A Survey of Rumex crispus for antitumor compounds.
A proposal submitted in partial fulfillment of the degree of Master of Science
by Eric Jeffrey Lawrence
To Graduate Committee Department of Biotechnology Stephen F. Austin State University July 2, 2002
Introduction The purpose of this study is to determine the presence of anti-tumor compounds in Rumex crispus. R. crispus is a common weed that is present on every continent.(1) R. crispus is known to contain several compounds with anti-tumor properties, but no in vitro studies have been performed using this plant.(2) R. crispus is commonly used as herbal remedy to treat anemia, as well as hepatic and dermatological disorders.(3,4) R. crispus was used in several anti-cancer formulas during the 19th
century.(5) Very little scientific research has been performed with this plant. This study hopes to achieve one of two outcomes: 1.) Identify a new anticancer compound, 2.) Be able to utilize this plant as a
R. crispus a common weed found throughout the world. The plant turns brown when it is mature in late spring.
source of known natural anti-cancer compounds. This project takes a novel approach to obtaining a dose-response curve from plant extracts. Upon finding a sample that has a positive response, the unknown “reactor” (compound) will already be classified. Samples to be tested are first separated by plant-part: rhizome, flower, root, leaf, or stem. Each plant part is then separated by solvent extraction: ethanol, methanol, or chloroform. Then each of these samples is further separated by solid phase extraction (SPE) in thirteen sorbents that separate compounds into general chemical classifications. Samples from each SPE column are then exposed to colon-
cancer cells (DLD-1) as well as a normal colon cell-line (CCD-112Co) as a control. A cell proliferation assay will then be performed in 96-well plates using an MTS colorimetric assay to measure cell viability. Data obtained from these assays will be used to construct a dose-response curve and LD50 for samples yielding positive results. Any samples yielding positive results will then have a time-assay performed and a dose vs. time plot for positive samples can be constructed. A time assay will also provide direction into the mode of action for positive samples, whether the anti-tumor compound’s action is during the stationary, or log phase of the cell cycle. These results would conclude this portion of the study, but this project will be continued by performing HPLC, GC, TLC, IR, NMR, and other chromatographic techniques on the unknown compound(s) for identification and concurrently determine the compounds’ mode of action.
Objectives The final objective of this project is to obtain dose-response activity from extracts of R. crispus and to characterize the class of possible compound(s). To accomplish this goal the following objectives must be met:
Objective 1: Obtain R. crispus, separate plant parts, and prepare for extraction. The whole plant(s) will be obtained from a known source and the plant will be separated into five parts: leaf, flower, stem, root, and rhizome. The fresh plant parts will then be ground to powder in a mortar and pestle with liquid N 2.
Objective 2: Extraction of each plant part using solvents of different polarity. Each ground plant part will be extracted in the Accelerated Solvent Extractor (ASE) from Dionex. Three extractions will be done on each plant part, each with a different extraction solvent. The solvents methanol, ethanol, and chloroform will be used to extract the samples. The samples will then be evaporated by spin drying them in a vacuum at 40 oC.
Objective 3: Solid Phase Extraction of samples. Each extraction sample will be further extracted using solid phase extraction. Approximately 13 different chromatography columns will be used to separate the extraction samples into specific classes of compounds. Evaporated ASE extracts will be resuspended in a solvent appropriate for the SPE column to
be used and following extraction from SPE the samples will evaporated by centrifugal evaporation.
Objective 4: Tissue culture of DLD-1’s and CCD-112Co cells. The colon adenocarcinoma DLD-1 cell line and the normal colon CCD112Co cell line purchased from American Type Culture Collection will be subcultured to several passages. Enough cells will be cultured to perform the cell proliferation assay on DLD-1 cells and the control CCD-112Co cells.
Objective 5: Cell proliferation assay with extract titrations. A cell proliferation assay will be performed with sample dilutions from 10 0 – 10-9 concentration of the evaporated SPE extracts resuspended in dimethylsulfoxide (DMSO). The assay will be performed in 96-well plates and detection of proliferation will be done using an MTS tetrazolium compound from Promega. The assay will be performed in triplicate with positive and negative controls.
Objective 6: Measure cell proliferation in a time assay using active samples. A follow-up cell proliferation assay will be performed using samples that tested positive for antitumor activity. The time assay will measure cell proliferation of active samples at 12, 24, 48, and 72 hours. Assay will be performed in duplicate with positive and negative controls.
Objective 7: Dose-response curve for active samples. A dose-response curve will be constructed for samples that are positive for antitumor activity with colon cancer cells. A dose-response curve will be used to determine the LD50 of the unknown compounds and the dose-response curve will be compared for all active compounds to determine the most promising extract to further pursue study on.
Literature Review R. crispus is a common weed of the Polygonacease family.(1) It grows between 30 – 160 cm. high with narrow leaves that have curly edges.(1,42) The tap root is large and fleshy and typically grows to 165 cm. deep.(1) Flowers are yellow to green and form clusters, and the seeds are winged and triangular(42) R. crispus has invaded every continent and the genus seems to follow the migratory patterns of man throughout the world.(1) It is a perennial that can be found in pastures, fields, and wastelands, but prefers a moist soil habitat.(1,42) Since R. crispus is almost ubiquitous it has been utilized for a wide variety of medicinal usage and is quite popular as a naturopathic treatment. R. crispus is well known for its treatment of skin ailments, as a poultice or taken internally. The leaves are used to treat skin ailments, such as: boils, hives, ringworm, itch, jaundice, acne, scabies, psoriasis, eczema, and other skin disorders.(3,42,43,44,45) The root is used for its well documented laxative properties, purportedly due to its high content of anthraquinones.(42,44,46,47) It has also been used widely as an alterative, that is, a remedy for syphilis or as a “blood cleanser”.(44,45,46,47) Alteratives are also used to treat anemia (iron deficiency in blood), R. crispus contains a high iron content.(3,47) The naturopathic use of R. crispus to treat cancer or as an antitumor agent is rare, but the root has been documented in several anticancer formulas from the 19th century, such as, Scudders cancer alterative, Kloss’ anticancer herbal, and Eli Jones Formula.(5) According to Dr. James Duke,(2,3) the plant can be used to treat cancer, and Table 1 lists some of the compounds that he has discovered in R. crispus to have proven antitumor
Table 1(2) Compounds in R. crispus that have proven antitumor acitivity. ( * = no quantitative results have been determined for this compound) Compound Ascorbic Acid Beta-carotene Emodin Erucic Acid Quercetin Antitumor activity Antitumor (lung) Antitumor Antitumor (breast) Antitumor Anticarcinomic (breast) IC50 = 1.5 uM Antitumor (bladder) Antitumor (breast) Antitumor (colon) Antitumor (ovary) Quercitrin Rutin Tannin Antitumor Antitumor Antitumor Plant Part Leaf Leaf Root Root Leaf Leaf Leaf Leaf Leaf Leaf Leaf Root Amounts
300-4,054 ppm 10.38-140 ppm * 9,000-121,612 ppm * * * * * * * 30,000-60,000 ppm.
properties.(2) A number of sources have identified medically active compounds that account for the uses described above. R. crispus has a high amount of anthraquinones with sources quoting between 3-4%, among the anthraquinones identified, include: nepodin, chrysophanol, physcion, emodin, chrysophanic acid, and rhein.(3,44,45,46,47) The flavonoid class of compounds has also been identified, including, quercitrin and quercetin.(44,45) Tannins and oxalates, which can be lethal in high doses are present in R. crispus, as well as several of their
derivatives.(3,44,45,47) Several naphthalene derivatives, napodin and lapodin, have also been identified.(44,45) A recent scientific study showed R. crispus to have a high degree of antioxidant activity and antimicrobial activity against Grampositive bacteria.(48) The study aconcluded that there was a relationship between the antioxidant activity of extracts and the amount of phenolic compounds detected.(48) Accelerated solvent extraction (ASE) was chosen as the solvent extraction method for the ground plant material. ASE uses high temperature and pressure to increase the speed and efficiency of extraction.(6) Results from ASE provide extractions comparable to conventional extraction methods when compared with analysis by UV-Vis spectroscopy and HPLC.(6) Increased temperature is used for extraction because as the temperature increases the solvent’s viscosity decreases, therefore, giving the solvent a greater ability to wet the matrix and solubilize analytes.(8) Elevated temperature also breaks bonds between the analyte and the matrix, and it encourages analytes to diffuse to the surface of the matrix.(8) Pressure is increased in ASE to move the mobile phase through the system rapidly, and to maintain the solvents in liquid phase while they are above their atmospheric boiling points.(8) Up to ten grams of sample can be extracted in less than twenty minutes using ASE.(6) Solvents used in the ASE were chosen based on their polarity and based on documentation of solvent extractions performed on other plant extracts.(6,7) Solvents of varying polarity can be used to extract a broad-range class of compounds.(8) Methanol, ethanol, and chloroform were chosen as solvents for ASE of the plant samples because of their solvent
properties. Methanol has high polarity and is aqueous, ethanol is_______ and aqueous, and chloroform is nonpolar and immiscible in water.(9) There are many advantages to using solid phase extraction (SPE) as a second step to separating compounds from the initial solvent extraction. A wide variety of sorbents, chemistries, and sizes of SPE columns are available. When combined with liquid extraction, solid-phase extraction concentrates specific phase extractions without sacrificing quantitative recoveries.(9) Further, SPE is rapid, easy to perform, can be automated, and solvent usage is minimal. (9) SPE method involves passing the sample (dissolved in appropriate solvent) through a conditioned matrix in which specific analytes are bound.(10) Interference compounds are washed from the matrix with a weak solvent, then the sample is eluted form the matrix with a small volume of strong solvent.(10) This produces a clean, concentrated, sample that is ready for a cell proliferation assay.(10) SPE uses the same analyte/matrix interactions that is used in the high performance liquid chromatography (HPLC) separation technique.(11) The SPE columns are silica gel-based, bonded phase packing that selectively retains specific classes of chemical compounds from within the given matrix.(11) Bonded silica sorbents are capable of having a variety of different functional groups readily bonded to the silica surface.(11) A description of the type of functional groups and what is retained by each sorbent to be used is in Figure 2.(9) Bonded silica also has a high surface area due to porosity, it’s a rigid support that will not shrink or swell, and it’s stable under a wide range of aqueous and organic solvent conditions. (11) Supelco silica-based packing has 40 μm particles with 60 Å pores.(9) The 3 mL
INSERT FIGURE 2 HERE: SPE PHASE TYPES
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columns are polypropylene columns which contain 500 mg of sorbent and is flanked by polyethylene frits.(10) Efficiency of SPE is particularly due to the vacuum manifold in which the SPE column resides in during extraction. The Visiprep vacuum manifold from Supelco enables up to 24 samples to be prepared simultaneously and utilizes a pressure gradient to pull volumes through the sorbent efficiently.(10) Disposable liners in the vacuum manifold eliminates the possibility of cross-contamination, and precise flow control is capable through each SPE tube by flow control valves.(10) SPE achieves clean-up, concentration, and solvent exchange of the analytes in four basic steps:
1.) Conditioning: The sorbent is prepared by solvating it with an appropriate volume of solvent that is similar in nature to the sample matrix.(11) Conditioning solvents depend on the tube packing and the application.(12) 2.) Load Sample: The dried sample that is dissolved in appropriate solvent is passed through the sorbent and analytes are retained by specific chemical interactions.(11) Some interference compounds may also be retained by weak chemical interactions.(11) 3.) Wash: Weakly retained materials are removed using a solvent with weak eluting strength, while the analyte is undisturbed in the sorbent bed.(12)
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4.) Elution: A small volume of strong eluting solvent is passed through the sorbent which disrupts analyte-sorbent chemical interactions yielding selective elution of a particular class of compounds.(11)
The classes of analytes extracted for each sorbent type are listed on Figure 2. Separation retention occurs by three methods, reversed phase extraction (LC-8, LC-18, LC-Ph, LC-CN), normal phase extraction (LC-Diol, LCCN, LC-NH2, LC-Si, LC-Florisil), and ion-exchange (LC-SAX, LC-SCX, LC-WCX, LC- NH2). Reversed phase separation uses hydrophobic interactions. The retention of analytes from a polar matrix is due to the attractive forces between the carbon-hydrogen bonds in the analyte and the functional groups bonded to the silica surface.(9) Nonpolar solvents disrupt the binding forces and are used to elute the adsorbed compounds.(9) Normal phase extraction utilizes hydrophilic interactions for separation.(9) Polar groups in the analyte interact with polar groups on the sorbent surface and are retained.(9) Solvents of higher polarity than the matrix disrupt the binding mechanism and elute the analytes.(9) Ionexchange is used to separate compounds that are charged when in solution. (9) The retention mechanism for ion-exchange is based on electrostatic attractions and elution of retained analytes is performed by displacement of the adsorbed compounds using a solution with a high ionic strength.(9) By separating the ASE samples into 13 different classes of compounds by SPE then the active anti-tumor compound(s) can be easily classified into a particular class of compounds. SPE seems to be the method to perform this
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separation, due to its reliability, precision, and efficiency. SPE has been used to separate a variety of drugs and organic compounds with excellent analytical results.(13) When compared to liquid-liquid extraction SPE has proven to yield the cleanest and best extraction results when separating 15 different organic compounds.(14) Colorectal cancer is the fourth most common form of cancer in men and it represents 8.9% of all new cancers per year worldwide and surgery is the only option available for treatment of patients with colorectal cancer.(15) The human cell-line chosen for this study is the DLD-1 colorectal adenocarcinoma. The DLD-1 cell-line was isolated in 1979 by D.L. Dexter and is available through the American Type Culture Collection (ATCC).(16,18) The DLD-1 cell-line is wellcharacterized in studies of the effects of polar solvents.(17) Doubling time for DLD-1 cells was determined to be 20 hours, and they are usually plated at 3 x 104 cells per 60 mm. dish.(16) Culturing methods of DLD-1’s are well documented, they are cultured in DMEM supplemented with 10% fetal calf serum (FCS), 100 U/mL penicillin, and 100 U/mL streptomycin at 37 oC and 5% CO2 in aiR.(15,19,20) DLD-1 cells are generally subcultured at confluency and are plated with a subculture ratio of 1:5 – 1:6.(18) CCD-112Co is a normal colon cell-line that is used as a control for this experiment. This normal colon cell-line is also available from the ATCC.(21) CCD-112Co is an adherent, fibroblast, that very little studies have been performed on, although information was obtained from other CCD colon cell-lines as reference for culturing CCD-112Co.(21) The recommended growth medium by
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the ATCC for the CCD-112Co cell-line is minimum essential medium (MEM) Eagle with 2 mM L-glutamine and Earle’s BSS adjusted to contain 1.5 g/L sodium bicarbonate, 0.1 mM non-essential amino acids, and 1.0 mM sodium pyruvate, with 10% fetal bovine serum.(21) It is recommended that normal cell-lines need to be subcultured at log growth phase just prior to confluency.(24) These normal cells are recommended to be split with a subculture ratio of 1:3 – 1:4.(21) OptiMEM reduced serum medium has shown in the lab to be able to culture the CCD-112Co cell-line. Opti-MEM is a modification of MEM (Eagle’s) that claims versatility and the ability to maintain equivalent growth rate of fibroblast cells in 2 – 4 % FBS supplementation as to basal media at 10% FBS. (25) Opti-MEM contains HEPES buffer, 2,400 mg/L sodium bicarbonate, hypoxanthine, thymidine, sodium pyruvate, L-glutamine, trace elements, growth factors, and phenol red reduced to 1.1 mg/L.(25) Characterization of the cell lines can be performed using a number of methods. The common method is by isoenzyme analysis, but cell-lines can be characterized using genetic analyses or fluorescent markers. Characterization can also be contracted out by companies that perform automated and reliable characterizations.(26,27) Mycoplasma contamination in eukaryotic cell-lines is the major concern of contamination for these cells. Antibiotics are generally added to media to prevent bacterial contamination and any other form of contamination besides mycoplasma virus would be visible, thus easily detectable. ATCC claim that cell samples purchased from them are mycoplasma-free, nevertheless, cultures can
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be contaminated anytime they are worked with. One method for mycoplasma detection is by growing a culture on agar plates with the Hayflick formula. (28) Mycoplasma can then be stained with Dienes stain and observed at 100x magnification.(28) Cells should also be maintained by making a working cell bank, that is, to freeze several cryovials of cells per passage as a precautionary measure. If the cell line were to be contaminated or die off for some reason, then they could easily be replaced from a preserved sample. This is more practical for the cancer cells since they grow quite readily, nevertheless, it is also recommended for the normal cell-line, too. A cell proliferation assay is the best means to determine activity of a suspected anticancer compound using an in vitro culture. Several methods of cell proliferation assays are available and used readily, including Trypan blue exclusion dye, MTT tetrazolium salt assay, and the [3H] thymidine incorporation assay. This experiment will use a fairly new method, the MTS assay, which is very similar to the MTT assay. Promega offers a Cell Titer 96® AQueous One Solution Cell Proliferation Assay which is used for microassays in 96-well plates, it is non-radioactive, and requires fewer steps than an MTT assay.(29) MTS is a tetrazolium compound [3(4.5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2Htetrazolium] that is reduced by active cells to a colored formazan product and is soluble in tissue culture medium (Figure 4).(29,30) After addition of the reagent, cells are cultured for 1 – 4 hours and then absorbance is recorded at 490 nm with
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a 96-well plate reader(29) When running an assay it is necessary to prepare a triplicate set of control wells with just culture medium and the MTS reagent as a standard to measure background absorbance.(29) Although the mechanism by which MTS is reduced is not fully understood, it is thought to occur as a result of the action of dehydrogenase enzymes generating reducing equivalents such as NADH or NADPH,(31) see Figure 4. The One Solution reagent contains MTS as well as phenozine ethosulfate (PES) which is the electron transfer reagent (ETR).(31) The amount of formazan produced and recorded at 490 nm is directly proportional to the amount of viable cells based on cellular metabolism. The MTS assay has been proven reliable for a variety of methods, such as: measuring cell proliferation, cytotoxicity, viability and apoptosis, cell adhesion, and chemotaxis, all using a variety of cultured mammalian cells.(30) The use of tetrazolium reagents is extensive and has been proven to be a reliable method.(30) The use of the DLD-1 cell-line with cell-proliferation assays is extensively reported on in the current scientific literature. Determination of several factors such as: DMSO percentage to dissolve plant extract in, number of cells to seed per well, whether or not to pre-incubate cells before addition of extract, volume per well, length of time for assay, all vary accordingly in the literature. To allow sufficient time for drug interaction with cells, a 72 hour assay will be employed and the total volume per well will be 100 uL to minimize reagent usage and coincide with Cell Proliferation Assay Kit (Promega) recommendations.(29) Evaporated samples from SPE are dissolved in DMSO and added to cell culture
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media at a 1:100 dilution.(7) Cells are then dispensed into 96-well plates at 5,000 cells per well.(29,32,33) A passage rate between P5 – P8 for DLD-1 and CCD112Co is commonly used for these cell types to perform the cell proliferation assay.( ) Promega claims that the incubation time after addition of MTS reagent varies with cell-line, but according to several papers using the MTT tetrazolium reagent for DLD-1 cell proliferation assays the incubation time was four hours.(15,32,33) Addition of the extract was also performed after a 24 hour preincubation period after the cells are added to the 96-well plates, according to several sources.(7,19,20,32,34) A positive control for tumor cell cytotoxicity was also considered to be used to compare effects of the test samples to a known antitumor compound. Doxorubicin is an antitumor compound readily available from Sigma Chemicals(35) and has shown strong antitumor activity in studies done on mice.(36) Doxorubicin is also used as a positive control for DLD-1 cell-line tumor assays by MDS Pharma at an IC50 of 0.35 uM.(37) VWR claims that doxorubicin has an IC50 equal to 0.1 uM. Doxorubicin’s activity is based upon inhibiting topoisomerase II and also intercalating into the DNA double helix, thus inhibiting nucleic acid synthesis.(38) The effect being measured in this experiment is cell death versus concentration (dose) and cell death versus time for the LD50 of the effective samples. First, a dose response curve will be generated by plotting the absorbance at 490 nm on the Y-axis versus the serial dilution of concentration of the sample on the X-axis.(39) As agonist (sample that causes a response)
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concentration increases on a dose response curve the curve should go downhill.(39) Doxorubicin as a standard is the full agonist (produces a full response) and will be a good comparison to agonist samples. When a doseresponse curve is generated for cell death versus concentration four parameters are defined on the curve. The baseline response is the bottom of the curve where the slope is equal to zero, and the maximum response is the top of the curve where the slope is equal to zero.(39) Both of these must be defined to define the LD50 (lethal dose) which is the response that occurs halfway between the baseline and maximum.(39) The fourth parameter to define is the slope of the curve, an example dose-response curve is Figure 5.1. For samples that yield an LD50, a graph of cell death versus time will be made based on the time assay, an example of a time assay graph is Figure 6.1. The toxicity of an antitumor drug, or any drug, is based on two factors, first, the concentration of the drug, and second, the amount of exposure to the drug.(40) A time assay of the effective samples also aids in determining the mode of action of the drug, for example, what stage of tumor growth the drug produces the greatest cytotoxicity. Dose response is an integral part of drug development, as it is important for determining the safety and effectiveness of a drug.(41) When determining dose response, the studies should be well-controlled and use accepted techniques to minimize bias.(41) A time assay has many benefits, and is easy to perform following up a dosage assay. There is a tight interaction between doseresponse and time, as in drug treatments the choice of the size of an individual dose is intertwined with the frequency of dosing.(41)
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Proposed Method of Study
Preparation of the cell-line will be the bottle-neck of this experiment. Enough cells are going to be needed to perform assays in over 400 96-well plates. Celllines must also continuously be kept free of contamination, and they also need to be characterized by karyotyping to insure there is no cross-contamination of celllines.
Cell proliferation assays will be performed with serial dilutions of the SPE sample from 1 – 10-7 of the 1:100 sample (resuspended in DMSO) in culture medium.(7) After a 24 hour pre-incubation period the extract dilutions will be added to the test well. After a 68 hour incubation the MTS reagent will be added to each well and incubated. The MTS tetrazolium salt will be allowed to react for 4 hours, then absorbance will be recorded at 490 nm in a 96-well plate readeR. Test samples that perform with positive results in the serial dilution assay will then be submitted to a time assay, in which the LD50 of the positive samples will be added to 96-well plates as before and cell proliferation will be measured at intervals of 12, 24, and 48 hours. Data from each of these tests will be recorded and graphed.
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DLD-1 w/ sample (duplicate) (duplicate)
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DLD-1 w/out sample
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O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O
DLD-1 w/ (+ control) (duplicate) CCD-18 w/ sample (duplicate) CCD-18 w/ out sample (duplicate) CCD-18 w/ (+ control) (duplicate)