Pharmacogn. Mag.
ORIGINAL ARTICLE
A multifaceted peer reviewed journal in the field of Pharmacognosy and Natural Products
www.phcog.com | www.phcog.net
Phytochemical and Biological Evaluations of Arum hygrophilum
Boiss. (Araceae)
Fatma U. Afifi, Violet Kasabri, Simona Litescu1, Ismail F. Abaza, Khalid Tawaha
School of Pharmacy, The University of Jordan, Amman, Jordan, 1National Institute for Biological Sciences, Bucharest, Romania
Submitted: 28‑03‑2016
Revised: 29‑04‑2016
Published: 18‑04‑2017
ABSTRACT
Background: Arum hygrophilum is a traditional medicinal plant indigenous
to Jordan. The present study explores its phytochemistry, antioxidative,
antidiabesity, and antiproliferative potentialities. Materials and Methods:
Column chromatography and HPLC-MS analysis were used for its
phytochemical evaluation. Using leaf crude water and ethanol extracts, the
antioxidative capacities, their modulation of pancreatic β-cell proliferation,
and insulin secretion as well as glucose diffusion and enzymatic bioassays
were evaluated. Results: Three flavonoids (luteolin, isoorientin, and vitexin)
and β-sitosterol have been isolated and their structures determined.
HPLC-MS analysis of the ethanol extract further revealed the presence of
caffeic, ferulic, gallic, and rosmarinic acids and quercetine-3-O-rhamnoside.
The ethanol extract exhibited DPPH and ABTS radical scavenging and
antioxidative capacities. A. hygrophilum (1), vitexin (2), and rosmarinic
acid (3) inhibited pancreatic lipase (PL) dose dependently with PL-IC50 (µg/
mL) values in an ascending order: (3); 51.28 ± 7.55 < (2); 260.9 ± 21.1 <
(1); 1720 ± 10. Comparable to GLP-1-enhanced β-cell proliferation in 2-day
treatment wells, a dose-dependent augmentation of BrdU incorporation
was obtained with the A. hygrophilum aqueous extract (AE) (0.5 and 1 mg/
mL, with respective 1.33- and 1.41-folds, P < 0.001). A. hygrophilum AE
was identified as an inhibitor of α-amylase/α-glucosidase with IC50 value of
30.5 ± 2.1 mg/mL but lacked antiproliferative effects in colorectal cancer
cell lines (HT29, HCT116, and SW620) and insulinotropic effects in β-cell
line MIN6. Conclusion: A. hygrophilum extracts inhibited gastrointestinal
enzymes involved in carbohydrate and lipid digestion and absorption.
Key words: A. hygrophilum Boiss, Araceae, HPLC-MS, pancreatic
lipase, α-amylase/α-glucosidase
SUMMARY
• Phytochemical evaluation of Arum hygrophilum recovered flavonoids (luteolin,
isoorientin and vitexin) and β-sitosterol
• HPLC-MS analysis of its antioxidative ethanol extract further revealed the
presence of caffeic-, ferulic-, gallic- and rosmarinic acids and quercetine-3O-rhamnoside
• A. hygrophilum inhibited α-amylase/α-glucosidase and pancreatic lipase dosedependently
• A. hygrophilum augmented β-cell proliferation dose dependently, but it lacked
antiproliferative effects in colorectal cancer cell lines (HT29, HCT116, and
SW620) and insulinotropic effects in β-cell line MIN6
INTRODUCTION
Plants have been long used for the ethnomedical integrative/
complementary treatment of cancer and obesity-diabetes in various
systems of medicine.[1-3] Type 2 diabetes and obesity, referred to as
diabesity, comprise global health threats with rising prevalence.[4]
Diverse studies were conducted to explore medicinal plants as potential
therapeutic agents for dual management of diabetes and hyperlipidemia
via digestive enzymes’ inhibition, namely pancreatic α-amylase, intestinal
α-glucosidase, and pancreatic lipase.[5,6] In the Jordanian traditional
medicine, the edible Arum species, referred with the common Arabic
name “Louf,” are recommended as a natural anticancer agent against
colon cancer.[7] Previously, different flavonoids, such as quercetin,
apigenin, vitexin, and isoorientin were isolated from A. palaestinum in
Abbreviations used: ABTS: 2,2’-Azino-Bis-3-Ethylbenzothiazoline-6Sulfonic Acid, AE: Aqueous Extract, ANOVA: Analysis Of Variance, AUC:
Area Under Curve, BrdU: 5-Bromo-2'-Deoxyuridine, DPPH: 2,2-Diphenyl1-Pycriylhydrazyl, ELISA: Enzyme Linked Immunosorbent Assay, GLP1:
Glucagon Like Peptide 1, GSIS: Glucose Stimulated Insulin Secretion,
HPLC-MS: High Performance Liquid Chromatography –Mass Spectrometry,
IC50: 50% Inhibitory Concentration, KRH: Krebs/Ringer/Hepes, MTT:
3-(4,5-Dimethylthiazol-2-Yl)-2,5-Diphenyltetrazolium Bromide, OGTT: Oral
Glucose Tolerance Test, ORAC: Oxygen Radical Antioxidant Capacity, OSTT:
Oral Starch Tolerance Test, PL: Pancreatic Lipase, SEM: Standard Error Of
The Mean, SRB: Sulforhodamine B, TEAC: Trolox Equivalent Antioxidant
Capacity, TLC: Thin Layer Chromatography
Access this article online
Website: www.phcog.com
Correspondence:
Prof. Fatma U. Afifi,
Department of Pharmaceutical Sciences, School
of Pharmacy, The University of Jordan, Queen
Rania Al-Abdullah Street, Amman, Jordan.
E-mail: fatueafi@ju.edu.jo
DOI: 10.4103/0973-1296.204551
Quick Response Code:
our laboratories and their antimicrobial activities were established.[8] In a
recent comparative study in in vitro and in vivo experiments, pancreatic
lipase (PL) and dual α-amylase/α-glucosidase inhibitory potentials were
This is an open access article distributed under the terms of the Creative Commons
Attribution‑Non Commercial‑Share Alike 3.0 License, which allows others to remix,
tweak, and build upon the work non‑commercially, as long as the author is credited
and the new creations are licensed under the identical terms.
For reprints contact: reprints@medknow.com
© 2017 Pharmacognosy Magazine | Published by Wolters Kluwer ‑ Medknow
Cite this article as: Afifi FU, Kasabri V, Litescu S, Abaza IF, Tawaha K. Phytochemical
and biological evaluations of Arum hygrophilum boiss. (Araceae). Phcog Mag
2017;13:275-80.
275
FATMA U. AFIFI, et al.: Arum hygrophilum Phytochemistry and Biology
demonstrated for the extracts of A. dioscorides and A. palaestinum as
well as for some of their isolated compounds.[9] Earlier, Karahan et al.[10]
reported free radical scavenging and ferric-reducing activities of ethanol,
methanol, acetone, and water extracts of A. dioscoridis leaves. The study
concluded that total phenolic and flavonoid contents were greatly
influenced by the extraction medium. Also, a literature survey indicated
that A. hygrophillum is the least evaluated Arum species, lacking on
both phytochemical and biological evaluations. This study investigates
the indigenous A. hygrophilum phytochemically and biologically.
The A. hygrophilum crude aqueous extract (AE) modulation of the
extrapancreatic digestive enzymes was examined in vitro. Additionally,
acute in vivo effects were investigated. Antiproliferative potential of this
species against colorectal cancer cell lines as well as possible pancreatic
effects in β-cell line was evaluated.
MATERIALS AND METHODS
Plant material
Aerial parts of the flowering A. hygrophilum were collected in February/
March 2013 in Zai, Salt, Jordan. Taxonomic identity of the collected
plants was established in comparison with herbarium specimens of
the School of Science, The University of Jordan. The identification was
confirmed by Prof. K. Tawaha. A vaucher specimen (FMJ-ARA2) was
kept in the Department of Pharmaceutical Sciences, School of Pharmacy,
The University of Jordan.
Extraction and chromatographic separation
The air-dried flowers and leaves were coarsly powdered and extracted
by soaking in EtOH for 3 weeks at RT. After solvent evaporation until
dryness, the syrupy residue was extracted successively with CHCl3, EtOAc,
and BuOH. Based on similar TLC profile, fractions of EtOAc and BuOH
were combined and chromatographed on Silica gel columns (Silicagel
60, Merck) successively. Chloroform/methanol gradients were used for
the extraction of flavonoids and plant acids. The isolated compounds
were purified by repeated crystallization in MeOH. For the biological
experiments, 10%% (w/v) AEs were prepared as reported earlier.[11]
HPLC analysis
Crude EtOH extract was evaluated by HPLC. The experiments were
based on the previously developed method published by Cristea et al.,[12]
adjusted to the current samples’ specificity for the measurement of the
plant acids and flavonoids.[12] The HPLC measurements were performed
using a complete HPLC SHIMADZU system, using a Nucleosil 100-3.5
C18 column,. The system was coupled to a MS detector, LCMS-2010
detector, equipped with an ESI interface. The mobile phase consisted of
formic acid in water (pH = 3.0) as solvent A and formic acid in acetonitrile
(pH = 3.0) as solvent B. The polyphenolic compounds separation was
performed using binary gradient elution: 0 min 5% solvent B; 0.01-20
min 5-30% solvent B; 20-40 min 30% solvent B; 40.01-50 min 30-50%
solvent B; 50.01-52 min 50-5% solvent B. The flow rate was 0-5 min 0.1
mL/min; 5.01-15 min 0.2 mL/min; 15.01-35 min 0.1 mL/min; 35.01-50
min 0.2 mL/min; 50-52 min 0.1 mL/min. The analyses were performed
at RT for the period of 70 min and the injection volume was 20 µL.
Initially full-scan acquisition mode was used in m/z range 50-800. Stock
solutions of the reference substances (1 mg/mL EtOH) were kept at 4°C
between the experiments.
Antioxidant efficacy and free radical scavenging
properties assessment
The radical scavenging activities of the ethanol extract of A. hygrophilum
were evaluated using 2,2-diphenyl-1-pycriylhydrazyl (DPPH) and
276
2,2'-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) radicalscavenging activity assays and expressed as Trolox Equivalent.
Antioxidant Capacity (TEAC). The antioxidant property was determined
using Oxygen Radical Antioxidant Capacity (ORAC).[13,14]
Insulin secretion static incubation experiments
Glucose-stimulated insulin secretion (GSIS) from MIN6 cells was
determined using a static incubation protocol as described earlier.[15]
Cell viability and proliferation assays
Cell viability was assessed by a MTT kit on 96-well plates using kit’s
manufacturer protocol. Proliferation of MIN6 cells was evaluated
with a colorimetric ELISA-based BrdU incorporation kit. Assays were
performed in accordance with manufacturer protocol instructions.[15]
Spectrophotometric quantification of PL activity
and assaying PL inhibition of test extracts and
compounds
The aqueous extract was evaporated until dryness under vacuum at 40°C
using a rotary evaporator and dissolved in Tris-HCl buffer. The reference
drug orlistat, rosmarinic acid, vitexin, and the AE were prepared in six
different concentration ranges and in vitro enzymatic PL activity was
assayed as described earlier in triplicates.[16,17]
In vitro enzymatic starch digestion assay
In vitro enzymatic starch digestion was assayed with acarbose as the
reference drug.[18] The extent of polysaccharide breakdown into glucose
was evaluated for the AE in seven concentrations (1, 5, 10, 12.5, 25, 50,
and 100 mg/mL). The effects of acarbose at 1000 μg/mL concentration
were performed in triplicates.
Glucose movement in vitro assay
In vitro glucose movement was assayed as described earlier with guar
gum (50 mg/mL) as a positive control.[15] A. hygrophilum AEs 10, 25,
and 50 mg/mL in 0.22 M glucose in triplicates were dialyzed against
0.15 M NaCl overnight at 37°C with gentle shaking. A parallel plant-free
(negative) control was included.[19]
In vivo confirmatory studies: Oral starch tolerance
test (OSTT) and oral glucose tolerance test (OGTT)
OSTT and OGTT were conducted as described earlier in the Experimental
Animal Laboratory of the School of Medicine, The University of Jordan
using rats (Rattus rattus) of both sexes weighing 220-260 g, applying
The University of Jordan ethical guidelines for animal protection.[20]
Experimental approval was obtained from the Scientific Research
Council at the Deanship of Academic Research. Aqueous extracts were
administered under mild anesthesia in doses 125, 250, and 500 mg/kg
body weight (n = 6 for each group).
In vitro antiproliferative assay
The cytotoxicity measurements with colorectal cell lines HT29, HCT116,
and SW620 were determined using sulforhodamine B (SRB) colorimetric
assay for cytotoxicity screening and mechanism of reduction of cell
viability as described previously.[21] Doxorubicin IC50 values were
calculated within treatment concentration range 0.1-50 μg/mL.
Statistical analysis
The values are presented as mean ± standard error of mean (SEM) of
three to six independent experiments. Statistical differences between
Pharmacognosy Magazine, Volume 13, Issue 50, April-June, 2017
FATMA U. AFIFI, et al.: Arum hygrophilum Phytochemistry and Biology
Figure 1: HPLC-MS chromatogram of Arum hygrophilum. (1) gallic acid;
(2) caffeic acid; (3) isoorientin; (4) vitexin; (5) ferulic acid; (6) quercetin-3O-rhamnoside; (7) luteolin
Table 1: Modulatory effects of A. hygrophilum AE on the viability of pancreatic
β-cells MIN6 in 48 h after seeding. Each result indicates the mean ± SEM of
four independent experiments
Treatment
MIN6 viability (%
control)
Control incubations (plant free)
99.5 ± 10.4
A. hygrophilum AE (0.01 mg/mL)
179.2 ± 7.3***
A. hygrophilum AE (0.05 mg/mL)
154.0 ± 15.2***
A. hygrophilum AE (0.1 mg/mL)
134.2 ± 16.7***
A. hygrophilum AE (0.5 mg/mL)
166.7 ± 4.2***
A. hygrophilum AE (1 mg/mL)
123.6 ± 17.1*
A. hygrophilum AE (5 mg/mL)
142.1 ± 4.8***
A. hygrophilum AE (10 mg/mL)
131.0 ± 16.3***
*P<0.05 and ***P<0.001 compared to control (plant-free) incubations.
control and different treatment groups and AUCs (incremental area
under 24-h glucose curve) were determined using Graph Pad Prism
one-way analysis of variance (ANOVA) followed by Dunnett’s posttest
whenever appropriate (version 3.02 for windows; Graph Pad Software,
San Diego, CA, USA). AUCs, also, were calculated by Graph Pad Prism.
Values were considered significantly different if P was less than 0.05 and
highly significantly different if P was less than 0.01 and less than 0.001.
RESULTS AND DISCUSSION
Arum is a genus of about 26 species of flowering plants in the family
Araceae, native to different parts of the globe with the highest species
diversity in the Mediterranean region. In Jordan, it is represented by
three species: A. palaestinum, A. dioscoridis, and A. hygrophilum.[22]
In our previous investigations with the former two species, several
flavonoids were isolated, and subsequently flavonoids, coumarins, and
plant acids were identified using LC-MS.[8,9] The isolated isoorientin
showed myolytic activity on smooth muscle containing preparations
from the rat and guinea pig.[23] Also, the antiproliferative activities of
both species were evaluated using different cancer cell lines.[9,24]
In the present study, using conventional column chromatography, from
the butanol and ethanol extracts of A. hygrophilum, three flavonoids
(luteolin, isoorientin, and vitexin) and rosmarinic acid were isolated.
Chloroform extract yielded β-sitosterol. The structures of the isolated
compounds were determined using their physical properties and
the different spectroscopic spectra (UV, IR, 1H-NMR, 13C-NMR).
The obtained results were in agreement with the values reported for
them.[25-28] For all isolated compounds, melting points and mixed melting
points with the reference substances were determined and confirmed.
HPLC-MS analysis of the ethanol extract revealed in addition to the isolated
flavonoids the presence of quercetin-3-O-rhamnoside (3.33 mg/mL)
and several plant acids, caffeic(3.33 mg/mL), ferulic (0.58 mg/mL), and
gallic acid (3.58 mg/mL) [Figure 1]. The in vitro antioxidant efficacy and
Pharmacognosy Magazine, Volume 13, Issue 50, April-June, 2017
Figure 2: Modulatory effects of A. hygrophilum AE (0.01–25 mg/mL) on
function of MIN6 pancreatic β-cells. Such augmentation of GSIS following
acute 1-h treatments was evaluated by rat insulin ELISA. A. hygrophilum
treatment wells were co-incubated in corresponding 5.6 mM glucose.
Each bar indicates the mean ± SEM of four determinations. *P < 0.05
compared to respective 5.6 mM glucose (negative) control wells; ∆P <
0.05 compared to respective treatment conditions in the presence of 2.5
mM Ca2+.
free radical scavenging properties assessment of A. hygrophilum ethanol
extract, expressed as trolox equivalent in micromols per milligram,
exhibited good correlation between the values obtained for TEAC
(115.29 ± 5.75), DPPH (4.44 ± 0.22), and ORAC (3.61 ± 0.18) and
identified polyphenolic compounds of the extract.
Glucose-dependent modulation of glucose
stimulated insulin secretion (GSIS) in pancreatic
β-cell by A. hygrophilum AEs
L-Alanine (10 mM) was used as a positive control, which enhanced
substantially (P < 0.05) GSIS in MIN6 by 178.5 ± 17.9% (n = 4) following
1-h incubations, compared to untreated (glucose only) controls
[Figure 2].[29] With obvious unlikeness to L-alanine, A. hygrophilum AE
doses lacked any marked augmentation of MIN6 GSIS in acute treatment
wells compared with controls [Figure 2]. Surprisingly, A. hygrophilum
AE (10 mg/mL) seemed to antagonize pancreatic GSIS significantly
(P < 0.05) [Figure 2]. Cell viability was unaffected, negating against plant
inflected cytotoxicity. Changes in β-cell cytosolic Ca2+ concentrations,
whether by an influx of extracellular Ca2+ or by release of Ca2+ from
intracellular stores, are thought to be a primary trigger for the initiation
of insulin exocytosis machinery. Figure 2 illustrates that the marked
insulinotropic trend of l-alanine was highly significantly (54.9 ± 8.6%,
P < 0.001) abolished in Ca2+ depleted KRH, as compared to corresponding
Ca2+ free glucose-only (negative control) wells. Apparently, Ca2+ depleted
A. hygrophilum treatments (0.01 mg/mL) had substantial reduction
(P < 0.05) in pancreatic secretory function, compared with respective
Ca2+ buffered conditions [Figure 2].
Pancreatic β-cell viability/expansion modulation by
A. hygrophilum AEs
Compared to control untreated cells, the MTT method revealed that
A. hygrophilum AEs (0.01-10 mg/mL) treatment in 48h post seeding
preserved β-cell integrity. A. hygrophilum induced highly significantly
pancreatic monolayers expansion by 1.23-1.76 folds (P<0.05-0.001
vs. basal plant-free control, Table 1). The higher concentrations,
277
FATMA U. AFIFI, et al.: Arum hygrophilum Phytochemistry and Biology
Table 2: Effect of ascending concentrations of A. hygrophilum (AE) (mg/mL) on percentage reduction of enzymatic starch digestion in vitro. Results expressed as
percentage decrease in control values are mean ± SEM (n = 3 independent replicates). *P < 0.05 and ***P < 0.001 compared to control (drug-free or plant-free)
incubations
Plant AE (mg/mL)
A. hygrophilum
0.1
0.3 ± 1.9
0.5
4.5 ± 1.2
1
7.0 ± 0.9***
Figure 3: In vitro inhibitory effects of A. hygrophilum (AE) (IC50 = 1.7 ± 0.01
mg/mL), vitexin (IC50 = 51.3 ± 7.6 μg/mL [0.6 ± 0.1 mM]), rosmarinic acid
(IC50 = 260.9 ± 21.1 μg/mL [0.1 ± 0.02 mM]), and orlistat (IC50 = 114.0 ± 4.0
ng/mL [0.2 ± 0.0 μM]) on pancreatic triacylglycerol lipase activity. Results
are mean ± SEM (n = 3 independent replicates)
however, proved ineffective and noncytotoxic (the same table). A
colorimetric immunoassay of BrdU incorporation into MIN6 β-cell
genome was recruited to ascertain proliferative principles of chronic
plants treatments. The gut hormone glucagon-like peptide-1 (GLP-1)
agonists have been shown to stimulate the growth and differentiation
of pancreatic cells, as well as to exert cytoprotective and antiapoptotic
effects on β-cells.[30] GLP-1 (500 nM) highly significantly promoted
a maximal extent of BrdU incorporation by 1.33-1.5-fold (P < 0.001,
n = 4) in comparison to basal BrdU incorporation (spontaneous
control). Similar to 48-h MTT findings, A. hygrophilum AE 0.5 and 1
mg/mL induced a highly substantial concentration related increase in
pancreatic BrdU incorporation (with respective 1.33 and 1.41 folds, P <
0.001 vs. basal controls). With its safety profile, pancreatic proliferative
capacities could be ascribed to A. hygrophilum AEs. This suggests the
plant's potential utility in diabetes regenerative therapeutics.[15] The
present study has revealed that water-soluble bioactive principles in
A. hygrophilum AEs lacked any glucose-evoked insulin-releasing effect
in pancreatic β-cells, unlike many other herbal remedies reputed for
substantial insulin secretagogue activity.[15,29]
In vitro inhibitory effects of A. hygrophilum AEs,
vitexin, and rosmarinic acid on PL activity
PL inhibition is one of the most widely studied mechanisms to determine
the potential efficacy of natural products and ethnomedicinal botanicals
as obesity-modulating agents, since they are generally considered to
be less toxic with less side effects than totally synthetic compounds.[16]
In this current study, the pancreatic triacylglycerol antilipase activity
profiles of the crude AE of A. hygrophilum, vitexin, and rosmarinic
acid were determined [Figure 3]. Orlistat's PL-IC50 value of 114.0 ± 4.0
ng/mL, equivalent to 0.2 ± 0.0 μM, is comparable to reported PL-IC50
values.[16] Comparable to orlistat performance, a marked concentrationdependent PL inhibition trend was obtained per tested extracts as well as
their isolated components [Figure 3]. PL-IC50 values obtained for triple
separate determinations are also illustrated [Figure 3]. The importance
of polyphenolic substances as potential inhibitors of PL were discussed
recently by Buchholz and Melzig.[31] Several flavonoids, as the largest
class of polyphenolic substances, have been evaluated and the studies
indicated that the flavonoids having hydroxyl or methoxy groups
278
1.25
6.9 ± 1.1***
2.5
7.5 ± 1.0***
5
17.4 ± 2.1***
10
22.2 ± 0.4***
Figure 4: In vitro effects of A. hygrophilum AE concentrations (mg/mL) on
the incremental AUC of 24-h glucose movement. ***P < 0.001 compared
to control (drug-free or plant-free) incubations
at C3’ and C4’ in ring B as well as C-glycosidic flavones favor the PL
inhibition.[31-33] Additionally, the efficacy of phenolic acids, namely
caffeic-, rosmarinic-, and ferulic acids, as PL inhibitors are well
described.[34,35] In effect, the results of the present study indicate that
the PL inhibitory efficacy of A. hygrophilum may be attributable to their
multiple phenolic components acting additively or synergistically.[36]
In vitro extrapancreatic inhibitory effects of A.
hygrophilum AE on α-amylase/α-glucosidase
In acarbose (0.1 mg/mL) incubations as the reference drug, glucose
liberation from starch was inhibited by 97.6% highly substantially (P <
0.001, vs. drug-free control incubations, n = 3). With an IC50 value of 30.5
± 2.1 mg/mL, the significant dose-related (P < 0.001) percentage decreases
in enzymatic starch hydrolysis by A. hygrophilum dosage gradient
(1-10 mg/mL) are summarized in Table 2. The overall dual α-amylase
and α-glucosidase inhibitory propensities of A. hygrophilum could be
the result of the combination of its several constituents performing in
concert. Additionally, similar digestive enzymes modulatory outcomes
were obtainable for both A. dioscoridis and A. palaestinum.[9,18,20]
Extrapancreatic modulation of glucose movement
in vitro by A. hygrophilum AEs
Using the diffusion model as described; mean AUCs (area under 24-h
glucose curve) for the viscous water-soluble gel-forming guar gum
(50 mg/mL) were decreased highly significantly by 30.8 ± 2.5%
(P < 0.001, n = 3) [Figure 4] compared to overnight negative control. The
efficacy of guar gum as a classical positive control has been elsewhere
detailed.[37] Incomparable to guar gum, A. hygrophilum AEs (10, 25,
and 50 mg/mL) lacked any marked glucose diffusional hindrances into
external solution across dialysis membrane (with respective 8.1 ± 4.1%,
8.8 ± 5.1% AUC % reductions in A. hygrophilum 10, 25, and 50 mg/mL
overnight incubations, P > 0.05) [Figure 4].
Confirmatory in vivo studies: OSTT and OGTT
The administration of acarbose 3 mg/kg body weight reduced highly
significantly the starch-induced postprandial hyperglycemia at 45,
90, and 135 min after corn starch load at 0 min, thus evoking highly
substantial reduction (P < 0.001 vs. untreated animals, n = 6) of the
overall glycemic excursion AUC compared to controls. Compared
Pharmacognosy Magazine, Volume 13, Issue 50, April-June, 2017
FATMA U. AFIFI, et al.: Arum hygrophilum Phytochemistry and Biology
to control normal rats, administration of A. hygrophilum AEs in
starch loaded fasting normoglycemic rats did not minimize in overall
glycemic excursions, neither did they reduce the acute starch-induced
postprandial hyperglycemia at any determination time point. In the
OGTT oral administration of A. hygrophilum AEs did not evoke any
marked improvement of glucose tolerance AUCs in comparison to
control determinations respective AUCs contrary to metformin and
glipizide therapeutic propensities metformin (300 mg/kg body weight)
or glipizide (0.6 mg/kg body weight). Equally A. dioscoridis and A.
palaestinum lacked in vivo efficacies in acute carbohydrate tolerance tests
performed in overnight fasting normoglycemic animals.[9]
Antiproliferative activity in colorectal cancer cell
lines
Like A. dioscoridis and A. palaestinum;[9] A. hygrophilum (25 μg/mL)
aqueous extract lacked on antiproliferative efficacies in any of the
colorectal carcinomas panel incubations; despite its notable activity
at the concentration 200 μg/mL. Doxorubicin respective IC50 (μg/mL)
values for the tested cell lines were 0.09 ± 0.01 (HT29); 0.11 ± 0.02
(HCT116), and 0.7 ± 0.01 (SW620).
CONCLUSIONS
Flavonoids and phenolic acids are considered valuable in the maintenance
of glucose homoeostasis by different mechanisms. In the present study, A.
hygrophilum AEs exhibited pancreatic MIN6 proliferative propensities.
A. hygrophilum AEs inhibited crucial gastrointestinal enzymes involved
in carbohydrate and lipid digestion and absorption. Our findings with
the colon cancer cell lines indicate that traditionally claimed anticancer
properties of A. hygrophilum have to be pharmacologically evidenced.
Still, further studies with other cancer cell lines are warranted.
Acknowledgement
This work was supported by a grant (UJ 1134) of the Deanship of
Academic Research, The University of Jordan and the Scientific Research
Fund, The Ministry of Higher Education (MPH/1/05/2014).
Financial support and sponsorship
Nil
9. Afifi F, Kasabri V, Litescu SC, Abaza IF. In vitro and in vivo comparison of the biological
activities of two traditionally and widely used Arum species from Jordan: Arum dioscoridis
Sibth and Sm and Arum palaestinum Boiss. Nat Prod Res 2015;1-10.doi:10.1080/14786419.
2015 1072713.
10. Karahan F, Kulak M, Urlu E, Gozuacik HG, Boyümez T, Şekeroğlu N, et al. Total phenolic
content, ferric reducing and DPPH scavenging activity of Arum dioscoridis. Nat Prod Res
2014;29:1678-83.
11. Hamdan II, Afifi FU. Studies on the in vitro and in vivo hypoglycemic activities of some
medicinal plants used in treatment of diabetes in Jordanian traditional medicine. J
Ethnopharmacol 2004;93:117-21.
12. Cristea V, Deliu C, Oltean B, Keul A, Brummer A, Albu C, et al. Soilless cultures for
pharmaceutical use and biodiversity conservation. Acta Horticul 2009;843:157-64.
13. Hammad HM, Matar SA, Litescu SC, Abuhamdah S, Al Jaber HI, Afifi FU. Biological activities
of the hydro-alcoholic and aqueous extracts of Achillea fragrantissima (Forssk.) grown in
Jordan. Nat Sci 2014;6:23-30.
14. Litescu SC, Eremia S, Radu GL. Methods for the determination of antioxidant capacity in
food and raw materials. Adv Exp Med Biol 2010;698:241-9.
15. Kasabri V, Abu-Dahab R, Afifi FU, Naffa N. Modulation of pancreatic MIN6 insulin secretion
and proliferation and extrapancreatic glucose absorption with Achillea santolina, Eryngium
creticum and Pistacia atlantica extracts: In vitro evaluation. J Exp Integ Med 2012;2:
245-54.
16. Habtemariam S. The antiobesity potential of sigmoidin A. Pharm Biol 2012;50:1519-22.
17. Bustanji Y, Issa A, Mohammad M, Hudaib M, Tawaha K, AlKhatib H, et al. Inhibition of
hormone sensitive lipase and pancreatic lipase by Rosmarinus officinalis extract and selected
phenolic constituents. J Med Plant Res 2010;4:2235-42.
18. Foddai M, Kasabri V, Petretto GL, Azara E, Sias A, Afifi FU, et al. In vitro inhibitory effects
of Limonium contortirameum (Mabille) Erben and Limonium virgatum (Willd) Fourr extracts
from Sardinia on α-amylase, α-glucosidase and pancreatic lipase. Nat Prod Commun
2014;9:181-4.
19. Gallagher AM, Flatt PR, Duggy G, Abdel-Wahab YHA. The effects of traditional antidiabetic
plants on in vitro glucose diffusion. Nutr Res 2003;23:413-24.
20. Kasabri V, Afifi FU, Hamdan II In vitro and in vivo acute antihyperglycemic effects of five
selected indigenous plants from Jordan used in traditional medicine. J Ethnopharmacol
2011;133:888-96.
21. Abu-Dahab R, Kasabri V, Afifi FU Evaluation of volatile oil composition and antiproliferative
activity of Laurus nobilis L (Lauraceae) on breast cancer cell line models. Rec Nat Prod
2014;8:136-47.
22. AL-Eisawi DM List of Jordan vascular plants. Mitt Bot München 1982;18:179-182.
23. Afifi FU, Khalil E, Abdalla S Effect of isorientin isolated from Arum palaestinum on uterine
smooth muscle of rats and guinea pigs. J Ethnopharmacol 1999;65:173-7.
Conflicts of interest
24. Abu-Dahab R, Afifi FU Aniproliferative activity of selected medicinal plants of Jordan against
a breast adenocarcinoma cell line. Sci Pharm 2007;75:121-36.
There are no conflicts of interest
25. Aggrawal PK Carbon-13 NMR of flavonoids. In Studies in Organic Chemistry, Vol.39.
REFERENCES
26. Nawwar MAM, El-Mousallamy AMD, Barakat HH, Buddrus J, Linscheid M Flavonoid lactates
1. Afifi-Yazar FU, Kasabri V, Abu-Dahab R. Medicinal plants from Jordan in the treatment
of diabetes: traditional uses vs. in vitro and in vivo evaluations-Part 2. Planta Med
2011;77:1210-20.
Amsterdam: Elsevier; 1989:320-21, 329-31.
from leaves of Marrubium vulgare. Phytochemistry 1989;28:3201-6.
27. Harborn JB The flavonoids: Advances in Research Since 1986. London: Chapman and Hall;
1994. 448-50.
2. Afifi FU, Wazaify M, Jabr M, Treish E. The use of herbal preparations as complementary
28. Zhang Y, Jiao J, Liu C, Wu X, Zhang Y. Isolation and purification of four flavone
and alternative medicine (CAM) in a sample of patients with cancer in Jordan. Complement
C-glycosides from antioxidant of bamboo leaves by macroporous resin column
Therap Clin Pract 2010;16:208-12.
chromatography and preparative high-performance liquid chromatography. Food
3. Wang CZ, Calway T, Yuan CS. Herbal medicines as adjuvants for cancer therapeutics. Am J
Chin Med 2012;40:657-69.
4. Tschop MH, DiMarchi RD. Outstanding scientific achievement award lecture 2011: defeating
diabesity: the case for personalized combinatorial therapies. Diabetes 2012;61:1309-14.
5. Rani N, Sharma SK, Vasudeva N. Assessment of antiobesity potential of Achyranthes aspera
Linn.seed. Evid Based Complement Alternat Med 2012 2012;715912.
6. Sun X, Zhang K, Ji X, Wang Y, Jeffrey Z, Tong Y, et al. Screening of pancreatic lipase and
alpha-glucosidase inhibitors from Chinese dietary herbs. Zhonqquo Zhonq Yao Za Zhi
2012;37:1319-23.
7. Abu-Irmaileh B, Afifi F. Treatment with medicinal plants in Jordan. Dirasat 2000;27:53-74.
8. Afifi FU, Shervington A, Darwish R. Phytochemical and biological evaluation of Arum
palaestinum Part 1: Flavone C-glycosides. Acta Technolog Legis Medicament 1997;VIII:105-11.
Pharmacognosy Magazine, Volume 13, Issue 50, April-June, 2017
Chem 2008;107:1326-36.
29. Kasabri V, Flatt PR, AbdelWahab Y In vitro modulation of pancreatic insulin secretion and
extrapancreatic insulin action, and peptide glycation by Curcuma longa aqueous extracts. J
Exp Integ Med 2014;4:187-93.
30. List JF, Habener JF. Glucagon-like peptide 1 agonists and the development of and growth of
pancreatic β-cells. Am J Physiol Endocrinol Metab 2004;286:E875-81.
31. Buchholz T, Melzig MF. Polyphenolic compounds as pancreatic lipase inhibitors. Planta Med
2015;81:771-83.
32. Lee EM, Lee SS, Chung BY, Cho JY, Lee IC, Ahn SR, et al. Pancreatic lipase inhibition by
C-glycosidic flavones isolated from Eremochloa ophiuroides. Molecules 2010;15:8251-9.
33. Shimura S, Itoh Y, Yamashita A, Kitano A, Hatano T, Yoshida T, et al. Inhibitory effects of
flavonoids on lipase. Nippon Shokuhin Kogyo Gakkaishi 1994;41:847-50.
279
FATMA U. AFIFI, et al.: Arum hygrophilum Phytochemistry and Biology
34. Dalar A, Türker M, Zabaras D, Konczak I. Phenolic composition, antioxidant and enzyme
inhibitory activities of Eryngium bornmuelleri leaf. Plant Foods Hum Nutr 2014;69:30-6.
36. Chuang CM, Wang HE, Peng CC, Chen KC, Peng RY. Hypolipidemic effects of different
angiocarp parts of Alpinia zerumbet. Pharm Biol 2011;49:1257-64.
35. Y-HS Wu, Chiu CH, Yang DJ, Lin YL, Tseng JK, Chen YC. Inhibitory effects of litchi (Litchi
37. Butt MS, Ahmad A, Sharif MK. Influence of pectin and guar gum composite flour on plasma
chinensis Sonn.) flower-water extracts on lipase activity and diet-induced obesity. J Funct
biochemical profile of streptozocin-induced diabetic male albino rats. Int J Food Prop
Foods 2013;5:923-9.
2007;10:345-61.
280
Pharmacognosy Magazine, Volume 13, Issue 50, April-June, 2017