EP1124991A1 - Phytomics: ein auf genomics basierender ansatz für pflanliche wirkstoffzummensetzungen - Google Patents

Phytomics: ein auf genomics basierender ansatz für pflanliche wirkstoffzummensetzungen

Info

Publication number
EP1124991A1
EP1124991A1 EP99971046A EP99971046A EP1124991A1 EP 1124991 A1 EP1124991 A1 EP 1124991A1 EP 99971046 A EP99971046 A EP 99971046A EP 99971046 A EP99971046 A EP 99971046A EP 1124991 A1 EP1124991 A1 EP 1124991A1
Authority
EP
European Patent Office
Prior art keywords
herbal
herbal composition
hbr
batch
array
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Withdrawn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
EP99971046A
Other languages
English (en)
French (fr)
Inventor
Patrick C. Kung
Yung-Chi Cheng
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Yale University
Original Assignee
Yale University
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Yale University filed Critical Yale University
Publication of EP1124991A1 publication Critical patent/EP1124991A1/de
Withdrawn legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6897—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids involving reporter genes operably linked to promoters
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6809—Methods for determination or identification of nucleic acids involving differential detection

Definitions

  • PHYTOMICS A GENOMIC-BASED APPROACH TO HERBAL
  • This invention relates to herbal compositions. More specifically, this invention provides tools and methodologies for improving the selection, testing, quality control and manufacture of herbal compositions, and to help guide the development of new herbal compositions and identify novel uses of existing herbal compositions.
  • Taxol is an antineoplastic drug obtained from the bark of the western yew tree. It is estimated that approximately 50 percent of the thousands of drugs commonly used and prescribed today are either derived from a plant source or contain chemical imitations of a plant compound (Mindell, E.R., 1992, Earl Mindell 's Herb Bible, A Fireside
  • Herbal medicines have been used for treating various diseases of humans and animals in many different countries for a very long period of time (see, e.g., LA. Ross, 1999, Medicinal Plants of the World, Chemical Constituents, Traditional and Modern Medicinal Uses, Humana Press; D. Molony, 1998, The American Associationof Oriental Medicine's Complete Guide to Chinese Herbal Medicine, Berkely Books; Kessler et al., 1996, The
  • Herbal Medicines There are many branches of herbal medicine around the world, such as Ayurveda, Unani, Sida and Traditional Chinese Medicine (TCM). While modern Western medicine typically consists of administering a single chemical entity capable of intervening a specific biochemical pathway, each formula of TCM typically contains hundreds of chemical entities from several herbs which are designed to interact with multiple targets in the body in a coordinated manner. Although empirical practice contributed in a significant way to the herbal composition and prescription of these ancient herbal medicines, they are also supported, to a varying degree, by a set of theories which all are distinct from that of modern Western medicine in terms of anatomy, pharmacology, pathology, diagnosis treatment, etc. Among the different herbal medicine fields, TCM has developed a more complete set of theories over several centuries which have been well documented and practiced by local physicians caring for a huge population (>1.3 billion people) in greater China and in East Asia including Korea and Japan.
  • TCM Western medicine generally uses purified compounds, either natural or synthetic, mostly directed towards a single physiological target.
  • compositions used in TCM are usually composed of multiple herbs and compounds which are aimed at multiple targets in the body based on unique and holistic concepts.
  • TCM mainly used processed crude natural products, with various combinations and formulations, to treat different conformations resulting in fewer side effects.
  • the great potential of TCM has yet to be realized for the majority of the world's people.
  • the herbs in a typical TCM prescription are assigned roles as the principal herb and the secondary herbs, including assistant, adjuvant and guiding herbs.
  • the principal herb produces the leading effects in treating the cause or the main symptom of a disease.
  • An assistant herb helps to strengthen the effect of the principal herb and produces leading effects in the treatment of the accompanying symptoms.
  • adjuvant herbs there are three types of adjuvant herbs: 1) those that enhance the therapeutic effects of the principal and assistant herbs or treat tertiary symptoms, 2) those that reduce or eliminate the toxicity and other side effects of the principal and the assistant herbs and 3) those which act on complementary target tissues not specifically affected by the principal herb.
  • a guiding herb directs the effect of other herbs to the affected site and/or coordinates and mediates the effects of the other herbs in the prescription or formulation.
  • the intended effects of TCM are directed at multiple tissues.
  • a well-known TCM recipe "Ephedra Decoction" used for treating asthma is composed of ephedra, cinnamon twig, bitter apricot kernel and licorice.
  • Ephedra is the principal herb, which expels cold, induces diaphoresis and facilitates the flow of the Lung Qi to relieve asthma, the main symptom.
  • Cinnamon twig as the assistant herb, enhances ephedra' s induction of diaphoresis and warms the Channels to ensure the flow of Yang Qi for reducing headache and pantalgia.
  • Bitter apricot kernel as the adjuvant herb, facilitates the adverse flow of the Lung Qi and strengthens the asthma relief by ephedra.
  • Licorice as the guiding herb moderates the effects of both ephedra and cinnamon to ensure a homeostasis of the vital Qi.
  • TCM The main theories of TCM that guide the treatment of sickness with herbal medicine and other means, such as acupuncture, are 1) the theory of Yin and Yang, 2) the theory of Five Elements, 3) the theory of Viscera and Bowels, 4. the theory of Qi, Blood and Body Fluid, and 5) the theory of Channels and Collaterals.
  • the first important aspect of making the proper diagnosis is to ascertain whether the disease is Yin or Yang.
  • those patients who have a fever, are thirsty, constipated or have a rapid pulse condition are of Yang character.
  • Those individuals who have an aversion to cold, are not thirsty, and diarrhea and a slow pulse condition are of Yin character.
  • the property, flavor and function of herbs can also be classified according to Ying and Yang theory. For example, herbs of cold and cool nature belong to Ying, while herbs which are warm and hot in nature belong to Yang. Herbs with sour, bitter and salty flavor belong to Ying, while herbs with pungent, sweet and bland flavor belong to Yang.
  • Herbs with astringent and subsiding function belong to Yin, while herbs with dispersing, ascending and floating function belong to Yang.
  • TCM the principles of treatment are based on the predominance or weakness of Yin and Yang.
  • Herbs are prescribed according to their property of Ying and Yang and their function for restoring the imbalance of the Ying and Yang. In so doing, the benefit of treatment is achieved.
  • the theory of Five Elements there are five basic substances that constitute the material world (i.e., wood, fire, earth, metal and water). In TCM, this theory has been used to explain the physiology and pathology of the human body and to guide clinical diagnosis and treatment.
  • Herbal physicians have applied the laws of generation, restriction, subjugation and reverse restriction of the five elements to work out many effective and specific treatment regimens, such as reinforcing earth to generate metal (strengthening the function of the spleen to benefit the lung), replenishing water to nourish wood (nourishing the essence of the kidney to benefit the liver), supporting earth to restrict the wood (supplementing the function of the spleen to treat the hyperactivity of the liver), and strengthening water to control fire (replenishing the essence of the kidney to treat hyperactivity of the heart).
  • the property of some herbs is assigned to each of the five Elements for the purposes of guiding the prescription of a TCM recipe.
  • the internal organs of the human body are divided into three groups: five Niscera (the Heart, the Liver, the Spleen, the Lung and the Kidney), Six Bowels (the Gall Bladder, the Stomach, the large Intestine, the Small Intestine, the Urinary Bladder, and the Triple Warmer), the Extraordinary Organs (the Brain, the Medulla, the Bone, the Blood Nessel, the Gall Bladder, and the Uterus).
  • the Niscera or the Bowel are not only anatomic units, but are also concepts of physiology and pathology about interactions between different organs.
  • the heart also refers to some of the mental functions and influence functions of blood, hair, tongue and skin. Ying- Yang and the Five Elements influence the interactions among these Niscera, Bowels and Organs.
  • the complexity of interplay of the theories is used to explain the pathology of diseases to which herbs are prescribed, as discussed below.
  • the prescription of herbal medicine in TCM starts with the diagnosis, which consists of four main items: interrogation, inspection, auscultation and olfaction, pulse taking and palpation.
  • interrogation much information is gathered, including the characteristics of the main symptoms. For instance, if the main symptom is characterized by dull pain of epigastric region, which may be relieved by warming and pressing, this suggests the insufficiency of the Spleen- Yang. Soreness and weakness of the loins and knees, intolerance of coldness with cold extremities manifests a weakness of the Kidney- Yang.
  • inspection observations are made for vitality, skin color and the general appearance and the condition of the tongue.
  • a pale complexion corresponds internally to the Lung of autumn, whose Qi is dry. This may occur when Yang Qi is lacking and the circulation of Qi and blood is impeded, or when the coldness in the channels and collaterals causes them to contract.
  • TCM it is from Qi, blood and body fluid that come energy needed by the Viscera and Bowels, Channels and Collaterals, tissues and other organs for carrying- out their physiological functions; and on which the formation and metabolism of Qi, blood and body fluid depend.
  • Prescriptions of TCM consider the herbal effects on Qi and blood for treatments. TCM holds that Channels, Collaterals and their subsidiary parts are distributed over the entire body It is through them that herbs exert influence on pathological targets and achieve the improvement of sickness.
  • ephedra acts on the Channels of the Lung and Urinary Bladder so as to induce sweat for relieving asthma and promoting diuresis.
  • clinical applications of acupuncture are also guided by the theory of Channels and Collaterals.
  • each herb in TCM may be assigned as Yin or Yang, and to one of the Five Elements, they act through Channels and Collaterals and are mediated via Qi, Blood and Fluid to yield therapeutic effects on targets, such as Viscera and Bowels.
  • Pathogenic factors may be disguised as decoy through the very same systems of Channels and Collaterals to adversely affect the functions of Viscera and Bowels and thus cause sickness.
  • the TCM terminology is as much of a philosophical concept as an anatomical one.
  • the Heart represents a host of tissues, organs or systems in the body that contribute to a function described in TCM.
  • the concept of the Heart requires a multiple dimension data set to describe each concept of TCM. Once this is accomplished, a molecular holistic medicine can be developed.
  • the FDA must approve each one of the chemical entities in a drug composition or cocktail, and then clinical trials must be undertaken so as to obtain separate FDA approval for marketing the drug. This process is extremely tedious and costly.
  • a molecular holistic medicine may require a less arduous evaluation since the previous use of a particular herbal composition as a botanical drug permits clinical trials with multiple chemicals at the outset (i.e., clinical trials using the herbal composition or specific components of the herbal composition).
  • the FDA has approved the testing of some herbal medicines in clinical trials as botanical drugs (FDA Guidance on Botanical Drugs, April, 1997). While these events represent a positive development for health care in general, it also raises important issues regarding the formulation, manufacturing and quality control of herbal medicines and dietary supplements, including the traditional Chinese medicines.
  • Herbal-based industries are coming under increasing pressure to upgrade their current practices (see, e.g., Angell et al, supra).
  • the need to apply scientific testing to the preparation and administration of herbal medicines and food supplements has been highlighted by several recent reports of toxicity resulting from ingesting herb-based formulations.
  • one patient who took an herbal-based dietary supplement experienced digitalis toxicity (Slifman et al, 1998, N. Engl. J. Med. 339:806-811). It was subsequently determined that the herb ingredient labeled as plantain in the supplement was actually contaminated with Digitalis lanata, an herb known to contain at least 60 cardiac glycosides.
  • Botanicals It is well known that the genetic identity (e.g., genera, species, cultivar, variety, clone), age of herbal growth, harvest time, the specific plant part utilized, processing method, geographical origin, soil type, weather patterns, type and rate of fertilizer, and other growth factors have a great impact on the particular chemical composition of any particular herb "harvested" from any particular area.
  • genetic identity e.g., genera, species, cultivar, variety, clone
  • age of herbal growth e.g., age of herbal growth, harvest time, the specific plant part utilized, processing method, geographical origin, soil type, weather patterns, type and rate of fertilizer, and other growth factors have a great impact on the particular chemical composition of any particular herb "harvested" from any particular area.
  • Mass spectrometry is an analytical method for determining the relative masses and relative abundances of components of a beam of ionized molecules or molecular fragments produced from a sample in a high vacuum.
  • MS unlike HPLC, is not optical density-dependent. In practice it is used in conjunction with HPLC or capillary electrophoresis (CE): the HPLC separates the chemicals and the MS then can be used to identify what they are.
  • HPLC capillary electrophoresis
  • Commercial systems are available which integrate MS and HPLC for biological uses. Mass spectrometry is limited to samples that are gaseous or volatile at low pressure, or that can be so rendered by derivatization. These steps are no longer adequate. Recent publications report a greater variation in the quality of herbs by specific suppliers, and the difficulty of providing biological equivalence of herbal extracts.
  • Ephedra intermedia from other species were used to distinguish between species of Phellodendron; and the contents of ginsenosides were used to differentiate between species of Panax.
  • these methods do not provide a direct measurement of the effect of the various herbs on the molecular, physiological or morphological responses following human treatment with the herbs.
  • G-CSF granulocyte colony-stimulating factor
  • Fc gamma 11/111 receptors and complement receptor 3 of macrophages were increased by treatment with Toki-shakuyakusan (TSS) (J. C. Cyong, 1997. Nippon Yakurigakn Zasshi 110(Suppl. l):87-92).
  • TSS Toki-shakuyakusan
  • Tetrandrine an alkaloid isolated from a natural Chinese herbal medicine, inhibited signal-induced NF-kappa B activation in rat alveolar macrophages (Chen et al, 1997, Biochem. Biophys. Res. Commun. 231(1):99-102).
  • IL-8 interleukin-8
  • Recent advances in cDNA microarray technology enable massive parallel mining of information on gene expression. This process has been used to study cell cycles, biochemical pathways, genome-wide expression in yeast, cell growth, cellular differentiation, cellular responses to a single chemical compound, and genetic diseases, including the onset and progression of the diseases (M. Schena et al, 1998, TIBTECH 16:301). No researchers to date, if any, have attempted to apply these new technologies to study the molecular effects of whole herbal treatments and supplements.
  • Treatment with NRl did not change urokinase-type plasminogen activator and PAI-1 antigen synthesis, nor did it effect the deposition of PAI-1 in the extracellular matrix.
  • TPA mRNA increased as much as twofold when HUVECs were treated with NRl, whereas expression of PAI-1 -specific mRNA was not significantly affected by NRl .
  • P. notoginseng have involved its mixture with other herbs, the researchers noted that it was difficult to assess how their results relate to the situation in vivo when is used therapeutically in humans (Id-, at 1045, second column, first paragraph).
  • the researchers only studied one major component of the herb it is not possible to ascertain the molecular effect of the whole herb or the interactions among components of the herb from this study.
  • Chronic heart failure rats with and without Astraglia treatment were compared for changes in various morphological characteristics (e.g., body weight, serum sodium concentration); physiological characteristics (e.g., mean arterial pressure, heart rate, hematocrit and plasma osmolality); mRNA expression levels (e.g., hypothalamic arginine vasopressin (AVP), AVP V,a receptor, renal AVP V 2 receptor, aquaporin-2
  • AVP hypothalamic arginine vasopressin
  • AVP V a receptor
  • renal AVP V 2 receptor e.g., aquaporin-2
  • AXP2 plasma atrial monophosphate peptide
  • cGMP urinary cyclic guanidino monophosphate
  • HBR Arrays Herbal BioResponse Arrays
  • the HBR Arrays of the present invention may include information on the plant-related parameters of the herbal constituents, marker information collected following the exposure of a biosystem to the herbal composition, and biological response information collected following the exposure of a biosystem to the herbal composition.
  • the present invention provides the tools and methodologies necessary for establishing standardized HBR Arrays for particular herbal compositions, wherein the standardized HBR Arrays are used as benchmarks by which to evaluate batches of similar or different herbal compositions.
  • the present invention further provides the tools and methodologies necessary to update and maintain the standardized HBR Arrays.
  • Particular embodiments of the present invention involve iterative processes whereby data for additional batches of the herbal composition, additional plant-related data, additional marker information, and/or additional BioResponse information is periodically added to the standardized HBR Arrays.
  • the present invention provides the tools and methodologies for creating, maintaining, updating and using HBR Arrays on an ongoing basis.
  • the present invention provides the tools and methodologies necessary to guide the standardization of herbal compositions, to determine which specific components of herbal compositions are responsible for particular biological activities, to predict the biological activities of herbal compositions, for the development of improved herbal therapeutics; for adjusting or modifying an herbal composition; for identifying specific molecules in the batch herbal composition which retain the desired biological activity; for determining which herbal components of a known herbal composition can be eliminated from the known herbal composition while maintaining or improving the desired biological activity of the known herbal composition; for identifying new uses and previously unknown biological activities for the batch herbal composition; and for using the predicted biological activity of the batch herbal composition to aid in the design of therapeutics which include herbal components and synthetic chemical drugs, including the design of therapeutics using the methods of combinatorial chemistry.
  • the present invention provides methods of establishing standardized Herbal BioResponse Arrays (HBR Arrays) for herbal compositions, wherein the methods comprise:
  • step 3 storing the marker data of step 2) as an HBR Array; 4). repeating steps 2) and 3) for one or more additional batches of the herbal composition using two or more of the same or different markers than used in step 2);
  • the present invention further provides such methods which further comprise exposing a biosystem to one or more batches of the herbal composition, collecting the data on one or more BioResponses, and adding the collected BioResponse data to the standardized HBR Array for that herbal composition.
  • the present invention provides methods of evaluating herbal compositions, wherein the methods comprise exposing a biosystem to a batch of the herbal composition and collecting data on two or more markers; and comparing the collected marker data with a standardized HBR Array for the same or a substantially same herbal composition as that of the batch herbal compositions.
  • the present invention provides a system for predicting the biological activity of an herbal composition comprising: 1). a biosystem comprising one or more different types of cells, tissues, organs or in vitro assays;
  • a computer processor including memory, for analyzing and storing the differential response measurements of the molecular markers so as to create an Herbal BioResponse Array (HBR Array) data set for the batch herbal composition;
  • HBR Array Herbal BioResponse Array
  • a computer processor including memory, for comparing the HBR Array of the batch herbal composition to one or more previously-stored HBR Arrays so as to predict the biological activity of the batch herbal composition, wherein the biological activities of the herbal compositions used to generate the one or more previously-stored HBR Arrays are known.
  • Figure 1 provides a schematic of the basic method steps for constructing a Standardized Herbal BioResponse Array (HBR Array) for any selected herbal composition.
  • HBR Array Standardized Herbal BioResponse Array
  • Figure 2 provides a schematic of the basic method steps for constructing a an Herbal BioResponse Array (HBR Array) for any batch herbal composition and for comparing this batch HRB Array to a selected subset of information from the Standardized HBR Array.
  • each of the pathways of the schematic can be done iteratively.
  • any information contained in one box can be used to guide decisions regarding gathering information for any other box. In this way, numerous feedback loops are possible throughout the scheme.
  • Figure 3 provides a schematic of the basic method steps for establishing and using a major data set. The figure is shown in its most basic form for ease of understanding. As discussed herein, each of the pathways of the schematic can be done iteratively. Furthermore, any information contained in one box can be used to guide decisions regarding gathering information for any other box. In this way, numerous feedback loops are possible throughout the scheme.
  • Figure 4 Western blot for various herbal compositions.
  • the present invention is directed to tools and methods useful for predicting the biological response of an herbal composition. More particularly, this invention provides methods of creating Herbal BioResponse Array (HBR Array) databases as well as methods for using such databases to improve the design of effective herbal-based therapeutics.
  • the goal of the present invention is the overall design, creation, improvement and use of HBR Arrays for the preparation, testing and administration of herbal compositions, and guide development of new herbal compositions and novel uses of existing herbal compositions.
  • Phytomics refers to using bioinformatics and statistical approaches to address the qualitative and quantitative aspects of the components of herbal compositions or to the actual data bases which are developed for addressing such aspects.
  • an HBR Array constitutes a data set of two or more observations or measurements associated with an herbal composition.
  • the HBR Array may include qualitative and quantitative data on the plants in the composition (plant-related data), marker information obtained after exposure of a biosystem to the herbal composition including a dose dependent study, and BioResponse data obtained after exposure of a biosystem to the herbal composition.
  • the data in any particular HBR Array can be statistically analyzed in either 2- or 3-dimensional space.
  • HBR Arrays may be designated as batch HBR Arrays and standardized HBR Arrays. Batch HBR Arrays are arrays of data associated with specific batches of an herbal composition.
  • Standardized HBR Arrays are arrays of data associated with a standardized herbal composition.
  • Major Data Set refers to the data set which acts as the baseline set of data by which various other sets of data are compared or otherwise analyzed for the same or different herbal compositions.
  • the major data set is created using biotechnological techniques to ascertain some genetic or protein aspect of the herbal compositions.
  • the major data set will usually, but not always, be based on a genomic or proteomic set of data.
  • DNA microarray results could be the major data set which is used to compare to other, dependent or minor data sets.
  • the "minor data set” or “dependent data set” refers to one or more data sets which are used for comparing to the major data set.
  • the minor data set will consist of information on an herbal composition which are collected by more traditional methods.
  • the minor, or dependent, data set may consist of a collection of plant- related data obtained by more conventional means. Examples of plant-related data include, but are not limited to, the genus/species of the herb(s) in the herbal composition, the particular plant parts of the herb(s) in the composition and the geographic location where the herb(s) were located.
  • Another example of a minor data set might consist of a set of biological responses of a cell, tissue, organ or organism after treatment with one or more different amounts of the herbal composition.
  • biological data or a whole organism may include, but are not limited to, cell toxicity studies, enzyme treatment studies, growth rates, weight gain or loss, changes in motor skills and changes in mental abilities.
  • an herb is a small, non-woody (i.e., fleshy stemmed), annual or perennial seed-bearing plant in which all the aerial parts die back at the end of each growing season. Herbs are valued for their medicinal, savory or aromatic qualities.
  • an "herb” refers to any plant or plant part which has a food supplement, medicinal, drug, therapeutic or life-enhancing use.
  • an herb is not limited to the botanical definition of an herb but rather to any botanical, plant or plant part used for such purposes, including any plant or plant part of any plant species or subspecies of the Metaphyta kingdom, including herbs, shrubs, subshrubs, and trees.
  • Plant parts used in herbal compositions include, but are not limited to, seeds, leaves, stems, twigs, branches, buds, flowers, bulbs, corms, tubers, rhizomes, runners, roots, fruits, cones, berries, cambium and bark.
  • Herbal Composition refers to any composition which includes herbs, herbal plants or herbal plant parts.
  • an herbal composition is any herbal preparation, including herbal food supplements, herbal medicines, herbal drugs and medical foods.
  • examples of herbal compositions include, but are not limited to, the following components: a whole plant or a plant part of a single plant species; whole plants or plant parts of multiple plant species; multiple components derived from a single plant species; multiple components derived from multiple plant species; or any combination of these various components.
  • Kee Chang Huang The Pharmacology of Chinese Herbs. CRC Press (1993), herein incorporated in its entirety. Representative examples of various herbal compositions are provided in the following paragraphs.
  • Herbal compositions which include the bark of the willow tree have been used to treat fever since the mid-eighteenth century in England.
  • the active ingredient in willow bark is a bitter glycoside called salicin, which on hydrolysis yields glucose and salicylic alcohol.
  • Aspirin acetylsalicylic acid
  • aspirin-like drugs e.g., ibuprofen
  • NSAIDs nonsteroidal antiinflammatory drugs
  • U.S. Patents have been issued for herbal compositions used for the treatment of various diseases and other health-related problems afflicting humans and animals.
  • U.S. Patent No. 5,417,979 discloses a composition comprising a mixture of herbs, including species of Stephania and Glycyrrhiza, as well as their extracts, which is used as an appetite stimulant and for the treatment of pain.
  • Herbal compositions which include Glycyrrhiza uralensis have been found useful for treating eczema, psoriasis, pruritis and inflammatory reactions of the skin (U.S. Patent No. 5,466,452).
  • U.S. Patent No. 5,683,697 discloses a pharmaceutical composition having anti-inflammatory, anti- fever, expectorant or anti-tussive action, wherein the composition includes plant parts from the species Melia, Angepica, Dendrobium, Impatiens, Citrus, Loranthus, Celosia,
  • Cynanchum and Glehnia An herbal composition which includes extracts of the roots, rhizomes, and/or vegetation of Alphinia, Smilax, Tinospora, Tribulus, Withania and Zingiber has been found to reduce or alleviate the symptoms associated with rheumatoid arthritis, osteoarthritis, reactive arthritis and for reducing the production of proinflammatory cytokines (U.S. Patent No. 5,683,698).
  • Herbal compositions are available in many forms, including capsules, tablets, or coated tablets; pellets; extracts or tinctures; powders; fresh or dried plants or plant parts; prepared teas; juices; creams and ointments; essential oils; or, as combinations of any of these forms.
  • Herbal medicines are administered by any one of various methods, including orally, rectally, parenterally, enterally, transdermally, intravenously, via feeding tubes, and topically.
  • Herbal compositions encompassed by the present invention include herbal compositions which also contain non-herbal components.
  • non- herbal components include, but are not limited to, whole insects and insect parts, worms, animal or insect feces, natural or petroleum oils, carbonate of ammonia, salt of tartar, liquor, water, glycerin, steroids, pharmaceuticals, vitamins, nutrient extracts, whey, salts, and gelatin.
  • the herbal compositions disclosed may take the form of, for example, tablets or capsules prepared by conventional means in admixture with generally acceptable excipients such as binding agents (e.g., pregelatinised maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystalline cellulose or calcium phosphate); lubricants (e.g., magnesium stearate, talc or silica); disintegrants (e.g., potato starch or sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulphate); glidants; artificial and natural flavors and sweeteners; artificial or natural colors and dyes; and solubilizers.
  • binding agents e.g., pregelatinised maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose
  • fillers e.g., lactose, microcrystalline cellulose or calcium phosphate
  • lubricants
  • the herbal compositions may be additionally formulated to release the active agents in a time- release manner as is known in the art and as discussed in U.S. Patent Nos. 4,690,825 and 5,055,300.
  • the tablets may be coated by methods well known in the art.
  • Liquid preparations for oral administration may take the form of, for example, solutions, syrups, suspensions, or slurries (such as the liquid nutritional supplements described in Mulchandani et al, 1992 U.S. Patent No. 5,108,767), or they may be presented as a dry product for reconstitution with water or other suitable vehicles before use.
  • Liquid preparations of folic acid, and other vitamins and minerals may come in the form of a liquid nutritional supplement specifically designed for ESRD patients.
  • Such liquid preparations may be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, methyl cellulose or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters or ethyl alcohol); preservatives (e.g., methyl or propyl p-hydroxybenzoates or sorbic acid); and artificial or natural colors and/or sweeteners.
  • suspending agents e.g., sorbitol syrup, methyl cellulose or hydrogenated edible fats
  • emulsifying agents e.g., lecithin or acacia
  • non-aqueous vehicles e.g., almond oil, oily esters or ethyl alcohol
  • preservatives e.g., methyl or propyl p-hydroxybenzoates or sorbic acid
  • herbal components may be combined in admixture with at least one other ingredient constituting an acceptable carrier, diluent or excipient in order to provide a composition, such as a cream, gel, solid, paste, salve, powder, lotion, liquid, aerosol treatment, or the like, which is most suitable for topical application.
  • a composition such as a cream, gel, solid, paste, salve, powder, lotion, liquid, aerosol treatment, or the like, which is most suitable for topical application.
  • Sterile distilled water alone and simple cream, ointment and gel bases may be employed as carriers of the herbal components.
  • Preservatives and buffers may also be added.
  • the formulation may be applied to a sterile dressing, biodegradable, absorbable patches or dressings for topical application, or to slow release implant systems with a high initial release decaying to slow release.
  • Standardized Herbal Composition refers to a particular herbal composition which is chosen as the standard herbal composition for evaluating batch herbal compositions which have the same, similar or different components as the components of the standardized herbal composition. Sometimes herein also referred to as the "master herbal composition.”
  • Standardized herbal compositions are generally herbal compositions which have been well characterized and which demonstrate the desired biological responses in a particular biosystem. Standardized herbal compositions are usually standardized by chemical tests well known to one skilled in the art and are properly stored for long term usage and reference. The standardized herbal composition is used to establish a standardized HBR Array based on observations and measurements for the plants (i.e., plant-related data), markers and BioResponses so as to characterize the herbal composition.
  • a "batch herbal composition” refers to any test herbal composition which is used to establish a HBR Array based on observations and measurements for the plants and markers so as to characterize the herbal composition. Sometimes herein also referred to as a "test” or “batch” herbal composition. Observations and measurements of BioResponses may or may not be included.
  • the herbal compositions used to establish the standardized herbal composition may also be referred to as "batch herbal compositions" until designated as "standardized herbal compositions.”
  • Batch refers to a particular quantity of an herbal composition which can be identified as to some particular attribute so as to distinguish it from any other particular quantity of that same herbal composition.
  • one batch of an herbal composition may differ from another batch of that same herbal composition in that one of the batches was harvested at a different time or in a different geographical location than the other batch.
  • compositions may include, but are not limited to, the following: 1) the particular plant part used (e.g., the root of an herb was used in one batch while the leaves of that same herb were used in a different batch); 2) the post-harvest treatment of the individual herbs or herbal composition (e.g., one batch may be processed with distilled water while a different batch may be processed with Hydrogen Chloride to simulate the acidity of the human stomach); and, 3) the relative proportions of the individual herbs in an herbal composition (e.g., one batch may have equal parts by weight or volume of three different herbs while another batch has proportionally more of one herb than the other two).
  • Biosystem refers to any biological entity for which biological responses may be observed or measured.
  • a biosystem includes, but is not limited to, any cell, tissue, organ, whole organism or in vitro assay.
  • Biological Activity As used herein, the "biological activity" of an herb refers to the specific biological effect peculiar to an herbal composition on a given biosystem.
  • Plant-Related Data refers to the data collected on the herbal composition including, but not limited to, data about the plants, their growing conditions and the handling of the plants during and after harvesting.
  • the plant-related data also includes the relative proportions of the components in an herbal compositions, wherein the components may be different plant parts, different plant species, other non-plant ingredients (e.g., insect parts, chemical drugs) or any combinations of these variables.
  • Plant-related data which may be gathered for an herbal composition includes, but is not limited to, the following: 1) the plant species (and, if available, the specific plant variety, cultivar, clone, line, etc.) and specific plant parts used in the composition; 2) the geographic origin of the herbs, including the longitude/latitude and elevation; 3) the growth conditions of the herbs, including fertilizer types and amounts, amounts and times of rainfall and irrigation, average microEinsteins received per day, pesticide usage, including herbicides, insecticides, miticides and fungicides, and tillage methods; 4) methods and conditions used for processing the herbs, including age/maturity of the herbs, soaking times, drying times, extraction methods and grinding methods; and 5) storing methods and conditions for the herbal components and the final herbal composition.
  • the standardized herbal composition may be analyzed chemically.
  • Chemical characterization may be accomplished by any chemical analysis method generally known by one skilled in the art. Examples of applicable chemical analyses include, but are not limited to, HPLC, TLC, chemical fingerprinting, mass spectrophotometer analyses and gas chromatography.
  • Bioinformatics refers to the use and organization of information of biological interest. Bioinformatics covers, among other things, the following: (1) data acquisition and analysis; (2) database development; (3) integration and links; and (4) further analysis of the resulting database. Nearly all bioinformatics resources were developed as public domain freeware until the early 1990s, and much is still available free over the Internet. Some companies have developed proprietary databases or analytical software. Genomic or Genomics. As used herein, the term “genomics” refers to the study of genes and their function. Genomics emphasizes the integration of basic and applied research in comparative gene mapping, molecular cloning, large-scale restriction mapping, and DNA sequencing and computational analysis. Genetic information is extracted using fundamental techniques, such as DNA sequencing, protein sequencing and PCR.
  • Gene function is determined (1) by analyzing the effects of DNA mutations in genes on normal development and health of the cell, tissue, organ or organism; (2) by analyzing a variety of signals encoded in the DNA sequence; and (3) by studying the proteins produced by a gene or system of related genes. Proteomic or Proteomics. As used herein, the term "proteomics”, also called
  • proteome research or "phenome” refers to the quantitative protein expression pattern of a genome under defined conditions.
  • proteomics refers to methods of high throughput, automated analysis using protein biochemistry.
  • the level of gene expression does not necessarily represent the amount of active protein in a cell.
  • the gene sequence does not describe post- tranlsational modifications which are essential for the function and activity of a protein.
  • the genome itself does not describe the dynamic cell processes which alter the protein level either up or down.
  • Proteome programs seek to characterize all the proteins in a cell, identifying at least part of their amino acid sequence of a isolated protein. In general, the proteins are first separated using 2D gels or HPLC and then the peptides or proteins are sequenced using high throughput mass spectrometry.
  • the output of the mass spectrometry can be analyzed so as to link a gene and the particular protein for which it codes. This overall process is sometimes referred to as "functional genomics".
  • proteomic services e.g., Pharmaceutical ProteomicsTM, The ProteinChipTM System from Ciphergen Biosystem; PerSeptive Biosystems.
  • signal transduction also known as cellular signal transduction, refers to the pathways through which cells receive external signals and transmit, amplify and direct them internally. Signaling pathways require intercommunicating chains of proteins that transmit the signal in a step wise fashion.
  • Protein kinases often participate in this cascade of reactions, since many signal transductions involve receiving an extracellular chemical signal, which triggers the phosphorylation of cytoplasmic proteins to amplify the signal.
  • post-translational modification is a blanket term used to cover the alterations that happen to a protein after it has been synthesized as a primary polypeptide.
  • post-translational modifications include, but are not limited to, glycosylation, removal of the N-terminal methionine (or N-formyl methionine), signal peptide removal, acetylation, formylation, amino acid modifications, internal cleavage of peptide chains to release smaller proteins or peptides, phosphorylation, and modification of methionine.
  • an "array” or “microarray” refers to a grid system which has each position or probe cell occupied by a defined nucleic acid fragment.
  • the arrays themselves are sometimes referred to as “chips”, “biochips”, “DNA chips” or “gene chips”. High-density DNA microarrays often have thousands of probe cells in a variety of grid styles.
  • markers refers to any biological-based measurement or observation for a particular herbal composition that is characteristic of a particular biosystem which is being exposed to a particular batch of an herbal composition.
  • the term “marker” encompasses both qualitative and qualitative measurements and observations of a biosystem.
  • the marker database constitutes a data set which characterizes gene expression patterns in response to herbal therapies, wherein the patterns show which genes are turned on, off, up or down in response to specific herbal compositions.
  • markers refers to any biologically-based measurement or observation whose up- and down- or temporal regulations, or qualitative or quantitative changes of expression levels in a biosystem are used to characterize differential biological responses of a biosystem to an herbal composition.
  • the particular batch of an herbal composition to which the biosystem is exposed may be an unknown herbal composition, a known herbal composition, or a standardized herbal composition.
  • markers useful in accomplishing the present invention include, but are not limited to, molecular markers, cytogenetic markers, biochemical markers or macromolecular markers.
  • Macromolecular markers include, but are not limited to, enzymes, polypeptides, peptides, sugars, antibodies, DNA, RNA, proteins (both translational proteins and post-translational proteins), nucleic acids, polysaccarides.
  • markers includes related, alternative terms, such as “biomarker” or “genetic marker” or “gene marker.” There may be one or more primary markers along with secondary markers, or a hierarchy of markers for achieving the purposes of increasing the discriminating power of a HBR array. Thus, selected molecular markers may be combined with various other molecular, cytogenetic, biochemical or macromolecular markers to enable an even more accurate, extended HBR Array.
  • a molecular marker comprises one or more microscopic molecules from one or more classes of molecular compounds, such as DNA, RNA, cDNA, nucleic acid fragments, proteins, protein fragments, lipids, fatty acids, carbohydrates, and glycoproteins.
  • molecular markers The establishment, generation and use of applicable molecular markers are well known to one skilled in the art. Examples of particularly useful technologies for the characterization of molecular markers include differential display, reverse transcriptase polymerase chain reactions (PCR), large-scale sequencing of expressed sequence tags (ESTs), serial analysis of gene expression (SAGE), Western immunoblot or 2D, 3D study of proteins, and microarray technology.
  • PCR reverse transcriptase polymerase chain reactions
  • ESTs large-scale sequencing of expressed sequence tags
  • SAGE serial analysis of gene expression
  • Western immunoblot or 2D, 3D study of proteins, and microarray technology One skilled in the art of molecular marker technology is familiar with the methods and uses of such technology (see, e.g., Bernard R. Glick and Jack J. Pasternak, Molecular Biotechnology, Principles and Applications of Recombinant DNA. Second Edition. ASM Press 09981: Mathew R. Walker and Ralph Rapley, Route Maps in Gene Technology. Blackwell Science
  • DNA, RNA and protein isolation and sequencing methods are well known to those skilled in the art. Examples of such well known techniques can be found in Molecular Cloning: A Laboratory Manual 2n Edition. Sambrook et al, Cold Spring
  • kits and tools available commercially for use in the present invention include, but are not limited to, those useful for RNA isolation, PCR cDNA library construction, retroviral expression libraries, vectors, gene expression analyses, protein antibody purification, cytotoxicity assays, protein expression and purification, and high- throughput plasmid purification (see, e.g., CLONTECHniques product catalog, XIII(3), 1-32 (1998) or www.clontech.com; AtlasTM cDNA Expression Assays product catalog (1998); STGMA® product catalog (1997)).
  • references applicable to the instant invention include, but are not limited to, those addressing the expression technologies, such as ESTs (see, e.g., Michael R. Fannon, Gene expression in normal and disease states - identification of therapeutic targets, TTBTECH 14, 294-298 (1996)); the generation of protein profiles (see, e.g., Robinson et al., A Tyrosine Kinase Profile of Prostate Carcinoma, Proc. Natl. Acad. Sci.
  • HPLCs see, e.g., Milton T. W. Hearn (Editor), HPLC of Proteins. Pepties r and Polynucleotides: Contemporary Topics and Applications (Analytical Techniques in Clinical Chemistry and Laboratory Manual). VCH Pub. (1991); electrophoresis (see, e.g., Westermeier et al, Electrophoresis in Practice: A Guide to Methods and Applications of DNA and Protein Separations. John
  • Cytogenetic parameters include, but are not limited to, karyotype analyses (e.g., relative chromosome lengths, centromere positions, presence or absence of secondary constrictions), ideograms (i.e., a diagrammatic representation of the karyotype of an organism), the behavior of chromosomes during mitosis and meiosis, chromosome staining and banding patterns, DNA-protein interactions (also known as nuclease protection assays), neutron scattering studies, rolling circles (A.M. Diegelman and E.T. Kool. Nucleic Acids Res 26(1 ):3235-3241 (1998): Backert et ⁇ l. Mol. Cell. Biol.
  • karyotype analyses e.g., relative chromosome lengths, centromere positions, presence or absence of secondary constrictions
  • ideograms i.e., a diagrammatic representation of the karyotype of an organism
  • DNA-protein interactions also
  • Biochemical parameters include, but are not limited to, specific pathway analyses, such as signal transduction, protein synthesis and transport, RNA transcription, cholesterol synthesis and degradation, glucogenesis and glycolysis.
  • Fingerprinting refers to the means of making a characteristic profile of a substance, particularly an herb, in order to identify it.
  • fingerprint refers to the display of the result of the particular means employed for the fingerprinting.
  • fingerprinting means examples include, but are not limited to, DNA fingerprinting, protein fingerprinting, chemical fingerprinting and footprinting.
  • DNA fingerprinting refers to a way of making a unique pattern from the DNA of particular biological source (e.g., a particular plant, plant species, genus of plant, plant part or plant tissue).
  • the DNA fingerprint, or profile can be used to distinguish that particular biological source from a different biological source.
  • the pattern obtained by analyzing a batch using microarrays, oligonucletide arrays, DNA chips or biochips are also referred to as "fingerprints".
  • Protein fingerprinting refers to generating a pattern of proteins in a cell, tissue, organ or organism, such as a plant, which provides a completely characteristic "fingerprint" of that cell, tissue, organ or organism at that time.
  • Chemical fingerprinting refers to the analysis of the low molecular weight chemicals in a cell and the resulting pattern used to identify a cell, tissue, organ or organism, such as a plant. The analysis is usually done using Gas Chromotography (GC), HPLC or mass spectrometry.
  • GC Gas Chromotography
  • HPLC HPLC
  • mass spectrometry mass spectrometry
  • Footprinting refers to a method of finding how two molecules stick together.
  • DNA a protein is bound to a labeled piece of DNA, and then the DNA is broken down, by enzymes or by chemical attack. This process produces a "ladder" of fragments of all sizes. Where the DNA is protected by the bound protein it is degraded less, and so the "ladder” appears fainter.
  • Footprinting is a common technique for homing in on where the proteins that regulate gene activity actually bind to the DNA. The means, or methods, used to accomplish each type of fingerprinting are described in detail elsewhere herein.
  • BioResponses refers to any observation or measurement of a bjQlogical response of a biosystem following exposure to an herbal composition. Sometimes herein also referred to as a "biological effect.”
  • a BioResponse is a qualitative or quantitative data point for the biological activity of a particular herbal composition.
  • BioResponse data includes both dosage and temporal information, wherein such information is well known to one skilled in the art of measuring responses of biosystems to various treatments.
  • BioResponse data includes information on the specific biological response of a specific biosystem to a specific dosage of herbal composition administered in a particular manner for a specific period of time.
  • BioResponses include, but are not limited to, physiological responses, morphological responses, cognitive responses, motivational responses, autonomic responses and post-translational modifications, such as signal transduction measurements. Many herbal compositions demonstrate more than one BioResponse (see, e.g., Kee Chang Huang, The Pharmacology of Chinese Herbs. CRC Press (1993)). Some particular BioResponses may be included in more than one of the delineated groups or have aspects or components of the response that encompass more than one group. BioResponses applicable to the instant invention are well known to one skilled in the art. The following references are representative of the state of art in the field: Kee Chang Huang, The Pharmacology of Chinese Herbs. CRC Press (1993); Earl Mindell, Earl Mindell's Herb Bible. Simon & Schuster (1992); Goodman & Gilman's The Pharmacological Basis of Therapeutics. Ninth Edition. Joel G. Hardman, et. al.
  • a “physiological response” refers to any characteristic related to the physiology, or functioning, of a biosystem. Physiological responses on a cellular, tissue or organ level include, but are not limited to, temperature, blood flow rate, pulse rate, oxygen concentration, bioelectric potential, pH value, cholesterol levels, infection state (e.g., viral, bacterial) and ion flux.
  • Physiological responses on a whole organism basis include gastrointestinal functioning (e.g., ulcers, upset stomach, indigestion, heartburn), reproductive tract functioning (e.g., physiologically-based impotence, uterine cramping, menstrual cramps), excretory functions (e.g., urinary tract problems, kidney ailments, diarrhea, constipation), blood circulation (e.g., hypertension, heart disorders), oxygen consumption, skeletal health (e.g., osteoporosis), condition of the cartilage and connective tissues (e.g., joint pain and inflammation), locomotion, eyesight (e.g., myopia, blindness), muscle tone (e.g.
  • gastrointestinal functioning e.g., ulcers, upset stomach, indigestion, heartburn
  • reproductive tract functioning e.g., physiologically-based impotence, uterine cramping, menstrual cramps
  • excretory functions e.g., urinary tract problems, kidney ailments, diarrhea, constipation
  • blood circulation e.g., hypertension,
  • wasting syndrome, muscle strains presence or absence of pain
  • epidermal and dermal health e.g., skin irritation, itching, skin wounds
  • functioning of the endocrine system cardiac functioning, nervous coordination, head-related health (e.g., headaches, dizziness), age (e.g., life span, longevity) and respiration (e.g., congestion, respiratory ailments).
  • head-related health e.g., headaches, dizziness
  • age e.g., life span, longevity
  • respiration e.g., congestion, respiratory ailments
  • a “morphological response” refers to any characteristic related to the morphology, or the form and structure, of a biosystem following exposure to an herbal composition. Morphological responses, regardless of the type of biosystem, include, but are not limited to, size, weight, height, width, color, degree of inflammation, general appearance (e.g., opaqueness, transparency, paleness), degree of wetness or dryness, presence or absence of cancerous growths, and the presence or lack of parasites or pests (e.g., mice, lice, fleas).
  • Morphological responses on a whole organism basis include, but are not limited to, the amount and location of hair growth (e.g., hirsutism, baldness), presence or absence of wrinkles, type and degree of nail and skin growth, degree of blot clotting, presence or absence of sores or wounds, and presence or absence of hemorrhoids.
  • a "cognitive response” refers to any characteristic related to the cognitive, or mental state, of a biosystem following exposure to an herbal composition. Cognitive responses include, but are not limited to, perceiving, recognizing, conceiving, judging, memory, reasoning and imagining.
  • a “motivational response” refers to any characteristic related to the motivation, or induces action, of a biosystem following exposure to an herbal composition.
  • Motivational responses include, but are not limited to, emotion (e.g., cheerfulness), desire, learned drive, particular physiological needs (e.g., appetite, sexual drive) or similar impulses that act as incitements to action (e.g., stamina, sex drive).
  • An "autonomic response” refers to any characteristic related to autonomic responses of a biosystem following exposure to an herbal composition. Autonomic responses are related to the autonomic nervous system of the biosystem. Examples of autonomic responses include, but art not limited to, involuntary functioning (e.g., nervousness, panic attacks), or physiological needs (e.g., respiration, cardiac rhythm, hormone release, immune responses, insomnia, narcolepsy).
  • BioResponses of cells, tissues, organs and whole organisms treated with various herbal compositions or herbal components are well known in the herbal arts.
  • the herbal compositions Sairei-to (TJ-114), alismatis rhizoma (Japanese name 'Takusha') and hoelen (Japanese name 'Bukuryou') were each found to inhibit the synthesis and expression of endothelin-1 in rats (Hattori et al, Sairei-to may inhibit the synthesis of endothelin-1 in nephritic glomeruli, Nippon Jinzo Gakkai Shi 39(2), 121- 128 (1997)).
  • Interleukin (IL)-l alpha production was significantly promoted by treatment of cultured human epidermal keratinocytes with the herbal medicine Sho- saiko-to (Matsumoto et al, Enhancement of interleukin- 1 alpha mediated autocrine growth of cultured human keratinocytes by sho-saiko-to, Jpn J. Pharmacol 73(4), 333- 336 (1997).
  • PA PA-specific mRNA increased after treatment of human umbilical vein endothelial cells (HUVECs) with the saponin astragaloside IV (AS-IV) purified from the Chinese herb Astragalus membranaceus (Zhang et al, Regulation of the fibrinolytic potential of cultured human umbilical vein endothelial cells: astragaloside IV down regulates plasminogen activator inhibitor- 1 and up regulates tissue-type plasminogen activator expression, J Vase Res 34(4), 273-280 (1997)).
  • AS-IV saponin astragaloside IV
  • Toki-shakuyakusan (Cyong, New BRM from kampo-herbal medicine, Nippon Yakurigaku Zasshi 110 Suppl 1, 87P-92P (1997)).
  • TSS Toki-shakuyakusan
  • Chen et al. Image analysis for intercellular adhesion molecule- 1 expression in MRI/lpr mice: effects of Chinese herb medicine, Chung Hua I Hsueh Tsa Chih 75(4), 204-206 (1995) found that the distribution intensity of intercellular adhesion molecule- 1 (ICAM-1), immunoglobulins and C3 were significantly decreased in MRL/lpr mice after treatment with the Chinese herb stragalin.
  • IAM-1 intercellular adhesion molecule- 1
  • Algorithm refers to a step-by-step problem- solving procedure, especially an established, recursive computational procedure with a finite number of steps.
  • Appropriate algorithms for two- and three-dimensional analyses of the plant-related, marker and BioResponse data sets are well known to one skilled in the computational arts. Such algorithms are useful in constructing the Herbal BioResponse Arrays of the present invention.
  • Jerrod H. Zar Biostatistical Analysis, second edition. Prentice Hall (1984); Robert A. Schowengerdt, Techniques for image processing and classification in remote sensing.
  • Combinatorial Chemistry refers to the numerous technologies used to create hundreds or thousands of chemical compounds, wherein each of the chemical compounds differ for one or more features, such as their shape, charge, and/or hydrophobic characteristics.
  • Combinatorial chemistry can be utilized to generate compounds which are chemical variations of herbs or herbal components. Such compounds can be evaluated using the methods of the present invention.
  • Basic combinatorial chemistry concepts are well known to one of ordinary skill in the chemical arts and can also be found in Nicholas K. Terrett, Combinatorial Chemistry (Oxford Chemistry, Masters). Oxford Univ. Press (1998); Anthony W. Czarnik and Sheila Hobbs Dewitt (Editors), A Practical Guide to Combinatorial Chemistry. Amer. Chemical Society (1997); Stephen R. Wilson (Editor) and Anthony
  • Example 1 Establishing a Standardized HBR Array for Selected Herbal Compositions.
  • Plant-related data includes, but is not limited to, the plant species, specific plant parts, geographic origin of the plants in the herbal composition, the growth conditions of the plants, the processing methods used to prepare the herbal components, storage methods and conditions, and various chemical analyses of the herbal composition.
  • Marker information includes qualitative and quantitative data for markers collected after exposure of a biosystem to the herbal compost.
  • Applicable markers include, but are not limited to, molecular markers, cytogenetic markers, biochemical markers and macromolecular markers.
  • BioResponse information includes qualitative and quantitative data for biological responses collected after exposure of a biosystem to the herbal composition.
  • Each type of data can be obtained using one or more assays on the same, similar, substantially similar, or different batches of the herbal composition of interest. Such different assays can be conducted at the same or different times. In addition, data can be collected for the same or different markers at the same or different times. Similarly, BioResponse data can be collected for the same or different biological responses at the same or different times. Thus, collection of the data for the HBR Array is either collected at one time or collected on an ongoing basis. Where a biosystem is exposed to an herbal composition so as to collect data, information is recorded on the administered dosages of the herbal composition as well as treatment times. BioResponse data may also consist of post-translational modifications, such as measurements of signal transduction.
  • the data is analyzed using algorithms so as to create 2- and/or 3-dimensional Herbal BioResponse Arrays.
  • Various statistical parameters may be calculated for the HBR Array and may become part of the HBR Array data set. These statistical parameters may include, but are not limited to, means, standard deviations, correlation or match (or mismatch) matrices, ratios, regression coefficients, and transformed values (e.g., arcsin percentage transformations of the raw data).
  • the HBR Array may consist of the raw data as well as certain calculations, distributions, graphical presentations and other data manipulations associated with the raw data. Particular examples of such information include, but are not limited to, digital images, scatter graphs, cluster analyses and large scale gene expression profiles for marker data.
  • the total accumulated data and resultant analyses constitute a standardized HBR Array for the particular herbal composition used to establish the HBR Array data set. Due to the iterative nature of the process used to establish and maintain an HBR Array for an herbal composition, such arrays can be viewed as either static at any one point in time or dynamic over time.
  • the resulting analyses can identify subsets of the standardized HBR Arrays which are correlated (positively or negatively) or associated (i.e., showing a general trend) with one or more specific biological activities of any particular herbal composition.
  • Example 2. Establishing a Batch HBR Array for Batch Herbal
  • the amount of data collected for a batch HBR Array will be less than that collected to establish a standardized HBR Array.
  • data collected for a batch herbal composition may be added to an established HBR Array or used to establish a new standardized HBR Array.
  • the only data collected for a batch herbal composition is that data which has been found to be highly correlated or associated with the desired biological activities of the herbal composition being tested. For example, if it has been determined that a particular subset of plant-related and marker data is highly correlated to a desired biological activity of a particular herbal composition (based on the standardized HBR Array data and analyses discussed above), it is only necessary to test the batch herbal composition for that subset of traits in order to determine whether or not the batch has the desired biological activity. By comparing the data obtained for that subset of traits obtained from the batch (i.e., the batch HBR Array) with the standardized HBR Array for that particular herbal composition, one skilled in the art can determine whether or not that particular batch has the desired biological activity.
  • Example 3 Establishing and Using a Major Data Set.
  • the first step is the establishment of a major data set for a selected herbal composition or batch herbal composition. This is accomplished by exposing a biosystem to the herbal composition and collecting the resultant marker information which will constitute the major data set.
  • the major data set will consist of genomics and/or proteomics data in the form of an array, such as an array obtained with a DNA biochip.
  • differential expression/results are necessary in order to generate meaningful algorithms in the next step.
  • Examples of such differential expression/results include, but are not limited to, indications that certain genes are up- or down-regulated in response to exposure to the herbal composition or that the levels of certain proteins have been increased or decreased in response to the exposure. If no meaningful or useful differential expression/results are obtained, then it is necessary to repeat the exposure and marker collection step.
  • the exposure/data collection step can be repeated with all of the variables the same as the first time (e.g., same biosystem, same marker set, same experimental protocol, etc.). However, it may be necessary to vary the biosystem sampling (e.g., type of cell utilized, stage of cell growth), use a different marker set and/or change the experimental protocol in order to get differential expression/result..
  • Example 4 Using HBR Array Information.
  • the HBR Array information discussed herein can be used for many different purposes including, but not limited to, the following: 1) evaluating the components of an herbal composition; 2) predicting the BioResponse of an herbal composition; 3) determining which marker information is most highly correlated with a particular BioResponse of an herbal composition; 3) determining what data set of information
  • the HBR Array technology of the present invention is used to correlate or to determine a substantial equivalence of a specific batch of an herbal composition (single herb or multiple herbs of a formula) to a standardized, or master, batch of a same or substantial similar herbal composition.
  • the HBR Arrays utilized in this process include the acceptable range of quantitative variation for each of the biological effects (i.e., BioResponse), and possibly a global score composed of weighted values assigned to each of the biological effects, which may consist of markers from multiple biochemical pathways of a biosystem.
  • Data mining refers to a process used to determine or select which subset of biological effects is the minimum number of biological effects required in any specific HBR Array.
  • the information for data mining results from exposing a biosystem (e.g., a cell line) in a dose dependent manner to a standardized herbal composition to establish a standardized HBR Array.
  • This standardized HBR Array can then be compared to various HBR Arrays established for test herbal compositions.
  • test herbal compositions include, but are not limited to, different batches prepared at different dates; different batches prepared from raw herbs collected at different times; and different batches prepared from raw herbs collected at different locations.
  • Example 6 Improving an Herbal Composition or Identifying New Uses for an Herbal Composition.
  • HBR Arrays are generated by exposing biosystems to either extracts from individual herbs of a formula, or to extracts from the whole formula, and examining the biological effects of the extracts.
  • the observed biological effects can be from multiple biochemical pathways of a biosystem and/or from multiple tissues of an animal, wherein various markers are evaluated for their corresponding qualitative and/or quantitative changes.
  • the resulting HBR Arrays can be compared to novel HBR Arrays or to similar HBR Arrays from different herbal compositions or herbal compositions prepared by different processes. This procedure is useful for selecting a given set of biological effects .and the minimum number of markers required to predict that a given batch herbal composition has the given set of biological effects.
  • HBR Arrays In order to construct HBR Arrays, one skilled in the art utilizes various data mining tools including, but are not limited to, statistical analyses, artificial intelligence, and database research on neural work.
  • the statistical methods of choice include, but are not limited to, basic exploratory data analysis (EDA), graphic EDA (such as bushing) and multivariate exploratory techniques (e.g., cluster analysis, discriminating factor analyses, stepwise linear on non-linear regression, classification tree) (see, e.g., STATISTICATM, software packages from StatSoft, Tulsa, OK 74104; Tel: 918-749- 1119; Fax: 918-749-2217; www.statsoft.com).
  • EDA basic exploratory data analysis
  • graphic EDA such as bushing
  • multivariate exploratory techniques e.g., cluster analysis, discriminating factor analyses, stepwise linear on non-linear regression, classification tree
  • Data mining tools are used to explore large amounts of HBR Array data in search of constructing an HBR Array and consistent pattern within, between or among various HBR Arrays.
  • the procedure consists of exploration, construction of an HBR array, and validation. This procedure is typically repeated iteratively until a robust HBR Array, or standardized HBR Array, is identified.
  • Ginseng batches will first be characterized by geographic origin, species, plant part (e.g., rhizome, root, leaf skin, seed, bud and flower); growth conditions, processing methods and storage conditions both before and after processing.
  • plant part e.g., rhizome, root, leaf skin, seed, bud and flower
  • Verification of chemical content for these batches will be performed by qualitative HPLC analysis for determination of ginsenoside saponins (e.g., Ro, Ral, Ra2, Rbl, Rb2, Rb3, Re, Rgl, Rg2, Rd, Re, Rf, Rhl, Rh2, NG-R2 and Z-Rl), including TLC qualitative analysis for lipophilic constituents (see, Elkin et al, Chumg Kuo Yao Li Hsueh Pao (1993) 14: 97-100 and Yoshikawa et al, Yakugaku Zasshi (1993) 113: 460-467).
  • the saponin content of different herbs should be between 2.1 •and 20.6% (by weight) depending on the species (see Table 1). These data will then be stored, preferably in the memory of a computer processor, for further manipulation.
  • Expression biomarkers for standard ginseng include the following: IL-8, IL-2, GM-CSF, Nfi B, ICAM-1, interferon gamma, choline acetyl transferase, trk A, nerve growth factor (Kim et al, Planta Med (1998) 64: 110-115; Sonoda et al, Immunopharmacology (1998) 38: 287-294; Baum et al, Eur J Appl Physiol (1997) 76: 165-169; Iwangawa et al, Free Radic Biol Med (1998) 24: 1256-1268; Rhind et al, Eur J Appl Physiol (1996) 74: 348-360).
  • the 400,000 oligonucleotide group/1.6 cm 2 chip of Affimatrix can be used (U.S. Pat. No.5, 556,752).
  • the expression biomarkers for standard ginseng will be prepared by microarray technology of cDNA using either photolithography, mechanical microspotting or ink jet application (see Schena et al, TIBTECH (1998) 16: 301-306). Selected sets of cells will be contacted with standard ginseng for varying periods of time, under varying conditions to generate multiple microarray sets.
  • microarray sets will then be analyzed by hybridization-based expression monitoring of biochemical extracts via deduction of steady state mRNA levels from fluorescence intensity at each position on the microarrays (Schena et al, Science (1995) 270: 467- 470; Schena et al, Proc Natl Acad Sci USA (1996) 93: 10614-10619; Lockhart et al.
  • Biochemical biomarkers for standard ginseng include quantitative analysis for increases in cycloheximide sensitive [ 3 H] -leucine incorporation proportional to protein synthesis and [ 3 H]-thymidine incorporation reflective of mitosis, (see Yamamoto et al, Arzneiffenforschung (1977) 27: 1 169-1 173).
  • bone marrow cells will be contacted with standard ginseng for varying time periods under varying conditions in the presence of [ 3 H]-thymidine (for DNA synthesis) or in the presence and absence of cycloheximide and [ 3 H]-leucine (for protein synthesis) to perform multiple quantitative analysis of biochemical biomarkers (i.e., BBM sets).
  • BBM sets are then input into algorithms to generate statistical biochemical biomarker values for standard ginseng.
  • Statistical data will then be stored, preferably in the memory of a computer processor, for further manipulation.
  • Biological response of a biosystem i.e. , BioResponses
  • ginseng batches will be exposed to specific cell types, including, but not limited to, fibroblasts, macrophages, monocytes, PMNL, LAK cells, B16-F10 melanoma cells, THP-1 cells and hippocampal neurons at a concentration of 0.5 mg/ml to 100 mg/ml.
  • fibroblasts including, but not limited to, fibroblasts, macrophages, monocytes, PMNL, LAK cells, B16-F10 melanoma cells, THP-1 cells and hippocampal neurons at a concentration of 0.5 mg/ml to 100 mg/ml.
  • 0.5-100 mg/kg of ginseng herbal extract will be administered orally, by intraperitoneal injection or subcutaneous injection.
  • human ovarian cancer cells will be inoculated into nude mice, which results in the formation of palpable tumors. After tumor formation the mice will be treated by co- administration of cis-diamminecichloroplatinum and standard ginseng. Mice will be examined for tumor growth inhibition, increase in survival time and lowered adverse side-effects on hematocrit values and body weight (Nakata et al, Jpn J Cancer Res (1998) 89:733-740). The assay will be repeated using various concentrations of standard ginseng to generate measures of central tendency, dispersion and variability for each variable.
  • the data collected will then be subjected to multidimensional analysis to generate multivariant normal distribution sets as a means of determining a baseline correlation between biological activity and standard ginseng (see Zar, J. H., in
  • the resulting arrays display the highest correlation between composition (including growth conditions), biomarker information and biological response for standardized ginseng.
  • composition including growth conditions
  • biomarker information e.g., biomarker information values
  • the values for biological response variables can be predicted for the test batch by comparing test values against Standardized HBR Array values for standardized ginseng. The resulting prediction will be used to evaluate the quality of a given ginseng batch without necessitating the use of an observed biological response of a biosystem (see Example 2).
  • Example 8 Evaluation of a Selected Herbal Composition of Ginseng Using a Subset of Variables Correlated with a Specific Biological Response.
  • a test batch herbal composition To evaluate the quality of a test batch herbal composition, data is first collected concerning the plant-related parameters for the herbs in the selected herbal composition (e.g., plant species, plant parts, geographic origin, growth conditions, processing methods and storage conditions). The selected herbal composition is then manipulated such that chemical analysis can be performed to determine the chemical content of the herb (see Elkin et al, Chumg Kuo Yao Li Hsueh Pao (1993) 14: 97-100 and Yoshikawa et al, Yakugaku Zasshi (1993) 113: 460-467). Previously obtained ginseng data has demonstrated a strong correlation between oxygen consumption during aerobic exercise performance and the presence of a subset of saponin components, especially Rgl and Rbl (Wang et al, Planfa Med (1998) 64: 130-133).
  • saponin components especially Rgl and Rbl
  • test cells including, but not limited to, fibroblasts, macrophages, monocytes, PMNL, LAK cells, B16-F10 melanoma cells, THP-1 cells and hippocampal neurons at a concentration of 0.5 mg/ml to 100 mg/ml to determine expression biomarker values.
  • mRNA is isolated from exposed cells which is subsequently manipulated to serve as a substrate for hybridization-based expression monitoring of biochemical extracts using microarrays comprising IL-8, IL-2 and Interferon gamma cDNA (Schena et al, Science (1995) 270: 467-470; Schena et al, Proc Natl Acad Sci USA (1996) 93: 10614-10619; Lockhart et al, Nat Biotechnol (1996) 14: 1675-1680; DeRisi et al, Nat Genet (1996) 14: 457-460; Heller et al, Proc Natl Acad Sci USA (1997) 94: 2150-2155).
  • Previously obtained ginseng data has demonstrated a strong correlation between oxygen consumption during aerobic exercise performance and the induction of the expression biomarkers IL-8, IL-2 and Interferon gamma in test cells (Venkatraman et al, Med Sci Sports Exerc (1997) 29: 333-344 and Wang et al, Planta Med (1998) 64: 130-133).
  • biomarkers IL-8, IL-2 and Interferon gamma for biochemical biomarkers, rat bone marrow cells will then be exposed to the test batch and assayed for [ 3 H]-thymidine incorporation reflective of mitosis.
  • Previously obtained ginseng data has demonstrated that Rbl and Rgl show a strong correlation with DNA synthesis in rat bone marrow cells (Yamamoto et al, Arzneiffenforschung (1978) 28: 2238- 2241).
  • test HBR array for the selected herbal composition based on the enumerated plant-related data, including chemical analyses, and data concerning the subset of biomarkers.
  • the quality of a test batch will be determined by comparing test HBR and standard ginseng Standardized HBR Array variables directed toward analysis of the above observations and subsets, wherein the demonstration of the induction of IL-2, IL-8 and INF gamma mRNA in vitro and an increase in [ 3 H]-thymidine incorporation in rat bone marrow cells (including data collected on growth conditions, origin, and verification of the saponins Rgl and Rbl) is predictive of an equivalent BioResponse effect of the test batch on oxygen consumption as that exhibited by standard ginseng. Based on this procedure it can be determined whether or not the test batch is of a similar or different quality than that of the standard for the given biological response or biological response of interest.
  • HL huang ling
  • standard huang ling is chosen to be Coptis chinesis France, from southwest Asia, wherein growth conditions are well known to one skilled in the art (see Huang in The Pharmacology of Chinese Herbs. (1993), pp 69 and 287-288, CRC Press, Boca Raton, FL).
  • Dried rhizomes of Coptis chinesis France will be verified for chemical content by quantitative chemical analysis for determination arsenic, berberine, caeruleic acid, columbamine, copsine, coptine, coptiside-I, coptiside-II, coptisine, coreximine, epiberberine, ferulic acid, greenlandicine, isocoptisine, lumicaerulic acid, magnoflorine, oxybererine, thalifendine, umbellatine, urbenine, worenine, palmatine, jatrorrhizine and colubamine
  • Expression biomarkers for standard HL include the following: Nfi B; bcl-2 analog, Al; zinc fmger protein, A20; IL-2 receptor; cell cycle probes; c-Ki-ras2; growth regulators probes and glucocorticoid receptor dependent apoptosis probes (see Chi et al, Life Sci (1994) 54: 2099-2107; Yang et al, Naunyn Schmiedebergs Arch Pharmacol (1996) 354: 102-108; Miura et al., Biochem Pharmacol (1997) 53; Chang K.S., J Formos Med Assoc (1991) 90: 10-14).
  • the 400,000 oligonucleotide group/1.6 cm 2 chip of Affimatrix can be used (U.S. Pat.
  • Biochemical biomarkers for standard HL include increase in glucocorticoid receptor and inhibition of alpha- fetoprotein secretion in HL exposed HepG2 cells (see Chi et al, Life Sci (1994) 54: 2099-2107).
  • BBM sets are generated and analyzed as described in Example 1.
  • Statistical data will then be stored, preferably in the memory of a computer processor, for further manipulation.
  • Biological response of a biosystem will be determined using cells and whole animals. Batches of the selected herbal composition will be exposed to specific cell types, including but not limited to, human HepG2 hepatoma cells, human embryonal carcinoma cells and thymocytes at concentrations from 0.1-lOOmg/ml. For animal treatments 0.1mg-2g/kg of Coptic herbal composition (i.e., HL) will be administered orally, by intraperitoneal injection or subcutaneous injection.
  • Coptic herbal composition i.e., HL
  • HL e.g. 2g/kg
  • stool volumes will be determined (see, e.g., Rabbani G.H., Dan Med Bull (1996) 43: 173-185).
  • the assay will be repeated to generate measures and analysis will be performed as described for ginseng in Example 1.
  • the distribution sets for each biological system are then put into algorithms to generate statistical values for standard HL.
  • Statistical data will then be stored, preferably in the memory of a computer processor, for further manipulation.
  • HBR arrays for standardized HL, wherein the resulting HBR arrays will then be used to predict biological activity and evaluate batch quality.
  • a Standardized HBR Array can be generated and updated periodically.
  • Example 10 Evaluation of a Selected Herbal Composition of Huang Ling Using a Subset of Variables Correlated with a Specific Biological Response.
  • test batch is then exposed to test cells including human embryonal carcinoma clone, NT2/D1 at a concentration starting at a non-toxic concentration (determination of which is within the skill of the ordinary artisan).
  • mRNA is isolated from exposed cells which is subsequently manipulated to serve as substrate for hybridization based expression monitoring of biochemical extracts using microarrays comprising IL-2 receptor and Nfi B; (see Chi et al, Life Sci (1994) 54: 2099-2107; Yang et al, Naunyn Schmiedehergs Arch Pharmacol (1996) 354: 102-108; Miura et al., Biochem Pharmacol (1997) 53; Chang K.S., J Formos Med Assoc (1991) 90: 10-14; U.S. Pat. No.5, 556,752), and which can be used to determine down regulation of c-Ki-ras2 gene expression in said cells.
  • HL data has demonstrated terminal differentiation of human embryonal carcinoma clones into neuronal-like cells is strongly correlated with induction of mitogen probes and down regulation of c-Ki-ras2 gene expression (see Chang K.S., J Formos Med Assoc (1991) 90: 10-14).
  • HepG2 cells are exposed to the test composition and cells are assayed for increase in glucocorticoid receptor and inhibition of alpha- fetoprotein secretion (see Chi et al, Life Sci (1994) 54: 2099-2107).
  • test HBR test HBR
  • standard HL HBR Array variables directed toward analysis of the above observations and subsets, wherein the demonstration of the induction of IL-2 receptor and NficB, the down regulation of c-Ki-ras2 gene expression, an increase in glucocorticoid receptor and inhibition of alpha- fetoprotein secretion for HepG2 cells (to including data collected on growth conditions, origin, and verification of berberine alkaloid) is predictive of an equivalent BioResponse effect of the test batch on terminal differentiation of human embryonal carcinoma clones into neuronal-like cells and inhibition of dexamethasone induced apoptosis as that exhibited by standard HL. Based on this procedure it can be determined whether or not the test batch is of a similar or different quality than that of the standard.
  • the Xiao Chai Hu Tang composition is made from a mixture of 6-7 herbal plants (Radix Bupeuri, Rhizoma Pinelliae, Rhizoma Zingiberis, Radix Scutellariae, Fructus Ziziphi, Codonopsis Pilosula, Radix Ginseng and Radix Glycyrrhizae, see Table 2 for relative amounts, by weight). Table 2. Composition of Xiao Chai Hu Tang.
  • the three "recipes” originate in either Singapore, Korea or Taiwan. Batches were evaluated for toxicity and for the ability to inhibit hepatitis B virus as detected by DNA quantitation or detection of hepatitis B surface antigen (HbsAg) (see Dong et al, Proc Natl Acad Sci USA (1991) 88: 8495-8499).
  • HbsAg hepatitis B surface antigen
  • the data presented in Tables 2 and 3 for the Taiwan herbal composition constitute the initial data for the standardized HBR Array for this herbal composition. Therefore, this data set would initially include the source of the herbal composition, the plant species and relative amounts of each the herbal compostion, and two BioResponses (i.e., cell growth inhibition and hepatitis B virus secretion from infected cells). Using the procedures set forth in the schematic of Figure 1 and in Examples 1 and 3, additional data can be collected on plant-related data, markers and BioResponses for the standard herbal composition. This additional data is added to the initial standardized HBR Array to generate an expanded standardized HBR Array.
  • the standardized protocol for the herbal extract preparation was as follows: The ingredients of herbal raw materials with proper ratios were placed in a jacketed reactor and extracted with water at an elevated constant temperature with mixing. The solid was separated from the liquid with a 120-mesh screen. The resultant filtrate was collected and then concentrated by evaporating the water under reduced pressure. The concentrated liquor was spray dried at elevated temperature to yield granulated powder. This bulk substance was then formulated into the desired dosage form.
  • Huang Qing Tang is an ancient Chinese botanical formula composed of four distinct herbs: Scutellariae (scute), Glycyrrhizae (licorice), Paeonie lactiflora pallus (white peony root), and Fructus zizipho (date). (Table 4). This herbal formula has been long used in Asia to treat a variety of gastrointestinal ailments since 300 AD. Table 4. Herbal Ingredients of TCM Formula HQT
  • HQT Two cell types were used to test for biological effects of each batch of HQT: a) Jurkat T cells (ATCC cat #TIB-152) and b) HepG2 cells (ATCC cat # HB-8065). One to fifty dilutions were used for each assay. Frozen cells (10 7 /ml) were quickly thawed in a water bath at 37 °C. The cells were then diluted in 10 ml of pre-warmed media (see Life Technologies, Inc., Catalogue and Reference Guide, 1998-1999, Cell Culture section) followed by centrifugation at 1500 rpm for 5 min. The supernatant was then discarded and the cells were cultured in 100 ml media at 37 °C, 5% CO 2 . After 2 days, the cells were counted (approximately 8 x lOVml, total 100 ml).
  • HQT HQT is known for its anti-diarrhea properties. Different HQT extracts were added to triplicate wells of a 96-well plate which contained O.lmM phenolphthalein glucuronidate, 70 mM Tris-HCl (pH 6.8) and 0.8 ng of dialyzed ⁇ -glucuronidase (from E. Coli, purchased from SigmaTM) to a final volume of 80 ⁇ l.
  • HQT sources A and B have relatively low toxicities combined with higher inhibitory activity relative to batch HQT C (i.e., approximately 5 fold greater toxicity toward HepG2 cells and 3.3 fold less inhibitory activity against ⁇ - glucuronidase than either HQT A or B, see Tabl 6).
  • Table 6 Biological Assay of Three Preparations of HQT"
  • HepG2 cells (1 x 10 6 ) were seeded in 25 cm 2 flasks in 3.0 ml of RPMI-1640 medium (see Life Technologies, Inc., Catalogue and Reference Guide, 1998-1999, Cell Culture section) 24 hr before the drug addition.
  • the cells were treated with or without herbal medicine, where the former is added at two final concentrations of 0.2 mg/ml or 4 mg/ml, respectively, and incubated at 37°C for 24 hours. The medium was removed and the cells were washed twice with cold PBS.
  • the cells were harvested into 1 ml of PBS and centrifuged at 10,000 rpm for 2 minutes, extracted on ice with a buffer containing 50 mM Tris-Cl (pH 7.5), 0.2 mM PMSF and 10% glycerol, followed by three freeze-thaw cycles. Potassium chloride was added to the cell lysate at a final concentration of 0.15 M prior to centrifugation. The protein concentration was determined and the cell extract was electrophoresed according to the method of Laemmli (Nature (1970) 227:680-685). Western blots were performed by standard techniques known in the art, see for example Sambrook, et al (1989). The antibodies used were directed to the following proteins: Topo I; Stat (20707); Cyclin Bl; MAPK (Ab2) and Nm 23 Hl.
  • Figure 4 demonstrates that the higher concentrations of HQT A or HQT B differentially effects the expression of cyclin Bl polypeptide.
  • the herbal batches were analyzed by HPLC with a Beckman ODS UltrasphereTM column (5 micron particles, 4.6 mm X 25 cm) and detected with a UN spectrophotometer (Perkin Elmer). The wavelengths for UN detection were monitored at 280 nm and 340 nm. The mobile phase was pumped at 1 ml/min and consisted of
  • Solvent A H 2 O and Solvent B: 20% MeOH with the following gradient: 1) the solvent was 100% solvent A for the first 5 minutes; 2) the solvent composition was changed to 10% solvent A / 90% solvent B for the next 10 minutes; and 3) the solvent was changed to 10% solvent A / 90% solvent B for the next 40 minutes. This was followed by the addition of 100 % solvent A for 5 minutes.
  • the HPLC markers are baicalin and baicalein.
  • the herbal extract was analyzed by MarinerTM ESI-TOF Mass Spectrometry (MS) from PE Biosystems. Control tracings were generated using baicalein and baicalin, two known active ingredients in HQT.
  • Example 14 Individual components A. Licorice- Licorice is useful for moistening the lungs and reducing coughs, helps to relax spasm and pain.
  • the properties of the licorice batches used in this example are presented in Table 7.
  • each herbal extract supernatant was assayed and the analysis was repeated three times.
  • 1 gram of herbal powder was dissolved in 10 ml of 80° C deionized water (neutral pH) in a polypropylene tube. The tube was then incubated as outlined in Table 7, then centrifuged to obtain the supernatant. Batches of licorice were tested against either HepG2 cells (ATCC cat # HB-8065) or Jurkat T cells (ATCC cat #TIB-152) or both. Cells were cultured for 24 hours as described above.
  • ⁇ -glucuronidase was assayed.
  • Different licorice extracts were added to triplicate wells of a 96-well plate which contained O.lmM phenolphthalein glucuronidate, 70 mM Tris-HCl (pH 6.8) and 0.8 ng of dialyzed beta-glucuronidase (from E. Coli, purchased from Sigma) to a final volume of 80 ⁇ l and assayed as above.
  • ⁇ Values represent IC 50 %, % of Control values.
  • Jurkat T cells were treated with herbal extract as follows: Jurkat cells (10 7 /ml) were quickly thawed in a water bath at 37 °C. The cells were then diluted in 10 ml of pre-warmed media (see Life Technologies, Inc., Catalogue and Reference Guide, 1998-1999, Cell Culture section) followed by centrifugation at 1500 rpm for 5 min. The supernatant was then discarded and the cells were cultured in 100 ml media at 37 °C, 5% CO 2 . After 2 days, the cells were counted (approximately 8 x lOVml, total 100 ml).
  • the herbal extract solution was prepared as outlined above (e.g., 2 g of an herbal powder to obtain 20 ml of sterile solution (0.1 g/ml).
  • the cells were divided into 3 flasks at a density of 2.5 x 10 5 /ml, 100 ml/each flask. Assays were carried out with control (no extract), and 10 ml of extract at 10 mg/ml, and 1 mg/ml. Again, toxicity results were used to determine the "high” and "low” concentrations for any given extract.
  • cell cultures were incubated for 24 hours under conditions as outlined above. The cells were counted and subsequently collected in 50 ml centrifuge tubes. The resulting cell pellet was treated with an RNA isolation means to extract mRNA (see, for example, Sambrook et al, 1989 at pages 7.3-7.39).
  • Microarray Microarray printing was carried out as follows:
  • mRNA was isolated as outlined above
  • the labeled samples were treated with alkali and the resulting labeled nucleic acids were precipitated prior to use in hybridization.
  • Membrane hybridization and washing were carried out using the labeled probes as disclosed in Chen et al. (1998).
  • ⁇ - galactosidase-conjugated streptavidin Strept-Gal
  • alkaline phosphatase- conjugated digoxigenin antibody anti-Dig- AP
  • image digitization using a imaging means was employed (e.g., a flatbed scanner or digital camera). Quantitative measurements were determined by computer analysis which uses a program that measures the integrated density of the primary color components of each spot, performs regression analysis of the integrated density data and locates statistical outliers as differentially expressed genes.
  • the extracts were prepared as outlined in Example 6.
  • the cells were divided into 24 well culture plates by adding 1 ml of Jurkat cells at a density of 5 x lOVml.
  • Assays were carried out with control (no extract), and 5 concentrations of extracts as described (see Table 9).
  • the high and low concentrations for the cell culture assays were varied between 10 mg/ml and 0.05 mg ml (i.e., mg dry weight of herbal extract per ml) depending on the toxicity of the extract to cells.
  • the toxicities at 10 mg/ml were such that "high” and "low” concentrations were adjusted downward, nevertheless, at least one order of magnitude between extremes was maintained.
  • onginal cell number is 5xl0 5 /ml and the number to lOxlOVml after 24h incubation. "-" desc ⁇ bes all dead cells.
  • Test 5 concentration per sample 10 mg/ml, 5 mg/ml, 2 mg/ml, 1 mg/ml, 0.5 mg/ml
  • HLA-A HLA-A28.-B40, -Cw3
  • HLA-A28.-B40, -Cw3 MHC class I protein
  • Homo sapiens androgen receptor associated protein 24 (A 60 23 4787 3496 3741 984 44 8 465 0794787 0580442 0621119 0163374 0743815 0772041 118235
  • Ribosomal protein L5 2615 336 263 5586 4859 3126 4329 1284895 1005736 2136138 1858126 1195411 1655449 511410
  • beta 1 f ⁇ bronectin receptor, beta polypeptide, an 0 092 0 413 184 667 1118 547624
  • the herbal batches were analyzed by HPLC with a Beckman ODS UltrasphereTM column (5 micron particles, 4.6 mm X 25 cm) and detected with a UN spectrophotometer (Perkin Elmer). The wavelengths for UN detection were monitored at 280 nm and 340 nm. The mobile phase was pumped at 1 ml/min and consisted of
  • Solvent A H 2 O and Solvent B: 20% MeOH with the following gradient: 1) the solvent was 100% solvent A for the first 5 minutes; 2) the solvent composition was changed to 10% solvent A / 90% solvent B for the next 10 minutes; and 3) the solvent was changed to 10% solvent A / 90% solvent B for the next 40 minutes. This was followed by the addition of 100 % solvent A for 5 minutes.
  • the HPLC marker is glycyrrhizin.
  • Scute has been found to be useful in reducing capillary permeability and inflammation. It can also be used treat enteritis and dysentery, increases the secretion of bile to treat jaundice; to relieve muscle spasms; to treat coughing and to expel parasites.
  • enteritis and dysentery increases the secretion of bile to treat jaundice; to relieve muscle spasms; to treat coughing and to expel parasites.
  • the properties of the scute batches used in this example are presented in Table 11.
  • ⁇ -glucuronidase For ⁇ -glucuronidase, different scute extracts were added to triplicate wells of a 96-well plate which contained O.lmM phenolphthalein glucuronidate, 70 mM Tris- HC1 (pH 6.8) and 0.8 ng of dialyzed ⁇ -glucuronidase (from E.Coli, purchased from Sigma) to a final volume of 80 ⁇ l. After 2 hr incubation at 37°C, the reactions were terminated with 200 ⁇ l of stopping solution which contained 0.2 M Glycine and 0.2 M NaCl (pH 10.4), and the OD was monitored with a kinetic microplate reader at 540 nm.
  • HepG2 cells (1 x 10 6 ) were seeded in 25 cm 2 flasks in 3.0 ml of RPMI-1640 medium (see Life Technologies, Inc., Catalogue and Reference Guide, 1998-1999, Cell Culture section) 24 hr before the extract addition.
  • the cells were treated with or without herbal medicine, where the former is added at two final concentrations of 0.2 mg/ml or 4 mg/ml, respectively, and incubated at 37 °C for 24 hours. The medium was removed and the cells were washed twice with cold PBS.
  • the cells were harvested into 1 ml of PBS and centrifuged at 10,000 rpm for 2 minutes, extracted on ice with a buffer containing 50 mM Tris-Cl (pH 7.5), 0.2 mM PMSF and 10% glycerol, followed by three freeze-thaw cycles. Potassium chloride was added to the cell lysate at a final concentration of 0.15 M prior to centrifugation. The protein concentration was determined and the cell extract was electrophoresed according to the method of Laemmli U.K. (Nature (1970) 227:680-685). Western blots were performed by standard techniques known in the art, see for example Sambrook, et al (1989). The antibodies used were directed to the following proteins: Topo I; Stat (20707); Cyclin Bl ; MAPK (Ab2) and Nm 23 HI .
  • Figure 4 demonstrates that scute batches A and B do not differentially affect the expression of the polypeptides resolved on Western blots.
  • the herbal batches were analyzed by HPLC with a Beckman ODS UltrasphereTM column (5 micron particles, 4.6 mm X 25 cm) and detected with a UN spectrophotometer (Perkin Elmer). The wavelengths for UN detection were monitored at 280 nm and 340 nm.
  • the mobile phase was pumped at 1 ml/min and consisted of Solvent A: H 2 O and Solvent B: 20% MeOH with the following gradient: 1) the solvent was 100% solvent A for the first 5 minutes; 2) the solvent composition was changed to 10% solvent A / 90% solvent B for the next 10 minutes; and 3) the solvent was changed to 10% solvent A / 90% solvent B for the next 40 minutes. This was followed by the addition of 100 % solvent A for 5 minutes.
  • the HPLC markers are baicalin and baicalein.
  • Peony is used to suppress and soothe pain. It is also known to soothe ligaments and purify the blood. The properties of the peony batches used in this example are presented in Table 13.
  • the herbal batches were analyzed by HPLC with a Beckman ODS Ultrasphere column (5 micron particles, 4.6 mm X 25 cm) and detected with a UN spectrophotometer (Perkin Elmer). The wavelengths for UN detection were monitored at 280 nm and 340 nm.
  • the mobile phase was pumped at 1 ml/min and consisted of Solvent A: H 2 O and Solvent B: 20% MeOH with the following gradient: 1) the solvent was 100% solvent A for the first 5 minutes; 2) the solvent composition was changed to 10%o solvent A / 90% solvent B for the next 10 minutes; and 3) the solvent was changed to 10% solvent A / 90% solvent B for the next 40 minutes. This was followed by the addition of 100 % solvent A for 5 minutes.
  • HPLC marker is paeoniflorin. Peony batches were analyzed by HPLC as shown in Figure 5.
  • the data collected form part of the multidimensional analysis used to generate multivariant normal distribution sets as a means of determining a baseline correlation between biological activity and standard peony chemical (HPLC), and origin growth characteristics.
  • HPLC peony chemical
  • Date has been used for diuretic properties and strengthening effects.
  • the properties of the date batches used in this example are presented in Table 15.
  • the herbal batches were analyzed by HPLC with a Beckman ODS Ultrasphere column (5 micron particles, 4.6 mm X 25 cm) and detected with a UN spectrophotometer (Perkin Elmer). The wavelengths for UN detection were monitored at 280 nm and 340 nm. The mobile phase was pumped at 1 ml/min and consist of
  • Solvent A H 2 O and Solvent B: 20% MeOH with the following gradient: 1) the solvent was 100% solvent A for the first 5 minutes; 2) the solvent composition was changed to 10% solvent A / 90% solvent B for the next 10 minutes; and 3) the solvent was changed to 10% solvent A / 90% solvent B for the next 40 minutes. This was followed by the addition of 100 % solvent A for 5 minutes.
  • HPLC markers for date are chelidonic acid and cAMP.
  • the data collected form part of the multidimensional analysis used to generate multivariant normal distribution sets as a means of determining a baseline correlation between biological activity and standard peony chemical (HPLC), and origin/growth characteristics.
  • HPLC peony chemical

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Zoology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Engineering & Computer Science (AREA)
  • Wood Science & Technology (AREA)
  • Analytical Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Immunology (AREA)
  • Biotechnology (AREA)
  • Biophysics (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Physics & Mathematics (AREA)
  • Medicines Containing Plant Substances (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Investigating Or Analysing Biological Materials (AREA)
EP99971046A 1998-10-23 1999-10-22 Phytomics: ein auf genomics basierender ansatz für pflanliche wirkstoffzummensetzungen Withdrawn EP1124991A1 (de)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US10543598P 1998-10-23 1998-10-23
US105435P 1998-10-23
PCT/US1999/024851 WO2000024934A1 (en) 1998-10-23 1999-10-22 Phytomics: a genomic-based approach to herbal compositions

Publications (1)

Publication Number Publication Date
EP1124991A1 true EP1124991A1 (de) 2001-08-22

Family

ID=22305836

Family Applications (1)

Application Number Title Priority Date Filing Date
EP99971046A Withdrawn EP1124991A1 (de) 1998-10-23 1999-10-22 Phytomics: ein auf genomics basierender ansatz für pflanliche wirkstoffzummensetzungen

Country Status (10)

Country Link
EP (1) EP1124991A1 (de)
JP (1) JP2003521226A (de)
KR (1) KR100470379B1 (de)
CN (1) CN1335892A (de)
AU (1) AU779424B2 (de)
CA (1) CA2346768A1 (de)
DE (1) DE1124991T1 (de)
ES (1) ES2171371T1 (de)
HK (1) HK1039964A1 (de)
WO (1) WO2000024934A1 (de)

Families Citing this family (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001066803A2 (en) * 2000-03-09 2001-09-13 Yale University Phytomics: a genomic-based approach to herbal compositions
JP2006000113A (ja) * 2004-05-20 2006-01-05 Masatomo Sakurai 漢方医療の診断に用いる遺伝子およびその利用方法
KR102680200B1 (ko) * 2016-06-22 2024-07-02 예일 유니버시티 식물 의약을 위한 메커니즘 기반 품질 관리
CN108841928B (zh) * 2018-06-11 2021-11-23 河北医科大学 一种人类线粒体基因组甲基化检测试剂盒及其应用
CN110646497B (zh) * 2018-06-27 2021-10-29 中国科学院沈阳应用生态研究所 一种通过稳定氮同位素比值鉴定有机农产品的方法
CN110907580B (zh) * 2019-12-20 2022-02-25 厦门中药厂有限公司 一种保元汤的hplc特征图谱的检测方法
CN112305133B (zh) * 2020-12-25 2022-05-24 成都普思生物科技股份有限公司 一种测定天葵子中唐松草酚定含量的方法
CN112575005A (zh) * 2021-01-04 2021-03-30 昆明理工大学 一种提高烟草重金属镉胁迫抗性和降低镉富集的方法
CN114864016A (zh) * 2022-03-25 2022-08-05 广西壮族自治区药用植物园 一种药用植物化合物信息数据库建立方法及系统
JP7654293B1 (ja) * 2024-06-17 2025-04-01 バイオ － セラピー、フィジオロジカル システムズ フォー ヘルス ソシエタ ペル アチオニ 天然の治療用の製品又は有益な製品がその治療的な効果又は有益な効果を生理的な作用機構を介して発揮する時期を決定するための方法

Family Cites Families (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997027317A1 (en) * 1996-01-23 1997-07-31 Affymetrix, Inc. Nucleic acid analysis techniques
US5569588A (en) * 1995-08-09 1996-10-29 The Regents Of The University Of California Methods for drug screening
US5777888A (en) * 1995-08-09 1998-07-07 Regents Of The University Of California Systems for generating and analyzing stimulus-response output signal matrices
WO1997013877A1 (en) * 1995-10-12 1997-04-17 Lynx Therapeutics, Inc. Measurement of gene expression profiles in toxicity determination
WO2000003725A1 (en) * 1998-07-14 2000-01-27 Paracelsian, Inc. Method for identifying and confirming consistent bio-functionality of natural compositions
US6780596B2 (en) * 1998-09-17 2004-08-24 Ashni Naturaceuticals, Inc. Methods for determining the activity of complex mixtures

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO0024934A1 *

Also Published As

Publication number Publication date
KR100470379B1 (ko) 2005-02-07
JP2003521226A (ja) 2003-07-15
AU1224900A (en) 2000-05-15
CN1335892A (zh) 2002-02-13
WO2000024934A1 (en) 2000-05-04
CA2346768A1 (en) 2000-05-04
KR20010099741A (ko) 2001-11-09
ES2171371T1 (es) 2002-09-16
HK1039964A1 (zh) 2002-05-17
AU779424B2 (en) 2005-01-27
DE1124991T1 (de) 2002-08-22

Similar Documents

Publication Publication Date Title
Yadav et al. Recent approaches in herbal drug standardization
US20030207270A1 (en) Phytomics: a genomic-based approach to herbal compositions
TW200300231A (en) Matrix methods for quantitatively analyzing and assessing the properties of botanical samples
Smelcerovic et al. Phytochemical analysis and genetic characterization of six Hypericum species from Serbia
US11896639B2 (en) Mechanism based quality control for botanical medicine
Wang et al. Modulatory effects of Echinacea purpurea extracts on human dendritic cells: a cell-and gene-based study
Wu et al. Accumulation and biosynthesis of hydroxyl-α-sanshool in varieties of Zanthoxylum bungeanum Maxim. by HPLC-fingerprint and transcriptome analyses
AU779424B2 (en) Phytomics: a genomic-based approach to herbal compositions
Wang et al. In vivo and in vitro study on the regulatory mechanism of XiaoChaiHu decoction on PANoptosis in sepsis-induced cardiomyopathy
Gao et al. De novo transcriptome assembly and metabolomic analysis of three tissue types in Cinnamomum cassia
AU4556501A (en) Phytomics: a genomic-based approach to herbal compositions
Long et al. Differential expression of skeletal muscle mitochondrial proteins in yak, dzo, and cattle: a proteomics-based study
Wen et al. Correlation analysis between genetic and chemical differences of Nardostachys jatamansi from different habitats in Ganzi Tibetan Autonomous Prefecture, Sichuan Province, China
Cho et al. Combination of UHPLC-MS/MS with context-specific network and cheminformatic approaches for identifying bioactivities and active components of propolis
AU2005201751A1 (en) Phytomics: a genomic-based approach to herbal compositions
TWI243210B (en) A quality control method for assessing herbal compositions
Antoshechkin et al. Analysis of effects of the herbal preparation Circulat on gene expression levels in cultured human fibroblasts
Jiang et al. Genetic Diversity and Population Structure of Traditional Chinese Herb “Chai-Hu” Resources Using Genome-Wide SNPs Through Genotyping-By-Sequencing
Zhang et al. Gel-forming Mucins in Oviductus Ranae Contribute to Swelling Capacity by iTRAQ Proteomics Analysis.
Olarte Guasca Array based genotyping of medicinal plants from the genera Salvia and Echinacea
Zhang et al. Elucidating the molecular biosynthetic mechanism of phenylethanoid glycosides in two new varieties of Forsythiae Fructus via a multi-omics analysis combined with WGCNA
Oseni et al. DNA Barcode Authentication and Improvement of Andrographolide Yield in Andrographis paniculata Plant
Lurie et al. From dirt to data: plant extraction modalities and techniques
HAKAMATSUKA et al. Screening for bioactive compounds targeting the cellular signal transduction pathway using an RT-PCR-based bioassay system
Javeria et al. Medicinal Plants and Molecular Techniques

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 20010510

AK Designated contracting states

Kind code of ref document: A1

Designated state(s): AT BE CH CY DE DK ES FI FR GB GR IE IT LI LU MC NL PT SE

AX Request for extension of the european patent

Free format text: AL;LT;LV;MK;RO;SI

TCAT At: translation of patent claims filed
TCNL Nl: translation of patent claims filed
EL Fr: translation of claims filed
DET De: translation of patent claims
REG Reference to a national code

Ref country code: ES

Ref legal event code: BA2A

Ref document number: 2171371

Country of ref document: ES

Kind code of ref document: T1

17Q First examination report despatched

Effective date: 20020812

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE APPLICATION IS DEEMED TO BE WITHDRAWN

18D Application deemed to be withdrawn

Effective date: 20060502

REG Reference to a national code

Ref country code: HK

Ref legal event code: WD

Ref document number: 1039964

Country of ref document: HK