as mentioned before , several trypanocidal and leishmanicidal drugs are currently been used in endemic areas . however , these drugs are highly toxic and depending on the dosage and frequency of treatment , children are the most likely victims . the “ ideal drug ” would achieve parasitological cure of both acute and chronic cases from all parts of latin america ( tropical disease research , world health organization pp . 125 – 133 , geneva , 1995 ). this “ ideal drug ” should : ( i ) be effective by the oral and , for cases of mega - oesophagus , the parenteral route ; ( ii ) be effective in one or a small number of daily doses ; and ( iii ) be affordable by those who suffer from the disease . at the doses used , it would be free of serious side - effects and of teratogenicity . in addition , it would not require patients to be hospitalized , and drug resistance would not develop rapidlly . but until now , despite intensive research and many leads , nothing had come close to the requirements for chagas disease set in an expert meeting in washington , d . c ., in 1981 , and the health system is still left only with the original and unsatisfactory drugs , nifurtimox and benznidazole . moreover , there is a consensus that patients in the chronic stage of the disease , with overt lesions , do not benefit from trypanocidal treatment . symptomatic treatment appears to be all that can be offered . referring to the auto - immune diseases , nonspecific immunosuppressants must be used with extreme caution in order to avoid complications related with severe immunossupression . thus , in order to come closer to the “ ideal drug ” and to the requirements set forth above , the present invention proposes new pharmaceutical compositions based on one or more physalins as active anti - protozoan and immunosuppressive substances , at concentrations ranging from about 10 to about 50 %, for the treatment of protozoan infections . moreover , in another embodiment of the present invention , it is described a novel isolation process to obtain physalins from plants . physalins belong to the group of c - 28 ergostane - type compounds : 13 , 14 - seco - 16 , 24 - cyclosteroid derivatives having the characteristics of : a ) two lactones , γ and δ , in positions 13 – 20 and 22 – 24 ; b ) an α , β unsaturated ketone at a ring ; c ) an ether ring between carbons 14 and 17 ; d ) a ketone at c - 15 , e ) an α - hydroxyl group at c - 13 and f ) optionally an oxyrane bridge between carbons 14 – 27 . the main characteristic of those steroids is the presence of 13 , 14 - seco - 16 , 24 cyclowithanolide framework or skeleton ( glotter , e ., withanolides and related ergostane type steroids , nat . prod . rep ., 8 : 415 , 1994 ). nineteen physalins ( a to s ) are known and they are divided into 8 chemotypes : a ) chemotype i is represented by physalins a and c , b ) chemotype ii by physalins l , m and o , c ) chemotype iii contains physalins b , d , e , f , h , i , j and n , d ) chemotype iv by physalin g , e ) chemotype v by physalins k and q , f ) chemotype vi physalin p , g ) chemotype vii is represented by physalin s , and h ) chemotype viii by physalin r . physalins a , b and q correspond to the matrices of skeletons that lead to the biosynthesis of the other related substances . physalin b is the biogenetic precursor of the majority of the other physalins . the structures of the physalins are hereinafter presented . despite an improved isolation route to obtain physalins is provided by the present invention , conventional methods may also be used to obtain physalins from leaves and stems of physalis species . it is important to emphasize that the process of the invention is simplified as compared with the known methods which involve several steps dealing with column chromatography . roots , and epigeal parts of physalis spp . were collected in order to obtain physalins . after being collected , roots or epigeal parts of p . angulata may be cut in small pieces and ground in a mixer . crude extract is treated with an aqueous or alcoholic solvent in a suitable extractor , at room or higher temperature , the later by heating for at least 24 hours . suitable alcoholic solvents include , but are not limited to methanol , ethanol , 1 - propanol , 2 - propanol , iso - butanol , sec - butanol and the like . the alcoholic extract when tested for anti t . cruzi activity showed 100 % of mortality . the aqueous or organic extract is further evaporated and the syrup material ( residue ) is washed with a suitable non - polar organic solvent which , may include but is not limited to , chloroform , dichloromethane , dichloroethane , diethyl ether , toluene , and other non - polar organic solvents known to those skilled in the art . then , the soluble fraction is partially evaporated and poured over a layer of silica h ( gel ). the extraction is performed by using a polarity gradient of organic solvents s 1 & lt ; s 2 & lt ; s 3 & lt ; s 4 , i . e . the polarity degree increases from s 1 to s 4 . after filtration , the s 2 fraction ( intermediary polarity ) is treated by a further chromatographic step . the physalins came out with , for example , s 1 / s 3 or s 2 / s 3 fraction depending on the physalin required . the withasteroids compounds obtained from roots , leaves and stems of physalis spp . and other closely related genera of the solanaceae family may be characterized by physicochemical means , e . g . spectroscopic methodology , using no more than routine experimentation . the isolation process of the present invention has the following advantages : ( 1 ) it involves quite simpler and faster procedures ; ( 2 ) it is particularly useful in developing countries due to its lower cost ; and ( 3 ) it reduces product losses during the whole process because there are less chromatography steps . the following examples are illustrative of the present invention and represent preferred embodiments . those skilled in the art may know , or be able to find using no more than routine experimentation , other appropriate materials and techniques , such as the mentioned extracting substances and measuring methods . 330 g of dried roots of p . angulata were cut in small pieces , ground and extracted with ethanol by heating in a soxhlet extractor . the obtained extract was concentrated to dryness under reduced pressure and the resulting syrup material was washed with chloroform , in a proportion of about 3 to 5 times of the obtained weight . then , the soluble fraction was partially evaporated and poured over a layer of silica h ( gel ) for filtration using , in a sequential fashion , hexane , dichloromethane , ethyl acetate and methanol as solvent extractor . the dichloromethane fraction obtained in example 1 was treated again in a chromatographic column with silica g ( gel ). physalin b was eluted with an ethyl acetate - hexane gradient and recrystallized . physalin b was characterized using no more than routine spectroscopic methodology and the data obtained are shown below . physalin b has a melting point in the range of 235 – 237 ° c . it shows a molecular ion peak at m / z 510 ( m +) and its 1 h nmr ( 200 mhz ) dmso - d 6 spectrum presents signals at δ 5 , 88 ( h 2 , dd , j = 10 hz ) and δ 6 , 90 ( h 3 , dd , j = 10 hz ) for the same olefinic protons . those values represent the absorptions of the hydrogen atoms linked to c 2 and c 3 which are attached to a ring . three methyl groups are showed at δ 1 , 19 ( h 19 , s ); δ 1 , 88 ( h 28 , s ) and δ 1 , 26 ( h 21 , s ). a pair of signals at δ 3 , 67 ( d , j = 13 hz ) and δ 4 , 32 ( dd , j = 13 , 2 hz ) could be attributed to the methylene protons at c - 27 , confirming the oxyrane bridge at cl 4 — o — c 27 . the hydroxylproton at δ 6 , 27 ( h 13 , s ) and a doublet at δ 5 , 68 ( h 6 , dd ) were a good tool to make sure that the molecule is physalin b . 13 c nmr ( 54 mhz ) in dmso - d 6 , showed twenty six signals . the missing two carbons from the c 28 structure , is due to the solvent , dimethyl sulfoxide that enveloped them at the range between 40 to 37 ppm . the peaks at 209 , 2 ppm ( c 15 ); 203 , 3 ppm ( c 1 ); 171 , 0 ppm ( c 18 ) and 167 , 1 ppm ( c 26 ) correspond to carbonyls from : ketone , ketone α – β unsaturated , two lactones ( furan and pyrane rings ). the signals at 126 , 8 and 146 , 1 ppm are due to the methylene c 2 and c 3 in the double bond of a ring , and the signals at 135 , 5 and 123 , 3 ppm are from carbons 5 and 6 of b ring . all the signals obtained from dept technique led us to the conclusion that the proposed compound is physalin b . the material obtained in example 1 was treated again in a chromatographic column with silica g ( gel ) by using a chloroform / ethyl acetate gradient . physalin d was obtained in the eluent fraction 1 : 1 v / v . physalin d was characterized using no more than routine spectroscopic methodology and the data obtained are shown below . physalin d has a melting point 242 – 245 ° c . it shows a molecular ion peak at m / z 544 ( m +, 3 %); 125 ( m - 419 , 100 %). its 1 h nmr ( 200 mhz ) dmso - d 6 spectrum are expressed in δ units and presented the following results : the protons attached to c - 2 and c - 3 show signals at 5 , 67 dd ( j 2 . 3 = 10 ) 6 , 58 ddd ( j 3 . 2 = 10 ) and a singlet at 4 , 13 corresponds to the hydrogen of hydroxyl group . a multiplet is showed at 3 , 49 for the proton attached at c - 6 , while the second hydroxyl group absorbs as a doublet at 4 , 55 . it was possible to distinguish two hydrogen atoms linked to carbon 12 with signals at 1 , 48 ( dd ) β and 2 , 13 ( d ). the three methyl groups ( ch 3 ) absorb at 1 . 12 , 1 . 18 , 1 . 16 as a singlet for c - 19 , c - 21 and c - 28 respectively . all these values correspond to the literature data for physalin d . it must be emphazised that physalin &# 39 ; s d 13 c nmr is described for the first time ( see fig1 ). the 13 c nmr spectrum of physalin d in dmso - d 6 ( 54 mhz ) presents peaks at 209 , 64 ppm for the carbonyl carbon at c - 15 ; 204 , 52 ppm for the carbon c - 1 ; 171 , 68 and 167 , 12 ppm for the carbons attached to quaternary center of the two lactone rings c - 18 / c - 26 ; 142 , 89 and 127 , 16 ppm for the double bond c - 2 and c - 3 of a ring . the signals at 106 , 87 ppm correspond to ( c - 14 ); 80 , 60 ( c - 20 ); 80 , 40 ( c - 17 ); 78 , 75 ( c - 13 ); 76 , 38 ( c - 22 ); 72 , 52 ( c - 6 ); 60 , 49 ( c - 27 ); 54 , 11 ( c - 16 ); 53 , 48 ( c - 10 ); 49 , 43 ( c - 25 ); 35 , 19 ( c - 8 ); 31 , 36 ( c - 23 ); 30 , 46 ( c - 24 ); 29 , 93 ( c - 9 ); 26 , 56 ( c - 4 ); 25 , 78 ( c - 12 ); 24 , 69 ( c - 11 ); 24 , 27 ( c - 28 ); 21 , 49 ( c - 21 ) and 13 , 24 ppm to the carbon attached at position 19 . in vitro studies using pure physalins are carried out with trypomastigotes of trypanosoma cruzi . t . cruzi y strain is maintained in mk2 strain cell culture in a rpmi medium + 10 % bovine fetal serum , at 37 ° c ., under 5 % co 2 . this material is centrifuged during 15 minutes at 1500 rpm . afterwards , the material is maintained in rest at 37 ° c . for 2 hours . the supernatant is centrifuged again during 30 minutes at 3000 rpm . the trypomastigotes ( 5 × 10 5 / well ) in rpmi medium with 10 % bovine fetal serum and antibiotic are incubated in wells with different concentrations of physalin b or physalin d . after 24 – 48 hours , the viable parasites maintained at a temperature of 4 ° c . are counted . each experiment was carried out in triplicate . the results are shown below . as showed in table i , parasite mortality is obtained after 24 hours when dealing with physalin b at a concentration of 2 , 5 mg / ml . moreover , 100 % of mortality is obtained after 24 hours by using only 40 μg / ml of physalin d . these results are very significant if we consider the low concentration used , the mortality of the parasite ( 100 %) and the efficacy of the operation time , i . e . 24 hours . in vivo test with experimental animals infected with t . cruzi y strain several samples from p . angulata l . extract were used in this experiment . fractions obtained from the process for isolating the physalins were also used . serum trypomastigotes were used to evaluate the trypanocidal activity of the p . angulata extract and physalins of the present invention . groups of three mice were intraperitoneally inoculated with 10 4 parasites ( serum trypomastigotes ). during 10 days , these animals were daily , since the day before infection , treated with 20 mg / animal of ( a ) methanolic extract obtained from the fruit of p . angulata l . ; ( b ) physalin mixture ( physalins b , d , g , h and l ) obtained from the leaves of p . angulata l . ; and ( c ) ethanolic extract obtained from the stem - bark of p . angulata l . the treatment was orally applied . preparations ( a ) and ( c ) were diluted with 5 % alcaholic solution containing 0 . 5 % of polyoxyethylenesorbitol monolaurate . three mice used as controls received the same treatment but without the active substance (( a ), ( b ) or ( c ) preparations ). an additional group of three infected mice was treated with a known trypanocidal drug ( rochegan ™). the quantity of this drug applied to each animal was the same as the above mentioned preparations ( a ), ( b ) and ( c ). ( p . angulata l . extracts and physalins ). after 10 days , all mice were examined to evaluate their parasitemia ( the presence of parasites in the blood ) levels . table ii presents the results . from table ii , it can be concluded that the best efficacy performance ( 84 %) was obtained when ethanolic extract from the stem - bark of p . angulata l . is applied in the treatment . the second best result ( efficacy of 67 %) is in the treatment with physalin mixture ( physalins b , d , g , h and l ) from the leaves of p . angulata l . the treatment with methanolic extract from the fruit of p . angulata l . showed an efficacy of 65 %. in vitro test to evaluate the antileishmanicidal activity of physalin b stationary phase promastigotes of leishmania amazonensis were obtained from parasite cultures in lit medium with 10 % bovine fetal serum . the assays were standardized in plates with 96 wells . promastigotes were plaque harbored , in triplicate , in 100 μl of lit medium . 10 μl of physalin b solution and 10 μl of a positive control ( amphotericin b ) are added to the wells . several dilutions were used . parasite viability was determined after incubation with mtt by reading the absorbance at 570 nm in elisa microplate . 100 % of parasite mortality was obtained when 20 μg / ml of physalin b was used . evaluation of the antileishmanicidal activity of physalin b in in vitro infection assay by using peritoneal macrophages peritoneal macrophages were incubated during 2 hours at 37 ° c . in 24 wells plates with laminas . after washing to remove non - bound cells , the wells were incubated with stationary phase promastigotes of leishmania amazonensis ( parasites / macrophages in a ratio of 10 : 1 ). after 18 hours , the cultures were washed to remove the free parasites . after 24 hours , physalin b solution is added to the wells . the laminas are collected , fixed with methanol and dyed with giemsa . the infection level was determined by counting parasite quantity and the infected macrophages percentage . 100 % in vitro reduction in the infection of peritoneal macrophages was obtained when 10 μg / ml of physalin b was used . in vitro tests to evaluate the immunosuppresive properties of physalin b the immunosuppresive properties of physalin b were evaluated by simulating the responses of the immune system against invading organisms and toxic molecules produced by them . in a first test , thyoglycollate - elicited peritoneal macrophages were stimulated with lps ( 500 ng / ml ) in the presence of various concentrations of physalin b . cell - free supernatants were collected 4 hours after stimulation and tested for tnα and il - 6 production by elisa . fig3 a and 3b show the inhibitory properties of physalin b concerning to tnfα and il - 6 , respectively , production by macrophages . in a second test , thyoglycollate - elicited peritoneal macrophages were stimulated with lps ( 500 ng / ml ), ifnγ ( 5 ng / ml ), or ifnγ + lps in the presence of various concentrations of physalin b . cell - free supernatants were collected 24 hours after stimulation and tested for nitrite production by griess assay . fig4 shows the inhibitory properties of physalin b concerning to nitric oxide production by macrophages . in a third test , thyoglycollate - elicited peritoneal macrophages were stimulated with lps ( 500 ng / ml )+ ifnγ ( 5 ng / ml ). physalin b ( 2 μg / ml ) was added 0 , 2 , 4 and 6 hours after ifnγ + lps stimulation . cell - free supernatants were collected 24 hours after stimulation and tested for nitrite production by griess assay . ifnγ + lps was used as control . fig5 illustrates the time - dependence of the inhibition of nitric oxide production by physalin b . in a fourth test , thyoglycollate - elicited peritoneal macrophages were stimulated with lps ( 500 ng / ml )+ ifnγ ( 5 ng / ml ) in the presence of physalin b ( 2 μg / ml ) or indomethacyn ( 1 μg / ml ). cell - free supernatants were collected 24 hours after stimulation and tested for nitrite production by griess assay . fig6 demonstrates the fact that the addition of indomethacyn does not block the inhibition of nitric oxide production by physalin b . in a fifth test , balb / c spleen cells were stimulated with cona ( 1 μg / ml ) in the presence of several concentrations of physalin b . after 24 hours , cultures were pulsed with 3 h thymidine . proliferation was assessed by measurement of thymidine uptake . fig7 demonstrates the fact that physalin b inhibits proliferation of cona stimulated splenocytes . in a sixth test , balb / c spleen cells were stimulated with cona ( 1 μg / ml ) in the presence of several concentrations of physalin b . cell - free supernatants were collected 24 hours after stimulation and tested for il - 2 production by elisa . fig8 demonstrates the fact that physalin b inhibits il - 2 production by cona stimulated splenocytes . in the last test , balb / c spleen cells or thyoglycollate - elicited peritoneal macrophages were cultured in the presence of several concentrations of physalin b . after one day ( fig9 b ) or three days ( fig9 a ) of culturing , plates were pulsed with mtt . cell - viability was evaluated 2 hours later by optical density measuring at 570 nm . in this test , it is demonstrated that physalin is non - toxic in vitro at concentrations below 5 μg / ml .
the present invention relates to a process for obtaining physalis angulata extracts , particularly to isolate ergostane - type steroidal derivatives , such as physalins , which are useful in the treatment of infections caused by protozoans . the physalins of the invention have also prophylactic and therapeutic properties for treating auto - immune diseases in mammals . the isolation process to obtain physalins comprises the steps of : grinding roots , leaves or stem - bark of physalis ssp ; extracting the material obtained in step with solvents selected from the group consisting of water and alcohols , such as methanol , ethanol , 1 - propanol , 2 - propanol , isobutanol ; evaporating the extract obtained in step and washing the syrup material with a suitable solvent ; rapid filtering of the extract of step and fractionating the components of the extract according to a polarity gradient of organic solvents ; and separating the steroidal components contained in the fractions by chromatography .