Application Note AN 101-01-D

Plant Drug Analysis by OPLC

Many compounds of pharmaceutical interest are derived from plants and are readily isolated from raw plant materials by OPLC. Typical examples include the separation bstract of alkaloids from plant extracts, cannabinoids from cannabis resin, and aglycones of phenolic compounds, flavonoids, anthocyanins and coumarins, which include an aromatic ring with one or more hydroxyl groups or phenolic ether groups from plants, grasses and fodder. OPLC provides the scientist with a rapid technique for analytical separations and the method can be rapidly scaled up for semi-preparative separations. Additional advantages of OPLC for the separation of plant extracts include simplicity, efficiency, reproducibility and relatively small volume of mobile phase. As an example, a broad variety of alkaloids, coumarins, flavanoids, anthrocyanines and cannabinoids can be separated from natural products with neat ethyl acetate; it is not necessary to use complex mobile phases or different mobile phases for different samples. This application note provides a few examples of the separations of a broad range of compounds from plant extracts. Alkaloids

The separation of alkaloids can be performed using ethyl acetate as the mobile phase. This is a significant simplification over commonly used techniques where it is necessary to use three or more components in the mobile phase. The separation of opium alkaloids is shown in Figure 1 using OPLC in the off-line detection mode with a densitometer.

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Figure 2: Separation of ethanolic extract of Hydrastatis Canadensis. Stationary Phase: silica, Mobile phase: ethyl acetate/acetic acid/tetrahydrofuran (60/20/20) Flow rate = 0.4 mL/min, Visualization using Dragensdorfs reagent. Compounds 1: Hydrastinine, 2: Hydrastine, 3: Berberine.

Quaternary Ammonium Salts and Alkaloids

Figure 1: Separation of Opium Extract by OPLC. Stationary Phase: Aluminum Oxide 60F254, Mobile Phase: Ethyl Acetate (0.4 mL/min). Visualization via Dragendorfs reagent, = 540 nm. Compounds: Morphine (1), Codeine (2), Thebaine (3), Papaverine (4), Noscapine (5)

The separation of quaternary ammonium salts can be readily performed on silica using ethyl acetate/acetic acid/tetrahydrofuran (60/20/20). Excellent separations could be obtained. Figure 2 shows a typical example of the separation of ethanolic extracts of Hydrastatis Canadensis and all the major alkaloids (berberine, hydrastine and hydrastinine). The alkaloids from Chelidonium Majus can be separated in a similar manner.

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Coumarins
Coumarins (e.g. coumarin, umbelliferone, aesculetin, scopoletin), which are commonly found in medicinal plants, grasses and fodder are readily separated via OPLC. These compounds contain the benzo[]pyrone group and are highly fluorescent. Since coumarins are fairly polar, chloroform is added to ethyl acetate to modify the polarity of the eluent. A typical separation is shown in Figure 3.

fig. 3

fig. 4

Flavonoids
The OPLC separation of flavonoids from an extract of orange flower (citrus aurantium var.amara) is shown in Figure 4. This chromatogram shows that the major flavonoid compounds can be readily separated in a short period of time using silica with a moderately polar mobile phase.

Conclusion
The separation of a broad range of plant extracts including coumarins, flavanoids, anthocyanins, and cannabinoids have been performed by OPLC. These separations use polar stationary phases and moderately polar mobile phases to provide rapid, well defined separations on the analytical as well as semipreparative scale, so that scale up and isolation of the active components is readily effected. Detection of the various compounds is directly performed on the OPLC sorbent bed via a densitometer, further simplifying the overall analysis. Reference 1 provides a detailed list of over 54 plant extracts that have been separated by OPLC. 39 different alkaloids, 5 quaternary ammonium salts, 8 coumarins, 11 flavonoids and furanochromones, 3 anthocyanins and 2 cannabinoids have been identified, clearly demonstrating the power of the technique.

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Figure 3: Separation of Coumarins from Aesculus Hippocastani. Stationary Phase: silica Mobile Phase Ethyl Acetate/Chloroform (60/40). Visualization via Fluorescence. Compounds: 1: Aesculetin, 2: Scopoletin Figure 4: Separation of Flavonoids from citrus aurantium var.amara. Stationary Phase: silica, Mobile phase ethyl acetate/chloroform(60/40) Flow rate = 0.4 mL/min, Visualization at 365 nm. Compounds: 1: Luteolin, 2:Eriodictyol, 3: Hesperetin, 4: Naringenin

References 1) Pothier J, Galand N, Viel C, Application of OPLC to Plant Drug Analysis Proceedings of the 10th International Symposium on Instrumental Planar Chromatography, May 1619, 1998, Hungary, and references quoted therein.

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