Bezalel, Lea (Beer Sheva, IL)
Ziv, Meira (Rehovot, IL)
Perry, Inon (Tel Aviv, IL)
2. A process according to claim 1, wherein said extract inhibits growth of seedlings.
3. A process according to claim 2, wherein said extract inhibits growth of cucumber seedlings.
4. A process according to any one of claims
5. A process according to claim 4, wherein said producer cells or tissue are cells or tissue obtained from Narcissus bulbs.
6. A process according to claim 4, wherein said fraction is obtained from fruit.
7. A process according to claim 4, wherein said producer tissue is a seed.
8. A process according to any one of claims
9. A process according to claim 8, wherein said animal is Artemia.
10. Use of an effective amount of an agent for the preparation of a pharmaceutical or cosmetic composition having an anti-proliferating effect on target cells or tissue of an individual, said agent being a fraction with a cell anti-proliferating activity obtained from producer cells or producer tissue or from a medium in which said cell or tissue were incubated characterized in that: (i) said producer cells or producer tissue originate from an organism which is xenogeneic to said individual; (ii) said xenogeneic organism is such which can enter into a state of dormancy; and (iii) said fraction is obtained from producer cell or tissue while in a dormant state.
11. A method according to claim 10, wherein said activity is substantially higher in said fraction than the same fraction obtained from said producer cells or tissue when in a non-dormant state.
12. Use according to claim 10 or
13. Use according to claim 12, wherein said producer cells or tissue are obtained from Narcissus bulbs.
14. Use according to claim 12, wherein said producer tissue is fruit.
15. Use according to claim 12, wherein said tissue is a seed.
16. Use according to claim 11, wherein said organism is an animal.
17. Use according to claim 16, wherein said animal is Artemia.
18. Use according to any one of claims
19. Use according to claim 18, wherein said preparation is intended for use in the maintenance of juvenile appearance of an individual's skin or for the treatment of age-related phenomena of the skin.
20. Use according to and one of claims
21. Use according to any one of claims
22. Use according to any one of claims
23. A method for inhibiting of proliferating target cells or target tissue in an individual, comprising administering to the individual an effective amount of an agent having a cell- anti-proliferative activity in a manner permitting said agent to reach the target cells or tissue, said agent having the following characteristics: i. it is produced from producer cells or producer tissue originating from the organism which is xenogeneic to said individual; ii. said xenogeneic organism is such which can enter into a state of dormancy; and iii. said fraction is obtained from the producer cells or tissue while in a dormant state.
24. A method according to claim 23, wherein said activity is substantially higher in said fraction than the same fraction obtained from said producer cells or tissue when in a non-dormant state.
25. A method according to claim 23, wherein said organism is a plant.
26. A method according to claim 25, wherein said cells or tissue are obtained from Narcissus bulbs.
27. A method according to claim 25, wherein said producer tissue is a fruit.
28. A method according to claim 25, wherein said producer cell or tissue is a seed.
29. A method according to claim 23, wherein said organism is an animal.
30. A method according to claim 29, wherein said animal is Artemia.
31. A method according to claim 23, wherein said agent is applied topically onto the individual's skin.
32. A method according to claim 31, for maintenance of juvenile appearance of the individual's skin or for treatment of age-related phenomena of the skin.
33. A method according to claim 31, for the administration to an individual having a benign or malignant tumor or being at a high risk of developing a benign or malignant tumor.
34. A method according to claim 31, for prolonging duration of a tan.
35. A method according to claim 23, wherein said preparation is intended for use in inhibition of alopecia, inhibition of hirsutism, for reduction of rate of nail growth, in combination with hair treatment for inhibition of scar formation or for the treatment of skin disorders.
36. A method for inhibiting growth of plant seedlings comprising applying onto the seedlings or their growth media an effective amount of a cell-proliferation inhibiting agent, which is obtained from producer cells or producer tissue or from a medium in which said cells or tissue were incubated, characterized in that: (i) said producer cells or producer tissue originate from an organism which is xenogeneic to said seedlings; (ii) said xenogeneic organism is such which can enter into a state of dormancy; and (iii) said fraction is obtained from producer cell or tissue while in a dormant state.
37. A method of producing rate of seed germination, comprising applying onto the seeds an effective amount of a cell-proliferation inhibiting agent obtained from producer cells or producer tissue or from a medium in which said cells or tissue were incubated, characterized in that: (i) said producer cells or producer tissue originate from an organism which is xenogeneic to said seed; (ii) said xenogeneic organism is such which can enter into a state of dormancy; and (iii) said fraction is obtained from producer cell or tissue while in a dormant slate.
38. A method of preservation of fresh produce, comprising applying onto the flesh produce an effective amount of a cell-proliferation inhibiting agent, obtained from producer cells or producer tissue or from a medium in which said cells or tissue were incubated, characterized in that: (i) said producer cells or producer tissue originate from an organism which is xenogeneic to said produce; (ii) said xenogeneic organism is such which can enter into a state of dormancy; and (iii) said fraction is obtained from producer cell or tissue while in a dormant state.
FIELD OF THE INVENTION
[0001] The present invention concerns exacts of cells or tissue or their supernatants which can inhibit proliferation of cells or tissue. The invention also provides compositions comprising such extracts as well as pharmaceutical, cosmetic and agricultural uses of the compositions and extracts.
BACKGROUND OF THE INVENTION
[0002] Dormancy is a phenomena which is found in representatives of the plant kingdom as well as the animal kingdom.
[0003] The germination of various grains and seeds comprising the necessary propagation organs is delayed under certain circumstances and yet the grains or seeds are capable of germinating after various periods of time. The period of time in which germination of such seeds may be delayed varies and depends both on intrinsic properties of the seed as well as on the nature and extremity of the environmental conditions. Seeds have been shown to be in dormancy for a few days, a year, several years and even for more than several centuries (as was discovered lately in the case of some nympheaceae and seeds of trees of the Leguminosae family (Shen-Miller, J., et al.,
[0004] In some cases, the capability to develop dormancy lies in the embryo envelopes. In such a case, the separation of the envelopes from the embryo, result in its immediate germination.
[0005] In other cases, chemical growth inhibitors capable of preventing germination are present in the embryo itself and thus even a bare embryo may remain dormant (such as in the case of Rosaceae plants such as Kerria, Peach, etc.).
[0006] In plants, a state of dormancy may be found in the whole plant or in one or more of its parts. Dormant plants are plants which have two main metabolic states in their growth cycle. In their dormant state, the plants' metabolism is extremely low, and the plant growth process is significantly inhibited although differentiation of certain cells may occur. In their active state, the plants' metabolism rate is higher, the cells divide and differentiate and there is significant growth of various parts of the plant. In some cases, the whole plant enters the dormant state. Such is the case in Narcissus plants in which during the dormant state the only remaining viable part is the bulb which is in its dormant state. In other cases, some parts of the plants may be active while other parts may be in dormancy such as, for example, is the case of apple trees.
[0007] Substances capable of inhibiting germination have also been shown to be present in the juice of fleshy fruits or in other plant organs which produce juice. Examples are tomatoes, grapes, kiwi, watermelon and grapefruit wherein pips present in the fruit do not germinate although their surroundings are suitable for germination due to the water within the fruit.
[0008] Several plant-derived substances having an effect on cell proliferation have been reported. For example, European Patent Application No. 0381514 describes compositions comprising both naturally derived as well as synthetically prepared sphingolipids which have growth inhibitory activity on various kinds of cells. Another well known plant-derived substance having an anti-mitotic effect on various kinds of human cells is the substance colchicine (Samson, F. E.,
[0009] Against this, many plant extracts having an opposite effect on cells, i.e., capable of augmenting their proliferation were also described such as, for example, the methanolic extract from the root of
[0010] The phenomena of dormancy may also be found in the animal kingdom, for example, in the small crustacean
[0011] Various plant derived compositions (such as retinoic acid (U.S. Pat. No. 5,438,073) and α-hydroxy acids (Ditre, C. M., et al.,
[0012] It is also believed today that normal human and animal cells have a finite capacity to replicate. It has been shown that the number of mitotic events that cultured normal animal cells can undergo appear to be inversely related to the age of the donor from which they were obtained (Hayflick, L.,
GLOSSARY
[0013] The following is the meaning of some terms of which will be used in the text below:
[0014] Dormancy—a state in which there is a marked decrease in the metabolic rate of cells or tissues resulting in the inhibition of growth and proliferation of the cells or tissue.
[0015] Dormans—substances naturally found in cells or tissue and which are capable of inducing cells or tissue to enter a state of dormancy or of maintaining the dormant state in cells or tissues that have already entered that state. The dormans may be obtained from a variety of plant parts which are capable of entering into a state of dormancy; from juice of various fruits which contain dormans capable of inhibiting germination of seeds within the fruit; from animals which are capable of entering a phase of dormancy in their life cycle, e.g. gastrula of certain crustaceans such as artemia or dafnia: etc. In some cases, the dormans are found within a dormant tissue, e.g. in a dormant seed or in the gastrula of artemia or dafnia; in other cases the dormans are found in a tissue surrounding the dorman tissue or organ, e.g. in a fruit juice surrounding a dormant seed.
[0016] Extract—At least one substance obtained by any of a variety of extraction methods known in the art. For example, the extract may be an aqueous extract, a glycolic extract an alcoholic extract, an oily extract, etc. The extract in accordance with the invention is obtained from cells or tissues from a part of a plant or animal capable of entering a state of dormancy. The cells or tissue may be obtained directly from the plant or animal and the extract may then be prepared therefrom. Alternatively, cell cultures may first be prepared from the plant or animal cells or tissues and then the cell cultures may be grown for various periods of time. In order to prepare an extract, the cells are then harvested from the cell cultures, the cells and their growth medium are separated and an extract may be prepared either from the cells themselves or from the growth medium (which will be referred to as “supernatant”) which contains substances secreted by the cells into their growth medium. Thus, the “extract” may be obtained directly from plant or animal tissue or from an animal or plant cell or tissue culture.
[0017] Dorman extract—an extract obtained from a plant cell or tissue, from fruit or from an animal cell or tissue which comprises dormans.
[0018] Enriched dorman preparation (EDP)—a preparation derived from a natural source which comprises dormans in a concentration larger than that which is found in a natural unprocessed extract. The EDP may be obtained by purification of a natural extract to obtain fractions which contained dormans in the larger concentration, e.g. by various chromatographic techniques, by filtration, etc., as well as by biological means including growing cells or tissue which are capable of producing dormans under conditions in which they produce dormans in relatively large quantities and collecting their secretion products. In order to determine whether a preparation is an enriched dorman preparation, the preparation may be assayed for a specific biological activity associated with dormans, as described below. EDP contains a substantially higher concentration of dormans as compared to the natural preparation, e.g. at least 1.5 folds, preferably 2 folds and typically at least 2.5 folds to the concentration of the dormans in the natural preparation.
[0019] Producer cells or producer tissue—cells or tissue which are capable of producing dormans which may thus be extracted therefrom.
[0020] Target cells or target tissue—cells or tissue which are contacted with dormans, in accordance with the invention and which thereby enter a state of inhibition of their growth or proliferation or maintain such a state as the result of contact with dormans.
[0021] Dorman analog—a substance, typically synthetic, which has a dorman-like activity in that it is capable of inducing dormancy in the same cells or tissue induced to dormancy by the dorman and which, in accordance with the invention, is also capable of inhibiting growth and proliferatin of target cells or tissue.
[0022] Dorman composition (DC)—a composition comprising, as active ingredient, an amount of dorman (e.g. as a dorman extract) or dorman analog effective in inhibiting growth and proliferation of target cells or tissue (“effective amount”). A dorman composition may comprise a naturally derived EDP, a composition comprising synthetic dormans as well as dorman analogs.
[0023] Active Extract (AE)—extracts obtained from cells or tissues from a part of a plant or animal capable of entering a state of dormancy during the non dormant state.
SUMMARY OF THE INVENTION
[0024] In accordance with the invention, use is made of a dorman composition. The composition may be used for inhibiting proliferation of cells, particularly cells xenogeneic to the producer cells or tissue. By one preferred embodiment of the invention, the compositions are used in human medicine and cosmetics. In accordance with another embodiment, the compositions are used for controlling plant growth. By yet a further embodiment, the compositions of the invention are used in food preservation.
[0025] By one aspect of the invention there is thus provided a preparation capable of inhibiting proliferation of target cells or target tissues, comprising a member selected from the group consiting of:
[0026] (i) an extract obtained from producer cells or producer tissue, said cells or tissue originating in an organism capable of entering a phase of dormancy in at least one of its parts, said extract comprising at least one substance which induces or maintains said state of dormancy in an organism from which said producer cells or tissues are derived,
[0027] (ii) a dormancy inducing fraction of said extract; and
[0028] (iii) a dormancy inducing substance derived from said extract or fraction.
[0029] By one embodiment the composition comprises an enriched dorman preparation (EDP) which, as defined above, comprises dormans in a concentration larger than that which is found in a natural unprocessed extract.
[0030] The producer cells or tissues from which the extract is obtained may be of the same origin as said target cells or tissues but are preferably of a different origin. In accordance with one embodiment of the invention, said target cells or tissue are human cells or tissue and said producer cells or tissue are plant or non human animal cells or tissue. In accordance with another embodiment of the invention, said target cells or tissue are plant cells or tissue.
[0031] In accordance with one preferred embodiment of the invention, the dorman composition is a pharmaceutical or cosmetic composition for inhibiting cell proliferation within the body. In accordance with another embodiment of the invention, said composition is used for inhibiting germination of seeds (being either natural or artificially prepared) or growth of seedlings for the purpose of maintaining seedlings in a dormant state for example during storage. In accordance with a further embodiment of the invention, the dorman composition is used in fresh food preservation.
[0032] By one embodiment, the dormans or the extracts used in accordance with the invention are derived from dormant plants.
[0033] Compositions in accordance with the invention obtained from bulbs of dormant plants while in their dormant state are capable of inhibiting the growth of seedlings as well as to inhibit the proliferation of various target cells, including various mammalian cells, e.g. human cells, to a significantly higher extent than preparations obtained under the same conditions from bulbs of the same plant being in their active state. It was also found that dorman compositions obtained from cell cultures prepared from various parts of dormant plants and induced into dormancy are also capable of inhibiting the growth of seedlings as well as to inhibit the proliferation of various cells, including various mammalian cells, e.g. human cells. These DCs, in a wide range of concentrations, had no noticeable toxic effect on the target cells, this being in contrast to most substances which have an anti-proliferating effect.
[0034] In accordance with the invention, any plant capable of entering a dormant state may be used for obtaining an DC. Some non-limiting examples of such plants, as well as the parts of such plants which enter a dormant state (designated “D-part”) and from which the DC may be obtained, are shown in the following Tables I and II:
TABLE I (Bulbs, corms, roots, rhizomes) Name Family D-part Allium Liliacease bulbs Amaryllis belladona Amaryllidacea bulbs Anemone Ranunculacea perennials & tuberous or fibrous roots Babiana Iridacca corms Brodiaea Liliaceae corms Chionodoxa Liliaceae bulbs Crocus Iridacea corms Ornithogallum Liliaceae bulbs Cyclamen Primulaceae perennials & tuberous roots Endymion Liliaceae bulbs Arum Araceae perennials & Tuberous roots Freesia Iridacea corms Fritillaria Liliaceae bulbs Galanthus Amaryllidacea bulbs Hippeastrum Amaryllidacea bulbs Hyancinthus Liliaceae bulbs Leek Liliaceae bulbs Ipheion uniflorum Amaryllidacea bulbs Iris Iridacea bulbs & rhizomes Ixia Iridacea corms Leucojum Amaryllidacea bulbs Lilium Liliaceae bulbs Muscari Liliaceae bulbs Narcissus Amaryllidacea bulbs Oxalis Oxalidaceae bulbs & rhizomes Paeonia Paeoniaceae tuberous perennials Puschkinia scilloides Liliaceae bulbs Ranuculus Ranunculacea tubers or perennials Radohypoxis Hypoxidaceae bulbs Rhodophiala Amaryllidacea bulbs Scilia Liliaceae bulbs Sparaxis Iridacca corms Raritimum Liliaceae bulbs Triteleia Amaryllidacea corms Tulipa Liliaceae bulbs Tritonia crocata Iridacea corms Watsonia pyramidata Iridacea corms Zantedeschia Araceae rhizomes Begonia Begoniceae rhizomes Caladium Araceae perennials & tuberous roots Canna Cannaceae perennials & tuberous roots Crocosmia Iridacea corms Dahlia Asteraceae perennials & tuberous roots Gladiolus Iridacea corms Gloriosa rothschildiana Liliaceae perennials & tuberous roots Homeria collina Iridacea corms Hymenocallis Amaryllidacea bulbs Liatris Asteraceae perennials Polianthes tuberosa Agavaceae perennials & tuberous roots Tigridia pavonia Iridacea bulbs Zantedeschia Araceae rhizomes Zephyranthes Amaryllidacea bulbs Colchicum Liliaceae corms Lycoris Amaryllidacea bulbs Sterenbergia lutea Amaryllidacea bulbs Pancratium Liliaceae bulbs
[0035]
TABLE II (Deciduous fruit trees, shrubs, seeds) Name Family D-part Malus - crabapple Malus Deciduous & fruit trees & shrubs Mangifera Anacardiaceae Deciduous & fruit trees & shrubs Peach Rosaceae Deciduous & fruit trees & shrubs Persimmon Ebenaceae Deciduous & fruit trees & shrubs Pistacia chinensis Anacardiaceae Deciduous & fruit trees & shrubs Prunus Rosaceae Deciduous & fruit trees & shrubs Fraxinus Oleaceae Deciduous & fruit trees & shrubs Pyrus Rosaceae Deciduous & fruit trees & shrubs Quercus Fagaceae Deciduous & fruit trees & shrubs Salix Salicaceae Deciduous & fruit trees & shrubs Actinidia Actinidiaceae Deciduous & fruit trees & shrubs Akebia quinata Lardizabalaceae Deciduous & fruit trees & shrubs Blueberry Vitaceae Deciduous & fruit trees & shrubs Apple Rosaceae Deciduous & fruit trees & shrubs Aloysia Verbenaceae Deciduous & fruit trees & shrubs Campsis Bignoniaceae Deciduous & fruit trees & shrubs Celastus Celastraceae Deciduous & fruit trees & shrubs Cellery Apiaceae seeds Clematis Ranunculaceae Deciduous & fruit trees & shrubs Grape Vitaceae Deciduous & fruit trees & shrubs Humulus Cannabaceae Deciduous & fruit trees & shrubs Fig Moraceae Deciduous & fruit trees & shrubs Wisteria Fabaceae Deciduous & fruit trees & shrubs Bean Fabaceae Deciduous & fruit trees & shrubs Lathyrus pea Fabaceae Deciduous & fruit trees & shrubs Tropaeolum Tropaeolaceae Deciduous & fruit trees & shrubs Amelanchier Rosaceae Deciduous & fruit trees & shrubs Cotoneaster Rosaceae Deciduous & fruit trees & shrubs Barberry Berberadaceae Deciduous & fruit trees & shrubs Enkianthus Ericaeae Deciduous & fruit trees & shrubs Eunymus Celastraceae Deciduous & fruit trees & shrubs Kerria japonica Rosaceae Dedicuous & fruit trees & shrubs Parsnip Apiaceae seeds Passiflora Passifloraceae Deciduous & fruit trees & shrubs Rhdodendron Ericaceae Deciduous & fruit trees & shrubs Acacia Fabaceae Deciduous & fruit trees & shrubs Albizia Fabaceae Deciduous & fruit trees & shrubs Almond Rosaceae Deciduous & fruit trees & shrubs Ampelopsis Vitaceae veciduous & fruit trees & shrubs Anethum Apiaceae seeds Annona cherimola Annonaceae Deciduous & fruit trees & shrubs Apricot Rosaceae Deciduous & fruit trees & shrubs Artemisia Asteraceae Deciduous & fruit trees & shrubs Asparagus Liliaceae seeds Blackberry Rosaceae Deciduous & fruit trees & shrubs Carrot Apiaceae seeds Carya pecan Juglandeaceae Deciduous & fruit trees & shrubs Cherry Rosaceac Deciduous & fruit trees & shrubs Corn Poaceae seeds Helianthus Asteraceae seeds Cucumber Cucurbitaceae seeds Filbert Betulaceae Deciduous & fruit trees & shrubs Gooseberry Saxifragaceae Deciduous & fruit trees & shrubs Gourd Cucurbitaceae Deciduous & fruit trees & shrubs Lettuce Asteraceae seeds Melon Cucurbitaceae seeds Okra Malvaceae seeds Onion Amaryllidaceae seeds or bulbs Peanut Fabaceae seeds Pear Rosaceae Deciduous & fruit trees & shrubs Pumpkin Cucurbitaceae seeds Punika garantium Punicaceae seeds Radish Cruciferae seeds Walnut Juglandeaceae Deciduous & fruit trees & shrubs Ziziphus jujuba Rhamanceae Deciduous & fruit trees & shrubs Raspberry Rosaceae Deciduous & fruit trees & shrubs Strawberry Rosaceae Deciduous & fruit trees & shrubs Turnip & rutabaga Cruciferae Deciduous & fruit trees & shrubs Malva Malvaceae seeds Verbascum Scrophulariaceae seeds Chenopodium Chenopodiaceae seeds Nelumbo Nelumbonaceae seeds Lupinus Papilonaceaee seeds
[0036] In accordance with the invention, DC is preferably obtained from plants which are in their dormant state either as a result of the natural process of dormancy or as a result of being externally induced into dormancy by exposure which induce a dormant state e.g. conditions as incubation at a dormancy inducing temperature for a sufficient period of time. The conditions for inducing dormancy in various dormant plants may vary (e.g. the incubation temperature and the duration of the incubation) and are known to a person versed in the art. Thus for example there are plants (such as Narcissus) induced into dormancy by their exposure to relatively high temperatures. Against this, other plants (such as tulip) will be induced into dormancy by their exposure to relatively low temperatures. Other factors such as light, humidity, concentration of various growth factors, etc. may also be used to induce dormancy.
[0037] In accordance with one preferred embodiment of the invention, the DC is obtained from a part of the plant capable of entering dormancy (D-part), e.g. from bulbs. Bulbs induced into their dormant state may either be used immediately for the preparation of DC or, alternatively, may be stored under conditions which maintain a dormant state, e.g. in the case of Narcissus these include high temperature and low humidity. In addition to bulbs, other parts of dormant plants such as combs, roots, seeds, etc., may also be induced into dormancy as explained above and then used for obtaining DC therefrom.
[0038] In accordance with another embodiment of the invention, the dorman extract is obtained from cell cultures which were prepared from any part of a dormant plant (e.g. bulbs) and induced into dormancy. The culture may be obtained by inoculation of bulbs of dormant plants having inflorescence stalk initials into a suitable medium to form callous cultures of the bulb extracts. The cell cultures are then typically grown to confluency and very small bulb parts are formed in the cell culture (termed “bulblets”). Induction of a dormant state in the cell cultures or bulblets is obtained by their exposure to conditions which induce dormancy conditions, such as incubation at a dormancy inducing temperature for a sufficient period of time. Dormancy may also be induced in vitro by exposing the cell cultures or bulblets to various types of chemical stresses (low or high concentrations of sugar, salts, etc.).
[0039] In addition to bulblets, cell cultures derived from other parts of dormant plants such as combs, roots, seeds, etc., may also be used for obtaining cell culture derived dorman extracts.
[0040] By yet another embodiment, the dorman analogs of the invention may be synthetically prepared by any one of the methods known in the art such as by recombinant DNA techniques, chemical synthesis,, combinational chemistry, etc., the dorman analogs maintaining substantially similar characteristics as far as their ability to induce dormancy and inhibit proliferation of target cells of the dormans on which they are based.
[0041] Preferably, a plant derived DC of the invention is obtained as an aqueous extract of the plant material. The aqueous extract may be prepared by homogenizing the plant material and then suspending the homogenate in an aqueous solution. However, non aqueous plant extracts obtained by any one of the extraction methods known per se, may also at times be used in accordance with the invention.
[0042] In accordance with a further embodiment of the invention, the EDPs are obtained from juice of fruits or other juice producing plant organs. Fruits typically contain dormans which inhibit germination of the seeds and pips while these are within the fruit. Examples of juices from which dormans may be purified are juice of citrus fruits, grapes, tomato, kiwi, etc. The fruit juice may be used as such or alternatively, the dormans can be purified from the fruit juice as explained below.
[0043] In accordance with yet another embodiment of the invention, the dormans are extracted from producer cells originating from animals capable of entering a dormant phase during their life cycle. During the dormant phase, the animals' metabolic rate is lowered to a minimum and there is an arrest in cell proliferation. Examples of such animals are various marine crustacea such as artemia, dafnia and cyclops.
[0044] As explained above with regards to plant derived DC, animal derived DC may also be obtained from animals which are in their dormant state as a result of the natural process of dormancy or as a result of being externally induced into dormancy by exposure to dormancy-inducing conditions such as dehydration (
[0045] The animal derived DC may be obtained from the animals or their organs as such. Alternatively, cell cultures may first be prepared from the dormant animal, and after maintaining the cells in culture for various periods of time, DC may then be extracted either from the supernatant or by harvesting the cells and/or extracting the DC therefrom.
[0046] The DC of the invention may be purified from the producer cells by a variety of methods known per se, for example by chromatography (e.g. TLC, HPLC, ion exchange) by size fractionation (e.g. dialysis, gel filtration), etc.
[0047] In accordance with the invention, it has been realized for the first time that, when administered to an individual, the anti-proliferative activity of said dorman composition may slow down the cell division rate of the cells present in the inner layers of the epidermis.
[0048] Another aspect of the invention is thus the use of said dorman compositions as a cosmetic or dermatological composition useful for the maintenance of the juvenile appearance of an individual's skin or for the treatment of age related skin changes.
[0049] In accordance with this latter aspect of the present invention, a dermatological or cosmetic composition is provided comprising from about 0.0001%, preferably from about 0.001%, typically from about 0.01% up to about 5% preferably up to about 1% by weight of dorman extract or dorman analog together with a dermatologically or cosmetically acceptable carrier.
[0050] The dermatological or cosmetic compositions of the invention may be administered in various forms such as in the form of a balm, an emulsified gel, an aqueous-alcoholic gel, anhydrous gel, an oil in water (O/W) type emulsion, a clear gel, cream containing liposomes, etc.
[0051] The cosmetic or dermatological compositions are typically topically administered. However, it may at times be advantageous to administer the compositions by other administration modes, such as, for example, by subcutane injections, by orally administered capsules or by iontophoresis (which involves the use of electric fields to increase the penetration of ionic active substances).
[0052] Due to their significant anti proliferative effect said dorman compositions may also be used for the treatment of various malignancies. As mentioned above, the cell division rate is a significant factor in determining the probability of a cell to become a premalignant or malignant cell. In addition, as known, the formation of a benign or malignant tumor is dependent, inter alia, on continuous divisions of the cells forming the tumor. Administration of the dorman compositions to an individual before the formation or at early stages of the formation of a benign or malignant tumor may result in the delay or prevention of the formation of a fully fledged tumor in the treated individual. Administration of said extracts to an individual suffering from a fully fledged benign or malignant tumor may result in the reduction of the tumor load in the treated individual and in the alleviation of the tumor-related symptoms. Said dorman compositions may be effective in the treatment of primary as well as secondary (metastatic) tumors. Said extracts may also be administered in combination with one or more known anti-tumorigenic treatments (e.g. chemotherapeutic agents, radiation etc.) to achieve a synergistic anti-tumorigenic effect. The doses of said extracts to be administered to an individual as well as the treatment modality will be dependent on characteristics of the treated individual (age, weight, medical history, etc.) as well as on characteristics of the developing or existing tumor (benign or malignant, size, origin, primary or secondary, etc.). In individuals having a high risk of developing a primary or secondary tumor, the dorman compositions may be administered routinely in order to reduce the probability of tumor formation.
[0053] The present invention thus further provides a composition comprising a dorman extract capable of inhibiting the proliferation of cells, for the administration to an individual having a benign or malignant tumor or being at a high risk of developing a tumor.
[0054] By yet an additional aspect of the invention, dorman compositions may be used to enhance the therapeutic index of chemotherapeutic and radiation treatments. In an individual receiving such treatments, normal dividing cells such as cells of the inner lining of the intestines, cells of hair follicles and hematopoietic cells are also harmed by the chemotherapeutic agents or radiation which are aimed at destroying the malignant cells of which a large percent are dividing cells. By administration of dorman compositions to an individual prior to or together with such treatments, it may be possible to inhibit the proliferation of a significant percent of the normal cells. As a result, toxic side effects due to the influence of the treatments on normal cells may be significantly reduced and when beneficial, higher concentrations of the chemotherapeutic or radiation treatments may be used. In order to facilitate the toxicity reducing affect of the dorman composition, it may at times be administered directly to a needing site, tissue or organ, e.g. onto the skin.
[0055] The present invention thus provides by a further of its aspects, a composition capable of inhibiting the proliferation of cells, for administration to an individual receiving chemotherapeutic or radiation treatments, comprising an effective amount of dorman, dorman extract or dorman analog.
[0056] Other therapeutic applications of the dorman compositions include inhibition of fibrosis, e.g. skin fibrosis, cirrhosis, and others. It should be noted that hitherto, fibrosis, which is an over proliferation of fibroblasts, has been treated by cytotoxic drugs, but with a limited application due to their general non specific toxicity. Inhibition of the fibroblast proliferation by the use of the dorman composition of the invention, provides a viable, less toxic alternative. In a similar manner, the dorman compositions of the invention may also be useful in the treatment of psoriasis which results from over proliferation of keratinocytes. Seborrheic keratosis, papilomas and warts may also be treated by the dorman compositions.
[0057] Another possible application of the dorman composition of the invention is in preservation of organs or tissue prior to their use for transplantation.
[0058] Other applications of said dorman compositions may be, for example, in the treatment of scalp baldness (Alopecia) which is many times one of the phenomenas associated with aging of the skin in an individual. In individuals suffering from Alopecia, the life span of scalp hair decreases substantially (e.g. from a life span of about 3 years in a normal individual to a life span of about one year in an individual suffering from Alopecia). Therefore, decreasing the rate of hair growth in an individual having a high probability of developing Alopecia or in an individual already showing for signs of scalp hair loss, will decrease the extent of such hair loss. Administration of the dorman compositions of the invention to such an individual may result in a partial or complete decrease of the hair loss. For this purpose, the dorman comprising compositions may be administered either topically at the site of scalp hair loss or, alternatively, in other cases may be administered systemically.
[0059] An additional phenomena which may be treated by administration of the dorman comprising compositions of the invention is associated with overgrowth of hair in various parts of an individual's body, such as arms, back, etc. (Hirsutism). Such undesired overgrowth of hair appears many times in aging individuals and, at times, is associated with loss of scalp hair in the same individual. Due to their ability to reduce cell growth, compositions of the invention may be useful in reducing such undesired overgrowth of hair.
[0060] The amount of the dorman comprising compositions to be administered for the above two indications, the administration regimes as well as their mode of application will again depend both on characteristics of the treated individual (age, size, gender, etc.) as well as on parameters associated with the phenomena to be treated (such as the extent of scalp hair loss, the specific body parts in which there is overgrowth of hair, etc.).
[0061] In addition, the dorman composition may be useful as a complementary agent administered in combination with or following hair removal treatments such as, for example, shaving (where said extract may be incorporated in an aftershave solution) or hair stripping (e.g. by wax).
[0062] Another application of the dorman composition may involve its administration to an individual during the period in which a scar is formed, e.g. after an operation in order to decrease scar formation. By slowing down the rate of the healing process in such an individual, the final scar may be much less apparent. In addition, the anti-fibrotic effect of the dorman compositions decreases the formation of cheloids which commonly appear after healing.
[0063] The dorman composition may also be useful for extending the duration of a tan in an individual. Following exposure to the sun, epidermal cells comprise a high concentration of melanin. During skin renewal such melanin comprising cells are shed. By slowing down the cell renewal process in the skin, the dorman composition causes the melanin comprising cells and thus the tan to remain for a longer period of time.
[0064] In addition to inhibiting proliferation of various cells, said dorman compositions are also capable of slowing seed germination and inhibiting growth of various plant seedlings. Following germination of seeds, roots and hypocotyls begin to develop in the seedling. Incubation of the plant seedlings with the dorman composition results in the inhibition of the elongation of the seedling roots and hypocotyls. The dorman compositions may therefore be used for weed control, wherein their administration at an appropriate concentration may result in the inhibition of growth of non desirable weeds while not affecting the growth of the desired plant. In view of the natural origin of the dormans, their administration has no noticeable toxic effect on cells or tissue on which they are induced to act or on the environment. In addition, at times it may be useful to use such compositions for long term storage of seeds and seedlings.
[0065] The present invention thus provides a dorman composition having the activity of slowing and inhibiting the growth of plant seeds and/or seedlings comprising said dorman extract.
[0066] A further application of the dorman composition of the invention is in the preservation of fresh produce, e.g. vegetables, fresh fish eggs, fish shells, etc.
[0067] The invention also provides a process for the preparation of an anti-proliferative composition comprising mixing dormans or DC with a carrier so as to yield an anti-proliferative composition with an anti-proliferative effective amount of dormans in said composition. Such a prepared composition may be used, depending on the nature of the carrier, in therapy, cosmetics, food preservation or agriculture. Such a process for preparing a pharmaceutical or cosmetic composition typically comprises preparing an DC, and mixing it with an appropriate pharmaceutical or cosmetic acceptable carrier, the amount of DC being such so as to yield a final therapeutically or cosmetically (as the case may be) effective amount of dormans in the composition. Also provided is use of dormans or an DC for the preparation of such a pharmaceutical or cosmetic composition.
[0068] As will be appreciated, the various applications of the dorman composition of the invention given above, are examples of a myriad of possible applications of these compositions, all having in common the inhibition of proliferation of target cells.
[0069] In the following, the invention will be illustrated by some non-limiting examples with occasional reference to the figures.
BRIEF DESCRIPTION OF THE FIGURES
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EXAMPLES
Example I
[0086] A: Effect of Narcissus bulb DC prepared from Narcissus field bulbs on growth of cucumber seedlings
[0087] (a) Inducing dormancy in Narcissus field bulbs
[0088] Narcissus field bulbs were obtained and subjected to hot water having a temperature of 45° C. for 2-4 hours. The bulbs were then either used immediately for the preparation of water soluble extracts or, alteratively, maintained in a dry room at a temperature of 30° C. for a maximal period of 8 months after which they were used for the preparation of the plant extract.
[0089] (b) Preparation of extracts from Narcissus bulbs
[0090] Active or dormant Narcissus field bulbs (induced into dormancy as explained above) were disinfected in soap water for a period of 1 hour. The bulbs were then cut and homogenized in distilled water (30 sec×3) using a Homogenizer Ultra-Turbo-turax. The homogenized preparation of the bulbs was then filtrated through a first 0.45 m sterile filter and then through a second 0.22 μm filter and the preparation which was not maintained on the filters was then collected. The concentration is defined as weight of original bulb (gr.) per final extract volume (ml).
[0091] (c) The effect of the Narcissus field bulb extracts on growth of cucumber seedlings
[0092] (i) Experimental assay:
[0093] Cucumber seeds cv. “
[0094] One layer of filter paper was placed in each of the petri dishes and 10 cucumber seeds, germinated as explained above, were placed on the paper. The petri dishes were incubated for 24 to 72 hours at 26° C. to 28° C. in the dark.
[0095] The effect of the tested bulb extracts in various concentrations were tested on two parameters of the cucumber seeds:
[0096] 1. Root elongation
[0097] 2. Hypocotyl elongation.
[0098] These two parameters were tested every 24 hours after the incubation of the seeds with the tested extract and every 24 hours after that.
[0099] (ii) Results:
[0100] As seen in Table 3 below, DC IBR-1 showed sufficient inhibitive activity on the seedlings' growth, as measured by the length of the roots and hypocotyls of seeds which were incubated with DC IBR-1compared to the length of the same organs incubated with sterile water. The inhibitive activity of DC IBR-1 was dose dependent.
TABLE 3 Growth of seeds with and without extract: 24 hrs 48 hrs Root Hypocotyl Root Hypocotyl Extract source (mm) (mm) (mm) (mm) Sterile Water 30 2 47 7 DC from Narcissus 0.2 gr./ml 4 0 4 1 0.1 gr./ml 5 0 5 2 0.05 gr./ml 6 0.5 10 2 0.01 gr./ml 16 1 26 4 0.005 gr./ml 22 2 35 7 0.001 gr./ml 25 2 41 7 Inhibition (%) of seed growth by extracts: 24 hrs 48 hrs (% inhibition) (% inhibition) Extract source Root Hypocotyl Root Hypocotyl Sterile Water 0 0 0 0 DC from Narcissus 0.2 gr./ml 87 100 91 86 0.1 gr./ml 83 100 89 71 0.05 gr./ml 80 75 79 71 0.01 gr./ml 47 50 45 43 0.005 gr./ml 27 0 25 0 0.001 gr./ml 17 0 13 0
Example I
[0101] B: Reversibility of the effect of Narcissus DC on growth of cucumber seedlings
[0102] (i) Experimental assay:
[0103] (a) The experiment was conducted in an identical manner to that described in I(A) above. The effect of the DC on growth of roots and hypocotyls of cucumber seeds was measured 24 and 72 hours after incubation. 72 hours after incubation, the seeds were washed with sterile distilled water and incubated with sterile distilled water for an additional 72 hours at 27° C. in the dark. The length of the roots and hypocotyls of the seeds was measured again 144 hours after the beginning of incubation (72 hours after washing away the DC).
[0104] (ii) Results:
[0105] As seen in Table 4 below, after washing away the Narcissus derived DC which had an inhibitive effect on the growth of roots and hypocotyls of cucumber seeds, the roots and hypocotyls began to grow again. Thus, the inhibitive effect of DC was reversible and non toxic. The same effect was apparent at lower concentrations of the DC incubated with the seeds as well (results not shown).
TABLE 4 144 H (72 Hours 24 H 72 H after washing) Time Root Hyptocotyl Root Hyptoctyl Root Hypocotyl Extract (mm) (mm) (mm) (mm) (mm) (mm) Sterile 44.9 6.5 111.7 17.5 — — water DC 0.2 2.6 1.5 2.7 6.2 43.3 22.9 gr/ml
Example II
[0106] Effect of Narcissus bulb derived DC on proliferation of keratinocytes
[0107] (a) Preparation of keratinocyte cultures
[0108] Human keratinocyte cultures were prepared as described in Ben Bassat H., et al.,
[0109] The cell suspensions prepared as described above, were inoculated at a concentration of 3-6×10
[0110] The flasks were incubated at 37° C. 10% CO
[0111] (b) Effect of Narcissus bulb derived DC on the proliferation of keratinocytes
[0112] (i) Experimental assay:
[0113] Narcissus bulb extracts were obtained as described in Example I(b) above from active and dormant bulbs. The secondary keratinocyte cell cultures seeded in microplates as described above, were further grown in Kmed for a period of 3-4 days. The microplates were then divided into the following main groups:
[0114] (1) keratinocytes which were continuously grown in Kmed;
[0115] (2) keratinocytes grown in Kmed containing the AE IBR-3 prepared from active Narcissus bulbs in several concentrations (from 0.5 μg/ml-10 μg/ml, each concentration forming a separate experimental group) and
[0116] (3) keratinocytes grown in Kmed medium comprising the DC IBR-1 obtained from dormant Narcissus bulbs at various concentrations (0.5 μg/ml-10 μg/ml each concentration forming a separate experimental group).
[0117] Each experimental group contained 5 wells. The growth medium containing DC IBR-1 or AE IBR-3 was changed every 24 hours for a period of 5 days after which the medium was removed from all of the wells and fresh Kmed medium without any plant extract was added and the cultures were grown in it for an additional 3 days.
[0118] The effect of the tested DC or AE on the proliferation of the keratinocytes was determined by the number of cells detected in a tested treated well as compared to the number of cells detected in a well in which the cells grew in Kmed medium without any DC or AE.
[0119] The number of cells in each well was determined by the microculture methylene blue assay as follows:
[0120] Extract treated cultures and controls were fixed in glutaraldehyde, 0.05% final concentration, for 10 mins. at room temperature. After washings the microplates were stained with methylene blue 1% in 0.1 M borate buffer pH 8.5 for 60 mins. at room temperature. Thereafter the plates were extensively and rigorously washed to remove excess dye and dried. The dye taken up by cells is eluted in 0.1 N HCl for 60 mins. at 37° C., and read at 620 nm.
[0121] In preliminary titration experiments linear readings were obtained for 1×10
[0122] (ii) Results:
[0123] As can be seen in
[0124] Against this, as seen in
Example III
[0125] Effect of DC obtained from dormant and active Narcissus bulbs on the proliferation of fibroblast in culture
[0126] (a) Preparation of fibroblast cell cultures:
[0127] Primary fibroblast cell cultures were initiated from small human skin specimens and prepared as described in Example II above regarding preparation of keratinocyte cultures except that the growth medium used was DMEM+20% fetal calf serum.
[0128] (b) Preparation of DC and AE
[0129] DC IBR-1 and AE IBR-3 were prepared from dormant and active Narcissus bulbs as described above.
[0130] (c) Effect of DC and AE on fibroblast proliferation:
[0131] (i) Experimental assay:
[0132] The above extracts were added to the fibroblast cultures at various concentrations (1 g-10 g/ml) and the number of fibroblasts in the cultures was determined 2 days, 5 days and 8 days after the addition of the extracts to the cells as described in Example II(a) above.
[0133] (ii) Results:
[0134] As seen in
[0135] As can be seen in
Example IV
[0136] Preparation of cosmetic and dermatological compositions comprising DC
[0137] The following are several specific examples of cosmetic and dermatological compositions which may be used in accordance with the invention for administration to an individual.
A. Balm (topical route): Ozokerite 10 gr. Isopropyl palmitate 9 gr. White vaseline 14 gr. Preserving agent 0.2 gr. Antioxidants 0.3 gr. Perfume 1 gr. DC prepared from bulb extract 0.00001 gr. Liquid paraffin qs 100 gr. B. Balm (topical route): Ozokerite 19 gr. Liquid purcellin oil 10 gr. White vaseline 15 gr. Preserving agent 0.2 gr. Antioxidant 0.3 gr. DC prepared from bulb extract 0.00002 gr. Liquid paraffin qs 100 gr. C. Emulsfied gel of O/W type (topical route): Carbopol ® 981 (marketed by Goodrich) 0.6 gr. Volatile silicone oil 3 gr. Purcellin oil 7 gr. Preserving agent 0.3 gr. Ethyl alcohol 15 gr. Perfume 0.4 gr. Triethanolamine 0.2 gr. DC prepared from bulb extract 0.04 gr. Demineralized water qs 100 gr. D. Aqueous-alcoholic gel (topical route): Carbopol ® 981 (marketed by Goodrich) 1 gr. Triethanolamine 1 gr. 95% Ethanol 60 gr. Glycerol 3 gr. Propylene glycol 2 gr. DC prepared from bulb extract 5 gr. Demineralized water qs 100 gr. E. Anhydrous gel (topical route): Absolute ethanol 61,1992 gr. Hydroxyethyl cellulose 0.8 gr. Propylene glycol 25 gr. Polyethylene glycol 12 gr. DC prepared from bulb extract 0.0008 g F. Emulsion of O/W type (topical route): Volatile silicone oil 10 gr. Liquid paraffin 6 gr. Liquid lanolin 3 gr. Arlacel ® 165 (marketed by Atlas) 6 gr. Tween ® 60 (marketed by Atlas) 2 gr. Cetyl alcohol 1.2 gr. Stearic acid 2.5 gr. Triethanolamine 0.1 gr. Preserving agent 0.3 gr. Antioxidants 0.3 gr. DC prepared from bulb extract 0.5 gr. Demineralized water qs 100 gr. G. Emulsion of O/W type (topical route): Propylene glycol 2 gr. PEG 400 3 gr. Preserving agent 0.3 gr. Carbopol ® 981 (marketed by Goodrich) 0.2 gr. Isopropyl myristate 1 gr. Cetyl alcohol 3 gr. Stearic acid 3 gr. Glycerol 3 gr. Corn oil 2 gr. Perfume 0.5 gr. DC prepared from bulb extract 0.001 gr. Demineralized water qs 100 gr. H. Clear gel (topical route) Oxyethylenated nonylphenol 5 gr. Carbopol ® 981 (marketed by Goodrich) 1 gr. Ethyl alcohol 30 gr. Triethanolamine 0.3 gr. Glycerine 3 gr. Perfume 0.3 gr. Preserving agent 0.3 gr. DC prepared from bulb extract 1 gr. Demineralized water qs 100 gr. I. Cream containing Ziposomes (topical route): Cetyl alcohol 4 gr. B-sitosterol 4 gr. Dicetyl phosphate 0.5 gr. Preserving agent 0.3 gr. Sunflower oil 35 gr. Perfume 0.6 gr. Carbopol ® 981 (marketed by Goodrich) 0.2 gr. Triethanolamine 0.2 gr. Sphingosine 0.05 gr. DC prepared from bulb extract 0.2 gr. Demineralized water qs 100 gr. J. Per as composition: Talc 5 mg Aerosil 200 5 mg Stearate de Zn 5 mg DC prepared from bulb extract 3 mg Lactoseqs 400 mg K. Liquid of Iontophoresis: Benzoate de sodium 2 mg Preserving agent 0.15 gr. DC prepared from bulb extract 1 gr. Water qs 100 gr. L. Emulsion W/O: Protegin (marketed by Goldschmidt) 19 gr. Vaseline oil 8 gr. Glycerine 3 gr. DC prepared from bulb extract 1 gr. Sulfate de Mg 0.5 gr. Perfume 0.8 gr. Preserving agent 0.2 gr. Water qs 100 gr.
Example V
[0138] Extracts prepared from Narcissus Bulblets (cell cultures) on cucumber seedling growth:
[0139] (a) Preparation of Narcissus bulb cell cultures
[0140] Active Narcissus bulbs from the field having inflorescence stalk initials were used to prepare duplicate inner scale explants. The explants were then inoculated into NR31 medium (NAA/10 μM BA: 5:0.5 μM) to initiate callus cultures. Four to 5 weeks after the initiation of the callus cultures, the cultures were transplanted into NR8 medium (6% sucrose) to form bulblets growth explants. For scaling up of the biomass, half bulblets were transplanted into bulblet column bioreactors with liquid basal media N4 comprising:
Murashige & Skoog (Sigma M-5525) 4.33 gr/L Myoinositol 100 mg/L Adenine sulfate 150 mg/L NaH 345 mg/L NAA 5 μM Agar Type A 7 gr/L BA 5 μm pH = 5.7 Pyridoxine 1 mg/L Glycine 2 mg/L Nicotinic acid 5 mg/L Thiamine HCl 0.5 mg/L Sucrose 30 gr/L
[0141] for a period of 4 weeks.
[0142] (b) Preparation of cell culture derived DC
[0143] The plant material prepared as described in (a) above was then shaken for 7-10 days on a gyratory shaker at about 35° C. (the column weight per medium was 0.1 gr/ml). Half of the cell cultures were induced into dormancy by their incubation at a high temperature (of about 35° C.). The DC prepared from such cultures was designated IBR-11. The remaining cell cultures were maintained in their active state by growing them in regular conditions and the AE prepared from them was designated IBR-10. AE IBR-10 or DC IBR-11 were prepared from the medium free bioimass which was washed with water, weighed and homogenized in an ultra-Turbo-turax. The homogenate was suspended and diluted in steril distilled water.
[0144] (c) Cucumber seeds cv. “
[0145] (d) The experimental assay:
[0146] (i) The effect of the above DC and AE prepared from Narcissus bulblets on root elongation and hypocotyl elongation of the cucumber seedlings was determined as follows. Each experimental group consisted of petri dishes each containing 10 seeds was incubated with:
[0147] 1. DC IBR-11 (dormant)
[0148] 2. AE IBR-10(active).
[0149] One or two layers of filter paper were placed in each of the petri dishes and 10 cucumber seeds, germinated as explained above, were placed on the filters. The Petri dishes were incubated for 72 hours at 27° C. to 30° C. in the dark.
[0150] The effect of extracts was tested on two parameters of the cucumber seeds:
[0151] 1. Root elongation
[0152] 2. Hypocotyl elongation.
[0153] These two parameters were tested after 72 hours of incubation of the seeds with the extract.
[0154] (ii) Results:
[0155] As seen in Table 5 below, 72 hours after incubation, DC IBR-11 had a significantly higher inhibition activity on both the root length and hypocotyl length of the seedlings as compared to the effect of AE on the seedlings' growth.
TABLE 5 % inhibition of DC AE DC compared to AE (IBR-10) (IBR-11)
Root length (mm) 72 h 86 31 58% Hypocotyl (mm) 72 h 81 49 40%
Example VI
[0156] Effect of Narcissus bulb cell culture derived DC on proliferation of keratinocytes
[0157] (a) Preparation of keratinocyte cultures
[0158] Human keratinocyte cultures were prepared as described in Example II(a) above.
[0159] (b) Effect of Narcissus bulblet derived DC on the proliferation of keratinocytes
[0160] (i) Experimental assay:
[0161] Narcissus bulb derived cell cultures were obtained as described above and DC IBR-11 was prepared from bulblets induced into dormancy (as is also described above).
[0162] The secondary keratinocyte cell cultures seeded in microplates as described above, were further grown in Kmed for a period of 3-4 days. The microplates were then divided into the following main groups:
[0163] (1) keratinocytes which were continuously grown in Kmed; and
[0164] (2) keratinocytes grown in Kmed containing DC IBR-11 in several concentrations (from 0.5 μg/ml-10 μg/ml, each concentration forming a separate experimental group).
[0165] Each experimental group contained 5 wells. The growth medium containing DC was changed every 24 hours for a period of 5 days after which the medium was removed from all of the wells and fresh Kmed medium without any plant extract was added and the cultures were grown in it for an additional 3 days.
[0166] The effect of the tested DC on the proliferation of the keratinocytes was determined by the number of cells detected in a tested treated well as compared to the number of cells detected in a well in which the cells grew in Kmed medium without any DC.
[0167] The number of cells in each well was determined by the microculture methylene blue assay as follows:
[0168] Extract treated cultures and controls were fixed in glutaraldehyde, 0.05% final concentration, for 10 mins. at room temperature. After washing, the microplates were stained with methylene blue 1% in 0.1 M borate buffer pH 8.5 for 60 mins. at room temperature. Thereafter the plates were extensively and rigorously washed to remove excess dye and dried. The dye taken up by cells is eluted in 0.1 N HCl for 60 mins. at 37° C., and read at 620 nm.
[0169] In preliminary titration experiments linear readings were obtained for 1×10
[0170] (ii) Results:
[0171] As can be seen in
Example VII
[0172] Effect of DC obtained from dormant Narcissus bulblets on the proliferation of fibroblast in culture
[0173] (a) Preparation of fibroblast cell cultures:
[0174] Primary fibroblast cell cultures were initiated from small human skin specimens and prepared as described in Example II above regarding preparation of keratinocyte cultures except that the growth medium used was DMEM+20% fetal calf serum.
[0175] (b) Preparation of cell cultured DC:
[0176] IBR-11 was prepared from dormant bulblets as described in Example V(b) above.
[0177] (c) Effect of DC on fibroblast proliferation:
[0178] (i) Experimental assay:
[0179] The above extracts were added to the fibroblast cultures at various concentrations (1 μg-10 μg/ml) and the number of fibroblasts in the cultures was determined 2 days, 5 days and 8 days after the addition of the extracts to the cells as described in Example V(a) above.
[0180] (ii) Results:
[0181] As can be seen in
Example VIII
[0182] Effect of fruit juice on cucumber seed growth
[0183] (a) Preparation of fruit juice
[0184] Grapefruit juice was produced from one fresh grapefruit and the produced juice was squeezed and filtrated through cotton cloth and then centrifuged at 10,000 rpm for 10 mins. at room temperature. The supernatant was then used for testing its effect on cucumber seed growth as described below.
[0185] (b) The effect of grapefruit juice on growth of cucumber seedlings:
[0186] (i) Experimental assay:
[0187] Cucumber seeds were prepared as described in Example 1 (c)(i) above. Each experimental group comprised ten Petri dishes which were filled with 1.8 ml of the following:
[0188] (1) dH
[0189] (2) Fruit juice obtained as in (a) above.
[0190] One or two layers of filter paper were placed in each of the petri dishes and 10 cucumber seeds, germinated as explained above, were placed on the filters. The petri dishes were incubated at 25° C. and the parameters of the cucumber seeds were measured at 24 hours and 72 hours after beginning of incubation.
[0191] The effect of the fruit juice was tested on two parameters of the cucumber seeds:
[0192] (1) Root elongation
[0193] (2) Hypocotyl elongation
[0194] (ii) Results:
[0195] As seen in Table 6 below, this experiment indicates that the fruit juice comprises inhibitory activity on the growth of cucumber seeds.
TABLE 6 72 hours 72 hours % inhibition Root Hypocotyl Hypocotyl Treatment mm mm Root mm mm dH 110 35 — — Grapefruit Juice 3 0 97 100
Example IX
[0196] Effect of dorman extract obtained from the crustacean
[0197] (a) Preparation of extracts from
[0198] The dorman extract designated IBR-4 was obtained by preparing an extract from
[0199] (b) The effect of the DC extract from Artemia (IBR-4) on growth of cucumber seedlings:
[0200] (i) Experimental assay:
[0201] Cucumber seeds were prepared and seeded into petri dishes as explained in Example 1 above. Each experimental group comprised a petri dish containing 10 cucumber seeds and the results shown below are an average of the parameters measured for the 10 seeds. The petri dishes were grown at 28° C. in the dark and the root length and hypocotyl length of the cucumber seeds were measured 24 hours and 48 hours after beginning of incubation with 1.8 ml of one of the following:
[0202] (1) dH
[0203] (2) DC IBR4 at a concentration of 0.02 gr./ml.
[0204] (ii) Results:
[0205] As seen in Table 7 below, the Artemia dorman extract IBR-4 had a significant inhibitory effect on cucumber seed growth which was apparent already after 24 hours of incubation of the seeds with the DC IBR4 but was most significant 48 hours after incubation (66% inhibition on root growth and 40% inhibition on hypocotyl growth).
TABLE 7 24 hours 48 hours Root Hypocotyl 24 hours % Inhibition Root Hypocotyl 48 hours % inhibition Treatment mm mm Root Hypocotyl mm mm Root Hypocotyl dH 35 4 0 0 61 10 0 0 IBR4 0.02 gr/ml 20 3 43 25 21 6 66 40
Example X
[0206] Effect of a dorman extract from a dormant plant (Narcissus derived IBR-1) and a dorman extract obtained from animals (Artemia derived IBR-4) on proliferation of mouse bladder carcinoma cells
[0207] (a) Preparation of the dorman extracts:
[0208] The Narcissus derived plant dorman extract IBR-1 and the Artemia derived animal dorman extract IBR-4 were prepared as explained in the Examples above.
[0209] (b) The effect of DC IBR-1 and DC IBR-4 on proliferation of mouse bladder carcinoma cells:
[0210] (i) Experimental assay:
[0211] The two experiments were carried out as follows:
[0212] T24P cells (mouse bladder carcinoma cells) (
[0213] DC IBR-1 and DC IBR-4 were added to the T24P cell cultures and IBR-1 was added also to the T50 cells at various concentrations (0 g/ml-25 g/ml) 24 hours after plating of the cells and 48 hours after the beginning of incubation of the cells with the dorman extracts. The number of cells per well were determined 48 hours after the beginning of incubation of the cells with the extracts using the microculture methylene blue assay (described in Example 2 above) and the dyed culture plates were read at 620 nm.
[0214] (ii) Results:
[0215] As seen in
Example XI
[0216] Inhibition of cucumber seed growth by Grape and Kiwi juice
[0217] (a) Preparation of fruit juice
[0218] Grape and Kiwi juices were produced from fresh fruit by blending the fruit in the blender cup for 3 mins. at high speed, filtrating the blend through a cheese cloth and centrifuging it at 6,500 rpm for 10 mins. at room temperature. The supernatant was then used for the experiment at a concentration of 1.2 gr/ml of the grape juice and 1.26 gr/ml of the kiwi juice. The concentration was determined by fruit original weight (gr.) for final volume (v). Several dilutions of each juice were prepared and used for testing on cucumber seed growth.
[0219] Preparation of the cucumber seeds and the experimental assay were carried out as described in Example I(c)(i) above. The length of the roots and hypocotyls were measured 24 and 48 hours after beginning of incubation of the seeds with each of the tested juices or controls at 28° C. As seen in Table 8 below, both the kiwi juice and the grape juice showed a very high percent of inhibition both on growth of cucumber roots as well as on hypocotyls. The most prominent inhibition was seen 48 hours after beginning of incubation wherein both juices inhibited the growth of the cucumber seeds.
TABLE 8 48 h 24 h Hypocotyl Root % Hypocotyl % Root % % Inhibition Inhibition Inhibition Inhibition dH 0 0 0 0 Kiwi juice 100 mg/ml 50 100 72 100 Kiwi juice 50 mg/ml 33 67 55 63 Kiwi juice 25 mg/ml 0 67 42 38 Kiwi juice 5 mg/ml 0 0 7 13 Grape juice 100 mg/ml 83 100 88 100 Grape juice 50 mg/ml 40 100 67 63 Grape juice 25 mg/ml 17 67 <