Method for producing a medium containing steroidal glycosides from plant cells of the genus <i>Hoodia</i>

Abstract

A method for producing an active ingredient-containing medium from cultured plant cells, selected from the Hoodia genus, is provided. The method comprises the steps of: (i) incubating a mixture of cultured plant cells of the genus Hoodia and a liquid media in light; (ii) separating the incubated mixture into a first portion and a second portion; (iii) passing the first portion through a tangential flow filter to produce a filtrate containing only media and a retentate containing cultured plant cells; (iv) passing the filtrate through a nano-filter to obtain a nano-retentate having a high concentration of steroidal glycosides; (v) reintroducing some of the nano-retentate into the second portion; and (vi) repeating steps (i) to (v) using the second portion. The medium may be either plant cells or a plant extract.

Claims

1. A method for producing a composition containing steroidal glycosides from plant cells of the genus Hoodia, the method comprising the steps of: incubating a mixture comprising cultured plant cells of the genus Hoodia and a liquid media in light; (ii) separating the incubated mixture into a first portion and a second portion; (iii) passing the first portion through a tangential flow filter to produce a filtrate comprising liquid media and a retentate comprising cultured plant cells; (iv) passing the filtrate through a nano-filter to obtain a nano-retentate comprising a saturation level of steroidal glycosides; (v) reintroducing a portion of the nano-retentate into the second portion; and (vi) repeating steps (i) to (v) using the second portion.

2. The method of claim 1, wherein the composition comprises cultured plant cells of the retentate of step (iii).

3. The method according to claim 2, further comprising the step: (iii-a) freeze-drying the retentate.

4. The method according to claim 3, further comprising the step: (iii-b) washing the freeze-dried retentate in water to remove remaining liquid media.

5. The method of claim 1, wherein the composition is a plant extract comprising the nano-retentate of step (iv).

6. The method according to claim 1, wherein the incubating step lasts for a duration of at least 5 days.

7. The method according to claim 1, wherein the tangential flow filter has a pore size membrane between 0.01 microns and 3.5 microns.

8. The method of claim 7, wherein the tangential flow filter has a pore size membrane between 0.1 and 0.3 microns.

9. The method of claim 7 wherein the tangential flow filter has a 0.2 micron pore size membrane.

10. The method according to claim 1, wherein a nano-filtration membrane of the nano-filter has a molecular weight cut off of 5,000 kDa.

11. The method according to claim 1, wherein the species of the Hoodia genus is selected from Hoodia gordonii, Hoodia currorii or Hoodia lugardi.

12. A composition produced by the method according to claim 1 for use as an appetite suppressant.

13. A foodstuff, beverage or supplement comprising the composition of claim 2.

Description

EXAMPLE

(1) Step 1: Initiation of Callus Cultures from Hoodia: Preparation of Callus Induction Media

(2) Callus Induction Media Solution

(3) Distilled H.sub.2O to 100%

(4) 3.0% sucrose

(5) 1.0% NAA (naphthalene acetic acid) 0.004% stock solution

(6) 0.44% Murashige and Skoog Basal powdered medium

(7) Equipment

(8) Glass bottle with cap

(9) Magnetic stirrer

(10) Sterile plastic plant culture dishes

(11) Glass pipettes

(12) pH meter

(13) Autoclave

(14) Laminar flow cabinet

(15) Balance

(16) Nescofilm

(17) Phytagel

(18) 1M NaOH solution

(19) 0.1M NaOH solution

(20) Method

(21) Callus induction media was prepared using Murashige and Skoog (MS) media obtained from Sigma, with 3% sucrose and 1% naphthalene acetic acid (from a concentrated stock solution of 0.004% w/v). The pH of the prepared media was adjusted to pH 5.75 and solidified with 0.2% phytagel. The media was autoclaved for 20 mins at 121 C. and then poured out into sterile plastic plant tissue culture dishes.

(22) Step 2: Initiation of callus cultures from Hoodia: sterilisation of plant tissue

(23) Reagents

(24) Media prepared previously (according to Example 1)

(25) Hoodia gordonii plant tissue

(26) Equipment

(27) Sterile glass beakers

(28) Sterile distilled water

(29) Sterile scalpel

(30) Sterile tweezers

(31) 10% bleach solution

(32) 70% ethanol solution

(33) 1M NaOH solution

(34) 0.1M NaOH solution

(35) Method

(36) Plant tissue of Hoodia was sterilised by immersion in 70% ethanol for 2 minutes, followed by immersion in 10% bleach solution for 10 minutes. Hoodia was then washed three times with sterile (autoclaved) distilled water. The sterile Hoodia was aseptically cut into disk shapes in a sterile laminar flow cabinet. Hoodia slices were placed onto the prepared plates containing callus induction media, and the 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.

(37) Step 3: Media Preparation for Established Cultures

(38) Reagents

(39) Distilled H.sub.2O to 100%

(40) 3% sucrose

(41) 0.44% Murashige and Skoog Basal powdered medium

(42) 1% NAA (naphthalene acetic acid) 0.004% stock solution

(43) 0.01% Vitamin solution (0.05% pyridoxal hydrochloride, 0.10% thiamine dichloride and

(44) 0.05% nicotinic acid)

(45) 1M NaOH solution

(46) 0.1M NaOH solution

(47) Equipment

(48) 1 L glass bottle

(49) Magnetic stirrer

(50) 20250 m conical flasks

(51) 20 sheets of foil approximately 2020 cm

(52) Glass pipettes

(53) pH meter

(54) Autoclave

(55) Laminar flow cabinet

(56) Balance

(57) Method

(58) Mix 3% sucrose, 0.44% MS powder, 1% NAA stock and 0.01% vitamin stock and prepare to 100% with distilled H.sub.2O. Mix using a magnetic stirrer until all dry components dissolved, then adjust the pH with 1M and 0.1M NaOH, to pH 5.75. Take 20 250 ml conical flasks. To each add 50 ml media and the seal neck of flask with foil. Sterilize in autoclave, at 121 C., 103 kPa, for 25 minutes. Immediately following sterilization, place the flasks in laminar flow cabinet and allow to cool to ambient temperature.

(59) Step 4: Inoculation, Subculture and Production of Active Ingredient of Established Cultures

(60) Reagents

(61) Friable callus

(62) 70% Ethanol

(63) Equipment

(64) Laminar flow cabinet

(65) Bunsen burner

(66) Prepared media

(67) 20 sterile sheets of foil approximately 2020 cm

(68) Several pairs of tweezers or small forceps

(69) Wide spatulas with holes

(70) Peristaltic pump

(71) Sartorius Viva flow 200 system 5000 MWCO (Fisher Scientific, Loughborough)

(72) Method

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

(74) Initial Inoculation:

(75) Remove foil from prepared media flask. 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 5 g tissue to 50 ml media (10% w/v). Flame the neck of the flask, and cover with a sterile sheet of foil. 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 (approximately 2 weeks).

(76) Subculture:

(77) Remove foil from prepared media flask. Remove foil from flask containing dispersed cell suspension cultures (produced by initial inoculation). Take wide spatula with holes, sterilize, allow to cool and scoop out the cells. Add these cells to the fresh media. Aim to add approximately 5 g tissue to 50 ml media. Flame the neck of the flask, and cover with a sterile sheet of foil. 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.

(78) Production of Medium

(79) Take the flasks after the 14 days incubation and place in daylight. Incubate in daylight for 5 days. Split the content of the flasks into a first portion and a second portion of substantially equal volumes. Retain the second portion in a separate flask for further use. Pass the first portion through a tangential filter with 0.2 micron pore size to obtain a filtrate and a retentate. The retentate will substantially consist of cultured plant cells with a high steroidal glycoside content. The retentate is freeze-dried then rinsed of any remaining media with water to avoid undesirable compounds in the media. The resulting free-dried cultured plant cells are then used as an appetite suppressant.

(80) Pass the filtrate through a viva flow membrane with MWCO 5,000 kDa under reduced flow rate and pressure (10% of the recommended flow rate). Collect the retentate from this filter and reintroduce into the second portion. Discard the filtrate. A portion of the retentate may be retained and used as a plant extract containing steroidal glycosides.

(81) Repetition of Previous Step

(82) Repeat method as set out immediately above using the second portion. If necessary add further liquid media and/or subculture as produced in the preceding step.