WO2017028813A1 - A composition for reducing lung and/or systemic inflammation associated with pm2.5 exposure and the use thereof - Google Patents

A composition for reducing lung and/or systemic inflammation associated with pm2.5 exposure and the use thereof Download PDF

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WO2017028813A1
WO2017028813A1 PCT/CN2016/096025 CN2016096025W WO2017028813A1 WO 2017028813 A1 WO2017028813 A1 WO 2017028813A1 CN 2016096025 W CN2016096025 W CN 2016096025W WO 2017028813 A1 WO2017028813 A1 WO 2017028813A1
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composition
vitamin
lung
subject
exposure
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Lin Xu
Riccardo ACCOLLA
Valerio PAZIENZA
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Lin Xu
Accolla Riccardo
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/115—Fatty acids or derivatives thereof; Fats or oils
    • A23L33/12—Fatty acids or derivatives thereof
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/15—Vitamins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/20—Carboxylic acids, e.g. valproic acid having a carboxyl group bound to a chain of seven or more carbon atoms, e.g. stearic, palmitic, arachidic acids
    • A61K31/202—Carboxylic acids, e.g. valproic acid having a carboxyl group bound to a chain of seven or more carbon atoms, e.g. stearic, palmitic, arachidic acids having three or more double bonds, e.g. linolenic
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
    • A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline 
    • A61K31/353—3,4-Dihydrobenzopyrans, e.g. chroman, catechin
    • A61K31/355—Tocopherols, e.g. vitamin E
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/365—Lactones
    • A61K31/375—Ascorbic acid, i.e. vitamin C; Salts thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/33—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing oxygen
    • A61K8/36—Carboxylic acids; Salts or anhydrides thereof
    • A61K8/361—Carboxylic acids having more than seven carbon atoms in an unbroken chain; Salts or anhydrides thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/67—Vitamins
    • A61K8/676—Ascorbic acid, i.e. vitamin C
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/67—Vitamins
    • A61K8/678—Tocopherol, i.e. vitamin E
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/08—Antiallergic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P39/00—General protective or antinoxious agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
    • A61K2800/10—General cosmetic use

Definitions

  • This invention relates to a nutritional composition for reducing lung and/or systemic inflammation given by air pollution, specifically by a high level of PM2.5 pollutants, and to the use thereof. This invention also relates to a method for reducing lung and/or systemic inflammation associated with PM2.5 exposure.
  • PM2.5 i.e., the particulate matters with aerodynamic diameter smaller than 2.5 micrometers
  • the average daily level of PM2.5 in Beijing is averaging 5x the maximum safety level of 25 ⁇ g/m 3 established by 2005 WHO guidelines, with peaks measured at even 20x (WHO, 2005, see: http: //www. who. int/phe/health_topics/outdoorair/outdoorair_aqg/en/) .
  • High PM2.5 level is associated with several health complications, which include an increased local and systemic inflammatory response and a decreased anti-oxidative capability, leading to cell damage and death.
  • the long-term sustainable solution consists in reducing PM2.5 emissions
  • the supplementation with specific nutrients could help preventing or at least delaying the onset of air pollution related inflammation, especially in populations at greater risk such as infants and children.
  • Vitamin E and Vitamin C display well determined antioxidant properties. Recent studies suggest their supplementation can help reduce oxidative stress and nasal inflammation associated with high PM2.5 level, but the results are not consistent (Possamai et al, Antioxidant intervention compensates oxidative stress in blood of subjects exposed to emissions from a coal electric-power plant in South Brazil, Environ Toxicol Pharmacol. 2010 Sep; 30 (2) : 175-80; Sienra-Monge et al, Antioxidant supplementation and nasal inflammatory responses among young asthmatics exposed to high levels of ozone, Clin. Exp. Immunol. 2004 Nov; 138 (2) : 317-22) .
  • omega-3 The anti-inflammatory and anti-oxidative effects of omega-3 have been recognized for a long time. Less evidence exists on the effect of fish oil (containing DHA–docosahexanoic acid) in reducing oxidative stress and improving heart rate variability (see Zhang W., et al. Nutrition Solutions to Counter Health Impact of Air Pollution: Scientific Evidence of Marine Omega-3 Fatty Acids and Vitamins Minimizing Some Harms of PM2.5, J. Food Nutr Sci , 2015, 2 (2) : 1-6) .
  • probiotics can help restore proper balance of the intestinal microbiota and improve gastrointestinal symptoms.
  • specific bacteria such as a probiotic strain Bifidobacterium animalis subsp. Lactis
  • increase the body’s resistance to common respiratory infections as well as reduce the incidence of acute respiratory tract infections Jungersen et al, The Science behind the Probiotic Strain Bifidobacterium animalis subsp. lactis Microorganisms, 2014, 2, 92-110; Mortaz et al, Probiotics in the Management of Lung Diseases, Mediators of Inflammation, vol. 2013, Article ID 751068, 10 pages, 2013.
  • the present invention relates to a composition for reducing lung and/or systemic inflammation associated with PM2.5 exposure, comprising docosahexanoic acid, a probiotic strain (such as Bifidobacterium animalis subsp. Lactis) , Vitamin C and Vitamin E.
  • a probiotic strain such as Bifidobacterium animalis subsp. Lactis
  • Vitamin C and Vitamin E Preferably, the composition according to the present invention contains docosahexanoic acid, a probiotic strain, Vitamin C and Vitamin E in a ratio of 50-500mg : 5 X 10 7 -5 X 10 10 CFU : 10mg-250mg : 1mg-100mg.
  • the composition according to the present invention contains docosahexanoic acid, a probiotic strain, Vitamin C and Vitamin E in a ratio of 75mg-150mg : 1 X 10 9 -1 X 10 10 CFU : 30mg-150mg : 4mg-20mg.
  • the present invention also relates to a supplement for food, medicament, cosmetics and beverage comprising the composition according to the present invention.
  • the invention also provides a method for reducing lung and/or systemic inflammation associated with PM2.5 pollutant exposure in a subject in need thereof, comprising administrating an effective amount of the composition according to the present invention.
  • the present invention further provides use of the composition according to the present invention in the manufacture of a supplement for food, medicament, cosmetics and beverage, for reducing lung and/or systemic inflammation associated with PM2.5 pollutant exposure in a subject in need thereof.
  • Figure 1 Gene expressions’ Clustergram of inflammatory relevant cytokines/chemokines
  • Figure 3A The synergistic effect of DHA and BB12 on gene expression is represented.
  • the mean fold changes produced by the single nutrients (Groups 2+3, white area) are compared to the fold changes of the combined nutrients (Group 5, black area) .
  • Figure 3B The synergistic effect of BB12 and Vit E/Vit C on gene expression is represented.
  • the mean fold changes produced by the single nutrients (Groups 3+4, white area) are compared to the fold changes of the combined nutrients (Group 6, black area) ;
  • Figure 3C The synergistic effect of DHA, BB12 and Vit E/Vit C on gene expression is represented.
  • the mean fold changes produced by the single nutrients (Groups 2+3+4, white area) are compared to the fold changes of the combined nutrients (Group 7, black area) ;
  • Figure 4 Lung sections representative of the 8 groups (Group 1-7 and Ctrl Group) .
  • the presence of small black dots represents infiltration by air pollution particles.
  • the other sections (Group 2-7) are rated according to PM2.5 particles presence with respect to Group 1;
  • Figure 5 IL-10 expression profile across Groups. Serum IL-10 levels were measured by an ELISA assay (eBioscience, cat. n. 88-7105) following manufacturer’s recommendations. At least two serum dilutions were assessed for IL-10 concentration and plotted on the standard curve generated with known concentrations of the cytokine. Results are expressed as picograms/ml (ordinate) in the different groups (abscissa) and represent the average of two separate tests.
  • the present invention relates to a composition for reducing lung and/or systemic inflammation associated with PM2.5 exposure, comprising docosahexanoic acid, a probiotic strains (such as Bifidobacterium animalis subsp. Lactis) , Vitamin C and Vitamin E.
  • the composition according to the present invention contains docosahexanoic acid, a probiotic strain, Vitamin C and Vitamin E in a ratio of 50-500mg : 5 X 10 7 -5 X 10 10 CFU : 10mg-250mg : 1mg-100mg.
  • the composition according to the present invention contains docosahexanoic acid, a probiotic strain, Vitamin C and Vitamin E in a ratio of 75mg-150mg : 1 X 10 9 -1 X 10 10 CFU : 30mg-150mg : 4mg-20mg.
  • composition according to the present invention can be in a variety of forms that are suitable for administration as food and supplement.
  • the composition can be in the form of prepared food e.g. biscuit, juice, drinking powder, candy, ice cream, dried fruits and vegetables, functional drink, capsule, tablet, caplet, bar, cream, gel, powder, solution, gum, etc.
  • the composition according to the present invention can further include additional components.
  • the additional components can be one or more additional components selected from probiotics, vitamins, phytonutrients, natural herbals e.g. lycopene, turmeric, polyphenol, or combinations thereof.
  • the present invention also relates to a supplement for food, medicament, cosmetics and beverage comprising the composition according to the present invention.
  • docosahexanoic acid an important member of the family of omega-3 polyunsaturated fatty acid, is from both vegetable and fish/krill source.
  • DHA docosahexanoic acid
  • probiotic strains can be used in the present invention for example Lactobacillus, Bifidobacterium, Saccharomyces, preferably Bifidobacterium, more preferably Bifidobacterium animalis subsp. Lactis.
  • a probiotic strain has been used in infant formula, dietary supplements and fermented milk products worldwide, wherein Bifidobacterium animalis subsp. Lactisis technologically well suited, expressing fermentation activity, high aerotolerance, good stability and a high acid and bile tolerance, also as freeze-dried products in dietary supplements.
  • Bifidobacterium animalis subsp for example, Bifidobacterium animalis subsp.
  • Lactis does not have adverse effects on taste, appearance or on the mouth feel of the food and is able to survive in the probiotic food until consumption.
  • Bifidobacterium animalis subsp. Lactis used in the present invention is commercially available from, for example, Chr. Hansen in a form of powder. It has been proved by the present invention that a probiotic strain, preferably, Bifidobacterium animalis subsp. Lactis increases the body’s resistance to common respiratory infections as well as reduces the incidence of acute respiratory tract infections.
  • Vitamin C also called as ascorbic acid, is for example commercially available.
  • Vitamin E also called tocopherol, with four subgroups ( ⁇ , ⁇ , ⁇ e ⁇ ) of which ⁇ -tocopherol is the most active and potent biologically or ⁇ -tocopherol, is for example commercially available.
  • composition according to the present invention acts synergistically to reduce lung and/or systemic inflammation given by high level of air pollution, especially for PM2.5 pollutants.
  • the composition according to the present invention helps reducing air-pollution related lung inflammation assessed by two complementary measures, the histological analysis, and the lung mRNA profile of the genes playing a key role in inflammation such as Adipoq, CCL-7, IL-2, IL-4, IL-5, IL-10, IL-17 (and others) , with and without supplementation.
  • the composition of the present invention also helps reduce air pollution related systemic inflammation, by modifying the levels of cytokines produced by, and involved in the polarization of lymphocyte populations responsible of the inflammatory process itself.
  • TH1-type cytokines such as IL-12, TNF-alpha and IFN-gamma and the anti-inflammatory TH2-type cytokines IL-4 and IL-10, TGF-beta as well as IgE immunoglobulins, associated to an allergic type of inflammation.
  • pro-inflammatory TH1-type cytokines are not particularly affected in the short period by distinct formulations of the composition, other parameters such as variation of allergogenic cytokine IL-10 are significantly affected by the different assortment of the composition.
  • suitable assortment of the composition according to the present invention could not only prevent lung and/or systemic inflammation but also reduce lung and/or systemic inflammation associated with PM2.5 exposure.
  • the present invention also provides a method for reducing lung and/or systemic inflammation associated with PM2.5 pollutant exposure in a subject in need thereof, comprising administrating an effective amount of the composition according to the present invention.
  • the effective amount is for DHA at a level between 50 and 500mg/day, preferably between 75 and 150mg/day, for probiotic strains (Bifidobacterium animalis subsp.
  • Lactis at a level between 5 x 10 7 and 5 x 10 10 CFU/day, preferably between 1 X 10 9 and 1 x 10 10 CFU/day, for Vitamin C at a level between 10 and 250 mg/day, preferably between 30 and 150 mg/day, and for Vitamin E, at a level between 1 and 100 mg/day, preferably between 4 and 20 mg/day.
  • the dosages are overall dosages for adults (70kg) .
  • the composition according to the present invention can be delivered to a mammal including a human subject, targeting all age group.
  • the term “deliver” generally refers to body intake of the composition.
  • the composition is delivered to a subject by oral administration.
  • the human subject with allergies for pollen, dust, asthma, allergic rhinitis and atopic dermatitis etc. are especially suitable to use the composition on a daily basis, wherein the subject is exposed to PM2.5 pollutants with a level between 25 and 500 ⁇ g/m 3 , especially between 100 and 500 ⁇ g/m 3 .
  • a human subject living in the particular megacities of developing countries is exposed to high level of PM2.5 pollutants.
  • the average daily level of PM2.5 in Beijing is 125 ⁇ g/m 3 , with peaks measured at even 500 ⁇ g/m 3 .
  • the present invention also provides a method for reducing lung and/or systemic inflammation associated with PM2.5 pollutant exposure in a subject in need thereof, including the step of: delivering to the subject DHA at a level between 50 and 500mg/day, preferably between 75 and 150mg/day, probiotic strains (Bifidobacterium animalis subsp. Lactis) at a level between 5 x 10 7 and 5 x 10 10 CFU/day, preferably between 1 X 10 9 and 1 x 10 10 CFU/day, Vitamin C at a level between 10 and 250 mg/day, preferably between 30 and 150 mg/day, and Vitamin E, at a level between 1 and 100 mg/day, preferably between 4 and 20 mg/day.
  • the subject is exposed to PM2.5 pollutants with a level between 25 and 500 ⁇ g/m 3 , especially between 100 and 500 ⁇ g/m 3 ; preferably, the subject is a human subject, targeting all age group.
  • the four nutrients i.e. DHA, probiotic strains, vitamin C and vitamin E
  • they are delivered simultaneously to a subject as a supplement, which is preferably in a form of capsule, tablet, caplet, bar, gum, cream, gel, powder, solution, or etc.
  • DHA and the Bifidobacterium animalis subsp. Lactis are delivered together after being blended and then encapsulated into a capsule, tablet, powder, or gel format, while Vitamin C and Vitamin E are delivered together after being blended into a gummy, chew, or food format.
  • DHA, Vitamin C and Vitamin E are delivered together after being dry-blended and then assembled in a gummy, chew or other food format, while Bifidobacterium animalis subsp. Lactis is delivered separately.
  • the above-mentioned processes, such as dry-blending and encapsulating, are standard methods well known by the person skilled in the art.
  • the present invention further provides use of the composition according to the present invention in the manufacture of a supplement for food, medicament, cosmetics and beverage for reducing lung and/or systemic inflammation associated with PM2.5 pollutant exposure in a subject in need thereof.
  • Raw material DHA; Bifidobacterium animalis subsp. Lactis; Vitamin C; and Vitamin E.
  • Example 1 DHA and the Bifidobacterium animalis subsp. Lactis are blended and encapsulated into a capsule, tablet, powder, gel format, while Vitamin C and Vitamin E are blended into a gummy, chew, or food format by the standard methods well known by the person skilled in the art.
  • Example 2 DHA, Vitamin C, and Vitamin E are dry-blended and then assembled in a gummy, chew or other food format by the standard methods well known by the person skilled in the art, while Bifidobacterium animalis subsp. Lactis is delivered in a capsule, tablet, food format.
  • Example 3 Bifidobacterium animalis subsp. Lactis, Vitamin C, and Vitamin E are blended and then assembled in a powder, gummy, chew or other food format by the standard methods well known by the person skilled in the art, while DHA is delivered in a capsule, oil, tablet, food format.
  • Examples 4-5 are prepared by the same method as Example 1-2 except for the different amounts of the four nutrients as listed in Table 1.
  • Table 1 Amounts of four nutrients in the Examples
  • mice were randomly assigned to 8 different treatment groups (5 mice per group, see Table 2) ensuring that each group’s mean body weight was well matched.
  • 7-8 weeks old mice were treated under different engineered diets, consisting of a base of typical mice diet, usually provided under pellet format, to which ingredients of the present composition were added as shown in Table 2 for 30d before the pharyngeal aspiration which was performed on days 0, 3 and 6, for a total of three aspirations with 100 mg of PM2.5 in 50 ml of isotonic saline solution, or 50 ml of isotonic saline solution (sham) .
  • sham isotonic saline solution
  • the animals were anesthetized with isoflurane and maintained in a vertical position, by means of a mechanical holder.
  • the tongue was gently grabbed and pulled out with forceps and held at the side of the mouth, in such a way to make visible the base of the tongue itself.
  • the treatment solution was delivered on the base of the tongue and at least the time of two entire respiratory cycles was allowed to elapse to ensure that the solution was aspired and not swallowed.
  • the animals were given the assigned diets back.
  • This protocol allows us to evaluate whether engineered diets protect lungs and liver from pollution. Upon sacrifice, 3 days after the last aspiration, lungs and serum samples were collected and rapidly frozen. After thawing, samples were processed for biochemical, immunochemical analyses and for assessing the levels of inflammatory cytokines. For histology, serial sections of the lungs were cut by a cryostat, mounted on slides and stained with Carazzi’s haematoxylin and eosin staining.
  • This protocol allows us to evaluate whether engineered diets protect lungs and liver from PM2.5 pollutants.
  • Cytokines Adipoq, Bmp7, Il10, Il11, Il17a, Il2, Il22, Il23a, Il24, Il4, Il5, Lif, Tnfrsf11b, Tnfsf10;
  • Chemokines Ccl1, Ccl12, Ccl17, Ccl2, Ccl20, Ccl7, Cxcl1, Cxcl10, Cxcl5, Cxcl9.
  • Serum Cytokines/Chemokines pre and post pharyngeal aspiration exposure: TGF-b, IFN ⁇ , TNF- ⁇ , IL-10, IL-5, IL-4, IL-6, IL-1beta.
  • composition of the present invention acts synergistically to reduce lung inflammation given by high level of air pollution and might also have a beneficial effect on TH2 allergic systemic response. It is believed that the composition according to the present invention could not only prevent lung and/or systemic inflammation but also reduce lung and/or systemic inflammation associated with PM2.5.
  • Figure 1 displays the clustergram representation of these genes’ expression across all groups. Cytokines/chemokines classified by literature evidence as pro-inflammatory or anti-inflammatory are highlighted in light grey or dark grey, respectively. The ones reported to exert both pro-and anti-inflammatory effects are highlighted in white.
  • Control Group and Group 1 have almost a mirroring pattern in terms of gene expression.
  • comparison between columns of Group 4 and especially Group 7 to Control Group indicates how the delivery of certain diets seems to be more effective to counteract the exposure to PM2.5, by bringing the expression level on inflammatory cytokines/chemokines back to the control levels.
  • a clearer visualization of the similarities across groups of the expression patterns is provided by the 3-color version of the clustergram (light gray-black-dark gray) inserted as supporting Figure 1.
  • CCL7 the product of Ccl7 gene
  • CCL7 is a chemokine classically known to attract monocytes/macrophages during inflammation.
  • MCP-3 Monocyte chemotactic protein-3
  • FAC fibroblast-induced cytokine
  • adiponectin a hormone-like protein produced by adipose tissue, whose role in inflammation is controversial. Indeed, although predominantly endowed with anti-inflammatory functions, conflicting evidences report a pro-inflammatory role for adiponectin under certain conditions (Ehling et al., The potential of adiponectin in driving arthritis. J. Immunol. 2006; 176 (7) : 4468-4478; Garcia and Sood, Adiponectin in pulmonary disease and critically ill patients. Curr Med Chem.
  • Il23a encodes the alpha (or p19) subunit of the heterodimeric cytokine IL23, which has a pathogenic role in a number of chronic inflammation diseases, including asthma (Nakajima and Hirose. Role of IL-23 and Th17 Cells in Airway Inflammation in Asthma. Immune Netw. 2010; 10 (1) : 1-4) .
  • IL23a mRNA was found upregulated in lung of mice exposed to allergic stimuli, where it elicited both a neutrophil-and an eosinophil-mediated inflammatory response (Nakajima and Hirose. Role of IL-23 and Th17 Cells in Airway Inflammation in Asthma. Immune Netw.
  • IL23 deficiency or neutralization resulted in a reduced airway inflammation (Peng et al., IL-23 signaling enhances Th2 polarization and regulates allergic airway inflammation.
  • Cell Res. 2010; 20 (1) : 62-71; IL-23 and Th17 cells enhance Th2-cell-mediated eosinophilic airway inflammation in mice. Am J Respir Crit Care Med. 2008; 178(10) : 1023-1032) .
  • the murine Ccl12 gene encodes a chemokine which shares a high homology with the human CCL2 (Sarafi et al., Murine monocyte chemoattractant protein (MCP) -5: a novel CC chemokine that is a structural and functional homologue of human MCP-1. J Exp Med. 1997; 185 (1) : 99-109) , whose expression is increased in inflammatory disorders of the lung (Rose et al, Significant involvement of CCL2 (MCP-1) in inflammatory disorders of the lung. Microcirculation. 2003; 10 (3-4) : 273-288) .
  • MCP Murine monocyte chemoattractant protein
  • Figure 2 summarizes the clustergram data, by showing the overall proximity of each Group to the Control condition. Two coefficients of similarity, are calculated by averaging in every group the Up-&Down-regulated gene expressions, respectively.
  • Lungs from sham (Control Group) and PM2.5 treated mice (Groups 1 to 7) were rinsed and properly collected and stored in liquid nitrogen. 10 ⁇ m serial sections were cut by a cryostat for each sham and PM2.5 exposed lung sample, mounted on slides and stained with Carazzi’s haematoxylin and eosin staining. A Nikon Eclipse E600 microscope was used for analysis. Pictures were captured at 40X magnification.
  • cytokines representing both so-called pro-inflammatory TH-1 cytokines, such as IFN-g, TNF-alpha, and IL-1, and TH-2 cytokines associated to the generation of IgE-mediated allergic reactions such as IL-4, IL-5 and IL-10, together with immunosuppressive cytokine TGF-beta have been already preliminary tested and measured in the serum of PM2.5 injected animals having undergone specific diet regimens.
  • serum concentration of IL-10 a fundamental marker of the allergic inflammation (Figure 5) . From the concentration levels, it can be deduced that pretreatment with certain diets (Group 3, Group 4 and especially the combination of the four ingredients in Group 7) can potentially prevent the onset of allergic inflammatory response.
  • MCP Murine monocyte chemoattractant protein

Abstract

Provided is a composition for reducing lung and/or systemic inflammation associated with PM2.5 exposure, comprising docosahexanoic acid, a probiotic strain(such as Bifidobacterium animalis subsp. Lactis), Vitamin C and Vitamin E. Also provided is the use of this composition for human subjects living in the environment with the average daily level of PM2.5 between 25 and 500 μg/m 3, especially between 100 and 500 μg/m 3.

Description

A COMPOSITION FOR REDUCING LUNG AND/OR SYSTEMIC INFLAMMATION ASSOCIATED WITH PM2.5 EXPOSURE AND THE USE THEREOF
FIELD OF INVENTION
This invention relates to a nutritional composition for reducing lung and/or systemic inflammation given by air pollution, specifically by a high level of PM2.5 pollutants, and to the use thereof. This invention also relates to a method for reducing lung and/or systemic inflammation associated with PM2.5 exposure.
BACKGROUND
The prevalence of asthma, allergic rhinitis and atopic dermatitis has been increasing over the last four decades, both in the industrialized and developing countries. Many studies point to a potential role of various environmental factors, such as increasing industrialization, dietary changes and air pollution, in the increase of these allergic and inflammatory diseases (Rutkowski et al, Allergic diseases: the price of civilisational progress, Postep Derm Allergol 2014； XXXI, 2: 77–83) .
The situation is especially alarming in China and other fast developing countries, where a booming economy contributed to create health-threatening pollution level. Among the major pollutants, the levels of PM2.5, i.e., the particulate matters with aerodynamic diameter smaller than 2.5 micrometers, have to be contained as they can carry toxic substances deep into lungs when inhaled. Nowadays, the average daily level of PM2.5 in Beijing is averaging 5x the maximum safety level of 25 μg/m3 established by 2005 WHO guidelines, with peaks measured at even 20x (WHO, 2005, see: http: //www. who. int/phe/health_topics/outdoorair/outdoorair_aqg/en/) . High PM2.5 level is associated with several health complications, which include an increased local and systemic inflammatory response and a decreased anti-oxidative capability, leading to cell damage and death.
Although the long-term sustainable solution consists in reducing PM2.5 emissions, the supplementation with specific nutrients could help preventing or at least delaying the onset of air pollution related inflammation, especially in populations at greater risk such as infants and children.
Vitamin E and Vitamin C display well determined antioxidant properties. Recent studies suggest their supplementation can help reduce oxidative stress and nasal inflammation associated with high PM2.5 level, but the results are not consistent (Possamai et al, Antioxidant intervention compensates oxidative stress in blood of subjects exposed to emissions from a coal electric-power plant in South Brazil, Environ Toxicol Pharmacol. 2010 Sep； 30 (2) : 175-80； Sienra-Monge et al, Antioxidant supplementation and nasal inflammatory responses among young asthmatics exposed to high levels of ozone, Clin. Exp. Immunol. 2004 Nov； 138 (2) : 317-22) .
The anti-inflammatory and anti-oxidative effects of omega-3 have been recognized for a long time. Less evidence exists on the effect of fish oil (containing DHA–docosahexanoic acid) in reducing oxidative stress and improving heart rate variability (see Zhang W., et al. Nutrition Solutions to Counter Health Impact of Air Pollution: Scientific Evidence of Marine Omega-3 Fatty Acids and Vitamins Minimizing Some Harms of PM2.5, J. Food Nutr Sci , 2015, 2 (2) : 1-6) .
There are relevant scientific evidences that certain probiotics can help restore proper balance of the intestinal microbiota and improve gastrointestinal symptoms. Also, in terms of immune function, clinical studies have shown that specific bacteria, such as a probiotic strain Bifidobacterium animalis subsp. Lactis, increase the body’s resistance to common respiratory infections as well as reduce the incidence of acute respiratory tract infections (Jungersen et al, The Science behind the Probiotic Strain Bifidobacterium animalis subsp. lactis
Figure PCTCN2016096025-appb-000001
Microorganisms, 2014, 2, 92-110； Mortaz et al, Probiotics in the Management of Lung Diseases, Mediators of Inflammation, vol. 2013, Article ID 751068, 10 pages, 2013) . However, no study so far examined the supplementation of probiotic strain in conditions of air pollution.
There is still a strong demand on the market for daily diet supplementations that can effectively reduce lung and/or systemic inflammation given by air pollution, specifically by a high level of PM2.5 pollutants.
SUMMARY OF THE DISCLOSURE
The present invention relates to a composition for reducing lung and/or systemic inflammation associated with PM2.5 exposure, comprising docosahexanoic acid, a probiotic strain (such as Bifidobacterium animalis subsp. Lactis) , Vitamin C and Vitamin E. Preferably, the composition according to the present invention contains docosahexanoic acid, a probiotic strain, Vitamin C and Vitamin E in a ratio of 50-500mg : 5 X 107-5 X 1010 CFU : 10mg-250mg : 1mg-100mg. Most preferably, the composition according to the present invention contains docosahexanoic acid, a probiotic strain, Vitamin C and Vitamin E in a ratio of 75mg-150mg : 1 X 109-1 X 1010 CFU : 30mg-150mg : 4mg-20mg.
The present invention also relates to a supplement for food, medicament, cosmetics and beverage comprising the composition according to the present invention.
Additionally, in one aspect, the invention also provides a method for reducing lung and/or systemic inflammation associated with PM2.5 pollutant exposure in a subject in need thereof, comprising administrating an effective amount of the composition according to the present invention.
In another aspect, the present invention further provides use of the composition according to the present invention in the manufacture of a supplement for food, medicament, cosmetics and beverage, for reducing lung and/or systemic inflammation associated with PM2.5 pollutant exposure in a subject in need thereof.
Other features and aspects of the present invention are discussed in greater detail below.
BRIEF DESCRIPTION OF THE FIGURES
Figure 1: Gene expressions’ Clustergram of inflammatory relevant cytokines/chemokines；
Figure 2: Weighted Averages of Up (dark bars) and Down (light bars) regulated gene expressions. The averages are multiplied by a weight that takes into account the number of genes showing a significant >3-fold change over the Ctrl group (1 ＝ no change) ；
Figure 3A: The synergistic effect of DHA and BB12 on gene expression is represented. The mean fold changes produced by the single nutrients (Groups 2+3, white area) are compared to the fold changes of the combined nutrients (Group 5, black area) .
Figure 3B: The synergistic effect of BB12 and Vit E/Vit C on gene expression is represented. The mean fold changes produced by the single nutrients (Groups 3+4, white area) are compared to the fold changes of the combined nutrients (Group 6, black area) ；
Figure 3C: The synergistic effect of DHA, BB12 and Vit E/Vit C on gene expression is represented. The mean fold changes produced by the single nutrients (Groups 2+3+4, white area) are compared to the fold changes of the combined nutrients (Group 7, black area) ；
Figure 4: Lung sections representative of the 8 groups (Group 1-7 and Ctrl Group) . The presence of small black dots represents infiltration by air pollution particles. We counted the PM2.5 particles in the Group 1 section and assigned “+++” . The other sections (Group 2-7) are rated according to PM2.5 particles presence with respect to Group 1；
Figure 5: IL-10 expression profile across Groups. Serum IL-10 levels were measured by an ELISA assay (eBioscience, cat. n. 88-7105) following manufacturer’s recommendations. At least two serum dilutions were assessed for IL-10 concentration and plotted on the standard curve generated with known concentrations of the cytokine. Results are expressed as picograms/ml (ordinate) in the different groups (abscissa) and represent the average of two separate tests.
DETAILED DESCRIPTION OF THE DISCLOSURE
The present invention relates to a composition for reducing lung and/or systemic inflammation associated with PM2.5 exposure, comprising docosahexanoic acid, a probiotic strains (such as Bifidobacterium animalis subsp. Lactis) , Vitamin C and Vitamin E. Preferably, the composition according to the present invention contains docosahexanoic acid, a probiotic strain, Vitamin C and Vitamin E in a ratio of 50-500mg : 5 X 107-5 X 1010CFU : 10mg-250mg : 1mg-100mg. Most preferably, the composition according to the present invention contains docosahexanoic acid, a probiotic strain, Vitamin C and Vitamin E in a ratio of 75mg-150mg : 1 X 109-1 X 1010CFU : 30mg-150mg : 4mg-20mg.
The composition according to the present invention can be in a variety of forms that are suitable for administration as food and supplement. For example, the composition can be in the form of prepared food e.g. biscuit, juice, drinking powder, candy, ice cream, dried fruits and vegetables, functional drink, capsule, tablet, caplet, bar, cream, gel, powder, solution, gum, etc. Accordingly, the composition according to the present invention can further include additional components. For example, the additional components can be one or more additional components selected from probiotics, vitamins, phytonutrients, natural herbals e.g. lycopene, turmeric, polyphenol, or combinations thereof.
The present invention also relates to a supplement for food, medicament, cosmetics and beverage comprising the composition according to the present invention.
According to the present invention, docosahexanoic acid (DHA) , an important member of the family of omega-3 polyunsaturated fatty acid, is from both vegetable and fish/krill source. The anti-inflammatory and anti-oxidative effects of omega-3 have been recognized for a long time.
Many kinds of probiotic strains can be used in the present invention for example Lactobacillus, Bifidobacterium, Saccharomyces, preferably Bifidobacterium, more preferably Bifidobacterium animalis subsp. Lactis. Nowadays, a probiotic strain has been used in infant formula, dietary supplements and fermented milk products worldwide,  wherein Bifidobacterium animalis subsp. Lactisis technologically well suited, expressing fermentation activity, high aerotolerance, good stability and a high acid and bile tolerance, also as freeze-dried products in dietary supplements. Furthermore, for example, Bifidobacterium animalis subsp. Lactis does not have adverse effects on taste, appearance or on the mouth feel of the food and is able to survive in the probiotic food until consumption. Bifidobacterium animalis subsp. Lactis used in the present invention is commercially available from, for example, Chr. Hansen in a form of powder. It has been proved by the present invention that a probiotic strain, preferably, Bifidobacterium animalis subsp. Lactis increases the body’s resistance to common respiratory infections as well as reduces the incidence of acute respiratory tract infections.
Vitamin C, also called as ascorbic acid, is for example commercially available.
Vitamin E, also called tocopherol, with four subgroups (α, β, γ e δ) of which α-tocopherol is the most active and potent biologically or α-tocopherol, is for example commercially available.
Unexpectedly, these combined above four types of nutrients in the composition according to the present invention acts synergistically to reduce lung and/or systemic inflammation given by high level of air pollution, especially for PM2.5 pollutants. In particular, the composition according to the present invention helps reducing air-pollution related lung inflammation assessed by two complementary measures, the histological analysis, and the lung mRNA profile of the genes playing a key role in inflammation such as Adipoq, CCL-7, IL-2, IL-4, IL-5, IL-10, IL-17 (and others) , with and without supplementation. The composition of the present invention also helps reduce air pollution related systemic inflammation, by modifying the levels of cytokines produced by, and involved in the polarization of lymphocyte populations responsible of the inflammatory process itself. Crucial to this aspect are for example the levels of pro-inflammatory TH1-type cytokines such as IL-12, TNF-alpha and IFN-gamma and the anti-inflammatory TH2-type cytokines IL-4 and IL-10, TGF-beta as well as IgE immunoglobulins, associated to an allergic type of inflammation. Interestingly, while pro-inflammatory TH1-type cytokines are not particularly affected in the short period by distinct formulations of the  composition, other parameters such as variation of allergogenic cytokine IL-10 are significantly affected by the different assortment of the composition. Although not intending to be limited to the following theory, it is believed that suitable assortment of the composition according to the present invention could not only prevent lung and/or systemic inflammation but also reduce lung and/or systemic inflammation associated with PM2.5 exposure.
In one aspect, the present invention also provides a method for reducing lung and/or systemic inflammation associated with PM2.5 pollutant exposure in a subject in need thereof, comprising administrating an effective amount of the composition according to the present invention. In a preferred embodiment, the effective amount is for DHA at a level between 50 and 500mg/day, preferably between 75 and 150mg/day, for probiotic strains (Bifidobacterium animalis subsp. Lactis) at a level between 5 x 107 and 5 x 1010 CFU/day, preferably between 1 X 109 and 1 x 1010 CFU/day, for Vitamin C at a level between 10 and 250 mg/day, preferably between 30 and 150 mg/day, and for Vitamin E, at a level between 1 and 100 mg/day, preferably between 4 and 20 mg/day. Preferably, the dosages are overall dosages for adults (70kg) .
The composition according to the present invention can be delivered to a mammal including a human subject, targeting all age group. In the context, the term “deliver” generally refers to body intake of the composition. Preferably, the composition is delivered to a subject by oral administration. The human subject with allergies for pollen, dust, asthma, allergic rhinitis and atopic dermatitis etc. are especially suitable to use the composition on a daily basis, wherein the subject is exposed to PM2.5 pollutants with a level between 25 and 500μg/m3, especially between 100 and 500μg/m3. A human subject living in the particular megacities of developing countries is exposed to high level of PM2.5 pollutants. For example, the average daily level of PM2.5 in Beijing is 125 μg/m3, with peaks measured at even 500 μg/m3.
The present invention also provides a method for reducing lung and/or systemic inflammation associated with PM2.5 pollutant exposure in a subject in need thereof, including the step of: delivering to the subject DHA at a level between 50 and 500mg/day,  preferably between 75 and 150mg/day, probiotic strains (Bifidobacterium animalis subsp. Lactis) at a level between 5 x 107 and 5 x 1010 CFU/day, preferably between 1 X 109 and 1 x 1010 CFU/day, Vitamin C at a level between 10 and 250 mg/day, preferably between 30 and 150 mg/day, and Vitamin E, at a level between 1 and 100 mg/day, preferably between 4 and 20 mg/day. Preferably, the subject is exposed to PM2.5 pollutants with a level between 25 and 500 μg/m3, especially between 100 and 500 μg/m3； preferably, the subject is a human subject, targeting all age group.
It can be understood that the four nutrients, i.e. DHA, probiotic strains, vitamin C and vitamin E, can be delivered simultaneously, individually or in any combination among them to a subject. In one embodiment of the present invention, they are delivered simultaneously to a subject as a supplement, which is preferably in a form of capsule, tablet, caplet, bar, gum, cream, gel, powder, solution, or etc. In a preferred embodiment of the present invention, DHA and the Bifidobacterium animalis subsp. Lactis are delivered together after being blended and then encapsulated into a capsule, tablet, powder, or gel format, while Vitamin C and Vitamin E are delivered together after being blended into a gummy, chew, or food format. It is also possible according to the present invention that DHA, Vitamin C and Vitamin E are delivered together after being dry-blended and then assembled in a gummy, chew or other food format, while Bifidobacterium animalis subsp. Lactis is delivered separately. The above-mentioned processes, such as dry-blending and encapsulating, are standard methods well known by the person skilled in the art.
In another aspect, the present invention further provides use of the composition according to the present invention in the manufacture of a supplement for food, medicament, cosmetics and beverage for reducing lung and/or systemic inflammation associated with PM2.5 pollutant exposure in a subject in need thereof.
EXAMPLES
Preparation of the inventive composition
Raw material: DHA； Bifidobacterium animalis subsp. Lactis； Vitamin C； and Vitamin E.
For Example 1, DHA and the Bifidobacterium animalis subsp. Lactis are blended and encapsulated into a capsule, tablet, powder, gel format, while Vitamin C and Vitamin E are blended into a gummy, chew, or food format by the standard methods well known by the person skilled in the art.
For Example 2, DHA, Vitamin C, and Vitamin E are dry-blended and then assembled in a gummy, chew or other food format by the standard methods well known by the person skilled in the art, while Bifidobacterium animalis subsp. Lactis is delivered in a capsule, tablet, food format.
For Example 3, Bifidobacterium animalis subsp. Lactis, Vitamin C, and Vitamin E are blended and then assembled in a powder, gummy, chew or other food format by the standard methods well known by the person skilled in the art, while DHA is delivered in a capsule, oil, tablet, food format.
Examples 4-5 are prepared by the same method as Example 1-2 except for the different amounts of the four nutrients as listed in Table 1.
Table 1: Amounts of four nutrients in the Examples
Figure PCTCN2016096025-appb-000002
The study of the effects in vivo caused by PM2.5 exposure based on animal model
As a first step we took advantage of a well established in vivo model for the study of pollution which consists in female BALB/c mice (7–8 weeks old) treated by pharyngeal aspiration with either isotonic saline aerosol (sham mice group) or PM2.5-containing aerosol (PM2.5-treated mice groups) . Fine particle matter PM2.5 (median of 2.8 μm)  mimicking atmospheric particulate matter of polluted suburban areas was purchased from the National Institute of Standards and Technologies (SRM 2786, NIST, Gaithersburg, MD, USA) . The collection, composition, and storage of the fine particle matter are described in the certificate of analysis (https: //www-s.nist. gov/srmors/view_cert. cfm？ srm＝2786) .
Mice were randomly assigned to 8 different treatment groups (5 mice per group, see Table 2) ensuring that each group’s mean body weight was well matched. 7-8 weeks old mice were treated under different engineered diets, consisting of a base of typical mice diet, usually provided under pellet format, to which ingredients of the present composition were added as shown in Table 2 for 30d before the pharyngeal aspiration which was performed on  days  0, 3 and 6, for a total of three aspirations with 100 mg of PM2.5 in 50 ml of isotonic saline solution, or 50 ml of isotonic saline solution (sham) . Here below is the description of the pharyngeal aspiration procedure.
For the duration of the treatment the animals were anesthetized with isoflurane and maintained in a vertical position, by means of a mechanical holder. The tongue was gently grabbed and pulled out with forceps and held at the side of the mouth, in such a way to make visible the base of the tongue itself. The treatment solution was delivered on the base of the tongue and at least the time of two entire respiratory cycles was allowed to elapse to ensure that the solution was aspired and not swallowed. Upon awakening, the animals were given the assigned diets back.
We chose the pharyngeal aspiration to mimic the effect of air pollution in the lungs, in the attempt to carry out the least invasive procedure, but proven to be as effective as other techniques such as intratracheal inoculation (in which one would proceed with more severe anesthesia followed by intratracheal intubation) .
This protocol allows us to evaluate whether engineered diets protect lungs and liver from pollution. Upon sacrifice, 3 days after the last aspiration, lungs and serum samples were collected and rapidly frozen. After thawing, samples were processed for biochemical, immunochemical analyses and for assessing the levels of inflammatory cytokines. For  histology, serial sections of the lungs were cut by a cryostat, mounted on slides and stained with Carazzi’s haematoxylin and eosin staining.
Table 2: Diets and air pollution levels for different mice groups
Group No. Diets Air pollution levels
Control group Ctrl Sham
Group
 1 Ctrl PM2.5, 100 μg/m3
Group 2 Ctrl + DHA PM2.5, 100 μg/m3
Group 3 Ctrl + BB12 PM2.5, 100 μg/m3
Group 4 Ctrl + Vit E/Vit C PM2.5, 100 μg/m3
Group 5 Ctrl + DHA + BB12 PM2.5, 100 μg/m3
Group 6 Ctrl + BB12 + Vit E/VitC PM2.5, 100 μg/m3
Group 7 Ctrl + DHA + BB12 + Vit E/Vit C PM2.5, 100 μg/m3
Evaluation protocol
This protocol allows us to evaluate whether engineered diets protect lungs and liver from PM2.5 pollutants.
Upon sacrifice, serum and lungs samples collected as described above were used for biochemical, histological, immunochemical analyses and to assess the levels of inflammatory cytokines. Lung biopsies of mice belonging to the different treatment groups and control mice were analyzed by H&E staining to precisely evaluate the changes in morphology and inflammation. Moreover, serum samples were subjected to biochemical analysis.
Inflammatory Biomarkers tested herein are listed as below:
Lung Gene expression of an array of mouse cytokines and chemokines (Qiagen plates, SABiosciences) , 84 markers in total, amongst which the following, very relevant to the inflammatory pathway:
Cytokines: Adipoq, Bmp7, Il10, Il11, Il17a, Il2, Il22, Il23a, Il24, Il4, Il5, Lif, Tnfrsf11b, Tnfsf10；
Chemokines: Ccl1, Ccl12, Ccl17, Ccl2, Ccl20, Ccl7, Cxcl1, Cxcl10, Cxcl5, Cxcl9.
Serum Cytokines/Chemokines (pre and post pharyngeal aspiration exposure) : TGF-b, IFNγ, TNF-α, IL-10, IL-5, IL-4, IL-6, IL-1beta.
Test results
The combination of these four types of nutrients, in the composition of the present invention, acts synergistically to reduce lung inflammation given by high level of air pollution and might also have a beneficial effect on TH2 allergic systemic response. It is believed that the composition according to the present invention could not only prevent lung and/or systemic inflammation but also reduce lung and/or systemic inflammation associated with PM2.5.
Gene expression in Lungs.
We first assessed the lung mRNA profile of the genes playing a key role in inflammation using a SABioscience standard plate PAMM150Z (Cytokines &Chemokynes) . A subset of 23 genes was selected among the 84 markers based on a fold change expression value higher than 3 in Group 1 (PM2.5, Ctrl Diet) vs Control Group (Clean Air, Ctrl Diet) .
Figure 1 displays the clustergram representation of these genes’ expression across all groups. Cytokines/chemokines classified by literature evidence as pro-inflammatory or anti-inflammatory are highlighted in light grey or dark grey, respectively. The ones reported to exert both pro-and anti-inflammatory effects are highlighted in white.
As expected from the selection criteria, Control Group and Group 1 have almost a mirroring pattern in terms of gene expression. Interestingly, comparison between columns of Group 4 and especially Group 7 to Control Group indicates how the delivery of certain diets seems to be more effective to counteract the exposure to PM2.5, by bringing the expression level on inflammatory cytokines/chemokines back to the control levels. A clearer visualization of the similarities across groups of the expression patterns is provided by the 3-color version of the clustergram (light gray-black-dark gray) inserted as supporting Figure 1.
Among the genes analyzed, exposure to PM2.5 dramatically increased the expression of Ccl7, AdipoQ and Il23a, whose levels were partially or totally restored upon the different diets. Consistent with our findings, for all these genes a role in driving inflammatory diseases of the airways has been described in literature, consistent with our findings.
CCL7, the product of Ccl7 gene, is a chemokine classically known to attract monocytes/macrophages during inflammation. Literature reports that exposure to allergens and oxidative stress induced by ozone increase the production of CCL7 by airway epithelium (Michalec et al., CCL7 and CXCL10 orchestrate oxidative stress-induced neutrophilic lung inflammation. J Immunol. 2002； 168 (2) : 846-852； Stafford et al., Monocyte chemotactic protein-3 (MCP-3) /fibroblast-induced cytokine (FIC) in eosinophilic inflammation of the airways and the inhibitory effects of an anti-MCP-3/FIC antibody. J Immunol. 1997； 158 (10) : 4953-4960) and this results in the recruitment of eosinophils and neutrophils in lung, which drive inflammation (Michalec et al., CCL7 and CXCL10 orchestrate oxidative stress-induced neutrophilic lung inflammation. J Immunol. 2002； 168 (2) : 846-852； Mercer et al., Proteinase-activated receptor-1, CCL2, and CCL7 regulate acute neutrophilic lung inflammation. Am J Respir Cell Mol Biol. 2014； 50 (1) : 144-157) .
As for Adipoq, this gene encodes adiponectin, a hormone-like protein produced by adipose tissue, whose role in inflammation is controversial. Indeed, although predominantly endowed with anti-inflammatory functions, conflicting evidences report a pro-inflammatory role for adiponectin under certain conditions (Ehling et al., The potential of adiponectin in driving arthritis. J. Immunol. 2006； 176 (7) : 4468-4478； Garcia and Sood, Adiponectin in pulmonary disease and critically ill patients. Curr Med Chem. 2012； 19 (32) : 5493-5500； Sood et al., Serum Adiponectin is Associated with Adverse Outcomes of Asthma in Men but Not in Women. Front Pharmacol. 2011； 2: 55) . Interestingly, it has been demonstrated that not only adipocytes but also airway epithelial cells express adiponectin, which is increased in chronic obstructive pulmonary disease (COPD) , a chronic inflammatory disease of the lung (Miller et al., Adiponectin and functional adiponectin receptor 1 are expressed by airway epithelial cells in chronic obstructive pulmonary disease. J Immunol. 2009； 182 (1) : 684-691) .
Il23a encodes the alpha (or p19) subunit of the heterodimeric cytokine IL23, which has a pathogenic role in a number of chronic inflammation diseases, including asthma (Nakajima and Hirose. Role of IL-23 and Th17 Cells in Airway Inflammation in Asthma. Immune Netw. 2010； 10 (1) : 1-4) . IL23a mRNA was found upregulated in lung of mice exposed to allergic stimuli, where it elicited both a neutrophil-and an eosinophil-mediated inflammatory response (Nakajima and Hirose. Role of IL-23 and Th17 Cells in Airway Inflammation in Asthma. Immune Netw. 2010； 10 (1) : 1-4； Peng et al., IL-23 signaling enhances Th2 polarization and regulates allergic airway inflammation. Cell Res. 2010； 20 (1) : 62-71； IL-23 and Th17 cells enhance Th2-cell-mediated eosinophilic airway inflammation in mice. Am J Respir Crit Care Med. 2008； 178 (10) : 1023-1032) .
Consistently, IL23 deficiency or neutralization resulted in a reduced airway inflammation (Peng et al., IL-23 signaling enhances Th2 polarization and regulates allergic airway inflammation. Cell Res. 2010； 20 (1) : 62-71； IL-23 and Th17 cells enhance Th2-cell-mediated eosinophilic airway inflammation in mice. Am J Respir Crit Care Med. 2008； 178(10) : 1023-1032) .
Noteworthy, an interesting trend was observed in the expression of the gene Ccl12 in response to PM2.5 and the tested nutrients. Indeed, although not dramatically increased in Group1 (cut-off less than 3) , it became strongly down-regulated in all other groups treated with the different diets (especially in Group 3, receiving BB12) .
The murine Ccl12 gene encodes a chemokine which shares a high homology with the human CCL2 (Sarafi et al., Murine monocyte chemoattractant protein (MCP) -5: a novel CC chemokine that is a structural and functional homologue of human MCP-1. J Exp Med. 1997； 185 (1) : 99-109) , whose expression is increased in inflammatory disorders of the lung (Rose et al, Significant involvement of CCL2 (MCP-1) in inflammatory disorders of the lung. Microcirculation. 2003； 10 (3-4) : 273-288) . Moreover, in mouse models of idiopathic pulmonary fibrosis CCL12 itself was wound to recruit fibrocytes and to mediate fibrotic response (Moore et al., The role of CCL12 in the recruitment of fibrocytes and lung fibrosis. Am J Respir Cell Mol Biol. 2006； 35 (2) : 175-181) . Long- term exposure to air pollutants can expose airways to chronic inflammation and subsequent fibrosis, preventing the proper functioning of lungs. Therefore, the constant consumption of nutrients that can antagonize pro-fibrotic agents could represent a promising strategy in the prevention or management of such diseases.
Figure 2 summarizes the clustergram data, by showing the overall proximity of each Group to the Control condition. Two coefficients of similarity, are calculated by averaging in every group the Up-&Down-regulated gene expressions, respectively.
Different combinations of the four nutrients showed an overall synergistic effect on gene expression, as represented in Figures 3A (Group 2 and Group 3 vs Group 5) , 3B (Group 3 and Group 4 vs Group 6) and 3C (Group 2, Group 3 and Group 4 vs Group 7) , respectively.
Lung histological analyses
Lungs from sham (Control Group) and PM2.5 treated mice (Groups 1 to 7) were rinsed and properly collected and stored in liquid nitrogen. 10 μm serial sections were cut by a cryostat for each sham and PM2.5 exposed lung sample, mounted on slides and stained with Carazzi’s haematoxylin and eosin staining. A Nikon Eclipse E600 microscope was used for analysis. Pictures were captured at 40X magnification.
Random (or sporadic) particulate matter was observed engulfed in alveolar macrophages especially in Group 1 (PM2.5-treated group) , with evidences visible also in sections across other groups (Fig. 4b, d and f) . Some evident morphological changes consisted in occasional infiltration by inflammatory cells.
Serum inflammatory cytochines
A series of cytokines representing both so-called pro-inflammatory TH-1 cytokines, such as IFN-g, TNF-alpha, and IL-1, and TH-2 cytokines associated to the generation of IgE-mediated allergic reactions such as IL-4, IL-5 and IL-10, together with immunosuppressive cytokine TGF-beta have been already preliminary tested and measured in the serum of PM2.5 injected animals having undergone specific diet  regimens. As an example we show the serum concentration of IL-10, a fundamental marker of the allergic inflammation (Figure 5) . From the concentration levels, it can be deduced that pretreatment with certain diets (Group 3, Group 4 and especially the combination of the four ingredients in Group 7) can potentially prevent the onset of allergic inflammatory response. Although we did not observe significative variations to serum cytokines strictly attributable to PM2.5 powder it should be stressed that normal Balb/c mice analyzed in this study expressed already average levels of IL-10 noticeably higher than usual (2-3 fold, see for ex. Schloot et al., Diabetes Metab Res Rev 2002； 18: 64–70. ) , rendering even more important the decrease of IL-10 in certain groups of mice undergoing specific diet regimens. Moreover, it should also be considered that the time-frame of PM2.5 administration, is relatively short to produce and/or to appreciate measurable amounts of additional cytokines, particularly TH2 cytokines such as IL-4 and IL-5 mimicking chronic allergic reactions.
It will be appreciated that details of the foregoing examples, given for purposes of illustration, are not to be construed as limiting the scope of this invention. Although only a few exemplary embodiments of this invention have been described in detail above, those skilled in the art will readily appreciate that many modifications are possible in the examples without materially departing from the novel teachings and advantages of this invention. For example, the features described in relation to one example may be incorporated into any other example of the invention.
Accordingly, all such modifications are intended to be included within the scope of this invention, which is defined in the following claims and all equivalents thereto. Further, it is recognized that many embodiments may be conceived that do not achieve all of the advantages of some embodiments, particularly of the desirable embodiments, yet the absence of a particular advantage shall not be construed to necessarily mean that such an embodiment is outside the scope of the present invention. As various changes could be made in the above constructions without departing from the scope of the invention, it is intended that all matter contained in the above description shall be interpreted as illustrative and not in a limiting sense.
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Claims (7)

  1. A composition for reducing lung and/or systemic inflammation associated with PM2.5 exposure, comprising docosahexanoic acid, a probiotic strain, Vitamin C and Vitamin E, preferably the probiotic strain is Bifidobacterium animalis subsp. Lactis.
  2. The composition according to claim 1, wherein the composition contains docosahexanoic acid, probiotic strain, Vitamin C and Vitamin E in a ratio of 50-500 mg : 5 X 107-5 X 1010 CFU : 10 mg-250 mg : 1 mg-100 mg, preferably in a ratio of 75 mg-150 mg : 1 X 109-1 X 1010 CFU : 30 mg-150 mg : 4 mg-20 mg.
  3. The composition according to claim 1 or 2, wherein the composition is suitable for administration as food and supplement, for example, the composition is in the form of biscuit, juice, drinking powder, candy, ice-cream, dried fruits and vegetables, functional drink, capsule, tablet, caplet, bar, cream, gel, powder, solution or gum, and preferably the composition includes additional components selected from probiotics, vitamins, phytonutrients, natural herbals e.g. lycopene, turmeric or polyphenol, or the combination thereof.
  4. A method for preventing and reducing lung and/or allergic status linked to systemic inflammation associated with PM2.5 pollutant exposure in a subject in need thereof, comprising administrating an effective amount of the composition according to claim 1 or 2.
  5. The method according to claim 4, wherein the subject is exposed to PM2.5 pollutants with a level between 25 and 500 μg/m3, especially between 100 and 500 μg/m3.
  6. The method according to claim 4 or 5, wherein the subject is a human subject living in the environment with the average daily level of PM2.5 at 125 μg/m3, with the peak measured at even 500 μg/m3.
  7. Use of the composition according to one of claim 1 to 3 in the manufacture of a supplement for food, medicament, cosmetics and beverage for reducing lung and/or allergic status linked to systemic inflammation associated with PM2.5 pollutant exposure in a subject in need thereof.
PCT/CN2016/096025 2015-08-19 2016-08-19 A composition for reducing lung and/or systemic inflammation associated with pm2.5 exposure and the use thereof WO2017028813A1 (en)

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