WO2022058422A1

WO2022058422A1 - Use of andrographis paniculata extract to protect against air pollution related diseases

Info

Publication number: WO2022058422A1
Application number: PCT/EP2021/075472
Authority: WO
Inventors: Igor Bendik; Bettina Boehlendorf; Pascale FUCHS BOSSERT; Hubert Paul HUG; Katharina KUENZLI; Bernd Mussler; Nathalie Richard; Guido Wahl
Original assignee: Dsm Ip Assets B.V.
Priority date: 2020-09-17
Filing date: 2021-09-16
Publication date: 2022-03-24

Abstract

The present invention relates to systemic detoxification and chronic inflammation by using methanolic extracts of Andrographis paniculata, that lead to a decrease of anti-inflammatory markers in human lung cell lines, which decreases the expression of inflammatory cytokines. Therefore, the extract can be used to reduce the adverse effects of air pollution generally (and especially of particulate air pollution), which includes: cardiovascular problems, respiratory diseases, and chronic inflammation of tissues that come into contact with air borne particles.

Description

USE OF ANDROGRAPHIS PANICULATA EXTRACT TO PROTECT AGAINST AIR POLLUTION RELATED DISEASES

BRIEF DESCRIPTION OF THE INVENTION

The present invention relates to systemic detoxification and inhibiting inflammation during exposure to air polluting agents by using methanolic extracts of a Andrographis paniculate/ (Kalmegh, creat, green chireta).

BACKGROUND OF THE INVENTION

Air pollution has been associated with morbidity and mortality mainly due to pulmonary and cardiovascular diseases.

Inflammation is a key process in the development of the diseases induced by the particulate matter of air pollution. Interleukin-8 (IL-8) is part of the innate immune system and important in the initiation of an immune response, but overstimulation and the resulting dysfunction of the recruited neutrophils within airways can result in the release of pro-inflammatory molecules resulting in the damage rather than protection of lung tissue.

Interleukin-6 (IL-6) is secreted by T-lymphocytes and macrophages and helps also to stimulate an immune response. IL-6 inhibits the actions of tumor necrosis factor a (TNF-a) and interleukin-1 (IL-1). It has been mainly connected with anti-inflammatory action but also some pro-inflammatory functions. Therefore, the benefit on its inhibition depends on the state of the infection. To counteract chronic inflammation, it is helpful to decrease the expression of IL- 6.

Monocyte chemoattractant protein-1 (MCP-1) recruits monocytes, memory T-lymphocytes and dendritic cells to the site of inflammation. Also, in chronic inflammation or inflammation mediated by air pollution it may be of advantage to decrease MCPO-1 expression.

Prostaglandin E2 (PGE2) is an inflammatory cytokine that increase pain caused by other inflammation mediators like bradykinin or histidine. It is also with other cytokines involved in the induction of fever. In addition is has other complex functions in many tissues. PGE2 is accepted as a general marker for inflammation.

An enhancement of the cellular detoxification pathway is considered to be helpful in conditions such as ageing, cardiovascular diseases, and lung diseases such as chronic obstructive pulmonary disease (COPD).

Andrographis paniculate is a herbaceous plant which is used in Traditional Chinese Medicine (TCM) and Ayurveda, and is widely used to treat sore throat, flu, and other respiratory tract infections. An active ingredient, andrographolide, has been investigated as an anti-cancer and cardiovascular disease medication.

It is desirable to have natural compounds or extracts which can be used as a nutraceutical, pharmaceutical or food additive that could work to protect against air pollution in addition to inhibit inflammation.

It would be desirable to have a natural plant extract which can combat the adverse effects of particulate air pollution.

DETAILED DESCRIPTION OF THE INVENTION

We have shown that methanolic Andrographis paniculata extracts can reduce Diesel particulate matter induced pro-inflammatory cytokines. Therefore, methanolic Andrographis paniculata extracts can be used to reduce the adverse effects of air pollution generally (and especially of particulate air pollution), which includes: cardiovascular problems, respiratory diseases, and chronic inflammation of tissues that encounter air borne particles.

Some anti-inflammatory effects of compounds found in Andrographis paniculata extracts have been detected and described (Villedieu-Percheron et al. Foods 2019, 8(12):683). To date, however, no effects of Andrographis paniculata in air pollution related diseases have been reported.. For the first time, we have found that methanolic Andrographis paniculate extracts anti-inflammatory actions in human lung cell lines treated with Diesel particulate matter.

DEFINITIONS "Healthy person" means a person who has not been diagnosed with, or experiences symptoms of any of the following diseases or conditions: cardiovascular disease (including having had a non-fatal heart attack, irregular heartbeat, and impaired circulatory system), diabetes type 2, and respiratory disease, asthma or aggravated asthma, decreased lung function, or other conditions which result in difficulty breathing).

"Particulate air pollution" means which air which contains particles which are classified as nanoparticles, or have a particle size of PM2.5 or less. These size particles can be the result of "natural sources" such as volcanic emission, dust storms, forest fires, smoke from grassland fires and the like, or as a result of human activity such as automotive emissions, manufacturing emissions or other activities, including cigarette smoking.

"Cardiovascular health" is defined as the absence of cardiovascular symptoms which otherwise can occur in response to exposure to particulate air pollution.

"Respiratory health" as used herein is defined as the absence of conditions associated with abnormal respiratory functioning associated with particulate matter-induced pro-inflammatory cytokines such as: asthma, emphysema, bronchitis, and chronic obstructive pulmonary disease. As used herein, it does not include diseases such as the common colds

"Air pollution", as used herein, refers to conditions where potentially harmful particulates, biological molecules or other substances have been introduced into the air. Examples of categories of pollutants include:

• Sulfur oxides such as those produced as a result of coal and petroleum combustion;

• Nitrogen oxides such as those produced from high temperature combustion, including nitrogen dioxide (one of the more prominent air pollutants, it is a reddish brown gas with a characteristic sharp odor);

• Carbon monoxide which can be produced by incomplete combustion of fuel and vehicular exhaust;

• Volatile organic compounds can include methane- or non-methane type compounds and are often referred to as greenhouse gases; • Particulates (also called particulate matter or PM) which are small solid or liquid particles which are suspended in the atmosphere. Origins may be "natural" such as from volcanic emissions, dust storms, or forest and grassland fires, or may be a result of human activities.

• Pollution in the form of soot, gases and other matter which are in the form of tiny particles, termed "respirable particulate matter". Respirable particulate matter is categorized by size, such as below 10 or 2.5 microns aerodynamic diameter (PMio or PM2.5, respectively), or as nanoparticles (less than 100 nm diameter, or PM0.1). These particles often come from vehicle emissions, particularly diesel fuel, or from diesel-powered machinery.

"Ameliorating the risk" of an adverse conditions means: protecting against the occurrence of the condition or symptom; delaying the onset of a condition or symptom; lessening the severity of a condition or symptom that has already occurred; shortening the time that the condition or symptom persists; and/or elimination of the condition or symptom.

"Prevention" of an adverse condition includes ameliorating the risk as well as not allowing the adverse condition to occur.

Particulates from human activities are linked to many health hazards, including heart disease and adverse respiratory conditions, including lung cancer.

Cigarette smoke also contains PMs as well as other chemicals which are also found in polluted air. Thus, another aspect of this invention is the use of a methanolic Andrographis paniculate/ extract to protect a person exposed or at risk of exposure to cigarette smoke; which includes the smoker and persons subject to "second hand" smoke. Another aspect is a method of lessening the risk of adverse conditions in a person exposed to cigarette smoke comprising administering to the person at risk an effective amount of Andrographis paniculata extracts, fractions, or compounds

PM includes dust, dirt, soot and smoke. Particles termed "inhalable coarse particles" have diameters larger than 2.5 micrometers, but smaller than 10 micrometers. "Fine particles" are smaller, having diameters less than 2.5 micrometers. They are typically responsible for reduced visibility and haze. Many of the fine particles are "secondary particles", which are the end products of chemical reactions in the atmosphere which occur when sulfur dioxides and nitrogen oxides are emitted by power plants, automobiles and other industrial activities.

Fine particles are particularly troublesome as they can get deep into the lungs and the bloodstream and can potentially cause serious health problems, including:

Premature death in people who have heart or lung disease, Non-fatal heart attacks Irregular heartbeat Asthma or aggravated asthma Decreased lung function Acute exacerbation of chronic obstructive pulmonary disease (COPD) Increased respiratory symptoms, such as irritation of the airways, coughing and/or difficulty breathing.

Thus one aspect of this invention is the use of a methanolic Andrographis paniculate/ extract to protect, ameliorate, or lessen the risk of inflammation, cardiovascular and/or respiratory adverse conditions resulting from the exposure to air pollution, preferably particulate matter air pollution, wherein the adverse condition is selected from the group consisting of: inflammation, premature death in people who have heart or lung disease, non-fatal heart attacks, irregular heartbeat, asthma or aggravated asthma, decreased lung function, acute exacerbation of chronic obstructive pulmonary disease (COPD), and increased respiratory symptoms, such as irritation of the airways, coughing and/or difficulty breathing. Another aspect is a method of lessening the risk of adverse conditions in a person exposed to air pollution, comprising administering a methanolic Andrographis paniculata extract to a person in need thereof and wherein the adverse condition is selected from the group consisting of: inflammation, premature death in people who have heart or lung disease, non-fatal heart attacks, irregular heartbeat, asthma or aggravated asthma, decreased lung function, acute exacerbation of chronic obstructive pulmonary disease (COPD), and increased respiratory symptoms, irritation of the airways, coughing and/or difficulty breathing.

Another aspect of this invention is a method of protecting, ameliorating or lessening the risk of inflammation, cardiovascular and/or respiratory adverse conditions resulting from the exposure to air pollution, preferably particulate matter air pollution, wherein the adverse condition is selected from the group consisting of: inflammation, premature death in people who have heart or lung disease, non-fatal heart attacks, irregular heartbeat, asthma or aggravated asthma, decreased lung function, acute exacerbation of chronic obstructive pulmonary disease (COPD), and increased respiratory symptoms, such as irritation of the airways, coughing and/or difficulty breathing, comprising administering a methanolic Andrographis paniculate/ extract to a person in need thereof or at risk of exposure to air pollution.

Another aspect of this invention is the use of a methanolic Andrographis paniculata extract in the manufacture of a medicament or nutraceutical to protect, ameliorate, or lessen the risk of a cardiovascular, inflammatory and/or respiratory adverse condition resulting from the exposure to air pollution, preferably particulate matter air pollution, wherein the adverse condition is selected from the group consisting of: premature death in people who have heart or lung disease, non-fatal heart attacks, irregular heartbeat, asthma or aggravated asthma, decreased lung function, acute exacerbation of chronic obstructive pulmonary disease (COPD), and increased respiratory symptoms, such as irritation of the airways, coughing and/or difficulty breathing.

In some embodiments the person receiving the extract is a cigarette smoker or a person at risk of exposure to second hand cigarette smoke.

COMBINATIONS WITH OTHER ACTIVE INGREDIENTS

A methanolic Andrographis paniculata extract of this invention may be combined with other active ingredients to make a composition which has beneficial results. Examples of further active ingredients include Vitamin E, water soluble tomato extract, resveratrol, Vitamin D, 25- hydroxy vitamin D3, hydroxytyrosol, polyunsaturated fatty acids (PUFAs), Vitamin A and mixtures thereof. Thus, this invention also includes the following combination of ingredients:

An Andrographis Paniculata extract and Vitamin E

An Andrographis Paniculata extract and water-soluble tomato extract (such as FRUITFLOW® available from DSM Nutritional Products, Switzerland)

An Andrographis Paniculata extract resveratrol

An Andrographis Paniculata extract and Vitamin D

An Andrographis Paniculata extract and 25-OH Vitamin D3 An Andrographis Paniculate/ extract and hydroxytyrosol

An Andrographis Paniculata extract and Polyunsaturated fatty acids (PUFAs)

An Andrographis Paniculata extract Vitamin A.

In each of the above cases, the amount of the Andrographis paniculata extract is as detailed in this specification, and the amount of the second ingredient is present in an amount which is the maximum daily amount known in the art for each ingredient.

DOSAGES

A recommended daily dose is a sufficient amount of a methanolic Andrographis paniculata extract would be up to 2 grams/day for an adult. In some embodiments, the dosage is 10-1000 mg/day. In another embodiment it is 50-500 mg/ day. In yet another embodiment it is 100- 250 mg per day.

For combinations of the active ingredients, the dosages may be adjusted so that the dosages of the combined ingredients are from at least 0.1 to 10 mg per day, but should not exceed 30 mg per day.

If desired, the daily intake can be divided into two or more dosages, such as twice a day tablets.

FORMULATIONS

The composition of the present invention is preferably in the form of nutritional composition, such as fortified food or fortified beverages, or in form of fortified liquid food/feed (such as drinks, or shots), pills or capsules.

The dietary and pharmaceutical compositions according to the present invention may be in any galenic form that is suitable for administering to the human body, especially in any form that is conventional for oral administration, e.g. in solid form, such as (additives/supplements for) food or feed, food or feed premix, fortified food, tablets, pills, granules, dragees, capsules, and effervescent formulations such as powders and tablets, or in liquid form such as solutions, emulsions or suspensions as e.g. beverages, pastes and oily suspensions. The pastes may be encapsulated in hard or soft-shell capsules, whereby the capsules feature e.g. a matrix of (fish, swine, poultry, cow) gelatin, plant proteins or lignin sulfonate. Examples for other application forms are forms for transdermal, parenteral or injectable administration. The dietary and pharmaceutical compositions may be in the form of controlled (delayed) release formulations. The compositions of the present invention are not administered topically, such as application to the nasal passage.

The dietary compositions according to the present invention may further contain protective hydrocolloids (such as gums, proteins, modified starches), binders, film forming agents, encapsulating agents/materials, wall/shell materials, matrix compounds, coatings, emulsifiers, surface active agents, solubilizing agents (oils, fats, waxes, lecithins etc.), adsorbents, carriers, fillers, co-compounds, dispersing agents, wetting agents, processing aids (solvents), flowing agents, taste masking agents, weighting agents, jellyfying agents, gel forming agents, antioxidants and antimicrobials.

Examples of food are cereal bars, dairy products, such as yoghurts, and bakery items, such as cakes and cookies. Examples of fortified food are cereal bars, and bakery items, such as bread, bread rolls, bagels, cakes and cookies. Examples of dietary supplements are tablets, pills, granules, dragees, capsules and effervescent formulations, in the form of non-alcoholic drinks, such as soft drinks, fruit juices, lemonades, near-water drinks, teas and milk-based drinks, in the form of liquid food, such as soups and dairy products (muesli drinks).

Beverages encompass non-alcoholic and alcoholic drinks as well as liquid preparations to be added to drinking water and liquid food. Non-alcoholic drinks are e.g. soft drinks, sport drinks, fruit juices, vegetable juices (e.g. tomato juice), lemonades, teas and milk-based drinks. Liquid foods are e.g. soups and dairy products (e.g. muesli drinks).

In addition to the methanolic Andrographis paniculata extract, pharmaceutical and nutraceutical compositions according to the present invention may further contain conventional pharmaceutical additives and adjuvants, excipients or diluents, including, but not limited to, water, gelatin of any origin, vegetable gums, ligninsulfonate, talc, sugars, starch, gum rabic, vegetable oils, polyalkylene glycols, flavoring agents, preservatives, stabilizers, emulsifying agents, buffers, lubricants, colorants, wetting agents, fillers, and the like.

The following non-limiting Examples are presented to better illustrate the invention.

EXAMPLE 1 Decrease of inflammatory markers induced by Diesel Particles

Methods:

The human bronchial epithelial cell line BEAS-2B was from ATCC (American Type Culture Collection, Manassas, VA) and cultured in Bronchial Epithelial cell Growth Medium (BEGM, Lonza, Wakersville, MD) in Cell BIN D® surface plastic flasks (Corning Inc., Corning, NY). The adenocarcinomic human alveolar basal epithelial A549 cell line was obtained from ATCC and cultured in Kaighn's Modification of Ham's F-12 Medium (F-12K medium) (Life Technologies, USA), supplemented with 10% FBS (Sigma, Saint-Louis, MO). These cells were cultured at 37 °C in a humidified atmosphere containing 5% CO2.

BEAS-2B cells were seeded in 12-well Cell Bl N D® surface culture plates (Corning Inc.) at 3 to 4 x 10⁵ cells per well. A549 cells were seeded in 12-well plates at 2 x 10⁵ cells per well.

Diesel Particulate Matter (Standard Reference Material SRM 1650b, National Institute of Standards & Technology, NIST, Gaithersburg, MD) at 80 mg/ml DMSO (100 %) were sonicated for 5 min and thereafter diluted 400fold in medium. This dilution was twofold further diluted for the assay.

After 24 h, cells were treated with the diluted Diesel Particulate Matter at 100 pg/ml and in the presence of different concentrations of a methanolic Andrographis paniculate/ extract, other plant extracts, fractions and compounds as indicated. The final DSMO concentrations were 0.175%. Untreated cells or cells treated with 0.175% DMSO were used as controls. After 24 h, cell supernatants were collected.

The concentrations of IL-6 and IL-8 in the supernatants were determined by Luminex kits (BIORAD Laboratories, Hercules, CA) and used in the LiquiChip Workstation IS 200 (Qiagen, Hilden, Germany). The data were evaluated with the LiquiChip Analyser software (Qiagen).

Cell survival assays of the BEAS-2B and A549 cells were performed with AlamarBlue® Cell Viability Reagent (ThermoFisher Scientific) according to the protocol of the manufacturer. Nontoxic concentrations of the extracts, fractions and single compounds were selected for the assays.

Secreted PGE2 was determined by Enzyme Immuno Assay (EIA) (Cayman Chemicals, Ann Harbor, Wl). Mean values, standard deviation and p-values with Student's t-test were calculated with Excel.

P-values greater than 0.05 were considered as indication for significance.

Results:

Andrographis paniculate/ extracts were tested for their ability to inhibit Diesel Particulate Matter (PM)-induced IL-6 secretion in human lung cell lines. Untreated BEAS-2B cells did not secrete IL-6 Ttable 1, row 2; table 3, row2). Treatment with the solvent DMSO resulted in a slight decrease in IL-6 secretion of BEAS-2B cells and therefore, all values of the tested Andrographis paniculata extracts have to be compared with the PM control in the presence of DMSO (Table 1, rows 1 and 3; table 3, rows 1 and 3). TiOz particles did not lead to an increase in IL-6 secretion which shows that a physical effect of the particles is not responsible for the effects (Table 1, row 5). Lipopolysaccharide (LPS) a known inducer of IL-6 had a strong effect; it was over 40 times stronger than PM and DMSO (Table 1, below, rows 1 and 4; table 3, row 4).

Next, an extracts of Andrographis paniculata was tested under the described conditions (Table 2). It showed a significant decrease of IL-6 secretion on BEAS-2B cells (Table 2, row 5).

Compared to the control, PM and DMSO, set to 100 %, IL-6 secretion decreased to 39 % in the presence of extract. All values were significant by t-test (Table 2).

Similarly, in the human lung cell line A549, IL-6 secretion in the presence of PM was decreased by the Andrographis paniculata extract. The results are shown in Table 3. In the presence of the extract IL-6 secretion decreased to 42 % in comparison to the control that was set to 100 % (Table 3). All relevant values were significant. The decrease of IL-6 in A549 cells was similar to the value obtained with BEAS-2B cells: 42 % and 39 %, respectively.

Also, IL-8 secretion in the presence of PM of the human lung cell line A549 was decreased by this Andrographis paniculata extract. However, the decrease of IL-8 secretion was not as strong as that of IL-6. In the presence of the extract IL-8 secretion went down to 88 % compared to the PM + DMSO control that was set to 100 %, but with significant values (table 4). Table 1: IL-6 secretion (pg/ml) of BEAS-2B cells treated with Diesel Particulate Matter (PM) in the presence of compounds as indicated. PM concentration was always 100 pg/ml. The IL-6 concentration of the positive control PM with DMSO was set to 100 % for comparison.

Table 2: IL-6 secretion (pg/ml) of BEAS-2B cells treated with Diesel Particulate Matter (PM) in the presence of different Andrographis paniculata extracts. PM concentration was always 100 pg/ml. The IL-6 concentration of the positive control PM with DMSO was set to 100 % for comparison. This experiment has been performed four times.

Table 3: IL-6 secretion (pg/ml) of A549 cells treated with Diesel Particulate Matter (PM) in the presence of an Andrographis paniculata extract as indicated. PM concentration was always 100 pg/ml. The IL-6 concentration of the positive control PM with DMSO was set to 100 % for comparison.

Table 4: IL-8 secretion (pg/ml) of A549 cells treated with Diesel Particulate Matter (PM) in the presence of an Andrographis paniculate/ extract as indicated. PM concentration was always 100 pg/ml. The IL-8 concentration of the positive control PM with DMSO was set to 100 % for comparison.

Claims

1. Use of a methanolic Andrographis paniculata extract to protect, ameliorate, or lessen the risk of cardiovascular, inflammatory and/or respiratory adverse condition resulting from the exposure to air pollution, wherein the adverse condition is selected from the group consisting of: inflammation, premature death in people who have heart or lung disease, non-fatal heart attacks, irregular heartbeat, asthma or aggravated asthma, decreased lung function, acute exacerbation of chronic obstructive pulmonary disease (COPD), and increased respiratory symptoms, such as irritation of the airways, coughing and/or difficulty breathing.

2. Use according to Claim 1 where the air pollution is particulate air pollution.

3. Use according to Claim 1 or 2 wherein the air pollution comprises diesel particles or cigarette smoke.

4. A method of lessening the risk of adverse conditions in a person exposed to air pollution, comprising administering a methanolic Andrographis paniculata extract to a person in need thereof and wherein the adverse condition is selected from the group consisting of: inflammation, premature death in people who have heart or lung disease, non-fatal heart attacks, irregular heartbeat, asthma or aggravated asthma, decreased lung function, acute exacerbation of chronic obstructive pulmonary disease (COPD), and increased respiratory symptoms, irritation of the airways, coughing and/or difficulty breathing.

5. The method of Claim 4 wherein the air pollution is particulate air pollution.

6.The method of Claim 4 wherein the air pollution comprises diesel particles or comprises diesel particles or cigarette smoke.