Compositions Comprising Boswellia Serrata and Aegle Marmelos

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(11) EP 2 303 298 B1

(12) EUROPEAN PATENT SPECIFICATION

(45) Date of publication and mention (51) Int Cl.: of the grant of the patent: A61K 36/324 (2006.01) A61K 36/75 (2006.01) 16.12.2015 Bulletin 2015/51 A61K 36/9068 (2006.01) A61K 36/38 (2006.01) A61K 31/19 (2006.01) A61P 11/00 (2006.01) (2006.01) (21) Application number: 09769808.8 A61K 45/06

(22) Date of filing: 02.06.2009 (86) International application number: PCT/IN2009/000318

(87) International publication number: WO 2009/157016 (30.12.2009 Gazette 2009/53)

(54) Compositions comprising Boswellia serrata and Aegle marmelos extracts for use in the treatment of asthma and similar diseases Zusammensetzungen enthaltend Boswellia serrata und Aegle marmelos Extrakte zur Verwendung in der Behandlung von Asthma und ähnlichen Erkrankungen Compositions comprenant des extraits de Boswellia serrata et d’Aegle marmelos pour l’utilisation dans le traitement d’asthme et des maladies similaires

(84) Designated Contracting States: • GOKARAJU, Ganga Raju AT BE BG CH CY CZ DE DK EE ES FI FR GB GR Vijayawada 520 010 HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL Andhra Pradesh (IN) PT RO SE SI SK TR (74) Representative: Rees, Kerry et al (30) Priority: 03.06.2008 US 58271 P WP Thompson 55 Drury Lane (43) Date of publication of application: London WC2B 5SQ (GB) 06.04.2011 Bulletin 2011/14 (56) References cited: (73) Proprietor: M/s Laila Nutraceuticals JP-A- 10 072 357 US-A1- 2006 040 000 Vijayawada 520 010 AP (IN) • DATABASE TKDL [Online] 1995, "Nasika Rogahara Yoga-2", XP002671003, Database (72) Inventors: accession no. AK14/315B & Arkaprakasah: In: • GOKARAJU, Rama, Raju Indradeva Tripathi: "Lankapatiravana", 1995, Vijayawada 520 010 Krishnadas Academy, Varanasis, India page 117, Andhra Pradesh (IN) * the whole document * • GOLAKOTI, Trimurtulu • DATABASE TKDL [Online] 1990, Vijayawada 520 008 "Pänalaverädikasäyah", XP002671004, Database Andhra Pradesh (IN) accession no. VS/2964 & D. V. Panditarao: 1990, • CHILLARA, Sivaramakrishna Govt of India: Sahastrayoga, New Delhi page 63, Vijayawada 520 008 * the whole document * Andhra Pradesh (IN) • SENGUPTA, Krishanu Vijayawada 520 008 Andhra Pradesh (IN) • BHUPATHIRAJU, Kiran Vijayawada 520 008 Andhra Pradesh (IN)

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• AMMON H P T: "BOSWELLIASAEUREN • ARUL, V. ET AL.: ’Mechanisms of the contractile (INHALTSSTOFFE DES WEIHRAUCHS) ALS effect of the alcoholic extract of Aegle marmelos WORKSAME PRINZIPIEN ZUR BEHANDLUNG Corr. on isolated guinea pig ileum and tracheal CHRONISCH ENTZUENDLICHER chain.’ PHYTOMEDICINE vol. 11, 2004, pages 679 ERKRANKUNGEN//Boswellic acids - 683, XP004956773 (components of frankincense) as the active • SAVITHRAMMA, N. ET AL.: ’Ethnobotanical principle in treatment of chronic inflammatory survey of plants used to treat asthma in Andhra diseases", WIENER MEDIZINISCHE Pradesh’ JOUMAL OF ETHNOPHARMACOLOGY WOCHENSCHRIFT, vol. 152, no. 15-16, 1 January vol. 113, 2007, INDIA., pages 54 - 61, XP022162817 2002 (2002-01-01), pages 373-378, XP009096034, • ULBRICHT, C.: ’Boswellia: An Evidence-Based SPRINGER WIEN, AT ISSN: 0043-5341, DOI: Systematic Review by the Naturasl Standard 10.1046/J.1563-258X.2002.02056.X Research Collaboration.’ JOURNAL OF HERBAL • CHEMICAL ABSTRACTS, 27 November 2009, PHARMACOTHERAPY vol. 4, no. ISS.3, April Columbus, Ohio, US; abstract no. 147:533852, 2005, pages 63 - 83, XP009067501 SHARMA, A. ET AL.: ’Phytochemical profile of • JAINAND SUMITA SRIVASTAVA S K: "Traditional Boswellia serrata: an overview.’ XP008144679 & uses of some Indian plants among islanders of PHARMACOGNOSY REVIEWS vol. 1, no. 1, 2007, the Indian Ocean", INDIAN JOURNAL OF pages 137 - 142 TRADITIONAL KNOWLEDGE, RESOURCES, • CHEMICAL ABSTRACTS, 27 November 2009, NEW DELHI, NEW DELHI - INDIA, vol. 4, no. 4, 1 Columbus, Ohio, US; abstract no. 146:350251, October 2005 (2005-10-01) , pages 345-357, WADA, K. ET AL.: ’Bioactivities of Boswellia gum XP018021348, ISSN: 0972-5938 resin.’ XP008144680 & AROMA RESEARCH vol. 7, no. 3, 2006, pages 234 - 241

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Description hypersensitivity. [0005] The enriched Boswellia composition (5-Loxin) Field of the invention used in the present inventive composition for use contains unique blend of triterpenes, selectively enriched [0001] The invention relates to anti-adipocyte fatty ac- 5 with the most active boswellic acid called 3-O-acetyl-11- id-binding protein (aP2), anti-5-lipoxygenase-activating keto-β-boswellic acid (AKBA) and characterized by the protein (FLAP) and anti-Cysteinyl Leukotriene (CysLT)- lack of significant quantities of the components present 1 receptor expression herbal compositions comprising in regular Boswellia serrata extracts except AKBA, which 3-O-acetyl-11-keto-β-boswellic acid (AKBA) enriched is a minor component in regular extracts and a major extract of Boswellia serrata and Aegle mannelos Bael 10 component in present invention. The components fruit ethanol extract for use in the treatment of disorders present in regular extracts are 1) β-boswellic acid, 2) 3- selected from asthma, allergic rhinitis, hay fever, type-1 O-acetyl-β-boswellic acid, 3) 11-keto- β-boswellic acid, 4) hypersensitivity and mild allergies, eczema, and psoria- 3-O-acetyl-11-keto-β-boswellic acid,5) 9-ene- β-boswell- sis, and optionally in combination with extracts/frac- ic acid, 6) α 3-hydroxyurs-9,11-diene-24-oic acid, 7) tions/pure isolates of Zingiber officinale and Garcinia 15 2α,3α-hydroxyurs-12-ene-24-oic acid. The components mangostana. The compositions can take the form of Di- of unique Boswellia enriched product used in the inven- etary supplements or Pharmaceutical tive composition for use are 1) 3-O-acetyl-11-keto-β- boswellic acid, 2) 3-O-acetyl-9(11)-dehydro-β-boswellic Back ground of the invention acid, 3) 3-O-acetyl-11-keto-β-amirin, 4) 3-O-acetyl-11- 20 keto-α-boswellic acid. An enriched extract containing [0002] The inflammatory processes are known to be more than 30% 3-O-acetyl-11-keto-β-boswellic acid is triggered by increased metabolic activity of arachidonic used in the present invention (5-Loxin). Extracts enriched acid. Arachidonic acid diverges down into two main path- to higher concentration of AKBA may also be used. ways during this process, the cyclooxygenase (COX) and [0006] Aegle marmelos (Bael) is a medium sized tree lipoxygenase (LOX) pathways. The COX pathways lead 25 that grows up to 40 ft. It is native to Central and Southern to prostaglandins and thromboxane production and the India, Pakistan, Bangladesh, and Burma. The flesh of LOX pathways leads to leukotrienes (LTS) and hydroxyl the Aegle marmelos fruit is eaten raw or processed into eicosatetraenoic acid (HETEs). These classes of inflam- drinks or flavoring. Unripe pulp is used to treat diarrhea matory molecules exert profound biological effects, and dysentery. All other parts of the plant are used for a which enhance the development and progression of hu- 30 wide variety of medicinal purposes. The fruit or leaf or man cancers. Inhibition of 5-lipoxygenase indirectly re- root or bark extract or the mixed is used in the present duces the expression of TNF-α. Both 5-Lipoxygenase invention. and TNF-α therefore, are the target enzymes useful for [0007] Zingiber officinale (Ginger) has been used as a identifying inhibitors, which have the potential to cope treatment for nausea, diarrhea and epigastric and joint with a variety of inflammations and hypersensitivity-35 pains inAyurvedic and traditional Chinese medicine. Gin- based human diseases including asthma, arthritis, bowel ger has antihistaminic activity. Ginger’s oleoresin con- diseases such as ulcerative colitis and circulatory disor- stituents have potent inhibitory effect on the biosynthesis ders such as shock and ischemia. of pro inflammatory prostaglandins and leukotrienes. [0003] Asthma is the most common disease of diffuse Shogaol and Paradol inhibit COX-2 activity and Gingerol airway inflammation caused by variety of stimuli and40 inhibits 5-LOX enzyme activity (Tjendraputra, E., et al., characterized by reversible and recurrent attacks of Bioorg. Chem., 29, 156-163,2001; Srivastava, K. C., et breathlessness and wheezing (Murali P M, et. al., Res- al., Med. Hypoth, 39, 342-348, 1992). In an uncontrolled piration 2006; 73:457-463). US Census Bureau; Popu- trial, all seven arthritis patients treated with half gram of lation Estimates and International Data Base, 2004 esti- powdered ginger per day reported pain relief and de- mated that globally 373,847,408 people suffer from Asth- 45 creased signs of inflammation (Srivastava, K. C., et al., ma. An approximate 30.2 million American citizens have Med. Hypoth, 39, 342-348, 1992). Ginger root extracts been diagnosed with asthma. Children / teenagers age standardized to 5 - 15% gingerols are used to prepare 5-17 years have the highest prevalence rates. In 2004, this composition. Extracts standardized to other concen- 14.01% children were diagnosed with asthma. trations and other components could also be used for [0004] The pathophysiology of asthma consists of50 making the present inventive compositions for use chronic inflammation of airways accompanied by a de- [0008] Garcinia mangostana L belongs to Guttiferae gree of airway remodeling, which results in decreased family. It is commonly known as Mangosteen. Mangos- airway caliber. Accumulation of inflammatory cells in to teen is a slow-growing topical, evergreen tree and can the airway lining, together with inflammatory cascade attain 6 -25 m in height with leathery glabrous leaves (B. contribute to the development of edema, hyper-secretion 55 N. Sastry, The Wealth of India Raw materials, Vol. IV: pp of mucus and epithelial cell shedding which results in 103-105, 1956). The tree is mainly found in India, Myan- airway plugging (Fenech, A., et. al., British J. Clin Phar- mar, Sri Lanka and Thailand. The edible fruits of this plant macol 2002; 53: 3-15). The elevated IgE levels results in are considered to be one of the best of all tropical fruits.

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In the ayurvedic system of medicine, the fruit hull of the Boswellia serrata extract selectively enriched in 3-O- plant finds wide application, mainly as an anti-inflamma- acetyl-11-keto-β-boswellic acid including but not limited tory agent and in the treatment of diarrhea (V. Peres, et. to 5-Loxin and extracts ofAegle marmelos, optionally al., Phytochemistry 55: pp 683-710, 2000; V. Peres et. containing one or more of extracts/fractions/pure isolates al., Phytochemistry. 44: pp 191-214, 1997). Garcinia5 of Zingiber officinale, Garcinia mangostana for the pre- mangostana alcohol extract or hydroalcohol standard- vention, treatment and control of inflammation and asth- ized to α-mangostin is used in the present invention. ma. The composition can also be useful for skin care. However Garcinia mangostana extracts selectively enriched in γ-mangostin may also be used. Description of the Drawings: [0009] The use of Boswellia extract is known as a di- 10 etary supplement to control inflammatory conditions. For [0014] example, US2006/040000 discloses a composition comprising a mixture of Boswellia extract, salts of glu- Figure 1A is a representative immuno blot showing cosamine and curcuminoids. The composition also op- 5-Loxin-induced inhibition of aP2 protein in human tionally contains bromelain, chondroitin, methylsulfonyl- 15 monocytes THP-1 cells derived macrophages. THP- methane, resveratrol, extracts of white willow and ginger 1 cells were differentiated to macrophages by Phor- and quercetin. bol myristate acetate. Macrophage cells were treat- [0010] Aegle marmelos extract is also known to treat ed with 100 ng/ml of LPS in either 0.1% DMSO (con- certain medical conditions. For example, Arul, V et al. trol) treated cells or pre-treated with Montelukast (1 (fighter medicine, 11, 679-683, 2004) discloses the use 20 mM), or NDGA (10 mM) or 5-Loxin® (5 mg/ml). The of the alcoholic extract of the leaves of Aegle marmelos aP2 protein expression was specifically detected in to treat asthma. Lankapatiravana (Indradeva Tripathi, cell lysates by Western-immunoblots and normal- 1995, Krishnadas Academy, Varanasis, India, page 117) ized with the expression of actin protein (lower pan- discloses a formulation containing extracts of both Aegle el). marmelos and Zingiber officinale along with a few other 25 ingredients used for the treatment of allergenic rhinitis Figure 1B is a representative immuno blot showing and vasomotor rhinorrhoea. Panditarao (1990, Govt of inhibition of aP2 protein in human monocytes THP- India: Sahastrayoga, New Delhi, page 63) discloses an- 1 cells derived macrophages by 5-Loxin,Aegle other formulation containing extracts of bothAegle marmelos and Zingiber officinale. marmelos and Zingiber officinale along with a few other 30 ingredients used for the treatment of bronchial asthma Figure 1C is immunoblot showing Garcinia mangos- through oral administration. tana -induced inhibition of aP2 protein in human monocytes THP-1 cells derived macrophages. Objects of the invention: 35 Figure 1D is bar diagrammatic representation of the [0011] The object of the present invention is to provide comparative percentage inhibition of aP2 protein by a pharmaceutical and dietary supplement composition 5-Loxin and extracts of Aegle marmelos, Zingiber having synergistic anti-adipocyte fatty acid-binding pro- officinale and Garcinia mangostana. tein (aP2), anti-5-Lipoxygenase Activating Protein (FLAP) and anti-Cysteinyl Leukotriene (Cys LT) -1 re- 40 Figure 2 depicts representative immuno blots show- ceptor activities comprising an effective amount of a ing 5-Loxin-induced inhibition of 5-Lipoxygenase Boswellia serrata extract or fraction selectively enriched (A), Cysteinyl LT1 (B) and FLAP (C) expressions in in 3-O-acetyl-11-keto-β-boswellic acid (AKBA) and Aegle human monocytes THP-1 cells. In each respective marmelos fruit ethanol extract for use in the treatment of panel, bar diagram represent the normalized densi- disorders selected from asthma, allergic rhinitis, hay fe- 45 tometric values (in arbitrary units). ver, type-1 hypersensitivity and mild allergies, eczema, and psoriasis. Figure 3 represents in vivo anti-asthma efficacy of [0012] The composition for use may optionally be com- 5-LOXIN. The bar diagram represents the reduction bined with Zingiber officinale and Garcinia mangostana of TNF- α (A & B) and IL-4 (C & D) levels in Sephadex for improved therapeutic effect. 50 LH-20 induced airway inflammatory model in Sprague-Dawley rats supplemented with 5-LOXIN ®. Summary of the invention The TNF-α and IL-4 concentrations were measured in serum (A & C) and lung tissue lysates (B & D) by [0013] The present invention discloses anti-adipocyte specific antibody coated ELISA plates. Experiment fatty acid-binding protein (anti-aP2), anti 5-Lipoxygenase 55 includes groups with saline (vehicle) inoculated in Activating Protein (anti-FLAP) and anti-Cysteinyl Leuko- naive animals (a), Sephadex inoculated in CMC (b), triene(anti-CysLT)-1 receptor pharmaceutical, nutraceu- 50 mg/kg 5-Loxin® (c) and 100 mg/kg 5-Loxin® (d) tical and dietary supplement compositions comprising supplemented rats, respectively. Each bar repre-

4 5 EP 2 303 298 B1 6 sents mean 6 SD (n=6). * and ** represents P< 0.05, grams represent the Th1 and Th2 cytokines in lung P< 0.01 (vs. Sephadex challenge in CMC fed group), tissue protein lysates. Experiment includes, CMC respectively. supplemented group challenged with vehicle(1); CMC supplemented group challenged with Sepha- Figure 4 exhibits synergistic inhibition of aP2 ex- 5 dex (2); 100 and 200 mg/kg body weight treatment pression by Composition-1 compared to its individ- groups of Composition-1 (3 and 4); 100 and 200 ual ingredients. Panel A & B show 5-Loxin, bael and mg/kg body weight treatment groups of Composi- composition 1 inhibit aP2 expression in LPS induced tion-2 (5 and 6); 200 mg/kg body weight treatment THP-1 human monocytes-macrophage cells. The group of Composition-3 (7); and 5 mg/kg of Monte- aP2 protein expressions were measured densito- 10 lukast supplemented group(8) challenged with metrically, normalized with actin expression and rep- Sephadex. Each bar represents mean 6 SD (n=6). resented as bar diagrams. Panel C shows bar dia- * represents P< 0.05, (vs. Sephadex challenge in grammatic representation on comparative data of CMC fed group). D, represents the bar diagrammatic percent inhibition in aP2 expression in THP-1 cells presentation of the ratio of IL-4 and IFN-γ in each treated with composition-1 and its individual ingre- 15 group, as indicated. dients. Figure 9 represents Modulation of TNFα (A), IL-4 Figure 5 exhibits synergistic inhibition of aP2 ex- (B), IFN-γ (C) in Sephadex LH-20 induced airway pression by Composition-2 compared to its individ- inflammatory model of Sprague-Dawley rats supple- ual ingredients. Panel A & B show 5-Loxin, ginger 20 mented with composition-1. Bar diagrams represent and composition-2 inhibit aP2 expression in LPS in- the Th1 and Th2 cytokines in lung tissue protein duced THP-1 human monocytes-macrophage cells. lysates. a, CMC control; b, Montelukast (5 mg/kg aP2 protein expressions were measured densito- body wt.); c, Composition 1 (100 mg/kg body wt.); d, metrically, normalized with actin expression and rep- Composition 1 (200 mg/kg body wt.). Each bar rep- resented as bar diagrams. Panel C shows bar dia- 25 resents mean 6 SD (n=6). Panel D, bar diagram- grammatic representation on comparative data of matic presentation of the ratio of IL-4 and IFN- y in percent inhibition in aP2 expression in THP-1 cells each group, as indicated. treated with composition-2 and its individual ingredients. Figure 10 Illustrates Bar diagrammatic representa- 30 tions of granulocyte percentage in whole blood after Figure 6 depicts representative immunoblots show- 12 hours after sephadex challenge. The bars repre- ing the inhibition of 5-Lipoxygenase (5-LOX)(A), sent, control, montelukast 5 mg/kg, Composition-1 FLAP (B) and Cys-LT1 (C) protein expression in LPS 100 mg/kg and composition-1 200 mg/kg. Each bar induced human THP-1 monocytes by composition- represents mean6SE. N = 6, *: p<0.05. 1. The immunoblots are shown in the right panel. a, 35 vehicle control; b, LPS; c, 30% AKBA + LPS, d, al- Figure 11 Illustrates Bar diagrammatic representa- cohol extract of Aegle marmelos fruit + LPS and e, tions of granulocyte percentage in whole blood, at composition-1 + LPS. The bar diagrams in the side 24 hours after sephadex challenge. The bars repre- panels represent the normalized densitometric units sent, control, montelukast 5 mg/kg, Composition-1 (in arbitrary units) of respective proteins. 40 100 mg/kg and composition-1 200 mg/kg. Each bar represents mean6SE. N = 6, *: p<0.05. Figure 7 is representative immunoblots showing synergistic inhibition of 5-LOX (A), FLAP (B) and Cys Figure 12 Illustrates Bar diagrammatic representa- LT1 (C) protein expression in LPS induced human tions of granulocyte percentage in BAL fluid, at 48 THP-1 monocytes by composition-2.1 is vehicle 45 hours after sephadex challenge. The bars represent, control; 2 represents LPS induction; 3, 4 and 5 rep- control, montelukast 5 mg/kg, composition-1 100 resents cells treated with, 10 ug/ml of 5-Loxin®, 10 mg/kg and composition-1 200 mg/kg. Each bar rep- ug/ml of alcohol extract of Zingiber officinale and 10 resents mean6SE. N = 6, *: p<0.05 and **:p<0.01. ug/ml of composition-2 respectively. The bar diagrams in the side panels represent the normalized 50 Figure 13 Illustrates Bar diagrammatic representa- densitometric units (in arbitrary units) of respective tions of Eosinophil percentage in peripheral blood proteins. smears at 0, 12 and 24 hours after sephadex challenge. The bars represent, control, montelukast 5 Figure 8 represents in vivo anti-asthma efficacy of mg/kg, Composition-1 100 mg/kg and composition- compositions-1, 2 and 3. The bar diagram represents 55 1 200 mg/kg. Each bar represents mean6SE. N = the reduction of TNF α (A), IL-4 (B), IFN-γ (C) in drug 6, *: p<0.05 and **:p<0.01. supplemented Sprague-Dawley rats in Sephadex LH-20 induced airway inflammatory model. Bar dia-

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Detailed description of the invention: that play important roles in the pathophysiology of asthma, allergic rhinitis, and other inflammatory conditions. [0015] Asthma is a complex chronic inflammatory dis- The cysteinyl-leukotrienes (LTC4, LTD4 and LTE4) act ease of the airways that involves the activation of many at their cell-surface receptors CysLT1 and CysLT2 on inflammatory and structural cells, all of which release in- 5 target cells to contract bronchial and vascular smooth flammatory mediators that result in the typical patho- muscle, to increase permeability of small blood vessels, physiological changes. A number of inflammatory medi- to enhance secretion of mucus in the airway and gut, and ators, such as kinins, cytokines, eicosanoids, enzymes to recruit leukocytes to sites of inflammation. As cysteinyl and adhesion molecules act on specific targets leading leukotrienes (CysLTs) contribute to many of the symp- to the local release of other mediators from leukocytes, 10 toms of asthma, blocking cysteinyl leukotrienes (CysLTs) and also attract leukocytes to the site of inflammation. or their receptor function is a novel approach to thera- Leukotrienes, especially, play key role in lung function peutic strategy of asthma. It was also proved that inhibi- and diseases and are responsible for a number of the tion of CysLT1 by inhibiting TNF-α production can be effects of asthma and allergies. Asthma can be controlled useful for treatment of Arthritis [Shiota N et al, Eur J Phar- effectively by inhibiting the formation of inflammatory me- 15 macol. 2006 Oct 24;548(1-3):158-66]. diators, such as eicosanoids, prostaglandins and leuko- [0020] Th1 and Th2 cells are important in regulation of trienes, which are produced primarily from arachidonic immune responses. The Th1 cells and the pathway they acid that has been released from the cell membranes. dominate are heavily reliant on IFNγ, where as the Th2 [0016] Cytokines play an integral role in the coordina- cells are more heavily reliant on interleukin-4 (IL-4). Asth- tion and persistence of the inflammatory process in the 20 ma is a complex inflammatory disease characterized by chronic inflammation of the airways in asthma. Among bronchial hyperresponsiveness and airway inflamma- these, pro-inflammatory cytokines such as TNF- α, IL-1β, tion. The CD4+ lymphocytes with Th2 cytokine pattern IL-6, GM-CSF and CD4+, Th2 subset derived IL-4, IL-5 play a pivotal role in the pathogenesis of asthma, espe- and IL-13 lymphokines are considered as the key factors cially airway hyperresponsiveness. The Th1 cells are re- of immunopathogenesis of asthma (Knight D A, et. al., 25 quired to for recruitment of adoptively transferred Th2 J. Allergy Clin. Immunol. 2001; 108: 797-803). They are cells to the lung. Under the conditions of asthma the Th2 capable of inducing many of the pro-inflammatory effects vs Th1 ratio is in favour of Th2. The Th2/Th1 balance is characteristic of this disease and are being recognized thus an important factor, which has profound implication as important targets for treatment. TNF-α is a central on the conditions associated with inflammation, especial- mediator of airway inflammation and bronchial hyper re- 30 ly respiratory disorders. The IL-4/IFNγ ratio in the lung sponsiveness in asthma (Oosterhout A J, et. al., Pulm tissue lysates is a measure of Th2/Th1 balance. Pharmacol 1993; 6: 225-236). [0021] Based on the above information, the inventors [0017] Adipocyte Fatty acid binding protein (aP2) is a have conducted several cell based in vitro anti-inflam- member of large family of fatty acid binding proteins matory studies, particularly anti-asthma studies on many (FABPs) with distinct patterns of tissue distribution. The 35 extractsor enriched extracts includingthose derived from aP2 expressed in adipocytes regulates systemic glucose Boswellia serrata, Aegle marmelos (Bael), Garcinia man- and lipid metabolism. The aP2 protein is also expressed gostana (Mangosteen) and Zingiber officinale (Ginger). by airway epithelial cells and up-regulated following stim- These studies have unexpectedly indicated potent anti- ulation of epithelial cells with Th2 cytokines. Recent stud- aP2 activity for these extracts. Further unexpectedly, the ies suggest that aP2 plays an important role in regulation 40 compositions comprising the extracts derived from these of immune responses in bronchial epithelial cells, the site plants also showed potent anti-TNF-α activity, anti-aP2 of allergic manifestations in asthma; in addition, aP2-de- activity, anti-FLAP and anti-CysLT1 receptor activities. ficient mice are resistant to develop allergic airway in- [0022] The inventors assessed the effect of Boswellia flammation.The infiltration of leukocytes,especially eosi- serrata extract enriched to 30% 3-O-acetyl-11-keto-β- nophils into the airways is highly dependent on aP2 func- 45 boswellic acid (AKBA) (5-Loxin) and extracts ofAegle tion. The aP2 protein regulates allergic airway inflamma- marmelos, Zingiber officinale, and Garcinia mangostana tion and provides a key link between arachidonic acid on adipocyte fatty acid-binding protein aP2 expression metabolism and asthma. Hence blocking aP2 function is in human monocytes-macrophage cells in a cell based a novel approach to therapeutic strategy of asthma. in vitro model. In this experiment, THP-1 human mono- [0018] The 5-Lipoxygenase activating protein, also50 cyte-macrophage cells were pre-treated with 5 or 10 known as FLAP, is an enzyme necessary for the activa- mg/ml of each ingredient for 2h and thereafter primed tion of 5-lipoxygenase and therefore for the production with LPS to induce the inflammatory response. The cel- of leukotrienes (Peters-Golden M, et. al., Prostaglandins lular proteins were extracted by cell lysis buffer and sub- Leukot Essent Fatty Acids, 2003; 69 (2-3): 99-109). It is jected to Immuno-Western blot to detect the modulation bound to the nuclear membrane. Hence blocking or down 55 of expression of aP2 protein. The data has shown unex- regulating the FLAP is also a novel approach to thera- pectedly that 5-Loxin could able to potently inhibit the peutic strategy of asthma. LPS induced aP2 expression in THP-1 human monocyte- [0019] Cysteinyl-LTs are key inflammatory mediators macrophage cells (figure 1A), The extracts ofAegle

6 9 EP 2 303 298 B1 10 marmelos, Zingiber officinale and Garcinia mangostana position-1, comprising 5-Loxin and extract Aegle of also showed potent aP2 expression inhibitory activity marmelos, composition-2, comprising 5-Loxin and ex- (figures 1B and 1C). The comparative inhibition of aP2 tract of Zingiber officinale and composition-3, comprising by 5-Loxin, extracts of Aegle marmelos, Zingiber offici- 5-Loxin and extract of Garcinia mangostana. The com- nale and Garcinia mangostana is summarized in figure 5 positions-1 and 2 were tested for aP2 inhibition and both 1D. of them unexpectedly showed synergistic inhibition of [0023] The effect of 5-Loxin on the other key interme- aP2 protein compared to the corresponding individual diary proteins of arachidonic acid mediated inflammatory ingredients in the composition as shown in figures 4 and pathway was assessed in LPS induced-THP-1 human- 5 respectively for composition-1 and composition-2. monocyte-macrophage cells in vitro by pre-treating THP- 10 These compositions also showed synergistic inhibition 1 cells with 5-Loxin and thereafter primed the cells with of FLAP and CysLT1 as shown in figure 6 for composi- LPS to induce the inflammatory response. The cellular tion-1 and as shown in figure 7 for the composition-2. proteins were extracted by cell lysis buffer and subjected [0027] The composition-4 comprising 5-Loxin, (Aegle to immuno-western blot to detect the modulation of the marmelos) Bael and (Zingiber officinale) ginger extracts expression of 5-LOX and Cysteinyl LT1 proteins. 5-Loxin 15 and composition-9 comprising 5-Loxin, bael and Gar-( significantly down regulated 5-LOX and CysLT1 expres- cinia mangostana) mangosteen extracts also showed sions in LPS induced THP-1 human-monocyte-macro- significant down regulation of aP2, FLAP and CysLT1 phage cells (figures 2A and 2B). protein expression in LPS induced human THP-1 mono- [0024] The inventors also examined the effect of 5- cyte cells. Loxin on LPS induced expression of the 5-lipoxygenase- 20 [0028] Emerging evidence from experimental and clin- activating protein (FLAP), which is critical for leukotriene ical studies suggests that TNF- α plays a major role in the synthesis in mononuclear phagocytes. Prolonged expo- pathogenesis of human asthma, thus emphasizing the sure to the bacterial component, lipopolysaccharide significance of TNF-α as an important therapeutic target (LPS), increased FLAP gene transcription, mRNA ex- in this common medical condition (Clinical Science pression, and protein expression in the human mono- 25 (2005) 109, 135-142). Inteleukin-4 (IL-4) is another im- cyte-like THP-1 cell line. Unexpectedly, 5-Loxin signifi- portant marker for asthma. IL-4 mediates important pro- cantly down regulated the expression of FLAP compared inflammatory functions in asthma including induction of to that in the untreated control group (figure 2C). the IgE isotype switch, expression of vascular cell adhe- [0025] An in vivo experiment was conducted to check sion molecule-1 (VCAM-1), promotion of eosinophil if the anti-aP2 effects shown by 5-Loxin in vitro are rightly 30 transmigration across endothelium, mucus secretion, translated into in vivo effects in a Sephadex induced asth- and differentiation of T helper type 2 (Th2) lymphocytes ma model in Sprague-Dawley rats. In this study airway leading to cytokine release. inflammation was induced in Sprague-Dawley rats by ad- [0029] The Adipocyte Fatty acid binding protein (aP2) ministering Sephadex as a suspension in sterile saline plays an important role in the regulation of immune re- for 3 days through intrathoracic route. The control group 35 sponses in bronchial epithelial cells, the site of allergic animals were administered with sterile saline. The study manifestations in asthma and infiltration of leukocytes, animals were supplemented with 5-Loxin for ten days especially eosinophils into the airways is highly depend- prior to the asthma induction. Animals were sacrificed 48 ent on aP2 function. The inventors thus hypothesized h after saline or Sephadex challenge; blood and lung that the above compositions having potent anti-aP2 ac- tissue samples were collected from respective animals. 40 tivity in particular and anti-FLAP and anti-CysLT1 in gen- Pro-inflammatory cytokines such as TNF- αand IL-4 were eral could be potential therapeutic agents for the preven- measured in serum and protein lysates prepared from tion, treatment and control of asthma. The inventors thus lung tissues by specific and sensitive ELISA kits (R&D conducted animal experiments to check if the potent aP2 Systems, USA). The results are summarized in figure 3. activity exhibited by the foregoing compositions is rightly The serum and lung tissue levels of TNFα and IL-4 are 45 translated into the anti-asthma activity in vivo. As the air- significantly enhanced in the sephadex treated animals way inflammation has been the primary symptom of asth- compared to those in the naïve group. These levels have ma, the inventors studied the anti-asthma effect of the been significantly reduced by 5-Loxin treatment. These compositions comprising 5-Loxin and one or more ex- results supports the in vitro aP2 inhibition shown by 5- tracts derived from Aegle marmelos, Zingiber officinale Loxin in THP-1 human monocyte-macrophage cells. 50 and Garcinia mangostana (compositions-1, 2 and 3) in [0026] The inventors further explored the possibility of an airway inflammation model in Sprague-Dawley rats. developing synergistic compositions containing 5-Loxin [0030] Airway inflammation was induced in Sprague- as the base ingredient. Several combinations of the in- Dawley rats by administering Sephadex as a suspension gredients comprising 5-Loxin and one or more of the ex- in sterile saline for 3 days through intratracheal route. tracts derived from Aegle marmelos, Zingiber officinale 55 The study animals were supplemented with composi- and Garcinia mangostana were prepared compositions- tions-1, 2 and 3 for ten days prior to the asthma induction. 1 to 10 and a few of them were tested for aP2 expression Animals were sacrificed 48h after saline or Sephadex inhibition activity. A few selected compositions are com- challenge; blood and lung tissue samples were collected

7 11 EP 2 303 298 B1 12 from respective animals. Pro-inflammatory cytokines exhibited dose dependent and highly significant reduc- such as TNF-α and IL-4 were measured in serum and tion in percent eosinophils in peripheral blood(Figure lung tissue lysate by specific and sensitive ELISA kits 13). (R&D Systems, USA). The lung TNF-α and IL-4 levels [0033] In summary, both doses of composition-1 sup- were highly elevated and IFN-γ level was significantly 5 plementation reduced airway inflammation as evidenced reduced (figure 8) in the sephadex induced Asthma rats by reduction in granulocytes infiltrated into BAL fluid and (b), compared to the control animals (a). However the reduction of granulocytes (figures 10, 11 and 12) and treatment withcompositions-1, 2 and 3 significantly amel- reduction of eosinophil percentages in peripheral blood iorated the lung TNF- α (8A) and IL-4 (8B) and IFN- γ (8C) smears (figure 13). In addition, significant reduction of levels towards their normal levels. More importantly, the 10 pro-inflammatory cytokine and improvement in altered ratio of lung IL-4/IFN-y was significantly reduced in all Th2/Th1 balance further substantiates the protective ef- the treatment groups (8D). This data confirmed the anti- ficacy of the composition-1. aP2 activity exhibited by the said compositions in vitro. [0034] These observations suggest that 5-Loxin or the [0031] A similar experiment was repeated again with compositions containing 5-Loxin possess potential ap- composition-1 comprising 5-Loxin and Aegle marmelos 15 plication for the prevention, treatment and control of TNF- (Bael) fruit extract to study its effect on percentage gran- α, aP2, FLAP and CysLT1 mediated diseases such as ulocytes in blood, eosinophil percentage in Peripheral inflammation and asthma. blood and percentage granulocytes in Bronchoalveolar [0035] More importantly, the combined supplementa- Lavage fluid under the conditions of asthma, in addition tion of 5-Loxin and extracts of Aegle marmelos, or op- to further confirm its effects on lung TNF- α, IL-4 and IFN- 20 tionally with one or more of extracts of Zingiber officinale γ (9C) levels. Further more improved Th2/Th1 cytokine and Garciniamangostana provide potent anti-asthma ac- balance derived from IL-4/IFN-γ ratio in composition-1 tivity. treated animals substantiates the anti-asthmatic activity [0036] The disclosure provides a Pharmaceutical/Die- of compositioncomprising 5-Loxinand Bael. The animals tary supplement herbal compositions with potent anti- of treatment groups were supplemented with composi- 25 inflammatory, especially anti-asthma activity comprising tion-1 at 100 mg/kg or 200 mg/kg body weight for ten Boswellia serrata extract or fraction selectively enriched days prior to the induction of airway inflammation, wherein 3-O-acetyl-11-keto-β-boswellic acid (AKBA) and ex- as, the control group of animals was treated with just 10 tract(s) or fraction(s) or pure isolate(s) derived from Aegle mL /kg bodyweight of 0.5% CMC (CarboxyMethylCellu- marmelos. lose) suspended in water. 30 [0037] The disclosure provides a Pharmaceutical/Die- [0032] The induction of airway inflammation intary supplement herbal compositions with potent anti- Sprague-Dawley rats through intratracheal instillation of inflammatory, especially anti-asthma activity comprising Sephadex as a suspension in sterile saline for 3 days 5-Loxin and extracts of Aegle marmelos optionally along significantly elevated granulocyte count in blood and with one or more ingredients selected from the ex- Bronchoalveolar Lavage (BAL) fluid. However, the treat- 35 tracts/fractions/pure isolates of Zingiber officinale and ment with composition-1 potently ameliorated all the pa- Garcinia mangostana. These compositions further con- rameters towards the normal values. The data revealed tain optionally pharmaceutically, dietically and nutraceu- statistically significant and dose related efficacy of com- tically acceptable additives or excipients position-1 in various parameters tested, in comparison [0038] The compositions comprising 5-Loxin and ex- to control group (Vehicle). Estimation of biomarkers re- 40 tracts of Aegle marmelos combined with one or more of vealed that composition-1 can significantly inhibit the el- the known anti-inflammatory or anti-asthma or immune evated TNF-α and IL-4 in the lung tissue samples (Figure modulating herbal extracts or fractions are also being 9A and 9B). The composition-1 also enhanced the IFN γ considered. levels in the lung tissue of asthma induced animals (Fig- [0039] The disclosure provides inhibition of adipocyte ure 9C). The composition-1 supplemented rats also45 fatty acid-binding protein (ap2) expression and/or inhibi- showed significant improvement in Th2/Th1 cytokine bal- tion of 5-Lipoxygenase Activating Protein (FLAP) expres- ance (IL-4 vs. IFNγ ratio) and the effect is comparable sion and/or inhibition of Cysteinyl Leukotriene (Cys LT) with the rats treated with Montelukast (figure 9D). Sta- -1 receptor expression by the compositions comprising tistically significant reduction of granulocytes in periph- Boswellia serrata extract or fraction selectively enriched eral blood was observed in the groups supplemented with 50 in 3-O-acetyl-11-keto-β-boswellic acid (AKBA) and ex- various doses of composition-1 (figures 10 and 11) when tract(s) or fraction(s) or pure isolate(s) derived from Aegle compared to the control. However, the 200 mg treated marmelos. group exhibited statistically significant efficacy. Compo- [0040] The anti-asthma compositions comprising sition-1 exhibited dose dependent reduction in percent Boswelliaserrata extract enrichedin AKBA and extract(s) granulocyte in Bronchoalveolar Lavage (BAL) fluid (Fig- 55 or fraction(s) derived from Aegle marmelos (Bael) fruit ure 12). However, the 200 mg treated group exhibited extract optionally along with one or more ingredients se- statistically significant and comparable efficacy with lected from Zingiber officinale and Garcinia mangostana Montelukast 5 mg/kg treated group. Composition-1 also may further optionally be combined with one or more of

8 13 EP 2 303 298 B1 14 the known anti-inflammatory or anti-asthma or immune [0047] The composition for use according to the inven- modulating herbal extracts or fractions are also being tion further comprises optionally effective amounts of considered. pharmaceutically or dietetically acceptable anti-oxidants, [0041] The known anti-inflammatory and anti-asthma trace metals or mixtures thereof to form a formulation. extracts or fractions include those derived from but not 5 [0048] In another embodiment, of the invention, the ad- limited to Achyranthes aspera, Annona squamosa, Ano- ministration of the composition can be through oral, geissus latifolia, Bauhinia purpurea Boerhaavia diffusa, parenteral, nasal, inhalation, including nebulizers, rectal, Calotropis gigantean, Cassia fistula, Chloroxylon swiet- vaginal, transdermal, occular and any other suitable enia, Cressa cretica, Curculigo orchioides, Cynodon doc- routes. tylon, Datura metel, Euphorbia hirta, Evolvulus alsi-10 [0049] In another embodiment, the invention provides noides, Ficus religiosa, Garuga pinnata, Justicia adhato- the Pharmaceutical/Dietary Supplement dosage form of da, Madhuca latifolia, Morinda pubescens, Moringa oleif- the composition suitable for oral administration such as era,. Ocimum basilicum, Ocimum tenuiflorum, Pegularia tablets, soft capsule, hard capsule, pills, granules, pow- daemia, Phyllanthus emblica, Piper longum, Piper ni- ders, emulsions, suspensions, sprays, syrups and pel- grum, Pistacia integerrima, Prosopis julifera, Quercus in- 15 lets. The Pharmaceutical specific form is suitable for fectoria, Randia dumetorum, Solanum surattense, Ter- parenteral administration such as injections, drops, sup- minalia chebula, Tinospora malabarica, Trianthema por- positories. tulacastrum, Tribulus terrestris, Tylophora Indica, Vitex [0050] In another embodiment, the invention provides negundo and Wrightia tinctoria. nutritional/dietary supplements comprising the composi- [0042] The known immune modulating herbal agents 20 tions can be contemplated/made in the dosage form of include those derived from but not limited to Centella asi- cereals, baby foods, healthy foods, or food for specified atica, Rehmannia, Astragalus, Echinaceae, Gynostem- health uses such as solid food like chocolate or nutritional ma, Glycerrhiza, Curcuma, Prunella vulgaris, Scutellaria bars, semisolid food like cream or jam, or gel and also barbata, Cannabis sativa, Ganoderma lucidum, and Gly- beverage and the like, such as refreshing beverage, lac- cine max/Soy isoflavones and/or their mixture thereof. 25 tic acid bacteria beverage, drop, candy, chewing gum, [0043] It is disclosed that the 3-O-acetyl-11-keto-beta- chocolate, gummy candy, yoghurt, ice cream, pudding, boswellic acid (AKBA) concentration in the enriched ex- soft adzuki bean jelly, jelly, cookie and the like. tracts and/or enriched fractions of Boswellia serrata var- [0051] In another embodiment of the invention, admin- ies from 10% to 99%, preferably 20% to 60% and more istration is done to a mammal or animal in need thereof preferably 25% to 45%. 30 in the form of effective formulation or beverage or food [0044] It is disclosed that the percentage of Boswellia in a range from 0.01 to 500 mg/kg body weight/day. serrata extract enriched 3-O-acetyl-11-keto-β-Boswellic [0052] The amount of composition that will be effective acid in the composition varies from 1-95% and percent- in the treatment of a particular disorder or condition will age of the extract of Aegle marmelos varies from 5-95 %. depend on the nature of the disorder of the condition, [0045] It is disclosed that the percentage of the en- 35 which can be determined by standard clinical techniques. riched Boswellia extract(s) or fraction(s) obtained from In addition, the in vitro and in vivo assays may optionally Boswellia serrata varies in the range of 1-75% and the be employed to help identify optimal dosage ranges. The extract(s) or fraction(s) of Aegle marmelos varies in the precise dose to be employed in the formulation will de- range of 5-75%,the extract(s) of Zingiber officinale varies pend on the route of administration, and the seriousness in the range of 1-50% and extract(s) of Garcinia mangos- 40 or advancement of the diseased condition, and should tana varies in the range of 1-50%. be decided according to the practitioner and each pa- [0046] The composition for use according to claim 1 in tient’s circumstances. Effective dosages may be extrap- humans or animals in need thereof, comprises supple- olated from dose response curves derived from in vitro menting the said humans or animals with an effective or animal model test systems. For example an effective amount of a composition comprising: 45 amount of the composition of the invention is readily determined by administering graded doses of the compo- i) an effective amount of an enriched Boswellia ex- sition and observing the desired effect. tract containing atleast 10% by weight of 3-O-acetyl- [0053] The following examples, which include pre- 11-keto-β-Boswellic acid (5-Loxin) along with one or ferred embodiments, will serve to illustrate the practice more ingredients selected fromAegle marmelos, 50 of this invention, using appropriate doses/units of the se- Zingiber officinale and Garcinia mangostana; lected individual ingredients for preparing the composi- ii) Optionally can be combined with one or more of tions. It is being understood that the particulars shown the known anti-inflammatory or anti-asthma or im- are by way of example and for purpose of illustrative dis- mune modulating herbal extracts or fractions. cussion of preferred embodiments of the invention. iii) Further optionally one or more pharmaceutically, 55 These illustrations are not to limit the scope of the inven- dietically and nutraceutically acceptable addition. tives/excipient.

9 15 EP 2 303 298 B1 16

Example 1 Example 10

[0054] A composition-1 was prepared by mixing unit [0063] A composition-10 was prepared by mixing unit doses of the following components: 5-Loxin (1 g) and doses of the following components: 5-Loxin (1 g), Bael (Aegle marmelos) Bael fruit ethanol extract (1 g). 5 fruit ethanol extract (0.5 g) and excipient (0.5 g).

Example 2 Example 11

[0055] A composition-2 was prepared by mixing unit [0064] Down-regulation of aP2 protein expression in doses of the following components: 5-Loxin (1 g) and 10 THP-1 cell line by 5-Loxin: Based on the existing infor- (Zingiber officinale) Ginger extract (1 g). mation, we sought to assess the effect of 5-Loxin on aP2 expression in human monocytes-macrophage cells. 5- Example 3 Loxin-induced aP2 protein expression was evaluated in cell based in vitro model. Briefly, THP-1 human mono- [0056] A composition-3 was prepared by mixing unit 15 cyte-macrophage cells were pre-treated with either 1 mM doses of the following components: 5-Loxin (1 g) and montelukast, a leukotriene receptor antagonist or 10 mM Garcinia mangostana extract (1g). Nordihydroguaiaretic Acid (NDGA), a non-selective lipooxygenase inhibitor or 5 mg/ml of 5-Loxin for 2h and Example 4 thereafter primed with LPS to induce the inflammatory 20 response. The cellular proteins were extracted by cell [0057] A composition-4 was prepared by mixing unit lysis buffer and subjected to immuno-western blot to de- doses of the following components: 5-Loxin (1 g), Bael tect the modulation of expression of aP2 protein. alcohol extract (1 g) and ginger hydroalcohol extract (1g). [0065] The data clearly shows that lipopolysaccharide (LPS) significantly induced aP2 in human monocytes- Example 5 25 macrophage cells. The treatment of the cells with 5-Loxin and positive controls montelukast and NDGA significant- [0058] A composition-5 was prepared by mixing unit ly reduced the LPS elevated aP2 levels back to the nor- doses of the following components: 5-Loxin (1 g) and mal base line values (figure 1A). Bael fruit ethanol extract (2 g). [0066] Similar experiment was also conducted on Ae- 30 gle marmelos, Zingiber officinale and G. mangostana ex- Example 6 tracts. The outcome of the studies have established that 5-Loxin, Aegle marmelos, Zingiber officinale and Garcin- [0059] A composition-6 was prepared by mixing unit ia mangostana are superior as potential inhibitors of aP2 doses of the following components: 5-Loxin (1 g) and expression (figures 1B and 1C). The comparative per- Ginger extract (2 g). 35 centage inhibitions of aP2 expression by 5-Loxin, Aegle marmelos, Zingiber officinale and G. mangostana ex- Example 7 tracts are summarized in figure 1D.

[0060] A composition-7 was prepared by mixing unit Example 12 doses of the following components: 5-Loxin (1 g) and 40 Ginger extract (0.5 g). [0067] Inhibition of 5-Lipoxygenase, FLAP and Cystei- nyl LT1 protein expression by 5-Loxin: The effect of 5- Example 8 Loxinon thekey intermediaryproteins ofarachidonic acid mediated inflammatory pathway was assessed in LPS [0061] A composition-8 was prepared by mixing unit 45 induced-THP-1 human-monocyte-macrophage cells in doses of the following components: 5-Loxin (1 g), Bael vitro. Briefly, THP-1 human monocyte-macrophage cells fruit ethanol extracts (1 g), Ginger extract (1 g) and Gar- were pre-treated with 10 mg/ml of 5-Loxin for 1h and cinia mangostana extract (1 g). thereafter, the cells were primed with LPS for 2h to induce the inflammatory response. The cellular proteins were Example 9 50 extracted by cell lysis buffer and subjected to immuno- western blot to detect the modulation of expression of 5- [0062] A composition-9 was prepared by mixing unit Lipoxygenase, FLAP and Cysteinyl LT1 protein expres- doses of the following components: 5-Loxin (1 g), Bael sions. 5-Loxin significantly inhibited 5-Lipoxygenase, fruit ethanol extract (1g) and Garcinia mangostana ex- FLAP and Cysteinyl LT1 protein expression in LPS in- tract (1 g). 55 duced-THP-1 human-monocyte-macrophage cells as summarized in figure 2.

10 17 EP 2 303 298 B1 18

Example 13 induce the inflammatory response. The cellular proteins were extracted by cell lysis buffer and subjected to im- [0068] Anti-asthma activity: Down-regulation of pro-in- muno-westernblot todetect themodulation of expression flammatory markers in Sephadex LH-20 induced airway of 5-Lipoxygenase, FLAP and Cysteinyl LT1 protein ex- inflammation in Sprague-Dawley rats by 5-Loxin®: 5 pressions. Composition-1 significantly inhibited 5-Lipox- ygenase, FLAP and Cysteinyl LT1 protein expression in Rats were fed either with CMC or 5-Loxin at 50 mg/kg LPS induced-THP-1 human-monocyte-macrophage or at 100 mg/kg for 10 days. Thereafter, vehicle (sa- cells as summarized infigure 6. Composition-1 also line, 1 ml/kg) or Sephadex LH-20 (5 mg/kg) was giv- showed synergistic activity and more potent inhibition en via intrathoracic route. Sephadex was prepared 10 compared to that of the individual ingredients, 5-Loxin as a suspension by soaking in sterile saline for 3 and Aegle marmelos. days. Animals were sacrificed 48 h after saline or Sephadex challenge; blood and lung tissue samples Example 16 were collected from respective animals. Pro-inflammatory cytokines such as TNF-α and IL-4 were 15 [0072] Synergistic inhibition of 5-Lipoxygenase, FLAP measured in serum and protein lysates prepared and Cysteinyl LT1 protein expressions by Composition- from lung tissues. TNF-α and IL-4 were measured 2: The effect of Composition-2 on the key intermediary quantitatively by specific and sensitive ELISA kits proteins of arachidonic acid mediated inflammatory path- (R&D Systems, USA), by following protocol supplied way was assessed in LPS induced-THP-1 human-mono- by the vendor. 20 cyte-macrophage cells in vitro. Briefly, THP-1 human monocyte-macrophage cells were pre-treated with 10 [0069] 5-Loxin significantly inhibited both serum and mg/ml of Composition-2 or 5-Loxin or ginger extract for lung tissue TNF-α and IL-4 levels in the animal model 1h and thereafter, the cells were primed with LPS for 2h (Figure 3). As one can see from the following figure, the to induce the inflammatory response. The cellular pro- TNF-α and IL-4 levels were highly increased in the Asth- 25 teins were extracted by cell lysis buffer and subjected to ma induced models (b), compared to the control animals immuno-western blot to detect the modulation of expres- (a). However, the 5-Loxin treated groups of animals (c sion of 5-Lipoxygenase, FLAP and Cysteinyl LT1 protein and d) showed significantly reduced TNF- α and IL-4 lev- expressions. Composition-2 significantly inhibited 5- els. Most importantly there is a dose relation between 50 Lipoxygenase, FLAP and Cysteinyl LT1 protein expres- mg/kg 5-Loxin and 100 mg/kg 5-Loxin groups. 30 sion in LPS induced-THP-1 human-monocyte-macrophage cells as summarized in figure 7. Composition-2 Example 14 also showed synergistic activity and more potent inhibition compared to that of the individual ingredients, 5- [0070] Synergistic inhibition of aP2 protein expression Loxin and ginger. by compositions-1 and 2 in THP-1 human monocyte- 35 macrophage cells: The experimental procedure de- Example 17 scribed in example 11 repeated with composition-1 comprising 5-Loxin and bael and composition-2 comprising [0073] Anti-asthma activity of compositions-1, 2 and 3 5-Loxin and ginger and evaluated the inhibition of aP2 in Sephadex LH-20 induced airway inflammation in expression with individual compositions. The composi- 40 Sprague-Dawley rats: In an in vivo study, the efficacy of tion-1 and 2 significantly inhibited the LPS induced aP2 composition-1, composition-2 and composition-3 in expression in human monocytes-macrophage cells. Sephadex LH-20 induced airway inflammation in Composition-1 and 2 also exhibited synergistic anti-aP2 Sprague-Dawley rats. Rats were fed either with Compo- expression activity and showed potent inhibitory activity sition-1 or composition-2 or composition-3 at 50 mg/kg compared to those of the individual ingredients as shown 45 and 100 mg/kg for 10 days. The control group was treated in Figures 4 and 5. with 0.5% CMC. Thereafter, vehicle (saline, 1 ml/kg) or Sephadex LH-20 (5 mg/kg) was given via intrathoracic Example 15 route. Sephadex was prepared as a suspension by soaking in sterile saline for 3 days. Animals were sacrificed [0071] Synergistic inhibition of 5-Lipoxygenase, FLAP 50 24h after saline or Sephadex challenge; blood and lung and Cysteinyl LT1 protein expressions by Composition- tissue samples were collected from respective animals. 1: The effect of Composition-1 on the key intermediary Pro-inflammatory cytokines such as TNF α and IL-4 were proteins of arachidonic acid mediated inflammatory path- measured in protein lysates prepared from lung tissues. way was assessed in LPS induced-THP-1 human-mono- TNFα and IL-4 were measured quantitatively by specific cyte-macrophage cells in vitro. Briefly, THP-1 human 55 and sensitive ELISA kits (R&D Systems, USA), following monocyte-macrophage cells were pre-treated with 10 protocol supplied by the vendor. The IFN- γ concentration mg/ml of Composition-1 or 5-Loxin or Bael extract for 1h and the Th2 vs Th1 cytokines balance (IL-4/IFN- γ ratio) and thereafter, the cells were primed with LPS for 2h to was also evaluated and the data is summarized in figure

11 19 EP 2 303 298 B1 20

8. Claims [0074] The TNF-α (8A) and IL-4 (8B) levels were highly increased in the Asthma induced models (2), compared 1. A pharmaceutical and dietary supplement composi- to the control animals (1). Compositions-1, 2 and 3 sig- tion having synergistic anti-adipocyte fatty acid-bind- nificantly reduced both TNF-α and IL-4 in the treatment 5 ing protein (aP2), anti-5-Lipoxygenase Activating groups (3-7) compared to the untreated controlled group Protein (FLAP) and anti-Cysteinyl Leukotriene (Cys (2). Similarly, the IFN-γ concentration was significantly LT) -1 receptor activities comprising an effective reduced in the asthma induced group (8C). These levels amount of a Boswellia serrata extract or fraction se- were enhanced in all the treatment groups supplemented lectively enriched in 3-O-acetyl-11-keto-β-boswellic with compositions-1, 2 and 3 at different dose levels. The 10 acid (AKBA) and Aegle marmelos Bael fruit ethanol compositions-1, 2 and 3 also improved Th2/Th1 cytokine extract for use in the treatment of disorders selected balance (8D). from asthma, allergic rhinitis, hay fever, type-1 hypersensitivity and mild allergies, eczema, and pso- Example 18 riasis. 15 [0075] Anti-asthma activity of compositions-1 in 2. The pharmaceutical and dietary supplement compo- Sephadex LH-20 induced airway inflammation in sition for use according to claim 1, further comprising Sprague-Dawley rats: The therapeutic efficacy of com- aneffective amount ofextract(s) orfraction(s) or pure position-1 at different doses was evaluated in Sephadex- isolate(s) derived from Zingiber officinale and/or induced airway inflammation model of Sprague-Dawley 20 Garcinia mangostana. rats. Healthy Sprague Dawley rats were acclimatized for 7 days prior to the initiation of the study. All animals were 3. The pharmaceutical and dietary supplement compo- fasted overnight at free access to water. Blood samples sition for use according to claims 1 or 2, further com- were drawn before initiation of treatment (baseline eval- prising one or more pharmaceutically, dietically and uation) and samples were submitted for hematology and 25 nutraceutically acceptable additives or excipients. DC. Animals were given daily oral treatment with composition-1 at doses 100 mg/kg body weight or 200 mg/kg 4. The pharmaceutical and dietary supplement compo- body weight or standard or control for 10 days. Treatment sition for use according to any one of claims 1 to 3, was continued after induction till sacrifice. Blood samples provided in combination with an effective amount of were collected again on 10 th day of treatment and before 30 one or more known anti-inflammatory or anti-asthma induction of airway inflammation. All animal were treated or immune modulating herbal extracts or fractions. intra-tracheally either with 20 mg/kg sephadex LH-20 in saline or saline alone (for Negative Control), blood sam- 5. The pharmaceutical and dietary supplement compo- ples were collected 12, and 24 and 48 hrs after sephadex sition for use according to any one of claims 1 to 4, induction. EDTA blood samples were submitted for he- 35 provided in combination with an effective amount of matology, plain bloods were centrifuge in refrigerated one or more pharmaceutically or dietetically accept- centrifuge and aliquots were stored in -80°C for biomar- able anti-oxidants, trace metals or mixtures thereof. ker analysis. All animals were sacrificed 48 hrs after induction under euthanasia, Bronchoalveolar Lavage 6. The pharmaceutical and dietary supplement compo- (BAL) fluids were collected by injecting 2 aliquots of 5 40 sition for use according to any one of claims 1 to 5, mL PBS in to lung. Lung tissue aliquots were collected adapted for administration through oral, parenteral, and freezed using liquid nitrogen and stored at -80°C for nasal, inhalation, including nebulizers, transdermal, biomarker analysis. Additional lung sample was fixed in ocular and any other suitable route. 10%buffered formalin and stored for possiblehistopatho- logical examination. Cytokines such as TNF- α, IFN-γ and 45 7. The pharmaceutical and dietary supplement compo- IL-4were measuredin proteinsamples isolated from lung sition for use according to any one of claims 1 to 6, tissue by cytokine ELISA kits (R&D Systems, USA). The wherein the composition is in a dosage form for oral results are summarized in figure 9.The percentage gran- administration, selected from tablets, soft capsules, ulocytes in peripheral blood was measured and the data hard capsules, pills, granules, powders, emulsions, is summarized in figures 10 and 11. BAL fluids were 50 suspensions, sprays, syrups and pellets. subjected for granulocyte count using a blood cell counter (Humcount). Sediment obtained from BAL fluid was 8. The pharmaceutical and dietary supplement compo- smeared and differential counts were obtained by micro- sition for use according to any one of claims 1 to 6, scopy and the data is summarized in figure 12. The eosi- wherein the composition is in a dosage form for nophil count in blood was evaluated and summarized in 55 parenteral administration, selected from injections, figure 13. drops and suppositories.

9. The pharmaceutical and dietary supplement compo-

12 21 EP 2 303 298 B1 22

sition for use according to any one of claims 1 to 6, samen Menge an einem/einer oder mehreren be- wherein the composition is in a dosage form, select- kannten entzündungshemmenden oder Anti-Asth- ed from cereals, baby foods, healthy foods, solid ma- oder immunmodulierenden pflanzlichen Extrak- food selected from chocolate or nutritional bars, ten oder Fraktionen bereitgestellt wird. semisolid food selected from cream, jam, or gel, bev- 5 erages, lactic acid bacteria beverage, drop, candy, 5. Pharmazeutikum- und Nahrungsergänzungsmittel- chewing gum, chocolate, gummy candy, yoghurt, ice Zusammensetzung für die Verwendung nach einem cream, pudding, soft adzuki bean jelly, jelly and der Ansprüche 1 bis 4, die zusammen mit einer wirk- cookie. samen Menge an einem oder mehreren pharmazeu- 10 tisch oder diätetisch verträglichen Antioxidationsmit- 10. The pharmaceutical and dietary supplement for use teln,Spurenmetallen oder Mischungen davon bereit- according to any one of claims 1 to 9, wherein a gestellt wird. therapeutically effective amount of the composition is administered to humans or animals in need thereof 6. Pharmazeutikum- und Nahrungsergänzungsmittel- as a pharmaceutical or nutraceutical or dietary sup- 15 Zusammensetzung für die Verwendung nach einem plement. der Ansprüche 1 bis 5, die für die Verabreichung auf oralem, parenteralem, nasalem, Inhalations-, ein- 11. The pharmaceutical and dietary supplement for use schließlich Vernebler, transdermalem, okularem according to claim 10, wherein the amount of the und jedwedem anderen geeigneten Weg angepasst composition or dosage form, when dependent upon 20 ist. any of claims 7 to 9, administered to a human or an animal in need thereof is in the range of 0.01 to 500 7. Pharmazeutikum- und Nahrungsergänzungsmittel- mg/kg body weight/day. Zusammensetzung für die Verwendung nach einem der Ansprüche 1 bis 6, worin die Zusammensetzung 25 in einer Dosierungsform für die orale Verabreichung Patentansprüche vorliegt, die aus Tabletten, Weichkapseln, Hartkap- seln,Pillen, Granulat, Pulvern, Emulsionen, Suspen- 1. Pharmazeutikum- und Nahrungsergänzungsmittel- sionen, Sprays, Sirupen und Pellets ausgewählt ist. Zusammensetzung, die synergistische Aktivitäten für Anti-Adipocyte Fatty Acid-Binding Protein (aP2), 30 8. Pharmazeutikum- und Nahrungsergänzungsmittel- Anti-5-Lipoxygenase-Activating Protein (FLAP) und Zusammensetzung für die Verwendung nach einem Anti-Cysteinyl-Leukotrien (Cys LT)-1-Rezeptor be- der Ansprüche 1 bis 6, worin die Zusammensetzung sitzt, die eine wirksame Menge eines/einer Boswellia in einer Dosierungsform für die parenterale Verab- serrata-Extrakts oder -Fraktion umfasst, der/die se- reichung vorliegt, die aus Injektionen, Tropfen und lektiv mit 3-O-Acetyl-11-keto-β-boswelliasäure (AK- 35 Suppositorien ausgewählt ist. BA) und Aegle marmelos Belfrucht-Ethanolextrakt angereichert ist, für die Verwendung bei der Behand- 9. Pharmazeutikum- und Nahrungsergänzungsmittel- lung von Störungen, die aus Folgenden ausgewählt Zusammensetzung für die Verwendung nach einem sind: Asthma, allergischer Rhinitis, Heuschnupfen, der Ansprüche 1 bis 6, worin die Zusammensetzung Typ-1 Hypersensitivität und leichten Allergien, Ek- 40 in einer Dosierungsform vorliegt, die aus Folgenden zem und Psoriasis. ausgewählt ist: Getreideprodukten, Babynahrung, gesunden Nahrungsmitteln, fester Nahrung, die aus 2. Pharmazeutikum- und Nahrungsergänzungsmittel- Schokoladen- oder Nahrungsriegeln ausgewählt ist, Zusammensetzung für die Verwendung nach An- halbfester Nahrung, die aus Sahne, Marmelade oder spruch 1, die weiter eine wirksame Menge an Ex- 45 Gel ausgewählt ist, Getränken, Milchsäurebakteri- trakt(en) oder Fraktion(en) oder reinem/reinen Iso- en-Getränk, Drops, Bonbons, Kaugummi, Schoko- lat(en) umfasst, die von Zingiber officinale und/oder lade,Gummisüßigkeiten, Joghurt,Eis, Pudding, wei- Garcinia mangostana stammen. chem Adzukibohnengelee, Gelee und Keks.

3. Pharmazeutikum- und Nahrungsergänzungsmittel- 50 10. Pharmazeutikum und Nahrungsergänzungsmittel Zusammensetzung für die Verwendung nach den für die Verwendung nach einem der Ansprüche 1 bis Ansprüchen 1 oder 2, die weiter ein/einen oder meh- 9, worin die therapeutisch wirksame Menge der Zu- rere pharmazeutisch,diätisch und nutrazeutisch ver- sammensetzung an Menschen oder Tiere, die diese trägliche Additive oder Hilfsstoffe umfasst. benötigen, als Pharmazeutikum oder Nutrazeutikum 55 oder Nahrungsergänzungsmittel verabreicht wird. 4. Pharmazeutikum- und Nahrungsergänzungsmittel- Zusammensetzung für die Verwendung nach einem 11. Pharmazeutikum und Nahrungsergänzungsmittel der Ansprüche 1 bis 3, die zusammen mit einer wirk- für die Verwendung nach Anspruch 10, worin die

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Menge der Zusammensetzung oder Dosierungs- se prête à une administration orale, parentérale, na- form, bei Abhängigkeit von einem der Ansprüche 7 sale, inhalée, y compris au moyen de nébuliseurs, bis 9, die an einen Menschen oder ein Tier, der/das transdermique, oculaire et par toute autre voie ap- diese benötigt, verabreicht wird, in dem Bereich von propriée. 0,01 bis 500 mg/kg Körpergewicht/Tag liegt. 5 7. Composition qui est une formulation pharmaceutique et un supplément diététique pour l’utilisation se- Revendications lon l’une quelconque des revendications 1 à 6, la composition étant sous une forme galénique pour 1. Composition qui est une formulation pharmaceuti- 10 administration orale sélectionnée parmi des compri- que et un supplément diététique ayant des activités més, capsules molles, capsules dures, pilules, gra- anti-aP2 (forme adipocytaire de la protéine de liaison nules, poudres, émulsions, suspensions, aérosols, aux acides gras), anti-FLAP (protéine activant la 5- sirops et pastilles. lipoxygénase) et anti-Cys-LT (récepteur des cystéi- nyl-leucotriènes) de type 1 synergiques et qui com- 15 8. Composition qui est une formulation pharmaceuti- prendune quantité efficaced’un extrait ou d’une frac- que et un supplément diététique pour l’utilisation se- tion de Boswellia serrata sélectivement enrichi(e) en lon l’une quelconque des revendications 1 à 6, la acide 3-O-acétyl-11-céto-β-boswellique (AKBA) et composition étant sous une forme galénique pour un extrait éthanolique du fruit du bael,Aegle mar- administration parentérale sélectionnée parmi des melos, pour une utilisation dans le traitement d’af- 20 préparations injectables, gouttes et suppositoires. fections sélectionnées parmi l’asthme, la rhinite al- lergique, le rhume des foins, l’hypersensibilité de ty- 9. Composition qui est une formulation pharmaceuti- pe 1 et les allergies peu sévères, l’eczéma et le pso- que et un supplément diététique pour l’utilisation se- riasis. lon l’une quelconque des revendications 1 à 6, la 25 composition étant sous une forme galénique sélec- 2. Composition qui est une formulation pharmaceuti- tionnée parmi les suivantes : céréales, aliments pour que et un supplément diététique pour l’utilisation se- bébé, aliments sains, aliments solides sélectionnés lon la revendication 1, qui comprend en outre une parmi des barres chocolatées ou nutritionnelles, ali- quantité efficace d’un ou de plusieurs extraits ou ments semi-solides sélectionnés parmi une crème, fractions ou isolatspurs dérivés de Zingiber officinale 30 une confiture ou un gel, boissons, boisson contenant et/ou de Garcinia mangostana. des bactéries lactiques, bonbon à sucer, bonbon, chewing-gum, chocolat, bonbon gomme, yaourt, 3. Composition qui est une formulation pharmaceuti- crème glacée, dessert, gelée sucrée de haricots que et un supplément diététique pour l’utilisation se- adzuki, gelée et biscuit. lon les revendications 1 ou 2, qui comprend en outre 35 un ou plusieurs additifs ou excipients acceptables 10. Formulation pharmaceutique et supplément diététi- sur le plan pharmaceutique, diététique et neutraceu- que pour l’utilisation selon l’une quelconque des re- tique. vendications 1 à 9, une quantité efficace sur le plan thérapeutique de la composition étant administrée à 4. Composition qui est une formulation pharmaceuti- 40 un humain ou un animal qui le requiert sous la forme que et un supplément diététique pour l’utilisation se- d’une formulation pharmaceutique ou nutraceutique lon l’une quelconque des revendications 1 à 3, qui ou un supplément diététique. est fournie en combinaison avec une quantité efficace d’un ou de plusieurs extraits ou fractions de 11. Formulation pharmaceutique et supplément diététi- plantes ayant un effet anti-inflammatoire ou antiasth- 45 que pour l’utilisation selon la revendication 10, la matique ou modulateur immunitaire connu. quantité de la composition ou de la forme galénique, quand dépendante de l’une quelconque des reven- 5. Composition qui est une formulation pharmaceuti- dications 7 à 9, administrée à un humain ou à un que et un supplément diététique pour l’utilisation se- animal qui le requiert étant dans la plage qui va de lon l’une quelconque des revendications 1 à 4, qui 50 0,01 à 500 mg/kg de poids corporel/jour. est fournie en combinaison avec une quantité efficace d’un ou de plusieurs antioxydants, métaux tra- ces ou mélanges de ceux-ci acceptables sur le plan pharmaceutique ou diététique. 55 6. Composition qui est une formulation pharmaceutique et un supplément diététique pour l’utilisation selon l’une quelconque des revendications 1 à 5, qui

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REFERENCES CITED IN THE DESCRIPTION

This list of references cited by the applicant is for the reader’s convenience only. It does not form part of the European patent document. Even though great care has been taken in compiling the references, errors or omissions cannot be excluded and the EPO disclaims all liability in this regard.

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