Separation of acidic compounds and determination of shikimic acid in water extracts of several conifers by HPLC

Abstract

The plants of Coniferae are rich resources that have been found to be of important medicinal value in recent years. The medicinal importance of pine needles prompted us to carry out phytochemical investigations on it [1]. In the past work, we isolated a variety of active compounds from the needles of Cedrus deodara (Roxb.) Loud [2]. In the present work, four small subclasses of acidic compounds have been isolated from the needles of Cedrus deodara (Roxb.) Loud; these were shikimic acid (1), vanillic acid (2), 3,4-dihydroxybenzoic acid (3), and p-hydroxybenzoic acid (4). Shikimic acid (1) is an important intermediate in the biosynthesis of aromatic amino acids in plants and microorganisms [3–6], which is the starting compound utilized for the manufacture of the drug Oseltamivir [3]. Although most autotrophic organisms produce shikimic acid, the isolation yield is low [7]. Shikimic acid is mostly obtained from the fruits of star anise [8]. Only one Chinese paper reported the determination of shikimic acid in the fruit of Illiciaceae plants by LC with diode-array detection [9]. Mardones et al. reported in 2005 [10] a CZE method for determination of shikimic acid in red wines. Garcia Romero et al. [11] described the LC method for the determination of organic acids in grape musts and vinegars. Fang et al. [12] characterized the phenolic constituents in dried plums by LC-MS/MS, and Bylund et al. [13] analyzed the organic acids in natural waters by tandem mass spectrometry. In the present work, we developed a simple and highly precise RP-HPLC method for the analysis of the bioactive molecules of shikimic acid in seven different conifers, viz. Cedrus deodara (Roxb.) Loud, Platycladus orientalis (Linn.) Franco, Pinus massoniana Lamb., Picea crassifolia Kom., Pinus tabulaeformis Carr., Juniperus chensis L., and Sabinacentrasiatica Kom. The method can also be used for quality control and standardization of the species and to identify the species used in a formulation. Pine leaves and branches of seven different conifers were obtained from Gansu Province in northwest China. The samples were identified by Prof. Fujiang He (Gansu Academy of Medical Science, Lanzhou, China). The specimens were authenticated and a voucher specimen of each (YKY0915) was deposited in the Herbarium. The pine needles of Cedrus deodara (3.5 kg) were extracted with 95% ethanol (10 times volume) twice to afford the ethanol extract (390 g), which was suspended in water and extracted with petroleum ether, ethyl acetate, and n-butanol, separately. The n-butanol extract (130 g) was chromatographed over Diaion HP-20 with H2O containing increasing amounts of MeOH. The 20% MeOH eluate (3.4 g) was chromatographed on Toyopearl HW-40 (coarse grade) developing with 20% MeOH 50% MeOH. The 20% MeOH eluate (1.5 g) was rechromatographed on silica gel to yield compound 1 (46 mg), compound 2 (11 mg), compound 3 (16 mg), and compound 4 (13 mg). Samples of air-dried (45–55 C) powdered pine leaves from the seven different conifer species (1.0 g) were sonicated in 100 mL of water for 20 min followed by centrifugation for 15 min at 3500 rpm. The clear supernatant solution was transferred to a 100 mL volumetric flask. The procedure was repeated thrice and the respective supernatants combined. The final volume was adjusted to 100 mL with water and mixed thoroughly. Quantitative analysis of shikimic acid by HPLC with UV detection showed that the amounts of shikimic acid were largest in Cedrus deodara (Roxb.) Loud. The mobile phase was methanol containing aqueous phosphoric acid (pH 3); the amount of the latter component was 90%. The flow rate was 1.0 mL·min–1. The results are summarized in Table 1.