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A bioactivity guided study on the antidiabetic activity of Juniperus oxycedrus subsp. oxycedrus L. leaves
Journal of Ethnopharmacology 140 (2012) 409–415
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A bioactivity guided study on the antidiabetic activity of Juniperus oxycedrus subsp. oxycedrus L. leaves Nilüfer Orhan a,∗ , Mustafa Aslan a , Betül Demirci b , Fatma Ergun a a b
Gazi University, Faculty of Pharmacy, Department of Pharmacognosy, 06330, Etiler Ankara, Turkey Anadolu University, Faculty of Pharmacy, Department of Pharmacognosy, Eskis¸ehir, Turkey
a r t i c l e
i n f o
Article history: Received 17 November 2011 Received in revised form 17 January 2012 Accepted 24 January 2012 Available online 2 February 2012 Keywords: Cupressaceae Diabetes mellitus Hypoglycaemic Juniperus oxycedrus ssp. oxycedrus Linoleic acid Linolenic acid Palmitic acid
a b s t r a c t Ethnopharmacological relevance: Juniperus (Cupressaceae) species are widely used as folk medicine in spreading countries. Decoction of Juniperus oxycedrus subsp. oxycedrus L. leaves is used internally to lower blood glucose levels in Turkey. Aim of the study: To determine hypoglycaemic and antidiabetic activities of Juniperus oxycedrus subsp. oxycedrus leaves and to identify active compounds through bioactivity guided isolation technique. Materials and methods: Ethanol and water extracts of Juniperus oxycedrus subsp. oxycedrus (Joso), leaves on oral administration were studied using in vivo models in normal, glucose-hyperglycemic and streptozotocin-induced diabetic rats. Through in vivo bioactivity-guided fractionation processes, a nonpolar fraction was separated from the n-hexane subextract by silica gel column chromatography as the main active fraction. Subfractions of this fraction was found to possess antidiabetic activity and their chemical composition was investigated by GC-FID and GC–MS, simultaneously. Results: This is the first report on the antidiabetic constituents of Joso leaves. Fatty acids, such as palmitic, linoleic and linolenic acid were found as the major compounds in subfractions. Conclusion: Results indicated that Joso leaf extract and its active constituents might be beneficial for diabetes mellitus. © 2012 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Historical accounts of diabetes mellitus first appeared in the medical texts of several ancient cultures over 2000 years ago. In addition, recorded treatments for diabetes included largely dietand plant-based remedies (Cheng, 2000). There is, only one example of an approved antidiabetic drug that was developed from a herb with a long history of use for diabetes: metformin from French lilac (Galega officinalis) (Marles and Farnsworth, 1995). Numerous other herbs remain candidates for antidiabetic drug development and clinical data are beginning to emerge, which support antidiabetic indications for several of these herbs. This viewpoint outlines the opportunity that exists for these herbs in the management of diabetes and the state of the evidence for their clinical antidiabetic efficacy (Vuksan and Sievenpiper, 2005). Juniperus is one of the most diverse genera of conifers belonging to the Cupressaceae family (Cypress family). It is widely distributed throughout the northern hemisphere from the Arctic Circle to the mountains of the African tropics, from sea level to above timberline (Adams, 2011). 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). Although some species have toxic effects, juniper plants have many uses in folk medicine in several parts of the world (Seca and Silva, 2006). 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, diabetes mellitus, kidney inflammation and to pass kidney stone (Tuzlacı and Erol, 1993; Yes¸ilada et al., 1995; Honda et al., 1996). Additionally decoction of Juniperus oxycedrus subsp. oxycedrus (Joso) leaves is used internally in E˘girdir and Isparta for diabetes mellitus (Tuzlacı and Erol, 1993). The present study aimed to evaluate hypoglycaemic activity of Joso leaves in normoglycaemic, glucose-hyperglycaemic and streptozotocin-induced diabetic rats and to determine its active constituents through bioactivity guided fractionation and isolation techniques. 2. Materials and methods 2.1. Plant material
∗ Corresponding author. Tel.: +90 312 2023176; fax: +90 312 2235018. E-mail addresses: [email protected], [email protected] (N. Orhan). 0378-8741/$ – see front matter © 2012 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2012.01.042
Juniperus oxycedrus L. ssp. oxycedrus L. (Joso) leaves were collected in July 2007 from Karapiri province of Akda˘gmadeni, Yozgat
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(Turkey). The plant was identified by N. Orhan and a voucher specimen (GUEF 2616) is stored in the Herbarium of Gazi University, Faculty of Pharmacy. 2.2. Extraction and fractionation of material 2.2.1. Preparation of water-extract Chopped dried leaves (1000 g) were extracted with hot distilled water (50 ◦ C, 20 l) for 3 h with continuous stirring. The extract was filtered and evaporated under reduced pressure at 40 ◦ C and then lyophilized (Water-Extract, yield 17.0%). 2.2.2. Preparation and fractionation of EtOH extract The chopped dried leaves (1000 g) were extracted with ethanol 80% (10 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 extract under reduced pressure (EtOH-Extract, yield 35.0%). Dried ethanol extract (300 g) was dissolved in 1000 ml methanol/water (7:3) mixture and extracted with n-hexane in a separatory funnel (20 × 500 ml). Hexane phases were collected and dried to give hexane subextract (yield 6.5%). Polar phase was distillated to evaporate methanol and then fractionated successively with chloroform (8 × 500 ml), ethyl acetate (20 × 500 ml), n-butanol/saturated with water (14 × 500 ml) to obtain subextracts. Each subextract was concentrated to dryness under reduced pressure on a rotary evaporator to give (CHCI3 -subextract, yield 6.4%), (EtOAc-subextract, yield 18.6%), (n-BuOH-subextract, yield 41.6%) and the remaining water subextract (R-Water subextract, yield 26.5%), respectively. All the obtained subextracts were used in animal experiments at the doses calculated according to their yields. The hexane subextract was also used in phytochemical studies. 2.2.3. Chromatographic studies and isolation of active constituent 10 g of hexane subextract was chromatographed on silica gel column (350 g Kieselgel 60, 063–0.2 mm, Merck, Art. No.7734) and eluted with hexane, mixtures of Hex:EtOAc (90:10 → 30:70) and CHCl3 :MeOH (50:50) successively. Eluents were combined into six subfractions A (fr.1–9: 18.71%), B (fr.10–13: 4.96%), C (fr.14–29: 23.19%), D (fr.30–65: 15.67%), E (fr.66–105: 13.61%), F (fr.106–113: 19.55%) according to TLC behavior using solvent system Hex:EtOAc (6:4) [spots were visualized under daylight, UV-light and after spraying 5% H2 SO4 or anisaldehyde-sulfuric acid reagent]. After bioactivity studies hexane F fraction (1 g) was subjected to silica gel (30 g) column and eluted with mixtures of CHCl3 :MeOH (90:10 → 25:75) and methanol to afford 58 fractions. Eluents were combined to obtain four subfractions [F1–2 (fr.1–9: 6.20%), F3 (fr.10–19: 13.36%), F4–6 (fr.20–42: 49.93%), F7–8 (fr.43–58: 21.25%)]. After studies on antidiabetic activity of subfractions, hexane subextact and subfractions F1–2 and F7–8, possessed potent activity, were applied to GC–MS to identify their contents. 2.2.4. GC-FID and GC–MS analysis of active subextract and subfractions Dried hexane subextract was dissolved in n-hexane, subfractions F1–2 and F7–8 were dissolved in n-hexane–methanol mixture and filtered from Millipore filter before the injection. Samples were analyzed subsequently by capillary GC-FID and GC–MS. The individual components characterized are given results section. The GC–MS analysis was carried out with an Agilent 5975 GCMSD system. Innowax FSC column (60 m × 0.25 mm, 0.25 m film thickness) was used with helium as a carrier gas (0.8 ml/min). GC oven temperature was kept at 60 ◦ C for 10 min and programmed to 220 ◦ C at a rate of 4 ◦ C/min, and kept constant at 220 ◦ C for 10 min and then programmed to 240 ◦ C at a rate of 1 ◦ C/min. Split ratio was adjusted at 40:1. The injector temperature was set at 250 ◦ C.
Mass spectra were recorded at 70 eV. Mass range was from m/z 35 to 450. The GC analysis was carried out using an Agilent 6890N GC system. FID detector temperature was 300 ◦ C. To obtain the same elution order with GC–MS, simultaneous auto-injection was done on a duplicate of the same column applying the same operational conditions. Relative percentage amounts of the separated compounds were calculated from FID chromatograms. Identification of the components were carried out by comparison of their relative retention times with those of authentic samples or by comparison of their relative retention index (RRI) to series of n-alkanes. Computer matching against commercial (Wiley GC/MS Library, MassFinder 3 Library) and in-house “Bas¸er Library of Essential Oil Constituents” built up by genuine compounds and components of known oils, as well as MS literature data was used for the identification (McLafferty and Stauffer, 1989; Joulain and Koenig, 1998; ESO 2000, 1999; Koenig et al., 2004). 2.3. Assay for hypoglycemic activity 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, b.w.) was used as the reference drug. Glipizide was purchased from Sigma (G1171g, St. Louis, MO 63103, USA). Animals in the control group received only the vehicle 0.5% aqueous carboxymethylcellulose (10 ml/kg, b.w.). 2.3.2. Animals Male Wistar-albino rats (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 1 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 (for 8 h) with free access to water. Control group of animals were received 0.5% CMC. 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. 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 (for 8 h) 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 levels 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 STZ-induced diabetic animals 2.4.3.1. Induction of diabetes. Experimental diabetes was induced by intraperitoneal (i.p.) injection of streptozotocin (STZ) at a dose
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Table 1 Hypoglycaemic effect of Water and EtOH-extracts of Juniperus oxycedrus ssp. oxycedrus leaves. Group
Dose (mg/kg)
Test model
Inhibition % 1/2 h
1h
2h
4h
6h
Water extract
500 1000
Diabetic Diabetic Normoglycaemic OGTT Diabetic Normoglycaemic OGTT Diabetic
0.2 10.9* 0.8 0.9 1.7 x 3.2 10.0***
16.9*** 16.3*** 12.2** 2.9 5.2* 10.9** 11.7*** 13.0***
8.4*** 12.2*** 14.2* 5.2 7.3* 11.6* 7.8 15.5***
9.3*** 6.3** 25.9*** 7.8 1.1 21.9*** 10.7* 18.0***
6.4 1.9 – – 0.7 – – 17.9***
500 EtOH extract 1000
OGTT: oral glucose tolerance test; (x) not measured; (–) no effect. * p < 0.05. ** p < 0.01. *** p < 0.001
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 activity on acute administration. Diabetic animals were fasted for 8 h (water ad libitum). Test samples were given orally using oral gastric gavages. The blood glucose levels were measured in all animals at the beginning of the study and the measurements were repeated 1/2, 1, 2, 4, and 6 h after the initial of the experiment. 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 “GraphPad Instat” statistic computer program. A difference in the mean values of p < 0.05 was considered to be statistically significant. Inhibition percentages were calculated as following: Inhibition % =
Test value × 100 − 100 Control value
3.2. Hypoglycaemic effect of subextracts from EtOH extract of Joso leaves 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 subextracts were tested at two doses and as shown in Table 2, hexane subextract at 260 mg/kg was the most active one (31.4–39.5%) among all. Reference drug glipizide showed a remarkable and statistically significant antidiabetic activity (17.4–39.7) in comparison to Joso leaf subextracts. 3.3. Hypoglycaemic effect of fractions from hexane subextract n-Hexane subextract of Joso leaf ethanol extract was subjected to column chromatography for separation and eluents were combined into six fractions after TLC analysis. Fractions were given to diabetic rats in doses estimated from their ratio in hexane subextract (Table 3). Fraction F was found to possess a potent effect between 30.5 and 44.4% at 106.2 mg/kg dose. All other fractions were found to be inactive in lowering blood glucose levels of diabetic rats. Thus, further studies were conducted on hexane F fraction.
3. Results
3.4. Hypoglycaemic effect of subfractions from hexane F fraction
3.1. Hypoglycaemic effects of Joso leaf extracts
Hexane F fraction was subjected to column chromatography and eluents were combined into four main (F1–2, F3, F4–6, F7–8) subfractions according to TLC control. Fractions were given to diabetic rats in doses estimated from their yield percentages (Table 4). Subfractions F1–2 and F7–8 were found to possess antidiabetic activity at calculated doses. Thus, they were given to diabetic animals at higher doses too. F3 and F4–6 subfractions were found to have a mild antidiabetic effect and lowered blood glucose levels with different inhibition percentages. Antidiabetic effect of F1–2 and F7–8 subfractions were increased by administration of higher doses. Because of having a significant and continuous antidiabetic effect; constituents of F1–2 and F7–8 subfractions were evaluated by gas chromatography.
Decoction of the leaves of the plant is used for diabetes internally in Turkey, so in order to prove the claimed hypoglycaemic effect of Joso leaves, water and ethanol extracts were tested in STZ induced diabetic animals. Hypoglycaemic effect of ethanol extracts was also investigated in normoglycaemic and glucose-hyperglycaemic 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 leaves showed moderate hypoglycaemic effect in 1st, 2nd and 4th hour measurements at both tested doses (6.3–16.9%) in STZ-diabetic rats. Antihyperglycaemic effect of ethanol extract was significant and continuous (10.0–17.9%) at 1000 mg/kg dose in STZ-diabetic rats. In normoglycaemic rats, ethanol extract showed an outstanding hypoglycaemic effect at both doses, 1–4 h after the administration. In oral glucose tolerance test, glucose solution was loaded to normal rats just after 30th minute measurement. The ethanol extract, given in 1000 mg/kg, was found more effective (3.2–11.7%) than the other dose (in Table 1 control and reference data were not given for clarification).
3.5. Results of GC-FID and GC–MS analysis Name of the compounds and their relative percentage amounts found in n-hexane subextract and its antidiabetic subfractions F1–2, F7–8 are given in Table 5. Twenty four different compounds (terpenoids, fatty acids and other hydrocarbons) were found in
412
Table 2 Hypoglycaemic effect of subextracts from EtOH extract of Joso leaves in STZ-diabetic rats. Dose (mg/kg)
Control Glipizide
– 10 130 260 130 260 370 740 830 1660 530 1060
Hexane subextract CHCl3 subextract EtOAc subextract n-BuOH subextract R-Water subextract * ***
Blood glucose concentration (mg/dl) ± standard error of the mean (inhibition %) Initial
1/2 h
1h
2h
4h
6h
380.0 ± 26.9 380.0 ± 14.8 378.7 ± 12.0 384.2 ± 20.4 380.0 ± 29.0 382.0 ± 36.1 380.0 ± 8.0 381.0 ± 46.5 378.5 ± 23.5 382.0 ± 26.4 380.0 ± 26.1 380.0 ± 28.6
405.5 ± 24.5 387.7 ± 12.6 (4.4%) 403.3 ± 17.3 (0.5%) 413.0 ± 31.3 412.2 ± 15.3 395.0 ± 41.0 (2.6%) 414.9 ± 14.6 375.5 ± 56.5 (7.4%) 389.0 ± 25.4 (4.1%) 410.0 ± 22.9 406.2 ± 19.5 420.5 ± 23.0
395.5 ± 18.6 373.7 ± 17.4 (5.5%) 359.7 ± 15.9 (9.1%) 379.7 ± 24.3 (4.0%) 390.9 ± 15.3 (1.2%) 383.2 ± 33.9 (3.1%) 391.2 ± 19.6 (1.1%) 352.4 ± 59.3 (10.9%) 384.5 ± 22.7 (2.8%) 388.0 ± 22.3 (1.9%) 396.3 ± 24.2 351.2 ± 21.9 (11.2%)
390.7 ± 10.8 322.7 ± 18.0* (17.4%) 359.7 ± 15.9 (7.9%) 347.3 ± 13.3 (11.1%) 371.4 ± 17.2 (4.9%) 371.9 ± 19.2 (4.8%) 354.3 ± 14.7 (9.3%) 343.8 ± 12.5 (12.0%) 358.3 ± 26.2 (8.3%) 366.9 ± 23.1 (6.1%) 366.3 ± 24.8 (6.3%) 348.1 ± 38.1 (10.9%)
375.0 ± 21.7 226.1 ± 15.5*** (39.7%) 331.0 ± 17.8 (11.7%) 257.3 ± 16.0*** (31.4%) 364.8 ± 15.3 (2.7%) 388.8 ± 17.8 349.3 ± 12.7 (6.9%) 352.1 ± 57.9 (6.1%) 340.0 ± 34.7* (9.3%) 343.5 ± 33.9 (8.4%) 360.3 ± 24.2 (4.0%) 337.1 ± 27.4* (10.1%)
357.4 ± 16.3 224.1 ± 18.1*** (37.3%) 321.7 ± 10.7 (10.0%) 216.2 ± 16.4*** (39.5%) 354.7 ± 7.2 (0.8%) 384.6 ± 30.5 342.5 ± 17.5 (4.2%) 333.1 ± 56.4 (6.8%) 333.5 ± 35.2 (6.7%) 397.7 ± 47.7 (16.7%) 358.7 ± 23.3 313.8 ± 12.6 (12.2%)
p < 0.05. p < 0.001.
Table 3 Hypoglycaemic effect of fractions from hexane subextract obtained by column chromatography on STZ-induced diabetic rats. Group
Dose (mg/kg)
Control A (1–9) B (10–13) C (14–29) D (30–65) E (66–105)
– 101.7 27 126 85.1 74.0 106.2 212.4
Blood glucose concentration (mg/dl) ± standard error of the mean (inhibition %) Initial
F (106–113) * ** ***
p < 0.05. p < 0.01. p < 0.001.
303.6 290.2 303.6 282.6 304.6 297.8 303.6 306.6
1/2 h ± ± ± ± ± ± ± ±
20.9 14.3 53.3 35.8 32.0 36.3 26.8 37.5
305.8 322.8 320.5 309.6 324.2 299.4 246.6 305.8
1h ± ± ± ± ± ± ± ±
18.4 13.9 53.2 34.5 36.8 28.4(2.1%) 14.1 (19.4%) 12.5 (4.2%)
329.6 320.8 319.0 304.8 316.6 287.2 238.2 297.3
2h ± ± ± ± ± ± ± ±
19.1 19.7 (2.7%) 40.3 (3.2%) 34.9 (7.5%) 32.2 (3.9%) 29.7 (12.9%) 18.6 (27.7%) 21.0 (9.8%)
282.6 299.4 299.9 282.6 299.8 273.2 196.3 245.9
4h ± ± ± ± ± ± ± ±
17.9 20.6 47.6 31.2 32.3 28.3 (3.3%) 12.6* (30.5%) 19.6 (13.0%)
262.6 293.6 289.3 276.6 280.4 262.8 145.9 239.8
6h ± ± ± ± ± ± ± ±
17.3 1.9 46.1 28.2 30.9 39.2 10.1** (44.4%) 25.8 (8.7%)
224.6 274.2 281.6 259.0 257.8 245.2 127.3 207.8
± ± ± ± ± ± ± ±
15.1 25.1 46.0 31.8 42.3 42.6 15.4*** (43.3%) 11.5 (7.5%)
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Group
N. Orhan et al. / Journal of Ethnopharmacology 140 (2012) 409–415
413
243.0 192.2 163.4 150.7 230.6 226.2 202.2 165.2
4. Discussion
282.4 228.2 219.4 177.6 268.6 247.4 222.4 201.6 354.8 321.5 291.0 275.7 307.6 285.0 279.4 280.7
2h
7.4 7.2 (6.0%) 5.4 (8.5%) 4.3** (11.4%) 6.0 (1.3%) 14.5** (11.3%) 8.6 (% 4.8) 5.5*** (% 20.4) ± ± ± ± ± ± ± ± 385.0 361.9 352.4 341.1 380.0 341.4 366.6 306.5
1h
5.3 5.8 (3.3%) 3.7*** (11.1%) 2.3*** (17.7%) 3.9 (5.9%) 6.3*** (16.1%) 6.2*** (% 15.9) 10.4*** (% 15.8) ± ± ± ± ± ± ± ± 424.0 410.0 376.8 349.0 398.8 355.8 356.4 357.0
1/2 h
7.4 7.1 5.4 10.7 5.7 6.9 2.6 7.0 ± ± ± ± ± ± ± ±
***
p < 0.05. p < 0.01. p < 0.001 *
**
F7–8 (43–58)
F1–2 (1–9)
F3 (10–19) F4–6 (20–42)
Initial
382.8 375.8 373.4 351.0 389.2 372.8 379.6 356.8 – 10 13 26 28 106 45 90 Control Glipizide
Blood glucose concentration (mg/dl) ± standard error of the mean (inhibition %) Dose (mg/kg) Group
Table 4 Hypoglycaemic effect of subfractions from F fraction of hexane subextract obtained by column chromatography on STZ-induced diabetic rats.
± ± ± ± ± ± ± ±
1.7 7.1* (9.4%) 7.5*** (18.0%) 5.7*** (22.3%) 6.6*** (13.3%) 9.1*** (19.7%) 8.4*** (% 21.3) 4.4*** (% 20.9)
4h
± ± ± ± ± ± ± ±
9.8 5.2*** (19.2%) 8.8*** (22.3%) 9.5*** (34.8%) 7.8 (4.9%) 8.6 (12.4%) 6.6*** (% 21.3) 5.5*** (% 28.6)
6h
± ± ± ± ± ± ± ±
7.8 3.6** (20.9%) 9.8*** (32.8%) 6.8*** (38.0%) 10.2 (5.1%) 9.0 (6.9%) 11.2* (% 16.8) 6.1*** (% 32.0)
n-hexane subextract. Fatty acids and their derivatives were the major compounds in F1–2 and F7–8 subfractions. None of the terpenoids found in n-hexane subextract were encountered in subextracts.
Since olden days, plants are used to treat many ailments, for instance several species of medicinal plants are used in the treatment of diabetes mellitus, a disease affecting large number of people worldwide. Ethnobotanical studies in Turkey revealed that many plant remedies are used to alleviate the symptoms of diabetes and hyperglycaemic condition. As mentioned before, decoction of Juniperus oxycedrus subsp. oxycedrus (Joso) leaves are used internally in E˘girdir, Isparta (Tuzlacı and Erol, 1993). In literature survey, it has been reported that the fruit extracts from different species of juniper have possessed hypoglycaemic effect (Swanston-Flatt et al., 1990; Sanchez de Medina et al., 1994; Loizzo et al., 2007; Ju et al., 2008). In our previous study on subacute effects of Joso berry and leaf extracts, antidiabetic activity was investigated in diabetic animals after 10 days administration. This study pointed out treatment of diabetic rats with Joso fruit and leaf crude extracts decreased blood glucose levels and lipid peroxidation in tissues remarkably (Orhan et al., 2011). The active constituents of Joso berries were isolated as shikimic acid, 4-O--D-glucopyranosyl ferulic acid and oleuropeic acid-8-O--D-glucopyranoside and their mechanism of action were also discussed in our recent study (Orhan et al., 2012). Since, subfractions of hexane subextract of Joso leaves were found to possess significant antidiabetic activity on diabetic rats, GC–MS analysis were done to enlighten the chemical composition of this non-polar subfractions. Methyl linoleate (32.26%), methyl palmitate (25.84%), methyl linolenat (13.79%) were found as the major compounds in F1–2 subfraction. Chemical composition of F7–8 subfraction was similar to F1–2 subfraction and fatty acids (palmitic acid 66.32%; linolenic acid-11.21%) were the most abundant compounds. It is known that arachidonic, docosanoic, eneicosanoic, capric, lauric, linoleic, linolenic, miristic, oleic, palmitic, palmitoleic, stearic acid and their hydroxylated derivatives exist in Juniperus species. According to Seca and Silva, arachidic, capric, lauric, linoleic, linolenic, miristic, oleic, palmitic and stearic acid exists in Juniperus oxycedrus (Seca and Silva, 2006). Hence, occurrence of fatty acids in our active fractions is not a strange finding and it is supported by previous studies. Many studies have been found evaluating the effect of lipids; free fatty acids, unsaturated fatty acids and polyunsaturated fatty acids on progression of diabetes, blood glucose concentrations, insulin levels and antioxidant parameters in tissues of diabetics (Garg, 1998; Mohan and Das, 2001; Sirtori and Galli, 2002; Suresh and Das, 2003; Shah et al., 2007). Among these, a meta-analysis of various studies comparing lowsaturated-fat, high-carbohydrate diets or high-monounsaturatedfat diet therapies in patients with type 2 diabetes revealed that high-monounsaturated-fat diets improve lipoprotein profiles as well as glycemic control. The improvement in the glycemic profile with high-monounsaturated-fat diets may not be related to changes in insulin sensitivity but to a reduction in the carbohydrate load, which patients with type 2 diabetes may not be able to handle readily because of severe insulin resistance and  cell defects (Garg, 1998). In another study, it is reported that oral supplementation with oils rich in -3 eicosapentaenoic acid and docosahexaenoic acid and -6 ␥-linolenic acid and arachidonic acid could protect Wistar albino rats against alloxan-induced diabetes mellitus (Mohan
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Table 5 Results of GC-FID and GC–MS analysis of n-hexane subextract of Joso leaf ethanol extract and its antidiabetic subfractions F1–2, F7–8. Name of the compound
Hexane subextract (+)/(−)
Tetradecane (E, Z)-2,4-Heptadienal Hexadecane Trans-verbenol Verbenone Germacrene-D ␦-Cadinene ␥-Cadinene Tetradecanal Caryophyllene oxide Humulene epoxide-II Hexahydrofarnesyle acetone ␣-Cedrol Methyl hexadecanoate Ethyl hexadecanoate Sandarocopimaradiene Unknown I Unknown II (M+ 142) Manoyl oxide Dihydroactinidiolide Methy octadecanoate Methyl oleate Dodecanoic acid Methyl linoleate Abietatriene Methyl linolenate Phytol Tetradecanoic acid Nonacosane Hexadecanoic acid Unknown III (Diterpene M+ 284) Hentriacontane Octadecanoic acid Oleic acid Linoleic acid Linolenic acid Identified %
− − − + + + + + + + + + + − + + + − + + − − + − + − + + + + + + − + + +
RRI
Subfraction F1–2 % RPA
1683 1725 1726 1773 1776 1933 2008 2071 2131 2143
3.1 1.7 1.7 1.9 1.7 1.3 5.1 2.4 0.4 2.1
2262 2357 2273
0.2 1.2 3.0
2376 2380
14.7 2.6
2503
1.4
2524
2.4
2622 2670 2900 2931 3010 3100
11.1 3.1 1.2 11.3 12.5 1.5
3200 3290 3300
1.8 0.8 6.9
Subfraction F7–8
(+)/(−)
RRI
+ − + − − − − − − − − − − + − − + − − − + + − + − + − − − + − − − − − −
1400
4.3
1600
6.0
2226
25.8
49.69
% RPA
1.49
2431 2456
1.6 7.8
2509
32.3
2583
13.8
2931
4.5
81.6
(+)/(−) − + − − − − − − − − − − − − − − − + − − − − − − − − − − − + − − + + + +
96.1
RRI
% RPA
1479
1.7
2320
9.79
2931
66.3
3151 3200 3290 3300
3.6 2.5 4.8 11.2 90.1
(+) Present; (−) absent; RRI: relative retention indices calculated against n-alkanes; RPA: relative percentage area, % calculated from FID data.
and Das, 2001). Similar results were evaluated by Rodriguez et al. High fat diets, the fat in which consisted primarily of saturated fatty acids, had a markedly protective action against the toxic and diabetogenic effects of alloxan as compared with a high carbohydrate diet on rats (Rodriguez et al., 1953). On the other hand, there are many studies on the effect of linoleic acid on the progression of diabetes. According to Ide, Kushiro and Takahashi, dietary ␥-linolenic acid increased glucose metabolism in response to insulin stimuli in isolated rat adipocytes (Ide et al., 2001). Ghafoorunissa et al. was studied on the effect of substituting dietary linoleic acid (18:2 n-6) with ␣-linolenic acid on sucroseinduced insulin resistance. This study showed that the substitution of one-third of dietary 18:2 n-6 with 18:2 n-3 results in lowered blood lipid levels and increases peripheral insulin sensitivity (Ghafoorunissa et al., 2005). Additionally Zhou et al. found that dietary conjugated linoleic acid increases peroxisomal proliferatoractivated receptor-␥ (PPAR-␥) gene expression in adipose tissue of obese rat, and improves insulin resistance (Zhou et al., 2008).
antihyperglycaemic effect. Therefore, in the present study three different experimental models have been used together. As shown in Table 1, Joso leaf ethanol extract has possessed hypoglycaemic activity on normoglycaemic (healthy) rats. This effect can be explained by the direct effect on insulin secretion or insulin like activity of the extract. Moreover, the extract and its fractions has shown long term inhibitory effect on blood glucose levels of diabetic rats. Thus, antidiabetic effect of Joso leaves might be related to both direct and indirect mechanisms of glucose homeostasis. As mentioned before, fatty acids might increase glucose metabolism in response to insulin stimulation and also peripheral insulin sensitivity by increasing peroxisomal proliferator-activated receptor-␥ gene expression. Active subfractions of Joso leaf extracts rich in unsaturated fatty acids, might have shown their antidiabetic activity by rising sensitivity of PPAR-␥ reseptors or increasing the release of insulin from beta cells of pancreas. So Joso leaf extract and its fractions support the amelioration of diabetes and its complications significantly. In order to reveal mechanism of action, further in vivo and in vitro studies must be performed on fatty acids which are the major constituents of active Joso leaf subfractions.
5. Conclusions Acknowledgement In the in-vivo antidiabetic activity studies three general models are used to determine whether the plant extract has antidiabetic activity or not. Normoglycaemic rats are used to reveal hypoglycaemic effect, glucose loaded rats for effect on glucose absorption from the intestines and STZ-diabetic rats to evaluate
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).
N. Orhan et al. / Journal of Ethnopharmacology 140 (2012) 409–415
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Journal of Ethnopharmacology journal homepage: www.elsevier.com/locate/jethpharm
A bioactivity guided study on the antidiabetic activity of Juniperus oxycedrus subsp. oxycedrus L. leaves Nilüfer Orhan a,∗ , Mustafa Aslan a , Betül Demirci b , Fatma Ergun a a b
Gazi University, Faculty of Pharmacy, Department of Pharmacognosy, 06330, Etiler Ankara, Turkey Anadolu University, Faculty of Pharmacy, Department of Pharmacognosy, Eskis¸ehir, Turkey
a r t i c l e
i n f o
Article history: Received 17 November 2011 Received in revised form 17 January 2012 Accepted 24 January 2012 Available online 2 February 2012 Keywords: Cupressaceae Diabetes mellitus Hypoglycaemic Juniperus oxycedrus ssp. oxycedrus Linoleic acid Linolenic acid Palmitic acid
a b s t r a c t Ethnopharmacological relevance: Juniperus (Cupressaceae) species are widely used as folk medicine in spreading countries. Decoction of Juniperus oxycedrus subsp. oxycedrus L. leaves is used internally to lower blood glucose levels in Turkey. Aim of the study: To determine hypoglycaemic and antidiabetic activities of Juniperus oxycedrus subsp. oxycedrus leaves and to identify active compounds through bioactivity guided isolation technique. Materials and methods: Ethanol and water extracts of Juniperus oxycedrus subsp. oxycedrus (Joso), leaves on oral administration were studied using in vivo models in normal, glucose-hyperglycemic and streptozotocin-induced diabetic rats. Through in vivo bioactivity-guided fractionation processes, a nonpolar fraction was separated from the n-hexane subextract by silica gel column chromatography as the main active fraction. Subfractions of this fraction was found to possess antidiabetic activity and their chemical composition was investigated by GC-FID and GC–MS, simultaneously. Results: This is the first report on the antidiabetic constituents of Joso leaves. Fatty acids, such as palmitic, linoleic and linolenic acid were found as the major compounds in subfractions. Conclusion: Results indicated that Joso leaf extract and its active constituents might be beneficial for diabetes mellitus. © 2012 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Historical accounts of diabetes mellitus first appeared in the medical texts of several ancient cultures over 2000 years ago. In addition, recorded treatments for diabetes included largely dietand plant-based remedies (Cheng, 2000). There is, only one example of an approved antidiabetic drug that was developed from a herb with a long history of use for diabetes: metformin from French lilac (Galega officinalis) (Marles and Farnsworth, 1995). Numerous other herbs remain candidates for antidiabetic drug development and clinical data are beginning to emerge, which support antidiabetic indications for several of these herbs. This viewpoint outlines the opportunity that exists for these herbs in the management of diabetes and the state of the evidence for their clinical antidiabetic efficacy (Vuksan and Sievenpiper, 2005). Juniperus is one of the most diverse genera of conifers belonging to the Cupressaceae family (Cypress family). It is widely distributed throughout the northern hemisphere from the Arctic Circle to the mountains of the African tropics, from sea level to above timberline (Adams, 2011). 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). Although some species have toxic effects, juniper plants have many uses in folk medicine in several parts of the world (Seca and Silva, 2006). 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, diabetes mellitus, kidney inflammation and to pass kidney stone (Tuzlacı and Erol, 1993; Yes¸ilada et al., 1995; Honda et al., 1996). Additionally decoction of Juniperus oxycedrus subsp. oxycedrus (Joso) leaves is used internally in E˘girdir and Isparta for diabetes mellitus (Tuzlacı and Erol, 1993). The present study aimed to evaluate hypoglycaemic activity of Joso leaves in normoglycaemic, glucose-hyperglycaemic and streptozotocin-induced diabetic rats and to determine its active constituents through bioactivity guided fractionation and isolation techniques. 2. Materials and methods 2.1. Plant material
∗ Corresponding author. Tel.: +90 312 2023176; fax: +90 312 2235018. E-mail addresses: [email protected], [email protected] (N. Orhan). 0378-8741/$ – see front matter © 2012 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2012.01.042
Juniperus oxycedrus L. ssp. oxycedrus L. (Joso) leaves were collected in July 2007 from Karapiri province of Akda˘gmadeni, Yozgat
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(Turkey). The plant was identified by N. Orhan and a voucher specimen (GUEF 2616) is stored in the Herbarium of Gazi University, Faculty of Pharmacy. 2.2. Extraction and fractionation of material 2.2.1. Preparation of water-extract Chopped dried leaves (1000 g) were extracted with hot distilled water (50 ◦ C, 20 l) for 3 h with continuous stirring. The extract was filtered and evaporated under reduced pressure at 40 ◦ C and then lyophilized (Water-Extract, yield 17.0%). 2.2.2. Preparation and fractionation of EtOH extract The chopped dried leaves (1000 g) were extracted with ethanol 80% (10 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 extract under reduced pressure (EtOH-Extract, yield 35.0%). Dried ethanol extract (300 g) was dissolved in 1000 ml methanol/water (7:3) mixture and extracted with n-hexane in a separatory funnel (20 × 500 ml). Hexane phases were collected and dried to give hexane subextract (yield 6.5%). Polar phase was distillated to evaporate methanol and then fractionated successively with chloroform (8 × 500 ml), ethyl acetate (20 × 500 ml), n-butanol/saturated with water (14 × 500 ml) to obtain subextracts. Each subextract was concentrated to dryness under reduced pressure on a rotary evaporator to give (CHCI3 -subextract, yield 6.4%), (EtOAc-subextract, yield 18.6%), (n-BuOH-subextract, yield 41.6%) and the remaining water subextract (R-Water subextract, yield 26.5%), respectively. All the obtained subextracts were used in animal experiments at the doses calculated according to their yields. The hexane subextract was also used in phytochemical studies. 2.2.3. Chromatographic studies and isolation of active constituent 10 g of hexane subextract was chromatographed on silica gel column (350 g Kieselgel 60, 063–0.2 mm, Merck, Art. No.7734) and eluted with hexane, mixtures of Hex:EtOAc (90:10 → 30:70) and CHCl3 :MeOH (50:50) successively. Eluents were combined into six subfractions A (fr.1–9: 18.71%), B (fr.10–13: 4.96%), C (fr.14–29: 23.19%), D (fr.30–65: 15.67%), E (fr.66–105: 13.61%), F (fr.106–113: 19.55%) according to TLC behavior using solvent system Hex:EtOAc (6:4) [spots were visualized under daylight, UV-light and after spraying 5% H2 SO4 or anisaldehyde-sulfuric acid reagent]. After bioactivity studies hexane F fraction (1 g) was subjected to silica gel (30 g) column and eluted with mixtures of CHCl3 :MeOH (90:10 → 25:75) and methanol to afford 58 fractions. Eluents were combined to obtain four subfractions [F1–2 (fr.1–9: 6.20%), F3 (fr.10–19: 13.36%), F4–6 (fr.20–42: 49.93%), F7–8 (fr.43–58: 21.25%)]. After studies on antidiabetic activity of subfractions, hexane subextact and subfractions F1–2 and F7–8, possessed potent activity, were applied to GC–MS to identify their contents. 2.2.4. GC-FID and GC–MS analysis of active subextract and subfractions Dried hexane subextract was dissolved in n-hexane, subfractions F1–2 and F7–8 were dissolved in n-hexane–methanol mixture and filtered from Millipore filter before the injection. Samples were analyzed subsequently by capillary GC-FID and GC–MS. The individual components characterized are given results section. The GC–MS analysis was carried out with an Agilent 5975 GCMSD system. Innowax FSC column (60 m × 0.25 mm, 0.25 m film thickness) was used with helium as a carrier gas (0.8 ml/min). GC oven temperature was kept at 60 ◦ C for 10 min and programmed to 220 ◦ C at a rate of 4 ◦ C/min, and kept constant at 220 ◦ C for 10 min and then programmed to 240 ◦ C at a rate of 1 ◦ C/min. Split ratio was adjusted at 40:1. The injector temperature was set at 250 ◦ C.
Mass spectra were recorded at 70 eV. Mass range was from m/z 35 to 450. The GC analysis was carried out using an Agilent 6890N GC system. FID detector temperature was 300 ◦ C. To obtain the same elution order with GC–MS, simultaneous auto-injection was done on a duplicate of the same column applying the same operational conditions. Relative percentage amounts of the separated compounds were calculated from FID chromatograms. Identification of the components were carried out by comparison of their relative retention times with those of authentic samples or by comparison of their relative retention index (RRI) to series of n-alkanes. Computer matching against commercial (Wiley GC/MS Library, MassFinder 3 Library) and in-house “Bas¸er Library of Essential Oil Constituents” built up by genuine compounds and components of known oils, as well as MS literature data was used for the identification (McLafferty and Stauffer, 1989; Joulain and Koenig, 1998; ESO 2000, 1999; Koenig et al., 2004). 2.3. Assay for hypoglycemic activity 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, b.w.) was used as the reference drug. Glipizide was purchased from Sigma (G1171g, St. Louis, MO 63103, USA). Animals in the control group received only the vehicle 0.5% aqueous carboxymethylcellulose (10 ml/kg, b.w.). 2.3.2. Animals Male Wistar-albino rats (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 1 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 (for 8 h) with free access to water. Control group of animals were received 0.5% CMC. 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. 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 (for 8 h) 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 levels 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 STZ-induced diabetic animals 2.4.3.1. Induction of diabetes. Experimental diabetes was induced by intraperitoneal (i.p.) injection of streptozotocin (STZ) at a dose
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Table 1 Hypoglycaemic effect of Water and EtOH-extracts of Juniperus oxycedrus ssp. oxycedrus leaves. Group
Dose (mg/kg)
Test model
Inhibition % 1/2 h
1h
2h
4h
6h
Water extract
500 1000
Diabetic Diabetic Normoglycaemic OGTT Diabetic Normoglycaemic OGTT Diabetic
0.2 10.9* 0.8 0.9 1.7 x 3.2 10.0***
16.9*** 16.3*** 12.2** 2.9 5.2* 10.9** 11.7*** 13.0***
8.4*** 12.2*** 14.2* 5.2 7.3* 11.6* 7.8 15.5***
9.3*** 6.3** 25.9*** 7.8 1.1 21.9*** 10.7* 18.0***
6.4 1.9 – – 0.7 – – 17.9***
500 EtOH extract 1000
OGTT: oral glucose tolerance test; (x) not measured; (–) no effect. * p < 0.05. ** p < 0.01. *** p < 0.001
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 activity on acute administration. Diabetic animals were fasted for 8 h (water ad libitum). Test samples were given orally using oral gastric gavages. The blood glucose levels were measured in all animals at the beginning of the study and the measurements were repeated 1/2, 1, 2, 4, and 6 h after the initial of the experiment. 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 “GraphPad Instat” statistic computer program. A difference in the mean values of p < 0.05 was considered to be statistically significant. Inhibition percentages were calculated as following: Inhibition % =
Test value × 100 − 100 Control value
3.2. Hypoglycaemic effect of subextracts from EtOH extract of Joso leaves 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 subextracts were tested at two doses and as shown in Table 2, hexane subextract at 260 mg/kg was the most active one (31.4–39.5%) among all. Reference drug glipizide showed a remarkable and statistically significant antidiabetic activity (17.4–39.7) in comparison to Joso leaf subextracts. 3.3. Hypoglycaemic effect of fractions from hexane subextract n-Hexane subextract of Joso leaf ethanol extract was subjected to column chromatography for separation and eluents were combined into six fractions after TLC analysis. Fractions were given to diabetic rats in doses estimated from their ratio in hexane subextract (Table 3). Fraction F was found to possess a potent effect between 30.5 and 44.4% at 106.2 mg/kg dose. All other fractions were found to be inactive in lowering blood glucose levels of diabetic rats. Thus, further studies were conducted on hexane F fraction.
3. Results
3.4. Hypoglycaemic effect of subfractions from hexane F fraction
3.1. Hypoglycaemic effects of Joso leaf extracts
Hexane F fraction was subjected to column chromatography and eluents were combined into four main (F1–2, F3, F4–6, F7–8) subfractions according to TLC control. Fractions were given to diabetic rats in doses estimated from their yield percentages (Table 4). Subfractions F1–2 and F7–8 were found to possess antidiabetic activity at calculated doses. Thus, they were given to diabetic animals at higher doses too. F3 and F4–6 subfractions were found to have a mild antidiabetic effect and lowered blood glucose levels with different inhibition percentages. Antidiabetic effect of F1–2 and F7–8 subfractions were increased by administration of higher doses. Because of having a significant and continuous antidiabetic effect; constituents of F1–2 and F7–8 subfractions were evaluated by gas chromatography.
Decoction of the leaves of the plant is used for diabetes internally in Turkey, so in order to prove the claimed hypoglycaemic effect of Joso leaves, water and ethanol extracts were tested in STZ induced diabetic animals. Hypoglycaemic effect of ethanol extracts was also investigated in normoglycaemic and glucose-hyperglycaemic 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 leaves showed moderate hypoglycaemic effect in 1st, 2nd and 4th hour measurements at both tested doses (6.3–16.9%) in STZ-diabetic rats. Antihyperglycaemic effect of ethanol extract was significant and continuous (10.0–17.9%) at 1000 mg/kg dose in STZ-diabetic rats. In normoglycaemic rats, ethanol extract showed an outstanding hypoglycaemic effect at both doses, 1–4 h after the administration. In oral glucose tolerance test, glucose solution was loaded to normal rats just after 30th minute measurement. The ethanol extract, given in 1000 mg/kg, was found more effective (3.2–11.7%) than the other dose (in Table 1 control and reference data were not given for clarification).
3.5. Results of GC-FID and GC–MS analysis Name of the compounds and their relative percentage amounts found in n-hexane subextract and its antidiabetic subfractions F1–2, F7–8 are given in Table 5. Twenty four different compounds (terpenoids, fatty acids and other hydrocarbons) were found in
412
Table 2 Hypoglycaemic effect of subextracts from EtOH extract of Joso leaves in STZ-diabetic rats. Dose (mg/kg)
Control Glipizide
– 10 130 260 130 260 370 740 830 1660 530 1060
Hexane subextract CHCl3 subextract EtOAc subextract n-BuOH subextract R-Water subextract * ***
Blood glucose concentration (mg/dl) ± standard error of the mean (inhibition %) Initial
1/2 h
1h
2h
4h
6h
380.0 ± 26.9 380.0 ± 14.8 378.7 ± 12.0 384.2 ± 20.4 380.0 ± 29.0 382.0 ± 36.1 380.0 ± 8.0 381.0 ± 46.5 378.5 ± 23.5 382.0 ± 26.4 380.0 ± 26.1 380.0 ± 28.6
405.5 ± 24.5 387.7 ± 12.6 (4.4%) 403.3 ± 17.3 (0.5%) 413.0 ± 31.3 412.2 ± 15.3 395.0 ± 41.0 (2.6%) 414.9 ± 14.6 375.5 ± 56.5 (7.4%) 389.0 ± 25.4 (4.1%) 410.0 ± 22.9 406.2 ± 19.5 420.5 ± 23.0
395.5 ± 18.6 373.7 ± 17.4 (5.5%) 359.7 ± 15.9 (9.1%) 379.7 ± 24.3 (4.0%) 390.9 ± 15.3 (1.2%) 383.2 ± 33.9 (3.1%) 391.2 ± 19.6 (1.1%) 352.4 ± 59.3 (10.9%) 384.5 ± 22.7 (2.8%) 388.0 ± 22.3 (1.9%) 396.3 ± 24.2 351.2 ± 21.9 (11.2%)
390.7 ± 10.8 322.7 ± 18.0* (17.4%) 359.7 ± 15.9 (7.9%) 347.3 ± 13.3 (11.1%) 371.4 ± 17.2 (4.9%) 371.9 ± 19.2 (4.8%) 354.3 ± 14.7 (9.3%) 343.8 ± 12.5 (12.0%) 358.3 ± 26.2 (8.3%) 366.9 ± 23.1 (6.1%) 366.3 ± 24.8 (6.3%) 348.1 ± 38.1 (10.9%)
375.0 ± 21.7 226.1 ± 15.5*** (39.7%) 331.0 ± 17.8 (11.7%) 257.3 ± 16.0*** (31.4%) 364.8 ± 15.3 (2.7%) 388.8 ± 17.8 349.3 ± 12.7 (6.9%) 352.1 ± 57.9 (6.1%) 340.0 ± 34.7* (9.3%) 343.5 ± 33.9 (8.4%) 360.3 ± 24.2 (4.0%) 337.1 ± 27.4* (10.1%)
357.4 ± 16.3 224.1 ± 18.1*** (37.3%) 321.7 ± 10.7 (10.0%) 216.2 ± 16.4*** (39.5%) 354.7 ± 7.2 (0.8%) 384.6 ± 30.5 342.5 ± 17.5 (4.2%) 333.1 ± 56.4 (6.8%) 333.5 ± 35.2 (6.7%) 397.7 ± 47.7 (16.7%) 358.7 ± 23.3 313.8 ± 12.6 (12.2%)
p < 0.05. p < 0.001.
Table 3 Hypoglycaemic effect of fractions from hexane subextract obtained by column chromatography on STZ-induced diabetic rats. Group
Dose (mg/kg)
Control A (1–9) B (10–13) C (14–29) D (30–65) E (66–105)
– 101.7 27 126 85.1 74.0 106.2 212.4
Blood glucose concentration (mg/dl) ± standard error of the mean (inhibition %) Initial
F (106–113) * ** ***
p < 0.05. p < 0.01. p < 0.001.
303.6 290.2 303.6 282.6 304.6 297.8 303.6 306.6
1/2 h ± ± ± ± ± ± ± ±
20.9 14.3 53.3 35.8 32.0 36.3 26.8 37.5
305.8 322.8 320.5 309.6 324.2 299.4 246.6 305.8
1h ± ± ± ± ± ± ± ±
18.4 13.9 53.2 34.5 36.8 28.4(2.1%) 14.1 (19.4%) 12.5 (4.2%)
329.6 320.8 319.0 304.8 316.6 287.2 238.2 297.3
2h ± ± ± ± ± ± ± ±
19.1 19.7 (2.7%) 40.3 (3.2%) 34.9 (7.5%) 32.2 (3.9%) 29.7 (12.9%) 18.6 (27.7%) 21.0 (9.8%)
282.6 299.4 299.9 282.6 299.8 273.2 196.3 245.9
4h ± ± ± ± ± ± ± ±
17.9 20.6 47.6 31.2 32.3 28.3 (3.3%) 12.6* (30.5%) 19.6 (13.0%)
262.6 293.6 289.3 276.6 280.4 262.8 145.9 239.8
6h ± ± ± ± ± ± ± ±
17.3 1.9 46.1 28.2 30.9 39.2 10.1** (44.4%) 25.8 (8.7%)
224.6 274.2 281.6 259.0 257.8 245.2 127.3 207.8
± ± ± ± ± ± ± ±
15.1 25.1 46.0 31.8 42.3 42.6 15.4*** (43.3%) 11.5 (7.5%)
N. Orhan et al. / Journal of Ethnopharmacology 140 (2012) 409–415
Group
N. Orhan et al. / Journal of Ethnopharmacology 140 (2012) 409–415
413
243.0 192.2 163.4 150.7 230.6 226.2 202.2 165.2
4. Discussion
282.4 228.2 219.4 177.6 268.6 247.4 222.4 201.6 354.8 321.5 291.0 275.7 307.6 285.0 279.4 280.7
2h
7.4 7.2 (6.0%) 5.4 (8.5%) 4.3** (11.4%) 6.0 (1.3%) 14.5** (11.3%) 8.6 (% 4.8) 5.5*** (% 20.4) ± ± ± ± ± ± ± ± 385.0 361.9 352.4 341.1 380.0 341.4 366.6 306.5
1h
5.3 5.8 (3.3%) 3.7*** (11.1%) 2.3*** (17.7%) 3.9 (5.9%) 6.3*** (16.1%) 6.2*** (% 15.9) 10.4*** (% 15.8) ± ± ± ± ± ± ± ± 424.0 410.0 376.8 349.0 398.8 355.8 356.4 357.0
1/2 h
7.4 7.1 5.4 10.7 5.7 6.9 2.6 7.0 ± ± ± ± ± ± ± ±
***
p < 0.05. p < 0.01. p < 0.001 *
**
F7–8 (43–58)
F1–2 (1–9)
F3 (10–19) F4–6 (20–42)
Initial
382.8 375.8 373.4 351.0 389.2 372.8 379.6 356.8 – 10 13 26 28 106 45 90 Control Glipizide
Blood glucose concentration (mg/dl) ± standard error of the mean (inhibition %) Dose (mg/kg) Group
Table 4 Hypoglycaemic effect of subfractions from F fraction of hexane subextract obtained by column chromatography on STZ-induced diabetic rats.
± ± ± ± ± ± ± ±
1.7 7.1* (9.4%) 7.5*** (18.0%) 5.7*** (22.3%) 6.6*** (13.3%) 9.1*** (19.7%) 8.4*** (% 21.3) 4.4*** (% 20.9)
4h
± ± ± ± ± ± ± ±
9.8 5.2*** (19.2%) 8.8*** (22.3%) 9.5*** (34.8%) 7.8 (4.9%) 8.6 (12.4%) 6.6*** (% 21.3) 5.5*** (% 28.6)
6h
± ± ± ± ± ± ± ±
7.8 3.6** (20.9%) 9.8*** (32.8%) 6.8*** (38.0%) 10.2 (5.1%) 9.0 (6.9%) 11.2* (% 16.8) 6.1*** (% 32.0)
n-hexane subextract. Fatty acids and their derivatives were the major compounds in F1–2 and F7–8 subfractions. None of the terpenoids found in n-hexane subextract were encountered in subextracts.
Since olden days, plants are used to treat many ailments, for instance several species of medicinal plants are used in the treatment of diabetes mellitus, a disease affecting large number of people worldwide. Ethnobotanical studies in Turkey revealed that many plant remedies are used to alleviate the symptoms of diabetes and hyperglycaemic condition. As mentioned before, decoction of Juniperus oxycedrus subsp. oxycedrus (Joso) leaves are used internally in E˘girdir, Isparta (Tuzlacı and Erol, 1993). In literature survey, it has been reported that the fruit extracts from different species of juniper have possessed hypoglycaemic effect (Swanston-Flatt et al., 1990; Sanchez de Medina et al., 1994; Loizzo et al., 2007; Ju et al., 2008). In our previous study on subacute effects of Joso berry and leaf extracts, antidiabetic activity was investigated in diabetic animals after 10 days administration. This study pointed out treatment of diabetic rats with Joso fruit and leaf crude extracts decreased blood glucose levels and lipid peroxidation in tissues remarkably (Orhan et al., 2011). The active constituents of Joso berries were isolated as shikimic acid, 4-O--D-glucopyranosyl ferulic acid and oleuropeic acid-8-O--D-glucopyranoside and their mechanism of action were also discussed in our recent study (Orhan et al., 2012). Since, subfractions of hexane subextract of Joso leaves were found to possess significant antidiabetic activity on diabetic rats, GC–MS analysis were done to enlighten the chemical composition of this non-polar subfractions. Methyl linoleate (32.26%), methyl palmitate (25.84%), methyl linolenat (13.79%) were found as the major compounds in F1–2 subfraction. Chemical composition of F7–8 subfraction was similar to F1–2 subfraction and fatty acids (palmitic acid 66.32%; linolenic acid-11.21%) were the most abundant compounds. It is known that arachidonic, docosanoic, eneicosanoic, capric, lauric, linoleic, linolenic, miristic, oleic, palmitic, palmitoleic, stearic acid and their hydroxylated derivatives exist in Juniperus species. According to Seca and Silva, arachidic, capric, lauric, linoleic, linolenic, miristic, oleic, palmitic and stearic acid exists in Juniperus oxycedrus (Seca and Silva, 2006). Hence, occurrence of fatty acids in our active fractions is not a strange finding and it is supported by previous studies. Many studies have been found evaluating the effect of lipids; free fatty acids, unsaturated fatty acids and polyunsaturated fatty acids on progression of diabetes, blood glucose concentrations, insulin levels and antioxidant parameters in tissues of diabetics (Garg, 1998; Mohan and Das, 2001; Sirtori and Galli, 2002; Suresh and Das, 2003; Shah et al., 2007). Among these, a meta-analysis of various studies comparing lowsaturated-fat, high-carbohydrate diets or high-monounsaturatedfat diet therapies in patients with type 2 diabetes revealed that high-monounsaturated-fat diets improve lipoprotein profiles as well as glycemic control. The improvement in the glycemic profile with high-monounsaturated-fat diets may not be related to changes in insulin sensitivity but to a reduction in the carbohydrate load, which patients with type 2 diabetes may not be able to handle readily because of severe insulin resistance and  cell defects (Garg, 1998). In another study, it is reported that oral supplementation with oils rich in -3 eicosapentaenoic acid and docosahexaenoic acid and -6 ␥-linolenic acid and arachidonic acid could protect Wistar albino rats against alloxan-induced diabetes mellitus (Mohan
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N. Orhan et al. / Journal of Ethnopharmacology 140 (2012) 409–415
Table 5 Results of GC-FID and GC–MS analysis of n-hexane subextract of Joso leaf ethanol extract and its antidiabetic subfractions F1–2, F7–8. Name of the compound
Hexane subextract (+)/(−)
Tetradecane (E, Z)-2,4-Heptadienal Hexadecane Trans-verbenol Verbenone Germacrene-D ␦-Cadinene ␥-Cadinene Tetradecanal Caryophyllene oxide Humulene epoxide-II Hexahydrofarnesyle acetone ␣-Cedrol Methyl hexadecanoate Ethyl hexadecanoate Sandarocopimaradiene Unknown I Unknown II (M+ 142) Manoyl oxide Dihydroactinidiolide Methy octadecanoate Methyl oleate Dodecanoic acid Methyl linoleate Abietatriene Methyl linolenate Phytol Tetradecanoic acid Nonacosane Hexadecanoic acid Unknown III (Diterpene M+ 284) Hentriacontane Octadecanoic acid Oleic acid Linoleic acid Linolenic acid Identified %
− − − + + + + + + + + + + − + + + − + + − − + − + − + + + + + + − + + +
RRI
Subfraction F1–2 % RPA
1683 1725 1726 1773 1776 1933 2008 2071 2131 2143
3.1 1.7 1.7 1.9 1.7 1.3 5.1 2.4 0.4 2.1
2262 2357 2273
0.2 1.2 3.0
2376 2380
14.7 2.6
2503
1.4
2524
2.4
2622 2670 2900 2931 3010 3100
11.1 3.1 1.2 11.3 12.5 1.5
3200 3290 3300
1.8 0.8 6.9
Subfraction F7–8
(+)/(−)
RRI
+ − + − − − − − − − − − − + − − + − − − + + − + − + − − − + − − − − − −
1400
4.3
1600
6.0
2226
25.8
49.69
% RPA
1.49
2431 2456
1.6 7.8
2509
32.3
2583
13.8
2931
4.5
81.6
(+)/(−) − + − − − − − − − − − − − − − − − + − − − − − − − − − − − + − − + + + +
96.1
RRI
% RPA
1479
1.7
2320
9.79
2931
66.3
3151 3200 3290 3300
3.6 2.5 4.8 11.2 90.1
(+) Present; (−) absent; RRI: relative retention indices calculated against n-alkanes; RPA: relative percentage area, % calculated from FID data.
and Das, 2001). Similar results were evaluated by Rodriguez et al. High fat diets, the fat in which consisted primarily of saturated fatty acids, had a markedly protective action against the toxic and diabetogenic effects of alloxan as compared with a high carbohydrate diet on rats (Rodriguez et al., 1953). On the other hand, there are many studies on the effect of linoleic acid on the progression of diabetes. According to Ide, Kushiro and Takahashi, dietary ␥-linolenic acid increased glucose metabolism in response to insulin stimuli in isolated rat adipocytes (Ide et al., 2001). Ghafoorunissa et al. was studied on the effect of substituting dietary linoleic acid (18:2 n-6) with ␣-linolenic acid on sucroseinduced insulin resistance. This study showed that the substitution of one-third of dietary 18:2 n-6 with 18:2 n-3 results in lowered blood lipid levels and increases peripheral insulin sensitivity (Ghafoorunissa et al., 2005). Additionally Zhou et al. found that dietary conjugated linoleic acid increases peroxisomal proliferatoractivated receptor-␥ (PPAR-␥) gene expression in adipose tissue of obese rat, and improves insulin resistance (Zhou et al., 2008).
antihyperglycaemic effect. Therefore, in the present study three different experimental models have been used together. As shown in Table 1, Joso leaf ethanol extract has possessed hypoglycaemic activity on normoglycaemic (healthy) rats. This effect can be explained by the direct effect on insulin secretion or insulin like activity of the extract. Moreover, the extract and its fractions has shown long term inhibitory effect on blood glucose levels of diabetic rats. Thus, antidiabetic effect of Joso leaves might be related to both direct and indirect mechanisms of glucose homeostasis. As mentioned before, fatty acids might increase glucose metabolism in response to insulin stimulation and also peripheral insulin sensitivity by increasing peroxisomal proliferator-activated receptor-␥ gene expression. Active subfractions of Joso leaf extracts rich in unsaturated fatty acids, might have shown their antidiabetic activity by rising sensitivity of PPAR-␥ reseptors or increasing the release of insulin from beta cells of pancreas. So Joso leaf extract and its fractions support the amelioration of diabetes and its complications significantly. In order to reveal mechanism of action, further in vivo and in vitro studies must be performed on fatty acids which are the major constituents of active Joso leaf subfractions.
5. Conclusions Acknowledgement In the in-vivo antidiabetic activity studies three general models are used to determine whether the plant extract has antidiabetic activity or not. Normoglycaemic rats are used to reveal hypoglycaemic effect, glucose loaded rats for effect on glucose absorption from the intestines and STZ-diabetic rats to evaluate
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).
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