( solanaceae ) , popularly known as wolf - fruit , is native to the brazilian cerrado and is used in folk medicine as an anti - inflammatory , a hypocholesterolemic , a hypoglycemic , a sedative , a diuretic , an antispasmodic , an antiepileptic , and to control obesity . this species belongs to the genus solanum , the largest genus of the family solanaceae . this genus is distinguished by its variety and productivity of alkaloids ; those combined with sugar moieties are known as glycoalkaloids [ 3 , 4 ] . certain glycoalkaloids , such as solasonine ( sn ) and solamargine ( sm ) , stand out economically because their chemical structures are very similar to steroidal hormones and therefore have been proposed to be used as an important source for the production of medicines , such as contraceptives and steroidal anti - inflammatory drugs . however , these compounds might also act as possible antiandrogenic agents and if they cross the placental barrier they could exert a possible effect on development of the embryo . from the alkaloidic extract of the fruits of s. lycocarpum was obtained a mixture of mostly the glycoalkaloids solasonine ( sn ) and solamargine ( sm ) ( figure 1 ) . these compounds bear the same aglycone , solasodine , and differ from each other only in the nature of the involved trioses , namely , solatriose for solasonine and chacotriose for solamargine . these glycoalkaloids have been studied for their antidiabetic , antifungal , antiparasitic , and mostly for their anticancer properties [ 10 , 11 ] , including in vivo human nonmelanoma anticancer . therefore , considering the potential of solanum lycocarpum as a medicinal plant , as a source of compounds for the synthesis of steroids and the development of new pharmaceuticals , as well as the lack of reliable quantitation methods for these compounds , the development of analytical methods for the quantitation of steroidal alkaloids in plant biomass , extracts , and final products would seem mandatory . the aim of this paper was to develop a validated method to quantify the glycoalkaloids solasonine and solamargine in different plant tissues : leaves , stem , ripe and unripe fruits , as well as in the crude plant extracts . hplc grade acetonitrile ( mecn ) and methanol ( meoh ) were obtained from mallinckrodt co. ( xalostoc , mexico ) . anhydrous disodium hydrogen phosphate was acquired from carlo erba reagents ( brazil ) , and deionized water was purified by milli - q - plus filter systems ( millipore , bedford , ma , usa ) . analytical grade ethanol ( etoh ) and methanol veratraldehyde ( 3,4-dimethoxybenzaldehyde ) , internal standard , was provided by merck ( darmstadt , germany ) . solanine , a glycoalkaloid used as secondary standard , was bought from sigma - aldrich inc . ( st . louis , mo , usa ) . authentic compounds , solamargine and solasonine , were kindly provided by dr . james d. mcchesney from ironstone separations , inc with purities estimated to be greater than 96% for both specimens . the leaves , stems , and ripe and unripe fruits of s. lycocarpum were collected in cajur , state of so paulo , brazil , in january of 2008 . milton groppo , department of botany , faculdade de filosofia cincias e letras de ribeiro preto , university of so paulo , sp , brazil , where a voucher specimen was deposited ( spfr : 11638 ) . chopped fruits , leaves , and branches were dried under air circulation in an oven at 45c and powdered in a hammer mill . the particle size was standardized ( mesh 35 ) , and the powdered plant materials were stored in a sealed container in the freezer ( 18c ) until use . a neutral extract was obtained by maceration of 100  g of powdered dried fruit biomass with 200  ml of ethanol / water ( 80% ) at room temperature for 72  h , three times , followed by percolation . the filtered extracts were combined and concentrated under vacuum furnishing 13.64  g of the crude hydroalcoholic extract ( 13.64% of extractable material ) . the alkaloidic extract was prepared using a selective extraction based on the method of henriques et al . . the powdered dried fruits ( 1.0  kg ) of s. lycocarpum were submitted to hydrochloric acid ( 0.2  m ) extraction overnight by maceration , followed by filtration . then , the aqueous acid extract was basified to ph 12.0 using 6.0  m  naoh . after precipitation the supernatant was removed ; the precipitated material was centrifuged and the pellet was suspended in ethanol with shaking . the ethanol soluble fraction was concentrated under vacuum and lyophilized to furnish the alkaloidic extract ( 15.8  g ) . the total ash content of dried plant biomass was obtained by incinerating the samples using the methodology employed by matos , . the total moisture content was determined by loss on drying as described in aoac , , and the data were collected in triplicate . a high performance liquid chromatography ( hplc ) , shimadzu ( kyoto , japan ) instrument consisting of a uv detector , multisolvent delivery system ( lc-10ad ) , autosampler ( sil-10advp ) , controller module ( scl-10avp ) , autosampler and class vp 5.02 software was used . a zorbax sb - c18 analytical reverse phase column ( 250  4.6  mm i.d . ; particule size 5  m ) ( agilent technologies , usa ) , coupled with a guard column from the same company was used . the sample analyses were carried out employing an isocratic elution system using a mobile phase composed of acetonitrile and sodium phosphate buffer ( ph 7.2 ; 0.01  m ) in a ratio of 36.5  :  63.5 ( v / v ) at a flow rate of 1  ml / min . a 20  l aliquot of each sample was injected and a run time of analysis of 20  min with detection at 200  nm was employed . for the quantitation of solasonine and solamargine in plant biomass , an aliquot of 250  mg of the powdered material were extracted in three replicates in a shaker ( 120  rpm/30c/2  h ) , using 20  ml of 80% aqueous etoh containing 3  g  ml of veratraldehyde ( is ) . for both the dried crude hydroalcoholic extract ( 42  mg ) and the dried alkaloidic extract ( 4  mg ) , the samples were directly dissolved in 20  ml of is solution . all the samples were filtered and analyzed by hplc - uv using the same conditions according to the analytical method developed . after analysis , the areas corresponding to veratraldehyde ( is ) , solamargine and solasonine were used to quantify the target compounds in the dried plant biomass . the results are reported as means  s.d ( standard error of the mean ) . the difference between the content in ripe and unripe fruits was determined using the unpaired t - test . significant differences were considered for p values < 0.05 . a validated analytical method was developed for the quantitation of the glycoalkaloids solasonine and solamargine in both plant biomass and extracts of s. lycocarpum , considering the parameters described by ribani et al . , anvisa , and ich , . thus , selectivity was performed by comparing the chromatographic profiles of the analytical standards in relation to those obtained for plant biomass samples . for that purpose , the identification of the peaks was assured according to their retention times and by coelution with authentic standards . veratraldehyde ( rt = 7.2  min ) was used as internal standard ( is ) , and it was added to the extracting solvent prior to the extraction ( figure 2 ) . the analytical curves of the standards were prepared in concentrations ranging between 0.77 and 1000.0  g  ml for both glycoalkaloids and between 1.62 and 12.5  g  ml for veratraldehyde . the correlation coefficients ( r ) were determined for each compound , and linear regression obtained and its correlations were used for the deduction of equations to quantify the glycoalkaloids . the limits of detection ( lod ) and quantification ( loq ) were determined based on the parameters of the analytical curves , considering standard deviation of the response ( s ) and the slope of the analytical curve ( s ) . thus , the curves were made in triplicate and values of s and s were applied at equations ld = 3.3  s / s and lq = 10  s / s . the precisions were determined by the evaluation of the repeatability ( intraday ) and by intermediate precision ( interday ) . repeatability was determined by preparation and analysis of the same sample in six replicates evaluated on the same day and by the same technician . intermediate precision was also performed in six replicates at intervals of one day with injections made by two different technicians . accuracy was evaluated by recovery studies using a method of spiking with the chemical markers a previously exhausted matrix consisting of dried and powdered s. lycocarpum leaves ( 250  mg ) . for that , 35  g of plant biomass were exhaustively extracted with 96% aqueous ethanol using a soxhlet apparatus to reduce the content of glycoalkaloids to trace amounts which was confirmed by hplc analysis . after that , the matrix was spiked by adding the glycoalkaloids in solution , in four replicates , at three levels of concentration : low , medium , and high , corresponding to 62.5 , 125.0 and 187.5  g  ml , respectively . then , the spiked matrix was extracted using 20  ml of extraction solvent consisting of 80% etoh containing 3  g  ml of veratraldehyde . after extraction , 1  ml of secondary standard , solanine , at 300  g  ml was added to aliquots of 5  ml of each extract . the resultant solutions were filtered and analyzed by hplc - uv , using the analytical method developed . for that , six factors including extraction time ( a ) , sample size ( b ) , particle size ( c ) extraction temperature ( d ) , stirring ( e ) , and extraction volume ( f ) were evaluated using a combination of eight factors to determine the possible variation in the sample preparation by using different conditions . in table 1 the nominal factors are codified by capital letters and the variations are codified by lower - case letters . as displayed in table 1 , the combination of 1 furnished the result s , and the consecutive results were obtained by taking into consideration the combinations 28 . to determine the variation of a factor , four values corresponding to capital letters and four values corresponding to lower - case letters were considered by statistical analysis comparing the average between the two groups . for example , for the influence of the factor a ( extraction time ) , was evaluated by comparison of the mean group with capital letters [ ( s + t + u + v)/4 ] and the mean group of lower - case [ ( w + x + y + z)/4 ] . phytochemical studies have shown that solanum lycocarpum fruit contain different classes of compounds , such as : phenols and tannins ; glycoalkaloids as solamargine , solasonine , 12-hydroxysolasonine , robeneoside a , and robeneoside b , as well as lobofrutoside and saponins , as lyconoside ia , ib , ii , iii , and iv . however , this method has been developed for the glycoalkaloids solasonine and solamargine , not only because they are major compounds in solanum lycocarpum fruits , but also because of their potential importance for the development of new pharmaceuticals . the determination of moisture and total ash content in plant biomass is of paramount importance for the quality control of medicinal plants , since high values of water favor the action of enzymes which can degrade the active ingredients , and enable the growth of microorganisms . total ash determination allows the verification of nonvolatile inorganic impurities , which may include contaminants such as sand coming from a careless handling during the processing of plant material . the contents of total ash and total moisture in the dried fruits of s. lycocarpum amounted to 2.52%   0.001 and 4.72%   0.774 , respectively . thus , considering the recommended values , the moisture content of our samples was acceptable .   to ensure the quality of plant materials and their products it is necessary to apply validated analytical methods and ensure that the developed methods are selective , accurate , reproducible , and robust for the purposes they are designed . evaluation for selectivity for solamargine and solasonine in the chromatograms of both standard compounds and hydroalcoholic extract revealed no peak of interference . the peaks were identified by comparing the retention times of the analyzed compounds with authentic standards . the retention times obtained for solasonine and solamargine were 10.08  min and 12.08  min , respectively ( figure 2 ) . also , the linear regression coefficients ( table 2 ) displayed values higher than 0.999 for all standard compounds . both glycoalkaloids presented a wide linear dynamic range of 0.77990.0  g  ml for sn and 0.781000.0  g  ml for sm . quantification of the alkaloids was carried out using the linear regression equations obtained for both sn and sm , and linear regression of internal standard ( table 2 ) . the limits of detection and quantification obtained were 0.29 and 0.86  g  ml for solasonine , and 0.57 and 1.74  g  ml for solamargine , respectively , rendering the method sufficiently sensitive for the present purposes . the values obtained for repeatability and intermediate precision ( in rsd % ) for precision ranged from 1.13 to 5.57% for concentrations , and between 2.16 and 2.55% for retention times of sn and sm , respectively ( table 3 ) . the obtained recoveries for solasonine , solamargine , and veratraldehyde ( table 4 ) were higher than 80.92% , reaching 91.71% of recovery with range rsd ( % ) of 0.77 to 5.14% , and error ranging between 8.29 and 19.08% , which indicates that the developed method displays good accuracy . thus , to assess the robustness of an analytical method it is necessary to evaluate the influence of small deliberate variations of the analytical method parameters on the result obtained . then , recognizing that sample preparation and manipulation by the technician is an important source of error , that parameter was considered in the variables described in table 1 . the influence of variability of each parameter was assessed by comparing the results obtained for the nominal conditions and for variations of these conditions . then , these groups were compared regarding quantification of solasonine and solamargine in results obtained with deliberate variations of the parameters . therefore , these data suggest that the developed method is reliable for the quantification of sn and sm considering the evaluated parameters . no detectable amounts of solasonine and solamargine were found in stems and leaves of the species studied sl . these compounds were found in leaves of other solanum species , such as : s. xanthocarpum , s. havanense , s. scabrum , s. lycopersicoides , and s. sodomaeum . however , the fruits of sl bear a significant amount of both glycoalkaloids . also , the amounts of glycoalkaloids were statistically different between ripe and unripe fruits . the unripe fruits displayed significantly higher concentrations of glycoalkaloids in comparison with ripe ones , which furnished , respectively , 1.04% and 0.83% of solasonine and 0.69% and 0.60% of solamargine ( table 5 ) . therefore , from the phytochemical point of view , these quantitative data suggest that when the objective is optimum retrieval attainment of glycoalkaloids , it is more profitable to harvest the fruit while it is still unripe . besides , solasonine and solamargine are present in more than 100 species of the genus solanum , as for instance : s. melongena   and s. incanum . however , it should be pointed out that the amounts of these alkaloids found in other solanum species are not comparable with the amounts found in s. lycocarpum . for instance , the contents of solasonine and solamargine in mg per 100  g in other solanum species fruits were , respectively , s. melongena ( 0.171 and 0.584.5 ) ; s. macrocarpum ( 1623 and 124197 ) ; s. aethiopicum , on wet basis ( 0.411 and 0.584.86 ) ; s. ptycanthum , on wet basis ( 490 and 330 ) ; s. sodomaeum , as total glycoalkaloids ( 830 for ripe fruits and 450 for unripe fruits ) . although the fruits of s. lycocarpum display comparable nutritional values for their contents of sugars , vitamin c , and iron , with other edible fruits , such as banana , pineapple , and orange , it should be taken into consideration that the safety limits for the intake of glycoalkaloids from potato are 0.02% of fresh samples therefore , the amount of total glycoalkaloids in dried fruits of s. lycocarpum , 1.73% and 1.43% , for unripe and ripe fruits , respectively , indicates that the ingestion of these fruits could be toxic . hence , the population should be advised of the risks associated with the intake of this fruit , especially for pregnant women , since there is reported fetotoxic effect in rats , which may affect fetus development . nevertheless , , s. lycocarpum has economical relevance not only for the synthesis of steroid derivatives , due to the presence of the steroidal moiety in these compounds , but also as raw material for the development of new pharmaceuticals . regarding the productivity and economic utilization of this species , we pointed out that sl plant can bear from 40 to 100 fruits per adult individual , and the weight of each fruit varies from 400 to 900  g . also , it bears fruits throughout the year , with the highest productivity between january and july [ 2 , 33 ] . this crop does not require large investments , since it is able to grow and thrive in unfavorable environmental conditions , including low nutrients and acidic soils , as well as its ability to withstand a harsh climate and periods of prolonged drought , as a plant characteristic of the brazilian cerrado . in addition , the fruit harvesting can be ecologically sustainable , since collecting fruits would not impact the environment , if well handled . phytochemical studies revealed that s. lycocarpum fruits , in addition to the glycoalkaloids , contain tannins and phenolics . also , sensorial studies showed that the bitterness or burning associated with the intake of solanum fruits are related to glycoalkaloid content and not with the phenolic compounds . high contents of glycoalkaloids would likely be noticed by humans making consumption of the fruits unacceptable . besides , other studies have reported that higher amount of glycoalkaloids in solanum makes the fruit more toxic and less palatable to frugivorous seed dispersers . therefore , from the ecological point of view , the ripe fruit should be favored by both frugivorous which feed on less toxic fruits and the plant species by having its viable mature seeds spread . the extraction protocol for alkaloids used in our work was very selective , because the content of glycoalkaloids in the obtained extract was quite impressive , corresponding approximately to 90% of the alkaloidic extract , while in both fruit dry biomass and its hydroalcoholic extract it corresponded to 1% and 10% of glycoalkaloids , respectively ( table 5 ) . in conclusion , we have developed a validated analytical method which is reliable and brings an important contribution to the field , since s. lycocarpum fruits do not only contain high amounts of glycoalkaloids and s. lycocarpum has a very good productivity of fruits which could also be used as a crop for the production of steroidal drugs and new pharmaceuticals .
solanum lycocarpum ( solanaceae ) is native to the brazilian cerrado . 
 fruits of this species contain the glycoalkaloids solasonine ( sn ) and solamargine ( sm ) , which display antiparasitic and anticancer properties . 
 a method has been developed for the extraction and hplc - uv analysis of the sn and sm in different parts of s. lycocarpum , mainly comprising ripe and unripe fruits , leaf , and stem . 
 this analytical method was validated and gave good detection response with linearity over a dynamic range of 0.771000.00  g  ml1 and recovery in the range of 80.9291.71% , allowing a reliable quantitation of the target compounds . 
 unripe fruits displayed higher concentrations of glycoalkaloids ( 1.04%    0.01 of sn and 0.69%    0.00 of sm ) than the ripe fruits ( 0.83%    0.02 of sn and 0.60%    0.01 of sm ) . 
 quantitation of glycoalkaloids in the alkaloidic extract gave 45.09%    1.14 of sn and 44.37%    0.60 of sm , respectively .