molecules

Article
Acute Hypoglycemic and Antidiabetic Effect of
Teuhetenone A Isolated from Turnera diffusa
Aída Parra-Naranjo 1,† , Cecilia Delgado-Montemayor 1,† , Alejandra Fraga-López 1 ,
Gabriela Castañeda-Corral 2 , Ricardo Salazar-Aranda 1 , Juan José Acevedo-Fernández 2 and
Noemi Waksman 1, *
1 Departmento de Química Analítica, Facultad de Medicina, Universidad Autónoma de Nuevo León,
Monterrey, N.L., C.P. 64460, Mexico; ap._183@hotmail.com (A.P.-N.);
ceciliadmontemayor@gmail.com (C.D.-M.); jany1524@hotmail.com (A.F.-L.);
salazar121212@yahoo.com.mx (R.S.-A.)
2 Depto. de Fisiología y Fisiopatología, Facultad de Medicina, Universidad Autónoma del Estado de Morelos,
Calle Leñeros s/n, Col. Los Volcanes, Cuernavaca Mor. C.P. 62350, Mexico;
gabriela.castaneda@uaem.mx (G.C.-C.); juan.acevedo@uaem.mx (J.J.A.-F.)
* Correspondence: nwaksman@gmail.com; Tel.: +52-8183294185
† Both authors contributed equally.

Academic Editor: Thomas J. Schmidt

Received: 10 February 2017; Accepted: 29 March 2017; Published: 8 April 2017

Abstract: Diabetes mellitus is a chronic degenerative disease that causes long-term complications
and represents a serious public health problem. Turnera diffusa (damiana) is a shrub that grows
throughout Mexico and is traditionally used for many illnesses including diabetes. Although a
large number of plant metabolites are known, there are no reports indicating which of these are
responsible for this activity, and this identification was the objective of the present work. Through
bioassay-guided fractionation of a methanolic extract obtained from the aerial part of T. diffusa,
teuhetenone A was isolated and identified as the main metabolite responsible for the plant’s
hypoglycemic activity. Alpha-glucosidase inhibitory activity and cytotoxicity of this metabolite
were determined. Hypoglycemic and antidiabetic activities were evaluated in a murine model of
diabetes in vivo, by monitoring glucose levels for six hours and comparing them with levels after
administering various controls. Teuhetenone A was not cytotoxic at the tested concentrations, and
did not show inhibitory activity in the glucosidase test, and the in vivo assays showed a gradual
reduction in glucose levels in normoglycemic and diabetic mice. Considering these results, we suggest
that teuhetenone A has potential as an antidiabetic compound, which could be further submitted to
preclinical assays.

Keywords: diabetes mellitus; damiana; teuhetenone A; antidiabetic; Turnera diffusa

1. Introduction
Type 2 diabetes mellitus (T2DM) is a chronic degenerative disease characterized by hyperglycemia,
mainly due to defects of insulin secretion and/or action. The disorder is associated with an imbalance
of the glycemic index and glucose intolerance, which increases the risk of individuals progressing to
T2DM. Complications from diabetes, such as coronary artery and peripheral vascular disease, stroke,
diabetic neuropathy, risk of ulcers and amputations, renal failure, sexual dysfunction, and blindness
are resulting in increasing disability, reduced life expectancy, and enormous health costs for virtually
every society [1,2].
T2DM is a leading cause of morbidity and mortality in industrialized and developing countries,
resulting in part from high-carbohydrate and high-fat diets that lead to excess weight and/or obesity,

Molecules 2017, 22, 599; doi:10.3390/molecules22040599 www.mdpi.com/journal/molecules

Molecules 2017, 22, 599 2 of 13

as well as reduced physical activity. The disease is endemic in low- and middle-income countries.
In 2015, around two-thirds of all individuals with diabetes lived in such countries. The consequences
of high-carbohydrate and high-fat diets are most severe in communities in developing countries, where
poverty represents an additional problem [3,4].
T2DM is a serious public health problem. According to the World Health Organization, more than
422 million people worldwide are affected [5]. Its prevalence is increasing rapidly, and it is estimated
that the number affected will double by 2040. Diabetes is one of the major causes of morbidity and
mortality worldwide. In 2015, five million people died from complications of diabetes, making the
disease and its complications the leading cause of death in most countries. In Mexico, 11.5 million
people have diabetes, and Mexico is among the 10 countries with the highest prevalence of this
disease. According to the National Institute of Statistics and Geography data of 2015, the percentage of
deaths attributed to diabetes in Mexico is 15%, placing it as one of the main causes of mortality in the
country [6].
T2DM is a complex disease, and a key component of the management process is to reinstate
glycemic control to as near normal levels as is practicable, safe, and comfortable for the patient.
This goal is achieved by lifestyle adaptation, particularly in regards to diet and exercise, health
education, and pharmacological treatment. The last approach includes insulin and hypoglycemic
agents (including sulfonylureas, biguanides, thiazolidinediones, and α-glucosidase inhibitors), and
focuses on increasing insulin levels, improving sensitivity to the hormone in tissues, or reducing the
rate of carbohydrate absorption from the gastrointestinal tract [7,8]. However, despite being effective,
these agents provide inadequate glycemic control, and/or are associated with side effects or non-
adherence. The most frequently-reported adverse effects of these agents include hypoglycemia, weight
gain, edema, and heart failure [9]. Alternatives are being sought for affected diabetic patients, for
whom the development of new hypoglycemic therapies is desirable.
Plants have formed the basis of traditional medicine systems around the world for thousands of
years. Even in modern systems, ethnobotanical treatments continue to play an important role in health
care. About 80% of the population in developing countries rely on traditional medicine for their health
care. In Mexico, there is a great diversity of medicinal plants. Andrade et al. reported on 306 species of
Mexican plants used as hypoglycemic agents [10]. Despite the large number of plants that have been
reported for their empirical use as antidiabetics, only a small percentage of these plants have been
studied in a systematic way, and the molecules responsible for the biological activity of most of them
are unknown.
Within the list of Mexican plants used as hypoglycemic agents, Turnera diffusa (damiana) attracted
our attention. This shrub grows in arid and semiarid regions of South America, Mexico, the United
States, and the West Indies. Throughout history, various properties have been attributed to this plant.
Usually, the leaves of the plant are used to prepare a decoction [11,12]. Phytochemical investigations
have been conducted to isolate and identify some components present in the plant, among which are
flavonoids, sesquiterpenes, triterpenes, polyterpenes, fatty acids, and xanthine-derived sugars [13,14].
However, few reports exist [15–18] that provide evidence of its use for hypoglycemia. Therefore, the
objective of the present study was to determine the basis of such use, by identifying the compounds
responsible for its hypoglycemic activity.

2. Results

2.1. Isolation and Identification

In the first pharmacological approach to T. diffusa derivatives, the hypoglycemic activity of
methanolic extracts of the plant was investigated in normoglycemic mice, as described in the
Materials and Methods section (Figure 1). The results obtained corroborated the use of this plant for
hypoglycemia. By means of a bioassay-guided fractionation, we determined that the terpenoid-rich
fraction was responsible for the activity. In this fraction, we found a main active component (Figure 2),
Molecules 2017, 22, 599 3 of 13

which was isolated and identified by spectroscopy, comparing spectral data with those reported in
Molecules
the 2017,
2017, 22,
literature.
Molecules 22, 599
599 33 of
of 13
13

Control
Control
1.1
1.1 Ext
Ext MeOH
MeOH
1.0
1.0

0.9

(normalized)
0.9
Glucose(normalized) 0.8
0.8

0.7
0.7
Glucose

0.6
0.6

0.5
0.5

0.4
0.4

0 1 2 3 4 5 6
0 1 2 3 4 5 6
Time
Time (hrs)
(hrs)
Time (h) Time (h)
Figure
Figure 1. Hypoglycemic effect of damiana methanolic extract on adult CD1 mice. Dosage
Dosageofof 0.5 mg/kg
Figure1.
1. Hypoglycemic
Hypoglycemic effect of damiana
effect of damiana methanolic
methanolicextract
extractononadult
adultCD1
CD1mice.
mice.Dosage of
0.50.5 mg/kg
mg/kg
administered
administered intraperitoneally. n = 5, Mean ± standard error (SE).
administeredintraperitoneally.
intraperitoneally. nn ==5,5,Mean
Mean±±standard
standarderror
error(SE).
(SE).

Figure 2. Structure
Figure 2. Structure of
of teuhetenone
teuhetenone A.
A.
25
Teuhetenone
Teuhetenone A
Teuhetenone A was
wasobtained
was obtainedas
obtained asaspale
pale yellow
paleyellow
yellowoil (AcOEt),
oiloil
(AcOEt),
(AcOEt),95.37%
95.37% chromatographic
chromatographic
95.37% chromatographic purity;
purity; [α]
[α]D25D
purity;
+31 (c 3, 400 MHz) δH 6.35 (1H, s, H-6), 2.57 (1H, ddd, J=17.9, 14.8, 5.2 Hz,
(c 0.1,
0.1, MeOH),
MeOH), 1H-NMR(CDCl
1
+3125 1
H-NMR(CDCl 3, 400 MHz) δH 6.35 (1H, s, H-6), 2.57 (1H, ddd, J=17.9, 14.8, 5.2 Hz,
[α] D + 31 (c 0.1, MeOH), H-NMR(CDCl3 , 400 MHz) δH 6.35 (1H, s, H-6), 2.57 (1H, ddd, J = 17.9,
H-8b),
H-8b),
14.8, 2.38(1H,
5.22.38(1H,
Hz, H-8b),m, H-8a),
m, H-8a), 1.98
2.38 (1H,1.98m, (1H,
(1H, m,
m, H-3b),
H-8a), H-3b), 1.90 (1H,
1.90 m,
1.98 (1H, (1H, m,
m, H-9b),
H-3b), 1.90 1.79
H-9b), 1.79 (1H,
(1H, (1H, m,
m, H-9a),
m, H-9b), 1.791.73
H-9a), (1H,(2H,
1.73 (2H, m,
m, H-
m, H-9a),H-
2),
2), 1.66
1.66 (1H,
(1H, m,
m, H-1b),
H-1b), 1.58
1.58 (1H,
(1H, m,
m, H-3a),
H-3a), 1.43
1.43 (3H,
(3H, s,
s, H-12),
H-12), 1.37
1.37 (1H,
(1H, m,
m, H-1a)
H-1a)
1.73 (2H, m, H-2), 1.66 (1H, m, H-1b), 1.58 (1H, m, H-3a), 1.43 (3H, s, H-12), 1.37 (1H, m, H-1a) y 1.31 y
y 1.31
1.31 (3H,
(3H, s,
s, H-11).
H-11).
13C-NMR (CDCl3, 400MHz) δC 200.7 (C, C-7), 175.3 (C, C-5), 122.7 (CH, C6), 72.6 (C, C-4), 42.4 (CH2,
(3H, s, H-11). 13 C-NMR
13C-NMR (CDCl 3, 400MHz)
(CDCl δC3 ,200.7 (C, C-7),
400 MHz) 175.3(C,
δC 200.7 (C,C-7),
C-5), 122.7
175.3 (C,(CH,
C-5),C6),
122.772.6 (C,C6),
(CH, C-4),
72.642.4
(C,(CH
C-4),2,
C-3),
42.4 41.0
41.02 ,(CH
C-3),(CH (CH 2, C-8), 40.9 (CH2, C-1), 36.4 (C, C-10), 34.1 (CH2, C-9), 29.9 (CH3, C12), 24.7 (CH3, C-11),
2, C-8),
C-3), 40.92 ,(CH
41.0 (CH 2, C-1),
C-8), 36.42(C,
40.9 (CH C-10),
, C-1), 36.434.1
(C, (CH 2, C-9),
C-10), 34.1 29.9
(CH2(CH 3, C12),
, C-9), 29.9 24.7
(CH3(CH 3, C-11),
, C12), 24.7
19.7
19.7 (CH
(CH 2, C-2). ESI/MS m
, C-2). ESI/MS m//zz 195
195 [M+H]
[M+H]
+.
m
(CH3 , C-11), 19.7 (CH2 , C-2). ESI/MS /z 195 [M + H] .
2 +. +

2.2.
2.2. Alpha-Glucosidase
2.2. Alpha-Glucosidase Inhibitory
Alpha-Glucosidase Inhibitory Activity
Inhibitory Activity of
Activity of Teuhetenone
of Teuhetenone
TeuhetenoneAA
A
The
The isolated
The isolated teuhetenone
teuhetenone A
isolated teuhetenone A was
A was analyzed
analyzed to
was analyzed to determine
determine its
to determine its inhibitory
its inhibitory activity
inhibitory on α-glucosidase.
activity on α-glucosidase.
α-glucosidase.
The
The results
The results of
resultsof this
ofthis experiment
thisexperiment are
experimentare shown
areshown in
shownin Table
inTable 1.
Table1.1.

Table
Table
Table1.1. Inhibitory
1.Inhibitory effect
Inhibitoryeffect ofof
effect teuhetenone
teuhetenone
of AA
teuhetenone onon
A on α-glucosidase
α-glucosidase
α-glucosidase (IC
(IC
(IC 50
50 50 , μg/mL)
, ,µg/mL)
μg/mL)a,b...
a,b
a,b

Compound
Compound
Compound IC
IC
IC 50
50 50
Teuhetenone
Teuhetenone
TeuhetenoneAA
A >330
>330
>330
Acarbose 210.5 ± 7.30
a Acarbose Acarbose210.5 ±as7.30 210.5 ± 7.30
Average of the triplicate (± SD). b Acarbose positive control.
a
a Average
Average of
of the
the triplicate
triplicate (±
(± SD).
SD). Acarbose
Acarbose as
as positive
positive control.
b
control.
b

2.3. Cytotoxic Activity of Teuhetenone A

2.3.
2.3. Cytotoxic
Cytotoxic Activity
Activity ofof Teuhetenone
Teuhetenone A A
The cytotoxicity of teuhetenone A was evaluated on a Vero cell line. No concentration tested was
toxic The
The cytotoxicity
so its CCof
cytotoxicity
to cells, teuhetenone
of was
50 teuhetenone AA was
considered was evaluated
than 500on
evaluated
greater on aa Vero
Vero cell
µg/mL. cell line.
line. No
No concentration
concentration tested
tested
was
was toxic
toxic to
to cells,
cells, so
so its
its CC
CC5050 was
was considered
considered greater
greater than 500 μg/mL.
than 500 μg/mL.
Molecules 2017, 22, 599 4 of 13

Molecules 2017, 22, 599 4 of 13

2.4. Hypoglycemic Activity of Teuhetenone A in CD1 Mice

2.4. Hypoglycemic Activity of Teuhetenone A in CD1 Mice
Under the experimental conditions used, levels of glycemia were decreased by about 20% for
Under the experimental conditions used, levels of glycemia were decreased by about 20% for six
six hours
hours of
of fasting
fasting in
in CD1
CD1 mice treatedwith
mice treated witha avehicle
vehiclecontrol,
control,andand 40%
40% after
after administration
administration of of
teuhetenone A intraperitoneally,
teuhetenone with
A intraperitoneally, no no
with difference in in
difference thethe
doses
dosesevaluated
evaluated(1(1and
and 55 mg/kg). Under the
mg/kg). Under
samethe
conditions, insulininsulin
same conditions, at 5 IU/kg had had
at 5 IU/kg a hypoglycemic
a hypoglycemic activity ofof50%
activity 50%(Figure
(Figure 3).

Control Glib 1 mg
Glib 5 mg Ins 5 UI
1.3
Teu A 1 mg Teu A 5 mg
1.2

1.1
Glucose (normalized)

1.0

0.9

0.8

0.7

0.6

0.5

0.4

0 1 2 3 4 5 6
Time
Time(h)
(hrs)
(a)
Control Glib 1 mg
200 Glib 5 mg Ins 5 UI
Teu A 1 mg Teu A 5 mg
180

160
Glucose (mg/dl)

140

120

100

80

60

0 1 2 3 4 5 6
Time (hrs)
Time (h)

(b)
Figure 3. Hypoglycemic effect of insulin, glibenclamide, and teuhetenone A in adult CD1 mice. Glib
Figure 3. Hypoglycemic effect of insulin, glibenclamide, and teuhetenone A in adult CD1 mice.
- glibenclamide; Teu A - teuhetenone A; Ins - insulin. n = 5, Mean: ±SE. (a) Graph with normalized
Glib -values;
glibenclamide;
(b) GraphTeu
withAunnormalized
- teuhetenonevalues. - insulin. n = blood
A; Ins Standardized 5, Mean: ±SE.for
glucose (a)baseline
Graph with normalized
glycemia to
values; (b) Graph with unnormalized values. Standardized
compare the kinetics of each experimental group. blood glucose for baseline glycemia to
compare the kinetics of each experimental group.
Molecules 2017, 22, 599 5 of 13
Molecules 2017, 22, 599 5 of 13

The kinetics obtained with teuhetenone A were similar to those observed after intraperitoneal
Molecules 2017, 22, 599 5 of 13
administration
The kineticsofobtained the insulin secretagogue
with teuhetenone glibenclamide
A were (1 and
similar to those 5 mg/kg);
observed however, the
after intraperitoneal
hypoglycemic
administration effect
The kinetics
of was higher
the obtained
insulin withthan
secretagoguethat
teuhetenonewith glibenclamide,
A were
glibenclamidesimilar to 5
(1 and a commercial
those observed
mg/kg); hypoglycemic
after
however, intraperitoneal agent
the hypoglycemic
administration
(Figure 4). of the insulin secretagogue glibenclamide (1 and 5 mg/kg);
effect was higher than that with glibenclamide, a commercial hypoglycemic agent (Figure 4). however, the
hypoglycemic effect was higher than that with glibenclamide, a commercial hypoglycemic agent
(Figure 4).
0.9

0.8
0.9

Glucose Reduction (normalized)

0.7
0.8
0.6

Glucose Reduction (normalized)

0.7
0.5
0.6

0.4 0.5

0.3 0.4

0.2 0.3

0.1 0.2
0.1
0.0
0.0 Control Ins Glib 1 Teu A 1 Glib 5 Teu A 5
Control Ins Glib 1 Teu A 1 Glib 5 Teu A 5

Figure 4. Maximum hypoglycemic effect of insulin, glibenclamide, and teuhetenone A in adult CD1
mice.
FigureVehicle: water; hypoglycemic
4. Maximum Ins – insulin; Glib 1 - glibenclamide (1 mg/kg); and
Glib teuhetenone
5 - glibenclamidein (5 mg/kg);
Figure 4. Maximum hypoglycemiceffect
effectof
ofinsulin, glibenclamide,
insulin, glibenclamide, and teuhetenone A inAadultadult
CD1 CD1
Teu A 1 - teuhetenone A (1 mg/kg); Teu A 5 - teuhetenone A (5 mg/kg). n = 5, Mean ± SE.
mice. Vehicle: water; Ins – insulin; Glib 1 - glibenclamide (1 mg/kg); Glib 5 - glibenclamide (5 mg/kg);
mice. Vehicle: water; Ins – insulin; Glib 1 - glibenclamide (1 mg/kg); Glib 5 - glibenclamide (5 mg/kg);
Teu ATeu
1 -Ateuhetenone A (1
1 - teuhetenone A mg/kg);
(1 mg/kg);Teu
TeuAA55--teuhetenone
teuhetenone AA(5(5mg/kg).
mg/kg).n =n5,= Mean
5, Mean ± SE.
± SE.
2.5. Antidiabetic Activity of Teuhetenone A
2.5. Antidiabetic
2.5. Antidiabetic Activity
Activity of TeuhetenoneAA
of Teuhetenone
In the murine model of diabetes used in the present study, insulin secretion was limited by
In the Inmurine
the pancreotoxicthe murine
effect
modelmodel of of
of alloxan, diabetes
diabetes usedin
so glycemia
used inthe
the present study,
increased
present from
study, insulin
140–155
insulin secretion
tosecretion
340–380waswaslimited
mg/dL by the
(Figure
limited 5),
by the
and pancreotoxic effect
insulin administration of alloxan,
(5 so so
IU/kg) glycemia
reducedincreased from
bloodfrom 140–155
glucose to
by 35% 340–380 mg/dL
at the time (Figure 5), and
of administration.
pancreotoxic effect of alloxan, glycemia increased 140–155 to 340–380 mg/dL (Figure 5), and
insulin administration (5 IU/kg) reduced blood glucose by 35% at the time of administration. As
As observed
insulin in normoglycemic
administration (5 IU/kg)mice, glucose
reduced bloodkinetics
glucoseobserved
by 35% after insulin
at the timeadministration
of administration. showed As
observed in normoglycemic mice, glucose kinetics observed after insulin administration showed that
that the
observed effect was maximal at the time of administration, and that glucose
in normoglycemic mice, glucose kinetics observed after insulin administration showed that levels reverted during the
the effect was maximal at the time of administration, and that glucose levels reverted during the
evaluation.
the effect However,
was
evaluation. maximal
However,in the mouse
atinthe
the time
mousemodel
ofmodelof diabetes,
administration, theand
of diabetes, vehicle
the that control
vehicleglucose
controlbarely
levels
barelyreduces
reverted
reduces glucose
during
glucose levels
the
by 10%. By
evaluation. contrast,
However, with
in the effect
mouse observed
model in
of normoglycemic
diabetes, the mice,
vehicle the effect
control of
barely
levels by 10%. By contrast, with the effect observed in normoglycemic mice, the effect of teuhetenone teuhetenone
reduces A was
glucose
dose-dependent
levelsAbywas10%. Byin diabetic
contrast,
dose-dependent in mice,
with thehaving
diabetic effect no effect
mice, observed
having at
no in 1 mg/kg
normoglycemic
effect but but
at 1 mg/kg being
mice,
beingactive
the atat5 5mg/kg.
effect
active of At the
teuhetenone
mg/kg. At
higher
A was dose,
the higherthedose,
dose-dependentantidiabetic effect was
in diabetic
the antidiabetic similar
mice,
effect having
was to that
no to
similar observed
effect
that at 1 with
mg/kg
observed insulin,
with and and
butinsulin,
being glucose
active atlevels
glucose did not
5 mg/kg.
levels At
didduring
reverse
the higher not reverse
dose, the duringevaluated,
theperiod the period
antidiabetic effect evaluated,
being
was maximal
similarbeing maximal
toatthat
six hours atafter
observed sixwith
hours after teuhetenone
teuhetenone
insulin, glucoseAlevels
A administration
and
administration
(Figures
did not 3reverse (Figures
and 5). during the3 and 5). evaluated, being maximal at six hours after teuhetenone A
period
administration (Figures 3 and 5).
Control Insulin 5 UI/kg
1.3 Teu A 1 mg/kg Teu A 5 mg/kg
Control Insulin 5 UI/kg
1.2
1.3 Teu A 1 mg/kg Teu A 5 mg/kg
1.1
1.2
1.0
Glucose (normalized)

1.1
0.9
1.0
Glucose (normalized)

0.8

0.9 0.7

0.8 0.6

0.7 0.5

0.6 0.4
0 1 2 3 4 5 6
0.5
Time (h)
Time (hrs)
0.4
(a)
0 1 2Figure3 5. Cont.4 5 6

Time
Time
Figure (h)
Cont.
5. (hrs)

(a)
Figure 5. Cont.
Molecules 2017, 22, 599 6 of 13
Molecules 2017, 22, 599 6 of 13

550 Control Insulin 5 UI/kg

Molecules 2017, 22, 599 6 of 13
Teu A 1 mg/kg Teu A 5 mg/kg
500
550 Control Insulin 5 UI/kg
450 Teu A 1 mg/kg Teu A 5 mg/kg
500
400

Glucose (mg/ml)
450
350
400

Glucose (mg/ml)
300
350
250
300
200
250
150
200
100
150 0 1 2 3 4 5 6

100 Time (h)

Time (hrs)
0 1 2
(b) 3 4 5 6
Time (h)
Time (hrs)
Figure 5. Antidiabetic effect of insulin and teuhetenone A in diabetic mice induced with 200 mg/kg of
Figure 5. Antidiabetic effect of insulin and teuhetenone (b) A in diabetic mice induced with 200 mg/kg of
alloxan. n = 5, Mean ±SE. (a) Graph with normalized values; (b) Graph with unnormalized values.
alloxan. n = 5, Mean ±SE. (a) Graph with normalized values; (b) Graph with unnormalized values.
Standardized blood glucose
Figure 5. Antidiabetic forof
effect baseline
insulinglycemia to compare
and teuhetenone the
A in kineticsmice
diabetic of each experimental
induced with 200group.
mg/kg of
Standardized blood glucose for baseline glycemia to compare the kinetics of each experimental group.
alloxan. n = 5, Mean ±SE. (a) Graph with normalized values; (b) Graph with unnormalized values.
Figure 6 shows
Standardized bloodthe maximum
glucose antidiabetic
for baseline effect
glycemia to obtained
compare for of
the kinetics each
eachexperimental group,
experimental group.
demonstrating that teuhetenone A has an activity comparable to insulin.
Figure 6 shows the maximum antidiabetic effect obtained for each experimental In the diabetic mice, the group,
effect Figure
of 6 shows
glibenclamide the
was maximum
not significantantidiabetic
(not shown). effect obtained for each experimental
demonstrating that teuhetenone A has an activity comparable to insulin. In the diabetic mice, the effect group,
demonstrating that teuhetenone A has an activity comparable to insulin. In the diabetic mice, the
of glibenclamide was not significant (not shown).
effect of glibenclamide was 1.0 not significant (not shown).
Glucose Reduction (normalized)

0.8 1.0
Glucose Reduction (normalized)

0.6 0.8

0.4 0.6

0.2 0.4

0.0 0.2
Control Ins 5UI Teu A 1 mg Teu A 5 mg

0.0
Figure 6. Maximum antidiabetic Control
effect of insulin and teuhetenone
Ins 5UI Teu A 1 mg A in
Teudiabetic
A 5 mg mice induced with

200 mg/kg of alloxan. n = 5, Mean ± SE.

Figure 6. Maximum
Figure 6. Maximum antidiabetic
antidiabeticeffect
effectof
ofinsulin and teuhetenone
insulin and teuhetenoneAA
in in diabetic
diabetic mice
mice induced
induced with with
3. 200
Discussion
mg/kg of alloxan.
200 mg/kg of alloxan. Mean±
n =n =5,5,Mean SE.
± SE.
Diabetic patients, even in urban areas in Mexico, use medicinal plants to control their disease,
3. Discussion
3. Discussion
sometimes with complementation by conventional therapies. The species used can be obtained fresh
Diabetic
from markets orpatients,
in packaged evenform.
in urban
This areas
is the in Mexico,
case with T.use medicinal
diffusa plants
(damiana), to control
which their disease,
is consumed and
Diabetic patients, even in urban areas in Mexico, use medicinal plants to control their disease,
sometimes
sold throughout withthecomplementation
country for various by conventional therapies.
health conditions The species used
and complications, can bediabetes.
including obtainedFor fresh
sometimes
from
this with
markets
reason, a complementation
or in packaged
study form.byThis
of its pharmacological conventional
is the therapies.
case with
properties The
isT.important.
diffusa species
In the used
(damiana), which can be
weobtained
is consumed
literature, found and fresh
from markets or
sold throughout
contradictory in packaged
the country
information about form.
forthe This
various is the case
health conditions
hypoglycemic with
effect of and T. diffusa (damiana),
complications,
T. diffusa. which
including
In 1984, Pérez is
et al.diabetes.consumed
evaluatedFor
andansold
this throughout
reason,
aqueous extract the
a study country
of
in mice itswith for various health
pharmacological
alloxan-induced conditions
properties
diabetes. is In andstudy,
important.
their complications,
In the including
theyliterature,
administered we found
thediabetes.
Forextract
this reason,
contradictory a information
study
either orally of itsabout
pharmacological
the hypoglycemic
or intraperitoneally, properties
and glucose effect of is
T. important.
was measured diffusa.
once In Pérez
In 1984,
using the literature, we found
et al. oxidase
a glucose evaluated
an aqueous
method
contradictory extractafter
fiveinformation
hours in mice the with
about alloxan-induced
extract was administered.
the hypoglycemic diabetes.
effect T. In
of The their
T. study,
diffusa
diffusa. In they
extract
1984, Pérezadministered
showed goodthe an
et al. evaluated
extract either orally or intraperitoneally, and glucose was measured
aqueous extract in mice with alloxan-induced diabetes. In their study, they administeredon
hypoglycemic activity by either method of administration [17]. once
Aguilar using
et al. a glucose
reported oxidase
thea extract
method
hypoglycemic five hours
effect after
from an the extract
aqueous was
extract administered.
of T. diffusa (4 The
mL/kg)T.
either orally or intraperitoneally, and glucose was measured once using a glucose oxidase method diffusa
in extract
rabbits that showed
were good five
made
hypoglycemic
temporarily activity by T.either
hyperglycemic. diffusamethod
extract of administration
significantly [17]. Aguilar
decreased et al. reported
the hyperglycemic peak onby a
hours after the extract was administered. The T. diffusa extract showed good hypoglycemic activity by
hypoglycemic effect from an aqueous extract of T. diffusa (4 mL/kg) in rabbits that were made
either method of administration [17]. Aguilar et al. reported on a hypoglycemic effect from an aqueous
temporarily hyperglycemic. T. diffusa extract significantly decreased the hyperglycemic peak by
extract of T. diffusa (4 mL/kg) in rabbits that were made temporarily hyperglycemic. T. diffusa extract
significantly decreased the hyperglycemic peak by 15.9% (p < 0.005) [15]. However, in 2002, the same
Molecules 2017, 22, 599 7 of 13

investigators reported that there was no significant decrease in glucose levels in normoglycemic mice
or mice with2017,
Molecules alloxan-induced
22, 599 diabetes after administration of a hydroalcoholic extract (500 mg/kg) of
7 of 13
T. diffusa [16]. Besides, a methanolic extract of leaves of Turnera ulmifolia at 400 mg/kg significantly
15.9% (p < 0.005) [15]. However, in 2002, the same investigators reported that there was no significant
reduced blood glucose levels in normal rats and in rats with alloxan-induced diabetes [19].
decrease in glucose levels in normoglycemic mice or mice with alloxan-induced diabetes after
We found the of
administration treatment of normal
a hydroalcoholic mice
extract (500with a methanolic
mg/kg) extract
of T. diffusa [16]. of T.adiffusa
Besides, promising,
methanolic extract because
their of
glucose
leaves of levels
Turnera were significantly
ulmifolia at 400 mg/kg reduced. Thereduced
significantly discrepancy between
blood glucose these
levels results
in normal ratsand those
reported in rats
and in thewith
literature could bediabetes
alloxan-induced the result
[19].of various factors, because the experimental conditions
We found the
between different treatment
trials of normal mice
were different. Forwiththisareason,
methanolic weextract of T. to
decided diffusa promising,
follow-up because
these experiments
their glucose levels were significantly reduced. The discrepancy
with a bioassay-guided fractionation of the extract to identify the compound(s) responsible between these results and those for the
reported in the literature could be the result of various factors, because the experimental conditions
hypoglycemic activity.
between different trials were different. For this reason, we decided to follow-up these experiments
After
with athe removal of chlorophyll
bioassay-guided fractionation with
of thesolid phase
extract extraction
to identify (SPE) cartridges
the compound(s) (Extract-Clean
responsible for the SPE
C18-HC; 1000 mg/8
hypoglycemic mL, Grace Davison-Alltech, Deerfield, IL), only the fraction eluted with 50%
activity.
MeOH showed After the activity;
removalvacuum chromatography
of chlorophyll with solid phase was performed
extraction with this (Extract-Clean
(SPE) cartridges fraction using SPEsolvents of
C18-HC; 1000 mg/8 mL, Grace Davison-Alltech, Deerfield, IL), only the fraction
increasing polarity. The extract obtained with ethyl acetate showed hypoglycemic activity. Surprisingly, eluted with 50%
MeOH showed activity; vacuum chromatography was performed with this fraction using solvents of
this fraction did not show antioxidant activity, and no strong presence of flavonoids was detected,
increasing polarity. The extract obtained with ethyl acetate showed hypoglycemic activity.
unlikeSurprisingly,
the AcOEt:MeOH fraction, in which most of the plant flavonoids were identified but were not
this fraction did not show antioxidant activity, and no strong presence of flavonoids
active.wasThe active fraction contained a fraction,
detected, unlike the AcOEt:MeOH major constituent,
in which mostwhich wasflavonoids
of the plant later identified as teuhetenone
were identified
A afterbutpurification
were not active. (Figure 2). A
The active chromatographic
fraction contained a major analysis of the
constituent, methanolic
which was later extract
identifiedshowed
as that
teuhetenone A after purification (Figure 2). A chromatographic analysis
teuhetenone A was present in the original extracts (retention time 24.5 min) and was not an artifact of the methanolic extract
showed
produced that teuhetenone
during A was present
the purification processin(Figure
the original
7). extracts (retention time 24.5 min) and was not
an artifact produced during the purification process (Figure 7).

(a)

(b)

Figure 7. (a) Chromatogram of the original extract of T. diffusa, conditions as in text; (b) Chromatogram
of teuhetenone A.
Molecules 2017, 22, 599 8 of 13

The structure for teuhetenone A was assigned on the basis of spectroscopic evidence, comparison
with literature values [13,20,21], and the following consideration: the 1 H-NMR spectrum showed the
presence of two uncoupled methyl groups at δ 1.43 and 1.31, and a singlet at 6.35 ppm. In DEPT
experiments, we found two primary and five secondary carbon atoms, and one tertiary carbon atom in
the olefin region; as well as four quaternary carbon atoms, one as a carbonyl and the other in the olefin
region. HSQC and HMBC experiments assisted the elucidation, and four diastereotopic carbon atoms
were revealed. Selective 1 H-NOESY to the methyl signals allowed us to confirm that C-11 and C-12
were on the same side of the plane. Irradiation of H-11 (1.31 ppm) dipolar coupling with H-1b, H-3a,
H-8b, H-9a, and H-12 was observed; moreover, irradiation of H-12 (1.43 ppm) dipolar coupling with
H-3b, H-6, and H-11 was found. Optical rotation was also similar to that reported for this molecule.
Teuhetenone A is a terpenoid that has been previously isolated from T. diffusa and other plants,
but to our knowledge, no hypoglycemic activity has been attributed to it thus far [13,20]. In the
literature, it is possible to find some references to its biological activity: it has low activity in an
antiaromatase test, low activity as an antifungal, it has slightly neuroprotective properties, and it
moderately inhibited the production of nitric oxide by macrophages (IC50 = 12.93 µg/mL, positive
control IC50 = 0.17 µg/mL) [22–25]. However, the literature is not conclusive.
Before evaluating the hypoglycemic activity of the isolated compound, the glycemic state of
normoglycemic murine models was determined. Mean basal glycemic levels in the mice were
in the range of 140–155 mg/dL, significantly higher than the 80–90 mg/dL after fasting for 12 h
(not shown). To avoid biochemical and endocrine alterations related to fasting, or hypoglycemia
induced by hypoglycemic agents and insulin that could be lethal in mice deprived of food, the mice
were maintained on water and food ad libitum during the night before the hypoglycemic test was
conducted. However, to avoid a hyperglycemic event from intake while evaluating the hypoglycemic
effect, food was withdrawn, maintaining the mice on water alone during the six-hour period of the test.
As seen in Figure 3, although the hypoglycemic activity of teuhetenone A and insulin appeared
similar, the observed kinetics for both compounds were very different. While insulin reached a
reversible maximum hypoglycemic activity one hour after administration, the decrease in glycemia
with teuhetenone A was gradual and did not reverse for at least six hours after administration,
potentially maintaining an “antidiabetic” effect for longer and thereby reducing the frequency of
administration required for long-term control of glycemia.
The hypoglycemic activity of teuhetenone A in the normoglycemic mice suggested possible
antidiabetic activity; however, their response may have resulted from an insulin-related mechanism.
Therefore, to discriminate the hypoglycemic effect of the endogenous insulin by teuhetenone A in the
mice and to determine the antidiabetic effect, diabetes was induced pharmacologically with 200 mg/kg
alloxan (intraperitoneally) in eight groups of five mice each. In these groups of mice, insulin secretion
was limited by the pancreotoxic effect of alloxan, so glycemia increased from 140–155 to 340–380 mg/dL
(Figure 5). At 1 mg/kg, teuhetenone A showed an antidiabetic effect similar to that observed with
insulin, but the glucose levels did not reverse during the period evaluated (Figures 3 and 5). Injection
of the mice with alloxan produced a significant increase in glucose levels compared with that in a
nondiabetic control group, because in these mice, insulin secretion was limited by the destruction of
pancreatic β cells by alloxan [21], which is the mechanism for the hyperglycemic state of the mice.
Therefore, this model is useful for evaluating antidiabetic effects. The results obtained in mice with
diabetes induced by alloxan suggest that the antidiabetic action of teuhetenone A is similar to insulin,
taking into account that with both substances, the glucose level decreases by approximately 35%.
Analysis of the area under the curve (AUC) regarding the kinetics of both insulin and
teuhetenone A (hypoglycemic and antidiabetic) (Figures 3 and 5) confirmed the effect described
for teuhetenone A. The AUCs obtained for the normoglycemic control mice were 5.17 ± 0.11 for
the vehicle and 4.92 ± 0.09 for insulin, which were similar to the values obtained for teuhetenone
A (4.93 ± 0.08). Moreover, in diabetic mice, the AUCs for vehicle, insulin, and teuhetenone A were
5.93 ± 0.04, 5.48 ± 0.05, and 5.43 ± 0.03, respectively. These results suggest that teuhetenone A
Molecules 2017, 22, 599 9 of 13

has hypoglycemic and antidiabetic effects very similar to those shown by insulin. Therefore, the
hypoglycemic activity shown by teuhetenone A is interesting because it is more potent than that of
glibenclamide (Figure 4) and as active as insulin (Figure 5).
Neither the extracts nor the isolated teuhetenone A significantly inhibited the activity of
α-glucosidase in vitro, which is consistent with the literature, so the mechanism of action of this
terpenoid is probably different from that of hypoglycemic agents that inhibit the absorption of
carbohydrates from the diet.
The isolated teuhetenone A was not toxic to Vero cells, consistent with previous reports of
inactivity on HepG2 and Hep3B liver, MCF-7 and MDA-MB-231 breast, and A-549 lung carcinoma cell
lines [26].
We conclude that teuhetenone A, or a standardized extract containing it, would be a good
candidate for an herbal-based antidiabetic therapy.

4. Materials and Methods

4.1. Biological Materials and Reagents

T. diffusa (leaves and stems) were collected in Montemorelos, Nuevo León, Mexico in November
2014 (latitude 25.1186, longitude −99.7865). A voucher specimen (No. 23569) was identified and
deposited in the herbarium at the Faculty of Biology, Universidad Autónoma de Nuevo León, Mexico.
Vero cells were used to assess cytotoxicity with Dulbecco’s modified Eagle’s medium (DMEM,
Advanced) supplemented with 10% fetal bovine serum, 1% antibiotics (penicillin G/streptomycin
10000 units/mL), and 1% L-glutamine 200mM. All the solvents used for extraction and purification
were analytical grade and were purchased from Fermont (Monterrey, Nuevo León, Mexico).
HPLC-grade methanol (MeOH) was purchased from JT Baker (Fisher Scientific, Fair Lawn, New Jersey).
Extract-Clean SPE C18-HC 1000 mg/8 mL cartridges were purchased from Grace Davison-Alltech
(Deerfield, Illinois). TLC was conducted on plates precoated with silica gel 60 F254 at a thickness
of 0.2 mm with an aluminum support (Merck, Darmstadt, Germany). MilliQ deionized water was
obtained with an Elga II MilliQ system (Veolia, Mexico). Mobile phases were filtered through a 0.45 µm
nylon filter before use (Waters Corp., Milford, MA). The samples were filtered through nylon Acrodiscs
(Waters Corp, Milford, MA, USA).

4.2. Isolation and Purification

Before extraction, the aerial parts of T. diffusa were dried in the shade at room temperature and
then crushed into a fine powder. Then, 500 g of the dried selected plant was crushed into a powder and
extracted with MeOH at room temperature (3 × 800 mL × 1 h × 200 rpm) with a Heidolph Unimax
1010 shaker. All the extracts were pooled together and evaporated to dryness in vacuo to yield a
viscous mass, which weighed 60 g. The chlorophyll contents were eliminated using SPE C18 cartridges,
and the samples were eluted with 8 mL of 50%, 70%, and 100% MeOH. Five grams of the fraction
obtained using 50% MeOH was subjected to silica vacuum liquid chromatography (silica gel 60 G for
thin layer chromatography Merck Millipore, 5 g for sample adsorption, 50 g for the column, Hirsch
funnel with pore size M using Cl2 CH2 , AcOEt, AcOEt:MeOH (1:1), and MeOH as eluents (400 mL
of each solvent). The fraction eluted with AcOEt (158 mg) was active and was further separated by
gravitational chromatography), eluted successively with hexane-AcOEt (3:1, 70 mL) and hexane-AcOEt
(2:1, 150 mL). We collected 20 fractions, each containing 10 mL. Fractions 17 and 18 were pooled and
evaporated to dryness in vacuo to yield 3.7 mg of yellow oil. The purity of the oil was assessed by
TLC, HPLC-DAD, and 1 H-NMR. The proposed structure was established based on the NMR results
and comparisons with published results.
Molecules 2017, 22, 599 10 of 13

4.3. Instrumental Analysis

Chromatograms were obtained using a Waters 600 Series HPLC system including
a 717 autosampler, 2996 diode array detector, and binary pump 1525. HPLC conditions were as
follows: Hypersil Gold C-18 column (5 µm, 150 × 4.6 mm; Thermo Scientific Waltham, MA, USA);
solvent system: A = 0.1% aqueous HCOOH solution, B = MeOH, eluted with a gradient from 70%
to 40% A over 20 min, held at 40% A for 5 min, after which the proportion of A was changed to
30% over 15 min before being returned to the initial conditions for 5 min; flow 0.4 mL/min; injection
volume 10 µL; and concentration of the sample 1 mg/mL in MeOH. Optical rotation was determined
using a Perkin-Elmer 341 polarimeter. To determine NMR spectra, a Bruker Avance DPX400 model
400 MHz NMR spectrometer was used. The mass of teuhetenone A was obtained in a UHPLC Ultimate
3000 (Dionex, Thermo Scientific) system including an auto sampler, quaternary pump, and variable
wavelength detector interfaced with an LCQFleet (Thermo Scientific) mass spectrometer equipped with
an ESI ionization chamber. Separation was performed on a Hypersil Gold C-18 column, 50 × 2.1 mm,
1.9 µm (Thermo Scientific) at 25 ◦ C using 1% aqueous HCOOH (A) in MeOH (B) as the mobile phase.
The gradient profile used was as follows: 30% to 45% of B over 1.8 min, changed to 60% B over 4.6 min,
and finally to 100% B over 2.2 min; flow 0.2 mL/min; injection volume 0.7 µL; and concentration of the
sample 1mg/mL in MeOH. The first detections were done at 280 and 350 nm, followed by a second
detection using mass spectrometry. Mass analyses were obtained in the negative ion mode. The mass
spectrometer was programmed to perform three consecutive scans: full mass (m/z 200–2000), MS2 of
the most abundant ion in the full mass, and MS3 of the most abundant ion in the MS2. The source
voltage was 5 kV, and the capillary voltage and temperature were 14 V and 275 ◦ C, respectively.
Nitrogen was used as the sheath and auxiliary gas at 30 and 20 units, respectively. The normalized
collision energy using helium as collision gas was 35%.

4.4. Alpha-Glucosidase Assay

The α-glucosidase assay was performed as described by Apostolidis et al. with some
modifications [27]. One milligram of the obtained fraction was dissolved in 500 µL of phosphate
buffer (100 mM, pH 6.8). Various concentrations of the solution (final concentrations between 20.6 to
330 µg/mL in 33 µL) were mixed with 17 µg of p-NPG (to give a final concentration 33.6 µg/mL)
and incubated for 5 min at 37 ◦ C. Subsequently, 17 µL of α-glucosidase was added (to give a
final concentration 46.75 mU/mL) and incubated for 17.5 min at 37 ◦ C. Finally, 133 µL of sodium
carbonate (final concentration 66.5 µm) was added. Absorbance was measured on a plate-reading
spectrophotometer at 405 nm. In each experiment, a 100% control of enzymatic activity was included,
in which 33 µL of phosphate buffer 100 mM, pH 6.8 was included, instead of sample or standard.
Also included was a sample blank or standard, containing only 17 µL of buffer but no enzyme.
The absorbance of the sample blank or standard was subtracted from the absorbance values of its
sample or standard. All experiments were performed in triplicate. The percentage of enzyme inhibition
was calculated using the following formula.

( Abs sample − Abs blank)

 
% enzyme inhibition = 100 − × 100 (1)
Abs control 100%

The percentage reduction was plotted according to sample concentrations. From the curve, the
concentration that inhibited 50% of the activity (IC50 ) was interpolated.

4.5. Cytotoxic Activity

Cytotoxicity evaluation was performed using a 3-(4,5-dimethylthiazol-2-yl)-2,3-diphenyltetrazolium
(MTT) bromide salt reduction test as described by Mosmann et al. in 1983 [28] with some modifications.
To perform the assay, 44,000 Vero cells per well were added to 96-well plates; after 24 h, the medium
was removed, and 100 µL of teuhetenone A dissolved in culture medium at concentrations of from
Molecules 2017, 22, 599 11 of 13

3.9 to 500 µg/mL was added. The plates were incubated at 37 ◦ C for 48 h under an atmosphere of 5%
CO2 , and the cells were then washed twice with phosphate-buffered saline. After washing, 200 µL
MTT 0.5 mg/mL was added and incubated with the cells for an additional three hours. After this time,
the supernatant was decanted, and 200 µL of DMSO was added; the mixture was shaken for eight
minutes at 25 rpm and read spectrophotometrically at 570 nm in a plate reader.

4.6. In Vivo Experiments

To assess hypoglycemic activity, both normoglycemic and diabetic CD1 mice (24 to 40 g) were
used. Male CD1 mice were maintained for seven days in an acclimation period before the experiment.
Mice were maintained in the animal experimental area under standard conditions (12 h light/dark
cycle, at 23 ± 2◦ C, 65% humidity) through the acclimation and during the experiment. Animals were
weighed and allocated in groups of five animals in polycarbonate cages of 44 × 28 × 15 cm according
with the treatment. Pelleted food and water were offered ad libitum. All experimental protocols were
approved by the Institutional Animal Care and Use Committee of the Faculty of Medicine (UAEM) and
comply with the applicable Mexican Official Norm (NOM-062-ZOO-1999), “Technical Specifications for
the Care and Use of Laboratory Animals”, as well as all applicable federal and institutional regulations.
Diabetes was induced by administering 200 mg/kg alloxan. A blood sample to measure glycemia was
obtained by cutting 1 mm from the tip of the tail. Glucose concentration was determined by the use of
a commercial glucometer (Optium Xceed, Abbott) and expressed as mg/dL. Mice with glycemia equal
to or greater than 250 mg/dL were considered diabetic. Five groups of five mice each were formed
for both normoglycemic and diabetic mice: vehicle control (water), insulin positive control 5 IU/kg,
a positive control of glibenclamide 5 mg/kg, and the isolated teuhetenone A (1 and 5 mg/kg) were
administered. Basal glycemia was measured before administration of the tested substance or vehicle
(control group). The test substances diluted to 100 µL were administered intraperitoneally, and blood
glucose readings were made every hour for six hours. Once the evaluation was over, the obtained
experimental data were normalized with respect to the basal glycemia of each mouse, and the means
and standard errors were plotted and adjusted to a sigmoid equation.

4.7. Statistical Analysis

All the results were processed using measures of central tendency (mean) and dispersion (standard
error). Statistical differences between results from each experimental group were evaluated with a
Student t test or one-way ANOVA using GraphPad Prism software, version 6.05.

Acknowledgments: The authors gratefully acknowledge CONACYT, Mexico for support through Red
Farmoquimicos (CONACYT N◦ 271520) and Red Metabolómica de plantas (PRODEP). We acknowledge the
technical assistance of Ivonne Carrera.
Author Contributions: A.P.N. and C.D.M. performed the experiments. A.F.L. performed the initial experiments
with animals. G.C.C. helped with all the in vivo experiments. N.W. and J.J.A. conceived and designed the
experiments and wrote the paper. R.S.A. analyzed the data and contributed to the writing of the paper.
Conflicts of Interest: The authors confirm that this article content has no conflict of interest.

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Sample Availability: Samples of the compounds are available from the authors.

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