WO1995005839A1 - Nigella sativa as a medicinal treatment - Google Patents
Nigella sativa as a medicinal treatment Download PDFInfo
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- WO1995005839A1 WO1995005839A1 PCT/US1994/009660 US9409660W WO9505839A1 WO 1995005839 A1 WO1995005839 A1 WO 1995005839A1 US 9409660 W US9409660 W US 9409660W WO 9505839 A1 WO9505839 A1 WO 9505839A1
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- sativa
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/71—Ranunculaceae (Buttercup family), e.g. larkspur, hepatica, hydrastis, columbine or goldenseal
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
Definitions
- the present invention is generally directed to the fields of medicine and pharmacology, and specifically directed to using the plant seed extract of Nigella sat ⁇ va Linn (N. satxva) in the treatment of cancer, viral diseases, protection from side effects of chemotherapy and growth factor for bone marrow in hematopoiesis.
- Nigella sat ⁇ va Linn N. satxva
- the primary targets for treatment is cancer.
- Anticancer remedies are available today which are effective in killing cancer cells. However, many of these medicaments also damage or kill off normal cells or have other serious side effects. It is therefore vitally important to develop an anticancer program which is specific for the cancerous growth in a body, but which is not toxic to the rest of the body system. Ideally, the program will include treatment using natural plant extracts.
- body or patient can include any warm-blooded mammal, but is specifically intended to refer to the human body.
- Patent 4, 986, 895 to Grossman et al . is directed to use of water-soluble plant extracts in the treatment of virus skin infections.
- U.S. Patent 5,178,865 to Ho et al . is directed to the use of Chinese herbal extracts in the treatment of HIV related disease in vi tro . A total of 56 herbal extracts were screened for anti-HIV activity using in vi tro techniques.
- Aruna (1990) also describes the use of spices, leafy vegetables and condiments having diverse medicinal properties. Products of 20 spices or leafy vegetables were screened for anti-carcinogenic activity using induction of glutathione-S-transferase. One of the plants utilized was Cuminum cyminum Linn (C. cyminum) , also known as cumin. All of the spices and leafy vegetables were tolerated well and no toxic effects were seen.
- Bi tter an et al . (1991) disclose a study that was performed on a population of 964 adult patients, of which 28% suffered from malignant diseases of the urinary tract and 72% from a wide spectrum of the nine neurologic diseases. The results conclude that the use of C. cyminum in the diet may contribute to the prevention of diseases mediated by peroxidation of lipids.
- N. sativa is an annual herb belonging to family Ranunculaceae.
- Other species of Nigella include Nigella arvensis and Nigella damascena . Induction of callus cultures indicates considerable chromosomal variations in callus tissues between the different species of Nigella (Datta, et al . 1983) .
- N. sativa is characterized by an erect branched stem and alternate finely divided, feathery, grayish-green leaves. The bluish-white, star-shaped flowers are terminal and solitary. Petals are absent.
- the fruit is a globose capsule with small, black, rough seeds.
- the plant is cultivated in India, Bangladesh, Turkey, Middle-east and the Mediterranean basin mainly for its seeds or "black cumin" which is almost entirely used for edible and medical purposes, such as spices and for treatment of various diseases.
- the ripe seeds of N. sativa also known as Kalajira or Kalaonji, are known to have a wide range of medicinal uses (Kirtikar et al . 1982, and Chopra et al . 1982) .
- the constituents of the seeds include saponin, an essential oil, a bitter compound (nigellone) and tanners. These substances have been shown to have diuretic (Nadkarni 1976) , cholagogic and antispasmodic ( Tennekoon, et al . 1991), carminative (Shayeb and Mabrouk, 1984), galactogogic ( Vihan 1987), antibacterial (Hassan, et al .
- the petroleum ether extract of the seeds at 1000-62.5 parts per million (ppm) concentrations was found to have the same activities -as growth regulating juvenile hormone when tested against the fifth instar larvae of Dysdercus similis ⁇ Kumar et al . 1987) . al -Awadi et al . (1981) is directed to the study of the effect of a plant's mixture extract containing N. sativa on liver gluconeogenesis. The researchers report that non-insulin dependent diabetes mellitus is treated in Kuwait by a plant mixture extract, which contains N. sativa . In this study, a powdered mixture of equal portions of N.
- N. sativa was tested in volunteers with a low helper T-cell to suppressor T-cell ratio. The results indicated an increase in the helper T-cell population in the experimental group. Further, the helper T-cell to suppressor T-cell ratio increased while the ratio within the control groups remain the same.
- Nair et al . (1991) investigated the effects of N. sativa as potential protective agents against cisplatin-induced toxicity in mice. Some protective effects were shown by the use of N. sativa extracts. Salomi , N. J. , et al . (1992) studied N. sativa seeds containing certain fatty acids for antitumor activities against Ehrlich ascites carcinoma (EAC) , Dalton's lymphonia ascites (DLA) and Sarcoma-180 (S-180) cells. The paper presents the results of in vi tro and in-vivo antitumor experiments. The active antitumor principle was isolated. It was found that the active principle was cytotoxic for EAC cells, KB cells and lymphocytes.
- EAC Ehrlich ascites carcinoma
- DLA Dalton's lymphonia ascites
- S-180 Sarcoma-180
- the present invention provides an anticancer remedy and treatment which has, as its active ingredient, the extract of the plant N. sativa .
- the medicament of the present invention is useful in treating cancer, preventing toxicity of anticancer drugs in human body and in increasing immune function.
- the present invention is specifically directed to a pharmaceutical composition for the treatment of cancer comprising a pharmaceutical preparation consisting essentially of an extract from Nigella sativa, at a concentration which is effective to destroy cancer cells in a patient.
- the present invention is directed to a pharmaceutical composition for the treatment of the side effects of anticancer therapy consisting essentially of an extract from Nigella sativa, at a concentration of the extract which is effective to reduce the side effects of anticancer therapy.
- the present invention is also directed to a method for treating humans suffering from the side effects of anticancer chemotherapy using the extract of Nigella sativa, comprising administering to humans effective doses of the composition described above.
- the present invention is directed to a method for increasing the immune function in humans comprising administering to humans effective doses of an extract from Nigella sativa, at a concentration of the extract which is effective to increase the immune function.
- the present invention is also directed to a method for protecting the normal cells from cytopathic effects of virus, comprising administering to humans effective doses of an extract from Nigella sativa, at a concentration of the extract which is effective to protect against the cytopathic effects of the virus.
- the present invention is still further directed to a method for increasing antibody producing B cells, comprising administering to humans an extract from Nigella sativa, at a concentration which is effective to increase the antibody-producing B cells.
- the present invention is also directed to a process for inhibiting tumor cells without affecting normal or nontumor cells in a patient comprising administering to a patient an extract from Nigella sativa, at a concentration which is effective to inhibit the tumor cells without affecting nontumor cells.
- the present invention is also directed to a method for stimulating bone marrow formation in humans comprising administering to humans effective doses of an extract from Nigella sativa, at a concentration of the extract which is effective to stimulate bone marrow formation.
- N. sativa extract helps stimulate bone marrow cells, protects the normal cells from cytopathic effects of virus, destroys tumor cells and increases antibody producing B cells. It protects the bone marrow against chemotherapy and at the same time, can act as an anticancer agent. All these factors makes N. sativa seed extract an ideal candidate to be used as vaccine for cancer prevention and cure.
- N. sativa plant extract is more effective than standard chemotherapeutic anti-cancer drugs.
- N. ' sativa extract stimulates bone marrow cells, protects the normal cells from cytopathic effects of virus, destroys tumor cells and increases antibody producing B cells. Further, it protects the bone marrow against chemotherapy and can act as an anti-cancer agent.
- N. sativa plant extract also has been found to help restore immune competent cells in immunosupressed cancer patients and to overstimulate bone marrow formation in normal individuals.
- N. sativa extract helps free tumor antigen binding sites on B cells.
- the administration of the N. sativa extract, rather than the increase of the immune competent cell number, has been found to help free tumor antigen binding sites on B cells, thereby elevating the CD19 and associated cell population.
- the antigen binding site on the immunoglobulin molecule on the surface of B cells is free because of N. sativa treatment, it binds to the tumor associated antigen thereby generating an immune response against the antigen.
- VSV VSV
- N. sativa plant extract was also observed upon administration of N. sativa plant extract. Additionally, the serum interferon level is found to increase; and, hence, the plant extract of N. sativa has interferon-like antiviral activity. This is an example of interferon level increasing in the circulation, preventing viral diseases and, in addition, possibly curing viral diseases.
- N. sativa promotes anti-tumor activity.
- Data from pharmacosensitivity screening indicates anti-tumor activity of N. sativa plant extract mainly against melanoma and colon cancer types.
- N. sativa plant extract destroys tumor cells and leaves normal cells alone, possibly because of its ability to bind to cell surface asialofeutin (lectin) in diseased cells, which causes aggregation and clumping of tumor cells. It also blocks enzymes and inappropriate gene products involved in nucleic acid synthesis and metabolism.
- FIG. 1 is a graph illustrating the elevation in CD19, HLADR, NK CD3-/CD56+ and CD38 populations of peripheral blood cells from cancer patients upon incubation with N. sativa extract over a 18 hour period at 37°C in 5% C0 2 incubator as shown in Experiment 2.
- Figure 2 is a graph illustrating the percent protection of 3.5 x 10 4 WISH cells from the cytopathic effects (CPE) of vesicular stomatitis virus (VSV) by serial dilutions of N. sativa extract as described in Experiment 3.
- CPE cytopathic effects
- VSV vesicular stomatitis virus
- B cells B cells or B lymphocytes secrete proteins/antibodies that protect the human body against infections.
- CD3 Cluster Differentiation 3. These are the antibodies which indicate Activated T lymphocytes which are used by the body for its protection against foreign harmful germs.
- CD19 Cluster Differentiation 19. These are antibodies which help detect B lymphocytes. Elevation in CD19 indicates an elevation in B lymphocytes and vice versa.
- CD56 Cluster Differentiation 56 are antibodies which inhibit Natural Killer target cell interactions in certain systems.
- HLADR Human Leukocyte antigen DR. DR designates a genetic locus or the antigen of the major histocompatibility complex corresponding to the locus. An increase in HLADR indicates an elevation in immunological parameters against the disease.
- NK Natural Killer cells. NK is an indication to detect the Natural Killer cells. NKCD3-/CD56+: Natural Killer Cluster
- the process for preparing the N. sativa extract comprises grinding the seeds and separating the extract of the N. sativa with appropriate solvents such as alcohol or water, removing lipids by extraction with ether or petroleum ether, crystallization or chromatographic fractionation and then mixing its components in the desired proportion.
- the N. sativa extract can then be prepared in 3 forms - oil, fluid and crystal - by processes known to the art.
- the preferred process for the preparation of N. sativa extract is described in Experiment 1.
- Administration The extract of N. sativa may be administered by itself or in admixture with an appropriate excipient or carrier.
- the preparation may be administered to the patient by enteral, such as oral or rectal, and parenteral, such as intraperitoneal, intramuscular, intravenous or subcutaneous route.
- enteral such as oral or rectal
- parenteral such as intraperitoneal, intramuscular, intravenous or subcutaneous route.
- the preparation may also be administered in combination with supplements, such as antiviral agents, immune modulators, antibodies, other chemotherapeutic agents, or combinations thereof.
- the preparation may additionally be administered in dosage form, such as by capsules, tablets, suppositories or the like. Dosages :
- N. sativa has been found to be most effective when administered at a dosage of 30g per day, with an effective range of 20-40g per day.
- N. sativa extract is also known to confer protection of human amniotic "WISH" cells against cytopathic effects of vesicular stomatitis virus (VSV) .
- VSV vesicular stomatitis virus
- a patient weighing 70 kg has about 7xl0 13 cells in its body and N. sativa will be useful at a dosage between about 20 and 40g per day and preferably about 30g per day to protect against viral attack in virus endemic areas.
- N. sativa was measured on bone marrow and peripheral white blood cells. In vi tro antiviral antitumor and growth factor like activity were also measured. N. sativa extract was incubated with bone marrow cells to determine the growth of the cells. The results were compared with that of bone marrow growth factors and biological response modifiers (GM-CSF, G-CSF, erythropoietin, interferon, IL-2, and STS) . Mouse connective cells and human amnion or "WISH" cells were also assayed.
- GM-CSF bone marrow growth factors and biological response modifiers
- % inhibition 1 - R ⁇ -R c X 100 in which R ⁇ is the average cell count of the experimental well with extract/Abrin control, R c is the average background cell count with control, R ⁇ is the average maximum inhibition without extract/Abrin control.
- FC Flow Cytometrv Direct Immunofluorescence Staining Automated Flow Cytometry
- Peripheral Blood is aseptically collected in an appropriate container such as a lavender top (EDTA anti-coagulant) vacutainer tube and delivered at room temperature preferably within 48 hours.
- the minimum amount required is approximately 2 ml whole blood per panel ordered.
- the optimum amount required is approximately 3 ml whole blood per panel ordered
- the bone marrow (BM) specimen is aseptically collected in a container such as a lavender top (EDTA) vacutainer tube and transported at room temperature within 48 hours.
- the minimum amount required is approximately 2 ml of BM specimen per panel ordered.
- the optimum amount is approximately 3 ml of BM specimen per panel ordered.
- Solid tumors include those from the breast, lymph node, colon, ovarian, lung, and skin. Reference is made to the HTCA Pharmacosensitivity Procedure (Procedure 5, infra . ) for a description of the procedures.
- the tumor should be delivered at room temperature within 48 hours of extraction.
- the minimum amount required is approximately 2 ml of cells at a concentration of 1 x 10 6 /ml per panel ordered.
- the optimum amount is approximately 3 ml of cells at a concentration of 1 x 10 6 /ml per panel ordered.
- the reagents are prepared as follows:
- PBS Phosphate Buffered Saline
- Ca++ Ca++
- Mg++ Gabco order #310-4190AJ
- repeater pipet (set on 4) add 1 ml of IX PBS to each tube and vortex each tube (use safety shield) .
- Repeater pipet (set on 2) add 0.5 ml of 0.5% paraformaldehyde. Vortex. Keep tubes covered with parafilm and in the refrigerator until ready for flow cytometric analysis. Specimen Analysis:
- CFU when stem cells are treated with different medications will help determine predominance of development of cell lines. It has been demonstrated that interferon prolongs myelopoietic differentiation; therefore, the number of CFU will increase. This will demonstrate efficiency of interferon on bone marrow recovery and confirm the protective activity of interferon to the bone marrow.
- CFU development for the different cell lines can be directed.
- Specimen Requirements Collection and Handling: Specimen Requirements: Bone marrow sufficient to yield a minimum of 4.0 ml and an optimum of 5.0 ml mononuclear cells at a concentration of 1.0 x 10 6 cells/ml.
- Bone Marrow collection procedure Prepare 4- 6 50 ml Falcon tubes containing 20 ml of tissue transport media. Transport the media in the cooler with frozen ice chips to the procedure. A mask, gloves and gown must be worn during the procedure. Inject 2500 units of preservative-free heparin into the media tubes using sterile technique. This must be done no earlier then 15 minutes before obtaining the bone marrow specimen. Mix well. When bone marrow is handed to the technologist in a syringe, carefully inject into the media tubes, rinsing with media twice. Discard syringe in the Sharps container. Cap tube and mix well. Label the specimen with the following: 1) Patient's full name; 2) Date specimen was received; 3) Time specimen was received; and 4) Initials of technologist who obtained the specimen. Place in cooler with ice chips for transportation. Materials:
- Penicillin/Streptomycin L-Glutamine [100X] Trace Elements [10OX 2-Mercaptoethanol Insulin Transferrin-Sodium Selenite Media
- Interleukin-2 (Boehringer Mannheim GMBH Cat #799068)
- GM-CSF (Amgen Cat #13050)
- IMDM/RMPI 1640 .1X1 Tissue Transport Media
- interleukin-2 Thaw interleukin-2 and take 5 ml of interleukin-2 containing 200 units per ml. Immediately freeze remaining interleukin-2 into 5 ml aliquots. Add 5 ml of RPMI (IX) media to 5 ml of interleukin-2 containing 200 units per ml to obtain a concentration of 100 units/ml. To 10 tubes containing 9 ml of the media, add 1 ml of interleukin-2 at a concentration of 100 units/ml to obtain a concentration of 10 units/ml. These aliquots are stable for 30 days at 4-8°C. For colony forming assay, use 0.5 ml (or 5 units) for each 25cm 2 flask containing 4.5 ml of medium and 0.5 ml of cells. Somatostatin:
- Somatostatin is available in single vials containing 50 ⁇ g/ml and 100 ⁇ g/ml from Sandoz Ltd. Store at 4-8°C.
- For the Colony Forming Assay use 1.0 ml of STS containing 50 ⁇ /ml for each 25 cm 2 flask containing 4.0 ml of media and 0.5 ml of cells. This procedure is written for 50 ⁇ g/ml vials. If 100 ⁇ g/ml vials are used, use 0.5 ml of Somatostatin and 4.5 ml of media for a 25 cm 2 flask.
- GM-CSF GM-CSF
- GM-CSF 50 units/ml (total 1,000 units) . Close 4 of these aliquots with sterile adhesive and store at 4°C. Aliquot #5 will be marked "IN USE" and stored at 4°C. For colony forming assay, use 50 ul (or 2.5 units) for 25 cm 2 flask containing 5.0 ml of medium and 0.5 ml of cells.
- G-CSF 50 units/ml (total 1,000 units) .
- a sterile pipet Using a sterile pipet, dispense 20-25 ml of histopaque into the appropriate number of capped sterile 50 ml conical tubes. Using another sterile pipet, gently layer the bone marrow at a 45°C angle onto the histopaque using a 1:1 ratio of histopaque to specimen. Centrifuge at 1600 RPM for 20 minutes in refrigerated centrifuge at 4°C (brake on 2) . Remove and discard the supernatant.
- Viable cell count X 100 % viability total cell count 3. Adjust volume to amount needed for the appropriate cell concentration for the assay (this assay requires 1 X 10 6 cells/ml) using the following formula:
- V ⁇ V 2 C 2
- ⁇ 10 ml
- V 2 ⁇ ?
- a colony is defined as 40 or more cells adhering to the bottom of the flask.
- Flasks may be discarded after the final report is reviewed by the Medical Director.
- the optimum amount is 3 ml at 1 x 10 6 cells per ml.
- Plasmapheresis specimen - 3 ml Both a whole blood specimen and a serum specimen are required for complete immunomodulatory evaluation. However, in the event that only one of the two specimens (WB or serum) is available, partial immunomodulatory testing may be performed. Collection, Processing, and Handling: The patient will be properly identified by the nurse or phlebotomist (i.e., check wristband, verify patient name) .
- Specimens will be aseptically collected by the nurse or a phlebotomist. Gloves and lab coat must be worn while drawing or handling the specimen and "universal precautions" must be observed.
- 1. Whole Blood a. Collection - Add 2500 units of preservative free heparin (Calciparine) to a 10 ml red top vacutainer tube. Perform venipuncture and draw the patient's blood into tube. Label all tubes with patient's name, date and time collected. Invert several times to mix anticoagulant. Refrigerate tube until ready for separation of the mononuclear layer by the density gradient procedure (Reference is made to Procedure 4, infra . , at part B-2) . Minimum amount whole blood 7 ml
- Optimum amount whole blood 10 ml b. Unacceptable Specimens - Clotted specimens, grossly hemolyzed specimens, or frozen specimens are unacceptable. c. Rejection of Specimens - When any criteria are not met, the unacceptable specimens may be tested if necessary.
- Serum a. Collection - Use a red top vacutainer tube to perform venipuncture and draw 10 ml of the patient's blood into the tube. Centrifuge the serum specimen at 2000 RPM for 10 minutes at 25°C. Refrigerate tube until testing. Aseptically draw off serum and place in a separate tube. Minimum amount serum 3 ml
- Optimum amount serum 5 ml b. Unacceptable Specimens - Specimens with excessive fibrin clotting are unacceptable. c. Rejection of Specimens - When any criteria are not met, the unacceptable specimens may be tested if necessary.
- Optimum amount of plasma 5 ml b. Unacceptable Specimens - Specimens with excessive fibrin clotting are unacceptable. c. Rejection of Specimens - When any criteria are not met, the unacceptable specimens may be tested if necessary.
- Bone Marrow a Collection - Not more than 15 minutes prior to the BM procedure, add 2500 units of Calciparine per 25 ml of tissue transport media using aseptic techniques. Using aseptic techniques, quickly add the BM specimens to the transport tube and mix well. Refrigerate the tube until ready for separation of the mononuclear layer by the density gradient procedure (see part IV, section B-2) .
- b Unacceptable Specimens - Grossly clotted specimens, grossly hemolyzed specimens, or frozen specimens are unacceptable.
- c Rejection of Specimens - When any criteria are not met, the unacceptable specimens may be tested if necessary.
- Test Tubes Capped (-5 ml, -10 ml)
- Lower layer (2.5% working solution) Weigh out 2.50 gm of Bacto-Agar and place in a 100 ml capped bottle. Add 100 ml distilled water. Swirl gently to mix.
- IL-2 in 10 ml capped tube: Remove working stock aliquot of IL-2 (10 units/ml) from refrigerator. If the aliquot is not available in refrigerator, follow the IL-2 aliquot procedure found in the aliquot procedure book. The stock tubes are kept in the -48°C freezer. The dosage for testing will be 0.1 ml, which will equal 1 unit of interleukin-2. Therefore, 1 unit of interleukin-2 will be tested in the immunomodulatory procedure. Interleukin-2, in this media, can be maintained for 30 days at 2-8°C.
- LI-8-ABIFN mAB to IFN alpha 2A
- remove working aliquot neutralizing effect of 1000 units/ml from freezer
- the dosage will be 0.1 ml in the assay to achieve a concentration of 100 units of neutralizing ABIFN.
- L929 cells microscopically examine the liquid culture flasks of the desired cell line and determine by confluency which flasks to use. Remove supernatant with pipet and discard. Add 5 ml Trypsin. Expose to cell layer and remove 2 ml. Incubate flasks with Trypsin for minute. Hit flask with the palm of your hand to knock cells off. Add 7 ml of L929 cell media to stop reaction. Transfer cells to a sterile 50 ml conical tube, add more media (to bring to 20 ml) . Centrifuge tube @ 1600 RPM for 5 minutes with brake on 2. Remove supernatant and resuspend pellet in L929 cell media. Centrifuge cells at 1600 RPM for 5 minutes with brake on 2. Remove supernatant, resuspend pellet in appropriate media. Preparing the Patient Cells (from whole blood with Calciparine, or BM Specimen with Calciparine) :
- a whole blood tube containing 8 ml of blood dispenses the appropriate volume of histopague (use equal amounts of histopaque to equal amounts of whole blood, i.e., if a whole blood tube containing 8 ml of blood is received, dispense 4 ml of histopaque and 4 ml ' of whole blood into each of two 15 ml capped tubes. If 75 ml of BM is received, dispense approximately 18.75 of histopaque and 18.75 of BM into four 50 ml conical tubes. Layer in the whole blood very slowly at a 45°C angle on the histopaque. Centrifuge at 1600 RPM for 20 minutes in centrifuge at 4°C, brake on 2.
- Ovarian2 X 10 4 Patientl X 10 6 Count the total number of cells (stained and unstained) in the 4 corner 1mm 2 squares. CALCULATION FOR CELL COUNT:
- % of viable cells X 100 % viability total # of cells
- V x 10 ml
- V 2
- a separate set of control tubes must be set up for each cell line assayed (colon, L cell, prostate) . Dispense the upper layer stock media into each tube, then add appropriate amounts of BRM, ABIFN, cell line and serum or patient whole blood. See Table 2 as follows:
- RULE 26 Add all components except agar to the appropriate tube. For the following, one tube should be finished before moving on to the next. With a 2.0 ml pipet, draw up 0.6 ml of 1.5% agar. Dispense the agar into the tubes one at a time, starting with tube #1. Mix by aspirating and dispensing twice. Draw up 2.2 ml of solution and dispense 1.0 ml into each gridded petri plate (on top of the other layer) taking care not to produce bubbles in the agar. Gently swirl plate to allow for even distribution. Allow agar to solidify, and then add distilled water (approximately 0.5 ml) to humidity plate and place in large labeled petri plate. Repeat steps 5-7 for each plate to be set up. Incubate the cultures at 37°C in a humid, 5% C0 2 -enriched atmosphere. REPORTING RESULTS: Reading semi-solid culture plates:
- Interferons are cytokines that have the ability to inhibit the growth of viruses and to protect infected cells against viral cytopathic effects.
- the immunoregulatory functions of " interferons such as the increase in natural killer (NK) lymphocyte activity, the increase in histocompatibility antigens, the activation of monocyte/macrophages, and B-cell function have also proven to be of clinical importance.
- the first natural interferon was discovered by Isaac and Linderman in 1975, and recombinant interferon alpha 2 was registered by the FDA in 1986 ushering in a new phase of biotherapy.
- the goal of this assay is to determine the level of interferon in international units/ml in patient's serum. Potency of the human interferon of serum samples and controls will be determined using WISH cells challenged with Vesicular Stomatitis Virus measuring the cytopathic effect.
- Specimens are labeled with the following information: 1) Patient name; 2) Date drawn; 3) Time drawn; 4) Phlebotomist's initials; 5) Test name. Serum - Amount required:
- the specimen must be aseptically collected in a "tiger" top vacutainer tube, allowed to clot, then centrifuged at room temperature for 10 minutes at 2000 rpm.
- the serum should be poured over into a sterile plastic tube, capped, appropriately labeled, and stored frozen until the assay is performed.
- Plasmapheresis - Amount required: Optimum 3.0 ml
- the specimen must be collected using sterile technique into a 10 ml red top vacutainer tube from the plasma bag obtained during the plasmapheresis procedure. Add 2500 units of calciparine to the 10 ml red top vacutainer tube containing the plasmapheresis sample and mix well. In addition to the information required above, the specimen should be labeled with the bag number from which it was obtained, i.e. #1, #2, etc.. The specimen must be poured over into a sterile plastic tube, capped, appropriately labeled, and stored frozen until the assay is performed. EQUIPMENT:
- Biohazardous waste can and bags Stainless steel pan with cover MATERIALS The following materials are needed for this procedure: Basal Medium Eagle IX (BME) , 500 ml Gibco Cat. No. 320-1010AJ
- Fetal Bovine Serum 100 ml Hyclone Cat. No. A-llll-D L-Glutamine, Cat. No. 320-5030AG
- Penicillin/Streptomycin WISH cells Human Amnion ATCC 25-CCL Vesicular Stomatitis Virus ATCC
- VSV Vesicular Stomatitis Virus
- C02 incubator Add 15 ml of the BME with 2% FBS to each flask and incubate at 37°C in a 5% C02 incubator for 1-3 days or until cell layers show nearly complete viral cytopathic effect (CPE) .
- CPE viral cytopathic effect
- Dye by adding 500 mg of red dye powder to 500 ml of distilled water. Mix with magnetic stir bar at room temperature for 1 hour. Autoclave for 10 minutes at 15 psi. Cool. Label and store stock Neutral Red Dye in a brown bottle and refrigerate. Staining Solution - prepare a 15% staining solution by adding 85 ml PBS with Ca + + and MG + + (Ph 6.85) to 15 ml stock Neutral Red Dye. Eluting Solution:
- VxCx V 2 C 2
- V 2 229 ml
- BME BME
- FBS FBS
- 189 ml of BME with 10% FBS to the original volume of 40 ml to obtain 3.5 x 10 5 cells/ml.
- Set aside diluted WISH cells until ready to be added to the microtiter plates.
- Bl and Cl of plate #1 each sample will be run in duplicate
- add 40 ul of the reference To well positions Dl and El add 40 ul of patient serum.
- To each successive pair of wells add 40 ul of the appropriate patient serum.
- Reading per plate - 1 Number of plates - 9 Filename - date (i.e., 0722) Remove cover of microtiter plate #1 and place in the reader. Select Start. After all the plates have been read, print the raw data reports as follows: Under file, select Open and Lotus Results. Arrow down to filename, or type in the file name and plate number. Select Open. If message appears "Save current results...," say NO. When Raw Data Report appears on the screen, under File, select Print. Select Raw Data Report and OK. Under file select Close. Repeat for each plate to be printed. RESULTS:
- the correlation coefficient (r) should be -1.0 + 0.1. 3. Enter O.D. 50%, press 2nd, then y'
- HTCA Human Tumor Colony Assay
- HTCA Using HTCA, the growth and chemosensitivity of clonogenic tumor cells present in fresh biopsy specimens of human tumors can be investigated. Since this technique was first described, there has been a marked increase in the direct study of human tumors in vi tro. Excellent evidence has been obtained which establishes that colonies grown in HTCA are comprised of tumor cells and that clonogenic cells within tumor colonies have the property of self- renewal (the defining property of a tumor stem cell) . Chemosensitivity testing with specific agents in HTCA has documented striking degrees of heterogeneity in drug sensitivity from patient to patient, even for tumors of the same histopathology. Clinical correlations have been made between in vi tro chemosensitivity and the response of patients with metastatic cancer to chemotherapy.
- HTCA has had a 71% true-positive rate and a 91- true-positive rate for predicting the drug sensitivity and resistance, respectively, of cancer patients to specific chemotherapeutic agents.
- the assay thus appears to be a prognostic factor which identifies chemosensitive patients and which may allow some individualization of chemotherapy.
- Tumor specimens either from solid tumor masses, malignant ascites, or bone marrow are mechanically disaggregated into a suspension which is as close to a "single-cell suspension- as possible.
- These cells along with chemotherapeutic agents, biological response modifiers, and hormones are suspended in an agar-containing culture medium and then layered or "plated” onto a semi-solid underlayer.
- the underlayer prevents normal human fibroblasts, a major tumor stromal component, from adhering to the culture dish bottom and forming colonies which might be confused with colonies of tumor-cell origin. After 7 days, 14 days, and 21 days of incubation in a humid, C02-enriched atmosphere at 37°C, colonies (greater than 20 cells) and aggregates (4-20 cells) are counted. A negative control and a positive control are set up for each tumor. From this data the effect of each specific drug on the tumor can be determined.
- This assay may be performed on the following specimen types:
- the sample should be sufficient to yield an optimum of 20 ml of 1.0 X 10 6 viable cells/ml. A minimum of 12 ml of 1.0 X 10 6 viable cells/ml is acceptable. All specimens should be labeled with patient name, collection date, tumor type, and initials of technician.
- Disposable Pipettes (sterile) (l ml; 2 ml; 5 ml; 10 ml, 25 ml) Select-A-Pette Pipetter (1 ml)
- Fetal Bovine Serum Test Tubes (12x75 Glass-disposable; 15 ml
- Petri Dishes (sterile) (Routine-100 x 15 mm; Gridded Plates-35 X 10 mm)
- Vortex Mixer Refrigerator (2 - 10°C)
- Thermometer (-20°C - 120°C) Beaker (500 ml)
- Lower layer 2.5% working solution: Measure out 2.5 gm of Bacto-Agar and place in a 100 ml capped bottle. Add 100 ml distilled water. Swirl gently to mix.
- Upper layer 1.5% working solution: Measure out 1.50 gm of Bacto-Agar and place in a 100 ml capped bottle. Add 100 ml distilled water. Swirl gently to mix.
- the agar should cool down to 50°C before use. Swirl agar to mix well before using. After preparation of each media, label the final product according to procedures previously described.
- Solid tumor In a sterile petri dish, using sterile scalpel and forceps, cut the tumor specimen into pea-size portions. Save the original transport media for centrifugation. Place the specimen in a glass conical tube that has a matched loose-fitting homogenizer. Add 5.0 ml of transport media. Gently homogenize the tumor pieces a small portion at a time and transfer the cell suspension to a sterile 50 ml conical tube. When all of the tumor has been homogenized, mix well and then allow cell suspension to settle, undisturbed, for one minute (this will allow clumps of tissue to settle out) .
- Liquid tumor (ascites, bone marrow, etc.) : Using a sterile pipet, dispense 20-25 ml of histopaque into the appropriate number of capped sterile 50 ml conical tubes. Using another sterile pipet, gently layer the ascites, bone marrow or cell suspension at a 45°C angle onto the histopaque using a 1:1 ratio of histopaque to specimen.
- V j 10 ml
- V 2 ?
- C 1 2 X 10 6 cells/ml
- C 2 1 X 10 6 cells/ml
- Ten ml of media are needed to have a volume of 1 X 10 6 cells/ml.
- Dried seeds are finely ground, and 5 grams (5g) of the powder is extracted with 95% ethanol 3 times, by soxhlet extraction.
- a Wheaton soxhlet extraction apparatus consists of 3 parts: the bottom part is the flask in which the 95% ethanol is heated; the middle portion is the extractor; and the top the Allihn condenser, in which there is an inlet and outlet for running cold water through the condenser.
- Dried N. sativa seed powder is packed in filter paper and placed in the extractor.
- the pooled extracts are evaporated under reduced pressure to a known volume of 10 ml and loaded onto a silica gel (Keiselgel 60: Fluka) column and eluted with 95% methanol/water (9:1).
- silica gel suspended in methanol/water is layered on sand.
- N. sativa extract is added on top of the silica gel, followed by the eluting solvent.
- the active fraction (as indicated by brown color) is collected and separated by preparing thin layer chromatography (TLC) .
- TLC thin layer chromatography
- the extract is then available as a soluble solution and is then stored at 2°C.
- Bone marrow sufficient to yield an optimum of 5.0 ml mononuclear cells at a concentration of l.OxlO 6 cells/ml was collected.
- the assay was performed according to a modified process Metcalf (1984, 1985) , which is incorporated herein by reference. The process is described in detail above in Procedure 2. To each 25 cm 2 flask, lxl0 6 bone marrow cells are added, and incubated at 37°C with 5% C0 2 and 100 ⁇ l of N. sativa plant extract.
- the assay was conducted according to processes disclosed in and partially modified from Karen (1989) and Merkel et al . (1987), which are incorporated herein by reference. The process is described in detail above in Procedure 1.
- the N. sativa plant extract was incubated with peripheral blood cells from cancer patients over an 18 hour period at 37°C in a 5% C0 2 incubator.
- Gates for the antibodies CD3, CD19, HLADR, LAK CD3+/CD56+, NK CD3- /CD56+, CD38, CD37, and CD33 surface markers were set on a Becton Dickinson FacScan serial No. 81326. These antibodies are designed to assist in the characteristics of the types of cells.
- the cluster designation is used to group two or more mABs that have statistically similar expression on normal -and neoplastic cells and cell lines, as well as similar recognition of the same antigen or binding site (epitope) . While mABs in a CD group usually define different epitopes on the same antigen, some CD groups, primarily non-lymphoid clusters, contain mABs that do not bind to the same antigen.
- each flask was scanned under a Reichart Jung Biostar inverted microscope.
- a colony is defined as 40 or more cells adhering to the bottom of the flask.
- IL-2 and GM-CSF gave the maximum elevation of 120 and 115% after incubation with bone marrow cells for 15 days. There was not much detection of CFU elevation post 21-day incubation with growth factor/biological response modifiers. Interferon showed marginal elevation after a 15-day incubation, but negligible detectable response after 21 days.
- N. sativa plant extract helps restore the immune competent cells in immunosupressed cancer patients.
- the colony forming cell unit assay indicates increase in CFU count when bone marrow cells from cancer patients were incubated with N. sativa plant extract as well as with different growth factors indicating that N. sativa plant extracts mimic the potential application of these several growth factors. Even though the plant extract was used without dissolving, it had no toxic effects against human bone marrow cells. Hence, N. sativa plant extract helps restore the immune competent cells in immunosupressed cancer patients and over- stimulates bone marrow in normal individuals.
- HLADR HLADR
- NK CD3-/CD56+ and CD38 population There was not much alteration in CD3, CD37 and CD33 surface marker index.
- a proliferation of connective tissue 'L' cells was observed upon incubation with cancer patient serum and N. sativa extract at 37° centigrade in 5% C0 2 incubator for 7 days.
- the mouse connective tissue L929 cells were obtained from the ATCC and grown in MEM medium (GIBCO) with 20% fetal equine serum (BioWhittaker) as described above.
- the assay was conducted according to the process described in Medenica, et al . (1990), which is incorporated herein by reference. Proliferation of 5xl0 5 'L' cells/ml was checked on a double layer agar procedure in which lower 1.5% agar layer media contained 125 ml FBS, 125 ml 2X G.G. fortified solution and 250 ml IX G.G. fortified solution.
- the upper layer stock media contained 1 50 ml FBS, 150 ml 2X G.G. fortified solution and 150 ml IX G.G. fortified solution. To 2.0 ml of upper layer, 0.1 ml of N.
- Connective tissue 'L' cells at a concentration of 5x10 s were incubated with IFN, IL-2 or plant extract at 37°C in a humid 5% C0 2 incubator for 7 days.
- the SD of the data was ⁇ 10%.
- Immunomodulatory evaluation indicates enhanced proliferation of the connective tissue 'L' cells in presence of N. sativa extract than in its absence, thereby indicating positive immunomodulatory effect of the extract. Similar results were also obtained with patient white blood cells.
- IIF interferon inhibitor factor
- LIF lymphokine inhibitor factor
- N. sativa has potential use in connective tissue diseases such as rheumatoid arthritis, lupus and autoimmune diseases.
- WISH human amnion or "WISH" cells were obtained from the American Type Cell Culture facility (ATCC) and grown in Basal Eagle Medium (BEM) (GIBCO) with 15% fetal bovine serum.
- BEM Basal Eagle Medium
- VSV Vesicular stomatitis virus
- FBS fetal bovine serum
- Assay The assay was conducted as described above in Procedure 4. For determining the amount of protection conferred to the "WISH" cells by N. sativa plant extract, complete viral cytopathic effect (CPE) was calculated by inoculating 8-10 ml per flask of the 1:1000 VSV into each WISH cell flask (containing 3.5xl0 5 cells/ml) such that it covered the entire cell layer. The flasks were incubated for 1 hour at 37°C in a 5% CO, incubator.
- CPE viral cytopathic effect
- N. sativa plant extract was serially diluted in a 96-well polystyrene plate. To 40 ⁇ l of the extract was added 100 ⁇ l of the 3.5xl0 5 cells/ml "WISH" cell suspension and the plates were incubated for approximately 18-24 hours at 37°C at 5% C0 2 . In the control wells, no extract was added. Next day (after 18-24 hours) the plates were observed under Nikon TMS microscope for confluency. Fifty ⁇ l VSV were added (in a concentration to yield 100% CPE) , and Incubated 24-48 hours at 37°C with 5% C0 2 .
- the plates were observed under a Nikon TMS microscope for 100% CPE in the viral control wells. Once satisfied, the plates were dumped and blotted. One hundred ⁇ l of 15% neutral red dye were added to all wells. The wells were incubated at 37°C in 5% C0 2 incubator for 45 minutes. The plates were dumped, blotted and rinsed with 200 ⁇ l PBS at pH 6.85. The plates were again dumped and blotted. The cells were eluted by adding 100 ⁇ l of the eluting solution to each well. The plates were read on Bio-Rad Model 3550. microplate reader.
- VSV vesicular stomatitis virus
- CPE cytopathic effect
- results depicted in Figure 2 indicate protection of "WISH" cells in the order of 25 -and 65% upon incubation with 1:10 and 1:100 diluted N. sativa plant extract. There was no noticeable cytopathic effect when N. sativa was added at a dilution of 1:1000. Approximate active compound concentrations were calculated according to materials and methods. Serum interferon levels were measured after plant extract administration according a modified procedure of Finter (1969) . Protection of Human Amniotic "WISH" cells from cytopathic effects of vesicular stomatitis virus (VSV) was observed upon administration of 1:1000 diluted N. sativa plant extract. This had an active compound concentration of 88 ng.
- VSV vesicular stomatitis virus
- N. sativa will be useful in the dosage of 20-40g to protect against viral attack in virus endemic areas.
- the serum interferon level is found to increase and hence N. sativa has interferon-like antiviral activity.
- the extract possibly blocks all receptor sites on "WISH" cells which are essential for attachment of the virus to cell membrane.
- the tumor specimens can be of 2 types: solid tumor and liquid tumor.
- Solid tumor 2 grams of tumor specimen were excised, cut into pea size portions and gently homogenized in a Potter-Elveh am homogenizer with 10% FBS, centrifuged at 1600 rpm for 20 minutes in a Mistral 3000i refrigerated centrifuge at 4°C. Cells were finally suspended in media RPMI 1640 with 10% FBS into a fine suspension.
- Liquid tumor The specimen was collected from the ascitic fluid of cancer patients. The ascitic fluid was gently layered on a histopaque using a 1:1 ratio of the histopaque to specimen, and centrifuged at 1600 rpm for 20 minutes in a Mistral 3000i refrigerated centrifuge at 4°C. Cells were finally suspended in RPMI with 10% FBS. Assay: The assay was performed according to the modified processes of Von Hoff, et al . (1981) and Salmon, et al . (1978), which are incorporated herein by reference, as described above in Procedure 5. Tumoricidal activity of N. sativa was checked on a double agar layer.
- the lower layer of warm 2.5% Bacto-agar working solution was poured into 35 mm grid petri dishes. The plates were then allowed to sit undisturbed on a flat surface until the agar solidified.
- Tumor specimens (2 grams) either from solid tumor masses or malignant ascites were mechanically homogenized into a suspension containing 8.2xl0 4 cells. Incubation of tumor cells at 37°C in a humid 5% C0 2 incubator for 7, 14 and 21 days with N. sativa plant extract indicated a percent inhibition of 35, 46 and 48, respectively. Incubation with Abrin in an ideally similar condition did not exhibit any noticeable inhibition.
- N. sativa promotes anti-tumor activity.
- Data from pharmacosensitivity screening indicates anti-tumor activity of N. sativa plant extract, as cell growth in culture was inhibited anywhere between 25-90%.
- the extract indicates anti-tumor function mainly against melanoma and colon cancer types.
- N. sativa plant extract destroys tumor cells and leaves normal cells alone, possibly because of its ability to bind to cell surface asialofeutin (lectin) in diseased cells, which causes aggregation and clumping of tumor cells. It also blocks enzymes and inappropriate gene products involved in nucleic acid synthesis and metabolism. It is understood that the invention is not confined to the particular construction and arrangement herein described, but embraces such modified forms thereof as come within the scope of the claims following the bibliographic citations.
- Kirtikar K.R. and Basu, B.D., 1982, Indian Medicinal Plants, Vol. I. Bishen Singh and Mahendra Pal Singh, eds., Dehra Dun, India. Kumar, B.H. and Thakur, S.S., 1989, "Effect of Certain Non-edible Seed Oils on Growth Regulation in Disdercus Si ilis (F) , J. Anim. Morphol. Phvsiol. 36(2) pp. 209-218.
- Nadkarni K.M., 1976, " Crocus sativus, Nigella sativa, " Indian Materia Medica, K.M. Nadkarni (ed) Bombay, India; Popular Prakashan, Vol 1, pg. 386- 411. Nair, S.C.; Salomi, M.J.; Panikkar, B. ;
Abstract
Description
Claims
Priority Applications (7)
Application Number | Priority Date | Filing Date | Title |
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EP94929736A EP0804212B1 (en) | 1993-08-25 | 1994-08-25 | Nigella sativa as a medicinal treatment |
AU78687/94A AU7868794A (en) | 1993-08-25 | 1994-08-25 | (nigella sativa) as a medicinal treatment |
HU9600442A HU226319B1 (en) | 1993-08-25 | 1994-08-25 | Anticancer medicinal composition comprising nigella sativa extract and use of the active component of said extract in the manufacture of a medicament |
DE69435091T DE69435091D1 (en) | 1993-08-25 | 1994-08-25 | NIGELLA SATIVA AS MEDICAL TREATMENT |
BR9407362A BR9407362A (en) | 1993-08-25 | 1994-08-25 | Nigella sativa as a medicinal treatment |
SI9420054A SI9420054B (en) | 1993-08-25 | 1994-08-25 | Nigella sativa as a medical treatment. |
PL94313223A PL182890B1 (en) | 1993-08-25 | 1994-08-25 | Nigella sativa as a therapeutic agent |
Applications Claiming Priority (2)
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US08/111,631 US5482711A (en) | 1993-08-25 | 1993-08-25 | Use of Nigella sativa to increase immune function |
US08/111,631 | 1993-08-25 |
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WO1995005839A1 true WO1995005839A1 (en) | 1995-03-02 |
WO1995005839B1 WO1995005839B1 (en) | 1995-05-04 |
Family
ID=22339582
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PCT/US1994/009660 WO1995005839A1 (en) | 1993-08-25 | 1994-08-25 | Nigella sativa as a medicinal treatment |
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US (2) | US5482711A (en) |
EP (1) | EP0804212B1 (en) |
AT (1) | ATE392902T1 (en) |
AU (1) | AU7868794A (en) |
BR (1) | BR9407362A (en) |
CA (1) | CA2169956A1 (en) |
DE (1) | DE69435091D1 (en) |
HU (1) | HU226319B1 (en) |
OA (1) | OA10572A (en) |
PL (1) | PL182890B1 (en) |
SI (1) | SI9420054B (en) |
WO (1) | WO1995005839A1 (en) |
Cited By (5)
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BE1009579A5 (en) * | 1996-07-04 | 1997-05-06 | Ismail Mohamed Tejeddine | Vibrating system for protection against radiation interference emitted by viewing screens and/or other sources of radiation, bio-energy balancing and geo-biological harmonisation or bio-cleaning of work places and homes |
GB2347859A (en) * | 1999-03-19 | 2000-09-20 | Yasin Nazir Karim Yakub | A herbal composition comprising the seeds of Nigella sativa |
GB2348132A (en) * | 1999-03-02 | 2000-09-27 | Nassief Nida Abdul Ghani | Uses for glycophosphopeptical |
WO2004004631A3 (en) * | 2002-07-09 | 2004-03-11 | Abdo Saeed Ahmed Shifa | Natural herbal antibiotic for the treatment of the infections of the upper respiratory system and various diseases in the body |
EP1709995A1 (en) | 1999-03-02 | 2006-10-11 | Al-Jassim, Rawaa | Asthma/allergy therapy using nigella sativa |
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US6042834A (en) * | 1997-01-29 | 2000-03-28 | Baraka; Mohamed Wasif | Herbal composition for diabetes and method of treatment |
WO1998041609A1 (en) * | 1997-03-20 | 1998-09-24 | Barnes Allen C | Micropathological patient replica based on unadulterated whole blood |
KR100239879B1 (en) * | 1997-11-05 | 2000-02-01 | 김상조 | Crude drug composition for treatment and prevention of liver cancer |
US6440448B1 (en) | 1998-03-16 | 2002-08-27 | Joseph Intelisano | Food supplement/herbal composition for health enhancement |
US6231866B1 (en) | 1998-04-30 | 2001-05-15 | Douglas G. Mann | Infused vegetable, fruit, herb, and/or seed fiber product and dietary supplements containing same |
US6218434B1 (en) | 1998-05-28 | 2001-04-17 | University Of Kentucky Research Foundation | Use of the naturally-occurring quinones thymoquinone and dithymoquinone as antineoplastic and cytotoxic agents |
DE19844022C1 (en) * | 1998-09-25 | 2000-05-25 | Sherif Sabry Ragab Mekkawi | Use of iron-binding glycoproteins and/or 10-hydroxy-2-decenoic acid in combination with thymoquinone for treating immunodeficiency diseases |
RU2172322C1 (en) | 1999-12-27 | 2001-08-20 | Энтофарм Ко., Лтд. | Allopherones as immunomodulating peptides |
US6948494B1 (en) * | 2000-05-10 | 2005-09-27 | Innovative Devices, Llc. | Medicament container with same side airflow inlet and outlet and method of use |
WO2003075685A2 (en) * | 2002-03-12 | 2003-09-18 | Council Of Scientific And Industrial Research | Bioavailability/bioefficacy enhancing activity of cuminum cyminum and extracts and fractions thereof |
US7744929B2 (en) * | 2003-09-15 | 2010-06-29 | Ambotan Pharma, Llc | Botanical drug compositions for treatments of liver and immunological disorders |
US20050214393A1 (en) * | 2004-03-26 | 2005-09-29 | Osama Kandil | Lipid fraction of Nigella sativa L. seeds |
US7722906B2 (en) * | 2004-03-26 | 2010-05-25 | Biopharm Research & Development Corporation Limited | Polyunsaturated fatty acid fractions of Nigella sativa L. seeds |
US20090175970A1 (en) * | 2005-01-26 | 2009-07-09 | Veritron Limited | Stabilized Plant Extract and Its Therapeutic Use |
GB0501654D0 (en) * | 2005-01-26 | 2005-03-02 | Veritron Ltd | Stabilised plant extract |
US7910361B2 (en) | 2006-08-10 | 2011-03-22 | Barnes Allen C | Portable biological testing device and method |
FR2922109B1 (en) | 2007-10-16 | 2010-08-20 | Bruno Eto | PHARMACEUTICAL OR DIETETIC COMPOSITION FOR INHIBITING INTESTINAL ABSORPTION OF SUGAR. |
CN109776669A (en) * | 2018-12-29 | 2019-05-21 | 广州动物园 | Brave interferon and its expressing gene, preparation method and application |
US11260097B2 (en) | 2020-07-06 | 2022-03-01 | COVImmune Pharma LLC | Immunomodulatory composition to treat and/or prevent COVID-19 illness |
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US4945115A (en) * | 1985-05-09 | 1990-07-31 | Yaguang Liu | Process for preparing ferulic acid |
US4687761A (en) * | 1985-05-09 | 1987-08-18 | Yaguang Liu | Pharmaceutical composition for increasing immunity and decreasing side effects of anticancer chemotherapy |
-
1993
- 1993-08-25 US US08/111,631 patent/US5482711A/en not_active Expired - Lifetime
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1994
- 1994-08-25 SI SI9420054A patent/SI9420054B/en not_active IP Right Cessation
- 1994-08-25 DE DE69435091T patent/DE69435091D1/en not_active Expired - Lifetime
- 1994-08-25 AU AU78687/94A patent/AU7868794A/en not_active Abandoned
- 1994-08-25 HU HU9600442A patent/HU226319B1/en not_active IP Right Cessation
- 1994-08-25 EP EP94929736A patent/EP0804212B1/en not_active Expired - Lifetime
- 1994-08-25 CA CA002169956A patent/CA2169956A1/en not_active Abandoned
- 1994-08-25 AT AT94929736T patent/ATE392902T1/en not_active IP Right Cessation
- 1994-08-25 WO PCT/US1994/009660 patent/WO1995005839A1/en active Application Filing
- 1994-08-25 BR BR9407362A patent/BR9407362A/en not_active Application Discontinuation
- 1994-08-25 PL PL94313223A patent/PL182890B1/en not_active IP Right Cessation
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- 1995-05-03 US US08/433,603 patent/US5653981A/en not_active Expired - Lifetime
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1996
- 1996-02-23 OA OA60779A patent/OA10572A/en unknown
Non-Patent Citations (3)
Title |
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AHMED ELKADI ET AL.: "NIGELLA SATIVA AND CELL-MEDIATED IMMUNITY", ARCHIVES OF AIDS RESEARCH, vol. 1, no. 2&3, 1987, pages 232 - 233 * |
MEDENICA, R. ET AL.: "IMMUNOMODULATORY AND ANTI-CANCER ACTIVITY OF NIGELLA SATIVA PLANT EXTRACT IN HUMANS.", PROCEEDINGS 85TH ANNUAL MEETING OF THE AMERICAN ASSOCIATION FOR CANCER RESEARCH, vol. 35, March 1994 (1994-03-01), pages 481 * |
N.J. SALOMI ET AL.: "ANTITUMOUR PRINCIPLES FROM NIGELLA SATIVA SEEDS", CANCER LETTERS, vol. 63, no. 1, 31 March 1992 (1992-03-31), pages 41 - 46 * |
Cited By (7)
Publication number | Priority date | Publication date | Assignee | Title |
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BE1009579A5 (en) * | 1996-07-04 | 1997-05-06 | Ismail Mohamed Tejeddine | Vibrating system for protection against radiation interference emitted by viewing screens and/or other sources of radiation, bio-energy balancing and geo-biological harmonisation or bio-cleaning of work places and homes |
GB2348132A (en) * | 1999-03-02 | 2000-09-27 | Nassief Nida Abdul Ghani | Uses for glycophosphopeptical |
GB2348132B (en) * | 1999-03-02 | 2004-08-04 | Nedaa Abdul-Ghani Nasif | Asthma/allergy therapy that targets t-lymphocytes and/or eosinophils |
EP1709995A1 (en) | 1999-03-02 | 2006-10-11 | Al-Jassim, Rawaa | Asthma/allergy therapy using nigella sativa |
GB2347859A (en) * | 1999-03-19 | 2000-09-20 | Yasin Nazir Karim Yakub | A herbal composition comprising the seeds of Nigella sativa |
GB2347859B (en) * | 1999-03-19 | 2004-02-25 | Yasin Nazir Karim Yakub | Herbal compositions |
WO2004004631A3 (en) * | 2002-07-09 | 2004-03-11 | Abdo Saeed Ahmed Shifa | Natural herbal antibiotic for the treatment of the infections of the upper respiratory system and various diseases in the body |
Also Published As
Publication number | Publication date |
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BR9407362A (en) | 1996-04-23 |
US5482711A (en) | 1996-01-09 |
EP0804212B1 (en) | 2008-04-23 |
US5653981A (en) | 1997-08-05 |
SI9420054B (en) | 2008-10-31 |
EP0804212A2 (en) | 1997-11-05 |
SI9420054A (en) | 1997-02-28 |
PL313223A1 (en) | 1996-06-10 |
HU226319B1 (en) | 2008-08-28 |
HUT74226A (en) | 1996-11-28 |
OA10572A (en) | 2002-06-03 |
AU7868794A (en) | 1995-03-21 |
PL182890B1 (en) | 2002-03-29 |
CA2169956A1 (en) | 1995-02-26 |
ATE392902T1 (en) | 2008-05-15 |
DE69435091D1 (en) | 2008-06-05 |
HU9600442D0 (en) | 1996-04-29 |
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