Determination Of Echinacoside in Water Extract Of Cistanche Herba By HPLC

Abstract： 

Objective An HPLC method was established for the determination of echinacoside in the water extract of Cistanche herba. 

Methods Echinacoside was determined by HPLC. The separation was performed on the Inertsil ODS-SP C18 column with a mobile phase consisting of acetonitrile(A)and 0.4% phosphoric acid solution(B), by gradient elution. The detection wavelength was set at 333 nm, and the column temperature was 30 ℃. The flow rate was 1.0 mL·min-1 and the injection volume was 10 μL. Results The linear relation of echinacoside within the concentration range of 44.939 7-539.276 4 μg ·mL- 1 was good(r = 0.999 8). The recoveries were 94.59%-105.31% (RSD= 2.93%,n = 9). The content of echinacoside in water extracts of Cistanche herba was 15.527,7.347,12.267 mg·g-1. Conclusion The established HPLC method with good repeatability is accurate, simple, and feasible. It is suitable for the determination of echinacoside in the water extract of Cistanche herba.

Keywords： HPLC; water extract of Cistanche herba; echinacoside; determination of content

Cistanche deserticola is a tonic traditional Chinese medicine commonly used in clinical practice and is known as "desert ginseng" [1]. The Cistanche deserticola recorded in the 2015 edition of the Chinese Pharmacopoeia is the dried fleshy stem with scaly leaves of Cistanche deserticola Y. C. Ma or Cistanche tubulosa (Schenk) Wight. It is warm, sweet and salty, and returns to the kidney and large intestine meridians. It has the effects of nourishing kidney yang, replenishing essence and blood, and moisturizing the intestines and laxatives [2]. "Shen Nong's Materia Medica," says it can "tonify the middle, nourish the five internal organs, strengthen yin and nourish essence". Cistanche deserticola mainly contains phenylethanoid glycosides, iridoids and their glycosides, lignans and their glycosides, polysaccharides, ester compounds and alkaloids [3]. Phenylethanol glycosides are the main active ingredients of Cistanche deserticola. Among them, echinacoside and verbascoside are listed in Cistanche deserticola in the Chinese Pharmacopoeia.

Indicative components for identification and content determination. Pharmacological studies [4] show that Cistanche deserticola has various pharmacological effects such as laxative, liver protection, anti-osteoporosis, antioxidant, anti-aging, and anti-fatigue. Cistanche deserticola aqueous extract can promote the expression of bone morphogenetic protein 2 gene in rat osteoblasts, tartrate-resistant acid phosphatase, osteocalcin in the serum of ovariectomized mice, and Smad1, mad5 genes, transforming growth factor-β, etc. in the bone marrow. , has anti-osteoporosis effects [5-6].

Echinacea can promote the proliferation of osteoblasts and promote the differentiation of bone marrow mesenchymal stem cells cultured in vitro into osteoblasts [7-9]. In recent years, there have been many research reports on the content determination of echinacoside and verbascoside in Cistanche deserticola ethanol extract and its compound preparations [10-12], but there are few reports on the content determination of index components in Cistanche deserticola aqueous extract. In this study, high-performance liquid chromatography (HPLC) was used to determine the content of echinaceaside in the aqueous extract of Cistanche deserticola.

1 material

1.1 Instruments

  LC-20AT high-performance liquid chromatography, SPD-20A detector, SIL-20A autosampler, CTO-10ASvp column oven, LC solution liquid chromatography workstation, Shimadzu Corporation of Japan; MES125P-OCE-DU electronic analytical balance, Sartorius Company, Germany; SHZ-D (Ⅲ) circulating water multi-purpose vacuum pump, Henan Yuhua Instrument Co., Ltd.; RC2B SO25 rotary evaporator cooling circulation system, RV 10 D S96 rotary evaporator, IKA Company, Germany; DK-S26

Electric constant temperature water bath, Shanghai Jinghong Experimental Equipment Co., Ltd.; UPR series ultrapure water machine, Sichuan Youpu Ultrapure Technology Co., Ltd.

1.2 Medicinal materials and reagents

Cistanche deserticola medicinal material was purchased from Guangzhou Lingnan Traditional Chinese Medicine Pieces Co., Ltd., batch number: 1801001, 1806001, 181001. Tu Xingming, the chief traditional Chinese pharmacist at the Third Affiliated Hospital of Guangzhou University of Chinese Medicine, identified it as the dried fleshy stem with scaly leaves of Cistanche deserticola Y. C.Ma. The sample is stored in the Drug Testing Room of the Third Affiliated Hospital of Guangzhou University of Chinese Medicine; Echinacea Glycoside reference substance, content 89.7%, China Institute of Food and Drug Control, batch number: 111670-

201706; acetonitrile is chromatographically pure (Merck, Germany); methanol and phosphoric acid are analytically pure (Tianjin Fuyu Fine Chemical Co., Ltd.); water is ultrapure water.

2 Methods and results

2.1 Preparation of water extract of Cistanche deserticola

  Take the coarse powder of Cistanche deserticola medicinal material, add 10 times the amount of distilled water, heat and reflux for extraction twice, 1 hour each time, filter, combine the filtrate, and concentrate the filtrate with a rotary evaporator to a crude drug content of 0.100 g·mL-1, and store it at 4°C for later use.

2.2 Chromatographic conditions and system suitability

Chromatographic column: Inertsil ODS SP C18 column (150 mm × 4.6 mm, 5 μm); mobile phase: acetonitrile (A)-0.4% phosphoric acid solution (B), gradient elution: 0 ~ 5 min, 17%

A; 5 ~ 25 min, 20% A; 25 ~ 30 min, 17% A; detection wavelength: 333 nm; column temperature: 30 ℃; flow rate: 1.0 mL·min-1;

Injection volume: 10 μL. Under these chromatographic conditions, the separation between echinacoside and adjacent component peaks is greater than 1.5, and the number of theoretical plates based on the echinaceaside peak should be no less than 4,000. The chromatogram is shown in Figure 1.

2.3 Preparation of solution

2.3.1 Reference substance solution Weigh an appropriate amount of echinacoside reference substance, weigh it accurately, place it in a 10 mL volumetric flask, add methanol to dissolve it, and prepare a reference substance solution containing 898.794 μg of echinaceaside per ml, 4 ℃ Save for later use.

2.3.2 Test solution

Precisely measure 2.0 mL of Cistanche deserticola aqueous extract, evaporate to dryness in a water bath, add methanol to dissolve, dilute to volume in a 10 mL volumetric flask, shake well, and filter with a 0.22 μm microporous membrane to obtain.

2.4 Investigation of linear relationship

Precisely measure 0.25, 0.50, 1.00, 2.00, 2.50, 3.00 mL of the reference solution under item 2.3.1, place it in a 5 mL volumetric flask, add methanol to make up to volume, and shake well to obtain a gradient concentration of 44.939 7, 89.879 4. 179.758 8, 359.517 6, 449.397 0, 539.276 4 μg·mL-1 reference solution, filtered through 0.22 μm microporous membrane. Measure according to the chromatographic conditions under 2.2, and record the peak area. Perform linear regression with the mass concentration of the reference substance as the abscissa (X, μg·mL-1) and the peak area as the ordinate (Y), and the regression equation is obtained: Y=13 185.0X-32 273.0, r = 0.999 8. The results show that echinacea has a good linear relationship within the concentration range of 44.939 7 ~ 539.276 4 μg·mL-1.

2.5 Precision test

Take 539.276 4 μg ·mL- 1 reference solution, inject and measure 6 times continuously according to the chromatographic conditions under 2.2, and record the peak area. The results showed that the RSD of the echinacoside peak area was 1.81%, indicating good instrument precision.

2.6 Stability test

Precisely measure 2.0 mL of Cistanche deserticola aqueous extract, evaporate to dryness in a water bath, add methanol to dissolve, dilute to a 10 mL volumetric flask, shake well, filter with a 0.22 μm microporous membrane, follow the chromatographic conditions under item 2.2, and place at 0 and 2 respectively. , 4, 8, 12, 24 h injection measurement, record the peak area. The results showed that the RSD of the echinacoside peak area was 1.16%, indicating that the test solution had good stability within 24 hours.

2.7 Repeatability test

Precisely measure 2.0 mL of Cistanche deserticola water extract and prepare 6 test solutions in parallel according to the method under 2.3.2. Inject samples for measurement according to the chromatographic conditions under 2.2 and record the peak area. The results showed that the RSD of the echinacoside mass concentration was 1.28%, indicating good repeatability of the method.

2.8 Sample recovery test

Precisely measure 9 portions of 1.0 mL of Cistanche deserticola water extract, add 0.8, 1.0, and 1.2 mL of reference solution (1.552 mg·mL-1) respectively, evaporate to dryness in a water bath, add methanol to dissolve, and adjust to a 10 mL volumetric flask; Shake well and filter through a 0.22 μm microporous membrane to prepare high, medium and low concentration test solutions. Prepare 3 copies of each concentration in parallel. Measure according to the chromatographic conditions under 2.2. Record the peak area and calculate the sample recovery. Rate. The results are shown in Table 1.

2.9 Determination of sample content

Precisely measure 3 portions of 2.0 mL of Cistanche deserticola aqueous extract, evaporate to dryness in a water bath, add methanol to dissolve, dilute to a 10 mL volumetric flask, shake well, filter with a 0.22 μm microporous membrane, and measure according to the chromatographic conditions under 2.2. Record the peak area and calculate the echinaceaside content. The results are shown in Table 2.

Table 1 Results of recovery tests of echinacoside

Table 2 Results of content determination of echinacoside in water extract of Cistanche herba