Studies on Chemical Constituents of Radix Angelicae pubescentis

—Backgound:Angelicae pubescentis is the root of Angelica pubescens Maxim. f. biserrata Shan et Yuan. The main active ingredients are coumarin and volatile oil, and it has a variety of pharmacological activities such as antibacterial, antioxidant and anti-inflammatory. In this paper, the active components of the CO 2 supercritical extract of Angelicae pubescentis were studied. The results of this study will provide a scientific basis for the study of the chemical composition of Angelicae pubescentis. Purpose: To study the chemical constituents of supercritical extract of Radix Angelicae pubescentis. Method: The supercritical extract of Radix Angelicae pubescentis was separated by silica gel column chromatography to obtain monomers. The structures were identified based on physicochemical properties and spectroscopic data. Result: Eight compounds were separated from the supercritical extract of Radix Angelicae pubescentis, six of which were identified.They are Osthol, Columbianedin, Columbianetin, Columbianetin Acetate, Xanthotoxin and Bergapten. Conclusion: Xanthotoxin and Bergapten were isolated, which structure were similar. The standard substance of Osthol and Columbianedin were producted in order to offer the good material base for quality evaluation and pharmacodynamic study of Radix Angelicae pubescentis. Their purification were more than 99%. The results of this study are of great significance for exploring the effective material basis and medicinal value of the supercritical extract of Radix Angelicae pubescentis.


INTRODUCTION
There are many chemical components of Radix Angelicae pubescentis, domestic and foreign scholars have made a lot of research on it. So far, it has been found to contain coumarin, volatile oil, terpenes, sterols, organic acids, sugars and other components.
The main component of Radix Angelicae pubescentis is coumarin. So far, more than 70 kinds of coumarins have been isolated from Ducuo. They include simple coumarins, barking coumarins, picrancoumarins, dicumarins, and some coumarin glycosides. The various coumarin structures are shown in Figure1.
In addition, there are also differences in the main chemical components in different varieties of Radix Angelicae pubescentis. The root of A. pubescens Maxim contains Angelol, Angelicone, Glabralactone, Bergapten, Osthol [1] , Umbelliferone, Scoppoletin, Angelic acid, Tiglic acid, Palmitic acid, stearic acid, oleic acid, linolenic acid, phytonol, glucose and a small amount of volatile oil [2] . The root of H. lanatum Michx contains Angelicin, Pimpinellin, Bergapten and other various barking coumarins. In addition to the above components, the leaves also contain psoralen and so on. Among them, the main barking coumarins are bovine parsnips [3] . It also contains 0.26-0.57% volatile oil. So far, the chemical components isolated from Radix Angelicae pubescentis have been far more than these. With the progress of the times and the gradual improvement of scientific and technological means, the chemical components in Radix Angelicae pubescentis will be studied more and more thoroughly.
In this experiment, we utilized column chromatography for the separation of coumarins on the basis of CO2 supercritical extract. We prepared to product the relevant standard substance, The results of this study are of great significance for exploring the effective material basis and medicinal value of the supercritical extract of Radix Angelicae pubescentis.  All chemicals for chromatography were of analytical grade. All chemicals for HPLC were of chromatographic grade.

Methods
In this experiment, we utilized column chromatography for the separation of coumarins on the basis of CO 2 supercritical extract.
1) Extract: We crushed the herbs of Radix Angelicae pubescentis. We take 1kg of crushed herbs between the 40-mesh screen and the 60-mesh screen, and then put them into the extraction tank. According to the CO 2 flow rate of 1.5 ml/min, extraction time of 20 minutes, pressure of 20 Mpa, temperature of 40℃, the operation was carried out to obtain the independent active supercritical extract.
2) Separation: Add 87g of supercritical extract of Radix Angelicae pubescentis into 100~200 mesh silica gel and stir them evenly. It was used for elution of mixture of petroleum ether and ethyl acetate. One stream fraction is collected every 500ml, and then solvent was recovered. After thin layer detection, flow fractions of similar composition were merged. a) 25 to 30 flow fractions were placed stationary after they were combined. After  f) 68 to 71 flow fractions were placed stationary after they were combined. After solvent was dried, the mixture of petroleum ether and acetone was recrystallized to give a white needle-like crystal. It was defined as compound Ⅲ .
The process of separation is showed in Figure 2.

RESULTS & DISCUSSION
In this experiment, we used CO 2 supercritical extraction technology to get the extract of monosacrum. We systematically separated the extracts and thus obtained eight compounds. We identified six of them by physicochemical properties and spectral data analysis. They includs five furan coumarins and one simple coumarins. They are Osthol, Columbianetin, Columbianetin Acetate, Xanthotoxin, Bergapten. The structures of the compounds were showed in Figure 3.  The physical constants and spectral data were consistent with those reported Osthol [4] . 1  The physical constants and spectral data were consistent with those reported Columbianedin [5] . 1 HNMR and 13 CNMR spectral data of Columbianedin were showed in Table 2.  The physical constants and spectral data were consistent with those reported Columbianetin [6] . 1 143.9, 128.8, 113.3, 112.9, 112.1, 106.6, 88.6, 82.0,  27.5, 22.2, 21.8, 20.8. The physical constants and spectral data were consistent with those reported Columbianetin Acetate [7] . 1 HNMR and 13 CNMR spectral data of Columbianetin Acetate were showed in Table 4.  The physical constants and spectral data were consistent with those reported Xanthotoxin [7] . 1 HNMR and 13 CNMR spectral data of Xanthotoxin were showed in Table 5. The physical constants and spectral data were consistent with those reported Bergapten [7] . 1 HNMR and 13 CNMR spectral data of Bergapten were showed in Table 6.

CONCLUSIONS
A systematic chemical separation of Radix Angelicae pubescentis CO 2 supercritical extracts was carried out, eight compounds were obtained, and their chemical composition was preliminarily studied. Six of them were identified by various chromatographic methods combined with spectral data. They are Osthol, Columbianedin, Columbianetin, Columbianetin Acetate, Xanthotoxin, Bergapten. Xanthotoxin and Bergapten were isolated,which structure were similar. The standard substance of Osthol and Columbianedin were producted in order to offer the good material base for quality evaluation and pharmacodynamic study of Radix Angelicae pubescentis. Their purification were more than 99%. The results of this study are of great significance for exploring the effective material basis and medicinal value of the supercritical extract of Radix Angelicae pubescentis.