Field of the Invention

The invention relates to plant cells of the genus Hoodia and uses thereof.

Introduction

Obesity and weight problems are a major health problem, particularly in the developed world. Globally, there are more than one billion overweight adults and at least 300 million of these are clinically obese. It is widely recognised that obesity and overweight can lead to serious disease such as diabetes, cardiovascular disease, stroke and certain types of cancer.

Accordingly, there is a great demand for treatments that can help to change the eating habits of patients. A number of appetite suppressants are available but they often have unwanted side effects. Furthermore, patients are often adverse to taking chemically synthesised drugs and prefer to use natural remedies or compounds derived from natural sources.

Hoodia (common name: Milkweed) is a flowering succulent plant which belongs to the family of Asclepiadaceae. Another name which is sometimes used synonymously with Hoodia is Trichocaulon. Hoodia grows in Namibia, South Africa and Botswana. Although it is difficult to cultivate the plant outside its natural habitat, the plant can be grown in the green house and is available for sale in Garden centres in

different countries and also by mail order through the World Wide Web.

Hoodia plants are known for their medicinal properties. A number of patent applications describe compositions having appetite suppressant activity comprising an extract of a plant of the genus Hoodia (CA 2283564, US2003086984, WO98/46243 and EP1166792) . All of these applications teach the preparation of an extract from plant material other than cultured plant cells such as the whole plant or parts thereof, for example stem and roots.

WO98/46243 discloses an extract of Hoodia comprising a steroidal trisaccharide (C ₄₇ H ₇₄ Oi ₅ M+878) having the formula (1) and having appetite suppressant activity. WO98/46243 also discloses active analogues or steroidal glycoside derivatives of the compound shown in formula (1) having appetite suppressant activity. This document also shows experimental evidence using animal models showing that the compounds identified have appetite suppressant activity.

Formula (1)

B

EP1166792 relates to the use of an extract of Hoodia in the manufacture of a medicament for the treatment of diabetes.

However, the use of a plant extract derived from an intact plant or parts of an intact plant has a number of disadvantages. Firstly, sufficient amounts of plant material have to be obtained for the extraction process. Harvesting is limited in those countries where Hoodia occurs naturally and outside the natural conditions, Hoodia have to be grown in specialised conditions in the green house. Furthermore, Hoodia plants have a long life cycle and therefore, growing large quantities of plant material is not only expensive but also time consuming.

It is well known in the art that plant cells can be maintained in tissue culture. Tissue culture is a term used in the art to define the growth of plant cells outside an intact plant in a suitable nutrient medium. Tissue culture is defined as a method wherein parts of a plant are transferred to an artificial environment in which they can continue to survive. The term tissue culture as understood in the art refers to cultured tissue which may consist of individual or groups of plant cells, protoplasts or whole or parts of a plant organ.

In tissue culture, plant cells can be grown on a solid . , - surface as pale brown lumps -called callus or as individual or small clusters of cells in suspension cultures. Cells grown in culture are actively dividing and can be maintained indefinitely in an undifferentiated state by transferring the cells to fresh media (subculturing) . Cultured cells can also be induced to re-differentiate into whole plants.

However, it has been shown that whole plants regenerated from callus cultures produce genetic variants (Philipps et al) .

Tissue culture is a method well known in the field of plant biology and has several applications, for example it is used to produce large quantities of plants or plant material by vegetative multiplication in a short period of time (micropropagation) .

Plant tissue cultures can be initiated from almost any part of the source plant (termed explant) , although younger parts of a plant are generally more useful as they contain a higher amount of dividing cells.

Although tissue culture is a method well known in the art, different plants may vary in the exact conditions for maintaining cells in culture.

Cells in tissue culture are generally different from in vivo cells (cells in an intact plant which have not been isolated from the plant and cultured) , for example they have a small ^' Vacuole, lack chloroplasts and photosynthetic pathways. It is also well known in the art that cultured plant cells produce different amounts and altered profiles of metabolites (Dicosmo and G. Delle Monache) .

The present inventors have surprisingly found that cultured plant cells of the genus Hoodia have a very similar profile of compounds having appetite suppressant activity compared to the profile of compounds having appetite suppressant activity found in extracts of a whole Hoodia plant or parts

thereof other than isolated cells. The inventors have prepared a culture of plant cells isolated from a Hoodia. plant and established a stable plant cell line in culture. As plant cells propagate within a few days, it is inexpensive to produce large quantities of the cultured cell by subculturing. Therefore, plant cell cultures of the genus Hoodia or Trichocaulon provide a convenient and inexpensive alternative to known appetite suppressants. Furthermore, the cells can also be used for the production of a medicament for the treatment and/or prevention of obesity.

Description of the invention

In accordance with a first aspect of the invention, there are provided cultured plant cells of the genus Hoodia characterised in that they have appetite suppressant activity.

The present invention will now be further described. In the following passages different aspects of the invention are defined in more detail . Each aspect so defined may be combined with any other aspect or aspects unless clearly indicated to the contrary. In particular, any feature indicated as being preferred or advantageous may be combined with any other feature or features indicated as being preferred or advantageous.

The cells of the invention are characterised in that they produce at least one pharmaceutically active compound having appetite suppressant activity. According to the invention, the term produce is used to describe that plant cells make a compound which may then be retained within the cell, for

example in the vacuole or in a storage organ, or which may be secreted.

The cells are also characterised in that they are maintained and propagated in culture.

The inventors have surprisingly found that cells isolated from a plant of the genus Hoodia which are maintained and propagated in culture according to known methods in the art have a very similar profile of compounds compared to the profile of compounds found in an extract of a whole Hoodia plant. The inventors have also shown that cells isolated from a plant of the genus Hoodia which are maintained and propagated in culture according to known methods in the art produce at least one compound having appetite suppressant activity. Accordingly, the inventors are the first to show that cultured Hoodia cells have appetite suppressant activity.

This is an unexpected and surprising result as it is well known in the art that plant cells which are maintained in culture produce different quantities of metabolites than in -vivo cells. In some cases, metabolites present in the intact plant are absent in cultured cells (Delle Monache) .

To verify that the cells have appetite suppressant activity, the active fraction in the cells can be identified using conventional chemical techniques. A person skilled in the art will appreciate that such techniques include, but are not limited to, chromatographic methods and nuclear magnetic resonance. Compounds having appetite suppressant activity in a Hoodia extract derived from a Hoodia plant and not derived .

from cultured cells are known in the art. These compounds may be used for comparative purposes to assess whether the correct compound(s) is/are produced by the cultured Hoodia cells. Therefore, the person skilled in the art will be able to identify the presence of compounds having appetite suppressant activity in the cultured cells of the invention using routine methods and knowledge available in the art.

The plant cells according to the invention have been isolated from their natural environment. A number of techniques for isolation of cells are known in the art. For example, the cells may be isolated by cutting off a small piece of plant tissue. A skilled person will appreciate that any of the methods known in the art can be used according to the invention. It will be understood by the skilled person that the invention can be carried out using cells isolated from different parts of one or more plants. For example, cells may be isolated from the mesoderm as exemplified in example 1.

It will also be understood by a person skilled in the art that the total number of cells isolated may vary. In -principle, there must be at least one cell as this cell will divide and multiply. However, starting from a single cell requires precise isolation of a single cell and is therefore time consuming. Accordingly, the total number of cells according to the invention may vary.

The terms cells "in culture" or "cultured cells" are used herein to refer to tissue culture of plant cells. Tissue culture refers to methods wherein plant cells derived from any part of the plant are grown in isolation from intact

plants in nutrient media under controlled and sterile conditions. Nutrient media commonly used in the art comprise carbohydrates as a source of energy, salts, vitamins, amino acids, minerals, plant growth hormones and other compounds. The media may also comprise antibacterial and fungicidal compounds to prevent contamination by bacteria and/or fungi.

A number of different tissue culture methods are well known in the art and a skilled person will appreciated that the cells according to the invention may be cultured according to any of these methods. Such methods include, for example, tissue culture using Petri dishes and solid agar medium. Another well known culture method is suspension culture wherein the cells are suspended in a liquid and stored in flasks. Furthermore, plant cells may also be cultured using adherent plant cell cultures wherein cells are immobilized on gels, foams or membranes.

According to the invention, the plant cells used are of the genus Hoodia. Preferably, the cells are from the species Hoodia gordinii. In another preferred embodiment, the cells are from the species Hoodia currorii or Hoodia lugardi. A skilled person will appreciate that the genus Hoodia is sometimes used in the art as a synonym for the genus Trichocaulon. Accordingly, in another preferred embodiment, plant cells of the genus Trichocaulon may be used. In another preferred embodiment, cells of the species Trichocaulon officinale or Trichocaulon piliferum may be used. Furthermore, the cells according to the invention may also comprise combinations of two or more species selected from the group of Hoodia gordinii, Hoodia currorii, Hoodia lugardi, Trichocaulon officinale or Trichocaulon piliferum.

In another aspect, the invention relates to cultured plant cells according to the first aspect of the invention obtainable by isolating cells from a whole plant or parts of a plant and maintaining the cells in a culture medium. As described above, methods for isolating and culturing plant cells are well known in the art.

The skilled person will appreciate that cultured plant cells may secrete compounds into the surrounding medium. Accordingly, in one embodiment, the cultured cells of the invention secrete the at least one compound having appetite suppressant activity into the culture medium. If the cells according to the invention secrete the compound into the surrounding medium, then the medium can be used as a pharmaceutical composition or as a food or beverage supplement . It is also possible to extract the active fraction from the medium and to use the resulting extract as a food or beverage supplement or in the manufacture of a medicament .

As explained above, one way of tissue culturing plant cells ¹ XS by suspension culture. In a suspension culture, small clusters of cells are grown in a flask suspended in a culture media. The culture or nutrient media typically comprise carbohydrates as a source of energy, salts, vitamins, amino acids, minerals, plant growth hormones and other compounds. The flasks containing the cells and the culture media are typically stored on a shaker to prevent the cells from settling at the bottom of the flask. Suspension cultures are sub-cultured at specified intervals, for example every three weeks, to provide fresh growth media

and to maintain the cells in an undifferentiated state. According to the invention, the media in which the cells are suspended and into which the at least one compound having appetite suppressant activity is secreted can be collected. The resulting liquid has appetite suppressant activity and can, for example, be used as a food or beverage supplement to provide appetite suppression when ingested. The liquid may be used as a dietary food or beverage supplement for non medical purposes to increase general well being but may also be used in the manufacture of a medicament .

In another preferred embodiment, the invention relates to cultured plant cells according to the invention characterised in that the cells are treated following culturing to change the physical nature of the cells. In one embodiment, the cells are treated by freeze-drying. In another embodiment, the cells are treated by spray-drying. In a further embodiment, the cells are treated by vacuum- drying. In yet a further embodiment, the cells are homogenised. Homogenisation may also be used prior to a method for drying. Methods of freeze-drying, spray-drying, vacuum-drying or homogenisation are well known in the art. ror example, for homogenisation, the cells may be mixed in a blender or vortexed. According to the invention, the ■ - ^• treatment of the cells by any method which may alter the physical nature of the cells will not alter the appetite suppressant activity of the fraction obtained by such treatment.

In another aspect, the invention relates to freeze-dried cells having appetite suppressant activity derived from cultured cells according to the invention. ^{' ■} -

In a further aspect, the invention also relates to a process for preparing an extract having appetite suppressant activity from cultured plant cells according to the invention comprising the steps of treating the cells with one or more solvents and collecting a fraction comprising at least one compound having appetite suppressant activity. The one or more solvent used is characterised in that it is preferably organic and polar. It is also useful if the one or more solvent is immiscible with water as this allows the easy separation of the active ingredients from other components which will be drawn out .

In a preferred embodiment, the organic solvent comprises an alcohol . Preferably, the alcohol is a Ci to C ₄ alcohol . Preferably, the Ci to C ₄ alcohol is a linear or alkyl alcohol. The alcohol may preferably be methanol, ethanol or propanol .

In another embodiment, the polar organic solvent comprises a haloalkane. Preferably, the haloalkane is a C _x to C ₄ haloalkane. Also preferably, the haloalkane comprises ^■ (--nlorine. The chlorine may be present as Cl _x to Cl ₄ . For example, the haloalkane may be trichloromethane (chloroform), chloromethane or dichloromethane. ...

Another polar organic solvent that may be used is a carbonyl alkane. Preferably, the carbonyl alkane comprises C _x to C ₄ . In a preferred embodiment, the carbonyl alkane is acetone.

It is also possible to use combinations of the polar organic solvents described above. For example, the solvent may

comprise an alcohol and a haloalkane, an alcohol and a carbonyl alkane or a carbonyl alkane and a haloalkane. In a preferred embodiment, the solvent comprises a mixture of methanol and chloroform. In another preferred embodiment, the mixture of methanol and chloroform contains both compounds in equal parts.

The resulting extract having appetite suppressant activity may be mixed together with other compounds, for example a pharmaceutical carrier, to form a final composition. For example, the resulting extract having appetite suppressant activity can be used as a medicament for the treatment of obesity. Furthermore, the extract may be used as a food supplement in a foodstuff or beverage.

In another aspect, the invention relates to an extract having appetite suppressant activity derived from cultured cells according to the invention.

In yet another aspect, the invention relates to cultured plant cells of the genus Hoodia for use as a medicament. The invention also relates to cultured plant cells for use as an ^■ appetite suppressant.

Obesity is a major health problem due to conditions associated with this disease, such as cardiovascular disease ^" and diabetes. However, weight problems which are not classified as obesity pose a problem for people affected, as overweight often results in lowered self esteem and finding less acceptance within society. Accordingly, the scope of the invention also relates to cultured cells according to- ' the invention for use as an appetite suppressant wherein the

appetite suppressant is not a medicament for the treatment of a disease. Rather, the appetite suppressant can used by- overweight people to increase their general well-being.

In another aspect of the invention, there is provided a foodstuff or beverage comprising the plant cells according to the invention as a dietary supplement for appetite suppression. As explained above, the cells according to the invention may be for use as an appetite suppressant for a non medical purpose. Many people prefer the use of natural or herbal remedies over chemically synthesized products. Accordingly, the use of cultured Hoodia cells having appetite suppressant activity as a natural remedy to suppress appetite and to increase general well-being is advantageous.

The cells according to the invention which may be used as a foodstuff or beverage may have been harvested and then further treated, for example they may have been dried by freeze-drying, spray-drying or vacuum-drying or they may have been homogenised. The cells which may have been further treated as described above may then be mixed with other -ingredients, for example with a carrier, to form a composition or final product. The final product may be a foodstuff or beverage. It is a feature of the invention that treatment of the cells will not lead to a loss of appetite suppressant activity of the resulting fraction.

In one embodiment, freeze-dried cells may be used as or in a food stuff or beverage. The inventors have found that freeze-dried cells according to the invention are heat resistant and retain their appetite suppressant activity in

baked foodstuff. Accordingly, the freeze-dried cells according to the invention can be used as an ingredient in baked or fried foodstuff, such as confectionary, bread and snacks.

Furthermore, freeze-dried cells according to the invention easily dissolve in liquid and are therefore easy to use as a supplement in beverages.

In another embodiment, the cells may have been subjected to an extraction step to extract the at least one compound with appetite suppressant activity before use as a food supplement .

In another aspect, the invention relates to the use of the plant cells according to the invention in the manufacture of a medicament for the treatment and/or prevention of obesity. In this aspect, the term "cultured cells of the invention" relates to cultured cells which may or may not have been treated following harvesting from culture. As explained above, the cells according to the invention may have been dried by, for example, freeze-drying, spray-drying or -vacuum-drying or they may have been homogenised or subjected to an extraction step.

It has also been shown that the compounds in Hoodia extracts of plant material other than cultured plant cells can be used in the manufacture of a medicament for the treatment and/or prevention of diabetes. Accordingly, a skilled person will also understand that the cultured cells according to the invention can be used in the manufacture of a medicament for the treatment and/or prevention of diabetes.

In another aspect, the invention also relates to a method for suppressing appetite by administration of the plant cells according to the invention. Furthermore, the invention also relates to a method for treating obesity by administration of the plant cells according to the invention.

The invention also encompasses pharmaceutical compositions comprising cultured plant cells of the genus Hoodia which are characterised in having appetite suppressant activity and also comprise at least one pharmaceutical carrier, diluent or excipient . The cells produce at least one compound having appetite suppressant activity. The pharmaceutical composition according to the invention comprises an amount of cells necessary to provide a sufficient amount of the compound(s) to achieve an appetite suppressant effect.

The use of cultured plant cells as a medicament provides an advantage over using an extract derived from dried plant material obtained by methods described in the art, as -cultured cells are easy to maintain and propagate in large quantities. In one embodiment, the compound may be further purified by using an extraction step. However, it is also possible to use the cells without further purification. The cells may simply be harvested and used for, for example, oral administration or as food supplements. In a preferred embodiment of the invention, dried cells may be used in the manufacture of the medicament .

Suitable dosage forms include, but are not limited to, solid dosage forms, for example tablets, capsules, powders, dispersible granules, cachets and suppositories, including sustained release and delayed release formulations. Powders and tablets will generally comprise from about 5% to about 70% active ingredient. Suitable solid carriers and excipients are generally known in the art and include, e.g. magnesium carbonate, magnesium stearate, talc, sugar, lactose, etc. Tablets, powders, cachets and capsules are all suitable dosage forms for oral administration.

The cells may also be formulated as liquid dosage forms which include solutions, suspensions and emulsions. Liquid form preparations may be administered by intravenous, intracerebral, intraperitoneal, parenteral or intramuscular injection or infusion. Sterile injectable formulations may comprise a sterile solution or suspension of the active agent in a non-toxic, pharmaceutically acceptable diluent or solvent. Liquid dosage forms also include solutions or sprays for intranasal, buccal or sublingual administration. In the dosage forms described above, cells may be present as, for example, dried cells.

Also encompassed are dosage forms for transdermal administration, including creams, lotions, aerosols and/or emulsions. These dosage forms may be included in transdermal patches of the matrix or reservoir type, which . are generally known in the art. ^' ,• ■.'

Pharmaceutical preparations may be conveniently prepared in unit dosage form, according to standard procedures of pharmaceutical formulation. The quantity of active compound

per unit dose may be varied according to the nature of the active compound and the intended dosage regime. However, a skilled person will understand that the dosage form may contain other pharmaceutically acceptable excipients for modifying conditions such as pH, osmolarity, taste, viscosity, sterility, lipophilicity, solubility etc. The choice of diluents, carriers or excipients will depend on the desired dosage form, which may in turn be dependent on the intended route of administration to a patient.

The active compounds present in the cultured cells are to be administered to human subjects in "therapeutically effective amounts" , which is taken to mean a dosage sufficient to provide a medically desirable result in the patient. The exact dosage and frequency of administration of a "therapeutically effective amount" of active agent will vary, depending on the condition which it is desired to treat, the stage and severity of disease, and such factors as the nature of the active substance, the dosage form and route of administration. A typical dosage range for compounds of general formula 1 is for example about 0.1-lOmg of compound per kg of mammal by weight, however this is ^■ given by way of example only and is not intended to limit the invention to this dosage range. The appropriate dosage regime for a given patient will generally be determined by a medical practitioner having regard to such factors as the severity of disease, and the age, weight and general physical condition of the patient, and the intended duration of treatment, as would be appreciated by those skilled in the art.

According to the invention, the plant cells of the genus Hoodia obtained from a cell culture - and/or pharmaceutical compositions containing these cells - may be used as an appetite suppressant for the prevention (e.g. prophylaxis) and/or treatment of a disease associated with weight gain (which for the purposes herein in its broadest sense also includes preventing, treating and/or alleviating the symptoms and/or complications of such disease) .

The invention will be further understood with reference to the following non limiting experimental examples.

Examples

Example 1 Induction and Maintenance of Hoodia cell suspension culture

Initiation of callus cultures from Hoodia, sterilisation of the starting plant tissue and preparation of callus induction media

Callus inducing media was prepared using MS media (Sigma) -with 3% sucrose and 1% Naphthalene acetic acid (from concentrated stock solution of 4mg/l00ml) . The media was made up and the pH was adjusted to between 4.6 and 5.0. ^■ After this, phytagel (lg/500ml media) was added to solidify the media. The media was then autoclaved for 20mins at 121°C and then poured out into sterile plastic plant tissue culture dishes. Water and glassware needed for the plant sterilisation were also autoclaved. Plant tissue derived from the mesoderm of Hoodia gordinii was sterilised by

immersion in 70% ethanol for 2 mins, followed by immersion in 10% bleach solution for 10 mins.

Hoodia tissue was then washed three times with sterile (autoclaved) water. The sterile Hoodia tissue was aseptically cut into disk shapes in a sterile laminar flow cabinet using scalpel and tweezers that had been dipped in 70% ethanol and flamed until red in the blue heat of a Bunsen burner. The slices of Hoodia tissue were placed onto the prepared plates containing callus inducing media, and plates were sealed with Nescofilm. The plates were placed in the dark at 27°C and callus formation began to appear after about 1 month.

Sterility and Aseptic techniques

Aseptic techniques were maintained at all times - all glass equipment, media and water were autoclaved in a gas autoclave for 20 minutes at 121 °C. Self indicating autoclave tape was used to identify if the correct conditions had been achieved inside the autoclave, as the tape changes colour from creamy white all over to having a jjlack stripe when correctly autoclaved.

Before initial use the laminar flow cabinet was sprayed with 70% ethanol and wiped clean. When using the laminar flow cabinet and handling any cultures inside the cabinet all watches, rings and clothing covering the arms were removed (including lab coats) . Hands were washed in antibacterial soap to ensure no contamination immediately before start of work. All work was carried out inside the sterile laminar flow cabinet which is only used for plant cell culture work.

Media preparation, for established callus cultures

Reagents

IL distilled water

3Og sucrose

4.4g Murashige and Skoog Basal powdered medium

5ml NAA (naphthalene acetic acid) stock solution

ImI Vitamin solution

IM NaOH solution

0. IM NaOH solution

Equipment

IL glass bottle

Magnetic stirrer

20 250m conical flasks

20 sheets of foil approximately 20 x 20 cm

Glass pipettes pH meter

Autoclave

Laminar flow cabinet

Balance

..he method used for -the media preparation comprises the following steps:

1. To 9994ml distilled H ₂ O add 30ml sucrose, 4.4g MS powder, 5ml NAA stock and ImI vitamin stock.

2. Mix using a magnetic stirrer until all dry components dissolved, then pH adjust with IM and 0. IM NaOH, to 5.75.

3. Take 20 250ml conical flasks. To each add 50ml media and seal neck of flask with foil . Sterilize in autoclave, at 121°C, 103 kPa, for 25 minutes.

4. Immediately following sterilization, place flasks in laminar flow cabinet and allow to cool to ambient temperature.

To make NAA stock solution, add 4mg NAA tolOOml distilled H ₂ O and stir until mixed. Store stock in refrigerator.

To make vitamin stock solution, add 50mg pyridoxalhydrochlorid, lOmg thiamine dichloride and 50mg nicotinic acid, to 100ml distilled H ₂ O. When mixed, transfer ImI into 100 1.5ml Eppendorf vials and freeze till use at -20 ⁰ C. Use 1 vial per 1000ml media.

Inoculation and sub-culturing of established callus cultures

Reagents : Friable callus 70% Ethanol

Equipment:

Laminar flow cabinet

Bunsen burner

Prepared media

20 sterile sheets of foil approximately 20 x 20 cm

Several pairs of tweezers or small forceps

Wide spatulas with holes

The method used for the inoculation and sub-culturing of established callus cultures comprises the following steps:

I. Sterilize inside of laminar flow cabinet with 70% ethanol .

2. Sterilize all tweezers and spatulas by dipping in 70% ethanol, then flaming till red hot. Allow to cool inside laminar flow cabinet.

3. Initial inoculation: Remove foil from prepared media flask.

4. Take sterilized tweezers and remove thumbnail sized pieces of friable callus from the plant tissue. Break up into finely dispersed cells and add to flask. Aim to add approximately 5g tissue to 50ml media.

5. Flame the neck of the flask, and cover with a sterile sheet of foil .

6. Place the flask on a shaker at 120 rpm, in a dark room heated to 27°C. Leave until a thick, dispersed cell suspension culture can be observed.

7. Subculture: remove foil from prepared media flask.

8. Remove foil from flask containing dispersed cell suspension cultures (see point 6)

9. Take wide spatula with holes, and scoop out the cells. Add these cells to the fresh media. Aim to add approximately 5g tissue to 50ml media.

10. Flame the neck of the flask, and cover with a sterile sheet of foil .

II. Place the flask on a shaker at 120 rpm, in a dark room heated to 27°C. After 14 days, use the cell suspension culture for further subcultures.

Example 2: Comparison of HPLC profiles

Cells were obtained from 3 week old cell suspension cultures of the cells as prepared in Example 1 (termed DMU-PR-I) ,

washed in distilled water and freeze dried before extraction.

Methods

Freeze-drying: The freeze dryer works by vacuum, once it has cooled down to O ⁰ C, frozen cell material can be placed inside the chamber. The vacuum is turned on and the pressure inside the chamber is reduced, this allows the frozen liquid to be removed from the cells into the ice trap which has been pre cooled to 0 ⁰ C. The vacuum is left on for 24 hours to allow all of the moisture in the cells to be dried out. Anything to freeze dry must be water based. If solvents are used, the oil inside the pump is diluted and this stops and slows the vacuum, also if the material is not frozen before use the sample will not dry.

Extraction: 5g freeze dried cells were allowed to stand for 1 hour in 10ml of 1:1 chloroform: methanol mixture. After this time, the cell: solvent mixture was poured into a pestle and mortar and ground to a smooth paste. The paste was centrifuged at 4400 rpm for 30 minutes. After this time, the solvent was decanted and dried in a rotavapour at ^' trOOmbar, 6O ⁰ C. Chloroform was used to remove dried residue ■- from the rotavapour flask, and this was placed into a glass vial. This chloroform-dissolved extract was washed 3 times with distilled water. The washed chloroform extract was then dried on a rotavapour as above, and the residue was redissolved in methanol. This methanol-dissolved extract was ^" used for HPLC analysis.

Another sample was prepared from a Hoodia gordinii plant by cutting the plant was cut into sections. The Hoodia plant ^'

had dried naturally in the air, but other than this, the extraction procedure was the same as for the cell line.

HPLC Analysis: 5ul of sample was used for HPLC analysis. The machine was a HPLC Agilent series 1100. The column was a C18 15cm x 0.46 cm reverse phase column.

The method used was as follows :

Gradient; A = 80Og H ₂ O, 154.6g acetonitrile, ImMoI phosphoric acid, B = 20Og H ₂ O, 625.6g acetonitrile, ImMoI phosphoric acid at a flow rate of lml/min, and UV detection was used.

Results

The peaks obtained in the HPLC analysis are shown in the table below .

Table 1. Retention times (in minutes) of HPLC-UV peaks found in dried Hoodia cactus, freeze-dried Hoodia DMU-PR-I cells, and fresh Hoodia sap. All peaks refer to those observed at 254 nm. Figures in bold refer to the largest peaks.

All samples show a peak at 1.2 minutes. The dried plant gave 8 peaks; the cell culture 9 peaks. The sap showed the most, st 14 peaks, but the peaks were small compared to the other two samples. There seem to be 4 peaks that the dried plant has in common with the cell line: 1.28, 2.4/2.3, 3.9/3.59, 5.7/5.58 (these last 3 peaks are not identical retention times, but sufficiently close and a skilled person will understand that minor variation can be due to slight differences in preparation of mobile phase on the different days that analyses were performed) .

Although there are some peaks observed in the cell lines that were not seen in the whole plant extract and also peaks

in the whole plant extract not observed in the cell line, the profiles are very similar. Differences may be due to the presence of chlorophyll or other dermis constituents, as the plant was not peeled before extraction.

It appears that the cell line does produce some metabolites, including some not produced by the whole plant. Accordingly, the inventors have produced a cell line that seems to produce largely the same compounds found in plant extracts .

Furthermore, the inventors have also found that the extracted fraction of the cell culture comprises a compound known in the art to have appetite suppressant activity. The peak obtained at 1.282 min in the extract derived from the freeze dried Hoodia cells according to the invention was isolated on the HPLC and the fraction was collected into a separate sterile vial. HPLC data showed that the peak was pure and it was then adsobed onto a clean Solid Phase Micro- Extraction (SPME) fibre. The fibre was then allowed to air dry for 30 minutes before being introduced into the sample inlet valve of the GCMS. The resulting fraction was further analysed by mass spectrometry. The resulting mass spectra ^• was then used to identify the peak. The spectra showed that there was a 95% probability that this fraction is the p57 compound which has been shown in the art to have appetite suppressant activity and is substantially as shown in formula 1.

References :

G. Delle Monache et al . Comparison between metabolite productions in cell culture and in whole plant of Madura

Pomifera. Phytochemistry, Vol. 39, No. 3, pages 575-5880.

1995

F. Dicosmo and M. Misawa: Plant cell and tissue culture: Alternatives for metabolite production. Biotechnology Advances, VoI 13, No. 3, pages 425-453. 1995

R. L. Philipps et al: Genetic instability of plant tissue culture: Breakdown of normal controls. Proc. Natl. Acad. Sci. USA, VoI 91, pages 5222-5226, 1994

A specimen of the cultured plant cells of the present invention was deposited at the following depository institution:

DSMZ - Deutsche Sammlung von Mikroorganismen und

Zellkulturen GmbH

Mascheroder Weg Ib u-38124 Braunschweig

Germany

The specimen was deposited at DSMZ before the filing date of the present application ( on 30 October 2004 ) .