Herbal anti-inflammatory composition

Abstract

The present invention relates to a composition based on a mixture of herbal extracts of nigella foenum-graecum (ranunculaceae), hemsleya amabilis (stephaniaceae), petasites tatewakianus (compositae) and purple perilla (labiatae) for use as an anti-inflammatory agent.

Description

Field of the Invention

[0001] The present invention relates to a composition based on a mixture of herbal extracts of Nigella sativa (Ranunculaceae), Tinospora cordifolia (Menstruaceae), Petasites hybridus (Asteraceae), Perilla frutescens (Lamiaceae) for use as an anti-inflammatory agent. Background of the Invention

[0003] Inflammation is a complex process, which is usually related to pain and involves events such as increased vascular permeability, increased protein denaturation and membrane changes. When cells in the body are damaged by microorganisms, physical agents or chemical agents, damage is in the form of stress. When tissue cells are damaged, they release kinins, prostaglandins and histamine. These events collectively act to cause vasodilation (capillary dilation) and increased capillary permeability. This results in an increase in blood flow to the injury site. Due to increased blood flow, the temperature at the injury site rises. In response to this, physiological protein denaturation and the cascade reaction of the release of inflammatory mediators are enhanced in addition (see N.Parrow et al. "Sequestration and Scavenging of Iron inInfection", Infection and Immunity 81 the 10th phase (2013): 3503-3514). Inflammation is a kind of physical response to damage, infection or destruction, characterized by heat, redness, pain, swelling and physiological dysfunction. It triggers chemical mediators to be released from damaged tissue and migratory cells. Inflammatory mediators include histamine, 5-HT (serotonin), bradykinin, prostaglandin E2 (e.g., PGE2), interleukins, substance P, nitrous oxide, etc. (see Edvinsson, L., Haanes, KA and Warfvinge, K. Does inflammation have a role in migraine?; Nat Rev Neurol 15, 483-490 (2019)). In inflammatory disorders, phagocytes are overactivated and produce O 2- OH -Free radicals and non-free radical species (H2O2) can cause serious damage to tissues by powerful direct oxidation or indirectly through lipid peroxidation leading to membrane damage (see SN Heendeniya, WD Ratnasooriya and RN Pathirana; In vitro investigation of anti-inflammatory activity and evaluation of phytochemical profile of Syzygium caryophyllatum, Journal of Pharmacognosy and Phytochemistry 2018; 7(1): 1759-1763). Tissue damage subsequently causes an inflammatory response by producing mediators and chemokines.

[0004] There are references to the use of fennel leaf nigella in allergic rhinitis. Some of them are discussed here. WO0051580 relates generally to the fields of medicine and pharmacology, and specifically to a pharmaceutical composition for the treatment of asthma / allergy, consisting essentially of a glycophosphopeptic agent (Glycosphosphopeptical) or pure fennel leaf nigella seed as an equivalent, which is active in stimulating type 1 T helper lymphocytes, thereby selectively shutting down eosinophilic inflammation, and also treating viral respiratory infections (flu and influenza), other viral infections, urinary tract infections, pelvic inflammatory diseases, especially neuroimmune appendicitis, cancer, Crohn's disease and facial paralysis. CN111568955 discloses a Chinese medicine composition for relieving allergic rhinitis, characterized in that the active ingredients include nigella sativa seeds, liquorice and fennel seeds; the mass ratio of nigella sativa seeds to liquorice to fennel seeds is 1-4:0.5-2:0.5 to 2. WO2020142054 discloses a nasal spray preparation containing fennel leaf black cumin oil, which is used to treat dry nose, nasal congestion and allergic rhinitis symptoms caused by dry air or influenza or common cold, characterized in that it contains purified water, fennel leaf black cumin oil, salt (NaCl), xylitol, eucalyptus oil, peppermint oil, an emulsifier and a microbial preservative.

[0005] JP2006070038 provides a natural product for use in a nutritional product, a dietary supplement, a food or a pharmaceutical composition, the product being used for treating allergies, wherein the composition comprises an extract of Tinospora cordifolia and discloses the use of the extract in treating allergies (including but not limited to seasonal or perennial or sporadic allergic rhinitis).

[0006] Some prior art references are discussed here. DE19702168 discloses the use of a Butterbur extract for the preparation of a medicament for the treatment of allergic rhinitis. DE102004039011 discloses an extract comprising Butterbur, which is free of pyrrolizidine alkaloids and furanostanes. The extract is used for gastrointestinal, analgesic, anti-migraine, anti-asthmatic, anticonvulsant, antitussive, anti-allergic, anti-inflammatory, anti-psoriatic, anti-ulcer, anti-arthritic, anti-rheumatic, immunosuppressive indications.

[0007] There are some references discussing the use of perilla in allergic rhinitis. Some of them are discussed here. US2019255135 relates to a composition for treating inflammatory diseases, comprising a composite plant extract including a perilla extract and a white atractylodes (Atractylodes macrocephala Koidzumi) extract. KR20200052628 discloses a pharmaceutical composition for preventing or treating inflammatory diseases or allergic diseases, which contains a supercritical defatted perilla powder ethanol extract as an active ingredient. JPH09291299 claims protection for oils containing ω3 polyunsaturated fatty acids, oils containing ω6 polyunsaturated fatty acids, and perilla leaf extracts, which are used as anti-allergic oil compositions. US10238707 claims protection for a composition comprising at least one sustained-release component, the sustained-release component comprising quercetin; and at least one rapid-release component, the rapid-release component comprising an extract from perilla or a component selected from the following: luteolin, rosmarinic acid, apigenin, catechins, acid, caffeic acid, or a mixture thereof. KR102048977 discloses an anti-allergic composition using Ulmus davidiana bark extract and Perilla extract, which can be used to treat atopic dermatitis, asthma, allergic rhinitis, allergic conjunctivitis, allergic dermatitis, allergic contact dermatitis or allergic keratitis. CN110613775 discloses a traditional Chinese medicine formula for treating nasal diseases, which is prepared from raw materials including Perilla and several other herbal extracts.

[0008] While individual herbs have been reported in the art for the treatment of allergic rhinitis, and / or have been disclosed for use in combination with other herbs, there is no disclosure in the art indicating that a composition containing a specific combination of Nigella cuminata (Ranunculaceae), Tinospora cordifolia (Mensternaceae), Butterbur (Asteraceae), and Perilla frutescens (Lamiaceae) is useful for treating inflammation.

[0009] Inflammation can lead to common conditions such as joint pain or arthritis, allergies, asthma, allergic rhinitis, autoimmune diseases, coeliac disease, glomerulonephritis, hepatitis, inflammatory bowel disease, pre-perfusion injury, transplant rejection, etc. The most common are allergies and allergic rhinitis, and joint pain. The usual standard therapy for such inflammation is drugs such as ibuprofen, acetaminophen, cetirizine, cinnarizine, fexofenadine, etc. The present invention now provides a multi-herbal mixture as an effective agent for treating inflammation such as allergic rhinitis and joint pain.

[0010] Purpose of the Invention

[0011] It is an object of the present invention to provide compositions based on herbal extracts which are useful as anti-inflammatory agents.

[0012] Another object of the present invention is to provide a herbal anti-inflammatory composition which is safe and does not suffer from the limitations of known compositions based on synthetic compounds which cause various side effects including drowsiness, acid reflux, etc.

[0013] Another object of the present invention is to provide a herbal anti-inflammatory composition which is a multi-herb extract which is effective at a concentration lower than the concentration required for each individual component of the multi-herb extract to provide an anti-inflammatory effect.

[0014] Another object of the present invention is to provide herbal anti-inflammatory compositions which are effective in treating and managing allergic reactions such as allergic rhinitis. SUMMARY OF THE INVENTION

[0016] The present invention provides an anti-inflammatory composition based on herbal extracts, wherein the composition comprises a mixture of—

[0017] (i) Nigella sativa (Fennel Leaf) aqueous extract,

[0018] (ii) Tinospora cordifolia water extract,

[0019] (iii) an aqueous extract of Butterbur,

[0020] (iv) Perilla frutescens aqueous extract.

[0021] In one embodiment, the anti-inflammatory composition comprises about 20% to about 35% w / w Nigella cumin aqueous extract, about 40% to about 60% w / w Tinospora cordifolia aqueous extract, about 5% to about 15% w / w Butterbur aqueous extract, and about 5% to about 20% w / w Perilla frutescens aqueous extract.

[0022] In another embodiment, the anti-inflammatory composition comprises about 0.12 μg to about 80 μg of Nigella cuminata, about 0.5 μg to about 10 μg of Tinospora cordifolia, about 0.12 μg to about 20 μg of Butterbur, and about 0.25 μg to about 10 μg of Perilla frutescens.

[0023] In another embodiment, the anti-inflammatory composition comprises a mixture of—

[0024] (i) about 20% to about 35% w / w of Nigella sativa extract,

[0025] (ii) about 40% to about 60% w / w Tinospora cordifolia extract,

[0026] (iii) about 5% to about 15% w / w of a Butterbur extract,

[0027] (iv) about 5% to about 20% w / w Perilla extract.

[0028] In further embodiments, a multi-herb extract comprising a mixture of Nigella Sativa, Tinospora cordifolia, Butterbur and Perilla frutescens is formulated into a suitable dosage form for oral administration for the treatment and / or prevention of inflammatory disorders. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] [ Figure 1 ] describe the inhibition of protein denaturation by Nigella sativa (Ranunculaceae; D1).

[0031] [ Figure 2 ] described the inhibition of protein denaturation by Tinospora cordifolia (Menamectinaceae; D2).

[0032] [ Figure 3 ] describe the inhibition of protein denaturation by Butterbur (Asteraceae; D3).

[0033] [ Figure 4 ] described the inhibition of protein denaturation by Perilla frutescens (Lamiaceae; D4).

[0034] [ Figure 5 ] described the inhibition of protein denaturation by ibuprofen.

[0035] [ Figure 6 ] described the inhibition of protein denaturation by cetirizine.

[0036] [ Figure 7 ] describe the inhibition of protein denaturation by fexofenadine.

[0037] [ Figure 8 ] describe the inhibition of protein denaturation by aqueous extracts of various herbs.

[0038] In the figure, the X-axis represents the concentration in μg, and the Y-axis represents the percentage of inhibition of protein coagulation, and n=3.

[0039] Therefore, the IC50 (i.e., x in the formula) value for each extract and standard was determined according to the formula of the graph (y=mx+c, where y is 50% inhibition of protein coagulation, m is the slope of the line, x is the IC50, and c is the y-intercept of the line). DETAILED DESCRIPTION OF THE INVENTION

[0041] The term "comprising," which is synonymous with "including," "containing," or "characterized by," is defined herein as being inclusive or open-ended, and does not exclude additional, unrecited elements or method steps unless the context clearly requires otherwise.

[0042] The term "about" means within 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1% or 0.05% of a given value or range.

[0043] Allergic rhinitis (AR) is the most common of all allergic diseases, afflicting approximately 400 million people worldwide and is associated with reduced quality of life, lower work productivity and school performance, and increased healthcare costs. Allergic rhinitis is an IgE-mediated inflammatory disease of the nasal mucosa caused by allergen interactions characterized by an inflammatory infiltrate consisting of eosinophils, T cells, mast cells, and basophils that release a variety of mediators, chemokines, and cytokines (of which histamine and cysteinyl leukotrienes are the main vasoactive mediators), modulation of local and systemic IgE synthesis, and communication with the immune system and bone marrow.

[0044] The present invention provides an anti-inflammatory composition based on herbal extracts, wherein the composition comprises a mixture of—

[0045] (i) fennel leaf nigella sativa extract,

[0046] (ii) Tinospora cordifolia extract,

[0047] (iii) Butterbur extract,

[0048] (iv) Perilla extract.

[0049] Fennel Leaf Nigella Sativa

[0050] It is commonly known as black fennel, black cumin, black seed, Roman coriander, fennel flower, black seed (nigella) or black fennel (kalonji), is a kind of annual flowering plant in the Ranunculaceae, originating in Eastern Europe (Bulgaria and Romania) and Western Asia (Cyprus, Turkey, Iran and Iraq). It is considered to be densely packed with nutrients, and is traditionally known to provide numerous health benefits in the treatment of respiratory disorders, cardiovascular disorders, neurasthenia, rheumatism, inflammation, back pain, anorexia, amenorrhea, paralysis, etc. It is regarded as a wonder drug by most traditional medicine practitioners. The seeds of fennel leaf black seed contain a large amount of protein (it contains amino acids such as glutamic acid, aspartic acid, methionine, arginine and cysteine), fiber, fat and carbohydrates and multiple vitamins and minerals. Fennel leaf black seed fixed oil is known to be used in inflammatory disorders such as sinusitis, otitis, bronchitis or asthma. The anti-inflammatory effects of Nigella sativa may be produced by reducing eicosanoid synthesis (thromboxane A2, prostaglandins, and leukotrienes) due to inhibition of COX and lipoxygenase.

[0051] Tinospora cordifolia

[0052] It is commonly known as gurjo, guduchi, guduchi or giloy and is an herbaceous vine of the family Menispermaceae, native to tropical regions of the Indian subcontinent. It has been used for centuries in traditional medicine for the treatment of a variety of disorders. All parts of the plant are very useful due to the presence of different compounds of medicinal importance belonging to different groups such as alkaloids, diterpenoid lactones, glycosides, steroids, sesquiterpenoids and phenolics. These compounds have pharmacological properties that impart antidiabetic, antipyretic, anti-inflammatory, antioxidant, hepatoprotective and immunomodulatory effects. The root extract of Tinospora cordifolia is known to have strong anti-inflammatory effects. The anti-inflammatory effects of Tinospora cordifolia are mediated by reducing the production of proinflammatory cytokines such as IL-1β, TNF-α, IL-6, and IL-17; the frequency of IL-17-producing T cells; and chemokines such as RANTES (Regulated upon Activation, Normal T Cell Expressed and Presumably Secreted).

[0053] Butterbur (P. hybridus)

[0054] It is commonly known as butterbur, a herbaceous perennial flowering plant in the Asteraceae family, native to Europe and North Asia. Although it has been used in traditional medicine for centuries to treat a variety of disorders, there is no approved medical use, but it is sold as a dietary supplement. It is traditionally used to treat malignant and cancerous ulcers, migraines, gastrointestinal, respiratory and genitourinary disorders. Butterbur is usually composed of a variety of ingredients, including sesquiterpenes belonging to three main categories - bakkenolide, eremophilane and furanoeremophilane. There are also pyrrolizidine alkaloids and phenolic compounds. Butterbur root extract is known to treat migraine in adults and has also shown potential in treating migraine in children. Other uses include the treatment of asthma, allergic rhinitis, etc. It is also used as an antispasmodic, neuroprotective, cardioprotective, anti-inflammatory, antioxidant, etc., and can be used as a functional food. The anti-inflammatory activity of Butterbur root extract has been attributed to its sesquiterpenoid components, such as peterin and isoperin. Butterbur reduces the production of inflammatory mediators prostaglandin E2 (PGE2), leukotriene B4 (LTB4), and cysteinyl leukotrienes (LT) in animal and human cell systems as well as purified enzyme preparations.

[0055] Perilla (P.frutescens)

[0056] It is commonly known as deulkkae, perilla or Korean perilla, and is a species of the genus Perilla in the mint familyLamiaceae. It is an annual plant native to Southeast Asia and the Indian highlands, and traditionally grown as a crop in the Korean Peninsula, southern China, Japan and India. The ingredients present in Perilla include rosmarinic acid, luteolin, quinacridone, quercetin, catechins, caffeic acid, ferulic acid and several other compounds. Perilla seeds contain oil, protein, sterols (such as phytosterols and tocopherols), squalene and polyunsaturated fatty acids. Its leaves contain abundant amounts of carotenoids. The oil obtained from the seeds shows antioxidant, anti-inflammatory, antimicrobial, antifungal and anticancer activities. Other pharmacological effects include antispasmodic, antidepressant, neuroprotective, insecticidal, hepatoprotective and antiallergic activities. Each of these activities can be attributed to the numerous plant components present. Several anti-inflammatory components of perilla leaves were also identified, including luteolin and potentilla acid, which are known to inhibit overproduction of tumor necrosis factor alpha, a cytokine important in immune and inflammatory responses.

[0057] Although individual herbs have been reported in the art for treating allergic rhinitis and / or inflammation, there is no disclosure in the art indicating that a composition containing a specific combination of Nigella cuminata (Ranunculaceae), Tinospora cordifolia (Mensternaceae), Butterbur (Asteraceae) and Perilla frutescens (Lamiaceae) is useful for treating inflammation.

[0058] Aqueous extracts of the four individual herbs, namely Nigella cuminata (Ranunculaceae), Tinospora cordifolia (Mensternaceae), Butterbur (Asteraceae) and Perilla frutescens (Lamiaceae), were obtained by conventional and commercially feasible methods in the art. The individual extracts were collected and stored under conditions such that they remained physically and chemically stable. Stable means that there was no microbial growth and no precipitation was observed during storage.

[0059] A multi-herb extract is obtained by mixing (i) about 20% to about 35% w / w of an aqueous extract of Nigella cuminata, (ii) about 40% to about 60% w / w of an aqueous extract of Tinospora cordifolia, (iii) about 5% to about 15% w / w of an aqueous extract of Butterbur frondosa, and (iv) about 5% to about 20% w / w of an aqueous extract of Perilla frutescens. The multi-herb extract was tested for its anti-inflammatory effect as described in the Examples below. The terms "multi-herb extract" and "multi-herb mixture" may be used interchangeably.

[0060] Reactive oxygen species are also known to activate matrix metalloproteinase damage seen in various arthritic tissues. Inflammation is a protective response to tissue damage, and it involves a series of complex enzyme activation, mediator release, cell migration, fluid extravasation, tissue decomposition and repair. The principle behind the implementation of this assay is that albumin denaturation can lead to the formation of antigens, which trigger type III hypersensitivity reactions, leading to inflammation. And therefore, the inhibition of the denaturation process by the agent shows its anti-inflammatory properties; the higher the degree of inhibition of protein denaturation, the greater its anti-inflammatory potential (see Jayashree V, Bagyalakshmi S, Manjula Devi K, Richard Daniel; In vitro anti-inflammatory activity of 4-benzylpiperidine, Asian JPharm Clin Res, Vol. 9, Supplement 2, 2016, 108-110).

[0061] Protein denaturation is the process by which proteins lose their tertiary and secondary structures due to the application of external stress or compounds such as strong acids or bases, concentrated inorganic salts, organic solvents or heat. Most biological proteins lose their biological function when denatured. Protein denaturation is a well-documented cause of inflammation. This principle is used to perform in vitro tests on single components and multi-herbal mixtures to determine IC50 values, the amount required to cause 50% inhibition of protein denaturation.

[0062] Select herbs according to quality inspection (raw material identification and appraisal, heavy metal analysis, pesticide residue inspection and active ingredient content analysis). Select the seeds of fennel leaf black cumin, the stem bark of heart leaf ox gall, the aerial parts of butterbur and the seeds of perilla for extracting their components. The sliced ​​herbal material (i.e. stem bark and aerial parts, in the case of use) is dried in an oven at a temperature not exceeding 50°C. After being crushed in a grinder, it is sieved through 80 meshes. After impregnation, the required amount of powdered medicine is impregnated with a sufficient amount of (one or more) suitable solvents. The seeds are crushed, powdered and impregnated, then suspended in a solvent and subjected to ultrasonic treatment (ultrasonification). The obtained extract is filtered, and the filtrate is concentrated to dryness. Although the dry powders of fennel leaf black cumin, heart leaf ox gall and butterbur are obtained, the semi-solid extract of perilla is obtained because it contains a large amount of oil.

[0063] Solvents suitable for obtaining the above-mentioned herbal extracts include, but are not limited to, water, ethanol, propanol, isopropanol, dimethylformamide, dimethyl sulfoxide, dichloromethane, acetone, hexane, ether and other solvents conventionally used for extracting herbal products. These solvents can be used alone or in combination in different amounts as needed. Technicians are well versed in the knowledge of evaluating suitable solvents required for extraction and can use these solvents (see Abubakar et al., J Pharm Bioallied Sci. 2020 January-March; 12(1): 1-10). In a preferred embodiment of the present invention, the solvent used is selected from water, alcohol or a mixture thereof. In a highly preferred embodiment, water is used as a solvent for extracting fennel leaf black grass, heart leaf bluegrass, beeswax and perilla. The individual extracts thus obtained are then mixed in a defined amount to obtain the multi-herb extract of the present invention. In a preferred embodiment, 0.12 μg to about 80 μg of Nigella cumin, about 0.5 μg to about 10 μg of Tinospora cordifolia, about 0.12 μg to about 20 μg of Butterbur, about 0.25 μg to about 10 μg of Perilla frutescens are mixed to obtain a multi-herb extract.

[0064] Macroscopic determination of herbal materials is based on shape, size, color, surface characteristics, texture, fracture characteristics, and cut surface appearance. However, because these characteristics are subjective and substitutes or adulterants may closely resemble the authentic material, it is often necessary to confirm findings by microscopy and / or physicochemical analysis. Herbal extracts are additionally evaluated for extraneous organic matter, total ash content, loss on drying, and extractable value by conventional methods known in the art.

[0065] Each individual extract is standardized to ensure the reproducibility of the final product to be used in the multi-herb extract of the present invention. Biomarkers for multi-herb extracts were determined and analyzed using standard methods in the art. For the multi-herb extract of the present invention, four biomarkers were selected for standardization, namely rosmarinic acid, berberine, butterbur and thymoquinone. Therefore, the multi-herb extract of the present invention has standardized amounts of these biomarkers, which are the biologically active components of the extract. Standardization ensures that the multi-herb extract contains the same amount of active substances each time it is prepared. This is important in herbal preparations because the amount of active substances can vary based on the content in the plant, stem bark, aerial parts or seeds (depending on the amount used), as well as based on environmental factors that can affect the natural content of active substances in these herbs. If the standardization process is not carried out, there is a high probability of batch-to-batch variation, i.e. the content of active substances may vary from batch to batch, thereby affecting the anti-inflammatory activity of the multi-herb extract. Therefore, the present invention provides for the first time standardized extracts of the four herbs for anti-inflammatory use.

[0066] The present invention provides a multi-herb extract containing Nigella Sativa, Tinospora cordifolia, Butterbur and Perilla frutescens, wherein the anti-inflammatory effect of the combination is significantly higher than the anti-inflammatory effect obtained with each individual extract. As can be seen in the examples provided below, the multi-herb extract provides a synergistic effect in the treatment of inflammation, i.e. the amount of the multi-herb extract required to provide an anti-inflammatory effect (measured from the perspective of IC50, as described above) will be much lower than the amount of each individual extract required to provide the same effect. In addition, the multi-herb extract of the present invention also provides a better anti-inflammatory effect than known standard drugs (such as ibuprofen, fexofenadine and cetirizine). Therefore, the multi-herb extract is expected to provide a better effect than the individual herbs as well as standard drugs.

[0067] The multi-herb extract of the present invention can be additionally formulated with suitable pharmaceutically acceptable excipients to obtain dosage forms such as capsules, tablets, granules, sachets of powder, oral liquid solutions, oral liquid suspensions, dry suspensions for reconstitution, etc. In general, excipients such as binders, diluents, disintegrants, lubricants, flow enhancers, solubility enhancers, permeability enhancers, sweeteners, flavors, coating agents, etc. can be used. The amount of these excipients can be appropriately set according to the type of excipients used, and as known in the pharmaceutical field.

[0068] In a preferred embodiment, the composition is formulated as a tablet.

[0069] In another preferred embodiment, the composition is formulated as a capsule. The capsule may contain powder, granules, pellets or tablets.

[0070] The composition may contain a binder, which may be selected from, but not limited to, starch, pregelatinized starch, sodium alginate, gelatin, polyvinyl pyrrolidone (PVP), methylcellulose, hydroxypropyl methylcellulose (HPMC), polymethacrylate, sodium carboxymethylcellulose, polyethylene glycol (PEG), and methylcellulose or a mixture thereof. The binder may be present in an amount of about 0.1% to about 5% by weight of the composition.

[0071] The composition may further contain a filler selected from disaccharides such as lactose and sucrose; polysaccharides and their derivatives such as starch, cellulose or modified cellulose such as microcrystalline cellulose (MCC) and cellulose ethers such as hydroxypropyl cellulose (HPC); sugar derivatives such as sorbitol, xylitol and mannitol, etc. The filler may be present in an amount of about 5% to about 95% by weight of the composition.

[0072] The composition may include a disintegrant selected from the group consisting of cross-linked carboxymethylcellulose sodium, starch, sodium starch glycolate, crospovidone, carboxymethylcellulose calcium, carboxymethylcellulose sodium, magnesium aluminum silicate, or a mixture thereof, and more preferably cross-linked carboxymethylcellulose sodium and / or starch. The disintegrant may be present in an amount of about 0.1% to about 20% by weight of the composition.

[0073] The composition may also include a flow enhancer in an amount of about 0.1% to about 5% by weight of the composition. The flow enhancer may be, for example, colloidal anhydrous silica, silicic acid, talc, or a mixture thereof.

[0074] The composition may also contain a lubricant in an amount of about 0.1% to about 5% by weight of the composition. Examples of lubricants for use according to the present invention include, but are not limited to, magnesium stearate, calcium silicate, talc, fumed silicon dioxide, calcium stearate, stearic acid, sodium stearyl fumarate. Preferably, the lubricant (if present) is magnesium stearate or stearic acid, more preferably magnesium stearate.

[0075] The composition may optionally contain a solubility enhancer such as a medium chain fatty acid or a salt, ester, ether or derivative of a medium chain fatty acid having a carbon chain length of about 4 to about 20 carbon atoms. The absorption enhancer, if present, may be selected from one or more of sodium decanoate, sodium dodecanoate, sodium palmitate, chitosan and its derivatives, fatty acids, surfactants, liposomes, triglycerides, polyethers, bile salts, triglycerides and mixtures thereof.

[0076] Sweeteners or sweeteners that can be used in the compositions of the present invention include any compound that provides sweetness. It includes but is not limited to nutritive sweeteners, non-nutritive sweeteners and mixtures thereof. Nutritive sweeteners can be selected from but are not limited to dextrose, fructose, sucrose, brown rice syrup, date sugar (date sugar), honey, maple syrup, coconut sugar, erythritol, maltitol, mannitol, sorbitol, xylitol, isomalt, lactitol and mixtures thereof. Non-nutritive sweeteners can be selected from but are not limited to acesulfame, aspartame, neotame (neotame), saccharin, saccharin sodium, sucralose, acesulfame potassium, stevioside and mixtures thereof.

[0077] Flavoring agents or spices may be added to enhance the taste or aroma of the compositions of the present invention. Non-limiting examples of suitable natural flavors, some of which can be easily simulated with synthetic agents or combinations thereof, include almond, fennel, apple, apricot, bergamot, blackberry, blackcurrant, blueberry, cocoa, caramel, cherry, cinnamon, clove, coffee, coriander, cranberry, cumin, dill, eucalyptus, fennel, fig, ginger, mango, grape, grapefruit, guava, hops, lemon, licorice, lime, malt, mandarin, molasses, nutmeg, mixed berry, orange, peach, pear, mint, pineapple, raspberry, rose, spearmint, strawberry, tangerine, tea, vanilla, winter green, and the like, and combinations thereof.

[0078] The compositions of the present invention may have one or more coatings, such as film coatings or sugar coatings. Useful coating agents in the coating process include, but are not limited to, polysaccharides, alkyl celluloses such as methyl or ethyl cellulose, hydroxyalkyl celluloses (e.g., hydroxypropyl cellulose or hydroxypropyl methyl cellulose); polyvinyl pyrrolidone, gum arabic, corn, sucrose, gelatin, shellac, polyvinyl alcohol (PVA), copolymers of vinyl pyrrolidone and vinyl acetate (e.g., sold under the brand name of plasdone).

[0079] Compositions of the present invention were evaluated using a milk-induced eosinophilia and leukocytosis animal model to assess anti-inflammatory activity.

[0080] Injecting milk into mice induces an allergic reaction, which in turn leads to leukocytosis. Leukocytosis is a condition in which there are large numbers of leukocytes in the blood. Leukocytes are white blood cells. Leukocytosis can be a temporary response to an allergy, infection, or injury while the body heals naturally, or it can be a sign of disease.

[0081] Cell viability is defined as the number of healthy cells in a sample. Measurement of cell viability plays an important role in all forms of cell culture. Cell viability assays are basically used to screen the response of cells to drugs or chemical agents. In particular, the pharmaceutical industry widely uses viability assays to evaluate the effects of developed agents on cells.

[0082] There are several types of assays that can be used to determine the number of viable cells. These assays are based on various functions of cells, including enzyme activity, cell membrane permeability, cell adhesion, adenosine triphosphate (ATP) production, coenzyme production, and nucleotide uptake activity. Although there are different classifications, cell viability assays can be broadly categorized into (a) dye exclusion assays, (b) colorimetric assays, (c) fluorescence assays, (d) luminescence assays, and (e) flow cytometry assays. Dye exclusion assays are the simplest method, which are based on the use of different dyes such as trypan blue, eosin, Congo red, and erythrosin B, which are excluded by viable cells but not by dead cells.

[0083] The trypan blue staining assay has been developed to measure viable cell counts and is used as a confirmatory test for measuring changes in viable cell numbers caused by drugs or toxins. Trypan blue stain is a negatively charged macromolecule and is one of the simplest assays for determining the number of viable cells in a cell suspension. The principle of the assay is that viable cells have intact cell membranes that reject the trypan blue stain, while dead cells do not. The cell suspension is mixed with the trypan blue stain and visually inspected under light microscopy to determine whether the cells contain or reject the stain. Viable cells will have a clear cytoplasm, while non-viable cells will have a blue cytoplasm.

[0084] The following examples illustrate the nature of the invention and are given solely for the purpose of illustrating the invention in more detail and are not limiting and relate to protocols which are particularly effective on a laboratory scale.

[0085] Example 1

[0086] Fennel leaf, Nigella sativa seed, Tinospora cordifolia stem bark, Butterbur aerial parts and Perilla frutescens seed were extracted with water. These extracts are described in Table 1 below:

[0087] [Table 1]

[0088]

[0089]

[0090] The multi-herb extract was obtained by mixing the 4 extracts in the amounts described in the following Table 2: [Table 2]

[0091] Extract Name Amount(%w / w) Fennel Leaf Nigella Sativa 28.59 Tinospora cordifolia 51.36 Butterbur 8.56 Perilla 11.47

[0092] Example 2

[0093] 1. Preparation of multi-herbal tablets

[0094] A. Preparation of Blends for Tablets

[0095] The extracts of all four herbs were mixed and the blend was passed through a #40 mesh screen to ensure uniformity of the particles. No excipients were added.

[0096] B. Pre-compression parameters of the blended mixture

[0097] The parameters of the multi-herb extract mixture such as bulk density, tap density, Carr's index, Hausner's ratio and angle of repose were evaluated by conventional methods to determine the flow properties and compressibility of the prepared blend.

[0098] [Table 3]

[0099]

[0100]

[0101] C. Evaluation of multi-herbal tablets

[0102] [Table 4]

[0103] Evaluation parameters result Weight change (mg) 100 Diameter(mm) 12 Thickness(mm) 4 Hardness(kg / cm2) 6 Friability (%) 0.3-0.4 Disintegration (seconds) 360

[0104] D. For dissolution study of tablets:

[0105] Use USP dissolution apparatus II to carry out in vitro drug dissolution study under sink condition. Use phosphate buffer solution with pH 6.8 as dissolution medium. Prepare the dissolution medium by dissolving 28.80g disodium hydrogen phosphate and 11.45g potassium dihydrogen phosphate in enough water to produce 1000ml. Stir the dissolution medium continuously under 100rpm at 37 ± 5 ℃. Extract 5ml sample and filter at different time intervals, and compensate with fresh medium of same volume. Then use UV spectrophotometer to measure sample at 282nm for three times.

[0106] Table 5: Dissolution results of multi-herb tablets

[0107] Time (minutes) Absorbance at 282 nm 0 0.0191 5 0.4470 10 0.6297 15 0.6852 20 0.7865 25 0.9436 30 1.0252 45 1.1365 60 1.1364

[0108] 2. Preparation of multi-herbal capsules

[0109] a. Formulation of powder blend for capsules:

[0110] The herbal extracts were weighed and mixed thoroughly to ensure uniformity of the particles. The mixture was then passed through a #40 mesh screen to achieve the desired particle size distribution. The powder was filled into capsule shells.

[0111] b. Evaluation of capsules

[0112] Capsule size and weight were within the recommended pharmacopoeial range. Individual capsule weights did not show significant differences compared to the average capsule weight.

[0113] c. Dissolution study of capsules:

[0114] In vitro drug dissolution studies were performed using USP dissolution apparatus I under sink conditions. Phosphate buffer solution with pH 6.8 was used as the dissolution medium. The dissolution medium was prepared by dissolving 28.80 g of disodium hydrogen phosphate and 11.45 g of potassium dihydrogen phosphate in sufficient water to produce 1000 ml. The dissolution medium was stirred continuously at 100 rpm at 37 ± 50 °C. 5 ml samples were drawn at different time intervals and filtered, and compensated with the same volume of fresh medium. The samples were then measured three times at 282 nm using a UV spectrophotometer.

[0115] Table 6: Dissolution results of multi-herb capsules

[0116] Time (minutes) Absorbance at 282 nm 0 -0.0063+0.0004 5 0.2945+0.0001 20 0.6070+0.0002 25 0.9917+0.0008 30 1.1666+0.0005 45 1.2463+0.0009 60 1.2053+0.0006

[0117] Example 3

[0118] The anti-inflammatory effects of individual plant extracts of Nigella cuminata, Tinospora cordifolia, Butterbur and Perilla frutescens as well as a multi-herb mixture of said four extracts (the composition of Example 1) were investigated in vitro. The ability of the individual extracts and multi-herb mixture to inhibit protein denaturation was investigated.

[0119] Stock solutions of each of the individual extracts were prepared by dissolving 200 mg of each extract in 100 ml of distilled water. Stock solutions of the multi-herb extracts were prepared by dissolving 200 mg of the multi-herb mixture (described in Example 1 above) in 100 ml of distilled water. The standard solutions were solutions of ibuprofen, cetirizine and fexofenadine each at a concentration of 100 μg per ml.

[0120] 5 gm of bovine serum albumin powder was dissolved in 100 ml of distilled water and adjusted to pH 7 using 0.1 M HCl to obtain a 5% solution. One tablet of phosphate buffered saline (PBS) was dissolved in 1000 ml of distilled water to obtain a PBS solution of pH 7.2.

[0121] 2 mL of 5% bovine albumin was mixed with 0.12, 0.25, 0.3, 0.5, 1, 5, 10, 20, 40 and 80 micrograms of each of the following: (1) the 4 individual extracts, (2) the multi-herb extract and (3) the standard solution, and the final volume was made up to 5 ml with PBS. The mixture was then incubated at 37°C in a water bath for 15 minutes and then heated at 70°C for 5 minutes. The turbidity (if any) was made uniform by using a vortex. The solution was cooled and the turbidity was measured at 660 nm using a UV / VIS spectrometer (Optima, SP-3000, Tokyo, Japan). PBS was used as a control. The percentage inhibition of protein denaturation was calculated by using the following formula:

[0122] Denaturation inhibition % = [(blank - sample) x 100] / blank

[0123] in

[0124] Blank = Absorbance of control

[0125] Sample = Absorption of each single extract and multi-herb extract test sample

[0126] The % inhibition for each concentration of each extract calculated as above was plotted on a graph (% inhibition of protein coagulation versus concentration in micrograms), and the IC50 value for each extract was thus determined by calculating the slope of the graph.

[0127] It was found that Nigella sativa (Ranunculaceae) inhibited protein denaturation by 2.31% to 84.34% at doses ranging from 0.12 μg to 80 μg, respectively. Figure 1 ]), it was found that protein denaturation was inhibited by 50% at a concentration of 10.97 μg.

[0128] It was found that the inhibition of protein denaturation by Tinospora cordifolia (Menispermaceae) ranged from 4.4% to 66.55% at doses ranging from 0.5 μg to 10 μg. Figure 2 ]), it was found that for Tinospora cordifolia (Menamectinaceae), protein denaturation was inhibited by 50% at 6.97 μg.

[0129] It was found that the inhibition of protein denaturation by Butterbur (Asteraceae) ranged from 2.31% to 78.12% at doses ranging from 0.12 μg to 20 μg. Figure 3 ]), it was found that for Butterbur (Asteraceae), protein denaturation was inhibited by 50% at 11.2 μg.

[0130] It was found that the inhibition of protein denaturation by Perilla frutescens (Lamiaceae) ranged from 0.4% to 66.66% at doses ranging from 0.25 μg to 10 μg. Figure 4 ]), it was found that for Perilla (Lamiaceae), protein denaturation was inhibited by 50% at 7 μg.

[0131] It was found that ibuprofen inhibited protein denaturation by 0.04% to 96.03% at doses ranging from 0.12 μg to 80 μg. Figure 5 ]), it was found that for ibuprofen, protein denaturation was inhibited by 50% at 9.73 μg.

[0132] It was found that the inhibition of protein denaturation by cetirizine ranged from 1 μg to 32 μg by 11.23% to 74.28%, respectively. Figure 6 ]), it was found that for cetirizine, protein denaturation was inhibited by 50% at 11 μg.

[0133] It was found that the inhibition of protein denaturation by fexofenadine ranged from 4.91% to 69.75% at doses ranging from 1 μg to 32 μg. Figure 7 ]), it was found that for fexofenadine, protein denaturation was inhibited by 50% at 16.83 μg.

[0134] It was found that the multi-herb aqueous extract of Example 1 inhibited protein denaturation by 9.04% to 86.84% at doses ranging from 0.1 μg to 10 μg. Figure 8 ]), it was found that for a multi-herb extract, protein denaturation was inhibited by 50% at 4.3 μg.

[0135] While all extracts were found to have significant anti-inflammatory effects, the multi-herb extract showed better anti-inflammatory effects than those produced by the individual extracts and better than the control standard. Details of the IC50 values ​​obtained are provided in Table 7 below. It is clear that the multi-herb extract showed a synergistic effect in inhibiting protein denaturation, i.e. the inhibitory effect was much greater than the additive effect of the four extracts.

[0136] Table 7: IC50 values ​​for individual extracts, multi-herb mixtures and control standards

[0137]

[0138]

[0139] Example 4

[0140] Evaluation of antiallergic / anti-inflammatory activities of herbal extracts.

[0141] The aim of this study was to evaluate the anti-allergic activity of the multi-herb extract of Example 1 by calculating eosinophil and leukocyte counts from mouse model studies in comparison with standard drugs i.e. levocetirizine and fexofenadine.

[0142] A study was conducted to examine the effect of the extract on milk-induced eosinophilia and leukocytosis in mice by determining the counts of eosinophils and leukocytes. For this model, 54 Swiss albino mice weighing in the range of 20-25 gm were used and divided into 9 groups (6 mice / group). Eosinophil and leukocyte counts were examined before and after treatment (i.e. 24 hours after administration with the multi-herbal preparation and standard drugs).

[0143] All animals were housed in an animal room under standard conditions: temperature (24 ± 1 ° C), relative humidity (45-55%), light (12 h) and dark (12 h) cycles, and free access to food and water was allowed ad libitum. After one week of acclimatization in the animal room, the animals were used for the experiment. The extracts were administered according to a standard protocol, and the signs and symptoms of the animals and their weekly body weights were observed.

[0144] Test item preparation:

[0145] A multi-herb extract containing 88 mg / kg Nigella cuminata, 158 mg / kg Tinospora cordifolia, 26 mg / kg Butterbur and 35 mg / kg Perilla frutescens was suspended in carboxymethylcellulose. The suspension thus contained 307 mg / kg of the extract.

[0146] Similarly, standard drugs levocetirizine and fexofenadine were prepared as required.

[0147] I] The animal groups and treatment models studied are provided in Table 8.

[0148] Table 8:

[0149]

[0150]

[0151] Treatments of multi-herbal extract and standard drug preparation were orally administered 1 hour before injection of boiled and cooled milk. After 24 hours, blood samples were collected from the retro-orbital plexus under ketamine-xylazine anesthesia. Total eosinophils per cubic millimeter and differential leukocyte counts were analyzed using a blood analyzer before and after treatment.

[0152] Table 9: Eosinophil and WBC counts

[0153]

[0154] Total Eosinophil Count: Eosinophil count in the disease control animal group increased significantly, while the multi-herb extract treated group effectively suppressed the increase in eosinophil count. The multi-herb extract was found to be more effective than the standard drugs levocetirizine and fexofenadine.

[0155] Total leukocyte count: The leukocyte count increased significantly in the disease control group of animals, while the multi-herb extract treated group effectively suppressed the increase in leukocyte count. The multi-herb extract showed a better anti-inflammatory effect than the control standard.

Claims

1. An anti-inflammatory composition comprising the following mixture: (i) about 20% to about 35% w / w of Nigella sativa extract, (ii) about 40% to about 60% w / w Tinospora cordifolia extract, (iii) about 5% to about 15% w / w of a Butterbur extract, (iv) about 5% to about 20% w / w Perilla extract.

2. The anti-inflammatory composition of claim 1, wherein the composition is in the form of a dosage form selected from the group consisting of capsules, tablets, granules, powders in sachets, oral liquid solutions, oral liquid suspensions, and dry suspensions for reconstitution.

3. The anti-inflammatory composition of claim 2, wherein the composition is a tablet.

4. The anti-inflammatory composition of claim 2, wherein the composition is a capsule.

5. The anti-inflammatory composition of claim 3, wherein the tablet further comprises one or more excipients selected from the group consisting of a binder, a diluent, a disintegrant, a lubricant, a flow enhancer, a solubility enhancer, a permeability enhancer, a sweetener, a flavor, and a coating agent.

6. The anti-inflammatory composition of claim 4, wherein the capsule further comprises one or more excipients selected from the group consisting of a binder, a diluent, a disintegrant, a lubricant, a flow enhancer, a solubility enhancer, a permeability enhancer, a sweetener, and a flavor.

7. The anti-inflammatory composition of claim 1, wherein the composition comprises about 0.12 μg to about 80 μg of Nigella Sativa.

8. The anti-inflammatory composition of claim 1, wherein the composition comprises about 0.5 μg to about 10 μg of Tinospora cordifolia.

9. The anti-inflammatory composition of claim 1, wherein the composition comprises about 0.12 μg to about 20 μg of Butterbur.

10. The anti-inflammatory composition of claim 1, wherein the composition comprises about 0.25 μg to about 10 μg of Perilla.

11. The anti-inflammatory composition of claim 1, wherein the composition is used for the treatment and management of allergies.

12. The anti-inflammatory composition of claim 1, wherein the composition comprises about 0.12 μg to about 80 μg of Nigella cumin, about 0.5 μg to about 10 μg of Tinospora cordifolia, about 0.12 μg to about 20 μg of Butterbur, and about 0.25 μg to about 10 μg of Perilla frutescens.

13. The anti-inflammatory composition of claim 12, wherein the composition has an IC50 value of about 4.3 μg.

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