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School of Biomedical Sciences, University of Ulster, Coleraine, Northern Ireland, BT52 1SA, United Kingdom
2To whom correspondence should be addressed.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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KEY WORDS: • elder (Sambucus nigra) • streptozotocin-diabetic mice • insulin • traditional plant treatments • diabetes
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). The development of new dietary adjuncts and novel
antidiabetic agents, which reinstate a normal metabolic environment,
thereby reducing the long-term complications associated with
diabetes is required. Such agents would both ideally stimulate the
secretion and improve the action of insulin (Bailey and Flatt 1995
).
Throughout the world, many traditional plant treatments for diabetes
exist and therein lies a hidden wealth of potentially useful natural
products for diabetes control (Bailey and Day 1989
,
Gray and Flatt 1997a
, Swanston-Flatt et al. 1991
). Despite this, few traditional antidiabetic plants
have received scientific or medical scrutiny, and the World Health
Organization (1980)
recommended accordingly that this area warrants
further evaluation.
Recent studies showed that aqueous extracts of agrimony (Agrimony
eupatoria), lucerne (Medicago sativa), coriander
(Coriandrum sativum), eucalyptus (Eucalyptus
globulus), edible mushroom (Agaricus campestris) and
mistletoe Viscum album) enhanced insulin secretion and
mimicked the effect of insulin on glucose metabolism in vitro
(Gray and Flatt 1997b
, Gray and Flatt 1998a
, b
, c
, Gray and Flatt 1999a
, b
). Such dual
pancreatic and extrapancreatic actions would prove to be an important
advance on existing therapies used to treat and control diabetes,
such as oral hypoglycemic drugs (which act either by enhancing
insulin secretion or by improving the action of insulin). These
combined findings illustrate the enormous potential of plants for
use as possible dietary adjuncts and the discovery of natural products
for diabetes therapy.
Sambucus nigra (elder) was reported to have a number
of potentially useful medicinal attributes including diuretic,
diaphoretic, purgative and hemostatic properties (Chiej 1988
, Lust 1986
). In addition, it was advocated
as an effective traditional remedy for diabetes in Europe
(Atkinson 1979
, Palaiseul 1983
). The
present study was undertaken to evaluate the possible presence of
antidiabetic components in elder flower. Effects on glucose transport
and metabolism by isolated abdominal muscle and on insulin secretion by
clonal ß-cells were studied to further evaluate the potential for
natural products in elder for the treatment of diabetes.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Dried elder flowers were obtained from a commercial source (The Health Food Center, Birmingham, United Kingdom). Flowers were homogenized to a fine powder and stored at room temperature (20 ± 2°C) until use. Aqueous extract of elder (AEE)3 was prepared by 15 min infusion of the powdered material at 250 g/L. In brief, 1 g of powdered material was placed in 40 mL of boiling water, removed from the heat source and allowed to infuse for 15 min. This suspension was filtered (Whatman no. 1) and the volume readjusted to 40 mL with distilled water. For in vitro studies, 1 mL aliquots of AEE were brought to dryness under vacuum (Savant Speedvac; Savant Instrumentation Inc., Framingdale, NY) and stored at -20°C until use. AEE doses are expressed as total plant material weight (rather than dry residue) per L of water. To account for possible differences in potency, test and control incubations within a single experiment using isolated muscle or insulin-secreting cells were always conducted using the same batch of extract. This allowed for variation in potency of different batches of extract apparent in the same insulin release experiments.
Glucose transport and glucose metabolism in vitro.
Recently weaned postprandial male mice (3–5 wk) derived from a colony
maintained at Aston University, Birmingham, United Kingdom
(Flatt and Bailey 1981
) were killed by cervical
dislocation and each freshly excised abdominal muscle was dissected
into eight individual pieces (~10–20 mg). Incubations were performed
using Krebs-Ringer bicarbonate buffer supplemented with 2 g/L of
insulin-free bovine serum albumin (KRB-BSA; 118 mmol/L of NaCl, 25
mmol/L of NaHCO3, 5 mmol/L of KCl, 1.28 mmol/L of
CaCl2, 1.18 mmol/L of MgSO4, 1.17 mmol/L of
KH2PO4). In order to replicate by mouse, pieces
of muscle from each mouse were designated to each of the treatment
groups.
Glucose uptake was determined as described previously (Gray and Flatt 1997b
). In brief, muscle pieces were
incubated at 30°C for 30 min in 1 mL KRB-BSA supplemented with 2
mmol/L of sodium pyruvate, 3.7 Bq/L of
2-deoxy-D-[1-3H]glucose, 0.37 Bq/L
L-[1-14C]glucose in the presence and absence
of 10-8 mol/L of human insulin and 0.5 g/L of AEE (2
x 2 factorial design). After incubation, tissue was hydrolyzed
using 1 mol/L of NaOH and counted for 3H and
14C radioactivity. The extracellular fluid volume of the
muscle was determined from the amount of the nontransported
L-[1-14C]glucose, and this was taken into
account in the calculation of tissue
2-deoxy-D-[1-3H]glucose uptake, expressed as
nBq/(mg wet muscle · h).
Oxidative glucose metabolism to CO2 and incorporation of
glucose into glycogen were determined as described previously
(Gray and Flatt 1998a
). In brief, muscles
were incubated at 37°C for 60 min in 1 mL of KRB-BSA supplemented
with 10 mmol/L of glucose, 18.5 Bq/L
D-[U-14C]glucose in the presence and absence
of 10-8 mol/L human insulin and 0.5 g/L of AEE (2 X 2
factorial design). Following incubation, carbon dioxide was captured
onto a NaOH saturated filter paper and muscles were removed for
glycogen analysis (Gray and Flatt 1999a
).
14C Radioactivity of the filter paper was counted and
CO2 production was expressed as nmoles CO2/(mg
wet muscle · h). The incorporation of glucose into glycogen was
expressed as nmoles glucose equivalents/(mg wet muscle · h).
This concentration of insulin (10-8 mol/L) was chosen to
represent a large but submaximal stimulation of glucose uptake and
oxidation within this preparation (O’Harte et al. 1997
).
All animal studies were performed in accordance with the Animals (Scientific Procedures) Act, 1996.
Insulin secretion in vitro.
Insulin secretion was evaluated using BRIN-BD11 cells, produced by
electrofusion of immortal RINm5F cell with New England Deaconess
Hospital rat pancreatic beta-cell (Gray and Flatt 1998b
, McClenaghan et al. 1996
).
Secretory characteristics of these cells are described elsewhere
(McClenaghan et al. 1996
). Cells were seeded at a
concentration of 0.2 x 106 cells/well in 24-well
plates (Falcon, NJ) cultured in RPMI-1640 containing 11.1 mmol/L of
glucose, 10 g/L of fetal calf serum and antibiotics (50,000
IU/L of penicillin-streptomycin) to allow attachment
overnight prior to acute tests. Cells were washed thrice with KRB (115
mmol/L of NaCl, 4.7 mmol/L of KCl, 1.28 mmol/L of CaCl2,
1.2 mmol/L of KH2PO4, 1.2 mmol/L of
MgSO4, 24 mmol/L of NaHCO3, 10 mmol/L of hepes
free acid, 1 g/L of BSA, 1.1 mmol/L of glucose; pH 7.4) and
preincubated for 40 min at 37°C. Unless otherwise stated, cells were
then incubated for 20 min with 1 mL of KRB at 1.1 mmol/L of glucose in
the absence and presence of plant extract, diazoxide (an established
opener of ß-cell K+-ATP channels) and other test agents
as indicated. Following incubation, aliquots were removed from each
well and stored at -20°C for insulin assay (Flatt and Bailey 1981
). Cell viability following incubations was evaluated by
modified neutral red assay (Hunt et al. 1987
).
To investigate the importance of heat during extract preparation, AEE was prepared by normal method of infusion (normal extract) or by cold infusion (cold extract; plant material placed in cold water, allowed to stand for 15 min, then filtered as before). Modified aqueous extract was freshly reconstituted in KRB and effect on insulin secretion evaluated at a concentration equivalent to 1 g/L compared with AEE.
Further studies on the nature of the insulin-releasing component(s) involved exposure of the AEE to heat, overnight dialysis, acid-alkali or acetone treatment. Heat: AEE was boiled for 1 h immediately after preparation. Dialysis: AEE was dialyzed overnight (Spectra/Por molecular weight cut-off 2000 Da;Spectrum,Los Angeles,CA) against Millipore water at 4°C. Acid-alkali treatment: aliquots of AEE were added to 5 mol/L of HCl or 5 mol/L of NaOH to produce 0.1 mol/L of HCl or 0.1 mol/L of NaOH, allowed to stand at room temperature overnight, then neutralized. Acetone treatment: 1 mL of AEE (1 g/L) was added to 10 mL ice-cold acetone, allowed to stand for 30 min and centrifuged (800 x g, 5 min) to obtain acetone-soluble and acetone-insoluble fractions. Aliquots of AEE and modified aqueous extracts were dried under vacuum. All modified aqueous extracts were freshly reconstituted in KRB and effects on insulin secretion at a concentration equivalent to 1 g/L were compared with AEE.
In another series of experiments, elder flowers were subjected to sequential extraction by increasingly polar solvents. 0.25 g of plant material was placed in 5 mL of hexane, agitated for 15 min, centrifuged (950 x g, 5 min). The precipitate was dried under vacuum and extracted with a further 5 mL of hexane and centrifuged (as before). The extraction supernatants were pooled, filtered (Whatman no. 1) and the volume adjusted to 10 mL with hexane. The extraction precipitate (dried under vacuum) was subsequently extracted (as above) with 2 x 5 mL vol of ethyl acetate, then methanol and finally with water. All extract fractions were freshly reconstituted in KRB, and effects on insulin secretion at a concentration equivalent to 1 g/L were compared with AEE.
A final experimental series was undertaken to evaluate potential
insulin-releasing actions of known chemical constituents isolated
from elder flower (Shoaib et al. 1972
, Willuhn and Richter 1997
). These included S. nigra
lectin, rutin, lupeol, ß-sitosterol, tannic acid and choline chloride
(Sigma Chemical Ltd., Poole, Dorset, United Kingdom). The concentration
range tested (0.5, 5, 25 and 50 mg/L) assumed that these constituents
represented at most 1% of the dried extract (Shoaib et al. 1972
, Willuhn and Richter 1997
). Lupeol and
ß-sitosterol were dissolved at 50 g/L in chloroform, and all natural
products were tested using KRB supplemented with 1.1 mmol/L of glucose.
We confirmed that the solvent did not influence insulin release at the
dilutions used.
Statistical analyses.
Data were evaluated using Student’s unpaired t test, one-way or two-way ANOVA where appropriate. Groups were considered to be significantly different if P < 0.05. When a significant F-value was obtained for ANOVA, the differences between all pairs were tested using Student-Newman Keuls multiple comparisons test. If the SD were significantly different (Bartlett’s test for homogeneity of variances), data were transformed (log10[x]).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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AEE (1 g/L) increased glucose uptake (70%), glucose oxidation (50%)
and glycogenesis (70%) during incubations without insulin but did not
significantly alter the stimulatory effect of
10-8 mol/L of insulin (Table 1
).
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AEE (0.25–1 g/L) exerted a dose-dependent stimulatory effect on
insulin secretion from BRIN-BD11 cells at 1.1 mmol/L of glucose
(Fig. 1
). At concentrations of 5 g/L of AEE and above, cell viability during
the test period was significantly diminished (P < 0.001) as evaluated by modified neutral red assay (Hunt et al. 1987
) (data not shown). Such an effect was not evident at
concentrations of 1 g/L of AEE and below. The presence of 0.5 mmol/L of
diazoxide inhibited the stimulatory effect of AEE (Fig. 2A
), also indicating that the enhancement of insulin release
was not a mere consequence of cellular damage. Consistent with lack of
toxicity at lower concentrations, prior exposure of BRIN-BD11 cells to
0.5 g/L of AEE for 20 min did not alter the subsequent insulin
secretory response to 10 mmol/L of L-alanine (Fig. 2B
).
|
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). In contrast, the extract did not augment insulin release
in cells stimulated by 10 mmol/L L-alanine, 1 mmol/L of
3-isobutyl-1-methylxanthine (IBMX), or depolarized by combination of
16.7 mmol/L of glucose and 25 mmol/L of KCl (Fig. 3B-D
).
|
). Overnight dialysis of AEE to remove components with a molecular mass
< 2000 Da reduced the insulin-releasing activity by 41%
(P < 0.01; Table 2
). Although significantly less
potent than AEE, the methanol and water fractions of elder (produced by
sequential extraction) exhibited insulin-releasing activity
(Fig. 4
). However, activity in the ethyl acetate fraction was significantly
reduced compared with AEE, and the hexane fraction did not alter basal
insulin release (Fig. 4)
.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, Palaiseul 1983
), scientific studies to
evaluate its efficacy and possible mode of action are lacking. The
present study reports for the first time that elder flowers contain
water-soluble natural products which directly stimulate glucose
metabolism by muscle and promote insulin secretion from clonal
pancreatic ß-cells.
The effect of elder extract on glucose transport and metabolism was
evaluated in vitro using an insulin-sensitive muscle preparation
(Gray and Flatt 1997b
). AEE (1 g/L)
significantly enhanced glucose uptake, glucose oxidation and glycogen
synthesis in a magnitude similar to 10-8 mol/L of
insulin. Although this effect was observed in the absence of added
insulin, it does not preclude involvement of residual insulin receptor
binding within the muscle preparation. However, the lack of significant
potentiation by AEE and insulin in combination implies that the extract
is likely to act via pathways (at least terminally) that are utilized
by insulin rather than entirely separate pathways. Notably, the effect
of AEE on glucose uptake is different from that of the established
antihyperglycemic drug, metformin, which enhances glucose uptake via
insulin-mediated enhanced peripheral glucose uptake (Bailey and Puah 1986
, Prager et al. 1986
). The ability
of AEE to enhance glucose uptake and oxidation in vitro is paralleled
by findings for other aqueous plant extracts, including agrimony,
lucerne, coriander, mushroom, mistletoe and eucalyptus (Gray and Flatt 1997b
, Gray and Flatt 1998a
,b
,c
,
Gray and Flatt 1999a
,b
).
Incubations were performed with glucose-responsive BRIN-BD11 cells
(McClenaghan et al. 1996
) to investigate the possible
effects of AEE on insulin secretion in vitro. This revealed a stepwise
dose-dependent stimulation of insulin secretion by AEE at low
(non-stimulatory) glucose concentration. Evaluation of cell viability
using modified neutral red assay and the insulin-releasing action
of L-alanine following exposure of clonal ß-cells to
extract argues against a simple cytotoxic action at or below 1 g/L of
extract.
Studies to evaluate the possible mechanisms underlying the
insulin-releasing action of elder indicated a similarity to the
sulfonylurea class of drugs currently used for diabetes therapy
(Bailey and Flatt 1995
). These agents enhance insulin
secretion by binding to sulfonylureas receptors on the ß-cell, with
subsequent closure of K+-ATP channels, membrane
depolarization and Ca2+ influx (Rorsman 1997
). Diazoxide inhibits the stimulatory action of
sulfonylureas by preventing closure of the K+-ATP
channels (Dunne et al. 1994
). In the present study the
insulin-releasing action of elder extract was inhibited by
diazoxide. Importantly, the action of AEE was potentiated by 16.7
mmol/L of glucose, suggesting that ß-cell glucose metabolism is able
to augment the insulinotropic effect. Consistent with this view,
L-alanine, which promotes insulin secretion through changes
in Na+ transport (Yada 1994
),
failed to augment the insulin-releasing effect of AEE. In contrast
to sulfonylureas (Eliasson et al. 1996
, Flatt et al. 1994
), AEE failed to stimulate insulin secretion from
beta-cells depolarized by 25 mmol/L of KCl, indicating absence of
similar K+-ATP channel-independent effects.
Interestingly, the phosphodiesterase inhibitor IBMX which increases
cyclic AMP and promotes insulin release (Sharp 1979
) did
not potentiate the insulin-releasing effect of AEE, raising the
possibility that AEE itself may inhibit islet phosphodiesterase
(Leibowitz et al. 1995
).
Elder flowers contain a number of established natural products
including lectin, the flavenoid rutin, choline tannin and lipophilic
triterpenoid and sterol compounds such as lupeol and ß-sitosterol
(Shoaib et al. 1972
, Willuhn and Richter 1997
). It was therefore of interest to evaluate if any of these
available compounds could account for the insulin-releasing action.
Since Agaricus bisporus (mushroom) lectin documented
insulinotropic activity (Ahmad et al. 1984a
and 1984b
;
Ewart et al. 1975
), it was particularly interesting to evaluate
whether the lectin component of elder might be important. However,
neither S. nigra lectin, rutin, lupeol, ß-sitosterol,
tannic acid or choline affected insulin release over a wide range of
concentrations approximating to 1% of active plant extract. Thus, the
chemical nature of potential antihyperglycemic components(s) of elder
remains to be established. In terms of insulin-releasing activity,
the present study indicates that the active principle(s) is
heat-stable, acetone-insoluble and unaffected by altered pH
environment. The approximate 40% decrease in the insulin-releasing
activity of AEE following overnight dialysis (to remove components with
a molecular mass < 2000 Da) suggests some involvement of smaller
molecules or ions in the insulin releasing effect. Sequential solvent
extractions point toward a cumulative effect of more than one active
constituent, likely to be more polar in nature. Other aqueous plant
extracts have also been shown to enhance insulin secretion using this
model (Gray and Flatt 1997b
, Gray and Flatt 1998a
,b
,c
, Gray and Flatt 1999a
and 1999b
).
However, the precise mechanisms by which these extracts enhance insulin
secretion and the chemical identity of biologically active component(s)
remain to be elucidated.
In conclusion, the traditional antidiabetic plant elder contains water-soluble components capable of stimulating insulin secretion and enhancing muscle glucose uptake and metabolism. Sambucus nigra therefore represents a possible dietary adjunct for the treatment of diabetes and a potential source for the discovery of new orally active agent(s) for future diabetes therapy.
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FOOTNOTES |
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3 Abbreviations used: AEE, aqueous extract of elder; KRB, Kreb’s-Ringer bicarbonate buffer; KRB-BSA, Kreb’s
Ringer bicarbonate buffer supplemented with 2 g/L insulin-free bovine serum albumin.
Manuscript received June 10, 1999. Initial review completed July 3, 1999. Revision accepted September 28, 1999.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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4. Bailey C. J., Day C. Traditional treatments for diabetes. Diabetes Care 1989;12:553-564[Abstract]
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6. Bailey C. J., Puah J. A. Effect of metformin on glucose metabolism in mouse soleus muscle. Diabète et Metabolisme 1986;12:212-218
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8. Dunne M. J., Harding E. A., Jaggar J. H., Ayton B. J., Squires P. E. Endogenous and chemical activators of ATP-regulated potassium channels in insulin-secreting cells: possible mechanisms and physiological significance. Flatt P. R. Lenzen S. eds. Frontiers of Insulin Secretion and Pancreatic B-Cell Research 1994:153-159 Smith-Gordon London
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P < 0.001 compared with
incubation with extract.
