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THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
Copyright © 1999 by The American Society for Pharmacology and Experimental Therapeutics
JPET 288:529 –534, 1999
Vol. 288, No. 2
Printed in U.S.A.
Novel Terpenoid-Type Quinones Isolated from
Pycnanthus angolensis of Potential Utility in the Treatment
of Type 2 Diabetes
JIAN LUO, JEANNE CHEUNG, EILEEN M. YEVICH, JOHN P. CLARK, JOYCE TSAI, PRISCILLA LAPRESCA,
ROSA P. UBILLAS, DIANA M. FORT, THOMAS J, CARLSON, RICHARD F. HECTOR, STEVEN R. KING,
CHRISTOPHER D. MENDEZ, S. D. JOLAD and GERALD M. REAVEN
Shaman Pharmaceuticals, Inc., South San Francisco, California
Accepted for publication September 10, 1998
This paper is available online at http://www.jpet.org
The tree Pycnanthus angolensis of the Myristicaceae family, also known as “African nutmeg,” is widely used for ethnomedical purposes (Akendengue and Louis, 1994). Of particular relevance to our drug discovery program (Oubré et al.,
1997) was information gathered during a field trip to Nigeria
on its use to treat chronic fungal infections, a clinical problem
commonly seen in patients with uncontrolled hyperglycemia
(Gill, 1991). Based on this information, extracts of the leaves
of P. angolensis were used to initiate an in vivo guided fractionation program (Oubré et al., 1997; Luo et al., 1998a) in an
effort to discover new drugs for the treatment of type 2
diabetes. These efforts resulted in the isolation of two compounds that lowered plasma glucose after the oral administration in mouse models of type 2 diabetes. These compounds, shown in Fig. 1, represent a new class of terpenoidtype quinones. Furthermore, they are structurally distinct
from the currently available oral compounds used to treat
type 2 diabetes: sulfonylureas, biguanides, disaccharidase
inhibitors, and thiazolidinediones. In this presentation, we
evaluated the pharmacological effects of these two compounds on various aspects of glucose and insulin metabolism
in mouse models of diabetes.
Received for publication April 21, 1998.
decreases in plasma insulin concentrations (p , .05), suggesting that both compounds lowered glucose by enhancing insulin-mediated glucose uptake. This was supported by the insulin
suppression test in ob/ob mice. Studies in hyperglycemic, insulin-deficient mice and in vitro experiments on 3T3-L1 adipocytes further supported this conclusion. As such, these two
terpenoid-type quinones represent a new class of compounds
of potential use in the treatment of type 2 diabetes.
Materials and Methods
Compounds SP-18904 and SP-18905 were isolated from an ethanolic extract of the leaves of P. angolensis with a series of in vivo
guided fractionation steps (Oubré et al., 1997; Luo et al., 1998a)
involving liquid/liquid partition, LH-20 column chromatography,
and high pressure liquid chromatography.
Male C57BL/6J-ob/ob mice (ob/ob) and C57BL/ks-db/db mice (db/
db) (7– 8 weeks old) were used to both guide the fractionation process
and evaluate the purified compound. Mice were purchased from the
Jackson Laboratory (Bar Harbor, ME), housed (4 mice/cage) in a
temperature- (22 6 3°C) and humidity- (50 6 20%) controlled room
with a 12-h light (6 AM– 6 PM)/dark cycle, and maintained on a diet
of Purina rodent chow and water ad libitum. In addition, experiments were also performed on C57BL/ks mice in whom insulin
deficiency was induced by the i.p. injection of streptozotocin (150
mg/kg) (Sigma Chemical, St. Louis, MO) according to Rossini et al.,
(1977).
Mice were bled and prescreened for plasma glucose. Mice selected
for study had glucose concentrations of 300 to 600 mg/dl. Each
treatment group consisted of five to eight mice, distributed so that
the mean glucose levels were equivalent in each group at the start of
each study. Mice were dosed orally once a day by gavage with either
vehicle, SP-18904, or SP-18905. Testing materials were delivered in
a liquid vehicle containing 0.25% (w/v) carboxymethylcellulose, 1%
Tween 60, and 10% dimethylsulfoxide (DMSO) (all from Sigma). The
amount of material given and the duration of treatment varied
ABBREVIATIONS: SSPG, steady-state plasma glucose; SSPI, steady-state plasma insulin; ANOVA, analysis of variance; DMEM, Dulbecco’s
modified Eagle’s medium; DMSO, dimethylsulfoxide.
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ABSTRACT
Using an ethnomedical-based drug discovery program, two
previously unknown compounds (SP-18904 and SP-18905)
from Pycnanthus angolensis were isolated that lower glucose
concentrations in mouse models of type 2 diabetes. SP-18904
and SP-18905 are terpenoid-type quinones that significantly
lowered plasma glucose concentration (p , .05) when given
orally to either ob/ob or db/db mice, both of which are hyperglycemic and hyperinsulinemic. The antihyperglycemic actions
of SP-18904 and SP-18905 were associated with significant
530
Luo et al.
Fig. 1. Chemical structures of SP-18904 and SP-18905
transport assays were initiated by the addition of 2-deoxy-D-[3H]glucose (0.5 mCi/ml; 100 mM final concentrations) to each well followed
by incubation for 10 min at 22°C. Assays were terminated by aspirating the media and rapidly washing the monolayer 2 times with
ice-cold phosphate-buffered saline solution. Cell monolayers were
solubilized in 0.1 N NaOH and transferred to scintillation vials, and
radioactivity was determined by liquid scintillation counting. All
data were corrected for nonspecific hexose uptake determined in
parallel samples treated for 5 min with 200 mM cytochalasin B.
Chemicals and medium used in the in vitro assay were from Sigma
Chemical (St. Louis, MO).
Plasma glucose concentrations were determined using the Glucose
Diagnostic Kit (Sigma 315), an enzyme colorimetric assay. Plasma
insulin levels were determined by using the Rat Insulin RIA Kit from
Linco Research Inc. (Cat. No. RI-13K; St. Charles, MO). Data are
expressed as mean 6 S.E.M., and one-way analysis of variance
(ANOVA) with Fisher’s PLSD post hoc test was used for assessing
statistical significance of differences, with a p value of ,.05 used as
measure of significance.
Results
Plasma glucose concentrations measured at baseline and
3 h after daily treatment with various doses of SP-18904 for
4 days in ob/ob mice are shown in Fig. 2 (top). These data
demonstrate that glucose concentrations were significantly
reduced after 1 day of the oral administration of SP-18904
and remained lower on every day of dosing. Furthermore, a
dose-response effect is evident. In contrast, plasma glucose
concentration did not fall in vehicle-treated mice.
Somewhat similar data were seen in ob/ob mice treated
with SP-18905 (Fig. 2, bottom), only in this instance data are
only available on days 2 and 3 after initiation of treatment.
Although the decline in plasma glucose concentration was
statistically significant on days 2 and 3 in response to 50 and
100 mg/kg SP-18905, this compound seems to be somewhat
less potent than SP-18904. As before, there was no change in
plasma glucose concentration in the vehicle-treated mice.
Food intake of all the groups of mice shown in Fig. 2 ranged
from a mean of 4.9 to 5.7 g/mouse/day, with no difference
between the groups. Similarly, weight gain over the 4 days
averaged ;1.0 g and was comparable in each of the groups of
mice.
Plasma insulin concentrations 3 h after the last dose of
SP-18904 (4 days of treatment) and SP-18905 (3 days of
treatment) to ob/ob mice are seen in Fig. 3. It is apparent that
the fall in plasma glucose concentration shown in Fig. 2 was
associated with a decrease in plasma insulin after treatment
with either SP-18904 (top) and SP-18905 (bottom). Furthermore, a dose-response effect is also obvious from insulin
concentrations.
The antihyperglycemic effect of these compounds were
evaluated in db/db mice, a second model that is hyperglycemic and hyperinsulinemic (;400 mU/ml), as well as in mice
made insulin deficient (;10 mU/ml) with streptozotocin.
These studies were performed after 2 days of treatment and
indicated that the plasma glucose concentration of db/db
mice fell significantly (p , .05) after oral administration of
either 100 mg/kg SP-18904 (411 6 22 mg/dl) SP-18905 (368 6
18 mg/dl) compared with vehicle (523 6 24 mg/dl)-treated
mice. In contrast, plasma glucose concentrations in insulindeficient mice did not decrease in response to treatment with
either SP-18904 (683 6 32 versus 657 6 25 mg/dl) or SP18905 (507 6 17 versus 523 6 14 mg/dl).
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among experiments, and details are given with the experimental
results. Blood samples were taken from the tail vein 3 h after the
dosing on the corresponding sampling day in nonfasted conditions
unless indicated otherwise. Individual body weight and mean food
consumption (each cage) were measured daily.
Estimates of insulin-mediated glucose uptake were obtained after
4 days of treatment with SP-18905 following the simplified insulin
suppression test method described by Luo et al., (1998b). Food was
removed on the day of the experiment, with the last oral dose of test
compounds was given 1 h later. Four hours after the withdrawal of
the food, mice were anesthetized with i.p. sodium pentobarbital
(Sigma) at 100 mg/kg. The abdominal cavity was opened, and the
main abdominal vein exposed and catheterized with a 24-gauge i.v.
catheter (Johnson-Johnson Medical Inc., Arlington, TX). The catheter was secured to muscle tissue adjacent to the abdominal vein, cut
on the bottom of the syringe connection, and hooked to a prefilled
PE50 plastic tube, which in turn was connected to a syringe with
infusion solution. The abdominal cavity then was sutured closed.
With this approach, there is no blockage of the back flow of the blood
from the lower part of the body. Mice were infused continuously with
glucose (20 mg/kg/min) and insulin (20 mU/kg/min) (both from
Sigma) at a rate 10 ml/min. Retro-orbital blood samples (70 ml each)
were taken 105, 120, and 135 min after the start of infusion for the
measurement of plasma glucose and insulin concentration. The
mean of these three samples was used to estimate the steady-state
plasma glucose (SSPG) and insulin (SSPI) concentrations for each
animal.
Murine 3T3-L1 preadipocytes (American Type Culture Collection
CL 173) were maintained in Dulbecco’s modified Eagle’s medium
(DMEM) containing 10% (v/v) supplemented calf serum, antibiotics,
and 25 mM glucose. Cells were seeded onto 24-well cluster plates
(10,000 cells/well), grown to confluence (typically 5 days), and induced to differentiate 2 days after confluence (day 0) according to the
standard protocol of Frost and Lane (1985). After differentiation,
adipocytes were maintained in DMEM containing 10% fetal bovine
serum and provided with fresh medium every 2 to 3 days. Adipocytes
used in this study were used on days 7 to 10 after differentiation. On
the day of the experiment, adipocytes were washed with phosphatebuffered saline and switched to serum-free DMEM. Adipocytes were
treated (in triplicate) for 18 h with 10 mM SP-18904 or SP-18905.
Concentrated stock solution of SP-18904 or SP-18905 was freshly
prepared in DMSO and diluted into culture medium. The final concentration of DMSO was 0.2% (v/v), which was also included in basal
conditions. After overnight (18 h) treatment, the culture medium
was aspirated, and the monolayers were washed with Krebs-RingerHEPES buffer. To assess the effects of the compounds on basal
glucose transport, 2-deoxy-D-glucose uptake (an indicator of glucose
transport) was measured in the absence of insulin stimulation. To
determine whether 18-h exposure to compounds potentiated the
stimulatory effect of insulin, adipocytes were further treated with 0.5
nM insulin (a submaximal concentration) for 30 min at 37°C. Glucose
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1999
Terpenoid-Type Quinones for Type 2 Diabetes
Because the chemical structure and the effects on plasma
glucose and insulin concentrations of both compounds were
similar, only SP-18905 was used to determine whether the
compound could counteract insulin resistance in ob/ob mice.
As shown in Fig. 4, SSPG concentrations (right) after the
administration of 50 mg/kg SP-18905 for 4 days were significantly lower compared with vehicle-treated mice. Because
the SSPI concentrations were similar in the two groups,
these data indicate that insulin-mediated glucose disposal
had increased in association with SP-18905 administration.
Both compounds were evaluated for their effects on 2-deoxy glucose transport in 3T3-L1 adipocytes. In the absence of
insulin, neither SP-18904 nor SP-18905 (10 mM) increased
glucose transport into adipocytes (Fig. 5). However, at the
same concentrations, in the presence of a submaximal insulin
concentration (5 nM), both SP-18904 and SP-18905 potentiated insulin-stimulated glucose uptake.
Discussion
To the best of our knowledge, there is no previous ethnomedical evidence that extracts of P. angolensis would be
useful in the treatment of hyperglycemia. Our decision to
explore this possibility was based on the observation of its
use as an antifungal agent and the knowledge that uncontrolled hyperglycemia increases the risk of fungal infections.
Based on this association, we evaluated the ability of crude
extracts of P. angolensis to lower plasma glucose concentrations in animal models of type 2 diabetes and, if so, to initiate
an in vivo based fractionation effort (Oubré et al., 1997; Luo
et al., 1998a) to identify the compound or compounds responsible for this effect. We used ob/ob and db/db mice to identify
and evaluate the antihyperglycemic compounds present in P.
angolensis. They are well recognized animal models of type 2
diabetes (Shafrir, 1992) that are used extensively in drug
discovery and evaluation of potential pharmacological approaches to treat the clinical syndrome (Chang et al., 1983;
Shafrir, 1992). This effort was successful, as attested to by
the results shown in Fig. 2 documenting the fact that two
compounds isolated from P. angolensis, SP-18904 and SP18905, were capable of significantly lowering plasma glucose
concentrations when given orally to a mouse model of type 2
diabetes.
The two antihyperglycemic compounds isolated from P.
angolensis are terpenoid-like quinones not previously identified. As such, they are both novel compounds, as well as
newly recognized antihyperglycemic agents. It is apparent
from the structures shown in Fig. 1 that both SP-18904 and
SP-18905 are chemically distinct from the four classes of
compounds currently approved to treat type 2 diabetes: sulfonylureas, biguanides, thiazolidinediones, and disaccharidase inhibitors. Consequently, the identification of these terpenoid-like quinones offers a new approach to the
development of drugs that may be useful in the treatment of
type 2 diabetes.
Although not the primary goal of this study, the results
presented also provide some insight into the means by
which SP-18904 and 18905 lower plasma glucose concentration. Specifically, the observation that the decline in
plasma glucose concentration was associated with lower
plasma insulin concentrations indicates that the compounds are not insulin secretagogues but seem to be enhancing the ability of insulin to stimulate glucose disposal.
This possibility is further supported by the infusion studies in which treated mice had similar SSPI concentrations
during the infusion but lower SSPG concentrations (Fig.
4). The fact that these compounds had little, if any, effect
on plasma glucose concentrations in insulin-deficient mice
provides further evidence that it is acting to enhance insulin-mediated glucose disposal. Furthermore, consistent
with the improvement of insulin action in in vivo, both
SP-18904 and SP-18905 were found to enhance insulinstimulated glucose uptake by 3T3-L1 adipocytes (Fig. 5).
These data suggest that SP-18904 and SP-18905 improve
insulin action at the cellular level, the underlying mechanism of which awaits further study. Given the extensive
evidence that insulin resistance is the basic defect in patients with type 2 diabetes (Reaven, 1995), it is not surprising that recent drug development for the treatment of
type 2 diabetes has focused on agents that reduce insulin
resistance (Saltrel and Olefsky, 1996; Imura, 1998). In this
context, the fact that SP-18904 and SP-18905 appear to
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Fig. 2. Dose-dependent effects of SP-18904 (top) and SP-18905 (bottom)
on plasma glucose concentrations in ob/ob mice. There were eight mice in
each group. *p , .05 (ANOVA, Fisher’s PLSD post hoc test).
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Luo et al.
Vol. 288
enhance insulin-mediated glucose disposal suggests that
they represent a new and physiologically relevant approach to the treatment of type 2 diabetes.
In summary, SP-18904 and SP-18905 represent a new
class of terpenoid-type quinones that have marked antidia-
betic effects in mouse models of type 2 diabetes. The antidiabetic effects of this class of compound may rely on their
ability to improve insulin-mediated glucose disposal. These
characteristics define compounds of potential great use in the
treatment of type 2 diabetes.
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Fig. 3. Dose-dependent effects of SP18904 (top) and SP-18905 (bottom) on
plasma insulin concentrations in ob/ob
mice. There were eight mice in each
group. *p , .05 (ANOVA, Fisher’s
PLSD post hoc test).
1999
Terpenoid-Type Quinones for Type 2 Diabetes
533
Fig. 5. Effects of SP-18904 and
SP-18905 on 2-deoxyglucose uptake in 3T3-L1 adipocytes with or
without the presence of insulin.
Results are the mean of three experiments, each experiment done
in triplicate. *p , .01 compared
with insulin-stimulated glucose
uptake (ANOVA, Fisher’s PLSD
post hoc test).
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Fig. 4. Effects of SP-18905 on SSPI and SSPG concentrations during the last 30 min of a 135-min infusion of insulin (20 mU/kg/min) and glucose (20
mg/kg/min) in ob/ob mice. There were eight and seven mice in the vehicle and treatment groups, respectively. *p , .05 (ANOVA, Fisher’s PLSD post
hoc test).
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Luo et al.
Acknowledgments
We thank Dr. Ray Cooper for his help and guidance in creating the
ethnobotanical drug discovery program that led to this publication
and Nancy Waldeck for her technical assistance in the in vitro
assays.
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Send reprint requests to: Gerald M. Reaven, M.D., Shaman Pharmaceuticals, Inc., 213 East Grand Avenue, South San Francisco, CA 94080-4812.
E-mail: [email protected]
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