Crude plant extracts

Chemical separations may first be accomplished by partitioning on the basis of polarity into a series of solvents from non-polar hexane to very polar compounds like methanol. Compounds may also be separated by molecular size, charge, or adsorptive characteristics, etc. Various chromatography methods are utilized, including columns, thin layer (TLC) gas-liquid (GLC), and more recently, high pressure liquid (HPLC) systems. HPLC has proven particularly useful for separations of water soluble compounds from relatively crude plant extracts. Previously, the major effort toward compound identification involved chemical tests to detect specific functional groups, whereas characterization is now usually accomplished by using a... 

Tarantilis, P. A., G. Tsoupras et al. (1995). Determination of saffron (Crocus sativa L.) components in crude plant extracts using hihg-performance liquid chromatography-UV-visible photodiode array detection-mass spectrometry. J. Chromat. A 699 107-118. 

Wolfender, J. L., Waridel, P., Ndjoko, K., Hobby, K. R., Major, H. J. and Hostettmann, K. (2000). Evaluation of Q-TOF-MS/MS and multiple stage IT-MSn for the dereplication of flavonoids and related compounds in crude plant extracts. Analysis 28 895-906A. 

Several soluble antigens that have been produced in plants using plant viruses as expression systems have shown immunogenic and protective properties in test animals [11]. The plant-derived proteins, which include known immunogens from pathogens or allergens from plants [38], have been tested either as crude plant extracts containing recombinant protein or as purified material from the infected plants. 

LC-NMR plays a central role in the on-line identification of the constituents of crude plant extracts (Wolfender and others 2003). This technique alone, however, will not provide sufficient spectroscopic information for a complete identification of natural products, and other hyphenated methods, such as LC-UV-DAD and LC-MS/MS, are needed for providing complementary information. Added to this, LC-NMR experiments are time-consuming and have to be performed on the LC peak of interest, identified by prescreening with LC-UV-MS. NMR applied to phenolic compounds includes H NMR,13 C NMR, correlation spectroscopy (COSY), heteronuclear chemical shift correlation NMR (C-H HECTOR), nuclear Overhauser effect in the... 

Giner-Chavez BI, Van Soest PJ, Robertson JB et al (1997) A method for isolating condensed tannins from crude plant extracts with trivalent ytterbium. J Sci Food Agric 74 359-368... 

Andrographolide, a labdane-diterpenoid lactone, is the principal bioactive chemical constituent of Andrographis paniculata (Brum. F.) Nees (family Acanthaceae) this prime constituent is mainly concentrated in leaves and can easily be isolated from the crude plant extracts as crystalline... 

We are exposed to plant products (many of the earlier drugs and most of the herbal pharmacopoeia in use today are crude plant extracts), some of which are potentially toxic, as, less commonly, are foods of animal origin. Medicinal drugs are just one of a set of chemicals which are exposed to the range of defence mechanisms put up by the body to protect it from the onslaught of foreign chemicals. 

Toxicity. Atractyloside, a diterpenoid glycoside that occurs naturally in plants, may be present at levels as high as 600 mg/kg of dried plant material. Gonsumption of plants containing atractyloside or carboxyatractyl-oside has caused fatal renal proximal tubule necrosis and/or centrilobular hepatic necrosis in man and farm animals. Although pure atractyloside and crude plant extracts disrupt carbohydrate homeostasis and induce similar pathophysiological lesions in the kidney and liver, it is also possible that the toxicity of atractyloside may be confounded by the presence of other natural constituents in plants. Atractyloside competitively inhibits the adenine nucleoside... 

LC-NMR can be operated in two different modes on-flow and stopped-flow. In the onflow mode, LC-NMR spectra are acquired continuously during the separation. The data are processed as a two-dimensional (2D) NMR experiment. The main drawback is the inherent low sensitivity. The detection limit with a 60 p.1 cell in a 500 MHz instrument for a compound with a molecular weight around 400 amu is 20 pig. Thus, on-flow LC-NMR runs are mainly restricted to the direct measurement of the main constituents of a crude extract and this is often under overloaded HPLC conditions. Typically, 1 to 5 mg of crude plant extract will have to be injected on-column.In the stopped-flow mode, the flow of solvent after HPLC separation is stopped for a certain length of time when the required peak reaches the NMR flow cell. This makes it possible to acquire a large number of transients for a given LC peak and improves the detection limit. In this mode, various 2D correlation experiments (COSY, NOESY, HSQC, HMBC) are possible. 

Wolfender, J.-L. et al.. Comparison of liquid chromatography/electrospray, atmospheric pressure chemical ionization, thermospray and continuous-flow fast atom bombardment mass spectrometry for the determination of secondary metabolites in crude plant extracts, J. Mass Spectrom. Rapid Commun. Mass Spectrom., S35, 1995. 

Hostettmann, K. et al.. Rapid detection and subsequent isolation of bioactive constituents of crude plant extract, Planta Med., 63, 2, 1997. 

Wolfender, J.-L. and Hostettmann, K., Liquid chromatographic-UV detection and liquid chromatographic-thermospray mass spectrometric analysis of Chironia (Gentianaceae) species. A rapid method for the screening of polyphenols in crude plant extracts, J. Chromatogr. A, 647, 191, 1993. 

Much data on the structure of flavonoids in crude or semipurified plant extracts have been obtained by HPLC coupled with MS, in order to obtain information on sugar and acyl moieties not revealed by ultraviolet spectrum, without the need to isolate and hydrolyze the compounds. In the last decade, soft ionization MS techniques have been used in this respect, e.g., thermospray (TSP) and atmospheric pressure ionization (API). However, the most used methods for the determination of phenols in crude plant extracts were the coupling of liquid chromatography (LC) and MS with API techniques such as electrospray ionization (ESI) MS and atmospheric pressure chemical ionization (APCI) MS. ESI and APCI are soft ionization techniques that generate mainly protonated molecules for relatively small metabolites such as flavonoids. 

The shrub Baeckea frutescens (Myrtaceae) has long been used in traditional medicine for the treatment of rheumatism and snake bites, and a recent examination of a dichloromethane extract of the aerial parts revealed the presence of a number of structurally unique compounds. The major component of the extract was shown to have structure 1. Another constituent of the mixture was shown to have structure 2, and it was tentatively suggested that 2 might be a degradation product of 1, formed either during the extraction process or during chromatographic separation of the crude plant extract. 

Analytical methods for PGS research have been greatly improved during this past decade. GLC-MS analysis has proven to be the method of choice, particularly when appropriate internal standards are used for accurate assessment of PGS recovery. HPLC, the most rapidly developing form of separation science, should substantially enhance present PGS analytical efforts. One advantage of HPLC is the substantial purification obtained for PGS compounds from crude plant extracts. For analytical identification by instrumentation, scrupulous purification is required, along with selective identification of the PGS. Preferably, two different analytical procedures should be utilized for positive identification of a given compound. 

Crude plant extracts contain ion-exchange resins, which combine with the... 

What are some considerations influencing a botanical investigational new drug (IND) application A botanical IND application should be filed if the proposed product contains crude plant extracts, partially purified plant extract fractions, or a combination of highly purified compounds from different plants. A botanical IND application should also be filed if a proposed product contains a single herb, multiple herbs, or a botanical alone or with additional active components such as vitamins, minerals, or animal parts. 

Small quantities of compounds in natural extracts are often a problem when these need to be evaluated in bioassays. Sometimes there is just not enough of the compound isolated to carry out the usual bioassay.20 Microassays have been developed10 1 to overcome this problem. Typically, a microassay is carried out on a thin-layer chromatography plate with a cellulose layer. A small droplet (1.5 pi) of the tested compound in a solvent (1-102 nmol cm-2) is then added on the plate. After the solvent has evaporated a small amount (5 pi) of sucrose solution lmoll-1 is added to the place where the compound was added. In the control the same procedure was followed on a different plate, but with the solvent alone, with no compound added. These two plates are then placed in a petri dish with the test insect species. In the past, when paper chromatography was widely used, a crude plant extract was placed on the origin of the paper and then eluted into bands. The paper was freed of solvent, sprayed with sugar solution, and used directly in a bioassay to see which parts of the paper were not eaten, and therefore of interest for further examination. 

Alkaloids are widespread in plants and include some very well-known poisons (notably coniine and strychnine), hallucinogens (morphine, cocaine and muscimol) and other potentially lethal compounds that are nevertheless used in medical practice (e.g. atropine, codeine, colchicine and morphine). As indicated by the preliminary snap-shot above, alkaloids typically have names ending in -ine and which are often related to the plant source or properties. Thus, morphine was named after Morpheus (the God of sleep) and coniine derives from Conium maculatum (hemlock), the plant used in the judicial murder of Socrates (399 BC). Various chemical tests for alkaloids are used as preliminary indicators of alkaloid presence in crude plant extracts. Finally, it should be noted that alkaloids can also exist as jVoxides of the alkaloid base. 

G. Bringmann, M. Wohharth, H. Rischer, M. Heubes, W. Saeb, S. Diem, M. Herderich, and J. Schlauer, A photometric screening method for dimeric naph-thylisoquinohne alkaloids and complete on-hne structural elucidation of a dimer in crude plant extract, by the LC-MS/LC-NMR/LC-CD triad. Anal. Chem. 73... 

The story has all the classic ingredients, starting with observation and reasoning, extending through chance into new areas, and characterized throughout by persistence and determination, particularly when biological activity had to be traced to very minor constituents in the crude plant extract. 

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