Bioactivity of alkamides isolated from Echinacea purpurea (L.) Moench.Summary
Alkamides from the roots of Echinacea purpurea (L.) Moench were examined for anti-inflammatory activity in an in vitro model system. Cyclooxygenase-I (COX-I) and cyclooxygenase-II (COX-II) inhibitory activities were assessed at pH 7 for alkamides isolated from E. purpurea roots to compare inhibitory activities between the two cyclooxygenase isozymes. At 100 [micro]g/ml, several E. purpurea alkamides inhibited COX-I and COX-II enzymes in the range of 36-60% and 15-46%, respectively, as compared to controls. Mosquitocidal activity was assessed at 100 and 10 [micro]g/ml, with 100% mortality against Aedes aegyptii L. larvae noted for several E. purpurea alkamides at 100 [micro]g/ml.
Key words: Echinacea purpurea, alkamides, cyclooxygenase, enzyme, Aedes aegyptii, mosquitocidal
Echinacea purpurea is one of three Echinacea echinacea (ĕk'ənā`shēə), popular herbal remedy, or botanical, believed to benefit the immune system. It is used especially to alleviate common colds and the flu, but several controlled studies using it as a cold medicine have species used widely for phytoceutical purposes. Initially used by North American Indians to treat various infections, the use of Echinacea spread to Europe where Echinacea-based products have flourished in the natural product supplement market (Bauer and Wagner, 1991). Following the establishment of Echinacea products in the European market, interest in Echinacea spp. has increased in the North American nutraceutical market, where the popularity of Echinacea products has grown as a complement to modern medicine (Borchers et al., 2000). Proof of safety and efficacy of herbal supplements would aid in consumer confidence.
Many adverse side-effects, including upper gastrointestinal irritation and ulceration, have been reported after long-term use of COX-I inhibitors such as aspirin (Loll, 1996). COX-I is the constitutive form of the enzyme responsible for basic regulatory functions in cells and is involved in the production of prostaglandins (Cryer CRYER, practice. An officer in a court whose duty it is to make various proclamations ordered by the court. and Dubois, 1998). Because prostaglandins are responsible for such physiological process as control of gastric secretions and maintenance of mucosal integrity (Ford-Hutchinson, 1996), inhibition of COX-I may lead to the adverse side-effects noted for long-term non-steroidal anti-inflammatory drug (NSAID NSAID: see nonsteroidal anti-inflammatory drug. ) use. COX-II, the cyclooxygenase isozyme isozyme /iso·zyme/ (i´so-zim) one of the multiple forms in which an enzyme may exist in an organism or in different species, the various forms differing chemically, physically, or immunologically, but catalyzing the same reaction. inducible in response to inflammation (Lipisky, 1999), is believed to possibly inhibit inflammation without the adverse side-effects noted for COX-I inhibitors (Pairet et al., 1996; Vane and Botting, 1996). Compounds with relatively greater COX-Il inhibition, as compared to COX-I, have demonstrated anti-inflammatory activity with a reduction of adverse side-effects in preclinical trials (Ford-Hutchinson, 1996). Specific of greater inhibition of COX-II as opposed to COX-I is a desirable target in developing pharmaceutical and phytoceutical products, due to the potential reduction in adverse side-effects.
Numerous bioactive compounds have been isolated from Echinacea spp. Extracts and compounds from various parts of Echinacea spp. plants have demonstrated anti-viral, antioxidant, and anti-inflammatory activites (Facino et al., 1995; Bauer and Wagner, 1991; Wagner et al., 1998; Luettig et al., 1989). Of particular interest are the alkamides isolated from E. purpurea and E. angustifolia, some of which have been examined for in vitro anti-inflammatory activity. Inhibition of 5-lipoxygenase and cyclooxygenase enzymes isolated from ram seminal vesicles by alkamides present in E. angustifolia and F. purpurea has been reported previously (Muller-Jakic et al., 1994). Two cyclooxygenase isoforms have been identified, COX-I and COXII; however, a comparative assessment of the COX-Iversus the COX-II-inhibitory properties of the alkamides has not been completed. In this paper we report the comparative COX-I- and COX-II-inhibition exhibited by E. purpurea alkamides.
In addition to anti-inflammatory properties, alkamides from a variety of plants have been reported to possess significant mosquitocidal activity. The mosquitocidal activity of dodeca-2E,4E,8E, 1OZ-tetraenoic acid isobutylamide, an alkamide isolated from Spilanthes mauritiana (Jondiko, 1986), is of interest due to its close structural similarity to an alkamide identified from E. purpurea. The mosquitocidal activites of the majority of alkamides from F. purpurea have not been determined. Therefore, the mosquitocidal properties of alkamides from E. purpurea were also evaluated.
Materials and Methods
Serving or tending to prepare or make ready; preliminary.
Something that prepares for or acts as a preliminary to something following. HPLS HPLS Hierarchical Partial Least Squares was performed using a Model LC-20 Preparative Recycling Liquid Chromatograph chromatograph /chro·mato·graph/ (kro-mat´o-graf)
1. the apparatus used in chromatography.
2. to analyze by chromatography.
1. to analyze by chromatography.
2. (Japan Analytical Instrument Co., Ltd., Tokyo, Japan) with UV detection at 260 nm. A gradient of methanol:water (50-80% methanol over 2 h) was used to separate the alkamides into crude fractions. Final purification was achieved using a gradient of acetonitrile acetonitrile /ac·e·to·ni·trile/ (as?e-to-ni´tril) a colorless liquid with an etherlike odor used as an extractant, solvent, and intermediate; ingestion or inhalation yields cyanide as a metabolic product. : water (40-60% acetonitrile over 2 h). [HNMR HNMR High Resolution Nuclear Magnetic Resonance .sup.1] NMR NMR: see magnetic resonance. spectra were recorded in [CDC See Control Data, century date change and Back Orifice.
CDC - Control Data Corporation 1.sub.3] on a Varian Inova 300 MHz (MegaHertZ) One million cycles per second. It is used to measure the transmission speed of electronic devices, including channels, buses and the computer's internal clock. A one-megahertz clock (1 MHz) means some number of bits (16, 32, 64, etc. spectrometer. Mass spectra were recorded using a Waters Alliance HT LC/MS system (Milford, Massachusetts) with a 2690 Separations Module and Micromass detector (ionization mode AP+ and cone voltage 40, mobile phase 40-80% acetonitrile over 30 mm). Identification of E. purpurea alkamides was made using H-[NMR.sup.1] experiments and comparison with literature (Bauer et al., 1988) and LC7MS values.
E. purpurea roots were obtained from Trout Lake Farm (Trout Lake, Washington Trout Lake is a census-designated place (CDP) in Klickitat County, Washington, United States. The population was 494 at the 2000 census. Geography
Trout Lake is located at (45.995522, -121.520645)GR1. ). Dried, milled E. purpurea roots (989.9 g) were extracted exhaustively using dichloromethane (4 1 x 3 days) and concentrated in vacuo. Following concentration, the dichloromethane extract (10.1 g) was separated into methanol-soluble (7.1 g) and insoluble (3.0 g) fractions, with the addition of methanol (750 ml x 3). The methanol-soluble portion was concentrated in vacuo, dissolved in chloroform (250 ml), and hexane hexane /hex·ane/ (hek´san) a saturated hydrogen obtained by distillation from petroleum.
n. was added. The supernatant, containing the crude alkamides, was concentrated (4.2 g) and subjected to separation through preparative high-performance liquid chromatography (HPLC HPLC high-performance liquid chromatography.
high performance liquid chromatography.
HPLC High-performance liquid chromatography Lab instrumentation A highly sensitive analytic method in which analytes are placed ).
Cyclooxygenase Inhibitory Assay
An in vitro COX-inhibition model system was used to assess the anti-inflammatory activity of alkamides isolated from E. purpurea. The COX-I used in the assay was prepared from ram seminal vesicles (Oxford Biomedical Research Inc., Oxford, MI). COX-I was obtained from microsomal microsomal
pertaining to or emanating from microsome. preparations of ram seminal vesicles according to methods reported previously (Laneuville et al., 1994; Meade et al., 1993). A microsomal preparation of recombinant human COX-II, obtained from an insect cell lysate ly·sate
The cellular debris and fluid produced by lysis. , was used as the source of COX-II enzyme for the assay. The assay was conducted in a 600-[micro]l Instech chamber (Instech Laboratory, Plymouth Meeting, PA) maintalned at 37 [degrees]C, containing a reaction buffer of 0.1 m Tris, 1 mM phenol, 100 [micro]M arachidonic acid, and 17 [micro]g hemoglobin. Alkamides were dissolved in DMSO DMSO dimethyl sulfoxide.
Dimethyl sulfoxide; a colorless hygroscopic liquid obtained from lignin, used as a penetrant to convey medications into the tissues.
n. such that a 10-[micro]l aliquot aliquot (al-ee-kwoh) adj. a definite fractional share, usually applied when dividing and distributing a dead person's estate or trust assets. (See: share) would yield a final assay concentration of 100 [micro]g/ml. The reaction was initiated using either a 10[micro]l aliquot of COX-I or a 20-[micro]1 aliquot of COX-I I. Oxygen uptake was monitored using a YSI YSI Yousendit (File Transfer Website)
YSI Youth Science Institute
YSI You Stupid Idiot model 5300 biological oxygen monitor (Yellow Springs Instruments, Inc., Yellow Springs, OH) and recorded using Quicklog for Windows, version 1.0 (Strawberry Tree, Inc., Sunnyvale, CA).
Aspirin, ibuprof en, naproxen naproxen and naproxen sodium, potent nonsteroidal anti-inflammatory drugs (NSAID) used to alleviate the minor pain of arthritis, menstruation, headaches, and the like, and to reduce fever. , celecoxib (Celebrex [TM]), and rofecoxib (Vioxx [TM]) were dissolved in DMSO and used as controls for both the COX-I and COX-II inhibitory assays. Aspirin was tested at 1000 [micro]M (180 [micro]g/ml), ibuprofen at 10 [micro]M (2.06 [micro]g/ml), and naproxen at 10 [micro]M (2.52 [micro]g/ml) for both COX-I and COX-II. Celecoxib (Celebrex [TM]) and rofecoxib (Vioxx [TM]) were obtained as physican's professional samples (Dr. Subash Gupta, Sparrow Pain Center, Sparrow Hospitals, Michigan), ground to fine powder, dissolved in DMSO, and tested at 1.6 [micro]g/ml for both COX-I and COX-II.
The mosquitocidal assay was performed according to methods published previously (Kelm et al., 1998; Nair et al., 1989). Aedes aegyptii L., provided by Dr. Alan Hayes (Department of Entomology entomology, study of insects, an arthropod class that comprises about 900,000 known species, representing about three fourths of all the classified animal species. , Michigan State University Michigan State University, at East Lansing; land-grant and state supported; coeducational; chartered 1855. It opened in 1857 as Michigan Agricultural College, the first state agricultural college. ) were hatched and raised in 500 ml of degassed distilled water, with approximately 5 mg of bovine liver powder added for nourishment. After the mosquito larvae had reached the fourth-instar (four days), the larvae were prepared for the bioassay. Ten to fifteen larvae in 980 [micro]l of degassed, distilled water were placed in 4-mi culture tubes. Stock solutions of the alkamides in DM80 were prepared such that a 20-[micro]l aliquot added to the mosquito larvae in the bioassay culture tubes yielded a final assay concentration of 100 [micro]g/ml or 10 [micro]g/ml. A 20-[micro]l aliquot of DM80 was used as the control. The alkamides were tested at 100 [micro]g/ml and 10 [micro]g/m1 in triplicate with the control set. Dead larvae were recorded at time zero, 1, 2, 4, 9, and 24 h, and reported in term s of percent mortality.
The bioactive alkamides isolated from dried E. purpurea roots (Figure 1) were identified as undeca-2E-4Zdien-8, 10-diynoic acid isobutylamide (1), undeca2Z,4E-dien-8, 10-diynoic acid isobutylamide (2), dodeca-2E,4Z-dien-8, 10-diynoic acid isobutylamide (3), undeca-2E,4Z-dien-8, 10-diynoic acid 2-methylbutylamide (4), dodeca-2E,4Z-dien-8, 10-diynoic acid 2-methylbutylamide (5), and a mixture of dodeca-2E,4E,8Z,1OEtetraenoic acid isobutylamide and dodeca2E,4Z,8Z, 10Z-tetraenoic acid isobutylamide (6/7). Compounds 6 and 7 were inseparable under varioius HPLC conditions. Yields obtained were 8 mg (1), 14 mg (2), 21 mg (3), 12 mg (4), 15 mg (5), and 40 mg (6/7).
The results of the mosquitocidal assay are summarized in Figure 2. The mixture of alkamides 6/7 proved to be the most effective in the mosquitocidal assay, with 87.5% mortality of mosquito larvae within 15 min when assayed at a concentration of 100 [micro]g/ml. Lowering the concentration to 10 [micro]g/ml still yielded significant mosquitocidal activity, with 63% mortality in one hour (Figure 2). Mosquito larvae that were still regarded as alive showed significant impairment at 1 h and beyond for alkamides 6/7. Compound 1 demonstrated slightly less activity with 71 and 100% mortality by 2 and 9 h, respectively. Compounds 2 and 3 showed mosquitocidal activity at the end of 9 h, with 78 and 50% mortality, respectively. Alkamides 4 and 5 proved to be the least active, with only 25 and 10% mortality at the end of 24 h.
COX-I and COX-II inhibitory activities of the alkamides are summarized in Figure 3 COX-I inhibitory activity was highest for alkamides 2, 4, and 5, with inhibitions of 60, 55, and 48%, respectively. Alkamides 1 and 3 both exhibited COX-I inhibition of 36% as compared to DMSO. The mixture of alkamides 6/7 did not, however, demonstrate inhibition of the COX-I enzyme (Figure 3).
The alkamides showed lower COX-II inhibitory activity as compared to COX-I. Compound 2 possessed the strongest COX-II inhibitory activity at 46%. Compounds 4 and 5 showed 39 and 31% inhibition of the COX-II enzyme, respectively. Compounds 1 and 3 had the lowest activities of the alkamides, with each exhibiting 15% inhibition. As with COX-I, the mixture of alkamides 6/7 did not demonstrate activity against COX-II.
Numerous plant species of the Asteraceae contain insecticidal alkamides (Greger, 1984), and the mosquitocidal properties of E. angustifolia have been reported previously (Hartzell, 1947; Greger, 1984). Dodeca-2E,4E,8Z, 10E-tetraenoic acid isobutylamide, an alkamide isolated from S. mauritiana (Jondiko, 1986) similar in structure to 6/7 identified from E. purpurea (Bauer et al., 1988) and E. angustifolia (Bauer and Wagner, 1991), showed 100% mortality at 24 h against A. aegyptii at [10.sup.-5] mg/ml (Jondiko, 1986). In our study, the high activity noted for alkamides 6 and 7 from E. purpurea supports the mosquitocidal activity indicated from E. angustifolia.
Interestingly, both 2-methylbutylamides, 4 and 5, demonstrated the lowest mosquitocidal activity in the assay as compared to the isobutylamides tested. The presence of an E/Z configuration of the double bonds is regarded as important for conveying the highest degree of insecticidal, and thus biological, activity (Greger, 1984). From the results of the mosquitocidal assay, it appears that the 2-methyl versus an isobutyl functionality may also play a role in the effectiveness of alkamides as mosquitocidal compounds.
Previously, eight alkamides from E. angustifolia D.C. and ten alkamides from various Achillea species were examined for cyclooxygenase and 5-lipoxygenase activities (Muller-Jackic et al., 1994). A mixture of 6/7, isolated from E. angustifolia roots, demonstrated 54% inhibition of COX from microsomal sheep seminal vesicle preparations at a concentration of 50 [micro]/ml. The mixture of 6/7 isolated from E. purpurea roots in our study did not show activity at 100 [micro]g/ml for either COX-I or COX-II. Alkamides 1-5 from E. purpurea roots have not been examined for cyclooxygenase inhibitory activity; however, undeca-2E,4E-diene-8, 10-diynoic acid isobutylamide, isolated from Achillea millefolium L., was shown to inhibit COX-I enzyme at 40% (Muller-Jakic et al., 1994), while compounds 1 and 2 exhibited 36 and 60% COX-I enzyme inhibition, respectively.
Compound 2 exhibited the highest inhibition against both COX-I and COX-II enzymes. The 2-methylbutylamides, compounds 4 and 5, demonstrated higher COX-I and COX-II inhibitory activity when compared to the majority of the isobutylamides tested, with the exception of compounds 2. Compounds 1 and 3, differing in one methyl group, demonstrated equal inhibitory activities for both COX-I and COX-II enzymes, indicating that the inhibition of COX-I and COX-II enzymes is unaffected by the addition of a single methyl group.
The E. purpurea alkamides examined in this study did not show selective inhibition of COX-II enzyme. Additional work in identifying the structure activity relationships of E. purpurea alkamides and alkamides of similar structure differing in E/Z configurations would aid in determining the potency of these alkamides for specific phytoceutical applications.
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[FIGURE 3 OMITTED]
The authors would like to thank Dr. Steve Missler for performing the LC/MS analysis and Dr. Subash Gupta of the Sparrow Pain Center (Lansing, Michigan) for providing the celecoxib (Celebrex[TM]) and rofecoxib (Vioxx[TM]) physician's professional samples.
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An agent that kills insect larvae.
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L. J. Clifford (1)
M. G. Nair (2)
J. Rana (3)
D. L. Dewitt (4)
(1.) Department of Food Science and Human Nutrition and National Food Safety and Toxicology Center, Michigan State University, East Lansing, Michigan East Lansing is a city in the U.S. state of Michigan. The city is located directly east of Lansing, Michigan, the state's capital. Most of the city is within Ingham County, though a small portion lies in Clinton County. , USA
(2.) Department of Horticulture and National Food Safety and Toxicology Center, Michigan State University, East Lansing, Michigan, USA
(3.) Alticor Corporation, Ada, Michigan, USA
(4.) "Department of Biochemistry, Michigan State University, East Lansing, Michigan, USA
M. G. Nair, Bioactive Natural Products and Phytoceuticals, 173 National Food Safety and Toxicology Center, Michigan State University, East Lansing, Michigan 48824, USA