Naturally occurring neuronal NO-synthase inactivators found in Nicotiana tabacum (Solanaceae) and other plants.
NO-synthase (NOS) is a heme-containing enzyme that catalyzes the oxidation of L-arginine to nitric oxide, an important cellular signaling molecule. Recently, it was found that aqueous extracts of tobacco cigarettes cause the inactivation of the neuronal isoform of NOS (nNOS) and that this may explain some of the toxicological effects of smoking. Although the exact identity of the chemical inactivator(s) is not known, we wondered if extracts prepared from other plants, including those closely related to tobacco, Nicotiana tabacum (Solanaceae), would similarly inactivate nNOS. We examined 33 plants, representing diverse members of the plant kingdom ranging from whisk fern, Psilotum nudum (Psilotaceae) to tobacco and discovered 18 plants that contain a chemical inactivator(s) of nNOS. Of these plants, 16 are members of the core asterids flowering plant group. Of these asterids, 6 are members of the Solanaceae family, of which tobacco is a member. Based on the phylogenetic relationship of the plants, it is possible that the same chemical or related chemical inactivator(s) exist. This, in turn, may help elucidate the structure of the chemical(s), as well as provide a source of a potentially novel inactivator of nNOS. The alkaloid nicotine can be excluded as putative nNOS inhibitor.
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Keywords: Nicotiana tabacum; Nitric oxide; Neuronal NO-synthase; Tobacco; Inactivation
Nitric oxide (NO) is a reactive, gaseous, lipophilic molecule that is a key intracellular signaling factor (Crane et al., 1997; Andrew and Mayer, 1999; Alderton et al., 2001; Vallance and Leiper, 2002). The rapidly diffusible NO regulates various physiological functions, including neuronal function (Crane et al., 1997; Andrew and Mayer, 1999; Rosen et al., 2002). This production of NO is due to NO synthase (NOS), a hemoprotein enzyme that catalyzes the conversion of L-arginine to L-citrulline and NO. There is great interest in developing selective NOS inhibitors for clinical use (Marletta, 1994; Snyder and Bredt, 1992; Bryk and Wolff, 1999; Hobbs et al., 1999). For example, a specific inhibitor of the neuronal isoform of NOS (nNOS) would be of potential clinical benefit for neuroprotection during stroke, attenuation of opiate withdrawal symptoms (Vaupel et al., 1995a, b; Kimes et al., 1993), and for the treatment of obesity (Morley and Flood, 1992).
It is established that aqueous extracts of cigarette and cigarette smoke cause the inactivation of nNOS (Demady et al., 2003) and this could be the molecular mechanism for some of the toxic effects of smoking. The nNOS inactivation is due to a low molecular weight, non-volatile, cationic, organic molecule(s) present in the extract (Demady et al., 2003). In particular, an alkaloid-containing fraction from tobacco was prepared by cation exchange chromatography and it was shown that this fraction contains the inhibitor(s) (Demady et al., 2003). The inhibitor(s) did not bind to anion exchange resin, consistent with the cationic nature of the inhibitor. The alkaloid-containing fraction contains nicotine in great abundance. However, we have shown with pure nicotine that it is not the inhibitor (Demady et al., 2003). Although the exact identity of the chemical(s) responsible is not known, we wondered if extracts prepared from other plants have a chemical(s) that could inactivate nNOS. It was thought that if the inactivator(s) of nNOS was found only in specific plants, that this may represent a novel chemical entity. In this respect, extracts made from lettuce-based cigarettes did not inactivate nNOS (Demady et al., 2003). Thus, we chose a variety of plants from diverse phylogeny to survey the prevalence of such nNOS inactivator(s).
In the course of these studies, we also discovered that plants contain a high molecular mass factor, most likely a protease, which leads to degradation of nNOS protein and interfered with the ability to survey for nNOS inactivators. We found that heat-denaturation of the plant extracts completely abrogates the effect of the high molecular weight factor, but has no effect on the chemical inactivator(s). By the use of this method, we examined 33 plants and discovered that tobacco, Nicotiana tabacum (Solanaceae), and 17 other plants contain a chemical(s) that can inactivate nNOS. The majority of the plants with the nNOS inactivator(s) are members of the core asterids flowering plant group. Although it remains to be determined if the same chemical inactivator is present in all the plant extracts, our findings strongly suggest that the chemical nNOS inactivator(s) is a natural product present in select families of plants.
Materials and methods
(6R)-5,6,7,8-Tetrahydro-L-biopterin (BH4) was purchased from Dr. Schirck's Laboratory (Jona, Switzerland). L-arginine, glucose-6-phosphate, glucose-6-phosphate dehydrogenase, superoxide dismutase, calmodulin, catalase, NADPH, and NADP+ were purchased from Sigma. Activated charcoal (Darco, G60) was purchased from Aldrich (Milwaukee, WI). Anion exchange resin (AG1-X10) and cation exchange resin (AG 50W-X8) were purchased from Bio-Rad (Hercules, CA). Tobacco, chocolate, chasteberry, bitter yam, yerba mate, beehive ginger, bread palm, common fig, silktree, black pepper, wintersweet, coconut, rehmannia, and whisk fern were obtained from the University of Michigan Matthaei Botanical Gardens. Eggplant, long leaf ground cherry, and Japanese belladonna were obtained from the W.J. Beal Botanical Gardens at Michigan State University. Tomato and white petunia were obtained from a local nursery or garden. Regular non-filter Camel Cigarettes (R.J. Reynolds Tobacco Co.), cilantro, spearmint, carrot, chard, cabbage, spinach, dandelion, fennel, parsley, and celery were purchased from a local grocery store.
Preparation of plant extracts
The leaves were removed from the stems and stalks of plants and minced with scissors. The minced leaves were weighed and then dried in a vacuum desiccator for 4 days to determine the dry weight. The dried leaves were ground to a fine powder with a mortar and pestle. Water was added to the ground leaves to attain a concentration of 55 mg dried weight of leaves/ml and transferred to 14 ml tubes. The tubes were placed on a rotator mixer for 24 h at room temperature. The samples were spun at 2000 x g for 4 min to remove debris and then vacuum-filtered through a series of Buchner funnels with glass fritted discs of porosities from coarse (40-60 [micro]m) to fine (4-5.5 [micro]m). To prepare the heat-denatured samples, the extracts were heated for 10min in a boiling water bath and cooled immediately on ice. The inactivator(s) was stable for at least 1 month at 4 [degrees]C. Extracts of cigarettes were prepared by the same process as above except the paper wrapper was removed.
Studies on the nature of the nNOS inactivator(s) in aqueous tobacco leaf extract
To test for volatility of the inactivator(s), we placed 0.5-ml aliquots of aqueous tobacco leaf extract into eppendorf tubes, which were placed in a SpeedVac, and dried to completeness (approximately 12 h). The residue was reconstituted in 0.5 ml of water and tested for the presence of the nNOS inactivator. This material was compared to the original solution of tobacco extract. We also determined if the inactivator(s) would pass through a Centricon (Amicon, 3000 molecular weight cut-off) filter. An aliquot (1.5 ml) of aqueous tobacco leaf extract was placed in the Centricon filter and processed according to the manufacturers instructions. The filter was spun at 3000 x g for 4 h at 4 [degrees]C. The retentate was reconstituted to a final volume of 1.5 ml with water. The filtrate and retentate were tested for their ability to inactivate nNOS. The aqueous tobacco leaf extract was also tested for binding to activated charcoal. A 250 [micro]l-aliquot of activated charcoal (5% w/v) was placed in an eppendorf tube and spun (16,000 x g) on a microcentrifuge for 5 min. The water was removed and 500 [micro]l of tobacco leaf extract was added and the tube was placed on a rotator for 30 min. The mixture was spun down again and the supernatant was tested for the presence of the inactivator(s).
The nature of the inactivator(s) was also tested by ion-exchange chromatography. Aqueous tobacco extract (1 ml) was loaded onto a cation exchange column (Bio-Rad AG 50W-X8, sodium form, 0.5 ml of resin loaded in a 5 3/4 inch disposable Pasteur pipette with a glass wool plug) equilibrated with water. Fractions were collected from the flow-through and tested for the presence of the inactivator. In other studies, aqueous tobacco extract was treated with an anion exchange column (Bio-Rad AG1-X10, chloride form) instead of the cation exchange column and the resulting fractions were tested for the inactivator.
Treatment of nNOS with extracts and the NOS activity assay
The rat nNOS was overexpressed in Sf9 insect cytosol and purified as previously described (Demady et al., 2003). The specific activity of the purified nNOS preparation was approximately 1050 nmol/min/mg of protein. For assessment of the presence of the inactivator(s), the purified nNOS (80 [micro]g/ml) was added to a 'first reaction mixture' of 40 mM potassium phosphate, pH 7.4, containing 0.2 mM Ca[Cl.sub.2], 500 unit/ml superoxide dismutase, 100 units/ml catalase, 80 [micro]g/ml calmodulin, 100 [micro]M [BH.sub.4], 1 mM dithiothreitol, the desired concentration of extract, and an NADPH-generating system composed of 0.4 mM [NADP.sup.+], 10 mM glucose 6-phosphate, and 1 unit of glucose 6-phosphate dehydrogenase/ml, expressed as final concentrations, in a total volume of 180 [micro]l. The reaction mixture was incubated at 30 [degrees]C for 60min and aliquots (10 [micro]l) were taken and transferred to an 'oxyhemoglobin assay mixture' containing 200 [micro]M Ca[Cl.sub.2], 250 [micro]M L-arginine, 100 [micro]M [BH.sub.4], 100 units/ml catalase, 10 [micro]g/ml calmodulin, 25 [micro]M oxyhemoglobin, and an NADPH-generating system composed of 0.4 mM [NADP.sup.+], 10 mM glucose 6-phosphate, and 1 unit of glucose 6-phosphate dehydrogenase/ml, expressed as final concentrations, in a total volume of 190 [micro]l of 40 mM potassium phosphate, pH 7.4. The assay mixture was incubated at 37 [degrees]C and the rate of NO-mediated oxidation of oxyhemoglobin was monitored by measuring the absorbance at [lambda]401-411 nm with a microtiter plate reader (SpectraMax Plus, Molecular Devices, Sunnydale, CA). The rate of oxidation was determined from the linear portion of the time-dependent changes in absorbance. The [IC.sub.50] for each extract was calculated from a non-linear regression analysis with the use of PRISM software (GraphPad Software Inc., San Diego, CA).
SDS-PAGE and Western blot analysis
An aliquot (20 [micro]l) of the first reaction mixture was quenched with an equal volume of sample buffer containing 5% SDS, 20% glycerol, 100 mM dithiothreitol, and 0.02% bromophenol blue in 125 mM Tris-HCl, pH 6.8. The samples were boiled for 3min and an aliquot (20 [micro]l) was submitted to 6% SDS-PAGE (10 x 8 cm). Proteins were then transferred to nitrocellulose membranes (0.2 [micro]m, BioRad) and probed with 0.1% anti-nNOS. An anti-rabbit IgG conjugated to peroxidase was used as a secondary antibody at a concentration of 0.01% (Chemicon International, Temecula, CA). Immunoreactive bands were visualized with the use of enhanced chemiluminescence reagent (Super Signal, Pierce) and X-Omat film (Eastman Kodak Co.).
HPLC fingerprint of the active extracts
HPLC was performed with the use of a Waters Alliance 2695 equipped with a 2996 photodiode array detector (Waters Corp., Milford, MA). An aliquot (10 [micro]l) of the plant extracts was injected onto an HPLC column (C18 Vydac 5 [micro]m 4.6 x 250 mm) equilibrated with 99% solvent A (water) and 1% solvent B (acetonitrile) at a flow rate of 1.0ml/min. After 10 min, a linear gradient was run to 20% solvent B over 20 min and then to 80% solvent B over 5 min. The absorbance at 260 nm was monitored.
Water-soluble extract of tobacco-based cigarettes contain a chemical(s) that inactivates nNOS in a time-dependent manner (Demady et al., 2003). The chemical(s) is of low molecular mass, non-volatile, cationic, and has the ability to bind to activated charcoal. We wondered if such an inactivator(s) is present in other plants especially those closely related to tobacco plants. We therefore planned to test water extracts from a variety of plants that represent diverse phylogeny. In the course of these studies, we discovered a factor unrelated to the chemical inactivator(s) that is most likely a protein, which is present in fresh plant leaves, that causes a time-dependent loss of nNOS protein. As shown in Fig. 1, aqueous extracts of tobacco leaves causes the loss of nNOS protein (cf. lane 3 with lane 1). This loss does not occur with cigarette extracts (lane 2) or when the tobacco leaf extract is heat-denatured (lane 4). This loss of nNOS protein caused by tobacco leaf extract prevented an accurate analysis of the chemical nNOS inactivator(s) in fresh plant leaf extracts. The loss of nNOS protein is not observed for cigarette extracts, perhaps due to the drying and curing process that denatures the putative protein factor. Since heat-denaturing of the fresh plant leaf extracts abrogated the loss of nNOS, we further explored this finding to develop a method so that the chemical nNOS inactivator(s) could be better analyzed. We chose to initially work out the method by comparison of fresh tobacco leaf extracts with cigarette extracts, which have been shown to contain a low molecular mass chemical inactivator(s) (Demady et al., 2003).
[FIGURE 1 OMITTED]
[FIGURE 2 OMITTED]
As shown in Fig. 2, aqueous tobacco leaf extracts (closed circles) cause the loss of nNOS activity in a concentration-dependent manner with an [IC.sub.50] of 0.9 mg/ml. However, the loss of activity of nNOS caused by tobacco leaf extract is much more potent than the inactivation of nNOS by cigarette extracts (closed squares) with an [IC.sub.50] of 17 mg/ml. The heat-denaturation of the extract causes a dramatic rightward shift in the dose response for tobacco leaf extract (open circles), but not for cigarette extract (open squares). Thus, it appears that the chemical inactivator(s) is heat-stable.
We further validated the heat-denaturation method by a more detailed analysis of the heated extracts. The chemical inactivator(s) derived from cigarettes is of low molecular mass and will pass through a 3000 molecular weight cutoff membrane. As shown in Fig. 3A, passing fresh tobacco leaf extract through a 3000 molecular weight cut-off membrane shows that both the retentate (hatched bars, Retentate), which comprises high molecular weight compounds, as well as the filtrate (hatched bars, Filtrate), which contains low molecular weight compounds, cause the loss of nNOS activity. Heat-denaturation of the tobacco leaf extract completely abrogates the effects of the retentate fraction (solid bars, Retentate) indicating that the high-molecular weight factor was denatured. This is entirely consistent with the lack of effect on nNOS protein described above of the heat-denatured tobacco leaf extract. The filtrate fraction from the heat-denatured extract had no effect (solid bars, Filtrate).
[FIGURE 3 OMITTED]
To further confirm that the heat-denatured tobacco extract represents a chemical inactivator(s), similar to that previously characterized (Demady et al., 2003), we examined three different chemical properties (Fig. 3B). We found that drying the sample on a SpeedVac apparatus and then reconstituting the sample in the original volume of water (SV) inactivated nNOS as well as the untreated extract (Initial). Thus, the inactivator(s) is non-volatile. The inactivator(s) in heat-denatured tobacco leaf extract bound to activated charcoal (Char), similar to that described previously for the chemical inactivator(s) in cigarettes. Furthermore, the inactivator(s) is cationic (FT, Cation) and not anionic (FT, anion), based on the ability of the flow-through fraction to inactivate nNOS. Thus, the heat-denaturation removes the effect of the high-molecular weight factor and allows for the accurate measure of the low molecular weight chemical inactivator(s) that previously was established to selectively inactivate nNOS (Demady et al., 2003).
As shown in Table 1, we tested for the presence of the inactivator(s) of nNOS in plants obtained from the University of Michigan Matthaei Botanical Gardens, the W.J. Beal Botanical Gardens at Michigan State University, a local grocery store, a local nursery, and a local home garden. We made a point to select organically grown plant leaves to reduce the influence of added pesticides. For those extracts found to inactivate nNOS, the concentration-dependence of the extract on inactivation of nNOS was determined. The data were fitted to non-linear curve function with the use of PRISM software to determine the [IC.sub.50].
The highest concentration tested was 50 mg/ml and if there was no effect seen under this condition, we indicated on Table 1 that there was no inactivation (NI). Of the 33 plants tested, 19 had no effect. These plants include edible leaves such as cabbage, spinach, and parsley. There were 14 plants that contain nNOS inactivator(s). For those that did inactivate nNOS, there are varying degrees to which the plant extracts inactivated nNOS based on the [IC.sub.50] values. In this group, some plants such as tobacco, Rehmannia, and white petunia have strong inactivating activity with [IC.sub.50] of less than 7 mg/ml. Other plants, such as eggplant and cilantro, have moderate inactivating activity with [IC.sub.50] of 11-30 mg/ml, whereas Japanese Belladonna had weak inactivating activity with an [IC.sub.50] of 45 mg/ml.
As shown in Fig. 4, a phylogenetic tree of the 33 plants tested was constructed according to the classification of plants by the University of California Museum of Paleontology and the [IC.sub.50] for nNOS inactivation is also indicated in parentheses. The plants covered a wide spectrum of families throughout the plant kingdom. We found that the three most basal flowering plants (bread palm, common fig, and black pepper) did not contain a chemical inactivator(s) of nNOS. We observe that many of the plants with nNOS inactivator(s) are plants in the core asterids flowering plant group, which comprises wintersweet, Chinese foxglove, dandelion, yerba mate, cilantro and its relatives, and tobacco and its relatives. In addition, some members of the Solanaceae family, which include tobacco, contain chemical(s) that were amongst the strongest inactivators of nNOS. The nNOS inactivator(s) also occurs in two other plants, the beehive ginger, which belongs to the Zingiberaceae family, and chard, a member of the Chenopodiaceae family.
We also measured the [IC.sub.50] of all the extracts before heat denaturation. This reflects the contribution of both the protein factor and chemical inactivator(s). As shown in Table 2, we found 9 plants, in addition to tobacco, contain the protein factor that leads to loss of nNOS protein. In other words, these are the plant extracts with very potent apparent [IC.sub.50]s that were then made less potent by heat-denaturation of the extracts. In all the other plants not shown in Table 2 but present in Table 1, there was either no inactivation or the [IC.sub.50] could be explained predominately by the chemical inactivator(s). Interestingly, five of the nine plants in Table 2 are members of the Solanaceae family, which is also the family of plants that contain the strongest chemical inactivator(s).
[FIGURE 4 OMITTED]
As shown in Fig. 5, the plant extracts that were found to inhibit nNOS were examined by reverse phase HPLC analysis. The profiles of the extracts at 260 nm are shown.
Aqueous extracts prepared from fruits, such as apples and grapes, and vegetables, such as broccoli and cauliflower, were found to promote endothelium-dependent vasorelaxation, which was inhibited by the nitric oxide synthase inhibitor [N.sup.G]-monomethyl-L-arginine, in rat aortas (Fitzpatrick et al., 1995). This indicates that plant extracts can enhance the biologically available NO. This could arise from activation of NOS, as well as from a variety of other downstream mechanism, including decreased degradation of NO. Other studies have shown that aqueous extracts prepared from plants, such as the Curcuma zedoaria, Satureja hortensis L. (summer savory), and Zingiber officinale, lower nitric oxide (NO) levels in animals and cells (Hong et al., 2002a, b; Uslu et al., 2003; Ippoushi et al., 2003). These studies were performed with the use of models for inflammation or lipopolysaccharide challenge that induce a specific isoform of NOS and thus these authors concluded that decreased NO levels were due to limiting the induction of NOS rather than direct inhibition of activity. Thus, it is clear that plants contain factors that modulate NOS in a variety of ways. Our current study focuses on the direct effects of plant chemicals on nNOS.
This work was prompted by an earlier study where the aqueous extracts of cigarettes have been shown to directly inactivate nNOS (Demady et al., 2003). This inactivation is due to a low molecular weight chemical(s) that is non-volatile, organic, and cationic (Demady et al., 2003). In this current work where we use an assay that measures the direct effects on nNOS, we show for the first time that certain plants contain a chemical(s) that directly inactivates nNOS. By analysis of 33 plant extracts, we found 18 plants with inactivators of nNOS. All these plants, except two, that contain a chemical inactivator(s) of nNOS are clustered among the core asterids flowing plants, which contain the Solonaceae family. In addition to the core asterids, the ability to inactivate nNOS occurs independently in two other plants, the beehive ginger and the chard. Thus, the preponderance of the plants in the core asterids group that contain the inactivator strongly argues for a common chemical or chemicals in these plants. Perhaps, a common metabolic pathway exists that leads to formation of these chemicals. The existence of an inactivator in beehive ginger and chard appear to be an independent occurrence. It is likely that beehive ginger and chard do not have the same chemical inactivator(s) as the core asterid group. On the other hand, not all members of the core asterid group possess the inactivator(s), perhaps indicating the disappearance of select metabolic pathways responsible. This argues that the pathways are not essential for plants in the asterid group.
We have also established that the inactivator(s) in cigarettes is neither created nor added during the processing of tobacco leaves to manufacture the cigarette (Demady et al., 2003). That additives are not responsible for inactivation is consistent with the finding that research grade cigarettes, which do not contain additives, inactivate nNOS. The inactivation is not due to nicotine (Demady et al., 2003), and thus we are not simply researching the nicotine content of different plants in our assay.
In the course of these studies on the chemical inactivator(s), we discovered a high molecular weight, heat labile factor that appears to degrade nNOS. This factor is likely a protease, although the specificity for nNOS is unknown. Interestingly, of the 33 plants tested, we detected 9 plants with the protein factor and five of these plants are members of the Solanaceae family.
We do not know why specific plants contain chemicals and protein modulators of nNOS. Since plants do contain a functional NOS (Wendehenne et al., 2003; Zemojtel et al., 2004), it is possible that the chemical or protein modulators evolved for regulation of plant NOS. Alternatively, the chemical inactivator(s) may have evolved to affect NOS in other organisms, perhaps insects, as a mechanism of defense. Whatever the reason for the existence of a plant-derived inactivator(s) of NOS, this discovery provides an opportunity to identify potential novel inactivators and regulators of NOS for pharmacological and biological use.
This work was supported in part by the Philip Morris External Research Program and the National Institutes of Health Grants ES08365 and DA22354. E.R.L. is a trainee under Pharmacological Science Training Program GM07767 from the National Institutes of Health. We thank Dr. Frank Telewski (W.J. Beal Botanical Gardens at Michigan State University) and Judith M. Birk (home garden) for supplying some of the plants used in this study. We thank Dr. Paul Fine of the University of Michigan for his input on the data analysis.
[FIGURE 5 OMITTED]
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Ezra R. Lowe (a), Andrew C. Everett (a), Miranda Lau (a), Anwar Y. Dunbar (a), David Michener (b), Yoichi Osawa (a,*)
(a) Department of Pharmacology, The University of Michigan Medical School, 1301 Medical Science Research Building III, Ann Arbor, MI 48109-0632, USA
(b) University of Michigan Matthaei Botanical Gardens and Nichols Arboretum, Ann Arbor, MI 48109, USA
Abbreviations: NOS, nitric oxide synthase; nNOS, neuronal nitric oxide synthase
*Corresponding author. Tel.: 1 + 734 763 5797; fax: + 1 734 763 4450.
E-mail address: firstname.lastname@example.org (Y. Osawa).
Table 1. The median inhibitory concentration ([IC.sub.50]) of aqueous extracts of heat-denatured plant leaves needed for inactivation of nNOS Order Family Plant name Author Scrophulariales Scrophulariaceae Rehmannia elata N.E. Br. ex Prain Solanales Solanaceae Petunia axillaris (Lam.) Britton, Stern & Poggenb. Solanales Solanaceae Nicotiana tabacum L. Solanales Solanaceae Solanum melongena L. Apiales Apiaceae Apium graveolens L. Apiales Apiaceae Coriandrum sativum L. Solanales Solanaceae Vitex agnus-castus L. Lamiales Lamiaceae Mentha spicata L. Zingiberales Zingiberaceae Zingiber spectabile Griff. Caryophyllales Chenopodiaceae Beta vulgaris L. Solanales Solanaceae Physalis longifolia Nutt. Aquifoliales Aquifoliaceae Ilex paraguensis St.Hilaire Solanales Solanaceae Lycopersicon Mill. esculentum Solanales Solanaceae Scopolia carniolica Jacq. Solanales Solanaceae Lycopersicon Mill. esculentum Apiales Apiaceae Daucus carota L. Solanales Solanaceae Lycopersicon Mill. esculentum Malvales Sterculiaceae Theobroma cacao L. Solanales Solanaceae Lycopersicon Mill. esculentum Gentianales Apocynaceae Acokanthera (Hochstetter) oblongifolia Codd Apiales Apiaceae Petroselinum sativum Hoffm. Cycadales Cycadaceae Cycas rumphii Miq. Urticales Moraceae Ficus carica L. Sapindales Sapindaceae Paullinia pinnata L. Fabales Fabaceae Albizia julibrissin Durazz. Piperales Piperaceae Piper nigrum L. Psilotales Psilotaceae Psilotum nudum (L.) P. Beauv. Arecales Arecaceae Cocos nucifera L. Brassicales Brassicaceae Brassica oleracea L. Caryophyllales Chenopodiaceae Spinacia oleracea L. Asterales Asteraceae Taraxacum officinale F.H. Wigg. Apiales Apiaceae Foeniculum vulgare Mill. Dioscoreales Dioscoreaceae Dioscorea bulbifera L. Order Common name [IC.sub.50] (mg/ml) Scrophulariales Chinese Foxglove 4.5 Solanales White Petunia 5.7 Solanales Tobacco 6.5 Solanales Eggplant 11.5 Apiales Celery 12 Apiales Cilantro 12.4 Solanales Chasteberry 17.3 Lamiales Spearmint 19.8 Zingiberales Beehive Ginger 20.5 Caryophyllales Chard 21.4 Solanales Long Leaf Ground 23.4 Cherry Aquifoliales Yerba Mate 26.1 Solanales Tomato (Black Krim) 28.7 Solanales Japanese Belladonna 44.7 Solanales Tomato NI Apiales Carrot NI Solanales Tomato (Chianti Rose) NI Malvales Chocolate NI Solanales Tomato (Watermelon) NI Gentianales Wintersweet NI Apiales Parsley NI Cycadales Bread Palm NI Urticales Common Fig NI Sapindales [no common name] NI Fabales Silktree, Mimosa NI Piperales Black Pepper NI Psilotales Whisk Fern NI Arecales Coconut NI Brassicales Cabbage NI Caryophyllales Spinach NI Asterales Dandelion NI Apiales Fennel NI Dioscoreales Bitter Yam NI Table 2. Plants that contain a heat-labile factor that degrades nNOS. The [IC.sub.50] was determined on aqueous extracts before heat- denaturation Order Family Plant name Apiales Apiaceae Coriandrum sativum Solanales Solanaceae Solanum melongena Solanales Solanaceae Nicotiana tabacum Solanales Solanaceae Lycopersicon esculentum Solanales Solanaceae Physalis longfolia Lamiales Lamiaceae Mentha spicata Solanales Solanaceae Scopolia carniolica Caryophyllales Chenopodiaceae Beta vulgaris Apiales Apiaceae Daucus carota Order Common name [IC.sub.50] (mg/ml) Apiales Cilantro 0.5 Solanales Eggplant 0.7 Solanales Tobacco 0.9 Solanales Tomato 1.0 Solanales Long Leaf Ground Cherry 1.1 Lamiales Spearmint 1.2 Solanales Japanese Belladonna 3.3 Caryophyllales Chard 3.4 Apiales Carrot 4.5