Oleanolic acid was from Wako Pure Chemical. Industries, Ltd. (Osaka). Human pancreas -amylase was from. Elastin Products
Food Sci. Technol. Res., 9 (1), 35–39, 2003
Identification of Anti--Amylase Components from Olive Leaf Extracts Eriko KOMAKI,1 Shinya YAMAGUCHI,1 Isafumi MARU,1 Mitsuhiro KINOSHITA,2 Kazuaki KAKEHI,2 Yasuhiro OHTA1 and Yoji TSUKADA1 1
Marukin Bio, Inc., 27 Monnomae, Todo, Uji, Kyoto 611-0013, Japan School of Pharmaceutical Sciences, Kinki University, 3-4-1 Kowakae, Higashi-Osaka 577-8502, Japan
2
Received June 12, 2002; Accepted October 18, 2002 Olive (Olea europaea L.) is recognized as a folk medicine for diabetes in Europe. The inhibitory action of an ethanol extract of olive leaves (OEE) on the activities of human amylases was examined in vitro. OEE inhibited the activities of -amylases from human saliva and pancreas with IC50 values of 4.0 and 0.02 mg/ml, respectively. Two anti-amylase components were purified from a 50% ethanol soluble fraction of OEE using Sephadex LH-20 column chromatography. One was identified as luteolin-7-O- glucoside and the other as luteolin-4¢¢-O- glucoside. The 50% ethanol insoluble fraction of OEE was dissolved in 98% ethanol and fractionated using Cosmosil C18-OPN column chromatography. The anti--amylase component purified by this chromatography was identified as oleanolic acid. Both luteolin and oleanolic acid have an inhibitory effect on postprandial blood glucose increase in diabetic rats. Olive leaves suppressed the elevation of blood glucose after oral administration of starch in borderline volunteers (fasting blood glucose: 110–140 mg/dl), and thus they may be a useful food supplement for the prevention of diabetes. Keywords: olive leaf, luteolin-7-O--glucoside, luteolin-4¢-O--glucoside, oleanolic acid, anti--amylase
Olive (Olea europaea L.) belongs to the botanical order of Ligustrals, of the Oleaceae family, which includes the genera Jasminum, Phillyea, Ligustrum, Syringa, Fraxinus and Olea. Olive trees are cultivated predominantly for their fruit which is used as table olives and also processed into oil. The medical value of olive oil has been recognized from ancient times. Olive oil contains a variety of compounds, and the phenolic compounds in olive oil have been found to prevent oxidative stress (Visioli & Galli, 1998). Olive leaves also have been used as a medical herb to treat diabetic hyperglycemia, hypertension and infectious diseases (Jacotot, 1993), and they are especially widely recognized as a folk medicine for diabetes and hypertension in Europe. The hypoglycemic (Gonzalez et al., 1992), vasodilator (Zaezuelo et al., 1991) and antiallergic (Kohyama et al., 1997) activities of olive leaves have been studied. It has also been reported that some components in these leaves are useful in preventing oxidative stress (Tutour & Guedon, 1992), radical-scavenging (Visioli et al., 1998) and protecting low density lipoprotein from oxidation (Visioli & Galli, 1994). The antimicrobial activities of phenolic compounds in olive leaves have also been confirmed in vitro (Bisignano et al., 1999; Bisignano et al., 2001; Juven et al., 1972; Koutsoumanis et al., 1998; Tassou et al., 1991; Tassou & Nychas, 1995). A typical component of olive leaves is oleuropein, which belongs to the secoiridoid glucoside (Gariboldi et al., 1986). Although many other components, such as triterpenes, polyphenols, flavonoids (Tutour & Guedon, 1992, Pieroni et al., 1996), sugars, sugar alcohols (Romani, 1994), sterols and lipids have been detected, any components responsible for hypoglycemic and hypotensive activities are mostly unknown. E-mail:
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In this study, we detected in vitro anti--amylase activities in hot water extracts (OWE) and ethanol extracts (OEE) of olive leaves and isolated the anti--amylase components. Hypoglycemic effects of olive leaves and the anti--amylase components luteolin and oleanolic acid were observed in diabetic rats; hypoglycemic effects of olive leaves were also recognized in humans. Materials and Methods Materials Olive leaves (Olea europaea L. ) were obtained from Olive Park on Shodoshima island in Kagawa prefecture, Japan. Luteolin, luteolin-7-O--glucoside, luteolin-4¢-O--glucoside and oleuropein were purchased from Extrasynthese S.A. (Genay, France). Oleanolic acid was from Wako Pure Chemical Industries, Ltd. (Osaka). Human pancreas -amylase was from Elastin Products Company, Inc. (Owensville, MS). Human salivary -amylase was from Sigma Chemical Co. (St. Louis, MO). Preparation of olive hot water extract (OWE) Dried olive leaves (1 kg) were extracted in 10 l of boiling water for 1 h and filtered through a filter paper. The filtrate was concentrated to dryness in vacuo and used as an olive water extract (OWE 120 g). Preparation of olive ethanol extract (OEE) Dried olive leaves (1 kg) were extracted with 10 l of 98% ethanol for 16 h at room temperature. The extract was filtered off and concentrated to dryness in vacuo at 40˚C (OEE 100 g). Isolation of the anti--amylase components from olive leaves A scheme of the isolation of the anti--amylase components from OEE is shown in Fig. 1. OEE (20 g) was dissolved in 50% ethanol (200 ml) and centrifuged at 10,000g for 30 min. The supernatant was fractionated by Sephadex LH-20 (2.5100 cm) chromatography and eluted with a linear gradient of 0 (3500 ml) to 50% ethanol (3500 ml). The active fractions were pooled and concentrated in vacuo. The concentrates were put on a col-
36
Fig. 1. Schematic diagram of procedure for isolation of anti--amylase components from olive leaf.
umn of Sephadex LH-20 again and eluted with a linear gradient of 0 (3500 ml) to 50% ethanol (3500 ml). The active fractions were concentrated in vacuo and freeze-dried. The precipitate (10 g) obtained from OEE (see above) was dissolved in methanol (500 ml), put on a column of Cosmosil C18-OPN (510.5 cm) and eluted with 70, 90 and 100% methanol by stepwise elution. The active fraction (90% methanol eluent) was pooled and concentrated to dryness in vacuo. -Amylase inhibitory activity assay Activity of -amylases from human pancreas and saliva were measured according to the method of Kotaru et al. (1987). -Amylase was allowed to react with soluble starch as substrate at 37˚C for 30 min in the presence of the fractions from olive leaves. Residual starch was quantitated by iodo-starch reaction method. Analysis of sugar The neutral sugar content in the sample was measured by phenol-sulfuric acid method (Dubois et al., 1956). Quantitative analysis of sugar in the sample was carried out by HPLC (Honda et al., 1989) after hydrolyzing (2 N HCl, 80˚C, 2 h), followed by labeling with 1-phenyl-3-methyl-5-pyrazolone (PMP). HPLC analysis for phenolic compounds from OEE Phenolic compounds of OEE were analyzed by HPLC (Ficarra & Ficarra, 1991). The Cosmosil 5C18-AR column (6 mm i.d. 150 mm, Nacalai Tesque) was used and detected at UV 280 nm, using a linear gradient of solvent B (acetonitrile) from 0% to 50% (v/v) in solvent A (0.1% (w/v) phosphoric acid in water (pH 2.0)) at a flow rate of 1.0 ml per min. Infrared analysis The infrared spectra of the samples were obtained by an FTIR-8100M Fourier transform infrared spectrophotometer (SHIMADZU). Measurement was made in a KBr pellet. Mass spectrometry and NMR analysis GC-MS analysis was performed with a GCMS-QP5050A equipment (SHIMADZU), using a DB-5 column (0.25 mm i.d. 30 m, filter thickness 0.25 m) with helium as the carrier gas in the split less mode at a temperature programmed from 100˚C for 1 min to 200˚C at 15˚C/min, followed by raising to 320˚C at 10˚C/min and held for 20 min. The samples were previously trimethylsilylated. The apparatus was operated at 70 eV in the split less mode. The samples were also analyzed by MALDI-TOFMS, which was performed on a Voyager DE-PRO (PE Biosystems, Framingham, MA). A nitrogen laser was used to irradiate sam-
E. KOMAKI et al.
ples with ultraviolet light (337 nm), and an average of 100 shots was taken. The instrument was operated in linear operation using positive polarity. An accelerating voltage of 13 kV was used. A sample (0.5 l) was applied to a polished stainless steel target, to which was added a solution of 0.5 l of 2,5-dihydroxybenzoic acid in a mixture of methanol-water (1:1). The mixture was dried in the atmosphere by keeping it for several hours at room temperature. 1 H and 13C NMR spectra were measured on a JEOL JNM GX-500 spectrometer operated at 500 MHz in d6-DMSO solution. The chemical shift was expressed as -scale (ppm) using tetramethylsilane as internal standard. Effect of olive leaves on postprandial blood glucose in diabetic rats Five-week-old male rats (Type II diabetes, GK/Jcl, Clea Japan, Inc., Tokyo), weighing about 80 g, were housed in cages in an air-conditioned room (232˚C). The rats were fed freely, given water and a basal diet (CE-2 diet, Clea Japan, Inc.) for one week to accustom them to their surroundings. Before the experiment, the hyperglycemic rats were divided into four groups of 5 animals each, which received the following oral treatment: 1. Starch (2 g/kg) 2. Olive leaf powder (20 mg/kg)starch (2 g/kg) 3. Oleanolic acid (1 mg/kg)starch (2 g/kg) 4. Luteolin (0.1 mg/kg)starch (2 g/kg) After each compound was administered by oesophageal catheter, blood samples were taken from the tail using a disposable cutter, and glucose levels of each sample were measured by a commercial glucose level reader (Medisafe Reader GR-101, TERUMO). Measurements were performed every 30 min up to 120 min. Effect of olive leaves on the glycemic responses to cooked rice loading in humans The 14 healthy adult volunteers offered themselves as subjects to be experimented upon. In the first experiment, the subjects who had fasted for 12 h or more were fed 300 g of cooked rice, and in the next experiment they were fed 1 g olive leaves and 300 g of cooked rice. Two experiments were performed at a one week interval. In both experiments, blood samples were taken from a finger and blood glucose levels
Fig. 2. The inhibitory effect of OWE and OEE on the activities of -amylase from human saliva and pancreas. , saliva; , pancreas; Dashed lines indicate OEE and solid lines indicate OWE.
37
Identification of Anti--Amylase Components from Olive Leaf Extracts
were immediately measured by commercial glucose level reader (Medisafe Reader GR-101, TERUMO). Measurements were performed every 30 min up to 120 min. Results and Discussion -Amylase inhibitory activity in the extract of olive leaves As shown in Fig. 2, both OWE and OEE inhibited -amylase from human saliva and human pancreas. IC50 values of OWE were estimated to be 67.0 mg/ml (against human salivary -amy-
Fig. 3. Chromatogram of anti--amylase active fraction in 1st Sephadex LH-20. , -amylase inhibition (%); , A280.
Table 1. Position 1¢ 2¢ 3' 4¢ 5¢ 6¢ 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30
1
Table 2.
H NMR data for compounds 1, 2 and 3. Compound 1 (ppm) Compound 2 (ppm) Compound 3 (ppm) 7.43–7.42
7.49–7.48
6.89–6.87 7.34–7.30
7.27–7.25 7.47
6.69
6.75
6.44
6.22 7.95 6.49–6.48
6.78–6.76
lase) and 70.2 mg/ml (against human pancreatic -amylase), respectively. IC50 values of OEE were estimated to be 4.0 and 0.02 mg/ml, respectively. Therefore, we attempted to isolate anti-amylase components from OEE. Isolation and characterization of the anti--amylase components As shown in Fig. 1, two components (compounds 1 and 2) were isolated by Sephadex LH-20 chromatography after 50% ethanol extraction of OEE (20 g). Figure 3 shows a typical chromatogram of anti--amylase fractions (compounds 1 and 2) in the 1st Sephadex LH-20 column. After rechromatography, 200 mg of compound 1 and 200 mg of compound 2 were obtained. Compound 3 (2.4 g) was isolated from OEE 50% ethanol insoluble fraction. The retention time by HPLC of compound 3 was the same as that of oleanolic acid. The IR spectrum of compound 3 was also identical to that of oleanolic acid. The molecular weights of compounds 1 and 2 were estimated as 450.7 (MH) by TOF-MS analysis, and the molecular weight of authentic luteolin glucoside is also 450.7 (MH). The above finding strongly indicated that compounds 1 and 2 were luteolin glucosides. To determine the carbonhydrate moiety in these compounds, HPLC analyses were carried out after hydrolysis and labeling with PMP of the respective compound. Both compounds provided glucose (date not shown). From these results, these compounds were identified as luteolin glucosides. To determine the linkage of glucose in each compound, GCMS analysis was performed. Compounds 1 and 2 were identified as luteolin-7-O--glucoside and luteolin-4¢-O--glucoside, re-
1.58 1.56 3.2
1.32 1.64 1.88 5.31 1.81 2.02 2.89 1.72 1.41 0.77 0.99 0.93 0.78 1.14 0.95 0.91
Position 1¢ 2¢ 3¢ 4¢ 5¢ 6¢ 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30
13
C NMR data for compounds 1, 2 and 3. Compound 1 (ppm) Compound 2 (ppm) Compound 3 (ppm) 119.1 113.6 145.8 149.9 116.1 121.5
118.7 113.8 147.2 148.6 116.7 125.2
163.1 103.2 181.8 156.9 94.9 164.6 99.7 161.2 105.5
163.4 104.1 181.8 157.6 94.2 164.3 99.1 162.6 104.3
38.6 27.3 79.1 38.8 55.4 18.4 33.1 39.5 47.8 37.2 23.6 122.8 143.7 41.8 27.8 23.1 46.7 41.2 46.1 32.6 33.9 30.7 15.6 28.2 15.4 17.2 25.9 182.7 23.5 32.8
38
E. KOMAKI et al.
spectively. 13 C and 1H NMR data of compounds 1, 2 and 3 are shown in Tables 1 and 2. Both 13C and 1H spectra of these three compounds coincided well with the spectra of luteolin-7-O--glucoside, luteolin-4¢-O--glucoside and oleanolic acid, respectively. Structures of the compounds are shown in Fig. 4. Compound 1 (luteolin-7-O--glucoside) and compound 2 (luteolin-4¢-O--glucoside) inhibited human pancreatic -amylase activity with IC50 values of 0.5 and 0.3 mg/ml, respectively. Human pancreatic -amylase inhibitory activity of luteolin, which was prepared from compound 1, was stronger than luteolin glucoside with IC50 of 0.01 mg/ml. It was reported that luteolin inhibited porcine pancreatic -amylase activity and its IC50 was in the range of 0.05 to 0.5 mg/ml (Kim et al., 2000). The order of -amylase inhibitory activity of luteolin was the same as our findings. Compound 3 (oleanolic acid) also inhibited human pancreatic -amylase activity and its IC50 was 0.1 mg/ml. Hypoglycemic effect of -amylase inhibitors in diabetic GK/Jcl rat Figure 5 shows the time course of hypoglycemic activity after a single oral administration of doses of 20 mg/kg olive leaves, 1 mg/kg oleanolic acid and 0.1 mg/kg luteolin with
Fig. 5. Effect of olive leaf, oleanolic acid and luteolin on postprandial blood glucose levels in soluble starch-loaded rats. Each sample and 2 g/kg of soluble starch were given simultaneously by oral administration to 5-weekold GK/Jcl rats. , control; , 20 mg/kg olive leaf; ,1 mg/kg oleanolic acid; , 0.1 mg/kg luteolin. The values are meanSE. *p0.05, **p0.01.
Fig. 4. The structure of compound 1–3.
Fig. 6. Effect of olive leaf on the glycemic responses to cooked rice loading in normal and borderline humans. Overnight fasted subjects were given 300 g of cooked rice and blood samples were taken at 0, 0.5, 1, 2 and 3 h after the loading. 1 g of olive leaf was administered to subjects before cooked rice loading. , cooked rice only; , cooked rice with 1g olive leaf. The values are meanSE. *p0.05, **p0.01.
Identification of Anti--Amylase Components from Olive Leaf Extracts
2 g/kg starch. Dietary flavonoid glycosides are mostly hydrolyzed to aglycones by intestinal microfloral -glucosidases and finally decomposed to low molecular weight compounds (Griffiths, 1987). Therefore, luteolin aglycone rather than luteolin glucoside was used in this test. As can be seen in Fig. 5, blood glucose levels were significantly lowered in all groups except the control group. The blood glucose level was significantly decreased after 0.5 h (p0.05) and 2 h (p0.05) in the group given olive leaves. Both groups given oleanolic acid and luteolin were significantly decreased in blood glucose level after 0.5 h (p0.01), 1 h (p0.05), 1.5 h (p0.05) and 2 h (p0.05). Thus, the elevation of glucose level was suppressed by olive leaves, luteolin and oleanolic acid. Hypoglycemic effect of olive leaves in human The subjects were divided into two groups with blood glucose levels of before loading with cooked rice (normal and borderline groups for diabetes). As shown in Fig. 6, the change in blood glucose level of the borderline group was decreased significantly at 0.5 h (p0.05) and 1 h (p0.05) after loading olive leaves and cooked rice in comparison with the control experiment, but in the normal group, no difference in blood glucose level was observed between pre- and post-loading of olive leaves and cooked rice. These findings strongly suggest that the suppression of blood glucose elevation is due to the inhibitory action of luteolin and/or oleanolic acid on intestinal and/or salivary -amylases. It was reported that oleuropein in olive leaf accelerated the intake of glucose to the cell (Gonzalez et al., 1992). In our examination of whether -amylase is inhibited by oleuropein, we found that although it did not inhibit -amylase (IC50 100 mg/ ml), oleuropein’s aglycon moiety did strongly inhibit the enzyme (IC50 0.03 mg/ml). This may suggest that oleuropein also reduces the blood glucose level by inhibiting the activity of -amylase in vivo. Furthermore, luteolin-7-O--glucoside, luteolin-4¢O--glucoside and oleanolic acid isolated from OEE exhibited an inhibitory effect on -glucosidases prepared from rat intestine in vitro (data not shown). The findings described in this study indicate that olive leaves are a useful food supplement for preventing diabetes. References Bisignano, G., Tomaino, A., Lo Cascio, R., Crisafi, G., Uccella, N. and Saija, A. (1999). On the in vitro antimicrobial activity of oleuropein and hydroxytyrosol. J. Pharm. Pharmacol., 51, 971–974. Bisignano, G., Lagana, M.G., Trombetta, D., Arena, S., Nostro, A., Uccella, N., Mazzanti, G. and Saija, K. (2001). In vitro antibacteroal activity of some aliphatic aldehydes from Olea europaea L. FEMS. Microbiol. Lett., 198, 9–13. Dubois, M., Gilles, K.A., Hamilton, J.K., Rebers, P.A. and Smith, F. (1956). Colorimetric method for determination of sugars and related substances. Anal. Chem., 28, 350–356. Ficarra, P. and Ficarra, R. (1991). HPLC analysis of Oleuropein and
39 some flavonoids in leaf and bud of Olea europaea L.. Il. Farmaco., 46, 803–815. Gariboldi, P., Jommi, G. and Verotta, L. (1986). Secoiridids from Olea europaea. Phytochemistry, 25, 865–869. Gonzalez, M., Zarzuelo, A., Ganez, M.J., Utrilla, M.P., Jimenez, J. and Osuna, I. (1992). Hypoglycemic activity of olive leaf. Planta Med., 58, 513–515. Griffiths, L.A. (1987). Mammalian metabolism of flavonoids. In “The Flavonoids, Advances in Research,” ed. by J.B. Harbone and T.J. Mabry, Chapman and Hall, London, pp. 681–718. Honda, S., Akao, E., Suzuki, S., Okuda, M., Kakehi, K. and Nakamura, L. (1989). High-performance liquid chromatography of reducing carbohydrates as strongly ultraviolet-absorbing and electrochemically sensitive 1-phenyl-3-methyl-5-pyrazolone derivatives. Anal. Biochem., 180, 351–357. Jacotot, B. (1993). “L’Huile D’Olive” Artulen, Paris, pp. 56 Juven, B., Henis, Y. and Jacoby, B. (1972). Studies on the mechanism of the antimicrobial action of oleuropein. J. Appl. Bacteriol., 35, 559–567. Kim, J., Kwon, C. and Son, K. (2000). Inhibition of alpha-glucosidase and amylase by luteolin, a flavonoid. Biosci. Biotechnol. Biochem., 64, 2458–2461. Kohyama, N., Nagata, T., Fujimoto, S. and Sekiya, K. (1997). Inhibition of arachidonate lipoxygenase activities by 2-(3,4-dihydroxyphenyl)ethanol, a phenolic compound from olives. Biosci. Biotechnol. Biochem., 61, 347–350. Kotaru, M., Yoshikawa, H., Ikeuchi, T., Saito, K., Iwami, K. and Ibuki, F. (1987). An alpha-amylase inhibitor from inhibition of mammalian pancreatic alpha-amylase and formation of a complex with the porcine enzyme. J. Nutr. Sci. Vitaminol., 33, 359–367. Koutsoumanis, K., Tassou, C.C., Taoukis, P.S. and Nychas, G.J. (1998). Modelling the effectiveness of a natural antimicrobial on Salmonella enteritidis as a function of concentration, temperature and pH, using conductance measurements. J. Appl. Microbiol., 84, 981–987. Pieroni, A., Heimler, D., Pieters, L., van Poel, B. and Vlietinck, A.J. (1996). In vitro anti-complementary activity of flavonoids from olive (Olea europaea L.) leaves. Pharmazie., 51, 765–768. Romani, A. (1994). Extraction, purification procedures and HPLC-RI analysis of carbohydrates in olive (Olea europaea L.) plants. Chromatographia, 39, 35–39. Tassou, C.C., Nychas, G.J. and Board, R.G. (1991). Effect of phenolic compounds and oleuropein on the germination of Bacillus cereus T spores. Biotechnol. Appl. Biochem., 13, 231–237. Tassou, C.C. and Nychas, G.J. (1995). Inhibition of Salmonella enteritidis by oleuropein in broth and in a model food system. Lett. Appl. Microbiol., 20, 120–124. Tutour, L.B. and Guedon, D. (1992). Antioxidative activities of Olea europaea leaves and related phenolic compounds. Phytochemistry, 31, 1173–1178. Visioli, F. and Galli, C. (1994). Oleuropein protects low density lipoprotein from oxidation. Life Sci., 55, 1965–1971. Visioli, F., Bellomo, G. and Galli, C. (1998). Free radical-scavenging properties of olive oil polyphenols. Biochem. Biophys. Res. Commun., 247, 60–64. Visioli, F. and Galli, C. (1998). Olive oil phenols and their potential effects on human health. J. Agric. Food. Chem., 46, 4292–4296. Zaezuelo, A., Duarte, J., Jimenez, J., Gomzalez, M. and Utrilla, M.P. (1991). Vasodilator effect of olive leaf. Planta Med., 57, 417–419.
