Liang Qingsong 1,2,4, Pan Yingjie 2,4, Chen Ziying 2,4, Zhang Ying 1,3, Wu Menghua 1,3, Cao Hui 1,3, Ma Zhiguo 1,3*

(1. School of Pharmacy, Jinan University, Guangzhou 510632, Guangdong; 2. Guangdong Yifang Pharmaceutical Co., Ltd., Foshan 528244, Guangdong; 3. Lingnan Traditional Chinese Medicine Research Center, Jinan University, Guangzhou 510632, Guangdong; 4. Key Laboratory of Guangdong Traditional Chinese Medicine Formula Granules Enterprise, Foshan 528244, Guangdong)

Abstract: Objective To prepare cistanche deserticola standard decoction and provide a reliable method for its quality control. Methods According to the preparation requirements of the standard decoction, 14 batches of Cistanche deserticola standard decoction were prepared. The contents of echinaside and piloside in the standard decoction were determined by HPLC, the transfer rate was calculated, the extraction rate and pH value of the decoction were measured, and the UPLC fingerprint of Cistanche deserticola standard decoction was established. Results The average transfer rates of echinaside and piloside in 14 batches of Cistanche deserticola standard decoction were 22.99% and 17.09%, respectively. The extraction rate ranged from 25.1% to 56.6%, and the pH value ranged from 4.30 to 5.15. There are 6 common peaks in the fingerprint, and among them, the No. 1 peak is identified as echinacea glycoside, the No. 3 peak is identified as angiocyanin A, the No. 4 peak is identified as piloside, and the No. 5 peak is identified as isopiloside. The similarity of 14 batches of Cistanche deserticola standard decoction was evaluated, and the similarity ranged from 0.789 to 0.999. Conclusion The preparation of Cistanche deserticola standard decoction is standardized, with good overall homogeneity, controllable quality, good accuracy, stability, and repeatability of the determination method, and high fingerprint similarity, which can provide a reference for the quality control of Cistanche deserticola standard decoction.

Key words: Cistanche deserticola; Standard decoction; Echinacoside; Piloside; Fingerprint; Quality evaluation

"Cistanche deserticola is a dry, scaly, fleshy stem of the genus Cistanche in the Orobaceae or Cistanche tubulosa ma. It has the effects of tonifying kidney yang, benefiting blood essence, moistening the intestines, and relieving constipation. It is widely used in impotence, infertility, waist and knee tenderness, muscle and bone weakness, and intestinal dryness and constipation."

cistanche powder health benefits—tonifying kidney

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It mainly contains phenylethanoid glycosides, benzyl alcohol glycosides, iridoids and their glycosides, lignin and its glycosides, and other compounds. Among them, phenylethanoid glycosides are considered to be the main effective components in Cistanche deserticola ma.

Phenylethanol glycoside is the main active component of Cistanche deserticola

The standard decoction of traditional Chinese medicine slices (i.e., the standard decoction) is a single decoction of traditional Chinese medicine slices prepared through standardized processes, guided by the theory of traditional Chinese medicine and based on clinical application, with reference to modern extraction methods. Due to the fact that the standard decoction has no excipient interference and does not undergo a drying process, it is consistent with traditional decoction for clinical use, and it is easy to test various ideal concentrations through the preparation of slices or extracts. Therefore, the standard decoction of traditional Chinese medicine slices, as the quality standard for classic Ming Fang preparations and the standard reference for measuring traditional Chinese medicine formula particles, is currently an effective form of ensuring the safety, effectiveness, and stability of traditional Chinese medicine in order to achieve consistency in clinical efficacy. Currently, there are no literature reports on the study of standard decoction of Cistanche deserticola decoction pieces. This experiment prepared corresponding standard decoction for 14 batches of Cistanche deserticola, and conducted research on the standard decoction for Cistanche deserticola, with a view to establishing core samples of quality markers for Cistanche deserticola, providing an experimental basis for the development of Cistanche deserticola formula granules.

Cistanche powder

1. Apparatus and Materials

1.1 Instruments and equipment

Waterse2695 High-Performance Liquid Chromatography (Waters); ME204E 1/10000 balance (Mettler Toledo); XP26 millionth scale (Mettler Toledo); WatersAcquire UPLC Ultra High-Performance Liquid Chromatography (WATSCH); KQ-500E Ultrasonic Cleaner (Kunshan Ultrasonic Instrument Co., Ltd.).

1.2 Reagents and materials

Echinacoside reference substance (batch number: S-003-190403), anthocyanin A reference substance (batch number: G-066-181216), Acteoside reference substance (batch number: M-011-181106), and isopiloside reference substance (batch number: Y-073-170325) were all purchased from Chengdu Ruifensi Biotechnology Co., Ltd., with a purity of more than 98.0%. Chromatographic pure formic acid, Tianjin Kemio Chemical Reagent Co., Ltd; Chromatographic pure methanol, Merck Co., Ltd; The experimental water is a laboratory ultrapure water system,Merck Co., Ltd; Other reagents are analytical pure. In this study, a total of 14 batches of prepared pieces of Cistanche deserticola were collected from the market and identified by Professor Ma Zhiguo of Jinan University as the dried, scaly, fleshy stems of the genus Cistanche deserticola or Cistanche tubulosa, a plant of the genus Cistanche in the family Orobaceae. Sample information (see Table 1).

Echinoside, the main chemical component of Cistanche deserticola

2 .Methods and Results

2.1 Preparation of standard decoction and test solution

2.1.1 Preparation of Cistanche deserticola standard decoction:

According to the Regulations on the Management of Traditional Chinese Medicine Decocting Room in Medical Institutions, Technical Requirements for Quality Control and Standard Formulation of Granules of Traditional Chinese Medicine Formulas, and Standard Soup of Traditional Chinese Medicine, take 100g of the prepared slices of Cistanche deserticola, place them in an electric ceramic pot, add water to decoct twice, add 9 times the water for the first time, soak for 30min, heat and boil with Wuhuo (500W), keep them slightly boiling under mild heat (200W), continue to decoct for 60min, and filter the decocted liquid through a 350 mesh sieve while it is hot. Add 7 times the amount of water for the second time, heat it over a warm fire, and bring it to a boil. Change to low heat and keep it slightly boiling. Continue to cook for 40 minutes. Filter the decoction with a 350 mesh sieve while it is hot, and combine the two decoctions. Transfer the decoction to a 2000mL round bottom flask, use a rotary evaporator to reduce pressure, and concentrate at low temperature (temperature: 60 ℃; vacuum degree: - 0.10MPa) to a constant volume of 500mL, and shake well.

Table 1 Sample Information of Herba Cistanche

2.1.2 Preparation of the test solution of Cistanche deserticola decoction pieces:

Take about 1g of the powder of Cistanche deserticola (sieved through a fourth sieve), accurately weigh it, place it in a 100mL brown volumetric flask, accurately add 50mL of 50% methanol, close the lid, shake well, weigh the weight, soak for 30min, ultrasonic treatment for 40min (power: 250W, frequency: 35kHz), cool it, weigh the weight again, add 50% methanol to make up the lost weight, shake well, let it stand, take the supernatant, and dilute it with 0.22 μ M microporous membrane filtration, obtained.

2.1.3 Preparation of the test solution of Cistanche deserticola standard decoction:

Take the standard cistanche soup, shake well, accurately measure 5mL to 50mL in a volumetric flask, add anhydrous ethanol to dilute to the scale, shake well, and dilute with 0.22 μ M microporous membrane filtration, obtained.

2.1.4 Preparation of reference solution:

Take an appropriate amount of echinacea glycoside control sample and poolside control sample, accurately weigh them, place them in a 10 mL brown volumetric flask, dissolve them with 50% methanol and dilute to the scale, shake well, and prepare a solution with the concentrations of 0.0200 and 0.02100 mg · mL - 1 for the above two components, respectively, to obtain a mixed control solution.

Acteoside, the main chemical component of Cistanche deserticola

2.2 Content of Echinoside and Piloside Components

The content determination method for Cistanche deserticola in Volume I of the Chinese Pharmacopoeia (2015 edition) was performed using Agilent ZORBAXB-C18 chromatographic column (4.6 × 150mm，5 μ m) Using methanol as the mobile phase A and 0.1% formic acid solution as the mobile phase B, gradient elution was performed (0 to 17 min, 26.5% A; 17 to 20 min, 26.5% → 29.5% A; 20 to 27 min, 29.5% A); Flow rate: 1.0mL · min-1; Column temperature: 30 ℃; Detection wavelength: 330 nm; The number of theoretical plates calculated based on the peak of echinacea should not be less than 3000. The contents of echinacea glycoside and poolside in the prepared pieces of Cistanche deserticola and their standard decoction were determined, and the results were shown in Table 2.

2.3 Determination of evaluation characteristic parameters of standard decoction of Cistanche deserticola decoction pieces

Table 2 Quality Analysis Results of Herba Cistanche and Its Standard Decoction (n=2)

2.3.1 Paste yield:

Shake the standard Cistanche deserticola soup prepared under 2.1.1 well, accurately suck 100mL, place it in a constant weight evaporation dish (weight: m1), evaporate it in a water bath, dry it in an oven at 105 ℃ for 3h, take it out, place it in a dryer to cool and dry for 30min, weigh it, record its weight as m2, and the weight of the medicinal material as M. According to the formula, "Cream yield=(m2 m1)" × 500/(100 × M) × Calculate the paste rate by "100%", and take the average value after 2 parallel copies of each sample. Results (see Table 2).

2.3.2 Transfer rate:

Based on the above measurement results of the content of echinacea glycosides and pilosides, the transfer rates of echinacea glycosides and pilosides were calculated according to the transfer rate formula (transfer rate=quality of indicator components in Cistanche deserticola standard decoction/quality of indicator components in decoction pieces × 100%), 2 parallel copies for each sample. Results (see Table 2).

2.3.3 pH value:

Shake the Cistanche deserticola standard decoction prepared under 2.1.1 evenly, take an appropriate amount, measure the pH value with a pH meter, and take an average value. Each sample is divided into two parts in parallel. Results (see Table 2).

2.4 Establishment of UPLC fingerprint of Cistanche deserticola standard decoction

2.4.1 Chromatographic conditions:

Using a WatersHSST3 column (2.1 × 100mm，1.8 μ m); Using methanol as mobile phase A and 0.1% formic acid solution as mobile phase B, gradient elution was performed (0 to 4 min, 34% A; 4 to 10 min, 34% → 40% A; 10 to 15 min, 40% → 46% A; 15 to 17 min, 46% → 34% A; 17 to 20 min, 34% A); Flow rate: 0.3mL · min-1; Column temperature: 30 ℃; Detection wavelength: 330 nm; Reference solution injection volume 1 μ 50. Injection amount of test solution 0.4 μ L。

2.4.2 Preparation of reference solution:

Take an appropriate amount of the control substances Echinacetin, Angioside A, Piloside, and Isopiloside, accurately weigh them, place them in a 10mL brown volumetric flask, dissolve them with 50% methanol, dilute to the scale, shake well, and prepare solutions with the concentrations of the above four components of 0.196, 0.234, 0.228, and 0.213 mg · mL - 1, respectively, to obtain a mixed control solution.

2.4.3 The preparation of the test solution is the same as that in Item 2.1.3.

2.4.4 Precision test:

Take the test solution of the same batch of Cistanche deserticola standard decoction (GH-3) and inject it six times continuously according to the chromatographic conditions in 2.4.1. Using poolside (peak 4) as the reference peak, calculate that the RSD of the relative retention time of each common peak is less than 0.2%, and the RSD of the relative peak area is less than 5.0%, indicating that the instrument has good precision.

Experimental Study on Cistanche deserticola

2.4.5 Stability test:

Take the test solution of the same batch of Cistanche deserticola standard decoction (GH-3) and inject it at 0, 2, 4, 8, 12, 16, 20, 24, and 28 hours after preparation according to the chromatographic conditions in 2.4.1. Using poolside (peak 4) as the reference peak, calculate that the RSD of the relative retention time of each common peak is less than 0.3%, and the RSD of the relative peak area is less than 4.8%, indicating that the test solution has good stability within 28 hours.

2.4.6 Repeatability test:

Take the same batch of Cistanche deserticola standard decoction (GH-3), prepare 6 parts of the solution in parallel according to the method in Item 2.1.3, analyze according to the chromatographic conditions in Item 2.4.1, record the chromatogram, and calculate that the RSD of the relative retention time of each common peak is less than 0.2%, and the RSD of the relative peak area is less than 3.8%. The results show that the repeatability of the method is good.

2.4.7 Establishment of fingerprint:

Prepare the test solution of 14 batches of Cistanche deserticola standard decoction according to Item 2.1.3, and inject the sample for determination according to the chromatographic conditions in Item 2.4.1 to obtain the UPLC fingerprint of 14 batches of Cistanche deserticola standard decoction. The UPLC fingerprint chromatograms of 14 batches of test solution were imported into the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" (Version 2012) software, using SI as the reference chromatogram, peak matching was performed with a time width of 0.1, and the median method was used to generate the reference fingerprint R of Cistanche deserticola standard soup (see Figure 1). At the same time, the similarity was calculated, and the results (see Table 3) were obtained. From the matching map, it can be seen that there are 6 common peaks in the standard decoction of Cistanche deserticola. According to the peak area and retention time of the six common peaks, the relative retention time (peak number (average relative retention time) was calculated as peak 1 (0.434), peak 2 (0.643), peak 3 (0.919), peak 4 (1.000), peak 5 (1.635), and peak 6 (1.858), respectively; Relative peak areas, [peak number (average relative peak area)] were peak 1 (6.340), peak 2 (0.890), peak 3 (0.295), peak 4 (1.000), peak 5 (0.674), and peak 6 (0.0190), respectively. Among them, peak 1 was identified as echinacea glycoside, peak 3 was identified as anthocyanin A, peak 4 was identified as poolside, and peak 5 was identified as isopiloside; The similarity analysis showed that the similarity between the 14 batches of standard decoction and the control map was between 0.789 and 0.999 (see Figure 2).

Table 3    14 Similarity of UPLC fingerprint of batch Cistanche deserticola standard decoction

Figure 1 UPLC chromatograms of mixed reference solution (A), cistanche deserticola standard decoction (B), and cistanche tubulosa standard decoction (C)

1. Echinoside; 3. Anthocyanin A; 4. Physalis glycosides; 5. Isopiloside

Figure 2    14 UPLC fingerprint of batch Cistanche deserticola standard decoction

3 .Discussion

3.1 Preparation of standard decoction

As a quality standard, the standard decoction of traditional Chinese medicine slices standardizes different clinical medication forms, which is currently one of the research hotspots in the field of traditional Chinese medicine. The preparation of standard decoction requires that the source of the decoction pieces be authentic and representative. Therefore, the Chinese Pharmacopoeia includes 9 batches of prepared Cistanche deserticola and 5 batches of prepared Cistanche deserticola. Before preparation, the prepared slices were tested and all met the requirements of the 2015 edition of the Chinese Pharmacopoeia.

3.2 Investigation of the injection amount of the test solution

In the content determination test of the index components in the standard decoction, the sample injection amount of the test article was 10 μ At L, there was no peak of poolside in the standard decoction chromatogram of Cistanche deserticola, except for echinacea glycoside. Although both echinacea glycoside and poolside appeared in the standard decoction chromatogram of Cistanche deserticola, the peak areas of both echinacea glycoside and poolside were small, and it was found that a high peak appeared at the solvent peak position in the chromatogram of the test article. The reason for the analysis is that the constant volume of the test sample is anhydrous ethanol without water, and it is inconsistent with the preparation solvent of the control sample, 50% methanol, resulting in a significant solvent effect, resulting in no retention of echinacea and poolside. Some samples have peaks of echinacea and provide due to their relatively high content and partial retention. Respectively adjust the injection amount of the test article to 5 and 3 μ L Investigate the elimination effect of solvent effect, and the result shows that the sample injection amount of the test article is 3 μ At L, the solvent effect can be eliminated, and the peak of echinacea can be normal, so the sample injection amount of the test article is set to 3 μ L。 Similarly, the solvent effect generated during the fingerprint determination of the standard decoction was eliminated by investigating the injection amount of the test solution, and the optimal injection amount was determined to be 0.4 μ L。

3.3 Reasons for the low similarity of fingerprint of Cistanche deserticola standard decoction

The similarity of fingerprints of 14 batches of Cistanche deserticola and Cistanche tubulosa standard decoction ranged from 0.789 to 0.999. The lowest similarity batch was RCR-9 with a standard soup of 0.789. The similarity of other batches is above 0.98. Compared with other samples, the overall similarity of the RCR-9 standard soup is not high. From the fingerprint chromatogram of the RCR-9 standard soup, it can be seen that it has two obvious peaks compared to other samples at retention times of 12.278 and 15.466. Due to the complex chemical components in traditional Chinese medicine, differences in a growth environment, harvesting time, processing methods, etc. lead to different material accumulation and component content of prepared slices. The low similarity of RCR-9 standard soup may be due to various factors that cause the material accumulation of this batch of prepared slices to be different from other batches.

3.4 Quality control of Cistanche deserticola standard decoction

This experiment analyzed the extraction rate, pH value, and transfer rates of echinacoside and poolside of 14 batches of cistanche deserticola standard decoction. The extraction rate of 14 batches of Cistanche deserticola standard decoction ranged from 25.1% to 56.6%. Due to the low sugar content of the prepared pieces of Cistanche deserticola compared to the prepared pieces of Cistanche deserticola, the extraction rate of the standard decoction of Cistanche deserticola deserticola was lower than that of the standard decoction of Cistanche deserticola. The pH value of 14 batches of Cistanche deserticola standard decoction ranged from 4.30 to 5.15, indicating that the pH value of Cistanche deserticola standard decoction was relatively stable. The average transfer rate of Echinacetin in 14 batches of Cistanche deserticola and Cistanche deserticola standard decoction was 22.99%, and the average transfer rate of poolside was 17.09%. Among them, the transfer rate of index components in the standard decoction prepared by Cistanche deserticola decoction pieces was relatively stable, while the transfer rate of index components in the standard decoction prepared by Cistanche deserticola decoction pieces was relatively large among batches.

4. Conclusion

In this study, the UPLC method was used to establish the fingerprint of Cistanche deserticola standard decoction, which can comprehensively reflect and evaluate the internal quality of Cistanche deserticola standard decoction. The results of the extraction rate, index component transfer rate, and fingerprint analysis of the standard cistanche decoction showed that the overall homogeneity of the 14 batches of cistanche standard decoction prepared was good, the preparation method was consistent with clinical practice, and the quality was controllable. This helps to standardize the different forms of cistanche medication, ensure the consistency of drug clinical efficacy, and solve the quality differences caused by different production processes and preparation methods.

Superman herbs cistanche

reference

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〔4〕Gao Yan, Guo Linong, Ma Shuangcheng, et al. Comparative study of three species of cistanche deserticola based on UPLC characteristic map and main component content ［ J ］. Chinese Journal of Traditional Chinese Medicine, 2019, 44 (17): 3749-3757

〔5〕Chen Shilin, Liu An, Li Qi, et al. Research strategy for standard decoction of traditional Chinese medicine slices ［ J ］. Chinese Journal of Traditional Chinese Medicine, 2016, 41 (08): 1367-1375