WO2020053750A1

WO2020053750A1 - Pre-diabetic therapeutic composition

Info

Publication number: WO2020053750A1
Application number: PCT/IB2019/057607
Authority: WO
Inventors: Patrick BOUIC
Original assignee: Cape Kingdom Nutraceuticals Pty Ltd
Priority date: 2018-09-10
Filing date: 2019-09-10
Publication date: 2020-03-19

Abstract

The invention discloses a pre-diabetic 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 causes decreased fasting serum glucose levels and insulin levels and thereby reversing metabolic syndrome in humans. The composition is adapted to lowering Leptin serum levels and to cause sensitization of the hypothalamus for it to be receptive to satiety.

Description

PRE-DIABETIC THERAPEUTIC COMPOSITION

FIELD OF INVENTION

The present invention relates to a pre-diabetic therapeutic composition.

More particularly, the present invention relates to a pre-diabetic 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 the WHO Factsheet, November 2017, the current estimation of people with diabetes has risen to an astounding 422 million worldwide. Diabetes is a serious and chronic disease, occurring when the pancreas does not produce enough insulin or when the body is unable to effectively utilize the available insulin. Both these conditions result in raised blood glucose levels that may, over time lead, to serious damage to the heart and vascular system, the eyes, kidneys and the nerves.

Three types of diabetes are recognized: (i) Type 1 diabetes (T1 D), formerly known as insulin dependent or juvenile diabetes; (ii) Type 2 diabetes (T2D), previously termed non-insulin-dependent or adult-onset diabetes and (iii) gestational diabetes. The latter is a condition where pregnancy results in raised blood glucose levels, but mostly this is resolved after delivery.

T1 D is an autoimmune disease and has a strong genetic component. It is characterized by a destruction of the pancreatic b— cells leading to insulin deficiency. It accounts for between 5% and 10% of diabetes but its prevalence is increasing globally at a rate of 2-3% per year. Although previously seen as a juvenile form of diabetes, it is currently estimated that more than 40% of all new cases of T1 D, occur over the age of 30.

In the aetiology of type 1 diabetes, the pancreatic islet cells are progressively destroyed by autoantibodies. It is currently a disease without cure and, although the understanding of the disease progression has grown, there is no prevention of disease development known. All prevention trials to date have failed and the only current treatment option is daily insulin injections. However, the interesting concept of pre-T1 D with a staging of disease development has been put forward by the American Diabetes Association. This staging includes an important presymptomatic stage characterized by a steady decline in functional b-cell mass. Even at the beginning of the symptomatic phase, there is still a portion of functional b-cells available. T2D on the other hand, describes the majority of people with diabetes and has a strong link to obesity and metabolic factors. However, the risk factors for the development of T2D also include genetic factors, ethnicity, physical inactivity, smoking and age. An unhealthy lifestyle including a high intake of total fat and total carbohydrates leading to overweight and obesity, are the strongest risk factors for development of T2D. Additionally, all these risk factors predisposes not only to the development of gestational diabetes, but also imprints a future risk of both obesity and T2D on the genome of the mother, that is carried into the next generation.

In the development of obesity-related T2D, the pre-diabetic state is characterized by peripheral insulin resistance (an inability to utilize available insulin). Insulin is the primary stimulus for the muscle to utilize available blood glucose, the liver to stop producing glucose ( de novo glucose synthesis) and the fat tissue to store any leftover glucose into fat. If this system malfunctions, the resultant raised blood glucose levels prompt a further increase in insulin secretion by the pancreas as glucose uptake and metabolism are the primary stimuli for insulin secretion. The pre-T2 diabetic state is therefore characterized by normal glucose levels but high insulin levels. The tipping point is when the pancreatic b-cells are not able to compensate further. The most recent paradigm for the progressive loss of function of the b— cell population lies in the concept of dedifferentiation of the b-cells caused by the post-prandial rise in blood glucose in the insulin resistant state. As all cells in the body, b-cells are derived from embryonic stem cells during the developmental stages. During this process, certain transcription factors and their downstream gene clusters are activated or repressed. With dedifferentiation, this process is driven backwards, leaving the pancreas with a lower concentration of mature b— cells and more precursor cells. The differentiated state of the cell populations are recognized by the specific transcription factors expressed at a given time point.

At the tipping point, the blood glucose levels start to rise to pathologic levels, turning pre-diabetes and insulin resistance into T2D. It has been shown that this happens when 50% of the b— cell population is not functional anymore. Because of this long, asymptomatic prelude to the development of obesity-related T2D, many of the detrimental consequences, e.g. cardiovascular complications, had a 5-10 year period to develop. Type 2D, is recognized as a chronic disease state with no cure, because, at the root, lies again the destruction of mature, insulin- secreting pancreatic b-cells, albeit by a totally different mechanism. Prevention is therefore not an option anymore at the time of diagnosis, but treatment is.

It is an object of the invention to suggest a pre-diabetic 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 pre-diabetic therapeutic composition comprising at least one pre-diabetic active ingredient originating from an Buchu extract or bio-active fraction thereof in a pharmaceutically acceptable form.

Preferably, the pre-diabetic therapeutic composition is a pharmaceutical composition comprising a therapeutically effective amount of at least one or more pre-diabetic 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 pre-diabetic 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 a pre-diabetic response, in particular a pre-diabetic 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 a pre-diabetic response, in particular a pre-diabetic response, in a mammal, preferably a human, in need thereof.

According to a further aspect of the invention, there is provided a method of pre treating a Type 1 and Type 2 diabetes 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: Figures 1.1 to 1.4: results of the Type 1 Diabetes experiments; and Figures 2.1 to 2.7: results of the Type 2 Diabetes experiments.

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.

Models

Experiments

In all instances, only male, outbred Wistar rats, obtained from Charles Rivers laboratories, Inc., Wilmington, MA and bred within the Central Research Facility of the University of Stellenbosch, were used. These rats were age and weight matched at the onset of experimentation. All animals were housed at the University of Stellenbosch Central Research Facility, which provided a simulated environment of the animals’ natural habitat, exposing them to a temperature controlled room (22 °C - 24 °C) and a 12-hour light/dark cycle. Animals had free access to water and food for the duration of the experiment. All animals were fed normal rat chow, unless otherwise stated/ specified, and specific groups in both diabetic models were treated with Buchu water. The projects were approved by the Ethics committee of the University of Stellenbosch (Faculty of Health Sciences - protocol number SU-ACUM1 1 -00003) and conformed to the principles revised in the South African National Standard for the Care and Use of Animals for Scientific Purposes (South African Bureau of Standards, SANS 10386, 2008).

1. Type 1 Diabetes:

The well-known Streptozotocin (Stz) model to induce T1 D by chemical destruction of the pancreatic b— cells, was utilized. We have previously determined that a dose of 40 mg/Kg of Stz that is injected in the adult rat, will result in a proportional destruction of b-cells, thereby representing the pre-T1 D state¹². Adult male Wistar rats (200-250 g) were injected with a single intraperitoneal dose of Stz in citrate buffer (pH 4.5) at 40 mg/Kg body weight. Vehicle injected animals represented the controls. This amount of Stz results in a 50% ablation of b— cells. Blood glucose levels, measured via a drop of blood obtained from a tail prick using a handheld glucometer were monitored daily for a 3-week period. Animals that spontaneously recovered normal blood glucose levels were removed from the groups. T1 D animals were separated into 2 groups, (i) with unfasted blood glucose levels below 20 mmol/L and (ii) with unfasted blood glucose levels between 20 and 30 mmol/L. Stz-injected animals received the diluted Buchu water from the 3-week time point. After 14 weeks, animals were fasted overnight to obtain fasting blood and perform an intra peritoneal glucose tolerance test (IPGTT).

2. Type 2 Diabetes:

A model of diet-induced obesity was used to render the animals insulin resistant. It is known that rats do not develop type 2 diabetes as result of obesogenic diets without a mechanism to also ablate some of the b— cell population. It is postulated that this is because a rat do not form and deposit islet amyloid polypeptide in the pancreatic b— cells. The animals in our experiments therefore represent the pre diabetic state.

Young rats weighing 190 ± 10 g (4-5 weeks old) were divided into a group receiving normal rat chow and a group receiving rat chow supplemented with sugar and condensed milk for a period of 16 weeks. Buchu water treatment was commenced 8 weeks afterwards when the animals have gained substantial weight and already presented with insulin resistance. Food and water intake and the weight of the animals were monitored throughout.

After 15 weeks, animals were fasted overnight to perform an IPGTT. After 16 weeks, animals were euthanized with 160 mg/Kg sodium pentobarbital. Blood was collected and the serum stored. The pancreata were harvested, snap frozen in liquid nitrogen and stored at -80°C for biochemical analyses. In addition, ventricular cardiomyocytes were prepared to determine effects on myocardial insulin resistance.

3. Assays performed:

3.1 On both models:

IPGTT: An intraperitoneal glucose tolerance test was routinely performed to determine whole-body glucose tolerance. Animals were fasted overnight where after the fasting blood glucose value was determined through a drop of blood collected by tail prick. At the same time, 1 ml_ of fasting blood was collected from the carotid artery, the serum separated and stored at -80°C. Here after, a dose of 1 mg/Kg sucrose was injected intraperitoneally and the disappearance of glucose from the blood monitored over a 2 hour period.

3.2 Only on Type 1 diabetic animals:

Daily monitoring of blood glucose levels: The blood glucose levels of these animals were followed daily for the full duration of 14 weeks by collecting a drop of blood from a tail prick in the morning and reading the value on a handheld glucometer.

3.3 Only on Type 2 diabetic animals:

Insulin R!A.

A commercially available RIA (Coat-A-Count® Radioimmunoassay (RIA) kit (Siemens Medical Solutions Diagnostics, Los Angeles, CA) was used to determine the fasting insulin levels in the serum collected.

C-Peptide ELISA:

A commercial rat-sensitive Elisa kit was obtained and used according to the manufacturer’s instructions to determine the C-peptide levels in the stored serum. C-peptide levels are an indication of newly secreted insulin from the pancreatic b-cells.

Cardiomvocvte Glucose uptake:

A. Preparation of cells: Calcium-tolerant adult ventricular myocytes in an unstimulated state were prepared as described previously. After isolation, myocytes were suspended in buffer A containing in mmol/L HEPES 10, KCL 6, NaH2P04 0.2, Na2HP04 1 , MgS04 1.4, NaCI 128, pyruvate 2, glucose 5.5, 2 % BSA (fraction V, fatty acid- free) plus 1.25 mmol/L calcium, pH 7.4. The cells were left for 1 -2 h under an oxygen atmosphere on a gently shaking platform to recover from the trauma of isolation. This procedure routinely rendered in excess of 80 % viable cells as measured by Trypan blue exclusion. After recovery, the cells were allowed to settle into a loose pellet, the supernatant was aspirated, and the cells washed twice with and suspended in a suitable volume of substrate -free buffer A (buffer B).

B. Determination of 2-deoxy-D-3[H1 Glucose (2DG) uptake:

Cardiomyocytes (~0.5 mg protein) were assayed in a total volume of 750 uL of oxygenated buffer B (pH 7.4) as described previously. The cells were pre incubated for 15 min in a shaking water bath (37°C) with or without phloretin (400 uM) for measurement of non-carrier mediated glucose uptake. Each experimental series was incubated with or without 100 nM insulin for 30 min after which glucose uptake was initiated by addition of 2-deoxy-D- [3H]glucose (1.5 uCi/mL; final concentration 1.8 uM). Glucose uptake was allowed to progress for a further 30 min before the reaction was stopped by adding 50 ul phloretin to give a final concentration of 400 uM. The cells were then microfuged for 1 min and the pellet dissolved in NaOH. 50 uL of this was used to assay protein by the method of Lowry et al. while the rest was counted for radioactivity. Results were calculated as pmole 2DG/mg protein/30min. Pancreatic tissue Western blotting of Transcription factors:

Frozen sections of pancreatic tissue were pulverized in a liquid nitrogen pre cooled mortar and pestle and extracted in a standard RIPA buffer containing in mM: Tris-HCI (pH 7.4) 20; EGTA 1 ; EDTA 1 ; NaCI 150; b-glycerophosphate 1 ; tetrasodium- pyrophosphate 2.5; sodium orthovanadate 1 with 1 % Triton-X100, 10 ug/mL Leupeptin, 10 ug/mL Aprotinin and 50 ug/mL PMSF. The tissue was homogenized using a PolyTron PT10 homogenizer twice for 5 sec at setting 4 and left on ice for 15 min to fully digest. The lysate was centrifuged at 15 000 rpm in a microfuge at 4oC and the pellet discarded. The protein content of the supernatant was determined according to the method of Bradford and diluted to equal protein per sample. A 3-times concentrated Laemmli sample buffer was added in a 1 :2 v/v ratio and the sampled boiled for 5 min.

Sixty micrograms of protein were subjected to PAGE using a BioRad Mini Protean III system and the resultant separated proteins transferred to PVDF membranes. Transfer and equal loading were confirmed by Pongeau Red reversible stain. The non-specific binding sites on the membranes were blocked for 2 hours at room temperature with 5% fat free milk dissolved in TBS-Tween. They were then incubated overnight at 4°C with the suitable primary antibody, diluted according to the manufacturer’s instructions. After rinsing thoroughly with TBST the next morning, the membranes were exposed for 1 hour at room temperature to the suitable secondary antibody coupled to horse radish peroxidase. The signal of the specific antibodies bound to the target protein, was captured by exposure to ECL chemiluminescent reagent reacting with the horse radish peroxidase to render a signal captured on film. The film was laser scanned and analysed using computer software (Un-Scan-lt, Silkscience, USA).

Statistical Analyses:

The statistical significance of results were analysed using GraphPad Prism 6. A 1 -way or 2-way analysis of variance were used as applicable followed by a Bonferroni post-hoc analysis to indicate how groups differed. Daily monitoring of blood glucose levels and the intraperitoneal glucose tolerance curves were analysed using a repeated measures 2-way ANOVA. A P-value of less than 0.05 was taken as statistically significant.

Referring to Figures 1.1 to 1.4 it can be stated that the ingestion of this aqueous extract of Buchu, given as a treatment option to animals with partially ablated pancreatic b— cells, resulted in normalization of intra-peritoneal glucose tolerance testing. In addition, a pre-type 1 diabetic state presenting with blood glucose levels of less than 20mmol/L was completely normalized in all measured parameters while the more robust ablation, leading to blood glucose levels of more than 20mmol/L was significantly lowered.

Referring to Figures 2.1 and 2.2, in this group of animals, fed a diet supplemented with sugar and condensed milk to induce hyperphagia, there was no significant increase in body weight of the obese animals. Flowever, they gained significantly more intraperitoneal fat weight. No differences in either food or water intake were observed. Ingestion of Buchu water as a treatment option, resulted in significantly less body weight gain by both the control and the DIO animals. In addition, the DIO animals ingesting the Buchu water, now did not gain intraperitoneal fat despite ingesting the obesogenic diet.

Referring to Figures 2.3 to 2.5, testing of the effects of ingestion of Buchu water as a treatment option in pre-type 2 diabetic animals, demonstrated highly significant effects on insulin sensitivity. Despite not observing a significant lowering of fasting plasma glucose levels, probably because of the wide scatter in data in the DIO group, the glucose tolerance curves presented in Figure 2.4, indicative of the post-prandial handling of blood glucose, showed a normalization of the curves. On organ level, this was substantiated by the data on the ability of isolated ventricular cardiomyocytes to respond to a concentration of 10 nM insulin as shown in Figure 2.5. In both control animals and DIO animals ingesting Buchu water, insulin sensitivity at organ level was significantly enhanced. It can therefore be concluded that the ingestion of Buchu water without a change in ingestion of an obesogenic diet, was able to enhance the glucose utilization at a given level of insulin secretion in peripheral insulin sensitive tissue.

Referring to Figures 2.6 and 2.7, because of the observed effects of ingestion of Buchu water on glucose homeostasis in models of both Type 1 and Type 2 pre diabetes, we investigated possible pancreatic effects. This was prompted by results showing enhanced insulin secretion in the DIO animals after ingestion of Buchu water. The two chosen transcription factors, Maf A and Pdx-1 , were both significantly downregulated in the DIO animals. Ingestion of Buchu water by DIO animals in combination with the obesogenic diet, resulted in significant upregulation of both Maf A and Pdx-1. In addition, the overall effects of Buchu ingestion, as indicated by a 2-way ANOVA analysis, showed a highly significant effect. The enhanced expression of these transcription factors is an indication of b— cell neogenesis, probably by means of redifferentiation of pancreatic progenitor cells or acinar cells.

The question was whether the aqueous extract of Agathosma possessed any anti-diabetic properties as a treatment option. The research project tested this question by utilizing 2 different animal models of diabetes simulating the Type 1 and Type 2 diabetic populations. In both these populations, it is recognized that there are stages of the disease that can be classified as pre-type 1 diabetes and pre-type 2 diabetes. The animal models used was manipulated to represent these so called pre-stages. In type 1 diabetes, this allows a remainder of pancreatic b-cells that has the potential to respond to treatment. If all b— cells were ablated, as is the scenario in end-stage type 1 diabetes, manipulation would be impossible. Similarly, the DIO rat model, induced by feeding a diet rich in sugar and fatty acids, results in animals that are insulin resistant but, according to their fasting blood glucose levels, not type 2 diabetic.

From the results obtained, it is evident that the Buchu water was able to alleviate the pre-type 1 diabetes condition, in all instances, to very near normal levels when used as a treatment option. The animal group presented with animals with either >20mmol/L or <20mmol/L fasting blood glucose values and were grouped accordingly. This is also representative of the measure to which the pancreas was damaged. Highly significant statistical results were obtained showing normalization of blood glucose levels in the <20mmol/L and a significant improvement in the >20mmol/L group (Figures 1 and 2). The IPGTT, used to measure post-prandial insulin responses to remove glucose from the circulation (Figure 1.3), clearly demonstrated normalization in both groups at the clinical relevant 2-hour post load values. This is deemed a novel and important demonstration that this aqueous extract of Buchu has the capability to be used as a treatment in newly diagnosed pre-type 1 diabetic patients.

Anti-hyperglycaemic effects of Buchu water were further substantiated in the diet- induced obese animals (Figure 2.3 and 2.4). In addition, it was observed that ingestion of the Buchu water resulted in a loss of intraperitoneal fat (IP fat). This loss of IP fat is also observed in a different rat model of obesity that also results in hypertension (the FIFD).

To ascertain that the effects on whole-body insulin sensitivity, as measured by the IPGTT assay, was also carried over to organ- and cell level, we made use of myocytes isolated from the ventricles of the hearts of the animals after treatment. This is a sensitive measure to confirm effects on the glucose utilization of peripheral insulin sensitive tissue that is known to be responsible for the removal of post-prandial glucose from the circulation. Figure 2.6 shows that ingestion of Buchu water as treatment of pre-diabetes was able to significantly improve the ability of cardiomyocytes to accumulate glucose after stimulation with insulin. The exact cellular mechanism that was involved in this improvement, is not elaborated on. As it was further noted in the DIO animals that ingestion of Buchu water stimulated insulin secretion (Figure 2.6 A and B), we measured pancreatic transcription factors that is associated with the regeneration of pancreatic b-cells.

Musculoaponeurotic fibrosarcoma homolog A (Maf A), is a transcription factor of the basic leucine zipper family. Maf A is a unique transcription factor expressed during late pancreatic development and is restricted to pancreatic b-cells. Maf A is known to regulate insulin gene expression. In addition, it has been shown that the transcriptional activity of Maf A has a synergistic interaction with Pdx-1 that will enhance gene expression.

Pdx-1 (pancreatic duodenal homeobox 1 protein) in turn, is essential for early pancreatic development and b— cell survival. Pdx-1 expression is restricted to pancreatic progenitor cells during early development but later becomes restricted to the b-cells. Low expression of Pdx-1 has been linked to a decline in b-cell population and impaired glucose tolerance. This decline in Pdx-1 is postulated to be because of enhanced DNA methylation induced by hyperglycaemia. Eventually, this leads to b-cell death via apoptosis. When substantial b-cell loss has occurred, it will result in permanent endocrine deficiency and irreversible diabetes. It has been shown that patients suffering from Type 2 diabetes, has a significantly lower b-cell population than healthy persons. Current research to remedy this situation, has a strong focus on different strategies to produce new endocrine islet cells. Preclinical studies have already shown Pdx-1 expression as a promising candidate in this arena as it was found that, when Pdx-1 progenitor cells were transplanted into mice, some of these cells differentiated into functional b-cells which could reverse diabetes. In addition, it was found that combination expression of Pdx-1 and Maf A amongst others, could efficiently convert pancreatic acinar cells into b-like cells when these transcription factors were supplied to mouse pancreata through adenoviral vectors.

In light of the fact that both Type 1 and Type 2 diabetes are the result of substantial b-cell loss with currently no treatment option to remedy or stop this loss, the results obtained by the current study is of high importance. If pharmacological means can be found to‘redifferentiate’ the dedifferentiated b- cells, it could constitute a new therapeutic approach for diabetes and may be viewed as a distinct form of regenerative therapy. The obesogenic diet fed to the rats in the current study, resulted in a significantly lower expression of both Maf A and Pdx-1 , indicative of a loss of b-cell mass. Ingestion of Buchu water as treatment option, significantly enhanced expression of both these transcription factors, indicating an increase in functional b— cell mass.

It is recognized that the different polyphenols in an extract like the one used in the current study, has very low levels. In addition, the bio-availability of most of these polyphenols is also very low. This has repeatedly posed the question in the past of how orally ingested polyphenols may change physiology. In this regard, a new paradigm has emerged centring on the ability of nutrition to alter the inherent microbiome of the host ingesting any form of food. This microbiome consists of various bacteria, viruses, protozoa and fungi and has an intricate relationship with the physiology of the host. It plays a major role in the way by which any substance ingested, influences the physiology and pathophysiology of the host.

With regards to effects on pancreatic regeneration, b-aminobutyric acid (GABA) has been recognized to act as facilitator to reprogram pancreatic b-cells. Treating mice with GABA was able to significantly increase pancreatic b-cell mass. A definite link between the gut microbiome and the regulation of brain neurotransmitters has repeatedly been demonstrated. Probiotic modulation of the gut microbiota of mice could induce neurochemical changes that was relayed through the vagus nerve connections. It was also shown the gut microbiota can act as an endocrine organ by directly producing metabolites that are transferred to the circulation. Amongst these neurotransmitters, both serotonin and GABA were identified and it was demonstrated that oral ingestion of Lactobacillus increased GABA levels also in the brain. If the aqueous extract of Agathosma utilized in the current study, could induce the gut microbiome to secrete substances like GABA that has been shown to act on the most important transcription factors involved in pancreatic redifferentiation, it could explain our findings of enhanced b-cell neogenesis despite the low levels of polyphenols that can be expected to reach the circulation.

These findings are deemed novel and may indeed lead to development of the pharmacological means to redifferentiate b-cells and treat both pre-type 1 as well as pre-type 2 diabetes. Retrospectively, determination of serotonin and GABA levels in the circulation, could have been performed to substantiate these arguments.

Claims

PATENT CLAIMS

1. A pre-diabetic 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. A pre-diabetic 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. A pre-diabetic therapeutic composition, which includes at least one pre diabetic active ingredient originating from an 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 pre-diabetic 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 pre-diabetic active ingredients.

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

24. A method of pre-treating a Type 1 and Type 2 diabetes 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. A pre-diabetic therapeutic method of reversing metabolic syndrome substantially as hereinbefore described with reference to the accompanying drawings.

30. A pre-diabetic 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 pre-treating a Type 1 and Type 2 diabetes 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.