EP1494689A1 - Procede d'extraction de composes organiques de racines de plantes - Google Patents

Procede d'extraction de composes organiques de racines de plantes

Info

Publication number
EP1494689A1
EP1494689A1 EP01274852A EP01274852A EP1494689A1 EP 1494689 A1 EP1494689 A1 EP 1494689A1 EP 01274852 A EP01274852 A EP 01274852A EP 01274852 A EP01274852 A EP 01274852A EP 1494689 A1 EP1494689 A1 EP 1494689A1
Authority
EP
European Patent Office
Prior art keywords
plant
process according
root system
aqueous medium
intact
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP01274852A
Other languages
German (de)
English (en)
Inventor
Barry L. Marrs
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Pharmaleads Inc
Original Assignee
Pharmaleads Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Pharmaleads Inc filed Critical Pharmaleads Inc
Publication of EP1494689A1 publication Critical patent/EP1494689A1/fr
Withdrawn legal-status Critical Current

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines

Definitions

  • This invention relates to a novel, non-destructive technique for separation of organic compounds from the roots of plants.
  • bioactive phytochemicals perform a number of functions on behalf of the plant. These functions are primarily protective of the plant against threats such as, inter alia, bacteria, fungi, other plants and insects.
  • the nature of the chemicals produced by a particular plant reflects not only the identity of the plant, but, to a great degree, the environment to which that plant is adapted.
  • bioactive phytochemicals are contained both within the body of the root and in a mucilaginous sheath formed about the exterior of the root.
  • mucilaginous sheath The nature of the mucilaginous sheath is not completely known, but is believed to be made up of hydrated polysaccharides and mucopolysaccharides. In fact, the great significance of the mucilaginous sheath with respect to the functioning of the bioactive phytochemicals has only recently been realized.
  • the invention to be described herein is a process for separating organic compounds from the root system of a plant, which process comprises immersing the root system of the plant, having the mucilaginous sheath surrounding said root system intact or substantially intact, in an aqueous medium containing one or more enzymes under conditions sufficient to break down the mucilaginous sheath, whereby organic compounds contained within the sheath are liberated into the aqueous medium.
  • Another embodiment of the invention encompasses a process for separating organic compounds from the root system of an intact or substantially intact living plant which comprises growing the plant in a hydroponic or aeroponic medium; immersing the root system of the plant having an intact or substantially intact mucilaginous sheath in an aqueous medium containing one or more enzymes under conditions sufficient to break down the mucilaginous sheath surrounding the roots whereby organic compounds are released into the aqueous medium; returning the plant to the growth medium; and recovering an aqueous solution containing the released organic compounds.
  • the plant can be exposed to at least one stimuli that would stimulate the plant to produce bioactive phytochemicals prior to immersing the root system of the plant into the aqueous medium containing at least one enzyme.
  • Yet another embodiment of the invention encompasses a process for separating organic compounds from the root system of a plant comprising immersing the root system of an intact or substantially intact plant, having an intact or substantially intact mucilaginous sheath surrounding the root system, in an aqueous medium containing one or more enzymes under conditions sufficient to break down the mucilaginous sheath, whereby organic compounds contained within the root system are released into the aqueous medium.
  • One of the advantages of the invention is that it can be carried out in a non-destructive manner. That is to say, it is not necessary to treat the plant in such a way that it is destroyed in the course of removing the desired organic compounds therefrom as in the case with present known bioactive phytochemical separation techniques.
  • the plant is intact or substantially intact, i.e. the plant is minimally damaged or altered such that the plant will continue to grow.
  • the root system of the plant can be placed in the aqueous medium for the enzyme treatment and, following the enzyme treatment, the plant can be replaced in its growth medium. During this process, the root system of the plant has an intact or substantially intact mucilaginous sheath.
  • substantially intact mucilaginous sheath means that the mucilaginous sheath of the root system may be damaged by handling, but the mucilaginous sheath has not been intentionally broken, cut, pulverized, chopped, slit, or damaged in excess of the damaged incurred by handling of the plant. The mucilaginous sheath is rapidly regrown.
  • a plant to be used in the practice of the invention can be grown in any medium in which plants are normally grown and to which the specific plant under consideration is amenable to growth. Thus it can be grown, e.g., hydroponically or aeroponically or in dirt or in potting soil mixtures. Any plant found in nature can thus be a candidate for use in the process since all plants generate specific compounds to perform necessary protective functions against specific agents from which protection is required. hi the practice of the invention, it is preferred that the plant be grown either hydroponically or aeroponically. Most preferably, the plant will be grown hydroponically.
  • the plant is grown in a medium that is neither aeroponic nor hydroponic, i.e., in a solid medium, it is necessary to clean all or substantially all of the growing medium from the root system prior to immersion in the aqueous enzyme containing-medium. This can be done by rinsing, but an extra handling step is thus required. Any extra handling step increases the likelihood of damaging the plant so that it cannot be returned to its growing medium for continued growth and reuse. Moreover, when a plant is returned to a solid growing medium, its resumption of growth is much slower and less certain than when it is returned to an aeroponic or hydroponic environment. As stated above, the preferred growth medium for plants to be employed in the invention is a hydroponic medium.
  • An aqueous medium for hydroponic cultivation of herbaceous plants typically consists essentially of undistilled tap water containing a water soluble plant food.
  • the specific concentrations of ingredients must be maintained within a range in which the plant will not be damaged.
  • a plant food having a nitrogen/phosphorus/potash composition can be used.
  • the characteristics of the plant food and the concentration of the food in the medium will, of course be determined case-by-case based on consideration of the particular plant being grown.
  • the plant does not encounter the threats to its well-being that are encountered in nature. Thus, it has little or no stimulus to generate the protective bioactive phytochemicals that are of interest in this invention.
  • the hydroponically grown plant is exposed to one or more elicitor compounds prior to the enzyme treatment. These are compounds that can imitate the challenges that the plant might have encountered in nature and thereby stimulate the plant to generate the bioactive phytochemical that would have responded to that threat in nature.
  • the plant does not encounter the threats to its well-being that are encountered in nature. Thus, it has little or no stimulus to generate the protective bioactive phytochemicals that are of interest in this invention.
  • the plant is exposed to one or more stimuli that stimulate the production of bioactive phytochemicals prior to treating the roots to an enzyme treatment.
  • Stimuli can present or imitate the challenges that the plant might have encountered in nature and thereby stimulate the plant to generate the bioactive phytochemical that would have responded to the threat in nature.
  • Stimuli include, but are not limited to, environmental conditions, naturally occurring pathogens, elicitors, or a combination thereof.
  • Environmental conditions include, but are not limited to, excessive heat, light, or water, cold, sparse light, drought, UV light, crowding by the same or other plants, poor soil conditions, pH, salt and/or unbalanced mineral concentrations, or other environmental factors that stress plant growth.
  • Naturally occurring pathogens include, but are not limited to, polypeptides, bacteria, fungi, insects, other plants, and the like.
  • Elicitors include, but are not limited to, chemical compounds that stimulate bioactive phytochemical productions such as arachidonic acid, aluminum ion, chitosan breakdown products, hydrogen peroxide, quorum sensing compounds( such as homo serine lactones used by many Gram-negative bacteria to signal population density information) or combinations thereof.
  • the exposure to the elicitor compound is typically continued for a time of about 1 to 2 days prior to the enzyme treatment.
  • the actual time of the treatment is not critical except that it must be sufficient to cause the plant to secrete a sufficient amount of the bioactive phytochemical to make the process economically practical.
  • One very active elicitor compound is arachidonic acid.
  • Other elicitor compounds are known such as, e.g. chitosan breakdown products, aluminum ion, hydrogen peroxide and U.V. light.
  • Other elicitor compounds for stimulating production of bioactive phytochemicals will be known to those skilled in the art.
  • the enzymes useful in the practice of the invention can be selected from any of the several classes of enzymes. Any water soluble or water dispersible enzyme can be used. In particular, it is preferred to employ water soluble enzymes from the classes known as hydrolases and lyases. These materials are well known and do not per se form a part of the invention. Examples of hydrolases that can be employed include, by way of example, proteases and carbohydrases such as mannanases, amylases and galacturonases. Examples of the lyases include pectin and pectate lyases.
  • the enzyme concentration required in the aqueous organic compound separation medium is dependent in part on the specific enzyme being employed, the specific plant root being treated and on the size of that root.
  • the amount of enzyme or enzyme mixture required can readily be determined on a case-by-case basis as experience in carrying out the invention is acquired.
  • the enzyme treatment can be carried out for a time period of a few minutes to several days. Time of treatment is to some degree dependent on the enzyme concentration in the treatment medium. Accordingly, enzyme concentration can be varied to effect digestion over shorter or longer time periods.
  • the temperature of treatment is not critical except as the temperature might affect the integrity of the plant roots being treated. Any temperature that matches the temperature activity profile of the enzyme employed and does not harm the root system can be used. Temperatures between about -5°C and 50°C can be employed successfully.
  • the pH at which the invention is carried out is likewise not critical so long as it is such that the plant is not harmed and the enzyme employed is active at the chosen pH level.
  • the ionic strength of the treatment medium resulting from minerals found in the treatment water and such materials as buffers that may be added thereto is preferably lower than about 1.0 M.
  • Butterfly Weed (Asclepias tuberosa) was grown hydroponically from seeds under Sylvania Gro Lux Wide Spectrum fluorescent lights in solution of an all purpose water soluble plant food (Schultz 30/20/20 All Purpose Plant Food Plus from A.Y. Schultz Company, St. Louis, MO 65043).
  • the plant food was present in a concentration of 2 tsp/2 gallons of water.
  • the plants were grown to a height of about 6 to 8 inches, at which time a plant was removed from the growth solution.
  • the plant's roots were blotted with a tissue to remove most of the growth solution and then placed in a solution of arachidonic acid and the Schultz all purpose plant food (0.75 tsp/2 gal) and placed under the growing lights for another 23 hours.
  • the plant was removed from the arachidonic acid/nutrient solution and placed in 50 ml of a solution of 0.1 ml of ENZECO ® Pectinase AJ (22,000 PGU/ml) and 0.1 ml of ENZECO ® Mannanase L (40,000 MGU/ml) in 100 ml of Schultz plant food solution and 0.05 M phosphate buffer at pH 6.0.
  • the aqueous enzyme solution was analyzed by High Performance Liquid Chromatography (HPLC) at 220 nm and 254 nm. The analysis was performed on a Hewlett Packard HPLC unit with a C-18 column at lml/min flow for 20 minutes with gradient of 30 to 90 methanol and 0.5 M aqueous phosphate buffer at pH 3.5. The injection volume was 25 ⁇ l.
  • the arachidonic acid, Schultz nutrient and enzyme solutions were also analyzed using the same method to establish controls against the possible introduction of UV absorbing compounds from other sources. The resulting spectra were overlaid electronically and it was found that 43 chemical compounds were present. Of these 43 compounds, 15 were well above background (more than 20 miliabsorbance units).
  • Example 2 Poppy (Eschscholtzia California) was grown hydroponically from seeds substantially as in Example 1 to a height of 4 to 6 inches. A single plant was treated substantially the same as in Example 1. Twenty six chemical entities were observed by HPLC and these were analyzed in the manner described in Example 1. Of these 26, 14 were significant (more than 20 milliabsorbance units). Of these 14, 7 absorbed strongly at both 220 nm and 254 nm, 7 absorbed strongly at 220 nm but weakly or not at all at 254 nm. The compounds were evenly distributed along the gradient demonstrating a wide variability in polarity.
  • Example 3
  • Example 1 Purple Cornflower (Echinea Purpurea) was grown hydroponically from seeds substantially as in Example 1 to a height of 4 to 6 inches. A single plant was treated substantially the same as in Example 1. The resulting new chemical entities were analyzed in the manner described in Example 1. Thirty five chemical entities were observed. Of these 35, 15 were significant (more than 20 milliabsorbance units). Of these 15, 9 absorbed strongly at both 220 nm and 254 nm, 6 absorbed strongly at
  • Example 4 Teasel (Dipsacus Sylvestrus) was grown hydroponically from seeds substantially as in Example 1 to a height of 3 to 5 inches. A single plant was treated substantially the same as in Example 1. The resulting new chemical entities were analyzed by HPLC in the manner described in Example 1. Thirty eight chemical entities were observed. Of these 38, 22 were significant (more than 20 milliabsorbance units). Of these 22, 5 absorbed strongly at both 220 nm and 254 nm, 13 absorbed strongly at 220 nm but weakly or not at all at 254 nm and 4 absorbed strongly at 254 nm but weakly or not at all at 220 nm. The compounds were evenly distributed along the gradient demonstrating a wide variability in polarity.
  • Teasel (Dipsacus Sylvestrus) was grown hydroponically from seeds substantially as in Example 1 to a height of 3 to 5 inches. A single plant was removed from the growth solution, the roots were blotted with tissue to remove most of the growth solution and placed in a 1 mM solution of AgNO Schultz' nutrients (0.75 tsp/2 gal) and replaced under the growing lights for 23 hours. The plant was then removed from the growth solution and placed in a solution of 0.5 ml of Pectinase AJ and 0.5 ml of Mannanase L in 50 ml of Schultz plant food solution at 0.04 M phosphate buffer at pH 6.0 for 21 hours. The aqueous solution was removed for analysis and the plant was returned to a fresh solution of nutrients for continued growth and further use.
  • the resulting chemical entities were analyzed in the same manner as described in Example 1. Thirty one chemical entities were observed. Of these 31, 1 absorbed strongly at both 220 nm and 254 nm, 8 absorbed strongly at 220 nm but weakly or not at all at 254 nm and 2 absorbed strongly at 254 nm but weakly or not at all at 220 nm. The compounds were distributed evenly along the gradient demonstrating a wide variability in polarity.
  • the purpose of this invention is to provide a process by which organic compounds, which include bioactive phytochemicals, can be separated from the root systems of plants. No attempt is made herein to identify specific compounds present in the enzyme solutions analyzed. Specific compounds can be isolated and recovered from the solutions for testing for their pharmaceutical or other utility by techniques known to the art.

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Alternative & Traditional Medicine (AREA)
  • Biotechnology (AREA)
  • Botany (AREA)
  • Medical Informatics (AREA)
  • Medicinal Chemistry (AREA)
  • Microbiology (AREA)
  • Mycology (AREA)
  • Natural Medicines & Medicinal Plants (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Epidemiology (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Agricultural Chemicals And Associated Chemicals (AREA)
  • Cultivation Of Plants (AREA)
  • Hydroponics (AREA)

Abstract

On récupère, dans le cadre de ce procédé, des composés organiques dans les racines de plantes vivantes en leur faisant subir un traitement d'extraction à l'aide d'enzymes. On retire la plante de son milieu de croissance, on la soumet au traitement d'extraction et on la rend à son milieu de croissance originel. Ce procédé est non destructeur et il est possible de soumettre une nouvelle fois la plante au traitement enzymatique lorsque les composés retirés se sont régénérés. Un milieu de croissance préféré est un milieu hydroponique. Ce procédé se révèle particulièrement utile, s'agissant de fournir des composés de tête à l'industrie de la recherche pharmaceutique.
EP01274852A 2001-11-13 2001-11-13 Procede d'extraction de composes organiques de racines de plantes Withdrawn EP1494689A1 (fr)

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/US2001/043071 WO2003045413A1 (fr) 2001-11-13 2001-11-13 Procede d'extraction de composes organiques de racines de plantes

Publications (1)

Publication Number Publication Date
EP1494689A1 true EP1494689A1 (fr) 2005-01-12

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EP01274852A Withdrawn EP1494689A1 (fr) 2001-11-13 2001-11-13 Procede d'extraction de composes organiques de racines de plantes

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EP (1) EP1494689A1 (fr)
AU (1) AU2002216655A1 (fr)
WO (1) WO2003045413A1 (fr)

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP4954523B2 (ja) * 2005-09-26 2012-06-20 長谷川香料株式会社 高濃度のｓ−アルケニル（またはアルキル）システインスルフォキサイド類を含有する抽出物の製造方法
US10806100B1 (en) 2019-10-18 2020-10-20 Mendel Systems, Inc. Grow cabinet and system for growing plants

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US6324785B1 (en) * 1999-07-12 2001-12-04 Pharmaleads, Inc. Process for harvesting organic compounds from plant roots

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WO2003045413A1 (fr) 2003-06-05

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