Comparing Content Of Phenylethanol Glycosides in Two Different Host Cistanches Herba After Different Drying Treatments

They were comparing the content of phenylethanol glycosides in two different host Cistanches Herba after different drying treatmentsGAO Peiwen1, GUO Yehong1 *, ZHAO Fafa1, ZHANG Dan2, ZHANG Fangming1 1(Agronomy College, Gansu Agricultural University, Lanzhou 730070, China) 2(State Key Laboratory of Aridland Crop Science, Lanzhou 730070, China)

Abstract To compare the content of 10 phenylethanoid glycosides in different processed Cistanches Herba from different hosts using ultra-high performance liquid chromatography-triple quadrupole mass spectrometry (UPLC-QQQ-MS). The dried Cistanches Herba was crushed, sieved through a 60-mesh sieve, and extracted by ultrasound with 50% methanol. Separation was performed on an Agilent Eclipse Plus C18 column (2.1 mm×150 mm, 1.8 μm) using gradient elution with mobile phase A (0.1% formic acid aqueous solution) and B (acetonitrile). Detection was carried out in dynamic multiple reaction monitoring mode using an electrospray ionization source in UPLC-QQQ-MS.

Results showed that significant variations were observed in the composition of 10 phenylethanoid glycosides among Cistanches Herba cultivated on different hosts and subjected to various drying treatments. Freeze-dried Cistanches Herba showed the highest content of these compounds for both host plants, followed by drying at 80 ℃. Although freeze-drying preserved the maximum content of 10 phenylethanoid glycosides in Cistanches Herba, drying at 80℃ was more cost-effective for large-scale production.

Keywords: ultra-high performance liquid chromatography-triple quadrupole mass spectrometry; Cistanches Herba; Haloxylon ammodendron (C.A.Mey.) Bunge; Atriplex canescens (Pursh) Nutt; phenylethanoid glycosides; drying method

Cistanche Raw material

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Cistanches Herba is the dry fleshy stem with scaly leaves of Cistanche deserticola Y.C.Ma or Cistanche tubulosa (Schenk) Wight. It was first seen in "Shen Nong's Materia Medica" and ranks among the top grade. It is a precious Chinese herbal medicine that can be used both as medicine and food. Because it mostly grows in arid areas such as Inner Mongolia, Xinjiang, Gansu, and Ningxia, it is also known as "desert ginseng." Cistanche deserticola is sweet, salty, and warm and returns to the kidney and large intestine meridians; it has the effects of nourishing kidney yang, replenishing essence and blood, moistening the intestines and laxative, etc., and is often used to treat diseases such as insufficient kidney yang, deficiency of essence and blood, impotence and infertility, and weakness of muscles and bones [1 -4]. Phenylethanol glycosides are the main active ingredients in Cistanche deserticola, and they are also the most reported substances in the research on Cistanche deserticola [5]

The traditional host plant of Cistanche deserticola is Haloxylon ammodendron (C.A.Mey.) Bunge in the family Chenopodiaceae. However, Haloxylon ammodendron grows slowly and has low economic value. Haloxylon ammodendron plants less than 3 years old are difficult to use for cultivating Cistanche deserticola. To alleviate the contradiction between supply and demand in the Cistanche deserticola medicinal market, increase the production of Cistanche deserticola, and achieve large-scale and standardized cultivation of Cistanche deserticola. Scientific researchers successfully introduced Atriplex canescens (Pursh) Nutt in Gansu, but whether this tree species can be used as a new host plant for Cistanche deserticola and whether its growth performance is better than Haloxylon ammodendron has become a hot issue of widespread concern [6]. Whether Atriplex truncatula can completely replace Haloxylon ammodendron in cultivating Cistanche deserticola still needs to be supported by broader, more comprehensive, and in-depth basic research results. In my country, although extensive research has been conducted on Atriplex truncatula from the aspects of introduction, seedling cultivation, and component analysis [7-10], there are very few reports on the inoculation of Atriplex truncatula with Cistanche deserticola. In the past, research mainly focused on the development and utilization characteristics of Cistanche deserticola [11-14]. However, the research on the active ingredients of two host Cistanche deserticola has not been reported, especially the two host Cistanche deserticola after using different drying processes. There are few studies on differences in the retention of active ingredients.

Ultra-high performance liquid chromatography-triple quadrupole mass spectrometry (UPLC-QQQ-MS) uses UPLC as the chromatographic separation system and triple quadrupole (QQQ) as the analyzer in series It is a liquid mass spectrometry technology that has good separation effect on multi-component complex systems and can quickly and accurately quantitatively analyze the complex components of traditional Chinese medicine [15-16]. In this experiment, UPLC-QQQ-MS technology was used to quantitatively analyze 10 phenylethanol glycosides, including echinaceaside, verbascoside, isorbascoside, and anthocyanoside A, in the dried products of two host species of Cistanche deserticola after different drying treatments. Comparing the contents of these components in Cistanche deserticola under different drying methods and different hosts provides a theoretical basis for the development and precise utilization of phenylethanol glycosides in Cistanche deserticola.

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1 Materials and instruments

Haloxylon ammodendron Cistanches and Cistanches tetrapterans were collected from the Cistanche deserticola planting base in Jingtai County, Gansu Province. The harvest time was March 2023. Professor Guo Yehong of Gansu Agricultural University identified the fleshy stems of Cistanche deserticola with scaly leaves. Reference substances echinacea side (C35H46O20, CAS No.: 82854-37-3, purity 98.30%), verbascoside (C29H36O15, CAS No.: 61276-17-3, purity 99.35%), tuberose A (C37H48O21, CAS No.: 112516-05-9, purity 99.74%), Isomurascoside (C29H36O15, CAS No.: 61303-13-7, purity 97.77%), gardeniposide (C17H24O10, CAS No.: 24512-63-8, purity 99.11 %), 2'-acetyl verbascoside (C31H38O16, CAS number: 94492-24-7, purity 97.12%), salidroside

(C14H20O7, CAS No.: 10338-51-9, purity 98.43%), Motherwort (C15H24O9, CAS No.: 52949-83-4, purity 97.64%), Cistancheside A (C36H48O20, CAS No.: 93236-42-1, purity 93.24%), angioside B (C31H38O16, CAS number: 112516-04-8, purity 98.57%), Chengdu Pufeide Biotechnology Co., Ltd.; methanol and acetonitrile are chromatographically pure, Shanghai Anpu Testing Technology Co., Ltd. Co., Ltd.; formic acid, Tianjin Comeo Chemical Reagent Co., Ltd.; water was ultrapure water.

DESKTOP-T2G6S78\6460 ultra-high performance liquid chromatography-triple quadrupole tandem mass spectrometer, Agilent Company of the United States; AL104 electronic analytical balance, Mettler-Toledo Instruments Co., Ltd., Switzerland; LyoQuest-85 freeze dryer, Telstar, Spain company.

2 Experimental methods

2.1 Preparation of Cistanche deserticola samples with different drying processes

Cut fresh Cistanche deserticola products from different hosts into 3 mm thick slices and process them as follows.

(1) Sun-drying: Steam the slices for 5 minutes and then lay them flat in a sunny place to dry. Check the changes in moisture content. When the moisture content is less than 10%, put them away for later use.

(2) Drying: Steam the slices for 5 minutes and then place them in an oven at 40, 60, or 80°C to dry until the moisture content is less than 10% and put them away for later use.

(3) Vacuum freeze-drying: Place the slices in a -80 ℃ refrigerator for 2 hours before pre-cooling, then place them in a vacuum freeze dryer (vacuum degree 650Pa, freezing temperature -55 ℃), dry for 48 hours, and put away for later use.

Cistanche tubulosa (5)

2.2 Chromatographic conditions

Agilent Eclipse Plus C18 column (2.1 mm × 150 mm, 1.8 μm); flow rate: 0.3 mL/min; injection volume 1 μL; mobile phase: phase A is 0.1% formic acid aqueous solution, and phase B is chromatographic acetonitrile. The elution speed of mobile phase A and B is 0~1.5 min, 10%~15% B; 1.5~2 in, 15%~20% B; 2~3 min, 20%~25% B; 3~4 min , 25%~30% B; 4~5 min, 30%~32% B; 5~6 min, 32%~34% B; 6~7 min, 34%~36% B; 7~8 min, 36 %~38% B; 8~9 min, 38%~40% B; 9~9.5 min, 0%~45% B; 9.5~9.6 min, 45%~10% B; 9.6~10 min, 10% B .

2.3 Optimized mass spectrometry conditions

Ion source type: electronic spray ion source; scanning method: negative ion scanning; ion source temperature: 350 ℃; nebulizer: 35 psi; multiple reaction monitoring mode and dynamic multiple reaction mode to monitor and analyze each compound. The optimized conditions for mass spectrometry of 10 substances are shown in Table 1.

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2.4 Preparation of reference solution and standard curve solution

Precisely weigh the standard powders of echinacoside, verbascoside, anorthosite A, isolebascoside, gardeniposide, 2'-acetyl verbascoside, salidroside, leonine, distances A and anorthosite B., add 50% methanol to make a mixed reference solution containing 0.2 mg per 1 mL; dilute with 50% methanol into a standard series of mixed solutions of 40.96, 102.4, 256, 640, 1 600, 4 000 μg/L, press " The conditions under 2.2" and 2.3" are sequentially injected from low concentration to high concentration, and then the quantitative software (agilent mass hunter) is used to process the detection results using the standard curve passing the origin. Perform linear regression with the substance concentration X (ng/mL) as the abscissa and the ion response value Y as the ordinate, and calculate the regression equation and correlation coefficient, as shown in Table 2. There is a good linear relationship with the ion response value within the range.

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