CA2615200A1 - Use of alkylphthalides for inducing phase 2 proteins - Google Patents

Abstract

Use of a therapeutically effective amount of an alkylphthalide for inducing phase 2 proteins is disclosed.

Description

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Field of the Invention [0001] The present invention is related to compounds having a biochemical effect, particularly to the use of alkylphthalides for inducing phase 2 proteins.

Background of the Invention

[0002] The potential of phytochemicals (plant natural products) to positively impact human health through prevention of disease is increasingly recognized (Surh 2003). Many phytochemicals with health-promoting activities, often termed nutraceuticals, exert their effects through the induction of an "indirect antioxidant"
response mediated through phase 2 proteins (Talalay 2000; Wu et al. 2004). Such proteins act to metabolize oxidative radicals, thereby attenuating inflammation, and to detoxify carcinogens. Phase 2 protein inducers may prove useful in chemoprevention of chronic diseases such as cancer, neurodegeneration and cardiovascular disease. Examples of phytochemicals with phase 2 protein inducing activity that have been isolated from food or spice plants are shown in Table 1. Such compounds induce expression of phase 2 protein genes by binding with the sensor protein Keap1 and resulting in the release of the transcription factor Nrf2 (Dinkova-Kostova et al. 2002). Nrf2 moves from the cytoplasm to the nucleus where it, together with small Maf proteins, interacts with antioxidant response elements (AREs) in promoter regions and activates transcription of phase 2 proteins.

[0003] Oxidative and electrophilic stresses are thought to be major contributing factors in the development of chronic diseases including cancer, atherosclerosis and neurodegeneration [Griendling et al. 2003; Valko et al. 2007]. A promising method to prevent damage caused by reactive oxygen species and other harmful electrophiles is through enhanced expression of cytoprotective genes. Transcription factor NF-E2-related factor 2(Nrf2) regulates the transcription of numerous detoxifying and antioxidant genes through the antioxidant responsive element (ARE) in their promoter regions.
Under non-inducing conditions the cytosolic actin-bound protein Kelch-like ECH-associated protein 1 (Keapl) binds to Nrf2 and directs its degradation [Cullinan et al. 2004].
Certain small molecules can alkylate cysteine residues on Keap1 thereby disrupting its association with Nrf2 and allowing Nrf2 to translocate to the nucleus where it directs the transcription and expression of cytoprotective genes [Wakabayashi et al. 2004]. Many of these genes encode classical phase 2 metabolizing enzymes, and the alkylating molecules involved are termed phase 2 protein inducers (P2PIs).

Table 1 Phytochemicals with Phase 2 Protein Inducing Activity Phytochemical Compound Plant Species Reference Class Sulforaphane Isothiocyanate Brassica oleracea (broccoli) Zhang et al. (1992) Xanthohumol Chalcone Humulus lupulus (hop) Miranda et al. (2000) Curcumin I Diarylheptanoid Curcuma longa (turmeric) 2-methoxy-1,3,6- Anthraquinone Morinda citrifolia (noni) Pawlus et al. (2005) trihydroxyanthraquinone Withaphysacarpin Withanolide Physalis philadelphica Kennelly et al.
(tomatillo) (1997) Carnosic acid catechol Rosmarinus officinalis Satoh et al. (2007) (Rosemary) p-Hydroxyphenethyl Phenylpropanoid Allium cepa (onion) Xiao and Parkin trans-ferulate (2006) Pinostrobin Flavonoid Boesenbergia pandurata Fahey and (Thai ginger) Stephenson (2002)

[0004] Although an increasing number of cytoprotective phytochemicals have been isolated (Kinghorn et al. 2004), many food and medicinal plants have not yet been investigated for their phase 2 protein inducing potential. Certainly very little of the flora of Canada has been screened for this activity. There are only a limited number of plant-derived compounds that have been shown to possess phase 2 protein activity.
Some of these are toxic, exhibit poor absorption or pharmacokinetics or are not present at high concentrations in plants.

[0005] Several potent P2PIs, including sulforaphane from broccoli [Zhang et al.
1992] and xanthohumol from hops [Dietz et al. 2005] (see Table 1), have been isolated from plants. It is likely that phytochemicals with phase 2 protein inducing activity contribute to the decrease in chronic diseases observed in epidemiological studies of fruit and vegetable consumption [Fahey et al. 2007].

[0006] Various bioactivities have been attributed to (Z)-ligustilide and other alkylphthalides. Mice with ischemic injury treated with (Z)-ligustilide have higher levels of glutathione peroxidase and superoxide dismutase than untreated controls (Kuang et al 2006). Both glutathione peroxidase and superoxide dismutase are classified as antioxidant proteins and not phase 2 proteins.

[0007] Further, Dietz et al. (2007) report a ligustilide rich fraction of an Angelica sinensis root extract with strong quinone reductase inducing properties (CD=6.8 1.2pM), but does not say that (Z)-ligustilide is the active principle.

[0008] Identification of compounds that induce phase 2 proteins may provide drugs that can be used to prevent chronic diseases. Furthermore, knowledge about plant-derived phase 2 protein inducers may be useful in informing consumers about foods and dietary supplements useful for chemoprevention.

Summary of the Invention

[0009] There is provided a use of a therapeutically effective amount of an alkylphthalide for inducing phase 2 proteins.

[0010] There is also provided a method for inducing phase 2 proteins in a subject requiring phase 2 protein induction comprising administering a therapeutically effective amount of an alkylphthalide to the subject.

[0011] Surprisingly, not only are alkylphthalides potent inducers of phase 2 proteins, they are also less toxic than other well known phase 2 protein inducers.

[0012] Alkylphthalides that are monofunctional phase 2 protein inducers are particularly useful. Some alkylphthalides useful in the present invention include, for example, (Z)-ligustilide, 6,7-dihydroxyligustilide (DHL), (Z)-6,7-epoxyligustilide, angelicide, (Z)-butylidenephthalide, butylphthalide, 2,4-dihydrophthalic anhydride, levistolide, cnidilide, tokinolide and mixtures thereof.

[0013] Alkylphthalides are present in many natural sources, especially plants, particularly plants of the family Apiaceae (also known as Umbelliferae).
Alkylphthalides for use in the present invention may be obtained from such natural sources.
Some examples of suitable plants that contain alkylphthalides are Ligusticum porteri, Ligusticum wallichii, Ligusticum chuanxiong, Angelica sinensis (common name "dong quai"
or "danggui") and Cnidium officinale. Many other sources are known to one skilled in the art.

[0014] Alkylphthalides from natural sources may be obtained through a variety of techniques. For example, whole plants or plant parts may be used as gathered, plants or plant parts may be dried and optionally ground into a powder, alkylphthalides may be extracted from plants or plant parts with one or more solvents (e.g. organic solvents such as methanol, chloroform, etc.) and isolated from the solvent by crystallization from and/or evaporation of the solvent. Such techniques are generally well known in the art.

[0015] Alkylphthalides may be used in pure or semi-pure form to induce phase 2 proteins in animals, for example mammals, (e.g. humans, rats, rabbits, monkeys, horses, cows, dogs, cats).

[0016] In the context of the present invention, alkylphthalides may be used as nutraceuticals or dietary supplements by administering them directly in pure form or as part of a pharmacologically acceptable composition. The composition may comprise an extract, for example a plant extract, and/or may be formulated with other pharmacologically acceptable ingredients, for example, carriers, excipients, diluents or other active principles. Carriers, excipients or diluents as well as the methods of formulating the compositions are well known to those skilled in the art.
Cellulose, maltodextrin and water are preferred carriers, excipients or diluents.

[0017] Pure alkylphthalides or compositions thereof are generally formulated in a dosage form. Dosage forms include powders, tablets, capsules, solutions, suspensions, emulsions and other forms that are readily appreciated by one skilled in the art.
Administration may be orally, parenterally, intravenously or by any other convenient method. Powders, capsules and tablets for oral administration are preferred.

[0018] Alkylphthalides or compositions thereof may also be admixed with a food or beverage and taken orally in such a manner. Foods or beverages may help mask undesirable flavours and are more palatable for consumption by humans or other animals. Fortified foods and beverages may be made by adding alkylphthalides or compositions thereof during the manufacturing of the food or beverage.
Alternatively, the consumer may add the alkylphthalides or composition thereof to the food or beverage near the time of consumption. Each ingredient of the composition may be added to the food or beverage together with the other ingredients or separately from the other ingredients. Examples of foods and beverages are cereals, snack bars, dairy products, fruit juices, powdered food and dry powder beverage mixes.

[0019] Effective daily dosages of alkylphthalides may be readily determined by one skilled in the art. Generally, daily doses may be 5 mg/kg or greater based on body weight of the subject. Daily doses of 10 mg/kg or greater may be more efficacious. The surprisingly low toxicity of alkylphthalides, as determined by tests with Hepa 1 c1 c7 mouse cell cultures, may permit the use of larger daily doses than with other phase 2 protein inducers. A daily dose of from 20 mg/kg to 500 mg/kg is of general therapeutic usefulness.

[0020] Daily dosage may be given all at once in a single dose or can be given incrementally in several smaller dosages. Thus, the dosage forms of the present invention can be formulated such that the recommended daily dose is achieved by the administration of a single dose or by the administration of several smaller doses.

[0021] It is apparent to one skilled in the art that the alkylphthalides or compositions thereof may be included in commercial packages together with instructions for use in inducing phase 2 proteins. Such packages may be in the form of sachets, bottles or blisterpacks but are not limited to such. Instructions are normally in the form of a written material but are not limited to such.

[0022] As phase 2 protein inducers, alkylphthalides are useful in a number of therapeutic indications related to oxidative stress, for example, cancer chemoprevention, neuroprotection (e.g. anti-ischemia), anti-inflammation (anti-hypertension and anti-atherosclerosis), cardiovascular protection and treatment, and treatment of dermatological damage and inflammation (e.g. sunburn).

[0023] Further features of the invention will be described or will become apparent in the course of the following detailed description.

Brief Description of the Drawings

[0024] In order that the invention may be more clearly understood, embodiments thereof will now be described in detail by way of example, with reference to the accompanying drawings, in which:

[0025] Fig. 1 depicts a flow chart for isolation of (Z)-ligustilide and DHL
from a methanolic extract of Ligusticum porteri.

[0026] Fig. 2 depicts a graph of induction of the phase 2 enzyme NQO-1 in Hepa-1 c1 c7 cells. Cells were seeded in 96 well plates, allowed to grow for 24 h and incubated for 48 hours with test compounds prepared in DMSO and diluted in the culture medium.
NQO-1 activity was measured [Prochaska et al. 1998] and compared to solvent treated controls to give a fold change in activity. (R)-sulforaphane was included as a positive control. Data are represented as the mean of six wells (two separate assays done in triplicate) plus or minus the standard deviation.

[0027] Fig. 3 depicts a graph showing changes in mRNA expression of phase 1 and 2 genes by (Z)-ligustilide and controls. Hepa-1 c1 c7 cells were exposed to test compounds for 24 hours and mRNA levels analyzed by quantitative real-time RT-PCR.
Values are given with respect to solvent treated controls, and are the mean of at least three experiments plus or minus the standard deviation.

Description of Preferred Embodiments

[0028] Two phase 2 protein inducers have been isolated from the roots of Ligusticum porteri (common name "Coulter and Rose", Apiaceae or "Osha", Umbelliferae). L. porteri is native to the southern Rocky Mountains where it grows in high-altitude environments. This plant has a long history of use by Native Americans and continues to find use as a herbal medicine [Foster et al. 2002; Appelt et al.
1985; Linares et al. 1987]. Its expanding popularity as an herbal medicine has put wild populations at risk from over harvesting [Panter et al. 2004]. The two inducers, (Z)-ligustilide and 6,7-dihydroxyligustilide (DHL) have structures as follows:

I ~ ~
~ HO
O OH O
(2)-Ligustilide 6,7-Dihydroligustilide

[0029] The two inducers were identified using fractionation of a methanolic extract of L. porteri guided by the quinone reductase bioassay. (Z)-ligustilide has been previously identified from L. porteri (Beck and Stermitz 1995). Cis-6,7-dihydroxyligustilide (DHL), which is present in the crude methanolic extract from L. porteri roots, is a degradation product of Z-Iigustilide.

[0030] Materials and Methods

[0031] Isolation and identification of (Z)-ligustilide and DHL:

[0032] One hundred and four grams of dried L. porteri roots (Richters Herbs, Goodwood, ON) were ground to a fine powder, extracted twice with 500 ml of methanol and concentrated by rotary evaporation. Water was added to 10% (v/v) and the extract defatted twice with hexane. The extract was concentrated, re-suspended in 25%
methanol and extracted twice with 1% NaCI and subsequently twice with chloroform. The chloroform fraction (2.66 g), which had the highest inducing activity in the bioassay, was separated by flash chromatography over a silica gel column (2.6 x 46 cm) using a gradient of 5-100% acetone in hexane (60 min at 20 ml/min). The fractions were combined based on TLC analysis to give 6 fractions. Fraction 2 was most active in the QR bioassay and was separated by flash chromatography using a reversed phase cartridge (1.2 x 7.5 cm, Buchi, Switzerland) and a gradient of 5-100%
acetonitrile in water. The most active sub-fraction, (F2-2) was purified by semi-preparative HPLC using a reversed phase column (5 pm, 10 x 250 mm, SunfireT ", Waters, Bedford, MA) with 70%
(v/v) MeOH in water as the mobile phase and yielded (Z)-Iigustilide as the major component. (Z)-Ligustilide was identified based on UV and NMR spectroscopy [Gijbels et al. 1982] and mass spectrometry [Li et al. 2003] as well as comparison with authentic (Z)-ligustilide (Chromadex, Irvine, CA). Cis-6,7-dihydroxy-ligustilide (DHL) was purified by HPLC from a degraded (Z)-ligustilide preparation and identified by comparing of UV, NMR and mass spectrometric data with published values [Li et al. 2003; Pushan et al.
19841. Coniferyl ferulate was isolated from a chloroform fraction (prepared as described above from 25 g of L. porteri roots) by flash chromatography followed by preparative TLC
and HPLC and was identified on basis of UV, mass spectral [Lin et al. 1998]
and H1 and C13 NMR data [Lu et al. 2004]. All NMR experiments were performed in CDCI3 using a Bruker AvanceT"" 500 MHz instrument.

[0033] Cell culture:

[0034] A mouse hepatoma cell line Hepa-1 c1 c7 was purchased from the American Type Culture Collection (Manassas, VA). Cells were maintained in 75 cm2 flasks using a-minimal essential medium, without nucleosides, with GlutamaxTM
(Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (FBS) in a humidified incubator at 37 C and 5% CO2. Cells were sub-cultured once they reached 75-90% confluency.

[0035] NAD(P)H: quinone-acceptor oxidoreductase (NQO-1) induction and cell viability assay.

[0036] Hepa-1c1c7 cells were plated in 96 well plates at approximately 10,000 cells per well in 200 pL of the normal cell culture medium. After 24 h the medium was replaced with medium supplemented with 100 U/ml penicillin G, 100 Ng/mI
streptomycin, and 10% charcoal treated FBS. Dilutions of test compounds or extracts dissolved in DMSO were prepared in this medium and added to a final DMSO concentration of 0.1%
(v/v). Each concentration was tested in triplicate and two independent experiments were conducted. After 48 h, medium was decanted and cell viability determined using CalceinTM

AM reagent (Invitrogen, Carlsbad, CA). Each well was incubated in the dark with 200 ul of 4 pM Calcein AM in D-PBS at 37 C for 20 min and the fluorescence was measured using excitation and emission wavelengths of 485 nm and 535 nm respectively.
This step was shown not to interfere with subsequent assays. The Calcein AM solution was removed and the plates incubated at for 10 minutes at 37 C followed by 10 minutes at room temperature with orbital shaking in the presence of 50 pl of a solution containing 0.08% digitonin, and 2 mM EDTA, pH 7.8. Thirty pl of lysate was used for the assay [Prochaska et al. 1988] and 20 pl was used for protein determination using bicinchoninic acid [Smith et al. 1985]. Experimental NQO-1 activities were compared to the activities of DMSO (0.1 %) treated controls.

[0037] Real-time quantitative RT-PCR:

[0038] Hepa-1 c1 c7 cells were treated with test compounds as described above, lysed after 24 h and cDNA synthesized using a FastlaneTM kit (Qiagen, Valencia, CA) according to the manufacturer's instructions. Quantitative PCR was performed on a StratageneT"' Mx3000P using QuantiFastT"" SybrGreenT^" Kit (Qiagen). The primers are summarized below. PCR conditions were: 5 min at 95 C, 40 cycles of 20 seconds at 95 C, 30 seconds at 60 C. Product amplification was monitored by melting curve analysis and agarose gel electrophoresis.

[0039] Primer sequences were as follows:

GAPDH: 5'-AGGTCGGTGTGAACGGATTTG-3' (SEQ ID NO: 1) 5'-TGTAGACCATGTAGTTGAGGTCA-3' (SEQ ID NO: 2) NQO-1: 5'-AGGATGGGAGGTACTCGAATC-3' (SEQ ID NO: 3) 5'-AGGCGTCCTTCCTTATATGCTA-3' (SEQ ID NO: 4) HO-1: 5'-AAGCCGAGAATGCTGAGTTCA-3' (SEQ ID NO: 5) 5'-CCGTGTAGATATGGTACAAGGA-3' (SEQ ID NO: 6) CYP1a1: 5'-GACCCTTACAAGTATTTGGTCGT-3' (SEQ ID NO: 7) 5'-GGTATCCAGAGCCAGTAACCT-3'. (SEQ ID NO: 8)

[0040] Statistics and calculations:

[0041] Figure data are presented as means standard deviation. Statistical significance was calculated using the student's t-test. Results were considered significant if P < 0.05. CD values were calculated using a logarithmic regression.

= = ~

[0042] Results and Discussion:

[0043] Isolation and activity of (Z)-ligustilide and DHL:

[0044] A large proportion of known P2PIs have been isolated using the Prochaska assay which measures the induction of NQO-1 activity in murine Hepa-1 c1 c7 cells [Prochaska et al. 1988]. Numerous plants were screened for NQO-1 inducing activity and the methanolic extract of L. porteri roots was found to be one of the most potent inducers (CD 1.83 pg/mI) (Fig. 2 and Table 2). Solvent-solvent partitioning with hexane, chloroform and water, led to increased NQO-1 activity in the hexane and chloroform partitions (CDs = 1.54 and 0.54 pg/mi, respectively). Further fractionation of the chloroform partition by flash chromatography, solid phase extraction and semi-preparative HPLC yielded (Z)-ligustilide, as a principal component responsible for NQO-1 induction (CD = 10.5 pm) (Fig. 2 and Table 2). A degradation product of (Z)-ligustilide, cis-6,7-dihydroxy-(Z)-ligustilide (DHL), also showed significant induction of NQO-1 (CD =
19.6 pM). The dose response of (Z)-ligustilide and DHL are shown in Fig 2. DHL
was also identified in a fresh methanolic extract at low levels. Neither (Z)-ligustilide or DHL reduced cell viability at the highest concentration tested (100 pM). In addition, we tested coniferyl ferulate, the second most abundant compound (by HPLC) in the methanolic extract for NQO-1 inducing activity but found that coniferyl ferulate, unlike some other ferulic acid derivatives, was inactive [Xiao et al. 2007; Xiao et al. 2006].

Table 2 Quinone reductase induction and toxicity of (Z)-ligustilide and DHL
Compound CDa (pM) IC50 b(PM) CIC
(Z)-Ligustilide 10.5 >100 >9.55 DHL 19.6 >100 >5.1 R-Sulforaphane 0.43 23.2 53.9 a Concentration to double quinone reductase activity b Median Inhibition Concentration Chemopreventive index (ratio of IC50 to CD)

[0045] Thiol reactivity of (Z)-ligustilide and DHL:

[0046] Compounds that solely increase the expression of phase 2 genes via modification of Keap1 thiol groups are referred to as monofunctional inducers.
Bifunctional inducers increase the expression of both phase 1 and 2 genes through a separate system, involving the Aryl hydrocarbon receptor and the transcriptional enhancer, the xenobiotic responsive element (XRE). Since phase 1 enzymes are responsible for the bioactivation of harmful compounds, monofunctional P2PIs are more desirable for cytoprotection. Monofunctional P2PIs belong to at least nine distinct chemical classes but share the ability to alkylate or otherwise modify thiol groups.

[0047] The thiol reactivity of (Z)-ligustilide has been demonstrated using the model biological thiol nucleophile methyl thiogycolate [Beck et al. 1995].
This study reported 1,6-addition at position 8 as well as addition at position 7.
Incubation of (Z)-ligustilide or DHL with GSH lead to formation of a product with a mass consistent with addition of one molecule of GSH to DHL but not to (Z)-ligustilide.

[0048] Quantitative real-time RT-PCR:

[0049] In order to confirm that (Z)-ligustilide increases NQO-1 activity by increasing transcription through the Keap1-Nrf2-ARE pathway changes in gene expression of the phase 1 enzyme, CYP1 a1 and two phase 2 enzymes, heme oxygenase 1(HO-1) and quinone reductase (NQO-1) were measured using quantitative real-time RT-PCR. Hepa-1 c1 c7 cells were treated with (Z)-ligustilide, (R)-sulforaphane (monofunctional inducer positive control), or R-napthoflavone (bifunctional inducer positive control) for 24 h before cell lysis and cDNA synthesis. As shown in Fig. 3, (Z)-ligustilide increased transcription of the phase 2 genes HO-1 and NQO-1 by factors of 1.9 and 1.8 fold respectively. This level of induction was similar to that observed in the R-sulforaphane treatment (2.2 and 1.8 fold for HO-1 and NQO-1 respectively) indicating that 25 pM (Z)-ligustilide is approximately as effective as 1 pM sulforaphane at increasing phase 2 gene transcription. [3-NF, the bifunctional inducer control, was the only compound to increase the expression of the phase 1 enzyme, CYP1 a1. The level of CYP1a1 mRNA was decreased in both (Z)-ligustilide and R-sulforaphane treatments although this difference was only significant in the R-sulforaphane treatment.
The above results indicate that like, R-sulforaphane, (Z)-ligustilide is a monofunctional P2PI.

[0050] (Z)-ligustilide is a common naturally occurring phthalide that has been identified in 43 plants, 40 of which belong to the Apiaceae [Beck et al.
2007].
(Z)-ligustilide was the most abundant compound in the present methanolic extract as judged by HPLC and has been identified as a major component of the non-polar extracts of Ligusticum chuanxiong Hort. [Li et al. 2003], and Angelica sinensis (Oliv.) Diels [Lin et al. 1998], two of the most widely used traditional Chinese medicines whose indications include treating cardiovascular ailments, menstrual disorders and cerebral ischemia [Huang 1999; China 2000].

[00511 Several studies have attempted to link the apparent cardiovascular protective effects of preparations rich in (Z)-ligustilide and related phthalides with biological activities including inhibition of inflammation by reducing tumor necrosis factor-a (TNF-a) expression [Liu et al. 2005] and anti-proliferative effects on smooth muscle cells [Kobayashi et al. 1993], although the underlying mechanisms responsible for these activities remained unclear. We propose that activation of the Keapl-Nrf2-ARE
pathway is responsible, at least in part, for these activities. P2PIs have been shown to inhibit the expression of TNF-a [Thimmulappa et al. 2006], as well as inhibit the proliferation of smooth muscle cells [Levonen et al. 2007; Siow et al. 2007]. Furthermore, the neuroprotective effects of (Z)-ligustilide in ischemic injury [Kuang et al.
2006] may also be due in large part to the phase 2 inducing activity of this compound. Several P2PIs have been shown to be neuroprotective in cerebral ischemic injury, and the involvement of the Keap1-Nrf2-ARE pathway in neuroprotection has been demonstrated [Shih et al.
2005;
Zhao et al. 2006].

[0052] Recent analysis of the pharmacokinetics of (Z)-ligustilide showed that this compound has a large volume of distribution and undergoes extensive first pass metabolism [Yan et al. 2007]. DHL was among the compounds shown to be metabolites of (Z)-Iigustilide. Thus, the phase 2 inducing activity of DHL may contribute to the activity of (Z)-Iigustilide in vivo.

[0053] Thus, it has now been found that (Z)-ligustilide and DHL are phase 2 protein inducers with relatively high activity. (Z)-ligustilide did not increase the expression of CYP1a1 mRNA and is therefore a monofunctional inducer. Due to their high abundance in L. porteri and other Apiaceae plants, this activity is very important to the understanding of the bioactivity of these extracts, as well as understanding the underlying mechanism controlling the observed bioactivities of (Z)-ligustilide and DHL.
Serum concentrations of (Z)-ligustilide have been shown to exceed its CD value in rabbits [Dong et al. 2007]. In addition, other phthalides, in particular those containing the same unsaturated lactone moiety, may contribute to the activity observed in the original L.
porteri extract, and may be working in other Apiaceae extracts. Although other activities exist for these compounds, the phase 2 inducing activity of these compounds may be emphasized as being largely important for their effects on cardiovascular health and neuroprotection.

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[0055] Other advantages that are inherent to the structure are obvious to one skilled in the art. The embodiments are described herein illustratively and are not meant to limit the scope of the invention as claimed. Variations of the foregoing embodiments will be evident to a person of ordinary skill and are intended by the inventor to be encompassed by the following claims.

Claims (31)

Claims:

1. Use of a therapeutically effective amount of an alkylphthalide for inducing phase 2 proteins.

2. The use of claim 1, wherein the alkylphthalide is used in a daily dose of 5 mg/kg or greater based on body weight.

3. The use of claim 1, wherein the alkylphthalide is used in a daily dose of 10 mg/kg or greater based on body weight.

4. The use of claim 1, wherein the alkylphthalide is used in a daily dose of from 20 mg/kg to 500 mg/kg based on body weight.

5. Use of a therapeutically effective amount of an alkylphthalide for preparing a medicament for inducing phase 2 proteins.

6. The use of any one of claims 1 to 5, wherein the alkylphthalide is a monofunctional inducer of phase 2 proteins.

7. The use of any one of claims 1 to 5, wherein the alkylphthalide is (Z)-ligustilide, 6,7-dihydroxyligustilide (DHL), (Z)-6,7-epoxyligustilide, angelicide, (Z)-butylidenephthalide, butylphthalide, 2,4-dihydrophthalic anhydride, levistolide, cnidilide, tokinolide or any mixture thereof.

8. The use of any one of claims 1 to 5, wherein the alkylphthalide is (Z)-ligustilide, 6,7-dihydroxyligustilide (DHL) or a mixture thereof.

9. The use of any one of claims 1 to 5, wherein the alkylphthalide is (Z)-ligustilide.

10. The use of any one of claims 1 to 9, wherein the alkylphthalide is formulated for use orally.

11. The use of any one of claims 1 to 10, wherein the alkylphthalide is formulated for use by a mammal.

12. The use of any one of claims 1 to 10, wherein the alkylphthalide is formulated for use by a human.

13. The use of any one of claims 1 to 12, wherein the alkylphthalide is formulated in a dosage form.

14. The use of claim 13, wherein the dosage form is a powder, tablet, capsule, solution, suspension or emulsion.

15. The use of claim 13, wherein the dosage form is a powder, tablet or capsule.

16. The use of any one of claims 1 to 12, wherein the alkylphthalide is formulated into a food or beverage.

17. A pharmacologically acceptable composition for inducing phase 2 proteins comprising a therapeutically effective amount of an alkylphthalide and a pharmacologically acceptable ingredient.

18. The composition of claim 17, which is a plant extract.

19. The composition of claim 17 or 18, wherein the alkylphthalide is a monofunctional inducer of phase 2 proteins.

20. The composition of claim 17 or 18, wherein the alkylphthalide is (Z)-ligustilide, 6,7-dihydroxyligustilide (DHL), (Z)-6,7-epoxyligustilide, angelicide, (Z)-butylidenephthalide, butylphthalide, 2,4-dihydrophthalic anhydride, levistolide, cnidilide, tokinolide or any mixture thereof.

21. The composition of claim 17 or 18, wherein the alkylphthalide is (Z)-ligustilide, 6,7-dihydroxyligustilide (DHL) or a mixture thereof.

22. The composition of claim 17 or 18, wherein the alkylphthalide is (Z)-ligustilide.

23. A commercial package comprising a therapeutically effective amount of an alkylphthalide or a composition as defined in claim 17 or 18 together with instructions for its use for inducing phase 2 proteins in a mammal.

24. The commercial package of claim 23, wherein the alkylphthalide is a monofunctional inducer of phase 2 proteins.

25. The commercial package of claim 23, wherein the alkylphthalide is (Z)-ligustilide, 6,7-dihydroxyligustilide (DHL), (Z)-6,7-epoxyligustilide, angelicide, (Z)-butylidenephthalide, butylphthalide, 2,4-dihydrophthalic anhydride, levistolide, cnidilide, tokinolide or any mixture thereof.

26. The commercial package of claim 23, wherein the alkylphthalide is (Z)-ligustilide, 6,7-dihydroxyligustilide (DHL) or a mixture thereof.

27. The commercial package of claim 23, wherein the alkylphthalide is (Z)-ligustilide.

28. The commercial package of any one of claims 23 to 27, wherein the mammal is a human.

29. The commercial package of any one of claims 23 to 28, wherein the instructions recite a daily dose in a range of 5 mg/kg or greater based on body weight of the mammal.

30. The commercial package of any one of claims 23 to 28, wherein the instructions recite a daily dose in a range of 10 mg/kg or greater based on body weight of the mammal.

31. The commercial package of any one of claims 23 to 28, wherein the instructions recite a daily dose in a range of from 20 mg/kg to 500 mg/kg based on body weight of the mammal.