WO2020053751A1 - Anti-obesity therapeutic composition - Google Patents

Abstract

The invention discloses an anti-obesity therapeutic composition which includes a buchu extract and being adapted to reverse metabolic syndrome by reduction of hyperglycaemia, obesity, hypertension and/or cholesterol. Consumption of the buchu extract cause decreased fasting serum glucose levels and insulin levels and thereby reversing metabolic syndrome in humans. The composition is also adapted to lowering Leptin serum levels and cause sensitization of the hypothalamus for it to be receptive to satiety.

Description

ANTI-OBESITY THERAPEUTIC COMPOSITION

FIELD OF INVENTION

The present invention relates to an anti-obesity therapeutic composition.

More particularly, the present invention relates to an anti-obesity therapeutic composition of Buchu plant material extracts.

BACKGROUND TO INVENTION

Buchu is one of the best known medicinal plants of South Africa and is indigenous to the Cedarberg Mountains and surrounding areas. Despite its popularity little scientific evidence exists about the various medicinal uses of this small fynbos shrub from the family Rustaceae. The two primary species of Buchu used commercially are Agasthoma betulina (round-leaf Buchu) and Agathosma crenulata (oval-leaf Buchu). Besides its medicinal properties, Buchu oil is also used in the flavourant and fragrance industry, currently the largest commercial use thereof. Buchu oil is typically prepared in a (high vacuum) low steam distillation process in which the Buchu oil required for the commercial market is extracted from the plant material and separated from the by-products of this steam distillation process.

According to the World Health Organization (WHO), 2016 more than 1.9 billion adults worldwide are currently overweight and more than 650 million of these individuals are obese. Obesity is associated with several health consequences, including cardiovascular diseases (CVDs), and type 2 diabetes (T2D). These conditions cause premature death or substantial disability, resulting in an economic burden.

Obesity refers to excessive fat accumulation due to an imbalance between energy intake vs. energy expenditure. Excess energy is stored in adipocytes causing hyperplasia (increase in fat cell number) and hypertrophy (increase in fat cell size).

With diet management, adipocyte size can decrease, but adipocyte numbers are constant during adulthood. Hypertrophied adipocytes lead to the activation of several macrophage-attracting chemokines followed by macrophage recruitment. Macrophages secrete cytokines, including, tumour necrosis factor alpha (TNF-a) and lnterleukin-6 (IL-6), causing an inflammatory cascade and thereby inducing insulin resistance in the mature adipocytes. Activated macrophages block pre adipocyte differentiation resulting in a larger population of hypertrophied fat cells (Figure 1 ). According to the adipose tissue expandability hypothesis, this leads to the development of insulin resistance within the adipose mass while adipose depots that consist of new or small adipocytes are associated with improved insulin sensitivity. Insulin resistant adipocytes cause an elevation in free fatty acids (FFAs) release and further macrophage activation, forming a vicious circle. It is postulated that insulin resistance in adipose tissue precedes development of whole-body insulin resistance and lipid accumulation in other organs. It has also been shown in humans, that even in non-obese individuals, adipocyte insulin resistance, characterized by altered adipokine profiles, is associated with adipocyte hypertrophy and whole-body insulin insensitivity.⁷ In addition, Bremer et al. found that subcutaneous fat depots of patients suffering from the metabolic syndrome were associated with increased biomarkers of both insulin resistance and low-grade inflammation. PPARs are important regulators of glucose and lipid storage and oxidation. In adipose tissue, PPARy is an important transcription factor controlling adipogenesis and adipocyte differentiation. PPARa then again, is associated with regulating the expression of genes controlling fatty acid oxidation. Sterol Regulatory Element-binding Proteins (SREBPs) in turn, regulate lipid homeostasis by acting as transcription factors for lipid synthesis. Ferreira et al. demonstrated upregulation of both PPARy and SREBP-1 (also termed Srebfl ) in adipose tissue of high fat fed mice while overexpression of Srebfl in adipose tissue of mice, resulted in adipocyte hypertrophy.

It is an object of the invention to suggest an anti-obesity therapeutic compositions of Buchu plant material extracts.

SUMMARY OF INVENTION

According to the invention, use of Buchu to reverse metabolic syndrome by reduction of hyperglycaemia, obesity, hypertension and cholesterol.

Also according to the invention, use of a Buchu extract by obese persons, adapted to obtain decreased body weight, intra-peritoneal fat deposits possibly by decreasing adipocyte hypertrophy without affecting water consumption.

The consumption of the Buchu extract may lead to decreased fasting serum glucose levels and insulin levels, thereby reversing the metabolic syndrome in humans. The effects of Buchu may be by lowering Leptin serum levels (even in control subjects but even more pronounced in obese individuals): this ultimately results in a sensitization of the hypothalamus to be receptive to satiety.

No detectable effects on serum Adiponectin, IL6 or TNFa may be measurable in the circulating serum.

The ingestion of Buchu may lead to the decrease in the expression of the transcription factor PPAR-gamma, the receptor that regulates fatty acid storage and glucose metabolism: this has a direct implication of the generation of obesity

The Buchu extract may be able to normalize total serum cholesterol and phospholipids and triglycerides in obese subjects

According to the invention, there is provided an anti-obesity therapeutic composition comprising at least one anti-obesity active ingredient originating from a Buchu extract or bio-active fraction thereof in a pharmaceutically acceptable form.

Preferably, the anti-obesity therapeutic composition is a pharmaceutical composition comprising a therapeutically effective amount of at least one or more anti-obesity active ingredient and one or more pharmaceutically acceptable carriers or additives.

The invention extends to a modified Buchu extract and/or bio-active fraction thereof comprising an effective amount of one or more anti-obesity active ingredients. The invention also extends to a therapeutic composition, pharmaceutical composition or modified Buchu extract or bio-active fraction thereof for use in a method of inducing an anti-obesity response, in particular an anti-obesity response, in a mammal, preferably a human, in need thereof.

The invention extends further to the use of a Buchu extract or bio-active fraction thereof in the manufacture of a medicament for use in a method of inducing an anti-obesity response, in particular an anti-obesity response, in a mammal, preferably a human, in need thereof.

According to a further aspect of the invention, there is provided a method of treating obesity comprising administering to a patient in need thereof a therapeutically effective amount of at least one active ingredient obtained from a Buchu extract or bio-active fraction thereof.

The Buchu extract may be obtained from the species Agasthoma betulina (round-leaf Buchu) and/or Agathosma crenulata (oval-leaf Buchu).

BRIEF DESCRIPTION OF DRAWINGS

The invention will now be described by way of example with reference to the accompanying schematic drawings.

In the drawings there is shown in:

Figure 1 : Adipose tissue expandability; Figure 2: Histological examination of fat cell size with n=3-5 biological replicates analysed with an n-value of 650-1050 individual cells per group, were measured and the data compile;

Figure 3: Plasma leptin (A), adiponectin (B), TNF-a (C) and IL-6 (D) levels from fasted buchu treated and untreated animals determined by ELISA assay kits (Abeam);

Figure 4: PCR analyses of the fat tissue; and

Figure 5: Liver analysis results

DETAILED DESCRIPTION OF DRAWINGS

With the concepts of T1 D and T2D as described in the background in mind, pre- clinical evaluations of the effectiveness of an aqueous extract of Agathosma (Buchu), to act as treatment option in both types of diabetes or to act as a preventative option for the development of obesity-related T2D were performed.

2.1 Animal model

Male Wistar rats, inbred from animals obtained from Charles River Laboratories Inc., Wilmington, MA, were used. Animals were housed at the Stellenbosch University Central Research Facility, Tygerberg, in temperature-controlled rooms (22 - 24°C) and kept on a 12-hour light/dark cycle. The animals received humane care in accordance with the principles of the South African National Standard for the care and use of animals for scientific purposes (South African Bureau of Standards, SANS 10386, 2008). Rats weighing 190±10 g were randomly divided into a control and HFD group and kept on the respective diets for 16 weeks (Table 1 ).

Table 1 Composition of diets

Protein Carbohydrates Fructose

Fat Cholesterol Sugar

kJ/100g

(g/1 OOg) (mg/100g) (9/1 OOg)

(%) (%) (g/1 OOg)

Control 4.8 3 17.1 34.6 6.6^. 0.5 1272

HFD ^.27.9^. 6.4^. 14.6 29.5 13.3^. 1 1 1829

Half of each animal group was treated with buchu extract diluted 4 times with tap water, by replacing their drinking water from day 1 of the diet period. To ensure that the buchu species were well blended over the 16-week period, mmultiple 1.5 L sealed buchu water bottles were received from Cape Kingdom Nutraceuticals and diluted on a weekly basis.

Animals had free access to food, undiluted water and diluted buchu extract water and the intake was monitored throughout the 16-week period. The buchu extract dilution was calculated according to the Equivalent Surface Area Dosage Conversion Factors as described by Freireich et al., assuming that an adult human (60 kg) will consume 250 ml_ of the buchu water per day while a rat weighing 250 g, consumes a mean of 30 ml_ water per day.

Fifteen weeks after the onset of the diet period, animals were fasted for 18 hours where after baseline glucose were determined and 1 ml fasting blood at baseline was collected from the carotid artery. Blood was left on ice to clot, followed by serum collection and storage at -80°C. Fasting serum was used to determine insulin levels.

Animals were left to recuperate from the metabolic insult for 1 week before experimentation.

Animals were anaesthetized by intra-peritoneal (IP) injection with 160mg/kg of Sodium pentobarbital and monitored until deep anesthesia, as determined by the lack of reaction on a foot pinch, was observed.

Body weight was recorded, and death induced by exsanguination. Unfasted blood was collected in SGVac gel serum collection tubes, left to clot on ice, followed by serum collection. Serum was stored at -80°C for biochemical analyses.

Both renal and gonadal fat pads were removed and weighed. A specific portion of fat with a piece of muscle attached was dissected from the renal pads for histological examination of fat cell size. Fifty mg fat was collected in RNA latei® (Thermo Fischer Scientific) for real time Polymerase chain reaction (PCR).

2.2 Histology

Fat was cryo-fixed, and eosin stained for the histological determination of fat cell size. Three areas (1 mm x 1 mm) per sample were selected using low magnification, a Zeiss Axioskop 2 microscope with AxioVision v4.7 software and an Axiocamera. Each area was enlarged 10X where after the diameter of every cell in the specific area was measured. Data from the different groups of animals were compiled and statistically analyzed.

2.3 Biochemical analyses

Enzyme-linked immunosorbent assay (ELISA) kits (Abeam®, Cambridge, UK) were used to determine unfasted serum leptin, adiponectin, TNF-a and IL-6 levels. Insulin levels were determined using fasting serum and a RIA coat-a- count assay (Siemens Medical Solutions Diagnostics, Los Angeles, CA)

2.4 PCR analysis

Total RNA was extracted from 50 mg of fat in RNA latei® using Qiazol reagent and purified using the RNeasy Mini Kit, according to the manufacturer’s instructions (Qiagen). RNA concentration and purity were determined spectrophotometrically using the Nanodrop, ND-1000 (Nanodrop Technologies) and integrity determined using the Bioanalyser 2100 (Agilent Technologies). DNase treated RNA (Turbo DNA-free DNase, Life Technologies) was reverse- transcribed to cDNA using the High Capacity Reverse Transcription (Life Technologies). Real-time quantitative PCR was performed in a total volume of 10 mI using cDNA templates and pre-formulated Taqman gene expression assays (Table 2) according to the manufacturer’s protocol (Life Technologies). The cycling conditions were: 50°C for 2 min, 95° for 10 min, followed by 40 cycles of 95°C for 15 sec and 60°C for 1 min. Quantification was carried out on the 7500 Real-time PCR system (Life Technologies). The default settings for the threshold cycle (Ct) and baseline were used. The mRNA levels of each gene were normalised to those of the housekeeping genes b-actin (ActB) and hypoxanthine phosphoribosyltransferase 1 (Hprt).

Table 2 Tagman gene expression assay

Taqman gene expression assay Assay number Srebfi ^. Rn01495769 mi

Fatty acid synthase (Fasn) Rn01463550 ml

PPARa Rn00566193 ml

PPARy Rn00440940 ml

ActB 4326315E

Tiprti Rn01527840 ml

Taqman gene expression assays performed according to the manufacturer’s protocol (Life Technologies). The mRNA levels of each gene were normalised to those of the housekeeping genes b-actin (ActB) and hypoxanthine phosphoribosyltransferase 1 (Hprt).

2.5 Total liver cholesterol, liver triglycerides, plasma phospholipids, and plasma triglycerides analysis

Plasma and liver samples were analysed for concentrations of lipids including total cholesterol, triglycerides and phospholipids

Total cholesterol, triglycerides and phospholipids concentrations were determined using enzymatic colorimetric kits: (LabAssay™ Cholesterol (catalogue number 294-65801 ), LabAssay™ Triglyceride (catalogue number 290- 63701 ) and LabAssay™ Phospholipid (catalogue number 296-63801 ), Wako Chemicals, Germany) using a SPECTRA-max Plus 384 spectrophotometer with SoftMax Pro 4.8 microplate data acquisition and analysis software (Molecular Devices, (US/Canada); Labotec Industrial Technologies, South Africa). The inter assay coefficients of variation (CV) for these analyses were all <3%.

Livers were stored at -80°C for lipid analysis. Liver tissue was homogenised on ice, using a VirTis handishear (The VirTis Company, Gardiner, NY, USA), in 10 volumes of 75m mM phosphate buffer containing 0.5M EDTA (Sigma-Aldrich), pH 7.4. Extraction of lipids was carried out by the Bligh & Dyer method, a modified method of Folch et al. Briefly, lipids were extracted into a monophasic mixture of chloroform and methanol (Merck (Pty) Ltd, South Africa). This mixture was finally separated into two phases, one of which was the organic phase containing the purified lipids. This layer was dried under nitrogen gas, after which it was dissolved in chloroform and divided into aliquots, dried once again under nitrogen headspace and stored at -80°C. For lipid analyses, the extracts were solubilised in ethanol (Merck (Pty) Ltd, South Africa) and Triton X-100 (Sigma- Aldrich (Pty) Ltd, South Africa), and this was followed by the addition of physiological saline. This was used for the determination of total cholesterol, triglyceride and phospholipid concentrations in the liver samples. Liver cholesterol, triglyceride and phospholipid concentrations were determined using the enzymatic colorimetric kits as described for plasma above. 2.6 Statistical analysis

All data was statistically analyzed using Graph Pad Prism 5. All values are given as Mean ± SEM. A 2-way ANOVA followed by a Bonferroni Post hoc test was used to determine significance and a p-value of <0.05 was taken as significant.

Results

3.1 Biometric data

Table 3 summarizes the biometric parameters and daily food and water consumption of the animals. The HFD significantly increased the animals body weight from 381.3±9.5 g to 451.8±15.1 g; (n=10/group; p<0.001 ) and IP fat from 8.6±0.4 g to 24.5±2.7 g (n=10/group; p<0.0001 ) when compared to the controls. However; buchu consumption resulted in less weight - and IP fat gain in the HFD animals (from 451.8±15.1 g to 394.1 ±14.0 g; n=10/group; p<0.01 and from 24.5±2.7 g to 15.9±1.3 g; n= 10/group; p<0.01 respectively). The effect of buchu consumption on lowering body weight gain was further indicated as highly significant (p=0.003).

In addition, similar analyses of IP fat accumulation, showed that the HFD had a significant effect (p<0.0001 ) while the effect of buchu consumption on lowering IP fat content was significant with p=0.007. These changes were not associated with differences in the amount of food consumed by the animals However; the HFD has a higher caloric density compared to the control diet (Table 1). A slightly diminished buchu extract intake was noted in the control animals, but not in the HFD animals. The HFD animals presented with raised fasting blood glucose levels (p<0.01 ) while buchu consumption had a significant overall effect on blood glucose levels (p<0.01 ) and prevented the rise in blood glucose in the HFD animals (p<0.05).

Table 3: Biometric parameters of different animal groups.

Control Control + HFD HFD + buchu buchu

Body weight (g)

381 .3±9.5 363.2±5.5 451 .8±15.1 ^*** 394.1 ±14.0^## n=10/group

IP fat weight (g)

8.610.4 8.7+0.4 24.5±2.7^**** 15.9±1 .3^## n=10/group

Blood glucose (mmol/L)

6.2±0.1 6.0±0.1 7.3±0.2^** 6.2±0.2^# n=5/group

Insulin (mlU/mL)

16.6±3.3 1 1 .8+4.7 21 .8±3.8 12.2±0.5 n=5/group

Food intake (g/rat/day)

21 .4±0.5 20.4±0.4 21 .7±1 .0 19.3±0.7 n=20/group

Water intake (mL/rat/day)

33.8+0.9 28.7+0.9 29.7±1 .7 30.411 .0 n=20/group Data are expressed as mean ± SEM. 2-Way ANOVA; n=5-20. ^* p<0.05 vs Control; ^**p<0.01 vs Control; ^***p<0.001 vs Control; ^****p<0.0001 vs Control; # p<0.05 vs HFD; ## p<0.01 vs HFD. The effect of buchu on: Body weight p=0.003; IP fat p=0.007; Blood glucose levels p<0.01. Effect of FIFD on: IP fat p<0.0001.

3.2 Adipocyte hypertrophy

In Figure 2, the FIFD animals presented with enlarged fat cells (p<0.0001 ) while ingestion of buchu by the FIFD animals decreased fat cell size (p<0.0001 ). Then again, fat cells in the control animals were overall significantly larger after buchu ingestion (p<0.001 ).

Figure 2: Adipocyte hypertrophy. Flistological examination of fat cell size with n=3-5 biological replicates analysed with an n-value of 650-1050 individual cells per group, were measured and the data compiled.

3.3 Biochemical analysis

Blood serum indicated that buchu ingestion had an overall significant effect on lowering leptin levels (p=0.0003, Figure 3a). This was accentuated by significantly lower leptin levels in the FIFD animals (p=0.0023). No significant differences were observed in the adiponectin (Figure 3b), TNF-a (Fig. 3c) and IL- 6 (Fig. 3d) levels.

Figure 3: Biochemical markers leptin, adiponectin, TNF-a and IL-6. Plasma leptin (A), adiponectin (B), TNF-a (C) and IL-6 (D) levels from fasted buchu treated and untreated animals determined by ELISA assay kits (Abeam). Values are given as mean ± SEM. n=5-8 biological replicates per group. Analyses were performed in triplicate. The overall effect of buchu on leptin levels were significant (p=0.0003).

3.4 Transcription factors

According to PCR analyses of the fat tissue: The HFD elevated the mRNA levels of PPARy (p<0.05, Figure 4a) while ingestion of buchu lowered these levels to control values (p<0.05). The overall effect of the FIFD on the mRNA of Srebfl was also indicated as significant (p=0.05, Figure 4c). These levels were not affected by ingestion of buchu. PPARa showed no significant changes (Figure 4b).

Figure 4: Transcription factors PPARy, PPARa and Srebfl For (A) PPARy, (B) PPARa and (C) Srebfl determination, total RNA was extracted from 50 mg of fat in RNAIater® for quantitative real-time PCR analysis. mRNA levels were normalised to the housekeeping genes b-actin (ActB) and hypoxanthine phosphoribosyltransferase 1 (Hprt). ^*p<0.05 with n=4 biological replicates per group. Additionally, a 2-way ANOVA indicated the effect of the FIFD on Srebfl levels to be significant with p=0.05.

3.6 Total liver cholesterol, liver triglycerides, plasma phospholipids, plasma triglycerides

Liver analysis indicated that treatment with buchu significantly decreased total cholesterol levels in both the control and FIFD animals (p=0.03). Furthermore, buchu treatment decreased plasma phospholipids with borderline significance (p=0.07) in both animal groups, and plasma triglycerides in the HFD animals (p=0.03).

Figure 5: Total liver cholesterol, liver triglyceride, plasma phospholipids and plasma triglycerides

Total liver cholesterol (A), liver triglyceride (B), plasma phospholipids (C) and plasma triglycerides (D) were determined using commercially available assays. n=3-5 biological replicates per group. A 2-way ANOVA indicated that buchu had an overall positive effect on total cholesterol levels (p=0.03) and plasma phospholipids (p=0.07, borderline significant). Buchu also significantly decreased plasma triglyceride levels in the FIFD animals (p=0.03).

The current pandemic of obesity is a known risk factor for several non- communicable diseases, including CVDs, T2D and various cancers. Treatment strategies include: lifestyle changes, drugs and bariatric surgery. Lifestyle changes have proved to be difficult to adhere to, drugs have at best modest benefits while bariatric surgery, although successful in affecting weight loss, has serious long-term consequences. Nutraceutical therapies have been explored with various degrees of success in this field and, if scientifically validated, can supply a readily available and safe alternative to pharmacotherapy. In the current study, we therefore tested the anti-obesity potential of an aqueous extract of buchu, a product currently commercially available.

The Agathosma plant, especially A. crenulata, contains one possible hepatotoxic substance, pulegone. Pulegone however, is not water soluble and has Food and Drug Administration (FDA) approval for use in the food industry (FEMA GRAS status and listed among the authorized synthetic flavouring substances CFR 21 - 172.515.) The no effect level of pulegone in beverages is 100mg/kg. The buchu extract contained 74.22±1.1 mg/L pulegone which is well below the acceptable level indicated by the FDA and the LC50 value of 25.91 mg/mL. Buchu treated animals in our study would have ingested a maximum of 1.7 mg/kg/day of pulegone and showed no observable signs of hepatotoxicity according to liver cholesterol and triglyceride analysis (Figure 5).

The water of buchu treated animals were replaced with a 4-times dilution of the aqueous buchu extract. This would then render a concentration in mg/L of Diosmin (0.0005), Quercetin (0.007), Flesperidin (0.001 ) and Rutin (0.0035), of which the animals consumed a mean of 30 mL per day. The ingestion of these bioflanonoids were therefore extremely low. Despite this, the FIFD buchu treated animals presented with significantly less IP fat weight gain and lower blood glucose values when compared to FIFD untreated animals (Table 3). In view of the adipose expandability hypothesis and the known effects of bioflavonoids on obesity, we examined changes brought about in the fat depots of the animals to understand the lesser weight gain.

As endocrine organ, fat secretes certain adipokines, amongst others leptin and adiponectin. In obese individuals, raised leptin levels increase macrophage phagocytic activity, along with their production of pro-inflammatory cytokines resulting in decreased small adipocyte formation. As shown in Figure 2, the FIFD resulted in increased adipocyte size while buchu ingestion resulted in significantly smaller adipocytes accompanied by significantly lower leptin secretion. Leptin acts as an appetite suppressant by inhibiting neuropeptide Y via negative feedback. However, the current study documented no difference in the amount of food consumed by the animals (Table 3), therefore indicating that, in the buchu treated HFD animals, the lower leptin secretion was because of smaller adipocytes, as larger cells secrete more adipokines.

Buchu ingestion significantly attenuated blood glucose levels in both animal groups, indicating improvement of insulin sensitivity (Table 3).

Thus, buchu ingestion in the HFD animals therefore decreased adipocyte size and leptin levels, thereby increasing insulin sensitivity and pre-adipocyte formation (Figure 2; 3a).

Adipogenesis and lipogenesis are regulated by multiple transcription factors coding for different enzymes modulating fat deposition and lipolysis. To further elucidate the anti-obesity effects of buchu, we determined the mRNA levels of three regulators of adipogenesis and fatty acid oxidation: PPARy, PPARa and Srebfl (Fig. 4a-c). No changes were observed in PPARa levels. PPARy has long-chain fatty acids as endogenous ligands and, as also found in the current study, is known to be increased in adipose tissue following a HFD. In accordance with findings in mice fed a HFD, the transcription factor Srebfl , known to be involved in lipid synthesis, was also elevated in the current study and associated with adipocyte hypertrophy.

PPARy expression is regulated amongst others, by the b-catenin pathway which in turn, is regulated by Wnt-GSK-3 signalling. As GSK-3 is overexpressed in adipose tissue of obese mice, it can be expected that b-catenin levels are low, leading to increased PPARy levels. The increased insulin sensitivity of the adipose tissue after buchu ingestion may therefore, via normalization of inhibition of GSK-3, lead to the decrease in PPARy levels. It is also known that Sirtl promotes fat loss by repressing PPARy through acetylation; however Sirtl expression was not determined in the current study. In contrast to the decrease in PPARy levels induced by buchu ingestion in the HFD animals, we observed no significance in the control animals’ PPARy levels, but an increase in adipocyte hypertrophy. This may be explained by an observation by Ferreira et al. that diet composition differentially affects adipocyte hypertrophy. In addition, despite this small increase in adipocyte size, no increase in the intraperitoneal-fat deposition was detected.

Reverse cholesterol transport (RCT) refers to the process where cholesterol from the peripheral tissues are returned to the liver via the plasma.

The first step entails the movement of peripheral cholesterol and phospholipids to the pre-Fligh-density lipoprotein (FIDL) via the ATP-binding cassette transporter (ABCA1 ). Following this, the cholesterol present on the pre-FIDL is converted by the lecithin-cholesterol acyltransferase (LCAT) enzyme, to cholesteryl esters, which are stored in the core of the FIDL. Consequently the cholesteryl esters will be exchanged for triglycerides from other lipoproteins (VLDL and LDL) via the cholesterylester transfer protein (CETP). The mature FIDL will bind to the Scavenger receptor class B type 1 (SRB1 ) present on the liver surface, followed by the release of cholesteryl esters. The cholesterol will be converted to bile and transferred to the intestine for excretion. Interestingly, in rats and mice, RCT is altered due to the sequences coding for functional CETP activity, being deficient. Thus, due to this deficiency, the plasma cholesterol, phospholipids and triglyceride levels of rats will be more accurate when measured in the plasma, when compared to the liver.

In the current study, buchu decreased (borderline significant) plasma phospholipids in both the animal groups and plasma triglyceride levels in the HFD animals (Figure 5C-D). Both these results are indicative of decreasing the risks associated with the metabolic syndrome.

Although it is recognized that the known bioflavonoids in the buchu extract, especially hesperidin and quercetin, have the capability of regulating the levels of PPARy and Srebfl the concentrations of these substances in even the original, undiluted buchu extract are so low in comparison to the concentrations used in these studies, that, in isolation, they cannot be deemed to elicit the physiological effects observed.

The buchu extract tested in this study has beneficial effects as nutraceutical to counteract obesity with all the accompanying health benefits of less weight gain. The exact mechanisms whereby this is accomplished is possibly multifactorial but include insulin sensitization of fat cells. The extract may contain a hitherto unrecognized substance that may have elicited the observed changes, or the combination of bioflavonoids may be responsible.

Claims

PATENT CLAIMS

1. An anti-obesity therapeutic composition which includes a buchu extract and being adapted to reverse metabolic syndrome by reduction of hyperglycaemia, obesity, hypertension and/or cholesterol.

2. A composition as claimed in claim 1 , which is adapted through consumption of the buchu extract to cause decreased fasting serum glucose levels and insulin levels and thereby reversing metabolic syndrome in humans.

3. A composition as claimed in claim 1 or claim 2, which is adapted to lowering Leptin serum levels.

4. A composition as claimed in claim 3, which is adapted to cause sensitization of the hypothalamus for it to be receptive to satiety.

5. A composition as claimed in any one of the preceding claims, which after consumption thereof shows no detectable effects on serum Adiponectin, IL6 or TNFa may be measurable in the circulating serum.

6. A composition as claimed in any one of the preceding claims, in which ingestion thereof is adapted to lead to the decrease in the expression of the transcription factor PPAR-gamma, the receptor that regulates fatty acid storage and glucose metabolism.

7. A composition as claimed in any one of the preceding claims, in which the buchu extract is adapted to normalize total serum cholesterol and phospholipids and triglycerides in obese subjects.

8. A composition as claimed in any one of the preceding claims, in which use of the Buchu extract by obese persons, is adapted to obtain decreased body weight, intra-peritoneal fat deposits possibly by decreasing adipocyte hypertrophy without affecting water consumption.

9. A composition as claimed in any one of the preceding claims, in which the buchu extract is obtained from the species Agasthoma betulina (round-leaf Buchu) and/or Agathosma crenulata (oval-leaf Buchu).

10. An anti-obesity therapeutic method of reversing metabolic syndrome, which includes the steps of administering a buchu composition which includes a buchu extract adapted to cause reduction of hyperglycaemia, obesity, hypertension and/or cholesterol.

1 1. A method as claimed in claim 10, which includes the step of decreasing fasting serum glucose levels and insulin levels and thereby reversing metabolic syndrome in humans.

12. A method as claimed in claim 10 or claim 1 1 , which includes the step of lowering Leptin serum levels.

13. A method as claimed in claim 12, which includes the step of causing sensitization of the hypothalamus for it to be receptive to satiety.

14. A method as claimed in any one of claims 10 to 13, which after consumption of the buchu composition shows no detectable effects on serum Adiponectin, IL6 or TNFa may be measurable in the circulating serum.

15. A method as claimed in any one of claims 10 to 14, which includes the step of causing a decrease in the expression of the transcription factor PPAR-gamma, the receptor that regulates fatty acid storage and glucose metabolism.

16. A method as claimed in any one of claims 10 to 15, which includes the step of normalizing total serum cholesterol and phospholipids and triglycerides in obese subjects.

17. A method as claimed in any one of claims 10 to 16, which includes the step causing decreased body weight in obese persons and intra-peritoneal fat deposits possibly by decreasing adipocyte hypertrophy without affecting water consumption.

18. A method as claimed in any one of claims 10 to 17, in which the buchu extract is obtained from the species Agasthoma betulina (round-leaf Buchu) and/or Agathosma crenulata (oval-leaf Buchu).

19. An anti-obesity therapeutic composition, which includes at least one anti obesity active ingredient originating from a Buchu extract or bio-active fraction thereof in a pharmaceutically acceptable form.

20. A composition as claimed in claim 19, which is a pharmaceutical composition comprising a therapeutically effective amount of at least one or more anti-obesity active ingredient and one or more pharmaceutically acceptable carriers or additives.

21. A modified Buchu extract and/or bio-active fraction thereof which includes an effective amount of one or more anti-obesity active ingredients.

22. A therapeutic composition, pharmaceutical composition or modified Buchu extract or bio-active fraction thereof for use in a method of inducing an anti-obesity response, in particular an anti-obesity response, in a mammal, preferably a human, in need thereof.

23. Use of a Buchu extract or bio-active fraction thereof in the manufacture of a medicament for use in a method of inducing an anti-obesity response, in particular an anti-obesity response, in a mammal, preferably a human, in need thereof.

24. A method of treating obesity comprising administering to a patient in need thereof a therapeutically effective amount of at least one active ingredient obtained from a Buchu extract or bio-active fraction thereof.

25. Use of Buchu, which is adapted to reverse metabolic syndrome by reduction of hyperglycaemia, obesity, hypertension and cholesterol.

26. Use of a Buchu extract by obese persons, which is adapted to obtain decreased body weight, intra-peritoneal fat deposits possibly by decreasing adipocyte hypertrophy without affecting water consumption.

27. Consumption of a Buchu extract, which is adapted to lead to decreased fasting serum glucose levels and insulin levels, thereby reversing the metabolic syndrome in humans.

28. A buchu composition substantially as hereinbefore described with reference to the accompanying drawings.

29. An anti-obesity therapeutic method of reversing metabolic syndrome substantially as hereinbefore described with reference to the accompanying drawings.

30. An anti-obesity therapeutic composition substantially as hereinbefore described with reference to the accompanying drawings.

31.A modified Buchu extract and/or bio-active fraction thereof substantially as hereinbefore described with reference to the accompanying drawings.

32. Use of a Buchu extract or bio-active fraction thereof substantially as hereinbefore described with reference to the accompanying drawings.

33. A method of treating obesity substantially as hereinbefore described with reference to the accompanying drawings.

34. Use of Buchu substantially as hereinbefore described with reference to the accompanying drawings.

35. Use of a Buchu extract by obese persons substantially as hereinbefore described with reference to the accompanying drawings.

36. Consumption of a Buchu extract substantially as hereinbefore described with reference to the accompanying drawings.