HPLC For Simultaneous Determination Of Four Phenylethanol Glycoside Components in Cistanche Herba Decoction Pieces And Cistanche Tubulosa Extracts

Abstract: The objective of this study is to establish a high-performance liquid chromatography (HPLC) method for the simultaneous determination of four phenylethanol glycosides in Cistanches Herba decoction pieces and Cistanche tubulosa extracts. The chromatographic conditions employed an Inertsil ODS-3 column (250 mm × 4.6 mm, 5 µm), with a mobile phase consisting of acetonitrile - 0.1% formic acid solution using gradient elution. The flow rate was set at 1.0 mL/min, the column temperature was maintained at 30°C, and the detection wavelength was 330 nm with an injection volume of 10 µL. The linearity of the peak area with concentrations ranged from 2.53 to 201.60 µg/mL for different glycosides, with correlation coefficients (r) greater than 0.9993. The limits of detection (LOD) and quantification (LOQ) were between 0.2249 to 1.1004 µg/mL and 0.3101 to 0.8161 µg/mL, respectively. The method demonstrated good precision, stability, and repeatability with RSD values less than 3.0%. The average recovery rates ranged from 97.70% to 100.95%, indicating the method's accuracy and effectiveness for the simultaneous quantification of these components in the samples.

Keywords: High-performance liquid chromatography, Cistanches Herba, decoction pieces, Cistanche tubulosa extracts, phenylethanol glycosides, content determination.

HETIAN DICHEN MICROBIAL LIMIT ROOM

Cistanche, also known as Rou Cong Rong and Desert Ginseng, belongs to the family Orobanchaceae and is used in traditional medicine for its fleshy stems covered with scales. It parasitizes the roots of Chenopodiaceae plants such as Haloxylon and Tamarix, and is primarily found in arid regions of Inner Mongolia, Qinghai, Xinjiang, and Ningxia in China. It was first listed in the classic herbal text "Shennong Bencao Jing" as a top-grade herb. Cistanche tastes sweet and salty, is warm in nature, and is associated with the kidney and large intestine meridians. It is known for its benefits in strengthening the kidneys and yang, moisturizing the intestines and relieving constipation, protecting the liver, anti-aging, combating fatigue, and enhancing immunity. Clinically, it is often used to treat conditions such as kidney yang deficiency, blood deficiency constipation, infertility in women, weak knees and back, and memory decline. Cistanche is revered as the "Ginseng of the Desert" and a "precious medicine."

The plant contains phenylethanol glycosides, iridoid terpenes, alkaloids, lignans, and other components, with echinacoside and acteoside being representative phenylethanol glycosides known for their effects in enhancing yang, anti-aging, antioxidant properties, and memory enhancement. These components are the primary basis for the medicinal effects of Cistanche and are also key indicators for quality evaluation and control in the 2020 edition of the Chinese Pharmacopoeia.

There have been reports on the determination of phenylethanol glycoside content in Cistanche species using high-performance liquid chromatography (HPLC), but these have been limited to raw Cistanche materials and slices. Currently, there are no national standards for Cistanche tubulosa extract, and its content determination has not been reported. Our preliminary research found that Cistanche tubulosa from Xinjiang mainly contains four representative phenylethanol glycosides: echinacoside, isoacteoside, acteoside, and tubuloside A. This study established an HPLC method for the simultaneous determination of these four components in both Cistanche decoction pieces and Cistanche tubulosa extracts, aiming to better evaluate the quality of Cistanche decoction pieces and to help establish quality standards for its extracts. The report is as follows.

1 Instruments and Reagents

1.1 Instruments

• LC-20AT high-performance liquid chromatograph, including a diode array detector (Shimadzu, Japan)

• AB135-S electronic balance (Mettler Toledo, Switzerland)

• KQ-500DE ultrasonic cleaner (Kunshan Ultrasonic Instruments Co., Ltd.)

• Aike ultra-pure water system (Chengdu Tangshi Corning Science and Technology Development Co., Ltd.)

1.2 Reagents

• Standard substances of Pinoresinol diglucoside (Batch number S-003-01904003), Isomucronulatol 7-O-glucoside (Batch number Y-073-11803024), Mucronulatol 7-O-glucoside (Batch number M-011-02103019), and Tubuloside A (Batch number G-066-11812016), all purchased from Beijing Inlay Technology Development Co., Ltd., with a purity of over 98%.

• Acetonitrile and methanol of chromatographic grade, formic acid of analytical grade, water was ultra-pure.

• Four batches of Cistanches Herba decoction pieces (code S1, S2 from Inner Mongolia; code S3, S4 from Xinjiang) were all purchased from four pharmacies in Urumqi.

• Cistanche tubulosa extracts (code S5, enzyme-inactivated dry slice water extraction; code S6, enzyme-inactivated dry slice alcohol extraction; code S7, enzyme-inactivated dry slice alcohol resin-bound extraction) were all purchased from Xinjiang Hetian Dichen Pharmaceutical Biotechnology Co., Ltd.

2 Methods and Results

2.1 Chromatographic Conditions

• Chromatographic column: Inertsil ODS-3 column (250 mm × 4.6 mm, 5 µm)

• Mobile phase: Acetonitrile (A) - 0.1% formic acid aqueous solution (B) with a gradient elution (from 17% A to 20% A over 0-12 min, 20% A to 22% A over 12-30 min, 22% A to 17% A over 30-35 min)

• Flow rate: 1.0 mL/min

• Detection wavelength: 330 nm

• Column temperature: 30°C

• Injection volume: 10 µL

  

Hetia Dichen Physico-chemical Laboratory

2.2 Solution Preparation

• Mixed standard solution: Accurately weigh each of the four standards, and transfer into separate 10 mL volumetric flasks, make up to volume with 50% methanol to obtain single standard solutions with concentrations of 1.008, 1.011, 1.012, and 1.012 mg/L for Pinoresinol diglucoside, Tubuloside A, Mucronulatol 7-O-glucoside, and Isomucronulatol 7-O-glucoside, respectively. Measure an appropriate amount of each, combine in a 10 mL volumetric flask, bring to volume with 50% methanol, mix well to prepare a mixed standard solution with concentrations of 201.60, 50.55, 101.20, and 50.60 µg/mL, respectively, and filter through a 0.45 µm membrane filter.

• Sample solution: Approximately 1.0 g of Cistanches Herba decoction piece powder (passed through a No. 4 sieve) is accurately weighed and transferred into a 100 mL amber volumetric flask. Add precisely 50 mL of 50% methanol, weigh, ultrasonically extract for 40 minutes with a power of 250 W and frequency of 35 kHz, cool, reweigh, and make up the lost mass with 50% methanol. Mix well, filter, and collect the filtrate. Pass through a 0.45 µm membrane filter to obtain the Cistanches Herba sample solution. Similarly, prepare the Cistanche tubulosa extract sample solution using approximately 0.2 g of the extract powder.

1. Echinacoside 2. Tubuloside A 3. Acteoside 4. Isoacteoside A. Mixed reference solution B. Test solution of Cistanches Herba decoction pieces C. Test solution of Cistanche Tubulosa extract

2.3 Methodological Examination

System Suitability Test: Appropriate volumes of three solutions from section 2.2 were sampled under the chromatographic conditions outlined in section 2.1, and chromatograms were recorded. The results indicated that the theoretical plate number for the echinacoside peak should be no less than 3000, with separation degrees greater than 1.5, indicating good baseline separation. See Figure 1 for details.

Linearity Evaluation: Precise volumes of the mixed reference solution from section 2.2 (0.5, 1.0, 2.5, 5.0, and 10.0 mL) were transferred into 10 mL volumetric flasks, brought to volume with 50% methanol, mixed well, and used to create a series of mixed reference solutions. Precisely 10 µL of these solutions were injected under the chromatographic conditions of section 2.1. Peak areas were recorded, and linear regression was performed using the concentrations of the components (X, µg/mL) as the independent variable and peak areas (Y) as the dependent variable. The regression equations and the linearity range can be found in Table 1.

Limits of Detection and Quantitation: Precise volumes of the mixed reference solution from section 2.2 were taken, diluted accordingly, and analyzed under the chromatographic conditions of section 2.1. Concentrations corresponding to signal-to-noise ratios of 3:1 and 10:1 were used as the detection and quantitation limits, respectively. Results are presented in Table 1.

Precision Test: An appropriate volume of the mixed reference solution from section 2.2 was analyzed six consecutive times under the chromatographic conditions of section 2.1. The RSD values for the peak areas of echinacoside, tubuloside A, acteoside, and isoacteoside were 1.31%, 0.91%, 1.58%, and 0.58%, respectively (n = 6), indicating good precision of the instrument.

Stability Test: Test sample solutions were prepared and left at room temperature for 0, 2, 4, 6, 8, 12, and 24 hours before being analyzed under the conditions of section 2.1. The RSD values for the peak areas of echinacoside, tubuloside A, acteoside, and isoacteoside were 1.84%, 2.18%, 1.05%, and 1.55%, respectively (n = 7), indicating that the test solutions were stable at room temperature for up to 24 hours.

Repeatability Test: A sample (ID: S2) was accurately weighed and divided into six portions. Each portion was prepared as a test sample solution according to the method in section 2.2 and analyzed under the chromatographic conditions of section 2.1. The mean contents of echinacoside, tubuloside A, acteoside, and isoacteoside were 0.779%, 0.020%, 0.155%, and 0.074%, respectively, with RSD values of 1.21%, 3.12%, 2.65%, and 2.96% (n = 6), demonstrating good repeatability of the method.

Recovery Test: A known quantity of sample (ID: S2) was taken and divided into nine portions. Each portion was spiked with low, medium, or high concentrations of the individual reference solutions, prepared according to the method in section 2.2, and analyzed under the chromatographic conditions of section 2.1. Peak areas were recorded, and recovery rates were calculated. Results are shown in Table 2.

Hetian DiChen HPLC Laboratory

2.4 Sample Content Determination

Appropriate volumes of each batch of samples were prepared as test solutions according to the method in section 2.2, then analyzed under the chromatographic conditions of section 2.1. Each sample was tested in triplicate, peak areas were recorded, and the content of the samples was calculated. Results are shown in Table 3.

3 Discussion

In this study, a diode array detector was employed, and scans of the mixed reference solutions and test sample solutions were performed within the wavelength range of 200 to 450 nm. The results showed that echinacoside and acteoside exhibited maximum absorption at 330 nm, tubuloside A at 331 nm, and isoacteoside at 327 nm. Additionally, referencing the 2020 edition of the Chinese Pharmacopoeia, which specifies a detection wavelength of 330 nm for echinacoside and acteoside, it was observed that many components had significant chromatographic peaks and high response values at 330 nm, leading to the selection of 330 nm as the final measurement wavelength.

From the results of the content determination of Cistanches Herba decoction pieces, it was found that echinacoside had the highest content in both Cistanche tubulosa and desert Cistanche, followed by acteoside, while tubuloside A and isoacteoside had relatively lower contents. There was a significant variability in the content of the four phenylethanol glycoside components, and this variability was also pronounced between different batches from the same location or different locations. This variation can be attributed to factors such as the geographical environment, temperature, climate, and harvesting processes of the raw materials, which affect the accumulation of chemical constituents in the plants. While the chemical components of Cistanche sourced from different origins are similar, the concentrations of these components vary, leading to differences in their effects. Therefore, it is appropriate to select raw materials based on their efficacy to achieve better clinical outcomes.

In summary, the method established in this study is simple, rapid, accurate, and effective. It can be used to simultaneously determine four phenylethanol glycoside components in Cistanches Herba decoction pieces and Cistanche tubulosa extracts. The results of the content determination can reflect the quality levels of different batches and different extracts from the same or different locations, providing a theoretical basis for establishing relevant quality standards.

Tab. 2 Results of the recovery test (n = 9)

Tab. 3 Results of content determination of four phenylethanoid glycosides in the samples(%,n = 3)

Cistanche Benefits

References：

1. 中文 (Chinese): 国家药典委员会．中华人民共和国药典(一部)［M］．北京：中国医药科技出版社,2020：140 - 141.
   英文 (English): National Pharmacopoeia Commission. Pharmacopoeia of the People's Republic of China (Vol. 1) [M]. Beijing: China Medical Science and Technology Press, 2020: 140-141.

2. 中文 (Chinese): 朱乃亮,徐荣,吴海峰,等. 荒漠肉苁蓉和管花肉苁蓉指纹图谱比较研究［J］. 中国药学杂志,2016,51(13)：1116-1119.
   英文 (English): Zhu Nailiang, Xu Rong, Wu Haifeng, et al. Comparative study on the fingerprints of Cistanche deserticola and Cistanche tubulosa [J]. Chinese Pharmaceutical Journal, 2016, 51(13): 1116-1119.

3. 中文 (Chinese): 闫暾,齐海平,李旻辉,等. 肉苁蓉和管花肉苁蓉的对比［J］. 包头医学院学报,2019,35(1)：120-121.
   英文 (English): Yan Tun, Qi Haiping, Li Minhui, et al. Comparison of Cistanche deserticola and Cistanche tubulosa [J]. Journal of Baotou Medical College, 2019, 35(1): 120-121.

4. 中文 (Chinese): 吴波,顾少菊,傅玉梅,等. 肉苁蓉和管花肉苁蓉通便与补肾壮阳药理作用的研究［J］. 中医药学刊,2003,21(4)：539-548.
   英文 (English): Wu Bo, Gu Shaoju, Fu Yumei, et al. Study on the laxative and tonifying kidney-yang effects of Cistanche deserticola and Cistanche tubulosa [J]. Journal of Chinese Medicine and Pharmacy, 2003, 21(4): 539-548.

5. 中文 (Chinese): 范亚楠,黄玉秋,贾天柱,等. 肉苁蓉炮制前后对衰老模型大鼠抗衰老及免疫功能的影响［J］. 中华中医药学刊,2017,35(11)：2882-2885.
   英文 (English): Fan Yanan, Huang Yuqiu, Jia Tianzhu, et al. Effects of Cistanche deserticola before and after processing on anti-aging and immune function in aging model rats [J]. Chinese Journal of Traditional Chinese Medicine and Pharmacy, 2017, 35(11): 2882-2885.

6. 中文 (Chinese): 吴波,付玉梅. 肉苁蓉总苷对亚急性衰老小鼠抗脂质过氧化作用的研究［J］. 中国药理学通报,2005,21(5)：639.
   英文 (English): Wu Bo, Fu Yumei. Study on the anti-lipid peroxidation effect of Cistanche glycosides on subacute aging mice [J]. Chinese Pharmacological Bulletin, 2005, 21(5): 639.

7. 中文 (Chinese): 胡余明,胡怡秀,刘秀英,等. 苁蓉总苷对正常小鼠学习记忆功能的影响研究［J］. 中国预防医学杂志,2007,8(4)：370-373.
   英文 (English): Hu Yuming, Hu Yixiu, Liu Xiuying, et al. Study on the effect of Cistanche glycosides on learning and memory function in normal mice [J]. Chinese Journal of Preventive Medicine, 2007, 8(4): 370-373.

8. 中文 (Chinese): 屠鹏飞,姜勇,郭玉海,等. 发展肉苁蓉生态产业推进西部荒漠地区生态文明［J］. 中国现代中药,2015,17(4)：297-301.
   英文 (English): Tu Pengfei, Jiang Yong, Guo Yuhai, et al. Developing Cistanche deserticola ecological industry to promote ecological civilization in western desert areas [J]. Modern Chinese Medicine, 2015, 17(4): 297-301.

9. 中文 (Chinese): 覃文婷,宿美凤,雒晓梅,等. 定性分析不同产地荒漠肉苁蓉苯乙醇苷类及多糖类成分［J］. 辽宁中医药大学学报,2018,20(7)：77-81.
   英文 (English): Qin Wenting, Su Meifeng, Luo Xiaomei, et al. Qualitative analysis of phenylethanoid glycosides and polysaccharides in Cistanche deserticola from different origins [J]. Journal of Liaoning University of Traditional Chinese Medicine, 2018, 20(7): 77-81.

10. 中文 (Chinese): 王力伟,曹瑞,房永雨,等. 超高效液相色谱-串联三重四极杆质谱联用法测定肉苁蓉中有效成分的含量［J］. 中药材,2017,40(2)：295-300.
    英文 (English): Wang Liwei, Cao Rui, Fang Yongyu, et al. Determination of active components in Cistanche deserticola by ultra performance liquid chromatography-tandem triple quadrupole mass spectrometry [J]. Journal of Chinese Medicinal Materials, 2017, 40(2): 295-300.

11. 中文 (Chinese): 初侨,席兴军,杨丽. 肉苁蓉中松果菊苷和毛蕊花糖苷闪提工艺研究［J］. 食品研究与开发,2015,36(16)：49-52.
    英文 (English): Chu Qiao, Xi Xingjun, Yang Li. Study on flash extraction technology of echinacoside and verbascoside in Cistanche deserticola [J]. Food Research and Development, 2015, 36(16): 49-52.

12. 中文 (Chinese): 余逸凡. 肉苁蓉属植物化学成分及药理活性研究进展［J］. 化工管理,2019(13)：111-112.
    英文 (English): Yu Yifan. Research progress on chemical constituents and pharmacological activities of Cistanche [J]. Chemical Management, 2019(13): 111-112.

13. 中文 (Chinese): 王小新,骆婷婷. 肉苁蓉对小鼠抗疲劳及记忆力的影响［J］. 内蒙古中医药,2014,33(22)：102–103.
    英文 (English): Wang Xiaoxin, Luo Tingting. Effect of Cistanche deserticola on anti-fatigue and memory in mice [J]. Inner Mongolia Journal of Traditional Chinese Medicine, 2014, 33(22): 102-103.

14. 中文 (Chinese): 随家宁,李芳婵,郭勇秀,等. 肉苁蓉化学成分,药理作用研究进展及其质量标志物预测分析［J］. 辽宁中医药大学学报,2021,23(1)：191-196.
    英文 (English): Sui Jianing, Li Fangchan, Guo Yongxiu, et al. Research progress on chemical constituents and pharmacological effects of Cistanche deserticola and prediction analysis of its quality markers [J]. Journal of Liaoning University of Traditional Chinese Medicine, 2021, 23(1): 191-196.

15. 中文 (Chinese): 刘涛,殷松娜,栾昕. 肉苁蓉对卵巢早衰大鼠免疫因子和凋亡相关蛋白的影响［J］. 中国临床药理学杂志,2019,35(23)：3084-3087.
    英文 (English): Liu Tao, Yin Songna, Luan Xin. Effect of Cistanche deserticola on immune factors and apoptosis-related proteins in rats with premature ovarian failure [J]. Chinese Journal of Clinical Pharmacology, 20