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Induction of insulin secretion by a component of Urtica dioica leave extract in perifused Islets of Langerhans and its in vivo effects in normal and streptozotocin diabetic rats
Journal of Ethnopharmacology 89 (2003) 47–53
Induction of insulin secretion by a component of Urtica dioica leave extract in perifused Islets of Langerhans and its in vivo effects in normal and streptozotocin diabetic rats Bijan Farzami∗ , D. Ahmadvand, S. Vardasbi, F.J. Majin, Sh. Khaghani Department of Medical Biochemistry, Tehran University of Medical Sciences, P.O. Box 14155-5399, Tehran, Iran Received 8 November 2002; accepted 19 June 2003
Abstract The blood glucose lowering effect of Urtica dioica (Stinging Nettle) as a medicinal plant has been noted in old writings such as those of Avicenna. Recently, there has also been other investigators that indicated the hypoglycemic effect of Urtica dioica. But so far, the mechanism of this effect has not been deduced. In this report, a perifusion system is arranged in which an exact number of Langerhans Islets were exposed to several fractions of extracts of Urtica dioica by TLC. The active ingredient fraction named F1 , caused a marked increase in insulin secretion. A simultaneous assay of glucose showed that the increase in insulin level was associated with a decrease in glucose level. Furthermore, the active component of Urtica dioica was found to increase the insulin content of blood sera in normal and streptozotocin diabetic rats that were injected intraperitoneally (i.p.) with the active ingredient of the extract. The in vivo studies presented in this report show that not only an increase in insulin level of blood sera was observed in rats after 30 min from the initial point of injection but a simultaneous decrease of blood sugar was detected when similar sera was tested for glucose. The increase in insulin level was six times during the 120 min of our determination. The decrease in blood sugar was found to be similar both in the level and time of initiation. On the basis of our findings, we assume that F1 is the active ingredient of plant leaves extract. The results show that the blood lowering effect of the extract was due to the enhancement of insulin secretion by Langerhance Isletes. © 2003 Elsevier Ireland Ltd. All rights reserved. Keywords: Urtica dioica; Perifusion; Insulin secretion; Hypoglycemic activity; Islets of Langerhans; Streptozotocin; Diabetes
1. Introduction The blood sugar lowering effect of Urtica dioica (UD) (Stinging Nettle) as a medicinal plant has been introduced in old script such as those written by Avicenna. There has been also other reports indicating the benefits of using the infusion or the extract of the leaves or other parts of the plant for the use in different conditions, i.e. diabetes (Roman Ramos et al., 1992; Swanston-Flatt et al., 1989) as well as other disorders like prostatic hyperplasia (Hirono et al., 1994; Krzeski et al., 1993; Schneider et al., 1995; Kayser et al., 1995) inflammation (Obertreis et al., 1996) rheumatoid arthritis, hypertension and allergic rhinitis (Miltman, 1990). It is noteworthy that the mode of the effect has not been determined to date. In our studies, the use of Langerhans-isolated Islets in perifusion experiments showed
that the blood lowering effect of the extract was solely due to the enhancement of insulin secretion by Langerhans Islets. On the basis of existing reports and our initial observations regarding the blood sugar lowering effect of UD, we undertook the task of investigating the mode of action by setting up a perifusion experiment. Furthermore, an in vivo study was carried out using the active component obtained by TLC and tested by perifusion experiment. Intraperitoneal injections of the active component of UD extract in normal and streptozotocin diabetic rats caused a significant rise in serum insulin accompanied by a drop in glucose level of blood sera.
2. Materials and methods 2.1. Plant material
∗ Corresponding
author. Tel.: +98-21-895-9745; fax: +98-21-895-9745. E-mail address: [email protected] (B. Farzami).
UD plant was collected from northern area of Iran, identified and authenticated at the Department of Biology, Tehran
0378-8741/$ – see front matter © 2003 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/S0378-8741(03)00220-4
48
B. Farzami et al. / Journal of Ethnopharmacology 89 (2003) 47–53
University. The leaves were separated, dried at room temperature and were grounded into powder. 2.2. Preparation of plant extract Ten grams of UD leave powder were boiled with 200 ml of distilled water for 15 min with an occasional stirring. The decoction preparation was then filtered through a gauz cloth followed by filtration through regular filter paper (Watman no. 1). The extract was evaporated to one-fifth of its original volume and kept at 4 ◦ C until its use within 1 week. 2.3. TLC separation One millimeter thick silica gel type G (Sigma Chemical Company, St. Louis, MO, USA) plates were prepared and were activated during 30 min at 110 ◦ C. Extract of Urtica dioica was applied several times in a narrow band to obtain sufficient concentration of the extract. Several elution mediums containing different proportions of isopropanol/water mixture ranging 10–90% (v/v) were used. The best separation was obtained with 70 (v/v) isopropanol/water mixture. The TLC time was 7–10 h. The plates were dried and detected by fluorescent light. Six distinct bands were present which were assigned F1 –F6 . Each band was then scraped off the plates and was eluted separately by similar mixture of isopropanol/water mixture. Each fraction was then separated by centrifugation at 3000 rpm to remove the solid gel. The supernatants were separated and the gels were washed twice with similar eluting solutions. Each fraction was then dried and weighed. The in between areas of fractions were eluted separately. 2.4. Animal experiment Male adult Albino rats (Wistar strain) of 220–370 g were fed on pellet diet and tap water for full acclimatization. The animals were kept 1 week in an air-conditioned animal room (22 ± 2 ◦ C) under a 12 h light/dark cycle prior to the experiment.
units of collagenase was added. The tubes were stoppered and was shaken by hand in 37 ◦ C water bath for 5 min. The tubes were then checked at each 30 s interval and the shaking was continued until no threads of tissue were observed. Ten milliliter of Krebs–Hepes containing 2.8 mM glucose and 36% BSA was then added and centrifuged three times for 30 s at 3000 rpm. The supernatant and the pink layer was removed from the pellet each time after centrifugation. After the final centrifugation the Islets were then identified and picked up by the aid of contrast microscope. Using stereomicroscope, the Islets could be detected as white particles against a darkened background of a petri dish. They can be identified from the rest of the tissues, the final separation was performed by a hand picking technique. This was done by a Pasteur pipette and application of a mild suction. With one pancreas a total of 100 Islets could be obtained. The Islets were separated into several equal portions in silicone-coated tubes and in Krebs–Hepes solution containing 2.8 mM glucose and 35% BSA (Lacy et al., 1967, 1972, 1976). A 1 ml suspension of Islets were placed in separate tubes under a flow of carbogene. One milliliter of the purified UD fraction was added to the tubes incubated at 37 ◦ C for 1 h and centrifuged at 3000 rpm, 0.5 ml of supernatant was taken and insulin was assayed by ELISA. The readings were compared with the control tubes containing, only Islets, or glucose, 16.7 mM and Islets. 2.6. In vivo studies 2.6.1. Animal preparation The animals were divided into two groups. Anesthesia was performed by i.p. injection of ketamine HCl, 50 mg/kg plus 0.2 ml chloroform. The first group of animals were given normal saline, served as control group, the second group were given fraction (F1 ) and normal saline. 2.6.2. GTT test All animals were fasted for 16 h. A concentration of 1.25 g/kg of glucose was injected intraperitonealy. Blood samples were obtained from retro-orbital vein plexus of the animal eye by hematocrit tubes 30 min before the injections and during the intervals of 30, 40, 90 and 120 min.
2.5. Isolation of Islets Islets were isolated from pancreas of rats by a technique described by Lacy and Kastianovsky. In this procedure, after a light ether anesthesia the Islets were efficiently extracted by injecting Hank’s solution in the pancreatic duct to produce distention. The pieces of pancreas weighing about 100 mg were chopped into smaller portions. The chopped pancreas was added to a 15 ml test tube containing Krebs–Hepes, 16.7 mM glucose in ice. The content of the test tube was centrifuged twice at 3000 rpm for 1 min. The supernatant was removed each time. An equal volume of Krebs–Hepes, 2.8 mM glucose solution was added and centrifuged. For each 1 ml of solution of Krebs–Hepes containing 2.8 mM glucose, 500
2.6.3. Blood sampling Approximately, 1 ml blood sample was obtained from each animal, placed in to an Eppendorf tube, centrifuged at 3000 rpm. The sera were collected and the blood glucose were estimated by glucose oxidase method. Insulin was assayed by an enzyme immunoassay ELISA kit (Dako). 2.7. Induction of diabetes The fasting animals (16 h) were given a single i.v. injection of streptozotocin, 40 mg/kg of body weight through the tail vein (the solution was prepared in cold citrate buffer, pH 4.5). The diabetic condition was found to be established
B. Farzami et al. / Journal of Ethnopharmacology 89 (2003) 47–53
49
Fig. 1. The effect of UD extract on insulin secretion in presence of 2.8 and 16.7 mM glucose at 30 and 60 min incubation time.
when the fasting blood sugar was in the range of 250 mg/dl. All the other conditions were the same as those of normal rats (Renold, 1969).
3. Results 3.1. In vitro studies 3.1.1. Secratogogue effect of aqueous extract of Urtica dioica on Islets of Langerhans by perifusion The results from perifusion by aqueous extract of UD leave showed an increase in insulin secretion both in 2.8 and 16.7 mM solutions of glucose containing Islets
(Fig. 1). In another set of experiment the optimal respiratory condition was maintained by purging carbogene (5/95% CO2 /O2 ). 3.1.2. Time factor for insulin induction Aliquots were taken from the medium during the incubation with aqueous extract, produced highest activity after a maximum of 60 min. The highest activity was obtained for solutions containing 16.7 mM glucose compared to that of 2.8 mM (Fig. 1). 3.1.3. Concentration factor The insulin secretion by the Islets was found to obey a concentration dependency both in glucose and in the
Fig. 2. Urtica dioica extract fractionation by TLC with silica gel (G60 ) that is detected by UV and fluorescence.
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B. Farzami et al. / Journal of Ethnopharmacology 89 (2003) 47–53
3.1.4. TLC results Fractionation by TLC showed six distinct fractions (F1 –F6 ) identified by UV and fluorescence (Rf F1 = 0.96, Rf F2 = 0.93, Rf F3 = 0.88, Rf F4 = 0.70, Rf F5 = 0.60, Rf F6 = 0.45) (Fig. 2). The weights of eluted fractions from 10 g of dried leaves were as follows: F1 = 14.75; F2 = 13.61; F3 = 7.95; F4 = 7.9; and F6 = 8.48 mg. The fraction with largest Rf value, assigned (F1 ), showed strongest effect in insulin secretion by perifusion (Fig. 3). This fraction was combined with similar fraction of F1 , obtained in several TLC separations.
Error Bars show 95.0% Cl of Mean Bars show Means 0.800
*
m U/ ml Insulin
0.600
0.400
3.2. In vivo results 0.200
0.000
F1
F2
F3
F4
F5
F6
control
Fig. 3. The effect of several TLC fractions of UD extract (F1 –F6 ) on insulin secretion by perifusion system.
extract concentration. 16.7 mM of glucose concentration in incubating medium, in presence of original concentration of extract produced a higher insulin secretary effect while in the presence of half concentrated solution of extract the effect was almost reduced to one-half (data not shown).
3.2.1. Insulin assay of rat sera The assay of insulin in normal and streptozotocin diabetic rat sera was performed by ELISA. The rats were injected i.p. by the active component of extract obtained from Urtica dioica leaves. A significant rise in the level of serum insulin was observed. This rise was primarily initiated after the lapse of 60 min from the initial point (time of sample injection), the rise was statistically significant after 60 min (P < 0.05) (Figs. 4 and 5). 3.2.2. Glucose assay of rat sera The glucose assay with glucose oxidase was carried out simultaneously in samples that were assayed for insulin. Glucose level showed a decrease initiated at 60 min from the time of sample injection. The trend continued in the last sample collection, 120 min (Figs. 6 and 7).
2.0
*
(a) (b) Bars show Means
mU/ml Insulin
1.5
1.0
* 0.5
*
0.0 0
30
60
90
120
Time (min) Fig. 4. The effect of intraperitoneal injection of F1 fraction on serum insulin level of rats as a function of time. (a) Normal serum insulin, as controls; (b) serum insulin containing fraction F1 .
B. Farzami et al. / Journal of Ethnopharmacology 89 (2003) 47–53
51
Fig. 5. The effect of intraperitoneal injection of F1 fraction on serum insulin level of diabetic rats as a function of time. (a) Serum insulin, as controls; (b) serum insulin containing fraction F1 .
600
mg/dl Glucose
500
(a) (b)
* *
400
*
300 200 100 0 time =0
time=30
Time=60
time=90
Time=120
vent mixture of 30/70% water/isopropanol was found most suitable for initial separations. The fractions with several Rf values, were tested by incubation in perifusion system, fraction F1 , showed strongest effect on insulin secretion. The most active fraction was then eluted with the same isopropanol/water solvent mixture. The in vivo studies were performed using the ultimate active fraction obtained from TLC. The fraction was dried in vacuum and re-dissolved in normal saline containing 1.25 g/kg glucose. An interaperitoneal injection of purified UD had a hypoglycemic effect in rats. This finding corresponded to our in vitro results.
Fig. 6. The effect of intraperitoneal injection of F1 fraction on serum glucose level of normal rats, as a function of time. (a) Serum glucose, as controls; (b) serum glucose containing fraction F1 .
600 500
The results of this study showed that the aqueous extract of Urtica dioica leaves (Stinging Nettle) could enhance the secratogogue function of Islets of Langerhans as detected by perifusion experiment, the highest insulin level was obtained at 60 min after the initial time of perifusion. The process was found to be concentration dependent. That is, the use of half concentrated extract decreased the insulin level to its half value (the results are not shown). The use of carbogen was effective in viability of Islet cells. For in vivo studies, purification of the preparation was further carried out by using TLC with several changes of solvent. A sol-
mg/dl Gluco s
4. Discussion
(a) (b)
* * *
400 300 200 100 0 time =0
time=30
Time=60
time=90 Time=120
Fig. 7. The effect of intraperitoneal injection of F1 fraction on serum glucose level of diabetic rats, as a function of time. (a) Serum glucose, as controls; (b) serum glucose containing fraction F1 .
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B. Farzami et al. / Journal of Ethnopharmacology 89 (2003) 47–53
(a) (b) 100
Pure effect of F1
80 60 40 20 0 -20 0
30
60
90
120
150
-40 -60 -80
Time (min) Fig. 8. The graph representing a relative changes in (a) serum insulin; (b) blood sugar, after an intraperitoneal injection of F1 fraction, as a function of time in normal rats.
(a) (b)
Pure effect of F1
20 10 0 -10 0
30
60
90
120
150
-20 -30 -40 -50
Time (min) Fig. 9. The graph representing a relative changes in (a) serum insulin; (b) blood sugar, after an intraperitoneal injection of F1 fraction, as a function of time in diabetic rats.
Although in in vivo studies, we examined this effect both in normal and STZ induced diabetic rats. In STZ diabetic rats the magnitude of changes were smaller than in normal rats, although in both cases the increase in insulin level and the decrease in glucose level were symmetrical (Figs. 7–9). We are hoping that the structure elucidation of the active component of UD will give more insight into the mechanism of insulin induction of the Islets of Langerhans.
References Hirono, T., Homma, M., Oka, K., 1994. Effects of Stinging Nettle root extracts and their steroidal components on the Na, K: ATPase of the benign prostatic hyperplasia. Planta Medica 60, 30–33. Kayser, K., Bubenzer, J., Kayser, G., Eichhorn, S., Bovin, N.V., Andres, S., Koopmann, J., Gabius, H.J., 1995. Expression of lectim, interleakin-2 and histopathologic blood group binding sites in prostate cancer and
its correlation with integrated optical density and syntactic structure analysis. Analytical and Quantitative Cytology and Histology 17, 135– 142. Krzeski, T., Kazon, M., Bordowski, A., witeska, A., Kuczera, J., 1993. Combined extracts of Urtica dioica and pygeum africanum in treatment of bening prostatic hyperplasia: double-blind comparison of two doses. Clinical Therapeutics 15, 1011–1020. Lacy, P.E., Kostianovsky, M., Louis, S., 1967. Method for the isolation of intact Islets of Langerhans from the rat pancreas. Diabetes 16, 35–39. Lacy, P.E., Walker, M.M., Fink, C.J., 1972. Perifusion of isolated rat Islets in vitro. Diabetes 21, 987–998. Lacy, P.E., Finke, E.H., Conant, S., Naber, S., 1976. Long-term perifusion of isolated rat Islets in vitro. Diabetes 25, 484–493. Miltman, P., 1990. Randomized double blind study of freeze dried Urtica dioica in the treatment of allergic rhinitis. Planta Medica 56, 44–47. Obertreis, B., Giller, K., Teucher, T., Behnke, B., Schmitz, H., 1996. Anti-inflammatory effect of Urtica dioica folia extract in comparison to caffeic malic acid. Arzneimittel Forschung 46, 52–56. Renold, A.E., 1969. Diabetogenic action of streptozotocin. The Journal of Clinical Investigation 48, 129.
B. Farzami et al. / Journal of Ethnopharmacology 89 (2003) 47–53 Roman Ramos, R., Alarcon Aguilar, F., Lara Lemus, A., Flores Saenz, J.L., 1992. Hypoglycemic effect of plants used in Mexico as antidiabetics. Archives of Medical Research 23, 59–64. Schneider, H.J., Honold, E., Masuhr, T., 1995. Treatment of benign prostatic hyperplasia. Results of a treatment study with the phytogenic combination of Sabal extract WS 1475 and Urtica extract WS
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1031 in urologic specialty practices. Fortschritte der Medizin 113, 37–40. Swanston-Flatt, S.K., Day, C., Flatt, P.R., Gould, B.J., Bailey, C.Y., 1989. Glyceamic effects of traditional European plant treatment for diabetes. Studies in normal and streptozotocin diabetic mice. Diabetes Research 10, 69–75.
Induction of insulin secretion by a component of Urtica dioica leave extract in perifused Islets of Langerhans and its in vivo effects in normal and streptozotocin diabetic rats Bijan Farzami∗ , D. Ahmadvand, S. Vardasbi, F.J. Majin, Sh. Khaghani Department of Medical Biochemistry, Tehran University of Medical Sciences, P.O. Box 14155-5399, Tehran, Iran Received 8 November 2002; accepted 19 June 2003
Abstract The blood glucose lowering effect of Urtica dioica (Stinging Nettle) as a medicinal plant has been noted in old writings such as those of Avicenna. Recently, there has also been other investigators that indicated the hypoglycemic effect of Urtica dioica. But so far, the mechanism of this effect has not been deduced. In this report, a perifusion system is arranged in which an exact number of Langerhans Islets were exposed to several fractions of extracts of Urtica dioica by TLC. The active ingredient fraction named F1 , caused a marked increase in insulin secretion. A simultaneous assay of glucose showed that the increase in insulin level was associated with a decrease in glucose level. Furthermore, the active component of Urtica dioica was found to increase the insulin content of blood sera in normal and streptozotocin diabetic rats that were injected intraperitoneally (i.p.) with the active ingredient of the extract. The in vivo studies presented in this report show that not only an increase in insulin level of blood sera was observed in rats after 30 min from the initial point of injection but a simultaneous decrease of blood sugar was detected when similar sera was tested for glucose. The increase in insulin level was six times during the 120 min of our determination. The decrease in blood sugar was found to be similar both in the level and time of initiation. On the basis of our findings, we assume that F1 is the active ingredient of plant leaves extract. The results show that the blood lowering effect of the extract was due to the enhancement of insulin secretion by Langerhance Isletes. © 2003 Elsevier Ireland Ltd. All rights reserved. Keywords: Urtica dioica; Perifusion; Insulin secretion; Hypoglycemic activity; Islets of Langerhans; Streptozotocin; Diabetes
1. Introduction The blood sugar lowering effect of Urtica dioica (UD) (Stinging Nettle) as a medicinal plant has been introduced in old script such as those written by Avicenna. There has been also other reports indicating the benefits of using the infusion or the extract of the leaves or other parts of the plant for the use in different conditions, i.e. diabetes (Roman Ramos et al., 1992; Swanston-Flatt et al., 1989) as well as other disorders like prostatic hyperplasia (Hirono et al., 1994; Krzeski et al., 1993; Schneider et al., 1995; Kayser et al., 1995) inflammation (Obertreis et al., 1996) rheumatoid arthritis, hypertension and allergic rhinitis (Miltman, 1990). It is noteworthy that the mode of the effect has not been determined to date. In our studies, the use of Langerhans-isolated Islets in perifusion experiments showed
that the blood lowering effect of the extract was solely due to the enhancement of insulin secretion by Langerhans Islets. On the basis of existing reports and our initial observations regarding the blood sugar lowering effect of UD, we undertook the task of investigating the mode of action by setting up a perifusion experiment. Furthermore, an in vivo study was carried out using the active component obtained by TLC and tested by perifusion experiment. Intraperitoneal injections of the active component of UD extract in normal and streptozotocin diabetic rats caused a significant rise in serum insulin accompanied by a drop in glucose level of blood sera.
2. Materials and methods 2.1. Plant material
∗ Corresponding
author. Tel.: +98-21-895-9745; fax: +98-21-895-9745. E-mail address: [email protected] (B. Farzami).
UD plant was collected from northern area of Iran, identified and authenticated at the Department of Biology, Tehran
0378-8741/$ – see front matter © 2003 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/S0378-8741(03)00220-4
48
B. Farzami et al. / Journal of Ethnopharmacology 89 (2003) 47–53
University. The leaves were separated, dried at room temperature and were grounded into powder. 2.2. Preparation of plant extract Ten grams of UD leave powder were boiled with 200 ml of distilled water for 15 min with an occasional stirring. The decoction preparation was then filtered through a gauz cloth followed by filtration through regular filter paper (Watman no. 1). The extract was evaporated to one-fifth of its original volume and kept at 4 ◦ C until its use within 1 week. 2.3. TLC separation One millimeter thick silica gel type G (Sigma Chemical Company, St. Louis, MO, USA) plates were prepared and were activated during 30 min at 110 ◦ C. Extract of Urtica dioica was applied several times in a narrow band to obtain sufficient concentration of the extract. Several elution mediums containing different proportions of isopropanol/water mixture ranging 10–90% (v/v) were used. The best separation was obtained with 70 (v/v) isopropanol/water mixture. The TLC time was 7–10 h. The plates were dried and detected by fluorescent light. Six distinct bands were present which were assigned F1 –F6 . Each band was then scraped off the plates and was eluted separately by similar mixture of isopropanol/water mixture. Each fraction was then separated by centrifugation at 3000 rpm to remove the solid gel. The supernatants were separated and the gels were washed twice with similar eluting solutions. Each fraction was then dried and weighed. The in between areas of fractions were eluted separately. 2.4. Animal experiment Male adult Albino rats (Wistar strain) of 220–370 g were fed on pellet diet and tap water for full acclimatization. The animals were kept 1 week in an air-conditioned animal room (22 ± 2 ◦ C) under a 12 h light/dark cycle prior to the experiment.
units of collagenase was added. The tubes were stoppered and was shaken by hand in 37 ◦ C water bath for 5 min. The tubes were then checked at each 30 s interval and the shaking was continued until no threads of tissue were observed. Ten milliliter of Krebs–Hepes containing 2.8 mM glucose and 36% BSA was then added and centrifuged three times for 30 s at 3000 rpm. The supernatant and the pink layer was removed from the pellet each time after centrifugation. After the final centrifugation the Islets were then identified and picked up by the aid of contrast microscope. Using stereomicroscope, the Islets could be detected as white particles against a darkened background of a petri dish. They can be identified from the rest of the tissues, the final separation was performed by a hand picking technique. This was done by a Pasteur pipette and application of a mild suction. With one pancreas a total of 100 Islets could be obtained. The Islets were separated into several equal portions in silicone-coated tubes and in Krebs–Hepes solution containing 2.8 mM glucose and 35% BSA (Lacy et al., 1967, 1972, 1976). A 1 ml suspension of Islets were placed in separate tubes under a flow of carbogene. One milliliter of the purified UD fraction was added to the tubes incubated at 37 ◦ C for 1 h and centrifuged at 3000 rpm, 0.5 ml of supernatant was taken and insulin was assayed by ELISA. The readings were compared with the control tubes containing, only Islets, or glucose, 16.7 mM and Islets. 2.6. In vivo studies 2.6.1. Animal preparation The animals were divided into two groups. Anesthesia was performed by i.p. injection of ketamine HCl, 50 mg/kg plus 0.2 ml chloroform. The first group of animals were given normal saline, served as control group, the second group were given fraction (F1 ) and normal saline. 2.6.2. GTT test All animals were fasted for 16 h. A concentration of 1.25 g/kg of glucose was injected intraperitonealy. Blood samples were obtained from retro-orbital vein plexus of the animal eye by hematocrit tubes 30 min before the injections and during the intervals of 30, 40, 90 and 120 min.
2.5. Isolation of Islets Islets were isolated from pancreas of rats by a technique described by Lacy and Kastianovsky. In this procedure, after a light ether anesthesia the Islets were efficiently extracted by injecting Hank’s solution in the pancreatic duct to produce distention. The pieces of pancreas weighing about 100 mg were chopped into smaller portions. The chopped pancreas was added to a 15 ml test tube containing Krebs–Hepes, 16.7 mM glucose in ice. The content of the test tube was centrifuged twice at 3000 rpm for 1 min. The supernatant was removed each time. An equal volume of Krebs–Hepes, 2.8 mM glucose solution was added and centrifuged. For each 1 ml of solution of Krebs–Hepes containing 2.8 mM glucose, 500
2.6.3. Blood sampling Approximately, 1 ml blood sample was obtained from each animal, placed in to an Eppendorf tube, centrifuged at 3000 rpm. The sera were collected and the blood glucose were estimated by glucose oxidase method. Insulin was assayed by an enzyme immunoassay ELISA kit (Dako). 2.7. Induction of diabetes The fasting animals (16 h) were given a single i.v. injection of streptozotocin, 40 mg/kg of body weight through the tail vein (the solution was prepared in cold citrate buffer, pH 4.5). The diabetic condition was found to be established
B. Farzami et al. / Journal of Ethnopharmacology 89 (2003) 47–53
49
Fig. 1. The effect of UD extract on insulin secretion in presence of 2.8 and 16.7 mM glucose at 30 and 60 min incubation time.
when the fasting blood sugar was in the range of 250 mg/dl. All the other conditions were the same as those of normal rats (Renold, 1969).
3. Results 3.1. In vitro studies 3.1.1. Secratogogue effect of aqueous extract of Urtica dioica on Islets of Langerhans by perifusion The results from perifusion by aqueous extract of UD leave showed an increase in insulin secretion both in 2.8 and 16.7 mM solutions of glucose containing Islets
(Fig. 1). In another set of experiment the optimal respiratory condition was maintained by purging carbogene (5/95% CO2 /O2 ). 3.1.2. Time factor for insulin induction Aliquots were taken from the medium during the incubation with aqueous extract, produced highest activity after a maximum of 60 min. The highest activity was obtained for solutions containing 16.7 mM glucose compared to that of 2.8 mM (Fig. 1). 3.1.3. Concentration factor The insulin secretion by the Islets was found to obey a concentration dependency both in glucose and in the
Fig. 2. Urtica dioica extract fractionation by TLC with silica gel (G60 ) that is detected by UV and fluorescence.
50
B. Farzami et al. / Journal of Ethnopharmacology 89 (2003) 47–53
3.1.4. TLC results Fractionation by TLC showed six distinct fractions (F1 –F6 ) identified by UV and fluorescence (Rf F1 = 0.96, Rf F2 = 0.93, Rf F3 = 0.88, Rf F4 = 0.70, Rf F5 = 0.60, Rf F6 = 0.45) (Fig. 2). The weights of eluted fractions from 10 g of dried leaves were as follows: F1 = 14.75; F2 = 13.61; F3 = 7.95; F4 = 7.9; and F6 = 8.48 mg. The fraction with largest Rf value, assigned (F1 ), showed strongest effect in insulin secretion by perifusion (Fig. 3). This fraction was combined with similar fraction of F1 , obtained in several TLC separations.
Error Bars show 95.0% Cl of Mean Bars show Means 0.800
*
m U/ ml Insulin
0.600
0.400
3.2. In vivo results 0.200
0.000
F1
F2
F3
F4
F5
F6
control
Fig. 3. The effect of several TLC fractions of UD extract (F1 –F6 ) on insulin secretion by perifusion system.
extract concentration. 16.7 mM of glucose concentration in incubating medium, in presence of original concentration of extract produced a higher insulin secretary effect while in the presence of half concentrated solution of extract the effect was almost reduced to one-half (data not shown).
3.2.1. Insulin assay of rat sera The assay of insulin in normal and streptozotocin diabetic rat sera was performed by ELISA. The rats were injected i.p. by the active component of extract obtained from Urtica dioica leaves. A significant rise in the level of serum insulin was observed. This rise was primarily initiated after the lapse of 60 min from the initial point (time of sample injection), the rise was statistically significant after 60 min (P < 0.05) (Figs. 4 and 5). 3.2.2. Glucose assay of rat sera The glucose assay with glucose oxidase was carried out simultaneously in samples that were assayed for insulin. Glucose level showed a decrease initiated at 60 min from the time of sample injection. The trend continued in the last sample collection, 120 min (Figs. 6 and 7).
2.0
*
(a) (b) Bars show Means
mU/ml Insulin
1.5
1.0
* 0.5
*
0.0 0
30
60
90
120
Time (min) Fig. 4. The effect of intraperitoneal injection of F1 fraction on serum insulin level of rats as a function of time. (a) Normal serum insulin, as controls; (b) serum insulin containing fraction F1 .
B. Farzami et al. / Journal of Ethnopharmacology 89 (2003) 47–53
51
Fig. 5. The effect of intraperitoneal injection of F1 fraction on serum insulin level of diabetic rats as a function of time. (a) Serum insulin, as controls; (b) serum insulin containing fraction F1 .
600
mg/dl Glucose
500
(a) (b)
* *
400
*
300 200 100 0 time =0
time=30
Time=60
time=90
Time=120
vent mixture of 30/70% water/isopropanol was found most suitable for initial separations. The fractions with several Rf values, were tested by incubation in perifusion system, fraction F1 , showed strongest effect on insulin secretion. The most active fraction was then eluted with the same isopropanol/water solvent mixture. The in vivo studies were performed using the ultimate active fraction obtained from TLC. The fraction was dried in vacuum and re-dissolved in normal saline containing 1.25 g/kg glucose. An interaperitoneal injection of purified UD had a hypoglycemic effect in rats. This finding corresponded to our in vitro results.
Fig. 6. The effect of intraperitoneal injection of F1 fraction on serum glucose level of normal rats, as a function of time. (a) Serum glucose, as controls; (b) serum glucose containing fraction F1 .
600 500
The results of this study showed that the aqueous extract of Urtica dioica leaves (Stinging Nettle) could enhance the secratogogue function of Islets of Langerhans as detected by perifusion experiment, the highest insulin level was obtained at 60 min after the initial time of perifusion. The process was found to be concentration dependent. That is, the use of half concentrated extract decreased the insulin level to its half value (the results are not shown). The use of carbogen was effective in viability of Islet cells. For in vivo studies, purification of the preparation was further carried out by using TLC with several changes of solvent. A sol-
mg/dl Gluco s
4. Discussion
(a) (b)
* * *
400 300 200 100 0 time =0
time=30
Time=60
time=90 Time=120
Fig. 7. The effect of intraperitoneal injection of F1 fraction on serum glucose level of diabetic rats, as a function of time. (a) Serum glucose, as controls; (b) serum glucose containing fraction F1 .
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B. Farzami et al. / Journal of Ethnopharmacology 89 (2003) 47–53
(a) (b) 100
Pure effect of F1
80 60 40 20 0 -20 0
30
60
90
120
150
-40 -60 -80
Time (min) Fig. 8. The graph representing a relative changes in (a) serum insulin; (b) blood sugar, after an intraperitoneal injection of F1 fraction, as a function of time in normal rats.
(a) (b)
Pure effect of F1
20 10 0 -10 0
30
60
90
120
150
-20 -30 -40 -50
Time (min) Fig. 9. The graph representing a relative changes in (a) serum insulin; (b) blood sugar, after an intraperitoneal injection of F1 fraction, as a function of time in diabetic rats.
Although in in vivo studies, we examined this effect both in normal and STZ induced diabetic rats. In STZ diabetic rats the magnitude of changes were smaller than in normal rats, although in both cases the increase in insulin level and the decrease in glucose level were symmetrical (Figs. 7–9). We are hoping that the structure elucidation of the active component of UD will give more insight into the mechanism of insulin induction of the Islets of Langerhans.
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