Antidiabetic activity and toxicity of Zizyphus spina-christi leaves

Journal of Ethnopharmacology 101 (2005) 129–138

Antidiabetic activity and toxicity of Zizyphus spina-christi leaves Ahmed O. Abdel-Zaher a,∗ , Safa Y. Salim a , Mahmoud H. Assaf b , Randa H. Abdel-Hady c a

Department of Pharmacology, Faculty of Medicine, Assiut University, Egypt Department of Pharmacognosy, Faculty of Pharmacy, Assiut University, Egypt Department of Forensic Medicine and Toxicology, Faculty of Medicine, Assiut University, Egypt b

c

Received 7 March 2005; received in revised form 7 March 2005; accepted 7 April 2005 Available online 11 July 2005

Abstract The effect of the butanol extract of Zizyphus spina-christi (L.), Willd (Rhamnaceae) leaves and its major saponin glycoside, christinin-A, on the serum glucose and insulin levels was studied in non-diabetic control, type-I (insulin-dependent) and type-II (non-insulin-dependent) diabetic rats. Pretreatment either with 100 mg/kg butanol extract or christinin-A potentiated glucose-induced insulin release in non-diabetic control rats. In type-II but not in type-I diabetic rats pretreatment with the butanol extract or christinin-A improved the oral glucose tolerance and potentiated glucose-induced insulin release. Treatment either with 100 mg/kg butanol extract or christinin-A reduced the serum glucose level and increased the serum insulin level of non-diabetic control and type-II diabetic rats but not of type-I diabetic rats. Effects of the butanol extract and christinin-A were similar. Pretreatment of non-diabetic control and type-II diabetic rats either with 100 mg/kg butanol extract or christinin-A enhanced the glucose lowering and insulinotropic effects of 5 g/kg glibenclamide. The hyperglycemic and hypoinsulinemic effects of 30 mg/kg diazoxide in non-diabetic control and type-II diabetic rats were inhibited and antagonized, respectively by pretreatment with the butanol extract or christinin-A. The relaxant effects of different concentrations of diazoxide on the isolated norepinephrine-contracted aortic strips were inhibited by 100 ␮mol/l christinin-A or 10 ␮mol/l glibenclamide. The combination of glibenclamide and christinin-A led to complete inhibition of the relaxant effects of different concentrations of diazoxide. At a dose level much higher than that required to produce satisfactory insulinotropic and hypoglycemic effects, the butanol extract of Zizyphus spina-christi leaves produced a depressant effect on the central nervous system in rats. Treatment of rats with 100 mg/kg butanol extract for 3 months produced no functional or structural disturbances in liver and kidney and no haematological changes. In addition, the oral LD50 of the butanol extract in mice was 3820 mg/kg, while that of glibenclamide was 3160 mg/kg. Thus, Zizyphus spina-christi leaves appears to be a safe alternative to lower blood glucose. The safe insulinotropic and subsequent hypoglycemic effects of Zizyphus spina-christi leaves may be due to a sulfonylurea-like activity. © 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: Zizyphus spina-christi (L.) Willd; Rhamnaceae; Type-I diabetic rats; Type-II diabetic rats; Glibenclamide

1. Introduction Zizyphus (Rhamnaceae) species are used in folk medicine for the treatment of some diseases, such as digestive disorders, weakness, liver complaints, obesity, urinary troubles, diabetes, skin infections, fever, diarrhea and insomnia (Kirtikar and Basu, 1984; Han and Park, 1986). From the different species of the genus Zizyphus, peptide and cyclopeptide alkaloids, flavonoids, sterols, tannins, ∗

Corresponding author. Fax: +20 88 332278. E-mail address: [email protected] (A.O. Abdel-Zaher).

0378-8741/$ – see front matter © 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2005.04.007

betulinic acid and triterpenoidal saponin glycosides have been isolated and chemically identified (Ikram et al., 1981; Higuchi et al., 1984; Nawwar et al., 1984; Han et al., 1990; Barboni et al., 1994; Abu-Zarga et al., 1995; Cheng et al., 2000; Shahat et al., 2001; Tripathi et al., 2001). From the butanol extract of the leaves of Zizyphus spinachristi (L.), Willd. (Rhamnaceae) growing in Egypt, four triterpenoidal saponin glycosides were isolated and named christinin-A, B, C and D, respectively. Christinin-A was the major saponin (Mahran et al., 1996). Some pharmacological screening studies indicated that the aqueous extract of Zizyphus spina-christi root bark has an

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antinociceptive activity in mice and rats (Adzu et al., 2001) and a central depressant effect in mice (Adzu et al., 2002) and the methanol extract of Zizyphus spina-christi stem bark has antidiarrheal effects in rats (Adzu et al., 2003). Furthermore, Glombitza et al. (1994) found that the butanol extract of Ziziphus spina-christi leaves or its main saponin glycoside, christinin-A, improved glucose utilization in diabetic rats, but not in normal rats after 1 and 4 weeks of treatment. Serum insulin level showed a significant increase in diabetic rats treated for a period of 4 weeks with the butanol extract. The antidiabetic effect of butanol extract was more pronounced than that of christinin-A. Since Zizyphus spina-christi is a wild tree commonly available in Egypt and its leaves are used in folk medicine for the treatment of diabetes mellitus, it is, therefore deemed interesting to reexamine the potential antidiabetic activity of these leaves. In addition, an attempt was undertaken to elucidate the possible mechanism(s) of the antidiabetic activity and to estimate the toxicity of these leaves, if present.

2. Materials and methods 2.1. Plant material The leaves of Zizyphus spina-christi (L.) Willd were collected from trees growing at the Experimental Station of Medicinal Plants, Faculty of Pharmacy, Assiut University, Egypt. The leaves were air dried and then powdered. 2.2. Preparation of the plant extract The powdered leaves (5 kg) were extracted with 20 l of 70% ethanol by percolation till complete exhaustion. The ethanol extract was filtered and concentrated under reduced pressure (150 g). The ethanol extract was then fractionated using n-hexane, ethyl acetate and finally with water-saturated

butanol. The butanol fraction was dehydrated over anhydrous sodium sulphate and concentrated under reduced pressure to afford 120 g of the butanol extract. 2.3. Isolation of saponins The triterpenoidal saponin glycosides were isolated from the butanol fraction of the leaves (1 g) using a Sephadex LH-20 column (Pharmacia, SR25/ 50) using methanol and methanol: water (80:20) as eluent, flow rate 20 ml/min and run time 200 min. The system used was chloroform: methanol: n-propanol: water (9:12:1:8). The obtained fractions were purified by means of preparative reversed phase HPLC. The HPLC system consists of a Knauer HPLC delivery pump 64, a Knauer variable wavelength spectrophotometric detector (VO890) monitored at 210 nm, Shimadzu CR6A chromatopac recording integrator, C-ODS-5 (25 cm × 20 mm i.d.). The mobile phase was methanol:water (80:20), flow rate 1.5 ml/min. The injected volume of the sample was 20 ␮l and run times were 7–25 min. The retention time for christinin-A was 15 min and for christininB was 7 min. The isolated christinin-B was further purified on the same HPLC column, with the mobile phase methanol:acetonitrile:water (4:2.5: 3.5). The retention time for christinin-C and D was 13 min. Christinin-C and D were not separated directly on HPLC. They were separated from each other using preparative thin layer chromatography plates, Kieselgel 60 F254 (Merck), chloroform:methanol:water (65:40:10) as solvent system. The separated substances were run on HPLC, mobile phase methanol:water (80:20) for final purification. From 120 g of the butanol extract of Zizyphus spina-christi leaves, the isolated amounts of christinin-A, B, C and D were 250, 10, 8 and 6 mg, respectively. The major saponin glycoside, christinin-A (Fig. 1), was crystalized from ether as white ◦ amorphous powder, mp 288–290 ◦ C, ([α]25 D , 23 C = 1.0, methanol). Thin layer chromatography was performed on silica gel G (254 nm) using the solvent system chloro-

Fig. 1. Christinin-A.

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131

form:methanol:water (65:40:10) and authentic christinin-A. The spots were visualized by spraying with 10% sulphuric acid followed by heating at 110 ◦ C for 10 min. The isolated christinin-A was identical and confirmed through comparison of its physical and chemical data with those reported for the authentic christinin-A.

trol animals received an equal volume of the vehicle. Blood samples were collected for glucose and insulin levels estimation before and 30, 60 and 120 min after each treatment.

2.4. Induction of diabetes mellitus in rats

The overnight fasted non-diabetic control, type-I and type-II diabetic rats were treated with 5 mg/kg glibenclamide (in saline/NaOH/ethanol) orally or with 30 mg/kg diazoxide (in saline/NaOH/ethanol) intraperitoneally. Control animals were treated likewise with the vehicle. Other groups of animals were treated orally either with 100 mg/kg butanol extract or christinin-A 30 min before administration of glibenclamide or diazoxide. Blood samples were collected for glucose and insulin levels estimation before and 30, 60 and 120 min after treatment of rats with glibenclamide or diazoxide.

The research was conducted in accordance with the internationally accepted principles for laboratory animal use and care as found in the European Community guidelines. Type-I diabetes (insulin-dependent diabetes mellitus) was induced by an intraperitoneal injection of 65 mg/kg streptozotocin in 9-week-old male Wistar rats (Burcelin et al., 1993). Streptozotocin was dissolved in isotonic saline immediately before use. Animals were used to study the effect of drugs under investigation 2 days after the injection of streptozotocin. Type-II diabetes (non-insulin-dependent diabetes mellitus) was induced in 1-day-old male Wistar rats by treating them with a single intraperitoneal injection of 95 mg/kg streptozotocin (Angel et al., 1996). After weaning, rats were individually housed with water and food available ad libitum. When rats were 9-week olds they were used to evaluate the effect of drugs under investigation. Control animals received only the vehicle. 2.5. Oral glucose tolerance test Groups, six animals each, of non-diabetic control, typeI and type-II diabetic rats were fasted overnight before the day of experiments with free access to water. These animals were treated with an oral d-glucose load of 2 g/kg by means of a stomach tube. Also, in this set of experiments, groups of non-diabetic control, type-I and type-II diabetic rats were treated orally with either the butanol extract of Zizyphus spina-christi leaves or its major saponin glycoside, christininA, at a dose level of 100 mg/kg, each for 30 min before the oral administration of the d-glucose load. Control animals were treated with the vehicle (isotonic saline). Blood samples were collected for glucose and insulin levels estimation from the orbital sinus of rats before and 30, 60 and 120 min after the administration of the oral glucose load. In this study, pentobarbital sodium was used to minimize the effect of the stress resulting from handling, injection and blood sampling on animals (Crofford and Davis, 1965). The intraperitoneal injection of 25 mg/kg pentobarbital sodium into non-diabetic control, type-I and type-II diabetic rats did not produce any significant changes in blood glucose and insulin levels of these animals. 2.6. Effect on serum glucose and insulin levels Groups, six animals each, of overnight fasted non-diabetic control, type-I and type-II diabetic rats were treated either with 100 mg/kg butanol extract or christinin-A orally. Con-

2.7. Effect on glibenclamide or diazoxide-induced alterations in serum glucose and insulin levels

2.8. Effect on isolated aortic strips Adult male rabbits weighing 1.5–2.5 kg were used in these experiments. Animals were killed by decapitation. The thorax was opened and the descending aorta was removed. The aorta was immediately placed in ice-cold oxygenated Kreb’sHenseleit solution. The composition of Kreb’s-Henseleit solution was as follows (mmo/l): NaCl 118; KCl 4.69; CaCl2 2.52; NaHCO3 25; MgSO4 ·7H2 O 1.18; KH2 PO4 1.17 and d-glucose 11.10. The vessel was cleaned of fat and loose connective tissue and cut into long continuous spiral strips (5 mm wide and 3–4 cm long, approximately) by threading a glass rod through the lumen of the aorta and with a pair of fine scissors. The aortic strips were suspended in an organ bath containing 20 ml of Kreb’s-Henseleit solution maintained at 37 ◦ C and bubbled with carbogen mixture (5% CO2 in O2 ). The strips were connected to a T2 isotonic transducer and an amplifier of a two-channel Oscillograph MD2 (BioScience, Kent, UK). Each preparation was allowed to equilibrate for 30 min before the experiment was started, during which time the physiological salt solution was renewed every 15 min. In these experiments, the aortic strips were first contracted with 5 ␮mol/l norepinephrine (from a stock solution of 1 mmo/l in saline). Tissues were then washed several times and allowed to relax to base line levels, before the same concentration of norepinephrine was reapplied. At the plateau of the norepinephrine contraction, tissues were exposed to graded concentrations (0.01–1 mmol/l) of diazoxide (from a stock solution of 5 mmol/l in saline/ethanol/ NaOH). The effects of diazoxide on norepinephrine-contracted strips were recorded 10 min after addition of each concentration. The percentage changes in the height of norepinephrineinduced contractions were determined and the cumulative concentration–response curve of diazoxide-induced changes in the aortic strip contractions elicited by norepinephrine was constructed. In addition, the effects produced by pretreatment with 100 ␮mol/l christinin-A (from

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a stock solution of 5 mmol/l) for 15 min on this cumulative concentration–response curve of diazoxide were evaluated. Also, the alterations produced by pretreatment with 10 ␮mol/l glibenclamide (from a stock solution of 5 mmol/l in saline/ ethanol/NaOH) alone or in combination with 100 ␮mol/l christinin-A for 15 min on the effects of different concentrations of diazoxide on the aortic strips contracted with norepinephrine were determined. One tissue always served as a control. The final concentration of ethanol in the bath fluid was always less than 0.2%, which had no effect on tissue responses. 2.9. Measurement of the spontaneous locomotor activity and pentobarbital sleeping time The spontaneous locomotor activity of rats was carried out at 9:00 AM to 1:00 PM using the activity cage (Ugo Basile, Comerio (VA), Italy) as previously reported (Reavill et al., 1990). Four groups of six male Wistar rats weighing 120–200 g were used. Water and food were allowed ad libitum. The rats were placed far from intense or sudden noise and bright light to avoid upsetting of animals. After 1 h of the oral administration of 100, 400 and 800 mg/kg butanol extract of Zizyphus spina-christi leaves or saline, the animals were placed singly in the activity cage and the number of counts over a period of 30 min was recorded. The mean sleeping time of pentobarbital was determined in groups of male Wistar rats treated with 30 mg/kg pentobarbital sodium intraperitoneally or pretreated orally with 100, 400 and 800 mg/kg butanol extract, 1 h before injection of the drug. 2.10. Determination of the median lethal dose (LD50 ) Groups of ten male adult albino mice weighing 20–28 g were housed under the same conditions. The animals were treated orally by means of a stomach tube with graded doses of the butanol extract of Zizyphus spina-christi leaves or glibenclamide. The mortality was determined 24 h later, in each group of animals. The LD50 and its 95% fiducial limits for the butanol extract or glibenclamide were calculated. Control groups of mice were treated with the pure solvents. 2.11. Effect on liver and kidney function and structure Two groups, six animals each, of male Wistar rats were used. Water and food were allowed ad libitum. Rats of group-I were treated once daily for 3 months with 100 mg/kg butanol extract of Zizyphus spina-christi leaves orally. Group-II rats were treated likewise with the vehicle. Twenty-four hours after the last dose, animals were sacrificed by decapitation. Blood, liver and kidney tissues were obtained from each animal for biochemical, haematological and morphological analysis.

2.12. Histopathological examination Liver and kidney tissue blocks for light microscopy were fixed in 10% neutral formalin. All samples were embeded in paraffin, cut in sections of 3–5 um thickness and stained with hematoxylin and eosin. 2.13. Haematology Blood samples were analyzed by H5-MSEAC, 5 parameter single probe cell counter (Italy) for estimation of red blood cells count, Haemoglobin value, Haematocrit value, mean corpuscular volume, and total white blood cells count. 2.14. Biochemical measurements The serum glucose level was assayed by an enzymatic colorimetric method using a commercially available Glucose Enzymatique PAP kit (Bio Merieux, France). The serum insulin concentration was measured by the radioimmunoassay method using a commercially available Coat-ACount Insulin kit (Diagnostic Products Corporation, USA). The activities of serum glutamyl-oxaloacetic transa-minase (SGOT), glutamyl-pyruvic transaminase (SGPT), alkaline phosphatase (SAP) and total billirubin level (i.e., liver function tests) as well as creatinine and urea nitrogen levels (i.e., kidney function tests) were measured spectrophotometrically using standardized commercially available Diamond kits (Modern Laboratory Chemical, Egypt). The serum sodium, potassium and calcium levels were determined by flame photometry using a Jenway flame photometer (England). 2.15. Chemicals The following chemicals were used: glibenclamide (Hoechst Co., Egypt); streptozotocin (Sigma Chemical Co., USA); diazoxide (Sigma Chemical Co., USA); d-Glucose (El Naser Pharmaceutical and Chemical Co., Egypt) and pentobarbital sodium (Sigma Chemical Co., USA). All other chemicals were of analytical grade. 2.16. Statistical analysis of results The variability of results is expressed as mean ± S.E. The significance of differences between mean values was determined by the student’s t-test.

3. Results 3.1. Oral glucose tolerance Results presented in Table 1 show that the oral administration of a glucose load of 2 g/kg to non-diabetic control rats produced a rapid increase in the serum glucose and insulin levels, followed by a progressive decline until they nearly

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Table 1 Effect of 30 min pretreatment either with 100 mg/kg butanol extract (BE) of Zizyphus spina-christi leaves or christinin-A (CA) orally on oral glucose (Glu) tolerance and oral glucose load-induced alterations in the serum insulin level of non-diabetic control, type-I and type-II diabetic rats Time (min)a

Serum glucose (mg/dl) Glu

Serum insulin (␮IU/ml) BE + Glu

CA + Glu

Glu

BE + Glu

CA + Glu

0 30 60 120

Non-diabetic control rats 98.00 ± 7.10 93.08 240.20 ± 11.50 190.02** 190.01 ± 12.02 155.02** 102.10 ± 9.10 90.01**

± ± ± ±

6.40 15.03 10.20 7.20

95.02 198.03** 16.01** 92.03**

± ± ± ±

6.60 16.02 11.04 7.10

15.00 40.32 27.20 16.12

± ± ± ±

0.80 2.14 2.21 1.20

16.10 55.41** 34.30** 17.22

± ± ± ±

0.90 3.22 2.11 1.10

16.20 54.31** 33.42** 17.21

± ± ± ±

0.82 3.10 2.12 1.12

0 30 60 120

Type-I diabetic rats 210.12 ± 12.04 525.10 ± 18.01 440.20 ± 15.21 402.30 ± 14.20

208.20 518.32 430.12 400.14

± ± ± ±

11.22 19.04 14.20 18.30

210.22 520.40 435.30 401.32

± ± ± ±

12.30 22.20 28.14 16.22

2.40 2.52 2.50 2.40

± ± ± ±

0.18 0.15 0.13 0.14

2.31 2.62 2.60 2.40

± ± ± ±

0.15 0.20 0.18 0.13

2.32 2.62 2.61 2.40

± ± ± ±

0.16 0.12 0.16 0.12

0 30 60 120

Type–II diabetic rats 150.12 ± 12.22 370.40 ± 18.31 310.24 ± 22.32 195.14 ± 14.22

140.10 210.12** 205.12** 145.14**

± ± ± ±

11.22 18.11 12.13 11.21

142.20 218.42** 208.31** 148.10**

± ± ± ±

10.40 16.21 14.22 13.21

11.01 19.11 17.20 12.08

± ± ± ±

0.50 1.12 1.20 0.85

11.10 32.20** 25.30** 14.10**

± ± ± ±

0.58 2.62 2.11 1.12

11.20 31.10** 24.30** 14.08**

± ± ± ±

0.72 2.41 2.20 1.20

Values are mean ± S.E. of six experiments. a After glucose load administration. ** P < 0.01 vs. corresponding Glu values.

reached control values at the end the of 120 min of investigation. In type-I diabetic rats, there was a marked intolerance to the oral glucose associated with a concomitant failure in induction of insulin secretion. In type-II diabetic rats, there was an intolerance to the oral glucose and a reduction in glucose-induced insulin secretion. Pretreatment either with 100 mg/kg butanol extract or christinin-A orally, 30 min before the oral glucose load reduced the serum glucose level of non-diabetic control and type-II diabetic rats and produced no change in the serum glucose level of type-I diabetic rats .The elevation in the serum insulin level induced in non-diabetic control and typeII diabetic rats by the oral glucose load was potentiated by pretreatment with butanol extract or christinin-A. In type-I diabetic rats, there was no change in serum insulin level. 3.2. Serum glucose and insulin levels Data listed in Table 2 illustrate that the oral administration of 100 mg/kg either butanol extract or christinin-A to nondiabetic control and type-II diabetic rats reduced the serum glucose level after 60 and 120 min of administration. The maximal percentages of reduction, that occurred after 60 min of butanol extract and christinin-A administration were 20 and 19% in control rats as well as 24 and 22% in typeII diabetic rats, respectively. In type-I diabetic rats, butanol extract or christinin-A produced no changes in the serum glucose level. The serum insulin level of non-diabetic control and type-II diabetic rats increased significantly after 60 and 120 min of oral administration of either butanol extract or christinin-A at a dose level of 100 mg/kg, each. The maximal percentages of increase, that occurred after 60 min of

butanol extract and christinin-A administration were 38 and 34% in control rats as well as 64 and 60% in type-II diabetic rats, respectively. 3.3. Glibenclamide or diazoxide-induced alterations in serum glucose and insulin levels Results presented in Table 3 illustrate that oral administration of 5 mg/kg glibenclamide to non-diabetic control and type-II diabetic rats produced a progressive reduction in the serum glucose level. This effect started after 30 min of glibenclamide administration. Oral administration of 5 mg/kg glibenclamide to type-I diabetic rats decreased the serum glucose level only after 120 min of its administration. The progressive reduction in the serum glucose level induced by administration of glibenclamide to non-diabetic control and type-II diabetic rats was potentiated by 30 min pretreatment either with 100 mg/kg butanol extract or christinin-A orally. The same pretreatment produced no change in type-I diabetic rats. The serum insulin level of non-diabetic control and typeII but not of type-I diabetic rats was increased after 30, 60 and 120 min of glibenclamide administration .The elevation in serum insulin level of non-diabetic control and type-II diabetic rats induced by administration of glibenclamide was enhanced by pretreatment for 30 min either with 100 mg/kg butanol extract or christinin-A orally. Pretreatment of type-I diabetic rats with the butanol extract or christinin-A produced no change in the serum insulin level. It is evident from the same table that the intraperitoneal injection of 30 mg/kg diazoxide into non-diabetic control and type-II diabetic rats produced a progressive increase and

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Table 2 Time course of changes in the serum glucose and insulin levels of non-diabetic control, type-I and type-II diabetic rats receiving 100 mg/kg either butanol extract (BE) of zizyphus spina-christi leaves or christinin-A(CA) orally Time (min)

Serum glucose (mg/dl)

Serum insulin (␮IU/ml)

BE

CA

0 30 60 120

Non-diabetic control rats 100.20 ± 7.52 95.11 ± 8.20 80.08** ± 6.60 88.31* ± 6.12

BE

CA

102.41 97.20 83.10** 90.20*

± ± ± ±

8.22 8.31 7.22 6.82

16.00 16.21 22.06** 17.80*

± ± ± ±

0.80 0.72 0.90 0.82

16.10 16.32 21.50** 17.80*

± ± ± ±

0.72 0.90 0.82 0.73

0 30 60 120

Type-I diabetic rats 208.42 ± 9.81 205.44 ± 8.92 204.32 ± 7.42 205.40 ± 8.62

208.32 204.51 203.22 204.42

± ± ± ±

9.22 8.22 9.14 8.82

2.20 2.30 2.31 2.30

± ± ± ±

0.17 0.18 0.15 0.16

2.22 2.32 2.30 2.31

± ± ± ±

0.18 0.15 0.14 0.17

0 30 60 120

Type-II diabetic rats 150.30 ± 8.12 142.21 ± 7.22 114.40** ± 8.23 121.40** ± 5.22

151.20 144.62 118.20** 124.30**

± ± ± ±

9.23 8.60 7.92 6.11

11.20 11.14 18.40** 14.20**

± ± ± ±

0.82 0.72 0.92 0.62

10.96 11.21 17.50** 13.10**

± ± ± ±

0.70 0.92 0.72 0.61

Values are means ± S.E. of six experiments. * P < 0.05, ** P < 0.01 vs. zero-time values.

decrease in the serum glucose and insulin levels, respectively. These effects started after 30 min. The same dose of diazoxide produced no change in the serum glucose and insulin levels of type-I diabetic rats over 120 min. Pretreatment of non-diabetic control rats either with 100 mg/kg butanol extract or christinin-A orally 30 min before diazoxide injection reduced the serum glucose level. The same pretreatment produced no change in the serum glucose level in type-I diabetic rats, and reduced the serum glucose level elevated by diazoxide in type-II diabetic rats. Diazoxide-induced reduction in the serum insulin level was antagonized by 30 min pretreatment of non-diabetic control and type-II diabetic rats either with 100 mg/kg butanol extract or christinin-A orally. Similar pretreatment produced no change in the serum insulin level of type-I diabetic rats. 3.4. Effect on isolated aortic strips Fig. 2 shows the cumulative concentration–response curve of the effects of diazoxide on the contractions of the isolated aortic strips of rabbit elicited by 5 ␮mol/l norepinephrine. It is evident from the figure that the contractions of the aortic strips were inhibited in a concentration-dependent manner by diazoxide. Addition of either 100 ␮mol/l christinin-A or 10 ␮mol/l glibenclamide to the fluid bathing isolated aortic strips contracted with 5 ␮mol/l norepinephrine, resulted after 15 min in inhibition of the relaxant effects of different concentrations of diazoxide on these strips. Results presented in the same figure also show that the concurrent addition of 100 ␮mol/l christinin-A with 10 ␮mol/l glibenclamide to the bath fluid completely inhibited the relaxant effects of different concentrations of diazoxide on the norepinephrine-contracted aortic strips. It is of inter-

est that, in preliminary experiments, the concentration levels of christinin-A and glibenclamide used in this study did not produce by themselves any significant change on norepinephrine-contracted aortic strips. 3.5. The spontaneous locomotor activity and pentobarbital sleeping time The butanol extract of Zizyphus spina-christi leaves at a dose level of 800 mg/kg produced a significant reduction in the locomotor activity of rats (245 ± 21 counts/30 min) compared with untreated saline group (295 ± 20 counts/30 min). The extract at dose levels of 100 and 400 mg/kg was inactive. Pretreatment of rats with 800 mg/kg butanol extract orally for 1 h prior to intraperitoneal injection of pentobarbital sodium increased the pentobarbital sleeping time (32.3 ± 2.1 min) compared with control value

Fig. 2. The cumulative concentration–response curves of diazoxide-induced relaxation of isolated aortic strips contracted with 5 ␮mol/l norepinephrine in the absence and in the presence of 100 ␮mol/l christinin-A; 10 ␮mol/l glibenclamide and their combination. Values are means ± S.E. of six experiments. *P < 0.05, **P < 0.01 vs. diazoxide values.

0.72 1.23 0.82 0.62

0.54 0.72 0.62 0.63

± ± ± ±

± ± ± ±

16.20 18.50** 16.50** 11.23

11.20 12.50** 10.50** 8.04**

CA+DI

135

Table 4 Effect of the oral administration of 100 mg/kg/day butanol extract (BE) of zizyphus spina-christi leaves to rats for 3 months on serum glucose, electrolytes and haematological parameters

0.62 0.81 0.62 0.54 ± ± ± ± 11.10 13.20** 11.10** 8.10** 0.71 0.63 0.41 0.32 ± ± ± ± 0.75 1.22 2.14 1.52 11.10 23.50** 32.40** 23.20** 0.62 1.64 2.22 1.72 ± ± ± ± 0.60 0.81 1.24 1.72 ± ± ± ± 11.08 14.22 22.42 20.43 12.23 10.24 13.42 11.80 ± ± ± ± 11.12 10.23 12.22 11.62

Saline Glucose (mg/dl) Electrolytes (mEq/l) Na+ K+ Ca2+ Haematological parameters Red blood cells count × 106 /␮l Haemoglobin value (g/dl) Haematocrit value (%) Mean corpuscular volume (␮3 ) Total white blood cells count × 103 /␮l

94.20 ± 6.10

82.40** ± 4.32

142.62 ± 4.80 4.90 ± 0.38 6.48 ± 0.44

139.94 ± 3.83 4.60 ± 0.32 6.46 ± 0.42

5.10 14.40 50.50 85.42 8.14

± ± ± ± ±

0.38 0.80 3.10 5.31 0.82

5.30 14.60 51.22 86.40 8.20

± ± ± ± ±

0.40 0.90 3.21 6.28 0.72

(21.6 ± 1.6 min). The extract at dose levels of 100 and 400 mg/kg did not increase the sleeping time of pentobarbital. 3.6. The median lethal dose (LD50 ) The LD50 of the butanol extract of Zizyphus spina-christi leaves in mice was 3820 (3440–4200) mg/kg while that of glibenclamide was 3160 (2850–3470) mg/kg. 3.7. Effect on rat liver and kidney function and structure Daily administration of 100 mg/kg butanol extract of Zizyphus spina-christi leaves to rats orally for 3 months produced no change in the SGOT, SGPT and SAP activities and serum total bilirubin level. Also, no changes in serum creatinine and urea nitrogen levels were detected. The histopathological examination of liver and kidney specimens obtained from these rats showed no pathological changes. Values are mean ± S.E. of six experiments. a After GL or DI administration. * P < 0.05, ** P < 0.01 vs. corresponding GL or DI values.

10.14 11.22 11.60 20.14 ± ± ± ± 152.20 185.40 210.21 245.22 9.31 7.21 5.32 6.23 ± ± ± ± 144.12 88.14** 84.12** 82.13** 10.20 6.21 6.24 5.32 ± ± ± ± Type-II diabetic rats 150.20 ± 8.31 142.10 130.30 ± 6.21 83.12** 115.22 ± 8.10 80.14** 102.22 ± 9.31 78.12** 0 30 60 120

BE

Values are mean ± S.E. of six experiments. ** P < 0.01 vs. saline values.

142.40 115.20** 155.24** 220.40*

± ± ± ±

144.10 118.12** 157.14** 220.62*

11.10 24.40** 33.60** 23.40**

± ± ± ±

11.22 7.52 7.12 6.53

0.82 1.12 0.92 0.73 ± ± ± ± 16.10 19.40** 17.40** 11.23 0.91 0.62 0.43 0.32 ± ± ± ± 15.82 13.23 12.24 11.12 0.62 1.64 2.21 1.42 ± ± ± ± 16.10 27.40** 37.60** 23.24 0.72 1.83 2.12 1.62 ± ± ± ± 16.20 28.40** 38.60** 25.42 0.60 0.82 1.22 1.32 ± ± ± ± 16.22 19.31 29.52 25.30 721 6.14 8.11 12.21 ± ± ± ± 95.12 88.22** 110.23** 143.14 6.21 5.20 9.22 12.22 ± ± ± ± 94.04 85.10** 108.20** 141.10 8.40 9.12 10.21 11.32 ± ± ± ± 100.04 115.40 130.30 150.22 8.10 6.11 5.22 5.11 0 30 60 120

± ± ± ± 7.12 5.31 4.63 4.81

GL BE+GL Non-diabetic control rats 100.40 ± 7.82 94.21 ± 86.24 ± 6.23 66.40** ± 80.42 ± 5.20 65.62** ± 70.08 ± 4.60 64.22* ±

99.02 70.42** 68.50** 66.32

BE+GL GL

Serum glucose (mg/dl)

CA+GL

DI

BE+DI

CA +DI

Serum insulin (␮IU /ml)

CA+GL

DI

BE+DI

Treatment

Time (min)a

Table 3 Effect of 30 min pretreatment either with 100 mg/kg butanol extract (BE) of zizyphus spina-christi leaves or christinin-A (CA) orally on glibenclamide (GL)or diazoxide (DI)-induced alterations in the serum glucose and insulin levels of non-diabetic control and type-II diabetic rats

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3.8. Effect on serum electrolytes and haematological parameters Oral treatment of rats with 100 mg/kg/day butanol extract for 90 consecutive days produced no disturbance in serum electrolyte levels and no changes in haematological parameters (Table 4).

4. Discussion In our phytochemical studies of the Zizyphus spina-christi leaves, four saponin glycosides have been isolated from the butanol extract. In accordance with Mahran et al. (1996), christinin-A was the major saponin glycoside. Therefore, the butanol extract and chistinin-A were used in this study to

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evaluate the potential antidiabetic activity and toxicity of Zizyphus spina-christi leaves. Previous studies demonstrated that neonatal streptozotocin injection induced in adult rat defects similar to those observed in mild type-II human diabetes: moderate increase in fasting blood glucose level, intolerance to oral glucose, hypoinsulinemia and reduced glucose-induced insulin secretion (Angel et al., 1996). This model of type-II diabetes was compared to that of rats treated with streptozotocin when adult, which results in destruction of pancreatic beta-cells and is accepted as a model of type-I insulinopenic diabetes (Burcelin et al., 1993). Rats in this study responded to streptozotocin in a similar manner. Treatment of adult rats with streptozotocin produced a profound increase and decrease in the fasting blood glucose and insulin levels, respectively (i.e., a rat model of type-I diabetes). On the other hand, treatment of 1-day-old rats with streptozotocin produced a relatively moderate increase and decrease in the fasting blood glucose and insulin levels, respectively (i.e., a rat model of type-II diabetes). Pretreatment of non-diabetic control rats with either the butanol extract of Zizyphus spina-christi leaves or its major saponin glycoside, christinin-A potentiated glucoseinduced insulin release. Also, pretreatment of type-II but not of type-I diabetic rats with either the butanol extract or christinin-A produced an improvement of oral glucose tolerance and potentiated glucose-induced insulin release. Thus, the antihyperglycemic effect of Zizyphus spinachristi leaves obtained in this study in non-diabetic control and type-II diabetic rats could be explained in terms of potentiating glucose-induced insulin secretion. This explanation is easily confirmed since the butanol extract and christinin-A neither improved oral glucose tolerance nor increased insulin secretion in response to the glucose load in type-I diabetic rats. This possible insulinotropic effect of these agents was further supported in the present study after estimating their effects on serum glucose and insulin levels of fasted nondiabetic control, type-I and type-II diabetic rats. Our results demonstrated that treatment of non-diabetic control and typeII diabetic rats with either the butanol extract of Zizyphus spina-christi leaves or christinin-A reduced the serum glucose level and increased the serum insulin level. On the other hand, treatment of type-I diabetic rats with either the butanol extract or christinin-A produced no change in both serum glucose and insulin levels. These findings indicate that Zizyphus spina-christi leaves decrease the serum glucose level by stimulation of insulin secretion and have no extrapancreatic effects. Similar observations demonstrated that the butanol extract of Zizyphus spina-christi leaves or its main saponin glycoside, christinin-A, improved glucose utilization in diabetic rats after 1 and 4 weeks of treatment. Serum insulin level showed a significant increase in diabetic rats treated for a period of 4 weeks with the butanol extract (Glombitza et al., 1994). Also, they found that the antidiabetic effect of the butanol extract was more pronounced than that of christinin-A. How-

ever, our results indicate that the effects of the butanol extract and christinin-A were nearly similar. Therefore, the antidiabetic activity of the butanol extract of Zizyphus spina-christi leaves might be due mainly to its major saponin glycoside, christinin-A. The pancreatic beta-cells KATP channels play a central role in glucose-induced insulin secretion by coupling betacells metabolism to elevation of the cytosolic free Ca2+ concentration. In response to an increase in plasma glucose, increased uptake and metabolism of glucose within the beta-cells increases ATP, bringing about the closure of KATP channels in the plasma membrane. This produces membrane depolarization leading to activation of voltage-dependent calcium channels. The ensuing Ca2+ influx elevates intracellular Ca2+ and initiates exocytosis of insulin granules (Ashcroft et al., 1992; Zhang et al., 1995). It has become evident from many studies that the gating of KATP channels in pancreatic beta-cell can be regulated by a variety of pharmacological agents. The most potent of these agents are the sulfonylureas (Nelson et al., 1992). The ability of sulfonylureas, e.g., glibenclamide to lower the serum glucose concentration in diabetics appears mainly to be due to their ability to stimulate insulin secretion. The ability of sulfonylureas to block the beta-cell KATP channels directly explains their stimulatory effect on insulin secretion (Satin, 1996; Doyle and Egan, 2003). On the other hand, diazoxide was found to inhibit insulin release by selectivity opening of KATP channels of pancreatic beta-cells, leading to hyperpolarization of the plasma membrane (Dunne, 1989; Sato et al., 1992; Dabrowski et al., 2003). In view of these considerations, the role of KATP channels in the glucose lowering activity of Zizyphus spina-christi leaves was evaluated in the present work by studying the interaction between their butanol extract or christinin-A and glibenclamide or diazoxide. The data obtained in this study, indicate that administration of glibenclamide to non-diabetic control and type-II diabetic rats produced a marked reduction in the serum glucose level and a marked increase in the serum insulin level. Pretreatment of animals with the butanol extract or christininA enhanced the glucose lowering and insulinotropic effects of glibenclamide. On the other hand, diazoxide produced an increase in the serum glucose level and a decrease in the serum insulin level in non-diabetic control and type-II but not in type-I diabetic rats. Pretreatment of animals with the butanol extract or christinin-A, inhibited the effect of diazoxide on the serum glucose level and antagonized its effect on the serum insulin level. In view of the previous considerations, these findings are in favor of the possibility that Zizyphus spinachristi leaves can block KATP channels in pancreatic beta-cell membranes. The first evidence that KATP channels exist in smooth muscle came from electrophysiological measurements of these channels in smooth muscle and the observation that vasodilating actions of diazoxide were inhibited by glibenclamide (Standen et al., 1989) Then the evidence has accumulated

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that KATP channels in vascular smooth muscle are the target of a variety of vasodilating stimuli (Nelson and Quayle, 1995; Yokoshiki et al., 1998; Fujita and Kurachi, 2000). The demonstration of KATP channels in arterial smooth muscle (Standen et al., 1989) argues for the presence of sulfonylurea binding sites in arterial smooth muscle (Kovacs and Nelson, 1991). In endothelium-denuded rings from rat aorta, a high affinity component of [3 H] glibenclamide binding mediates the block of KATP channels by sulfonylureas in rat aorta; hence, it represents the sulfonylurea receptor in this vessel. The pharmacological properties of this binding site resemble those of the binding site for the openers of KATP channels (Loeffler and Quast, 1997). It has been found that glibenclamide antagonized, in a concentration-dependent manner, the vasorelaxant effects of the KATP channel openers, cromakalim, minoxidil and diazoxide on isolated rabbit aorta contracted with angiotensinII, norepinephrine or methoxamine (Winquist et al., 1989.). Furthermore, diazoxide, a potent activator of KATP channels in pancreatic beta-cells, fails to discriminate between vascular smooth muscle and beta-cell KATP channels (Antoine et al., 1992; De Tullio et al., 1996). In the present study, in vitro experiments using isolated rabbit aortic strips, illustrate further the role of KATP channels in the hypoglycemic effect of Zizyphus spina-christi leaves. Our results demonstrate that the relaxant effects of different concentrations of diazoxide on the isolated norepinephrinecontracted aortic strips were inhibited by christinin-A or glibenclamide. The combination between glibenclamide and christinin-A led to complete inhibition of the relaxant effects of different concentrations of diazoxide. In this study, experiments were carried out to assess the effect of the butanol extract of Zizyphus spina-christi leaves on the central nervous system. Our results indicate that the butanol extract at a dose level much higher than that required producing sufficient insulinotropic and hypoglycemic effects, decreased the locomotor activity and prolonged pentobarbital sleeping time in rats. The inhibition of motor activity and potentiation of hypnotic action of pentobarbital are in favor of the possibility that the butanol extract of Zizyphus spina-christi leaves, at high dose levels, produced a depressant effect on the central nervous system (Adzu et al., 2002). Liver is the primary site of biotransformation and detoxification of xenobiotics. Thus, the liver is especially vulnerable to toxic injury (Delaney, 1998). Also, the kidneys as the principal organs for the excretion of xenobiotics and their metabolites are particularly prone to their toxic effects (Mathew, 1992). In addition, many substances produce adverse haematologic effects when given at therapeutic doses or at intentionally toxic doses. The response to toxic influences manifests itself primarily in a reduction in the number of circulating blood cells, and in functional and structural abnormalities of the blood cells (Max, 1996). In the present study, our findings indicate that chronic administration of the butanol extract of Zizyphus spina-christi

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leaves orally to rats did not show any signs of hepatotoxicity and nephrotoxicity as assessed by biochemical measurements and by histopathological examination. Also, this treatment produced no haematological changes. It is of interest that, in preliminary experiments, treatment of rats orally with a single high dose of the butanol extract also did not produce hepatotoxicity, nephrotoxicity or haematological changes. In addition, results of acute toxicity tests, in this work, show that the butanol extract of Zizyphus spina-christi leaves is safe, having a relatively high LD50 value in mice. Taken together, our results suggest that Zizyphus spinachristi leaves decrease the serum glucose level in control and type-II diabetic rats. This effect is mediated by releasing insulin. The insulinotropic effect of Zizyphus spina-christi leaves, may be due to blockade of KATP channels in pancreatic beta-cell membranes. Zizyphus spina-christi leaves may potentially be safe for use as an antidiabetic agent.

References Abu-Zarga, M., Sabri, S., AL-Aboudi, A., 1995. New cyclopeptide alkaloids from Zizyphus lotus. Journal of Natural Products 58, 504–511. Adzu, B., Amos, S., Amizan, M.B., Gamaniel, K., 2003. Evaluation of the antidiarrheal effects of Zizyphus spina-christi stem bark in rats. Acta Tropica 7, 245–250. Adzu, B., Amos, S., dzarma, S., Wambebe, C., Gamaniel, K., 2002. Effect of Zizyphus spina-christi Willd aqueous extract on the central nervous system in mice. Journal of Ethnopharmacology 79, 13–16. Adzu, B., Amos, S., wambebe, C., Gamaniel, K., 2001. Antinociceptive activity of Zizyphus spina-christi root bark extract. Fitoterapia 72, 344–350. Angel, I., Burcelin, R., Girard, J., Salomon, Z., 1996. Normalization of insulin secretion by selective alpha-2 adrenoceptor antagonist receptors GLUT-4 glucose transporter expression in adipose tissue of typeII diabetic rats. Endocrinology 137, 2022–2027. Antoine, M., Berkenboom, G., Herchuelz, A., Lebrun, P., 1992. Mechanical and ionic response of rat aorta to diazoxide. European Journal of Pharmacology 216, 299–306. Ashcroft, F.M., Williams, B., Smith, P.A., Fewtrell, C.M.S., 1992. Ion channels involved in the regulation of nutrient-stimulated insulin secretion. In: Flatt, P.R. (Ed.), Nutrient Regulation of Insulin Secretion. Portland Press, London, pp. 193–212. Barboni, L., Gariboldi, P., Torregiani, E., Verotta, L., 1994. Cyclopeptie alkaloid from Zizyphus mucronata. Phytochemistry 35, 1579–1583. Burcelin, R., Printz, R.L., Kande, J., Assan, R., Granner, D.K., Girard, J., 1993. Regulation of glucose transporter and hexokinase-II expression in tissues of diabetic rats. American Journal of Physiology 265, 392–E401. Cheng, G., Bai, Y., Zhao, Y., Tao, J., Liu, Y., Tu, G., Ma, L., Liao, N., Xu, X., 2000. Flavonoids from Zizyphus jujuba Mill var. spinosa. Tetrahedron 56, 8915–8920. Crofford, O.B., Davis, C.K., 1965. Evaluation of a new technique of multiple serial sampling. Metabolism 14, 271–275. Dabrowski, M., Larsen, T., Ashcroft, F.M., Bondo Hansen, J., Wahl, P., 2003. Potent and selective activation of the pancreatic beta-cell type of KATP channel by two novel diazoxide analogues. Diabetologia 5, 71–75. De Tullio, P., Pirotte, B., Antoine, M., Delarge, J., 1996. 3 and 4 substituted 4 H-pyrid thiadiazine dioxides as potassium channel openers synthesis, pharmacyological evaluation and structure activity relationships. Journal of Medical Chemistry 47, 4525–4529.

138

A.O. Abdel-Zaher et al. / Journal of Ethnopharmacology 101 (2005) 129–138

Delaney, K., 1998. Hepatic principles. In: Goldfrank, et al. (Eds.), Goldfranks Toxicologic Emergencies. National Academy Press, Washington, DC, pp. 213–228. Doyle, M.E., Egan, J.M., 2003. Pharmacological agents that directly modulate insulin secretion. Pharmacological Review 55, 105–131. Dunne, M.J., 1989. Protein phosphorylation is required for diazoxide to open ATP-sensitive K+ channels in insulin (RINm5F) secreting cells. FEBS Letters 250, 262–266. Fujita, A., Kurachi, Y., 2000. Molecular aspects of ATP-sensitive K+ channel in the cardiovascular system and K+ channels openers. Pharmacology and Therapeutics 85, 39–53. Glombitza, K.W., Mahran, G.H., Mirhom, Y.W., Ichel, K.G., Motawi, T.K., 1994. Hypoglycemic and antihyperglycemic effects of Zizyphus spina-christi in rats. Planta Medica 60, 244–247. Han, B.H., Park, M.H., 1986. Folk Medicine: The Art and Science. The American Chemical Society, Washington, DC, p. 205. Han, B.H., Park, M.H., Han, Y.N., 1990. Cyclic peptide and peptide alkaloids from seeds of Zizyphus vulgaris. Phytochemistry 29, 3315–3319. Higuchi, R., Kubota, S., Komori, T., Kawasaki, T., Pardey, V.B., Singh, J.P., Shah, A.H., 1984. Triterpenoid saponins from the bark of Zizyphus joazeiro. Phytochemistry 23, 2597–2600. Ikram, M., Ogihara, Y., Yamasaki, K., 1981. Structure of a new saponin from Zizyphus Vulgaris. Journal of Natural products 44, 91–93. Kirtikar, K.R., Basu, B.D., 1984. Indian Medicinal Plants, Lalit Mohan Basu, Allahabad, p. 593. Kovacs, R.J., Nelson, M.T., 1991. ATP-sensitive K+ channels from aortic smooth muscle incorporated into planar lipid bilayers. American Journal of Physiology 261, H1604–H1609. Loeffler, C., Quast, U., 1997. Pharmacological characterization of the sulphonylurea receptor in rat isolated aorta. British Journal of Pharmacology 120, 476–480. Mahran, G.H., Glombitza, K.W., Mirhom, Y.W., Hartmann, R., Michel, C.G., 1996. Novel saponins from Zizyphus spina-christi growing in Egypt. Planta Medica 62, 163–165. Mathew, T.H., 1992. Drug-induced renal disease. The Medical Journal of Australia 156, 724–756. Max, J.J.M., 1996. Toxicology of the blood: pathophysiology, toxicological pathology and mechanistic aspects. In: Niesink, R.J.M., deVries, J., Hollinger, M.A. (Eds.), Toxicology. Principles and Applications. CRC Press Inc, Florida, pp. 817–839.

Nawwar, M.M., Ishak, M.S., Michael, H.N., Buddrus, J., 1984. Leaf flavonoid of Zizyphus spina-christi. Phytochemistry 23, 2110– 2111. Nelson, D.A., Aguilar-Bryan, L., Raef, H., Boyd, A., 1992. Molecular mechanisms of sulphonylurea in the pancreatic beta-cells. In: Flatt, P.R. (Ed.), Nutrient Regulation of Insulin Secretion. Portland Press, London, pp. 319–340. Nelson, M.T., Quayle, J.M., 1995. Physiological roles and properties of potassium channels in arterial smooth muscle. American Journal of Physiology 268, C799–C822. Reavill, C., Walters, J.A., Stolerman, I.P., Garacha, H.S., 1990. Behavioural effects of the nicotinic antagonists N-C3-pyridylmethyl pyridoline and isourecolone in rats. Psychopharmacology 102, 521–528. Satin, L.S., 1996. New mechanisms for sulphonylurea control of insulin secretion. Endocrine 4, 191–198. Sato, Y., Aizawa, T., Komatsu, M., Okada, N., Yamada, T., 1992. Dual functional role of membrane depolarization Ca2+ influx in rat pancreatic beta-cell. Diabetes 41, 438–443. Shahat, A.A., Pieters, L., Apers, S., Nazeif, N.M., Abdel-Azim, N.S., Bergh, D.V., Vlienk, A.J., 2001. Chemical and biological investigations on Zizyphus spina-christi L. Phytotherapy Research 15, 593– 597. Standen, N.B., Quayle, J.M., Davies, N.W., Nelson, M.T., 1989. Hyperpolarizing vasodilators activate ATP-sensitive K+ channels in arterial smooth muscle. Science 245, 177–180. Tripathi, M., Pandey, M.B., Jha, R.N., Pandey, V.B., Tripathi, P.N., Singh, J.P., 2001. Cyclopeptide alkaloids from Zizyphus jujuba. Fitoterapia 72, 507–510. Winquist, R.J., Heaney, L.A., Wallace, A.A., Kaczarowski, G.J., 1989. Glyburide Blocks the relaxation response to BRL 3415 (cromakalim), minoxidil, sulfate and diazoxide in vascular smooth muscle. Journal of Pharmacology and Experimental Therapeutics 248, 149– 156. Yokoshiki, H., Sunagawa, W., Seki, T., Sperelakis, N., 1998. ATPsensitive K+ channels in pancreatic, cardiac and vascular smooth muscle cells. American Journal of Physiology 274, C25–C37. Zhang, Y., Warren-Perry, M., Sakura, H., Turner, R., 1995. No evidence for Mutations in a putative beta-cell. ATP-sensitive K+ channel subunit in MODY, NIDDM or GDM. Diabetes 44, 597–600.