PLANT EXTRACTS ENRICHED WITH IPOLAMIIDE DERIVATIVES AS IMMUNOSUPPRESSANTS FOR TREATING IMMUNOLOGICAL DISORDERS

Abstract

The invention describes extracts from plants of the genus Stachytarpheta enriched with ipolamiide derivatives as immunosuppressants for treating immunological disorders. The isolated active compounds and a process of producing these are also disclosed.

Claims

1. Compounds comprising the general formula: ##STR00014## wherein R corresponds to H, OH, OGlyc (Glycoside); R.sub.1, R.sub.1, R.sub.1 correspond to H, OH; R.sub.2 corresponds to H, COOH, COOCH.sub.3, CH.sub.3, CHO; R.sub.3 corresponds to H, OH, CH.sub.3; R.sub.4, R.sub.4 correspond to H, OH, CH.sub.2OH, CH.sub.3; R.sub.5, R.sub.5 correspond to H, CH.sub.3, COOCH.sub.3, CHO, CH.sub.2OH; R.sub.6 corresponds to CHO, COOH, COOCH.sub.3; R.sub.7 corresponds to H, CH.sub.3; R.sub.8, R.sub.8, R.sub.8 correspond to CHO, CH.sub.3, CH.sub.2OH, COOH and the dashed bonds represent single (CC) or double (CC) bonds between carbons (up to two double bonds per structure).

2. Compounds, according to claim 1, having immunosuppressive activity.

3. Method of treatment of immune disorders, comprising the step of administering to a patient one compound of general formula (I), (II) and/or (III), in sufficient amount to provide immunosuppressive effect.

4. Method of treatment, according to claim 3, for the treatment of patients with vitiligo.

5. Pharmaceutical composition comprising compounds derived from ipolamiide for the treatment of immune disorders, wherein the compounds comprise at least one compound selected from the group that comprises: ##STR00015## wherein R corresponds to H, OH, OGlyc (Glycoside); R.sub.1, R.sub.1, R.sub.1 correspond to H, OH; R.sub.2 corresponds to H, COOH, COOCH.sub.3, CH.sub.3, CHO; R.sub.3 corresponds to H, OH, CH.sub.3; R.sub.4, R.sub.4 correspond to H, OH, CH.sub.2OH, CH.sub.3; R.sub.5, R.sub.5 correspond to H, CH.sub.3, COOCH.sub.3, CHO, CH.sub.2OH; R.sub.6 corresponds to CHO, COOH, COOCH.sub.3; R.sub.7 corresponds to H, CH.sub.3; R.sub.8, R.sub.8, R.sub.8 correspond to CHO, CH.sub.3, CH.sub.2OH, COOH and the dashed bonds represent single (CC) or double (CC) bonds between carbons (up to two double bonds per structure); and d) pharmaceutically acceptable vehicle.

6. Pharmaceutical composition, according to claim 5, further comprising the ipolamiide compound.

7. Pharmaceutical composition, according to claim 5, comprising at least one of the following compounds: ##STR00016##

8. Pharmaceutical composition, according to claim 5, additionally comprising at least one of the following compounds: ##STR00017##

9. Process for production of active compounds comprising the step of subjecting at least one ipolamiide compound to a heating step at high temperatures in a suitable solvent for a time sufficient to obtain the ipolamiide derivatives of formula (I), (II) and/or (III).

10. Process for production, according to claim 9, wherein the active compounds comprise immunosuppressive activity.

11. Process for production, according to claim 9, wherein the solvent is water.

12. Process for production, according to claim 9, allowing to obtain high concentrations of the compounds of formula (I), (II) and/or (III).

13. Process for production, according to claim 9, wherein the high concentrations comprise about 0.5% to about 45% of the compounds of formula (I), (II) and/or (III) in a mixture.

15. Process for production, according to claim 9, wherein the high temperatures comprise temperatures above 35 C.

16. Process for production, according to claim 15, wherein the high temperatures comprise temperatures between 35 C. and 165 C.

17. Process for production, according to claim 9, comprising the hydrolysis step.

18. Process for production, according to claim 17, comprising an acid hydrolysis step.

19. Process for production, according to claim 17, comprising a basic hydrolysis step.

20. Process for production of extract enriched with compounds derived from ipolamiide comprising the steps of: a) selecting input vegetal biomass with a content of ipolamiide between 2.5% and 3.5% obtained from plants of the genus Stachytarpheta; b) submitting the selected biomass from a) to oven drying at temperature between 40 to 80 C., until obtaining the humidity stabilization between 10 to 12%; c) milling the vegetal biomass; d) performing the extraction of the vegetal biomass through the steps of: i. heating of the vegetal biomass at a temperature between 70 to 100 C., with constant stirring; ii. maceration of the vegetal biomass at room temperature; iii. heating of the vegetal biomass with temperature between 70 to 100 C.

21. Process for production of extract, according to claim 20, further comprising the steps of: iv. filtering and concentration of the extract; v. drying in Spray Dryer, during 1 to 60 seconds, with inlet temperature between 155 and 165 C. and outlet temperature between 85 to 95 C., coupled to a dehumidifier.

22. Process for production of extract, according to claim 20, in which the vegetal biomass heating of d) occur for 5 to 30 minutes.

23. Process for production of extract, according to claim 20, in which the vegetal biomass heating of d) occur for 5 to 15 hours.

24. Process for production of extract, according to claim 20, in which the input vegetal biomass comprises the aerial parts of the plants of the genus Stachytarpheta.

25. Process for production of extract, according to claim 20, in which the plant is Stachytarpheta cayennensis.

26. Process for production of extract, according to claim 20, in which the process enables an extraction yield of about 8 to about 10%.

27. Process for production of the extract, according to claim 20, in which the process comprises an aqueous extraction.

28. Process for production of extract, according to claim 20, in which the process provides an extract containing about 1% to about 20% of ipolamiide and derivatives.

29. Process for production of extract, according to any one of claims 20 to 28, comprising the step of predicting the theoretical content of ipolamiide and derivatives in the extract from the actual content of ipolamiide in the input vegetal biomass using Equation I:
% Theoretical content of ipolamiide and derivatives in the extract=% Actual content of ipolamiide in the vegetal biomassDER/(<actual content of ipolamiide and derivatives in the extract/actual content of ipolamiide in the input vegetal biomass>)standard deviation(Equation I).

30. Standardized extracts, wherein the extracts are obtained from the plants of the genus Stachytarpheta and comprises at least one compound of general formula (I), (II) and/or (III).

31. Extracts enriched with compounds derived from ipolamiide obtained by the process as defined in any of the claims 20 to 28.

32. Enriched standardized extract fractions from plants of the genus Stachytarpheta comprising at least one compound of general formula (I), (II) and/or (III).

33. Use of extract and/or standardized fractions comprising compounds derived from ipolamiide of general formula (I), (II) and/or (III), for the manufacture of a medicament with immunosuppressive activity for the treatment of immunological disorders.

Description

DETAILED DESCRIPTION OF THE FIGURES

[0056] FIG. 1Summary flowchart describing the production process of the active extract enriched with ipolamiide derivatives obtained from Stachytarpheta cayennensis.

[0057] FIG. 2Effect of the aqueous extract of Stachytarpheta cayennensis (3, 10 and 30 M, concentration expressed in ipolamiide) and isolated ipolamiide (3, 10 and 30 M) on the proliferation of CD8+ T cells activated by CD3/CD28 (A) and IFN production (B). The effect of the pool of compounds (IV to VIII) and the five novel isolated compounds (IV to VIII) generated after the acid hydrolysis of ipolamiide (30 M) was also evaluated in the same experiments, proliferation of CD8+ T cells activated by CD3/CD28 (C) and IFN production (D). Tacrolimus (0.5 M) was used as the positive control for all experiments. The data are the meanSD of three replicates.

[0058] FIG. 3Effect of the acid hydrolysis of ipolamiide on the formation of its derivatives. Chromatogram of intact ipolamiide (blue); Ipolamiide hydrolyzed at 0.1 N HCl at 40 C. for 1 h (green); Ipolamiide hydrolyzed at 0.1 N HCl at 40 C. for 2 h (red); Ipolamiide hydrolyzed at 0.1 N HCl at 40 C. for 5 h (magenta). IPO=Ipolamide.

[0059] FIG. 4Chromatogram of the Stachytarpheta cayennensis extract obtained from the production process claimed herein. The figure illustrates the ipolamiide marker and its specific derivatives at retention times: 5.5; 9.7; 12.0; 14.3; 17.3 min. IPO=Ipolamiide.

DETAILED DESCRIPTION OF THE INVENTION

[0060] The examples shown herein are for the sole purpose of exemplifying one of several ways of carrying out the invention, however, without limiting the scope thereof.

[0061] Active Compounds

[0062] The present invention presents novel and inventive compound groups, comprising the following general formulas:

##STR00006##

[0063] wherein R corresponds to H, OH, OGlyc (Glycoside); R.sub.1, R.sub.1, R.sub.1 correspond to H, OH; R.sub.2 corresponds to H, COOH, COOCH.sub.3, CH.sub.3, CHO; R.sub.3 corresponds to H, OH, CH.sub.3; R.sub.4, R.sub.4 correspond to H, OH, CH.sub.2OH, CH.sub.3; R.sub.5, R.sub.5 correspond to H, CH.sub.3, COOCH.sub.3, CHO, CH.sub.2OH; R.sub.6 corresponds to CHO, COOH, COOCH.sub.3; R.sub.7 corresponds to H, CH.sub.3; R.sub.8, R.sub.8, R.sub.8 correspond to CHO, CH.sub.3, CH.sub.2OH, COOH and the dashed bonds represent single (CC) or double (CC) bonds between carbons (up to two double bonds per structure).

Example 1

[0064] The compounds of general formula (I) comprise the following structures.

##STR00007##

Example 2

[0065] The compounds of general formula (II) comprise the following structures.

##STR00008##

Example 3

[0066] The compounds of general formula (III) comprise the following structures.

##STR00009##

[0067] Those above mentioned structures exemplify chemical structures backbones included in formulas (I), (II) or (III).

[0068] Immunological Disorders

[0069] The term immune disorders of the present invention comprises any dysfunction of the immune system. Commonly the disorders can be characterized by the components of the immune system that are affected or by the level of activity of the immune system. In the present invention, preferably, the immune disorders refer to diseases that have some evidence of autoimmunity. We can consider in the present invention vitiligo as being even more preferably chosen among the possible immune disorders.

[0070] Method of Treatment of Immune Disorders

[0071] The present invention describes a method of treatment of immune disorders, comprising administering to a patient a compound of general formula (I), (II) and/or (III), in sufficient amount to provide immunosuppressive effect. It should be noted that, for the purpose of the present patent application, the treatment of immune disorders can be achieved using ipolamiide derivatives and/or fractions and/or extracts containing ipolamiide derivatives, any of these having immunosuppressive activity. In a preferred embodiment, the method of treatment is intended for the treatment of vitiligo.

[0072] Pharmaceutical Composition Comprising Isolated Compounds Derived from Ipolamiide.

[0073] In one embodiment, the pharmaceutical composition of the present invention comprises isolated ipolamiide derivatives, used alone or in combination, for the treatment of immune disorders, wherein the compounds comprise at least one compound selected from the group comprising:

##STR00010##

[0074] wherein R corresponds to H, OH, OGlyc (Glycoside); R.sub.1, R.sub.1, R.sub.1 correspond to H, OH; R.sub.2 corresponds to H, COOH, COOCH.sub.3, CH.sub.3, CHO; R.sub.3 corresponds to H, OH, CH.sub.3; R.sub.4, R.sub.4 correspond to H, OH, CH.sub.2OH, CH.sub.3; R.sub.5, R.sub.5 correspond to H, CH.sub.3, COOCH.sub.3, CHO, CH.sub.2OH; R.sub.6 corresponds to CHO, COOH, COOCH.sub.3; R.sub.7 corresponds to H, CH.sub.3; R.sub.8, R.sub.8, R.sub.8 correspond to CHO, CH.sub.3, CH.sub.2OH, COOH and the dashed bonds represent single (CC) or double (CC) bonds between carbons (up to two double bonds per structure); and

[0075] d) pharmaceutically acceptable vehicle.

[0076] In an optional embodiment, the above pharmaceutical composition further comprises the ipolamiide compound.

[0077] In a preferred embodiment, the pharmaceutical composition of the present invention comprises the compounds:

##STR00011##

[0078] In this preferred embodiment, the compound of general formula (I) comprises the compounds of formula (IV), (V), (VIII) and (IX), the compound of general formula (II) comprises the compound of formula (VII), (X) and (XI) and the compound of general formula (III) comprises the compound of formula (VI).

[0079] The pharmaceutical composition of the present invention may additionally comprise the following compounds:

##STR00012##

[0080] and pharmaceutically acceptable vehicle.

[0081] Pharmaceutically Acceptable Vehicle.

[0082] To carry out their activity, the compounds of general formula (I), (II) and/or (III) should be administered to an animal organism, a mammal, particularly a human, preferably in the form of a pharmaceutical composition, i.e., associated with pharmaceutically acceptable vehicles which are suitable for each route of administration.

[0083] The pharmaceutical compositions of the present invention contain as active ingredient one or more compounds proposed herein, associated with one or more pharmaceutically acceptable vehicles. The active ingredient is commonly mixed, diluted or encapsulated with at least one vehicle.

[0084] When the vehicle is a diluent, it may be in the solid, semi-solid or liquid form, acting as a carrier, excipient or medium for the active ingredient. Therefore, the composition can be in the form of tablets, pills, powders, sachets, suspensions, emulsions, solutions, aerosols (in solid or liquid medium), creams, hard or soft capsules, suppositories, injectable solutions.

[0085] In the present invention, it is preferably considered as pharmaceutically suitable vehicle any substance different from the compound of general formula (I), (II) or (III), which has been intentionally added thereto to produce a pharmaceutical dosage form appropriate to a route of administration. Non-limiting examples of pharmaceutical excipients suitable for the preparation of pharmaceutical compositions are described in Handbook of Pharmaceutical Manufacturing FormulationsVol. 1 to 62004Sarfaraz K. NiaziCRC Press e Remington's Pharmaceutical Sciences, Mack Publishing.

[0086] Non-limiting examples of routes of administration of the composition comprising the compound of general formula (I), (II) or (III) are the topical, oral, parenteral, nasal, rectal, transmucosal, transdermal routes.

[0087] The therapeutic dose to be employed of the compounds of the present invention should be planned and calculated according to the route of administration chosen, the age, the weight and condition of the patient and the severity of the treated disorder. In general, the compounds of the present invention are administered in therapeutically effective doses. Effective doses can be extrapolated from dose-response curves, derived from in vitro or animal models. Typically, the clinician will administer the compound until an appropriate dose to achieve the desired effect.

[0088] Process of Production of Active Compounds

[0089] The present invention describes in detail the process of production of active compounds of general formula (I), (II) and/or (III). Essentially, the process of production of isolated compounds comprises the step of subjecting at least one ipolamiide compound to at least one heating step at high temperatures, in suitable solvent, for a sufficient time to obtain the compounds derived from ipolamiide of the general formula (I), (II) and/or (III).

[0090] In an optional embodiment, the composition of the present invention further comprises the ipolamiide compound.

[0091] In a preferred embodiment, the solvent of the present invention comprises water.

[0092] Additionally, the present invention describes the process of production of the compounds of general formula (I), (II) and/or (III), comprising the step of subjecting at least one ipolamiide compound to a heating step at high temperatures, in suitable solvent, for a sufficient time to obtain the compounds derived from ipolamiide of the general formula (I), (II) and/or (III).

[0093] In a preferred embodiment, high concentrations of the compounds of formula (I), (II) and/or (III) of the present invention are obtained from the total conversion (100%) of the content of ipolamiide used in the respective production process.

[0094] Even more preferably, the high concentrations of the present invention comprise about 0.5% to about 45% of each compound (IV to VIII) in a mixture. In this case, the concentrations comprise about 1 to about 5% of the compound IV, preferably 4%; about 15 to about 25% of the compound V, preferably 19%; about 1% to about 6% of the compound VI, preferably 3%; about 35% to about 45% of the compound VII or its isomers (e.g. compound X or compound XI), preferably 37%; about 0.5% to about 4% of the compound VIII or its isomers (e.g. compound IX), preferably 2%.

[0095] In a preferred embodiment, the high temperatures of the present invention comprise temperatures above 35 C., more preferably between 35 C. and 165 C.

[0096] In another preferred embodiment, the solvent of the present invention comprises other suitable solvents.

[0097] In a preferred embodiment, the process of production of the active compounds derived from ipolamiide comprises the step of hydrolysis or solvolysis of ipolamiide.

[0098] Even more preferably, the hydrolysis of ipolamiide may be of the acid type. Among the acids suitable for acid hydrolysis of ipolamiide, hydrochloric acid, hydrochloric, sulfuric, nitric, phosphoric and acetic acid can be mentioned, without any specific restrictions to any of them. In a preferred embodiment, in the present invention hydrochloric acid is used.

[0099] In an optional embodiment, the hydrolysis of ipolamiide may be of the basic/alkaline type. Among the bases suitable for basic/alkaline hydrolysis of ipolamiide, the alkali-metal hydroxides can be mentioned, without specific restrictions to any of them. In a preferred embodiment, in the present invention sodium hydroxide is used.

[0100] In an even more preferred embodiment, the hydrolysis in acidic medium is followed by a hydrolysis in basic/alkaline medium. As an example, we can mention the production process comprising a step of submitting at least one ipolamiide compound to high temperatures and 0.1 N (eq/L) of hydrochloric acid, followed by incubation for different time intervals that can range from 0 to 120 minutes. The hydrolysis is then interrupted by a process of neutralization using, preferably, sodium hydroxide 0.1 N.

[0101] In an optional embodiment, the production process of the compounds of general formula (I), (II) and/or (III) comprises a step of hydrolysis of ipolamiide in basic medium, being carried out with aqueous sodium hydroxide solution 0.1 N, maintained at 40 C. for 2 hours.

[0102] Production Process of Active Fractions/Extracts Enriched with Ipolamiide Derivatives

[0103] It was verified by the inventors that certain isolated active compounds of the present invention can be obtained by techniques of molecular design and synthesis. At the same time, we have also specified the advantages of obtaining herbal medicines for the treatment of diseases, since these compound production systems allow a series of productive interactions between the components of the plant and the active compounds, often even synergistically. Thus, to additionally obtain an herbal medicament comprising such active compounds, we have developed an unique production process which allows to obtain an extract enriched with compounds of interest.

[0104] As described below, the extract production process of the present invention comprises unique steps that lead to extracts enriched with the ipolamiide derivatives with immunosuppressive activity. We verified the relevance of preselecting input vegetal biomasses containing between 2.5% and 3.5% of ipolamiide, resulting in an extract enriched with ipolamiide and compounds derived from ipolamiide from about 8.5% to about 11.5%. As previously presented, the vegetal biomass containing ipolamiide will be used as starting material for the production process of the extract. Only with the production process of the present invention it is possible to obtain an extract enriched with specific compounds derived from ipolamiide. This enriched extract, further, presents immunosuppressive activity.

[0105] Thus, the present invention provides a process to produce standardized extract enriched with ipolamiide derivatives from plants of the genus Stachytarpheta.

[0106] It is, therefore, an additional object of the present invention a process for production of extract enriched with compounds derived from ipolamiide, comprising essentially the steps of:

[0107] a) selecting input vegetal biomass with a content of ipolamiide between 2.5% and 3.5% obtained from plants of the genus Stachytarpheta;

[0108] b) submitting the selected biomass from a) to oven drying at temperature between 40 to 80 C., until obtaining the humidity stabilization between 10 to 12%;

[0109] c) milling the vegetal biomass;

[0110] d) performing the extraction of the vegetal biomass through the steps of:

[0111] i. heating of the vegetal biomass at a temperature between 70 to 100 C., with constant stirring;

[0112] ii. maceration of the vegetal biomass at room temperature;

[0113] iii. heating of the vegetal biomass with temperature between 70 to 100 C.

[0114] In a preferred embodiment, the process for production of the present invention further comprises the steps of:

[0115] iv. filtering and concentration of the extract;

[0116] v. drying in Spray Dryer, during 1 to 60 seconds, with inlet temperature between 155 and 165 C. and outlet temperature between 85 to 95 C., coupled to a dehumidifier.

[0117] In a preferred embodiment, the process for extraction of the present invention is an aqueous or hydroalcoholic process, even more preferably an aqueous process.

[0118] Therefore, the process for production of the present invention allows to obtain a standardized extract enriched with compounds derived from ipolamiide, preferably with a yield of about 8% to about 10%.

[0119] It is, therefore, an additional object of the present invention the extract enriched with compounds derived from ipolamiide obtained by the above-mentioned procedure. The standardized extract enriched with compounds derived from ipolamiide of the present invention comprises, preferably, the compounds of formula (I), (II) and/or (III).

[0120] The vegetal biomass of the present invention comprises all parts of plants of the genus Stachytarpheta. Preferably, the vegetal biomass comprises the aerial parts of the plants, more preferably, the leaves.

[0121] In a preferred embodiment, the input vegetal biomass comprises at least one vegetal biomass with uniform content of ipolamiide between 2.5% and 3.5%. In an optional embodiment, the input vegetal biomass comprises more than one vegetal biomass, wherein the different vegetal biomasses have different contents of ipolamiide independently, but together achieve an uniform content of ipolamiide (between 2.5% and 3.5%).

[0122] In another preferred embodiment, the actual content of ipolamiide in the input vegetal biomass can be used as a parameter for predicting the theoretical content of ipolamiide and derivatives in the extract obtained. This prediction can be accomplished by a method comprising the step of applying Equation I to some parameters obtained experimentally to find the ideal proportions of ipolamiide in the input vegetal biomass, which preferably projects the content of ipolamiide and derivatives in the extract from 8.5% to 11.5% of. The Equation I is defined below:

% Theoretical content of ipolamiide and derivatives in the extract=% Actual content of ipolamiide in the vegetal biomassDER/(<actual content of ipolamiide and derivatives in the extract/actual content of ipolamiide in the input vegetal biomass>)standard deviation(Equation I).

[0123] In this way, it is possible to predict the theoretical content of ipolamiide and derivatives in the extract from the actual content of ipolamiide in the input vegetal biomass. Preferably, the ratio between the actual content of ipolamiide and derivatives in the extract/content of ipolamiide in the input vegetal biomass is between about 3.0 and about 3.5.

[0124] The present invention further claims standardized extracts enriched with compounds derived from ipolamiide from plants of the genus Stachytarpheta obtained from the production process described above.

[0125] In an embodiment even more preferred, the plants of the present invention comprise Stachytarpheta cayennensis.

[0126] The standardized extracts from plants of the genus Stachytarpheta are preferably obtained by the production process described above resulting in an extract enriched with ipolamiide and compounds derived from ipolamiide from about 1% to about 20%, preferably from about 8.5% to about 11.5% of content of ipolamiide and derivatives.

[0127] It is, therefore, an additional object of the present invention the use of standardized extracts of plants of the genus Stachytarpheta containing compounds derived from ipolamiide for the manufacture of a medicament with immunosuppressive activity. More specifically, standardized extracts of plants of the genus Stachytarpheta containing compounds derived from ipolamiide of general formula (I), (II) and/or (III) for the manufacture of a medicament with immunosuppressive activity.

[0128] It is, therefore, an additional object of the present invention at least one active fraction of extract enriched with compounds derived from ipolamiide. Preferably, at least one fraction comprises at least one compound derived from ipolamiide of formula (I), (II) and/or (III).

[0129] In an optional embodiment, the active fraction of enriched extract further comprises ipolamiide.

[0130] It is, therefore, an additional object of the present invention the use of at least one standardized fraction enriched with compounds derived from ipolamiide, obtained from plants of the genus Stachytarpheta for the manufacture of medicament with immunosuppressive activity.

EXAMPLESPREFERRED EMBODIMENT

[0131] The examples described in the experimental part have the sole purpose of exemplifying one of several ways of carrying out the invention, however, without limiting the scope thereof.

[0132] Process of Production and Identification of Active Compounds

[0133] The isolated compounds of the present invention are obtained by subjecting the ipolamide compound to 0.1 N hydrochloric acid, at 40 C. and 100 C., for 1 h, 2 h and 5 h. FIG. 3 illustrates the condition at 40 C. From this experiment, we could observe several products from this hydrolysis and, based on chromatograms, identify several derivatives of ipolamiide, such as, for example, the structures described below, as illustrated on FIG. 3.

##STR00013##

[0134] Alternatively, the hydrolysis can be carried out by varying the hydrochloric acid concentration between 0.1 to 1 N and the experimental temperature may vary between 35 C. and 165 C. In addition, the hydrolysis time can vary between 1 minute and 24 hours to facilitate the formation of higher concentrations of certain ipolamiide derivatives.

[0135] Process for Obtaining the Extract

[0136] According to the present invention, the process for obtaining aqueous extract of Stachytarpheta cayennensis rich in ipolamiide derivatives mainly comprised the steps of producing a standardized extract illustrated in the flowchart of FIG. 1 to obtain material for the development of pre-clinical research in immunology.

[0137] At first, seeds preferably selected by genotyping of Stachytarpheta cayennensis were submitted to a process of seeding for two months in a controlled environment regarding temperature, humidity and light. The seeding was carried out in expanded polystyrene trays, filled with substrate, and kept in this protected environment with controlled irrigation. The seedlings began to appear in 10 to 15 days. The trays remained in these conditions until the seedlings reached size and ideal conditions for permanent transplantation.

[0138] The seedlings with an approximate size of 5 to 8 cm in height and with 2 to 3 pairs of definitive leaves were transplanted to the growing site, which preferably had an annual average temperature of 30 C., annual average relative humidity of less than 55%, and in which the soil had preferably, but not limited to, the results of specific chemical and physical soil analyses, for example, acidity, calcium, nitrogen and use of organic fertilization in all areas.

[0139] After planting the seedlings, the first harvest was carried out after 6 months, and the other regrowth every 4 months, thus guaranteeing an optimized life cycle for the shrub aiming at maximizing ipolamiide content in the input vegetal biomass.

[0140] Following the planting, the vegetal biomass was stabilized through a greenhouse drying process, under defined conditions of temperature and humidity. The plants were dried in dryers with heat exchanger, forced air circulation, and temperature ranging from 50 to 70 C., preferably 60 C. The drying consisted of the passage of hot air through the plants, removing the humidity, until the vegetal biomass was stabilized with humidity between 10 to 12%. In a preferred embodiment, the drying time occurs from 8 to 20 hours.

[0141] As shown in FIG. 1, the vegetal biomass was subjected to the milling process by means of a hammer mill with 1800 RPM and a 19 mm sieve, obtaining a productivity of 50 to 150 kg/hour at room temperature. After milling, the vegetal biomass was submitted to an aqueous extraction at temperature between 80 to 90 C., with constant stirring for 15 min. The amount of extractive solution used should be 10 the amount of vegetal biomass used, guaranteeing exhaustive extraction of the substance of interest in the vegetal biomass.

[0142] After the previous step, the material was submitted to the maceration process for 10 h at room temperature. Subsequently, the material was again heated at 80 to 90 C. for 15 min.

[0143] The material was filtered on a rotary filter with polyester mesh of 40 um. After the filtration step, the material was concentrated on a Bernauer evaporator and/or falling film evaporator to about 30% of total solids.

[0144] The product of this step was submitted to drying in Spray Dryer, with inlet temperature between 155 to 165 C. and outlet temperature between 85 to 95 C. coupled to a dehumidifier, preferably of the Bry-Air type, during 20 to 40 seconds, aiming at obtaining the lowest residual humidity content possible (Table 1). This process substantially improves the quality of the enriched extract, since the residual humidity in the material initially compromises the stability of the components of interest, during the shelf life of the vegetal extract/derivative. This extractive process resulted in a ratio of 10 to 12:1 and yield varying between 8 to 10%.

TABLE-US-00001 TABLE 1 Humidity content in the extract after drying only and after drying with humidifier Humidity content in the extract Humidity content in the extract after drying with SD after drying with SD + dehumidifier 4.61% 1.77%

[0145] The extractive process described above guaranteed the exhaustive extraction of ipolamiide to assure the immunosuppressive activity of the extract due to the presence of ipolamiide derivatives generated in the process. Therefore, it was necessary to establish process controls for the input vegetal biomass, so that the actual content of ipolamiide and derivatives in the extract was between 8.5 and 11.5%, as follows in Table 2. The control of ipolamiide in the vegetal biomass ensures the presence of ipolamiide derivatives with immunosuppressive activity in the extract, generated by the process described herein.

TABLE-US-00002 TABLE 2 Content of ipolamiide in the vegetal biomass and in the extract. Theoretical content Actual content Actual content of of ipolamiide and of ipolamiide Selection of ipolamiide in the in- derivatives in the and derivatives input vegetal put vegetal biomass extract in the extract.sup.a biomass 1.57% 5.23% 4.47% Not selected (4.44-6.02) 2.20% 7.33% 7.62% Not selected (6.23-8.43) 3.00% 10% 10.80% Selected (8.5-11.5) 3.30% 11% 11.20% Selected (9.35-12.65) 3.07% 10.2% 8.90% Selected (8.67-11.73) 3.65% 12.2% 12.40% Not selected (10.37-14.03) .sup.aThe agreement between the theoretical and actual contents of ipolamiide and its derivatives in the extract demonstrates the applicability of Equation I.

[0146] With this, it was possible to determine a method for predicting the theoretical content of ipolamiide and its derivatives in the enriched active extract. For this purpose, it was used an equation to project the content that would be obtained by the extraction process (theoretical) from the actual content of ipolamiide in the vegetal biomass (Equation I).

% Theoretical content of ipolamiide and derivatives in the extract=% Actual content of ipolamiide in the vegetal biomassDER/(<actual content of ipolamiide and derivatives in the extract/actual content of ipolamiide in the input vegetal biomass>)standard deviation(Equation I).

[0147] In the above-described equation, DER can be understood as the amount of vegetal biomass required to obtain 1 kilo of native extract. The ratio between the actual content of ipolamiide and derivatives on the extract/content of ipolamiide in the input vegetal biomass is, preferably, between about 3.0 and about 3.5.

[0148] Thus, in a prospective way, using the above equation, it is possible to select only the vegetal biomass (whose content of ipolamiide is obtained by analytical methods, such as HPLC) that projects an adequate theoretical content of ipolamiide and derivatives in the extract between 8.5 and 11.5% and discard those that will not project this range. In a novel and inventive manner, it has been verified in the present invention that the relationship between the actual content of ipolamiide and derivatives in the extract and the content of ipolamiide in the input vegetal biomass will, preferably, be between 3.0 and 3.5.

[0149] The HPLC analysis comprised the steps of preparing sample solutions and standards, and elution thereof, as follows:

[0150] 1. Solutions Preparation

[0151] 1.1Formic Acid Solution 0.1% (Mobile Phase A):

[0152] In a 1000 mL volumetric flask containing approximately 800 ml of ultra-pure water, it was added 1 ml of formic acid. The flask volume was filled with ultra-pure water and well homogenized.

[0153] 1.2Formic Acid Diluent Solution: Methanol (1:1):

[0154] In a beaker, it was mixed 50 mL of the 0.1% formic acid solution with 50 mL of methanol.

[0155] 2. Sample Preparation:

[0156] Raw-material (Herbal): 1.0 g of the ground herbal was weighed and transferred to an amber 250 ml Erlenmeyer flask or covered with aluminum foil. 50 mL of distilled water was added and extracted under reflux at 80 C. for 2 hours. The solution was paper-filtered into a 50 mL volumetric flask and filled up with distilled water. It was filtered through 0.22 OR 0.45 m membrane to an HPLC vial.

[0157] 3. Standards Preparation:

[0158] Ipolamiide standard 100 ppm: 1.0 mg of ipolamiide standard was weighed and transferred to an amber 10 mL volumetric flask. 5 ml of the diluent was added, which was left in an ultrasonic bath for 10 minutes or until complete dissolution. It was filled with the diluent and then there was the homogenization. Humidity analysis by Karl Fischer was performed for the ipolamiide standard.

[0159] 4. Analysis by HPLC:

[0160] 4.1Parameters/Equipment

[0161] Column: Zorbax SB-C18 (250 mm4 mm; 5 um)

[0162] Mobile phase: (A) 0.1% formic Acid; [0163] (B) Methanol.

TABLE-US-00003 TABLE 3 Ipolamiide Elution Gradient Time (min) (%) A (%) B 0 80 20 27 58 42 32 58 42 32.1 0 100 36 0 100 36.1 80 20 45 80 20

[0164] Flow rate: 1.0 mL/min

[0165] Detection: 254 nm.

[0166] Analysis time: 45 minutes

[0167] Injection volume: 30 l

[0168] 4.2Calculation of Ipolamiide Content

[0169] Content of ipolamiide (%)=A sampleM standardP standardD sample(100U standard);

[0170] A standardM sampleD standard10000/100;

[0171] wherein:

[0172] A sample: Peak area of ipolamiide in the sample

[0173] M standard: Mass of the standard ipolamiide in mg

[0174] P standard: Purity of the standard in decimal

[0175] D sample: Dilution of the sample in mL

[0176] A standard: Peak area of ipolamiide in the standard

[0177] M sample: Mass of the sample used in g

[0178] D standard: Dilution of the standard in L

[0179] U standard: Humidity of the standard quantified by Karl Fischer

[0180] 10000: Conversion of units

[0181] The result of the content in the extract was given in dry base, that is, the humidity was discounted. Therefore the calculation for the mass of the extract was:

[0182] M sample=Mass(100U sample)/100;

[0183] wherein:

[0184] Mass=Mass of dry extract (in grams)

[0185] U sample=Humidity of the dry extract in percentage, according to iT2-052.

[0186] 5. Analysis by HPLC for Ipolamiide Derivatives

[0187] 5.1Parameters/Equipments

[0188] Column: Eclipse XDB AgilentC18 (1504.6 MM) 5 MICRONS

[0189] Mobile phase: (A) 0.1% formic Acid buffer; [0190] (B) Acetonitrile

TABLE-US-00004 TABLE 4 Ipolamiide Derivatives Elution Gradient Time (min) (%) A (%) B 0 95 5 5.00 90 10 8.00 80 20 10.00 90 10 12.00 95 5

[0191] Flow: 1.2 mL/min

[0192] Detection: DAD (205-280 nm).

[0193] Analysis time: 15 minutes

[0194] Injection volume: 20 L

[0195] 5.2 Calculation of Content for Ipolamiide Derivatives

[0196] For analysis of a given ipolamiide derivative (Y), we submitted it to a solution with defined concentration. This concentration is directly correlated with the area observed in the chromatogram. This area, when compared with the total area of the chromatogram, multiplied by the concentration previously defined, give us the percentage value of the said derivative (Y) in the sample, as can be seen below:

[00001]$\mathrm{Sample}.Math..Math.\mathrm{concentration}\frac{\mathrm{Derivative}.Math..Math.Y.Math..Math.\mathrm{area}}{\mathrm{Total}.Math..Math.\mathrm{area}}100=\%.Math..Math.\mathrm{of}.Math..Math.\mathrm{derivative}.Math..Math.Y.Math..Math.\mathrm{content}.Math.\left(.Math..Math.\mathrm{content}.Math..Math.\mathrm{of}.Math..Math.\mathrm{derivatives}=\%.Math..Math.\mathrm{total}.Math..Math.\mathrm{content}.Math..Math.\mathrm{of}.Math..Math.\mathrm{ipolamiide}.Math..Math.\mathrm{derivatives}.Math..Math.\mathrm{in}.Math..Math.\mathrm{the}.Math..Math.\mathrm{sample}.\right)$

[0197] Biological/Immunosuppressive Activity

[0198] The evaluation of the in vitro biological activity of the aqueous extract of Stachytarpheta cayennensis as well as of ipolamiide and its derivatives was conducted as described below.

[0199] The extract of Stachytarpheta cayennensis, ipolamiide and derivatives were studied in an experimental in vitro immunological model involving CD8+ T cells and IFN. The objective of this study was to evaluate whether the extract and other substances, isolated or mixed, act by blocking the activation of CD8+ T cells and the consequent secretion of IFN.

[0200] Peripheral blood mononuclear cells (PBMCs) from healthy volunteers were isolated from leukocyte layers by Ficoll-Hystopaque centrifugation. Thereafter, human CD8+ T cells were isolated using the CD8+ T cell isolation kit (Miltenyi Biotec, #130-096-495). These cells (310.sup.5 cells/well) were incubated in RPMI+10% of FBS medium, activated with CD3/CD28 (1 g/mL) and treated with different concentrations of aqueous extract of Stachytarpheta cayennensis standardized in 10% of ipolamiide, isolated ipolamiide, and ipolamiide derivatives obtained by acid hydrolysis to study their effect on the prevention of the CD8+ T cell activation and secretion of IFN. For evaluation of cell proliferation, bromodeoxyuridine (BrdU), a thymidine analogue commonly used for proliferation assays, was used as a marker for the proliferation. Specifically, it was evaluated the incorporation of BrdU through the Biotrak ELISA System (GE Healthcare, RPN250) per the manufacturer's instructions. The methodology for quantification of interferon gamma comprised the use of the Human IFN ELISA Ready-SET-Go kit (eBiosciences 88-7316-88) and followed the manufacturer's instructions. As illustrated in the graph set of FIG. 2, the aqueous extract of Stachytarpheta cayennensis enriched with ipolamiide derivatives blocked the activation of CD8+ T cells induced by CD3/CD28. Isolated ipolamiide had no effect on the proliferation of CD8+ T cells induced by CD3/CD28, or the secretion of IFN. However, the compounds derived from ipolamiide in a mixture obtained for its acid hydrolysis significantly reduced the CD8+ T cell proliferation and the secretion of IFN. The ipolamiide derivatives isolated from this mixture also demonstrated a statistically significant reduction of CD8+ T cell proliferation and of secretion of IFN. Tacrolimus, a well-known immunosuppressive agent, was used as a reference compound and as a positive control for the experiment. The effect obtained by tacrolimus was similar to that obtained with hydrolyzed ipolamiide in both cell proliferation and IFN production.

[0201] Thus, the results showed that the immunosuppressive activity derives from the ipolamiide derivatives and not from the intact molecule. It is important to reinforce that the compounds obtained by the experimental condition of acid hydrolysis of isolated ipolamiide are present in the aqueous extract of Stachytarpheta cayennensis, as shown in FIG. 4. However, the presence of such compounds is due to the extraction process used in the present invention.

[0202] Those skilled in the art will appreciate the knowledge presented herein and may reproduce the invention in the embodiments presented and in other embodiments, falling within the scope of the appended claims.