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WO2002005830A2 - Extraits de plantes a photosynthese de type 'cam' et leurs utilisations - Google Patents

Extraits de plantes a photosynthese de type 'cam' et leurs utilisations Download PDF

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WO2002005830A2
WO2002005830A2 PCT/IN2001/000132 IN0100132W WO0205830A2 WO 2002005830 A2 WO2002005830 A2 WO 2002005830A2 IN 0100132 W IN0100132 W IN 0100132W WO 0205830 A2 WO0205830 A2 WO 0205830A2
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plant
oil
extract
cam
water
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WO2002005830A3 (fr
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Shantaram Govind Kane
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Priority to AU2001280087A priority Critical patent/AU2001280087A1/en
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Priority to US10/338,405 priority patent/US20040156920A1/en
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N65/00—Biocides, pest repellants or attractants, or plant growth regulators containing material from algae, lichens, bryophyta, multi-cellular fungi or plants, or extracts thereof
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N65/00—Biocides, pest repellants or attractants, or plant growth regulators containing material from algae, lichens, bryophyta, multi-cellular fungi or plants, or extracts thereof
    • A01N65/06—Coniferophyta [gymnosperms], e.g. cypress
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N65/00—Biocides, pest repellants or attractants, or plant growth regulators containing material from algae, lichens, bryophyta, multi-cellular fungi or plants, or extracts thereof
    • A01N65/08—Magnoliopsida [dicotyledons]
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N65/00—Biocides, pest repellants or attractants, or plant growth regulators containing material from algae, lichens, bryophyta, multi-cellular fungi or plants, or extracts thereof
    • A01N65/40—Liliopsida [monocotyledons]
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N65/00—Biocides, pest repellants or attractants, or plant growth regulators containing material from algae, lichens, bryophyta, multi-cellular fungi or plants, or extracts thereof
    • A01N65/40—Liliopsida [monocotyledons]
    • A01N65/42—Aloeaceae [Aloe family] or Liliaceae [Lily family], e.g. aloe, veratrum, onion, garlic or chives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/20—Milk; Whey; Colostrum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/28—Asteraceae or Compositae (Aster or Sunflower family), e.g. chamomile, feverfew, yarrow or echinacea
    • A61K36/286—Carthamus (distaff thistle)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/41—Crassulaceae (Stonecrop family)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/87—Vitaceae or Ampelidaceae (Vine or Grape family), e.g. wine grapes, muscadine or peppervine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/88—Liliopsida (monocotyledons)
    • A61K36/886—Aloeaceae (Aloe family), e.g. aloe vera
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/88—Liliopsida (monocotyledons)
    • A61K36/889—Arecaceae, Palmae or Palmaceae (Palm family), e.g. date or coconut palm or palmetto
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/14—Drugs for dermatological disorders for baldness or alopecia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/06—Antihyperlipidemics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the plants of different genera and species of the Crassulaceae family have been used to combat inflammation, promote healing, and improve overall well being.
  • the Crassulaceae fix carbon via Crassulacean Acid Metabolism (CAM) ⁇ in the dark.
  • CAM Crassulacean Acid Metabolism
  • plants temporally separate the two pathways of carbon fixation, C 3 and C , while using both cycles within the same cells.
  • the initial fixation of carbon dioxide, C 4 pathway occurs at night (via cytosolic PEP carboxylase), while the C 3 pathway functions during the day.
  • CAM plants have been reported in at least 23 families of flowering plants, mostly eudicots, including maternity plant, wax plant, snake plant. Less succulent CAM plants include pineapple and Spanish moss. Interestingly, some nonflowering plants also show CAM activity, including the gymnosperm Welwitschia mirabilisi, quillwort (Isoetes), and some ferns (Raven et al., 1999).
  • CAM plants are adapted to high stress conditions such as arid zones, including hot and cold deserts, and high altitudes.
  • CAM plants can be found in many genera and are not limited to succulents; these include Kalanchoe, Bryophyllum, Sedum, Sempervium, Rhodiola, Crassulaceae, Aloe, and Cissus sp.
  • CAM plants have been used for many human applications. Most often, plant parts, such as leaves, or plant juices are orally administered.
  • the typical dosage is very high, above 100 mg/kg body weight per day (Blazovics et al.,, 1993; Boikova and Akulova, 1995; Botha et al., 1997; Da Silva et al., 1995; Da Suva et al., 1999; Lans and Brown, 1998; Nadkarni, 1982; Nassis et al., 1992; Obaseiki-Ebor, 1985; Pal et al., 1992; Sendl et al., 1993; Verma et al., 1986; Yoshikawa et al., 1997).
  • the useful medicinal agent is believed to be released in the juice from ground leaves, or the decoction of other plant parts; the plant residues remaining after extraction were not added to the composition.
  • Kalanchoe is perhaps the most widely known genus in folk medicine. Kalanchoe pinnata (Lam.) pers; Bryophullum calicinum Salis; Cotyledon pinnata and Bryophullum pinnatum are synomyous, and have been extensively studied. Folk medicine has bestowed nicknames, such as "Wonder Plant” and descriptions such as "Divine", thus illustrating their importance.
  • Sedum and Sempervivum are more commonly known in folk medicine of Europe whereas Rhodiola and other genera are known more in China and the Far East. Although the uses of these genera are not as far-reaching as that of Kalanchoe, specific uses have been reported, such as for protecting the liver and lowering lipids for Sempervivum which are not reported so far for Kalanchoe.
  • Aloe sp. has been used to promote health worldwide for thousands of years. Aloe vera is the most commonly used species throughout the world. The plant is used both by external topical application and by internal dose. These applications include promotion of general health; specifically, wound and burn healing, surgery recovery, bone growth, immunoprotection against cancer, health in HTV-infected subjects, protection against frostbite, reducing arthritic swelling, bowel inflammation, blood sugar, and protection of superoxide dismutase and glutathione from radiation.
  • the effective dosage for Aloe preparations required is typical for many herbal preparations. Dosage is high: an oral dose of 100 mg/kg per day in animal studies for wound healing (Davies et al., 1989) and 150 mg/kg per day has been prescribed to treat arthritis (Davies et al., 1992). For humans, the reported dose of the extract or juice ranges from 2 g/day (1/2 teaspoon) to over 100 g/day. Direct topical application also requires several grams per application. Anthraquinones and other low molecular weight compounds in Aloe are also reported to have cytotoxicity(Avilaetal., 1997; Mueller and Stopper, 1999). Cissus quadrangularis is the most commonly used species throughout Asia and Africa.
  • Oral dose of juice is 10 to 20 grams/day.
  • Typical dosages of dry stem powder are 2 to 4 grams.
  • Topical applications in the form of paste of dried parts is usually applied at least 10 grams or more.
  • the usefulness of this plant is diminished: the various previously l ⁇ iown compositions are reported to have mutagenic (including clastogenic) activity (Balachandran et al., 1991; Sivaswamy et al., 1991).
  • Table 1 summarizes the common uses of CAM plants; for a comprehensive review of Bryophyllum, Kalanchoe, Aloe and Cissus regarding uses, see ( adkarni, 1982). Table 1 CAM plants and their uses
  • toxins from CAM plants that might be otherwise negligible can adversely affect a subject.
  • Crassulaceae juices and aqueous extracts from various plants have cytotoxic substances (Avila et al., 1997; Balachandran et al, 1991; Botha et al., 1997; Mueller and Stopper, 1999; Sivaswamy et al., 1991).
  • shelf life and potency The traditional methods of preparation, juice extraction and ground leaf, suffer from poor shelf life, especially fresh plant juice, which ferments readily if not sterilized or stored properly. Even when dry leaf or stem powder is used, the shelf life of such products is 6 months to one year. Because preparation methods have not been optimized, potency varies by preparation, and thus each preparation may have different effective doses.
  • Medicated oils using herbal materials are known in Indian traditional medicine.
  • the base oils used for such preparations are sesame oil and ghee (clarified butter), hi South Indian practice coconut oil may replace sesame oil.
  • Sharangdharsamhita an ancient treatise by
  • Juice based or decoction based preparation are the commonly used preparation in practice for fresh or dried succulents.
  • the standard preparations for succulents call for a very high ratio of juice to ground paste (32: 1) or decoction to ground paste (24: 1) in making the medicated oil extracts.
  • the effective substances were thought to be present only in the fresh juice or decoction; the bulky residue from ground paste was considered unimportant.
  • This standard procedure is practiced in Ayurveda, the traditional medicine of India. These oil preparations are thus characterized by predominant use of juice or decoction and a high dose.
  • Classical treatises and other references specify particular plants for such methods since the belief is that the various healing substances are liberated from the plants in very specific ways (Nanal, 1995).
  • Such Kalanchoe preparations are usually prepared from the juice of the plant; with only a small amount of leafy residue. These juice-based preparations are seldom used because of serious toxic side effects at very low doses, and when used, are only topically (as opposed to internally) administered.
  • Aloe extracts are used commercially in hair oils, often as part of multi-herb medicated oils. Such preparations use extracts prepared from fresh Aloe juice or decoctions of dried Aloe pulp . Aloe oils, by themselves, are not generally recommended for topical or internal use; instead, Aloe liquids, gels or pulp are used. The use of oil extracts of Cissus is unknown.
  • leafy paste or leafy residue as the predominant component compared to juice or decoction in extractions is contrary to the teachings of traditional medicine.
  • the methods of the invention allow for the preparation of compositions that have an enormous potential to improve health by mining the beneficial effects and minimizing toxicity of CAM plants. These methods also produce compositions of high potency at very low doses, thus further reducing any potential for toxicity.
  • the invention circumvents the problems of toxicity and shelf life by providing compositions that incorporate oil extracts of Crassulaceae plants. These compositions are useful for a wide variety of applications, including human, veterinary and plant applications, for both known and novel uses. These applications include broad general effects such as disease resistance, stress resistance, general promotion in health and growth, delaying senescence and special effects such as wound healing, skin repair, stimulation of hair growth, bone repair and lipid lowering.
  • This invention relates to herbal compositions comprising CAM plant extracts to be used in human, veterinary and agricultural applications.
  • the novel herbal compositions are prepared from CAM plants by detaching plant parts, washing them with water, cutting them into pieces, mixing them with water, homogenizing the mixture, and filtering the homogenate to obtain two fractions: juice (J) fraction (as the filtrate) and the leafy residue (or stem, plant part cell mass, etc; LR) fraction.
  • the fractions may be mixed together, or kept separate as J or LR fractions. Any form and any proportion of the fractions may be mixed with oil or fat, removing the water by boiling, cooling the mixture, and filtering the mixture to separate any residue and obtain the first residue.
  • a second extract from the particular fraction is obtained by washing the corresponding first residue with oil and filtering to obtain a second extract. The two extracts are combined.
  • the composition can be used to treat a variety of O 02/05830
  • novel herbal compositions of the invention is prepared by a method wherein one part of fresh leaves, stem, and or other plant parts are homogenized, adding water as required.
  • the total homogenate (kalka) is filtered to separate the juice fraction (J) from the concentrated stem leaf/plant parts residue (LR).
  • the two fractions may be added separately, mixed in any proportion or together as total homogenate to one part of oil and additional water as required; the water is then removed by boiling.
  • any part or parts of the plant can be used to prepare a range of extracts can be obtained.
  • the concentrated stem/leaf/plant parts residue may be used to prepare LR fraction; or, only the juice fraction may be used to obtain J fraction.
  • the combined extract comprises both the J and LR fractions.
  • the starting plant material including plant, plant parts, etc.
  • the admixing of J and LR fractions, and filtration provides the preparation of extracts with a variable biological activity that are suitable for specific applications (see Examples).
  • the drawbacks of traditional methods of preparation such as cytotoxicity and excessively high doses, are circumvented.
  • the compositions of the invention have unexpected and useful results, including high potency coupled with low toxicity, an exceptionally long shelf life, and a wide range of usefulness.
  • Potency Doses of less than 0.1 mg/kg body weight/day on the basis of total fresh leaf or stem or plant parts weight for human (and mammalian) internal use is sufficient to produce significant therapeutic effects compared to greater than 50 mg/kg body weight therapeutic dosage traditionally used.
  • a 5 to 10 mg plant equivalent is sufficient for topical applications, compared to the traditional use of 5 to 10 g of juice or homogenate.
  • a dose level of less than 0.5 mg/kg body weight per day of plant equivalent is effective in poultry applications, compared to approximately 100 mg/kg body weight per day as habitually used.
  • Oil extracts oi Kalanchoe pinnata when prepared according to the methods of the invention, are not toxic when given in doses of 50 mg/kg/day for 6 months to Sprague-Dawley rats. Even at doses of 500 mg/kg/day, changes in mortality rates or histopathology are not observed.
  • the compositions are not cytotoxic in vitro when administered to 60 different tumor cell lines at doses up to 250 ppm. Thus, compared to the toxicity levels reported for the traditionally prepared compositions, the toxicity of the compositions of the present invention is negligible, even at high doses.
  • compositions of the invention have a wide variety of human applications. A summary of examples of the many embodiments is given in Table 2.
  • compositions of the invention may be used to treat respiratory disorders and skin conditions, modulate the immune system, lower blood lipid levels, improve digestion, promote healing, regulate menstruation and ovulation, and may be used as an anti-inflammatory agent. Dosages are unexpectedly low when compared to traditional apphcations, from 100 to 1000 times less.
  • compositions may also be used prophylactically.
  • compositions of the invention may also be used to improve livestock productivity, treat animals for a variety of conditions, and improve animal health. Additionally, other benefits may be realized, such as an early onset of maturity, improvement in the shelf life of buffalo milk, an improvement in feed conversion efficiency (more production for less feed), and a decrease in mortality. Table 3 summarizes examples of embodiments in which the compositions of the invention may be used on animals.
  • compositions of the present invention extends to all areas of the Plant Kingdom.
  • the compositions of the invention have beneficial effects on vegetables, ornamentals, flowers, fruits, trees, cereals, legumes, herbs and medicinal plants.
  • Table 4 summarizes examples of embodiments in wliich the compositions of the invention may be used in plants.
  • CAM involves the use of both the C 3 and C 4 pathways of carbon fixation. However, unlike C plants, CAM plants temporally separate, as opposed to spatially separate, the C 3 and C 4 cycles.
  • the C 3 cycle (Calvin cycle) takes place in the stroma of the chloroplasts, starts and ends with the five carbon sugar, ribulose 1,5-bisphosphate (RuBP).
  • the Calvin cycle occurs in three stages. (1) Carbon dioxide enters the cycle and is enzymatically combined (fixed) to RuBP. The resultant six-carbon compound, an unstable enzyme-bound intermediate, is immediately hydrolyzedto generate two molecules of 3-phosphoglycerate or 3-phosphoglyceric acid (PGA). Each PGA molecule contains three carbon atoms. RuBP carboyxlase/oxygenase (Rubisco) catalyzes this reaction.
  • oxaloacetate is either reduced to malate or transaminated to aspartate through the addition of an amino group .
  • the malate or aspartate then releases the carbon dioxide for use in the Calvin cycle.
  • C plants spatially separate the C 3 and C 4 cycles.
  • Kranz leaf anatomy clearly identifies most C 4 plants, wherein mesophyll cells are orderly arranged around a layer of large bundle-sheath cells, so that together, the two form concentric layers around the vascular bundle.
  • CAM plants are distinguished by their ability to fix carbon dioxide in the dark through the activity of PEP carboxylase in the cytosol.
  • the initial carboxylation product is oxaloacetate, which is immediately reduced to malate.
  • the malate is stored as malic acid in the vacuole.
  • the malic acid is recovered from the vacuole, decarboxylated, and the carbon dioxide transferred to RuBP of the Calvin cycle within the same cells.
  • CAM plants have cells with large vacuoles (for aqueous storage of malic acid), and chloroplasts, where the carbon dioxide obtained from the malic acid can be transformed into carbohydrates.
  • CAM plants are largely dependent upon nighttime accumulation of carbon dioxide for their photosynthesis because their stomata are closed during the day to retard water loss. " In general, CAM plants, while able to survive harsh environmental conditions, grow more slowly and if forced to compete with C 3 and C 4 species (in favorable environments), will compete poorly (Raven et al., 1999).
  • CAM plants include Crassula sp., Faucaria sp., ithops sp. Rhodia sp., Cactaceae, Euphorbiaceae, Agave sp, , Spanish moss, epiphytic bromeliads, pineapple, and vanilla orchids. Other examples are given in Table 5.
  • a cell extract is most simply a preparation that is in a different form than its source.
  • a cell extract may be as simple as mechanically lysed cells. Such preparations maybe clarified by centrifugation or filtration to remove insoluble debris.
  • Extracts also comprise those preparations that involve the use of a solvent.
  • solvents are water, a detergent, an oil or an organic compound. Extracts may be concentrated, removing most of the solvent and/or water; and may also be fractionated, using any method common to those of skill in the art (such as a second extraction, filtration, size fractionation by gel filtration or gradient centrifugation, etc.). i addition, extracts may also contain substances added to the mixture to preserve some components, such as the case with protease inhibitors to prolong protein life, or sodium azide to prevent microbial contamination.
  • oils are used as a solvent, generally all oils are meant and that are appropriate for the application.
  • oils include vegetable (com, hempnut, mustard, rapeseed, safflower, sesame, sunflower, flaxseed, canola, soybean, olive, grape seed, walnut, peanut, anise, balm, bay, bergamont, borage, cajeput, castor (including Turkey Red (sulfated castor)), cedarwood, cinnamon, clove, coconut, cottonseed, evening primrose, jojoba bean, linseed (boiled or not), macadeamia, orignaum (thyme), Tea Tree, wheat germ, Neem (Azadirachta indic ⁇ ), Karanj (Pongamia glabrd) and almond), animal (lard, fish, and butterfat from milk from various species), and those produced by the extraction industries (mineral, immersion, halocarbon and . Purified oil components (lipids) may also be used. While all combinations of such oils and fats ⁇ can
  • cell or tissue extracts are made to isolate a component from the intact source; for example, growth factors, surface proteins, nucleic acids, lipids, polysaccharides, etc. , or even different cellular compartments, including Golgi vesicles, lysosomes, nuclei, mitochondria and chloroplasts may be extracted from cells.
  • a plant extract may be made from any part of, or the entire, plant.
  • Plant parts include leaves, stems, flowers, inflorescences, shoots, cotyledons, etc. The various parts may be dehydrated or used fresh. Often, the plant parts are washed before processing. Fractionation with organic solvents may be desired to separate out organic-soliihles cnmnnnents, such as chlorophyll.
  • CAM plant extract in the context of the current invention refers to any extract, made from a CAM plant, that has at least one activity of the CAM plant extracts and compositions of the invention.
  • a CAM plant extract activity is one that is evident throughout the description of the invention, including, but not limited to, Tables 2, 3, and 4.
  • Vigor refers to the active, healthy, and well-balanced growth of plants or animals.
  • a “vigorous” plant has a fast growth rate coupled with a non-etiolated habit and copious reproduction (seed or spore).
  • a vigorous animal also has a fast growth rate coupled with adequate body strength.
  • Resistance is of two types.
  • a plant or animal may resist pests or opportunistic infections.
  • a plant or animal may also show resistance or tolerance to environmental stresses, such as heat, drought, frost, osmotic stresses and sudden fluctuations in the environment.
  • Production refers to the aspect of a plant or animal that is used for human purposes.
  • tomato plants are grown for their tomatoes; a tomato variety that produces many fruits per plant is more "productive" than one that produces few fruit but many leaves.
  • a lettuce plant with many leaves is more productive than one that bolts early.
  • Yield refers the actual production per unit, unit referring to an organism, such as a plant or animal.
  • Feed conversion is tied into production and yield. Feed conversion refers to the ability of an animal to efficiently produce per amount of feed.
  • Quantality refers to subjective criteria that are used commercially to distinguish goods. For example, a high “quality” apple is one of a certain weight, certain shape, free of blemishes, ripened and has a desired coloration, flavor, and texture. Qualitative assessments are well known to those of skill in the various arts.
  • Longevity refers to criteria that define delaying of senescence such as a longer green life of a leaf or longer shelf life of flower or fruit. HJ. Using the invention A. Extraction
  • any extract comprising regeneration activities can be similarly prepared from any CAM plant, such as Aloe vera or Cissus quadrangularis.
  • Such extracts will have at least one activity of the compositions of the invention (see Examples).
  • a mixture of small, medium and large leaves (1205 g) of Kalanchoe pinnata (Lam.) are plucked. After washing in water, the leaves are blended in a household blender, adding water to the mixture to allow the blades of the blender to contact the leaves such that the leaves are reduced to a pulp.
  • water equal to half the weight of fresh leaves suffices 1205 g of sesame oil is heated to 100 -120°C, but well below the smoke point of the oil in a stainless steel pot.
  • the leaf mixture is charged to the pot and brought to boil. Boiling is continued until only fine bubbles or fine foam is formed, and bubbling nearly ceases. When the oil just starts to smoke, the extract is sufficiently free of water and is ready for filtration.
  • the boiling time may be anywhere from 25 minutes to over 6 hours, depending on a variety of variables, including the starting material, volumes of water, etc..
  • Heating is then stopped, the mixture cooled and filtered through cheesecloth to separate the first extract from the leafy residue.
  • the leafy residue is mixed with sesame oil, 0 to 1 times the weight of the filtrate and filtered through a double layer of cheesecloth to obtain a second extract.
  • the two extracts are combined, and additional sesame oil is added to adjust the total weight to 1205 g.
  • the composition is based on lOO g of leaf equivalent per lOO g of total final extract.
  • the starting material may consist of leaves, stems, shoots, or the entire plant.
  • juice that has been manually extracted, or expressed, from the plant or plant parts may also be used.
  • a blender to homogenize the plant tissues a mortar and pestle may be used, or any other device that can destroy the integrity of the plant tissue.
  • Boiling time may range from 25 minutes to 6 hours without losing efficacy.
  • the oil may be any known in t e art, including coconut, sesame, mineral and butterfat. It will be apparent to one of skill in the art to adjust other variables as appropriate, as, for example, when large-scale preparations are desired.
  • the compositions thus made may also be further diluted with oils to achieve extracts of different strengths that are suitable for various applications. Dilution serves important functions, including reducing any irritants and providing convenient doses.
  • compositions of the invention can be incorporated into pharmaceutical compositions.
  • Such compositions typically comprise the CAM plant extracts of the invention.
  • a “pharmaceutically acceptable carrier” includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration (Gennaro, 2000).
  • Preferred examples of such carriers or diluents include, but are not limited to, water, saline, Finger's solutions, dextrose solution, and 5% human serum albumin.
  • Liposomes and non-aqueous vehicles such as fixed oils may also be used Except when a conventional media or agent is incompatible with an active compound, use of these compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions.
  • compositions for the administration of the active compounds may conveniently be presented in dosage unit form and may be prepared by any of the methods well known in the art of pharmacy. All methods include the step of bringing the active compound or CAM plant extracts into association with the carrier that constitutes one or more accessory ingredients, hi general, the pharmaceutical compositions are prepared by uniformly and intimately bringing the active compound into association with a liquid carrier or a finely divided solid carrier or both, and then, if necessary, shaping the product into the desired formulation.
  • the active compound is included in an amount sufficient to produce the desired effect upon the process or condition of diseases.
  • a pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration, including intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (i.e., topical), transmucosal and rectal administration.
  • Solutions or suspensions used for parenteral, intradermal, or subcutaneous appUcation can include: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetates, citrates or phosphates, and agents for the adjustment of tonicity such as sodium chloride or dextrose.
  • the »H can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide.
  • the parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
  • compositions suitable for injection include sterile aqueous solutions
  • compositions for intravenous administration, suitable carriers include physiological saline, bacteriostatic water, CREMOPHOR EL TM (BASF, Parsippany, N. J.) or phosphate buffered saline (PBS).
  • suitable carriers include physiological saline, bacteriostatic water, CREMOPHOR EL TM (BASF, Parsippany, N. J.) or phosphate buffered saline (PBS).
  • PBS phosphate buffered saline
  • the composition must be sterile and should be fluid so as to be administered using a syringe.
  • Such compositions should be stable during manufacture and storage and must be preserved against contamination from microorganisms such as bacteria and fungi.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (such as glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures.
  • Proper fluidity can be maintained, for example, by using a coating such as lecithin, by maintaining the required particle size in the case of dispersion and by using surfactants.
  • Various antibacterial and antifungal agents for example, parabens, chlorobutanol, phenol, ascorbic acid, and thimerosal, can contain microorganism contamination.
  • Isotonic agents for example, sugars, polyalcohols such as manitol, sorbitol, and sodium chloride can be included in the composition.
  • Compositions that can delay absorption include agents such as aluminum monostearate and gelatin.
  • Sterile injectable solutions can be prepared by incorporating the active compound or composition, such as CAM plant extracts, in the required amount in an appropriate solvent with one or a combination of ingredients as required, followed by sterilization.
  • dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium, and the other required ingredients as discussed Sterile powders for the preparation of sterile injectable solutions, methods of preparation include vacuum drying and freeze-drying that yield a powder containing the active ingredient and any desired ingredient from a sterile solutions.
  • Oral compositions generally include an inert diluent or an edible carrier, They can be enclosed in gelatin capsules or compressed into tablets.
  • the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules.
  • Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is annlied oral! .
  • Tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline ceUulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, PRIMOGEL, or corn starch; a lubricant such as magnesium stearate or STEROTES; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring./
  • a binder such as microcrystalline ceUulose, gum tragacanth or gelatin
  • an excipient such as starch or lactose, a disintegrating agent such as alginic acid, PRIMOGEL, or corn starch
  • a lubricant such as magnesium stearate or STEROTES
  • a glidant such as colloidal
  • compositions for inhalation For administration by inhalation, the compounds are delivered as an aerosol spray from a nebulizer or a pressurized container that contains a suitable propellant, e.g., a gas such as carbon dioxide.
  • a suitable propellant e.g., a gas such as carbon dioxide.
  • Systemic administration can also be transmucosal or transdermal.
  • penetrants that can permeate the target barrier(s) are selected.
  • Transmucosal penetrants include, detergents, bile salts, and fusidic acid derivatives.
  • Nasal sprays or suppositories can be used for transmucosal administration.
  • the active compounds are formulated into ointments, salves, gels, or creams. Creams are useful for a variety of external applications such as on chapped lips, cracked feet, heat rash, face cream, pimples, arm and body lotion to restore darkened skin after sun exposure, etc.
  • the compounds can also be prepared in the form of suppositories (e.g., with bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.
  • suppositories e.g., with bases such as cocoa butter and other glycerides
  • retention enemas for rectal delivery.
  • CAM plant extracts are prepared with carriers that protect the compound against rapid elimination from the body, such as a controUed release formulation, including implants and microencapsulated delivery systems.
  • a controUed release formulation including implants and microencapsulated delivery systems.
  • Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycoUc acid, coUagen, polyorthoesters, and polylactic acid.
  • Such materials can be obtained commercially from ALZA Corporation (Mountain View, CA) and NOVA Pharmaceuticals, Inc. (Lake Elsinore, CA), or prepared by one of skill in the ar Liposomal suspensions can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, such as in (Eppstein et al., US Patent No. 4,522,811, 1985).
  • Unit dosage can be prepared according to methods known to those skilled in the art, such as in (Eppstein et al.,
  • Unit dosage form refers to physicaUy discrete units suited as single dosages for the subject to be treated, containing a therapeuticaUy effective quantity of active compound in association with the required pharmaceutical carrier.
  • the specification for the unit dosage forms of the invention are dictated by, and directly dependent on, the unique characteristics of the active compound and the particular desired therapeutic effect, and the inherent limitations of compounding the active compound.
  • compositions and method of the present invention may further comprise other therapeuticaUy active compounds, such as CAM plant compositions, as noted herein that are usually applied in the treatment of wounds or other associated pathological conditions.
  • therapeuticaUy active compounds such as CAM plant compositions, as noted herein that are usually applied in the treatment of wounds or other associated pathological conditions.
  • an appropriate dosage level wiU generaUy be about 0.01 to 10 mg per kg patient body weight per day which can be administered in single or multiple doses.
  • the dosage level wiU be about 0.01 to about 10 mg/kg per day; more preferably about 0.01 to about 1.0 mg/kg per day, and most preferably 0.01 to about 0.1 mg/kg per day.
  • a suitable dosage level may be about 0.001 to 10 mg/kg per day, about 0.01 to 1 mg/kg per day, or about 0.01 to 50 mg/kg per day. Within this range the dosage may be 0.05 to 0.5, 0.5 to 5 or 5 to 50 mg/kg per day.
  • the compositions are preferably provided in the form of tablets containing 0.1 to 10 milligrams of the active ingredient, particularly 0.1, 0.2, 0.5, 1.0, 1.5, 2.0, 2.5, 5.0, 7.5 and 10.0 milligrams of the active ingredient.
  • the compounds may be administered 1 to 4 times per day, preferably once or twice per day.
  • the composition may have a dosage of about 0.001%to 5%, more preferably 0.01% to 1%, deUvering 0.5 mg to 50 mg per 5 g appUcation.
  • the compositions may be administered 1 to 8 times per day, preferably once or twice per day.
  • pads and other materials may be impregnated with such compositions and held in contact to the surface of the subject for chronic application.
  • the dosages outlined above are also suitable for veterinary apphcations. It will be understood, however, that the specific dose level and frequency of dosage for any particular subject may be varied and wiU depend upon a variety of factors including the activity of the specific compound employed, the metaboUc stability and length of action of that compound, the age, body weight, general health, sex, diet, mode and time of administration, rate of excretion, drug combination, the severity of the particular condition, and the host undergoing therapy. In addition, the site of deUvery wiU also impact dosage and frequency. Also understood, however, is that dosage for livestock may also differ. A skilled artisan wiU know how to adjust the unit dosage.
  • the pharmaceutical compositions can be included in a kit, container, pack, or dispenser together with instructions for administration.
  • the different components of the composition may be packaged in separate containers and admixed immediately before use. Such packaging of the components separately may permit long-term storage without losing the active components' functions.
  • Containers or vessels The reagents included in the kits can be supphed in containers of any sort such that the life of the different components are preserved, and are not adsorbed or altered by the materials of the container.
  • sealed glass ampoules may contain lyophilized CAM plant extracts or buffer that have been packaged under a neutral, non-reacting gas, such as nitrogen.
  • Ampoules may consist of any suitable material, such as glass, organic polymers, such as polycarbonate, polystyrene, etc., ceramic, metal or any other material typically employed to hold reagents.
  • suitable containers include simple bottles that may be fabricated from similar substances as ampoules, and envelopes, that may consist of foil-lined interiors, such as aluminum or an aUoy.
  • Other containers include test tubes, vials, flasks, bottles, syringes, or the like.
  • Containers may have a sterile access port, such as a bottle having a stopper that can be pierced by a hypodermic injection needle.
  • Other containers may have two compartments that are separated by a readily removable membrane that upon removal permits the components to mix.
  • Removable membranes maybe glass, plastic, rubber, etc.
  • Instructional materials Kits may also be supphed with instructional materials. Instructions may be printed on paper or other substrate, and/or may be supphed as an electronic-readable medium, such as a floppy disc, CD-ROM, DVD-ROM, Zip disc, videotape, audiotape, etc. Detailed instructions may not be physicaUy associated with the kit; instead, a user may be directed to an internet web site specified by the manufacturer or distributor of the kit, or supphed as electronic mail.
  • composition of the invention such as CAM plant extracts
  • Interstitial space of the tissues comprises the interceUular, fluid, mucopolysaccharide matrix among the reticular fibers of organ tissues, elastic fibers in the walls of vessels or chambers, coUagen fibers of fibrous tissues, or that same matrix within connective tissue ensheathing muscle cells or in the lacunae of bone. It is similarly the space occupied by the plasma of the circulation and the lymph fluid of the lymphatic channels. They may be conveniently delivered by injection into the tissues comprising these ceUs. They are preferably deUvered to sites of injury, preferably to Uve ceUs and extracellular matrices directly adjacent to dead and dying tissue.
  • compositions of the invention may be delivered to the site of injury mterstitially. These include, but are not limited to, syringes, stents and catheters.
  • any apparatus known to the skiUed artisan in the medical arts may be used to deUver the compositions of the invention to the circulation system. These include, but are not limited to, syringes, stents and catheters.
  • One convenient method is delivery via intravenous drip.
  • Another approach would comprise implants, such as transdermal patches, that deliver the compositions of the invention over prolonged periods of time. Such implants may or may not be absorbed by the subject over time.
  • compositions of the invention may be deUvered in a way that is appropriate for the surgery, including by bathing the area under surgery, implantable drug deUvery systems, and matrices (absorbed by the body over time) impregnated with the compositions of the invention.
  • Superficial deUvery Direct application of the compositions of the invention, such as CAM plant extracts, may be used.
  • gauze impregnated with CAM plant extracts or active components may be directly applied to the site of damage, and may be held in place, such as by a bandage or other wrapping.
  • the compositions of the invention may be applied in salves, creams, or other pharmaceutical compositions known in the art meant for topical appUcation.
  • compositions suitable for application to plants In its simplest fonn, CAM plant extract compositions that are suitable for agricultural compositions are simply diluted in water. Oil, powder and tablets of the CAM plant extract compositions may be used.
  • Wettable powders are preparations which are uniformly dispersible in water and which, besides the active substance, also comprise ionic and or nonionic surfactants (wetting agents, dispersants), for example polyoxyethylated alkylphenols, polyoxyethylated fatty alcohols, polyoxyethylated fatty amines, fatty alcohol polyglycol ether sulfates, alkanesulfbnates, alkylbenzenesulfonates, sodium Ugnosulfonate, sodium 2,2 , -dinaphthyimethane 6,6'-disulfonate, sodium dibutylnaphthalene-sulfonate, or else sodium oleoylmethyltaurinate, in addition to a diluent or inert substance.
  • ionic and or nonionic surfactants for example polyoxyethylated alkylphenols, polyoxyethylated fatty alcohols, polyoxyethyl
  • Emulsifiable concentrates are prepared by dissolving the CAM plant extracts in an organic solvent, for example butanol, cyclohexanone, dimethylformamide, xylene, or else higher-boiUng aromatics or hydrocarbons, or mixtures of the organic solvents with the addition of one or more ionic and/or nonionic surfactants (emulsifiers).
  • organic solvent for example butanol, cyclohexanone, dimethylformamide, xylene, or else higher-boiUng aromatics or hydrocarbons, or mixtures of the organic solvents with the addition of one or more ionic and/or nonionic surfactants (emulsifiers).
  • substances which can be used as emulsifiers are: calcium alkylarylsulfbnates such as calcium dodecylbenzenesulfonate, or nonionic emuslifiers such as fatty acid polyglycol esters, alkylaryl polyglycol ethers, fatty alcohol polyglycol ethers, propylene oxide/ethylene oxide condensates, alkyl polyethers, sorbitan esters, for example sorbitan fatty acid esters, or polyoxyethylene sorbitan esters, for example polyoxyethylene sorbitan fatty acid esters.
  • calcium alkylarylsulfbnates such as calcium dodecylbenzenesulfonate
  • nonionic emuslifiers such as fatty acid polyglycol esters, alkylaryl polyglycol ethers, fatty alcohol polyglycol ethers, propylene oxide/ethylene oxide condensates, alkyl polyethers, sorbitan esters, for example
  • Dusts are obtained by grinding or mixing the CAM plant extracts with finely distributed solid substances, for example, talc, natural clays such as kaolin, bentonite and pyrophylhte, or diatomaceous earth.
  • solid substances for example, talc, natural clays such as kaolin, bentonite and pyrophylhte, or diatomaceous earth.
  • Suspension concentrates can be water-based or oil-based. They can be prepared, for example, by wet grinding using commerciaUy available bead mills with or without an addition of surfactants, for example those that have already been mentioned above in the case of the other formulation types.
  • Emulsions for example oil-in-water emulsions (EW)
  • EW oil-in-water emulsions
  • Granules can be prepared either by spraying the active substance onto adsorptive, granulated inert material or by applying active substance concentrates to the surface of carriers such as sand, ka ⁇ linites or granulated inert material with the aid of binders, for example polyvinyl alcohol, sodium polyacrylate or else mineral oils.
  • Suitable active substances can also be granulated in the manner that is conventional for the preparation of fertilizer granules, if desired as a mixture with fertilizers.
  • water-dispersible granules are prepared by the customary processes such as spray drying, fluidized-bed granulation, disk granulation, mixing with high-speed mixers, and extrusion without solid inert material.
  • the concentration of active substance is, for example, approximately 0.01% to 90% by weight, more preferably 0.01% to 0.5%, the remainder to 100% by weight being composed of customary formulation components.
  • the concentration of active substance may be approximately 0.01% to 90%, preferably 0.01% to 0.5% by weight.
  • Formulations in the form of dusts comprise 0.01% to 30% by weight of active substance, in most cases preferably 0,01% to 0.5% by weight of active substance; sprayable solutions comprise approximately 0.01% to 80%, preferably 0.01% to 0.5% by weight of active substance.
  • the active substance content depends partly on whether the active compound is in liquid or soUd form and on which granulation auxiliaries, fibers and the like are being used.
  • the active substance content of the water-dispersible granules is, for example, between 0.01% and 95% by weight, preferably between 0.01% and 0.5% by weight.
  • the rate of appUcation of an active CAM plant extract is 2 to 20 g per hectare per year, apphed in 4 to 20 sprays per year (or 2-5 sprays ,per season). More preferably, 3 to 12 g per hectare per year are applied.
  • the extract concentration is increased to 25 to 500 g per hectare per year.
  • the abovementioned formulations of active substances may comprise, if appropriate, the adhesives, wetting agents, dispersants, emulsifiers, penetrants, preservatives, antifreeze agents, solvents, fttlers, carriers, colorants, antifoams, evaporation inhibitors and pH and viscosity regulators which are customary in each case.
  • the formulations that are in commerciaUy available form are, if desired, diluted in the customary manner, for example using water in the case of wettable powders, emulsifiable concentrates, dispersions and water-dispersible granules. Preparations in the form of dusts, granules and sprayable solutions are usuaUy not diluted any further with other inert substances prior to use.
  • the necessary rate of application of the safeners varies with the external conditions such as temperature and humidity.
  • Components that can also be present in CAM plant extract compositions suitable for plant (agricultural) application include natural enzymes, growth hormones such as the gibberellins (gibberelUc acid and gibbereUin plant growth hormones), and control agents including Pesticides such as acaracides and molluskicides, insecticides, fungicides, nematocides, and the like, depending of course on their compatibility with CAM plant extracts..
  • control agents that can be used in the compositions of the invention, depending on CAM plant extract compatibility, include inorganic compounds such as elementary sulfur and inorganic sulfur compounds, e.g., calcium polysulfide and sodium thiosulfate, which are effective fungicides, copper, zinc, and other metal in organics such as copper carbonate copper oxychloride, copper sulfate, and copper zinc sulfate.
  • OrganometaUic compounds such as iron and tin compounds, e.g., triphenyl tin hydroxide exhibit both insecticidal and pesticidal activity.
  • Saturated higher alkyl alcohols, either straight or branched chain, such as nonyl and decyl alcohol, can be present as insecticides.
  • Aldehydes such as metaldehyde, are effective moUuskicides, e.g., useful against snails.
  • Carbonic acid derivatives, especiaUy their mixed esters, are potent acaracides and fungicides; when sulfur is also present, e.g., mixed esters of thio- and di-thiocarbonic acids, activity is enhanced, 6-me ylqumoxal e-2,3-(hthiocyclocarbonate is an effective acaricide, fungicide, and insecticide.
  • Carbamic acid derivatives such as aryl esters of N-methylcarbamnic acid, e.g., 1-naphthyl-N-methylcarbamate can also be used.
  • Halogen substituted aUphatic monobasic and dibasic carboxyUc acids are effective pesticides. Natural pyrethrins and their synthetic analogs are also effective pesticides. Salicylanilide is effective against leaf mold and tomato brown spot. HetercycUc compounds possessing insecticidal and/or fungicidal activity can also be used. Halogen derivatives of benzene, such as paradichlorobenzene, are effective pesticides, often used against the sugarbeet weevil. Chitm-cciitaining products are effective menatocides.
  • ahphatic mercaptans having four or fewer carbon atoms organic sulfides and thioacetals, nitro compounds such as chloropicrin dichloronitroethane, and chloronitropropane
  • copper and zinc inorganic and organic compounds e.g., copper Unoleate, copper naphthenate, etc., organophosphorous compounds of which there are well over a hundred, e.g., DDVP, tris-(2,4-diphenoxyethyl) phosphite, derivatives of mono- and dithiophosphoric acids, such as 0,0-diethyl S (2-ethylthio)-ethyl)phosphorodithioate, phosphoric acid derivatives, pyrophosphoric acid derivatives and phosphonic acid derivatives, quin ⁇ nes, sulfonic acid derivatives, thiocyanates and isocyanates, phytoalexins, insect killing soaps such as potassium fatty acid salts
  • CAM plant extract compositions suitable for plant appUcation can comprise from 0.001 to 10% or more by weight of the CAM plant extract compositions suitable for plant appUcation,
  • alkaUzing agents such as ground limestone and aci(Ufying agents such as inorganic acids or acid salts can be added as needed or desired.
  • the CAM plant extract compositions suitable for plant application can be in solid form or in the form of an aqueous solution.
  • Solid forms include powders and larger particulate forms, e.g., from 20 to 200 mesh.
  • the CAM plant extract compositions can be separately encapsulated in water soluble coatings, e.g., dyed or undyed gelatin spheres or capsules, or by micro-encapsulation to a free flowing powder using one or more of gelatin, polyvinyl alcohol, ethylcellulose, ceUulose acetate phthalate, or styrene maleic anhydride.
  • the separately encapsulated CAM plant extracts can then be mixed with the powder or larger particulates of another unencapsulated component and any optional components.
  • CAM plant extracts in the compositions suitable for agricultural use provides further enhancement of plant growth, and where appUcable, crop production, i.e., by further enhancement is meant benefits in plant growth and crop production in addition to the benefits provided by the components other than CAM plant extracts, and/or provides control of pest damage and resistance to stress.
  • CAM plant extracts also improve the effectiveness of beneficial microorganisms, and promote nutrient absorption and assimilation.
  • CAM plant extracts may be added to herbicides, known in the art, to increase their effectiveness; as such, CAM plant extracts can also be used to control unwanted proliferation of weeds and other vegetative growth.
  • a mixture of small, medium and large leaves (1205 g) of Kalanchoe pinnata (Lam.) pers. were harvested.
  • the leaves were washed with water, and blended in a household blender by addition of water, approximately half the weight of plant material, 600 g (or 600 ml).
  • an equal weight of sesame oil was heated in a stainless steel pot.
  • the blended mixture of leaves and water was charged to the pot and boiled for about 2 hours and 45 minutes until a very fine foam appeared. Heating was stopped, the mixture cooled and filtered through a once-folded (double) layer of cheesecloth to separate the first extract from leafy residue.
  • the leafy residue was stirred with sesame oil equal to half the weight of the wet residue and filtered through a double layer of cheesecloth to obtain a second extract.
  • the two extracts were combined and sesame oil was added to adjust the total weight of the final extract to 1205 g.
  • This composition is based on 100 g of leaf equivalent per 100 g of total extract.
  • the final extract was named R-100.
  • Example 2 Illustrating smaller starting amounts of materials and shorter boiling times
  • Large, thick leaves (380 g) of Kalanchoe pinnata (Lam.) pers. were harvested and processed as in Example 1, except for a boiling time of 25 minutes.
  • the final extract weight was adjusted with sesame oil to 380 g. This extract was named R-100.
  • Example 3 Illustrating smaller amounts of starting materials and longer boiling times Leaves of Kalanchoe pinnata (Lam) pers. (2000 g.) were harvested. Procedure as outlined in Example 1 was followed, but with a boiling time of 6 hours. The final extract weight was adjusted with sesame oil to 2000 g. This extract was named R-100
  • Leaves (1380 g) of Kalanchoe pinnata (Lam.) pers. were harvested and washed with water. Leaves were blended as in Example 1. Separately, 2000 g of refined coconut oil was heated in a stainless steel pot. The total leaf homogenate was charged to the pot and brought to boil. After boiling for 4 hours and 45 minutes, until a very fine foam formed and started to subside, heating was stopped. The mixture was cooled and filtered through a double layer of cheesecloth to separate the first extract from leafy residue. The extract was adjusted to 1380 g by adding coconut oil. This composition, based on 100 g of leaf equivalent per 100 g of total final extract, was named R-100.
  • Leaves (600 g) of Kalanchoe pinnata (Lam.) pers. were harvested and washed with water. Leaves were then ground in a pestle and mortar, adding water as in Example 1 to produce a leaf homogenate. Separately, 400 g of safflower oil was heated in a stainless steel pot. The total leaf homogenate was charged to the pot and boiled for 45 minutes until a fine foam formed and subsided. The mixture was cooled and filtered a double layer of cheesecloth to separate the first oil extract. The leafy residue was stirred with an equal amount by weight of safflower oil. The two extracts were combined, and additional safflower oil was added to a final total weight of 600 g This composition, based on 100 g of leaf equivalent per 100 g of total final extract, was named R- 100.
  • Example 5 General procedure for Aloe vera extraction 800 g of Aloe vera leaves were plucked and washed with water. Leaves were blended in a household blender by addition of 200 ml water as in Example 1 to produce a total homogenate. This homogenate was filtered over a cloth to separate the juice (J fraction) from the leafy residue concentrate (LR fraction). Separately, sesame oil was heated in two separate stainless steel pots. The LR f action was charged to one of the pots containing 800 g of sesame oil; 400 ml of water was then added, and the mixture was boiled for 45 minutes until a fine foam formed and subsided. The mixture was cooled and filtered through double layer of cheesecloth to separate the first extract.
  • the J fraction was charged to a second stainless steel pot containing 800 g of sesame oil and the mixture was boiled for 1 hour and 20 minutes until the foam subsided.
  • This extract was filtered through a double layer of cheesecloth and additional sesame oil added to a final weight of 800 g.
  • This composition from the J fraction based on 100 g of initial total leaf equivalent per 100 g of final extract is designated as A-l 00 MINUS .
  • Example 6 General procedure for Cissus quadrangularis extraction The stem portion of Cissus quadrangularis (650 g) was harvested and washed with water. The stems were then blended in a household blender by addition of water as in Example 1 to produce a total homogenate. The homogenate was filtered over a double layer of cheesecloth to separate the juice (J fraction) from the fibrous stem residue concentrate (LR fraction). Separately, sesame oil was heated in two separate stainless steel pots.
  • the LR fraction was charged to one of the pots containing 650 g of sesame oil. 600 ml of water was then added, and the mixture boiled for 30 minutes until a fine foam formed and subsided. The mixture was cooled and filtered a double layer of cheesecloth to separate the extract. Additional sesame oil was added to a final total weight of 650 g.
  • This composition from the LR fraction based on 100 g of initial stem equivalent per 100 g of total final extract, was named C-100.PLUS.
  • the J fraction was charged to a second stainless steel pot containing 640 g of sesame oil, and the mixture was boiled for 1 hour and 20 minutes until the foam subsided. This extract was filtered, and additional sesame oil added to a final weight of 650 g
  • This composition from the J fraction based on 100 g of initial total stem equivalent per 100 g of final extract, is designated as C-100 MINUS.
  • Leaves of Kalanchoe pinnata were harvested and washed with water. Leaves were blended in a household blender by adding water as in Example 1 to produce a leaf homogenate. Separately, 2000 g of butterfat (ghee) was heated in a stainless steel pot. The total leaf homogenate was charged to the pot and brought to boil. Boiling was continued for 1 hour and 30 minutes to drive off the water, The mixture was cooled and filtered through a double layer of cheesecloth while warm to recover butterfat. The final composition, weighing 1600 g and based on 50 g of leaf equivalent per 100 g of total final extract was named R-50.
  • Extracts by using the general procedures outlined in Examples 1 to 7 can also be made from all other CAM plants.
  • Extracts made by the general procedure described in Examples 1-7 were further diluted with sesame oil to make extracts of different strengths .
  • extract made by mixing one part of R-100 with 9 parts of sesame oil was named R-10.
  • R-10 extract made by mixing one part of R-100 with 9 parts of sesame oil
  • R-5 and R- 2 respectively.
  • one part of R-100 with 19 parts of sesame oil, and one part of R-100 with 49 parts of sesame oil were designated R-5 and R- 2, respectively.
  • R-5 and R- 2 respectively.
  • one can make diluted oil extracts of any desired strength such as R-1, R-2, R-5, R-10, etc. as convenient and stable dosage forms.
  • R-5 can be used effectively on simple burns and open wounds. However, applications in more sensitive skin application require a much lower concentration such as R-1 or one may have to go even lower and use skin lotions as described in subsequent examples. R-5 to R-1 range can also be conveniently given in the form of one or more drops as such or in drinking water.
  • R-10 to R-1 constitute a more convenient dosage form for addition to feed or drinking water.
  • R-5 to R-1 can be conveniently added to the root zone or added to the water for spraying purposes. Extracts from other plants and from J or LR fractions were also diluted to different strengths and designated by the degree of dilution and the fraction used, such as A-5, A-5 PLUS, A-5 MINUS, C-2, C-2 PLUS, C-2 MINUS.
  • Example 9 Admixing CAM plant extracts with pharmaceutical carriers
  • R-100 extract made by the general procedure described in Example 1 is further diluted by mixing it with carriers such as sucrose, lactose, or other sugars.
  • R-100 can be absorbed on porous supports such as precipitated calcium carbonate, talc, precipitated silica, etc.
  • Powders made by mixing one part of R-100 with 9 parts of solids was named R-10(P).
  • R-5(P) and R-2(P) one part of R-100 with 19 parts of solids and one part of R-100 with 49 parts of solids are named R-5(P) and R-2(P), respectively.
  • R-5(P) and R-2(P) one part of R-100 with 49 parts of solids.
  • R-1(P) and lower strengths up to R- 0. l(P) can be used effectively in talcum powder formulations, dental preparations or other powder formulations for dusting apphcations on skin.
  • R-5(P) to R-1(P) can be conveniently added to the root zone or added to the water for spraying purposes.
  • Tablets of different strength can be made as convenient and stable dosage forms for a variety of appUcations.
  • a few typical recipes for tablet making are given in the examples below.
  • a variety of other excipients may also be used, with or without other adjuvants, for tablet making.
  • T-l represents one of the convenient forms as the typical human dose is one T-l per day for an adult.
  • T-10 to T-l constitute a more convenient dosage form for addition to feed.
  • T-5 in 5 liters water is a very convenient spray dose per 100 m 2 field area.
  • one T-5 at the root zone is the typical dose for a new tree sapling. Larger trees need root zone application dose in multiples of T-5.
  • T-l and multiples can be conveniently used at the root zone for small potted plants and one T-l per liter is a useful spray solution.
  • R-10(P) 100 g was mixed with 900 g sucrose and homogenized in a pestle and mortar to make R-10(P).
  • R-10(P) was then mixed with other components, processing aids and binding agents in the proportion of 1 kg R-10(P), 0.3 kg gum Arabic, 0.3 kg gelatine, 1 , 15 kg magnesium stearate, 0.3 kg talc, and 11.95 kg sucrose (for a total of 15 kg) and made into tablets weighing 150 mg in a tablet making machine.
  • 250 mg tablets 100 g of R-100 is mixed with 900 g sucrose and homogenized in a pestle and mortar to make R-10(P).
  • R-10(P) was then mixed with other components, processing aids and binding agents in the proportion of 1.0 kg R-10(P), 0.1 kg gum Arabic, 0.1 kg gelatine, 0.35 kg magnesium stearate, 0.1 kg talc, and 3.35 kg potassium chloride (for a total of 5.0 kg) and made into tablets weighing 250 mg in a standard tablet making machine.
  • Mixture A consisted of 3% stearic acid, 40% mineral oil (70 viscosity), 7% lanolin, 10% petrolatum (USP), 2% cetyl alcohol, 2% microcrystalline wax and 0.10 R-100.
  • Mixture B consisted of 5% MgAl silicate (as a 5% dispersion), 1.78% triethanoloamine, and 29.22% water.
  • Mixtures A and B were heated separately to 70°C. Mixture B was then added to mixture A and stirred continuously. Then, the mixtures was cooled to 35-40°C. A negligeable amount of fragrance (such as lavendar) and preservatives (such as parabens) was then added, and the mixture mixed until dispersion was complete. Evaporation loss was replaced with water.
  • fragrance such as lavendar
  • preservatives such as parabens
  • Mixture A consisted of 2.5% stearic acid, 2% mineral oil (70 viscosity), 1% glyceryl onostearate, 2% isopropyi palmitate, 1% petrolatum (USP), 1% cetyl alcohol, 0.25% PEG 40 stearate wax and 0, 10% R-100.
  • Mixture B consisted of 7% Carbomer 934 (as a 2% dispersion), 5% glycerine, 1% tiiethanolamine (as 99% solution), and 77% deionized water.
  • Mixtures A and B were heated separately to 70°C. Mixture B was then added to mixture A and then agitated. Then, the mixtures was mixed to 35°C. A neghgible amount of lavender and parabens were added for fragrance and stabiUty, respectively, and the mixture mixed until dispersion was complete.
  • Oil, powder, or tablets made as described in the preceding examples were used in aU cases for internal human admimstration. Oils, creams or lotions were made as described in Examples 8- 11 and were used in aU topical appUcations. These formulations were based on R-100 oil extracts made according to Examples 1-3. hi aU of these reported cases in Table 6, the dose administered internally was 1 to 2 drops of R-5 oil or between 1 to 4 tablets per day each ccaitaining 1 mg of leaf equivalent per day, i.e. 1 to 4 mg of R-100 per day, and in a vast majority of cases, 1 tablet per day or 1 drop of R-5 oil per day. The topical appUcation (1 to 2 times a day) using oil or body lotion was also less than or equal to 5 mg of R-100 or leaf equivalent per day.
  • Diabetic, leprotic, varicose ulcers; bedsores and burns were treated successfully with the daily application of one to four drops of R-5 oil to the ulcer (1 to 6 mg of leaf equivalent) .
  • the whiteness near the top of the wound changes to a healthy pink color by topical appUcation of R-5 oil, indicating local promotion of angiogenesis.
  • Faster growth of a tougher collagen layer in healing of diabetic ulcer was also observed.
  • Infected diabetic wounds were cleared by topical appUcation.
  • Administration was accompUshed by a variety of means, including direct appUcation to the root zone, foliar spray, appUcation of a solution at the root zone after dissolving/dispersing tablet/oil in water, injection in to the trunks or stems, application to terminal buds, addition to tissue culture medium, etc.
  • the typical dosage of extract for field crops was 0.5 to 1 g of R-
  • the number of sprays can be typically at a frequency of once every one to three weeks.
  • the dose for tree crops varied from 5mg to 50 mg per tree of R-100 per year, depending on the size of the sapling/tree.
  • the Onion Root Tip Assay was used to study genotoxicity profile of CAM plant extracts.
  • the results of this test can be usefully related to the expected cytotological profile in animal ceUs or human lymphocytes (Meenakumari, 1995; Rothkutty, 1980).
  • root tips were recovered and fixed in acetic acid-alcohol (1:3).
  • root tips were hydrolyzed in 1 N HC1 and squashed in 1% acetocarmine. Slides were examined under a microscope. Cells were observed and scored QTable 8), and the status of ceUs with respect to mitosis and various other physiological (clumping, stray and lagging chromosomes) and clastogenic (anaphase, fragments, binucleate) aberrations was recorded. The total number of roots and the average length of the roots were also measured, and sprouting from the tip of the bulb was also noted.
  • Roots that formed at higher concentrations of R-100 MINUS were short, yellowish and had curved tips. There was exceUent sprouting on top of the bulb in case of control and 10 ⁇ l/liter of R-100 PLUS. Sprouting was moderate with 10 ⁇ l/liter of R-100 and 30 ⁇ l/liter of R-100 PLUS. There was no sprouting at aU in any of the other sets.
  • major aberrations were physiological and mainly clumping of chromosomes. However, the onset of aberrations was shown by R-100 MTNUS-treated root tips at 10 ⁇ l liter whereas R-100 PLUS-treated root tips began to show aberrations at 100 ⁇ l/liter, a tenfold higher concentration.
  • R-100 PLUS at least up to 30 ⁇ l/liter promoted ceU division, rooting and sprouting.
  • R-100 MINUS began to act as a mitogen and root system inhibitor beyond 10 ⁇ l Uter.
  • root tips were recovered and fixed in acetic acid-alcohol (1 :3).
  • root tips were hydrolyzed in 1 HC1 and squashed in 1% acetocarmine. Slides were examined under a microscope. Cells were observed and scored OTable 9), and the status of ceUs with respect to mitosis and various other physiological (clumping, stray and lagging chromosomes) and clastogenic (anaphase, fragments, binucleate) aberrations was recorded. The results are summarized in Table 9.
  • the PLUS fraction appeared to be an excellent promoter of mitosis or ceU proliferation compared to sesame oil controls. In some cases (R-100 PLUS and A-100 PLUS), this activity was retained up to 100 ⁇ l/liter concentration inthis assay.
  • the PLUS fractions also had lower toxicity than the corresponding MINUS (J) fractions in terms of mitogenic activity inhibition and genotoxicity.
  • the use of the PLUS fraction and exclusion of the MINUS fraction for medicated oil preparations is contradicts the teachings of traditional medicine.
  • the metliod of the invention improves the overaU potential of health promotion and broadens the safe operating range. This method also aUows compositions of high potency at low dose; thus further reducing the toxicity potential.
  • Example 16 R-100 PLUS vs. R-100 MINUS of Kalanchoe pinnata on Phaseolus mungo Ger ination experiments were carried out with R-100 PLUS vs. R-100 MINUS and also with the standard R-100 extract. 25 seeds of Phaeolus mungo were placed in a plate with 5 ml of distilled water containing various concentrations of the R-100 extracts. On the 7 ⁇ day after initiation of experiment, mean values of 11 seedlings were taken and reported. The results are summarized below in Table 11.
  • AU fractions showed significant biological activity at very low doses. Both R-100 and R- 100 PLUS show significant promotion of root and shoot growth and biomass weight at the end of 7 days compared to sesame oil controls at 1 and 3 mg levels. The PLUS fraction showed the best overaU promotional effect. R-100 MINUS did show shoot growth compared to sesame oil control. However, there was no root growth and no increase in dry biomass weight up to 3 mg. At the higher dose level, R- 100 also showed a sharper drop in root length and biomass retention.
  • Example 17 Effect of CAM plant fractions on germination of Phaseolus mungo 25 seeds of Phaseolus mungo were placed in a plate with 5 ml of distiUed water, containing various concentrations of oil extracts or plain base oil. On the 7 th day after initiation of experiment, mean values of 10 seedlings were taken and reported in Table 12.
  • Dry Weight(gm/10 seedlings) mean of two observations. Numbers in parenthesis indicate ⁇ range of the two observations.
  • A-100 PLUS and MINUS fractions of both CAM plants promoted auxin-like (rooting promotion), gibberellin-Uke (shooting promotion) and cytokine-Uke fbiomass preservation/growth) activity up to 1 ⁇ l/50 ml DW.
  • A-100 PLUS and MINUS both promoted rooting and a higher dry biomass at the end of 7 days, even at the higher concentration of 10 ⁇ l/50ml distilled water.
  • A-100 PLUS and MINUS promote a wide range of endogenous hormones at a low concentration and this promotional effects continue up to a high concentration.
  • C-100 PLUS and MINUS had a different activity profile.
  • Leaf area (LA), and leaf dry weight (LW) increased in both hydroponics and soU cultures at flowering (mean of three plants) at all concentrations compared to control (Table 14).
  • Chlorophyll levels are in mg/lOOg fresh weight (FW) of leaves. Numbers in parenthesis are values for standard deviation.
  • Table 17 Increase in carbohydrates, proteins and polyphenols in leaves (mean of three plants)
  • R-100 appears to act at a very fundamental level in aU stages of plant growth. For example, higher chlorophyU level and altered metaboUc activities caused by R-100 might have increased the RuBp-case activity and resulted in a higher carbohydrate level in the leaves. Induction of endogenous phytohormone synthesis by R-100 may be responsible for increase in height and leaf area and UA oxidase level.
  • Induced auxin and cytokine levels and higher peroxidase activity may have reduced hydrogen peroxide levels and delayed senescence.
  • R-100 was much more pronounced in soil culture than in hydroponics medium. This may be a result of a synergistic interaction of R-100 with the rhizosphere microflora (fungi, yeast, actinomycetes, etc.)
  • Plant height increased for treated plants in soil culture (mean of 10 plants) at all concentrations compared to control.
  • Example 1 Yield, productive life, and pest resistance in tomato, brinjal and okra
  • Tomato Licopersicum esculentum
  • Golden variety Golden variety
  • Brinjal Solanum melangona
  • Kalptharu variety Kalptharu variety
  • Okra Almoschus esculentus L. Parbhani kranti variety.
  • the plants were administered a concentration of 1 T-5 (250 mg) tablet /5 liters at 30, 60 and 90 days after transplantation.
  • the solution was used at 3, 4, and 5 liters/100 sq. ft for sprays 1, 2 and 3, respectively. 50 plants were used per experimental condition; the results are reported in Table 18.
  • Tomato Sucking pest attack reduced Brinjal: Fruit borer attack was reduced. Fruit soft, tender. Okra: Leaf curling reduced.
  • Example 20 Yield in Capsicum annuum and okra (Abelmoschus esculenuts L.) Trials were carried out at Dapoli, Maharashtra, India; 3 replicates were used for each treatment. R-10 oil (Batch 910318) was used, and a total of three sprays were applied. Controls were given water sprays. The results are presented in Table 19,
  • Leaf area (LA), and leaf dry weight (LW) increased in both hydroponic and soU culture at flowering (mean of three plants) at all concentrations compared to controls.
  • AU treated plants had dark green glossy leaves and higher chlorophyU a and b level particularly up to 3 mg R-5 level (Table 21)
  • Chlorophyll levels are in mg/lOOgm fresh weight (FW) of leaves. Numbers in parenthesis are values of Standard Deviation.
  • Example 22 Fruit: higher yield, pest resistance and shelf life
  • T-5 (250mg) tablets were carried out near Pune, India on a variety of fruit trees. Observations were recorded with respect to control trees. 10 trees of each type were used for measurements at the end of the season. Two T-5 tablets were dissolved in a minimum of 2 liters of water. This solution was used per spray per tree. The results are reported in Table 23.
  • Table 23 Fruit yield, resistance, quality, ripening and shelf life
  • Example 23 Yield and size in Strawberry (Fragaria x ananasa) (ChandUar variety)
  • Example 24 Growth, chlorophyU, nutrients, phenols and solasodine in Solanum khasianum Trials were carried out on plants grown in soil at Pune with T-l (150mg) tablets (batch
  • Tab 3 1/3 0 6 7 Spray Method 10 ml/plant, twice a month up to fruiting.
  • the amount of R- 100 or leaf equivalent used per plant per spray was approx. 0.03 mg, 0.01 mg and 0.0033 mg.
  • Treatments were initiated 30 days after seedling (30 day old seedlings) transplanting.
  • the average results of the combined three sets for Tabl, Tab2, 05 and 06 are summarized in Tables 25-27.
  • PLANT Average values Height, cm 56.0 (2.31) 77.0 (1,31) 92.7 (1.3) 64.5 (65.5) 66.3 (0.85)
  • Treatment Control Tabl Tab2 0 5 0 6 (mg/100 g of Fresh Wt. of leaves) Chlorophyll, 132.6(3.39) 138.3(4.62) 140.4(1.57) 133.8(1.84) 136.8(2.1) ( g / 100 g of Fresh Wt.of leaves)
  • Carbohydrates2.5 (0.07) 3.1(0.11) 2.9(0.05) 2.8(0.04) 2.6(0.05)
  • Example 25 Cotton Trials were carried out at Dharwad, Karnataka, India in Kharif on cotton. Plants were cultivated on 18,2 m 2 plots; 3 rephcates were used for each set.
  • R-2 oil (batch 910608) was used, and a total of 3 sprays were applied: 65, 83, 113 days after sowing. The results are reported in Table 28.
  • Plants (Glycine max L. cvMacs) (winter variety) were cultivated in soil in pots 20 cm x 30 cm x 40 cm;
  • Spray Method 100 ml/pot, twice a month up to fruiting.
  • the amount of R-100 used per plant per spray was approx.0.3 mg, 0.1 mg and 0.033 mg.
  • the first treatment was appUed 40 days after sowing.
  • LAD Leaf Area Duration
  • e Average number of days for which leaves remain green 60.5(2.54) 80.8(1.81) 85.6(2.08) 85.7(1.98) 85.2(1/56)
  • R-100 induced increases in a variety of growth parameters such as height, number of branches, total leaf area, chlorophyU, etc.
  • growth parameters such as height, number of branches, total leaf area, chlorophyU, etc.
  • increase in total proteins and carbohydrates and particularly in non-reducing sugars increase in number of pods and seeds per pod and with a considerable increase in LAD, higher yield of oilseeds can be expected.
  • the increase in LAD or delaying of leaf senescence was particularly significant for legumes as they otherwise suffer from monocarpic senescence leading to lower overall yield in comparison with cereals.
  • the data from the two tables highUght the differential activity possible by fractionating extracts.
  • the plant height data shows the early onset of toxicity of the R-5 MINUS oil compared to R-5 PLUS oil. This observation is further corroborated by the data on the number of pods per plant, average seed weight and the yield per plant.
  • R-5 PLUS shows excellent promotional effects, even at 10 mg per spray, up to harvesting.
  • Fruit were selected from mature bunches. Hands of 14-18 fruit each, unifbnn in size were selected. Test solutions were made with 0, 20, 60, 100 and 200 micro-liter of R-5 oil per 1000 ml distilled water. One hand was treated with each test solution for 30 minutes. Green life and yeUow life were estimated by visual examination. Acidity and total soluble solids (TSS) were measured at the end of yeUow life. Change of peel color from green to yellow indicated end of green life.
  • TSS total soluble solids
  • Example 28 Effect of oil medium on germination of Sorghum vulgare (cv. M, 35-1) R-100 was made by the methods in examples described above with commercial grade coconut oil, and safflower oU, respectively. 20 seeds of Sorghum vulgare (Jowar) were placed in a plate with 5ml of distiUed water containing various concentrations of R-100 oU or plain base oU (controls). On the 7 th day after initiation of the experiments, mean values for several variables of 11 seedlings were taken. As shown in Table 34, R-100 made in coconut and safflower oil media promoted both rooting and shooting in germination up to 1 ⁇ l/50 ml distilled water. At the higher concentration of 10 ⁇ l/50 ml DW, both R-100 oils showed a marked decline in root and shoot length unlike the plain coconut or safflower oU medium.
  • Example 29 Effect of CAM plant extracts in seed germination in monocots.
  • R-100 and C-100 were used. 20 seeds of Sorghum vulgare (cv.M, 35- ⁇ )(Jowar) were placed in a plate with 5ml of distilled water containing different concentrations of R-100 oU. On the 7 th day after initiation of experiment, mean values of several variables of 11 seedlings were taken. As shown in Table 35, R-100 and C-100 promoted both rooting and shooting in germination compared to controls (0.0 concentration and sesame oU at 1 ⁇ l/50 ml distiUed water).
  • Example 32 Marigold Trials with R-5 oil were carried out near Vietnamese, India on Marigold, Tagates erecta. Each set had 5 plants; measurements were the average for each set. Concentration of 1 T-5 tablet (250 mg) per 5 liters was used, and 25 ml spray/plant was applied on mature, flowering plants. Results are reported in Table 38.
  • Plant spacing 30 cm x 30 cm (1000 plants each in TEST and CONTROL groups)
  • Plant spacing 90 cm x 90 cm (100 plants each in TEST and CONTROL groups)
  • T-5 tablets were dissolved in water to the indicated solution concentration and used as a foliar spray. Solution was sprayed once every two months, commencing on 19 th Nov 1990. Data from two plants are reported for each set in Table 40.
  • ROOT ZONE APPLICATION T-5 tablets were kept in a small basin 15 cm away from the tree and 5cm deep. Only one appUcation of tablets was made. Controls (0), 1, 2 and 3 tablets were used. Two plants were used in each set. Plant height was recorded in cm and is reported in Table 41.
  • T-5 tablet was applied to the root zone.
  • a 5 g piece of cotton soaked in a 500 ppm solution of T-5 tablet was also placed on top of the teminal bud once every 3 months. 5 test and 5 control plants were used. The results are given in Table 42.
  • Mushrooms from the treated beds were large, more uniform in size and with a thicker stalk.
  • R-100 oil (batch 881206) did not show any antibacterial activity against Staphylococcus aereus and Proteus vulgaris, even at the high ratio of 1:10 of R-100: nutrient broth.
  • Example 38 Anti-mutagenicity in bacteria hi a standard Ames test, the following results were obtained: AMES TEST: Salmonella typhimurium (S9)
  • R-100 oil did not act as a mutagen when added at 1 :200 or 1 :500 dilution; R-100 oil was anti-mutagenic or prophylactic for the mutagenicity of Benz(o)pyrene in both TA98 and TA100 type of mutations.
  • R-10 powder A chronic toxicity study of R-10 powder was carried out on Albino rats (Wister strain). There were 20 animals in each group, evenly distributed by sex. Dose levels of 0, 500, 1000 and 2000 mg/kg/day of R-10(P) powder were used. These are equivalent to 0, 50, 100 and 200 mg/kg/day of leaf equivalent or R-100 oil.
  • Example 40 Faster cartilage tissue growth and low toxicity
  • R-100 oil A chronic toxicity study of R-100 oil (batch 930425) was carried out on in Sprague Dawley rats. There were 10 animals in each group (5 males and 5 females). Dose levels of 0, 5, 50 and 500 mg/kg/day of leaf equivalent in the form of R-100 oil were used. R-100 oil was mixed with com and administered to rats for 180 days. This was followed by a recovery period of 28 days. Hematology, blood biochemistry, urine analysis and histopathology of all major organs were performed at the end of 180 days, showing no toxic effects. There was no remarkable change in gross pathology or no remarkable changes in the histopathology. Dose levels of 5 and 50 mg kg day did not induce any toxicity. At 500 mg/kg/day, nasal secretions, polyurea, diarrhoea, drowsiness, ataxia, alopecia were observed for some male and female animals. These signs of intoxication subsided during the recovery period of 28 days.
  • mice bone marrow micronucleus test R-100 oil (batch 910217) was given to mice in drinking water at 2 ppm (v/v) level for 15 days as a prophylactic before challenging them with B(a)P ( Benz(o)pyrene). Results are reported as per cent micronucleated ceUs (% MNPCE) in Table 46,
  • R-100 exhibited prophylactic activity against an important carcinogen, Benz(0)pyrene in a mammaUan system as weU as in bacteria.
  • Example 42 Low toxicity in topical appUcation (skin irritation and dermal toxicity) R-100 (batch 920814) in the amount of 0.5 ml was appUed to the shorn back skin both intact and abraded site of three rabbits per sex. Each site was observed and reaction recorded by Draize method (States, 1979). No erythema or edema of skin was observed in rabbits after appUcation of test substance.
  • R-100 oil did not cause any irritation to the skin of rabbits.
  • R-100, R-5 and R-1 oU (batch 920814) were appUed to the shaven back skin of New Zealand White rabbits at the rate of 3ml/kg body weight.
  • Control animals were treated with sesame oU. 6 animals (3 males and 3 females) were used at each dose level.
  • the extract was kept in contact with the shaven intact skin for 6 hours per day, 5 days a week for 3 weeks. The foUowing results were observed at 21 days:
  • R-1 oil showed no observable effects at the dose levels tested.
  • R-100 oU sample was screened at the Frederick Cancer Research and Development Center of the National Cancer Institute, (Bethesda, MD; USA) according to (Boyd and Pauli, 1995). There was no ceU mortaUty up to a high concentration of 250 mg/liter of R-100 tested in vitro in 60 different cancer cell lines. Thus, the extract showed very low cytotoxicity.
  • the test birds had produced 23683 eggs with a feed consumption of 2842 kg, i. e. , with an FCR of 120 g feed egg.
  • the control birds had produced 258074 eggs with a total feed consumption of 40272 kg, i.e., with an FCR of 156 g feed/egg.
  • MortaUty in the test group during this 26 week period was 9.5%, whereas mortaUty in the control group during this period was 12.2 %. Thus, survival of the test birds was definitely improved.
  • the average weekly feed intake during the laying period was approximately 0.8 kg.
  • the approximate weekly consumption of R-100 at the higher dose of 5mg/kg feed was 4 mg./bird.
  • the average weight of birds during the laying period was 1.6 to 1.8 kg.
  • the intake was 0.135 mg R-100 kg body weight per day.
  • TYPE OF BIRDS HISEX DOSE: R-10 OIL 50 ml MT Feed, for TRIALS 1,2 and 3
  • the feed was 50% red maize, 20% roasted soybean, 10% groundnut cake, 7% fish meal and 3% minerals.
  • the feed was given ad lib. Three consecutive batches, two control and one experimental, were run. The results are given in Table 48.
  • Total consumption of R-100 was 20000 mg on a total feed of 5000 kg. Thus, the average level was 4 mg/kg feed. T his is a range similar to that used in Example 45 for Layers.
  • the total weight of broilers is about 2000 kg at the end of 50 days. Thus, using an average weight of 1000 kg for 50 days, the average R-100 dose was 0.4 mg/kg body wt/day.
  • Example 47 Lower acidity and bacterial count in buffalo milk Four Murrah buffaloes were given 2 drops per day of R-5 oU in drinking water over a 2.5 month period. The milk quahty was tested on the day foUowing the final administration of R-5. Dosage was approximately 3.5 mg per day of R-100 oil or leaf equivalent per animal.
  • the acidity (expressed as wt% lactic acid equivalent ) of 10ml milk after incubation at 37°C for 4 hours was 0.18 acidity units for the control animal and 0.14 to 0.15 for the test animals.
  • the SPC by standard plate count was 54000 for control vs. 43000 to 49000 for the test animals.
  • a coliform test (Durham) showed gas formation in the control animal sample and no visible gas formation in aU the test animal samples. Thus, the coliform level in the milk of treated animals was reduced and shelf life of milk improved (acidity formation slowed down).
  • Aloe vera J. Am. Pod. Med. Assoc. 79:559-562. Davies, R., G. Strewart, and P. Bregman, 1992. Aloe vera and the inflamed synovial pouch model.
  • Uposomes enhances the anti-infective activity of muramyldipeptides. 1985. Gennaro, A.R. 2000. Remington: The science and practice of pharmacy. Lippincott, Williams &
  • Rhodiolae Radix On the histamine release inhibitors from the underground parts of Rhodiola sacra (Prain ex Hamet) S.H.Fu (Crassulaceae). Chem. Oharm. Bull. 45:1498-1503,

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  • Dermatology (AREA)
  • Obesity (AREA)
  • Diabetes (AREA)
  • Pulmonology (AREA)
  • Hematology (AREA)
  • Rheumatology (AREA)
  • Endocrinology (AREA)
  • Reproductive Health (AREA)
  • Pain & Pain Management (AREA)
  • Biomedical Technology (AREA)
  • Cell Biology (AREA)

Abstract

L'invention concerne des extraits d'huile de plantes à photosynthèse de type CAM (acronyme de Crassulaceae Acid Metabolism) utiles dans des applications humaines, vétérinaires et agricoles. L'invention concerne également des méthodes de préparation de ces extraits sous forme d'huile ainsi que des méthodes d'application et d'administration de ces extraits.
PCT/IN2001/000132 2000-07-14 2001-07-04 Extraits de plantes a photosynthese de type 'cam' et leurs utilisations Ceased WO2002005830A2 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
AU2001280087A AU2001280087A1 (en) 2000-07-14 2001-07-04 Extracts from crassulacean acid metabolism (cam) mechanism plants and uses thereof
US10/338,405 US20040156920A1 (en) 2000-07-14 2003-01-07 Extracts from plant and non-plant biomass and uses thereof

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
IN653/MUM/2000 2000-07-14
IN653MU2000 IN188857B (fr) 2000-07-14 2000-07-14
IN654MU2000 2000-07-14
IN654/MUM/2000 2000-07-14

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* Cited by examiner, † Cited by third party
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WO2002085394A1 (fr) * 2001-04-24 2002-10-31 Bakulesh Mafatlal Khamar Procede de production de preparation topique anti-inflammatoire et analgesique
WO2003086430A3 (fr) * 2002-04-16 2003-12-18 Swedish Herbal Inst Carpediol, extrait d'herbes medicinales utilise dans le traitement de la depression
WO2006024143A1 (fr) * 2004-08-31 2006-03-09 Yu, Ru-Chein Utilisation pharmaceutique de graptopetalum et de plantes apparentees
EP1731158A4 (fr) * 2004-03-31 2009-03-18 Morinaga Milk Industry Co Ltd Medicaments, nourritures et boissons destines à ameliorer l'hyperglycemie
US7588776B2 (en) 2004-08-31 2009-09-15 Shih-Lan Hsu Pharmaceutical use of water-soluble fraction of Graptopetalum
CN101558771B (zh) * 2009-05-26 2010-12-29 刘兴健 一种活性杀虫剂
WO2012012390A1 (fr) 2010-07-19 2012-01-26 Marvphyt Development Llc Composition botanique et procédés de fabrication et utilisation
WO2012028758A1 (fr) * 2010-08-31 2012-03-08 Servicio Andaluz De Salud Utilisation de crassula pour le traitement de maladies prolifératives
WO2012162471A3 (fr) * 2011-05-26 2013-01-17 Biortec, Llc Extrait végétal de pachycereus et ses procédés d'utilisation
WO2017081654A1 (fr) * 2015-11-12 2017-05-18 ISN Pharma Sp. z.o.o. Composition pour le soin de la peau
WO2018164592A1 (fr) * 2017-03-06 2018-09-13 Gibowskl Zygmunt Procédé de repousse des cheveux sur la tête
WO2018197668A1 (fr) * 2017-04-28 2018-11-01 Isn Pharma Sp.Z.O.O. Composition pour soins de la peau
WO2020201847A1 (fr) * 2019-03-29 2020-10-08 Koshy Santy Composition à base d'herbes pour le traitement de brûlures
CN112841134A (zh) * 2021-02-22 2021-05-28 广西壮族自治区畜牧研究所 一种使用芒果培育优质鸡的方法
FR3122991A1 (fr) * 2021-05-20 2022-11-25 Patrinove Extrait de vegetal a metabolisme acide crassulaceen pour utilisation dans le traitement de la dysmenorrhee et/ou des douleurs liees aux spasmes et crampes menstruels

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DATABASE BIOSIS [Online] BIOSCIENCES INFORMATION SERVICE, PHILADELPHIA, PA, US; 1994 DEKA D K ET AL: "Effect of Cissus quadrangularis in accelerating healing process of experimentally fracture radius-ulna of dog: A preliminary study." Database accession no. PREV199497274282 XP002229918 & INDIAN JOURNAL OF PHARMACOLOGY, vol. 26, no. 1, 1994, pages 44-45, ISSN: 0253-7613 *
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Cited By (21)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002085394A1 (fr) * 2001-04-24 2002-10-31 Bakulesh Mafatlal Khamar Procede de production de preparation topique anti-inflammatoire et analgesique
WO2003086430A3 (fr) * 2002-04-16 2003-12-18 Swedish Herbal Inst Carpediol, extrait d'herbes medicinales utilise dans le traitement de la depression
US8338403B2 (en) 2004-03-31 2012-12-25 Morinaga Milk Industry Co., Ltd. Drug for improving hyperglycemia
EP1731158A4 (fr) * 2004-03-31 2009-03-18 Morinaga Milk Industry Co Ltd Medicaments, nourritures et boissons destines à ameliorer l'hyperglycemie
US7754704B2 (en) 2004-03-31 2010-07-13 Morinaga Milk Industry Co., Ltd. Method for treating hyperglycemia
WO2006024143A1 (fr) * 2004-08-31 2006-03-09 Yu, Ru-Chein Utilisation pharmaceutique de graptopetalum et de plantes apparentees
US7364758B2 (en) 2004-08-31 2008-04-29 Shih-Lan Hsu Pharmaceutical use of Graptopetalum and related plants
US7588776B2 (en) 2004-08-31 2009-09-15 Shih-Lan Hsu Pharmaceutical use of water-soluble fraction of Graptopetalum
AU2005279655B2 (en) * 2004-08-31 2009-10-29 Veterans General Hospital Pharmaceutical use of graptopetalum and related plants
CN101558771B (zh) * 2009-05-26 2010-12-29 刘兴健 一种活性杀虫剂
WO2012012390A1 (fr) 2010-07-19 2012-01-26 Marvphyt Development Llc Composition botanique et procédés de fabrication et utilisation
WO2012028758A1 (fr) * 2010-08-31 2012-03-08 Servicio Andaluz De Salud Utilisation de crassula pour le traitement de maladies prolifératives
ES2377612A1 (es) * 2010-08-31 2012-03-29 Servicio Andaluz De Salud. Consejer�?A De Salud. Junta De Andaluc�?A Uso de crassula para el tratamiento de enfermedades proliferativas.
WO2012162471A3 (fr) * 2011-05-26 2013-01-17 Biortec, Llc Extrait végétal de pachycereus et ses procédés d'utilisation
WO2017081654A1 (fr) * 2015-11-12 2017-05-18 ISN Pharma Sp. z.o.o. Composition pour le soin de la peau
WO2018164592A1 (fr) * 2017-03-06 2018-09-13 Gibowskl Zygmunt Procédé de repousse des cheveux sur la tête
WO2018197668A1 (fr) * 2017-04-28 2018-11-01 Isn Pharma Sp.Z.O.O. Composition pour soins de la peau
CN110831566A (zh) * 2017-04-28 2020-02-21 艾斯恩制药有限责任公司 皮肤护理组合物
WO2020201847A1 (fr) * 2019-03-29 2020-10-08 Koshy Santy Composition à base d'herbes pour le traitement de brûlures
CN112841134A (zh) * 2021-02-22 2021-05-28 广西壮族自治区畜牧研究所 一种使用芒果培育优质鸡的方法
FR3122991A1 (fr) * 2021-05-20 2022-11-25 Patrinove Extrait de vegetal a metabolisme acide crassulaceen pour utilisation dans le traitement de la dysmenorrhee et/ou des douleurs liees aux spasmes et crampes menstruels

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