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 andrographolide and rosemary extract.
BACKGROUND OF INVENTION
[002] Reactive oxygen species (ROS) are molecules produced as a by-product of 5 oxygen metabolism. These molecules contain oxygen and are highly reactive as they can initiate chain reactions leading to further ROS production. ROS can serve as intercellular and intracellular messengers and recent studies have also found roles of these molecules in cell signalling and signal transduction. Overtime, as by-products of metabolism, ROS accumulate within the body. In addition, exogenous factors can
10 also result in the accumulation of ROS, including exposure to pollutants, tobacco and radiation. This excessive accumulation of ROS results in oxidative stress. [003] Oxidative stress has several damaging effects in the body. It has been linked to several degenerative diseases including Alzheimer’s and is thought to contribute to the process of aging. Further, the accumulation of ROS can also cause damage to
15 DNA and RNA. This results in the accumulation of mutations which overtime can result in the development of cancer and cause cell apoptosis.
[004] A method to counteract the accumulation of ROS is through the use of antioxidants, which delay the accumulation of ROS. Some common antioxidants include Vitamin C, selenium, carotenoids, flavonoids and quercetin among others.
20 Thus, plant-derived sources are particularly rich in antioxidants.
[005] US5462946A describes a pharmaceutical composition, comprising an anti-oxidative stress effective amount of a compound selected from the group consisting of a metal-independent nitroxide, an oxazolidine compound.
2

[006] US20060257502A1 describes a method for relieving stress, preventing and improving stress-related disorders comprising orally use of a composition containing mitochondrial nutrients including cyanocobalamin, thiamin, niacin, pyridoxine, pantothenate, folic acid, alpha-tocopherol, ascorbic acid, and others. 5 [007] FR2905827A1 antioxidant additive for incorporation in a food, a food supplement or a dietary supplement for the animal, in particular a ruminant comprising at least one plant extract selected from the group consisting of artichoke extract (Cynara scolymus L.), rosemary extract (Rosmarinus officinalis L.), extract of marigold (Tagetes spp. (T. minuta, T. erecta)), turmeric extract (Curcuma Tonga L)
10 extract of citrus (citrus), in particular the grapefruit extract of nettle (Urtica dioica), extract of garlic (Allium sativum), and others.
[008] The multi-factorial and delayed progression of ROS-associated diseases has made it difficult to determine a comprehensive solution to remedy these effects. Thus, there still remains a pressing need to find further solutions to reduce ROS levels in
15 the body.
SUMMARY OF THE INVENTION
[009] In an aspect of the present disclosure, there is provided a composition comprising: (a) andrographolide; and, (b) rosemary extract, wherein andrographolide to the rosemary extract w/w ratio in said composition is in the range of 1:0.2- 1:0.7. [0010] In an aspect of the present disclosure, there is provided a method for preparing 20 a composition comprising: (i) andrographolide; and (ii) rosemary extract, said method comprising:
(a) obtaining andrographolide;
(b) obtaining the rosemary extract; and
(c) contacting andrographolide, and the rosemary extract to obtain said composition. 25 [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
3

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
5 [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 cytotoxic effects of andrographolide and rosemary extract 10 on Neuro 2a cells, in accordance with an embodiment of the present disclosure.
[0014] Figure 2 shows the percentage ROS inhibition by andrographolide and rosemary extract, in accordance with an embodiment of the present disclosure. [0015] Figure 3 shows the percentage ROS inhibition by the combination of andrographolide and rosemary extract at different w/w ratios
DETAILED DESCRIPTION OF THE INVENTION
15 [0016] The present disclosure relates to a composition to inhibit reactive oxygen species (ROS).
[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.
20 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.
4

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 5 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. 10 [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] Carriers are substances that serve as mechanisms to improve the delivery and
the effectiveness of drugs. 15 [0022] A diluent (also referred to as filler, dilutant, or thinner) is a diluting agent.
[0023] An excipient is an inactive substance that serve as the vehicle or medium for a
drug or other active substance. Excipients include colouring agents, humectants,
preservatives, emollients, and combinations thereof.
[0024] Throughout this specification, unless the context requires otherwise the word 20 “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.
[0025] The term “including” is used to mean “including but not limited to”.
“Including” and “including but not limited to” are used interchangeably. 25 [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
5

disclosure, the preferred methods, and materials are now described. All publications mentioned herein are incorporated herein by reference.
[0027] Reactive oxygen species such as hydroxyl radical, superoxide anion radical, hydrogen peroxide, singlet molecular oxygen hypochlorite, nitric oxide radical and 5 peroxynitrite are capable of damaging DNA, protein, lipid and carbohydrate. These free radicals damage cells through oxidation. Eventually, the damage caused by them can cause several chronic illnesses.
[0028] Several studies have shown that antioxidants through diet help people's immune systems, protect against free radicals, and lower the risk of developing
10 cancer and heart disease.
[0029] Andrographis paniculata leaves have been shown to possess significant immunostimulant, cerebroprotective and nootropic activities [Radhika, et al., Indian Journal of Medical Research, 2012, 135 (5)]. Andrographolide is the active ingredient found in Andrographis paniculata. It is an apolar compound of low molecular weight
15 that acts on the central nervous system (CNS) in doses of 1 mg/kg and that can cross the blood–brain barrier; thus, andrographolide is an efficient molecule with a potential property for various treatments (Serrano1, et al., Molecular Neurodegenaration, Biomed Central, 2014). [0030] Constituents in rosemary have shown a variety of pharmacological activities
20 for cancer chemoprevention and therapy in in vitro and in vivo models (Cheung and Tai, Spandidos publications, 2007, DOI: 17.6.1525, 1525-1531) [0031] Carnosic acid, a diterpene, is abundant in the herb rosemary and has potent antioxidant activity in vitro. An extract of rosemary leaf has strong antioxidant activity due to carnosic acid and carnosol. Since antioxidants in food protect cells
25 against free-radical species and oxidative stress, they have been studied extensively. Carnosic acid has a neuroprotective effect in vitro and in vivo (Kayashima and Matsubara, 2014, Taylor and Francis).
6

[0032] In the present disclosure, andrographolide and rosemary extract have been tested to determine the effect of the combination of the two in managing oxidative stress. This disclosure provides a solution to this problem in the form of a composition comprising andrographolide and rosemary extract which at specific w/w 5 ratios and concentrations can inhibit ROS production.
[0033] 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.
10 [0034] In an embodiment of the present disclosure, there is provided a composition comprising: (a) andrographolide; and (b) rosemary extract, wherein andrographolide to the rosemary extract w/w ratio in said composition is in the range of 1:0.2- 1:0.7. [0035] In an embodiment of the present disclosure, there is provided a composition comprising: (a) andrographolide; and (b) rosemary extract, wherein andrographolide
15 to the rosemary extract w/w ratio in said composition is in the range of 1:0.33- 1:0.50. [0036] In an embodiment of the present disclosure, there is provided a composition comprising: (a) andrographolide; and (b) rosemary extract, wherein andrographolide to the rosemary extract w/w ratio in said composition is 1:0.25. [0037] In an embodiment of the present disclosure, there is provided a composition
20 comprising: (a) andrographolide; and (b) rosemary extract, wherein andrographolide to the rosemary extract w/w ratio in said composition is 1:0.33. [0038] In an embodiment of the present disclosure, there is provided a composition comprising: (a) andrographolide; and (b) rosemary extract, wherein andrographolide to the rosemary extract w/w ratio in said composition is 1:0.50.
25 [0039] In an embodiment of the present disclosure, there is provided a composition comprising: (a) andrographolide; and (b) rosemary extract, wherein andrographolide to the rosemary extract w/w ratio in said composition is 1:0.60.
7

[0040] In an embodiment of the present disclosure, there is provided a composition comprising: (a) andrographolide; and (b) rosemary extract, wherein andrographolide to the rosemary extract w/w ratio in said composition is 1:0.70. [0041] In an embodiment of the present disclosure, there is provided a composition 5 comprising: (a) andrographolide; and (b) rosemary extract, wherein andrographolide w/w percentage in said composition is in the range of 0.000013- 0.000030%, the rosemary extract w/w percentage in said composition is in the range of 0.000005-0.000012%, and andrographolide to the rosemary extract w/w ratio in said composition is in the range of 1:0.2- 1:0.7. 10 [0042] In an embodiment of the present disclosure, there is provided a composition comprising: (a) andrographolide; and (b) rosemary extract, wherein andrographolide w/w percentage in said composition is in the range of 0.000013- 0.000030%, the rosemary extract w/w percentage in said composition is in the range of 0.000005-0.000012%, and andrographolide to the rosemary extract w/w ratio in said 15 composition is in the range of 1:0.33- 1:0.50.
[0043] In an embodiment of the present disclosure, there is provided a composition comprising: (a) andrographolide; and (b) rosemary extract, wherein andrographolide w/w percentage in said composition is in the range of 0.0000195- 0.0000234%, the rosemary extract w/w percentage in said composition is in the range of 0.00000975-20 0.0000078%, and andrographolide to the rosemary extract w/w ratio in said composition is in the range of 1:0.2- 1:0.7.
[0044] In an embodiment of the present disclosure, there is provided a composition comprising: (a) andrographolide; and (b) rosemary extract, wherein andrographolide w/w percentage in said composition is in the range of 0.0000195- 0.0000234%, the 25 rosemary extract w/w percentage in said composition is in the range of 0.00000975-0.0000078%, and andrographolide to the rosemary extract w/w ratio in said composition is in the range of 1:0.33- 1:0.50.
8

[0045] In an embodiment of the present disclosure, there is provided a composition comprising: (a) andrographolide; and (b) rosemary extract, wherein andrographolide w/w percentage in said composition is in the range of 0.000013- 0.000030%, the rosemary extract w/w percentage in said composition is in the range of 0.000005-5 0.000012%, and andrographolide to the rosemary extract w/w ratio in said composition is 1:0.33.
[0046] In an embodiment of the present disclosure, there is provided a composition comprising: (a) andrographolide; and (b) rosemary extract, wherein andrographolide w/w percentage in said composition is in the range of 0.000013- 0.000030%, the
10 rosemary extract w/w percentage in said composition is in the range of 0.000005-0.000012%, and andrographolide to the rosemary extract w/w ratio in said composition is 1:0.50.
[0047] In an embodiment of the present disclosure, there is provided a composition comprising: (a) andrographolide; and (b) rosemary extract, wherein andrographolide
15 w/w percentage in said composition is in the range of 0.0000195- 0.0000234%, the rosemary extract w/w percentage in said composition is in the range of 0.00000975-0.0000078%, and andrographolide to the rosemary extract w/w ratio in said composition is 1:0.33. [0048] In an embodiment of the present disclosure, there is provided a composition
20 comprising: (a) andrographolide; and (b) rosemary extract, wherein andrographolide w/w percentage in said composition is in the range of 0.0000195- 0.0000234%, the rosemary extract w/w percentage in said composition is in the range of 0.00000975-0.0000078%, and andrographolide to the rosemary extract w/w ratio in said composition is 1:0.50.
25 [0049] In an embodiment of the present disclosure, there is provided a composition comprising: (a) andrographolide; and (b) rosemary extract, wherein andrographolide w/w percentage in said composition is 0.0000234%, the rosemary extract w/w
9

percentage in said composition is 0.0000078%, and andrographolide to the rosemary extract w/w ratio in said composition is 1:0.33.
[0050] In an embodiment of the present disclosure, there is provided a composition comprising: (a) andrographolide; and (b) rosemary extract, wherein andrographolide 5 w/w percentage in said composition is 0.0000195%, the rosemary extract w/w percentage in said composition is 0.00000975%, and andrographolide to the rosemary extract w/w ratio in said composition is 1:0.50.
[0051] In an embodiment of the present disclosure, there is provided a composition comprising: (a) andrographolide; (b) rosemary extract; (c) at least one carrier; (d) at
10 least one diluent; and (e) at least one excipient, wherein andrographolide to the rosemary extract w/w ratio in said composition is in the range of 1:0.2- 1:0.7. [0052] In an embodiment of the present disclosure, there is provided a composition comprising: (a) andrographolide; (b) rosemary extract; (c) at least one carrier; (d) at least one diluent; and (e) at least one excipient, wherein andrographolide to the
15 rosemary extract w/w ratio in said composition is in the range of 1:0.33- 1:0.50.
[0053] In an embodiment of the present disclosure, there is provided a composition comprising: (a) andrographolide; (b) rosemary extract; (c) at least one carrier; (d) at least one diluent; and (e) at least one excipient, wherein andrographolide w/w percentage in said composition is in the range of 0.000013- 0.000030%, the rosemary
20 extract w/w percentage in said composition is in the range of 0.000005- 0.000012%, and andrographolide to the rosemary extract w/w ratio in said composition is in the range of 1:0.2- 1:0.7.
[0054] In an embodiment of the present disclosure, there is provided a composition comprising: (a) andrographolide; (b) rosemary extract; (c) at least one carrier; (d) at
25 least one diluent; and (e) at least one excipient, wherein andrographolide w/w percentage in said composition is in the range of 0.000013- 0.000030%, the rosemary extract w/w percentage in said composition is in the range of 0.000005- 0.000012%,
10

and andrographolide to the rosemary extract w/w ratio in said composition is in the range of 1:0.33- 1:0.50.
[0055] In an embodiment of the present disclosure, there is provided a composition comprising: (a) andrographolide; (b) rosemary extract; (c) at least one carrier; (d) at 5 least one diluent; and (e) at least one excipient, wherein andrographolide w/w percentage in said composition is in the range of 0.0000195- 0.0000234%, the rosemary extract w/w percentage in said composition is in the range of 0.00000975-0.0000078%, and andrographolide to the rosemary extract w/w ratio in said composition is in the range of 1:0.2- 1:0.7. 10 [0056] In an embodiment of the present disclosure, there is provided a composition comprising: (a) andrographolide; (b) rosemary extract; (c) at least one carrier; (d) at least one diluent; and (e) at least one excipient, wherein andrographolide w/w percentage in said composition is in the range of 0.0000195- 0.0000234%, the rosemary extract w/w percentage in said composition is in the range of 0.00000975-15 0.0000078%, and andrographolide to the rosemary extract w/w ratio in said composition is in the range of 1:0.33- 1:0.50.
[0057] In an embodiment of the present disclosure, there is provided a composition comprising: (a) andrographolide; (b) rosemary extract; (c) at least one carrier; (d) at least one diluent; and (e) at least one excipient, wherein andrographolide w/w 20 percentage in said composition is in the range of 0.000013- 0.000030%, the rosemary extract w/w percentage in said composition is in the range of 0.000005- 0.000012%, and andrographolide to the rosemary extract w/w ratio in said composition is 1:0.33. [0058] In an embodiment of the present disclosure, there is provided a composition comprising: (a) andrographolide; (b) rosemary extract; (c) at least one carrier; (d) at 25 least one diluent; and (e) at least one excipient, wherein andrographolide w/w percentage in said composition is in the range of 0.000013- 0.000030%, the rosemary extract w/w percentage in said composition is in the range of 0.000005- 0.000012%, and andrographolide to the rosemary extract w/w ratio in said composition is 1:0.50.
11

[0059] In an embodiment of the present disclosure, there is provided a composition comprising: (a) andrographolide; (b) rosemary extract; (c) at least one carrier; (d) at least one diluent; and (e) at least one excipient, wherein andrographolide w/w percentage in said composition is in the range of 0.0000195- 0.0000234%, the 5 rosemary extract w/w percentage in said composition is in the range of 0.00000975-0.0000078%, and andrographolide to the rosemary extract w/w ratio in said composition is 1:0.33.
[0060] In an embodiment of the present disclosure, there is provided a composition comprising: (a) andrographolide; (b) rosemary extract; (c) at least one carrier; (d) at
10 least one diluent; and (e) at least one excipient, wherein andrographolide w/w percentage in said composition is in the range of 0.0000195- 0.0000234%, the rosemary extract w/w percentage in said composition is in the range of 0.00000975-0.0000078%, and andrographolide to the rosemary extract w/w ratio in said composition is 1:0.50.
15 [0061] In an embodiment of the present disclosure, there is provided a composition comprising: (a) andrographolide; (b) rosemary extract; (c) at least one carrier; (d) at least one diluent; and (e) at least one excipient, wherein andrographolide w/w percentage in said composition is 0.0000234%, the rosemary extract w/w percentage in said composition is 0.0000078%, and andrographolide to the rosemary extract w/w
20 ratio in said composition is 1:0.33.
[0062] In an embodiment of the present disclosure, there is provided a composition comprising: (a) andrographolide; (b) rosemary extract; (c) at least one carrier; (d) at least one diluent; and (e) at least one excipient, wherein andrographolide w/w percentage in said composition is 0.0000195%, the rosemary extract w/w percentage
25 in said composition is 0.00000975%, and andrographolide to the rosemary extract w/w ratio in said composition is 1:0.50.
12

[0063] In an embodiment of the present disclosure, there is provided a method of preparing a composition comprising: (i) andrographolide; and (ii) rosemary extract, said method comprising: (a) obtaining andrographolide; (b) obtaining the rosemary extract; and (c) contacting andrographolide, and the rosemary extract to obtain said 5 composition, wherein andrographolide to the rosemary extract w/w ratio in said composition is in the range of 1:0.2- 1:0.7.
[0064] In an embodiment of the present disclosure, there is provided a method of preparing a composition comprising: (i) andrographolide; and (ii) rosemary extract, said method comprising: (a) obtaining andrographolide; (b) obtaining the rosemary
10 extract; and (c) contacting andrographolide, and the rosemary extract to obtain said composition, wherein andrographolide to the rosemary extract w/w ratio in said composition is in the range of 1:0.33- 1:0.50.
[0065] In an embodiment of the present disclosure, there is provided a method of preparing a composition comprising: (i) andrographolide; and (ii) rosemary extract,
15 said method comprising: (a) obtaining andrographolide; (b) obtaining the rosemary extract; and (c) contacting andrographolide, and the rosemary extract to obtain said composition, wherein andrographolide to the rosemary extract w/w ratio in said composition is 1:0.33. [0066] In an embodiment of the present disclosure, there is provided a method of
20 preparing a composition comprising: (i) andrographolide; and (ii) rosemary extract, said method comprising: (a) obtaining andrographolide; (b) obtaining the rosemary extract; and (c) contacting andrographolide, and the rosemary extract to obtain said composition, wherein andrographolide to the rosemary extract w/w ratio in said composition is 1:0.50.
25 [0067] In an embodiment of the present disclosure, there is provided a method of preparing a composition comprising: (i) andrographolide; and (ii) rosemary extract, said method comprising: (a) obtaining andrographolide; (b) obtaining the rosemary extract; and (c) contacting andrographolide, and the rosemary extract to obtain said
13

composition, wherein andrographolide w/w percentage in said composition is in the range of 0.000013- 0.000030%, the rosemary extract w/w percentage in said composition is in the range of 0.000005- 0.000012%, and andrographolide to the rosemary extract w/w ratio in said composition is in the range of 1:0.2- 1:0.7. 5 [0068] In an embodiment of the present disclosure, there is provided a method of preparing a composition comprising: (i) andrographolide; and (ii) rosemary extract, said method comprising: (a) obtaining andrographolide; (b) obtaining the rosemary extract; and (c) contacting andrographolide, and the rosemary extract to obtain said composition, wherein andrographolide w/w percentage in said composition is in the
10 range of 0.000013- 0.000030%, the rosemary extract w/w percentage in said composition is in the range of 0.000005- 0.000012%, and andrographolide to the rosemary extract w/w ratio in said composition is in the range of 1:0.33- 1:0.50. [0069] In an embodiment of the present disclosure, there is provided a method of preparing a composition comprising: (i) andrographolide; and (ii) rosemary extract,
15 said method comprising: (a) obtaining andrographolide; (b) obtaining the rosemary extract; and (c) contacting andrographolide, and the rosemary extract to obtain said composition, wherein andrographolide w/w percentage in said composition is in the range of 0.0000195- 0.0000234%, the rosemary extract w/w percentage in said composition is in the range of 0.00000975- 0.0000078%, and andrographolide to the
20 rosemary extract w/w ratio in said composition is in the range of 1:0.2- 1:0.7.
[0070] In an embodiment of the present disclosure, there is provided a method of preparing a composition comprising: (i) andrographolide; and (ii) rosemary extract, said method comprising: (a) obtaining andrographolide; (b) obtaining the rosemary extract; and (c) contacting andrographolide, and the rosemary extract to obtain said
25 composition, wherein andrographolide w/w percentage in said composition is in the range of 0.0000195- 0.0000234%, the rosemary extract w/w percentage in said composition is in the range of 0.00000975- 0.0000078%, and andrographolide to the rosemary extract w/w ratio in said composition is in the range of 1:0.33- 1:0.50.
14

[0071] In an embodiment of the present disclosure, there is provided a method of preparing a composition comprising: (i) andrographolide; and (ii) rosemary extract, said method comprising: (a) obtaining andrographolide; (b) obtaining the rosemary extract; and (c) contacting andrographolide, and the rosemary extract to obtain said 5 composition, wherein andrographolide w/w percentage in said composition is in the range of 0.000013- 0.000030%, the rosemary extract w/w percentage in said composition is in the range of 0.000005- 0.000012%, and andrographolide to the rosemary extract w/w ratio in said composition is 1:0.33. [0072] In an embodiment of the present disclosure, there is provided a method of
10 preparing a composition comprising: (i) andrographolide; and (ii) rosemary extract, said method comprising: (a) obtaining andrographolide; (b) obtaining the rosemary extract; and (c) contacting andrographolide, and the rosemary extract to obtain said composition, wherein andrographolide w/w percentage in said composition is in the range of 0.000013- 0.000030%, the rosemary extract w/w percentage in said
15 composition is in the range of 0.000005- 0.000012%, and andrographolide to the rosemary extract w/w ratio in said composition is 1:0.50.
[0073] In an embodiment of the present disclosure, there is provided a method of preparing a composition comprising: (i) andrographolide; and (ii) rosemary extract, said method comprising: (a) obtaining andrographolide; (b) obtaining the rosemary
20 extract; and (c) contacting andrographolide, and the rosemary extract to obtain said composition, wherein andrographolide w/w percentage in said composition is in the range of 0.0000195- 0.0000234%, the rosemary extract w/w percentage in said composition is in the range of 0.00000975- 0.0000078%, and andrographolide to the rosemary extract w/w ratio in said composition is 1:0.33.
25 [0074] In an embodiment of the present disclosure, there is provided a method of preparing a composition comprising: (i) andrographolide; and (ii) rosemary extract, said method comprising: (a) obtaining andrographolide; (b) obtaining the rosemary extract; and (c) contacting andrographolide, and the rosemary extract to obtain said
15

composition, wherein andrographolide w/w percentage in said composition is in the range of 0.0000195- 0.0000234%, the rosemary extract w/w percentage in said composition is in the range of 0.00000975- 0.0000078%, and andrographolide to the rosemary extract w/w ratio in said composition is 1:0.50. 5 [0075] In an embodiment of the present disclosure, there is provided a method of preparing a composition comprising: (i) andrographolide; and (ii) rosemary extract, said method comprising: (a) obtaining andrographolide; (b) obtaining the rosemary extract; and (c) contacting andrographolide, and the rosemary extract to obtain said composition, wherein andrographolide w/w percentage in said composition is
10 0.0000234%, the rosemary extract w/w percentage in said composition is 0.0000078%, and andrographolide to the rosemary extract w/w ratio in said composition is 1:0.33.
[0076] In an embodiment of the present disclosure, there is provided a method of preparing a composition comprising: (i) andrographolide; and (ii) rosemary extract,
15 said method comprising: (a) obtaining andrographolide; (b) obtaining the rosemary extract; and (c) contacting andrographolide, and the rosemary extract to obtain said composition, wherein andrographolide w/w percentage in said composition is 0.0000195%, the rosemary extract w/w percentage in said composition is 0.00000975%, and andrographolide to the rosemary extract w/w ratio in said
20 composition is 1:0.50.
[0077] In an embodiment of the present disclosure, there is provided a method of preparing a composition comprising: (i) andrographolide; and (ii) rosemary extract, said method comprising: (a) obtaining a solution A of andrographolide; (b) obtaining a solution B of rosemary extract; and (c) contacting said solution A, and said solution
25 B to obtain said composition, wherein andrographolide to the rosemary extract w/w ratio in said composition is in the range of 1:0.2- 1:0.7.
[0078] In an embodiment of the present disclosure, there is provided a method of preparing a composition comprising: (i) andrographolide; and (ii) rosemary extract,
16

said method comprising: (a) obtaining a solution A of andrographolide; (b) obtaining a solution B of rosemary extract; and (c) contacting said solution A, and said solution B, wherein andrographolide to the rosemary extract w/w ratio in said composition is in the range of 1:0.33- 1:0.50. 5 [0079] In an embodiment of the present disclosure, there is provided a method of preparing a composition comprising: (i) andrographolide; and (ii) rosemary extract, said method comprising: (a) obtaining a solution A of andrographolide; (b) obtaining a solution B of rosemary extract; and (c) contacting said solution A, and said solution B, wherein andrographolide w/w percentage in said composition is in the range of
10 0.000013- 0.000030%, the rosemary extract w/w percentage in said composition is in the range of 0.000005- 0.000012%, and andrographolide to the rosemary extract w/w ratio in said composition is in the range of 1:0.2- 1:0.7.
[0080] In an embodiment of the present disclosure, there is provided a method of preparing a composition comprising: (i) andrographolide; and (ii) rosemary extract,
15 said method comprising: (a) obtaining a solution A of andrographolide; (b) obtaining a solution B of rosemary extract; and (c) contacting said solution A, and said solution B, wherein andrographolide w/w percentage in said composition is in the range of 0.000013- 0.000030%, the rosemary extract w/w percentage in said composition is in the range of 0.000005- 0.000012%, and andrographolide to the rosemary extract
20 w/w ratio in said composition is in the range of :0.33- 1:0.50.
[0081] In an embodiment of the present disclosure, there is provided a method of preparing a composition comprising: (i) andrographolide; and (ii) rosemary extract, said method comprising: (a) obtaining a solution A of andrographolide; (b) obtaining a solution B of rosemary extract; and (c) contacting said solution A, and said solution
25 B, wherein andrographolide w/w percentage in said composition is 0.0000234%, the rosemary extract w/w percentage in said composition is 0.0000078%, and andrographolide to the rosemary extract w/w ratio in said composition is 1:0.33.
17

[0082] In an embodiment of the present disclosure, there is provided a method of preparing a composition comprising: (i) andrographolide; and (ii) rosemary extract, said method comprising: (a) obtaining a solution A of andrographolide; (b) obtaining a solution B of rosemary extract; and (c) contacting said solution A, and said solution 5 B, wherein andrographolide w/w percentage in said composition is 0.0000195%, the rosemary extract w/w percentage in said composition is 0.00000975%, and andrographolide to the rosemary extract w/w ratio in said composition is in the range of 1:0.50. [0083] In an embodiment of the present disclosure, there is provided a method of
10 preparing a composition comprising: (i) andrographolide; and (ii) rosemary extract, said method comprising: (a) obtaining a solution A of andrographolide; (b) obtaining a solution B of rosemary extract; and (c) contacting said solution A, and said solution B, wherein said solution A is prepared in an organic solvent. [0084] In an embodiment of the present disclosure, there is provided a method of
15 preparing a composition comprising: (i) andrographolide; and (ii) rosemary extract, said method comprising: (a) obtaining a solution A of andrographolide; (b) obtaining a solution B of rosemary extract; and (c) contacting said solution A, and said solution B, wherein said solution A is prepared in an inorganic solvent. [0085] In an embodiment of the present disclosure, there is provided a method of
20 preparing a composition comprising: (i) andrographolide; and (ii) rosemary extract, said method comprising: (a) obtaining a solution A of andrographolide; (b) obtaining a solution B of rosemary extract; and (c) contacting said solution A, and said solution B, wherein said solution B is prepared in an organic solvent. [0086] In an embodiment of the present disclosure, there is provided a method of
25 preparing a composition comprising: (i) andrographolide; and (ii) rosemary extract, said method comprising: (a) obtaining a solution A of andrographolide; (b) obtaining a solution B of rosemary extract; and (c) contacting said solution A, and said solution B, wherein said solution B is prepared in an inorganic solvent.
18

[0087] In an embodiment of the present disclosure, there is provided a method of preparing a composition comprising: (i) andrographolide; (ii) rosemary extract; (iii) at least one carrier; (iv) at least one diluent; and (v) at least one excipient, said method comprising: (a) obtaining andrographolide; (b) obtaining the rosemary extract; (c) 5 obtaining the at least one carrier, the at least one diluent, and the at least one excipient; and (d) contacting andrographolide, the rosemary extract, the at least one carrier, the at least one diluent, and the at least one excipient to obtain said composition, wherein andrographolide to the rosemary extract w/w ratio in said composition is in the range of 1:0.2- 1:0.7.
10 [0088] In an embodiment of the present disclosure, there is provided a method of preparing a composition comprising: (i) andrographolide; (ii) rosemary extract; (iii) at least one carrier; (iv) at least one diluent; and (v) at least one excipient, said method comprising: (a) obtaining andrographolide; (b) obtaining the rosemary extract; (c) obtaining the at least one carrier, the at least one diluent, and the at least one
15 excipient; and (d) contacting andrographolide, the rosemary extract, the at least one carrier, the at least one diluent, and the at least one excipient to obtain said composition, wherein andrographolide to the rosemary extract w/w ratio in said composition is in the range of 1:0.33- 1:0.50. [0089] disclosure, there In an embodiment of the present disclosure, there is provided
20 a method of preparing a composition comprising: (i) andrographolide; (ii) rosemary extract; (iii) at least one carrier; (iv) at least one diluent; and (v) at least one excipient, said method comprising: (a) obtaining andrographolide; (b) obtaining the rosemary extract; (c) obtaining the at least one carrier, the at least one diluent, and the at least one excipient; and (d) contacting andrographolide, the rosemary extract, the at least
25 one carrier, the at least one diluent, and the at least one excipient to obtain said composition, wherein andrographolide to the rosemary extract w/w ratio in said composition is 1:0.33.
19

[0090] In an embodiment of the present disclosure, there is provided a method of preparing a composition comprising: (i) andrographolide; (ii) rosemary extract; (iii) at least one carrier; (iv) at least one diluent; and (v) at least one excipient, said method comprising: (a) obtaining andrographolide; (b) obtaining the rosemary extract; (c) 5 obtaining the at least one carrier, the at least one diluent, and the at least one excipient; and (d) contacting andrographolide, the rosemary extract, the at least one carrier, the at least one diluent, and the at least one excipient to obtain said composition, wherein andrographolide to the rosemary extract w/w ratio in said composition is 1:0.50.
10 [0091] In an embodiment of the present disclosure, there is provided a method of preparing a composition comprising: (i) andrographolide; (ii) rosemary extract; (iii) at least one carrier; (iv) at least one diluent; and (v) at least one excipient, said method comprising: (a) obtaining andrographolide; (b) obtaining the rosemary extract; (c) obtaining the at least one carrier, the at least one diluent, and the at least one
15 excipient; and (d) contacting andrographolide, the rosemary extract, the at least one carrier, the at least one diluent, and the at least one excipient to obtain said composition, wherein andrographolide w/w percentage in said composition is in the range of 0.000013- 0.000030%, the rosemary extract w/w percentage in said composition is in the range of 0.000005- 0.000012%, and andrographolide to the
20 rosemary extract w/w ratio in said composition is in the range of 1:0.2- 1:0.7.
[0092] In an embodiment of the present disclosure, there is provided a method of preparing a composition comprising: (i) andrographolide; (ii) rosemary extract; (iii) at least one carrier; (iv) at least one diluent; and (v) at least one excipient, said method comprising: (a) obtaining andrographolide; (b) obtaining the rosemary extract; (c)
25 obtaining the at least one carrier, the at least one diluent, and the at least one excipient; and (d) contacting andrographolide, the rosemary extract, the at least one carrier, the at least one diluent, and the at least one excipient to obtain said composition, wherein andrographolide w/w percentage in said composition is in the
20

range of 0.000013- 0.000030%, the rosemary extract w/w percentage in said composition is in the range of 0.000005- 0.000012%, and andrographolide to the rosemary extract w/w ratio in said composition is in the range of 1:0.33- 1:0.50. [0093] In an embodiment of the present disclosure, there is provided a method of 5 preparing a composition comprising: (i) andrographolide; (ii) rosemary extract; (iii) at least one carrier; (iv) at least one diluent; and (v) at least one excipient, said method comprising: (a) obtaining andrographolide; (b) obtaining the rosemary extract; (c) obtaining the at least one carrier, the at least one diluent, and the at least one excipient; and (d) contacting andrographolide, the rosemary extract, the at least one
10 carrier, the at least one diluent, and the at least one excipient to obtain said composition, wherein andrographolide w/w percentage in said composition is in the range of 0.0000195- 0.0000234%, the rosemary extract w/w percentage in said composition is in the range of 0.00000975- 0.0000078%, and andrographolide to the rosemary extract w/w ratio in said composition is in the range of 1:0.2- 1:0.7.
15 [0094] In an embodiment of the present disclosure, there is provided a method of preparing a composition comprising: (i) andrographolide; (ii) rosemary extract; (iii) at least one carrier; (iv) at least one diluent; and (v) at least one excipient, said method comprising: (a) obtaining andrographolide; (b) obtaining the rosemary extract; (c) obtaining the at least one carrier, the at least one diluent, and the at least one
20 excipient; and (d) contacting andrographolide, the rosemary extract, the at least one carrier, the at least one diluent, and the at least one excipient to obtain said composition, wherein andrographolide w/w percentage in said composition is in the range of 0.0000195- 0.0000234%, the rosemary extract w/w percentage in said composition is in the range of 0.00000975- 0.0000078%, and andrographolide to the
25 rosemary extract w/w ratio in said composition is in the range of 1:0.33- 1:0.50.
[0095] In an embodiment of the present disclosure, there is provided a method of preparing a composition comprising: (i) andrographolide; (ii) rosemary extract; (iii) at least one carrier; (iv) at least one diluent; and (v) at least one excipient, said method
21

comprising: (a) obtaining andrographolide; (b) obtaining the rosemary extract; (c) obtaining the at least one carrier, the at least one diluent, and the at least one excipient; and (d) contacting andrographolide, the rosemary extract, the at least one carrier, the at least one diluent, and the at least one excipient to obtain said 5 composition, wherein andrographolide w/w percentage in said composition is in the range of 0.000013- 0.000030%, the rosemary extract w/w percentage in said composition is in the range of 0.000005- 0.000012%, and andrographolide to the rosemary extract w/w ratio in said composition is 1:0.33. [0096] In an embodiment of the present disclosure, there is provided a method of
10 preparing a composition comprising: (i) andrographolide; (ii) rosemary extract; (iii) at least one carrier; (iv) at least one diluent; and (v) at least one excipient, said method comprising: (a) obtaining andrographolide; (b) obtaining the rosemary extract; (c) obtaining the at least one carrier, the at least one diluent, and the at least one excipient; and (d) contacting andrographolide, the rosemary extract, the at least one
15 carrier, the at least one diluent, and the at least one excipient to obtain said composition, wherein andrographolide w/w percentage in said composition is in the range of 0.000013- 0.000030%, the rosemary extract w/w percentage in said composition is in the range of 0.000005- 0.000012%, and andrographolide to the rosemary extract w/w ratio in said composition is 1:0.50.
20 [0097] In an embodiment of the present disclosure, there is provided a method of preparing a composition comprising: (i) andrographolide; (ii) rosemary extract; (iii) at least one carrier; (iv) at least one diluent; and (v) at least one excipient, said method comprising: (a) obtaining andrographolide; (b) obtaining the rosemary extract; (c) obtaining the at least one carrier, the at least one diluent, and the at least one
25 excipient; and (d) contacting andrographolide, the rosemary extract, the at least one carrier, the at least one diluent, and the at least one excipient to obtain said composition, wherein andrographolide w/w percentage in said composition is in the range of 0.0000195- 0.0000234%, the rosemary extract w/w percentage in said
22

composition is in the range of 0.00000975- 0.0000078%, and andrographolide to the rosemary extract w/w ratio in said composition is 1:0.33.
[0098] In an embodiment of the present disclosure, there is provided a method of preparing a composition comprising: (i) andrographolide; (ii) rosemary extract; (iii) at 5 least one carrier; (iv) at least one diluent; and (v) at least one excipient, said method comprising: (a) obtaining andrographolide; (b) obtaining the rosemary extract; (c) obtaining the at least one carrier, the at least one diluent, and the at least one excipient; and (d) contacting andrographolide, the rosemary extract, the at least one carrier, the at least one diluent, and the at least one excipient to obtain said
10 composition, wherein andrographolide w/w percentage in said composition is in the range of 0.0000195- 0.0000234%, the rosemary extract w/w percentage in said composition is in the range of 0.00000975- 0.0000078%, and andrographolide to the rosemary extract w/w ratio in said composition is 1:0.50. [0099] In an embodiment of the present disclosure, there is provided a method of
15 preparing a composition comprising: (i) andrographolide; (ii) rosemary extract; (iii) at least one carrier; (iv) at least one diluent; and (v) at least one excipient, said method comprising: (a) obtaining andrographolide; (b) obtaining the rosemary extract; (c) obtaining the at least one carrier, the at least one diluent, and the at least one excipient; and (d) contacting andrographolide, the rosemary extract, the at least one
20 carrier, the at least one diluent, and the at least one excipient to obtain said composition, wherein andrographolide w/w percentage in said composition is 0.0000234%, the rosemary extract w/w percentage in said composition is 0.0000078%, and andrographolide to the rosemary extract w/w ratio in said composition is 1:0.33.
25 [00100] In an embodiment of the present disclosure, there is provided a method
of preparing a composition comprising: (i) andrographolide; (ii) rosemary extract; (iii) at least one carrier; (iv) at least one diluent; and (v) at least one excipient, said method comprising: (a) obtaining andrographolide; (b) obtaining the rosemary
23

extract; (c) obtaining the at least one carrier, the at least one diluent, and the at least one excipient; and (d) contacting andrographolide, the rosemary extract, the at least one carrier, the at least one diluent, and the at least one excipient to obtain said composition, wherein andrographolide w/w percentage in said composition is 5 0.0000195%, the rosemary extract w/w percentage in said composition is 0.00000975%, and andrographolide to the rosemary extract w/w ratio in said composition is 1:0.50.
[00101] In an embodiment of the present disclosure, there is provided a
composition for use in preparing formulations, said composition comprising: (a)
10 andrographolide; and (b) rosemary extract, wherein andrographolide to the rosemary
extract w/w ratio in said composition is in the range of 1:0.2- 1:0.7.
[00102] In an embodiment of the present disclosure, there is provided a
composition for use in preparing formulations, said composition comprising: (a) andrographolide; and (b) rosemary extract, wherein andrographolide to the rosemary
15 extract w/w ratio in said composition is in the range of 1:0.33- 1:0.50.
[00103] In an embodiment of the present disclosure, there is provided a
composition for use in preparing formulations, said composition comprising: (a) andrographolide; and (b) rosemary extract, wherein andrographolide w/w percentage in said composition is in the range of 0.000013- 0.000030%, the rosemary extract
20 w/w percentage in said composition is in the range of 0.000005- 0.000012%, and andrographolide to the rosemary extract w/w ratio in said composition is in the range of 1:0.2- 1:0.7.
[00104] In an embodiment of the present disclosure, there is provided a
composition for use in preparing formulations, said composition comprising: (a)
25 andrographolide; and (b) rosemary extract, wherein andrographolide w/w percentage in said composition is in the range of 0.000013- 0.000030%, the rosemary extract w/w percentage in said composition is in the range of 0.000005- 0.000012%, and
24

andrographolide to the rosemary extract w/w ratio in said composition is in the range of 1:0.33- 1:0.50.
[00105] In an embodiment of the present disclosure, there is provided a
composition for use in preparing formulations, said composition comprising: (a) 5 andrographolide; and (b) rosemary extract, wherein andrographolide w/w percentage in said composition is in the range of 0.0000195- 0.0000234%, the rosemary extract w/w percentage in said composition is in the range of 0.00000975- 0.0000078%, and andrographolide to the rosemary extract w/w ratio in said composition is in the range of 1:0.2- 1:0.7.
10 [00106] In an embodiment of the present disclosure, there is provided a
composition for use in preparing formulations, said composition comprising: (a) andrographolide; and (b) rosemary extract, wherein andrographolide w/w percentage in said composition is in the range of 0.0000195- 0.0000234%, the rosemary extract w/w percentage in said composition is in the range of 0.00000975- 0.0000078%, and
15 andrographolide to the rosemary extract w/w ratio in said composition is in the range of 1:0.33- 1:0.50.
[00107] In an embodiment of the present disclosure, there is provided a
composition for use in preparing formulations, said composition comprising: (a) andrographolide; (b) rosemary extract; (c) at least one carrier; (b) at least one diluent;
20 and (c) at least one excipient, wherein andrographolide to the rosemary extract w/w ratio in said composition is in the range of 1:0.2- 1:0.7.
[00108] In an embodiment of the present disclosure, there is provided a
composition for use in preparing formulations, said composition comprising: (a) andrographolide; (b) rosemary extract; (c) at least one carrier; (b) at least one diluent;
25 and (c) at least one excipient, wherein andrographolide to the rosemary extract w/w ratio in said composition is in the range of 1:0.33- 1:0.50.
[00109] In an embodiment of the present disclosure, there is provided a
composition for use in preparing formulations, said composition comprising: (a)
25

andrographolide; (b) rosemary extract; (c) at least one carrier; (b) at least one diluent; and (c) at least one excipient, wherein andrographolide w/w percentage in said composition is in the range of 0.000013- 0.000030%, the rosemary extract w/w percentage in said composition is in the range of 0.000005- 0.000012%, and 5 andrographolide to the rosemary extract w/w ratio in said composition is in the range of 1:0.2- 1:0.7.
[00110] In an embodiment of the present disclosure, there is provided a
composition for use in preparing formulations, said composition comprising: (a) andrographolide; (b) rosemary extract; (c) at least one carrier; (b) at least one diluent;
10 and (c) at least one excipient, wherein andrographolide w/w percentage in said composition is in the range of 0.000013- 0.000030%, the rosemary extract w/w percentage in said composition is in the range of 0.000005- 0.000012%, and andrographolide to the rosemary extract w/w ratio in said composition is in the range of 1:0.33- 1:0.50.
15 [00111] In an embodiment of the present disclosure, there is provided a
composition for use in preparing formulations, said composition comprising: (a) andrographolide; (b) rosemary extract; (c) at least one carrier; (b) at least one diluent; and (c) at least one excipient, wherein andrographolide w/w percentage in said composition is in the range of 0.0000195- 0.0000234%, the rosemary extract w/w
20 percentage in said composition is in the range of 0.00000975- 0.0000078%, and andrographolide to the rosemary extract w/w ratio in said composition is in the range of 1:0.2- 1:0.7.
[00112] In an embodiment of the present disclosure, there is provided a
composition for use in preparing formulations, said composition comprising: (a)
25 andrographolide; (b) rosemary extract; (c) at least one carrier; (b) at least one diluent; and (c) at least one excipient, wherein andrographolide w/w percentage in said composition is in the range of 0.0000195- 0.0000234%, the rosemary extract w/w percentage in said composition is in the range of 0.00000975- 0.0000078%, and
26

andrographolide to the rosemary extract w/w ratio in said composition is in the range of 1:0.33- 1:0.50.
[00113] Although the subject matter has been described with reference to
specific embodiments, this description is not meant to be construed in a limiting 5 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
10 [00114] The disclosure will now be illustrated with working examples, which
is intended to illustrate the working of disclosure and not intended to take 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
15 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.
20 Example 1
Material and Methods
[00115] 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% CO2. DMEM (Dulbecco's Modified Eagle's
25 Medium) medium with 10% FBS (Fetal Bovine Serum) and 1% of suitable
27

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 1X PBS (Phosphate Buffer Saline) and stained with 25 μM DCFDA (2’, 7’ – dichlorofluorescein diacetate) for 30 minutes at 37 °C. After this step cells were 5 washed with 1X PBS followed by incubating cells with 1mM 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.
10 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’, 7’ – dichlorofluorescein (DCF).
15 [00116] Rosemary extract was obtained commercially from NATUREX and
has a Rosemarinic Acid concentration of at least 2%. Andrographolide was obtained commercially from Sigma Chemicals, product no. 365645.
Example 2
20 Cytotoxicity analysis of andrographolide and rosemary extract
[001] Andrographolide and rosemary extract were both tested to determine the cytotoxic effects of the two active ingredients. As seen in Figure 1, andrographolide was tested in a concentration ranging from 1.56 μg/ml to 50 μg/ml. The active did not show any demonstrable toxic effects up to the concentration of 6.25 μg/ml. At
25 concentrations higher than this, the percent cytotoxicity increased sharply. Thus concentrations of 6.25 μg/ml and below of andrographolide was used in all the following experiments.
28

[002] Cytotoxicity analysis of rosemary extract showed that this active had in general low toxicity levels. Concentrations of 6.25 μg/ml upwards had higher toxicity levels, for instance, a 3-fold increase in toxicity levels was seen from the concentration of 6.25 μg/ml to the next tested concentration of 12.5 μg/ml. Based on 5 these results, concentrations of 6.25 μg/ml and lower were tested for the following experiments.
Example 3
Effect of andrographolide and rosemary extract on intracellular ROS inhibition
[00117] In order to determine whether andrographolide and rosemary extract
10 could inhibit intracellular reactive oxygen species (ROS), the actives were tested at different individual concentrations using the DCFDA dye assay described above. As is seen in Figure 2, both the actives could inhibit ROS, and the degree of inhibition varied according to the concentrations used. ROS inhibition by andrographolide was within the range of 5-17%. The maximum inhibition at 17% was seen at 1.56 μg/ml 15 and 0.78 μg/ml.
[00118] Rosemary extract at different concentrations also showed
concentration-dependent ROS inhibition ability. ROS inhibition by this active ranged
between 7- 18%, with the maximum inhibition of 18% obtained at 1.56 μg/ml of
rosemary extract.
20 [00119] Overall, it can be inferred that both andrographolide and rosemary
extract are able to inhibit ROS production individually. However, as compared to the positive control quercetin, the inhibition levels demonstrated by both the actives were approximately two-fold lower.
25 Example 3
Effect of the combination of andrographolide and rosemary extract on intracellular ROS inhibition
29

[00120] As both andrographolide and rosemary extract could inhibit ROS
production, the combination of both the actives was tested to determine whether this composition demonstrated any enhanced inhibitory ability. Based on the data obtained in Figure 2, a maximum individual concentration of 0.39 μg/ml of 5 andrographolide and 0.195 μg/ml of rosemary extract were chosen for further analysis. All further concentrations tested in the different w/w ratios are a percentage of the maximum individual concentrations; for example, a 80:20 ratio of andrographolide to the rosemary extract represents 80% of 0.39 μg/ml, (which is calculated to be 0.312 μg/ml andrographolide), and 40% of 0.195 μg/ml (which is 10 calculated to be 0.039 μg/ml). The w/w ratios used in the composition have been described in Table 1 below.

Ratio
(andrographolide:
Rosemary) Concentration

andrographolide (0.39 ug/ml) Rosemary
extract
(0.195 ug/ml) w/w Ratio
(100:0) 0.39 0
(80:20) 0.312 0.039 1:0.125
(60:40) 0.234 0.078 1:0.33
(50:50) 0.195 0.0975 1:0.50
(40:60) 0.156 0.117 1:0.75
(20:80) 0.078 0.156 1:2
(0:100) 0 0.195
Table 1
[00121] As seen in Figure 3, inhibition of ROS production was observed at
15 varying degrees; however, not all combinations demonstrated synergism in inhibition of ROS production. Surprisingly, however, a 1:0.50 ratio and 1:0.33 ratio of andrographolide to the rosemary extract demonstrated a surprising synergistic effect in ROS inhibition. A 1:0.50 ratio of andrographolide to the rosemary extract showed
30

16% inhibition which is a two fold increase compared to either active in isolation, and further this percentage inhibition is higher than the expected values obtained by a mere admixture of the two active ingredients. Additionally, at 11% inhibition, a 1:0.33 ratio shows a 1.3 times increase in the inhibition of ROS production as 5 compared to the actives in isolation.
[00122] Advantages of present disclosure: Overall the present disclosure
provides a composition comprising andrographolide and rosemary extract which demonstrates an enhanced effect in the inhibition of ROS species. The synergism in the combination of the two active ingredients is particularly apparent at the w/w
10 ratios of 1:0.50 and 1:0.33. The 1:0.50 ratio of andrographolide to the rosemary extract, for instance, shows a two fold increase compared to either active in isolation, and is also demonstrably higher than expected values if a mere admixture of the two active ingredients is obtained. Further, the synergism of this combination is only apparent at specific ratios and combinations and is not seen at all combinations.
15 Therefore, it is not obvious to a person skilled at the art to arrive at this effective composition without undue experimentation.
31

I/We Claim:
1. A composition comprising:
(a) andrographolide; and
(b) rosemary extract,
5 wherein andrographolide to the rosemary extract w/w ratio in said composition is in the range of 1:0.2- 1:0.7.
2. The composition as claimed in claim 1, wherein andrographolide to the
rosemary extract w/w ratio in said composition is 1:0.5.
3. The composition as claimed in any of the claims 1-2, said composition further
10 comprising: (a) at least one carrier, (b) at least one diluent, and (c) at least one
excipient.
4. A method for preparing the composition as claimed in any of the claims 1-2,
said method comprising:
(a) obtaining andrographolide; 15 (b) obtaining the rosemary extract; and
(c) contacting andrographolide, and the rosemary extract to obtain said composition.
5. A method for preparing the composition as claimed in any of the claims 1-3, said
method comprising:
(a) obtaining andrographolide; 20 (b) obtaining the rosemary extract;
(c) obtaining the at least one carrier, the at least one diluent, and the at least one
excipient; and
(d) contacting andrographolide, the rosemary extract, the at least one carrier, the at
least one diluent, and the at least one excipient to obtain said composition.
25
32

6. The composition as claimed in any of the claims 1-5, wherein said composition manages oxidative stress responses.