In the Laboratory
Investigation of Secondary Metabolites in Plants A General Protocol for Undergraduate Research in Natural Products Jonathan Cannon, Du Li, Steven G. Wood, Noel L. Owen,* Alexandra Gromova,† and Vladislav Lutsky† Department of Chemistry and Biochemistry, Brigham Young University, Provo, UT 84602; *
[email protected] Secondary metabolites are compounds found in higher plants that are not involved in the major metabolic pathways. These compounds are however, believed to play important roles in plants. For example, some of them are toxic and help in defending the plant from predators; others have odors that attract insects and thus facilitate pollination. Many of these compounds have important medicinal properties and are useful drugs. In fact, a large percentage of all known medicines have their origins in natural products found in plants. A number of pharmaceutical companies and universities have research groups that investigate the bioactivity and potential medicinal uses of plant secondary metabolites. We have been engaged in such research over the course of the past ten years or so and much of the work in our laboratory has been done by undergraduate students. Plant secondary metabolite research cuts across many scientific disciplines such as botany, microbiology, organic chemistry, biochemistry, and other chemical sciences. Consequently such research provides a unique opportunity to expose students to working in an interdisciplinary environment. In this paper we (i) outline typical experimental procedures that can be used to extract and isolate individual chemical constituents from a plant, (ii) suggest some simple procedures to test for selected bioactivity, and (iii) explain how the molecular structures of the natural products may be determined using spectroscopic techniques. We use the Siberian plant Astragalus danicus as our example. The work done on this plant will illustrate the multitude and complexity of the chemical components found in plants. The general scheme and approaches outlined in this paper may be adapted for undergraduates at most colleges and universities. The Plant Source We have found that plants having a history of medicinal use by indigenous peoples to treat various illnesses are the most interesting candidates for study. However, any convenient plant material such as commercial herbal products would be suitable for this exercise. Considerable work has already been done on many common medicinal plants. Therefore, it is very valuable (imperative for serious research work) to check the literature on the chosen plant in order to avoid duplication of effort (1). To obtain a sufficient amount of a purified secondary metabolite for total structure determination, approximately 0.5–2 kg of dried plant material is required. For some plants, the season in which they are collected governs to a large degree the nature and quantities of the metabolites present in the stems or leaves. The best method of collecting the plant material is † Permanent address: Irkutsk Institute of Chemistry, Siberian Branch of the RAS, ul. Favorskogo, 664033 Irkutsk, Russian Federation.
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freezing in the field using solid CO2 or liquid N2. The frozen plant material may then be stored frozen or subsequently freeze-dried. Alternatively, the plants may be carefully dried in the shade (drying in the sun may result in photodegradation of the constituents). Before extraction, the plant material should be macerated if fresh or frozen, or ground up if dried. If the sample is small or the plant material is fresh or frozen, a pestle and mortar is sufficient; otherwise, a blender, mechanical grinder, or mill works well. Initial Extraction Scheme We have worked out a scheme to yield three extracts of differing polarity. A small-scale initial extraction is sufficient to provide enough extract for bio-testing. The dried, ground plant material (5 g) is covered with two volumes of methylene chloride and stirred for 20 min. The solvent is filtered off, the extraction is repeated, and the collected solvent is taken to dryness. If the plant material is fresh or frozen, the initial extraction is carried out with methylene chloride/methanol (50/50) and the subsequent extractions are carried out as described. This process is repeated utilizing methanol as the solvent. The methanol extracts are added to the residue from methylene chloride extractions and the resulting solution is extracted three times with equal volumes of hexane (addition of a small amount of water may be necessary to facilitate phase separation). The phases are separated and pooled. After removal of the solvents, the hexane and methanol phases yield nonpolar and polar organic extracts, respectively. Since in most instances any native use of these plants for medicinal purposes involves aqueous extraction or poultices, a hot water (90 °C) extract may be prepared from the extracted plant material or, if preferred, from a new sample, resulting in a third (aqueous) extract. There may be instances when the indigenous ethnobotanical procedure involves acidic or basic conditions, but in the majority of cases neutral water can be used. The Isolation and the Purification Scheme A larger-scale extraction of the plant material based on the nature of the initial extract demonstrating the bioactivity is initiated. An appropriate extraction solvent is chosen and a 500-g plant sample is extracted three times with two volumes of solvent. The solvent is removed (a rotary evaporator is useful here) and the resulting crude extract is tested to confirm the bioactivity. The crude extract is analyzed by TLC and on the basis of the result, a silica gel column is often run to subfractionate the crude extract. The subfractions are then tested for bioactivity and the active subfraction is chosen for further purification. The active compounds are usually obtained in pure form following additional chromatographic steps. If
Journal of Chemical Education • Vol. 78 No. 9 September 2001 • JChemEd.chem.wisc.edu
In the Laboratory
the compounds are in the right polarity range, silica gel chromatography works satisfactorily. Both preparative and analytical HPLC, if available, are extremely useful to separate mixtures and determine the purity of the isolated compounds. The Bio-testing Procedures We have found the use of relatively simple and straightforward cytotoxicity and antimicrobial assays to be effective in identifying and confirming the biological activity throughout the extract fractionation and purification procedures. Cytotoxicity testing is carried out using standard 96-well microtiter plates seeded (ca. 5 × 105 cells) with HeLa cells (epithelial carcinoma, cervix, human, ATTC CCL2). The plates are incubated for at least 4 h and the plant extracts are tested in triplicate at initial concentrations of 200, 100, and 50 mg/mL. After a 36-h incubation, the results are determined using sulphorhodamine B dye to stain protein as described by Skehan et al. (2). This method involves fixing the viable cells to the plate with a 0.1% perchloric acid solution, washing several times with water, and staining the cells with a solution of the dye in 1% acetic acid (4 mg/mL). Excess dye is removed with subsequent washings of 1% acetic acid and the absorbance of the dye at 540 nm (directly proportional to the number of viable cells) is determined by taking up the bound dye in Tris buffer (10 mM, pH 7.4). The percent viability is determined relative to untreated control wells. Antimicrobial testing is done using a gram-positive organism, Staphylococcus aureus (ATCC 6538P), a gram-negative organism, Pseudomonas aeruginosa (ATTC 27853), and a yeast, Candida albicans (ATCC 90028). These tests are also run in 96-well microtiter plates as described by Donaldson (3). The plant extract samples are added in triplicate to plates containing appropriate growth media (water and methanol extracts 200 to 0.1 µg/mL, hexane extracts 500 to 0.5 µg/mL) and an inoculum of the microorganism is added. Growth inhibition is determined by comparing the optical density (600 nm) of the plate read directly after inoculation to the optical density read after a 24-h incubation.
spectroscopy. The former is the method of choice if goodquality crystals can be obtained and if one has access to the appropriate equipment. 1H and 13C 1-D and 2-D NMR experiments are usually sufficient to obtain a structure of a small molecule (under 1000 MW). A quantitative 13C experiment provides the total number of carbons in a compound. These data, in conjunction with high-resolution mass spectral data, are usually sufficient to provide the molecular formula of the unknown compound. The majority of natural products extracted from plants are composed only of carbon, hydrogen, and oxygen, with the exception of some classes of nitrogen-containing compounds such as alkaloids. A NMR DEPT experiment identifies all quaternary carbon atoms as well as those connected to one, two, or three hydrogen atoms. The 2-D NMR experiments, COSY and HETCOR, show the connectivity of adjacent hydrogen atoms and hydrogen atoms to carbon atoms, respectively. Occasionally, more sophisticated experiments such as NOESY, HMBC, or INADEQUATE are necessary for complete structure determination. However, these techniques are often only available in laboratories that have well-trained spectrometer operators. A routine infrared or ultraviolet spectrum will always provide useful information concerning the presence or absence of specific functional groups in the compound under investigation, provided enough sample is available. An Example Plants of the Astragalus genus (the largest genus of the Fabaceae family) are found worldwide. Astragalus danicus grows wild in eastern Siberia (Russia) and has been used for
Aerial parts of the plant
#1 H2O extract
The Structure Determination After a pure chemical component is obtained, one of the first steps in ascertaining its structure is the determination of its molecular mass. If a high-resolution mass spectrometer (HRMS) is available, then a precise determination of the mass of the parent ion will often indicate the most likely molecular formulas. For many pure natural products, especially glycosides and related compounds, fast atom bombardment (FAB) is the mass spectrometric method of choice for analysis, but for more volatile compounds electron impact (EI) and chemical ionization (CI) can give useful data. An analysis of the mass spectral fragmentation pattern will, in many instances suggest the molecular formulas of the main breakdown fragments, which is helpful in determining the total structure. Tandem techniques such as GC–MS or HPLC–MS can facilitate the simultaneous separation and the identification of compounds of interest. Electrospray MS techniques are useful for compounds with molar masses above about 1000. The most useful techniques for structure determination of natural products are, however, X-ray diffraction and NMR
H 2O MeOH
#2 MeOH extract
CH 2Cl2
#3 CH2Cl2 extract
#6 Waxes, fats, lipids, etc.
#4 CHCl3 extract
#5 H2O soluble
#7 H2O residue
SIO2 chromatography (hexane/acetone)
1-BuOH/H2O partition
#8 1-BuOH soluble SIO2 chromatography (CHCl3/MeOH/H2O) prep HPLC (MeOH/H2O)
prep HPLC
#9 Fatty acid fraction linolenic acid (1)
CHCl3/H2O partition
#10 D-3-Omethyl-chiroinositol (2)
#11 Saponin fraction monoside (3) biosides (4, 5) triosides (6, 7)
Figure 1. Extraction scheme for Astragalus danicus.
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In the Laboratory Table 1. Data for Compounds Isolated from Astragalus danicus Compound No. Name
Formula
Mol. Ion Exptl (HRMS)
Calcd
Lit
Exptl
Value
Ref
1
Linolenic acid
C18H30O2
[M–H] ᎑
278.224
278.225 —
᎑11
4
2
D-3-O-Methyl-chiro-inositol
C7H14O6
[M+H]+
195.086
195.087
186–187
188
10
3
Soyasapogenol 3-β-D-glucuronide C36H58O9
[M–H] ᎑
633.399
633.400
242–244
—
—
3a
Soyasapogenol B
+
458.375
458.376
252–253
258–260
᎑
C30H50O3
[M]
8
4
Cloversaponin IV (bioside)
C41H66O13
[M–H]
765.442
765.443
220–222
—
5
Azukisaponin II (bioside)
C42H68O14
[M–H] ᎑
795.454
795.453
283–285
285–287 11
6
Astragaloside VIII (trioside)
C47H76O17
[M–H] ᎑
911.499
911.500
223–225
223–224 12
7
Hespidacin (trioside)
C48H78O18
[M–H] ᎑
941.511
941.511
240–242
245–247 13
centuries by healers and native peoples of that area to cure headaches, to treat post-menstrual symptoms and fatigue, to increase stamina, etc. The above-ground parts of the plant were collected during the summer flowering season and airdried. The crushed plant material (ca. 1 kg) was extracted by a method similar to that outlined above, but the methylene chloride and methanol extracts were kept separate (see Fig. 1). A sample of each of the three extracts was tested for bioactivity against HeLa cells (human cervical cancer), Candida albicans (fungus), Pseudomonas aeruginosa (gram-negative bacterium), and Staphylococcus aureus (gram-positive bacterium). The MeOH and the CH2Cl2 extracts were cytotoxic to HeLa cells and inhibited the growth of S. aureus. The chloroform-soluble portion (#4, Fig. 1) of the methanol extract was combined with methylene chloride extract (#3) on the basis of both TLC and the S. aureus bioassay results. This combined material (#6) was subjected to SiO2 column chromatography and then preparative HPLC [C-18, 5 µm, column; solvents: MeCN/H2O (45:55 to 100:0, v/v)] providing a fraction that inhibited bacterial growth by 50% at a concentration of 30 µg/mL. An IR spectrum of this fraction showed a strong carbonyl group absorption at 1712 cm᎑1, indicative of a COOH group. The major component of this fraction was easily identified from the 13C NMR spectrum of this fraction as linolenic acid (1) (4) (Table 1). A subsequent literature search (5–7) confirmed that the bioactivity of this fraction (both antibacterial and cytotoxicity) could be completely accounted for by this polyunsaturated fatty acid. A second, non-biologically-active compound was also isolated from fraction #6. This compound was identified on the basis of its IR, MS, and 1H and 13C NMR spectra as D-3-O-methylchiro-inositol (2). The genus Astragalus contains a class of plant secondary metabolites known as saponins, which have a terpenoid framework with one or more sugar groups attached. Although these compounds were not active in our bioassays, we were interested in comparing the structures of the saponins present in A. danicus with those found in other Astragalus species. A butanol/water separation technique (Fig. 1) effective in isolating such compounds was employed on the CHCl3-extracted aqueous potion of the initial MeOH extract. The saponincontaining butanol extract (#11) was further separated by column chromatography on silica gel (CHCl3/MeOH/H2O solvent system) and produced three fractions. Five saponins (Fig. 2) were subsequently isolated from these fractions. The first fraction contained a single component, 3. After preparative 1236
Melting Point/°C
Mass Data ( m /z )
—
HPLC (C-18; 5 µ m, column 21.4 × 250 mm; solvents MeCN/H2O 45:55, v/v) fractions two and three yielded compounds 4 and 5, and 6 and 7, respectively. We will consider in detail the structural elucidation of two of these saponins, 3 and 4, to illustrate methodology and briefly comment on the elucidation of the remaining three. The MS data indicated that molecular mass of 4 was greater than that of 3 by 132 mass units. The proton and carbon NMR spectra of both compounds were consistent with the suspected saponin structure: elements of both the cyclic hydrocarbon terpenoid ring structure and the characteristic patterns of the oxygenated carbon atoms of sugar groups were clearly discernible. Many plant secondary metabolites have attached sugar groups. The removal of these groups often aids in the determination of both the genin (nonsugar portion) and the individual sugar structures. To this end, acid hydrolysis (3.5% H2SO4; dioxane/water 1:1, 80 °C for 6 h) of compound 4 resulted in the isolation of two components from the nonaqueous part of the hydrolysate by silica gel chromatography. The first of these (3a) was found to have the molecular
3a 3 4 5 6 7
Monoside Bioside Bioside Trioside Trioside
R
R1 (on sugar unit)
H S1 S2 S2 S3 S3
H CH2OH H CH2OH
20 22 12
OH
17
1 3 4
RO
CH2OH
S1
S2
COOH O OH
OH OH
OH
R1
O
OH OH
OH
Xyl or Glc
β-D-glucopyranosiduronic acid β-D-xylopyranosil β-D-glucopyranosil α-L-rhamnopyranosil
R1 OH
O O
Xyl or Glc
OH O
OH
O CH3 OH OH
Figure 2. Triterpenoid saponins of Astragalus danicus.
Journal of Chemical Education • Vol. 78 No. 9 September 2001 • JChemEd.chem.wisc.edu
Glc A
OH
Glc A
O
Glc A: Xyl: Glc: Rha:
O
O OH
Glc A
OH
S3 COOH
COOH
Rha
In the Laboratory
formula C30H50O3 from analysis of the HRMS. This formula corresponds to a degree of unsaturation of 6, indicating a structure with 6 double bonds or rings. The 1H and 13C NMR spectra of this compound showed the presence of one trisubstituted double bond (δ 5.41; 1H), 7 methyl groups, and 1 primary and 2 secondary hydroxyl groups. These data were consistent with a pentacyclic triterpenoid with one double bond. The location and the relative configuration of the three oxygen-containing substituents (at positions 3β, 4β, and 22β on the triterpenoid ring system) were determined by further inspection of NMR chemical shift and coupling-constant values (Fig. 2). A search of the literature revealed that compound 3a is the known compound soyasapogenol B (8), previously isolated from soybean. The second component (a white amorphous powder) isolated from the hydrolysate of 4 was identified as compound 3 by comparison of their mass spectra (C36H58O9), NMR spectra, and TLC traces. The NMR spectra of 3 indicated that the genin portion of this compound has the same structure as 3a. However, the spectra showed that compounds 3 and 3a differ by the presence of six additional carbon atoms in 3 and a concomitant increase of 176 in mass number. Infrared and NMR data also indicated the presence in 3 of a COOH group (at 1716 cm᎑1 and δ 175.3 ppm, respectively). These features indicated that compound 3 is the monoside of genin 3a, glycosylated by glucuronic acid. The place of attachment of the glucuronic acid at C-3 of the genin was determined by a long-range NMR coupling experiment (HMBC), and coupling constant data confirmed that the conformation of this sugar derivative is β-D-glucuronic acid. Hence, the hydrolysis of compound 4 produced both compound 3 and the genin 3a. Compounds 3 and 4 differed by 132 mass units, corresponding to a pentose sugar unit. The presence of five additional carbon atoms in the 13C NMR spectrum of 4 confirmed this, and the pentose moiety was identified as xylose from its 13C chemical shift values. Analysis of the long-range NMR coupling constants showed that the xylose was attached to C-2 of the glucopyranosiduronic acid of compound 3. The three other saponin compounds (5, 6, and 7) isolated from the butanol extract of the plant were all based on the same genin, 3a. Compound 5 contained two sugar groups and compounds 6 and 7 each contained three sugars. The structures of all the saponins are shown in Figure 2. Table 1 summarizes the physical properties of all the compounds isolated from A. danicus. None of these saponins were active in our bioassays, although similar oleanene glucuronides have shown hepatoprotective activity (9). Conclusions The example cited above is among the more difficult cases encountered, but it illustrates the approach and techniques involved in the isolation and structural elucidation of secondary plant metabolites. The student involved in this work was well supervised, but other less involved projects have been successfully completed by students with minimal outside help.
Many positive attributes are associated with a project such as this one. Some of these are summarized below. 1. Discovering what chemicals are found in medicinally important plants is an exciting adventure to most students, and their enthusiasm for this kind of project contributes significantly to their success. 2. The project involves a multidisciplinary approach, and students benefit from exposure to a wide range of analytical and bioorganic techniques. 3. This kind of project allows students to carry out more sophisticated spectral studies in a context that they find interesting and encouraging to independent learning. 4. Students are introduced to the discovery processes for new and potentially important medicines.
Acknowledgments We wish to thank David M. Grant of the University of Utah for his encouragement and financial support of this work, and also Brigham Young University for financial assistance. Literature Cited 1. Duke, J. A. CRC Handbook of Medicinal Herbs; CRC Press: Boca Raton, FL, 1985; Handbook of Biologically Active Phytochemicals and Their Activities; CRC Press: Boca Raton, FL, 1992. Society for Medicinal Plant Research; http:// www.uni-duesseldorf.de/WWW/GA/inhalt.htm. Agricultural Research Service. Dr. Duke’s Phytochemical and Ethnobotanical Databases; http://www.ars-grin.gov/duke/ (accessed May 2001). 2. Skehan, P.; Storeng, R.; Scudiero, D.; Monks, A.; McMahon, J.; Vistica, D.; Warren, J. T.; Bokesch, H.; Kenny, S.; Boyd, M. R. J. Natl. Cancer Institute 1990, 82, 1107–1112. 3. Donaldson, J. R. A New Approach: Comparing Ethnobotany and Chemical Ecology Approach in the Search for Medicinal Plants; Master’s Thesis, Department of Botany, Brigham Young University April 2000. 4. Beilstein 1961, Suppl. Series 3, Vol. 2, p 1508. 5. Raychhowdhury, M. K.; Goswami, R.; Chakrabarti, P. J. Appl. Bacteriol. 1985, 59, 183. 6. Sagar, P. S.; Das, U. N.; Koratkar, R.; Ramesh, G.; Padma, M.; Kumar G. S. Cancer Lett. 1992, 63, 189. 7. Ramesh, G.; Das, U. N. Cancer Lett. 1998, 123, 207. 8. Kitagawa, I.; Yoshikawa, M.; Yosioka, I. Chem. Pharm. Bull. 1976, 24, 121. 9. Kinjo, J.; Aoki, K.; Okawa, M.; Shii, Y.; Hirakawa, T.; Nohara, T.; Nakajima, Y.; Yamazaki, T.; Hosono, T.; Someya, M.; Niiho, Y.; Kurashige, T. Chem. Pharm. Bull. 1999, 47, 708. 10. Beilstein 1967, Suppl. Series 3, Vol. 6, p 6927. 11. Fukunaga T.; Nishiya, K.; Takeya, T. Chem. Pharm. Bull. 1987, 35, 1610. 12. Kitagawa, I.; Wang, H. K.; Yoshikawa, M. Chem. Pharm. Bull. 1983, 31, 716. 13. Mahato, S. B. Phytochemistry 1991, 30, 3389.
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