WO2020153909A1

WO2020153909A1 - Natural compound compositions for treating medical conditions

Info

Publication number: WO2020153909A1
Application number: PCT/SG2020/050037
Authority: WO
Inventors: Edward Kai-Hua CHOW; Masturah MOHD ABDUL RASHID; Lissa HOOI
Original assignee: National University Of Singapore
Priority date: 2019-01-24
Filing date: 2020-01-24
Publication date: 2020-07-30

Abstract

The invention relates to natural compound compositions comprising at least three natural compounds. In particular, the natural compound compositions comprise fruit extract from Baobab and at least two compounds selected from the group consisting of Quercetin dihydrate, Emodin, Resveratrol, Lycium and betaine. The invention also relates to methods and uses of said natural compound compositions for treating fatty liver disorders, in particular non-alcoholic fatty liver disease and non-alcoholic steatohepatitis.

Description

NATURAL COMPOUND COMPOSITIONS FOR TREATING

MEDICAL CONDITIONS

Field of Invention

The invention relates generally to natural compound compositions comprising at least three natural compounds. The invention also relates generally to methods and uses of said compositions in treating medical conditions.

Background

Drug compounds are constantly being developed and used to treat various indications and diseases. However, while these drug compounds show efficacy in treating the targeted diseases, they often come with unavoidable and undesirable side effects. For example, stomach upset, nausea, drowsiness, skin irritation and hair loss are some common side effects that are associated with medications. Memory loss has been associated with benzodiazepines, statins, certain seizure medications, opioids, and incontinence drugs. Another example is some blood pressure and cholesterol drugs when taken together with fruit juice such as grapefruit juice, can affect the drug concentration in the blood which is undesirable. Accordingly, there is often a need for an alternative treatment regime.

Natural-based compounds have been well recognized to have health benefits, and are used as health supplements, food additives, cosmetics, or as leads to the development of therapeutics. These natural compounds can be found in food and beverages and can be sourced naturally. While some of the beneficial effects of natural compounds are known, they are often used in monotherapy and accordingly their effect can be limited.

Further, it is recognised in the art that the beneficial or therapeutic effect of some of these natural compounds can be limited as they are not optimised for use, and hence one trend of using natural compounds is to increase the dosage. However, this is inefficient and also translates to higher raw material and production costs. Non-alcoholic fatty liver disease (NAFLD) is growing to become the most common liver condition and is often associated with diabetes, high blood pressure, kidney disease and obesity. NAFLD is known to correlate with economic prosperity. With over 1 billion people afflicted with NAFLD, NAFLD can be considered an emerging global epidemic. A study reports an average of 20-30% of the population in US, Europe and Asia Pacific having NAFLD. In Asia, other studies have shown that NAFLD is prevalent in 54% of elderly female in Taiwan, 60% of people aged 45 or older in Malaysia, and at least 40% of the population in urbanised areas such as Beijing, Shanghai and Hong Kong.

NAFLD can be characterised by the presence of hepatic steatosis with no evidence of hepatocellular injury. Hepatic steatosis is a consequence of lipid acquisition exceeding lipid disposal; the diminished export results in hepatic lipid overload and subsequent intracellular lipid accumulation, leading to steatosis, lipotoxicity and liver damage, and promoting disease progression and fibrosis. Risk factors includes obesity, diabetes, arterial hypertension, dyslipidemia, insulin resistance and genetic factors. NAFLD can progress to nonalcoholic steatohepatitis (NASH).

While NAFLD and NASH are reversible conditions, there are currently no clinically approved pharmacological methods for preventing or treating NAFLD, with weight loss and lifestyle changes being the only clinically proven methods for alleviating or preventing progression of NAFLD. Nutritional supplements have been identified in small clinical studies and preclinical studies to have some therapeutic effect, however, clinical studies of monotherapy with these compounds rarely results in a sufficient increase in efficacy to warrant long-term use and does not outperform normal diet and lifestyle changes. For example, results from clinical trials of milk thistle for liver diseases have been mixed, and two rigorously designed studies have found no benefit. In another randomised trial of 99 patients, Silymarin did not statistically reduce NASH scores compared to placebo. This is likely due to the complex nature of NAFLD disease progression, complex interconnected networks of molecular mechanisms that regulate liver biology as well as the limited mechanisms by which single molecules can effect changes on such a robust biological process. As such, there is a need to develop improved treatment methodologies. Summary of the Invention

The present invention is predicated on the discovery that certain combinations of natural compounds are particularly advantageous in the treatment of fatty liver disorders and/or diseases, in particular NAFLD or NASH. In this regard, the inventors have found that certain combinations have a synergistic (or at least additive) effect for preventing and/or treating fatty liver disorders and/or diseases.

In an aspect, the present invention discloses a natural compound composition comprising Baobab, and at least two of the following compounds selected from the group consisting of Quercetin dihydrate, Emodin, Resveratrol, Lycium and betaine.

In an embodiment to the above aspect,

wherein Baobab is from about 4 mg/kg to about 12 mg/kg;

when Quercetin dihydrate is present, Quercetin dihydrate is from about 0.16 mg/kg to about 0.65 mg/kg;

when Emodin is present, Emodin is from about 1.6 mg/kg to about 6.4 mg/kg;

when Resveratrol is present, Resveratrol is from about 0.8 mg/kg to about 3.2 mg/kg; when Lycium is present, Lycium is from about 1.6 mg/kg to about 6.4 mg/kg; and when betaine is present, betaine is from about 2.4 mg/kg to about 7.2 mg/kg.

In some embodiment, the present invention provides a natural compound composition comprising:

a) Baobab;

b) Quercetin dihydrate; and

c) at least one or more further compound selected from Emodin, Resveratrol, Lycium and betaine.

a) Baobab; b) Quercetin dihydrate; and

c) Emodin.

a) Baobab;

b) Quercetin dihydrate;

c) Emodin; and

d) Resveratrol.

In all embodiments, Baobab is present from about 4 mg/kg to about 12 mg/kg.

In all embodiments, Quercetin dihydrate is present from about 0.16 mg/kg to about 0.65 mg/kg.

In some embodiments, the wt/wt ratio of Baobab to Quercetin dihydrate is from about 5 to about 50.

In some embodiments, Emodin is present from about 1.6 mg/kg to about 6.4 mg/kg.

In some embodiments, Resveratrol is present from about 0.8 mg/kg to about 3.2 mg/kg.

In other embodiments, Emodin and Resveratrol are both present, wherein Emodin is present from about 1.6 mg/kg to about 6.4 mg/kg and Resveratrol is present from about 0.8 mg/kg to about 3.2 mg/kg.

In other embodiments, the natural compound composition comprises:

a) Baobab from about 170 mg to about 400 mg;

b) Quercetin dihydrate from about 9 mg to about 20 mg; and

c) Emodin from about 9 mg to about 20 mg;

wherein the wt/wt ratio of Baobab to Quercetin dihydrate is from about 5 to about 50. In other embodiments, the natural compound composition comprises:

a) Baobab from about 170 mg to about 400 mg;

b) Quercetin dihydrate from about 9 mg to about 20 mg;

c) Emodin from about 90 mg to about 200 mg; and

d) Resveratrol from about 40 mg to about 100 mg;

wherein the wt/wt ratio of Baobab to Quercetin dihydrate is from about 5 to about 50.

a) Baobab;

b) Lycium; and

c) betaine.

In some embodiments, Lycium is present from about 1.6 mg/kg to about 6.4 mg/kg.

In some embodiments, betaine is present from about 2.4 mg/kg to about 7.2 mg/kg.

In other embodiments, the natural compound composition comprises:

a) Baobab from about 90 mg to about 200 mg;

b) Lycium 90 mg to about 200 mg; and

c) betaine from about 110 mg to about 240 mg;

the wt/wt ratio of Baobab to Lycium is from about 0.5 to about 5.

In certain embodiments, Baobab is a fruit extract derived from Adansonia genus of deciduous trees.

In certain embodiments, the natural compounds in the composition are plant based extracts.

In certain embodiments, the natural compounds in the composition are GRAS (Generally Recognised As Safe). In another embodiment, the composition further comprises a pharmaceutically acceptable excipient.

In other embodiments, the composition is administrable orally, intravenously or transdermally.

In an aspect, the present invention discloses a method of treating a fatty liver disorder and/or disease, comprising administering a natural composition, the composition comprising Baobab and at least two of the following compounds selected from the group consisting of Quercetin dihydrate, Emodin, Resveratrol, Lycium and betaine.

In an embodiment, the liver disorder or disease is non-alcoholic fatty liver disease (NAFLD) or non-alcoholic steatohepatitis (NASH).

In another embodiment, the non-alcoholic fatter liver disease is selected from simple fatty liver (hepatic steatosis).

In another aspect, the present invention provides a natural compound composition for use as a medicament in the treatment of a fatty liver disorder and/or disease, the composition comprising Baobab and at least two of the following compounds selected from the group consisting of Quercetin dihydrate, Emodin, Resveratrol, Lycium and betaine.

In another aspect, the present invention provides a use of a natural compound composition in the manufacture of a medicament for the treatment of a fatty liver disorder or disease, the composition comprising Baobab and at least two of the following compounds selected from the group consisting of Quercetin dihydrate, Emodin, Resveratrol, Lycium and betaine.

Brief Description of Drawings

Embodiments of the present invention will now be described, by way of non-limiting example, with reference to the drawings in which: Figure 1 illustrates an overview of the methodology for identifying suitable natural compound combinations for preventing and/or treating NAFLD;

Figure 2 illustrates an example of high content screening of lipid droplets in FFA- induced steatosis cells by Nile Red staining;

Figure 3 shows a representative plot of high content screening of 131 natural compounds (step 2);

Figure 4 illustrates a representative in vitro analysis for natural compounds orthogonal array composite design combination sets (step 3);

Figure 5 illustrates representative (A) antagonistic and (B) synergistic response surface maps;

Figure 6 shows an overview of in vivo mice model methodology for assessing natural compound orthogonal array composite design combination sets (step 4); Figure 7 illustrates the results from the in vivo mice model methodology of Figure 5, tabulating 30 representative orthogonal array composite design combination sets with respect to a control;

Figure 8 presents representative micrograph images of in vivo NAFLD progression from the standards and after combination treatment from Figure 6;

Figure 9 presents quantification of histological Oil-red O staining and gross images

(upper row of images) and micrograph images (lower row of images) of in vivo NAFLD progression from the standards and after combination treatment with selected 3-5 compound combinations; and

Figure 10 presents in vivo interventional results of mice after 4 and 8 months of high fat diet with and without treatment

Detailed Description

Drug combinations have been the mainstay for many indications and complex human diseases. However, the extensive drug dosing space has made it arduous to come up with the most optimal drug combinations coupled with their corresponding drug concentrations. Further, the skilled person would know that a combination of drug compounds can produce either an antagonistic (cancels out each other), additive or synergistic (to produce a combined effect greater than sum of separate effects), and such responses are not predictable in the art. With particular reference to NAFLD and/or NASH, determining the best combination of compounds to treat NAFLD and/or NASH remains a challenge due to unknown interacting effects between compounds that may decrease overall efficacy as well as antagonize and impair each other. It should further be appreciated that NAFLD and/or NASH disease progression is of a complex nature; complex interconnected networks of molecular mechanisms exist that regulate liver biology as well as the limited mechanisms by which single molecules can effect changes on such a robust biological process.

The inventors are of the opinion that natural products continue to play an important role as precursors or leads to active metabolites for synthetic drugs. For example, from 1981 to 2006, 62% of new small molecules were derived from natural products or their derivatives. Considering the pivotal role of natural compounds in the drug development space, the inventors envisioned that repurposing and combining these natural compounds (i.e. not the synthetic drug products) for different indications or diseases would stretch the potential of these natural compounds. Further, as these natural compounds are derived or extracted from natural and safe sources, they can be more readily available and can be manufactured at a lower cost. These natural compounds are also known to have no or less serious side effects.

In this regard, the inventors have found that particular combinations of natural compounds are beneficial for treating fatty liver disorder and/or disease. In particular, the inventors have found that certain combinations of natural compounds may exhibit an additive or synergistic effect with respect to treating NAFLD and/or NASH when subjected to in vitro and in vivo testing. These natural compounds combinations can be formulated as compositions, which can be utilised as health supplements or as medicament for treating NAFLD and/or NASH.

As used herein, 'natural compound' refers to a substance which is produced by a living organism. In this regard, natural compounds can be extracted from microorganism, plants and/or animals. This definition also includes derivatives of natural compounds, which can include simple modification or functionalisation for, for example, increasing the solubility or compounding ability of the natural compounds. This definition also includes the pure compound. While natural compounds are usually produced and extracted from organisms, these compounds, once known, can also be prepared by chemically synthesis (both semisynthesis and total synthesis) via known methodologies.

The natural compound compositions of the present invention are provided at effective amounts and which is suitable for use in treating fatty liver disease. In this regard, the present invention includes within its scope, natural compound compositions having at least an additive effect from the overall combination of the compounds in the composition. Preferentially, the composition has a synergistic effect from the overall combination of the compounds in the composition. Accordingly, compositions having an overall antagonistic effect are not suitable for use in treating fatty liver disorders are excluded from this invention.

The present invention discloses a natural compound composition comprising at least three of the following compounds selected from the group consisting of Baobab, Quercetin dihydrate, Emodin, Resveratrol, Lycium and betaine.

The inventors have found that Baobab can advantageously be used for treating NAFLD and/or NASH. In particular, the inventors have found that certain compositions, when comprising Baobab, have a synergistic (or at least additive) effect for treating NAFLD and/or NASH. As is known in the art, Baobab is the common name of a genus of trees (Adansonia). Derived from the baobab tree that is native to Africa, baobab fruit powder is rich in anti oxidants and a range of known vitamins and minerals that are considered nutritionally beneficial. For the first time and after preliminary studies, and without wanting to be bound by theory, the inventors are of the opinion that oral consumption of baobab fruit powder is able to provide hepatoprotective effects. In this regard, it is believed that Baobab provides nutrients for the proper functioning of the body such as vitamin C, vitamin B6, Niacin, Iron, Potassium, Magnesium, and Calcium. Further, it is high in fiber, which helps curb excessive calorie intake which may cause the liver to work excessively. The inventors have also found that the beneficial effects from Baobab and other components in the natural compounds composition as disclosed herein can be amplified based on the additive or synergistic combinations. In this regard, the baobab-containing combinations shown in Table 5 and the validation of those combinations in Figure 9 are believed to be the first reported evidence of the use of dried baobab fruit powder as a synergistic or additive combination. The skilled person would know that dried baobab fruit powder is widely available off the shelf and it does not represent a purified specific molecule. For example, Baobab can be sourced from Powbab, USA. Other sources of Baobab are FGO and Aduna (purchasable from Amazon), MRM (purchasable from iHerb) and LVL (purchasable from Lazada).

Accordingly, in all embodiments, the natural compound composition comprises Baobab (or Baubab). The Baobab can be in a dry powder form. The Baobab powder can be from the pulp of the Baobab fruit and/or the Baobab seed. In certain embodiments, Baobab is a fruit extract derived from Adansonia genus of deciduous trees.

Accordingly, the present invention provides a natural compound composition comprising Baobab and at least two of the following compounds selected from the group consisting of Quercetin dihydrate, Emodin, Resveratrol, Lycium and betaine.

In an embodiment,

wherein Baobab is from about 4 mg/kg to about 12 mg/kg;

when Emodin is present, Emodin is from about 1.6 mg/kg to about 6.4 mg/kg

when Resveratrol is present, Resveratrol is from about 0.8 mg/kg to about 3.2 mg/kg when Lycium is present, Lycium is from about 1.6 mg/kg to about 6.4 mg/kg

when betaine is present, betaine is from about 2.4 mg/kg to about 7.2 mg/kg.

In some embodiments, the composition comprises:

a) Baobab; and

b) at least two or more further compound selected from Emodin, Resveratrol, Quercetin dihydrate, Lycium and betaine. The inventors have found that certain combinations of compounds provide a beneficial effect for treating fatty liver disease. In this regard, Table 5 shows a list of compositions that the inventors have found to have an overall additive or synergistic effect.

In some embodiments, the composition comprises:

a) Baobab;

b) Emodin; and

c) at least one or more further compound selected from Quercetin dihydrate, Resveratrol, Lycium and betaine.

In some embodiments, the composition comprises:

a) Baobab;

b) Resveratrol; and

c) at least one or more further compound selected from Quercetin dihydrate, Emodin, Lycium and betaine.

a) Baobab;

b) Quercetin dihydrate; and

c) Emodin.

a) Baobab;

b) Quercetin dihydrate;

c) Emodin; and

d) Resveratrol.

In some embodiments, the composition comprises:

a) Baobab; b) Lycium; and

c) at least one or more further compound selected from Quercetin dihydrate, Resveratrol, Emodin and betaine.

In some embodiments, the composition comprises:

a) Baobab;

b) betaine; and

c) at least one or more further compound selected from Quercetin dihydrate, Resveratrol, Lycium and Emodin.

a) Baobab;

b) Lycium; and

c) betaine.

Preferentially, in some embodiments, the present invention discloses a natural compound composition comprising:

a) Baobab;

b) Quercetin dihydrate; and

Advantageously, the inventors have found that the inclusion of Baobab and Quercetin can provide a strong synergistic effect to the composition. Further, this synergistic effect (when at the correct compounds concentration) can be stronger than all the other combined effect from the interaction of the other compounds, which thus accordingly provide for an overall synergistic interaction for the composition. Without wanting to be bound by theory, it is believed that the polyphenols in Quercetin is protected from degradation when used in combination with Baobab. Further, the polyphenols in Quercetin form a complex with the components of Baobab (for example the hydrophilic fibers) such that its anti-oxidant effect can be enhanced. In this regard, it is believed that this brings about the synergistic effect of the combination.

In some embodiments, food grade Baobab is used. In particular, the fruit of Baobab is used. In other embodiments, >99% purified Baobab is used. In other embodiments, Baobab is purified to >98%, >97%, >96%, >95%, >94%, >93%, >92%, >91%, >90%, >85%, >80%, >75% or >70%. In some embodiments, when dosed in mouse, Baobab is present from about 50 mg/kg to about 150 mg/kg. In other embodiments, when dosed in mouse, Baobab is present from about 60 mg/kg to about 140 mg/kg, about 70 mg/kg to about 130 mg/kg or about 80 mg/kg to about 120 mg/kg. The skilled person would understand that a mouse dosage can be converted to a human equivalent dosage. Accordingly, in another embodiment, when dosed in humans, Baobab is present from about 4 mg/kg to about 12 mg/kg, or from about 4.05 mg/kg to about 12.15 mg/kg. In another embodiment, when dosed in humans, Baobab is present from about 4.86 mg/kg to about 11.34 mg/kg, about 5.67 mg/kg to about 10.53 mg/kg or about 6.48 mg/kg to about 9.72 mg/kg.

In some embodiments, the natural compound composition comprising:

a) Baobab from about 4 mg/kg to about 12 mg/kg;

b) Quercetin dihydrate; and

In some embodiment, the natural compound composition comprising:

a) Baobab from about 4 mg/kg to about 12 mg/kg;

b) Quercetin dihydrate; and

c) Emodin.

In some embodiment, the natural compound composition comprising:

a) Baobab from about 4 mg/kg to about 12 mg/kg;

b) Quercetin dihydrate;

c) Emodin; and d) Resveratrol.

In some embodiments, the natural compound composition comprises Quercetin dihydrate. Quercetin is a plant pigment (flavonoid). It is found in many plants and foods, such as red wine, onions, green tea, apples, berries, Ginkgo biloba, St. John's wort, American elder, and others. Buckwheat tea has a large amount of quercetin. An example of Quercetin used in the present invention is Quercetin dihydrate (2-(3,4-Dihydroxyphenyl)-3,5,7-trihydroxy-4H-l- benzopyran-4-one dihydrate). Quercetin dihydrate is obtainable as a solid (powder) from, for example, Sigma- Aldrich. In some embodiments, food grade quercetin dihydrate is used. In other embodiments, >99% purified Quercetin dihydrate is used. In other embodiments, Quercetin dihydrate is purified to >98%, >97%, >96%, >95%, >94%, >93%, >92%, >91%, >90%, >85%, >80%, >75% or >70%. In some embodiments, when dosed in mouse, Quercetin dihydrate is present from about 2 mg/kg to about 8 mg/kg. In other embodiments, when dosed in mouse, Quercetin dihydrate is present from about 3 mg/kg to about 8 mg/kg, about 3 mg/kg to about 7 mg/kg or about 4 mg/kg to about 7 mg/kg. The skilled person would understand that a mouse dosage can be converted to a human equivalent dosage. Accordingly, in another embodiment, when dosed in humans, Quercetin dihydrate is present from about 0.16 mg/kg to about 0.65 mg/kg. In another embodiment, when dosed in humans, Quercetin dihydrate is present from about 0.24 mg/kg to about 0.65 mg/kg, about 0.24 mg/kg to about 0.56 mg/kg or about 0.32 mg/kg to about 0.56 mg/kg.

In some embodiments, the natural compound composition comprising:

a) Baobab;

b) Quercetin dihydrate from about 0.16 mg/kg to about 0.65 mg/kg; and

In other embodiments, the natural compound composition comprising:

a) Baobab from about 4 mg/kg to about 12 mg/kg;

b) Quercetin dihydrate from about 0.16 mg/kg to about 0.65 mg/kg; and c) at least one or more further compound selected from Emodin, Resveratrol, Lycium and betaine.

In some embodiment, the natural compound composition comprising:

a) Baobab from about 4 mg/kg to about 12 mg/kg;

b) Quercetin dihydrate from about 0.16 mg/kg to about 0.65 mg/kg; and

c) Emodin.

In some embodiment, the natural compound composition comprising:

a) Baobab from about 4 mg/kg to about 12 mg/kg;

b) Quercetin dihydrate from about 0.16 mg/kg to about 0.65 mg/kg;

c) Emodin; and

d) Resveratrol.

For the synergistic effect to be maximised, the wt/wt ratio of Baobab to Quercetin can be in a specific range. In some embodiments, amount of Baobab is in excess to the amount of Quercetin. In some embodiments, the wt/wt ratio of Baobab to Quercetin dihydrate is from about 5 to about 50. In other embodiments, the wt/wt ratio is from about 10 to about 50, about 10 to about 45, about 10 to about 40, about 12 to about 40, about 14 to about 40, about 16 to about 40, about 18 to about 40, about 20 to about 40 or about 25 to about 40. In some embodiments, the wt/wt ratio is about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45 or about 50.

In some embodiments, the natural compound composition comprising:

a) Baobab from about 4 mg/kg to about 12 mg/kg;

b) Quercetin dihydrate from about 0.16 mg/kg to about 0.65 mg/kg; and

c) at least one or more further compound selected from Emodin, Resveratrol, Lycium and betaine;

In some embodiment, the natural compound composition comprising: a) Baobab from about 4 mg/kg to about 12 mg/kg;

b) Quercetin dihydrate from about 0.16 mg/kg to about 0.65 mg/kg; and

c) Emodin;

In some embodiment, the natural compound composition comprising:

a) Baobab from about 4 mg/kg to about 12 mg/kg;

b) Quercetin dihydrate from about 0.16 mg/kg to about 0.65 mg/kg;

c) Emodin; and

d) Resveratrol;

In some embodiments, the natural compound composition comprises at least one or more further compound selected from Emodin, Resveratrol, Lycium and betaine. Without wanting to be bound by theory, the inventors believe that the spaced apart phenol structure of Emodin and Resveratrol is advantageous for the targeted indication.

In some embodiments, the natural compound composition further comprises Emodin. Emodin (6-methyl-l,3,8-trihydroxyanthraquinone) is a chemical compound that can be isolated from rhubarb, buckthorn, and Japanese knotweed (Fallopia japonica syn. Polygonum cuspidatum). It is also produced by many species of fungi, including members of the genera Aspergillus, Pyrenochaeta, and Pestalotiopsis, inter alia. The common name is derived from Rheum emodi (Himalayan rhubarb) and synonyms include emodol, frangula emodin, rheum emodin, Schuttgelb, and Persian Berry Lake. Emodin can, for example, be obtained from Sigma-Aldrich as a solid extracted from Frangula bark with >90% purity (HPLC). In some embodiments, food grade Emodin is used. In other embodiments, >98% purified Emodin is used. In other embodiments, Emodin is purified to >97%, >96%, >95%, >94%, >93%, >92%, >91%, >90%, >85%, >80%, >75% or >70%.

In some embodiments, when dosed in mouse, Emodin is present from about 20 mg/kg to about 80 mg/kg. In other embodiments, when dosed in mouse, Emodin is present from about 20 mg/kg to about 75 mg/kg, about 20 mg/kg to about 70 mg/kg, about 25 mg/kg to about 70 mg/kg, about 30 mg/kg to about 70 mg/kg, about 30 mg/kg to about 65 mg/kg or about 35 mg/kg to about 65 mg/kg. The skilled person would understand that a mouse dosage can be converted to a human equivalent dosage. Accordingly, in another embodiment, when dosed in humans, Emodin is present from about 1.6 mg/kg to about 6.5 mg/kg. In another embodiment, when dosed in humans, Emodin is present from about E6 mg/kg to about 6.1 mg/kg, about E6 mg/kg to about 5.7 mg/kg, about 2 mg/kg to about 5.7 mg/kg, about 2.4 mg/kg to about 5.7 mg/kg, about 2.4 mg/kg to about 5.3 mg/kg or about 2.8 mg/kg to about 5.3 mg/kg.

Accordingly, in some embodiment, the natural compound composition comprises:

a) Baobab;

b) Quercetin dihydrate;

c) Emodin; and

d) optionally one or more further compound selected from Resveratrol, Lycium and betaine.

In some embodiments, the natural compound composition comprising:

a) Baobab from about 4 mg/kg to about 12 mg/kg;

b) Quercetin dihydrate from about 0.16 mg/kg to about 0.65 mg/kg; and

c) Emodin; and

In other embodiments, the natural compound composition comprising:

a) Baobab from about 4 mg/kg to about 12 mg/kg;

b) Quercetin dihydrate from about 0.16 mg/kg to about 0.65 mg/kg; and

c) Emodin from about 1.6 mg/kg to about 6.4 mg/kg; and

d) optionally one or more further compound selected from Resveratrol, Lycium and betaine. In some embodiment, the natural compound composition comprising:

a) Baobab from about 4 mg/kg to about 12 mg/kg;

b) Quercetin dihydrate from about 0.16 mg/kg to about 0.65 mg/kg; and

c) Emodin from about 1.6 mg/kg to about 6.4 mg/kg;

In some embodiment, the natural compound composition comprising:

a) Baobab from about 4 mg/kg to about 12 mg/kg;

b) Quercetin dihydrate from about 0.16 mg/kg to about 0.65 mg/kg;

c) Emodin from about 1.6 mg/kg to about 6.4 mg/kg; and

d) Resveratrol;

In some embodiments, the wt/wt ratio of Baobab to Emodin is from about 1 to about 5. In other embodiments, the wt/wt ratio is from about 1 to about 4, about 1 to about 3, or about 1 to about 2. In other embodiments, the wt/wt ratio is about 1, about 2, about 3, about 4 or about 5.

In some embodiment, the natural compound composition comprising:

a) Baobab from about 4 mg/kg to about 12 mg/kg;

b) Quercetin dihydrate from about 0.16 mg/kg to about 0.65 mg/kg; and

c) Emodin from about 1.6 mg/kg to about 6.4 mg/kg;

wherein the wt/wt ratio of Baobab to Quercetin dihydrate is from about 5 to about 50; wherein the wt/wt ratio of Baobab to Emodin is from about 1 to about 5.

In some embodiment, the natural compound composition comprising:

a) Baobab from about 4 mg/kg to about 12 mg/kg;

b) Quercetin dihydrate from about 0.16 mg/kg to about 0.65 mg/kg;

c) Emodin from about 1.6 mg/kg to about 6.4 mg/kg; and

d) Resveratrol;

In some embodiments, the natural compound composition can comprise Resveratrol. As is known in the art, Resveratrol (3,5,4'-trihydroxy-trans-stilbene) is a stilbenoid, which is a type of natural phenol. Sources of resveratrol in food include the skin of grapes, blueberries, raspberries, mulberries, and peanuts. Resveratrol, for example, can be obtained from Sigma Aldrich as a solid. In some embodiments, food grade Resveratrol is used. In other embodiments, >98% purified Resveratrol is used. In other embodiments, Resveratrol is purified to >97%, >96%, >95%, >94%, >93%, >92%, >91%, >90%, >85%, >80%, >75% or >70%.

In some embodiments, when dosed in mouse, Resveratrol is present from about 10 mg/kg to about 40 mg/kg. In other embodiments, when dosed in mouse, Resveratrol is present from about 12 mg/kg to about 38 mg/kg, about 14 mg/kg to about 38 mg/kg, about 14 mg/kg to about 36 mg/kg, about 14 mg/kg to about 34 mg/kg, about 16 mg/kg to about 34 mg/kg, about 18 mg/kg to about 34 mg/kg, about 20 mg/kg to about 34 mg/kg, about 22 mg/kg to about 34 mg/kg, about 24 mg/kg to about 34 mg/kg, about 26 mg/kg to about 34 mg/kg, about 28 mg/kg to about 34 mg/kg, about 30 mg/kg to about 34 mg/kg or about 32 mg/kg to about 34 mg/kg. The skilled person would understand that a mouse dosage can be converted to a human equivalent dosage. Accordingly, in another embodiment, when dosed in humans, Resveratrol is present from about 0.8 mg/kg to about 3.2 mg/kg, or from about 0.8 mg/kg to about 3.24 mg/kg. In other embodiments, when dosed in humans, Resveratrol is present from about 0.97 mg/kg to about 3 mg/kg, about 1.1 mg/kg to about 3 mg/kg, about 1.1 mg/kg to about 2.9 mg/kg, about 1.1 mg/kg to about 2.8 mg/kg, about 1.3 mg/kg to about 2.8 mg/kg, about 1.4 mg/kg to about 2.8 mg/kg, about 1.6 mg/kg to about 2.8 mg/kg, about 1.8 mg/kg to about 2.8 mg/kg, about 1.9 mg/kg to about 2.8 mg/kg, about 2.1 mg/kg to about 2.8 mg/kg, about 2.2 mg/kg to about 2.8 mg/kg, about 2.4 mg/kg to about 2.8 mg/kg or about 2.6 mg/kg to about 2.8 mg/kg.

Accordingly, in some embodiment, the natural compound composition comprises:

a) Baobab; b) Quercetin dihydrate;

c) Resveratrol; and

d) optionally one or more further compound selected from Emodin, Lycium and betaine.

In some embodiments, the natural compound composition comprising:

a) Baobab from about 4 mg/kg to about 12 mg/kg;

b) Quercetin dihydrate from about 0.16 mg/kg to about 0.65 mg/kg; and

c) Resveratrol; and

In other embodiments, the natural compound composition comprising:

a) Baobab from about 4 mg/kg to about 12 mg/kg;

b) Quercetin dihydrate from about 0.16 mg/kg to about 0.65 mg/kg; and

c) Resveratrol from about 0.8 mg/kg to about 3.2 mg/kg; and

In some embodiments, the natural compound composition comprises both Emodin and Resveratrol. The concentration of Emodin and Resveratrol are as disclosed herein.

Accordingly, in some embodiment, the natural compound composition comprises:

a) Baobab;

b) Quercetin dihydrate;

c) Emodin;

d) Resveratrol; and

e) optionally one or more further compound selected from Lycium and betaine.

In some embodiments, the natural compound composition comprising:

a) Baobab from about 4 mg/kg to about 12 mg/kg; b) Quercetin dihydrate from about 0.16 mg/kg to about 0.65 mg/kg; and

c) Emodin;

d) Resveratrol; and

e) optionally one or more further compound selected from, Lycium and betaine.

In some embodiments, the natural compound composition comprising:

a) Baobab from about 4 mg/kg to about 12 mg/kg;

b) Quercetin dihydrate from about 0.16 mg/kg to about 0.65 mg/kg; and

c) Emodin from about E6 mg/kg to about 6.4 mg/kg;

d) Resveratrol from about 0.8 mg/kg to about 3.2 mg/kg; and

e) optionally one or more further compound selected from Lycium and betaine.

In some embodiment, the natural compound composition comprising:

a) Baobab from about 4 mg/kg to about 12 mg/kg;

b) Quercetin dihydrate from about 0.16 mg/kg to about 0.65 mg/kg;

c) Emodin from about 1.6 mg/kg to about 6.4 mg/kg; and

d) Resveratrol from about 0.8 mg/kg to about 3.2 mg/kg;

In some embodiments, the wt/wt ratio of Baobab to Resveratrol is from about 3 to about 10. In other embodiments, the wt/wt ratio is from about 3 to about 9, about 3 to about 8, about 3 to about 7, about 3 to about 6, or about 3 to about 5. In other embodiments, the wt/wt ratio is about 3, about 4, about 5, about 6, about 7, about 8 or about 9.

In some embodiment, the natural compound composition comprising:

a) Baobab from about 4 mg/kg to about 12 mg/kg;

b) Quercetin dihydrate from about 0.16 mg/kg to about 0.65 mg/kg;

c) Emodin from about 1.6 mg/kg to about 6.4 mg/kg; and

d) Resveratrol from about 0.8 mg/kg to about 3.2 mg/kg;

wherein the wt/wt ratio of Baobab to Quercetin dihydrate is from about 5 to about 50; wherein the wt/wt ratio of Baobab to Emodin is from about 1 to about 5; wherein the wt/wt ratio of Baobab to Resveratrol is from about 3 to about 10.

The inventors have also advantageously found another synergistic effect in the combination of Baobab and Lycium. In this regard, and without wanting to be bound by theory, it is believed the hydrophilic fibers in Baobab can form complexes with the glyconjugates (carbohydrates covalently linked with other chemical species such as proteins, peptides, lipids and saccharides) in Lycium due to the like-like attraction. This prevents or slow down the degradation of Lycium when ingested.

In some embodiments, the natural compound composition comprises Lycium. As is known in the art, Lycium, is commonly known as goji berry. The inventors believe that oral consumption of Lycium is able to provide hepatoprotective effects. Examples of Lycium is lycium barbarum and/or lyceum chinense. Lycium barbarum is, for example, available as a powder obtained from the dried fruit. Lycium Barbarum Polysaccharides can also be used, which is obtainable from Lycium bararum powder after purification by for example ion- exchange and/or gel chromatography. In some embodiments, food grade Lycium is used. In other embodiments, >55% purified Lyucium is used. In other embodiments, Lycium is purified to >50%, >45%, >40%, >35% or >30%.

In some embodiments, when dosed in mouse, Lycium is present from about 20 mg/kg to about 80 mg/kg. In other embodiments, when dosed in mouse, Lycium is present from about 25 mg/kg to about 80 mg/kg, about 30 mg/kg to about 80 mg/kg, about 30 mg/kg to about 75 mg/kg, about 30 mg/kg to about 70 mg/kg, about 30 mg/kg to about 67 mg/kg, about 35 mg/kg to about 67 mg/kg, about 40 mg/kg to about 67 mg/kg, about 45 mg/kg to about 67 mg/kg, about 50 mg/kg to about 67 mg/kg or about 55 mg/kg to about 67 mg/kg. The skilled person would understand that a mouse dosage can be converted to a human equivalent dosage. Accordingly, in another embodiment, when dosed in humans, Lycium is present from about 1.6 mg/kg to about 6.4 mg/kg. In other embodiments, when dosed in humans, Lycium is present from about 2 mg/kg to about 6.4 mg/kg, about 2.4 mg/kg to about 6.4 mg/kg, about 2.4 mg/kg to about 6.1 mg/kg, about 2.4 mg/kg to about 5.7 mg/kg, about 2.4 mg/kg to about 5.4 mg/kg, about 2.8 mg/kg to about 5.4 mg/kg, about 3.2 mg/kg to about 5.4 mg/kg, about 3.6 mg/kg to about 5.4 mg/kg, about 4 mg/kg to about 5.4 mg/kg or about

4.5 mg/kg to about 5.4 mg/kg.

Accordingly, in some embodiment, the natural compound composition comprises:

a) Baobab;

b) Quercetin dihydrate;

c) Emodin;

d) Lycium; and

e) optionally one or more further compound selected from Resveratrol and betaine.

In some embodiments, the natural compound composition comprising:

a) Baobab from about 4 mg/kg to about 12 mg/kg;

b) Quercetin dihydrate from about 0.16 mg/kg to about 0.65 mg/kg; and

c) Emodin from about 1.6 mg/kg to about 6.4 mg/kg;

d) Lycium from about 1.6 mg/kg to about 6.4 mg/kg; and

In some embodiments, the natural compound composition comprises:

a) Baobab;

b) Quercetin dihydrate;

c) Emodin;

d) Lycium; and

e) Resveratrol.

In some embodiments, the natural compound composition comprising:

a) Baobab from about 4 mg/kg to about 12 mg/kg;

b) Quercetin dihydrate from about 0.16 mg/kg to about 0.65 mg/kg; and

c) Emodin from about 1.6 mg/kg to about 6.4 mg/kg;

d) Lycium from about 1.6 mg/kg to about 6.4 mg/kg; and

e) Resveratrol from about 0.8 mg/kg to about 3.2 mg/kg. In some embodiment, the natural compound composition comprising:

a) Baobab from about 4 mg/kg to about 12 mg/kg;

b) Lycium 1.6 mg/kg to about 6.4 mg/kg; and

c) betaine.

In some embodiments, the wt/wt ratio of Baobab to Lycium is from about 0.5 to about 5. In other embodiments, the wt/wt ratio is about 0.5 to about 4, about 0.5 to about 3, about 0.5 to about 2, or about 0.5 to about 1. In other embodiments, the wt/wt ratio is about 0.5, about 1, about 1.5, about 2, about 2.5, about 3, about 3.5, about 4, about 4.5 or about 5.

In some embodiment, the natural compound composition comprising:

a) Baobab from about 4 mg/kg to about 12 mg/kg;

b) Lycium 1.6 mg/kg to about 6.4 mg/kg; and

c) betaine;

wherein the wt/wt ratio of Baobab to Lycium is from about 0.5 to about 5.

In some embodiments, the natural compound composition further comprises betaine. One form of betaine is N,N,N-trimethylglycine, which refers to a small N-trimethylated amino acid, existing in zwitterionic form at neutral pH. It was named after its discovery in sugar beet (Beta vulgaris subsp. vulgaris) in the nineteenth century. In the 21st century, this substance is called glycine betaine to distinguish it from other betaines that are widely distributed in microorganisms, plants, and animals. In this regard, a betaine is any neutral chemical compound with a positively charged cationic functional group such as a quaternary ammonium or phosphonium cation (generally: onium ions) that bears no hydrogen atom and with a negatively charged functional group such as a carboxylate group that may not be adjacent to the cationic site. A betaine thus may be a specific type of zwitterion. For example, betaine is available as a solid from Sigma- Aldrich. In some embodiments, betaine anhydrous is used. In other embodiments, food grade betaine is used. In other embodiments, >99% purified betaine is used. In other embodiments, betaine is purified to >98%, >97%, >96%, >95%, >94%, >93%, >92%, >91%, >90%, >85%, >80%, >75% or >70%. In some embodiments, when dosed in mouse, betaine is present from about 30 mg/kg to about 90 mg/kg. In other embodiments, when dosed in mouse, betaine is present from about 35 mg/kg to about 90 mg/kg, about 40 mg/kg to about 90 mg/kg, about 40 mg/kg to about 85 mg/kg, about 40 mg/kg to about 80 mg/kg, about 45 mg/kg to about 80 mg/kg, about 50 mg/kg to about 80 mg/kg, about 55 mg/kg to about 80 mg/kg, about 60 mg/kg to about 80 mg/kg or about 65 mg/kg to about 80 mg/kg. The skilled person would understand that a mice dosage can be converted to a human equivalent dosage. Accordingly, in another embodiment, when dosed in humans, betaine is present from about 2.4 mg/kg to about 7.2 mg/kg. In other embodiments, when dosed in humans, betaine is present from about 2.8 mg/kg to about 7.2 mg/kg, about 3.2 mg/kg to about 7.2 mg/kg, about 3.2 mg/kg to about 6.8 mg/kg, about 3.2 mg/kg to about 6.4 mg/kg, about 3.6 mg/kg to about 6.4 mg/kg, about 4 mg/kg to about 6.4 mg/kg, about 4.4 mg/kg to about 6.4 mg/kg, about 4.8 mg/kg to about 6.4 mg/kg or about 5.2 mg/kg to about 6.4 mg/kg.

Accordingly, in some embodiment, the natural compound composition comprises:

a) Baobab;

b) Quercetin dihydrate;

c) Emodin;

d) betaine; and

e) optionally one or more further compound selected from Resveratrol and Lycium.

In some embodiments, the natural compound composition comprising:

a) Baobab from about 4 mg/kg to about 12 mg/kg;

b) Quercetin dihydrate from about 0.16 mg/kg to about 0.65 mg/kg; and

c) Emodin from about 1.6 mg/kg to about 6.4 mg/kg;

d) betaine from about 2.4 mg/kg to about 7.2 mg/kg; and

In some embodiment, the natural compound composition comprises:

a) Baobab;

b) Quercetin dihydrate; c) Emodin;

d) betaine; and

e) Resveratrol.

In some embodiments, the natural compound composition comprising:

a) Baobab from about 4 mg/kg to about 12 mg/kg;

b) Quercetin dihydrate from about 0.16 mg/kg to about 0.65 mg/kg; and

c) Emodin from about 1.6 mg/kg to about 6.4 mg/kg;

d) betaine from about 2.4 mg/kg to about 7.2 mg/kg; and

e) Resveratrol from about 0.8 mg/kg to about 3.2 mg/kg.

In some embodiment, the natural compound composition comprising:

a) Baobab from about 4 mg/kg to about 12 mg/kg;

b) Lycium from about 1.6 mg/kg to about 6.4 mg/kg; and

c) betaine from about 2.4 mg/kg to about 7.2 mg/kg.

In some embodiments, the wt/wt ratio of Baobab to betaine is from about 0.5 to about 5. In other embodiments, the wt/wt ratio is about 0.5 to about 4, about 0.5 to about 3, about 0.5 to about 2, or about 0.5 to about 1. In other embodiments, the wt/wt ratio is about 0.5, about 1, about 1.5, about 2, about 2.5, about 3, about 3.5, about 4, about 4.5 or about 5.

In some embodiment, the natural compound composition comprising:

a) Baobab from about 4 mg/kg to about 12 mg/kg;

b) Lycium 1.6 mg/kg to about 6.4 mg/kg; and

c) betaine from about 2.4 mg/kg to about 7.2 mg/kg;

wherein the wt/wt ratio of Baobab to Lycium is from about 0.5 to about 5;

wherein the wt/wt ratio of Baobab to betaine is from about 0.5 to about 5.

In some embodiments, the composition comprises at least three natural compounds. In other embodiments, the composition comprises three to five natural compounds. In other embodiments, the composition comprises three, four, five natural compounds. In this regard, some examples of natural compound composition having three natural compounds are given below:

Some examples of natural compound composition having four natural compounds are given below:

Some examples of natural compound composition having five natural compounds are given below:

As mentioned above, a combination of compounds can produce either an antagonistic, additive or synergistic, and such responses are not predictable in the art. The inventors have found that certain compositions provide for an overall interactive effect which is overall synergistic or at least additive. In particular, the inventors have found that certain Baobab containing compositions provide for an overall interactive effect which is overall synergistic or at least additive. To this end, and without wanting to be bound by theory, the inventors have performed experiments and analysed the results from single compound and 2-6 compound compositions to further derive the overall combinatory effects from 3-5 compound compositions. The results indicate that the prevailing assumption in natural compound supplementation that all compounds known to have beneficial effects can be put together in a single combination for at least an additive effect is incorrect. In this regard, it is the overall combination that matters which can be resultant from the individual linear and second order effects of each compound. When a wholistic approach is taken by considering compound interactions (and also their relative concentrations) to understand the complex interactions, the overall effect from the combination of compounds can be deduced. In this regard, the linear, bilinear and quadratic combination effects must be considered. To this end, Table 5 tabulates a series of combinations that the inventors have found to have an overall beneficial effect against NAFLD and/or NASH (i.e. overall synergistic or at least overall additive).

Advantageously, due to the additive or synergistic effect, lesser amount of natural compounds can be provided to a subject in need thereof. Accordingly, it translates to lower raw material cost and production cost.

In a preferred embodiment, the natural compound composition comprises:

a) Baobab from about 4 mg/kg to about 12 mg/kg;

b) Quercetin dihydrate from about 0.16 mg/kg to about 0.65 mg/kg; and

c) Emodin from about 1.6 mg/kg to about 6.5 mg/kg.

In certain embodiments, the natural compounds in the composition are all plant based extracts. In this regard, the natural compounds are extracted and derived from a plant. The natural compounds, after extraction from the plant, can be formed into a powder or a liquid.

In certain embodiments, the natural compounds in the composition are GRAS (Generally Recognised As Safe). Generally recognized as safe is a United States Food and Drug Administration designation that a chemical or substance added to food is considered safe by experts, and so is exempted from the usual Federal Food, Drug, and Cosmetic Act food additive tolerance requirements. The skilled person would know how to convert the human equivalent dosage to an actual dosage for treating liver conditions. For example, if an average human has a weight from about 60 kg to about 120 kg, the natural compound composition can have:

Baobab from about 240 mg to about 1440 mg;

when Quercetin dihydrate is present, Quercetin dihydrate from about 10 mg to about 80 mg; when Emodin is present, Emodin from about 100 mg to about 770 mg;

when Resveratrol is present, Resveratrol from about 50 mg to about 380 mg;

when Lycium is present, Lycium from about 100 mg to about 770 mg; and

when betaine is present, betaine from about 140 mg to about 860 mg.

For example, if an average human is about 60 kg, the natural compound composition will comprises:

a) Baobab from about 240 mg to about 720 mg;

b) Quercetin dihydrate from about 9.6 mg to about 39 mg; and

c) Emodin from about 96 mg to about 390 mg.

In some embodiments, if an average human has a weight from about 60 kg to about 120 kg and based on the formulation of Ex 5 (Table 5), the natural compound composition comprises:

a) Baobab from about 170 mg to about 400 mg;

b) Quercetin dihydrate from about 9 mg to about 20 mg; and

c) Emodin from about 9 mg to about 20 mg.

In other embodiments, the natural compound composition comprises:

a) Baobab from about 170 mg to about 400 mg;

b) Quercetin dihydrate from about 9 mg to about 20 mg; and

c) Emodin from about 9 mg to about 20 mg;

In another preferred embodiment, the natural compound composition comprises:

a) Baobab from about 4 mg/kg to about 12 mg/kg; b) Quercetin dihydrate from about 0.16 mg/kg to about 0.65 mg/kg;

c) Emodin from about 1.6 mg/kg to about 6.5 mg/kg; and

d) Resveratrol from about 0.8 mg/kg to about 3.2 mg/kg.

In this regard, when converted to a dose for a 60 kg human, the natural compound composition will comprises:

a) Baobab from about 240 mg to about 720 mg;

b) Quercetin dihydrate from about 9.6 mg to about 39 mg;

c) Emodin from about 96 mg to about 390 mg; and

d) Resveratrol from about 48 mg to about 192 mg.

In other embodiments, if an average human has a weight from about 60 kg to about 120 kg and based on the formulation of Ex 1 (Table 5), the natural compound composition comprises:

a) Baobab from about 170 mg to about 400 mg;

b) Quercetin dihydrate from about 9 mg to about 20 mg;

c) Emodin from about 90 mg to about 200 mg; and

d) Resveratrol from about 40 mg to about 100 mg.

In other embodiments, the natural compound composition comprises:

a) Baobab from about 170 mg to about 400 mg;

b) Quercetin dihydrate from about 9 mg to about 20 mg;

c) Emodin from about 90 mg to about 200 mg; and

d) Resveratrol from about 40 mg to about 100 mg;

In some embodiment, if an average human has a weight from about 60 kg to about 120 kg and based on the formulation of Ex 13 (Table 5), the natural compound composition comprises:

a) Baobab from about 90 mg to about 200 mg;

b) Lycium 90 mg to about 200 mg; and c) betaine from about 110 mg to about 240 mg.

In other embodiments, the natural compound composition comprises:

a) Baobab from about 90 mg to about 200 mg;

b) Lycium 90 mg to about 200 mg; and

c) betaine from about 110 mg to about 240 mg;

the wt/wt ratio of Baobab to Lycium is from about 0.5 to about 5.

The natural compound composition of the present invention may be administered in a single dose or a series of doses. For example, the composition may be administered as a single dose or in two doses over the duration of 24 hr. Such method of formulation of the composition is well known to those skilled in the art. The composition may contain any suitable carriers, diluents or excipients, which can further be pharmaceutically acceptable. These include all conventional solvents, dispersion media, fillers, solid carriers, coatings, antifungal and antibacterial agents, dermal penetration agents, surfactants, isotonic and absorption agents and the like. It will be understood that the compositions of the invention may also include other supplementary physiologically active agents.

In an embodiment, suitable carriers, diluents or excipients include, but are not limited to, other natural compounds such as Milk Thistle (Sylimarin), Dandelion Root (Taraxacum Officinale), Globe Artichoke (Cynara Scolymus) and Turmeric (Curcuma longa). In another embodiment, salts, sugars, preservatives and binders are used. It is believed that the use of these substances can improve the deliverability and overall efficacy of the composition.

The carrier must be pharmaceutically "acceptable" in the sense of being compatible with the other ingredients of the composition and not injurious to the patient. The compositions may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. Such methods include the step of bringing into association the natural compounds with the carrier which constitutes one or more accessory ingredients. In general, the compositions are prepared by uniformly and intimately bringing into association the natural compounds with liquid carriers or finely divided solid carriers or both, and then if necessary shaping the product.

Injectables for such use can be prepared in conventional forms, either as a liquid solution or suspension or in a solid form suitable for preparation as a solution or suspension in a liquid prior to injection, or as an emulsion. Carriers can include, for example, water, saline (e.g., normal saline (NS), phosphate-buffered saline (PBS), balanced saline solution (BSS)), sodium lactate Ringer's solution, dextrose, glycerol, ethanol, and the like; and if desired, minor amounts of auxiliary substances, such as wetting or emulsifying agents, buffers, and the like can be added. Proper fluidity can be maintained, for example, by using a coating such as lecithin, by maintaining the required particle size in the case of dispersion and by using surfactants. By way of example, the compound, composition or combination can be dissolved in a pharmaceutically effective carrier and be injected into the vitreous of the eye with a fine gauge hollow bore needle (e.g., 30 gauge, 1/2 or 3/8 inch needle) using a temporal approach (e.g., about 3 to about 4 mm posterior to the limbus for human eye to avoid damaging the lens).

A person skilled in the art will appreciate that other means for injecting and/or administering the composition can also be used. These devices and methods can include, for example, biodegradable polymer delivery members that are inserted subcutaneously for long term delivery of medicaments.

The present invention also includes other modes of administration including topical administration. The composition of the invention may be suitable for topical administration to the skin may comprise the compounds dissolved or suspended in any suitable carrier or base and may be in the form of lotions, gel, creams, pastes, ointments and the like. Suitable carriers include mineral oil, propylene glycol, polyoxyethylene, polyoxypropylene, emulsifying wax, sorbitan monostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water. Transdermal patches may also be used to administer the compounds of the invention. For example, solutions or suspensions of the compound, composition or combinations of the invention may be formulated as a paste or cream or ointment, or as a membranous ocular patch, which is applied directly to the surface of the skin. Topical application typically involves administering the compound of the invention in an amount between 0.1 ng and 10 mg.

The composition of the invention may be suitable for topical administration in the mouth including lozenges comprising the natural compounds in a flavoured base, usually sucrose and acacia or tragacanth gum; pastilles comprising the natural compounds in an inert basis such as gelatine and glycerin, or sucrose and acacia gum; and mouthwashes comprising the active ingredient in a suitable liquid carrier.

The composition of the invention may also be suitable for intravenous administration. For example, a composition may be administered intravenously at a dose of up to 16 mg/m².

The composition of the invention may also be suitable for oral administration and may be presented as discrete units such as capsules, sachets or tablets each containing a predetermined amount of the active ingredient; as a powder or granules; as a solution or a suspension in an aqueous or non-aqueous liquid; or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion. The composition may also be presented as a bolus, electuary or paste. In another embodiment, the composition is orally administrable.

A tablet may be made by compression or moulding, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing in a suitable machine the active ingredient in a free-flowing form such as a powder or granules, optionally mixed with a binder (e.g inert diluent, preservative disintegrant (e.g. sodium starch glycolate, cross- linked polyvinyl pyrrolidone, cross-linked sodium carboxymethyl cellulose) surface-active or dispersing agent. Moulded tablets may be made by moulding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent. The tablets may optionally be coated or scored and may be formulated so as to provide slow or controlled release of the active ingredient therein using, for example, hydroxypropylmethyl cellulose in varying proportions to provide the desired release profile. Tablets may optionally be provided with an enteric coating, to provide release in parts of the gut other than the stomach. It will also be understood that if a single dose is provided in the format of multiple tablets, the skilled person would know how to alter the total dosage of the composition such that a subset may be provided by each tablet.

Accordingly, in some embodiments, the composition is providable as a tablet or a capsule. In other embodiments, the composition is providable in a fruit chew form.

The composition of the invention may be suitable for parenteral administration include aqueous and non-aqueous isotonic sterile injection solutions which may contain anti oxidants, buffers, bactericides and solutes which render the compound, composition or combination isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. The composition may be presented in unit-dose or multi-dose sealed containers, for example, ampoules and vials, and may be stored in a freeze-dried (lyophilised) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described.

Preferred unit dosage composition or combinations are those containing a daily dose or unit, daily sub-dose, as herein above described, or an appropriate fraction thereof, of the natural compounds.

It should be understood that in addition to the natural compounds particularly mentioned above, the composition of this invention may include other agents conventional in the art having regard to the type of composition or combination in question, for example, those suitable for oral administration may include such further agents as binders, sweeteners, thickeners, flavouring agents disintegrating agents, coating agents, preservatives, lubricants and/or time delay agents. Suitable sweeteners include sucrose, lactose, glucose, aspartame or saccharine. Suitable disintegrating agents include cornstarch, methylcellulose, polyvinylpyrrolidone, xanthan gum, bentonite, alginic acid or agar. Suitable flavouring agents include peppermint oil, oil of wintergreen, cherry, orange or raspberry flavouring. Suitable coating agents include polymers or copolymers of acrylic acid and/or methacrylic acid and/or their esters, waxes, fatty alcohols, zein, shellac or gluten. Suitable preservatives include sodium benzoate, vitamin E, alpha-tocopherol, ascorbic acid, methyl paraben, propyl paraben or sodium bisulphite. Suitable lubricants include magnesium stearate, stearic acid, sodium oleate, sodium chloride or talc. Suitable time delay agents include glyceryl monostearate or glyceryl distearate.

In another aspect, the present invention also provides a method of treating a fatty liver disorder or disease, comprising administering a natural compound composition. As used herein, treating a disease includes the act of preventing a disease. In some embodiments, the composition is as provided herein. In an embodiment, a method of preventing a fatty liver disorder or disease by administering a natural compound composition is provided. In another embodiment, a method of treating a fatty liver disorder or disease by administering a natural compound composition is provided.

Fatty liver disease is an accumulation of fat in the liver. This is usually characterised as an accumulation of fat at more than about 10% of the liver's weight. Non-alcoholic fatty liver disease (NAFLD) is a liver disorder that affects up to one-third of the adult population who drink little or no alcohol.

In some embodiments, the liver disorder or disease is non-alcoholic fatty liver disease (NAFFD) or non-alcoholic steatohepatitis (NASH).

In an embodiment, the liver disorder or disease is selected from simple fatty liver (hepatic steatosis). In other embodiments, NAFFD is selected from simple fatty liver (hepatic steatosis).

In an embodiment, the present invention provides a method of short-term interventional treatment of NAFFD and/or NASH or long-term prevention of NAFFD and/or NASH. In another embodiment, the present invention provides a method for improving the general liver health by administering the composition as a supplement. It is envisioned that these compositions can be sold as retail health supplements, or clinically approved prescribed therapeutics or clinically approved over-the-counter therapeutics.

Given that there is currently no standard treatment for NAFLD and/or NASH, the current course of action is to treat the underlying condition, such as obesity, high cholesterol and type 2 diabetes. Accordingly, in an embodiment, the composition can be administered in combination with treatment for these indications. In another embodiment, composition is administered for controlling weight; i.e. promoting weight loss or preventing weight gain.

In another aspect, the present invention provides a natural compound composition for use as a medicament in the treatment of a fatty liver disorder and/or disease. In some embodiments, the composition is as provided herein.

In another aspect, the present invention provides a use of a natural compounds composition in the manufacture of a medicament for the treatment of a fatty liver disorder and/or disease. In some embodiments, the composition is as provided herein.

Throughout this specification and the claims which follow, 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 integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

The reference in this specification to any prior publication (or information derived from it), or to any matter which is known, is not, and should not be taken as an acknowledgment or admission or any form of suggestion that that prior publication (or information derived from it) or known matter forms part of the common general knowledge in the field of endeavour to which this specification relates.

Those skilled in the art will appreciate that the invention described herein in susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications which fall within the spirit and scope. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any two or more of said steps or features.

Certain embodiments of the invention will now be described with reference to the following examples which are intended for the purpose of illustration only and are not intended to limit the scope of the generality hereinbefore described.

Example 1: Methodology

Figure 1 illustrates the methodology used to identify natural compounds suitable for NAFLD. Using a 3-stage optimization process following a high content screen of 131 natural compounds, a series of natural compound combinations for use in the prevention and treatment of NAFLD were identified. High content analysis was performed in normal hepatocyte cells (THLE-2) to evaluate the ability of single natural compounds to prevent hepatic lipid accumulation in vitro (i.e. prevention of NAFLD) (Step 2; Fig 2 and 3). From this screen, candidate natural compounds (8 in total) were selected as inputs for in vitro combinatory optimization (Step 3). The components in the combinations are shown in Table 1 below.

The optimization process (Step 3 and 4) utilizes both in vitro and in vivo optimization to identify natural compounds within a orthogonal array composite design (OACD) set as well as the appropriate dosage ratios. The combinations were then analysed computationally to evaluate their efficacy. In step 3, in vitro experimental studies followed by computational analysis was performed on 8 compounds over 3 different dosages of these compounds (Fig 4). The experimental combinations that were carried out are listed in Table 1. The analysis identified combinations optimized in vitro for free fatty acid (FFA) accumulation which are efficacious, as demonstrated by the exemplary top ranked 2-drug combinations resulting from the analysis (Table 2). These rankings are also reflective of the interactions between the natural compounds, where a high ranked combination, Quercetin and Emodin (Table 2), exhibit enhanced lower fatty acid accumulation as the concentrations of the compounds increase, suggesting a synergistic interaction (Fig 5b). Comparatively, a low ranked combination, Triptolide and Emodin (Table 2), does not demonstrate a marked decrease upon increasing the concentrations of the drugs, reflective of an antagonistic interaction (Fig 5a) .

Table 1. List of experimental combinations for in vitro optimization with 8 drugs, 3 concentrations (where -1, 0 and 1 corresponds to inhibitory concentrations (IC) of 0% (ICo), 10% (ICio) and 20% (IC20) of observed cell death for the respective compounds).

Table 2: Exemplary top-ranked 2-drug combinations for in vitro NAFLD analysis (8 compounds, 3 concentrations)

As used herein, 'inhibitory concentration (IC)' is defined as the concentration of a compound that will result in cell death after overnight incubation. For example, IC20 refers to the concentration of a compound that will result in 20% cell death after overnight incubation.

Three natural compounds (Quercetin dihydrate, Emodin and Resveratrol) were identified for in vivo analysis that had potential in optimized combinations. In addition to these 3 compounds, 3 more compounds (Baobab, Lycium and betaine) were added that were not present in the initial library. Following identification of appropriate compounds to develop optimized combinations, in vivo analysis was performed utilizing a validated clinically relevant preclinical mouse model of NAFLD (Fig 6). The analysis was performed utilizing an orthogonal array composite design 30 combination set with 6 drugs at 3 concentrations identified in Table 3.

Table 3: Natural compounds used for in vivo analysis

In this NAFLD model (Fig 6), hepatic steatosis developed after 3 months on a choline- deficient high-fat diet. NAFLD progression was quantified as an output of lipid droplet total area following Oil Red O staining of histological samples following 4 months of diet and test combination treatment (Fig 7 and 8). The compound combination numbers in Fig 7 correlate to the numbered staining images in Fig 8, of which the combinations are as provided in Table 4. There was a wide range of variance amongst test combinations which revealed optimized combinations that are capable of complete prevention of NAFLD progression. From these orthogonal array composite design combination sets, a list of optimized 3-5 compound combinations can be generated computationally by factoring in higher order mixing (i.e. antagonistic, additive or synergistic effects).

Table 4. Exemplary list of combinations for in vivo optimization with 6 drugs, 3 concentrations (where -1, 0 and 1 corresponds to ICo, ICio and IC20 of the respective compounds).

The results indicate that the prevailing assumption in natural compound supplementation that all compounds known to have beneficial effects can be put together in a single combination for at least an additive effect is incorrect. In this regard, it is the overall combination that matters as well as individual linear and second order effects of each compound. A wholistic approach should be taken in considering compound interactions (and also their relative concentrations) to understand the complex interactions that can ultimately provide an overall synergistic effect (or at least additive effect). In this regard, linear, bilinear, and quadratic effects have to be considered.

Example 2

Experimentations with male mice have shown that the use of Triptolide in these compositions has a certain degree of toxicity and is not ideal. To solve this toxicity issue, the inventors have further tested more compounds and Baobab was identified. Traditionally, Baobab has been shown to exert antioxidative effects. For the first time ever, the inventors believe that Baobab fruit is able to provide hepatoprotective effects. The inventors have also found that the therapeutic effects from Baobab can be further amplified based on the synergistic combinations disclosed in Table 5.

The results from the NAFLD in vivo analysis (Figure 9) shows that Quercetin and Baobab have a positive interaction (synergistic or additive effect). Synergy is also observed with the following combinations: Quercetin and Emodin, Baobab and Emodin, Baobab and Resveratrol, Emodin and Lycium, and Emodin and Betaine.

In particular, this synergism is more pronounced in Ex 1, Ex 5 and Ex 13. While synergy is shown for 2 compound combinations, for higher order combinations, there is a further need to calculate the contribution of linear, bilinear and quadratic effects of the individual compounds and binary pairs to get to the overall effect (i.e. additive or synergistic) of the composition as described above. For example, while synergism may be present between Baobab and Quercetin, and between Baobab and Lycium in Ex 40, there may be other effects between the other compounds that causes a reduction in the observed overall effect and hence shows an additive effect (as opposed to synergistic). However, it is noted that these combinations are still effective.

Table 5: Combinations (in mg/kg) for in vivo NAFLD analysis showing overall additive or overall synergistic effects

The 40 combinations listed in Table 5 are determined to be overall additive or overall synergistic and can be useful against NAFLD and/or NASH. Traditionally, the skilled person would only consider binary combination in testing the compositions and may arrive at a result of antagonistic effect. For instance, a combination of Triptolide and Emodin was found to be antagonistic and/or toxic, the use of which needs to be avoided. However, by using the invention as described, when in combination, the inventors have found that by taking into account complex interactions, the compositions can ultimately result in an overall additive or overall synergistic effect within the dosage levels evaluated. In vivo results show that 3- drug combinations (Ex 5 and Ex 13) (p-value < 0.01 and p-value < 0.001) resulted in a significant decrease in total lipid droplet accumulation when compared against the fatty liver control, as quantified via Oil Red O staining (Fig 9). Ex 40 (p-value < 0.01) also resulted in a decrease in total lipid droplet accumulation. This is attributed to an additive effect. Comparing Ex 5 and Ex 40, this analysis demonstrates that additivity does not directly translate to overall efficacy, where we see that the 3 -drug combination performs as well as the 5-drug combination (due to an overall synergistic effect) (Fig 9). The representative liver images and their respective Oil Red O histological staining correspond to the quantified normalized mean fatty liver area. The results demonstrate the robustness of this approach in identifying the most optimal drug combination as the validation of these combinations in vivo reflect the combinations from the analysis. Thus, this computational approach identifies specific combinations of natural compounds that are effective in preventing NAFLD progression that cannot be intuitively determined based on existing knowledge. Example 3

The interventional benefits of the exemplary compositions was investigated further to it preventative benefits. Figure 10 shows the in vivo results (liver) of mice after 4 months of high fat diet (top two images). As control, the exemplary compositions were not given to the mouse after this 4 months (4 months of high fat diet). The image on the bottom right shows the in vivo results of a mouse after another 4 months of high fat diet together with composition treatment (8 months of high fat diet). The image on the bottom left shows the in vivo results of a mouse after 8 months of high fat diet with no composition treatment (8 months of high fat diet). The results show that after treatment, an improvement was observed.

Human Equivalent Dosage Calculations

The dosage in the mice model can be converted to a human equivalent. Several methods are known and these methods are easily available to the skilled person. For example, one such reference is M. L. Macheda et. al. J. Cellular Physio., 2005, 202, 654-662. As used herein, the dosage (mg/kg) in mice can be converted to its human equivalent (mg/kg) by multiplying the mice dosage with a factor of 0.081. This factor is based on standard allometric scaling where the Mouse k_m = 3 and Human k_m = 37.

Claims

1. A natural compound composition comprising Baobab and at least two of the following compounds selected from the group consisting of Quercetin dihydrate, Emodin, Resveratrol, Lycium and betaine.

2. The natural compound composition of claim 1,

wherein Baobab is from about 4 mg/kg to about 12 mg/kg;

wherein when Quercetin dihydrate is present, Quercetin dihydrate is from about 0.16 mg/kg to about 0.65 mg/kg;

wherein when Emodin is present, Emodin is from about 1.6 mg/kg to about 6.4 mg/kg; wherein when Resveratrol is present, Resveratrol is from about 0.8 mg/kg to about 3.2 mg/kg;

wherein when Lycium is present, Lycium is from about 1.6 mg/kg to about 6.4 mg/kg; and wherein when betaine is present, betaine is from about 2.4 mg/kg to about 7.2 mg/kg.

3. A natural compound composition comprising:

a) Baobab;

b) Quercetin dihydrate; and

4. The composition of any of claims 1 to 3, the natural compound composition comprising:

a) Baobab;

b) Quercetin dihydrate; and

c) Emodin.

5. The composition of any of claims 1 to 3, the natural compound composition comprising:

a) Baobab;

b) Quercetin dihydrate; c) Emodin; and

d) Resveratrol.

6. The composition of any of claims 1 to 5, wherein Baobab is present from about 4 mg/kg to about 12 mg/kg.

7. The composition of any one of claims 1 to 6, wherein Quercetin dihydrate is present from about 0.16 mg/kg to about 0.65 mg/kg.

8. The composition of any of claims 1 to 7, wherein the wt/wt ratio of Baobab to Quercetin dihydrate is from about 5 to about 50.

9. The composition of any of claims 1 to 8, wherein the compound is Emodin from about 1.6 mg/kg to about 6.4 mg/kg.

10. The composition of any of claims 1 to 3 and 5 to 9, wherein the compound is Resveratrol from about 0.8 mg/kg to about 3.2 mg/kg.

11. The composition of any of claims 1 to 3 and 5to 10, wherein the compounds are Emodin from about 1.6 mg/kg to about 6.4 mg/kg and Resveratrol from about 0.8 mg/kg to about 3.2 mg/kg.

12. The natural compound composition of any of claims 1 to 4, comprises:

a) Baobab from about 170 mg to about 400 mg;

b) Quercetin dihydrate from about 9 mg to about 20 mg; and

c) Emodin from about 9 mg to about 20 mg;

13. The natural compound composition of any of claims 1 to 3 and 5, comprises:

a) Baobab from about 170 mg to about 400 mg;

b) Quercetin dihydrate from about 9 mg to about 20 mg; c) Emodin from about 90 mg to about 200 mg; and

d) Resveratrol from about 40 mg to about 100 mg;

14. The natural compound composition of claims 1 or 2, comprising:

a) Baobab;

b) Lycium; and

c) betaine.

15. The composition of any of claims 1 or 14, further comprising Lycium from about 1.6 mg/kg to about 6.4 mg/kg.

16. The composition of any of claims 1, 14 or 15, further comprising betaine from about 2.4 mg/kg to about 7.2 mg/kg.

17. The natural compound composition of any of claims 1, 2, 14 to 16, comprises: a) Baobab from about 90 mg to about 200 mg;

b) Lycium 90 mg to about 200 mg; and

c) betaine from about 110 mg to about 240 mg;

the wt/wt ratio of Baobab to Lycium is from about 0.5 to about 5.

18. The natural compound composition of any of claims 1 to 17, wherein Baobab is a fruit extract derived from Adansonia genus of deciduous trees.

19. The natural compound compositions of any of claims 1 to 18, wherein the natural compounds are plant based extracts.

20. The natural compound compositions of any of claims 1 to 19, wherein the natural compounds are GRAS (Generally Recognised As Safe).

21. The composition of any of claims 1 to 20, further comprising a pharmaceutically acceptable excipient.

22. The composition of any of claims 1 to 21, the composition is administrable orally or intravenously or transdermally.

24. A method of treating a fatty liver disorder and/or disease, comprising administering a natural compound composition, the composition comprising Baobab and at least two of the following compounds selected from the group consisting of Quercetin dihydrate, Emodin, Resveratrol, Lycium and betaine.

25. The method of claim 24, wherein the liver disorder or disease is non-alcoholic fatty liver disease (NAFLD) or non-alcoholic steatohepatitis (NASH).

26. The method of claim 25, wherein NAFLD is selected from simple fatty liver (hepatic steatosis).

27. A natural compounds composition for use as a medicament in the treatment of a fatty liver disorder and/or disease, the composition comprising Baobab and at least two of the following compounds selected from the group consisting of Quercetin dihydrate, Emodin, Resveratrol, Lycium and betaine.

28. Use of a natural compounds composition in the manufacture of a medicament for the treatment of a fatty liver disorder and/or disease, the composition comprising Baobab and at least two of the following compounds selected from the group consisting of Quercetin dihydrate, Emodin, Resveratrol, Lycium and betaine.