mirabilis jalapa l. ( nyctaginaceae ;  z m l ) is a traditional medicine widely used in many parts of the world for the treatment of various diseases .
it is a perennial herb , which reaches a height of 50100  cm from the tuberous root .
it produces beautiful flowers that usually open at around 4 oclock in the afternoon , and hence , its common name is four o'clock plant . it is a popular ornamental plant grown worldwide for the beauty of its flowers ( which can be white , pink , yellow , or multicolored ) , which have a sweet fragrance . traditionally , the tuberous root of m.  jalapa has been used for treatment of carbuncles ( a skin infection caused by staphylococcus aureus ) .
phytochemical investigation of the extracts from this plant showed that it is rich in many active compounds including triterpenes , proteins , flavonoids , alkaloids , and steroids .
alanine , alpha - amyrins , arabinose , beta - amyrins , campesterol , daucosterol , and dopamine were the other compounds reported from extracts of this plant .
gas chromatography ( gc)/mass spectral analysis of dichloromethane and methanol extracts of m.  jalapa tubers indicated that oleic acid and beta - sitosterol , respectively , were the major compounds present .
liquid chromatography / mass spectroscopy analysis of the aqueous extract of the tuberous root of the plant showed a high content of flavonols .
phenolic acids such as ferulic acid and caffeic acid were also detected in the plant extracts .
it has been also reported that the water extract of m.  jalapa tubers , containing higher amounts of flavonoids , exhibits antimicrobial and antioxidant activities . in comparison with terpenoid and flavonoid fractions from other parts of the plant , better antioxidant capacity and antimicrobial activity of these fractions from the tuberous root of m.  jalapa
thus , it was observed that most studies were carried out using a crude extract of a particular solvent or a fraction to determine either various functional activities or the content of major compounds . in this investigation
thin - layer chromatography ( tlc ) silica gel 60 f254 , chemicals , and solvents of analytical grade were purchased from merck , himedia , and fisher scientific ( mumbai , maharastra , india ) .
mirabilis jalapa l. (  z m l ) was collected from locally grown flower gardens in tezpur ( 9148e and 2638n ) , assam .
voucher specimen was authenticated with the help of the botanical survey of india , shillong ( no .
the tuberous roots collected were thoroughly washed with tap water and finally rinsed with distilled water .
approximately 50  g of ground sample was extracted with 500  ml of 80% methanol for 48 hours .
extraction was repeated and the extracts were pooled and filtered through whatman number 1 filter paper .
the filtrate was concentrated under reduced pressure in a rotary vacuum evaporator ( rv10 control , ika , germany ) .
the concentrated extract was air dried to a constant weight ( 16.1% yield ) at room temperature .
approximately 10  g of aqueous methanolic extract of m.  jalapa was dissolved in 100  ml of methanol , which was concentrated to one tenth of its volume , and acidified with 10  ml of 2 m h2so4 .
the remaining aqueous methanol solution was basified with nh4oh and extracted three times with ethyl acetate .
the remaining basified aqueous methanol was further extracted three times with isoamyl alcohol and this extract was named fraction 3 ( f3 ) .
the remaining extract was termed as fraction 4 ( f4).4 , 5 phytochemical screening indicated steroids and triterpenoids as the major compounds in f1 , and flavonoids as the major compound in f3 . f1 and f3 of aqueous methanolic extract were subjected to tlc studies for isolation of potential free - radical scavenging and antimicrobial spots .
silica gel h without caso4 was used as the adsorbent for collection of spots in tlc .
the tlc plates were prepared and heated for activation in an oven for 30 minutes at 110c .
the precoated tlc plates were also used for observation of spots after collection . approximately 10  mg / ml of f1 and f3 in methanol and 5 
mg / ml of standard solutions ( -sitosterol , betulinic acid , ursolic acid , oleanolic acid , rutin hydrate , quercetin , kaempferol , and chlorogenic acid ) in methanol were prepared for spotting on the prepared tlc plates .
the plates were spotted with 510 l of samples ( f1 and f3 ) and standards , and dried with a light stream of air while spotting .
the following six different solvent systems were used as developing systems : hexane : ethyl acetate : methanol ( 5:3:2 ) ; benzene : methanol ( 45:55 ) ; chloroform : methanol : acetic acid : water ( 5:4:0.2:1 ) ; 1-butanol : acetic acid : water ( 4:1:5 ) ; petroleum ether : chloroform : methanol for terpenoids ; and chloroform : ethyl acetate : formic acid for flavonoids.5 , 6 the plates were air dried and visualized by spraying with vanillin 
sulfuric acid spray followed by heating for 5 minutes at 110c for detection of steroids and triterpenoids .
spots were separated by running on preparative tlc plates in the selected solvent systems and collected by scrapping silica from the tlc plate for further study .
precoated tlc plates ( tlc silica gel 60 f254 ) were used for detection of free - radical scavenging activity of the collected spots . the petroleum ether : chloroform :
the plates were developed by spraying with 0.2% of 2,2-diphenyl-1-picrylhydrazyl ( dpph ) solution in methanol , followed by 30-minute incubation in the dark .
yellow or colorless spots against a purple background showed the presence of free - radical scavenging activity of the spots.8 , 9 the experiment was conducted in n  = 4 for each sample .
potential free - radical scavenging spots were further tested against s.  aureus ( mtcc338 ) and candida albicans ( mtcc854 ) by agar diffusion method .
the presence of a clear zone against a full - grown pathogen plate shows the antimicrobial activity of the spot .
gc ( perkin elmer , germany ) of the active spot was conducted by applying the following run conditions : temperature , 80c for 5 minutes ; ramp , 5c / min to 300c ; hold time , 10 minutes ; inlet temperature , 320c ; volume injected , 1  l ; split , 50:1 ; carrier gas , helium ; solvent delay , 8 minutes .
fourier transmission infrared spectra ( ftir ) of potential antioxidant spots were recorded on a modified ftir spectrophotometer ( attenuated total reflectance ; nicolet is5 ; thermo fisher scientific , madison , wi , usa ) with a scanning speed of 1  cm / s , a number of 32 scans , and a resolution power of 4 .
spots having both free - radical scavenging and antimicrobial activities were tested with ethanolic ferric chloride ( fecl3 ) for flavone / flavonol content : 50 l of sample dissolved in methanol was taken to which 10 l of 10% ethanolic fecl3 was added .
approximately 50 l of sample in methanol was treated with 0.2  ml of concentrated h2so4 , and shinoda test ( flavones / flavonol / flavanone ) was carried out .
magnesium ribbons were added to 50 l of the sample in methanol followed by addition of concentrated h2so4 .
aluminum chloride colorimetric method was performed to detect the presence of keto and hydroxyl groups in the ring of flavone nucleus .
the f1 and f3 fractions of mirabilis jalapa l. radix (  z m l gn ) showed the presence of terpenoids and flavonoid , respectively . better separation of f1  was found in the solvent system that included petroleum ether , chloroform , and methanol ( petroleum ether : chloroform : methanol  = 49:50:1 ) .
the presence of steroids and terpenoids ( pink / purple spots ) was detected upon spraying the plates with vanillin 
f3 was separated in the solvent system containing chloroform , ethyl acetate , and formic acid ( chloroform : ethyl acetate : formic acid  = 5:4:1 ) and the fraction showed the presence of flavonoids , which was detected on exposure to ultraviolet rays at 365  nm ( fig . 
the rf of each spot was also calculated and the values are presented in table  1 .
the fifth spot of f1 ( 5f1 ) and the first spot of f3 ( 1f3 ) showed free - radical scavenging activity , as indicated by the development of yellow or colorless spots ( circled spots in fig . 
2 ) against a purple background when sprayed with dpph solution.7 , 8 potential free - radical scavenging spots were further tested against s.  aureus and c.  albicans ( fig . 
the 5f1 spot was active against s.  aureus , but it was not active against c.  albicans .
the 1f3 spot showed activity against c.  albicans , but it was found to be only partially active against s.  aureus ( fig . 
the 1f3 spot was chosen for the gc analysis to evaluate its purity . in the gc analysis
, the 1f3 spot showed a single sharp peak with a retention time of 55.15 minutes in gc chromatogram ( fig . 
the 1f3 spot was brownish yellow , highly amorphous , and soluble in water / methanol .
data on ir spectral transmission bands of the standard ( oleanolic acid ) , 5f1 , and 1f3 spots are presented in table  2 and fig . 
further chemical characterization of the 1f3 spot showed positive results for the shinoda test and for concentrated h2so4 , alcl3 , and fecl3 tests .
these results indicated the presence of a flavone nucleus , which may have a hydroxyl group at c-5 of the a ring ( maximum absorbance at 395  nm and 415420  nm with the aluminum chloride colorimetric test ; fig . 
in this study , the f3 fraction of the hydromethanolic extract of mirabilis jalapa l. radix (  z m l gn ) was selected for isolation of bioactive components based on its antioxidant capacity and antibacterial activity .
the f1 fraction was selected because it was detected in good amounts while screening for major phytochemicals .
many studies have also reported terpenoids from the aerial parts and tubers of m.  jalapa.1 , 2 tlc is a very basic and easy method for separation of phytochemicals .
however , to achieve proper separation of different phytochemicals , one has to experiment using different solvents and combinations ( trial and error ) to standardize the tlc developing system suitable for a particular plant extract .
we have tried various combinations of different solvents , and better separation of triterpenoids and steroids in f1 was achieved with petroleum ether : chloroform : methanol ( 49:50:1 ) compared with hexane : ethyl acetate ( 1:1 ) .
presence of -sitosterol was detected in f1 with petroleum ether : chloroform : methanol ( 4.9:5:0.1 ) in our investigation , which is in line with earlier reports.1 , 3 the -sitosterol was reported as a major component in the aerial part .
separation of f3 components was achieved using the chloroform : ethyl acetate : formic acid ( 5:4:1 ) solvent system .
tlc - separated spots of f1 and f3 were subjected to free - radical scavenging and antimicrobial screening .
the fifth spot of f1 ( 5f1 ) and the first spot of f3 ( 1f3 ) showed free - radical scavenging activity ( fig . 
2 ) , which was in line with the findings of hajji et  al , who reported that m.  jalapa radix can be used for the preservation of food stuffs due to its antioxidant and antimicrobial activities .
we also tested the effects of 5f1 and 1f3 against s.  aureus and c.  albicans .
although the 5f1 spot was active against s.  aureus in accordance with an earlier report , it was not active against c.  albicans ( fig .  3 ) .
various studies have demonstrated the better antioxidant capacity and antimicrobial activity of flavonoid - rich fractions from the tuberous root of m.  jalapa compared with fractions from other parts,2 , 4 which supports our finding that the 1f3 spot of flavonoid fraction was active against both c.  albicans and s.  aureus . according to gogoi et  al , although the f1 and f3 fractions were active against these pathogens , f3 showed a lower minimum inhibition concentration and bigger zone of inhibition against c.  albicans than f1 .
the antimicrobial activity of 5f1 and 1f3 followed the same trend as that of the f1 and f3 fractions .
better antimicrobial activity in the f3 fraction than in the separated spot ( 1f3 ) might be due to the synergistic effect of other components .
the ir spectra of the 5f1 spot and standard showed similar stretching and bending band patterns at 2918.58 cm , 2848.99 cm , 1725.54 cm , 1630.25 cm , 1560.18 cm , 1444.04 cm and 2941.36 cm , 2869.75 cm , 1719.11 cm , 1690.83 cm , 1466.56 cm , respectively .
the ir spectra are interpreted by observing the following banding patterns : stretching bands at 30002800 cm indicate the presence of alkyl groups and shoulder peaks of the carbonyl group are seen in the 28502750 cm range .
a peak of the carbonyl group of the aromatic three- to four - membered ring is observed between 1750 cm and 1700 cm .
peaks between 1600 cm and 1400 cm indicate the presence of aromatic c = c groups . by observing these patterns
, it can be said that the 5f1 spot may be a triterpenoid having a carbonyl group .
the ir spectra of the 1f3 spot indicated that it may be an aromatic compound ( 2929.38 cm and 1560.02 cm ) having carbonyl ( 1725.25 cm ) and c  co  c groups ( 1122.28 cm and 1286.88 cm ) .
a search of the ir spectra of 1f3 in the national institute of standards and technology library indicated that it may be a flavone , which is hydroxylated in ring a. cultured m.  jalapa has been reported to contain 5,7-dihydroxy-2-methoxy-6-methylisoflavone ( 2-o - methylabronisoflavone ) as one of the components showing inhibitory activity against c.  albicans .
flavone ( 1f3 ) was identified as one of the components of m.  jalapa tuberous root in this study .
the site(s ) and number of hydroxyl groups on the phenol group are thought to be related to their relative toxicity to microorganisms .
existing evidence suggests that increased hydroxylation results in increased toxicity owing to the formation of intramolecular hydrogen bridges , which increase the lipophilic character , and thus , allows for easier penetration through the cell wall.15 , 16 the hydrophilicity ( free hydroxyl group ) of kaurene diterpenoids was increased by adding a methyl group .
thus , in comparison with the terpenic / steroid component ( 5f1 ) of f1 , the flavone molecule ( 1f3 ) of f3 showed better free - radical scavenging and antimicrobial activities , which may be due to the presence of the free hydroxyl group .
1f3 exhibited both antibacterial and antifungal activities , but 5f1 was active only against bacteria .
the ir spectral analysis coupled with the ir library search indicated 1f3 and 5f1 to be a flavone and a triterpenoid , respectively .
further studies on 1f3 may be taken up to explore the possible use of these compounds in the treatment of skin afflictions caused by s.  aureus and other pathogens .
 the present investigation was carried out to isolate and characterize bioactive components from mirabilis jalapa l. radix (  z m l gn ) . 
 thin - layer chromatography was used for the separation of spots from fractions of the crude extract . 
 separated spots were collected for identification of their activities . 
 free - radical scavenging activity was evaluated by spraying thin - layer chromatography plates ( spotted with fractions ) with 0.2% of 2,2-diphenyl-1-picrylhydrazyl solution . 
 activity against human pathogens such as staphylococcus aureus and candida albicans were determined using the agar diffusion method . 
 potential spots were subjected to infrared ( ir ) analysis and gas chromatography for characterization . 
 two spots ( 5f1 and 1f3 ) showed free - radical scavenging activity . 
 the 1f3 spot was active against both s.  aureus and c.  albicans , whereas the 5f1 spot was active against s.  aureus only . 
 ir spectral analysis indicated that 5f1 spot to be a triterpenoid . using ir spectral analysis and an ir library search , 
 the 1f3 spot was identified to be a flavone , which may have a hydroxyl group in ring  a  of the flavone nucleus . 
 our results indicated that the 1f3 and 5f1 spots are potential free - radical scavengers . 
 both 1f3 and 5f1 exhibited antimicrobial activity . 
 ir spectral analysis coupled with an ir library search indicated 1f3 and 5f1 to be a flavone and a triterpenoid , respectively . 
Introduction
Materials and methods
Results
Discussion
Conclusion
Conflicts of interest