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type 2 diabetes or hyperglycemia , which is characterized by high blood glucose levels and glycosuria , is an endocrine disorder .
it may occur for two main reasons : ( 1 ) deficient / insufficient secretion of insulin by the pancreatic beta cells ; or ( 2 ) decreased or lost sensitivity of the insulin receptors .
these two factors are preceded by stress , obesity , and a sedentary lifestyle.3 , 4 at present , diabetes management is a prime concern in the medical community . as a result of various factors ,
diabetes management is a challenging problem.5 , 6 , 7 , 8 however , many medicinal plants in ancient indian medicine ( ayurveda ) have been identified and used to reduce the hyperglycemic condition in the body .
the herbs control the hyperglycemic condition and effectively maintain normal glucose levels from a long - term perspective .
many medicinal plants have recently been evaluated for their antihyperglycemic property and have been successfully proven for their effects.9 , 10 , 11 numerous polyherbal formulations are also being evaluated for effective diabetes management .
thus , a new formulation has been developed that combines six different herbs that are used in daily life as foods and have significant antidiabetic activity .
we combined the six plants based on previously available literature , indigenous knowledge , and various preliminary studies .
our aim was to investigate the antidiabetic potential of the polyherbal formulation ( i.e. , adj6 ) and prove its synergistic effect against various aspects involved in diabetes management .
chemicals such as soluble starch and porcine pancreatic -amylase ( ppa ) were purchased from srl pvt . ltd .
-glucosidase , 3,5-dinitrosalicylicacid ( dnsa ) , and 2,4-dinitrophenyl hydrazine ( dnph ) were obtained from himedia laboratories ( mumbai , india ) .
para - nitrophenyl -d - glucopyranoside and standards such as gallic acid , quercetin , and ascorbic acid were purchased from sigma - aldrich co. ( st .
the herbs were collected from the medicinal farm frontier mediville ( elavur , gummidipoondi , india ) and were submitted to the plant anatomy research centre ( tamil nadu , india ) for authentication .
the plant names , families , and parts used and their medicinal properties are listed in table  1 . the plant parts were minced individually using a mixer .
the individual plants were then freeze - dried using a lyophilizer to minimize the loss of bioactive components .
the freeze - dried powder was stored in an air - tight container at room temperature until further use .
the traditional ayurvedic methods of preparing herbal formulations are primarily aqueous ( it is likely that the peptides , proteins and glycans possessing antidiabetic activity would be denatured in organic solvents . ) .
the powders of each plant were mixed together in a specific proportion , soaked in water for 24 hours , and filtered .
the assay mixture consisted of 500  l of 0.02 m sodium phosphate buffer [ containing 6 mm sodium chloride ( nacl ) , ph 6.9 ] , 0.05 units of ppa solution , and adj6 at a concentration of 0.11.5  mg / ml ( w / v ) .
after incubation , 250  l of 0.5% ( v / v ) starch solution in the aforementioned buffer was added to the tubes and incubated for 15 minutes at 37c .
the reaction was terminated by adding 1  ml of dinitrosalicylic acid reagent and then incubated in a boiling water bath for 10 minutes .
the tubes were cooled and the absorbance was measured at 540  nm ( shimadzu uv - vis 1800 spectrophotometer ; shimadzu corporation , kyoto , japan ) . a tube with ppa but without adj6 served as the control with 100% enzyme activity .
the assay mixture consisted of 150  l of 0.1 m sodium phosphate buffer ( containing 6 mm nacl , ph 6.9 ) , 0.1 unit of -glucosidase , and adj6 at a concentration of 0.11.5  mg / ml ( w / v ) . the assay mixture was preincubated at 37c for 10 minutes .
after incubation , 50  l of 2 mm para - nitrophenyl -d - glucopyranoside in 0.1 m sodium phosphate buffer was added to the mixture and incubated at 37c for 25 minutes . the reaction was terminated by adding 50  l of 0.1 m sodium carbonate ( na2co3 )
. the yellow color that developed was measured at 405  nm ( bio - rad microplate reader ; bio - rad laboratories , california , usa ) . the tube with -glucosidase but without adj6 served as the control with 100% enzyme activity , and acarbose served as the positive control.%  inhibition = absorbance  of  controlabsorbance  of  extractabsorbance  of  control    100 the formulation adj6 was analyzed for the presence of amino acids , steroids , cardiac glycosides , phenols , tannins , terpenoids , alkaloids , flavonoids , saponins , carbohydrates , reducing sugar , and anthrones .
one milliliter of sample ( 0.21  mg / ml ) was added with 0.5  ml of folin  ciocalteau reagent and incubated at room temperature for 10 minutes .
furthermore , 2.5  ml of saturated na2co3 solution was added and incubated at room temperature for 30 minutes .
the resultant color was measured at 750  nm versus a blank containing distilled water and folin  ciocalteau reagent .
total phenolic content was expressed in gallic acid equivalents using the equation t  =  cxv / m ( t  =  total phenolic content ( mg / g ) of extract as gallic acid equivalents , c  =  concentration of gallic acid established from the calibration curve , v  =  volume of the extract solution in ml , m  =  weight of extract in grams ) .
one milliliter of sample ( 0.21  mg / ml ) was added with 0.5  ml of 1.2% aluminum chloride in 10% methanol , 0.5  ml of 1 m potassium acetate , and made up to 3  ml with distilled water .
the mixture was incubated for 30 minutes in the dark , and the absorbance was read at 415  nm .
vitamin c was determined using the method described by roe , but with modification , using ascorbic acid as the reference compound .
fifty microliters of sample ( 0.21  mg / ml ) was added with 150  l of 5% tca ( trichloroacetic acid ) .
the mixture was added with 500  l of 10 mm dnph and 500  l of 4% thiourea and incubated at room temperature for 3 hours .
postincubation 2.5  ml of 85% sulfuric acid was added and the absorbance was measured at 530  nm .
the data are expressed as the mean    the standard deviation ( sd ) .
-amylase ( ec 3.2.1.1 ) randomly cleaves the -(14 ) glycosidic linkages of amylase to yield dextrin , maltose , or maltotriose , whereas -glucosidase ( ec 3.2.1.20 ) hydrolyzes the terminal nonreducing 1 - 4 linked -glucose to release glucose molecules .
incubation of the -amylase with the substrate leads to the generation of maltose ; however , the addition of the adj6 [ inhibitory effect ( ic50 ) , 0.41    0.03  mg / ml ) significantly inhibited the liberation of maltose in a dose - dependent manner , compared with acarbose ( ic50 , 0.2    0.01  mg / ml ) ( fig .  1 ) .
the ic50 of -glucosidase was 0.51    0.01  mg / ml , whereas that of acarbose was 0.39    0.02  mg / ml ( fig .  2 ) .
the aqueous extract of the formulation also showed the presence of major phytochemical constituents such as phenols , flavonoids , steroids , cardiac glycosides , and saponins ( table  2 ) .
the aqueous extract of the formulation was estimated for total phenol , flavonoids , and vitamin c content .
the amount of phenols was 340    2.5  mg / g equivalents of gallic acid .
the amount of flavonoids was 235.4    8.3  mg / g of quercetin equivalents , whereas the level of vitamin c was the highest at 470.8    16.6  mg / g of ascorbic acid equivalents ( fig . 
the analyses suggested that the ic50 values of adj6 against -glucosidase and -amylase at various concentrations were less than those for the standard drug acarbose .
acarbose is a potent inhibitor of -amylase and -glucosidase , but adverse effects have occurred in the gastrointestinal tract because of excessive inhibition of the amylase enzyme that results in flatulence and diarrhea .
use of acarbose for the treatment of diabetes by dietary control has reportedly resulted in hepatitis.52 , 53 , 54 acarbose treatment also elevates liver enzyme levels , whereas its withdrawal normalizes levels .
there has recently been a push to find an alternative source of treatment for type 2 diabetes , especially from medicinal plants that have high potency and fewer adverse effects than preexisting drugs.56 , 57 , 58 , 59 , 60 , 61 excessive inhibition of pancreatic amylase can lead to abnormal bacterial fermentation of carbohydrate foods in the colon , which may lead to adverse digestive disorders.51 , 62 plant - based compounds and products derived from natural sources have been reported for their potent inhibitory activities of the enzymes , but have fewer or no side effects.56 , 57 , 58 , 59 , 60 , 61 the polyherbal formulation adj6 , although it has slightly less inhibitory activity than acarbose , will likewise have fewer or no adverse effects because it is completely derived from plant sources . because adj6 is rich in phytochemical constituents ( i.e. , phenols , flavonoids , and vitamin c )
, it will be helpful in the management of diabetes and will decrease the risk of other chronic disorders that are associated with diabetes .
the present study elucidates that the polyherbal formulation adj6 can be used for effectively managing type 2 diabetes mellitus .
the activities of the individual components of the plants are well known for their insulin mimetic role and for their role in reducing insulin resistance .
the plants that are used in the formulation have been long used for food and medicinal purposes .
our results suggest that adj6 inhibits the enzymes -amylase and -glucosidase and contains a high amount of phytochemical constituents ( i.e. , total phenols , flavonoids , and vitamin c ) .
thus , adj6 may be used in the management of type 2 diabetes mellitus with few or no side effects . however ,
further studies of adj6 using in  vitro and in  vivo models need to be performed to elucidate its insulin mimetic activity and reduction of insulin resistance .
there has recently been much advancement in the diagnosis , treatment , and research of metabolic disorders , especially diabetes . 
 current research around the world is focused on finding an alternative source of treatment from natural resources for diabetic management , apart from the available synthetic medicines . 
 the present study is a preliminary study of a polyherbal formulation using edible natural resources and an assessment of its antidiabetic activity . 
 the formulation was screened for its phytochemical constituents , total phenols , flavonoids , and vitamin c content . 
 it was also analyzed for its inhibitory effect against the digestive enzymes -amylase and -glucosidase , compared with the standard drug acarbose . 
 the formulation showed the presence of major constituents such as steroids , cardiac glycosides , phenols , flavonoids , and saponins . 
 it also had a high level of phenols ( 340    2.5  mg / g ) , flavonoids ( 235.4    8.3  mg / g ) , and vitamin c ( 470.8    16.6  mg / g ) , and showed a half - maximal inhibitory concentration ( ic50 ) value of 0.41    0.03  mg / ml and 0.51    0.01  mg / ml for amylase and glucosidase , respectively . 
 the results showed that adj6 had a significant inhibitory activity on -amylase and -glucosidase ; however , its inhibitory activity was less than that of acarbose . 
 the plants that are formulated in adj6 possess potent antidiabetic activity . 
 thus , we found that adj6 is a potent lead for effective diabetic management ; however , an evaluation of the formulation must be illustrated using an in  vivo model .