Research ArticleMiconia sp Increases mRNA Levels of PPAR Gamma andInhibits Alpha Amylase and Alpha Glucosidase
David Mizael Ortiacutez-Martinez1 Catalina Rivas-Morales1
Myriam Angelica de la Garza-Ramos2 Maria Julia Verde-Star1
Maria Adriana Nuntildeez-Gonzalez1 and Catalina Leos-Rivas1
1Facultad de Ciencias Biologicas Universidad Autonoma de Nuevo Leon Pedro de Alba sn Ciudad Universitaria66455 San Nicolas de los Garza NL Mexico2Facultad de Odontologia Universidad Autonoma de Nuevo Leon Eduardo Aguirre Pequeno y Silao sn Mitras Centro64460 Monterrey NL Mexico
Correspondence should be addressed to Catalina Leos-Rivas catalinaleosrivasyahoocom
Received 8 April 2016 Revised 2 June 2016 Accepted 12 June 2016
Academic Editor Menaka C Thounaojam
Copyright copy 2016 David Mizael Ortız-Martinez et al This is an open access article distributed under the Creative CommonsAttribution License which permits unrestricted use distribution and reproduction in any medium provided the original work isproperly cited
Diabetes mellitus is a public health problem worldwide For this reason ethanolic extract ofMiconia sp fromOaxaca Mexico wasselected in search of an alternative against this diseaseThe effect ofMiconia sp onmRNA expression of PPAR120574 on cell line 3T3-L1its effect on alpha amylase and alpha glucosidase lipid accumulation during adipogenesis and cell viability on VERO cells wereevaluated The mRNA levels of PPAR120574 increased on 1393 plusmn 0008 folds lipid accumulation was increased by 2955 withMiconiasp extract and 3457with rosiglitazone and 120572-amylase and 120572-glycosidase were inhibited with IC
50values from 2823plusmn215 120583gmL
and 195 plusmn 015 120583gmL respectively the IC50on antiproliferative activity on VERO cells was 31454 plusmn 4540 120583gmL In case of 120572-
amylase and120572-glycosidase assays IC50(inhibitory concentration 50) refers to necessary extract amounts to inhibit 50of enzymatic
activity On the other hand on antiproliferative activity IC50(inhibitory concentration 50) refers to necessary extract amounts to
inhibit 50 of cell proliferation It was concluded that the compounds present in Miconia sp ethanolic extract increase mRNAexpression of PPAR120574 inhibit 120572-amylase and 120572-glucosidase and increase lipid accumulation It constitutes an alternative as adjuvantin diabetes mellitus treatment therefore we recommend continuing identifying the compounds responsible for its promising invivo antidiabetic activity
1 Introduction
Diabetes mellitus is a chronic metabolic disease considered aserious global public health problem In 2010 approximately285million people suffered from this disease and this amountis expected to double up within the next 20 years [1] Diabetesmellitus type 2 (DM2) is the most common form of diabetesIt is a complex metabolic alteration characterized by aninsulin combination resistance (IR low sensitivity of one ormultiple tissues to insulin) and insulin secretion alteration[2]
The search for new drugs that act against peroxi-some proliferator-activated receptor gamma (PPAR120574) is very
important because ligands of these transcription factorsexhibit multiple biological responses such as decreasing theIR and avoiding high levels of plasm glucose It has beenshown that the adipogenesis process is under the controlof a complex cascade of transcriptional regulatory factorsin which PPAR120574 and other transcriptional factors of CEBPfamily play a fundamental role [3 4]
Enzymes 120572-amylase and 120572-glucosidase found in salivaand the brush border of the small intestine respectively acton hydrolysis oligosaccharides and disaccharides to produceeasy absorption monosaccharides such as glucose For theabove mentioned delaying absorption of glucose throughinhibition of enzymatic hydrolysis of carbohydrates carried
Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2016 Article ID 5123519 6 pageshttpdxdoiorg10115520165123519
2 Evidence-Based Complementary and Alternative Medicine
out by alpha amylase and alpha glucosidase could be anotherform of combating DM [4]
About 80 of the population worldwide use medicinalplants to treat various diseases These constitute a majorsource of drugs about 25 of prescribed drugs worldwideoriginate from plant species [5]
A species of the genre Miconia in Mexican traditionalmedicine is used in south Mexico as an alternative fordiabetes mellitus treatment However such fact has not beenscientifically confirmed yetMiconia is a genus of about 1000species distributed in tropical America and belongs to theMelastomataceae family which has about 4300 species dis-tributed in 166 genera Very few studies on biological activityhave been performed on this genre but it has been shownthat extracts and compounds isolated from species of thegenusMiconia have antibiotic antitumor analgesic and anti-malarial activities A phytochemical analysis of methanolicextract ofMiconia cabucu reveals the presence of glycosylatedflavonoids (quercetin myricetin and kaempferol all withdifferent glycosides) a tannin and rare bioflavonoid Thephytochemical research of M rubiginosa extract led to theidentification of several glycosylated forms such as quercetingallic acid and epicatechin Similarly quercetin myricetinand catechin derivativesM stenostachya were found [6]
2 Materials and Methods
21 Preparation of the Extract Miconia sp aerial part wascollected in Oaxaca Mexico The plant was dried in shadeat room temperature and extraction with ethanol was per-formed by the Soxhlet method (PYREX) for 48 h solvent wasremoved under reduced pressure using a rotary evaporator(Yamato RE-200) Extract yield was determined and thenstored at room temperature until it was used
22 Cell Culture Preparation and Adipocyte DifferentiationMurine cell line 3T3-L1 preadipocytes were used DMEMmedium supplemented with 10 fetal bovine serum (FBS)was used for propagation it was incubated at 37∘C at 5 CO
2
atmosphere until reaching a confluence of 90 The 100confluency was used for adipocyte differentiation It couldbe DMEM with 10 FBS 1 120583M dexamethasone 05mM 3-isobutyl-1-methylxanthine and 10 120583gmL insulin (all brandsfrom Santa Cruz Biotechnology) After 48 hours of incuba-tion (day 2) the differentiation medium was removed andreplaced every 48 hours until maturation of adipocytes (days9ndash11) with maturation medium of adipocytes (DMEM with10 FBS and insulin 10 120583gmL called MM) The maturationwas confirmed by microscope observation of the typicalmorphology of an adipocyte [7ndash10]
23 Expression of PPAR120574 in Adipocytes The study wasperformed in the cell line 3T3-L1 differentiated follow-ing the methodology described above using microplate 6-well culture with an inoculum of 100000 cells per wellin a final volume of 2mL with the corresponding culturemedium At day 10 adipocyte differentiation was observedand replaced in the medium (MM)The following treatmentswere added Group 1 DMEM with 01 ethanol (expression
control group) Group 2 MM supplemented with 1 120583M ofrosiglitazone (positive control) Group 3 MM supplementedwith Miconia sp extract (40 120583gmL) and Group 4 MMonly to observe the effect of the medium After 24 hoursof treatment application of the total RNA extraction theretrotranscription (cDNA) and PCR were performed in realtime to determine the effect on mRNA expression [8ndash10]
The total RNA isolation of treated adipocytes was per-formed with an extraction kit SV Total RNA Isolation SystemZ3100 Promega (following themanufacturerrsquos instructions)and stored atminus80∘Cuntil use Total RNA integrity purity andquantification analysis was made in NanoDrop 8000 Spec-trophotometer withThermo Scientific and an electrophoresisgel (15 agarose)
CDNA synthesis was done with the ImProm-II ReverseTranscription System kit Promega A3800 with 500 ng ofRNA isolated The cDNA was stored at minus20∘C until use
Analysis of mRNA expression PPAR120574 was performedby qRT-PCR using LightCycler 480 II Roche equipmentand 50 ng of cDNA 05 120583M of the primers 125 120583L ofMaxima SYBRGreen qPCRMasterMix (2x) K0251ThermoScientific and the proper amount of nuclease-free water tohave a final volume of 25 120583L The 36B4 was used [Thomson]as reference gene The sequence of the primers used isPPAR120574 forward 51015840-CTGGCCTCCCTGATGAATAAAG-31015840reverse 51015840-AGGCTCCATAAAGTCACCAAAG-31015840 36B4 for-ward 51015840-ACTGGTCTAGGACCCGAGAAG-31015840 and reverse51015840-TCAATGGTGCCTCTGGAGATT-31015840 The relative mRNAexpression was calculated based on the 2minusΔΔCt method
24 Lipid Accumulation in Adipocytes Lipid accumulationwas evaluated on the cell line 3T3-L1 using Oil Red O withmodifications to the method described by other authors Themethod described above was used for cell differentiationMicroculture plates were used having 24 wells with aninoculum of 30000 cells per well leading to a final volumeof 500120583L with culture medium At 48 h after confluence(day 0) the differentiation medium (Dm) was applied tostimulate adipogenesis supplemented with the substances tobe evaluated treatment 1 [Dm (control reference)] treatment2 [Dm supplementedwith 40120583gmL extractMiconia sp] andtreatment 3 [Dm supplemented with 1120583Mrosiglitazone (pos-itive control)] Subsequently the microplate was incubatedfor 48 h with the established conditions After that Dm wasreplaced by MM (day 2) and incubated for 8 d The culturemedium was removed and formalin 10 was used for 1 h tofix monolayer cells After monolayer cells had been washedtwice with distilled water the dye Oil Red O (05 in 60isopropanol) was applied for 15min at room temperatureThecells were washed three times with distilled water the dyeinside the cells was removed with 100 isopropanol and readthe optical density at 540 nm [9ndash12]
25 Inhibitory Activity of 120572-Amylase The enzyme inhibitionwas evaluated by the method of dinitrosalicylic acid 35-(DNS) with some modifications The Miconia sp extractwas applied at different concentrations (25 50 75 100and 125 120583gmL) as a vehicle using phosphate buffer with
Evidence-Based Complementary and Alternative Medicine 3
20mM sodium 67mM sodium chloride at pH 69 120572-Amylase enzyme (Sigma-Aldrich A9857-250KU) was usedat 05UmL concentration with the same buffer as a vehicleStarch 05 in phosphate buffer was used as a substrateAcarbose at different concentrations (3125 625 1250 2500and 5000 120583gmL) was used as a positive control AdditionallyDNS solution was used at 96mM In 15mL conical tubes50120583L of substances was placed to be evaluated treatment1 Miconia sp extracts treatment 2 buffer phosphate with2 ethanol (negative control) and treatment 3 acarbose(positive control) Briefly to the above treatments 50 120583L ofthe 120572-amylase was added and incubated at 37∘C for 10minAfter that to initiate the reaction 50 120583L of the substratewas added immediately and incubated at 37∘C for 15minThen the reaction was stopped by adding 50 120583L of DNS andheated in a water bath at 95∘C for 5min the tubes wereleft to cool at room temperature and the optical densitywas measured at 540 nm The absorbance rates were used tocalculate the percent inhibition of each treatment using thefollowing equation
Inhibition = (Abs control minus Abs treatment)Abs control
times 100 (1)
In this equation ldquoAbs treatmentrdquo is the productrsquos lightabsorption of the enzyme-substrate reaction in the presenceof the Miconia sp or negative control as appropriate ldquoAbscontrolrdquo is the reaction productrsquos light absorption of enzyme-substrate in the presence of phosphate buffer as a treatmentThe percentage inhibition using the statistical package (SPSSv20) allows us to calculate the inhibitory concentration 50(IC50) is the amount required to inhibit 50 of the enzyme
(Sigma-Aldrich A8980-1G) [5 13ndash15]
26 120572-Glucosidase Inhibitory Activity Inhibition of theenzyme was evaluated by the pNPG method (p-nitrophenyl-120572-D-glucopyranoside) with some modificationsMiconia spextract at different concentrations (1 2 3 4 and 5120583gmL)wasused as a vehicle a buffer of sodium phosphate mentionedabove Acarbose at different concentrations (625 125 250500 and 1000 120583gmL) is positive control using buffer sodiumphosphate as a vehicle 120572-Glucosidase enzyme (Sigma-Aldrich G5003-100UN) at a concentration of 02UmLusing phosphate buffer as a vehicle Additionally a prepara-tion of pNPG 2mM (Sigma-Aldrich N1377-1G) was usedas a substrate in the same vehicle After that in a 96-well microplate 25120583L of the substances was mixed withthe enzyme to the assay treatment 1 [Miconia sp extracts]treatment 2 [phosphate buffer with 1 ethanol (negativecontrol)] and treatment 3 [acarbose (positive control)]Besides the above treatments 25120583L of the enzyme suspensionwas added and incubated at 37∘C for 15min immediatelyafter that 50 120583L of pNPG was added and incubated at 37∘Cfor 10min then the reaction was stopped by adding 50 120583Lsodium carbonate (02M)
Finally the optical density at 405 nm was measured Theabsorbance rates were used to calculate the percent inhibitionof each treatment using the following equation
Inhibition = (Abs control minus Abs treatment)Abs control
times 100 (2)
In this equation ldquoAbs treatmentrdquo is the productrsquos lightabsorption of the enzyme-substrate reaction in the presenceof the Miconia sp acarbose or negative control as appro-priate ldquoAbs controlrdquo is the productrsquos light absorption ofthe enzyme-substrate reaction in the presence of phosphatebuffer as a treatment Percent inhibitions using a statisticalpackage (SPSS v20) were used to determine the inhibitoryconcentration 50 (IC
50) that is the amount of treatment
necessary to inhibit the enzyme 50 [4 13ndash15]
27 Cell Proliferation Assay Cell proliferation with MTTcolorimetric method (3-(45-dimethylthiazol-2-y1)-25-diphenyltetrazolium bromide) was evaluatedThe VERO cell(monkey kidney epithelial) lines are widely used in researchto evaluate the effect of chemicals and toxins in mammals[16] 1 times 105 cellswell were seeded in a 96-well microplate andadjusted to a final volume of 200120583L with culture medium(DMEM supplemented with 10 FBS) and incubated at37∘C in an atmosphere of 5 CO
2 After 24 h and until
reaching a layer of 80 confluence the culture mediumwas removed and cells were washed with phosphates (PBS)a buffered saline solution further substances were addedand evaluated treatment 1 [extracts of Miconia sp (625125 250 500 and 1000 120583gmL)] treatment 2 [DMEM with1 ethanol (negative control)] and treatment 3 [cells withculture medium (reference of proliferation)] After that themicroplate was incubated for 24 h at the above conditionsThen 10 120583L MTT (5mgmL) were added to each well andincubated again for 4 h the culture medium was removedand cells were washed with PBS Immediately afterwards200120583L dimethyl sulfoxide (DMSO) was added then theoptical density was measured at 570 nm By the followingformula the percentage of cell proliferation was determined
cell proliferation = Abs treatmentAbs control
times 100 (3)
In this equation ldquoAbs treatmentrdquo is the productrsquos light absorp-tion of the treated cells and ldquoAbs controlrdquo is the productrsquos lightabsorption of cells used as proliferation referenceThe IC
50is
calculated with the percentages of cell proliferation and withthe support of SPSS v20 [17 18]
3 Results
In this study different biological activities of ethanolic extractof Miconia sp were evaluated in their effect on expressionof mRNA PPAR120574 lipid accumulation during adipogenesisthe activity of 120572-amylase and 120572-glucosidase and the effect onproliferation of VERO cell line
In determining the expression level ofmRNAof PPAR120574 inmature mouse adipocytes Group 1 was used as an expressioncontrol which was normalized to a value of 1 Therefore the
4 Evidence-Based Complementary and Alternative Medicine
EC expression controlMM maturation medium
EC MMMicRos06070809
11112131415
mRN
A ex
pres
sion
leve
l (fo
ld ch
ange
)
Ros rosiglitazone 1120583M
lowast
lowastlowast
Mic Miconia sp 40120583gmL
Figure 1 Effect of different compounds on the expression of mRNAPPAR120574 On day 10 the treatments were applied Group 1 DMEMwith 01 ethanol (EC) Group 2 MM and rosiglitazone (Ros) asa positive control Group 3 MM and Miconia (Mic) and Group4 MM On day 11 of adipocyte differentiation the total RNA wasisolated lowast119875 le 005 there is a significant difference with the controlof expression lowastlowast119875 le 005 there is a significant difference with thecontrol of expression and rosiglitazone (119899 = 3)
values above 1 indicate an overexpression or upregulation InGroup 2 the value in the expression levels was 1166 plusmn 0007fold change at the same time in Group 3 a value was 1393 plusmn0008 and finally Group 4 showed a value of 0746plusmn0034 foldchange (Figure 1)
Miconia sp 40 120583gmL produced upregulation in theexpression of mRNA of PPAR120574 with a value greater thanthat of the drug rosiglitazone which increases the expressionof PPAR120574 as expected The use of maturation medium withboth extracts as rosiglitazone did not increase by itself theexpression levels of the gene PPAR120574
In the lipid accumulation test during adipogenesis withdifferent treatments Group 1 (Dm) showed absorbance of0137 this value represents the accumulated lipids duringadipogenesis induced differentiation medium already estab-lished At the same time Group 2 (Dm + Miconia sp40 120583gmL) showed absorbance of 0184 Group 3 (Dm +rosiglitazone 1 120583M) showed an absorbance value of 0177
Taking the absorbance value from Group 1 as 100 lipidaccumulation when extract Miconia sp was added to thedifferentiation medium lipid accumulation was increased to3457 compared to Group 1 Like the Miconia sp extractadding the drug rosiglitazone to differentiation medium itcauses an increase of 2955 compared to Group 1 Theincreased absorbance of the extract and the drug is significantcompared with Dm Miconia sp presented a very similarresult to rosiglitazone difference between them not of greatsignificance (Figure 2)
In the 120572-amylase inhibition assay different treatmentsto the mixture of enzyme-substrate reaction were addedobtaining the following results for treatment 1 Miconia sppresented IC
50of 2823 plusmn 215 120583gmL At the same time
for treatment 2 phosphate buffer and 2 ethanol (negativecontrol) showed no inhibition by the vehicle used for
(abs
orba
nce510
nm)
Accu
mul
atio
n of
lipi
ds
Dm + rosiglitazone1120583M
Dm + Miconia sp40120583gmL
Dm
04
035
03
025
02
015
01
005
0
lowast lowast
Figure 2 Effect of different substances in lipid accumulation duringadipogenesis in 3T3-L1 cellsThe treatments were applied at day 0 ofdifferentiation and withdrawn at day 2 On day 10 when adipocytesreached maturity the staining was performed lowast119875 le 005 significantdifference between treatments and Dm (119899 = 3)
Table 1 Enzymatic inhibition from acarbose and Miconia spextract
Treatment120572-Amylase(IC50plusmn DE
120583gmL)
120572-Glucosidase(IC50plusmn DE
120583gmL)Miconialowast 2823 plusmn 215 195 plusmn 015Acarbose (positive control) 99384 plusmn 15713 33100 plusmn 7208Phosphate buffer with 1ethanol (negative control) NI NI
Data are expressed as mean plusmn the SD (119899 = 3) NI no inhibition lowast119875 le 005significant difference with acarbose
(positive control)According to the results and despite being a complete
extract the extract of Miconia sp reveals a greater capacitythan acarbose to inhibit 50 of enzyme activity this differ-ence between the two treatments is statistically significant(Table 1)
In the inhibition assay of 120572-glucosidase the followingresults were obtained for treatment 1 Miconia sp extractprovided IC
50of 195 plusmn 015 120583gmL At the same time for
treatment 2 phosphate buffer with 1 ethanol (negativecontrol) did not show any inhibitory effect on the enzymefor treatment 3 acarbose (positive control) showed IC
50of
33100 plusmn 7208 120583gmL (Table 1)Miconia sp extract also has a greater inhibition than
acarbose and acarbose is used as a reference compound in theinhibition of these enzymesThe differences in the treatmentsare statistically significant
In determining the proliferation on the VERO cell lineIC50of 31454 plusmn 4540mgmL considered toxic was obtained
forMiconia sp extract
4 Discussion
PPAR120574 is a nuclear receptor that acts as a transcription factorit improves insulin sensitivity by the cells and enhances
Evidence-Based Complementary and Alternative Medicine 5
glucose utilization [19] A diverse set of natural and syntheticmolecules is classified as ligands and can induce activationand that expression of PPAR120574 These ligands include nutri-ents endogenous ligands and drugs [20] one of those drugsis thiazolidinediones (TZDs) such as rosiglitazone Miconiasp showed an increased mRNA expression of PPAR120574 evenmore than rosiglitazone Moreover Miconia sp was ableto increase lipid accumulation during adipogenesis in 3T3cell line L-1 similar to positive control rosiglitazone It isknown that PPAR120574 is the master regulator of adipocytedifferentiation and during adipogenesis PPAR120574 is induced[21] PPAR120574 activation in adipocytes ensures proper balanceand secretion of adipokines such as leptin and adiponectinthey are mediators in insulin action in peripheral tissueswhich causes insulin sensitivity throughout the body [22]The products mentioned above that stimulate the productionof PPAR120574 are candidates to induce the proper functioningof insulin and recognition considering them as potentialantidiabetic agents
The results suggest that the presence of secondarymetabolites could be involved in upregulation of PPAR120574 genQuercetin catechin and kaempferol have been reported insome species of Miconia sp and these compounds could actas ligands of PPAR120574 [6 21] just as rosiglitazone does SomeTZDs as rosiglitazone have been associated with a significantincrease of cardiovascular diseases For this reason FDArestricted their prescription in the United States [23]
Avoiding the increase of postprandial glucose is impor-tant to keep the glycemic levels low in diabetic patientsThe inhibition of 120572-amylase and 120572-glucosidase present inthe gastrointestinal tract could keep the levels of glycemialow Drugs inhibit these enzymes such as acarbose miglitolvoglibose nojirimycin and 1-deoxynojirimycin which allowthe slow absorption of carbohydrates [23] The ethanolicextract of Miconia sp inhibits 120572-amylase by 50 to a lessconcentration than the acarbose drug Additionally the 120572-glucosidase is inhibited byMiconia sp at low concentrationslower than the drug Therefore it is believed that in thepresence of molecules with antihyperglycemic effect in invitro model these compounds could be an alternative toexisting treatments or adjunctive to them which may haveundesired side effects
Miconia sp showed cytotoxicity at a greater concentrationthan necessary to increase expression of PPAR120574 increaselipid accumulation and inhibit 120572-glucosidase and 120572-amylaseThere are reports that the species of the genus MiconiaM stenostachya M cabucu M albicans and M rubiginosalack cytotoxicity or mutagenicity at lower concentrations of100 120583gmL [6]
5 Conclusions
The ethanolic extract of Miconia sp showed increase of thelevel of mRNA expression of PPAR120574 at a significantly higherlevel than rosiglitazone (drug) Also Miconia sp showedinhibition of the enzymes 120572-glucosidase and 120572-amylase withIC50lower than acarbose (drug) and furthermore increase the
capacity of lipid accumulation during adipogenesis similar tothe drug rosiglitazone At the same timeMiconia sp showed
cytotoxicity on VERO cells with a concentration higherthan that presenting biological activity For this reason thecompounds present in the ethanolic extract ofMiconia sp canbe an alternative for the treatment of diabetes mellitus or likean adjunctive with the recommendation of continuing within vivo tests and elucidation of bioactive compounds
Competing Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
References
[1] J Chen Y Wu J Zou and K Gao ldquo120572-Glucosidase inhibitionand antihyperglycemic activity of flavonoids from Ampelop-sis grossedentata and the flavonoid derivativesrdquo Bioorganic ampMedicinal Chemistry vol 24 no 7 pp 1488ndash1494 2016
[2] J F Ascaso ldquoDiabetes mellitus tipo 2 nuevos tratamientosrdquoMedicina Clınica vol 143 no 3 pp 117ndash123 2014
[3] M E Rafols ldquoTejido adiposo heterogeneidad celular y diver-sidad funcionalrdquo Endocrinologıa y Nutricion vol 61 no 2 pp100ndash112 2014
[4] S Ghosh and L Rangan ldquoMolecular docking and inhibitionstudies of120572-amylase activity by labdane diterpenes fromAlpinianigra seedsrdquo Medicinal Chemistry Research vol 23 no 11 pp4836ndash4852 2014
[5] A Josabad Alonso-Castro J Jose Maldonado-Miranda AZarate-Martinez et al ldquoMedicinal plants used in the HuastecaPotosina Mexicordquo Journal of Ethnopharmacology vol 143 no1 pp 292ndash298 2012
[6] J M Serpeloni G R Mazzaron M Prates et al ldquoExperimentaland toxicologic pathology cytotoxic and mutagenic evaluationof extracts from plant species of the Miconia genus and theirinfluence on doxorubicin-induced mutagenicity an in vitroanalysisrdquo Experimental and Toxicologic Pathology vol 63 pp499ndash504 2011
[7] A J Richard T P Burris D Sanchez-Infantes Y Wang DM Ribnicky and J M Stephens ldquoArtemisia extracts activatePPAR120574 promote adipogenesis and enhance insulin sensitivityin adipose tissue of obese micerdquo Nutrition vol 30 no 7-8 ppS31ndashS36 2014
[8] C-S Kong J-A Kim S-S Bak H-G Byun and S-K KimldquoAnti-obesity effect of carboxymethyl chitin by AMPK andaquaporin-7 pathways in 3T3-L1 adipocytesrdquo Journal of Nutri-tional Biochemistry vol 22 no 3 pp 276ndash281 2011
[9] R Chaiittianan P Chayopas A Rattanathongkom P Tip-payawat and K Sutthanut ldquoAnti-obesity potential of cornsilks relationships of phytochemicals and antioxidation anti-pre-adipocyte proliferation anti-adipogenesis and lipolysisinductionrdquo Journal of Functional Foods vol 23 pp 497ndash5102016
[10] X C Tan K H Chua M Ravishankar Ram and U RKuppusamy ldquoMonoterpenes novel insights into their biologicaleffects and roles on glucose uptake and lipidmetabolism in 3T3-L1 adipocytesrdquo Food Chemistry vol 196 pp 242ndash250 2016
[11] H-L Kim J ParkH Park et al ldquoPlatycodon grandiflorumA decandolle ethanolic extract inhibits adipogenic regulators in 3T3-L1 cells and inducesmitochondrial biogenesis in primary brownpreadipocytesrdquo Journal of Agricultural and Food Chemistry vol63 no 35 pp 7721ndash7730 2015
6 Evidence-Based Complementary and Alternative Medicine
[12] F M Siraj S Natarajan M A Huq Y J Kim and D CYang ldquoStructural investigation of ginsenoside Rf with PPAR120574major transcriptional factor of adipogenesis and its impact onadipocyterdquo Journal of Ginseng Research vol 39 no 2 pp 141ndash147 2015
[13] H S Nyambe J A Villa I Ifie et al ldquoInhibition of human 120572-amylase by dietary polyphenolsrdquo Journal of Functional Foodsvol 19 pp 723ndash732 2015
[14] C-W Liu Y-C Wang H-C Lu and W-D Chiang ldquoOpti-mization of ultrasound-assisted extraction conditions for totalphenols with anti-hyperglycemic activity from Psidium guajavaleavesrdquo Process Biochemistry vol 49 no 10 pp 1601ndash1605 2014
[15] H Dehghan Y Sarrafi and P Salehi ldquoAntioxidant and antidia-betic activities of 11 herbal plants from Hyrcania region IranrdquoJournal of Food and Drug Analysis vol 24 no 1 pp 179ndash1882016
[16] N C Ammerman M Beier-Sexton and A F Azad ldquoGrowthand maintenance of Vero cell linesrdquo Current Protocols inMicrobiology 2008
[17] B Kling D Bucherl P Palatzky et al ldquoFlavonoids flavonoidmetabolites and phenolic acids inhibit oxidative stress in theneuronal cell line HT-22 monitored by ECIS and MTT assaya comparative studyrdquo Journal of Natural Products vol 77 no 3pp 446ndash454 2014
[18] M Boncler M Rozalski U Krajewska A Podsędek and CWatala ldquoComparison of PrestoBlue and MTT assays of cellularviability in the assessment of anti-proliferative effects of plantextracts on human endothelial cellsrdquo Journal of Pharmacologicaland Toxicological Methods vol 69 no 1 pp 9ndash16 2014
[19] T Kariharan G Nanayakkara K Parameshwaran et al ldquoCen-tral activation of PPAR-gamma ameliorates diabetes inducedcognitive dysfunction and improves BDNF expressionrdquo Neuro-biology of Aging vol 36 no 3 pp 1451ndash1461 2015
[20] S N Lewis J Bassaganya-Riera and D R Bevan ldquoVirtualscreening as a technique for PPARmodulator discoveryrdquo PPARResearch vol 2010 Article ID 861238 10 pages 2010
[21] L Wang B Waltenberger E-M Pferschy-Wenzig et al ldquoNatu-ral product agonists of peroxisome proliferator-activated recep-tor gamma (PPAR120574) a reviewrdquo Biochemical Pharmacology vol92 no 1 pp 73ndash89 2014
[22] C Janani and B D Ranjitha ldquoPPAR gamma genemdasha reviewrdquoDiabetes and Metabolic Syndrome Clinical Research andReviews vol 9 no 1 pp 46ndash50 2015
[23] G Oboh O B Ogunsuyi M D Ogunbadejo and S A Ade-fegha ldquoInfluence of gallic acid on 120572-amylase and 120572-glucosidaseinhibitory properties of acarboserdquo Journal of Food and DrugAnalysis 2016
2 Evidence-Based Complementary and Alternative Medicine
out by alpha amylase and alpha glucosidase could be anotherform of combating DM [4]
About 80 of the population worldwide use medicinalplants to treat various diseases These constitute a majorsource of drugs about 25 of prescribed drugs worldwideoriginate from plant species [5]
A species of the genre Miconia in Mexican traditionalmedicine is used in south Mexico as an alternative fordiabetes mellitus treatment However such fact has not beenscientifically confirmed yetMiconia is a genus of about 1000species distributed in tropical America and belongs to theMelastomataceae family which has about 4300 species dis-tributed in 166 genera Very few studies on biological activityhave been performed on this genre but it has been shownthat extracts and compounds isolated from species of thegenusMiconia have antibiotic antitumor analgesic and anti-malarial activities A phytochemical analysis of methanolicextract ofMiconia cabucu reveals the presence of glycosylatedflavonoids (quercetin myricetin and kaempferol all withdifferent glycosides) a tannin and rare bioflavonoid Thephytochemical research of M rubiginosa extract led to theidentification of several glycosylated forms such as quercetingallic acid and epicatechin Similarly quercetin myricetinand catechin derivativesM stenostachya were found [6]
2 Materials and Methods
21 Preparation of the Extract Miconia sp aerial part wascollected in Oaxaca Mexico The plant was dried in shadeat room temperature and extraction with ethanol was per-formed by the Soxhlet method (PYREX) for 48 h solvent wasremoved under reduced pressure using a rotary evaporator(Yamato RE-200) Extract yield was determined and thenstored at room temperature until it was used
22 Cell Culture Preparation and Adipocyte DifferentiationMurine cell line 3T3-L1 preadipocytes were used DMEMmedium supplemented with 10 fetal bovine serum (FBS)was used for propagation it was incubated at 37∘C at 5 CO
2
atmosphere until reaching a confluence of 90 The 100confluency was used for adipocyte differentiation It couldbe DMEM with 10 FBS 1 120583M dexamethasone 05mM 3-isobutyl-1-methylxanthine and 10 120583gmL insulin (all brandsfrom Santa Cruz Biotechnology) After 48 hours of incuba-tion (day 2) the differentiation medium was removed andreplaced every 48 hours until maturation of adipocytes (days9ndash11) with maturation medium of adipocytes (DMEM with10 FBS and insulin 10 120583gmL called MM) The maturationwas confirmed by microscope observation of the typicalmorphology of an adipocyte [7ndash10]
23 Expression of PPAR120574 in Adipocytes The study wasperformed in the cell line 3T3-L1 differentiated follow-ing the methodology described above using microplate 6-well culture with an inoculum of 100000 cells per wellin a final volume of 2mL with the corresponding culturemedium At day 10 adipocyte differentiation was observedand replaced in the medium (MM)The following treatmentswere added Group 1 DMEM with 01 ethanol (expression
control group) Group 2 MM supplemented with 1 120583M ofrosiglitazone (positive control) Group 3 MM supplementedwith Miconia sp extract (40 120583gmL) and Group 4 MMonly to observe the effect of the medium After 24 hoursof treatment application of the total RNA extraction theretrotranscription (cDNA) and PCR were performed in realtime to determine the effect on mRNA expression [8ndash10]
The total RNA isolation of treated adipocytes was per-formed with an extraction kit SV Total RNA Isolation SystemZ3100 Promega (following themanufacturerrsquos instructions)and stored atminus80∘Cuntil use Total RNA integrity purity andquantification analysis was made in NanoDrop 8000 Spec-trophotometer withThermo Scientific and an electrophoresisgel (15 agarose)
CDNA synthesis was done with the ImProm-II ReverseTranscription System kit Promega A3800 with 500 ng ofRNA isolated The cDNA was stored at minus20∘C until use
Analysis of mRNA expression PPAR120574 was performedby qRT-PCR using LightCycler 480 II Roche equipmentand 50 ng of cDNA 05 120583M of the primers 125 120583L ofMaxima SYBRGreen qPCRMasterMix (2x) K0251ThermoScientific and the proper amount of nuclease-free water tohave a final volume of 25 120583L The 36B4 was used [Thomson]as reference gene The sequence of the primers used isPPAR120574 forward 51015840-CTGGCCTCCCTGATGAATAAAG-31015840reverse 51015840-AGGCTCCATAAAGTCACCAAAG-31015840 36B4 for-ward 51015840-ACTGGTCTAGGACCCGAGAAG-31015840 and reverse51015840-TCAATGGTGCCTCTGGAGATT-31015840 The relative mRNAexpression was calculated based on the 2minusΔΔCt method
24 Lipid Accumulation in Adipocytes Lipid accumulationwas evaluated on the cell line 3T3-L1 using Oil Red O withmodifications to the method described by other authors Themethod described above was used for cell differentiationMicroculture plates were used having 24 wells with aninoculum of 30000 cells per well leading to a final volumeof 500120583L with culture medium At 48 h after confluence(day 0) the differentiation medium (Dm) was applied tostimulate adipogenesis supplemented with the substances tobe evaluated treatment 1 [Dm (control reference)] treatment2 [Dm supplementedwith 40120583gmL extractMiconia sp] andtreatment 3 [Dm supplemented with 1120583Mrosiglitazone (pos-itive control)] Subsequently the microplate was incubatedfor 48 h with the established conditions After that Dm wasreplaced by MM (day 2) and incubated for 8 d The culturemedium was removed and formalin 10 was used for 1 h tofix monolayer cells After monolayer cells had been washedtwice with distilled water the dye Oil Red O (05 in 60isopropanol) was applied for 15min at room temperatureThecells were washed three times with distilled water the dyeinside the cells was removed with 100 isopropanol and readthe optical density at 540 nm [9ndash12]
25 Inhibitory Activity of 120572-Amylase The enzyme inhibitionwas evaluated by the method of dinitrosalicylic acid 35-(DNS) with some modifications The Miconia sp extractwas applied at different concentrations (25 50 75 100and 125 120583gmL) as a vehicle using phosphate buffer with
Evidence-Based Complementary and Alternative Medicine 3
20mM sodium 67mM sodium chloride at pH 69 120572-Amylase enzyme (Sigma-Aldrich A9857-250KU) was usedat 05UmL concentration with the same buffer as a vehicleStarch 05 in phosphate buffer was used as a substrateAcarbose at different concentrations (3125 625 1250 2500and 5000 120583gmL) was used as a positive control AdditionallyDNS solution was used at 96mM In 15mL conical tubes50120583L of substances was placed to be evaluated treatment1 Miconia sp extracts treatment 2 buffer phosphate with2 ethanol (negative control) and treatment 3 acarbose(positive control) Briefly to the above treatments 50 120583L ofthe 120572-amylase was added and incubated at 37∘C for 10minAfter that to initiate the reaction 50 120583L of the substratewas added immediately and incubated at 37∘C for 15minThen the reaction was stopped by adding 50 120583L of DNS andheated in a water bath at 95∘C for 5min the tubes wereleft to cool at room temperature and the optical densitywas measured at 540 nm The absorbance rates were used tocalculate the percent inhibition of each treatment using thefollowing equation
Inhibition = (Abs control minus Abs treatment)Abs control
times 100 (1)
In this equation ldquoAbs treatmentrdquo is the productrsquos lightabsorption of the enzyme-substrate reaction in the presenceof the Miconia sp or negative control as appropriate ldquoAbscontrolrdquo is the reaction productrsquos light absorption of enzyme-substrate in the presence of phosphate buffer as a treatmentThe percentage inhibition using the statistical package (SPSSv20) allows us to calculate the inhibitory concentration 50(IC50) is the amount required to inhibit 50 of the enzyme
(Sigma-Aldrich A8980-1G) [5 13ndash15]
26 120572-Glucosidase Inhibitory Activity Inhibition of theenzyme was evaluated by the pNPG method (p-nitrophenyl-120572-D-glucopyranoside) with some modificationsMiconia spextract at different concentrations (1 2 3 4 and 5120583gmL)wasused as a vehicle a buffer of sodium phosphate mentionedabove Acarbose at different concentrations (625 125 250500 and 1000 120583gmL) is positive control using buffer sodiumphosphate as a vehicle 120572-Glucosidase enzyme (Sigma-Aldrich G5003-100UN) at a concentration of 02UmLusing phosphate buffer as a vehicle Additionally a prepara-tion of pNPG 2mM (Sigma-Aldrich N1377-1G) was usedas a substrate in the same vehicle After that in a 96-well microplate 25120583L of the substances was mixed withthe enzyme to the assay treatment 1 [Miconia sp extracts]treatment 2 [phosphate buffer with 1 ethanol (negativecontrol)] and treatment 3 [acarbose (positive control)]Besides the above treatments 25120583L of the enzyme suspensionwas added and incubated at 37∘C for 15min immediatelyafter that 50 120583L of pNPG was added and incubated at 37∘Cfor 10min then the reaction was stopped by adding 50 120583Lsodium carbonate (02M)
Finally the optical density at 405 nm was measured Theabsorbance rates were used to calculate the percent inhibitionof each treatment using the following equation
Inhibition = (Abs control minus Abs treatment)Abs control
times 100 (2)
In this equation ldquoAbs treatmentrdquo is the productrsquos lightabsorption of the enzyme-substrate reaction in the presenceof the Miconia sp acarbose or negative control as appro-priate ldquoAbs controlrdquo is the productrsquos light absorption ofthe enzyme-substrate reaction in the presence of phosphatebuffer as a treatment Percent inhibitions using a statisticalpackage (SPSS v20) were used to determine the inhibitoryconcentration 50 (IC
50) that is the amount of treatment
necessary to inhibit the enzyme 50 [4 13ndash15]
27 Cell Proliferation Assay Cell proliferation with MTTcolorimetric method (3-(45-dimethylthiazol-2-y1)-25-diphenyltetrazolium bromide) was evaluatedThe VERO cell(monkey kidney epithelial) lines are widely used in researchto evaluate the effect of chemicals and toxins in mammals[16] 1 times 105 cellswell were seeded in a 96-well microplate andadjusted to a final volume of 200120583L with culture medium(DMEM supplemented with 10 FBS) and incubated at37∘C in an atmosphere of 5 CO
2 After 24 h and until
reaching a layer of 80 confluence the culture mediumwas removed and cells were washed with phosphates (PBS)a buffered saline solution further substances were addedand evaluated treatment 1 [extracts of Miconia sp (625125 250 500 and 1000 120583gmL)] treatment 2 [DMEM with1 ethanol (negative control)] and treatment 3 [cells withculture medium (reference of proliferation)] After that themicroplate was incubated for 24 h at the above conditionsThen 10 120583L MTT (5mgmL) were added to each well andincubated again for 4 h the culture medium was removedand cells were washed with PBS Immediately afterwards200120583L dimethyl sulfoxide (DMSO) was added then theoptical density was measured at 570 nm By the followingformula the percentage of cell proliferation was determined
cell proliferation = Abs treatmentAbs control
times 100 (3)
In this equation ldquoAbs treatmentrdquo is the productrsquos light absorp-tion of the treated cells and ldquoAbs controlrdquo is the productrsquos lightabsorption of cells used as proliferation referenceThe IC
50is
calculated with the percentages of cell proliferation and withthe support of SPSS v20 [17 18]
3 Results
In this study different biological activities of ethanolic extractof Miconia sp were evaluated in their effect on expressionof mRNA PPAR120574 lipid accumulation during adipogenesisthe activity of 120572-amylase and 120572-glucosidase and the effect onproliferation of VERO cell line
In determining the expression level ofmRNAof PPAR120574 inmature mouse adipocytes Group 1 was used as an expressioncontrol which was normalized to a value of 1 Therefore the
4 Evidence-Based Complementary and Alternative Medicine
EC expression controlMM maturation medium
EC MMMicRos06070809
11112131415
mRN
A ex
pres
sion
leve
l (fo
ld ch
ange
)
Ros rosiglitazone 1120583M
lowast
lowastlowast
Mic Miconia sp 40120583gmL
Figure 1 Effect of different compounds on the expression of mRNAPPAR120574 On day 10 the treatments were applied Group 1 DMEMwith 01 ethanol (EC) Group 2 MM and rosiglitazone (Ros) asa positive control Group 3 MM and Miconia (Mic) and Group4 MM On day 11 of adipocyte differentiation the total RNA wasisolated lowast119875 le 005 there is a significant difference with the controlof expression lowastlowast119875 le 005 there is a significant difference with thecontrol of expression and rosiglitazone (119899 = 3)
values above 1 indicate an overexpression or upregulation InGroup 2 the value in the expression levels was 1166 plusmn 0007fold change at the same time in Group 3 a value was 1393 plusmn0008 and finally Group 4 showed a value of 0746plusmn0034 foldchange (Figure 1)
Miconia sp 40 120583gmL produced upregulation in theexpression of mRNA of PPAR120574 with a value greater thanthat of the drug rosiglitazone which increases the expressionof PPAR120574 as expected The use of maturation medium withboth extracts as rosiglitazone did not increase by itself theexpression levels of the gene PPAR120574
In the lipid accumulation test during adipogenesis withdifferent treatments Group 1 (Dm) showed absorbance of0137 this value represents the accumulated lipids duringadipogenesis induced differentiation medium already estab-lished At the same time Group 2 (Dm + Miconia sp40 120583gmL) showed absorbance of 0184 Group 3 (Dm +rosiglitazone 1 120583M) showed an absorbance value of 0177
Taking the absorbance value from Group 1 as 100 lipidaccumulation when extract Miconia sp was added to thedifferentiation medium lipid accumulation was increased to3457 compared to Group 1 Like the Miconia sp extractadding the drug rosiglitazone to differentiation medium itcauses an increase of 2955 compared to Group 1 Theincreased absorbance of the extract and the drug is significantcompared with Dm Miconia sp presented a very similarresult to rosiglitazone difference between them not of greatsignificance (Figure 2)
In the 120572-amylase inhibition assay different treatmentsto the mixture of enzyme-substrate reaction were addedobtaining the following results for treatment 1 Miconia sppresented IC
50of 2823 plusmn 215 120583gmL At the same time
for treatment 2 phosphate buffer and 2 ethanol (negativecontrol) showed no inhibition by the vehicle used for
(abs
orba
nce510
nm)
Accu
mul
atio
n of
lipi
ds
Dm + rosiglitazone1120583M
Dm + Miconia sp40120583gmL
Dm
04
035
03
025
02
015
01
005
0
lowast lowast
Figure 2 Effect of different substances in lipid accumulation duringadipogenesis in 3T3-L1 cellsThe treatments were applied at day 0 ofdifferentiation and withdrawn at day 2 On day 10 when adipocytesreached maturity the staining was performed lowast119875 le 005 significantdifference between treatments and Dm (119899 = 3)
Table 1 Enzymatic inhibition from acarbose and Miconia spextract
Treatment120572-Amylase(IC50plusmn DE
120583gmL)
120572-Glucosidase(IC50plusmn DE
120583gmL)Miconialowast 2823 plusmn 215 195 plusmn 015Acarbose (positive control) 99384 plusmn 15713 33100 plusmn 7208Phosphate buffer with 1ethanol (negative control) NI NI
Data are expressed as mean plusmn the SD (119899 = 3) NI no inhibition lowast119875 le 005significant difference with acarbose
(positive control)According to the results and despite being a complete
extract the extract of Miconia sp reveals a greater capacitythan acarbose to inhibit 50 of enzyme activity this differ-ence between the two treatments is statistically significant(Table 1)
In the inhibition assay of 120572-glucosidase the followingresults were obtained for treatment 1 Miconia sp extractprovided IC
50of 195 plusmn 015 120583gmL At the same time for
treatment 2 phosphate buffer with 1 ethanol (negativecontrol) did not show any inhibitory effect on the enzymefor treatment 3 acarbose (positive control) showed IC
50of
33100 plusmn 7208 120583gmL (Table 1)Miconia sp extract also has a greater inhibition than
acarbose and acarbose is used as a reference compound in theinhibition of these enzymesThe differences in the treatmentsare statistically significant
In determining the proliferation on the VERO cell lineIC50of 31454 plusmn 4540mgmL considered toxic was obtained
forMiconia sp extract
4 Discussion
PPAR120574 is a nuclear receptor that acts as a transcription factorit improves insulin sensitivity by the cells and enhances
Evidence-Based Complementary and Alternative Medicine 5
glucose utilization [19] A diverse set of natural and syntheticmolecules is classified as ligands and can induce activationand that expression of PPAR120574 These ligands include nutri-ents endogenous ligands and drugs [20] one of those drugsis thiazolidinediones (TZDs) such as rosiglitazone Miconiasp showed an increased mRNA expression of PPAR120574 evenmore than rosiglitazone Moreover Miconia sp was ableto increase lipid accumulation during adipogenesis in 3T3cell line L-1 similar to positive control rosiglitazone It isknown that PPAR120574 is the master regulator of adipocytedifferentiation and during adipogenesis PPAR120574 is induced[21] PPAR120574 activation in adipocytes ensures proper balanceand secretion of adipokines such as leptin and adiponectinthey are mediators in insulin action in peripheral tissueswhich causes insulin sensitivity throughout the body [22]The products mentioned above that stimulate the productionof PPAR120574 are candidates to induce the proper functioningof insulin and recognition considering them as potentialantidiabetic agents
The results suggest that the presence of secondarymetabolites could be involved in upregulation of PPAR120574 genQuercetin catechin and kaempferol have been reported insome species of Miconia sp and these compounds could actas ligands of PPAR120574 [6 21] just as rosiglitazone does SomeTZDs as rosiglitazone have been associated with a significantincrease of cardiovascular diseases For this reason FDArestricted their prescription in the United States [23]
Avoiding the increase of postprandial glucose is impor-tant to keep the glycemic levels low in diabetic patientsThe inhibition of 120572-amylase and 120572-glucosidase present inthe gastrointestinal tract could keep the levels of glycemialow Drugs inhibit these enzymes such as acarbose miglitolvoglibose nojirimycin and 1-deoxynojirimycin which allowthe slow absorption of carbohydrates [23] The ethanolicextract of Miconia sp inhibits 120572-amylase by 50 to a lessconcentration than the acarbose drug Additionally the 120572-glucosidase is inhibited byMiconia sp at low concentrationslower than the drug Therefore it is believed that in thepresence of molecules with antihyperglycemic effect in invitro model these compounds could be an alternative toexisting treatments or adjunctive to them which may haveundesired side effects
Miconia sp showed cytotoxicity at a greater concentrationthan necessary to increase expression of PPAR120574 increaselipid accumulation and inhibit 120572-glucosidase and 120572-amylaseThere are reports that the species of the genus MiconiaM stenostachya M cabucu M albicans and M rubiginosalack cytotoxicity or mutagenicity at lower concentrations of100 120583gmL [6]
5 Conclusions
The ethanolic extract of Miconia sp showed increase of thelevel of mRNA expression of PPAR120574 at a significantly higherlevel than rosiglitazone (drug) Also Miconia sp showedinhibition of the enzymes 120572-glucosidase and 120572-amylase withIC50lower than acarbose (drug) and furthermore increase the
capacity of lipid accumulation during adipogenesis similar tothe drug rosiglitazone At the same timeMiconia sp showed
cytotoxicity on VERO cells with a concentration higherthan that presenting biological activity For this reason thecompounds present in the ethanolic extract ofMiconia sp canbe an alternative for the treatment of diabetes mellitus or likean adjunctive with the recommendation of continuing within vivo tests and elucidation of bioactive compounds
Competing Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
References
[1] J Chen Y Wu J Zou and K Gao ldquo120572-Glucosidase inhibitionand antihyperglycemic activity of flavonoids from Ampelop-sis grossedentata and the flavonoid derivativesrdquo Bioorganic ampMedicinal Chemistry vol 24 no 7 pp 1488ndash1494 2016
[2] J F Ascaso ldquoDiabetes mellitus tipo 2 nuevos tratamientosrdquoMedicina Clınica vol 143 no 3 pp 117ndash123 2014
[3] M E Rafols ldquoTejido adiposo heterogeneidad celular y diver-sidad funcionalrdquo Endocrinologıa y Nutricion vol 61 no 2 pp100ndash112 2014
[4] S Ghosh and L Rangan ldquoMolecular docking and inhibitionstudies of120572-amylase activity by labdane diterpenes fromAlpinianigra seedsrdquo Medicinal Chemistry Research vol 23 no 11 pp4836ndash4852 2014
[5] A Josabad Alonso-Castro J Jose Maldonado-Miranda AZarate-Martinez et al ldquoMedicinal plants used in the HuastecaPotosina Mexicordquo Journal of Ethnopharmacology vol 143 no1 pp 292ndash298 2012
[6] J M Serpeloni G R Mazzaron M Prates et al ldquoExperimentaland toxicologic pathology cytotoxic and mutagenic evaluationof extracts from plant species of the Miconia genus and theirinfluence on doxorubicin-induced mutagenicity an in vitroanalysisrdquo Experimental and Toxicologic Pathology vol 63 pp499ndash504 2011
[7] A J Richard T P Burris D Sanchez-Infantes Y Wang DM Ribnicky and J M Stephens ldquoArtemisia extracts activatePPAR120574 promote adipogenesis and enhance insulin sensitivityin adipose tissue of obese micerdquo Nutrition vol 30 no 7-8 ppS31ndashS36 2014
[8] C-S Kong J-A Kim S-S Bak H-G Byun and S-K KimldquoAnti-obesity effect of carboxymethyl chitin by AMPK andaquaporin-7 pathways in 3T3-L1 adipocytesrdquo Journal of Nutri-tional Biochemistry vol 22 no 3 pp 276ndash281 2011
[9] R Chaiittianan P Chayopas A Rattanathongkom P Tip-payawat and K Sutthanut ldquoAnti-obesity potential of cornsilks relationships of phytochemicals and antioxidation anti-pre-adipocyte proliferation anti-adipogenesis and lipolysisinductionrdquo Journal of Functional Foods vol 23 pp 497ndash5102016
[10] X C Tan K H Chua M Ravishankar Ram and U RKuppusamy ldquoMonoterpenes novel insights into their biologicaleffects and roles on glucose uptake and lipidmetabolism in 3T3-L1 adipocytesrdquo Food Chemistry vol 196 pp 242ndash250 2016
[11] H-L Kim J ParkH Park et al ldquoPlatycodon grandiflorumA decandolle ethanolic extract inhibits adipogenic regulators in 3T3-L1 cells and inducesmitochondrial biogenesis in primary brownpreadipocytesrdquo Journal of Agricultural and Food Chemistry vol63 no 35 pp 7721ndash7730 2015
6 Evidence-Based Complementary and Alternative Medicine
[12] F M Siraj S Natarajan M A Huq Y J Kim and D CYang ldquoStructural investigation of ginsenoside Rf with PPAR120574major transcriptional factor of adipogenesis and its impact onadipocyterdquo Journal of Ginseng Research vol 39 no 2 pp 141ndash147 2015
[13] H S Nyambe J A Villa I Ifie et al ldquoInhibition of human 120572-amylase by dietary polyphenolsrdquo Journal of Functional Foodsvol 19 pp 723ndash732 2015
[14] C-W Liu Y-C Wang H-C Lu and W-D Chiang ldquoOpti-mization of ultrasound-assisted extraction conditions for totalphenols with anti-hyperglycemic activity from Psidium guajavaleavesrdquo Process Biochemistry vol 49 no 10 pp 1601ndash1605 2014
[15] H Dehghan Y Sarrafi and P Salehi ldquoAntioxidant and antidia-betic activities of 11 herbal plants from Hyrcania region IranrdquoJournal of Food and Drug Analysis vol 24 no 1 pp 179ndash1882016
[16] N C Ammerman M Beier-Sexton and A F Azad ldquoGrowthand maintenance of Vero cell linesrdquo Current Protocols inMicrobiology 2008
[17] B Kling D Bucherl P Palatzky et al ldquoFlavonoids flavonoidmetabolites and phenolic acids inhibit oxidative stress in theneuronal cell line HT-22 monitored by ECIS and MTT assaya comparative studyrdquo Journal of Natural Products vol 77 no 3pp 446ndash454 2014
[18] M Boncler M Rozalski U Krajewska A Podsędek and CWatala ldquoComparison of PrestoBlue and MTT assays of cellularviability in the assessment of anti-proliferative effects of plantextracts on human endothelial cellsrdquo Journal of Pharmacologicaland Toxicological Methods vol 69 no 1 pp 9ndash16 2014
[19] T Kariharan G Nanayakkara K Parameshwaran et al ldquoCen-tral activation of PPAR-gamma ameliorates diabetes inducedcognitive dysfunction and improves BDNF expressionrdquo Neuro-biology of Aging vol 36 no 3 pp 1451ndash1461 2015
[20] S N Lewis J Bassaganya-Riera and D R Bevan ldquoVirtualscreening as a technique for PPARmodulator discoveryrdquo PPARResearch vol 2010 Article ID 861238 10 pages 2010
[21] L Wang B Waltenberger E-M Pferschy-Wenzig et al ldquoNatu-ral product agonists of peroxisome proliferator-activated recep-tor gamma (PPAR120574) a reviewrdquo Biochemical Pharmacology vol92 no 1 pp 73ndash89 2014
[22] C Janani and B D Ranjitha ldquoPPAR gamma genemdasha reviewrdquoDiabetes and Metabolic Syndrome Clinical Research andReviews vol 9 no 1 pp 46ndash50 2015
[23] G Oboh O B Ogunsuyi M D Ogunbadejo and S A Ade-fegha ldquoInfluence of gallic acid on 120572-amylase and 120572-glucosidaseinhibitory properties of acarboserdquo Journal of Food and DrugAnalysis 2016
Evidence-Based Complementary and Alternative Medicine 3
20mM sodium 67mM sodium chloride at pH 69 120572-Amylase enzyme (Sigma-Aldrich A9857-250KU) was usedat 05UmL concentration with the same buffer as a vehicleStarch 05 in phosphate buffer was used as a substrateAcarbose at different concentrations (3125 625 1250 2500and 5000 120583gmL) was used as a positive control AdditionallyDNS solution was used at 96mM In 15mL conical tubes50120583L of substances was placed to be evaluated treatment1 Miconia sp extracts treatment 2 buffer phosphate with2 ethanol (negative control) and treatment 3 acarbose(positive control) Briefly to the above treatments 50 120583L ofthe 120572-amylase was added and incubated at 37∘C for 10minAfter that to initiate the reaction 50 120583L of the substratewas added immediately and incubated at 37∘C for 15minThen the reaction was stopped by adding 50 120583L of DNS andheated in a water bath at 95∘C for 5min the tubes wereleft to cool at room temperature and the optical densitywas measured at 540 nm The absorbance rates were used tocalculate the percent inhibition of each treatment using thefollowing equation
Inhibition = (Abs control minus Abs treatment)Abs control
times 100 (1)
In this equation ldquoAbs treatmentrdquo is the productrsquos lightabsorption of the enzyme-substrate reaction in the presenceof the Miconia sp or negative control as appropriate ldquoAbscontrolrdquo is the reaction productrsquos light absorption of enzyme-substrate in the presence of phosphate buffer as a treatmentThe percentage inhibition using the statistical package (SPSSv20) allows us to calculate the inhibitory concentration 50(IC50) is the amount required to inhibit 50 of the enzyme
(Sigma-Aldrich A8980-1G) [5 13ndash15]
26 120572-Glucosidase Inhibitory Activity Inhibition of theenzyme was evaluated by the pNPG method (p-nitrophenyl-120572-D-glucopyranoside) with some modificationsMiconia spextract at different concentrations (1 2 3 4 and 5120583gmL)wasused as a vehicle a buffer of sodium phosphate mentionedabove Acarbose at different concentrations (625 125 250500 and 1000 120583gmL) is positive control using buffer sodiumphosphate as a vehicle 120572-Glucosidase enzyme (Sigma-Aldrich G5003-100UN) at a concentration of 02UmLusing phosphate buffer as a vehicle Additionally a prepara-tion of pNPG 2mM (Sigma-Aldrich N1377-1G) was usedas a substrate in the same vehicle After that in a 96-well microplate 25120583L of the substances was mixed withthe enzyme to the assay treatment 1 [Miconia sp extracts]treatment 2 [phosphate buffer with 1 ethanol (negativecontrol)] and treatment 3 [acarbose (positive control)]Besides the above treatments 25120583L of the enzyme suspensionwas added and incubated at 37∘C for 15min immediatelyafter that 50 120583L of pNPG was added and incubated at 37∘Cfor 10min then the reaction was stopped by adding 50 120583Lsodium carbonate (02M)
Finally the optical density at 405 nm was measured Theabsorbance rates were used to calculate the percent inhibitionof each treatment using the following equation
Inhibition = (Abs control minus Abs treatment)Abs control
times 100 (2)
In this equation ldquoAbs treatmentrdquo is the productrsquos lightabsorption of the enzyme-substrate reaction in the presenceof the Miconia sp acarbose or negative control as appro-priate ldquoAbs controlrdquo is the productrsquos light absorption ofthe enzyme-substrate reaction in the presence of phosphatebuffer as a treatment Percent inhibitions using a statisticalpackage (SPSS v20) were used to determine the inhibitoryconcentration 50 (IC
50) that is the amount of treatment
necessary to inhibit the enzyme 50 [4 13ndash15]
27 Cell Proliferation Assay Cell proliferation with MTTcolorimetric method (3-(45-dimethylthiazol-2-y1)-25-diphenyltetrazolium bromide) was evaluatedThe VERO cell(monkey kidney epithelial) lines are widely used in researchto evaluate the effect of chemicals and toxins in mammals[16] 1 times 105 cellswell were seeded in a 96-well microplate andadjusted to a final volume of 200120583L with culture medium(DMEM supplemented with 10 FBS) and incubated at37∘C in an atmosphere of 5 CO
2 After 24 h and until
reaching a layer of 80 confluence the culture mediumwas removed and cells were washed with phosphates (PBS)a buffered saline solution further substances were addedand evaluated treatment 1 [extracts of Miconia sp (625125 250 500 and 1000 120583gmL)] treatment 2 [DMEM with1 ethanol (negative control)] and treatment 3 [cells withculture medium (reference of proliferation)] After that themicroplate was incubated for 24 h at the above conditionsThen 10 120583L MTT (5mgmL) were added to each well andincubated again for 4 h the culture medium was removedand cells were washed with PBS Immediately afterwards200120583L dimethyl sulfoxide (DMSO) was added then theoptical density was measured at 570 nm By the followingformula the percentage of cell proliferation was determined
cell proliferation = Abs treatmentAbs control
times 100 (3)
In this equation ldquoAbs treatmentrdquo is the productrsquos light absorp-tion of the treated cells and ldquoAbs controlrdquo is the productrsquos lightabsorption of cells used as proliferation referenceThe IC
50is
calculated with the percentages of cell proliferation and withthe support of SPSS v20 [17 18]
3 Results
In this study different biological activities of ethanolic extractof Miconia sp were evaluated in their effect on expressionof mRNA PPAR120574 lipid accumulation during adipogenesisthe activity of 120572-amylase and 120572-glucosidase and the effect onproliferation of VERO cell line
In determining the expression level ofmRNAof PPAR120574 inmature mouse adipocytes Group 1 was used as an expressioncontrol which was normalized to a value of 1 Therefore the
4 Evidence-Based Complementary and Alternative Medicine
EC expression controlMM maturation medium
EC MMMicRos06070809
11112131415
mRN
A ex
pres
sion
leve
l (fo
ld ch
ange
)
Ros rosiglitazone 1120583M
lowast
lowastlowast
Mic Miconia sp 40120583gmL
Figure 1 Effect of different compounds on the expression of mRNAPPAR120574 On day 10 the treatments were applied Group 1 DMEMwith 01 ethanol (EC) Group 2 MM and rosiglitazone (Ros) asa positive control Group 3 MM and Miconia (Mic) and Group4 MM On day 11 of adipocyte differentiation the total RNA wasisolated lowast119875 le 005 there is a significant difference with the controlof expression lowastlowast119875 le 005 there is a significant difference with thecontrol of expression and rosiglitazone (119899 = 3)
values above 1 indicate an overexpression or upregulation InGroup 2 the value in the expression levels was 1166 plusmn 0007fold change at the same time in Group 3 a value was 1393 plusmn0008 and finally Group 4 showed a value of 0746plusmn0034 foldchange (Figure 1)
Miconia sp 40 120583gmL produced upregulation in theexpression of mRNA of PPAR120574 with a value greater thanthat of the drug rosiglitazone which increases the expressionof PPAR120574 as expected The use of maturation medium withboth extracts as rosiglitazone did not increase by itself theexpression levels of the gene PPAR120574
In the lipid accumulation test during adipogenesis withdifferent treatments Group 1 (Dm) showed absorbance of0137 this value represents the accumulated lipids duringadipogenesis induced differentiation medium already estab-lished At the same time Group 2 (Dm + Miconia sp40 120583gmL) showed absorbance of 0184 Group 3 (Dm +rosiglitazone 1 120583M) showed an absorbance value of 0177
Taking the absorbance value from Group 1 as 100 lipidaccumulation when extract Miconia sp was added to thedifferentiation medium lipid accumulation was increased to3457 compared to Group 1 Like the Miconia sp extractadding the drug rosiglitazone to differentiation medium itcauses an increase of 2955 compared to Group 1 Theincreased absorbance of the extract and the drug is significantcompared with Dm Miconia sp presented a very similarresult to rosiglitazone difference between them not of greatsignificance (Figure 2)
In the 120572-amylase inhibition assay different treatmentsto the mixture of enzyme-substrate reaction were addedobtaining the following results for treatment 1 Miconia sppresented IC
50of 2823 plusmn 215 120583gmL At the same time
for treatment 2 phosphate buffer and 2 ethanol (negativecontrol) showed no inhibition by the vehicle used for
(abs
orba
nce510
nm)
Accu
mul
atio
n of
lipi
ds
Dm + rosiglitazone1120583M
Dm + Miconia sp40120583gmL
Dm
04
035
03
025
02
015
01
005
0
lowast lowast
Figure 2 Effect of different substances in lipid accumulation duringadipogenesis in 3T3-L1 cellsThe treatments were applied at day 0 ofdifferentiation and withdrawn at day 2 On day 10 when adipocytesreached maturity the staining was performed lowast119875 le 005 significantdifference between treatments and Dm (119899 = 3)
Table 1 Enzymatic inhibition from acarbose and Miconia spextract
Treatment120572-Amylase(IC50plusmn DE
120583gmL)
120572-Glucosidase(IC50plusmn DE
120583gmL)Miconialowast 2823 plusmn 215 195 plusmn 015Acarbose (positive control) 99384 plusmn 15713 33100 plusmn 7208Phosphate buffer with 1ethanol (negative control) NI NI
Data are expressed as mean plusmn the SD (119899 = 3) NI no inhibition lowast119875 le 005significant difference with acarbose
(positive control)According to the results and despite being a complete
extract the extract of Miconia sp reveals a greater capacitythan acarbose to inhibit 50 of enzyme activity this differ-ence between the two treatments is statistically significant(Table 1)
In the inhibition assay of 120572-glucosidase the followingresults were obtained for treatment 1 Miconia sp extractprovided IC
50of 195 plusmn 015 120583gmL At the same time for
treatment 2 phosphate buffer with 1 ethanol (negativecontrol) did not show any inhibitory effect on the enzymefor treatment 3 acarbose (positive control) showed IC
50of
33100 plusmn 7208 120583gmL (Table 1)Miconia sp extract also has a greater inhibition than
acarbose and acarbose is used as a reference compound in theinhibition of these enzymesThe differences in the treatmentsare statistically significant
In determining the proliferation on the VERO cell lineIC50of 31454 plusmn 4540mgmL considered toxic was obtained
forMiconia sp extract
4 Discussion
PPAR120574 is a nuclear receptor that acts as a transcription factorit improves insulin sensitivity by the cells and enhances
Evidence-Based Complementary and Alternative Medicine 5
glucose utilization [19] A diverse set of natural and syntheticmolecules is classified as ligands and can induce activationand that expression of PPAR120574 These ligands include nutri-ents endogenous ligands and drugs [20] one of those drugsis thiazolidinediones (TZDs) such as rosiglitazone Miconiasp showed an increased mRNA expression of PPAR120574 evenmore than rosiglitazone Moreover Miconia sp was ableto increase lipid accumulation during adipogenesis in 3T3cell line L-1 similar to positive control rosiglitazone It isknown that PPAR120574 is the master regulator of adipocytedifferentiation and during adipogenesis PPAR120574 is induced[21] PPAR120574 activation in adipocytes ensures proper balanceand secretion of adipokines such as leptin and adiponectinthey are mediators in insulin action in peripheral tissueswhich causes insulin sensitivity throughout the body [22]The products mentioned above that stimulate the productionof PPAR120574 are candidates to induce the proper functioningof insulin and recognition considering them as potentialantidiabetic agents
The results suggest that the presence of secondarymetabolites could be involved in upregulation of PPAR120574 genQuercetin catechin and kaempferol have been reported insome species of Miconia sp and these compounds could actas ligands of PPAR120574 [6 21] just as rosiglitazone does SomeTZDs as rosiglitazone have been associated with a significantincrease of cardiovascular diseases For this reason FDArestricted their prescription in the United States [23]
Avoiding the increase of postprandial glucose is impor-tant to keep the glycemic levels low in diabetic patientsThe inhibition of 120572-amylase and 120572-glucosidase present inthe gastrointestinal tract could keep the levels of glycemialow Drugs inhibit these enzymes such as acarbose miglitolvoglibose nojirimycin and 1-deoxynojirimycin which allowthe slow absorption of carbohydrates [23] The ethanolicextract of Miconia sp inhibits 120572-amylase by 50 to a lessconcentration than the acarbose drug Additionally the 120572-glucosidase is inhibited byMiconia sp at low concentrationslower than the drug Therefore it is believed that in thepresence of molecules with antihyperglycemic effect in invitro model these compounds could be an alternative toexisting treatments or adjunctive to them which may haveundesired side effects
Miconia sp showed cytotoxicity at a greater concentrationthan necessary to increase expression of PPAR120574 increaselipid accumulation and inhibit 120572-glucosidase and 120572-amylaseThere are reports that the species of the genus MiconiaM stenostachya M cabucu M albicans and M rubiginosalack cytotoxicity or mutagenicity at lower concentrations of100 120583gmL [6]
5 Conclusions
The ethanolic extract of Miconia sp showed increase of thelevel of mRNA expression of PPAR120574 at a significantly higherlevel than rosiglitazone (drug) Also Miconia sp showedinhibition of the enzymes 120572-glucosidase and 120572-amylase withIC50lower than acarbose (drug) and furthermore increase the
capacity of lipid accumulation during adipogenesis similar tothe drug rosiglitazone At the same timeMiconia sp showed
cytotoxicity on VERO cells with a concentration higherthan that presenting biological activity For this reason thecompounds present in the ethanolic extract ofMiconia sp canbe an alternative for the treatment of diabetes mellitus or likean adjunctive with the recommendation of continuing within vivo tests and elucidation of bioactive compounds
Competing Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
References
[1] J Chen Y Wu J Zou and K Gao ldquo120572-Glucosidase inhibitionand antihyperglycemic activity of flavonoids from Ampelop-sis grossedentata and the flavonoid derivativesrdquo Bioorganic ampMedicinal Chemistry vol 24 no 7 pp 1488ndash1494 2016
[2] J F Ascaso ldquoDiabetes mellitus tipo 2 nuevos tratamientosrdquoMedicina Clınica vol 143 no 3 pp 117ndash123 2014
[3] M E Rafols ldquoTejido adiposo heterogeneidad celular y diver-sidad funcionalrdquo Endocrinologıa y Nutricion vol 61 no 2 pp100ndash112 2014
[4] S Ghosh and L Rangan ldquoMolecular docking and inhibitionstudies of120572-amylase activity by labdane diterpenes fromAlpinianigra seedsrdquo Medicinal Chemistry Research vol 23 no 11 pp4836ndash4852 2014
[5] A Josabad Alonso-Castro J Jose Maldonado-Miranda AZarate-Martinez et al ldquoMedicinal plants used in the HuastecaPotosina Mexicordquo Journal of Ethnopharmacology vol 143 no1 pp 292ndash298 2012
[6] J M Serpeloni G R Mazzaron M Prates et al ldquoExperimentaland toxicologic pathology cytotoxic and mutagenic evaluationof extracts from plant species of the Miconia genus and theirinfluence on doxorubicin-induced mutagenicity an in vitroanalysisrdquo Experimental and Toxicologic Pathology vol 63 pp499ndash504 2011
[7] A J Richard T P Burris D Sanchez-Infantes Y Wang DM Ribnicky and J M Stephens ldquoArtemisia extracts activatePPAR120574 promote adipogenesis and enhance insulin sensitivityin adipose tissue of obese micerdquo Nutrition vol 30 no 7-8 ppS31ndashS36 2014
[8] C-S Kong J-A Kim S-S Bak H-G Byun and S-K KimldquoAnti-obesity effect of carboxymethyl chitin by AMPK andaquaporin-7 pathways in 3T3-L1 adipocytesrdquo Journal of Nutri-tional Biochemistry vol 22 no 3 pp 276ndash281 2011
[9] R Chaiittianan P Chayopas A Rattanathongkom P Tip-payawat and K Sutthanut ldquoAnti-obesity potential of cornsilks relationships of phytochemicals and antioxidation anti-pre-adipocyte proliferation anti-adipogenesis and lipolysisinductionrdquo Journal of Functional Foods vol 23 pp 497ndash5102016
[10] X C Tan K H Chua M Ravishankar Ram and U RKuppusamy ldquoMonoterpenes novel insights into their biologicaleffects and roles on glucose uptake and lipidmetabolism in 3T3-L1 adipocytesrdquo Food Chemistry vol 196 pp 242ndash250 2016
[11] H-L Kim J ParkH Park et al ldquoPlatycodon grandiflorumA decandolle ethanolic extract inhibits adipogenic regulators in 3T3-L1 cells and inducesmitochondrial biogenesis in primary brownpreadipocytesrdquo Journal of Agricultural and Food Chemistry vol63 no 35 pp 7721ndash7730 2015
6 Evidence-Based Complementary and Alternative Medicine
[12] F M Siraj S Natarajan M A Huq Y J Kim and D CYang ldquoStructural investigation of ginsenoside Rf with PPAR120574major transcriptional factor of adipogenesis and its impact onadipocyterdquo Journal of Ginseng Research vol 39 no 2 pp 141ndash147 2015
[13] H S Nyambe J A Villa I Ifie et al ldquoInhibition of human 120572-amylase by dietary polyphenolsrdquo Journal of Functional Foodsvol 19 pp 723ndash732 2015
[14] C-W Liu Y-C Wang H-C Lu and W-D Chiang ldquoOpti-mization of ultrasound-assisted extraction conditions for totalphenols with anti-hyperglycemic activity from Psidium guajavaleavesrdquo Process Biochemistry vol 49 no 10 pp 1601ndash1605 2014
[15] H Dehghan Y Sarrafi and P Salehi ldquoAntioxidant and antidia-betic activities of 11 herbal plants from Hyrcania region IranrdquoJournal of Food and Drug Analysis vol 24 no 1 pp 179ndash1882016
[16] N C Ammerman M Beier-Sexton and A F Azad ldquoGrowthand maintenance of Vero cell linesrdquo Current Protocols inMicrobiology 2008
[17] B Kling D Bucherl P Palatzky et al ldquoFlavonoids flavonoidmetabolites and phenolic acids inhibit oxidative stress in theneuronal cell line HT-22 monitored by ECIS and MTT assaya comparative studyrdquo Journal of Natural Products vol 77 no 3pp 446ndash454 2014
[18] M Boncler M Rozalski U Krajewska A Podsędek and CWatala ldquoComparison of PrestoBlue and MTT assays of cellularviability in the assessment of anti-proliferative effects of plantextracts on human endothelial cellsrdquo Journal of Pharmacologicaland Toxicological Methods vol 69 no 1 pp 9ndash16 2014
[19] T Kariharan G Nanayakkara K Parameshwaran et al ldquoCen-tral activation of PPAR-gamma ameliorates diabetes inducedcognitive dysfunction and improves BDNF expressionrdquo Neuro-biology of Aging vol 36 no 3 pp 1451ndash1461 2015
[20] S N Lewis J Bassaganya-Riera and D R Bevan ldquoVirtualscreening as a technique for PPARmodulator discoveryrdquo PPARResearch vol 2010 Article ID 861238 10 pages 2010
[21] L Wang B Waltenberger E-M Pferschy-Wenzig et al ldquoNatu-ral product agonists of peroxisome proliferator-activated recep-tor gamma (PPAR120574) a reviewrdquo Biochemical Pharmacology vol92 no 1 pp 73ndash89 2014
[22] C Janani and B D Ranjitha ldquoPPAR gamma genemdasha reviewrdquoDiabetes and Metabolic Syndrome Clinical Research andReviews vol 9 no 1 pp 46ndash50 2015
[23] G Oboh O B Ogunsuyi M D Ogunbadejo and S A Ade-fegha ldquoInfluence of gallic acid on 120572-amylase and 120572-glucosidaseinhibitory properties of acarboserdquo Journal of Food and DrugAnalysis 2016
4 Evidence-Based Complementary and Alternative Medicine
EC expression controlMM maturation medium
EC MMMicRos06070809
11112131415
mRN
A ex
pres
sion
leve
l (fo
ld ch
ange
)
Ros rosiglitazone 1120583M
lowast
lowastlowast
Mic Miconia sp 40120583gmL
Figure 1 Effect of different compounds on the expression of mRNAPPAR120574 On day 10 the treatments were applied Group 1 DMEMwith 01 ethanol (EC) Group 2 MM and rosiglitazone (Ros) asa positive control Group 3 MM and Miconia (Mic) and Group4 MM On day 11 of adipocyte differentiation the total RNA wasisolated lowast119875 le 005 there is a significant difference with the controlof expression lowastlowast119875 le 005 there is a significant difference with thecontrol of expression and rosiglitazone (119899 = 3)
values above 1 indicate an overexpression or upregulation InGroup 2 the value in the expression levels was 1166 plusmn 0007fold change at the same time in Group 3 a value was 1393 plusmn0008 and finally Group 4 showed a value of 0746plusmn0034 foldchange (Figure 1)
Miconia sp 40 120583gmL produced upregulation in theexpression of mRNA of PPAR120574 with a value greater thanthat of the drug rosiglitazone which increases the expressionof PPAR120574 as expected The use of maturation medium withboth extracts as rosiglitazone did not increase by itself theexpression levels of the gene PPAR120574
In the lipid accumulation test during adipogenesis withdifferent treatments Group 1 (Dm) showed absorbance of0137 this value represents the accumulated lipids duringadipogenesis induced differentiation medium already estab-lished At the same time Group 2 (Dm + Miconia sp40 120583gmL) showed absorbance of 0184 Group 3 (Dm +rosiglitazone 1 120583M) showed an absorbance value of 0177
Taking the absorbance value from Group 1 as 100 lipidaccumulation when extract Miconia sp was added to thedifferentiation medium lipid accumulation was increased to3457 compared to Group 1 Like the Miconia sp extractadding the drug rosiglitazone to differentiation medium itcauses an increase of 2955 compared to Group 1 Theincreased absorbance of the extract and the drug is significantcompared with Dm Miconia sp presented a very similarresult to rosiglitazone difference between them not of greatsignificance (Figure 2)
In the 120572-amylase inhibition assay different treatmentsto the mixture of enzyme-substrate reaction were addedobtaining the following results for treatment 1 Miconia sppresented IC
50of 2823 plusmn 215 120583gmL At the same time
for treatment 2 phosphate buffer and 2 ethanol (negativecontrol) showed no inhibition by the vehicle used for
(abs
orba
nce510
nm)
Accu
mul
atio
n of
lipi
ds
Dm + rosiglitazone1120583M
Dm + Miconia sp40120583gmL
Dm
04
035
03
025
02
015
01
005
0
lowast lowast
Figure 2 Effect of different substances in lipid accumulation duringadipogenesis in 3T3-L1 cellsThe treatments were applied at day 0 ofdifferentiation and withdrawn at day 2 On day 10 when adipocytesreached maturity the staining was performed lowast119875 le 005 significantdifference between treatments and Dm (119899 = 3)
Table 1 Enzymatic inhibition from acarbose and Miconia spextract
Treatment120572-Amylase(IC50plusmn DE
120583gmL)
120572-Glucosidase(IC50plusmn DE
120583gmL)Miconialowast 2823 plusmn 215 195 plusmn 015Acarbose (positive control) 99384 plusmn 15713 33100 plusmn 7208Phosphate buffer with 1ethanol (negative control) NI NI
Data are expressed as mean plusmn the SD (119899 = 3) NI no inhibition lowast119875 le 005significant difference with acarbose
(positive control)According to the results and despite being a complete
extract the extract of Miconia sp reveals a greater capacitythan acarbose to inhibit 50 of enzyme activity this differ-ence between the two treatments is statistically significant(Table 1)
In the inhibition assay of 120572-glucosidase the followingresults were obtained for treatment 1 Miconia sp extractprovided IC
50of 195 plusmn 015 120583gmL At the same time for
treatment 2 phosphate buffer with 1 ethanol (negativecontrol) did not show any inhibitory effect on the enzymefor treatment 3 acarbose (positive control) showed IC
50of
33100 plusmn 7208 120583gmL (Table 1)Miconia sp extract also has a greater inhibition than
acarbose and acarbose is used as a reference compound in theinhibition of these enzymesThe differences in the treatmentsare statistically significant
In determining the proliferation on the VERO cell lineIC50of 31454 plusmn 4540mgmL considered toxic was obtained
forMiconia sp extract
4 Discussion
PPAR120574 is a nuclear receptor that acts as a transcription factorit improves insulin sensitivity by the cells and enhances
Evidence-Based Complementary and Alternative Medicine 5
glucose utilization [19] A diverse set of natural and syntheticmolecules is classified as ligands and can induce activationand that expression of PPAR120574 These ligands include nutri-ents endogenous ligands and drugs [20] one of those drugsis thiazolidinediones (TZDs) such as rosiglitazone Miconiasp showed an increased mRNA expression of PPAR120574 evenmore than rosiglitazone Moreover Miconia sp was ableto increase lipid accumulation during adipogenesis in 3T3cell line L-1 similar to positive control rosiglitazone It isknown that PPAR120574 is the master regulator of adipocytedifferentiation and during adipogenesis PPAR120574 is induced[21] PPAR120574 activation in adipocytes ensures proper balanceand secretion of adipokines such as leptin and adiponectinthey are mediators in insulin action in peripheral tissueswhich causes insulin sensitivity throughout the body [22]The products mentioned above that stimulate the productionof PPAR120574 are candidates to induce the proper functioningof insulin and recognition considering them as potentialantidiabetic agents
The results suggest that the presence of secondarymetabolites could be involved in upregulation of PPAR120574 genQuercetin catechin and kaempferol have been reported insome species of Miconia sp and these compounds could actas ligands of PPAR120574 [6 21] just as rosiglitazone does SomeTZDs as rosiglitazone have been associated with a significantincrease of cardiovascular diseases For this reason FDArestricted their prescription in the United States [23]
Avoiding the increase of postprandial glucose is impor-tant to keep the glycemic levels low in diabetic patientsThe inhibition of 120572-amylase and 120572-glucosidase present inthe gastrointestinal tract could keep the levels of glycemialow Drugs inhibit these enzymes such as acarbose miglitolvoglibose nojirimycin and 1-deoxynojirimycin which allowthe slow absorption of carbohydrates [23] The ethanolicextract of Miconia sp inhibits 120572-amylase by 50 to a lessconcentration than the acarbose drug Additionally the 120572-glucosidase is inhibited byMiconia sp at low concentrationslower than the drug Therefore it is believed that in thepresence of molecules with antihyperglycemic effect in invitro model these compounds could be an alternative toexisting treatments or adjunctive to them which may haveundesired side effects
Miconia sp showed cytotoxicity at a greater concentrationthan necessary to increase expression of PPAR120574 increaselipid accumulation and inhibit 120572-glucosidase and 120572-amylaseThere are reports that the species of the genus MiconiaM stenostachya M cabucu M albicans and M rubiginosalack cytotoxicity or mutagenicity at lower concentrations of100 120583gmL [6]
5 Conclusions
The ethanolic extract of Miconia sp showed increase of thelevel of mRNA expression of PPAR120574 at a significantly higherlevel than rosiglitazone (drug) Also Miconia sp showedinhibition of the enzymes 120572-glucosidase and 120572-amylase withIC50lower than acarbose (drug) and furthermore increase the
capacity of lipid accumulation during adipogenesis similar tothe drug rosiglitazone At the same timeMiconia sp showed
cytotoxicity on VERO cells with a concentration higherthan that presenting biological activity For this reason thecompounds present in the ethanolic extract ofMiconia sp canbe an alternative for the treatment of diabetes mellitus or likean adjunctive with the recommendation of continuing within vivo tests and elucidation of bioactive compounds
Competing Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
References
[1] J Chen Y Wu J Zou and K Gao ldquo120572-Glucosidase inhibitionand antihyperglycemic activity of flavonoids from Ampelop-sis grossedentata and the flavonoid derivativesrdquo Bioorganic ampMedicinal Chemistry vol 24 no 7 pp 1488ndash1494 2016
[2] J F Ascaso ldquoDiabetes mellitus tipo 2 nuevos tratamientosrdquoMedicina Clınica vol 143 no 3 pp 117ndash123 2014
[3] M E Rafols ldquoTejido adiposo heterogeneidad celular y diver-sidad funcionalrdquo Endocrinologıa y Nutricion vol 61 no 2 pp100ndash112 2014
[4] S Ghosh and L Rangan ldquoMolecular docking and inhibitionstudies of120572-amylase activity by labdane diterpenes fromAlpinianigra seedsrdquo Medicinal Chemistry Research vol 23 no 11 pp4836ndash4852 2014
[5] A Josabad Alonso-Castro J Jose Maldonado-Miranda AZarate-Martinez et al ldquoMedicinal plants used in the HuastecaPotosina Mexicordquo Journal of Ethnopharmacology vol 143 no1 pp 292ndash298 2012
[6] J M Serpeloni G R Mazzaron M Prates et al ldquoExperimentaland toxicologic pathology cytotoxic and mutagenic evaluationof extracts from plant species of the Miconia genus and theirinfluence on doxorubicin-induced mutagenicity an in vitroanalysisrdquo Experimental and Toxicologic Pathology vol 63 pp499ndash504 2011
[7] A J Richard T P Burris D Sanchez-Infantes Y Wang DM Ribnicky and J M Stephens ldquoArtemisia extracts activatePPAR120574 promote adipogenesis and enhance insulin sensitivityin adipose tissue of obese micerdquo Nutrition vol 30 no 7-8 ppS31ndashS36 2014
[8] C-S Kong J-A Kim S-S Bak H-G Byun and S-K KimldquoAnti-obesity effect of carboxymethyl chitin by AMPK andaquaporin-7 pathways in 3T3-L1 adipocytesrdquo Journal of Nutri-tional Biochemistry vol 22 no 3 pp 276ndash281 2011
[9] R Chaiittianan P Chayopas A Rattanathongkom P Tip-payawat and K Sutthanut ldquoAnti-obesity potential of cornsilks relationships of phytochemicals and antioxidation anti-pre-adipocyte proliferation anti-adipogenesis and lipolysisinductionrdquo Journal of Functional Foods vol 23 pp 497ndash5102016
[10] X C Tan K H Chua M Ravishankar Ram and U RKuppusamy ldquoMonoterpenes novel insights into their biologicaleffects and roles on glucose uptake and lipidmetabolism in 3T3-L1 adipocytesrdquo Food Chemistry vol 196 pp 242ndash250 2016
[11] H-L Kim J ParkH Park et al ldquoPlatycodon grandiflorumA decandolle ethanolic extract inhibits adipogenic regulators in 3T3-L1 cells and inducesmitochondrial biogenesis in primary brownpreadipocytesrdquo Journal of Agricultural and Food Chemistry vol63 no 35 pp 7721ndash7730 2015
6 Evidence-Based Complementary and Alternative Medicine
[12] F M Siraj S Natarajan M A Huq Y J Kim and D CYang ldquoStructural investigation of ginsenoside Rf with PPAR120574major transcriptional factor of adipogenesis and its impact onadipocyterdquo Journal of Ginseng Research vol 39 no 2 pp 141ndash147 2015
[13] H S Nyambe J A Villa I Ifie et al ldquoInhibition of human 120572-amylase by dietary polyphenolsrdquo Journal of Functional Foodsvol 19 pp 723ndash732 2015
[14] C-W Liu Y-C Wang H-C Lu and W-D Chiang ldquoOpti-mization of ultrasound-assisted extraction conditions for totalphenols with anti-hyperglycemic activity from Psidium guajavaleavesrdquo Process Biochemistry vol 49 no 10 pp 1601ndash1605 2014
[15] H Dehghan Y Sarrafi and P Salehi ldquoAntioxidant and antidia-betic activities of 11 herbal plants from Hyrcania region IranrdquoJournal of Food and Drug Analysis vol 24 no 1 pp 179ndash1882016
[16] N C Ammerman M Beier-Sexton and A F Azad ldquoGrowthand maintenance of Vero cell linesrdquo Current Protocols inMicrobiology 2008
[17] B Kling D Bucherl P Palatzky et al ldquoFlavonoids flavonoidmetabolites and phenolic acids inhibit oxidative stress in theneuronal cell line HT-22 monitored by ECIS and MTT assaya comparative studyrdquo Journal of Natural Products vol 77 no 3pp 446ndash454 2014
[18] M Boncler M Rozalski U Krajewska A Podsędek and CWatala ldquoComparison of PrestoBlue and MTT assays of cellularviability in the assessment of anti-proliferative effects of plantextracts on human endothelial cellsrdquo Journal of Pharmacologicaland Toxicological Methods vol 69 no 1 pp 9ndash16 2014
[19] T Kariharan G Nanayakkara K Parameshwaran et al ldquoCen-tral activation of PPAR-gamma ameliorates diabetes inducedcognitive dysfunction and improves BDNF expressionrdquo Neuro-biology of Aging vol 36 no 3 pp 1451ndash1461 2015
[20] S N Lewis J Bassaganya-Riera and D R Bevan ldquoVirtualscreening as a technique for PPARmodulator discoveryrdquo PPARResearch vol 2010 Article ID 861238 10 pages 2010
[21] L Wang B Waltenberger E-M Pferschy-Wenzig et al ldquoNatu-ral product agonists of peroxisome proliferator-activated recep-tor gamma (PPAR120574) a reviewrdquo Biochemical Pharmacology vol92 no 1 pp 73ndash89 2014
[22] C Janani and B D Ranjitha ldquoPPAR gamma genemdasha reviewrdquoDiabetes and Metabolic Syndrome Clinical Research andReviews vol 9 no 1 pp 46ndash50 2015
[23] G Oboh O B Ogunsuyi M D Ogunbadejo and S A Ade-fegha ldquoInfluence of gallic acid on 120572-amylase and 120572-glucosidaseinhibitory properties of acarboserdquo Journal of Food and DrugAnalysis 2016
Evidence-Based Complementary and Alternative Medicine 5
glucose utilization [19] A diverse set of natural and syntheticmolecules is classified as ligands and can induce activationand that expression of PPAR120574 These ligands include nutri-ents endogenous ligands and drugs [20] one of those drugsis thiazolidinediones (TZDs) such as rosiglitazone Miconiasp showed an increased mRNA expression of PPAR120574 evenmore than rosiglitazone Moreover Miconia sp was ableto increase lipid accumulation during adipogenesis in 3T3cell line L-1 similar to positive control rosiglitazone It isknown that PPAR120574 is the master regulator of adipocytedifferentiation and during adipogenesis PPAR120574 is induced[21] PPAR120574 activation in adipocytes ensures proper balanceand secretion of adipokines such as leptin and adiponectinthey are mediators in insulin action in peripheral tissueswhich causes insulin sensitivity throughout the body [22]The products mentioned above that stimulate the productionof PPAR120574 are candidates to induce the proper functioningof insulin and recognition considering them as potentialantidiabetic agents
The results suggest that the presence of secondarymetabolites could be involved in upregulation of PPAR120574 genQuercetin catechin and kaempferol have been reported insome species of Miconia sp and these compounds could actas ligands of PPAR120574 [6 21] just as rosiglitazone does SomeTZDs as rosiglitazone have been associated with a significantincrease of cardiovascular diseases For this reason FDArestricted their prescription in the United States [23]
Avoiding the increase of postprandial glucose is impor-tant to keep the glycemic levels low in diabetic patientsThe inhibition of 120572-amylase and 120572-glucosidase present inthe gastrointestinal tract could keep the levels of glycemialow Drugs inhibit these enzymes such as acarbose miglitolvoglibose nojirimycin and 1-deoxynojirimycin which allowthe slow absorption of carbohydrates [23] The ethanolicextract of Miconia sp inhibits 120572-amylase by 50 to a lessconcentration than the acarbose drug Additionally the 120572-glucosidase is inhibited byMiconia sp at low concentrationslower than the drug Therefore it is believed that in thepresence of molecules with antihyperglycemic effect in invitro model these compounds could be an alternative toexisting treatments or adjunctive to them which may haveundesired side effects
Miconia sp showed cytotoxicity at a greater concentrationthan necessary to increase expression of PPAR120574 increaselipid accumulation and inhibit 120572-glucosidase and 120572-amylaseThere are reports that the species of the genus MiconiaM stenostachya M cabucu M albicans and M rubiginosalack cytotoxicity or mutagenicity at lower concentrations of100 120583gmL [6]
5 Conclusions
The ethanolic extract of Miconia sp showed increase of thelevel of mRNA expression of PPAR120574 at a significantly higherlevel than rosiglitazone (drug) Also Miconia sp showedinhibition of the enzymes 120572-glucosidase and 120572-amylase withIC50lower than acarbose (drug) and furthermore increase the
capacity of lipid accumulation during adipogenesis similar tothe drug rosiglitazone At the same timeMiconia sp showed
cytotoxicity on VERO cells with a concentration higherthan that presenting biological activity For this reason thecompounds present in the ethanolic extract ofMiconia sp canbe an alternative for the treatment of diabetes mellitus or likean adjunctive with the recommendation of continuing within vivo tests and elucidation of bioactive compounds
Competing Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
References
[1] J Chen Y Wu J Zou and K Gao ldquo120572-Glucosidase inhibitionand antihyperglycemic activity of flavonoids from Ampelop-sis grossedentata and the flavonoid derivativesrdquo Bioorganic ampMedicinal Chemistry vol 24 no 7 pp 1488ndash1494 2016
[2] J F Ascaso ldquoDiabetes mellitus tipo 2 nuevos tratamientosrdquoMedicina Clınica vol 143 no 3 pp 117ndash123 2014
[3] M E Rafols ldquoTejido adiposo heterogeneidad celular y diver-sidad funcionalrdquo Endocrinologıa y Nutricion vol 61 no 2 pp100ndash112 2014
[4] S Ghosh and L Rangan ldquoMolecular docking and inhibitionstudies of120572-amylase activity by labdane diterpenes fromAlpinianigra seedsrdquo Medicinal Chemistry Research vol 23 no 11 pp4836ndash4852 2014
[5] A Josabad Alonso-Castro J Jose Maldonado-Miranda AZarate-Martinez et al ldquoMedicinal plants used in the HuastecaPotosina Mexicordquo Journal of Ethnopharmacology vol 143 no1 pp 292ndash298 2012
[6] J M Serpeloni G R Mazzaron M Prates et al ldquoExperimentaland toxicologic pathology cytotoxic and mutagenic evaluationof extracts from plant species of the Miconia genus and theirinfluence on doxorubicin-induced mutagenicity an in vitroanalysisrdquo Experimental and Toxicologic Pathology vol 63 pp499ndash504 2011
[7] A J Richard T P Burris D Sanchez-Infantes Y Wang DM Ribnicky and J M Stephens ldquoArtemisia extracts activatePPAR120574 promote adipogenesis and enhance insulin sensitivityin adipose tissue of obese micerdquo Nutrition vol 30 no 7-8 ppS31ndashS36 2014
[8] C-S Kong J-A Kim S-S Bak H-G Byun and S-K KimldquoAnti-obesity effect of carboxymethyl chitin by AMPK andaquaporin-7 pathways in 3T3-L1 adipocytesrdquo Journal of Nutri-tional Biochemistry vol 22 no 3 pp 276ndash281 2011
[9] R Chaiittianan P Chayopas A Rattanathongkom P Tip-payawat and K Sutthanut ldquoAnti-obesity potential of cornsilks relationships of phytochemicals and antioxidation anti-pre-adipocyte proliferation anti-adipogenesis and lipolysisinductionrdquo Journal of Functional Foods vol 23 pp 497ndash5102016
[10] X C Tan K H Chua M Ravishankar Ram and U RKuppusamy ldquoMonoterpenes novel insights into their biologicaleffects and roles on glucose uptake and lipidmetabolism in 3T3-L1 adipocytesrdquo Food Chemistry vol 196 pp 242ndash250 2016
[11] H-L Kim J ParkH Park et al ldquoPlatycodon grandiflorumA decandolle ethanolic extract inhibits adipogenic regulators in 3T3-L1 cells and inducesmitochondrial biogenesis in primary brownpreadipocytesrdquo Journal of Agricultural and Food Chemistry vol63 no 35 pp 7721ndash7730 2015
6 Evidence-Based Complementary and Alternative Medicine
[12] F M Siraj S Natarajan M A Huq Y J Kim and D CYang ldquoStructural investigation of ginsenoside Rf with PPAR120574major transcriptional factor of adipogenesis and its impact onadipocyterdquo Journal of Ginseng Research vol 39 no 2 pp 141ndash147 2015
[13] H S Nyambe J A Villa I Ifie et al ldquoInhibition of human 120572-amylase by dietary polyphenolsrdquo Journal of Functional Foodsvol 19 pp 723ndash732 2015
[14] C-W Liu Y-C Wang H-C Lu and W-D Chiang ldquoOpti-mization of ultrasound-assisted extraction conditions for totalphenols with anti-hyperglycemic activity from Psidium guajavaleavesrdquo Process Biochemistry vol 49 no 10 pp 1601ndash1605 2014
[15] H Dehghan Y Sarrafi and P Salehi ldquoAntioxidant and antidia-betic activities of 11 herbal plants from Hyrcania region IranrdquoJournal of Food and Drug Analysis vol 24 no 1 pp 179ndash1882016
[16] N C Ammerman M Beier-Sexton and A F Azad ldquoGrowthand maintenance of Vero cell linesrdquo Current Protocols inMicrobiology 2008
[17] B Kling D Bucherl P Palatzky et al ldquoFlavonoids flavonoidmetabolites and phenolic acids inhibit oxidative stress in theneuronal cell line HT-22 monitored by ECIS and MTT assaya comparative studyrdquo Journal of Natural Products vol 77 no 3pp 446ndash454 2014
[18] M Boncler M Rozalski U Krajewska A Podsędek and CWatala ldquoComparison of PrestoBlue and MTT assays of cellularviability in the assessment of anti-proliferative effects of plantextracts on human endothelial cellsrdquo Journal of Pharmacologicaland Toxicological Methods vol 69 no 1 pp 9ndash16 2014
[19] T Kariharan G Nanayakkara K Parameshwaran et al ldquoCen-tral activation of PPAR-gamma ameliorates diabetes inducedcognitive dysfunction and improves BDNF expressionrdquo Neuro-biology of Aging vol 36 no 3 pp 1451ndash1461 2015
[20] S N Lewis J Bassaganya-Riera and D R Bevan ldquoVirtualscreening as a technique for PPARmodulator discoveryrdquo PPARResearch vol 2010 Article ID 861238 10 pages 2010
[21] L Wang B Waltenberger E-M Pferschy-Wenzig et al ldquoNatu-ral product agonists of peroxisome proliferator-activated recep-tor gamma (PPAR120574) a reviewrdquo Biochemical Pharmacology vol92 no 1 pp 73ndash89 2014
[22] C Janani and B D Ranjitha ldquoPPAR gamma genemdasha reviewrdquoDiabetes and Metabolic Syndrome Clinical Research andReviews vol 9 no 1 pp 46ndash50 2015
[23] G Oboh O B Ogunsuyi M D Ogunbadejo and S A Ade-fegha ldquoInfluence of gallic acid on 120572-amylase and 120572-glucosidaseinhibitory properties of acarboserdquo Journal of Food and DrugAnalysis 2016
6 Evidence-Based Complementary and Alternative Medicine
[12] F M Siraj S Natarajan M A Huq Y J Kim and D CYang ldquoStructural investigation of ginsenoside Rf with PPAR120574major transcriptional factor of adipogenesis and its impact onadipocyterdquo Journal of Ginseng Research vol 39 no 2 pp 141ndash147 2015
[13] H S Nyambe J A Villa I Ifie et al ldquoInhibition of human 120572-amylase by dietary polyphenolsrdquo Journal of Functional Foodsvol 19 pp 723ndash732 2015
[14] C-W Liu Y-C Wang H-C Lu and W-D Chiang ldquoOpti-mization of ultrasound-assisted extraction conditions for totalphenols with anti-hyperglycemic activity from Psidium guajavaleavesrdquo Process Biochemistry vol 49 no 10 pp 1601ndash1605 2014
[15] H Dehghan Y Sarrafi and P Salehi ldquoAntioxidant and antidia-betic activities of 11 herbal plants from Hyrcania region IranrdquoJournal of Food and Drug Analysis vol 24 no 1 pp 179ndash1882016
[16] N C Ammerman M Beier-Sexton and A F Azad ldquoGrowthand maintenance of Vero cell linesrdquo Current Protocols inMicrobiology 2008
[17] B Kling D Bucherl P Palatzky et al ldquoFlavonoids flavonoidmetabolites and phenolic acids inhibit oxidative stress in theneuronal cell line HT-22 monitored by ECIS and MTT assaya comparative studyrdquo Journal of Natural Products vol 77 no 3pp 446ndash454 2014
[18] M Boncler M Rozalski U Krajewska A Podsędek and CWatala ldquoComparison of PrestoBlue and MTT assays of cellularviability in the assessment of anti-proliferative effects of plantextracts on human endothelial cellsrdquo Journal of Pharmacologicaland Toxicological Methods vol 69 no 1 pp 9ndash16 2014
[19] T Kariharan G Nanayakkara K Parameshwaran et al ldquoCen-tral activation of PPAR-gamma ameliorates diabetes inducedcognitive dysfunction and improves BDNF expressionrdquo Neuro-biology of Aging vol 36 no 3 pp 1451ndash1461 2015
[20] S N Lewis J Bassaganya-Riera and D R Bevan ldquoVirtualscreening as a technique for PPARmodulator discoveryrdquo PPARResearch vol 2010 Article ID 861238 10 pages 2010
[21] L Wang B Waltenberger E-M Pferschy-Wenzig et al ldquoNatu-ral product agonists of peroxisome proliferator-activated recep-tor gamma (PPAR120574) a reviewrdquo Biochemical Pharmacology vol92 no 1 pp 73ndash89 2014
[22] C Janani and B D Ranjitha ldquoPPAR gamma genemdasha reviewrdquoDiabetes and Metabolic Syndrome Clinical Research andReviews vol 9 no 1 pp 46ndash50 2015
[23] G Oboh O B Ogunsuyi M D Ogunbadejo and S A Ade-fegha ldquoInfluence of gallic acid on 120572-amylase and 120572-glucosidaseinhibitory properties of acarboserdquo Journal of Food and DrugAnalysis 2016
![PPAR: Bangladesh: Ganges-Kobadak Irrigation Rehabilitation ... · Title: PPAR: Bangladesh: Ganges-Kobadak Irrigation Rehabilitation Project (Loan 671-BAN[SF]) Author: Asian Development](https://static.documents.pub/doc/80x56/5f87bd77888d5524a31f3544/ppar-bangladesh-ganges-kobadak-irrigation-rehabilitation-title-ppar-bangladesh.jpg)
