JP2001521876A - Pharmaceutical grade medicinal carrot - Google Patents

Abstract

(57) Abstract The present invention relates to a common medicinal carrot material and a method for converting the material into a pharmaceutically acceptable and pharmaceutically acceptable form. In particular, the present invention uses compositional and active fingerprints in the treatment of medicinal carrot materials and is suitable for use in clinical or veterinary settings for the purpose of treating and / or reversing a disease, disorder or condition. Preparing a compound that can be identified as a certain pharmaceutical grade compound.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001] This application is a pending co-pending US patent application Ser.
/ 956,616 (the entire disclosure of which is incorporated herein by reference), which is part of concurrently pending U.S. Patent Application Serial No. 08 / 838,198, filed April 15, 1997. Continuation, which is ongoing at 1 April 1996
5 is a continuation-in-part of US Patent Application Serial No. 08 / 632,273,
Is a continuation-in-part of US Patent Application Serial No. 08 / 421,993, filed April 14, 5
U.S. Patent Application Serial No. 08 / 774,550, filed February 4, 997, has been abandoned.

[0002] 1. TECHNICAL FIELD The present invention relates generally to plant materials and methods of converting the materials to therapeutically useful and pharmaceutically acceptable forms. More particularly, the present invention relates to the use of compositional and active fingerprints in the processing of medicinal carrots, wherein said pharmaceutical grade composition is suitable for use in a clinical setting to treat and / or ameliorate diseases, disorders and / or conditions. Pertains to the use of making botanicals defined as:

[0003] 2. BACKGROUND ART Pharmaceutical manufacturing is based on the modulation of composition and biological activity for each manufacturing batch. This standardization and adjustment provides a material that can be remanufactured in predictable and consistent patient treatment. There are unique problems with herbs arising from plant material, which are the adjustments, reproducibility, and standardization desired by the manufacturer, which are necessary for medicine. This problem is primarily due to the multiple components contained in the herbs, and the diverse composition and efficacy is due to the increase, recovery and processing of the raw material.

[0004] Plants have always been the source of various pharmaceutical compounds. For centuries,
Various types of plant derived materials have been used to treat myriad different diseases. The botanical material is typically in powder form made from one or more plants or plant parts, or an extract obtained from whole plants or selected plant parts. These powders and extracts are most often complex mixtures of both biologically active and biologically inactive compounds.

[0005] While plant powders and extracts are widely used for pharmaceutical purposes, their use as pharmaceuticals has many problems. For example, the complex chemistry of plant materials makes the use of plant materials difficult in any type of control and predictable method. Possible variations in the chemical composition of different batches of material obtained from another plant harvest render the material unsuitable for clinical use.

As a good feature, multiple groupings of bioactive ingredients have typically been found in plant materials with potential for synergistic or additional bioactivity profiles. However, these possible increases in medical effect are unpredictable because the nature of these composite materials is not known.

[0007] The above problems with the inherent chemical complexity of medicinal herbs require a great deal of effort aimed at separating and isolating biologically active ingredients from many medically important plant materials. . Efforts in this field are rapidly expanding with many improvements in chemical separation and analytical techniques. Once isolated and purified, the various active ingredients can be used in clinical settings to establish the medicinal effect of a particular ingredient. Separation and purification of the individual components from plant material is central to this type of drug development method. Once purified,
The suspended active ingredient is typically mixed with a pharmaceutically acceptable carrier, for further study in research animals and finally for clinical trials in humans. In proving clinical efficacy,
These types of drugs are considered pharmaceutical grade. Single or in most cases
This is because a small number of well-characterized compounds contain known amounts of compounds.

[0008] Pharmaceutical grade drugs are advantageous in that the effects of individual compounds can be carefully tracked in treatment protocols. In addition, the drug dose can be adjusted with care,
Provides relatively predictable medicinal effects. The disadvantage of the relative purity of the pharmaceutical grade drug is that the potential for complex and co-biological activity provided by naturally occurring plant material is reduced due to the isolation of the drug from the natural environment. is there. Studies of isolated products are also artifacts created by disruption of sensitive biological / botanical complexes. The potential benefits offered by its joint activity are believed by many industry experts to be more important than the clinical risks associated with the use of poorly characterized or uncontrolled complex plant materials in clinical settings. ing.

Although the isolation and purification of single compounds from plant material is a common type in drug research and development, the study of complex plant extracts characterized by pharmaceutical quality is also of interest. ing. There are many complex plant materials and extracts, which may be medicinal ingredients, but are relatively unpredictable. These materials are in most cases useless in clinical settings. It is because batch consistency has not been established,
Because of the inherent risks involved in treating patients with poorly characterized materials that vary widely in composition. Therefore, there is a need to provide a method for standardizing complex plant material for effective use in clinical research and patient treatment.

2.1. Background of Medicinal Carrots Medicinal carrots are dry roots of Asian or American medicinal carrots, both of the medicinal carrot family. Panax ginseng is also known as Korean carrot. Wild medicinal carrots are rare, but are widely grown in China and Korea. Medicinal carrots are said to be "the most stress effective in the world" (Mowrey, 1990, Next Generation Herbal Medicine,
Keats Publishing, New Canaan, CT), has also been used as a libido stimulant in China for centuries. Although the use of medicinal carrots is well documented, medicinal carrots are generally popular for enhancing physical strength, including physical and psychological abilities. Contraindications,
No known side effects or interactions with other drugs. The recommended dose is 1
One to two grams of root per day, tea is prepared from 1.75 g of medicine and is taken once or twice daily. Other capsules containing 250 mg of root may also be used (T
yler, 1994, Herbs of Choice, Haworth Press, NY).

The first use of medicinal carrots is for nervous and cardiovascular effects.
Medicinal carrots are thought to be effective in promoting mental activity and intellectual performance, especially productivity and accuracy (Fulder, 1984, About Ginseng, Thorsens Publishers,
New York). It is thought that learning ability is promoted. Medicinal carrots are reportedly helping reduce the physiological effects of stress and protect the stomach from stress-induced ulcers. Interestingly, studies in animals show that the physiological response to medicinal carrots is lost after removal of the adrenal glands (Mowrey, 1990, Next Generation Herbal
Medicine, Keats Publishing, New Canaan, CT). Decrease in heart speed and blood pressure (
Chen, 1982, Chung Hua Hsin Hsueh Kuan Ping Tsa Chih, 10 (2): 182-187) have also been reported, as well as increased vascular tone.

Medicinal carrots have also been reported to have the following effects on the host: increased endurance, enhanced fatigue and weakness, accelerated protein and lipid synthesis, stimulated the immune system, Increasing fertility, increasing anti-toxin radiation gland resistance, protecting against nitrogen and mustard gas poisoning, increasing antibody production, including anti-inflammatory, anti-inflammatory, anti-pyretic, anti-analgesic, natural aging Used for increasing lifespan and disease resistance and for accelerating recovery. Medicinal carrots are reportedly anticancer active and are also good at treating other conditions such as diabetes, asthma, headache, anemia, indigestion, impotence, depression and menstrual disorders (Mowrey, 1990, Next Generation Herbal Medicine, Keats Publishi
ng, Nwq Canaan, CT).

[0013] 3. SUMMARY OF THE INVENTION The present invention provides a method for producing pharmaceutical grade medicinal carrots. This method is a method of Pharma Printing (registered trademark). In one embodiment, the method comprises: providing a plant material, eg, a medicinal carrot, comprising a plurality of biologically active components; collecting a representative aliquot from the plant material; separating the aliquot into a plurality of marker fractions. (Each marker fraction comprises at least one active ingredient); measuring the degree of bioactivity imparted to each marker fraction to provide a bioactive fingerprint of the aliquot; An organism established for a pharmaceutical grade medicinal carrot to provide a bioactive fingerprint comparison for determining whether a botanical material is a pharmaceutical grade medicinal carrot based on the bioactive fingerprint comparison Includes comparison of active fingerprint standards.

The present invention also provides: providing a plant material having biological activity, wherein the plant material is comprised of a plurality of components; Separation (at least one of the marker fractions contains at least one active ingredient); determination of the degree of bioactivity imparted to each marker fraction to provide a bioactive fingerprint of a representative aliquot; Bioactive fingerprint of the target aliquot and a pharmaceutical grade medicinal carrot that has been established to determine if the botanical material is a pharmaceutical grade medicinal carrot,
Includes a step of comparison with a bioactive fingerprint standard.

[0015] In one embodiment, one or more marker fractions comprises one active ingredient.

The method also includes: determining the amount of the active ingredient in each marker fraction to provide a quantitative composition fingerprint of the aliquot, and determining whether the botanical material is a pharmaceutical grade medicinal carrot. And an additional step of comparing the quantitative composition and bioactive fingerprint standards with both the quantitative composition and bioactive fingerprint standards. The method may also include an additional step of: determining the total biological activity of the aliquot of the botanical material and comparing the total biological activity of the aliquot with the standard total biological activity established for pharmaceutical grade medicinal carrots.

The present invention also provides: a plant material of medicinal carrot comprising a plurality of components having a certain biological activity, each active component having a standardized bioactivity profile; taking a representative aliquot from the plant material Separation of aliquots into multiple marker fractions (each marker fraction contains at least one active ingredient); determination of the biological activity present in each marker fraction; calculation of the amount and aliquot of each active ingredient present Bioactivity calculation based on the standardized component bioactivity profile to provide a customized bioactivity fingerprint; based on a comparison of the calculated bioactivity fingerprint of the aliquot and the bioactivity fingerprint, the plant material is Provides a bioactivity fingerprint comparison to determine if a grade of medicinal carrot was obtained For, comprising the comparison of the biological activity fingerprint standards that have been established for a pharmaceutical grade ginseng to provide a method for producing ginseng pharmaceutical grade.

The present invention is useful for producing pharmaceutical grade medicinal carrots from suitable botanical materials having a certain or desired biological activity. Preferably, the plant material is an extract comprising a plant material such as an aqueous extract or an organic extract such as an alcoholic extract or a supercritical carbon dioxide extract or an organic solvent extract that may be subjected to further processing. It is. Alternatively, the plant material is a powdered plant material, seed oil, essential oil or the product of steam distillation. In one embodiment, the plant material is a homogeneous material in one physical state, such as oil or solution. A plant material may be a pure material that is solely derived from the plant of interest.

In the present invention, the active ingredient may include, but is not limited to, one or more of the following chemical classes: ginsenosides, acetogenins, alkaloids, carbohydrates, carotenoids, cinnamic acid derivatives, fatty acids, fatty acids Esters, flavonoids, glycosides, isoprenoids, lipids, macrocyclic antibiotics, nucleic acids, penicillins, peptides, phenols, polyacetylenes, polyketides, polyphenols, polysaccharides, proteins, prostaglandins, steroids and terpenoids.

In one embodiment, the present invention provides a method for producing a pharmaceutical grade medicinal carrot, wherein one or more marker fractions comprises two active ingredients. In one embodiment, the present invention provides a method wherein the at least one marker fraction comprises ginsenoside Rb1
And at least one marker component selected from the group consisting of ginsenoside Rg1, gamma-aminobutyric acid, glutamic acid and glutamine. In yet another aspect, the present invention provides a method for producing a pharmaceutical grade medicinal carrot, wherein the active ingredient is selected from the group consisting of ginsenoside Rb1, ginsenoside Rg1, and γ-aminobutyric acid.

The biological activity / clinical indication of a medicinal carrot may relate to a disease, disorder or condition in humans or other animals. Accordingly, the present methods are useful for the manufacture of pharmaceutical grade medicinal carrots for the treatment and / or alleviation and / or prevention of human and / or domestic diseases, diseases or conditions. Exemplary indications are adrenal insufficiency, allergic disorders, cardiovascular disorders, cancer or central nervous system disorders, endocrine disorders, gastrointestinal disorders, inflammatory disorders, metabolic disorders, nausea or diseases induced by microorganisms or viruses, and stress. Including, but not limited to, the treatment of sexually transmitted diseases.

In these methods, aliquots can be separated into biologically active and inactive components. Further, the marker fraction may include a class of related components.

The present invention also provides a method of preparing PharmaPrint® for pharmaceutical grade plants, for example, medicinal carrots. Further, the present invention provides a pharmaceutical grade plant, for example, a medicinal carrot, prepared by a method described herein.

In another embodiment, the medicinal carrot (Asian or Siberian species) is one or more of: aloe, astragalus, lingonberry, bourdac, camille, chestnut, Coriols versicolo, bermudagrass, clamp bulk, Dandelion roots, donkai, giant grizzly bear, primrose, kogomegusa, feres unicorn roots,
Feverfew, garlic, ginger, ginkgo, hydrastis, gotha kola, grape seed extract, green tea, gugu lipids, hawthorn, hops, ivy, kawa, licorice, nogeshi, mistletoe (American, Asian and European species), Motherwalt,
Selected from Oat, Osha, Passiflora, Pumpkin, Pigaeum, Purple Clover, Rosemary, Sarasparilla, Saw Palmetto, Pomfret, St. John's Walt, Nettle, V. Italian Carrot, Wildflower, Yamanoimo, and Jerba Mansa Can be combined with vegetable materials. The method of the present invention for producing a medicament comprises the steps of PharmaPrinting®
Includes medicinal carrots and the method of adding one or more plants listed above, and pharmaceutical grade medicinal products containing medicinal carrots and one or more plants listed above. In one embodiment, the medicinal carrot may be combined with Astragalus, liquorice and / or salaspilla. For the purpose of illustration and not limitation, and in no way limiting, medicinal grade medicinal carrots may include echinacea, valerian and / or
Or it can be combined with a pharmaceutical grade plant material such as black cohosh. US Patent Application, Oct. 1997, which is hereby incorporated by reference in its entirety.
Application No. 08 / 956,603 (Attorney Docket 9117-015), filed on Nov. 23, 2008, and Application No. 08 / 956,615 (Attorney), named "Pharmaceutical Grade Echinacea" Docket 9117-016); also entitled "Pharmaceutical Grade Black Kohosh" filed on Oct. 23, 1997, filed 08 / 956,611 (Attorney), incorporated herein by reference in its entirety. Docket 91
17-018).

3.1. Definitions As used herein, the term "pharmaceutical grade" refers to certain absolutely specific and / or inactive ingredients in botanical medicine.
Or within a relative concentration range and / or means that the components must exhibit a certain level of activity as measured in a disease, disease or condition specific bioactivity assay. The disease, disorder or condition results in human or animal suffering.

As will be appreciated by those skilled in the art, the term “pharmaceutical grade” refers to a botanical that is provided, for example, under a formula, ie, an “Rx” product or marketed product, ie, It does not mean adapting only to regulated products, such as "OTC". This term is equally applied as a prescription, product offering, or food additive, or "DSHEA".

As used herein, “ingredient” refers to a pharmaceutical grade botanical that naturally exists in a botanical or has an ingredient within a defined biological activity range and / or compositional range. Means a separate component (ie, a chemical material) that is added to a botanical medicine in order to dispense it.

As used herein, “active ingredient” means that the sum of the individual component activities in a disease-specific bioassay accounts for a substantial portion of the observed biological activity of the plant material. One or more components are meant. Preferably, the sum of the active ingredient activities accounts for the majority or 50% or more of the biological activity observed.

As used herein, a “fraction” is typically defined parameters such as solubility, molecular weight range, polar range, adsorption coefficient, binding properties, chemical reactivity or selective solubility. Or a group of structurally similar components.
Most often, the fractions are subjected to selective solvent solubility and pH-dependent separation, chromatographic separation methods, i.e., flash chromatography, preparative high performance liquid chromatography (HPLC), preparative gas chromatography, partition chromatography. Separation methods (i.e., liquid-liquid extraction, including preparative thin-layer chromatography, affinity chromatography, size exclusion chromatography, liquid-liquid chromatography, e.g., countercurrent chromatography or centripetal or centrifugal chromatography). ) Product.

The present invention will be more fully understood from the detailed description of the invention and examples of specific embodiments in the following sections and the accompanying drawings. FIG. 1 shows a schematic of the method of the invention used to establish a standard chemistry and / or bioactive fingerprint comparing a subsequently processed botanical material during the manufacture of a pharmaceutical grade drug. FIG. 2 shows a schematic diagram of the method of the invention used to process botanical materials into pharmaceutical grade drugs. FIG. 3 shows a schematic diagram of a method for isolating different types of biologically active ingredients. FIG. 4 is a comparison of medicinal carrot products as percentages (%) of the indicated specific substances (compounds) as measured with six products, namely Brands AF. Abbreviation: R
g1, Ginsenosaido Rg _1; Re, Ginsenosaido Re; Rb1, Ginsenosa id Rb _1; Rc, Ginsenosaido Rc; Rb2, Ginsenosaido Rb _2; Rd,
Ginsenoside Rd; Rf, ginsenoside Rf (not shown).

[0031] 5. DETAILED DESCRIPTION OF THE INVENTION 5.1. Pharma Printing ™ Method The present invention provides a method for producing botanical drugs that can be classified as belonging to pharmaceutical grade. This method is called Pharma Printing ™. The pharmaceutical grade botanicals produced by the method of the present invention are particularly suitable for use in clinical trials and, more importantly, for treatment of patients. By this method,
The drug used for the particular protocol has consistent quality as a prophylactic or therapeutic agent for human and veterinary medicine, ensuring that it is consistently suitable for its use.

The present invention allows for tight control of the quality, dose and clinical efficacy of botanical extracts and other botanical raw materials such as botanical materials and mammalian tissue-derived biological preparations. One aspect of the present invention involves establishing chemical and / or biologically active fingerprint standards for various plant materials. Once established, the use of fingerprint standards in the drug manufacturing process ensures that the plant material meets the requirements of pharmaceutical grade. As yet another aspect of the present invention, there are provided specific quantitative and biological fingerprints established for some plant materials. These fingerprints are useful for determining whether a particular botanical material meets the level of pharmacological activity and composition requirements for a particular treatment regime. Such measurements are important to ensure that clinical trials and patient treatments with botanical ingredients are based on consistent and verifiable extract composition parameters.

The present invention is useful for providing well-characterized, botanical ingredients whose composition is consistent between batches so that they can be accurately administered and used effectively in a clinical setting. obtain. The methods described herein provide assurance that the results of a clinical trial are reproducible.

First, a sample of the plant material of interest is obtained. Many botanicals are commercially available as raw materials or processed extracts. It is often a botanical extract or other composition intended for use as a medicament. The processed raw material may include a plurality of active ingredients that exhibit a given biological activity and a plurality of inert ingredients that do not directly exhibit the biological activity of interest. In one aspect, an aliquot is removed from the plant material and subjected to a quality assurance or standardization method.
Preferably, the aliquot is a typical aliquot of a homogeneous plant material. The method comprises separating an aliquot of the plant material into a plurality of marker fractions, each of which comprises at least one active ingredient or optionally one of the inactive ingredients. Measuring the amount of active or inactive component in each of the marker fractions provides a quantitative fingerprint of the aliquot. Measuring the degree of bioactivity for each of the marker fractions also provides a bioactivity fingerprint for the aliquot. The chemical and / or bioactive fingerprint of this aliquot is then compared to the corresponding fingerprint established for the pharmaceutical grade drug. If the fingerprint of the botanical matches the standard fingerprint, the botanical is identified as a pharmaceutical grade botanical.
If not, modifying the botanical may provide compatibility with the standard fingerprint or be rejected.

5.1.1. Method of developing PharmaPrint (TM) The method of developing PharmaPrint (TM) for botanicals begins with a review of the literature when the range of putative active ingredients is known. This would include a review of the chemical and biological literature, published bioassays and clinical data on botanicals. Particularly useful sources of information are the NAPRALERT computer database, Loing and Foster, managed by Dr. Norman Farnsworth in the Chicago, University of Illinois, collaborative program in pharmaceutical sciences, Loing and Foster, Encyclopedia of Common Natural Ingredients Used in Foo
d, Drugs and Cosmetics, 2nd edition, John Willie and Sons, New York, New York, 1996, Herbal Drugs and Phytopharmaceuticals, N
.G. Bisset, CRC Press, Boca Reiton, Florida, 1994, Duke, Handbook of Biologically Active Phytochemicals and Their Activities, CRC Press, Boca Reiton, Florida, 1992, Tyler and Foster, in the Handbook of Nonprescription Drugs, "Herbs and Phytomedicinal Pr
oducts, edited by Bellardi et al., United Book Press, Inc .: Washington, DC, 1996. For certain indications, studying the literature indicates that the putative active ingredient is actually associated with the condition. In addition, if there is a bioassay known for the putative active ingredient and known for the indication, the bioassay must be consistent with both the indication and the putative active ingredient. Any bioassay (s) leads to a clinically relevant endpoint (s) .The bioassay (s) should be quantitative over a wide concentration range. , IC ₅₀ curve (50% inhibitory concentration), EC ₅₀ (50% effective concentration) or appropriate K _I or K _d (
A dissociation constant) curve for the enzyme and its inhibitor is generated. A thorough chemical and biological analysis of both the putative active ingredient and the chromatographic fraction of the botanical is then performed. By analyzing the results, a quantitative analysis table of biological activity is created for each of the chemical components in the sample. The biological activity of the sample as a whole is then compared to the biological activity of the individual components. At this point, the individual chemical components may correlate with a clinically relevant endpoint. Similar methodology can be applied to bioassays that measure stimulatory or inhibitory effects.

Obtaining knowledge of the total activity, based on the activity of the components individually, naturally accounts for a significant portion of the biological activity when these components are combined. In general, the combined activities account for at least 25% of the total activity.

Preferably, the sum of the activities of the individual active ingredients accounts for the majority or more than 50% of the biological activity observed. More preferably, the individual components isolated are responsible for more than 70% of the activity. Even more preferably, the individual components isolated are responsible for more than 80% of the biological activity.

As an alternative, the idea is to select as few active ingredients as possible to be part of PharmaPrint ™. Low active ingredients are important for practical considerations in raw material acceptability and manufacturing. In the present invention, a correlation has been established between related chemical components and biological activity. Once a satisfactory correlation has been established, it may not be necessary to perform a biological fingerprint on each sample. Rather, a chemical analysis of the appropriate components and / or marker fractions of each sample of the botanical of interest will explain most of the biological activity and confirm that a given botanical sample is of pharmaceutical grade. Sufficient to establish.

In one aspect, the invention can include one of the following methods. One method is schematically illustrated in FIG. 1 and involves the establishment of a composition and bioactive fingerprint standard for a given pharmaceutical grade botanical. Once the fingerprint standard is established, the actual processing of botanicals into pharmaceutical grade drugs can be performed as shown schematically in FIG.

In the first step in establishing a chemical and / or biologically active fingerprint for a given botanical, the extract or powder may be treated with one or more extracts as shown by step 1 in FIG. Separate into groups. These groups are separated and identified on the basis of their ability as markers (with or without active ingredients) for the fingerprints established for the processed plant material. The putative component or group of putative components that are selected and identified as potential markers will vary greatly depending on the botanical drug being processed and the pharmaceutical application. At least two putative markers should be selected for each botanical. The number of potential markers can be greater than 5, and even 15-20 or more for complex plant extracts or powders. Potential markers are mostly
Identified and selected based on their potential biological activity or contribution to biological activity for a given pharmaceutical application. For different indications, specific bioactive ingredients can be optimized by using the same botanicals for the production of extracts in different extraction methods. Markers that do not show overt biological activity on their own can be isolated and included as markers used in fingerprints. These "surrogate" markers may be desirable as an internal standard, in which case the presence of the marker will indicate that other active ingredients required to provide a substantial portion of the overall biological activity observed with the botanical drug. It is an indicator. They are also an aid in ensuring the proper plant identity of the drug (ie, chemical taxonomy).

The initial separation of botanicals into various putative marker groups involves simple affinity extraction and partitioning, and complex affinity chromatography techniques such as gel filtration chromatography, flash silica gel chromatography and reverse phase chromatography. Achieved by a wide range of conventional separation techniques. Once the putative marker is identified for a given botanical, the biological activity of each of the markers is measured as depicted by step 2 in FIG. The particular bioassay used to measure the biological activity of the botanical drug is selected based on the intended use for the botanical drug. The bioassay preferably provides an image of the biological activity of the putative marker for the condition or indication being treated with the botanical.

By using the bioassay results obtained in step 2, the component having the desired biological activity (step 3) and the less active or essentially inert component (step 4)
) Is identified. The amount of each identified component present in each group is then determined by quantitative analysis of each of the groups identified in steps 3 and 4. The results of the bioassay and the quantitative composition assay are then used to produce a bioassay fingerprint and / or chemical fingerprint for the botanical as depicted by step 5 of FIG. As part of establishing a fingerprint for a botanical drug, an acceptable range of biological activity and / or chemical composition is measured. This is based primarily on the establishment of a biological activity and a quantitative amount of an acceptable range for each marker that provides the desired pharmacological activity of the processing material as a whole.

In addition, by evaluating various combinations of active and inactive marker fractions, a potential increase in the desired biological activity resulting from the combination of active and inactive components can be established.

With the bioassay and quantitative fingerprint established in step 5,
Accurate identification of botanicals that can be used to establish drug dosing and treatment schedules required for clinical use is provided. Drug administration and treatment schedules are established using routine clinical methods routinely used in new drug research. The raw materials used in determining drug dosing and treatment schedules must meet and meet the fingerprint requirements established in Step 5. This method ensures that the drug dosing method and treatment schedule is effective and reproducible, because all the raw materials used in the drug dosing and scheduling tests have the same fingerprint according to the present invention.

The bioassays and quantitative fingerprints determined by the schematic method shown in FIG. 1 are used as part of a manufacturing method for producing pharmaceutical grade botanical drugs. By using the fingerprint as part of a quality assurance or standardization method, certain botanicals contain the appropriate compounds and are accurately processed, resulting in raw materials standardized and tested according to the method shown in FIG. The provision of a botanical drug that functions in a clinically similar manner is ensured.

One example of a method for producing a pharmaceutical grade botanical according to the present invention is schematically illustrated in FIG. The plant material 21 of interest is first processed by extraction, milling or other manufacturing steps to form a processed plant material 22. Then
By analyzing a sample of the processing material, it is established whether it meets the fingerprint requirements established during the standardization process of FIG. This quality assurance or standardization method is depicted in step 23 of FIG. If the processing ingredient meets the pre-established fingerprint requirements for the particular ingredient, it is recognized as having a pharmaceutical grade as represented by step 24.
If the raw material is close to the standard fingerprint but does not fit perfectly, it is adapted to the fingerprint by modifying it as necessary (step 25).
Modification of the processing material to match the fingerprint standard can be done in a variety of ways. Still other botanical treatment methods include, as needed, additional extraction, selective extraction, selective processing, and combination of batches (eg, production of pharmaceutical grade ingredients by mixing high and low dose batches). ) Or the addition of various compounds. If the botanical is substantially outside the fingerprint range for both bioactive and quantitative markers, the batch is rejected (
Step 26).

In one aspect, the quality assurance standardization stage 23 used to determine whether a given botanical is pharmaceutical grade, comprises a uniform sample, preferably a homogeneous sample, of the botanical to be tested, Or get an aliquot. The sample should contain active ingredients that contribute to the observed biological activity of the raw material and produce a predetermined standard biological activity and / or chemical fingerprint. An inert component is one that cannot have a directly measurable biological activity. Inactive ingredients are those that have too little activity to explain the next category, a substantial part of the activity, whose presence indicates the presence of other biologically active ingredients and may serve as close markers for these ingredients Component, which contains an inert component that is chemically or biologically inert in the relevant assay. The sample is preferably only a small aliquot of the plant material being tested. It is therefore important to obtain a uniform sample, preferably a homogeneous sample, representing the entire batch of raw material.

A more detailed scheme is shown in FIG. 3, which shows the initial separation of the different components present in the aqueous extract of the botanical. By using continuous extraction and precipitation, the active ingredient in the aqueous or organic phase is isolated. The diagram in FIG. 3 is particularly suitable for separating water-soluble active ingredient species from botanicals, such as mistletoe.

One example of a general method for separating plants into the main types of chemical components is illustrated in FIG. Dry ingredients may be used, but primarily fresh plants (including leaves, roots, flowers, berries and stems) should be used. Specific plant parts, such as leaves, flowers, stems or roots, may be used if desired.

In this method, specific parts or whole plants can be frozen at liquid nitrogen temperature. This facilitates milling and preserves the integrity and potency of the active ingredient.

The micronized powder is repeatedly extracted using distilled water. If desired, the extraction can be performed with warm water, alcohol, other organic solvents, aqueous alcohol, dilute acetic acid, or a combination thereof. The actual temperature selected is preferably at or near the boiling temperature of water. It is preferred to first determine the overall biological activity of the extract. The extracts are combined and subjected to a specific bioassay, for example, a test that inhibits bacterial growth in Petri dishes when the agent is used as an antimicrobial agent. Alternatively, if the agent is intended for use as an anti-cancer agent, a test is preferably performed on the cell culture of cancer cells. From these data, the units of biological activity contained in the extract per ml are calculated (units of biological activity are the dilutions of this extract required for 50% growth inhibition of bacteria or cancer cells in the test system) Defined). Similarly, a biological activity with respect to a stimulating effect, for example an immunostimulation, can be calculated.

To establish a pharmaceutical fingerprint (Pharmaprint ™) according to the present invention, plants are extracted according to the method shown in FIG. (Alkaloids, nucleic acids, proteins and carbohydrates). Each separated group of components is tested for biological activity as needed. This may be evidence of activity (eg, in protein and alkaloid fractions, as in the case of the mistletoe mistletoe (Viscum album)). The type (s) exhibiting activity of the compound is further separated into individual compounds by affinity chromatography, high performance liquid chromatography, gas chromatography or other chromatography. Components that primarily affect biological activity are quantified based on weight and specific biological activity units. These ingredients provide a fingerprint that establishes the pharmaceutical requirements for the original herbal extract. Bioactive units per ml of pharmaceutical grade extract provide a way to establish accurate dosages for clinical trials.

Once the sample has been separated into individual marker fractions and at least one with at least one active ingredient, by analyzing each fraction,
The amount of active ingredient present therein is measured and a quantitative fingerprint of the sample is provided. Quantification of each fraction can be achieved using any of the known quantitative analysis methods. Examples of quantification methods include the use of gravimetric analysis, spectral analysis or quantitative detection devices, such as those used in gas chromatography or high performance liquid chromatography and other separation systems. Other suitable quantitative analysis methods include enzyme analysis, radiometry, colorimetric analysis, elemental analysis, spectrophotometry, fluorescence or phosphorescence and antibody assays, such as enzyme-linked immunosorbent assay (ELISA) or radio There is an immunoassay (RIA).

In one aspect, a quantitative fingerprint of the sample is produced by using the results of the quantitative analysis of each fraction. The fingerprint is constituted by the amounts of the components and the identity of the components in each of the marker fractions. This quantitative fingerprint is then compared to a known standard fingerprint that has been established (FIG. 1) so that the raw material is considered to be of pharmaceutical grade. If the quantitative fingerprint of the sample falls within the range indicated for the pharmaceutical grade fingerprint, the material can be identified as being pharmaceutical grade.

As another part of a quality assurance assay, individual marker fractions can be subjected to a biological assay. The bioassays used to test the various fractions are the same as those used for the standard fingerprint and will depend on the particular clinical application intended for this material.

The bioactive fingerprint generated for this material is compared to a standard bioactive fingerprint established such that the material is considered to be of pharmaceutical grade. If the biological activity fingerprint of the sample falls within the range of biological activities shown for the pharmaceutical grade fingerprint, the material is identified and approved as having pharmaceutical grade.

5.1.2. Alternative PharmaPrint ™ Development Method [0057] The method of developing PharmaPrint ™ for botanicals where the putative active ingredient is unknown also begins with a review of the literature. In that case, review the available chemical literature, biological literature, published bioassays or clinical data for the botanical or related botanical or botanical with related activity. Based on the pathology, a series of appropriate bioassays are selected. The activity of all samples or extracts is analyzed using a bioassay. The fraction of plant material whose putative active ingredient is not yet known is then analyzed by using a bioassay showing activity. Fractionation is based on conventional methods, such as separation by relative permittivity, biological affinity, polarity, size, solubility or adsorptivity. The fractions are then analyzed to determine which fractions are involved in the activity. Assuming activity is found, by refractionating each active fraction, the individual putative active ingredient, ie, the pure chemical compound, is isolated. Know individual chemical compounds,
Based on knowing their quantitative biological activity, a quantitative potency curve can be drawn and a 50% inhibitory concentration (IC ₅₀ ) can be determined for each individual chemical component. If the putative active ingredient is an agonist, other parameters (binding, activation, response) may be required. In the general case, a bioassay consists of an appropriate test of the stimulatory or inhibitory effects of the components, fractions or whole extracts, followed by an appropriate quantitative evaluation of their effects. For the most promising (or typical) assays in which a standard (or radiolabeled) agonist or antagonist elicits a measurable effect, inhibition and / or stimulation by the subject material is typically IC ₅₀ , EC ₅₀ It can be evaluated and represented by a value such as ₅₀ , or other suitable measurement (eg, _Ki , _Kd , _Km, etc.). The activities of the individual putative active ingredients are then summed and the sum is compared with the activity in the unfractionated plant sample. Where these ingredients account for a substantial portion of the activity, they are those that have an initial fingerprint of the "active ingredient" for botanicals for which the active ingredient is unknown.

5.1.3. Additional Variations on PharmaPrint ™ Development Method The general method outlined above for the botanical drug PharmaPrinting ™, whose putative active ingredient is unknown, is described in the analytical process. Some modifications will be made if the complexity of the system increases. A variant is made when the sum of the individual components does not represent a substantial part of the biological activity of the botanical. In this respect, there are several promising reasons why the activity of the individual components is reduced, firstly the decomposition or disintegration of the active components or secondly a synergistic effect. In another possible scenario, no significant or greatly reduced activity can be seen from any of the fractions, but the whole botanical or extract shows activity in the bioassay. Non-specific matrix action can also reduce total extract activity when compared to a standard.

Determining whether the active ingredient has degraded during the course of the assay is relatively straightforward. Simply combine all of the fractions and compare the activity of the combined fractions to the activity of the raw material. If substantial activity is lost, the problem is likely to be degradation. The chromatographic analysis of the crude botanical is compared to that of the combined fractions to determine which active ingredient has been degraded. Missing peaks or reduced magnitude indicate that the components may be degraded. There are many ways to overcome the degradation. Typically, mild extraction / fractionation methods such as liquid-liquid chromatography (counter-current chromatography) or supercritical carbon dioxide extraction or chromatography can be used.

Another explanation for the activity of individual fractions that do not represent a substantial part of the expected total activity is the synergistic effect between one or more active ingredients or with the inactive ingredients. To determine that synergy is taking place, it is necessary to analyze the fractions combined in pairs. If the combined fractions show higher activity than the individual fractions, it is possible that two or more individual components in the fraction are acting synergistically. For example, 3
There are two fractions, each alone responsible for 10% of the biological activity (ie their uncombined additive biological activity is 30%), but when combined they are responsible for 100% of the activity May be. In that case, the fractions act synergistically. Consideration of combinatorial repetitions or large fractions in fraction pairings reveals some synergistic activity. Once the two fractions show a synergistic effect, they are refractionated as described above and by testing individual fraction pairs or isolated component pairs, individual components that act synergistically are found. Also, three-way comparisons of individual components or fractions can be tested.

What happens if a fraction has no activity in a bioassay in which a botanical is active? Illustrative herein include many active ingredients that have no activity in degradation, synergy, or individual fractions. The first step is to fractionate each initial fraction and see if the active ingredient appears in the bioassay. If that does not work, the fractions should be combined and assayed to determine if activity has been degraded. If decomposition is taking place, the appropriate measures described above should be taken. If no degradation is present, another fractionation method should be tried. In practice, a sufficiently large or appropriately sized or selected fraction indicates activity. If synergy is a suspected problem, proceed as described above for synergy.

5.2. Methods of Producing and Extracting Vegetable Raw Materials By processing the vegetable raw materials, aqueous or organic extracts of whole plants or selected parts of plants can be formed. Vegetable raw materials can also be wholly or partially processed to form a powder. Many of the botanical materials of interest are commercially available as powders, aqueous extracts, organic extracts or oils. In one aspect, an extract of a plant material is preferred because it is easily dissolved in a liquid pharmaceutical carrier. However, powdered plant materials are preferred for many applications where the drug is administered in solid form, eg, tablets or capsules. These methods are familiar to those skilled in the art. In addition, many of the plant raw materials and / or extracts are commercially available. The following examples are provided as examples of processing and extraction of botanicals. Additional examples are provided in the Detailed Description section.

For a typical root, the root may be sliced, frozen or ground. If powdered, shake it with a suitable solvent and filter (Tanabe et al., 1991, Shoyakugaku
Zassi, 45 (4): 316-320). Alternatively, the following method is used. The roots are homogenized, extracted with acetone and filtered. When steam-distilling botanicals,
Essential oils are obtained and the distillate can be dissolved in acetone-water or a suitable solvent. Alternatively, the cut rhizome is frozen and / or freeze-dried, and the resulting powder is extracted with acetone-water (Tanabe et al., 1991, Shoyakugaku Zassi, 45 (4): 321-326). Another method for producing botanicals is water extraction with water at 100 ° C. (Yamahara et al., 1985, J. Ethnopharmacology 13: 217-225). The initial solvent extract by the above method can be further extracted using liquid / liquid extraction with a suitable solvent. The botanicals can be extracted in two stages using polar or non-polar solvents, respectively. The solvents are then concentrated and the fractions are combined (Nagabhusan et al., 198).
7, Cancer Let. 36: 221-233). Vegetables can also be processed as pastes or powders that can be cooked (Zhang et al., 1994, J. of Food Sc
ience 59 (6): 1338-1343).

By using various solvents, for example, acetone, acetonitrile, dichloromethane, ethyl acetate, ethanol, hexane, isopropanol, methanol, other alcohols and supercritical carbon dioxide, dried vegetable drugs can be extracted (Cipro et al. , 1990, Int. J. of Food Science and Technology 25: 566-575 and references described therein).

In the case of other botanicals, for example Saw Palmetto, the pharmaceutical product is seed oil or dried berries. Typical products produce hexane or supercritical carbon dioxide extracts. Many saw palmetto products are commercially available, for example Permixone ™ or Talso ™. For an example of supercritical carbon dioxide extraction of botanicals, see Indena, EP 025095.
See 3B1. Alternatively, botanicals can be milled and extracted with a suitable solvent (90%) in a Soxhlet extractor (El Gamley et al., 1969, Experien).
tia 25 (8): 828-829). Vegetable drugs can also be extracted with ethanol (Visa et al., 1996, The Prostate 28: 300306).

The dry raw materials include freeze-drying, microwave drying, liquid nitrogen cooling and grinding,
It can be manufactured in a variety of ways including vacuum drying at 70 ° C. for 0 hours, or in the shade, or air blown with a hot air blast (Wrist and Schmidt, Hagers Handbuch der Pharma
zeutischen Praxis, Springer-Verlag: New York, 1993, 1973-79, Alaya et al., 1981, Journal of Comparative Pathology, 1
35-141). What is also known as tea, dilute aqueous extract, leachate is 6
It can be prepared in water at 0-100 ° C (Nozel and Schircher, 1990).
Infusions can also be used. Extraction is even more effective if the particle size is less than .25 mm (Wrist and Schmidt, Phytopharmaceutical Technology, CR
C Press: Boca Leyton, Florida, 1989).

Various guidelines are available for making oil extracts of botanicals. Vegetables can be digested (macerated) in oils, although recommended for 10 days at 45 ° C. and 12-24 hours at 70 ° C. (Hobbs, 1989, Herbal Gr.
am 18/19: 24-33, Smith et al., Quality Validation Laboratory Herb.
Pharm: Williams, Oregon, 1996). St. John's Wort reports, for example, that exposing the product to sunlight during the extraction process increases the flavonoid content, calculated as quercetin, by a factor of 4 (Meisenberger and Kovar, 1992). In addition, in the case of St. John's Walt, a two-fold increase in hypericin was reported in oil products in which the raw material was further alcohol extracted and mixed with oil (Georgiev et al., 1983, Nauchn).
i Tr.-Vissh Inst. Plovid. 30: 175-183).

Alternatively, alcohol-water products can be manufactured from botanical drugs (Dukova, 1985, Farmitsiya 34: 71-72, Georgiev et al., 1985, Nauch
ni Tr.-Vissh Inst. Plovid. 32: 257-263, Wagner and Bratt, 1994, Kovalevski et al., 1981, Herba Pol. 27: 295-302). According to Hagers Handbuch, tinctures of botanicals, such as St. John's Walt, can be manufactured using drugs or by freezing the ethanol-soaked botanicals, filtering and storing them in dark bottles (list and list). Hellhammer, 1
993).

[0069] Some botanicals, such as St. John's Walt, are sensitive to both temperature and light. For botanicals of this type, the ingredients should be dry-packed or refrigerated with a coolant and protected from light and air. Cent
In the case of Jones Walt, the hypericin content is 60 ° C. for more than 6 weeks.
Powder extracts, tablets and juice products have been shown to have a significant reduction when stored at a temperature of -140 ° C. Dry extracts stored at 20 ° C. were found to remain stable for at least one year (Adamski et al., 1971, Farm. Po.
l. 27: 237-241, Benigni et al., Hypericum. Plante Medicinali: Chi.
mica, Farmacologia e Terapia, Milan: Inverni and Della Beffa, 1971). Other St. John's Walt components found in oil products, hyperperforin and adhiperforin, are very unstable, especially when exposed to light, and can decompose in as little as 14 days (Meisenberger et al., 1992; Pl
anta Med., 351-354). Stability (in the absence of air) was increased for products extracted with ethanol up to 6 months. Similarly, the detection of up to four xanthones and several flavonoids, including quercetin and I3 ', II8-biapigenin, suggests that they may be included in the active ingredients in external products (Bistrov 1975, Tetrahedron Letters 32: 2791-27.
94).

5.2.1. Plant Raw Materials and Liquid Extracts of Powdered Plant Material Medicinal carrots are provided as an aqueous or alcoholic extract of powdered roots, or as a botanical material that is a crude medicinal plant. One common form of a liquid extract of vegetable material is "tea." Tea can be manufactured through a brewing or brewing process. Tea is generally an effective means of extracting water-soluble components from dried or raw botanicals.

Another common form of liquid vegetable extract is a tincture. Vegetable tinctures are alcoholic or hydroalcoholic solutions, typically made from raw or dried botanicals. Usually, it is produced by a filtration or maceration process.

Powerful botanical tincture and homeopathic tincture mother liquor can correspond to 10 g botanical (dry weight) in 100 ml tincture. Common botanicals include botanicals equivalent to 20 g in 100 ml tincture. The respective ratios of dry plant drug to solvent in these products are 1:10 and 1: 5, respectively. Although these concentrations are officially recognized by the United States National Drug Collection, it is common for tinctures to be manufactured at 1: 4 and other concentrations.

As compared to crude plant extracts, tinctures may have a reduced microbial load and a longer shelf life. This is largely due to the presence of alcohol at a concentration of 20% or more in the extract. Liquid extracts may be made with glycerin and water as solvents. These glycerites usually require the presence of glycerin at a rate of at least 50% to prevent microbial contamination. Glycerite also concentrates alcohol and replaces glycerin with "
It can be manufactured from a tincture by "adding back".

Another type of liquid extract is a “flow extract”. The fluid extract is Extract 1
It is a liquid product of a botanical that exhibits the pharmaceutical properties of 1 g of dry botanical in ml. The certified version is made by a filtration process according to a certified monograph that determines the solvent used.

Liquid extracts, which are usually concentrated by evaporation of the solvent, may form virtually oily, semi-solid or solid extracts. Dry powdered extracts may be prepared by absorbing the liquid extract, oil or semi-solid into a suitable carrier before removing the solvent. Alternatively, the dry powdered extract is
By removing the solvent directly from the liquid extract, a solid extract can be provided that can be made and pulverized.

5.3. Separation of Fractions Once the sample extract has been manufactured and / or otherwise purchased as a commercially available extract, an aliquot needs to be subjected to fractional analysis. If the fingerprint is already established, separate the sample or aliquot into the same multiple marker fractions present in the standard fingerprint. Each of the marker fractions contains one or more active or inactive components. The marker fraction is established on an individual basis for each plant material being tested. Depending on the raw material, only a small fraction of the marker is required. For other more complex raw materials, a large number of marker fractions may be present. For example,
In the case of mistletoe, mistletoe mistletoe (Viscum album L.) protein extracts, the preferred protein marker fraction is the fraction that is separated based on the sugar binding affinity of the fraction. However, various parameters for identifying a raw material and separating it into a marker fraction can be established based on the types of components present in the plant raw material. Separation of the sample into marker fractions can be achieved by any of the conventional separation techniques, including liquid chromatography and extraction methods. The same method used to establish the standard fingerprint should be used. Since various fractions can be tested for biological activity, it is preferable to utilize non-destructive separation techniques. Liquid column chromatography is a useful separation technique by affinity chromatography based on the specific binding ability of the compounds (eg, carbohydrates and target enzymes) used in particular.

After fractionation, the solvent is removed and the starting material is dissolved in a medium suitable for bioassay. Examples of suitable media include DMSO, ethanol, various detergents, water and suitable buffers. The choice of solvent will depend on the chemistry of the component being analyzed and its compatibility with the assay system.

5.4. Establishment of a suitable bioassay The biological assay can be any cell proliferation assay, such as L1210 cell inhibition,
It can include the measurement of inhibition of a critical enzyme associated with immune activity or a specific disease. Examples of other transformed cell lines that can be used in bioassays include HDLM-3 Hodgkin's lymphoma and large Burkitt's lymphoma, hepatome cell line, one of the human / animal cell lines bearing specific cell receptors or enzymes. There are next or established cultures.

Using the results of the bioassay, the raw bioactive fingerprinting is produced. Fingerprinting can be as simple as assaying the two selected marker fractions. Conversely, a fingerprint may include a number of different bioassays performed on a number of different fractions. The same assay may sometimes be performed on different marker fractions. Also, different assays can be performed on the same marker fraction. The combination of bioassays will depend on the complexity of a given botanical source and its intended clinical use. The bioassay is the same as that performed when establishing the bioactivity fingerprint of the standard material.

5.4.1. Enzyme and Receptor Based Assays Enzyme and receptor based assays are preferred when practicing this invention. Assays are selected based on accepted enzyme assays for clinical disease, or they are selected from assays appropriate for a given clinical disease. It is important to select an appropriate bioassay that can be confirmed. Ideally, a bioassay should be robust, ie, reproducible over time, and show a quantitative dose response over a wide concentration range. A problem encountered with botanicals where the active ingredient is not known is the selection of an appropriate bioassay. Here, human therapeutic applications are useful as a guide to select assays known in the art based on possible mechanisms of action. The mechanism of action should be consistent with a clinically relevant endpoint. There is a wide array of clinically relevant assays based on enzyme activity, receptor binding activity, cell culture activity, activity on tissues, and gross animal in vivo activity.

This section deals with enzyme and receptor binding assays. There are many books on enzyme or receptor assays, for example, Methods in Enz by Academic Press.
ymology or Boyers, The Enzymes. Bioactive Natural Products, Detec
tion, Isolation, and Structural Determination, SM Colgate and RJ Molineux, CRC Press (1993) also discuss specific bioassays. Methods in Cellular Immunology, R.A. Rafael Fernandez-Botlan and V.A. Vetvica, CRC Press (1995)
Assays from immune cell activation and cytokine receptor assays are reported. “Screening Microbial Metabolites for New Drugs-Theoretical and Pr
Actical Considerations "reports additional methods of finding pharmaceutically suitable bioassays (Yalbrough et al., (1993) J. Antibiotics 46 (4): 536-544). Panlabs, Paracelsian and Nova There are many commercial contract research vendors, including screens.

For example, in the case of botanicals useful for the treatment of neurological diseases, bioassay methods
Ray contains adrenergic receptors, cholinergic receptors, dopamine receptors
, GABA receptor, glutamate receptor, monoamine oxidase, nitric oxide
It may include an intestase, an opiate receptor or a serotonin receptor. Cardiovascular disease
In patients, the assay array contains adenosine A₁Agonism and antagonism; adrenergic alpha₁, Α_Two, Β₁Agonism and antagonism; angiotensin I inhibition; platelet aggregation; calcium channel blockade; ileal contractile response;
Heart rhythm abnormalities; Cardiac muscle inotropy (inotropy); Blood pressure; Heart rate; Cycle
Variability (chronotropy); contractile; hypoxic, hypotensive; hypoxic, KCN; portal vein, potassium depolarization; portal vein, spontaneous activation; or thromboxane A _Two , Platelet aggregation. For metabolic disorders, the following bioassays, namely cholesterol, serum HDL, serum total, serum HDL / cholesterol ratio, H
DL / LDL ratio, glucose, serum-glucose load, or renal function, potassium
Urine, salt excretion and urine volume changes can be used. For allergic / inflammatory diseases, the following
Bioassays, ie allergy, arthus reaction, passive cutaneous anaphylaxis
ー, bradykinin B_Two, Contractile, trachea, histamine H₁Antagonism, inflammation, ma
Effect of carrageenin on clophage migration, leukotriene D_FourAntagonism, Neurokinin NK₁Antagonism, or platelet activating factor, platelet aggregation or important inflammatory mediators (eg, interleukin IL-1, IL-6, tumor destruction
Biosynthesis induction of death factors or arachidonic acid) can be used. For gastrointestinal disorders,
Bioassay, ie cholecystokinin CCK_AAntagonism, cholinergic antagonism, peripheral, gastric acidity, pentagastrin, gastric ulcer, etano
Ileal electrical stimulation modulation, ileal electrical stimulation spasticity or serotonin 5-HT_ThreeAntagonism may be used. Antimicrobial, antifungal or anti-trichomonas disease
In cases where: Candida albicans, Escherichia coli
Escherichia coli, Klebsiella pneumoniae
ella pneumonaie), Mycobacterium ranae, professional
Proteus vulgaris, Pseudomonas aeruginosa (Ps
eudomonas aeruginosa, Staphylococcus aureus
ureus), methicillin resistant, Trichomonas foetus, or Trichophyton mentagrophytes. For other indications, a person skilled in the art
You should be able to select the appropriate list in A.

Specific examples of enzyme or receptor based assays include acetylcholinesterase, aldol-reductase, angiotensin converting enzyme (ACE),
Adrenergic α, β, rat androgen receptor, CNS receptor, cyclooxygenase 1 or 2 (Cox1, Cox2), DNA repair enzyme, dopamine receptor, endocrine bioassay, estrogen receptor, fibrinogenase, GAB
A A or GABA B, β-glucuronidase, lipoxygenase, e.g.
- lipoxygenase, monoamine oxidase (MAO-A, MAO-B ), phospholipase A _2, platelet activating factor (PAF), potassium channel assay, prostacyclin cyclin, prostaglandin synthetase, serotonin assays, for example 5-HT activity or other Has a serotonin receptor subtype, a serotonin reuptake activity, a steroid / superthyroid receptor, and a thromboxane synthesis activity. Specific enzyme assays can be obtained from a variety of sources, including PanLabs ™ Inc. (Bozel, Washington) and Novascreen ™ (Baltimore, MD). Additional assays include adenosine triphosphatase inhibition, benzopyrene hydroxylase inhibition, HMG-CoA reductase inhibition, phosphodiesterase inhibition, protease inhibition, protein biosynthesis inhibition, tyrosine hydroxylase and kinase inhibition, testosterone-5
There are α-reductase and cytokine receptor assays.

5.4.2. Cell culture and other assays In addition to enzyme and receptor assays, there are other biological assays. Preferably, these assays are performed in cell culture, but can also be performed on whole organisms. Cell culture assays include activity in cultured hepatocytes and liver cancer (cholesterol levels, LDL-cholesterol receptor levels and LDL / HDL
Cholesterol ratio), L1210, HeLa or MC
Anti-cancer activity on F-cells, modulation of receptor levels in PC12 human neuroblastoma cells, modulation of primary cell culture activity of luteinizing hormone (LH), follicle-stimulating hormone (FSH) or prolactin, mast cells Cell culture assays for Ca ²⁺ influx, phagocytosis, lymphocyte activity or TNF release, platelet aggregation activity or HDL
Activity against M-3 Hodgkin's lymphoma and large Burkitt's lymphoma cells,
Includes antimitotic activity, antiviral activity in infected cells, antibacterial activity (bacterial cell culture) and antifungal activity. Tissue or gross animal assays, including anti-inflammatory mouse otitis dermatitis, rat paw swelling, muscle contractility assays, passive cutaneous anaphylaxis, vasodilatory assays or gross animal carbon clearance tests can also be used. These assays are available from Panlabs ™ Inc. (Bozel,
(Washington).

5.4.3. Anti-cancer activity The anti-cancer effect of a drug can be tested in various cell culture systems. These include mouse leukemia, L1210, P388, L1578Y and the like. Tumor cell lines of human origin, such as KB and HeLa cells, can also be used. In a typical assay, tumor cells are grown in a suitable cell culture medium, such as RPMI-1640 containing 10% fetal calf serum. Exponentially growing cells are treated with different concentrations of test material for 14-72 hours depending on the cell cycle period of the cell line. At the end of the incubation, cell growth is assessed by counting the number of cells in the untreated and treated groups. Cell viability can be confirmed by trypan blue exclusion test or reduction of tetrazolium dye by mitochondrial dehydrogenase. The fact that an agent can inhibit cell growth in culture can be considered to have potential anti-cancer effects. These effects can be demonstrated in tumor-bearing animals, a model of human disease (Kwaja, TA et al. (1986) Oncology).
, 43 (Supplement 1): 42-50).

The most economical way to evaluate a drug's anti-cancer effect is to test its effect on tumor cell growth in minimal essential medium (MEM) containing 10% fetal calf serum.
Drug-exposed cells (in duplicate) were incubated at 37 ° C according to the population doubling time of the tumor cells.
For 2-4 days in a humidified CO ₂ incubator. At the end of the incubation period, cells are counted and the degree of cell growth inhibition is calculated from comparison with untreated control cells grown under the same conditions. The type of cell line used varied from laboratory to laboratory depending on individual needs. The National Cancer Institute (NCI) in the United States recommends the use of KB cells (human nasopharyngeal carcinoma) for in vitro evaluation of anticancer agents. Cell growth inhibition is measured by assessing the protein content of drug-treated and untreated controls (Lowry method). NCI also
For evaluation of anti-cancer efficacy of plant extracts and related natural products, see mouse leukemia P38
We recommend the use of 8 suspension cultures.

Mouse leukemia L1210 cells cultured in microtiter plates are routinely used in in vitro assays for anti-cancer activity. The cell population doubling time of leukemia L1210 is 10-11 hours, and 48 hours (3-4 generations of logarithmic growth) of drug exposure is used to evaluate its anticancer activity. For the growth inhibition test, all stock solutions and diluents are made with sterile 0.9% NaCl solution. Cell cultures are seeded in microtiter plates (0.18 ml / well) in duplicate for each inhibitor concentration at a rate of 2-5 x 10 cells / ml. The inhibitor is added in a volume of 0.02 ml to make a 1:10 dilution in each case. The coated microtiter plate is
Incubate for 48 hours in a humidified CO ₂ incubator containing 5% CO _{2 in} air. At the end of the incubation period, an aliquot of each well is added to a measured volume of isotonic saline and counted on an electronic cell counter. Cell viability is measured by trypan blue exclusion. The results were calculated by plotting the percent cell growth inhibition (compared to the cell density of the saline-treated control) versus the log of the drug concentration, as the concentration (IC ₅₀ ) that elicits a 50% inhibition of cell growth measured from the graph. Express.

[0088] The cytotoxic effect of an agent on a tumor cell line can also be evaluated. However, these experiments require long test times and are expensive. In these tests, the drug is washed from drug-treated cells and then placed in soft agar or a suitable medium, and the viability of the cells is assessed by their ability to increase viable cells and form microscopic colonies. Cell killing or cytotoxic activity is assessed by comparing the number of cell colonies obtained with certain drugs to the number obtained from untreated controls. In tests with mistletoe extracts, weakly adherent cultures of EMT-6 cells (mouse mammary adenocarcinoma) were used. These cells are cultured in Eagle MEM (F14) containing 10% dialyzed fetal calf serum and antibiotics. Spin the cell suspension,
The pellet was suspended in a spinner medium supplemented with 10% dialyzed fetal bovine serum (70 cells / ml).
), Put in plastic Petri dishes and incubate for 2 hours to allow cells to attach. At this point, the cells are exposed to various concentrations of the extract for 2-24 hours. The medium is then removed, replaced with drug-free medium, and the dishes are incubated for 5-7 days. Colonies were methylene blue (0.33% in 0.01% KOH)
) And count with an automatic colony counter. The plating efficiency of EMT-6 cells was 46
%. (Kwaja et al., 1986, Oncology, 43 (Addendum 1): 42-50).

5.4.4. Antiviral Activity The antiviral activity of various agents can be confirmed in human cell lines, such as cell cultures of HeLa or H9 lymphoma cells. These cells are infected with the virus and the virus is propagated in cell culture. The situation in which the virus can cause cell lysis or cytopathic effects is considered an endpoint. For example, HIV infection of H9 cells produces multinucleated cells. These cytopathic effects, if reduced or eliminated by certain concentrations of the drug, indicate potential as anti-HIV drugs. These results can be confirmed by evaluation of viral enzymes in cell culture, for example, by examining viral reverse transcriptase expression levels. Reduction of viral enzyme expression confirms the antiviral effect of drug treatment (Kwaja, TA, US Patent No. 5,565,200, J. Levy et al., 1984, Science 2).
25: 840).

5.5. Analytical methods for analyzing chemical components Gas chromatography (GC), mass spectroscopy (MS), GC-MS, high performance liquid chromatography (HPLC), HPLC-MS, thin layer chromatography (TLC), high speed TLC There are many ways to separate and analyze individual chemical components, including (HPTLC) gel chromatography and reverse phase chromatography (RPC). These chromatographic methods can be performed on an analytical or preparative scale. To determine the actual chemical structure of the unknown component, a nuclear magnetic resonance (NM)
R) and mass spectral fragmentation analysis are commonly used.

[0091] The determination of the type of chromatography will depend on the chemical component which is most likely to be involved in the biological activity. For example, if the biological activity appears to be due to fatty acids, the fatty acids are esterified and the esters are analyzed by GC. In the case of organic compounds with alcohol groups, they are modified to produce ethers, silyl derivatives or other less polar functional groups. In that case, these derivatives are suitable for GC analysis (Steinke et al., 1993, Planta Med. 59: 155-160, Breu et al., 1992, A
rzneim.-Forsch / Drug Res. 42 (1): 547-551). HPLC is a particularly suitable method if the activity appears to be due to flavonoids. Reversed phase HPLC (R
P-HPLC) has been used to analyze flavonoids from various botanicals, specifically hawthorn, passiflora, chamomile and ginkgo (Pietta et al., 1).
989, Chromatographia 27 (9/10): 509-512). Plant constituents are measured quantitatively by TLC (Van Heren and Van Heren-Fastre, 1983, J. Chromatography 281: 263-271), similar to MS-analysis on garlic. "Analysis of Lipids", K.C. D. Mukhelje, “Analysis and Characterization of Steroids”, H.C. Rampartic, J.A. Shelma, and “High-Performance Liquid Chromatography of Peptides
and Proteins ", a chromatographic CRC handbook on CT cloak and RS Hojes, is available and describes columns and solvent systems.

5.6. Analysis of Fractions In another embodiment, Pharmaprint (TM) uses a specified fraction or type of compound rather than based on a pharmaceutical fingerprint (PharmaPrint (TM)) based on individual chemical components of known biological activity. Can be established. From a practical point of view, PharmaPrint ™ can be used to form compounds rather than individual components, since some of the chemical components in botanicals form a very complex mixture of closely related components. Preferably, it is based on fraction or type. Examples of these types of components are lectins (glycoproteins) or glycoproteins and silymarin in Nogesi. There are many examples of fractional analysis ("Principles and Methods of Gel Filtration", Pharmacia Biotech, Rams y Lund, Utsumi, Sweden, 1987, J. Biochem. 101: 1199-1208).

5.7. Method of Using Pharma Printed Materials After the fingerprints of the plant materials have been established, individual samples are analyzed to determine if they fall within acceptable standards. Once a sample is approved, it is suitable for a variety of human and animal related diseases. Because the materials are useful in clinical trials, they provide consistent quality and accurate dosages of the material for the test formulation. Pharmaprinted ™ ingredients are also useful in toxicological testing in animals, where again the consistency of the ingredients is useful for quantitative toxicological analysis. In many cases, it is useful as a control material for analytical or biological applications.

PharmaPrinted ™ botanical materials are useful for conditions involving botanical drugs. See, for example, Loing and Foster, 1996 and "Herbal Drugs a
nd Phytopharmaceuticals "1994. More specific examples of medical conditions or therapeutic indications are AIDS, adaptogens, mild-moderate depression, anti-arthritic, anti-cancer, anti-diarrheal, anthelmintic, anti-inflammatory. Drugs, gastrointestinal anti-emetic, anti-rheumatic, antispasmodic, anti-ulcer, angina, antibacterial, antimutant, antioxidant, antiviral, arteriosclerosis, arthritis, asthma, blood pressure, benign prostatic hyperplasia ( BPH)
, Bronchial asthma, bronchitis, sedatives, cough, cerebral circulation disorder, cholesterol lowering, cirrhosis, dermatological anti-inflammatory drugs, diabetes, diuretics, laxatives, dysmenorrhea, indigestion, emphysema, environmental stress, expectorant, Free radical remover, GI difficulties, hemorrhoids, hepatitis, liver protection, hypertension,
Hyperlipidemia, hyperprolactinemia, immunomodulatory activity, increased fibrinolysis, resistance to bacterial infection, inflammation, insomnia, lactation, liver protection, longevity, menstrual cycle regulation, migraine, myalgia, osteoarthritis, pain , Peripheral vascular disease, platelet aggregation, PMS, menstruation promotion, prostate disease, triglyceride reduction, menstrual pain relief, respiratory infection (RTI), retinopathy, sinusitis, rheumatism, sedatives, sleep promoting agents, throatitis, There is hair growth irritation, superficial wound healing, tinnitus, local eczema (dermatitis), urinary tract infection (UTI), varicose veins, venous insufficiency or wound healing.

Other indications include hemostatics, antibacterials, anthelmintics, antipyretics, inotropics, carminatives, bile secretions, protectives, antiperspirants, emetics, menstrual stimulants, emollients, antipyretics , Lactation, hepatic, hypnotic, laxative, sedative, pulmonary, redness, stimulant, tonic, wound healing, canker stores, pyorrhea, gingivitis, gastritis, ulcer, Gallstones, intermittent, cold, influenza, pharyngitis, headache, shingles, cystitis, kidney stones, atopic vaginitis, uterine fibroids, osteoporosis and gout.

Preferred indications for PharmaPrinted ™ medicinal carrot medicine include anti-stress, aphrodisiacs, increased vitality, nervous system disease, cardiovascular system disease, increased intelligence, increased productivity, increased accuracy, learning ability To promote and reduce stress, anti-ulcer, adrenal disease, low heart rate, reduced blood pressure, increased vascular tone, increased persistence, stimulation of immune system, promotion of fertility, antitoxin, anti-inflammatory, anti-fever, analgesia, aging Includes, but is not limited to, delayed processes, accelerated recovery, anti-cancer treatment, diabetes, asthma, headache, anemia, dyspepsia, impotence, depression and menstrual disorders.

5.8 Medicinal Carrot Pharmaprint ™ Medicinal Carrot Biological and Chemical Pharmaprint ™ are provided in this section using the values specified in Tables 1-10 below. A description of the assay and references to its use can be found in Section 6 below.

5.8.1. Medicinal Carrot Biological Pharmaprint ™ The Medicinal Carrot Biological Pharmaprint ™ is characterized by the biological activity profile set forth in Tables 1-8 below. The activity (%) of PharmaPrint ™ extracts, fractions and target compounds is shown. The calculation of extracts and fractions is 20
Based on an estimate of the molecular weight of gold to zero. In all tables, the concentration of extract or object tested to obtain the indicated percent inhibition is indicated in parentheses. If the concentration is indicated both above the column and as a percentage of the specific inhibition value, the concentration is adapted together with the specific percentage inhibition; when the concentration is indicated at the top of the column, Only the percent inhibition values in that column that do not occur with the specific percent inhibition value are applied.

[0099]

[Table 1]

[0100]

[Table 2]

[0101]

[Table 3]

[0102]

[Table 4]

[0103]

[Table 5]

[0104]

[Table 6]

Using the values from Tables 1-8, as an example, Pharmaprint ™ of medicinal carrots was obtained from fractions 3, 4, 13, and 17 and total extract activity and: preferably in the GABA _A receptor assay. AMPA, PAF-R,
Adrenergic β-nonselective, glutamate NMDA glycine, and MA
O _A may be based on one or more of the assays for biological activity is selected from the assay.

[0106]

[Table 7]

In another embodiment, PharmaPrint ™ can be developed based on biological activity that is equal to or greater than the lower end of the range of biological activity values shown in Tables 1-8, for example. As an illustration of this embodiment, the PharmaPrint ™ value (100 ± 20) based on the biological activity of all extracts in the GABA _A assay is at least 80% inhibition at 10 ⁻⁴ mol (Table 1).

[0108]

[Table 8]

5.8.2. Medicinal Carrot Chemical Pharmaprint ™ The development of Chemical Pharmaprint ™ for medicinal carrots is shown in Tables 9 and 1
Promoted by looking at zero, these specify the range of values for the particular medicinal carrot marker component shown. In many cases, these specific components are tested and show biological activity in the specific bioassays described herein.

[0110]

[Table 9]

Table 10. Chemical Pharma Print

[Table 10]

5.8.3. Conversion ratio PharmaPrint (TM) developed using a dry powdered extract of plant material
The values can be converted to values for the dry weight of the crude plant material using the ratios shown in Table 11 below. That is, the PharmaPrint ™ value based on the dry powdered extract is divided by the appropriate factor in Table 11 to convert it to a value for dry plant material.

[0113]

[Table 11]

The following examples are for illustrative purposes only and are not intended to limit the scope of the invention in any way.

[0115] 6. Examples: PHARMAPRINTING ™ Medicinal Carrot (Asian), Panax Ginseng 6.1. Commercially Available Medicinal Carrots Medicinal carrots are probably the most popular plant products available worldwide. Some products and supplies are Ginsun ™ by Murdock Madaus Schwabe (Springville, UT), GS-500 ™ by Enzymatic Therapy (Green Bay, WI), and Natural Factors Nutritional
Ginseng Softgels (TM) by Products, Ltd. (Burnaby, British Columbia, Canada). Powdered medicinal carrot extract is available from Zuellig Bo
Available from Botanicals International, a division of tanicals, Inc. (Germany). Medicinal carrot dry extract IDB is available from Indena sa (Milano, Italy) and is standardized to contain 7% ginsenoside. Medicinal carrots are also available through the following companies: Shaklee, Richtwer, Sunsourc
e, Nature's Resource, Herbal Choice-Botalia, Nature's Way, Natu
raLife, Herbal Harvest, Bootalia Gold, and PhytoPharmica.

6.2. Fraction analysis Panax Ginseng powder (15 g) was dissolved in deionized water (40 ml) and LiChropre
Load onto a column of pRP-18 (40-63 μm) (2.5 × 92 cm, 450 ml column volume). The column was pre-packed and equilibrated with deionized water. As shown in Table 12 below, the column was batch developed with a water / methanol mixture and finally with ethyl acetate. Aliquots (0.5 ml) from each of the 22 fractions shown in Table 12 were removed for HPLC analysis. The remaining fraction was then evaporated and the residue weighed. The volume of the collection and the weight of the residue of each fraction are listed in Table 12. The total weight of the collected column fractions was 14.6074 g, or 97% of the applied material.

[0117]

[Table 12] Table 12. LiChroprep RP-18 column fraction

[Table 13]

Next, 0.5 ml aliquots of each of the 22 column fractions were further analyzed by HPLC. These HPLC conditions were as follows. Waters μ Bondpak C-18 ODS column (250 cm × 4.6 cm ID) and PDA detection (200-400 nm) with a solvent gradient from 18% acetonitrile to 40% acetonitrile for 75 minutes followed by 100% acetonitrile for 5 minutes Retained.

6.3. Biological activity analysis The putative biological mode of action of medicinal carrots has two aspects. For one thing,
It has an adaptive effect that results in a nonspecific increase in the body's defenses against exogenous stressors and potentially toxic chemicals (Kim
Et al., 1992, Biochem. Biophys. Res. Commun. 189: 670-676).
. On the other hand, it promotes an overall improvement in physical and mental performance (Mohri et al., 1992, Planta Medica 58: 321-323). The immunomodulatory activity of medicinal carrots appears to explain some of its adaptive effects. Intraperitoneal administration of medicinal carrot extract in mice stimulates cell-mediated immunity and results in increased antibody levels against sheep red blood cells and natural killer cells (Singh et al., 1984, Planta Medica 50: 459). According to Ginsenosaido Rg ₁ and Rb _1, normal animals, furthermore, the improvement of memory and learning in animals of cognitive impairment has been reported (Foster, 1996, American Botanical Council , is an overview by Botanical Series-303).

6.3.1. GABA _{A / B} Receptor Antagonism GABA (γ-aminobutyric acid) is the major inhibitory neurotransmitter in the mammalian central nervous system (CNS). When GABA is released from the presynaptic site, it can bind to the receptor or be taken up and metabolized by cells. There are two classes of GABA receptors, GABA _A and GABA _B. While the GABA _A receptor activates chloride channels, it regulates Ca ²⁺ and K ⁺ channels by interacting with intracellular second messengers such as G proteins or adenylate cyclase (Kimura et al. , 1994, Gen. Pharmacol. 25: 193-199). Ginsenosides Rb ₁ , Rb ₂ , Rc, Re, Rt, and Rg all inhibit [ ³ H] muscimol binding to high affinity GABA _A sites. In contrast, only every saponin fraction Contact and Ginsenosaido R _c are you inhibit ^[3 H] baclofen binding to the site.

The GABA _A bioassay for compounds uses a receptor partially purified from bovine cerebellar membrane. The concentration was set to 5 nM [ ³ H] -GABA and 50 mM
The reaction is carried out in Tris buffer (pH 7.4) at 0-4 ° C for 60 minutes. Non-specific binding is measured in reactions with 1 μM GABA. The reaction is terminated by rapid vacuum filtration onto a glass fiber filter. [ ³ H] -GAB to GABA _A receptor
To determine the degree of competition for A-binding, the radioactivity captured on the filter is measured by scintillation counting and compared to a control compound (Enna et al., 197).
7, Brain Research 124: 185-190). Literature compounds and assay properties are listed below. Ginseng extract and reference compound in the fraction is Ginsenosaido _{Rb 1, Rb 2, Rc,} Re, Rf, and Rg (Sigma Chemical Co
mpany or Indofine Chemical Company).

<Control Compound> <Ki (nM)> Muscimol 4.4 Isogubacin 9.5 GABA 23.1 THIP 25.1

<Assay Characteristics> K _D (binding affinity): 370 nM B _MAX (receptor number): 0.7 pmol / mg (protein)

Inhibition of binding to the receptor uses a partially purified receptor obtained from rat cortical membrane. [ ³ H] -GABA ligand is used at a concentration of 5 nM, to which 100 μM isogubacin is added to block binding to GABA _A.
The reaction is carried out in 50 mM Tris (pH 7.4) containing 2.5 mM CaCl ₂ and incubated at 25 ° C. for 60 minutes. The reaction is terminated by rapid vacuum filtration onto a glass fiber filter. The amount of radioactivity captured on the filter was determined by scintillation counting and the value was compared to a control to determine the [ ³ H]
]-Determine whether any competition for GABA binding has occurred (Scherer et al., 1).
988, Brain Res. Bulletin 21: 439-443). Reference compounds are listed below. Extract and fraction standards are the same as those used for the GABA _A assay described above.

[0125]

[Table 14]

[0126]

[Table 15]

6.3.2 Platelet Aggregation Inhibitory Activity It has become clear in recent years that platelets play an important role in the development of atherosclerosis. When endothelial damage occurs, subendothelial collagen is exposed to circulating blood cells, resulting in the accumulation of macrophages,
Platelets collect at the site of injury. In this process, platelets secrete chemicals such as vasoactive substances and platelet-derived growth factor (PDGF). This leads to cell proliferation and smooth muscle cell migration, leading to the development of atherosclerotic disorders

It has been reported that 70% methanol extract of medicinal carrots and ginsenoside Ro prevent the progress of blood coagulation and enhance fibrinolysis (Kuo et al., 1).
990, Planta Medica 56: 164-167). The medicinal carrot extract and its fractions are assayed for inhibition of platelet aggregation as follows.

6.3.2.1. Platelet Aggregation Assay Briefly, venous blood collected from male or female New Zealand-derived albino (albino) rabbits weighing 2.5-3.0 kg was subjected to a tenth volume of trisodium citrate (0.13 mol). And then centrifuged at 220 G for 10 minutes at room temperature. The resulting supernatant is platelet rich plasma (PRP). It is subjected to irreversible aggregation with 200 μM sodium arachidonic acid incubated at 37 ° C. Aggregation is measured with an optical platelet aggregometer. 30 μM test material was added to 5
Incubate with PRP for minutes. The percent inhibition on platelet aggregation is determined. Reference standards are listed below. The assay is based on a study by Barthele et al. (1983, Science 220: 517-519).

[0130]

[Table 16]

Indicates the standard reference drug used. BW-755C = 3-amino-1- [3-
(Trifluoromethyl) phenyl] -2-pyrazolin, CGS-12970 = 3
-Methyl-2- (3-pyridinyl) -1H-indole-1-octanoic acid, ND
GA = Nordihydroguaiaretic acid.

In addition to inducing platelet aggregation by using 200 μM sodium arachidonic acid,
A 5 nanomolar platelet activating factor-aceser (PAF-aceser) is used (Nunetsu, 1986, Eur. J. Pharmacol 123: 197-205). Reference compounds are listed below.

[0133]

[Table 17]

CGS-12970 = 3-methyl-2- (3-pyridinyl) -1H-indole-1-octanoic acid, CV-3988 = 3- (4-hydroxy-7-methoxy-
10-oxo-3,5,9-trixa-11-aza-4-phosphanonacos-1-
Yl) -thiazolinium, L-652731 = 2R, 5R-di (3,4,5-trimethoxyphenyl).

Other assays described in the literature may also be used. Keith Wetter et al. (1
991, Int'l J. Clin. Pharm. Ther. & Toxicol. 29: 151-155) report on other techniques for assessing thrombocoagulation. Another assay for venous insufficiency is a clinical indicator of vasodilator inhibitory effect. This is done by examining the contractile response of coronary artery segments to acetylcholine (Bettini et al., 1991, Fi
toterapia 62 (1): 15-28).

6.3.2.2 PAF Receptor (PAF-R) Assay In this assay, the binding of [ ³ H] -platelet activating factor (PAF) to the PAF receptor was measured to extract ginseng extracts and fractions. Assay. 1 sample
Screen at 0 μM. Albinola rabbit from New Zealand (weight 2.5-
3.0 kg) of male or female platelets are prepared in modified Tris-HCl (pH 7.5) buffer using standard techniques. A 50 μg aliquot of the membrane is incubated for 60 minutes at 25 ° C. with 0.4 nM [ ³ H] -PAF. Non-specific binding is assessed in the presence of 1 μM PAF. The membrane is filtered, washed three times, and the filters are measured to assay for [ ³ H] -PAF specific binding. As a description of the assay, Hwang et al. (1983
Biochemistry 22: 4756-4763).

Similarly, in a bioassay, 1 nM [ ³ H] -hexadecyl-2-acetyl-sn-glycerol-3-phosphorylcholine and 50 mM HEPES buffer (pH 7.0) containing 0.25% BSA. At 0 ° C. for 2 hours. Unbound ligand is separated from bound by passing through a glass filter. The trapped radioactivity was measured by scintillation (modified from Hwang et al., 1985, J. Biological Chemistry 260: 15639-15645).

6.3.3. Activation of lymphocytes and PC12 cells The lipophilic fraction of medicinal carrots differentiates PC12 cells (Mohri et al., 1992, Plas.
nta Medica 58: 321-323) and alcohol-stimulated cell extraction of the immune system (Singh
et al., 1984, Planta Medica 50: 459).

For assaying the para-neuronal cell line PC12, cells were plated in 35 mm collagen-coated sterile tissue culture plates with Dulbecco's modified basal medium supplemented with 5% horse serum and 5% fetal calf serum. (DMEM). Extracts or fraction compounds are diluted in DMSO and tested at the appropriate concentration. To assess neurite outgrowth, the entire range of cells was measured daily using a computational image processor with a phase contrast microscope (XL-500, Olympus, Japan) for the process from the PC12 cell test (neurites) to assess neurite outgrowth. Quantify. As many as 20 to 30 selected cells, up to five ranges, are assayed in the appropriate sample (Mohri, 1992, Planta Medica 58:32).
1-323).

The modulation of T and B lymphocyte proliferation is assayed as follows. T-cells isolated from mouse thymus using standard techniques are cultured in DMEM at 37 ° C. at an initial cell concentration of 5 × 10 ⁶ cells / ml. The test compound is evaluated by diluting 10-fold from 10 μM to 0.001 μM. After incubating at 37 ° C for 15 hours,
Add 2 μCi [ ³ H] -thymidine to the medium. After an additional 48 hours of incubation, cells are harvested and the amount of incorporated thymidine is measured by scintillation (Day
ton et al., 1992, Mol. Pharmacol. 41: 671-676). The literature reference compounds are described below. Literature reference excerpts and fractional compounds are approximately the same section on antagonism of GABA _{A / B} receptors.

[0141]

[Table 18]

To assay for changes in B cell growth by a medicinal carrot derived compound, cells are isolated from mouse spleens using standard techniques. Cells are placed in a DMEM suspension
Culture at a concentration of 10 ⁶ cells / ml. Compounds are diluted in DMSO and tested at 10-fold dilutions ranging from 10 μM to 0.001 μM. After incubation for 15 hours, 2 μCi [ ³ H] -thymidine is added. Cells are harvested 48 hours later and thymidine incorporation is measured by scintillation (Dayton, 1992, Mol. Pharmacol. 41: 671-676). The literature reference compounds are described below. The same excerpt and fraction compounds are assayed as described in the section above on antagonism of GABA _{A / B} receptors.

[0143]

[Table 19]

6.3.4. Antidepressant (tetrabenazine) assay Thirty minutes after injection of tetrabenazine methanesulfonic acid (100 mg / kg intraperitoneally), medicinal carrot, medicinal carrot extract, medicinal carrot extract fraction or vehicle control was
Groups of ICR-induced mice weighing 22 ± 2 g are orally administered (30 mg / kg po). Body temperature is recorded after 60 minutes. A 50% or greater reduction in tetrabenazine-induced hypothermic response is considered important and may exhibit antidepressant activity (Gylys et al., 196
3, Annals NY Acad. Sci. 107: 899-913).

[Table 20] * Indicates the standard reference drug used

6.3.5. Anti-Stress Assay Biological analysis of medicinal carrots for clinical indication to relieve stress using PC
Performed in assays that measure activation of 12 cells. The procedure to follow is described in Mohri et al.,
1992, Planta Med. 58: 321-323. They reported that the lipophilic component of medicinal carrots could activate neurons in their model.

Alternatively, the anti-stress activity of medicinal carrots is evaluated in an in vivo rat or mouse model. Experiments to study the medicinal carrot components in these animal models
Nabata et al., 1973, Japan J. Pharmacol. 23: 29-41. They reported the effects of various active assays using mice and rats, including condition avoidance tests, motor coordination tests and pole climb tests.
ng test).

In addition, a receptor binding assay that evaluates a medicinal carrot-induced anti-stress material may be performed. Target receptors include, but are not limited to, GABA _A and GABA _B receptors as described above. Receptor binding assays can be performed by any of a myriad of methods known to those skilled in the art, for example, Kimura et al., 1994, Gen. Pharma.
col. 25 (1): 193-199.

6.3.6. GABA _A and PAF Receptor Bioassay Summary Table Bioassay using two extracts, 18 fractions and 7 reference standards, G
Either the antagonism of the binding of the ligand GABA to the ABA _A receptor or the antagonism of PAF to the receptor was measured (Table 13). The first extract, PG101, consists of protrusions and fibers (PE 5% N, East Earth Harb, Eugene,
OR) is a Chinese medicinal carrot root extract prepared from The second extract is E
Medicinal carrot dry extract obtained from uromed, USA (Pittsburgh, PA). An extract fraction was obtained from Hauser (Boulder, co) and pure ginsenoside was obtained from Sigma (S
t. Louis, MO) or from the Indofine Chemical Company (Sommerville, NJ).

Table 13. Bioassay summary table reporting reported extracts, fractions and% inhibition by reference compound in GABA _A receptor and PAF receptor assays

[Table 21]

[Table 22]

[Table 23]

6.3.7. Additional Bioassays 26 additional bioassays were performed on medicinal carrot extracts or reference compounds as described in the following paragraphs. The results are summarized in Tables 14-18 below. The dashes in Tables 13 and 14-18 above indicate that no activity was observed (0% inhibition or activity) at that test concentration.

Inhibition of [ ³ H] -pirenzepine on muscarinic M1 receptor by substances
The measurement was performed using a receptor partially purified from bovine striatal membrane. Final hot ligand concentration was 1 nM and non-specific binding was measured in the presence of 100 nM atropine. The reaction was performed in 25 mM HEPES (pH 7.4) at 25 ° C. for 60 minutes. The reaction was stopped by rapid vacuum filtration of the reaction mixture through a glass fiber filter. Bound radioactivity was measured by liquid scintillation counting (Watson
et al., 1983, Life Sciences 32: 3001-3011).

To assay for glutamate receptors, two assays were performed. In the first assay, the agonist site of the glutamate receptor was studied (NMDA). Here, the receptor was a partially purified substance produced from rat forebrain. The radioligand has a final ligand concentration of 2 nM [ ³ H] CGP-
39653. Non-specific binding was measured using 1 mM NMDA. 5
The assay reaction was performed in 0 nM Tris-acetate (pH 7.4) at 0-4 ° C. for 60 minutes. The reaction was terminated by rapid vacuum filtration of the reaction mixture through a glass fiber filter (Lehmann, J. et al., 1988, J. Pharmac. Exp. T.
her. 246: 65-75). In a second assay for glutamate receptors, reactions were performed using [ ³ H] -AMPA at a final concentration of 5 nM. 100
Non-specific binding was measured using μM AMPA. During _{10mM K 2 HPO 4/1 00mM} KSCN (pH 7.5), was performed the assay reaction for 60 minutes at 0-4 ° C.. The reaction was terminated by rapid vacuum filtration through a glass fiber filter
(Murphy et al., 1987, Neurochem. Res. 12: 775-781).

To determine the inhibition of the cholecystokinin receptor of the central nervous system (CCK _B ), a receptor preparation partially purified from the mouse forebrain membrane was prepared. Final concentration 0
. Nonspecific binding was determined using 02 nM [ ¹²⁵ I] -cholecystokinin in the presence of 1 μM cholecystokinin sulfate 8. The reaction was performed with 360 mM NaCl, 15 mM K
This was performed at 25 ° C. for 120 minutes in 20 mM HEPES containing Cl, 5 mM MgCl ₂ , 1 mM EGTA and 0.25 mg / ml bacitracin (pH 6.5). The reaction was stopped by rapid vacuum filtration through a glass fiber filter (Wennogl
e et al., 1985, Life Sciences 36: 1485-1492).

Inhibition of MAO _A enzyme activity (MAO _A ) was measured using rat liver mitochondrial membrane as a partially purified enzyme source. The substrate was [ ¹⁴ C] -serotonin, and the non-specific activity was measured using 1 μM Ro 41-1049. The reaction depends on the substrate [ ¹⁴ C]-
Conversion to 5-hydroxyindole acetaldehyde + NH ₄ ⁺ is included. Simply, the enzyme is converted to the target substance and the special-specific blocker diprenyl (300 nM
) And preincubation at 37 ° C. for 60 minutes in 100 mM KPO ₄ (pH 7.2). Add substrate and incubate for another 10 minutes. The reaction is 2M
Stop by adding 0.5 ml of citric acid. The radioactive product is extracted into toluene / ethyl acetate fluor and compared to control samples using scintillation spectrophotometry (Otsuka, S. and Kobayashi, Y., 1964, Biochem.
Pharmacol. 13: 995-1006). Inhibition of MAO _B activity (MAO _B) was also measured using a rat liver mitochondrial membranes as the enzyme source of partially purified. The substrate was [ ¹⁴ C] -phenylethylamine. Non-specific activity was measured in the presence of 1 μM Ro 166491. Simply enzymatically transform the enzyme into a specific-selective blocker clorgyline (300
nM) and 100 mM KHPO ₄ (pH 7.2) for 60 minutes at 37 ° C. The substrate is then added and incubated for 7 minutes. The reaction is
Stop by adding 0.5 ml of 2M citric acid. Then, the radioactivity produced was assayed for MAO _A enzyme (Otsuka, S. and Kobayashi, Y. , 1964,
Biochem. Pharmacology 13: 995-1006).

In order to measure the inhibitory activity of the target substance on dopamine receptor (dp), the following assay was performed. The partially purified receptor from bovine striatal membranes, [H ^3] - and used at a final concentration of 0.3nM as ligand spiperone was prepared. Cold spiperone was tested at 1 μM to determine non-specific binding. 120
5 containing 5 mM NaCl, 5 mM KCl, 2 mM CaCl ₂ and 1 mM MgCl ₂
The reaction was carried out in 0 mM Tris HCl (pH 7.7) at 37 ° C. for 60 minutes. The reaction was terminated by rapid vacuum filtration of the reaction mixture through a glass fiber filter (leysen
et al., 1978, Biochem. Pharmacol. 27: 307-316).

The inhibitory properties of substances that are ligands that bind to the adenosine receptor (ADNS)
Measurements were made using a partially purified receptor made from bovine striatal membrane. The radioactive ligand used was [ ³ H] -5′-N-ethylcarboxyamide adenosine (NECA) at a final concentration of 4 nM. Non-specific binding was measured in the presence of 10 μM NECA. The reaction was performed in 50 mM Tris-HCl (pH 7.7) for 60 minutes at 25 ° C. The reaction was stopped by rapid vacuum filtration of the reaction mixture through a glass fiber filter (Bruns, R. et al., 1986 Pharmacology 29: 331-346).

To measure the inhibitory activity of the opiate receptor (Opiate) substance, a partially purified product derived from rat forebrain was used. 1 nM [ ³ H] -naloxone was the ligand used. Non-specific binding was measured in the presence of 1 μM naloxone. The assay was performed in 50 mM Tris-HCl (pH 7.4) at 25 ° C. for 90 minutes. The reaction was stopped by rapid vacuum filtration of the reaction mixture through a glass fiber filter (Pert, C. and
Snyder, SH, 1974, Mol. Pharmacology 19: 868-879).

To measure inhibition of the corticotropin releasing factor (CRF) receptor, a partially purified receptor from rat cortical membrane was used. The radioligand used was [ ¹²⁵ I] -Tyr-CRF with a final ligand concentration of 0.1 nM. Non-specific binding was measured in the presence of 1 μM Tyr-CRF. The reaction was 10 mM MgCl ₂ , 2 m
Performed at 25 ° C. for 120 minutes in 50 mM HEPES containing MEGTA, 0.12 TIU / ml aprotinin and 0.3% BSA. The reaction is stopped by centrifugation at 4 ° C. for 15 minutes. After repeated washing, the resulting pellet is dissolved, and the radioactivity is measured using a gamma counter (De Souza, EB, 1987 J
Neuroscience 7: 88-100).

To determine the effects mediated by the adrenergic α ₂ receptor (adrenergic α ₂ ), partially purified receptor preparations were prepared from whole rat brain. Radioligand used was, [6,7- ³ H] - triamcinolone acetonide, by a final ligand concentration 1 nM. Non-specific binding was determined in the presence of 10 μM triamcinolone acetonide. Assay reaction is carried out in 10mM sodium molybdate and 10mMα- monothioglycerol containing _{50mM KH 2 PO 4 (pH7.4)} , was carried out for 16 hours at 0 ° C.. The reaction was stopped by rapid vacuum filtration of the reaction through a glass fiber filter. Bound radioactivity was determined by liquid scintillation counting (Da Han et al., 1994, Neurochem. Int. 24: 339-
348).

The inhibitory activity of a substance on the thromboxane A ₂ (thromboxane A ₂ ) receptor is:
The measurement was performed using a partially purified receptor preparation from human platelets. The radioligand [ ³ H] -SQ29,548 was used at a final concentration of 2 nM. Non-specific binding is 1
It was determined by adding 0 μM pinane-thromboxane. The assay response is 138
The reaction was performed at 25 ° C. for 60 minutes in 25 mM Tris-HCl (pH 7.4) containing mM NaCl, 5 mM KCl, 5 mM MgCl ₂ , 5.5 mM dextrose and 2 mM EDTA. The reaction was stopped by rapid vacuum filtration of the reaction through a glass fiber filter. Bound radioactivity was measured by liquid scintillation counting (Hedbert, A. et al., 1988, J. Pharmacol. Exp. Ther. 245: 786-792).
).

[0161] was prepared from guinea pig spleen membranes, using partially purified preparations were measured inhibitory substance for leukotriene B ₄ receptor. Radioactive Li ligands used were the final concentration was in a ^[3 H] leukotriene B ₄ 0.5 nM. The nonspecific binding was determined in the addition of 500nM leukotriene B _4. The assay reaction was performed at 0 ° C. for 2 hours in a phosphate buffer (pH 7.4) containing NaCl, MgCl ₂ , EDTA, and bacitrin. The reaction was terminated by rapid vacuum filtration of the reaction through a glass fiber filter. Bound radioactivity was determined by liquid scintillation counting (Gardiner, PJ, et al., Eur. J. Pharmac. 182: 291-299, 1990).

The anti-inflammatory effect of the substance was further demonstrated by the phospholipase A partially purified from porcine pancreas. _Two Tested by analysis of enzyme inhibition. Briefly, the enzyme,¹⁴C] -3-Phosphatidylcholine in 100 mM glycine-NaOH buffer (pH 9) for 10 min.
Incubate with Reaction was performed at 2.5 mM Ca⁺⁺And incubate for 5 minutes. The reaction is stopped by adding 200 mM EDTA. Raw
The product is extracted with acidic hexane and the amount of radioactivity present is used for scintillation counting.
(Katsumata, M. et al., Anal. Biochem. 154: 676-681, 1986).

To determine how the substance affects the binding of interleukin-8 (IL-8) to its receptor, a partially purified preparation of the receptor was prepared from human neutrophils. The reaction is performed in a modified Tris-HCl (pH 7.5) buffer. Briefly, a 30 μg preparation of the crude receptor was added to 15 pM [ ¹²⁵ I] IL-8 for 120 minutes at 0 ° C.
Incubate with Non-specific activity is measured by reactions containing 250 nM IL-8. The membrane is filtered through a glass filter and washed three times before measuring the amount of trapped radioactivity (Grob, PM et al., J. Biol. Chem. 265: 8311-83).
16, 1990).

Another assay for investigating the anti-inflammatory properties of a substance involves the investigation with the enzyme 5-lipoxygenase (5-lipoxygenase). Crude enzyme is prepared from rabbit basophil leukemia cells (RB-1). The material is pre-incubated with the crude enzyme preparation for 5 minutes at 25 ° C. The reaction is then started by the addition of [ ¹⁴ C] -arachidonic acid.
After 8 minutes, the reaction is stopped by the addition of citric acid. The amount of radiolabeled 5-HETE is determined by radioimmunoassay (RIA) (Shimizu, T. et al., Proc. Natl. Aca
d. Sci. USA 81: 689-693, 1984). The leukotriene C ₄ (LTC ₄₎ synthase is related enzyme is assayed using the same tissue source with respect to crude enzyme preparation. L
Methyl ester of TC _4, albumin and Serinboreto presence, 15 minutes, incubated with crude enzyme at 15 ° C.. The reaction is stopped by the addition of ice-cold methanol. The formation of LTC _4, using the RIA readout method, take as an index of enzyme activity (Bach et al, Biochem Pharmacol 34 :... 2695-2704, 1985).

The inhibitory activity of the substance was seen at two channel receptors. First, Ca²⁺ Test for activated, volt-insensitive potassium channel receptor (K channel)
did. Crude receptor preparations were prepared from rat forebrain and a final concentration of 0.05 nM [ ¹²⁵ I] -apamine ligand was used. Non-specific binding was determined with 100 nM apamin
Measured in the presence. The assay reaction was performed in 50 mM Tris-HCl (pH 7.4) containing 0.1% BSA and 5 mM KCl at 0-4 ° C. for 60 minutes. The reaction was stopped by rapid vacuum filtration of the reaction through a glass fiber filter. Conclusion
Radioactivity was measured by gamma counting (Seager, M. et al., J. Neuroscience 7:56
5-570, 1987).

The second channel where activity was seen is the sodium channel, site 2 (Na channel). Crude receptor preparations were prepared from rat forebrain. Radioligand [H ³ ] -batrachotoxin was used at a final concentration of 2 nM. Non-specific binding was determined in the presence of 100 nM aconitine. 50 nM H containing 130 mM choline chloride
The assay reaction was performed in EPES (pH 7.4) at 37 ° C. for 60 minutes. The reaction was terminated by quick vacuum filtration of the reaction mixture through a glass fiber filter and the specific activity was measured by scintillation (Creveling, CR Mol. Pharmacology 23: 35).
0-358, 1983).

An additional bioassay for the activity exhibited by the substance is angiotensin II, type 2,
Central (AT ₂ ). Partially purified receptors were prepared from bovine cerebellar membranes using [ ¹²⁵ I] -Tyr ⁴ -angiotensin as a radiolabeled ligand at a final concentration of 0.1 nM. Non-specific binding was determined in the presence of 50 nM of human angiotensin II. The assay reaction was carried out at 37 ° C. for 60 minutes in a phosphate buffer (pH 7.4) containing NaCl, EDTA, and BSA. The reaction mixture was passed through a glass fiber filter, the reaction was terminated by rapid vacuum filtration, and the specific activity was measured by gamma (Bennett J
.P. And Synder, J. Bil. Chem. 251: 7423-7430, 1976).

Another set of receptors that exhibit activity on substances are histamine, H ₁ (H1) and histamine H ₂ (H2). Crude receptor preparations for the H1 receptor were prepared from bovine cerebellar membrane. A final concentration of 2 nM radioligand [ ³ H] -pyrylamine was used. Non-specific binding was determined in the presence of 10 μM triprolidine.
The assay reaction was performed at 25 ° C. in 50 mM Na-KPO ₄ (pH 7.5) buffer at 60 ° C.
Went in minutes. The reaction was stopped by rapid vacuum filtration of the reaction through a glass fiber filter and specific activity was counted by liquid scintillation counting (Chang, et al.
J. Neurochemistry 32: 1653-1663, 1979).

H2 crude receptor preparation was prepared from guinea pig striatal membrane. Radioligand [H ^3] tiotidine the (tiotdine), used at a final concentration of 4 nM, Non-specific binding was determined in the presence of 10mM cimetidine. The assay reaction was performed in the same buffer as the H1 receptor. The reaction was terminated by rapid vacuum filtration of the reaction mixture through a glass fiber filter and specific activity was determined by liquid scintillation counting (Gajtkowski,
et al., Nature 304: 65-67, 1983). Crude receptor preparations were prepared from rat cortical membranes for determination of the activity of the substance on the β-adrenergic, non-selective, receptor assay (adrenergic β, NS). The radioligand used was [ ³ H] -DHA at a final concentration of 2 nM. 10 μM
It was measured in reactions performed in the presence of alprenolol. The assay reaction was run at 50 mM
Performed in Tris-HCl (pH 7.4) at 37 ° C. for 60 minutes. The reaction was stopped by rapid vacuum filtration of the reaction through a glass fiber filter and specific activity was counted by liquid scintillation counting (Riva, M. and Creese, I. Mol. Pharmacol. 36:21).
1-218, 1989).

The final receptor assay showing inhibition by some of the substances was the serotonin receptor. Crude receptor preparations were prepared from rat cortical membranes. Radioligand [ ³ H] -lysergic acid diethylamide with a final ligand concentration of 5 nM was used. Assay reactions were performed in 50 mM Tris-HCl (pH 7.4) containing 4 mM CaCl ₂ , 0.1 mM pargyline and 0.1% ascorbic acid at 37 ° C. for 60 minutes. The reaction was stopped by rapid vacuum filtration of the reaction through a glass fiber filter and specific activity was counted by liquid scintillation counting (Peroutka, SJ and Sny
der, SH Mol. Pharmacology 16: 687-699, 1979).

Chemical analysis of medicinal carrots was performed by HPLC or TLC. Chromatographic peaks may be further analyzed using mass spectroscopy. Medicated carrots
Contains various components, including but not limited to: sterols (β-sitosterol and β-glucoside), 7-9% medicinal carrot polysaccharide, panaxins AU, pectin, free sugar, Biomin, polyacetylin and polypeptide. Sappinins are called ginsenosides by Japanese researchers and panaxosides by Russian researchers. There are at least 18 sappines found in Asian medicinal carrots. It is all a triterpenide. Six types of panaxoside have been reported. It has also been reported that medicinal carrot oils contain sesquiterpenes, and there are at least 56 closely related saponins called ginosaponins [Leung and Foster, 199
6].

An example of a chemical analysis of ginsenoside is shown below. Korean carrot extract capsule (
Six samples (6) of Panax Ginseng Extract Capseule) were represented as follows: Brand A, extract soft gel capsule number 611251; Brand B, extract soft gel capsule number GG 10527; Brand C, root Powder soft gel capsule No. HC 10999; brand D, extract (4%) soft gel capsule no. 50959AA; brand E, extract (4: 1) soft gel capsule no. 7B04201; and extract soft gel capsule no. 5N03338; brand F.

The HPLC measurement of ginsenoside in the capsule was performed as follows. The average weight of the capsule was measured. Hauser Part Number 4129.000 (dissolved in extraction solvent and modified to eliminate the solid phase washing step during gelatin capsule sample preparation)
Analyzed by HPLC by Boulder, Co.). It was based on the column response to each of the six ginsenoside standards (Rg1, Re, Rb1, Rc, Rb2, Rd) obtained from Indofine Chemical Company (So
mmervill, NJ). The result is shown in FIG.

The concentrations shown in FIG. 4 represent the average of two independent measurements based on the average capsule content weight for each sample. Comparing the results obtained by including the above solid phase extraction washing step, the results do not differ by more than 10%. The values plotted in FIG. 4 are shown in Table 19 below.

[0175]

[Table 24]

[Table 25]

[0176]

[Table 26]

[Table 27]

[0177]

[Table 28]

[Table 29]

[0178]

[Table 30]

[Table 31]

[0179]

[Table 32]

[Table 33]

[0180]

[Table 34]

The description and claims of the invention herein are not intended to limit its scope by the specific embodiments disclosed in the specification, which illustrate some aspects of the invention. It is intended. Any equivalent embodiments are intended to be within the scope of this invention. Indeed, various modifications of the invention in addition to those shown or described herein will become apparent to those skilled in the art from the foregoing description. Also, such modifications are intended to be within the scope of the appended claims. Throughout the specification, various publications and patents are referenced in parentheses. Here, its contents are incorporated by reference in this specification.

[Brief description of the drawings]

FIG. 1 shows a schematic representation of the method of the invention used to establish a standard chemistry and / or bioactive fingerprint comparing a subsequently processed botanical material during the manufacture of a pharmaceutical grade drug.

FIG. 2 shows a schematic of the method of the invention used to process botanical raw materials into pharmaceutical grade drugs.

FIG. 3 shows a schematic of a method for isolating different types of biologically active ingredients.

FIG. 4 is a comparison of six commercial products, namely medicated carrot products, expressed as percentages (%) of the indicated specific substances (compounds) as measured by brands AF. Abbreviations: Rg1, ginsenoside Rg ₁ ; Re, ginsenoside Re; Rb1, ginsenoside Rb ₁ ; Rc, ginsenoside Rc; Rb2, ginsenoside Rb ₂ ; R
d, ginsenoside Rd; Rf, ginsenoside Rf (not shown).

──────────────────────────────────────────────────続 き Continuation of front page (81) Designated country EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE ), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CU, CZ, DE, DK, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IS, JP, KE, KG , KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, UA, UG, US, UZ, VN, YU, ZW (72) Inventor Tasneem A. Kwaja Corona del Mar, California 92625 California , Monterrey Circle 7 (72) Inventor Elliott P. Friedman Belize 2160, Montezito, California 93108 F-term (reference) 4C086 AA01 AA02 EA08 MA01 MA04 NA20 ZA18 ZA36 ZA66 ZB11 ZC21 4C088 AB18 AC11 BA12 BA13 BA32 BA33 CA03 CA05 CA06 CA09 MA02 NA20 ZA18 ZA36 ZA66 ZB11 ZC21

Claims (33)

[Claims] 1. A process for the manufacture of a pharmaceutical grade carrot, comprising the steps of: combining a representative aliquot of a carrot material comprising a plurality of components with at least one marker fraction having at least one active ingredient. Separating the marker fractions, assaying the intensity of the bioactivity of each marker fraction to obtain a bioactive fingerprint of the representative aliquot, and converting the bioactive fingerprint of the representative aliquot to a pharmaceutical grade medicinal carrot. Compared to established bioactivity fingerprint standards for
Determining whether the medicinal carrot material is a pharmaceutical grade medicinal carrot. 2. The process for producing a pharmaceutical grade Korean ginseng according to claim 1, wherein one or more marker fractions comprises at least one active ingredient. . 3. As an additional step, assaying the amount of active ingredient in at least one marker fraction to obtain a quantitative composition fingerprint of said representative aliquot, and a quantitative composition fingerprint of said representative aliquot. Comparing the print to a quantitative composition fingerprint standard established for a given pharmaceutical grade medicinal carrot to determine if the medicinal carrot material is a pharmaceutical grade medicinal carrot. 1. A method for producing a pharmaceutical grade medicinal carrot according to 1. . 4. As an additional step, assaying the total biological activity of a representative aliquot of the medicinal carrot material and comparing the total biological activity of the representative aliquot to the total biological activity of the biological activity fingerprint standard. Determining whether the medicinal carrot material is a medicinal carrot. The method of claim 1, wherein the medicinal carrot material is a medicinal carrot. 5. The production of a pharmaceutical grade medicinal carrot according to claim 1, wherein said medicinal carrot material is a supercritical carbon dioxide extract, an ethanol extract, an aqueous extract, an organic extract, an oily substance or a powdered plant. Law. 6. The method for producing a pharmaceutical grade medical carrot according to claim 1, wherein said medical carrot material is a homogeneous material. . 7. The method for producing a pharmaceutical grade carrot according to claim 1, wherein said carrot material is a mixture of plants. 8. The process for producing pharmaceutical grade medicinal carrots according to claim 7, wherein the plant mixture comprises at least 10% by weight of medicinal carrot roots. 9. The method of claim 1, wherein the at least one active ingredient is ginsenoside, a carbohydrate,
5. The method for producing pharmaceutical grade medicinal carrot according to claim 1,2,3 or 4 selected from the group consisting of fatty acids, fatty acid esters, phenols and terpenoids. 10. The bioactive fingerprint treats an abnormality or disease selected from the group consisting of a stress disease, an inflammatory disease, a cardiovascular disease, a gastrointestinal disease, a metabolic disease and an adrenal disease, Or direct its use to improve,
A process for producing a pharmaceutical grade medicinal carrot according to claim 1. 11. A process for producing a pharmaceutical grade carrot, comprising a plurality of components having a predetermined biological activity, each component comprising a medicinal carrot material having a standard bioactivity profile, wherein a representative of the medicinal carrot material. Dividing the target aliquot into a plurality of marker fractions, wherein at least one marker fraction has at least one active ingredient, determining the amount of each active ingredient in each marker fraction, and determining the amount of each active ingredient present Calculating the bioactivity of each marker fraction based on the amount and the standard bioactivity profile to obtain a calculated bioactivity fingerprint of the representative aliquot, and a calculated bioactivity fingerprint of the representative aliquot The print is compared to a bioactive fingerprint standard established for pharmaceutical grade medicinal carrots and the medicinal carrot is Material to determine whether the ginseng pharmaceutical grade, which method comprises. 12. As an additional step, assaying the total biological activity of a representative aliquot of the medicinal carrot material and comparing the total biological activity of the representative aliquot to a standard total biological activity. 12. A method for producing a pharmaceutical grade medicinal carrot according to claim 11, comprising determining whether the material is a pharmaceutical grade medicinal carrot. 13. The method for producing a pharmaceutical grade carrot according to claim 11, wherein said medicinal carrot material is an extract. 14. The method for producing a pharmaceutical grade medicinal carrot according to claim 13, wherein said extract is an aqueous or organic extract. 15. The medicinal carrot material is a powdered plant.
For producing pharmaceutical grade medicinal carrots. 16. The method for producing pharmaceutical grade medical carrot according to claim 11, wherein said medical carrot material is a homogeneous material. 17. The medicinal carrot material is a mixture of plants.
1. A method for producing a pharmaceutical grade medicinal carrot according to 1. 18. The method of producing a pharmaceutical grade of carrot according to claim 17, wherein the mixture of plants comprises at least 10% by weight of the root of the carrot. 19. The method for producing a pharmaceutical grade medicinal carrot according to claim 11 or 12, wherein said active ingredient is selected from the group consisting of ginsenoside, carbohydrate, fatty acid, fatty acid ester, phenol and terpenoid. 20. The biological activity treats or ameliorates an abnormality or disease selected from the group consisting of a stress disease, an inflammatory disease, a cardiovascular disease, a gastrointestinal disease, a metabolic disease and an adrenal disease. A process for the manufacture of a pharmaceutical grade medicinal carrot according to claim 11, wherein the use is indicated for use. 21. A method for producing a pharmaceutical grade carrot, comprising using a medicinal carrot material having a predetermined biological activity and comprising a plurality of components, wherein at least one representative aliquot of the medicinal carrot material is used. Dividing the marker fraction into a plurality of marker fractions containing at least one active ingredient, assaying the strength of the predetermined biological activity of each marker fraction to obtain a bioactive fingerprint of the representative aliquot; Comparing the bioactive fingerprint of the representative aliquot to a bioactive fingerprint standard established for pharmaceutical grade medicinal carrots and determining whether the medicinal carrot material is a medicinal grade medicinal carrot. Method. 22. The method of producing a pharmaceutical grade carrot according to claim 21, wherein said active ingredient is selected from the group consisting of ginsenoside, carbohydrates, fatty acids, fatty acid esters, phenols and terpenoids. 23. A method of making pharmaceutical grade medicinal carrots, comprising the steps of determining the total biological activity of a representative aliquot by a platelet activating factor receptor assay, GAB
The medicinal carrot material is assayed by a bioassay selected from the group consisting of an A _A receptor assay, a glutamate receptor assay, and a phospholipase A ₂ assay, and comparing the total biological activity of the representative aliquot to its standard. Determining whether it is a pharmaceutical grade medicinal carrot. 24. The method of producing a pharmaceutical grade medicinal carrot according to claim 1, 11, 21 or 23, wherein one or more marker fractions comprises a group of related components. 25. A method for producing pharmaceutical grade carrots, comprising assaying the amount of active ingredient in at least one of the plurality of marker fractions of the medicinal carrot material to determine the quantitative composition fingerprint of the representative aliquot. And comparing the quantitative composition fingerprint of the representative aliquot to a quantitative composition fingerprint standard established for pharmaceutical grade medicinal carrots, to determine whether the medicinal carrot material is a pharmaceutical grade medicinal carrot. How to determine if a method, including steps. 26. A method of making a pharmaceutical grade carrot, comprising: assaying the total bioactivity of a representative aliquot of the medicinal carrot material; and measuring the total bioactivity of the representative aliquot with a total bioactivity fingerprint standard. Comparing to determine whether the medicinal carrot material is a pharmaceutical grade medicinal carrot. 27. Ginsenoside is Ginsenoside Rc, Ginsenoside Re,
Ginsenoside Rb1, Ginsenoside Rf, Ginsenoside Rd, Ginsenoside Rb2
And a method for producing medicinal grade medicinal carrots according to claim 9, which is selected from the group consisting of ginsenoside Rg1. . 28. Ginsenoside is Ginsenoside Rc, Ginsenoside Re,
Ginsenoside Rb1, Ginsenoside Rf, Ginsenoside Rd, Ginsenoside Rb2
20. The method for producing a medicinal grade carrot according to claim 19, wherein the carrot is selected from the group consisting of ginsenoside Rg1. 29. The ginsenoside is ginsenoside Rc, ginsenoside Re,
Ginsenoside Rb1, Ginsenoside Rf, Ginsenoside Rd, Ginsenoside Rb2
23. A method for producing a medicinal grade medicinal carrot according to claim 22, which is selected from the group consisting of ginsenoside Rg1. . 30. Pharmaceutical grade medicinal carrot produced by the method of claim 1, 11, 21, 23, 25 or 26. 31. The method for producing a pharmaceutical grade medicinal carrot according to claim 1, wherein one or more marker fractions comprises at least two active ingredients. 32. The method according to claim 1, wherein the at least one marker fraction comprises at least one component selected from the group consisting of ginsenoside Rb1, ginsenoside Rg1, γ-aminobutyric acid, glutamic acid, glutamine and proline. A method for producing pharmaceutical grade medicinal carrots. 33. The method of producing a pharmaceutical grade of a medicinal carrot according to claim 1, 11 or 21, wherein the at least one active ingredient is selected from the group consisting of ginsenoside Rb1, ginsenoside Rg1 and γ-aminobutyric acid. .