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Life Sciences 74 (2003) 663–673

Aniseed oil increases glucose absorption and reduces urine

output in the rat

Sawsan Ibrahim Kreydiyyeha,*, Julnar Ustab, Khuzama Knioa,Sarine Markossiana, Shawky Daghera

aDepartment of Biology, Faculty of Arts and Sciences, American University of Beirut, Beirut, LebanonbDepartment of Biochemistry, Faculty of Medicine, American University of Beirut, Beirut, Lebanon

Received 20 March 2003; accepted 18 July 2003

Abstract

Anise (Pimpinella anisum) has been used as a traditional aromatic herb in many drinks and baked foods because

of the presence of volatile oils in its fruits commonly known as seeds. Hot water extracts of the seeds have been

used also in folk medicine for their diuretic and laxative effect, expectorant and anti-spasmodic action, and their

ability to ease intestinal colic and flatulence. The aim of this work was to study the effect of aniseed oil on

transport processes through intestinal and renal epithelia and determine its mechanism of action. The essential oils

were extracted from the seeds by hydrodistillation and analyzed by gas chromatography. Aniseed oil enhanced

significantly glucose absorption from the rat jejunum and increased the Na+-K+ ATPase activity in a jejunal

homogenate in a dose dependent manner. The oil, however, exerted no effect on water absorption from the colon

and did not alter the activity of the colonic Na+-K+ ATPase. When added to drinking water, it reduced the volume

of urine produced in the rat and increased the activity of the renal Na+-K+ ATPase even at extremely low

concentrations. It was concluded that aniseed oil increases glucose absorption by increasing the activity of the Na+-

K+ ATPase and consequently the sodium gradient needed for the sugar transport. Its anti-diuretic effect is also

mediated through a similar mechanism in the kidney whereby a stimulation of the Na+-K+ pump increases tubular

sodium reabsorption and osmotic water movement. The colonic Na+-K+ ATPase was however, resistant to the oil.

D 2003 Elsevier Inc. All rights reserved.

Keywords: Aniseed; Glucose; Na+-K+ ATPase; Antidiuretics

205/$ - see front matter D 2003 Elsevier Inc. All rights reserved.

.1016/j.lfs.2003.07.013

orresponding author. Fax: +961-1744461.

ail address: sawkreyd@aub.edu.lb (S.I. Kreydiyyeh).

S.I. Kreydiyyeh et al. / Life Sciences 74 (2003) 663–673664

Introduction

Traditional herbal medicine is gaining recently more popularity and phytotherapy has become a

more acceptable scientific approach to disease treatment and prevention. Plant extracts are

considered nowadays, as potential bioactive agents that can interfere and alter positively or

negatively different cellular processes. In most instances, volatile oils constitute the most common

active ingredients in medicinal herbs. Anise (Pimpinella anisum) is a plant that produces fruits

commonly referred to as seeds which are rich in such oils. The oils are used in perfumery,

medicine, soaps and beverages (Duke, 1985). Aniseed and aniseed oils are employed also in food

industry as flavoring agents (e.g., Greek Ouzo, Lebanese arak, anisette) and a concoction of seeds

in hot water is used as a carminative, antiseptic, diuretic, digestive, and a folk remedy to insomnia

and constipation (Bisset, 1994). Most of these reported biological effects have rarely been supported

by any scientific background and rely mainly on empirical information. Because aniseeds are highly

used in Lebanon in baked goods and beverages, a study of their physiological properties was

deemed necessary and informative.

This work aims at studying, in the rat, the effect of aniseed oil on the absorption of glucose from the

jejunum, and water from the colon and kidney tubules.

Materials and methods

Isolation of the essential oils

The fruits of the anise plant(Pimpinella anisum), commonly known and thereafter referred to as

aniseeds, were obtained from the local market and allowed to dry at room temperature for 24 hours. The

essential oils were extracted by hydrodistillation using a ratio of 1:6 (g/ml) of seed weight to volume of

distilled water.

The distillation process was carried out over a period of 6 hours using a round bottom container, and a

receiver for oils lighter than water to collect the essential oils. The collected oil was measured and dried

over anhydrous sodium sulfate.

Gas chromatography/mass spectroscopy

Essential oil composition was analyzed using a HP6890 gas chromatograph hooked to HP 5972 mass

selective detector. A fused silica column loaded with 5% diphenyl 95% dimethylpoly siloxane was used.

Column dimensions were: length 30 m, i.d. 0.25 mm. The operating conditions were as follows: linear

velocity 36 cm/sec; split ratio 1:100; ionization voltage 70 eV; ionization current 50 AA; scanning speed

30–500 amus/sec; volume of injection 1 AL of pure oil.

Components of the essential oil were screened using the libraries: Wiley 275 and NBS.

Animal treatments

Male Sprague-Dawley rats (Rattus norvegicus) weighing 150–200 g were used and handled all

through in accordance with the Guide for Laboratory Animal Facilities and Care, US Department of

Health, Education and Welfare.

S.I. Kreydiyyeh et al. / Life Sciences 74 (2003) 663–673 665

Measurement of 14C glucose absorption by the upper jejunum

Rats were anesthetized by i.p. injection of pentobarbital (5 mg/100 g body weight) and laid dorsally

on a heating pad maintained at 37 jC throughout the experiment. The abdomen was opened to expose

the intestines and a 10 cm segment of the upper jejunum situated 15 cm from the pylorus was isolated

keeping the mesenteries and vasculature intact. An ‘‘L-shaped’’ inlet and outlet catheter were

introduced at each end of the segment and in the same plane of the segment, to allow for buffer

infusion and collection. The perfusion of the segment followed a single pass method and the perfusion

rate was maintained all through at 0.8 ml/min. The intestinal segment was perfused first for 15 min

with oxygenated (95% O2/5%CO2) Krebs improved Ringer buffer pH 7.5 (KIRB) (123.3 mM NaCl;

6.17 mM KCl; 3.29 mMCaCl2�7H2O; 0.78 mM MgSO4�7H2O; 32.14 mM NaHCO3; 1.54 mM

KH2PO4; 6.4 mM sodium pyruvate; 6.4 mM sodium glutamate; 7 mM sodium fumarate; 11.1 mM

glucose) to clear it from wastes. In a second step the jejunum was perfused for 30 min at the same

rate, with KIRB to which 180 AM D[U-14C]glucose (9.9 GBq/mmol; Sigma) was added.The effluent

buffer was collected at different time intervals and its volume measured and compared to the volume

of buffer infused during the same period. When the effect of aniseed oil was studied it was dissolved

in DMSO and added to the buffer during both perfusion steps at a concentration of 5 � 10� 4 Al oil/Al buffer. The oil was added to the first perfusion buffer to give it enough time to exert its effect, if

such an effect is present, before measurements of glucose absorption are started. The same volume of

the vehicle was added in the control treatment. At the end of the experiment the perfused intestinal

segment was isolated, cut longitudinally and opened to expose the mucosal surface. The width and

length of the obtained rectangle were measured, and its surface area calculated. Samples were

withdrawn from the effluent buffers collected at different time intervals during the second perfusion

period and assayed for radioactivity together with samples from the infused buffer. From the

radioactivity data obtained and the measured volumes of the effluent and infused buffers the total

radioactivity entering and leaving the intestine during each interval were computed. Absorption of

glucose was calculated as the difference between the total radioactivity infused and the total

radioactivity appearing in the collected buffer divided by the area of the segment perfused.

Measurement of luminal water movements in the rat colon

The effect of the oil on water movements in the colon was studied on another group of animals. The

abdomen of anesthetized rats was opened through a mid line incision to expose this time the colon. The

whole colon was perfused in a two-step process, and as described before for the jejunum, with KIRB.

The volume of the effluent buffer collected during the second perfusion step was measured and

compared to the volume of infused buffer. Anise oil when tested was added to the two perfusion steps at

the same concentration as for the jejunum (0.5 ml/L). All buffers used were oxygenated (95%O2,

5%CO2) at 37 jC and their pH adjusted to 7.4.

At the end of the second perfusion, the colon was excised, cut longitudinally, its width and length

measured, and its surface area determined. Water movements during the second 30-min perfusion step

were calculated as the difference in volume between the infused and collected buffers divided by the

surface area of the perfused colon.

Measurement of the volume of urine eliminated

Rats of similar weights were housed in metabolic cages and after an overnight fast were

allowed free access to water and food. The volume of water consumed and the volume of urine

S.I. Kreydiyyeh et al. / Life Sciences 74 (2003) 663–673666

produced over 24 hours were measured. The animals were then offered water to which aniseed oil

dissolved in DMSO was added at a concentration of 0.5 ml oil/L water. Water intake and urine

production were measured as before. Thus every animal served as it own control. In the control

treatment a similar volume of DMSO was added to the drinking water. The ratio of urine

produced/water intake was calculated and considered as a standardized indicator of urine

elimination.

In vitro assay of the Na+-K+ ATPase activity In a homogenate of the jejunum and colon

The whole colon and an upper jejunal segment were excised from three rats that have not

received any treatment. The segments were opened longitudinally and washed well with KIRB. The

mucosal surface was then scraped with a glass slide and the scrapings were suspended in a buffer

with the following composition (200 mM NaCl; 5 mM MgCl2�6H2O; 2 mM EGTA; 5 mM KCL;

200 mM Tris-HCl, pH 7.4), and homogenized in a glass-Teflon homogenizer with 20 strokes at

2100 rpm (Arthur Thomas Scientific Apparatus, Philadelphia,PA). Samples from the homogenate

were diluted with the same buffer to a concentration of 2 mg protein/ml and used to assay for the

Na+-K+ ATPase activity. After a 30-min incubation period with saponin (0.02% final concentration)

at room temperature, samples (50 Al) were pre-incubated at 37 jC for 10 min in presence or

absence of ouabain (4 mM final concentration). The reaction was then initiated by addition of ATP

to a final concentration of 1.25 mM and terminated after a one hour incubation at 37 jC by

addition of trichloroacetic acid (200 Al, 11%). The samples were centrifuged at 3000 g for 5 min

and the amount of inorganic phosphate liberated in the supernatant was measured colorimetrically

according to the method of Taussky and Shorr (Taussky and Shorr, 1953). Since ouabain is a

specific inhibitor of the Na+-K+ ATPase, the activity of the enzyme was determined by measuring

the ouabain-inhibitable inorganic phosphate released.

Results are reported as percentage of the enzymatic activity in control, as follows:

PiðoilÞ � Piðoil þ ouabainÞPiðcontrolÞ � Piðcontrol þ ouabainÞ � 100

In vitro assay of the Na+-K+ ATPase in kidney homogenate

The abdomen of anesthetized rats was opened through a lateral incision and the intestine and

stomach were pushed aside to expose the left kidney. A heparinized catheter was then introduced

in the left renal vein to allow for drainage, and ligated to fix it in place. Another heparinized

catheter was introduced in the aorta just above the level of the left renal artery to allow for the

infusion of the buffer. The mesenteric artery and the right renal artery were both ligated. The left

kidney was perfused with gassed Krebs improved Ringer buffer (KIRB) for 10 min at a rate of 5

ml/min to clear it from blood and then excised, its capsule removed, cut into small pieces and

homogenized in KIRB buffer in a glass-Teflon homogenizer with 20 strokes at 2100 rpm (Arthur

Thomas Scientific Apparatus, Philadelphia, PA). Its protein content was determined using the Bio-

Rad protein assay (Bio-Rad Laoratories, 2000 Alfred Nobel Drive, Hercules, CA 94547, USA) and

samples were taken to assay for the Na+-K+ ATPase activity which was conducted as described

above.

S.I. Kreydiyyeh et al. / Life Sciences 74 (2003) 663–673 667

Statistical analysis

Results are reported as means F SEM. Statistical significance between ratios of water intake/urine

volume was tested by a paired Student t test and by a one-way analysis of variance followed by a Tukey-

Kramer multiple comparisons test for all remaining values.

Results

Essential oil composition

The extraction procedure resulted in a yield of 1.4 ml oil/100 g dry sample. Relative percentage

composition of essential oils extracted from aniseeds appears in Table 1. From the compounds

identified in aniseed oil, trans-anethole (76.7%) was found to be the most abundant followed by

anisalacetone (7.1%), estragole (6.1%) and anisaldehyde (1.5%). Linalool, germacrene-d, ar-

curcumene and h-bisabolene were the least abundant and were all present in the same amount

(0.2%).

Glucose absorption

Aniseed oil added to the perfusion buffer at a final concentration of 0.05% increased significantly

(Fig. 1) jejunal glucose absorption during a 15 (P < 0.002), 25 (P < 0.002) and 30 (P < 0.003) min.

perfusion period.

Effect of aniseed oil on the volume of water reabsorbed from the colon

Aniseed oil at a 0.05% concentration did not exert any significant effect on the volume of water

absorbed from the colon (Fig. 2).

Table 1

Relative percentage composition of essential oils isolated from aniseeds

Name RT(min) CAS number %F SEM

Linalool 13.55 000078-70-6 0.2 F 0.049

Estragole 16.92 000140-67-0 6.1 F 2.001

Anisaldehyde 18.92 000123-11-5 1.5 F 0.601

Trans-anethole 19.87 004180-23-8 76.7 F 12.79

Anisalacetone 23.12 000122-84-9 7.1 F 6.59

Germacrene-d 26.00 023986-74-5 0.2 F 0.049

Ar-curcumene 26.15 000644-30-4 0.2 F 0.098

h-bisabolene 26.86 000495-61-4 0.2 F 0.049

Identified 92.1

Yield (ml oil/100 g dry sample) 1.4

Values are Means F SEM of 3 observations.

Fig. 1. Effect of aniseed oil on the absorption of glucose over different perfusion periods. Values are means F SEM of 6

observations i.e., every group had 6 rats. Statistical significance was tested by a one-way analysis of variance followed by a

Tukey-Kramer multiple comparisons test. *: Significantly different from the control (P < 0.002). **: Significantly different

form the control (P < 0.003).

S.I. Kreydiyyeh et al. / Life Sciences 74 (2003) 663–673668

Effect of aniseed oil on the volume of urine eliminated in vivo

Aniseed oil added to drinking water at a 0.05% concentration, exerted a significant anti-diuretic effect

(Fig. 3). Aniseed oil did not exert any significant effect on water intake (33.78 F 9 ml in control vs.

35.94 F 9 ml in aniseed group) but decreased significantly the urine to water intake ratio from 47.36 F4.5 in control to 27.7 F 5.78 in the aniseed treatment (P < 0.027).

Fig. 2. Effect of aniseed oil on water absorption from the colon. Values are means F SEM of 6 observations. Values were not

significantly different from each other as demonstrated by the Student t test.

Fig. 5. Effect of aniseed oil on the Na+-K+ ATPase activity in colonic homogenate. Values are means F SEM of three

observations. No significant differences were detected between the different values as demonstrated by the Tukey-Kramer

multiple comparisons test.

Fig. 3. Urine/water intake in control rats and rats given aniseed oil. Values are means F SEM. Every group had 5 rats and every

animal served as its own control. *: Significantly different from the control (P < 0.027) as demonstrated by the Student t-test.

Fig. 4. Effect of different concentrations of aniseed oil on the Na+-K+ ATPase activity in jejunal homogenates. All samples

contained 0.8 mg protein. Statistical difference between the different values was tested by a one-way analysis of variance

followed by a Tukey-Kramer multiple comparisons test. *: Significantly different from the control (P < 0.01). **: Significantly

different from the control (P < 0.05).

S.I. Kreydiyyeh et al. / Life Sciences 74 (2003) 663–673 669

Fig. 6. Na+-K+ ATPase activity in kidney homogenate. All values were significantly different from the control P < 0.01 except

the points marked with * as demonstrated by the Tukey-Kramer multiple comparisons test. The enzymatic assay was run in

triplicates, and was repeated three times on homogenates prepared from three different rats.

S.I. Kreydiyyeh et al. / Life Sciences 74 (2003) 663–673670

Effect of aniseed oil on the Na+-K+ ATPase activity in jejunal and colonic homogenates

Aniseed oil stimulated significantly the Na+-K+ ATPase activity in jejunal homogenates at all

concentrations used (Fig. 4). This stimulation ranged between 2.7 to 3.2 folds for the concentrations

used. The oil did not have, however, any effect on the in vitro activity of the Na+-K+ ATPase in colonic

homogenates (Fig. 5).

Effect of aniseed oil on the Na+-K+ ATPase activity in kidney homogenates

Aniseed oil increased significantly (Fig. 6) in kidney homogenate the Na+-K+ ATPase activity at all

concentrations between 0.025 and 12500 nl/L with a peak appearing at 250 nl/L resulting in a 4 folds

increase in the enzyme activity.

Discussion

This work demonstrated a significant increase in jejunal glucose absorption and a reduction in urine

output by aniseed oil. The oil enhanced also the activity of the Na+-K+ ATPase in both intestinal and

kidney homogenates.

Effect on jejunal glucose absorption

The jejunum is the main site of glucose absorption. The sugar is transported across the mucosal

membrane by a sodium-dependent secondary active process that relies on the sodium gradient

established by the Na+-K+ ATPase (Schultz and Curran, 1970; Crane, 1965) also known as the Na+-

K+ pump. The Na+-K+ATPase couples the exchange of three cytoplasmic Na+ ions with two

S.I. Kreydiyyeh et al. / Life Sciences 74 (2003) 663–673 671

extracellular K+ ions, to the hydrolysis of one molecule of ATP (Berribi-Bertran et al., 2001) resulting in

the establishment of an electrochemical gradient across the plasma membrane. It has been estimated that

the pump consumes around 25% of the energy spent by a cell (Lingrel and Kuntzweiler, 1994). The

sodium gradient created by the pump provides the driving force for several secondary active transport

processes like the one responsible for the absorption of glucose from the lumen of the intestine and

kidney tubules.

Any change in the activity of the Na+-K+ ATPase is expected to increase or decrease the sodium

gradient and consequently alter glucose absorption.

Aniseed oil increased significantly glucose absorption in the rat jejunum. To see if this is a

consequence of an increase in the Na+-K+ ATPase activity, the direct in vitro effect of the oil on the

enzyme activity in jejunal homogenates was studied. Aniseed oil increased in a dose-dependent manner,

the ATPase activity, and the effect appeared at a concentration as low as 0.025%.

The data suggest that the observed increase in the intestinal absorption of glucose with aniseed oil is

mediated through a stimulation of the Na+-K+ ATPase which increases the sodium gradient that gears the

mucosal glucose transport.

Effect on water absorption from the colon

One of the aims of this work was to demonstrate the laxative effect of aniseed oil if present.

A laxative agent may decrease net water absorption from the colon by either decreasing electrolyte

absorption, or increasing electrolyte secretion, or both. These absorptive and secretory processes are

regulated by different carriers and result from the transport of different ions (Halm and Frizzel, 1991).

The mammalian colon absorbs sodium and chloride, and secretes potassium and bicarbonate (Binder and

Sandle, 1994). The neutral NaCl mucosal transport is the predominant sodium absorptive mechanism in

the rat (Binder et al., 1987; Zimmerman and Binder, 1984) and involves the dual ion exchangers Na/H

and Cl/HCO3 which are coupled by intracellular pH (Goldfarb et al., 1988) and energized by the sodium

gradient created by the Na+-K+ ATPase (Binder and Sandle, 1994). Active potassium secretion is geared

also by the Na+-K+ pump (Binder and Sandle, 1994). The Na+-K+ ATPase is thus the key player in the

regulation of electrolytes and consequently water movements.

Aniseed oil added at a 0.05% concentration did not have any significant effect on colonic water

absorption. This finding was supported further by the absence of any effect of the oil on the in vitro

activity of the Na+-K+ ATPase in homogenates from the colon.

It seems thus that the ATPase in the colon is resistant to the oil effect. The Na+-K+-ATPase is a highly-

conserved integral membrane protein that is expressed in virtually all cells of higher organisms. The

ATPase has two functional subunits: a and h. In mammals, four alpha-subunit and at least three beta-

subunit isoforms have been identified in addition to two gamma-subunits in the kidney. Different

combinations of subunit isoforms are present in different cells (Ewart and Klip, 1995). The alpha subunit

(f 113 kD) is the action hero of the pair. It binds ATP and both sodium and potassium ions, and contains

the phosphorylation site. The smaller beta subunit (f 35 kDa glycoprotein) is absolutely necessary for

the activity of the complex (Lingrel and Kuntzweiler, 1994), and appears to be critical in facilitating

plasma membrane localization and activation of the alpha subunit.

The resistance of the colonic enzyme to the oil effect may be due to the presence in the colon of

different isoform combinations than in the jejunum in which the binding site for the active component is

absent or not accessible.

S.I. Kreydiyyeh et al. / Life Sciences 74 (2003) 663–673672

The use of aniseed concoctions as a traditional remedy for constipation is not in contradiction with our

findings since the concoctions may contain some active ingredients extracted with water but not

appearing in the oil.

Effect on urine volume

Aniseed oil added to drinking water caused a significant reduction in the volume of urine produced

and thus exerted an anti-diuretic effect. Water movements in the kidney tubules are usually secondary to

movements of electrolytes. An increase in electrolytes reabsorption would increase the osmotic gradient

that drives water movements in the same direction leading to a decrease in the volume of urine produced.

Sodium is the major electrolyte reabsorbed, and the process is geared by by the Na+-K+ pump. By

extruding sodium from the cytosol, the pump builds up a concentration gradient for sodium across the

luminal membrane that favors its entry to the inside of the cell. Intracellular sodium is then removed by

the Na+-K+ ATPase to the extracellular space and water follows.

Aniseed oil decreased significantly the volume of urine produced and increased the in vitro activity of

the Na+-K+ ATPase in kidney homogenates. It could be concluded that the anti-diuretic effect of the oil is

due to a stimulation of the Na+-K+ pump.

The renal Na+-K+ ATPase as compared to the jejunal one, seems however to be much more sensitive

to the oil. While a two fold stimulation was observed with a concentration of 0.1% in the jejunum, a

concentration of only 0.025 nl/L produced the same effect in the kidney. Here again this may be ascribed

to the presence of different isoforms in the two tissues.

This work has demonstrated a stimulation of the Na+-K+ ATPase activity in the jejunum and kidney

which resulted in an increase in glucose absorption and a decrease in the volume of urine produced. The

oil did not exert however any effect on water absorption from the colon nor on its ATPase activity. The

incorporation of aniseed oil in the diet will thus make a greater portion of ingested glucose available in

blood for use by the cells, and in a hot and dry weather, the oil would help conserve water in the body

and prevent dehydration.

The work did not determine, however, the nature of the active ingredient(s).Trans-anethole constituted

76.7% of the total oils extracted from aniseeds This value is consistent with the 70–90% range reported

in the literature (Bisset, 1994). Since the active ingredient may not necessary be the most abundant one

in the oil mixture, each of the identified components in addition to trans-anethole may be responsible for

the observed physiological effects. Identification of the active ingredient(s) will be conducted in a

follow-up project.

Acknowledgements

This work was supported by a grant from the University Research Board.

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