ES2426174T3 - Herbaceous composition for inflammatory disorders - Google Patents

Abstract

A pharmaceutical composition for use in the treatment of an inflammatory disorder mediated by TNF-a, interleukins (IL-1, IL-6, IL-8) and intercellular adhesion molecule 1 (ICAM-1), adhesion molecule 1 vascular cell (VCAM-1) and E-Selectin, wherein said composition comprises, as an active ingredient, a therapeutically effective amount of an extract of flora and fruit chapters of the Sphaeranthus indicus plant, together with pharmaceutically acceptable carriers.

Description

Herbaceous composition for inflammatory disorders

5 Field of the invention

[001] The present disclosure refers to an herbaceous composition comprising an extract of flora and fruit chapters of a plant, Sphaeranthus indicus. Additionally it refers to a herbaceous composition containing an extract obtained from the flora and fruit chapters of Sphaeranthus indicus, which as a bioactive marker comprises a compound, 3a-hydroxy-5a, 9-dimethyl-3-methylene-3a, 4,5 , 5a, 6,7,8,9b-octahydro-3Hnapfto [1,2-b] furan-2-one (7-hydroxy-4,11 (13) -edudesmadien-12,6-olida) (compound 1) and optionally another active (or active) for the effective treatment of inflammatory disorders. The present disclosure also refers to a pharmaceutical composition which as an active ingredient comprises 3a-hydroxy-5a, 9-dimethyl-3-methylene3a, 4,5,5a, 6,7,8,9b-octahydro-3H-naphtho [ 1,2-b] furan-2-one (compound 1) and pharmaceutically acceptable carriers,

15 for use in the treatment of inflammatory disorders. It also refers to a method of manufacturing the compositions. The compositions of the present invention are adapted for the treatment of inflammatory disorders. The invention also relates to the inhibitory activity of tumor necrosis factor-α (TNF-α) and interleukin (IL-1, IL-6, IL-8) of the compositions. The present invention further relates to the inhibition of the expression of intracellular adhesion molecule 1 (ICAM-1), cell-vascular adhesion molecule 1 (VCAM1) and E-Selectin by the -compositions.

The invention also discloses medical uses of the compositions for the treatment of inflammatory disorders. Optionally, the extract or composition comprising compound 1 can be used in combination with at least any other anti-inflammatory agent.

Background of the invention

[003] Inflammation plays a fundamental role in host defenses and in the development of immune-mediated disease. The inflammatory response begins in response to lesions (for example trauma, ischemia and foreign particles) and infection (for example bacterial or viral infection) by multiple events, including chemical mediators (for example cytokines and prostaglandins) and inflammatory cells (for example leukocytes ). It is characterized by increased blood flow in the tissues, which produces pyrexia, erythema, hardening and pain. A delicate, well-balanced interaction between humoral and cellular immune elements in the inflammatory response allows the elimination of harmful agents and the beginning of the restoration of the

35 damaged tissue. When this delicate balanced interaction is altered, the inflammatory response can cause considerable damage to normal tissues and may be more harmful than the original aggression that initiates the reaction. In these cases of uncontrolled inflammatory responses, clinical intervention is needed to prevent tissue injury and organ dysfunction. Diseases such as rheumatoid arthritis, osteoarthritis, Crohn's disease, asthma, allergies, septic shock syndrome, atherosclerosis, inflammatory bowel disease, among other clinical conditions, are characterized by chronic inflammation.

[004] Cytokines, especially IL-1β, IL-6, IL-8 and TNF-α, play an important role in the inflammatory process.

[005] Macrophages mainly produce TNF-α, a pleiotropic cytokine, but other types of cells can also produce it. TNF-α demonstrates beneficial as well as pathological activities. It has both growth stimulating and growth inhibiting properties, as well as being self-regulating. The beneficial functions of TNF-α include the maintenance of homeostasis regulating the circadian rhythm of the organism, generating an immune response against bacterial, viral, fungal and parasitic infections, replacing or remodeling injured tissue stimulating fibroblast growth and as the name suggests , destroying certain tumors.

[006] Although TNF-α plays a critical role in innate and acquired immune responses, inappropriate production of TNF-α can cause pathological changes that result in inflammation.

55 chronic and tissue damage. TNF-α has been shown to play a crucial role in the pathogenesis of many chronic inflammatory diseases such as inflammatory bowel disease, rheumatoid arthritis, juvenile rheumatoid arthritis, psoriatic arthritis, osteoarthritis, refractory rheumatoid arthritis, chronic non-rheumatoid arthritis, osteoporosis, resorption Bone, coronary heart disease, vasculitis, ulcerative colitis, psoriasis, adult respiratory distress syndrome, diabetes, delayed skin hypersensitivity disorders and Alzheimer's disease. Interleukin-1 (IL-1) is an important part of the innate immune system, which regulates functions of the immunoadaptive system. The balance between IL-1 and IL-1 receptor antagonists (IL-1ra) in local tissues influences the possible development of an inflammatory disease and resulting structural damage. In the presence of an excess amount of IL-1, inflammatory and immune disorders can develop in joints, lungs, gastrointestinal tract, central nervous system (CNS) or blood vessels.

65 [007] Among the various inflammatory disorders, rheumatoid arthritis (RA) is an autoimmune disorder. RA is a chronic, systemic, articular inflammatory disease of unknown etiology. In RA, the normally thin synovial wall of the joints is replaced by an invasive, inflammatory, highly vascularized fibrocolagenase tissue (cloth), which destroys both cartilage and bone. The areas that may be affected

5 include the joints of the hands, wrists, neck, jaw, elbows, knee, feet and ankles. The destruction of cartilage in RA is related to aberrant cytokine expression and growth factors in the affected joints.

[008] Two clinically important cytokines released in the synovial membrane are IL-1β and TNF-α. TNF-α

10 can positively regulate its own expression as well as facilitate the expression of other genes involved in RA, including IL-1β, IL-6, IL-8, cyclooxygenase-2 (COX-2), inducible nitric oxide synthetase (iNOS), intracellular adhesion molecule 1 (ICAM-1), vascular-cell adhesion molecule 1 (VCAM-1) and E-Selectin. This type of positive regulatory loop can amplify and perpetuate local inflammatory responses. Therefore, inappropriate overexpression or expression of TNF-α leads to a coordinated increase in the expression of many genes whose

15 products mediate inflammatory and immune responses and therefore lead to clinical manifestations of RA.

[009] The accumulation and retention of leukocytes is a critical event in the pathogenesis of all chronic inflammatory disorders including RA. In addition, the adhesion of circulating leukocytes, especially monocytes, in the vascular endothelium is also a crucial event in the development of atherosclerosis. This process depends on the interaction between adhesion molecules expressed on the surface of endothelial cells such as ICAM-1, VCAM-1 and E-Selectin and their related leukocyte ligands. Therefore, ICAM-1, VCAM-1 and E-Selectin are responsible for the accumulation of inflammatory cells, such as neutrophils, eosinophils and T lymphocytes, from circulation to the site of inflammation. These adhesion proteins are normally

25 at low level on the surface of endothelial cells but they are greatly induced by various proinflammatory cytokines such as TNF-α.

[0010] The most common therapy for the treatment of inflammatory disorders involves the use of non-steroidal anti-inflammatory drugs (NSAIDs) for example naproxen, diclofenac, ibuprofen to relieve such symptoms.

30 as pain. However, despite the extensive use of NSAIDs, many individuals cannot tolerate the doses necessary to treat the disorder for a prolonged period of time since it is known that NSAIDs produce gastric ulcers. In addition, NSAIDs only treat the symptoms of the disorder and not the cause.

[0011] When patients do not respond to NSAIDs, other drugs such as methotrexate, gold salts, D-penicillamine and corticosteroids are used. These drugs also have significant toxic effects.

[0012] Monoclonal antibody drugs such as infliximab, etanercept and adalimumab are useful as anti-inflammatory agents, but they have drawbacks such as the route of administration (parenteral only), high cost, induction of allergies, latent tuberculosis activation, increased risk of cancer and heart disease

40 congestive

[0013] Therefore, there is a need for the development of improved and alternative medications that reduce the side effects for the prevention and treatment of inflammatory disorders caused by an increase in IL-1 and TNF-α.

[0014] Herbaceous plants are known and used throughout the world for the treatment of many conditions. It is obvious that plant-derived products have possible pharmacological and therapeutic effects in mammals and tend to have less harmful side effects compared to synthetic drugs.

[0015] The present invention describes a new herbaceous composition, comprising extracts from flora and fruit chapters of the Sphaeranthus indicus plant. The composition can be used for the treatment of various inflammatory disorders with minimal side effects.

[0016] Sphaeranthus indicus is a common weed found in rice fields. It belongs to the family

55 Asteraceae and in the Ayurveda bibliography, it is known as mahamundi or gorakhmundi. The plant, available in India, is a branched herbaceous plant with purple flowers. It is used in liver and gastric disorders. It is used in traditional medicine as a remedy for various ailments including dysentery, pain in the uterus and vagina, thoracic diseases, purification and enrichment of blood, urinary tract infections, wound healing and various other diseases.

[0017] In order to counteract immunodeficient disorders (Indian Journal of Pharmacology, 33, 454-55, (2001)) a poly-herbaceous formulation "RV-08" has been developed, containing Sphaeranthus indicus.

[0018] The isolation of a new sesquiterpene glycoside, sphaeranthanolide, from 65 flowers of Sphaeranthus indicus has been described. The isolated compound, Sphaeranthanolide, has immunostimulatory activity. (Phytochemistry, 29, 2573-76, (1990)).

[0019] The immunomodulatory activity of methanol extract from the floral chapters of Sphaeranthus indicus has been described (Ars Pharmaceutica 45: 3; 281-91, (2004)).

[0020] The aqueous extract obtained from the roots of Sphaeranthus indicus is indicated to be moderately active in the negative regulation of the production of TNF-α and IL-8 induced in Propionibacterium acnes. Sphaeranthus indicus produces a smaller, however significant, suppression of reactive oxygen species (Phytomedicine, 10 (1), 34-38, (2003)).

[0021] As far as is known, there are no descriptions of any medicament containing extract of flora and fruit chapters of Sphaeranthus indicus for the treatment of inflammatory disorders. To overcome the problems of side effects of the current treatment line, such as induction of allergy, activation of latent tuberculosis, myelosuppression, increased risk of cancer and congestive heart disease, associated with

15 compositions of the prior art, the authors of the present invention have prepared a new herbaceous composition effective against inflammation, which has inhibitory activity against TNF-α, interleukins (IL-1, IL-6, IL-8) and expression of intercellular adhesion molecule 1 (ICAM-1), vascular-cell adhesion molecule 1 (VCAM-1) and E-Selectin. The compositions of the present invention can also be used in combination with at least one other anti-inflammatory agent.

Objects of the invention

[0022] An object of the present disclosure relates to providing a herbaceous composition comprising, as an active ingredient, a therapeutically effective amount of an extract of floral and fruiting chapters of

25 Sphaeranthus indicus together with pharmaceutically acceptable vehicles.

[0023] Another aspect is to provide a composition comprising, as active ingredient, a therapeutically effective amount of 3a-hydroxy-5a, 9-dimethyl-3-methylene-3a, 4,5,5a, 6,7,8,9b -octahydro-3H-naphtho [1,2-b] furan-2-one (compound 1) together with pharmaceutically acceptable carriers, for the treatment of inflammatory disorders.

[0024] Another additional aspect of the present disclosure is to provide a method of manufacturing the compositions.

[0025] An object of the present invention is to provide a composition comprising an amount

Therapeutically effective of the active ingredient selected from any extract of Sphaeranthus indicus or compound 1 for use in the treatment of disorders mediated by TNF-α and interleukins (IL-1, IL-6, IL-8).

[0026] Yet a further object of the present invention is to provide a composition comprising a therapeutically effective amount of the active ingredient selected from any extract of Sphaeranthus indicus

or compound 1, for use in the treatment of disorders mediated by intercellular adhesion molecule 1 (ICAM-1), vascular-cell adhesion molecule 1 (VCAM-1) and E-Selectin.

[0027] Another object of the present disclosure is to provide a composition comprising a therapeutically effective amount of the active ingredient selected from any extract of Sphaeranthus indicus or the

45 compound 1 for use in the treatment of inflammatory disorders.

[0028] Still another object of the present disclosure is to provide a composition comprising a therapeutically effective amount of the active ingredient selected from any extract of Sphaeranthus indicus or compound 1, to treat inflammatory disorders mediated by TNF-α.

[0029] Still another object of the present disclosure is to provide a composition comprising an effective amount of the active ingredient selected from any extract of Sphaeranthus indicus or compound 1, to treat inflammatory disorders mediated by interleukins (IL-1, IL-6, IL-8).

[0030] Still another object of the present invention is to provide a composition comprising a therapeutically effective amount of the active ingredient selected from any extract of Sphaeranthus indicus or compound 1, to treat inflammatory disorders mediated by intercellular adhesion molecule 1 (ICAM) -1), vascular cell adhesion molecule 1 (VCAM-1) and E-Selectin.

[0031] Still another object of the present invention is to provide a composition comprising a therapeutically effective amount of the active ingredient selected from any extract of Sphaeranthus indicus or compound 1 in combination with at least one bioactive substance to obtain a synergistic effect.

[0032] Still another object of the present invention is to provide the use of said compositions alone or in

65 combination with at least one other anti-inflammatory agent to treat inflammatory disorders, including rheumatoid arthritis.

[0033] Other additional objects and areas of applicability of the present invention will be obvious from the following detailed description.

5 Summary of the invention

[0034] Therefore, in accordance with one aspect of the present invention, a herbaceous composition is provided comprising, as an active ingredient, a therapeutically effective amount of an extract of flora and fruit chapters of Sphaeranthus indicus together with pharmaceutically acceptable carriers.

[0035] According to another aspect of the present disclosure, as an active ingredient, a composition is provided comprising a therapeutically effective amount of 3a-hydroxy-5a, 9-dimethyl-3-methylene3a, 4,5,5a, 6, 7,8,9b-octahydro-3H-naphtho [1,2-b] furan-2-one (compound 1) together with pharmaceutically acceptable carriers, for the treatment of inflammatory disorders.

[0036] In accordance with a further aspect of the present disclosure, a method of manufacturing the compositions is provided.

[0037] According to a further aspect of the present disclosure, a composition is provided comprising a therapeutically effective amount of the active ingredient selected from any extract of Sphaeranthus indicus or compound 1 for the treatment of inflammatory disorders.

[0038] In accordance with a further aspect of the present disclosure, a composition is provided comprising an amount of the active ingredient selected from any extract of Sphaeranthus indicus or the

Compound 1, to treat inflammatory disorders mediated by TNF-α.

[0039] In accordance with a further aspect of the present disclosure, a composition comprising a therapeutically effective amount of the active ingredient selected from any extract of Sphaeranthus indicus or compound 1, for treating inflammatory disorders mediated by interleukins (IL-1) is provided. , IL-6, IL8).

[0040] In accordance with a further aspect of the present disclosure, a composition is provided comprising a therapeutically effective amount of the active ingredient selected from any extract of Sphaeranthus indicus or compound 1, for treating inflammatory disorders mediated by the adhesion molecule 1

35 intercellular (ICAM-1), vascular adhesion molecule 1 (VCAM-1) and E-Selectin.

[0041] According to another additional aspect, a composition is provided comprising a therapeutically effective amount of the active ingredient selected from any extract of Sphaeranthus indicus or compound 1 in combination with at least one bioactive substance to obtain a synergistic effect. According to another additional aspect, a use of the compositions alone or in comparison with at least one other anti-inflammatory agent to treat inflammatory disorders including rheumatoid arthritis is provided.

Detailed description of the invention

[0042] Based on the description herein, a person skilled in the art can use the present invention at its highest level. The following specific embodiments should be considered as merely illustrative and in no way at all not limiting the remaining disclosure.

[0043] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as normally understood by a person skilled in the art to which the invention pertains.

[0044] The term "inflammatory disorder," as used herein, refers to a disease.

or condition characterized by chronic inflammation that includes, but is not limited to, rheumatoid arthritis, osteoarthritis,

55 juvenile rheumatoid arthritis, psoriatic arthritis, refractory rheumatoid arthritis, chronic non-rheumatoid arthritis, osteoporosis / bone resorption, coronary heart disease, atherosclerosis, vasculitis, ulcerative colitis, psoriasis, Crohn's disease, adult respiratory distress syndrome, septic shock syndrome and inflammatory bowel disease

[0045] The term "pharmaceutically acceptable", as used herein, means that the vehicle, diluent, excipients, and / or salts must be compatible with the rest of the ingredients in the formulation and not harmful to its receptor.

[0046] The term "pharmaceutically acceptable carrier", as used herein, means a

65 encapsulating material, a diluent, a semi-solid, a non-toxic solid, solid, inert or an auxiliary formulation of any kind. Some examples of materials that can serve as pharmaceutically acceptable carriers are sugars such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; malt; jelly; talcum powder; as well as other compatible non-toxic lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweeteners, flavoring agents

5 and perfumes; preservatives and antioxidants may also be present in the composition, according to the formulator's criteria.

[0047] The expression, "therapeutically effective amount", as used herein, means an amount of the compound or composition (eg, the extract of Sphaeranthus indicus) sufficient to significantly induce a positive modification in the condition to be regulated. or to treat, but low enough to prevent side effects, if any (at a reasonable benefit / risk ratio), within the scope of good medical judgment. The therapeutically effective amount of the compound or composition will vary with the particular condition to be treated, the age and physical condition of the end user, the severity of the condition to be treated / prevented, the duration of treatment, the nature of the simultaneous therapy. , the compound or composition

15 specific employees, the particular pharmaceutically acceptable vehicle used and similar factors. As used herein, all percentages are by weight unless otherwise specified.

[0048] The term "bioactive marker" is used herein to define a characteristic (or a phytochemical profile) that correlates with an acceptable degree of pharmaceutical activity.

[0049] The "maximum practicable dose" is the largest amount of a drug that an adult can safely take.

[0050] It should be noted that, as used in the specification and in the appended claims, the singular forms "a", "one", "a" and "the", "the" include plural referents, except that the context clearly indicates otherwise.

[0051] "Sphaeranthus indicus extract" or "an extract of Sphaeranthus indicus", mentioned herein, means a combination of compounds present in the Sphaeranthus indicus plant. Sayings

30 compounds are extracted from dried plant and fruiting chapters of the plant using extraction procedures well known in the art (for example, the use of organic solvents, such as lower alcohols, alkyl esters, alkyl ethers, alkyl ketones, chloroform, petroleum ether, hexane and / or inorganic solvents, such as water). The present process for the extraction of phytoconstituent derivatives from flora and fruit chapters of Sphaeranthus indicus can be gradually increased for a large-scale preparation.

[0052] The term "active ingredient", as used herein, refers to "Sphaeranthus indicus extract" or "compound 1" or "an enriched extract of Sphaeranthus indicus containing a mixture of two or more active compounds ”.

[0053] Sphaeranthus indicus extract can be standardized using conventional techniques such as High Performance Liquid Chromatography (HPLC) or High Performance Fine Layer Chromatography (HPTLC).

[0054] In one embodiment, a herbaceous composition is provided comprising a standardized extract of Sphaeranthus indicus together with pharmaceutically acceptable carriers.

[0055] From the floral and fruitful chapters of Sphaeranthus indicus, bioactive marker compounds can be isolated by column chromatographic purification guided by bioactivity and preparative HPLC. Compounds can be characterized by spectral data analysis.

[0056] The herbaceous composition comprises, as a bioactive marker, an extract of floral and fruitful chapters of Sphaeranthus indicus, which comprises 2-9% of 3a-hydroxy-5a, 9-dimethyl-3-methylene 3, 4,5, 5a, 6,7,8,9b-octahydro-3H-naphtho [1,2-b] furan-2-one (compound 1) and optionally another active (or active).

[0057] In one embodiment, the disclosure provides a composition comprising 3a-hydroxy-5a, 9-dimethyl-355 methylene-3a, 4,5,5a, 6,7,8,9b-octahydro-3H-naphtho [1 , 2-b] furan-2-one (compound 1) as an active ingredient, together with pharmaceutically acceptable carriers.

[0058] In one embodiment, the use of the composition comprising 3a-hydroxy-5a, 9-dimethyl-3-methylene-3a, 4,5,5a, 6,7,8,9b-octahydro-3H-naphtho is provided [ 1,2-b] furan-2-one (compound 1) for the manufacture of a medicament for the treatment of inflammatory disorders.

[0059] The disclosure further relates to a method of manufacturing compositions useful for the treatment of inflammatory disorders. The standardized extract of Sphaeranthus indicus is mixed with pharmaceutically acceptable carriers and formulated in therapeutic dosage forms. The dose to be administered

65 daily must be selected to meet the desired effect.

[0060] In one embodiment, the herbaceous composition comprising the standardized extract of Sphaeranthus indicus for the treatment of inflammatory disorders is provided. [0061] In another embodiment, the composition comprising 3a-hydroxy-5a, 9-dimethyl-3-methylene3a, 4,5,5a, 6,7,8,9b-octahydro-3H-naphtho [1,2] is provided -b] furan-2-one (compound 1), together with pharmaceutical vehicles

5 acceptable, for the treatment of inflammatory disorders.

[0062] Compound 1, 3a-hydroxy-5a, 9-dimethyl-3-methylene-3a, 4,5,5a, 6,7,8,9b-octahydro-3H-naphtho [1,2-b] furan -2-one, was isolated from the extract of Sphaeranthus indicus by a procedure known in the related art and was characterized by Nuclear Magnetic Resonance (NMR) and Mass spectrometry. The composition comprises the compound 1, 3a-hydroxy-5a, 9-dimethyl-3-methylene-3a, 4,5,5a, 6,7,8,9b-octahydro-3H-naphtho [1,2-b] furan -2-one, whose compound can also be obtained from other plant sources or can be manufactured by conventional synthetic methods known to one skilled in the art.

[0063] Accordingly, the present disclosure includes, within its scope, a pharmaceutical composition that

15 comprises compound 1, which can be obtained from other sources, for use in the treatment of inflammatory disorders.

[0064] In a further embodiment, a method of manufacturing a pharmaceutical composition comprising 3a-hydroxy-5a, 9-dimethyl-3-methylene-3a, 4,5,5a, 6,7,8,9b- is provided. octahydro-3H-naphtho [1,2-b] furan-2-one (compound 1) by mixing compound 1 with one or more pharmaceutically acceptable carriers and formulating in therapeutic dosage forms. The dose to be administered daily is selected to satisfy the desired effect.

[0065] The compositions of the present disclosure may be administered orally, for example, in the form of pills, tablets, coated tablets, capsules, granules, elixirs or syrup.

[0066] The extract of flora and fruit chapters of Sphaeranthus indicus is used to prepare oral preparations containing 3-70% by weight of the extract, which is carefully combined on a conventional basis as will be described in detail later in this document. . The extract of flora and fruit chapters containing, as a bioactive marker, 2-9% (w / w) of compound 1, is sufficient to achieve the desired results.

[0067] Compound 1, 3a-hydroxy-5a, 9-dimethyl-3-methylene-3a, 4,5,5a, 6,7,8,9b-octahydro-3H-naphtho [1,2-b] furan -2-one, is used to prepare oral preparations containing 3-99% by weight of the compound, which is combined

35 carefully on a conventional basis as will be described in detail later in this document.

[0068] The compositions can be used for topical and transdermal administration. Topical compositions useful in the present invention involve formulations suitable for topical application to the skin. The combinations can be formulated in a wide variety of product types that include, but are not limited to, lotions, creams, gels, bars, sprayers or ointments.

[0069] The extract of flora and fruit chapters of Sphaeranthus indicus is used to prepare topical preparations containing 1-15% by weight of the extract that is carefully combined on a conventional basis as will be described in detail later in this document. The extract of flora and fruitful chapters

Sphaeranthus indicus, which contains, as a bioactive marker, approximately 2-9% (w / w) of compound 1, is sufficient to achieve the desired results.

[0070] In one embodiment, the compositions are provided for the treatment of inflammatory disorders mediated by TNF-α and interleukins (IL-1, IL-6, IL-8).

[0071] In one embodiment the compositions are provided for the treatment of inflammatory disorders mediated by intercellular adhesion molecule 1 (ICAM-1), vascular / cell adhesion molecule 1 (VCAM-1), and E-Selectin.

[0072] Actual dosage levels of the active ingredient, "Sphaeranthus indicus extract" or compound 1 in the compositions of the present invention can be modified to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a patient, composition and particular mode of administration.

[0073] The dosage level selected will depend on various factors, including the activity of the particular active ingredient, of the "Sphaeranthus indicus extract" or "compound 1" employed, the route of administration, the time of administration, the rate of excretion. of the particular composition to be treated, the duration of treatment, the use in combination with the other extracts, the age, sex, weight, condition, general health and previous medical history of the patient to be treated and similar factors well known in The medical technique.

[0074] In another embodiment, the invention provides a composition comprising the active ingredient, "Sphaeranthus indicus extract" or compound 1, in combination with at least one other herbaceous extract that exhibits anti-inflammatory activity to obtain a synergistic effect. Such other plants can be selected from plants such as Curcuma longa and Zingiber officinale.

[0075] In another additional embodiment, the composition also comprises the active ingredient, "Sphaeranthus indicus extract" or compound 1, in combination with at least one bioactive substance to obtain a synergistic effect.

[0076] In another additional embodiment, the composition of the present invention comprising the active ingredient,

10 "Sphaeranthus indicus extract" or compound 1, may optionally contain at least any other anti-inflammatory agent or may also be used in combination with a conventional anti-inflammatory agent. The anti-inflammatory agent can be selected from steroids such as prednisolone, hydrocortisone; disease-modifying antirheumatic drugs (DMARDs), such as methotrexate, sulfasalazine; or NSAIDs, such as naproxen, diclofenac, ibuprofen and the like.

[0077] In one embodiment, the herbaceous composition comprising the active ingredient, "Sphaeranthus indicus extract" or compound 1 isolated from the Sphaeranthus indicus extract, is provided for the treatment of rheumatoid arthritis.

[0078] Another embodiment of the present disclosure also relates to the inhibitory activity of TNF-α and interleukin (IL-1, IL-6, IL-8) of the compositions comprising the active ingredient.

[0079] Another embodiment also relates to the inhibition of cell surface expression of adhesion molecules such as intercellular adhesion molecule 1 (ICAM-1), vascular-cell adhesion molecule 1 (VCAM-1) and E-Selectin by the compositions comprising the active ingredient.

[0080] The compositions of the present disclosure are suitable for use in the treatment of both acute and chronic forms of inflammatory disorders mediated by TNF-α, interleukins (IL-1, IL-6, IL-8) and ICAM-1 , VCAM-1 and E-Selectin, in particular, rheumatoid arthritis, juvenile rheumatoid arthritis, psoriatic arthritis, osteoarthritis, 30 refractory rheumatoid arthritis, chronic non-rheumatoid arthritis, osteoporosis / bone resorption, coronary heart disease, vasculitis, ulcerative colitis, psoriasis, syndrome of adult respiratory distress, Alzheimer's disease in humans. In addition, the compositions of the present invention can be used for the treatment of inflammation in diseases such as inflammatory bowel disease, Crohn's disease, septic shock syndrome, atherosclerosis and various autoimmune diseases among other clinical conditions. The present disclosure

35 also refers to a method of treatment of inflammatory disorders comprising the administration of the compositions selectively orally, by topical application, by transdermal application.

[0081] The following examples illustrate, but do not limit, the scope of the invention. Those of ordinary skill in the art should understand that the present analysis is only of exemplary embodiments, and is not intended to limit the 40 broader aspects of the present invention, which are performed in the exemplary construction.

Example 1

Preparation of methane extract from Sphaeranthus indicus.

[0082] Flora and fruit chapters of Sphaeranthus indicus dried (200 g) were sprayed. The pulverized material was extracted using methanol (2.5 L) with stirring at 60 ° C for 3 hours. The extract was filtered under vacuum. This extraction process was repeated two more times. The extracts were combined and concentrated.

[0083] Yield: 23.29 g (11.65% w / w).

[0084] The extract of example 1 was found to contain 6% of compound 1 (as described in example 4), calculated by HPTLC.

55 Example 2

Preparation of Sphaeranthus indicus ethyl acetate extract.

[0085] Flora and fruit chapters of Sphaeranthus indicus (350 g) were pulverized. The pulverized material was extracted using ethyl acetate (3 L) with stirring at 60 ° C for 3 hours. The extract was filtered under vacuum. This extraction process was repeated two more times. The extracts were combined and concentrated.

[0086] Yield: 19 g (9.5% w / w).

65 Example 3

Preparation of aqueous extract of Sphaeranthus indicus.

[0087] Spraeranthus indicus dried flora and fruit chapters were sprayed (200 g). The pulverized material was extracted using water (1.2 L) with stirring at 80 ° C-90 ° C for 3 hours. The extract was filtered under vacuum. This 5 extraction process was repeated. The extracts were combined and concentrated to remove water. Additionally, the crude extract was dried by cryo drying.

[0088] Yield: 21 g (10.5% w / w).

10 Example 4

Isolation of 3a-hydroxy-5a, 9-dimethyl-3-methylene-3a, 4,5,5a, 6,7,8,9b-octahydro-3H-naphtho [1,2-b] furan-2-one ( compound 1).

[0089] The methanol extract (32 g), prepared by the method described in example 1, was purified by column chromatography (silica gel, methanol in chloroform). Final purification was achieved by preparative HPLC (silica column, hexane: isopropanol, 95: 5) to obtain the indicated compound. 1 H NMR (CDCI3, 500 MHz): δ1.085 (3H, CH3), 4.997 (1 H, s), 5.801 (1 H, s), 6.270 (1 H, s); MS: m / e (ES) 248 (M +).

[0090] The compound was characterized by comparing the spectral data obtained with the published literature (Indian Journal of Chemistry, Vol. 25B, 233-238, (1986); J. Chem Soc. Perkin Trans. 1: (2), 157 -160, (1988); J. Chem. Research (M), 0501-0509, 1989).

Pharmacological results

[0091] The efficacy of the plant extracts of the present invention, compounds isolated by purification of the extract and formulations, in terms of inhibition of TNF-α activity and interleukins (IL-1, IL-6 and IL-8 ) was determined by various pharmacological assays, well known in the art and described below.

30 In vitro examination to identify TNF- inhibitors

Example 5

[0092] Primary exploration - human peripheral blood mononuclear cells (CMSPh). The production of

TNF-α by lipopolysaccharides (LPS) in the CMSPh was measured according to the method described by Jansky, L. et al (Physiol. Res. 52: 593-598, (2003)). Blood was drawn from healthy donors in vacutainer tubes (BD vacutainer) with Potassium EDTA. The CMSPs were isolated using gradient centrifugation in Histopaque-1077 solution (Sigma). The isolated CMSPs were suspended in RPMI 1640 culture medium (Gibco BRL, Pasley, UK) containing 10% fetal bovine serum (SBF) (Hyclone, Utah, United States), 100 U / ml penicillin (Sigma Chemical Co. St Louis

40 MO) and 100 μg / ml streptomycin (Sigma Chemical Co. St Louis, MO). The cell concentration was adjusted to 1x106 cells / ml. The viability, determined by exclusion with trypan blue dye, was uniformly ≥98%. The cell suspension (100 μl) was added to the wells of a 96-well culture plate. After plating the cells, 79 μl of the culture medium and 1 μl of eight different concentrations of the test samples (final concentration 0.03, 0.1, 0.3, 1, 3, 10, 30, 100 μg / ml) dissolved in DMSO (dimethylsulfoxide, Sigma, MO, States

45) were added to the cells. The final concentration of DMSO was adjusted to 0.5%. The vehicle was used as control (0.5% DMSO). Rolipram (100, 300 μM) was used as the standard. The plates were incubated for 30 minutes at 37 ° C in a 5% CO2 atmosphere. Finally, 20 μl (10 μg / ml) per well of LPS, (Escherchia coli 0127: B8, Sigma Chemical Co., St. Louis, MO), was added for a final concentration of 1 μg / ml. The plates were incubated at 37 ° C for 5 hours in a 5% CO2 atmosphere. To evaluate the cytotoxicity effect of the extracts

50 vegetables, cell viability assay was performed using MTS reagent (3- (4,5-dimethylthiazol-2-yl) -5- (3-carboxymethoxyphenyl) -2- (4-sulfonyl) -2H-tetrazolium) after 5 h of incubation. The supernatants were recovered and tested for TNF-α by ELISA as described by the manufacturer (OptiEIA ELISA sets, BD Biosciences, Pharmingen). % Inhibition was recorded. The percentage of cytotoxicity of the test samples compared to the control was evaluated.

55 [0093] Table 1 summarizes the results.

Table 1: TNF-α inhibition in human peripheral blood mononuclear cells

Sample

Concentration (μg / ml) % TNF inhibition Toxicity% at 5h

Excerpt from Example 1

0.1 0.0 0

one

14.0 0

10

96.0 7

Sample

Concentration (μg / ml) % TNF inhibition Toxicity% at 5h

100

97.0 0

Excerpt from Example 2

0.1 0.0 twenty

one

34.0 13

10

97.0 22

100

97.0 8

Excerpt from Example 3

0.1 0.0 0

one

0.0 eleven

10

4.0 0

100

96.0 0

Rolipram (μM)

100 84.0 2

300

90.0 twenty-one

Example 6

Example on proinflammatory cytokines released by CMSPh stimulated with LPS

[0094] Effect of plant extract on proinflammatory cytokines: TNF-α, interleukin-1β (IL-1β), interleukin-6 (IL-6) and interleukin-8 (IL-8) were measured using supernatants generated in the primary screening trial. The levels of these cytokines were calculated by ELISA as described by the manufacturer. (OptiEIA ELISA sets, BD Biosciences, Pharmaginen). 50% inhibitory concentration values (IC50) were calculated

10 by a non-linear regression method using the GraphPad software (Prism 3.03).

Table 2: Effect of the extract of Example 1, on proinflammatory cytokines

Mr. Nº

Proinflammatory cytokines Extract from Example 1, IC50 μg / ml, (CMSPh)

01

TNF-α 5.1

02

IL 1β 4.9

03

IL-6 26.8

04

IL-8 31.0

[0095] Conclusion: It was found that the extract of Example 1 inhibited proinflammatory cytokines (TNF-α, IL-1β, IL-6 and IL-8) released by CMSPh stimulated by LPS.

Example 7

Effect of compound 1 on proinflammatory cytokines released by CMSPh stimulated by LPS

[0096] Compound 1 was obtained using the procedure of Example 4. The bioactivity evaluation was performed according to the procedure of Example 6.

[0097] The effect of Compound 1 on proinflammatory cytokines: TNF-α, interleukin-1β (IL-1β), interleukin

25 6 (IL-6) and interleukin-8 (IL-8) was measured using the supernatants generated in the primary screening test. The levels of these cytokines were calculated by ELISA as described by the manufacturer. (OptiEIA ELISA sets, BD Biosciences, Pharmaginen). The 50% inhibitory concentration values (IC50) were calculated by a non-linear regression method using the GraphPad software (Prism 3.03). Table 3 summarizes the results.

30 Table 3: Effect of compound 1 on proinflammatory cytokines [0098] Conclusion: Compound 1 was found to inhibit proinflammatory cytokines (TNF-α, IL-1β, IL-6 and IL8) released by CMSPh stimulated by LPS .

Mr. Nº

Proinflammatory cytokines Compound 1, IC50 μM (CMSPh)

01

TNF-α 0.7

02

IL 1β 0.4

03

IL-6 1.6

04

IL-8 8.9

5 Example 8

Effect on proinflammatory cytokines produced by synovial cells obtained from a patient with RA.

[0099] The production of cytokines by synovial cells obtained from a patient with rheumatoid arthritis (RA)

10 undergoing knee arthroplasty was measured according to the method described by Brennan, F. M. et al (The Lancet. July 29: 244-247, (1989)). The synovial membrane tissue was digested in DMEM (Gibco) containing 10% SBF, 100 U / ml penicillin and 100 μg / ml streptomycin, 4 mg / ml type I collagenase (Worthington), 1 , 5 μg / ml Dnasa type I (Sigma) and 15 U / ml heparin and incubated at 37 ° C for 3 hours. After incubation, the digested tissue was filtered through a 70 µm membrane and the cells washed.

15 times in complete medium (DMEM with 10% SBF). Synovial cells were cultured at 1 x 10 6 cells / ml in the presence / absence of the test sample for 10 hours. Supernatants were collected by centrifugation and cytokine levels (TNF-α, IL-1β, IL-6, IL-8) were measured by ELISA. To evaluate the cytotoxic effect of plant extracts, a cell viability assay was performed using MTS reagent. 50% inhibitory concentration values (IC50) were calculated using a non-linear regression method using the software

20 GraphPad (Prism 3.03).

[0100] Conclusion: It was found that the extract of Example 1 inhibited pro-inflammatory cytokines (TNF-α, IL-1β, IL-6 and IL-8) produced by synovial cells obtained from a patient with RA.

25 Example 9

[0101] Effect of compound 1 on proinflammatory cytokines produced by synovial cells obtained from a patient with RA.

[0102] The effect on proinflammatory cytokines produced by synovial cells obtained from a patient with RA for compound 1 was studied as described by the procedure of Example 8.

[0103] Table 4 summarizes the results.

Table 4: effect of compound 1 on proinflammatory cytokines produced by synovial cells

Mr. Nº

Proinflammatory cytokines Compound 1, IC50 (μM), synovial cells

01

TNF-α 0.8

03

IL-6 1.4

04

IL-8 10.9

[0104] Conclusion: Compound 1 was found to inhibit proinflammatory cytokines (TNF-α, IL-6 and IL-8) produced by synovial cells obtained from a patient with RA.

40 Example 10

Cellular ELISA for the expression of adhesion molecules

[0105] The assay was designed based on the Transplantation reference, Vol 63 (5), 759-764, 1997 with 45 modifications.

Cell Culture and Reagents

[0106] Umbilical Vein Human Endothelial Cells (CEHVU) were obtained in Cascade Biologies and obtained

50 conserved in M200 (Cascade Biologies, Portland, Or) supplemented with growth supplement with low serum content (CCBS) at 37 ° C in a 5% CO2 incubator. U937 cells (ATCC, Manassas, VA) were grown in RPMI 1640 medium supplemented with 10% SBF (Hyclone, Logan, UT). Recombinant human TNFα, antibodies against VCAM-1, ICAM-1, E-Selectin were obtained in R&D Systems and LPS was obtained in Sigma (St. Louis, MO).

55 Cellular ELISA for the expression of adhesion molecules

[0107] CEHVUs were plated at 7x105 cells / well in 96-well plates coated with fibronectin. Cells were stimulated with TNF-α (10 μg / ml) or LPS (1 μg / ml), 30 min after the addition of

test compound After stimulation, the cells (E-Selectin and ICAM-1) were fixed with paraformaldehyde in phosphate buffered saline (PBS). Non-specific binding was blocked with 2% bovine serum albumin (ASB) in phosphate buffered saline (PBS) for 1 h, and the cells were incubated with primary antibody for 2 h. For the detection of VCAM-1 cells were blocked, incubated with primary antibody overnight and then fixed. Cells were washed with 0.1% ASB in PBS, and incubated with mouse antibody (Ab) against peroxidase-conjugated mouse immunoglobulin G (IgG) were added for 90 min. After washing, seven times, 3,3'5,5'-tetramethylbenzidine liquid substrate (TMB substrate) was added and the optical density of each well at 450 nm was determined using a microtiter plate reader (Spectramax, Molecular Devices , CA). As standard, BAY 11-7082 [(E) -3- (4-methylphenylsulfonyl) -2-propenonitrile] was used.

10 and DMSO as vehicle control. The percent inhibition of the test sample was evaluated compared to the control. The 50% inhibitory concentration values (IC50) of each sample compared to the control were determined by a non-linear regression method. Table 5 summarizes the results.

Table 5: Cellular ELISA for expression of adhesion molecules for the extract of example 1 and compound 1

Extract from Example 1 IC50 (μg / ml)

Compound 1, IC50 (μM)

ICAM-1

7.6 0.52

VCAM-1

6.4 0.4

E-Selectin

3.5 0.2

15 Conclusion:

[0108] The extract of example 1 and compound 1 reduced, in a dose-dependent manner, the surface expression induced by TNF-α of endothelial cell adhesion molecules such as ICAM-1, VCAM-1 and E

20 Selectina.

Example 11

Adhesion of THP-1 mononuclear cells to CEHVU monolayers

[0109] Adhesion studies were performed with the THP-1 promonocytic cell line, which had been established as a useful model for monocytes in adhesion studies in Circ. Res., 97, 236-243, 2005, with modifications. THP-1 cells were washed twice with marker medium (M200 plus LSGS). THP-1 cells (6x105 cells per ml) were labeled with 10 μg / ml acetoxymethyl ester of biscarboxyethylcarboxyfluorescein (a fluorescent probe,

30 BCECF-AM; Sigma) for 30 min at RT. After inactivating with 0.1% BSA, the sediment was resuspended in marker medium. To assess monocyte adhesion, CEHVU monolayers were treated with TNF-α (1 ng / ml) in the presence or absence of various concentrations of test samples. Media were removed, washed and labeled THP-1 cells (6x104 cells per well) were added to the wells and incubated for 10 minutes at RT in the dark. After co-incubation, the wells were washed, loaded with lysis buffer (Triton-X

0.1% 35 in 1.5M Tris buffer) and incubated for 30 min. Fluorescence was measured using a fluorescence reader (PolarStar Optima, BMG Labtech) at a maximum excitation of 485 nm and a maximum emission of 520 nm. Values are means + ETM, which represent fluorescence adhesion data.

[0110] As standard BAY 11-7082 [(E) -3- (4-methylphenylsulfonyl) -2-propenonitrile] and DMSO were used as control vehicle. Table 6 summarizes the results.

Table 6: THP-1 mononuclear cell adhesion to CEHVU monolayers for the extract of example 1 and compound 1

Mr. Nº

Test sample Concentration Fluorescence intensity Control times

01

Excerpt from Example 1 1 (μg / ml) 3 (μg / ml) 10 (μg / ml) 30 (μg / ml) 39768 35302 13183 10236 23 21 6 5

02

Compound 1 0.1 (μM) 0.3 (μM) 1 (μM) 3 (μM) 33421 34024 10195 5728 13 13 4 2

Mr. Nº

Test sample Concentration Fluorescence intensity Control times

03

BAY 11-7082 0.5 (μM) 1 (μM) 18442 14271 10 8

04

Control not stimulated with DMSO Control stimulated with DMSO 2544 30791 1 18

[0111] Conclusion: The extract of example 1 and compound 1 inhibited the adhesion of monocytic THP-1 cells stimulated with TNF-α to CEHVU at 10 μg / ml and 1 μM respectively.

[0112] Therefore, since these compounds inhibit cell surface expression of adhesion molecules in CEHVU as well as monocyte adhesion to CEHVU, they can stop leukocyte migration, which is a key event in chronic inflammatory diseases. and they can prove beneficial in various inflammatory disorders.

In vivo studies

Example 12

[0113] Release of tumor necrosis factor (TNF) -α induced by lipopolysaccharide (LPS) in BALB / c mice.

[0114] The protocol described by Fukuda T. et al (Eur. J. Pharmacol., 391: 317-320, (2000)) was followed. BALB / c mice were divided into groups of ten each. The test sample, suspended in Tween 80 and in 0.5% carboxymethylcellulose (CMC), was administered orally (p.o.) to the mice. One hour later, 20 LPS dissolved in sterile apyrogenic saline was administered by i.p. at the dose of 1 mg / kg. The negative control group received saline solution as an i.p. injection, while the other groups received LPS. Rolipram (30 mg / kg, p.o.) was used as the standard drug. An hour and a half later, under anesthesia with urethane (1.5 g / kg, i.p.), blood was drawn from the abdominal artery using a 1 ml syringe rinsed with heparin (500 Ul / ml). Heparin (5 μl) was used as an anticoagulant in blood collection tubes. Plasma was separated by centrifugation at 10,000

25 rpm at room temperature, divided into aliquots and stored at -70 ° C until analysis. TNF-α levels in blood samples were tested using ELISA and the percentage inhibition of TNF-α release was calculated compared to the control group. Table 7 summarizes the results.

Table 7: Release of tumor necrosis factor (TNF) -α induced by lipopolysaccharide (LPS) in BALB / c 30 mice for the extract of Example 1 and for compound 1

Mr. Nº.

Test sample Dose mg / kg % inhibition

01

Excerpt from Example 1 100 43.21 ± 14.52

02

Compound 1 10 28.69 ± 13.71

30

39.98 ± 10.32

100

87.10 ± 3.67

[0115] Conclusion: the extract of example 1 and compound 1 inhibit the release of TNF-α in BALB / c mice.

Example 13

35 Collagen-induced arthritis (AIC) in DBA / 1J mice

Male DBA / 1J mice, 8-10 weeks old, were immunized with 200 μg of Collagen

40 [0116] Type II as an emulsion in Freund's Complete Adjuvant, by intradermal injection at the base of the tail. Twenty-one days later, the mice were given a 100 μg Type II Collagen booster injection. A set of untreated mice was also maintained with these mice.

[0117] From day 23 onwards, the appearance of rheumatoid arthritis was evaluated in mice using, as

45 a parameter, the Articular Index. Mice were induced in the study with a minimum score of 2 on the hind legs. The extract of Example 1 was administered at a dose of 400 m.p.k (milligram per kilogram of body weight) orally twice daily for 12 days. Compound 1 was administered at a dose of 50 m.p.k. and 100

m.p.k. orally twice a day for 12 days. Enbrel (3 mg / kg) was used as a standard and was given subcutaneously once a day. The volume and articular index of the legs was recorded daily. The statistical significance of the data was analyzed.

[0118] At the end of the experiment the legs of the mice were processed for histopathological evaluation. [0119] Table 8 summarizes the data on the reduction of the thickness of the legs and reduction of the articular index of the legs

Table 8: efficacy of the extract of example 1 and compound 1 in the AIC model

Test sample

Parameters

AIC

Dose mg / kg

Leg thickness reduction Joint Index Reduction

Excerpt from Example 1

400 Statistically significant over control at a significance level of 0.01 Statistically significant over the control between the level of significance of 0.05 and 0.06.

Compound 1

fifty Statistically significant over control at a significance level of 0.05 Statistically significant over control at a significance level of 0.05

100

Statistically significant over control at a significance level of 0.01 Statistically significant over control at a significance level of 0.01

10 Histopathological analysis:

[0120] The beneficial effect of the extract of example 1 and compound 1 on the pathology of DBA / 1J arthritic mice (AIC) was evaluated. Microscopy was performed after staining with Hematoxylin and Eosin as well as staining with

15 Safranine O of the synovial joints. Histopathological analysis showed that both the extract of example 1 and compound 1 exerted beneficial effects in reducing cartilage destruction, bone destruction and synovitis compared to the vehicle-treated group.

[0121] Conclusion: both the extract of example 1 and compound 1 exerted beneficial effects in the AIC arthritis model.

Toxicity studies Example 14

25 Acute oral toxicity

[0122] The extract of Example 1 was tested for acute oral toxicity in Sprague Dawley rats according to the guidelines determined in "Scheme Y" of Drugs and Cosmetics Act, 1940. (India)

[0123] The extract of Example 1, suspended in 0.5% Tween 80 in water, was administered orally by probe as a single dose to a group of five male rats and five female rats at a maximum practicable dose of 2000 mg / kg body weight. The animals were observed with respect to mortality and symptoms of intoxication for a period of 14 days after dosing and their body weights were also recorded. At the end of

35 study was performed necropsy of all rats.

[0124] Conclusion: In this study, simple oral administration of extract from Example 1 to Sprague Dawley rats at the maximum practicable dose of 2000 mg / kg did not cause any mortality in the treated rats.

[0125] The average lethal dose (LD50) of the extract of Example 1, after oral administration as a single dose in Sprague Dawley rats, both in females and males, was found to be greater than 2000 mg / kg body weight .

Example 15

45 Subcutaneous oral toxicity

[0126] A subcutaneous oral toxicity study (day 28) of the extract of example 1 was conducted in Sprague Dawley rats in accordance with the guidelines set forth in “Scheme Y” of Drugs and Cosmetics Act, 1940. (India)

50 [0127] Groups of six male and six female Sprague Dawley rats were given daily doses of 0, 250, 500 or 1000 mg / kg body weight of Example 1 extract by oral probe for 28 days and the Day 29 to assess its toxicity. The rats were examined daily for toxicity symptoms. Body weight and feed intake were recorded in individual rats during the period

5 experimental along with all mortality frequencies and disease symptoms. At the end of the study clinical blood tests were performed.

[0128] When finally all animals were sacrificed, they underwent a complete necropsy and the weights of some organs were recorded. Histopathological evaluation was performed on all tissues listed.

10 in the protocol in all animals of the control and high dosage groups.

[0129] All animals that received extract from Example 1 and up to a dose of 1000 mg / kg survived during the treatment period. No clinical symptoms of toxicity were observed in any of the treated animals. Data on body weight gain and food consumption indicated that there were no effects

15 adverse events due to the test article and up to a dose of 1000 mg / kg.

[0130] Conclusion: based on the findings of this study, it was found that the level of non-observable adverse effects (NOAEL) of the extract of example 1 in rats, after oral administration for 28 days, was greater than 1000 mg / kg of body weight.

Formulations

Example 16

25 Capsule preparation

[0131] General procedure: ingredients 01 to 05 in a specified amount were weighed and transferred to a suitable mixer. The contents were mixed well and ingredients 09, 10 and 11 were added and mixing continued. To this combination the ingredients 06, 07 and 08 were added and the dough was mixed for 30

30 40 minutes The combination was passed through a 40 mesh screen, and was used to fill capsules.

Table 9: Sphaeranthus indicus capsule formulation

Each capsule contains

MR. No.

INGREDIENT QUANTITY% in P / P

01

Excerpt from Example 1 69.72

02

Methyl Sodium Paraben 0.39

03

Propyl Sodium Paraben 0.13

04

Bromerol 0.18

05

Sodium benzoate 0.39

06

talcum powder 2.61

07

Magnesium stearate 1.74

08

Spray 0.87

09

Sodium Starch Glycolate 2.18

10

Lactose 8.72

eleven

Dibasic calcium phosphate 13.07

Example 17

35 Tablet Preparation

[0132] General procedure: Ingredients 01 to 05 in a specified amount were weighed and transferred to a suitable mixer. Ingredient 13 was added and the wet mass mixed well. To this, they were added

Ingredients 09, 10, 11 and 12 and mixing continued until a homogenized mass was obtained. This wet mass was passed through a 16 mesh screen and the wet granules were dried at 70 ° C ± 5 ° C. Ingredients 06, 07 and 08 were added to the above granules and the dough was mixed for 30-45 minutes. The combination was passed through a 40 mesh screen and the tablets were compressed using a suitable die.

Table 10: Sphaeranthus indicus tablet formulation

Each tablet contains

MR. No.

INGREDIENT QUANTITY% in P / P

01

Excerpt from Example 1 66.53

02

Methyl Sodium Paraben 0.37

03

Propyl Sodium Paraben 0.12

04

Bromerol 0.17

05

Sodium benzoate 0.37

06

talcum powder 2.50

07

Magnesium stearate 1.66

08

Spray 0.83

09

Sodium Starch Glycolate 2.50

10

Lactose 8.32

eleven

Dibasic calcium phosphate 12.47

12

Starch 4.16

13

Isopropanol *

* only for granulation

Example 18

5 Syrup Preparation

General procedure

[0133] Ingredient 01 was weighed and ingredient 15 was added with continuous stirring. To this they were added

10 quantities measured by weight of ingredients 03, 04, 05, 06, 08, 09, 10, 11, 12 and 14 with continuous stirring to dissolve. Ingredients 02 and 13 were weighed and dissolved in ingredient 07. To this was added purified water to adjust the volume to 10 ml. The solution obtained was filtered through a filter press / nylon cloth.

Table 11: Sphaeranthus indicus syrup formulation

Each 10 ml of syrup contain

MR. No.

INGREDIENT QUANTITY% in P / P

01

Excerpt from Example 1 4

02

Peppermint Powder 0.025

03

Honey 0.25

04

Sugar fifty

05

70% sorbitol solution 5

06

Liquid glucose 10

07

Propylene glycol 5

08

Citric acid monohydrate 0.5

09

Methyl Sodium Paraben 0.2

10

Propyl Sodium Paraben 0.02

eleven

Sodium benzoate 0.2

12

Bronopol 0.02

13

Essence of fresh mint "S" 0.25

Each 10 ml of syrup contain

MR. No.

INGREDIENT QUANTITY% in P / P

14

Sugary caramel coloring 0.75

fifteen

Purified water c.s up to 10 ml

Example 19

Cream formulation preparation 5 General procedure

[0134] Ingredient 01 was weighed and suspended in ingredient 17. Ingredients 02 to 07 were combined. Ingredients 08, 09, 10, 11, 13 and 14 were weighed and mixed with part of 18. Ingredient 12 was weighed and added to the remaining part of ingredient 18 and mixed with ingredients 15 and 16. The content of all phases was mixed at 55 ° C and homogenized, allowed to cool and packaged in a suitable tube.

Table 12: Sphaeranthus indicus cream formulation

Each 100 g of cream contains

MR. No.

INGREDIENT QUANTITY% in P / P

01

Excerpt from Example 1 05.00

02

Ketostearyl Alcohol - 12.0 g 12.00

03

Ketomacragol - 1000 03.00

04

Sorbitan Monooleate 02.00

05

Glycerol monostearate S.E. 03.00

06

Isopropyl myristate 02.50

07

Stearic acid 02.50

08

Methyl Sodium Paraben 00.40

09

Propyl Sodium Paraben 00.08

10

Phenoxyethanol 00.52

eleven

Disodium EDTA 00.02

12

Carbomer - 940 00.75

13

Sodium Lauryl Sulfate 00.75

14

Simethicone 01.00

fifteen

Triethanolamine 01.00

16

Propylene glycol 05.00

17

Isopropanol 10.00

18

Water 50.48

15 Example 20

Gel formulation preparation

General Procedure 20 [0135] Ingredient 01 was weighed and suspended in ingredient 06. Ingredient 04 was dissolved in ingredient

07. Ingredients 05 and 08 were mixed. Ingredients 02 and 03 were mixed. The combination was mixed well and packed in a suitable tube.

Table 13: Sphaeranthus indicus gel formulation

Each 100 g of gel contains

MR. No.

INGREDIENT QUANTITY% in P / P

01

Excerpt from Example 1 05.00

02

Butylated Hydroxytoluene 00,025

03

Butylated Hydroxyanisole 00,025

04

Carbopol - 940 02.95

05

Polyethylene Glycol - 400 30.00

06

Isopropanol 05.00

07

Propylene glycol 55.00

08

Sorbitan Monooleate 02.00

Example 21

5 Preparation of ointment formulation General procedure [0136] Ingredients 02 to 06 were weighed and combined in a suitable container. To this, the

Ingredient 01. To this combination were added ingredients 07 and 08. The contents were mixed well and packed in a suitable tube. Table 14: Sphaeranthus indicus ointment formulation

Each 100 g of ointment contains

MR. No.

INGREDIENT QUANTITY% in P / P

01

Excerpt from Example 1 05.00

02

White beeswax 15.00

03

Hard paraffin 25.00

04

Microcrystalline wax 15.00

05

Soft white paraffin 30.00

06

Light liquid paraffin 09.95

07

Butylated Hydroxytoluene 0.025

08

Butylated Hydroxyanisole 0.025

15 Example 22

Tablet preparation

General procedure

[0137] Ingredients 01 and 02 were weighed separately, screened through a 20 mesh and mixed. Ingredients 03 to 07 were weighed and screened through a 40 mesh. Ingredients 03, 04, 05 and 07 were mixed and ingredients 01 and 02 were added to this mixture of ingredients. To this combination was added ingredient 06 and It mixed. The lubricated combination obtained was compressed with a mechanical tool

25 adequate.

Table 15: Compound formulation of compound 1

Each tablet contains

MR. No.

INGREDIENT QUANTITY% in P / P

01

Compound 1 58.33

02

Microcrystalline cellulose 35.97

03

talcum powder 2.50

04

Sodium Starch Glycolate 1.60

05

Colloidal Silicon Dioxide 0.80

06

Magnesium stearate 0.50

07

Yellow Quinolein Coloring 0.30

Example 23

5 Preparation of tablets

General procedure

[0138] Ingredients 01 and 02 were weighed separately and screened through a 20 mesh. The ingredient

10 04 dissolved in ingredient 08 while stirring. The above combination was granulated using binder solution. The wet mass was passed through a suitable sieve. The sieved mass was dried at room temperature (25 ° C) and then at approximately 40 ° C. The dry mass was screened through a suitable sieve. Ingredients 03, 05 and 07 were sieved separately through a 40 mesh and mixed. To this the dry mass was added and mixed. To this combination was added ingredient 06 and the lubricated combination was compressed with a

15 suitable mechanical tool.

Table 16: Compound formulation of compound 1

Each tablet contains

MR. No.

INGREDIENT QUANTITY% in P / P

01

Compound 1 61.40

02

Lactose monohydrate 35.15

03

Croscarmellose sodium 1.40

04

Polyvinylpyrrolidone 0.85

05

Colloidal Silicon Dioxide 0.35

06

Magnesium stearate 0.50

07

Yellow Quinolein Coloring 0.35

08

Isopropyl alcohol Sufficient quantity

Example 24

20 Capsule Preparation

General procedure

[0139] Ingredients 01 and 02 were weighed separately and screened through a 20 mesh and mixed. Ingredient 03 was weighed and screened through a 40 mesh. All ingredients were mixed and lubricated using ingredient 04. The combination was loaded into empty hard gelatin capsules using suitable mechanical tools.

Table 17: Capsule formulation of compound 1

Each capsule contains

MR. No.

INGREDIENT QUANTITY% in P / P

01

Compound 1 98.59

02

Microcrystalline cellulose 0.75

03

Colloidal Silicon Dioxide 0.47

04

Magnesium stearate 0.19

Claims (11)

1. A pharmaceutical composition for use in the treatment of an inflammatory disorder mediated by TNF-α, interleukins (IL-1, IL-6, IL-8) and intercellular adhesion molecule 1 (ICAM-1), molecule 1 vascular adhesion 5 cellular (VCAM-1) and E-Selectin, wherein said composition comprises, as active ingredient, a therapeutically effective amount of an extract of flora and fruit chapters of the Sphaeranthus indicus plant, together with pharmaceutically acceptable carriers. 2. A pharmaceutical composition for use in the treatment of an inflammatory disorder according to the 10 claim 1, wherein the extract of Sphaeranthus indicus contains 3a-hydroxy-5a, 9-dimethyl-3-methylene3a, 4,5,5a, 6,7,8,9b-octahydro-3H-napfto [1,2 -b] furan-2-one (compound 1) as a bioactive marker. 3. A pharmaceutical composition for use in the treatment of an inflammatory disorder according to the claim 2, wherein the extract of Sphaeranthus indicus contains 2-9% of compound 1. 4. A pharmaceutical composition for use in the treatment of an inflammatory disorder mediated by TNF-α, interleukins (IL-1, IL-6, IL-8) and intercellular adhesion molecule 1 (ICAM-1), molecule 1 of Vascular cell adhesion (VCAM-1) and E-Selectin, wherein said composition comprises a therapeutically effective amount of 3a-hydroxy-5a, 9-dimethyl-3-methylene-3a, 4,5,5a, 6,7, 8,9b-octahydro-3H-napfto [1,2-b] furan-2-one (compound 1) as a 20 active ingredient together with pharmaceutically acceptable carriers. 5. A pharmaceutical composition for use in the treatment of an inflammatory disorder according to any one of claims 1 to 4, wherein said composition is formulated for oral, topical or transdermal administration. 6. A pharmaceutical composition for use in the treatment of an inflammatory disorder according to any one of claims 1 to 5, wherein the inflammatory disorder is selected from the group consisting of inflammatory bowel disease, rheumatoid arthritis, juvenile rheumatoid arthritis , psoriatic arthritis, osteoarthritis, refractory rheumatoid arthritis, chronic non-rheumatoid arthritis, osteoporosis / bone resorption, coronary heart disease, 30 atherosclerosis, vasculitis, ulcerative colitis, psoriasis and adult respiratory distress syndrome. 7. A pharmaceutical composition for use in the treatment of an inflammatory disorder according to any one of claims 1 to 6, wherein the inflammatory disorder is rheumatoid arthritis. A pharmaceutical composition for use in the treatment of an inflammatory disorder according to any one of claims 1 to 6, wherein the inflammatory disorder is inflammatory bowel disease. 9. A pharmaceutical composition for use in the treatment of an inflammatory disorder according to a any one of claims 1 to 6, wherein the inflammatory disorder is ulcerative colitis. 40 10. A pharmaceutical composition for use in the treatment of an inflammatory disorder according to any one of claims 1 to 6, wherein the inflammatory disorder is atherosclerosis. 11. A pharmaceutical composition for use in the treatment of an inflammatory disorder according to any one of claims 1 to 6, wherein the inflammatory disorder is psoriasis. 12. A pharmaceutical composition for use in the treatment of an inflammatory disorder, as claimed in any one of claims 1 to 6, further comprising at least one anti-inflammatory agent. 13. A pharmaceutical composition for use in the treatment of an inflammatory disorder, as claimed in claim 12, wherein the anti-inflammatory agent is selected from prednisolone, hydrocortisone, methotrexate, sulfasalazine, naproxen, diclofenac or ibuprofen.