The animals were handled in accordance with the criteria outlined in the “Guide for the Care and Use of Laboratory Animals” (http://www.nap.edu/readingroom/ books/labrats/). Male Wistar rats, weighing 180-250 g were selected and maintained at a constant temperature of 22˚C ± 2˚C with a fixed 12:12-h light-dark cycle. Nutritionally balanced pellets and water were freely available. The animals were divided into five groups (10 rats in each group): non-diabetic control (NDC); acute diabetic (AD); chronic diabetic (CD); Psidium guavatreated chronic diabetic (PSG-CD), and; Psidium guavatreated control (PSG-C).

Diabetes was induced with and IP injection of 60 mg/kg STZ. At 10 days after diabetes induction, fasting blood glucose level was determined and the presence of diabetes was confirmed by blood glucose levels of above 250 mg/dl. Animals in which the diabetes lasted for 10 days were classed as “acute diabetic” and those in which the diabetes lasted for 8 weeks were defined as “chronic diabetic”. The PSG-CD and PSG-C groups received 1g/l of aqueous and ethanol extract of Psidium guava leaf added to the drinking water, 10 days after diabetes induction, for 8 weeks. Animals were monitored for blood glucose concentrations and body weight every week over the course of the experiment. Blood glucose was measured with an Ascensia ELITE XL glucometer and Ascensia Elite blood glucose test strips.

Fresh leaves of Psidium guava were collected from open grassland in Menab (Southern Iran). The plant was identified by the Taxonomist at the ShahidBeheshti University of Medical Sciences. A kilogramme of fresh Psidium guava leaves was air dried under shade at room temperature (26˚C ± 1˚C) for 2 weeks. The dried leaves were then milled into a fine powder in a Waring commercial blender. The powdered leaves were extracted with 3 volumes of 95% ethanol for 3 days. The extract was filtered through filter paper (Whatman No. 1) and concentrated with a rotary vacuum evaporator (Eyela N-3000 Nseries, Tokyo Rikakikai, Tokyo, Japan). The concentrate was then freeze-dried (Model 77500-00 M, Labconco Co., MO) and stored at −80˚C before use as per Shen et al. [1]. One gram of Psidium guava leaf extract was contain 75.7 mg dried substance.

For IPGTT, animals in the NDC, CD and PSG-CD groups were fasted overnight for 15 h, and were given 1.5 g/kg body weight glucose via IP injection. Blood was drawn from the tail vein at 0, 10, 20, 30, 60, 90 and 120 min after glucose administration.

Animals were decapitated after anesthesia induction with ketamine HCl. Blood samples were taken from the neck vascular trunk in order to determine the triglycerides, total cholesterol, HDL-cholesterol, magnesium and calcium levels by spectrophotometer (UV 3100, Shimadzu) and appropriate kits (Zistshimi, Tehran, Iran). LDL and VLDL cholesterol were calculated by the following formulae as per Saunders et al. (1999) [17]:

VLDL = Triglycerides/5 LDL = Total cholesterol – HDL cholesterol – VLDL

The animals in the NDC, CD and PSG-CD groups were anesthetized by IP injection of ketamine HCl 50 mg/kg and the mesenteric vascular beds were prepared as originally described by McGregor (1965) [18]: the abdominal wall was opened, the superior mesenteric artery was exposed and cannulated, then gently flushed with modified Krebs-Henseleit solution (containing [in mM]: NaCl: 118, KCl: 4.7, CaCl₂; 2.5, MgSO₄; 1.2, glucose; 2, NaHCO₃; 2.5, NaHPO₄; 1.2) concomitantly bubbled with a mixture of 95% O₂ and 5% CO₂ (final pH 7.4), and warmed to 37˚C. The mesentery was isolated from the intestine and placed in a water-jacketed perfusion chamber maintained at 37˚C. The preparation was perfused at 1 ml/min with modified Krebs-Henseleit solution by a peristaltic pump (Meredos GmbH). The tissue was prevented from drying by superfusion with 0.1 ml/min modified Krebs-Henseleit solution. Perfusion pressure was monitored via a T-tube inserted between the pump and the inflow cannula. This was connected to a pressure transducer (MLT0380 AD Instruments). The recording was performed by Power Lab System (16SP, AD Instruments). Tissues were used for experiment after equilibration of 30-minutes. After the 30-minute equilibration, the vascular bed was constricted by Krebs-Henseleit solution containing phenylephrine, an α₁-adrenoceptor agonist, from 0.0001 M to 0.1 M and perfusion pressure was recorded. Drug concentrations were increased every 15 minutes.

The following drugs were used: STZ from Sigma (USA) dissolved in 1 ml normal saline immediately before use. Phenylephrine from Sigma (St. Louis, MO, USA) and ketamine HCl from Rotexmedica (Trittau, Germany).

Data were expressed as mean ± S.E.M. Differences among groups were evaluated by one-way and two-way ANOVA with the Tukey post-hoc test. P < 0.05 was selected for acceptance of statistical significance.

Changes in feeding plasma glucose were measured in all groups (Figure 1). Diabetes induction caused plasma glucose concentration to increase (440 ± 71.99) and blood glucose continued to be elevated (442.5 ± 58.93) 8 weeks after diabetes induction. Administration of Psidium guava for 8 weeks (from day 10) caused the plasma glucose concentrations in the PSG-CD group to return to normal levels (118.88 ± 9.44) but there were no changes in blood glucose in the PSG-C group.

Before initiation of Psidium guava and STZ administration, the IPGTT patterns for 3 groups (NDC, CD and

PSG-CD) were similar and there was no significant difference between the groups when the glycemic response was expressed as the area under the curve (AUC) (data was not shown). However, after 8 weeks, the CD group displayed severe glucose intolerance, which was significantly improved in the PSG-CD group to a degree that was judged effective (Figure 2(b), AUC: NDC vs. CD vs. PSG-CD = 26606 ± 1430.9 vs. 55273.5 ± 6764.68 vs. 20627 ± 1894.27 P < 0.0001).

Changes in plasma calcium (Ca), magnesium (Mg) and the Ca/Mg ratio were measured in all groups (Figures 3(a) to 3(c)) and 8 weeks after the administration of Psidium guava leaves the plasma calcium level in the PSG-CD group was unchanged in comparison with the

NDC and CD groups (NDC vs. AC vs. CD vs. PSG-CD = 9.22 ± 0.12 vs. 8.98 ± 0.19 vs. 9.76 ± 0.16 vs. 9.7 ± 0.41 vs.). However a significant increase (p < 0.001) was seen in the PSG-C group (10.1 ± 0.16) in comparison to the NDC group (Figure 3(a)).

The plasma magnesium level (0.35 ± 0.035) significantly (P < 0.001) decreased compared to the NDC group (1.73 ± 0.01) 10 days after diabetes induction and the plasma magnesium level was still decreased (0.46 ± 0.08) 8 weeks after diabetes induction. Administration of Psidium guava for 8 weeks (from day 10) caused plasma magnesium concentrations in the PSG-CD group to increase although these did not reach normal levels (0.75 ± 0.11). It is interesting that Psidium guava administration in the PSG-C group (0.54 ± 0.03) caused the plasma magnesium level to decrease significantly (P < 0.001) in comparison to the NDC group.

The Ca/Mg ratio in the CD and PSG-C groups increased significantly (P < 0.001) 8 weeks after induction of diabetes compared to the NDC group (NDC vs. AC vs. CD vs. PSG-C vs. PSG-CD = 3.31 ± 0.07 vs. 23.32 ± 1.6 vs. 19.3 ± 2.65 vs. 18.5 ± 1.21 vs. 12.97 ± 1.41).

Changes in plasma triglycerides, total cholesterol, HDL, LDL and VLDL concentrations were measured in all groups (Figures 4(a) to 4(e)). LDL, VLDL, total cholesterol and triglyceride concentrations were elevated significantly (P < 0.01) in the diabetic control group 8 weeks after induction of diabetes, while HDL concentration decreased significantly (P < 0.01) compared to the NDC group. Administration of Psidium guava for 8 weeks (from day 10) caused plasma VLDL and triglyceride concentrations to return to normal levels but LDL and total cholesterol levels were lower than in the NDC group. The plasma HDL level in PSG-CD animals was significantly higher than in the NDCs, but plasma HDL in the PSG-C group was lower than in the NDC group.

Baseline perfusion pressure for the CD group was significantly (p < 0.001) higher than for the NDC and PSGCD groups, but there was no significant difference between NDC animals and the PSG-CD group (Figure 5).

After equilibration of 20 minutes, a cumulative concentration-response curve was plotted for phenylephrine, an α1 adrenoceptor agonist, (from 0.0001 to 0.1 M) in intact mesenteric beds. Phenylephrine was added into the medium and perfusion pressure (15 min for each concentration) was recorded (Figure 6). The phenylephrine induced concentration-dependent vasoconstriction in mesenteric beds manifested as an increase in perfusion pressure (Figure 6). Maximum vasoconstriction was achieved at Phenylephrine concentration of 0.01 M.

Contractile responses to Phenylephrine at concentrations higher than 0.006 M were found to be significantly increased in the mesenteric beds of CD rats compared to the NDC and PSG-CD groups. However, there was no significant difference between the NDC and PSG-CD groups at high doses of Phenylephrine.