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Research ArticleIdentification of Digestive Enzyme Inhibitors fromLudwigia octovalvis (Jacq) PHRaven

Dulce Morales 12 Guillermo Ramirez2 Armando Herrera-Arellano 1

Jaime Tortoriello 2 Miguel Zavala 3 and Alejandro Zamilpa 2

1Facultad de Medicina Universidad Autonoma del Estado de Morelos Cuernavaca 62350 Mexico2Centro de Investigacion Biomedica del Sur Instituto Mexicano del Seguro Social Xochitepec 62790 Mexico3Departamento de Sistemas Biologicos UAMndashXochimilco Mexico City 04960 Mexico

Correspondence should be addressed to Alejandro Zamilpa azamilpa 2000yahoocommx

Received 10 April 2018 Revised 6 June 2018 Accepted 26 June 2018 Published 16 July 2018

Academic Editor Mohammed S Razzaque

Copyright copy 2018 Dulce Morales et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Current antiobesity and antidiabetic tools have been insufficient to curb these diseases and frequently cause side effects thereforenew pancreatic lipase and 120572ndashglucosidase inhibitors could be excellent aids for the prevention and treatment of these diseases Theaim of this study was to identify quantify and characterize the chemical compounds with the highest degree of inhibitory activityof these enzymes contained in a Ludwigia octovalvis hydroalcoholic extract Chemical purification was performed by liquidndashliquidseparation and column chromatography Inhibitory activities were measured in vitro employing acarbose orlistat and a Camelliasinensis hydroalcoholic extract as references For structural elucidation Nuclear Magnetic Resonance was carried out and HighPerformance LiquidChromatographywas used to quantify the compounds For120572ndashglucosidases L octovalvis hydroalcoholic extractand its ethyl acetate fraction showed halfndashmaximal Inhibitory Concentration (IC50) values of 700 and 250 120583gmL for lipase 480and 718 120583gmL while C sinensis showed 260 and 587 120583gmL The most active compounds were identified as ethyl gallate (1 IC50832 120583M) and gallic acid (2 IC50 969 120583M) both displayed competitive inhibition of 120572ndashglucosidases and isoorientin (3 IC50 201 120583M)which displayed uncompetitive inhibition of lipase These data could be useful in the development of a novel phytopharmaceuticaldrug

1 Introduction

Although 120572ndashglucosidase inhibitors such as acarbose and pan-creatic lipase inhibitors such as orlistat are one of the safestantiobesity and antidiabetic drugs for weight loss and reg-ulation of several metabolic and cardiovascular parametersin adults [1ndash3] these drugs have unpleasant gastrointestinalside effects that frequently result in therapy abandonment[4] Therefore it is necessary to continue the search for newalternatives to120572ndashglucosidase and pancreatic lipase inhibitorswithmilder side effects andwhich contribute to the treatmentof obesity and type 2 diabetes mellitus in conjunction withcurrent therapies

Treatment with acarbose brings forth benefits in theregulation of HbA1c blood pressure coagulation factorsthickness of the intimal layer of the carotid endothelial

dysfunction serum glucose and postprandial insulin [2]being especially useful in the treatment of diabetic patientswith adequate baseline control but persistent postprandialhyperglycaemia [1] While orlistat treatment not only pro-duces a reduction in body weight and waist diameter it alsodecreases HbA1c blood pressure and cholesterol [5] reduc-ing the incidence of type 2 diabetes mellitus In additionorlistat is currently the only drug approved by the Food andDrug Administration (FDA) for the treatment of obesity inchildren [3]

Ludwigia octovalvis (Jacq) PHRaven (Onagraceae) [synJussiaea suffruticosa L Jussiaea pubescens L and Jussi-aea angustifolia Lamk] is an helophyte erect herb withoblongndashlanceolate leaves and solitary flowers of four yellowpetals [6] According to Mexican data this species is noton a protection status [7] Almost all parts of the plant

HindawiEvidence-Based Complementary and Alternative MedicineVolume 2018 Article ID 8781352 11 pageshttpsdoiorg10115520188781352

2 Evidence-Based Complementary and Alternative Medicine

have been reported as having several medicinal uses [8 9]among them the antidiabetic use by Mexican and Indianhealers [10 11] in which the boiled extract or the juiceof the whole plant is used Previous phytochemical studieshave described the presence of flavonoids phenolic acidspolyphenols saponins sterols tannins and triterpenoids[12ndash15] in different organs of this medicinal plant Severalpharmacological effects such as hypoglycaemic [8] anti-hyperglycaemic [16 17] and antiproliferative in 3T3ndashL1adipocytes [18] have been described through variousmodelsMoreover the hydroalcoholic extract of L octovalvis leaveswas themost effective in the inhibition of 120572ndashglucosidases andpancreatic lipase in a screening of 23 extracts of medicinalplants reported as traditional treatments for type 2 diabetesmellitus [10] In addition a report also exists on L octovalvisantidiarrheal activity probably mediated by regulation ofgastrointestinal motility [19] this activity could help reducesome of the side effects of intestinal enzyme inhibition suchas faecal urgency or abdominal pain

The aim of this work was to isolate identify quantifyand characterize the compounds with the greatest inhibitoryactivity of 120572ndashglucosidases and pancreatic lipase in thehydroalcoholic extract of L octovalvis leaves through itsbioassayndashguided fractionation

2 Materials and Methods

21 General All chemicals were of analyticalndashreagent gradeCorn starch (S4126) 23ndashdimercaptondash1ndashpropanol tributyrate(DMPTB 97 282413) 551015840ndashdithiobis(2ndashnitrobenzoic acid)(DTNB ge98 D8130) lipase from porcine pancreas (PPLtype II 100ndash500 unitsmg L3126) Triton Xndash100 (X100) SDS(ge985 L3771) glycerol (ge995 GE17ndash1325ndash01) DMSO(ge999 547239) polyethylene glycol (PEG 1546580)2ndashaminoethyl diphenylborinate (97 D9754) isoorientin(ge98 I1536) and gallic acid (ge97 27645) were purchasedfrom SigmandashAldrich (St Louis MO) Miscellaneous solventswere purchased fromMerck KGaA (Darmstadt Germany)

Orlistat (Lysthin PsicoFarmaMexico City) and acarbose(Sincrosa Alpharma Mexico City) were purified by silicachromatography and crystallized to be used as positivecontrols for enzyme inhibition assays

Thin layer chromatography (TLC) was performed usingsilica gel 60 RPndash18 F254s aluminium sheets (105560 MerckKGaA) TLC plates were analysed under UV light at 254 and360 nm using the Natural ProductsndashPEG reagent (NPndashPEG1 methanolic solution of diphenylboryloxyethylamine fol-lowed by 5 ethanolic PEG) as chemical detection system[20]

Melting points were obtained on a Thermo ScientificIA9000 series melting point apparatus (ElectrothermalEssex UK)

Nuclear Magnetic Resonance (NMR) 1H (400 MHz) andNMR 13C (100 MHz) spectra were obtained with VarianINOVAndash400 equipment (Varian Co Palo Alto CA) usingtetramethylsilane as internal standard

22 Plant Material and Preparation of Extracts Leaves ofL octovalvis were collected at Xochitepec Morelos Mexico

(18∘471015840407010158401015840 N 99∘111015840492710158401015840 W) between September andOctober of 2012 A voucher of plant material was depositedunder code number 34667 at the HUMO Herbarium in theCentro de Investigacion en Biodiversidad y Conservacion of theAutonomous University of the State of Morelos (UniversidadAutonoma del Estado de MorelosndashCIByCndashUAEM MorelosMexico)

Camellia sinensis (L) Kuntze (Theaceae) commercialground leaves purchased at a Japanese specialty store (Yama-motoyama Pomona CA) was used as a positive vegetalcontrol Plant names were checked and updated with theonline website httpwwwtheplantlistorg [21]

Fresh leaves of L octovalviswere washed and dried underdark conditions at room temperature and then milled to 4ndash6mm Ground material (1 kg) was extracted (110 ratio wv)with a 60 ethanol aqueous solution at 25∘C for 24 h Theliquid extract was paper-filtered concentrated in a rotaryevaporator Laborota 4000 (Heidolph Schwabach Germany)under reduced pressure at 50∘C and freeze-dried to obtain337 g of brown powder (324 yield) This dry extract(LoHAE) was stored at 4∘C until its pharmacological andphytochemical analysis C sinensis hydroalcoholic extract(CsHAE) was identically prepared

23 Fractionation of LoHAE and Purification of Active Frac-tions One hundred and ninety grams of LoHAE was sub-jected to a liquidndashliquid separation process using water andethyl acetate The solvent of both fractions was eliminatedby low pressure distillation to obtain an organic fraction(LoEAF) and an aqueous fraction (LoAqF)

The less polar fraction (LoEAF 25 g) was subjected to achromatographic silica gel 60 column (109385 Merck KGaA)using dichloromethanemethanol gradient system as mobilephase to give 69 samples of 150 mL each The separationprocess was monitored by TLC and all the samples weregrouped into 20 final fractions The most representativefractions (yields ge5 C1F1ndashC1F6) were subjected to bothassays

The active fractions C1F4 and C1F6 were fraction-ated using column chromatography with silica gel LiChro-prep RPndash18 (113900 Merck KGaA) and a mixture ofwateracetonitrile All the fractions were analysed by TLCand the samples with similar chemical composition weregrouped

From C1F4 (186 mg) 10 final fractions were obtained ofwhich C2F1 produced a white precipitate which was found tobe a pure compound by TLC and High Performance LiquidChromatography (HPLC)

From C1F6 (11 g) 19 final fractions were obtained themost representative (yields ge5) were C3F1 C3F2 C3F3and C3F4 Fraction C3F3 was purified obtaining fractionsC4F1 C4F2 C4F3 C4F4 C4F5 and C4F6 Fraction C4F4produced an orangeyellow precipitate (C4F4ndashP 12 mg)All these fractions (see Scheme 1) were subjected to thepharmacological assay

24 HPLC Analysis HPLC analysis was performed on achromatographic system equipped with a Waters AllianceSeparationModule (2695Waters CorporationMilfordMA)

Evidence-Based Complementary and Alternative Medicine 3

LoHAELoEAF

C1F1

C1F2

C1F3

C1F4 C2F1

C1F5

C1F6

C3F1

C3F2

C3F3

C4F1

C4F2

C4F3

C4F4 C4F4-P

C4F5

C4F6

C3F4

LoAqF

Scheme 1 Fractionation of L octovalvis hydroalcoholic extract (LoHAE) The isolation process of the active compounds is illustrated bycolors green for ethyl gallate blue for gallic acid and yellow for isoorientin

and a photodiode array detector (2996Waters Corporation)employing Empower Pro software (Waters Corporation)Separation was carried out using a Supelcosil LCndashF HPLCcolumn (59158 Supelco Bellefonte PA) The mobile phaseconsisted of amixture of trifluoroacetic acid solution (solventA 05) and acetonitrile (solvent B)with the following ratiosAB = 1000 (0ndash1 min) 955 (2ndash3 min) 7030 (4ndash7min) 5050(8ndash22 min) 2080 (23 min) 0100 (24ndash26 min) 1000 (27ndash30min) The sample injection volume was 10 mL with a 09mLmin flow rate during 30 min The detection wavelengthwas 190ndash600 nm

Quantification of the isolated compounds was achievedusing calibration curves and LoHAE or LoEAF HPLCanalysis The calibration curve was made using ascendantconcentrations (25 50 100 and 200 120583gmL) of the isolatedcompounds which were injected by triplicate at 10 120583L inthe previously described HPLC method A chromatographicprofile of each concentration was obtained at 254 or 360 nmwavelength and data on area under curve peak were used toobtain the respective straightndashline equations

25 Enzymatic Inhibition Assays Pancreatic lipase inhibitionassay was previously reported [22] Briefly the absorbanceof a mixture of DTNB 02 mM DMPTB 08 mM NaCl01M CaCl2 2 mM Triton Xndash100 004 porcine lipase 65120583gmL and the sample (dissolved in DMSO and water) at025 mgmL was followed with a Thermo Scientific Genesys20 Visible Spectrophotometer (Fisher Scientific 4001000Hampton NH) at 412 nm every 20 s for five minutes andplotted (Excel Microsoft) to obtain initial velocity value Thelipase was prepared as a stock at 10 mgmL in TrisndashHCl 25mM pH 62 with 01 M NaCl SDS 2 mM and 250 120583LmL ofglycerol A control assay without substrate was carried out todiscard nonspecific reactions with DTMB All reactions weretested by triplicate

The 120572ndashglucosidase assay was previously reported [10] Inbrief corn starch (4 mgmL) was digested by crude enzymeat 37∘C during 10minutes in a phosphate buffer pH 7 solution

at a sample concentration of 06 mgmL (dissolved in DMSOand water) Subsequently released glucose was quantified bya glucose oxidase-based clinical reagent with the GODndashPODTrinder kit (Spinreact Girona Spain) following manufac-turerrsquos directions All tests were performed in quadruplicateCrude enzymewas obtained directly fromhealthyWistar rats(12 h fasting) The small intestine was flushed several timeswith ice-cold isotonic buffer pH 7 and after the scraping ofthe mucosa it was homogenized and stored at -20∘C Animalcare and management were carried out under the guidelinesof Mexican Official Standard NOMndash062ndashZOOndash1999

For both assays percentage of inhibitions was calculatedas the residual enzymatic activity of the negative control(DMSO and water) by using

119894119899ℎ119894119887119894119905119894119900119899 = 100 minus (119860119887119904119900119903119887119886119899119888119890119904119886119898119901119897119890119860119887119904119900119903119887119886119899119888119890119888119900119899119905119903119900119897 times 100) (1)

Concentrations of extracts resulting in 50 inhibition ofenzyme activity (IC50 values) were determined graphicallyquantifying enzymatic activities at ascendant concentrationsof each sample (6ndash3600 120583gmL for 120572ndashglucosidases and5ndash2500 120583gmL for pancreatic lipase) The logarithm of theconcentration was plotted on the x-axis and the percentageof enzymatic inhibitory activity on the y-axis to obtain asemilogarithmic graphic

The type of inhibition was determined quantifyingthe activity with and without inhibitor at different sub-strate concentrations (5ndash035 mgmL for 120572ndashglucosidasesand 005ndash02 120583gmL for pancreatic lipase) and comparingLineweaverndashBurk plots (inverse substrate concentration [S]and inverse reaction velocity V) In the case of the determi-nation of 120572ndashglucosidase type of inhibition the substrate waschanged from corn starch to maltodextrin (MD100 LuzhouBiondashChem Technology Co Shandong China) in order tohave greater uniformity in the reaction

MichaelisndashMenten constant (Km) and apparent Km(Km

app) were obtained analysing the LineweaverndashBurk plotsThese values allowed to obtain the inhibition constant (Ki)


Table 1 Enzyme inhibition of hydroalcoholic extract fractions and compounds isolated from L octovalvis leaves

SampleInhibition percentage

120572ndashglucosidases06 mgmL

Pancreatic lipase025 mgmL

Acarbose 500 plusmn 16lowast NAOrlistat NA 500 plusmn 26lowastlowastCsHAE 808 plusmn 11 348 plusmn 25LoHAE 589 plusmn 57 236 plusmn 25LoEAF 828 plusmn 36 312 plusmn 19LoAqF 768 plusmn 19 156 plusmn 25C1F1 (ethyl gallate) 984 plusmn 20 232 plusmn 30C1F2 601 plusmn 55 225 plusmn 36C1F3 399 plusmn 56 43 plusmn 35C1F4 989 plusmn 16 200 plusmn 23C1F5 842 plusmn 53 282 plusmn 27C1F6 798 plusmn 38 453 plusmn 06C2F1 (gallic acid) 989 plusmn 06 NAC3F1 NA 109 plusmn 03C3F2 NA 293 plusmn 36C3F3 NA 435 plusmn 43C3F4 NA 364 plusmn 40C4F1 NA 414 plusmn 32C4F2 NA 166 plusmn 45C4F3 NA 458 plusmn 51C4F4ndashP (isoorientin) NA 551 plusmn 31C4F5 NA 535 plusmn 37C4F6 NA 491 plusmn 38Luteolin 663 plusmn 56 NAThe data is indicated as the mean plusmn standard deviationNA = not analysed lowast evaluated at 58 120583M lowastlowast evaluated at 16 120583M

for competitive inhibitors using (2) where [I] representsinhibitor concentration

119870119898119886119901119901 = 119870119898 (1 + [119868]119870119894 ) (2)

26 Statistical Analysis Experimental enzymatic inhibitionactivity values are expressed as the percentage of inhibitionAll biological assays were analysed by ANOVA followed bya Tukey postndashtest with statistical differences established atplt005 using the SPSS100 program

3 Results

31 Fractionation of Hydroalcoholic Extract The liquidndashliq-uid separation of LoHAE produced LoAqF (823 yield 156g) and LoEAF (171 32 g) Samples of these materials andCsHAE were analysed in the in vitro models of enzymeinhibition at 06 mgmL in the case of 120572ndashglucosidases and at025 mgmL in the case of pancreatic lipase (see Table 1)

LoHAE inhibited the 120572ndashglucosidases by 589 and thepancreatic lipase by 236 while CsHAE produced an 808inhibition of 120572ndashglucosidases and 348 of pancreatic lipase

The organic fraction LoEAF had more inhibitory activitythan LoAqF fraction or LoHAE extract in both assays withan 828 inhibition of 120572ndashglucosidases and 312 inhibitionof pancreatic lipase

High Performance Liquid Chromatography spectra anal-ysis of LoEAF (see Figure 1(a)) indicated the presence offlavonoids and organic acids [20 23] The first chromatog-raphy separation of LoEAF afforded 60 fractions which weregrouped in six (C1F1ndashC1F6) where C1F1 and C1F4 fractionsdisplayed the highest inhibitory effect on 120572ndashglucosidaseswhile C1F6 was the most active for lipase (see Table 1)

32 Identification of 120572ndashGlucosidase Inhibitors Fraction C1F1produced a white precipitate (melting point = 160∘C) thatwas analysed by HPLC (see Figure 1(b)) and its chemicalstructure was corroborated by comparison of spectroscopic1H and 13C NMR data (see Table 2 and Figures S1ndashS2 inthe Supplementary Material) indicating that this compoundcorresponds to ethyl gallate [24] (see Figure 2)

Fraction C1F4 produced Fraction C2F1 which also pro-duced a white precipitate (melting point= 260∘C) HPLCUV spectra (see Figures 1(c)ndash1(d)) and spectroscopic 1H


(a)

(b)

(c)

(d)

(e)

(f)

Inhibitionndashglucosidases 828pancreatic lipase 312



Inhibitionndashglucosidases 828


Inhibitionpancreatic lipase 551

500 600 700 800 1400 1500 1600 1700 1800Minutes

300200100

000

300200100

000

300200100

000

300

200

100

000

150

100

050

000

AU

AU

AU

AU

AU

20000 30000 40000nm

90

90

99

71

71

100

118

97

73

88 89

90

94

97

101

20000 30000 40000nm

20000 30000 40000nm

20000 30000 40000

20000 30000 40000nm

1935

21692749

22392710

22392710

1923

2545

3658

2099

2698 3482

1300120011001000900

nm

Figure 1 High Performance Liquid Chromatography chromatograms UV spectra (at 270 nm) and enzymatic inhibition percentage ofdifferent L octovalvis fractions (a) Ethyl acetate fraction LoEAF (b) Fraction C1F1 (c) Fraction C1F4 (d) Fraction C2F1 (e) Fraction C1F6(f) Fraction C4F4ndashP

HO

HO

OH

O

O

RR

ethyl gallategallic acid -H

O

OH

OH

O

HO

OH

O

OH

HO

HOHO

isoorientin

-（2-（3

Figure 2 Chemical structure of the most active compounds identified in L octovalvis hydroalcoholic extract


Table 2 Nuclear Magnetic Resonance (NMR) 13C data of the compounds contained in C1F1 and C4F4ndashP fractions and previously reporteddata for ethyl gallate and isoorientin

Carbon position Chemical shifts (ppm)Ethyl gallate C1F1 Isoorientin C4F4ndashP

1 1219 12195 ndash ndash2 1101 11018 16344 163613 1464 14657 10238 102784 1396 13979 18145 181845 1464 14657 16059 160676 1101 11018 10888 108867 1686 16869 16344 163238 616 6181 9373 93469 146 1473 15627 1561610 ndash ndash 10279 1033811015840 ndash ndash 12156 121421015840 ndash ndash 11882 1189531015840 ndash ndash 11600 1160241015840 ndash ndash 15044 1496851015840 ndash ndash 14595 1457261015840 ndash ndash 11292 11329110158401015840 ndash ndash 7318 7302210158401015840 ndash ndash 7050 7060310158401015840 ndash ndash 7895 7893410158401015840 ndash ndash 7019 7017510158401015840 ndash ndash 8135 8156610158401015840 ndash ndash 6134 6148

NMR analysis (see Figure S3 in the Supplementary Material)indicated that this fraction corresponds to gallic acid [24] (seeFigure 2)

According to HPLC analysis (see Figure S4 in theSupplementary Material) LoHAE and LoEAF containedrespectively 07 and 46 of ethyl gallate and 19 and 25of gallic acid

33 Identification of Pancreatic Lipase Inhibitors FractionC1F6 was analysed by HPLC where several kinds of organicconstituents were observed (see Figure 1(e)) Subsequentchromatographic separations of this fraction followed byinhibitory activity evaluation (see Table 1) allowed us toobtain 11 fractions (see Scheme 1)with different chemical pro-files but similar inhibitory activitiesThemost active fractionC4F4ndashP (melting point = 245∘C) was evaluated by HPLC(see Figure 1(f)) and elucidated by 1H NMR 13C NMRand twondashdimensional NMR spectroscopy experiments (seeTable 2 and Figures S5ndashS9 in the Supplementary Material)and corresponded to isoorientin [25] (see Figure 2)Theotheractive fractions are constituted mainly by flavonoids andother nonidentified compounds

According to HPLC analysis (see Figure S4 in the Supple-mentary Material) LoHAE and LoEAF contained 02 and01 of isoorientin respectively

34 Calculating HalfndashMaximal Inhibitory Concentration andDetermining Type of Inhibition

341 120572ndashGlucosidases All graphs corresponding to con-centrationndashresponse curves in the 120572ndashglucosidase inhibitionmodel are shown (see Figure 3) CsHAE displayed a value ofhalfndashmaximal Inhibitory Concentration (IC50) 260 120583gmLwhile LoHAE produced IC50 700 120583gmL Ethyl gallate (C1F1)and gallic acid (C2F1) IC50 values were 832 120583M and 969 120583Mrespectively Luteolin (Sigma L9283) was used as a naturallyoccurring reference displaying an IC50 = 12577 120583M

Both compounds ethyl gallate and gallic acid make Km(intersection x-axis) increase but maximal velocity (Vmaxintersection y-axis) remains the same as expected for acompetitive enzymatic inhibition (see Figures 4(a)ndash4(b))

For the particular conditions of this assay the calculatedKm was 460 plusmn 3 120583M In the case of Ki constants for ethylgallate at 625 120583M Ki = 636120583M and at 1250 120583M Ki = 315 120583Mfor gallic acid at 625 120583M Ki = 436 120583M and at 1250 120583M Ki =208 120583M

342 Pancreatic Lipase The positive vegetal control Csinensis displayed an IC50 value of 587 120583gmL while LoHAEdisplayed 480 120583gmL LoEAF 718 120583gmL and isoorientin 201120583M (see Figure 5)


CsHAELoHAELoEAFC1F1C2F1

000

2500

5000

7500

10000

Inhi

bitio

n

10 100 1000 100001Concentration [gml]

Figure 3 Concentrationndashresponse graphics for halfndashmaximal Inhibitory Concentration (IC50) determination of CsHAE LoHAE LoEAFC1F1 (isolated ethyl gallate) and C2F1 (isolated gallic acid) in the inhibition model of 120572ndashglucosidases X-axis values are presented in 120583gmL(real values are logarithmic) The error bars represent the standard deviation of 2 measurements in four separate sample runs (n = 8)

2

4

6

8

10

12

2 4 6 8

1V

(mm

olm

in)

1[S] (mgmL)

C1F1 0 M C1F1 625 MC1F1 1250 M

(a)

2

4

6

8

10

12

2 4 6 8

1V

(mm

olm

in)

1[S] (mgmL)

C2F1 0 M C2F1 625 MC2F1 1250 M

(b)

Figure 4 Determination of enzymatic inhibition type by LineweaverndashBurk plots curves in the 120572ndashglucosidase inhibition model (a) C1F1(isolated ethyl gallate) (b) C2F1 (isolated gallic acid)

As observed in the graph (see Figure 6) isoorientinchanged both Vmax and Km (both intersection axes) so itproduced uncompetitive enzymatic inhibition of pancreaticlipase [26]

4 Discussion

According to several studies postprandial hyperglycaemiaperiods even the relative shortndashlasting ones contribute to thedevelopment of chronic diabetes complications even morethan basal hyperglycaemia [27] Moreover the management

of postprandial hyperglycaemia is more difficult to achievethan basal glucose control even with a satisfactory HbA1ccontrol [28] making it one of the main problems in dia-betes treatment [1] Of all the available antidiabetic drugs120572ndashglucosidase inhibitors are currently the most effective andsafest for postprandial glycaemia control as well as intradayand interday glucose fluctuation [29] On the other handchanges have also been found in postprandial lipaemia andplasma free fatty acids (fasting and postprandial) in patientswith type 2 diabetes mellitus which increase macrovasculardamage [30] and also may cause 120573ndashcell dysfunction [31]


00

250

500

750

1000

Inhi

bitio

n

1 10 100 1000 10000Concentration [gml]

CsHAELoHAELoEAFC4F4ndashP

Figure 5 Concentrationndashresponse graphics for halfndashmaximal InhibitoryConcentration (IC50) determination ofCsHAE LoHAE LoEAF andC4F4ndashP (isolated isoorientin) in the inhibitionmodel of pancreatic lipase x-axis values are represented in120583gmL (real values are logarithmic)The error bars represent the standard deviation of 2 measurements in three separate sample runs (n = 6)

00050

00150

00250

10 20

1V

(mm

olm

in)

1[S] (mgmL)

C4F4ndashP 0 MC4F4ndashP 1125 MC4F4ndashP 2250 M

Figure 6 Determination of enzymatic inhibition type of C4F4ndashP(isolated isoorientin) by LineweaverndashBurk plots curves in thepancreatic lipase inhibition model

What is worse when high levels of free fatty acids couplewith glycaemic fluctuations they not only cause endotheliumdamage [32] but also have a prooxidant effect on pancreatic120573cells leading to 120573ndashcell exhaustion [33] this phenomenon hasbeen called glucolipotoxicityHowever it has been shown thatorlistat a lipase inhibitor significantly improves postprandiallipaemia and free fatty acid levels in nondiabetic hyperlipi-demic subjects and also in overweight type 2 diabetic patients[34 35]

L octovalvis hydroalcoholic extract has the advantage ofdisplaying both 120572ndashglucosidase and pancreatic lipase inhibi-tion activities This is the first time that these mechanism

modes are described for this species Besides L octovalvis isan interesting option as antidiabetic because it was describedas innocuous according to the OECD [12]

In this study the concentration of low and intermedi-ate polarity compounds contained in LoEAF considerablyincreased the inhibition of both digestive enzymes althoughan increase of 120572ndashglucosidase inhibition was also observed inLoAqF indicating the presence of other polar compoundswith high inhibitory activity of these enzymes Neverthelessaccording to HPLC quantitative analysis the bipartitionprocess successfully increased the concentration of the two120572ndashglucosidase inhibitors in the organic fraction Thereforeit would be proper to design an extraction or separa-tion method that concentrates these polyhydroxy benzoicacid derivatives Although gallic acid has been previouslydescribed for L octovalvis [15] this is the first time that itsethyl ester derivative (ethyl gallate) is identified and relatedto the biological activity The inhibition of these compoundsusing intestinal rat enzyme and starch as substrate was foundhigher than that produced by the natural product referenceluteolin (IC50 asymp 12577 120583M)which has been described as goodinhibitor of 120572ndashglucosidases [36ndash38]

The inhibitory activity of carbohydrate degradingenzymes by gallic acid and its esters such as ethyl gallatehas been described with inconsistent results According tosome authors gallic acid showed very low or no inhibitoryactivity on porcine and Bacillus sp 120572ndashamylase on rat andSaccharomyces sp 120572ndashglucosidases on rat maltase [39ndash43]However other studies report that this compound shows highinhibitory activity on rat [42 44] and yeast 120572ndashglucosidases[45] and on porcine 120572ndashamylase [43] Moreover it wasfound that gallic acid was able to inhibit mouse rabbitand rat sucrose as well as rat maltase and trehalase [46]Furthermore the IC50 values of gallic acid and ethyl gallatein the inhibition of maltase (390 120583M 415 120583M) and sucrase


(130 120583M 660 120583M) in rat were considered significantly highvalues [40]

This inconsistency of results could be due in part to thediversity of enzymes and substrates used for these tests ithas been shown that the effect of 120572ndashglucosidase inhibitorsvaries according to the origin of the enzymes and the typeof substrate used According to Oki et al [47] to performthe best evaluation of possible 120572ndashglucosidase inhibitors forclinical use mammalian enzymes and natural substrates ofeach type of enzyme should be used Results of this workstrengthen the hypothesis that these phenolic compounds(gallic acid and ethyl gallate) could be active in the inhibitionof human 120572ndashglucosidases

In this study ethyl gallate and gallic acid displayed acompetitive enzymatic inhibition in which the inhibitorcompetes directly with the substrate for the binding site inthe active site of the enzyme [27] This is one of few studiesin which the enzymatic inhibition type and Ki of naturallyoccurring compounds are described on digestive enzymes[48]

In the case of lipase inhibition the most active com-poundswere enriched in the organic LoEAF fraction Furtherpurification by silica chromatography allowed us to obtaina Cndashglycosylated flavone isoorientin [13] This flavonoiddisplayed the best inhibitory effect and most of the fractionsthat produced significant activity (C1F6 C4F3 C4F5 andC4F6) contain high levels of isoorientin

These kinds of Cndashglycosylated flavonoids have shownhigh inhibition of pancreatic lipase and according to someauthors glycosylation in position Cndash8 seems to significantlyincrease this biological activity [42ndash44]

Considering that it is desirable to have reference com-pounds to standardize a phytopharmaceutical drug isoori-entin could fulfil this purpose in L octovalvis extracts withpancreatic lipase inhibitory action

According to a toxicity analysis of this plant an alcoholicextract from L octovalvis did not display acute toxicity inmicewhen itwas tested at 5000mgkg nor subacute toxicity at400mgkg during 28 days [12] which is essential in the devel-opment of new phytomedicines Furthermore it is worthmentioning that the findings of the present study validatethe traditional use of this plant species in the treatment ofdiabetes and also as an alternative to synthetic drugs such asacarbose and orlistat since L octovalvis displayed at least twomechanisms of antidiabetic and antiobesity action which aresynergistic and complementary

Although none of the L octovalvis treatments wereas potent as the reference drugs there are reports wherein vitro digestive enzyme inhibition of naturally occurringcompounds is lower than acarbose or orlistat but when testedon in vivo models they produced similar pharmacologicalactivities [49 50]

5 Conclusions

The chemical separation of L octovalvis hydroalcoholicextract which is bioactive in 120572ndashglucosidase and pancre-atic lipase inhibition allowed the identification and phar-macological characterization of one flavone (isoorientin)

with considerable inhibitory effect of pancreatic lipase andtwo isolated compounds with high inhibitory effect of the120572ndashglucosidases (ethyl gallate and gallic acid) These findingsbear out one of the possible mechanisms of action bywhich this medicinal plant could help in the prevention andtreatment of type 2 diabetes and obesity therefore thesedata will be useful in the development of a potential novelphytomedicine

Data Availability

The data used to support the findings of this study areavailable from the corresponding author upon request

Conflicts of Interest

The authors declare that there are no conflicts of interestregarding the publication of this paper

Acknowledgments

The authors are indebted to Gabriel Flores curator of HUMOHerbarium for his support identifying L octovalvis andalso to Ernesto Sanchez for his technical spectroscopicsupport of NMR The technical assistance of Arturo Perezand Jonathan Orduno is also acknowledged This workwas supported by the Consejo Nacional de Ciencia y Tec-nologıa (CONACYT) [Grant no 598815] and CISndashIMSS[(FISIMSSPROTMD171693) Grant no 99187804] Ale-jandro Zamilpa thanks Fundacion IMSS

Supplementary Materials

Figure S1 Nuclear Magnetic Resonance (NMR) 1H spectrumof C1F1 and structure of the identified compound ethylgallate Figure S2 Nuclear Magnetic Resonance (NMR) 13Cspectrum of C1F1 and structure of the identified compoundethyl gallate Figure S3 Nuclear Magnetic Resonance (NMR)1H spectrum of C2F1 and structure of the identified com-pound gallic acid Figure S4 calibration curves of the HPLCanalysis of the isolated compounds and their straightndashlineequations Figure S5 Nuclear Magnetic Resonance (NMR)1H spectrum of C4F4ndashP and structure of the identified com-pound isoorientin Figure S6 Nuclear Magnetic Resonance(NMR) 13C spectrum of C4F4ndashP and structure of the iden-tified compound isoorientin Figure S7 Correlation Spec-troscopy (COSY) of C4F4ndashP Figure S8 Heteronuclear Sin-gle Quantum Coherence Spectroscopy (HSQC) of C4F4ndashPFigure S9 Heteronuclear Multiple Bond Correlation Spec-troscopy (HMBC) of C4F4ndashP (Supplementary Materials)

References

[1] M C Riddle ldquoBasal glucose can be controlled but the prandialproblem persistsditrsquos thenext targetrdquo Diabetes Care vol 40 no3 pp 291ndash300 2017

[2] M A Esquivel and M C Lansang ldquoOptimizing diabetestreatment in the presence of obesityrdquo Cleveland Clinic Journalof Medicine vol 84 no 1 pp S22ndashS29 2017


[3] V Shettar S Patel and S Kidambi ldquoEpidemiology of Obesityand Pharmacologic Treatment Optionsrdquo Nutrition in ClinicalPractice vol 32 no 4 pp 441ndash462 2017

[4] U Ghani ldquoRe-exploring promising 120572-glucosidase inhibitors forpotential development into oral anti-diabetic drugs findingneedle in the haystackrdquo European Journal of Medicinal Chem-istry vol 103 pp 133ndash162 2015

[5] R S Padwal and S R Majumdar ldquoDrug treatments for obesityorlistat sibutramine and rimonabantrdquoThe Lancet vol 369 no9555 pp 71ndash77 2007

[6] L J Cumana-Campos ldquoClave para especies de Ludwigia L(Onagraceae) de la regilen nor-oriental e insular de Venezueladepositadas en el herbario IRBRrdquoActa Bot Venez vol 33 no 2pp 299ndash327 2010

[7] ldquoSEMARNAT lsquoNorma Oficial Mexicana NOM-059-SEMARNAT-2010 Proteccion ambiental-Especies nativasde Mexico de flora y fauna silvestres-Categorıas de riesgo yespecificaciones para su inclusion exclusion o cambio-Lista deespecies en riesgorsquo Diario Oficial de la Federacion Mexico pp1ndash78 2006rdquo

[8] T Murugesan S Sinha M Pal and B Saha ldquoReview onPhytochemical and Medicinal Aspects of Jussiaea SuferuticosaLinnrdquo Ancient Science of Life vol 21 no 3 pp 205ndash207 2002

[9] ldquoUNAM Atlas de las Plantas de la Medicina Tradicional Mex-icanarsquo Biblioteca Digital de la Medicina Tradicional Mexicanardquohttpwwwmedicinatradicionalmexicanaunammxatlasphp

[10] G Ramırez M Zavala J Perez and A Zamilpa ldquoIn vitroscreening of medicinal plants used in Mexico as antidiabeticswith glucosidase and lipase inhibitory activitiesrdquo Evidence-Based Complementary and Alternative Medicine vol 2012 pp1ndash6 2012

[11] M H Khan and P S Yadava ldquoAntidiabetic plants used inThoubal district of Manipur Northeast Indiardquo Indian Journalof Traditional Knowledge vol 9 no 3 pp 510ndash514 2010

[12] H Kadum Yakob A Manaf Uyub and S Fariza SulaimanldquoToxicological evaluation of 80 methanol extract of Ludwigiaoctovalvis (Jacq) PH Raven leaves (Onagraceae) in BALBcmicerdquo Journal of Ethnopharmacology vol 142 no 3 pp 663ndash668 2012

[13] J E Averett E M Zardini and P C Hoch ldquoFlavonoid sys-tematics of ten sections of Ludwigia (Onagraceae)rdquo BiochemicalSystematics and Ecology vol 18 no 7-8 pp 529ndash532 1990

[14] C-I Chang C-C Kuo J-Y Chang and Y-H Kuo ldquoThreeNewOleanane-Type Triterpenes from Ludwigia octovalvis withCytotoxic Activity against Two Human Cancer Cell LinesrdquoJournal of Natural Products vol 67 no 1 pp 91ndash93 2004

[15] J Yan and X W Yang ldquoStudies on the chemical constituents inherb of Ludwigia octovalvisrdquo China Journal of Chinese MateriaMedica vol 30 no 24 pp 1923ndash1926 2005

[16] W-S Lin J-Y Chen J-CWang et al ldquoThe anti-aging effects ofLudwigia octovalvis on Drosophila melanogaster and SAMP8micerdquo AGE vol 36 no 2 pp 689ndash703 2014

[17] W-S Lin J-H Lo J-H Yang et al ldquoLudwigia octovalvisextract improves glycemic control andmemory performance indiabetic micerdquo Journal of Ethnopharmacology vol 207 pp 211ndash219 2017

[18] S-J Wu L-T Ng G-H Wang Y-J Huang J-L Chen and F-M Sun ldquoChlorophyll a an active anti-proliferative compoundof Ludwigia octovalvis activates the CD95 (APO-1CD95)system and AMPK pathway in 3T3-L1 cellsrdquo Food and ChemicalToxicology vol 48 no 2 pp 716ndash721 2010

[19] T Murugesan L Ghosh K Mukherjee J Das M Pal andB P Saha ldquoEvaluation of antidiarrhoeal profile of Jussiaeasuffruticosa Linn extract in ratsrdquo Phytotherapy Research vol14 no 5 pp 381ndash383 2000

[20] H Wagner and S Bladt lsquoFlavonoid Drugsrsquo in Plant druganalysis A Thin Layer Chromatography Atlas Springer-VerlagBerlin Germany 2nd edition 1996

[21] ldquoThe Plant Listrdquo httpwwwtheplantlistorg[22] G Ramirez A Zamilpa M Zavala J Perez D Morales and J

Tortoriello ldquoChrysoeriol and other polyphenols from Tecomastans with lipase inhibitory activityrdquo Journal of Ethnopharma-cology vol 185 pp 1ndash8 2016

[23] T J Mabry K R Markham and M B Thomas Reagents andprocedures for the Ultraviolet Spectral Analysis of FlavonoidsSpringer Berlin Heidelberg Heidelberg Germany 1st edition1970

[24] S Uzuner and D Cekmecelioglu ldquoA rapid screening approachto factors affecting dilute acid hydrolysis of hazelnut shellsrdquoInternational Proceedings of Chemical BiologicalampEnvironmen-tal Engineering vol 50 pp 180ndash185 2013

[25] J Peng G Fan Z Hong Y Chai and Y Wu ldquoPreparativeseparation of isovitexin and isoorientin from Patrinia villosaJuss by high-speed counter-current chromatographyrdquo Journal ofChromatography A vol 1074 no 1-2 pp 111ndash115 2005

[26] H Bisswanger lsquoEnzyme Kineticsrsquo in Enzyme kinetics Principlesand Methods Ringgold Inc Portland Ore USA 2nd edition2008

[27] D S H Bell J H OrsquoKeefe and P Jellinger ldquoPostprandialdysmetabolism the missing link between diabetes and cardio-vascular eventsrdquo Endocrine Practice vol 14 no 1 pp 112ndash1242008

[28] T Shiraiwa H Kaneto T Miyatsuka et al ldquoPostprandialhyperglycemia is a better predictor of the progressionrdquoDiabetesCare vol 28 no 11 pp 2806-2807 2005

[29] G Derosa and P Maffioli ldquo120572-Glucosidase inhibitors and theiruse in clinical practicerdquo Archives of Medical Science vol 8 no5 pp 899ndash906 2012

[30] M P Hermans ldquoDiabetes and the endotheliumrdquo Acta clinicaBelgica vol 62 no 2 pp 97ndash101 2007

[31] AGastaldelliMGaggini andRADeFronzo ldquoRole of adiposetissue insulin resistance in the natural history of type 2 diabetesResults from the san antonio metabolism studyrdquo Diabetes vol66 no 4 pp 815ndash822 2017

[32] M Brownlee ldquoThe pathobiology of diabetic complications aunifying mechanismrdquo Diabetes vol 54 no 6 pp 1615ndash16252005

[33] J Kim and K Yoon ldquoGlucolipotoxicity in Pancreatic 120573-CellsrdquoDiabetes ampMetabolism Journal vol 35 no 5 pp 444ndash450 2011

[34] K C B Tan AWK Tso S C F Tam RWC Pang andK S LLam ldquoAcute effect of orlistat on post-prandial lipaemia and freefatty acids in overweight patients with Type 2 diabetes mellitusrdquoDiabetic Medicine vol 19 no 11 pp 944ndash948 2002

[35] J B Reitsma M C Cabezas T W A de Bruin and DW Erke-lens ldquoRelationship between improved postprandial lipemiaand low-density lipoprotein metabolism during treatment withtetrahydrolipstatin a pancreatic lipase inhibitorrdquo Metabolismvol 43 no 3 pp 293ndash298 1994

[36] K Tadera Y Minami K Takamatsu and T Matsuoka ldquoInhibi-tion of 120572-glucosidase and 120572-amylase by flavonoidsrdquo Journal ofNutritional Science and Vitaminology vol 52 no 2 pp 149ndash1532006


[37] H Li F Song J Xing R Tsao Z Liu and S Liu ldquoScreeningand Structural Characterization of 120572-Glucosidase Inhibitorsfrom Hawthorn Leaf Flavonoids Extract by Ultrafiltration LC-DAD-MSn and SORI-CIDFTICRMSrdquo Journal ofTheAmericanSociety forMass Spectrometry vol 20 no 8 pp 1496ndash1503 2009

[38] S V Reddy A K Tiwari U S Kumar R J Rao and J M RaoldquoFree radical scavenging enzyme inhibitory constituents fromantidiabetic ayurvedic medicinal plant Hydnocarpus wightianablumerdquo Phytotherapy Research vol 19 no 4 pp 277ndash281 2005

[39] S Ochir M Nishizawa B Jae Park et al ldquoInhibitory effectsof Rosa gallica on the digestive enzymesrdquo Journal of NaturalMedicines vol 64 no 3 pp 275ndash280 2010

[40] O Kamiyama F Sanae K Ikeda et al ldquoIn vitro inhibition of 120572-glucosidases and glycogen phosphorylase by catechin gallates ingreen teardquo Food Chemistry vol 122 no 4 pp 1061ndash1066 2010

[41] A Ishikawa H Yamashita M Hiemori et al ldquoCharacterizationof inhibitors of postprandial hyperglycemia from the leaves ofNerium indicumrdquo Journal of Nutritional Science and Vitaminol-ogy vol 53 no 2 pp 166ndash173 2007

[42] A Kam K M Li V Razmovski-Naumovski et al ldquoA com-parative study on the inhibitory effects of different parts andchemical constituents of pomegranate on 120572-amylase and 120572-glucosidaserdquo Phytotherapy Research vol 27 no 11 pp 1614ndash1620 2013

[43] L Kakarla S Katragadda A Tiwari et al ldquoFree radicalscavenging 120572-glucosidase inhibitory and anti-inflammatoryconstituents from Indian sedges Cyperus scariosus RBr andCyperus rotundus Lrdquo PharmacognosyMagazine vol 12 supple-ment 4 no 47 pp S488ndashS496 2016

[44] J Li Y Lu X Su et al ldquoA norsesquiterpene lactone and abenzoic acid derivative from the leaves of Cyclocarya paliurusand their glucosidase and glycogen phosphorylase inhibitingactivitiesrdquo Planta Medica vol 74 no 3 pp 287ndash289 2008

[45] J D Wansi M-C Lallemand D D Chiozem et al ldquo120572-Glucosidase inhibitory constituents from stem bark of Termi-nalia superba (Combretaceae)rdquo Phytochemistry vol 68 no 15pp 2096ndash2100 2007

[46] N Gupta S Gupta and A Mahmood ldquoGallic acid inhibitsbrush border disaccharidases in mammalian intestinerdquo Nutri-tion Research vol 27 no 4 pp 230ndash235 2007

[47] T Oki T Matsui and Y Osajima ldquoInhibitory effect of 120572-glucosidase inhibitors varies according to its originrdquo Journal ofAgricultural and Food Chemistry vol 47 no 2 pp 550ndash5531999

[48] A I Martinez-Gonzalez A G Dıaz-Sanchez L A De La Rosaet al ldquoPolyphenolic compounds and digestive enzymes In vitronon-covalent interactionsrdquo Molecules vol 22 no 4 article no669 2017

[49] G-N Kim M-R Shin S H Shin et al ldquoStudy of AntiobesityEffect through Inhibition of Pancreatic Lipase Activity ofDiospyros kaki Fruit and Citrus unshiu Peelrdquo BioMed ResearchInternational vol 2016 Article ID 1723042 pp 1ndash7 2016

[50] L Liu Y-L Yu J-S Yang et al ldquoBerberine suppresses intestinaldisaccharidases with beneficial metabolic effects in diabeticstates evidences from in vivo and in vitro studyrdquo Naunyn-Schmiedebergrsquos Archives of Pharmacology vol 381 no 4 pp 371ndash381 2010

Stem Cells International

Hindawiwwwhindawicom Volume 2018


MEDIATORSINFLAMMATION

of

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018

Immunology ResearchHindawiwwwhindawicom Volume 2018

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine


Behavioural Neurology

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary andAlternative Medicine

Volume 2018Hindawiwwwhindawicom

Submit your manuscripts atwwwhindawicom


have been reported as having several medicinal uses [8 9]among them the antidiabetic use by Mexican and Indianhealers [10 11] in which the boiled extract or the juiceof the whole plant is used Previous phytochemical studieshave described the presence of flavonoids phenolic acidspolyphenols saponins sterols tannins and triterpenoids[12ndash15] in different organs of this medicinal plant Severalpharmacological effects such as hypoglycaemic [8] anti-hyperglycaemic [16 17] and antiproliferative in 3T3ndashL1adipocytes [18] have been described through variousmodelsMoreover the hydroalcoholic extract of L octovalvis leaveswas themost effective in the inhibition of 120572ndashglucosidases andpancreatic lipase in a screening of 23 extracts of medicinalplants reported as traditional treatments for type 2 diabetesmellitus [10] In addition a report also exists on L octovalvisantidiarrheal activity probably mediated by regulation ofgastrointestinal motility [19] this activity could help reducesome of the side effects of intestinal enzyme inhibition suchas faecal urgency or abdominal pain

The aim of this work was to isolate identify quantifyand characterize the compounds with the greatest inhibitoryactivity of 120572ndashglucosidases and pancreatic lipase in thehydroalcoholic extract of L octovalvis leaves through itsbioassayndashguided fractionation

2 Materials and Methods

21 General All chemicals were of analyticalndashreagent gradeCorn starch (S4126) 23ndashdimercaptondash1ndashpropanol tributyrate(DMPTB 97 282413) 551015840ndashdithiobis(2ndashnitrobenzoic acid)(DTNB ge98 D8130) lipase from porcine pancreas (PPLtype II 100ndash500 unitsmg L3126) Triton Xndash100 (X100) SDS(ge985 L3771) glycerol (ge995 GE17ndash1325ndash01) DMSO(ge999 547239) polyethylene glycol (PEG 1546580)2ndashaminoethyl diphenylborinate (97 D9754) isoorientin(ge98 I1536) and gallic acid (ge97 27645) were purchasedfrom SigmandashAldrich (St Louis MO) Miscellaneous solventswere purchased fromMerck KGaA (Darmstadt Germany)

Orlistat (Lysthin PsicoFarmaMexico City) and acarbose(Sincrosa Alpharma Mexico City) were purified by silicachromatography and crystallized to be used as positivecontrols for enzyme inhibition assays

Thin layer chromatography (TLC) was performed usingsilica gel 60 RPndash18 F254s aluminium sheets (105560 MerckKGaA) TLC plates were analysed under UV light at 254 and360 nm using the Natural ProductsndashPEG reagent (NPndashPEG1 methanolic solution of diphenylboryloxyethylamine fol-lowed by 5 ethanolic PEG) as chemical detection system[20]

Melting points were obtained on a Thermo ScientificIA9000 series melting point apparatus (ElectrothermalEssex UK)

Nuclear Magnetic Resonance (NMR) 1H (400 MHz) andNMR 13C (100 MHz) spectra were obtained with VarianINOVAndash400 equipment (Varian Co Palo Alto CA) usingtetramethylsilane as internal standard

22 Plant Material and Preparation of Extracts Leaves ofL octovalvis were collected at Xochitepec Morelos Mexico

(18∘471015840407010158401015840 N 99∘111015840492710158401015840 W) between September andOctober of 2012 A voucher of plant material was depositedunder code number 34667 at the HUMO Herbarium in theCentro de Investigacion en Biodiversidad y Conservacion of theAutonomous University of the State of Morelos (UniversidadAutonoma del Estado de MorelosndashCIByCndashUAEM MorelosMexico)

Camellia sinensis (L) Kuntze (Theaceae) commercialground leaves purchased at a Japanese specialty store (Yama-motoyama Pomona CA) was used as a positive vegetalcontrol Plant names were checked and updated with theonline website httpwwwtheplantlistorg [21]

Fresh leaves of L octovalviswere washed and dried underdark conditions at room temperature and then milled to 4ndash6mm Ground material (1 kg) was extracted (110 ratio wv)with a 60 ethanol aqueous solution at 25∘C for 24 h Theliquid extract was paper-filtered concentrated in a rotaryevaporator Laborota 4000 (Heidolph Schwabach Germany)under reduced pressure at 50∘C and freeze-dried to obtain337 g of brown powder (324 yield) This dry extract(LoHAE) was stored at 4∘C until its pharmacological andphytochemical analysis C sinensis hydroalcoholic extract(CsHAE) was identically prepared

23 Fractionation of LoHAE and Purification of Active Frac-tions One hundred and ninety grams of LoHAE was sub-jected to a liquidndashliquid separation process using water andethyl acetate The solvent of both fractions was eliminatedby low pressure distillation to obtain an organic fraction(LoEAF) and an aqueous fraction (LoAqF)

The less polar fraction (LoEAF 25 g) was subjected to achromatographic silica gel 60 column (109385 Merck KGaA)using dichloromethanemethanol gradient system as mobilephase to give 69 samples of 150 mL each The separationprocess was monitored by TLC and all the samples weregrouped into 20 final fractions The most representativefractions (yields ge5 C1F1ndashC1F6) were subjected to bothassays

The active fractions C1F4 and C1F6 were fraction-ated using column chromatography with silica gel LiChro-prep RPndash18 (113900 Merck KGaA) and a mixture ofwateracetonitrile All the fractions were analysed by TLCand the samples with similar chemical composition weregrouped

From C1F4 (186 mg) 10 final fractions were obtained ofwhich C2F1 produced a white precipitate which was found tobe a pure compound by TLC and High Performance LiquidChromatography (HPLC)

From C1F6 (11 g) 19 final fractions were obtained themost representative (yields ge5) were C3F1 C3F2 C3F3and C3F4 Fraction C3F3 was purified obtaining fractionsC4F1 C4F2 C4F3 C4F4 C4F5 and C4F6 Fraction C4F4produced an orangeyellow precipitate (C4F4ndashP 12 mg)All these fractions (see Scheme 1) were subjected to thepharmacological assay

24 HPLC Analysis HPLC analysis was performed on achromatographic system equipped with a Waters AllianceSeparationModule (2695Waters CorporationMilfordMA)


LoHAELoEAF

C1F1

C1F2

C1F3

C1F4 C2F1

C1F5

C1F6

C3F1

C3F2

C3F3

C4F1

C4F2

C4F3

C4F4 C4F4-P

C4F5

C4F6

C3F4

LoAqF








119894119899ℎ119894119887119894119905119894119900119899 = 100 minus (119860119887119904119900119903119887119886119899119888119890119904119886119898119901119897119890119860119887119904119900119903119887119886119899119888119890119888119900119899119905119903119900119897 times 100) (1)












119870119898119886119901119901 = 119870119898 (1 + [119868]119870119894 ) (2)


3 Results








(a)

(b)

(c)

(d)

(e)

(f)







500 600 700 800 1400 1500 1600 1700 1800Minutes

300200100

000

300200100

000

300200100

000

300

200

100

000

150

100

050

000

AU

AU

AU

AU

AU

20000 30000 40000nm

90

90

99

71

71

100

118

97

73

88 89

90

94

97

101

20000 30000 40000nm

20000 30000 40000nm

20000 30000 40000

20000 30000 40000nm

1935

21692749

22392710

22392710

1923

2545

3658

2099

2698 3482

1300120011001000900

nm


HO

HO

OH

O

O

RR


O

OH

OH

O

HO

OH

O

OH

HO

HOHO

isoorientin

-（2-（3

















000

2500

5000

7500

10000

Inhi

bitio

n



2

4

6

8

10

12

2 4 6 8

1V

(mm

olm

in)

1[S] (mgmL)

C1F1 0 M C1F1 625 MC1F1 1250 M

(a)

2

4

6

8

10

12

2 4 6 8

1V

(mm

olm

in)

1[S] (mgmL)

C2F1 0 M C2F1 625 MC2F1 1250 M

(b)



4 Discussion




00

250

500

750

1000

Inhi

bitio

n




00050

00150

00250

10 20

1V

(mm

olm

in)

1[S] (mgmL)

















5 Conclusions



Data Availability




Acknowledgments




References

























































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of




















LoHAELoEAF

C1F1

C1F2

C1F3

C1F4 C2F1

C1F5

C1F6

C3F1

C3F2

C3F3

C4F1

C4F2

C4F3

C4F4 C4F4-P

C4F5

C4F6

C3F4

LoAqF








119894119899ℎ119894119887119894119905119894119900119899 = 100 minus (119860119887119904119900119903119887119886119899119888119890119904119886119898119901119897119890119860119887119904119900119903119887119886119899119888119890119888119900119899119905119903119900119897 times 100) (1)












119870119898119886119901119901 = 119870119898 (1 + [119868]119870119894 ) (2)


3 Results








(a)

(b)

(c)

(d)

(e)

(f)







500 600 700 800 1400 1500 1600 1700 1800Minutes

300200100

000

300200100

000

300200100

000

300

200

100

000

150

100

050

000

AU

AU

AU

AU

AU

20000 30000 40000nm

90

90

99

71

71

100

118

97

73

88 89

90

94

97

101

20000 30000 40000nm

20000 30000 40000nm

20000 30000 40000

20000 30000 40000nm

1935

21692749

22392710

22392710

1923

2545

3658

2099

2698 3482

1300120011001000900

nm


HO

HO

OH

O

O

RR


O

OH

OH

O

HO

OH

O

OH

HO

HOHO

isoorientin

-（2-（3

















000

2500

5000

7500

10000

Inhi

bitio

n



2

4

6

8

10

12

2 4 6 8

1V

(mm

olm

in)

1[S] (mgmL)

C1F1 0 M C1F1 625 MC1F1 1250 M

(a)

2

4

6

8

10

12

2 4 6 8

1V

(mm

olm

in)

1[S] (mgmL)

C2F1 0 M C2F1 625 MC2F1 1250 M

(b)



4 Discussion




00

250

500

750

1000

Inhi

bitio

n




00050

00150

00250

10 20

1V

(mm

olm

in)

1[S] (mgmL)

















5 Conclusions



Data Availability




Acknowledgments




References

























































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of


























119870119898119886119901119901 = 119870119898 (1 + [119868]119870119894 ) (2)


3 Results








(a)

(b)

(c)

(d)

(e)

(f)







500 600 700 800 1400 1500 1600 1700 1800Minutes

300200100

000

300200100

000

300200100

000

300

200

100

000

150

100

050

000

AU

AU

AU

AU

AU

20000 30000 40000nm

90

90

99

71

71

100

118

97

73

88 89

90

94

97

101

20000 30000 40000nm

20000 30000 40000nm

20000 30000 40000

20000 30000 40000nm

1935

21692749

22392710

22392710

1923

2545

3658

2099

2698 3482

1300120011001000900

nm


HO

HO

OH

O

O

RR


O

OH

OH

O

HO

OH

O

OH

HO

HOHO

isoorientin

-（2-（3

















000

2500

5000

7500

10000

Inhi

bitio

n



2

4

6

8

10

12

2 4 6 8

1V

(mm

olm

in)

1[S] (mgmL)

C1F1 0 M C1F1 625 MC1F1 1250 M

(a)

2

4

6

8

10

12

2 4 6 8

1V

(mm

olm

in)

1[S] (mgmL)

C2F1 0 M C2F1 625 MC2F1 1250 M

(b)



4 Discussion




00

250

500

750

1000

Inhi

bitio

n




00050

00150

00250

10 20

1V

(mm

olm

in)

1[S] (mgmL)

















5 Conclusions



Data Availability




Acknowledgments




References

























































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of




















(a)

(b)

(c)

(d)

(e)

(f)







500 600 700 800 1400 1500 1600 1700 1800Minutes

300200100

000

300200100

000

300200100

000

300

200

100

000

150

100

050

000

AU

AU

AU

AU

AU

20000 30000 40000nm

90

90

99

71

71

100

118

97

73

88 89

90

94

97

101

20000 30000 40000nm

20000 30000 40000nm

20000 30000 40000

20000 30000 40000nm

1935

21692749

22392710

22392710

1923

2545

3658

2099

2698 3482

1300120011001000900

nm


HO

HO

OH

O

O

RR


O

OH

OH

O

HO

OH

O

OH

HO

HOHO

isoorientin

-（2-（3

















000

2500

5000

7500

10000

Inhi

bitio

n



2

4

6

8

10

12

2 4 6 8

1V

(mm

olm

in)

1[S] (mgmL)

C1F1 0 M C1F1 625 MC1F1 1250 M

(a)

2

4

6

8

10

12

2 4 6 8

1V

(mm

olm

in)

1[S] (mgmL)

C2F1 0 M C2F1 625 MC2F1 1250 M

(b)



4 Discussion




00

250

500

750

1000

Inhi

bitio

n




00050

00150

00250

10 20

1V

(mm

olm

in)

1[S] (mgmL)

















5 Conclusions



Data Availability




Acknowledgments




References

























































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of


































000

2500

5000

7500

10000

Inhi

bitio

n



2

4

6

8

10

12

2 4 6 8

1V

(mm

olm

in)

1[S] (mgmL)

C1F1 0 M C1F1 625 MC1F1 1250 M

(a)

2

4

6

8

10

12

2 4 6 8

1V

(mm

olm

in)

1[S] (mgmL)

C2F1 0 M C2F1 625 MC2F1 1250 M

(b)



4 Discussion




00

250

500

750

1000

Inhi

bitio

n




00050

00150

00250

10 20

1V

(mm

olm

in)

1[S] (mgmL)

















5 Conclusions



Data Availability




Acknowledgments




References

























































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of





















000

2500

5000

7500

10000

Inhi

bitio

n



2

4

6

8

10

12

2 4 6 8

1V

(mm

olm

in)

1[S] (mgmL)

C1F1 0 M C1F1 625 MC1F1 1250 M

(a)

2

4

6

8

10

12

2 4 6 8

1V

(mm

olm

in)

1[S] (mgmL)

C2F1 0 M C2F1 625 MC2F1 1250 M

(b)



4 Discussion




00

250

500

750

1000

Inhi

bitio

n




00050

00150

00250

10 20

1V

(mm

olm

in)

1[S] (mgmL)

















5 Conclusions



Data Availability




Acknowledgments




References

























































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of




















00

250

500

750

1000

Inhi

bitio

n




00050

00150

00250

10 20

1V

(mm

olm

in)

1[S] (mgmL)

















5 Conclusions



Data Availability




Acknowledgments




References

























































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of




























5 Conclusions



Data Availability




Acknowledgments




References

























































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of









































































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of






































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of























of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of



















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