient eluent having two or more gradient volumes, the composition of which can be the same or different, or the compositon of the gradient eluent can be varied continuously during elution. The composition of the elution volume that is a gradient eluent can vary step-wise, linearly, sigmoidally, exponentially, logarithmically, parabolically, or hypyperbolically during elution. The limits of concentration of gradient eluents are determined by the concentration of polar organic solvent necessary to elute products from the stationary phase and by the requirement that the polar organic solvent be miscible to form a single phase at the required concentration.
- In certain embodiments of the present invention the initial alcohol concentration in the elution volume is 10 volume percent (10 vol-%) or less and is increased as separation and purification proceeds.
- The reverse phase liquid chromatography systems used to practice the present invention may be either preparative or analytical. Preparative columns have larger loading capacity and are typically larger in size.
- With regards to the dimensions of the reverse phase liquid chromatographic column, the loading of the column, the temperature, and flow rate, one skilled in the art will know to vary these parameters based primarily upon practical considerations known in the art. For example, flow rates of the eluent are adjusted according to the column dimensions, the degree of separation desired, the particle size of the stationary phase, and the back pressure in the column. The separation is typically carried out at 20° C. to 30° C. However, a temperature up to about 45° C. can be used. The separation may be carried out at high pressure (500-200 psi) or moderate pressures (100-500 psi) or, preferably, at lower pressures (10-100 psi).
- Prior to use, the reverse phase liquid chromatography column can be conditioned by eluting the column with a conditioning volume of a conditioning liquid, preferably an aqueous solvent, more preferably water. The conditioning volume is preferably between about 1 and about 10 column volumes.
- The material to be treated is applied to the preferably conditioned reverse phase chromatography column as a solution, a slurry, or a loading concentrate obtained by evaporating an aqueous solvent, preferably alcohol, from an extraction composition containing the product. If the product to be treated is solid, it may be mixed with a suitable solid carrier, for example treated or untreated silica gel, and the solid mixture placed on top of the solid support. Loading of the column is accomplished by eluting the solution, slurry, or loading concentrate through the column; or, when the product to be treated is admixed with silica gel, by eluting the column with a loading elution volume. Preferably, elution of the solution, slurry, loading concentrate, or loading elution volume is followed by elution with a washing elution volume comprising an aqueous solvent having the same composition as the aqueous solvent of the solution, slurry, or loading concentrate used to load the column stationary phase. The washing elution volume, when one is used, is preferably between about 1 and about 10 column volumes.
- Starting material for this adapted first process is a first Lepidium composition made by the previously described first process in which the aqueous solvent is an alcohol-water mixture having between about 65 vol-% and about 85 vol-%, preferably about 75 vol-% of alcohol, preferably SDA. The stationary phase of the RPLC column is a styrenic resin, preferably a crosslinked styrene-divinylbenzene resin such as Amberlite® XAD-16HP available from Rohm and Haas. The first Lepidium composition from a first process, combined with water (5-7 L per kg of first composition), is eluted through an RPLC column to apply the first composition to the column. The column is then eluted with an elution volume effective to elute a first effluent containing a first Lepidium composition, the solids component of which includes about 45% or more of a Lepidium polysaccharide component. Typically the elution volume contains aqueous solvent that is preferably water or an alcohol-water mixture having at least about 90 vol-% water and the elution volume amounts generally to 4 to 7, preferably 6, column volumes. The effluent is collected and concentrated to yield a first Lepidium composition having a Lepidium polysaccharide component of about 45% or more.
- The first process can be farther adapted to produce a second effluent by including, after elution of a first elution volume, the step of eluting a second elution volume that includes an aqueous solvent. Second Lepidium composition is obtained by concentrating the second effluent. In this or any other embodiment, the concentrating can be by any suitable means as know in the art such as evaporation, distillation, lyophilization, and the like. Generally, the second elution volume is typically to 4 to 10 column volumes. The second effluent contains a second Lepidium composition having between about 0.3% and about 12%, preferably between about 2% and about 10%, more preferably between about 5% and about 10% of benzyl isothiocyanate; between about 0.3% and about 2.7%, preferably between about 1% and about 2.5% of a Lepidium sterol fraction; and between about 10% to about 65%, preferably between about 10% and about 25% of a Lepidium fatty acid component.
- In one embodiment of the adapted first process, the second elution volume has at least two gradient volumes, each of which contains an aqueous solvent. The identity or composition of the aqueous solvent in each of the two or more gradient volumes can be the same or it can be different. Preferably it is different. The second effluent resulting from the elution of the second elution volume can be fractionate collected into at least as many gradient fractions as there are gradient volumes eluted. The fractionate collected gradient fractions can be combined, or they can be maintained separately. The gradient fractions can be concentrated to obtain second Lepidium compositions. It will be apparent to one skilled in the art that the identity and amounts of constituents of second Lepidium compositions made by this process can be varied by varying the number and composition of gradient volumes eluted, the number and volume of gradient fractions that are fractionate collected, and the manner in which gradient fractions are combined.
- In a preferred embodiment of the adapted first process, the second elution volume is an alcohol-water mixture having 70 vol-% and preferably 80 vol-% or more alcohol, preferably SDA. The second Lepidium composition obtained in this process contains between about 0.2% and about 10%, preferably between about 2% and about 5%, of benzyl isothiocyanate; between about 0.15% and about 3%, preferably between about 0.2% and about 1%, of a Lepidium sterol component; between about 1% and about 65%, preferably between about 10% and about 25% of a Lepidium fatty acid component; and between about 0.3% and about 0.5% of a macamide component, wherein the macamide component comprises about 30% or more macamide B and 20% or more macamide C. Second Lepidium compositions typically have less than about 5%, preferably less than about 1%, of either a Lepidium polysaccharide component or a Lepidium amino acid component.
- A second Lepidium composition can also be obtained from a second extraction composition from a second process. In a second process, residual Lepidium plant material that remains after separating the first extraction composition is contacted in a second contacting step with an aqueous solvent, preferably a mixture of an alcohol and water having about 80 vol-% or more, preferably about 90 vol-% or more alcohol, preferably SDA, to produce a second extraction composition or liquor. The process of contacting in the second contacting step can be the same as that in the first contacting step of a first process or it can be different. The liquor is separated from residual Lepidium plant material and concentrated to obtain a second Lepidium composition. The separating can be by any process known in the art; for example centrifugation, filtration, or decanting.
- The second Lepidium composition obtained by a second process R-A) has between about 7% and about 9% of benzyl isothiocyanate, a Lepidium sterol component of between about 1% and about 3%/o, a Lepidium fatty acid component between about 10% and about 20%, and a macamide component of between about 0.3% and about 0.5%. Generally, a Lepidium sterol component includes about 50% or more β-sitosterol and also contains stigmasterol and campesterol. Generally, a Lepidium fatty acid component contains between about 0.5% and about 1.2% 9,16-dioxo-10E,12E,14E-octadecatrieneoic acid between about 1.5% and about 3.5% of 16-hydroxy-9-oxo-10E,12E,14E-octadecatrieneoic acid between about 30% and about 35% linoleic acid, and between about 15% and about 22% linolenic acid. Generally, a Lepidium fatty acid component also contains oleic acid. In preferred embodiments, the second composition also contains about 0.2% or more of a macamide component, wherein the macamide component comprises 1% or less macamide A, about 10% or more macamide B, and about 20% or more macamide C.
- The present invention also provides a third Lepidium composition that contains a mixture of a pharmaceutically acceptable excipient with a first Lepidium composition, a second Lepidium composition, or with both a first and second Lepidium composition. Pharmaceutically acceptable excipients are any materials that do not interfere with the pharmacological activity of the third composition or degrade the bodily functions of the animal to which it can be administered, but facilitate fabrication of dosage forms or actual administration of the composition; for example by improving palatability of oral dosage forms. Examples of pharmaceutically acceptable excipient include but are not limited to maltodextrin, calcium phosphate, and fused silica. Pharmaceutically acceptable excipients also include flavorants.
- Third compositions of the present invention can be made, for example, by combining about 25% to about 50% (dry weight) of a first or second Lepidium composition of the present invention and about 75% to about 25% (dry weight) of one or more pharmaceutically acceptable excipients, combining this mixture with water (5 liter per kg of solids) and homogenizing the mixture. A Silverson Model 14 RT-A homogenizer (Silverson Corporation, East Longmeadow, Mass.) is an example of an apparatus suitable for carrying-out the homogenization. The homogenized composition is then dried to obtain a third Lepidium composition. The drying may be carried-out by any means as are known in the art; for example spray drying, oven dying, rotary vacuum drying, or lyophilization.
- In yet other embodiments, the present invention provides novel amides of fatty acids, namely; N-benzyl octanamide (also called macamide A or MA-3), racemic and enantiomerically pure N-benzyl-16-hydroxy-9-oxo-10e,12e,14e-octadecatrienamide (also called macamide B or MA-S-4), and N-benzyl 9,16-dioxo-10e,12e,14e-octadecatrienamide (also called macamide C or MA-9). All of these N-substituted amides are members of the class macamides, as that term is used herein. The N-benzyl amides of the present invention, which are useful in the prevention or treatment of carcinomas, can be synthetically prepared, or obtained from Lepidium plant material, preferablyLepidium meyenii, by chromatographic processes. The chromatographic process is particular useful for obtaining enantiomerically pure N-benzyl-16(S)-hydroxy-9-oxo-10e,12e, 14e-octadecatrienamide.
- N-benzyl amides of the present invention can be synthetically prepared by various methods (See Barstaw, L. E. et al., J. Org. Chem., 36, 1305,(1971)). For example, N-benzyl amides of the present invention can be made by refluxing the corresponding carboxylic acid and benzyl amine with triphenylphosphene and bromotrichloromethane.
- The N-benzyl amides of the present invention can be isolated from Lepidium plant material by c chromatographic process. Starting material for isolation of the N-benzyl amides of the present invention by chromatographic processes is a dry powder loading composition formed by mixing with silica gel (60-100 mesh) the residue from rotary vacuum concentration of an ethyl acetate extract of an aqueous suspension of a first Lepidium composition from a first process in which the aqueous solvent contains a mixture of about 75% SDA and about 25% water. The dry powder loading composition is applied to a chromatography column that is the eluted with a series of gradient volumes. The resulting gradient eluents are fractionate collected and compared by thin layer chromatography (TLC). Fractionate collected gradient eluents having similar TLC patterns are combined and combined fractions are further treated by column chromatography.
- The methods of preventing or treating carcinomas, libido-related male sexual dysfunction, male impotence, and muscle fatigue comprise administering or dosing an effective amount of a composition, which can be a third Lepidium composition, that contains a first or second Lepidium composition, or both. The meaning of effective amount will be recognized by clinicians but includes an amount effective to either (1) reduce the symptoms of the disease or condition sought to be treated or prevented (i.e. cancer, sexual dysfunction), (2) induce a pharmacological change relevant to treating or preventing the disease sought to be treated or prevented, or (3) prevent the occurrence of the disease or condition.
- The Lepidium compositions used in the method of the present invention can be administered by any route. Compositions of the present invention are administered alone, or are combined with a pharmaceutically-acceptable carrier or excipient according to standard pharmaceutical procedures. Preferably, Lepidium compositions are administered orally as a third Lepidium composition. For the oral mode of administration, the compositions of the present invention are used in the form of tablets, capsules, chewing gum, and the like. In the case of tablets, various disintegrants such as starch, and lubricating agents such as magnesium stearate and talc can be used.
- Compositions of the invention can include pharmaceutically acceptable acid addition salts, particularly those obtained with mineral acids, for example hydrochloric or hydrobromic acid. However, organic acids, for example tartaric acid, can also be used.
- The amount dosed will depend upon the composition used and the disease or condition to be treated or prevented. Generally, the compositions are dosed at between 0.1 g and 10 g per kg of body weight per day (is this about right?).
- The present invention is illustrated by the following non-limiting examples.
- In this and other examples, HPLC analysis of Maca product were performed on a Hewlett Packard Series 1100 HPLC using an phenomenex, Luna C-8 column. GC/MS analysis of Maca product was performed on an HP-5973 MSD using a Supelco SAC-5 capillary column.
- Roots ofLepidium meyenii (4.6 Kg) were cut to a nominal dimension of about 1 cm and contacted with an aqueous solvent (75 vol-% SDA and 25 vol-% water; 25 L per kg root) at 45° C. by percolation. Decoction was separated from the plant material and concentrated to yield a first composition (2.1 Kg), denoted NE, having a solids content of 77% (i.e. 1.6 Kg on a dry basis). The solids component included, based on the solids present; 0.89% benzyl isothiocyanate, 0.079% of a Lepidium sterol component, 1.46% of a Lepidium fatty acid component, 8.72% of a Lepidium amino acid component, and 41.9% of a Lepidium polysaccharide component.
- Roots ofLepidium meyenii (500 g) were reduced in size to a nominal dimension of 0.5 cm and contacted with 14 L water by percolation. The aqueous phase was separated from residual plant material and evaporated to dryness to obtain 20 g of a tacky product. The tacky product had a Lepidium sterol component of <0.01%, a Lepidium fatty acid component of <0.1%, a Lepidium amino acid component of 9%, and A Lepidium polysaccharide component of 44%.
- The residual plant material from example 2 was contacted with 15 L of 100% SDA by percolation to form a liquor. The liquor was separated and concentrated to yield 10 g of a second Lepidium composition having 7.8% benzyl isothiocyanate, a Lepidium sterol component of 1.8%, a Lepidium fatty acid component of 22%, and a macamide component of 12%. No Lepidium amino acid component was found in the composition.
- A first Lepidium composition (1.6 Kg) obtained according to the process of example 1 (i.e. NE), was slurried with water (8.8 L per kg of dry NE) for about 30 min. The slurry was applied to a RPLC column (15 cm.×100 cm packed with Amberlite® XAD-16 (Rohm and Haas Co.) that had been preconditioned with 20 column volumes of water. The column was eluted with a first elution volume (6 column volumes) of 100 vol-% water. The resulting first effluent was collected and evaporated to dryness to yield 1.5g of a first Lepidium composition having 0.15% benzyl isothiocyanate, 0.07% of a Lepidium sterol component, 1.8% of a Lepidium fatty acid component, 10% of a Lepidium amino acid component, and 48% of a Lepidium polysaccharide component. The composition had less than 1% of a macamide component.
- The RPLC of example 4 was eluted with a second elution volume (6 column volumes) that was made-up of 100% SDA. The resulting effluent was collected and evaporated to dryness to yield 162 g of a second Lepidium composition having 4.1% benzyl isothiocyanate, 0.4% of a Lepidium sterol component, 12% of a Lepidium fatty acid component, 4.4% of a macamide component, and no Lepidium amino acid component or Lepidium polysaccharide component.
- A first Lepidium composition (1.6 Kg NE on a dry basis), obtained according to the method of example 1, was slurried with water (6.3 L per kg of first Lepidium composition) for about 30 min. The slurry was applied to a RPLC column (15 cm×100 cm) packed with Amberlite® XAD-16 resin (Rohm and Haas Co) that had been preconditioned with about 20 column volumes of water. The column was eluted with a first elution volume of 4 column volumes of water. The column was then eluted with a second elution volume that was made-up of five gradient volumes, each having a volume equal to 4 column volumes and each made-up of a mixture of SDA and water. The gradient fractions had, respectively, 20 vol-%, 40 vol-%, 60 vol-%, 80 vol-%/o, and 100 vol-% SDA. The gradient fractions corresponding to each of the gradient volumes were fractionate collected and analyzed (HPLC and GC). The gradient fractions were free of both an Lepidium amino acid component and a Lepidium polysaccharide component. The gradient fractions contained other components as set-out below in Table 1.
TABLE 1 Content of Various Lepidium Components in Gradient fractions of a RPLC Process. Fr.* Fr. Fr. Fr. Fr. M-F1 M-F2 M-F3 M-F4 M-F5 Benzyl isothiocyanate - % 0.7 0.4 2.5 2.5 5.8 Lepidium sterol component - % 0.36 0.18 0.67 0.87 2.2 Lepidium fatty acid component - % 0.29 0.14 0.54 0.70 1.8 Macamide component - % 0 0 0.2 1.6 0 - The gradient fractions were combined and concentrated to yield 125 g of a second Lepidium composition having 2.3% benzyl isothiocyanate, 0.8% of a Lepidium sterol component, 17.2% of a Lepidium fatty acid component, and 0.4% of a macamide component.
- The tests were performed according to the MTT assay (See, Mosmann, T.,J. Immun. Meth. 65, 55(1983).
- Cells were planted in 96 well flat bottom plates with low evaporation lids. Three cell lines per plate were seeded in 0.2 ml medium per well. Each cell line was planted at the optimum concentration for its particular growth rate: HT-29 and A-549, 5000 c/ml; MCF-7, 15000 c/ml; A-498, 10,000 c/ml; PC-3, 15000 c/ml; and PACA-2, 10,000 c/ml. Products were tested at various dilutions (at least ten) to determine the ED50.
- The results are given in table 2.
TABLE 2 6-Cell Line Clinical Test Results, Expressed as ED50 values (in μg/ml) Sample Kidney Prostate Pancreatic Lung Breast Colon Sample Code Description A-498 PC-3 PACA-2 A-549 MCF-7 HT-29 MA-1 24.88 85.77 17.66 28.66 >100 54.65 MA-2 9.08 4.05 3.06 17.97 29.33 22.97 MA-3 5.83 6.42 4.33 7.57 28.45 19.02 macamide A MA-7 51.79 64.22 31.62 63.37 93.49 61.55 SY-197A Maca NE 56.85 94.67 37.60 56.99 97.51 64.17 M-F1 Purified >100 >100 >100 >100 >100 49.24 Product Fraction #1 (20% SDA) M-F2 Purified >100 >100 >100 >100 >100 40.57 Product Fraction #2 (40% SDA) M-F3 Purified >100 >100 >100 >100 >100 40.57 Product Fraction #3 (60% SDA) M-F4 Purified 38.29 29.59 29.43 17.03 34.41 6.10 Product Fraction #4 (80% SDA) M-F5 Purified 26.54 20.66 24.94 25.37 32.57 3.09 Product Fraction #5 (100% SDA) M-F2-5 Purified >100 30.96 37.56 33.26 56.18 41.08 Product Fraction #2˜5 Adriamycin 3.59 × 10−3 2.81× 10−2 5.22 × 10−3 3.16 × 10−3 1.07 × 10−1 2.20 × 10−2 - Ten healthy rats were dosed by oral gavage with 5 g of third Lepidium composition per kilogram of body weight. The animals were observed for signs of gross toxicity for 14 days. The body weights of the animals were checked on the 7th and 14th days. Body weight data is collected in Table 3. Gross necropsy findings at terminal sacrifice were unremarkable.
TABLE 3 Individual Bodyweight and Dosage. Bodyweight (g) Animal No. Sex Initial Day 7 Day 14 Dose* 5025 M 246 309 349 1.6 5026 M 236 297 332 1.6 5027 M 251 340 383 1.7 5028 M 263 341 379 1.7 5029 M 242 308 335 1.6 5030 F 197 230 253 1.3 5031 F 200 231 261 1.3 5032 F 185 227 247 1.2 5033 F 193 230 250 1.3 5034 F 186 231 249 1.2 - Sufficient mice (22±1.5 g) were dosed for 21 days with Ig of either of two third Lepidium compositions. One group (group I) was dosed with a third Lepidium composition made with a first Lepidium composition obtained according to example 1. A second group group (II) was dosed with a third Lepidium composition made with a second Lepidium composition obtained according to example 5. One hour after the dosing on the 21st day, each mouse was individually placed in water (25±2° C.) and observed. The elapsed time at which a mouse remained submerged. The results are collected in Table 4.
TABLE 4 Muscle Fatigue Results. Animal Duration of Swim Time In Number Seconds Group (n) (mean ± SD) p Control Group 15 110.07 ± 2.58 Group I 15 124.07 ± 3.30 <0.01 Group II 15 144.13 ± 3.52 <0.01 - Sufficient male mice (22±1.5 g; 15 per group) were dosed by oral gavage for 21 days with 1 g/day of either of two third Lepidium compositions. Male mice in one group (group-I) were dosed with a third Lepidium composition made with a first Lepidium composition obtained according to example 1. Male mice in a second group (group II) were dosed with a third Lepidium composition made with a second Lepidium composition obtained according to example 5. A control group received no third Lepidium composition. Mice were ear-tagged or color coded for identification. On the 21st day, ½ hour after dosing, each male was placed in a cage with two mice and observed under darkroom conditions. The mice were observed for three hours and the number intromissions were recorded. Intromission was indicated by a characteristic rearward lunge by the male terminating coitus. Results are summarized in table 5 below.
TABLE 5 Intromission Results. Number of Intromissions Group (mean ± SD) p Control 16.33 ± 1.78 Group I 46.67 ± 2.39 <0.01 Group II 67.01 ± 2.55 <0.01 - Testectomies were performed on a sufficient number of male rats using pentobarbital, 45 mg/kg, as anesthetic. Rats were treated postoperatively for 3 days with sodium penicillin, 2,000 U/kg. Rats were earcoded and color-coded for identification and divided into 7 groups. Three groups of 10 rats each (Set A) were dosed by oral gavage for 21 days with a third Lepidium composition made with a first Lepidium composition obtained according to example 1. Rats in each of the three groups received a different dosage. A second set of three groups of 10 rats each (Set B) were dosed for 21 days with a third Lepidium composition made with a second Lepidium composition obtained according to example 5. Rats in each of the three groups received a different dosage. A control group of 10 rats received no third Lepidium composition.
- On the 21st day, ½ hour after dosing, rats were restrained and an electric pulse of 20 V was applied to the penis using an electrode of a YSD4G multifunction instrument. The time to achieve full erection was monitored. Results are collected in table 6 below.
TABLE 6 Incubation Period of Erection (IPE) in Testicle-Removed Rats with Oral Administration of M-PE and MC-A PE. Incubation Period of Dose Animal Number Erection in Seconds Group (mg/kg) (n) (mean ± SD) Surgical Group 10 137.4 ± 81.6 Set A Low dose 45 10 121.4 ± 51.3 Middle dose 180 10 54.0 ± 25.8 High dose 1800 10 90.5 ± 80.2 Set B Low dose 45 10 71.2 ± 32. Middle dose 180 10 73.2 ± 39. High dose 1800 10 80.9 ± 85.1 - The N-benzyl amides of the present invention can be isolated from Lepidium plant material by chromatographic process. Starting material for isolation of the N-benzyl amides of the present invention by chromatographic processes is a dry powder loading composition formed by mixing with silica gel (60-100 mesh) the residue from rotary vacuum concentration of an ethyl acetate extract of an aqueous suspension of a first Lepidium composition from a first process in which the aqueous solvent contains a mixture of about 75% SDA and about 25% water. The dry powder loading composition is applied to a silica gel column (130-270 mesh) and the column is then eluted with, in sequence, five elution volumes, each about 8 column volumes that contain mixtures of n-hexane and acetone in the following ratios (vol-% n-hexane:vol-% acetone): 10:1, 5:1, 2:1, 1:1, and 0:1. The column effluent is divided into 18 fractions. The 25th through 27th liter of effluent make-up a sixth fraction. The 41st through 49½th liter of effluent make-up a ninth fraction. The 63rd through 70th liter of effluent make-up a twelfth fraction.
- N-benzyl octanamide can be obtained by chromatographic treatment of the sixth fraction by treating the sixth fraction on a silica gel column using a n-hexane-acetone mobile phase (3:1). N-benzyl octanamide can be isolated from the effluent (all of it? What part?) by removing mobile phase from the effluent.
-
- N-benzyl-9,16 dioxo-10E,12E14E-octadecatriene amide (MA-9, macamide C) can be isolated from the ninth fraction by further chromatographic treatment of the fraction on a silica gel column using 30 elution volumes of hexane/acetone as mobile phase. Fractions 15-20 (i.e. effluent from elution volumes 15-20) were further treated by preparative IPLC (Dynamax-60A column) using acetonitrile-water gradient eluent (5:95 to 95:5 vol. ratio, acetonitrile:water) to obtain N-benzyl-9,16-dioxo-10E,12E,14E-octadecatrienamide. The structure of macamide C can be represented, without regard to stereochemistry, as:
-
- N-benzyl-16(S)-hydroxy-9-oxo-10E,12E,14E-octadecatrienamide (MA-S-4, macamide B) can be isolated from the twelfth fraction by preparative BPLC (Dynamax C-18 column) of the twelfth fraction using an acetonitrile-water gradient mobile phase (10:90 to 90:10 volume ratio acetonitrile:water).
- N-benzyl-16(S)-hydroxy-9-oxo-10E,12E,14E-octadecatrienamide has the following physical and spectroscopic properties: White powder, m.p. 95-96° C., UVλmax(MeOH)=314 nm (ε=36392). IRγKBr (cm−1): 3368, 2930, 2850, 1677, 1622, 1598, 1583, 1240, 1108, and 1057. Molecular formula (elemental analysis): C25H35NO3. CI MS m/z, 397.
- Experimental details are given below.
- Contacting ofLepidium meyenii was carried out by percolation accordibg to the following procedure. The roots were grounded into pieces shorter than 3 cm in length. The ground material was then equally divided and loaded into three similarly-sized percolators. For the first cover, 75% SDA was loaded into each percolator at a ratio of 4.2:1 (volume of solvent in liter: weight of material in kg). After the solution had been circulated for 5 hr. at room temperature, the extract was transferred to a still and concentrated in vacuo at a temperature below 65° C. For the second cover, fresh 75% SDA was added into percolator #1. After the solution had been circulated for 5 hrs, the extract was transferred to percolator #2. After the solution in percolator #2 had been circulated for 5 hrs, the extract was transferred to percolator #3. The solution in percolator #3 was circulated for 5 hrs and the extract was transferred to the same still (for the first cover) and concentrated in vacuo at a temperature below 65° C. The procedure as described above was repeated three more times to give a total of 5 covers, which were concentrated until the residue had a total solid dry base of 72.9%.
- 3.5 kg of the extract was suspended in 1.8 liter water, and extracted with EtOAc (3×1000 ml). The combined extract was concentrated in vacuo at 40° C. to dryness (241 g). The residue was mixed with silica gel (60-100 mesh, 200 g) and air-dried, and then applied to a silica gel column (7.5×60 cm, 800 g, 130-270 mesh) eluted with five 20 L hexane/acetone gradient volumes (10:1-5:1-2:1-1:1-0:1, total volume 100 l). Every 500 ml was fractionate collected and concentrated, a total of 200 portions were collected. All portions were analyzed by thin layer chromatography (TLC) and portions having similar TLC patterns were combined to give 18 fractions. The sixth fraction (1.8 g from 25th through 27th liters collected) was chromatographed over silica gel (60 g) using hexane/acetone (3:1, 2 l) as the mobile phase to give 25 mg of N-benzyloctanamide (macamide A or MA-3). The ninth fraction (3.4 g; 41st through 49th liters collected) was initially separated over silica gel column (120 g, 130-270 mesh) into 30 fractions (100 nm each). The fractions (frs 15-20) which mainly contained N-benzyl-9,16dioxo-10E,12E,14E-octadecatrienamide (MA-9 or macamide C) were combined and subjected to preparative HPLC (column: Dynamax-60A C18, 2.14 I 25 cm i.d., 8 μm) using Acetonitrile/0.1% HOAc gradient system (0-60 min, from 5% Acetonitrile to 95% Acetonitrile) as the mobile phase with a flow rate of 10 ml/min to give 10 mg of MA-9. The twelfth fraction (10 g) was chromatographed on silica gel eluting with CHCl3/EtOAc (2:1, 200 ml per fraction, total 5 l). The fractions (10-15) mainly contained N-benzyl-16(S)-hydroxy-9-oxo-10E,12E,14E-octadecatrienamide (MA-S-4 or macamide B) were combined and purified by preparative HPLC run on a Dynamax C-18 column using an acetonitrile/0.1% HOAc gradient system (0-60 min; from 10% Acetonitrile to 90% Acetonitrile) as eluent to give 33 mg of MA-S4.
- MA-3 showed the similar UV and IR spectra as those of N-benzyl-hexadecamide (MA-1) indicating that MA-3 was a fatty amide. The13C resonance of a carbonyl at δ 173.2 supported the amide structure. The 1H signals at δ 4.41 (2H, d, J=4.4 Hz), 5.71 (br s), 7.25 (5H), and 13C signals at δ 127.5 (1C, d), 127.8 (2C, d), 128.7 (2C, d), and 43.6 (1C, t) suggested that MA-3 had a same amine group as that of MA-1. The molecular fromula, C15H23NO, was derived from the El mass spectrum and indicated five degrees of unsaturation. The benzene ring and the amide group accounted for all the five unsaturation degrees. Therefore, the fatty acid moiety was acyclic.
- The1H NMR spectrum displayed a terminal methyl group at δ 0.86, and the COSY spectrum revealed coupling of this group with an unresolved eight proton proton complex at δ 1.28. One methylene group was observed as triplet (J=7.2 Hz) at δ 2.17, indicating that it was adjacent to the carbonyl group. In the COSY spectrum, this methylene group was found to be coupled to another methylene group at δ 1.59 which in turn was coupled to the methylene signals at ca δ 1.18. It was suggested that the fatty acid moiety had eight carbons. Treatment of this compound by 6N HCl followed by CH2N2 gave methyl caprylate that was identified by GC-MS analysis. Thus, the fatty acid moiety was unambiguously determined as octanoyl. Therefore, MA-3 was identified as N-benzylotanamide, also designated herein as macamide A.
- Compound N-benzyl-16(S)-hydroxy-9-oxo-10E,12E,14E,-octadecatrienamide (MA-S4) showed maximum absorption at 314 nm (ε=36392), suggesting a conjugated trienone. It possessed a molecular formula C25H35NO3 by mass spectrum and the NMR data (Table 1), which requires eight unsaturation equivalents. The 1H signals at δ 4.73 (2H, d, J=6 Hz), 9.00 (br s, NH), 7.35 (5H), and 13C signals at δ 127.3 (1C, d), 128.1 (2C, d), 128.9 (2C, d), and 43.4 (1C, t) suggested that MA-S4 had the same amine group as that of MA-I and MA-3. The 13C NMR spectrum revealed two carbonyl, a benzene ring and three other olefins, accounting for all the degrees of unsaturation and indicating MA-S4 had an acyclic fatty acid moiety. The DEPT and 13C NMR spectra showed the signals for total eighteen carbons, including one methyl, eight methylenes, seven methines, two quaternary carbons for the fatty acid moiety. Thus, a C18 fatty acid moiety was proposed.
- The COSY, with aid of TOSCY, assigned the all proton resonances. The isolated terminal methyl group at δ 1.07 (H-18) and methylene groups at δ 2.45 (H-2) and 2.54 (H-8) were chosen as the starting points for the analysis of COSY and TOCSY. The correlations between H-18 (δ 1.07) and H-17 (δ 1.74), H-17 and H-16 (δ 4.41), H-16 and H-15 (δ 6.25), H-15 and H-14 (δ 6.71), H-14 and H-13 (6.82), H-13 and H-12 (δ 6.44), H-12 and H-11 (δ 7.47), H-11 and H-10 (δ 6.33) led to the assignment of the protons continuing from H-18 to H-10. The correlations between H-2 and H-3 (δ 1.63), H-3 and H4 (δ 1.24), H-8 and H-7 (δ 1.83), H-7 and H-6 (δ 1.34) led to the assignment of protons from H-2 to H-8 except for H-5. The overlaped proton systems at (δ 1.24) contained four protons. Hence, two protons were assigned to H-5 (δ 1.24). In HMBC spectrum, the long-range correlations between H-2 (δ 2.45) and C-1 (δ 173.1), and H-8 (2.54) and C-9 (δ 200.0) confirmed the assignment of H-2 and H-8. The13C NMR data were then assigned by HSQC spectrum. The 13C resonance of C-16 at δ 72.7 indicated that one hydroxy group was attached to C-16.
- The large couplings between the olefinic protons (J10,11=15.6 Hz, J12,13=14.0 Hz, and J14,15=15.6 Hz) revealed that three of the double bonds had E configurations. The absolute stereochemistry of C-16 secondary alcohol was determined by optical rotations which were of the same sign and magnitude ([α]D+) as those of coalital ([α]D+21°, C=0.63, Me2CO) and 16(S)-hydroxy-9oxo-10E,12E,14E,-octadecatrienoic acid ([α]D+11.7°, C=0.2, Me2CO), assigning a S configuration to C-16. The stereochemistry of coalital was determined by exciton chirality method reported by Bernart et al., J. Nat. Prod. 56:245(1993)).
- Therefore, MA-S-4 was determined to be N-benzyl-16(S)-hydroxy-9-oxo-10E,12E,14E-octadecatrienamide.
- Compound N-benzyl-9,16-dioxo-10E,12E,14E-octadecatdenaide (MA-9) was isolated as a light yellow powder, m p. 115-6° C. The UV spectrum showed maximum absorption at 317 nm (ε=13847), suggesting a conjugated trienone. The strong absorption bands at 2950 (aliphatic), 1715, 1680 (conjugated ketone), 3297, 1640, 1545 (amide), and 1003 cm−1 (trans double bond) in the IR spectrum indicated that this compound was a fatty amide. The EI mass spectrum of MA-9 showed a molecular ion peak at m/z 395 consistent with the molecular formula C25H33NO3. The 13C resonance of the carbonyl carbon at δ 173.3 favored that MA-9 was a fatty amide.
- The1H signals at δ 4.62 (2H, d, J=4.4 Hz), 8.83 (br s), 7.30 (5H), and 13 C signals at δ 127.6 (1C, d), 128.4 (2C, d), 129.2 (2C, d), and 43.6 (1C, t) suggested that MA-9 had a same amine group as those of MA-1, MA-3 and MA-S4. The intense fragment ion peaks at m/z 91 and 106 supported the above inference.
- All proton and carbon signals of the fatty acid moiety were unambiguously assigned using COSY, TOCSY, HMQC and HMBC techniques. The isolated terminal methyl group at δ 1.01 (H1-18), methylene groups at δ 2.38 (H-2) and 2.50 (H-8), and olefinic protons for H-10 at δ 6.30 and H-15 at δ 6.34 were chosen as the starting points for the analysis of COSY and TOCSY. The methyl group (Me-18) were coupled with H-17 (δ 2.61). The correlations between H-2 (δ 2.38) and H-3 (δ 1.62), H-8 (δ 2.50) and H-7 (δ 1.23) were observed. The correlations between H-3 and H-4 (δ 1.19), H-7 and H-6 (δ 1.19) were observed. The proton complex at δ 1.19 had six protons. The H-5 signals were regarded to account the left two protons. The correlations between H-10 (δ 6.30) and H-11 (δ 7.30), H-15 (δ 6.34) and H-14 (δ 7.30), H-11 and H-12 (δ 6.68), H-14 and H-13 (δ 6.68) confirmed the triene structure.
- In HMBC, the correlations between H-2 (δ 2.33) and C-1 (δ 173.3), H-8 (δ 2.56) and one ketone (C-9, δ 200.5) confirmed the assignment of H-2 and H-8. The correlation between H-17 (δ 2.61) and another ketone (C-16, 67 200.3) confirmed the position of another ketone at C-16. The positions of the two ketone groups were supported by the daughter ion peaks observed in the EI mass spectrum. The ions at m/z 366 and 338 derived from the cleavage of C-17-C16 bond, and C-16-C-15 bond, respectively, indicated that a ketone was located at C-16. The ions at m/z 135, 163 and 260 derived from the rupture of C-9-C-10 bond and C-8-C-9 bond, respectively, revealed another ketone at C-9.
- The double bonds at C-10 and C-14 were determined to be E configurations based on the large coupling constants (J10,11=15.3 Hz, and J15,14=15.3 Hz). Because the multiplets of H-11, H-12, H-13 and H-14 are not first order, and may be AA′BB′ system, they do not display substantive couplings each other. The configuration of the double bond at C-12 could not be determined by coupling constant. However, it might be suggested to be E configuration based on the inference that MA-9 be a oxidized product of MA-S4.
- Therefore, MA-9 was determined to be N-benzyl-9,16-dioxo-10E,12E,14E-octadecatrienamide. MA-9 is a new compound and named macamide C.
Claims (3)
Priority Applications (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US10/138,030 US6552206B1 (en) | 1999-03-03 | 2002-05-02 | Compositions and methods for their preparation from lepidium |
US11/943,867 USRE43005E1 (en) | 1999-03-03 | 2007-11-21 | Compositions and methods for their preparation from lepidium |
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US09/261,806 US6267995B1 (en) | 1999-03-03 | 1999-03-03 | Extract of Lepidium meyenii roots for pharmaceutical applications |
US09/878,141 US20020042530A1 (en) | 1999-03-03 | 2001-06-08 | Compositions and methods for their preparation from lepidium |
US10/138,030 US6552206B1 (en) | 1999-03-03 | 2002-05-02 | Compositions and methods for their preparation from lepidium |
Related Parent Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US09/878,141 Continuation US20020042530A1 (en) | 1999-03-03 | 2001-06-08 | Compositions and methods for their preparation from lepidium |
Related Child Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US11/943,867 Reissue USRE43005E1 (en) | 1999-03-03 | 2007-11-21 | Compositions and methods for their preparation from lepidium |
Publications (2)
Publication Number | Publication Date |
---|---|
US20030068388A1 true US20030068388A1 (en) | 2003-04-10 |
US6552206B1 US6552206B1 (en) | 2003-04-22 |
Family
ID=22994957
Family Applications (5)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US09/261,806 Expired - Lifetime US6267995B1 (en) | 1999-03-03 | 1999-03-03 | Extract of Lepidium meyenii roots for pharmaceutical applications |
US09/878,141 Abandoned US20020042530A1 (en) | 1999-03-03 | 2001-06-08 | Compositions and methods for their preparation from lepidium |
US10/002,757 Expired - Lifetime US6428824B1 (en) | 1999-03-03 | 2001-10-19 | Treatment of sexual dysfunction with an extract of Lepidium meyenii roots |
US10/138,030 Ceased US6552206B1 (en) | 1999-03-03 | 2002-05-02 | Compositions and methods for their preparation from lepidium |
US11/943,867 Expired - Lifetime USRE43005E1 (en) | 1999-03-03 | 2007-11-21 | Compositions and methods for their preparation from lepidium |
Family Applications Before (3)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US09/261,806 Expired - Lifetime US6267995B1 (en) | 1999-03-03 | 1999-03-03 | Extract of Lepidium meyenii roots for pharmaceutical applications |
US09/878,141 Abandoned US20020042530A1 (en) | 1999-03-03 | 2001-06-08 | Compositions and methods for their preparation from lepidium |
US10/002,757 Expired - Lifetime US6428824B1 (en) | 1999-03-03 | 2001-10-19 | Treatment of sexual dysfunction with an extract of Lepidium meyenii roots |
Family Applications After (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US11/943,867 Expired - Lifetime USRE43005E1 (en) | 1999-03-03 | 2007-11-21 | Compositions and methods for their preparation from lepidium |
Country Status (5)
Country | Link |
---|---|
US (5) | US6267995B1 (en) |
EP (1) | EP1180006A4 (en) |
AU (1) | AU3864900A (en) |
CA (1) | CA2362858C (en) |
WO (1) | WO2000051548A2 (en) |
Families Citing this family (22)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6267995B1 (en) | 1999-03-03 | 2001-07-31 | Pure World Botanicals, Inc. | Extract of Lepidium meyenii roots for pharmaceutical applications |
US20070054001A1 (en) * | 2003-12-22 | 2007-03-08 | Santerra Pharmaceuticals, Inc. | Methods and compositions to enhance endogenous igf production and their uses |
US20040137131A1 (en) * | 1999-09-03 | 2004-07-15 | Bobrowski Paul J. | Maca products and their uses |
CN1320886C (en) | 2002-05-17 | 2007-06-13 | 杜克大学 | Method for treating obesity |
WO2004071437A2 (en) * | 2003-02-07 | 2004-08-26 | Barmensen, Inc. | Compositions for enhancing sexual responsiveness |
TW200529864A (en) * | 2004-01-28 | 2005-09-16 | Suntory Ltd | Method for producing maca extract |
JP3747049B2 (en) * | 2004-03-30 | 2006-02-22 | サントリー株式会社 | Alcoholic beverage containing maca extract |
JP2005281271A (en) * | 2004-03-31 | 2005-10-13 | Suntory Ltd | Skin moisturizing ability improving agent |
JP2005281272A (en) * | 2004-03-31 | 2005-10-13 | Suntory Ltd | Peripheral blood stream improving agent |
JP2007112782A (en) * | 2004-12-22 | 2007-05-10 | Towa Corporation 株式会社 | Estrogen-increasing composition, estrogen-increasing food, estrogen-increasing medicament, and progesterone-increasing composition |
JP4627477B2 (en) * | 2005-10-07 | 2011-02-09 | サントリーホールディングス株式会社 | Maca extract-containing food and drink containing acetic acid or vinegar |
KR100814133B1 (en) | 2006-12-05 | 2008-03-18 | 주식회사 내추럴하우스 | Method for maca extracts with high content of macamide |
KR101346884B1 (en) * | 2011-10-17 | 2014-01-06 | (주)풀무원홀딩스 | Composition for preventing and improving inflammatory bowel diseases and anti-fatigue comprising extract of maca as an active ingredient |
ES2604356T3 (en) | 2011-12-14 | 2017-03-06 | Yeda Research And Development Co. Ltd. | Subpopulation of human monocytes for the treatment of lesions of the central nervous system |
CN103275012B (en) * | 2013-06-08 | 2015-07-01 | 中国科学院过程工程研究所 | Preparation method and application of Maca alkaloid |
CN104513173B (en) * | 2013-09-30 | 2018-03-06 | 中国科学院过程工程研究所 | A kind of Maca extract, Its Preparation Method And Use |
CN103864949B (en) * | 2014-03-20 | 2016-02-03 | 北京中科健宇生物科技有限公司 | Membrane separation technique is utilized to prepare maca polysaccharide, the alkaloidal method of agate coffee |
CN105146511A (en) * | 2015-07-31 | 2015-12-16 | 昆明博尚生物技术有限公司 | Processing method for improving specific active components of Peruvian ginseng |
CN105367677A (en) * | 2015-12-01 | 2016-03-02 | 中国药科大学 | Maca polysaccharide of immunoregulatory activity and preparation method therefor |
CN105348357B (en) * | 2015-12-03 | 2017-06-09 | 中国科学院过程工程研究所 | A kind of maca saponin, its preparation method and application |
CN105853491B (en) * | 2016-04-29 | 2019-12-03 | 杭州纳趣实业有限公司 | A method of the composition is prepared with the composition for promoting fecundity function and by raw material of maca |
DE202023001867U1 (en) | 2023-09-03 | 2023-10-17 | Penta Phi Eg | Formulation comprising at least one type of cress |
Family Cites Families (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPH0812565A (en) * | 1994-06-29 | 1996-01-16 | Shiseido Co Ltd | Skin external preparation |
US6267995B1 (en) | 1999-03-03 | 2001-07-31 | Pure World Botanicals, Inc. | Extract of Lepidium meyenii roots for pharmaceutical applications |
-
1999
- 1999-03-03 US US09/261,806 patent/US6267995B1/en not_active Expired - Lifetime
-
2000
- 2000-03-03 CA CA2362858A patent/CA2362858C/en not_active Expired - Fee Related
- 2000-03-03 EP EP00917719A patent/EP1180006A4/en not_active Ceased
- 2000-03-03 WO PCT/US2000/005607 patent/WO2000051548A2/en active Application Filing
- 2000-03-03 AU AU38649/00A patent/AU3864900A/en not_active Abandoned
-
2001
- 2001-06-08 US US09/878,141 patent/US20020042530A1/en not_active Abandoned
- 2001-10-19 US US10/002,757 patent/US6428824B1/en not_active Expired - Lifetime
-
2002
- 2002-05-02 US US10/138,030 patent/US6552206B1/en not_active Ceased
-
2007
- 2007-11-21 US US11/943,867 patent/USRE43005E1/en not_active Expired - Lifetime
Also Published As
Publication number | Publication date |
---|---|
US6428824B1 (en) | 2002-08-06 |
WO2000051548A3 (en) | 2001-11-15 |
EP1180006A4 (en) | 2004-06-16 |
US6552206B1 (en) | 2003-04-22 |
US20020042530A1 (en) | 2002-04-11 |
EP1180006A2 (en) | 2002-02-20 |
CA2362858A1 (en) | 2000-09-08 |
WO2000051548A2 (en) | 2000-09-08 |
CA2362858C (en) | 2010-12-21 |
US6267995B1 (en) | 2001-07-31 |
AU3864900A (en) | 2000-09-21 |
USRE43005E1 (en) | 2011-12-06 |
US20020061341A1 (en) | 2002-05-23 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
USRE43005E1 (en) | Compositions and methods for their preparation from lepidium | |
US20030171613A1 (en) | Method for the preparation of oleanolic acid and/or maslinic acid | |
CH695661A5 (en) | Pharmaceutical composition. | |
US20080057117A1 (en) | Pharmaceutical composition made up of cannibus extracts | |
US20140065174A1 (en) | Pharmaceutical composition for enhanching immunity, and extract of poria | |
CN101130561B (en) | Method for producing salidroside and injection containing the same | |
JP2015189745A (en) | Extract composition of fermented food | |
Ma et al. | Study of antimalarial activity of chemical constituents from Diospyros quaesita | |
CN112194698B (en) | Triterpenoid compound and preparation method and application thereof | |
CN111533772A (en) | Preparation method of iridoid compound, iridoid compound and application | |
NO179640B (en) | Alkaloids from Mappia foetida, their use and formulations containing them | |
DE3228006C2 (en) | Deoxybouvardin derivatives, process for their preparation and their use | |
KR101895430B1 (en) | Method of separating fukinolic acid from petasites japonicus | |
KR100519080B1 (en) | Fungicidal composition comprising the extract from Cornu cervi | |
CN112194697A (en) | Novel triterpenoid and application thereof in preparation of medicine for treating cardiovascular diseases | |
CN109180632A (en) | A kind of noval chemical compound isolated from tripterygium wilfordii and preparation method thereof and medical usage | |
HAJI et al. | Phytochemical study of Swertia longifolia | |
CN112047988B (en) | Paederoside monomer compound, preparation method and application thereof | |
Steyn et al. | Alkaloids from the Crinum variabile (Amaryllidaceae)-including a full house of lycorine and its acylated derivatives | |
KR100458003B1 (en) | New anticancer compound and its purification from Asparagus oligoclonos | |
Mohanraj | Phytochemicals in Macrotyloma uniflorum–A Review | |
US7223792B2 (en) | Cytotoxic annonaceous acetogenins from Annona muricata | |
CN106083802A (en) | The pharmaceutical composition of dihydrocodeine bitartrate and the application in biological medicine thereof | |
KR0176415B1 (en) | Farnesyl transferase inhibitor and derivative, process for the preparation thereof and composition containing same | |
JPH1192492A (en) | Method for separating 3-o-glucopyranosyl-gamma-butyrolactone |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
STCF | Information on status: patent grant |
Free format text: PATENTED CASE |
|
AS | Assignment |
Owner name: FLEET CAPITAL CORPORATION, NEW JERSEY Free format text: SECURITY INTEREST;ASSIGNOR:PURE WORLD BOTANICALS, INC., A DELAWARE CORPORATION;REEL/FRAME:014892/0662 Effective date: 20031222 |
|
FEPP | Fee payment procedure |
Free format text: PAYOR NUMBER ASSIGNED (ORIGINAL EVENT CODE: ASPN); ENTITY STATUS OF PATENT OWNER: SMALL ENTITY |
|
FPAY | Fee payment |
Year of fee payment: 4 |
|
AS | Assignment |
Owner name: NATUREX INC., NEW JERSEY Free format text: CHANGE OF NAME;ASSIGNOR:PURE WORLD BOTANICALS, INC.;REEL/FRAME:020112/0301 Effective date: 20051130 |
|
RF | Reissue application filed |
Effective date: 20071121 |
|
FPAY | Fee payment |
Year of fee payment: 8 |