Identification of hypoglycaemic compounds from berries of Juniperus oxycedrus subsp. oxycedrus through bioactivity guided isolation technique

Journal of Ethnopharmacology 139 (2012) 110–118

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Identification of hypoglycaemic compounds from berries of Juniperus oxycedrus subsp. oxycedrus through bioactivity guided isolation technique Nilüfer Orhan a,∗ , Mustafa Aslan a , Mert Pekcan b , Didem Deliorman Orhan a , Erdal Bedir c , Fatma Ergun a a b c

Gazi University, Faculty of Pharmacy, Department of Pharmacognosy, 06330, Etiler, Ankara, Turkey Ankara University, Faculty of Veterinary Medicine, Department of Biochemistry, 06110, Dıs¸kapı, Ankara, Turkey Ege University, Faculty of Engineering, Department of Bioengineering, 35100, Bornova, Izmir, Turkey

a r t i c l e

i n f o

Article history: Received 24 August 2011 Received in revised form 7 October 2011 Accepted 20 October 2011 Available online 29 October 2011 Keywords: Antioxidant Diabetes mellitus Ferulic acid Hypoglycaemic Juniperus oxycedrus ssp. oxycedrus L. (Cupressaceae) Oleuropeic acid Shikimic acid

a b s t r a c t Ethnopharmacological relevance: Decoction of Juniperus oxycedrus subsp. oxycedrus L. (Cupressaceae) berries is used internally as tea and pounded fruits are consumed to lower blood glucose levels in Turkey. Aim of the study: To evaluate hypoglycaemic and antidiabetic activity of J. oxycedrus subsp. oxycedrus berries and to identify active compounds through bioactivity guided isolation technique. Material and methods: Hypoglycaemic effect of J. oxycedrus subsp. oxycedrus (Joso) berry extracts on oral administration was studied using in vivo models in normal, glucose-hyperglycaemic rats. Streptozotocin induced diabetic rats were used to examine antidiabetic activity of Joso extracts, subextracts, fractions, subfractions and shikimic acid (SA). Results: Through in vivo bioactivity-guided fractionation processes, shikimic acid, 4-O-ˇ-dglucopyranosyl ferulic acid and oleuropeic acid-8-O-ˇ-d-glucopyranoside were isolated from the n-butanol subextract by silica gel and reverse phase column chromatography as the main active ingredient of the active subfraction. After 8 days administration of the major compound shikimic acid, blood glucose levels (24%), malondialdehyde levels in kidney tissues (63–64%) and liver enzymes (AST, ALT, ALP) of diabetic rats were decreased. Conclusion: Results indicated that Joso berry extract and its active constituents might be beneficial for diabetes and its complications. © 2011 Elsevier Ireland Ltd. All rights reserved.

1. Introduction For thousands of years, plants and their derivatives are being used for treatment of diabetes. More than 400 plants incorporated in approximately 700 recipes are used to treat diabetes mellitus in almost two thirds of the world population. Application of modern science to traditional system of medicine has also given birth to compound like metformin. A large number of animal studies to test the claimed activity have demonstrated the hypoglycaemic property of many of these plants. In addition, clinical trials have shown some plants as useful antidiabetic agents, but the pure chemical compounds isolated from the crude extracts of these plants do not bear structural resemblance to the antidiabetic drugs in current clinical use nor have they similar mechanisms of action. But still the search for a novel antidiabetic drug advocates the utilization of plants as a potential source and can be achieved by application of modern scientific technology and recent knowledge on the physiological changes in case of diabetes (Rout et al., 2009).

∗ Corresponding author. Tel.: +90 312 2023176; fax: +90 312 2235018. E-mail address: [email protected] (N. Orhan). 0378-8741/$ – see front matter © 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2011.10.027

Juniperus oxycedrus L. (Cupressaceae) (prickly juniper, plum juniper, cade juniper, red-berry juniper, cada) is a species of juniper, native across the Mediterranean region growing on a variety of rocky sites from sea level up to 1600 m altitude. Juniper berries are used as a spice, particularly in European cuisine, which are the only spice derived from conifers. The berries are used in northern European and particularly Scandinavian cuisine to impart a sharp, clear flavor to meat dishes (Loizzo et al., 2007). Junipers are used in folk medicine for different purposes all over the spreading countries. In Turkey; juniper tar, leaves and fruits are widely used to heal wounds, abdominal pain and stomachic disorders, gynecological diseases, hemorrhoids, common cold, cough, bronchitis, calcinosis in joints, against fungal infections, kidney inflammation and to pass kidney stone (Tuzlacı and Erol, 1993; Yes¸ilada et al., 1995; Honda et al., 1996). Additionally infusion of Juniperus oxycedrus subsp. oxycedrus (Joso) berries and leaves are used internally and pounded berries are consumed for treatment of diabetes in Isparta and Afyon (Tuzlacı and Erol, 1993; Honda et al., 1996). The present study was performed to evaluate hypoglycaemic activity of Joso berries in normoglycaemic, glucose-hyperglycaemic and streptozotocin-induced diabetic rats and to determine its

N. Orhan et al. / Journal of Ethnopharmacology 139 (2012) 110–118

active constituents through bioactivity guided fractionation and isolation techniques. 2. Materials and methods 2.1. Plant materials Juniperus oxycedrus L. ssp. oxycedrus L. (Joso) berries were collected in October 2007 from Karapiri province of Akda˘gmadeni, Yozgat (Turkey). The plant was identified by N. Orhan and a voucher specimen (GÜEF 2616) was stored in the Herbarium of Gazi University, Faculty of Pharmacy.

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water (100% → 25%) and methanol to obtain 77 fractions. Fr. 2–4 gave 250 mg “shikimic acid (3,4,5-trihydroxycyclohex-1-ene-1carboxylic acid)”, fr. 6–10 and 15–18 were applied to Sephadex LH-20 column (25–100 ␮m, Sigma Chem. Co., Lot. 12H0053) with methanol. Subfraction 22–28 of fr. 6–10 was purified; the major molecule was isolated and elucidated as “4-O-ˇ-d-glucopyranosyl ferulic acid (3-{3-methoxy-4-[(3,4,5,6-tetrahydroxytetrahydroacid)” (10 mg). 2H-pyran-2-yl)methoxy]phenyl}propanoic Subfraction 10–12 of fr. 15–18 was elucidated as a monoterpene glycoside “oleuropeic acid-8-O-ˇ-d-glucopyranoside” (4-(1-ˇ-dglucopyranosyloxy-1-methyl)ethyl-1-cyclohexene-1-carboxylic acid) (20 mg).

2.2. Extraction and fractionation of material 2.2.1. Water extract Chopped dried berries (1000 g) were extracted with hot distilled water (50 ◦ C, 20 l) for 3 h with continuous stirring on a water bath at 50 ◦ C. The extract was filtered and evaporated under reduced pressure and then lyophilized (water extract, yield 26.0%). 2.2.2. Preparation and fractionation of EtOH extract The chopped dried berries (1000 g) were extracted with ethanol 80% (20 l) by mixer for 8 h individually. The day after, extract was filtrated and the residue was extracted by the same procedure with ethanol again. The filtrates were pooled and evaporated to yield dry extracts under reduced pressure (EtOH-extract, yield 33.0%). Dried ethanol extract (250 g) was dissolved in 500 ml methanol/water (1:1) mixture and extracted with n-hexane in a separatory funnel (20× 500 ml). n-Hexane phases were collected and dried to give nhexane subextract (yield 14.8%). Aqueous phase was distillated to remove methanol and then fractionated successively with chloroform (6× 500 ml), ethyl acetate (12× 500 ml), n-butanol/saturated with water (12× 500 ml) to obtain subextracts. Each subextract was concentrated to dryness under vacuum on a rotary evaporator to give (CHCI3 -subextract, yield 0.9%), (EtOAc-subextract, yield 8.3%), (n-BuOH-subextract, yield 18.4%) and the remaining water subextract (R-Water subextract, yield 53.7%), respectively. All the obtained subextracts were used in animal experiments at the doses calculated according to their yields. The n-BuOH subextract was also used in phytochemical studies. 2.2.3. Chromatographic studies and isolation of active constituents 10 g of n-BuOH subextract was applied to silica gel column (260 g Kieselgel 60, 063–0.2 mm, Merck, Art. No. 7734) and eluted with mixtures of CHCl3 –MeOH–H2 O (90:10:1 → 10:90:5), and methanol successively. Eluents were combined into six subfractions A (fr.1–18: 1.83%), B (fr.19–23: 3.91%), C (fr.24–29: 9.61%), D (fr.30–32: 7.36%), E (fr.33–41: 52.05%), F (fr.42–60: 19.63%) according to TLC behavior using two solvent systems CHCl3 :MeOH:H2 O (61:32:7) and BuOH:AcOH:H2 O (4:1:5-upper layer) [spots were visualized under daylight, UV-light and after spraying 5% H2 SO4 ]. After bioactivity studies n-BuOH C fraction (2 g) was subjected to silica gel (100 g) column and eluted with mixtures of CH2 Cl2 :MeOH:H2 O (90:10:1 → 40:60:1) and methanol to afford 75 fractions. Eluents were combined to obtain seven subfractions [C1 (fr. 1–22: 1.10%), C2 (fr. 23–40: 0.63%), C3 (fr. 41–47: 2.01%), C4 (fr. 48–50: 6.02%), C5 (fr. 51–58: 37.70%), C6 (fr. 59–63: 21.66%), C7 (fr. 64–75: 25.68%)]. After studies on antidiabetic activity of the subfractions, it was observed that antidiabetic activity of n-BuOH C fraction was detected in its polar subfractions (C5, C6, C7). In order to separate polar part of n-BuOH C fraction, it was dissolved in water and partitionated with EtOAc. Aqueous part was dried (1.7 g) and chromatographed on reverse phase column (RP-18 Cosmosil 75 C18 , Nacalai USA Inc., Cat. No. 37842-66) with

2.2.4. Determination of the chemical structures Structures of isolated compounds were elucidated by extensive spectroscopic methods including 1D-(1 H, 13 C) and 2D NMR (HMQC, HMBC) experiments as well as LC–TOF MS analysis. The 1 H and 13 C NMR experiments were recorded at 400 MHz and 100 MHz on a Varian Oxford AS400 Spectrometer, respectively. For NMR measurements, samples were dissolved in D2 O, CD3 OD or DMSO. Waters Micromass TOF-MS was used for confirmation of molecular weights of the isolated molecules. All data were compared with those in previous literatures (Baderschneider and Winterhalter, 2001; Nakanishi et al., 2005; Enrich et al., 2008).

2.3. Assay for hypoglycaemic effect 2.3.1. Preparation of test samples Extracts and fractions were suspended in 0.5% aqueous carboxymethylcellulose (CMC-suspension in distilled water) prior to oral administration to animals. Glipizide (10 mg/kg, body weight [b.w.]) was used as the reference drug. Glipizide was purchased from Sigma (G117-1 g, St. Louis, MO 63103 USA). Animals in the control group received only the vehicle 0.5% CMC solution (10 ml/kg, b.w.).

2.3.2. Animals Male Wistar-albino rats used in the experiments (150–200 g) were obtained from the Animal House of Gazi University (Ankara, Turkey). Prior to the experiments, rats were fed with standard food for one week in order to adapt to the laboratory conditions. Institutional Animal Ethical Committee of the Gazi University approved (G.Ü.ET-06.087) the experimental protocol used in the present study.

2.3.3. Determination of the blood glucose levels The rats were fasted 12 h before the determination of blood glucose levels, but allowed free access to water. Blood glucose concentrations (mg/dl) were determined using an Ascensia-Elite commercial test (Serial No. 9123232, Bayer), based on the glucose oxidase method. Blood samples were collected from the tip of tail at the defined time patterns.

2.4. Experimental procedure 2.4.1. Effect in normoglycaemic animals Fasting blood glucose level of each animal was determined at initial time, after overnight fasting with free access to water. Control group was received 0.5% CMC (10 ml/kg b.w.). Animals in test groups were treated with the test samples suspended in the same vehicle. Blood samples were collected at 1/2, 1, 2 and 4 h after the oral administration of test samples.

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Table 1 Acute hypoglycaemic effect of water and EtOH-extracts of Juniperus oxycedrus ssp. oxycedrus fruits. Group

Water Extract

Dose (mg/kg)

500 1000 500

EtOH extract 1000

Test model

Diabetic Diabetic Normoglycaemic OGTT Diabetic Normoglycaemic OGTT Diabetic

Inhibition % 1/2 h

1h

2h

4h

6h

5.3 – – – 5.4 – – 13.0***

15.1*** 10.4*** 6.7 11.0*** 7.7** 6.1 10.0*** 12.4***

12.1*** 7.6** 6.1 19.0*** 11.3*** 7.1 3.9 20.8***

2.7 – 12.8** 15.6*** 15.3** 13.0* 4.0 19.5***

1.6 1.4 x x 23.6*** x x 17.8**

OGTT: oral glucose tolerance test, x: not measured, –: no effect. * p < 0.05. ** p < 0.01. *** p < 0.001.

2.4.2. Effect in glucose-hyperglycaemic animals (glucose-loaded model, oral glucose tolerance test, OGTT) Fasting blood glucose level of each rat was determined at initial time, after overnight fasting with free access to water. Glucose (2 g/kg b.w.) was orally administered 30 min after an oral administration of the test sample or vehicle (for control). Blood glucose concentrations were measured just before and 1/2, 1, 2 and 4 h after the oral administration of the test samples. 2.4.3. Effect in diabetic animals (non-insulin dependent diabetes model – NIDDM) 2.4.3.1. Induction of diabetes. Experimental diabetes was induced by intraperitoneal (i.p.) injection of streptozotocin (STZ) at a dose of 65 mg/kg b.w. dissolved in distilled water (1 ml/kg). Three days after the injection, the blood glucose levels were measured and the animals with blood glucose levels higher than 300 mg/dl were considered as diabetic. 2.4.3.2. Determination of hypoglycaemic effect on acute administration. Diabetic animals were fasted for 12 h (water ad libitum). Test samples were given orally using oral gastric gavages. The blood glucose concentrations were measured in all animals at the beginning of the study and the measurements were repeated 1/2, 1, 2, 4 h and 6 h after the initial of the experiment. 2.4.3.3. Determination of hypoglycaemic effect after subacute administration of shikimic acid. Three days after STZ injection, diabetic animals were chosen and animals were divided into 4 groups. Shikimic acid (15 and 30 mg/kg), CMC and glipizide were administered for 8 successive days (once in a day) by gastric gavage. Fasted blood glucose levels and body weights were determined at 10:00 a.m. on the 1st and 8th days. 2.4.4. Effect of shikimic acid on biochemical parameters after subacute administration Blood samples (4–5 ml) were collected into heparinised tubes. Plasma was obtained by cold (4 ◦ C) centrifugation at 1000 × g for 15 min and kept at −70 ◦ C until determination of insulin, triglyceride and total cholesterol levels and to test aspartate transferase (AST), alanine transaminase (ALT) and alkaline phosphatase (ALP) activities as well. Insulin levels were determined by ELISA using Linco Research Rat/Mouse Insulin ELISA Kits (MO, USA) and were expressed as ng/dl. Lipid measurements were evaluated by using commercial kits from IBL Turkiye in Erba XL 600 autoanalyser. Total cholesterol and triglyceride levels were expressed as mg/dl. IBLTurkiye reagents were also used in measurement of AST, ALT and ALP levels in plasma samples. All enzyme levels were expressed as U/l.

2.4.5. Invivo antioxidant effect of shikimic acid after subacute administration 2.4.5.1. Determination of lipid peroxidation (TBARS) in tissues. Rats were anesthetized with intramuscular ketamine injection (i.m.) on the 9th day of the experiment. Liver, kidneys and heart were immediately excised and chilled in ice-cold 0.9% NaCl. After washing with 0.9% NaCl, 0.5 g wet tissues were weighted exactly and homogenised in 4.5 ml of 0.25 M sucrose using a Teflon homogeniser to obtain a 10% suspension. Subsequently, the method of Ohkawa et al. (1979) as modified by Jamall and Smith (1985) was used to determine malondialdehyde (MDA) levels in tissue samples as described before (Aslan et al., 2010) and MDA levels were expressed as nmol/g wet tissue. 2.4.5.2. Determination of non-protein sulfhydryl groups in tissues. For determination of non-protein sulfhydryl groups (cellular GSH) in tissues, 200 mg of liver, 400 mg of heart and kidney were homogenised in 8 ml of 0.02 M EDTA in an ice bath. The homogenates were kept in an ice bath until use. The method of Sedlak and Lindsay was used to determine cellular GSH in tissues, details of the method are described elsewhere (Sedlak and Lindsay, 1968; Aslan et al., 2010). GSH levels were expressed as ␮mol/g wet tissue. 2.5. Statistical analysis Values were presented as means ± standard error of the mean (S.E.M.). Statistical differences between the treatments and the controls were tested by one-way analysis of variance (ANOVA) followed by the Student–Newman–Keuls test using the “Instat” statistic computer program. A difference in the mean values of p < 0.05 was considered to be statistically significant (GraphPad InStat statistical program). Inhibition and change percentages were calculated as following: Inhibition or change % =

test value × 100 − 100. control value

3. Results 3.1. Hypoglycaemic effects of Joso berry extracts In order to prove the claimed hypoglycaemic effect of the Joso berries, water and ethanol extracts were prepared and tested in STZ induced diabetic animals. Hypoglycaemic effect of ethanol extracts was also investigated in normoglycaemic and glucosehyperglycaemic rats at 500 and 1000 mg/kg doses. The data obtained from normal, glucose-hyperglycaemic and diabetic rats were shown in Table 1. Water extract of Joso berries exhibited mild

± ± ± ± ± ± 338.7 370.0 337.6 255.0 302.8 272.7 29.1 (12.2%) 10.9 (6.6%) 44.3 (9.9%) 11.7*** (26.4%) 43.7 (7.3%) 26.1(13.6%) ± ± ± ± ± ± 363.1 386.6 372.6 304.3 383.4 357.2 20.0 (0.7%) 15.9 (6.7%) 30.9 (0.7%) 8.1** (18.3%) 15.0 13.5 ± ± ± ± ± ± 409.7 385.0 409.4 337.2 417.4 425.4 21.2 (6.1%) 16.4 (7.6%) 40.3 (0.3%) 12.3 (12.2%) 17.4 19.9

**

***

*

R-Water subextract

n-BuOH subextract

a

41.4 27.4 72.7 11.7 37.3 19.0 ± ± ± ± ± ± 450.0 452.8 453.6 450.0 450.4 450.6 170 340 370 740 1080 2160

CHCl3 subextract + hexane subextracta EtOAc subextract

Since the yield % chloroform subextract was so poor, it was administered to animals with hexane subextract. p < 0.05. p < 0.01. p < 0.001.

± ± ± ± ± ± 425.6 412.2 444.6 391.7 461.8 490.2 17.8 (12.2%) 17.5 (1.1%) 36.7 (0.7%) 20.0 (9.5%) 33.5 15.6 ± ± ± ± ± ±

413.4 213.9 395.6 401.0 383.4 412.7 336.1 413.0 392.0 410.6 16.0 17.4 (10.5%) 10.8 (17.0%) 33.7 (3.9%) 28.1 (3.6%) ± ± ± ± ± 446.1 399.3 370.2 428.6 430.0 20.7 12.6 (7.3%) 8.4 (6.5%) 37.7 (5.4%) 25.8 (2.9%) ± ± ± ± ± 453.2 420.1 420.1 428.8 440.2 23.4 14.8 13.9 32.8 24.0 ± ± ± ± ± 450.0 450.0 450.2 450.6 450.4 – 10 150 600 20 + 300

After isolation process, three different molecules were identified. Shikimic acid was the one found in the highest amount, and 4-O-glucoside of ferulic acid and 8-O-glucoside of oleuropeic acid were the others. Hence, shikimic acid is the major molecule found in the active fraction, acute and subacute antidiabetic activities of shikimic acid were tested on STZ-diabetic rats at two different doses (15 and 30 mg/kg). Shikimic acid possessed a promising

Control Glipizide Hexane subextract

3.5. Acute hypoglycaemic effect of shikimic acid

Dose (mg/kg)

n-BuOH C fraction was subjected to column chromatography and eluents were combined into seven main subfractions according to TLC control. Fractions were given to diabetic rats in doses estimated from their ratio in n-BuOH C fraction (Table 4). Subfractions C 1–4 were combined and given to animals at 59 mg/kg dose and all other subfractions were given to diabetic rats at two different doses. As seen from Table 4, except subfraction C 1–4, all tested subfractions lowered blood glucose levels with different inhibition percentages. For this reason further studies were planned to isolate the active components of n-BuOH C fraction.

Group

3.4. Acute hypoglycaemic effect of subfractions from n-BuOH C fraction

Table 2 Acute hypoglycaemic effect of subextracts from EtOH extract of Joso fruits in STZ-diabetic rats.

3.3. Acute hypoglycaemic effect of fractions from n-BuOH subextract

Blood glucose concentration (mg/dl) ± standart error of the mean (inhibition %) 1/2 h 1h Initial

2h

± ± ± ± ±

11.3 18.0** (18.6%) 12.4 18.0 (5.1%) 17.4 (0.5%)

4h

± ± ± ± ±

Further studies were conducted on EtOH extract. First it was subjected to bioassay-guided fractionation process. As the first step fractionation, EtOH extract was subjected to successive solvent extractions with hexane, chloroform, ethylacetate and n-butanol. The hypoglycaemic effect of each subextract was studied using diabetic rats. Doses of subextracts were calculated according to their yield percentages. The sub-extracts were tested at two doses and as shown in Table 2, n-BuOH subextract at 740 mg/kg was the most active one (9.5–31.0%) among all. Reference drug glipizide showed a remarkable and statistically significant antidiabetic activity (18.6–48.3%) in comparison to Joso berry subextracts. Even though R-Water subextract exhibited a remarkable effect at 6th h, this was occurred after a long time.

398.0 448.2 450.0 410.2 498.2 526.9

3.2. Acute hypoglycaemic effect of subextracts from EtOH extract of Joso berries

n-BuOH subextract was subjected to column chromatographic separation and eluents were combined into six fractions according to TLC control. Fractions were given to diabetic rats in doses estimated from their ratio in n-BuOH subextract (Table 3). Fractions C (150.6 mg/kg) and D (115.4 mg/kg) were found to possess moderate hypoglycaemic effect between 7.7–12.7% and 7.2–16.6% respectively. When higher doses were tested, fraction C showed a remarkable and statistically significant antidiabetic activity between 18.6 and 26.4%. Thus further studies were conducted on n-BuOH C fraction.

369.5 192.6 331.4 341.8 312.0 12.4 15.5*** (48.3%) 17.9 (4.2%) 18.4 (3.0%) 20.0 (7.3%)

6h

± ± ± ± ±

16.4 18.1*** (47.9%) 5.1 (10.3%) 18.8 (7.5%) 11.5* (15.6%)

hypoglycaemic effect in 1st and 2nd h measurements at both tested doses (7.6–15.1%) where ethanol extract showed higher and continuous hypoglycaemic effect (7.7–23.6%) in STZ-diabetic rats. In normal rats, ethanol extract exhibited a moderate hypoglycaemic effect (12.8–13.0%) only at 4th h measurement at both doses. In oral glucose tolerance test, glucose solution was administered to normal rats just after 30 min measurement. The ethanol extract, given in 500 mg/kg, was found more effective (11.0–19.0%). (In Table 1 control and reference data were not given for clarification).

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50.2 (8.3%) 6.8 54.3 (8.6%) 6.9*** (31.0%) 11.8 (18.1%) 7.2*** (26.2%)

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N. Orhan et al. / Journal of Ethnopharmacology 139 (2012) 110–118

hypoglycaemic effect when administered to diabetic rats at 30 mg/kg dose (Table 5).

11.0 13.3 (20.1%) 9.2 (3.1%) 10.5 4.8 (12.7%) 9.0*** (26.4%) 14.8 (12.0%) 31.6 (3.1%) 15.7 16.6 (11.2%)

114

323.2 258.0 313.2 349.4 282.2 214.6 284.4 282.6 325.2 287.1

Shikimic acid was administered to diabetic rats for 8 days to evaluate its subacute antidiabetic effect at 15 and 30 mg/kg doses. The blood glucose concentrations were measured at the 8th day of the experiment. Shikimic acid (at 30 mg/kg) was more effective than reference antidiabetic drug glipizide compared to control. The body weight of diabetic rats was measured at the beginning and at the end of the experiment. Although 8 day is a short period, rats in control and glipizide groups lost weight (0.3 and 5.4%) but rats in shikimic acid groups gain weight (0.2 and 1.8%) compared to the first day (Table 6).

337.2 285.8 323.6 352.6 303.2 264.6 290.8 307.6 329.4 320.5

4h

± ± ± ± ± ± ± ± ± ±

7.8 8.4* (15.2%) 4.7 (4.2%) 7.0 16.6 (10.1%) 6.6*** (20.0%) 4.7 (13.8%) ± 16.4 (6.9%) 14.8 (2.3%) 12.1 (5.0%)

6h

± ± ± ± ± ± ± ± ± ±

3.6. Subacute hypoglycaemic effect of shikimic acid

5.2 20.6 (3.4%) 8.7 8.9 12.4 (5.1%) 5.5* (18.6%) 11.0 (7.2%) 30.0 (3.6%) 12.6 6.9

3.7. Effect of shikimic acid on some biochemical parameters after subacute administration

334.4 323.2 338.4 365.6 317.4 306.0 310.2 362.2 383.2 356.2 7.3 15.4 (6.5%) 11.0 (8.8%) 15.1 (3.6%) 9.6* (11.8%) 26.4 (6.5%) 9.0** (16.6%) 28.4 20.4 12.3 ± ± ± ± ± ± ± ± ± ± 398.6 372.8 363.6 384.2 351.6 359.8 332.4 389.4 431.0 398.6

1h

11.2 15.6 (6.4%) 9.7 (5.6%) 17.1 (1.5%) 14.1 (7.7%) 27.4 (0.4%) 8.6* (12.7%) 26.5 21.8 5.3 (1.1%) ± ± ± ± ± ± ± ± ± ± 409.0 382.8 386.0 403.0 377.6 388.0 357.0 410.8 458.6 404.6

1/2 h

9.2 22.1 11.0 18.3 17.1 21.8 26.8 22.3 18.6 24.6 ± ± ± ± ± ± ± ± ± ± 386.2 375.2 378.8 392.0 363.2 365.4 369.2 370.0 370.4 385.0

***

p < 0.05. p < 0.01. p < 0.001. *

n-BuOH E (33–41) n-BuOH F (42–60)

n-BuOH D (30–32)

4. Discussion

**

Initial

– 10 28.6 61.4 150.6 301.2 115.4 230.8 816.2 307.8 Control Glipizide n-BuOH A (0-18) n-BuOH B (19–23) n-BuOH C (24–29)

Blood glucose concentration (mg/dl) ± standart error of the mean (inhibition %) Dose (mg/kg) Group

Table 3 Hypoglycaemic effect of fractions from n-BuOH subextract obtained by column chromatography on STZ-induced diabetic rats.

2h

± ± ± ± ± ± ± ± ± ±

After subacute administration; plasma insulin levels, total cholesterol, triglyceride, AST, ALT and ALP levels were determined. It is seen in Table 7 that shikimic acid did not elevated insulin levels remarkably compared to control thence it can be said that antidiabetic effect of shikimic acid is not arising from the increased insulin secretion. Effect of shikimic acid on plasma total cholesterol (TC) and triglyceride (TG) concentrations of diabetic rats was examined. Although TC levels were not lowered remarkably in shikimic acid groups, TG levels were reduced significantly (10.3–30.6%). Therefore, it could be concluded that shikimic acid have a positive effect on lipid metabolism. Additionally plasma AST, ALT and ALP levels were also measured after subacute administration of shikimic acid. Enzyme activities were low in all test groups compared to diabetic control. Hence, these enzyme activities were significantly lowered by shikimic acid; it might have a curative or protective effect against tissue and organ damages caused by diabetes-induced oxidative stress. In connection with this hypothesis liver, kidney and heart MDA and GSH levels were investigated (Table 8). It is known that MDA levels are elevated in diabetic patients because of oxidative stress inducedlipid peroxidation. Anti-lipid peroxidative effect of shikimic acid at 15 mg/kg on kidney and heart tissues is highly pronounced (64.5–63.2%). GSH is one of the guardians to free radicals and other oxidants in living organisms. Shikimic acid at 30 mg/kg elevated GSH levels moderately in kidney and heart tissues. Consequently, in the light of biochemical parameters it can be said that subacute administration of shikimic acid ameliorated complications of diabetes.

Ethnobotanical studies in Turkey revealed that a number of plant remedies were used to alleviate the symptoms of diabetes (Tabata et al., 1993, 1994). Among these, decoction of Juniperus oxycedrus berries is used internally as tea and pounded berries are consumed to lower blood glucose levels (Tuzlacı and Erol, 1993; Honda et al., 1996). According to literature survey, it has been reported that the fruit extracts from different species of juniper have possessed hypoglycaemic effect. Antidiabetic activity of ethanol and water extracts of Juniperus chinensis berries was investigated on alloxaninduced diabetic rats. Ethanol extract exhibited more remarkable hypoglycaemic effect than the reference drug glibenclamide (Ju et al., 2008). In another study, decoction and infusion of Juniperus communis fruits significantly decreased blood glucose levels in normoglycaemic rats, and also in STZ-induced diabetic rats and mice (Swanston-Flatt et al., 1990; Sanchez de Medina et al., 1994).

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Table 4 Hypoglycaemic effect of subfractions from C fraction of n-BuOH subextract obtained by column chromatography on STZ-induced diabetic rats. Group

Dose (mg/kg)

Blood glucose concentration (mg/dl) ± standart error of the mean (inhibition %) Initial

Control Glipizide C 1–4 (1–50) C5 (51–58) C6 (59–63) C7 (64–75) * ** ***

– 10 59 227 454 131 262 155 310

345.0 339.9 343.8 342.0 324.0 343.2 329.7 344.2 316.9

1/2 h ± ± ± ± ± ± ± ± ±

10.6 0.7 12.9 5.8 7.0 8.5 7.3 8.8 4.8

1h

371.2 348.6 374.6 348.4 334.1 358.6 329.3 318.4 305.1

± ± ± ± ± ± ± ± ±

7.6 1.1 (6.1%) 8.5 9.1 (6.1%) 8.3** (10.0%) 8.1 (3.4%) 7.1** (11.3%) 6.0*** (14.2%) 4.9*** (17.8%)

350.8 325.9 348.0 318.4 343.1 343.0 324.1 303.4 298.9

2h ± ± ± ± ± ± ± ± ±

7.5 1.4 (7.1%) 10.8 (0.8%) 8.6* (9.2%) 7.1 (2.2%) 7.6 (2.2%) 7.4 (7.6%) 4.4*** (13.5%) 4.1*** (14.8%)

310.4 291.8 316.0 301.8 263.2 327.4 229.7 286.4 230.0

4h ± ± ± ± ± ± ± ± ±

2.7 2.6 (6.0%) 8.4 5.2 (2.8%) 5.8*** (15.2%) 8.0 7.4*** (26.0%) 10.1 (7.7%) 4.5*** (25.9%)

6h

263.6 242.5 264.4 265.6 245.4 272.0 223.0 233.2 221.7

± ± ± ± ± ± ± ± ±

9.0 2.6 (8.0%) 5.5 6.6 9.0 (6.8%) 1.6 6.6*** (15.4%) 6.5** (11.5%) 5.8*** (15.9%)

± ± ± ±

6.0 3.8 (2.6%) 5.5 4.8** (14.8%)

225.4 218.9 246.4 238.6 195.0 233.0 202.0 195.8 172.4

± ± ± ± ± ± ± ± ±

5.7 1.0 (2.9%) 6.8 7.1 6.9* (13.5%) 5.4 7.0* (10.4%) 9.5* (13.1%) 5.7*** (23.5%)

± ± ± ±

4.9 3.1* (9.6%) 3.5 6.9*** (24.0%)

p < 0.05. p < 0.01. p < 0.001.

Table 5 Acute hypoglycaemic effect of shikimic acid on STZ-induced diabetic rats. Group

Dose (mg/kg)

Control Glipizide

– 10 15 30

Shikimic acid * ** ***

Blood glucose concentration (mg/dl) ± S.E.M. (inhibition %) Initial

1/2 h

334.0 ± 5.4 339.7 ± 3.7 335.3 ± 7.0 324.7 ± 3.6

346.8 331.1 332.8 317.3

1h ± ± ± ±

3.5 6.2 (4.5%) 3.1 (4.0%) 7.8* (8.5%)

330.5 312.4 318.5 303.3

2h ± ± ± ±

5.3 8.6 (5.5%) 4.2 (3.6%) 7.6 (8.2%)

289.9 270.7 271.0 252.3

4h ± ± ± ±

9.5 9.5 (6.6%) 7.9 (6.5%) 5.1* (13.0%)

6h

235.5 229.4 243.0 200.7

195.9 177.1 199.8 148.9

p < 0.05. p < 0.01. p < 0.001.

Loizzo et al. investigated in vitro hypoglycaemic activity of Juniperus oxycedrus ssp. oxycedrus (Joso) berry and wood essential oils by the inhibition of ˛-amylase, wood oil was found to be active (Loizzo et al., 2007). In our previous study on subacute effects of Joso berry and leaf ethanol extracts, antidiabetic activity was investigated in diabetic animals. This study indicates treatment of diabetic rats for 10 days with Joso fruit and leaf crude extracts decreased blood glucose levels. Moreover, extracts decreased levels of lipid peroxidation in liver and kidney tissues remarkably and augmented Zn concentrations in liver (Orhan et al., 2011). Juniperus genus has attracted a great interest in the scientific communities and it has been the subject of more than 320 published phytochemical studies in the last 40 years. It is mainly a source of sesquiterpenes, diterpenes and lignans, some of them with very promising biological activities. The compounds identified from Juniperus species during the period 1970–2004, except essential oils fractions, are compiled in a review together with the biological activities of the Juniperus extracts and of the isolated pure compounds by Seca and Silva. According to this manuscript, coumarins (umbelliferone), flavonoids (amentoflavone, cupressuflavone, hinokiflavone, rutin), sterols (campesterol, cholesterol, ␤sitosterol, stigmasterol), terpenes (monoterpenes, sesquiterpenes, diterpenes), aliphatic compounds (alkanes, fatty acids, waxes)

exist in different parts of Juniperus oxycedrus (Seca and Silva, 2006). In this study; shikimic acid, ferulic acid glucoside and oleuropeic acid glucoside were isolated from Juniperus oxycedrus ssp. oxycedrus berries by bioactivity guided fractionation technique. Occurrence of these three known molecules in Juniperus species was searched through the previously published articles. (−)-Shikimic acid was first isolated in 1885 by Eykman from the fruit of lllicium religiosum (Bohm, 1965) and later studies have shown that in various organisms, shikimic acid pathway appears to be an important route for the biosynthesis of the C6 –C3 units(phenylpropane derivatives) from carbohydrates. Also recent studies proved occurance of shikimic acid in Juniperus chinensis, phoenicea and virginiana (Bohm, 1965; Aboul-Ela et al., 2005). Ferulic (coniferic) acid is one of the key molecules that has an important role in the formation of lignin of Gymnosperms and lignans like podophyllotoxin and pinoresinol (Evans, 2009). A monoterpene glucoside (−)-oleuropeic acid 8-O-ˇ-D-glucopyranoside was isolated from aerial parts (nBuOH subextract of methanol extracts) of Juniperus communis var. depressa (Nakanishi et al., 2005). Ou and Kwok reported that ferulic acid (FA) has many physiological functions, including antioxidant, antimicrobial, antiinflammatory, anti-thrombosis, and anti-cancer activities. It also

Table 6 Effect of shikimic acid on blood glucose concentrations and body weights after 8 days administration to STZ-diabetic rats. Group

Control Glipizide Shikimic acid ** ***

p < 0.01. p < 0.001.

Dose (mg/kg)

– 10 15 30

Blood glucose conc. ± S.E.M. (inhibition %)

Body weight ± S.E.M. (change % to 1st day)

1st day

8th day

334.0 ± 5.4 339.7 ± 3.7 335.3 ± 7.0 324.7 ± 3.6

309.6 264.3 279.0 259.9

± ± ± ±

1st day 3.7 6.6*** (14.6%) 7.1** (9.9%) 7.3*** (16.1%)

178.4 185.1 187.0 189.9

± ± ± ±

8th day 5.9 5.3 6.9 4.8

177.9 175.1 187.3 193.3

± ± ± ±

6.7 (−0.3%) 10.0 (−5.4%) 7.6 (+0.2%) 6.2 (+1.8%)

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Table 7 Effect of shikimic acid on some biochemical parameters after 8 days administration to STZ-diabetic rats. Group

Dose (mg/kg)

Plasma insulin ± SEM

Control Glipizide

– 10 15 30

50.78 51.93 51.22 51.01

Shikimic acid

± ± ± ±

0.3 0.8 0.3 0.6

Total cholesterol ± SEM (change %) 21.0 18.7 24.2 20.4

± ± ± ±

1.7 3.4 (−10.0%) 3.5 (+15.2%) 0.4 (−2.9%)

Triglyceride ± SEM (change %) 196.3 182.9 176.0 136.3

± ± ± ±

AST ± SEM (change %)

4.4 2.3* (−6.8%) 3.2** (−10.3%) 3.6*** (−30.6%)

184.0 170.3 155.0 128.4

± ± ± ±

ALT ± SEM (change %)

4.1 4.0* (−7.4%) 4.3*** (−15.8%) 1.7*** (−30.2%)

152.4 136.9 116.5 78.7

± ± ± ±

ALP ± SEM (change %)

6.1 3.2** (−10.2%) 2.8*** (−23.6%) 1.7*** (−48.4%)

576.9 382.9 434.3 405.3

± ± ± ±

10.4 8.1*** (−33.6%) 10.0*** (−24.7%) 9.9*** (−29.7%)

N. Orhan et al. / Journal of Ethnopharmacology 139 (2012) 110–118

AST aspartate transaminase, ALT alanine transaminase, ALP alkaline phosphatase. * p < 0.05. ** p < 0.01. *** p < 0.001.

Table 8 Effect of shikimic acid on tissue malondialdehyde and glutathione levels after 8 days administration to STZ-diabetic rats. Group Control Glipizide Shikimic acid * ***

p < 0.05 p < 0.001.

Dose (mg/kg) – 10 15 30

Malondialdehyde levels ± S.E.M. (inhibition %) Liver Kidney 136.3 67.5 139.6 146.7

± ± ± ±

3.4 2.7*** (−50.2%) 1.7 (+2.4%) 4.2 (+7.6%)

386.8 122.0 137.3 142.2

± ± ± ±

9.1 8.3*** (−68.5%) 7.9*** (−64.5%) 4.3*** (−63.2%)

Glutathione levels ± S.E.M. (inhibition %) Liver Kidney

Heart 39.6 11.0 15.1 37.3

± ± ± ±

1.8 2.2*** (−72.2%) 1.9*** (−61.9%) 2.6 (−5.8%)

152.2 131.1 143.4 150.2

± ± ± ±

4.9 4.3* (−13.9%) 6.1 (−5.8%) 5.5 (−1.3%)

63.9 62.8 63.5 66.0

± ± ± ±

1.3 1.8 (−1.7%) 1.7 (−0.6%) 1.6 (+3.3%)

Heart 34.2 34.3 35.4 38.2

± ± ± ±

1.1 1.8 (+0.3%) 1.7 (+3.5%) 1.3 (+11.7%)

N. Orhan et al. / Journal of Ethnopharmacology 139 (2012) 110–118

has a protective effect against coronary disease, lowers cholesterol and increases sperm viability (Ou and Kwok, 2004). Hypoglycaemic effect of FA was studied on STZ-induced (type I) and KK-Ay (type II) diabetic mice. The blood glucose level in STZ-induced diabetic animals is reduced by the administration of FA. FA helps to neutralize the free radicals produced by STZ in the pancreas. This may help the beta cells to proliferate and secrete more insulin, which can cause increased utilization of glucose by the extra hepatic tissues and thereby decrease the blood glucose level (Balasubashini et al., 2004). Noumura et al. reported that amide compounds of FA exhibited their stimulatory abilities on insulin secretion in rat pancreatic RIN-5F cells (Noumura et al., 2003). Administration of FA at a dose of 0.01 and 0.1% of basal diet showed it can suppress the blood glucose levels in STZ induced diabetic mice. In KK-Ay mice, 0.05% of FA suppressed the blood glucose level significantly (Ohnishi et al., 2004). Hence antidiabetic activity of ferulic acid is well known and shikimic acid (SA) is the major molecule found in the active fraction, acute and subacute antidiabetic activities of SA were tested on STZ-diabetic rats at two different doses (15 and 30 mg/kg). SA possessed a promising hypoglycaemic effect when applied to diabetic rats at 30 mg/kg dose (Table 5). Subacute antidiabetic effect of SA (at 30 mg/kg) was more effective than reference antidiabetic drug glipizide compared to control. After subacute administration; plasma insulin levels, total cholesterol, triglyceride, AST, ALT and ALP levels were determined. Although TG levels (10.3–30.6%) and enzyme levels were reduced significantly, SA did not elevated insulin and TC levels. Anti-lipid peroxidative effect of SA at 15 mg/kg on kidney and heart tissues is so pronounced (64.5–63.2%) and SA at 30 mg/kg increased GSH levels moderately in kidney and heart tissues. Consequently, biochemical parameters suggest that subacute administration of SA could ameliorate complications of diabetes. Approaches to the control of blood glucose and prevention of hyperglycaemia are central to the treatment of diabetes mellitus. Appetite suppressants (vanadium compounds, antiobesity agents), inhibitors of digestion (˛-glucosidase inhibitors, guar gum, polysaccharides), insulin secretagogues (sulphonylureas), insulin potentiators (sulphonylureas, metformin, thiazolidinediones, growth hormone), stimulants of glucose utilization (nicotinic acid), insulin mimetics (incretins, thiazolidinediones, trace elements, vanadium compounds), inhibitors of gluconeogenesis and glucogenolysis (sulphonylureas, metformin, thiazolidinediones, nicotinic acid) are used to balance blood glucose. At present, none of these therapies either alone or in combination can reinstate normal blood glucose homeostasis, and many limitations exist in the use of anti-diabetic drugs. New therapies are needed which reinstate a normal metabolic environment and prevent long-term complications of diabetes (Gray and Flatt, 1997). Effect of SA on plasma insulin levels after subacute administration was examined to enlighten the mechanism of action. SA did not elevated insulin levels compared to control, thus SA is not an insulin secretagogue and might have another mechanism of action. In literature survey on the pharmacological effect of SA, a study on the effect of Illicium anisatum extracts on B6C3HF1 mouse vibrissae follicles revealed that follicules treated with SA grew significantly longer than controls. Data demonstrated that SA also induced mRNA expression of insulin-like growth factor-1, keratinocyte growth factor and vascular endothelial growth factor in the hair follicles (Sakaguchi et al., 2004). Insulin growth factor (IGF1) also known as somatomedin C, is a hormone similar in molecular structure to insulin. It plays an important role in childhood growth and continues to have anabolic effects in adults. IGF-1 binds to at least two cell surface receptors; the IGF-1 receptor and the insulin receptor. It has growth-promoting effects on almost every cell in the body, especially skeletal muscle, cartilage, bone, liver, kidney, nerves, skin, hematopoietic cell and lungs. In addition to regulation

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of cell growth especially in nerve cells as well as cellular DNA synthesis, it has insulin like effect as an insulin potentiator. Results of clinical trials evaluating the efficacy of IGF-1 in type 1 diabetes and type 2 diabetes showed great promise in reducing hemoglobin A1c (HbA1c) levels as well as daily insulin consumption (Moses et al., 1996; Amiel et al., 1984; Wüster and Rosen, 2001). It is a common belief that oxidative stress results from an imbalance between the generation of oxygen derived radicals and the organism’s antioxidant reserve. Various studies have shown that diabetes mellitus is associated with increased formation of free radicals and decrease in antioxidant potential. Such as decrease in glutathione levels and elevation in thiobarbituric acid reactive substances are consistently observed in diabetes. Due to occurrence of these events, the balance normally present in cells between radical formation and protection against them is disturbed which leads to oxidative damage of cell components such as proteins, lipids, and nucleic acids. In both insulin dependent (type 1) and noninsulin-dependent diabetes (type 2) oxidative stress is increased. It has been suggested that enhanced production of free radicals and oxidative stress is a central event in the development of diabetic complications like cardiovascular disease, nephropathy, neuropathy, retinopathy etc. Therefore, it seems reasonable that antioxidants can play an important role in the improvement of diabetes. There are many reports on effects of antioxidants in the management of diabetes (Rahimi et al., 2005). In our study, malondialdehyde (MDA) that is widely used as an indicator of lipid peroxidation is measured in tissue samples after subacute administration of SA. SA at 15 mg/kg reduced MDA levels on kidney and heart tissues significantly (64.5–63.2%). Additionally, at 30 mg/kg, MDA levels in kidney were decreased (63.2%) and GSH levels in kidney and heart tissues were increased moderately. According to reduction in MDA levels and elevation in GSH levels in tissues it could be said that SA has a promising antioxidant activity. Due to these findings and previous studies, it could be hypothesized that SA exhibits its antidiabetic activity by increasing insulin like growth factor-1. In addition to insulin like effect as an insulin potentiator, IGF-1 regulates cell growth in cells as well as cellular DNA synthesis, thus it may have a curative effect on the tissue damage occurred in diabetic animals because of oxidative stress. As a result, antioxidant effect of SA supports the antidiabetic activity. In conclusion, the results of antidiabetic activity studies support the traditional use of Juniperus oxycedrus ssp. oxycedrus berries as a folk remedy in the treatment of diabetes in Turkey. Further studies are necessary to prove the mechanism of action, to obtain toxicological data and to observe and evaluate long-term effects of shikimic acid. Acknowledgements This study was a part of Nilüfer Orhan’s PhD thesis called “Pharmacognosic Inverstigations on the Juniperus Species Used for Diabetes Mellitus in Folk Medicine” and financially supported by the Research Fund of Gazi University (02/2007-07). References Aboul-Ela, M., El-Shaer, N., El-Azim, T.A., 2005. Chemical constituents and hepatotoxic effect of the berries of Juniperus phoeniciea, Part II. Natural Product Sciences 11, 240–247. Amiel, S.A., Sherwin, R.S., Hintz, R.L., Gertner, J.M., Press, C.M., Tamborlane, W.V., 1984. Effects of diabetes and its control on insulin-like growth factors in the young subject with type I diabetes. Diabetes 33, 1175–1179. Aslan, M., Orhan, N., Deliorman Orhan, D., Ergun, F., 2010. Hypoglycemic activity and antioxidant potential of some medicinal plants traditionally used in Turkey for diabetes. Journal of Ethnopharmacology 128, 384–389. Baderschneider, B., Winterhalter, P., 2001. Isolation and characterization of novel benzoates, cinnamates, flavonoids, and lignans from riesling wine and screening for antioxidant activity. Journal of Agricultural and Food Chemistry 49, 2788–2798.

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