DESC:Field of the Invention
The present invention relates to a synergistic composition comprising chrysin and xanthohumol. The composition is delivered through various routes, preferably through an aerosol route and product formats which include, combustible, non-combustible and consumable product formats containing tobacco / nicotine to offer multiple cellular protection for users.
Background and the Prior Art
In a biological system, cells are constantly exposed to various exo- and endogenous stressors, which are stimulated by various physical, chemical, and biological sources triggering a cascade of events to maintain cellular homeostasis. In order to establish cellular homeostasis, the cells are required to trigger stress response pathways including heat shock response, unfolded protein response, autophagy and inflammatory response to rapidly preserve redox balance, recycle the antioxidants and clear the abnormal proteins. For instance, when lung cells are exposed to smoke produced from various sources including but not limited to cigarette smoke, fumes, exhaust gases, gases produced during cooking using wood, charcoal etc, which may contain microbes, particulate matter, allergens and pollutants might induce cellular stress, misfolding of proteins and/or damaged proteins.
Such an assault is likely to aggravate the accumulation of misfolded or damaged proteins inside the Endoplasmic Reticulum (ER), a condition known as ER stress or proteostasis imbalance.
Normally, in order to maintain the 'cellular (or) proteostasis balance' (also known as cellular homeostasis), autophagy is one of the mechanisms, naturally happens at the intracellular level as a response to the stimuli arising from ER. Under such stressed conditions; the cells may also undergo an aberrant autophagy which may eventually lead to cell death including normal cells instead of attacking only the cells involved in ER.
Available literature evidences suggest that in order to establish cellular homeostasis, the cells are required to activate pathways including unfolded protein response (UPR), autophagy, antioxidant recycling and DNA repair mechanism in order to remove abnormal proteins and maintain consistent overall cellular redox balance (Rattan SI 2007 Ann N Y Acad Sci, 1100:424-30; Rattan SI 2013 Dose Response, 11:99-108). Further, yet another pathway such as Notch1 signaling pathway which is highly conserved across system has a very important role in regulating cell proliferation, cell death and acquisition of specific cell fate. (Kopan R, Ilagan MX. Cell. 2009; 137:216–233).
Any defect in the ability of the cells to activate any one of the above said specific pathways would have a significant impact on the health state of the biological system.
Various systems and methods for delivering pharmaceutically or therapeutically active agents have been reported in the art in order to provide health benefits such as cellular protection, modulating autophagy, inhibition of autophagosome maturation, enhanced apoptosis etc.
However, to attain cellular homeostasis and avert proteostasis imbalance through a pulmonary delivery route is ever challenging. Further, identifying the right bioactives without any adverse effects to be delivered via pulmonary route is yet another challenge. Due to the high safety of the phytochemicals at the cellular level a number of applications in the area of health care are emerging (Nakamura Y et al Mol Cancer. 2009 Nov 12; 8:100). In order to achieve desirable cellular benefits, normally, high doses may be required when a "single" active is used. In order to offer enhanced cellular protection, effective combinations of two or more bioactive molecules each one at relatively low but effective concentrations may hold the key.
Pulmonary route broadly covers, the aerosol format which can be both combustible and non-combustible smoking articles based on ingredients such as tobacco, nicotine or the like. More particularly, attaining cellular homeostasis more specifically through various aerosol routes such as thermal, non-thermal, pyrolysis to offer cellular benefits such as modulating aberrant autophagy and/or to enhance apoptosis through Notch 1signaling of ER stressed cells.
There remains a need for a synergistic composition comprising a bioactive through various routes, wherein the product format includes, combustible, non-combustible product formats containing tobacco, nicotine or the like to offer cellular protection.
Object of the present invention
An object of the present invention is to overcome the drawbacks of the prior art.
Another object of the present invention is to provide a synergistic composition comprising bioactives such as chrysin and xanthohumol.
Yet another object of the present invention is to provide a synergistic composition comprising bioactives such as chrysin and xanthohumol to deliver biological effects at the cellular and molecular level.
Yet another object of the present invention is to provide a process of incorporating the bioactives onto a combustible and/or non-combustible product formats through a pulmonary route, which contains tobacco and/or nicotine or the like.
The mechanism of delivery is primarily by the respiratory route.
Summary of the present invention

An aspect of the present invention provides a composition comprising
a. Chrysin and
b. Xanthohumol and
c. organoleptic agents
Wherein ratio of chrysin and xanthohumol ranges from 5:4 to 4:3
Wherein the concentration of chrysin and xanthohumol ranges from 0.1 to 5 wt%.
Brief Description of Accompanying Drawings

Figure 1 illustrates the inhibitory effects of chrysin and xanthohumol on cell viability in A549 cells.

Figure 2: Experiment to demonstrate that the actives of the present invention at a critical concentration to offer cellular and/or molecular benefits.
Figure 3: Criticality data by scratch assay
Figure 4: Chrysin and Xanthohumol ratio on cell viability in A549 cells
Figure 5: Assessment of apoptosis by Annexin V-FITC
Figure 6: Post smoking effect of chrysin and xanthohumol on cell viability in A549 cells
Description of the Invention

The present invention relates to a composition comprising bio-actives at a critical concentration Chrysin + Xanthohumol to modulate aberrant autophagy and/or to enhance apoptosis through Notch 1 signaling of ER stressed cells. The examples of actives include chrysin and xanthohumol which are phytoextracts.
Chrysin (5,7-dihydroxyflavone) is present at high levels in honey and propolis. It has been reported to be cytotoxic with EC50 value of 100 µM in wide range of cell lines such as breast (MCF-7, MDA-MB-231 cells), colon (Lovo, DLD-1) and prostate cancer cells (Androutsopoulos VP et al., Bioorg Med Chem 2011, 19:2842–2849; Gan YH et al., Oral Oncol 2009, 45:e150–e154). Moreover it has been implicated as Notch 1 signaling activator in anaplastic Thyroid Carcinoma (ATC) on a routine high put screening vide publication (Xiao- Min Yu et al., Development 136, 2297-2307;2009)
Xanthohumol (XN) is the principle prenylated chalcone of the hop plant an ingredient of beer. XN exerts a broad spectrum of chemo preventive actions towards malignant tumors in vitro and in vivo (Gerhauser, C et al., 2002, Mol. Cancer Ther. 1, 959–969, Gerhauser, C. 2005 Eur. J. Cancer 41, 1941–1954). Recently, XN has been reported to activate the pro-apoptotic arm of the unfolded protein response in chronic lymphocytic leukemia (Lust, S et al., 2009. Anticancer Res. 29, 3797–3805).
Combustible, non-combustible product formats containing tobacco, nicotine or the like to offer cellular protection.
Cigarettes used in the present invention: Kentucky reference cigarette 3R4F is a commercially available reference cigarette available, for example, from the College of Agriculture, Tobacco Research & Development center at the University of Kentucky. Lexington, KY.
Cigarette smoke condensate (CSC) refers to the sticky particles comprised of thousands of chemicals created by burning tobacco. It is the particulate component obtained from mainstream or sidestream tobacco smoke that is often used in scientific research to test its biological effects. It does not form part of the composition. It is meant for testing or analysis purposes.
To prepare CSC, cigarette smoke is generated from 3R4F reference cigarettes, using a smoking machine and the particulate matter is collected using Cambridge filter pad. The particulate matter trapped on the pad is eluted using dimethyl sulfoxide (DMSO) according to International Organization for Standardization (ISO) 4387:2000 and (Cooperation Centre for Scientific Research Relative to Tobacco) CORESTA Method Number 23. Once collected, CSC are diluted in an appropriate diluent (e.g. culture medium) and applied to the cell surface.
An embodiment of the present invention provides a composition comprising the bio-actives along with the other components in order to achieve desired results.

Combustible articles such as cigarette comprise tobacco of about 700-750mg.

Chrysin in an amount ranging from 0.1 to 5% by wt is added to the composition. Xanthohumol (XN) in an amount ranging from 0.1 to 5% by wt is added to the composition. The concentration of CH and XN present in composition is dependent on the total amount of tobacco present in the composition. Here the concentration of CH and XN must be within the range of from about 1 part CH : 5 parts tobacco to 3 parts CH : 5 parts tobacco and from about 1 part XN : 5 parts tobacco to 3 parts XN : 5 parts tobacco.
The organoleptic agents and/or acceptable excipients are selected from Flavors / flavorants include menthol, citrus flavors and the like, taste enhancers, natural extract such as clove, elaichi, etc.
These organoleptic agents are present in an amount ranging from 0.1 to 2 wt% wt.
The product formats includes, cigarettes, heat not burn formats, thermal aerosol, electronic or electrically heated cigarettes, inhalers, sprays, dermal patches and the like. The composition of the present invention is delivered through various routes including but not limited to pulmonary, nasal, dermal and buccal routes to offer cellular benefits to the consumers.
Protypes/ illustrative examples of the product formats by which the composition of the current invention may be provided

· Sprayed on the cigarette wrapper finished product
· Added to tobacco and converted to shreds in the form of reconstituted tobacco
· Sprayed onto the foil
· Filled in a capsule and embedded inside the cigarette filter
· Thread coated with synergistic composition and placed inside the cigarette filter
· Coaxial cigarette coated with synergistic composition
· Synergistic composition sprayed onto the tobacco flakes and rolled into regular cigarettes
· Synergistic composition sprayed onto the tobacco leaves / stem before it cut into flakes
· Cavity filters, filled with synergistic composition
· Plug wrap coated with synergistic composition
· e-juice containing nicotine + synergistic composition and heated through a battery
· Tobacco sheet and heated to charcoal
· Tobacco sheet and heated with a battery
· Spray using pressurized gas
· Spray solution using a liquid and generation of an aerosol

Yet another embodiment of the present invention provides a system to modulate aberrant autophagy. “System” refers to any product format not necessarily confined to tobacco containing smoking articles such as cigarettes, electrically or electronically heated cigarettes, heat not burn formats and so forth to deliver the actives of the present invention to modulate aberrant autophagy.
“System” also refers to “cigarette filter” wherein said active shall be made into capsule or thread or filter itself wherein it holds the actives and made it suitable for pulmonary delivery.
The present invention is now illustrated by means of non limiting examples:
Example 1:

Various concentrations of chrysin and xanthohumol individually, co-incubated with cigarette smoke condensate (CSC) and exposed to A549 cells did not show any significant decrease in cell viability whereas the synergistic effect emerged when CSC co-incubated with chrysin and xanthohumol at a specific concentration, e.g. 20µM and 15µM respectively (fig 1).
Equal number of A549 cells (10,000) per well was seeded in a 96 wells plate in DMEM (medium to grow A549 cells) containing 10% Fetal Calf Serum (FCS) and penicillin (100 µg/ml), streptomycin (100 µg/ml) and incubated at 37°C in humidified chamber under 95% carbon dioxide and 5% oxygen for 18 h. Later cells were exposed to 50 µg/ml of cigarette smoke condensate (CSC) and incubated with various concentrations of chrysin (CH) and xanthohumol (XN), individually or in combination as mention in the figure, in DMEM containing 10% FCS and co-incubated further for 24 hours. The cells viability was assayed by WST dye uptake measured at 450nm and the results are expressed as % of viable cells as compared to untreated cells. As presented in the bar diagrams, combination of 20 µM chrysin and 15 µM Xanthohumol in A549 cells showed the highest decrease in residual cell viability. In the current experiment, Total Particulate Matter (TPM) was generated from reference cigarettes smoked under ISO conditions. The TPM was re-suspended in DMSO to obtain CSC. Solvent control – DMSO. T-test * < 0.05; ** < 0.005

Result and observation: Modified Tobacco Products (MTP) of the present invention is suitable to offer cellular benefits to the consumers such as to offer cellular and/or molecular benefits, through pulmonary route of delivery system.
Example 2:
The proposed invention contemplates a mechanism to deliver the said actives, predominantly through aerosol route to the consumers, which poses a formidable challenge. The very objective of the immediate invention is to deliver the actives to offer cellular and/or molecular level, pulmonary route of delivery is the most efficient method. Contextually, "efficiency" refers to the rate at which the 'active' reaches the target site and resort the anomaly.

Whereas, it is possible that the same actives might be delivered through other modes such as dermal / sub-cutaneous, buccal however, there is a fair chance that these actives might get metabolized easily before it reaches out to rescue the victimized cells.

Example 3: Comparative data substantiating criticality of composition
Figure 2 is an outcome of the experiment to demonstrate that the actives of the present invention at a critical concentration attempts to offer cellular and/or molecular benefits.
In one of the embodiment shown below demonstrates the modulation of autophagy aberration by the actives of the present invention.
A) Control. A549 cell nucleus stained with Propidium iodide-PI (Red). Also A549 stained against ubiquitin (marker of misfolded protein, naturally present along with misfolded protein) which is green colored.
B) Untreated cells: A549 cells exposed with smoke condensate (e.g. cigarette smoke) to form misfolded proteins, identified by the presence of ubiquitin. Further, the cells were stained against ubiquitin (green) and PI (red) to demonstrate that there is misfolded protein formation. The more green color suggests that there is more ubiquitin which signifies that the misfolded proteins are present in abundance.
C) Treated cells: A549 cells when exposed with smoke condensate and treated with Chrysin (20µM) and Xanthohumol (15µM) and stained against ubiquitin and PI that modulates aberrant autophagy. This clearly demonstrates that there is a substantial decrease in the formation of misfolded proteins when compared with untreated cells (B) v. treated cells (C). The less green or no green suggests that there is none or reduced misfolded proteins and hence there is none or less ubiquitin, because of the synergistic action of the given actives.
Example 4: Criticality data of composition (scratch assay)
Scratch assay is carried out in vitro using cell lines to estimate the migration and proliferation rates of cells after and before exposed to the specific condition. In particular this method mimics metastasis suppression or metastatic colonization of the damaged or diseased cell in vivo. The assay generally involves first growing a confluent cell monolayer. A small area is then disrupted and a group of cell destroyed or displaced by scratching a line through the layer. The open gap is then inspected microscopically over time as the cells move in and fill the damaged area. This “migration” can take from several hours to over a day depending on many factors that alter the motility and/or growth of the cell.
Figure 3 illustrates the result of scratch assay
Result and observation: the experiment demonstrates the heavy migration of untreated A549 cells involving aberrant autophagy (labels 1, 2, 3, 4). Even the results shows the negative effect of A549 cells when treated with chrysin and xanthohumol individually (labels 5 and 6). However, when chrysin and xanthohumol used together, at 20µM and 15µM, respectively (label 7), it clearly shows that migration of A549 cells is arrested substantially.
Example 5: Chrysin and Xanthohumol ratio on cell viability in A549 cells
Equal number of A549 cells (10,000) per well was seeded in a 96 wells plate in DMEM (medium to grow A549 cells) containing 10% Fetal Calf Serum (FCS) and penicillin (100 µg/ml), streptomycin (100 µg/ml) and incubated at 37°C in humidified chamber under 5% carbon dioxide and 95% oxygen for 18 h. Later cells were exposed to 50 µg/ml of cigarette smoke condensate (CSC) and incubated with various concentrations of chrysin (CH) and xanthohumol (XN), individually or in combination as mentioned in the figure, in DMEM containing 10% FCS and co-incubated further for 24 hours. The cell viability was assayed by WST dye uptake measured at 450nm and the results are expressed as % of viable cells as compared to untreated cells. Figure 4 illustrates the same.
The working ratio of CH to XN is 5:4 to 4:3. Figure 4 demonstrates that outside the mentioned range for example 2:1 (above the stated ratio range) and 3: 4 (below the stated ratio range), the combinations of CH and XN do not provide synergistic effects. Further the concentration of CH and XN are critical. The concentration of CH must always be higher than the concentration of XN. The concentration of chrysin and xanthohumol ranges from 0.1 to 5 wt% Furthermore concentration of CH and XN present in composition is dependent on the total amount of tobacco present in the composition. Here the concentration of CH and XN must be within the range of from about 1 part CH : 5 parts tobacco to 3 parts CH : 5 parts tobacco and from about 1 part XN : 5 parts tobacco to 3 parts XN : 5 parts tobacco.
Result and observation: As presented in the bar diagrams, synergistic effect of chrysin and Xanthohumol in A549 cells are in the range of 20-25:15-20.
Example 6: Assessment of apoptosis by Annexin V-FITC
Apoptotic cell death was measured using a flouresin isothiocynate (FITC)-conjugated Annexin V/PI assay kit (BD Pharmingen™) by flow cytometry. Briefly, 5 x105 cells were washed with ice–cold PBS, resuspended in 100 µl binding buffer, and stained with 5 µl of FITC- conjugated Annexin V (10 µg/ml) and 10 µl of PI (50 mg/ml). The cells were incubated for 15 minat room temperature in the dark, 400 ml of binding bufferwas added, and the cells were analysed (FACScan, Becton-Dickinson, USA). The A549 cells were gated separatelyaccording to their granularity and size on forward scatter (FSC) versus Side Scatter (SSC) plots. Early and lateapoptosis was evaluated on fluorescence 2 (FL2 forpropidium iodide) versus fluorescence 1 (FL1 for Annexin) plots. The data were analysed using Flow Jo software. The results were interpreted in the following fashion: cells in the lower-left quadrant (Annexin-V-/PI-) represent living cells; those in the lower-right quadrant (Annexin-V+/PI-) represent early apoptotic cells; those in the upper-right quadrant (Annexin-V+/PI+) represent late apoptotic cells; those in the upper-left quadrant (Annexin-V-/PI+) represent necrotic cells. Figure 5 illustrates the same.
Result and observation: The quantitative analysis using dot plot showed the synergistic effect of Chrysin and Xanthohumol by increasing the percentage of early stage apoptotic cells (Annexin V+/PI-) from 1.45% (Chrysin only; B), 6.15% (Xanthohumol only; C) to 26 % (synergetic effect; D) and reducing necrosis.
Example 7: Post smoking effect of chrysin and xanthohumol on cell viability in A549 cells
5X concentration of chrysin: xanthohumol was directly injected into the 3R4F cigarette rod (A) and filter (B) and stored overnight at ambient temperature. Simultaneously same concentration of chrysin: xanthohumol mixture was mixed with electronic cigarette e juice (C) composed of propylene glycol: vegetable glycerin 80:20 and 1% pure nicotine. Both cigarettes without actives were used as control. The cigarettes were smoked under ISO conditions. Then the condensate was collected from both the pads samples A and B using DMSO and were used to determine cell viability. Briefly, equal number of A549 cells (10,000) per well was seeded in a 96 wells plate in DMEM (medium to grow A549 cells) containing 10% Fetal Calf Serum (FCS) and penicillin (100 µg/ml), streptomycin (100 µg/ml) and incubated at 37°C in humidified chamber under 5% carbon dioxide and 95% oxygen for 18 h. Later cells were exposed to 50 µg/ml of cigarette smoke condensate (CSC) and incubated with various concentrations of chrysin (CH) and xanthohumol (XN), individually or in combination as mentioned in the figure, in DMEM containing 10% FCS and co-incubated further for 24 hours. The cells viability was assayed by WST dye uptake measured at 450nm and the results are expressed as % of viable cells as compared to untreated cells. Figure 6 illustrates the same.
Result and observation: As presented in the bar diagrams, the cigarette smoke condensate from (A) rod, (B) filter (C) e-juice fortified with actives showed 20, 32 and 25% of cell death simultaneously when compared with the negative control.
,CLAIMS:1. A composition comprising
a) Chrysin and
b) Xanthohumol and
c) organoleptic agents
Wherein ratio of chrysin and xanthohumol ranges from 5:4 to 4:3
Wherein the concentration of chrysin and xanthohumol ranges from 0.1 to 5 wt %
2. The composition as claimed in claim 1, wherein said chrysin is present in an amount ranging from 0.1 to 5%.
3. The composition as claimed in claim 1, wherein said Xanthohumol (XN) is present in an amount ranging from 0.1 to 5%.
4. The composition as claimed in claim 1, wherein said organoleptic agents are present in an amount ranging from 0.1 to 2 wt% wt.
5. The composition as claimed in claim 4, wherein said organoleptic agents are selected from Flavors / flavorants chosen from a group comprising menthol, citrus flavors and the like, taste enhancers, natural extract such as clove, elaichi and the like.
6. The composition as claimed in claim 1, wherein said composition is included in product formats selected from group comprising cigarettes, heat not burn formats, thermal aerosol, electronic or electrically heated cigarettes, inhalers, sprays, dermal patches and the like.
7. The composition as claimed in claim 6, wherein tobacco in the said product format is present in an amount ranging from 700-750mg.

8. The composition as claimed in any one of the preceding claims, wherein said composition is delivered through various routes selected from but not limited to pulmonary, nasal, dermal and buccal routes to offer cellular benefits.