Novel Compounds and Pharmaceutical Preparations
20220184165 · 2022-06-16
Assignee
Inventors
- Jarl Erik Sylvester Wikberg (Sigtuna, SE)
- Philippe Rasoanaivo (Antananarivo, MG)
- Rasolondratovo Benoit (Tolaria, MG)
- Andriantiaray Solofoniaina Razafimhefa (Antananarivo, MG)
Cpc classification
A61K2236/00
HUMAN NECESSITIES
A61K36/58
HUMAN NECESSITIES
C07D493/22
CHEMISTRY; METALLURGY
International classification
A61K36/58
HUMAN NECESSITIES
Abstract
Extracts and pharmaceuticals form Neobeguea mahafalensis, the procedures for their preparation, and their use for eliciting sexual enhancing effect and for treatment of sexual dysfunction, in particular erectile dysfunction and hypoactive desire disorders are disclosed. The structures of the chemical compounds in these extracts causing sexual enhancing effect, the preparation of these compounds and the pharmaceuticals prepared from them are disclosed.
Claims
1. A process for producing an extract from a Neobeguea spp. root tissue, the process for producing the extract involving at least one of the steps: (i) isolating a lipid soluble fraction, and (ii) isolating a high molecular weight fraction.
2. The process according to claim 1 wherein a lipophilic solvent is used at least once, the lipophilic solvent preferably being selected from the group of pentane, n-pentane, 2-methylbutane, 2,2-dimethylpropane, hexane, n-hexane, 2-methylpentane, 3-methylpentane, 2,3-dimethylbutane, 2,2-dimethylbutane, heptane, n-heptane, 2-methylhexane, 3-methylhexane, 2,2-dimethylpentane, 2,3-dimethylpentane, 2,4-dimethylpentane, 3,3-dimethylpentane, 3-ethylpentane, 2,2,3-trimethylbutane, octane, n-octane, 2-methylheptane, 3-methylheptane, 4-methylheptane, 3-ethylhexane, 2,2-dimethylhexane, 2,3-dimethylhexane, 2,4-dimethylhexane, 2,5-dimethylhexane, 3,3-dimethylhexane, 3,4-dimethylhexane, 2-methyl-3-ethylpentane, 3-methyl-3-ethylpentane, 2,2,3-trimethylpentane, 2,2,4-trimethylpentane, isooctane, 2,3,3-trimethylpentane, 2,3,4-trimethylpentane, 2,2,3,3-tetramethylbutane, cyclohexane, benzonitrile, chlorobenzene, diethyl ether, methyl-tert-butyl ether, methylenechloride, dichloromethane, chloroform, carbon tetrachloride, 1,2-dichloroethane, perchloroethylene, trichloroethylene, 1,1,1-trichloroethane, trichloroethene, perchloroethylene, tetrachloroethene, vinylchloride, ethylacetate, methyl ethylketone, propyl acetate, iso-propyl acetate, butyl lactate, n-butyl lactate, iso-butyl lactate, tert-butyl lactate, sec-butyl lactate, butyl acetate, n-butyl acetate, iso-butyl acetate, tert-butyl acetate, sec-butyl acetate, triacetin, 1,2,3-triacetoxypropane, diacetin, glycerol 1,3-diacetate, glycerol 1,2-diacetate, benzene, toluene, xylene, o-xylene, m-xylene, p-xylene, 2,2,4-trimethylpentane, butanone, 2-butanone, pentan-3-one, pentan-2-one, 3-pentanone, 2-pentanone, cyclopentanone, butan-1-ol, butan-2-ol, n-butanol, 1-butanol, sec-butanol, 2-butanol, isobutanol, 2-methyl-1-propanol, 2-methyl-2-propanol, pentanol, 1-pentanol, 3-methyl-1-butanol, 2-methyl-1-butanol, 2,2-dimethyl-1-propanol, 3-pentanol, 2-pentanol, 3-methyl-2-butanol, 3-methyl-2-butanol, 2-methyl-2-butanol, hexanol, heptanol, octanol, monohydroxylated hydrocarbon(s), diethylene glycol, dimethyl sulfoxide, methyl t-butyl ether, N-methyl-2-pyrrolidinone, nitromethane, tetrahydrofuran, triethyl amine, benzylalcohol, carboxylic acid methyl ester, carboxylic acid ethyl ester, fatty acid methyl ester, vegetable oil, animal oil, triglyceride(s), mineral oil, wood turpentine, petroleum ether, naphtha, hydrocarbon solvent, chlorinated hydrocarbon solvent, fiuorinated hydrocarbon solvent, halogenated hydrocarbon solvent, freon, 1-bromo-3-chloropropane, [2-(2-butoxyethoxy)ethyl] acetate, 2-butoxyethyl acetate, cumene, cyclohexanol, cyclohexanone, decahydronaphthalene, n-decane, dibenzyl ether, 1,2-dichlorobenzene, 1,3-dichlorobenzene, 1,4-dichlorobenzene, 1,2-dichloroethane, dimethyl carbonate, diethylene glycol dibutyl ether, diethylene glycol diethyl ether, diethylene glycol mono-n-hexyl ether, diethylene glycol monobutyl ether, diethylene glycol monoethyl ether, diethylene glycol monomethyl ether, ethylbenzene, ethyl formate, 2-ethyl-1-hexanol, dimethyl sulfoxide, 1,1,1,3,3,3-hexafluoro-2-propanol, isoamyl acetate, 2-methylbutyl acetate, 3-methylbutyl acetate, isoamyl butyrate, isobutyl acetate, isopropyl acetate, isopropyl methyl ketone, 1,3,5-trimethylbenzene, (1-methoxy-2-propyl)acetate, methyl acetate, methyl cyclohexane, methylcyclohexanol, 5-methyl-3-heptanone, 3-methyltetrahydropyran, 2-methylpentane, 2-methyl-1-butanol, n-nonane, nitroethane, propylacetate, 1,2-propylene glycol diacetate, propylene carbonate, tetrahydrofuran, tetrahydrofurfuryl alcohol, 1,2,3,4-tetrahydronaphthalene, triethylene glycol, dimethyl ether, methyl ethyl ether, liquid ethane, liquid ethene, liquid propane, liquid n-butane, liquid iso-butane, liquid carbon dioxide, liquid trifluoromethane, liquid chlorotrifluoromethane, liquid trichlorofluoromethane, liquid ammonia.
3. The process according to any one of claims 1 or 2 for producing extracts from a Neobeguea spp. root tissue, the process comprising the steps: (i) extracting the Neobeguea spp. root tissue with a lipophilic solvent to produce a lipid soluble first extract, and (ii) optionally extracting the Neobeguea spp. root tissue from step (i) with a hydrophilic solvent to produce a second extract, (iii) optionally combining the extracts from steps (i) and (ii) to produce a united extract.
4. The process according to any one of claims 1 or 2 for producing extracts from a Neobeguea spp. root tissue, the process comprising the steps: (i) extracting the Neobeguea spp. root tissue with a hydrophilic solvent to produce a first extract, and (ii) extracting the Neobeguea spp. root tissue from step (i) with a lipophilic solvent to produce a lipid soluble second extract, (iii) optionally combining the extracts from steps (i) and (ii) to produce a united extract.
5. The process according to any one of claims 1 or 2 for producing extracts from a Neobeguea spp. root tissue, the process comprising the steps: (i) extracting a Neobeguea spp. root tissue with a hydrophilic solvent to produce a first extract, (ii) extracting the first extract with a lipophilic solvent to produce a lipid soluble second extract.
6. The process according to any one of claims 1 or 2 for producing extracts from a Neobeguea spp. root tissue, the process comprising the steps: (i) extracting the Neobeguea spp. root tissue with a lipophilic solvent to produce a lipophilic first fraction, and (ii) exhausting the lipophilic first fraction with a hydrophilic solvent and isolating the materials not being solubilized by the hydrophilic solvent, thereby producing a lipid soluble extract.
7. The process according to any one of claims 1 or 2 for producing extracts from a Neobeguea spp. root tissue, the process comprising the steps: (i) extracting a Neobeguea spp. root tissue with water to produce a water soluble fraction, and (ii) exhausting the water soluble fraction with a lipophilic solvent that is preferably partially miscible with water, and then isolating the water phase to produce a water soluble extract, and (iii) optionally isolating the high molecular weight fraction from the water soluble extract of step (ii) to produce a high molecular weight fraction, and/or (iv) optionally extracting either the water soluble extract isolated in step (ii) or the high molecular weight fraction isolated in step (iii) with a lipophilic solvent to produce a lipid soluble fraction.
8. The process according to any one of claims 1 or 2 for producing extracts from a Neobeguea spp. root tissue, the process comprising the steps: (i) optionally extracting a Neobeguea spp. root tissue with a lipophilic solvent, and (ii) extracting a Neobeguea spp. root tissue or the Neobeguea spp. root tissue from step (i) with a hydrophilic solvent, to produce a fraction, and (iii) isolating the high molecular weight fraction from the fraction of step (ii) to produce a high molecular weight fraction, and (iv) optionally extracting the high molecular weight fraction from step (iii) with a lipophilic solvent to produce a lipid soluble fraction.
9. A process as in any one of claims 1 or 7 or 8 wherein the high molecular weight fraction is a fraction with molecular weight at least above 900, even more preferably above 1000, more preferably above 1200, even more preferably above 1500, even more preferably above 1800, even more preferably above 2000, even more preferably above 2500, even more preferably above 3000 and most preferably above 5000 daltons.
10. A process as in any one of claims 1 or 7 to 9 wherein the high molecular weight fraction is isolated by chromatography.
11. A process as in any one of claims 1 or 7 to 10 wherein the high molecular weight fraction is isolated using a Sephadex column, most preferably a G-25 column.
12. A process as in any one of claims 1 or 7 to 9 wherein the high molecular weight fraction is isolated by dialysis or ultrafiltration.
13. A process according to any one of claims 1 or 2 for producing a fraction having sexual enhancing activity from a Neobeguea spp. root tissue, the process comprising the steps: (i) extracting a Neobeguea spp. root tissue with a hydrophilic and/or lipophilic solvent to produce an extract or fraction, and (ii) applying the extract or fraction of step (i) to a chromatographic column, and (iii) eluting the bound material in fractions, and selecting an eluted fraction having sexual enhancing activity, the eluted fraction preferably being lipid soluble.
14. A process for producing a fraction having sexual enhancing activity from a Neobeguea spp. root tissue, the process comprising the steps: (i) applying a fraction or extract obtained by any of the processes of claims 1 to 13 to a chromatographic column, and (ii) eluting the bound material in fractions, and selecting an eluted fraction having sexual enhancing activity, the eluted fraction preferably being lipid soluble.
15. A process for producing a lipid soluble fraction having sexual enhancing activity from a Neobeguea spp. root tissue, the process comprising the steps: (i) applying a fraction or extract obtained by any of the processes of claims 1 to 14 to a polar interaction column, and (ii) eluting the bound material in fractions, and selecting an eluted fraction having sexual enhancing activity, the eluted fraction being lipid soluble.
16. A process according to any one of claims 1 or 2 for producing a lipid soluble fraction having sexual enhancing activity from a Neobeguea spp. root tissue, the process comprising the steps: (i) extracting a Neobeguea spp. root tissue with a hydrophilic solvent and/or lipophilic solvent to produce a first extract, and (ii) applying the first extract to a polar interaction column, and (iii) eluting the bound material in fractions, and selecting an eluted fraction having sexual enhancing activity, the eluted fraction being lipid soluble.
17. A process according to any one of claim 15 or 16 wherein the stationary phase of the polar interaction column is selected from the group of silica, alumina, hydroxyapatite, cellulose, vinylalkohol bonded silica, polyamine bonded silica, silanol bonded silica, diol bonded silica, amino bonded silica, anionic bonded silica, amide bonded silica, cationic bonded silica, zwitterionic bonded silica, or any other bonded silica.
18. A process according to any one of claims 15 to 17 wherein the mobile phase is comprised by a lipophilic solvent, or a mixture of lipophilic solvents, with optional additive(s).
19. The process according to claim 18 wherein the lipophilic solvent(s) are selected from the group of pentane, n-pentane, 2-methylbutane, 2,2-dimethylpropane, hexane, n-hexane, 2-methylpentane, 3-methylpentane, 2,3-dimethylbutane, 2,2-dimethylbutane, heptane, n-heptane, 2-methylhexane, 3-methylhexane, 2,2-dimethylpentane, 2,3-dimethylpentane, 2,4-dimethylpentane, 3,3-dimethylpentane, 3-ethylpentane, 2,2,3-trimethylbutane, octane, n-octane, 2-methylheptane, 3-methylheptane, 4-methylheptane, 3-ethylhexane, 2,2-dimethylhexane, 2,3-dimethylhexane, 2,4-dimethylhexane, 2,5-dimethylhexane, 3,3-dimethylhexane, 3,4-dimethylhexane, 2-methyl-3-ethylpentane, 3-methyl-3-ethylpentane, 2,2,3-trimethylpentane, 2,2,4-trimethylpentane, isooctane, 2,3, 3-trimethylpentane, 2,3,4-trimethylpentane, 2,2,3,3-tetramethylbutane, cyclohexane, benzonitrile, chlorobenzene, diethyl ether, methyl-tert-butyl ether, methylenechloride, dichloromethane, chloroform, carbon tetrachloride, 1,2-dichloroethane, perchloroethylene, trichloroethylene, 1,1,1-trichloroethane, trichloroethene, perchloroethylene, tetrachloroethene, vinyl chloride, ethylacetate, methylethylketone, propyl acetate, iso-propyl acetate, butyl lactate, n-butyl lactate, iso-butyl lactate, tert-butyl lactate, sec-butyl lactate, butyl acetate, n-butyl acetate, iso-butyl acetate, tert-butyl acetate, sec-butyl acetate, triacetin, 1,2,3-triacetoxypropane, diacetin, glycerol 1,3-diacetate, glycerol 1,2-diacetate, benzene, toluene, xylene, o-xylene, m-xylene, p-xylene, 2,2,4-trimethylpentane, butanone, 2-butanone, pentan-3-one, pentan-2-one, 3-pentanone, 2-pentanone, cyclopentanone, butan-1-ol, butan-2-ol, n-butanol, 1-butanol, sec-butanol, 2-butanol, isobutanol, 2-methyl-1-propanol, 2-methyl-2-propanol, pentanol, 1-pentanol, 3-methyl-1-butanol, 2-methyl-1-butanol, 2,2-dimethyl-1-propanol, 3-pentanol, 2-pentanol, 3-methyl-2-butanol, 3-methyl-2-butanol, 2-methyl-2-butanol, hexanol, heptanol, octanol, monohydroxylated hydrocarbon(s), diethylene glycol, dimethyl sulfoxide, methyl t-butyl ether, N-methyl-2-pyrrolidinone, nitromethane, tetrahydrofuran, triethyl amine, benzylalcohol, carboxylic acid methyl ester, carboxylic acid ethyl ester, fatty acid methyl ester, vegetable oil, animal oil, triglyceride(s), mineral oil, wood turpentine, petroleum ether, naphtha, hydrocarbon solvent, chlorinated hydrocarbon solvent, fluorinated hydrocarbon solvent, halogenated hydrocarbon solvent, freon, 1-bromo-3-chloropropane, [2-(2-butoxyethoxy)ethyl]acetate, 2-butoxyethyl acetate, cumene, cyclohexanol, cyclohexanone, decahydronaphthalene, n-decane, dibenzyl ether, 1,2-dichlorobenzene, 1,3-dichlorobenzene, 1,4-dichlorobenzene, 1,2-dichloroethane, dimethyl carbonate, diethylene glycol dibutyl ether, diethylene glycol diethyl ether, diethylene glycol mono-n-hexyl ether, diethylene glycol monobutyl ether, diethylene glycol monoethyl ether, diethylene glycol monomethyl ether, ethylbenzene, ethyl formate, 2-ethyl-1-hexanol, dimethyl sulfoxide, 1,1,1, 3,3, 3-hexafluoro-2-propanol, isoamyl acetate, 2-methylbutyl acetate, 3-methylbutyl acetate, isoamyl butyrate, isobutyl acetate, isopropyl acetate, isopropyl methyl ketone, 1,3,5-trimethylbenzene, (I-methoxy-2-propyl)acetate, methyl acetate, methyl cyclohexane, methylcyclohexanol, 5-methyl-3-heptanone, 3-methyltetrahydropyran, 2-methylpentane, 2-methyl-1-butanol, n-nonane, nitroethane, propylacetate, 1,2-propylene glycol diacetate, propylene carbonate, tetrahydrofuran, tetrahydrofurfuryl alcohol, 1,2,3,4-tetrahydronaphthalene, triethylene glycol, dimethyl ether, methyl ethyl ether.
20. A process for producing a lipid soluble fraction having sexual enhancing activity from a Neobeguea spp. root tissue, the process comprising the steps: (i) applying a fraction or extract obtained by any of the processes of claims 1 to 19 to a hydrophobic interaction column, and (ii) eluting the bound material in fractions, and selecting an eluted fraction having sexual enhancing activity, the eluted fraction being lipid soluble.
21. A process according to any one of claims 1 or 2 for producing a lipid soluble fraction having sexual enhancing activity from a Neobeguea spp. root tissue, the process comprising the steps: (i) extracting a Neobeguea spp. root tissue with a hydrophilic solvent and/or lipophilic solvent to produce a first extract, and (iii) applying the first extract to a hydrophobic interaction column, (iv) eluting the bound material in fractions, and selecting an eluted fraction having sexual enhancing activity, the eluted fraction being lipid soluble.
22. A process according to any one of claims 1 or 2 for producing a lipid soluble fraction having sexual enhancing activity from a Neobeguea spp. root tissue, the process comprising the steps: (i) optionally extracting a Neobeguea spp. root tissue with a hydrophilic solvent to produce a first extract, (ii) extracting a Neobeguea spp. root tissue or the first extract of step (i) with a lipophilic solvent to produce a second extract, (iii) applying the second extract from step (ii) to a hydrophobic interaction column, (iv) eluting the bound material in fractions, and selecting an eluted fraction having sexual enhancing activity, the eluted fraction being lipid soluble.
23. A process according to any one of claims 20 to 22, wherein the hydrophobic interaction column is derivatized with C.sub.4-22 groups, preferably C.sub.18 groups.
24. A process according to any one of claims 3 to 23 wherein at least one of the hydrophilic solvent(s) used is selected from the group of methanol, ethanol, acetonitrile, propionitrile, propanol, propan-1-ol, propan-2-ol, dimethyl sulfoxide, formamide, dimethylformamide, acetone, tetrahydrofurane, glycol, glycerol, dioxane, 1,4-dioxane, formic acid, acetic acid, propionic acid, butyric acid, 2-methylpropanoic acid, 3-oxobutanamide, N,N-diethylacetamide, N,N-diethyl acetoacetamide, propylene glycol, methylsulfonylmethane, ethanol amine, tert-butyl alcohol, diethylene glycol, dimethyl ether, 1,2-dimethoxy-ethane, ethylene glycol, hexamethylphosphoramide, hexamethylphosphorous triamide, pyridine, 2-methyltetrahydrofuran, 3-methyl tetrahydropyran, 2-methylpyridine, 1,3-propanediol, sulfolane, triethylene glycol, tetraethylene glycol, tetrahydrofurfuryl alcohol, triethanolamine, triethyl phosphate, triethylene glycol, triethylene glycol dimethyl ether, triethylene glycol monomethyl ether, N-methylpyrrolidone, liquid carbon dioxide.
25. A process according to any one of claims 3 to 24 wherein at least one of the hydrophilic solvent(s) used is water.
26. A process as claimed in any one of claims 1 to 25 wherein the extract or fraction has sexual enhancing activity.
27. A process as claimed in any one of claims 1 to 26 wherein the extract or fraction contains at least 0.05%, more preferably at least 0.1%, even more preferably at least 0.15%, even more preferably at least 0.2%, and even more preferably at least 0.25%, and most preferably at least 0.3% of a chemical as defined in any one of claims 40 to 42.
28. A process as claimed in any one of claims 1 to 27 wherein the lipid soluble fraction or extract is entirely soluble with an amount of at least 1 mg/ml, more preferably at least 2 mg/ml, even more preferably at least 4 mg/ml, even more preferably at least 8 mg/ml, even more preferably at least 12 mg/ml, even more preferably at least 16 mg/ml, and most preferably at least 18 mg/ml in sun flower oil and/or in octan-1-ol at the temperature 20° C.
29. A process as claimed in any one of claims 1 to 28 wherein one or more of Neobeguea spp. stem bark, seeds, branches, trunk, leaves or fruit are used instead of the root tissue.
30. A process for making a formulation, comprising combining an extract or fraction obtained by or obtainable by a process as claimed in any one of claims 1 to 29 with any one of a pharmaceutically acceptable filler, carrier, excipient, dissolvent, diluent, lubricant, glidant, antioxidant and/or additive, preferably those being selected from the group of pharmaceutically acceptable oil (such as peanut oil, corn oil, sesame oil, cotton oil, olive oil, soybean oil), triglyceride, diglyceride, cocoa butter, coconut fat, hydrogenated fat, hydrogenated animal oil, hydrogenated vegetable oil, solid triglycerides, solid fat, malleable fat, Fattibase, Wecobee bases, Witespol bases, cetyl ester wax, beeswax, glycerol, polysorbate 80, polyethylene glycol, propylene glycol, isopropyl alcohol, magnesium stearate, stearic acid, talc, silicon dioxide, lauryl sulphate, sodium starch glycolate, detergent, oil detergent mixture, oil surfactant mixture, emulsifiable oil, self-emulsifying system, Neobee M5, tri caprylic/capric triglyceride ester, Miglyol 810, propyleneglycol dicaprylate, Sefsol 228, ethoxylated plant fats, ethanol, polyvinyl pyrrolidone, Tween, polyoxyethylenesorbitan monolaurate, triacetin, diacetin, ascorbic acid, ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, hypophosphorous acid, monothioglycerol, propyl gallate, sodium ascorbate, sodium bisulphite, sodium formaldehyde sulfoxylate, sodium meta bisulphite, butylparaben, ethyl paraben, benzoic acid, propylparaben, sodium benzoate, sodium propionate, benzalkonium chloride, benzethonium chloride, benzyl alcohol, cetylpyridinium chloride, chlorobutanol, phenol, phenylethyl alcohol, phenylmercuric nitrate, thiomersal, lactose, microcrystalline cellulose, starch, powdered sucrose, calcium phosphate, acacia, alginic acid, carboxymethylcellulose sodium, compressible sugar, ethylcellulose, gelatine, liquid glucose, methylcellulose, povidone, pregelatinized starch, croscarmellose, crospovidone, cation exchange resin, silica, colloidal silica, cornstarch, calcium stearate, mineral oil, anis oil, cinnamon oil, cocoa, menthol, orange oil, peppermint oil, vanillin, ginger, colorant.
31. A process a claimed in any one of the previous claims wherein the Neobeguea spp. is collected in the wild or cultivated.
32. A process a claimed in any one of the previous claims wherein the Neobeguea spp. is subjected to stress.
33. A process as claimed in any one of the previous claims wherein the content of a chemical compound as defined in any one of claims 40 to 42 is assayed.
34. A process as claimed in claim 33 wherein the content of the chemical substance is assayed by use of chromatography and/or mass spectrometry.
35. A composition comprising: (i) a lipid soluble extract from a Neobeguea spp. tissue and/or (ii) a high molecular weight fraction from a Neobeguea spp. tissue, wherein the Neobeguea spp. tissue is preferably a root tissue.
36. A composition comprising an extract or fraction obtained by or obtainable by the process of any one of claims 1 to 34.
37. A composition as claimed in claim 35 or 36, which additionally comprises any one of a pharmaceutically acceptable filler, carrier, excipient, dissolvent, diluent, lubricant, glidant, antioxidant and/or additive, preferably those being selected from the group of pharmaceutically acceptable oil (such as peanut oil, corn oil, sesame oil, cotton oil, olive oil, soybean oil), triglyceride, diglyceride, cocoa butter, coconut fat, hydrogenated fat, hydrogenated animal oil, hydrogenated vegetable oil, solid triglycerides, solid fat, malleable fat, Fattibase, Wecobee bases, Witespol bases, cetyl ester wax, beeswax, glycerol, polysorbate 80, polyethylene glycol, propylene glycol, isopropyl alcohol, magnesium stearate, stearic acid, talc, silicon dioxide, lauryl sulphate, sodium starch glycolate, detergent, oil detergent mixture, oil surfactant mixture, emulsifiable oil, self-emulsifying system, Neobee M5, tri caprylic/capric triglyceride ester, Miglyol 810, propyleneglycol dicaprylate, Sefsol 228, ethoxylated plant fats, ethanol, polyvinyl pyrrolidone, Tween, polyoxyethylenesorbitan monolaurate, triacetin, diacetin, ascorbic acid, ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, hypophosphorous acid, monothioglycerol, propyl gallate, sodium ascorbate, sodium bisulphite, sodium formaldehyde sulfoxylate, sodium meta bisulphite, butylparaben, ethyl paraben, benzoic acid, propylparaben, sodium benzoate, sodium propionate, benzalkonium chloride, benzethonium chloride, benzyl alcohol, cetylpyridinium chloride, chlorobutanol, phenol, phenylethyl alcohol, phenylmercuric nitrate, thiomersal, lactose, microcrystalline cellulose, starch, powdered sucrose, calcium phosphate, acacia, alginic acid, carboxymethylcellulose sodium, compressible sugar, ethylcellulose, gelatine, liquid glucose, methylcellulose, povidone, pregelatinized starch, croscarmellose, crospovidone, cation exchange resin, silica, colloidal silica, cornstarch, calcium stearate, mineral oil, anis oil, cinnamon oil, cocoa, menthol, orange oil, peppermint oil, vanillin, ginger, colorant.
38. A composition comprising a formulation obtained by or obtainable by the process of claim 30.
39. A composition as claimed in any one of claims 35 to 38 comprised in the form of any one of an oral administrable, parenteral administrable, systemically administrable, capsule, tablet, powder, granulate, suppository, insert, lozenge, troche, buccal tablet, sublingual tablet, compressed tablet, multiple compressed tablet, molded tablet, chewable tablet, effervescent tablet, tablet triturate, sugar coated tablet, film-coated tablet, gelatine-coated tablet, enteric-coated tablet, dispensing tablet, hypodermic tablet, extended release tablet, instant disintegrating tablet, immediate release tablet, dermal formulation, ointment, cream, gel, transdermal formulations, solutions, tincture, injectable, parenteral, implantat, formulation for urethral administration, formulations for topical administration, ophthalmic solution, vaginal formulation, inhalant, disperse system, emulsion, multitablet system, microencapsulated drug system, osmotic pump, subdermal implant, ocular system, parenteral system, vaginal system, coated bead system, granule, microsphere, modified-release system, extended-release system, delayed-release system, repeat action system, targeted release system, ion-exchange resin system, slowly eroding and/or hydrophilic matrix system, inert plastic matrix embedded system, ointment, cream, gel, solution, water solution, tincture, oil solution, pill or infusion package, or tea bag.
40. A chemical compound having the general formula of structure III: ##STR00037## wherein R1 is a substituent having from one to 30 atoms of any type(s), more preferably one to 16 atoms, more preferably one to 12 atoms and most preferably one to 10 atoms, with hydrogen, oxygen, carbon, sulphur, nitrogen, phosphorous and halogen atoms being preferred; the R1 substituent being a hydrogen and/or linear, branched and/or cyclic structure; the R1 substituent preferably comprising of from zero to 10 heavy atoms, even more preferably zero to 6 heavy atoms, even more preferably zero to 4 heavy atoms, even more preferably zero to 3 heavy atoms, and most preferably zero to 2 heavy atoms; most preferably the substituent being comprised of any one of hydrogen, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, cyclopropyl, cyclopropenyl, cyclobutyl, cyclopentyl, cyclohexyl, alkyl, cyclic alkyl, halogenated alkyl, halogenated cyclic alkyl, propenyl, halogenated propenyl, alkenyl, halogenated alkenyl, halogenated cyclic alkenyl, alkynyl, halogenated alkynyl, aryl, halogenated aryl, with methyl being most preferred, and wherein R2 is a substituent having from one to 30 atoms of any type(s), more preferably one to 20 atoms, and most preferably one to 16 atoms, with hydrogen, oxygen, carbon, sulphur, nitrogen, phosphorous and halogen being preferred; the R2 substituent being a hydrogen or a linear, branched and/or cyclic structure; the R2 substituent preferably comprising of from zero to 10 heavy atoms, even more preferably zero to 8 heavy atoms, even more preferably zero to 6 heavy atoms, even more preferably zero to 5 heavy atoms; most preferably the substituent being composed of any one of hydrogen, methyl, ethyl, propyl, isopropyl, cyclopropyl, cyclopropenyl, butyl, isobutyl, cyclobutyl, alkyl, halogenated alkyl, cyclic alkyl, halogenated cyclic alkyl, propenyl, halogenopropenyl, alkenyl, halogenated alkenyl, cyclic alkenyl, halogenated cyclic alkenyl, alkynyl, halogenated alkynyl, aryl, halogenated aryl, acetyl, halogenoacetyl, propionyl, halogenopropionyl, butyryl, halogenobutyryl, isobutyryl, halogenoisobutyryl, alkyryl, halogenated alkyryl, cyclic alkyryl, halogenated cyclic alkyryl, benzoyl, aryryl, with a hydrogen or acetyl group or isobutyryl group being most preferred, and wherein R3 is a substituent having from one to 30 atoms of any type(s), more preferably one to 20 atoms, and most preferably one to 16 atoms, with hydrogen, oxygen, carbon, sulphur, nitrogen, phosphorous and halogen being preferred; the R3 substituent being a hydrogen or a linear, branched and/or cyclic structure; the R3 substituent preferably comprising of from zero to 10 heavy atoms, even more preferably zero to 8 heavy atoms, even more preferably zero to 6 heavy atoms, even more preferably zero to 5 heavy atoms; most preferably the substituent being composed of any one of hydrogen, methyl, ethyl, propyl, isopropyl, cyclopropyl, cyclopropenyl, butyl, isobutyl, cyclobutyl, alkyl, halogenated alkyl, cyclic alkyl, halogenated cyclic alkyl, propenyl, halogenopropenyl, alkenyl, halogenated alkenyl, cyclic alkenyl, halogenated cyclic alkenyl, alkynyl, halogenated alkynyl, aryl, halogenated aryl, acetyl, halogenoacetyl, propionyl, halogenopropionyl, butyryl, halogenobutyryl, isobutyryl, halogenoisobutyryl, alkyryl, halogenated alkyryl, cyclic alkyryl, halogenated cyclic alkyryl, benzoyl, aryryl, with a hydrogen or acetyl group or isobutyryl group being most preferred, and wherein R4 is a substituent connected by single or double bond having from one to 32 atoms, more preferably one to 18 atoms, more preferably one to 15 atoms more preferably one to 12 atoms, and most preferably one to 9 atoms with hydrogen, oxygen, carbon, sulphur, nitrogen, phosphorous and halogen atoms being preferred; the R4 substituent preferably being a hydrogen or a linear or branched and/or cyclic structure; the R4 substituent preferably comprising between 0 to 12 heavy atoms, even more preferably between 0 to 11 heavy atoms, even more preferably between 0 to 10 heavy atoms, even more preferably between 0 to 9 heavy atoms, even more preferably between 0 to 8 heavy atoms, even more preferably between 0 to 7 heavy atoms, even more preferably between 0 to 6 heavy atoms, and most preferably between 0 to 5 heavy atoms; most preferably the substituent being comprised of any of hydrogen, halogeno, oxo, hydroxy, methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, alkoxy, halogenated alkoxy, ethenyloxy, propenyloxy, alkenyloxy, halogenated alkenyloxy, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, alkyl, halogenated alkyl, hydroxymethyl, hydroxyethyl, hydroxypropyl, hydroxyispropyl, hydroxybutyl, hydroxyisobutyl, hydroxyalkyl, halogenenated alkyl, methylene, ethenyl, propenyl, isopropenyl, butenyl, isobutenyl, alkenyl, halogenated alkenyl, acetyl, halogenoacetyl, propionyl, butyryl, isobutyryl, alkyryl, halogenated alkyryl, acetyloxy, halogenoacetyloxy, propionyloxy, isopropionyloxy, butyryloxy, isobutyryloxy, alkyryloxy, halogenated alkyryloxy, 2-oxy-2-methyl-ethyl, hydroxy-oxomethyl, 2-hydroxy-2-oxoethyl, 3-hydroxy-3-oxopropionyl, methoxy-oxomethyl, ethoxy-oxomethyl, propoxy-oxomethyl, isopropoxy-oxomethyl, butoxy-oxomethyl, isobutoxy-oxomethyl, alkoxy-oxomethyl, 2-methoxy-2-oxoethyl, 2-ethoxy-2-oxoethyl, 2-propoxy-2-oxoethyl, 2-isopropoxy-2-oxoethyl, 2-butoxy-2-oxoethyl, 2-isobutoxy-2-oxoethyl, 2-alkoxy-2-oxoethyl, hydroxymethylene, 1-hydroxyethylidene, 1-hydroxypropylidene, 1-hydroxy-2-methylpropylidene, 1-acetyloxy-2-methylpropylidene, 1-halogenoacetyloxy-2-methylpropylidene, 1-alkyryloxy-2-methylpropylidene, 1-halogenoalkyryloxy-2-methylpropylidene, with hydrogen or isobutyryl or 1-hydroxy-2-methylpropylidene being most preferred, and wherein R5 is a substituent connected by single or double bond having from one to 32 atoms, more preferably one to 18 atoms, more preferably one to 15 atoms, more preferably one to 12, more preferably or one to 10 atoms, more preferably one to 8 atoms and most preferably one to 7 atoms, with hydrogen, oxygen, carbon, sulphur, nitrogen, phosphorous and halogen atoms being preferred; the R5 substituent preferably being a hydrogen or an oxygen or a linear or branched and/or cyclic structure; the R5 substituent preferably comprising between 0 to 12 heavy atoms, even more preferably between 0 to 11 heavy atoms, even more preferably between 0 to 10 heavy atoms, even more preferably between 0 to 9 heavy atoms, even more preferably between 0 to 8 heavy atoms, even more preferably between 0 to 7 heavy atoms, even more preferably between 0 to 6 heavy atoms, more preferably between 1 to 5 heavy atoms and most preferably between 1 to 4 heavy atoms; most preferably the substituent being comprised of any of hydrogen, halogeno, oxo, hydroxy, methoxy, ethoxy, propoxy, butoxy, isobutoxy, alkoxy, halogenated alkoxy, ethenyloxy, propenyloxy, alkenyloxy, halogenated alkyloxy, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, alkyl, halogenated alkyl, hydroxymethyl, hydroxyethyl, hydroxypropyl, hydroxyisopropyl, hydroxybutyl, hydroxyisobutyl, hydroxyalkyl, methylene, ethenyl, propenyl, isopropenyl, butenyl, isobutenyl, alkenyl, halogenated alkenyl, acetyl, halogenoacetyl, propionyl, isopropionyl, butyryl, isobutyryl, alkyryl, halogenated alkyryl, acetyloxy, halogenoacetyloxy, propionyloxy, halogenopropionyloxy, butyryloxy, halogenobutyryloxy, isobutyryloxy, halogenoisobutyryloxy, alkyryloxy, halogenated alkyryloxy, 2-oxy-2-methyl-ethyl, hydroxy-oxomethyl, 2-hydroxy-2-oxoethyl, 3-hydroxy-3-oxopropionyl, methoxy-oxomethyl, ethoxy-oxomethyl, propoxy-oxomethyl, isopropoxy-oxomethyl, butoxy-oxomethyl, isobutoxy-oxomethyl, alkoxy-oxomethyl, 2-methoxy-2-oxoethyl,2-ethoxy-2-oxoethyl, 2-propoxy-2-oxoethyl, 2-isopropoxy-2-oxoethyl, 2-butoxy-2-oxoethyl, 2-isobutoxy-2-oxoethyl, 2-alkoxy-2-oxoethyl, hydroxymethylene, 1-hydroxyethylidene, 1-hydroxypropylidene, 1-hydroxy-2-methylpropylidene, with oxo and acetyloxy being preferred, or a pharmaceutically acceptable salt thereof, and which is optionally capable of eliciting a sexual enhancing effect.
41. A chemical compound having the general formula of structure III: ##STR00038## wherein R1 is any one of hydrogen, methyl or ethyl, with methyl being most preferred, and wherein R2 is any one of hydrogen, methyl, ethyl, acetyl, halogenoacetyl, propionyl, butyryl, isobutyryl, with a hydrogen or acetyl group or isobutyryl group being most preferred, and wherein R3 is any one of hydrogen, methyl, ethyl, acetyl, halogenoacetyl, propionyl, butyryl, isobutyryl, with a hydrogen or acetyl group or isobutyryl group being most preferred, and wherein R4 is any one of hydrogen, isobutyryl, 1-hydroxy-2-methylpropylidene, 1-methoxy-2-methylpropylidene, 1-acetyloxy-2-methylpropylidene, 1-halogenoacetyloxy-2-methylpropylidene, with hydrogen or isobutyryl or 1-hydroxy-2-methylpropylidene being most preferred, and wherein R5 is any one of oxo, hydroxy, methoxy, ethoxy, acetyloxy, halogenoacetyloxy, propionyloxy, butyryloxy, isobutyryloxy, with oxo and acetyloxy being preferred, or a pharmaceutically acceptable salt thereof, and which chemical substance (chemical compound) is optionally capable of eliciting a sexual enhancing effect.
42. A compound according to claim 40 or 41 having any one of the structures of ##STR00039## ##STR00040## ##STR00041## ##STR00042## ##STR00043## ##STR00044## ##STR00045## ##STR00046## ##STR00047## ##STR00048## or a pharmaceutically acceptable salt thereof, and which is optionally capable of eliciting a sexual enhancing effect.
43. A chemical compound according to any of claims 40 to 42 having the summary formula C.sub.37H.sub.46O.sub.13 or the summary formula C.sub.37H.sub.46O.sub.14, and which is optionally having sexual enhancing effect.
44. A chemical compound comprising a glycone of the chemical compound according to any of claims 40 to 43, and which is optionally having sexual enhancing effect.
45. A chemical compound according to any of claims 40 to 44 obtained from or being obtainable from Neobeguea spp. plant tissue, and which is optionally having sexual enhancing effect.
46. A chemical compound according to any of claims 40 to 45 which sexual enhancing activity amounts to at least 0.1 U/mg, even more preferably to at least 0.3 U/mg, even more preferably to at least 1 U/mg, even more preferably to at least 3 U/mg and most preferably to at least 10 U/mg, the unit activity preferably being estimated as U.sub.mnt.
47. A chemical compound comprising a pro-drug of the compound according to any of claims 40 to 46.
48. A chemical compound according to any one any one of claims 40 to 47 obtained from or being obtainable from a natural source, the natural source preferably being a species belonging to the Meliaceae family.
49. A chemical compound according to of any one of claims 40 to 48 obtained by or being obtainable by chemical synthesis or chemical semi-synthesis.
50. A chemical compound according to any one of claims 40 to 49 which is synthesized starting from a raw-material originating from a natural source, the natural source preferably being a species belonging to the Meliaceae family.
51. A chemical compound according to of any one of claims 40 to 50 obtained by chemical synthesis or chemical semi-synthesis, by a process that at least in part involves hydrolysis and/or esterification and/or alkylation.
52. A chemical compound according to any one of claims 40 to 50 being obtained using a raw material comprising any one of a limonoid, phragmalin, neobeguin, leandreanin A, leandreanin B, leandreanin C, pseudrelone A2, bussein A, B, C, D, E, F, G, H, J, K, L, M, the compound optionally having a sexual enhancing effect.
53. The chemical compound according to any one of claims 40 to 52 in radioactive form.
54. A pharmaceutical composition comprising the chemical compound of any one of claims 40 to 53, optionally together with one or more pharmaceutically acceptable adjuvants, carriers, disolvants or diluents.
55. A pharmaceutical composition comprising the chemical compound of any one of claims 40 to 53 in pharmaceutically acceptable oil, fat and/or triglyceride, which optionally in addition contains pharmaceutically acceptable additives, adjuvants, carriers, disolvants, preservatives or diluents.
56. A pharmaceutical composition as claimed in claim 54 or 55, which additionally comprises any one of a pharmaceutically acceptable filler, carrier, excipient, dissolvent, diluent, lubricant, glidant, antioxidant and/or additive, preferably those being selected from the group of pharmaceutically acceptable oil (such as peanut oil, corn oil, sesame oil, cotton oil, olive oil, soybean oil), triglyceride, diglyceride, cocoa butter, coconut fat, hydrogenated fat, hydrogenated animal oil, hydrogenated vegetable oil, solid triglycerides, solid fat, malleable fat, Fattibase, Wecobee bases, Witespol bases, cetyl ester wax, beeswax, glycerol, polysorbate 80, polyethylene glycol, propylene glycol, isopropyl alcohol, magnesium stearate, stearic acid, talc, silicon dioxide, lauryl sulphate, sodium starch glycolate, detergent, oil detergent mixture, oil surfactant mixture, emulsifiable oil, self-emulsifying system, Neobee M5, tri caprylic/capric triglyceride ester, Miglyol 810, propyleneglycol dicaprylate, Sefsol 228, ethoxylated plant fats, ethanol, polyvinyl pyrrolidone, Tween, polyoxyethylenesorbitan monolaurate, triacetin, diacetin, ascorbic acid, ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, hypophosphorous acid, monothioglycerol, propyl gallate, sodium ascorbate, sodium bisulphite, sodium formaldehyde sulfoxylate, sodium meta bisulphite, butylparaben, ethyl paraben, benzoic acid, propylparaben, sodium benzoate, sodium propionate, benzalkonium chloride, benzethonium chloride, benzyl alcohol, cetylpyridinium chloride, chlorobutanol, phenol, phenylethyl alcohol, phenylmercuric nitrate, thiomersal, lactose, microcrystalline cellulose, starch, powdered sucrose, calcium phosphate, acacia, alginic acid, carboxymethylcellulose sodium, compressible sugar, ethylcellulose, gelatine, liquid glucose, methylcellulose, povidone, pregelatinized starch, croscarmellose, crospovidone, cation exchange resin, silica, colloidal silica, cornstarch, calcium stearate, mineral oil, anis oil, cinnamon oil, cocoa, menthol, orange oil, peppermint oil, vanillin, ginger, colorant.
57. A pharmaceutical composition as claim in any one of claims 54 to 56 in form of oral administrable, parenteral administrable, systemically administrable, capsule, tablet, powder, granulate, suppository, insert, lozenge, troche, buccal tablet, sublingual tablet, compressed tablet, multiple compressed tablet, molded tablet, chewable tablet, effervescent tablet, tablet triturate, sugar coated tablet, film-coated tablet, gelatine-coated tablet, enteric-coated tablet, dispensing tablet, hypodermic tablet, extended release tablet, instant disintegrating tablet, immediate release tablet, dermal formulation, ointment, cream, gel, transdermal formulations, solutions, tincture, injectable, parenteral, implantat, formulation for urethral administration, formulations for topical administration, ophthalmic solution, vaginal formulation, inhalant, disperse system, emulsion, multitablet system, microencapsulated drug system, osmotic pump, subdermal implant, ocular system, parenteral system, vaginal system, coated bead system, granule, microsphere, modified-release system, extended-release system, delayed-release system, repeat action system, targeted release system, ion-exchange resin system, slowly eroding and/or hydrophilic matrix system, inert plastic matrix embedded system, ointment, cream, gel, solution, water solution, tincture, oil solution, pill.
58. A Neobeguea spp. root tissue or extract thereof for use as a medicament or for use in therapy.
59. A Neobeguea spp. tissue or extract thereof, preferably a Neobeguea spp. root tissue or extract thereof, for use in eliciting a sexual enhancing effect.
60. A Neobeguea spp. tissue or extract thereof, preferably a Neobeguea spp. root tissue or extract thereof, for use in the treatment of any one of sexual dysfunction, erectile dysfunction, ejaculatory dysfunction or hypoactive sexual desire disorder.
61. A Neobeguea spp. tissue or extract thereof as claimed in any one of claims 58 to 60 wherein the composition is administered systemically or non-topically.
62. A composition as claimed in any one of claims 35 to 39 or 54 to 57 or a chemical compound as claimed in any one of claims 40 to 53 for use as a medicament or for use in therapy.
63. A composition as claimed in any one of claims 35 to 39 or 54 to 57 or a chemical compound as claimed in any one of claims 40 to 53 for use in eliciting a sexual enhancing effect.
64. A composition as claimed in any one of claims 35 to 39 or 54 to 57 or a chemical compound as claimed in any one of claims 40 to 53 for use in treatment of any one of sexual dysfunction, erectile dysfunction, ejaculatory dysfunction or hypoactive sexual desire disorder.
65. A composition as claimed in any of claims 62 to 64 wherein the composition is administered systemically or non-topically.
66. A method of eliciting a sexual enhancing effect comprising administering to a subject a pharmaceutically effective amount of a Neobeguea spp. tissue or extract thereof, preferably a Neobeguea spp. root tissue or extract thereof.
67. A method of treating any one of sexual dysfunction, erectile dysfunction, ejaculatory dysfunction or hypoactive sexual desire disorder comprising administering to a subject a pharmaceutically effective amount of a Neobeguea spp. tissue or extract thereof, preferably a Neobeguea spp. root tissue or extract thereof.
68. A method of eliciting a sexual enhancing effect comprising administering to a subject a pharmaceutically effective amount of a composition as claimed in any one of claims 35 to 39 or 54 to 57 or a chemical compound as claimed in any one of claims 40 to 53.
69. A method of treating any one of sexual dysfunction, erectile dysfunction, ejaculatory dysfunction or hypoactive sexual desire disorder comprising administering to a subject a pharmaceutically effective amount of a composition as claimed in any one of claims 35 to 39 or 54 to 57 or a chemical compound as claimed in any one of claims 40 to 53.
70. A method as claimed in any one of claims 66 to 69, wherein the tissue, extract or composition is administered systemically or non-topically.
71. Use of a Neobeguea spp. tissue or extract thereof, preferably a Neobeguea spp. root tissue or extract thereof, in the manufacture of a medicament for eliciting a sexual enhancing effect.
72. Use of a Neobeguea spp. tissue or extract thereof, preferably a Neobeguea spp. root tissue or extract thereof, in the manufacture of a medicament for treatment of any one of sexual dysfunction, erectile dysfunction, ejaculatory dysfunction or hypoactive sexual desire disorder.
73. Use of a composition as claimed in any one of claims 35 to 39 or 54 to 57 or a chemical compound as claimed in any one of claims 40 to 53 in the manufacture of a medicament for eliciting a sexual enhancing effect.
74. Use of a composition as claimed in any one of claims 35 to 39 or 54 to 57 or a chemical compound as claimed in any one of claims 40 to 53 in the manufacture of a medicament for treatment of any one of sexual dysfunction, erectile dysfunction, ejaculatory dysfunction or hypoactive sexual desire disorder.
75. A use as claimed in any one of claims 71 to 74, wherein the tissue, extract or composition is administered systemically or non-topically.
76. Use of a Neobeguea spp. tissue or extract thereof, preferably a Neobeguea spp. root tissue or extract thereof, for eliciting a sexual enhancing effect.
77. Use of a Neobeguea spp. tissue or extract thereof, preferably a Neobeguea spp. root tissue or extract thereof, for treatment of any one of sexual dysfunction, erectile dysfunction, ejaculatory dysfunction or hypoactive sexual desire disorder.
78. Use of a composition as claimed in any one of claims 35 to 39 or 54 to 57 or a chemical compound as claimed in any one of claims 40 to 53 for eliciting a sexual enhancing effect.
79. Use of a composition as claimed in any one of claims 35 to 39 or 54 to 57 or a chemical compound as claimed in any one of claims 40 to 53 of any one of sexual dysfunction, erectile dysfunction, ejaculatory dysfunction or hypoactive sexual desire disorder.
80. A use as claimed in any one of claims 76 to 79, wherein the tissue, extract or composition is administered systemically or non-topically.
Description
BRIEF DESCRIPTION OF FIGURES
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EXAMPLES
[0456] All samples of Neobegua mahfalensis used herein were collected from Madagascar with permit from Ministere de l′evironment, des eaux et forets et du tourisme, Repoblikan′i Madagaskara, B.P. 243-Nanisana-ANTANANARIVO-101, Madagascar.
Example 1: Water Extract of Stem Bark oïNeobeguea mahafalensis
[0457] 50-100 g of stem bark from Neobeguea mahafalensis was placed in 200-1000 ml of water at room temperature for 3 days. The water turned brown. After removing the water by decantation the water solution was ingested orally as a pharmaceutical.
Example 2: Tincture of Root of Neobeguea mahafalensis
[0458] 0.5 g of ground root from Neobeguea mahafalensis was placed in 2-4.5 ml of ethanol/water (between 50-99% ethanol was used) and allowed stand for 3 days with gentle shaking. The root was sedimented and the supernatant decanted and filtered. The sediment was pressed to yield additional solution, which was also filtered. Portions of the combined supernatant solution (i.e. REtOH extract) were ingested orally as a pharmaceutical.
Example 3: Ethanol Extract of Root of Neobeguea mahafalensis
Example 3 a
[0459] 0.5 g of ground root from Neobeguea mahafalensis was placed in 4.5 ml of 50% aqueous ethanol and gently shaken for 3 days at room temperature. The mixture was then filtered through a lump of cotton wool placed in a conical funnel; afterwards the extracted root on the lump was rinsed with the same solvent (2×1.5 ml). The combined filtrates were evaporated to dryness; the residue was dissolved in 30% acetonitrile in water (10 ml) and freeze-dried. A brown, fluffy powder formed. Yield 133.2 mg. The residual root remaining after extraction was dried in the air keeping it on an open Petri dish. Residual dried root weighed 332 mg.
Example 3b
[0460] 0.5 g of ground root from Neobeguea mahafalensis was placed in 4.0 ml of 70% aqueous ethanol and gently shaken for 3 days at room temperature. The procedures following were then as described for Example 3a. Yield of lyophilized powder was 127.5 mg. Residual dried root weighed 352 mg.
Example 3c
[0461] 0.5 g of ground root from Neobeguea mahafalensis was placed in 2.5 ml of commercial 95.5% ethanol and gently shaken for 3 days at room temperature. The procedure following was then as described for Example 3a. Yield of lyophilized powder was 57.5 mg. Residual dried root weighed 425 mg.
[0462] Herein ethanol extracts according to Examples 3 a, 3b and 3 c are collectively termed REtOH.
Example 4: Acetone Extract of Root of Neobeguea mahafalensis
[0463] 0.5 g of ground root from Neobeguea mahafalensis was placed in 2.5 ml of acetone and gently shaken for 3 days at room temperature. The mixture was then filtered through a lump of cotton wool placed in a conical funnel; afterwards the extracted root on the lump was rinsed with the same solvent (2×1.5 ml). The combined filtrates were evaporated to dryness; the residue was dissolved in 30% acetonitrile in water (10 ml) and freeze-dried. powder, herein termed extract RT, was 28.7 mg. Residual root weighed 458 mg.
Example 5: Ethanol Extract of Stem Bark of Neobeguea mahafalensis
Example 5a
[0464] 0.5 g of ground stem bark from Neobeguea mahafalensis was placed in 2.5 ml of 50% aqueous ethanol and gently shaken for 3 days at room temperature. The mixture was filtered through a circle of filter paper (diameter about 10 mm) placed in a glass filtering funnel; afterwards the extracted stem bark on the filter was rinsed with the same solvent (2×1 ml). The combined filtrates were evaporated to dryness, the residue dissolved in 30% acetonitrile in water (10 ml) and freeze-dried. A brown, fluffy powder formed. Yield 99.1 mg. The residual stem bark after extraction was dried in the air keeping it on an open Petri dish. Residual dried root weighed 366 mg.
Example 5b
[0465] 0.5 g of ground stem bark from Neobeguea mahafalensis was placed in 2.5 ml of 70% aqueous ethanol and gently shaken for 3 days at room temperature. The procedure following was then as described for Example 5a. Yield of lyophilized powder was 82.9 mg. Residual dried stem bark weighed 391 mg.
Example 5 c
[0466] 0.5 g of grinded stem bark from Neobeguea mahafalensis was placed in 2.5 ml of commercial 95.5% ethanol and gently shaken for 3 days at room temperature. The procedure following was then as described for Example 5a. Yield of lyophilized powder was 44.0 mg. Residual dried stem bark weighed 439 mg.
Example 5d
[0467] 0.5 g of grinded stem bark from Neobeguea mahafalensis was placed in 2.5 ml of commercial 99.5% ethanol and gently shaken for 3 days at room temperature. The procedure following was then as described for Example 5a. Yield of lyophilized powder was 35.1 mg. Residual dried stem bark weighed 450 mg.
[0468] Herein ethanol extracts according to Examples 5a, 5b, 5c and 5d are collectively termed SEtOH.
Example 6: Chloroform Extract of Root of Neobeguea mahafalensis
[0469] 0.5 g of ground root from Neobeguea mahafalensis was placed in 10 ml of chloroform and gently shaken for 3 days at room temperature. The mixture was filtered through a lump of cotton wool placed in a conical funnel; afterwards the extracted root on the lump was rinsed with the same solvent (2×5 ml).
[0470] The combined filtrates (colourless) were evaporated to dryness. A wax-like residue, herein termed extract RCH, formed. Yield 18 mg. The root after extraction was dried in the air keeping it on an open Petri dish. Residual root weighed 480 mg.
Example 7: Chloroform Extract of Stem Bark of Neobeguea mahafalensis
[0471] 0.5 g of ground stem bark from Neobeguea mahafalensis was placed in 10 ml of chloroform and gently shaken for 3 days at room temperature. The mixture was filtered through a lump of cotton wool placed in a conical funnel; afterwards the extracted stem bark on the lump was rinsed with the same solvent (2×5 ml). The combined filtrates (slightly yellow-green) were evaporated to dryness. A wax-like residue, herein termed SCH, formed. Yield 40 mg. The stem bark after extraction was dried in the air keeping it on open Petri dish. Residual stem bark weighed 460 mg.
Example 8: Freeze Dried Water Extract of Root of Neobeguea mahafalensis
[0472] A tablespoon of dried ground roots (5.0 g) from Neobeguea mahafalensis was put in 0.5 litre of boiling water. The mixture was boiled for 2 min, then left to cool down (slowly, overnight). It was then filtered (through a wool plug placed into a glass funnel under water jet pump vacuum) into two round-bottom flasks. The filtrates were then liquid shell frozen and freeze-dried, yielding 1.45 g of reddish-brown fluffy powder (herein termed ‘RW extract). The reddish-brown powder was used further in the manufacture of pharmaceuticals or used directly without further processing as a pharmaceutical.
Example 9: Enriched Extract from Root of Neobeguea mahafalensis
[0473] Dried ground root (5.0 g, 1 table spoon) from Neobeguea mahafalensis was put in boiling water (500 ml). The mixture was boiled for 2 min, and then allowed to cool down for 2-3 h. The concoction was filtered, and the filtrate lyophilized. A light-brown powder (RW extract) formed. Yield 1.50 g.
[0474] The powder was placed in a separation funnel; water (100 ml) was first added, solubilizing the RW powder, and chloroform (100 ml) was then added. The mixture was intensely shaken for 2 min, then left to stand for 20 h (separation of phases proceeds very slowly, part of the mixture remains as an unseparated zone containing a precipitate between the water and chloroform layers). The chloroform and water layers were collected in separate portions, and the remaining intermediate zone was placed in plastic tubes resistant to chloroform and centrifuged at 4000 rpm for 30 min. Chloroform and water layers formed in the tubes after the centrifugation and were separately collected by use of a pipette. 20 ml each of chloroform and water were equally divided and added to all the tubes, the tubes were vortexed, centrifugation repeated, and the new chloroform and water layers were collected. This procedure of addition of solvents, vortexing, centrifugation and collection of layers was repeated twice.
[0475] The unresolved central layer was evaporated. A brown-red powder (R2P) remained. Yield 28.0 mg.
[0476] The united chloroform extracts were evaporated to dryness, the residue was dissolved in acetonitrile-water (volume proportion 4:6; 50 ml), and freeze-dried. A white powder (R2C) formed. Yield 32.3 mg.
[0477] The united water phases were freeze-dried. A brown, fluffy powder (R2W) formed. Yield 1.09 g.
[0478] The separation procedure is further clarified in
Example 10: Enriched Extract from Stem Bark of Neobeguea mahafalensis
[0479] Dried and ground stem bark (6.0 g, 1 table spoon) was put into boiling water (500 ml). The mixture was boiled for 2 min, and then allowed to cool down for 2-3 h. The concoction was filtered, and the filtrate was lyophilized. A light-brown powder (herein termed ‘SW) formed. Yield 1.16 g.
[0480] All of the above SW the powder was placed in a separation funnel and water (100 ml) and chloroform (100 ml) were added. The mixture formed was intensely shaken for 2 min, then left to stand for 20 h (separation of phases proceeded very slowly, part of the mixture remains as an unseparated zone containing a precipitate between the water and chloroform layers). The chloroform and water layers, as well as the intermediate zone were collected separately. The intermediate zone was placed in plastic tubes resistant to chloroform and centrifuged at 4000 rpm for 30 min. The chloroform and water layers, formed in the tubes after the centrifugation, were collected separately using a pipette. 20 ml each of chloroform and water were equally divided and added to the tubes, the tubes were vortexed, the centrifugation repeated, and the new chloroform and water layers were again collected. This addition of solvents, vortexing, centrifugation and collection of layers was repeated twice. The unresolved central layer was evaporated. A dirty-yellow powder (herein termed ‘S2P’) remained. Yield 35 mg. The united chloroform extracts were evaporated to dryness, the residue was dissolved in acetonitrile-water (volume proportion 4:6, 50 ml), and freeze-dried. A white powder (herein termed ‘S2C’) formed. Yield 69 mg. The united water phases were freeze-dried. A light brown, fluffy powder (herein termed ‘S2W) formed. Yield 0.90 g.
Example 11: Capsules Prepared from Stem Bark of Neobeguea mahafalensis
[0481] Dried coarsely ground stem bark of Neobeguea mahafalensis was carefully further ground in a mortar into a fine powder. The powder was then directly filled into vegetarian capsules obtained from Capsuline, Inc, P.O. Box 667260, Pompano Beach, Fla. 33066, USA.
a) Size 0 capsules were filled with the stem bark powder, the powder was gently packed and the capsules were closed with their caps. Each capsule contained 300 mg of stem bark.
b) Size 00 capsules were filled with the stem bark powder, the powder was gently packed and the capsules were closed with their caps. Each capsule contained 465 mg of stem bark.
[0482] Following their filling the capsules were stored in capped plastic vessels.
Example 12: Capsules Prepared from Root of Neobeguea mahafalensis
[0483] Dried coarsely ground root of Neobeguea mahafalensis was carefully further ground in a mortar into a fine powder. The powder was then directly packed into vegetarian capsules obtained from Capsuline, Inc, P.O. Box 667260, Pompano Beach, Fla. 33066, USA.
a) Size 0 capsules were filled with the root powder, the powder was gently packed and the capsules were closed with their caps. Each capsule contained 220 mg of root.
b) Size 00 capsules were filled with the root powder, the powder was gently packed and the capsules were closed with their caps. Each capsule contained 350 mg of root.
[0484] Following their filling the capsules were stored in capped glass vessels.
Example 13: Capsules Prepared from Water Extract SW of Stem Bark of Neobeguea mahafalensis
[0485] The freeze-dried water extract SW of stem bark of Neobeguea mahafalensis comprising a fluffy powder (prepared as described in Example 10) was carefully ground to a fine compacted powder in a mortar.
[0486] The fine compacted powder was then directly packed into vegetarian capsules from Capsuline, Inc, P.O. Box 667260, Pompano Beach, Fla. 33066, USA, as follows;
a) Size 0 capsules were filled with the fine compacted powder, the fine compacted powder was gently packed and the capsules were closed with their caps. Each capsule contained 105 mg of SW extract.
b) Size 00 capsules were filled with the fine compacted powder, the fine compacted powder was gently packed and the capsules were closed with their caps. Each capsule contained 160 mg of SW extract.
[0487] Following their filling the capsules were stored in capped glass vessels.
Example 14: Capsules Prepared from Water Extract RW of Root of Neobeguea mahafalensis
[0488] The freeze-dried water extract RW of root of Neobeguea mahafalensis comprising a fluffy powder (prepared as described in Example 8) was carefully ground to a fine compacted powder in a mortar. The powder was then directly packed into vegetarian capsules from Capsuline, Inc, P.O. Box 667260, Pompano Beach, Fla. 33066, USA, as follows:
a) Size 0 capsules were filled with the fine compacted powder, the fine compacted powder was gently packed and the capsules were closed with their caps. Each capsule contained 70 mg of RW extract. b) Size 00 capsules were filled with the fine compacted powder, the fine compacted powder was gently packed and the capsules were closed with their caps. Each capsule contained 100 mg of RW extract.
[0489] Following the filling capsules were stored in capped glass vessels.
Example 15: Capsules Prepared from Enriched Extract of Stem Bark of Neobeguea mahafalensis
[0490] 1 g of the S2C extract of stem bark from Neobeguea mahafalensis was prepared according to Example 10 and thoroughly mixed with 30 g corn starch. Size 0 vegetarian capsules (Capsuline, Inc, P.O. Box 667260, Pompano Beach, Fla. 33066, USA) were filled with the powder mixture, the powder was gently packed and the capsules were closed with their caps. Each capsule contained 10 mg of S2C extract.
Example 16: Capsules Prepared from Enriched Extract of Root of Neobeguea mahafalensis
[0491] 1 g of the R2C extract from root from Neobeguea mahafalensis was prepared according to Example 9 and thoroughly mixed with 30 g corn starch. Size 0 vegetarian capsules (Capsuline, Inc, P.O. Box 667260, Pompano Beach, Fla. 33066, USA) were filled with the powder mixture, the powder was gently packed and the capsules were closed with their caps. Each capsule contained 10 mg of R2C extract.
Example 17: Influence of Extract from Neobeguea mahafalensis on Sexual Activity in Male Rats
Methods
[0492] Behavioral tests were conducted during the dark phase of the day/night cycle (16.00-20.00 h) under dim light illumination. Sexually naïve Wistar rats (220-250 g) of both sexes were housed under standard conditions. After a 7-day adaptation period they were used in the tests.
[0493] On the time for evaluation each male rat was placed individually into a clear plastic observation chamber (36×24×33 cm, length×width×height) for a 30 min adaptation period prior to the introduction of a receptive, sexually inexperienced female.
[0494] Before use the females rats were brought into full sexual receptivity by sequential treatment with estradiol benzoate (12 ag/rat; 48 h pre-session injection time) and progesterone (500 ag/rat; 4.5 h pre-session injection time). Estradiol and progesteron were prepared in olive oil and injected subcutaneously in a volume of 0.1 ml/rat.
Sexual Behaviour Test (SBT)
[0495] The following indices of male sexual behavior were registered: [0496] (1) Mounting the female. The male normally mounts from the rear, sometimes posing his forelegs over the female's back, and makes rapid anteroposterior pelvic thrusts for about 300 ms. He then dismounts rather slowly. After a mount, the male frequently licks his genital region. Mounts are performed in bouts with short (5-10 s) inter-mount intervals. A bout is defined as a sequence of mounts, with or without vaginal penetration, uninterrupted by any behavior that is not oriented towards the female, except genital self-grooming. The number of mounts within a bout varies usually between 1 and 5 (mean number about 2). Between each bout there is a longer (20-80 seconds) pause, during which time the male may engages in other behaviors. Before, at the beginning of, or during the mount the female assumes a lordosis posture. If no lordosis is displayed, the female is immediately replaced. An inexperienced male, and sometimes also an experienced male, may mount the female with incorrect orientation, e.g. on the flanks, the head, etc. Such mounts are not scored. [0497] (2) Intromission (vaginal penetration). This behavior starts with a mount, but suddenly the male makes a deep thrust forward and stops pelvic thrusting. He then vigorously withdraws and always licks his genitals. A male will never mount again immediately after an intromission. [0498] (3) Ejaculation. This behavior starts with an intromission, but after vaginal penetration (the deep forward thrust) the male remains on the female for 1-3 s. Rhythmic contraction of the posterior abdomen are clearly visible. He then slowly rises with his forelegs held open. At ejaculation, it is the female that moves away from the male. He then licks his genitals and remains inactive for several (4-7) min.
Recorded Elements of SBT
[0499] The elements recorded from the sexual behavior tests were used to calculate all standard measures of sexual behavior, providing a general characterization of the copulatory cycle. The following were thus recorded: [0500] 1. Latencies of the first mount and intromission [0501] 2. Total number of ejaculations [0502] 3. Series in a complete copulatory cycle.
[0503] Additionally, for each copulatory series (n, number of series), the following measures were calculated: [0504] 1. Copulatory efficacy—average number. of finished copulatory cycles (i.e., cycles that end with ejaculation) in the group [0505] 2. Duration of the series (DSn, time interval between the first mount or intromission to the ejaculation; often labeled as ejaculatory [0506] 3. Number of mounts and intromissions preceding each successive ejaculation (NMn and Nin). [0507] 4. Post-ejaculatory interval (the time interval between the ejaculation and first successive mount/intromission). [0508] 5. Number of active couples (also termed number of active rats). As active couples are counted all couples that started sexual activity during each test period.
[0509] (Note that herein by ‘Copulatory efficacy’ and ‘Copulatory efficacy index’ is mutatis mutandis intended to mean the same thing.)
[0510] The screening tests are normally ended at the first ejaculation (if there no special reason for recording the post-ejaculatory interval). If a male does not intromit within 30 min, the test is ended for that male and he is considered sexually inactive. If a male has intromitted but not ejaculated within 30 min, he is given 30 min after the first intromission to ejaculate. Otherwise he is considered inactive.
Test Protocol
[0511] Experimental groups: Control group and RW-group (8 male rats each). The RW extract of Example 8, was dissolved in a small amount of water and administered per os at a dose of 60 mg/kg/day for 3 consecutive days. Controls received an equal volume of waterier os. The experimental protocol is clarified in Table 1, Example 17:
TABLE-US-00001 TABLE 1 Example 17 Day Procedure Comments 0 Sexual behavior test (SBT) Assses normal (basal) level 1 Extract of Example 8 (RW) Test if there is any ‘viagra’ type dose 60 mg/kg, or Control activity treatment, SBT after ~1 hr 2 RW 60 mg/kg or Control treatment 3 RW 60 mg/kg or Control treatment 4 SBT Evaluation 24 hrs after 3.sup.rd dose 7 SBT Evaluation 4 days 3.sup.rd dose 14 SBT Test for long-term effect
Results
[0512] One hour after administration of the first RW-dose there was no appreciable influence on the sexual activity among the male rats. After 3 days of administration of RW the sexual activity was tested again on the day 4, 24 hrs after the last RW administration. The results show that the number of active couples in the RW-treated group (given in % of the respective proper test group and occasion) was statistically significantly (p<0.05) higher on the 4th day compared to the respective control (
[0513] On the 7:th and 14:th day there were also statistically significant (p<0.05) increases in the number of active couples (
[0514] The copulatory efficacy, or the average number of finished copulatory cycles in the group, was also measured on the 4:th, 7:th and 14:th day (
[0515] Furthermore the copulatory cycle length decreased on the 1:st and 14:th day as compared to the control group and corresponding 0:th day group in the RW-treated group (
[0516] We also observed that the number of mounts and intromissions decreased on the 7:th and 14:th day in the RW-treated rats (Table 2, Example 17).
[0517] The full summary of the data from this test series is given in Table 2, Example 17.
Conclusions
[0518] No effect of RW was observed after a single administration (test performed 1 hr after RW administration). However, following the 3:d day of administration of RW a significant increase in the number of sexually active couples was seen; the effect was statistically significant even at the 14:th day of the experiment (i.e., 11 days after the last administration of RW). On the other hand, RW decreased the length of the copulatory cycle, as well as the number of mounts and intromissions in the cycle, but this effect was statistically significant only versus the level on day 0 of the corresponding group.
[0519] In conclusion, after treatment with RW rats are more sexually active, but the length of copulatory cycle is shorter (less intromissions and mounts). Thus, RW has a sexual enhancing effect.
TABLE-US-00002 TABLE 2 Example 17 Control RW Control RW Control RW Control RW Control RW 0.sup.th day 1.sup.st day 4.sup.th day 7.sup.th day 14.sup.th day No Sexual behavior Mean Mean Mean Mean Mean Mean Mean Mean Mean Mean 1 Copulatory 0.4 0.4 0.0 0.4 0.8 1.9 0.6 1.6 0.9 2.4 Efficacy, Index StErr 0.4 0.4 0.0 0.4 0.5 0.8 0.4 0.5 0.6 0.7 ANOVA 9 Number of active 25% 38% 25% 63% 38% 88%** 38% 88%** 38% 88%** rats, % 2 The Copulatory 1980.0 1691.3 1016.7 1756.3 1603.5 911.6 1423.8 1415.0 891.4 Cycles Length, Sec StErr 395.1 64.9 124.6 421.3 212.5 229.0 241.4 225.5 86.6 ANOVA 3 Latencies of the 160.5 210.7 495.0 648.6 193.0 271.6 32.3 109.1 24.0 27.1 first Mount or Intromission, sec StErr 64.5 79.0 365.0 454.0 156.0 117.0 23.9 37.8 10.5 10.3 ANOVA 4 The Number of 3.0 4.3 4.3 2.7 4.4 2.0 2.1 3.0 1.4* Mounts in the Cycle StErr 0.6 0.3 0.9 0.9 0.5 0.9 0.5 0.6 0.3 ANOVA 5 The Number of 43.7 32.7 21.3 23.0 28.3 20.4 20.0 17.3 13.7 Intromissions in the Cycle StErr 7.2 3.7 1.9 7.5 2.5 6.8 2.8 1.9 1.1 ANOVA 6 The Number of 46.7 37.0 25.7 27.2 33.2 24.7 21.7 20.3 15.2 Mounts and Intromissions in the Cycle StErr 7.7 3.5 2.7 4.5 3.3 13.7 2.6 2.4 1.2 ANOVA 7 Postejaculatory 452.0 472.0 591.5 412.2 714.7* 638.4 660.7 811.0 671.3 interval, sec StErr 88.0 55.0 133.5 112.4 74.8 108.6 82.0 156.4 79.3 ANOVA 8 The first 1502.0 1792.0 1261.0 2424.0 2016.8 1218.0 1625.4 1767.5 1261.8 Copulatory Cycle Length, Sec StErr 200.0 472.9 123.0 291.5 105.5 209.9 ANOVA P < 0.05 vs corresponding 0.sup.th day, t-test *P < 0.05 vs Control, t-test **P < 0.05 vs Control, Chi-Square Test
Example 18: Effect of the Pharmaceutical Preparation Prepared from an Extract of Neobeguea mahafalensis in a 40 Year Old Male
[0520] The subject is married and has two children, 3 and 5 years old. He is healthy and he takes no medicines.
[0521] His sexual performances had declined gradually over the years, during the period before treatment. He had also since long time been afraid to have sex with a woman due to fear of loosing erection. He volunteered to take totally nine capsules 00 prepared according to Example 14 each capsule comprising 100 mg of the RW extract according to Example 8. (Each capsule corresponding to about 300 mg of dried root). The subject was told that the capsules contained extract from Madagscarian plant but he was not revealed from which species. He took 3 capsules each day for 3 consecutive days.
[0522] Already at the first day of treatment, after having taken the first capsule, the subject noticed a positive effect on his sexual ability. During the 2:nd and 3:d day this effect increased even more. He describes the effect as an increase in sexual excitement when he wants to have sex.
[0523] He also describes the effect as an improved sexual performance and that he is no longer afraid to have sex. He obtains a stronger erection that is maintained during the whole period of intercourse. He also experiences an increased feeling of pleasure while making love. He also has increased the duration of the intercourse to approximately 30 minutes; while earlier it was about 15 min.
[0524] Remarkably, the effect of the treatment remains still after 3% years.
[0525] The subject states that he has not experienced any other effect of the drug. His memory is not affected; he experiences no change of sleeping pattern and no change in motor behaviour. He has no problems of urination.
Example 19: Effect of the Pharmaceutical Preparation Prepared from an Extract of Neobeguea mahafalensis in a 37 Year Old Male
[0526] The subject is married and has tree children, 4, 6 and 8 years old. He states that he is healthy and that he takes no medicines.
[0527] The subject did not earlier have any erection problems. He volunteered to take totally 12 capsules 00 prepared according to Example 14 each capsule comprising 100 mg of the RW extract according to Example 8. (Each capsule corresponding to about 300 mg of dried root). The subject was told that the capsules contained extract from Madagscarian plant but he was not revealed from which species. He took 3 capsules each day for 4 consecutive days.
[0528] The subject states that after the treatment he experiences a stronger sexual appetite when he is together with a woman. During intercourse he experiences now a longer time before ejaculation. After ejaculation he experiences now also a shorter period of sleepiness than before taking the drug. Moreover, at the day following having sex he also feels stronger than earlier, being capable to renewed sexual activity to an extent that he was not capable of prior to obtaining the treatment.
[0529] However, if he tries to force the ejaculation during intercourse he experiences exhaustion, as he finds it more difficult to ejaculate fast than earlier. However, if he allows it to take the time it needs he does not experience this type of exhaustion.
[0530] Since he obtained the capsules for almost 1% year ago the effect still remains, although there has been some progressive reduction in the extent of the effect. In particular the prolongation of the time for ejaculation that he experienced in the beginning has be come less pronounced.
[0531] The subject states that he don't have any problems to urinate, and that he never experience any other noticeable effects from the treatment. He does not have any problems with memory, sleeping or performing motor tasks.
Example 20: Effect of the Pharmaceutical Preparation of the Extract of Neobeguea mahafalensis in a 55 Year Old Male Volunteer
[0532] The subject is married and has 15 children, the youngest is only a few months while the oldest is 33 years. He states that he is over all healthy and that he takes no medicines. However, he had experienced a decreased sexual ability being able to make love only 3 times per months, or even being totally unable. Due to his incapacity he lost his relation with his woman. Earlier he had had many wives (in accordance with local tradition) and got many children. However, the 14 first children was born when he was young, i.e. while he was aged 21 to 35 years and due to his increasing incapacity did not receive any more children for long time. He volunteered to take totally nine capsules 00 prepared according to Example 14 each capsule comprising 100 mg of the RW extract according to Example 8. (Each capsule corresponding to about 300 mg of dried root). The subject was told that the capsules contained extract from Madagscarian plant but he was not revealed from which species. He took 3 capsules each day for 3 consecutive days.
[0533] After the treatment he started to notice a sexual excitement during closeness with a woman that he was not able to feel before the treatment. After the treatment he became able to make love once a day. He obtains more rapid erections now compared to what he was able to obtain earlier before the treatment (this effect is still seen more than one year after the treatment). He now also obtains spontaneous erections, something that he did not have before the treatment. The volume of his ejaculations is larger than they were before the treatment. He sexual performance is now even better than it was when he was young. The duration of intercourse is now usually 5-10 minutes and this duration has not changed since before the treatment. The effect is still, since slightly more than one year following the treatment, clearly noticeable and shows only slight decline.
[0534] Shortly after the treatment, due to his regain of sexual ability, he married a young wife who became pregnant and recently gave him a child.
[0535] He states that he has not experienced any other effects of the drug. He sleeps normal, has no problems with the memory and no change in motor behaviour. He does not experience any problems with urination.
Example 21: Effect of the Pharmaceutical Preparation of the Extract of Neobeguea mahafalensis in a 57 Year Old Male Volunteer
[0536] The subject is an academic holding an administrative position. He is married and has 4 children, the youngest is 20 and the oldest is 30 years old.
[0537] The subject states that he is overall healthy although he suffers from a slightly elevated blood pressure that is under control by medication.
[0538] The subject had noticed a progressive decline in his sexual performance from the age he was 45 years old. His erections had since then become progressively weaker and he required more sexual stimulation to obtain erection. Still he had been able to have sex with a woman but to a much lesser extent than when he was young.
[0539] He volunteered to take totally 14 capsules 0 prepared according to Example 14 each capsule comprising 70 mg of the RW extract according to Example 8. (Each capsule corresponding to about 200 mg of dried root). The subject was told that the capsules contained extract from Madagscarian plant but he was not revealed from which species. He took 2 capsules each day for 7 consecutive days.
[0540] Already at the first day of treatment he noticed an effect as a sensation of the desire to make love. At the second day that desire was further increased and in contact with a woman he obtained a strong erection. Immediately after the completion of the treatment he gained a dramatic increase in sexual performance being able to have sex with a woman 3 times a day. He also now experienced an increased time for ejaculation during intercourse. Earlier it took him a few minutes to ejaculate; now this time had increased to 5-10 minutes. The subject also describes the effect from the drug as the Wteries has become charged again’. He experiences an increase sexual desire; the drugs effect reminds him of the performance in his youth.
[0541] Following the treatment the subject did not receive any further treatment during a period of 3 years. Even after this long time, however, the effect remains although it has gradually declined since this time. After three years following the treatment he has sex about 3 times per week. However, his performance is still better compared to the situation before the treatment.
[0542] The drug has not given him any noticeable side effects. His memory is good, he sleeps well and his motor functions are normal. His work capacity is normal and was not affected by the drug. He does not experience any problems with urination.
Example 22: Evaluation of Behavioral Effects of R2W, R2P and R2C Extracts from Neobeguea mahafalensis
[0543] The activity of the extracts R2W, R2P and R2C of Example 9 were evaluated using essentially the same method as detailed in Example 17. The sexual behaviour test (SBT) and recorded elements of SBT were the same as in Example 17.
Test Protocol
[0544] The experimental groups comprised: Control, R2W, R2C and R2P groups. Each group contained 6 male rats. Each extract was administered per os to the rats, suspended or dissolved in a small volume of water (R2W dissolved in water, whereas R2C and R2P formed suspensions). Control rats were given the same volume of water.
[0545] The extracts were administered as follow: R2W—43 mg/kg body weight; R2C—4 mg/kg body weight; and R2P—4 mg/kg body weight. These doses were administered once a day for 3 consecutive days. The protocol is further detailed in Table 1, Example 22:
TABLE-US-00003 Day Procedure 1 Drug per os administration at the doses: R2W (6 rats) - 43 mg/kg; R2C (6 rats) - 4 mg/kg; R2P (6 rats) - 4 mg/kg; Controls received water per os only 2 Drug administration - same as for day 1 3 Drug administration - same as for day 1 7 Sexual behavior test (SBT)
Results
[0546] Four days following the last administration (i.e., on day 7) the sexual activity was tested. Extracts R2W (43 mg/kg, p.o.) and R2P (4 mg/kg, p.o.) did not appreciably increase any of the recorded indices of sexual activity. For example, for the R2W group the copulatory efficacy (i.e., the average number of finished copulatory cycles in the group) was similar to that of the control group; for the R2P group it was even lover than in control group (
[0547] The number of mounts was drastically and statistically significantly increased in R2C group as is illustrated in
[0548] The full data from this series test is given in Table 2, Example 22. (Note that while rats in the R2P group started activity and therefore are included in the ‘Number of active rats’ none of them completed any cycles; i.e., none of the rats ejaculated. Therefore the copulatory cycle length, number of mounts in the cycle, number of intromission in the cycle, number of mounts and intromissions in the cycle, postejaculatory interval and first copulatory cycle length are left blank for the R2P group.)
Conclusion
[0549] R2W and R2P do not influence male rat sexual behavior at the doses used. Extract R2C induces an increase of sexual activity. Thus, R2C has a sexual enhancing effect.
TABLE-US-00004 TABLE 2 Example 22 R2W R2C R2P Control 43 mg/kg 4 mg/kg 4 mg/kg No Sexual behavior Mean Mean Mean Mean 1 Copulatory Efficacy, 0.5 0.8 2.0 0.0 index StErr 0.5 0.8 0.7 0.0 ANOVA 0.2967 2 Number of active rats, 33% 33% 67% 50% % 3 The Copulatory Cycles 1540.0 1560.0 1522.9 lenght, sec StErr 429.6 229.4 256.8 ANOVA 0.9961 4 Latencies of the first 104.0 669.0 226.3 509.0 Mount or Intermission, sec StErr 47.0 513.0 49.4 415.5 ANOVA 0.2809 5 The Number of Mounts 0.3 0.4 in the Cycle StErr 0.3 0.2 0.4 ANOVA 0.0062 6 The Number of 16.0 17.4 17.1 Intromissions in the Cycle StErr 3.1 3.1 2.3 ANOVA 0.9648 7 The Number of Mounts 16.3 17.8 19.4 and Intromissions in the Cycle StErr 3.3 3.2 2.5 ANOVA 0.8173 8 Postejaculatory interval, 778.0 592.8 619.2 sec StErr 83.2 24.4 34.3 ANOVA 0.0809 9 The first Copulatory 1759.0 1806.0 2502.0 Cycle lenght, sec StErr 279.8 ANOVA
P < 0.05 vs Control, Chi-Square Test
Example 23: Assay of Activity of Water Extract RW from Root of Neobeguea mahafalensis
[0550] The activity of the extract RW, prepared as described in Example 8, was assayed for activity in essence using the same method as detailed in Example 17. The sexual behaviour test (SBT) and recorded elements of SBT were the same as in Example 17.
Test Protocol
[0551] The test protocol comprised 5 groups of rats, wherein each group contained 8 rats. The test drug (RW) comprised the extract of Example 8. Prior to administration it was dissolved in a small volume of water. On day 1 group 1 was given 3 rng/kg body weight (bw) of RW per os; group 2, 10 mg/kg bw; group 3, 30 mg/kg bw; group 4, 100 mg/kg bw. Group 5 was given waterier os only. This administration was repeated on day 2 and 3. Sexual behaviour test was performed on day 7.
TABLE-US-00005 TABLE 1 Example 23: Day Procedure 1 Drug: Extract RW pf Example 8 a) 1 mg/kg body weight (8 male rats) b) 3 mg/kg body weight (8 male rats) c) 10 mg/kg body weight (8 male rats); d) 30 mg/kg body weight (8 male rats) e) 100 mg/kg body weight (8 male rats) Control: Water (8 male rats) 2 Drug administration - same as for day 1 3 Drug administration - same as for day 1 7 Sexual behavior test (SBT)
Results
[0552] Of the recorded element of SBT the number of active couples (in %) and the copulatory efficacy index for each group was as follows:
TABLE-US-00006 TABLE 2 Example 23 Dose Number of active Copulatory efficiency Group (mg/kg bw) couples (%) index 1 1 25 0.0 2 3 38 0.8 3 10 50 0.8 4 30 75 1.9 5 100 88 2.0 6 0 38 0.4
[0553] The data for the number of active couples was then plotted in a semi logarithmic diagram as shown in
[0554] Non-linear curve fitting of the data to the logistic equation:
where Y is the number of active couples, A is the starting level of the dose response curve (i.e., Y level without drug), B the ending level of the dose response curve (i.e., the maximally possible attainably Y level with high doses of the drug), K the ED.sub.50, and P the Hill coefficient (i.e., slope of the curve) was afforded by using GraphPad Prism software (GraphPad Software, Inc.l 1452 El Camino Real, #215 San Diego, Calif. 92130 USA).
[0555] The results were as follows:
A=32%
B=91%
[0556] K (ED.sub.50)=16.3 mg/kg body weight
P=1.67
[0557] Accordingly using the Equation:
where ED.sub.50 is expressed in mg/kg body weight, the activity of the RW extract of Example 8 can be calculated to 1/16.3=0.061 U/mg. (These units correspond to U.sub.sac; i.e. the activity of the extract was estimated to be 0.061.sub.sac/mg). (It shall be observed that the activity obtained and the data obtained that underlies this activity is given only as an example with the purpose to illustrate the method for assaying activity. The exact activity obtained of a sample may be subjected to variation due to experimental error, variation due to sample size and variation due to variations in extraction procedures and variation due to the different specimens of Neobeguea mahafalensis and differences in collections and storage of the specimen, drug and extract thereof).
[0558] The data for the copulatory efficacy index was also plotted in a semi logarithmic diagram as shown in
[0559] The data was fitted to the logistic equation using the same approach as above. The results were as follows:
A=0.36
B=2.08
[0560] K (ED.sub.50)=14.1 mg/kg body weight
P=2.19
[0561] Applying the same approach as above the activity of the extract could then be computed as 1/14.1=0.071 U/mg (i.e. 0.071 U.sub.cei/mg). (It shall be observed that the activity obtained and the data obtained that underlies this activity is given only as an example with the purpose to illustrate the method for assaying activity. The exact activity obtained of a sample maybe subjected to variation due to experimental error, variation due to sample size and variation due to variations in extraction procedures and variation due to the different specimens of Neobeguea mahafalensis and differences in collections and storage of the specimen, drug and extract thereof).
Example 24: Identification of Characteristic Mass-Peaks in Extracts of Neobeguea mahafalensis
Introduction
[0562] Extracts prepared from Neobeguea mahafalensis were analysed by mass spectrometry in order to demonstrate the presence of characteristic mass-peaks. Two instruments were applied:
[0563] One instrument was a PE SCIEX API 150EX single quadrupole mass spectrometer (Perkin Elmer, 45 William Street, Wellesley, Mass. 02481-4078, USA). This is a general purpose instrument with a mass range up to 3000 amu (amu=atomic mass units), which can be used as a mass detector, when connected to HPLC. A choice of inlets is available, including atmospheric pressure chemical ionisation (APCI) ion source (Heated Nebulizer). This ion source provides a better ionization than the more common electrospray source for less polar analytes. API 150EX allows fast, reliable, sensitive and fully automated data acquisition and analysis.
[0564] The other instrument was a Q-Tof2 from Micromass (Macromass/Waters; Waters Corporation, 34 Maple Street, Milford Mass. 01757, USA). This is a high resolution hybrid quadrupole time-of-flight mass spectrometer, equipped with Z-spray electrospray ionization inlet (i.e., the Micromass nanoflow interface together with glass capillary option was used). This instrument utilizes a high performance, research grade quadrupole mass analyzer (MS1) and an orthogonal acceleration time-of-flight mass measuring part (MS2). A hexapole collision cell, placed between two mass analysers, is useful to induce fragmentation, which is necessary for structural investigations; (however, the hexapole collision cell option was not active in the experiments of this example). Ions coming from mass analysers are detected by a microchannel plate detector and ion counting system.
Methods
[0565] Using the PE SCIEX API 150EX single quadrupole mass spectrometer the R2C sample of root prepared according to Example 9, and the S2C sample from stem bark prepared according to Example 10 were analysed as follows:
[0566] The R2C or S2C sample (1.0 mg) was dissolved in acetonitrile (0.3 ml), diluted with water to 1.0 ml volume and centrifuged. The clear supernatant (50 μl) was introduced into the LC/MS system, containing a 4×250 mm Silasorb C18 HPLC column. Analysis time was 80 minutes using a linear gradient made from solution A (5% acetonitrile in water with 5 mM ammonium acetate additive) and solution B (94% acetonitrile in water with 5 mM ammonium acetate additive). Upon starting the analysis the eluent contained 80% solution A and 20% solution B; at the end of the 80 minutes the eluent was composed of 20% solution A and 80% solution B. The eluate from the column was introduced into an APCI ion source integrated with the PE SCIEX API 150EX mass spectrometer at a flow rate of 1.0 ml/min.
[0567] Using the Q-Tof2 mass spectrometer the R2C and S2C samples were prepared similarly as above (i.e., 0.2 mg of each was dissolved in 0.2 ml acetonitrile then diluted with 1% formic acid to 1.0 ml volume and centrifuged) and were then introduced via an electrospray needle and infused into the Micromass Q-Tof2 mass spectrometer. Change of polarity was applied to induce positive or negative ionization. Analysis of raw data was attained using MassLynx V4.0 software.
Results
[0568] Examples of mass spectra of root extract R2C using the PE SCIEX API 150EX spectrometer with atmospheric pressure chemical ionisation are shown in
[0569] Upon analyzing all separate scans between 0-80 min the following characteristic root mass-peaks were identified:
[0570] Positive ionization, root extract R2C (accuracy approximately ±0.1 amu):
305.7, 323.7, 324.2, 342.2, 378.3, 378.7, 383.2, 384.2, 391.2, 391.7, 408.2, 413.2, 413.7, 417.2, 425.7, 432.7, 441.7, 442.2, 465.3, 477.3, 481.3, 491.3, 507.3, 523.3, 537.3, 539.3, 540.3, 551.3, 565.3, 567.3, 579.3, 620.3, 639.3, 652.4, 655.4, 658.4, 662.4, 667.4, 671.4, 683.4, 694.4, 699.4, 700.4, 703.4, 706.4, 710.4, 713.4, 715.4, 716.4, 717.4, 727.4, 730.4, 731.4, 732.4, 732.9, 734.4, 741.4, 743.4, 744.4, 745.4, 759.4, 761.4, 762.4, 775.4, 785.4, 788.4, 789.4, 803.4, 819.4
[0571] Negative ionization, root extract R2C (accuracy approximately ±0.1 amu):
311.2, 335.2, 346.7, 347.2, 352.7, 353.2, 359.2, 364.7, 373.2, 388.7, 389.2, 401.2, 406.7, 407.2, 412.7, 418.7, 419.2, 425.2, 428.7, 450.7, 460.8, 463.3, 465.3, 485.3, 497.3, 498.8, 510.8, 513.3, 522.8, 525.3, 541.3, 551.3, 553.4, 559.3, 569.3, 573.3, 574.8, 575.3, 585.3, 587.3, 588.8, 599.3, 601.3, 603.3, 611.3, 617.3, 629.3, 633.3, 643.3, 645.3, 655.4, 657.4, 659.4, 661.4, 669.4, 671.4, 675.4, 681.4, 685.4, 687.4, 691.4, 695.4, 697.4, 701.4, 703.4, 706.9, 712.9, 713.4, 715.4, 719.4, 721.4, 725.4, 727.4, 729.4, 734.9, 738.9, 739.4, 741.4, 745.4, 748.9, 755.4, 759.4, 761.4, 772.9, 773.4, 787.4
[0572] Examples of mass spectra of root extract S2C using the PE SCIEX API 150EX with atmospheric pressure chemical ionisation are shown in
[0573] Analyzing all separate scans between 0-80 min the following characteristic stem bark mass-peaks were identified: Positive ionization, stem bark extract S2C (accuracy approximately ±0.1 amu):
359.7, 378.3, 413.2, 465.3, 477.3, 507.3, 509.3, 523.3, 539.3, 555.3, 569.3, 577.3, 595.3, 599.3, 639.3, 646.3, 655.4, 657.4, 659.4, 662.4, 672.4, 675.4, 687.4, 688.4, 689.4, 694.4, 699.4, 703.4, 704.4, 706.4, 715.4, 716.4, 717.4, 722.4, 731.4, 732.4, 733.4, 734.4, 738.4, 745.4, 746.4, 747.4, 748.4, 749.4, 759.4, 761.4, 764.4, 765.4, 773.4, 775.4, 776.4, 777.4, 790.4, 792.4, 803.4, 819.4, 833.4, 850.5
[0574] Negative ionization, stem bark extract S2C (accuracy approximately ±0.1 amu):
337.2, 373.2, 411.2, 451.2, 457.3, 467.3, 497.3, 499.3, 543.3, 551.3, 553.4, 573.3, 585.3, 601.3, 611.3, 629.3, 637.3, 641.3, 655.4, 657.4, 661.4, 669.4, 671.4, 675.4, 685.4, 687.4, 697.4, 703.4, 713.4, 719.4, 721.4, 727.4, 745.4, 748.9, 849.5
[0575] Examples of mass spectra of root extract R2C using the Q-To f2 mass spectrometer are shown in drawing section
[0576] Examples of mass spectra of stem bark extract S2C using the Q-To f2 mass spectrometer are shown in drawing section
[0577] In the above lists, mass-peaks were detected with electrospray ionization on Q-To f2 as well and are shown in bold face, underlined. The lower number of mass-peaks detected is attributed to the lower efficiency of ionization with the electrospray ionization compared to atmospheric pressure chemical ionisation.
Example 25: Example of a Suitable Tablet Formulation
[0578]
TABLE-US-00007 Constituent Per tablet R2C extract, prepared according to Example 9 10 mg Potato starch 90 mg Colloidal silica 10 mg Talc 20 mg Magnesium stearate 2 mg 5% aqueous solution of gelatin 25 mg Final weight of tablet 135 mg
Example 26: Example of a Formulation of an Injectable (Suspension for Subcutaneous Injection)
[0579]
TABLE-US-00008 Constituent Per 1 ml R2C extract, prepared according to Example 9 10 mg Sodium chlorid 4 mg Aqua ad inject q.s.
Example 27: Extract of Stem Bark of Neobeguea mahafalensis by Use of Soxhlet Procedure
[0580] 16.04 g of stem bark prepared from Neobeguea mahafalensis by grinding was placed in a Soxhlet apparatus and extracted with boiling methylene chloride (200 ml) for 15 h, as schematically shown in drawing section,
[0581] (The remaining materials of stem bark after extraction weighed 14.3 g, after thorough drying.)
Example 28: Extract of Root of Neobeguea mahafalensis by Use of Soxhlet Procedure
[0582] 8.0 g of root materials derived from Neobeguea mahafalensis by grinding was placed in a Soxhlet apparatus and extracted with boiling methylene chloride (200 ml) for 15 h essentially as schematically shown in
[0583] (The remaining materials of root after extraction weighed 7.56 g after thorough drying.)
Example 29: Enriched Extract of Root of Neobeguea mahafalensis Prepared by Combined Water and Ethylacetate Extraction
[0584] Twenty gram of dried root materials derived from Neobeguea mahafalensis by grinding was poured into boiling water (1 litre), the mixture boiled for 2 min, then allowed to cool down and left for 3 hours at room temperature. The mixture was filtered and divided into two portions, about 500 ml each. One of the 500 ml portion was evaporated to dryness using a Rotavapor, resulting in ‘Crude aqueous extract1 (RW). The other 500 ml portion was extracted (‘exhausted’) with ethyl acetate (500 ml). After separation of phases the ethyl acetate fraction was evaporated to dryness using a Rotavapor, resulting in ‘EtOAc fraction’ (RWEtOAc). After separation of phases the water phase was also evaporated to dryness, resulting in ‘Exhausted aqueous fraction1 (RWExh), comprising the desired enriched root extract of Neobeguea mahafalensis.
Example 30: Assay of Root Extracts of Neobeguea mahafalensis in Mice Sexual Behaviour Test
Animals
[0585] Mice of 9 weeks age weighing 30±2 g were used throughout the study. They were all sexually unskilled.
[0586] At 4 weeks of age they had been separated from their parents and males and females had been placed in separated cages. The mice were fed with pellets enriched with proteins and lipids.
Sexual Behaviour Test
[0587] Prior to the tests, male and female mice received specific treatments, as follows:
[0588] Each male mouse received one of the extracts of Example 29, ‘Crude aqueous extract’, ‘EtOAc fraction’ and ‘Exhausted aqueous fraction’ (i.e., respectively, RW, EWEtOAc and RWExh extracts) by oral administration using a syringe and a cannula for esophageal insertion. Each extract was given at a dose of 60 mg/kg dissolved in a small amount of water during three consecutive days (D-1, D-2 and D-3) at the same time of the day (3.00 μm). Male control mice received the identical treatment schedule with water only.
[0589] Female mice were first treated with 50 ag/kg β-estradiol 48 hours before the test, and then with 2 mg/kg progesterone 4 Vi hours before the test, each by subcutaneous injection (compounds dissolved in olive oil) at a volume of 1 ml/kg, in order to make them sexually receptive.
[0590] The behavioural test was performed in a dark room at ambient temperature, during the last enlightened period of the day (3.00 μm) and the first dark period of the night (7.00 μm). Each observation period lasted 4 hours.
[0591] At the fourth, seventh and fourteenth day (D-4, D-7 and D-14), the male control mice and the treated male mice were individually placed in Plexiglass cages, 30 min before the start of the test.
[0592] At 3.00 μm, a female mouse was introduced into each cage hosting a male mouse. The number of mounts of the male mouse, defined as a tentative of lateral or dorsal mounting on the female mouse with or without penis intromission, was then counted throughout the 4 hour observation period and used as a simple measure of sexual behaviour activity.
[0593] In case a female was sexually non-receptive it was immediately replaced with a new female mouse.
Results
[0594] The results are summarized in
[0595] The number of mounts during the four hour test period amounted to 45 on an average in non-treated male mice and is represented in the figure as the four bars of equal height at day 0.
[0596] At day four (D-4) and seven (D-7) the number of mounts increased for both the Crude aqueous extract (RW) and the Exhausted aqueous fraction (RWExh) treated mice, compared to the control. For the EtOAc fraction (RWEtOAc) treated mice the number of mounts was substantially lower compared with the control at D-4, whereas it was not substantially different from the control at D-7.
[0597] Also at day 14 (D-14) the RW and RWExh treated mice showed a higher number of mounts than the control and RWEtOAc treated mice.
[0598] The sexual enhancing effect peaked for RWExh at D-7 when it amounted to 138 mounts during the 4 hour test period—an increase by a factor of three compared to the control. Accordingly RWExh is a highly preferred extract of the invention.
Example 31: Preparation of Enriched Extract RW1 from Root from Neobeguea mahafalensis by Molecular Size Fractionation Using G-25 Chromatography
[0599] One gram of dried ground root of Neobeguea mahafalensis was poured into boiling water (50 ml) and the mixture was allowed to boil for 2 min, then allowed to cool down to room temperature and then left overnight at +5° C. The mixture was filtered (weight of residual roots after extraction, filtration and drying was 0.66 g), and the filtrate obtained was extracted with ethyl acetate (3×50 ml). (To improve separation of layers, the water-ethyl acetate emulsions were transferred to glass tubes with conical bottom and centrifuged.) The obtained clear organic extracts were then evaporated giving 14.9, 5.6 and 4.2 mg of dark brown oily residues for the 1st, 2″ and 3 rd extractions, respectively. The remaining water layer was evaporated to 20 ml volume (using a Rotavapor) and introduced onto a transparent plastic column (3.3×88.5 cm) filled with Sephadex G-25 in aqueous suspension. The column was eluted with distilled water under hydrostatic pressure, flow rate 3.4 ml/min. The high molecular fraction, i.e. the dark brown zone, emerging after about 280 ml of colourless eluate had passed, corresponding to the void volume of the G-25 column, was collected and dried by freeze drying until completely dried. A brown fluffy powder, belonging to the type RW1, was obtained.
Example 32: Assay of Enriched Extract RW1 from Neobeguea mahafalensis in Mice Sexual Behaviour Test
[0600] The activity if RW1 prepared according to Example 31 was assayed for sexual enhancing activity essentially using the method describe in Example 30, the only difference being that the mice were treated per orally with 60 mg/kg RW1 dissolved in a small amount of water for three consecutive days. Controls were subject to the same treatment schedule but given water only per os.
[0601] The number of mounts increased substantially when assayed on the fourth (D-4) and seventh day (D-7) of the experiment, as shown schematically in
[0602] The sexual enhancing effect of RW1 is seen as an increase in the number of mounts counted during the 4 hour observation period.
Example 33: Enriched Extract from Root of Neobeguea mahafalensis Prepared by Solvent Extractions and Reversed Phase Chromatography
Boiling Water Extraction
[0603] Twenty five grams (25.0 g) of dried ground root from Neobeguea mahafalensis were put in boiling water (2500 ml). The mixture was boiled for 2 min, and then allowed to slowly cool and the mixture left for 16 h. The concoction was then filtered. The filtrate was freeze-dried. A light-brown fluffy powder (RW) formed, but to save the time the process was interrupted when about 10% of ice from start was still present. [As a control the residual solid residue (RR) was also collected and dried in the open air until constant weight (15.8 g); however RR was not used further].
Chloroform Extractions
[0604] The partly freeze dried filtrate RW was thawed and water was added to 500 ml final volume. Chloroform (500 ml) was then added and the mixture was put at stand to allow the residual ice to melt. The mixture was then placed into a separation funnel, intensely shaken for 2 min, then left to stand for 20 h (separation of phases proceeded very slowly, part of the mixture remained as an unseparated zone between water and chloroform layers, containing precipitate). The chloroform and water layers were separately collected. The intermediate zone was placed in plastic tubes resistant to chloroform and centrifuged at 3000 rpm for 15 min. Chloroform and water layers, as well as an unresolved layer in between the chloroform and water layers formed in the tubes. After completion of the centrifugation the chloroform and water layers were separately collected by pipette and united with the previous water and chloroform phases, whereas the unresolved middle layer was retained in the tube. Twenty ml each of water and chloroform were then added to each tube, the tubes were again vortexed, the centrifugation repeated, and the new chloroform and water layers were again collected, again retaining the middle layer. Addition of solvents, vortexing, centrifugation, collection of layers and uniting the chloroform and water layers with the previous chloroform and water layers was repeated four times. The unresolved middle layers were also united. The combined water layers were placed in a separation funnel and fresh chloroform (500 ml) was added and the mixture intensely shaken for 2 minutes. After separation three layers formed as before, and were collected.
[0605] The middle layer was combined with the previous united middle layers. After evaporation the united middle layers weigh 195 mg comprising a brown-red powder (R2P). R2P was not used further.
[0606] The chloroform phase was united with all the previous chloroform phases and evaporated to dryness, the residue treated with acetonitrile (40 ml), trying to dissolve it. Some part of the residue (a wax-like material) was not soluble, and was removed upon decantation. To the decantate (i.e., the acetonitrile solution) water (60 ml) was added. An oily precipitate formed. The mixture was filtered, and the filtrate was freeze-dried. A white powder, R2C (yield 190 mg) formed.
[0607] The wax-like material (R2X1) (constituting only a small fraction) and the oily precipitate (R2X2; obtained from the chloroform phase after having filtrated of R2C and evaporating off the chloroform) were first dried under vacuum in presence of P205, then dissolved in chloroform (10 ml for each sample), the solutions combined and evaporated. Residual colorless oil (R2X) weighed 30 mg. (R2X was not used further in this example).
[0608] United water phases obtained after extraction with chloroform were freeze-dried. A brown, fluffy powder (R2W) formed. Yield 6.39 g. (R2W was not used further in this example).
Further Fractionation of R2C Using Reversed Phase Liquid Chromatography
[0609] R2C (190 mg) was dissolved in acetonitrile (15 ml), and then water (35 ml) was added. A slightly turbulent solution formed. The turbulent solution was applied to a glass column (33×420 mm) filled with LiChroprep RP-18 (Merck Chemical Co., Germany) equilibrated with cool (+5° C.) 30% acetonitrile in water+0.1% trifluoroacetic acid. The column (which was maintained in a cold room with all eluents at +5° C.) was eluted first with 30% acetonitrile in water+0.1% trifluoroacetic acid (1 liter), then with similar solutions where the acetonitrile content was sequentially changed to respectively 40%, 50%, 60% (volume of each eluent portion 1 liter) and finally to 70% (1.5 liter). Flow rate was about 1.5 ml/min. Eluate fractions of 12 ml volumes each were collected in glass tubes (each tube being given an ordinal number in the order they were collected starting from tube 1 and ending with tube 440). 30% acetonitrile content in eluent corresponded to the eluted 1-84 fractions (i.e. tube 1-84), 40%- to 85-179 (i.e. tube 85-179), 50%- to 180-267 (i.e. tube 180-267), 60%- to 268-345 (i.e. tube 268-345), and 70% to 346-440 fractions (i.e. tube 346-440).
[0610] Every 10th fraction (i.e. content of every 10:th tube) was then analyzed by HPLC (LiChroprep RP-18 2.1×100 mm column, eluent—linear gradient from 20% acetonitrile in 5 mM ammonium acetate to 80% acetonitrile in 5 mM ammonium acetate in 60 min, flow rate 0.2 ml/min, detection at 220 and 260 run). (Later on some fractions were analyzed further to determine the partition of certain UV absorbing peaks between fractions).
[0611] Three bigger fractions were then created by pooling the contents of tubes 1 to 257, 258 to 350 and 351 to 440, respectively. The three pools were freeze dried. The three freeze dried portions were then dissolved in 50% (for the pool of tubes 1 to 257), 50% (for the pool of tubes 258-350) and 70% (for the tubes 351-440) acetonitrile in water (volume of each solution 100 ml) and the solution formed were again freeze dried. Extracts RA (43.5 mg) (for pool of tube 1-257), RB (83.9 mg) (for pool of tube 258-350) and RC (54.6 mg) (for pool of tube 351-440) were obtained as white fluffy powders. (Besides, an additional portion of RA (9.6 mg) was contaminated with material used in the laboratory practice (i.e. dye from marker used for labelling on glass surface) and collected separately and measured for its weight; adding up this materials with the RA collected the yield of RA was 53.1 mg.) (The weights above for RA, RB and RC are approximate, as the samples were weighed in plastic tubes, not on small pieces of paper or aluminum foil, because of suspected hygroscopic properties and static electricity).
Exemplary Analytical HPLC Chromatograms of Extracts
[0612] RA, RB and RC, along with the starting material R2C, are shown in Drawing section
[0613] Assay of the sexual enhancing activities of extracts R2C, RA, RB and RC in mice is given in Example 34. Assay of the sexually enhancing activity of R2C was given in Example 22. Extracts R2C and RB are particularly preferred sexual enhancing extracts of the invention.
Example 34: Assay of Enriched Extracts Prepared from Neobeguea mahafalensis on Sexual Behavior in Mice
[0614] The activity of extract R2C, RA, RB and RC, prepared according to Example 33, was assayed for sexual enhancing activity essentially using the method describe in Example 30, the only difference being that the mice were treated with subcutaneous injections of extracts R2C, RA, RB or RC for three consecutive days at a dose of 4 mg/kg for each day. For the sake of administration extracts R2C, RB and RC were dissolved in sun flower oil and injected subcutaneously in a volume of 1 ml/kg of mice body weight. RA was dissolved in water and injected subcutaneously in a volume of 1 ml/kg. Controls received the same amount of sun flower oil subcutaneously for three consecutive days.
[0615] As seen from the
Example 35: A Formulation of a Pharmaceutical Prepared from Extract RB and R2C from Neobeguea mahafalensis Comprising Oil Solution for Intramuscular or Subcutaneous Injection
A)
[0616]
TABLE-US-00009 RB extract, prepared according to Example 33 20 mg Cotton oil Ad 1 ml
B)
[0617]
TABLE-US-00010 R2C extract, prepared according to Example 33 20 mg Cotton oil Ad 1 ml
Example 36: Formulation of a Pharmaceutical Comprising Oil Solution of Extract RB and R2C from Neobeguea mahafalensis for soft gelatin capsules intended for per oral administration
A)
[0618]
TABLE-US-00011 RB extract, prepared according to Example 33 20 mg Peanut oil Ad 1 ml
[0619] The oil solution was filled into an open soft gelatin capsule and the capsule was sealed by adding a drop of hot liquidized gelatin to the opening and allowing the drop to cool down.
B)
[0620]
TABLE-US-00012 R2C extract, prepared according to Example 33 20 mg Peanut oil Ad 1 ml
[0621] The oil solution was filled into an open soft gelatin capsule and the capsule was sealed by adding a drop of hot liquidized gelatin to the opening and allowing the drop to cool down.
Example 37: Tea Bag with Ground Root of Neobeguea mahafalensis
[0622] Heat-sealable tea bag paper was obtained from Glatfelter Corporation—Schoeller Hoesch (Papierfabrik Schoeller & Hoesch, GmbH & Co. KG, Postfach 1155, D-76584 Gernsbach, Germany). The paper was cut to 14×8 cm and was folded in the middle of the long direction so as to yield a double paper layer of 14×4 cm. The paper was then sealed by applying heat along the open long edge so as to form a tube 14×4 cm.
[0623] Root of Neobeguea mahafalensis was carefully ground into a fine powder and the tube was filled with 2 g of the powder. The tube was then folded again in the middle along the short direction thus forming a double layered tube with the powder inside and the openings to the same side. The tube was then closed in its open ends by pursing them together and sealing with a stapler. A string with a small paper label attached to its opposite was also attached under the staple to allow immersing and stirring the bag in hot water.
Example 38: Preparation of Biological Sample for Assay of Characteristic Mass-Peaks
[0624] Minipigs (Ellegaard Gδttingen Minipigs ApS, Góttingen, Germany) were treated with 4, 40 or 400 mg/kg of extract R2C (prepared as described in Example 33) or the same quantities of extract RB (prepared as described in example 33) by subcutaneous injection dissolved in sun flower oil. 24 hours post-injection blood was drawn into heparinized Vacutainer glass tubes (Becton Discinson Co.) and the plasma immediately prepared by centrifugation and stored at −20° C. until time of further sample preparation.
[0625] Prior to use, all glassware was silanized by treatment with dimethyldichlorosilane and then rinsed with toluene and dried. Since the characteristic mass-peak arise from different compounds (components) several different extraction procedures were used in the preparation of samples in order to extract them optimally.
Preparation Procedure 1
[0626] Ten ml aliquots of plasma were transferred to glass tubes with teflon-lined screw caps. Twenty ml of acetonitrile were added to each tube, the samples vortexed for 30 seconds and then centrifuged at 2,000 rpm for 10 minutes. The supernatants were carefully decanted into clean tubes and the volumes reduced to less than 0.1 ml by evaporation at 40° C. under a dry nitrogen stream for use in subsequent mass-spectrometric analysis.
Preparation Procedure 2:
[0627] Ten ml aliquots of plasma were transferred to glass tubes with teflon-lined screw caps. Twenty ml of acetonitrile were added to each tube, the samples vortexed for 30 seconds and then centrifuged at 2,000 rpm for 10 minutes. The supernatants were carefully decanted into clean tubes and the volume reduced to less than 1 ml by evaporation at 40° C. under a dry nitrogen stream. Ten ml of 0.2 N NaOH and 20 ml of a hexane-ethylacetate mixture (9:1, v/v) were added to each tube. The tubes were shaken for 30 minutes on a reciprocating shaker at 60 cycles/minute followed by centrifugation at 2,000 rpm for 5 minutes. The organic phases were combined and evaporated to dryness. The samples were dissolved in small volumes of acetonitrile for use in subsequent HPLC and/or mass-spectrometric analysis.
Preparation Procedure 3:
[0628] Ten ml aliquots of plasma were transferred to glass tubes with teflon-lined screw caps. Twenty ml of acetonitrile were added to each tube, the samples vortexed for 30 seconds and then centrifuged at 2,000 rpm for 10 minutes. The supernatants were carefully decanted into clean tubes and the volume reduced to less than 1 ml by evaporation at 40° C. under a dry nitrogen stream. Ten ml of 0.1 N HCl and 20 ml of a hexane-ethylacetate mixture (9:1, v/v) were added to each tube. The tubes were shaken for 30 minutes on a reciprocating shaker at 60 cycles/minute followed by centrifugation at 2,000 rpm for 5 minutes. The organic phases were combined and evaporated to dryness. The samples were dissolved in small volumes of acetonitrile for use in subsequent HPLC and/or mass-spectrometric analysis.
Preparation Procedure 4:
[0629] Ten ml aliquots of plasma were transferred to glass tubes with teflon-lined screw caps. Twenty ml of acetonitrile were added to each tube, the samples vortexed for 30 seconds and then centrifuged at 2,000 rpm for 10 minutes. The supernatants were carefully decanted into clean tubes and the volume reduced to less than 1 ml by evaporation at 40° C. under a dry nitrogen stream. Ten ml of 0.2 N NaOH and 20 ml of a hexane-ethylacetate mixture (9:1, v/v) were added to each tube. The tubes were shaken for 30 minutes on a reciprocating shaker at 60 cycles/minute followed by centrifugation at 2,000 rpm for 5 minutes. The organic phases were transferred to a clean glass tubes and 10 ml of 0.1 N HCl added. The tube was again agitated (15 minutes) and again centrifuged (10 minutes). The organic phases were combined and evaporated to dryness. The samples were dissolved in small volumes of acetonitrile for use in subsequent HPLC and/or mass-spectrometric analysis.
Preparation Procedure 5:
[0630] Ten ml aliquots of plasma were transferred to glass tubes with teflon-lined screw caps. Twenty ml of acetonitrile were added to each tube, the samples vortexed for 30 seconds and then centrifuged at 2,000 rpm for 10 minutes. The supernatants were carefully decanted into clean tubes and the volume reduced to less than 1 ml by evaporation at 40° C. under a dry nitrogen stream. Ten ml of distilled water and 20 ml of chloroform were added to each tube. The tubes were shaken for 30 minutes on a reciprocating shaker at 60 cycles/minute followed by centrifugation at 2,000 rpm for 5 minutes. The samples were dissolved in small volumes of acetonitrile for use in subsequent HPLC and/or mass-spectrometric analysis.
Preparation Procedure 6:
[0631] 500 μl plasma samples were diluted with 700 μl of acetonitrile. After vortex mixing during 30 s, the samples were ultra centrifuged for 8 min at 10,500 rpm. The supernatant was mixed in a 5 ml glass tube, with 800 Il of 1-octanosulfonic acid sodium salt solution at 0.2 M during 30 s.
[0632] The extraction procedure of the obtained solution was preformed on solid-phase extraction (SPE) cartridges using a vacuum manifold. Three milliliter per hundred milligrams Upti-Clean CN-S (INTERCHIM, Asniere sur seine, France) cartridges were used.
[0633] The mixture was loaded under vacuum on an extraction cartridge previously activated with 2 ml of methanol and 2 ml of water. The cartridge was then washed with 2 ml of water and dried under pressure for 5 min. Elution was carried out with 2×0.4 ml of methanol (sometimes ethylacetate or chloroform was used as eluant). The eluant was evaporated under a stream of nitrogen in water bath at 40° C. The residue was reconstituted in 200 Il aliquot of acetonitrile. A 50 μl aliquot was then analyzed in subsequent HPLC and/or mass-spectrometric analysis.
Sample Analysis:
[0634] Samples prepared according to preparation procedures 1-6 were introduced into Q-To f2 mass spectrometer using flow injection approach. The instrument was configured corresponding to either ESI or APPI analysis. Injection volume was 10 ml, flow rate—100 ml/min. Alternatively, an HPLC column was connected to Q-Tof2 in order to perform LC/MS. The analysis was performed as described under Example 24 and 39. Characteristic set of mass peaks, corresponding to those listed in Examples 24 and 39, were detected.
Example 39: Accurate Mass Measurements of RB Extract Using Electrospray Ionization and APPI on a Q-Tof2
[0635] To analyze the presence of characteristic mass-peaks in the RB extract, the RB extract prepared according to Example 33 was subjected to repeated fractionation on a semi-preparative (10×250 mm) HPLC column filled with LiChrosorb RP 18, particle size 10 microns (Merck Chemical Co., Germany). Eluent for this fractionation was 52% acetonitrile in water+0.1% trifluoroacetic acid. Eluate fractions, containing ten different separated components were further investigated by analytical HPLC performed on an HPLC column (4.6×250 mm) filled with LiChrosorb RP 18, particle size 5 microns, eluted by linear gradient from 20 to 80% acetonitrile in water+0.1% trifluoroacetic acid, flow rate 1.0 ml/min. A photo diode array (PDA) detection during HPLC analysis allowed us also to obtain characteristic UV spectra of separated RB components (obtained data for retention times on the HPLC column and UV maxima of the separate components are shown in the table in the drawing section,
[0636] Each one of the ten freeze dried components were dissolved separately to yield an analyte concentration of about 1 ng/mL in acetonitrile-water-formic acid (50:50:0.2). The solution was then mixed with a similar solution containing a reference substance with known molecular mass (preferably the molecular mass of the known substance was close to the mass peak of the analute), and the mixture was introduced into the Q-To f2 mass spectrometer using electrospray needle. ESI spectrum of the mixture was recorded. Composition of the mixture was optimized in terms of proportions of the components and their concentration, trying to obtain peaks of equal intensity comprising about 200 counts in one scan. When such a situation was attained the mass spectrum was recorded. The mass-peak value of the reference substance was used to correct the masses of the analytes, which was done automatically by the built in software of the Q-To f2: MassLynx. The thus obtained mass values, representing the masses of the characteristic mass-peaks associated with the respective component of RB and are listed in the table of
[0637] Accurate mass measurements using electrospray ionization in negative mode were made in a similar fashion as above for each of the 10 components, however the solvent used for the components was acetonitrile-water (50:50) without the addition of formic acid. The mass values thus obtained, representing the masses of the characteristic mass-peaks associated with the respective component of RB, are listed in the table of
[0638] Accurate mass measurements using atmospheric pressure photo ionization (APPI) were made in the similar fashion as above, by injecting 10 μL of a solution of each of the ten freeze dried components mixed a with reference substance in methanol-toluene (95:5) using a syringe pump (flow rate 100 μL/min). The mass-peaks thus obtained are presented in the table of
Example 40: Comparison of Sexual Activity Enhancing Activity of Extracts from Stem Bark and Root of Neobeguea mahafalensis
[0639] The activity of extracts R2C and S2C, prepared in essentially the identical fashion as described to Examples 9 and 10, from respectively stem bark and root of Neobeguea mahafalensis were assayed for sexual enhancing activity essentially using the method describe in Example 30, the only difference being that the mice were treated with subcutaneous injections with either of the extracts R2C or S2C at a dose of 4 mg/kg for each day for 3 consecutive days. Extracts R2C and S2C were dissolved in sun flower oil and injected in a volume of 1 ml/kg of mice body weight. Controls received the same injection schedule of sunflower oil. Each of the treatment group comprised 4 male mice. The sexual activity test was performed only on D-4 (i.e., fourth day after start of treatment of the male mice). As seen from
Conclusion
[0640] The extract of root from Neobeguea mahafalensis is highly active in enhancing sexual activity and thus comprise a highly desired embodiment of the invention—i.e. being an extract comprising sexual enhancing effect. However, the extract of the stem bark from Neobeguea mahafalensis is inactive and accordingly not very desired for administration to animal or human.
[0641] Because of the finding that S2C is devoid of sexual enhancement activity, S2C is not a desired part of the present invention. Accordingly the use of stem bark of Neobeguea mahafalensis is specifically excluded from all aspects of the present invention.
Example 41: Preparation of R306 and R310 from Root of Neobeguea mahafalensis by Hot Water and Chloroform Extraction and Chromatography
[0642] Dried ground root (722 g) from Neobeguea mahafalensis was divided into 144 portions (about 5 g in each). Each portion was put in boiling water (500 ml). The mixtures were boiled for 2 min, and then allowed to slowly cool down for 16 h. The concoctions were filtered through cotton wool plugs placed into glass funnels. The solid residues (RR) were collected, united and dried on the open air until constant weight (405.4 g). The united filtrates were freeze-dried. A light-brown powder (RW) formed, but to save time the process was interrupted when about 10% of starting ice was still present.
Extraction Procedure:
[0643] To about 1/15 part of the above material water (to 500 ml volume) and chloroform (500 ml) were added allowing melting the residual ice. The mixture was placed into a separation funnel and intensely shaken for 2 min. Then it was left to stand for 20 h (separation of phases proceeded very slowly, part of the mixture remained as an unseparated zone between water and chloroform layers containing precipitate). The chloroform and water layers were separately collected, but the intermediate zone was placed in plastic tubes resistant to chloroform and centrifuged at 3000 rpm for 15 min. Chloroform and water layers formed in the tubes after the centrifugation were separately collected by pipette. 20 ml of each solvent were equally divided and added to the tubes, tubes were vortexed, centrifugation repeated, and the new chloroform and water layers were again collected. Addition of water and chloroform solvents, vortexing, centrifugation, collection of layers was repeated four times. The unresolved central layer was evaporated. A brown-red powder (R2P) remained.
[0644] The procedure was repeated using combined water layers from the previous extraction, adding a fresh chloroform portion (500 ml). A new R2P portion was collected, which was combined with the previous one.
[0645] All 15 portions of RW were fractionated this way; summary yield of R2P was 14.4 g.
[0646] The united chloroform extracts were evaporated to dryness, the residue treated with acetonitrile (1000 ml), trying to dissolve it. Some part of residue (R2X, a wax-like material) was not soluble. The acetonitrile solution was separated by decantation and evaporated to dryness. Yield of oily residue (R2C) was 6.68 g. R2X weighed 146 mg.
[0647] United water phases obtained after extraction with chloroform were freeze-dried. A brown, fluffy powder (R2W) formed. Yield 101.1 g.
Fractionation of R2C Using Reversed Phase Liquid Chromatography
[0648] R2C (6.68 g) was dissolved in acetonitrile (500 ml), and water (1000 ml) was added. A slightly turbulent solution formed. The turbulent solution was applied to a cooled (+5° C.) glass column (10×24.5 cm) filled with LiChroprep RP-18 (Merck) and equilibrated with 30% acetonitrile in water+0.1% trifluoroacetic acid. The column was then placed in the cold room and eluted first with 30% acetonitrile in water+0.1% trifluoroacetic acid (4 liters), then with similar solutions, changing the acetonitrile content subsequently to 40%, 50%, 60% and 70% (volume of each eluent portion 4 liter). Flow rate was about 10 ml/min. Just after starting of application of the 70% eluent about 60 ml of eluate were collected separately for further isolation of substantially pure compounds. This eluate fraction was diluted with an equal volume of water, frozen and freeze dried. A white powder (R3004) was obtained. Yield 250 mg. The column was regenerated by washing with isopropanol (4 liter) and before storage 40% ethanol (1 liter) was passed through it.
Preparation of Crude R306 and R310
[0649] R3004 (about 20 mg) was placed into 1.5 ml volume centrifuge tube. 0.7 ml of acetonitrile were added, and vortexed until dissolution. Then 0.7 ml of water were added, the tube shaken and centrifuged. The clear supernatant obtained was introduced into a semipreparative Lichrosorb C18 column (10×250 mm) (Merck) previously equilibrated with 62% acetonitrile in water. The column was eluted with the same buffer, flow rate 5 ml/min, UV detection at 270 nm. The eluate fraction corresponding to the peak that follows the highest peak in the peak group (retention time 30.6 min) was separately collected in order to obtain crude R306, and similarly eluent fraction corresponding to the last, remotely placed peak (retention time 46.8 min) was collected in order to obtain crude R310. In such a way R3004 was repeatedly applied to the Lichrosorb C18 column and the eluate fractions containing crude R306 and R310 were pooled, diluted with an equal volume of water, frozen and freeze-dried. Fluffy, white powders formed. Yield 58 mg of powder for crude R306 and 24 mg of powder for crude R310.
Preparation of Substantially Pure R306 (Grade 3)
[0650] Crude R306 from the previous step (about 2 mg) was placed into 1.5 ml volume centrifuge tubes, 0.5 ml isopropanol was added and the tubes were vortexed until dissolution. 1.0 ml of water was added, the mixture was shaken and centrifuged. The clear supernatant obtained was introduced into a semipreparative Chirobiotic V column (10×250 mm) (Astec; obtained from Supelco/Sigma-Aldrich, 595 N. Harrison Road, Bellefonte, Pa. 16823-0048, USA) previously equilibrated with 34% isopropanol in water. The column was eluted with the same buffer, flow rate 1 ml/min, UV detection at 220 nm. The eluate fraction corresponding the central part of the main peak was separately collected. In such a way 20 mg of crude R306 were applied for purification. The appropriate eluate fractions were pooled and freeze-dried. Yield of substantially pure R306 (Grade 3) (white, fluffy powder) was 12 mg.
Preparation of Substantially Pure 306 (Grade 4)
[0651] R306 (Grade 3) from the previous step (about 1.2 mg) was placed into 1.5 ml volume centrifuge tubes, 0.5 ml isopropanol was added and the tubes were vortexed until dissolution of the substance. 1.0 ml of water was added, the mixture was shaken and centrifuged. The clear supernatant obtained was introduced into analytical Lichrospher C18 column (4.6×250 mm; Merck) previously equilibrated with 40% isopropanol in water. The column was eluted with the same buffer, flow rate 1 ml/min, UV detection at 220 nm. The eluate fraction corresponding to the central part of the main peak was separately collected. In such a way 12 mg of R306 (Grade 3) were repeatedly applied for purification. The appropriate eluate fractions were pooled and freeze-dried. According to HPLC and mass-spectrometry a 100% pure product R306 (Grade 4) was obtained as a white, fluffy powder. Yield 7.6 mg. High-resolution mass-spectrometry 699.2991 (M+H+).
[0652] Analytical HPLC of substantially pure R306 (Grade 4) was performed on a Waters system (Millenium32 Workstation, 2690 Separation Module, 996 Photodiode Array Detector) equipped with LISPRPI 8-5-3627 column (Hichrom Ltd, England) using water/2-propanol (60:40) as eluent (isocratic regime) at flow rate −0.7 mL/min and detection at 220 nm yielded one single peak with retention time 9.957 min (
[0653] The UV spectrum of R306 was rerecorded on-line with the diode array detector yielding absorption maxima at 198, 215 and 261 nm (Drawing section,
Preparation of Substantially Pure R306 (Crystalline)
[0654] R306 (Grade 4) (1.3 mg) was placed into a small thick-wall glass vial. Isopropanol (300 microlitres) was added, the vial was closed with a polypropylene cap and vortexed until dissolution. The solvent was then evaporated under a gentle argon stream till dryness. Formation of a white, crystalline residue occurred. Isopropanol (100 microlitres) was then added and the vial was placed in the refrigerator overnight. Then liquid was removed by pipette and the remaining crystals were dried in a desiccator. Yield 1.0 mg.
Preparation of Substantially Pure R310
[0655] Crude R310 obtained above was subjected to purification essentially as described above for R306 in Example 41 by first applying the crude R310 into the semipreparative Chirobiotic V column (10×250 mm) previously equilibrated with 34% isopropanol in water and eluted with the same buffer, flow rate 1 ml/min, and after pooling and freeze-drying the main peak the materials was further applied into a Lichrospher C18 4.6×250 mm column equilibrated with water/2-propanol (65:35) and eluted with the same buffer (isocratic regime) at a flow rate of 1 mL/min and detection at 268 nm, which yielded essentially only one peak of substantially pure R310 eluted at the retention time 37.1 min, which was collected and freeze-dried. High-resolution mass-spectrometry 715.2953 (M+H+). The UV spectrum of R310 was rerecorded on-line with the diode array detector yielding absorption maxima at 210 and 270 nm. In addition the thus obtained R310 was further purified on Lichrospher C18 column (4.6×250 mm; Merck) using essentially the same procedure as for R306 above, under “Preparation of substantially pure 306 (Grade 4)”. The very pure R310 thus obtained was used for NMR determination of the structures the tautomeric forms R310A and R310B, of R310.
Analysis of Substantial Pure R310 by HPLC and UV-Spectrometry
[0656] The sample of the very pure R310 from the previous step was analyzed by HPLC. It was then observed that the compound gradually formed two peaks on the chromatographs, which correspond to the keto and enol forms R310A and R310B, respectively; the keto form being the initial one purified and also residing in larger quantity in the equilibrium formed.
[0657] On LiChrosorb RP 18-5 (2.1 mm×100 mm, 5 μm) using a gradient formed from water and acetonitrile (from 20% to 90% MeCN) with 5 mM ammonium acetate additive; flow rate 0.2 mL/min during 60 min; detection at 220 nm, two peaks formed with retention times 37.0 (presumed R310B) and 42.1 (presumed R310A) min.
[0658] On LiChrospher RP 18-5 (4.6 mm×250 mm, 5 am), using a Millenium32 Workstation, 2690 Separation Module, 996 Photodiode Array Detector, using acetonitrile (30:70) with 0.1% trifluoroacetic acid additive a eluent; flow rate 0.7 mL/min during 35 min (isocratic regime); detection at 220 nm, two peaks formed at 11.8 minutes (presumed R310B) and 16.4 minutes (presumed R310A).The UV spectra of the peaks were recorded with the Photodiode Array Detector; the presumed R310A form of R310 showing two UV absorption maxima at 208 and 269 nm, and the presumed R310B form of R310 showing two UV absorption maxima at 191 and 209 nm.
Example 42: Preparation of R306 from Root of Neobeguea mahafalensis by Chloroform Extraction and Chromatography
[0659] To dried ground root of Neobeguea mahafalensis (124 g) chloroform (1.2 liter) was added. The mixture was placed in the cold room (+5° C.) and shaken overnight. The suspension was then filtered under vacuum through a porous glass filter, and the material on the filter was washed with chloroform (300 ml). The united filtrates were evaporated on rotary evaporator till dryness. A glass like material RCH (3.59 g) formed. The glass like material was agitated with acetonitrile (300 ml) for 30 min, trying to dissolve it. A precipitate formed. The mixture was vacuum filtered through a cotton wool plug placed into a glass funnel. The filtrate was diluted with an equal volume of water, and the emulsion formed was applied to a cooled (+5° C.) glass column (10×24.5 cm) filled with LiChroprep RP-18 and equilibrated with 60% acetonitrile in water. The column was then placed in the cold room (+5° C.) and eluted, first with 60% acetonitrile in water (4 liters), then with 70% acetonitrile in water (4 liters). Flow rate was about 10 ml/min. Just after starting of application of the 70% eluent, 100 ml size eluate fractions were collected. The content of R306 characteristic mass-peaks in these fractions was monitored using mass spectrometer with electrospray needle, looking for ion 699.3 (M+H*). Substantial content of this ion was found in fractions 6 to 10 (i.e. 600-1000 ml of 70% eluent). These fractions were diluted with the equal volumes of water and freeze dried. The obtained fluffy powders were placed into 1.5 ml volume centrifuge tubes, each dissolved in 1.0 ml acetonitrile, 0.5 ml water added and centrifuged. The clear supernatant obtained was introduced into two semipreparative columns connected in sequence comprised of a Lichrospher C18 column and a Lichrosorb C18 column (each 10×250 mm) previously equilibrated with 65% acetonitrile in water. The columns were eluted with the same buffer, flow rate 5 ml/min, UV detection at 260 nm. The eluate fractions corresponding to the descending slope of the main peak (including shoulder, monitoring the 699.3+ ion continuously) were separately collected, united and freeze dried affording 19.5 mg of white, fluffy powder. The powder was suspended in isopropanol (1.5 ml). The suspension was distributed between 5 centrifuge tubes (1.5 ml volume) 0.3 ml in each. To each tube 0.2 ml isopropanol and 1.0 ml water were added, shaken and centrifuged. The clear supernatant obtained was introduced into a semipreparative Chirobiotic V column (10×250 mm) previously equilibrated with 34% isopropanol in water. The column was eluted with the same buffer, flow rate 1 ml/min, UV detection at 220 nm. The eluate fraction corresponding the former part of the main peak (monitoring the 699.3+ ion continuously) was separately collected. The appropriate eluate fractions from the following purification runs were pooled and freeze-dried. Yield of crude R306 (white, fluffy powder) was 2.4 mg.
[0660] The crude R306 sample from the previous step (about 1.2 mg) was placed into 1.5 ml volume centrifuge tube, 0.5 ml isopropanol added and the mixture was vortexed until dissolution. 1.0 ml of water was added, mixture shaken and centrifuged. The clear supernatant obtained was introduced into an analytical Lichrospher C18 column (4.6×250 mm) previously equilibrated with 40% isopropanol in water. The column was eluted with the same buffer, flow rate 0.7 ml/min, UV detection at 220 nm. The eluate fraction corresponding to the main peak (monitoring the 699.3+ ion continuously) was separately collected. In such a way all of the remaining crude R306 from the previous step was applied for purification. The appropriate eluate fractions were pooled and freeze-dried. Yield of according to HPLC and mass-spectrometry 100% pure product R306 (Grade CH 4) (white, fluffy powder) was 1.0 mg. Mass-spectrometry data: 699.3 (M+H+), 716.3 (M+NH4+), 721.3 (M+Na+), 737.3 (M+K+).
Example 43: Dose and Time Response of R306 and R310 on Sexual Activity in Mice
[0661] The sexual enhancing effect of R306 (Grade CH 4; i.e. better than 99% pure R306) from Example 42 was assessed using the method described in Example 30. Mice were injected subcutaneously with either of 0.4, 0.04 and 0.004 mg/kg of R306 (Grade CH 4) dissolved in sun-flower oil each day for three consecutive days. Control mice received sun-flower oil only. Each group contained 3 mice.
[0662] The number of mounts of each male mouse onto the female mouse was then counted over a 3 hour period on respectively the 4:th day (D-4), the 7:th day (D-7) and the 14:th day (D-14) after the first days injection of R306 for each couple and the average of number of mounts was calculated for each group.
[0663] Results are shown in Drawing section
[0664] Based on these data, using the 7:th day as a basis for computation, the activity of the substantially pure R306 was estimated to be about 15 U/mg (i.e. 15 Umnt/mg; i.e. computed by fitting the mounting data of
[0665] The sexual enhancing activity of R310 obtained as described in Example 41 was assessed using the same method as described in Example 30. Mice were injected subcutaneously with 4, 0.4 or 0.04 mg/kg of R310 dissolved in sun flower-oil each day for three consecutive days. Control mice received sun-flower oil only. Each group contained 3 mice.
[0666] The number of mounts of the female over a 3 hour period were then counted on day 4 (D-4), day 7 (D-7) and day 14 (D-14) following the first injection of R310. Results are shown in Drawing section
[0667] Significances in this example were assessed with Student's t-test, two-tailed.
Example 44: Pharmaceutical Preparations of R306
Preparation of R306 for Oral Use:
[0668]
TABLE-US-00013 R306 (Grade 4) 15 mg Cotton oil Ad 1 ml
Preparation of R306 for Subcutaneous Injection:
[0669]
TABLE-US-00014 R306 (Grade 4) 5 mg Peanut oil Ad 1 ml
Example 45: Pharmaceutical Preparation of R310
Preparation of R310 for Oral Use:
[0670]
TABLE-US-00015 Substantially pure R310 prepared according to Example 41 15 mg Cotton oil Ad 1 ml
Preparation of R310 for Subcutaneous Injection:
[0671]
TABLE-US-00016 Substantially pure R310 prepared according to Example 41 5 mg Peanut oil Ad 1 ml
Example 46: Pharmaceutical Preparation Containing a Mix of R306 and R310 for Injection
[0672]
TABLE-US-00017 R306 (Grade 4) 5 mg Substantially pure R310 prepared according to Example 41 10 mg Cotton oil Ad 1 ml
Example 47: Determination of the Structure of R306, R310A and R310B by NMR
Methods
[0673] NMR spectra were recorded with a Varian UNITY INOVA 600 MHz spectrometer equipped with a cryoprobe in CDCl.sub.3 solution at 25° C. Chemical shifts are reported in ppm relative to residual solvent signal (δ(.sup.1H) 7.25 ppm, δ(.sup.13C) 70.0 ppm). Two-dimensional spectra recorded included DQF-COSY, NOESY, TOCSY, sensitivity-enhanced .sup.13C-HSQC and .sup.13C-1H HMBC. Pulsed-field gradients were used for all .sup.13C correlation spectra. The NOESY mixing time was 800 ms, the TOCSY one 70 ms. .sup.13C-HMBC spectra were recorded with coupling evolution delay for the generation of multiple-bond correlations set to 62.5 ms. All 2D spectra were run with 4096*1024 points data matrix, giving T.sub.2max=250 ms for 1H nucleus in acquisition dimension and τ.sub.1max=100 ms for 1H or τ.sub.1max=50 ms for .sup.13C for indirect dimension. Prior to Fourier transform the data matrix was zero-filled twice and multiplication by shifted sine-bell window function applied. For .sup.1H-.sup.13C HMBC the magnitude spectra were calculated. Graphs from the spectra recorded are shown in drawing section
Results
[0674] The structure elucidations were accomplished on the basis of two-dimensional .sup.1H-.sup.1H and .sup.1H-.sup.13C spectra of the compounds taken in CDCl.sub.3 solutions. According to the spectra R306 was stable during several weeks, but R310 consisted of two isomers which slowly interchanged. One of the R310 isomers had a very low field signal at 13.84 ppm being characteristic for the intramolecularly heated OH proton in β-diketones when the enolized proton is hydrogen-bonded to the ester function. Thus, the initial proposal was that R310 should contain a β-diketone moiety that gives two forms in the spectrum due to slow keto-enol equilibrium. Analysis of low field part of the .sup.1H spectrum showed that both of the R306 and R310 compounds contain a 3-substituted furane cycle. For R306 this furane cycle was attached to the rest of molecule through a carbonyl function in position 17, whereas in R310 instead of a carbonyl group there was a 17-CH(OCOCH.sub.3) substituent.
[0675] Careful analysis of the .sup.1H-.sup.13C HMBC spectra allowed us to propose the structure depicted in the drawing section
[0676] In R310A and R310B the β-diketone moiety is incorporated as side chain in position-15. Unequal populations of keto- and enol-forms allowed us to make the assignments of .sup.1H and .sup.13C resonances for both forms of R310 separately (Table 1, Example 47). The R-configuration of 17-CH in R310A and R310B was deduced on basis of very strong NOEs to 30-CH and 5-CH, as well as NOEs between the furane ring and 12-CH2 and NOEs between 17-OAc methyl and isopropyl group.
TABLE-US-00018 TABLE 1 .sup.1H (600 MHz) and .sup.13C (150.9 MHz) NMR Data for Compunds 306 and 310 AB in CDCl3 R306 R310A (keto) R310B (enol) No. δ(.sup.1H), (J, inHz) δ(.sup.13C) δ(.sup.1H), (J, inHz) δ(.sup.13C) δ(.sup.1H), (J, inHz) δ(.sup.13C) 1 — 84.4 — 84.0 — 84.0 2 — 83.3 — 78.2 — 77.7 3 5.37 s 80.4 4.83 s 82.2 4.84 s 82.9 4 — 46.2 — 45.5 — 45.4 5 2.54dd (2.4; 9.8) 35.9 2.84dd (3.2; 8.8) 37.1 3.00dd (2.3; 9.7) 37.0 6 pro-R2.08dd (9.8; 15.5) 33.6 2.37dd (3.2; 15.5) 34.1 2.26dd (3.2; 16.7) 33.7 pro-S 2.32dd (2.4; 15.5) 2.44dd (8.8; 15.5) 2.46dd (9.7; 16.7) 7 — 172.3 — 172.6 — 172.5 8 — 79.7 — 79.4 — 80.5 9 — 83.7 — 83.8 — 84.8 10 — 46.6 — 46.0 — 45.9 11 pro-R 1.39 m 25.0 1.96 m 24.3 1.92 m 23.3 pro-S 2.17 m 2.19 m 2.03 m 12 pro-R 1.83 m 31.4 1.50 m 32.0 1.50 m 30.9 pro-S 2.11 m 1.42 m 1.16 m 13 — 51.6 — 38.7 — 39.8 14 1.96dd (1.7; 8.1) 49.1 2.79 d (0.8) 50.6 2.66 s 44.8 15 2.71; 2.73 m 28.4 3.80 d (0.8) 52.1 — 90.8 16 — 169.0 — 167.2 — 170.5 17 — 199.0 6.00 s 69.6 5.84 s 70.6 18 1.48 s 24.5 1.18 s 21.4 1.22 s 21.6 19 1.10 s 16.0 1.15 s 15.3 1.15 s 15.3 20 — 124.6 — 121.4 — 122.1 21 8.06dd (1.4; 0.8) 147.1 7.55dd (1.7; 0.8) 141.3 7.49dd (1.7; 0.8) 140.7 22 6.79dd (1.9; 0.8) 110.1 6.35dd (2.0; 0.8) 109.3 6.39dd (2.0; 0.8) 109.7 23 7.41dd (1.9; 1.4) 143.4 7.35dd (2.0; 1.7) 142.8 7.32dd (2.0; 1.7) 142.5 28 0.90 s 15.0 0.96 s 14.8 0.95 s 14.6 29 pro-R 2.00 (11.1) 40.8 1.96 d (10.9) 39.6 1.92 d (10.9) 39.4 pro-S 1.80 (11.1) 1.79 d (10.9) 1.81 d (10.9) 30 5.87 s 74.8 5.25 s 75.8 5.32 s 74.4 31 — 118.6 — 118.6 — 118.5 32 1.62 s 21.3 1.55 s 20.7 1.57 s 20.9 CH.sub.3O— 3.51 s 51.7 3.73 s 52.0 3.69 s 51.9 2- isopropilcarboxyl- 2- acetyl- 2-acetyl- C═O — 175.3 C═O — 170.6 C═O 170.4 CH 2.59 septet (6.9) 35.1 CH.sub.3 2.22 s 20.3 CH.sub.3 2.27 s 20.5 CH.sub.3 1.19 d (6.9) 19.0 1.16 d (6.9) 18.5 17-acetyl- 17-acetyl- 3- isopropilcarboxyl- C═O 169.5 C═O 169.3 C═O — 175.8 CH.sub.3 2.01 s 21.1 CH.sub.3 1.97 s 20.5 CH 2.78 septet (7.4) 34.8 CH.sub.3 1.38 d (7.4) 18.6 15-C(O)CH(CH.sub.3).sub.2 15- ═C(OH)CH(CH3)2 1.37 d (7.4) 18.9 C═O — 208.4 ═C(OH) 13.84 182.8 CH 3.15 septet (6.8) 37.9 CH 2.94 septet (6.8) 29.9 CH.sub.3 1.16 d (6.8) 19.5 CH.sub.3 1.26 d (6.8) 18.2 1.04 d (6.8) 18.9 1.12 d (6.8) 20.4
Example 48: Determination of the Content of R306 in R2C and S2C Preparations
[0677] All experiments were curried out using a Perkin Elmer PE SCIEX API 150EX LC/MS masspectrometer equipped with a turboionspray ion source (PerkinElmer Life and Analytical Sciences); samples were injected onto a LiChrosorb RP18-5 (2.1 mm×100 mm, 5 μm) HPLC column (Merck Chemical Co., Germany) attached to the mass spectrometer and eluted with a gradient formed from water and acetonitrile (from 20% to 90% acetonitrile) with a 5 mM ammonium acetate additive during a 60 min period at a flow rate of 0.2 mL/min; eluted peaks were detecting by monitoring for the positive ion around 699.3 amu corresponding to the computed molecular mass of R306. In addition peaks were also detected by UV detection at 220 nm, besides monitoring with the masspectromter. R306 eluted with a peak at about 36.8 min. (Note that in the following all ion-currents were detected at 699.4 amu—the difference to the 699.3 amu calculated from the R306 structure being due to the inability of the masspectrometer to determine molecular masses at higher resolution than about ±0.1 amu.)
Standard Curve
[0678] The accurate weight of 0.106 mg of pure R306 (prepared according to Example 41) was dissolved in 0.707 mL of a mixture of acetonitrile/water (1:1) thereby yielding a stock solution with a concentration of 0.15 mg/mL of R306. Using this solution six experiments were curried out by injecting different amounts of the R306 sample onto the HPLC column and detecting the response occurring at 699.4 amu about 36.8 min following the injection.
[0679] Experiment 1: 20 μL of the R306 stock solution was injected onto the HPLC column; i.e. 3.0 μg of R306 was loaded onto the column.
[0680] Experiment 2: 10 μL of the R306 stock solution was injected onto the HPLC column; i.e. 1.5 μg of R306 was loaded onto the column.
[0681] Experiment 3: The above stock solution of R306 was diluted two-fold with a mixture of acetonitrile/water (1:1) yielding a solution of 0.075 mg/mL of R306, and 10 μL of this solution was injected onto the HPLC column; i.e. 0.75 μg of R306 was loaded onto the column.
[0682] Experiment 4: The diluted R306 solution of Experiment 3 was diluted further two-fold with a mixture of acetonitrile/water (1:1) and 10 μL of this solution was injected; i.e. 0.375 μg of R306 was loaded onto the column.
[0683] Experiment 5: The diluted R306 solution of Experiment 4 was diluted further twofold with a mixture of acetonitrile/water (1:1) and 10 μL of this solution was injected; i.e. 0.1875 μg of R306 was loaded onto the column.
[0684] Experiment 6: The diluted R306 solution of Experiment 5 was diluted further twofold with a mixture of acetonitrile/water (1:1) and 10 μL of this solution was injected; i.e. 0.09375 μg of R306 was loaded onto the column.
[0685] The peak responses from the masspectrometer above the base-line at 699.4 amu occurring at about 36.8 min following the injections were monitored in cps (counts per seconds) and were determined as follows:
Results from Assays of R306, Pure Sample:
TABLE-US-00019 Peak response Weight of R306 for positive ion Experiment injected (μg) at 699.4 amu (cps) 1 3.0 6.84 × 10.sup.6 2 1.5 5.64 × 10.sup.6 3 0.75 4.89 × 10.sup.6 4 0.375 3.89 × 10.sup.6 5 0.1875 2.89 × 10.sup.6 6 0.09375 1.99 × 10.sup.6
[0686] (Exemplary recordings from these assays are also shown graphically in the Drawing section,
[0687] Although the curve is non-linear the amount of R306 in a sample can thus be estimated by using the standard curve from the measured peak ion-current at 699.4 amu occurring close to about 36.8 min following the sample's infection onto the HPLC column.
[0688] Experiments with R2C Two samples of R2C were prepared from roots of Neobeguea mahafalensis collected from two different trees. Sample 1 was from an old stressed tree with heavily damaged stem bark collected in November 2005 and Sample 2 was from a young healthy tree obtained on Feb. 8, 2008. The R2C samples were prepared using the procedure described in Example 9. (Exemplary recordings from these assays are shown in Drawing section,
Sample 1 of R2C
[0689] R2C (0.304 mg) was dissolved in 0.760 mL of a mixture of acetonitrile/water (1:1) yielding a concentration of 0.4 mg/mL of R2C. Three experiments were carried out as follows:
[0690] Experiment 1: 50 μL of the R2C solution (0.4 mg/mL) was injected onto the HPLC column; i.e. 20 μg of R2C was loaded onto the column.
[0691] Experiment 2: 25 μL of the R2C solution (0.4 mg/mL) was injected; i.e. 10 μg of R2C was loaded onto the column.
[0692] Experiment 3: 10 μL of the R2C solution (0.4 mg/mL) was injected; i.e. 4 μg of R2C was loaded onto the column.
[0693] The following results were obtained and after using the above standard curve the content of R306 in R2C was determined:
TABLE-US-00020 Total amount of R306 in Peak response injected sample for positive ion estimated by at 699.4 amu at reading from Weight of R2C about 36.8 min standard curve, Content of R306 in injected, μg in cps in μg R2C, μg/mg (%) 20 3.95 × 10.sup.6 0.39 19.5 (1.95) 10 2.85 × 10.sup.6 0.19 19.0 (1.90) 4 1.35 × 10.sup.6 0.065 16.25 (1.625)
[0694] The average content of R306 in Sample 1 of R2C is (19.5+19+16.25)/3=18.25 μg/mg; i.e. Sample 1 of R2C contained 1.825% R306.
Sample 2 of R2C
[0695] R2C (0.320 mg) from Sample 2 was dissolved in 0.8 mL of a mixture of acetonitrile/water (1:1) yielding a concentration of 0.4 mg/mL of R2C. The solution was injected onto the HPLC column in three experiments in the same way as for Sample 1 above; the results obtained were as follows:
TABLE-US-00021 Total amount of R306 in Peak response injected sample for positive ion estimated by at 699.3 amu at reading from Weight of R2C about 36.8 min standard curve, Content of R306 in injected, μg in cps in μg R2C, μg/mg (%) 20 2.19e6 0.12 6.0 (0.6) 10 1.28e6 0.057 5.7 (0.57) 4 0.63e6 0.027 6.75 (0.65)
[0696] The average content of R306 in Sample 1 of R2C is (6+5.7+6.75)/3=6.15 μg/mg; i.e. 0.615%.
[0697] Conclusion: Because of the difference in contents of the active component R306 in R2C samples from different specimens of Neobeguea mahafalensis a very important aspect of the invention is to determine the content of R306 in extracts prior to their use for preparation of pharmaceutical and prior to administration to human subjects. This assures quality control and is used to determine the dosage of extract to be administered to human subjects as well as Neobeguea mahafalensis specimens can be collected which contain large amounts of R306.
Experiments with S2C
[0698] S2C was prepared from stem-bark of Neobeguea mahafalensis as described in Example 10. 20 μg of the S2C sample was injected onto the HPLC column and assayed as for the other samples. The results from these assays are exemplified in Drawing section
Example 49: Comparison of the Sexual Enhancing Effect of Oral and Sub-Cutaneous Administration of R2C to Male Mice
[0699] The enriched extract R2C was prepared using the procedure described in Example 9 and assayed for its content of R306 using the procedure of Example 48, which was determined to be 0.62%.
[0700] R2C was dissolved in olive oil and administered to male mice, either orally or by subcutaneous injection for three consecutive days and the sexual enhancing activity was then assessed on the 4:th and 7:th day following the start of the experiment essentially using the method describe in Example 30. Controls were subject to the same treatment schedule but given the same amounts of olive oil—per os or by subcutaneous injection, respectively. Each group of treatment comprised 3 animals.
[0701] For oral treatments the doses given each of the tree days were 0.4, 4 and 40 mg/kg. At the 4:th day the sexual behaviour was assessed; all doses increased the number of mountings during a 3 hour period with the effect being highly significant at 4 and 40 mg/kg. These results are illustrated in
[0702] At the 7:th day following the start of treatments an enhancement of the mountings were also observable; the increase in number of mounts being significant at all doses of R2C administered. The results are illustrated in
[0703] The subcutaneous administration of R2C was assessed at a dose of 4 mg/kg. A significant enhancement of the number of mounts was seen both at the 4:th and 7:th day, as illustrated in
[0704] Assessing the effects at 4 mg/kg for oral and subcutaneous administration at the 4:th and 7:th day there was no statistical difference in the increase in the number of mounts seen by the two different modes of administration of R2C.
[0705] Conclusion: Oral and subcutaneous administration of R2C when administered dissolved in vegetable oil are equally effective modes of administration for achieving a sexual enhancing effect.
[0706] Note: Statistics were by Student's non-paired t-test, two tailed
Estimation of Sexual Enhancing Activity of R2C
[0707] Based on these data, using the 7:th day of oral administration of R2C as a basis for computation the EC50 of R2C was estimated to be about 0.91 mg/kg. This gives an activity for this R2C preparation (which infact contains 0.61% R306) amounting to 1.1 U.sub.mnt/mg; i.e. 1/0.91=1.098901.
Example 50: Preparation of DCM, 01DG2 and D-Acl Extracts
[0708] 500 g of dried and powdered roots of Neobeguea mahafalensis were extracted with 1 L of dichloromethane during 1 h by shaking at room temperature. The dichloromethane solution was filtered off and the remainder of the roots were once again extracted with 1 L dichloromethane in the same way, and the two filtrates were pooled and evaporated to dryness under reduced pressure, yielding 7.8 g of crude dichloromethane extract, herein termed “DCM”. The remainder of the “dichloromethane exhausted” roots were extracted twice with 1 L acetone using the same procedure; after evaporation of the acetone 5.2 g of crude acetone extract (herein termed “01DG2”) had been obtained.
[0709] Minor samples of the DCM and 01DG2 extracts were taken away for testing of their sexual enhancing effects (see further below) while the major parts of these extracts were combined (i.e. yielding in total 13 g) and 8 g of this combined extract was dissolved in 5 mL dichloromethane and applied to a silica gel column (length 42 cm, internal diameter 2.5 cm; 97 g of silica 60 from Merck, Germany).
[0710] Elutions then proceeded in steps applying 300 mL of solvent or solvent mixture with increasing polarity (i.e. dichloromethane and methanol/dichloromethane mixtures) in each step, collecting 10 mL fractions and subjecting these fractions to silica gel thin layer chromatography (TLC; using Merck TLC aluminium sheets Silica gel 60 F.sub.254 no. 1.05554.0001) using 1% methanol in dichloromethane as mobile phase and searching after a TLC profile matching that of the R2C extract prepared according to Example 9.
[0711] Specifically three major UV absorbing spots with R.sub.f-values 0.14, 0.32 and 0.40 which are clearly detectable in R2C samples by illumination with a UV lamp at 254 nm were looked for (the spots with R.sub.f0.14 and 0.40 being quite narrow while that of 0.32 being quite broad). After each step the column was washed with additionally 100 mL of the same solvent or solvent mixture and then elutions proceeded with the next step.
[0712] Step 1 comprised elutions with 300 mL of dichloromethane only and none of the fractions collected yield the desired TLC profile; all spots showing up having too high Rf values as being detectable on the TLC, and these eluents were therefore discarded; the column was then washed with additionally 100 mL of dichloromethane and the resulting eluent was also discarded.
[0713] Step 2 comprised elutions with 300 mL of 1% methanol in dichloromethane and did also not yield the desired TLC profile, with spots of too high Rf values being detectable on the TLC, and these eluents were accordingly discarded; the column was then washed with additionally 100 mL of 1% methanol in dichloromethane and the eluent discarded.
[0714] Step 3 comprised elutions with 300 mL of 2% methanol in dichloromethane and did also not yield the desired TLC profile with spots of too high Rf values being detectable on the TLC and these eluents were also discarded; the column was then washed with additionally 100 mL of 2% methanol in dichloromethane and the eluent discarded.
[0715] Step 4 comprised elutions with 300 mL of 3% methanol in dichloromethane and collecting 10 mL fractions. Starting from fraction 7 and onward to fraction 18 the UV absorbing TLC spots with the desired Rf values 0.14, 0.32 and 0.40, with a pattern closely resembling that of R2C, in parallel TLC runs, became detectable and these fractions were then combined and the solvent evaporated under reduced pressure yielding the desired D-Acl extract at a final yield of 4.6029 g.
Measurement of Contents of R306 in DCM. 01DG2 and D-Acl Extracts
[0716] The contents of R306 were measured using the method described in Example 48 and were found to be as follows: DCM 0.37%, 01DG2 0.57% and D-Acl 0.55%. By calculation the average content of R306 in the combined DCM+01DG2 extract was 0.45%.
Assay of Sexual Enhancing Effect of DCM. 01DG2 and D-Acl Extracts
[0717] The sexual enhancing effects of DCM, 01DG2 and D-Acl were assessed essentially using the method describe in Example 30. Moreover, for comparison a dichloromethane extract of the stem bark of Neobeguea mahafalensis had been prepared in exactly the same way as the dichloromethane extract of the root, DCM, prepared above.
[0718] The extracts were dissolved in olive oil and administered subcutaneously, D-Acl at 0.4 mg/mg; all the others at 4 mg/mg. Mountings were recorded during 1 h following introduction of the male mice the female mice. Each group comprised 3-6 male mice. Results were as shown in
Example 51: Solubility of R2C Extract
[0719] The solubility of R2C extract prepared according to Example 9 was tested for its solubility in sun flower oil, octan-1-ol and hexane as follows.
Sun Flower Oil:
[0720] R2C, 1 mg, was added to 0.1 ml sun-flower oil at 20° C. A clear solution was obtained. Additionally 1 mg R2C was added. A clear solution was formed. Additionally 0.1 mg of R2C was added and a clear solution was formed. When adding additionally 0.1 mg of R2C some insolubilized material remained.
[0721] Conclusion: R2C is soluble up to a concentration of 21 mg/ml in sun flower oil at 20° C.
Octan-1-ol:
[0722] R2C, 0.75 mg, was added to 0.05 mL octan-1-ol at 20° C. A clear solution was obtained. Additionally 0.75 mg of R2C was added and a clear solution was again obtained.
[0723] Conclusion: R2C is soluble in octan-1-ol at 20° C. up to a concentration of at least 30 mg/ml.
Hexane:
[0724] R2C, 0.5 mg, was added to 0.5 mL n-hexane at 20° C. A turbulent solution was obtained. Addition of additional 0.5 mL n-hexane did not still give a clear solution. T
[0725] Conclusion: R2C is only partially soluble in »-hexane.
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