WO2006045171A2

WO2006045171A2 - Process for obtaining dry steroids derivative from ergostane

Info

Publication number: WO2006045171A2
Application number: PCT/BR2005/000223
Authority: WO
Inventors: Therezinha Coelho Barbosa Tomassini; Ivone Maria Ribeiro; Ana Cláudia Fernandes AMARAL; Milena Botelho Pereira Soares; Ricardo Ribeiro Dos Santos
Original assignee: Fundação Oswaldo Cruz (Fiocruz)
Priority date: 2004-10-25
Filing date: 2005-10-25
Publication date: 2006-05-04
Also published as: BRPI0404635B1; BRPI0404635A; BRPI0404635B8; WO2006045171A3

Abstract

The present invention refers to a process to obtain ergostane derivative dry steroids, such as physalins, including steps of: (a) grinding of dried parts of plant (s) from the Solanaceae species; (b) treatment of the said plant (s)' parts from step (a) with sodium chloride saline solution (physiological buffer) for a proper period of time; (c) filtration of the resulting solute of step (b) and, then, its partition in methylene chloride or solvent which is unsolvable in water, such as chlorophorm or ethyl acetate; (d) washing of the resulting organic phase of step (c) with, distilled water and further filtration in anhydrous magnesium sulfate or any other drier, which is unsolvable in water; (e) dehydration of the resulting solution of step (d) , resulting in an enriched-steroid fraction from ergostane derivative dry steroids; (f) isolation of the ergostane derivative dry steroids by means of medium pressure liquid chromatography (MPLC) and subsequent identification by spectroscopy.

Description

"PROCESS FOR OBTAINING DRY STEROIDS DERIVATIVE FROM

ERGOSTANE"

This invention refers to a process for obtaining ergostane derivative dry steroids, such as physalins, in which there occurs the extraction of said steroids from dried and crushed plant(s) of the Solanaceae family, such as the Physalis species, and the separation of the said steroids by using the Medium Pressure Liquid Chromatography (MPLC) technique. BACKGROUND OF THE INVENTION

Vita-steroids are ergostane derived steroids and were isolated, principally, from plants of the Solanaceae family, and specifically, of the types Physalis, Withania, Dυralia, Datura, Jaborosa, Deprea and N±candria. The popular use of these plants is known worldwide. Their extracts, for example, are used in the treatment of asthma, hepatic dysfunctions, various inflammatory processes, cancer and even as a hypnotic drug. Vitajardin A, B, C and D and their derivatives are vitanolides isolated from the stem and the leaves of Deprea orinocensis and are active as immune-modulators of the immune system (Patent US 5,681,95.0} .

The chemo type III physalins are dry ergostanes derivative open in C13-C14, cycled in C16-C24 with position 14 functionalized by a hydroxyl or in an oxirane bridge in C28. They are constituents of the steroid _.type present in the species Physalis, comprising, among the most extensively studied: P. angulata, P. alkekengi var franchetir P- ixocarpa, P. laninfolia, P. minima, P. peruviana, P. phyladelphia_r P. pυbescens and P. v±scosa. As a result of their polyoxifunctional structures, the physalins can be classified as the most advanced group, among the vita-steroids, in terms of the biogenetic 5 oxidation level.

The physalins are normally present in the root and the aerial parts of the Physalis angulata L. in a proportion that varies from 30 to 500 ppm. This plant, which belongs to the Solanaceae family, is tropically cosmopolitan and is

10 found in Brazil from Para to Rio de Janeiro (Braga, R. , Plants from the Northeast, specially from Ceara, Mossorό, Brazil, ESAM, 540, 1976) . In this country, it is popularly known as Bate-Testa, Bucho de Ra, Mata-Fome, Jua ou Jua de Capote and, more frequently, Camapύ (Pio Correa, Dictionary

15 of Useful Plants of Brazil and of the Exotic Plants Cultivated, Rio de Janeiro, Min. of Agriculture, Vol. I, pp. 10,1962) .

The use of basic extracts of Physalis spp. by Indian tribes is ^"present in literature. According to Sanchez et al

20.^' (Sanchez, E.G., Silva, M.T.G., Ribeiro, I.M., Tomassini, T.C.B., Evolutions of the antibacterial activity of Physalis a-ngulata L. , in Abstracts of the 1^st Congress of Pharmaceuticals Sciences, Ribeirao Preto, S. Paulo,■ Brazil, - Index. Bolletino Chimico Farmaceυtico, Vol. 136, pp. 154,-

25 1997), extracts and isolated constituents of Physalin show biological activity, including the anti-bacterial activity of root, stem and leaf extracts of P. angulata L.

Chiang et al (Chiang, H.C, Jaw, S.M., Chen, CF. and Kan, W.S., Anticancer Research 12, 837, 1992.) demonstrated that the physalins D and F extracted from Physalis spp. have shown activity in laboratory tests, in vitro and in vivo, against diverse types of human tumors, such as: hepatoma, uterine cervix, lungs and colon cells. The patent document BRPI9904363-7 shows medicinal compositions with immuno-modulating activity, the active principles being represented by extracts of Physalis spp or by physalins obtained from these extracts.

The patent document BRPI 9904635 also describes another type of activity of the physalins, that is, the anti-protozoan activity. Parallel, it has a process of isolation of physalins from the Solanaceae family of plants (as in the case of the Physalis) , characterized by the following steps: (a) crushing of the roots, leaves and stems of Physalis spp.; (b) extraction of the said roots, leaves and stems of step (a) using a alcohol solvent selected from the group consisting of methanol, ethanol, 1- propanol, 2-propanol, iso-butanol, dry-butanol, among others, hot or cold, for a pre-determined time period; (c) the evaporation of the organic extract of step (b) and the washing of the syrup like material with a non-polar organic solvent selected from a group consisting of chloroform, methylene chloride, ethylene chloride, diethyl ether, toluene or similar; (d) purification of the extract previously washed in step (c) by quick filtration in an of silica gel H absorbent, followed by a polar gradient with organic solvents; and the separation of the dry steroids by the use of chromatography.

However, the process described in the previously mentioned patent document (BRPI9904635) has the disadvantage of the extraction not being completed in a single step, and therefore, the physalin mixture is only achieved after the extraction with an alcohol solvent, evaporation and washing with a non-polar organic solvent and, after subsequent purification and filtration steps, submission to a polar gradient with organic solvents.

It is also important to mention the disadvantages of the well-known Mabry Technique (Mabry et aJ. The structure of Psilocostachyin, a new sesquieterpene dilactone from Ambrosia Psilostachya. Tetrahedron, 22, 1139-1146, 1966), which uses lead acetate for the possible extraction of tanins, chlorophylls and aromatic molecules in general. The mentioned _. organic salt is toxic and insoluble in water at room temperature. Additionally, it is difficult to remove from the medium, requiring the raising of the temperature, which can lead to structural modifications. Also, this technique uses activated coal for filtration. This activated coal is also absorbent. The original Mabry technique also uses celite, which increase the price and consequently reduces the yield; it possesses a larger number of steps than the usual processes, and, therefore, increases the operational costs.

The other usual techniques are characterized by the preparation of alcohol extracts (ethanol or methanol), followed by successive chromatography in ^' open columns, which has the following disadvantages: (a) use of large volumes of solvents; (b) long elution time and (c) high number of fractions. A classic example was shown by Matsuura T. et al (Matsuura T.; et al; The Structure of Physalins F. and J from Physalis angulata and P.lancefolia, Phytochemistry, vol.17, 1647-1650, 1978), where, in the extraction of 5 physalin F, 86 500-ml-each fractions are collected (each corresponding to 430 liters of solvent), followed by evaporation; which, by consequence, reduces the yield due to the high number of fractions to be evaporated and re- purified.

10 It is also relevant to cite the work of the researcher Kawai M. et al (Kawai M. et al; Physalin L isolated from P mlnimia, Phytochemistry, vol 43, n°3, 661-663, 1996) , which points out the possibility of the appearance of artifacts (a substance which is not the same initially elaborated by 15 the plant), through the long exposure of the botanic material to absorbents and polar solvents. SUMMARY OF THE INVENTION

The object of this invention is to obtain ergostane derivative dry steroids, such as physalins, through a 20. process that produces high yields, does not use toxic substances, is fast and has low operational cost.

The realization of this invention is related to a process for obtaining ergostane derivative dry steroids, such as physalins, encompassing the following steps: 25 (a) Crushing of the dry parts of the plant (s) of the

Solanaceae family;

(b) Treatment of such parts of the plant (s) of step (a) with a saline sodium chloride solution (physiologic buffer) during a pre-determined time period; (c) Filtration of the solution obtained in the previous step (b) and, then, its partitioning in methylene chloride or a solvent immiscible in water, such as chloroform or ethyl acetate; (d) Washing of the organic phase resulting from the previous step (c) with distilled water and then filtering in anhydrous magnesium sulfate or any other drying agent insoluble in water;

(e) Evaporation of the solution resulting of step (d) , providing a fraction enriched in ergostane derivative dry steroids;

(f) Separation of the ergostane derivative dry steroids by medium pressure liquid chromatography (MPLC) and the subsequent spectroscopic characterization. BRIEF DESCRIPTION OF DRAWINGS

FIGURE 1: Presents a comparative graph between the yields of physalins obtained from Physalis angulata L. through the modified Mabry Technique (patent application BRPI 9904363-

7) and the process demonstrated in this invention. FIGURE 2: Corresponds to the Infrared spectrum of physalin

B.

FIGURE 3: Shows the Infrared spectrum of physalin D.

FIGURE 4: Corresponds to the Infrared spectrum of physalin

F. FIGURE 5: Shows the Infrared spectrum of physalin G.

FIGURE 6: Corresponds to the Ultra Violet spectrum of physalin B.

FIGURE 7: Shows the Ultra Violet spectrum of physalin D.

FIGURE 8: Corresponds to the Ultra Violet spectrum of physalin F .

FIGURE 9: Shows the Ultra Violet spectrum of physalin G.

FIGURE 10: Corresponds to the mass spectrum of physalin B.

FIGURE 11: Shows the mass spectrum of physalin D. FIGURE 12: Corresponds to the mass spectrum of physalin F.

FIGURE 13: Shows the mass spectrum of physalin G.

FIGURE 14: Corresponds to the ¹³C RMN spectrum of physalin B.

FIGURE 15: Shows the ¹³C RMN spectrum of physalin D.

FIGURE 16: Corresponds to the ¹³C RMN spectrum of physalin F. FIGURE 17: Shows the ¹³C RMN spectrum of physalin G.

FIGURE 18: Corresponds to the ¹H RMN spectrum of physalin B.

FIGURE 19: Shows the ¹H RMN spectrum of physalin D.

FIGURE 20: Corresponds to the ¹H RMN spectrum of physalin F.

FIGURE 21: Shows the ¹H RMN spectrum of physalin G. DETAILED DESCRIPTION OF THE INVENTION

The physalins are steroids of the dry ergostanes derivative group, open at C13-C14 and cycled in C16-C24 having the following characteristics: a) 2 lactones, γ and δ, in the positions C13-C20 and C22-C24; b) ^'a saturated α, ^■ β ketone in ring A; c) an ethereal ring between carbons 14 and 17; d) a ketone in the position C15; e) a group α- hydroxyl in C13 and f) optionally, a oxirane bridge between carbons C14-C27. The main characteristic of these dry steroids is the presence of the vita-steroid skeleton with open positions C13-C14 and cycled in C16-C24 (Glotter, E.,

Withanolides and related ergostane type steroids, Nat. Prod. Rep., 8, 415, 1994).

There are twenty known physalins (A through T) , which are divided in 8 chemo-types: a) chemo-type I is represented by the physalins A and C, b) chemo-type II by the physalins L, M and O, c) chemo-type III composed of physalins B, D, E, F, H, I, J and N, d) chemo-type IV physalin G, e) chemo-type V physalins K and Q, f) chemo- type VI physalin P, g) chemo-type^' VII represented by physalin S, and h) chemo-type VIII by physalin R. The physalins A, B and Q correspond to the matrixes of the skeletons which lead to the biosynthesis of other related substances, the physalin B being the bio-genetic precursor of most of the other physalins. Following, we will show the structures of the previously mentioned physalins:

Physalin A Physalin B

Physalin C Physalin D

Physalin E Physalin F

Physalin G Physalin H

Physalin I Physalin J

Physalin K Physalin L

Physalin M Physalin N

Physalin O Physalin P

Physalin Q Physalin R

This invention describes a new process to obtain ergostane-derivative dry steroids (e.g.: physalins) . In this process, such steroids are extracted from dried and ground plants of the Solanaceae species, as in the case of species of the Physalis genus, and the isolation of the previously obtained steroids by using the method of Medium Pressure Liquid Chromatography (MPLC) .

The process to obtain ergostane-derivative dry steroids, which is presented in the this invention, when compared with those traditionally used to obtain the said dry-steroids derivatives, has several advantages, namely:

- The migration of the ground plant constituents is carried out in saline solution, which solute contains the fraction enriched in physalins. The fraction enriched in physalins in the above solute is treated with an organic solvent: methylene chloride (chlorophorm and ethyl acetate can also be used) or solvents insoluble in water.

- The "extraction" is conducted in a single step using 0.9% sodium chloride. The following step is partition, since the physalins have already been extracted in the solute obtained through physiological buffer.

- High output (the segregated amounts of physalins are 2 to 3 times higher than those noted with other processes used to such purpose: (Row, L.R., Reddy, K.S., Sarma, N.S., Matsuura, T., Nakashima, R., New Physalins from Physalis angulata and Physalis lancifolia, structure and reactions of Physalins D, I, G and K. Phytochemistry, vol. 19, 1175- 1181, 1980; Vasina, O.E et al, Withaesteroids of Physalis VII Vamonolide, Khim. Prir. Soed., n⁰ 6, 856-858,1987; Kawai, M. Yamamoto, T., Makino, B. Yamamura, H. Araki, S., Butsugan, Y., Saito, K., The structure of Physalin T from Physalls alkekengi Var franchet±, JNPR, vol. 3, 199-205, 2001) . - It does not use toxic material.

- The speed in which the extraction is carried out (6 to 8 working hours) . For comparison purposes, we can mention the study made by Chiang et al (Chiang, H.C, Jaw, S.M., Chen, CF, Kan, W.S., Antitumor agent, Physalin F from Physalis angulata L., Anticancer Research, vol. 12, 837-844, 1992), in which the intended extraction to isolate physalin F takes one week to each solvent. The whole extracting process amounts to a three-week period, and thus the difference between the present invention duration is clearly evidenced (6 to 8 hours) . Another study which can be used as a comparison is the one by Row et al (Row, L. R., Sarma, N.S., Matsura, T. Nakashima, R., Physalins E and H, NEW Physalins from Physalis angulata and P. lanclfolia, Phytochemistry, vol. 17, 1641-1645, 1978); - It has low operating costs (for example, due to its using of chemical sodium chloride, whose unit price is lower than those used in other processes with the same purpose) .

This claim can be evidenced by considering the following studies: (Kawai, M., Ogura, T. Marino, B. MATSUMOTO, A., Yamamura, H. Butsugan, Y., Hayashi, M., Physalins N and from Physalis alkekengi Phytochemistry, vol. 31 n⁰ 12, 4299-4302, 1992; Mulchandani, N.B., Benjamin, B.D., Isolation of Physalins D, a 13,14 - seco - 16,24, cyclo - steroid from tissue cultures of Physalis minima, Planta Medica, Short Comminications, 88-89, 1978) . These studies use, when carrying out the extraction, organic solvents such as: chloroform, ethanol, methane, ethanol, ethyl acetate, which are quite more expensive than the sodium chloride buffer solution.

To carry out this invention, plant parts of the Solanaceae species may be collected to provide ergostane- derivativ^p dry steroids, as in the case of roots and/or stems and/or leaves and/or fruit capsules from species of the Physalis genus in order to obtain the physalins.

In the present invention, such plant parts from the Solanaceae species, as in the case of roots and/or stems and/or leaves, are dried, cut in small pieces and ground. Later, the migration of physalin into a sodium chloride saline solution (physiologic buffer) is conducted for a proper period of time.

In addition, in the present invention, the saline concentration of sodium chloride is (or physiologic serum) / 0.9% (w/v) . The pH from the physiologic buffer ranges from 7.2 to 7.4. The constituent migration in the saline solution can be conducted with hot buffer, the temperature ranging from 36^°C to 38^°C, within the period of 6 to 8 hours, while mixing. The following step consists of filtering the resulting solute from the previous step, that is, the extraction, and to fraction it into methylene chloride, or chloroform, or ethyl acetate. The partition is a different step, since the extraction has already been carried out by the buffer solution. The resulting organic phase is washed out with distilled water and dried with anhydrous magnesium sulfate

(or any other drier which is insoluble in water) . The resulting solution is dehydrated, resulting in a ergostane

5 derivative dry steroids mixture, of which chromatograms are taken using the MPLC method.

In this invention, the above mentioned partition can be conducted using methylene chloride, as well as chloroform and ethyl acetate. Please note that it is not

10 the solvent percentage range but this solvent is indeed the only partitioning agent, that is, 100% of methylene chloride, for example.

In the present invention, the isolation step from ergostane derivative dry steroids can be accomplished by

15 using the method of medium pressure liquid chromatography (MPLC) with systems intended to such purpose.

In a preferred embodiment of the present invention, roots and/or stems and/or leaves and/or fruit capsules of Physalis angulata L., which are dried and ground, are

20_. treated with a 0,9% sodium chloride saline solution (physiologic buffer with pH ranging from 7.2 to 7.4). The resulting product of this extraction is already the physalin-enriched fraction, which suffers, in the following step, a partition with methylene chloride, or chloroform,

25 or ether or ethyl acetate. Chromatograms of this physalin- enriched fraction are then obtained using the MPLC method, and the eluted material is identified by spectroscopy.

Still in this preferred embodiment of the invention, the MPLC step is carried out in a device specifically intended to such purpose, which uses silica as adsorbent. This adsorbent is previously treated, being washed out with methanol to be activated (it is reactivated in stove) . The hexane/ethyl acetate solvent mixture then undergoes a 5 suspension process and the resulting adsorbent (silica) is placed into the column, under pressure and for the required time to achieve the isolation of the physalin mixture.

In the preferred embodiment of the present invention, the column elution process is conducted is carried out with

10 a ratio hexane/ethyl acetate system.

The ergostane derivative dry steroids obtained in accordance with the process outlined in present invention, such as the physalins, can be characterized by physic- chemical .data using only routine procedures, such as

15 spectroscopy.

The following examples illustrate the invention and represent the preferred embodiments. It is important to stress that the invention is not restricted to those examples, but also includes variances and changes within

20. the limits in which it works.

EXZiMPLE 1: Physalin extraction steps from Physalis angulata L.

The dried and ground plant underwent an extraction with hot physiological serum (0,9%) within a temperature

25 range of, approximately, 36⁰C - 38⁰C for 6 - 8 hours, while it was stirred. After this time, the solute was filtered and fractionated with 100% methylene chloride and the resulting organic phase, after being washed out by distilled water, was dried in anhydrous magnesium sulfate. The methylene chloride solution was then dehydrated resulting in the physalin-enriched fraction. EXAMPLE 2: Physalin isolation obtained from Physalis angulata L. - Preparation of adsorbent and sample: 5 Approximately 1 Kg of Lichroprep Si 60 silica was previously washed with methanol and activated over 24 hours, in a stove, at 100⁰C. This material underwent then a suspension process in a hexane/ethyl acetate solution (70:30v/v⁷) and the resulting product was used to fill the 10 chromatographic column, which was set, under a 20 bar pressure (Pump operating conditions: Flow rate: 18mL/minute, Pressure limits: 20 bar), for 40 minutes.

The pastille preparation with the enriched fraction

(physalin mixture obtained according to the previous

15 example) was conducted in a rotating drier, using silica gel in a 5% ratio related to the sample weight to be adsorbed (w/w' ) .

EXAMPLE 3: Physalin isolation obtained from Physalis angulata L. - Chromatographic process:

20. The MPLC process was carried out for four reproducible analysis, using samples which varied from 2.9g to 15.Og of the enriched fractions, which included the resulting physalins from Physalis angulata L' s stems.

The MPLC equipment used was from BUCHI, with a 25 manageable flow rate ranging from 16 to 160 mL/minute according to the selected chromatographic column type and dimensions. This equipment is fitted with one model 688 binary flow rate Pump (line A and B), one model 684 fraction Sampler, one model 687 ratio generator System, one model 685 3.3 borosilicate Column (46cm x 10 cm) and 50 mL capacity collecting Tubes.

The column elution process started with hexane/ethyl acetate (70:30 v/v' ) in binary system, the most polar solvent being progressively increased in 5% with larger ranges, within a 40% to 60% critical range of the program.

50-mL fractions were sampled which provided 1.2g of four individual chemical material, which were identified by spectroscopy, for example, as B, D, F and G physalins (from 2.9g of the enriched fraction). When 15.0 g of the enriched fraction was used, the quantity of obtained physalins was 7.Ig. These results are shown in Table 1.

Table 1: Physalin Isolation by the present invention process.

It is important to note that the identification was conducted at first by means of TLC (Thin Layer Chromatography) , using as reference B, D, F and G individual material, and confirmed by literature results of equivalent values for ¹H and ¹³C melting point, infrared, ultraviolet, Masses and Nuclear Magnetic Resonance. Figures 2 to 21 show the relevant spectra.

The Infrared data are:

Equipment: Nexus 670/ FT- IR - Thermo Nicolet Physalins: v KBR cm^"1 B - 3430 0ITs

-1780 γ lactone; 1763 saturated ketone -1721 δ lactone; 1656 unsaturated α β ketone D -3470 OETs

-1763 overlapping δ lactone and saturated ketone; -1733 γ lactone; 1666 unsaturated α β ketone

F -3502 3460 OITs

-1780 γ lactone; 1762 saturated ketone -1739 δ lactone; 1670 unsaturated α β ketone G _.-3554,3419 OITs -1780 γ lactone; 1769 saturated ketone

-1739 δ lactone; 1655 unsaturated α β ketone - 1620 C=C unsaturation The ultraviolet data are: Equipment: Hitachi U.2000 ; Concentration: 0.01 mg/ mL

The Mass Spectrometry data are: EQUIPMENT: LC MASS/MASS - WATERS B Physalin: 20mg/mL in methanol (cone) Injection: 10μL m/z 510 (M+) D Physalin: 40mg/mL in methanol (cone) Injection: 25μL m/z 544 (M+)

F Physalin: 20mg/mL in methanol (cone) Injection: 10μL

M/z 526 (M+)

6 Physalin: 20mg/mL in methanol (cone) Injection: 10μL

M/z 526 (M+) ^■ , The ¹³C RMN spectral data are:

¹³C RMN spectral data with chemical displacement (δ/ppm) of B, D, F and G physalins at DMSO-d₆- The high absorption levels in the 39ppm region are due to the Bruker 100MHz Equipment . solvent.

(Cont .

13 C spectrum is new - it was submitted to be published by the Farmanguinhos Group - PN2 and Analytical Center.

¹H RMN spectral data with chemical displacement (δ/ppm) , B, D, F and G physalin typical signs at DMSO-d₆. The high absorption levels in the 39ppm region are due to the Bruker 400MHz Equipment solvent-

(Cont . )

Bibliography

B PHYSALIN MakinoB., Kawai M., Kito K., Yamamura H., Butsugan Y., New Physalins -Possessing An Additional Carbon-Carbon Bond from Physalis alkekengi var. francheti,_. Tetrahedron, 51, 1259- 12538, 1995. D PHYSALIN Kawai M., Yamamoto T., Makino B., Yamamura H., Araki S., Butsugan Ϋ., Saito K., The Structure of Physalin T from Phys.alis alkekengi var. francheti, JANPR, 3, 199-205, 2001. F Physalin Chiang H., Jaw S., Chen C, Kan W., Antitumor Agent, Physalin F from Physalis angυlata L, Antitumor Research, 12, 837-844, 1992.

Kawai M., Makino B., Taga T., Miwa Y., Yamamoto T., Furuta T., Yamamura H., Butsugan Y., Ogawa K., Hayashi M., Crystal Structures of 5α, 6ά-Epoxy and 2,3-Dihydro Derivatives of Physalin B, a 13, 14-Seco-16,24-cyclosteroid, and Their ¹H NMR Spectral Analysis, Bull. Chem. Soc. Jpn., 67, 222-226, 1994.

G Physalin

Row L.R., Reddy K. S., Sarma N. S., Matsuura T._f Nakashima

R. , New Physalins from Physalis angulata and Physalis Landfolia. Structure and Reactions af Physalins D, I, G and K. , Phytochemistry, 19, 1175-81, 1980.

EXAMPLE 4: isolation step for Physalin obtained from

Physalis angulata L. - Absorbent reuse:

Intending to provide an economic use of the chromatographic process, the absorbent (Merck Lichroprep Si

60 Silica, 40- 63μm particle, code number 113905} was treated and tested for efficiency.

The adopted operating procedure included a methanol washing of the column, over two hours, under a pressure of a pump similar to that described in the initial conditions.

After that period of time, the adsorbent was dried and activated inside a stove, at 100 ⁰C, for eight hours. The resulting material was stored in an amber colored bottle.

EXAMPLE 5: Comparison between the obtained physalin yields according to the changed Mabry Technique (patent application BRPI 9904363-7) and according to the process described in the present invention.

In order to see the obtained physalin yields according to the changed Mabry Technique and according to the process described in the present invention, which uses physiological serum, comparative trials were conducted.

The physalins obtained according to the process described in the present invention resulted from the methodology used in the previous examples. The physalins obtained according to the changed Mabry Technique uses lead acetate to remove chlorophylls, tanins, aromatics, substances whose presence delay and makes more expensive the purification steps.' After two hours under intense stirring, an active charcoal is placed into it and the resulting material is filtered and conveyed to organic phase with chlorophorm. The following step includes washing with water and dehydration of the organic phase (see references, patent application BRPI9904363-7)-.

The percentage results are shown in Figure 1 and Table 2. Examining this figure and table, it is possible to confirm that, for each obtained physalin, the yield is greater when the present invention process is used. Table 2: Yield percentage of P. angυlata L isolated Physalin

Although the present invention has been described in relation to its preferred embodiments, it is evident to an experienced specialist in the art that several variances and changes are possible without moving from the present invention scope, which is established by the attached claims.

Claims

1. Process to obtain Ergostane derivative dry steroids characterized by the inclusion of the following steps:

(a) grinding of dried parts of Solanaceae species' 5 plant(s) ;

(b) treatment of the said plant(s)' parts of step (a) with sodium chloride saline solution (physiological buffer) for a proper period of time;

(c) filtration of the resulting solid phase ^•from step (b) 10 and, then, its partition in methylene chloride or solvent immiscible in water, such as chlorophorm or ethyl acetate;

(d) Washing of the resulting organic phase from step (c) with distilled water and further filtration in anhydrous magnesium. sulfate or any other drier which is unsolvable in

15 water;

(e) Dehydration of the resulting solution from step (d) , providing an enriched fraction with ergostane derivative dry steroids;

(f) Isolation of the ergostane derivative dry steroids by 20.^' means of medium pressure liquid chromatography (MPLC) and subsequent characterization by spectroscopy.

2. Process according to claim 1 characterized by the fact that dried parts of Solanaceae species's plant(s) used in step (a) are roots and/or stems and/or leaves and/or

25 fruit capsules.

3. Process according to claim 2 characterized by the fact that roots and/or stems and/or leaves and/or fruit capsules of plants from Physalis genus are used in step (a).

4. Process according to claim 3 characterized by the fact that Physalis angulata L. stem is used in step (a) .

5. Process according to claim 1 characterized by the fact that the extraction referred to in step (b) is carried out with sodium chloride saline solution, whose concentration is 0.9%.

6. Process according to claim 1 characterized by the fact that the physiological buffer used in step (b) has a pH varying from 7.2 to 7.4.

7. Process according to claim 1 characterized by the fact that the extraction related to step (b) is conducted in high temperature, which varies from 36⁰C to 38⁰C, for 6 to 8 hours, while stirring.

8. Process according to claim 1 characterized by the fact that the referred partition of step (c) is conducted with methylene chloride.

9. Process according to claim 1 characterized by the fact that the referred MPLC of step (f) is conducted by using silica as absorbent, which can be reused at least three times, after washing it with methanol.

10. Process according to claim 1 characterized by the fact that the referred elution process of the column of step (f) is carried out using an hexane/ethyl acetate ratio system.