Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07J—STEROIDS
  • C07J63/00—Steroids in which the cyclopenta(a)hydrophenanthrene skeleton has been modified by expansion of only one ring by one or two atoms

Definitions

• Birch bark is a low-value waste product in the forest industry today. Ekman, R., Holzaba, (1983) 37, 205. Approximately 230,000 tons of birch bark are generated per year. For example, a single paper mill can generate 70 tons of birch bark per day. Thus, vast quantities of birch bark and its chemical components are available. Birch bark is a potential source of a variety of organic chemicals. Several triterpenoids have been identified in birch bark extracts.
• lupeol, betulin, betulinic aldehyde, betulinic acid, methyl betulinate, lupenone, betulonic aldehyde, betulonic acid, /3-amyrin, erythiOdiol, oleanolic aldehyde, oleanolic acid, methyl leanolate and acetyl oleanolic acid are all present in the outer bark o ⁇ Betula verrucosa. Eckerman, C, (1985) Paperi ja Puu, No. 3, 100.
• Betulin is also a useful starting material for preparing alobetulin and derivatives thereof, which posses useful pharmacological properties. Betulin can be converted to betulinic acid, which is useful as a therapeutic agent.
• Pisha E. et al., (1995) J. M. Nature Medicine, 1,
• betulinic acid has anti-tumor activity against human melanoma, e.g., MEL-1, MEL-2 and MEL-4.
• Fujioka, T. et al, J. Nat. Prod.. (1994) 57, 243-247 discloses that betulinic acid has anti-HIV activity in H9 lymphocytic cells.
• Current methods for isolating the chemical constituents of birch bark are deficient in several ways. For example, betulin has been extracted from the bark of white-barked birches in amounts up to 20%, based on the dry weight of the bark.
• Russian Patent Nos. RU2175326 discloses methods of preparing betulin, and derivatives thereof.
• the methods disclosed in Russian Patent No. RU2192879 include birch bark milling, separation of birch bark fibers, extraction of birch bark, separation of solution from extracted birch bark, and solvent removal from solution.
• the birch bark extraction is carried out with toluene at temperature of 90°C-110°C for 1.5-3.0 h, and the solution is filtered at a temperature of 40°C-50°C.
• the toluene betulin solution is cooled for 6-10 h to a temperature of 15°C - 5°C.
• the methods described in the '776 patent application are not able to effectively remove triterpenoids such as betulin-3-caffeate, betulinic acid, lupeol, esters of fatty acids, fatty acids, polyphenols and tannins, from the birch bark or birch bark extract.
• the process described in the '776 patent application is unsuitable for the industrial scale recovery of betulin, as well as lupeol and betulinic acid.
• the yields and purities of betulin disclosed in the '776 patent application are believed to be erroneous, utilizing the processes described therein. Even if accurate, the yields and purities of betulin disclosed in the '776 patent application can be improved.
• the processes described therein cannot be practiced on larger, industrial scales (e.g., kilograms or tons). A need therefore exists for safer, more cost-efficient and/or more efficient methods to obtain commercial quantities (e.g., tons) of betulin; as well as commercial quantities (e.g., kg) of lupeol and betulinic acid from birch bark.
• Summary of the Invention The process of the present invention is suitable for obtaining betulin, lupeol and betulinic acid from birch bark, in a yield of about 10 wt.% to about 12 wt.%, about 2.5 wt.% and about 2 wt.%, respectively.
• the betulin, lupeol and betulinic acid obtained using the methods of the present invention are relatively pure.
• the betulin will typically include less than about 0.02 wt.% of betulinic acid.
• commercial quantities (e.g., tons) of betulin; as well as commercial quantities (e.g., kg) of lupeol and betulinic acid can be obtained from birch bark.
• the methods of the present invention can also be employed to effectively remove betulin-3-caffeate, betulinic acid, esters of fatty acids, fatty acids, polyphenols and tannins from the birch bark or birch bark extract.
• betulin-3-caffeate and esters of fatty acids are effectively hydro lyzed upon contact and heating with the base.
• the betulin-3-caffeate is converted to betulin, which increases the overall yield of betulin; and the esters of fatty acids provide methanol and a salt of betulinic acid, which precipitates and can be removed from the reaction mixture.
• the betulinic acid precipitates as a basic salt, and can be removed from the reaction mixture.
• the polyphenols also precipitate as polyphenolates, which can be removed from the reaction mixture.
• the present invention provides a method for obtaining a natural product from plant material.
• the method includes: (a) contacting plant material with a solvent to provide a first mixture; (b) separating the plant material from the solvent to provide a first extract; (c) contacting the first extract with an aqueous base to provide a second mixture; (d) heating the second mixture in an organic solvent that (i) is water-immiscible, (ii) is capable of forming an azeotropic mixture with water, or (iii) has a boiling point of at least 100°C (e.g., at least about 120°C, at least about 140°C, or at least about 160°C), effective to distill off water present in the second mixture, thereby providing a third mixture; (e) separating solids from the third mixture to provide a fourth mixture; and (f) concentrating the fourth mixture, or precipitating solids from the fourth mixture, to provide a natural product.
• an organic solvent that (i) is water-immiscible, (ii) is capable of forming an azeotropic mixture with water, or (i
• the present invention also provides a method for selectively obtaining one or more non-acidic compounds from plant tissue in the presence of one or more acidic compounds.
• the method includes: (a) contacting plant material with a solvent to provide a first mixture; (b) separating the plant material from the solvent to provide a first extract; (c) contacting the first extract with an aqueous base to provide a second mixture; (d) heating the second mixture in an organic solvent that (i) is water-immiscible, (ii) is capable of forming an azeotropic mixture with water, or (iii) has a boiling point of at least 100°C (e.g., at least about 120°C, at least about 140°C, or at least about 160°C), effective to distill off water present in the second mixture, thereby providing a third mixture; (e) separating solids from the third mixture to provide a fourth mixture; and (f) concentrating the fourth mixture, or precipitating solids from the fourth mixture, to provide a natural product.
• the present invention also provides a method for obtaining betulin from birch bark.
• the method includes: (a) contacting birch bark with a solvent to provide a first mixture; (b) separating the birch bark from the solvent to provide a first extract; (c) contacting the first extract with an aqueous base to provide a second mixture; (d) heating the second mixture in an organic solvent that (i) is water-immiscible, (ii) is capable of forming an azeotropic mixture with water, or
• (iii) has a boiling point of at least 100°C (e.g., at least about 120°C, at least about
• the present invention also provides a method for obtaining lupeol from birch bark.
• the method includes: (a) contacting birch bark with a solvent to provide a first mixture; (b) separating the birch bark from the solvent to provide a first extract; (c) contacting the first extract with an aqueous base to provide a second mixture; (d) heating the second mixture in an organic solvent that (i) is water-immiscible, (ii) is capable of forming an azeotropic mixture with water, or (iii) has a boiling point of at least 100°C (e.g., at least about 120°C, at least about 140°C, or at least about 160°C), effective to distill off water present in the second mixture, thereby providing a third mixture; (e) separating solids from the third mixture to provide a fourth mixture; (f) precipitating solids from the fourth mixture; (g) separating the solids from the fourth mixture to provide a mother liquor; (h) concentrating the mother liquor to provide a fifth mixture; (i) washing the fifth mixture with a polar organic solvent to provide
• the process can optionally further include (1) recrystallizing the lupeol from a polar organic solvent to provide pure lupeol and (m) drying the pure lupeol.
• the present invention also provides a method for obtaining betulinic acid from birch bark.
• the method includes: (a) contacting birch bark with a solvent to provide a first mixture; (b) separating the birch bark from the solvent to provide a first extract; (c) contacting the first extract with an aqueous base to provide a second mixture; (d) heating the second mixture in an organic solvent that (i) is water-immiscible, (ii) is capable of forming an azeotropic mixture with water, or (iii) has a boiling point of at least 100°C (e.g., at least about 120°C, at least about 140°C, or at least about 160°C), effective to distill off water present in the second mixture, thereby providing a third mixture; (e) separating solids from the third mixture; (f) washing the solid with water to provide a second solids; (g) neutralizing or acidifying the second solids with an aqueous acid, thereby providing a fourth mixture; (h) separating crude betulinic acid from the fourth mixture.
• an organic solvent that (i)
• the process can optionally further include (i) crystallizing the crude betulinic acid from polar organic solvent to provide pure betulinic acid and (j) drying the pure betulinic acid.
• the present invention also provides a method for purifying an extract of a natural product.
• the method includes: (i) contacting an extract of a natural product with an aqueous base, to provide a first mixture; (ii) heating the first mixture in an organic solvent that (i) is water-immiscible, (ii) is capable of forming an azeotropic mixture with water, or (iii) has a boiling point of at least 100°C (e.g., at least about 120°C, at least about 140°C, or at least about 160°C), effective to distill off water present in the first mixture, thereby providing a second mixture; (iii) separating solids from the second mixture, to provide a third mixture; and (iv) concentrating the third mixture or precipitating solids from the third mixture, to provide a p ⁇ rified natural product.
• an organic solvent that (i) is water-immiscible, (ii) is capable of forming an azeotropic mixture with water, or (iii) has a boiling point of at least 100°C (e.g., at least
• the present invention also provides a compound obtained from the method of the present invention.
• the present invention also provides a pharmaceutical composition that includes a pharmaceutically acceptable carrier and a compound obtained from the method of the present invention.
• the present invention also provides a cosmetic composition that includes a cosmetically acceptable carrier and a compound obtained from the method of the present invention.
• a cosmetic composition that includes a cosmetically acceptable carrier and a compound obtained from the method of the present invention.
• Figure 1 illustrates a block flow diagram depicting the isolation of betulin, lupeopl, and/or betulinic acid from birch bark, employing distillation.
• Figure 2 illustrates a block flow diagram depicting the isolation of acidic components (natural products) and non-acidic components (natural products) from plant material, employing distillation.
• Specific values listed below for ranges are for illustration only; they do not exclude other defined values or other values within defined ranges.
• triterpene or “triterpenoid” refers to a plant secondary metabolite that includes a hydrocarbon, or its oxygenated analog, that is derived from squalene by a sequence of straightforward cyclizations, functionalizations, and sometimes rean-angement. Triterpenes or analogues thereof can be prepared by methods known in the art, i.e., using conventional synthetic techniques or by isolation from plants. Suitable exemplary triterpenes and the biological
• triterpene refers to one of a class of compounds having approximately 30 carbon atoms and synthesized from six isoprene units in plants and other organisms. Triterpenes consist of carbon, hydrogen, and optionally oxygen. Most triterpenes are secondary metabolites in plants. Most, but not all, triterpenes are pentacyclic.
• triterpenes examples include betulin, allobetulin, lupeol, friedelin, and all sterols, including lanosterol, stigmasterol, cholesterol, jS-sitosterol, and ergosterol.
• betulin refers to 3/3,28-dihydroxy-lup-20(29)-ene.
• Betulin is a pentacyclic triterpenoid derived from the outer bark of paper birch trees (Betula papyrifera, B. pendula, B. verucosa, etc.). It can be present at concentrations of up to about 24% of the bark of white birch. Merck Index, twelfth edition, page 1236 (1996). Stmcturally, betulin is shown below:
• betulinic acid refers to 9-hydroxy-l-isopropenyl- 5a,5b,8,8,l la-pentamethyl-eicosahydro-cyclopenta[a]chrysene-3a-carboxylic acid. Structurally, betulinic acid is shown below:
• lupeol refers to lup-20 (29)-en-3 /3-ol. Lupeol is also found in birch bark and in other plant sources. Lupeol is present at concentrations of about 1.5-3% of the birch bark and at up to about 8.2% in Canavalia ensifomiis, a plant widespread in the humid tropics of Asia and Africa. Structurally, lupeol is shown below:
• natural product refers to naturally occurring compounds that are end products of secondary metabolism; often, they are unique compounds for particular plants or species of plants.
• the compounds can be derived from plant material, e.g., birch bark. Such compounds can include, e.g., triterpenes or triterpenoids.
• the natural product can include a single compound, or can include one or more different compounds. Additionally, these one or more different compounds can be structurally related or stmcturally unrelated.
• a “non-acidic compound” refers to a natural product that comprises lupeol, betulin, taxol, paclitaxel, echinacea extract, valerian root extract, ginkgolide A, ginkgolide B, ginlcgolide C, bilobalide, garlic extract, ginseng extract, aloe gel, barbaloin, cranbeny extract, eleutheroside A, eleutheroside B, eleutheroside C, eleutheroside D, eleutheroside E, eleutheroside
• an "acidic compound” refers to a natural product that acidic compounds comprises betulin acid, betulin-3-caffeate, tannin, lipid, phenol, caffeic acid, cichoric acid, valerenic acid, isovaleric acid, flavonoid, quercetin, kaempferol, catechin, lignin, shikimic acid, succinic acid, amino acid, nicotinic acid, pantothenic acid, anthraquinone, acidic galactan, benzoic acid, quinic acid, malic acid, citric acid, hippuric acid, phenolic acid, feralic acid, .
• plant material or “plant tissue” refers to a collection of similar cells of a plant, that typically act together to perform a particular function.
• the term refers to the tissue of any organism of the plant kingdom, as opposed to one of the animal kingdom or of the kingdoms of Fungi, Protista, or Monera.
• the plant tissue can be any portion or portions of the plant (e.g., bark, roots, leaves, flowers, needles, bulbs, benies, rhizomes, rootstocks, stems, and seeds), as well as the entire plant.
• the tissues of a plant (“plant tissue”) generally fall into three main categories: dermal tissue, ground tissue, and vascular tissue.
• Dermal tissue refers to the "skin" layer of all plant organs and is responsible for environmental interaction (light passage, gas exchange, pathogen recognition and protection, color display, etc.).
• Demial tissue is composed of epidermal cells, closely packed cells that secrete a waxy cuticle that aids in the prevention of water loss.
• Ground tissue lies between dermal tissue and vascular tissue.
• the ground tissue comprises the bulk of the primary plant body. Parenchyma, collenchyma, and sclerenchyma cells are common in the ground tissue, h roots, the ground tissue may store sugars or starches to fuel the spring sap flow; in leaves, the ground tissue is the layer responsible for photosynthesis (the mesophyll).
• Vascular tissue transports food, water, hormones and minerals within the plant.
• Vascular tissue includes xylem, phloem, parenchyma, and cambium cells.
• bark refers to the dry, dead outer covering of woody branches, stems and roots of plants that is very distinct and separable from the wood itself. It includes all tissue outside the cambium (growth layer between bark and wood).
• leaf or leafaves refer to those parts of a plant which grow along the sides of branches or stems or at the bases of plants. Most are green and contain chlorophyll, though they vary in their shapes and sizes. Leaves are the part of the plant that ordinarily performs photosynthesis (the process that converts sunlight and carbon dioxide into energy).
• needle generally refers to a narrow stiff leaf, such as those of conifers (e.g., pine trees).
• root refers to the part of a plant, normally underground, that absorbs nutrients and anchors the plant into the ground.
• bulb refers to a spheroidal body growing from a plant either above or below the ground (usually below), which is usually a bud, consisting of a cluster of partially developed leaves, and producing, as it grows, a stem above, and roots below, (e.g., the onion or tulip bulb).
• a true bulb is a complete package containing next year's plant (flower) already forming inside.
• the contents of the bulb are often enclosed in protective, fleshy scales, which are held together by a small basal plate.
• the scales are modified leaves that contain enough nutrients to sustain the plant through dormancy and early growth. They may be loose and open like those of a lily, or tightly closed like those of a hyacinth.
• a paper-thin tunic protects the scales (lilies don't have a tunic). Roots will grow from the bulb's basal plate.
• "beny” refers to any small fruit that is pulpy or succulent throughout, having seeds loosely imbedded in the pulp, such as the cunant, grape, or bluebeny.
• Beny can be further defined as an indehiscent fruit derived from a single ovary and having the whole wall fleshy, such as the grape or tomato. Furthermore, benies come in various stmctures including simple, such grape; bluebeny, cranberry, or aggregate, such as blackbeny; raspbeny, strawbeny mulbeny.
• rhizome refers to a horizontal, usually underground stem that often sends out roots and shoots from its nodes (also called rootstalk or rootstock).
• rootstock refers to a robust plant that provides the root system in grafting, also known as a stock. Scions and buds are grafted and budded to a rootstock or stock.
• Rootstock also refers to the elongated and often thick rhizomes of certain perennial herbaceous plants such as the Iris, Aspidistra and Solomon's Seal. .
• stem refers to the main (usually aerial) axis (sometimes refened to as the trunk or stalk) of a tree, shrub, or plant.
• stem also refers to the part of the plant that supports the leaves, flowers or fruits of a plant, such as the peduncle of a fruit or the pedicel of a flower.
• seed refers to a ripened ovule, consisting of an embryo with one or more integuments, or coverings, such as an apple seed, a cunant seed, dill seed, or kola nut seed. By germination, most seeds produces a new plant. "Seed” also refers to any small seedlike fruit, though it may consist of a pericarp, or even a calyx, as well as the seed proper, such as a parsnip seed or thistle seed. The seed proper has an outer and an inner coat, and within these the kernel or nucleus. The kernel is either the embryo alone, or the embryo enclosed in the albumen, which is the material for the nourishment of the developing embryo. The scar on a seed, left where the stem parted from it, is called the hilum, and the closed orifice of the ovule, the micropyle.
• the plant can be a bryophyte or vascular plant. More specifically, the plant can be grass, flower or a tree and the plant tissue can be any part of the grass, flower or tree.
• Specific plants, flowers, and trees include, e.g., Moss (e.g., Club Moss), Horsetail, Fern, Conifer, Cycad, Ginkgo biloba (Ginkgo), Taxus yunnanesis (yew tree), Echinacea spp., Valeriana officinalis, Allhmi sativum (garlic), Panax ginseng, aloe vera, Vaccinium macrocarpon, Eleutherococcus senticosus, Piper methysticum, dill, kola nut, and cinchona.
• Moss e.g., Club Moss
• Horsetail Fern, Conifer, Cycad
• Ginkgo biloba Ginkgo
• Taxus yunnanesis yew tree
• birch refers to any of the several deciduous tress of the genus Betula.
• the birches comprise the family Betulaceae in the order Fagales.
• Birch trees include, for example, white birch, B. alba; sweet, black or cheny birch, B. lenta; monarch birch, B. maximowicziana; dwarf or arctic birch; B. nana; Japanese white birch, B. platphyla japonica; smooth-bark birch, B. pubescens; yellow birch, B.
• alleghaniensis paper, white or canoe birch, B. papyrifera; gray birch, B. populifolia; river birch, B. nigra; and the European birches, B. pubescens; B. alba and B. pendula.
• birch can be B. alba, B. lenta, B. maximowicziana, B. nana, B. platyphylajaponica, B. pubescens, B. alleghaniensis, B. papyrifera, B. populifolia, B. nigra or B. pendula.
• a specific birch for use in the processes of the present invention is B. papyrifera.
• Taxus or “yew” refers to plants belonging to Taxaceae Gymnopenmae. There are 11 species and five sub-species of Taxus in the world, mainly found in East Asia, North America, and Europe; , "Echinacea spp.” Refers to a perennial native to North American which resembles a black-eyed Susan and is called Dchinacea, purple coneflower or snake root; "Valeriana offlcinalis” or “valerian” refers to the plant Valeriana of ⁇ cinalis of the valerianaceae family, which is also known as valerian, phu, allheal, great wild valerian, amantilla, setwall, setewale, capon's tail; "Allium sativum” refers to garlic; “Panax ginseng” refers to ginseng, commonly called Korean ginseng, Chinese ginseng or American ginseng. Asian ginseng is a member of the
• Araliaceae family which also includes the closely related American ginseng, Panax quinquefolius, and less similar Siberian ginseng; "Eleutherococcus senticosus,” refers to “eleuthero” (which contains eleutheroside A, eleutheroside B (syringin), eleutheroside C, eleutheroside D, eleutheroside E (syringaresinol di-O- ⁇ -D-glucoside, liriodendrin), and eleutheroside G, among other constituents); "Aloe” refers to any of the over 500 different species of Aloe.
• Aloe Vera is a member of the Lily family and is very-cactus like in its characteristics. This unique plant also belongs to a larger plant family called "Xeroids". Of the 500+ species of Aloe, Aloe barbaclensis miller (Aloe Vera species) is prefened; "Vaccinium macrocarpon” refers to cranbeny; “Piper methysticum,” a member of the pepper family, refers to a plant native to the South Sea Islands of Micronesia, Melanesia and Polynesia; "Kola vera,” of the family N.O.
• Sterculiaceae also known as "Kola nut” refers to the tree that grows about 40 feet high and has yellow flowers spotted with purple; and "Cinchona,” belongs to the family N.O. Rubiaceae and refers to Peruvian bark (Cinchona succimbra) which is an evergreen tree that grows 15 to 45 feet in height.
• Non-acidic compounds and acidic compounds The specific non-acidic compounds and acidic compounds that can be isolated from the plant tissue will depend, in part, upon the specific plant tissue that is being extracted.
• the bark of Taxus yunnanesis can be extracted employing the methods described herein to provide taxol (paclitaxel) as the non-acidic compound and tannin, fatty acids, and phenols as the acidic compounds
• the needles of the Yew tree can be extracted employing the methods described herein to provide taxol (paclitaxel) as the non-acidic compound, and tannin, fatty acids, and phenols as the acidic compounds
• the root of the Echinacea spp the bark of Taxus yunnanesis can be extracted employing the methods described herein to provide taxol (paclitaxel) as the non-acidic compound and tannin, fatty acids, and phenols as the acidic compounds
• the root of the Echinacea spp the bark of Taxus yunnanesis
• Echinacea extract as the non-acidic compound and tannin, caffeic acid, and cichoric acid as the acidic compounds
• the root of the Valeriana officinalis can be extracted employing the methods described herein to provide Valerian root extract as the non-acidic compound and valerenic acid, isovaleric acid, and tannins as the acidic compounds
• the roots, bark, leaves, or any combination thereof of the Ginkgo biloba can be extracted employing the methods described herein to provide Ginkgolide A, Ginkgolide B, Ginkgolide C, and bilobalide as the non-acidic compounds and tannins, flavonoids (e.g., quercetin, kaempferol, catechin), lignins, shikimic, and succinic acids as the acidic compounds
• the bulb of the Allium sativum can be extracted employing the methods described herein to provide garlic extract as the non-acidic compound and fatty acids and amino acids as the acidic compounds
• Table 1 Non-acidic compounds and acidic compounds that can be isolated from specific plant tissue.
• “Paclitaxel” refers to [2aR-[2a ⁇ ,4 ⁇ ,4a ⁇ ,6 ⁇ ,9 ⁇ ( ⁇ R*, ⁇ S*),- l l, ⁇ l2 ⁇ ,12a ⁇ ,12b ⁇ ]]- ⁇ -(Benzoylamino)- ⁇ -hydroxybenzenepropanoic acid 6,12b-bis(acetyloxy)-12-(benzoyloxy)-2a,3,4,4a,5,6,9,10,l l,12,12a,12b- dodecahydro-4,1 l,-dihydroxy-4a,8, 13, 13-tetramethyl-5-oxo-7,l 1-methano-lH- cyclodeca[3,4]benz[l,2-b]oxet-9-yl ester.
• Echinacea extract is believed to include essential oil, polysaccharides, such as inulin, polyacetylenes, betain, glycoside, sesquiterpenes and caryophylene. Echinacea extract is also believed to contain copper, iron, tannins, protein, fatty acids, fat-soluble alkylamides, caffeic acid glycoside (echinacoside), and vitamins A, C, and E.
• valeriana officinalis extract is a very effective sedative and is used most often to help insomnia, especially due to stress. It has an advantage over prescription sedatives in that it is not habit forming. Valerian has many actions besides its well-known sedative effects. It strengthens the heart and in some cases lower blood pressure.
• valerian which vary in potency and can be used similarly, although V. officinalis is the prefened plant.
• Other constituents are a volatile oil, which includes isovalerianic acid and bomeol; choline; flavonoids; sterols and several alkaloids, including actinidine, valerianine, valerine, and chatinine.
• Valepotriates are not water-soluble, but valeric acid is and may be the constituent most likely to produce valerian's sedative effect, especially when used as it was traditionally in water extracts (teas) or water/alcohol extracts (tinctures).
• the genus Ziziphus (ber, jujube) belongs to the buckthorn familiy (Rhamnaceae). It is a genus of about 100 species of deciduous or evergreen trees and shrubs distributed in the tropical and subtropical regions of the world. Some species, like Z. mauritiana and Z. jujuba, occur on nearly every continent, whereas other species, like Z. nummularia, Z. spina-christi and Z.
• Ziziphus species can grow either as trees and shrubs (Z. mauritiana, Z. rotundifolia, Z. jujuba, Z. mucronata) or exclusively as small shrubs or bushes (Z. nummularia, Z. lotus, Z. spina-christi, Z. obtusifolia). This is a tree that may be used for manufacturing betulinic acid.
• Many studies have provided clinical evidence that ginkgo prevents many problems throughout the entire body. Ginkgo is gaining recognition as a brain tonic that enhances memory because of its positive effects on the vascular system, especially in the cerebellum.
• Ginkgo may help to counteract the effects of aging, including mental fatigue and lack of energy.
• Ginkgo has two groups of active substances, flavonoids (a three-ringed molecule with hydroxyl (OH) groups attached) and terpene lactones, including ginkgolides A, B, and C, bilobalide (a sesquiterpene), quercetin (a flavonoid), and kaempferol (a flavonoid).
• the constituents of gingko include terpenoids (bilobalide), diterpenoids (ginkgolide A, ginkgolide B, ginkgolide C, ginkgolide J, ginkgolide M), flavonoids: flavones (luteolin, tricetin, 2-hydroxyluteolin), biflavones (amentoflavone, ginkgetin, isoginkgetin, sciadoptysin, 5-methoxybilobetin, bilobetin), flavonols
• Ginkgolides have been shown to control allergic inflammation, anaphylactic shock and asthma.
• Ginkgo extract is generally derived from dried ginkgo leaves, but also may be derived from gingko root or bark.
• Garlic contains compounds that are antibacterial, antifungal and reduce blood clotting. In order for the active ingredient that gives garlic its characteristic odor and its therapeutic effects to be released, the garlic clove (or bulb) must be cut or crashed. This releases an enzyme that causes the formation of allicin, the component responsible for garlic's odor and medicinal activity.
• Active constituents present in garlic include the sulphur compound allicin, produced by crashing or chewing fresh garlic, which in turn produces other sulphur compounds: ajoene, allyl sulfides, and vinyldithiins.
• “Ginseng” is believed to increase energy, counter the effects of stress, and enhance intellectual and physical perfomiance. Thirteen ginsenosides have been identified in ginseng, including ginsenosides Rgl and Rbl.
• Other constituents include the panaxans, which are believed to help lower blood sugar, and the polysaccharides (complex sugar molecules), which are believed to support immune function. Also, long-term intake may be linked to a reduced risk of cancer.
• aloe is a mild anesthetic, relieving itching, swelling, and pain: it also is antibacterial and antifungal, increases blood flow to wounded areas, and stimulates fibroblasts, the skin cells responsible for wound healing.
• "Cranbeny” has astringent applications for the urinary tract and is a traditional remedy for bladder infections and kidney-related disorders. Two components of cranbeny juice have been shown to inhibit the adherence of E. coli to uroepithelial cells. The first is fructose. The second is proanthocyanidin, the chemical structure of which has been elucidated. Fructose inhibits the adherence of type-1 fimbriated E.
• Kavalactones consists of an oleoresin from which kavalactones originate, starch, sugars, proteins, vitamins Bl, B2, B3, B6, folic acid and E, potassium, manganese, biotin, choline, inositol, fat, glycynhizin, lecithin, pantothenic acid, para-aminobenzoic acid, pentacyclic terpenes, phosphorous, and a yellow dye.
• Kavalactones are considered the most active constituents in the plant. The main use for kava today is in the treatment of anxiety. It is also an excellent muscle relaxant and has diuretic and urinary antiseptic properties, so it may be useful in urinary cystitis and prostatitis.
• Kava also shows pain-relieving properties.
• "Kola vera” or “Cola vera” seeds are said to contain a glucoside, Kolanin (this substance may be a mixture of Kola red and caffeine). The seeds also contain starch, fatty matter, sugar, and a fat decomposing enzyme acting on various oils.
• "Dill seed” is an herbal medicine that is used to reduce gas, upset stomach, and colic pains. It is also used to promote the flow of milk in breastfeeding mothers, and to help control bad breath and hiccups. Other names for Dill Seed include: Anethum Graveolens, Dill, and Dillweed.
• tannin refers to tannic acid or gallotannic acid.
• Tannin varies somewhat in composition, depending on the source, having the approximate empirical formula C 76 H 52 0 6 . Tannic acid is a colorless to pale yellow solid; it is believed to be a glucoside in which each of the five hydroxyl groups of the glucose molecule is esterified with a molecule of digallic acid. Tannin is used in tanning animal skins to make leather; it transforms certain proteins of animal tissue into compounds that resist decomposition. It is also used in manufacturing inks, as a mordant in dyeing, and in medicine as an astringent and for treatment of burns.
• fatty acids refers to a long-chain of carboxylic acids that may either be saturated (without double bond) or non-saturated (with double bond). It refers to any acid derived from fats by hydrolysis (e.g., oleic acid, palmitic acid, or stearic acid); any long-chain monobasic organic acid.
• phenols refers to compounds that include a C 6 H 5 OH backbone. They are aromatic alcohols that are optionally substituted by one or more substituents. Phenols exhibits weak acidic properties and are sometimes called carbolic acids, especially when in water solution.
• caffeic acid refers to 3-(3,4-Dihydroxyphenyl)-2- propenoic acid.
• valeric acid refers to pentanoic acid; valerianic acid; and propylacetic acid.
• isovaleric acid refers to 3-Methylbutanedioic acid and isovalerianic.
• flavonoid refers to polyphenols that have a carbon skeleton. They have an acidic nature due to the phenol groups.
• quercetin refers to 2-(3,4-Dihydroxyphenol)-3,5,7- trihydroxy-4H-l -benzopyran-4-one.
• kaempferol refers to 3,5,7-Trihydroxy-2-(4- hydroxyphenyl)-4H- 1 -benzopyran-4-one.
• catechin refers to (2R-trans)-2-(3,4-dihydroxyphenyl)- 3,-4-dihydro-2H-l-benzopyran-3,5,7-triol.
• lignin refers to a highly polymerized and complex chemical compound especially common in woody plants. The cellulose walls of the wood become impregnated with lignin, a process called lignification, which i greatly increases the strength and hardness of the cell and gives the necessary rigidity to the tree.
• amino acids refers to any one of a class of simple organic compounds containing carbon, hydrogen, oxygen, nitrogen, and in certain cases sulfur. These compounds are the building blocks of proteins. They are characterized by the presence of a carboxyl group (COOH) and an amino group (NH 2 ).
• the 20 amino acids commonly found in animals are alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. More than 100 less common amino acids also occur in biological systems, particularly in plants. Every amino acid except glycine can occur as either of two optically active stereoisomers, D or L; the more common isomer in nature is the L-fonn.
• shikimic acid refers to [3R-(3 ⁇ ,4 ⁇ ,5 ⁇ ]-3,4,5- Trihydrooxy- 1 -cyclohexene- 1 -carboxylic acid.
• succinic acid refers to butanedioic acid (HOOCCH 2 CH 2 COOH).
• nicotinic acid refers to 3-Pyridinecarboxylic acid.
• pantothenic acid refers to "(R)-N-(2,4-Dihydroxy-3,3- dimethyl- 1 -oxobutyl)- ⁇ -alanine.
• anthraquinone refers to 9,10-anthracenedione.
• acidic galactan refers to a poly sugar with attached carboxylic groups.
• benzoic acid refers to benzoic acid, C 6 H 5 CO 2 H. It is the simplest aromatic carboxylic acid.
• benzyl alcohol e.g., benzyl alcohol, benzaldehyde, toluene, and phthalic acid
• resins notably gum benzoin. It is used largely for making its salts and esters, most notably sodium benzoate, which is widely used as a preservative in foods and beverages and as a mild antiseptic in mouthwashes and toothpastes.
• quinic acid refers to [lR-(l ⁇ ,3 ⁇ ,4 ⁇ ,5 ⁇ ]-l,3,4,5- Tetrahydroxycyclohexanecarboxylic acid.
• malic acid refers to hydroxybutanedioic acid.
• citric acid refers to citric acid or 2-hydroxy- 1,2,3 - propanetricarboxylic acid, H0 2 CCH 2 C(OH)(CO 2 H)CH 2 CO 2 H, an organic carboxylic acid containing three carboxyl groups. It is responsible for the tart taste of various fraits in which it occurs, e.g., lemons, limes, oranges, pineapples, and goosebenies.
• hippuric acid refers to N-Benzoylglycine.
• feralic acid refers to 3-(4-Hydroxy-3-methoxyphenyl)- 2-propenoic acid.
• chlorogenic acid refers to [lS-(l ⁇ ,3 ⁇ ,4 ⁇ ,5 ]-3-[[3- (3,4-Dihydroxyphenyl)-l -oxo-2-propenyl]oxy]- 1 ,4,5,trihydroxycyclohexanecarboxylic acid.
• carboxylic acid refers to 3-phenyl-2-propenoic acid.
• Betulinic acid can also be obtained from African Tree Uapaca nitida Mull-Arg (Euphorbiacea), leaves and bark o ⁇ Bacopa monniera( West Bengal), all species of Dilleniacea (Acrotrema arnothianum Wight, Dillenia andamanica Parkinson, D. aurea Smith, D. bracteata Wight, D. indica Linn, D. pentagina Roxb, D. retusa Thunb, D. scabtalla (D. Don) Roxb, ex Wall, Tetracera (Houtt. exChrism.& Panz., Men.), Tetracera akara (Burm.f.) Men., T. indica (Houtt.
• separating refers to the process of removing solids from a mixture.
• the process can employ any technique l ⁇ iown to those of skill in the art, e.g., decanting the mixture, filtering the solids from the mixture, or a combination thereof.
• filtering refers to the process of removing solids from a mixture by passing the liquid through a filter, thereby suspending the solids on the filter.
• aqueous base refers to a solution of water, and a substance that produces OH ions in the aqueous solution.
• the aqueous base can include water and at least one of a lithium ion (Li + ), a sodium ion (Na + ), a potassium ion (K + ), a calcium ion (Ca” + ), and a barium ion (Ba + ).
• the aqueous base can include water and at least one of sodium hydroxide (NaOH) and potassium hydroxide (KOH).
• alkaline metal refers to metals of Group IA of the
• the present invention employs a solvent that: (i) is water-immiscible, (ii) is capable of forming an azeotropic mixture with water, or (iii) has a boiling point of at least 100°C.
• the solvent is water-immiscible.
• the solvent is capable of forming an azeotropic mixture with water.
• the solvent has a boiling point of at least 100°C.
• the solvent is water-immiscible and is capable of forming an azeotropic mixture with water.
• the solvent is water-immiscible and has a boiling point of at least 100°C.
• the solvent is capable of forming an azeotropic mixture with water and has a boiling point of at least 100°C.
• the solvent having a boiling point of at least 100°C can have a boiling point of at least about 120°C, at least about 140°C, or at least about 160°C. It is appreciated that those of skill in the art understand that the solvent should not chemically react with any of the starting materials or reagents present in the reaction mixture, to any significant degree, under the reaction conditions employed.
• water-immiscible solvent refers to a solvent that is not miscible (i.e., not capable of mixing in all proportions) with water.
• Suitable specific water-immiscible solvents include, e.g., aromatic hydrocarbons such as xylenes, oxylene, nt-xylene, ⁇ xylene, toluene, benzene, and combinations thereof; chlorinated solvents such as chloroform and methylene chloride; as well as other organic solvents such as ethyl-tert-butyl ether and ethyl acetate.
• water-miscible solvent refers to a solvent that is miscible (i.e., is capable of mixing in all proportions) with water. Suitable specific water-miscible solvents include, e.g., methanol, ethanol, z ' so-propanol, tert-butanol, ethylene glycol, acetone, 1-propanol and propylene glycol.
• distill or “distillation” refers to the process of extracting the volatile components of a mixture by the condensation and collection of the vapors that are produced as the mixture is heated. The process includes the evaporation and subsequent collection of a liquid by condensation.
• concentrating or “condensing” refers to the process whereby the volume is reduced, by the removal of liquid.
• mother liquor refers to the liquid obtained after solids are removed from a mixture or a solution of solids in a liquid. As such, the mother liquor will not include an appreciable amount of these solids.
• birch refers to any of the several deciduous trees of the genus Betula. The birches comprise the family Betulaceae in the order Fagales. Birch trees include, for example, white birch, B. alba; sweet, black or cheny birch, B. lenta; monarch birch, B.
• the birch can be B. alba, B. lenta, B. Maximowicziana, B. Nana, B. Platyphyla Japonica, B. Pubescens, B. alleghaniensis, B.
• birch bark refers to inner birch bark and outer birch bark.
• Inner birch bark is more dense and granular than outer birch bark, while outer birch bark is more flexible and fibrous than inner birch bark.
• Outer birch bark is light in color, thin (1-5 mm), tough, and of low water-content relative to inner birch bark. The inner bark is darker in color, thicker (3-10 mm) and non- fibrous relative to the outer bark.
• the inner bark is the portion of the tree wherein significant water transport occurs (i.e., an area of high water content). Due to the differences in the physical properties of inner birch bark and outer birch bark, fragmentation produces outer birch bark shreds and inner birch bark chunks. Outer birch bark shreds can be separated from the inner birch bark chunks using any suitable means. The separation can conveniently be accomplished by screening the mixture through a mesh having openings intermediate in size between the smaller inner bark chunks and the larger outer bark shreds. The smaller inner bark chunks fall through the screen and are separated from the outer bark.
• the “mesh” can be a unit comprising one or more open spaces in a cord, thread, or wire network in which the cords, threads or wires sunound the spaces.
• Any mesh suitable to separate inner birch bark from outer birch bark can be employed.
• the mesh is a wire mesh containing openings of about 54 of an inch by 54 of an inch, or smaller.
• mesh can conveniently contain openings of about 1/4 of an inch by about 1/4 of an inch.
• the size of the mesh can be about 20 mm by about 20 mm, or about 10 mm by about 10 mm, or about 6 mm by about 6 mm. More specifically, the size of the mesh can be about 3 mm by about 3 mm.
• the inner birch bark chunks and outer birch bark shreds may be separated with the use of an air classifier.
• an "air classifier” is a device which operates on the principle of the differing properties of the two components (e.g., imier and outer birch bark) in an air stream to effect a physical separation.
• the less dense outer bark travels a greater distance in the air stream than the more dense imier bark.
• the inner birch bark and the outer birch bark can be separated.
• outer birch bark of about 10 wt.%) to about 45 wt.% based on initial birch bark content is typically obtained and inner birch bark of about 55 wt.% to about 85 wt.% is typically obtained.
• birch bark shreds less than about 10 mm in diameter can conveniently be used. More specifically, outer birch bark shreds less than about 6 mm in diameter, less than about 4 mm in diameter, or less than about 2 mm in diameter, can be used.
• aromatic hydrocarbon refers to a compound having at least one phenyl or naphthyl ring, wherein the compound contains carbon and hydrogen atoms.
• the aromatic hydrocarbon can optionally be substituted, e.g., with one or more groups selected from the group of alkyl (e.g., methyl), hydroxyl, halo, alkoxy, cyano, carboxyl, sulfonyl, and amino.
• Suitable specific aromatic hydrocarbons include, e.g., xylenes, o-xylene, -xylene, p-xylene, toluene, benzene, and combinations thereof.
• "reflux” refers to the process of boiling a liquid in a vessel attached to a condenser so that the vapors continuously condense for reboiling.
• washing refers to the process of purifying a solid mass (e.g., crystals) by passing a liquid over and/or through the solid mass, as to remove soluble matter.
• the process includes passing a solvent, such as distilled water, over and/or through a precipitate obtained from filtering, decanting, or a combination thereof.
• a solvent such as distilled water
• washing includes contacting solids with water, vigorously stirring (e.g., for two hours), and filtering.
• the solvent can be water, can be an aqueous solvent system, or can be an organic solvent system.
• the washing can be ca ied out with the solvent having any suitable temperature.
• the washing can be carried out with the solvent having a temperature between about 0°C and about 100°C.
• a "binder" refers to a substance that can effectively bind
• Suitable binders useful in the present invention include, e.g., metal hydrides (e.g., lithium hydride (LiH), sodium hydride (NaH), potassium hydride (KH), calcium hydride (CaH ), and lithium aluminum hydride (LiAlH ); metal alcoholates (e.g., sodium methoxide (NaOMe), sodium ethoxide (NaOEt), potassium methoxide (KOMe), potassium ethoxide (KOEt), aluminum iso- propoxide [Al(i-OPr) 3 ], aluminum tert-butoxide [Al(t-OBu) 3 ], and aluminum methoxide [Al(OMe) 3 ]), ortho-esters (e.g., ethylorthocarbonate), dialkoxysulfates (e.g., dimethylsulfate and diethylsulfate), alumina and silica.
• metal hydrides e.g
• metal hydride refers to a binary compound of hydrogen and a metal. Suitable metal hydrides include e.g., lithium hydride (LiH), sodium hydride (NaH), potassium hydride (KH), calcium hydride (CaH 2 ), and lithium aluminum hydride (LiAlH ).
• metal alcoholate or “alcoholate” refers to an organic alcohol wherein the hydroxy hydrogen has been replaced with a metal, e.g., (CH CH 0) 3 A1.
• Aluminum alcoholates are suitable reagents for triterpene purification because it is believed that aluminum alcoholates bind strongly and ineversibly to acids and tannins, therefore providing complete discoloration of the total extract.
• Suitable specific metal alcoholates include, e.g., sodium methoxide (NaOMe), sodium ethoxide (NaOEt), potassium methoxide (KOMe), potassium ethoxide (KOEt), aluminum w ⁇ -propoxide [Al(i-OPr) 3 ], aluminum tert-butoxide [Al(t-OBu) 3 ], and aluminum methoxide [Al(OMe) ].
• “hydro lyze” or “hydrolysis” refers to the process of converting a carboxylic ester to the conesponding carboxylic acid, with the addition of water.
• the reaction i.e., hydrolysis
• the reaction can be canied out in a suitable solvent and at a suitable temperature and pressure, under basic conditions, neutral conditions, or acidic conditions.
• Suitable reagents and reaction conditions are disclosed, e.g., in Advanced Organic Chemistry, Part B: Reactions and Synthesis, Carey and Sundberg,
• the hydrolysis can be carried out at a pH of greater than about 7.0 (e.g., a pH of about 7-8, 8-9, 9-10, 10-11, or 11-12).
• Suitable bases include metal hydroxides and metal alkoxides.
• Suitable metal hydroxides include sodium hydroxide, potassium hydroxide, lithium hydroxide, calcium hydroxide and barium hydroxide.
• Suitable metal alkoxides include lithium methoxide, lithium ethoxide, lithium isopropoxide, lithium tert-butoxide, sodium methoxide, sodium ethoxide, sodium isopropoxide, sodium tert-butoxide, potassium methoxide, potassium ethoxide, potassium isopropoxide, potassium tert-butoxide, magnesium methoxide, magnesium ethoxide, barium methoxide, barium ethoxide, calcium methoxide and calcium ethoxide.
• a specific base suitable for the processes of the present invention i.e., hydrolysis of birch bark
• the hydrolysis can be carried out at a pH of less than about
• 7.0 e.g., a pH of about 1-2, 2-3, 3-4, 4-5, 5-6, or 6-7.
• suitable acids include, for example, hydrochloric acid, phosphoric acid, formic acid, hydi'obromic acid, sulfuric acid, nitric acid, acetic acid, and combinations thereof.
• a specific acid suitable for the processes of the present invention i.e., hydrolysis of birch bark
• the hydrolysis can be earned out at a pH of about 6.5 to about 7.5 (i.e., neutral conditions).
• natural ester refers to an organic compound (e.g., triterpenes or triterpenoids) having at least one carboxylic ester group.
• azeotropic distillation refers to the process of boiling off or distilling any liquid mixture having constant minimum and maximum boiling points and distilling off without decomposition and in a fixed ratio, as with benzene and water, or as with toluene and water.
• the azeotropic distillation can include the co -distillation of water and at least one of xylenes, oxylene, -xylene, p-xy ⁇ ene, toluene, and benzene.
• a distillation can be canied out at a temperature and a period of time, effective to distill or boil more than about 90% of the water, more than about 95% of the water, more than about 98% of the water, or up to about 100 wt.% of the water.
• azeotropic mixture refers to a mixture which would undergo azeotropic distillation.
• agitating refers to the process of putting a mixture into motion with a turbulent force. Suitable methods of agitating include, e.g., stining, mixing, and shaking.
• precipitating refers to the process of causing a solid substance (e.g., crystals) to be separated from a solution.
• the precipitating can include, e.g., crystallizing.
• polar organic solvent refers to an organic solvent having a measurable dipole. Specifically, it refers to an organic solvent having a dielectric constant of at least about 15, at least about 20, or between about 20 and about 30.
• non-polar organic solvent refers to an organic solvent having no measurable dipole. Specifically, it refers to an organic solvent having a dielectric constant of less than about 15, less than about 10, or between about 6 and about 10.
• drying refers to the removal of water and/or solvent, such that the water and/or solvent content is below about 5 wt.%, below about 2 wt.% or below about 1 wt.%.
• neutralizing refers to the process of changing or bringing the pH to about 7 ⁇ 1.
• the neutralizing can include bringing the pH to about 6 to about 8.
• the neutralizing can include bringing the pH to about 6.5 to about 7.5 (i.e., to a pH of about 7 ⁇ 0.5).
• the neutralizing can include bringing the pH to about 6.75 to about 7.25 (i.e., to a pH of about 7 ⁇ 0.25).
• acidifying refers to the process of lowering the pH to below 7.0.
• the acidifying includes lowering the pH to below about 6.0.
• the acidifying includes lowering the pH to below about 5.0.
• the acidifying includes lowering the pH to below about 4.0.
• purifying refers to the process of ridding a solid substrate (e.g., crystals) of impurities. Suitable methods of purifying include, e.g., washing, recrystallizing and drying. In cases where compounds are sufficiently basic or acidic to form stable nontoxic acid or base salts, administration of the compounds as salts maybe appropriate.
• Examples of pharmaceutically acceptable salts are organic acid addition salts formed with acids, which form a physiological acceptable anion, for example, tosylate, methanesulfonate, acetate, citrate, malonate, tartarate, succinate, benzoate, ascorbate, ⁇ -ketoglutarate, and oglycerophosphate.
• a physiological acceptable anion for example, tosylate, methanesulfonate, acetate, citrate, malonate, tartarate, succinate, benzoate, ascorbate, ⁇ -ketoglutarate, and oglycerophosphate.
• Suitable inorganic salts may also be formed, including hydrochloride, sulfate, nitrate, bicarbonate, and carbonate salts.
• Pharmaceutically acceptable salts may be obtained using standard procedures well known in the art, for example by reacting a sufficiently basic compound such as an amine with a suitable acid affording a physiologically acceptable anion.
• Alkali metal (for example, sodium, potassium or lithium) or alkaline earth metal (for example calcium) salts of carboxylic acids can also be made.
• compositions that include a compound obtained by the processes described herein can be fonnulated as pharmaceutical compositions and/or cosmetic compositions, and administered to a mammalian host, such as a human patient in a variety of forms adapted to the chosen route of administration, i.e., orally or parenterally, by intravenous, intramuscular, topical or subcutaneous routes.
• a mammalian host such as a human patient
• the present compositions can be systemically administered, e.g., orally, in combination with a pham aceutically acceptable vehicle such as an inert diluent or an assimilable edible canier. They may be enclosed in hard or soft shell gelatin capsules, may be compressed into tablets, or may be incorporated directly with the food of the patient's diet.
• compositions may be combined with one or more excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like.
• excipients for oral therapeutic administration, the compositions may be combined with one or more excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like.
• Such preparations should contain at least 0.1 % of the triterpene compound.
• the percentage of the compositions can, of course, be varied and may conveniently be between about 2 to about 60%) of the weight of a given unit dosage form.
• the amount of active compound (i.e., triterpene compound) in such therapeutically useful compositions is such that an effective dosage level will be obtained.
• the tablets, troches, pills, capsules, and the like may also contain the following: binders such as gum tragacanth, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as com starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; and a sweetening agent such as sucrose, fructose, lactose or aspartame or a flavoring agent such as peppermint, oil of wintergreen, or cheny flavoring may be added.
• a liquid canier such as a vegetable oil or a polyethylene glycol.
• any material used in preparing any unit dosage form should be pharmaceutically acceptable and substantially non-toxic in the amounts employed.
• the active compound i.e., triterpene
• the active compound may be incorporated into sustained-release preparations and devices.
• compositions may also be administered intravenously or intraperitoneally by infusion or injection.
• Solutions of the triterpene can be prepared in water, optionally mixed with a nontoxic surfactant.
• Dispersions can also be prepared in glycerol, liquid polyethylene glycols, triacetin, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
• the pharmaceutical dosage forms suitable for injection or infusion can include sterile aqueous solutions or dispersions or sterile powders comprising the active ingredient, which are adapted for the extemporaneous preparation of sterile injectable or infusible solutions or dispersions, optionally encapsulated in liposomes.
• the liquid canier or vehicle can be a solvent or liquid dispersion medium comprising, for example, water, ethanol, a polyol (for example, glycerol, propylene glycol, liquid polyethylene glycols, and the like), vegetable oils, nontoxic glyceryl esters, and suitable mixtures thereof.
• the proper fluidity can be maintained, for example, by the formation of liposomes, by the maintenance of the required particle size in the case of dispersions or by the use of surfactants.
• the prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like.
• isotonic agents for example, sugars, buffers or sodium chloride.
• Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.
• Sterile injectable solutions are prepared by incorporating the triterpene in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filter sterilization.
• the prefened methods of preparation are vacuum drying and the freeze-drying techniques, which yield a powder of the triterpene, plus any additional desired ingredient present in the previously sterile-filtered solutions.
• the present compositions may be applied in pure form, i.e., when they are liquids. However, it will generally be desirable to administer them to the skin as compositions or formulations, in combination with a dermatologically acceptable canier, which may be a solid or a liquid.
• a dermatologically acceptable canier which may be a solid or a liquid.
• Useful solid carriers include finely divided solids such as talc, clay, microcrystalline cellulose, silica, alumina and the like.
• Useful liquid carriers include water, alcohols or glycols or water-alcohol/glycol blends, in which the triterpene can be dissolved or dispersed at effective levels, optionally with the aid of non-toxic surfactants.
• Adjuvants such as fragrances and additional antimicrobial agents can be added to optimize the properties for a given use.
• the resultant liquid compositions can be applied from absorbent pads, used to impregnate bandages and other dressings, or sprayed onto the affected area using pump-type or aerosol sprayers.
• Thickeners such as synthetic polymers, fatty acids, fatty acid salts and esters, fatty alcohols, modified celluloses or modified mineral materials can also be employed with liquid caniers to form spreadable pastes, gels, ointments, soaps, and the like, for application directly to the skin of the user.
• useful dermatological compositions which can be used to deliver the compositions of the triterpene, to the skin, are known to the art; for example, see Jacquet et al. (U.S. Pat. No. 4,608,392), Geria (U.S. Pat. No. 4,992,478), Smith et al. (U.S. Pat. No. 4,559,157) and Wortzman (U.S. Pat. No.
• compositions of the triterpene can be determined by comparing their in vitro activity, and in vivo activity in animal models. Methods for the extrapolation of effective dosages in mice, and other animals, to humans are known to the art; for example, see U.S. Pat. No. 4,938,949.
• concentration of the compositions of the triterpene in a liquid composition, such as a lotion will be from about 0.1-25 wt-%, preferably from about 0.5-10 wt-%.
• concentration in a semi-solid or solid composition such as a gel or a powder will be about 0.1-5 wt-%, preferably about 0.5-2.5 wt- %.
• a suitable dose will be in the range of from about 0.5 to about 100 mg/kg, e.g., from about 10 to about 75 mg/kg of body weight per day, such as 3 to about 50 mg per kilogram body weight of the recipient per day, preferably in the range of 6 to 90 mg/kg/day, most preferably in the range of 15 to 60 mg/kg/day.
• the composition is conveniently administered in unit dosage form; for example, containing 5 to 1000 mg, conveniently 10 to 750 mg, most conveniently, 50 to 500 mg of triterpene per unit dosage form.
• the composition should be administered to achieve peak plasma concentrations of the triterpene of from about 0.5 to about 75 ⁇ M, preferably, about 1 to 50 ⁇ M, most preferably, about 2 to about 30 ⁇ M. This maybe achieved, for example, by the intravenous injection of a 0.05 to 5% solution of the triterpene, optionally in saline, or orally administered as a bolus containing about 1-100 mg of the triterpene.
• Desirable blood levels may be maintained by continuous infusion to provide about 0.01-5.0 mg/kg/hr or by intermittent infusions containing about 0.4-15 mg/kg of the triterpene(s).
• the desired dose may conveniently be presented in a single dose or as divided doses administered at appropriate intervals, for example, as two, three, four or more sub-doses per day.
• the sub-dose itself may be further divided, e.g., into a number of discrete loosely spaced administrations; such as multiple inhalations from an insufflator or by application of a plurality of drops into the eye.
• compositions of the invention may be determined using pharmacological models which are well known to the art.
• the compositions of the invention maybe also be useful as pharmacological tools for the further investigation of the mechanism of their anti-fungal, anti-bacterial, and/or anti- viral action.
• the compositions of the invention can also be administered in combination with other therapeutic agents that are effective to treat, e.g., fungal infections, bacterial infections, and/or viral infections; or to inhibit or kill a fungus, bacterium, and/or virus.
• the processing includes extracting (3) outer birch bark (2) with an organic solvent to provide a birch bark extract (4).
• Suitable solvents, techniques and conditions for the extracting (3) are known to those of skill in the art.
• the extracting (3) will preferably employ suitable solvent(s) in which the desired compound(s) are relatively soluble.
• the extracting (3) can be canied out under conditions (e.g., temperature, pressure and period of time), effective to extract the desired compound(s) in a relatively high yield, while minimizing any decomposition of the desired compound(s).
• the extracting (3) effectively provides birch bark extract (4), which can subsequently be refluxed (21) with a metal hydroxide (5), distilled (6), and filtered/decanted (7), to provide the solids (8) and filtrate (9).
• the refluxing (21) of the birch bark extract (4) with the metal hydroxide (5) can be carried out under conditions (e.g., temperature and period of time) and with reagents and solvents, effective to reflux (21) the reaction mixture, while minimizing any decomposition of the desired compound(s).
• Suitable metal hydroxides (5) are l ⁇ iown to those of skill in the art.
• the reaction mixture can then be distilled (6), to effectively remove water present in the reaction mixture.
• This distillation (6) will remove not only water, but also a discrete amount or organic solvent (e.g., xylenes, toluene and/or benzene) present in the reaction mixture.
• the distillation (6) will also not remove any appreciable amounts of metal hydroxides (5) along with the water. Additionally, no appreciable amounts of triterpenes or triterpenoids are removed with the water during the distillation (6).
• the metal hydroxide reacts with acids (e.g., triterpenoid acids) while in solution, to provide the conesponding salts. These salts are relatively insoluble in the organic solvent employed. As such, the salts will precipitate from the solution after removal of the water during the distillation (6).
• the solids (8) which can be visible as a black tar, precipitates from solution, and can subsequently be separated (e.g., filtered or decanted (7)) from solution to provide the solid (8) and filtrate (9).
• the solid (8) contains betulinic acid (15), and can be purified accordingly.
• the filtrate (9) can be treated with binder (25) to provide a mixture.
• the mixture can be filtered (27) to provide a second filtrate.
• the second filtrate can be concentrated (29), and crystallized (30), to provide betulin (17) and a filtrate (16).
• This filtrate (16) can be concentrated (31), washed (33), crystallized (35), concentrated (36) and crystallized (37) to provide lupeol (20).
• the solid (8) described above can be washed with water (38), neutralized/acidified (10), filtered/decanted (11), and subsequently crystallized (12) to provide betulinic acid (15).
• plant matter processing (100) utilizing distillation (106) is illustrated.
• Plant matter (101) can be purified, to provide acidic components of plant mater extract (117) and non-acidic components of plant matter extract (120).
• the plat matter (101) is extracted (102) to provide plant matter extract (103).
• the plant matter extract (103) can b e refluxed with a metal hydroxide (105), distilled (106), and filtered/decanted (107), to provide solids (108) and filtrate (109).
• the solids (108) can be washed (130), neutralized/acidified (110), filtered/decanted (111), and crystallized (112), to provide acidic components of plant matter extract (117).
• the filtrate (109) can be contacted with binder (131), filtered (132), concentrated (113), and crystallized (114), to provide filtrate (115) and non-acidic component of plant matter extract (116).
• the filtrate (115) can be concentrated (140), washed (141), and crystallized (142) to provide non-acidic component of plant matter extract (120).
• Example 1 Extraction of outer birch bark with xylenes Air-dry pellets of outer birch bark (200 g) were extracted with p-xylene (1 x 1 L) and then with p-xylene (3 x 300ml). Each time the extraction mixture was refluxed for 20 min and then filtered at 90°C. The solvent of the combined organic extracts was evaporated and the residue was dried at 80°C, in vacuum, to give 44 g (yield is 22 wt.%>) of dry birch bark extract. The p-xylene extract was used without further purification.
• Example 2 Isolation of betulin
• the birch bark extract (44 g) obtained from the procedure in Example 1 was dissolved in xylenes (1.1 L) at 80°C.
• Sodium hydroxide (NaOH) (8.8g) was dissolved in 80°C water (17.4 ml).
• the two mixtures were combined, vigorously stined and refluxed for 1 hr.
• Water (17.4 ml) was removed from the reaction mixture by azeotropic distillation on a Dean-Stark trap, and the reaction mixture was filtered to remove the black tar that precipitated from solution.
• This black tar includes the crude sodium salts of triterpenoid acids (19.8 g), including betulinic acid, as shown in Example 4 below.
• Example 3 Isolation of lupeol
• the mother liquid from Example 2 was evaporated to afford 11 g (25 %) of crade lupeol / betulin mixture. 20 ml of acetone was added to this mixture, stined at 60°C for one hour and cooled down 5-10°C for two hours. The yellowish precipitate (4.5 g) was filtered and crystallized from xylene. The mother liquid after xylene filtering was removed and the yellowish solid crystals of lupeol were crystalyzed from cyclohexane.
• Example 5 Azeotropic distillation sing sodium hydroxide, water and xylenes A solution of NaOH (90g) in 180 ml of water was added to the vigorously stined solution of birch bark extract in xylenes* (13.5 L) at 90°C.
• the stined solution was kept at 100°C for 1 hour. Azeotropic mixture of water and xylenes (total volume 4.0 L) was distilled (xylene recycling). The xylenes solution (9.5 L) was then filtered at 90-100°C into the heated up to 100°C reactor and transfened to example 7. The black solid product was dried at 80°C for 5 hours in vacuum. This product (196g) was transfened to example 9 for betulinic acid separation and purification. * - The cheapest Xylene isomers mixture can be used for this purpose
• Example 6 Azeotropic Distillation A solution of KOH (126g in 180 ml of water) was added to the ⁇ vigorously stined solution of birch bark extract (456 g in 13.5 L of xylenes*) at 90°C. The stined solution was kept at 100°C for 1 hour. Azeotropic mixture of water and xylenes (total volume 4.0 L) was removed (xylene recycling). The solution was then filtered at 90-100°C. The black solid product was transfened from filter and dried at 80°C for 5 hours in vacuum. This product (221 g) was transfened to example 9 for betulinic acid separation and purification. * - The cheapest Xylene isomers mixture may be used for this purpose (Aldrich 24,764-2; 18 L, $93.80)
• Example 7 Treatment with Dimethyl Sulfate and crystallization Recycled betulin (33 g) and 18.0 g of K2CO3 were added to the stined hot filtrate of treated with NaOH birch bark extract (350 g) in xylenes (9.5 L) (xylenes solution from example 5). This mixture was stined for 10 minutes at 100°C. Dimethyl sulfate (12 ml) was added and the resulting mixture was stined for 2 hrs. Then 180 ml of water was added and mixture was stined at 100°C for 0.5 hour. Xylenes (5.5 L) and water (170 ml) were removed by azeotropic distillation.
• Example 8 Lupeol separation and betulin recycling Xylenes were evaporated from and the filtrate solution (the filtrate solution from example 7). Acetone (0.5 L) was then added and the mixture was vigorously stined and refluxed for 1 hr. Then acetone solution was cooled down to room temperature and filtered. Operation with acetone was repeated three times. White solid material (83 g) after washing with acetone was dried and crystallized from 1.8 L of xylene. After filtration 37 g of solid white material (75% of betulin and 10% of lupeol, 15% others) was received and used in Example 7 as recycling betulin product.
• the yield of lupeol is 0.47% calculated from starting outer birch bark. Combined acetone mother liqueurs were evaporated. Yellow solid (27.6 g) product (50% of lupeol) was obtained and can be used for lupeol separation in recycling process.
• Example 9 Betulinic acid separation and purification Dry black solid product from step 1 (196 g) was stined in 3.5 L of water and filtered. This operation was repeated two times using 1 L of water each time. A solution of 2% HCl (0.8 L) was then added to a suspension of solid product in 250 ml of water and stined for 0.5 hours, filtered. Solid product was then washed with water (2x200 ml) and dried at 40°C for 5 hours in vacuum drier to give 32 g of crade betulinic acid.
• Crade betulinic acid (32 g) was dissolved in 0.95 L of refluxed 2- propanol, filtered and evaporated to 300 ml, and left for crystallization at 35°C for 5 hours. Precipitate was filtered at 35°C and dried at 40°C for 5 hours in vacuum drier to give 17.6 g of crade betulinic acid.
• Cmde betulinic acid (17.6 g) was refluxed with 500 ml of 2-propanol and filtered. 2-Propanol was evaporated to volume of 170 ml. After crystallization at room temperature white crystals were filtered and 15.5 g of betulinic acid (92 %>+ purity) was obtained.
• Betulinic acid (15.5 g) was dissolved in 500 ml of refluxed xylenes and a solution of NaOH (5 g) in 10 ml of water was added. This solution was refluxed with a good stining for 1 hour. Xylenes and water (total volume is 90 ml) were removed by azeotropic distillation from reaction mixture. Sodium salt of betulinic acid was filtered on hot filter and dried at 80oC for 3 hours. White solid product was then added into water (50 ml) and a 1% solution (200 ml) of HCl in water was added dropwise, and stined for 0.5 hour, filtered, washed with water (2x50 ml) and dried. 14 g of betulinic acid (98%) purity) was obtained. The yield of betulinic acid from starting outer birch bark was 0.7%.
• Example 10 Betulin separation from birch bark extract
• Raw Material Amount Birch Bark pellets 100 g Xylenes 1065 ml KOH 6.125 g Water 8.75 ml Celite 521 (Aldrich) 20 g Aluminum Oxide (basic) 2.5 g
• the approximate expected retention time for Betulinic Acid is 7.7 minutes. Specificity is determined by an analysis of the diluent blank sample. Specificity of the instrument is acceptable when there is no interference of the blank sample with quantitation of the target sample. In order to test the linearity of the relationship between the target sample and concentration, five standards are prepared covering the range of 10-120% of the 50 ⁇ g/mL target concentration for Betulinic Acid. The peak area response versus concentration is evaluated by linear regression. The slope, intercept and residual sum of squares are reported to provide a linear conelation of not less than 0.99 (1 being perfectly linear). The sensitivity samples have target concentrations of 12.5 ⁇ g/mL and 5 ⁇ g/mL Betulinic Acid.

Landscapes

• Chemical & Material Sciences (AREA)
• Organic Chemistry (AREA)
• Health & Medical Sciences (AREA)
• General Health & Medical Sciences (AREA)
• Steroid Compounds (AREA)
• Medicines Containing Plant Substances (AREA)
• Compounds Of Unknown Constitution (AREA)

Applications Claiming Priority (2)

Publications (1)

Family

ID=34590334

Family Applications (1)

Country Status (2)

Cited By (1)

Families Citing this family (8)

Family Cites Families (3)

• 2004
  • 2004-11-10 WO PCT/US2004/038252 patent/WO2005047304A2/en not_active Application Discontinuation
  • 2004-11-10 EP EP04811101A patent/EP1687326A2/de not_active Withdrawn

Non-Patent Citations (1)

Also Published As

Similar Documents

Legal Events