ABSTRACT
Water extract of Brazilian propolis showed anti-emetic activity on copper sulfate–induced emesis in young chicks. Bioassay-guided fractionation of the extract was carried out, and dehydrohautriwaic acid (1), (E.)-3-(2,2-dimethyl-2H.-1-benzopyran-6-yl)-2-propenoic acid (2), dihydrocinnamic acid (3), (Z.)-3-(2,2-dimethyl-2H.-1-benzopyran-6-yl)-2-propenoic acid (4), aromadendrane-4β,10α-diol (5), and lupeol (6) were isolated as the active components.
Introduction
Propolis is popular as a health supplement to improve health and prevent diseases. The alcohol extracts of propolis have been used widely, but the water extract is now available. Propolis is a natural product collected by honeybees from parts of plants such as buds, bark, and exudates to use as a part of the inside of their hives. As the chemical composition of propolis depends on the plants from which honeybees collected it, it is known that Brazilian propolis is rich in cinnamic acid derivatives, whereas that of other countries such as China, Japan, Australia, and so forth, are rich in flavonoids (Tazawa et al., Citation2000). Several biological activities in propolis and their constituents, including antioxidant (Hayashi et al., Citation1999), anticancer (Luo et al., Citation2001), antibacterial (Velikova et al., Citation2000), antimicrobial (Hashimoto et al., Citation1998), and cytotoxic effects (Banskota et al., Citation1998), have already been reported.
On the other hand, anticancer drugs, for example cisplatin, frequently cause nausea and vomiting as the side effect. In the course of our research searching for new anti-emetic compounds, we noticed the principles of propolis so far reported are similar to the anti-emetic compounds that have already been reported to Chinese Materia Medica by our laboratory. In addition, the anticancer and the antibiotic effects were reported as mentioned above. If propolis is useful to prevent this, it may be important to increase the quality of life for patients. In this paper, we describe the anti-emetic activity of water extract and some active compounds in the extract of Brazilian propolis.
Materials and Methods
Materials
Alecrin-propolis was purchased in the Minas Gerais region in Brazil by CELSO DALL AGNOL.com in June 2003. Alecrin-propolis (200 g) was extracted with water (2 l). A voucher specimen (Gross Propolis Alecrin-2003) is deposited in our laboratory.
Spectrometry and chromatography
The IR spectra were measured with a JASCO A-102 IR spectrophotometer. Melting points were determined on a Yanagimoto MP micromelting point apparatus. The [α]D values were determined with a JASCO DIP-140 digital polalimeter (Tokyo, Japan). 1H and 13C NMR spectra were recorded using a JEOL AL-400 or JNM LA-500 spectrometer (Tokyo, Japan) in CDCl3 or DMSO-d.6 with tetramethylsilane as an internal standard. Kieselgel-60F254 (Merck, Tokyo, Japan) or RP-18F254S (Merck) precoated plates were employed for thin-layer chromatography (TLC). Column chromatography was carried out on 70-230 mesh silica gel (Merck) and prep ODS-7515-12A. High performance liqued chromatography (HPLC) was performed using an PU-2080 Plus pump with an JASCO UV-2075 Plus UV detector. High resolution fast atom bombardment mass spectra (HREIMS) and High resolution electron ionzation mass spectra (HRFABMS) were obtained using a JEOL JMS-700.
Animals
Young male chicks (2–4 days of age) weighing 30–40 g each were purchased from Goto Franjo Co. Ltd. (Saitama, Japan).
Bioassay of anti-emetic activity
The young chicks were divided into 1–3 groups consisting of six each, and each young chick was set aside for stabilization in large beakers at 25°C for 10 min. The sample was dissolved in 0.9% saline containing 5% DMSO and 1% Tween 80 and injected abdominally at a volume of 10 ml/kg. After 10 min, copper sulfate anhydride was administered orally at 50 mg/kg, then the number of retches (an emetic action without vomiting gastric materials) was recorded during the next 10 min. The results were judged by the decrease in number of retches in contrast with those of control. The inhibition (%) was calculated as follows:
where A. is control frequency of retching and B. is frequency retching after sample treatment.
Statistical analysis
All numerical data are expressed as the mean ± SEM. The statistical significance of the difference was assessed using analysis of variance (ANOVA) followed by Bonferroni's test.
Preparation of extract
The H2O extract of Brazilian propolis was lyophilized and extracted four-times successively with 2 l MeOH at room temperature for 24 h. The MeOH extract was concentrated to use for bioassay.
Isolation and purification of the anti-emetic principles from Brazilian propolis
The H2O extract of Brazilian propolis was lyophilized (177 g) and extracted successively with MeOH (2 l × 4 times). Each extract was examined for the anti-emetic activity using young chicks (Tables and ). The MeOH extract (115 g) which showed the anti-emetic activity (50.9%) was treated with 50% MeOH aq. with 0.5% AcOH, and the insoluble portion (56.9 g) was filtered off.
Table 1.. Anti-emetic effects of metoclopramide on copper sulfate–induced emesis in young chicks.
Table 2.. Anti-emetic effects of each extract on copper sulfate–induced emesis in young chicks.
The 50% aq. MeOH-soluble fraction (67.4 g) was chromatographed on an octadecyl silica gel column [H2O: MeOH (1:1–0:1) with 0.5% AcOH], and 7 fractions (fr. 1–7) were obtained. Each fraction was tested for anti-emetic activity, and fr. 1 (40.1 g), fr. 4 (3.5 g), fr. 5 (82.9 mg), fr. 6 (6.6 g), and fr. 7 (2.8 g) showed positive activities ().
Table 3.. Anti-emetic effects of chromatographic fractions on copper sulfate–induced emesis in young chicks.
Fraction 6 (3.0 g) was chromatographed on a silica gel column [CHCl3:MeOH (100:1–0:1)] and 6 fractions (fr. I–VI) were obtained. Each fraction was tested for the anti-emetic activity, and fr. II (309.3 mg) and fr. III (81.8 mg) showed positive activities (). Fraction II was chromatographed on an octadecylsilysilica gel (ODS) column [H2O:MeOH (1:1–0:1)], and 6 fractions (fr. A–F) were obtained. Each fraction was tested for the anti-emetic activity, and fr. B (9.0 mg) and fr. E (38.0 mg) showed positive activities ().
Table 4.. Anti-emetic effects of chromatographic fractions on copper sulfate–induced emesis in young chicks.
Fraction E was chromatographed on a HPLC using a silica gel column [Silica 60-5 10 ϕ × 250 mm, CHCl3-MeOH (50:1)]. Compound 1 (18.4 mg) was obtained and identified as dehydrohautriwaic acid by comparison with published spectral data (Saad et al., Citation1988).
Fraction 4 (3.0 g) was chromatographed on a silica gel column [CHCl3:MeOH (100:1–0:1)], and 6 fractions (fr. a–f) were obtained. Each fraction was tested for the anti-emetic activity, and fr. a (75.9 mg), fr. b (542 mg), and fr. d (56.3 mg) showed positive activities (). Fraction b was treated with MeOH, and the insoluble portion (85.1 mg) was separated by filtration.
Table 5.. Anti-emetic effects of chromatographic fractions on copper sulfate–induced emesis in young chicks.
The MeOH-insoluble fraction of fraction b was subjected to HPLC using a silica gel column treated with oxalic acid [Silica 60-5 10 ϕ × 250 mm, CH2Cl2-acetone (100:1)], and compound 2 (42.2 mg) and compound 3 (15.0 mg) were obtained. Compounds 2 and 3 were identified as (E.)-3-(2,2-dimethyl-2H.-1-benzopyran-6-yl)-2-propenoic acid by comparison with published spectral data (Brown, Citation1994) and dihydrocinnamic acid by comparison with spectral data of the authentic sample.
The MeOH-soluble fraction (170.1 mg) of fraction b was subjected to HPLC using silica gel column treated with oxalic acid [Silica 60-5 10 ϕ × 250 mm, CH2Cl2-acetone (100:1)]. Compounds 2 (15.6 mg) and 4 (2.2 mg) were obtained. Compound 4 was identified as (Z.)-3-(2,2-dimethyl-2H.-1-benzopyran-6-yl)-2-propenoic acid by comparison with published spectral data (Brown, Citation1994).
An active fraction, fr. d, was chromatographed on a silica gel column [n.-hexane:AcOEt (100:1–0:1) and n.-hexane:acetone (100:1–4:1)]. Compound 5 (9.6 mg) was obtained and identified as aromadendrane-4β,10α-diol by comparison with published spectral data (Nagashima et al., Citation1994) and nuclear Overhauser effect difference spectroscopy (NOESY).
Fraction 7 was chromatographed on a silica gel column [CHCl3:MeOH (100:1–1:1), n.-hexane:AcOEt (100:1–0:1) and n.-hexane:acetone (100:1–4:1)], HPLC using silica gel column [Silica 60-5 10 ϕ × 250 mm, n.-hexane:acetone (5:1)], and HPLC using ODS column [ODS 10 ϕ × 250 mm, CH3CN-MeOH (1:20)]. Compound 6 (4.9 mg) was obtained and identified as lupeol by comparison with published spectral data (Wenkert et al., Citation1978).
Results and Discussion
On investigating the anti-emetic compounds from Chinese crude drug materials, we isolated many compounds belonging to monoterpenes, sesquiterpenes, triterpenes, phenyl propanoids, lignans, flavonoids (Kinoshita et al., Citation1996), and diarylheptanoids (Yang et al., Citation2002) as active principles. We noticed that some phenylpropanoid compounds among them, for example eugenol and methyl eugenol (Kawai et al., Citation1994), showed anti-emetic activities, and it was reported that the analogues of their compounds, for example cinnamic acid, caffeic acid, and ferulic acid, are in Brazilian propolis, (Tazawa, Citation2002). However, there were no reports for the anti-emetic acivity in any propolis. Three cinnamic acid derivatives, cinnamic acid, caffeic acid, and ferulic acid, from Brazilian propolis were examined for the anti-emetic activity using young chicks, and anti-emetic activities at the dose of 20 mg/kg (36.6%, 30.4%, and 33.9%) were shown.
Metoclopramide, which has already been reported for anti-emetic activity throught acceleration of gastrointestinal tract movement, was examined for anti-emetic activity using young chicks (Akita et al., Citation1998). As shown in , metoclopramide inhibited the emesis induced by copper sulfate at a dose of 50 mg/kg (72.3%).
As shown in , water extract of Brazilian propolis significantly inhibited the emesis induced by copper sulfate in young chicks. Bioassay-guided fractionation of the extract was carried out, and dehydrohautriwaic acid (1), (E.)-3-(2,2-dimethyl-2H.-1-benzopyran-6-yl)-2-propenoic acid (2), dihydrocinnamic acid (3), (Z.)-3-(2,2-dimethyl-2H.-1-benzopyran-6-yl)-2-propenoic acid (4), aromadendrane-4β,10α-diol (5), and lupeol (6) were isolated. Compounds 1 and 5 are the first isolated from propolis.
As shown in , compounds 1–6 also showed anti-emetic activity. Dehydrohautriwaic acid (1) and (Z.)-3-(2,2-dimethyl-2H.-1-benzopyran-6-yl)-2-propenoic acid (4) significantly inhibited the emetic action at doses of 2 and 4 mg/kg, respectively. Aromadendrane-4β,10α-diol (5) was active at doses of 5 mg/kg and 10 mg/kg, respectively. (E.)-3-(2,2-dimethyl-2H.-1-benzopyran-6-yl)-2-propenoic acid (2) was active at a dose of 20 mg/kg. Dihydrocinnamic acid (3) was active at doses of 25–100 mg/kg, respectively. Lupeol (6) was active at three doses of 2.5–10 mg/kg, respectively. They showed dose-dependent inhibition.
Table 6.. Anti-emetic effects of compounds 1–6 on copper sulfate–induced emesis in young chicks.
By analyses of their NMR data, compounds 2 and 4 were determined as geometric isomers of each other; E.- and Z.-isomers. It is very interesting that compound 4 showed approximately 10-fold stronger anti-emetic activity compared with compound 2 ().
This is the first result to show that water extract of Brazilian propolis is effective for the emesis induced by copper sulfate. Compounds 1–6 might play roles in the anti-emetic effect in water extract of Brazilian propolis. This result suggests that water extract of Brazilian propolis could have a new use other than the traditionally known usage.
References
- Akita Y, Yang Y, Kawai T, Kinoshita K, Koyama K, Takahashi K (1998): New assay method for surveying anti-emetic compounds from natural sources. Nat Prod Sci 4: 72–77.
- Banskota AH, Tezuka Y, Prasain JK, Matsushige K, Saiki I, Kadota S (1998): Chemical constituents of Brazilian propolis and their cytotoxic activities. J Nat Prod 61: 896–900. [PUBMED], [INFOTRIEVE], [CROSSREF], [CSA]
- Brown GD (1994): Phenylpropanoids and other secondary metabolites from Baccharis linearis.. Phytochemistry 35: 1037–1042. [CROSSREF]
- Hashimoto T, Aga H, Tabuchi A, Shibuya T, Chaen H, Fukuda S, Kurimoto M (1998): Anti-Helicobactor pylori. compounds in Brazilian propolis. Nat Med 52: 518–520.
- Hayashi K, Komura S, Isaji N, Ohishi, N, Yagi K (1999): Isolation of antioxidative compounds from Brazilian propolis: 3,4-Dihydroxy-5-prenylcinnamic acid, a novel potene antioxidant. Chem Pharm Bull 47: 1521–1524.
- Kawai T, Kinoshita K, Koyama K, Takahashi K (1994): Anti-emetic principles of Magnolia obovata. bark and Zingiber officinale. rhizome. Planta Med 60: 17–20. [PUBMED], [INFOTRIEVE], [CSA]
- Kinoshita K, Kawai T, Imaizumi T, Akita Y, Koyama K, Takahashi K (1996): Anti-emetic principles of Inula linariaefolia. flowers and Forsythia suspense. fruits. Phytomedicine 3: 51–58.
- Luo J, Soh JW, Xing WQ, Mao Y, Matsuno T, Weinstein IB (2001): PM-3, a benzo-γ-pyran derivative isolated from propolis, inhibits growth of MCF-7 human breast cancer cell. Anticancer Res 21: 1665–1672. [PUBMED], [INFOTRIEVE], [CSA]
- Nagashima F, Tanaka H, Toyota M, Hashimoto T, Kan Y, Takaoka S, Tori M, Asakawa Y (1994): Sesqui- and diterpenoids from Plagiochila. species. Phytochemistry 36: 1425–1430. [CROSSREF]
- Saad JR, Davicino JG, Giordano OS (1988): A diperpene and flavonoids of Baccharis flabellata.. Phytochemistry 27: 1884–1887. [CROSSREF]
- Tazawa S, Warashina T, Noro T (2000): On the chemical evaluation of propolis. Nat Med 54: 306–313.
- Tazawa S (2002): Analysis of the constituents, chemical evaluation and tyrosinase inhibition of propolis. Fragrance J 3: 25–32.
- Velikova M, Bankova V, Tsvetkova I, Kujumagiev A, Marcucci MC (2000): Antibacterial ent.-kaurene from Brazilian propolis of native stingless bees. Fitoterapia 71: 693–696. [PUBMED], [INFOTRIEVE], [CROSSREF], [CSA]
- Wenkert E, Baddeley GV, Burfitt IR, Moreno LN (1978): Carbon-13 nuclear magnetic resonance spectroscopy of naturally-occurring substances. LVII. Triterpenes related to lupane and hopane. Org Mag Res 11: 337–343.
- Yang Y, Kinoshita K, Koyama K, Takahashi K, Kondo S, Watanabe K (2002): Structure-antiemetic-activity of some diarylheptanoids and their analogues. Phytomedicine 9: 146–152. [PUBMED], [INFOTRIEVE], [CSA]