Determination,of,β-sitosterol,in,Plumbago,zeylanica,L.,by,HPLC-ELSD

Hongyu ZHAO

Market Supervision Service Center of Dandong City, Dandong 118001, China

Abstract [Objectives] A method for the determination of β-sitosterol in Plumbago zeylanica L. was established and the content of β-sitosterol in different medicinal parts, different producing areas and different harvest periods were compared. [Methods] High performance liquid chromatography (HPLC)-evaporative light scattering detector assay was used. The chromatographic column was Kromasil C18 column (250 mm × 4.6 mm, 5 μm); the mobile phase was pure methanol; the flow rate was 1.0 mL/min; the column temperature was 30 ℃. The detection parameters of evaporative light scattering detector were as follows: drift tube temperature was 40 ℃, carrier gas (N2) pressure was 3.5 bar. [Results] There was a good linear relationship between β-sitosterol (1.08 0-4.86 0 μg) and the natural logarithm of peak area (r=0.999 5). The average recovery rate was 99.80%. The content of β-sitosterol in root and stem was 0.207 4 and 0.406 4 mg/g, respectively, but it was not found in leaves; the content of β-sitosterol in P. zeylanica L. in Guangxi was generally lower than that in Yunnan, and the content of β-sitosterol in P. zeylanica L. in Xishuangbanna was the highest; the content of β-sitosterol in the stem of P. zeylanica L. was stable at a relatively high level in different harvest periods. [Conclusions] The method is simple, accurate and reproducible, and can be used as one of the methods to control the quality of P. zeylanica L.

Key words Plumbago zeylanica L., β-sitosterol, Content, HPLC-ELSD method

PlumbagozeylanicaL. is a plant of the family Plumbaginaceae. The whole grass or root ofP.zeylanicaL. is bitter, slightly warm in nature and poisonous, with the effects of dispelling wind and relieving pain, dispersing stasis and detumescence. It is clinically used for the treatment of skin itching, nameless swelling, injuries from falls, tinea of hands and feet, women’s postpartum bleeding and some malignant diseases. The active constituents are plumbago danquinone, β-sitosterol, vanillic acid, Plumbagic acid and so on[1-2]. It is a common medicinal material for many countries in Southeast Asia and many ethnic groups in China. Modern pharmacological studies have shown thatP.zeylanicaL. extract has the anti-tumor, anti-malaria parasite, antibacterial, anti-allergy, and anti-hyperglycemia effects[3-11]. During a series of studies onP.zeylanicaL., the monomer β-sitosterol was isolated, which had obvious anticancer, antibacterial, antitussive, cholesterol-lowering effects[12]. However, there is no report on the determination of β-sitosterol inP.zeylanicaL. Therefore, the content of β-sitosterol in stems of different medicinal parts, different producing areas and different harvest periods was determined by HPLC-ELSD.

2695 high performance liquid chromatography system (Waters, USA); Sedex-75 evaporative light scattering detector (Sedex, France); reference substance β-sitosterol (self-made, purity of 98.12%); the samples of different medicinal parts were collected from the South Pharmaceutical Garden of Xishuangbanna, Yunnan Province. The samples ofP.zeylanicaL. from different areas can be shown in Section3.2.

The samples of medicinal materials in different harvest periods were collected from West China Specimen Garden of Sichuan University, and all of them were identified as the whole grass or root ofP.zeylanicaL.; methanol was chromatographically pure and other reagents were analytically pure.

3.1 Selection and optimization of experimental conditions

3.1.1Chromatographic conditions. The chromatographic column was C18column (250 mm × 4.6 mm, 5 μm), the mobile phase was pure methanol, the flow rate was 1.0 mL/min, and the column temperature was 30 ℃. The drift tube temperature of the evaporative light scattering detector was 40 ℃ and the carrier gas (N2) pressure was 3.5 bar. Under this chromatographic condition, the peak of β-sitosterol and other components in the sample reached baseline separation, the peak shape was symmetrical, the tR was about 22 min, and there was no interference (Fig.1).

Note: *represents β-sitosterol.Fig.1 HPLC chromatograms of β-sitostro control solution (A), stem solution (B), root solution (C) and leaf solution (D) of Plumbago zeylanica L

3.1.2Preparation of solution. An appropriate amount of β-sitosterol reference substance was precisely weighed and methanol was added to make 0.108 mg/mL reference solution. About 5 g of crude powder ofP.zeylanicaL. was weighed and 100 mL of chloroform was added. After 3-h Soxhlet extraction, the solvent was removed, dissolved with methanol and transferred to a 25 mL flask. The sample solution was obtained by adding methanol to a fixed volume and filtering with 0.45 μm microporous membrane.

3.1.3Preparation of standard curve. 10, 15, 20, 25, 30, 35, 40 and 45 μL of reference solutions were weighted accurately and determined according to the chromatographic conditions in Section3.1.1. The linear regression equation was obtained by taking the natural logarithm of reference substance weight as the abscissa and the natural logarithm of peak area as the ordinate. The regression equation wasY=1.370X+3.556 (r=0.999 5,n=8), and the linear range of sample injection volume was 1.08～4.86 μg.

3.1.4Precision test. After precise absorption of 30 μL of 0.108 mg/mL reference solution and repeated injection for 6 times, theRSDof the chromatographic peak area was determined to be 0.8%. The results showed that the precision was good.

3.1.5Reproducibility test. 5 g of crude powder of medicinal materials from the same place (Guangxi Botanical Garden) was precisely weighed, with a total of 6 samples. The test solution was prepared according to the method of Section3.1.2. It was determined according to the chromatographic conditions in Section3.1.1(RSD=1.50%). The results showed that the reproducibility was good.

3.1.6Stability test. 5 g of crude powder of sample was weighed precisely, and the sample solution was prepared according to the method in Section3.1.2. The samples were injected at 0, 2, 4, 6 and 8 h, respectively, and theRSDof the peak area was measured to be 1.65%. The results showed that the solution of the sample was stable within 8 h.

3.1.7Recovery test. The crude powder of 2.5 g of medicinal materials from the same place (Guangxi Botanical Garden) was precisely weighed, with a total of 9 samples, and 4.80, 9.50, 14.20 mL of reference solutions were precisely added (3 for each sample). The test solution was prepared according to the method in Section3.1.2and determined according to the chromatographic conditions in Section3.1.1. The average recovery rate was 99.8% andRSDwas 1.15%.

3.2 Determination of samplesThe content of β-sitosterol in the sample was determined according to the method in Section3.1.1, and 30 μL of each sample was injected. The peak area external standard method was used to calculate the content. The content of β-sitosterol in root and stem was 0.207 4 and 0.406 4 mg/g, respectively, but it was not found in leaves; the content of β-sitosterol inP.zeylanicaL. from different producing areas and different harvest periods are shown in Table 1-2.

Table 1 The content of β-sitosterol in the stem of Plumbago zeylanica L. from different producing areas (n=3)

Table 2 The content of β-sitosterol in the stem of Plumbago zeylanica L. in different harvest periods (n=3)

The Soxhlet extraction of chloroform and ethanol was investigated. The results showed that the content of β-sitosterol in the sample was the highest when chloroform was used as solvent.

Ultrasonic treatment, heating reflux extraction and Soxhlet extraction were used in the experiment. The results showed that the content of β-sitosterol in the sample was the highest in Soxhlet extraction, so the Soxhlet extraction method was adopted. Different ratios of methanol-water, acetonitrile-water and pure methanol mobile phase systems were used in the experiment[12]. By comparison, pure methanol was selected as the mobile phase, the retention time of β-sitosterol was shortened and the separation effect was better.

For the different producing areas ofP.zeylanicaL., the content of β-sitosterol produced in Yunnan was higher than that in Guangxi, and it was not detected in one of the producing areas in Guangxi. This is consistent with the results of the previous study on the content of plumbagin and vanillic acid, suggesting that the ecological environment of Yunnan is more suitable for the growth ofP.zeylanicaL.

In accordance with the early determination of the content of characteristic components (plumbagin and vanillic acid) ofP.zeylanicaL. in different harvest periods, it was found that the regularity was not very strong in different harvest periods, but the content was stable at a relatively high level. the best harvest time was still in the early flowering period (before September to October).