Grinding And Extraction Protocols

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7.3.1 General Extraction Protocols for Biologically Important Organic Compounds

The primary ways for extraction of organic molecules of interest to biologists and medical investigators involve breaking open the cells of the organism under investigation. Cell rupturing is accomplished in a variety of ways. The method used depends on the type of organism being considered and the type of tissue used. For bacterial cells, one usually uses a French press so as to break open the cell walls. This involves the use of a heavy cylinder with high pressure applied to a piston that compresses the cells into a successively smaller volume within the free cylinder. As the cells leave the cylinder, the rapid drop in pressure causes the cells to lyse. Such a procedure can also be used for plant cells grown in suspension culture or for plant callus tissue. A sonicator can also be used for this purpose. In this case, repeated high frequency pulses of ultrasonic vibrations rupture the cell membranes. Animal cells and plant cells grown in culture can be ruptured with a glass tissue homogenizer. Highly lignified or silicified plant tissues within organs such as leaves, stems, roots, seeds, or fruits, are usually frozen and pulverized using liquid nitrogen in a mortar and a pestle. (Figure 7.5). Softer plant tissues can be ground in a small volume of buffer in a mortar, using white washed sand and a pestle to rupture the cells.

Mortar Pestle Liquid Nitrogen Extraction
FIGURE 7.5 Gregg Roslund, undergraduate student at the University of Michigan, grinding a frozen plant sample with liquid nitrogen and a ceramic mortar and pestle, in preparation of analysis of medicinal products of medicinal value in this sample. (Photo by David Bay.)

Once the cells have been ruptured, the actual extraction is performed using techniques that depend on the chemical properties of the compound(s) of interest. Water-soluble compounds and proteins are extracted in water or buffers. Organically soluble compounds are extracted with organic solvents. For example, since taxanes are miscible in methanol, this solvent is often used as the extraction reagent. This is by no means the only solvent that will work. Some compounds, such as cell-wall constituents, have no need to be solubilized for extraction because they can be obtained in pellet form by filtration or centrifu-gation followed by washing with buffer solutions. It is worth noting that integral membrane proteins often require the use of strong detergents, such as Triton x 100, to be extracted from the membranes.

Two steps are usually critical for good extraction. First, ruptured cells should be ground or homogenized in the extraction solvent, depending on the cell rupturing technique chosen. For example, taxanes (terpenoid compounds derived from plants) are extracted by grinding the plant tissue in organic solvent (methanol) in the same mortar and pestle that is used for the liquid nitrogen to rupture the cells. Waxes, on the other hand, can be removed from the aqueous phase coming from a French press by homogenizing and partitioning with chloroform and methanol. Second, once ground or homogenized, the extraction mixture should be allowed to stand undisturbed for 0.5 to 24 h at a temperature that will not allow degradation of the compound(s) or pro tein(s) of interest to occur (e.g., 4°C). This is done simply to allow time for the extraction solvent to penetrate all parts of the ruptured cells.

After these two critical steps, the resulting slurry is filtered to obtain a filtrate free of particulates or it is centrifuged in order to obtain a cell wall and/or membrane pellet fraction and a cell cytosol containing supernatant fraction. Such crude fractions can be used directly for enzyme reaction assays, or they can be subjected to further purification and clean-up procedures in order to separate and identify the compounds of interest. For example, C-18 Sep Pak columns can be used to remove chlorophylls, which interfere with subsequent analysis of taxane extractions (compare Figure 7.6A and B).

For the extraction of natural products of potential medicinal or other value in plant samples, as used in the author's laboratory, the following specific protocols have been employed:

Protocol (1): Hot-Water Extraction of Water-Soluble Medicinal

Compounds from Plants Protocol (2): Organic-Solvent Extraction of Organic Solvent-Soluble

Medicinal Compounds from Plants Protocol (3): Extraction of Taxanes (Diterpenoid Compounds) From

Yew (Taxus spp.) Needles and Stems Protocol (4): Extraction of Cuticular Wax from Needles of Yew (Taxus spp.) Plants

FIGURE 7.6 HPLC taxane analysis results showing chart recordings resulting from C-18 Sep-Pak™ cleaning (A) vs. no cleaning (B) of extracts from yew (Taxus spp.) needles and stems.

7.3.2 Hot-Water and Organic-Solvent Extraction of Water-Soluble and Organic Solvent-Soluble Medicinal Compounds from Plants

There are so many different ailments and diseases, it is a wonder we are ever healthy. Today, the development of drugs and medicines is a growing industry. U.S. companies are losing the race to be the leading developer of medicines. What are other countries doing that the U.S. is not? Most other countries have made the trend back to nature. They started looking at plants and trees for natural medicines. The U.S. has just joined this trend, but has not been able to produce as many drugs as some other countries, such as Japan. Unfortunately, it takes a lot of money to develop any new drug, and the risk of the drug failing is great.

Many of the plants that have known medicinal values have been used for centuries by different cultures. Natural medicines have been used for centuries in many parts of Asia, such as China and India. In particular, Native Americans have had a profound influence on the natural medicines of today. The Indians of North America have made the most contribution to this field.

A project conducted in Peter Kaufman's laboratory at The University of Michigan by DaRhon Conner and Nina Jain has involved the preparation of plant extracts from plants that contain natural products of know medicinal value. These extracts are prepared to be screened by Parke-Davis/Warner-Lambert Pharmaceutical Co. in Ann Arbor, MI, U.S. The plants come from a variety of sources ranging from botanical gardens to grocery stores. The plants are cut and stored in a deep freeze at -80°C. These extracts are tested in an 80-sample screen for medicinal compounds in order to determine which are effective against viruses, bacteria, mycoplasms, and fungi that are pathogenic to humans. Many of the plants are naturally toxic and harmful to humans. However, if they are administered in small amounts, the effects on humans are beneficial.

There are two different types of extracts used in this project. One is done using hot water and is called an aqueous extract. The other uses organic solvents, such as methylene chloride, and is called an organic extract. There are two very specific procedures that have been developed for each type of extraction. These procedures are delineated in Sections 7.3.2.1 and 7.3.2.2.

7.3.2.1 Hot Water Extraction of Water-Soluble Medicinal Compounds from Plants (Protocol 1)

First, we weigh out a 0.5-g sample of a given plant. This is then ground to a fine powder using liquid nitrogen. Next, we place the powder into a Corex centrifuge tube with hot (80°C) water and place the tube into a hot water bath for 10 min. The tube is centrifuged at 3000 x g for 10 min. By centrifuging it, all of the particulate plant materials from the grinding get pelleted to the bottom of the tube, leaving a relatively clear liquid (the supernatant) containing the water-soluble compounds of interest. This liquid is filtered to make sure that no plant particulates remain in the filtrate. Next, the filtrate is placed in a Petri dish and frozen by placing it on dry ice. After the sample is completely frozen, it is placed in a freeze-drying apparatus and lyophilized. The sample could take anywhere from 2 to 12 h to lyophilize. Freeze-drying is done in order to remove all moisture. This yields a powdered residue and it is this powder that we send to Park-Davis/Warner-Lambert. Figure 7.7 outlines this procedure.

7.3.2.2 Organic Solvent Extraction of Organic Solvent-Soluble Medicinal Compounds from Plants (Protocol 2)

As mentioned above, this procedure uses an organic solvent to extract the compounds of interest. At least one very toxic solvent (methylene chloride) is used and therefore needs to be monitored carefully and with caution. The temperature of this extraction must also be carefully watched due to the special equipment that is used. We make use of a Soxhlet extractor which is basically a specialized glass refluxing unit that is used for organic solvent extractions. The temperature must be maintained at 80°C for 18 h in order to obtain complete extraction of a given sample. If the temperature falls below this, extraction will be slow. If the temperature goes above this, the risk of degrading the compounds of interest becomes great. Figure 7.8 outlines this procedure.

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FIGURE 7.7 Hot water extraction protocol for water-soluble plant natural product compounds that have potential medicinal value.

7.3.2.3 Case Studies on the Purification of Crude Extracts Prior to Chromatographic Separation — Taxol and Cuticular Wax Extractions from (Yew) Plants

For bioseparation, cleanup procedures are usually necessary before samples are analyzed by high-performance liquid chromotography (HPLC) or by any of the other above-mentioned chromatographic techniques. One application that we have employed is for the analysis of taxol. Taxol is a unique taxane diterpene amide that possesses antitumor and antileukemic properties. Kilograms of this cancer chemotherapeutic agent are needed for clinical treatment of patients having breast cancer; however, taxol exists in only minute quantities — 0.01% of the inner bark and needles of Yew (Taxus) species. Until recently, taxol could not be synthesized, and even now, the most economical source of taxol is still from the Pacific yew (Taxus brevifolia). For this reason, large areas of Pacific yew forests in the Pacific northwest of the U.S. were destroyed in order to obtain this anticancer drug. The taxol extraction methods used by researchers commonly involve complicated partitioning methods in which the plant is first extracted with methanol and H2O and then partitioned using methylene chloride to remove chlorophyll and other unwanted compounds. In the process, taxol molecules move from the aqueous methanol to the more hydrophobic methylene chloride. Unfortunately, methylene chloride is a suspected carcinogen and it seems counterproductive, in our opinion, to extract a cancer chemotherapeutic with a substance that could cause cancer! Hence, it has been our motive in studying taxol to find a cheap, efficient, and easy way to separate taxol from the thousands of other organic compounds in yew tissues.

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FIGURE 7.8 Organic solvent extraction protocol for organic solvent-soluble plant natural product compounds that have potential medicinal value.

One of the primary difficulties, in this case, is the removal of chlorophylls from methanolic extracts. Chlorophylls absorb at the same wavelength as taxol and often occur in such large quantities that their resulting peaks interfere with taxane peaks. For this purpose, we use a C-18 reverse phase Sep-Pak™ column. The protocol for doing this follows. The main point here, however, is that preparatory columns are also useful to remove many unwanted compounds before the actual chromatographic analysis is performed. Figure 7.9 shows the small size of the C-18 Sep-Pak™ set-up as used in our laboratory. HPLC results using C-18 Sep-Pak™ cleaning vs. no cleaning are shown in Figures 7.6A and 7.6B, respectively.

A new, simple and rapid method that successfully works for extraction and HPLC separation of taxol from crude extracts of Taxus cuspidata (Japanese Yew) needles and stems has recently been developed in our laboratory and tested by 10 groups independently with repeated success. It requires 2 h to perform steps 1 through 21 and 70 min per sample to run them in an automated Shimadzu HPLC apparatus. This long run time is used to separate the multitude of peaks that result at 228-nm spectrophotometric monitoring when not using the C-18 Sep-Pak™ cleanup method. The advantage of not using cleanup

FIGURE 7.9 Leland Cseke is shown with the C-18 Sep-Pak™ column (lower corner) used in our laboratory to partially purify yew (Taxus spp.) plant extracts in preparation for HPLC analysis of taxane-type diterpenes found in these plants.

procedures is that the researcher saves time in preparing extracts. In this case, the researcher can go home and sleep while the Shimadzu Sil-6a autoinjector does the work. Figures 7.10A and 7.10B illustrate Shimadzu and JASCO automated HPLC apparatuses, respectively, that can be used in this procedure.

FIGURE 7.10A Shimadzu HPLC apparatus in Peter Kaufman's laboratory at University of Michigan.
FIGURE 7.10B JASCO HPLC apparatus with three independent pump systems, three detectors, controller, and recorder in Dr. Akira Okubo's laboratory at the University of Tokyo.

In some analytical situations, or for cases where publication is desired, it may be necessary to clean the crude extract with a C-18 Sep-Pak™ column. The clean-up procedure is given following the crude extraction protocol described in Section 7.3.2.3. [Note: This is by no means the only cleanup procedure for extracts. Any form of chromatography can be used to cleanup the extracts (see discussion of chromatographic methods).] The basic trick is to find the fraction eluted from the cleanup column which contains the compound of interest. In the case of taxol, this was accomplished experimentally, using purified taxol standards dissolved in 10% methanol. At this concentration of methanol, it was found that taxol binds to the C-18 packing material in the column. Then, the concentration of methanol was arbitrarily increased in several steps up to 100% methanol. A fraction of eluted mobile phase (2 to 3 ml) was collected at each concentration increase, and HPLC was performed on each fraction to determine where taxol had eluted off the column. After repeated experiments, the specific concentration of methanol that elutes taxol was identified.

In some cases, chromatography is not necessary for cleanup of the sample. Addition of adsorptive particles such as activated charcoal to the crude extracts followed by filtration may be all that is necessary to remove unwanted compounds.

7.3.2.3.1 Taxol Extraction from Fresh Taxus spp. (Yew) Tissue Resulting in Crude Extract Samples that Can be Used for HPLC (Protocol 3)

1. Obtain Taxus spp. plant specimens.

2. For each sample, weigh out 0.5 g of needles + 0.5 g of stems.

3. Grind to a fine powder using liquid N2 in a mortar and pestle.

4. Let powder warm to room temperature.

5. Add 1 ml of 100% MeOH and grind vigorously.

6. Transfer this slurry to a 15 ml Corex centrifuge tube.

7. Add 2 ml of 100% MeOH and grind the remaining plant material in the mortar.

8. Add this to the tube and label.

9. Add another 1 ml of 100% MeOH to the mortar and rinse again.

10. Add this to the tube and label.

11. Place Parafilm™ over the tube and vortex or shake for 10 min.

13. Balance the tubes by adding 100% MeOH to the lighter tube.

14. Centrifuge at 12,000 rpm for 15 min.

15. Take off the supernatant and place in a clean, acid-washed tube using a Pasteur pipette; then label.

16. Put this tube into a waterbath at 55°C.

17. Blow N2 gas gently into tubes until the samples are completely dry.

18. Add 0.5 ml of ice-cold HPLC grade 100% MeOH and vortex for several min. with periodic placement in an ice bath. This allows one to later quantify the amount of taxol extracted because a known volume of sample has been created.

19. When the MeOH is very cool, place the 0.5 ml into a syringe and filter through a 0.2-pm filter into an HPLC bottle.

20. Add an aluminum septum to the top of the bottle and cap.

21. Parafilm™ well to prevent evaporation and label.

22. Inject sample into HPLC and run through the Curosil G column (from Phenomenex Corp.), collecting data at 228 nm. Run each sample for 70 min. at 1 ml • min1 in 36.5% acetonitrile + 63.5% 10 mM ammonium acetate at pH 4.0.

23. Use purified taxol to make several known concentrations of taxol. Run these standards on HPLC, as in step 22, to make a standard curve. Figure 7.11 shows an example of a typical standard curve for taxol.

24. Compare your data to the standard curve for taxol and calculate the percent of taxol per unit (gram) of fresh weight of tissue.

7.3.2.3.2 Taxol Extraction from Fresh Taxus Spp. (Yew) Tissue Resulting in Cleaned Extracts that Can Be Used for HPLC — Use of the C-18 Sep-Pak™ Column (Protocol 4)

1. Obtain Taxus spp. plant specimens.

3. Grind to a fine powder using liquid N2 in a mortar and pestle.

4. Let powder warm to room temperature.

5. Add 1 ml of 100% MeOH and grind vigorously.

6. Transfer this slurry to a 15-ml Corex centrifuge tube.

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FIGURE 7.11 A typical standard curve for taxol as determined by HPLC analysis of taxol standards at a wavelength of 228 nm.

7. Add 2 ml of 100% MeOH and grind the remaining plant material in the mortar.

8. Add this to the tube and label.

9. Add another 1 ml of 100% MeOH to the mortar and rinse again.

10. Add this to the tube and label.

11. Place Parafilm™ over the tube and vortex or shake for 10 min.

13. Balance the tubes by adding more 100% MeOH to the lighter tube.

14. Centrifuge at 12,000 rpm for 15 min.

15. Take off the supernatant and place in a clean, acid-washed tube using a Pasteur pipette; then label.

16. Dilute the volume of MeOH supernatant 10-fold with ddH2O to produce a 10% MeOH sample.

17. Run all of this diluted sample through a C-18 Sep-Pak™ column (activated as per instructions from supplier). Taxol-type molecules will bind to the column at this concentration of MeOH.

18. In the case of taxol itself, wash the column first with 3 ml of 35% MeOH, then with 3 ml of 55% MeOH. This washes compounds that have less affinity for the C-18 absorbent than taxol out of the column, while the taxol remains bound.

19. Now wash the C-18 column with 2 ml of 65% MeOH and collect the sample. All taxane species of compounds (including taxol, 7-epi-taxol, 7-epi-10 deacetyl taxol, and cephalomanine) are released from the column during this elevation in MeOH concentration, leaving almost all of the problematic chlorophyll still bound to the column adsorbent phase.

20. The 2 ml of taxol-containing sample is completely dried under a stream of N2 gas in a 55°C waterbath while the C-18 column can be cleaned with 100% MeOH and reused. (Note: No detectable amount of taxol is lost during this procedure.)

21. Add 0.5 ml of ice-cold HPLC grade 100% MeOH and vortex for several min. with periodic placement in an ice bath.

22. When the MeOH is very cool, place the 0.5 ml into a syringe and filter through a 0.2-pm filter into an HPLC bottle.

23. Add an aluminum septum to the top of the bottle and cap.

24. Parafilm™ well to prevent evaporation; then label.

25. Inject sample into HPLC and run through a Curosil G column (from Phenomenex Corp.), collect data at 228 nm. Run each sample for 30 min. at 1 ml • min1 in 40% acetonitrile + 60% 10 mM ammonium acetate at pH 4.0.

26. Use purified taxol to make several known concentrations of taxol. Run these standards on HPLC as in Step 22 to make a standard curve (Figure 7.11).

27. Compare your data to the standard curve for taxol and calculate the percent of taxol per unit (gram) of fresh weight of tissue.

Note: The run time can be reduced to as little as 10 min by increasing the percentage of acetonitrile. The taxol peak is still clearly resolved in this case.

7.3.2.3.3 Extraction of Cuticular Wax from Needles of Yew (Taxus) Plants

Waxes are lipids synthesized by plants and animals and function to keep out infectious organisms (e.g., ear wax in animals) and to prevent desiccation and serve as a barrier against fungal and bacterial pathogens in plants. In yew plants (Taxus spp.), waxes are synthesized in increased amounts in response to physical stresses. The protocol we have successfully used to quantitatively analyze cuticular waxes from Taxus needles is listed. This procedure should be adaptable for any species of plant or animal.

1. Weigh out 1.0 g of Taxus needles.

2. Dice needles with a razor blade and place in a mortar and pestle.

3. Add 2:1 mixture of methanol:chloroform to the mortar and grind thoroughly.

4. Transfer the resulting slurry to a centrifuge tube and centrifuge at 12,000 rpm for 15 min.

5. Remove the supernatant and place in a separate collection flask. This supernatant contains the lipids from the Taxus needles.

6. Place the pellet back into a mortar and pestle.

7. Add a 2:1:0.8 mixture of methanol:chloroform:water, and grind thoroughly.

8. Centrifuge this slurry at 12,000 rpm for 15 min.

9. Transfer the supernatant to the above collection flask.

10. Regrind the pellet from the centrifuge tube using the original 2:1 mixture of methanol:chloroform mixture, and repeat Steps 8 and 9.

11. While collecting the rinse liquid, vacuum filter the pellet, rinsing with 100% chloroform.

12. Transfer this rinse liquid to the collection flask.

13. Transfer all of the collected liquid to a separatory funnel and add a 1:1 mixture of chloroform:water. This will produce two phases. The lower phase will contain the dissolved lipid.

14. Empty the bottom layer of chloroform from the separatory funnel and dry it over solid sodium acetate. This absorbs water from the sample.

15. Weigh a clean rotary evaporator flask (round bottom flask) and record the weight.

16. Pipette the chloroform solution into the rotary evaporator flask.

17. Vacuum filter the sodium acetate solid, rinsing with chloroform. Add the collected rinse liquid to the rotary evaporator flask.

18. Evaporate the chloroform using a rotary evaporator until the sample is completely dry.

19. Weigh the rotary evaporator flask with the sample and subtract the recorded initial weight for the empty flask. This will give the amount of cuticular wax from the original sample of Taxus needles.

7.3.3 Square Wave Electrical Fractionations

This is a relatively new procedure that allows one to fractionate plant tissues physically without any grinding. It gives a nice powder preparation that can be used for hot water or organic solvent extractions. It will virtually break-up cell walls in plant tissues and pulverize the tissue to a powder. It relies on the same principle used in ultrasound sonicating apparatus, where such vibrations can loosen particulate matter adhering to glass walls of collection vials or HPLC vials. This new protocol is being used commercially by biotech and pharmaceutical companies because it is much easier than grinding samples in liquid nitrogen and works well for both woody (lignified) and herbaceous plant tissues.

7.3.4 Use of Detergents, Alcohols, and Dimethylsulfoxide to Extract Compounds of Interest without Killing the Tissue

This is a relatively new concept being used experimentally in our laboratory to achieve maximal extraction of the metabolites of interest without actually killing the plant. It is done with seeds or green, living plants. No grinding of tissues is involved. The method relies on the partition coefficients of the chemicals used, the polarity of molecules to be extracted, and the ease with which the solvents can penetrate the tissues without killing them. Dimethylsulfoxide (DMSO) is a penetrant used in human medicine and works well in getting compounds into plants, like alcohols (methanol, ethanol, or long-chain alcohols) and detergents (which are good wetting agents and good at liberating molecules like proteins from plant membranes). In testing such compounds in various combinations, one has to run viability tests with the plants being treated to extract compounds of interest. This method is being developed at the University of Michigan in the Biomedical Engineering Program, and the Departments of Chemical Engineering and Biology by Professors Henry Wang and Peter Kaufman and Ph.D. students, Kittinan Komolpis and Artiwan Shotipruk from Thailand.

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Responses

  • felix
    Why we use grind plant parts to extract the becteria pathogen in palnt?
    2 years ago
  • ISIDORO
    What happened when plant parts is being grind?
    2 years ago

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