FIELD OF INVENTION
[001] The present disclosure relates to the field of formulations for disease management in general and oxidative stress management in particular. There is provided a composition comprising sulforaphane, bacoside A, and phloridzin for inhibition of reactive oxygen species production.
BACKGROUND OF THE INVENTION
[002] Oxidative stress is caused when there is an accumulation of reactive oxygen species (ROS) in the body. ROS molecules which are found in the body include hydroxyl radicals, superoxide anion radicals, hydrogen peroxide, singlet molecular oxygen hypochlorite, nitric oxide radical and peroxynitrite among others. ROS are sometimes also referred to as free radicals. When these molecules occur in profusion in the body, they are capable of damaging DNA, proteins, lipids, and carbohydrates, which can in turn lead to several chronic illnesses such as cancer, heart failure, weakening of the immune system, and Alzheimer's disease among others. [003] There are various ways in which ROS are produced in human body. Broadly ROS can be classified into two main classes, in terms of their source: ones having an internal source and the others having an external source. Internal sources include processes like enzymatic and non-enzymatic reactions. Enzymatic reactions comprise reactions which form a part of respiratory chain, phagocytosis, prostaglandin synthesis, and cytochrome P-450 system. Some non-enzymatic reactions also result in ROS generation. External sources of ROS contain factors such as exposure to UV radiation, cigarette smoking, air pollution, and industrial pollution. [004] As mentioned above, oxidative stress occurs when the concentration of ROS in human body exceeds acceptable limits, and this happens when the ROS-antioxidant balance gets tilted in favour of the ROS. Thus, increasing levels of

antioxidants in the body is considered as one of the better ways of maintaining
healthy levels of ROS in human body. This, however, has remained a particularly
difficult problem to solve, largely because of toxicity of synthetic antioxidants like
butylated hydroxytoluene and butylated hydroxyanisole, which have been tried as
antioxidants to reduce levels of ROS.
[005] Thus, although studies are already going on to determine effective
compositions against oxidative stress, problems like hazards associated with known
synthetic antioxidants, and the multifaceted ill-effects of ROS, have made it difficult
to determine an effective strategy to inhibit the production of these molecules within
the body.
[006] US6303139B1 relates to a dietary product comprising ubiquinone, stabilized
vitamin E, phospholipids, selenuim in an organic form and L-methionin, which is
effective to combat cell oxidative stress and cellular decay.
[007] US5916912A describes a method comprising orally administering to the host
an effective dosage of a carnitine, such as acetyl-L-carnitine, and a mitochondrially
active antioxidant, such as lipoic acid.
[008] US7241461B2 describes an antioxidant-promoting composition comprising
Bacopa monniera extract; milk thistle extract, ashwagandha powder, green tea
extract, Gotu kola powder, Ginko biloba leaf extract; Aloe vera powder; turmeric
extract; and N-acetyl cysteine that increases antioxidant defense potential in a subject.
SUMMARY OF THE INVENTION
[009] In an aspect of the present disclosure there is provided a composition for inhibiting reactive oxygen species (ROS) production, said composition comprising: (a) sulforaphane; (b) bacoside A; and (c) phloridzin.
[0010] In an aspect of the present disclosure there is provided a method for preparing a composition comprising: (a) sulforaphane; (b) bacoside A; and (c) phloridzin, said method comprising:

(a) obtaining sulforaphane;
(b) obtaining bacoside A;
(c) obtaining phloridzin; and
(d) contacting sulforaphane, bacoside A, and phloridzin to obtain said composition. [0011] These and other features, aspects, and advantages of the present subject matter will be better understood with reference to the following description and appended claims. This summary is provided to introduce a selection of concepts in a simplified form. This summary is not intended to identify key features or essential features of the claimed subject matter, nor is it intended to be used to limit the scope of the claimed subject matter.
BRIEF DESCRIPTION OF ACCOMPANYING DRAWINGS
[0012] The following drawings form a part of the present specification and are included to further illustrate aspects of the present disclosure. The disclosure may be better understood by reference to the drawings in combination with the detailed description of the specific embodiments presented herein.
[0013] Figure 1 shows the effect of different individual concentrations of sulforaphane, bacoside A, and phloridzin on the inhibition of reactive oxygen species (ROS) production in accordance with an embodiment of the present disclosure. [0014] Figure 2 shows the effect of a combination of different concentration ratios of sulforaphane, bacoside A, and phloridzin on the inhibition of reactive oxygen species (ROS) production in accordance with an embodiment of the present disclosure. [0015] Figure 3 shows the cytotoxicity of different concentrations of sulforaphane, bacoside A, and phloridzin in accordance with an embodiment of the present disclosure

DETAILED DESCRIPTION OF THE INVENTION
[0016] The present disclosure relates to a composition for managing oxidative stress by inhibition of reactive oxygen species.
[0017] Those skilled in the art will be aware that the present disclosure is subject to variations and modifications other than those specifically described. It is to be understood that the present disclosure includes all such variations and modifications. The disclosure also includes all such steps, features, compositions, and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any or more of such steps or features.
Definitions
[0018] For convenience, before further description of the present disclosure, certain
terms employed in the specification, and examples are delineated here. These
definitions should be read in the light of the remainder of the disclosure and
understood as by a person of skill in the art. The terms used herein have the meanings
recognized and known to those of skill in the art, however, for convenience and
completeness, particular terms and their meanings are set forth below.
[0019] The articles "a", "an" and "the" are used to refer to one or to more than one
(i.e., to at least one) of the grammatical object of the article.
[0020] The terms "comprise" and "comprising" are used in the inclusive, open sense,
meaning that additional elements may be included. It is not intended to be construed
as "consists of only".
[0021] Throughout this specification, unless the context requires otherwise the word
"comprise", and variations such as "comprises" and "comprising", will be understood
to imply the inclusion of a stated element or step or group of element or steps but not
the exclusion of any other element or step or group of element or steps.

[0022] The term "including" is used to mean "including but not limited to".
"Including" and "including but not limited to" are used interchangeably.
[0023] Carriers are substances that serve as mechanisms to improve the delivery and
the effectiveness of drugs.
[0024] A diluent (also referred to as filler, dilutant, or thinner) is a diluting agent.
[0025] An excipient is an inactive substance that serves as the vehicle or medium for
a drug or other active substance. Excipients include colouring agents, preservatives,
humectants, and stabilizers.
[0026] Unless defined otherwise, all technical and scientific terms used herein have
the same meaning as commonly understood by one of ordinary skill in the art to
which this disclosure belongs. Although any methods and materials similar or
equivalent to those described herein can be used in the practice or testing of the
disclosure, the preferred methods, and materials are now described. All publications
mentioned herein are incorporated herein by reference.
[0027] Oxidative stress, caused by the over-production of reactive oxygen species
(ROS) is a major cause of several diseases. A prior art search shows that studies to
reduce damage caused by the production of ROS have been ongoing, however,
particular formulations to inhibit ROS production have not been effective. The
present disclosure uses a unique combination of ingredients which facilitate enhanced
inhibition of intracellular ROS production. The present disclosure also provides an
unique combination of ingredients at specific ratios and concentrations.
[0028] The present disclosure is not to be limited in scope by the specific
embodiments described herein, which are intended for the purposes of
exemplification only. Functionally-equivalent products, compositions, and methods
are clearly within the scope of the disclosure, as described herein.
[0029] Sulforaphane [l-isothiocyanato-4-(methylsulfinyl)-butane] belongs to
isothiocyanate class of organic compounds. Several studies have shown that
sulforaphane has anti-inflammatory, antioxidant, anticancer activity, and
chemopreventive activity. This compound occurs predominantly in the plant family

Brassicaceae which includes crops such as oilseed rape, cabbage, and broccoli (Chunliu Li et al., 2014, PLoS One, 9(2): e90520).
[0030] Bacopa monnieri is a herb found in the wetlands of southern and eastern India. It is known to improve memory and cognition. The active constituent responsible for its pharmacological effects is bacoside A, a mixture of dammarane-type tri terpenoid saponins containing sugar chains linked to a steroid aglycone skeleton (Ramasamy et al., PLoS One, 2015; 10(5): e0126565). [0031] Phloridzin (phlorizin or phloretin 29-O-glucoside), a diydrochalcone, is a phenolic flavonoid glucoside found primarily in the vegetative tissues of Mains sp. or apple. It is known for its anti- diabetic properties, but has shown to have other biological activities such as inhibition of lipid peroxidation, prevention of bone loss in rats and enhancement of memory in mice (Nair et al., PLOS One, 2014; 9(9): el07149j.
[0032] In an embodiment of the present disclosure, there is provided a composition
for inhibiting reactive oxygen species (ROS) production, said composition
comprising: (a) sulforaphane; (b) bacoside A; and (c) phloridzin.
[0033] In an embodiment of the present disclosure, there is provided a composition
for inhibiting reactive oxygen species (ROS) production, said composition
comprising: (a) sulforaphane; (b) bacoside A; and (c) phloridzin, wherein
sulforaphane to bacoside A to phloridzin w/w ratio in said composition is in the range
of 1:1:0.04- 1:3:0.2.
[0034] In another embodiment of the present disclosure, there is provided a
composition for inhibiting reactive oxygen species (ROS) production, said
composition comprising: (a) sulforaphane; (b) bacoside A; and (c) phloridzin,
wherein sulforaphane to bacoside A to phloridzin w/w ratio in said composition is
1:1.7:0.2.
[0035] In an embodiment of the present disclosure, there is provided a composition
for inhibiting reactive oxygen species (ROS) production, said composition

comprising: (a) sulforaphane; (b) bacoside A; and (c) phloridzin, wherein
sulforaphane to bacoside A to phloridzin w/w ratio in said composition is 1:1.7:
0.089.
[0036] In an embodiment of the present disclosure, there is provided a composition
for inhibiting reactive oxygen species (ROS) production, said composition
comprising: (a) sulforaphane; (b) bacoside A; and (c) phloridzin, wherein
sulforaphane to bacoside A to phloridzin w/w ratio in said composition is 1:2.3:0.06.
[0037] In an embodiment of the present disclosure, there is provided a composition
for inhibiting reactive oxygen species (ROS) production, said composition
comprising: (a) sulforaphane; (b) bacoside A; (c) phloridzin; and (d) suitable carriers,
diluents, and excipients,, wherein sulforaphane to bacoside A to phloridzin w/w ratio
in said composition is in the range of 1:1:0.04- 1:3:0.2.
[0038] In an embodiment of the present disclosure, there is provided a composition
for inhibiting reactive oxygen species (ROS) production, said composition
comprising: (a) sulforaphane; (b) bacoside A; (c) phloridzin; and (d) suitable carriers,
diluents, and excipients, wherein sulforaphane to bacoside A to phloridzin w/w ratio
in said composition is 1:1.7:0.2.
[0039] In an embodiment of the present disclosure, there is provided a composition
for inhibiting reactive oxygen species (ROS) production, said composition
comprising: (a) sulforaphane; (b) bacoside A; (c) phloridzin; and (d) suitable carriers,
diluents, and excipients, wherein sulforaphane to bacoside A to phloridzin w/w ratio
in said composition is 1:1.7: 0.089.
[0040] In an embodiment of the present disclosure, there is provided a composition
for inhibiting reactive oxygen species (ROS) production, said composition
comprising: (a) sulforaphane; (b) bacoside A; (c) phloridzin; and (d) suitable carriers,
diluents, and excipients, wherein sulforaphane to bacoside A to phloridzin w/w ratio
in said composition is 1:2.3:0.06.
[0041] In an embodiment of the present disclosure, there is provided a method of
preparing a composition for inhibiting reactive oxygen species (ROS) production,

said composition comprising: (a) sulforaphane; (b) bacoside A; and (c) phloridzin, said method comprising the steps of: (a) obtaining sulforaphane; (b) obtaining bacoside A; (c) obtaining phloridzin; and (d) contacting sulforaphane, bacoside A and phloridzin to obtain said composition.
[0042] In an embodiment of the present disclosure, there is provided a method of preparing a composition for inhibiting reactive oxygen species (ROS) production, said composition comprising: (a) sulforaphane; (b) bacoside A; and (c) phloridzin, said method comprising the steps of: (a) obtaining sulforaphane; (b) obtaining bacoside A; (c) obtaining phloridzin; and (d) contacting sulforaphane and bacoside A to obtain said composition, wherein sulforaphane to bacoside A to phloridzin w/w ratio in said composition is in the range of 1:1:0.04-1:3:0.2.
[0043] In an embodiment of the present disclosure, there is provided a method of preparing a composition for inhibiting reactive oxygen species (ROS) production, said composition comprising: (a) sulforaphane; (b) bacoside A; and (c) phloridzin, said method comprising the steps of: (a) obtaining sulforaphane; (b) obtaining bacoside A; (c) obtaining phloridzin; and (d) contacting sulforaphane, bacoside A and phloridzin to obtain said composition, wherein sulforaphane to bacoside A to phloridzin w/w ratio in said composition is 1:1.7:0.2.
[0044] In an embodiment of the present disclosure, there is provided a method of preparing a composition for inhibiting reactive oxygen species (ROS) production said composition comprising: (a) sulforaphane; (b) bacoside A; and (c) phloridzin, said method comprising the steps of: (a) obtaining sulforaphane; (b) obtaining bacoside A; (c) obtaining phloridzin; and (d) contacting sulforaphane, bacoside A and phloridzin to obtain said composition, wherein sulforaphane to bacoside A to phloridzin w/w ratio in said composition is 1:1.7: 0.089.
[0045] In an embodiment of the present disclosure, there is provided a method of preparing a composition for inhibiting reactive oxygen species (ROS) production said composition comprising: (a) sulforaphane; (b) bacoside A; and (c) phloridzin, said method comprising the steps of: (a) obtaining sulforaphane; (b) obtaining

bacoside A; (c) obtaining phloridzin; and (d) contacting sulforaphane, bacoside A and phloridzin to obtain said composition, wherein sulforaphane to bacoside A to phloridzin w/w ratio in said composition is 1:2.3:0.06.
[0046] In an embodiment of the present disclosure, there is provided a method of preparing a composition for inhibiting reactive oxygen species (ROS) production said composition comprising: (a) sulforaphane; (b) bacoside A; and (c) phloridzin, said method comprising the steps of: (a) obtaining sulforaphane; (b) obtaining bacoside A; (c) obtaining phloridzin; (d) obtaining suitable carriers, diluents, and excipients; and (e) contacting sulforaphane, bacoside A, phloridzin, and suitable carriers, diluents, and excipients, to obtain said composition.
[0047] In an embodiment of the present disclosure, there is provided a method of preparing a composition for inhibiting reactive oxygen species (ROS) production comprising: (a) sulforaphane; (b) bacoside A; (c) phloridzin; and (d) suitable carriers, diluents, and excipients, said method comprising the steps of: (a) obtaining sulforaphane; (b) obtaining bacoside A; (c) obtaining phloridzin; (d) obtaining suitable carriers, diluents, and excipients; and (e) contacting sulforaphane, bacoside A, phloridzin, and suitable carriers, diluents, and excipients, to obtain said composition, wherein sulforaphane to bacoside A to phloridzin w/w ratio in said composition is in the range of 1:1:0.04- 1:3:0.2.
[0048] In an embodiment of the present disclosure, there is provided a method of preparing a composition for inhibiting reactive oxygen species (ROS) production comprising: (a) sulforaphane; (b) bacoside A; (c) phloridzin; and (d) suitable carriers, diluents, and excipients, said method comprising the steps of: (a) obtaining sulforaphane; (b) obtaining bacoside A; (c) obtaining phloridzin; (d) obtaining suitable carriers, diluents, and excipients; and (e) contacting sulforaphane, bacoside A, phloridzin, and suitable carriers, diluents, and excipients, to obtain said composition, wherein sulforaphane to bacoside A to phloridzin w/w ratio in said composition is 1:1.7:0.2.

[0049] In an embodiment of the present disclosure, there is provided a method of preparing a composition for inhibiting reactive oxygen species (ROS) production comprising: (a) sulforaphane; (b) bacoside A; (c) phloridzin; and (d) suitable carriers, diluents, and excipients, said method comprising the steps of: (a) obtaining sulforaphane; (b) obtaining bacoside A; (c) obtaining phloridzin; (d) obtaining suitable carriers, diluents, and excipients; and (e) contacting sulforaphane, bacoside A, phloridzin, and suitable carriers, diluents, and excipients, to obtain said composition, wherein sulforaphane to bacoside A to phloridzin w/w ratio in said composition is 1:1.7: 0.089.
[0050] In an embodiment of the present disclosure, there is provided a method of preparing a composition for inhibiting reactive oxygen species (ROS) production comprising: (a) sulforaphane; (b) bacoside A; (c) phloridzin; and (d) suitable carriers, diluents, and excipients, said method comprising the steps of: (a) obtaining sulforaphane; (b) obtaining bacoside A; (c) obtaining phloridzin; (d) obtaining suitable carriers, diluents, and excipients; and (e) contacting sulforaphane, bacoside A, phloridzin, and suitable carriers, diluents, and excipients, to obtain said composition, wherein sulforaphane to bacoside A to phloridzin w/w ratio in said composition is 1:2.3:0.06.
[0051] In an embodiment of the present disclosure, there is provided a composition as described herein, wherein the composition manages oxidative stress by inhibiting intracellular ROS production.
[0052] In an embodiment of the present disclosure, there is provided a composition to manage oxidative stress responses comprising: (a) sulforaphane; (b) bacoside A; and (c) phloridzin, wherein sulforaphane to bacoside A to phloridzin w/w ratio in said composition is in the range of 1:1:0.04-1:3:0.2.
[0053] In another embodiment of the present disclosure, there is provided a composition to manage oxidative stress responses comprising: (a) sulforaphane; (b) bacoside A; and (c) phloridzin, wherein sulforaphane to bacoside A to phloridzin w/w ratio in said composition is 1:1.7:0.2.

[0054] In an embodiment of the present disclosure, there is provided a composition to manage oxidative stress responses comprising: (a) sulforaphane; (b) bacoside A; and (c) phloridzin, wherein sulforaphane to bacoside A to phloridzin w/w ratio in said composition is 1:1.7: 0.089.
[0055] In an embodiment of the present disclosure, there is provided a composition to manage oxidative stress responses comprising: (a) sulforaphane; (b) bacoside A; and (c) phloridzin, wherein sulforaphane to bacoside A to phloridzin w/w ratio in said composition is 1:2.3:0.06.
[0056] Although the subject matter has been described with reference to specific embodiments, this description is not meant to be construed in a limiting sense. Various modifications of the disclosed embodiments, as well as alternate embodiments of the subject matter, will become apparent to persons skilled in the art upon reference to the description of the subject matter. It is therefore contemplated that such modifications can be made without departing from the spirit or scope of the present subject matter as defined.
EXAMPLES
[0057] The disclosure will now be illustrated with working examples, which is intended to illustrate the working of disclosure and not intended to be taken restrictively to imply any limitations on the scope of the present disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice of the disclosed methods and compositions, the exemplary methods, devices and materials are described herein. It is to be understood that this disclosure is not limited to particular methods, and experimental conditions described, as such methods and conditions may vary.

Example 1
Material and Methods
[0058] Testing inhibition of intracellular ROS production: Neuro 2a cells were obtained from NCCS, Pune. Cells were seeded in 96 well black clear bottom plate and incubated at 37 °C and 5% C02. DMEM (Dulbecco's Modified Eagle's Medium) medium with 10% FBS (Fetal Bovine Serum) and 1% of suitable antibiotics were used for culturing. After overnight incubation the cells were treated with the active ingredients under study. The next day cells were washed with IX PBS (Phosphate Buffer Saline) and stained with 25 uM DCFDA (2', 7' -dichlorofluorescein diacetate) for 30 minutes at 37 °C. After this step washed cells with IX PBS followed by incubating cells with ImM H2O2 for 10 minutes. Fluorescence was measured with excitation wavelength at 485 nm and emission wavelength at 535 nm using a Thermo Fischer plate reader. Percentage inhibition was calculated as, % inhibition = (Absorbance control - Absorbance Test)/ Absorbance Control X 100. [0059] DCFDA dye was used in this assay to measure intracellular ROS production. It is a fluorogenic dye that measures hydroxyl, peroxyl and other ROS activity within the cell. After diffusion into the cell, DCFDA is deacetylated by cellular esterases to a non-fluorescent compound, which is later oxidized by ROS into 2', T -dichlorofluorescein (DCF). Sulforaphane, bacoside A , phloridzin were obtained from Sigma Aldrich, catalog numbers S4441, 76091, and P3449 respectively.
Example 2
Effect of sulforaphane, bacoside A, and phloridzin on intracellular ROS
inhibition

[0060] In order to determine whether sulforaphane, bacoside A, and phloridzin, had
any effect in inhibiting ROS within cells, the three actives were tested individually
using the DCFDA dye protocol described above.
[0061] As is seen in Figure 1, sulforaphane showed the highest percentage ROS
inhibition amongst the three actives with a maximum inhibition at 0.44 ug/ml
concentration showing approximately 40% inhibition.
[0062] Both of the remaining actives bacoside A and phloridzin showed lower
inhibitory activity around a mean value of approximately 20% inhibition. Bacoside A
showed a maximum inhibitory capacity at the concentrations of 0.78 ug/ml and 0.39
ug/ml. Phloridzin showed a marginally higher inhibitory effect on ROS production
than bacoside A. The inhibitory effect was strongest at the concentrations of 0.0295
ug/ml, 0.0147 ug/ml and 0.00736 ug/ml.
[0063] Thus, from these data it can be seen that the actives in isolation do not exhibit
very high ROS inhibitory activity.
Example 3
Effect of a combination of sulforaphane, bacoside A, and phloridzin on
intracellular ROS inhibition
[0064] As the individual actives did not show any marked ability to repress ROS production, the actives were tested in different combinations as seen in Table 1. Based on the results obtained from the effect of the individual actives, 0.11 ug/ml sulforaphane, 0.195 ug/ml bacoside A and 0.0074 ug/ml phloridzin were chosen as the maximum individual concentrations.

[0065] The ratios mentioned in the first column of Table 1, rows 3-9, are with reference to the individually tested concentrations of the three components as mentioned in the first three rows. For instance, a ratio of 33.3:33.3:33.3 for sulforaphane: bacoside A: phloridzin (row 3, Table 1) means concentration of sulforaphane is 33.3% of individually tested concentration of sulforaphane, i.e., 0.11 ug/ml, which comes to 0.036 ug/ml; concentration of bacoside A is 33.3% of individually tested concentration of bacoside A, i.e., 0.195 ug/ml, which comes to 0.065 ug/ml; and the concentration of phloridzin is 33.3% of individually tested concentration of phloridzin, i.e., 0.0074 ug/ml, which comes to 0.00246 ug/ml. Thus, when the ratio is calculated based on actual concentrations of the three components it comes to 1:1.8:0.07. All other ratios in column 1 of Table 1 are to be interpreted similarly.

[0066] Combinations at different ratios of sulforaphane, bacoside A, and phloridzin were tested and compared with the individual ingredients. Remarkably, at certain specific ratios, the compositions comprising all the three actives sulforaphane, bacoside A, and phloridzin showed significantly enhanced inhibitory activity against ROS production (Figure 2).
[0067] At the ratios of 30:40:30, 30:30:40, and 20:20:60 of sulforaphane to bacoside A to phloridzin, respectively, a distinct synergistic effect was observed which together was more than the effect of each individual constituents (Figure 2). The highest inhibition was observed using a 30:30:40 ratio of sulforaphane to bacoside A to phloridzin, which showed an approximately two fold increase in inhibitory effect. [0068] Further, the synergism of all three components is only evident at certain specific ratios, and changes to these does not result in the same effect. In other words, a mere aggregation of the three components cannot produce the desired results. For instance, a 60:20:20 or a 33.3:33.3:33.3 ratio of sulforaphane to bacoside A to phloridzin does not show any inhibitory activity and thus the combinations lie beyond the ranges where synergism is observed.
[0069] Therefore, within a range of 1:1:0.04- 1:3:0.2, a synergistic effect of a combination of sulforaphane, bacoside A, and phloridzin is observed, with significantly enhanced synergism at the ratios of 1:1.7:0.2, 1:1.7:0.089 and 1:2.3:0.06 of sulforaphane: bacoside A: phloridzin.
Example 3
Assessment of cytotoxicity of a sulforaphane, bacoside A, and phloridzin
[0070] The data indicate that sulforaphane, bacoside A, and phloridzin together show a significant effect in inhibiting ROS production in cells. In order to determine whether the concentrations at which the active ingredients used in the compositions

demonstrate any downstream cytotoxic effects, the actives were supplied to Neuro 2a cells and these were monitored for cell viability.
[0071] As observed in Figure 3, no cytotoxic effects were observed on cells supplied with either of the three actives. The maximum cytotoxicity was observed at 0.88 ug/ml sulforaphane and 6.25 ug/ml bacoside A. However, these are not at highly significant values and also outside the ranges at which the constituents are used within the composition disclosed.
[0072] Overall, the present disclosure provides a composition of sulforaphane, bacoside A, and phloridzin, which exhibits a surprising and unexpected synergistic effect on the inhibition of ROS production. This apparent synergism of the said combination is not readily apparent at all concentration combinations (as discussed previously), suggesting that the results are not a result of mere admixture of the three or routine in nature. Further, the identification of this novel combination to manage oxidative stress cannot be arrived at without undue experimentation, and is not obvious to a person skilled in the art.

I/We Claim:
1. A composition for inhibiting reactive oxygen species (ROS) production, said
composition comprising:
(a) sulforaphane;
(b) bacoside A; and
(c) phloridzin.
2. The composition as claimed in claim 1, wherein sulforaphane: bacoside A:
phloridzin w/w ratio in said composition is in the range of 1:1:0.04- 1:3:0.2.
3.The composition as claimed in claim 2, wherein sulforaphane: bacoside A: phloridzin w/w ratio in said composition is 1:1.7:0.2.
4. The composition as claimed in claim 2, wherein sulforaphane: bacoside A: phloridzin w/w ratio in said composition is 1:1.7: 0.089.
5. The composition as claimed in claim 2, wherein sulforaphane: bacoside A: phloridzin w/w ratio in said composition is 1:2.3:0.06.
6. The composition as claimed in any of the claims 1-5, said composition further comprising suitable carriers, diluents, and excipients.
7. A method for preparing the composition as claimed in claim 1, said method comprising the steps of:

(a) obtaining sulforaphane;
(b) obtaining bacoside A;
(c) obtaining phloridzin; and
(d) contacting sulforaphane, bacoside A, and phloridzin to obtain said composition.

8. A method for preparing the composition as claimed in any of the claims 1-6, said
method comprising the steps of:
(a) obtaining sulforaphane;
(b) obtaining bacoside A;
(c) obtaining phloridzin;
(d) obtaining suitable carriers, diluents, and excipients; and
(e) contacting sulforaphane, bacoside A, phloridzin, and suitable carriers, diluents, and excipients to obtain said composition.
9. The composition as claimed in any of the claims 1-6 wherein said composition
manages oxidative stress responses.