WO2016093692A1

WO2016093692A1 - Standardised extract of labisia pumila for weight reduction and nanoformulation thereof

Info

Publication number: WO2016093692A1
Application number: PCT/MY2015/050152
Authority: WO
Inventors: Zhari ISMAIL; Hooi Kheng BEH; Mohd Shahrul Ridzuan HAMIL; Gheniya GHAFAR; Mohammed Ali Ahmed SAEED; Abdul Hakeem MEMON; Suzana HASHIM
Original assignee: Universiti Sains Malaysia; Kementerian Pertanian & Industri Asas Tani
Priority date: 2014-12-08
Filing date: 2015-12-08
Publication date: 2016-06-16
Also published as: MY193591A

Abstract

There is provided an extract of Labisia pumila that comprises: gallic acid; caffeic acid; rutin; and one or more additional extract components, wherein on a dry weight basis: the weight/weight ratio of gallic acid to caffeic acid is from 10:1 to 75:1; the weight/weight ratio of gallic acid to rutin is from 10:1 to 50:1; and the weight/weight ratio of rutin to caffeic acid is from 1:0.3 to 1:4. There is also provided a nano-formulated liposome containing said extract and methods of preparing these compositions, as well as methods of using the same.

Description

STANDARDISED EXTRACT OF LABISIA PUMILA FOR WEIGHT REDUCTION AND NANO-

FORMULATION THEREOF

TECHNICAL FIELD

The present invention relates to a process for obtaining an extract of Labisia pumila, a nano- formulated extract of Labisia pumila, the extract and nano-formulated extracts thereof, and their uses.

BACKGROUND

The listing or discussion of an apparently prior-published document in this specification should not necessarily be taken as an acknowledgement that the document is part of the state of the art or is common general knowledge.

Obesity is a growing health problem worldwide and is associated with many life-threatening diseases such as cardiovascular disease, renal disease, hypertension, stroke, infertility, respiratory dysfunction, and type 2 diabetes.

Obesity is a complex condition which involves various poorly understood pathways and as a result the development of anti-obesity drugs has proven to be extremely difficult. Previous attempts to treat obesity include the identification of drugs which reduce/suppress the appetite of a patient, for example the centrally acting serotonin-norepinephrine reuptake inhibitor, sibutramine. However drugs, such as sibutramine, have been associated with increased cardiovascular events and strokes and have now been withdrawn from the market in many countries. Although many anti-obesity drugs currently on the market do not suffer from an increased cardiovascular/stroke incidence, they do however cause undesirable side effects such as dry mouth, anorexia, constipation, insomnia, dizziness and nausea.

Hence, there is a need to explore new sources for anti-obesity agents. Medicinal plants continue to supply the scientific community with new chemical compositions for the treatment of various diseases/disorders.

One medicinal plant that has recently gained lots of interest for its utility in medicine is Labisia pumila, also known as Kacip Fatima in Malay. Labisia pumila is a plant that can be found growing wild in the shade of tropical forest floors. It has been used for hundreds of years to enhance vitality, improve blood circulation and firm and tone muscles after child-birth.

l However, despite the increased interest in Labisia pumila there is still a lack/shortage of extraction processes capable of obtaining therapeutically safe, active and stable extracts from the Labisia pumila plant material.

SUM MARY OF INVENTION

In a first aspect of the invention, there is provided an extract of Labisia pumila that comprises: gallic acid;

caffeic acid;

rutin; and

one or more additional extract components, wherein on a dry weight basis:

the weight/weight ratio of gallic acid to caffeic acid is from 10: 1 to 75: 1 ;

the weight/weight ratio of gallic acid to rutin is from 10: 1 to 50: 1 ; and

the weight/weight ratio of rutin to caffeic acid is from 1 :0.3 to 1 :4,

(e.g. the weight/weight ratio of gallic acid to caffeic acid is from 25: 1 to 50: 1 ; the weight/weight ratio of gallic acid to rutin is from 25: 1 to 35: 1 ; and

the weight/weight ratio of rutin to caffeic acid is from 1 : 1 to 1 :2).

In certain embodiments, the one or more additional extract components may comprise chlorogenic acid, kaempferol-3-O-rutinoside and 2,4-Di-te/f-butylphenol. For example, on a dry weight basis:

the weight/weight ratio of rutin to chlorogenic acid is from 1 :2 to 1 : 14; and/or the weight/weight ratio of rutin to kaempferol-3-O-rutinoside is from 1 :0.15 to 1 :5.5; and/or

the weight/weight ratio of rutin to 2,4-Di-te/f-butylphenol is from 1 : 1.6 to 1 :0.04,

(e.g. the weight/weight ratio of rutin to chlorogenic acid is from 1 :5 to 1 :9; and/or the weight/weight ratio of rutin to kaempferol-3-O-rutinoside is from 1 :0.5 to 1 :2.5;

and/or

the weight/weight ratio of rutin to 2,4-Di-te/f-butylphenol is from 1 : 1 to 1 :0.2).

In further embodiments of the invention, the extract may comprise on a dry weight basis:

gallic acid in an amount of from 32.94 to 51.12 μg/mg;

caffeic acid in an amount of from 1.03 to 2.70 μg/mg;

rutin in an amount of from 0.73 to 2.79 μg/mg; and

one or more additional extract components that take the total weight of the extract to 1 mg. In yet further embodiments of the invention, on a dry weight basis of the extract, the one of more additional extract components may comprise:

chlorogenic acid in an amount of from 5.62 to 10.00 μg/mg;

kaempferol-3-O-rutinoside in an amount of from 0.51 to 3.97 μg/mg; and

2,4-Di-te/f-butylphenol in an amount of from 0.14 to 1.16 μg/mg.

In yet further embodiments of the invention, the extract may exhibit the following peaks in its Fourier Transform infra-red spectrum: 1044 cm^-1 , 1392 cm^-1 , 1589 cm^-1 , 2930 cm^-1 , and 3350 cm^-1.

In a second aspect of the invention, there is provided a nano-formulated liposome extract of Labisia pumila comprising:

a liposome formed from a lipid; and

an extract of Labisia pumila within the liposome, as defined in the first aspect of the invention and in any combination of its embodiments.

In certain embodiments of the invention, the lipid may be a phospholipid. For example, the phospholipid may be a soybean phospholipid (e.g. a mixture of one or more phospholipids derived from soybean).

In yet further embodiments, the liposome may have an average particle size of from 100 nm to 200 nm, optionally wherein the average particle size is from 170 nm to 176 nm.

In a third aspect of the invention, there is provided a pharmaceutical formulation comprising an extract of Labisia pumila according to the first aspect of the invention (and its embodiments) and/or a nano-formulated liposome extract of Labisia pumilia according to the second aspect of the invention (and its embodiments) in admixture with a pharmaceutically acceptable adjuvant, diluent or carrier.

In a fourth aspect of the invention, there is provided an extract or formulation according any of the first to third aspects of the invention (and their embodiments) for use in medicine.

In a fifth aspect of the invention, there is provided an extract or formulation according to any of the first to third aspects of the invention (and their embodiments) for use in the manufacture of a medicament for the treatment of obesity. In a sixth aspect of the invention, there is provided a method of treating obesity comprising administration of therapeutically effective amount of an extract or formulation according to any of the first to third aspects of the invention (and their embodiments), to a patient in need of such treatment.

In a seventh aspect of the invention, there is provided a process to obtain an extract of Labisia pumila according to the first aspect of the invention (and its embodiments), comprising: i. obtaining a Labisia pumila plant sample;

ii. reducing the water content of the plant sample to provide a dried sample;

iii. subjecting the dried sample to extraction with a solvent containing a mixture of an alcohol and water to provide an alcoholic extract;

iv. removing the solvent from the alcoholic extract to obtain the extract of Labisia pumila.

In certain embodiments of the invention the plant sample may be derived from the leaves of a Labisia pumila plant, optionally the plant sample may be additionally or alternatively derived from the roots and/or stems of a Labisia pumila plant. A particular variety that may be mentioned herein is the Labisia pumila var. alata strain.

In certain embodiments, the step of reducing the water content in step (ii) may be performed for from 24 hours to 72 hours at from 40°C to 60°C, optionally wherein:

(a) reducing the water content is performed for 72 hours at 55°C; and/or

(b) the dried sample is ground into a powder before use in step (iii).

In certain embodiments, extraction step (iii) may comprise the use of a soxhlet extracting technique, optionally wherein the solvent containing a mixture of an alcohol and water is replaced at least once to form at least two alcoholic extracts that are combined together. In additional or alternative embodiments, extraction step (iii) may be performed for from 5 hours to 240 hours, optionally, wherein the extraction step is performed for 120 hours.

In certain embodiments of the invention, the solution may contain a mixture of an alcohol and water has a volumetric ratio (alcohol: water) of from 4: 1 to 1 :4, optionally wherein the volumetric ratio is 1 : 1. In additional or alternative embodiments, the alcohol may be a lower aliphatic alcohol having from 1 to 6 carbon atoms, such as a lower aliphatic alcohol selected from any one of the group consisting of methanol, ethanol, isopropanol, n-propanol, f-butanol n-butanol, and any combination thereof, optionally wherein the lower aliphatic alcohol is ethanol.

In certain embodiments, the step of removing the solvent from the alcoholic extract in step (iv) is performed by spray drying, freeze drying or evaporation.

In an eighth aspect of the invention, there is provided a process to obtain a nano-formulated liposome extract of Labisia pumila according to the second aspect of the invention (and its embodiments), comprising:

i. providing a dry Labisia pumila extract prepared in accordance with the seventh aspect of the invention (and its embodiments) and dissolving the same in a first solvent to provide an extract solution;

ii. providing a phospholipid solution comprising phospholipids and a second solvent;

iii. mixing the extract solution and phospholipid solution together and removing the first and second solvents to form the nano-formulated liposome extract of Labisia pumila.

In embodiments of the invention, the first solvent may be ethanol and/or the second solvent may be chloroform.

In a ninth aspect of the invention, there is provided a process to obtain an extract of Labisia pumila comprising: i. obtaining a Labisia pumila plant sample;

ii. reducing the water content of the plant sample to provide a dried sample;

iii. subjecting the dried sample to extraction with a solvent containing a mixture of an alcohol and water to provide an alcoholic extract; and

In certain embodiments, the plant sample may be derived from the leaves of a Labisia pumila plant, optionally the plant sample may be additionally or alternatively derived from the roots and/or stems of a Labisia pumila plant. In further embodiments, reducing the water content in step (ii) is performed for from 24 hours to 72 hours at from 40°C to 60°C. For example, reducing the water content can be performed for 72 hours at 55°C. In certain embodiments, the dried sample is ground into a powder before use in step (iii).

In yet further embodiments, extraction step (iii) may comprise the use of a soxhlet extracting technique.

In yet further embodiments, the solvent containing a mixture of an alcohol and water is replaced at least once to form at least two alcoholic extracts that are combined together.

In yet further embodiments, extraction step (iii) may be performed for from 5 hours to 240 hours. For example, the extraction step is performed for 120 hours.

In yet further embodiments, the solution containing a mixture of an alcohol and water may have a volumetric ratio (alcohol: water) of from 4: 1 to 1 :4 (e.g. the volumetric ratio may be 1 : 1).

In still further embodiments, the alcohol is a lower aliphatic alcohol may have from 1 to 6 carbon atoms. For example, the lower aliphatic alcohol is selected from any one of the group consisting of methanol, ethanol, isopropanol, n-propanol, f-butanol n-butanol, and any combination thereof (e.g. wherein the lower aliphatic alcohol is ethanol).

In yet still further embodiments, the step of removing the solvent from the alcoholic extract in step (iv) may be performed by spray drying, freeze drying or evaporation.

In yet still further embodiments, the process may further comprise the steps of: v. dissolving the Labisia pumila extract in a solvent to provide a Labisia pumila extract solution;

vi. adding a polymer to the Labisia pumila extract solution to provide a polymer-extract mixture; and

vii. adding the polymer-extract mixture to a surfactant to obtain a nano- formulated extract of Labisia pumila.

In yet still further embodiments, the nano-formulated extract of Labisia pumila may have an average particle size of from 300nm to 500nm (e.g. the average particle size is from 400nm to 420nm). In certain embodiments, the solvent may be a mixture of a polar organic solvent and water. For example, the polar organic solvent may be selected from any one of the group consisting of a polar aprotic solvent, a polar protic solvent, or a mixture thereof (e.g. the solvent is a mixture of a polar aprotic solvent, a polar protic solvent and water, having a volumetric ratio of X:Y:Z, where X is from 1 to 3, Y is from 1 to 3 and Z is from 1 to 3, such as wherein the mixture of an aprotic polar solvent, a protic polar solvent and water has a volumetric ratio of 3: 1 : 1).

In further embodiments, the polymer is selected from any one of the group consisting of poly(N-vinyl pyrrolidone), poly(methyl acrylate), poly(ethyl acrylate), poly(acrylic acid), polyvinyl alcohol), polyacrylamide, poly(ethylene glycol), any copolymer thereof, and any combination thereof. For example, the polymer is an ethyl acrylate-methyl acrylate copolymer, optionally wherein the polymer is EUDRAGIT™ RL PO.

In yet still further embodiments, the surfactant may be a zwitterionic surfactant, a cationic surfactant, an anionic surfactant, or a non-ionic surfactant. For example, the surfactant may be a non-ionic surfactant selected from the group consisting of any one or more of a polyethylene glycol, a polypropylene glycol, a glucoside alkyl ether, a glycerol alkyl ester, a polysorbate, a poloxamer and any combination thereof (e.g. the non-ionic surfactant is a poloxamer, optionally the poloxamer is poloaxamer-188). In certain embodiments, the surfactant may be provided in an aqueous solution.

In still further embodiments, the surfactant may be present in the aqueous solution in a concentration of from 0.001 g/mL to 0.1 g/mL (e.g. the aqueous surfactant mixture is an aqueous poloaxamer-188 solution with a concentration of 0.005 g/mL).

In yet further embodiments, the polymer-extract mixture may be added to the surfactant at a temperature of from 40°C to 100°C (e.g. 50°C) in at least one portion.

In yet still further embodiments, the nano-formulated extract may be provided as a suspension.

In a tenth aspect of the invention, there is provided a Labisia pumila extract or a nano- formulated extract of Labisia pumilia obtained and/or obtainable by the process of the ninth aspect of the invention. In a eleventh aspect of the invention, there is provided an extract of Labisia pumila according and/or a nano-formulated extract of Labisia pumilia according to the tenth aspect of the invention in admixture with a pharmaceutically acceptable adjuvant, diluent or carrier.

In a twelfth aspect of the invention, there is provided a pharmaceutical formulation according to the tenth aspect of the invention for use in medicine.

In a thirteenth aspect of the invention, there is provided an extract or formulation according to the tenth to twelfth aspects of the invention for use in the manufacture of a medicament for the treatment of obesity.

In a fourteenth aspect of the invention, there is provided a provided an extract or formulation according to the tenth to twelfth aspects of the invention for use in the treatment of obesity.

In a fifteenth aspect of the invention, there is provided a method of treating obesity comprising administration of therapeutically effective amount of provided an extract or formulation according to the tenth to twelfth aspects of the invention to a patient in need of such treatment.

In a sixteenth aspect of the invention, there is provided a nano-formulated suspension of a Labisia pumila extract comprising:

an extract of Labisia pumila as defined in the first aspect of the invention (and its embodiments);

a solvent;

a polymer; and

a surfactant.

In certain embodiments of the invention:

(a) the nano-formulated extract of Labisia pumila may have an average particle size of from 300nm to 500nm, optionally wherein the average particle size is from 400nm to 420nm;

(b) the solvent may be a mixture of a polar organic solvent and water, optionally wherein the polar organic solvent is selected from any one of the group consisting of a polar aprotic solvent, a polar protic solvent, or a mixture thereof;

(c) the solvent may be a mixture of a polar aprotic solvent, a polar protic solvent and water, having a volumetric ratio of X: Y: Z, where X is from 1 to 3, Y is from 1 to 3 and Z is from 1 to 3, optionally wherein the mixture of an aprotic polar solvent, a protic polar solvent and water has a volumetric ratio of 3: 1 : 1 ; (d) the polymer may be selected from any one of the group consisting of poly(N- vinyl pyrrolidone), poly(methyl acrylate), poly(ethyl acrylate), poly(acrylic acid), polyvinyl alcohol), polyacrylamide, poly(ethylene glycol), any copolymer thereof, and any combination thereof, optionally wherein the polymer is an ethyl acrylate-methyl acrylate copolymer, optionally wherein the polymer is EUDRAGIT™ RL PO;

(e) the surfactant may be a zwitterionic surfactant, a cationic surfactant, an anionic surfactant, or a non-ionic surfactant, optionally wherein the surfactant is a non-ionic surfactant selected from the group consisting of any one or more of a polyethylene glycol, a polypropylene glycol, a glucoside alkyi ether, a glycerol alkyi ester, a polysorbate, a poloxamer and any combination thereof (e.g. the non-ionic surfactant is a poloxamer, such as poloaxamer-188);

(f) the surfactant is provided in an aqueous solution, optionally wherein the surfactant is present in the aqueous solution in a concentration of from 0.001 g/mL to 0.1 g/mL (e.g. the aqueous surfactant mixture is an aqueous poloaxamer-188 solution with a concentration of 0.005 g/mL).

Additional aspects and embodiments of the invention are described in the numbered clauses below.

1. A process to obtain an extract of Labisia pumila comprising: i. obtaining a Labisia pumila plant sample; ii. reducing the water content of the plant sample to provide a dried sample; iii. subjecting the dried sample to extraction with a solvent containing a mixture of an alcohol and water to provide an alcoholic extract; iv. removing the solvent from the alcoholic extract to obtain the extract of Labisia pumila.

2. The process according to Clause 1 , wherein the plant sample is derived from the leaves of a Labisia pumila plant, optionally the plant sample may be additionally or alternatively derived from the roots and/or stems of a Labisia pumila plant. 3. The process according to Clause 1 or Clause 2, wherein reducing the water content in step (ii) is performed for from 24 hours to 72 hours at from 40°C to 60°C, optionally wherein:

(a) reducing the water content is performed for 72 hours at 55°C; and/or

(b) the dried sample is ground into a powder before use in step (iii).

4. The process according to any one of the preceding clauses, wherein extraction step (iii) comprises the use of a soxhiet extracting technique, optionally wherein the solvent containing a mixture of an alcohol and water is replaced at least once to form at least two alcoholic extracts that are combined together.

5. The process according to any one of the preceding clauses, wherein extraction step (iii) is performed for from 5 hours to 240 hours, optionally, wherein the extraction step is performed for 120 hours.

6. The process according to any one of the preceding clauses, wherein the solution containing a mixture of an alcohol and water has a volumetric ratio (alcohol: water) of from 4: 1 to 1 :4, optionally wherein the volumetric ratio is 1 :1.

7. The process according to any one of the preceding clauses, wherein the alcohol is a lower aliphatic alcohol having from 1 to 6 carbon atoms.

8. The process according to Clause 7, wherein the lower aliphatic alcohol is selected from any one of the group consisting of methanol, ethanol, isopropanol, n-propanol, f-butanol, n- butanol, and any combination thereof, optionally wherein the lower aliphatic alcohol is ethanol.

9. The process according to any one of the preceding clauses, wherein the step of removing the solvent from the alcoholic extract in step (iv) is performed by spray drying, freeze drying or evaporation.

10. The process of any one of the preceding clauses, wherein the process further comprises the steps of: v. dissolving the Labisia pumila extract in a solvent to provide a Labisia pumila extract solution; vi. adding a polymer to the Labisia pumila extract solution to provide a polymer-extract mixture; and vii. adding the polymer-extract mixture to a surfactant to obtain a nano- formulated extract of Labisia pumila.

11. The process according to Clause 10, wherein the nano-formulated extract of Labisia pumila has an average particle size of from 300nm to 500nm, optionally wherein the average particle size is from 400nm to 420nm.

12. The process according to Clause 10 or Clause 11 , wherein the solvent is a mixture of a polar organic solvent and water.

13. The process according to Clause 12, wherein the polar organic solvent is selected from any one of the group consisting of a polar aprotic solvent, a polar protic solvent, or a mixture thereof.

14. The process according to Clause 13, wherein the solvent is a mixture of a polar aprotic solvent, a polar protic solvent and water, having a volumetric ratio of X: Y: Z, where X is from 1 to 3, Y is from 1 to 3 and Z is from 1 to 3, optionally wherein the mixture of an aprotic polar solvent, a protic polar solvent and water has a volumetric ratio of 3: 1 : 1.

15. The process according to any one of Clauses 10 to 14, wherein the polymer is selected from any one of the group consisting of poly(N-vinyl pyrrolidone), poly(methyl acrylate), poly(ethyl acrylate), poly(acrylic acid), polyvinyl alcohol), polyacrylamide, poly(ethylene glycol), any copolymer thereof, and any combination thereof.

16. The process according Clause 15, wherein the polymer is an ethyl acrylate-methyl acrylate copolymer, optionally wherein the polymer is EUDRAGIT™ RL PO.

17. The process according to any one of Clauses 10 to 16, wherein the surfactant is a zwitterionic surfactant, a cationic surfactant, an anionic surfactant, or a non-ionic surfactant.

18. The process according to Clause 17, wherein the surfactant is a non-ionic surfactant selected from the group consisting of any one or more of a polyethylene glycol, a polypropylene glycol, a glucoside alkyi ether, a glycerol alkyi ester, a polysorbate, a poloxamer and any combination thereof. 19. The process according to Clause 18, wherein the non-ionic surfactant is a poloxamer, optionally wherein the poloxamer is poloaxamer-188.

20. The process according to any one of Clauses 10 to Clause 19, wherein the surfactant is provided in an aqueous solution.

21. The process according to Clause 20, wherein the surfactant is present in the aqueous solution in a concentration of from 0.001 g/mL to 0.1 g/mL.

22. The process according to Clause 21 , wherein the aqueous surfactant mixture is an aqueous poloaxamer-188 solution with a concentration of 0.005 g/mL.

23. The process according to any one of Clauses 10 to 22, wherein the polymer-extract mixture is added to the surfactant at a temperature of from 40°C to 100°C (e.g. 50°C) in at least one portion.

24. The process according to any one of Clauses 10 to 23, wherein the nano-formulated extract is provided as a suspension.

25. A Labisia pumila extract obtained or obtainable by the process of any one of Clauses 1 to 9.

26. A nano-formulated extract of Labisia pumila obtained or obtainable by the process of any one of Clauses 10 to 24.

27. A pharmaceutical formulation comprising an extract of Labisia pumila according to Clause 25 and/or a nano-formulated extract of Labisia pumilia according to Clause 26 in admixture with a pharmaceutically acceptable adjuvant, diluent or carrier.

28. An extract or formulation according to any one of Clauses 25 to 27 for use in medicine.

29. An extract or formulation according to any one of Clauses 25 to 27 for use in the manufacture of a medicament for the treatment of obesity.

30. A method of treating obesity comprising administration of therapeutically effective amount of an extract or formulation according to any one of Clauses 25 to 27, to a patient in need of such treatment. BRIEF DESCRIPTION OF FIGURES

Figure 1 provides microscopic images that illustrate the fragment of vessels with annular and spiral thickenings (20X magnification) (a), peltate trichrome (20X magnification) (b), a rosette of calcium oxalate crystal (20X magnification) (c), a group of fibres (20X magnification) (d) and sclerids (20X magnification) (e) of the Labisia pumila leaf powder.

Figure 2 provides a UV absorbance spectrum of a 50% water-ethanol extract of Labisia pumila according to the current invention.

Figure 3 provides an FTIR spectrum of a 50% water-ethanol extract of Labisia pumila according to the current invention.

Figure 4 provides a HPTLC profile for Labisia pumila extract compared to gallic acid. L1 is gallic acid, L2 is a 50% water-ethanol extract of Labisia pumila according to the current invention.

Figure 5 provides a HPLC chromatogram for the Labisia pumila standard (gallic acid, chlorogenic acid, caffeic acid, rutin, kaempferol-3-O-rutinoside, kaempferol and 2,4-Di-te/f- butylphenol) of 50% water-ethanol extract of Labisia pumila.

Figure 6.0 provides FTIR spectra of Labisia pumila extract, soybean phospholipids and liposomes.

Figure 6.1 provides a FTIR spectrum of Labisia pumila extract. Figure 6.2 provides a FTIR spectrum of soybean phospholipids. Figure 6.3 provides a FTIR spectrum of liposomes.

Figure 6.4 provides a FTIR spectrum of mixture of standard compounds (gallic acid, caffeic acid and rutin).

Figure 7 provides a microscopic image of blood vessel growth on rat aortic rings when treated with the negative and positive controls in the rat aortic ring assay. Figure 8 provides a microscopic image of blood vessel growth on rat arotic rings when treated with 50% water-ethanol extracts of Labisia pumila.

Figure 9 provides a bar chart showing blood vessel growth inhibition (%) in the rat aortic ring assay for a standard 50% water-ethanol extract of Labisia pumila (also known as Kacip Fatimah), a nano-formulated 50% water-ethanol extract of Kacip Fatimah (liposomal formulation), and the positive control, suramin.

Figure 10 illustrates the incubated plate 5 minutes after p-NPB was added in the porcine pancreatic lipase activity assay.

Figure 11 is a bar chart showing inhibition of pancreatic lipase activity (%) for 50% water- ethanol extracts of Labisia pumila, KF is Labisia pumila (also known as Kacip Fatimah), EW is 50% water-ethanol extract.

Figures 12a and Figure 12b provide the in vitro inhibition activity and IC50 of kacip fatimah extract and nano-formulated Kacip Fatimah extract on 3T3 L1 cells.

Figure 13 provides a flow diagram depicting the in vivo anti-obesity experimental model.

Figures 14-27 are bar charts depicting the effect of the 45 day treatment on the amount of food intake; percentage body weight increase; bodymass index; the triglyceride level cholesterol level; high density lipoprotien (HDL) level; low density lipoprotien (LDL) level; aspartate transaminase (AST) level; Alanine transaminase (ALT) level; heart organ weight relative to body weight; lung organ weight relative to body weight; kidney organ weight relative to body weight; liver organ weight relative to body weight; and adipose tissue weight relative to body weight measured for animal groups 1 to 8, respectively. In each of Figures 14-27, the symbol (*) indicates that the statistical difference P-value is less than 0.05, (**)indicates that the statistical difference P-value is less than 0.01 and (***) indicates that the statistical difference P-value is less than 0.001 compared to group 8.

Figure 28 depicts the liver histology of diet induced rats subjected to: (A) Regular diet (control group) and without treatment; (B) High fat diet and treated with kacip fatimah (125mg/kg) (C) High fat diet and treated with kacip fatimah (250mg/kg); and (D) High fat diet and treated with kacip fatimah (500mg/kg) (E) High fat diet and treated with nano-kacip fatimah (500mg/kg) , (F) High fat diet and treated with herb-based positive control (Zenoctil at 500mg/kg); (G) High fat diet and treated with orlistat (at 28 mg/kg/day); (H) High fat diet and without treatment. Figure 29-31 shows the mean plasma concentration-time profiles of gallic acid, caffeic acid and rutin, respectively for both a normal formulation and a nano-liposome formulation.

Figure 32-34 illustrates the area under the curve (AUC 0→∞), peak concentration (Cmax) and time to reach peak concentration (Tmax) respectively, for gallic acid for both a normal formulation and a nano-liposome formulation.

Figure 35-37 illustrates the area under the curve (AUC 0→∞), peak concentration (Cmax) and time to reach peak concentration (Tmax) respectively, for caffeic acid for both a normal formulation and a nano-liposome formulation.

Figure 38-40 illustrates the area under the curve (AUC 0→∞), peak concentration (Cmax) and time to reach peak concentration (Tmax) respectively, for rutin for both a normal formulation and a nano-liposome formulation.

Figure 41 depicts the inhibition percentage achieved for a water-based extract compared to the 50:50 water: ethanol extract in antiangiogenesis, inhibition of pancreatic lipase and in the inhibition of 3T3-L1 cell line.

DETAILED DESCRIPTION

As described above, the current invention relates to a new formulation of Labisia pumila with surprising medicinal properties, its medical applications and processes for the production of said extract.

Thus, there is provided an extract of Labisia pumila that comprises:

gallic acid;

caffeic acid;

rutin; and

one or more additional extract components, wherein on a dry weight basis:

the weight/weight ratio of gallic acid to caffeic acid is from 10: 1 to 75: 1 ;

the weight/weight ratio of gallic acid to rutin is from 10: 1 to 50: 1 ; and

the weight/weight ratio of rutin to caffeic acid is from 1 :0.3 to 1 :4.

It will be appreciated that the extract of Labisia pumila referred to herein contains more than just gallic acid, rutin and caffeic acid. For example, the extract may also comprise chlorogenic acid, kaempferol-3-O-rutinoside and 2,4-Di-te/f-butylphenol, amongst other compounds present in the extract. When gallic acid, rutin and caffeic acid are present, one or more of these compounds may be present on a dry weight basis such that:

the weight/weight ratio of rutin to 2,4-Di-te/f-butylphenol is from 1 : 1.6 to 1 :0.04.

For the avoidance of doubt, the compounds mentioned hereinbefore as being part of the extract of Labisia pumila are non-limiting and the extract may contain further additional compounds as well. While not explicitly mentioned, these compounds would be expected to be present in an extract of Labisia pumila that exhibits the following peaks in its Fourier Transform infra-red (FTIR) spectrum: 1044 cm^"1 , 1392 cm^"1 , 1589 cm^"1 , 2930 cm^"1 , and 3350 cm^-1.

In particular embodiments of the invention that may be mentioned herein, the extract may comprise on a dry weight basis:

gallic acid in an amount of from 32.94 to 51.12 μg/mg;

caffeic acid in an amount of from 1.03 to 2.70 μg/mg;

rutin in an amount of from 0.73 to 2.79 μg/mg; and

one or more additional extract components that take the total weight of the extract to 1 mg.

In additional or alternative embodiments, the one of more additional extract components may comprise:

chlorogenic acid in an amount of from 5.62 to 10.00 μg/mg;

kaempferol-3-O-rutinoside in an amount of from 0.51 to 3.97 μg/mg; and

2,4-Di-te/f-butylphenol in an amount of from 0.14 to 1.16 μg/mg.

For example, the extract may comprise:

on a dry weight basis:

gallic acid in an amount of from 32.94 to 51.12 μg/mg;

caffeic acid in an amount of from 1.03 to 2.70 μg/mg;

rutin in an amount of from 0.73 to 2.79 μg/mg;

chlorogenic acid in an amount of from 5.62 to 10.00 μg/mg;

kaempferol-3-O-rutinoside in an amount of from 0.51 to 3.97 μg/mg;

2,4-Di-te/f-butylphenol in an amount of from 0.14 to 1.16 μg/mg; and one or more additional extract components that take the total weight of the extract to 1 mg. Where the one or more additional extract compounds does not contain chlorogenic acid, kaempferol-3-O-rutinoside or 2,4-Di-te/f-butylphenol.

While the extract mentioned hereinbefore may be useful as it is, or when formulated into a pharmaceutical formulation, the extract may be further improved by the manufacture of a nano- formulation.

Thus, there is provided a nano-formulated liposome extract of Labisia pumila comprising:

a liposome formed from a lipid; and

an extract of Labisia pumila as defined herein within the liposome.

It will be appreciated that any suitable phospholipid material may be used to make the liposome. A particular example that may be mentioned herein includes a soybean phospholipid (e.g. a mixture of one or more phospholipids derived from soybean).

In particular embodiments of the invention, the liposome may have an average particle size of from 100 nm to 200 nm, optionally wherein the average particle size is from 170 nm to 176 nm.

Alternatively, the Labisia pumila extract may be provided as a nano-formulated suspension of a Labisia pumila extract comprising:

a solvent;

a polymer; and

a surfactant.

In the nano-formulated suspension:

(c) the solvent may be a mixture of a polar aprotic solvent, a polar protic solvent and water, having a volumetric ratio of X: Y: Z, where X is from 1 to 3, Y is from 1 to 3 and Z is from 1 to 3, optionally wherein the mixture of an aprotic polar solvent, a protic polar solvent and water has a volumetric ratio of 3: 1 : 1 ;

(d) the polymer may be selected from any one of the group consisting of poly(N- vinyl pyrrolidone), poly(methyl acrylate), poly(ethyl acrylate), poly(acrylic acid), polyvinyl alcohol), polyacrylamide, poly(ethylene glycol), any copolymer thereof, and any combination thereof, optionally wherein the polymer is an ethyl acrylate-methyl acrylate copolymer, optionally wherein the polymer is EUDRAGIT™ RL PO;

(e) the surfactant may be a zwitterionic surfactant, a cationic surfactant, an anionic surfactant, or a non-ionic surfactant, optionally wherein the surfactant is a non-ionic surfactant selected from the group consisting of any one or more of a polyethylene glycol, a polypropylene glycol, a glucoside alkyl ether, a glycerol alkyl ester, a polysorbate, a poloxamer and any combination thereof (e.g. the non-ionic surfactant is a poloxamer, such as poloaxamer-188);

The extract of Labisia pumila may be prepared by the process comprising:

i. obtaining a Labisia pumila plant sample;

ii. reducing the water content of the plant sample to provide a dried sample;

It will be appreciated that the order of steps presented above need to be conducted in the sequence presented in order to provide the desired extract.

While the plant sample is preferably derived from the leaves of the Labisia pumila plant, it may be additionally or alternatively derived from the roots and/or stems of said plant instead. It will be appreciated that any variety of Labisia pumila may be used to obtain the extract described herein, provided that the extract complies with the quantities and/or FTIR peaks mentioned hereinbefore. However, a particular variety of Labisia pumila that may be mentioned herein is the Labisia pumila var. alata strain. It will be appreciated that the removal of water from the plant may be accomplished by simple drying of the plant sample. For example, reducing the water content in step (ii) may be accomplished by subjecting the plant sample to a temperature of from 40°C to 60°C for from 24 hours to 72 hours (e.g. reducing the water content is performed for 72 hours at 55°C).

While not essential, it has been found that if the dried sample obtained from step (ii) is ground into a powder may make the extraction easier to perform.

The extraction of step (iii) above may be performed using any suitable technique. For example, the extraction of step (iii) may be performed using the soxhlet extraction technique. It will be appreciate, that replacing the extraction solvent at least once may lead to an overall increase in the material obtained from the extraction. Therefore, in certain embodiments of the invention, the solvent containing a mixture of an alcohol and water is replaced at least once to form at least two alcoholic extracts that are combined together.

In general, the extraction step (iii) is performed for from 5 hours to 240 hours (e.g. the extraction step is performed for 120 hours).

The solvent used in step (iii) is a mixture or an alcohol and water. In certain embodiments, the volumetric ratio (alcohol: water) of the solvent may be from 4: 1 to 1 :4 (e.g. the volumetric ratio is 1 : 1). While any suitable alcohol may be used, it is preferred that the alcohol is a lower aliphatic alcohol having from 1 to 6 carbon atoms. For example, the lower aliphatic alcohol may be selected from any one of the group consisting of methanol, ethanol, isopropanol, n- propanol, f-butanol n-butanol, and any combination thereof. Most preferably, the lower aliphatic alcohol is ethanol.

While any suitable method can be used to remove the solvent from the alcoholic extract in step (iv), the preferred methods involve spray drying, freeze drying or evaporation. It will be appreciated that these methods may involve the use of elevated temperatures and/or reduced pressure to assist in the removal of the solvent.

While the extract generated by the method described above may be used as-is. It is preferred to subject the extract to further processing in order to generate a nano-formulated material.

As such, there is provided a process to obtain a nano-formulated liposome extract of Labisia pumila according to the second aspect of the invention (and its embodiments), comprising: i. providing a dry Labisia pumila extract prepared as described hereinbefore and dissolving the same in a first solvent (e.g. ethanol) to provide an extract solution;

ii. providing a phospholipid solution comprising phospholipids and a second solvent (e.g. chloroform);

It will be appreciated that the Labisia pumila extract used herein complies with the requirements of the first aspect of the invention (and its embodiments).

As noted hereinbefore, the nano-formulation may alternatively be a nano-formulated suspension of a Labisia pumila extract, the process comprising:

a. providing a Labisia pumila extract (as described hereinbefore) and dissolving it in a solvent to provide a Labisia pumila extract solution; b. adding a polymer to the Labisia pumila extract solution to provide a polymer-extract mixture; and

c. adding the polymer-extract mixture to a surfactant to obtain a nano- formulated extract of Labisia pumila.

The process of generating the nano-formulated extract of Labisia pumila provides a formulation having an average particle size of from 300nm to 500nm. More preferably, the process provides a nano-formulated extract where the average particle size is from 400nm to 420nm.

The solvent added to the extract in step (a) may be any suitable solvent to aid in the generation of a nano-formulation. A suitable solvent contains a mixture of a polar organic solvent and water. The polar organic solvent may be selected from any one of the group consisting of a polar aprotic solvent, a polar protic solvent, or a mixture thereof. For example, the solvent is a mixture of a polar aprotic solvent, a polar protic solvent and water, having a volumetric ratio of X: Y: Z, where X is from 1 to 3, Y is from 1 to 3 and Z is from 1 to 3 (e.g. the mixture of an aprotic polar solvent, a protic polar solvent and water has a volumetric ratio of 3: 1 : 1). The polymer of step (b) may be selected from any one of the group consisting of poly(N-vinyl pyrrolidone), poly(methyl acrylate), poly(ethyl acrylate), poly(acrylic acid), polyvinyl alcohol), polyacrylamide, poly(ethylene glycol), any copolymer thereof, and any combination thereof. For example, the polymer is an ethyl acrylate-methyl acrylate copolymer, optionally wherein the polymer is EUDRAGIT™ RL PO.

The surfactant of step (c) may be a zwitterionic surfactant, a cationic surfactant, an anionic surfactant, or a non-ionic surfactant. For example, the surfactant is a non-ionic surfactant selected from the group consisting of any one or more of a polyethylene glycol, a polypropylene glycol, a glucoside alkyi ether, a glycerol alkyi ester, a polysorbate, a poloxamer and any combination thereof. In certain embodiments, the non-ionic surfactant is a poloxamer, optionally wherein the poloxamer is poloaxamer-188. Preferably, the surfactant is provided as an aqueous solution. While any suitable concentration may be used, a suitable concentration range is from 0.001 g/mL to 0.1 g/mL. For example, a suitable aqueous surfactant mixture is an aqueous poloaxamer-188 solution with a concentration of 0.005 g/mL.

In certain embodiments, the polymer-extract mixture is added to the surfactant at a temperature of from 40°C to 100°C (e.g. 50°C) in at least one portion.

The resulting nano-formulated extract may be provided in any suitable form. For example, a suitable form for the nano-formulated extract is as a suspension.

Also provided is a Labisia pumila extract obtained or obtainable by the processes described herein. As will be appreciated, the potential extracts desired above include a Labisia pumila extract that has not been subjected to further processing further to its formation (e.g. a dried extract, or one that has been reconstituted in a solvent) and a nano-formulated extract of Labisia pumila obtained or obtainable as described herein by means of additional down-stream processing (e.g. solvation of the dried extract, followed by the addition of a polymer and a surfactant).

While the Labisia pumila extracts described herein may be used to treat a subject without the addition of further materials, in some embodiments, the extracts may be formulated in admixture with a pharmaceutically acceptable adjuvant, diluent or carrier. That is, to provide a pharmaceutical formulation. The Labisia pumila extracts, nano-formulations and pharmaceutical formulations thereof described hereinbefore may be provided for use in medicine, said extracts and nano- formulations and pharmaceutical formulations may also be used:

(a) in the manufacture of a medicament for the treatment of obesity;

(b) in the treatment of obesity; and

(b) as a method of treating obesity comprising administration of therapeutically effective amount of the extracts, nano-formulations and pharmaceutical formulations to a patient in need of such treatment.

For the avoidance of doubt, in the context of the present invention, the term "treatment" includes references to therapeutic or palliative treatment of patients in need of such treatment, as well as to the prophylactic treatment and/or diagnosis of patients which are susceptible to the relevant disease states.

The terms "patient" and "patients" include references to mammalian (e.g. human) patients. As used herein the terms "subject" or "patient" are well-recognized in the art, and, are used interchangeably herein to refer to a mammal, including dog, cat, rat, mouse, monkey, cow, horse, goat, sheep, pig, camel, and, most preferably, a human. In some embodiments, the subject is a subject in need of treatment or a subject with a disease or disorder. However, in other embodiments, the subject can be a normal subject. The term does not denote a particular age or sex. Thus, adult and newborn subjects, whether male or female, are intended to be covered.

The term "effective amount" refers to an amount of the extract, which confers a therapeutic effect on the treated patient (e.g. sufficient to treat or prevent the disease). The effect may be objective (i.e. measurable by some test or marker) or subjective (i.e. the subject gives an indication of or feels an effect).

The extracts described herein may be administered by any suitable route, but may particularly be administered orally, intravenously, intramuscularly, cutaneously, subcutaneously, transmucosally (e.g. sublingually or buccally), rectally, transdermal^, nasally, pulmonarily (e.g. tracheally or bronchially), topically, by any other parenteral route, in the form of a pharmaceutical preparation comprising the extract in a pharmaceutically acceptable dosage form. Particular modes of administration that may be mentioned include oral, intravenous, cutaneous, subcutaneous, nasal, intramuscular or intraperitoneal administration. The extract will generally be administered as a pharmaceutical formulation in admixture with a pharmaceutically acceptable adjuvant, diluent or carrier, which may be selected with due regard to the intended route of administration and standard pharmaceutical practice. Such pharmaceutically acceptable carriers may be chemically inert to the active compounds and may have no detrimental side effects or toxicity under the conditions of use. Suitable pharmaceutical formulations may be found in, for example, Remington The Science and Practice of Pharmacy, 19th ed., Mack Printing Company, Easton, Pennsylvania (1995). For parenteral administration, a parenterally acceptable aqueous solution may be employed, which is pyrogen free and has requisite pH, isotonicity, and stability. Suitable solutions will be well known to the skilled person, with numerous methods being described in the literature. A brief review of methods of drug delivery may also be found in e.g. Langer, Science (1990) 249, 1527.

Otherwise, the preparation of suitable formulations may be achieved routinely by the skilled person using routine techniques and/or in accordance with standard and/or accepted pharmaceutical practice.

The amount of extract in any pharmaceutical formulation used in accordance with the present invention will depend on various factors, such as the severity of the condition to be treated, the particular patient to be treated, as well as the compound(s) which is/are employed. In any event, the amount of compound of formula I in the formulation may be determined routinely by the skilled person.

For example, a solid oral composition such as a tablet or capsule may contain from 1 to 99 % (w/w) active ingredient (i.e. the extract); from 0 to 99% (w/w) diluent or filler; from 0 to 20% (w/w) of a disintegrant; from 0 to 5% (w/w) of a lubricant; from 0 to 5% (w/w) of a flow aid; from 0 to 50% (w/w) of a granulating agent or binder; from 0 to 5% (w/w) of an antioxidant; and from 0 to 5% (w/w) of a pigment. A controlled release tablet may in addition contain from 0 to 90 % (w/w) of a release-controlling polymer.

A parenteral formulation (such as a solution or suspension for injection or a solution for infusion) may contain from 1 to 50 % (w/w) active ingredient; and from 50% (w/w) to 99% (w/w) of a liquid or semisolid carrier or vehicle (e.g. a solvent such as water); and 0-20% (w/w) of one or more other excipients such as buffering agents, antioxidants, suspension stabilisers, tonicity adjusting agents and preservatives.

Depending on the disorder, and the patient, to be treated, as well as the route of administration, the extracts described herein may be administered at varying therapeutically effective doses to a patient in need thereof. However, the dose administered to a mammal, particularly a human, in the context of the present invention should be sufficient to affect a therapeutic response in the mammal over a reasonable timeframe. One skilled in the art will recognize that the selection of the exact dose and composition and the most appropriate delivery regimen will also be influenced by inter alia the pharmacological properties of the formulation, the nature and severity of the condition being treated, and the physical condition and mental acuity of the recipient, as well as the potency of the specific compound, the age, condition, body weight, sex and response of the patient to be treated, and the stage/severity of the disease.

Administration may be continuous or intermittent (e.g. by bolus injection). The dosage may also be determined by the timing and frequency of administration. In the case of oral or parenteral administration the dosage can vary from about 0.01 mg to about 1000 mg per day of an extract of the current invention.

In any event, the medical practitioner, or other skilled person, will be able to determine routinely the actual dosage, which will be most suitable for an individual patient. The above- mentioned dosages are exemplary of the average case; there can, of course, be individual instances where higher or lower dosage ranges are merited, and such are within the scope of this invention.

The aspects of the invention described herein (e.g. the above-mentioned extracts, formulations, methods and uses) may have the advantage that, in the treatment of the conditions described herein, they may be more convenient for the physician and/or patient than, be more efficacious than, be less toxic than, have better selectivity over, have a broader range of activity than, be more potent than, produce fewer side effects than, or may have other useful pharmacological properties over, similar compounds, combinations, methods (treatments) or uses known in the prior art for use in the treatment of those conditions or otherwise.

EXPERIMENTAL

Sourcing and characterisation of raw material samples

Raw material of Labisia pumila was purchased from HERBagus Sdn Bhd. The raw material was characterised by microscopy of the leaf powders, as shown in Figure 1.

I Quality of raw materials

Specification:

Name of plant: Labisia pumila var. alata, family (Myrsinaceae), part of plant used: whole plant, and the other specifications listed below.

Foreign matter

Table 1 : Foreign matter contain of raw material

Ash content of raw material

Table 2: Acid Insoluble Ash content of raw material

Loss on drying

Table 3: Loss on drying of raw material

Determination of ash

About 3 g of ground material, accurately weighed was placed in a suitable tared dish (for example, of silica or platinum), previously ignited, cooled and weighed. The material was incinerated by gradually increasing the heat up to, but not exceeding 450°C, until free from carbon; cool, and weigh. The content in mg of ash per g of air-dried material can be calculated.

Determination of acid-insoluble ash

The ash obtained above was boiled for 5 minutes with 25 mL of hydrochloric acid (2 N; the insoluble matter was collected in a sintered crucible, or on an ashless filter-paper, # washed with hot water, and ignited at about 500°C to constant weight. The content in mg of acid- insoluble ash per g of air-dried material was calculated. Plant extraction method

Preparation of 50% water-ethanol extracts

The plant samples were cleaned with water and dried using a conventional oven at 55°C for 3 days to afford dried leaves (powdered). The dried powdered leaves of Labisia pumila (500 g) were Soxhlet-extracted with a (50% v/v) water-ethanol solution (5.0 L)for 5 days (the 50% water-ethanol solution (5.0 L) was replaced with a second batch of fresh 50% water-ethanol solution (5.0 L) at 2.5 days). After 5 days of extraction, the respective pooled 50% water- ethanolic extracts were then evaporated to yield 70.04 g (14.01 %) of plant extract.

Hot and cold extract values

Table 4: Hot/cold extract value of raw material

The value for hot-extraction provided in Table 4 was obtained using a modified version of the process above, wherein the plant sample was extracted for one hour at 80°C using reflux extraction. The extract was then evaporated.

The cold extract was extracted at room temperature using maceration for 7 hr after which the extract was left to stand for 24 hr. The extract was then evaporated.

Analytical methods used

Standardization of plant material extracts was performed using high-performance liquid chromatography (HPLC), high-performance thin layer chromatography (HPTLC), Fourier Transform- Infrared (FTIR) spectroscopy and Ultraviolet-visible spectrophotometry (UV-vis) and Chemometric methods. These techniques are now described in more detail below. HPLC

Table 5: HPLC conditions

HPLC chromatograms for the Labisia pumila standard compounds (gallic acid, chlorogenic acid, caffeic acid, rutin, kaempferol-3-O-rutinoside, kaempferol and 2,4-Di-te/f-butylphenol) of the 50% water-ethanol extract of Labisia pumila are shown in Figure 5.

Range of marker compounds in Kacip Fatimah 50% ethanol extract derived from selected plants

The acceptable ranges of the key marker compounds (*) and lesser marker compounds that may also be beneficial are provided below in Table A. It is noted that, the presence of at least some additional compounds not listed in Table A may be essential to the extract to provide the level of activity demonstrated herein, and these additional compounds may also facilitate the pharmacokinetic properties of marker compounds or may improve or protect them in biological processes. It should be noted that particular extract compositions that may be mentioned herein include those wherein the FTI R spectrum contains the following peaks: 1044 cm^-1 , 1392 cm^-1 , 1589 cm^-1 , 2930 cm^-1 , and 3350 cm^"1. Without these key peaks the extract may not work satisfactorily.

Table 6: HPTLC conditions

HPTLC profiles for Labisia pumila extracts are shown in Figure 4.

UV-Vis spectrophotometry

5 mg of plant material extract was dissolved in 10 mL of methanol and the resulting solution's concentration was adjusted so that the absorbance falls between about 0.05 to about 1 .0. UV- Vis spectra were recorded within a wavelength range of 200-500 nm. Figure 2 provides a UV spectrum of a 50% water-ethanol extract of Labisia pumila. FTIR

The plant material extracts were analysed using a single-bounce Attenuated Total Reflection FTIR (ATR-FTIR) scan technique. Three milligrams (mg) of plant material extract was placed on an optic window with a diamond crystal, the compression clamp was set to 6 psi to ensure good contact between the extract sample and the diamond crystal. IR spectra were recorded within a wavelength range of 4000-650 cm^-1 (mid-IR range).

Figure 3 provides an I R spectrum of a 50% water-ethanol extract of Labisia pumila.

Chemometric Data Analysis (Principle Component Analysis, PCA)

Prior to data analysis, each spectrum was baseline corrected and the absorbance was normalized so that peak absorbance of the most intense band is set to unity. The spectra were transferred into the statistical software program The Unscrambler®10.0.

Preparation of Labisia pumila nano suspension

A Labisia pumila 50% water-ethanol extract was obtained in accordance with the procedure provided above.

A sample of the Spray dried Labisia pumila 50% water-ethanol extract (20mg) was dissolved in a 30 mL mixture of acetone: ethanol: water (3: 1 : 1 v:v) under sonication for 30 minutes at room temperature (25°C) to provide an extract solution. The solution was then filtered using Whatman™ filter paper to provide a filtered solution. Eudragit™ RL PO (0.2 g) was dissolved in the filtered solution and sonicated for 10 minutes before being labelled as mixture A. Pluronic™ F-68 (or Poloxamer-188) non-ionic surfactant (0.4 g) was dissolved in 80 mL of water whilst sonicating for 20 minutes at room temperature (25°C) before being labelled as mixture B. Mixture A was added to mixture B in a drop-wise fashion and stirred using magnetic stirrer at 50°C to provide the Labisia pumila nano suspension with a final volume of 30m L. The formulation was analysed using a zetasizer to check particle size.

A dynamic light scattering (DLS) technique was used for analysis of zeta potential and size distribution. The Labisia pumila nano suspension (5-10 μί) was diluted to a volume of 1 mL using double distilled deionised water before being filtered and prepared for analysis. The size distribution was recorded on a Nano-ZS Zetasizer instrument (Malvern Instrument Ltd) at 25°C in back scattering mode. The z-average was recorded to be 409 nm. The remaining Labisia pumila nano suspension was lyophilized (by evaporation of solvent and freeze-drying) and stored at 20°C.

Preparation of nano liposomes of Labisia pumila extract in soybean phospholipids

Preparation of soybean phospholipids

The unpurified soybean phospholipids were prepared from food grade soybean lecithin. In brief; crude lecithin (600 g) was refluxed in 96% ethanol (3 L) for 30 min and cooled to room temperature. Subsequently, the supernatant was collected by decantation and concentrated at 60°C using rotavapor. The residue was then washed 5* with acetone (1 L) to give 56 g of semi solid material; this phospholipid was called PH-Et. Abdalrahim (2014).

Preparation of Labisia pumila liposomes

Liposomes of Labisia pumila extract (LP-L) were prepared by the film method as the following; soybean phospholipids was dissolved in chloroform and LP extract was dissolved in ethanol, the solutions were mixed, and the solvent was evaporated under vacuum using rotary evaporator at 45°C for 30 min, followed by drying in oven at 60°C for 1 hr.

Fourier transform infrared spectroscopy

FTIR analysis of LP extract, soybean phospholipids and LP liposomes was carried out using Spectrum 400 spectrometer (Perkin Elmer, USA). The IR spectra were recorded in the range of 4000 - 400 cm^"1 (n = 6).

FTIR spectra of soybean phospholipid, LP extract, and LP liposomes were studied in order to get insights into occurrence of interaction between LP extract and phospholipids (Figures 6.0- 6.3). In PH-Et phospholipids extract the broad band centred at 3358 cm^-1 represents the OH stretching, the principal bands at 2853 cm^-1 and 2924 cm^-1 correspond to the symmetric and anti-symmetric stretching in the CH2 groups of alkyl chains, the strong band centred at 1738 cm^-1 corresponds to the stretching vibrations of the ester carbonyl groups, the band centred at 1649 cm^-1 is assigned to C=0 stretching, and the scissoring vibrations of the CH2 groups are represented by the band at 1465 cm^-1. The characteristic phosphate group vibrational band assigned to the PO^2- anti-symmetric stretching mode is centred at 1221 cm^-1 and the PO^2- symmetric stretching mode PO^2- at 1062 cm^-1. In the Labisia pumila extract, the broad band centred at 3350 cm^-1 corresponds to OH stretching, vibration at 2930 cm^-1 correspond to C-H stretching, the bands centred at 1589 cm^-1 correspond to symmetric and asymmetric of C=C, the vibration at 1392 cm^-1 correspond to N=0 bend, and the vibration at 1044 cm^-1 corresponds to C=0 stretch. In liposomes, remarkable changes can be seen in the infrared absorption spectra due to incorporation of Labisia pumila extract in phospholipids; the broad band corresponding to OH group is shifted from 3350 cm^-1 to 3262 cm^-1 , the vibration at 2930 cm^-1 shifted to 3009 cm^-1 , the vibrations at 1589 cm^-1 , 1392 cm^-1 and 1044 cm^-1 disappeared.

Heavy metal limit test for dried Labisia pumila leaves

Table 7: Heavy metal test data

The data in Table 7 above was determined as follows.

A Labisia pumila was analysed for content of lead (Pb), cadmium (Cd), arsenic (As) and mercury (Hg) using Atomic Absorption Spectroscopy (AAS) Perkin Elmer model AANALYST 800, auto sampler, as per the standard method set out by the British Pharmacopoeia Commission (2008). Approximately 0.5g of ground powder dried leaf was accurately weighed and transferred to Teflon vessel. Then 10 mL of HNO3 was added to the sample vessel. The samples were diluted to 50 mL with distilled water in plastic disposable tubes and filtered with 2 μηι Teflon FilterMate. This high dirt trapping FilterMate is especially suitable for trace level analysis and is supplied with lot certification for trace metals. The filtered samples were transferred directly to be analyzed by AAS using nitric acid as blank solution, and standard lead, cadmium, arsenic and mercury solution made with 2% nitric acid as reference.

The powder of Labisia pumila was subjected to the Microbial Limit Test (MLT) as per the United State Pharmacopoeia method consists of total aerobic microbial counts, test for Staphylococcus aureus and Pseudomonas aeruginosa, test for Salmonella sp and Escherichia coli and total combined molds and yeasts count (United State Pharmacopoeia, 2009). The results are shown in Table 8 below. Microbial limit test for Labisia pumila 50% water-ethanol extract

Table 8: Microbial test data

Procedure for total aerobic microbial, yeast and mold count (via pour plate)

For preparation of test solution, 90 mL phosphate buffer (pH 7.2) was added to 10g of powdered dried of Labisia pumila. Then 1 mL of test solution was added into the Petri dish (9 - 10cm in diameter) aseptically, and after that 15 - 20 mL of sterilized molten agar medium (Soybean - Casein Digest agar (SCD)) for aerobic organisms and Sobouraud Dextrose Agar (SDA) for Yeast and Mold organisms) were added to the Petri dishes. The plates were incubated at 20 - 25°C for 5 - 7 days. After that plates was removed from incubation and the colonies were counted.

Procedures for specific microorganisms test

1. Escherichia coli

One mL of test solution was incubated at 30 - 35°C for 18 - 24 hours and then it was added to 100 mL of Mac Conkey broth, incubated at 42 - 44°C for 24 - 28 hours, streak onto Mac Conkey agar. After 18 - 27 hours incubation at 30 - 35°C the plate was observed for microbial growth.

2. Salmonella sp.

Ten mL of test solution was incubated at 30 - 35°C for 18 - 24 hours. Then 0.1 mL of test solution was added to 10 mL of RVSEB (RAPPAPORT-VASSILIADIS SALMONELLA ENRICHMENT BROTH), incubated at 30 - 35°C for 18 - 24 hours, streak onto XLD (xylose lysine deoxycholate) agar. The plates were incubated at 30 - 35°C for 18 - 48 hours and observed for microbial growth. Pseudomonas aeruginosa and Staphylococcus aureus

One ml_ of test solution was incubated at 30 - 35°C for 18 - 24 hours and streak onto agar (CET for P. aeruginosa and MSA for S. aureus). After 18 - 27 hours incubation at 30 - 35°C, it was observed for microbial growth.

Establishment of the diet-induced rat model

Rat aortic ring assay

Adult male Sprague Dawley (SD) rats 12 to 14 weeks of age and weighing around 160 to 180 g were used in the experiments. Animals were killed by using carbon dioxide (CO2) gas followed by exsanguination. The animals were excised to isolate the thoracic aorta and placed in phosphate buffered saline (PBS). The freshly excised thoracic rat aorta was rinsed with Hank's balanced salt solution containing 2.5 μg/mL amphotericin B (Sigma-Aldrich, Germany). The tissue specimens (i.e. thoracic rat aorta) were then cleaned of adipose tissue materials and residual blood clots. The rat aortic ring was then cut into small rings of 1 to 2 mm cross- section segments under a dissecting microscope (Motic®Taiwan). The assay was performed in a 48 well tissue culture plate (Nunclon™, Denmark). A volume of 500 μΙ_ of 3 mg/mL fibrinogen (Calbiochem, USA) in serum free M199 growth medium (Gibco™, USA) was added to each well with 5 mg/mL of aprotinin (Sigma-Aldrich, Germany), to prevent fibrinolysis of the vessel fragments (i.e. rat aortic rings segments). The clean rat aortic ring segments were rinsed five times with M199 growth media and placed in the centre of the each well (1 ring/well) of the 48 well tissue culture plates. Then, 15 μΙ_ of thrombin (50 NIH units/mL) (Sigma-Aldrich, Germany) in 0.15 M sodium chloride (NaCI) were added to each well. Bovine serum albumin plasma (Sigma-Aldrich, Germany) was then added to the wells and mixed rapidly with fibrinogen. Immediately after embedding the vessel fragment in the fibrin gels, 0.5 ml_ of a medium M199 supplemented with 20% heat inactivated fetal calf serum (Gibco®, USA), 0.1 % ε-aminocaproic acid (Sigma-Aldrich, Germany), 1 % L-glutamine (Sigma-Aldrich, Germany), 1 % amphotricin B (Sigma-Aldrich, Germany), 0.6% gentamycin (Sigma-Aldrich, Germany) was added to each well. Next the 50% water-ethanol extracts (standard and liposime nano-formulation) and control solutions (said solutions prepared by dissolving the extracts/controls in DMSO (dimethyl sulfoxide)) were added to the tissue culture wells by micropipette (to provide 100 ppm of extract in the well, calculated as 100 μg/mL of the extract).

Suramin (a well-known anti-angiogenic agent) was used as a positive control. A solution of equivalent concentration of the supplemented M199 medium without the sample (i.e. rat aortic ring segment) was added into the well and served as negative control. Vessels were cultured at 37°C in a humidified CB150 incubator (Binder, Germany) for 5 days. Fresh medium was added on day four of the experiment. Briefly, the length of the tiny blood vessel outgrowths from the primary ex-plant was measured under a microscope using an inverted Olympus LH 50A microscope camera (Olympus, Japan) on day five of the procedure. The pictures of the vessels were captured with the aid of a camera (Lieca CCD, Japan) and software packages (Lieca QWin) connected with an Intel Pentium 4 desktop computer. The percentage of blood vessels growth inhibition was determined according to the following formula:

Blood vessels growth inhibition (%) = 1 - (Sample growth / Control growth) ^χ 100

The microscopic images of Figure 7 illustrate the blood vessel growth on rat aortic rings when treated with the negative and positive controls.

The microscopic images of Figure 8 illustrate the blood vessel growth on rat arotic rings when treated with 50% water-ethanol extracts of Labisia pumila.

The bar chart of Figure 9 illustrates blood vessel growth inhibition (%) in the rat aortic ring assay for the standard 50% water-ethanol extract of Labisia pumila (also known as Kacip Fatimah), liposomal nano-formulated Kacip Fatimah, and the positive control suramin.

Porcine pancreatic lipase activity assay

Porcine pancreatic lipase (PPL, type II) activity was measured using p-nitrophenyl butyrate (p- NPB) as a substrate. PPL stock solutions (1 mg/mL) were prepared in Tris-HCI and the solutions were stored at -20°C. Plant extract samples having a concentration of 100 mg/mL, a positive control sample (Orlistat 100mg/ml) and a negative control sample (with and without an inhibitor) were pre-incubated with PPL stock solutions at 30°C before measuring the PPL inhibitory activity. The assay was started by adding 10 of p-NPB which brought the final volume of each assay sample/well to 210 μί. After incubation at 30 °C for 5 min, the amount of p-NPB was measured at 405 nm using a UV-Visible spectrophotometer. The PPL inhibitory activity was calculated according to the following formula:

Inhibitory activity (%) = 100 - ((B - b) /(A - a) 100)

Where A is the activity without inhibitor; a is the negative control without inhibitor; B is the activity with inhibitor; and b is the negative control with inhibitor. The photographic image of Figure 10 illustrates the incubated plate 5 minutes after p-NPB was added.

The bar chart of Figure 1 1 illustrates inhibition of pancreatic lipase activity (%) for 50% water- ethanol extracts of Labisia pumila. When referring to Figure 11 , KF is Labisia pumila (also known as Kacip Fatimah), EW is 50% water-ethanol extract.

As shown, the plant extracts caused some inhibition of porcine pancreatic lipase, but did not inhibit the lipase as strongly as orlistat (the positive control for inhibition).

Figures 12a and 12b show that kacip fatimah extract at 200 μg/mL has a potent anti- adipogenic effect in 3T3-L1 cells due to the inhibition of adipocyte differentiation and adipogenesis with IC50 = 156.56 μg/mL. And nano-formulated kacip fatimah extract showing improvement in the activity compare to kacip fatimah extract with IC50 = 134.68 μg/mL.

Anti-obesity and anti-hyperlipidemic studies of the 50% water-ethanol extract of Labisia pumila using the diet-induced obese rat model

The 50% water-ethanol extracts of Labisia pumila were selected for anti-obesity and anti- hyperlipidemic studies based on the results from the in vitro assays (i.e. porcine pancreatic lipase activity assay and the rat aortic ring assay). The effects of the 50% water-ethanol extracts of Labisia pumila were investigated using diet-induced obese rats as described below.

In vivo anti-obesity experimental model

A summary of this in vivo anti-obesity experimental model is illustrated in the scheme of Figure 13.

Animals

Male Sprague Dawley rats, 10 weeks old were used. The rats were obtained from the animal house of Universiti Sains Malaysia and were housed in cages whilst being allowed to adapt to the animal transit room in the school of pharmacy.

Dietary conditions/regime

Standard food pellets (Gold Coin, Penang, Malaysia) were used as a regular diet regime (normal control). These pellets have a calorie ratio of protein-fat-carbohydrate of 22:3:46. Pellets for the high fat diet (HFD) regime were made by mixing the standard food pellets with margarine at a ratio of 3:2 by weight. The HFD had a calorie ratio of protein-fat-carbohydrate of 13:35:28.

Preparation of test samples

The plant extracts were dissolved in 10% Tween 20 to enhance solubility. Sonication or vortexing may also be applied in order to promote solubility. The extracts were given orally to the rats by oral gavage. A commercial natural product containing a mixture of Garcinia cambogia, banaba, green tea and green coffee (Zenoctil™; also dissolved in 10% Tween 20) was selected as positive control and was dosed at 500mg/kg per day.

Experimental design

The animals were divided into 8 groups as summarised in the table below.

Table 9: Details of animal test groups

The animals were fed using the regular or high fat diet regime for 45 days and were treated with the plant extracts at 500mg/kg/day, vehicle alone (10% tween 20) and the positive control (Zenoctil at 500mg/kg/day). The animal weights and food consumption were recorded weekly throughout the experiment. The food was supplied to the animals 30 minutes after dosing the animals with test samples (i.e. plant extract etc). The percentage change in body weight and the body mass index of the animals were then calculated (Brandt et al., 2002). Blood analysis

All the animals were fasted overnight (12 hours) before 3ml_ of blood sample were collected through a cardiac puncture. Blood samples were centrifuged at 2500rpm for 15 minutes and the plasma was obtained before being kept at -80°C. The plasma samples were analysed for triglycerides (TG), total cholesterol (TC), high-density lipoprotein (HDL), low-density lipoprotein (LDL), Aspartate transaminase (AST) and Alanine transaminase (ALT).

Tissue preparation and relative organ weight

After the blood collection, the rats were sacrificed by carbon dioxide inhalation. The animals were dissected and the organs (heart, liver, lung and kidney) were immediately collected, weighed and recorded. The liver and adipose tissue samples were preserved in 10% neutral buffered formalin for histology purposes.

Statistical analysis

The experimental results were represented as mean ± S.D. The data was analysed for statistical significance using one-way ANOVA (SPSS 18.0) followed by the Tukey's multiple range tests. P values less than 0.05 were considered to be significant.

Figures 14-27 illustrate in graphical form the effect of the study on each of the four animal groups tested. Figures 28 (A)-(H) depicts the liver histology of diet induced rats in groups 1-8, respecively. It is noted that the groups treated with the 50% water-ethanol extract of Labisia pumila, nano-Kacip Fatimah and orlistat consistently appear to be amongst the best performing extracts across the entire range of analysis conducted.

In contrast, a comparison of the data reported for the use of pure gallic acid in an anti-obesity study (Chin and Gow 2007) shows that the 50:50 extract used here possess better results/activity in terms of anti-obesity parameters, even though this extract contains a much smaller amount of gallic acid (a dose of 125 mg/kg of the extract contains 20 - 31.25 mg/g of gallic acid in the extract) as compared to the dose used in the Chin study (1 g/kg of gallic acid in food plus 50 mg/kg oral and 2 g/kg of gallic acid in food plus 100 mg/kg oral). This clearly indicates that the current extract contains compounds other than gallic acid in the Labisia pumila extract that help to provide a synergistic increase in anti-obesity activity for this formulation. Pharmacokinetics of Kacip Fatimah extract and nano-formulated Kacip Fatimah extract

Male Sprague Dawley rats weighing 350-395 g were used in this experiment. The animals were fasted overnight with free access to water prior to the experimentation. Food is only allowed after the sampling of blood at 4hr. On the first occasion, eight rats (group I) were randomized to receive 500 mg/kg of Kacip Fatimah 50% ethanolic extract administered orally, while the other eight rats (group II) received orally 500 mg/kg of nano-formulated Kacip Fatimah extract in the form of liposomes.

The Kacip Fatimah 50% ethanolic extract and nano-formulated Kacip Fatimah extract were prepared in deionized water. The volume for oral route was 5 rnUkg of body weight. The rats were placed in animal restraining cages during blood collection and blood samples of 0.20 mL were withdrawn from the tail vein at 0.00, 0.50, 1.00, 1.50, 2.00, 2.50, 3.00, 4.00, 5.00, 6.00, 8.00, 12.00 and 24.00 hr after oral administration.

After oral administration of Kacip Fatimah extract, the marker compounds achieve maximum plasma concentration (C_max) of 3.99, 0.52 and 0.41 μg/mL in 5.25, 5.25 and 6.00 hr for gallic acid, caffeic acid and rutin, respectively. For nano-formulated Kacip Fatimah extract, the marker compounds achieve maximum plasma concentration (Cmax) of 8.09, 0.83 and 0.61 μg/mL in 3.83, 4.33 and 5.67 hr for gallic acid, caffeic acid and rutin, respectively. These results indicated that the nano-formulated Kacip Fatimah extract showed improvement in the mean plasma concentration of the marker compounds and also improvement in the time to reach peak concentration. Overall the nano-formulated Kacip Fatimah extract represented an improvement in the pharmacokinetic parameters.

Comparison to Water-Based Extract of Labisia Pumilla

A water extract of Labisia pumila as detailed above was compared to a 50:50 water: ethanol extract in various biological experiments. As depicted in Figure 41 , the water-based extract provided less anti-obesity activity (in terms of inhibition of pancreatic lipase, inhibition on 3T3- L1 cell line and antiangiogenesis activities) as compared to the 50:50 water: ethanol extract.

Without wishing to be bound by theory, it is suspected that the difference in activity is not just down to better extraction of the marker compounds identified above, but in maintaining a particular balance of the secondary compounds as well within the mixture. References

Abdalrahim FA Aisha, Amin Malik Shah Abdul Majid and Zhari Ismail. 2014. Preparation and characterization of nano liposomes of Orthosiphon stamineus ethanolic extract in soybean phospholipids. BMC Biotechnology, 14:23

Alemayehu Toma, Eyasu Makonnen, Yelamtsehay Mekonnen, Asfaw Debella andSirichai Addisakwattana. 2014.1 ntestinal a-glucosidase and some pancreatic enzymes inhibitory effect of hydroalcholic extract of Moringa stenopetala leaves. Complementary and Alternative Medicine. 14:180.

Bultmann; Kant, IJmert; Kasl, Stanislav V.; Schroer, Kees A. P.; Swaen, Gerard M.H.; van den Brandt, Piet A. 2002. Lifestyle Factors as Risk Factors for Fatigue and Psychological Distress in the Working Population: Prospective Results from the Maastricht Cohort Study. Journal of Occupational & Environmental Medicine. 44, 2, 1 16-124.

H. Van de VenC Paulussen' P.B. Feijens A. Matheeussen' P. Rombaut P. Kayaert G. Van den Mooter W. Weyenberg' P. Cos' L. Maes A. Ludwig. 2012. PLGA nanoparticles and nanosuspensions with amphotericin B: Potent in vitro and in vivo alternatives to Fungizone and AmBisome. Journal of Controlled Release, Volume 161 , Issue 3, 10, Pages 795-803.

Khalid Hussain', Zhari Ismail, Amirin Sadikun, Amin Malik Abdual Majid Shah, Abida Latif and Furqan Khurshid Hashmi. 2012. Antiangiogenic Activity and Bioassay-guided Isolation of Aqueous Extract of Orthosiphon stamineus. Journal of the Chinese Chemical Society, Volume 59, Issue 9, pages 1 137-1143.

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Mustafa Tuzen. Determination of heavy metals in soil, mushroom and plant samples by atomic absorption spectrometry. 2003. Microchemical Journal. Volume 74, Issue 3, Pages 289- 297. The United States Pharmacopeia, 34th edition, The National Formulary, 29th ed., The Official Compendia of Standards, United States Pharmacopoeial Convention, Inc.: asian ed. Rockville, MD; 2011. pp. 779-82, 3675-6.

Van de Ven. Paulussen' P.B. Feijens' A. Matheeussen' P. Rombaut P. Kayaert' G. Van den Mooter W. Weyenberg' P. Cos' L. Maes' A. Ludwig. 2012. PLGA nanoparticles and nanosuspensions with amphotericin B: Potent in vitro and in vivo alternatives to Fungi zone and AmBisome. Journal of Controlled Release, Volume 161 , Issue 3, 10, Pages 795-803.

Chin-Lin Hsu and Gow-Chin Yen. Effect of gallic acid on high fat diet-induced dyslipidaemia, hepatosteatosis and oxidative stress in rats. British Journal of Nutrition (2007), 98, 727- 735.

Claims

1. An extract of Labisia pumila that comprises:

gallic acid;

caffeic acid;

rutin; and

one or more additional extract components, wherein on a dry weight basis:

the weight/weight ratio of gallic acid to caffeic acid is from 10: 1 to 75: 1 ;

the weight/weight ratio of gallic acid to rutin is from 10: 1 to 50: 1 ; and

the weight/weight ratio of rutin to caffeic acid is from 1 :0.3 to 1 :4.

2. The extract of Claim 1 , wherein the one or more additional extract components comprise chlorogenic acid, kaempferol-3-O-rutinoside and 2,4-Di-terf-butylphenol.

3. The extract of Claim 2, wherein on a dry weight basis:

4. The extract of Labisia pumila according to any one of Claims 1 to 3, wherein, on a dry weight basis the extract comprises:

gallic acid in an amount of from 32.94 to 51.12 μg/mg;

caffeic acid in an amount of from 1.03 to 2.70 μg/mg;

rutin in an amount of from 0.73 to 2.79 μg/mg; and

one or more additional extract components that take the total weight of the extract to

1 mg.

5. The extract of Labisia pumila according to Claim 4, wherein on a dry weight basis of the extract the one of more additional extract components comprises:

chlorogenic acid in an amount of from 5.62 to 10.00 μg/mg;

kaempferol-3-O-rutinoside in an amount of from 0.51 to 3.97 μg/mg; and

2,4-Di-te/f-butylphenol in an amount of from 0.14 to 1.16 μg/mg.

6. The extract of Labisia pumila according to any one of Claims 1 to 5, wherein the extract exhibits the following peaks in its Fourier Transform infra-red spectrum: 1044 cm^-1 , 1392 cm^"1 , 1589 cm^"1 , 2930 cm^"1 , and 3350 cm^"1.

7. A nano-formulated liposome extract of Labisia pumila comprising:

a liposome formed from a lipid; and

an extract of Labisia pumila as defined in any one of Claims 1 to 5 within the liposome.

8. The liposome of Claim 7, wherein the lipid is a phospholipid.

9. The liposome of Claim 8, wherein the phospholipid is a soybean phospholipid.

10. The liposome according to any one of Claims 7 to 9, wherein the liposome has an average particle size of from 100 nm to 200 nm, optionally wherein the average particle size is from 170 nm to 176 nm.

11. A pharmaceutical formulation comprising an extract of Labisia pumila according to any one of Claims 1 to 5 and/or a nano-formulated liposome extract of Labisia pumilia according to any one of Claims 6 to 9 in admixture with a pharmaceutically acceptable adjuvant, diluent or carrier.

12. An extract or formulation according to any one of Claims 1 to 1 1 for use in medicine.

13. An extract or formulation according to any one of Claims 1 to 11 for use in the manufacture of a medicament for the treatment of obesity.

14. A method of treating obesity comprising administration of therapeutically effective amount of an extract or formulation according to any one of Claims 1 to 11 , to a patient in need of such treatment.

15. A process to obtain an extract of Labisia pumila according to any one of Claims 1 to 6, comprising: i. obtaining a Labisia pumila plant sample; ii. reducing the water content of the plant sample to provide a dried sample; iii. subjecting the dried sample to extraction with a solvent containing a mixture of an alcohol and water to provide an alcoholic extract; iv. removing the solvent from the alcoholic extract to obtain the extract of Labisia pumila.

16. The process according to Claim 15, wherein the plant sample is derived from the leaves of a Labisia pumila plant, optionally the plant sample may be additionally or alternatively derived from the roots and/or stems of a Labisia pumila plant.

17. The process according to Claim 15 or Claim 16, wherein reducing the water content in step (ii) is performed for from 24 hours to 72 hours at from 40°C to 60°C, optionally wherein:

(a) reducing the water content is performed for 72 hours at 55°C; and/or

(b) the dried sample is ground into a powder before use in step (iii).

18. The process according to any one of Claims 15 to 17, wherein extraction step (iii) comprises the use of a soxhlet extracting technique, optionally wherein the solvent containing a mixture of an alcohol and water is replaced at least once to form at least two alcoholic extracts that are combined together.

19. The process according to any one of Claims 15 to 18, wherein extraction step (iii) is performed for from 5 hours to 240 hours, optionally, wherein the extraction step is performed for 120 hours.

20. The process according to any one of Claims 15 to 19, wherein the solution containing a mixture of an alcohol and water has a volumetric ratio (alcohol: water) of from 4: 1 to 1 :4, optionally wherein the volumetric ratio is 1 : 1.

21. The process according to any one of Claims 15 to 20, wherein the alcohol is a lower aliphatic alcohol having from 1 to 6 carbon atoms.

22. The process according to Claim 21 , wherein the lower aliphatic alcohol is selected from any one of the group consisting of methanol, ethanol, isopropanol, n-propanol, f-butanol n-butanol, and any combination thereof, optionally wherein the lower aliphatic alcohol is ethanol.

23. The process according to any one of Claims 15 to 22, wherein the step of removing the solvent from the alcoholic extract in step (iv) is performed by spray drying, freeze drying or evaporation.

24. A process to obtain a nano-formulated liposome extract of Labisia pumila according to any one of Claims 7 to 10, comprising: i. providing a dry Labisia pumila extract prepared in accordance with any one of Claims 15 to 23 and dissolving the same in a first solvent to provide an extract solution; ii. providing a phospholipid solution comprising phospholipids and a second solvent; iii. mixing the extract solution and phospholipid solution together and removing the first and second solvents to form the nano-formulated liposome extract of Labisia pumila.

25. The process according to Claim 24, wherein the first solvent is ethanol.

26. The process according to Claim 24 or Claim 25, wherein the second solvent is chloroform.