Introduction

Hyperlipemia is a risk factor for arteriosclerosis. Control of the cholesterol level in the body is effective for prevention and improvement of hyperlipemia. Currently, HMG-CoA reductase inhibitors are used clinically for the treatment of hyperlipemia. The HMG-CoA reductase inhibitors, however, might cause simultaneous reduction of the physiologically essential non-steroidal isoprenoid metabolites such as dolichol, ubiquinone and prenylated proteins because HMG-CoA reductase is located in the upstream of the cholesterol biosynthetic pathway. Therefore, the long-term administration of the inhibitors of the enzyme may cause unexpected side effects. Lanosterol synthase is considered to be a more selective target for suppression of cholesterol biosynthesis, since it is located in the middle stage of the biosynthetic pathway of the cholesterol in mammals. Lanosterol synthase-inhibiting compounds were isolated from microbial cultures using in vitro assay with recombinant human enzyme [1, 2].

In recent years, a tertiary function of foods contributing to healthy life by modulating human homeostasis has been recognized in addition to their nutritional and organoleptic functions. From the viewpoint of this function, we have investigated a new function of foods and foodstuffs, and found that apple-condensed tannins had an antiallergic effect on type I allergic symptoms [3]; extracts of cabbage, red cabbage, tomato, and watercress showed antiallergic effects [4]; and some constituents in watercress inhibited histamine release from RBL-2H3 cells [5].

In order to find the foods which function to reduce the cholesterol level, 130 kinds of vegetable extracts were examined for inhibition of human lanosterol synthase in our previous study. Among them, the extract of Colocasia esculenta (taro) exhibited the most potent activity. Monogalactosyl diacylglycerol (MGDG) and digalactosyl diacylglycerol (DGDG) were isolated as active components [6]. In this paper, we report inhibitory activities of another 37 kinds of foods and foodstuffs on human lanosterol synthase and isolation of the active compounds from the extract of Laurus nobilis L., which showed potent inhibitory activity among the tested samples.

Materials and methods

Materials

Dried powder of laurel (L. nobilis L.) was purchased from Yasuma (Tokyo, Japan). Other plant materials for screening were gifts from San-Ei Gen F.F.I., Inc. (Osaka, Japan).

Screening of human lanosterol synthase-inhibiting food and foodstuff

Thirty-seven foods and foodstuffs, Glycyrrhiza glabra L. (roots), Coriandrum sativum L. (fruits), Cistanche salsa G. Beck (whole plant), Cichorium intybus L. (roots), Dimocarpus longan Lour (fruits), Litchi chinensis Sonn. (fruits), Cymbopogon citratus Stapf. (leaves), Melissa officinalis L. (leaves), Uncaria gambir Roxburgh (leaves), Piper nigrum L. (fruits), L. nobilis L. (leaves), Pimpinella anisum L. (fruits), Acanthopanax senticosus Harms (bark), Diospyros kaki Thunberg (leaves), Urtica platyphylla Wedd. (aerial part), Elettaria cardamomum Maton (seeds), Citrus unshiu Markovich (fruits), Aloe arborescens Mill. (leaves), Lycium chinense Mill. (fruits), Syzygium aromaticum (L.) Merr. et Perry (buds), Gentiana lutea L. (roots), Jasminum grandiflorum L. (flowers), Illicium verum Hook. fil. (fruits), Mentha spicata L. (aerial part), Salvia officinalis L. (leaves), Curcuma longa L. (rhizomes), Myristica fragrans Houttuyn (seeds), Petroselium sativum L. (aerial part), Foeniculum vulgare Mill. (fruits), Brassica juncea (L.) Czerniak (seeds), Porphyra tenera Kjellman. (whole plant), Momordicae grosvenori Swingle (fruits), Achillea millefolium L. (buds), Zanthoxylum bungeanum Maxim. (fruits), Origanum majorana L. (leaves), Zizyphus jujuba Mill. var. inermis Rehd. (fruits), and Chrysanthemum morifolium Ramat. (flowers) were extracted with 95% ethanol, 50% ethanol or water on a water bath at 80°C for 2 h and adjusted to 5 mg/ml with ethanol to afford 64 sample solutions. Sixty μl (300 μg) of each sample solution was tested for inhibition of human lanosterol synthase as described in previous papers [1, 2].

Isolation of components from L. nobilis L.

The laurel powder (800 g) was extracted by reflux with methanol (3.0 l×3). The solvent was removed under reduced pressure to yield 328.7 g of a methanol extract. The methanol extract (100 g) was suspended in water and partitioned with dichloromethane, ethyl acetate, and 1-butanol, successively, and resulted in three crude fractions, dichloromethane (8.6 g), ethyl acetate (16.1 g) and 1-butanol (20.1 g). The ethyl acetate soluble fraction was chromatographed over a silica gel column eluting with hexane and ethyl acetate, followed by MPLC and HPLC, to give kaemperol 3-O-[2″,4″-O-di-E-p-coumaroyl-α-l-pyranorhamnoside] (1; 13.3 mg) [7]. The dichloromethane soluble fraction was subjected to activated charcoal column chromatography (methanol, methanol-chloroform and chloroform) to afford four fractions (fractions 1–4). The third fraction was further separated by silica gel column chromatography using ethyl acetate-hexane, followed by HPLC, and yielded 3,3′,4′,5,6,7,8-heptamethoxyflavone (2, 24.3 mg) [8, 9]; 3′,4′,5,6,7,8-hexamethoxyflavone (nobiletin) (3, 2.9 mg) [8, 9]; 4′,5,6,7,8-pentamethoxyflavone (tangeretin) (4, 2.2 mg) [8, 9]. Fraction 1 was further separated by silica gel column chromatography using ether–hexane, followed by HPLC (80% MeOH) , and yielded eremanthine (5, 4.1 mg) [10], dehydrocostus lactone (6, 8.6 mg) [11] and costunolide (7, 8.1 mg) [12]. Fraction 2 was further separated by silica gel column chromatography using ether-hexane, followed by HPLC (80% MeOH), and yielded zaluzanin C (8, 31.7 mg) [13], zaluzanin D (9, 10.3 mg) [14], and reynosin (10, 5.1 mg) [15]. These compounds were identified by comparison of their spectral data with those published (Figs. 1, 2).

Fig. 1
figure 1

Chemical structures of compounds 1–4

Full size image
Fig. 2
figure 2

Chemical structures of compounds 5–10

Full size image

Results and discussion

Among 37 tested samples, five samples (L. nobilis, A. millefolium, U. gambri, P. higrum and C. unshiu) showed inhibitory activity (more than 5% inhibition) on human lanosterol synthase (Table 1). Among them, the active constituents of L. nobilis were investigated in this study. L. nobilis is an evergreen tree, family Lauraceae, native to Europe. The leaves of L. nobilis, bay leaf or laurel, are usually used as a spice and are known as a folk medicine for rheumatism in Europe [16, 17]. The constituents of L. nobilis have bioactivities such as nematocidal activity [18], ethanol-absorption inhibitory activity [19], trypanocidal activity [20] and inhibitory activity on nitric oxide production in lipopolysaccharide-activated mouse peritoneal macrophages [21].

Table 1 Foods and foodstuffs showing inhibitory activity on recombinant human lanosterol synthase
Full size table

The inhibitory activity of extracts of laurel with several solvents (95% aqueous ethanol, 50% aqueous ethanol and water) was 55, 22 and 0%, respectively, suggesting that the active components of L. nobilis were hydrophobic. Therefore, laurel was first extracted with methanol, and the methanol extract was partitioned with water and organic solvents (dichloromethane, ethyl acetate and 1-butanol, successively), and then the active compounds in the dichloromethane and ethyl acetate layers were further investigated. From the dichloromethane layer, we isolated three flavonoids (2–4) and six sesquiterpenes (5–10) which were identified to be 3,3′,4′,5, 6,7,8-heptamethoxyflavone (2); 3′,4′,5,6,7,8-hexamethoxyflavone (nobiletin) (3); 4′,5,6,7,8-pentamethoxyflavone (tangeretin) (4); eremanthine (5), dehydrocostus lactone (6); costunolide (7); zaluzanin C (8); zaluzanin D (9) and reynosin (10). Kaemperol 3-O-[2″,4″-O-di-E-p-coumaroyl-α-l-pyranorhamnoside] (1) was isolated from the ethyl acetate layer. These isolated non-glycoside flavonoids (2–4) were not new compounds, but were isolated from L. nobilis for the first time.

The inhibitory activity of compounds 1–10 is shown in Table 2. Among them, 5 showed the most potent inhibitory activity (70% at 500 μM) followed by 6 (63% at 500 μM). These compounds are bicyclic, guaiane-type sesquiterpenes, and the difference is in the geometry of the double bond at C10. Inhibitory activity was reduced in compounds 8 and 9, analogs of 6 with hydroxyl and acetoxyl at C3, suggesting that no substitution group at C3 is required for significant inhibitory activity. On the other hand, costunolide (7), a monocyclic, germacrane-type sesquiterpen, showed no enzyme inhibitory activity. Since the structure of 7 has a ten-membered ring, conformation of 7 is considerably flexible, compared to that of other sesquiterpenes listed in Fig. 2. These results suggested that a particular rigid conformation is a factor for high inhibitory activity.

Table 2 Inhibitory activity of 1–10 on human lanosterol synthase
Full size table

Three flavonoids, 2–4, were isolated as active compounds (20–40% inhibitory activity) in addition to the sequiterpenes. Compound 1, a glycoside of flavonoid isolated from the ethyl acetate layer, however, showed no activity. The structures of 2–4 are characteristic in that all hydroxy groups are methylated. These results indicated that hydrophobicity of the molecule is indispensable for significant inhibitory activity. This is the first report to describe flavonoids as having lanosterol synthase inhibitory activity.

As shown in Table 1, the ethanol extract of A. millefolium L. and C. unshiu showed inhibitory activity. Since it has been reported that A. millefolium L. [22] and C. unshiu [23] contain non-glycosidic flavonoids, flavonoids are deduced to be the active components of A. millefolium L. and C. unshiu.

Five sesquiterpenes and three flavonoids were isolated from L. nobilis as lanosterol synthase inhibitors. The inhibitory activity of each compound was not as high as the known inhibitors, lauryldimethylamine N-oxide (LDAO), AMO 1618, lanopylins A1, B1, A2 and B2, and epohelmins A and B, for which the IC50 values were 0.84, 120, 15, 18, 33, 41, 10 and 6.0 μM, respectively, under the same assay conditions [1, 2]. However, the fact that laurel contains multiple compounds with moderate inhibitory activity and is ingested repeatedly may make laurel a functional food for reducing the amount of cholesterol in the human body by inhibition of its de novo biosynthesis, even though the activity of each compound is not so high.