Methods for the quality control of Cistanche deserticola

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Shi Ziyi, Wu Yun, Zhu Yuemei & et al.

The traditional Chinese medicine Cistanche is the succulent stem of the Cistanche deserticola Y.C. Ma or Cisanche tubulosa (Schenk) Wight with dry scaly leaves. It has the functions of tonifying kidney yang, nourishing essence and blood, moistening intestines and laxatives, etc. It is mostly used for deficiency of kidney yang, deficiency of essence and blood, impotence and infertility, soreness and weakness of waist and knees, weakness of muscles and bones, intestinal dryness and constipation. Modern pharmacological research shows that Cistanche has many functions such as improving sexual function, resisting Alzheimer's disease and Parkinson's disease, improving learning and memory ability, anti-ageing, anti-fatigue, protecting the liver, and laxative [4]. A large number of studies have confirmed that the phenylethanoid glycosides in Cistanche deserticola have obvious effects on improving learning and memory ability, resisting Alzheimer's disease and Parkinson's disease (4, oligosaccharides and polyols have the effect of laxative and laxative 64 ). The "Pharmacopoeia of the People's Republic of China" only measures the content of phenylethanoid glycosides echinacoside and verbascoside in Cistanche deserticola, but does not measure the active components of its laxative effect, so it is difficult to achieve a comprehensive analysis of the medicinal materials of Cistanche deserticola. Quality control. In order to overcome this limitation and establish a multi-index quality evaluation system related to the main functions and indications of Cistanche deserticola, this study used HPLCELSDP4 to establish a quantitative analysis method for betaine, mannitol, fructose, glucose and sucrose in the medicinal materials of Cistanche deserticola. In addition, 58 batches of Cistanche deserticola medicinal materials from different origins and 10 batches of medicinal materials of Cistanche in the Hotan area of Xinjiang were tested, which can provide a reference for the comprehensive quality control and evaluation of medicinal materials of Cistanche deserticola in the future.

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1 Instrument and reagents

1.1 Instruments

Sartorius electronic balance BT25 S, BS 124 S (Beijing Sartorius Balance Co., Ltd.); Agilent 1100 High-Performance Liquid Chromatography; ALLTECH 2000ES Evaporative Light Scattering Detector (EISD); Ultrasonic Cleaner KQ 250 (Kunshan Ultrasonic Instrument Ltd). 1.2 Reagents

Control substance: betaine (batch: 110894-201604, purity: 99.2%), mannitol (batch: 10053-201304, purity: 99.0%), D-fructose (batch: 111504-201703, purity: 99.8%), D - Anhydrous glucose (batch number: 110833-201506, purity: 99.9%), sucrose (batch number: 111507-201303, purity: 99.8%), all purchased from China National Institute for Food and Drug Control.

The medicinal materials of Cistanche deserticola were collected in Inner Mongolia, Xinjiang, Gansu and other places, with a total of 58 batches. They were identified by Professor Tu Pengfei of Peking University School of Pharmacy as the dried fleshy stems of Cistanche deserticola Y.C.Ma of the Lidangaceae plant, as shown in Table 3.

The medicinal materials of Cistanche deserticola were collected in the Hotan area, Xinjiang, in a total of 10 batches. They were identified by Professor Tu Pengfei from the School of Pharmacy of Peking University as the dried fleshy stems with scaly leaves of Cistanche tubulosa (Schenk) Wight of the Liedangaceae plant, as shown in Table 4.

1.3 Reagents

Acetonitrile is chromatographic pure (Tianjin Boshiqi Technology Development Co., Ltd.), methanol is chromatographic (Tianjin Saifu Century Technology Development Co., Ltd.), and water is ultrapure water.

2 Methods and results

2.1 Chromatographic conditions

The chromatographic column was a ShodexAshaipak NH2P-50 4E polymer gel column (250 mm × 4.6 mm, 5 μm); the mobile phase was acetonitrile-water (77∶23); the flow rate was 0.7 mL·min-1; the column temperature was 25 °C; Light scattering detector (ELSD): The drift tube temperature was 100 °C, and the carrier gas flow rate was 3 L·min-1.

2.2 Preparation of reference solution

Take an appropriate amount of betaine, mannitol, fructose, glucose, and sucrose reference substance, accurately weigh it, add water to make a mixed solution containing 0.25 mg of betaine, mannitol, fructose, glucose, and sucrose per 1 mL, shake well, and get.

2.3 Preparation of the test solution

Take about 1.0 g of Cistanche medicinal powder (passed through a No. 4 sieve), accurately weigh it, put it in a 100 mL brown volumetric flask, add 50 mL of 50% methanol precisely, plug it tightly, shake it up, weigh it, soak it for 30 min, and then ultrasonically (power 250 W, frequency 35 kHz) for 40 min, take it out, let it cool, weigh it again, add 50% methanol to reduce the mass, shake well, and let it stand. Precisely aspirate 5 mL of supernatant, put it in a 25 mL volumetric flask, dilute to the mark with 50% methanol, shake well, and filter through a 0.2 μm microporous membrane.

2.4 Determination method

Precisely draw 5 and 10 μL of the reference solution and 5 μL of the test solution respectively, inject them into the liquid chromatograph, record the peak area, and calculate according to the logarithmic equation of the external standard two-point method.

2.5 System suitability test

According to the chromatographic conditions under item 2.1, accurately draw 5 μ of the reference solution and the test solution, respectively, and inject them into the high-performance liquid chromatography, measure and record the chromatogram, as shown in Figure 1. The chromatographic peaks of the test sample showed the same retention time as the reference substance chromatographic peaks, the resolution was good, and the chromatographic conditions were feasible.

Note: A. Control substance; B. Cistanche No. 49 sample; 1. betaime ; 2. fnuckese ; 3. mannibol ; 4. glucose ; 5. sucreve

Fig. 1 HPLC profiles of the reference substance and the sample of Cistanche No. 49

2.6 Examination of Linear Relationships

Take appropriate amounts of betaine, mannitol, fructose, glucose, and sucrose as reference substances, accurately weigh them, and add water to make each 1 mL containing betaine 0.496 0 mg, mannitol 0.994 0 mg, fruit 1.0126 mg, glucose 1.0882 mg, and sucrose 0.9922 mg. 0.0992, 0.2480, 0.4960, 0.9920, 1.9840, 2.9760 μ, mannitol 0.1988, 0.4970, 0.9940, 1.9880, 3.9760, 5.9640 μ, fructose 0.5063, 1.012 6. 2.025 2. 4.050 4. 5.063 0. Chromatographic conditions were injected and detected, and the peak area was recorded; then the logarithm value X (μg) of each reference substance injection amount was used as the abscissa, and the logarithm value Y of the peak area was the ordinate, and the standard curve was drawn. The results are shown in Table 1.

Table 1 The results of the linear relationship investigation

2.7 Precision test

Accurately draw the test solution (sample No. 49), under the above chromatographic conditions, continuously inject 6 times, measure the peak area of each chromatographic peak, and calculate its RSD value. Results The RSDs of the peak areas of betaine, mannitol, fructose, glucose and sucrose were 0.84%, 2.56%, 1.27%, 2.62% and 1.52%, respectively, indicating that the precision of the instrument used in this study was good. 2.8 Repeatability test

Take 6 samples of Cistanche deserticola (sample No. 49), prepare the test solution according to the method under item 2.3, measure the peak area of each chromatographic peak, and calculate the corresponding content. Results The average contents of betaine, mannitol, fructose, glucose and sucrose (n=6) were 7.93%, 7.27%, 8.74%, 2.75% and 4.81%, and the RSDs were 2.83%, 1.94%, 0.97% and 2.60, respectively. %, 1.40%, indicating that the method has good repeatability. 2.9 Stability test

Take the test solution (sample No. 49), inject it at 0, 4, 8, 12, 16, and 24 h after preparation for measurement, and calculate the RSD value of each chromatographic peak. Results The RSDs of betaine, mannitol, fructose, glucose and sucrose were 0.61%, 1.92%, 1.34%, 2.83% and 0.69%, respectively, indicating that the test solution had good stability within 24 hours. 2.10 Recovery test

Take 6 samples of Cistanche deserticola with known content (sample No. 49), accurately weigh 0.25g, put them in a 50mL brown volumetric flask, and accurately draw 25mL of the mixed reference solution at a concentration of 1:1 (which contains 0.7900 mg of betaine). mL-, fructose 0.7236 mg"mL-1, mannitol 0.8812 mg·mL-1, glucose 0.2708 mg·mL-1, sucrose 0.4883 mg'mL-') were added to the sample, and the test solution was prepared according to the method under 2.3 Detected according to the above-mentioned chromatographic conditions, calculated its recovery, the results are shown in Table 2, indicating that the method has good accuracy.

Table 2: Measurement results of the sample recovery rate of 5 components to be tested (n=6)

3 Discussion

3.1 Selection of index components

The total oligosaccharides of Cistanche are the effective parts of Cistanche for laxatives. The author analyzed the chemical composition of this part by LC-MS and the reference substance, and determined that this part is mainly composed of betaine, mannitol, fructose, glucose and sucrose. Therefore, These 5 components were selected as the index components of Cistanche deserticola, and the content determination method was established.

3.2 Selection of chromatographic columns

The five components to be tested, betaine, mannitol, fructose, glucose and sucrose, are relatively polar and cannot be retained in the reversed-phase Cn column. Generally, peaks appear within 1-2 minutes, and the components cannot be separated. Amino columns Waters Bonoapar NH, (300 mm × 4.6 mm, 10 μm) and Phenomenex Bondclone 10 NH, (300 mm × 3.9 mm, 10 μm) for sugar and polyol separation, and polymer-based amino columns Prevail Carbohydrate ES ( 250 mm×4.6 mm, 5 μm), ShodexAshaipak NH2P-50 4E (250 mm×4.6 mm, 5 μm). The results show that the polymer matrix amino column can effectively separate these five components.

3.3 Detector selection

The five components to be tested have no obvious UV absorption, and the content of the UV detector cannot be directly determined. Therefore, this paper established a quantitative analysis method for the sugar alcohol components of Cistanche deserticola using HPLC-EISD. The results show that the method has high sensitivity, good repeatability and high accuracy, and can be used for the quality control of Cistanche deserticola and other medicinal materials containing betaine and sugar alcohols.

3.4 Investigation of the preparation method of the test solution

Different extraction solvents including water, 30% methanol, 50% methanol, 70% methanol, methanol, etc. were compared respectively. The results showed that except for the slightly lower extraction rate of 70% methanol and methanol solvent, there was no significant difference in other solvents, so the direct selection was made. 50% methanol was used as the solvent under the item "Determination of Content" of Cistanche in the Pharmacopoeia of the People's Republic of China, and it was prepared according to the preparation method of the relevant test solution, and then diluted 5 times to determine the sugar alcohol components.

3.5 Detection and analysis of samples with different bases

In this study, the contents of 5 components in 58 batches of Cistanche deserticola and 10 batches of Cistanche deserticola medicinal materials were determined. The mass fractions of betaine, mannitol, fructose, glucose and sucrose in 58 batches of Cistanche deserticola samples were 2.73%-17.65%, 2.46%-16.34%, 2.52%-22.74%, 0.97%-14.06%, 3.15%-27.16 %; Mannitol, fructose and glucose were 1.00%-13.04%, 2.98%-8.85%, 1.09%-5.33% in the 10 batches of Cistanche samples, respectively, and betaine and sucrose were not detected. The contents of medicinal materials differ greatly among batches, and there is no rule to follow, suggesting the necessity of establishing the content determination standard to control the quality of medicinal materials. In addition, this method can effectively distinguish two kinds of Cistanche base plants, namely, Cistanche does not contain betaine and sucrose, while the content of these two components is higher in Cistanche deserticola. This also provides a basis for analyzing the differences in texture and habitat of the two medicinal materials of Cistanche deserticola. Compared with the medicinal material of Cistanche deserticola, the texture is softer, and it is locally known as "Soft Dayun", while Cistanche is called "Chai Dayun" because it contains sucrose (3.15%~27.16%). ). Cistanche deserticola is mainly distributed in Alxa in Inner Mongolia, while Cistanche is mainly distributed in Hotan area of Xinjiang, where the temperature is higher than that in Alxa area, and drip irrigation is often used in the local area, but once the temperature drops, Cistanche will suffer serious damage. Freezing damage, on the one hand, has a certain relationship with its high water content, and on the other hand, it has something to do with the ingredients it contains. Betaine 243 is an important osmotic adjustment substance, which plays an important role in enhancing the stress resistance of plants, such as resistance to salinity, drought, low temperature, and strong light. It is precisely because of the high content of betaine contained in Cistanche deserticola that it has better resistance to salt-alkali, drought and freezing damage than Cistanche, and has a relatively wide distribution.

3.6 Analysis of Cistanche deserticola samples from different origins

Among the 58 batches of desert cistanche medicinal material samples, 45 batches were produced from Alxa League, Inner Mongolia, 3 batches were from Wangyedi (Dengkou County), Inner Mongolia, and 1 batch was unknown. All 49 batches of medicinal materials had different contents of 5 components. However, there is no significant difference. The reason for the analysis may be that the medicinal materials are all produced in the same desert area, the ecology and geographical environment are similar, and the product quality is relatively uniform and stable. There are 3 batches produced in Minqin County, Gansu, and 6 batches from Xinjiang. According to the analysis in Figure 2, compared with the medicinal materials produced in Inner Mongolia, the main difference is reflected in the content of fructose and sucrose. The sucrose content of the medicinal materials was relatively highest, but given the low amount of samples from the two origins, this conclusion needs to be verified by collecting more samples. 3.7 Summary

In this study, HPLC Wang ISD was established for the simultaneous determination of 5 active ingredients of laxatives, which provided a more convenient, efficient and accurate method for the quality control of Cistanche deserticola, and provided a basis for improving the quality standard and further development of Cistanche deserticola. meaningful reference.

References

【1】 State Pharmacopoeia Commission. Pharmacopoeia of the People's Republic of China: A 【M】. Beijing: China Pharmaceutical Science and Technology Press, 2015;135.

【2】 Song Zhihong, Lei Li, Tu Pengfei. Research progress on the pharmacological activity of the Cistanche Exhibition. Chinese Herbal Medicine, 2003, 34(9); 113415.

【3】 Xue Haiyan. Jiao Danyuan, Yao Jun. Research status of the pharmacological effects of total glucosides in Cilantro [J]. Chinese Journal of Clinical Pharmacology, 2018, 34(4): 486-488.

【4】 An Chunna, Zhang Hongning, Pu Xiaoping. Research progress of neuropharmacology of Cistanche deserticola Exhibition [J]. Chinese Journal of Pharmacy, 2011, 46(12): 887-890.

【5】 Gao Yuhong, Ke Zunji. Research progress on the pharmacological effects of Cistanche deserticola in the treatment of Alzheimer's disease []. Shanghai Journal of Traditional Chinese Medicine, 2018, 52(1): 109-112.

【6】 Zhang Baishun, Zhao Xuewen, Chen Shuanghou, et al. Laxative effect of isolated parts of Cistanche deserticola Experimental Research □. China Journal of Traditional Chinese Medicine, 2003, 10(1I1): 31-32.

【7】 Zhang Baishun, Chen Shuanghou, Zhao Xuewen, et al. Cistanche deserticola extract galactitol A dose-response study on the action of stool [J]. China Journal of Traditional Chinese Medicine, 2003, 10 (12): 28-29.

【8】 Gao Yunjia, Jiang Yong, Dai Fang, et al. Research on the medicinal substances of Cistanche deserticola for laxatives Research [J]. Modern Chinese Medicine in China, 2015, 17(4): 307-311.

【9】 Bao Zhong, Cai Hong, Jiang Yong, et al. Three activities of Cistanche deserticola under different influencing factors Quantitative Analysis of Components J. Chinese Herbal Medicine, 2013, 44 (22): 32233230.

【10】 Bao Zhong, Cai Hong, Jiang Yong, et al. Determination of Cistanche deserticola by HPLCEISD Content of Galactitol.Journal of Pharmaceutical Analysis, 2007, 27(7): 1004-1006.

【11】 Zhao Kuijun, Liang Lijuan, Bi Dan, et al. HPLCELSD determination of Cistanche and tube Content of galactitol in Cistanche [J]. China Journal of Traditional Chinese Medicine, 2012, 19(8): 52-54.

【12】 Wang Guiping, Xue Xiaomin, Lu Chao, et al. The effect of betaine on improving plant stress resistance and its mechanism of action.Jiangxi Agricultural Journal, 2014, 26(8): 2226.

【13】 Wang Qingjie, Wang Li. A review of plant cold resistance genes research. Rural economy and Technology, 2018, 29(8): 23-25.