EP1708724A1 - Composition comprising xanthoceras sorbifolia extracts, compounds isolated from same, methods for preparing same and uses thereof - Google Patents

Composition comprising xanthoceras sorbifolia extracts, compounds isolated from same, methods for preparing same and uses thereof

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Publication number
EP1708724A1
EP1708724A1 EP04815530A EP04815530A EP1708724A1 EP 1708724 A1 EP1708724 A1 EP 1708724A1 EP 04815530 A EP04815530 A EP 04815530A EP 04815530 A EP04815530 A EP 04815530A EP 1708724 A1 EP1708724 A1 EP 1708724A1
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EP
European Patent Office
Prior art keywords
compound
composition
cancer
extract
profile
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP04815530A
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German (de)
French (fr)
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EP1708724A4 (en
Inventor
Pui-Kwong Chan
May Sung Mak
Yun Wang
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Pacific Arrow Ltd
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Pacific Arrow Ltd
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Priority claimed from PCT/US2004/033359 external-priority patent/WO2005037200A2/en
Application filed by Pacific Arrow Ltd filed Critical Pacific Arrow Ltd
Publication of EP1708724A1 publication Critical patent/EP1708724A1/en
Publication of EP1708724A4 publication Critical patent/EP1708724A4/en
Withdrawn legal-status Critical Current

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/045—Hydroxy compounds, e.g. alcohols; Salts thereof, e.g. alcoholates
    • A61K31/05—Phenols
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/555—Heterocyclic compounds containing heavy metals, e.g. hemin, hematin, melarsoprol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7012—Compounds having a free or esterified carboxyl group attached, directly or through a carbon chain, to a carbon atom of the saccharide radical, e.g. glucuronic acid, neuraminic acid
    • 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)
    • A61K36/77—Sapindaceae (Soapberry family), e.g. lychee or soapberry
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/10—Drugs for disorders of the urinary system of the bladder
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A61P15/10—Drugs for genital or sexual disorders; Contraceptives for impotence
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/04—Centrally acting analgesics, e.g. opioids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
    • A61P25/16—Anti-Parkinson drugs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/06—Antihyperlipidemics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/18—Antivirals for RNA viruses for HIV
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/04—Inotropic agents, i.e. stimulants of cardiac contraction; Drugs for heart failure
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/14—Vasoprotectives; Antihaemorrhoidals; Drugs for varicose therapy; Capillary stabilisers

Definitions

  • This invention relates to bioactive compounds isolated and/or purified from the extracts of a plant called Xanthoceras Sorbifolia, commonly referred to as Wenguanguo in Traditional Chinese Medicine) , their usages and functions, and methods of their preparation.
  • This invention is related to the use the compounds purified from the extracts of Wenguanguo. These compounds can prevent patients from enuresis and frequency micturition, improve the functions of the central nervous system of signaling from the bladder for wakening from deep sleep, relaxes the bladder so that it can store more urine.
  • the compounds identified and purified from Wenguanguo may be used to relax the detrusor tension caused by aging, stress, nervousness, over-activity, instability, hyper-reflexia, and uninhibited bladder.
  • the extracts of Wenguanguo may be used for relaxing the contracted bladder tissue induced by acetylcholine, ACh.
  • the compounds identified and purified from Wenguanguo may be used as acetylcolinesterase, AChE inhibitor, for regulating Antidiuretic hormone (ADH) , which reduces the volume of urine and as an anti-inflammatory agent.
  • AChE inhibitor for regulating Antidiuretic hormone (ADH) , which reduces the volume of urine and as an anti-inflammatory agent.
  • This invention provides a process of producing saponins from the husks, kernels, leaves, fruit-stems, branches, stems, roots, seed's shells and barks of Wenguanguo and their applications.
  • This invention provides a composition comprising saponins from the husks, kernels, leaves, fruit- ste s, branches, stems, roots, seed's shells and barks of Wenguanguo.
  • Wenguanguo saponins may be used for accelerating the growth of bladder, for suppressing deep sleep, for increasing alterness in a sleeping subject, for modulating the release, breakdown and uptake of Antidieuretic hormone (ADH) and its receptors, for modulating the secretion, breakdown and uptake of Adrenocorticotropic hormone (ACTH) and its receptors, for modulating the release, breakdown and uptake of 5-hydroxytryptamine and its receptors, for modulating the release, breakdown and uptake of Acetycholine (Ach) and its receptors, for modulating the release, breakdown and uptake of Adrenaline (AD) and its receptors, for modulating the release, breakdown and uptake of Dopamine (DA) and its receptors, for modulating the release, breakdown and uptake of Norepinephrine (NE) and its receptors, for preventing sleep paralysis, for modulating the formation, release, breakdown and activity of neuropeptides and their receptors, for curing cancer, including but not limited to breast cancer, leukocyte
  • This invention provides compounds comprising a triterpene backbone or Sapogenin, in which the sugar moiety attached to Carbon 3 of the triterpene backbone; an acetyl and/or sugar moiety linkage to Carbon 21 and 22; and angeloyl groups attached to the sugar moiety. These functional groups operatively linked to the triterpene or Sapogenin to form the compound.
  • This invention provides compounds comprising the following structures:
  • This invention provides a salt of the above-described compounds.
  • This invention provides a composition comprising the above-described compounds and a suitable carrier.
  • This invention provides a pharmaceutical composition comprising an effective amount of the above-described compounds and a pharmaceutically acceptable carrier (s).
  • This invention provides a method for isolating compounds from Xanthoceras Sorbifolia comprising steps of: extracting Xanthoceras Sorbifolia powder with an appropriate amount of one or more organic solvents for an appropriate amount of time to form an organic extract; collecting the organic extract; refluxing the organic extract to form a second extract; removing the organic solvent from the second extract; drying and sterilizing the second extract to form a Xanthoceras Sorbifolia extract powder; fractionating the extract powder to obtain one or more components of the extract powder; identifying the bioactive components of the extract powder; purifying one or more bioactive components of the extract powder with FPLC to obtain one or more fraction of the bioactive component; and isolating the compound with preparative HPLC.
  • This invention provides a compound, hereinafter referred to as compound Yl and having its structure verified by ID-, 2D- NMR spectral data, as shown in Figures 7, 8, 9 and 10.
  • This invention provides a compound, hereinafter referred to as compound Rl and having its structure verified by 1D-NMR (-NMR, C13-NMR) , 2D-NMR (HMBC, HMQC) and MS spectral analysis.
  • the data or profiles of this compound are presented in Figures 21, 22, 23, 24 and 25.
  • This invention provides the chemical features of a compound Yl having the formula C65H100O27 and the chemical name:
  • the compounds or compositions of the present invention regulate the receptors or components of cell such as G- protein receptor, Fas protein, receptor Tyrosine' Kinases, Mitogen, mitogen receptor.
  • the compounds can be isolated from the plant called Xanthoceras Sorbifolia or can be synthesized chemically, or extracted from other biological sources .
  • This invention provides a compound Yl identified and purified from Xanthoceras Sorbifolia against cancer growth.
  • the cancer includes, but is not limited to bladder cancer, cervix cancer, prostate cancer, lung cancer, breast cancer, leukocytes cancer, colon cancer, liver cancer, bone cancer, brain cancer, and ovary cancer.
  • Figure 1 shows the separation of an extract of Xanthoceras Sorbifolia components by HPLC with a ⁇ bondapak C18 column.
  • Figure 2 shows screening of cell growth activity of FPLC fractions (conducted with bladder cells) .
  • Figure 2A shows the elution fractions obtained from FPLC. These fractions were subsequently used in MTT assay to determine which fraction is active.
  • Figures 2B shows that different components of Xanthoceras Sorbifolia extracts (as fractionated by FPLC) cause either growth or inhibition effects on cells. Only fraction 5962 (Y components) causes cell inhibition. Abscissa: concentration (ug/ml) . Ordinate:
  • Figure 3 shows elution profile of Fraction 5962 with 64% acetonitrile isocratic elution. Two major FPLC fractions X and Y are separated. Ordinate: optical density (254nm) . Abscissa: fraction number (lml/fraction) .
  • Figure 4 shows the comparison of inhibition activity in bladder cells by Fractions X (2021) and Y (2728) . Only Y has inhibition activity.
  • Figure 5 shows the HPLC profile of Fraction Y with 35% acetonitrile isocratic elution. It shows that the Y (2728) fraction contains 4-5 components (Y0, Yl, Y2, Y3 and Y4).
  • Figure 6 shows HPLC profile of Fraction Ys with 45%
  • Acetonitrile isocratic elution in a preparative C18 column (Delta Pak C18) . Under these conditions, fractions Yl and Y2 are well separated from each other and they are collected individually.
  • Figure 7 shows the Proton NMR spectrum of Yl .
  • Figure 8 shows 2D NMR spectrum of Yl (HMQC) .
  • Figure 9 shows the 2D NMR HMBC profile of Yl .
  • Figure 10 shows COSY-NMR profile of Yl .
  • Figure 11 shows the proton NMR spectrum of Y2.
  • Figure 12 shows 2D NMR spectrum of Y2 (HMQC) .
  • Figure 13 shows proton NMR spectrum of Y5.
  • Figure 14 shows 2D NMR spectrum of Y5 (HMQC) .
  • Figure 15 shows four possible chemical structures of Yl.
  • Figure 16 shows the structure of Yl.
  • Figure 17 shows the inhibition of the Yl and Y2 on Ovarian cancer cells' growth.
  • Figure 18 shows purification of compound R from with FPLC.
  • Figure 19 shows the HPLC analysis of fractions #9, #10 and #11 obtained from FPLC.
  • FIG. 20 shows purification of component-R with HPLC
  • Figure 21 shows Proton-NMR spectra of compound Rl .
  • Figure 22 shows 2D NMR (HMQC) spectra of compound Rl .
  • Figure 23 shows 2D NMR (HMBC) spectra of compound Rl .
  • Figure 24 shows 2D NMR (COSY) spectra of compound Rl .
  • Figure 25 shows C13 NMR spectra of compound Rl .
  • Figure 26 shows chemical structure of compound Rl .
  • Figure 27 shows chemical structure of Y-a.
  • R5 B or C or SI (see note 1) ;
  • Rl A or B or C;
  • R2 A or B or C;
  • R4 B or C.
  • A angeloyl;
  • B acetyl;
  • C H;
  • Sl chain with one or more sugar such as
  • D- glucose, D-galactose, L-rhamnose, L-arabinose, D-xylose, and alduronic acid such as D- glucuronic acid, D- galacturonic acid, and their derivatives.
  • Figure 28 shows chemical structure of Y-b.
  • R5 B or C or SI (see note 1) ;
  • Rl A or B or C;
  • R2 A or B or C;
  • R4 B or C.
  • A angeloyl;
  • B acetyl;
  • C H;
  • Sl chain with one or more sugar such as D- glucose, D-galactose, L-rhamnose, L-arabinose, D-xylose, and alduronic acid such as D- glucuronic acid, D- galacturonic acid, and their derivatives.
  • Figure 29 shows chemical structure of Y-c.
  • Figure 30 shows chemical structure of Yl-a.
  • R5 B or C or SI (see note 1);
  • Rl A or B or C;
  • R2 A or B or C;
  • R4 B or C.
  • A angeloyl;
  • B acetyl;
  • C H;
  • Sl chain with one or more sugar such as D- glucose, D-galactose, L-rhamnose, L-arabinose, D-xylose, and alduronic acid such as D- glucuronic acid, D- galacturonic acid, and their derivatives.
  • Figure 31 shows chemical structure of Yl-b.
  • Figure 32 shows chemical structure of Yl-c.
  • A angeloyl
  • B acetyl
  • C H
  • Sl chain with one or more sugar such as D- glucose, D-galactose, L-rhamnose, L-arabinose, D-xylose, and alduronic acid such as D- glucuronic acid, D- galacturonic acid, and their derivatives.
  • Figure 33 shows Fractionation of component-0 from FPLC with HPLC with 20% acetonitrile isocratic elution (isO-20) .
  • Figure 34 shows Rechromatography of #28 (from iso-20) .
  • Figure 35 shows Rechromatography of #34 (from iso-20) .
  • Figure 36 shows Rechromatography of #54 (from iso-20) .
  • This invention provides the crude saponins from the husks, fruit-stems or seed's shell, leaves, branches and stems, kernels and roots of Wenguanguo, in the form of Wenguanguo powder.
  • the methods for preparing the crude saponins from Wenguanguo comprise the following steps: extracting Wenguanguo powder with an organic solvent, i.e., ethanol, methanol and others, at ratio of 1:2 for 4-5 times for 20-35 hours each time to form an organic extract; collecting and refluxing the organic extract 2-3 times at 80°C to form second extracts; resolving the second extracts in water to form an aqueous solution; extract the aqueous solution by n- butanol to form a n-butanol extracts; performing chromatography the n-butanol extracts to form the crude saponins.
  • the crude extract or Wenguanguo extract comprises saponins .
  • the Wenguanguo extracts of the present invention can also be used to treat the enuresis, frequent micturition and urinary incontinence. However, the Wenguanguo extracts cure the enuresis, frequent micturition and urinary incontinence through the "bladder pathway" to regulate water metabolism of human's body and urination.
  • the Wenguanguo extracts of the present invention stimulate the growth of the bladder. See Figure 9 and Figure 23B.
  • the Wenguanguo extracts of th'e present invention increase the capacity of bladder and function of bladder controlling the urination. For example, see Experiment 5 on page 101, lines 14-29, to page 106, lines 1-21 in International PCT Application NO. PCT/US04/33359.
  • Wenguanguo extracts when used with the "kidney pathway” herbs to treat the enuresis, frequent micturition and urinary incontinence, will strengthen both the pathways of kidney and bladder, and then will produce better treatment results .
  • This invention provides the medicines or health foods which further comprises Vitamin B, Vitamin D, Vitamin K, grape seed extract and other antioxidants, Cordyceps or its extract, gingko or its extract, Panax ginseng and P. quinquefolium or their extracts, Huangpi (Clausena lansium) or its extracts, Echinacea or its extract, St John's Wort (Hypericum perforatum) or its extract, herbs (Pueraria lobata) or its extract, Tianma (Gastrodia elata) or its extract, Armillariella mellea or its extract, Danshen (Salvia iltiorrhiza) or its extract, Sanqi (Panax notoginsen) or its extract, Monascus or Honqu (Red yeast rice) , Huanqi (Hedysarum polybotrys) or its extract, D ihuang (Rehmannia glutinosa) or its extract, Danggui (Angelica
  • Nocetile, folic acid, amino acids, creatine, fiber supplement, or any combination thereof Nocetile, folic acid, amino acids, creatine, fiber supplement, or any combination thereof.
  • This invention provides the extract of Wenguanguo for inhibiting the uptake of 5-hydroxytryptamine (5HT) in a subject.
  • 5-HT controls and modulates a sleep factor that sustains and increases deep sleep. Inhibiting the uptake of 5HT will decrease deep sleep. People who spend too much time in SWS 3 and SWS 4 are unable to awaken from their sleep when their bladder is full because their sleep is too deep. This is the reason that enuresis often occurs during SWS 3 and SWS 4. See page 38, lines 1-35 in International PCT Application NO. PCT/US04/33359.
  • This invention provides the compounds of Wenguanguo for increasing the activity of Dopamine in a subject thereby making the central nerve system of said subject alert.
  • This invention provides the compounds of Wenguanguo for increasing the secretion of antidiuretic hormone (ADH) in a subject, which reduces urine in said subject.
  • ADH antidiuretic hormone
  • This invention provides the compounds of Wenguanguo for modulating the release, breakdown and uptake of Acetylcholine (Ach) and its receptors in a subject.
  • the said extracts of this invention inhibits the deep sleep created by 5HT and increase REM sleep.
  • This invention provides the compounds of Wenguanguo for preventing sleep paralysis in a subject.
  • This invention provides the compounds of Wenguanguo for providing alertness to a sleeping subject.
  • This invention provides the compounds for helping the growth of the bladder and sphincter.
  • An immature bladder and sphincter cannot control the process and action of urination. By accelerating the growth of the bladder and the sphincter, this problem will be overcome, and enuresis will not occur.
  • the cancer includes, but is not limited to bladder cancer, cervix cancer, prostate cancer, lung cancer, breast cancer, leukocytes cancer, colon cancer, liver cancer, bone cancer, brain cancer, and ovary cancer.
  • This invention provides compounds comprising a triterpene backbone or Sapogenin, in which the sugar moiety attached to Carbon 3 of the triterpene backbone; an acetyl and/or sugar moiety linkage to Carbon 21 and 22; and angeloyl groups attached to the sugar moiety. These functional groups operatively linked to the triterpene or Sapogenin to form the compound.
  • This invention provides a compound comprising the following structures :
  • This invention provides a compound comprising the following structures :
  • the compounds of the present invention comprise the structures as shown in Figures 15A, 15B, 15C 15D, 27, 28, 29, 30, 31 and 32.
  • This invention provides a salt of the above-described compounds .
  • This invention provides a composition comprising the above- described compounds and a suitable carrier.
  • This invention provides a pharmaceutical composition comprising an effective amount of the above-described compounds and a pharmaceutically acceptable carrier.
  • This invention provides an anti-ovarian cancer and composition comprising the above-described composition.
  • HTB-9 bladedder
  • HeLa-S3 cervix
  • DU145 prostate
  • H460 lung
  • MCF-7 breast
  • K562 leukocytes
  • HCTll ⁇ colon
  • HepG2 liver
  • U20S bone
  • T98G brain
  • OVCAR-3 ovary
  • Cells were grown in culture medium (HeLa-S3, DU145, MCF-7, Hep-G2 and T98G in MEN (Earle's salts); HTB-9, H460, K562, OVCAR-3 in RPMI- 1640; HCT-116, U20S in McCoy-5A) supplemented with 10% fetal calf serum, glutamine and antibiotics in a 5% C02 humidified incubator at 37oC.
  • culture medium HeLa-S3, DU145, MCF-7, Hep-G2 and T98G in MEN (Earle's salts); HTB-9, H460, K562, OVCAR-3 in RPMI- 1640; HCT-116, U20S in McCoy-5A
  • MTT Assay The procedure for MTT assay followed the method described in (Carmichael et al., 1987) with only minor modifications.
  • Cells were seeded into a 96-wells' plate at concentrations of 10,000/well (HTB-9, HeLa, H460, HCT116, T98G, OVCAR-3), 15,000/well (DU145, MCF-7, HepG2, U20S) , or 40,000/well (K562), for 24 hours before drug-treatment. Cells were then exposed to drugs for 48 hours (72 hours for HepG2, U20S, and 96 hours for MCF-7) . After the drug- treatment, MTT (0.5 mg/ml) was added to cultures for an hour.
  • %G (TD-T0 / TC-T0) x 100 (1) ,
  • TC or TD represent O.D. readings of control or drug- treated cells.
  • LC cytotoxicity
  • %LC (TD-T0 / TO) x 100 (2) .
  • This invention provides the cellular signaling pathways interacted by compounds isolated from Xanthoceras Sorbifolia.
  • a compound has the formula C65H100O27 and the chemical name:
  • the compounds of the present invention comprise the chemical structures designated herein as "Structure Y", “Structure Rl”, “Structure 1 to 4", “Structure Y-a to Y-c” and “Structure Yl-a to Yl-c", “Structure Yl-3 to Yl-4" and their derivatives. See Figures 15, 27-32. These components described above regulate the receptors or components of cells. These' compounds can be isolated from the plant called Xanthoceras Sorbifolia or can be synthesized.
  • the Xanthoceras Sorbifolia compound or its derivatives work in the Wnt (Wingless-type MMTV integration site family member) signaling pathway.
  • the Wnt signaling pathway is evolutionarily conserved and controls many events during the embryogenesis. This pathway regulates cell morphology, proliferation, motility and as well as cell apoptosis. It also plays an important role during tumorigenesis .
  • the Wnt pathway has also been observed as inappropriately activated in several different types of cancers in humans .
  • the target genes for Wnt signaling are normally kept silent by an inhibitory complex of gene regulatory proteins, e.g. the Groucho corepressor protein bound to the gene regulatory protein LEF-I/TCF.
  • gene regulatory proteins e.g. the Groucho corepressor protein bound to the gene regulatory protein LEF-I/TCF.
  • some ⁇ -cartenin is bound to the cytosolic tail of cadherin proteins, and any cytosolic ⁇ - cartenin that becomes bound by the APC-axin-GSK-3 ⁇ will trigger its ubiquitylation and degradation in proteasomes. The result is the decrease of intracellular amount of ⁇ - cartenin.
  • Frizzled a seven transmembrane receptor
  • LRP Low density lipoprotein Receptor
  • APC ⁇ - catenin-axin-adenomatous-polyposis coli
  • GSK glycogen synthase kinase
  • Dvl When stimulated by Wnt, Dvl recruits the GSK-3 binding protein, GBP, to the multiprotein complex of ⁇ -catenin-axin-adenomatous-polyposis coli (APC) -glycogen synthase kinase (GSK)-3 ⁇ . GBP then titrates GSK- ⁇ from axin, and in this way, phosphorylation of ⁇ -catenin is inhibited. Then, axin is sequestrated by LRP at the cell membrane. The result of all of this is an accumulation of cytosolic ⁇ -catenin. In the nucleus, ⁇ - catenin binds to LEF-I/TCF, displaces Groucho, and acts a co-activator to stimulate the transcription of Wnt target genes .
  • GBP GSK-3 binding protein
  • Xanthoceras Sorbifolia compositions regulate the components related to Wnt pathways or its receptors, thereby stopping the proliferation of cancer cells.
  • the compound or its derivatives work in the Mitogens, Ras and a MAP (Microtubule associated protein) kinase pathway. Mitogens stimulate cell division. The binding of mitogens to cell-surface receptors leads to the activation of Ras and a MAP kinase cascade.
  • MAP Microtubule associated protein
  • One effect of this pathway is the increased production of the gene regulatory protein Myc. Myc increases the transcription of several genes, including the gene encoding cyclin D and a subunit of the SCF ubiquitin ligase.
  • the resulting increase in Gl-Cdk and Gl/S-Cdk activities promotes Rb phosphyorylation and activation of the gene regulatory protein E2F, resulting in S-phase entry, in which Gl-Cdk activity initiates Rb phosphorylation, in turn inactivating Rb and freeing E2F to activate the transcription of S-phase genes including the genes for a Gl/S-cyclin(cyclin E) and S-cyclin (cyclin A).
  • the resulting appearance of Gl/S-Cdk and S-Cdk further enhances Rb phosphorylation, forming a positive feedback loop, and the E2F acts back to stimulate the transcription of its own gene, forming another positive feedback loop.
  • Myc may also promote E2F activity directly by stimulating the transcription of the E2F gene. The result is the increased transcription of genes entry into S phase. However if this pathway is overactive, it will cause cancer cell growth.
  • the compound or its derivatives work in Ras-dependent or Myc pathway.
  • mutation of amino acid in Ras causes the protein to become permanently overactive, stimulating the Ras-dependent signal pathways overactive in absence of mitogenic stimulation.
  • mutations that cause an overexpression of Myc promote excessive cell growth, which in turn promotes the development of cancer.
  • the compound or its derivatives reactivate the abnormal cell checkpoint mechanism.
  • a checkpoint mechanism which detects abnormal mitogenic stimulation and causes abnormally overactive cells to go into apopto'sis.
  • this mechanism is not active in cancer cells due to mutations in the genes that encode essential components of the checkpoint responses. If the mutation happens in the checkpoint mechanism, the cancer cell will grow and divide endlessly.
  • Compounds or compositions derived from the plant Xanthoceras Sorbifolia reactivate the checkpoint mechanism to stop the cancer cell growth.
  • the compound or its derivatives affect the extracellular growth signaling pathways.
  • the extracellular growth factors that stimulate cell growth are bound to receptors on the cell surface and activate intracellular signaling pathways. It activates the enzyme PI3-kimase, which promotes protein synthesis, at least partly through the activation of eif4e and phosphorylated S6 1 kinase, resulting in increased mRNA translation and then a stimulation of cell growth.
  • Compounds or compositions derived from the plant Xanthoceras Sorbifolia regulate the components' or receptor relate to extracellular growth. It binds the receptor of ovarian cancer cells so as to stop the cancer cell growth.
  • Compounds or compositions derived from the plant Xanthoceras Sorbifolia regulate the components relating to Ras and MAP Kinase, which ceases ovarian cancer cell growth.
  • the compound or its derivatives affect the intracellular mechanism.
  • Cell division is also controlled by an intracellular mechanism that can limit cell proliferation.
  • the Myc protein acts in the nucleus as a signal for cell proliferation. Large quantities of Myc can cause the cell to proliferate in excess and form a tumor.
  • Compounds or compositions derived from the plant Xanthoceras Sorbifolia regulate the components or receptor of the Myc cell's proliferation to stop the tumor cells from dividing.
  • TGF-alpha is produced by keratimcytes, macrophages, hepatocytes, and platelets. Its synthesis is stimulated by the infection by viruses.
  • TGF-Alpha induces the long term proliferation of murine and chicken immature hematopoietic progenitor cell such as BFU-E without causing differentiation. It also induces the terminal differentiation of BFU-Ecell into erythrocytes .
  • TGF-Alpha stimulates the proliferation of cultured endothelial cells. It plays an importance role in the vascularisation of tumor tissues .
  • Compounds or compositions derived from the plant Xanthoceras Sorbifolia regulate the components or receptor of TGF-alpha to suppress ovarian cancer and bladder cancer cell growth.
  • TGF-beta affects the TGF-beta signaling pathway.
  • TGF-beta regulates growth and proliferation of cells, blocking growth of many cell types.
  • TGF-beta receptors There are two TGF-beta receptors: Type 1 and Type 2. They are serine-threonine kinases that signal through the SMAD (Protein named after the first two identified, Sma in C. elegans and Mad in Drosophila) family of transcriptional regulators. The TGF-beta pathway and mutation in SMADs are associated with cancer in humans. Compounds or compositions derived from the plant Xanthoceras Sorbifolia regulate the components or receptor of TGF-beta to suppress the ovarian cancer and bladder cancer cell growth.
  • the compound or its derivatives reactivate the cell functions which are damaged by DNA viruses.
  • DNA tumor viruses cause cancer by interfering with cell cycle control Rb protein and the p53 protein. Mutation in p53 gene will allow cancer cells to survive and proliferate despite DNA damage.
  • the papillomanius uses the proteins E6 and E7 to sequence the p53 and Rb respectively. This action activates mutated cells, allowing them to survive and then divide and accumulate. The accumulation of damaged cells can lead to cancer.
  • the compound or its derivatives affect the p53 signaling pathway.
  • p53 helps multi-cellular organisms cope safely with DNA damage and other stressful cellular ' events, stopping cell proliferation in circumstances where it would be dangerous. Cancer cells tend to contain large quantities of mutant p53 protein, suggesting that the genetic accidents they undergo or the stresses of growth in an inappropriate environment created the signals that normally activate the p53 protein. Thus, the loss of p53 activity can be extremely dangerous in relation to cancer because it allows mutant cells to continue through the cell cycle. It also allows them to escape apoptosis. So, if their DNA is damaged, some cells will die but the cells which survive will carry on dividing without pausing to repair the damage.
  • the compound or its derivatives affect the cell suicide signaling pathway. All cells with a nucleus contain various inactive procaspases, awaiting a signal before destroying the cell.
  • Each suicide protease is made as an inactive proenzyme called procaspase. It is usually activated by proteolytic cleavage by another member of the caspase family. Two of the cleaved fragments come together to form the active part of the caspase, and the active enzyme is thought to be a tetramer of two of these two parts.
  • Each activated caspase molecule can cleave many procaspase molecules, which in turn activates more molecules. Through a chain reaction or cascade, this leads to the explosive action of a large number of procaspase molecules. Then, some of the activated procaspases cleave a number of key proteins in the cell, including specific cytosolic proteins and nuclear-lamins leading to the controlled death of the cell.
  • Activating the death receptor on the outside of the cell can also trigger inactive procaspases.
  • killer lymphocytes can cause apoptosis by producing the protein Fas on the surface of the targeted cell. These clusters of Fas protein then recruit intracellular adaptor proteins that bind and aggregate procaspase-8 molecules. These then cleave and activate one another. The activated caspase-8 molecules then activate downstream procaspases to induce apoptosis.
  • This invention provides compounds comprising a triterpene backbone or Sapogenin, in which the sugar moiety attached to Carbon 3 of the triterpene backbone; an acetyl and/or sugar moiety linkage to Carbon 21 and 22; and angeloyl groups attached to the sugar moiety. These functional groups operatively linked to the triterpene or Sapogenin to form the compound.
  • This invention provides a method for inhibiting tumor cell growth comprising contacting an amount of the above- described compounds.
  • This invention provides a method for inhibiting tumor cell growth in a subject comprising administering to the above- described subject,
  • Experiment 1 Herb Extraction (a) extracting Xanthoceras Sorbifolia powder of husks or branches or stems or leaves or kernels or roots or barks with organic solvent at ratio of 1:2 for 4-5 times for 20-35 hours for each time to form an organic extract; (b) collecting the organic extract; (c) refluxing the organic extract for 2-3 times at 80°C to form second extracts; (d) removing the organic solvent from the second extract; and (e) Drying and sterilizing the extract to form a Xanthoceras
  • Sorbifolia extract powder is Sorbifolia extract powder.
  • HPLC HPLC. About 60-70 peaks can be accounted for in the profile. Among them four are major peaks, 10 are medium size and the rest are small fractions. The major peaks are labelled with a to z following increased concentration of acetonitrile elution. See Figure 1.
  • HTB-9 bladedder
  • HeLa-S3 cervix
  • DU145 prostate
  • H460 lung
  • MCF-7 breast
  • K562 leukocytes
  • HCT116 colon
  • HepG2 liver
  • U20S bone
  • T98G brain
  • OVCAR-3 ovary
  • Cells were grown in culture medium (HeLa-S3, DU145, MCF-7, Hep-G2 and T98G in MEN (Earle's salts); HTB-9, H460, K562, OVCAR-3 in RPMI- 1640; HCT-116, U20S in McCoy-5A) supplemented with 10% fetal calf serum, glutamine and antibiotics in a 5% C02 humidified incubator at 37oC.
  • culture medium HeLa-S3, DU145, MCF-7, Hep-G2 and T98G in MEN (Earle's salts); HTB-9, H460, K562, OVCAR-3 in RPMI- 1640; HCT-116, U20S in McCoy-5A
  • MTT assay The procedure for MTT assay followed the method described in (Car ichael et al., 1987) with only minor modifications.
  • Cells were seeded into a 96-wells plate at concentrations of 10,000/well (HTB-9, HeLa, H460, HCT116, T98G, OVCAR-3), 15,000/well (DU145, MCF-7, HepG2 , U20S) , or 40,000/well (K562) , for 24 hours before drug-treatment. Cells were then exposed to drugs for 48 hours (72 hours for HepG2, U20S, and 96 hours for MCF-7). After the drug- treatment, MTT (0.5 mg/ml) was added to cultures for an hour.
  • %G (TD-TO / TC-TO) x 100 (1) where TC or TD represent O.D. readings of control or drug- treated cells.
  • LC cytotoxicity
  • FPLC fractions are grouped into 7 pools and analyzed for cell growth activity with bladder cells with MTT assay. It was found only one pool (#5962) contains inhibition activity. See Figure 2B.
  • the purified fraction Y (5962) of the Xanthoceras Sorbifolia plant extract of the present invention does not have the growth stimulating activity but maintained the inhibition activity.
  • Fraction Collector 1 ml fraction (collect the first 90 fractions)
  • Instrument AKTA-FPLC, P920 pump; Monitor UPC-900; Frac-900.
  • Fraction 5962 was further separated with an open ODS-C18 column using isocratic 64% acetonitrile elution. Two major fractions, i.e., X and Y, were collected. See Figure 3. MTT assay showed that only the 'Y fraction has the inhibition activity. See Figure 4.
  • the pure Compound Yl is amorphous white powder, soluble in aqueous alcohol (methanol, ethanol), 50% acetonitrile and 100% pyridine.
  • NMR analysis The pure compound Yl of Xanthoceras Sorbifolia were dissolved in pyridine-D5 with 0.05% v/v TMS. All NMR spectra were acquired using a Bruker Avance 600 MHz NMR spectrometer with a QXI probe (1H/13C/15N/31P) at 298 K. The numbers of scans for ID 1H spectra were 16 to 128, depending on the sample concentration. 2D HMQC spectra were recorded with spectral widths of 6000 x 24,000 Hz and data points of 2024 x 256 for t2 and tl dimensions, respectively. The numbers of scans were 4 to 128.
  • 2D HMBC were acquired with spectral widths of 6000 x 30,000 Hz and data points of 2024 x 512 for t2 and tl dimensions, respectively.
  • the numbers of scans were 64.
  • the 2D data were zero-filled in tl dimension to double the data points, multiplied by cosine-square-bell window functions in both tl and t2 dimensions, and Fourier- transformed using software XWIN-NMR.
  • the final real matrix sizes of these 2D spectra are 2048 x 256 and 2048 x 512 data points (F2 x FI) for HMQC and HMBC, respectively.
  • Mass spectral analysis The mass of samples was analyzed by MALDI-TOF Mass Spectrometry.
  • Samples for MALDI-TOF were first dissolved in acetonitrile, then mixed with the matrix CHCA (Alpha-cyano-4- hydroxycinnamic acid, lOmg CHCA/mL in 50:50 water/acetonitrile and 0.1% TFA in final concentration). The sample was dissolved completely in acetonitrile and stayed dissolved after mixing with the matrix. The molecular weight was determined by the high resolution mass spectroscope analysis with standards.
  • CHCA Alpha-cyano-4- hydroxycinnamic acid
  • the profile of the proton NMR of Yl is presented in Figure 7.
  • the profiles of 2D NMR (HMQC) of Yl is presented in Figure 8.
  • the profiles of 2D NMR (HMBC) of Yl is presented in Figure 9.
  • the profiles of 2D NMR (COSY) of Yl is presented in Figure 10.
  • the active compound Yl isolated from extract of Xanthoceras Sorbifolia is a triterpenoid saponins with four sugars and biangeloyl groups attached to the sugar moiety.
  • the formula of Yl is C65H100O27, and the structure and the chemical name of Yl is:
  • the linkage of oligosaccharide can be cleaved by partial acid hydrolysis and by specific enzyme hydrolysis.
  • the 1 ⁇ 4 linkage of arabinofuranosyl can be removed by ⁇ -amylase.
  • Other enzymes such as ⁇ -amylase, isoamylase, glucose oxidase, mannanse and pullulanase can be used to cleave individual saccharide in saponins.
  • Angeloyl groups of the structures can be removed by alkaline hydrolysis.
  • compound Y is dissolved in IM NaOH and stirred for 2-3 hours at room temperature. The solution is then acidified or neutralized with 2M HCI and hydrolyzed saponin can be extracted with ethyl acetate. Further purification can be achieved with HPLC using C18 column.
  • enzyme hydrolysis of Structure Y the following structures can be obtained:
  • Fraction Collector 5 ml fraction (collect the first 90 fractions)
  • Fractions 9-11 contain a major component with' a few minor components. See Figure 19. These components were further separated into 4-5 components with 30% acetonitrile isocratic elution in a DeltaPak column. The fraction designated herein as "Rl" is the major component. See Figure 20A. This fraction Rl was subsequently isolated by collecting from the column. See Figure 20B.
  • the pure Rl is amorphous white powder, soluble in aqueous alcohol (methanol, ethanol) , 50% acetonitrile and 100% pyridine.
  • the compound Rl isolated from extract of Xanthoceras Sorbifolia is a triterpenoid saponins with five sugars and one angeloyl group attached to the sugar moiety.
  • the formula of compound Rl is C65H106O29, and the chemical name of Rl is:
  • Rl Chemical name of structure Rl is: 3-0- [angeloyl- (1 (3) - (-D- glucopyranosyl- (1(6) ] - (-D-glucopyranosyl-28-O- [ (-L- rhamnopyranosyl- (1 (2) - (-D-glucopyranosyl- (1(6)- (-D- glucopyranosyl-3 ( , 21 (, 22 (, 28-tetrahydroxyolean-12-ene
  • Sample loading 1-2 ml, concentration: 100 mg/ml in 10% acetonitrile/TFA.
  • Gradient elution 10-80% acetonitrile in a total volume of 500 ml.
  • Fraction Collector 5 ml/fractions (collect from 10% to 72% acetonitrile, total 90 fractions)
  • Instrument AKTA-FPLC, P920 pump; Monitor UPC-900; Frac-900.
  • Fractions including 28, 34 and 54 were collected and lyophilized. Re-chromatographic analysis of these fractions were performed under same conditions.

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Abstract

This invention provides methods and process of identification and producing pure, biologically active compound(s) from Xanthoceras sorbifolia. This invention also provides the composition of the pure compound(s) of interest from Xanthoceras sorbifolia. The purified compounds comprise saponins. The composition can be used for treating cancer, arthritis, rheumatism, poor circulation, arteriosclerosis, Raynaud’s syndrome, angina pectoris, cardiac disorder, coronary heart disease, headache, dizziness, kidney disorder, impotence and premature ejaculation; for preventing cerebral aging; for improving memory, cerebral functions; or for curing enuresis, frequent micturition, urinary incontinence, dementia, weak intelligence and Alzheimer’s disease, autism, brain trauma, Parkinson’s disease or other diseases caused by cerebral dysfunctions, and treating arthritis, rheumatism, poor circulation, arteriosclerosis, Raynaud’s syndrome, angina pectoris, cardiac disorder, coronary heart disease, headache, dizziness, kidney disorder. This invention provides compounds comprising a triterpene backbone or Sapogenin, in which the sugar moiety attached to Carbon 3 of the triterpene backbone; an acetyl and/or sugar moiety linkage to Carbon 21 and 22; and angeloyl groups attached to the sugar moiety. These functional groups operatively linked to the triterpene or Sapogenin to form the compound.

Description

COMPOSITION COMPRISING XANTHOCERAS SORBIFOLIA EXTRACTS, COMPOUNDS ISOLATED FROM SAME, METHODS FOR PREPARING SAME AND USES THEREOF
This application claims priority of International - Application No. PCT/US04/33359, filed October 8, 2004, which claims benefit of U.S. Serial No. 60/532,101, Filed December 23, 2003; which claims priority of U.S. Serial No. 60/617,379, filed October 8, 2004; U.S. Serial No. 60/613,811, filed September 27, 2004; and U.S. Serial No. 60/607,858, filed September 7, 2004. The contents of these preceding applications are hereby incorporated in their entireties by reference into this application.
Throughout this application, various publications are referenced. Disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this invention pertains.
FIELD OF THE INVENTION I This invention relates to bioactive compounds isolated and/or purified from the extracts of a plant called Xanthoceras Sorbifolia, commonly referred to as Wenguanguo in Traditional Chinese Medicine) , their usages and functions, and methods of their preparation.
BACKGROUND OF THE INVENTION For a more detailed discussion of the background or relevant art of the present invention please refer to page 1, lines 25-38, to page 13 in International PCT Application NO. PCT/US04/33359, filed October 8, 2004, which is incorporated herein in its entirety by reference.
SUMMARY OF THE INVENTION
In accordance with these and other objects of the invention, a brief summary of the present invention is presented. Some simplifications and omission may be made in the following summary, which is intended to highlight and introduce some aspects of the present invention, but not to limit its scope. Detailed descriptions of a preferred exemplary embodiment adequate to allow those of ordinary skill in the art to make and use the invention concepts will follow in later sections.
This invention is related to the use the compounds purified from the extracts of Wenguanguo. These compounds can prevent patients from enuresis and frequency micturition, improve the functions of the central nervous system of signaling from the bladder for wakening from deep sleep, relaxes the bladder so that it can store more urine.
The compounds identified and purified from Wenguanguo may be used to relax the detrusor tension caused by aging, stress, nervousness, over-activity, instability, hyper-reflexia, and uninhibited bladder. In another embodiment, the extracts of Wenguanguo may be used for relaxing the contracted bladder tissue induced by acetylcholine, ACh.
The compounds identified and purified from Wenguanguo may be used as acetylcolinesterase, AChE inhibitor, for regulating Antidiuretic hormone (ADH) , which reduces the volume of urine and as an anti-inflammatory agent. This invention provides a process of producing saponins from the husks, kernels, leaves, fruit-stems, branches, stems, roots, seed's shells and barks of Wenguanguo and their applications. This invention provides a composition comprising saponins from the husks, kernels, leaves, fruit- ste s, branches, stems, roots, seed's shells and barks of Wenguanguo.
Wenguanguo saponins may be used for accelerating the growth of bladder, for suppressing deep sleep, for increasing alterness in a sleeping subject, for modulating the release, breakdown and uptake of Antidieuretic hormone (ADH) and its receptors, for modulating the secretion, breakdown and uptake of Adrenocorticotropic hormone (ACTH) and its receptors, for modulating the release, breakdown and uptake of 5-hydroxytryptamine and its receptors, for modulating the release, breakdown and uptake of Acetycholine (Ach) and its receptors, for modulating the release, breakdown and uptake of Adrenaline (AD) and its receptors, for modulating the release, breakdown and uptake of Dopamine (DA) and its receptors, for modulating the release, breakdown and uptake of Norepinephrine (NE) and its receptors, for preventing sleep paralysis, for modulating the formation, release, breakdown and activity of neuropeptides and their receptors, for curing cancer, including but not limited to breast cancer, leukocyte cancer, liver cancer, ovary cancer, bladder cancer, prostate cancer and brain cancer, and for improving the functions of the lung and the bladder.
This invention provides compounds comprising a triterpene backbone or Sapogenin, in which the sugar moiety attached to Carbon 3 of the triterpene backbone; an acetyl and/or sugar moiety linkage to Carbon 21 and 22; and angeloyl groups attached to the sugar moiety. These functional groups operatively linked to the triterpene or Sapogenin to form the compound.
This invention provides compounds comprising the following structures:
Compound Y-l
3-0- [ (-D-galactopyranosyl (1(2)]- (-L- arabinofuranosyl (1 (3) - (-D-glucuronopyranosyl-21-O- (3, 4-diangeloyl) - (-L-rhamnop yranosyl-22-O-acetyl- 3(,16(, 21 (, 22 (, 28-pentahydroxyolean-12-ene
Compound Rl
Structure Rl
3-0- [angeloyl- (1 (3) - (-D-glucopyranosyl- (1(6)]- (-D- glucopyranosyl-28-O- [ (-L-rhamnopyranosyl- (1(2)- (-D- glucopyranosyl- (1 (6) - (-D-glucopyranosyl-3 ( , 21 (, 22 (, 28- tetrahydroxyolean-12-ene
This invention provides a salt of the above-described compounds. This invention provides a composition comprising the above-described compounds and a suitable carrier.
This invention provides a pharmaceutical composition comprising an effective amount of the above-described compounds and a pharmaceutically acceptable carrier (s). This invention provides a method for isolating compounds from Xanthoceras Sorbifolia comprising steps of: extracting Xanthoceras Sorbifolia powder with an appropriate amount of one or more organic solvents for an appropriate amount of time to form an organic extract; collecting the organic extract; refluxing the organic extract to form a second extract; removing the organic solvent from the second extract; drying and sterilizing the second extract to form a Xanthoceras Sorbifolia extract powder; fractionating the extract powder to obtain one or more components of the extract powder; identifying the bioactive components of the extract powder; purifying one or more bioactive components of the extract powder with FPLC to obtain one or more fraction of the bioactive component; and isolating the compound with preparative HPLC.
This invention provides a compound, hereinafter referred to as compound Yl and having its structure verified by ID-, 2D- NMR spectral data, as shown in Figures 7, 8, 9 and 10.
This invention provides a compound, hereinafter referred to as compound Rl and having its structure verified by 1D-NMR (-NMR, C13-NMR) , 2D-NMR (HMBC, HMQC) and MS spectral analysis. The data or profiles of this compound are presented in Figures 21, 22, 23, 24 and 25.
This invention provides the chemical features of a compound Yl having the formula C65H100O27 and the chemical name:
3-0- [/?-D-galactopyranosyl (1→2) ] -or-L-arabinofuranosyl (l-»3) - ?-D-glucuronopyranosyl-21-0- (3, 4-diangeloyl) -cc-L- rhamnophyranosyl-22-0-acetyl-3?, 16α, 21/0, 22α, 28- pentahydroxyolean-12-ene, and its derivatives which are effective against cancer. The compounds or compositions of the present invention regulate the receptors or components of cell such as G- protein receptor, Fas protein, receptor Tyrosine' Kinases, Mitogen, mitogen receptor. The compounds can be isolated from the plant called Xanthoceras Sorbifolia or can be synthesized chemically, or extracted from other biological sources .
This invention provides a compound Yl identified and purified from Xanthoceras Sorbifolia against cancer growth. The cancer includes, but is not limited to bladder cancer, cervix cancer, prostate cancer, lung cancer, breast cancer, leukocytes cancer, colon cancer, liver cancer, bone cancer, brain cancer, and ovary cancer.
In accordance with these and other objects of the invention, a brief summary of the present invention is presented. Some simplifications and omission may be made in the following summary, which is intended to highlight and introduce some aspects of the present invention, but not to limit its scope. Detailed descriptions of a preferred exemplary embodiment adequate to allow those of ordinary skill in the art to make and use the invention concepts will follow in later sections.
DETAILED DESCRIPTION OF THE FIGURES
Figure 1 shows the separation of an extract of Xanthoceras Sorbifolia components by HPLC with a μbondapak C18 column.
Figure 2 shows screening of cell growth activity of FPLC fractions (conducted with bladder cells) . Figure 2A shows the elution fractions obtained from FPLC. These fractions were subsequently used in MTT assay to determine which fraction is active. Figures 2B shows that different components of Xanthoceras Sorbifolia extracts (as fractionated by FPLC) cause either growth or inhibition effects on cells. Only fraction 5962 (Y components) causes cell inhibition. Abscissa: concentration (ug/ml) . Ordinate:
% Cell Growth (determined by MTT assay) .
Figure 3 shows elution profile of Fraction 5962 with 64% acetonitrile isocratic elution. Two major FPLC fractions X and Y are separated. Ordinate: optical density (254nm) . Abscissa: fraction number (lml/fraction) .
Figure 4 shows the comparison of inhibition activity in bladder cells by Fractions X (2021) and Y (2728) . Only Y has inhibition activity.
Figure 5 shows the HPLC profile of Fraction Y with 35% acetonitrile isocratic elution. It shows that the Y (2728) fraction contains 4-5 components (Y0, Yl, Y2, Y3 and Y4).
Figure 6 shows HPLC profile of Fraction Ys with 45%
Acetonitrile isocratic elution in a preparative C18 column (Delta Pak C18) . Under these conditions, fractions Yl and Y2 are well separated from each other and they are collected individually.
Figure 7 shows the Proton NMR spectrum of Yl .
Figure 8 shows 2D NMR spectrum of Yl (HMQC) .
Figure 9 shows the 2D NMR HMBC profile of Yl . Figure 10 shows COSY-NMR profile of Yl .
Figure 11 shows the proton NMR spectrum of Y2.
Figure 12 shows 2D NMR spectrum of Y2 (HMQC) .
Figure 13 shows proton NMR spectrum of Y5.
Figure 14 shows 2D NMR spectrum of Y5 (HMQC) . Figure 15 shows four possible chemical structures of Yl. A: structure Yl-1; B: structure Yl-2; C: structure Yl-3 and D: structure Yl-4.
Figure 16 shows the structure of Yl. Figure 17 shows the inhibition of the Yl and Y2 on Ovarian cancer cells' growth.
Figure 18 shows purification of compound R from with FPLC.
Figure 19 shows the HPLC analysis of fractions #9, #10 and #11 obtained from FPLC.
Figure 20 shows purification of component-R with HPLC
(Delta-Pak C18) . A: Extract from fraction #10 of FPLC (iso-
30) was further separated by HPLC (conditions are (1) column: DeltaPak C18 column; (2) elution: 30% acetonitrile isocratic; (3) Flow rate: lml/min;monitored at 207 nm; O.D. scale: -0.128. B: Re-chromatogram of the major R component under same condition as described in A.
Figure 21 shows Proton-NMR spectra of compound Rl .
Figure 22 shows 2D NMR (HMQC) spectra of compound Rl . Figure 23 shows 2D NMR (HMBC) spectra of compound Rl .
Figure 24 shows 2D NMR (COSY) spectra of compound Rl .
Figure 25 shows C13 NMR spectra of compound Rl .
Figure 26 shows chemical structure of compound Rl .
Figure 27 shows chemical structure of Y-a. R5 = B or C or SI (see note 1) ; Rl = A or B or C; R2 = A or B or C; R4 = B or C. Note: A = angeloyl; B = acetyl; C = H; Sl= chain with one or more sugar such as
D- glucose, D-galactose, L-rhamnose, L-arabinose, D-xylose, and alduronic acid such as D- glucuronic acid, D- galacturonic acid, and their derivatives.
Figure 28 shows chemical structure of Y-b.
R5 = B or C or SI (see note 1) ; Rl = A or B or C; R2 = A or B or C; R4 = B or C. Note: A = angeloyl; B = acetyl; C = H; Sl= chain with one or more sugar such as D- glucose, D-galactose, L-rhamnose, L-arabinose, D-xylose, and alduronic acid such as D- glucuronic acid, D- galacturonic acid, and their derivatives.
Figure 29 shows chemical structure of Y-c.
Figure 30 shows chemical structure of Yl-a. R5 = B or C or SI (see note 1); Rl = A or B or C; R2 = A or B or C; R4 = B or C. Note: A = angeloyl; B = acetyl; C = H; Sl= chain with one or more sugar such as D- glucose, D-galactose, L-rhamnose, L-arabinose, D-xylose, and alduronic acid such as D- glucuronic acid, D- galacturonic acid, and their derivatives. Figure 31 shows chemical structure of Yl-b.
R5 = B or C or SI (see note 1) ; Rl = A or B or C; R2 = A or B or C; R4 = B or C. Note: A = angeloyl; B = acetyl; C = H; Sl= chain with one or more sugar such as D- glucose, D-galactose, L-rhamnose, L-arabinose, D-xylose, and alduronic acid such as D- glucuronic acid, " D- galacturonic acid, and their derivatives. Figure 32 shows chemical structure of Yl-c. R5 = B or C or SI (see note 1); Rl = A or B or C; R2 = A or B or C; R4 = B or C. Note: A = angeloyl; B = acetyl; C = H; Sl= chain with one or more sugar such as D- glucose, D-galactose, L-rhamnose, L-arabinose, D-xylose, and alduronic acid such as D- glucuronic acid, D- galacturonic acid, and their derivatives.
Figure 33 shows Fractionation of component-0 from FPLC with HPLC with 20% acetonitrile isocratic elution (isO-20) . Figure 34 shows Rechromatography of #28 (from iso-20) . Figure 35 shows Rechromatography of #34 (from iso-20) . Figure 36 shows Rechromatography of #54 (from iso-20) .
DETAILED DESCRIPTION OF THE INVENTION
This invention provides the crude saponins from the husks, fruit-stems or seed's shell, leaves, branches and stems, kernels and roots of Wenguanguo, in the form of Wenguanguo powder. The methods for preparing the crude saponins from Wenguanguo comprise the following steps: extracting Wenguanguo powder with an organic solvent, i.e., ethanol, methanol and others, at ratio of 1:2 for 4-5 times for 20-35 hours each time to form an organic extract; collecting and refluxing the organic extract 2-3 times at 80°C to form second extracts; resolving the second extracts in water to form an aqueous solution; extract the aqueous solution by n- butanol to form a n-butanol extracts; performing chromatography the n-butanol extracts to form the crude saponins. The crude extract or Wenguanguo extract comprises saponins .
According to the theory of traditional Chinese medicine, enuresis, frequent micturition and urinary incontinence are caused by "deficiency in kidney ("shen")". Therefore, they are treated by using Chinese herbs which can tone the kidney, such as Ginseng Bajitian, Roucongrong Duzhong and Cordyceps. These tonifying herbs can strengthen function of the kidney and regulate water metabolism of human's body through the "kidney pathway" that will help with curing the enuresis, frequent micturition and urinary incontinence.
The Wenguanguo extracts of the present invention can also be used to treat the enuresis, frequent micturition and urinary incontinence. However, the Wenguanguo extracts cure the enuresis, frequent micturition and urinary incontinence through the "bladder pathway" to regulate water metabolism of human's body and urination. The Wenguanguo extracts of the present invention stimulate the growth of the bladder. See Figure 9 and Figure 23B. The Wenguanguo extracts of th'e present invention increase the capacity of bladder and function of bladder controlling the urination. For example, see Experiment 5 on page 101, lines 14-29, to page 106, lines 1-21 in International PCT Application NO. PCT/US04/33359. In another aspect of the present invention, Wenguanguo extracts, when used with the "kidney pathway" herbs to treat the enuresis, frequent micturition and urinary incontinence, will strengthen both the pathways of kidney and bladder, and then will produce better treatment results .
This invention provides the medicines or health foods which further comprises Vitamin B, Vitamin D, Vitamin K, grape seed extract and other antioxidants, Cordyceps or its extract, gingko or its extract, Panax ginseng and P. quinquefolium or their extracts, Huangpi (Clausena lansium) or its extracts, Echinacea or its extract, St John's Wort (Hypericum perforatum) or its extract, Gegen (Pueraria lobata) or its extract, Tianma (Gastrodia elata) or its extract, Armillariella mellea or its extract, Danshen (Salvia iltiorrhiza) or its extract, Sanqi (Panax notoginsen) or its extract, Monascus or Honqu (Red yeast rice) , Huanqi (Hedysarum polybotrys) or its extract, D ihuang (Rehmannia glutinosa) or its extract, Danggui (Angelica sinensis) , Yuanzhi (Polygala tenuifoila) or its extract, Lingzhi (Ganoderma spp.) or its extracts, Fuling
(Poria cocos) or its extract, enokitake (Flammulina velutipes) or its extract, Gan Cao (Glycyrrhiza uralensis
Fisch) or its extract, Huperzine A, Lacithin, Metrifonate,
Nocetile, folic acid, amino acids, creatine, fiber supplement, or any combination thereof.
This invention provides the extract of Wenguanguo for inhibiting the uptake of 5-hydroxytryptamine (5HT) in a subject.
5-HT controls and modulates a sleep factor that sustains and increases deep sleep. Inhibiting the uptake of 5HT will decrease deep sleep. People who spend too much time in SWS 3 and SWS 4 are unable to awaken from their sleep when their bladder is full because their sleep is too deep. This is the reason that enuresis often occurs during SWS 3 and SWS 4. See page 38, lines 1-35 in International PCT Application NO. PCT/US04/33359.
This invention provides the compounds of Wenguanguo for increasing the activity of Dopamine in a subject thereby making the central nerve system of said subject alert.
This invention provides the compounds of Wenguanguo for increasing the secretion of antidiuretic hormone (ADH) in a subject, which reduces urine in said subject.
This invention provides the compounds of Wenguanguo for modulating the release, breakdown and uptake of Acetylcholine (Ach) and its receptors in a subject. The said extracts of this invention inhibits the deep sleep created by 5HT and increase REM sleep.
This invention provides the compounds of Wenguanguo for preventing sleep paralysis in a subject.
This invention provides the compounds of Wenguanguo for providing alertness to a sleeping subject.
This invention provides the compounds for helping the growth of the bladder and sphincter. An immature bladder and sphincter cannot control the process and action of urination. By accelerating the growth of the bladder and the sphincter, this problem will be overcome, and enuresis will not occur.
This invention provides the compounds of Wenguanguo against cancer growth. The cancer includes, but is not limited to bladder cancer, cervix cancer, prostate cancer, lung cancer, breast cancer, leukocytes cancer, colon cancer, liver cancer, bone cancer, brain cancer, and ovary cancer.
This invention provides compounds comprising a triterpene backbone or Sapogenin, in which the sugar moiety attached to Carbon 3 of the triterpene backbone; an acetyl and/or sugar moiety linkage to Carbon 21 and 22; and angeloyl groups attached to the sugar moiety. These functional groups operatively linked to the triterpene or Sapogenin to form the compound.
This invention provides a compound comprising the following structures :
Structure Yl
3-0- [β-D-galactopyranosyl (l-2) ] -α-L-arabinofuranosyl (l-»3) - β-D-glucuronopyranosyl-21-0- (3, 4-diangeloyl) -α-L- rhamnophyranosyl-22-0-acetyl-3β, 16α, 21β, 22α, 28- pentahydroxyolean-12-ene
This invention provides a compound comprising the following structures :
Strucutre Rl
3-0- [angeloyl- (l-3) -β-D-glucopyranosyl- (l→δ) ] - (-D- glucopyranosyl-28-O- [ (-L-rhamnopyranosyl- (1(2)- (-D- glucopyranosyl- (1 (6) - (-D-glucopyranosyl-3 (, 21 (, 22 (, 28- tetrahydroxyolean-12-ene In another embodiment, the compounds of the present invention comprise the structures as shown in Figures 15A, 15B, 15C 15D, 27, 28, 29, 30, 31 and 32.
This invention provides a salt of the above-described compounds .
This invention provides a composition comprising the above- described compounds and a suitable carrier.
This invention provides a pharmaceutical composition comprising an effective amount of the above-described compounds and a pharmaceutically acceptable carrier.
This invention provides an anti-ovarian cancer and composition comprising the above-described composition.
The following methods and materials were used in the examples and/or experiments described in this application.
Cells. Human cancer cell lines were obtained from American Type Culture Collection: HTB-9 (bladder) , HeLa-S3 (cervix) , DU145 (prostate), H460 (lung), MCF-7 (breast), K562 (leukocytes), HCTllβ (colon), HepG2 (liver), U20S (bone), T98G (brain) and OVCAR-3 (ovary) . Cells were grown in culture medium (HeLa-S3, DU145, MCF-7, Hep-G2 and T98G in MEN (Earle's salts); HTB-9, H460, K562, OVCAR-3 in RPMI- 1640; HCT-116, U20S in McCoy-5A) supplemented with 10% fetal calf serum, glutamine and antibiotics in a 5% C02 humidified incubator at 37oC.
MTT Assay. The procedure for MTT assay followed the method described in (Carmichael et al., 1987) with only minor modifications. Cells were seeded into a 96-wells' plate at concentrations of 10,000/well (HTB-9, HeLa, H460, HCT116, T98G, OVCAR-3), 15,000/well (DU145, MCF-7, HepG2, U20S) , or 40,000/well (K562), for 24 hours before drug-treatment. Cells were then exposed to drugs for 48 hours (72 hours for HepG2, U20S, and 96 hours for MCF-7) . After the drug- treatment, MTT (0.5 mg/ml) was added to cultures for an hour. The formation of formazan (product of the reduction of tetrazolium by viable cells) was dissolved with DMSO and the O.D. at 490nm was measured by an ELISA reader. The MTT level of cells before drug-treatment was also measured (TO) . The % cell-growth (%G) is calculated as:
%G = (TD-T0 / TC-T0) x 100 (1) ,
where TC or TD represent O.D. readings of control or drug- treated cells. When TO > TD, then the cytotoxicity (LC) expressed as % of the control is calculated as:
%LC = (TD-T0 / TO) x 100 (2) .
This invention provides the cellular signaling pathways interacted by compounds isolated from Xanthoceras Sorbifolia.
In a further embodiment, a compound has the formula C65H100O27 and the chemical name:
3-0- [β-D-galactopyranosyl (l-2) ] -α-L-arabinofuranosyl (l-»3) - β-D-glucuronopyranosyl-21-0- (3, 4-diangeloyl) -α-L- rhamnophyranosyl-22-0-acetyl-3β,16α, 21β, 22α, 28- pentahydroxyolean-12-ene, designated herein as "Structure Yl", and derivative compounds which are effective against cancer. In a further embodiment, the compounds of the present invention comprise the chemical structures designated herein as "Structure Y", "Structure Rl", "Structure 1 to 4", "Structure Y-a to Y-c" and "Structure Yl-a to Yl-c", "Structure Yl-3 to Yl-4" and their derivatives. See Figures 15, 27-32. These components described above regulate the receptors or components of cells. These' compounds can be isolated from the plant called Xanthoceras Sorbifolia or can be synthesized.
There are many components and pathways monitoring cell proliferation. The Xanthoceras Sorbifolia compound or its derivatives work in the Wnt (Wingless-type MMTV integration site family member) signaling pathway. The Wnt signaling pathway is evolutionarily conserved and controls many events during the embryogenesis. This pathway regulates cell morphology, proliferation, motility and as well as cell apoptosis. It also plays an important role during tumorigenesis . The Wnt pathway has also been observed as inappropriately activated in several different types of cancers in humans .
In the nucleus, the target genes for Wnt signaling are normally kept silent by an inhibitory complex of gene regulatory proteins, e.g. the Groucho corepressor protein bound to the gene regulatory protein LEF-I/TCF. In the absence of a Wnt signal, some β-cartenin is bound to the cytosolic tail of cadherin proteins, and any cytosolic β- cartenin that becomes bound by the APC-axin-GSK-3β will trigger its ubiquitylation and degradation in proteasomes. The result is the decrease of intracellular amount of β- cartenin. However, when the Wnt binding to Frizzled (a seven transmembrane receptor) and LRP (Low density lipoprotein Receptor) activates Dishevelled (a cytoplasmic signaling protein) by a mechanism, this leads to the inactivation of GSK-β3 in the degradation complex by a mechanism which requires casein kinase I, as well as casein kinase II. The activity of the multiprotein complex of β- catenin-axin-adenomatous-polyposis coli (APC) -glycogen synthase kinase (GSK)-3β, which targets β-catenin by phosphorylation for degradation by the proteasome, is then inhibited by Dsh/Dvl (Dishevelled, dsh homolog 1) . This then inhibits priming of β-catenin, and indirectly prevents the GSK-3β phosphorylation of β-catenin. When stimulated by Wnt, Dvl recruits the GSK-3 binding protein, GBP, to the multiprotein complex of β-catenin-axin-adenomatous-polyposis coli (APC) -glycogen synthase kinase (GSK)-3β. GBP then titrates GSK-β from axin, and in this way, phosphorylation of β-catenin is inhibited. Then, axin is sequestrated by LRP at the cell membrane. The result of all of this is an accumulation of cytosolic β-catenin. In the nucleus, β- catenin binds to LEF-I/TCF, displaces Groucho, and acts a co-activator to stimulate the transcription of Wnt target genes .
Xanthoceras Sorbifolia compositions regulate the components related to Wnt pathways or its receptors, thereby stopping the proliferation of cancer cells.
The compound or its derivatives work in the Mitogens, Ras and a MAP (Microtubule associated protein) kinase pathway. Mitogens stimulate cell division. The binding of mitogens to cell-surface receptors leads to the activation of Ras and a MAP kinase cascade. One effect of this pathway is the increased production of the gene regulatory protein Myc. Myc increases the transcription of several genes, including the gene encoding cyclin D and a subunit of the SCF ubiquitin ligase. The resulting increase in Gl-Cdk and Gl/S-Cdk activities promotes Rb phosphyorylation and activation of the gene regulatory protein E2F, resulting in S-phase entry, in which Gl-Cdk activity initiates Rb phosphorylation, in turn inactivating Rb and freeing E2F to activate the transcription of S-phase genes including the genes for a Gl/S-cyclin(cyclin E) and S-cyclin (cyclin A). The resulting appearance of Gl/S-Cdk and S-Cdk further enhances Rb phosphorylation, forming a positive feedback loop, and the E2F acts back to stimulate the transcription of its own gene, forming another positive feedback loop. Myc may also promote E2F activity directly by stimulating the transcription of the E2F gene. The result is the increased transcription of genes entry into S phase. However if this pathway is overactive, it will cause cancer cell growth.
Compounds or compositions derived from the plant Xanthoceras Sorbifolia regulate the Ras-MAP kinase cascade so that the pathway is not overactive.
The compound or its derivatives work in Ras-dependent or Myc pathway. Sometimes the mutation of amino acid in Ras causes the protein to become permanently overactive, stimulating the Ras-dependent signal pathways overactive in absence of mitogenic stimulation. Similarly, mutations that cause an overexpression of Myc promote excessive cell growth, which in turn promotes the development of cancer.
Compounds or compositions derived from the plant Xanthoceras Sorbifolia regulate the components of the Ras-dependent or Myc pathway to make sure it is not overactive.
The compound or its derivatives reactivate the abnormal cell checkpoint mechanism. Inside the cell, there is a checkpoint mechanism which detects abnormal mitogenic stimulation and causes abnormally overactive cells to go into apopto'sis. However this mechanism is not active in cancer cells due to mutations in the genes that encode essential components of the checkpoint responses. If the mutation happens in the checkpoint mechanism, the cancer cell will grow and divide endlessly.
Compounds or compositions derived from the plant Xanthoceras Sorbifolia reactivate the checkpoint mechanism to stop the cancer cell growth.
The compound or its derivatives affect the extracellular growth signaling pathways. The extracellular growth factors that stimulate cell growth are bound to receptors on the cell surface and activate intracellular signaling pathways. It activates the enzyme PI3-kimase, which promotes protein synthesis, at least partly through the activation of eif4e and phosphorylated S61 kinase, resulting in increased mRNA translation and then a stimulation of cell growth.
Compounds or compositions derived from the plant Xanthoceras Sorbifolia regulate the components' or receptor relate to extracellular growth. It binds the receptor of ovarian cancer cells so as to stop the cancer cell growth.
Compounds or compositions derived from the plant Xanthoceras Sorbifolia regulate the components relating to Ras and MAP Kinase, which ceases ovarian cancer cell growth.
The compound or its derivatives affect the intracellular mechanism. Cell division is also controlled by an intracellular mechanism that can limit cell proliferation.
In normal cells, the Myc protein acts in the nucleus as a signal for cell proliferation. Large quantities of Myc can cause the cell to proliferate in excess and form a tumor.
Compounds or compositions derived from the plant Xanthoceras Sorbifolia regulate the components or receptor of the Myc cell's proliferation to stop the tumor cells from dividing.
The compound or its derivatives affect the TGF-alpha signaling pathway. TGF-alpha is produced by keratimcytes, macrophages, hepatocytes, and platelets. Its synthesis is stimulated by the infection by viruses. TGF-Alpha induces the long term proliferation of murine and chicken immature hematopoietic progenitor cell such as BFU-E without causing differentiation. It also induces the terminal differentiation of BFU-Ecell into erythrocytes . TGF-Alpha stimulates the proliferation of cultured endothelial cells. It plays an importance role in the vascularisation of tumor tissues .
Compounds or compositions derived from the plant Xanthoceras Sorbifolia regulate the components or receptor of TGF-alpha to suppress ovarian cancer and bladder cancer cell growth.
The compound or its derivative compounds affect the TGF-beta signaling pathway. TGF-beta regulates growth and proliferation of cells, blocking growth of many cell types.
There are two TGF-beta receptors: Type 1 and Type 2. They are serine-threonine kinases that signal through the SMAD (Protein named after the first two identified, Sma in C. elegans and Mad in Drosophila) family of transcriptional regulators. The TGF-beta pathway and mutation in SMADs are associated with cancer in humans. Compounds or compositions derived from the plant Xanthoceras Sorbifolia regulate the components or receptor of TGF-beta to suppress the ovarian cancer and bladder cancer cell growth.
The compound or its derivatives reactivate the cell functions which are damaged by DNA viruses. DNA tumor viruses cause cancer by interfering with cell cycle control Rb protein and the p53 protein. Mutation in p53 gene will allow cancer cells to survive and proliferate despite DNA damage. The papillomanius uses the proteins E6 and E7 to sequence the p53 and Rb respectively. This action activates mutated cells, allowing them to survive and then divide and accumulate. The accumulation of damaged cells can lead to cancer.
Compounds or compositions derived from the plant Xanthoceras Sorbifolia regulate the proteins E6 and E7 and release the proteins Rb and p53, which will prevent abnormal cells from dividing. It also regulates or reacts with the protein, causing the cancer cells to die.
The compound or its derivatives affect the p53 signaling pathway. p53 helps multi-cellular organisms cope safely with DNA damage and other stressful cellular ' events, stopping cell proliferation in circumstances where it would be dangerous. Cancer cells tend to contain large quantities of mutant p53 protein, suggesting that the genetic accidents they undergo or the stresses of growth in an inappropriate environment created the signals that normally activate the p53 protein. Thus, the loss of p53 activity can be extremely dangerous in relation to cancer because it allows mutant cells to continue through the cell cycle. It also allows them to escape apoptosis. So, if their DNA is damaged, some cells will die but the cells which survive will carry on dividing without pausing to repair the damage. This may cause the cells to die, or they could survive and proliferate with a corrupted genome, which could lead to loss of both tumor suppressor genes and the activation of oncogenes, for example by gene amplification. Gene amplification could enable cells to develop resistance against therapeutic drugs.
Compounds or compositions derived from the plant Xanthoceras Sorbifolia regulate the components and receptor of the p53 pathway, which stops the cancer cells from dividing.
The compound or its derivatives affect the cell suicide signaling pathway. All cells with a nucleus contain various inactive procaspases, awaiting a signal before destroying the cell. Each suicide protease is made as an inactive proenzyme called procaspase. It is usually activated by proteolytic cleavage by another member of the caspase family. Two of the cleaved fragments come together to form the active part of the caspase, and the active enzyme is thought to be a tetramer of two of these two parts. Each activated caspase molecule can cleave many procaspase molecules, which in turn activates more molecules. Through a chain reaction or cascade, this leads to the explosive action of a large number of procaspase molecules. Then, some of the activated procaspases cleave a number of key proteins in the cell, including specific cytosolic proteins and nuclear-lamins leading to the controlled death of the cell.
Activating the death receptor on the outside of the cell can also trigger inactive procaspases. For example, killer lymphocytes can cause apoptosis by producing the protein Fas on the surface of the targeted cell. These clusters of Fas protein then recruit intracellular adaptor proteins that bind and aggregate procaspase-8 molecules. These then cleave and activate one another. The activated caspase-8 molecules then activate downstream procaspases to induce apoptosis.
However in cancer cells, the signal to destroy the cell is blocked, due to gene mutation. This means that the cancer cells continue to divide, thereby causing a tumor.
Compounds or compositions derived from the plant Xanthoceras Sorbifolia unblock the suicide signals, allowing cancer cells to destroy themselves.
This invention provides compounds comprising a triterpene backbone or Sapogenin, in which the sugar moiety attached to Carbon 3 of the triterpene backbone; an acetyl and/or sugar moiety linkage to Carbon 21 and 22; and angeloyl groups attached to the sugar moiety. These functional groups operatively linked to the triterpene or Sapogenin to form the compound. |
This invention provides a method for inhibiting tumor cell growth comprising contacting an amount of the above- described compounds.
This invention provides a method for inhibiting tumor cell growth in a subject comprising administering to the above- described subject,
This invention will be better understood from the examples which follow. However, one skilled in the art will readily appreciate that the specific methods and results discussed are merely illustrative of the invention as described more fully in the claims which follow thereafter. EXPERIMENTAL DETAILS
Experiment 1 : Herb Extraction (a) extracting Xanthoceras Sorbifolia powder of husks or branches or stems or leaves or kernels or roots or barks with organic solvent at ratio of 1:2 for 4-5 times for 20-35 hours for each time to form an organic extract; (b) collecting the organic extract; (c) refluxing the organic extract for 2-3 times at 80°C to form second extracts; (d) removing the organic solvent from the second extract; and (e) Drying and sterilizing the extract to form a Xanthoceras
Sorbifolia extract powder.
Experiment 2 : Analysis of Xanthoceras Sorbifolia Extract Components by HPLC Chromatography
Methods
HPLC. A C-18 reverse phase μbondapak column (Water P/N
27324) was equilibrated with 10% acetonitrile, 0.005% Trifluoroacetic acid (equilibration solution) . An extract of Xanthoceras Sorbifolia prepared using the methods of the present invention, such as the method described above, or using other methods known to one of ordinary skill in the art was dissolved in equilibration solution (1 mg/ml) before being applied onto the column. 20ug of samples was applied into column.
Elution conditions. Fractions were eluted (flow rate 0.5 ml/min) . with acetonitrile (concentration gradient from 10% to 80% in 70 min) and then remains at 80% for 10 min (70-80 min) . The acetonitrile then dropped to 10% (80-85 min) and remained at 10% for 25 min (85-110 min) . The fractions were monitored at 207 nm with a chart speed 0.25 cm/min and OD full scale of 0.128. Instruments. Waters Model 510 Solvent Delivery System; Waters 484 tunable Absorbance Detector; Waters 745/745B Data Module.
Results
HPLC. About 60-70 peaks can be accounted for in the profile. Among them four are major peaks, 10 are medium size and the rest are small fractions. The major peaks are labelled with a to z following increased concentration of acetonitrile elution. See Figure 1.
Experiment 3 : Screening of Cytotoxicity of Xanthoceras Sorbifolia Extract toward Cancer Cells Derived from Different Human Organs using MTT Assay
Methods and Materials
Cells. Human cancer cell lines were obtained from American Type Culture Collection: HTB-9 (bladder) , HeLa-S3 (cervix) , DU145 (prostate), H460 (lung), MCF-7 (breast), K562 (leukocytes), HCT116 (colon), HepG2 (liver), U20S (bone), T98G (brain) and OVCAR-3 (ovary) . Cells were grown in culture medium (HeLa-S3, DU145, MCF-7, Hep-G2 and T98G in MEN (Earle's salts); HTB-9, H460, K562, OVCAR-3 in RPMI- 1640; HCT-116, U20S in McCoy-5A) supplemented with 10% fetal calf serum, glutamine and antibiotics in a 5% C02 humidified incubator at 37oC.
MTT assay. The procedure for MTT assay followed the method described in (Car ichael et al., 1987) with only minor modifications. Cells were seeded into a 96-wells plate at concentrations of 10,000/well (HTB-9, HeLa, H460, HCT116, T98G, OVCAR-3), 15,000/well (DU145, MCF-7, HepG2 , U20S) , or 40,000/well (K562) , for 24 hours before drug-treatment. Cells were then exposed to drugs for 48 hours (72 hours for HepG2, U20S, and 96 hours for MCF-7). After the drug- treatment, MTT (0.5 mg/ml) was added to cultures for an hour. The formation of formazan (product of the reduction of tetrazolium by viable cells) was dissolved with DMSO and the O.D. at 490nm was measured by an ELISA reader [Dynatech. Model MR700] . The MTT level of cells before drug-treatment was also measured (TO) . The % cell-growth (%G) is calculated as:
%G = (TD-TO / TC-TO) x 100 (1) where TC or TD represent O.D. readings of control or drug- treated cells. When TO > TD, then the cytotoxicity (LC) expressed as % of the control is calculated as: %LC = (TD-TO / TO) x 100.
Results. Among the 10 cell lines studied, their sensitivity toward Xanthoceras Sorbifolia extract can be divided into four groups (most sensitive: Ovary. Sensitive: bladder, bone, prostate, and leukocyte, marginal sensitive: liver, breast, and brain; and lease sensitive: colon, cervix, and lung) See Figure 23 and Table 8.1 of International Application No. PCT/US04/33359. Table A. IC50 values of Xanthoceras Sorbifolia Extract Determined in Different Cancer Cells Cancer cells from different IC50 determined by MTT organs assay (ug/ l)
Ovary (most sensitive) 15-15
Bladder (sensitive) 45-50
Bone 40-55
Prostate 40-50
Leukocyte 45-50
Liver (marginal sensitive) 45-65 Breast 65
Brain . 70-85
Colon (least sensitive) 90
Cervix 115
Lung 110
Among these cell line studied, it was found that low concentrations of the Xanthoceras Sorbifolia plant extract stimulate cell growth of bladder, bone and lung cells. See page 109, lines 17-25, to page 110, lines 1-13 of International Application No. PCT/US04/33359.
Experiment 4 : Purification and structural determination of Active Component-Yl in the Xanthoceras Sorbifolia Extract
(A) Fractionation of Xanthoceras Sorbifolia extracts components with FPLC
Methods Column. Octadecyl functionalized silica gel; column dimension: 2cm x 28cm; equilibrated with 10% acetonitrile - 0.005% TFA. Sample loading: 1-2 ml, concentration: 100rαg/ml in 10% acetonitrile/TFA. Gradient elution: 10-80% acetonitrile in a total volume of 500 ml. Monitor absorption wavelength: at 254nm. Fraction Collector: 5 ml/fractions (collect from 10% to 72% acetonitrile, total 90 fractions) Instrument: AKTA-FPLC, P920 pump; Monitor UPC-900; Frac-900.
Results. The elution profile shows 4-5 broad fractions. See Figure 2A. These fractions were analyzed with HPLC. Specific components, i.e., a-z as specified in Figure 1, are then assigned in the FPLC fractions.
FPLC fractions are grouped into 7 pools and analyzed for cell growth activity with bladder cells with MTT assay. It was found only one pool (#5962) contains inhibition activity. See Figure 2B. The purified fraction Y (5962) of the Xanthoceras Sorbifolia plant extract of the present invention does not have the growth stimulating activity but maintained the inhibition activity.
(B) Fractionation of Fraction #5962 with FPLC by a C18 Open Column with 64% Acetonitrile Isocratic Elution
Methods
Column. Octadecyl-functionalized silica gel; 50 ml; 2cm x
28cm; equilibrated with 64% acetonitrile - 0.005% TFA.
Sample loading: 0.2 ml, with concentration: 1-2 mg/ml in 65% acetonitrile/TFA. Elution: 64% acetonitrile isocratic.
Monitor absorption wavelength: at 254nm.
Fraction Collector: 1 ml fraction (collect the first 90 fractions)
Instrument: AKTA-FPLC, P920 pump; Monitor UPC-900; Frac-900.
Results. Fraction 5962 was further separated with an open ODS-C18 column using isocratic 64% acetonitrile elution. Two major fractions, i.e., X and Y, were collected. See Figure 3. MTT assay showed that only the 'Y fraction has the inhibition activity. See Figure 4.
(C) Analysis of Fraction Y with HPLC
Methods Column. Waters μbondapak C18 (3.9 mm x 300 cm) .
Elution: 35% or 45% isocratic elution.
Flow rate: 0.5 ml/min; monitored at 207 nm with O.D. Scale of 0.128; chart speed: 0.25 cm/mim.
Results. On 35% isocratic analysis, the Y fraction contains 4-5 components (Y0, Yl, Y2, Y3, Y4 and Y5) . See Figure 5.
(D) Final Isolation of Active Ys Component was Achieved with Preparative HPLC
Methods
Column. A preparative HPLC column (Waters Delta Pak C18- 300A) . Elution: 45% acetonitrile isocratic with flow rate of 1 ml/min. Monitor at 207nm.
Results. Yl and Y2 are well separated from each other and are collected individually. Y3/4 and Y5 are not well separated. See Figure 6. Peak fraction of Yl was collected and lyophilized (Compound-Yl) . '
Appearance and solubility. The pure Compound Yl is amorphous white powder, soluble in aqueous alcohol (methanol, ethanol), 50% acetonitrile and 100% pyridine.
Inhibition activity of Compound Yl and Y2 with MTT assay, Results indicated that both Yl and Y2 have anticancer activity. See Figure 17.
(E) Determination of the Chemical Structure of Compound Yl of Xanthoceras Sorbifolia Extract
Methods NMR analysis. The pure compound Yl of Xanthoceras Sorbifolia were dissolved in pyridine-D5 with 0.05% v/v TMS. All NMR spectra were acquired using a Bruker Avance 600 MHz NMR spectrometer with a QXI probe (1H/13C/15N/31P) at 298 K. The numbers of scans for ID 1H spectra were 16 to 128, depending on the sample concentration. 2D HMQC spectra were recorded with spectral widths of 6000 x 24,000 Hz and data points of 2024 x 256 for t2 and tl dimensions, respectively. The numbers of scans were 4 to 128. 2D HMBC were acquired with spectral widths of 6000 x 30,000 Hz and data points of 2024 x 512 for t2 and tl dimensions, respectively. The numbers of scans were 64. The 2D data were zero-filled in tl dimension to double the data points, multiplied by cosine-square-bell window functions in both tl and t2 dimensions, and Fourier- transformed using software XWIN-NMR. The final real matrix sizes of these 2D spectra are 2048 x 256 and 2048 x 512 data points (F2 x FI) for HMQC and HMBC, respectively.
Mass spectral analysis. The mass of samples was analyzed by MALDI-TOF Mass Spectrometry.
Samples for MALDI-TOF were first dissolved in acetonitrile, then mixed with the matrix CHCA (Alpha-cyano-4- hydroxycinnamic acid, lOmg CHCA/mL in 50:50 water/acetonitrile and 0.1% TFA in final concentration). The sample was dissolved completely in acetonitrile and stayed dissolved after mixing with the matrix. The molecular weight was determined by the high resolution mass spectroscope analysis with standards.
Results. The profile of the proton NMR of Yl is presented in Figure 7. The profiles of 2D NMR (HMQC) of Yl is presented in Figure 8. The profiles of 2D NMR (HMBC) of Yl is presented in Figure 9. The profiles of 2D NMR (COSY) of Yl is presented in Figure 10.
Conclusion. Based on the chemical shift analysis data, as shown in Table 1, the active compound Yl isolated from extract of Xanthoceras Sorbifolia is a triterpenoid saponins with four sugars and biangeloyl groups attached to the sugar moiety. The formula of Yl is C65H100O27, and the structure and the chemical name of Yl is:
3-0- [β-D-galactopyranosyl (1—2) ] -α-L-arabinofuranosyl (1—»3) ■ β-D-glucuronopyranosyl-21-0- (3, 4-diangeloyl) -α-L- rhamnophyranosyl-22-0-acetyl-3β, 16α, 21β, 22α, 2i pentahydroxyolean-12-ene
Table 1. 13C and 1H NMR data for compound yl (in Pyridine- d5)a
a The data were assigned based on HMQC and HMBC correlations . The mass of Yl as determined by MALDI-TOF is 1358.71 which agree with the theoretical mass of Yl (1312.64) plus 2 Na and a proton (M+2Na+H) .
Results of Y2 analysis The profile of the proton NMR of Y2 is presented in Figure 11. The profiles of 2D NMR (HMQC) of Y2 is presented in Figure 12.
Results of Y5 analysis The profile of the proton NMR of Y5 is presented in Figure 13. The profiles of 2D NMR (HMQC) of Y5 is presented in Figure 14.
Experiment 5 : Acid Hydrolysis of Compound Y Compound Y is identified and purified from Xanthoceras Sorbifolia in International Application No. PCT/US04/33359. See page 107, lines 5-31, to page 108, lines 1-10, and page 110, lines 15-30, and page 136, lines 1-14. Acid Hydrolysis of compound Y generates the following compound with the following structure, designated herein as Yl-c. See Figure 29. Methods: 5mg of Y is dissolved in 3ml of MeOH and then treated with 3ml of 3N HCI. Hydrolysis of saponins will be conducted under reflux for 4 hr. After hydrolysis, the solution was neutralized with 5% Na2C03 and extracted with EtOAc three times to afford an aqueous layer and organic layer, containing sugars and aglycon, respectively. Aglycon from the organic layer will be further purified on Silica gel chromatography in (CHC13: MeOH, 1:9) or with C18 ODS HPLC chromatography. About 2mg of compound with the structure Y-c, shown below, can be obtained.
Method reference: Essentials of Carbohydrate Chemistry. By John F. Robyt, (Springer, 1998) .
The linkage of oligosaccharide can be cleaved by partial acid hydrolysis and by specific enzyme hydrolysis. For example, the 1 → 4 linkage of arabinofuranosyl can be removed by α-amylase. Other enzymes such as β-amylase, isoamylase, glucose oxidase, mannanse and pullulanase can be used to cleave individual saccharide in saponins.
Angeloyl groups of the structures can be removed by alkaline hydrolysis. For example, compound Y is dissolved in IM NaOH and stirred for 2-3 hours at room temperature. The solution is then acidified or neutralized with 2M HCI and hydrolyzed saponin can be extracted with ethyl acetate. Further purification can be achieved with HPLC using C18 column. By enzyme hydrolysis of Structure Y, the following structures can be obtained:
Acid Hydrolysis of compound Y generates the following compound with the following structure, designated herein as structure Y-c:
Experiment 6: Purification and structural determination of component Rl from Xanthoceras Sorbifolia Extract (A) Fractionation of Xanthoceras Sorbifolia extracts components with FPLC (gradient elution)
Methods
Column: Octadecyl functionalized silica gel; column dimension: 2cm x 28cm; equilibrated with 10% acetonitrile - 0.005% TFA. Sample loading: 1-2 ml, concentration: 100 mg/ml in 10% acetonitrile/TFA.
Gradient elution: 10-80% acetonitrile in a total volume of 500 ml. Monitor absorption wavelength: at 254nm. Fraction Collector: 5 ml/fractions (collect from 10% to 72% acetonitrile, total 90 fractions) Instrument: AKTA-FPLC, P920 pump; Monitor UPC-900; Frac-900.
Results. The elution profile shows 4-5 broad fractions. See Figure 2A. These fractions were analyzed with HPLC. Specific components, i.e., a-z, are then keyed back in the FPLC profile and fractions. See Figure 1.
(B) Fractionation of R with FPLC with 30% Acetonitrile Isocratic Elution
Methods
Column: Octadecyl-functionalized silica gel; 50 ml; 2cm x 28cm; equilibrated with 30 acetonitrile - 0.005% TFA. Sample loading: 0.2 ml, with concentration: 1-2 mg/ml in 30% acetonitrile/TFA. Elution: 30% acetonitrile isocratic.
Monitor absorption wavelength: at 254nm.
Fraction Collector: 5 ml fraction (collect the first 90 fractions)
Instrument: AKTA-FPLC, P920 pump; Monitor UPC-900; Frac-900. Results. Fraction No. 39-41 from gradient elution of FPLC (Figure 2A) were pooled and further purified with an open ODS-C18 column with isocratic 30% acetonitrile elution. Six identifiable fractions in two groups were collected. See Figure 18. Fractions 6-13 were further characterized with HPLC.
(C) Analysis and Isolation of R with HPLC
Methods
Column: Waters μbondapak C18 (3.9 x 300 nm) and Waters DeltaPak C18 (7.8mm x 30 cm).
Elution: Gradient (10-80%) and 30% isocratic elution. Flow rate: 0.5 ml/min; 207 nm; attenuation 0.128; chart speed: 0.25 cm/mim.
Results. On HPLC gradient elution analysis, Fractions 9-11 contain a major component with' a few minor components. See Figure 19. These components were further separated into 4-5 components with 30% acetonitrile isocratic elution in a DeltaPak column. The fraction designated herein as "Rl" is the major component. See Figure 20A. This fraction Rl was subsequently isolated by collecting from the column. See Figure 20B.
Appearance and solubility. The pure Rl is amorphous white powder, soluble in aqueous alcohol (methanol, ethanol) , 50% acetonitrile and 100% pyridine.
(D) Determination of the chemical structure of Rl isolated from Xanthoceras Sorbifolia extract
Methods NMR Analysis. The procedure for NMR analysis of pure Rl fraction of Xanthoceras Sorbifolia is same as described in Experiment . Mass spectral analysis. The mass of samples was analyzed by (A) MALDI-TOF Mass Spectrometry as described in previous section.
Results. The pure Rl proton NMR is presented in Figure 21. The 2D NMR (HMQC) spectra of Rl were presented in Figure 22. The 2D NMR (HMBC) spectra of Rl were presented in Figure 23. The 2D COSY spectrum was presented in Figure 24. The Carbon 13 NMR spectrum was presented in Figure 25. Based on all the data presented above, the following table, i.e., Table 2, summarizes the results of the structural analysis and the assignment of the functional groups of compound Rl . Table 2. 13C and IH NMR Data for Rl (in Pyridine-d5)a
a The data were assigned based on COSY, HMQC and HMBC correlations. b,c,d,f The data with the same labels in each column were overlapped. e The data with the same labels in each column may be interchanged.
Conclusion. Based on the chemical shift analysis, the compound Rl isolated from extract of Xanthoceras Sorbifolia is a triterpenoid saponins with five sugars and one angeloyl group attached to the sugar moiety. The formula of compound Rl is C65H106O29, and the chemical name of Rl is:
Chemical name of structure Rl is: 3-0- [angeloyl- (1 (3) - (-D- glucopyranosyl- (1(6) ] - (-D-glucopyranosyl-28-O- [ (-L- rhamnopyranosyl- (1 (2) - (-D-glucopyranosyl- (1(6)- (-D- glucopyranosyl-3 ( , 21 (, 22 (, 28-tetrahydroxyolean-12-ene
Experiment 7 : Purification of component-0 from Xanthoceras Sorbifolia extract
(A) Fractionation of Xanthoceras Sorbifolia extracts components with FPLC (gradient elution)
Methods
Column: Octadecyl functionalized silica gel; column dimension: 2cm x 28cm; equilibrated with 10% acetonitrile -
0.005% TFA.
Sample loading: 1-2 ml, concentration: 100 mg/ml in 10% acetonitrile/TFA. Gradient elution: 10-80% acetonitrile in a total volume of 500 ml.
Monitor absorption wavelength: at 254nm.
Fraction Collector: 5 ml/fractions (collect from 10% to 72% acetonitrile, total 90 fractions)
Instrument: AKTA-FPLC, P920 pump; Monitor UPC-900; Frac-900.
Results. The elution profile shows 4-5 broad fractions (Figure 2A) . These fractions were analyzed with HPLC Specific components (a-z) are then keyed back in the FPLC profile and fractions.
(B) Fractionation of component-0 with HPLC with 20% acetonitrile isocratic elution.
Methods
Column: A preparative HPLC column (Waters Delta Pak C18-
300A) ;
Samples: Fraction #28 from the gradient elution of FPLC were pooled and applied into the HPLC.
Elution: 20% acetonitrile isocratic with flow rate of 1 ml/min. Fractions were collected. Monitored at 207nm.
Fractions including 28, 34 and 54 were collected and lyophilized. Re-chromatographic analysis of these fractions were performed under same conditions.
Results. Sixteen identifiable fractions were observed in the elution profiles (Figure 33) . Fractions 28, 34 and 54 were further characterized with HPLC using same condition. Figures 34, 35 and 36 show a single peak elution of fractions 28, 34 and 54, respectively, indicating they are homogeneous (pure) . Appearance and solubility: The purified 0-28 and 0-34 are light yellow amorphous powder, soluble in aqueous alcohol (methanol, ethanol), 50% acetonitrile and 100% pyridine. The purified 0-54 is white amorphous powder, soluble in aqueous alcohol (methanol, ethanol) , 50% acetonitrile and 100% pyridine.
(C) Structure analysis of compounds purified from component-O
Methods
NMR Analysis. The procedure for NMR analysis of pure Rl fraction of Xanthoceras Sorbifolia is same as described in Experiment 4.
Mass spectral analysis. The mass of samples was analyzed by (A) MALDI-TOF Mass Spectrometry as described in Experiment 4.
Although the present invention has been described in detail with particular reference to preferred embodiments thereof, it should be understood that the invention is capable of other different embodiments, and its details are capable of modifications in various obvious aspects. As is readily apparent to those skilled in the art, variations and modifications can be affected while remaining within the spirit and scope of the invention. Accordingly, the foregoing disclosure, description, and figures are for illustrative purpose only, and do not in any way limit the invention which is defined only by the claims. REFERENCES
1. Chen, Q. 1995. Methods of study on pharmacology of Chinese medicines. Press of People's Public Health, Beijing. p892. 2. Huang, Zh. Sh., Liu, M. P., Chen, Ch. Zh. 1997. Study on effects of Yangshou Dan on improving learning and retention. Chinese Journal of combination of Chinese and west medicine, 9(17): 553.
3. Zhang, Y., Zhang, H. Y., Li, W. P. 1995. Study on effects of Anjifu on improving intelligence, Chinese
Bulletin of Pharmacology, 11 (3) : 233.
4. Yang, J., Wang, J. , Feng, P. A. 2000. Study on effects of Naokkangtai capsule on improving learning and retention in mice, New Chinese Medicine and Clinical Pharmacology, 1(11) : 29.
5. Yang, J. , Wang, J. , Zhang, J. Ch. 2000. Study on' effects of Crude saponins of peonies on improving learning and retention in mice, Chinese journal of Pharmacology, 2(16) : 46. 6. Xia, W. J., Jin, M. W., Zhang, L. 2000. Study on treatment of senile dementia caused by angio-aging with Didang tang, Pharmacology and Clinical of Chinese Medicines, 16 (4) .
7. Bian, H. M., Yu, J. Z., Gong, J. N. 2000. Study on effects of Tongmai Yizhi capsule on improving learning and retention in mice, Pharmacology and Clinical of Chinese Medicines, 16 (5): 40.
8. Wei, X. L., Zhang, Y. X. 2000. Study of animal model for studying senile dementia, Chinese journal of Pharmacology, 8(16) : 372.
9. Bureau of Medicinal Police, Department of Public Health. Guide line for study of effect of medicines for treatment of nervous system diseases, in Guidebook of study of new medicine. p45. 10. Zhang, D. Sh. , Zhang, J. T. 2000. Effects of crude Ginseng saponins on improving impairment induced by B- peptide, Chinese journal of Pharmacology, 8(16): 22.

Claims

What is claimed is :
1. A composition comprising compounds from husks or fruit- stems or seed' s shells or leaves or branches or stems or kernels or roots or barks of Wenguanguo or Xanthoceras sorbifolia.
2. The composition of claim 1, further comprising saponins .
3. A medicine or health food according to claims 1 or 2 for curing enuresis.
4. A medicine or health food according to claims 1 or 2 for relaxing the bladder.
5. A medicine or health food according to claims 1 or 2 for improving the response of cerebral cortex and cerebellum.
6. A medicine or health food according to claims 1 or 2 for preventing people from sleeping in the extra deep level.
7. A medicine or health food according to claims 1 or 2 for improving the sensory stretch receptor in the bladder wall to give a collect signal to central nerves system.
8. A medicine or health food according to claims 1 or 2 for inhibiting AChE. 9. A medicine or health food according to claims 1 or 2 for use as an anti-inflammatory agent.
10. A medicine or health food according to claims 1 or 2 for inhibiting of virus.
11. A medicine or health food according to claims 1 or 2 for use against human immunodeficiency virus.
12. A medicine or health food according to claims 1 or 2 for inhibiting cancer.
13. A medicine or health food according to claims 1 or 2 for decreasing cholesterol or triglyceride level in blood.
14. The composition according to claims 1 or 2 for preventing cerebral aging; for improving memory; for improving cerebral functions; for curing enuresis, frequent micturition, urinary incontinence, dementia, weak intelligence, Alzheimer's disease, autism, brain trauma, Parkinson's disease or other diseases caused by cerebral dysfunctions; for treating arthritis, rheumatism, poor circulation, arteriosclerosis, Raynaud's syndrome, angina pectoris, cardiac disorder, coronary heart disease, headache, dizziness, kidney disorder; or for treating impotence or premature ejaculation.
15. The medicines or health food according to claims 1 or 2, further comprising in any combinations of: Vitamin B, Vitamin D, Vitamin K, grape seed extract or other antioxidants, Cordyceps or its extracts, gingko or its extracts, Panax ginseng, P. quinquefolium or their extracts, Huangpi (Clausena lansium) or its extracts, Echinacea or its extracts, St John's Wort (Hypericum perforatum) or its extracts, Gegen (Pueraria lobata) or its extracts, Tianma (Gastrodia elata) or its extracts, Armillariella mellea or its extracts, Danshen (Salvia miltiorrhiza) , or its extracts, Sanqi (Panax notoginsen) or its extracts, Monascus or Hongu (Red yeast rice) , Huanqi (Hedysarum polybotrys) or its extracts, Dihuang (Rehmannia glutinosa) or its extracts, Danggui (Angelica sinensis) , Yuanzhi (Polygala tenuifoila) or its extracts, Lingzhi (Ganoderma spp.) or its extracts, Fuling (Poria cocos) or its extracts, Gan Cao (Glycyrrhiza uralensis Fisch) or its extracts, Huperzine A, Lacithin, Metrifonate, Nocetile, folic acid, amino acids, creatine, fiber supplement, or any combination thereof.
16. The medicines or health foods according to claims 1 or 2, in capsule, pill, powder, liquid and other forms.
17. The composition of claim 2, wherein the crude saponins from Wenguanguo or Xanthoceras sorbifolia husks or fruit-stems or seed's shell, or leaves or branches or stems or roots or barks are obtained by methods comprising the steps of:
(a) extracting Wenguanguo powder of the husks or fruit- stems or seed' s shell or leaves or branches or stems or roots or barks with an organic solvent at a ratio of herbs to solvent = 1:2 for 4-5 times, for 20-35 hours each time to obtain an organic extract; (b) collecting and refluxing the organic extract for 2- 3 times at 80°C to obtain the second extract; (c) dissolving the second extract with water to obtain an aqueous solution; (d) extracting the aqueous solution with n-butanol to obtain a n-butanol extract; and (e) separating Saponins from the n-butanol extract by chromatography to obtain the crude saponins.
18. The method of claim 17, wherein the organic solvent is ethanol or methanol. 19. The composition Of claim 2, wherein the crude saponins from Wenguanguo or Xanthoceras sorbifolia kernels are obtained by a method comprising the steps of:
(a) removing oil for the kernels by pressing the kernels to form kernel cakes; (b) grounding and extracting the kernel cakes with n- exane to from n-hexane extract; (c) removing the n-hexane from the n-hexane extract; (d) drying the n-hexane extract to form a kernel powder; (e) extracting the kernel powder with an organic solvent at a ratio of 1:2 for 4-5 times, for 20-35 hours each time to form an organic extract; (f) collecting and refluxing the organic extract for 2- 3 times at 80°C to form the second extract; (g) dissolving the second extract in water to form an aqueous solution; (h) Extract the aqueous solution with n-butanol to form an n-butanol extract; and (i) separating the Saponins from the n-butanol extracts using chromatography to obtain the crude saponins.
20. The method of claim 19, wherein the organic solvent is ethanol or methanol.
21. The medicine or health food according to claims 1 or 2, capable of accelerating the growth of bladder; suppressing deep sleep; increasing the wake up alert; modulating the release, break down and uptake of anti- diuretic hormone or ADH or its receptors; modulating the secretion, break down and uptake of adrenocorticotropic hormone or ACTH or its receptors; modulating the release, break down and uptake of 5- hydroxytryptamine or its receptors; modulating the release, break down and uptake of acetylcholine or Ach or its receptors; modulating the release, break down and uptake of adrenaline or AD or its receptors; modulating the release, break down and uptake of dopamine orDA or its receptors; or modulating the release, break down and uptake of norepinephrine (NE) or its receptors.
22. The medicine or health food according to claims 1 or 2, capable of preventing sleep paralysis, or modulating the composition, release, break down and activity of the neuropeptides or their receptors. 23. The medicine or health food according to claims 1 or 2, capable of curing cancer.
24. The medicine or health food of claim 23, where the cancer includes breast cancer, leukocyte cancer, liver cancer, ovary cancer, bladder cancer, prostate cancer, colon cancer, bone cancer, cervical cancer, skin cancer or brain cancer.
25. The medicine or health food according to claims 1 or 2, capable of improving the functions of the lung and the bladder; accelerating the growth of bone; reducing the frequent of urination or volume of urine; or deferring the urination.
26. The medicine or health food according to claims 1 or 2, further comprising Ginseng, Bajitian, Roucongrong, Duzhong or Cordyceps. 27. A compound comprising the following structure (Structure Yl) :
3-0- [β-D-galactopyranosyl ( l-»2 ) ] -α-L-arabinofuranosyl ( 1 (3 ) - (-D-glucuronopyranosyl-21-O- ( 3 , 4-diangeloyl) - (-L- rhamnoρhyranosyl-22-0-acetyl-3 ( , 16 ( , 21 ( , 22 ( , 28- pentahydroxyolean-12-ene .
28 . A compound comprising :
a triterpene backbone or Sapogenin; a sugar moiety attached to Carbon 3 of triterpene backbone; an acetyl and/or sugar moiety linkage to Carbon 21 and 22 ; and angeloyl groups attached to the sugar moiety, wherein the sugar moiety and angeloyl groups are operatively linked to the triterpene or Sapogenin to form the compound.
29. A compound of claim 28 comprising at least 2 sugars.
30. A compound of claim 28 comprising at least one angeloyl group.
31. A compound comprising the structure (Yl-1) as shown in Figure 15A.
32. A compound comprising the structure (Yl-2) as shown in Figure 15B.
33. A compound comprising the structure (Yl-3) as shown in Figure 15C. 34. A compound comprising the structure (Yl-4) as shown in Figure 15D.
35. A compound comprising the structure (Y-a) as shown in Figure 27.
36. A compound comprising the structure (Y-b) as shown in Figure 28.
37. A compound comprising the structure (Y-c) as shown in Figure 29.
38. A compound comprising the structure (Yl-a) as shown in Figure 30. 39. A compound comprising the structure (Yl-b) as shown in Figure 31.
40. A compound comprising the structure (Yl-c) as shown in Figure 32.
41. A compound comprising the structure (Structure Rl) with the following chemical nomenclature:
3-0- [angeloyl- (1(3)- (-D-glucopyranosyl- (1(6) ] - (-D- glucopyranosyl-28-O- [ (-L-rhamnopyranosyl- (1(2)- (-D- glucopyranosyl- (1 (6) - (-D-glucopyranosyl-3 (, 21 (, 22 (, 28- tetrahydroxyolean-12-ene.
42. A compound comprising the following structure:
, A compound comprising a NMR spectral data or Proton NMR profile as shown in Figure 7. A compound comprising a NMR spectral data or Heteronuclear Multiple Quantum Correlation (HMQC) profile as shown in Figure 8. , A compound having a NMR spectral data or Heteronuclear Multiple Bond Correlation (HMBC) profiled as shown in Figure 9. , A compound having a NMR spectral' data (COSY) profile as shown in Figure 10. A compound comprising a proton NMR spectral data profile as shown in Figure 11. A compound having a NMR spectral data or Heteronuclear Multiple Quantum Correlation (HMQC) profile as shown in Figure 12. A compound comprising a NMR spectral data or Proton NMR profile as shown in Figure 13.
50. A compound comprising a NMR spectral data or Heteronuclear Multiple Quantum Correlation (HMQC) profile as shown in Figure 14.
51. A compound comprising a NMR spectral data or Proton NMR profile as shown in Figure 21.
52. A compound comprising a NMR spectral data or Heteronuclear Multiple Quantum Correlation (HMQC) profile as shown in Figure 22.
53. A compound having a NMR spectral data or Heteronuclear Multiple Bond Correlation (HMBC) profile as shown in Figure 23.
54. A compound having a NMR spectral data (COSY) profile as shown in Figure 24.
55. A compound comprising a carbon-13 NMR spectral data profile as shown in Figure 25.
56. A compound comprising a proton NMR spectral data profile as shown in Figure 37.
57. A compound having a NMR spectral data • or Heteronuclear Multiple Quantum Correlation (HMQC) profile as shown in Figure 38.
58. A compound comprising a proton NMR spectral data profile as shown in Figure 39.
59. A compound having a NMR spectral data or Heteronuclear Multiple Quantum Correlation (HMQC) profile as shown in Figure 40.
60. A compound comprising a proton NMR spectral data profile as shown in Figure 41.
61. A compound having a NMR spectral data or Heteronuclear Multiple Quantum Correlation (HMQC) profile as shown in Figure 42.
62. A compound having a NMR spectral data or Heteronuclear Multiple Quantum Correlation (HMBC) profile as shown in Figure 43.
63. A salt of the compound of any one of claims 27 to 62.
64. A composition for inhibiting tumor cell growth, comprising the compound of any one of claims 27 to 62.
65. A composition for inhibiting tumor cell growth, comprising the compound of any one of claims 27 to 62 and a suitable carrier.
66. A composition for inhibiting tumor cell growth, comprising the compound of any one of claims 27 to 62 and a pharmaceutically suitable carrier. 67. A method for treating ovarian cancer in a subject, comprising administering an effective amount of the composition of claim 27 to said subject.
68. A method for curing enuresis in a subject, comprising administering an effective amount of the composition of any one of claims 27 to 62 to said subject.
69. A composition for relaxing the bladder, comprising the compound of any one of the claims 27 to 62.
70. A composition for improving the response of cerebral cortex or cerebellum, comprising the compound of any one of the claims 27 to 62.
71. A composition for preventing extra deep level sleep, comprising the compound of any one of the claims 27 to 62.
72. A composition for improving the sensory stretch receptor in the bladder wall to give a collect signal to the central nerves system, comprising the compound of any one of the claims 27 to 62.
73. A composition for relaxing the bladder tissue, comprising the compound of any one of the claims 27 to 62.
74. A composition for inhibiting AChE, comprising the compound of any one of the claims 27 to 62.
75. A composition for reducing inflammation, comprising the compound of any one of the claims 27 to 62.
76. A composition for inhibiting virus, comprising the compound of any one of the claims 27 to 62.
77. A composition for use against human immunodeficiency virus protease, comprising the compound of any one of the claims 27 to 62.
78. A composition for inhibiting cancer cells, comprising the compound of any one of the claims 27 to 62.
79. A composition of claim 78, wherein the cancer is breast cancer, leukocyte cancer, liver cancer, ovary cancer, bladder cancer, prostate cancer, bone cancer or brain cancer.
80. A composition for decreasing cholesterol or triglyceride level in the blood, comprising the compound of any one of the claims 27 to 62. 81. A composition for preventing cerebral aging, for improving memory, for improving cerebral functions, for curing enuresis, frequent micturition, urinary incontinence, dementia, weak intelligence, Alzheimer's disease, autism, brain trauma, Parkinson's disease or other diseases caused by cerebral dysfunctions, for treating arthritis, rheumatism, poor circulation, arteriosclerosis, Raynaud's syndrome, angina pectoris, cardiac disorder, coronary heart disease, headache, dizziness, kidney disorder, impotence or premature ejaculation, comprising the compound of any one of the claims 27 to 62. 82. A composition for improving the function of bladder or for inhibiting bladder cancer, comprising the compound of any one of the claims 27 to 62. 83. A composition for increasing the wake up alert, comprising the compound of any one of the claims 27 to 62.
84. A composition for modulating the release, break down or uptake of anti-diuretic hormone orADH or its receptors, comprising the compound of any one of the claims 27 to 62.
85. A composition for modulating the secretion, break down or uptake of adrenocorticotropic hormone or ACTH or its receptors, comprising the compound of any one of the claims 27 62.
86. A composition for modulating the release, break down or uptake of 5-hydroxytryptamine or its receptors, comprising the compound of any one of the claims 27 to 62.
87. A composition for modulating the release, break down or uptake of acetylcholine or Ach or its receptors, comprising the compound of any one of the claims 27 to 62. 88. A composition for modulating the release, break' down or uptake of adrenaline or AD or its receptors, comprising the compound of any one of the claims 27 to 62.
89. A composition for modulating the release, break down or uptake of dopamine or DA or its receptors, comprising the compound of any one of the claims 27 to 62.
90. A composition for modulating the release, break down or uptake of norepinephrine or NE or its receptors, comprising the compound of any one of the claims 27 to 62. 91. A composition for modulating the formation, release, break down or activity the neuropeptides or their receptors, comprising the compound of any one of the claims 27 to 62.
92. A composition for improving the functions of the lung and the bladder, comprising the compound of any one of the claims 27 to 62.
93. A method for isolating compounds from Xanthoceras Sorbifolia herb comprising the steps of: (a) extracting Xanthoceras Sorbifolia powder with organic solvents;
(b) collecting the organic extract;
(c) refluxing the organic extract to obtain a second extract;
(d) removing the organic solvent from the second extract;
(e) drying and sterilizing the second extract to obtain a Xanthoceras sorbifolia crude extract powder;
(f) fractionating the crude extract powder into components using HPLC and FPLC chromatography with silica gel, C18 and other equivalent solid phase materials;
(g) monitoring absorption wavelength at 207nm or 254nm;
(h) identifying the bioactive components of the crude extract powder; (i) purifying one or more bioactive components of the crude extract powder with FPLC to obtain one or more fraction of the bioactive component; and
(j) isolating the bioactive components with preparative HPLC.
94. The method of claim 93, wherein the Xanthoceras Sorbifolia powder is prepared from the husks, branches, stems, leaves, kernels, roots, barks or seed shells of the Xanthoceras Sorbifolia herb. 95. The method of claim 93, wherein the organic solvent is ethanol, methanol, ether, chloroform, alcohol or acetone.
96. The method of claim 93, wherein the ratio of the Xanthoceras Sorbifolia powder to the organic solvent is 1:2.
97. The method of claim 93, wherein the extraction step in (a) is performed 4-5 times for 20-35 hours each time.
98. The method of claim 93, wherein the refluxing step (c) is performed 2-3 times.
99. The method of claim 93, wherein the refluxing step (c) is performed at 80°C.
100. The method of claim 93, wherein the identifying step (h) is performed with MTT Assay. 101. The method of claim 93, wherein the one or more fraction of steps (i-j) has the HPLC profile as shown in Figures 5 to 6. 102. The method of claim 93, wherein the compound has the HPLC profile as shown in Figure 19-20. 103. The method of claim 93, wherein the compound has the HPLC profile as shown in Figure 20.
104. A composition for treating bladder cancer, comprising the compound of any one of the claims 27 to 62.
105. A method for inhibiting the growth of ovarian cancer cells comprising contacting said cells with an effective concentration of the composition of claim 27 to 62.
106. A method for treating or preventing ovarian cancer in a subject comprising administering an effective amount of the composition of claim 27 to 62 to the subject.
107. A composition capable of regulating the components related to Wnt pathways or its receptors to stop the proliferation of Cancer in a subject comprising an effective amount of the compound of any one of claims 27 to 62.
108. A composition capable of regulating the components or receptors of the Ras-MAP kinase cascade comprising an effective amount of the compound of any one of claims 27 to 62. 109. A composition capable of regulating the components of the Ras-dependent or Myc pathway comprising an effective amount of the compound of any one of claims 27 to 62.
110. A composition capable of reactivating the checkpoint mechanism to stop cancer cell growth in a subject comprising an effective amount of the compound of any one of claims 27 to 62. 111. A composition capable of regulating the components or receptor related to extra cellular growth comprising an effective amount of the compound of any one of claims 27 to 62.
112. A composition capable of regulating the components relating to Ras and MAP Kinase to stop ovarian cancer cell growth in a subject comprising an effective amount of the compound of any one of claims 27 to 62.
113. A composition capable of regulating the components or receptor of the Myc cell's proliferation to stop the tumor cells from dividing comprising an effective amount of the compound of any one of claims 27 to 62.
114. A composition capable of regulating the components or receptor of TGF-alpha to suppress ovarian cancer or bladder cancer cell growth in a subject comprising an effective amount of the compound of any one of claims 27 to 62.
115. A composition capable of regulating the components or receptor of TGF-beta to suppress the ovarian cancer and bladder cancer cell growth in a subject comprising an effective amount of the compound of any one of claims 27 to 62.
116. A composition capable of regulating the proteins E6 and E7 and releasing proteins Rb and p53 to prevent abnormal cells from dividing comprising an effective amount of the compound of any one of claims 27 to 62.
117. A composition capable of regulating or reacting with the proteins to cause cancer cell death comprising an effective amount of the . compound of any one of claims 27 to 62.
118. A composition- capable of regulating the components or receptor of the p53 pathway to stop cancer cells from dividing comprising an effective amount of the compound " of any one of claims 27 to 62.
119. A composition capable of unblocking the suicide signals in a cell comprising an effective amount of the compound of any one of claims 27 to 62.
120. A composition capable of regulating the components or receptors of the BMP, FGF, Jak-Jnk-STAT or Jun-fos comprising an effective amount of the compound of any one of claims 27 to 62.
121. A composition capable of regulating the glucocorticoid receptor, Oestrogen receptor, Estrogen receptor, Tyrosine kinase receptor, G-protein linked receptor or EGF receptor comprising an effective amount of the compound of any one of claims 27 to 62.
122. A method for inhibiting tumor cell growth comprising contacting said cell with an effective amount of a compound, as shown below:
3-0- [β-D-galactopyranosyl (l- 2) ] -α-L-arabinofuranosyl (l- 3) - β-D-glucuronopyranosyl-21-0- (3, 4-diangeloyl) -α-L- rhamnophyranosyl-22-0-acetyl-3β, 16α, 21β, 22α, 2£ pentahydroxyolean-12-ene.
EP04815530A 2003-12-23 2004-12-23 Composition comprising xanthoceras sorbifolia extracts, compounds isolated from same, methods for preparing same and uses thereof Withdrawn EP1708724A4 (en)

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US53210103P 2003-12-23 2003-12-23
US60785804P 2004-09-07 2004-09-07
US61381104P 2004-09-27 2004-09-27
US61737904P 2004-10-08 2004-10-08
PCT/US2004/033359 WO2005037200A2 (en) 2003-10-09 2004-10-08 Composition comprising xanthoceras sorbifolia extracts, compounds isolated from same, methods for preparing same and uses thereof
PCT/US2004/043465 WO2005063273A1 (en) 2003-12-23 2004-12-23 Composition comprising xanthoceras sorbifolia extracts, compounds isolated from same, methods for preparing same and uses thereof

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