HK1026373B

HK1026373B - Pharmaceutical compositions having appaetite suppressant activity

Info

Publication number: HK1026373B
Application number: HK00105778.7A
Authority: HK
Inventors: 希尔丹 F‧R‧范; R‧乌莱加尔; R‧M‧霍拉克; R‧A‧里尔蒙斯; V‧玛哈耶; R‧D‧维塔尔
Original assignee: Csir公司
Priority date: 1997-04-15
Filing date: 1998-04-15
Publication date: 2009-01-30

Description

Pharmaceutical composition with appetite suppressant activity

The present invention relates to steroid glycosides, to compositions comprising such steroid glycosides, to novel uses of these steroid glycosides and compositions comprising them. Furthermore, the present invention relates to a process for the extraction and isolation of these steroid glycosides from plant material, to a process for the synthesis of these steroid glycosides, and to the products of said extraction process and synthesis process.

In particular, the present invention relates to an appetite suppressant, to a process for synthesizing said appetite suppressant, to a process for extracting the appetite suppressant from a plant material, to an appetite suppressant composition comprising the appetite suppressant, and to a method of suppressing appetite.

The present invention provides a process for the preparation of an extract of a plant of the genus Trichocaulon or Hoodia, which extract comprises an appetite suppressant, which process comprises the steps of: treating the collected plant material with a solvent to extract a component having appetite suppressant activity, separating the extract from the remainder of the material, removing the solvent from the extract, and recovering the extract. The recovered extract may be further purified, such as by a suitable solvent extraction step.

The present invention also provides a plant extract prepared from a plant comprising a plant of the genus Trichocaulon or Hoodia and having appetite suppressant activity.

The extract can be prepared from plant material such as stem and root of the plant of Trichocaulon or Hoodia. Plants of the genus Trichocaulon or leoideus include succulent plants grown in arid regions, such as those found in south africa. In the present invention, the active appetite suppressant extract may be obtained from the species Trichocaulon piliferum. Trichocaulon officinalis species can also be used to provide active appetite suppressant extracts. In the present invention, the active appetite suppressant extract may also be obtained from the species Pyroluarii, Pyroluaria gordonii and Pyrolugardii. Biological tests carried out by the applicant on rats have shown that some extracts have appetite suppressant activity.

The plant material may be homogenised in the presence of a suitable solvent, such as methanol/dichloromethane, using a device such as a Waring blender. The extract may then be separated from the residual plant material by a suitable separation step, such as filtration or centrifugation. The solvent is then removed by means of a rotary evaporator, preferably in a water bath at 60 ℃. The separated crude extract can be further extracted with dichloromethane and water to separate into dichloromethane extract and water extract. The solvent can be removed from the dichloromethane extract, preferably by means of a rotary evaporator, and the resulting extract can be purified again by means of methanol/hexane extraction. The methanol/hexane extract product can be further separated into methanol extract and hexane extract. The methanol extract is evaporated to remove the solvent to obtain a partially purified active extract.

The partially purified active extract was dissolved in methanol and chromatographed by column chromatography using silica gel as the adsorption medium and chloroform/30% methanol mixture as the eluent. A plurality of different fractions are available, each of which can be evaluated using appropriate biological testing procedures to determine appetite suppressant activity.

The fraction having appetite suppressant activity is preferably further subjected to column chromatography using silica gel as the adsorbent medium and a 9: 1 chloroform: methanol solvent as the eluent, and the resulting sub-fractions are subjected to biological testing to determine appetite suppressant activity. If desired, the subfractions exhibiting appetite suppressant activity may be further subjected to chromatographic separation and purification, conventionally using a column chromatography procedure, silica gel as the adsorbent medium and a 9: 1 chloroform: methanol solvent as the solvent. The resulting purified fraction can then be evaluated for appetite suppressant activity by a suitable biological testing procedure.

The applicant has also found that at least one of said purified fractions has excellent appetite suppressant activity and that the active principle in the fraction can be identified by conventional chemical techniques including nuclear magnetic resonance and found to be a compound having the following formula:

according to S.I. nomenclature, the active ingredient (1) is the compound 3-O- [ -beta-D-pyrane oleander glycosyl- (1 → 4) -beta-D-pyrane-magnecosyl]-12 β -O-methylcrotonyloxy-14-hydroxy-14 β -pregn-50-en-20-one (C)₄₇H₇₄O₁₅M⁺878)。

According to another aspect of the present invention there is provided a process for the preparation of an extract from a plant of the genus Trichocaulon or Hoodia, said extract comprising an appetite suppressant, the process comprising the steps of: squeezing the collected plant material to separate a juice from the solid plant material, and recovering the juice free of the solid plant material to form an extract.

The extract can be dried to remove water to form a free-flowing powder, for example, by spray drying, freeze drying or vacuum drying.

The present invention also provides a composition having appetite suppressant activity comprising an extract as described above.

The compositions may contain a pharmaceutically acceptable excipient, diluent or carrier, and may optionally be prepared in unit dosage form.

The invention also provides the use of the above extract in the manufacture of a medicament having appetite suppressant activity, provides an extract for use as a medicament having appetite suppressant activity, and provides a method of suppressing appetite by administering to a human or animal an effective amount of the above composition.

The compound (1) is a novel compound, and the invention provides the compound (1) with appetite suppressant property and some analogues or derivatives of steroid trisaccharide. The molecule chosen as analogue or derivative should influence the properties of the steroidal trisaccharide by increasing the activity of the active ingredient. When selecting the analogs, the following effects should be considered:

(i) hydrophobic interactions and oleophilicity

Functional group modifications of reactive molecules will alter the hydrophobicity and lipophilicity of the molecules. Increased lipophilicity results in increased biological activity, decreased water solubility, enhanced deoximation/cell solubility, enhanced storage in tissues, more rapid metabolism and elimination, enhanced plasma protein binding, and increased rate of onset of activity.

(ii) Electrical properties and ionization constants

Modification of the functional groups of the molecule will also alter the acidity and basicity, which plays a major role in controlling the transfer of the compound to its site of action and binding at that target site.

(iii) Hydrogen bonds

Modification of carboxyl and carbonyl functional groups in reactive molecules alters the interaction of proteins with chemically modified functional groups in biological systems.

(iv) Spatial parameters

The purpose of altering the spatial characteristics of the molecule is to increase binding to the receptor and thus increase its biological activity.

The following chemical modifications to the molecule will have an effect on the hydrophobicity and oleophilicity of the molecule, the electronic properties, hydrogen bonding and steric parameters:

(a) chemical modification and ester functionality of the C-12 group;

(b) chemical modifications of the 5, 6-double bond, such as hydrogenation and migration;

(c) chemical modification of C-20 carbonyl and C-17 acetyl;

(d) chemical modification of the "D" ring or the glycosidic ring of the steroid;

(e) chemical modification of the sugar of the trisaccharide.

Thus, the present invention provides a compound having the formula

Wherein R is alkyl;

R₁is hydrogen, alkyl, methylcrotonyl, benzoyl or any other organic ester group;

R₂is hydrogen, or one or more 6-deoxy sugars, or one or more 2, 6-dideoxy sugars, or a glucose molecule, or a combination thereof;

wherein the dotted line represents the optional presence of another bond between C4-C5 or C5-C6.

The present invention also provides a compound as described above wherein there is another bond between C5 and C6, R is methyl, R is₁Is methylcrotonyl, R₂Is 3-O- [ -beta-D-pyrane oleander glycosyl- (1 → 4) -beta-D-pyrane-glyco-l]The compound has the following structural formula

The active analogue or derivative of the appetite suppressant compound (1) is a compound with the following structural formula

Wherein R is alkyl;

R₁is hydrogen, benzoyl, methylcrotonyl, or any other organic ester group;

wherein R is alkyl;

R₁is hydrogen, methylcrotonyl, benzoyl or any other organic ester group;

wherein R is alkyl;

R₁is hydrogen, methylcrotonyl, benzoyl or any other organic ester group;

wherein R is alkyl;

R₁is hydrogen, methylcrotonyl, benzoyl or any other organic ester group;

wherein R is alkyl;

R₁is hydrogen, methylcrotonyl, benzoyl or any other organic ester group;

wherein R is alkyl;

wherein the dotted line represents the optional presence of another bond between C4-C5 or C5-C6;

wherein R is alkyl;

wherein the dotted line represents the presence of another bond between C4-C5 or C5-C6;

wherein R is alkyl;

wherein the dotted line represents the optional presence of another bond between C4-C5, C5-C6 or C14-C15;

wherein R is alkyl;

wherein the dotted line represents the optional presence between C4-C5, C5-C6 or C14-C15

Another bond; r₃Is hydrogen, alkyl, aryl, acyl or glucoxy (glucoxy).

Wherein R is hydrogen, alkyl, aryl, or a steroid radical having a beta hydroxy group at the C14 position, or a beta hydroxy functional group at the C12 position, or an acyl group at the C17 position, or an olefinic bond at the C5-C6 positions, or combinations thereof.

The invention also provides methods for synthesizing compounds having appetite suppressant activity.

The process employs as a starting material (or as an intermediate or precursor) a steroid having the formula

Steroid (15) can be prepared from a compound of formula (22) by a process comprising the steps of:

(i) treating a progesterone of the formula

To produce the compound 12 beta, 15 alpha-dihydroxyprogesterone of the formula

(ii) Treatment of compound (17) with tosyl chloride and pyridine yields compound 12 β -hydroxy-15 α - (p-toluenesulfonyl) -progesterone of the formula

(iii) Treating compound (18) with collidine at 150 ℃ to give compound 12 β -hydroxy- Δ of the formula¹⁴-progesterone

(iv) Treating compound (19) with acetyl chloride and acetic anhydride at 120 ℃ to give the compound 3, 12 β -diacetoxypregna-3, 5, 14-trien-20-one of the formula

(v) Treating compound (20) with ethylene glycol and a catalytic amount of p-toluenesulfonic acid to yield the compound 3, 12 β -diacetoxy-20, 20-ethylenedioxypregna-3, 5, 14-triene of the formula

(vi) With NaBH₄Treating compound (21) to produce the compound 3 beta, 12 beta-dihydroxy-20, 20-ethylenedioxypregna-5, 14-diene-12-acetate of the formula

In a first alternative process, the process of the invention for the preparation of steroid (15) comprises the following steps:

(a) treating compound (22) with a reducing agent such as lithium aluminum hydride to produce the compound 3 beta, 12 beta-dihydroxy-20, 20-ethylenedioxypregna-5, 14-diene of the formula

(b) Treatment of compound (23) with N-bromoacetamide (NBA) and a base such as pyridine yields the compound 3 β,12 β -dihydroxy-14, 15-epoxy-20, 20-ethylenedioxypregn-5-ene of the formula

(c) Treating compound (24) with a reducing agent such as lithium aluminum hydride at reflux to produce the compound 3 β,12 β, 14 β -trihydroxy-20, 20-ethylenedioxypregn-5-ene of the formula

(d) Treatment of compound (25) with an acid such as acetic acid and water produces the steroid intermediate 3 β,12 β, 14 β -trihydroxy-pregn-5-ene (15).

Scheme A illustrates the "first alternative procedure" according to the present invention, a procedure for the preparation of steroidal intermediate (15) from compound (22), which for illustrative purposes, includes the preparation of compound (22) from compound (16).

Reaction scheme A

In a second alternative process, the process of the invention for the preparation of steroid (15) comprises the following steps:

(a) treatment of compound (22) (3 β,12 β -dihydroxy-20, 20-ethylenedioxypregna-5, 14-diene-12-acetate) with p-toluenesulfonyl chloride and a base such as pyridine yields the compound 3 β,12 β -dihydroxy-20, 20-ethylenedioxypregna-5, 14-diene-3-tosyl-12-acetate of the formula

(b) Treatment of compound (26) with potassium acetate in a solvent such as acetone yields the compound 6 β,12 β -dihydroxy-20, 20-ethylenedioxy-3, 5 α -cyclopent-14-ene-12-acetate of the formula

(c) Treatment of compound (27) with a reducing agent such as lithium aluminium hydride and, for example, tetrahydrofuran, produces the compound 6 beta, 12 beta-dihydroxy-20, 20-ethylenedioxy-3, 5 alpha-cyclopent-14-ene of the formula

(d) Treatment of compound (28) with N-bromoacetamide, optionally acetic acid and a base such as pyridine, yields the compound 6 beta, 12 beta-dihydroxy-14, 15-epoxy-3, 5 alpha-cyclopregna

(e) Treatment of compound (29) with a reducing agent such as lithium aluminum hydride and, for example, tetrahydrofuran, produces the compound 6 β,12 β, 14 β -trihydroxy-20, 20-ethylenedioxy-3, 5 α -cyclopregna

(f) Compound (30) is treated with an acid such as hydrochloric acid and a solvent such as acetone to give compound (15).

Scheme B shows the "second alternative" according to the present invention, a process for preparing steroid intermediate (15) from compound (22).

Reaction scheme B

Compound (1) can be synthesized from the first saccharide intermediate in the form of an activated monosaccharide, which can be prepared from the compound of formula (36). Compound (36) can be prepared by a process comprising the steps of:

(i) treatment of methyl-alpha-D-glucose of the formula with benzaldehyde and zinc chloride

To give the compound methyl-4, 6-O-benzylidene-alpha-D-glucopyranoside of the formula

(ii) Treatment of compound (32) with tosyl chloride and pyridine at 0 deg.C yields the compound methyl-4, 6-O-benzylidene-2-O-tosyl-alpha-D-glucopyranoside of the formula

(iii) Treatment of compound (33) with sodium methoxide at 100 ℃ gives the compound methyl-4, 6-O-benzylidene-3-O-methyl-alpha-D-atropine pyranoside of formula

(iv) Treatment of compound (34) with N-bromosuccinimide (NBS) yields the compound 6-bromo-4-O-benzoyl-3-O-methyl-6-deoxy-alpha-D-atropine pyrane glycoside of the formula

(v) Treatment of compound (35) with sodium borohydride and nickel dichloride yields the compound methyl 4-O-benzoyl-3-O-methyl-6-deoxy-alpha-D-atratoside of the formula

The invention also provides a process for the preparation of a saccharide intermediate in the form of a monosaccharide, maghemin, comprising the steps of:

(i) using PhSSiMe₃、ZnI₂And Bu4⁺I^-Treating compound (36) to produce the compound 4-O-benzoyl-3-O-methyl-6-deoxy-alpha beta-D-thiophenylazepinoside of formula

(ii) Optionally, compound (37) is treated with diethylaminosulfur trifluoride (DAST), e.g. at 0 deg.C, to give the compound 4-O-benzoyl-3-O-methyl-2-phenylthio-2, 6-dideoxy-. alpha. -D-fluoropyranocymoside of the formula

Or

(iii) Optionally, compound (37) is treated with a solvent such as tert-butyldimethylsilyl chloride in pyridine and imidazole to yield the compound 4-O-benzoyl-3-O-methyl-2-O-tert-butyldimethylsilyl-alpha beta-D-phenylthioaaltrose of the formula

Wherein, Z ═ TBDMS ═ tert-butyldimethylsilyl;

(iv) treatment of compound (39) with a base such as sodium methoxide gives the compound 3-O-methyl-2-O-tert-butyldimethylsilyl-alpha beta-D-phenylthioaltoreq glucoside of the formula

Wherein, Z is TBDMS is tert-butyldimethylsilyl.

Scheme C shows the synthesis of the activated monosaccharide, maginose (40), from compound (36) according to the present invention (for illustrative purposes, including the preparation of compound (36) from compound (31)).

Reaction scheme C

Z-tert-butyldimethylsilyl

Alkyl radical

The synthesis of compound (1) can also employ a second sugar intermediate in the form of an activated monosaccharide oleander sugar, which can be prepared from the compound of formula (47). Compound (47) can be prepared by a process comprising the steps of:

(i) treatment of alpha-D-glucose of the formula with acetone and sulfonic acid

To produce compound 1,2 of the formula: 5, 6-di-O-isopropylidene-alpha-D-glucopyranose

(ii) Treating compound (42) with sodium hydride and methyl iodide to yield compound 1,2 of the formula: 5, 6-di-O-isopropylidene-3-O-methyl-alpha-D-glucopyranose

(iii) Treating compound (43) with acetic acid to produce the compound 3-O-methyl-alpha beta-D-glucopyranose of the formula

(iv) Treating compound (44) with methanol and hydrochloric acid to produce the compound methyl 3-O-methyl-alpha beta-D-glucopyranoside of the formula

(v) Treating compound (45) with benzaldehyde and zinc chloride to produce the compound methyl 4, 6-O-benzylidene-3-O-methyl-alpha beta-glucopyranoside of the formula

(vi) Treating compound (46) with N-bromosuccinimide, nickel chloride and sodium borohydride to produce the compound methyl 4-O-benzoyl-3-O-methyl-6-deoxy-alpha beta-glucopyranoside of the formula

The invention provides a preparation method of activated monosaccharide yellow oleander sugar, which comprises the following steps:

(i) treatment of compound (47) with phenylthiotrimethylsilane and trimethylsilyl triflate yielded the compound 4-O-benzoyl-3-O-methyl-1-phenylthio-6-deoxy-alpha beta-glucopyranoside of the formula

(ii) Treating compound (48) with pivaloyl chloride and a solvent such as pyridine to produce the compound 4-O-benzoyl-3-O-methyl-2-O-pivaloyl-1-thiophenyl-6-deoxy-alpha-beta-glucopyranoside of the formula

(iii) Treatment of compound (49) with a brominating agent such as N-bromosuccinimide and diethylaminosulfur trifluoride yields the compound 4-O-benzoyl-3-O-methyl-2-O-pivaloyl-1-fluoro-6-deoxy-beta-glucopyranoside of the formula

Scheme D shows the process of synthesizing the activated monosaccharide, maginose ((50A) and 50(B)) from compound (48) according to the present invention (for illustrative purposes, including the process of preparing compound (47) from compound (41)).

Reaction scheme D

According to another aspect of the present invention there is provided a method of synthesizing a compound of formula (1) and analogues and derivatives thereof, the method comprising the steps of: suitable steroid intermediates or precursors are synthesized and the desired number of monosaccharides are coupled to the steroid intermediate.

The invention also provides a coupling method of monosaccharide-maginose and a steroid intermediate, which comprises the following steps:

(i) reacting the magnetoephedrine of formula (38) with the steroidal intermediate of formula (15) in a solvent such as diethyl ether, for example at-15 ℃ in the presence of tin chloride to give the compound 3-O- [ 4-O-benzoyl-2-phenylthio- β -D-pyraclostrobin-12, 14- β -dihydroxypregn-5-en-20-one of the formula

And (ii) treating compound (51) with methylcrotonyl chloride in pyridine followed by a base such as sodium methoxide to produce the compound 3-O- [ 2-phenylthio- β -D-glucopyranosyl ] -12 β -methylcrotonyl-14 β -hydroxypregn-5-en-20-one of the formula

The invention provides a method comprising the steps of: the monosaccharide, maghemin, is coupled to the monosaccharide, oleander sugar, and the resulting disaccharide is coupled to the bound steroid product (52) to form compound (1).

The method of coupling the monosaccharide, maginose, to the monosaccharide, oleander sugar, and coupling the formed disaccharide to the bound steroid product (52) may comprise the steps of:

(i) coupling of the selectively protected magadiulose (40) with the selectively protected oleander (50A) using tin chloride and silver triflate, e.g., at-15 deg.C, yields a compound of the formula

Wherein, Z ═ TBDMS ═ tert-butyldimethylsilyl;

(ii) treatment of compound (53) with tetrabutylammonium fluoride yields a compound of the formula

(iii) For example, treatment of compound (54) with diethylaminosulfur trifluoride at 0 ℃ gives a compound of the formula

(iv) Reacting compound (55) with compound (52) to produce a compound of the formula

And (v) treating compound (56) in a raney nickel reaction with a base such as sodium methoxide to give compound (1) as described above.

Scheme E shows the procedure used to synthesize intermediates (52) and (55) and couple them to form compound (56).

Reaction scheme E

According to the present invention, there is provided a further process for coupling a maghemin and a oleander saccharide to form a trisaccharide and coupling the trisaccharide to a steroid derivative to form a compound of formula (1).

A method of coupling a maghemite and a oleander to form a trisaccharide, and coupling the trisaccharide to a steroid derivative to form a compound of formula (1) comprising the steps of:

(i) using tin chloride, AgOTf, Cp₂ZrCl₂Coupling the selectively protected magnetoephedrine (40) with a compound (45) to produce a compound of the formula

Wherein, Z ═ TBDMS ═ tert-butyldimethylsilyl;

(ii) treatment of compound (57) with tetrabutylammonium fluoride and diethylaminosulfur trifluoride yields a trisaccharide compound of the formula

(iii) Using tin chloride, AgOTf, Cp₂ZrCl₂Coupling a trisaccharide of formula (58) with a steroidal intermediate of the formula

To produce compound (1).

Reaction scheme F shows a process for synthesizing trisaccharide (58) and a process for synthesizing compound (1) by coupling trisaccharide (58) with steroid intermediate (59).

Reaction scheme F

Intermediates (23), (24), (25), (27), (28), (29), (30), (37), (38), (39), (40), (48), (49), (50), (51), (53), (54), (55), (56), (57) and (58) as described above are novel compounds, and the present invention provides these compounds.

It has now been found that the compound (1)3-O- [ - β -D-pyranosyloleandosyl- (1 → 4) - β -D-pyranosylyl ] -12 β -O-methylcrotonyl-14-hydroxy-14 β -pregn-5-en-20-one and various analogs and derivatives thereof have appetite suppressant activity.

The present invention also provides a composition or a preparation having an appetite suppressant activity, wherein the active ingredient is an extract obtained from a plant of the genus Trichocaulon or Hoodia.

The active ingredient may be a compound of formula (1) or a derivative thereof extracted from a plant of the genus Trichocaulon or Hoodia. The plant may be of the species Trichocaulon piliferum and Trichocaulon officinalis, or of the species Currorii, Gordonii and Lugardii.

The present invention also provides compositions or formulations having appetite suppressant activity, wherein the active ingredient is a synthetic compound of formula (1) or a derivative or analogue thereof, such as the aforementioned compounds (2) - (14).

According to another aspect of the present invention there is provided a method of suppressing appetite comprising administering to a human or animal an appetite suppressant comprising an extract of a plant of the genus Trichocaulon or Hoodia in a suitable dosage. The extract may be incorporated into a composition or formulation, which may also contain other pharmaceutically acceptable ingredients.

The appetite suppressant is an isolated natural or synthetic compound having the formula or a derivative or analogue thereof as described above

An appetite suppressant composition or formulation may consist of the appetite suppressant together with a pharmaceutically acceptable excipient, diluent or carrier. Other suitable additives including stabilizers may also be added as desired.

The invention also provides the use of compound (1) or a derivative or analogue thereof in the manufacture of a medicament having appetite suppressant activity.

The present invention also provides the compound (1) or a derivative or analog thereof as described above for use as a medicament having appetite suppressant activity.

The invention also provides a method of suppressing appetite comprising administering to a human or animal an effective amount of a composition as described above.

The present invention describes a process for the extraction of steroidal glycosides having appetite suppressant activity from plant material obtained from plants of the genus Trichocaulon or Hoodia. Accordingly, the present invention provides an extract obtained from plant material of the genus Trichocaulon or Hoodia, comprising a substantial amount of pure steroidal glycosides of formula (1).

The present invention also provides a food or drink comprising an appetite suppressant-effective amount of a steroidal glycoside of formula (1) as defined above or a derivative or analogue thereof.

Molecular genetic studies have led to a significant increase in awareness of appetite, safety, and regulation of body weight. These studies have revealed a wide variety of neuropeptide-mediated central regulatory pathways. Maintaining a normal volume can be achieved by a complex balance between energy intake, food consumption and energy expenditure. The homeostasis of energy is affected by various factors and is ultimately controlled by the brain. Different signals include, for example, odor and taste, and gastrointestinal signals such as gastrointestinal distension, chemical signals to the gastric mucosa and to blood-forming metabolites such as fatty acids and glucose.

In general, neuropeptide "Y" (NPY), negatively regulated by leptin, has been considered as a positive regulator of eating behavior. The expression of endogenous antagonists of melanocortin (melanocortin) receptors has also been shown to underlie obesity in a particular model (ob/ob mice). The apparent deficiency in the melanocortin receptor of MC4 is completely consistent with obesity. Other mediator mediators that have been shown to have a role in energy balance include bombesin, galonin, glucagon-like peptide-1.

While not being bound by theory, applicants believe that the compounds as described above, and analogs thereof, may act as agonists at the melanocortin 4 receptor. This effect can modulate NPY but also increases cholecystokinin. Among them, cholecystokinin acts to inhibit gastric emptying.

Thus, the present invention provides a composition having appetite suppressant activity comprising a melanocortin 4 receptor agonist.

The agonist may be an extract or a compound as described previously, in particular a compound of formula (1). The compositions may contain a pharmaceutically acceptable excipient, diluent or carrier, and are optionally prepared in unit dosage form.

The invention also provides the use of a melanocortin 4 receptor agonist in the manufacture of a medicament having appetite suppressant activity, provides a melanocortin 4 receptor agonist for use as a medicament having appetite suppressant activity, and provides a method of suppressing appetite comprising administering to a human or animal an effective amount of a composition as hereinbefore described comprising a melanocortin 4 agonist, and provides the use of a melanocortin 4 receptor agonist in suppressing appetite and/or reducing weight in a human or animal.

The invention and its effectiveness will be further described below with reference to the following examples and the accompanying drawings, which are not to be construed as limiting the invention.

In the accompanying drawings, FIG. 1 shows a flow chart of a general process for extracting a first crude appetite suppressant extract and a purified appetite suppressant extract from a plant material of the genus Trichocaulon or Hoodia;

FIG. 2 shows a graphical illustration of a bioassay using a partially purified methanol extract of Trichocaulon piliferum in rats;

FIGS. 3 and 4 each show a schematic illustration of a method for producing an extract from a plant material of the genus Trichocaulon or Hydridanum according to a preferred embodiment of the present invention;

figures 5 and6 show graphical illustrations of the percent weight change in repeat dose studies of rats in different groups on days-7 to 7and 0-7, respectively, using sap extract and spray dried sap extract from fire land sub-gordonii plant material.

Example 1

The general process for extracting a first crude appetite suppressant extract and a purified appetite suppressant extract from a plant material of the genus Trichocaulon or Hoodia is illustrated by the flow chart of FIG. 1.

Example 2

Biological tests on rats using the partially purified methanol extract obtained in the process described in example 1 showed that the extract indeed showed appetite suppressant activity. The appetite suppressant activity of the active extract can be illustrated by means of figure 2 by a typical example of the effect of a methanol extract of trichocaulon on rats.

From fig. 2, it was confirmed that the rats of the test group administered with the extract showed a substantial decrease in food intake at day 5 compared to 2 days later, while the control group did not show a comparable decrease in food intake. From day 8 onwards, the food intake of the test groups was normal and in fact increased.

Example 3

A preferred embodiment of the method of the invention for producing a peptide having appetite suppressant activity is illustrated by figures 3 and 4, which together illustrate a complex process. However, various other processes may be employed, as is well known to those skilled in the art.

Referring to fig. 3, a plant material of the genus Trichocaulon or geotrichum is fed through a feed pipe 1 to a mixer 3 such as a Waring mixer, and a solvent in the form of a dichloromethane/methanol solution is fed through a feed pipe 2. The homogenized product is fed via line 4 to a separation device 5, such as a filter or centrifuge, from which the remainder of the plant material is removed via line 27.

The solvent/extract mixture is fed via line 6 to an evaporation unit 7 where the solvent is removed, for example using a rotary evaporator. The dried crude extract is fed via line 8 to a further extraction unit 9 and further extracted via line 29 with dichloromethane/water solution and then via line 11 to a separation unit 13, wherein the aqueous fraction is removed via line 31. The dissolved extract fraction is fed via line 15 to a drying unit 17 where the solvent is evaporated, for example by means of a rotary evaporator.

Referring to fig. 4, the dried extract is fed via line 10 to an extraction apparatus 12. The dried extract is further purified by adding a methanol/hexane solution to extraction apparatus 12 via line 14. The extract/methanol/hexane mixture is fed via line 16 to separation unit 18, the hexane fraction is removed via line 20, and the methanol/extract mixture is then fed via line 22 to drying unit 24. In the drying device 24, the solvent is removed by an evaporation process such as a rotary evaporator.

The dried and partially purified active extract is fed via line 26 to the dissolution unit 30 along with the remainder of the methanol via line 28 and the dissolved fraction is fed via line 36 to a chromatographic column 38.

In the chromatographic column 38, the methanol-soluble fraction was further subjected to chromatography using silica gel and chloroform/30% methanol solvent, and separated into different fractions as schematically illustrated by fractions I to V. According to the actual chromatographic separation carried out by the applicant, the chromatographic separation yields the following weight fractions: i (3.9 g); II (2.6 g); III (2.1 g); IV (1.1g) and V (2.0 g). These fractions are individually evaluated by a suitable biological testing procedure (in a step not shown) and these fractions are identified as fractions I and II, which exhibit outstanding appetite suppressant activity, and fractions I and II are fed through feed lines 40 and 42, respectively, to columns 44 and 46 where they are further chromatographed and purified by column chromatography, again using silica gel and a 9: 1 chloroform: methanol system.

The subfractions ii (a) - (C) obtained from column 44 show significant appetite suppressant activity when measured and can be recycled for chromatographic processing.

While subfractions i (a) - (L) obtained from column 46 were evaluated (by steps not shown), subfractions i (c) showed outstanding appetite suppressant activity.

Subfraction I (C) is chromatographed and purified via line 48 on column 50 using silica gel and 9: 1 ethyl acetate hexane as eluent. Of the resulting purified fractions, fraction I (C), (ii) was found to have outstanding appetite suppressant activity after testing.

The purified product was identified by nmr spectroscopy (see tables 1 and 2 below) to be compound (1).

Table 1: CDCl of Compound (1)₃Data:¹H(300.13MHz)n.m.r.

hydrogen atom	Compound (1) J (HH)/Hz	δ/p.p.m.
hydrogen atom	Compound (1) J (HH)/Hz	δ/p.p.m.	Aglycone-361217181921345Cym-1′2′2′3′4′5′6′3′-OMe1″2″2″3″4″5″6″3″-OMeThev-1234563*-OMe	--11.5，4.19.3，9.3---7.1，1.57.1，1.21.6，1.29.4，2.113.8，3.7，2.113.8，9.4，2.63.7，2.9，2.69.4，2.96.3，9.46.3-9.4，2.113.8，3.7，2.113.8，9.4，2.63.7，2.9，2.69.4，2.96.3，9.46.3-7.77.7，8.08.0，2.99.3，2.96.3，9.36.3-	3.522m5.381m4.607dd3.157dd1.029s0.951s2.164s6.888qq1.806dq1.853dq4.816dd2.055ddd1.552ddd3.776ddd3.179dd3.821dd1.279d3.408s4.730dd2.108ddd1.601ddd3.755ddd3.239dd3.898dd1.243d3.392s4.273d3.469dd3.099dd3.179dd3.351dd1.183d’3.622s

In each column a, b, c are interchangeable, in each column d, e are interchangeable

Atom of a methyl crotonic acid ester group

Table 2: in CDCl₃Relative to Compound (1) in¹³C (75.25MHz) n.m.r. data

Aglycone moieties		Sugar moieties
Aglycone moieties		Sugar moieties		Carbon (C)	δ/p.p.m.	Carbon (C)	δ/p.p.m.
12345678910111213141516171819202112345	37.04T29.44T77.24D38.62T138.95S131.90D27.30T35.30D43.04D37.22S26.04T75.88D53.71S85.69S34.36T24.31T57.18D9.85Q19.27Q216.85S33.01Q167.60S128.69D137.66D14.41Q12.08Q	cym -1′2′3′4′5′6′3′-OMe1″2″3″4″5″6″3″-OMeThev-1234563*-OMe	95.84D35.57T77.05D82.57D68.48D18.14Q57.93Q99.54D35.17T76.99D82.52D68.30D18.36Q57.09Q104.28D74.62D85.30D74.62D71.62D17.75Q60.60Q	Carbon (C)	δ/p.p.m.	Carbon (C)	δ/p.p.m.

Atom of a methyl crotonic acid ester group

Compound (1)

IR data: 3440cm^-1(OH)，2910cm^-1(CH)，1700cm^-1(C＝0)

[α_D]²⁰ ₅₈₉＝12,67°(C＝3，CHCl₃)

m.p.147℃-152℃

Examples 4-13 illustrate synthetic procedures whereby the intermediate compound and steroid (15) may be prepared according to the "first alternative procedure".

Example 4

12 beta, 15 alpha-dihydroxyprogesterone (17)

Comprises sucrose (900g), dipotassium hydrogen carbonate (30g),The culture medium of Chua's culture medium concentrated solution (300mL), corn soaking solution (300mL) and distilled water (30L) is inoculated to prepareRed shell of Decolor(ATCC 14767) (150X 500mL flask). After shaking for 5 days at 26 ℃, a suspension of progesterone (16) (150g) in tween 80 (0.1% solution, 1.5L) was added to the flask. The culture was further cultured for 5 days, and then centrifuged, decanted, extracted with chloroform, and evaporated to give dihydroxyprogesterone (17) (75g, 45%).

¹H NMR(CDCl₃)：5,71(1H，s，H-4)；4,12-4,22(1H，m，H-15)

4,43(1H，br，s，OH)；3,46-3,53(1H，dd，J＝4,6Hz，H-12)；2,16Hz(3H，s，H-21)；1,18(3H，s，H-19)；0,74(3H，s，H-18)

Example 5

12 beta-hydroxy-15 alpha- (p-toluenesulfonyl) -progesterone (18)

Dydroxyprogesterone (17) (75g, 0.22mo1) was dissolved in anhydrous pyridine (300mL) and cooled to 0 ℃. P-toluenesulfonyl chloride (46g, 0.24mol) in anhydrous pyridine (200mL) was added dropwise to the reaction mixture at 0 ℃. The reaction mixture was stirred at 0 ℃ overnight and water (500mL) was added to stop the reaction. The aqueous layer was extracted with ethyl acetate (1L), and the organic layer was washed with hydrochloric acid (6M, 3X 1L), a saturated aqueous solution of sodium hydrogencarbonate (500mL), a saturated aqueous solution of sodium chloride (500mL) and water (500 mL). The organic layer was dried over magnesium sulfate, filtered and evaporated to give p-tosylated progesterone (18) (98g, 92%) as a viscous dark yellow oil.

¹H NMR(CDCl₃)：7,7(2H，d，J＝14Hz，H-2,6)；7,34(2H，d，J＝8,4Hz，H-3,5)；5,67(1H，s，H-4)；4,86-4,93(1H，m，H-15)；3,45-3,50(1H，dd，J＝4,6Hz，H-12)；2,44(3H，s，H-4Me)；2,15(3H，s，H-21)1,13(3H，s，H-19)；0,74(3H，s，H-18).

Example 6

12 beta-hydroxy-delta¹⁴-progesterone (19)

A solution of tosylated progesterone (18) (98g, 0.19mol) in 2,4, 6-trimethylpyridine (500mL) was refluxed at 150 ℃ for 3 hours. The reaction mixture was cooled and poured into water (500 mL). The aqueous layer was extracted with ethyl acetate (1L), and thereafter, the organic layer was washed with hydrochloric acid (6M, 3X 1L), a saturated aqueous solution of sodium hydrogencarbonate (500mL), a saturated aqueous solution of sodium chloride (500mL) and water (500 mL). After drying over magnesium sulfate and filtration, ethyl acetate is evaporated off, the crude mixture is chromatographed on silica gel, eluting with acetone/chloroform (1: 10) to give Δ¹⁴Progesterone (19) (50g, 78%) as dark red oil.

¹H NMR(CDCl₃)：5,73(1H，s，H-4)，5,28(1H，dd，J＝2,2Hz，H-15)，4,41(1H，br，s，OH)，3,49-3,52(1H，dd，J＝4,3Hz，H-12)，2,80-2,84(1H，dd，J＝9,2Hz，H-17)，2,14(3H，s，H-21)，1,19(3H，s，H-19)，0.89(3H，s，H-18).

Example 7

3, 12 beta-diacetoxy-pregna-3, 5, 14-trien-20-one (20)

Will be delta¹⁴A solution of progesterone (19) (50g, 0.15mol) in acetyl chloride (1.5L) and acetic anhydride (750mL) was refluxed for 2 hours. The reaction mixture was poured into cold ethyl acetate (1L) and saturated aqueous sodium bicarbonate solution was added with stirring until bubbling ceased. The ethyl acetate layer was separated from the sodium bicarbonate layer and washed with a portion of aqueous sodium bicarbonate (3X 700mL), then saturated aqueous sodium chloride (700mL), and finally water (700 mL). The organic layer was dried over magnesium sulfate, filtered and evaporated to give 3, 12 β -diacetoxy-pregn-3, 5, 14-trien-20-one (20) (60g, 93%) as an orange oil.

¹H NMR(CDCl₃)：5,68(1H，s，H-4)，5,44(1H，m，H-6)，5,31(1H，dd，J＝2,2Hz，H-15)，4,82-4,86(1H，dd，J＝4,5Hz，H-12)，3,10-3,18(1H，t，J ＝9,5Hz，H-17)，2,18(3H，s，3-Ac)，2,11(3H，s，12-Ac)，2,08(3H，s，H-21)，1,02(3H，s，H-19)，1,01(3H，s，H-18)

Example 8

3, 12 beta-diacetoxy-20, 20-ethylenedioxypregna-3, 5, 14-triene (21)

The diacetoxy compound (20) (60g, 0.14mol) was dissolved in benzene (1L), and ethylene glycol (60mL) and p-toluenesulfonic acid (1g) were added. (benzene was previously refluxed with a Dean-Stark trap). The mixture was refluxed with stirring and the water was removed by azeotropic boiling for 16 hours. To the cooled solution was added saturated aqueous sodium bicarbonate (500 mL). Then, it was washed with brine (500mL) and water (500mL) and dried over magnesium sulfate. The solvent was distilled off, and the crude mixture was purified by silica gel column chromatography eluting with ethyl acetate: hexane (2: 8) to give ethylenedioxypregn-3, 5, 14-triene (21) (35g, 53%).

¹H NMR(CDCl₃): 5,68(1H, s, H-4), 5,45(1H, m, H-6), 5,31(1H, dd, J ═ 2,2Hz, H-15), 4,73-4,85(1H, dd, J ═ 4,4Hz, H-12), 3,78-3,98(4H, m, ethylenedioxy), 2,16(3H, s, 3-Ac), 2,04(3H, s, 12-Ac), 1,29(3H, s, H-21), 1,12(3H, s, H-19), 1,02(3H, s, H-18).

Example 9

3 beta, 12 beta-dihydroxy-20, 20-ethylenedioxypregna-5, 14-diene-12-acetate (22)

Dienol acetate (21) (35g, 0.077mol) was suspended in ethanol (500mL) and sodium borohydride (2.8g, 0.074mol) was added at 0 ℃. The mixture was warmed to room temperature and stirred overnight. Most of the solvent was removed in vacuo and the mixture was diluted with water (500mL), extracted with ethyl acetate (500mL) and chromatographed on silica gel eluting with acetone/chloroform (1: 10) to give 3 β -alcohol (22) (25g, 80%).

¹H NMR(CDCl₃): 5,41(1H, m, H-6), 5,28(1H, dd, J-2, 2Hz, H-15), 4,72-4,81(1H, dd, J-4, 4Hz, H-12), 3,82-4,02(4H, m, ethylenedioxy), 3,45-3,59(1H, m, H-3), 2,03(3H，s，12-Ac)，1,28(3H，s，H-21)，1,10(3H，s，H-19)，1,01(3H，s，H-18).

example 10

3 beta, 12 beta-dihydroxy-20, 20-ethylenedioxypregna-5, 14-diene (23)

3 β -alcohol (22) (25g, 60.2mmol) in anhydrous tetrahydrofuran (300mL) was added dropwise to a suspension of lithium aluminum hydride (2.7g, 72.2mmol) in anhydrous tetrahydrofuran (500 mL). The reaction mixture was stirred at room temperature for 24 hours, after which water (2.7mL) was added carefully and stirring was continued for 10 minutes. Sodium hydroxide (15% solution, 2.7mL) was added and the suspension was stirred. After 10 min, water (8.1mL) was added and the suspension was stirred for a further 10 min, filtered and the solvent was evaporated off with drying (magnesium sulphate) to give 3 β,12 β -dihydroxy-pregna-diene (23) (20g, 90%).

¹H NMR(CDCl₃): 5,36(1H, m, H-6), 5,23(1H, dd, J ═ 2,2Hz, H-15), 3,94-4,06(4H, m, ethylenedioxy), 3,41-3,52(1H, m, H-3), 3,32-3,36(1H, dd, J ═ 4,3Hz, H-12), 1,31(3H, s, H)1,01(3H, s, H-19), 0,96(3H, s, H-18).

¹³C NMR(CDCl₃): 152,4(C-14), 140,2(C-5), 121,1(C-15)119,7(C-6), 111,1(C-20), 79,8(C-12), 71,6(C-3), 63,7and63,6 (ethylenedioxy), 58,8(C-17), 19,0(C-19), 11,9(C-18).

3 beta, 12 beta-dihydroxy-14, 15-epoxy-20, 20-ethylenedioxypregn-5-ene and 3 beta, 12 beta-dihydroxy-5, 6-epoxy-20, 20-ethylenedioxypregn-14-ene

N-bromoacetimide (211mg, 1.5mmol) was added to a stirring solution of 5, 14-diene (23) (500mg, 1.34mol) in acetone (100mL), acetic acid (2.5mL) and water (5mL) at 0 ℃. After 15 minutes, sodium sulfite (5% solution, 50mL) was added to the reaction mixture. The acetone was distilled off and the aqueous layer was extracted with dichloromethane (3X 50 mL). The organic layer was dried over magnesium sulfate, filtered and evaporated. Pyridine (1mL) was added to the product and stirred for 0.5 h. Then, dichloromethane (100mL) was added to the reaction mixture, and the dichloromethane phase was washed with citric acid (5% solution, 3 × 100mL), saturated sodium bicarbonate (50mL), and water (50 mL). The organic layer was dried over magnesium sulfate, filtered and evaporated to give a mixture of 14, 15-and 5, 6-epoxides (360mg, 69%) as a white foam. Mixtures of epoxides cannot be separated by silica gel column chromatography.

Example 11

3 beta, 12 beta-dihydroxy-14, 15-epoxy-20, 20-ethylenedioxypregn-5-ene (24)

A mixture of 14, 15-and 5, 6-epoxides in dry tetrahydrofuran (200mL) (14.4g, 37.0mmol) was added to a suspension of lithium aluminum hydride (1.69g, 44.4mmol) in dry tetrahydrofuran (300 mL). The reaction mixture was stirred at room temperature for 24 hours, after which it was treated as before by adding water (1.69mL) and sodium hydroxide (15% solution, 1.69 mL). After filtration, the solvent was evaporated off and the crude product was purified by silica gel column chromatography using methanol/chloroform (1: 9) as the solvent to give unreacted 14, 15-epoxy-20, 20-ethylenedioxypregn-5-ene (24) (300mg, 2.1%).

¹H NMR(CDCl₃): 5,31(1H, m, H-6), 3,82-3,98(4H, m, ethylenedioxy), 3,43-3,52(1H, m, H-3), 3,41(1H, s, H-15), 3,31-3,35(1H, dd, J ═ 4,3Hz, H-12), 1,29(3H, s, H-21), 1,17(3H, s, H-19), 1,02(3H, s, H-18).

¹³C NMR(CDCl₃)：139,8(C-5)，120,8(C-6)，112,1(C-20)，77,2(C-12)，75,4(C-14)，61,0(C-15)，22,3(C-21)，19,2(C-19)，9,5(C-18).

Example 12

3 beta, 12 beta, 14 beta-trihydroxy-20, 20-ethylenedioxypregn-5-ene (25)

14, 15-epoxide (300mg, 0.77mmol) in dry tetrahydrofuran (10mL) was added to a suspension of lithium aluminum hydride (300mg, 7.89mmol) in tetrahydrofuran and the reaction mixture was refluxed for 48 hours. After addition of water (0.3mL), sodium hydroxide (15% solution, 0.3mL) and filtration as before, the mixture was purified by silica gel column chromatography using methanol: chloroform (1: 9) as solvent to give trihydroxypregnene (25) (250mg, 83%).

¹H NMR(CDCl₃): 5,38(1H, m, H-6), 3,98(4H, m, ethylenedioxy), 3,43-3,53(1H, m, H-3), 3,25-3,32(1H, dd, J ═ 4,1Hz, H-12), 1,32(3H, s, H-21), 1,01(3H, s, H-19), 0,98(3H, s, H-18)

¹³C NMR CDCl₃)：139,1(C-5)，122,1(C-6)，112,2(C-20)，85,1(C-14)，75,1(C-12)，71,6(C-3)，23,4(C-21)，19,4(C-19)，8,9(C-18)

Example 13

3 beta, 12 beta, 14 beta-trihydroxy-pregn-5-ene (15)

Ethylenedioxypregnene (25) (250mg, 0.64mmol) was dissolved in acetic acid (13.4mL) and water to give, after lyophilization, the trihydroxysteroid (15) (200mg, 89%), m.p.: 228 ℃ and 235 ℃ below (litl 225 ℃ and 235 ℃ below), M⁺348，[α_D]²⁰+35°(lit[α_D]²⁰+39°)。

¹H NMR(CDCl₃)：5,39(1H，m，H-6)，3,56-3,62(1H，t，J＝8,1Hz，H-17)，3,42-3,51(1H，m，H-3)，3,28-3,39(1H，dd，J＝4,3Hz，H-12)，2,23(3H，s，H-21)，1,01(3H，s，H-19)，0,90(3H，s，H-18)

¹³C NMR(CDCl₃)：217,7(C-20)，138,9(C-5)，122,2(C-6)，85,5(C-14)，73,6(C-12)，71,6(C-3)，57,0(C-17)，55,1(C-13)，43,6(C-9)，42,1(C-4)，37,3(C-1)，36,8(C-10)，35,9(C-8)，34,5(C-15)，32,9(C-21)，31,5(C-16)，30,1(C-2)，27,4(C-7)，24,4(C-11)，19,4(C-19)，8,3(C-18).

Examples 14-19 illustrate synthetic procedures whereby the intermediate compound and steroid (15) may be prepared according to the "second alternative procedure".

Example 14

20, 20-ethylenedioxy-3 β -toluene-p-sulfonyloxypregna-5, 14-diene-12 β -ol acetate (26)

A solution of p-toluenesulfonyl chloride (650mg, 3.4mmol) in pyridine (10mL) was added dropwise to a mixture of 20, 20-ethylenedioxypregna-5, 14-diene-3 β,12 β -diol 12-acetate (22) (1.3g, 3.1mmol) in pyridine (15mL) at 0 ℃. The reaction mixture was stirred at room temperature for 24 hours, after which water was added to the reaction mixture. The solution was extracted with ethyl acetate (2X 50mL), and the ethyl acetate layer was washed with citric acid (5X 50mL), saturated aqueous sodium bicarbonate (100mL), saturated aqueous sodium chloride (100mL), and water (100 mL). The ethyl acetate layer was dried over magnesium sulfate, filtered, evaporated and purified by flash column chromatography using hexane-ethyl acetate (8: 2v/v) as eluent to give β -O-tosyl steroid (26) (1.5g, 84%) as a yellow oil (C)₃₂H₄₂O₇M found value of S: 570.271, calculated as: 570.273).

δ_H1.021(3H, s, 19-H), 1.131(3H, s, 18-H), 1.282(3H, s, 21-H), 2.021 (acetate group OCH)₃)，2.431(3H，s，Ar-CH₃)，3.883(4H，m，OCH₂CH₂O)，4.750(1H，dd，³J 10.8Hz，5.2Hz，12-H)，4.890(1H，m，30H)，5.281(1H，dd，³J4.2Hz，2.1Hz，15-H)，5.388(1H，m，6-H)，7.341(2H，d，³J 8.2Hz，ArH)，7.746(2H，d，³J 8.2Hz，ArH).

δ_C13.493Q (C-18), 19.002Q (C-19), 21.612Q (Ar-methyl)^＊，21.671Q(C-21)^＊24.175Q (acetate methyl), 63.401T (ethylenedioxy), 63.498T (ethylenedioxy), 71.531S (C-13), 80.912D (C-12), 82.531D (C-3), 111.363S (C-20), 120.881D (C-15), 121.461D (C-6), 123.715-133.917 (aryl), 139,903S (C-14), 151,722S (C-5), 170.819S (ester carbonyl).

^＊Interchangeable

Example 15

20, 20-ethylenedioxy-3 α, 5-cyclo-5 α -pregn-14-ene-6 β,12 β -diol-12-acetate (27)

A solution of 3 β -toluene-p-sulfonyloxypregna-5, 14-diene (26) (1.2g, 2.1mmol) and potassium acetate (2.2g, 22.4mmol) in water (250mL) and acetone (500mL) was refluxed at 60 ℃ for 16 h. The acetone was distilled off, and the aqueous layer was extracted with ethyl acetate (200 mL). The ethyl acetate layer was dried over magnesium sulfate, filtered and evaporated. The mixture was subjected to flash chromatography using chloroform-acetone (9: 1v/v) as an eluent to give 3 α, 5-cyclic derivative (27) (530mg, 61%) as a yellow oil (C)₂₅H₃₆O₅M found value of (a): 416.262, calculated as: 416.263).

δ_H0.288(1H，dd，³J 8.1Hz，4.9Hz，4-H_a)，0.477(1H，dd，³J 4.4Hz，4.4Hz，4-H_b) 1.025(3H, s, 19-H), 1.121(3H, s, 18-H), 1.256(3H, s, 21-H), 1.989(3H, s, acetate-CH)₃)，3.302(1H，dd，³J 2.8Hz 2.8Hz，6-H)，3.784-3.947(4H，m，OCH₂CH₂O)，4.721(1H，dd，³J8.5Hz，5.6Hz，12-H)，5.232(1H，dd，³J 3.9Hz，1.9Hz，15-H).

δ_C11.678T (C-4), 12.298Q (C-18), 19.971Q (C-19), 23.623Q (C-21), 24.153Q (acetate methyl), 63.700T (ethylenedioxy), 63.788T (ethylenedioxy), 73.591D. (C-6), 80.551D (C-12), 111.126S (C-20), 118.778D (C-15), 152.959S (C-14), 170.991S (ester carbonyl).

Example 16

20, 20-ethylenedioxy-3 α, 5-cyclo-5 α -pregn-14-ene-6 β,12 β -diol (28)

A solution of the 3 α, 5-ring derivative (27) (500mg, 1.2mmol) in tetrahydrofuran (20mL) was added dropwise to lithium aluminum hydride (50mg, 1).3mmol) of tetrahydrofuran (10 mL). The reaction mixture was stirred for 4 hours and water (50. mu.L) was added to stop the reaction. After 30 minutes, sodium hydroxide (15% solution, 50 μ L) was added and stirring was continued for 30 minutes. Water (150. mu.L) was added and the reaction mixture was filtered. Tetrahydrofuran was dried over magnesium sulfate, filtered, evaporated and purified by flash chromatography using chloroform-acetone (8: 2v/v) as eluent to give diol (28) (370mg, 83%) as an oil (C)₂₃H₃₄O₄M found value of (a): 374.250, calculated as: 374.252).

δ_H0.298(1H，dd，³J 8.1Hz，4.9Hz，4-H₂)，0.510(1H，dd，³J 4.4Hz，4.4Hz，4-H_b)，0.985(3H，s，19-H)，1.055(3H，s，18-H)，1.325(3H，s，21-H)，3.318(1H，dd，³J 3.0Hz，3.0Hz，6-H)，)，3.363(1H，dd，³J 11.4Hz，4.2Hz，12-H)，4.019(4H，m，OCH₂Ch₂O)4.622(1H，s，OH)，5.255(1H，dd，³J 3.9Hz，1.9Hz，15-H).

δ_C11.681T (C-4), 12.243Q (C-18), 19.844Q (C-19), 23.604Q (C-21), 63.620T (ethylenedioxy), 63.733T (ethylenedioxy), 73.569D (C-6), 77.478D (C-12), 111.125S (C-20), 118.702D (C-15), 152.912S (C-14).

Example 17

20, 20-ethylenedioxy-14, 15 beta-epoxy-3 alpha, 5-cyclo-5 alpha, 14 beta-pregnane-6 beta, 12 beta-diol (29)

N-bromoacetimide (150mg, 1.1mmol) was added to a solution of 20, 20-ethylenedioxy-3 α, 5-cyclo-5 α -pregn-14-ene-6 β,12 β -diol (28) (340mg, 0.91mmol) in acetone (20mL), water (0.25mL) and acetic acid (0.25mL) at 0 ℃. After 15 minutes, sodium sulfite (5% solution, 20mL) was added to the reaction mixture. The acetone was distilled off under reduced pressure and the residual solution was extracted with dichloromethane (3X 30 mL). The dichloromethane layer was dried over magnesium sulfate, filtered and evaporated to a concentrated volume (50 mL). Pyridine (0.5mL) was added to the mixture and stirring was continued for 1 hour, after which the dichloromethane layer was washed with lemonCitric acid solution (5%, 3 × 30mL), saturated aqueous sodium bicarbonate (30mL), and water (30 mL). The dichloromethane layer was dried over magnesium sulfate, filtered, evaporated, purified by flash column chromatography using chloroform-methanol (9.5: 0.5v/v) as eluent to give epoxide (29) (180mg, 51%) as a foam (C)₂₃H₃₄O₂M found value of (a): 390.245, calculated as: 390.247).

δ_H0.287(1H，dd，³J 8.1Hz，4.9Hz，4-H_a)，0.501(1H，dd，³J 4.4Hz，4.4Hz，4-H_b)，0.978(3H，s，19-H)，1.048(3H，s，18-H)，1.321(3H，s，21-H)，3.318(1H，dd，³J 3.1Hz，3.1Hz，6-H)，)，3.355(1H，dd，³J11.2Hz，4.1Hz，12-H)，3.491(1H，s，15-H)，4.001(4H，m，OCH₂Ch₂O)，4.901(1H，s，OH).

δ_C11.668T (C-4), 11.973Q (C-18), 19.515Q (C-19), 23.519Q (C-21), 59.910D (C-15), 63.601T (ethylenedioxy), 63.713T (ethylenedioxy), 72.501S (C-14), 73.571D (C-6), 77.471D (C-12), 111.085S (C-20).

Example 18

20, 20-ethylenedioxy-6 beta, 12 beta, 14-trihydroxy-3 alpha, 5-cyclo-5 alpha, 14 beta-pregnane (29)

A solution of epoxide (29) (170mg, 0.44mmol) in tetrahydrofuran (10mL) was added to a suspension of lithium aluminum hydride (20mg, 0.53mmol) in tetrahydrofuran (5 mL). The reaction mixture was refluxed for 2 hours, after which water (20 μ L) was added and stirring was continued for 0.5 hour. Sodium hydroxide (15%, 20 μ L) was added and stirred for an additional 0.5 hours. Water (60. mu.L) was added thereto and the suspension was stirred for 1 hour. After filtration, the suspension was dried over magnesium sulfate, filtered and the tetrahydrofuran was distilled off. The resulting mixture was subjected to flash chromatography, eluting with chloroform-methanol (9: 1v/v) to give the desired triol (30) (90mg, 53%) as a clear oil (C)₂₃H₃₈O₅M found value of (a): 392.261, calculated as: 392.263).

δ_H0.287(1H，dd，³J 8.1Hz，4.9Hz，4-H₂)，0.510(1H，dd，³J 4.4Hz，4.4Hz，4-H_b)，0.971(3H，s，19-H)，1.042(3H，s，18-H)，1.319(3H，s，21-H)，3.321(1H，dd，³J 3.0Hz，3.0Hz，6-H)，3.321(1H，dd，³J 11.1Hz，3.9Hz，12-H)，3.561(1H，s，OH)，4.084(4h，m，OCH₂Ch₂O)4.671(1H，s，OH).

δ_C11.668T (C-4), 11.971Q (C-18), 19.511Q (C-19), 23.520Q (C-21), 63.612T (ethylenedioxy), 63.711T (ethylenedioxy), 73.483D (C-6), 76.051D (C-12), 84.307S (C-14), 111.099S (C-20).

Example 19

3 beta, 12 beta, 14-trihydroxy-14 beta-pregn-5-en-20-one (15)

A mixture of triol (30) (80mg, 0.20mmol) in acetone (20mL) and hydrochloric acid (1M, 10mL) was refluxed at 60 ℃ for 2 hours. The reaction mixture was cooled and saturated sodium bicarbonate solution (20mL) was added. The acetone was evaporated, the aqueous layer extracted with chloroform (3X 20mL), the chloroform layer dried over magnesium sulfate, filtered and evaporated to give the epimeric trihydroxysteroid (15a, 15b) (42mg, 61%). The epimer mixture (15a, 15b) (15mg) was separated by flash chromatography (using chloroform: methanol 9: 1(v/v) as eluent) to give pure 17 β -epimer (15a) (10mg), m.p.224-229 ℃ (acetone), (lit 226-₂₁H₃₂O₄Measured value of (d): m348.234; c72.32; h9.21 percent; calculated C72.38; h9.26%; m348.236); 17 α -epimer (15b) (10mg), m.p.183-191 ℃ were also obtained (acetone), (lit 184-196 ℃).

3 beta, 12 beta, 14-trihydroxy-14 beta-pregn-5-en-20-one (15a)

δ_H 0.963(1H，s，19-H)，1.192(3H，s，18-H)，2.236(3H，s21-H)，3.325(1H，dd，³J 11.2Hz，3.9Hz，12-H)，3.464(1H，s，OH)，3.5140(1H，m，3-H)，3.598(1H，dd，³J 9.6Hz，9.6Hz，17-H)，4.255(1H，s，OH)，5.383(1H，m，5-H).

δ_C8.275Q(C-18)，19.414Q(C-19)，24.400T(C-11)24.581T(C-16)，27.443T(C-7)，30.062T(C-2)，32.972Q(C-21)，34.543T(C-15)，35.864D(C-8)，36.975S(C-10)，37.337T(C-1)，42.144T(C-4)，43.565D(C-9)，55.101S(C-13)，57.038D(C-17)，71.597D(C-3)，73.558D(C-12)，85.566S(C-14)，122.223D(C-6)，138.932S(C-5)，217.011S(C-20).

3 beta, 12 beta, 14-trihydroxy-14 beta-pregn-5-en-20-one (15b)

δ_H0.996(1H，s，19-H)，1.144(3H，s，18-H)，2.221(3H，s21-H)，3.339(1H，dd，³J 9.4Hz，9.4Hz，17-H)，3.492(1H，m，3-H)，3.629(1H，dd，³J 11.1Hz，3.9Hz，12-H)，3.712(1H，s，OH)，4.325(1H，s，OH)，5.383(1H，m，5-H).

Examples 20-28 illustrate the process by which intermediate compounds can be prepared to form the first monosaccharide (40).

Example 20

Methyl-4, 6-O-benzylidene-alpha-D-glucopyranoside (32)

A mixture of methyl- α -D-glucopyranoside (30g, 0.15mol), benzaldehyde (70mL) and zinc chloride (20g) was stirred at room temperature for 24 hours. The reaction product was poured into ice water and stirring was continued for 15 minutes. The white precipitate was filtered off and washed with diethyl ether. The solid material was stirred with sodium metabisulfite (10% solution) for 15 minutes, filtered and washed with water. The solid material was crystallized from chloroform and diethyl ether to give the benzylidene product (32) (31g, 72%).

Example 21

Methyl-4, 6-O-benzylidene-2-O-tosyl-alpha-D-glucopyranoside (33)

P-toluenesulfonyl chloride (25g, 1.2 equiv.) in pyridine (100mL) was added dropwise to a solution of benzylidene glucose (32) (31g, 0.12mol) in pyridine (100mL) at 0 ℃. The reaction mixture was stirred at room temperature for 48 hours. Ice was added to the reaction mixture. The resulting white solid was washed with water and recrystallized from hot ethanol to give tosylated glucose (33) (28g, 60%).

Example 22

Methyl-4, 6-O-benzylidene-3-O-methyl-alpha-D-atropine pyranoside (34)

Tosylate (33) (28g, 64mmol) in a solution of sodium (7g) in methanol (150mL) was heated at 110 ℃ for 48 h in an autoclave. The reaction vessel was cooled and solid carbon dioxide was added to the reaction mixture. After filtration, the methanol is distilled off and the solid material is then absorbed into water. The aqueous layer was extracted three times with chloroform. The chloroform layer was dried over magnesium sulfate, filtered and evaporated. The crude mixture was purified by column chromatography on silica gel eluting with chloroform: acetone (9: 1) to give coronene glycoside (34) (10g, 52%).

Example 23

methyl-6-bromo-4-O-benzoyl-3-O-methyl-6-deoxy-alpha-D-atropine pyranoside (35)

Benzylidene altrose glycoside (34) (10g, 33mmol) was added to a solution of N-bromosuccinimide (7.6g) and barium carbonate (20g) in carbon tetrachloride and the reaction mixture was refluxed at 75 ℃ for 3 hours. The reaction mixture was filtered and the carbon tetrachloride layer was washed with water. The organic layer was dried over magnesium sulfate, filtered, and evaporated to give 6-bromo-altrose glycoside (35) (9g, 69%).

Example 24

methyl-4-O-benzoyl-3-O-methyl-6-deoxy-alpha-D-atropine pyrane glycoside (36)

Sodium borohydride (18g) in water (30mL) was added dropwise to a solution of bromoaltrose glycoside (35) (9g, 23mmol) and nickel chloride (18g) in ethanol (300mL) at 0 deg.C. The reaction mixture was refluxed at 75 ℃ for 1 hour, and then, it was filtered. The ethanol was evaporated, the residual aqueous layer was extracted three times with chloroform, and the chloroform layer was dried over magnesium sulfate, filtered, and evaporated to give 6-deoxy-altrose glycoside (36) (5g, 72%).

Example 25

4-O-benzoyl-3-O-methyl-6-deoxy-alpha beta-D-phenylthiopyran altrose glycoside (37)

Phenylthiotrimethylsilane (5mL) and trimethylsilyltrifluoromethanesulfonate (2mL) were added to a solution of 6-deoxy-altrose glycoside (36) (5g, 17mmol) in dichloromethane (200mL) at 0 deg.C. The reaction mixture was stirred at room temperature for 6 hours. To the reaction mixture was added a saturated sodium bicarbonate solution. The dichloromethane layer was dried over magnesium sulfate, filtered and evaporated. The crude mixture was purified by column chromatography on silica gel eluting with chloroform: acetone (9: 1) to give α β -D-phenylthioaaltrose glycoside (37) (4g, 63%).

Example 26

4-O-benzoyl-3-O-methyl-2-phenylthio-2, 6-dideoxy-alpha beta-D-fluoropyran cymaroside (38)

Diethylaminosulfur trifluoride (0.65g) was added to a solution of α β -D-phenylthioaaltrose (37) (0.5g, 1.33mmol) in dichloromethane at 0 ℃. The reaction mixture was stirred at 0 ℃ for 0.5 h, then saturated sodium bicarbonate solution was added. The dichloromethane layer was separated from the aqueous layer, dried over magnesium sulfate, filtered and evaporated to give α β -fluoro-ephedrine (38) (450mg, 90%).

Example 27

4-O-benzoyl-3-O-methyl-2-O-tert-butyldimethylsilyl-alpha beta-D-phenylthio-altrose glycoside (39)

6-deoxyaltrose glycoside (5g) was silylated with tert-butyldimethylsilyl chloride (3g) and imidazole (3g) in pyridine (50 mL). The reaction mixture was worked up by extraction with ethyl acetate, the ethyl acetate layer was washed with hydrochloric acid (6N), then with sodium bicarbonate solution and then with water. The ethyl acetate layer was dried over magnesium sulfate, filtered and evaporated to give silylated benzoylphenylthio altrose glycoside (39) (80%).

Example 28

3-O-methyl-2-O-tert-butyldimethylsilyl-alpha beta-D-phenylthioaaltrose (40)

Siloxylated benzoylphenylthio altrose (39) (6g) was treated with sodium methoxide (100mL) for 4 h. Methanol was distilled off, and water was added to the reaction mixture. The aqueous layer was acidified (pH5, ACOH) and extracted with ethyl acetate. The ethyl acetate layer was washed with water, dried over magnesium sulfate, filtered and evaporated to give silylated methylphenylsulfanylalnoside (40) (75%).

Examples 29-37 illustrate synthetic procedures that can prepare intermediate compounds to form a second monosaccharide (50).

Example 29

1,2: 5, 6-di-O-isopropylidene-alpha-D-glucofuranose (42)

Sulfuric acid (40mL) was added dropwise to a solution of α -D-glucose (41) (50g, 0.28mol) in acetone (1L) at 0 ℃. The reaction mixture was stirred for 24 hours and then neutralized with sodium hydroxide (6M). The acetone was distilled off and the aqueous layer was extracted twice with chloroform. The chloroform layer was dried over magnesium sulfate, filtered and evaporated. Crystallization from cyclohexane yielded diisopropylidene glucose (42) (41g, 57%).

Example 30

1,2: 5, 6-bis-O-isopropylidene-3-O-methyl-alpha-D-glucofuranose (43)

α -D-glucopyranose (42) (41g, 0.16mol) in tetrahydrofuran (300mL) was added dropwise to a suspension of sodium hydride (5g) in tetrahydrofuran (200 mL). After 0.5 hour, methyl iodide (25g) in tetrahydrofuran (100mL) was added dropwise to the reaction mixture, followed by stirring for 24 hours. Water was added to the reaction mixture, followed by extraction with diethyl ether three times. The ether layer was dried over magnesium sulfate, filtered and evaporated to give methyl protected glucose (43) (38g, 83%).

Example 31

3-O-methyl-alpha beta-D-glucopyranose (44)

Methyldiisopropylene compound (43) (38g, 0.14mol) was dissolved in acetic acid (50%, 700mL), and the solution was refluxed for 18 hours. After cooling, acetic acid was distilled off. The crude product was purified by column chromatography eluting with chloroform, methanol, acetone, water (70: 27: 2: 1) to give 3-O-methyl-. alpha. -D-glucopyranose (44) (13g, 50%).

Example 32

Methyl 3-O-methyl-alpha beta-D-glucopyranose (45)

3-O-methyl-. alpha. -D-glucopyranose (44) (10g) was dissolved in methanol (50mL) and hydrochloric acid (concentrated hydrochloric acid, 1mL) and refluxed overnight. Sodium bicarbonate was added and the reaction mixture was filtered. The methanol was distilled off to leave 1, 3-di-O-methyl-. alpha. beta. -D-glucopyranoside (45) (95%).

Example 33

Methyl 4, 6-O-benzylidene-3-O-methyl-alpha beta-glucopyranoside (46)

Glucopyranoside (45) (8g) was stirred in a solution of benzaldehyde (20mL) and zinc chloride (5g) at room temperature. After 24 hours, ice was added and the aqueous layer was extracted with chloroform. The chloroform layer was dried over magnesium sulfate, filtered and evaporated. Benzaldehyde was removed by vacuum distillation and the product was purified by silica gel column chromatography eluting with acetone: chloroform (0.5: 9.5) to give benzylidene- α β -glucopyranoside (46) (60%).

Example 34

Methyl 4-O-benzoyl-3-O-methyl-6-deoxy-alpha beta-glucopyranoside (47)

The benzylidene compound (46) (5g) was refluxed in a mixture of N-bromosuccinimide (3.7g) and barium carbonate (4g) in carbon tetrachloride at 80 ℃. After 4 hours, the reaction mixture was filtered, the carbon tetrachloride layer washed with water, dried over magnesium sulfate, filtered and evaporated to give the bromo compound (70%).

The bromo compound (4.3g) was dissolved in a solution of ethanol (300mL) and nickel chloride (8.6g) at 0 ℃. To this solution was added dropwise sodium borohydride (8.6g) in water (50mL) over 15 minutes. The reaction mixture was refluxed at 100 ℃ for 45 minutes, cooled, filtered and evaporated. Chloroform was added, and the chloroform layer was washed with water, dried over magnesium sulfate, filtered, and evaporated to give 6-deoxy sugar (47) (70%).

Example 35

4-O-benzoyl-3-O-methyl-1-thiophenyl-6-deoxy-alpha beta-glucopyranoside (48)

6-deoxyglucopyranoside (47) (3g) was dissolved in methylene chloride (50 mL). To this solution was added phenylthiotrimethylsilane (2g) and trimethylsilyl trifluoromethanesulfonate (0.2 mL). The solution was stirred at room temperature overnight, after which saturated sodium bicarbonate solution was added. The dichloromethane layer was dried over magnesium sulfate, filtered and evaporated. The product was purified by column chromatography on silica gel eluting with ethyl acetate: hexane (2: 8) to give compound (48) (60%).

Example 36

4-O-benzoyl-3-O-methyl-2-O-pivaloyl-1-thiophenyl-6-deoxy-alpha beta-glucopyranoside (49)

To a solution of glucopyranoside (48) (2g) in pyridine (20mL) was added pivaloyl chloride (2 mL). The solution was stirred at room temperature overnight, after which water was added. The aqueous layer was extracted with ethyl acetate, and the organic layer was washed with hydrochloric acid (6N). The organic layer was dried over magnesium sulfate, filtered and evaporated to give pivaloyl ester (49) (89%).

Example 37

4-O-benzoyl-3-O-methyl-2-O-pivaloyl-1-fluoro-6-deoxy-beta-glucopyranoside (50)

N-bromosuccinimide (1.2g) and diethylaminosulfur trifluoride (1.2g) were added to a solution of pivaloyl ester (49) (2g) in dichloromethane (100mL) at 0 ℃. After 1 hour, saturated sodium bicarbonate solution was added. The dichloromethane was dried over magnesium sulfate, filtered and evaporated. The beta-fluoropyranoside (50) was purified by silica gel column chromatography, eluting with ethyl acetate: hexane (2: 8).

Example 38 illustrates a synthetic procedure for the preparation of the compound 3-O- [ 4-O-benzoyl-2-phenylthio- β -D-pyraclostrobin ] -12, 14- β -dihydroxypregn-5-en-20-one (51).

Example 38

3-O- [ 4-O-benzoyl-2-phenylthio-beta-D-pyranosyl ] -12, 14 beta-dihydroxypregn-5-en-20-one (51)

Tin chloride (190mg, 1mmol) was added to 3, 12, 14 β -trihydroxypregn-5-en-20-one (15) (100mg, 0.28mmol) and fluoro-pyranyl-cymaroside (38) (210mg, 0.56mmol) in anhydrous ether and 4 Å molecular sieves at-15 ℃. The reaction mixture was kept at-15 ℃ for 3 days. To the reaction mixture was added a saturated sodium bicarbonate solution. The ether layer was dried over magnesium sulfate, filtered and evaporated. The product was purified by silica gel column chromatography eluting with chloroform: methanol (9.5: 0.5) to give glucoside (51) (30mg, 15%).

Examples 39-41 illustrate the synthesis of conjugated magnetoleprose and oleander sugars.

Example 39

Yellow oleander sugar-magnetic sesame sugar disaccharide (53)

A solution of yellow oleander sugar (50A) (1.5g), maghemite (40) (1.3g) and molecular sieve 4 Å in methylene chloride was stirred at room temperature for 1 hour. The reaction mixture was cooled to-15 ℃ and tin (II) chloride (0.8g) and silver triflate (1.1g) were added. The mixture was stirred at-15 ℃ for 16 h, after which triethylamine (0.5mL) was added. The reaction product was filtered and dichloromethane was distilled off. The disaccharide was purified by column chromatography on silica gel eluting with ethyl acetate: hexane (2: 8) in 15% yield.

Example 40

Yellow oleander sugar-magnetic sesame sugar disaccharide (54)

To a solution of disaccharide (53) (200mg) in tetrahydrofuran (20mL) was added tetrabutylammonium fluoride (0.4 mL). The mixture was stirred at room temperature for 1 hour, after which saturated sodium bicarbonate solution was added. The reaction mixture was extracted with ethyl acetate and the ethyl acetate layer was dried over magnesium sulfate, filtered and evaporated. The disaccharide was purified by silica gel column chromatography eluting with acetone: chloroform (0.5: 9.5) at a yield of 60%.

EXAMPLE 41

Yellow oleander sugar-magnetic sesame sugar disaccharide (55)

Diethylaminosulf trifluoride (80. mu.L) was added to a solution of disaccharide (54) (80mg) in dichloromethane (10mL) at 0 ℃. After stirring for 0.5 h at 0 ℃, saturated sodium bicarbonate solution and more dichloromethane were added. The dichloromethane layer was dried over magnesium sulfate, filtered and evaporated. Purification by silica gel column chromatography (ethyl acetate: hexane 1: 9) gave disaccharide (55) (65%).

Example 42

The following biological test results for three appetite suppressants are given below.

a) Irwin test;

b) acute toxicity test; and

c) oral administration for anorexia test.

a) Irwin test

The purpose of this test is to evaluate the appetite suppressant of the invention produced from the aforementioned plant extracts, by means of a simplified animal Irwin test for calming and sedative effects.

Procedure of experiment

Appetite suppressants were extracted from plant material by the applicant of the present application by the method described previously and administered to two of four groups of animals, three animals per group, one untreated, one receiving the solvent dimethyl sulfoxide (DMSO), one receiving 50mg/kg of test samples, and one receiving 300mg/kg of test samples. Treatment was performed by intraperitoneal injection, with observations made at specific time intervals until five hours after treatment. Symptoms other than those observed in animals of the DMSO-treated group were used for interpretation of the results.

Results

It is clear that the solvent DMSO has a significant effect on animals, especially on the thermal regulation mechanism. Animals dosed with all solvents, including DMSO alone or with test samples, had a significant drop in body temperature.

As with all other groups including the control group, the animals in the low dose group showed a significant decrease in cage dispersion. Hypopneas were observed 15-60 minutes after dosing. Ptosis (eyelid closure) was observed to a greater extent than in the DMSO group. Auricle (ear) responses were also seen, and a positive finger response was also observed, indicating a fear. After dosing, body temperature dropped to 32.7 ℃.

As in the other groups, animals in the high dose group showed reduced dispersibility in the initial cages and reduced locomotor activity, but showed increased dispersibility and locomotor activity before death, and died about 1 hour after administration. Severe clonic symmetric tics occurred 30 minutes after dosing. Hypopnea begins to appear but increases before death. Auricle (ear) responses were delayed and positive finger responses were observed, indicating fear, which was also observed in the low dose group of animals. After administration, the body temperature was reduced to 30.7 ℃. Increased positional passivity and decreased body elasticity were also observed. Abnormal limb rotation, reduced grip strength, absence of painful reactions, and loss of righting reflex were observed.

Discussion of the related Art

Animals receiving low doses (50mg/kg) showed only a reduction in respiration and an increase in ptosis when compared to animals in the control and DMSO-administered groups. Whereas animals receiving high doses (300mg/kg) of the test samples responded very strongly, showing twitching and death. All other observations made in these animals can be attributed to animal twitching and death. No suggestive signs of sedation, such as a marked reduction in cage dispersion, reduced locomotor activity and apathy, were observed in the test group, which could be attributed to the test samples. Thus, it can be concluded that the test samples are lethal factors leading to 300mg/kg group of rats and have respiratory depression effect on 50mg/kg group of rats when administered intraperitoneally using DMSO as a solvent.

b) Acute toxicity test

The purpose of this test is to obtain information about the toxicity of the test sample.

Procedure of experiment

The plant extract having appetite suppressant effect prepared according to the aforementioned process of the present invention was purified, and a test sample was orally administered to rats at an increased dose. Two rats were used for each dose group, except that only one rat was used for the highest dose group. The rats were examined for good physical condition and their body weights were measured on the day of administration.

The dosage range is 100mg/kg to 3028.5 mg/kg. The dose was calculated and mixed into the prepared potato starch so that each rat received a total dose of 0.2 mL. Rat 13 received 0.25 mL. The potato starch is prepared by: 20g of starch was mixed into a small amount of cold water, to which hot water was added to make up the volume to 1 liter. The suspension was cooled to room temperature before dosing.

Rats in group 1 and group 2 were dosed on the same day. They were observed for 24 hours and if there were no signs of toxicity, the next group was administered. The same study procedure was continued until all rats were dosed. This procedure was performed sequentially to ensure that rats were not dosed any more when the acute toxic dose had been reached in the previous group.

Clinical signs of immediate intoxication (1-2 hours) were observed after dosing and daily thereafter. Body weight was measured once a week and total food and water intake was measured for each rat.

Penbarbiton sodium (commercially available) was injected intraperitoneally on day 14 of the experimentTrade name Euthanaze, Centaur^®) Surviving rats were sacrificed. Necropsy was performed on these rats, as were dead rats during the experiment. Samples for histopathology were collected.

Results

Group 1 (control group)

No clinical signs of toxicity were observed over the 14 day observation period. Food and water intake were within normal ranges. The change in body weight was also within the normal range. There were no pathological histological changes in liver samples.

Group 2 (100mg/kg)

No clinical signs of toxicity were observed over the 14 day observation period. Food and water intake were within normal ranges. The change in body weight was also within the normal range. No macroscopic lesions were observed in liver samples, nor were there any changes in pathology histology or morphology.

Group 3 (200mg/kg)

During the observation period, the rats of this group showed no clinical signs of toxicity. Food and water intake were within normal ranges. The change in body weight was also within the normal range. No macroscopic lesions were observed, but histopathological changes were detected in the liver samples. In rat No. 6, hepatocytes were slightly turbid, but in rat No. 5, moderate turbid. Moderate edema degeneration also occurred in rat No. 5.

Group 4 (400mg/kg)

During the observation period, the rats of this group showed no clinical signs of toxicity. During necropsy, no macroscopic lesions were observed. Moderate edema and slight edema changes were found upon histological observation.

Animal 7 water and food intake was normal and weight gain was normal. Animal 8 consumed almost twice as much food as animal 7 (144.6 g and 73.9g, respectively), but gained only 0.81g in weight compared to 2.7 g.

Group 5 (800mg/kg)

One rat (10 th rat) died after 3 hours of administration and did not show any particular signs. The other rat (9 th rat) survived the entire observation period without any signs of toxicity. The water intake of the surviving rats was normal (42.42mL) while the food intake was increased (134.2 g). Body weight gain was 2.85g, which is the highest value among all rats in the experiment.

Pulmonary congestion was found at necropsy in rats 10 (dead short after oral administration). No other external body reactions occurred, indicating inhalation of the test material. Rat 9 found no macroscopic lesions. There was slight cytoplasmic vacuole formation (hydropsy degeneration) in rat 10, but moderate in rat 9. The appearance of the liver gland cytoplasm was moderate in both rats.

Group 6 (1600mg/kg)

During the experiment, there was no evidence of clinical toxicity. At necropsy, no macroscopic lesions were observed, but moderate degeneration in the liver of rat 11 was observed in the histophysiological examination. Rat 12 showed moderate nephelocity and slight hepatocellular hydropsy. Food and water intake was normal and body weight increased during the experiment.

Group 7 (3028.5mg/kg)

Only one rat was dosed at this dose. During the observation period, no sign of toxicity was observed in the rats, nor was macroscopic lesions observed. In tissue physiology assays, moderate degrees of nephelomas and hepatocellular hydropsy are observed. The rats showed a decrease in body weight (-0.82g) during the observation period, but normal food and water intake.

Discussion of the related Art

Since only a small number of animals were used for the experiment at each dose, it was difficult to draw any conclusions. The fact that only one rat died at low doses did not show any signs, indicating that the death was not related to the test samples, due to stress during and/or after the dosing. This hypothesis is further supported by the fact that no rats died or showed any signs of toxicity in the high dose group.

The observed increase in food intake in rats 8 was likely the result of excessive food spill (spill), and this was reflected by only a small increase in body weight. It is noted that all rats in this experiment were dosed only once, and thus, it was not possible that the appetite suppressant had a significant effect on food or water intake or volume within 14 days, as well as from the experimental results.

From histophysiological experiments on liver samples, it is clear that pathological changes are dose-dependent, and rats receiving high doses show more intense changes. The pathology observed is not naturally metabolized, but may be tested for induction by the sample. The change is only a kind of degeneration and is thus reversible. No sign of irreversible hepatocyte changes was observed.

Thus, it can be concluded that only one rat died at the low dose (800mg/kg), but that its death may not be related to the test sample. The other rats in any dose group had no signs of toxicity, or death due to dosing, during the observation period of 14 days after dosing. A single oral dose of the test sample will produce reversible dose-related changes in hepatocytes.

c) Oral drug administration anorexia test

The aim of this test is to determine the activity and the minimum effective dose of the plant extracts prepared according to the invention, and to study possible side effects such as respiratory depression, as in the case of the Irwin test (as described above).

Procedure of experiment

Rats were divided into several dosing groups using a random selection. Each administration group consisted of three rats, 6 rats in the control group. The test samples were administered to environmentally-adapted female pups (rats) 100-150g for three consecutive days. Rats were identified simply by means of a metallic ear marker and a potassium permanganate skin marker. Rats were housed individually in standard rodent polycarbonate cages, water and powdered commercial rodent chow were supplied ad libitum. Water and food intake values were measured and calculated daily. To find the minimum effective dose for the test sample, five doses were measured. The test samples were suspended in potato starch by oral gavage.

The substance tested was compound (1), a granulated powder prepared according to the invention from an extract of a plant material, and a test amount of the test sample was mixed with the prepared potato starch and formed into a dosage form. The mixing with potato is carried out immediately before the daily administration. The suspension was mixed well with Vortex at the time of dose volume discharge for each rat.

Five dosage ranges were tested, with the control group receiving carrier substance only. The choice of the dose is based on the effects observed in the above-mentioned Irwin test, which are:

group 1: 0.00mg/kg (control group)

Group 2: 6.25mg/kg

Group 3: 12.50mg/kg

Group 4: 25.00mg/kg

Group 5: 37.50mg/kg

Group 6: 50.00mg/kg

Results

The administration did not affect the health of the rats during the study. Throughout the study period, the test sample-administered rats showed a significant reduction in the mean weight gain value in all dose groups, with three of the five dose groups actually losing weight.

The average food intake for all dosing groups decreased during the study. The rats in the high dose group showed increased consumption of effluent.

Rats in each dose group did not show a significant effect on respiration rate.

Rats in all dose groups had fragile livers at necropsy but no macroscopic lesions.

Discussion of the related Art

Data collected during acclimation confirmed that all rats used in the experiment were healthy and that body weight was comparable between rats.

The reduction of the volume increase and even the weight loss and the reduction of food intake in some rats strongly showed an appetite centre inhibitory effect. The decrease in food intake and weight gain values occurred even in the lowest dose group (6.25mg/kg), with the actual weight loss occurring in the 12.50mg/kg group.

It is important that the water consumption was increased in all the administration groups while the feed consumption was decreased (fig. 2). This may be due to the diuretic effect of the test sample or to the stimulation of the craving center in the brain.

The fact that no respiratory depression was observed in the above-described acute toxicity test using the intraperitoneal route can be regarded as a positive aspect. This may be due to reduced absorption from the gastrointestinal tract, thereby reducing bioavailability. However, bioavailability in the oral dose tested is sufficiently effective for the test sample. A slight decrease in respiratory rate after 1 hour of administration in most groups can be seen as due to filling of the stomach with dose volume and consequent passive behavior of the rats.

The fragile liver observed in the administered group was probably due to a weaker change in energy metabolism than a decrease in food intake, resulting in increased fat metabolism and overload of liver burden. If this is true, these changes may be considered temporary, will recover after reaching a steady state, or will recover after the test sample is expelled. The possible effects on the liver also require further investigation.

Since the study was only preliminary to screening tests, few groups of test animals were used. This is difficult to satisfactorily interpret the data throughout, particularly when individual animals differ in response overall. However, the data also show that the test samples had appetite suppression even at the lowest dose tested (6.25 mg/kg). No clinical signs of respiratory depression were observed at the doses tested.

Example 43

Naturally occurring or artificially cultivated plants of the fire land subplants are first stored at 4 ℃ for up to 48 hours after harvesting. The plants were washed in tap water and then cut into pieces of + -1 cm. The pieces after slicing were combined and pressed through a hydraulic press at 300 bar pressure for a minimum of 0.5 hour per press. During the squeezing process, the plant juices are collected separately. The juice was stored at-18 ℃ until further processing was required.

Spray drying the juice under suitable conditions to obtain a free flowing powder. The moisture content of the powder after spray drying is preferably less than 5% and, if desired, further drying may be carried out in a vacuum oven or by means of a fluid bed dryer.

Both the juice and the spray-dried material were shown to be effective as appetite suppressants in biological tests in rats.

Experiment of

50kg of fire land sub gordonii was washed with tap water and then cut into 1cm pieces. The sliced plants were pressed with a hydraulic press at 300 bar for a minimum of 0.5 hours per press. The sap weight was 10kg when the natural fire ground gordonii was used, whereas 20kg was obtained from the artificially cultured fire ground sub gordoni plants. The juice (500g) was spray dried using the following conditions:

flow rate: 285mL/min

Inlet temperature: 110 deg.C

Outlet temperature: 70 deg.C

Temperature of the working chamber: 78 deg.C

The spray-dried powder obtained was a free-flowing powder (22g) with a water content of 6.9%.

The spray-dried powder was analyzed for active ingredient content using HPLC techniques. The content of active ingredient was determined to be 13g/kg of spray-dried powder.

HPLC analysis method

Eluent: acetonitrile/water (7: 3), gradient-free elution

Column: reversed phase C-18

UV absorption: 225nm

Flow rate: 1mL/min

Injection volume: 10 μ l

Method

The spray dried powder (10mg) was dissolved in water (0.5mL) and acetonitrile (0.5 mL). Mu.l of this solvent was injected into HPLC, and the concentration of active compound (1) was determined using a standard curve prepared from pure compound (1).

Example 44

The results of studies designed to evaluate the possible anorexic effect of compound (1) in rats are given below. Among the samples tested were pure sap (sample 1), spray-dried sap (sample 2) and active ingredient (sample 3). Samples 1 and 2 were respectively sap and spray dried sap as described in example 43. Sample 3 is solvent extracted compound (1) with a purity of 95% or more.

Samples 1-3 were administered to male Wistar rats in a single oral dose. Two control groups were dosed with vehicle (distilled water or DMSO). Fenfluramine (7.5mg/kg) was administered orally as a reference standard.

Sample 1 (pure sap) was administered orally and, as a statistical result, produced a statistically significant reduction in the amount of food consumed, depending on the dose, at and above 1600mg/kg compared to the control administered with vehicle. A corresponding decrease in body weight (or rate of growth) was also recorded. During the administration, a statistically significant increase in water consumption was recorded after 3 hours (6400 and 10000mg/kg) and6 hours (10000mg/kg) of administration. At doses of 3200mg/kg and above 24-48 hours after administration, a statistically significant decrease in water consumption was recorded.

Sample 2 (spray dried sap) also produced a statistically significant reduction in food consumption and body weight when administered orally at 76mg/kg compared to vehicle-administered rats. No statistically significant effect on water consumption was recorded.

Sample 3 (active ingredient) produced a statistically significant reduction in food consumption when administered orally at 5.0 mg/kg. The active ingredient had no statistically significant effect on body weight compared to rats dosed with vehicle, although the data from the assay showed a slight delay in growth. No statistically significant effect on water consumption was recorded.

The reference standard fenfluramine (7.5mg/kg) had a statistically significant reduction in food consumption after 6 and 24 hours of administration, compared to the vehicle-administered control group. No statistically significant effect on water consumption was recorded.

No effects on the liver associated with drug administration were recorded.

Test substance

Characteristics of	Sample 1 (pure juice)	Sample 2 (spray dried juice)	Sample 3 (active ingredient)
Characteristics of	Sample 1 (pure juice)	Sample 2 (spray dried juice)	Sample 3 (active ingredient)	Apparent storage condition purity carrier	Pure juice distilled water at-20 deg.C in dark	Pure spray-dried juice distilled water in dark powder at room temperature	White powder more than or equal to 95% dimethyl sulfoxide (DMSO) at 4 ℃ in dark

Procedure of experiment

55 male Wistar rats were used in this study.

Body weight, food consumption (food funnel fill) and water consumption (bottle weight) were recorded at the same time each day from the start of the experiment to the end of the study.

On day 1, rats were given a single oral (gavage) dose according to the following table:

group of	n	Oral administration	Dosage (mg/kg)
group of	n	Oral administration	Dosage (mg/kg)	123456789101112	545555555533	Carrier (distilled water) sample 1 (pure sap) sample 2 (spray dried sap) sample 3 (active ingredient) fenfluramine carrier (DMSO)	-8001600320064001000038762.55.07.5-

Groups 1-8 were dosed at a stable dose volume of 10mL/kg, and groups 9-12 were dosed at a dose volume of 1 mL/kg.

Food consumption and water consumption were measured at 1,3 and6 hours after the administration on day 1.

After measurement on day 8, rats were sacrificed by carbon dioxide asphyxiation, livers were excised, and placed in 10% buffered formalin before microdissection studies.

Paraffin sections of liver were taken at 4-5 μm and stained with hematoxylin and eosin. A12 μm section was taken in a cryostat and the fat was stained with Oil Red O (ORO).

Data analysis

Food and water consumption measurements and body weights of P57-dosed rats at each time point after dosing were compared to corresponding data for related control groups similarly dosed with vehicle using analysis of variance, followed by William test for control group comparison.

The data of fenfluramine-dosed rats were compared with the data of vehicle-dosed control groups using the t-test method.

Results

The experimental results are listed in the tables below.

Sample 1 (pure sap) administered orally produced a significant dose-related reduction in daily food consumption. The duration and magnitude of this reduction in food consumption is dose dependent. Sample 1 (neat sap) produced a statistically significant reduction in food consumption at doses of 1600mg/kg and higher after 24 hours of administration compared to vehicle-administered controls. The highest dose (10000mg/kg) of sample 1 (sap) produced a statistically significant reduction in daily food consumption up to 5 days after administration.

Sample 2 (spray dried sap) and sample 3 (active ingredient) produced significant and statistically significant reductions at oral doses of 76 and 5.0mg/kg, respectively. In both cases, the effect may last up to 48 hours after administration.

The reference standard fenfluramine (7.5mg/kg, p.o.) produced a statistically significant reduction in food consumption after 6 and 24 hours of administration compared to the vehicle-administered control group (group 12).

Sample 2 (spray dried juice) and sample 3 (active ingredient) did not have a significant dose-related effect on water consumption. On the day of administration, the pure juice produced a statistically significant increase in water consumption after 3 hours (6400 and 10000mg/kg) and6 hours (10000mg/kg) of administration. However, two days after administration, the water consumption of the rats receiving sample 1 (sap) was statistically reduced at 3200, 6400 and 10000 mg/kg. However, these decreases are not clearly identified as dose-related, but only within 1-2 days after administration. Thus, the biological significance of these effects remains unclear.

Sample 1 (neat sap) produced a dose-related statistically significant effect compared to the vehicle dosed control group (group 1). When administered orally at a dose of 3200mg/kg and higher, sample 1 (pure sap) had a statistically significant reduction in body weight or growth rate compared to rats administered with vehicle. These effects were statistically significant from 48 hours of administration until the end of the study.

Sample 2 (spray dried sap) also produced a statistically significant reduction in rat growth when administered orally at 76mg/kg compared to the vehicle-administered control group (group 1). These effects were statistically significant both during day 3 (48 hours post-dose) and day 5.

Although sample 3 (active ingredient) showed a delay in rat production at the highest dose (5.0mg/kg) compared to the vehicle-dosed control group (group 12), this effect was not statistically significant.

Fenfluramine (7.5mg/kg) did not have a significant or statistically significant effect on water consumption or body weight compared to the vehicle-administered control group (group 12).

No relevant effect of treatment on the liver was recorded.

TABLE 1a

Effect of food consumption on oral administration to rats (initial daily dose data)

Group of	Oral administration	Dosage (mg/kg)	Average food consumption per group (g. + -. sd) between two days
								-6--5	-5--4	-4--3	-3--2	-2--1
			1	Carrier (Water)	-	27.8±1.54	24.2±1.83	-6--5	-5--4	-4--3	-3--2	-2--1	27.6±3.67	28.3±3.50	29.4±2.66
2	Sample 1 juice	800	1	Carrier (Water)	-	27.8±1.54	24.2±1.83	28.3±1.43	24.9±0.82	27.7±0.76	28.4±1.51	30.1±0.27	27.6±3.67	28.3±3.50	29.4±2.66
2	Sample 1 juice	800	3	Sample 1 juice	1600	29.0±1.39	26.0±2.16	28.3±1.43	24.9±0.82	27.7±0.76	28.4±1.51	30.1±0.27	27.4±1.96	28.8±0.61	29.6±1.55
4	Sample 1 juice	3200	3	Sample 1 juice	1600	29.0±1.39	26.0±2.16	27.2±2.33	25.1±2.46	26.0±2.52	28.5±2.29	27.6±1.15	27.4±1.96	28.8±0.61	29.6±1.55
4	Sample 1 juice	3200	5	Sample 1 juice	6400	28.7±1.64	25.3±1.73	27.2±2.33	25.1±2.46	26.0±2.52	28.5±2.29	27.6±1.15	27.3±1.46	29.2±1.09	30.3±0.90
6	Sample 1 juice	10000	5	Sample 1 juice	6400	28.7±1.64	25.3±1.73	28.5±2.38	23.7±2.73	26.0±2.31	27.0±3.50	28.7±2.26	27.3±1.46	29.2±1.09	30.3±0.90
6	Sample 1 juice	10000	7	Sample 2 spray dried juice	38	28.1±1.24	23.9±1.79	28.5±2.38	23.7±2.73	26.0±2.31	27.0±3.50	28.7±2.26	24.5±2.30	27.6±1.61	28.5±1.87
8	Sample 2 spray dried juice	76	7	Sample 2 spray dried juice	38	28.1±1.24	23.9±1.79	28.7±0.91	26.5±1.55	27.1±1.01	28.7±1.99	28.9±1.37	24.5±2.30	27.6±1.61	28.5±1.87
8	Sample 2 spray dried juice	76	9	Sample 3 active ingredient	2.5	28.8±1.49	26.4±3.1 2	28.7±0.91	26.5±1.55	27.1±1.01	28.7±1.99	28.9±1.37	29.0±1.99	29.4±1.76	29.5±2.81
10	Sample 3 active ingredient	5.0	9	Sample 3 active ingredient	2.5	28.8±1.49	26.4±3.1 2	28.3±2.1	25.8±1.86	28.1±2.65	28.0±2.65	28.5±3.03	29.0±1.99	29.4±1.76	29.5±2.81
10	Sample 3 active ingredient	5.0	11	Fenfluramine	7.5	29.1±0.66	25.3±4.03	28.3±2.1	25.8±1.86	28.1±2.65	28.0±2.65	28.5±3.03	27.0±1.53	30.8±0.54	29.7±2.84
12	Carrier (DMSO)	-	11	Fenfluramine	7.5	29.1±0.66	25.3±4.03	27.9±1.8	26.7±2.11	28.7±1.99	28.1±4.06	30.5±2.54	27.0±1.53	30.8±0.54	29.7±2.84

sd: standard deviation of

TABLE 1b

Effect of oral administration to rats on food consumption (initial daily dose data)

Group of	Oral administration	Dosage (mg/kg)	Average food consumption per group (g. + -. sd) between two days
										1-2	2-3	3-4	4-5	5-6	6-7	7-8
			1	Carrier (Water)	-	29.5±3.15	29.6±2.84	30.6±3.49	31.8±3.21	1-2	2-3	3-4	4-5	5-6	6-7	7-8	30.7±2.24	31.7±3.03	32.9±3.18
2	Sample 1 juice	800	1	Carrier (Water)	-	29.5±3.15	29.6±2.84	30.6±3.49	31.8±3.21	26.1±0.98	29.3±1.49	30.7±1.15	30.9±0.60	33.3±1.69	32.7±0.80	40.1±13.40	30.7±2.24	31.7±3.03	32.9±3.18
2	Sample 1 juice	800	3	Sample 1 juice	1600	22.6±3.17	26.9±2.06	30.9±2.54	30.9±1.22	26.1±0.98	29.3±1.49	30.7±1.15	30.9±0.60	33.3±1.69	32.7±0.80	40.1±13.40	34.1±1.36	33.7±1.69	33.8±1.61
4	Sample 1 juice	3200	3	Sample 1 juice	1600	22.6±3.17	26.9±2.06	30.9±2.54	30.9±1.22	20.1±1.39	19.0±1.88	22.8±1.77	28.0±3.14	31.4±2.82	32.3±2.91	33.0±3.01	34.1±1.36	33.7±1.69	33.8±1.61
4	Sample 1 juice	3200	5	Sample 1 juice	6400	18.2±4.18	14.8±1.75	18.40.97	22.4±3.01	20.1±1.39	19.0±1.88	22.8±1.77	28.0±3.14	31.4±2.82	32.3±2.91	33.0±3.01	26.9±2.81	31.0±2.31	32.0±2.34
6	Sample 1 juice	10000	5	Sample 1 juice	6400	18.2±4.18	14.8±1.75	18.40.97	22.4±3.01	16.1±2.98	12.4±2.61	16.0±3.15	19.7±4.31	22.6±5.70	30.1±4.79	32.6±5.90	26.9±2.81	31.0±2.31	32.0±2.34
6	Sample 1 juice	10000	7	Sample 2 spray dried juice	38	25.6±2.85	27.3±0.95	30.3±2.06	31.0±2.13	16.1±2.98	12.4±2.61	16.0±3.15	19.7±4.31	22.6±5.70	30.1±4.79	32.6±5.90	31.8±1.63	31.1±1.94	31.8±2.45
8	Sample 2 spray dried juice	76	7	Sample 2 spray dried juice	38	25.6±2.85	27.3±0.95	30.3±2.06	31.0±2.13	24.2±3.25	25.2±3.24	29.9±1.05	30.2±2.28	31.2±2.26	32.3±1.44	33.1±0.61	31.8±1.63	31.1±1.94	31.8±2.45
8	Sample 2 spray dried juice	76	9	Sample 3 active ingredient	2.5	26.8±3.33	29.1±3.43	31.7±3.00	34.0±2.95	24.2±3.25	25.2±3.24	29.9±1.05	30.2±2.28	31.2±2.26	32.3±1.44	33.1±0.61	34.4±4.32	33.1±4.11	34.8±3.71
10	Sample 3 active ingredient	5.0	9	Sample 3 active ingredient	2.5	26.8±3.33	29.1±3.43	31.7±3.00	34.0±2.95	22.1±2.19	21.0±3.07	27.6±5.26	30.5±3.33	33.0±3.16	32.4±3.25	33.0±3.84	34.4±4.32	33.1±4.11	34.8±3.71
10	Sample 3 active ingredient	5.0	11	Fenfluramine	7.5	22.4±3.19	31.9±0.84	32.7±2.50	33.0±2.55	22.1±2.19	21.0±3.07	27.6±5.26	30.5±3.33	33.0±3.16	32.4±3.25	33.0±3.84	30.4±0.23	32.7±1.90	32.4±1.60
12	Carrier (DMSO)	-	11	Fenfluramine	7.5	22.4±3.19	31.9±0.84	32.7±2.50	33.0±2.55	29.9±3.36	30.6±4.43	30.1±4.17	32.4±5.26	31.8±3.08	32.8±3.98	33.3±3.76	30.4±0.23	32.7±1.90	32.4±1.60

sd: standard deviation of

Groups 2-8 compared to vector group 1:^*p＜0.05，^**p₁＜0.01

groups 9-11 compared to vehicle group 12: p is less than 0.05 and p is less than 0.01

TABLE 2a

Effect of oral administration on Water consumption in rats (data before daily administration)

Group of	Oral administration	Dosage (mg/kg)	Average water consumption per group (g. + -. sd) between two days
								-6--5	-5--4	-4--3	-3--2	-2--1
			1	Carrier (Water)	-	40.9±4.61	34.8±4.15	-6--5	-5--4	-4--3	-3--2	-2--1	37.6±5.63	33.5±7.42	32.2±6.32
2	Sample 1 juice	800	1	Carrier (Water)	-	40.9±4.61	34.8±4.15	38.6±1.96	37.1±9.74	36.4±4.81	28.1±1.83	30.4±4.75	37.6±5.63	33.5±7.42	32.2±6.32
2	Sample 1 juice	800	3	Sample 1 juice	1600	43.4±10.53	35.9±3.84	38.6±1.96	37.1±9.74	36.4±4.81	28.1±1.83	30.4±4.75	38.4±4.56	31.1±4.47	36.5±5.39
4	Sample 1 juice	3200	3	Sample 1 juice	1600	43.4±10.53	35.9±3.84	40.1±5.58	33.3±3.01	37.3±4.46	31.3±3.48	31.7±3.18	38.4±4.56	31.1±4.47	36.5±5.39
4	Sample 1 juice	3200	5	Sample 1 juice	6400	43.8±8.57	36.3±9.02	40.1±5.58	33.3±3.01	37.3±4.46	31.3±3.48	31.7±3.18	35.4±8.18	34.0±8.62	35.1±5.72
6	Sample 1 juice	10000	5	Sample 1 juice	6400	43.8±8.57	36.3±9.02	37.4±5.34	32.7±3.35	33.2±4.86	29.0±5.11	32.2±3.27	35.4±8.18	34.0±8.62	35.1±5.72
6	Sample 1 juice	10000	7	Sample 2 spray dried juice	38	40.0±4.36	35.8±4.92	37.4±5.34	32.7±3.35	33.2±4.86	29.0±5.11	32.2±3.27	34.7±3.20	30.2±1.88	31.4±2.98
8	Sample 2 spray dried juice	76	7	Sample 2 spray dried juice	38	40.0±4.36	35.8±4.92	38.6±1.98	37.0±1.96	48.8±21.6	31.6±4.56	39.0±17.27	34.7±3.20	30.2±1.88	31.4±2.98
8	Sample 2 spray dried juice	76	9	Sample 3 active ingredient	2.5	42.0±6.70	37.0±5.05	38.6±1.98	37.0±1.96	48.8±21.6	31.6±4.56	39.0±17.27	34.1±3.16	28.0±2.58	31.6±3.12
10	Sample 3 active ingredient	5.0	9	Sample 3 active ingredient	2.5	42.0±6.70	37.0±5.05	40.9±4.48	34.2±3.00	32.7±1.26	28.2±1.65	33.1±4.82	34.1±3.16	28.0±2.58	31.6±3.12
10	Sample 3 active ingredient	5.0	11	Fenfluramine	7.5	47.0±5.3	35.5±7.49	40.9±4.48	34.2±3.00	32.7±1.26	28.2±1.65	33.1±4.82	34.7±3.73	30.9±2.12	31.6±2.80
12	Carrier (DMSO)	-	11	Fenfluramine	7.5	47.0±5.3	35.5±7.49	43.3±5.67	34.5±4.97	35.2±4.34	28.3±4.64	31.4±6.44	34.7±3.73	30.9±2.12	31.6±2.80

sd: standard deviation of

TABLE 2b

Oral administration	Dosage (mg/kg)	Average water consumption per group (g. + -. sd) between two days
									1-2	2-3	3-4	4-5	5-6	6-7	7-8
		Carrier (Water)	-	34.9±5.45	36.9±6.06	38.0±7.59	37.2±8.18	37.7±5.54	1-2	2-3	3-4	4-5	5-6	6-7	7-8	35.3±2.86	36.5±5.85
Sample 1 juice	800	Carrier (Water)	-	34.9±5.45	36.9±6.06	38.0±7.59	37.2±8.18	37.7±5.54	30.9±3.77	34.4±8.12	38.2±13.71	35.9±13.51	39.5±11.20	28.8±1.22	31.8±5.58	35.3±2.86	36.5±5.85
Sample 1 juice	800	Sample 1 juice	1600	29.2±1.66	31.7±5.35	41.3±11.21	34.6±4.10	48.1±12.27	30.9±3.77	34.4±8.12	38.2±13.71	35.9±13.51	39.5±11.20	28.8±1.22	31.8±5.58	37.8±7.28	36.9±9.28
Sample 1 juice	3200	Sample 1 juice	1600	29.2±1.66	31.7±5.35	41.3±11.21	34.6±4.10	48.1±12.27	35.9±5.88	26.2±2.66	30.5±2.44	34.1±4.80	45.8±18.54	51.0±35.21	42.6±13.88	37.8±7.28	36.9±9.28
Sample 1 juice	3200	Sample 1 juice	6400	33.4±12.04	27.4±8.13	32.6±10.67	35.4±10.78	45.2±8.72	35.9±5.88	26.2±2.66	30.5±2.44	34.1±4.80	45.8±18.54	51.0±35.21	42.6±13.88	36.2±6.72	35.9±9.58
Sample 1 juice	10000	Sample 1 juice	6400	33.4±12.04	27.4±8.13	32.6±10.67	35.4±10.78	45.2±8.72	31.7±12.74	28.5±8.85	32.4±8.87	36.6±6.50	40.7±11.51	38.0±6.66	37.5±6.21	36.2±6.72	35.9±9.58
Sample 1 juice	10000	Sample 2 spray dried juice	38	36.0±6.02	34.5±1.79	38.2±7.16	39.6±7.09	42.7±9.74	31.7±12.74	28.5±8.85	32.4±8.87	36.6±6.50	40.7±11.51	38.0±6.66	37.5±6.21	45.6±17.15	46.1±9.49
Sample 2 spray dried juice	76	Sample 2 spray dried juice	38	36.0±6.02	34.5±1.79	38.2±7.16	39.6±7.09	42.7±9.74	46.0±19.03	39.1±16.69	46.9±18.34	35.9±3.40	41.9±12.37	36.9±8.47	38.1±8.93	45.6±17.15	46.1±9.49
Sample 2 spray dried juice	76	Sample 3 active ingredient	2.5	32.2±4.01	36.1±12.42	38.3±11.71	41.5±16.60	34.7±7.57	46.0±19.03	39.1±16.69	46.9±18.34	35.9±3.40	41.9±12.37	36.9±8.47	38.1±8.93	33.0±4.20	35.3±8.70
Sample 3 active ingredient	5.0	Sample 3 active ingredient	2.5	32.2±4.01	36.1±12.42	38.3±11.71	41.5±16.60	34.7±7.57	33.9±2.40	31.5±8.12	35.1±3.82	37.7±5.99	39.5±7.78	37.4．±11.07	37.8±6.42	33.0±4.20	35.3±8.70
Sample 3 active ingredient	5.0	Fenfluramine	7.5	34.1±3.60	37.2±1.48	36.7±3.92	33.8±2.89	33.7±5.43	33.9±2.40	31.5±8.12	35.1±3.82	37.7±5.99	39.5±7.78	37.4．±11.07	37.8±6.42	32.1±1.93	33.6±2.50
Carrier (DMSO)	-	Fenfluramine	7.5	34.1±3.60	37.2±1.48	36.7±3.92	33.8±2.89	33.7±5.43	40.7±9.10	33.8±9.37	32.9±7.07	35.2±11.49	33.8±9.82	32.3±7.44	32.0±7.22	32.1±1.93	33.6±2.50

sd: standard deviation of

Groups 1-8 compared to vector group 1:^*p＜0.05

groups 9-11 compared to vehicle group 12 (no significance)

TABLE 3a

Effect of oral administration to rats on body weight (initial daily dose data)

Group of	Oral administration	Dosage (mg/kg)	Average body weight per group per day (g. + -. sd)
			Average body weight per group per day (g. + -. sd)					-5	-4	-3	-2	-1
			1	Carrier (Water)	-	130.9±5.56	150.7±5.37	-5	-4	-3	-2	-1	157.3±5.29	168.1±6.20	177.5±6.70
2	Sample 1 juice	800	1	Carrier (Water)	-	130.9±5.56	150.7±5.37	131.6±4.34	150.1±4.84	158.5±4.35	169.6±4.99	177.7±4.10	157.3±5.29	168.1±6.20	177.5±6.70
2	Sample 1 juice	800	3	Sample 1 juice	1600	130.1±4.3	148.6±6.59	131.6±4.34	150.1±4.84	158.5±4.35	169.6±4.99	177.7±4.10	156.7±6.38	167.5±6.04	176.6±6.37
4	Sample 1 juice	3200	3	Sample 1 juice	1600	130.1±4.3	148.6±6.59	130.8±6.19	147.7±7.56	154.4±8.06	165.2±8.43	175.8±9.10	156.7±6.38	167.5±6.04	176.6±6.37
4	Sample 1 juice	3200	5	Sample 1 juice	6400	132.6±7.01	151.3±7.23	130.8±6.19	147.7±7.56	154.4±8.06	165.2±8.43	175.8±9.10	158.4±8.50	169.0±8.79	178.1±7.75
6	Sample 1 juice	10000	5	Sample 1 juice	6400	132.6±7.01	151.3±7.23	132.3±6.75	151.8±9.08	157.3±9.37	167.1±10.41	175.4±10.90	158.4±8.50	169.0±8.79	178.1±7.75
6	Sample 1 juice	10000	7	Sample 2 spray dried juice	38	131.7±8.28	149.0±5.85	132.3±6.75	151.8±9.08	157.3±9.37	167.1±10.41	175.4±10.90	156.2±5.81	166.7±5.54	175.6±8.42
8	Sample 2 spray dried juice	76	7	Sample 2 spray dried juice	38	131.7±8.28	149.0±5.85	130.0±6.99	146.1±6.00	155.9±6.59	166.0±6.87	175.1±6.55	156.2±5.81	166.7±5.54	175.6±8.42
8	Sample 2 spray dried juice	76	9	Sample 3 active ingredient	2.5	132.6±7.63	148.9±8.51	130.0±6.99	146.1±6.00	155.9±6.59	166.0±6.87	175.1±6.55	157.3±8.91	169.8±8.96	179.4±8.71
10	Sample 3 active ingredient	5.0	9	Sample 3 active ingredient	2.5	132.6±7.63	148.9±8.51	133.5±6.45	150.5±9.55	158.8±8.48	171.0±7.72	179.0±9.20	157.3±8.91	169.8±8.96	179.4±8.71
10	Sample 3 active ingredient	5.0	11	Fenfluramine	7.5	133.2±9.21	152.7±9.09	133.5±6.45	150.5±9.55	158.8±8.48	171.0±7.72	179.0±9.20	160.0±9.82	170.0±9.15	182.8±10.21
12	Carrier (DMSO)	-	11	Fenfluramine	7.5	133.2±9.21	152.7±9.09	129.1±3.17	147.3±4.37	155.0±6.29	166.0±5.91	174.8±8.26	160.0±9.82	170.0±9.15	182.8±10.21

sd: standard deviation of

TABLE 3b

Effect of oral administration to rats on body weight (initial daily dose data)

Group of	Oral administration	Dosage (mg/kg)	Average body weight per group per day (g. + -. sd)
			Average body weight per group per day (g. + -. sd)								Predose (1)	2	3	4	5	6	7	8
			1	Carrier (Water)	-	185.4±7.77	192.6±7.16	202.0±10.17	211.2±7.98	220.2±10.36	Predose (1)	2	3	4	5	6	7	8	227.2±10.28	235.8±11.82	242.8±11.97
2	Sample 1 juice	800	1	Carrier (Water)	-	185.4±7.77	192.6±7.16	202.0±10.17	211.2±7.98	220.2±10.36	186.0±4.90	187.0±4.55	198.5±4.20	206.8±5.91	214.8±4.65	222.8±4.99	231.5±3.70	240.0±3.65	227.2±10.28	235.8±11.82	242.8±11.97
2	Sample 1 juice	800	3	Sample 1 juice	1600	185.0±6.67	186.0±8.28	193.2±6.42	204.0±6.40	212.4±5.81	186.0±4.90	187.0±4.55	198.5±4.20	206.8±5.91	214.8±4.65	222.8±4.99	231.5±3.70	240.0±3.65	223.0±6.33	232.6±7.70	240.4±6.66
4	Sample 1 juice	3200	3	Sample 1 juice	1600	185.0±6.67	186.0±8.28	193.2±6.42	204.0±6.40	212.4±5.81	181.8±9.18	184.6±8.88	186.2±8.67	189.8±9.99	199.2±9.34	210.6±10.21	219.0±11.29	228.4±12.18	223.0±6.33	232.6±7.70	240.4±6.66
4	Sample 1 juice	3200	5	Sample 1 juice	6400	186.8±7.96	185.8±6.39	183.8±6.87	185.2±9.18	191.2±7.89	181.8±9.18	184.6±8.88	186.2±8.67	189.8±9.99	199.2±9.34	210.6±10.21	219.0±11.29	228.4±12.18	201.0±6.89	213.0±6.98	222.0±7.94
6	Sample 1 juice	10000	5	Sample 1 juice	6400	186.8±7.96	185.8±6.39	183.8±6.87	185.2±9.18	191.2±7.89	182.8±12.22	181.4±14.06	179.8±15.85	180.6±13.85	185.6±11.28	192.2±10.99	203.4±11.68	212.4±11.35	201.0±6.89	213.0±6.98	222.0±7.94
6	Sample 1 juice	10000	7	Sample 2 spray dried juice	38	183.4±8.11	185.8±9.23	195.8±7.79	205.6±9.79	214.4±9.61	182.8±12.22	181.4±14.06	179.8±15.85	180.6±13.85	185.6±11.28	192.2±10.99	203.4±11.68	212.4±11.35	222.6±9.34	231.4±10.62	239.8±11.46
8	Sample 2 spray dried juice	76	7	Sample 2 spray dried juice	38	183.4±8.11	185.8±9.23	195.8±7.79	205.6±9.79	214.4±9.61	180.6±6.47	183.4±7.57	188.6±6.73	198.2±8.50	206.0±9.43	214.0±9.51	222.0±9.49	232.2±9.68	222.6±9.34	231.4±10.62	239.8±11.46
8	Sample 2 spray dried juice	76	9	Sample 3 active ingredient	2.5	188.2±9.42	191.2±11.15	200.0±11.25	209.6±12.28	219.6±12.95	180.6±6.47	183.4±7.57	188.6±6.73	198.2±8.50	206.0±9.43	214.0±9.51	222.0±9.49	232.2±9.68	229.4±13.69	238.4±14.50	247.0±14.35
10	Sample 3 active ingredient	5.0	9	Sample 3 active ingredient	2.5	188.2±9.42	191.2±11.15	200.0±11.25	209.6±12.28	219.6±12.95	186.4±10.02	192.0±9.93	192.4±9.84	201.0±11.27	209.4±12.70	219.8±11.86	228.2±12.28	236.0±13.95	229.4±13.69	238.4±14.50	247.0±14.35
10	Sample 3 active ingredient	5.0	11	Fenfluramine	7.5	190.3±9.71	190.3±10.97	197.7±7.37	207.7±7.23	217.7±10.69	186.4±10.02	192.0±9.93	192.4±9.84	201.0±11.27	209.4±12.70	219.8±11.86	228.2±12.28	236.0±13.95	224.3±10.12	234.3±12.70	243.3±9.24
12	Carrier (DMSO)	-	11	Fenfluramine	7.5	190.3±9.71	190.3±10.97	197.7±7.37	207.7±7.23	217.7±10.69	183.3±8.33	190.3±10.26	199.0±10.82	207.7±12.66	215.7±14.05	222.3±14.84	230.7±15.95	239.0±17.35	224.3±10.12	234.3±12.70	243.3±9.24

sd: standard deviation of

Groups 1-8 compared to vector group 1:^*p＜0.05

groups 9-11 compared to vehicle group 12 (no significance)

Histopathology report

Histopathological examination is limited to the liver. No changes were detected in samples 1 (liquid), 2 (spray dried sap), 3 (active ingredient), fenfluramine or DMSO controls as appropriate for administration.

Watch (A)

Summary of incidence of microscopic pathology

Sex: male Studies on Male animals	Group 10mg/kg 55	Group 2 800mg/kg44	Group 3 1600mg/kg55	4 groups 3200mg/kg55	5 groups 6400mg/kg55	6 groups 10000mg/kg55
Sex: male Studies on Male animals	Group 10mg/kg 55	Group 2 800mg/kg44	Group 3 1600mg/kg55	4 groups 3200mg/kg55	5 groups 6400mg/kg55	6 groups 10000mg/kg55	Liver examination did not detect abnormally parenchymal inflammatory cell foci (total) minimum hepatocyte fat size-lobe center (total) minimum bulbar hematopoiesis (total) minimum hepatocyte necrosis foci (total) lymphoid of the minimum portalMinimal eosinophilic hepatocyte cell proliferation (total) -foci (total) minimal hepatic fibrosis (total) minimal hepatic (ORO) pigmented plaques) liver test no abnormal hepatocyte fat-minimal lobular center (total) minimal hepatocyte fat-minimal periportal (total) liver cell was detected	5000002211331100523300	4011000000440000431100	5100000000440011522211	5200000000330000541100	5300000000220000532200	50331100000220000532200

Watch (A)

(continuation)

Sex: male Studies on Male animals	7 groups 38mg/kg55	76mg/kg55 in group 8	9 groups 2.5mg/kg55	10 groups 5mg/kg55	11 groups 7.5mg/kg33	12 groups 0mg/kg33
Sex: male Studies on Male animals	7 groups 38mg/kg55	76mg/kg55 in group 8	9 groups 2.5mg/kg55	10 groups 5mg/kg55	11 groups 7.5mg/kg33	12 groups 0mg/kg33	Liver test did not detect abnormal parenchymal inflammatory cell foci (total) minimal hepatocyte hypertrophy-lobular center (total) minimal hepatocellular necrotic foci (total) minimal portal lymphoproliferation of minimal portal white blood cells (total) minimal liver (ORO pigmented macules) abnormal hepatocyte adipo-lobular center (total) minimal liver (total) abnormal hepatocyte focal length	52000033005500	52000033005322	50001155115322	51000044005322	30000033003211	32110011003210

Example 45

Another biological experiment using the same test sample as described in example 44 is described below. Animals in this study received a strict diet, i.e., animals only fed between 12:00 and 3:00 pm daily. This is different from all other biological tests currently performed, so that food can be supplied to rats in pounds. Rats were acclimatized during the seven days (day-7 to day-1) and dosed by oral gavage at 9 am daily from day 0 to day 6. The recovery period ranged from day 7 to day 13. The dose groups are described in table 1 below. Note that the actual control group was labeled as group 09. Group 5 was a control group receiving a diet equivalent to group 4. The purpose of this group was to evaluate the effect of a strictly restricted diet on the rat liver.

Results

The results generated during the study indicated that the acclimatization period was too short. The rats eat mostly at night, and a sudden change to strict eating within 3 hours of the day results in little food intake daily. In most groups, the daily food intake values still increased at the end of the acclimatization period when dosing with the test items began. For this reason, the effect of the test material on the food intake values of the rats during the administration period becomes insignificant.

Table D1 and table D2 show the average body weight for different groups from day-7 to day-1. The effect of different doses of sap and spray dried sap is shown in the figure, where figure 5 relates to the change in body weight from day 0 to day 7and figure 6 relates to the change in body weight from day-7 to day 7. It is readily apparent that the reduction in body weight is dose-related, especially for higher doses.

Histopathological examination of the liver did not reveal any meaningful condition in the groups receiving the test item.

Food product

Food consumption was measured daily during acclimation and during the study. Food was served 3 hours daily, starting at 12 o' clock and starting at 3 p.m. For the rest of the time, animals were fasted. Animals in group 5 received a measured amount of food on day 1, corresponding to the average food consumption of group 4 on day 0. This controlled feeding profile for group 5 was performed on days 1-7 as determined by the average food consumption of the day prior to group 4.

Water (W)

The water is provided in a standard container. Water (Magalies Water Board Tap Water, suitable for human consumption) is available at will. After the food consumption was measured, the water consumption was measured once at the same time every day.

Adaptive to environment

Animals were acclimated for 7 days prior to study initiation, during which time food and water consumption was determined as described previously. During this period, body weight was measured daily.

Research design and procedure

TABLE 1

Design of research

Group of	Test of	Number of	Dosage form	Test items
Group of	Test of	Number of	Dosage form	Test items	01	6	001-006	100mg/kg	Frozen juice
02	6	007-012	400mg/kg	Frozen juice	01	6	001-006	100mg/kg	Frozen juice
02	6	007-012	400mg/kg	Frozen juice	03	6	013-018	1600mg/kg	Frozen juice
04	6	019-024	3200mg/kg	Frozen juice	03	6	013-018	1600mg/kg	Frozen juice
04	6	019-024	3200mg/kg	Frozen juice	05	6	025-030	Control	Elga Option4 purified water
06	6	031-036	2.2mg/kg	Spray-dried juice	05	6	025-030	Control	Elga Option4 purified water
06	6	031-036	2.2mg/kg	Spray-dried juice	07	6	037-042	8.8mg/kg	Spray-dried juice
08	6	043-048	35mg/kg	Spray-dried juice	07	6	037-042	8.8mg/kg	Spray-dried juice
08	6	043-048	35mg/kg	Spray-dried juice	09	6	049-054	Control	Elga Option4 purified water

Mode of administration

The test item was dosed daily using a gastric feeding tube. Animals were fasted for 18 hours (starting at 9 o' clock) before the first dose.

Duration of administration

Animals were dosed for 7 consecutive days (from day 0 to day 6). Three animals of each group were sacrificed 24 hours after the last dose (day 7). The remaining 3 animals were sacrificed 7 days after the last administration (day 13). This procedure was adapted to all groups except three animals in group 5 were sacrificed 24 hours after the last controlled meal (day 8) and the remaining three animals were sacrificed 7 days after the last dose (day 13).

Body weight

Body weight was measured at approximately the same time of day during the study, including the acclimation period.

Death by tranquilization

Three animals in each group were sacrificed 24 hours after the last dose (day 7).

The remaining three animals were sacrificed 7 days after the last dose. This procedure was adapted to all groups except three animals in group 5 were sacrificed 24 hours after the last controlled meal (day 8) and the remaining three animals were sacrificed 7 days after the last dose (day 13). Animals were euthanized at the end of the study with carbon dioxide gas.

Ophthalmoscopy method

All animals of all groups were subjected to ophthalmoscopy using an ophthalmoscope before the first dose of the test item and at the end of the test.

Macroscopic pathology

At the end of the study, each euthanized animal was necropsied by whole-body necropsy.

Histopathology

Histopathological experiments were performed on the livers of each animal.

TABLE D.1

Mean body weight/group/week

Group of	Oral administration	Dosage (mg/kg)	Mean body weight and standard deviation
			Mean body weight and standard deviation							Day 7	Day 6	Day 5	Day 4	Day 3	Day 2	Day 1
			01	Sample 1 (juice)	100	203.38±95.39	197.13±90.63	192.75±89.49	188.62±86.75	Day 7	Day 6	Day 5	Day 4	Day 3	Day 2	Day 1	184.95±84.80	182.48±83.47	182.25±82.57
02	Sample 1 (juice)	400	01	Sample 1 (juice)	100	203.38±95.39	197.13±90.63	192.75±89.49	188.62±86.75	192.53±65.60	183.92±61.20	178.25±59.37	173.17±58.10	170.82±57.42	168.25±58.40	169.37±59.25	184.95±84.80	182.48±83.47	182.25±82.57
02	Sample 1 (juice)	400	03	Sample 1 (juice)	1600	149.25±54.80	142.87±51.89	136.85±52.17	132.37±49.64	192.53±65.60	183.92±61.20	178.25±59.37	173.17±58.10	170.82±57.42	168.25±58.40	169.37±59.25	131.50±49.50	129.67±48.89	131.12±48.22
04	Sample 1 (juice)	3200	03	Sample 1 (juice)	1600	149.25±54.80	142.87±51.89	136.85±52.17	132.37±49.64	224.15±80.70	214.45±77.25	207.10±76.38	201.82±75.42	198.25±74.82	194.83±75.34	196.77±74.56	131.50±49.50	129.67±48.89	131.12±48.22
04	Sample 1 (juice)	3200	05	Elga Option4 purified water (control)	-	214.55±74.90	204.85±72.41	198.57±71.79	193.48±68.49	224.15±80.70	214.45±77.25	207.10±76.38	201.82±75.42	198.25±74.82	194.83±75.34	196.77±74.56	192.40±67.48	190.87±67.39	190.15±65.24
06	Sample 2 (spray dried juice)	2.2	05	Elga Option4 purified water (control)	-	214.55±74.90	204.85±72.41	198.57±71.79	193.48±68.49	208.65±65.74	199.37±62.49	193.18±61.18	188.25±60.89	186.22±59.98	184.55±58.86	185.97±58.76	192.40±67.48	190.87±67.39	190.15±65.24
06	Sample 2 (spray dried juice)	2.2	07	Sample 2 (spray dried juice)	8.8	256.95±77.55	246.02±73.67	237.47±73.53	232.62±71.73	208.65±65.74	199.37±62.49	193.18±61.18	188.25±60.89	186.22±59.98	184.55±58.86	185.97±58.76	229.78±71.76	228.07±69.88	228.45±68.81
08	Sample 2 (spray dried juice)	35	07	Sample 2 (spray dried juice)	8.8	256.95±77.55	246.02±73.67	237.47±73.53	232.62±71.73	194.37±43.74	185.83±42.70	177.53±41.10	172.05±40.13	170.10±39.49	167.25±37.61	168.00±38.83	229.78±71.76	228.07±69.88	228.45±68.81
08	Sample 2 (spray dried juice)	35	09	Elga Option4 purified water (control)	-	171.52±69.81	162.67±62.68	154.95±61.83	151.38±59.48	194.37±43.74	185.83±42.70	177.53±41.10	172.05±40.13	170.10±39.49	167.25±37.61	168.00±38.83	149.63±57.66	148.30±57.12	149.07±56.01

TABLE D.2

Average body weight/group/week (continue)

Group of	Oral administration	Dosage (mg/kg)	Mean body weight and standard deviation
			Mean body weight and standard deviation							Day 0	Day 1	Day 2	Day 3	Day 4	Day 5	Day 6
			01	Sample 1 (juice)	100	183.87±83.33	175.83±81.82	175.72±79.05	175.48±77.54	Day 0	Day 1	Day 2	Day 3	Day 4	Day 5	Day 6	175.53±76.20	177.95±73.99	178.43±72.68
02	Sample 1 (juice)	400	01	Sample 1 (juice)	100	183.87±83.33	175.83±81.82	175.72±79.05	175.48±77.54	173.45±60.73	184.58±58.52	164.75±58.37	166.22±57.69	166.55±57.79	169.93±57.47	171.77±57.29	175.53±76.20	177.95±73.99	178.43±72.68
02	Sample 1 (juice)	400	03	Sample (juice)	1600	134.38±46.01	129.20±44.74	127.53±43.20	127.20±41.36	173.45±60.73	184.58±58.52	164.75±58.37	166.22±57.69	166.55±57.79	169.93±57.47	171.77±57.29	126.70±39.19	128.00±39.22	128.07±38.66
04	Sample (juice)	3200	03	Sample (juice)	1600	134.38±46.01	129.20±44.74	127.53±43.20	127.20±41.36	199.60±75.16	196.38±73.96	192.20±71.20	189.05±69.11	186.57±66.29	186.05±67.45	185.68±65.73	126.70±39.19	128.00±39.22	128.07±38.66
04	Sample (juice)	3200	05	Elga Option4 purified water (control)	-	194.27±67.46	187.93±65.48	181.97±65.01	177.53±64.73	199.60±75.16	196.38±73.96	192.20±71.20	189.05±69.11	186.57±66.29	186.05±67.45	185.68±65.73	174.73±61.08	172.85±58.63	171.45±56.79
06	Sample 2 (spray dried juice)	2.2	05	Elga Option4 purified water (control)	-	194.27±67.46	187.93±65.48	181.97±65.01	177.53±64.73	189.07±60.15	181.52±58.99	181.48±57.79	184.42±55.64	185.75±55.29	189.35±54.66	189.68±53.70	174.73±61.08	172.85±58.63	171.45±56.79
06	Sample 2 (spray dried juice)	2.2	07	Sample 2 (spray dried juice)	8.8	230.28±69.32	221.55±68.02	220.17±66.63	221.80±63.88	189.07±60.15	181.52±58.99	181.48±57.79	184.42±55.64	185.75±55.29	189.35±54.66	189.68±53.70	222.82±63.56	224.82±62.38	224.90±62.05
08	Sample 2 (spray dried juice)	35	07	Sample 2 (spray dried juice)	8.8	230.28±69.32	221.55±68.02	220.17±66.63	221.80±63.88	169.10±38.40	164.42±38.03	162.50±36.81	162.75±36.36	162.52±36.93	164.30±37.69	164.22±37.18	222.82±63.56	224.82±62.38	224.90±62.05
08	Sample 2 (spray dried juice)	35	09	Elga Option4 purified water (control)	-	151.02±55.45	146.55±53.77	148.10±52.67	149.70±52.05	169.10±38.40	164.42±38.03	162.50±36.81	162.75±36.36	162.52±36.93	164.30±37.69	164.22±37.18	152.58±50.37	155.82±49.91	157.85±49.70

TABLE D.3

Mean body weight/group/week

Group of	Oral administration	Dosage (mg/kg)	Mean body weight and standard deviation
			Mean body weight and standard deviation							Day 7	Day 8	Day 9	Day 10	Day 11	Day 12	Day 13
			01	Sample 1 (juice) (GHA l 35A)	100	185.38±72.64	234.73±62.44	236.73±62.39	234.07±82.09	Day 7	Day 8	Day 9	Day 10	Day 11	Day 12	Day 13	236.33±62.31	239.07±60.24	238.43±59.85
02	Sample 1 (juice) (GHA l 35A)	400	01	Sample 1 (juice) (GHA l 35A)	100	185.38±72.64	234.73±62.44	236.73±62.39	234.07±82.09	178.83±58.24	225.63±13.05	277.13±14.18	227.10±14.03	229.43±16.97	234.93±18.35	236.20±15.97	236.33±62.31	239.07±60.24	238.43±59.85
02	Sample 1 (juice) (GHA l 35A)	400	03	Sample 1 (juice) (GHA l 35A)	1600	132.22±37.08	133.80±55.17	135.23±455.74	134.53±54.96	178.83±58.24	225.63±13.05	277.13±14.18	227.10±14.03	229.43±16.97	234.93±18.35	236.20±15.97	138.30±53.03	139.30±51.10	142.80±49.51
04	Sample 1 (juice) (GHA 935A)	3200	03	Sample 1 (juice) (GHA l 35A)	1600	132.22±37.08	133.80±55.17	135.23±455.74	134.53±54.96	188.57±66.14	199.63±61.07	198.90±57.48	198.70±54.55	194.73±52.78	194.93±50.78	197.93±51.57	138.30±53.03	139.30±51.10	142.80±49.51
04	Sample 1 (juice) (GHA 935A)	3200	05	Elga Option4 purified water (control)	-	173.97±54.29	172.98±52.06	157.80±58.62	158.87±57.76	188.57±66.14	199.63±61.07	198.90±57.48	198.70±54.55	194.73±52.78	194.93±50.78	197.93±51.57	160.80±57.67	163.40±56.27	167.80±58.49
06	Sample 2 (spray dried juice) (GHA l 59)	2.2	05	Elga Option4 purified water (control)	-	173.97±54.29	172.98±52.06	157.80±58.62	158.87±57.76	196.00±53.09	190.27±27.78	190.27±29.54	192.60±29.09	194.73±29.68	196.97±29.04	198.60±30.18	160.80±57.67	163.40±56.27	167.80±58.49
06	Sample 2 (spray dried juice) (GHA l 59)	2.2	07	Sample 2 (spray dried juice) (GHA l 59)	8.8	231.30±61.91	177.27±24.48	178.17±23.79	180.67±25.04	196.00±53.09	190.27±27.78	190.27±29.54	192.60±29.09	194.73±29.68	196.97±29.04	198.60±30.18	182.03±25.31	185.10±24.60	189.73±23.58
08	Spray dried juice (GHA l 59)	35	07	Sample 2 (spray dried juice) (GHA l 59)	8.8	231.30±61.91	177.27±24.48	178.17±23.79	180.67±25.04	167.48±36.75	164.90±22.54	166.63±23.08	168.43±22.66	171.67±24.42	174.90±25.70	178.57±23.58	182.03±25.31	185.10±24.60	189.73±23.58
08	Spray dried juice (GHA l 59)	35	09	Elga Option4 purified water (control)	-	165.50±49.27	193.73±22.37	196.87±21.86	198.07±21.02	167.48±36.75	164.90±22.54	166.63±23.08	168.43±22.66	171.67±24.42	174.90±25.70	178.57±23.58	199.83±20.21	204.93±18.65	207.13±18.22

Table 1: histological evaluation of liver sections from male rats

Sample 1

Group 1: 100mg/kg sample 1			Group 2: 400mg/kg sample 1
Group 1: 100mg/kg sample 1			Group 2: 400mg/kg sample 1				Animal number	Liver damage		Animal number	Liver damage
Day 7	01	NPL		07	FHS1+		Animal number	Liver damage		Animal number	Liver damage
Day 7	01	NPL		07	FHS1+		02	NPL		08	NPL C1+
	03	NPL C1+		09	NPL		02	NPL		08	NPL C1+
	03	NPL C1+		09	NPL	Day 13	04	NPL MLC	Day 7	10	DHS1+
	05	FHS1+		11	NPL	Day 13	04	NPL MLC	Day 7	10	DHS1+
	05	FHS1+		11	NPL		06	NPL		12	DHS1+

Group 3: 1600mg/kg sample 1			Group 4: 3200kg/kg sample 1
Group 3: 1600mg/kg sample 1			Group 4: 3200kg/kg sample 1			Animal number	Liver damage			Animal number	Liver damage
Day 7	13	NPL		19	NPL	Animal number	Liver damage			Animal number	Liver damage
Day 7	13	NPL		19	NPL		14	NPL		20	NPL
	15	NPL		21	NPL		14	NPL		20	NPL
	15	NPL		21	NPL	Day 13	16	NPL	Day 13	22	DHS1+
	17	DHS1+		23	FHS1+	Day 13	16	NPL	Day 13	22	DHS1+
	17	DHS1+		23	FHS1+		18	NPL		24	NPL

Group 5: control group: ELGA OPTION purified Water: strict food intake

Description of the drawings:

table 2: histological evaluation of liver sections from male rats

Sample 2

Group 6: 2.2mg/kg sample 2			Group 7: 8.8mg/kg sample 2
Group 6: 2.2mg/kg sample 2			Group 7: 8.8mg/kg sample 2				Animal number	Liver damage		Animal number	Liver damage
Day 7	31	NPL		37	NPL		Animal number	Liver damage		Animal number	Liver damage
Day 7	31	NPL		37	NPL		32	NPL MLC		38	NPL
	33	FHS1+		39	NPL C1+		32	NPL MLC		38	NPL
	33	FHS1+		39	NPL C1+	Day 13	34	NPL	Day 13	40	DHS1+
	35	DHS1+		41	NPL	Day 13	34	NPL	Day 13	40	DHS1+
	35	DHS1+		41	NPL		36	NPL		42	MLC FHS1+

And 8: 35mg/kg sample 2
And 8: 35mg/kg sample 2				Animal number	Liver damage
Day 7	43	NPL		Animal number	Liver damage
Day 7	43	NPL		44	NPL
	45	NPL		44	NPL
	45	NPL	Day 13	46	NPL
	47	NPL C1+	Day 13	46	NPL
	47	NPL C1+		48	MLC FHS1+

Group 9: control group: ELGA OPTION4 purified Water: strict food intake

Description of the drawings:

no particular damage was noted in liver sections of experimental rats receiving frozen sap and spray-dried sap, which was administered orally. Edema cell swelling recorded in both control and experimental rats indicates swelling of normal metabolic cells and changes in hypoxia. Very few perivascular sets of lymphocytes were found in some rats, most likely an occasional observation. In several rats, there was a slight degree of congestion in the sinusoids of the liver, which should be seen as a haphazard observed phenomenon.

An important feature of the present invention, as demonstrated by the results of this study, is that it does not exhibit tolerance to either sample throughout the duration of the test. This may provide important functions, particularly in the treatment of obesity, using the compounds and compositions of the present invention.

The compounds and compositions of the present invention are described primarily as appetite suppressants, it being noted that the description of "appetite suppressants" is used herein to suggest that appetite is restricted and/or satiety is increased, and therefore, total caloric intake is reduced; thus, obesity can be offset. Accordingly, the invention extends to a method of treating, preventing or combating obesity in a human or non-human animal which comprises administering to said human or non-human animal a compound of formula (2) in an amount effective to treat, prevent or combat obesity. A preferred embodiment of this aspect of the invention utilizes a composition or extract containing a compound of formula (1).

The term "animal" in the present invention extends to, but is not limited to, companion animals such as domestic pets and domesticated animals; non-limiting examples of such animals include: cattle, sheep, ferrets, pigs, camels, horses, poultry, fish, rabbits, goats, dogs, and cats.

As an anorectic agent for use in the treatment or prevention of obesity in humans, the compound of formula (2) is preferably a compound of formula (1), or a composition as defined in any of claims 9 and 25 to 31, which compounds or compositions are preferably administered in a dose of from about 0.01 mg/kg/day to about 10 mg/kg/day. A preferred dosage range is from 0.05 mg/kg/day to about 0.5 mg/kg/day. When spray-dried powders from the extracts of the present invention are used, the preferred dosage range is from 0.1 mg/kg/day to about 20 mg/kg/day. A preferred dosage range is from 0.5 mg/kg/day to about 5 mg/kg/day.

Claims

A process for preparing an extract of a plant of the genus Trichocaulon or geotrichum, said extract comprising an appetite-suppressing effective amount of a steroidal glycoside appetite suppressant having the following formula I:

the method comprises the following steps: treating the plant material with a solvent to extract the components having appetite suppressant activity, separating the extract solution from the remaining plant material, removing the solvent from the extract solution, and recovering the extract.
2. The method according to claim 1, wherein the genus Trichocaulon is selected from the group consisting of Trichocaulon piliferum and Trichocaulon offibile, and the genus Hoodia is selected from the group consisting of Pyroluri, Pyrolunia and Pyroludriii.
3. A process according to claim 2 wherein the plant is selected from the group consisting of fireland subcurrorii, fireland sub gordonii and fireland sub lugardii.
4. A process according to any one of claims 1 to 3, comprising the further step of extracting with a solvent to concentrate the active agent in the extracted material.
5. A process according to any one of claims 1 to 3, wherein the solvent employed in the solvent extraction step is one or more of dichloromethane, water, methanol, hexane, ethyl acetate or mixtures thereof.
6. A process according to any one of claims 1 to 3, including the step of concentrating the active agent in the extracted material by chromatographic separation.
7. The process according to claim 6, wherein the chromatographic separation method uses one or more of chloroform, methanol, ethyl acetate, hexane as eluent.
8. The method of claim 7, comprising: performing chromatographic separation on the chromatographic column, collecting the eluate from the fractions from the chromatographic column, evaluating the fractions to determine their appetite suppressant activity, and selecting at least one fraction containing appetite suppressant.
9. A process according to any one of claims 1 to 3, wherein the extract is treated to form a free-flowing powder.
10. An appetite suppressant-containing extract produced by the method of any one of claims 1-3.
11. A composition having appetite suppressant activity comprising the extract of claim 10.
12. The composition according to claim 11, wherein the composition is a food or beverage.
13. A composition according to claim 11 which comprises a pharmaceutically acceptable excipient, diluent or carrier.
14. A composition according to claim 13, which is prepared in unit dosage form.
15. Use of the extract of claim 10 in the manufacture of a medicament having appetite suppressant activity.
16. Use of the extract of claim 10 in the manufacture of a medicament for the treatment, prevention or management of obesity.
17. A non-therapeutic method of suppressing appetite or reducing total caloric intake comprising administering to a human or animal an effective dose of the composition of any one of claims 11-14.
18. Use of a composition according to any one of claims 14 in the manufacture of a medicament having appetite suppressant activity or for use in the treatment, prevention or combating obesity.
A process for preparing an extract of a plant of the genus Trichocaulon or geotrichum, said extract comprising an appetite-suppressing effective amount of a steroidal glycoside appetite suppressant having the following formula I:

the method comprises the following steps: squeezing the plant material to separate the juice from the solid plant material, and recovering the juice that does not contain the solid plant material to form the extract.
20. The method of claim 19, wherein the extract is dried to form a free-flowing powder.
21. An appetite suppressant-containing extract produced by the method of claim 19 or 20.
22. A composition having appetite suppressant activity comprising the extract of claim 21.
23. The composition of claim 22, wherein the composition is a food or beverage.
24. A composition according to claim 22 which comprises a pharmaceutically acceptable excipient, diluent or carrier.
25. A composition according to claim 22 or 24, which is prepared in unit dosage form.
26. Use of the extract of claim 21 in the manufacture of a medicament having appetite suppressant activity.
27. Use of an extract according to claim 21 in the manufacture of a medicament for the treatment, prevention or combating of obesity.
28. A non-therapeutic method of suppressing appetite or reducing total caloric intake comprising administering to a human or animal an effective dose of the composition of any one of claims 22-25.
29. Use of a composition as claimed in any one of claims 22 to 25 in the manufacture of a medicament having appetite suppressant activity or for use in the treatment, prevention or management of obesity.
30. An extract obtainable from a plant of the genus Trichocaulon or geotrichum, comprising an appetite suppressant having the formula:
31. an extract according to claim 30, wherein said extract comprises an appetite suppressant having the formula I as defined in claim 1 in an inhibitory effective amount.
32. An extract according to claim 30 or 31, wherein the plant of the genus Trichocaulon is selected from Trichocaulon piliferum and Trichocaulon officinalis, and the plant of the genus Hoodia is selected from Hoodia curcorii, Hoodia gordonii and Hoodia lugardii.
33. An extract according to claim 32, wherein the plant is selected from the group consisting of fireland subcurrorii, fireland sub gordonii and fireland sub lugardii.
34. The extract according to claim 32 or 33, wherein substantially all inactive impurities are removed.
35. An extract according to claim 30, which is processed into a free-flowing powder.
36. A composition having appetite suppressant activity comprising the extract of any one of claims 30-35.
37. The composition of claim 36, wherein the composition is a food or beverage.
38. A composition according to claim 36 which comprises a pharmaceutically acceptable excipient, diluent or carrier.
39. A composition according to claim 36 or 38, which is prepared in unit dosage form.
40. Use of an extract according to any one of claims 30 to 35 in the manufacture of a medicament having appetite suppressant activity.
41. Use of an extract according to any one of claims 30 to 35 in the manufacture of a medicament for the treatment, prevention or management of obesity.
42. A non-therapeutic method of suppressing appetite or reducing total caloric intake comprising administering to a human or animal an effective dose of the composition of any one of claims 36-39.
43. Use of a composition as claimed in any one of claims 36 to 39 in the manufacture of a medicament having appetite suppressant activity or for use in the treatment, prevention or management of obesity.
44. A compound having the formula

Wherein R is alkyl;

R₁is hydrogen, alkyl or an organic ester group;

R₂is hydrogen, or one or more 6-deoxy sugars, or one or more 2, 6-dideoxy sugars, or a glucose molecule, or a combination thereof;

wherein the dotted line represents the optional presence of a further bond between C4-C5 or C5-C6, excluding the following compounds:

(a)R＝CH₃，R₁h or benzoyl, R₂H and a double bond between C5-C6, and

(b)R＝C_1-4alkyl radical, R₁＝H，R₂H and the bond between C5-C6 is saturated.
45. The compound of claim 44, wherein there is a double bond between C5 and C6, R is methyl, R is₁Is methylcrotonyl, R₂Is 3-O- [ -beta-D-pyrane oleander glycosyl- (1 → 4) -beta-D-pyrane-glyco-l]The compound has the following structural formula
46. A compound having the formula

Wherein R is alkyl; and is

R₁Hydrogen, alkyl or organic ester groups.
47. A compound having the formula

Wherein R is alkyl; and is

R₁Hydrogen, alkyl or organic ester groups.
48. A compound having the formula

Wherein R is alkyl; and is

R₁Hydrogen, alkyl or organic ester groups.
49. A compound having the formula

Wherein R is alkyl; and is

R₁Is hydrogen, an alkyl group or an organic ester group, and the dotted line represents the optional presence of another bond between C4-C5 or C5-C6.
50. A compound having the formula

Wherein R is alkyl; and is

R₁Is hydrogen, an alkyl group or an organic ester group, and the dotted line represents the optional presence of a further bond between C4-C5 or C5-C6,

but excluding the following compounds:

(a)R＝CH₃，R₁h or benzoyl and a double bond between C5-C6, and

(b)R＝C_1-4alkyl radical, R₁H and the bonds between C5-C6 are saturated.
51. A compound having the formula

Wherein R is alkyl; and is

R₁Is hydrogen, alkyl or an organic ester group;

R₂is hydrogen, or one or more 6-deoxy sugars, or one or more 2, 6-dideoxy sugars, or a glucose molecule, or a combination thereof;

wherein the dotted line represents the optional presence of another bond between C4-C5 or C5-C6, excluding the following compounds:

R＝CH₃，R₁h or benzoyl, a double bond between C5-C6, and R₂＝H。
52. A compound having the following structural formula

Wherein R is alkyl; and is

R₁Is hydrogen, alkyl or an organic ester group;

R₂is hydrogen, or one or more 6-deoxy sugars, or one or more 2, 6-dideoxy sugars, or a glucose molecule, or a combination thereof;

wherein the dotted line represents the optional presence of a further bond between C4-C5 or C5-C6, excluding the following compounds:

(a)R＝CH₃，R₁＝H，R₂glycopyranoluteotoxin or H; and double bonds are between C5 and C6;

(b)R＝CH₃，R₁h, benzoyl, methylcrotonyl or angeloyl, R₂H and a double bond between C5-C6;

(c)R＝CH₃，R₁h or benzoyl, R₂H, and the bonds between C5-C6 are saturated;

(d)R＝CH₃，R₁(ii) benzoyl, R₂6-deoxy-3-O-methyl- β -D-pyranoallosyl- (1 → 4) - β -D-pyranosylactopyranosyl; and

(e)R＝CH₃，R₁(ii) benzoyl, R₂6-deoxy-3-O-methyl- β -D-arabinopyranosyl- (1 → 4) - β -D-oleanoline pyranosyl- (1 → 4) - β -D-magnetopyranosyl.
53. A compound having the formula

Wherein R is alkyl; and is

R₁Is hydrogen, alkyl or an organic ester group;

R₂is hydrogen, one or more of 6-deoxysugar or one of 2, 6-dideoxysugar

One or more, or a glucose molecule, or a combination thereof;

wherein the dotted line represents the optional presence of another bond between C4-C5 or C5-C6,

but excluding the following compounds:

(a)R＝CH₃，R₁＝H，R₂glycopyranoluteotoxin or H; and double bonds are between C5 and C6;

(b)R＝CH₃，R₁h, benzoyl, methylcrotonyl or angeloyl, R₂H and a double bond between C5-C6;

(c)R＝CH₃，R₁h or benzoyl, R₂H, and the bonds between C5-C6 are saturated;

(d)R＝CH₃，R₁(ii) benzoyl, R₂6-deoxy-3-O-methyl- β -D-pyranoallosyl- (1 → 4) - β -D-pyranosylactopyranosyl(ii) a And

(e)R＝CH₃，R₁(ii) benzoyl, R₂6-deoxy-3-O-methyl- β -D-arabinopyranosyl- (1 → 4) - β -D-oleanoline pyranosyl- (1 → 4) - β -D-magnetopyranosyl.
54. A compound having the formula

Wherein R is alkyl; and is

R₁Is hydrogen, alkyl or an organic ester group;

R₂is hydrogen, or one or more 6-deoxy sugars, or one or more 2, 6-dideoxy sugars, or a glucose molecule, or a combination thereof;

wherein the dotted line represents the optional presence of another bond between C4-C5, C5-C6 or C14-C15.
55. A compound having the formula

Wherein R is alkyl; and is

R₁Is hydrogen, alkyl or an organic ester group;

R₂is hydrogen, or one or more 6-deoxy sugars, or one or more 2, 6-dideoxy sugars, or a glucose molecule, or a combination thereof;

wherein the dotted line represents the optional presence of another bond between C4-C5, C5-C6 or C14-C15,

but excluding the following compounds:

R＝CH₃，R₁＝H，R₂h and the bond between C5-C6 is saturated.
56. A compound having the formula

Wherein R is alkyl; and is

R₁Is hydrogen, alkyl or an organic ester group;

R₂is hydrogen, or one or more 6-deoxy sugars, or one or more 2, 6-dideoxy sugars, or a glucose molecule, or a combination thereof;

wherein the dotted line represents the optional presence of another bond between C4-C5, C5-C6 or C14-C15; and R is₃Is hydrogen, alkyl, aryl, acyl or glucoxy (glucoxy).
57. The compound of any one of claims 44, 46-56, wherein R₁Is methyl crotonyl or benzoyl.
58. A process for the preparation of steroidal intermediates of the formula

The method comprises the following steps:

(a) treating the following compounds with a reducing agent:

to produce the compound 3 beta, 12 beta-dihydroxy-20, 20-ethylenedioxypregna-5, 14-diene of the formula

(b) Treatment of compound (23) with N-bromoacetamide (NBA) and a base produces the compound 3 β,12 β -dihydroxy-14, 15-epoxy-20, 20-ethylenedioxypregn-5-ene of the formula

(c) Treating compound (24) with a reducing agent to produce the compound 3 beta, 12 beta, 14 beta-trihydroxy-20, 20-ethylenedioxypregn-5-ene of the formula

And is

(d) Treating compound (25) with an acid and water to produce compound (15).
59. A process for preparing compound (15), comprising the steps of:

(a) treating a compound (22) as shown in claim 58 with p-toluenesulfonyl chloride and a base to give the compound 3 β,12 β -dihydroxy-20, 20-ethylenedioxypregna-5, 14-diene-3-tosyl-12-acetate of the formula

(b) Treating compound (26) with potassium acetate in a solvent to give the compound 6 beta, 12 beta-dihydroxy-20, 20-ethylenedioxy-3, 5 alpha-cyclopenta-14-ene-12-acetate of the formula

(c) Treating compound (27) with a reducing agent to produce the compound 6 beta, 12 beta-dihydroxy-20, 20-ethylenedioxy-3, 5 alpha-cyclopregn-14-ene of the formula

(d) Treating compound (28) with N-bromoacetamide and a base to produce a compound of the formula 6 beta, 12 beta-dihydroxy-14, 15-epoxy-3, 5 alpha-cyclopregnane

(e) Treating compound (29) with a reducing agent to produce the compound 6 beta, 12 beta, 14 beta-trihydroxy-20, 20-ethylenedioxy-3, 5 alpha-cyclopregna

(f) Treating compound (30) with an acid and a solvent to produce compound (15) as set forth in claim 58.
60. A process for the preparation of a saccharide intermediate in the form of a monosaccharide marshmallow, the process comprising the steps of:

(i) using PhSSiMe₃And trimethylsilyl fluoromethanesulfonate treatment of a compound of the formula

Wherein Ph ═ phenyl

To yield the compound 4-O-benzoyl-3-O-methyl-6-deoxy-alpha-D-phenylthioaltoreq glucoside of the formula

Wherein Ph ═ phenyl

(ii) Optionally, treating compound (37) with diethylaminosulfur trifluoride (DAST) to give the compound 4-O-benzoyl-3-O-methyl-2-thiophenyl-2, 6-dideoxy- α β -D-fluoropyraclostrobin of the formula

Wherein Ph ═ phenyl

Or

(iii) Optionally, treating compound (37) with t-butyldimethylsilyl chloride and imidazole in a solvent to produce the compound 4-O-benzoyl-3-O-methyl-2-O-t-butyldimethylsilyl- α β -D-phenylthioaaltrose glycoside of the formula

Wherein Ph ═ phenyl, Z ═ TBDMS ═ tert-butyldimethylsilyl;

(iv) treating compound (39) with a base to produce the compound monosaccharide 3-O-methyl-2-O-t-butyldimethylsilyl- α β -D-phenylthioaltoreq glycoside of the formula

Wherein Ph ═ phenyl, Z ═ TBDMS ═ tert-butyldimethylsilyl.
61. A process for the preparation of an activated yellow oleander sugar form of a sugar intermediate, the process comprising the steps of:

(i) treatment of a compound of the formula with phenylthiotrimethylsilane and trimethylsilyl triflate

Ph ═ phenyl

To produce the compound 4-O-benzoyl-3-O-methyl-1-thiophenyl-6-deoxy-alpha-beta-glucopyranoside of the formula

Wherein Ph ═ phenyl

(ii) Treating compound (48) with pivaloyl chloride and a solvent to produce the compound 4-O-benzoyl-3-O-methyl-2-O-pivaloyl-1-thiophenyl-6-deoxy-alpha-beta-glucopyranoside of the formula

Wherein Ph is phenyl and Pv is pivaloyl, and

(iii) treating compound (49) with a brominating agent and diethylaminosulfur trifluoride to give compound 4-O-benzoyl-3-O-methyl-2-O-pivaloyl-1-fluoro-6-deoxy-beta-glucopyranoside of the formula

Wherein Ph is phenyl and Pv is pivaloyl.
62. A saccharide intermediate of formula (40) produced by the process of claim 60.
63. A saccharide intermediate of formula (50A) or formula (50B) produced by the process of claim 61.
64. A method of coupling a monosaccharide, maginose, to a steroidal intermediate, the method comprising the steps of:

(i) reacting the magnetoephedrine of formula (38) with the steroidal intermediate of formula (15) as defined in claim 58 in a solvent in the presence of tin chloride to give the compound 3-O- [ 4-O-benzoyl-2-phenylthio- β -D-glucopyranosyl ] -12, 14- β -dihydroxypregn-5-en-20-one of the formula

Wherein Ph is phenyl and Pv is pivaloyl, and

(ii) treatment of compound (51) with methylcrotonyl chloride in pyridine and subsequent treatment with a base, yields the compound 3-O- [ 2-phenylthio- β -D-pyranosylyl ] -12 β -methylcrotonyloxy-14-hydroxy-14 β -pregn-5-en-20-one of the formula

Wherein Ph is phenyl.
65. A compound of formula (52) produced by the process of claim 64.
66. A method for coupling the monosaccharide marshmallow to the monosaccharide oleander sugar and coupling the disaccharide formed to the compound of formula (52) according to claim 64, comprising the steps of:

(i) coupling of the selectively protected magnetolepinose of formula (40) as defined in claim 60 with the monosaccharide oleander saccharide of formula (50A) as defined in claim 61 using tin chloride and silver triflate to yield the compound of formula

Wherein Ph ═ phenyl, Pv ═ pivaloyl, Z ═ TBDMS ═ tert-butyldimethylsilyl;

(ii) treatment of compound (53) with tetrabutylammonium fluoride yields a compound of the formula

Wherein Ph ═ phenyl, Pv ═ pivaloyl,

(iii) treatment of compound (54) with diethylaminosulfur trifluoride yields a compound of the formula

Wherein Ph ═ phenyl, Pv ═ pivaloyl,

(iv) reacting compound (55) with compound (52) of claim 64 to produce a compound of the formula

Wherein Ph ═ phenyl, Pv ═ pivaloyl,

(v) treating compound (56) in a raney nickel reaction, followed by treatment with a base, to produce compound (1) of claim 41.
67. A method of forming a trisaccharide and coupling the trisaccharide formed with a steroidal intermediate, the method comprising the steps of:

(i) using tin (II) chloride, AgOTf, Cp₂ZrCl₂Coupling the selectively protected magnetoephedrine of formula (40) as defined in claim 60 with the compound (55) as defined in claim 66 to give the compound of formula

Wherein Ph ═ phenyl, Pv ═ pivaloyl, Z ═ TBDMS ═ tert-butyldimethylsilyl;

(ii) treatment of compound (57) with tetrabutylammonium fluoride and diethylaminosulfur trifluoride yields a trisaccharide compound of the formula

Wherein Ph ═ phenyl, Pv ═ pivaloyl, and

(iii) using tin (II) chloride, AgOTf, Cp₂ZrCl₂Coupling a trisaccharide of formula (58) with a steroidal intermediate of the formula

Producing compound (1) according to claim 45.
68. A composition having appetite suppressant activity comprising a compound of any one of claims 44-57.
69. The composition of claim 68, wherein the compound is a compound of formula (1) according to claim 45.
70. The composition of claim 68 or 69 which is a food or beverage.
71. A composition according to claim 68 or 69 which comprises a pharmaceutically acceptable excipient, diluent or carrier.
72. The composition according to claim 68 or 69, which is prepared in unit dosage form.
73. Use of a compound as claimed in any one of claims 44 to 57, but without regard to the exclusion conditions therein, in the manufacture of a medicament having appetite suppressant activity.
74. Use of a compound as claimed in any one of claims 44 to 57, but without regard to the exclusion conditions therein, in the manufacture of a medicament for the treatment, prevention or management of obesity.
75. The use of a compound of formula (1) according to claim 45 as defined in claim 73.
76. A process for preparing a composition as claimed in claim 71 or 72, which comprises mixing a compound as claimed in any one of claims 44 to 57 with a pharmaceutically acceptable excipient, carrier or diluent.
77. A non-therapeutic method of suppressing appetite or reducing total caloric intake comprising administering to a human or animal an effective dose of the composition of any one of claims 68-72.
78. Use of a composition according to any one of claims 68 to 72 in the manufacture of a medicament having appetite suppressant activity or for use in the treatment, prevention or combating obesity.
79. A food or beverage comprising an effective amount of a compound as claimed in any one of claims 44 to 57, but without regard to the conditions excluded therein, which has an appetite suppressant effect when ingested.
80. A food or drink according to claim 79, wherein the compound is a compound of formula (1) as claimed in claim 45.
81. A non-therapeutic method of reducing total caloric intake or suppressing appetite in a human or animal comprising administering to the human or animal a food or beverage of claim 79 or 80.
82. Use of a compound of formula (1) as defined in claim 45, isolated from a plant of the genus Trichocaulon or Hoodia, in the manufacture of a medicament having appetite suppressant activity.
83. Use of a compound of formula (1) as claimed in claim 45, isolated from a plant of the genus Trichocaulon or Hoodia, in the manufacture of a medicament for the treatment, prevention or management of obesity.
84. The use according to claim 82 or 83, wherein the compound is isolated from Trichocaulon piliferum or Trichocaulon officinale, or from Pyrolunia recurori, Pyrolunia gordonii or Pyrolunia recurodi.
85. The use of claim 84, wherein the compound is isolated from Pyroluaria, or Pyrolugariii.
86. A food or drink having appetite suppressant activity, which comprises a compound of formula (1) isolated from a plant of the genus Trichocaulon or hordeia.
87. The food or beverage of claim 86, wherein the compound is isolated/purified from a Trichocaulon piliferum or Trichocaulon officinalis plant, or from a Pyrolunia eurorii, Pyrolunia gordonii or Pyrolunia lugarii plant.
88. The food or beverage of claim 87, wherein the compound is isolated from a fire land sub curorii, fire land sub gordonii or fire land sub lugardii plant.
89. The food or beverage of claim 86, wherein the compound is isolated/purified from an extract from a Trichocaulon piliferum or Trichocaulon officinale plant, or from a Pyrolunia eurorii, Pyrolunia gordonii or Pyrolunia lugardii plant.
90. The food or beverage of claim 89, wherein the compound is isolated/purified from an extract from a plant of the species Pyrolurolii, Pyroluonii or Pyrolugariii.
91. Use of a composition comprising a compound of formula (1) isolated from a plant of the genus Trichocaulon or Hoodia having appetite suppressant activity in the manufacture of a medicament for suppressing appetite.
92. The use according to claim 91, wherein the compound is isolated/purified from the plant Trichocaulon or Trichocaulon officinalis, or from the plant Currorii, Gordonii or Lugardii.
93. The use of claim 92, wherein the compound is isolated/purified from a fireland sub-curorii, fireland sub-gordonii or fireland sub-lugardii plant.
94. Use according to claim 91, wherein the compound is isolated/purified from an extract from the plant Trichocaulon piliferum or Trichocaulon officinale, or from the plant Currorii, Gordonii or Lugardii fire land.
95. The use of claim 94, wherein the compound is isolated/purified from an extract from the plant species fireland sub-curorii, fireland sub-gordonii or fireland sub-lugardii.
96. The use according to any one of claims 91-95, comprising a pharmaceutically acceptable excipient, diluent or carrier.
97. The use according to claim 96, prepared in unit dosage form.
98. Use of a food or beverage according to any one of claims 86 to 90 in the manufacture of a medicament for the treatment, prevention or management of obesity.
99. A non-therapeutic method of suppressing appetite or reducing total caloric intake comprising administering to a human or animal an effective dose of the food or beverage of any one of claims 86-90.
100. A compound having the formula
101. A compound having the formula

Wherein, Bz is benzoyl, and ph is phenyl.
102. A compound having the formula

Wherein, ph is phenyl.
103. A compound having the formula

Wherein, ph is phenyl and Pv is pivaloyl.