NZ761390B2 - Boysenberry, apple, and blackcurrant compositions and methods of preparation and use therefor - Google Patents

Abstract

The present disclosure encompasses compositions prepared from Boysenberry and apple, as well as compositions prepared from Boysenberry, apple and blackcurrant. Also encompassed are methods of preparing these compositions and methods of using these compositions, in particular, for treating or preventing disorders of the respiratory system, including amongst others: inflammation, asthma, chronic obstructive pulmonary disease, allergic airways inflammation, reactive airway disease, airway fibrosis, and airway remodelling.

Description

BOYSENBERRY, APPLE, AND BLACKCURRANT COMPOSITIONS AND METHODS OF PREPARATION AND USE THEREFOR RELATED APPLICATION This application claims the benefit of New Zealand patent application number 734440 filed on 8 August 2017, the entire ts of which are incorporated herein by reference.

FIELD OF THE INVENTION The t disclosure relates to compositions prepared from Boysenberry and apple, and compositions prepared from Boysenberry, apple and blackcurrant.

The present disclosure relates also to methods of preparing such itions, and methods of using such itions, including methods of treating or ting disorders of the respiratory tract, such as in?ammatory ions of the respiratory tract, including asthma, chronic obstructive pulmonary e, allergic airways in?ammation, reactive airway disease, airway fibrosis, and airway remodelling and the physiological conditions that lead to these conditions.

BACKGROUND OF THE INVENTION Airway remodelling is understood as a progressive and irreversible decline in airway function due to chronic in?ammatory processes that result in structural changes in the airway walls (67). Remodelling of the airways may e all layers of the airway walls and can occur anywhere along the respiratory tract, from the large to the small airways.

Remodelling leads to key changes in epithelial tissue (68). Damaged epithelial cells release profibrotic cytokines, including EGF and TGF—B, which leads to fibroblast proliferation, myofibroblast activation, and ultimately to the formation of subepithelial fibrosis (69).

Airway smooth muscle hypertrophy and hyperplasia lead to an increase in airway wall thickness. In turn, this leads to accelerated lung function decline and irreversible or only partially reversible air?ow obstruction.

Acute disorders causing airway in?ammation include asthma and COPD. It is estimated that 150 million people are affected by asthma worldwide, with a 5-15% ence in children (61). The ence of COPD is ted to be between 15—20%, and it is estimated to cause 2.75 million deaths per annum (86). In the case of chronic asthma there is evidence of cumulative tissue remodelling, ?brosis, and consequent loss of lung on (45, 59). Fibrosis and remodelling are also associated with COPD. Remodelling manifests as a progressive increase in symptoms such as dyspnoea and a ponding decrease in odilator responsiveness (67). Current asthma treatments are designed to manage ation and mitigate the symptoms and severity of asthma attacks (30, 43).

COPD treatments are also designed to control inflammation and improve air?ow. r, no asthma or COPD medications are known to prevent airway remodelling (70-74), and there are no current treatments available to t aberrant remodelling.

Asthma pathogenesis and lung tissue remodelling have been linked to an increase in pro?brotic, arginase—positive, alternatively activated macrophages (AAMs) in the lung (27, 29, 34). However, temporal depletion of macrophage populations in a model of bleomycin—induced pulmonary ?brosis illustrates that lung macrophages may also develop ?brolytic ons that contribute toward the resolution of ?brosis (14).

Mediators of tissue remodelling, such as the matrix metalloproteinases (MlVlPs), play an important role in regulating ?brosis (5, 7, 8, 10, 38). Of these, lV?VlP—9 is widely reported to increase in conditions of lung in?ammation and s and is ated with improved symptoms in asthma sufferers (25, 32, 33). MMP—9, in concert with other MMPs, exerts ?brolytic activity that leads to the breakdown of denatured collagens that could moderate inappropriate lung remodelling (5, 60). As such, MMP-9 may represent a le therapeutic target to limit lung damage in chronic asthma as well as other ary diseases.

Large epidemiological studies have found that increased fruit and vegetable consumption correlates with reduced asthma symptoms (39, 46, 47). These population studies have ?ed foods high in polyphenols such as apples, pears (13, 51, 62), carrots, es (46—48), and citrus (ll) as having inverse correlations with frequency and severity of reported asthma symptoms, in particular wheeze and cough symptoms (1 1, 13, 46, 47). r, the effect of fruits high in polyphenols on lung ?brosis and tissue lling is unknown. To date, no generally successful methods for preventing airway remodelling have been established.

Given the occurrence of respiratory disorders in the tion, including allergic airways in?ammation associated with asthma, COPD, reactive airway disease, airway fibrosis, and airway remodelling, there is a need for new compositions, particularly compositions derived from natural sources, for restoring and maintaining respiratory health.

SUMMARY OF THE INVENTION In one , the invention encompasses a method of treating or preventing in?ammation in the respiratory tract, comprising: administering to a subject a composition comprising a Boysenberry and apple trate or a Boysenberry, apple and blackcurrant concentrate, thereby treating or preventing the in?ammation in the respiratory tract in the subject.

Also encompassed is a composition, for example, a nutraceutical composition, comprising a Boysenberry and apple trate or a berry, apple and blackcurrant concentrate for treating or preventing inflammation in the respiratory tract in a subject.

In one other aspect, the invention encompasses a method of treating or preventing asthma, comprising: administering to a subject a composition comprising a Boysenberry and apple concentrate or a Boysenberry, apple and blackcurrant concentrate, thereby treating or preventing the asthma in the t.

Also encompassed is a composition, for example, a nutraceutical composition, comprising a Boysenberry and apple concentrate or a Boysenberry, apple and blackcurrant concentrate for treating or preventing asthma in a subject.

In yet one other aspect, the invention encompasses a method of treating or preventing chronic obstructive pulmonary disease, comprising: administering to a subject a composition comprising a Boysenberry and apple concentrate or a Boysenberry, apple and blackcurrant trate, thereby treating or preventing the chronic obstructive pulmonary disease in the subject.

Also encompassed is a composition, for example, a nutraceutical composition, comprising a berry and apple concentrate or a berry, apple and urrant concentrate for treating or ting chronic obstructive pulmonary disease in a subject.

In still one other , the invention encompasses a method of treating or ting aberrant collagen deposition or fibrosis in the respiratory tract, comprising: administering to a subject a composition comprising a Boysenberry and apple concentrate or a Boysenberry, apple and blackcurrant concentrate, thereby treating or preventing the aberrant collagen deposition or fibrosis in the respiratory tract of the subject.

Also encompassed is a composition, for example, a nutraceutical composition, comprising a Boysenberry and apple concentrate or a Boysenberry, apple and blackcurrant concentrate for treating or preventing aberrant collagen deposition or fibrosis in a subject.

In even one other aspect, the invention encompasses a method of treating or preventing airway remodelling, sing: administering to a subject a composition comprising a Boysenberry and apple concentrate or a Boysenberry, apple and blackcurrant concentrate, thereby treating or preventing the airway lling in the t.

Also encompassed is a composition, for example, a nutraceutical composition, comprising a Boysenberry and apple concentrate or a Boysenberry, apple and urrant trate for treating or preventing airway lling in a subject.

In various aspects: The composition comprises Boysenberry juice concentrate, Boysenberry puree, or Boysenberry powder.

The composition comprises apple juice trate, apple puree, or apple powder.

The composition comprises blackcurrant juice concentrate, blackcurrant puree, or blackcurrant powder.

The composition comprises a dosage unit comprising about 5 to about 500 mg total anthocyanins.

For the nutraceutical composition comprising the Boysenberry and apple concentrate, the composition comprises a dosage unit comprising about 5 to about 500 mg total Boysenberry anthocyanins.

For the nutraceutical composition comprising the Boysenberry, apple and urrant concentrate, the composition ses a dosage unit comprising about 5 to about 500 mg total Boysenberry and blackcurrant anthocyanins.

The ition is formulated for enteral administration.

The composition is ated for oral administration.

The composition is formulated as a syrup or as drops.

The composition is formulated as a gel or jelly.

The composition is ated as a tablet or capsule.

The composition is formulated for administration at a dosage of about 0.1 mg/kg to about 10 mg/kg total anthocyanins/subj ect’s body .

For the nutraceutical composition comprising the Boysenberry and apple concentrate, the composition is ated for administration at a dosage of about 0.1 mg/kg to about 10 mg/kg total Boysenberry anthocyanins/subject’s body weight.

For the nutraceutical composition comprising the Boysenberry, apple and blackcurrant concentrate, the composition is formulated for administration at a dosage of about 0.1 mg/kg to about 10 mg/kg total Boysenberry and urrant yanins/subj ect’ s body weight.

The composition is formulated for administration at a dosage of about 10 mg to about 1000 mg total anthocyanins per day.

For the nutraceutical composition comprising the Boysenberry and apple concentrate, the ition is formulated for administration at a dosage or about 10 mg to about 1000 mg total Boysenberry anthocyanins per day.

For the nutraceutical composition comprising the Boysenberry, apple and blackcurrant concentrate, the composition is formulated for administration at a dosage of or about 10 mg to about 1000 mg total Boysenberry and blackcurrant yanins per day.

Alternatively, the dosage is about 10 mg to about 200 mg total anthocyanins per day, or about 50 mg total anthocyanins per day.

For the nutraceutical composition comprising the berry and apple concentrate, the dosage is about 10 mg to about 200 mg total Boysenberry anthocyanins per day, or about 50 mg total Boysenberry anthocyanins per day.

For the nutraceutical composition comprising the Boysenberry, apple and blackcurrant concentrate, the dosage is about 10 mg to about 200 mg total berry and blackcurrant anthocyanins per day, or about 50 mg total Boysenberry and blackcurrant anthocyanins per day.

The composition comprises added polyphenols.

The composition is formulated for co-administration with a further respiratory aid.

The composition is formulated for co—administration with one or more treatments for a chronic respiratory disorder.

The in?ammation is associated with a chronic respiratory disorder.

The ation is associated with one or more of: asthma, chronic obstructive pulmonary disease, allergic airways in?ammation, reactive airway disease, airway fibrosis, and airway remodelling.

The asthma is atopic or non—atopic.

The asthma is ated with airway fibrosis or airway lling.

The c obstructive pulmonary disease is associated with smoking or The chronic obstructive pulmonary disease is associated with airway fibrosis or airway remodelling.

The aberrant en deposition or the fibrosis is associated with a chronic respiratory disorder.

The aberrant collagen deposition or the fibrosis is associated with asthma or chronic obstructive pulmonary e.

The airway remodelling is associated with a chronic respiratory disorder.

The airway lling is associated with one or more of: asthma and chronic obstructive pulmonary e.

In still one further aspect, the invention comprises the use of a composition comprising a Boysenberry and apple concentrate or a Boysenberry, apple and blackcurrant concentrate for preparing a nutraceutical composition for: (i) treating or preventing in?ammation in a respiratory tract in a subject; (ii) treating or preventing asthma in a subject; (iii) ng or preventing chronic obstructive pulmonary disease in a subject; (iv) treating or preventing allergic airways in?ammation in a subject; (v) treating or preventing reactive airway disease in a subject; (vi) treating or preventing aberrant collagen deposition in a subject; (vii) ng or preventing fibrosis in a respiratory tract in a subject; (viii) treating or preventing airway remodelling in a subject.

In various aspects, the therapeutic use employs the compositions, dosages, and ations, and relates to the various conditions, as noted above.

The ing brief summary broadly describes the features and technical ages of certain embodiments of the present invention. Further technical advantages will be described in the detailed description of the invention and examples that follows.

Novel es that are ed to be characteristic of the invention will be better understood from the detailed description of the invention when considered in connection with any accompanying figures and examples. r, the s and examples provided herein are intended to help illustrate the invention or assist with developing an tanding of the invention, and are not intended to limit the invention's scope.

BRIEF DESCRIPTION OF THE DRAWINGS Fig. lA—D. Therapeutic oral Boysenberry treatment reduces OVA—induced chronic lung in?ammation. A: 6—week—old male C57Bl/6 mice (n = 10 per group) were primed i.p. with OVA/alum (day 0) then challenged in with OVA every 7 days for 10 weeks. From weeks 6 to 10 Boysenberry juice was administered orally (gavage) l h prior to, and 2 days after, each in. OVA challenge. B: representative H&E staining of lung tissue from naive, k OVA challenge only (OVA), 10—week OVA challenge with therapeutic Boysenberry (OVA BoysB) treatment, and Boysenberry alone (BoysB)—treated mice.

Arrows and * indicate immune cell in?ltrate. Magni?cation X4 (top) and X10 (bottom). C: representative AB—PAS staining of lung tissue. Arrows indicate dark purple mucus—positive ioles. Magni?cation X4 (top) and X20 (bottom). D: total cells per m1 BALF and ?ow cytometric quanti?cation of tage of eosinophils in BALF following ?nal OVA challenge. **P < 0.01, ***P < 0.001 (n = 10 per group) one—way ANOVA with Tukey’s post hoc test ed with naive and OVA challenge with therapeutic Boysenberry treatment and Boysenberry alone—treated mice.

Fig. 2A—D. Boysenberry treatment increases arginase expression and macrophage accumulation in lung tissue during OVA—induced chronic lung in?ammation.

A: representative H&E staining of lung tissue from 10—week OVA—challenged mice, with and without Boysenberry treatment. Arrows indicate macrophages. Magni?cation X100, scale 200 um. B: entative Western blot analysis of iNOS (135 kDa) and arginase (37 kDa) expression in lung . Noncontiguous bands from the same Western blot are shown.

C and D: quanti?cation of iNOS and arginase Western blot signals normalized to B—actin signal. **P < 0.01 (n = 10 per group) one—way ANOVA with Tukey’s post hoc test.

Fig. 3A—B. Boysenberry treatment increases the accumulation of arginase+ alternatively activated hages during OVA—induced chronic lung in?ammation.

Representative immuno?uorescent labelling of lung tissue from lO—week OVA—challenged mice with and with—out Boysenberry treatment. A: CD68+CD206+macrophages identi?ed by *. B: CD206+arginase+macrophages identi?ed by *. DAPI nuclear stain (dark blue).

Magni?cation X40, scale 200 pm.

Fig. 4A—E. Boysenberry treatment decreases collagen deposition and increases MMP—9 protein expression in lung tissue during duced chronic lung in?ammation. A: representative Masson’s trichrome staining. Magni?cation X40, scale 200 um. B: hydroxyproline levels (mg/g lung tissue); >"**P < 0.001 (n = 10) y ANOVA with Tukey’s post hoc test. C: lung TGFB tration as determined by ELISA; >"P 0.05 (n 10 per group) one—way ANOVA with Tukey’s post hoc test. D: Western blot is of MMP—9 (pro 105 kDa; active 92 kDa) and TIMP—l (29 kDa) expression (noncontiguous bands from the same Western blot are shown) in lung tissue from lO-week OVA-challenged mice with and without Boysenberry treatment. E: ratio of TIMP—l/MMP—9 protein expression normalized to B—actin loading control; >"*P < 0.01 (n = 10) one—way ANOVA with Tukey’s post hoc test compared with naive and OVA plus berry treatment.

Fig. 5A-B. Boysenberry treatment increases MMP—9 expression by alternatively activated macrophages in lung tissue during OVA-induced chronic lung in?ammation. A: DAB labelling of MMP—9+ macrophages (arrows). B: immuno?uorescent labelling of MMP—9+macrophages (*). DAPI nuclear stain (dark blue).

Magni?cation X40, scale 200 pm.

Fig. 6A—B. Depletion of lung macrophages d the effect of oral Boysenberry treatment on OVA—induced chronic lung ation. A: 6—week—old male C57Bl/6 mice (n = 10 per group) were primed i.p. with OVA/alum (day 0) then challenged in. with OVA every 7 days for 5 weeks. From weeks 6 to 7 hages were depleted using clodronate liposomes (CloLip) the day before Boysenberry juice was administered orally (gavage). B: ?ow cytometric quanti?cation of percentage of macrophages in BALF following ?nal clodronate macrophage depletion; >"P < 0.05 (n = 10 per group) one—way ANOVA with Tukey’ s post hoc test. C: hydroxyproline levels (mg/g lung ) in the lung; *P < 0.05 (n = 10 per group) one-way ANOVA with Tukey’s post hoc test.

Fig. 7A-G. lactic oral Boysenberry treatment s OVA-induced chronic lung in?ammation and collagen deposition. A: —old male C57Bl/6 mice (n = per group) were primed i.p. with OVA/alum then challenged in. with OVA every 7 days for 5 weeks. Boysenberry juice was administered orally (gavage) l h prior and 2 days after each in. OVA challenge. B: lung tissue was stained with total cells per ml BALF and ?ow cytometiic quanti?cation of percentage of eosinophils in BALF following ?nal OVA challenge; >"P < 0.05, **P < 0.01 (n = 10 per group) one—way ANOVA with Tukey’s post hoc test. C: AB—PAS, dark purple positive bronchioles (arrows); magni?cation X20, scale 200 um. D: Masson’s trichrome; magni?cation X40; scale 200 um. E: hydroxyproline levels (mg/g lung tissue) in the lung. *P < 0.05, **P < 0.01 (n = 10 per group) one—way ANOVA with Tukey’s post hoc test. F: Western blot analysis of iNOS; arginase; MMP—9, and TIMP-l lung tissue. Noncontiguous bands from the same Western blot are shown. G: ratio of TIMP—l/MMP—9 protein levels normalized to B—actin loading control. *P < 0.05, (n = 10 per group) one—way ANOVA with Tukey’s post hoc test. Figure 4: tic of trial treatments, washouts, and sampling points.

Fig. 8. tics for experiments testing Boysenberry treatment combinations in a model of acute allergic airways in?ammation.

Figure 9. Treatment utilising BerriQiTM Boysenberry with apple administration reduced immune cell numbers in model of acute allergic s in?ammation. Total cell infiltration into the lung following min (OVA)-induced allergic airways infiltration. Total bronchioalveolar lavage ?uid (BALF) cell numbers were determined 4 days post—OVA challenge (n210 per intervention group).

Fig. 10. Treatment utilising BerriQiTM Boysenberry with apple administration reduced eosinophil numbers in model of acute allergic airways in?ammation.

Eosinophil infiltration into the lung following an min (OVA)—induced allergic airways infiltration. Total bronchioalveolar lavage ?uid (BALF) eosinophil cell numbers were determined 4 days post—OVA challenge (n=10 per intervention group). iTM concentrates and the other concentrates are described in Example 2, below.

Fig. 11. Treatment utilising iTM Boysenberry with apple stration in model of acute allergic airways in?ammation. oxylin and eosin staining of lung tissues following apple treatment.

Fig. 12. Treatment utilising BerriQiTM Boysenberry with apple administration in model of acute ic airways in?ammation. Haematoxylin and eosin staining of lung tissues following Boysenberry treatment.

Fig. 13. Treatment utilising BerriQiTM Boysenberry with apple administration in model of acute allergic s in?ammation. Haematoxylin and eosin staining of lung tissues following BerriQiTM berry with apple treatment.

Representative 10X (A—D) and 20X (E—H) images of (A, E) naive, (B, F) OVA, (C, G) BerriQiTM Boysenberry with apple 10, and (D, H) BerriQiTM Boysenberry with apple 1.

Immune cells appear as dark pinldpurple clusters.

Fig. 14. Treatment ing BerriQiTM Boysenberry with apple stration d neutrophil numbers in model of acute allergic airways in?ammation.

Neutrophil infiltration into the lung following ovalbumin (OVA)-induced allergic airways infiltration. Total bronchioalveolar lavage ?uid (BALF) neutrophil cell numbers were determined 4 days post—OVA challenge (n=lO per intervention group).

Fig. 15. Treatment utilising BerriQiTM Boysenberry with apple administration reduced monocyte numbers in model of acute allergic airways in?ammation.

Monocyte infiltration into the lung ing an ovalbumin (OVA)-induced allergic airways infiltration. Total bronchioalveolar lavage ?uid (BALF) monocyte cell numbers were determined 4 days post—OVA challenge (n=lO per intervention group).

Fig. 16. Treatment utilising BerriQiTM Boysenberry with apple administration reduced antigen presenting cells in model of acute allergic airways in?ammation. Antigen Presenting Cell (APC) ration into the lung following an min (OVA)—induced allergic airways infiltration. Total bronchioalveolar lavage ?uid (BALF) APC numbers were determined 4 days post—OVA challenge (n=lO per intervention group).

Fig. 17. Treatment utilising BerriQiTM berry with apple administration in model of acute allergic airways in?ammation. Alcian blue/periodic acid Schiff diastase staining of lung tissues following apple treatment.

Fig. 18. Treatment utilising BerriQiTM Boysenberry with apple administration in model of acute allergic airways in?ammation. Alcian eriodic acid Schiff se staining of lung tissues following Boysenberry treatment.

Fig. 19. ent utilising BerriQiTM Boysenberry with apple administration in model of acute allergic airways ation. Alcian blue/periodic acid Schiff diastase staining of lung tissues following BerriQiTM Boysenberry with apple treatment. entative 10X (A—D) and 20X (E—H) images of images of (A, E) naive, (B, F) OVA, (C, G) BerriQiTM Boysenberry with apple 10, and (D, H) BerriQiTM Boysenberry with apple 1. Mucous positive goblet cells are dark .

Fig. 20. Treatment utilising BerriQiTM Boysenberry with apple administration in model of acute allergic airways ation. Masson’ s trichrome ng of lung tissues following apple treatment.

Fig. 21. Treatment utilising BerriQiTM Boysenberry with apple administration in model of acute allergic airways in?ammation. ’ s trichrome ng of lung tissues following Boysenberry treatment.

Fig. 22. Treatment utilising BerriQiTM Boysenberry with apple administration in model of acute allergic airways in?ammation. Masson’ s trichrome ng of lung tissues following BerriQiTM Boysenberry with apple treatment. Representative 10X (A—D) and 20X (E—H) images of images of (A, E) na‘1've, (B, F) OVA, (C, G) BerriQiTM Boysenberry with apple 10, and (D, H) BerriQiTM berry with apple 1.

Fig. 23. Treatment utilising BerriQiTM Boysenberry with apple administration in model of acute allergic airways in?ammation. Granulocyte—macrophage colony—stimulating factor levels following Boysenberry, apple, and BerriQiTM Boysenberry with apple treatments.

Fig. 24. ent utilising BerriQiTM Boysenberry with apple administration in model of acute allergic airways in?ammation. CCLll levels following Boysenberry, apple, and BerriQiTM Boysenberry with apple treatments.

Fig. 25. Treatment utilising Bern'QiTM Boysenberry with apple (BA) administration reduced immune cell numbers in model of c allergic airways in?ammation. Total cell infiltration into the lung following chronic ovalbumin (OVA)- induced allergic airways infiltration. Total bronchioalveolar lavage ?uid (BALF) cell numbers were determined 4 days following final OVA challenge. Data are mean i SEM (n=20 per intervention group). P<0.05, P<0.001 compared to OVA; P<0.01 compared to naive.

Fig. 26. Treatment utilising BerriQiTM Boysenberry with apple (BA) administration reduced eosinophil numbers in model of chronic allergic airways in?ammation. Eosinophil infiltration into the lung ing chronic ovalbumin (OVA)— induced allergic airways infiltration. Number of eo sinophils in bronchioalveolar lavage ?uid (BALF) cell was determined 4 days following final OVA challenge. Data are mean i SEM (n=20 per intervention group). P<0.001 ed to naive.

Fig. 27. Treatment utilising iTM Boysenberry with apple (BA) administration reduced antigen presenting cell s in model of chronic allergic airways in?ammation. n presenting cell (APC) infiltration into the lung following chronic ovalbumin (OVA)—induced ic airways infiltration. Number of APCs in bronchioalveolar lavage ?uid (BALF) cell was determined 4 days following final OVA challenge. Data are mean i SEM (n=20 per intervention group). P<0.01 ed to naive.

Fig. 28. Treatment utilising BerriQiTM Boysenberry with apple (BA) stration reduced monocyte numbers in model of chronic allergic airways in?ammation. Monocyte infiltration into the lung following chronic ovalbumin (OVA)— induced allergic airways infiltration. Number of monocytes in bronchioalveolar lavage ?uid (BALF) cell was determined 4 days following final OVA challenge. Data are mean i SEM (n=20 per intervention group).

Fig. 29. ent utilising BerriQiTM Boysenberry with apple (BA) stration in model of c allergic airways in?ammation. Photomicrographs of lung tissue sections stained with haematoxylin and eosin staining following chronic ovalbumin (OVA)—induced allergic airways ration. Mice were primed with OVA/Alum intraperitoneally and then challenged 7 days later with OVA intranasally for 10 weeks. After weeks mice were orally d with nothing (A — na‘1‘ve) water (B — OVA control) 100% (C) 50% (D) or 25% (E) BeriiQiTM Boysenberry with apple 2 days piior, 1 hour before an OVA challenge and again 2 days post—challenge for 5 weeks.

Fig. 30. Treatment utilising BerriQiTM Boysenberry with apple (BA) administration in model of chronic allergic airways in?ammation. Photomicrographs of lung tissue sections stained with Alcian blue and periodic chiff staining of lung tissue following chronic ovalbumin (OVA)-induced allergic airways infiltration. Mice were primed with OVA/Alum intraperitoneally and then challenged 7 days later with OVA intranasally for 10 weeks. After 5 weeks mice were orally d with nothing (A — naive) water (B — OVA l) 100% (C) 50% (D) or 25% (E) Bern'QiTM Boysenberry with apple 2 days prior, 1 hour before an OVA challenge and again 2 days post—challenge for 5 weeks.

Fig. 31. Treatment utilising BerriQiTM berry with apple (BA) administration in model of chronic allergic airways ation. Photomicrographs of lung tissue sections stained with Masson’s Trichrome staining of lung tissue following chronic ovalbumin (OVA)—induced allergic airways infiltration. Mice were primed with OVA/Alum intraperitoneally and then challenged 7 days later with OVA intranasally for 10 weeks. After weeks mice were orally gavaged with nothing (A — naive) water (B — OVA control) 100% (C) 50% (D) or 25% (E) BerriQiTM Boysenberry with apple 2 days prior, 1 hour before an OVA challenge and again 2 days post—challenge for 5 weeks.

Fig. 32. ent utilising BerriQiTM Boysenberry with apple (BA) administration in model of chronic allergic airways in?ammation. Quantification of collagen in the lung following chronic ovalbumin (OVA)—induced allergic airways infiltration. Mice were primed with OVA/Alum eritoneally and then challenged 7 days later with OVA intranasally for 10 weeks. After 5 weeks mice were orally gavaged with g (naive), water (OVA control), 100%, 50%, or 25% BerriQiTM berry with apple (BA) 2 days prior, 1 hour before an OVA challenge and again 2 days post—challenge for 5 weeks.

Collagen was determined using the hydroxyproline assay 4 days following the final OVA challenge. Data are mean i SEM (n=20 per ention group). P<0.001 compared to all other treatment groups.

Fig. 33. Treatment utilising BerriQiTM Boysenberry with blackcurrant and apple (BBA) administration reduced immune cell numbers in model of chronic allergic airways inflammation. Total cell ration into the lung following chronic ovalbumin (OVA)—induced allergic airways infiltration. Total ioalveolar lavage fluid (BALF) cell numbers were determined 4 days following final OVA challenge (n=20 per intervention group).

Fig. 34. Treatment utilising BerriQiTM Boysenberry with blackcurrant and apple (BBA) administration reduced phil numbers in model of chronic allergic airways in?ammation. Eosinophil infiltration into the lung following chronic ovalbumin (OVA)—induced allergic airways infiltration. Number of eosinophils in bronchioalveolar lavage ?uid (BALF) cell was determined 4 days following final OVA challenge (n=20 per intervention group). P<0.001 compared to naive.

Fig. 35. Treatment utilising BerriQiTM Boysenberry with blackcurrant and apple (BBA) administration reduced antigen presenting cell numbers in model of c allergic airways in?ammation. Antigen presenting cell (APC) infiltration into the lung following chronic ovalbumin (OVA)—induced allergic airways ration. Number of APCs in bronchioalveolar lavage ?uid (BALF) cell was determined 4 days following final OVA challenge (n=20 per intervention group). P<0.001, P<0.01 compared to OVA.

Fig. 36. Treatment utilising BerriQiTM Boysenberry with blackcurrant and apple (BBA) administration reduced monocyte numbers in model of chronic allergic airways inflammation. Monocyte infiltration into the lung ing chronic min (OVA)— induced allergic airways infiltration. Number of monocytes in ioalveolar lavage ?uid (BALF) cell was ined 4 days following final OVA challenge (n=20 per intervention group).

Fig. 37. Treatment utilising BerriQiTM Boysenberry with urrant and apple (BBA) stration in model of chronic allergic airways in?ammation.

Haematoxylin and eosin staining of lung tissue ing min (OVA)—induced allergic airways infiltration. Mice were primed with OVA/Alum intraperitoneally and then challenged 7 days later with OVA intranasally for 10 weeks. After 5 weeks mice were orally gavaged with nothing (naive) water (OVA control) or 100% BerriQiTM Boysenberry with apple and blackcurrant (OVA + 100%) 2 days prior, 1 hour before an OVA challenge and again 2 days post-challenge for 5 weeks.

Fig. 38. Treatment utilising iTM Boysenberry with blackcurrant and apple (BBA) administration in model of chronic allergic airways in?ammation. Alcian blue and periodic acid—Schiff staining of lung tissue following ovalbumin (OVA)—induced allergic airways infiltration. Mice were primed with OVA/Alum intraperitoneally and then nged 7 days later with OVA intranasally for 10 weeks. After 5 weeks mice were orally gavaged with nothing (naive) water (OVA control) or 100% BerriQiTM Boysenberry with apple and urrant (OVA+100%) 2 days prior, 1 hour before an OVA challenge and again 2 days post—challenge for 5 weeks.

Fig. 39. Treatment utilising BerriQiTM Boysenberry with blackcurrant and apple (BBA) administration in model of chronic allergic airways in?ammation. Masson’s ome staining of lung tissue following ovalbumin (OVA)—induced allergic airways infiltration. Mice were primed with OVA/Alum intraperitoneally and then challenged 7 days later with OVA intranasally for 10 weeks. After 5 weeks mice were orally gavaged with nothing (naive) water (OVA control) or 100% BerriQiTM Boysenberry with apple and blackcurrant 00%) 2 days prior, 1 hour before an OVA challenge and again 2 days post—challenge for 5 weeks.

Fig. 40. ent utilising BerriQiTM Boysenberry with blackcurrant and apple (BBA) administration in model of chronic allergic airways inflammation.

Quantification of collagen in the lung following ovalbumin (OVA)—induced allergic airways infiltration. Mice were primed with OVA/Alum intraperitoneally and then challenged 7 days later with OVA intranasally for 10 weeks. After 5 weeks mice were orally gavaged with 100% BerriQiTM Boysenberry with apple and blackcurrant (100% BBA) 2 days prior, 1 h before an OVA challenge and again 2 days post—challenge for 5 weeks. en was determined using the hydroxyproline assay was determined 4 days post—OVA challenge (n=20 per intervention group).

DETAILED DESCRIPTION OF THE ION The following ption sets forth numerous exemplary configurations, parameters, and the like. It should be ised, r, that such description is not intended as a limitation on the scope of the present invention, but is instead provided as a description of exemplary embodiments.

All references, including patents and patent applications, cited in this specification are hereby incorporated by reference. No admission is made that any reference constitutes prior art. Nor does discussion of any reference constitute an admission that such reference forms part of the common general knowledge in the art, in New Zealand or in any other country. tions In each instance herein, in ptions, embodiments, and examples of the present invention, the terms "comprising77 (L 7 including", etc., are to be read ively, without limitation. Thus, unless the context clearly requires otherwise, throughout the description and the claims, the words "comprise77 (L 7 comprising", and the like are to be construed in an inclusive sense as to opposed to an exclusive sense, that is to say in the sense of "including but not limited to".

The term "consisting essentially of’, as used herein, may refer to the presence of a concentrate in a composition. For example, the concentrate may be at least 80% by weight of the composition, or at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.8%, or at least 99.9% by weight of the composition (% w/w). For liquids, the concentrate may be at least 80% by volume of the composition volume, or at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.8%, or at least 99.9% by volume of the composition volume (%v/v).

In the present ption, the articles "a" and "an" are used to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" can be taken to mean one element or more than one element.

Throughout this description, the term "about" is used to te that a value includes the standard deviation of error for the method being employed to ine the value, for example, levels of compounds or dosage levels, as described in detail . In particular, the term "about" encompasses a 10% to 15% deviation (positive and negative) in the stated value or range, particularly 10% deviation ive and negative) in the stated value or range.

"Airway lling", also referred to as tissue or lung lling, refers to the development of specific structural changes in the airway wall. This may include, for example, remodelling of the fibrous connective tissue in the lining of the airways, for example, in the lungs. Airway remodelling may include one or more of subepithelial fibrosis, myofibroblast accumulation, airway smooth muscle hyperplasia, and hypertrophy, mucous gland and goblet cell hyperplasia, and epithelial tion. ms may include decreased airway sibility (i.e., stiffer airways), diminished elastic recoil, progressive decline in FEVl (forced expiratory volume 1), and FVC (forced vital capacity), accelerated lung function decline, irreversible or only partially reversible air?ow obstruction, dyspnoea, and decreased responsiveness to respiratory y (e.g., asthma or COPD therapeutics).

"Asthma" refers to an in?ammatory disorder of the airways of the lungs, characterized by variable and recurring breathing ment, including air?ow obstruction and bronchospasm. Air?ow obstruction may be defined as reduced FEVl and/or reduced FEVl/VC ratio. The air?ow obstruction in asthma may be reversible with or without medication. Symptoms of asthma may include one or more of wheezing, ng, chest tightness or pain, and shortness of breath. Included herein are atopic (e. g., allergen or antigen induced) and non—atopic forms of asthma, as well as exercise—induced , occupational asthma, aspirin—induced asthma, and alcohol—induced asthma.

] A "respiratory aid" is a composition that assists with airway function or other aspects of the respiratory system, e.g., medicines, herbal compositions, essential oils, and various compositions for inhalation.

"Airway", "respiratory tract", and "respiratory system" refer to any of the organs, tissues, or ar components involved in gas exchange (i.e., breathing). This includes the upper respiratory tract, trachea, bronchi, bronchioles, alveoli, lungs, pleura and pleural cavity, and the nerves and muscles of breathing. It will be understood that "airways" describes the various structural components (e.g., cellular components, tissues, and organs) as well as the space where gas exchange . " as used herein encompasses any fruit of the genus Malus, and any hybrid, variety, and c tive thereof. Included, ically, are Malus pumila, as well as the particular cultivars of ‘Gala’ ‘Golden Supreme’ ‘Mclntosh’ ‘Transparent’ ‘Primate’ ‘Sweet Bough’ ‘Duchess’ ‘Fuji’ ‘Jonagold’ ‘Golden Delicious’ ‘Red Delicious’ ‘Chenango 7 ‘Gravenstein’ osh’ ‘Snow’ ‘Blenheim’ ‘Winesap 7 ‘Granny Smith’ ‘King’ er’ ‘Swayzie’ ‘Greening’ and ‘Tolman Sweet’. Included also are the cultivars ‘Alice’ sia’ ‘Ananasrenette’ ‘Aroma’ ‘Discovery’ ‘Envy’ ‘Braeburn’ ‘Bramley’ ‘Arkansas Black’ ‘Dougherty’/‘Red Dougherty’ ‘Goldrenette’/‘Reinette’ ‘Jazz’ ‘Jonagold’ ‘James Grieve’ ‘Yellow arent’ ‘Paci?c rose’ ‘Lobo’ ‘Sampion’/‘Shampion’ ‘Sonya’ ‘Splendour’/‘ Splendor’ ‘Summerred’ ‘Pink Lady’ ‘Belle de Boskoop’ ‘Cox Pomona’ ‘Cox’s Orange Pippin’ ‘Kidd's Orange Red’ and ‘SugarBee’.

Further ed are the cultivars of ‘Haralson’ ‘Wealthy’ ‘Honeygold’ and ‘Honeycrisp’.

Included as well are crabapples, apple-crabapple hybrids, and cooking apples.

"Blackcurrant" as used herein encompasses any black/dark ed Ribes fruit from the family Grossulariaceae, which includes but is not limited to that of Ribes arzigrmn, Ribes nigrzmz L, Ribes ame?carmm, Ribes hudsoniamam, Ribes laxz?onmz, and Bikes X nidigmlaria. Any hybrid, variety, and genetic derivative of these are also included. Included amongst suitable blackcui‘rant cultivars are those of ‘ Andega’, ‘Ben Ard", ‘Ben Alder’, ‘Ben Dorain’, ‘Ben Gairn’, ‘Ben Hope’, ‘Ben ’, ‘Ben More’, ‘Ben Rua’, ‘Ben Sarek’, ‘Ben ’, ‘Blackadder’, ‘Black Down’, ‘Burga’, ‘Orcs’, ‘Magnus’, ‘Murchison’, ‘Sefton’, ‘Wellington’, ‘lnvigo’, "E‘itania’, ‘(iionsort? ‘Cmsader’, "Gearit de Boskoop’, ‘Noir de Bourgogne’, ‘Royal de Naples’ op Giant’, ‘Tenah’, ‘Tiben’, ‘Tines’, ‘Tisel’, and. ‘V?lloughby’.

"Boysenberry" as used herein encompasses a Rubus hybrid berry, which includes but is not limited to a berry obtained from the plant identified as Rubus ursinus var loganobaccus cv Boysenberry, Rubus ursinus x Rubus idaeus, Rubus loganbaccus x baileyanus Britt, and Rubus idaeus x Rubus ulmifolius. Generally speaking, a Boysenberry may be derived from a cross between raspberry and blackberry plants, or between raspberry, blackberry, and loganberry . Included are various Boysenberry hybrids, varieties, and genetic derivatives thereof. Boysenberries are referred to herein as berries or, more y, as fruits. ic ctive pulmonary disease", or COPD, refers to a lung disorder associated with progressive obstruction of the airways and poor air?ow. w obstruction may be defined as a reduction in FEVl and/or a reduction in FEVl/VC ratio. The air?ow obstruction in chronic obstructive ary disease may not be fully reversible. Symptoms include but are not limited to shortness of breath, cough, and sputum production (i.e., phlegm). COPD may be associated with smoking, air pollution, poorly ventilated cooking or heating fires. A genetic component may also be ed in COPD. The disorder is also known as chronic obstructive lung disease , chronic obstructive airway disease (COAD), chronic itis, pulmonary ema, amongst other known terminology.

"Concentrate", for example, in relation to a Boysenberry, apple, or blackcurrant concentrate, or any combination f, refers to a composition where the liquid component (e.g., juice) has been partly or substantially removed. Removal of a liquid component may be by evaporation or any other means. A concentrate may be prepared, for example, as a puree, paste, or powder, or may be prepared from a Boysenberry, apple, or blackcurrant juice, or any ation thereof, e.g., prepared as a juice concentrate.

A der" of respiratory tract includes a disease or other condition affecting any of the , tissues, or cellular components involved in gas exchange (i.e., breathing), as noted herein. The disorders may be an acute or chronic condition, such as inflammation and conditions that are ated with inflammation. Particular disorders of interest include asthma, chronic obstructive pulmonary disease, reactive airway disease, airway fibrosis, and airway remodelling. Other ers are described in detail herein.

A ic derivative" of a plant refers to offspring, sports, or other ars that are obtained from the parent stock. This includes offspring ed from a genetic cross with the parent, e.g., Fl progeny or F2 progeny. The term "genetic derivative" may refer to the derived plant, itself, or to its fruit.

"Fibrosis", as in airway or pulmonary ?brosis, refers to a disruption in the tion of collagen and other extracellular matrix components in the respiratory tract. In the airways of patients with fibrosis, there may be increased extracellular matrix deposition, such as in the lar basement membrane , lamina propria, and/or submucosa. Scar formation and the accumulation of excess s connective tissue leads to thickening of the airway walls. Symptoms may include reduced oxygen supply, shortness of breath, chronic cough, fatigue and/or weakness, chest fort including chest pain, loss of appetite, and weight loss. Included are idiopathic forms of airway is, as well as airway fibrosis associated with smoking, air ion, connective tissue disease (e.g., rheumatoid arthritis, sarcoidosis, etc), infections, medications (e.g., methotrexate, bleomycin, etc), and radiation therapy.

"In?ammation" refers to a condition characterised by one or more of: vasodilation, heat, redness, discomfort, swelling, edema, lesions, fissures, ulcerations, leukocyte extravasation, and loss of function. Included are both acute and chronic forms of in?ammation, such as acute airways in?ammation, and other atory disorders, e.g., autoimmune diseases or allergic conditions. Particularly included are asthma, chronic obstructive pulmonary disease, airway fibrosis, reactive airway disease, and airway remodeling. Other atory disorders are described elsewhere in this document.

As noted herein, the terms "lyophilising" and "freeze drying" are used mously. It will be understood that the terms "freeze drying"/"lyophilising" do not exclude the use of higher atures (i.e., higher than freezing temperatures). For example, higher temperatures may be used for removing residual moisture during the secondary drying phase for lyophilisation/freeze drying procedures.

A "nutraceutical" refers to a standardised composition for administration to a subject. It may be a pharmaceutical grade composition, and may maintain or improve the health of a subject, or may treat or prevent one or more disorder in a subject. ive airway disease" refers to an in?ammatory airway er characterised by reversible airway narrowing due to al stimuli. The term can encompass other known disorders such as asthma, chronic obstructive pulmonary disease, upper respiratory tract infections, etc, or can refer to conditions that are similar to these disorders but not directly diagnosed as such, e.g., having asthma—like syndrome or asthma— like symptoms. Subjects with reactive airway disease may show one or more symptoms of coughing, wheezing, or shortness of breath upon exposure to particular stimuli, for example, smoke, vapour, fume, or other irritants.

As used herein, a "subject" may be a human or non—human animal, particularly a mammal, ing cattle, sheep, goats, pigs, horses, and other livestock, including, as well, dogs, cats, and other domesticated pets. In particular aspects, the subject is a human being.

"Treating" as used herein is meant as reducing, ameliorating, or resolving a disorder, for example a respiratory er, such as a disease or other condition of the atory . A treatment will result in the reduction, amelioration, or elimination of one or more symptoms of the disorder.

] "Preventing" as used herein is meant as ng or delaying the onset of a disorder, for example a respiratory disorder, such as a disease or other condition of the respiratory system. A preventative measure will result in the stoppage or delay of one or more symptoms of the disorder, or a lessening of symptoms if such do arise. It should be understood that the term "treating or preventing" does not exclude the ility of obtaining both treatment and prevention (e. g., at the same time or at different times) of a disorder in any given subject. In the same way treatment of "asthma or fibrosis" does not exclude the ility of obtaining treatment (e.g., simultaneous or not simultaneous) of both disorders.

Compositions comprising Boysenberry and the associated bioactivity of these compositions The inventors have found that ption of a Boysenberry ition reduces allergen-induced lung lling in a chronic model of asthma. For these experiments, the effect of Boysenberry consumption was tested on lung ?brosis, lung hage phenotype, and MMP—9 sion in a chronic model of allergic airway in?ammation. The results demonstrated that oral Boysenberry treatment supports the development of lung macrophages that express a mixed anti?brotic, AAM (alternatively activated macrophages) phenotype with the capacity to ameliorate ?brosis and promote balanced lung repair (74; incorporated herein by reference in its entirety). Further to this, the inventors have found that combined stration of Boysenberry and apple compositions, and combined administration of Boysenberry, apple and blackcurrant compositions, can be used to reduce numbers of immune cells associated with airways inflammation. This can e one or more of eosinophils, neutrophils, monocytes, and antigen ting cells. Surprisingly, and advantageously, the combined Boysenberry and apple compositions are effective at low dosages.

] Boysenberries are known to be high in Vitamin C and fibre and contain high levels of anthocyanins (120—160 mg/100 g) that give Boysenberries their deep, dark colour.

The ORAC (oxygen radical absorption capacity, i.e., antioxidant level) for Boysenberries is 42 umoles/TE/g almost double that of blueberries, a well known antioxidant food.

Boysenberries contain notable amounts of ellagic acid, a phenolic compound. The c acid level in Boysenberries is 5.98 mg/g of dry weight. Boysenberries also have a high ratio of free ellagic acid to total ellagitannins. The inventors have tested concentrates to confirm biological activity.

As described in Example 2, these solutions included: Boysenberry 10 (Boysenberry juice solution), 6.7%, Boysenberry 1 (Boysenberry juice solution), 0.67%, apple 10 (apple juice solution), 18.7%, apple 1 (apple juice solution), Boysenberry and apple (BerriQiTM Boysenberry with apple; combined juice solution), 6.7%/18.7%, Boysenberry and apple 1 (BerriQiTM Boysenberry with apple; combined juice solution), 1.87%.

Additional oral compositions are described in Examples 3—5, including BerriQiTM Boysenberry with apple ition at 100%, 50% or 25% concentration, and iTM Boysenberry with apple and blackcurrant composition at 100% tration. The 100% BerriQiTM Boysenberry with apple composition includes Boysenberry juice concentrate at 27% and apple juice concentrate at 73%. The 100% BerriQiTM Boysenberry with apple and blackcurrant composition includes Boysenberry juice concentrate at 13.5%, apple juice concentrate at 7%, and urrant juice concentrate at 13.5%. Further details are provided in the Examples section, below.

From the inventors’ results it is evident that berries and apples and Boysenberry, apple and blackcurrant may be used in compositions for treating or ting in?ammation of the respiratory tract, treating or ting asthma, treating or preventing chronic obstructive pulmonary disease, treating or ting allergic airways in?ammation, treating or ting fibrosis of the respiratory tract, or treating or preventing airway remodelling. In particular, as to airway remodelling, the compositions of the invention may be used to treat or prevent one or more of: the thickening of the walls of the alveoli, increasing of collagen fibres in the airway, spreading of en fibres into the airway tissues, and collapsing or closing of the airspace(s). In addition, the compositions of the invention may be used to reduce tissue in?ammation and collagen deposition that leads to airway remodelling.

In addition, from the results shown herein, it will be understood that berry and apple itions and Boysenberry, apple and blackcurrant compositions may be used to restore, e, or maintain the health of the respiratory , for example, in one or more activities of: decreasing collagen deposition, ting aberrant collagen deposition, decreasing cellular infiltration into the s, decreasing airway damage due to cellular infiltration, reducing cells in the lung ?uid, e.g., in?ammatory cells, reducing mucus production, reducing mucus—positive cells, decreasing hydroxyproline levels, sing matrix metallopeptidase expression levels, e.g., protein levels, increasing MMP—9 sion levels, e.g., protein levels, increasing TGFB expression levels, e.g., protein levels, decreasing the ratio level of TIMP—l/MMP—9, e.g., protein ratio levels, decreasing the activation or number of in?ammatory cells, increasing the number or activity of alternatively activated macrophages, increasing the number or activity of arginase+ macrophages, increasing the number or activity of CD68+/CD206+/arginase+ macrophages, or decreasing iNOS expression , e.g., protein levels. Further uses for these compositions are described in detail herein.

Methods of producing compositions comprising Boysenberry and apple and compositions comprising Boysenberry, apple and urrant The present invention relates lly to a composition prepared from Boysenberry and apple, as well as a composition prepared from Boysenberry, apple and blackcurrant. While specific combinations of Boysenberry and apple, and Boysenberry, apple and blackcurrant, are described herein, it is tood that the composition of the invention can include any combination of Boysenberry, apple, and blackcurrant therein.

In one particular aspect, the composition is prepared from Rubus ursirms var loganobaccus cv Boysenberry. In other aspects, one or more genetic derivatives from this Boysenberry plant may be used. For e, it may be desirable to use F1 or F2 progeny from a genetic cross that includes the parent stock of the Boysenberry plant. atively, any sports or other cultivars obtained from the parent may be used. It may be ble to source the Boysenberries from New Zealand, in particular, or alternatively, from Chile.

The composition is preferably prepared as a Boysenberry concentrate, for example, a Boysenberry puree, a Boysenberry pomace, Boysenberry paste, Boysenberry powder, Boysenberry juice concentrate. The Boysenberry and apple composition may comprise an apple concentrate, for example, an apple puree, an apple pomace, an apple paste, an apple , or apple juice concentrate. If an apple juice concentrate is used, this may have ated from cloudy apple juice or clear apple juice. The Boysenberry, apple and blackcurrant composition may comprise a blackcurrant concentrate, for example, a blackcurrant puree, a blackcurrant pomace, a blackcurrant paste, a blackcurrant powder, a blackcurrant juice trate. It will be understood that berry, apple, or blackcurrant pomaces, in ular, may be used, which include the solid remains of the fruit after pressing for juice. The pomace may encompass one or more of the skins, pulp, seeds, and stems of the fruit. In addition, it is possible to include one or more of a Boysenberry juice, an apple juice, and a blackcurrant juice in the compositions disclosed herein.

Accordingly, the composition may be prepared in liquid or powdered form, for example, a lyophilised powder, or in any other suitable dosage form. The composition may be formulated as a tonic, t, elixir, s, concentrate, syrup, solution, suspension, emulsion, draught, puree, paste, or as drops. In other aspects, the composition may be formulated as a gel or jelly, or a capsule, for example, with liquid or semi—liquid contents.

The ition may be provided in sachet form, for example, a powder sachet, or a gel or jelly sachet. Included also are formulations comprising thin strips, or comprising solids in a capsule to mix with food or drink. Other formulas are also possible, as described herein below.

In certain aspects, it may be ble to formulate the Boysenberry and apple composition (e.g., Boysenberry and apple juice concentrate or puree) or the Boysenberry, apple and blackcurrant composition (e.g., Boysenberry, apple and blackcurrant juice concentrate or puree) into a powder. As specific exemplifications, apple powder may comprise apple pomace powder or apple pectin powder. cial Boysenberry, apple, and blackcurrant powders are known and available, as noted herein.

The powder may be formulated as tablets (including rapid dissolve tablets) or capsules (including extended e capsules). The tablets may be scored tablets, le tablets, escent tablets, orally disintegrating tablets, or tablets for forming a suspension. The capsules may be gel capsules, for example, and may include powdered contents. This includes gel capsules made by single piece gel encapsulation and two piece gel encapsulation. Non—gelatine capsules are also included, as well as caplets. The powder may be provided in free ?owing form or as a solid cake. The composition may be provided as a powder for forming a suspension, powder for forming a solution, bulk oral granules, or bulk oral powder.

] The compositions of the invention may be prepared from Boysenberry, apple, or blackcurrant juice trate or puree obtained from one or more commercial sources. For example, commercial sources of New Zealand Boysenberry products include Boysenberries New Zealand Ltd, Nelson, and Tasman Bay Berries, Nelson. Commercially available products e individually quick frozen berries, Boysenberry puree, block frozen berries, Boysenberry juice concentrate, and Boysenberry powder. Commercial sources of New d apple and blackcurrant products include those from EnzaFoods New Zealand Ltd, Hastings, New Zealand, as well as Juice Products New Zealand, Timaru, New Zealand, NZ Blackcurrant rative Ltd, Nelson, New Zealand, Fruit Solutions, FSL Foods, Nelson, New Zealand, and Reso, Auckland, New Zealand, and encompass cloudy apple juice concentrate, clear apple juice concentrate, apple pomace, apple puree, blackcurrant juice trate, and blackcurrant puree. In addition, apple and blackcurrant powders may be obtained commercially from TreeTop®, FutureCeuticals, Nutradry, Sujon, ViberiTM, ZeaberryTM, Waitaki Biosciences, amongst other suppliers. Specific suppliers of apple and urrant juice concentrate include Infruit Ltd, nd, New Zealand, and RD2 International Ltd, Auckland, New Zealand, while manufacturers include Profruit (2006) Ltd, Hastings, 4175, New Zealand, as noted herein.

] The pH of the juice trate or puree, for example, the combined Boysenberry and apple juice concentrate, and combined Boysenberry, apple and blackcurrant juice concentrate, may range from 3.2 to 3.8; or 3.0 to 4.0; or 3.1 to 3.9; or may be about 3.1, about 3.2, about 3.3, about 3.4, about 3.5, about 3.6, about 3.7, about 3.8, about 3.9, or about 4.0. For the apple juice concentrate that is used to make the combined concentrate, the pH may range from 2.8 to 4.4; 2.9 to 4.3; 3.0 to 4.2; or 3.1 to 4.0; or may be about 2.8, about 2.9, about 3.0, about 3.1, about 3.2, about 3.3, about 3.4, about 3.5, about 3.6, about 3.7, about 3.8, about 3.9, about 4.0, about 4.1, about 4.2, about 4.3, about 4.4, or about 4.5. For the blackcurrant juice concentrate that is used to make the combined concentrate, the pH may range from 1.0 to 5.0; 2.0 to 4.0; 1.5 to 3.5; 2.1 to 3.4; or 2.3 to 3.3; or may be about 1.8, about 1.9, about 2.0, about 2.1, about 2.2, about 2.3, about 2.4, about 2.5, about 2.6, about 2.7, about 2.8, about 2.9, about 3.0, about 3.1, about 3.2, about 3.3, about 3.4, about 3.5, about 3.6, about 3.7, about 3.8, about 3.9, or about 4.0.

For the Boysenberry juice concentrate, the acidity (% w/w citric acid anhydrous) may be about 1 to about 20, about 1.5 to about 15, about 2 to about 12, about 5 to about 10, about 6 to about 9, about 10, about 9, about 8.5, about 8.3, about 8.2, about 8.17, about 8.1, about 8, about 7, about 6, or about 5. For the apple juice concentrate, the acidity (% w/w malic) may be about 0.5 to about 4.5, about 0.8 to about 4.2, about 1.0 to about 4.0, about 1.2 to about 3.5, or about 0.5, about 0.7, about 0.9, about 1, about 1.2, about 1.5, about 1.7, about 1.9, about 2, about 2.2, about 2.5, about 2.7, about 2.9, about 3, about 3.2, about 3.5, about 3.7, about 3.9, about 4, about 4.2, or about 4.5. For the blackcurrant juice concentrate, the acidity (citric acid g/100 g) may be about 5 to about 20, about 8 to about 18, about 7 to about 17, or about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, or about 25.

In some circumstances, it may be desirable to adjust the pH of the puree or that of the final composition to approximate physiological levels. In particular, it may be useful to obtain a pH range from 6.0 to 8.0; or 6.5 to 7.5; or 6.8 to 7.2; or a pH of about 6.5, about 6.7, about 6.8, about 6.9, about 7.0, about 7.1, about 7.2, about 7.3, about 7.4, or about In certain s, the itions of the invention may be prepared by "soft pulping" technology referred to in New Zealand Patent No. 235972 (incorporated by reference herein), which can be d to produce a "soft" Boysenberry, apple, or blackcurrant puree. It may be useful to prepare the puree to have seeds d. It may also be useful to prepare the puree with a sieve size of about 1 mm or less.

A juice concentrate may be prepared as a natural sugar solution that is extracted or pressed and filtered from the skin and pulp, and may include the seeds. The solution may be depectinized, filtered, and evaporated under vacuum to a specified Brix level. For example, the juice concentrate may be folded about two to about seven times the original Brix value. In particular, the trate may be folded about two times, about three times, about four times, about five times, about six times, or about seven times the original Brix value.

In certain aspects, the Boysenberry juice trate may be manufactured from sound, ripe graded berries (e.g., Rubus ursinus var loganobaccus cv Boysenberry). In particular aspects, the Boysenberry juice concentrate may have a final sugar level ranging from 55° to 75° Brix; or 59° to 69° Brix; or 61° to 66° Brix; or about 60°, about 61°, about 62°, about 63°, about 64°, about 65°, about 654°, about 655°, about 656°, about 66°, about 67°, about 68°, about 69°, about 70°, or about 71° Brix. In various aspects, the ed Boysenberry and apple juice concentrate, and the combined Boysenberry, apple and blackcurrant juice concentrate may have the Brix values as noted directly above. Similarly, the apple juice concentrate and blackcurrant juice concentrate that is used to make the combined concentrate may have the Brix values as noted above, when uncorrected for acidity.

The juice concentrate may be produced by milling, mashing and pressing into single strength juice which is centrifuged, pasteurized, depectinised, filtered and then concentrated by evaporation with aroma returned in the rdisation process. The standardised concentrate may then be packed through the hygienic filler head into the required pack style without r heat treatment. The concentrate can be checked for compliance with the tion of a pure fruit juice, for example, as defined by the FSANZ— Food Standards Australia New Zealand.

It is expected that the Boysenberry juice concentrate will be rich in ation. For example, the berry juice concentrate may have a colour ratio (absorbance 520 nm/ ance 430 nm) of about 1.5 to about 3.0, about 1.8 to about 2.8, about 1.9 to about 2.2, or about 1.9, about 2, about 2.01, about 2.05, about 2.1, or about 2.2.

In addition, the juice concentrate may have a colour intensity (utilising Chroma meter) of about 15 to about 30, about 20 to about 28, about 21 to about 25, about 22 to about 24, or about 22, about 23, about 23.2, about 23.5, about 23.7, about 24, or about 25. The juice concentrate is also expected to be relatively clear in appearance, for example, with clarity levels of about 0.01 to about 0.1, about 0.02 to about 0.08, about 0.03 to about 0.06, about 0.04 to about 0.05, or about 0.03, about 0.04, about 0.045, about 0.047, about 0.048, about 0.05, or about 0.06.

The blackcurrant concentrate that is used to make the combined concentrate will also have deep colouration. For example, the blackcurrant concentrate may have a colour ratio (absorbance 520 nm/ absorbance 430 nm at pH 3) of about 1.5 to about 4.0, about 1.8 to about 3.8, or about 2.0 to about 3.0; or a colour ratio of about 1.8, or about 1.9, or about 2.0, or about 2.1, or about 2.2, or about 2.3, or about 2.4, or about 2.5, or about 2.6, or about 2.7, or about 2.9, or about 3.0, or about 3.2, or about 3.3, or about 3.4, or about 3.5.

In contrast, the apple juice concentrate that is used to make the combined concentrate may be relatively colourless, for example, less than 0.35 abs at 420 nm 12 Bx, or at least less than 0.45 abs at 420 nm 12 Bx. For apple juice concentrate, this can also be express as a general range of about 0.15 to about 0.45 abs, about 0.10 to about 50 abs, or about 0.19 to about 45 at 440 nm and 11.5°Brix. The apple juice concentrate may be used as a clear concentrate, e.g., free from haze. The specific gravity of the various juice concentrates may be about 1.2 to about 1.4, about 1.29 to about 1.39, or about 1.32 to about 1.36, or about 1.2, about 1.3, about 1.31, about 1.32, about 1.33, about 1.35, about 1.36, about 1.37, about 1.38, about 1.39, or about 1.4 at 20°C. The various measurement methodologies, e.g., colour ratios, clarity, etc, are known in the art, and may be found, for example, in the Al]N code of practice in the International Fruit Juice Federation Handbook of Analysis, 1996, International Fruchtsaft—Union, Zug, Switzerland.

In initial preparatory stages, the Boysenberry, apple, or blackcurrant may undergo a pre—treatment process which may include the well known steps of ripening, inspecting, grading, and/or sorting of the berries/fruit. With regard to ripening, it is preferable to use ripe or mature Boysenberry, apple, or blackcurrant when producing the compositions of the invention; however, rotted or decaying material is ably avoided.

Ripeness can be ed using widely known and used s in the art. Ripeness can be measured prior to picking or processing the Boysenberry, apple, or urrant. In particular, ripeness may be measured using the Brix system, as noted . Boysenberry, apple, or blackcurrant that is overly mature or fermenting may not produce an ideal composition. Boysenberry, apple, or blackcurrant with a Brix level below the ideal may be artificially ripened before use.

As part of the processing, the Boysenberry, apple, or urrant may be ised. The fruit may be passed through an assembly having one or more roller brushes for ng any ng foreign matter. Conventional washing techniques may then be employed. For example, it is possible to use a series of spray nozzles to wash the Boysenberry, apple, or blackcurrant. Wash additives aiding cleansing or reducing the bacteria count on the berry, apple, or blackcurrant may be employed ing to local regulations and requirements. For example, the Boysenberry, apple, or blackcurrant may be washed by a chlorine wash and/or an ozone nated water wash followed by a fresh water rinse.

The sterilized Boysenberry, apple, or blackcurrant may then be conveyed into a hopper. This can be d to form a funnel to direct the berries or fruit to a pressing assembly. The pressing assembly may be d to perform a pulping or comminution process. Such process can be relatively mild and gentle ("soft pulping") compared to conventional fruit g ques. With soft pulping, no icant disintegration or lysis of fruit cells or components. Preferably, only a minor proportion (generally less than —10%) of seeds is fragmented by this s.

In one embodiment, the pressing assembly performs the soft pulping of the Boysenberry, apple, or blackcurrant by pressing between a twin converging belt press. The press belts may be multiple loops rotated about a series of s. The distance separating the press belts may decrease in the direction of travel of the Boysenberry, apple, or blackcurrant. In this way, increased force may be exerted upon the berry, apple, or blackcurrant as it travels along the length of the pressing assembly. This can produce pulping without icant damage to the seeds. This in turn prevents seeds from contaminating the pulp.

The pulp generated from the pressing assembly may be directed to a screening process, in order to separate the seeds from the pulp. In particular, the pulp may be separated from the seed using a soft mechanical screening technique. For example, a pulp finisher may be used. This includes a rotating ?exible er which is rotated within a cone shaped screen having apertures of a predetermined size. In particular aspects, the size of the apertures is selected to permit the pulp and juice to pass through the screen while retaining a substantial portion, if not all, of the seeds within the interior cavity defined by the screen.

In certain aspects, it may be preferable to use a paste rather than a puree from the Boysenberry, apple, or blackcurrant. A paste may be made as a concentrate. For example, the fruit may be heated for several hours, strained, and reduced to a thick, concentrated form.

The fruit may be heated after removing the skins, or after the pulping or pureeing process.

The fruit can be heated gradually, and then kept heated at a moderate temperature, with mixing. Upon thickening, the paste can be spread on a ?at sheet, or transferred to a packaging, for example, a bag, tube, jar, bottle, or other container. The paste may be transferred aseptically, such that it is suitable for human consumption. It may be desired to prepare the paste from mature berries/fruits. The paste may be prepared from pulped fruit.

The paste may be a smooth preparation.

The pulp (e.g., in paste or puree form) or juice concentrate may be processed by a freezing step. This may be followed by or used in conjunction with a drying step. In an alternative embodiment, the pulp is dried and processed to a powder without an intervening freezing step. For example, methods involving drum drying may be used. In the drum—drying process, a puree or paste may be dried at relatively low temperatures over rotating, high— ty drums that produce sheets of drum—dried product. In certain aspects, an ve may be used to accelerate or otherwise assist the drying process. For example, pea starch or other drying aids may be utilised. The dried product may then be milled to a finished ?ake or powder form. Advantageously, drum drying ques may be used to produce a dried ition that retains its key ents, e.g., phenolic compounds, and can be easily reconstituted using liquid. For example, drum dried products may be made to be cold water e. As further alternatives, belt drying or convection drying may be used. Such drying methods are widely known and used in the field.

If ng is used, it is preferable to freeze the pulp or juice concentrate as soon as possible after it is produced to maintain freshness. However, freezing may be carried out within 24 or 48 hours, as needed. Freezing methodologies are well known and need not be described in significant detail herein. Blast freezing is particularly preferred for use with the ion. The pulp or juice concentrate may be frozen in standard sized pales, which are used to collect the fresh pulp/concentrate after processing. The pulp or juice concentrate can be stored frozen (e.g., at —18°C) until it is required.

The frozen pulp or juice concentrate may be freeze dried, i.e., lyophilised.

Freeze drying techniques are widely known and commonly used. The freeze drying cycle may be about 48 hours; or ranging from 40 to 56 hours; or 12 to 36 hours; or 36 to 60 hours; or about 40 hours, about 42 hours, about 44 hours, about 46 hours, about 48 hours, about 50 hours, about 52 hours, or about 54 hours. A longer freeze drying cycle, e.g., at least 48 hours ("gentle freeze "), may be used to retain maximal ty. In particular aspects, the s may be carried out to such that water formation is avoided, and the moisture content is minimised during processing.

It may be desirable to use a particular lyophilisation process for obtaining the dried product. For example, a lyophilisation drying program may be used as part of an automated drying system. The lyophilisation process may include multiple drying steps, e.g., with step—wise ses and reductions in temperature. Preferably, a primary drying setting is used for sublimation, followed by one or more secondary drying settings that are used to remove residual moisture. In particular aspects, the top temperature of the lyophilisation process does not exceed 70°C. In other aspects, the temperature of the lyophilisation process ranges between -10°C to 70°C. In one other aspect, up to 48 hours of lyophilisation is utilised.

The resulting dried product may then be milled into a powder which can then be utilised as appropriate. Milling methods are well known and widely used in the art.

Standard mesh sizes may be used to produce the , for example, US 20, US 23, US , US 35, US 40, US 45, or US 50 mesh sizes may be used. The sieve size for the powder may range from 1.0 to 0.3 mm; or 0.84 to 0.4 mm; or 0.71 to 0.5 mm; or may be about 1.0 mm, about 0.84 mm, about 0.71 mm, about 0.59 mm, about 0.5 mm, about 0.47 mm, about 0.465 mm, about 0.437 mm, about 0.4 mm, about 0.355 mm, or about 0.3 mm.

To ensure minimal degradation of ingredients, the preparation process may be performed at a temperature of less than 40°C. In various embodiments, the s is performed at a temperature ranging from —4°C to 40°C; or from —l°C to 10°C; or from 1°C to 6°C; or at about 0°C, about 1°C, about 2°C, about 3°C, about 4°C, about 5°C, or about 6°C. These temperatures may be kept during the entire preparation process, including the storage of the whole fruit, prior to it being broken open, and during the pulping/pureeing process. For optimal results, these temperatures are kept at least from the point that the fruit has been broken open. Use of such temperatures avoids oxidation of the fruit and the use of ng agents. In n circumstances, it may be possible to obtain organic certification.

The processing method is preferably performed so as to prevent or at least minimise any damage or effects on the active material in the fruit. To ensure l production methods, the resulting compositions can be monitored for activity, for e, for anthocyanin levels, polyphenol levels, and/or antioxidant activity.

Assays for polyphenols are well known in the art and are also bed below. In particular, it is possible to measure gallic acid equivalents (GAE) to determine total polyphenol content. For example, the Folin-Ciocalteu method (employing the Folin— Ciocalteu t, also called Folin's phenol reagent or Folin—Denis reagent) may be used for colorimetric in vitro assays of phenolic nds (75). It is ed that the total polyphenol content of a Boysenberry juice concentrate will be relatively high, for example, about 500 to about 5000 mg GAE/100 g FW, about 1000 to about 3000 mg GAE/100 g FW, about 1500 to about 2500 mg GAE/100 g FW, about 3000, about 2500, about 2000, about 1500, or about 1000 mg GAE/100 g FW. It is noted that FW indicates the fresh weight of the juice concentrate.

Anthocyanins may be quantified by HPLC. This can be used give breakdown of individual compounds and expressed as cyanidin 3-glucoside lents (76). For example, HPLC eluted components may be red at 530 nm for anthocyanins. A standard curve may be ed using a cyanidin—3—glucoside rd (for example, from Extrasynthese) and total anthocyanins may be calculated on this basis. Other phenolics may also be analysed by HPLC, for example at 250—700 nm. A range of rds may be run, including gallic acid, ellagic acid, quercetin, rutin and n. Absorbance spectra and retention time of the standards may be compared with unknowns in the HPLC traces. This analysis can include measurements for ellagic acid. As non—limiting examples, the total anthocyanin content of a berry juice concentrate ssed as cyanidin 3—glucoside equivalents) may be about 1000 to about 10,000 mg/100 g FW, about 2000 to about 8000 mg/100 g FW, about 4000 to about 7000 mg/100 g FW, about 5500 to about 6500 mg/100 g FW, or about 8000, about 7000, about 6500, about 6800, about 6000, about 5000, about 4000, or about 3000 mg/100 g FW.

For the combined Boysenberry and apple compositions, it is expected that the total anthocyanins may account for about 40—50% of the total enol content that is present in these compositions, or at least 40%, at least 41%, at least 42%, at least 43%, at least 44%, at least 45%, at least 46%, at least 47%, at least 48%, at least 49%, at least 50%, at least 51%, at least 52%, at least 53%, at least 54%, or at least 55% of the total polyphenol content that is present in these compositions. For the combined and Boysenberry, apple and blackcurrant compositions, it is expected that the total anthocyanins may account for about 70—80% of the total polyphenol content that is present in these compositions, or at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, or at least 80%, of the total polyphenol content that is present in these compositions.

In addition, for the combined Boysenberry and apple compositions and the Boysenberry, apple and blackcurrant compositions, it is expected that the total polyphenols (including anthocyanins) may t for about 80—90% of the total polyphenol content that is present in these compositions, or at least 70%, at least 71%, at least 72%, at least 73%, at least at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, or at least 95% of the total polyphenol content that is present in these compositions.

Further to this, for the combined Boysenberry and apple itions and the Boysenberry, apple and blackcurrant compositions, it is expected that the hydrolysable tannins will account for about 25% to about 35% of the total polyphenols in the composition.

For the Boysenberry, apple and blackcurrant compositions, it is expected that the hydrolysable tannins will account for about 8% to about 12% of the total polyphenols in the composition. An exemplary method for measuring ysable s is LC—MS (liquid chromatography—mass spectrometry) analysis, as described in detail herein.

Antioxidant capacity may be measured by ORAC and/or DPPH assays. The oxygen radical absorbance capacity assay is one of the most widely ed assays to test the antioxidant potential of foods. The ORAC assay measures antioxidant inhibition of peroxyl radical—induced oxidation (77, 78, 84). Trolox, a water—soluble analogue of vitamin E, may be used as a control standard. In an additional assay, DPPH (2,2-diphenyl picrylhydrazyl) may be used to show the kinetic behaviour of polyphenols as free radical scavengers. The higher the antioxidant activity, the larger the decrease of DPPH- concentration. A methanolic solution of the DPPH radical changes from purple to colourless when quenched by idants. The se in DPPH- is measured at 515 nm against standard curves, e.g, Trolox and DPPH- (79, 80).

] As particular exemplifications, the antioxidant capacity for the Boysenberry juice concentrate may be about 10,000 to about 100,000 ORAC value (umol Trolox/100 g FW), about 20,000 to about 80,000 ORAC value, about 30,000 to about 70,000 ORAC value, about 40,000 to about 50,000 ORAC value, or about 80,000, about 70,000, about 60,000, about 50,000, about 40,000, about 30,000, or about 20,000 ORAC value. As r ifications, the antioxidant capacity for the Boysenberry juice concentrate may be measured with the DPPH assay (at 100% MeOH) as about 1000 to about 6000 umol 00 g FW, about 2000 to about 5000 umol TEAC/100 g FW, about 2500 to about 2900 umol TEAC/100 g FW, or about 5000, about 4000, about 3000, about 2800, about 2500, about 2000, or about 1000 umol TEAC/100 g FW.

Alternatively or additionally, the compositions can be tested for other components, e.g., sugars, folate, and Vitamin C. The corresponding assays are widely known. For example, folate levels of the Boysenberry juice concentrate may be measured using standard methodologies (see, e.g., 83), and may be about 20 u g/100 g FW, about 30 tug/100 g FW, about 40 pig/100 g FW, or about 50 pig/100 g FW, about 60 tug/100 g FW, about 70 ug/lOO g FW, or about 80 ug/lOO g FW, or about 10 to about 100 ug/lOO g FW, about 20 to about 80 u g/100 g FW, about 30 to about 70 u g/100 g FW, about 20 to about 50 gig/100 g FW, or about 50 to about 70 pig/100 g FW.

It will be understood that other known assays may also be used to analyse the disclosed compositions (see, e.g., 85), and the invention is not limited to one particular assay for bioactive compounds, including phenolics, yanins, antioxidants, Vitamins, ydrates, etc. It will be understood also that the levels identified herein for juice concentrates can be readily extrapolated to powdered forms, as well as puree and paste forms.

In some circumstances, it may be le to use genetic derivative of the plant to obtain the compositions of the invention. It is expected that a composition obtained from such derivative would share one or more of the characteristics of the compositions obtained from the original stock. Exemplary features include: polyphenol levels and polyphenol profiles, including anthocyanidin levels and profiles, vitamin levels, and reduction of OVA—induced in?ammation, as noted above and sed in detail herein.

Regarding the fruit itself, it is expected that the fruit obtained from a genetic derivative would share a similar compositional makeup as the parent fruit.

Compositions comprising Boysenberry and apple and itions comprising Boysenberry, apple and blackcurrant ] The inventors have found that Boysenberry itions e beneficial ingredients that are useful for maintaining the health of the respiratory system, as well as treating and preventing respiratory problems. The inventors have shown that a Boysenberry concentrate is ularly efficacious for reducing airway inflammation and fibrosis. Also cious are a combined Boysenberry and apple concentrate, and a combined Boysenberry, apple and blackcurrant concentrate, as described herein. As such, the Boysenberry compositions, including combined berry and apple compositions, and combined Boysenberry, apple and urrant compositions, disclosed herein can be used to support or improve overall respiratory health and/or to treat or prevent various disorders or other conditions of the respiratory tract, including in?ammation, , chronic obstructive pulmonary e, airway fibrosis, and airway remodelling. In this way, the disclosed compositions are understood to be anti—inflammatory compositions, and also anti— asthmatic compositions, as well as being compositions that are active against chronic obstructive pulmonary disease, reactive airway disease, airway fibrosis, and airway remodelling.

As described herein, a Boysenberry composition may se a juice concentrate or a powder concentrate prepared from Boysenberries. The composition may r comprise a juice concentrate or a powder concentrate prepared from apples, or may further comprise a juice concentrate or a powder concentrate prepared from blackcurrants.

As various alternatives, the ition may consist of, or may consist essentially of: a juice concentrate or a powder concentrate prepared from Boysenberries and a juice concentrate or a powder concentrate prepared from apples, or a juice concentrate or a powder concentrate prepared from Boysenberries and a juice concentrate or a powder concentrate prepared from blackcurrants.

Generally speaking, the Boysenberry and apple trate may include various ratios of Boysenberry concentrate to apple concentrate. As exemplifications, the percentages of Boysenberry concentrate to apple trate (having a combined tage of 100% v/v) may include about 17% Boysenberry concentrate to about 83% apple concentrate, about 18% Boysenberry concentrate to about 82% apple concentrate, about 19% Boysenberry concentrate to about 81% apple concentrate, about 20% Boysenberry concentrate to about 80% apple concentrate, about 21% Boysenberry concentrate to about 79% apple concentrate, about 22% Boysenberry trate to about 78% apple concentrate, about 23% Boysenberry trate to about 77% apple concentrate, about 24% Boysenberry concentrate to about 76% apple concentrate, about 25% Boysenberry concentrate to about 75% apple concentrate, about 26% Boysenberry trate to about 74% apple concentrate, about 27% Boysenberry concentrate to about 73% apple concentrate, about 28% berry concentrate to about 72% apple concentrate, about 29% Boysenberry concentrate to about 71% apple concentrate, about 30% Boysenberry concentrate to about 70% apple concentrate, about 31% Boysenberry concentrate to about 69% apple concentrate, about 32% berry concentrate to about 68% apple concentrate, about 33% Boysenberry concentrate to about 67% apple concentrate, or about 34% Boysenberry concentrate to about 66% apple concentrate, these tages being representative of v/v values.

In the same way, the percentages of Boysenberry and blackcurrant concentrate to apple concentrate (having a combined percentage of 100% v/v) may include about 17% Boysenberry and urrant concentrate to about 83% apple concentrate, about 18% Boysenberry and blackcurrant concentrate to about 82% apple concentrate, about 19% Boysenberry and blackcurrant concentrate to about 81% apple concentrate, about 20% Boysenberry and blackcurrant concentrate to about 80% apple concentrate, about 21% berry and blackcurrant concentrate to about 79% apple concentrate, about 22% Boysenberry and blackcurrant trate to about 78% apple concentrate, about 23% Boysenberry and blackcurrant trate to about 77% apple concentrate, about 24% Boysenberry and urrant concentrate to about 76% apple concentrate, about 25% Boysenberry and blackcurrant concentrate to about 75% apple concentrate, about 26% Boysenberry and blackcurrant concentrate to about 74% apple concentrate, about 27% Boysenberry and blackcurrant concentrate to about 73% apple concentrate, about 28% Boysenberry and blackcurrant concentrate to about 72% apple concentrate, about 29% Boysenberry and blackcurrant concentrate to about 71% apple concentrate, about 30% Boysenberry and blackcurrant concentrate to about 70% apple concentrate, about 31% berry and blackcurrant concentrate to about 69% apple concentrate, about 32% Boysenberry and blackcurrant concentrate to about 68% apple concentrate, about 33% Boysenberry and blackcurrant concentrate to about 67% apple concentrate, or about 34% Boysenberry and blackcurrant concentrate to about 66% apple concentrate, these percentages being representative of v/v values.

] As non-limiting examples, the tage of blackcurrant concentrate in the combined Boysenberry, apple and urrant concentrate may be about 5% to about 20%, or about 8% to about 18%, or about 10% to about 15%, or a percentage of about 5%, about 8%, about 10%, about 13.5%, about 15%, about 18%, or about 20%, these percentages being representative of v/v values. In particular aspects, the percentage of the blackcurrant concentrate is the same or substantially the same as the percentage of Boysenberry concentrate in the ed Boysenberry, apple and blackcurrant concentrate.

The Boysenberry and apple concentrate and the Boysenberry, apple and blackcurrant trate (having a combined percentage of 100% W/v) may include less than 1% of a preservative, for example, about 0.005% to about 0.5%, or about 0.05% to about 0.15%, or may include about 0.04%, about 0.06%, about 0.08%, about 0.1%, about 0.12%, about 0.14%, about 0.16%, about 0.18%, or about 0.2% of a preservative, these percentages being representative of w/v values. Useful preservatives include but are not limited to sorbic acid, sodium sorbate, potassium e, citric acid, ascorbic acid, malic acid, ic acid, propionic acid, and c acid, for e, in the form of its sodium salt, e.g., sodium benzoate.

] The composition may be formulated as a liquid, for example, a juice concentrate, syrup, suspension, or tonic for oral administration, or as a solution for enteral administration. Alternatively, the composition may be formulated as a powder to be encapsulated, tableted, or added to or incorporated in other products. Particularly encompassed are delayed release formulas, extended release formulas, as well as formulas for rapid disintegration. Capsules, for example gel capsules, are speci?cally encompassed, as well as sachets and chewable tablets. Additionally ed are combination formulas, which include the powder of the invention mixed with other beneficial agents, e.g., one or more respiratory aids. In various aspects, the composition may be prepared as a nutraceutical composition, a pharmaceutical composition, a functional food or beverage, a natural ingredient (e.g., a natural additive), or a natural supplement (e.g., a dietary supplement).

It is expected that the Boysenberry ition, including the combined Boysenberry and apple composition and the combined Boysenberry, apple and blackcurrant composition, will be prepared to e high levels of yanins. For example, the composition may include about 2 to about 50,000 mg/ml total anthocyanins or total Boysenberry and blackcurrant anthocyanins, or about 20 to about 40,000 mg/ml, about 25 to about 35,000 mg/ml, about 30 to about 30,000 mg/ml, about 40 to about 25,000 mg/ml, about 50 to about 20,000 mg/rnl, about 60 to about 15,000 mg/ml, about 70 to about 10,000 mg/ml, about 80 to about 8000 mg/ml, about 90 to about 6000 mg/ml, about 100 to about 5000 mg/ml, about 10 to about 1000 mg/ml, about 20 to about 800 mg/ml, about 30 to about 600 mg/ml, about 50 to about 200 mg/ml, or about 50,000, about 40,000, about 35,000, about 25,000, about 20,000, about 15,000, about 12,000, about 10,000, about 8000, about 7500, about 5000, about 2500, about 2000, about 1000, about 1500, about 1200, about 1000, about 750, about 500, about 250 mg/ml, about 200 mg/ml, about 150 mg/ml, about 100 mg/ml, about 75 mg/ml, about 50 mg/ml, about 25 mg/ml, about 20 mg/ml, or about 10 mg/ml total yanins, or total berry and blackcurrant anthocyanins, or a dry weight equivalent thereof.

In specific aspects, the Boysenberry composition, including the combined berry and apple composition and the combined Boysenberry, apple and blackcurrant composition, may be administered at a dosage unit of about 1 mg to about 20,000 mg total anthocyanins or total Boysenberry and blackcurrant yanins, or about 1 mg to about 2000 mg total anthocyanins or total Boysenberry and blackcurrant anthocyanins, or about 5 mg to about 5000 mg, about 10 mg to about 3000 mg, about 10 to about 1000, about 15 mg to about 1500 mg, about 20 mg to about 1000 mg, about 25 mg to about 850 mg, about 30 mg to about 600 mg, about 35 mg to about 550 mg, about 50 to about 500 mg, about 5 to about 500, about 10 mg to about 200 mg, about 1 to about 400 mg, about 1 to about 200 mg, about 40 mg to about 400 mg, about 40 to about 200 mg, about 20 mg to about 80 mg, about mg to about 60 mg, about 45 mg to about 55 mg, or about 20,000 mg, about 15,000 mg, about 12,000 mg, about 10,000 mg, about 7500 mg, about 5000 mg, about 4000 mg, about 3000 mg, about 2000 mg, about 1500 mg, about 1200 mg, about 1000 mg, about or about 500 mg, about 400 mg, about 300 mg, about 200 mg, about 100 mg, about 90 mg, about 95 mg, about 80 mg, about 75 mg, about 70 mg, about 65 mg, about 60 mg, about 55 mg, about 50 mg, about 45 mg, about 40 mg, about 35 mg, about 30 mg, about 25 mg, about 20 mg, or about 10 mg total anthocyanins or total Boysenberry and blackcurrant anthocyanins. In particular aspects, the dosage unit may be about 50 mg to about 500 mg total anthocyanins or total berry and blackcurrant anthocyanins.

The dosage units as noted above may be administered once per day, twice per day, or three times per day, or more as needed. An exemplary, and non—limiting, daily dosage may be about 10 mg to about 1000 mg total anthocyanins or total Boysenberry and blackcurrant anthocyanins. The dosage may be adjusted for pediatric, geriatric, ight, eight, or other patients, where required.

If a Boysenberry juice, apple juice, or blackcurrant juice concentrate is made by standard commercial production methods (large or small , or obtained from commercial sources, the juice trate, including the combined Boysenberry and apple juice concentrates and the combined Boysenberry, apple and blackcurrant juice concentrates, may be administered at a dosage unit of about 0.5 to about 50 ml, about 0.5 to about 20 ml, about 0.5 to about 10 ml, about 1 to about 9 ml, about 2 to about 8 ml, about 3 to about 7 ml, about 4 to about 6 ml, or about 50, about 40, about 30, about 20, about 15, about 12.5, about 10, about 9, about 8, about 7, about 6, about 5, about 4, about 3, about 2, about 1, or about 0.5 ml of juice concentrate. In particular aspects, the dosage unit may be about 5 ml ofjuice concentrate. The s dosage units may be administered once per day, twice per day, or three times per day, or more as needed. Dosage modi?cation can be made for patient size and age in accordance with known methods.

Each of the Boysenberry, apple, and blackcurrant trates will be rich sources of phenolics, anthocyanins, and other bene?cial components. For example, a blackcurrant juice concentrate will be expected to include a total anthocyanin content (g/100 g) of at least 0.8, at least 0.9, at least 1.0, at least 1.1, at least 1.2, at least 1.3, at least 1.4, or at least 1.5. For exemplary anthocyanin quantitation methods, see (98) and (99). The blackcurrant juice concentrate will also be expected to include Vitamin C (ascorbic acid; mg/100 g) levels of at least 500, at least 550, at least 600, at least 650, at least 700, at least 750, at least 780, at least 800, at least 850, or at least 900. The blackcurrant juice concentrate will further be expected to include a total phenolics content (g/100 g) of at least 3.0, at least 3.2, at least 3.4, at least 3.6, at least 3.8, at least 4.0, at least 4.2, at least 4.4, at least 4.6, at least 4.8, or at least 5.0. For exemplary phenolics quantitation methods, see (100).

In certain circumstances, it may be desirable to isolate or enrich the polyphenols from the fruits being used. In particular, it may be advantageous to use the Boysenberry, apple, or blackcurrant to obtain polyphenol enriched compositions, phenolic concentrates, or compositions comprising isolated phenolics, e.g., isolated anthocyanins. For example, the compositions of the invention may be enriched for polyphenols such that their tration is increased ve to the other components of the fruit, e.g., sugars. In particular aspects, the compositions of the invention may include polyphenols that have been isolated away from (e.g., purified from) the other components of the fruit. The particular polyphenols for isolation or ment are described in detail herein.

Methods of ing and extracting polyphenols are widely known in the art (e. g., 81, 82). The resulting composition may e at least 2 times, at least 3 times, at least 4 times, at least 5 times, or at least 10 times the amount of enols compared to the composition prepared without polyphenol enrichment or isolation steps. The polyphenol enriched compositions, phenolic concentrates, and compositions comprising isolated phenolics may be dried as a powder, and used in accordance with the present ion.

] The dosage form may contain excipients, for example, one or more anti— adherents, binders, gs, disintegrants, s, colours, sweeteners, lubricants, ts, ?ow agents, anti—caking agents, sorbents, or preservatives. Useful excipients include but are not d to: stearin, magnesium stearate, and stearic acid; saccharides and their derivatives, e. g., disaccharides: sucrose, lactose; polysaccharides and their derivatives, e.g., starches, cellulose or modified cellulose such as microcrystalline cellulose and cellulose ethers such as hydroxypropyl cellulose; sugar alcohols such as isomalt, xylitol, sorbitol and maltitol; proteins such as gelatin; synthetic polymers such as nylpyrrolidone, polyethylene glycol; fatty acids, waxes, shellac, plastics, and plant fibres, e.g., corn protein zein; hydroxypropyl methylcellulose; crosslinked polymers, e.g., crosslinked polyvinylpyrrolidone (crospovidone), and crosslinked sodium carboxymethyl ose (croscarmellose sodium); sodium starch glycolate; silicon dioxide, fumed silica, talc, and magnesium carbonate.

It is expected that the Boysenberry compositions disclosed herein will include various components, for example, carbohydrates and polyphenols, and in particular, anthocyanidins. Anthocyanidins of interest include cyanidins and rutinosides, such as cyanidin—3—O—sophoroside, cyanidin—3—O—glucoside, epicatechin, cyanidin—3—O— glucosylrutinoside, cyanidin—3-O—rutinoside, cyanidin—3—(6’—p—coumaryl)glycoside—5— glycoside, cyanidin—3—O—glycoside, cyanidin—3,5—diglycoside, and cyanidin—3—O—2G— glucosylrutinoside. Also of interest are hydrolysable tannins such as ellagitannins and ellagic acid. The Boysenberry itions of the invention may also e various carbohydrates, and in particular, various sugars, including neutral sugars. As to neutral sugars, the Boysenberry compositions may include one or more of fructose and glucose, as well as sucrose. rly, the apple compositions as disclosed herein will encompass various components, including various carbohydrates and polyphenols. Of particular interest are phenolic compounds such as flayonoids and cinnamic and benzoic acid derivatives.

Included are catechins, procyanidins, and hydroxycinnamates, and more particularly ed are included are 3—hydroxy ids such as anthocyanins, flavanols, and ?avan— 3—ols. Key individual compounds include epicatechin, in, nidin dimer B2, 5— quuinic acid, and quercetin glycosides. Dihydrochalcones such as phloridzin are particularly ed as flavonoid precursors found in apples. Apple peels can be used to obtain high levels of polyphenols and flavonoids such as tin glycosides and cyanidin.

Apple ?esh and cores can be used to obtain high levels of genic acid. Particular cultivars can be used to se polyphenol levels. In particular, cider apples can be used to maximise procyanidins that are responsible for their gency and bitterness. The apple compositions of the invention may also include various ydrates, and in particular, WO 31972 2018/050109 s sugars, including neutral sugars. As to neutral , the apple compositions may e one or more of fructose, glucose, and sucrose.

As disclosed herein, the blackcurrant itions of the invention will include various components, including sugars and polyphenols. Noted specifically are hydroxycinnamates such as chlorogenic and p—coumaroquuinic acids, and also anthocyanins, and ?avonol glycosides. Of particular interest are enol compounds such as delphinidin 3—O—glucoside; delphinidin 3—O—rutinoside; delphinidin-3—O—(6 —p— coumaroyl)glucoside; cyanidin 3—O—glucoside; cyanidin 3—O—rutinoside; cyanidin 3—0— glucoside-6—p—cumaryl; peonidin 3—O— rutinoside; malvidin 3—O— rutinoside; neochlorogenic acid; p—coumaric acid glucoside; myricetin derivatives; quercetin 3—O—rutinoside; quercetin 3-O—galactoside; and tin 3—O—glucoside. For example, the skin may be used to obtain high levels of anthocyanins. The blackcurrant compositions of the invention may also include various carbohydrates, and in particular, various sugars, including l sugars.

As to neutral sugars, the compositions in the ion may include one or more of fructose, glucose, and sucrose.

Methods of using compositions comprising Boysenberry and apple and compositions comprising Boysenberry, apple and blackcurrant As noted above, the Boysenberry compositions disclosed herein, including the compositions comprising Boysenberry and apple, and the itions comprising Boysenberry, apple and blackcurrant, can be used to support or improve overall respiratory health and/or to treat or prevent various conditions of the respiratory tract, including in?ammation, and atory disorders associated with in?ammation, such as asthma, chronic obstructive pulmonary disease, ve airway disease, airway fibrosis, and airway remodelling. Other conditions associated with in?ammation in the respiratory tract include: allergy or ic disorders, ema, bronchitis, respiratory bronchiolitis, interstitial lung disease, in?ammatory airway disease, fibrosing alveolitis, intrinsic alveolitis, pulmonary eosinophilia, pulmonary vasculitis, pneumonia, interstitial pneumonia, desquamative interstitial pneumonia, lymphoid interstitial pneumonia, nonspecific interstitial pneumonia, eosinophilic nia, pneumonitis, pleurisy (pleuritus), pleural effusion, cystic fibrosis, primary ciliary dyskinesia, acute respiratory ss syndrome (ARDS), sarcoidosis, dermatomyositis, toxocariasis, Wegener's omatosis, Langerhans cell histiocytosis, Sjogren’s syndrome, Kartagener syndrome, vocal cord dysfunction, spasmodic croup, autoimmune disease such as lupus, ve vasomotor disease, and autonomic disorders. Additional factors associated with in?ammation in the respiratory tract e smoking, air pollution, allergens, infection (e.g., Viral or ial), certain medication (e.g., chemotherapeutic agents), radiation treatment, l devices (e.g., ventilators), and surgery.

The compositions of the ion find use for treating or preventing respiratory tract ation, asthma, chronic obstructive pulmonary disease, airway fibrosis, airway remodelling, or other conditions described herein. As exemplary dosages, the compositions may be stered at dosages to obtain about 0.1 to about 200 mg/kg, about 0.2 to about 180 mg/kg, about 0.25 to about 150 mg/kg, about 0.5 to about 125 mg/kg, about 0.6 to about 100 mg/kg, about 0.7 to about 90 mg/kg, about 0.1 to about 50 mg/kg, about 0.1 to about 20 kg/mg, about 0.1 to about 10 mg/kg, about 0.1 to about 5 mg/kg, about 0.1 to about 1 mg/kg, about 1 to about 20 mg/kg, about 1 to about 10 mg/kg, 1 to about 5 mg/kg, or about 200 mg/kg, about 100 mg/kg, about 90 mg/kg, about 80 mg/kg, about 70 mg/kg, about 60 mg/kg, about 50 mg/kg, about 40 mg/kg, about 30 mg/kg, about 20 mg/kg, about 10 mg/kg, about 9 mg/kg, about 8 mg/kg, about 7 mg/kg, about 6 mg/kg, about 5 mg/kg, about 4 mg/kg, about 3 mg/kg, about 2 mg/kg, about 1 mg/kg, about 0.9 mg/kg, about 0.8 mg/kg, about 0.7 mg/kg, about 0.6 mg/kg, about 0.5 mg/kg, about 0.4 mg/kg, about 0.3 mg/kg, about 0.2 mg/kg, or about 0.1 mg/kg, of total anthocyanins or total Boysenberry anthocyanins in relation to patient body weight. In particular aspects, the dosage may be about 0.1 mg/kg to about 10 mg/kg. The dosages as indicated above may be administered once per day, twice per day, three times per day, or more, as needed. Administration may be made with food, or before a meal. The appropriate dosage and dosage form will be readily determined by a person of skill in the art.

Various routes of administration may be used for the compositions, including enteral administration and oral administration. Oral administration may be by tablet, capsule, sachet, drops, elixir, linctus, solution, emulsion, suspension, draught, puree, paste, syrup, gel, jelly, tonic, or other known means. Enteral administration may be by al tubing or gastric tubing, ing nasogastric tubing. Different means of administration are known in the art and may be utilised by a skilled person. The compositions disclosed herein are not limited to a particular form for administration.

It may be useful to add one or more phenolic nds to the compositions of the invention, to further supplement the phenolic activity therein. Exemplary nds e but are not limited to: phenolic derivatives such as phenolic acid, and ?avonoids such as lignins, proanthocyanidins, anthocyanins, anthocyanidins, isoflavones, catechins, tannins, quercetin, naringenin, and hesperidin. Specific anthocyanin compounds of st are described herein. ularly encompassed are phenolic compounds extracted from one or more of: tea, cocoa, wine, soybeans, feijoa, citrus fruits, apples, pears, grapes, s, and kiwifruit. Specific phenolics include but are not limited to: ellagic acid, chlorogenic acid, catechin, epicatechin, kaemferol, E—caffeoyl—3—glucoside, E—caffeoyl—4—glucoside, neochlorogenic acid, phlorizin, procyanidin B1 and B2, quercetin, quercetin side, and quercetin rutinoside.

As additional aspects, the compositions of the invention may be co— administered with one or more respiratory aids. A respiratory aid may be a medication, prescription or non—prescription, or an alternative treatment, such as a herbal remedy, or an essential oil, e.g., for vaporisation and/or inhalation. Of particular interest is use of the composition of the invention as a respiratory treatment during and/or following other respiratory treatments. For example, the composition may be formulated as a combined dosage form with one or more medicines or alternative treatments. Alternatively, the berry ition may be administered as a separate dosage form along with one or more tions or ative treatments. A respiratory aid may have one or more logical effects, for example, anti—in?ammatory, anti—spasmodic, bronchodilation, and/or muscle relaxation effects. Any respiratory aid may be long or short acting, and may be directed to a particular disorder, such as asthma, chronic obstructive pulmonary disease, GtC .

Exemplary medications include but are not limited to bronchodilators, including short—acting bronchodilators such as albuterol (e.g., Vospire ER), levalbuterol (e.g., Xopenex), ipratropium (e.g., Atrovent), albuterol/ipratropium (e.g., Combivent), corticosteroids such as fluticasone (e.g., Flovent, Flovent Diskus, Flovent HFA), budesonide (e. g., Pulmicort, Pulmicort Flexhaler), mometasone (e.g., Asmanex), beclomethasone (e.g., QVAR), ?unisolide (e.g., Aerospan), prednisolone, methylprednisolone, and hydrocortisone, xanthines such as theophylline (e.g., ron, Theo-24, Elixophyllin), long—acting bronchodilators such as aclidinium (e.g., Tudorza), arformoterol (e.g., Brovana), formoterol (e.g., l, omist), glycopyrrolate (e.g., Seebri Neohaler), indacaterol (e.g., Arcapta), erol (e.g., Striverdi Respimat), salmeterol (e.g., nt), tiotropium (e.g., Spiriva), and umeclidinium (e.g., Incruse Ellipta), combinations of two or more long—acting bronchodilators such as glycopyrrolate/formoterol (e.g., Bevespi Aerosphere), glycopyrrolate/indacaterol (e.g., Utibron Neohaler), pium/olodaterol (e. g., Stiolto Respimat), umeclidinium/vilanterol (e.g., Anoro Ellipta).

Further ary medications e but are not limited to combinations of inhaled corticosteroid(s) and long—acting bronchodilator(s) such as nide/formoterol (e.g., Symbicort), ?uticasone/salmeterol (e.g., , Advair Diskus), and ?uticasone/vilanterol (e.g., Breo Ellipta), phosphodiesterase—4 inhibitors such as ro?umilast (e.g., Daliresp), beta agonists, including short—acting beta agonists such as albuterol (e.g., ProAir HFA, Ventolin HFA), and levalbuterol (e.g., Xopenex HFA), anticholinergics such as ipratropium bromide (e.g., Atrovent HFA), long-acting beta antagonists (LABAs) such as formoterol (Perforomist), and salmeterol (e.g., Serevent Diskus), leukotriene modifiers such as montelukast (Singulair), zafirlukast (Accolate), and on (e.g., Zy?o, Zy?o CR), modulators such as zurnab (Nucala), omalizumab (e.g., Xolair), reslizumab (e.g., Cinqair), bronchodilators such as epinephrine (e. g., Primatene Mist, Bronkaid, Asthmahaler), ephedrine, and ylline—ephedrine (e.g., Primatene tablets).

EXAMPLES The examples described herein are provided for the purpose of illustrating specific ments of the invention and are not intended to limit the invention in any way.

Example 1: OVA-induced chronic airway ation and oral treatment with Boysenberry Overview Lung ?brosis negatively impacts on lung function in chronic asthma and is linked to the development of pro?brotic macrophage phenotypes. Epidemiological studies have found that lung function s from increased consumption of fruit high in polyphenols. However, previous studies have not investigated Boysenberry compositions, or effects on ic or remodelling in airway systems.

The inventors investigated the effect of berry ption, in both therapeutic and prophylactic treatment strategies in a mouse model of chronic antigen— induced airway in?ammation. Boysenberry consumption reduced collagen deposition and ameliorated tissue remodelling alongside an increase in the presence of CD68+CD206+ arginase alternatively activated macrophages in the lung tissue. The decrease in tissue remodelling was associated with sed expression of olytic matrix metalloproteinase-9 protein in total lung tissue.

The inventors identi?ed alternatively activated macrophages in the mice that ed Boysenberry as a source of the matrix metalloproteinase—9. The inventors esise that oral Boysenberry treatment moderate chronic tissue remodelling by supporting the development of olytic alternatively activated macrophages expressing matrix metalloproteinase-9. Regular Boysenberry consumption therefore has the ability to moderate chronic lung remodelling and ?brosis in asthma and other chronic pulmonary Materials ] ctin (clone AC—l5), ovalbumin (OVA), 4% formalin, Tween 20, trans— hydroxyproline, 3,3’—diaminobenzidine (DAB) substrate, ketamine/xylazine, and all other chemicals were obtained from Sigma (Auckland, NZ). Alum was obtained from Serya (Heidelberg, Germany). The Boysenberry juice was obtained as New Zealand 65 Brix Boysenberry juice trate kindly provided by Berryfruit Export NZ, currently trading as Boysenberries New Zealand Ltd (Nelson, New Zealand). The 65 Brix Boysenberry juice concentrate from Berryfruit Export NZ was diluted in sterile water to obtain a concentrate of 100 mg/ml total anthocyanins. From this, a r dilution was prepared to obtain a dosage of 10 mg/kg of total anthocyanins. This further dilution is noted as Boysenberry solution.

Anti—mouse polyclonal inducible nitric oxide synthase (iNOS) (ab3523), arginase, TIMP—l (ab38978), and MMP—9 (ab38898) were obtained from Abcam (Cambridge, UK). Antibodies against mouse CD68 (clone FA—ll) CD3e, CD8a, CD4, CD1 lb, CDl lc, and Gr—l were obtained from BioLegend (San Diego, CA) and anti—CD206 (clone MR5D3) was ed from AbDSerotec (Oxford, UK). Anti—mouse SiglecF, MHCII, and CD45 were from BD Biosciences (San Jose, CA).

TGFB ELISA kit was obtained from R&D Systems (Minneapolis, MN).

Vectastain Elite ABC staining kit was from Vector Laboratories ngame, CA). Bio— Plex multiplex cytokine assays for IL—4, IL—5, IL—6, IL-l3, and IFNv, DC Lowry n assay kit, and PVDF membrane were from Bio—Rad (Hercules, CA). BSA, NuPage 4-12% gels, MES running buffer, sample loading buffer, Novex sharp prestained, and MagicMark XP protein standards and all other buffers were from Life Technologies (Auckland, NZ).

Animals C57BL/6J male mice were bred and group housed (5 per cage) in conventional polycarbonate cages with a ?lter top, in a speci?c pathogen-free animal facility at the Malaghan Institute of Medical Research, Wellington, New Zealand. All experimental procedures were ed by the Victoria University of Wellington Animal Ethics Committee (approval number 2011R3M).

Mice were maintained on a 12-h light-dark cycle, at 21 i 2°C ambient ature with freely available irradiated rd laboratory rodent chow (Specialty Feeds, Glen Forrest, WA, Australia) and acidi?ed water.

OVA-induced c airway in?ammation and oral Boysenberry treatment Six—week—old mice were randomized into experimental groups (n = 10 per group) and primed intraperitoneally (i.p.) with 100 ug OVA in 200 pl alum adjuvant on day 0. On day +7 mice were challenged intranasally (i.n.) with 100 ug OVA or PBS.

To establish chronic e the in. challenge was repeated weekly (Figs. 1A and 6A). Four days following the last in. OVA challenge mice were euthanized (ketamine/xylazine overdose) and bronchial—alveolar lavage ?uid (BALF), serum, mediastinal lymph nodes and lung tissue were collected.

For the treatment studies mice were fasted overnight before being orally gavaged with 250 pl of Boysenberry solution (see above); dosage at 10 mg/kg of total anthocyanins) or e water on the day of OVA challenge and again 2 days post—OVA challenge (Figs. 1A and 6A).

Clodronate liposome ion of lung macrophages and tissue analysis Clodronate liposomes were prepared as previously described (58). Chronic OVA—induced tissue damage was established over 5 weeks. Mice were then treated intranasally with 100 pl clodronate mes the day prior to each oral gavage with 250 pl of Boysenberry solution (see above; dosage at 10 mg/kg of total anthocyanins) or sterile water (Fig. 6A). Two days following the last oral gavage mice were euthanized (ketamine/xylazine overdose) and BALF, serum, tinal lymph nodes, and lung tissue were ted.

Cells ed from the BALF were stained for key surface markers to identify monocytes/macrophages (CD45+/CD11b+/Cd11c+/MHCIIlow) and eosinophils (CD45+/CDl lb+/siglecF+) by ?ow cytometry as previously described (52). TGFB ELISA and Bio—Plex multiplex cytokine assays were performed on lung tissue supematants following the manufacturer’s instructions. Lung tissue was ?xed in 4% formalin, ned, and stained with hematoxylin and eosin (H&E), Masson’s Trichrome or Alcian blue— periodic chiff S) stains (Dept. of Pathology, Wellington School of Medicine, University of Otago, Wellington, NZ).

Further sections were cut for immunological labelling. Lung sections were incubated with biotin—conjugated MMP—9, then labelled with DAB and counter—stained with hematoxylin. Other tissue sections were incubated with ?uorescently labelled CD68 (31), CD206 (57), and arginase or MMP-9 (44), then counterlabelled with DAPI—containing mounting medium.

All sections were imaged on an Olympus BXS 1 compound microscope and captured by using cellSens us NZ) software, bright light in colour and fluorescence in grayscale. Fluorescence images were processed (cropped, false coloured, and merged) in Pixelmator image software (Vilnius, Lithuania). Fluorescently ed cells were quanti?ed by four independent, d observers. Cells were counted in random ?elds from multiple animals and scored as negative, single positive, or double positive for CD68, CD206, arginase, or MMP—9. Data were expressed as a percentage of total cells counted.

Biochemical and molecular ical tissue is and statistical analysis Biochemical and molecular biological tissue is. Lung tissue was snap frozen and stored at —70°C. Lung collagen was quanti?ed by the hydroxyproline assay as usly described (2). For Western blotting, tissue was homogenized in protein lysis buffer (Tris-HCl, NaCl, 10% t P-40, 10% sodium deoxycholate, 100 nM EDTA, pH 7.4 with se and phosphatase inhibitors). Protein concentration was quanti?ed by a Lowry n assay as per the manufacturer’s instruction.

Samples (30 ug protein) were separated by SDS—PAGE gel electrophoresis under reducing conditions and transferred onto PVDF membrane. Nonspeci?c n binding was blocked with 3% BSA (10 mM PBS with 0.2% Tween 20) and the membranes were probed overnight with primary antibodies speci?c to iNOS (64), arginase (53), MMP— 9 (44), and TIMP—l (55), or B—actin (12) loading control (4°C). Membranes were washed and incubated with horseradish peroxidase—conjugated secondary antibodies and visualized by chemiluminescence on a Carestream Gel Logic Pro 6000 imager. Protein expression was densitometrically quanti?ed and normalized to B—actin with ’s Gel analysis tool (50).

Images were processed and cropped in Pixelmator image software.

Data were analysed by one-tailed Student’s t-test for isons between two groups or one—way ANOVA with Tukey’s post hoc test for comparisons between three or more groups as indicated (Prism, GraphPad, San Diego, CA). P < 0.05 or less was considered statistically signi?cant.

Results - Boysenberry consumption ameliorates OVA-induced chronic airway in?ammation To igate the effect of Boysenberry treatment on established lung remodelling, mice were challenged weekly with intranasal OVA for 5 weeks, then challenged weekly with OVA for an additional 5 weeks alongside weekly oral treatment with Boysenberry (Fig. IA).

As shown in Fig. 1B, lung tissue from OVA-challenged mice exhibited increased cellular infiltrate and loss of lung structure. OVA—induced cellular in?ltrate and lung damage were sed in berry—treated mice (Fig. 1B). Staining of lung tissue for mucus production identi?ed fewer positive cells in OVA—challenged mice ing Boysenberry treatment compared with OVA only—challenged mice (Fig. 1C).

Boysenberry treatment alone had no effect on cellular ation, lung structure, or mucus production.

Results - Boysenberry treatment ses AAMs in the lung of OVA-challenged mice H&E—stained lung tissue sections showed more macrophages in OVA/Boysenberry—treated mice compared with OVA mice (Fig. 2A). blot analysis of lung tissue identi?ed a decrease in iNOS expression in the lung tissue of OVA/Boysenberry—treated mice compared with OVA challenge alone (Fig. 2, B and C). At the same time, an increase was observed in arginase expression in allenged mice (Fig. 2, B and D). that was further enhanced in OVA—challenged mice treated with Boysenberry. Arginase expression was not affected by Boysenberry treatment alone.

AAMs expressing arginase are closely associated with lung remodelling (29). To determine whether the observed lung macrophages were alternatively activated, lung tissue was stained with ?uorescently labelled antibodies for the macrophage marker CD68 and the AAM markers CD20 and arginase.

Lung tissue from OVA/Boysenberry—treated mice showed an increase in CD68+CD206+arginase+ macrophages ed with OVA-challenged mice (Fig. 3).

Quantitative is of the CD68+CD206+arginase+ macrophages further con?rmed a signi?cant increase in the percentage of CD68+CD206+arginase+ macrophages in the lung tissue of OVA/Boysenberry-treated mice compared with OVA-challenged mice (60.00 i 3.54% compared with 23.47 i 5.61%, P < 0.001, one—tailed t’s t—test). Together these data identify an increase in the number of lung macrophages sing an alternatively activated phenotype in OVA—challenged mice receiving Boysenberry treatment. s - Boysenberry treatment decreases OVA-induced collagen deposition and increases MMP-9 expression in the lung Increased AAMs and arginase expression are commonly associated with tissue ?brosis (14, 27, 66); ore the effect of Boysenberry ent was investigated for OVA—induced collagen tion in the lung. Following this the levels of hydroxyproline were measured in the lung tissue as a surrogate marker of collagen deposition (2, 63).

OVA challenge alone resulted in abnormal collagen deposition in the airways with signs of collagen invasion throughout the lung tissue that was abrogated in the lungs of OVA/Boysenberry—treated mice (Fig. 4). In addition, there was a signi?cant drop in the levels of hydroxyproline in the lungs of OVA—challenged mice treated with berry, con?rming that Boysenberry treatment ameliorated OVA—induced collagen deposition (Fig. 4B). Boysenberry restored the OVA—induced decrease in the levels of TGFB in the lung (Fig. 4C) but did not affect the levels of IL-4, IL-5, IL-6, IL-13, or IFNy (data not shown).

] To determine how Boysenberry treatment could be moderating lung ?brosis the expression of MMP-9 was measured in the lung tissue by immunoblot.

It was determined that MMP—9 expression was increased in OVA—challenged mice treated with berry compared with mice challenged with OVA alone (Fig. 4D).

Boysenberry treatment alone did not affect MMP—9 levels in the lung. Tissue inhibitor of matrix metalloproteinases-l (TIMP— 1) is the endogenous inhibitor of MMP—9 (49). The ratio of TIMP—l/MMP—9 sion signi?cantly increased in the lung tissue of chronic OVA— challenged mice and this increase was reversed with Boysenberry treatment (Fig. 4E). These results indicate that Boysenberry—mediated ion in collagen deposition and tissue W0 2019l031972 remodelling was ated with elevated production of ?brolytic MMP—9 and a subsequent rebalance in the ratio of TIMP— l/MMP—9.

Results - Alternatively activated macrophages are a source of MMP-9 protein in the lungs of ysenberry-treated mice ] Lung tissue slides were analysed to identify potential cellular sources of MMP—9. DAB—MMP—9 staining identi?ed a high degree of MMP+ cells exhibiting macrophage logy in ysenberry-treated mice compared with OVA—treated controls (Fig. 5A). Immuno?uorescent staining (Fig. 5B) and quantitative analysis of the lung tissue con?rmed that there were more MMP—9+/CD206+/CD68+ cells present in OVA/Boysenberry-treated lungs than those challenged with OVA alone (39.30 i 6.39 vs. 21.07 i 5.82%; P < 0.05, one—tailed Student’ s t—test). These results identify CD206+/CD68+ AAMs as a source of the increased MMP-9 protein levels.

Results - Depletion of lung macrophages reduces the bene?cial effect of Boysenberry ption on tissue remodelling in established chronic lung in?ammation Next, the inventors looked at the effect of depleting lung macrophages on the bene?cial effects of Boysenberry on chronic lung in?ammation. Macrophages were depleted by administration of clodronate liposomes after ishing chronic lung in?ammation and remodelling, and prior to administration of each Boysenberry treatment (Fig. 6A). It was con?rmed that signi?cant depletion of the lung macrophages had been ed by ?ow try (Fig. 6B) and that this was associated with a cant reduction in hydroxyproline levels in the lung of OVA—challenged mice d with Boysenberry (Fig. 6C). These data indicate that Boysenberry requires macrophages to mediate its bene?cial effects on lung tissue remodelling.

Results - Boysenberry treatment prophylactically prevents OVA-induced airway in?ammation Finally, the effect of Boysenberry treatment was tested using a prophylactic dosing regimen (Fig. 7A). Again, Boysenberry treatment resulted in abrogation of OVA— induced tissue remodelling and signi?cantly reduced cells in the lung lavage ?uid (Fig. 7, B-D), This was associated with lower levels of hydroxyproline in the lung tissue and a decrease in the ratio of TIMP—l/MMP—9 expression (Fig. 7, E—G).

Discussion ] Fruit consumption has been linked with improved lung function in asthma sufferers and the amelioration of acute airway in?ammation in experimental models (l6, 19, 40). However, no findings have been established in these studies in relation to Boysenberry compositions, airway fibrosis, or airway remodelling, and it is well established that other known asthma treatments have failed to address airway remodelling.

] It is demonstrated herein that consumption of a Boysenberry composition moderates c lung remodelling and s in both a therapeutic and a prophylactic g. Furthermore, the data indicate that macrophages play an important role in Boysenberry-mediated protection and that this protection may result from modulation of AAMs and increased MMP—9 activity.

An increase in both se activity (26, 27, 41) and AAMs (4, 9) is often linked with asthma pathogenesis. However, there is evidence that the presence of AAMs does not speci?cally underpin the development of allergic asthma (3 7), which indicates that AAMs may play an alternative role.

As shown herein, the Boysenberry treatment increased the population of arginase—positive AAMs alongside a drop in iNOS expression in the lung tissue of c OVA—challenged mice. Arginase and iNOS play an interactive role in regulating lung in?ammation and repair (34, 35, 66). Where iNOS activity is associated with active in?ammation, arginase expression is indicative of a switch toward in?ammatory resolution (35, 63). Boysenberry consumption therefore appears to rebalance the lung environment, supporting in?ammation resolution by modulating the functional phenotype ofAAMs in the lung.

The presence of AAMs has been associated with decreased Th-2 cytokine production in lung in?ammation (36, 42). However, it was determined that no changes in the levels of Th—2 cytokines IL—4, IL—5, and IL— 13 with Boysenberry consumption following OVA challenge. This indicated that inhibition of proin?ammatory Th—2 cytokine production by AAMs was not contributing to the protective effect of berry ent.

Clinical and animal data indicate that the role of MMP—9 in asthma is multifaceted. Lung macrophages producing MMP—9 have been identi?ed in both experimental and clinical settings (1, 5, 49). Elevated levels of active MMP—9 have been found in plasma and sputum samples from patients with , compared with y controls (3, 23). Increased MMP—9 sion has been correlated with acute asthma exacerbation, including sed lung eosinophilia (6, 23). sely, an increase in MMP—9 levels has been associated with improved lung function in airway disease (25, 65).

MMP—9 overexpression has also been shown to have bene?cial effects in a model of pulmonary ?brosis (5). In contrast, data from MlVIP—9 knockout mice show a partial reduction in the development of asthma ms and reduced remodelling but, in some cases, a lack of MMP—9 has been shown to exacerbate disease (15, 24, 32).

MMP—9 exerts many downstream effects on different immune parameters, including the activation of both pro— and anti—in?ammatory cytokines (15). Nevertheless, the data shown herein indicate that Boysenberry—induced protection of lung tissue from chronic collagen deposition and ?brosis is orchestrated, in part, through the generation of ?brolytic AAM ing MMP—9. Consistent with this, the data show that depletion of macrophages during the resolution phase of in?ammation leads to sed collagen tion with Boysenberry consumption. A similar resolution—promoting role for macrophages has been illustrated in bleomycin—induced ary ?brosis (14).

Matrix metalloproteinases are regulated by their natural inhibitors TIMPs, and high TIMP—l/MMP-9 ratios are proposed to favour collagen deposition and lung remodelling (21, 28, 38). Here a signi?cant increase was observed in the ratio of expression ofTIMP—l/MMP—9 in the lung tissue of chronic OVA—challenged mice and this was reversed by Boysenberry treatment. The drop in the ratio of TIMP-l/MMP-9 in Boysenberry-treated mice therefore represents a potentially bene?cial re—adjustment in the regulation of collagen deposition and breakdown.

TGFB is associated with both normal (20) and pathological (17, 22, 56) tissue repair processes h its role in extracellular matrix production. In this study, it was observed that chronic OVA challenge led to a decrease in TGFB levels that was reversed by Boysenberry consumption. There is evidence that TGFB lowers the TlMP—l/MMP—9 ratio, thus favouring a more ?brolytic environment (18, 54, 56). As such the increase in TGFB levels observed in the lungs of OVA—challenged mice following Boysenberry treatment could serve to limit excessive tissue s and inappropriate remodelling during lung repair by lowering the TIMP—l/MMP—9 ratio. TGFB is also known to stimulate ?broblast contraction for normal tissue repair (20), which could se contribute toward the bene?cial effects of Boysenberry treatment. As such the ion of TGFB has the ial to promote an anti—in?ammatory, pro—resolution environment within the lung via multiple mechanisms.

The results from these studies show that berry stration exhibits a bene?cial effect on chronic lung ?brosis in both a therapeutic and a prophylactic setting. This indicates that Boysenberry consumption may help avoid inappropriate ?brotic remodelling in cases of both poorly controlled and well—controlled asthma. Finally, these ?ndings provide the ?rst ce that berry consumption could be used to support the development of ?brolytic AAMs with the potential to regulate appropriate lung remodelling in asthma and other lung conditions exhibiting ?brotic pathologies.

In summary, these gs have showed that Boysenberry compositions may be used to decrease in?ammation and aberrant collagen deposition in the respiratory tract, and thereby find use in the ent and prevention of various disorders of the airways, including asthma, chronic obstructive pulmonary disease, reactive airway disease, airway fibrosis, and airway remodelling.

Example 2: OVA-induced acute airway in?ammation and oral treatment with berry and apple Materials and methodology Six—week—old mice treated and assessed as in Example 1, noted above with the ing cations. The tested solutions included: Boysenberry 1 and Boysenberry , 0.67% or 6.7%, respectively, apple 1 and apple 10 10, 1.87% and 18.7%, respectively, BerriQiTM Boysenberry with apple 1 and BerriQiTM Boysenberry with apple 10, 1.87% and 6.7%/18.7%, respectively. Commercial Boysenberry juice concentrate was obtained as described in Example 1. Apple juice trate was supplied from Infruit Ltd (Titirangi, Auckland, New d), as manufactured by Profruit (2006) Ltd (Hastings, New Zealand).

For the 18.7% solutions, 18.7 g juice concentrate was diluted in 100 g water.

For the 6.7% solutions, 6.7 g juice concentrate was diluted with 100 g water. For the 1.87% solutions, 1.87 g juice concentrate was diluted in 100 g water. For the 0.67% ons, 0.67 g juice concentrate was diluted in 100 g water. For the combined 6.7%/18.7% solutions, 6.7 g and 18.7 g for the respective juice concentrate was diluted in 100 g water. For the combined 0.67%/1.87% solutions, 0.67 g and 1.87 g for the respective juice concentrate was diluted in 100 g water. In reference to the combined solutions, the Boysenberry to apple percentage was 27% to 73%. Prepared in el, BerriQiTM Boysenberry with apple test solutions were heated for 8 hours at 80°C prior to administration to test for deactivation of anti— in?ammatory activity.

The protocol for testing the combined administration of Boysenberry with apple administration was based on previously published methods for inducing allergic airways in?ammation (52). The protocol for acute in?ammation utilised an 11 day model.

To evaluate the efficacy of Boysenberry, apple, combined Boysenberry and apple administration, and determine the effect of diluting the treatments 10-fold the following treatment groups were tested: 1) Baseline control (no disease); 2) Disease l; 3) Apple (at 18.7%) + disease; 4) Apple 1 (at 1.87%) + disease; 5) Boysenberry 10 (at 6.7%) + disease; 6) Boysenberry 1 (at 0.67%) + disease; 7) Bern'QiTM Boysenberry with apple 10 (at 6.7/18.7%) + e; 8) iTM berry with apple 1 (at 0.67/l.87%) + Disease; 9) d’ BerriQiTM Boysenberry with apple (at 6.7/18.7%) + disease. Each group included animals. Experiments were repeated three times. tical analysis was performed as described in e 1.

As per prior employment of this model, the following dosing/challenge regimen was used. Mice were primed for allergic airways in?ammation 7 days prior to the challenge and samples were collected 4 days post challenge. Mice were administered (treated) with 250 pl of the noted treatments or water (control) on day 0 and day +2 (see . For the Boysenberry 10 test solutions (Boysenberry 10 and BerriQiTM Boysenberry with apple 10), the dosage was administered to r 4.87 mg/kg total anthocyanins. For the Boysenberry 1 test solutions nberry 1 and BerriQiTM Boysenberry with apple 1), the dosage was administered to deliver 0.487 mg/kg total anthocyanins.

Twat? T13 33‘ '} 52‘: :- {1 ‘ "or": Chaleeqr Erdasmt The following samples were collected: 1) lung wash; 2) lung tissue; 3) mediastinal lymph node; 4) blood. These samples were analysed for the following as per the methodologies outlined in (52): 1) total cell counts (and cellular composition): lung wash, lung tissue, and lymph node; 2) cytokine and chemokine production including IL—4, IL—5, IL-6, IL-10, IL-13, CCL11, IFNy: lung wash, lung tissue and blood; 3) IgE, IgGl: blood only. Cytokine levels were tested using BioLegend LEGENDplexTM multi—analyte ?ow assay kit in accordance with the manufacturer’s instructions.

Results Fig. 8 shows the testing schematic. From the results obtained, ovalbumin challenge increased the ance of in?ammatory cells within the lung. This increase was reduced by ular treatments back to naive levels, i.e., without OVA addition.

The results demonstrated sed total immune cell infiltration into the lung in mice challenged intranasally with the allergen, OVA (Fig. 9). Treatment with apple alone did not reduce the cellular infiltration. iTM Boysenberry with apple reduced the cellular in?ltration at both concentrations , and this was reversed when the iTM Boysenberry with apple was heated to 80°C for 8 hours.

The results showed that apple treatment alone had little effect on phil numbers, neutrophils, monocytes, or antigen presenting cells (APCs; Figs. 10, 14—16). In contrast to this, combined BeriiQiTM Boysenberry with apple treatment substantially reduced the number of eosinophils, phils, and monocytes at low dosage levels (Figs. , 14, 15). Heating the BerriQiTM Boysenberry with apple solution prior to treatment reversed this effect (Figs. 10, 14, 15). Combined BerriQiTM Boysenberry with apple ent also reduced the number of APCs (Fig. 16). Heating ed this effect (Fig. 16).

Haematoxylin and eosin staining showed that the ovalbumin challenge resulted in tissue swelling and immune cell infiltration, while combined BerriQiTM Boysenberry with apple treatment appeared to reduce tissue swelling compared to OVA alone (Fig. 13).

AB—PAS staining showed mucous production was variable between the different treatments (Figs. 17—19). None of the treatments made mucous production worse, although there were more mucous-producing cells ed in the lowest fruit concentrations, and none of the treatments appeared to prevent increased mucous production (Figs. 17—19). These results were attributed to the short duration and small number of administrations (two) for the acute experimental model. This sted to the longer experimental testing period and additional administrations noted in Example 1, noted above.

It was proposed that effects on mucus production could be better observed given longer testing times and additional s.

Masson’s trichrome staining showed that acute OVA challenge did not substantially increase fibrosis. There was not an increase in collagen deposition within the lung tissue (Figs. 20—22). The results from this histology indicated that the effect of ents on fibrosis could not be tested in this acute experimental model. This confirmed that the short duration of the acute experimental model did not provide sufficient time to see effects for longer-term ms such as mucus production and collagen deposition.

Lung ?uid obtained from test and control s was assessed for cytokine levels, including granulocyte—macrophage colony—stimulating factor (GM—CSF), lFNy, IFNB, IL-ld, IL-lB, TNFd, IL-12(p40), IL-10, IL-6, IL—27, CXCLl, and CCL2.

Boysenberry treatment alone reduced GM—CSF levels (Fig. 23), while apple treatment and combined BerriQiTM Boysenberry with apple treatment did not reduce GM—CSF levels (Fig. 23). CCLll levels did not appear to be significantly affected (Fig. 24). IFNy, IFNB, IL—ld, IL—lB, TNFd, lL-12(p40), IL—lO, IL—6, IL—27, CXCLl, and CCL2 were not detected in the samples tested (data not . Based on this, it was postulated that the effects of the BerriQiTM Boysenberry with apple treatment were not mediated by CCLll secretion.

Discussion From the results, it was concluded that combined iTM Boysenberry with apple treatment reduced the number of infiltrating phils at both the standard and lower dosage levels. This effect was dependent on ature-sensitive elements of the BerriQiTM Boysenberry with apple composition. Treatment using apple alone had little effect on numbers of phils, tes, or APCs. Combined BerriQiTM Boysenberry with apple ent reduced the number of neutrophils and monocytes at lower dosage levels, and reduced the number of APCs at standard and lower dosage levels. These s were dependent on temperature—sensitive elements. It is clear, then, that combined administration of Boysenberry and apple compositions can be used to reduce numbers of immune cells associated with allergic airways ation.

] The assessment of mucous production was variable between the different ents, leading to the conclusion that additional or alternative measures of mucous suppression such as IL—l3, IL—9 production may provide further clarity. Analysis of lung tissue supernatant to identify changes in proin?ammatory cytokines showed that CCLll levels were unaffected by BerriQiTM Boysenberry with apple treatment, suggesting that the decrease in phils was not mediated via inhibition of CCLl 1. Additional analysis may be used to identify the particular agents that are involved in this process.

Overall, the results of this acute study were positive, showing that BerriQiTM Boysenberry with apple treatment reduced the cellular infiltration at both concentrations tested, whereas apple alone did not at either concentrations tested. berry alone also reduced cellular infiltration which was similar to what was found in the previous research described herein (see, e.g., e 1 and (74)). Notably, the acute model of allergic— s in?ammation used in this study did not sufficiently promote the development of tissue ?brosis, so it could not be determined if BerriQiTM Boysenberry with apple treatment was able to promote the development of anti—fibrosis macrophages and prevent tissue . To determine the presence of such effects, it has been necessary to carry out further studies on chronic allergic—airways ation.

Example 3: OVA-induced chronic airways in?ammation and oral treatment with Boysenberry and apple Overview ] While consumption of certain fruits and vegetables has been studied in relation to bene?cial health effects (87, 88, 91-94), the experiments described herein have been the first to show a substantial beneficial effect for specific Boysenberry compositions.

See Example 1 and 2, and also (74). The key findings from the studies of Example 1 include: 1) Boysenberry consumption significantly reduced allergen—induced airways in?ammation through decreased cell infiltration and increased anti—in?ammatory n production; 2) Boysenberry reduced collagen deposition and assisted in the repair of damaged tissue repair by supporting the pment of fibrolytic macrophages, a type of immune cell; and 3) Boysenberry treatment prophylactically prevents ovalbumin (OVA)-induced airways in?ammation.

The effect of different apple varieties on key cytokines for allergic airways disease has also been igated (96), and cytokine inhibitory ability has been established for apple varieties in a cell culture model of allergic asthma induction. The inhibitory ability was correlated to the presence of the procyanidin polyphenols (96), and it has been shown that these compounds, in isolation, are potent tors of key allergic chemokines CCLll (90) and CCL26 (89). The aim of the current project was to evaluate BerriQiTM Boysenberry with apple treatment, a novel ation of berry and apple juice concentrates and water, in an animal model of chronic OVA—induced allergic airways in?ammation.

Materials and methodology To perform this study, the previously ished mouse model and an oral dosing strategy of chronic duced allergic airways ation was utilised as in e 1 (see also, (52), (97)). Brie?y, mice were primed with OVA/Alum intraperitoneally (i.p.) and then challenged 7 days later with OVA intranasally (in). These in. challenges were performed every week for 10 weeks. After 5 weeks of OVA challenges, the BerriQiTM interventions were begun. For these interventions, mice were fasted for 4 hours before being orally gavaged with water (disease and vehicle control), iTM Boysenberry with apple at 100%, 50% or 25%, at 2 days prior, at 1 hour prior to in. OVA challenge, and at 2 days post OVA challenge.

The intervention groups were: 1) Naive (baseline control); 2) +OVA (disease and water vehicle control); 3) +OVA+BerIiQiTM 100% (disease plus 100% BerriQiTM Boysenberry with apple) containing New Zealand sourced 70°Brix apple juice concentrate and New Zealand sourced 65°Brix Boysenberry juice concentrate; 4) erriQiTM 50% (disease plus 50% BerriQiTM Boysenberry with apple) containing New Zealand sourced 70°Brix apple juice concentrate and New Zealand sourced 65°Brix Boysenberry juice concentrate; and 5) erIiQiTM 25% se plus 25% iTM Boysenberry with apple) containing New Zealand sourced 70°Brix apple juice concentrate and New Zealand sourced 65°Brix Boysenberry juice concentrate. The Boysenberry juice concentrate was ed from Boysenberry NZ (Nelson, New Zealand). The apple juice concentrate was supplied by RD2 International (Auckland, New Zealand), and manufactured by Profruit (Hastings, New Zealand).

The concentration of Boysenberry in BerriQiTM Boysenberry with apple was calculated to deliver 0.73 mg/kg berry anthocyanins per serve for a 70 kg human, this being equivalent to 10 mg/kg in mouse. This dose was ed based on the previous studies (see Example 1 and (52)) that determined that consumption of 10 mg/kg Boysenberry anthocyanins resulted in reduced in?ammation and tissue fibrosis in a mouse model of chronic allergic airways in?ammation. 100% BerriQiTM Boysenberry with apple provided mg/kg total berry anthocyanins for a 25 g mouse; 50% and 25% BerriQiTM Boysenberry with apple provided 5 mg/kg and 2.5 mg/kg total Boysenberry anthocyanins for a 25 g mouse, respectively. Details for the dosages are provided in Example 5, below.

Mice were euthanised 4 days ing in OVA challenge. The following parameters were ed as described in Example 1: l) cellular infiltration: eosinophils, neutrophils, monocytes, and antigen presenting cells (APCs); 2) histological changes: oxylin and eosin (H&E), Alcian blue and periodic acid—Schiff (AB—PAS), Masson’s trichrome staining; and 3) collagen production using the hydroxyproline assay. For the hydroxyproline assay, the commercial colorimetric kit was used (AbCam ab22294l).

The results showed an increased total immune cell infiltration into the lung in mice nged in. with the allergen, OVA (Fig. 25). BerriQiTM Boysenberry with apple treatment significantly reduced the cellular infiltration at the 50% and 25% concentrations tested (Fig. 25). The 100% concentration BerriQiTM berry with apple treatment appeared to have no effect on the OVA—induced increase in immune cells in the lung. This was postulated as showing a therapeutic window for the BerriQiTM Boysenberry with apple treatment.

The immune cells were identified as eosinophils (Fig. 26), antigen ting cells (Fig. 27), and monocytes (Fig. 28). The number of eosinophils showed a significant increase in mice challenged with OVA (Fig. 26). A decrease in eosinophil number was ed by the 50% BerriQiTM Boysenberry and apple treatment (Fig. 26).

Antigen presenting cells showed a significant se with the OVA challenge, and a decrease in APC number was obtained by the 50% and the 25% BerriQiTM Boysenberry and apple treatment (Fig. 27). The number of monocytes was increased in mice challenged with OVA, and a decrease in monocyte number was obtained by the 50% and the 25% BerriQiTM Boysenberry and apple treatment (Fig. 28).

] Haematoxylin and eosin ng showed that the ovalbumin challenge resulted in tissue swelling and confirmed the immune cell infiltration, and that this was decreased by BerriQiTM Boysenberry and apple treatment (Fig. 29). AB—PAS staining showed that the ovalbumin challenge resulted in increased mucous production that was decreased in a dose-dependent manner by BerriQiTM Boysenberry and apple treatment (Fig.

Masson’s trichrome staining showed that repeated OVA challenges resulted in diffuse blue staining of collagen fibres within the airways (Fig. 31). BerriQiTM Boysenberry and apple treatment reduced the appearance of these blue collagen fibres within the lung (Fig. 31).

] Quantification of collagen levels with the hydroxyproline assay showed no increase in collagen with OVA challenges (Fig. 32), which was contrary to us results.

No significant s in collagen levels were seen with 100% or 50% BerriQiTM Boysenberry and apple ent (Fig. 32). However, a significant increase in collagen levels was seen with 25% BerriQiTM Boysenberry and apple treatment (Fig. 32). This is discussed in more detail, below.

Discussion The in. OVA challenge resulted in the ance of sed atory cells within the lung, which was reduced by 50% and 25% BerriQiTM Boysenberry with apple treatment. The 100% BerriQiTM Boysenberry with apple treatment had no effect on the number of infiltrating immune cells compared to the OVA challenged mice, indicating that there may be an l concentration for this treatment.

] Mucous production was reduced by BerriQiTM Boysenberry with apple treatment in a dose-dependent manner, with the 25% BerriQiTM berry with apple treatment mediating the greatest reduction in mucous production.

] Analysis of lung tissue to quantify the changes in total collagen showed that the quantity of collagen following OVA challenges was unchanged, which is inconsistent with the results from the original hydroxyproline assay (see, e.g., Example 1 and (74)). The assay used in this Example employed measurement reagents obtained from an ate source as ed to the original assay.

Notably, Masson’s trichrome staining indicated that OVA challenges resulted in collagen infiltrating into the airways, and BerriQiTM Boysenberry and apple treatment helped to reverse this infiltration. Thus, histological methods established that the on of the collagen around the tissues is altered by OVA, and this can be addressed by BerriQiTM Boysenberry and apple treatment.

] Further research is needed to fully elucidate the meaning of the present hydroxyproline assay results. One possible explanation is the assay differences, as noted. In addition, it is possible that, although there was no change in the quantity of total collagen from OVA challenge, the location of the collagen has been altered, and this is being addressed by the BerriQiTM Boysenberry with apple and blackcurrant treatment. It is also possible that the increase in the amount collagen coupled with the reduction of collagen staining in the airways, as seen with the 25% iTM Boysenberry and apple treatment, is a result of tissue remodelling that occurs when the in?ammation is being resolved. e 4: OVA-induced chronic airways ation and oral treatment with Boysenberry, apple and blackcurrant Materials and methodology To perform this study, the mouse model and an oral dosing strategy of chronic duced allergic airways in?ammation was utilised as in Example 1. See also, (52), (97). Brie?y, mice were primed with OVA/Alum intraperitoneally (i.p) and then challenged 7 days later with OVA intranasally (in), this in challenge every week for 10 weeks. After 5 weeks of OVA challenges, the interventions were begun. For the interventions, mice were fasted for 4 hours before being orally d with water (disease and vehicle control), or BerriQiTM Boysenberry with apple and blackcurrant at 100%, at 2 days prior, at l h prior to in. OVA challenge, and at 2 days post OVA challenge.

The intervention groups were: 1) Naive (baseline control); 2) +OVA (disease and water vehicle control); 3) +OVA+Ber1iQiTM Boysenberry with apple and blackcurrant 100% (disease plus 100% BerriQiTM Boysenberry with apple and blackcurrant) containing New Zealand d 70 Brix apple juice concentrate, New Zealand sourced blackcurrant juice, and New Zealand sourced 65 Brix Boysenberry juice trate. The Boysenberry juice concentrate was obtained from Boysenberry NZ (Nelson, New Zealand).

The apple juice concentrate was supplied by RD2 International (Auckland, New Zealand), and manufactured by Profruit (Hastings, New Zealand). The blackcurrant juice concentrate was obtained from New Zealand Blackcurrant Co-operative Ltd n, New Zealand).

The concentration of Boysenberry in iTM berry with apple and blackcurrant oral composition was calculated to deliver 0.73 mg/kg total anthocyanins per serve for a 70 kg human, this being lent to 10 mg/kg in mouse. This dose was selected based on the previous studies (see Example 1 and (52)) that determined that consumption of mg/kg Boysenberry anthocyanins resulted in reduced ation and tissue fibrosis in a mouse model of chronic allergic airways in?ammation. The 100% BerriQiTM Boysenberry with apple and blackcurrant oral composition provided 10 mg/kg total anthocyanins for a g mouse. Details for the dosages are provided in Example 5, below.

Thus, in this study, it was tested whether a d concentration of Boysenberry anthocyanins could be utilised by supplementing the Boysenberry juice concentrate with an equal amount of blackcurrant juice concentrate to make the total concentration of anthocyanins to 10 mg/kg.

Mice were euthanized 4 days following i.n. OVA nge. The following parameters were measured as described in Example 1: l) cellular infiltration: eosinophils, monocytes and antigen presenting cells (APCs); 2) histological changes: haematoxylin and eosin (H&E), Alcian blue and periodic acid—Schiff (AB—PAS), and Masson’s trichrome staining; and 3) collagen production using the hydroxyproline assay. For the hydroxyproline assay, the commercial colorimetric kit was used (AbCam ab22294l).

The results showed sed total immune cell ration into the lung in mice challenged i.n. with the allergen, OVA (Fig. 33). BerriQiTM Boysenberry with apple and blackcurrant treatment significantly d the cellular infiltration at the concentrations tested (Fig. 33).

The infiltrating cells were made up of phils (Fig. 34), antigen presenting cells (Fig. 35), and monocytes (Fig. 36). The number of eosinophils was increased in mice challenged with OVA, but this increase was not affected by BerriQiTM Boysenberry with apple and blackcurrant treatment (Fig. 34). The number of antigen ting cells was significantly increased by OVA challenge, and the APC number was signi?cantly decreased by BerriQiTM Boysenberry with apple and blackcurrant treatment (Fig. 35). Monocytes trended towards increased s in mice challenged with OVA, and the monocyte numbers were decreased by BerriQiTM Boysenberry with apple and blackcurrant treatment (Fig. 36).

Haematoxylin and eosin staining showed that the ovalbumin challenge resulted in tissue swelling and immune cell ration, which was decreased by iTM Boysenberry with apple and blackcurrant treatment (Fig. 37). AB—PAS ng showed that the ovalbumin challenge resulted in increased mucous production that was unaffected by BerriQiTM Boysenberry with apple and blackcurrant treatment (Fig. 38).

Masson’s trichrome staining showed that repeated OVA challenges resulted in diffuse blue staining of collagen fibres within the s (Fig. 39). BerriQiTM Boysenberry with apple and blackcurrant treatment reduced the appearance of these blue collagen fibres within the lung (Fig. 39).

The hydroxyproline assay showed that there was no increase in total collagen with OVA challenges (Fig. 40), contrary to the original findings. In this assay, the 100% BerriQiTM Boysenberry with apple and blackcurrant treatment produced a significant W0 2019l031972 increase in the quantity of collagen compared to the other groups (Fig. 40). This is sed in more detail, below.

Discussion The in. OVA challenge resulted in the appearance of increased in?ammatory cells within the lung. The number of eosinophils in the lung were unaffected by BerriQiTM Boysenberry with apple and blackcurrant treatment, but the number of APCs was cantly decreased by iTM Boysenberry with apple and blackcurrant treatment. Monocytes trended s an increase by OVA challenge and d towards a decrease with BerriQiTM Boysenberry with apple and blackcurrant treatment. is of lung tissue to quantify the changes in en showed that the quantity of total en following OVA challenges was unchanged, which is inconsistent with the findings from the original assay (see, e. g., Example 1 and (74)). However, Masson’s trichrome staining indicated that OVA challenges resulted in en infiltrating into the airways, which was counteracted by the 100% BerriQiTM Boysenberry with apple and blackcurrant treatment.

The differences in results for the hydroxyproline assays could be due to the differences in these assays, as already noted. In addition, it is possible that although there was no change in the quantity of total collagen from OVA nge, the location of the collagen has been altered, and this is being addressed by the BerriQiTM Boysenberry with apple and blackcurrant treatment. It is also possible that the observations made for this this BerriQiTM berry with apple and blackcurrant treatment, including the increase in the amount collagen, coupled with the reduction of the appearance of collagen staining within the airways, is a result of tissue lling that occurs when in?ammation is undergoing resolution.

Example 5: Dosage calculations for oral treatments The oral treatment dosages for Examples 3 and 4 were calculated in accordance with the following information.

Table 1: Dose calculations for chronic nge mouse studies Four treatment groups: Dose rate in mice (mg/kg Human Equivalent Sample and description total anthocyanins TAC) Dose * (mg/kg) -(Boysenberry + Apple)BerriQiTM concentrate (l) 100% — 0729 W0 31972 2018/050109 See Example 3 BerriQiTM concentrate (2) (Boysenberry + Blackcurrant + Apple) See Exam-le 4 Dosing of BerriQiTM concentrate based on 200 uL for a 25 g mouse being equivalent for a 70 kg human (in total anthocyanins) lSee Example 1 and (74) *HED 2 animal dose in mg/kg x (animal weight in kg / human weight in kg)0'33 For example: 2 10 mg/kg mouse dose x (0.025 kg mouse / 70 kg human)033 2 0.73 mg/kg total anthocyanins for 70 kg human Table 2: Starting concentrates and their composition 27% Boysenberry juice trate 65 Brix (Boysenberries NZ Ltd); Sample BerriQiTM Concentrate (l) 72.88% Apple juice concentrate (clear) (l) nberry + Apple) 70 Brix (RD2 International); 0.12% Potassium sorbate 13.5% Boysenberry juice concentrate 65 Brix (Boysenbeiries NZ Ltd); BerriQiTM Concentrate (2) 13.5% Blackcurrant juice concentrate Sample (Boysenberry + Blackcurrant 65 Brix (NZ Blackcurrant Co—op); (2) + Apple) 72.88% Apple juice concentrate (clear) 70 Brix (RD2 ational); 0.12% Potassium sorbate Table 3: Calculations for anthocyanin concentrations Total anthocyanins Sample Description Calculation (as cyanidin- 3— lucoside) Specific gravity 2 1.34 g/ml BerriQiTM 145 mg/100 1 g juice concentrate = 0.746 ml Sample Concentrate (l) g juice 1 ml juice concentrate = 1.34 g (l) (Boysenberry + concentrate Therefore: Apple) 1,450 pg/g (1.45 mg/g) or Specific gravity 2 1.34 g/ml BerriQiTM 1 g juice trate = 0.746 ml Concentrate (2) 236 mg/100 1 ml juice concentrate = 1.34 g (Boysenberry + g juice Therefore: Blackcurrant + concentrate 2,360 pg/g (2.36 mg/g) or Apple) 3,162 pg/ml juice concentrate Table 4: Dosage calculations based on anthocyanin concentrations Volume of BerriQiTM Dose rate in mice (mg/kg Sample Description Concentrate requ1red 1n. . total anthocyanins TAC) 200 L dose ( 9L) Concentrate (l) BerriQiTM Concentrate (2) (Boysenberry + 100% 10 79 urrant + A "16) Detailed calculations for dosages in Table l for BerriQiTM concentrate (l) at 100% as utilised in Example 3 (Boysenberry with apple): ation to determine mouse dosage Desire a dose of 10 mg anthocyanin/kg for a 25 g mouse (Example 1 and (74)) - 100% A 10 mg anthocyanin/kg dose for a 25 g mouse would require 0.25 mg of anthocyanin Therefore: 0.172 g BerriQiTM conc (1) required to deliver a 0.25 mg dose of anthocyanin = 0.129 mL BerriQiTM cone (1) ed to deliver a 0.25 mg dose anthocyanin = 128.667 uL BerriQiTM conc (1) required to deliver a 0.25 mg dose anthocyanin Sp Gravity 1.34 g/ml Preparation of oral composition for mouse trial Take 128.667 uL BerriQiTM conc (l) and make it up to 200 uL with H20 There is now 0.25 mg of anthocyanin in 200 uL mouse dose, 10 mg/kg for a 25 g mouse Detailed calculations for dosages in Table l for BerriQiTM concentrate (2) at 100% as utilised in Example 4 (Boysenberry with apple and urrant): Calculation to ine mouse dose Desire a dose of 10 mg anthocyanin/kg for a 25 g mouse (Example 1 and (74)) - 100% A 10 mg anthocyanin/kg dose for a 25 g mouse would require 0.25 mg of anthocyanin Therefore: 0.106 g BerriQiTM cone (2) required to deliver a 0.25 mg dose of yanin = 0.079 mL BerriQiTM conc (2) required to deliver a 0.25 mg dose of anthocyanin = 79.054 uL BerriQiTM cone (2) required to deliver a 0.25 mg dose of anthocyanin Sp Gravity 1.340 g/ml Preparation of oral composition for mouse trial Take 79.054 uL BerriQiTM cone (2) and make it up to 200 uL with H20 There is now 0.25 mg of anthocyanin in 200 uL mouse dose, 10 mg/kg for a 25 g mouse Alternative dosage calculation details for BerriQiTM concentrate (1) (Boysenberry + Apple): Mouse weight 25 g Dose (total anthocyanins) 10 mg/kg (or 0.1 mg/10 g) Therefore: dose 2 0.25 mg per mouse BerriQiTM (Boysenberry + Apple) total yanins 145 mg/100 g (or 1.45 mg/g) Weight product needed 0.25 mg/1.45 mg = 0.172 g BerriQiTM Able to do weight of t in water e specific gravity is known Specific Gravity 1.34 kg/L (or 1.34 g/mL) Volume needed 0.172 g /1.34 g 2 0,128.66 mL This equates to 129 uL per mouse plus 71 uL water (200 pl minus 129 pl) Example 6: Compositional analysis of treatment formulations BerriQiTM liquid formulations were ted to chemical analysis to determine anthocyanin and phenolic ition. The formulations tested included: Sample 1, BerriQiTM with apple concentrate 1 (BB + AP); Sample 2, BerriQiTM with apple plus blackcurrant concentrate 2 (BB + BC + AP). See Example 5. d aliquots of the samples were diluted 5-fold with 10% formic aCidaq for is by ultra high pressure liquid chromatography (UHPLC). For analysis of other phenolics by liquid chromatography mass spectrometry (LC-MS), samples were diluted 10—fold with 0.1% formic acidaq. Sample density was ined and samples taken for dry matter calculations.

UHPLC analysis of anthocyanins: Anthocyanin concentrations were measured using a Dionex UltiMate 3000 Series UHPLC (ThermoFisher Scienti?c, San Jose, CA, USA) with PDA (photodiode array) detection at 520 nm. Compound separation was achieved using a i 4p hydro-RP 80A column, 4.6 x 250 mm (Phenomenex, Torrance, CA, USA), maintained at 40°C. Solvents were (A) 5:5:90 itrilezformic acidzwater v/v/v and (B) 5:95 v/v formic acidzacetonitrile and the flow rate was 1 mL/min. The initial mobile phase, 100% A was held for 1 min, then ramped linearly to 84% A in 16 min, followed by a column ?ush at 5% A before resetting to the al conditions. The sample injection volume was 0.5 uL. Detected anthocyanins were quantified by UHPLC using a pure standard of cyanidin 3—O—glucoside and all the results for individual and total anthocyanins are sed as cyanidin 3-O—glucoside equivalents.

LC—MS confirmation: LC—MS employed an LTQ linear ion trap mass ometer fitted with an ESI interface (ThermoFisher Scientific, San Jose, CA, USA) coupled to an Ultimate 3000 UHPLC and PDA or (Dionex, Sunnyvale, CA, USA).

Anthocyanin confirmation: Anthocyanin compound separation was achieved using a Poroshell 120 SB-C18 column, 2.7 u 2.1 x 150 mm (Agilent, Torrance, CA, USA), maintained at 70°C. Solvents were (A) 5:3:92 acetonitrilezformic acidzwater v/v/v and (B) acetonitrile + 0.1% formic acid, and the ?ow rate was 200 uL/min. The initial mobile phase, 100% A was held for 2 min before being ramped linearly to 88% A at 14 min, % A at 15 min and held for 4 min before resetting to the original conditions. The sample injection volume was 10 uL. MS data were acquired in the positive mode using a data— dependent LC—MS3 . This method isolates and fragments the most intense parent ion to give MS2 data (daughter ions), then isolates and fragments the most intense daughter ion (Ms3 data).

Other phenolics: Other phenolic nd separation was achieved using a il GOLD aQ 1.9u C18 175A (Thermo Scientific, Waltham, Massachusetts USA), 150 x 2.1 mm column ined at 45°C. Solvents were (A) water + 0.1% formic acid and (B) acetonitrile + 0.1% formic acid, and the ?ow rate was 200 ul/min. The initial mobile phase, 95% A/ 5% B, was ramped linearly to 85% A at 10 min, held for 3.75 min, then ramped linearly to 75% A at 18 min, 67.2% A at 25 min, 50% A at 28 min, 3% A at 29 min and held for 4 min before resetting to the original conditions. The sample injection volume was 4 uL. UV—vis detection was by absorbance at 200—600 nm. MS data were acquired in both negative and positive modes with ESI ionisation using three data—dependent LC—MS3 methods, the first using mass range [m/z 0] optimised for detection of low lar weight phenolic compounds, the second using mass range [m/z 150—2000]optimised for detection of ellagitannins and the third using mass range [m/z 150—4000] optimised for W0 2019l031972 detection of higher molecular weight soluble tannins. MS data were also acquired in both negative and positive modes with APCI ionisation.

Phenolic acids, gallic acid, protocatechuric acid, chlorogenic acid (3— caffeoquuinic acid), caffeic acid were quantified by LC—MS using pure standards of these compounds. Detected derivatives of coumaric acid were quantified by LC-MS using p- coumaric acid, and are expressed as p-coumaric acid equivalents. The ?avan—3—ols, epi— catechin and catechin, and procyanidin B2, were quantified by LC—MS using pure standards of these compounds. Unknowns m/z 563 and m/z 579 were quantified by LC—MS using epi— catechin, and expressed as techin equivalents. ysable tannins were quantified by LC—MS using a standard of Sanguiin H6 that had been isolated previously (>98% purity by LC—MS). Other detected tannins and unknown m/z 639 were quantified by LC—MS as Sanguiin H6 equivalents. Ellagic acid was quantified by LC-MS using a standard of ellagic acid. Detected ?avonol glycosides were quantified by LC—MS using a pure rd of quercetin 3-O—glucoside and are expressed as quercetin 3-O—glucoside equivalents. The non— glycosylated flavanols, tin and myricetin, and the chalcones, phloretin and phloretin— 2—O—glucoside were quantified by LC—MS using pure standards of these compounds. The preservative sorbic acid was quantified by LC—MS using a pure standard of this nd.

The s for the analysis are shown as follows.

Table 5. Density and dry matter data for BerriQiTM samples BerriQiTM concentrate (1) BB + AP 1 346 70.24 BerriQiTM trate (2) BB + BC + AP 1.351 71.37 Table 6. Quantitation summary for detected phenolics in BerriQiTM samples 1 and 2 expressed in pg/mL and pg/g dry weight (DW) yanins nidin 3—O-glucoside Cyanidin 3—O—sophoroside Delphinidin 3—O—rutinoside Cyanidin 3—O—glucoside 597 63 1 248 258 Cyanidin 3-O-sambubioside 2 -__6 Phenolic acids —---m —____ 4—p—C0umaroquuinic acid 20 2 1 -p-Coumaroquuinic acid II3 ols and procyanidins Procyanidin B2 Catechin Epi—catechin H \] p—A OO y—A p-A p—A [\) Hydrolysable tannins Sanguiin H10 isomer 1 p—A W p—A Q) Sangui sorbic acid dilactone ?ll H £11 [\D Galloyl-SH6 \] O 00 4; l\)4; L11 Sanguiin H10 isomer 2 \1 O 00 4;4; 4: L11 Lambertian C (minus ellagic acid) p—k A Lambertian C N\l l\) \D ,_i O Sanguiin H6 c acid Flavanols Quercetin 3—O—rutinoside [\J O\4; Quercetin 3—O—galactoside >—‘ 00 >—‘ \D O’\ Quercetin 3—O—glucuronide [\J .h £11 Quercetin 3—O—g1ucoside H DJ H .P O'\ Quercetin 3—O—pentoside 1 ,_. O [\J y—A tin 3—O—pentoside 2 y—A p—A y—t [\J 4; L11 —Quercetin 3—O—pentoside 3 Quercetin an1noside DJ Quercetin U) U) £11 00 MyricetinO-rutinoside Q 5Q ,_. oo 4; Myricetin—3—O—glucoside Q 5Q b) b) £11 Myricetin—malonylglucoside Q BQ Myricetin DQ BQ Aureusidin—glucoside 5Q. :3Q.

Kaempferol—3—O—rutinoside 5Q. BQ.

Kaempferol—3—O-glucoside .5Q BQ Chalcones Phloretin 2—O—Xylo—gluco side >—‘ 0 >—‘ O \I O'\ Hi>—‘ Phloretin 2—O—glucoside 0U1 Unknowns Unknown m/Z 563# ,_. O ,_. ,_. 4k 4k Unknown m/z 639 U1 U1 4; U] 0\ Unknown m/z 579 3 3 U.) Totals 4043 4275 3645 3784 nd 2 not detected # = detected as [M+formate]—adduct Persons of ordinary skill can utilise the disclosures and teachings herein to produce other embodiments and ions without undue experimentation. All such embodiments and ions are considered to be part of this invention. ingly, one of ordinary skill in the art will readily appreciate from the disclosure that later cations, substitutions, and/or variations performing substantially the same function or ing substantially the same result as embodiments described herein may be utilised according to such related embodiments of the present invention. Thus, the invention is intended to encompass, within its scope, the cations, substitutions, and variations to ses, manufactures, compositions of matter, compounds, means, methods, and/or steps disclosed herein.

The description herein may contain subject matter that falls outside of the scope of the claimed invention. This subject matter is included to aid understanding of the invention.

In this specification, where reference has been made to external sources of information, including patent specifications and other documents, this is generally for the purpose of providing a context for discussing the features of the present invention. Unless stated otherwise, reference to such sources of information is not to be construed, in any jurisdiction, as an admission that such sources of information are prior art or form part of the common general knowledge in the art.

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Claims (2)

WHAT IS D IS: 1. Use of a composition comprising a Boysenberry and apple concentrate, or a Boysenberry, apple, and blackcurrant concentrate, for preparing a medicament for: (i) ng or preventing acute or chronic allergic airway inflammation in a (ii) treating or preventing immune cell infiltration in lung tissue in a subject; or (iii) ng or preventing accumulation of collagen fibres in lung tissue in a subject, wherein the Boysenberry and apple concentrate comprises at least 27% Boysenberry concentrate, wherein the Boysenberry and apple concentrate comprises at least 91 µg/g chalcones t due to apple concentrate, wherein the Boysenberry and apple concentrate comprises at least 145 mg/100 g anthocyanin content, and wherein anthocyanins in the Boysenberry and apple concentrate account for at least 40% of the total polyphenol content of the Boysenberry and apple concentrate; and wherein the Boysenberry, apple, and urrant concentrate comprises at least 13.5% Boysenberry concentrate and at least 10% blackcurrant concentrate, wherein the Boysenberry, apple, and urrant concentrate comprises at least 91 µg/g chalcones content due to apple concentrate, wherein the Boysenberry, apple, and urrant concentrate comprises at least 236 mg/100 g anthocyanin t, and wherein anthocyanins in the Boysenberry, apple, and blackcurrant concentrate account for at least 65% of the total polyphenol content of the Boysenberry, apple, and blackcurrant concentrate. 2. The use composition of claim 1, wherein the composition comprises one or more of: Boysenberry juice concentrate, berry , apple juice concentrate, apple powder, blackcurrant juice concentrate, blackcurrant powder. 3. The use of claim 1 or claim 2, wherein the composition is formulated for enteral or oral administration. 4. The use of any one of claims 1 to 3, wherein the composition is formulated as a syrup, drops, gel, jelly, tablet, or capsule. 5. The use of any one of claims 1 to 4, wherein the composition includes added polyphenols. 6. The use of any one of claims 1 to 5, wherein the composition is formulated for administration with a further respiratory aid. 7. The use of claim 6, wherein the further respiratory aid is a medication, a herbal remedy, or an essential oil. 8. The use of claim 6 or claim 7, wherein the further atory aid has one or more of: anti-inflammatory, anti-spasmodic, odilation, or muscle relaxation effects. 9. The use of any one of claims 1 to 8, wherein the immune cell is selected from the group ting of monocytes, eosinophils, any combination of thereof. 10. The use of any one of claims 1 to 9, n the subject has a chronic respiratory disorder. 11. The use of any one of claims 1 to 10, wherein the subject has asthma, chronic obstructive pulmonary disease, or reactive airway disease. 12. The use of any one of claims 1 to 11, wherein the subject has airway fibrosis or airway remodelling. 13. The use of any one of claims 1 to 12, n the composition is formulated for administration once per day. 14. The use ition of any one of claims 1 to 13, wherein the ition is formulated as a dosage unit comprising about 5 to about 500 mg total anthocyanins. 15. The use of any one of claims 1 to 13, wherein the composition is formulated for administration at a dosage of about 0.1 mg/kg to about 5 mg/kg total anthocyanins/subject’s body weight. 16. The use of any one of claims 1 to 13, wherein the composition is formulated for administration at a dosage of about 10 mg to about 1000 mg total anthocyanins per day. 17. The use of any one of claims 1 to 13, wherein the composition is formulated for administration at a dosage of 10 mg to 200 mg total Boysenberry anthocyanins per day. 18. The use of any one of claims 1 to 13, wherein the composition is formulated for stration at a dosage of 10 mg to 200 mg total Boysenberry and blackcurrant anthocyanins per day. 19. The use of any one of claims 1 to 18, wherein the composition includes less than 1% w/v preservative. ]?I(}; 1 A " Ln OVA shall Day enge 0 +7 +14 +21 +28 +35 +42 #49 +56 +63 +70 +74 +5a +35 +72 nt Ora! Boysenbeny treatment OVA i! BaysB BoyIB alone an" # ”g M mm m g WW lmhuphlt BALF E in ‘g 519% [- Panama: m - + 1* Hana - - + mm;mm:-- 135 kn- ?—acin) (I! to was [numbed Ei??ii OD their: aa h{mmaizaed =§§§ ovnm++- H38: + .4. Name OVA B n OVA - + + - 3% 2 MW‘B :ggskléga t ‘ gt . i? “MP-«Ej-EJ-m OVA - + + - —?km TGF?cmnta?an g 5: 3 trawl-1 E3 a: TWPJMIWSMG iiiifS + OVA - + + H mm mum Merge A . m OVA challenge Ln Group m 0 +7 +14 +21 +28 +35 +43 +45 +4? +50 +52 +55 tVNAlum +1; +4|a +51I +53| End point Prime i.p 0m} Baysenberry treatment warming: a a wmwm Wemantam1 Peres-m A in OVAma?enge a +7 +14 +21 +28 +35 +39 +9 +16 +23 +30 +37 OVA! Oral beny treatment End point Prime 6.9, , g m- .L * é w: 3‘ £” '3 u a m w 5’“ g “.3 *3 g; m PW: 5'; + m 0V1. 4» ¢ _ _ _ BwsB m w 4 - - 4 Emma ratio P—Q ‘ TWP-1M 9

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