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Hypoglycaemic effect of Teucrium polium: studies with rat pancreatic islets
Journal of Ethnopharmacology 95 (2004) 27–30
Hypoglycaemic effect of Teucrium polium: studies with rat pancreatic islets Mohammad Ali Esmaeili, Razieh Yazdanparast∗ Institute of Biochemistry and Biophysics, P.O. Box 13145-1384, University of Tehran, Tehran, Iran Received 14 July 2003; received in revised form 31 May 2004; accepted 2 June 2004 Available online 11 August 2004
Abstract The aquous extract of Teucrium polium (Labiatae) has long being used in Iran as a hypoglycaemic aid without any knowledge about its probable side effects and mode of action. In an effort to assess the claimed hypoglycaemic property of the crude drug and to get some knowledge about the mechanism of action, the crude extract (0.5 g plant powder per kg body weight) was administered orally to a group of streptozotocin diabetic rats for six consecutive weeks. A significant decrease (64%) in blood glucose concentration was observed in the treated animals compared to the untreated diabetic rats, without any measurable effects on the major biochemical factors. In addition, the crude extract significantly enhanced the blood insulin level by almost 160% compared to the untreated diabetic rats. The insulinotropic property of the Teucrium polium extract was further assessed by an in vitro investigation using isolated pancreatic rat islets. Our data indicated that Teucrium polium crude extract is able to enhance insulin secretion by almost 135% after a single dose of plant extract (equivalent to 0.1 mg plant leaf powder per mL of the culture medium) at high glucose concentration (16 mmol/L). Meanwhile, the time pattern of insulin secretion was not affected by the plant extract compared to the untreated islets. These data clearly show that the plant extract, probably without metabolic transformation, is able to reduce high blood glucose levels through enhancing insulin secretion by the pancreas. © 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Hypoglycaemic; Insulin; Pancreatic islets; Teucrium polium
1. Introduction The use of medicinal plants for the treatment of diabetes mellitus dates back to the Ebers papyrus of about 1550 BC (Bailey and Day, 1989). Even after the discovery and use of insulin and other modern oral hypoglycaemic agents, the search for safer and more effective drugs of plant origin for the treatment of diabetes has continued (Lin, 1992; Bailey and Day, 1989). The aquous extract of Teucrium polium L. (Labiatae) has long being used in Iran as an hypoglycaemic aid without any knowledge about its probable side effects and its mode of action. In order to get some knowledge about the mechanism of action of this medicinal plant and in order to use it safely, ∗ Corresponding author. Tel.: +98-21-6113382; fax: +98-21-6404680. E-mail address: [email protected] (R. Yazdanparast).
0378-8741/$ – see front matter © 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2004.06.023
we evaluated the hypoglycaemic activity of Teucrium polium extract on the insulin secretion in streptozotocin (STZ) diabetic rats and also in the isolated pancreatic islets of rats at two different glucose concentrations (2 and 16 mM). The time pattern of insulin secretion by the isolated islets under the influence of the plant extract has been assessed.
2. Methodology 2.1. Plant material Aerial parts of Teucrium polium L. were collected from Fars province during spring, 1999. A voucher herbarium specimen (No. 570) was deposited in the Herbarium of the School of Pharmacy, Shaheed Beheshti University of Medical Sciences, Tehran, Iran. The plant aerial parts were air-dried
28
M.A. Esmaeili, R. Yazdanparast / Journal of Ethnopharmacology 95 (2004) 27–30
protected from direct sunlight and then powdered. The powder was kept in a closed container at 10 ◦ C. 2.2. Extraction The powdered plant material (250 g) was extracted three times with ethanol–water (7:3, v/v), at room temperature. The combined extracts were concentrated under reduced pressure and the volume was adjusted to 500 mL (equivalent to 0.5 g plant powder per mL). The concentrated extract was divided into 25 mL aliquots and kept at −20 ◦ C for further investigation. The w/w yield in terms of dry starting material was 6%. 2.3. Animals Male Wistar albino rats (30), 5–7 months old with a weight of 200–250 g were purchased from the Pasteur Institute, Tehran, Iran. They were housed under conventional conditions and were allowed access to food and water ad libitum. 2.4. Induction of experimental diabetes Streptozotocin (STZ; Sigma, USA; 40 mg/kg body weight) was dissolved in 0.1 M sodium citrate buffer at pH 4.5 just before use (Elias et al., 1994) and injected intraperitoneally to 22 rats. Control rats (8) received an equivalent volume of citrate buffer. One week after STZ administration, the diabetic rats with blood glucose levels between 16 and 25 mmol/L were selected and distributed into two groups (I and II). 2.5. Oral administration of the plant extract The plant extract was administrated by gavage (i.g.) to 12 rats of group II daily in a dose of 1 mL/rat (equivalent to 0.5 g plant powder/kg body weight) for 6 consecutive weeks. The control healthy rats (group III, n = 8) and the control diabetic rats (group I, n = 8) received the same volume of distilled water i.g. The blood glucose levels of rats in each group were determined weekly using a Glucomen kit (A. Menarini, diagnostics, Germany).
creases were isolated under sodium pentobarbital anesthesia (50 mg/kg body weight i.p.). Each pancreas was cut into small pieces and were subjected to enzymatic (collagenase) digestion for 20 min at 37 ◦ C on a magnetic stirrer. The medium consisted of Dublecco’s modified essential medium (DMEM) supplemented with collagenase type V (1 mg/mL), soybean trypsin inhibitor (STI) (2 mg/mL), and bovine serum albumin (BSA) fraction V (2%). The digested tissue was then centrifuged at 1000 rpm for 10 min, washed twice with phosphate buffer saline (pH 7.2) and seeded in culture flasks containing RPMI-1640 supplemented with 10% fetal calf serum (FCS) and incubated at 37 ◦ C in an incubator with 5% CO2 . After 24 h of incubation, islets were separated by hand picking under an inverted microscope. 2.8. Insulin secretion: static incubation Batches of 13 rat islets were placed into tubes containing 0.6 mL of the modified Krebs-bicarbonate medium with 20 mmol/L Hepes pH 7.4 and 2 mg/mL albumin (Ashcroft et al., 1986) along with various plant extract concentration. After 2 hours of incubation in a CO2 incubator at 37 ◦ C, samples were centrifuged at 5000 rpm to sediment the islets. The supernatant was collected and stored at −20 ◦ C for insulin measurement. 2.9. Insulin secretion: time dependent The triplicate culture flasks containing islets were transfered to tissue culture dishes and incubated for 1 h at 37 ◦ C in basal medium consisting of the modified Krebs-bicarbonate buffer with 2 mmol/L glucose. Doublicate Samples were collected for insulin measurement every 5 min up to 50 min. Then, the culture media was changed by the modified Krebsbicarbonate buffer containing 16 mmol/L glucose along with the plant extract (0.1 mg/mL). At 5 min intervals doublicate aliquots were taken for insulin measurements up to 90 min. The medium was then changed back to the basal medium containing 2 mmol/L glucose. Doublicate Samples were again collected for insulin determination at 5 min intervals up to 120 min. 2.10. Statistics
2.6. Insulin assay The Insulin level of each blood sample was measured by an enzyme-linked immunosorbent assay using a commercial kit (Rat Insulin ELISA; DRG Instruments GmbH, Germany), based on direct sandwich technique in which two monoclonal antibodies are directed against separate antigenic determinants on the insulin molecule.
The significance of differences between the control and the test groups was established by the paired Student’s t-test with the significance level set at P < 0.05.
3. Results
2.7. Pancreatic islet isolation
3.1. The effect of Teucrium polium extract on blood glucose level
The islet isolation was carried out according to Shewade’s method (Shewade et al., 1999). Briefly, six healthy pan-
One week after STZ injection, the diabetic rats with fasting blood glucose level in the range of 16–25 mmol/L, were
M.A. Esmaeili, R. Yazdanparast / Journal of Ethnopharmacology 95 (2004) 27–30
29
Table 1 Effect of EtOH–H2 O extract of Teucrium polium (0.5 g/kg body weight) on the blood glucose and body weight of diabetic rats Days −7
0
14
21
28
35
42
Normal rats
Blood glucose (mmol/L) 4.9 ± 0.1 Body weight (g) 238.0 ± 17.5
4.9 ± 0.1 265.0 ± 17.6
5.0 ± 0.8 276.0 ± 22.1
4.8 ± 0.2 293.0 ± 15.5
5.1 ± 0.4 296.0 ± 24.9
5.1 ± 0.2 306.0 ± 22.2
5.1 ± 0.1 310.0 ± 22.4
Diabetic rats
Blood glucose (mmol/L) 5.0 ± 0.1∗ Body weight (g) 215.0 ± 8.6
23.1 ± 3.5 227.0 ± 5.2
22.0 ± 1.2 202.0 ± 11.2
24.0 ± 2.7 185.0 ± 13.5
22.0 ± 0.9 173.0 ± 4.6
22.6 ± 1.4 172.0 ± 4.6
23.2 ± 3.4∗ 178.0 ± 2.5
Treated rats
Blood glucose (mmol/L) 4.9 ± 0.1∗ Body weight (g) 228.0 ± 24.2
23.7 ± 3.2 237.0 ± 27.6
19.5 ± 3.2 227.0 ± 24.8
1.7 ± 2.0 218.0 ± 23.2
15.2 ± 2.0∗ 224.0 ± 30.3
13.0 ± 17 238.0 ± 24.9
7.6 ± 3.1 253.0 ± 21.0
Each number represents the average of triplicate measurements ± S.D. ∗ P < 0.05
selected for further studies. Table 1 shows that prior to the extract administration, there was no significant difference between the blood glucose levels of the two diabetic groups of animals (I and II). However, after 6 weeks, the blood glucose levels of the treated rats were significantly lower than the controls (6.2 ± 1.64 mmol/L and 27.7 ± 5.1 mmol/L, respectively; P < 0.05). In contrast, the blood glucose level of the untreated diabetic rats remained high throughout the experimental period. The blood glucose level of the healthy group (III) remained unchanged during the course of the investigation. In addition, the blood insulin levels of the treated diabetic rats, after six consecutive weeks of treatments, were significantly enhanced (by 161%) compared to the untreated diabetic rats (8.96 ± 1.1 g/L and 3.42 ± 0.48 g/L, respectively; P < 0.05). Table 1 shows that, in contrast to the normal rats, there was a significant (P < 0.05) weight loss among group I and II, two weeks after STZ administration. However, Teucrium polium-treated rats showed signs of recovery in body weight
Fig. 1. Stimulation of rat islet insulin release by Teucrium polium EtOH–H2 O extract. Teucrium polium extract induced a dose dependent initiation of basal (2 mmol/L glucose, ) and stimulated (16 mmol/L glucose, ) insulin secretion, the data present the mean ± S.E.M. of three individual preparations of rat islets. Statistical significances of the observed differences in insulin release compared to the appropriate controls are ∗ P < 0.05, ∗∗ P < 0.001.
gain three weeks after treatments (224.0 ± 30.2 g) compared to diabetic control rats (173.0 ± 8.6 g). 3.2. The effect of Teucrium polium extract on insulin release by the rat islets Fig. 1 shows that Teucrium polium extract, in a dose range equivalent to 0.001–1 mg of the plant powder/mL, is capable of enhancing basal insulin release (at 2 mmol/L glucose) by a factor of 1.5 compared to the untreated islets. The insulinotropic effect of Teucrium polium was more evident at high glucose concentration (16 mM) (Fig. 2). Incubation of islets with 16 mmol/L glucose induced stimulation of basal insulin release, the rate of which fell rapidly when the high glucose medium was replaced by 2 mmol/L glucose. The addition of Teucrium polium extract (0.1 mg/mL) to high glucose medium resulted in an essentially similar pattern of insulin release. However, it was characterized
Fig. 2. Effect of Teucrium polium on the insulin secretion by the rat pancreatic islets with respect to time. Islets were incubated in basal (2 mmol/L glucose) medium for 1 h, the medium was then changed with either 16 mmol/L glucose () or 16 mmol/L glucose plus 0.1 mg/mL Teucrium polium extract () for 30 min, followed by incubation under basal condition for another 30 min, the data present the mean ± S.E.M. of three individual preparations of rat islets. Statistical significances of the observed differences in insulin release compared to the appropriate controls are ∗ P < 0.001.
30
M.A. Esmaeili, R. Yazdanparast / Journal of Ethnopharmacology 95 (2004) 27–30
by a more marked increase of secretion during stimulation period. 4. Discussion In the present investigation we have shown that STZ diabetic rats fed with a single dose of Teucrium polium extract (equivalent to 0.5 g plant powder per kg body weight) per day for six consecutive weeks exhibited significant blood glucose reduction (by 64%) and increase of blood insulin level by 160%. This hypoglycaemic potential, besides of confirming the traditional use of this plant as a blood glucose lowering agent has been reported in literature (Gharaibeh et al., 1988; Pushparaj et al., 2001; Noor and Ashcroft, 1998). Traditionally, the hyperglycaemic patients orally takes half a cup of a decoction of the plant aerial parts prepared with 50 g of the crude drug per liter of water once a day for 4–5 consecutive weeks. Phytochemical investigations on Teucrium spp. have shown the presence of compounds such as diterpene derivatives (Mini et al., 1991), fatty acid esters (Fonta et al., 1999), flavonoids (Rizk et al., 1986) and steroids (Ulubelen et al., 1994). However, so far, the biological activity of these compounds with respect to the hypoglycaemic effect of the plant has not been reported. Our results clearly indicated for the first time that Teucrium polium crude extract is able to produce a dose-dependant stimulation of basal insulin release and also to potentiate glucose-stimulated release of insulin in rat islets with no significant and detectable effects on the time pattern of insulin secretion. These data clearly indicate that the plant extract, probably without metabolic transformation, is capable of reducing high blood glucose mainly through enhancing insulin secretion. The insulinotropic agent of Teucrium polium has been purified to homogeneity in our lab and further work is in progress to elucidate its chemical structure.
Acknowledgements We are thankful to the Research Council of the University of Tehran for financial support of this investigation.
References Ashcroft, S.J.H., Hammonds, P., Harrison, D.E., 1986. Insulin secretory responses of clonal cell line of simian virus 40-transformed -cells. Diabetologia 29, 727–732. Bailey, C.J., Day, C., 1989. Traditional treatment for diabetes. Diabetes Care 12, 553–564. Elias, D., Prigozin, H., Polak, N., Rapaport, M., Lohse, A.W., Cohen, I.R., 1994. Autoimmune diabetes induced by the -cell toxin STZ. Diabetes 43, 992–998. Fonta, G., Savona, G., Redriguez, B., 1999. Usual 6 -fatty acid esters of (24S-24-ethyl-cholesta-5,25-dien-3 -yl--D-Glucopyranoside) from Teucrium fruticans. Phytochemistry 50, 283–285. Gharaibeh, M.N., Elayan, H.H., Salhab, A.S., 1988. Hypoglycemic effects of Teucrium polium. Journal of Ethnopharmacology 24, 93–99. Lin, C.C., 1992. Crude drugs used for the treatment of diabetes mellitus in Taiwan. American Journal of Chinese Medicine 20, 269– 279. Mini, D.A., Zhang, P., Zhao, X., Wang Chong, S., Zheno, Q., 1991. A neo clerodane diterpene from Teucrium japonicum. Phytochemistry 30, 4175–4177. Noor, H., Ashcroft, S.J.H., 1998. Pharmacological characterisation of the antihyperglycaemic properties of Tinospora crispa extract. Journal of Ethnopharmacology 62, 7–13. Pushparaj, P.N., Huat Tan, B.K., Hong Tan, C., 2001. The mechanism of hypoglycaemic action of semi- purified fractions of Averrhoa bilimbi in streptozotocin-diabetic rat. Life Sciences 70, 535–547. Rizk, A.M., Hammouda, F.M., Kamel, A., 1986. Iridoids and flavonoids of Teucrium polium herb. Planta Medica 43, 324–326. Shewade, Y.M., Umrani, M., Bhonde, R.R., 1999. Large scale isolation of islets by tissue culture of adult mouse pancreas. Transplantation Proceeding 31, 1721–1723. Ulubelen, K., Topcu, G., Kaya, U., 1994. Steroidal compounds from Teucrium chamaedrys subsp. chamaedrys. Phytochemistry 36, 171– 173.
Hypoglycaemic effect of Teucrium polium: studies with rat pancreatic islets Mohammad Ali Esmaeili, Razieh Yazdanparast∗ Institute of Biochemistry and Biophysics, P.O. Box 13145-1384, University of Tehran, Tehran, Iran Received 14 July 2003; received in revised form 31 May 2004; accepted 2 June 2004 Available online 11 August 2004
Abstract The aquous extract of Teucrium polium (Labiatae) has long being used in Iran as a hypoglycaemic aid without any knowledge about its probable side effects and mode of action. In an effort to assess the claimed hypoglycaemic property of the crude drug and to get some knowledge about the mechanism of action, the crude extract (0.5 g plant powder per kg body weight) was administered orally to a group of streptozotocin diabetic rats for six consecutive weeks. A significant decrease (64%) in blood glucose concentration was observed in the treated animals compared to the untreated diabetic rats, without any measurable effects on the major biochemical factors. In addition, the crude extract significantly enhanced the blood insulin level by almost 160% compared to the untreated diabetic rats. The insulinotropic property of the Teucrium polium extract was further assessed by an in vitro investigation using isolated pancreatic rat islets. Our data indicated that Teucrium polium crude extract is able to enhance insulin secretion by almost 135% after a single dose of plant extract (equivalent to 0.1 mg plant leaf powder per mL of the culture medium) at high glucose concentration (16 mmol/L). Meanwhile, the time pattern of insulin secretion was not affected by the plant extract compared to the untreated islets. These data clearly show that the plant extract, probably without metabolic transformation, is able to reduce high blood glucose levels through enhancing insulin secretion by the pancreas. © 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Hypoglycaemic; Insulin; Pancreatic islets; Teucrium polium
1. Introduction The use of medicinal plants for the treatment of diabetes mellitus dates back to the Ebers papyrus of about 1550 BC (Bailey and Day, 1989). Even after the discovery and use of insulin and other modern oral hypoglycaemic agents, the search for safer and more effective drugs of plant origin for the treatment of diabetes has continued (Lin, 1992; Bailey and Day, 1989). The aquous extract of Teucrium polium L. (Labiatae) has long being used in Iran as an hypoglycaemic aid without any knowledge about its probable side effects and its mode of action. In order to get some knowledge about the mechanism of action of this medicinal plant and in order to use it safely, ∗ Corresponding author. Tel.: +98-21-6113382; fax: +98-21-6404680. E-mail address: [email protected] (R. Yazdanparast).
0378-8741/$ – see front matter © 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2004.06.023
we evaluated the hypoglycaemic activity of Teucrium polium extract on the insulin secretion in streptozotocin (STZ) diabetic rats and also in the isolated pancreatic islets of rats at two different glucose concentrations (2 and 16 mM). The time pattern of insulin secretion by the isolated islets under the influence of the plant extract has been assessed.
2. Methodology 2.1. Plant material Aerial parts of Teucrium polium L. were collected from Fars province during spring, 1999. A voucher herbarium specimen (No. 570) was deposited in the Herbarium of the School of Pharmacy, Shaheed Beheshti University of Medical Sciences, Tehran, Iran. The plant aerial parts were air-dried
28
M.A. Esmaeili, R. Yazdanparast / Journal of Ethnopharmacology 95 (2004) 27–30
protected from direct sunlight and then powdered. The powder was kept in a closed container at 10 ◦ C. 2.2. Extraction The powdered plant material (250 g) was extracted three times with ethanol–water (7:3, v/v), at room temperature. The combined extracts were concentrated under reduced pressure and the volume was adjusted to 500 mL (equivalent to 0.5 g plant powder per mL). The concentrated extract was divided into 25 mL aliquots and kept at −20 ◦ C for further investigation. The w/w yield in terms of dry starting material was 6%. 2.3. Animals Male Wistar albino rats (30), 5–7 months old with a weight of 200–250 g were purchased from the Pasteur Institute, Tehran, Iran. They were housed under conventional conditions and were allowed access to food and water ad libitum. 2.4. Induction of experimental diabetes Streptozotocin (STZ; Sigma, USA; 40 mg/kg body weight) was dissolved in 0.1 M sodium citrate buffer at pH 4.5 just before use (Elias et al., 1994) and injected intraperitoneally to 22 rats. Control rats (8) received an equivalent volume of citrate buffer. One week after STZ administration, the diabetic rats with blood glucose levels between 16 and 25 mmol/L were selected and distributed into two groups (I and II). 2.5. Oral administration of the plant extract The plant extract was administrated by gavage (i.g.) to 12 rats of group II daily in a dose of 1 mL/rat (equivalent to 0.5 g plant powder/kg body weight) for 6 consecutive weeks. The control healthy rats (group III, n = 8) and the control diabetic rats (group I, n = 8) received the same volume of distilled water i.g. The blood glucose levels of rats in each group were determined weekly using a Glucomen kit (A. Menarini, diagnostics, Germany).
creases were isolated under sodium pentobarbital anesthesia (50 mg/kg body weight i.p.). Each pancreas was cut into small pieces and were subjected to enzymatic (collagenase) digestion for 20 min at 37 ◦ C on a magnetic stirrer. The medium consisted of Dublecco’s modified essential medium (DMEM) supplemented with collagenase type V (1 mg/mL), soybean trypsin inhibitor (STI) (2 mg/mL), and bovine serum albumin (BSA) fraction V (2%). The digested tissue was then centrifuged at 1000 rpm for 10 min, washed twice with phosphate buffer saline (pH 7.2) and seeded in culture flasks containing RPMI-1640 supplemented with 10% fetal calf serum (FCS) and incubated at 37 ◦ C in an incubator with 5% CO2 . After 24 h of incubation, islets were separated by hand picking under an inverted microscope. 2.8. Insulin secretion: static incubation Batches of 13 rat islets were placed into tubes containing 0.6 mL of the modified Krebs-bicarbonate medium with 20 mmol/L Hepes pH 7.4 and 2 mg/mL albumin (Ashcroft et al., 1986) along with various plant extract concentration. After 2 hours of incubation in a CO2 incubator at 37 ◦ C, samples were centrifuged at 5000 rpm to sediment the islets. The supernatant was collected and stored at −20 ◦ C for insulin measurement. 2.9. Insulin secretion: time dependent The triplicate culture flasks containing islets were transfered to tissue culture dishes and incubated for 1 h at 37 ◦ C in basal medium consisting of the modified Krebs-bicarbonate buffer with 2 mmol/L glucose. Doublicate Samples were collected for insulin measurement every 5 min up to 50 min. Then, the culture media was changed by the modified Krebsbicarbonate buffer containing 16 mmol/L glucose along with the plant extract (0.1 mg/mL). At 5 min intervals doublicate aliquots were taken for insulin measurements up to 90 min. The medium was then changed back to the basal medium containing 2 mmol/L glucose. Doublicate Samples were again collected for insulin determination at 5 min intervals up to 120 min. 2.10. Statistics
2.6. Insulin assay The Insulin level of each blood sample was measured by an enzyme-linked immunosorbent assay using a commercial kit (Rat Insulin ELISA; DRG Instruments GmbH, Germany), based on direct sandwich technique in which two monoclonal antibodies are directed against separate antigenic determinants on the insulin molecule.
The significance of differences between the control and the test groups was established by the paired Student’s t-test with the significance level set at P < 0.05.
3. Results
2.7. Pancreatic islet isolation
3.1. The effect of Teucrium polium extract on blood glucose level
The islet isolation was carried out according to Shewade’s method (Shewade et al., 1999). Briefly, six healthy pan-
One week after STZ injection, the diabetic rats with fasting blood glucose level in the range of 16–25 mmol/L, were
M.A. Esmaeili, R. Yazdanparast / Journal of Ethnopharmacology 95 (2004) 27–30
29
Table 1 Effect of EtOH–H2 O extract of Teucrium polium (0.5 g/kg body weight) on the blood glucose and body weight of diabetic rats Days −7
0
14
21
28
35
42
Normal rats
Blood glucose (mmol/L) 4.9 ± 0.1 Body weight (g) 238.0 ± 17.5
4.9 ± 0.1 265.0 ± 17.6
5.0 ± 0.8 276.0 ± 22.1
4.8 ± 0.2 293.0 ± 15.5
5.1 ± 0.4 296.0 ± 24.9
5.1 ± 0.2 306.0 ± 22.2
5.1 ± 0.1 310.0 ± 22.4
Diabetic rats
Blood glucose (mmol/L) 5.0 ± 0.1∗ Body weight (g) 215.0 ± 8.6
23.1 ± 3.5 227.0 ± 5.2
22.0 ± 1.2 202.0 ± 11.2
24.0 ± 2.7 185.0 ± 13.5
22.0 ± 0.9 173.0 ± 4.6
22.6 ± 1.4 172.0 ± 4.6
23.2 ± 3.4∗ 178.0 ± 2.5
Treated rats
Blood glucose (mmol/L) 4.9 ± 0.1∗ Body weight (g) 228.0 ± 24.2
23.7 ± 3.2 237.0 ± 27.6
19.5 ± 3.2 227.0 ± 24.8
1.7 ± 2.0 218.0 ± 23.2
15.2 ± 2.0∗ 224.0 ± 30.3
13.0 ± 17 238.0 ± 24.9
7.6 ± 3.1 253.0 ± 21.0
Each number represents the average of triplicate measurements ± S.D. ∗ P < 0.05
selected for further studies. Table 1 shows that prior to the extract administration, there was no significant difference between the blood glucose levels of the two diabetic groups of animals (I and II). However, after 6 weeks, the blood glucose levels of the treated rats were significantly lower than the controls (6.2 ± 1.64 mmol/L and 27.7 ± 5.1 mmol/L, respectively; P < 0.05). In contrast, the blood glucose level of the untreated diabetic rats remained high throughout the experimental period. The blood glucose level of the healthy group (III) remained unchanged during the course of the investigation. In addition, the blood insulin levels of the treated diabetic rats, after six consecutive weeks of treatments, were significantly enhanced (by 161%) compared to the untreated diabetic rats (8.96 ± 1.1 g/L and 3.42 ± 0.48 g/L, respectively; P < 0.05). Table 1 shows that, in contrast to the normal rats, there was a significant (P < 0.05) weight loss among group I and II, two weeks after STZ administration. However, Teucrium polium-treated rats showed signs of recovery in body weight
Fig. 1. Stimulation of rat islet insulin release by Teucrium polium EtOH–H2 O extract. Teucrium polium extract induced a dose dependent initiation of basal (2 mmol/L glucose, ) and stimulated (16 mmol/L glucose, ) insulin secretion, the data present the mean ± S.E.M. of three individual preparations of rat islets. Statistical significances of the observed differences in insulin release compared to the appropriate controls are ∗ P < 0.05, ∗∗ P < 0.001.
gain three weeks after treatments (224.0 ± 30.2 g) compared to diabetic control rats (173.0 ± 8.6 g). 3.2. The effect of Teucrium polium extract on insulin release by the rat islets Fig. 1 shows that Teucrium polium extract, in a dose range equivalent to 0.001–1 mg of the plant powder/mL, is capable of enhancing basal insulin release (at 2 mmol/L glucose) by a factor of 1.5 compared to the untreated islets. The insulinotropic effect of Teucrium polium was more evident at high glucose concentration (16 mM) (Fig. 2). Incubation of islets with 16 mmol/L glucose induced stimulation of basal insulin release, the rate of which fell rapidly when the high glucose medium was replaced by 2 mmol/L glucose. The addition of Teucrium polium extract (0.1 mg/mL) to high glucose medium resulted in an essentially similar pattern of insulin release. However, it was characterized
Fig. 2. Effect of Teucrium polium on the insulin secretion by the rat pancreatic islets with respect to time. Islets were incubated in basal (2 mmol/L glucose) medium for 1 h, the medium was then changed with either 16 mmol/L glucose () or 16 mmol/L glucose plus 0.1 mg/mL Teucrium polium extract () for 30 min, followed by incubation under basal condition for another 30 min, the data present the mean ± S.E.M. of three individual preparations of rat islets. Statistical significances of the observed differences in insulin release compared to the appropriate controls are ∗ P < 0.001.
30
M.A. Esmaeili, R. Yazdanparast / Journal of Ethnopharmacology 95 (2004) 27–30
by a more marked increase of secretion during stimulation period. 4. Discussion In the present investigation we have shown that STZ diabetic rats fed with a single dose of Teucrium polium extract (equivalent to 0.5 g plant powder per kg body weight) per day for six consecutive weeks exhibited significant blood glucose reduction (by 64%) and increase of blood insulin level by 160%. This hypoglycaemic potential, besides of confirming the traditional use of this plant as a blood glucose lowering agent has been reported in literature (Gharaibeh et al., 1988; Pushparaj et al., 2001; Noor and Ashcroft, 1998). Traditionally, the hyperglycaemic patients orally takes half a cup of a decoction of the plant aerial parts prepared with 50 g of the crude drug per liter of water once a day for 4–5 consecutive weeks. Phytochemical investigations on Teucrium spp. have shown the presence of compounds such as diterpene derivatives (Mini et al., 1991), fatty acid esters (Fonta et al., 1999), flavonoids (Rizk et al., 1986) and steroids (Ulubelen et al., 1994). However, so far, the biological activity of these compounds with respect to the hypoglycaemic effect of the plant has not been reported. Our results clearly indicated for the first time that Teucrium polium crude extract is able to produce a dose-dependant stimulation of basal insulin release and also to potentiate glucose-stimulated release of insulin in rat islets with no significant and detectable effects on the time pattern of insulin secretion. These data clearly indicate that the plant extract, probably without metabolic transformation, is capable of reducing high blood glucose mainly through enhancing insulin secretion. The insulinotropic agent of Teucrium polium has been purified to homogeneity in our lab and further work is in progress to elucidate its chemical structure.
Acknowledgements We are thankful to the Research Council of the University of Tehran for financial support of this investigation.
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