WO1999017609A1 - Extrait non toxique de larrea tridentata et son procede de production - Google Patents

Extrait non toxique de larrea tridentata et son procede de production Download PDF

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Publication number
WO1999017609A1
WO1999017609A1 PCT/US1998/019817 US9819817W WO9917609A1 WO 1999017609 A1 WO1999017609 A1 WO 1999017609A1 US 9819817 W US9819817 W US 9819817W WO 9917609 A1 WO9917609 A1 WO 9917609A1
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extract
åes
ndga
virus
larrea tridentata
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PCT/US1998/019817
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English (en)
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Robert A. Sinnott
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Larreacorp, Ltd.
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Priority claimed from PCT/US1997/018103 external-priority patent/WO1998015184A1/fr
Application filed by Larreacorp, Ltd. filed Critical Larreacorp, Ltd.
Priority to AU97754/98A priority Critical patent/AU9775498A/en
Publication of WO1999017609A1 publication Critical patent/WO1999017609A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)

Definitions

  • the present invention relates generally to a nontoxic extract of Larrea tridentata plant material having therapeutic value and a method of making the same.
  • the present invention also relates to the field of nontoxic, stable NDGA products used as food additives and therapeutic agents.
  • Larrea tridentata also known as Larrea divaricata, Larrea, chaparral, or creosote bush, is a shrubby plant which dominates some areas of the desert southwest in the United States and Northern Mexico as well as some desert areas of Argentina. Tea made from the leaves of Larrea tridentata has long been used in folk medicine to treat digestive disorders, rheumatism, venereal disease, sores, bronchitis, chicken pox, and the common cold.
  • NDGA is known as a powerful antioxidant compound. However, NDGA can itself be oxidized to toxic oxidation products by chemical means or by oxidation during processing and storage.
  • a highly reactive and toxic oxidation product of NDGA is nordihydroguaiaretic acid ortho di-a-b-unsaturated quinone ("NDGA quinone"), which according to TJ. Mabry et al., The Natural Products Chemistry of Larrea in CREOSOTE BUSH: BIOLOGY AND CHEMISTRY OF LARREA IN THE NEW WORLD DESERTS, ch. 5, pp.
  • NDGA quinone is found in chaparral (Larrea tridentata) and is suspected to be a causative agent of the toxic effects associated with consumption of chaparral products.
  • NDGA is the dominant lignan present in Larrea tridentata.
  • NDGA is known to possess a variety of biological effects including anti-tumor activity, enzyme inhibition activity and antimicrobial activity according to W. Donald MacRae & G.H. Neil Towers, Biological Activities of Lignans , PHYTOCHEMISTRY, vol. 23, pp. 1207-1220 (Pergamon Press 1984) (incorporated herein by reference).
  • NDGA and other antioxidants have been shown to be potent inhibitors of the human immunodeficiency virus type 1 (HIN) transcription.
  • HIN human immunodeficiency virus type 1
  • binding sites for the transcription factor Spl have been identified in the D ⁇ A promoter regions of at least two important viral genes of the Hepatitis B virus (HBV) which may be involved in the coordinate regulation of HBV transcription by transcription factor Spl .
  • HBV Hepatitis B virus
  • Kaposi's Sarcoma a cancer that frequently occurs among AIDS patients, has recently been implicated to be caused by a new herpes virus, human herpes virus-8 (HHV-8).
  • HHV-8 human herpes virus-8
  • Matt Crenson Kaposi's Sarcoma is tied to herpes, THE PHILADELPHIA INQUIRER, p. A4 (July 31, 1996), and Lawrence K.
  • Cancerous cells are commonly treated using various forms of chemotherapy, with some degree of success in many patients.
  • Human glioma cells are among the brain tumor cells that have been treated using chemotherapy with limited success.
  • Human glioma cells can be derived from a glioblastoma multiforme and cultured to become a drug resistant line.
  • these same cells have survived treatments of 70ug/ml BCNU, currently, the most common chemotherapeutic agent used in the treatment of glioblastoma multiforme.
  • Estrogens are known to promote the growth of breast cancers. Therefore, inhibiting the production of estrogens through the use of aromatase inhibitors is an important pharmaceutical accomplishment toward the treatment and/or prevention of breast cancers. Cancer Research 53, 4563-4566, October 1, 1993. Lignans and flavonoids have been shown to be competitive inhibitors of human preadipocyte aromatase. Certain naturally-occurring lignans and flavonoids have been shown to be moderate inhibitors of aromatase with K; values of between 1.3 and 27.2 micromolar concentration.
  • Aromatase functions in the conversion of androstene and testosterone to estrogens.
  • lignans and flavonoids function as competitive inhibitors for the binding of the aromatase substrates, androstene and testosterone to the active site of the aromatase enzyme.
  • Inhibition of aromatase enzyme activity resulting in reduction of estrogen synthesis and subsequent decrease in circulating plasma estrogen levels, is thought to contribute to the prevention of estrogen- dependent cancers, such as breast cancer.
  • Herpes zoster also known as shingles, afflicts approximately 300,000 to 500,000 people each year in the United States. Herpes zoster most often affects elderly and immunocompromised patients, such as those with AIDS, cancer, infections, physical trauma or those undergoing certain drug therapies. It is estimated that zoster will occur at some time in ten to 20 percent of the population. Men and women are affected equally by zoster and there are no apparent seasonal variations or racial predilections. Herpes zoster occurs at a constant rate of two to three cases per 1,000 between 50 and 80 years and increases again to 10 cases per 1,000 in persons over 80 years of age.
  • Herpes zoster occurs due to the reactivation of the varicella-zoster virus that has been dormant in the basal root ganglia.
  • Studies of acute herpes zoster have shown that the dorsal root ganglia sustain the brunt of the damage due to viral reactivation and replication.
  • the damage is accompanied by localized acute inflammation, tissue necrosis and often hemorrhaging. In more severe cases, inflammation can spread to adjacent motor and sensory roots. In exceptional cases, inflammation may spread to anterior and posterior horns or posterior columns.
  • Herpes zoster is usually present as a painful, unilateral vesicular eruption within a single dermatotome. In young, immunocompetent individuals, the course of zoster is usually benign. In elderly patients, however, acute neuritis and postherpetic neuralgia (PHN) may develop. Severe cases of zoster and PHN can be debilitating. Immunocompromised persons are at risk for more serious disease and increased morbidity. PHN is usually defined as pain persisting for more than a month after the date of the original zoster eruption. Overall, about 10% of patients presenting with an acute case of herpes zoster will subsequently get PHN.
  • Postherpetic neuralgia is most commonly associated with herpes zoster although neuralgia may be associated with other types of herpes virus infections. Besides he ⁇ es zoster, other members of the herpes virus family that may cause human disease include herpes simplex 1 and he ⁇ es simplex 2, cytomegalovirus (CMV),
  • Epstein-Barr virus human he ⁇ es virus 6, and human he ⁇ es virus 8. New members of the human he ⁇ es virus class continue to be discovered and undoubtedly many more exist in the global population. Recently, the inflammatory process associated with
  • Alzheimer's disease has been linked with the presence of he ⁇ es simplex virus in the brain of susceptible individuals (The Lancet, 1997, Vol. 349, p. 241-244).
  • Human he ⁇ es virus 6 has recently been linked to multiple sclerosis (Proceedings of the
  • Cytomegalovirus has been linked with the inflammatory process which may lead to hypertension, arteriosclerosis, atherosclerosis and coronary artery disease.
  • Epstein-Barr virus has been linked with human cancers including, Burkitt's lymphoma, naspharyngeal cancer and B- lymphomas (Science, 1991, Vol. 254, p. 1167-1173).
  • antiviral agents such as acyclovir, valciclover, famiciclover, cytarabine, idoxuridine, and vidarabine may be indicated (The New
  • Acyclovir Food and Drug Administration for the treatment of zoster. Most clinicians consider acyclovir to be the drug of choice because of its relatively low toxicity. Acyclovir has been shown to accelerate lesions healing and lessen the pain of the infection's acute stage.
  • prednisolone or prednisone which function as anti-inflammatory agents, in combination with antiviral therapy, can resolve episodes of acute pain and improve quality of life for the elderly immonocompetent patient (The Lancet, 1990, Vol. 336, p. 537-538; Emergency Medicine, April 1997, p. 125-126; Annals of Internal Medicine, 1996, Vol. 125, p. 376-383; Annals of Internal Medicine, 1997, Vol. 126, p. 831-832).
  • tricyclic antidepressant compounds i.e. amiti ⁇ tylene; possibly combined with a neuroleptic agent, i.e. a phenothiazine compound), anticonvulsant compounds (i.e. carbamazepine), neuroaugmentation with topical ethyl chloride spray to control pain
  • a substance P antagonist i.e. topically applied capsaicin
  • transcutaneous nerve stimulation strong analgesics and narcotics (i.e. acetaminophen, codeine), nonsteroidal anti-inflammatory agents (i.e.
  • ibuprofen topical anti-inflammatory agents
  • topical anti-inflammatory agents i.e. benzydamine
  • corticosteroid therapy i.e. trimacinolone, prednisolone, prednisone
  • nerve blocks i.e. lidocaine, bupivacaine, procaine; possibly combined with a steroid compound or noradrenaline
  • topical local anesthetics i.e. Eutectic Mixture of Local Anesthetics
  • NSAIDs nonsteroidal anti-inflammatory drugs
  • substances containing certain naturally occurring, anti-inflammatory fatty acids such as gamma-linolenic acid, alpha-linolenic acid and omega-3-fatty acids.
  • nonsteroidal anti-inflammatory drugs include aspirin, ibuprofen, phenylbutazone and indomethacin as well as others. All the commonly used NSAIDs exert their analgesic and anti-inflammatory effects, at least in part, by inhibition of cyclooxygenase, the enzyme that initiates transformation of arachidonic acid to prostaglandins, prostacyclin and thromboxanes.
  • cyclooxygenase the enzyme that initiates transformation of arachidonic acid to prostaglandins, prostacyclin and thromboxanes.
  • NSAIDs are generally safe, well tolerated and effective when properly administered. NSAIDs are widely used for treatment of pain and inflammation and are often formulated with other pharmaceutical agents, such as caffeine or opioids, to achieve specific pharmaceutical effects. Formulations containing NSAIDs are represented in both prescription and over- the-counter drugs.
  • omega-3 -fatty acids especially the omega-3 -fatty acids and gamma-linolenic acid (GLA)
  • GLA gamma-linolenic acid
  • Eicosapentaenoic acid EPA
  • docosahexaenoic acid DHA
  • LNA alpha-linolenic acid
  • Gamma-linolenic acid is not an omega-3 fatty acid, but rather, it is an omega-6 fatty acid like arachidonic acid (AA). Most, but not all eicosanoids potentiate the inflammatory pathway which leads to inflammatory diseases. The "1-" series prostaglandins produced by the metabolism of gamma-linolenic acid in place of arachidonic acid actually possess anti-inflammatory properties. Additionally, GLA also inhibits the production of inflammatory eicosanoids by competing with arachidonic acid for eicosanoid synthesis enzymes. A particularly rich source of gamma-linolenic acid is evening primrose oil.
  • Capsaicin a natural product extracted from hot peppers of the botanical genus Capsicum, has been used in the treatment of posthe ⁇ etic neuralgia, other types of painful nerve disorders and inflammatory conditions.
  • Hot peppers and their extractives have been used in food products and herbal medicine since ancient times. Extractives of Capsicum peppers contain differing amounts of capsaicin depending on the variety of pepper extracted and the extraction conditions employed. Capsaicin does not necessarily need to be purified from the other components of hot pepper extracts in order to be useful in pharmaceutical preparations.
  • Hot pepper extracts including oleoresins and alcoholic extracts have been used in herbal medicine.
  • the warming or burning sensation evoked by the topical application of capsaicin or an extract of Capsicum peppers may be partially masked by pretreatment or simultaneous application of a topical anesthetic (i.e. lidocaine).
  • topical anesthetics there are many topical anesthetics to choose from which may be formulated with natural or synthetic antiviral agents for treatment of he ⁇ es virus infections and the pain associated with them.
  • Two topical anesthetics commonly used in over-the-counter preparations are benzocaine and lidocaine.
  • a naturally occurring topical anesthetic, cocaine could also be used in place of the synthetic anesthetic compounds although, given the bad reputation associated with widespread abuse of cocaine, use of this compound is questionable in actual practice.
  • Certain flavonoid compounds especially members of the chemical classes flavones and flavonols can inhibit HTV activation at fairly low concentrations (See , J. William Critchfield et al., Inhibition of HTV Activation in Latently Infected Cells by Flavonoid Compounds" in AIDS Research and Human Retroviruses, AIDS RESEARCH
  • Larrea tridentata contains an abundance of these classes of antiviral flavonoids, particularly methyl ethers of flavonols.
  • these antiviral lignans and flavonoid compounds appear to work synergistically with other unresolved compounds present in crude extracts of Larrea tridentata.
  • the identity and mode of action of these synergistic compounds is unknown but they may facilitate abso ⁇ tion of the antiviral lignans or otherwise enhance the specific physiological, antiviral effects.
  • NDGA is known to be a potent inhibitor of the enzyme 5-lipoxygenase.
  • One of the enzymatic products of 5-lipoxygenase is 5-hydroperoxyeicosatetraenoic acid (HPETE) which is the precursor compound for the biosynthesis of very potent chemical mediators of inflammation, known as leukotrienes.
  • HPETE 5-hydroperoxyeicosatetraenoic acid
  • 5- lipoxygenase is limited to a specific number of myeloid cells including: neutrophils, eosinophils, monocytes, macrophages, mast cells, basophils and B-lymphocytes.
  • Leukotrienes are chemicals which induce prolonged muscle contraction, especially in the bronchioles of the lungs, and also increase vascular permeability and attract neutrophils and eosinophils to the site of inflammation. The leukotrienes play a major role in the inflammatory response to injury.
  • Leukotrienes have also been implicated in the pathogenesis of several inflammatory diseases including: asthma, psoriasis, rheumatoid arthritis and inflammatory bowel disease.
  • the role of leukotrienes as mediators of inflammatory diseases makes them attractive targets for therapeutic drugs to treat these diseases.
  • viral diseases are intended to include all diseases, attributed to a pathological virus of humans or animals, in which the causative viral agent which requires the Spl class of proteins to initiate viral replication, including certain viral agents of venereal diseases such as the He ⁇ es viruses (the He ⁇ esviridae), HSV-1 and HSV-2, viral hepatitis (the Hepadnaviridae) such as hepatitis B, and members of the retrovirus family (the retroviridae) including Varicella-Zoster viruses, cytomegalovirus (CMV), the human T-lymphotrophic viruses 1 and 2 (HTLV-1 and (HTLV-2) the human immunodeficiency viruses 1 and 2 (HIV-1 and HTV-2) and the cancer Kaposi's Sarcoma.
  • the He ⁇ es viruses the He ⁇ esviridae
  • HSV-1 and HSV-2 the Hepadnaviridae
  • retrovirus family the retroviridae
  • retroviridae members of the retrovirus family including Varicella
  • Inflammatory diseases throughout the specification and claims, are intended to include all diseases in which leukotrienes are known to play a major role or have been implicated including: asthma, allergic rhinitis, psoriasis, rheumatoid arthritis, inflammatory bowel disease, inflammatory pain, cystic fibrosis, adult respiratory distress syndrome, glomerulonephritis, inflammation of the skin, and virally induced inflammation (caused by CMV and other members of the He ⁇ esviridae) leading to atherosclerosis/ arteriosclerosis and subsequent coronary artery disease.
  • Larrea tridentata extract which contains a high concentration of both the identified antiviral lignans (NDGA and Mai. 4), flavonoids, and a wide variety of other associated organic compounds from the leaf resin, which may contribute synergistic antiviral and lipoxygenase inhibitory activity.
  • Larrea tridentata extract for the pu ⁇ ose of toxicological safety, it is of critical importance that the Larrea tridentata extract, to be used for medical applications, be processed to reduce the concentration of the toxic compound, NDGA quinone, which is reported to occur naturally in Larrea tridentata plant tissues. There is also a need to inhibit the natural production of toxic oxidation products, such as NDGA quinone, in the Larrea tridentata extract during processing and storage of the extract and formulated products and to facilitate the processing of the concentrated extract as either a liquid, slurry, or solid.
  • toxic oxidation products such as NDGA quinone
  • Additional value may be added if a combination of antiviral therapy and therapy to control the pain and inflammation associated with he ⁇ es viruses could be formulated into convenient topical and oral usage forms. Still, additional value may be added if one or more components of the pharmaceutical composition are naturally occurring substances since natural compounds may offer cost savings, increased customer acceptance and more rapid progression through the testing and marketing process.
  • the invention may comprise:
  • a method of preparing a nontoxic extract of Larrea tridentata plant material comprising the steps of: extracting endogenous antiviral and anti-inflammatory lignans and flavonoids and other synergistic compounds from Larrea tridentata plant material with a polar solvent, preferably acetone, to produce an extract by recirculating the solvent over the plant material a plurality of times; filtering the extract; adding an emulsifying and stabilizing agent, preferably polysorbate 80, to the filtered extract; reducing the NDGA quinone in the extract with ascorbic acid, ascorbic acid esters (i.e. ascorbyl palmitate), or ascorbic acid salts (i.e. sodium ascorbate), preferably ascorbic acid, by passing the extract through a bed of ascorbic acid powder; boiling the organic solvent out of the compound; and adding additional amounts of one of the above listed reducing agents to the compound subsequent to the step of reducing.
  • a polar solvent preferably acetone
  • the present invention may also comprise: A nontoxic, therapeutic agent comprising: an extract of Larrea tridentata plant material comprising NDGA and Mal.4; and ascorbic acid, ascorbic acid esters (i.e. ascorbyl palmitate), or ascorbic acid salts (i.e. sodium ascorbate).
  • a nontoxic, therapeutic agent comprising: an extract of Larrea tridentata plant material comprising NDGA and Mal.4; and ascorbic acid, ascorbic acid esters (i.e. ascorbyl palmitate), or ascorbic acid salts (i.e. sodium ascorbate).
  • the present invention may also comprise formulations of the described therapeutic agent in the embodiment of a lotion, liquid, powder or pill.
  • the formulations may be combined with one or more of a corticoid steroid compound, a local anesthetic compound, a non-steroidal anti inflammatory drug, an anti inflammatory fatty acid, and a substance P antagonist.
  • corticosteroid compound is combined with the formulations including the Larrea genus extract
  • suitable corticosteroids include amcinonide, betamethasone dipropionate, betamethasone valerate, clocortolone pivalate, corticosterone, corticosterone 21 -acetate, desonide, desoximetasone, diflorasone diacetate, halcinonide, hydrocortisone, hydrocortisone acetate, hydrocortisone, butyrate, hydrocortisone hemisuccinate, hydrocortisone sodium succinate, hydrocortisone valerate, cortisone, cortisone acetate, dexamethasone, dexamethasone acetate, dexamethasone sodium phosphate, fluadrenolide, fludrocortisone, fludrocortisone acetate, flunisolide, flucinolone acetonide, flucinolone acet
  • Suitable anesthetic compound include benzocaine, a benzocaine salt, cocaine, a cocaine salt, cocaine hydrochloride, cocaine nitrate, cocaine sulfate, lidocaine, a lidocaine salt, lidocaine hydrochloride, prilocaine, prilocaine hydrochloride, a prilocaine salt, procainamide hydrochloride, procaine, a procaine salt, procaine hydrochloride, tetracaine, tetracaine hydrochloride, and a tetracaine salt.
  • This list is not considered comprehensive, and the listed anesthetics are not necessarily considered to be equivalent to each other. It is simply stated that the above anesthetics are suitable to accomplish the objectives of one embodiment of the present invention.
  • NSAID is combined with the formulations including the Larrea genus extract
  • examples of a suitable NSAID include acetaminophen, aspirin, benzydamine, ibuprofen, indomethacin, ketoprofen, naproxen, naproxen sodium, and salicin. This list is not considered comprehensive, and the listed NSAIDs are not necessarily considered to be equivalent to each other. It is simply stated that the above NSAIDs are suitable to accomplish the objectives of one embodiment of the present invention.
  • a suitable anti-inflammatory fatty acids include gamma-linolenic acid (GLA), eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), and alpha-linolenic acid (LNA).
  • GLA gamma-linolenic acid
  • EPA eicosapentaenoic acid
  • DHA docosahexaenoic acid
  • LNA alpha-linolenic acid
  • examples of a suitable substance P antagonist include capsaicin, capsicum extract, capsicum tincture, oleoresins capsicum, and an extract from a plant which is a species of the Capsicum genus.
  • the present invention may also comprise a method of treating human glioma cells, comprising administering to a human in need thereof, an effective amount of a pharmaceutical composition of an extract of Larrea tridentata.
  • the present invention may also comprise a method of treating viral diseases caused by viruses from the retrovirus family of viruses, the Hepatitis B virus, the He ⁇ esviridae family of viruses, and viruses which require S l class proteins to initiate viral replication, inflammatory diseases which are mediated by the effects of leukotrienes, and virally induced inflammation leading to posthe ⁇ etic neuralgia, multiple sclerosis, Alzheimer's disease, cancer, including inhibition of human aromatase enzymes, atherosclerosis, hypertension, arteriosclerosis, and subsequent coronary artery disease using a nontoxic, therapeutic agent comprising an extract of Larrea tridentata plant material comprising NDGA and Mai. 4; and ascorbic acid, ascorbic acid esters (i.e. ascorbyl palmitate), and or ascorbic acid salts (i.e. sodium ascorbate).
  • a nontoxic, therapeutic agent comprising an extract of Larrea tridentata plant material comprising NDGA and Mai. 4
  • the extract may also include a corticosteroid compound, which may be selected from the above list, at a therapeutically effective concentration, generally 0.01 to 2.5 % when the formulation is topically applied, and generally 5 to 60 milUgrams per day for orally ingested steroids.
  • a corticosteroid compound which may be selected from the above list, at a therapeutically effective concentration, generally 0.01 to 2.5 % when the formulation is topically applied, and generally 5 to 60 milUgrams per day for orally ingested steroids.
  • the extract may also include a local anesthetic compound, which may be selected from the above list, at a therapeutically effective concentration, generally about 1 to 30 % when the formulation is topically applied.
  • a local anesthetic compound which may be selected from the above list, at a therapeutically effective concentration, generally about 1 to 30 % when the formulation is topically applied.
  • the extract may also include an NSAID, which may be selected from the above list, at a therapeutically effective concentration.
  • the extract may also include an anti-inflammatory fatty acid, which may be selected from the above list, at a therapeutically effective concentration, generally about 1 to 20 grams per day for oral formulations, and about 1 to 80 % (weight/weight) for topically applied formulations.
  • the extract may also include a substance P antagonist, which may be selected from the above list, at a therapeutically effective concentration, generally about 0.025 to 0.1 % for the liquid base, lotion base or cream base containing the extract of Larrea tridentata.
  • a substance P antagonist which may be selected from the above list, at a therapeutically effective concentration, generally about 0.025 to 0.1 % for the liquid base, lotion base or cream base containing the extract of Larrea tridentata.
  • Figure 1 is a high performance liquid chromatography ("HPLC") tracing of HPLC
  • NDGA as a purified compound (reported by the manufacturer to have a minimum purity of 90%). Slight amounts of unidentified impurities, probably methylated NDGA compounds, are indicated. However, no NDGA quinone is detectable in the sample.
  • Figure 2 is a HPLC tracing of NDGA as a purified compounds described in reference to Figure 1 which has been treated with the strong oxidizing agents sulfuric acid and potassium dichromate.
  • NDGA quinone the oxidation product of NDGA, is identified in the chromatogram as the large peak at 16.5 minutes.
  • the tracing was taken at 280 nm absorbance.
  • Figure 3 is an HPLC tracing of a purified NDGA sample as described in reference to Figure 1 and treated with oxidizing agents as described in reference to
  • Figure 4 is an HPLC tracing of a concentrated Larrea tridentata extract, which has been treated with the strong oxidizing agents sulfuric acids and potassium dichromate. A peak corresponding with NDGA quinone is identified in the chromatogram as the small peak occurring at 30 minutes. The tracing was taken at 375 nm absorbance.
  • Figure 5 is an HPLC tracing of a concentrated Larrea tridentata extract, produced according to the methods of the present invention. No NDGA quinone peak is observed at 30 minutes. The tracing was taken at 375 nm absorbance.
  • the present invention includes at least two products. (1) a nontoxic extract of Larrea tridentata plant material having a high concentration of NDGA and little or no NDGA quinone which can be used as a therapeutic agent; and
  • NDGA for use as a food additive or therapeutic agent, which does not oxidize into NDGA quinone during storage or processing.
  • An important principle of the invention is that ascorbic acid, when combined with NDGA, reduces any NDGA quinone present into NDGA, and prevent the NDGA from oxidizing and producing more NDGA quinone.
  • Other compounds and agents may be theoretically used to reduce NDGA, and to prevent oxidation of NDGA into an oxidation product.
  • Such agents include ascorbic acid esters (i.e. ascorbyl palmitate), ascorbic acid salts (i.e.
  • sodium ascorbate sodium ascorbate
  • butylated hydroxyanisole BHA
  • butylated hydroxytoluene BHT
  • hydrogen sulfide hypophosphorous acid (phosphinic acid), monothioglycerol (3 mercapto-l,2-propanediol)
  • potassium bisulfite potassium bisulfite (potassium metabisulfite, potassium pyrosulfite), propyl gallate
  • sodium bisulfite sodium metabisulfite, sodium pyrosulfite
  • sodium hydrosulfite sodium dithionite
  • sodium thiosulfate sodium hyposulfite
  • sulfur dioxide sulfurous acid
  • a tocopherol or vitamin E (DL-alpha-tocopherol).
  • ascorbic acid was found to be an unexpectedly powerful reducing agent for converting NDGA quinone to NDGA.
  • ascorbic acid, sodium hydrosulfite, and sodium bisulfite were added at 100 mg/ml to separate methanolic solutions of oxidized NDGA.
  • Ascorbic acid proved to be the fastest reducing agent (approximately 1 minute).
  • Subsequent chromatography of the mixtures showed that when sodium hydrosulfite or sodium bisulfite is used as the reducing agent, NDGA quinone is still detectable.
  • a nontoxic extract of Larrea tridentata having a high concentration of NDGA and very little or no NDGA quinone can be prepared by saturating the extract with ascorbic acid. Additional amounts of one of the ascorbic acid may be added to the extract or products formulated therefrom to inhibit the natural oxidation of component NDGA into NDGA quinone during processing and storage.
  • a nontoxic extract is prepared by the following method.
  • Larrea tridentata plant material consisting mostly of whole leaves and stems, but also possibly containing a small amount of whole flowers and fruits, i.e. whole plant material, is air dried.
  • the dried whole plant material is extracted using a suitable solvent.
  • the solvent is an organic solvent, preferably acetone.
  • the organic solvent is recirculated three times over the correct ratio of plant material to completely dissolve the organic compounds on the surface of the plant material and to produce a crude Larrea tridentata extract.
  • the resulting crude extract is filtered through cellulosic media (i.e. qualitative grade filter paper) to remove dirt and particulates.
  • the clarified extract is then passed through a bed of ascorbic acid powder (5 grams of F.C.C. grade ascorbic acid powder for each liter of extract passed through) in a manner which facilitates contact of the extract with the ascorbic acid powder and results in saturation of the extract with ascorbic acid.
  • ascorbic acid results in conditions which favor the chemical reduction of the toxic, oxidative metabolites of NDGA, which are present in Larrea tridentata plant tissues and the resulting extracts.
  • the toxic NDGA quinones are reduced to NDGA hydroquinone which is NDGA itself.
  • the extract is transferred to a water jacketed, stainless steel tank, which is heated to approximately 100 degrees C by circulation of a suitable solvent (i.e. water). While the extract is heated in the tank, the organic solvent is boiled out of the extract and may be recovered by condensation for reuse. The extract is heated in the tank to approximately 10 degrees above the boiling point of the organic extraction solvent. This rise in temperature of the extract indicates that the concentrated Larrea tridentata extract is substantially free of the extraction solvent.
  • the concentrated extract approximately 1/10 the volume of the original Larrea tridentata extract, is then removed from the tank and packaged in tightly sealed plastic drums for storage and shipping.
  • Another aspect of the present invention includes mixing the concentrated Larrea tridentata extract with effective, additional amounts of ascorbic acid. Formulation of the extract with excess amounts of ascorbic acid prevents subsequent formation of NDGA quinone during subsequent processing, storage, and formulating of the extract, and functions as a synergistic antioxidant in vivo with NDGA and other endogenous antiviral compounds.
  • a further aspect of the present invention includes mixing the concentrated Larrea tridentata extract with therapeutically effective amounts of at least one anti- inflammatory corticosteroid compound.
  • This may be accomplished by a direct mixing of the corticosteroid compound, preferably prednisone for oral formulations and betamethasone dipropionate for topical formulations, at the proper concentration, generally 0.1 to 2.5% for topically applied corticosteroids and generally 5 to 60 milligrams per day for orally ingested steroids, with the liquid base, lotion base, cream base or dry excipient ingredients containing the extract of Larrea tridentata.
  • the corticosteroid compound may be dissolved or dispersed in a suitable, pharmaceutically acceptable solvent, preferably USP/NF grade anhydrous ethanol, prior to mixing in to the liquid base, lotion base, cream base or dry excipient ingredients.
  • a suitable, pharmaceutically acceptable solvent preferably USP/NF grade anhydrous ethanol
  • the mixture resulting from the formulations containing dry excipient materials can be prepared in a manner suitable for milling into a uniform powder and encapsulation in standard, hard gelatin capsules.
  • Still a further aspect of the present invention includes addition of a local anesthetic to the concentrated extract of Larrea tridentata, at a therapeutically effective concentration.
  • a local anesthetic to the concentrated extract of Larrea tridentata, at a therapeutically effective concentration.
  • This may be accomplished by direct mixing of the anesthetic compound, preferably lidocaine base, at the proper concentration, generally 1 to 30 % concentration, with the liquid base, lotion base or cream base containing the extract of Larrea tridentata.
  • the anesthetic compound may be dissolved or dispersed in a suitable, pharmaceutically acceptable solvent, preferably USP/NF grade isopropanol, prior to mixing into the liquid base, lotion base, or cream base.
  • Liquid, lotion and cream based formulations, containing pharmaceutically ingredients may be used for topical application to regions of the body affected by he ⁇ es virus infections.
  • Another aspect of the present invention includes addition of a non-steroidal anti- inflammatory compound (NSAID) at a therapeutically effective concentration, to the concentrated extract of Larrea tridentata.
  • NSAID non-steroidal anti- inflammatory compound
  • This may be accomplished by direct mixing of the NSAID, preferably benzydamine at 3% (weight/weight) concentration for topical formulations, and ibuprofen at 100 milligrams per 50 milligrams of Larrea tridentata extract for oral formulations, with the liquid base, lotion base, cream base or dry excipient ingredients containing the extract of Larrea tridentata.
  • the NSAID may be dissolved or dispersed in a suitable, pharmaceutically acceptable solvent, preferably USP/NF grade ethanol, prior to mixing in to the liquid base, lotion base, cream base or dry excipient ingredients.
  • a suitable, pharmaceutically acceptable solvent preferably USP/NF grade ethanol
  • the mixture resulting from the formulations containing dry excipient materials can be prepared in a maimer suitable for milling into a uniform powder and encapsulation in standard, hard gelatin capsules.
  • the lipid or lipid containing substance may include at least one of the anti-inflammatory fatty acids, GLA, EPA, DHA, and LNA. This can be accomplished by direct mixing of the lipid or lipid containing substance, preferably marine fish oils that are rich in EPA and DHA or flax seed oil which is rich in LNA, at about 1 to 20 grams per day for oral formulations, and about 1 to 80 % (weight/weight) for topically applied formulations, with the liquid base, lotion base, cream base or dry excipient ingredients containing the extract of Larrea tridentata.
  • the resulting liquid formulations may be encapsulated in soft gelatin capsules and the mixture resulting from the formulations containing dry excipient materials can be prepared in a manner suitable for milling into a uniform powder and subsequent tableting or encapsulation in standard, hard gelatin tablets.
  • Yet another aspect of the present invention includes addition of a substance P antagonist at a therapeutically effective concentration, to the concentrated extract of Larrea tridentata. This can be accomplished by direct mixing of the substance P antagonist, preferably natural capsaicin, at the proper therapeutic concentration, generally 0.025 to 0.1 %, with the liquid base, lotion base or cream base containing the extract of Larrea tridentata.
  • the substance P antagonist preferably natural capsaicin
  • a suitable, pharmaceutically acceptable solvent preferably USP/NF grade isopropanol
  • Liquid, lotion and cream based formulations, containing pharmaceutically ingredients may be used for topical application to regions of the body affected by he ⁇ es virus infections.
  • the present invention is based in part on the discovery that a Larrea tridentata extract, which is produced according to the methods of the present invention, contains no detectable amount of NDGA quinone when analyzed by high performance liquid chromatography.
  • the non-toxic extract of Larrea tridentata contains an abundance of biologically active lignans and flavonoids. Because of the chemical similarities between the flavonoids and lignans tested in the scientific publications cited previously, and the phenolic compounds (predominantly lignans and flavonoids) contained in the non-toxic extract of Larrea tridentata of the present invention, extracts of Larrea tridentata can function as inhibitors of human aromatase enzymes.
  • a pharmaceutically useful dosage for the partial inhibition of aromatase in human preadipocyte tissues can be as low as 100 to 500 milligrams of Larrea tridentata extract per day taken over the course of several years prior to the onset of menopause.
  • supplementation with a small dose of Larrea tridentata extract only several hundred milligrams per day prior to the onset of menopause, can significantly decrease aromatase levels in human preadipocyte tissues and thereby prevent or diminish the incidence of breast cancer in these individuals.
  • the present invention therefore provides methods for the medically useful and effective extract of Larrea tridentata and formulations for production of pharmaceutical agents which have immediate and commercially important utility in the medical treatment of viral and inflammatory diseases.
  • Plant material (consisting of mostly leaves but with some small branches and a small amount of fruits and flowers) was harvested from Larrea tridentata shrubs growing in the Arizona desert, southwest of Gila Bend, Arizona. The plant material was air dried in the shade at ambient temperature and humidity for one week before processing. 100 kg of plant material was extracted with 100 liters of F.C.C. grade acetone by recirculating the acetone over the plant material three times. The resulting Larrea tridentata extract was filtered through Whatman #1 filter paper. 5 mils of F.C.C. grade polysorbate 80 was added to 50 liters of the extract and the extract was slowly passed through a glass column packed with 250 g of powdered ascorbic acid.
  • the extract saturated with ascorbic acid, was concentrated by boiling off the acetone solvent in a water-jacketed stainless steel tank, which was heated to approximately 100 degrees Celsius by circulating hot water.
  • the resulting concentrated extract in the form of a viscous liquid, was collected in plastic drums and used to prepare medical formulations.
  • a further formulation was prepared by adding betamethasone dipropionate at a concentration of 0.05% (weight/weight) to a 3% Larrea tridentata extract lotion prepared according to the principles of the present invention, in a pharmaceutically acceptable lotion base.
  • This novel combination of a natural antiviral agent and an anti- inflammatory corticosteroid was produced for use by human volunteers and in clinical trials.
  • a further formulation was prepared by adding prednisone at a concentration of 10 milligrams per capsule to a Larrea tridentata extract prepared according to the principles of the present invention, where the Larrea tridentata extract is also encapsulated at 50 milligrams per capsule, along with pharmaceutically acceptable excipients.
  • This novel combination of a natural antiviral agent and an anti-inflammatory corticosteroid was produced for use by human volunteers and in clinical trials.
  • a further formulation was prepared by adding lidocaine at a concentration of 5%
  • Larrea tridentata extract lotion prepared according to the principles of the present invention, in a pharmaceutically acceptable lotion base.
  • This novel combination of a natural antiviral agent and an anesthetic compound was produced for use by human volunteers and in clinical trials.
  • a further formulation was prepared by adding benzydamine at 3%
  • Larrea tridentata extract lotion prepared according to the principles of the present invention, in a pharmaceutically acceptable lotion base.
  • This novel combination of a natural antiviral agent and an NSAID was produced for use by human volunteers and in clinical trials.
  • a further formulation was prepared by adding ibuprofen at a concentration of 100 milligrams per capsule to a Larrea tridentata extract prepared according to the principles of the present invention, where the Larrea tridentata extract is also encapsulated at 50 milligrams per capsule, along with pharmaceutically acceptable excipients.
  • This novel combination of an NSAID agent and an anti-inflammatory corticosteroid was produced for use by human volunteers and in clinical trials.
  • a further formulation was prepared by adding flax seed oil at a concentration of 10 % (weight/weight) to a 3% Larrea tridentata extract lotion prepared according to the principles of the present invention, in a pharmaceutically acceptable lotion base.
  • a further formulation was prepared by adding marine fish oil at a concentration of 250 milligrams per capsule to a Larrea tridentata extract prepared according to the principles of the present invention, where the Larrea tridentata extract is also encapsulated at 50 milligrams per capsule, along with pharmaceutically acceptable excipients.
  • a further formulation was prepared by adding natural capsaicin at a concentration of 0.025 % (weight/weight) to a 3% Larrea tridentata extract lotion prepared according to the principles of the present invention, in a pharmaceutically acceptable lotion base.
  • This novel combination of a natural antiviral agent and a natural lipid compound containing a substance P antagonist, was produced for use by human volunteers and in clinical trials.
  • Example 2 Several formulations of concenfrated Larrea extract suitable for encapsulation were prepared by thoroughly mixing the concenfrated extract, according to the methods of the present invention, with dry excipient materials including starch, sucrose, fructose, and ascorbic acid powder. In some cases, other ingredients including antiviral and anti- inflammatory agents or herbal extracts i.e. Echinacea, Podophyllin, etc. were also combined in formulations. In all cases, the mixtures consisted of one part concenfrated Larrea tridentata extract with 5 to 10 parts dry excipient material. These formulations were used to treat HTV opportunistic infections, and He ⁇ es virus infections described in this specification. The treatment consisted of administering several capsules containing 50 to 100 mg of Larrea tridentata extract per capsule which were ingested daily.
  • dry excipient materials including starch, sucrose, fructose, and ascorbic acid powder.
  • other ingredients including antiviral and anti- inflammatory agents or
  • Extracts of Larrea tridentata prepared according to the methods of the present invention, have been shown to possess pronounced antiviral and anti-inflammatory properties when tested with human subjects. Reports by clinicians using these formulations show effective resolution of disease conditions caused by he ⁇ es viruses including: he ⁇ es simplex 1, he ⁇ es simplex 2 (genital he ⁇ es), he ⁇ es zoster, and Kaposi's Sarcoma (human he ⁇ es virus 8).
  • the fast and complete success in treating most of the he ⁇ es virus infections with Larrea-based formulations performed well where all other therapies including Zovirax and aggressive radiation therapy had failed.
  • Larrea-based antiviral therapy is shown to be superior to currently available prescription antiviral drug therapy, since widely-used antiviral drugs like acyclovir are often not considered practical in treating he ⁇ es virus infections in immunocompromised individuals.
  • Example 7 A human glioma cell line was used to assess the efficacy of an exfract of
  • Larrea tridentata prepared according to the principles of the present invention, to kill tumor cells.
  • the cells were derived from a glioblastoma multiforme and cultured to become a drug resistant line. These same cells have survived treatments of 70ug/ml BCNU, currently, the most common chemotherapeutic agent used in the treatment of glioblastoma multiforme (cell line obtained from Barrow Neurological Institute, Phoenix, Arizona).
  • An in vitro MTT was utilized to assess the Larrea tridentata extract's affect on growth and survival of the tumor cells. Cells were plated at a concenfration of 2.000 cells per well in 96 well dishes.
  • Varying concentrations of the Larrea tridentata extract solution were applied to the cells on day 1, ranging from 5 micrograms per milliliter to 125 micrograms per milliliter. 5 replicate wells of each drug concentration were analyzed. Cells were observed on Days 1, 3, 6 and 9 after drug freatment. Absorbance values directly correlate to cell survival and growth by MTT assay.
  • a pharmaceutically useful dosage for the freatment of brain tumors could be as low as 5 to 25 micrograms per gram of body weight or 340 to 1700 milligrams for an average 68 kilogram man.
  • Stimulators of IL-6 production have utility for stimulating the immune responses for treatment of infections (including viral infections), cancer, immimosupressive disorders (including AIDS and chronic fatigue syndrome) and physical trauma (including burns).
  • infections including viral infections
  • cancer including immimosupressive disorders (including AIDS and chronic fatigue syndrome)
  • physical trauma including burns.
  • This immune system-enhancing activity combined with the powerful anti- viral activity of the Larrea tridentata exfract, makes it a very useful compound for the treatment of viral infections in general (including he ⁇ es viruses), as well as cancer, immimosupressive conditions (including AIDS) and physical trauma.
  • NDGA quinone present initially in the extract. If another, perhaps even toxic, reducing agent is used to reduce the NDGA quinone naturally occurring in the Larrea tridentata extract, ascorbic acid can then be added to the resulting product to produce a nontoxic exfract which will not develop quantities of NDGA quinone through oxidation during processing and storage. In such a process, an additional step to remove the reducing agent would likely be necessary.
  • NDGA has many known uses as an antioxidant food additive and as a therapeutic agent.
  • NDGA for these pu ⁇ oses can be produced by extraction from natural sources, such as the Larrea tridentata, or can be synthesized.
  • the NDGA is produced, during processing and storage it oxidizes to produce NDGA quinone and is therefore toxic when ingested. Accordingly, it is within the scope of the invention to combine NDGA, whether extracted or synthesized, with ascorbic acid to produce a stable, nontoxic NDGA product that can be used as a food additive or therapeutic agent.

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Abstract

Un agent non toxique thérapeutique présentant une activité pharmacologique comprenant un extrait concentré de matières végétales tiré de Larrea tridentata ainsi qu'un acide ascorbique est produit selon un procédé dans lequel la matière végétale est extraite à l'aide d'un solvant organique, et ensuite saturée d'acide ascorbique afin de réduire l'acide nordihydroguaïarétique quinone toxique, se produisant naturellement dans la matière végétale, en acide nordihydroguaïarétique (ANDG) lui-même. On a ajouté des quantités additionnelles d'acide ascorbique à l'extrait afin d'inhiber l'oxydation naturelle de l'ANDG en ANDG quinone toxique in vivo, ou pendant le traitement ou le stockage. L'extrait obtenu est utile dans le traitement de maladies virales provoquées par des virus de la famille des Herpèsviridae ou des virus nécessitant la classe Sp1 de protéines pour initier des réplications virales. On peut également utiliser le composé obtenu en tant qu'anti-inflammatoire lorsque les maladies anti-inflammatoires sont induites par les effets de leucotriènes. Les agents de réduction listés peuvent aussi être utilisés pour stabiliser l'ANDG en tant qu'agent thérapeutique ou en tant qu'additif alimentaire.
PCT/US1998/019817 1997-10-07 1998-09-14 Extrait non toxique de larrea tridentata et son procede de production WO1999017609A1 (fr)

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US6480397P 1997-10-20 1997-10-20
US6480497P 1997-10-20 1997-10-20
US6480597P 1997-10-20 1997-10-20
US6480297P 1997-10-20 1997-10-20
US6467497P 1997-10-20 1997-10-20
US60/064,803 1997-10-20
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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7713984B2 (en) * 1998-08-25 2010-05-11 Novartis Ag Pharmaceutical uses

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