optical rotations were 
 measured on an auto pol iii automatic polarimeter ( rudolph research , 
 flanders , nj , usa ) at room temperature .
1d and 
 2d nmr data were recorded on a varian 500 mhz instrument with tms 
 as internal standard .
hresims data were acquired using a synapt g2-s 
 qtof mass spectrometer ( waters , milford , ma , usa ) .
semipreparative 
 hplc separations were performed on a hitachi elite lachrom system 
 consisting of an l2130 pump , an l-2200 autosampler , an l-2455 diode 
 array detector , and a phenomenex luna c18 column ( 250  
 10 mm , s-5 m ) , all operated by ezchrom elite software .
all 
 solvents were of acs or hplc grade and were obtained from sigma - aldrich 
 ( st .
louis , mo , usa ) through wilkem scientific ( pawcatuck , ri , usa ) . 

silica gel ( 230400 mesh , sorbent technologies ) , sephadex lh-20 
 gel ( amersham biosciences ) , and mci gel ( chp20p , 63150 m , 
 m & m industries inc . )
standards 
 of d - glucose and metformin were purchased from sigma - aldrich 
 ( st . louis , mo , usa ) .
n. sativa seeds were 
 collected and authenticated by matching the macroscopic and microscopic 
 characteristics to internal monograph and botanical reference standards 
 by verdure sciences ( noblesville , in , usa ) . a voucher specimen ( vs - nssp-001 ) 

has been deposited in the heber - youngken herbarium and greenhouse , 
 college of pharmacy , university of rhode island .
the air - dried , ground powder 
 of n. sativa seeds ( 2.0 kg ) was extracted with n - hexane ( 4 l  3 ) by maceration at room temperature 
 ( 3 days each time ) to afford 298.5 g of crude n - hexane 
 extract .
the residue was dried and extracted with meoh ( 4 l  
 3 ) by maceration at room temperature ( 3 days each time ) to yield 123.1 
 g of meoh extract . to further defat this extract , a portion ( 122.0 
 g )
was reconstituted in meoh ( 500 ml ) and partitioned with n - hexane ( 500 ml  3 ) to yield n - hexane 
 ( 12.3 g ) and meoh ( 110.0 g ) extracts , respectively .
the meoh extract 
 ( 108.0 g ) was chromatographed over a column of mci gel ( meoh  h2o , 50:50 to 100:0 , v / v ) to yield five fractions ( a  e ) . 

fraction b ( 5.2 g ) was subjected to separation on a sephadex lh-20 
 column ( 3  70 cm ) , eluted with isocratic meoh , to obtain three 
 fractions , b1b3 . fraction b2 ( 1.3 g )
was separated by silica 
 gel chromatography ( cc ) ( 4.5  40 cm ) eluted with a gradient 
 of chcl3meoh ( 10:1 to 1:1 v / v ) to obtain six subfractions , 
 b2a  b2f .
purification of subfraction b2c ( 194.4 mg ) by semipreparative 
 hplc , eluting with meoh 
( 016 min : 10:90 
 to 41:59 ; 1617 min : 41:59 to 100:0 ; 1718 min : 100:0 ; 
 1819 min : 100:0 to 10:90 ; 1926 min : 10:90 ; v / v , 3 
 ml / min ) , yielded nigeglanine ( 5 ) ( 1.8 mg ) .
fraction d 
 ( 3.5 g ) was purified by semipreparative hplc by eluting with meoh 
h2o ( 017 min : 20:80 to 55:45 ; 1718 min : 55:45 
 to 100:0 ; 1820 min : 100:0 ; 2021 min : 100:0 to 20:80 ; 
 2128 min : 20:80 ; v / v , 3 ml / min ) to yield compounds 1 ( 9.8 mg ) , 2 ( 2.0 mg ) , nigellidine ( 3 ) 
 ( 3.6 mg ) , 4-o - methylnigellidine ( 4 ) 
 ( 7.4 mg ) , and 4-o - methylnigeglanine ( 6 ) ( 12.6 mg ) .
yellow , amorphous 
 solid ; [ ]d 11 ( c 0.002 , meoh ) ; uv ( meoh ) max ( log  ) 330 
 ( 4.52 ) , 278 ( 4.39 ) , 228 ( 3.91 ) nm ; ir max 3345 , 
 1618 , 1581 , 1456 , 1382 , 860 cm ; h 
 nmr and c nmr data , see table 1 ; hresims m / z 471.2129 [ m ] ( calcd for c25h31n2o7 , 471.2126 ) .
colorless , 
 amorphous powder ; 
 [ ]d 14 ( c 0.008 , 
 meoh ) ; uv ( meoh ) max ( log  ) 285 ( 4.28 ) , 226 
 ( 3.95 ) nm ; ir max 3415 , 1678 , 1613 , 1598 , 1385 , 
 878 cm ; h nmr and c nmr 
 data , see table 1 ; hresims m / z 313.1554 [ m ] ( calcd for c18h21n2o3 , 313.1547 ) . compound 1 ( 2 mg ) was added to a mixture of concentrated 
 hcl ( 0.5 ml ) , h2o ( 1.5 ml ) , and dioxane ( 3 ml ) and refluxed 
 for 2 h. after completion of the reaction ( monitored by tlc ) , h2o was added to the reaction mixture , which was extracted with 
 chcl3 ( 3  5 ml ) .
the aqueous layer was neutralized 
 with nahco3 , concentrated to dryness under reduced pressure , 
 and purified by sephadex lh-20 chromatography to give a sugar fraction . 

the sugar fraction was determined to be d - glucose by co - tlc 
 and comparison of specific rotation with an authentic sample ( rf = 0.45 , chcl3meoh 
h2o , 1:1:0.1 , v / v / v , positive value for specific rotation ) .
hepg2 cells purchased from american type 
 culture collection ( manassas , va , usa ) were maintained in a high - glucose 
 ( 4.5
g / l ) dulbecco s modified eagle s medium culture 
 medium supplemented with 10% fbs , 2 mm glutamine , 1000 u / l penicillin , 
 and 100 mg / l streptomycin at 37 c , 5% co2 .
cells were detached from 
 the culture flask with a solution of 0.25% trypsin and 1 mm edta . 

the 
 cells were seeded into a 96-well plate at a density 4.0  10 cells / well and cultured for 8 h. the cells were incubated 
 with the low - glucose ( 1 mg / l ) detection medium supplemented with 2 
 mm glutamine and 1% fbs . after overnight incubation in the detection 
 media ,
the cells were treated for 7 h with metformin ( 1 mm ) or the 
 isolates ( at 25 m ; stock solutions made in dmso ) diluted in 
 the detection medium .
the glucose concentration in the medium was 
 determined by a glucose assay kit ( eton bioscience ) as per the manufacturer s 
 instructions .
absorbance was measured at 490 nm , and the assay was 
 performed using 3 replicates per test sample .
the cells were 
 seeded into a 6 well plate for 8 h followed by overnight incubation 
 in low glucose media .
after 24 h treatment with metformin ( 1 mm ) or 
 the test compounds 1 and 6 ( at 25 and 100 
 m ) , 
 total proteins were isolated using ripa buffer and quantified by the 
 bichonic assay ( pierce , rockford , il ) .
protein homogenates ( 20 g / lane ) 
 were electrophoretically separated by 8% sds - page and then transblotted 
 onto immobilion pvdf membrane ( millipore emd corporation , billerica , 
 ma ) .
membranes were blocked in 5% non - fat dry milk followed by incubation 
 with primary antibodies p - ampk , ampk and -actin ( cell signaling 
 technologies , danvers , ma ) overnight .
membranes were washed 3 times 
 with tris - buffered saline with 0.1% tween 20 ( tbst ) followed by incubation 
 with respective secondary horse radish peroxidase - conjugated antibodies 
 ( sigma - aldrich , st .
louis , mo ) for 1 h. after washing the membranes 
 3 times with tbst , bands were detected on x - ray films using an ecl 
 chemiluminescence detection kit ( ge healthcare , piscataway , nj ) according 
 to the manufacturer s protocol ..
 six rare naturally occurring indazole - type 
 alkaloids including 
 two new compounds , 17-o-(-d - glucopyranosyl)-4-o - methylnigellidine ( 1 ) and nigelanoid ( 2 ) , and four known compounds ( 36 ) were isolated from a defatted extract of nigella 
 sativa ( black cumin ) seeds . 
 17-o-(-d - glucopyranosyl)-4-o - methylnigellidine ( 1 ) increased glucose consumption by liver hepatocytes ( hepg2 
 cells ) through activation of amp - activated protein kinase ( ampk ) . 
 
 also , this is the first report of compounds 4 and 6 from a natural source . 
Experimental Section