WO2002005830A2

WO2002005830A2 - Extracts from crassulacean acid metabolism (cam) mechanism plants and uses thereof

Info

Publication number: WO2002005830A2
Application number: PCT/IN2001/000132
Authority: WO
Inventors: Shantaram Govind Kane
Original assignee: Shantaram Govind Kane
Priority date: 2000-07-14
Filing date: 2001-07-04
Publication date: 2002-01-24
Also published as: WO2002005830A3; AU2001280087A1

Abstract

Novel oil extracts from Crassulaceae Acid Metabolism (CAM) plants useful in human, veterinary and agricultural applications are described. Methods of preparation of these extracts in oil and methods of application and administration are also described.

Description

EXTRACTS FROM "CAM" MECHANISM PLANTS AND USES THEREOF

RELATED APPLICATIONS

This application claims priority to India application Serial Nos.653/MUM/2000 and 654/MUM 2000, both filed 07/14/2000 which are incorporated herein by reference in their entirety.

BACKGROUND OF THE INVENTION

Introduction

The use of plants and herbs for general welfare and effectively treating a variety of conditions and ailments dates back to ancient times. However, using plants can also be dangerous because of endogenous toxins. Effective doses are often achieved when large volumes of plant material are used, thus aggravating toxic effects.

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 [so called because originally found in the Crassulaceae family (stonecrops, comprising mostly succulents such as cacti)], plants temporally separate the two pathways of carbon fixation, C₃ and C , while using both cycles within the same cells. The initial fixation of carbon dioxide, C₄ pathway, occurs at night (via cytosolic PEP carboxylase), while the C₃ pathway functions during the day. Consequently, the stomata of CAM plants are closed during the day, thus enabling them to withstand brutal environmental conditions, such as drought and low temperatures, and are open at night to take in carbon dioxide. In contrast, C plants have open stomata during the day and closed during night. 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. However, 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). Whenever fresh juice or decoction of a particular plant is given internally according to traditional or folk medicine, 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. It has been used to promote fracture healing both by external topical application and by internal dose. Other uses include treating rheumatic back pain and body pain, irregular menstruation, stomachache and whooping cough. Most parts of the plant have been used, including the stem, leaves, and tender shoots. In addition, the entire plant, dried, is used in Arabic countries as a "Cure -All" medicine. Pastes made from leaves, stem or entire plants have been used for external applications.

The dosage levels required are typical for many herbal preparations. 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. However, the usefulness of this plant is diminished: the various previously lαiown 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

Toxicity

Because of the relatively high effective doses and the traditional methods of preparation, toxins from CAM plants that might be otherwise negligible can adversely affect a subject. For example, 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). High therapeutic doses of leaf juice for internal use (more thanlOO mg/kg body weiglit daily) coupled with high mammaϋan toxicity close to the habitual level of use (LD₅o in mice is 230 mg/kg and in rat is 560 mg/kg, respectively (Verma et al., 1986)) render these compositions less useful.

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.

Previous oil extraction methods

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

Sharangdharacharya (1961), a standard reference treatise of traditional medicine describes a standard method of preparing such "medicated oils".

In traditional practice, such extracts are made by boiling together a mixture of kalka (ground paste or homogenate), oil and other liquid substances. The recommended ratio of Azz/to:oil:liquid substances changes with the nature of liquid substance used. Water, plant derived liquids and juice are the three types of liquid substance described. Plant derived liquids used for making oil extracts are decoctions of plant parts in water. The decoctions are the filtrates prepared by boiling plant part in water, and filtering to obtain a clear liquid or decoction. The ratios of kalka:oil:liquid substance for these three cases are 1:4:16; 1:6:24; and 1:8:32, respectively. Therefore, in these three cases the overall ratio of water to kalka, plant derived Uquids to kalka and juice to kalka are recommended to be 16:1, 24:1 and 32:1

Juice based or decoction based preparation are the commonly used preparation in practice for fresh or dried succulents. Thus, 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. Traditionally, the effective substances were thought to be present only in the fresh juice or decoction; the bulky residue from ground paste was considered unimportant. Furtl ennore, the typical recommended dose of such medicated oils is as high as 4 tola (1 tola = 11.4 g). 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).

However, Nanal (1995), in reviewing the use of Kalanchoe in the context of theory and practice, remarks that Parnabeeja (Kalanchoe) is not mentioned in any Ayurvedic texts. Nanal mentions several different preparations from Kalanchoe that includes oils, both in sesame and in clarified butter, but does not specify the usefulness of such preparations, and he does not recommend dosages. 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.

The use of leafy paste or leafy residue as the predominant component compared to juice or decoction in extractions is contrary to the teachings of traditional medicine. However, surprisingly, 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.

BRIEF SUMMARY OF THE INVENTION

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

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human and animal ailments, and has manifold applications in agriculture, using exceptionally low doses and without toxic side effects. These uses will become apparent as the various embodiments of the invention are discussed.

DETAILED DESCRIPTION The 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. By varying 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). Because various factors can be adjusted during the preparation of the compositions of the invention (plant parts, ratio of leaf, stem, plant parts, J fraction, LR fraction, oil, etc.), the drawbacks of traditional methods of preparation, such as cytotoxicity and excessively high doses, are circumvented. In addition, 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.

Low toxicity O 02/05830

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Oil extracts oi Kalanchoe pinnata (Lam.), 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.

Shelfhfe Fresh juice or extracts prepared by traditional methods ferments rapidly. However, the compositions of the invention remain potent, even after at least 7 years,

I. Embodiments

A. Human The compositions of the invention have a wide variety of human applications. A summary of examples of the many embodiments is given in Table 2.

The 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.

The compositions may also be used prophylactically.

Table 2 Human embodiments of uses for the compositions of the invention

B. Veterinary

The 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.

Table 3 Veterinary embodiments of uses for the compositions of the invention

C. Agricultural

The utility of the compositions of the present invention extends to all areas of the Plant Kingdom. For example, 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.

Table 4 Embodiments of uses for the compositions of the invention

Other embodiments of the invention will be apparent to those of skill in the art.

TJ. Definitions Crassulacean Acid Metabolism (CAM)

"CAM" involves the use of both the C₃ and C₄ pathways of carbon fixation. However, unlike C plants, CAM plants temporally separate, as opposed to spatially separate, the C₃ and C₄ cycles.

The C₃ 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. (2) In the second stage, 3-phosphoglycerate is reduced to glyceraldehydes 3 -phosphate, or 3-phosphoglyceraldhyde (PGAL), requiring NADPH as the nucleotide cofactor for reduction. (3) In the third stage, five of the six molecules of clyceraldehyde 3-phosphate are used to regenerate three molecules of ribulose 1,5-bisphosphate. Many plants use only the C₃ cycle. The C cycle (Hatch-Slack pathway) involves a first step of fixing carbon dioxide to phosphoenolpyruvate (PEP) by the enzyme PEP carboxylase. PEP carboxylase uses the hydrated form of carbon dioxide, bicarbonate ion. Depending on the species, the resulting 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. Plants that are C₄ spatially separate the different steps of carbon fixation: oxaloacetate and malate (or aspartate) are produced in the mesophyll cells, but then the malate (or aspartate) moves to bundle-sheath cells, where decarboxylation occurs and the Calvin cycle. Hence, C plants spatially separate the C₃ and C₄ cycles. Kranz leaf anatomy clearly identifies most C₄ 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. During the following light period, the malic acid is recovered from the vacuole, decarboxylated, and the carbon dioxide transferred to RuBP of the Calvin cycle within the same cells. Structurally, 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 C₃ and C₄ species (in favorable environments), will compete poorly (Raven et al., 1999).

Examples of 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.

Table 5 Examples of CAM plants

Extract

An "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. Examples of 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.

When oils are used as a solvent, generally all oils are meant and that are appropriate for the application. Examples 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 indicά), 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 ι can be used, it is preferred to avoid those oils and oil combinations that polymerize or fonn gum during the extraction procedure that would interfere with extraction and fractionation.

Often, 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. The term "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

"Vigor" refers to the active, healthy, and well-balanced growth of plants or animals. For example, 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

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, yield, and feed conversion

Production refers to the aspect of a plant or animal that is used for human purposes. For example, 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. On the other hand, 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.

Quality

"Quality" 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

" 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

The following describes the preparation of an extract prepared from Kalanchoe pinnata (Lam.) . Also see Examples. It will be apparent to one of skill in the art that many variations of the following procedure may yield extracts with similar activities, hi general, any extract produced from Kalanchoe pinnata (Lam.) that has at least one of the activities of the extract (see examples) is contemplated by the inventors.

However, 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. Generally, 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.

Several variables can be adjusted to achieve extracts of equal potency. For example, the starting material may consist of leaves, stems, shoots, or the entire plant. Alternatively, juice that has been manually extracted, or expressed, from the plant or plant parts may also be used. Instead of 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.

B. Pharmaceutical compositions

The 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.

The pharmaceutical compositions for the administration of the active compounds, such as those of CAM plant extracts, 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. In the pharmaceutical composition the active compound is included in an amount sufficient to produce the desired effect upon the process or condition of diseases.

General considerations

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.

Injectable formulations Pharmaceutical compositions suitable for injection include sterile aqueous solutions

(where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, CREMOPHOR EL^™ (BASF, Parsippany, N. J.) or phosphate buffered saline (PBS). In all cases, 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. Generally, 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

Oral compositions generally include an inert diluent or an edible carrier, They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, 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! . Pharmaceutically compatible binding agents, and/or adjuvant materials can be included 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./

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.

Systemic administration, including patches Systemic administration can also be transmucosal or transdermal. For transmucosal or transdermal administration, 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. For transdermal 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.

Carriers

In one embodiment, 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. 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

Oral formulations or parenteral compositions in unit dosage form can be created to faciUtate administration and dosage uniformity. 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.

Dosage

The pharmaceutical composition 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.

In the treatment of human conditions which require the CAM compositions of the invention, 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. Preferably, 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.

For oral a<hninistraticn, 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.

For topical applications, 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. Alternatively, 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.

Kits for pharmaceutical compositions

The pharmaceutical compositions can be included in a kit, container, pack, or dispenser together with instructions for administration. When the invention is supphed as a kit, 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. For example, 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. Other examples of 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.

Delivery methods Interstitial delivery The composition of the invention, such as CAM plant extracts, may be dehvered to the interstitial space of tissues of the animal body, including those of muscle, skin, brain, lung, liver, spleen, bone marrow, thymus, heart, lymph, blood, bone, cartilage, pancreas, kidney, gaU bladder, stomach, intestine, testis, ovary, uterus, rectum, nervous system, eye, gland, and connective tissue. 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.

Any apparatus known to the skiUed artisan in the medical arts may be used to deliver the compositions of the invention to the site of injury mterstitially. These include, but are not limited to, syringes, stents and catheters. Systemic deUvery

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.

Surgical delivery The 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. For example, 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. Alternatively, the compositions of the invention may be applied in salves, creams, or other pharmaceutical compositions known in the art meant for topical appUcation.

C. Agricultural/horticultural compositions 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.

It is also possible to prepare combinations with other pesticidally active substances, fertilizers and/or growth regulators, for example in the form of a ready mix or a tank mix. These can be thought of us to be "carriers" for the CAM plant extracts.

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.

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). Examples of 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.

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.

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), can be prepared, for example, by means of stirrers, colloid miUs and/or static mixers using aqueous organic solvents in the presence or absence of surfactants, for example, in the case of the other formulation types.

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, kaσlinites 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.

As a rule, 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.

For the preparation of disk, fluidized-bed, extruder and spray granules see, for example (1973; 1979). h wettable powders, 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. In the case of emulsifiable concentrates, 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. In the case of water-dispersible granules, 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. Alternatively, 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. For herbicidal effects or for control of excessive growth mediated by CAM plant extracts, the extract concentration is increased to 25 to 500 g per hectare per year. Besides, 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.

For use, 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, such as fertilizers or pesticides, 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.. Examples of 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. Other compounds that can be used include 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, quinσnes, sulfonic acid derivatives, thiocyanates and isocyanates, phytoalexins, insect killing soaps such as potassium fatty acid salts, and antiaUatotropins such as 7-me oxy-2,2-dimethylchromene and the 6,7-dimethoxy analog. Diatomaceous earth can be used, which kills crawling insects.

These components can comprise from 0.001 to 10% or more by weight of the CAM plant extract compositions suitable for plant appUcation, Also, 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. Where the CAM plant extract compositions are in soUd form and CAM plant extracts are sensitive to light, air, or compounds in the composition, orto optional added components, 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.

The presence of 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.

EXAMPLES

The following examples are included to demonstrate preferred embodiments of the present invention. It should be appreciated by those of skiU in the art that the techniques disclosed in the examples that follow represent techniques discovered by the inventors to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments that are disclosed and still obtain a like or similar result without departing form the spirit and scope of the invention.

I. Examples of CAM plant extraction

The following examples illustrate CAM plant extractions; however, one of skill in the art wiU know how to vary the various variables to obtain extracts with the activity of the CAM plant extracts of the invention. Table A summarizes the designations for the various extracts used throughout the following examples. hi all of the foUowing examples of CAM plant extraction, final extract weight is the same as the starting fresh plant weight; hence all extracts are equivalent on fresh weight basis and have equivalent potencies .

Using Kalanchoe pinnata Example 1 General extract procedure

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). Separately, 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

Example 4 Illustrating the use of different oils Coconut oil

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.

Safflower oil; also demonstrating different homogenization

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.

Using Aloe vera

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 residue was stirred with an equal weight of sesame oil. The two extracts were combined and additional sesame oil was added to a final total weight of 800 g. This composition from the LR fraction based on 100 g of initial leaf equivalent per 100 g of total final extract was named A-100 PLUS.

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 . Using Cissus quadrangularis

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.

Example 7 Illustrating use of animal fat

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.

Table A Designations of the various extracts

Extracts by using the general procedures outlined in Examples 1 to 7 can also be made from all other CAM plants.

II. Useful preparations of CAM plant extracts

The foUowing examples iUustrate compositions of plant extracts for a variety of applications; however, they are not meant to be limiting. It will be apparent to one of skill in the art how to modify the various preparations for specific applications,

Example 8 Dilution of extracts

Extracts made by the general procedure described in Examples 1-7 were further diluted with sesame oil to make extracts of different strengths . For example, extract made by mixing one part of R-100 with 9 parts of sesame oil was named R-10. Similarly, 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. Thus, 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.

In veterinary applications, R-10 to R-1 constitute a more convenient dosage form for addition to feed or drinking water. hi crop health applications, too, 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. Alternatively, 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). Similarly, 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. Thus, one can make these diluted powders of any desired strength such as R-1(P), R-2(P), R-5(P), R-10(P), etc. as convenient and stable dosage forms.

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.

For internal veterinary appUcations, the quantities required are so smaU that R-10(P) to R- 1(P) dilutions can be conveniently used directly for feed mixing in the conventional mixing equipment.

In crop health applications, too, R-5(P) to R-1(P) can be conveniently added to the root zone or added to the water for spraying purposes.

Example 10 Tablet compounding

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. However, a variety of other excipients may also be used, with or without other adjuvants, for tablet making.

Tablets of any desired strength of mg of R-100 can be made. In the following examples, they were named accordingly as T-l, T-2, T-5, T-10, etc.. Tablet making also allows convenient dose metering. Thus, for internal human consumption, T-l represents one of the convenient forms as the typical human dose is one T-l per day for an adult. In veterinary applications also, for the same reason, T-10 to T-l constitute a more convenient dosage form for addition to feed.

In crop health appUcations, one T-5 in 5 liters water is a very convenient spray dose per 100 m² field area. Also, 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.

150 mg tablets

100 g of R-100 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.

Example 11 Cream formulations

The notations to describe the strength of oil, powder and tablet in terms of leaf equivalent are also appUcable to creams.

All purpose cream

Two mixtures were prepared. 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.

Hand and body lotion (oil in water emulsion)

Two mixtures were prepared. 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.

III. Human appUcations Methods

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.

Results Example 12

Table 6 Results of human applications of CAM plant oil extracts

As an example, none of the persons whose LDL andtriglyceride levels have responded to the use of these compositions made any special changes in their diet or their lifestyle during the trial period (Table 6). At a dose level of 2 to 4 T-l tablets/day, or at a dose level of 2 to 4 mg leaf equivalent/day, levels of serum LDL and/or serum triglyceride were reduced considerably within a few months (Table 7).

Table 7 Case Summary: Lipid-lowering effect of the compositions of the invention CASE SUMMARY -LIPID PROFILE mg/dl mg/dl mg/dl mg/dl T-l/day

CASENO./Dates Sex/Age(yrs.) Cholesterol HDL LDL Trigly Dose/ Remarks

1.

26 / 05 / 93 Male(50) 170 58 96 80 Base Data

09 / 12 / 93 168 55 98 77 Base Data 16/11/94 138 62 67 49 2 taken for two months bef. test

10 / 07 / 95 Male(40) 219 44 179 2 dose start 23 / 09 / 95 177 40 125 123 2 27 / 02 / 96 185 43 116 127 1-2 10/08/96 185 41 118 127 3.

23 / 09 / 95 Fem(12) 204 48 138 90 1 dose start 19 / 06 / 97 151 46 90 78 Taken a total ^" 150 tabs on/off

4.

09/10/95 Male(45) 268 37 176 273 1 dose start

30/1/96 247 38 151 247

5.

21 / 09 / 96 Male(60) 230 47 120 313 1 started from 26 / 02 / 97 225 41 130 269 4^th may 1996

26 / 03 / 98 228 53 194 Intermittently 6.

27 / 08 / 93 Male(40) 258 53 151 270 1 dose started 01 / 03 / 97 245 40 169 180 taken a total 150 tabs from 5/5/96

7..

15/11/97 Male(35) 240 4 dose started 06 / 02 / 98 148 from 8/12/97 8. 06 / 10 / 98 Fem(50) 220 60 137 115 2 dose started 18 / 12 / 98 170 60 89 105

06 / 10 / 98 Fem(24) 245 65 166 70 2 dose started 18 / 12 / 98 195 70 115 50

10.

06 / 11 / 98 Male(80) 310

01 / 03 / 99 174 2 Taken for two months before test

Notes on Table 6:

Stress resistance /energy level As reported in Table 6, a number of persons experienced effects such as reduction in stress-induced asthma, increase in sound sleep, etc. These effects were obtained by a daily intake of one T-l tablet for 2 to 4 weeks. A number of elderly persons (over 70 years) found an enhanced sense of well-being, higher energy levels, a general reduction in stomach upsets and a reduction in seasonal coughs and colds by daily intake of one T-l tablet.

Healthy growth - Height/weight gain in children

As reported in Table 6, several children who were otherwise had a lack of appetite, routine headaches, low hemoglobin, fatigue, etc, responded positively to the intake of one T-l tablet per day and started registering healthy height and weight gain with alleviation of these other symptoms. Significant improvement was noted after treatment for more than one month.

Healing response

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) . hi case of deep leprotic ulcers, 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.

Skin inflammation and repair hi cases of eczema and psoriasis, topical appUcation gave relief from the inflammatory process. In case of lichenplanus, the lesions healed readily. Tan caused by sunburn on exposed arms was eUminated by topical appUcation of a 0.1% R-100 body lotion. Inflammation of pimples reduced by facial appUcation of a 0, 1% R-100 body lotion. In a few cases, inflammation and wound due to piles was also controlled.

Hair health

As reported in Table 6, several persons losing hair on account of iU health or poor hair health, the loss was arrested and vigorous re-growth of hair started by the intake of one T-l tablet per day. Observable effects were noted within two weeks of starting the intake. AppUcation of hair oil containing 0.1% by weight of R-100 to the scalp produced the same effect.

Eyes hi a few cases, rapid vision recovery post-macular surgery by oral intake of T-l tablets was noted. Recovery from sty infection and dry corneas were achieved by topical application.

Reproductive system

A few cases of oligospermia/sperm motility were corrected by oral intake of T-l tablets for three months.

IV. Agricultural applications Methods R-5 or R-2 oil , R-10(P) powder and tablets made as per examples above were used in aU of the following examples. Plants were either grown in hydroponic or soil media.

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. In the following examples, the typical dosage of extract for field crops was 0.5 to 1 g of R-

100 oil or leaf equivalent per hectare per spray. 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.

Multiple high dose sprays of particular preparations such as R-100 MINUS (5 to 25 g per spray per hectare) act to reduce the flower set, total seeds produced and the size of seeds. This effect can be used to control the propagation of hardy weeds such as "Congresss Grass" (Parthium sp.), Lantana sp. , Cyperus sp. and others.

Toxicity

Example 13 R100 PLUS vs. R-100 MINUS of Kalanchoe pinnata in Onion Root Tip

Assay

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; Mercykutty, 1980).

Bulbs of Allium cepa,L. cvN-2-4-l(2n=16) were used. Selected bulbs were washed and the root systems of the bulbs were kept in 100 ml of aqueous solutions in cavity blocks containing different amounts of herbal oil extract. Roots were treated for 48 hours.

At the end of 48 hours, the root tips were recovered and fixed in acetic acid-alcohol (1:3). For cytological preparations, 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.

Table 8 Effect of R-100 PLUS vs. R-100 MINUS on Onion Root Tip

Concentration, No. cell I Mitotic Index No. cells showing aberrations Root no./ μl/liter scored - % dividing - Physiological Clastogenic (length, cm)

0 (CONTROL) 1120 36.25 0 0 42 (4.3)

R-100 PLUS

10 1105 39.12 0 0 50 (4.6)

30 1121 42.58 0 0 52 (4.7)

100 1085 40.12 8 4 44 (3.9)

300 1048 30.23 58 8 42 (3.6)

R-100

10 1012 38.56 0 0 48 (4.5)

30 1025 40.12 12 0 45 (4.1)

100 1045 25.26 87 7 38 (3.8)

300 1005 18.23 126 14 36 (1.6)

R-100 MINUS 10 1052 38.36 8 12 43 (4.2) 30 930 25.25 49 31 38 (3.2) 100 936 8.25 487 45 26(1.2) 300 856 1.22 671 69 12 (0.9)

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 μl/liter of R-100 PLUS. Sprouting was moderate with 10 μl/liter of R-100 and 30 μl/liter of R-100 PLUS. There was no sprouting at aU in any of the other sets. In the above data, 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 μl liter whereas R-100 PLUS-treated root tips began to show aberrations at 100 μl/liter, a tenfold higher concentration. The mitotic index, and the number and average root length also confirmed this observation. R-100 PLUS, at least up to 30 μl/liter promoted ceU division, rooting and sprouting. In contrast, R-100 MINUS began to act as a mitogen and root system inhibitor beyond 10 μl Uter.

Example 14 CAM plant fractions in Onion Root Tip Assay

Experiments were carried out using A-100 PLUS, A-100 MINUS, C-100 PLUS, C-100 MINUS, R-100 PLUS, R-100 MINUS and sesame oil at the solution concentrations given in Table 9.

Bulbs of Allium cepa, . cvN-2-4-l(2n=16) were used. Selected bulbs were washed and the root systems of the bulbs were kept in 100 ml aqueous solution in cavity blocks containing different amount of herbal oil extract. Roots were treated for 12 hours and then recovered in 10% glucose for another 12 hours.

At the end of recovery, the root tips were recovered and fixed in acetic acid-alcohol (1 :3). For cytological preparations, 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.

Table 9 Effect of CAM Plant Fractions on Onion Root Tip

Concentration, No. ceU Mitotic Index % cells showing aberrations μl/liter scored - % dividing - Physiological Clastogenic Sesame oil

0 1552 30.12 0 0

100 1230 31.23 0.08 0

300 1452 32.59 0.34 0

R-100 PLUS

30 1530 32.24 0 0

100 1547 33.56 0.52 0

300 1531 18.63 1.31 0.2

R-100 MINUS

30 1498 28.27 0.13 0

100 1521 14.45 1.78 0.26

300 1530 9.63 3.01 0.92

A-100 PLUS

30 1521 32.21 0 0

100 1511 33.52 0 0

300 1505 31.89 0.8 0

A-100 MINUS

30 1563 31.25 0 0

100 1524 32.68 0.2 0

300 1541 28.30 0.97 0

C-100 PLUS

30 1621 32.33 0.06 0

100 1563 26.23 1.09 0

300 1518 11.14 1.98 0.4

C-100 MINUS

30 1546 31.28 0.45 0

100 1543 12.65 1.94 0.32

300 1532 8.25 3.26 0.91

In all cases, the PLUS fraction (LR) 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 μl/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. Surprisingly, 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.

These positive effects, particularly in case of Kalanchoe and Cissus, have greatly extended their safe operating range, considerably improved their efficacy/toxicity ratio and therefore extended their utiUty in applications that hitherto were restricted due to the toxicity of the use of juice with a high dosage. The higher toxicity of the juice-based extract can be used in appUcations to eUminate unwanted vegetation or control plant growth.

Applications to dicotyledonous plants

Germination promotion Example 15 Germination of Phaseolus mungo using R-10(P)

Using R-10 (P) (batch 881128) in Phaseolus mungo, a legume, the seeds were soaked in a solution of R-10 (P) and observations were taken at 24, 48 and 120 hours after soaking. The results are summarized in Table 10.

Table 10 Germination promotion

Concentration, ppm, ROOT LENGTH(cm), RANGE

R-10(P) 24Hrs 48 Hrs 120 Hrs

O(Control) 0.2- 1.0 3.5- 6.0 4,2- 8.6

1 0.2- 2.0 4.0- 6.2 3,5-12.5 5 0.2- 2.5 4.0- 6.8 5.7-12.1

20 2.0- 2.8 4.5- 7.1 13.9-17.8

500 0.5- 2.5 0.5- 4.7 3.6-11.2

Thus, germination promotion is observed with increasing concentration up to 20 ppm of R-10(P). At 20 ppm of R-10(P) or 2 ppm of R-100 equivalent, there is a particularly strong promotion of germination.

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^ώ day after initiation of experiment, mean values of 11 seedlings were taken and reported. The results are summarized below in Table 11.

Table 11 Germination in Phaeolus mungo comparison of different forms of extract

Note: Concentration in the medium is in μl/50 ml distilled water. Values in parenthesis are standard deviation.

Concentration R-100 PLUS R- 100 R-100 MINUS Sesame oil

Root Length (cm)

.0 4.62(0.26) 4.62(0.26) 4.62(0.26) 4.62(0.26)

0.33 5.22(0.08) 5.18(0.40) 4.76(0.11) 4.52(0.29)

1.0 5.38(0.08) 5.30((0.10) 4.86(0.39) 4.92(0.18)

3.0 5.62(0.08) 5.56(0.11) 4.94(0.11) 4.96(0.19)

30.0 5.30(0.07) 5.12(0.09) 4.68(0.11) 5.28(0.22)

Shoot Length (cm)

0.0 9.08(0.29) 9.08(0.29) 9.08(0.29) 9.08(0.29)

0,33 11.48(0.24) 11.48(0.53) 13.64(0.38) 10.56(0.23)

1.0 12.44(0.23) 13.50(0.07) 15.02(0.22) 10.96(0.18)

3.0 13.70(0.37) 13.56(0.23) 15.70(0.20) 11.84(0.17)

30.0 13.46(0.15) 11.4(0.16) 15.12(0.49) 12.54(0.11)

Dry weight, g (10 seedlings) - mean of two observations

0.0 0.33 0.33 0.33 0.33

0.33 0.35 0.34 0.32 0.33

1.0 0.42 0.41 0.34 0.33

3. 0.40 0.38 0.31 0.34

30 0.35 0.32 0.27 0.35

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.

Thus, this data corroborates the contrasting behavior of LR and the J fractions-based compositions from Kalanchoe described in Example 14 above.

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.

Table 12 Effect of CAM plant fractions on seed germination activity

Note: Concentration in the medium is in μl of oil /50 ml distiUed water. Values in parenthesis indicate standard deviation.

Sesame Oil A-100 A-100 C-100 C-100

Concentration Plain PLUS MINUS PLUS MINUS

Root Length(cm)

0.0 5.09(0.065) 5.12(0.065) 5.12(0.065) 5.12(0.065) 5.12(0.065)

1.0 5.20(0.048) 6.33(0.068) 6.12(0.087) 6.75(0.055) 5.42(0.063)

3.0 5.30(0.052) 6.51(0.052) 6.39(0.047) 4,33(0.065) 5.23(0.071)

10.0 5.67(0.061) 6.69(0.061) 6.41(0.045) 4.67(0.062) 4.51(0.042)

Shoot Length(cm)

0.0 10.35(0.058) ^' 10.35(0.058) 10.35(0.058) 10.35(0.058) 10.35(0.058)

1.0 12.08(0.062) 12.12(0.054) 11.86(0.062) 13.35(0.064) 13.62(0.048)

3.0 12.98(0.047) 13.12(0.062) 13.02(0.068) 12.33(0.56) 12.65(0.052)

10.0 13.56(0.054) 13.56(0.057) 13.12(0.077) 11.38(0.054) 12.17(0.051)

Dry Weight(gm/10 seedlings) mean of two observations. Numbers in parenthesis indicate ± range of the two observations.

0.0 0.332(0.0023) 0.332(0.0023) 0.332(0.0023) 0.332(0.0023) 0.332(0.0023} 1.0 0.351(0.0023) 0.371(0.0022) 0.384(0.0022) 0.352(0.0022) 0.364(0.0018)

3.0 0.363(0.0018) 0.408(0.0023) 0.397(0.0021) 0.382(0.0023) 0.377(0.0019)

10.0 0.362(0.0019) 0.427(0.0019) 0.415(0.0023) 0.336(0.0021) 0.322(0.0023)

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 μl/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 μl/50ml distilled water. Thus, 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. However, C-100 PLUS and MINUS had a different activity profile. They showed auxin, gibbereUin and cytokine-Uke activities at 1 μl 50 ml DW as well. However, at higher concentrations (10 μl/50 ml distilled water), a reversal in all activities was observed. Thus, this process of inhibition of activity started earlier with C-100 than the other extracts.

Effects on fruits and vegetables

Example 18 Promotion of plant defense, growth, and enzymes in okra (Abelmoschus esculentus L CVPusa Savani)

Experiments were carried out at Pune, India, using R-5 oU (batch 920814). Plants were cultured in both hydroponic (sand culture with Modified Hoagland media) and normal soil (loamy soil and farmyard manure (3 : 1), 12 kg/pot) media. The three treatment levels used were 1 mg, 3 mg and 10 mg per Uter of R-5, and was applied at a rate of one liter per pot per application. Thus, the amount of R-100 oil equivalent added per treatment/pot was 0.05 mg, 0.15 mg and 0.50 mg. Plants were treated for 15 and 30 days after sowing. The results are summarized in Table 13.

Table 13 Increase in plant height (cm)

Cone, mg R-5 oil Initial @ 40 days @ 60 days

Control 8.3 42.3 52.6

1 8.2 51.4 61.7

3 8.3 53.3 67.5

10 8.2 54.6 69.4

Stand. Error. 0.41 1.02 0,83

CD. @ 1% 1.52 3.78 3.09

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).

Table 14 Increase in leaf area and leaf dry weight

Cone, mg,R-5oU LA LW

(cm²/plant) g/plant HYDROPONICS (54 days) Std.Deviation. Std. Deviation • 46

0(Control) 356.0 1.85 1.49 0.5

1 396.3 1.62 2.15 0.1

3 392.1 1.68 2.01 0.47

10 374.0 0.87 2.35 0.67

(58 days)

0(Control) 636.3 2.09 1.93 0.26

1 744.1 2.16 2.87 0.12

3 834.1 1.5 3.01 0.19

10 756.3 2.04 2.63 0.05

All treated plants had dark green glossy leaves and higher chlorophyU level, particularly those of chlorophyll-b (mean of three samples), summarized in Table 15.

Table 15 Leaf chlorophyll levels

Cone, mg,R-5 @30 days @60 days HYDROPONICS chloro-a chloro-b chloro-a chloro-b

0 (Control) 101.9(0.56) 127.6(0.85) 97.4(0,64) 123.6(0.55)

1 104.2(1.94) 129.1(1.65) 101.5(0.52) 133,9(0.76)

3 106.2((1.47) 143.8(1.03) 103.9(0.77) 141.8(0.52)

10 93.5(0.87) 131,8(0.68) 104.0(0.59) 130.1(0.88)

SOIL

0 (Control) 96.4 (1.26) 117.4 (1.20) 103.8 (1.09) 132.8 (1.09)

1 104.9 (0.93) 127.5 (1.29) 105.4 (0.76) 134.7 (0.80)

3 115.9 (1.14) 156.4 (1.05) 116.8 (0.95) 172.1 (0.89)

10 103.2 (1.76) 129.5 (1.22) 101.2 (1.02) 144.1 (0.67)

CD. (1%) 5.87 5.86 3.51 4.1

NOTE: Chlorophyll levels are in mg/lOOg fresh weight (FW) of leaves. Numbers in parenthesis are values for standard deviation.

Activity of photosynthetic enzyme, ribulose phosphate (RuBp) and oxidative enzymes

(Peroxidase, Polyphenol oxidase (PPO) and IAA oxidase (IAAO)) increased in treated okra plants, as summaraized in Table 16.

Table 16 Leaf enzyme activity

Cone, mg,R-5 RuBp IAAO PPO Peroxidase HYDROPONICS® 52 days

0(Control) 0.083 0.87 0.086 0.62

1 0.092 0.91 0.102 0.89

3 0.098 0.98 0.127 0.96

10 0.099 0.89 0.088 0.66

SOIL@ 52 days

0(Control) 0.092 0.94 0.087 0.88

1 1.025 1.29 0.092 0.93

3 1,058 1.44 0.111 1.27

10 1.098 1.21 0.102 1.14

NOTE: Enzyme activity for IIAO, PPO and Peroxidase is expressed as change in optical density/ming protein

RuBp enzyme: specific activity = rrucromoles/min/mg protein.

In addition, the level of carbohydrates, proteins and polyphenols increased in leaves of treated plants, summarized in Table 17.

Table 17 Increase in carbohydrates, proteins and polyphenols in leaves (mean of three plants)

Cone, mg,R-5 Carbohydrates Proteins Polyphenol HYDROPONICS® 62 days mg/g Fresh Wt. mg/g Fresh Wt mg/g Wt

0(Control) 30.1 (1.21) 56.3 (0.81) 6.2 (0.98)

1 52.2 (1.38) 57.9 (0.98) 16.0 (0.92)

3 59.8 (0.98) 58.1 (0.76) 18.0 (1.02) 10 68.2 (1.08) 58.3 (0.56) 11.1(0.78) NOTE: Numbers in parenthesis are values of standard deviation.

SOIL@ 68 days

0(Control) 38.7 61.1 8.3

1 98.4 65.1 10.6

3 88.1 66.1 14.3

10 102.4 65.3 12.9

Stand. Error 4.04 0.77 0.84

CD. @ 1% 14.96 2.85 3.1 QuaUtative observations included larger flowers and higher fruit yield in treated plants. Thus, 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.

Effect of 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.)

Growth parameters and biochemical status were also affected. 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

Trials were carried out near Daund, Maharashtra, India on Tomato (Lycopersicum esculentum), Golden variety; Brinjal (Solanum melangona), Kalptharu variety; and Okra (Abelmoschus 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.

Table 18 Yield, productive life, and pest resistance

O OBBSSEEERVATIONS TOMATO BRINJAL OKRA

Test Control Test Control Test Control

Yield, kg

T'Harvest 80 60 55 40 35 35

2^nd Harvest (31 days after 1^st harvest) 30 20 15 12 15 04

3^rd Harvest, (61 days after 1^st harvest) 20 06 10 02 14 07

TOTAL 130 86 80 54 64 46

Other observations relative to control plants were: 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,

Table 19 Yield

OBSERVATIONS RED CHILLIES OKRA

CONTROL SET I SET II CONTROL SET I SET II

R-10 oil Cone, ml ha 0 10 20 0 10 20

Yield / plant, g 63.0 81.3 103.1

Yield/Ha, quintals 35.03 45.10 57.33 168.3 180.0 188.80

Example 21 Shelf life of R-5 treated okra

Experiments were carried out at Pune, India in November 1999 using R-5 oil (batch 920814) and R-5 oil (batch 990509) from R-100 oil preparation. Abelmoschus esculentus L CV Lucy was grown in loamy soil and farmyard manure (3:1), 20 kg/pot (Plastic tubs with 25 cm radius and 25 cm high). The three treatment levels used were 1 mg, 3 mg and 10 mg per liter of R- 5. Per application, one Uter of solution was applied per pot. Thus, the amount of R-100 oU equivalent added per treatment/pot was 0.05 mg, 0.15 mg and 0.50 mg. Plants were treated 15 days and 30 days after sowing.

The growth parameters and biochemical status were examined OTable 20). Plant height increased for treated plants (mean of 10 plants) compared to controls at up to 3 mg of R-5. At higher concentrations, there was a reversal observed in both sets. The results of treatment with older (7 years old) and newer batch of R-100 did not show significant differences.

Table 20 Increase in plant height (cm) R-100 BATCH DATE 14^th August, 1992 9* May, 1999

Cone, mg R-5 oil Initial @ 60 days Initial @ 60 days

Control 8.6 49.9 8.2 46.6

1 8.6 53.6 8.2 52.3

3 8.6 54.8 8.3 56.3 10 8.6 51.5 8.3 48.6 Stand. Error. 0.26 0.38 0,35 0.91 CD. (1%) 1.52 1.41 1.29 3.39

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)

Table 21 Chlorophyll levels

R-100 DATE 14^ώ August, 1992 9^th May, 1999

Cone, mg,R-5 @60 days @60 days

SOIL chloro-a chloro-b chloro-a chloro-b

O(Control) 107.4(0.93) 141.4(0.96) 105.8(0.63) 136.5(0.67)

1 111.5(0.83) 152.1(0.35) 115.5(0.91) 152.9(0.77)

3 114.6(0.76) 154.3(0.42) 116.8(0.74) 172.1(0.70)

10 114.0(0.47) 148.4(0.49) 107.5(0.39) 143.3(0.60)

CD. (1%) 3.72 2.56 3.0 3,46

NOTE: Chlorophyll levels are in mg/lOOgm fresh weight (FW) of leaves. Numbers in parenthesis are values of Standard Deviation.

Levels of reducing sugars increased in treated plants at flowering both with old and new composition. Increases in non-reducing sugars were not highly significant OTable 22).

Table 22 Effect on non-reducing and reducing sugars

R-100 DATE 14^th August, 1992 9* May, 1999 SOIL® -62 days Reducing Non-Reducing Reducing Non-Reducing mg/g FW mg/g FW mg/g FW mg/g FW

Cone, mg of R-5 oil 0(Control) 17.9 16.8 14.3 17.12

1 22.4 18.8 22.6 20.08

3 26.4 19.4 22.7 21.6

10 22,4 18.7 21.9 19.3

Stand. Error 0.81 0.71 0.77 0.52 - CD. @ 1% 3.01 2.65 2.88 1.91 Other observations included larger flower and higher fruit yield in treated plants with both the new and old composition. Thus, R-100 appeared to act at a very fundamental level in aU stages of plant growth. R-100 activity is substantiaUy retained in samples that are 7 years old.

Example 22 Fruit: higher yield, pest resistance and shelf life

Trials with 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.

Mango, Pomegranate, Ber, Sapota: Two sprays were given at a 20 day interval during the flowering.

Lime and Guava: Three sprays were given at 30 day interval during flowering.

Table 23 Fruit yield, resistance, quality, ripening and shelf life

TEST CONTROL

MANGO (Mangifera indica) Number of fruit 370 300

Fruit quahty Shiny, attractive

Ripening delayed by 10-12 days

Resistance: Leaf curling reduced

POMEGRANATE (Punica granatum) Number of fruit 197 150

Fruit quaUty Redness increased

Black spots reduced Fruit drop and decay reduced Resistance: Pomegranate butterfly attack reduced BER (Zizyphus jujubά)

Fruit /tree, kg 6 4

Fruit quality Shiny, Ready for harvest early

Longer shelf life Resistence: sucking pest/fruit borer attack reduced SAPOTA (Achras sapota) Number of fruit 333 300

Fruit quahty healthy looking

Late ripening and.

Longer shelf life LIME (Citrus aurantifoliea) Yield increase 25 %

Fruit quality Larger size

Reduced fruit drop and decay of fallen fruit Resistance: Black leaf eating caterpillar reduced

GUAVA (Psidum guava) Yield increase 20% increase

Fruit quality Larger

Attractive color development on ripe fruit

Example 23 Yield and size in Strawberry (Fragaria x ananasa) (ChandUar variety)

Trials with R-5 oU were carried out at Panchgani, Maharashtra, India. Spray volume was 300 liter/ha. Three treatments with R-5 oil rate of 3 ml/ha, 10 ml/ha and 30 ml/ha were used. This corresponds to R-5 concentration of 33 ppm,100 ppm and 300 ppm solutions. A total of four sprays were given (one at 31 days, 42 days, 138 days and 156 days after planting). Each plot was 1 m² with 5 plants. Randomized Block design with 5 repUcates was used.

The first flush was washed out due to rain. The fruit were coUected from the next ten flushes. The results are reported in Table 24.

Table 24 Yield and size

Level of application

OBSERVATION CONTROL 10 ppm 33 ppm 100 ppm S.E. CD@ 5%

Av. Wt. of Fruit/plot, g 257.4 279.8 546.0 472.4 16.23 50.01 A Avv,, NNoo.. ooff FFrruuiitt//pplloott 2 233..44 23.4 25.0 29.4 1.27 3.92

Av. Wt. of Fruit, g 11.0 11.9 21.8 16.0

OTHER OBSERVATIONS

LEAVES glossy glossy glossy

FRUIT shiny shiny shiny Thus, there was a substantial increase in yield and size at concentrations of 33 and 100 ppm.

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

930417) and R-5 (batch 920814) from R-100 preparations that were more than 6 years old. Seeds were obtained from Mahatma Phule Agricultureal University, Rahuri, Maharasshtra, India. The plants were cultured in plots 1 m x 1.5 m, using the ridges and furrow method. Plants were space 30 cm within a rows 60 cm apart. Five plants/treatment, each in a row, were used. Solution concentrations used: (Control: distiUed water)

Treatment T-l(150) Tablets lit. Treatment R-5 Oil, μl/Uter

Tab l 3 04 60

Tab 2 1 0 5 20

Tab 3 1/3 0 6 7 Spray Method: 10 ml/plant, twice a month up to fruiting. Thus, 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.

Table 25 Plant growth parameters:

Observations taken 58-60 days after first treatment; Average values of three plants per set for three sets were measured

Treatment Control Tabl Tab2 05 06

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)

Branches 33.5 (2.07) 45.0 (1.410) 56.5 (1.1) 52.1 (1.49) 50.4 (0.72)

LEAF (Average values)

Spines (upper) 30.3 (0.85) 14.8 (0.30) 12.8 (0.91) 13.4 (0.53) 16.2 (0.66)

Spines (lower) 37.5 (0.70) 16.4 (1.14) 14.2 (0.60) 17.4 (0.53) 16.2 (0.79) Values in parenthesis indicate standard deviations.

Table 26 Pigments, proteins and polyphenols in leaves

60 days after transplanting; Average values of three plants each from three sets are reported 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)

Proteins 3.4(0.06) 4.1)0.63) 3.8(0.28) 4.3(0.20) 3.7(0.17) Total Phenols 2.82(0.09) 3.23(0.12) 3.1(0.07) 3.41(0.06) 3.02(0.15)

Table 27 Fruit and seeds yield

Average value of three plants each for three sets are reported.

Treatment Control Tabl Tab2 05 06 Fresh Wt. of Fruit

/plant, g 77.3(0.68) 82.1(0.44) 90.3(1.3) 85.7(1.01) 80.2(1.78)

Solasodine 31.4(0.092) 40.2(0.60) 45.3(1.23) 46.1(0.54) 45.1(0.27)

(mg / 100 gDW of Fruit) gm/100 seeds 0.20(0.02) 0,25(0.02) 0.30(0.04) 0.30(0.04) 0.28(0.02) seeds/fruit 181.5(3.62) 210.2(1.24) 215.3(4.20) 217.3(1.41) 215.2(3.21)

Thus, plant height and number of branches were enhanced by appUcation of tablets and oil. At these treatment levels, carbohydrate, protein and phenol levels/FW of leaves increased marginally in treated plants. However, the spines were reduced by more than 50% at aU treatment levels used. This makes harvesting easier. Fruit yield was higher and at this higher yield, solasodine levels are 40% to 50% higher in treated plants than in controls. Thus, the medicinaUy important alkaloid levels have been increased/plant.

Example 25 Cotton Trials were carried out at Dharwad, Karnataka, India in Kharif on cotton. Plants were cultivated on 18,2 m² 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.

Table 28 Cotton yield

CONTROL CONTROL TEST

Without Water With Water

R-2 oil Cone, ml/ha 0 0 50

Yield/Ha, quintals 14.27 13.73 16.98 % Increase 3.92 0 23.46 Example 26 Growth, resistance, leaf active life, and yield in soybean

Trials were carried out at the Pune University campus with T-l (150 mg) tablets (batch

930417) and R-5 (batch 920814) from R-100 prφarations that were more than 6 years old. Plants (Glycine max L. cvMacs) (winter variety) were cultivated in soil in pots 20 cm x 30 cm x 40 cm;

(farmyard manure and garden soil in 1:3 ratio). Seed were obtained from Agharkar Research

Institute (Pune, Maharasshtra, India). Four plants were grown in each pot, and each treatment consisted of 3 pots. Control solution was distiUed water. Treatments were:

Treatment T-l(150) Tablets/lit. Treatment R-5 Oil, microht./Ut. Tabl 3 04 60

Tab 2 1 05 20

Tab 3 1/3 06 7

Spray Method: 100 ml/pot, twice a month up to fruiting. Thus, 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.

Two sets of treatment were carried out. Average values of the combined set are reported in

Tables 29-31 (Values in parentheses indicate standard deviations).

Table 29 Growth parameters (Observations just before flowering)

Control Tabl Tab2 05 06

PLANT (Av. Value)

Height, cm 27.1(0.28) 30.3(1.27) 32.4(2.55) 32.5(1.27) 31.3(0.71)

Branches 5.2(0.21) 8.6(0.24) 9.2(0.18) 9.1(0.22) 8.2(0.24) Leaves 9.1(0.42) 12.3(0.24) 12.8(0.38) 12,6(0.35) 12.5(0,39)

Leafarea(cm²)27.5(1.12) 36.6(1,28) 36.8(1,35) 36.3(1.23) 36.5(1.27)

Table 30 Yield and leaf productivity

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)

Pods/Plant 5.5(0.07) 10.1(0.71) 12.2(0.49) 12.1(0.35) 11.3(1.13)

Seeds/Pod 2.4(0.014) 2.7(0.07) 2.9(0.09) 3.2(0.12) 2.7(0.16)

Gm/ 25 seeds 3.4(0.15) 3.9(0.18) 4.0(0.21) 4.1(0.13) 3.8(0.28)

Husk,gm 0.20(0.12) 0.23(0.03) 0.23(0.01) 0.22(0.02) 0.21(0.04) (Pod-Seeds) Table 31 Biochemical parameters at flowering mg/100 g Fresh Wt. of Leaves

Chlorophyll-a 101.2 (3.12) 122.7 (3.24) 134.0 (2.82) 128.9 (2.08)) 126.2 (2.58) ChlorophyU-b 108.5 (3.45) 137.6 (2.98) 140.1 (2.54) 139.3 (2.92) 130.1 (2.53) Proline 16.2 (0.65) 25.2 (0.82) 30.1 (0.18) 26.5 (0.62) 26.6 (0.54)

Polyphenols 23.68 (1.24) 34.12 (1.02) 46.98 (1.08) 46.01 (0.68) 45.01 (1.15) ChlorophyU 0.64 (0.03) 0.72 (0.04) 0.88 (0.02) 0.86 (0.03) 0.83 (0.02) StabiUty Index g/100 g Fresh Wt. of leaves Reducing Sugars

0.56 (0.05) 0.62 (0.04) 0.74 (0.05) 0.68 (0.02) 0.69 (0.04) Non-reducing Sugars

0.67 (0.08) 1.28 (0.06) 1.9 (0.05) 1.8 (0.05) 1.7 (0.06) Proteins

2.26 (0.12) 2.28 (0.2) 3.2 (0.18) 2.6 (0.12) 2.8 (0.18)

Thus, R-100 induced increases in a variety of growth parameters, such as height, number of branches, total leaf area, chlorophyU, etc. With an 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 level of defense chemicals, polyphenols, was also considerably enhanced. Higher proline levels and chlorophyU stabihty index are both strong indicators of environmental stress resistance against drought, frost, etc. Thus, an increase in biotic and abiotic stress tolerance was noted in this trial. Plants showed higher resistance to pest damage and also higher tolerance to water stress.

Example 31 Effect of CAM plant extracts in flowering and seed Production

Trials were carried out at the Pune University campus with R-5 PLUS (batch 000930) and R-5 MINUS (batch 000930) oil. Brassica juncea L. plants were cultivated in soil (farmyard manure and garden soil in 1 :3 ratio) in pots 20 cm x 20 cm x 40 cm. Fifteen plants were grown in each pot, and each treatment group consisted of 2 pots. Control solution was distiUed water. Spray solutions of different concentrations were made in 100 ml distiUed water.100 ml of solution was used per spray per pot. Spraying was done on 8^th 28^th and 48^th day after sowing, No spraying was done after flowering.

Two sets of treatment were carried out. Average values of the combined set are reported in Tables B and C Values in parentheses below indicate standard deviations.

Table B Plant Height (cm) (days (d) after treatment)

R-5 PLUS R-5 MINUS

Spray Cone ^' 20 d 60 d 90 d 20 d 60d 90d mg/100 ml 0 6.38 48.65 92.36 6.38 48.65 92.36

(0.52) (1.21) (2.03) (0.58) (1.21) (2.03)

3 7.82 53.92 106.86 7.95 52.36 88.54

(0.82) (1.55) (2.120 (0.61) (1.08) (2.88)

10 9.38 43.25 62.36 8.56 38.58 68.98 (0.57) (1.03) (3.21) (0.82) (1.31) (3.24)

Note: Above values are mean of five plants from each set.

Table C Yield

Note: D. F. = Days to Flowering; Seed wt is in mg per 100 seeds; Yield is in g/plant R-5 PLUS R-5 MINUS

Spray Cone D. F. Pods/ Seed Yield D. F. Pods/ Seed Yield. mg/100 ml Plant mg. g Plant mg g

0 65.32 106.9 436.4 4.85 65.32 106.9 436.4 4.85

(0,56) (3.25) (5.36) (0.45) (0.56) (3.25) (5.36) (0.45) 3 63.88 124.2 532.2 5.68 65.52 110.3 502.4 5.02

(0.87) (3.78) (4.98) (0,47) (0.85) (4.56) (5.21) (0.84)

10 63.51 117,3 494.8 5.12 62.35 78.21 411.21 3.69

(0,96) (3.21) (5.33) (0.89) (0.75) (4.89) (4.98) (0.98)

100 62.25 98.65 431.25 4,36 60.21 61.25 408.23 2.56 (o.87) (4.56) (5.41) (0.56) (0.71) (3.21) (5.69) (0.85)

Note: The above values are mean of five plants from each set. Yield, no.of pods and seed weight are taken at the time of harvest (110 days after sowing).

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.

What is particularly striking is the strongly negative effect MINUS extract has on flowering, as seen from the number of pods and the detrimental effect on seed size.

However, R-5 PLUS shows excellent promotional effects, even at 10 mg per spray, up to harvesting.

The negative effects with R-5 MINUS would have been even greater if the spraying was continued beyond the third spray.

Example 27 Post-harvest ripening of Banana, Musa paradisii Cv. Basrai

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.

Weakening of the fruit stalk, causing the fruit to be detachable, indicated end of yeUow life. As shown in Table 32, the shelf life of banana was extended by treatment up to 100 μl/liter, with the maximum extension at 60 μl/hter. Acidity development was slowed, and TSS levels steadily increased with concentration.

Table 32 E Effffeecctt on ripening and shelf life

Treatment TSS Acidity Green Life(G) YeUow Lif Y) Total Life(G+Y)

C Coonnttrrooll 2 233..8888 0 0..6633 8 8 5 13

20 24.58 0.61 9 6 15

60 24.75 0.57 9 7 16

100 25.05 0,54 9 6 15

200 25.25 0.52 7 12

Life extension was likely due to a progressively slower accumulation of α-amylase activity, with increasing concentrations up to 100 μl/hter. The peak of α-amylase activity coincided with the end of yellow Ufe. Afterwards, the activity declined rapidly. At the highest concentration, this process was reversed: amylase activity peaked early, as did the end of yeUow life. Thus, partial inhibition of α-amylase activity results in longer shelf Ufe of fruit OTable 33). Table 33 Effect of R-5 on α-amylase activity during ripening

Treatment 1^st Day 3^rd Day 5^th Day 7^th Day 9^th Day μl Uter

Control 63.5 115.3 190.5 150.5 34.5

20 50.4 102.7 168.5 170.6 60.3

60 48.6 98.7 165,1 178.3 62.4

100 47.6 98.0 172.2 164.8 52.3

200 48.7 101.3 199.5 120.2 20.3 Note: Days counted during yeUow Ufe.

Activity expressed as change of O.D./min/mg protein

Applications to monocotyledonous plants

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 μl/50 ml distilled water. At the higher concentration of 10 μl/50 ml DW, both R-100 oils showed a marked decline in root and shoot length unlike the plain coconut or safflower oU medium.

Table 34 Effect of oil medium on R-100 activity in germination Note: Concentration in the medium is in μl of R-100 oil /50 ml distilled water.

Values in parenthesis indicate standard deviation.

R-100 Control R-100 Contix )1

BASE: Coconut oU Coconut oil Safflower oil Safflower oil

Cone Root Length(cm)

0.0 5.12(0.78) 5.12(0.78) 5.12(0.78) 5.12(0.78)

0.33 6.13(0.75) 5,28(0,48) 6.78(0.42) 5.86(0.82)

1.0 7.12(0.65) 6.24(0.57) 7.25(0.40) 6.12(0.67)

3.0 8.26(0.76) 6.76(0.45) 8.56(0.38) 6.92(0.82)

10.0 5.46(0.72) 7.02(0.23) 5.98(0.37) 7.21(0.0.92) Shoot Length(cm) 0.0 2.58(0.72) 2.58(0.72) 2.58(0.78) 2.58(0.78)

0.33 3.12(0.72) 2.65(0,45) 3.12(0.36) 2.94(0.56)

1.0 3.62(0.78) 3.14(0.38) 3.84(0.46) 3.28(0.82)

3.0 4.16(0.98) 3.42(0.56) 4.56(0.42) 3.83(0.74) 10.0 2.83(0.82) 3.62(0.31) 3.23(0.39) 3.78(0.62)

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-ϊ)(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 μl/50 ml distiUed water).

Table 35 Effect of R-100 and C-100 on germination Note: Concentration in the medium is in μl of oU /50 ml distilled water.

Values in parenthesis indicate standard deviation.

Cone C-100 oil R-100 oϋ Sesame Oil

Root Length(cm)

0.0 5.12(0.78) 5.12(0.78) 5.12(0.78)

0.04 6.98(0.52) 6.52(0.74)

0.20 8.45(0.12) 7.98(0.63)

1.0 7.55(0.23) 8,02(0.65) 6.14(0.68)

Shoot Length(cm)

0.0 , 2.58(0.72) 2.58(0,72)

0.04 3.56(0.51) 3.29(0.71)

0.20 4.52(0,58) 4.18(0.74) 3.14(0.47)

1.0 4.28(0.56) 4.12(0.75)

Example 30 Effects on Rice Trials were carried out at Kalyani, W. Bengal in the Kharif. Rice plants were cultivated on

40 m² plots; 3 repUcates were used for each treatment. R-2 oil (batch 910608) was used, and a total of 2 sprays were appUed on the 30^th and 60^th day after sowing. Results are shown in Table 36.

Table 36 Yield UNTREATED CONTROL TEST R-2 oU Cone, ml/ha 0 50

Yield/Ha, quintals 44 55.5

% Increase 0 25

Example 31 Wheat

Trials were carried out at Dakore, Gujrat, India on wheat cultivated in 100 m² plots. R-2 oil (batch 910608) was used, and a total of 2 sprays were applied on the 29^th and 60^th day after sowing.

Table 37 Yield

CONTROL CONTROL TEST w/o WATER w/ WATER

R-2 oU Cone, ml/ha 0 0 25 50

Yield/Ha, quintals 21.8 22.5 37.62 32.75

% Increase -0.7 0 67 46

Plants grown for flowers (monocots anddicots)

Example 32 Marigold Trials with R-5 oil were carried out near Pune, 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.

Table 38 Marigold yield and quality

Test Control Spray Date

Flowering starts Flowering starts Plant height, cm 45 45 1^st appUcation

No. of flowers 50 30

Size of flowers Large SmaU Branches 17 12

Insect attack Low High (Aphids/jassids) 2^nd appUcation (31 days after 1^st) No. of flowers 120 60

Av.Flower wt., g 8 6

The number and size (by 25%) of flowers was increased. Resistance to sucking pests was also noted.

Example 33 Flowering plants

Trials with T-5 (250mg) tablets were taken at Daund, near Pune, Maharashtra, India. Dosages and other detaUs are given below.

JASMINE (Jasminum sambac) :

Concentration 1 T-5 (250 mg) tablet per 5 Uter; spray volume of 5 liters/20 plants

Frequency: Twice a month. Total sprays = 6.

First spray was appUed 15 days after pπining. Plant spacing: 1 m x 1 m (20 plants each in TEST and CONTROL groups)

GLADIOLA (Gladiolus sp.)

Concentration: 1 T-5 (250 mg) tablet per 5 liter; spray volume was lOliter/100 m² (1000 plants) Frequency: 30, 50 AND 70 days after bulb opening.

Plant spacing: 30 cm x 30 cm (1000 plants each in TEST and CONTROL groups)

ROSES (Rosa indica)

Concentration: 1 T-5 (250 mg) tablet per 5 liter; spray volume was 5 liter/100 m² (100 plants)

Frequency: once every 10 days (total of 9 sprays)

Plant spacing: 90 cm x 90 cm (100 plants each in TEST and CONTROL groups)

The observations compared to control are reported in Table 39.

Table 39 Yield and quality of flowers

OBSERVATIONS TEST CONTROL

JASMINE

Harvesting 10 to 15 days early normal

Flowering span wrthin ό days 15 days Total flower wt., kg 1.5 Other observations Large, uniform Compared to control Complete opening Longer shelf life Export quality

GLADIOLA

Number of flowers 7500 6000 Other observations Attractive, longer stick Compared to control Opens completely

ROSES

Number of flowers 1400 890

Example 34 Growth rate in forest trees (monocots and dicots)

A trial was carried out at Thane-belapur road, Maharashtra, India using T-5 tablets (250 mg) on a variety of forest species. Both fohar spray and root zone appUcation were employed.

FOLIAR SPRAY

Dendrocalamus strictus (two plants were used in each set)

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.

Table 40 Growth of forest trees

Date 100 ppm solution 50ppm solution control Height Branch Height Branch Height Branch

1990 cm number cm number cm number

19.Nov 35, 40 2, 1 30, 40 1, 2 41, 40 3, 2

1991

19.Jan 52, 64 5, 3 41, 63 3, 4 52, 59 6, 5 19.Mar 94, 79 12,13 69, 89 12,13 69, 78 11,12

19.Mayl38,135 22,28 113,128 41,49 119,128 28,31

19Jul 235,199 62,69 179,189 51,63 150,168 33,40

LEAVES YELLOW DARK GREEN LIGHT GREEN Thus, after 19^th March 1991, the 50 ppm solution set (1 ppm of R-100 oil) demonstrated an excellent spurt in growth and branching along with the development of a dark green foliage. However, the growth spurt with 100 ppm solution was even higher, although leaves were yellow.

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.

Table 41 Tree height Eucalyptus hybrid

Date Control 1 Tablet 2 Tablets 3 Tablets

1991 l.Jun 30, 30 30, 30 30, 30 30, 30 l.Sept 60, 62 70, 82 58, 65 45, 51 l.Dec 71, 79 100,125 84, 97 58, 60

1992 l.Jan 90, 100 145,155 110,130 70, 84 l.Feb 120,156 189,190 145,155 89,121

Tectona grandis 1991 l.Jun 20, 20 20, 20 20, 20 20, 20

1992 l.Feb 59, 62 60, 69 74, 70 54, 47

Dendrocalamus strictus 1991 l.Jun 60, 60 60, 60 60, 60 60, 60

1992 l.Feb 120,129 120,138 135,140 132,128

Leucena leucephalia 1991 l.Jun 64, 64 64, 64 64, 64 64, 64

1992 l.Feb 89, 92 94,105 78, 72 71, 80 Thus, depending on the plant system, one and/or two T-5 tablets appUed at the root zone lead to a considerable acceleration of height gain in forest trees and at optimal concentrations, the fohage turned a dark green.

ROOT ZONE AND TERMINAL BUD APPLICATION

One T-5 tablet was applied to the root zone. In addition, 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.

Table 42 Tree growth with terminal bud soaking

Casuarina equisetifolia

Date Test Control

Height Range,cm Average,cm Height Range,cm Average,cm

1^st app. 45- 60 51.8 42- 60 52.6

3 mos. 65- 80 74.0 65- 84 73.6

5 mos. 84- 98 92.2 74- 92 84.6

7 mos. 115-130 120,4 91- 105 99.2

Thus, for the first three months there was no difference. However, during the next four months the test trees grew at a much faster rate than the control trees.

Example 35 Mushroom yield

Trials were carried out at Chunchale, near Nashik, Maharastra, India on Pleurotus osteatus in rows of 15 beds each. Each bed was started with 1.5 kg of straw, dal and mushroom spawn tied up in polyethylene bags. The bags were cut open at the end of 3 weeks. From this point onwards, each bed was watered twice a day. 20 ppm solution of R-10 powder was sprayed once in three days (approx. 150 ml solution per bed). Mushrooms were harvested from the control and treated beds over the next 45 days. The total weight of the mushrooms from the control row of beds was 0.85 kg/bed. The weight of mushrooms from each of the three treated rows was 1.35 to 1.45 kg/bed.

Mushrooms from the treated beds were large, more uniform in size and with a thicker stalk.

Animal applications Toxicitv Example 37 Bacterial toxicity

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)

Tested against the carcinogen B(a)P (Benz(o)pyrene) at 2microg/plate according to the methods of (Ames et al., 1973; Ames et al., 1975). R-100 oil (batch 910217) duuted 1:200 or 1:500 in water

Concentration of water extract tested: 2 μl/plate

Results expressed as mean number of revertants/plate in Table 43.

Table 43 Anti - mutagenicity in bacteria

STRAIN CONTROL DILUTION DILUTION

1:500 1:200

TA98 + S9 25 23.5 35.1

± 1.6 ± 3.6

TA98 + S9 + B(a)P 235.5 119 145.3

±11.0 ± 11.8 ± 12.9

TA100 + S9 161 142.8 76

TA100 + S9 + B(a)P 489.5 124.4 162.5

± 5.0 ± 13.2 ± 2.5

Thus, 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.

Example 39 Low mammalian toxicity and faster growth

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.

Hematology, blood biochemistry and histopathology of aU major organs were performed at the end of 41 weeks; no toxic effects were observed. There was no remarkable change in gross pathology or in the histopathology. The average body weight (g) and standard error of estimate (number in parenthesis) at 0, 14, and 41 weeks in the study for aU dose levels are reported in Table 44.

Table 44 Growth rate and toxicity in rats DOSE MALE FEMALE

Mg/kg/day of 0 500 1000 2000 0 500 1000 2000

R-10(P) START 92.0 91.8 96.8 92.4 88.2 95.8 86.4 91.4

(1.71) (1.87) (1.77) (2.18) (2.20) (1.48) (1.63) (2.50) 14 WEEKS 176.4 193.4 193.0 200.8 143.0 151.4 144.0 150.0

(6.20) (6.48) (4.78) (6.67) (3.51) (2.72) (4.00) (5.30)

41 WEEKS 220.8 227.8 209.2 217.5 156.8 165.6 157.6 159.2

(8.31) (14,64) (8.13) (12.91) (5.4) (3.49) (6.72) (5.7)

Note: The numbers in parenthesis indicate standard deviation. The intake of R-10(P) did not resulted in any adverse chronic effects and did not effect weight after fuU maturity (41 weeks). However, between the 7^th to the 21^st week or during the early development, the intake accelerated the rate of weight gain or caused an increased, but healthy growth, in experimental animals.

Example 40 Faster cartilage tissue growth and low toxicity

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.

During a four week period, 6^th to 10^th week of study, there was a faster increase in tail length of test animals (both male and female) from various treatment groups compared to controls, as shown in Table 45.

Table 45 Tail length gain in rats ( 6^th to 10^th week of stnHy ), nun Dose, mg kg 'day of R-100 Male Female

Control 4.8 5.8

5 6.5 θ,9

50 10.0 9,0

500 12.2 7.3

This period coincided with the onset of sexual maturity. Faster increase in tail length test animals indicated a faster growth of cartilage tissue due to the intake of R-100 oil.

Example 41 Anti-mutagenicity in mammals

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,

Table 46 Anti - mutagenicity in mammals

R- 100 ,ppm %MNCPE

In drinking water

Solvent Control nil 0.7 ± o.04 n = 4 Solvent Control 2 0.48 ± 0.025 n = 4

Solvent Control + B(a)P nil 2.0 ± 0.1 n = 4

Solvent Control + B(a)P 2 ppm 0.7 ± 0.04 n = 4

Thus, 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.

Thus, R-100 oil did not cause any irritation to the skin of rabbits.

The R-100 oil, 0.1ml, was introduced on the penile and vaginal mucous membrane of male and female rabbits. No erythema or edema was observed as scored by the Draize method after 24, 48 and 72 hours. 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:

1. Elevated alkaline phosphatase levels was observed with the R-100 dose set

2. Moderate to severe, weU defined, and very slight erythema was observed with R-100, R-5 and R-1 oil, respectively.

3. Higher platelet values were observed in the blood of animals treated with R-100 and male animals treated with R-5 oil .

At the end of 14 day recovery period,

1. Serum alkaline phosphatase levels returned to normal

2. Erythema in aU cases subsided

3. However, elevation of platelet level persisted. Except for these effects, no other macroscopic effects were observed during necropsy.

Thus, R-1 oil showed no observable effects at the dose levels tested.

Example 43 Low cytotoxicity: in vitro cancer cell line screen

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.

AppUcations to livestock Example 44 Reduced Feed conversion ratio (FCR) and low mortaUty in poultry (layer birds)

Experiments were carried out with Babcock (BV300) Layer birds near Panvel, Maharashtra, India. In the 20^th week after hatching, the birds were transferred to layer cages. In one typical experiment, one row of 168 birds served as test birds, whereas 2250 remaining birds served as controls. Test birds were fed 100 g/day/bird, and the control birds were given 1 lOg/day/bird of feed consisting of 33% red maize, 35% of de-oiled soya and groundnut cakes, 15% de-oiled rice pohsh, 5% rice pohsh and calciferous material, fish meal, etc. Feed of test birds contained 400 ppm (v/w) R-10 oil (approx. 40 mg/kg feed of leaf equivalent).

At the end of 47^th week, 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. Thus, there was a 23% reduction in the FCR.

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.

Example 45 Lower Feed Conversion Ratio (FCR) and Higher Egg Production in poultry

Experiments were conducted at SangU, Maharashtra, India, with HISEX layer birds. 5 mg R-100 oil /kg feed and 2 mg R-100 / kg feed were used for test birds. The birds were moved to layer cages after 20 weeks. Control and test birds were fed identical feed, except that the test birds received the R-100 oil.

The average weekly feed intake during the laying period was approximately 0.8 kg. Hence, 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. Hence the approximate daily dose in test birds was 4/(1.7x7) = 0,335 mg of R-100 kg body weight per day. At the lower dose, the intake was 0.135 mg R-100 kg body weight per day. These results are summarized in Table 47.

The first trial where 5mg/kg feed of R-100 oil (5 mg leaf equivalent / kg feed) was given only after attaining 20 weeks; the reduction in feed consumption/egg (FCR ratio) was 7.0 %, along with a 4.7 % increase in the number of eggs produced.

In the second trial R-100 was given from birth. In this case, improvement in FCR was much higher: 13.4 % with a lower (2%) increase in total egg production.

In the third trial at the lower dose of 2 mg/kg feed of R-100 equivalent, there was stiU a 7.75 % improvement in FCR ratio.

Table 47 Summary of 'layer' bird trials

TYPE OF BIRDS: HISEX DOSE: R-10 OIL 50 ml MT Feed, for TRIALS 1,2 and 3

R-10 OIL 20 ml/MT Feed for TRIAL 4

TRIAL NO 1 2 3

Date of Hatch Mar 1,.90 Sφl7. 90 Dec22, 90

Trial Started on Jun22, 90 Sep 17, 90 Dec22, 90

From - week 20 0 0

To - week 82 61 52

Duration, weeks 62 61 52 O 02/05830

71 -

Test Birds.(T) 1350 1514 1399

Control Birds,(C) 1744 3000 3200

CUMULATIVE Test Control Test Control Test Control

RESULTS

Feed, kg /Bird 49.246 50.925 38.912 43.196 31.244 33.739

Eggs /Bird 296.15 282.92 210.09 205,96 157.87 157.0

Feed /Egg, g 166.3 178.0 147.6 167.4 153.5 165.4

( > 20 weeks ) (> 20 weeks )

FCR, % reduction 7.03 13.4 7.75

Eggs, % increase 4.67 2.00 0.55

The reported use of direct leaf extract is at the level of 70 leaves per day per 2000 birds. Assuming 5 g weight per leaf (these leaves were generaUy turgid), and 1.7 kg as the average weight per bird, this is approx. 350 g per 3400 kg per day or 100 mg/kg body weight per day. Thus, use of R-100 in layer birds results in a combination of improvement in FCR ratio and increase in egg production. This effect is novel and is produced at a leaf equivalent or R-100 dose levels considerably lower (0.335 and 0.135 mg/kg) than the reported use (100 mg/kg) of direct leaf extract dose in the literature.

Example 46 Broiler productivity and mortality

The experiment was conducted near Panvel, Maharashtra, India. 1 g of R-100 oil (batch 910316) was solubuized in one liter of 10% polysorbate. This solution (1000 ppm of R-100) was added to drinking water to the test batch at the rate of 1 ml Uter, creating approximately a 1 ppm solution of R-100. During the study, each batch consumed approximately 20000 liters of άήakmg water, or 20 gm of R-100 oil.

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.

Table 48 Broiler productivity and mortality

CONTROL I TEST CONTROL π

Total Days 56 49 53 Initial No. of Birds 1529 1530 1500 MortaUty 120 72 217 Total Feed , kg 4425 5025 4575

Total Broiler Wt.,kg 1969 2223 1709

Ave BroUer(end), kg 1.40 1.52 1.33

Feed Con. Ratio, kgkg 2,25 2.26 2.68

Productivity, kg meat/day 35.16 45.37 32.24

Thus, there was a considerable reduction in the mortality of birds. Also, the growth rate of broilers was much faster resulting in shed productivity for the test group being 30 to 40 % higher than the two control runs.

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).

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Claims

CLAIMS I claim:

1. An oil extract of a CAM plant other than Kalanchoe pinnata or Aloe vera ,

2. The oil extract of claim 1, wherein the CAM plant is Cissus quadrangularis.

3. An oU extract of Kalanchoe pinnata or Aloe vera, made from a ratio ofwater to kalka of less than 16:1.

4. An oil extract of Kalanchoe pinnata or Aloe vera, made from a ratio of decoction of plant part in water to kalka of less than 24: 1..

5. An oil extract of Kalanchoe pinnata or Aloe vera, made from a ratio of juice to kalka of less than 32:1..

6. The oil extract of claims 1-5 wherein the oil is vegetable, mineral or animal.

7. The oil extract of claim 1-5 wherein the oil is sesame oil, safflower, butterfat, mineral oU or coconut oU.

8. A composition comprising an oil extract of a CAM plant and a carrier.

9. A method of preparing an oil extract of a CAM plant comprising: mixing at least one CAM plant part with water, homogenizing the aqueous mixture, adding the homogenate to an oil, boiling the otf mixture, and filtering the oU mixture to produce a first extract and a first residue, such that the first extract is the oil extract of a CAM plant.

10. The method of claim 9, wherein the plant part is a leaf.

11. The method of claim 9, wherein the plant part is a decoction of plant part in water with kalka in the ratio of decoction of plant part in water to kalka of less than 24:1..

12. The method of claim 9, wherein the plant part is expressed juice with kalka in the ratio of expressed juice to kalka of less than 32:1.

13. The method of claim 9, wherein the entire plant is mixed with water.

14. The method of claim 9, wherein the plant part comprises a stem, leaf, shoot, flower, root, or cotyledon.

15. The method of claim 14, comprising mixing at least two CAM plant parts with water.

16. The method of claim 14, comprising mixing at least three CAM plant parts with water.

17. The method of claim 14, comprising mixing at least four CAM plant parts with water.

18. The method of claim 14, comprising mixing at least five CAM plant parts with water.

19. The method of claim 14, comprising mixing at least six CAM plant parts with water.

20. The method of claim 9, wherein the boiling is performed for about 25 to about 360 minutes.

21. The method of claim 9, 11 or 12 wherein the CAM plant is Kalanchoe pinnata, Aloe vera, or Cissus quadrangularis.

22. The method of claim 9, wherein a ratio of the CAM plant homogenate to the oil is 0.1 to 1.5.

23. The method of claim 9, further comprising washing the first residue with oil, filtering the oil to produce a second extract and second residue, and adding the second extract to the first extract.

24. A method of preparing an oU extract of a CAM plant comprising: mixing at least one CAM plant part with water, homogenizing the aqueous mixture, filtering the mixture to obtain a leafy residue fraction and a juice fraction, combining the leafy residue fraction and the juice fraction in any proportion with an oil to create an oil mixture, boiling the oil mixture, and filtering the oil mixture to produce a first extract and a first residue, such that the first extract is the oU extract of a CAM plant.

25. The method of claim 24, wherein the plant part is a leaf.

26. The method of claim 24, wherein the entire plant is mixed with water.

27. The method of claim 24, wherein the plant part comprises a stem, leaf, shoot, flower, root, or cotyledon.

28. The method of claim 27, comprising mixing at least two CAM plant parts with water.

29. The method of claim 27, comprising mixing at least three CAM plant parts with water.

30. The method of claim 27, comprising mixing at least four CAM plant parts with water.

31. The method of claim 27, comprising mixing at least five CAM plant parts with water.

32. The method of claim 27, comprising mixing at least six CAM plant parts with water.

33. The method of claim 27, wherein the boiling is performed for about 25 to about 360 minutes.

34. The method of claim 24, wherein the CAM plant is Kalanchoe pinnata, Aloe vera, or Cissus quadrangularis.

35. The method of claim 24, further comprising washing the first residue with oil, filtering the oil to produce a second extract and second residue, and adding the second extract to the first extract.

36. The method of claim 24, wherein said proportion of the leafy residue fraction to the juice fraction is 1:0.

37. The method of claim 24, wherein said proportion of the leafy residue fraction to the juice fraction is 0:1.

38. The method of any one of claims 9-36 or 37 wherein the ratio of filtered residue to the oil is 0.05 to 0.5.

39. The method of any one of claims 9-36 or 37 wherein the ratio of CAM plant juice to the oil is 0.5 to 10.

40. A method to treat a human disease or condition comprising administering an oil extract of a CAM plant.

41. The method of claim 40, wherein the human disease or condition is respiratory, high stress, energy level, digestive system, high blood lipid levels, wound, inflammatory, general pain, swelling, reproductive, hair loss, cancer, tumors or vision.

42. The method of claim 40, wherein the human disease or condition is cough, cold, congestion, aUergy-induced asthma, stress-induced asthma, sleeplessness, poor feet circulation, stomach acidity, stomach upsets, poor appetite, poor growth in chUdren, poor height growth in children, poor weight gain, high blood LDL cholesterol, high blood triglycerides, bruises, cuts, diabetic ulcers, leprotic ulcers, varicose ulcers, bedsores, bums, piles, fistula, spondulitis, arthritis, gingivitis, toothache, oligospermia, poor sperm motiUty, poor ovulation, menstrual discomfort, menstrual pain, menorrahgia, skin inflammation, pimples, Uchenplanus, eczema, dermatitis, psoriasis, poor hair growth, macular surgery, dry cornea or sty.

43. A method to enhance plant germination comprising applying an oil extract of a CAM plant to a seed.

44. The method of claim 43, wherein the seed is from monocotyledonous or dicotyledonous plant.

45. The method of claim 43, wherein the plant is mung bean or cane sugar.

46. A method to enhance plant vigor and defenses comprising applying an oil extract of a CAM plant to a portion of the plant.

47. The method of claim 46, wherein said applying comprises spraying on foliage, applying to terminal buds, fruits, flowers, injecting into phloem, or applying to soil at root zone.

48. The method of claim 46, wherein the plant is a monocot or dicot.

49. The method of claim 46, wherein the plant is okra, tomato, brinjal,. red chiU, marigold, jasmine, gladiola, rose, mango, pomegranate, ber, sapota, lime, guava, strawberry,

Solanum khasianum, rice, wheat, cotton, soybean, Dendrocalamus strictus, Eucalyptus sp., Leucina leucephalia, Casuarina equisetifolia or mung bean.

50. A method to enhance, from a plant, the yield of a seed, tuber, rhizome, vegetable, fruit, flower or secondary metabolite, comprising applying an oil extract of a CAM plant to a portion of the plant.

51. The method of claim 50, wherein the seed, tuber, rhizome, vegetable, fruit, flower or secondary metaboUte is produced by a monocot or dicot.

52. The method of claim 51, wherein the plant is tomato, brinjal, okra, red chili, marigold, jasmine, gladiola, rose, mango, pomegranate, ber, sapota, lime, guava, strawberry, Solanum khasianum, rice, wheat, cotton or soybean,

53. The method of claim 51 , wherein said applying comprises spraying on foilage, applying to terminal buds, fruits, flowers, injecting into phloem, or applying to soil at root zone.

54. A method to extend the shelf Ufe of a fruit or flower, comprising applying an oil extract of a CAM plant to a portion of the plant.

55. The method of claim 54, wherein said applying comprises spraying on foliage, applying to terminal buds, fruits, flowers, injecting into phloem, or applying to oU at root zone.

56. The method of claim 54, wherein the fruit or flower is produced by a monocot or dicot.

57. The method of claim 54, wherein the fruit or flower is selected from the group consisting of tomato, brinjal, okra, mango, pomegranate, ber, sapota, Ume, guava, okra, banana, rose, jasmine, or marigold.

58. A method to enhance the growth of a mushroom, applying an oil extract of a CAM plant to the mushroom.

59. A method to enhance feed conversion in livestock, comprising administering an oil extract of a CAM plant to the Uvestock.

60. The method of claim 59, wherein the livestock is a HISEX layer bird or broϋer.

61. The method of claim 59, wherein the Uvestock is a BABCOCK layer bird or broUer.

62. A method to decrease mortality in livestock, comprising administering an oil extract of a CAM plant to the Uvestock.

63. The method of claim 59, wherein the livestock is a HISEX layer bird or broUer.

64. The method of claim 59, wherein the Uvestock is a BABCOCK layer bird or broiler.

65. A method to decrease milk acidity in a mammal, comprising administering an oU extract to the mammal.

66. The method of claim 65, wherein the mammal is a buffalo.

67. A method of accelerating the onset of maturity in an animal, comprising administering to the animal an oil extract of a CAM plant.

68. The method of claim 67, wherein the animal is a rat.

69. A method to decrease mutations in an individual, comprising administering a CAM plant oil extract to the individual.

70. The method of claim 71 , wherein the individual is human, animal or plant.

71. A method to U plants comprising applying a CAM plant extract to said plants.

72. A method to control growth of plants comprising applying a CAM plant extract to said plants.

73. The method of claims 71 or 72, wherein the concentration of the CAM plant extract is 25 to 500 g/hectare.

74. The method of claim 71 or 72, wherein said applying comprises spraying on foliage, applying to terminal buds, fruits, flowers, injecting into phloem, or applying to soil at root zone.

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