Research Article(Diabetes and Metabolism Disorders Medicinal PlantsA Glance at the Past and a Look to the Future 2018)Antihyperglycemic Activity of Hamelia patens Jacq Extracts
Elvia Becerra-Martinez 3 Mariacutea del Carmen Cruz-Loacutepez1
Victor E Loacutepez-y-Loacutepez 1 AaroacutenMendieta-Moctezuma 1
Ignacio E Maldonado-Mendoza4 and Fabiola E Jimeacutenez-Montejo 1
1Centro de Investigacion en Biotecnologıa Aplicada del Instituto Politecnico Nacional Tlaxcala Mexico2Escuela Nacional de Medicina y Homeopatıa del Instituto Politecnico Nacional Ciudad de Mexico Mexico3Centro de Nanociencias y Micro y Nanotecnologıa del Instituto Politecnico Nacional Ciudad de Mexico Mexico4Centro Interdisciplinario de Investigacion para el Desarrollo Integral RegionalUnidad Sinaloa del Instituto Politecnico Nacional Sinaloa Mexico
Correspondence should be addressed to Fabiola E Jimenez-Montejo fejimenezmipnmx
Received 9 April 2018 Revised 21 June 2018 Accepted 31 July 2018 Published 27 August 2018
Academic Editor Akhilesh K Tamrakar
Copyright copy 2018 Catalina Rugerio-Escalona 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 is one the worldrsquos most widespread diseases affecting over 327 million people and causing about 300000 deaths annuallyDespite great advances in prevention and therapy existing treatments for this disorder have serious side effects Plants used intraditional medicine represent a valuable source in the search for new medicinal compounds Hamelia patens Jacq has been usedfor treating diabetes and so far no reports have been made on the in vivo antihyperglycemic activity of this plant The presentstudy on H patens aimed to test the antihyperglycemic effect of repeated administrations of the crude and fractional methanolicextracts (CME and FME respectively) on rats with hyperglycemia induced by streptozotocin After 10 administrations (20 days)each extract had lowered blood glucose to a normal levelThe extracts produced effects similar tometforminOf the five compoundsidentified by chromatographic analysis of the extracts epicatechin and chlorogenic acid demonstrated antihyperglycemic effectTheantioxidant activity of the extracts was evidenced by their IC50 values (517 and 507120583gmL respectively) The LD50 ge2000mgKgsuggests low toxicity for both CME and FME Thus considering that the antihyperglycemic and antioxidant effects of metforminand extracts from H patens were comparable the latter may be efficacious for treating diabetes
1 Introduction
Diabetes mellitus (DM) characterized by hyperglycemia andrelated to metabolic disorder [1ndash3] is a worldwide healthproblem and still on the increase According to the Inter-national Diabetes Federation (IDF) 327 million people cur-rently suffer fromDM a figure estimated to reach 438millionby 2045 Diabetes type 2 (DM2) is the most common formof this disorder representing 90 of the total affected popula-tion [4 5]
Today therapeutic alternatives include various drugs ad-ministered orally such as sulfonylureas (metformin) bigua-nides (glybenclamide) troglitazones (pioglitazone) and in-hibitors of DPP-4 (gliptins) SGLT2 (gliflozin) and 120572-gluco-sidase (acarbose) [6] However the secondary effects of thesetreatments (eg gastrointestinal disorders and hepatotoxic-ity) [7] have led diabetic patients to seek natural alternatives[8] At least 1200 species of medicinal plants are used intraditional medicine for their antidiabetic attributes A smallproportion (450 plants) of such plants have been studied to
HindawiEvidence-Based Complementary and Alternative MedicineVolume 2018 Article ID 7926452 9 pageshttpsdoiorg10115520187926452
2 Evidence-Based Complementary and Alternative Medicine
explore their effect and of these only 109 have had theiraction mechanism analyzed [9ndash11] It has been reported thatinMexico more than 383 plant species are employed for DM2treatment [12]
Hamelia patens Jacq (Rubiaceae commonly known asldquobayetilla coralillo firebush or scarlet bushrdquo) is a subtropicaland tropical shrub native to the Americas (widespreadfrom Florida to Argentina) Endemic to Mexico it is usedin traditional medicine for headaches diarrhea stomachache wound healing and diabetes among other applicationsBiological studies have demonstrated radical scavenging [1314] as well as anti-inflammatory [15] antibacterial [16] andcytotoxic [17] activity by H patens In an oral glucose toler-ance test this plant demonstrated inhibition of 120572-glucosidase[18] and a hypoglycemic effect [19] Phytochemical analysis ofthe plant has established the presence of isopteropodin rum-berin palmirin maruquin 24-methylenecicloartan-3szlig-ol24-methylcicloart-24-en-3szlig-ol 2E-37111519-pentamethyl-2-eicosaen-1-ol stigmasterol szlig-sitosterol ursolic acid aricin(+)-catechin (-)-epicatechin and(-)-hammelin [20ndash22]How-ever some of these compounds have not been shown to havebiological effects
The aim of the present study was to evaluate the antihy-perglycemic effect of methanolic extracts of H patens Jacqby assessing repeated administrations to a hyperglycemiamurine model induced by streptozotocin (STZ)The extractswere subjected to quantitative phytochemical and chromato-graphic analysis as well as examination of their antioxidantactivity
2 Materials and Methods
21 Reagents All reagents were purchased and used withoutfurther purification The Folin-Ciocalteu phenol reagent(F9252) (plusmn)-catechinhydrate (C1788) (-)-epicatechin (E1753)22-diphenyl-1-picrylhydrazyl (D9132) 120572-glucosidase (fromSaccharomyces cerevisiae) type I (G5003) 4-nitrophenyl 120572-D-glucopyranoside (N1377) quercetin (Q4951) and tannicacid (403040)were acquired fromSigma-Aldrich Aluminumchloride was obtained from Honeywell Fluka and gallic acidfrom Fermont
22 Plant Material H patens leaves were collected in themunicipality of Xicotepec de Juarez in the State of PueblaMexico The aerial parts were dried in the shade at roomtemperature and ground to a fine powder for extraction
23 Extract Preparation One kg of plant material (leaves)was subjected to a fractional extraction using solvents ofincreasing polarity (hexane dichloromethane and methanol)Extraction from another kg of plant material was madeseparately usingmethanol In each case the solvent was evap-orated under reduced pressure to dryness and subsequentlyextracts were lyophilized and stored at 4∘C until use Weworked with methanolic extracts specifically the fractionalmethanol extract (FME) and the crude methanol extract(CME)
24 Total Phenolic Content Total content of phenols wasmeasured using the Folin-Ciocalteu method [23] with cer-tain modifications The reaction mixture was prepared with02mLof extract (5 mgmL) 2mLof solutionA (2Na2CO31 CuSO4 and 27 potassium sodium tartrate) and 04mLof NaOH (5 N) Subsequently 02mL of the Folin-Ciocalteusolution (11 vv) was added and the mixture was allowedto stand for 30 minutes at room temperature Absorbancewasmeasured at 750 nmThe standard curve was constructedbased on various concentrations of gallic acidThe content oftotal phenols was expressed as milligram equivalents of gallicacid per gram of dried extract (mg EGAg dried extract)
25 Total Flavonoid Content Total flavonoid content wasevaluated using an aluminum chloride method with slightmodifications [23] In brief the reaction mixture consistedof 01mL of extract 030mL of absolute ethanol 002mLAlCl3 (10) 002mL CH3COOK and 056mL of distilledwater This was left to stand for 30min at room temperatureand the reading was taken at 415 nm Flavonoid contentwas determined from a calibration curve of quercetin andexpressed as mg equivalents of quercetin per gram of driedextract (mg EQg dried extract)
26 Condensed Tannins Content Tannins content was deter-mined using the vanillinHCl method [24] 2mL of extractwas placed in a tube and heated in a water bath at 30∘C for20min 04mL were removed from this sample and then2mL of vanillin solution (1 in methanol) were added Thetube was placed in a water bath once again at 30∘C for 20minFinally the reading was made at 550nm and tannins contentwas expressed asmg of catechin per gramof dried extract (mgECatg dried extract) from a standard curve for catechin
27 Antioxidant Activity (DPPH Assay) Antioxidant activitywas measured by employing the DPPH method describedby Cevallos [25] with a number of modifications Con-centrations of 4 04 and 004mgmL from each of theextracts were prepared to which a solution of 22-diphenyl-1-picrylhydrazyl radical (DPPH) (13333 120583M) was added ata ratio of 13 (vv) The mixture was incubated at 37∘C for30 minutes and read at 517 nm Antioxidant activity wasexpressed as mean effective concentration (EC50)
28 In Vitro 120572-Glucosidase Inhibitory Activity Theevaluationof 120572-glucosidase inhibition was determined using Salehirsquosmethod [26] with slight modifications The mixture con-taining 480 120583L of phosphate buffer (01M pH 69) 40120583L ofextract (4mgmL) and 80 120583L of 120572-glucosidase (05 UmL)was incubated at 37∘C for 15min in 96-well plates Thereaction was initiated by placing 80 120583L p-nitrophenyl 120572-D-glucopyranoside solution (p-NPG 5mM) in phosphatebuffer (01M pH 69) After 15min of incubation at 37∘C thereactionwas terminated by adding 320 120583L ofNa2CO3 (02M)120572-Glucosidase inhibition was determined by measuring theyellow-colored p-nitrophenolate ion released from p-NPGat 405 nm with a spectrophotometer The inhibition of 120572-glucosidase by the extracts was expressed as the IC50 and
Evidence-Based Complementary and Alternative Medicine 3
compared to the value found for chlorogenic acid quercetinand epicatechin
29 Qualitative High-Performance Liquid Chromatography(HPLC) Analysis Analytical HPLC was carried out usingan Agilent 1100 series apparatus equipped with a diodearray detector (DAD) (Agilent Technologies) using a XBD-C18 analytical column 46 times 150mm 35120583m particle size(Agilent Technologies) and a column temperature of 60∘CElution was performed at a flow rate of 1mLmin with02 formic acid (solvent A) and acetonitrile (solvent B)for the mobile phase The samples were eluted by applyingthe following gradient 100 A as the initial condition for3min 90 A and 10 B for 3min 85 A and 15 B for3min 80 A and 20 B for 3min 70 A and 30 B for3min 60 A and 40 B for 3min and finally 50 A and50 B for 15min At 253 290 and 349 nm compoundswere detected and identified by comparing them to knownstandards (caffeic acid chlorogenic acid trans-cinnamic acidtrans-ferulic acid syringic acid caffeine (plusmn)-catechin (-)-epicatechin kaempferol naringenin quercetin and rutin)based on retention times and UV spectra
210 Experimental Animals Male (27plusmn2 g) and female(20plusmn2 g) ICR mice and male Wistar rats (180plusmn30 g) wereobtained from the Facultad de Estudios Superiores AcatlanUniversidad Nacional Autonoma de Mexico The mice werehoused under standard conditions and given a standard pelletfeed and water ad libitum All experiments with animals wereauthorized by the Ethics Committee of the Escuela Nacionalde Medicina y Homeopatıa of the Instituto PolitecnicoNacional and complied with national and international prin-ciples for the care and use of lab animals
211 Acute Oral Toxicity Testing Toxicity assessment adheredto OECD (Organization of Economic Co-operation andDevelopment) guidelines for testing chemicals (Acute OralToxicityndashAcute Toxic Class Method section 423) Threefemale and three male ICRmice (20-30 g) were given a singleoral administration of 300mgkg of the plant extracts afterovernight fasting [27]The control group received only waterMice were observed for symptoms and weight variation atpostadministration intervals of 1 3 and 4 h and then twiceper day for the subsequent 14 days Animals were kept at23plusmn2∘C and 50 humidity under a 1212 h lightdarknesscycle They were provided with standard feed and water adlibitum throughout the study Results made it possible toclassify the substance according to the Globally HarmonizedSystem (GHS)
212 Streptozotocin-Induced Diabetic Rats This parameterwas explored among 110 male Wistar rats They were keptat a temperature of 23plusmn3∘C and on a 1212 h lightdarknesscycle with free access to food and water For the induction ofhyperglycemia all groups of rats (except the healthy control)were administered a single dose of 50mgkg of streptozotocin(STZ) intraperitoneally After three days of administrationthe glucose level was measured on reactive strips and animals
showing a blood glucose level of ge 200mgdL were selectedfor the test groups
213 Antihyperglycemic Activity of H patens Extracts Extractconcentrations necessary to induce the desired pharmacolog-ical responses were taken from previous studies Rats weredivided into eleven groups each consisting of ten animalsGroup I healthy control (water sterile) group II diabeticcontrol (STZ) group III vehicle (300120583L propylene glycolPPG) group IV metformin (100mgkg) group V acarbose(10mgkg) groups VI-VIII FME extracts at doses of 35 75and 150mgkg groups IX-XI CME extracts at doses of 3575 and 150mgkg were all administered intragastrically everythird day monitoring the peripheral glucose 48 h postad-ministration After the 15th administration animals wereeuthanized and a blood sample was obtained for biochemicalanalysis [28]
214 Biochemical Determinations Serum samples were col-lected in order to measure glucose concentration (SG)insulin (SIN) total cholesterol (TC) low-density lipoproteins(LDL) high-density lipoproteins (HDL) triglycerides (Tg)creatinine (SCr) urea (SUr) blood urea nitrogen (BUN) ala-nine aminotransferase (AlT) and aspartate aminotransferase(AsT) Detection was carried out following the manufac-turerrsquos protocol for each diagnostic kit
215 Statistical Analysis Data are expressed as the mean plusmnstandard error Statistical differenceswere evaluated using theTukey test with the SAS version 9 program For the evaluationof antihyperglycemic activity and biochemical parametersthe comparison ofmultiple variancewas analyzed by applyingthe Holm-Bonferroni method utilizing GraphPad Prismsoftware (version 50) In all cases statistical significance wasconsidered at plt005
3 Results and Discussion
CME yield was 89 and 54 for FME Chemical com-positions of CME and FME are presented in Table 1 Asapparent CME and FME ofH patens are an excellent sourceof tannins flavonoids and phenols Significant differencesbetween CME and FME were apparent in the total contentof phenols and flavonoids The latter extract displayed thehighest content of these metabolites We relate this to theway extracts were obtained These results concur with thosereported by Flores-Sanchez in 2017 [29]
Pathogenesis of DM has been shown to relate to thegeneration of free radicals especially reactive oxygen species(ROS) glucose oxidation increased lipid peroxidation andgreater insulin resistance Recent studies have shown thatphenolic compounds are well known for their great capacityfor radical scavenging especially flavonoids which may beeffective in the management and prevention of diabetesmellitus due to interference in the absorption digestion andmetabolism of carbohydrates [30 31] When evaluating theantioxidant activity of both extracts (Table 2) no significantdifferences were observed between CME and FME in terms
4 Evidence-Based Complementary and Alternative Medicine
Table 1 Metabolic content of the methanolic extracts of H patens
Data are expressed as the mean plusmn SD ECat equivalents of catechin EGA equivalents of gallic acid EQ equivalents of quercetin Themean values labeled withuppercase letters differ significantly compared to the control
Table 2 DPPH radical scavenging activity and 120572-glucosidase inhibition by methanolic extracts ofH patens
of their EC50 values (507 and 517120583gmL respectively) How-ever both extracts presented a better EC50 value than BHTwhich is a synthetic phenolic antioxidant currently used infood despite the evidence that it causes enzymatic or lipidalterations as well as carcinogenic effects and mutagenicactivity [32 33]120572-Glucosidase inhibitors are targeted to delay carbohy-
drate absorption and reduce postprandial glucose Severalphenolic compounds containing a flavonoid nucleus in theirstructure are reportedly useful for the control of diabetes byimproving glucose and lipid levels [30] Moreover studieshave demonstrated that quercetin epicatechin kaempferoland naringenin effectively inhibit the 120572-glucosidase enzyme[31 34] Consequently the 120572-glucosidase inhibition assay wasthe first stage in the identification of antidiabetic agentsHigh 120572-glucosidase inhibitory activity was found in bothextracts (FME IC50 = 678 and CME IC50 = 783120583gmLTable 2) Thus the extracts are more active than acar-bose (IC50= 49966 120583gmL) Contrastingly our low polarityextracts showed inhibitory activity of less than 10 at aconcentration of 4000120583gmL However in 2016 Jimenez [18]established that hexanic and methanol-ethyl acetate extractsof H patens manifest better 120572-glucosidase inhibitory effectwith IC50 = 2607120583gml and 3018120583gml respectively thandichloromethane-ethyl acetate and methanol-water extractswhich did not exhibit activity Their results coincide withthose reported by other authors who have demonstrated thathigh polarity extracts are more active than acarbose [35ndash37]
The compounds in the extracts which produce antag-onistic activity are polar in nature and a chromatographicHPLC analysis was performed to determine their compo-sition and identify the possible active principles By usingdifferent standards (Figure 1) the resulting retention times(Table 3) reveal that chlorogenic acid is the main componentin both extracts constituting 135 of CME and 195 of
FME Catechin and epicatechin were also detected in theextracts concurring with Wongrsquos description of H patensin 2017 [38] Looking for a possible explanation for thisbehaviour we decided to evaluate the inhibitory activity forthe compounds epicatechin chlorogenic acid and quercetinpreviously identified in the extractsThefirst two showed IC50values that were higher than those obtained from FME andCME with IC50 = 2826 120583gmL for epicatechin and IC50 ge3000120583gmL for chlorogenic acid Notably our results forchlorogenic acid differ from those previously reported (IC50= 24 461 1000 and gt2000120583gmL) [39ndash42] Although chloro-genic acid is the main component the antagonistic activityagainst the enzyme is low which does not correlate with theactivity that both extracts manifested Quercetin previouslyisolated fromH patens presented a similar inhibitory activity(IC50 = 27120583gmL) to the one described by Indrianingsihin 2015 (IC50 = 42120583gmL) [2] A peak at 14025 minuteswas displayed in the spectrum for CME (107) and at14075 minutes for FME (45) Likewise the peak with aretention time of 1921 indicated a higher concentration forCME (59) than for FME (09) As these two componentsof low polarity may partly explain the perceived differencein activity further research is necessary to isolate them forfurther structural characterization and biological evaluationUsing column chromatography a light brown amorphoussolid with a melting point of 236-238∘C was isolated fromCME Nuclear magnetic resonance (NMR) analysis made itpossible to identify a 3-flavonol skeleton and the 1H and 13CNMR spectra and two-dimensional spectra established thestructure as (-)-epicatechin concurring with a recent reportfor H patens [43]The chemical shifts for this compound inthe 1H and 13C NMR are presented in Table 4
The second stage of the current study intended to explorethe antihyperglycemic effect of the extracts in an in vivomurinemodelThe extractswere administered 15 times to rats
Evidence-Based Complementary and Alternative Medicine 5
140
120
100
80
60
40
20
0
minus20
mAU
4
1
5
5 10 15 20 25
lowast
+
min
(a) FME
175
150
100
125
75
50
25
0
minus25
mAU
4
1
5
5 10 15 20 25
lowast
+
min
(b) CME
Figure 1 HPLC chromatogram of fractional (FME) and crude (CME) methanolic extracts of H patens (1) chlorogenic acid (4) catechinand (5) (-) epicatechin lowastDifferent component +Different concentration
Table 3 The retention time of phenolic compounds at 290 nm
with hyperglycemia induced by STZ (glucose levels higher300mgdL) FME and CME extracts produced a reduction in
Table 5 Glucose levels and serum insulin in experimental rats
Sample SG (mgdL) SIN (mUImL)HC 11270 plusmn 2211 210 plusmn 127DC 43750 plusmn 071 250 plusmn 131DCV 42900 plusmn 283 195 plusmn 007Metformin 12390 plusmn 3399lowastlowastlowast 174 plusmn 006Acarbose 14230 plusmn 3199lowastlowastlowast 215 plusmn 039CME 150 9325 plusmn 2022lowastlowastlowast 163 plusmn 071CME 75 15600 plusmn 1513lowastlowastlowast 133 plusmn 015CME 35 15300 plusmn 3036lowastlowastlowast 232 plusmn 132FME 150 11750 plusmn 4135lowastlowastlowast 147 plusmn 055FME 75 16880 plusmn 1167lowastlowastlowast 200 plusmn 087FME 35 16200 plusmn 2877lowastlowastlowast 275 plusmn 035(SG) concentration of glucose (SIN) insulin HC healthy control DCdiabetic control DCV diabetic control vehicle CME 150 crude methanolicextract at a concentration of 150 mgkg CME 75 crude methanolic extractat a concentration of 75 mgkg CME 35 crude methanolic extract at aconcentration of 35 mgkg FME 150 fractional methanolic extract at aconcentration of 150 mgkg FME 75 fractional methanolic extract at aconcentration of 75 mgkg FME 35 fractional methanolic extract at aconcentration of 35 mgkg For the corresponding mean plusmn SD P lt 0001compared to the healthy control and lowastlowastlowastP lt 001 compared to the diabeticcontrol
glucose concentration (Table 5) which reached anormal levelafter 10 administrations At a concentration of 150mgkgextracts showed a greater decrease in glucose level Likewisethe extracts and metformin exhibited a decrease in seruminsulin compared to the diabetic control However in thediabetic control as well as in acarbose this behaviour wasnot observed Some studies explain that this increase ininsulin levels is due to resistance to insulin which leadsto peripheral hyperglycemia and major insulin secretion aprocess known as compensatory hyperinsulinemia [44 45]Levels of serum creatinine (SCr) serum urea (SUr) andblood urea nitrogen (BUN) were measured in order to assessthe effects on the kidney as this damage is one of the maincollateral effects of hyperglycemia The malfunction of thisorgan leads to an increase in metabolic waste products in theblood [46] The 150mgkg concentration of CME and FMEresults in the best protective effect in relation to SCr and no
6 Evidence-Based Complementary and Alternative Medicine
Table 6 Kidney and liver profile of experimental rats
Sample SCr (mgdL) SUr (mgdL) BUN (mgdL) AlT (mgdL) AsT (120583UmL)HC 120 plusmn 003 4783 plusmn 1520 2394 plusmn 650 6533 plusmn 2172 15620 plusmn 6050DC 105 plusmn 002 8250 plusmn 2469plusmn 3853 plusmn 1149 9933 plusmn 2627 20850 plusmn 9326DCV 075 plusmn 001 7600 plusmn 2713 3208 plusmn 1624 6133 plusmn 1320 18880 plusmn 8149Metformin 074 plusmn 002 3450 plusmn 1063z 2053 plusmn 504z 6550 plusmn 1222 14540 plusmn 2660Acarbose 083 plusmn 031 4833 plusmn 153 2267 plusmn 116 5950 plusmn 1457 13600 plusmn 3617CME 150 063 plusmn 002 3125 plusmn 171zzz 1455 plusmn 081z 7325 plusmn 2468 13800 plusmn 2207CME 75 090 plusmn 010 4300 plusmn 361z 2007 plusmn 165 6967 plusmn 1115 13930 plusmn 3118CME 35 137 plusmn 038 5433 plusmn 752 2650 plusmn 366 7325 plusmn 1406 15780 plusmn 2849FME 150 067 plusmn 005 3150 plusmn 407zzz 1468 plusmn 239zz 4550 plusmn 507z 14100 plusmn 3640FME 75 120 plusmn 032 5600 plusmn 577 2468 plusmn 402 7625 plusmn 1928 13630 plusmn 2229FME 35 110 plusmn 014 4700 plusmn 1086z 2193 plusmn 507 7675 plusmn 2604 13400 plusmn 3123(SCr) serum creatinine (SUr) serumurea (BUN) bloodurea nitrogen (AlT) alanine aminotransferase (AsT) aspartate aminotransferase HC healthy controlDC diabetic control DCV diabetic control vehicle CME 150 crude methanolic extract at a concentration of 150 mgkg CME 75 crude methanolic extract ata concentration of 75 mgkg CME 35 crude methanolic extract at a concentration of 35 mgkg FME 150 fractional methanolic extract at a concentration of150 mgkg FME 75 fractional methanolic extract at a concentration of 75 mgkg FME 35 fractional methanolic extract at a concentration of 35 mgkg Forthe corresponding mean plusmn SD plusmnP lt 005 compared to health control zP lt 005 compared to the diabetic control zzP lt 005 compared to the diabetic controland zzzP lt 005 compared to the diabetic control
Table 7 Lipid profile (mgdL) among experimental rats
FME 75 5975 plusmn 1242 7650 plusmn 2869 2667 plusmn 306 2300 plusmn 100FME 35 6075 plusmn 900 7575 plusmn 4842 2967 plusmn 551z 2233 plusmn 351(TC) total cholesterol (Tg) triglycerides (LDL) low-density lipoproteins (HDL) high-density lipoproteins HC healthy control DC diabetic control DCVdiabetic control vehicle CME 150 crudemethanolic extract at a concentration of 150 mgkg CME 75 crude methanolic extract at a concentration of 75 mgkgCME 35 crude methanolic extract at a concentration of 35 mgkg FME 150 fractional methanolic extract at a concentration of 150 mgkg FME 75 fractionalmethanolic extract at a concentration of 75 mgkg FME 35 fractional methanolic extract at a concentration of 35 mgkg For the corresponding mean plusmn SD zPlt 005 compared to the diabetic control zzP lt 005 compared to the diabetic control plusmnP lt 005 compared to health control 998771P lt 005 compared to acarboselozP lt 005 compared to metformin
difference was observed between the two extracts (Table 6)Enzymes AlT and AsT are an indicator of liver injury asenzymes leaking from cytosol liver cells into bloodstreamcorrelate with insulin resistance diabetes and inflammatoryprocesses of the liver [47 48] FME induced a markeddecrease in AlT making further histopathological studiesnecessary to confirm whether there is protection or damageto the liver as a result of the treatments The effect of theextracts on the lipid profile was also examined (Table 7)Patients with DM type 2 have been characterized by hightriglycerides levels low high-density lipoprotein cholesterol(HDL) levels normal low-density lipoprotein (LDL) levelsand normal or slightly increased total cholesterol levels [49ndash51] This characteristic was observed in the group treatedwith extracts and diabetic control where no significantdifferences were evident between treatments in terms of
total cholesterol nevertheless a sharp decline in triglyceridevalue was perceived in the acarbose and CME treatments(75 and 35mgkg) The capacity of acarbose for loweringtriglycerides has been described previously finding that200mg constitutes an effective dose for reducing the riskof cardiovascular events in humans implying a minimalrisk of hypoglycemia [52 53] Various studies have providedevidence that phenolic compounds such as flavonoids may beinvolved in decreased glucose levels in the in vivo model Ithas been suggested that the flavonoids naringenin and hes-peretin may produce antiatherogenic effects partly by meansof the activation of the peroxisome proliferator activatedreceptor gamma PPAR-120574 and the upregulation of adiponectinexpression in adipocytes Numerous reports document theantidiabetic effects of flavan-3-ols especially epigallocatechingallate (EGCG) in animals and cell-cultures EGCG can elicit
Evidence-Based Complementary and Alternative Medicine 7
various changes that are associated with beneficial effects fordiabetes including improvements in insulin secretion glu-cose uptake insulin resistance glucose tolerance oxidativestress inflammation and mitochondrial function [54 55]CME and FME comprise amixture of compounds and appearto inhibit the 120572-glucosidase enzyme however the inhibitoryactivity of the identified compounds suggests that this is notthe only way to reduce hyperglycemia As this is a mixtureof multitarget molecules it is impossible to assess the anti-hyperglycemic mechanism as this would imply identifyingthe two additional compounds and performing complemen-tary studies For the acute toxicity study which involved dos-ing ge2000mgkg there was no apparent mortality suggest-ing low toxicity within category 5 according to the OECDGlobally Harmonized Classification System (GHS) (2014)[27] This is advantageous should you wish to concoct anherbal preparation
4 Conclusions
These results reveal that extracts of Hamelia patens with ahigh content of phenolic compounds elicit 120572-glucosidaseinhibition and an antihyperglycemic effect At a concentrationof 150mgkg they produce an equivalent effect tometforminEpicatechin and chlorogenic acid contribute to the antihyper-glycemic activity ofH patens however it is necessary to carryout the structural identification of any other componentspresent Further research is necessary to elucidate the activitymechanisms
Data Availability
The data used to support the findings of this study are avail-able from the corresponding author upon request
Conflicts of Interest
The authors declare that there are no conflicts of interest re-garding the publication of this paper
Acknowledgments
The authors are grateful for technical support provided bythe personnel at the Laboratory of Natural Products andMolecular Biology of the ENMyH at IPN Catalina Rugerio-Escalona thanks CONACYT for the graduate scholarshipsawarded (264517) and SIP-IPN (BEIFI) for a complementaryscholarship Cynthia Ordaz-Pichardo Fabiola E Jimenez-Montejo Ignacio EMaldonado-MendozaMarıa del CarmenCruz-Lopez and Victor E Lopez-y-Lopez are Fellows of theEstımulos al Desempeno de los Investigadores (EDI)-IPNCynthia Ordaz-Pichardo Ignacio E Maldonado-MendozaMarıa del Carmen Cruz-Lopez and Victor E Lopez-y-Lopez thank the Comision de Operacion y Fomento deActividades Academicas (COFAA) an IPN program thatprovides support to researchers This work was financiallysupported by a project (20161781) from the Secretarıa deInvestigacion y Posgrado Instituto Politecnico Nacional
References
[1] F A Matough S B Budin Z A Hamid N Alwahaibi and JMohamed ldquoThe role of oxidative stress and antioxidants in dia-betic complicationsrdquo SQU Medical Journal vol 12 no 1 pp 5ndash18 2012
[2] AW Indrianingsih S Tachibana R TDewi andK Itoh ldquoAnti-oxidant and 120572-glucosidase inhibitor activities of natural com-pounds isolated fromQuercus gilva Blume leavesrdquoAsian PacificJournal of Tropical Biomedicine vol 5 no 9 pp 748ndash755 2015
[3] M Gondi and U J S Prasada Rao ldquoEthanol extract of mango(Mangifera indica L) peel inhibits 120572-amylase and 120572-glucosidaseactivities and ameliorates diabetes related biochemical param-eters in streptozotocin (STZ)-induced diabetic ratsrdquo Journal ofFood Science andTechnology vol 52 no 12 pp 7883ndash7893 2015
[4] ARayarandRManivannan ldquoIn-vitro alpha-amylase and alpha-glucosidase inhibition activity of umbelliferone and beta-ionone isolated from Coriandrum sativum LinnrdquoWorld Journalof Pharmacy and Pharmaceutical Sciences vol 5 no 1 pp 1280ndash1289 2016
[5] Atlas de la Diabetes de la FID-Octava edicion InternationalDiabetes Federation 2017
[6] American Diabetes Association ldquoPharmacologic Approachesto Glycemic Treatmentrdquo Journal of Clinical and Applied Resarchand Education vol 40 no 1 pp S64ndashS76 2017
[7] J J Marın-Penalver I Martın-Timon C Sevillano-Collantesand F J del Canizo-Gomez ldquoUpdate on the treatment of type2 diabetes mellitusrdquoWorld Journal of Diabetes vol 7 no 17 pp354ndash395 2016
[8] H-Y Chang M Wallis and E Tiralongo ldquoUse of comple-mentary and alternative medicine among people living withdiabetes literature reviewrdquo Journal of AdvancedNursing vol 58no 4 pp 307ndash319 2007
[9] A B Medagama and R Bandara ldquoThe use of Complementaryand AlternativeMedicines (CAMs) in the treatment of diabetesmellitus Is continued use safe and effectiverdquo Nutrition Journalvol 13 no 1 2014
[10] B Baharvand-Ahmadi M Bahmani P Tajeddini N Naghdiand M Rafieian-Kopaei ldquoAn ethno-medicinal study of medic-inal plants used for the treatment of diabetesrdquo Journal ofNephropathology vol 5 no 1 pp 44ndash50 2016
[11] S Verma M Gupta H Popli and G Aggarwal ldquoDiabetesmellitus treatment using herbal drugsrdquo International Journal ofPhytomedicine vol 10 no 1 pp 1ndash10 2018
[12] B Ovalle-Magallanes O N Medina-Campos J Pedraza-Cha-verri and R Mata ldquoHypoglycemic and antihyperglycemic ef-fects of phytopreparations and limonoids from Swietenia humi-lisrdquo Phytochemistry vol 110 pp 111ndash119 2015
[13] S Singh andM Vyas ldquoComparative in-vitro biological study ofaerial parts of plant Hamelia patensrdquo International Journal ofPharmaceutical Sciences and Research vol 7 no 4 pp 1793ndash1808 2016
[14] F Ruiz-Teran A Medrano-Martınez and A Navarro-OcanaldquoAntioxidant and free radical scavenging activities of plantextracts used in traditional medicine in Mexicordquo African Jour-nal of Biotechnology vol 7 no 12 pp 1886ndash1893 2008
[15] S Sosa M J Balick R Arvigo et al ldquoScreening of the topicalanti-inflammatory activity of some Central American plantsrdquoJournal of Ethnopharmacology vol 81 no 2 pp 211ndash215 2002
[16] A Camporese M J Balick R Arvigo et al ldquoScreening ofanti-bacterial activity of medicinal plants from Belize (Central
8 Evidence-Based Complementary and Alternative Medicine
America)rdquo Journal of Ethnopharmacology vol 87 no 1 pp 103ndash107 2003
[17] G Mena-Rejon E Caamal-Fuentes Z Cantillo-Ciau R Cedil-lo-Rivera J Flores-Guido and R Moo-Puc ldquoIn vitro cytotoxicactivity of nine plants used in Mayan traditional medicinerdquoJournal of Ethnopharmacology vol 121 no 3 pp 462ndash465 2009
[18] V Jimenez-Suarez A Nieto-Camacho M Jimenez-Estradaand B Alvarado Sanchez ldquoAnti-inflammatory free radical scav-enging and alpha-glucosidase inhibitory activities of Hameliapatens and its chemical constituentsrdquo Pharmaceutical Biologyvol 54 no 9 pp 1822ndash1830 2016
[19] A Andrade-Cetto S Escandon-Rivera and V Garcia-LunaldquoHypoglycemic effect of Hamelia patens Jacq aerial part inSTZ-NA-induced diabetic ratsrdquo Pharmacologyonline vol 3 pp65ndash69b 2015
[20] A Ahmad A Pandurangan N Singh and P Ananad ldquoA minireview on chemistry and biology of Hamelia Patens (Rubi-aceae)rdquo Pharmacognosy Journal vol 4 no 29 pp 1ndash4 2012
[21] J Bano S Santra and EMenghani ldquoHamelia patens a potentialplant from Rubiaceae family A reviewrdquo International Journalof Scientific amp Engineering Research vol 6 no 12 pp 960ndash9732015
[22] E C Cruz and A Andrade-Cetto ldquoEthnopharmacological fieldstudy of the plants used to treat type 2 diabetes among theCakchiquels in Guatemalardquo Journal of Ethnopharmacology vol159 pp 238ndash244 2015
[23] D A Sampietro M A Sgariglia J R Soberon E N QuirogaandM A Vattuone ldquoColorimetric reactionsrdquo in Isolation Iden-tification and Characterization of AllelochemicalsNatural Prod-ucts chapter 4 pp 78ndash82 2009
[24] H P S Makkar and K Becker ldquoVanillin-HCl method for con-densed tannins effect of organic solvents used for extraction oftanninsrdquo Journal of Chemical Ecology vol 19 no 4 pp 613ndash6211993
[25] B A Cevallos-Casals and L Cisneros-Zevallos ldquoStoichiometricand kinetic studies of phenolic antioxidants from Andean pur-ple corn and red-fleshed sweetpotatordquo Journal of Agriculturaland Food Chemistry vol 51 no 11 pp 3313ndash3319 2003
[26] P Salehi B Asghari M A Esmaeili H Dehghan and I Ghazildquo120572-Glucosidase and120572-amylase inhibitory effect and antioxidantof ten plant extracts traditionally used in Iran for diabetesrdquoJournal of Medicinal Plants Research vol 7 no 6 pp 257ndash2662013
[27] OECDOCDE 423 OECD Guideline for testing of chemicalsAcute Oral Toxicity ndash Acute Toxic Class Method EnvironmentDirectorate Organisation for Economic Co-Operation and De-velopment Paris France 2001
[28] B L Furman ldquoStreptozotocin-induced diabetic models in miceand ratsrdquo Current Protocols in Pharmacology vol 70 pp 5471ndash54720 2015
[29] I J Flores-Sanchez and A C Ramos-Valdivia ldquoA review frompatents inspired by two plant genera Uncaria and HameliardquoPhytochemistry Reviews vol 16 no 4 pp 693ndash723 2017
[30] R Vinayagam and B Xu ldquoAntidiabetic properties of dietaryflavonoids A cellular mechanism reviewrdquo Journal of Nutritionand Metabolism vol 12 no 60 pp 1ndash20 2015
[31] S Marella ldquoFlavonoids-Themost potent poly-phenols as antidi-abetic agents an overviewrdquoModernApproaches in Drug Design-ing vol 1 no 3 pp 2ndash5 2017
[32] AUllah A Khan and I Khan ldquoDiabetesmellitus and oxidativestress-A concise reviewrdquo Saudi Pharmaceutical Journal vol 24pp 547ndash553 2016
[33] D Shasha CMagogo and P Dzomba ldquoReversed phase HPLC-UV Quantitation of BHA BHT and TBHQ in food items soldin bindura supermarkets Zimbabwerdquo International ResearchJournal of Pure amp Applied Chemistry vol 4 no 5 pp 578ndash5842014
[34] M Vessal M Hemmati and M Vasei ldquoAntidiabetic effects ofquercetin in streptozocin-induced diabetic ratsrdquo ComparativeBiochemistry and Physiology Part C Toxicology amp Pharmacol-ogy vol 135 no 3 pp 357ndash364 2003
[35] P-C Hsieh G-J Huang Y-L Ho et al ldquoActivities of antioxi-dants 120572-glucosidase inhibitors and aldose reductase inhibitorsof the aqueous extracts of four Flemingia species in TaiwanrdquoBotanical Studies vol 51 no 3 pp 293ndash302 2010
[36] W Kang Y Song and X Gu ldquo120572-glucosidase inhibitory in vitroand antidiabetic activity in vivo ofOsmanthus fragransrdquo Journalof Medicinal Plants Research vol 6 no 14 pp 2850ndash2856 2012
[37] N Zahratunnisa B Elya and A Noviani ldquoInhibition of Alpha-glucosidase and antioxidant test of stem bark extracts ofGarcinia fruticosa Lauterbrdquo Pharmacognosy Journal vol 9 no2 pp 273ndash275 2017
[38] J E Wong C Rubio A Reyes C N Aguilar and M L Carril-lo ldquoPhenolic content and antibacterial activity of extracts ofHamelia patens obtained by different extraction methodsrdquo Bra-zilian Journal of Microbiology vol 49 no 3 pp 656ndash661 2017
[39] G Oboh O M Agunloye S A Adefegha A J Akinyemi andA O Ademiluyi ldquoCaffeic and chlorogenic acids inhibit key en-zymes linked to type 2 diabetes (in vitro) a comparative studyrdquoJournal of Basic and Clinical Physiology and Pharmacology vol26 no 2 pp 165ndash170 2015
[40] S-Y Chiou J-M Sung P-W Huang and S-D Lin ldquoAntiox-idant Antidiabetic and Antihypertensive Properties of Echi-nacea purpurea Flower Extract and Caffeic Acid DerivativesUsing in Vitro Modelsrdquo Journal of Medicinal Food vol 20 no2 pp 171ndash179 2017
[41] C-M Ma M Hattori M Daneshtalab and L Wang ldquoChloro-genic acid derivatives with alkyl chains of different lengths andorientations Potent120572-glucosidase inhibitorsrdquo Journal of Medic-inal Chemistry vol 51 no 19 pp 6188ndash6194 2008
[42] C Tan QWang C Luo S Chen Q Li and P Li ldquoYeast 120572-glu-cosidase inhibitory phenolic compounds isolated from Gynuramedica leafrdquo International Journal of Molecular Sciences vol 14no 2 pp 2551ndash2558 2013
[43] A I Suarez B Diaz S Tillett E Valdivieso and R S Com-pagnone ldquoLeishmanicidal activity of alkaloids from Hameliapatensrdquo Ciencia vol 16 no 2 pp 148ndash155 2008
[44] C Gutierrez-Rodelo A Roura-Guiberna and J A Olivares-Reyes ldquoMecanismos moleculares de la resistencia a la insulinauna actualizacionrdquo Gaceta Medica de Mexico vol 153 pp 215ndash228 2017
[45] J Rojas V Berm E Leal V Bermudez et al ldquoInsulinoresisten-cia e hiperinsulinemia como factores de riesgo para enfermedadcardivascularrdquo Revista Latinoamericana de Hipertension vol 2no 6 pp 179ndash190 2007
[46] SABamanikar A A Bamanikar andAArora ldquoStudy of serumurea and creatinine in diabetic and non-diabetic patients in atertiary teaching hospitalrdquoThe Journal of Medical Research vol2 no 1 pp 12ndash15 2016
[47] M Music A Dervisevic E Pepic et al ldquoMetabolic Syndromeand Serum Liver Enzymes Level at Patients with Type 2Diabetes MellitusrdquoMedical Archives vol 69 no 4 pp 251ndash2552015
Evidence-Based Complementary and Alternative Medicine 9
[48] S Kazemi S Asgary J Moshtaghian et al ldquoLiver-protectiveeffects of hydroalcoholic extract of Allium hirtifolium Boiss inrats with alloxan-induced diabetes mellitusrdquo ARYA Atheroscle-rosis Journal vol 6 no 1 pp 11ndash15 2010
[49] G A Bonneau M S Castillo Rascon R A Sanchez W RPedrozo and C Castro Olivera ldquoColesterol-IDL y parametroslipıdicos en diabeticos tipo 2rdquo Revista Argentina de Endocri-nologıa y Metabolismo vol 44 no 4 pp 215ndash222 2007
[50] A Husni D Purwanti and Ustadi ldquoBlood glucose level andlipid profile of streptozotocin-induceddiabetes rats treatedwithsodiumalginate from Sargassum crassifoliumrdquo Journal of Biolog-ical Sciences vol 16 no 3 pp 58ndash64 2016
[51] S Palazhy and V Viswanathan ldquoLipid abnormalities in type 2diabetes mellitus patients with overt nephropathyrdquo Diabetes ampMetabolism Journal vol 41 no 2 pp 128ndash134 2017
[52] J J DiNicolantonio J Bhutani and J H OrsquoKeefe ldquoAcarbosesafe and effective for lowering postprandial hyperglycaemia andimproving cardiovascular outcomesrdquo Open Heart vol 2 no 12015
[53] S Ogawa K Takaeuchi and S Ito ldquoAcarbose lowers serum tri-glyceride and postprandial chylomicron levels in type 2 dia-betesrdquo Diabetes Obesity and Metabolism vol 6 no 5 pp 384ndash390 2004
[54] L Liu S Shan K Zhang Z-Q Ning X-P Lu and Y-Y ChengldquoNaringenin andhesperetin two flavonoids derived fromCitrusaurantium up-regulate transcription of adiponectinrdquoPhytother-apy Research vol 22 no 10 pp 1400ndash1403 2008
[55] E P Cai and J-K Lin ldquoEpigallocatechin gallate (EGCG) andrutin suppress the glucotoxicity through activating IRS2 andAMPK signaling in rat pancreatic 120573 cellsrdquo Journal of Agricul-tural and Food Chemistry vol 57 no 20 pp 9817ndash9827 2009
Computational and Mathematical Methods in Medicine
Hindawiwwwhindawicom Volume 2018
Behavioural Neurology
OphthalmologyJournal of
Hindawiwwwhindawicom Volume 2018
Diabetes ResearchJournal of
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
Research and TreatmentAIDS
Hindawiwwwhindawicom Volume 2018
Gastroenterology Research and Practice
Hindawiwwwhindawicom Volume 2018
Parkinsonrsquos Disease
Evidence-Based Complementary andAlternative Medicine
Volume 2018Hindawiwwwhindawicom
Submit your manuscripts atwwwhindawicom
2 Evidence-Based Complementary and Alternative Medicine
explore their effect and of these only 109 have had theiraction mechanism analyzed [9ndash11] It has been reported thatinMexico more than 383 plant species are employed for DM2treatment [12]
Hamelia patens Jacq (Rubiaceae commonly known asldquobayetilla coralillo firebush or scarlet bushrdquo) is a subtropicaland tropical shrub native to the Americas (widespreadfrom Florida to Argentina) Endemic to Mexico it is usedin traditional medicine for headaches diarrhea stomachache wound healing and diabetes among other applicationsBiological studies have demonstrated radical scavenging [1314] as well as anti-inflammatory [15] antibacterial [16] andcytotoxic [17] activity by H patens In an oral glucose toler-ance test this plant demonstrated inhibition of 120572-glucosidase[18] and a hypoglycemic effect [19] Phytochemical analysis ofthe plant has established the presence of isopteropodin rum-berin palmirin maruquin 24-methylenecicloartan-3szlig-ol24-methylcicloart-24-en-3szlig-ol 2E-37111519-pentamethyl-2-eicosaen-1-ol stigmasterol szlig-sitosterol ursolic acid aricin(+)-catechin (-)-epicatechin and(-)-hammelin [20ndash22]How-ever some of these compounds have not been shown to havebiological effects
The aim of the present study was to evaluate the antihy-perglycemic effect of methanolic extracts of H patens Jacqby assessing repeated administrations to a hyperglycemiamurine model induced by streptozotocin (STZ)The extractswere subjected to quantitative phytochemical and chromato-graphic analysis as well as examination of their antioxidantactivity
2 Materials and Methods
21 Reagents All reagents were purchased and used withoutfurther purification The Folin-Ciocalteu phenol reagent(F9252) (plusmn)-catechinhydrate (C1788) (-)-epicatechin (E1753)22-diphenyl-1-picrylhydrazyl (D9132) 120572-glucosidase (fromSaccharomyces cerevisiae) type I (G5003) 4-nitrophenyl 120572-D-glucopyranoside (N1377) quercetin (Q4951) and tannicacid (403040)were acquired fromSigma-Aldrich Aluminumchloride was obtained from Honeywell Fluka and gallic acidfrom Fermont
22 Plant Material H patens leaves were collected in themunicipality of Xicotepec de Juarez in the State of PueblaMexico The aerial parts were dried in the shade at roomtemperature and ground to a fine powder for extraction
23 Extract Preparation One kg of plant material (leaves)was subjected to a fractional extraction using solvents ofincreasing polarity (hexane dichloromethane and methanol)Extraction from another kg of plant material was madeseparately usingmethanol In each case the solvent was evap-orated under reduced pressure to dryness and subsequentlyextracts were lyophilized and stored at 4∘C until use Weworked with methanolic extracts specifically the fractionalmethanol extract (FME) and the crude methanol extract(CME)
24 Total Phenolic Content Total content of phenols wasmeasured using the Folin-Ciocalteu method [23] with cer-tain modifications The reaction mixture was prepared with02mLof extract (5 mgmL) 2mLof solutionA (2Na2CO31 CuSO4 and 27 potassium sodium tartrate) and 04mLof NaOH (5 N) Subsequently 02mL of the Folin-Ciocalteusolution (11 vv) was added and the mixture was allowedto stand for 30 minutes at room temperature Absorbancewasmeasured at 750 nmThe standard curve was constructedbased on various concentrations of gallic acidThe content oftotal phenols was expressed as milligram equivalents of gallicacid per gram of dried extract (mg EGAg dried extract)
25 Total Flavonoid Content Total flavonoid content wasevaluated using an aluminum chloride method with slightmodifications [23] In brief the reaction mixture consistedof 01mL of extract 030mL of absolute ethanol 002mLAlCl3 (10) 002mL CH3COOK and 056mL of distilledwater This was left to stand for 30min at room temperatureand the reading was taken at 415 nm Flavonoid contentwas determined from a calibration curve of quercetin andexpressed as mg equivalents of quercetin per gram of driedextract (mg EQg dried extract)
26 Condensed Tannins Content Tannins content was deter-mined using the vanillinHCl method [24] 2mL of extractwas placed in a tube and heated in a water bath at 30∘C for20min 04mL were removed from this sample and then2mL of vanillin solution (1 in methanol) were added Thetube was placed in a water bath once again at 30∘C for 20minFinally the reading was made at 550nm and tannins contentwas expressed asmg of catechin per gramof dried extract (mgECatg dried extract) from a standard curve for catechin
27 Antioxidant Activity (DPPH Assay) Antioxidant activitywas measured by employing the DPPH method describedby Cevallos [25] with a number of modifications Con-centrations of 4 04 and 004mgmL from each of theextracts were prepared to which a solution of 22-diphenyl-1-picrylhydrazyl radical (DPPH) (13333 120583M) was added ata ratio of 13 (vv) The mixture was incubated at 37∘C for30 minutes and read at 517 nm Antioxidant activity wasexpressed as mean effective concentration (EC50)
28 In Vitro 120572-Glucosidase Inhibitory Activity Theevaluationof 120572-glucosidase inhibition was determined using Salehirsquosmethod [26] with slight modifications The mixture con-taining 480 120583L of phosphate buffer (01M pH 69) 40120583L ofextract (4mgmL) and 80 120583L of 120572-glucosidase (05 UmL)was incubated at 37∘C for 15min in 96-well plates Thereaction was initiated by placing 80 120583L p-nitrophenyl 120572-D-glucopyranoside solution (p-NPG 5mM) in phosphatebuffer (01M pH 69) After 15min of incubation at 37∘C thereactionwas terminated by adding 320 120583L ofNa2CO3 (02M)120572-Glucosidase inhibition was determined by measuring theyellow-colored p-nitrophenolate ion released from p-NPGat 405 nm with a spectrophotometer The inhibition of 120572-glucosidase by the extracts was expressed as the IC50 and
Evidence-Based Complementary and Alternative Medicine 3
compared to the value found for chlorogenic acid quercetinand epicatechin
29 Qualitative High-Performance Liquid Chromatography(HPLC) Analysis Analytical HPLC was carried out usingan Agilent 1100 series apparatus equipped with a diodearray detector (DAD) (Agilent Technologies) using a XBD-C18 analytical column 46 times 150mm 35120583m particle size(Agilent Technologies) and a column temperature of 60∘CElution was performed at a flow rate of 1mLmin with02 formic acid (solvent A) and acetonitrile (solvent B)for the mobile phase The samples were eluted by applyingthe following gradient 100 A as the initial condition for3min 90 A and 10 B for 3min 85 A and 15 B for3min 80 A and 20 B for 3min 70 A and 30 B for3min 60 A and 40 B for 3min and finally 50 A and50 B for 15min At 253 290 and 349 nm compoundswere detected and identified by comparing them to knownstandards (caffeic acid chlorogenic acid trans-cinnamic acidtrans-ferulic acid syringic acid caffeine (plusmn)-catechin (-)-epicatechin kaempferol naringenin quercetin and rutin)based on retention times and UV spectra
210 Experimental Animals Male (27plusmn2 g) and female(20plusmn2 g) ICR mice and male Wistar rats (180plusmn30 g) wereobtained from the Facultad de Estudios Superiores AcatlanUniversidad Nacional Autonoma de Mexico The mice werehoused under standard conditions and given a standard pelletfeed and water ad libitum All experiments with animals wereauthorized by the Ethics Committee of the Escuela Nacionalde Medicina y Homeopatıa of the Instituto PolitecnicoNacional and complied with national and international prin-ciples for the care and use of lab animals
211 Acute Oral Toxicity Testing Toxicity assessment adheredto OECD (Organization of Economic Co-operation andDevelopment) guidelines for testing chemicals (Acute OralToxicityndashAcute Toxic Class Method section 423) Threefemale and three male ICRmice (20-30 g) were given a singleoral administration of 300mgkg of the plant extracts afterovernight fasting [27]The control group received only waterMice were observed for symptoms and weight variation atpostadministration intervals of 1 3 and 4 h and then twiceper day for the subsequent 14 days Animals were kept at23plusmn2∘C and 50 humidity under a 1212 h lightdarknesscycle They were provided with standard feed and water adlibitum throughout the study Results made it possible toclassify the substance according to the Globally HarmonizedSystem (GHS)
212 Streptozotocin-Induced Diabetic Rats This parameterwas explored among 110 male Wistar rats They were keptat a temperature of 23plusmn3∘C and on a 1212 h lightdarknesscycle with free access to food and water For the induction ofhyperglycemia all groups of rats (except the healthy control)were administered a single dose of 50mgkg of streptozotocin(STZ) intraperitoneally After three days of administrationthe glucose level was measured on reactive strips and animals
showing a blood glucose level of ge 200mgdL were selectedfor the test groups
213 Antihyperglycemic Activity of H patens Extracts Extractconcentrations necessary to induce the desired pharmacolog-ical responses were taken from previous studies Rats weredivided into eleven groups each consisting of ten animalsGroup I healthy control (water sterile) group II diabeticcontrol (STZ) group III vehicle (300120583L propylene glycolPPG) group IV metformin (100mgkg) group V acarbose(10mgkg) groups VI-VIII FME extracts at doses of 35 75and 150mgkg groups IX-XI CME extracts at doses of 3575 and 150mgkg were all administered intragastrically everythird day monitoring the peripheral glucose 48 h postad-ministration After the 15th administration animals wereeuthanized and a blood sample was obtained for biochemicalanalysis [28]
214 Biochemical Determinations Serum samples were col-lected in order to measure glucose concentration (SG)insulin (SIN) total cholesterol (TC) low-density lipoproteins(LDL) high-density lipoproteins (HDL) triglycerides (Tg)creatinine (SCr) urea (SUr) blood urea nitrogen (BUN) ala-nine aminotransferase (AlT) and aspartate aminotransferase(AsT) Detection was carried out following the manufac-turerrsquos protocol for each diagnostic kit
215 Statistical Analysis Data are expressed as the mean plusmnstandard error Statistical differenceswere evaluated using theTukey test with the SAS version 9 program For the evaluationof antihyperglycemic activity and biochemical parametersthe comparison ofmultiple variancewas analyzed by applyingthe Holm-Bonferroni method utilizing GraphPad Prismsoftware (version 50) In all cases statistical significance wasconsidered at plt005
3 Results and Discussion
CME yield was 89 and 54 for FME Chemical com-positions of CME and FME are presented in Table 1 Asapparent CME and FME ofH patens are an excellent sourceof tannins flavonoids and phenols Significant differencesbetween CME and FME were apparent in the total contentof phenols and flavonoids The latter extract displayed thehighest content of these metabolites We relate this to theway extracts were obtained These results concur with thosereported by Flores-Sanchez in 2017 [29]
Pathogenesis of DM has been shown to relate to thegeneration of free radicals especially reactive oxygen species(ROS) glucose oxidation increased lipid peroxidation andgreater insulin resistance Recent studies have shown thatphenolic compounds are well known for their great capacityfor radical scavenging especially flavonoids which may beeffective in the management and prevention of diabetesmellitus due to interference in the absorption digestion andmetabolism of carbohydrates [30 31] When evaluating theantioxidant activity of both extracts (Table 2) no significantdifferences were observed between CME and FME in terms
4 Evidence-Based Complementary and Alternative Medicine
Table 1 Metabolic content of the methanolic extracts of H patens
Data are expressed as the mean plusmn SD ECat equivalents of catechin EGA equivalents of gallic acid EQ equivalents of quercetin Themean values labeled withuppercase letters differ significantly compared to the control
Table 2 DPPH radical scavenging activity and 120572-glucosidase inhibition by methanolic extracts ofH patens
of their EC50 values (507 and 517120583gmL respectively) How-ever both extracts presented a better EC50 value than BHTwhich is a synthetic phenolic antioxidant currently used infood despite the evidence that it causes enzymatic or lipidalterations as well as carcinogenic effects and mutagenicactivity [32 33]120572-Glucosidase inhibitors are targeted to delay carbohy-
drate absorption and reduce postprandial glucose Severalphenolic compounds containing a flavonoid nucleus in theirstructure are reportedly useful for the control of diabetes byimproving glucose and lipid levels [30] Moreover studieshave demonstrated that quercetin epicatechin kaempferoland naringenin effectively inhibit the 120572-glucosidase enzyme[31 34] Consequently the 120572-glucosidase inhibition assay wasthe first stage in the identification of antidiabetic agentsHigh 120572-glucosidase inhibitory activity was found in bothextracts (FME IC50 = 678 and CME IC50 = 783120583gmLTable 2) Thus the extracts are more active than acar-bose (IC50= 49966 120583gmL) Contrastingly our low polarityextracts showed inhibitory activity of less than 10 at aconcentration of 4000120583gmL However in 2016 Jimenez [18]established that hexanic and methanol-ethyl acetate extractsof H patens manifest better 120572-glucosidase inhibitory effectwith IC50 = 2607120583gml and 3018120583gml respectively thandichloromethane-ethyl acetate and methanol-water extractswhich did not exhibit activity Their results coincide withthose reported by other authors who have demonstrated thathigh polarity extracts are more active than acarbose [35ndash37]
The compounds in the extracts which produce antag-onistic activity are polar in nature and a chromatographicHPLC analysis was performed to determine their compo-sition and identify the possible active principles By usingdifferent standards (Figure 1) the resulting retention times(Table 3) reveal that chlorogenic acid is the main componentin both extracts constituting 135 of CME and 195 of
FME Catechin and epicatechin were also detected in theextracts concurring with Wongrsquos description of H patensin 2017 [38] Looking for a possible explanation for thisbehaviour we decided to evaluate the inhibitory activity forthe compounds epicatechin chlorogenic acid and quercetinpreviously identified in the extractsThefirst two showed IC50values that were higher than those obtained from FME andCME with IC50 = 2826 120583gmL for epicatechin and IC50 ge3000120583gmL for chlorogenic acid Notably our results forchlorogenic acid differ from those previously reported (IC50= 24 461 1000 and gt2000120583gmL) [39ndash42] Although chloro-genic acid is the main component the antagonistic activityagainst the enzyme is low which does not correlate with theactivity that both extracts manifested Quercetin previouslyisolated fromH patens presented a similar inhibitory activity(IC50 = 27120583gmL) to the one described by Indrianingsihin 2015 (IC50 = 42120583gmL) [2] A peak at 14025 minuteswas displayed in the spectrum for CME (107) and at14075 minutes for FME (45) Likewise the peak with aretention time of 1921 indicated a higher concentration forCME (59) than for FME (09) As these two componentsof low polarity may partly explain the perceived differencein activity further research is necessary to isolate them forfurther structural characterization and biological evaluationUsing column chromatography a light brown amorphoussolid with a melting point of 236-238∘C was isolated fromCME Nuclear magnetic resonance (NMR) analysis made itpossible to identify a 3-flavonol skeleton and the 1H and 13CNMR spectra and two-dimensional spectra established thestructure as (-)-epicatechin concurring with a recent reportfor H patens [43]The chemical shifts for this compound inthe 1H and 13C NMR are presented in Table 4
The second stage of the current study intended to explorethe antihyperglycemic effect of the extracts in an in vivomurinemodelThe extractswere administered 15 times to rats
Evidence-Based Complementary and Alternative Medicine 5
140
120
100
80
60
40
20
0
minus20
mAU
4
1
5
5 10 15 20 25
lowast
+
min
(a) FME
175
150
100
125
75
50
25
0
minus25
mAU
4
1
5
5 10 15 20 25
lowast
+
min
(b) CME
Figure 1 HPLC chromatogram of fractional (FME) and crude (CME) methanolic extracts of H patens (1) chlorogenic acid (4) catechinand (5) (-) epicatechin lowastDifferent component +Different concentration
Table 3 The retention time of phenolic compounds at 290 nm
with hyperglycemia induced by STZ (glucose levels higher300mgdL) FME and CME extracts produced a reduction in
Table 5 Glucose levels and serum insulin in experimental rats
Sample SG (mgdL) SIN (mUImL)HC 11270 plusmn 2211 210 plusmn 127DC 43750 plusmn 071 250 plusmn 131DCV 42900 plusmn 283 195 plusmn 007Metformin 12390 plusmn 3399lowastlowastlowast 174 plusmn 006Acarbose 14230 plusmn 3199lowastlowastlowast 215 plusmn 039CME 150 9325 plusmn 2022lowastlowastlowast 163 plusmn 071CME 75 15600 plusmn 1513lowastlowastlowast 133 plusmn 015CME 35 15300 plusmn 3036lowastlowastlowast 232 plusmn 132FME 150 11750 plusmn 4135lowastlowastlowast 147 plusmn 055FME 75 16880 plusmn 1167lowastlowastlowast 200 plusmn 087FME 35 16200 plusmn 2877lowastlowastlowast 275 plusmn 035(SG) concentration of glucose (SIN) insulin HC healthy control DCdiabetic control DCV diabetic control vehicle CME 150 crude methanolicextract at a concentration of 150 mgkg CME 75 crude methanolic extractat a concentration of 75 mgkg CME 35 crude methanolic extract at aconcentration of 35 mgkg FME 150 fractional methanolic extract at aconcentration of 150 mgkg FME 75 fractional methanolic extract at aconcentration of 75 mgkg FME 35 fractional methanolic extract at aconcentration of 35 mgkg For the corresponding mean plusmn SD P lt 0001compared to the healthy control and lowastlowastlowastP lt 001 compared to the diabeticcontrol
glucose concentration (Table 5) which reached anormal levelafter 10 administrations At a concentration of 150mgkgextracts showed a greater decrease in glucose level Likewisethe extracts and metformin exhibited a decrease in seruminsulin compared to the diabetic control However in thediabetic control as well as in acarbose this behaviour wasnot observed Some studies explain that this increase ininsulin levels is due to resistance to insulin which leadsto peripheral hyperglycemia and major insulin secretion aprocess known as compensatory hyperinsulinemia [44 45]Levels of serum creatinine (SCr) serum urea (SUr) andblood urea nitrogen (BUN) were measured in order to assessthe effects on the kidney as this damage is one of the maincollateral effects of hyperglycemia The malfunction of thisorgan leads to an increase in metabolic waste products in theblood [46] The 150mgkg concentration of CME and FMEresults in the best protective effect in relation to SCr and no
6 Evidence-Based Complementary and Alternative Medicine
Table 6 Kidney and liver profile of experimental rats
Sample SCr (mgdL) SUr (mgdL) BUN (mgdL) AlT (mgdL) AsT (120583UmL)HC 120 plusmn 003 4783 plusmn 1520 2394 plusmn 650 6533 plusmn 2172 15620 plusmn 6050DC 105 plusmn 002 8250 plusmn 2469plusmn 3853 plusmn 1149 9933 plusmn 2627 20850 plusmn 9326DCV 075 plusmn 001 7600 plusmn 2713 3208 plusmn 1624 6133 plusmn 1320 18880 plusmn 8149Metformin 074 plusmn 002 3450 plusmn 1063z 2053 plusmn 504z 6550 plusmn 1222 14540 plusmn 2660Acarbose 083 plusmn 031 4833 plusmn 153 2267 plusmn 116 5950 plusmn 1457 13600 plusmn 3617CME 150 063 plusmn 002 3125 plusmn 171zzz 1455 plusmn 081z 7325 plusmn 2468 13800 plusmn 2207CME 75 090 plusmn 010 4300 plusmn 361z 2007 plusmn 165 6967 plusmn 1115 13930 plusmn 3118CME 35 137 plusmn 038 5433 plusmn 752 2650 plusmn 366 7325 plusmn 1406 15780 plusmn 2849FME 150 067 plusmn 005 3150 plusmn 407zzz 1468 plusmn 239zz 4550 plusmn 507z 14100 plusmn 3640FME 75 120 plusmn 032 5600 plusmn 577 2468 plusmn 402 7625 plusmn 1928 13630 plusmn 2229FME 35 110 plusmn 014 4700 plusmn 1086z 2193 plusmn 507 7675 plusmn 2604 13400 plusmn 3123(SCr) serum creatinine (SUr) serumurea (BUN) bloodurea nitrogen (AlT) alanine aminotransferase (AsT) aspartate aminotransferase HC healthy controlDC diabetic control DCV diabetic control vehicle CME 150 crude methanolic extract at a concentration of 150 mgkg CME 75 crude methanolic extract ata concentration of 75 mgkg CME 35 crude methanolic extract at a concentration of 35 mgkg FME 150 fractional methanolic extract at a concentration of150 mgkg FME 75 fractional methanolic extract at a concentration of 75 mgkg FME 35 fractional methanolic extract at a concentration of 35 mgkg Forthe corresponding mean plusmn SD plusmnP lt 005 compared to health control zP lt 005 compared to the diabetic control zzP lt 005 compared to the diabetic controland zzzP lt 005 compared to the diabetic control
Table 7 Lipid profile (mgdL) among experimental rats
FME 75 5975 plusmn 1242 7650 plusmn 2869 2667 plusmn 306 2300 plusmn 100FME 35 6075 plusmn 900 7575 plusmn 4842 2967 plusmn 551z 2233 plusmn 351(TC) total cholesterol (Tg) triglycerides (LDL) low-density lipoproteins (HDL) high-density lipoproteins HC healthy control DC diabetic control DCVdiabetic control vehicle CME 150 crudemethanolic extract at a concentration of 150 mgkg CME 75 crude methanolic extract at a concentration of 75 mgkgCME 35 crude methanolic extract at a concentration of 35 mgkg FME 150 fractional methanolic extract at a concentration of 150 mgkg FME 75 fractionalmethanolic extract at a concentration of 75 mgkg FME 35 fractional methanolic extract at a concentration of 35 mgkg For the corresponding mean plusmn SD zPlt 005 compared to the diabetic control zzP lt 005 compared to the diabetic control plusmnP lt 005 compared to health control 998771P lt 005 compared to acarboselozP lt 005 compared to metformin
difference was observed between the two extracts (Table 6)Enzymes AlT and AsT are an indicator of liver injury asenzymes leaking from cytosol liver cells into bloodstreamcorrelate with insulin resistance diabetes and inflammatoryprocesses of the liver [47 48] FME induced a markeddecrease in AlT making further histopathological studiesnecessary to confirm whether there is protection or damageto the liver as a result of the treatments The effect of theextracts on the lipid profile was also examined (Table 7)Patients with DM type 2 have been characterized by hightriglycerides levels low high-density lipoprotein cholesterol(HDL) levels normal low-density lipoprotein (LDL) levelsand normal or slightly increased total cholesterol levels [49ndash51] This characteristic was observed in the group treatedwith extracts and diabetic control where no significantdifferences were evident between treatments in terms of
total cholesterol nevertheless a sharp decline in triglyceridevalue was perceived in the acarbose and CME treatments(75 and 35mgkg) The capacity of acarbose for loweringtriglycerides has been described previously finding that200mg constitutes an effective dose for reducing the riskof cardiovascular events in humans implying a minimalrisk of hypoglycemia [52 53] Various studies have providedevidence that phenolic compounds such as flavonoids may beinvolved in decreased glucose levels in the in vivo model Ithas been suggested that the flavonoids naringenin and hes-peretin may produce antiatherogenic effects partly by meansof the activation of the peroxisome proliferator activatedreceptor gamma PPAR-120574 and the upregulation of adiponectinexpression in adipocytes Numerous reports document theantidiabetic effects of flavan-3-ols especially epigallocatechingallate (EGCG) in animals and cell-cultures EGCG can elicit
Evidence-Based Complementary and Alternative Medicine 7
various changes that are associated with beneficial effects fordiabetes including improvements in insulin secretion glu-cose uptake insulin resistance glucose tolerance oxidativestress inflammation and mitochondrial function [54 55]CME and FME comprise amixture of compounds and appearto inhibit the 120572-glucosidase enzyme however the inhibitoryactivity of the identified compounds suggests that this is notthe only way to reduce hyperglycemia As this is a mixtureof multitarget molecules it is impossible to assess the anti-hyperglycemic mechanism as this would imply identifyingthe two additional compounds and performing complemen-tary studies For the acute toxicity study which involved dos-ing ge2000mgkg there was no apparent mortality suggest-ing low toxicity within category 5 according to the OECDGlobally Harmonized Classification System (GHS) (2014)[27] This is advantageous should you wish to concoct anherbal preparation
4 Conclusions
These results reveal that extracts of Hamelia patens with ahigh content of phenolic compounds elicit 120572-glucosidaseinhibition and an antihyperglycemic effect At a concentrationof 150mgkg they produce an equivalent effect tometforminEpicatechin and chlorogenic acid contribute to the antihyper-glycemic activity ofH patens however it is necessary to carryout the structural identification of any other componentspresent Further research is necessary to elucidate the activitymechanisms
Data Availability
The data used to support the findings of this study are avail-able from the corresponding author upon request
Conflicts of Interest
The authors declare that there are no conflicts of interest re-garding the publication of this paper
Acknowledgments
The authors are grateful for technical support provided bythe personnel at the Laboratory of Natural Products andMolecular Biology of the ENMyH at IPN Catalina Rugerio-Escalona thanks CONACYT for the graduate scholarshipsawarded (264517) and SIP-IPN (BEIFI) for a complementaryscholarship Cynthia Ordaz-Pichardo Fabiola E Jimenez-Montejo Ignacio EMaldonado-MendozaMarıa del CarmenCruz-Lopez and Victor E Lopez-y-Lopez are Fellows of theEstımulos al Desempeno de los Investigadores (EDI)-IPNCynthia Ordaz-Pichardo Ignacio E Maldonado-MendozaMarıa del Carmen Cruz-Lopez and Victor E Lopez-y-Lopez thank the Comision de Operacion y Fomento deActividades Academicas (COFAA) an IPN program thatprovides support to researchers This work was financiallysupported by a project (20161781) from the Secretarıa deInvestigacion y Posgrado Instituto Politecnico Nacional
References
[1] F A Matough S B Budin Z A Hamid N Alwahaibi and JMohamed ldquoThe role of oxidative stress and antioxidants in dia-betic complicationsrdquo SQU Medical Journal vol 12 no 1 pp 5ndash18 2012
[2] AW Indrianingsih S Tachibana R TDewi andK Itoh ldquoAnti-oxidant and 120572-glucosidase inhibitor activities of natural com-pounds isolated fromQuercus gilva Blume leavesrdquoAsian PacificJournal of Tropical Biomedicine vol 5 no 9 pp 748ndash755 2015
[3] M Gondi and U J S Prasada Rao ldquoEthanol extract of mango(Mangifera indica L) peel inhibits 120572-amylase and 120572-glucosidaseactivities and ameliorates diabetes related biochemical param-eters in streptozotocin (STZ)-induced diabetic ratsrdquo Journal ofFood Science andTechnology vol 52 no 12 pp 7883ndash7893 2015
[4] ARayarandRManivannan ldquoIn-vitro alpha-amylase and alpha-glucosidase inhibition activity of umbelliferone and beta-ionone isolated from Coriandrum sativum LinnrdquoWorld Journalof Pharmacy and Pharmaceutical Sciences vol 5 no 1 pp 1280ndash1289 2016
[5] Atlas de la Diabetes de la FID-Octava edicion InternationalDiabetes Federation 2017
[6] American Diabetes Association ldquoPharmacologic Approachesto Glycemic Treatmentrdquo Journal of Clinical and Applied Resarchand Education vol 40 no 1 pp S64ndashS76 2017
[7] J J Marın-Penalver I Martın-Timon C Sevillano-Collantesand F J del Canizo-Gomez ldquoUpdate on the treatment of type2 diabetes mellitusrdquoWorld Journal of Diabetes vol 7 no 17 pp354ndash395 2016
[8] H-Y Chang M Wallis and E Tiralongo ldquoUse of comple-mentary and alternative medicine among people living withdiabetes literature reviewrdquo Journal of AdvancedNursing vol 58no 4 pp 307ndash319 2007
[9] A B Medagama and R Bandara ldquoThe use of Complementaryand AlternativeMedicines (CAMs) in the treatment of diabetesmellitus Is continued use safe and effectiverdquo Nutrition Journalvol 13 no 1 2014
[10] B Baharvand-Ahmadi M Bahmani P Tajeddini N Naghdiand M Rafieian-Kopaei ldquoAn ethno-medicinal study of medic-inal plants used for the treatment of diabetesrdquo Journal ofNephropathology vol 5 no 1 pp 44ndash50 2016
[11] S Verma M Gupta H Popli and G Aggarwal ldquoDiabetesmellitus treatment using herbal drugsrdquo International Journal ofPhytomedicine vol 10 no 1 pp 1ndash10 2018
[12] B Ovalle-Magallanes O N Medina-Campos J Pedraza-Cha-verri and R Mata ldquoHypoglycemic and antihyperglycemic ef-fects of phytopreparations and limonoids from Swietenia humi-lisrdquo Phytochemistry vol 110 pp 111ndash119 2015
[13] S Singh andM Vyas ldquoComparative in-vitro biological study ofaerial parts of plant Hamelia patensrdquo International Journal ofPharmaceutical Sciences and Research vol 7 no 4 pp 1793ndash1808 2016
[14] F Ruiz-Teran A Medrano-Martınez and A Navarro-OcanaldquoAntioxidant and free radical scavenging activities of plantextracts used in traditional medicine in Mexicordquo African Jour-nal of Biotechnology vol 7 no 12 pp 1886ndash1893 2008
[15] S Sosa M J Balick R Arvigo et al ldquoScreening of the topicalanti-inflammatory activity of some Central American plantsrdquoJournal of Ethnopharmacology vol 81 no 2 pp 211ndash215 2002
[16] A Camporese M J Balick R Arvigo et al ldquoScreening ofanti-bacterial activity of medicinal plants from Belize (Central
8 Evidence-Based Complementary and Alternative Medicine
America)rdquo Journal of Ethnopharmacology vol 87 no 1 pp 103ndash107 2003
[17] G Mena-Rejon E Caamal-Fuentes Z Cantillo-Ciau R Cedil-lo-Rivera J Flores-Guido and R Moo-Puc ldquoIn vitro cytotoxicactivity of nine plants used in Mayan traditional medicinerdquoJournal of Ethnopharmacology vol 121 no 3 pp 462ndash465 2009
[18] V Jimenez-Suarez A Nieto-Camacho M Jimenez-Estradaand B Alvarado Sanchez ldquoAnti-inflammatory free radical scav-enging and alpha-glucosidase inhibitory activities of Hameliapatens and its chemical constituentsrdquo Pharmaceutical Biologyvol 54 no 9 pp 1822ndash1830 2016
[19] A Andrade-Cetto S Escandon-Rivera and V Garcia-LunaldquoHypoglycemic effect of Hamelia patens Jacq aerial part inSTZ-NA-induced diabetic ratsrdquo Pharmacologyonline vol 3 pp65ndash69b 2015
[20] A Ahmad A Pandurangan N Singh and P Ananad ldquoA minireview on chemistry and biology of Hamelia Patens (Rubi-aceae)rdquo Pharmacognosy Journal vol 4 no 29 pp 1ndash4 2012
[21] J Bano S Santra and EMenghani ldquoHamelia patens a potentialplant from Rubiaceae family A reviewrdquo International Journalof Scientific amp Engineering Research vol 6 no 12 pp 960ndash9732015
[22] E C Cruz and A Andrade-Cetto ldquoEthnopharmacological fieldstudy of the plants used to treat type 2 diabetes among theCakchiquels in Guatemalardquo Journal of Ethnopharmacology vol159 pp 238ndash244 2015
[23] D A Sampietro M A Sgariglia J R Soberon E N QuirogaandM A Vattuone ldquoColorimetric reactionsrdquo in Isolation Iden-tification and Characterization of AllelochemicalsNatural Prod-ucts chapter 4 pp 78ndash82 2009
[24] H P S Makkar and K Becker ldquoVanillin-HCl method for con-densed tannins effect of organic solvents used for extraction oftanninsrdquo Journal of Chemical Ecology vol 19 no 4 pp 613ndash6211993
[25] B A Cevallos-Casals and L Cisneros-Zevallos ldquoStoichiometricand kinetic studies of phenolic antioxidants from Andean pur-ple corn and red-fleshed sweetpotatordquo Journal of Agriculturaland Food Chemistry vol 51 no 11 pp 3313ndash3319 2003
[26] P Salehi B Asghari M A Esmaeili H Dehghan and I Ghazildquo120572-Glucosidase and120572-amylase inhibitory effect and antioxidantof ten plant extracts traditionally used in Iran for diabetesrdquoJournal of Medicinal Plants Research vol 7 no 6 pp 257ndash2662013
[27] OECDOCDE 423 OECD Guideline for testing of chemicalsAcute Oral Toxicity ndash Acute Toxic Class Method EnvironmentDirectorate Organisation for Economic Co-Operation and De-velopment Paris France 2001
[28] B L Furman ldquoStreptozotocin-induced diabetic models in miceand ratsrdquo Current Protocols in Pharmacology vol 70 pp 5471ndash54720 2015
[29] I J Flores-Sanchez and A C Ramos-Valdivia ldquoA review frompatents inspired by two plant genera Uncaria and HameliardquoPhytochemistry Reviews vol 16 no 4 pp 693ndash723 2017
[30] R Vinayagam and B Xu ldquoAntidiabetic properties of dietaryflavonoids A cellular mechanism reviewrdquo Journal of Nutritionand Metabolism vol 12 no 60 pp 1ndash20 2015
[31] S Marella ldquoFlavonoids-Themost potent poly-phenols as antidi-abetic agents an overviewrdquoModernApproaches in Drug Design-ing vol 1 no 3 pp 2ndash5 2017
[32] AUllah A Khan and I Khan ldquoDiabetesmellitus and oxidativestress-A concise reviewrdquo Saudi Pharmaceutical Journal vol 24pp 547ndash553 2016
[33] D Shasha CMagogo and P Dzomba ldquoReversed phase HPLC-UV Quantitation of BHA BHT and TBHQ in food items soldin bindura supermarkets Zimbabwerdquo International ResearchJournal of Pure amp Applied Chemistry vol 4 no 5 pp 578ndash5842014
[34] M Vessal M Hemmati and M Vasei ldquoAntidiabetic effects ofquercetin in streptozocin-induced diabetic ratsrdquo ComparativeBiochemistry and Physiology Part C Toxicology amp Pharmacol-ogy vol 135 no 3 pp 357ndash364 2003
[35] P-C Hsieh G-J Huang Y-L Ho et al ldquoActivities of antioxi-dants 120572-glucosidase inhibitors and aldose reductase inhibitorsof the aqueous extracts of four Flemingia species in TaiwanrdquoBotanical Studies vol 51 no 3 pp 293ndash302 2010
[36] W Kang Y Song and X Gu ldquo120572-glucosidase inhibitory in vitroand antidiabetic activity in vivo ofOsmanthus fragransrdquo Journalof Medicinal Plants Research vol 6 no 14 pp 2850ndash2856 2012
[37] N Zahratunnisa B Elya and A Noviani ldquoInhibition of Alpha-glucosidase and antioxidant test of stem bark extracts ofGarcinia fruticosa Lauterbrdquo Pharmacognosy Journal vol 9 no2 pp 273ndash275 2017
[38] J E Wong C Rubio A Reyes C N Aguilar and M L Carril-lo ldquoPhenolic content and antibacterial activity of extracts ofHamelia patens obtained by different extraction methodsrdquo Bra-zilian Journal of Microbiology vol 49 no 3 pp 656ndash661 2017
[39] G Oboh O M Agunloye S A Adefegha A J Akinyemi andA O Ademiluyi ldquoCaffeic and chlorogenic acids inhibit key en-zymes linked to type 2 diabetes (in vitro) a comparative studyrdquoJournal of Basic and Clinical Physiology and Pharmacology vol26 no 2 pp 165ndash170 2015
[40] S-Y Chiou J-M Sung P-W Huang and S-D Lin ldquoAntiox-idant Antidiabetic and Antihypertensive Properties of Echi-nacea purpurea Flower Extract and Caffeic Acid DerivativesUsing in Vitro Modelsrdquo Journal of Medicinal Food vol 20 no2 pp 171ndash179 2017
[41] C-M Ma M Hattori M Daneshtalab and L Wang ldquoChloro-genic acid derivatives with alkyl chains of different lengths andorientations Potent120572-glucosidase inhibitorsrdquo Journal of Medic-inal Chemistry vol 51 no 19 pp 6188ndash6194 2008
[42] C Tan QWang C Luo S Chen Q Li and P Li ldquoYeast 120572-glu-cosidase inhibitory phenolic compounds isolated from Gynuramedica leafrdquo International Journal of Molecular Sciences vol 14no 2 pp 2551ndash2558 2013
[43] A I Suarez B Diaz S Tillett E Valdivieso and R S Com-pagnone ldquoLeishmanicidal activity of alkaloids from Hameliapatensrdquo Ciencia vol 16 no 2 pp 148ndash155 2008
[44] C Gutierrez-Rodelo A Roura-Guiberna and J A Olivares-Reyes ldquoMecanismos moleculares de la resistencia a la insulinauna actualizacionrdquo Gaceta Medica de Mexico vol 153 pp 215ndash228 2017
[45] J Rojas V Berm E Leal V Bermudez et al ldquoInsulinoresisten-cia e hiperinsulinemia como factores de riesgo para enfermedadcardivascularrdquo Revista Latinoamericana de Hipertension vol 2no 6 pp 179ndash190 2007
[46] SABamanikar A A Bamanikar andAArora ldquoStudy of serumurea and creatinine in diabetic and non-diabetic patients in atertiary teaching hospitalrdquoThe Journal of Medical Research vol2 no 1 pp 12ndash15 2016
[47] M Music A Dervisevic E Pepic et al ldquoMetabolic Syndromeand Serum Liver Enzymes Level at Patients with Type 2Diabetes MellitusrdquoMedical Archives vol 69 no 4 pp 251ndash2552015
Evidence-Based Complementary and Alternative Medicine 9
[48] S Kazemi S Asgary J Moshtaghian et al ldquoLiver-protectiveeffects of hydroalcoholic extract of Allium hirtifolium Boiss inrats with alloxan-induced diabetes mellitusrdquo ARYA Atheroscle-rosis Journal vol 6 no 1 pp 11ndash15 2010
[49] G A Bonneau M S Castillo Rascon R A Sanchez W RPedrozo and C Castro Olivera ldquoColesterol-IDL y parametroslipıdicos en diabeticos tipo 2rdquo Revista Argentina de Endocri-nologıa y Metabolismo vol 44 no 4 pp 215ndash222 2007
[50] A Husni D Purwanti and Ustadi ldquoBlood glucose level andlipid profile of streptozotocin-induceddiabetes rats treatedwithsodiumalginate from Sargassum crassifoliumrdquo Journal of Biolog-ical Sciences vol 16 no 3 pp 58ndash64 2016
[51] S Palazhy and V Viswanathan ldquoLipid abnormalities in type 2diabetes mellitus patients with overt nephropathyrdquo Diabetes ampMetabolism Journal vol 41 no 2 pp 128ndash134 2017
[52] J J DiNicolantonio J Bhutani and J H OrsquoKeefe ldquoAcarbosesafe and effective for lowering postprandial hyperglycaemia andimproving cardiovascular outcomesrdquo Open Heart vol 2 no 12015
[53] S Ogawa K Takaeuchi and S Ito ldquoAcarbose lowers serum tri-glyceride and postprandial chylomicron levels in type 2 dia-betesrdquo Diabetes Obesity and Metabolism vol 6 no 5 pp 384ndash390 2004
[54] L Liu S Shan K Zhang Z-Q Ning X-P Lu and Y-Y ChengldquoNaringenin andhesperetin two flavonoids derived fromCitrusaurantium up-regulate transcription of adiponectinrdquoPhytother-apy Research vol 22 no 10 pp 1400ndash1403 2008
[55] E P Cai and J-K Lin ldquoEpigallocatechin gallate (EGCG) andrutin suppress the glucotoxicity through activating IRS2 andAMPK signaling in rat pancreatic 120573 cellsrdquo Journal of Agricul-tural and Food Chemistry vol 57 no 20 pp 9817ndash9827 2009
Computational and Mathematical Methods in Medicine
Hindawiwwwhindawicom Volume 2018
Behavioural Neurology
OphthalmologyJournal of
Hindawiwwwhindawicom Volume 2018
Diabetes ResearchJournal of
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Research and TreatmentAIDS
Hindawiwwwhindawicom Volume 2018
Gastroenterology Research and Practice
Hindawiwwwhindawicom Volume 2018
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Evidence-Based Complementary andAlternative Medicine
Volume 2018Hindawiwwwhindawicom
Submit your manuscripts atwwwhindawicom
Evidence-Based Complementary and Alternative Medicine 3
compared to the value found for chlorogenic acid quercetinand epicatechin
29 Qualitative High-Performance Liquid Chromatography(HPLC) Analysis Analytical HPLC was carried out usingan Agilent 1100 series apparatus equipped with a diodearray detector (DAD) (Agilent Technologies) using a XBD-C18 analytical column 46 times 150mm 35120583m particle size(Agilent Technologies) and a column temperature of 60∘CElution was performed at a flow rate of 1mLmin with02 formic acid (solvent A) and acetonitrile (solvent B)for the mobile phase The samples were eluted by applyingthe following gradient 100 A as the initial condition for3min 90 A and 10 B for 3min 85 A and 15 B for3min 80 A and 20 B for 3min 70 A and 30 B for3min 60 A and 40 B for 3min and finally 50 A and50 B for 15min At 253 290 and 349 nm compoundswere detected and identified by comparing them to knownstandards (caffeic acid chlorogenic acid trans-cinnamic acidtrans-ferulic acid syringic acid caffeine (plusmn)-catechin (-)-epicatechin kaempferol naringenin quercetin and rutin)based on retention times and UV spectra
210 Experimental Animals Male (27plusmn2 g) and female(20plusmn2 g) ICR mice and male Wistar rats (180plusmn30 g) wereobtained from the Facultad de Estudios Superiores AcatlanUniversidad Nacional Autonoma de Mexico The mice werehoused under standard conditions and given a standard pelletfeed and water ad libitum All experiments with animals wereauthorized by the Ethics Committee of the Escuela Nacionalde Medicina y Homeopatıa of the Instituto PolitecnicoNacional and complied with national and international prin-ciples for the care and use of lab animals
211 Acute Oral Toxicity Testing Toxicity assessment adheredto OECD (Organization of Economic Co-operation andDevelopment) guidelines for testing chemicals (Acute OralToxicityndashAcute Toxic Class Method section 423) Threefemale and three male ICRmice (20-30 g) were given a singleoral administration of 300mgkg of the plant extracts afterovernight fasting [27]The control group received only waterMice were observed for symptoms and weight variation atpostadministration intervals of 1 3 and 4 h and then twiceper day for the subsequent 14 days Animals were kept at23plusmn2∘C and 50 humidity under a 1212 h lightdarknesscycle They were provided with standard feed and water adlibitum throughout the study Results made it possible toclassify the substance according to the Globally HarmonizedSystem (GHS)
212 Streptozotocin-Induced Diabetic Rats This parameterwas explored among 110 male Wistar rats They were keptat a temperature of 23plusmn3∘C and on a 1212 h lightdarknesscycle with free access to food and water For the induction ofhyperglycemia all groups of rats (except the healthy control)were administered a single dose of 50mgkg of streptozotocin(STZ) intraperitoneally After three days of administrationthe glucose level was measured on reactive strips and animals
showing a blood glucose level of ge 200mgdL were selectedfor the test groups
213 Antihyperglycemic Activity of H patens Extracts Extractconcentrations necessary to induce the desired pharmacolog-ical responses were taken from previous studies Rats weredivided into eleven groups each consisting of ten animalsGroup I healthy control (water sterile) group II diabeticcontrol (STZ) group III vehicle (300120583L propylene glycolPPG) group IV metformin (100mgkg) group V acarbose(10mgkg) groups VI-VIII FME extracts at doses of 35 75and 150mgkg groups IX-XI CME extracts at doses of 3575 and 150mgkg were all administered intragastrically everythird day monitoring the peripheral glucose 48 h postad-ministration After the 15th administration animals wereeuthanized and a blood sample was obtained for biochemicalanalysis [28]
214 Biochemical Determinations Serum samples were col-lected in order to measure glucose concentration (SG)insulin (SIN) total cholesterol (TC) low-density lipoproteins(LDL) high-density lipoproteins (HDL) triglycerides (Tg)creatinine (SCr) urea (SUr) blood urea nitrogen (BUN) ala-nine aminotransferase (AlT) and aspartate aminotransferase(AsT) Detection was carried out following the manufac-turerrsquos protocol for each diagnostic kit
215 Statistical Analysis Data are expressed as the mean plusmnstandard error Statistical differenceswere evaluated using theTukey test with the SAS version 9 program For the evaluationof antihyperglycemic activity and biochemical parametersthe comparison ofmultiple variancewas analyzed by applyingthe Holm-Bonferroni method utilizing GraphPad Prismsoftware (version 50) In all cases statistical significance wasconsidered at plt005
3 Results and Discussion
CME yield was 89 and 54 for FME Chemical com-positions of CME and FME are presented in Table 1 Asapparent CME and FME ofH patens are an excellent sourceof tannins flavonoids and phenols Significant differencesbetween CME and FME were apparent in the total contentof phenols and flavonoids The latter extract displayed thehighest content of these metabolites We relate this to theway extracts were obtained These results concur with thosereported by Flores-Sanchez in 2017 [29]
Pathogenesis of DM has been shown to relate to thegeneration of free radicals especially reactive oxygen species(ROS) glucose oxidation increased lipid peroxidation andgreater insulin resistance Recent studies have shown thatphenolic compounds are well known for their great capacityfor radical scavenging especially flavonoids which may beeffective in the management and prevention of diabetesmellitus due to interference in the absorption digestion andmetabolism of carbohydrates [30 31] When evaluating theantioxidant activity of both extracts (Table 2) no significantdifferences were observed between CME and FME in terms
4 Evidence-Based Complementary and Alternative Medicine
Table 1 Metabolic content of the methanolic extracts of H patens
Data are expressed as the mean plusmn SD ECat equivalents of catechin EGA equivalents of gallic acid EQ equivalents of quercetin Themean values labeled withuppercase letters differ significantly compared to the control
Table 2 DPPH radical scavenging activity and 120572-glucosidase inhibition by methanolic extracts ofH patens
of their EC50 values (507 and 517120583gmL respectively) How-ever both extracts presented a better EC50 value than BHTwhich is a synthetic phenolic antioxidant currently used infood despite the evidence that it causes enzymatic or lipidalterations as well as carcinogenic effects and mutagenicactivity [32 33]120572-Glucosidase inhibitors are targeted to delay carbohy-
drate absorption and reduce postprandial glucose Severalphenolic compounds containing a flavonoid nucleus in theirstructure are reportedly useful for the control of diabetes byimproving glucose and lipid levels [30] Moreover studieshave demonstrated that quercetin epicatechin kaempferoland naringenin effectively inhibit the 120572-glucosidase enzyme[31 34] Consequently the 120572-glucosidase inhibition assay wasthe first stage in the identification of antidiabetic agentsHigh 120572-glucosidase inhibitory activity was found in bothextracts (FME IC50 = 678 and CME IC50 = 783120583gmLTable 2) Thus the extracts are more active than acar-bose (IC50= 49966 120583gmL) Contrastingly our low polarityextracts showed inhibitory activity of less than 10 at aconcentration of 4000120583gmL However in 2016 Jimenez [18]established that hexanic and methanol-ethyl acetate extractsof H patens manifest better 120572-glucosidase inhibitory effectwith IC50 = 2607120583gml and 3018120583gml respectively thandichloromethane-ethyl acetate and methanol-water extractswhich did not exhibit activity Their results coincide withthose reported by other authors who have demonstrated thathigh polarity extracts are more active than acarbose [35ndash37]
The compounds in the extracts which produce antag-onistic activity are polar in nature and a chromatographicHPLC analysis was performed to determine their compo-sition and identify the possible active principles By usingdifferent standards (Figure 1) the resulting retention times(Table 3) reveal that chlorogenic acid is the main componentin both extracts constituting 135 of CME and 195 of
FME Catechin and epicatechin were also detected in theextracts concurring with Wongrsquos description of H patensin 2017 [38] Looking for a possible explanation for thisbehaviour we decided to evaluate the inhibitory activity forthe compounds epicatechin chlorogenic acid and quercetinpreviously identified in the extractsThefirst two showed IC50values that were higher than those obtained from FME andCME with IC50 = 2826 120583gmL for epicatechin and IC50 ge3000120583gmL for chlorogenic acid Notably our results forchlorogenic acid differ from those previously reported (IC50= 24 461 1000 and gt2000120583gmL) [39ndash42] Although chloro-genic acid is the main component the antagonistic activityagainst the enzyme is low which does not correlate with theactivity that both extracts manifested Quercetin previouslyisolated fromH patens presented a similar inhibitory activity(IC50 = 27120583gmL) to the one described by Indrianingsihin 2015 (IC50 = 42120583gmL) [2] A peak at 14025 minuteswas displayed in the spectrum for CME (107) and at14075 minutes for FME (45) Likewise the peak with aretention time of 1921 indicated a higher concentration forCME (59) than for FME (09) As these two componentsof low polarity may partly explain the perceived differencein activity further research is necessary to isolate them forfurther structural characterization and biological evaluationUsing column chromatography a light brown amorphoussolid with a melting point of 236-238∘C was isolated fromCME Nuclear magnetic resonance (NMR) analysis made itpossible to identify a 3-flavonol skeleton and the 1H and 13CNMR spectra and two-dimensional spectra established thestructure as (-)-epicatechin concurring with a recent reportfor H patens [43]The chemical shifts for this compound inthe 1H and 13C NMR are presented in Table 4
The second stage of the current study intended to explorethe antihyperglycemic effect of the extracts in an in vivomurinemodelThe extractswere administered 15 times to rats
Evidence-Based Complementary and Alternative Medicine 5
140
120
100
80
60
40
20
0
minus20
mAU
4
1
5
5 10 15 20 25
lowast
+
min
(a) FME
175
150
100
125
75
50
25
0
minus25
mAU
4
1
5
5 10 15 20 25
lowast
+
min
(b) CME
Figure 1 HPLC chromatogram of fractional (FME) and crude (CME) methanolic extracts of H patens (1) chlorogenic acid (4) catechinand (5) (-) epicatechin lowastDifferent component +Different concentration
Table 3 The retention time of phenolic compounds at 290 nm
with hyperglycemia induced by STZ (glucose levels higher300mgdL) FME and CME extracts produced a reduction in
Table 5 Glucose levels and serum insulin in experimental rats
Sample SG (mgdL) SIN (mUImL)HC 11270 plusmn 2211 210 plusmn 127DC 43750 plusmn 071 250 plusmn 131DCV 42900 plusmn 283 195 plusmn 007Metformin 12390 plusmn 3399lowastlowastlowast 174 plusmn 006Acarbose 14230 plusmn 3199lowastlowastlowast 215 plusmn 039CME 150 9325 plusmn 2022lowastlowastlowast 163 plusmn 071CME 75 15600 plusmn 1513lowastlowastlowast 133 plusmn 015CME 35 15300 plusmn 3036lowastlowastlowast 232 plusmn 132FME 150 11750 plusmn 4135lowastlowastlowast 147 plusmn 055FME 75 16880 plusmn 1167lowastlowastlowast 200 plusmn 087FME 35 16200 plusmn 2877lowastlowastlowast 275 plusmn 035(SG) concentration of glucose (SIN) insulin HC healthy control DCdiabetic control DCV diabetic control vehicle CME 150 crude methanolicextract at a concentration of 150 mgkg CME 75 crude methanolic extractat a concentration of 75 mgkg CME 35 crude methanolic extract at aconcentration of 35 mgkg FME 150 fractional methanolic extract at aconcentration of 150 mgkg FME 75 fractional methanolic extract at aconcentration of 75 mgkg FME 35 fractional methanolic extract at aconcentration of 35 mgkg For the corresponding mean plusmn SD P lt 0001compared to the healthy control and lowastlowastlowastP lt 001 compared to the diabeticcontrol
glucose concentration (Table 5) which reached anormal levelafter 10 administrations At a concentration of 150mgkgextracts showed a greater decrease in glucose level Likewisethe extracts and metformin exhibited a decrease in seruminsulin compared to the diabetic control However in thediabetic control as well as in acarbose this behaviour wasnot observed Some studies explain that this increase ininsulin levels is due to resistance to insulin which leadsto peripheral hyperglycemia and major insulin secretion aprocess known as compensatory hyperinsulinemia [44 45]Levels of serum creatinine (SCr) serum urea (SUr) andblood urea nitrogen (BUN) were measured in order to assessthe effects on the kidney as this damage is one of the maincollateral effects of hyperglycemia The malfunction of thisorgan leads to an increase in metabolic waste products in theblood [46] The 150mgkg concentration of CME and FMEresults in the best protective effect in relation to SCr and no
6 Evidence-Based Complementary and Alternative Medicine
Table 6 Kidney and liver profile of experimental rats
Sample SCr (mgdL) SUr (mgdL) BUN (mgdL) AlT (mgdL) AsT (120583UmL)HC 120 plusmn 003 4783 plusmn 1520 2394 plusmn 650 6533 plusmn 2172 15620 plusmn 6050DC 105 plusmn 002 8250 plusmn 2469plusmn 3853 plusmn 1149 9933 plusmn 2627 20850 plusmn 9326DCV 075 plusmn 001 7600 plusmn 2713 3208 plusmn 1624 6133 plusmn 1320 18880 plusmn 8149Metformin 074 plusmn 002 3450 plusmn 1063z 2053 plusmn 504z 6550 plusmn 1222 14540 plusmn 2660Acarbose 083 plusmn 031 4833 plusmn 153 2267 plusmn 116 5950 plusmn 1457 13600 plusmn 3617CME 150 063 plusmn 002 3125 plusmn 171zzz 1455 plusmn 081z 7325 plusmn 2468 13800 plusmn 2207CME 75 090 plusmn 010 4300 plusmn 361z 2007 plusmn 165 6967 plusmn 1115 13930 plusmn 3118CME 35 137 plusmn 038 5433 plusmn 752 2650 plusmn 366 7325 plusmn 1406 15780 plusmn 2849FME 150 067 plusmn 005 3150 plusmn 407zzz 1468 plusmn 239zz 4550 plusmn 507z 14100 plusmn 3640FME 75 120 plusmn 032 5600 plusmn 577 2468 plusmn 402 7625 plusmn 1928 13630 plusmn 2229FME 35 110 plusmn 014 4700 plusmn 1086z 2193 plusmn 507 7675 plusmn 2604 13400 plusmn 3123(SCr) serum creatinine (SUr) serumurea (BUN) bloodurea nitrogen (AlT) alanine aminotransferase (AsT) aspartate aminotransferase HC healthy controlDC diabetic control DCV diabetic control vehicle CME 150 crude methanolic extract at a concentration of 150 mgkg CME 75 crude methanolic extract ata concentration of 75 mgkg CME 35 crude methanolic extract at a concentration of 35 mgkg FME 150 fractional methanolic extract at a concentration of150 mgkg FME 75 fractional methanolic extract at a concentration of 75 mgkg FME 35 fractional methanolic extract at a concentration of 35 mgkg Forthe corresponding mean plusmn SD plusmnP lt 005 compared to health control zP lt 005 compared to the diabetic control zzP lt 005 compared to the diabetic controland zzzP lt 005 compared to the diabetic control
Table 7 Lipid profile (mgdL) among experimental rats
FME 75 5975 plusmn 1242 7650 plusmn 2869 2667 plusmn 306 2300 plusmn 100FME 35 6075 plusmn 900 7575 plusmn 4842 2967 plusmn 551z 2233 plusmn 351(TC) total cholesterol (Tg) triglycerides (LDL) low-density lipoproteins (HDL) high-density lipoproteins HC healthy control DC diabetic control DCVdiabetic control vehicle CME 150 crudemethanolic extract at a concentration of 150 mgkg CME 75 crude methanolic extract at a concentration of 75 mgkgCME 35 crude methanolic extract at a concentration of 35 mgkg FME 150 fractional methanolic extract at a concentration of 150 mgkg FME 75 fractionalmethanolic extract at a concentration of 75 mgkg FME 35 fractional methanolic extract at a concentration of 35 mgkg For the corresponding mean plusmn SD zPlt 005 compared to the diabetic control zzP lt 005 compared to the diabetic control plusmnP lt 005 compared to health control 998771P lt 005 compared to acarboselozP lt 005 compared to metformin
difference was observed between the two extracts (Table 6)Enzymes AlT and AsT are an indicator of liver injury asenzymes leaking from cytosol liver cells into bloodstreamcorrelate with insulin resistance diabetes and inflammatoryprocesses of the liver [47 48] FME induced a markeddecrease in AlT making further histopathological studiesnecessary to confirm whether there is protection or damageto the liver as a result of the treatments The effect of theextracts on the lipid profile was also examined (Table 7)Patients with DM type 2 have been characterized by hightriglycerides levels low high-density lipoprotein cholesterol(HDL) levels normal low-density lipoprotein (LDL) levelsand normal or slightly increased total cholesterol levels [49ndash51] This characteristic was observed in the group treatedwith extracts and diabetic control where no significantdifferences were evident between treatments in terms of
total cholesterol nevertheless a sharp decline in triglyceridevalue was perceived in the acarbose and CME treatments(75 and 35mgkg) The capacity of acarbose for loweringtriglycerides has been described previously finding that200mg constitutes an effective dose for reducing the riskof cardiovascular events in humans implying a minimalrisk of hypoglycemia [52 53] Various studies have providedevidence that phenolic compounds such as flavonoids may beinvolved in decreased glucose levels in the in vivo model Ithas been suggested that the flavonoids naringenin and hes-peretin may produce antiatherogenic effects partly by meansof the activation of the peroxisome proliferator activatedreceptor gamma PPAR-120574 and the upregulation of adiponectinexpression in adipocytes Numerous reports document theantidiabetic effects of flavan-3-ols especially epigallocatechingallate (EGCG) in animals and cell-cultures EGCG can elicit
Evidence-Based Complementary and Alternative Medicine 7
various changes that are associated with beneficial effects fordiabetes including improvements in insulin secretion glu-cose uptake insulin resistance glucose tolerance oxidativestress inflammation and mitochondrial function [54 55]CME and FME comprise amixture of compounds and appearto inhibit the 120572-glucosidase enzyme however the inhibitoryactivity of the identified compounds suggests that this is notthe only way to reduce hyperglycemia As this is a mixtureof multitarget molecules it is impossible to assess the anti-hyperglycemic mechanism as this would imply identifyingthe two additional compounds and performing complemen-tary studies For the acute toxicity study which involved dos-ing ge2000mgkg there was no apparent mortality suggest-ing low toxicity within category 5 according to the OECDGlobally Harmonized Classification System (GHS) (2014)[27] This is advantageous should you wish to concoct anherbal preparation
4 Conclusions
These results reveal that extracts of Hamelia patens with ahigh content of phenolic compounds elicit 120572-glucosidaseinhibition and an antihyperglycemic effect At a concentrationof 150mgkg they produce an equivalent effect tometforminEpicatechin and chlorogenic acid contribute to the antihyper-glycemic activity ofH patens however it is necessary to carryout the structural identification of any other componentspresent Further research is necessary to elucidate the activitymechanisms
Data Availability
The data used to support the findings of this study are avail-able from the corresponding author upon request
Conflicts of Interest
The authors declare that there are no conflicts of interest re-garding the publication of this paper
Acknowledgments
The authors are grateful for technical support provided bythe personnel at the Laboratory of Natural Products andMolecular Biology of the ENMyH at IPN Catalina Rugerio-Escalona thanks CONACYT for the graduate scholarshipsawarded (264517) and SIP-IPN (BEIFI) for a complementaryscholarship Cynthia Ordaz-Pichardo Fabiola E Jimenez-Montejo Ignacio EMaldonado-MendozaMarıa del CarmenCruz-Lopez and Victor E Lopez-y-Lopez are Fellows of theEstımulos al Desempeno de los Investigadores (EDI)-IPNCynthia Ordaz-Pichardo Ignacio E Maldonado-MendozaMarıa del Carmen Cruz-Lopez and Victor E Lopez-y-Lopez thank the Comision de Operacion y Fomento deActividades Academicas (COFAA) an IPN program thatprovides support to researchers This work was financiallysupported by a project (20161781) from the Secretarıa deInvestigacion y Posgrado Instituto Politecnico Nacional
References
[1] F A Matough S B Budin Z A Hamid N Alwahaibi and JMohamed ldquoThe role of oxidative stress and antioxidants in dia-betic complicationsrdquo SQU Medical Journal vol 12 no 1 pp 5ndash18 2012
[2] AW Indrianingsih S Tachibana R TDewi andK Itoh ldquoAnti-oxidant and 120572-glucosidase inhibitor activities of natural com-pounds isolated fromQuercus gilva Blume leavesrdquoAsian PacificJournal of Tropical Biomedicine vol 5 no 9 pp 748ndash755 2015
[3] M Gondi and U J S Prasada Rao ldquoEthanol extract of mango(Mangifera indica L) peel inhibits 120572-amylase and 120572-glucosidaseactivities and ameliorates diabetes related biochemical param-eters in streptozotocin (STZ)-induced diabetic ratsrdquo Journal ofFood Science andTechnology vol 52 no 12 pp 7883ndash7893 2015
[4] ARayarandRManivannan ldquoIn-vitro alpha-amylase and alpha-glucosidase inhibition activity of umbelliferone and beta-ionone isolated from Coriandrum sativum LinnrdquoWorld Journalof Pharmacy and Pharmaceutical Sciences vol 5 no 1 pp 1280ndash1289 2016
[5] Atlas de la Diabetes de la FID-Octava edicion InternationalDiabetes Federation 2017
[6] American Diabetes Association ldquoPharmacologic Approachesto Glycemic Treatmentrdquo Journal of Clinical and Applied Resarchand Education vol 40 no 1 pp S64ndashS76 2017
[7] J J Marın-Penalver I Martın-Timon C Sevillano-Collantesand F J del Canizo-Gomez ldquoUpdate on the treatment of type2 diabetes mellitusrdquoWorld Journal of Diabetes vol 7 no 17 pp354ndash395 2016
[8] H-Y Chang M Wallis and E Tiralongo ldquoUse of comple-mentary and alternative medicine among people living withdiabetes literature reviewrdquo Journal of AdvancedNursing vol 58no 4 pp 307ndash319 2007
[9] A B Medagama and R Bandara ldquoThe use of Complementaryand AlternativeMedicines (CAMs) in the treatment of diabetesmellitus Is continued use safe and effectiverdquo Nutrition Journalvol 13 no 1 2014
[10] B Baharvand-Ahmadi M Bahmani P Tajeddini N Naghdiand M Rafieian-Kopaei ldquoAn ethno-medicinal study of medic-inal plants used for the treatment of diabetesrdquo Journal ofNephropathology vol 5 no 1 pp 44ndash50 2016
[11] S Verma M Gupta H Popli and G Aggarwal ldquoDiabetesmellitus treatment using herbal drugsrdquo International Journal ofPhytomedicine vol 10 no 1 pp 1ndash10 2018
[12] B Ovalle-Magallanes O N Medina-Campos J Pedraza-Cha-verri and R Mata ldquoHypoglycemic and antihyperglycemic ef-fects of phytopreparations and limonoids from Swietenia humi-lisrdquo Phytochemistry vol 110 pp 111ndash119 2015
[13] S Singh andM Vyas ldquoComparative in-vitro biological study ofaerial parts of plant Hamelia patensrdquo International Journal ofPharmaceutical Sciences and Research vol 7 no 4 pp 1793ndash1808 2016
[14] F Ruiz-Teran A Medrano-Martınez and A Navarro-OcanaldquoAntioxidant and free radical scavenging activities of plantextracts used in traditional medicine in Mexicordquo African Jour-nal of Biotechnology vol 7 no 12 pp 1886ndash1893 2008
[15] S Sosa M J Balick R Arvigo et al ldquoScreening of the topicalanti-inflammatory activity of some Central American plantsrdquoJournal of Ethnopharmacology vol 81 no 2 pp 211ndash215 2002
[16] A Camporese M J Balick R Arvigo et al ldquoScreening ofanti-bacterial activity of medicinal plants from Belize (Central
8 Evidence-Based Complementary and Alternative Medicine
America)rdquo Journal of Ethnopharmacology vol 87 no 1 pp 103ndash107 2003
[17] G Mena-Rejon E Caamal-Fuentes Z Cantillo-Ciau R Cedil-lo-Rivera J Flores-Guido and R Moo-Puc ldquoIn vitro cytotoxicactivity of nine plants used in Mayan traditional medicinerdquoJournal of Ethnopharmacology vol 121 no 3 pp 462ndash465 2009
[18] V Jimenez-Suarez A Nieto-Camacho M Jimenez-Estradaand B Alvarado Sanchez ldquoAnti-inflammatory free radical scav-enging and alpha-glucosidase inhibitory activities of Hameliapatens and its chemical constituentsrdquo Pharmaceutical Biologyvol 54 no 9 pp 1822ndash1830 2016
[19] A Andrade-Cetto S Escandon-Rivera and V Garcia-LunaldquoHypoglycemic effect of Hamelia patens Jacq aerial part inSTZ-NA-induced diabetic ratsrdquo Pharmacologyonline vol 3 pp65ndash69b 2015
[20] A Ahmad A Pandurangan N Singh and P Ananad ldquoA minireview on chemistry and biology of Hamelia Patens (Rubi-aceae)rdquo Pharmacognosy Journal vol 4 no 29 pp 1ndash4 2012
[21] J Bano S Santra and EMenghani ldquoHamelia patens a potentialplant from Rubiaceae family A reviewrdquo International Journalof Scientific amp Engineering Research vol 6 no 12 pp 960ndash9732015
[22] E C Cruz and A Andrade-Cetto ldquoEthnopharmacological fieldstudy of the plants used to treat type 2 diabetes among theCakchiquels in Guatemalardquo Journal of Ethnopharmacology vol159 pp 238ndash244 2015
[23] D A Sampietro M A Sgariglia J R Soberon E N QuirogaandM A Vattuone ldquoColorimetric reactionsrdquo in Isolation Iden-tification and Characterization of AllelochemicalsNatural Prod-ucts chapter 4 pp 78ndash82 2009
[24] H P S Makkar and K Becker ldquoVanillin-HCl method for con-densed tannins effect of organic solvents used for extraction oftanninsrdquo Journal of Chemical Ecology vol 19 no 4 pp 613ndash6211993
[25] B A Cevallos-Casals and L Cisneros-Zevallos ldquoStoichiometricand kinetic studies of phenolic antioxidants from Andean pur-ple corn and red-fleshed sweetpotatordquo Journal of Agriculturaland Food Chemistry vol 51 no 11 pp 3313ndash3319 2003
[26] P Salehi B Asghari M A Esmaeili H Dehghan and I Ghazildquo120572-Glucosidase and120572-amylase inhibitory effect and antioxidantof ten plant extracts traditionally used in Iran for diabetesrdquoJournal of Medicinal Plants Research vol 7 no 6 pp 257ndash2662013
[27] OECDOCDE 423 OECD Guideline for testing of chemicalsAcute Oral Toxicity ndash Acute Toxic Class Method EnvironmentDirectorate Organisation for Economic Co-Operation and De-velopment Paris France 2001
[28] B L Furman ldquoStreptozotocin-induced diabetic models in miceand ratsrdquo Current Protocols in Pharmacology vol 70 pp 5471ndash54720 2015
[29] I J Flores-Sanchez and A C Ramos-Valdivia ldquoA review frompatents inspired by two plant genera Uncaria and HameliardquoPhytochemistry Reviews vol 16 no 4 pp 693ndash723 2017
[30] R Vinayagam and B Xu ldquoAntidiabetic properties of dietaryflavonoids A cellular mechanism reviewrdquo Journal of Nutritionand Metabolism vol 12 no 60 pp 1ndash20 2015
[31] S Marella ldquoFlavonoids-Themost potent poly-phenols as antidi-abetic agents an overviewrdquoModernApproaches in Drug Design-ing vol 1 no 3 pp 2ndash5 2017
[32] AUllah A Khan and I Khan ldquoDiabetesmellitus and oxidativestress-A concise reviewrdquo Saudi Pharmaceutical Journal vol 24pp 547ndash553 2016
[33] D Shasha CMagogo and P Dzomba ldquoReversed phase HPLC-UV Quantitation of BHA BHT and TBHQ in food items soldin bindura supermarkets Zimbabwerdquo International ResearchJournal of Pure amp Applied Chemistry vol 4 no 5 pp 578ndash5842014
[34] M Vessal M Hemmati and M Vasei ldquoAntidiabetic effects ofquercetin in streptozocin-induced diabetic ratsrdquo ComparativeBiochemistry and Physiology Part C Toxicology amp Pharmacol-ogy vol 135 no 3 pp 357ndash364 2003
[35] P-C Hsieh G-J Huang Y-L Ho et al ldquoActivities of antioxi-dants 120572-glucosidase inhibitors and aldose reductase inhibitorsof the aqueous extracts of four Flemingia species in TaiwanrdquoBotanical Studies vol 51 no 3 pp 293ndash302 2010
[36] W Kang Y Song and X Gu ldquo120572-glucosidase inhibitory in vitroand antidiabetic activity in vivo ofOsmanthus fragransrdquo Journalof Medicinal Plants Research vol 6 no 14 pp 2850ndash2856 2012
[37] N Zahratunnisa B Elya and A Noviani ldquoInhibition of Alpha-glucosidase and antioxidant test of stem bark extracts ofGarcinia fruticosa Lauterbrdquo Pharmacognosy Journal vol 9 no2 pp 273ndash275 2017
[38] J E Wong C Rubio A Reyes C N Aguilar and M L Carril-lo ldquoPhenolic content and antibacterial activity of extracts ofHamelia patens obtained by different extraction methodsrdquo Bra-zilian Journal of Microbiology vol 49 no 3 pp 656ndash661 2017
[39] G Oboh O M Agunloye S A Adefegha A J Akinyemi andA O Ademiluyi ldquoCaffeic and chlorogenic acids inhibit key en-zymes linked to type 2 diabetes (in vitro) a comparative studyrdquoJournal of Basic and Clinical Physiology and Pharmacology vol26 no 2 pp 165ndash170 2015
[40] S-Y Chiou J-M Sung P-W Huang and S-D Lin ldquoAntiox-idant Antidiabetic and Antihypertensive Properties of Echi-nacea purpurea Flower Extract and Caffeic Acid DerivativesUsing in Vitro Modelsrdquo Journal of Medicinal Food vol 20 no2 pp 171ndash179 2017
[41] C-M Ma M Hattori M Daneshtalab and L Wang ldquoChloro-genic acid derivatives with alkyl chains of different lengths andorientations Potent120572-glucosidase inhibitorsrdquo Journal of Medic-inal Chemistry vol 51 no 19 pp 6188ndash6194 2008
[42] C Tan QWang C Luo S Chen Q Li and P Li ldquoYeast 120572-glu-cosidase inhibitory phenolic compounds isolated from Gynuramedica leafrdquo International Journal of Molecular Sciences vol 14no 2 pp 2551ndash2558 2013
[43] A I Suarez B Diaz S Tillett E Valdivieso and R S Com-pagnone ldquoLeishmanicidal activity of alkaloids from Hameliapatensrdquo Ciencia vol 16 no 2 pp 148ndash155 2008
[44] C Gutierrez-Rodelo A Roura-Guiberna and J A Olivares-Reyes ldquoMecanismos moleculares de la resistencia a la insulinauna actualizacionrdquo Gaceta Medica de Mexico vol 153 pp 215ndash228 2017
[45] J Rojas V Berm E Leal V Bermudez et al ldquoInsulinoresisten-cia e hiperinsulinemia como factores de riesgo para enfermedadcardivascularrdquo Revista Latinoamericana de Hipertension vol 2no 6 pp 179ndash190 2007
[46] SABamanikar A A Bamanikar andAArora ldquoStudy of serumurea and creatinine in diabetic and non-diabetic patients in atertiary teaching hospitalrdquoThe Journal of Medical Research vol2 no 1 pp 12ndash15 2016
[47] M Music A Dervisevic E Pepic et al ldquoMetabolic Syndromeand Serum Liver Enzymes Level at Patients with Type 2Diabetes MellitusrdquoMedical Archives vol 69 no 4 pp 251ndash2552015
Evidence-Based Complementary and Alternative Medicine 9
[48] S Kazemi S Asgary J Moshtaghian et al ldquoLiver-protectiveeffects of hydroalcoholic extract of Allium hirtifolium Boiss inrats with alloxan-induced diabetes mellitusrdquo ARYA Atheroscle-rosis Journal vol 6 no 1 pp 11ndash15 2010
[49] G A Bonneau M S Castillo Rascon R A Sanchez W RPedrozo and C Castro Olivera ldquoColesterol-IDL y parametroslipıdicos en diabeticos tipo 2rdquo Revista Argentina de Endocri-nologıa y Metabolismo vol 44 no 4 pp 215ndash222 2007
[50] A Husni D Purwanti and Ustadi ldquoBlood glucose level andlipid profile of streptozotocin-induceddiabetes rats treatedwithsodiumalginate from Sargassum crassifoliumrdquo Journal of Biolog-ical Sciences vol 16 no 3 pp 58ndash64 2016
[51] S Palazhy and V Viswanathan ldquoLipid abnormalities in type 2diabetes mellitus patients with overt nephropathyrdquo Diabetes ampMetabolism Journal vol 41 no 2 pp 128ndash134 2017
[52] J J DiNicolantonio J Bhutani and J H OrsquoKeefe ldquoAcarbosesafe and effective for lowering postprandial hyperglycaemia andimproving cardiovascular outcomesrdquo Open Heart vol 2 no 12015
[53] S Ogawa K Takaeuchi and S Ito ldquoAcarbose lowers serum tri-glyceride and postprandial chylomicron levels in type 2 dia-betesrdquo Diabetes Obesity and Metabolism vol 6 no 5 pp 384ndash390 2004
[54] L Liu S Shan K Zhang Z-Q Ning X-P Lu and Y-Y ChengldquoNaringenin andhesperetin two flavonoids derived fromCitrusaurantium up-regulate transcription of adiponectinrdquoPhytother-apy Research vol 22 no 10 pp 1400ndash1403 2008
[55] E P Cai and J-K Lin ldquoEpigallocatechin gallate (EGCG) andrutin suppress the glucotoxicity through activating IRS2 andAMPK signaling in rat pancreatic 120573 cellsrdquo Journal of Agricul-tural and Food Chemistry vol 57 no 20 pp 9817ndash9827 2009
Data are expressed as the mean plusmn SD ECat equivalents of catechin EGA equivalents of gallic acid EQ equivalents of quercetin Themean values labeled withuppercase letters differ significantly compared to the control
Table 2 DPPH radical scavenging activity and 120572-glucosidase inhibition by methanolic extracts ofH patens
of their EC50 values (507 and 517120583gmL respectively) How-ever both extracts presented a better EC50 value than BHTwhich is a synthetic phenolic antioxidant currently used infood despite the evidence that it causes enzymatic or lipidalterations as well as carcinogenic effects and mutagenicactivity [32 33]120572-Glucosidase inhibitors are targeted to delay carbohy-
drate absorption and reduce postprandial glucose Severalphenolic compounds containing a flavonoid nucleus in theirstructure are reportedly useful for the control of diabetes byimproving glucose and lipid levels [30] Moreover studieshave demonstrated that quercetin epicatechin kaempferoland naringenin effectively inhibit the 120572-glucosidase enzyme[31 34] Consequently the 120572-glucosidase inhibition assay wasthe first stage in the identification of antidiabetic agentsHigh 120572-glucosidase inhibitory activity was found in bothextracts (FME IC50 = 678 and CME IC50 = 783120583gmLTable 2) Thus the extracts are more active than acar-bose (IC50= 49966 120583gmL) Contrastingly our low polarityextracts showed inhibitory activity of less than 10 at aconcentration of 4000120583gmL However in 2016 Jimenez [18]established that hexanic and methanol-ethyl acetate extractsof H patens manifest better 120572-glucosidase inhibitory effectwith IC50 = 2607120583gml and 3018120583gml respectively thandichloromethane-ethyl acetate and methanol-water extractswhich did not exhibit activity Their results coincide withthose reported by other authors who have demonstrated thathigh polarity extracts are more active than acarbose [35ndash37]
The compounds in the extracts which produce antag-onistic activity are polar in nature and a chromatographicHPLC analysis was performed to determine their compo-sition and identify the possible active principles By usingdifferent standards (Figure 1) the resulting retention times(Table 3) reveal that chlorogenic acid is the main componentin both extracts constituting 135 of CME and 195 of
FME Catechin and epicatechin were also detected in theextracts concurring with Wongrsquos description of H patensin 2017 [38] Looking for a possible explanation for thisbehaviour we decided to evaluate the inhibitory activity forthe compounds epicatechin chlorogenic acid and quercetinpreviously identified in the extractsThefirst two showed IC50values that were higher than those obtained from FME andCME with IC50 = 2826 120583gmL for epicatechin and IC50 ge3000120583gmL for chlorogenic acid Notably our results forchlorogenic acid differ from those previously reported (IC50= 24 461 1000 and gt2000120583gmL) [39ndash42] Although chloro-genic acid is the main component the antagonistic activityagainst the enzyme is low which does not correlate with theactivity that both extracts manifested Quercetin previouslyisolated fromH patens presented a similar inhibitory activity(IC50 = 27120583gmL) to the one described by Indrianingsihin 2015 (IC50 = 42120583gmL) [2] A peak at 14025 minuteswas displayed in the spectrum for CME (107) and at14075 minutes for FME (45) Likewise the peak with aretention time of 1921 indicated a higher concentration forCME (59) than for FME (09) As these two componentsof low polarity may partly explain the perceived differencein activity further research is necessary to isolate them forfurther structural characterization and biological evaluationUsing column chromatography a light brown amorphoussolid with a melting point of 236-238∘C was isolated fromCME Nuclear magnetic resonance (NMR) analysis made itpossible to identify a 3-flavonol skeleton and the 1H and 13CNMR spectra and two-dimensional spectra established thestructure as (-)-epicatechin concurring with a recent reportfor H patens [43]The chemical shifts for this compound inthe 1H and 13C NMR are presented in Table 4
The second stage of the current study intended to explorethe antihyperglycemic effect of the extracts in an in vivomurinemodelThe extractswere administered 15 times to rats
Evidence-Based Complementary and Alternative Medicine 5
140
120
100
80
60
40
20
0
minus20
mAU
4
1
5
5 10 15 20 25
lowast
+
min
(a) FME
175
150
100
125
75
50
25
0
minus25
mAU
4
1
5
5 10 15 20 25
lowast
+
min
(b) CME
Figure 1 HPLC chromatogram of fractional (FME) and crude (CME) methanolic extracts of H patens (1) chlorogenic acid (4) catechinand (5) (-) epicatechin lowastDifferent component +Different concentration
Table 3 The retention time of phenolic compounds at 290 nm
with hyperglycemia induced by STZ (glucose levels higher300mgdL) FME and CME extracts produced a reduction in
Table 5 Glucose levels and serum insulin in experimental rats
Sample SG (mgdL) SIN (mUImL)HC 11270 plusmn 2211 210 plusmn 127DC 43750 plusmn 071 250 plusmn 131DCV 42900 plusmn 283 195 plusmn 007Metformin 12390 plusmn 3399lowastlowastlowast 174 plusmn 006Acarbose 14230 plusmn 3199lowastlowastlowast 215 plusmn 039CME 150 9325 plusmn 2022lowastlowastlowast 163 plusmn 071CME 75 15600 plusmn 1513lowastlowastlowast 133 plusmn 015CME 35 15300 plusmn 3036lowastlowastlowast 232 plusmn 132FME 150 11750 plusmn 4135lowastlowastlowast 147 plusmn 055FME 75 16880 plusmn 1167lowastlowastlowast 200 plusmn 087FME 35 16200 plusmn 2877lowastlowastlowast 275 plusmn 035(SG) concentration of glucose (SIN) insulin HC healthy control DCdiabetic control DCV diabetic control vehicle CME 150 crude methanolicextract at a concentration of 150 mgkg CME 75 crude methanolic extractat a concentration of 75 mgkg CME 35 crude methanolic extract at aconcentration of 35 mgkg FME 150 fractional methanolic extract at aconcentration of 150 mgkg FME 75 fractional methanolic extract at aconcentration of 75 mgkg FME 35 fractional methanolic extract at aconcentration of 35 mgkg For the corresponding mean plusmn SD P lt 0001compared to the healthy control and lowastlowastlowastP lt 001 compared to the diabeticcontrol
glucose concentration (Table 5) which reached anormal levelafter 10 administrations At a concentration of 150mgkgextracts showed a greater decrease in glucose level Likewisethe extracts and metformin exhibited a decrease in seruminsulin compared to the diabetic control However in thediabetic control as well as in acarbose this behaviour wasnot observed Some studies explain that this increase ininsulin levels is due to resistance to insulin which leadsto peripheral hyperglycemia and major insulin secretion aprocess known as compensatory hyperinsulinemia [44 45]Levels of serum creatinine (SCr) serum urea (SUr) andblood urea nitrogen (BUN) were measured in order to assessthe effects on the kidney as this damage is one of the maincollateral effects of hyperglycemia The malfunction of thisorgan leads to an increase in metabolic waste products in theblood [46] The 150mgkg concentration of CME and FMEresults in the best protective effect in relation to SCr and no
6 Evidence-Based Complementary and Alternative Medicine
Table 6 Kidney and liver profile of experimental rats
Sample SCr (mgdL) SUr (mgdL) BUN (mgdL) AlT (mgdL) AsT (120583UmL)HC 120 plusmn 003 4783 plusmn 1520 2394 plusmn 650 6533 plusmn 2172 15620 plusmn 6050DC 105 plusmn 002 8250 plusmn 2469plusmn 3853 plusmn 1149 9933 plusmn 2627 20850 plusmn 9326DCV 075 plusmn 001 7600 plusmn 2713 3208 plusmn 1624 6133 plusmn 1320 18880 plusmn 8149Metformin 074 plusmn 002 3450 plusmn 1063z 2053 plusmn 504z 6550 plusmn 1222 14540 plusmn 2660Acarbose 083 plusmn 031 4833 plusmn 153 2267 plusmn 116 5950 plusmn 1457 13600 plusmn 3617CME 150 063 plusmn 002 3125 plusmn 171zzz 1455 plusmn 081z 7325 plusmn 2468 13800 plusmn 2207CME 75 090 plusmn 010 4300 plusmn 361z 2007 plusmn 165 6967 plusmn 1115 13930 plusmn 3118CME 35 137 plusmn 038 5433 plusmn 752 2650 plusmn 366 7325 plusmn 1406 15780 plusmn 2849FME 150 067 plusmn 005 3150 plusmn 407zzz 1468 plusmn 239zz 4550 plusmn 507z 14100 plusmn 3640FME 75 120 plusmn 032 5600 plusmn 577 2468 plusmn 402 7625 plusmn 1928 13630 plusmn 2229FME 35 110 plusmn 014 4700 plusmn 1086z 2193 plusmn 507 7675 plusmn 2604 13400 plusmn 3123(SCr) serum creatinine (SUr) serumurea (BUN) bloodurea nitrogen (AlT) alanine aminotransferase (AsT) aspartate aminotransferase HC healthy controlDC diabetic control DCV diabetic control vehicle CME 150 crude methanolic extract at a concentration of 150 mgkg CME 75 crude methanolic extract ata concentration of 75 mgkg CME 35 crude methanolic extract at a concentration of 35 mgkg FME 150 fractional methanolic extract at a concentration of150 mgkg FME 75 fractional methanolic extract at a concentration of 75 mgkg FME 35 fractional methanolic extract at a concentration of 35 mgkg Forthe corresponding mean plusmn SD plusmnP lt 005 compared to health control zP lt 005 compared to the diabetic control zzP lt 005 compared to the diabetic controland zzzP lt 005 compared to the diabetic control
Table 7 Lipid profile (mgdL) among experimental rats
FME 75 5975 plusmn 1242 7650 plusmn 2869 2667 plusmn 306 2300 plusmn 100FME 35 6075 plusmn 900 7575 plusmn 4842 2967 plusmn 551z 2233 plusmn 351(TC) total cholesterol (Tg) triglycerides (LDL) low-density lipoproteins (HDL) high-density lipoproteins HC healthy control DC diabetic control DCVdiabetic control vehicle CME 150 crudemethanolic extract at a concentration of 150 mgkg CME 75 crude methanolic extract at a concentration of 75 mgkgCME 35 crude methanolic extract at a concentration of 35 mgkg FME 150 fractional methanolic extract at a concentration of 150 mgkg FME 75 fractionalmethanolic extract at a concentration of 75 mgkg FME 35 fractional methanolic extract at a concentration of 35 mgkg For the corresponding mean plusmn SD zPlt 005 compared to the diabetic control zzP lt 005 compared to the diabetic control plusmnP lt 005 compared to health control 998771P lt 005 compared to acarboselozP lt 005 compared to metformin
difference was observed between the two extracts (Table 6)Enzymes AlT and AsT are an indicator of liver injury asenzymes leaking from cytosol liver cells into bloodstreamcorrelate with insulin resistance diabetes and inflammatoryprocesses of the liver [47 48] FME induced a markeddecrease in AlT making further histopathological studiesnecessary to confirm whether there is protection or damageto the liver as a result of the treatments The effect of theextracts on the lipid profile was also examined (Table 7)Patients with DM type 2 have been characterized by hightriglycerides levels low high-density lipoprotein cholesterol(HDL) levels normal low-density lipoprotein (LDL) levelsand normal or slightly increased total cholesterol levels [49ndash51] This characteristic was observed in the group treatedwith extracts and diabetic control where no significantdifferences were evident between treatments in terms of
total cholesterol nevertheless a sharp decline in triglyceridevalue was perceived in the acarbose and CME treatments(75 and 35mgkg) The capacity of acarbose for loweringtriglycerides has been described previously finding that200mg constitutes an effective dose for reducing the riskof cardiovascular events in humans implying a minimalrisk of hypoglycemia [52 53] Various studies have providedevidence that phenolic compounds such as flavonoids may beinvolved in decreased glucose levels in the in vivo model Ithas been suggested that the flavonoids naringenin and hes-peretin may produce antiatherogenic effects partly by meansof the activation of the peroxisome proliferator activatedreceptor gamma PPAR-120574 and the upregulation of adiponectinexpression in adipocytes Numerous reports document theantidiabetic effects of flavan-3-ols especially epigallocatechingallate (EGCG) in animals and cell-cultures EGCG can elicit
Evidence-Based Complementary and Alternative Medicine 7
various changes that are associated with beneficial effects fordiabetes including improvements in insulin secretion glu-cose uptake insulin resistance glucose tolerance oxidativestress inflammation and mitochondrial function [54 55]CME and FME comprise amixture of compounds and appearto inhibit the 120572-glucosidase enzyme however the inhibitoryactivity of the identified compounds suggests that this is notthe only way to reduce hyperglycemia As this is a mixtureof multitarget molecules it is impossible to assess the anti-hyperglycemic mechanism as this would imply identifyingthe two additional compounds and performing complemen-tary studies For the acute toxicity study which involved dos-ing ge2000mgkg there was no apparent mortality suggest-ing low toxicity within category 5 according to the OECDGlobally Harmonized Classification System (GHS) (2014)[27] This is advantageous should you wish to concoct anherbal preparation
4 Conclusions
These results reveal that extracts of Hamelia patens with ahigh content of phenolic compounds elicit 120572-glucosidaseinhibition and an antihyperglycemic effect At a concentrationof 150mgkg they produce an equivalent effect tometforminEpicatechin and chlorogenic acid contribute to the antihyper-glycemic activity ofH patens however it is necessary to carryout the structural identification of any other componentspresent Further research is necessary to elucidate the activitymechanisms
Data Availability
The data used to support the findings of this study are avail-able from the corresponding author upon request
Conflicts of Interest
The authors declare that there are no conflicts of interest re-garding the publication of this paper
Acknowledgments
The authors are grateful for technical support provided bythe personnel at the Laboratory of Natural Products andMolecular Biology of the ENMyH at IPN Catalina Rugerio-Escalona thanks CONACYT for the graduate scholarshipsawarded (264517) and SIP-IPN (BEIFI) for a complementaryscholarship Cynthia Ordaz-Pichardo Fabiola E Jimenez-Montejo Ignacio EMaldonado-MendozaMarıa del CarmenCruz-Lopez and Victor E Lopez-y-Lopez are Fellows of theEstımulos al Desempeno de los Investigadores (EDI)-IPNCynthia Ordaz-Pichardo Ignacio E Maldonado-MendozaMarıa del Carmen Cruz-Lopez and Victor E Lopez-y-Lopez thank the Comision de Operacion y Fomento deActividades Academicas (COFAA) an IPN program thatprovides support to researchers This work was financiallysupported by a project (20161781) from the Secretarıa deInvestigacion y Posgrado Instituto Politecnico Nacional
References
[1] F A Matough S B Budin Z A Hamid N Alwahaibi and JMohamed ldquoThe role of oxidative stress and antioxidants in dia-betic complicationsrdquo SQU Medical Journal vol 12 no 1 pp 5ndash18 2012
[2] AW Indrianingsih S Tachibana R TDewi andK Itoh ldquoAnti-oxidant and 120572-glucosidase inhibitor activities of natural com-pounds isolated fromQuercus gilva Blume leavesrdquoAsian PacificJournal of Tropical Biomedicine vol 5 no 9 pp 748ndash755 2015
[3] M Gondi and U J S Prasada Rao ldquoEthanol extract of mango(Mangifera indica L) peel inhibits 120572-amylase and 120572-glucosidaseactivities and ameliorates diabetes related biochemical param-eters in streptozotocin (STZ)-induced diabetic ratsrdquo Journal ofFood Science andTechnology vol 52 no 12 pp 7883ndash7893 2015
[4] ARayarandRManivannan ldquoIn-vitro alpha-amylase and alpha-glucosidase inhibition activity of umbelliferone and beta-ionone isolated from Coriandrum sativum LinnrdquoWorld Journalof Pharmacy and Pharmaceutical Sciences vol 5 no 1 pp 1280ndash1289 2016
[5] Atlas de la Diabetes de la FID-Octava edicion InternationalDiabetes Federation 2017
[6] American Diabetes Association ldquoPharmacologic Approachesto Glycemic Treatmentrdquo Journal of Clinical and Applied Resarchand Education vol 40 no 1 pp S64ndashS76 2017
[7] J J Marın-Penalver I Martın-Timon C Sevillano-Collantesand F J del Canizo-Gomez ldquoUpdate on the treatment of type2 diabetes mellitusrdquoWorld Journal of Diabetes vol 7 no 17 pp354ndash395 2016
[8] H-Y Chang M Wallis and E Tiralongo ldquoUse of comple-mentary and alternative medicine among people living withdiabetes literature reviewrdquo Journal of AdvancedNursing vol 58no 4 pp 307ndash319 2007
[9] A B Medagama and R Bandara ldquoThe use of Complementaryand AlternativeMedicines (CAMs) in the treatment of diabetesmellitus Is continued use safe and effectiverdquo Nutrition Journalvol 13 no 1 2014
[10] B Baharvand-Ahmadi M Bahmani P Tajeddini N Naghdiand M Rafieian-Kopaei ldquoAn ethno-medicinal study of medic-inal plants used for the treatment of diabetesrdquo Journal ofNephropathology vol 5 no 1 pp 44ndash50 2016
[11] S Verma M Gupta H Popli and G Aggarwal ldquoDiabetesmellitus treatment using herbal drugsrdquo International Journal ofPhytomedicine vol 10 no 1 pp 1ndash10 2018
[12] B Ovalle-Magallanes O N Medina-Campos J Pedraza-Cha-verri and R Mata ldquoHypoglycemic and antihyperglycemic ef-fects of phytopreparations and limonoids from Swietenia humi-lisrdquo Phytochemistry vol 110 pp 111ndash119 2015
[13] S Singh andM Vyas ldquoComparative in-vitro biological study ofaerial parts of plant Hamelia patensrdquo International Journal ofPharmaceutical Sciences and Research vol 7 no 4 pp 1793ndash1808 2016
[14] F Ruiz-Teran A Medrano-Martınez and A Navarro-OcanaldquoAntioxidant and free radical scavenging activities of plantextracts used in traditional medicine in Mexicordquo African Jour-nal of Biotechnology vol 7 no 12 pp 1886ndash1893 2008
[15] S Sosa M J Balick R Arvigo et al ldquoScreening of the topicalanti-inflammatory activity of some Central American plantsrdquoJournal of Ethnopharmacology vol 81 no 2 pp 211ndash215 2002
[16] A Camporese M J Balick R Arvigo et al ldquoScreening ofanti-bacterial activity of medicinal plants from Belize (Central
8 Evidence-Based Complementary and Alternative Medicine
America)rdquo Journal of Ethnopharmacology vol 87 no 1 pp 103ndash107 2003
[17] G Mena-Rejon E Caamal-Fuentes Z Cantillo-Ciau R Cedil-lo-Rivera J Flores-Guido and R Moo-Puc ldquoIn vitro cytotoxicactivity of nine plants used in Mayan traditional medicinerdquoJournal of Ethnopharmacology vol 121 no 3 pp 462ndash465 2009
[18] V Jimenez-Suarez A Nieto-Camacho M Jimenez-Estradaand B Alvarado Sanchez ldquoAnti-inflammatory free radical scav-enging and alpha-glucosidase inhibitory activities of Hameliapatens and its chemical constituentsrdquo Pharmaceutical Biologyvol 54 no 9 pp 1822ndash1830 2016
[19] A Andrade-Cetto S Escandon-Rivera and V Garcia-LunaldquoHypoglycemic effect of Hamelia patens Jacq aerial part inSTZ-NA-induced diabetic ratsrdquo Pharmacologyonline vol 3 pp65ndash69b 2015
[20] A Ahmad A Pandurangan N Singh and P Ananad ldquoA minireview on chemistry and biology of Hamelia Patens (Rubi-aceae)rdquo Pharmacognosy Journal vol 4 no 29 pp 1ndash4 2012
[21] J Bano S Santra and EMenghani ldquoHamelia patens a potentialplant from Rubiaceae family A reviewrdquo International Journalof Scientific amp Engineering Research vol 6 no 12 pp 960ndash9732015
[22] E C Cruz and A Andrade-Cetto ldquoEthnopharmacological fieldstudy of the plants used to treat type 2 diabetes among theCakchiquels in Guatemalardquo Journal of Ethnopharmacology vol159 pp 238ndash244 2015
[23] D A Sampietro M A Sgariglia J R Soberon E N QuirogaandM A Vattuone ldquoColorimetric reactionsrdquo in Isolation Iden-tification and Characterization of AllelochemicalsNatural Prod-ucts chapter 4 pp 78ndash82 2009
[24] H P S Makkar and K Becker ldquoVanillin-HCl method for con-densed tannins effect of organic solvents used for extraction oftanninsrdquo Journal of Chemical Ecology vol 19 no 4 pp 613ndash6211993
[25] B A Cevallos-Casals and L Cisneros-Zevallos ldquoStoichiometricand kinetic studies of phenolic antioxidants from Andean pur-ple corn and red-fleshed sweetpotatordquo Journal of Agriculturaland Food Chemistry vol 51 no 11 pp 3313ndash3319 2003
[26] P Salehi B Asghari M A Esmaeili H Dehghan and I Ghazildquo120572-Glucosidase and120572-amylase inhibitory effect and antioxidantof ten plant extracts traditionally used in Iran for diabetesrdquoJournal of Medicinal Plants Research vol 7 no 6 pp 257ndash2662013
[27] OECDOCDE 423 OECD Guideline for testing of chemicalsAcute Oral Toxicity ndash Acute Toxic Class Method EnvironmentDirectorate Organisation for Economic Co-Operation and De-velopment Paris France 2001
[28] B L Furman ldquoStreptozotocin-induced diabetic models in miceand ratsrdquo Current Protocols in Pharmacology vol 70 pp 5471ndash54720 2015
[29] I J Flores-Sanchez and A C Ramos-Valdivia ldquoA review frompatents inspired by two plant genera Uncaria and HameliardquoPhytochemistry Reviews vol 16 no 4 pp 693ndash723 2017
[30] R Vinayagam and B Xu ldquoAntidiabetic properties of dietaryflavonoids A cellular mechanism reviewrdquo Journal of Nutritionand Metabolism vol 12 no 60 pp 1ndash20 2015
[31] S Marella ldquoFlavonoids-Themost potent poly-phenols as antidi-abetic agents an overviewrdquoModernApproaches in Drug Design-ing vol 1 no 3 pp 2ndash5 2017
[32] AUllah A Khan and I Khan ldquoDiabetesmellitus and oxidativestress-A concise reviewrdquo Saudi Pharmaceutical Journal vol 24pp 547ndash553 2016
[33] D Shasha CMagogo and P Dzomba ldquoReversed phase HPLC-UV Quantitation of BHA BHT and TBHQ in food items soldin bindura supermarkets Zimbabwerdquo International ResearchJournal of Pure amp Applied Chemistry vol 4 no 5 pp 578ndash5842014
[34] M Vessal M Hemmati and M Vasei ldquoAntidiabetic effects ofquercetin in streptozocin-induced diabetic ratsrdquo ComparativeBiochemistry and Physiology Part C Toxicology amp Pharmacol-ogy vol 135 no 3 pp 357ndash364 2003
[35] P-C Hsieh G-J Huang Y-L Ho et al ldquoActivities of antioxi-dants 120572-glucosidase inhibitors and aldose reductase inhibitorsof the aqueous extracts of four Flemingia species in TaiwanrdquoBotanical Studies vol 51 no 3 pp 293ndash302 2010
[36] W Kang Y Song and X Gu ldquo120572-glucosidase inhibitory in vitroand antidiabetic activity in vivo ofOsmanthus fragransrdquo Journalof Medicinal Plants Research vol 6 no 14 pp 2850ndash2856 2012
[37] N Zahratunnisa B Elya and A Noviani ldquoInhibition of Alpha-glucosidase and antioxidant test of stem bark extracts ofGarcinia fruticosa Lauterbrdquo Pharmacognosy Journal vol 9 no2 pp 273ndash275 2017
[38] J E Wong C Rubio A Reyes C N Aguilar and M L Carril-lo ldquoPhenolic content and antibacterial activity of extracts ofHamelia patens obtained by different extraction methodsrdquo Bra-zilian Journal of Microbiology vol 49 no 3 pp 656ndash661 2017
[39] G Oboh O M Agunloye S A Adefegha A J Akinyemi andA O Ademiluyi ldquoCaffeic and chlorogenic acids inhibit key en-zymes linked to type 2 diabetes (in vitro) a comparative studyrdquoJournal of Basic and Clinical Physiology and Pharmacology vol26 no 2 pp 165ndash170 2015
[40] S-Y Chiou J-M Sung P-W Huang and S-D Lin ldquoAntiox-idant Antidiabetic and Antihypertensive Properties of Echi-nacea purpurea Flower Extract and Caffeic Acid DerivativesUsing in Vitro Modelsrdquo Journal of Medicinal Food vol 20 no2 pp 171ndash179 2017
[41] C-M Ma M Hattori M Daneshtalab and L Wang ldquoChloro-genic acid derivatives with alkyl chains of different lengths andorientations Potent120572-glucosidase inhibitorsrdquo Journal of Medic-inal Chemistry vol 51 no 19 pp 6188ndash6194 2008
[42] C Tan QWang C Luo S Chen Q Li and P Li ldquoYeast 120572-glu-cosidase inhibitory phenolic compounds isolated from Gynuramedica leafrdquo International Journal of Molecular Sciences vol 14no 2 pp 2551ndash2558 2013
[43] A I Suarez B Diaz S Tillett E Valdivieso and R S Com-pagnone ldquoLeishmanicidal activity of alkaloids from Hameliapatensrdquo Ciencia vol 16 no 2 pp 148ndash155 2008
[44] C Gutierrez-Rodelo A Roura-Guiberna and J A Olivares-Reyes ldquoMecanismos moleculares de la resistencia a la insulinauna actualizacionrdquo Gaceta Medica de Mexico vol 153 pp 215ndash228 2017
[45] J Rojas V Berm E Leal V Bermudez et al ldquoInsulinoresisten-cia e hiperinsulinemia como factores de riesgo para enfermedadcardivascularrdquo Revista Latinoamericana de Hipertension vol 2no 6 pp 179ndash190 2007
[46] SABamanikar A A Bamanikar andAArora ldquoStudy of serumurea and creatinine in diabetic and non-diabetic patients in atertiary teaching hospitalrdquoThe Journal of Medical Research vol2 no 1 pp 12ndash15 2016
[47] M Music A Dervisevic E Pepic et al ldquoMetabolic Syndromeand Serum Liver Enzymes Level at Patients with Type 2Diabetes MellitusrdquoMedical Archives vol 69 no 4 pp 251ndash2552015
Evidence-Based Complementary and Alternative Medicine 9
[48] S Kazemi S Asgary J Moshtaghian et al ldquoLiver-protectiveeffects of hydroalcoholic extract of Allium hirtifolium Boiss inrats with alloxan-induced diabetes mellitusrdquo ARYA Atheroscle-rosis Journal vol 6 no 1 pp 11ndash15 2010
[49] G A Bonneau M S Castillo Rascon R A Sanchez W RPedrozo and C Castro Olivera ldquoColesterol-IDL y parametroslipıdicos en diabeticos tipo 2rdquo Revista Argentina de Endocri-nologıa y Metabolismo vol 44 no 4 pp 215ndash222 2007
[50] A Husni D Purwanti and Ustadi ldquoBlood glucose level andlipid profile of streptozotocin-induceddiabetes rats treatedwithsodiumalginate from Sargassum crassifoliumrdquo Journal of Biolog-ical Sciences vol 16 no 3 pp 58ndash64 2016
[51] S Palazhy and V Viswanathan ldquoLipid abnormalities in type 2diabetes mellitus patients with overt nephropathyrdquo Diabetes ampMetabolism Journal vol 41 no 2 pp 128ndash134 2017
[52] J J DiNicolantonio J Bhutani and J H OrsquoKeefe ldquoAcarbosesafe and effective for lowering postprandial hyperglycaemia andimproving cardiovascular outcomesrdquo Open Heart vol 2 no 12015
[53] S Ogawa K Takaeuchi and S Ito ldquoAcarbose lowers serum tri-glyceride and postprandial chylomicron levels in type 2 dia-betesrdquo Diabetes Obesity and Metabolism vol 6 no 5 pp 384ndash390 2004
[54] L Liu S Shan K Zhang Z-Q Ning X-P Lu and Y-Y ChengldquoNaringenin andhesperetin two flavonoids derived fromCitrusaurantium up-regulate transcription of adiponectinrdquoPhytother-apy Research vol 22 no 10 pp 1400ndash1403 2008
[55] E P Cai and J-K Lin ldquoEpigallocatechin gallate (EGCG) andrutin suppress the glucotoxicity through activating IRS2 andAMPK signaling in rat pancreatic 120573 cellsrdquo Journal of Agricul-tural and Food Chemistry vol 57 no 20 pp 9817ndash9827 2009
with hyperglycemia induced by STZ (glucose levels higher300mgdL) FME and CME extracts produced a reduction in
Table 5 Glucose levels and serum insulin in experimental rats
Sample SG (mgdL) SIN (mUImL)HC 11270 plusmn 2211 210 plusmn 127DC 43750 plusmn 071 250 plusmn 131DCV 42900 plusmn 283 195 plusmn 007Metformin 12390 plusmn 3399lowastlowastlowast 174 plusmn 006Acarbose 14230 plusmn 3199lowastlowastlowast 215 plusmn 039CME 150 9325 plusmn 2022lowastlowastlowast 163 plusmn 071CME 75 15600 plusmn 1513lowastlowastlowast 133 plusmn 015CME 35 15300 plusmn 3036lowastlowastlowast 232 plusmn 132FME 150 11750 plusmn 4135lowastlowastlowast 147 plusmn 055FME 75 16880 plusmn 1167lowastlowastlowast 200 plusmn 087FME 35 16200 plusmn 2877lowastlowastlowast 275 plusmn 035(SG) concentration of glucose (SIN) insulin HC healthy control DCdiabetic control DCV diabetic control vehicle CME 150 crude methanolicextract at a concentration of 150 mgkg CME 75 crude methanolic extractat a concentration of 75 mgkg CME 35 crude methanolic extract at aconcentration of 35 mgkg FME 150 fractional methanolic extract at aconcentration of 150 mgkg FME 75 fractional methanolic extract at aconcentration of 75 mgkg FME 35 fractional methanolic extract at aconcentration of 35 mgkg For the corresponding mean plusmn SD P lt 0001compared to the healthy control and lowastlowastlowastP lt 001 compared to the diabeticcontrol
glucose concentration (Table 5) which reached anormal levelafter 10 administrations At a concentration of 150mgkgextracts showed a greater decrease in glucose level Likewisethe extracts and metformin exhibited a decrease in seruminsulin compared to the diabetic control However in thediabetic control as well as in acarbose this behaviour wasnot observed Some studies explain that this increase ininsulin levels is due to resistance to insulin which leadsto peripheral hyperglycemia and major insulin secretion aprocess known as compensatory hyperinsulinemia [44 45]Levels of serum creatinine (SCr) serum urea (SUr) andblood urea nitrogen (BUN) were measured in order to assessthe effects on the kidney as this damage is one of the maincollateral effects of hyperglycemia The malfunction of thisorgan leads to an increase in metabolic waste products in theblood [46] The 150mgkg concentration of CME and FMEresults in the best protective effect in relation to SCr and no
6 Evidence-Based Complementary and Alternative Medicine
Table 6 Kidney and liver profile of experimental rats
Sample SCr (mgdL) SUr (mgdL) BUN (mgdL) AlT (mgdL) AsT (120583UmL)HC 120 plusmn 003 4783 plusmn 1520 2394 plusmn 650 6533 plusmn 2172 15620 plusmn 6050DC 105 plusmn 002 8250 plusmn 2469plusmn 3853 plusmn 1149 9933 plusmn 2627 20850 plusmn 9326DCV 075 plusmn 001 7600 plusmn 2713 3208 plusmn 1624 6133 plusmn 1320 18880 plusmn 8149Metformin 074 plusmn 002 3450 plusmn 1063z 2053 plusmn 504z 6550 plusmn 1222 14540 plusmn 2660Acarbose 083 plusmn 031 4833 plusmn 153 2267 plusmn 116 5950 plusmn 1457 13600 plusmn 3617CME 150 063 plusmn 002 3125 plusmn 171zzz 1455 plusmn 081z 7325 plusmn 2468 13800 plusmn 2207CME 75 090 plusmn 010 4300 plusmn 361z 2007 plusmn 165 6967 plusmn 1115 13930 plusmn 3118CME 35 137 plusmn 038 5433 plusmn 752 2650 plusmn 366 7325 plusmn 1406 15780 plusmn 2849FME 150 067 plusmn 005 3150 plusmn 407zzz 1468 plusmn 239zz 4550 plusmn 507z 14100 plusmn 3640FME 75 120 plusmn 032 5600 plusmn 577 2468 plusmn 402 7625 plusmn 1928 13630 plusmn 2229FME 35 110 plusmn 014 4700 plusmn 1086z 2193 plusmn 507 7675 plusmn 2604 13400 plusmn 3123(SCr) serum creatinine (SUr) serumurea (BUN) bloodurea nitrogen (AlT) alanine aminotransferase (AsT) aspartate aminotransferase HC healthy controlDC diabetic control DCV diabetic control vehicle CME 150 crude methanolic extract at a concentration of 150 mgkg CME 75 crude methanolic extract ata concentration of 75 mgkg CME 35 crude methanolic extract at a concentration of 35 mgkg FME 150 fractional methanolic extract at a concentration of150 mgkg FME 75 fractional methanolic extract at a concentration of 75 mgkg FME 35 fractional methanolic extract at a concentration of 35 mgkg Forthe corresponding mean plusmn SD plusmnP lt 005 compared to health control zP lt 005 compared to the diabetic control zzP lt 005 compared to the diabetic controland zzzP lt 005 compared to the diabetic control
Table 7 Lipid profile (mgdL) among experimental rats
FME 75 5975 plusmn 1242 7650 plusmn 2869 2667 plusmn 306 2300 plusmn 100FME 35 6075 plusmn 900 7575 plusmn 4842 2967 plusmn 551z 2233 plusmn 351(TC) total cholesterol (Tg) triglycerides (LDL) low-density lipoproteins (HDL) high-density lipoproteins HC healthy control DC diabetic control DCVdiabetic control vehicle CME 150 crudemethanolic extract at a concentration of 150 mgkg CME 75 crude methanolic extract at a concentration of 75 mgkgCME 35 crude methanolic extract at a concentration of 35 mgkg FME 150 fractional methanolic extract at a concentration of 150 mgkg FME 75 fractionalmethanolic extract at a concentration of 75 mgkg FME 35 fractional methanolic extract at a concentration of 35 mgkg For the corresponding mean plusmn SD zPlt 005 compared to the diabetic control zzP lt 005 compared to the diabetic control plusmnP lt 005 compared to health control 998771P lt 005 compared to acarboselozP lt 005 compared to metformin
difference was observed between the two extracts (Table 6)Enzymes AlT and AsT are an indicator of liver injury asenzymes leaking from cytosol liver cells into bloodstreamcorrelate with insulin resistance diabetes and inflammatoryprocesses of the liver [47 48] FME induced a markeddecrease in AlT making further histopathological studiesnecessary to confirm whether there is protection or damageto the liver as a result of the treatments The effect of theextracts on the lipid profile was also examined (Table 7)Patients with DM type 2 have been characterized by hightriglycerides levels low high-density lipoprotein cholesterol(HDL) levels normal low-density lipoprotein (LDL) levelsand normal or slightly increased total cholesterol levels [49ndash51] This characteristic was observed in the group treatedwith extracts and diabetic control where no significantdifferences were evident between treatments in terms of
total cholesterol nevertheless a sharp decline in triglyceridevalue was perceived in the acarbose and CME treatments(75 and 35mgkg) The capacity of acarbose for loweringtriglycerides has been described previously finding that200mg constitutes an effective dose for reducing the riskof cardiovascular events in humans implying a minimalrisk of hypoglycemia [52 53] Various studies have providedevidence that phenolic compounds such as flavonoids may beinvolved in decreased glucose levels in the in vivo model Ithas been suggested that the flavonoids naringenin and hes-peretin may produce antiatherogenic effects partly by meansof the activation of the peroxisome proliferator activatedreceptor gamma PPAR-120574 and the upregulation of adiponectinexpression in adipocytes Numerous reports document theantidiabetic effects of flavan-3-ols especially epigallocatechingallate (EGCG) in animals and cell-cultures EGCG can elicit
Evidence-Based Complementary and Alternative Medicine 7
various changes that are associated with beneficial effects fordiabetes including improvements in insulin secretion glu-cose uptake insulin resistance glucose tolerance oxidativestress inflammation and mitochondrial function [54 55]CME and FME comprise amixture of compounds and appearto inhibit the 120572-glucosidase enzyme however the inhibitoryactivity of the identified compounds suggests that this is notthe only way to reduce hyperglycemia As this is a mixtureof multitarget molecules it is impossible to assess the anti-hyperglycemic mechanism as this would imply identifyingthe two additional compounds and performing complemen-tary studies For the acute toxicity study which involved dos-ing ge2000mgkg there was no apparent mortality suggest-ing low toxicity within category 5 according to the OECDGlobally Harmonized Classification System (GHS) (2014)[27] This is advantageous should you wish to concoct anherbal preparation
4 Conclusions
These results reveal that extracts of Hamelia patens with ahigh content of phenolic compounds elicit 120572-glucosidaseinhibition and an antihyperglycemic effect At a concentrationof 150mgkg they produce an equivalent effect tometforminEpicatechin and chlorogenic acid contribute to the antihyper-glycemic activity ofH patens however it is necessary to carryout the structural identification of any other componentspresent Further research is necessary to elucidate the activitymechanisms
Data Availability
The data used to support the findings of this study are avail-able from the corresponding author upon request
Conflicts of Interest
The authors declare that there are no conflicts of interest re-garding the publication of this paper
Acknowledgments
The authors are grateful for technical support provided bythe personnel at the Laboratory of Natural Products andMolecular Biology of the ENMyH at IPN Catalina Rugerio-Escalona thanks CONACYT for the graduate scholarshipsawarded (264517) and SIP-IPN (BEIFI) for a complementaryscholarship Cynthia Ordaz-Pichardo Fabiola E Jimenez-Montejo Ignacio EMaldonado-MendozaMarıa del CarmenCruz-Lopez and Victor E Lopez-y-Lopez are Fellows of theEstımulos al Desempeno de los Investigadores (EDI)-IPNCynthia Ordaz-Pichardo Ignacio E Maldonado-MendozaMarıa del Carmen Cruz-Lopez and Victor E Lopez-y-Lopez thank the Comision de Operacion y Fomento deActividades Academicas (COFAA) an IPN program thatprovides support to researchers This work was financiallysupported by a project (20161781) from the Secretarıa deInvestigacion y Posgrado Instituto Politecnico Nacional
References
[1] F A Matough S B Budin Z A Hamid N Alwahaibi and JMohamed ldquoThe role of oxidative stress and antioxidants in dia-betic complicationsrdquo SQU Medical Journal vol 12 no 1 pp 5ndash18 2012
[2] AW Indrianingsih S Tachibana R TDewi andK Itoh ldquoAnti-oxidant and 120572-glucosidase inhibitor activities of natural com-pounds isolated fromQuercus gilva Blume leavesrdquoAsian PacificJournal of Tropical Biomedicine vol 5 no 9 pp 748ndash755 2015
[3] M Gondi and U J S Prasada Rao ldquoEthanol extract of mango(Mangifera indica L) peel inhibits 120572-amylase and 120572-glucosidaseactivities and ameliorates diabetes related biochemical param-eters in streptozotocin (STZ)-induced diabetic ratsrdquo Journal ofFood Science andTechnology vol 52 no 12 pp 7883ndash7893 2015
[4] ARayarandRManivannan ldquoIn-vitro alpha-amylase and alpha-glucosidase inhibition activity of umbelliferone and beta-ionone isolated from Coriandrum sativum LinnrdquoWorld Journalof Pharmacy and Pharmaceutical Sciences vol 5 no 1 pp 1280ndash1289 2016
[5] Atlas de la Diabetes de la FID-Octava edicion InternationalDiabetes Federation 2017
[6] American Diabetes Association ldquoPharmacologic Approachesto Glycemic Treatmentrdquo Journal of Clinical and Applied Resarchand Education vol 40 no 1 pp S64ndashS76 2017
[7] J J Marın-Penalver I Martın-Timon C Sevillano-Collantesand F J del Canizo-Gomez ldquoUpdate on the treatment of type2 diabetes mellitusrdquoWorld Journal of Diabetes vol 7 no 17 pp354ndash395 2016
[8] H-Y Chang M Wallis and E Tiralongo ldquoUse of comple-mentary and alternative medicine among people living withdiabetes literature reviewrdquo Journal of AdvancedNursing vol 58no 4 pp 307ndash319 2007
[9] A B Medagama and R Bandara ldquoThe use of Complementaryand AlternativeMedicines (CAMs) in the treatment of diabetesmellitus Is continued use safe and effectiverdquo Nutrition Journalvol 13 no 1 2014
[10] B Baharvand-Ahmadi M Bahmani P Tajeddini N Naghdiand M Rafieian-Kopaei ldquoAn ethno-medicinal study of medic-inal plants used for the treatment of diabetesrdquo Journal ofNephropathology vol 5 no 1 pp 44ndash50 2016
[11] S Verma M Gupta H Popli and G Aggarwal ldquoDiabetesmellitus treatment using herbal drugsrdquo International Journal ofPhytomedicine vol 10 no 1 pp 1ndash10 2018
[12] B Ovalle-Magallanes O N Medina-Campos J Pedraza-Cha-verri and R Mata ldquoHypoglycemic and antihyperglycemic ef-fects of phytopreparations and limonoids from Swietenia humi-lisrdquo Phytochemistry vol 110 pp 111ndash119 2015
[13] S Singh andM Vyas ldquoComparative in-vitro biological study ofaerial parts of plant Hamelia patensrdquo International Journal ofPharmaceutical Sciences and Research vol 7 no 4 pp 1793ndash1808 2016
[14] F Ruiz-Teran A Medrano-Martınez and A Navarro-OcanaldquoAntioxidant and free radical scavenging activities of plantextracts used in traditional medicine in Mexicordquo African Jour-nal of Biotechnology vol 7 no 12 pp 1886ndash1893 2008
[15] S Sosa M J Balick R Arvigo et al ldquoScreening of the topicalanti-inflammatory activity of some Central American plantsrdquoJournal of Ethnopharmacology vol 81 no 2 pp 211ndash215 2002
[16] A Camporese M J Balick R Arvigo et al ldquoScreening ofanti-bacterial activity of medicinal plants from Belize (Central
8 Evidence-Based Complementary and Alternative Medicine
America)rdquo Journal of Ethnopharmacology vol 87 no 1 pp 103ndash107 2003
[17] G Mena-Rejon E Caamal-Fuentes Z Cantillo-Ciau R Cedil-lo-Rivera J Flores-Guido and R Moo-Puc ldquoIn vitro cytotoxicactivity of nine plants used in Mayan traditional medicinerdquoJournal of Ethnopharmacology vol 121 no 3 pp 462ndash465 2009
[18] V Jimenez-Suarez A Nieto-Camacho M Jimenez-Estradaand B Alvarado Sanchez ldquoAnti-inflammatory free radical scav-enging and alpha-glucosidase inhibitory activities of Hameliapatens and its chemical constituentsrdquo Pharmaceutical Biologyvol 54 no 9 pp 1822ndash1830 2016
[19] A Andrade-Cetto S Escandon-Rivera and V Garcia-LunaldquoHypoglycemic effect of Hamelia patens Jacq aerial part inSTZ-NA-induced diabetic ratsrdquo Pharmacologyonline vol 3 pp65ndash69b 2015
[20] A Ahmad A Pandurangan N Singh and P Ananad ldquoA minireview on chemistry and biology of Hamelia Patens (Rubi-aceae)rdquo Pharmacognosy Journal vol 4 no 29 pp 1ndash4 2012
[21] J Bano S Santra and EMenghani ldquoHamelia patens a potentialplant from Rubiaceae family A reviewrdquo International Journalof Scientific amp Engineering Research vol 6 no 12 pp 960ndash9732015
[22] E C Cruz and A Andrade-Cetto ldquoEthnopharmacological fieldstudy of the plants used to treat type 2 diabetes among theCakchiquels in Guatemalardquo Journal of Ethnopharmacology vol159 pp 238ndash244 2015
[23] D A Sampietro M A Sgariglia J R Soberon E N QuirogaandM A Vattuone ldquoColorimetric reactionsrdquo in Isolation Iden-tification and Characterization of AllelochemicalsNatural Prod-ucts chapter 4 pp 78ndash82 2009
[24] H P S Makkar and K Becker ldquoVanillin-HCl method for con-densed tannins effect of organic solvents used for extraction oftanninsrdquo Journal of Chemical Ecology vol 19 no 4 pp 613ndash6211993
[25] B A Cevallos-Casals and L Cisneros-Zevallos ldquoStoichiometricand kinetic studies of phenolic antioxidants from Andean pur-ple corn and red-fleshed sweetpotatordquo Journal of Agriculturaland Food Chemistry vol 51 no 11 pp 3313ndash3319 2003
[26] P Salehi B Asghari M A Esmaeili H Dehghan and I Ghazildquo120572-Glucosidase and120572-amylase inhibitory effect and antioxidantof ten plant extracts traditionally used in Iran for diabetesrdquoJournal of Medicinal Plants Research vol 7 no 6 pp 257ndash2662013
[27] OECDOCDE 423 OECD Guideline for testing of chemicalsAcute Oral Toxicity ndash Acute Toxic Class Method EnvironmentDirectorate Organisation for Economic Co-Operation and De-velopment Paris France 2001
[28] B L Furman ldquoStreptozotocin-induced diabetic models in miceand ratsrdquo Current Protocols in Pharmacology vol 70 pp 5471ndash54720 2015
[29] I J Flores-Sanchez and A C Ramos-Valdivia ldquoA review frompatents inspired by two plant genera Uncaria and HameliardquoPhytochemistry Reviews vol 16 no 4 pp 693ndash723 2017
[30] R Vinayagam and B Xu ldquoAntidiabetic properties of dietaryflavonoids A cellular mechanism reviewrdquo Journal of Nutritionand Metabolism vol 12 no 60 pp 1ndash20 2015
[31] S Marella ldquoFlavonoids-Themost potent poly-phenols as antidi-abetic agents an overviewrdquoModernApproaches in Drug Design-ing vol 1 no 3 pp 2ndash5 2017
[32] AUllah A Khan and I Khan ldquoDiabetesmellitus and oxidativestress-A concise reviewrdquo Saudi Pharmaceutical Journal vol 24pp 547ndash553 2016
[33] D Shasha CMagogo and P Dzomba ldquoReversed phase HPLC-UV Quantitation of BHA BHT and TBHQ in food items soldin bindura supermarkets Zimbabwerdquo International ResearchJournal of Pure amp Applied Chemistry vol 4 no 5 pp 578ndash5842014
[34] M Vessal M Hemmati and M Vasei ldquoAntidiabetic effects ofquercetin in streptozocin-induced diabetic ratsrdquo ComparativeBiochemistry and Physiology Part C Toxicology amp Pharmacol-ogy vol 135 no 3 pp 357ndash364 2003
[35] P-C Hsieh G-J Huang Y-L Ho et al ldquoActivities of antioxi-dants 120572-glucosidase inhibitors and aldose reductase inhibitorsof the aqueous extracts of four Flemingia species in TaiwanrdquoBotanical Studies vol 51 no 3 pp 293ndash302 2010
[36] W Kang Y Song and X Gu ldquo120572-glucosidase inhibitory in vitroand antidiabetic activity in vivo ofOsmanthus fragransrdquo Journalof Medicinal Plants Research vol 6 no 14 pp 2850ndash2856 2012
[37] N Zahratunnisa B Elya and A Noviani ldquoInhibition of Alpha-glucosidase and antioxidant test of stem bark extracts ofGarcinia fruticosa Lauterbrdquo Pharmacognosy Journal vol 9 no2 pp 273ndash275 2017
[38] J E Wong C Rubio A Reyes C N Aguilar and M L Carril-lo ldquoPhenolic content and antibacterial activity of extracts ofHamelia patens obtained by different extraction methodsrdquo Bra-zilian Journal of Microbiology vol 49 no 3 pp 656ndash661 2017
[39] G Oboh O M Agunloye S A Adefegha A J Akinyemi andA O Ademiluyi ldquoCaffeic and chlorogenic acids inhibit key en-zymes linked to type 2 diabetes (in vitro) a comparative studyrdquoJournal of Basic and Clinical Physiology and Pharmacology vol26 no 2 pp 165ndash170 2015
[40] S-Y Chiou J-M Sung P-W Huang and S-D Lin ldquoAntiox-idant Antidiabetic and Antihypertensive Properties of Echi-nacea purpurea Flower Extract and Caffeic Acid DerivativesUsing in Vitro Modelsrdquo Journal of Medicinal Food vol 20 no2 pp 171ndash179 2017
[41] C-M Ma M Hattori M Daneshtalab and L Wang ldquoChloro-genic acid derivatives with alkyl chains of different lengths andorientations Potent120572-glucosidase inhibitorsrdquo Journal of Medic-inal Chemistry vol 51 no 19 pp 6188ndash6194 2008
[42] C Tan QWang C Luo S Chen Q Li and P Li ldquoYeast 120572-glu-cosidase inhibitory phenolic compounds isolated from Gynuramedica leafrdquo International Journal of Molecular Sciences vol 14no 2 pp 2551ndash2558 2013
[43] A I Suarez B Diaz S Tillett E Valdivieso and R S Com-pagnone ldquoLeishmanicidal activity of alkaloids from Hameliapatensrdquo Ciencia vol 16 no 2 pp 148ndash155 2008
[44] C Gutierrez-Rodelo A Roura-Guiberna and J A Olivares-Reyes ldquoMecanismos moleculares de la resistencia a la insulinauna actualizacionrdquo Gaceta Medica de Mexico vol 153 pp 215ndash228 2017
[45] J Rojas V Berm E Leal V Bermudez et al ldquoInsulinoresisten-cia e hiperinsulinemia como factores de riesgo para enfermedadcardivascularrdquo Revista Latinoamericana de Hipertension vol 2no 6 pp 179ndash190 2007
[46] SABamanikar A A Bamanikar andAArora ldquoStudy of serumurea and creatinine in diabetic and non-diabetic patients in atertiary teaching hospitalrdquoThe Journal of Medical Research vol2 no 1 pp 12ndash15 2016
[47] M Music A Dervisevic E Pepic et al ldquoMetabolic Syndromeand Serum Liver Enzymes Level at Patients with Type 2Diabetes MellitusrdquoMedical Archives vol 69 no 4 pp 251ndash2552015
Evidence-Based Complementary and Alternative Medicine 9
[48] S Kazemi S Asgary J Moshtaghian et al ldquoLiver-protectiveeffects of hydroalcoholic extract of Allium hirtifolium Boiss inrats with alloxan-induced diabetes mellitusrdquo ARYA Atheroscle-rosis Journal vol 6 no 1 pp 11ndash15 2010
[49] G A Bonneau M S Castillo Rascon R A Sanchez W RPedrozo and C Castro Olivera ldquoColesterol-IDL y parametroslipıdicos en diabeticos tipo 2rdquo Revista Argentina de Endocri-nologıa y Metabolismo vol 44 no 4 pp 215ndash222 2007
[50] A Husni D Purwanti and Ustadi ldquoBlood glucose level andlipid profile of streptozotocin-induceddiabetes rats treatedwithsodiumalginate from Sargassum crassifoliumrdquo Journal of Biolog-ical Sciences vol 16 no 3 pp 58ndash64 2016
[51] S Palazhy and V Viswanathan ldquoLipid abnormalities in type 2diabetes mellitus patients with overt nephropathyrdquo Diabetes ampMetabolism Journal vol 41 no 2 pp 128ndash134 2017
[52] J J DiNicolantonio J Bhutani and J H OrsquoKeefe ldquoAcarbosesafe and effective for lowering postprandial hyperglycaemia andimproving cardiovascular outcomesrdquo Open Heart vol 2 no 12015
[53] S Ogawa K Takaeuchi and S Ito ldquoAcarbose lowers serum tri-glyceride and postprandial chylomicron levels in type 2 dia-betesrdquo Diabetes Obesity and Metabolism vol 6 no 5 pp 384ndash390 2004
[54] L Liu S Shan K Zhang Z-Q Ning X-P Lu and Y-Y ChengldquoNaringenin andhesperetin two flavonoids derived fromCitrusaurantium up-regulate transcription of adiponectinrdquoPhytother-apy Research vol 22 no 10 pp 1400ndash1403 2008
[55] E P Cai and J-K Lin ldquoEpigallocatechin gallate (EGCG) andrutin suppress the glucotoxicity through activating IRS2 andAMPK signaling in rat pancreatic 120573 cellsrdquo Journal of Agricul-tural and Food Chemistry vol 57 no 20 pp 9817ndash9827 2009
FME 75 5975 plusmn 1242 7650 plusmn 2869 2667 plusmn 306 2300 plusmn 100FME 35 6075 plusmn 900 7575 plusmn 4842 2967 plusmn 551z 2233 plusmn 351(TC) total cholesterol (Tg) triglycerides (LDL) low-density lipoproteins (HDL) high-density lipoproteins HC healthy control DC diabetic control DCVdiabetic control vehicle CME 150 crudemethanolic extract at a concentration of 150 mgkg CME 75 crude methanolic extract at a concentration of 75 mgkgCME 35 crude methanolic extract at a concentration of 35 mgkg FME 150 fractional methanolic extract at a concentration of 150 mgkg FME 75 fractionalmethanolic extract at a concentration of 75 mgkg FME 35 fractional methanolic extract at a concentration of 35 mgkg For the corresponding mean plusmn SD zPlt 005 compared to the diabetic control zzP lt 005 compared to the diabetic control plusmnP lt 005 compared to health control 998771P lt 005 compared to acarboselozP lt 005 compared to metformin
difference was observed between the two extracts (Table 6)Enzymes AlT and AsT are an indicator of liver injury asenzymes leaking from cytosol liver cells into bloodstreamcorrelate with insulin resistance diabetes and inflammatoryprocesses of the liver [47 48] FME induced a markeddecrease in AlT making further histopathological studiesnecessary to confirm whether there is protection or damageto the liver as a result of the treatments The effect of theextracts on the lipid profile was also examined (Table 7)Patients with DM type 2 have been characterized by hightriglycerides levels low high-density lipoprotein cholesterol(HDL) levels normal low-density lipoprotein (LDL) levelsand normal or slightly increased total cholesterol levels [49ndash51] This characteristic was observed in the group treatedwith extracts and diabetic control where no significantdifferences were evident between treatments in terms of
total cholesterol nevertheless a sharp decline in triglyceridevalue was perceived in the acarbose and CME treatments(75 and 35mgkg) The capacity of acarbose for loweringtriglycerides has been described previously finding that200mg constitutes an effective dose for reducing the riskof cardiovascular events in humans implying a minimalrisk of hypoglycemia [52 53] Various studies have providedevidence that phenolic compounds such as flavonoids may beinvolved in decreased glucose levels in the in vivo model Ithas been suggested that the flavonoids naringenin and hes-peretin may produce antiatherogenic effects partly by meansof the activation of the peroxisome proliferator activatedreceptor gamma PPAR-120574 and the upregulation of adiponectinexpression in adipocytes Numerous reports document theantidiabetic effects of flavan-3-ols especially epigallocatechingallate (EGCG) in animals and cell-cultures EGCG can elicit
Evidence-Based Complementary and Alternative Medicine 7
various changes that are associated with beneficial effects fordiabetes including improvements in insulin secretion glu-cose uptake insulin resistance glucose tolerance oxidativestress inflammation and mitochondrial function [54 55]CME and FME comprise amixture of compounds and appearto inhibit the 120572-glucosidase enzyme however the inhibitoryactivity of the identified compounds suggests that this is notthe only way to reduce hyperglycemia As this is a mixtureof multitarget molecules it is impossible to assess the anti-hyperglycemic mechanism as this would imply identifyingthe two additional compounds and performing complemen-tary studies For the acute toxicity study which involved dos-ing ge2000mgkg there was no apparent mortality suggest-ing low toxicity within category 5 according to the OECDGlobally Harmonized Classification System (GHS) (2014)[27] This is advantageous should you wish to concoct anherbal preparation
4 Conclusions
These results reveal that extracts of Hamelia patens with ahigh content of phenolic compounds elicit 120572-glucosidaseinhibition and an antihyperglycemic effect At a concentrationof 150mgkg they produce an equivalent effect tometforminEpicatechin and chlorogenic acid contribute to the antihyper-glycemic activity ofH patens however it is necessary to carryout the structural identification of any other componentspresent Further research is necessary to elucidate the activitymechanisms
Data Availability
The data used to support the findings of this study are avail-able from the corresponding author upon request
Conflicts of Interest
The authors declare that there are no conflicts of interest re-garding the publication of this paper
Acknowledgments
The authors are grateful for technical support provided bythe personnel at the Laboratory of Natural Products andMolecular Biology of the ENMyH at IPN Catalina Rugerio-Escalona thanks CONACYT for the graduate scholarshipsawarded (264517) and SIP-IPN (BEIFI) for a complementaryscholarship Cynthia Ordaz-Pichardo Fabiola E Jimenez-Montejo Ignacio EMaldonado-MendozaMarıa del CarmenCruz-Lopez and Victor E Lopez-y-Lopez are Fellows of theEstımulos al Desempeno de los Investigadores (EDI)-IPNCynthia Ordaz-Pichardo Ignacio E Maldonado-MendozaMarıa del Carmen Cruz-Lopez and Victor E Lopez-y-Lopez thank the Comision de Operacion y Fomento deActividades Academicas (COFAA) an IPN program thatprovides support to researchers This work was financiallysupported by a project (20161781) from the Secretarıa deInvestigacion y Posgrado Instituto Politecnico Nacional
References
[1] F A Matough S B Budin Z A Hamid N Alwahaibi and JMohamed ldquoThe role of oxidative stress and antioxidants in dia-betic complicationsrdquo SQU Medical Journal vol 12 no 1 pp 5ndash18 2012
[2] AW Indrianingsih S Tachibana R TDewi andK Itoh ldquoAnti-oxidant and 120572-glucosidase inhibitor activities of natural com-pounds isolated fromQuercus gilva Blume leavesrdquoAsian PacificJournal of Tropical Biomedicine vol 5 no 9 pp 748ndash755 2015
[3] M Gondi and U J S Prasada Rao ldquoEthanol extract of mango(Mangifera indica L) peel inhibits 120572-amylase and 120572-glucosidaseactivities and ameliorates diabetes related biochemical param-eters in streptozotocin (STZ)-induced diabetic ratsrdquo Journal ofFood Science andTechnology vol 52 no 12 pp 7883ndash7893 2015
[4] ARayarandRManivannan ldquoIn-vitro alpha-amylase and alpha-glucosidase inhibition activity of umbelliferone and beta-ionone isolated from Coriandrum sativum LinnrdquoWorld Journalof Pharmacy and Pharmaceutical Sciences vol 5 no 1 pp 1280ndash1289 2016
[5] Atlas de la Diabetes de la FID-Octava edicion InternationalDiabetes Federation 2017
[6] American Diabetes Association ldquoPharmacologic Approachesto Glycemic Treatmentrdquo Journal of Clinical and Applied Resarchand Education vol 40 no 1 pp S64ndashS76 2017
[7] J J Marın-Penalver I Martın-Timon C Sevillano-Collantesand F J del Canizo-Gomez ldquoUpdate on the treatment of type2 diabetes mellitusrdquoWorld Journal of Diabetes vol 7 no 17 pp354ndash395 2016
[8] H-Y Chang M Wallis and E Tiralongo ldquoUse of comple-mentary and alternative medicine among people living withdiabetes literature reviewrdquo Journal of AdvancedNursing vol 58no 4 pp 307ndash319 2007
[9] A B Medagama and R Bandara ldquoThe use of Complementaryand AlternativeMedicines (CAMs) in the treatment of diabetesmellitus Is continued use safe and effectiverdquo Nutrition Journalvol 13 no 1 2014
[10] B Baharvand-Ahmadi M Bahmani P Tajeddini N Naghdiand M Rafieian-Kopaei ldquoAn ethno-medicinal study of medic-inal plants used for the treatment of diabetesrdquo Journal ofNephropathology vol 5 no 1 pp 44ndash50 2016
[11] S Verma M Gupta H Popli and G Aggarwal ldquoDiabetesmellitus treatment using herbal drugsrdquo International Journal ofPhytomedicine vol 10 no 1 pp 1ndash10 2018
[12] B Ovalle-Magallanes O N Medina-Campos J Pedraza-Cha-verri and R Mata ldquoHypoglycemic and antihyperglycemic ef-fects of phytopreparations and limonoids from Swietenia humi-lisrdquo Phytochemistry vol 110 pp 111ndash119 2015
[13] S Singh andM Vyas ldquoComparative in-vitro biological study ofaerial parts of plant Hamelia patensrdquo International Journal ofPharmaceutical Sciences and Research vol 7 no 4 pp 1793ndash1808 2016
[14] F Ruiz-Teran A Medrano-Martınez and A Navarro-OcanaldquoAntioxidant and free radical scavenging activities of plantextracts used in traditional medicine in Mexicordquo African Jour-nal of Biotechnology vol 7 no 12 pp 1886ndash1893 2008
[15] S Sosa M J Balick R Arvigo et al ldquoScreening of the topicalanti-inflammatory activity of some Central American plantsrdquoJournal of Ethnopharmacology vol 81 no 2 pp 211ndash215 2002
[16] A Camporese M J Balick R Arvigo et al ldquoScreening ofanti-bacterial activity of medicinal plants from Belize (Central
8 Evidence-Based Complementary and Alternative Medicine
America)rdquo Journal of Ethnopharmacology vol 87 no 1 pp 103ndash107 2003
[17] G Mena-Rejon E Caamal-Fuentes Z Cantillo-Ciau R Cedil-lo-Rivera J Flores-Guido and R Moo-Puc ldquoIn vitro cytotoxicactivity of nine plants used in Mayan traditional medicinerdquoJournal of Ethnopharmacology vol 121 no 3 pp 462ndash465 2009
[18] V Jimenez-Suarez A Nieto-Camacho M Jimenez-Estradaand B Alvarado Sanchez ldquoAnti-inflammatory free radical scav-enging and alpha-glucosidase inhibitory activities of Hameliapatens and its chemical constituentsrdquo Pharmaceutical Biologyvol 54 no 9 pp 1822ndash1830 2016
[19] A Andrade-Cetto S Escandon-Rivera and V Garcia-LunaldquoHypoglycemic effect of Hamelia patens Jacq aerial part inSTZ-NA-induced diabetic ratsrdquo Pharmacologyonline vol 3 pp65ndash69b 2015
[20] A Ahmad A Pandurangan N Singh and P Ananad ldquoA minireview on chemistry and biology of Hamelia Patens (Rubi-aceae)rdquo Pharmacognosy Journal vol 4 no 29 pp 1ndash4 2012
[21] J Bano S Santra and EMenghani ldquoHamelia patens a potentialplant from Rubiaceae family A reviewrdquo International Journalof Scientific amp Engineering Research vol 6 no 12 pp 960ndash9732015
[22] E C Cruz and A Andrade-Cetto ldquoEthnopharmacological fieldstudy of the plants used to treat type 2 diabetes among theCakchiquels in Guatemalardquo Journal of Ethnopharmacology vol159 pp 238ndash244 2015
[23] D A Sampietro M A Sgariglia J R Soberon E N QuirogaandM A Vattuone ldquoColorimetric reactionsrdquo in Isolation Iden-tification and Characterization of AllelochemicalsNatural Prod-ucts chapter 4 pp 78ndash82 2009
[24] H P S Makkar and K Becker ldquoVanillin-HCl method for con-densed tannins effect of organic solvents used for extraction oftanninsrdquo Journal of Chemical Ecology vol 19 no 4 pp 613ndash6211993
[25] B A Cevallos-Casals and L Cisneros-Zevallos ldquoStoichiometricand kinetic studies of phenolic antioxidants from Andean pur-ple corn and red-fleshed sweetpotatordquo Journal of Agriculturaland Food Chemistry vol 51 no 11 pp 3313ndash3319 2003
[26] P Salehi B Asghari M A Esmaeili H Dehghan and I Ghazildquo120572-Glucosidase and120572-amylase inhibitory effect and antioxidantof ten plant extracts traditionally used in Iran for diabetesrdquoJournal of Medicinal Plants Research vol 7 no 6 pp 257ndash2662013
[27] OECDOCDE 423 OECD Guideline for testing of chemicalsAcute Oral Toxicity ndash Acute Toxic Class Method EnvironmentDirectorate Organisation for Economic Co-Operation and De-velopment Paris France 2001
[28] B L Furman ldquoStreptozotocin-induced diabetic models in miceand ratsrdquo Current Protocols in Pharmacology vol 70 pp 5471ndash54720 2015
[29] I J Flores-Sanchez and A C Ramos-Valdivia ldquoA review frompatents inspired by two plant genera Uncaria and HameliardquoPhytochemistry Reviews vol 16 no 4 pp 693ndash723 2017
[30] R Vinayagam and B Xu ldquoAntidiabetic properties of dietaryflavonoids A cellular mechanism reviewrdquo Journal of Nutritionand Metabolism vol 12 no 60 pp 1ndash20 2015
[31] S Marella ldquoFlavonoids-Themost potent poly-phenols as antidi-abetic agents an overviewrdquoModernApproaches in Drug Design-ing vol 1 no 3 pp 2ndash5 2017
[32] AUllah A Khan and I Khan ldquoDiabetesmellitus and oxidativestress-A concise reviewrdquo Saudi Pharmaceutical Journal vol 24pp 547ndash553 2016
[33] D Shasha CMagogo and P Dzomba ldquoReversed phase HPLC-UV Quantitation of BHA BHT and TBHQ in food items soldin bindura supermarkets Zimbabwerdquo International ResearchJournal of Pure amp Applied Chemistry vol 4 no 5 pp 578ndash5842014
[34] M Vessal M Hemmati and M Vasei ldquoAntidiabetic effects ofquercetin in streptozocin-induced diabetic ratsrdquo ComparativeBiochemistry and Physiology Part C Toxicology amp Pharmacol-ogy vol 135 no 3 pp 357ndash364 2003
[35] P-C Hsieh G-J Huang Y-L Ho et al ldquoActivities of antioxi-dants 120572-glucosidase inhibitors and aldose reductase inhibitorsof the aqueous extracts of four Flemingia species in TaiwanrdquoBotanical Studies vol 51 no 3 pp 293ndash302 2010
[36] W Kang Y Song and X Gu ldquo120572-glucosidase inhibitory in vitroand antidiabetic activity in vivo ofOsmanthus fragransrdquo Journalof Medicinal Plants Research vol 6 no 14 pp 2850ndash2856 2012
[37] N Zahratunnisa B Elya and A Noviani ldquoInhibition of Alpha-glucosidase and antioxidant test of stem bark extracts ofGarcinia fruticosa Lauterbrdquo Pharmacognosy Journal vol 9 no2 pp 273ndash275 2017
[38] J E Wong C Rubio A Reyes C N Aguilar and M L Carril-lo ldquoPhenolic content and antibacterial activity of extracts ofHamelia patens obtained by different extraction methodsrdquo Bra-zilian Journal of Microbiology vol 49 no 3 pp 656ndash661 2017
[39] G Oboh O M Agunloye S A Adefegha A J Akinyemi andA O Ademiluyi ldquoCaffeic and chlorogenic acids inhibit key en-zymes linked to type 2 diabetes (in vitro) a comparative studyrdquoJournal of Basic and Clinical Physiology and Pharmacology vol26 no 2 pp 165ndash170 2015
[40] S-Y Chiou J-M Sung P-W Huang and S-D Lin ldquoAntiox-idant Antidiabetic and Antihypertensive Properties of Echi-nacea purpurea Flower Extract and Caffeic Acid DerivativesUsing in Vitro Modelsrdquo Journal of Medicinal Food vol 20 no2 pp 171ndash179 2017
[41] C-M Ma M Hattori M Daneshtalab and L Wang ldquoChloro-genic acid derivatives with alkyl chains of different lengths andorientations Potent120572-glucosidase inhibitorsrdquo Journal of Medic-inal Chemistry vol 51 no 19 pp 6188ndash6194 2008
[42] C Tan QWang C Luo S Chen Q Li and P Li ldquoYeast 120572-glu-cosidase inhibitory phenolic compounds isolated from Gynuramedica leafrdquo International Journal of Molecular Sciences vol 14no 2 pp 2551ndash2558 2013
[43] A I Suarez B Diaz S Tillett E Valdivieso and R S Com-pagnone ldquoLeishmanicidal activity of alkaloids from Hameliapatensrdquo Ciencia vol 16 no 2 pp 148ndash155 2008
[44] C Gutierrez-Rodelo A Roura-Guiberna and J A Olivares-Reyes ldquoMecanismos moleculares de la resistencia a la insulinauna actualizacionrdquo Gaceta Medica de Mexico vol 153 pp 215ndash228 2017
[45] J Rojas V Berm E Leal V Bermudez et al ldquoInsulinoresisten-cia e hiperinsulinemia como factores de riesgo para enfermedadcardivascularrdquo Revista Latinoamericana de Hipertension vol 2no 6 pp 179ndash190 2007
[46] SABamanikar A A Bamanikar andAArora ldquoStudy of serumurea and creatinine in diabetic and non-diabetic patients in atertiary teaching hospitalrdquoThe Journal of Medical Research vol2 no 1 pp 12ndash15 2016
[47] M Music A Dervisevic E Pepic et al ldquoMetabolic Syndromeand Serum Liver Enzymes Level at Patients with Type 2Diabetes MellitusrdquoMedical Archives vol 69 no 4 pp 251ndash2552015
Evidence-Based Complementary and Alternative Medicine 9
[48] S Kazemi S Asgary J Moshtaghian et al ldquoLiver-protectiveeffects of hydroalcoholic extract of Allium hirtifolium Boiss inrats with alloxan-induced diabetes mellitusrdquo ARYA Atheroscle-rosis Journal vol 6 no 1 pp 11ndash15 2010
[49] G A Bonneau M S Castillo Rascon R A Sanchez W RPedrozo and C Castro Olivera ldquoColesterol-IDL y parametroslipıdicos en diabeticos tipo 2rdquo Revista Argentina de Endocri-nologıa y Metabolismo vol 44 no 4 pp 215ndash222 2007
[50] A Husni D Purwanti and Ustadi ldquoBlood glucose level andlipid profile of streptozotocin-induceddiabetes rats treatedwithsodiumalginate from Sargassum crassifoliumrdquo Journal of Biolog-ical Sciences vol 16 no 3 pp 58ndash64 2016
[51] S Palazhy and V Viswanathan ldquoLipid abnormalities in type 2diabetes mellitus patients with overt nephropathyrdquo Diabetes ampMetabolism Journal vol 41 no 2 pp 128ndash134 2017
[52] J J DiNicolantonio J Bhutani and J H OrsquoKeefe ldquoAcarbosesafe and effective for lowering postprandial hyperglycaemia andimproving cardiovascular outcomesrdquo Open Heart vol 2 no 12015
[53] S Ogawa K Takaeuchi and S Ito ldquoAcarbose lowers serum tri-glyceride and postprandial chylomicron levels in type 2 dia-betesrdquo Diabetes Obesity and Metabolism vol 6 no 5 pp 384ndash390 2004
[54] L Liu S Shan K Zhang Z-Q Ning X-P Lu and Y-Y ChengldquoNaringenin andhesperetin two flavonoids derived fromCitrusaurantium up-regulate transcription of adiponectinrdquoPhytother-apy Research vol 22 no 10 pp 1400ndash1403 2008
[55] E P Cai and J-K Lin ldquoEpigallocatechin gallate (EGCG) andrutin suppress the glucotoxicity through activating IRS2 andAMPK signaling in rat pancreatic 120573 cellsrdquo Journal of Agricul-tural and Food Chemistry vol 57 no 20 pp 9817ndash9827 2009
Computational and Mathematical Methods in Medicine
Hindawiwwwhindawicom Volume 2018
Behavioural Neurology
OphthalmologyJournal of
Hindawiwwwhindawicom Volume 2018
Diabetes ResearchJournal of
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
Research and TreatmentAIDS
Hindawiwwwhindawicom Volume 2018
Gastroenterology Research and Practice
Hindawiwwwhindawicom Volume 2018
Parkinsonrsquos Disease
Evidence-Based Complementary andAlternative Medicine
Volume 2018Hindawiwwwhindawicom
Submit your manuscripts atwwwhindawicom
Evidence-Based Complementary and Alternative Medicine 7
various changes that are associated with beneficial effects fordiabetes including improvements in insulin secretion glu-cose uptake insulin resistance glucose tolerance oxidativestress inflammation and mitochondrial function [54 55]CME and FME comprise amixture of compounds and appearto inhibit the 120572-glucosidase enzyme however the inhibitoryactivity of the identified compounds suggests that this is notthe only way to reduce hyperglycemia As this is a mixtureof multitarget molecules it is impossible to assess the anti-hyperglycemic mechanism as this would imply identifyingthe two additional compounds and performing complemen-tary studies For the acute toxicity study which involved dos-ing ge2000mgkg there was no apparent mortality suggest-ing low toxicity within category 5 according to the OECDGlobally Harmonized Classification System (GHS) (2014)[27] This is advantageous should you wish to concoct anherbal preparation
4 Conclusions
These results reveal that extracts of Hamelia patens with ahigh content of phenolic compounds elicit 120572-glucosidaseinhibition and an antihyperglycemic effect At a concentrationof 150mgkg they produce an equivalent effect tometforminEpicatechin and chlorogenic acid contribute to the antihyper-glycemic activity ofH patens however it is necessary to carryout the structural identification of any other componentspresent Further research is necessary to elucidate the activitymechanisms
Data Availability
The data used to support the findings of this study are avail-able from the corresponding author upon request
Conflicts of Interest
The authors declare that there are no conflicts of interest re-garding the publication of this paper
Acknowledgments
The authors are grateful for technical support provided bythe personnel at the Laboratory of Natural Products andMolecular Biology of the ENMyH at IPN Catalina Rugerio-Escalona thanks CONACYT for the graduate scholarshipsawarded (264517) and SIP-IPN (BEIFI) for a complementaryscholarship Cynthia Ordaz-Pichardo Fabiola E Jimenez-Montejo Ignacio EMaldonado-MendozaMarıa del CarmenCruz-Lopez and Victor E Lopez-y-Lopez are Fellows of theEstımulos al Desempeno de los Investigadores (EDI)-IPNCynthia Ordaz-Pichardo Ignacio E Maldonado-MendozaMarıa del Carmen Cruz-Lopez and Victor E Lopez-y-Lopez thank the Comision de Operacion y Fomento deActividades Academicas (COFAA) an IPN program thatprovides support to researchers This work was financiallysupported by a project (20161781) from the Secretarıa deInvestigacion y Posgrado Instituto Politecnico Nacional
References
[1] F A Matough S B Budin Z A Hamid N Alwahaibi and JMohamed ldquoThe role of oxidative stress and antioxidants in dia-betic complicationsrdquo SQU Medical Journal vol 12 no 1 pp 5ndash18 2012
[2] AW Indrianingsih S Tachibana R TDewi andK Itoh ldquoAnti-oxidant and 120572-glucosidase inhibitor activities of natural com-pounds isolated fromQuercus gilva Blume leavesrdquoAsian PacificJournal of Tropical Biomedicine vol 5 no 9 pp 748ndash755 2015
[3] M Gondi and U J S Prasada Rao ldquoEthanol extract of mango(Mangifera indica L) peel inhibits 120572-amylase and 120572-glucosidaseactivities and ameliorates diabetes related biochemical param-eters in streptozotocin (STZ)-induced diabetic ratsrdquo Journal ofFood Science andTechnology vol 52 no 12 pp 7883ndash7893 2015
[4] ARayarandRManivannan ldquoIn-vitro alpha-amylase and alpha-glucosidase inhibition activity of umbelliferone and beta-ionone isolated from Coriandrum sativum LinnrdquoWorld Journalof Pharmacy and Pharmaceutical Sciences vol 5 no 1 pp 1280ndash1289 2016
[5] Atlas de la Diabetes de la FID-Octava edicion InternationalDiabetes Federation 2017
[6] American Diabetes Association ldquoPharmacologic Approachesto Glycemic Treatmentrdquo Journal of Clinical and Applied Resarchand Education vol 40 no 1 pp S64ndashS76 2017
[7] J J Marın-Penalver I Martın-Timon C Sevillano-Collantesand F J del Canizo-Gomez ldquoUpdate on the treatment of type2 diabetes mellitusrdquoWorld Journal of Diabetes vol 7 no 17 pp354ndash395 2016
[8] H-Y Chang M Wallis and E Tiralongo ldquoUse of comple-mentary and alternative medicine among people living withdiabetes literature reviewrdquo Journal of AdvancedNursing vol 58no 4 pp 307ndash319 2007
[9] A B Medagama and R Bandara ldquoThe use of Complementaryand AlternativeMedicines (CAMs) in the treatment of diabetesmellitus Is continued use safe and effectiverdquo Nutrition Journalvol 13 no 1 2014
[10] B Baharvand-Ahmadi M Bahmani P Tajeddini N Naghdiand M Rafieian-Kopaei ldquoAn ethno-medicinal study of medic-inal plants used for the treatment of diabetesrdquo Journal ofNephropathology vol 5 no 1 pp 44ndash50 2016
[11] S Verma M Gupta H Popli and G Aggarwal ldquoDiabetesmellitus treatment using herbal drugsrdquo International Journal ofPhytomedicine vol 10 no 1 pp 1ndash10 2018
[12] B Ovalle-Magallanes O N Medina-Campos J Pedraza-Cha-verri and R Mata ldquoHypoglycemic and antihyperglycemic ef-fects of phytopreparations and limonoids from Swietenia humi-lisrdquo Phytochemistry vol 110 pp 111ndash119 2015
[13] S Singh andM Vyas ldquoComparative in-vitro biological study ofaerial parts of plant Hamelia patensrdquo International Journal ofPharmaceutical Sciences and Research vol 7 no 4 pp 1793ndash1808 2016
[14] F Ruiz-Teran A Medrano-Martınez and A Navarro-OcanaldquoAntioxidant and free radical scavenging activities of plantextracts used in traditional medicine in Mexicordquo African Jour-nal of Biotechnology vol 7 no 12 pp 1886ndash1893 2008
[15] S Sosa M J Balick R Arvigo et al ldquoScreening of the topicalanti-inflammatory activity of some Central American plantsrdquoJournal of Ethnopharmacology vol 81 no 2 pp 211ndash215 2002
[16] A Camporese M J Balick R Arvigo et al ldquoScreening ofanti-bacterial activity of medicinal plants from Belize (Central
8 Evidence-Based Complementary and Alternative Medicine
America)rdquo Journal of Ethnopharmacology vol 87 no 1 pp 103ndash107 2003
[17] G Mena-Rejon E Caamal-Fuentes Z Cantillo-Ciau R Cedil-lo-Rivera J Flores-Guido and R Moo-Puc ldquoIn vitro cytotoxicactivity of nine plants used in Mayan traditional medicinerdquoJournal of Ethnopharmacology vol 121 no 3 pp 462ndash465 2009
[18] V Jimenez-Suarez A Nieto-Camacho M Jimenez-Estradaand B Alvarado Sanchez ldquoAnti-inflammatory free radical scav-enging and alpha-glucosidase inhibitory activities of Hameliapatens and its chemical constituentsrdquo Pharmaceutical Biologyvol 54 no 9 pp 1822ndash1830 2016
[19] A Andrade-Cetto S Escandon-Rivera and V Garcia-LunaldquoHypoglycemic effect of Hamelia patens Jacq aerial part inSTZ-NA-induced diabetic ratsrdquo Pharmacologyonline vol 3 pp65ndash69b 2015
[20] A Ahmad A Pandurangan N Singh and P Ananad ldquoA minireview on chemistry and biology of Hamelia Patens (Rubi-aceae)rdquo Pharmacognosy Journal vol 4 no 29 pp 1ndash4 2012
[21] J Bano S Santra and EMenghani ldquoHamelia patens a potentialplant from Rubiaceae family A reviewrdquo International Journalof Scientific amp Engineering Research vol 6 no 12 pp 960ndash9732015
[22] E C Cruz and A Andrade-Cetto ldquoEthnopharmacological fieldstudy of the plants used to treat type 2 diabetes among theCakchiquels in Guatemalardquo Journal of Ethnopharmacology vol159 pp 238ndash244 2015
[23] D A Sampietro M A Sgariglia J R Soberon E N QuirogaandM A Vattuone ldquoColorimetric reactionsrdquo in Isolation Iden-tification and Characterization of AllelochemicalsNatural Prod-ucts chapter 4 pp 78ndash82 2009
[24] H P S Makkar and K Becker ldquoVanillin-HCl method for con-densed tannins effect of organic solvents used for extraction oftanninsrdquo Journal of Chemical Ecology vol 19 no 4 pp 613ndash6211993
[25] B A Cevallos-Casals and L Cisneros-Zevallos ldquoStoichiometricand kinetic studies of phenolic antioxidants from Andean pur-ple corn and red-fleshed sweetpotatordquo Journal of Agriculturaland Food Chemistry vol 51 no 11 pp 3313ndash3319 2003
[26] P Salehi B Asghari M A Esmaeili H Dehghan and I Ghazildquo120572-Glucosidase and120572-amylase inhibitory effect and antioxidantof ten plant extracts traditionally used in Iran for diabetesrdquoJournal of Medicinal Plants Research vol 7 no 6 pp 257ndash2662013
[27] OECDOCDE 423 OECD Guideline for testing of chemicalsAcute Oral Toxicity ndash Acute Toxic Class Method EnvironmentDirectorate Organisation for Economic Co-Operation and De-velopment Paris France 2001
[28] B L Furman ldquoStreptozotocin-induced diabetic models in miceand ratsrdquo Current Protocols in Pharmacology vol 70 pp 5471ndash54720 2015
[29] I J Flores-Sanchez and A C Ramos-Valdivia ldquoA review frompatents inspired by two plant genera Uncaria and HameliardquoPhytochemistry Reviews vol 16 no 4 pp 693ndash723 2017
[30] R Vinayagam and B Xu ldquoAntidiabetic properties of dietaryflavonoids A cellular mechanism reviewrdquo Journal of Nutritionand Metabolism vol 12 no 60 pp 1ndash20 2015
[31] S Marella ldquoFlavonoids-Themost potent poly-phenols as antidi-abetic agents an overviewrdquoModernApproaches in Drug Design-ing vol 1 no 3 pp 2ndash5 2017
[32] AUllah A Khan and I Khan ldquoDiabetesmellitus and oxidativestress-A concise reviewrdquo Saudi Pharmaceutical Journal vol 24pp 547ndash553 2016
[33] D Shasha CMagogo and P Dzomba ldquoReversed phase HPLC-UV Quantitation of BHA BHT and TBHQ in food items soldin bindura supermarkets Zimbabwerdquo International ResearchJournal of Pure amp Applied Chemistry vol 4 no 5 pp 578ndash5842014
[34] M Vessal M Hemmati and M Vasei ldquoAntidiabetic effects ofquercetin in streptozocin-induced diabetic ratsrdquo ComparativeBiochemistry and Physiology Part C Toxicology amp Pharmacol-ogy vol 135 no 3 pp 357ndash364 2003
[35] P-C Hsieh G-J Huang Y-L Ho et al ldquoActivities of antioxi-dants 120572-glucosidase inhibitors and aldose reductase inhibitorsof the aqueous extracts of four Flemingia species in TaiwanrdquoBotanical Studies vol 51 no 3 pp 293ndash302 2010
[36] W Kang Y Song and X Gu ldquo120572-glucosidase inhibitory in vitroand antidiabetic activity in vivo ofOsmanthus fragransrdquo Journalof Medicinal Plants Research vol 6 no 14 pp 2850ndash2856 2012
[37] N Zahratunnisa B Elya and A Noviani ldquoInhibition of Alpha-glucosidase and antioxidant test of stem bark extracts ofGarcinia fruticosa Lauterbrdquo Pharmacognosy Journal vol 9 no2 pp 273ndash275 2017
[38] J E Wong C Rubio A Reyes C N Aguilar and M L Carril-lo ldquoPhenolic content and antibacterial activity of extracts ofHamelia patens obtained by different extraction methodsrdquo Bra-zilian Journal of Microbiology vol 49 no 3 pp 656ndash661 2017
[39] G Oboh O M Agunloye S A Adefegha A J Akinyemi andA O Ademiluyi ldquoCaffeic and chlorogenic acids inhibit key en-zymes linked to type 2 diabetes (in vitro) a comparative studyrdquoJournal of Basic and Clinical Physiology and Pharmacology vol26 no 2 pp 165ndash170 2015
[40] S-Y Chiou J-M Sung P-W Huang and S-D Lin ldquoAntiox-idant Antidiabetic and Antihypertensive Properties of Echi-nacea purpurea Flower Extract and Caffeic Acid DerivativesUsing in Vitro Modelsrdquo Journal of Medicinal Food vol 20 no2 pp 171ndash179 2017
[41] C-M Ma M Hattori M Daneshtalab and L Wang ldquoChloro-genic acid derivatives with alkyl chains of different lengths andorientations Potent120572-glucosidase inhibitorsrdquo Journal of Medic-inal Chemistry vol 51 no 19 pp 6188ndash6194 2008
[42] C Tan QWang C Luo S Chen Q Li and P Li ldquoYeast 120572-glu-cosidase inhibitory phenolic compounds isolated from Gynuramedica leafrdquo International Journal of Molecular Sciences vol 14no 2 pp 2551ndash2558 2013
[43] A I Suarez B Diaz S Tillett E Valdivieso and R S Com-pagnone ldquoLeishmanicidal activity of alkaloids from Hameliapatensrdquo Ciencia vol 16 no 2 pp 148ndash155 2008
[44] C Gutierrez-Rodelo A Roura-Guiberna and J A Olivares-Reyes ldquoMecanismos moleculares de la resistencia a la insulinauna actualizacionrdquo Gaceta Medica de Mexico vol 153 pp 215ndash228 2017
[45] J Rojas V Berm E Leal V Bermudez et al ldquoInsulinoresisten-cia e hiperinsulinemia como factores de riesgo para enfermedadcardivascularrdquo Revista Latinoamericana de Hipertension vol 2no 6 pp 179ndash190 2007
[46] SABamanikar A A Bamanikar andAArora ldquoStudy of serumurea and creatinine in diabetic and non-diabetic patients in atertiary teaching hospitalrdquoThe Journal of Medical Research vol2 no 1 pp 12ndash15 2016
[47] M Music A Dervisevic E Pepic et al ldquoMetabolic Syndromeand Serum Liver Enzymes Level at Patients with Type 2Diabetes MellitusrdquoMedical Archives vol 69 no 4 pp 251ndash2552015
Evidence-Based Complementary and Alternative Medicine 9
[48] S Kazemi S Asgary J Moshtaghian et al ldquoLiver-protectiveeffects of hydroalcoholic extract of Allium hirtifolium Boiss inrats with alloxan-induced diabetes mellitusrdquo ARYA Atheroscle-rosis Journal vol 6 no 1 pp 11ndash15 2010
[49] G A Bonneau M S Castillo Rascon R A Sanchez W RPedrozo and C Castro Olivera ldquoColesterol-IDL y parametroslipıdicos en diabeticos tipo 2rdquo Revista Argentina de Endocri-nologıa y Metabolismo vol 44 no 4 pp 215ndash222 2007
[50] A Husni D Purwanti and Ustadi ldquoBlood glucose level andlipid profile of streptozotocin-induceddiabetes rats treatedwithsodiumalginate from Sargassum crassifoliumrdquo Journal of Biolog-ical Sciences vol 16 no 3 pp 58ndash64 2016
[51] S Palazhy and V Viswanathan ldquoLipid abnormalities in type 2diabetes mellitus patients with overt nephropathyrdquo Diabetes ampMetabolism Journal vol 41 no 2 pp 128ndash134 2017
[52] J J DiNicolantonio J Bhutani and J H OrsquoKeefe ldquoAcarbosesafe and effective for lowering postprandial hyperglycaemia andimproving cardiovascular outcomesrdquo Open Heart vol 2 no 12015
[53] S Ogawa K Takaeuchi and S Ito ldquoAcarbose lowers serum tri-glyceride and postprandial chylomicron levels in type 2 dia-betesrdquo Diabetes Obesity and Metabolism vol 6 no 5 pp 384ndash390 2004
[54] L Liu S Shan K Zhang Z-Q Ning X-P Lu and Y-Y ChengldquoNaringenin andhesperetin two flavonoids derived fromCitrusaurantium up-regulate transcription of adiponectinrdquoPhytother-apy Research vol 22 no 10 pp 1400ndash1403 2008
[55] E P Cai and J-K Lin ldquoEpigallocatechin gallate (EGCG) andrutin suppress the glucotoxicity through activating IRS2 andAMPK signaling in rat pancreatic 120573 cellsrdquo Journal of Agricul-tural and Food Chemistry vol 57 no 20 pp 9817ndash9827 2009
Computational and Mathematical Methods in Medicine
Hindawiwwwhindawicom Volume 2018
Behavioural Neurology
OphthalmologyJournal of
Hindawiwwwhindawicom Volume 2018
Diabetes ResearchJournal of
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
Research and TreatmentAIDS
Hindawiwwwhindawicom Volume 2018
Gastroenterology Research and Practice
Hindawiwwwhindawicom Volume 2018
Parkinsonrsquos Disease
Evidence-Based Complementary andAlternative Medicine
Volume 2018Hindawiwwwhindawicom
Submit your manuscripts atwwwhindawicom
8 Evidence-Based Complementary and Alternative Medicine
America)rdquo Journal of Ethnopharmacology vol 87 no 1 pp 103ndash107 2003
[17] G Mena-Rejon E Caamal-Fuentes Z Cantillo-Ciau R Cedil-lo-Rivera J Flores-Guido and R Moo-Puc ldquoIn vitro cytotoxicactivity of nine plants used in Mayan traditional medicinerdquoJournal of Ethnopharmacology vol 121 no 3 pp 462ndash465 2009
[18] V Jimenez-Suarez A Nieto-Camacho M Jimenez-Estradaand B Alvarado Sanchez ldquoAnti-inflammatory free radical scav-enging and alpha-glucosidase inhibitory activities of Hameliapatens and its chemical constituentsrdquo Pharmaceutical Biologyvol 54 no 9 pp 1822ndash1830 2016
[19] A Andrade-Cetto S Escandon-Rivera and V Garcia-LunaldquoHypoglycemic effect of Hamelia patens Jacq aerial part inSTZ-NA-induced diabetic ratsrdquo Pharmacologyonline vol 3 pp65ndash69b 2015
[20] A Ahmad A Pandurangan N Singh and P Ananad ldquoA minireview on chemistry and biology of Hamelia Patens (Rubi-aceae)rdquo Pharmacognosy Journal vol 4 no 29 pp 1ndash4 2012
[21] J Bano S Santra and EMenghani ldquoHamelia patens a potentialplant from Rubiaceae family A reviewrdquo International Journalof Scientific amp Engineering Research vol 6 no 12 pp 960ndash9732015
[22] E C Cruz and A Andrade-Cetto ldquoEthnopharmacological fieldstudy of the plants used to treat type 2 diabetes among theCakchiquels in Guatemalardquo Journal of Ethnopharmacology vol159 pp 238ndash244 2015
[23] D A Sampietro M A Sgariglia J R Soberon E N QuirogaandM A Vattuone ldquoColorimetric reactionsrdquo in Isolation Iden-tification and Characterization of AllelochemicalsNatural Prod-ucts chapter 4 pp 78ndash82 2009
[24] H P S Makkar and K Becker ldquoVanillin-HCl method for con-densed tannins effect of organic solvents used for extraction oftanninsrdquo Journal of Chemical Ecology vol 19 no 4 pp 613ndash6211993
[25] B A Cevallos-Casals and L Cisneros-Zevallos ldquoStoichiometricand kinetic studies of phenolic antioxidants from Andean pur-ple corn and red-fleshed sweetpotatordquo Journal of Agriculturaland Food Chemistry vol 51 no 11 pp 3313ndash3319 2003
[26] P Salehi B Asghari M A Esmaeili H Dehghan and I Ghazildquo120572-Glucosidase and120572-amylase inhibitory effect and antioxidantof ten plant extracts traditionally used in Iran for diabetesrdquoJournal of Medicinal Plants Research vol 7 no 6 pp 257ndash2662013
[27] OECDOCDE 423 OECD Guideline for testing of chemicalsAcute Oral Toxicity ndash Acute Toxic Class Method EnvironmentDirectorate Organisation for Economic Co-Operation and De-velopment Paris France 2001
[28] B L Furman ldquoStreptozotocin-induced diabetic models in miceand ratsrdquo Current Protocols in Pharmacology vol 70 pp 5471ndash54720 2015
[29] I J Flores-Sanchez and A C Ramos-Valdivia ldquoA review frompatents inspired by two plant genera Uncaria and HameliardquoPhytochemistry Reviews vol 16 no 4 pp 693ndash723 2017
[30] R Vinayagam and B Xu ldquoAntidiabetic properties of dietaryflavonoids A cellular mechanism reviewrdquo Journal of Nutritionand Metabolism vol 12 no 60 pp 1ndash20 2015
[31] S Marella ldquoFlavonoids-Themost potent poly-phenols as antidi-abetic agents an overviewrdquoModernApproaches in Drug Design-ing vol 1 no 3 pp 2ndash5 2017
[32] AUllah A Khan and I Khan ldquoDiabetesmellitus and oxidativestress-A concise reviewrdquo Saudi Pharmaceutical Journal vol 24pp 547ndash553 2016
[33] D Shasha CMagogo and P Dzomba ldquoReversed phase HPLC-UV Quantitation of BHA BHT and TBHQ in food items soldin bindura supermarkets Zimbabwerdquo International ResearchJournal of Pure amp Applied Chemistry vol 4 no 5 pp 578ndash5842014
[34] M Vessal M Hemmati and M Vasei ldquoAntidiabetic effects ofquercetin in streptozocin-induced diabetic ratsrdquo ComparativeBiochemistry and Physiology Part C Toxicology amp Pharmacol-ogy vol 135 no 3 pp 357ndash364 2003
[35] P-C Hsieh G-J Huang Y-L Ho et al ldquoActivities of antioxi-dants 120572-glucosidase inhibitors and aldose reductase inhibitorsof the aqueous extracts of four Flemingia species in TaiwanrdquoBotanical Studies vol 51 no 3 pp 293ndash302 2010
[36] W Kang Y Song and X Gu ldquo120572-glucosidase inhibitory in vitroand antidiabetic activity in vivo ofOsmanthus fragransrdquo Journalof Medicinal Plants Research vol 6 no 14 pp 2850ndash2856 2012
[37] N Zahratunnisa B Elya and A Noviani ldquoInhibition of Alpha-glucosidase and antioxidant test of stem bark extracts ofGarcinia fruticosa Lauterbrdquo Pharmacognosy Journal vol 9 no2 pp 273ndash275 2017
[38] J E Wong C Rubio A Reyes C N Aguilar and M L Carril-lo ldquoPhenolic content and antibacterial activity of extracts ofHamelia patens obtained by different extraction methodsrdquo Bra-zilian Journal of Microbiology vol 49 no 3 pp 656ndash661 2017
[39] G Oboh O M Agunloye S A Adefegha A J Akinyemi andA O Ademiluyi ldquoCaffeic and chlorogenic acids inhibit key en-zymes linked to type 2 diabetes (in vitro) a comparative studyrdquoJournal of Basic and Clinical Physiology and Pharmacology vol26 no 2 pp 165ndash170 2015
[40] S-Y Chiou J-M Sung P-W Huang and S-D Lin ldquoAntiox-idant Antidiabetic and Antihypertensive Properties of Echi-nacea purpurea Flower Extract and Caffeic Acid DerivativesUsing in Vitro Modelsrdquo Journal of Medicinal Food vol 20 no2 pp 171ndash179 2017
[41] C-M Ma M Hattori M Daneshtalab and L Wang ldquoChloro-genic acid derivatives with alkyl chains of different lengths andorientations Potent120572-glucosidase inhibitorsrdquo Journal of Medic-inal Chemistry vol 51 no 19 pp 6188ndash6194 2008
[42] C Tan QWang C Luo S Chen Q Li and P Li ldquoYeast 120572-glu-cosidase inhibitory phenolic compounds isolated from Gynuramedica leafrdquo International Journal of Molecular Sciences vol 14no 2 pp 2551ndash2558 2013
[43] A I Suarez B Diaz S Tillett E Valdivieso and R S Com-pagnone ldquoLeishmanicidal activity of alkaloids from Hameliapatensrdquo Ciencia vol 16 no 2 pp 148ndash155 2008
[44] C Gutierrez-Rodelo A Roura-Guiberna and J A Olivares-Reyes ldquoMecanismos moleculares de la resistencia a la insulinauna actualizacionrdquo Gaceta Medica de Mexico vol 153 pp 215ndash228 2017
[45] J Rojas V Berm E Leal V Bermudez et al ldquoInsulinoresisten-cia e hiperinsulinemia como factores de riesgo para enfermedadcardivascularrdquo Revista Latinoamericana de Hipertension vol 2no 6 pp 179ndash190 2007
[46] SABamanikar A A Bamanikar andAArora ldquoStudy of serumurea and creatinine in diabetic and non-diabetic patients in atertiary teaching hospitalrdquoThe Journal of Medical Research vol2 no 1 pp 12ndash15 2016
[47] M Music A Dervisevic E Pepic et al ldquoMetabolic Syndromeand Serum Liver Enzymes Level at Patients with Type 2Diabetes MellitusrdquoMedical Archives vol 69 no 4 pp 251ndash2552015
Evidence-Based Complementary and Alternative Medicine 9
[48] S Kazemi S Asgary J Moshtaghian et al ldquoLiver-protectiveeffects of hydroalcoholic extract of Allium hirtifolium Boiss inrats with alloxan-induced diabetes mellitusrdquo ARYA Atheroscle-rosis Journal vol 6 no 1 pp 11ndash15 2010
[49] G A Bonneau M S Castillo Rascon R A Sanchez W RPedrozo and C Castro Olivera ldquoColesterol-IDL y parametroslipıdicos en diabeticos tipo 2rdquo Revista Argentina de Endocri-nologıa y Metabolismo vol 44 no 4 pp 215ndash222 2007
[50] A Husni D Purwanti and Ustadi ldquoBlood glucose level andlipid profile of streptozotocin-induceddiabetes rats treatedwithsodiumalginate from Sargassum crassifoliumrdquo Journal of Biolog-ical Sciences vol 16 no 3 pp 58ndash64 2016
[51] S Palazhy and V Viswanathan ldquoLipid abnormalities in type 2diabetes mellitus patients with overt nephropathyrdquo Diabetes ampMetabolism Journal vol 41 no 2 pp 128ndash134 2017
[52] J J DiNicolantonio J Bhutani and J H OrsquoKeefe ldquoAcarbosesafe and effective for lowering postprandial hyperglycaemia andimproving cardiovascular outcomesrdquo Open Heart vol 2 no 12015
[53] S Ogawa K Takaeuchi and S Ito ldquoAcarbose lowers serum tri-glyceride and postprandial chylomicron levels in type 2 dia-betesrdquo Diabetes Obesity and Metabolism vol 6 no 5 pp 384ndash390 2004
[54] L Liu S Shan K Zhang Z-Q Ning X-P Lu and Y-Y ChengldquoNaringenin andhesperetin two flavonoids derived fromCitrusaurantium up-regulate transcription of adiponectinrdquoPhytother-apy Research vol 22 no 10 pp 1400ndash1403 2008
[55] E P Cai and J-K Lin ldquoEpigallocatechin gallate (EGCG) andrutin suppress the glucotoxicity through activating IRS2 andAMPK signaling in rat pancreatic 120573 cellsrdquo Journal of Agricul-tural and Food Chemistry vol 57 no 20 pp 9817ndash9827 2009
Computational and Mathematical Methods in Medicine
Hindawiwwwhindawicom Volume 2018
Behavioural Neurology
OphthalmologyJournal of
Hindawiwwwhindawicom Volume 2018
Diabetes ResearchJournal of
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
Research and TreatmentAIDS
Hindawiwwwhindawicom Volume 2018
Gastroenterology Research and Practice
Hindawiwwwhindawicom Volume 2018
Parkinsonrsquos Disease
Evidence-Based Complementary andAlternative Medicine
Volume 2018Hindawiwwwhindawicom
Submit your manuscripts atwwwhindawicom
Evidence-Based Complementary and Alternative Medicine 9
[48] S Kazemi S Asgary J Moshtaghian et al ldquoLiver-protectiveeffects of hydroalcoholic extract of Allium hirtifolium Boiss inrats with alloxan-induced diabetes mellitusrdquo ARYA Atheroscle-rosis Journal vol 6 no 1 pp 11ndash15 2010
[49] G A Bonneau M S Castillo Rascon R A Sanchez W RPedrozo and C Castro Olivera ldquoColesterol-IDL y parametroslipıdicos en diabeticos tipo 2rdquo Revista Argentina de Endocri-nologıa y Metabolismo vol 44 no 4 pp 215ndash222 2007
[50] A Husni D Purwanti and Ustadi ldquoBlood glucose level andlipid profile of streptozotocin-induceddiabetes rats treatedwithsodiumalginate from Sargassum crassifoliumrdquo Journal of Biolog-ical Sciences vol 16 no 3 pp 58ndash64 2016
[51] S Palazhy and V Viswanathan ldquoLipid abnormalities in type 2diabetes mellitus patients with overt nephropathyrdquo Diabetes ampMetabolism Journal vol 41 no 2 pp 128ndash134 2017
[52] J J DiNicolantonio J Bhutani and J H OrsquoKeefe ldquoAcarbosesafe and effective for lowering postprandial hyperglycaemia andimproving cardiovascular outcomesrdquo Open Heart vol 2 no 12015
[53] S Ogawa K Takaeuchi and S Ito ldquoAcarbose lowers serum tri-glyceride and postprandial chylomicron levels in type 2 dia-betesrdquo Diabetes Obesity and Metabolism vol 6 no 5 pp 384ndash390 2004
[54] L Liu S Shan K Zhang Z-Q Ning X-P Lu and Y-Y ChengldquoNaringenin andhesperetin two flavonoids derived fromCitrusaurantium up-regulate transcription of adiponectinrdquoPhytother-apy Research vol 22 no 10 pp 1400ndash1403 2008
[55] E P Cai and J-K Lin ldquoEpigallocatechin gallate (EGCG) andrutin suppress the glucotoxicity through activating IRS2 andAMPK signaling in rat pancreatic 120573 cellsrdquo Journal of Agricul-tural and Food Chemistry vol 57 no 20 pp 9817ndash9827 2009
