Difference Of Phenylethanol Glycosides in Table Cistanches Herba From 2 Hosts BaDifference Of Phenylethanol Glycosides in Table Cistanches Herba From 2 Hosts Based On UPLC-QQQ-MS And PLS-DA Analysissed On UPLC-QQQ-MS And PLS-DA Analysis
May 30, 2025
Cistanche deserticola is a perennial holoparasitic plant belonging to the family Orobanchaceae. It is the dried fleshy stem covered with scale-like leaves of Cistanche deserticola Y. C. Ma [1-2]. This plant is mainly distributed in Inner Mongolia, Xinjiang, and Gansu, where it parasitizes Haloxylon ammodendron (C. A. Mey.) Bunge, a Chenopodiaceae xerophytic desert plant [3-4]. The primary active components of Cistanche deserticola include phenylethanoid glycosides, iridoids, and polysaccharides [5-7], which exhibit properties such as reproductive function protection, antioxidation, and liver protection [8-10]. With over 2,000 years of usage in China, it ranks first in traditional Chinese medicine prescriptions aimed at strengthening vitality and second in anti-aging formulations [11]. However, due to overharvesting and environmental destruction, wild resources of Cistanche deserticola are on the brink of extinction, leading to its classification as a nationally protected plant (Category II) and its inclusion in the International List of Wild Plant Protection [12]. To address the supply-demand imbalance in the medicinal market, artificial cultivation has become essential for the sustainable development of the Cistanche deserticola industry.
Cistanche deserticola Extract For Sale
Recent studies have reported successful inoculation of Cistanche deserticola on Atriplex canescens [13], an evergreen shrub of the Chenopodiaceae family. Commonly referred to as the "biological desalinator," Atriplex canescens is a high-quality tree species for saline-alkali soil improvement [14]. Cultivating Cistanche deserticola on Atriplex canescens offers significant advantages, such as high yield and superior quality. As a result, evaluating the quality of Atriplex canescens-Cistanche deserticola has become a research hotspot [15-16]. Previous research has mainly focused on the medicinal properties of dried Cistanche deserticola, while studies on the active components in its fresh form remain scarce. Particularly, following the inclusion of Cistanche deserticola as a food and medicinal substance under the trial management system (Notice No. 9 of the National Health Commission, 2023), its development and utilization as a dual-purpose medicinal herb have gained significant attention in the field of traditional Chinese medicine [17]. Fresh Cistanche deserticola is favored for its excellent taste and convenience, making it highly promising for development. Fresh medicinal materials refer to unprocessed fresh plant, animal, or fungal substances with therapeutic or health-promoting properties. Among the 2,000 commonly used Chinese medicinal herbs, 486 are fresh varieties [18].
The ultra-performance liquid chromatography-triple quadrupole tandem mass spectrometry (UPLC-QQQ-MS) method is highly sensitive, making it suitable for detecting low-concentration and complex components [19]. To address the issue of high-water content in Cistanche deserticola, where phenylethanoid glycosides are prone to enzymatic hydrolysis resulting in reduced content, an enzyme-inactivation drying method effectively preserves the phenylethanoid glycoside content [20]. This study employs UPLC-QQQ-MS to evaluate 8 phenylethanoid glycosides, including echinacoside and acteoside, as key active components in fresh Cistanche deserticola from two host plants [21-22]. The study examines differences in the component contents of juice and residue from fresh Cistanche deserticola grown on two hosts. Additionally, the residue was subjected to enzyme-inactivation treatment at 80°C for 5 minutes, followed by drying, to further compare the retention of active components in Cistanche deserticola. The findings aim to provide scientific evidence and technical support for the dual-purpose value of fresh Cistanche deserticola and the optimization of its processing techniques.
1 Instruments and Materials
1.1 Instruments
1290-6460 ultra-performance liquid chromatography-triple quadrupole tandem mass spectrometer (Agilent Technologies, USA).
AL104 electronic analytical balance (Mettler-Toledo, Switzerland).
H-2050R centrifuge (Changsha Xiangyi Centrifuge Instrument Co., Ltd.).
SB25-12DTD ultrasonic cleaner (Ningbo Xinzhi Biological Technology Co., Ltd.).
1.2 Materials
The Atriplex canescens-Cistanche deserticola and Haloxylon ammodendron-Cistanche deserticola samples were collected in March, April, and May of 2024 from the Cistanche deserticola cultivation base in Xiquan Town, Jingtai County, Gansu Province. The samples were identified by Professor Sun Xuegang from Gansu Agricultural University as the fleshy stems of Cistanche deserticola Y. C. Ma with scale leaves.
Reference standards:
Echinacoside (batch number: 23031605; purity: 99.74%).
Acteoside (batch number: 21102103; purity: 98.83%).
Cistanoside A (batch number: 23041703; purity: 98.30%).
Isoacteoside (batch number: 23052508; purity: 99.90%).
2'-Acetylacteoside (batch number: 210053102; purity: 97.12%).
Salidroside (batch number: 22051208; purity: 98.43%).
Cistanoside B (batch number: 20120903; purity: 98.57%).
Tubuloside A (batch number: 21060302; purity: 93.24%).
All were purchased from Chengdu Pfeide Biotechnology Co., Ltd.
Reagents:
Methanol (batch number: O1140576, chromatographically pure).
Acetonitrile (batch number: S3641440, chromatographically pure). Both were purchased from Shanghai Anpu Experimental Technology Co., Ltd.
Formic acid (batch number: 20230710, chromatographically pure) was purchased from Tianjin Kermio Chemical Reagent Co., Ltd.
Purified water (Hangzhou Wahaha Group Co., Ltd.).
2 Methods and Results
2.1 Methods
2.1.1 Sample Preparation
Three batches of Atriplex canescens-Cistanche deserticola (batch numbers: 20240330-1, 20240420-1, 20240510-1) and three batches of Haloxylon ammodendron-Cistanche deserticola (batch numbers: 20240330-2, 20240420-2, 20240510-2), totaling six batches, with five plants per batch, were collected. The plants were washed clean with water.
Processing:
The samples were crushed and juiced, resulting in juice and residue from the two host-derived Cistanche deserticola.
The residues were dried under two conditions:
Without enzyme inactivation.
With enzyme inactivation (5 minutes at 80°C in an oven).
The dried residues were stored for further analysis. Each batch of medicinal material was divided into four groups, as shown in Table 1.
Tab. 1 Sample grouping information
| Group | Type | Atriplex canescens-Cistanche deserticola | Haloxylon ammodendron-Cistanche deserticola |
|---|---|---|---|
| ① | Residue | C1 | S1 |
| ② | Juice | C2 | S2 |
| ③ | Enzyme-inactivated residue (5 min drying, crushed) | C3 | S3 |
| ④ | Non-enzyme-inactivated residue (dried and crushed) | C4 | S4 |
2.1.2 Chromatographic Conditions
Column: Agilent Zorbax Eclipse Plus C18 column (2.1 mm × 150 mm, 1.8 μm).
Flow rate: 0.3 mL/min.
Injection volume: 1 μL.
Column temperature: 35°C.
Mobile phase: 0.1% formic acid in water (A) and acetonitrile (B).
Analysis time: 12 minutes.
Elution gradient:
0–1.5 min: 10% → 20% B.
1.5–3 min: 20% → 30% B.
3–5 min: 30% → 32% B.
5–8 min: 32% → 38% B.
8–9.6 min: 38% → 10% B.
9.6–12 min: 10% B.
The chromatograms of the mixed reference standards and test samples are shown in Figure 1. (Please confirm whether the mobile phase is 0.01% formic acid in water (A) and acetonitrile (B).)
Fig. 1 Chromatograms of reference substances(A) and sample(B) 1−salidroside; 2−echinacoside; 3−cistanosideA; 4−tubulosideA; 5−acteoside; 6−isoacteoside; 7−2'-acetylacteoside; 8-tubuloside B.
2.1.3 Mass Spectrometry Conditions
Ion source type: Electrospray ionization (ESI).
Mode: Multiple reaction monitoring (MRM) in negative ion mode.
Ion source temperature: 350°C.
Nebulizer pressure: 35 psi.
Collision gas: Nitrogen, set to medium level.
The mass spectrometry parameters for the components to be analyzed are shown in Table 2.
2.1.4 Preparation of Mixed Standard Solution
Accurately weigh appropriate amounts of the reference standards echinacoside, acteoside, cistanoside A, isoacteoside, 2'-acetylacteoside, salidroside, cistanoside B, and tubuloside A, and place them in 10 mL amber reagent bottles. Add 50% methanol to prepare a mixed standard solution with a concentration of 0.2 mg·mL⁻¹ and store it for later use.
2.1.5 Preparation of Test Solutions
Accurately weigh 0.2 g of fresh Atriplex canescens-Cistanche deserticola residue (C1), fresh Haloxylon ammodendron-Cistanche deserticola residue (S1), dried Atriplex canescens-Cistanche deserticola residues (C3, C4), and dried Haloxylon ammodendron-Cistanche deserticola residues (S3, S4). Place the samples in 10 mL amber volumetric flasks, add 50% methanol, bring to volume, and record the mass. Soak for 0.5 hours, ultrasonicate for 40 minutes (250 W, 35 kHz), cool to room temperature, and record the mass again. Use 50% methanol to compensate for any weight loss, mix well, centrifuge, and allow the solution to settle. Filter the supernatant through a 0.22 μm microporous membrane to obtain the filtrate.
For fresh Atriplex canescens-Cistanche deserticola juice (C2) and fresh Haloxylon ammodendron-Cistanche deserticola juice (S2), take 10 mL of each, weigh accurately, mix well, centrifuge, and allow the solution to settle. Filter the supernatant through a 0.22 μm microporous membrane to obtain the filtrate.
2.1.6 Linearity Study
Appropriate amounts of the mixed standard solution prepared in section 2.1.4 were diluted to create a concentration gradient of 51.2, 128.0, 320.0, 800.0, 4,000.0, and 20,000.0 μg·L⁻¹. Samples were injected sequentially from low to high concentrations following the conditions described in sections 2.1.2 and 2.1.3. A linear regression was performed, using the injection concentration as the X-axis and the peak area as the Y-axis. The linear relationships of the eight components are shown in Table 3.
Tab. 3 Linear relationship examination results of 8 components in table Cistanches Herba
| Component | Regression Equation | r | Linear Range (μg·L⁻¹) |
|---|---|---|---|
| Echinacoside | Y = 16.410 × X − 0.804 | 0.9996 | 51.2–20,000.0 |
| Acteoside | Y = 102.794 × X − 55.793 | 0.9998 | 51.2–20,000.0 |
| Isoacteoside | Y = 31.007 × X − 45.169 | 0.9999 | 51.2–20,000.0 |
| 2'-Acetylacteoside | Y = 110.235 × X − 8.953 | 0.9992 | 51.2–20,000.0 |
| Salidroside | Y = 19.678 × X − 0.557 | 0.9993 | 51.2–20,000.0 |
| Tubuloside A | Y = 26.785 × X − 13.630 | 0.9997 | 51.2–20,000.0 |
| Tubuloside B | Y = 71.810 × X − 0.993 | 0.9998 | 51.2–20,000.0 |
| Cistanoside A | Y = 0.578 × X + 1.531 | 0.9994 | 51.2–20,000.0 |
2.1.7 Precision Test
The mixed standard solution prepared under the conditions in section 2.1.4 was diluted with 50% methanol and injected six consecutive times under the conditions of sections 2.1.2 and 2.1.3. The relative standard deviations (RSDs) of the peak areas for echinacoside, acteoside, isoacteoside, 2'-acetylacteoside, salidroside, cistanoside B, tubuloside A, and cistanoside A were 0.95%, 1.47%, 0.56%, 1.05%, 0.93%, 1.08%, 1.19%, and 0.91%, respectively, indicating good precision.
2.1.8 Stability Test
A single sample of Cistanche deserticola (S4) was prepared under the conditions described in section 2.1.5. The test solution was injected and analyzed at 0, 2, 4, 8, 12, and 24 hours under the conditions described in sections 2.1.2 and 2.1.3. The RSDs of the peak areas for echinacoside, acteoside, isoacteoside, 2'-acetylacteoside, salidroside, cistanoside B, tubuloside A, and cistanoside A were 1.46%, 0.79%, 0.95%, 1.29%, 1.22%, 0.56%, 0.49%, and 1.18%, respectively, indicating that the sample solution was stable within 24 hours.
2.1.9 Repeatability Test
A single batch of Cistanche deserticola (S4) sample (batch number: 20240420-2) was prepared in six parallel test solutions under the conditions described in section 2.1.5. The samples were injected and analyzed under the conditions described in sections 2.1.2 and 2.1.3. The RSDs of the content for echinacoside, acteoside, isoacteoside, 2'-acetylacteoside, salidroside, cistanoside B, tubuloside A, and cistanoside A were 1.26%, 0.98%, 1.55%, 1.06%, 1.33%, 0.99%, 1.65%, and 0.95%, respectively, demonstrating good repeatability.
2.1.10 Recovery Test
Nine samples from the same batch of Cistanche deserticola (S4, batch number: 20240420-2) were prepared, with 0.1 g per sample. The samples were divided into three groups, with three replicates per group. Each group was spiked with 80%, 100%, and 120% of the reference standard content in the Cistanche deserticola sample. The test solutions were prepared following the method described in section 2.1.5 and analyzed under the conditions in sections 2.1.2 and 2.1.3. The recoveries of the eight phenylethanoid glycosides were calculated, and the results are shown in Table 4.
Tab. 4 Recoveries rates of 8 tested components in Cistanches Herba
| Component | Sample Content (mg) | Spiked (mg) | Measured (mg) | Average Recovery (%) | RSD (%) |
|---|---|---|---|---|---|
| Echinacoside | 0.513 8 | 0.410 1 | 0.911 2 | 99.85 | 1.37 |
| Echinacoside | 0.513 8 | 0.513 8 | 1.015 2 | 99.88 | 0.95 |
| Echinacoside | 0.513 8 | 0.616 5 | 1.129 4 | 99.85 | 1.06 |
| Acteoside | 0.421 5 | 0.337 2 | 0.759 6 | 99.90 | 1.02 |
| Acteoside | 0.421 5 | 0.421 5 | 0.843 5 | 99.87 | 1.18 |
| Acteoside | 0.421 5 | 0.505 8 | 0.926 7 | 99.84 | 1.24 |
| Isoacteoside | 0.327 1 | 0.261 7 | 0.588 6 | 99.85 | 0.86 |
| Isoacteoside | 0.327 1 | 0.327 1 | 0.654 2 | 99.86 | 1.05 |
| Isoacteoside | 0.327 1 | 0.392 5 | 0.719 1 | 99.84 | 1.13 |
| 2'-Acetylacteoside | 0.110 2 | 0.088 2 | 0.198 3 | 99.87 | 1.15 |
| 2'-Acetylacteoside | 0.110 2 | 0.110 2 | 0.220 1 | 99.86 | 1.09 |
| 2'-Acetylacteoside | 0.110 2 | 0.132 2 | 0.242 4 | 99.85 | 1.21 |
| Salidroside | 0.081 6 | 0.065 3 | 0.146 7 | 99.84 | 1.18 |
| Salidroside | 0.081 6 | 0.081 6 | 0.163 1 | 99.85 | 1.02 |
| Salidroside | 0.081 6 | 0.097 9 | 0.179 7 | 99.84 | 1.17 |
| Tubuloside B | 0.072 3 | 0.057 9 | 0.130 0 | 99.86 | 1.09 |
| Tubuloside B | 0.072 3 | 0.072 3 | 0.144 1 | 99.86 | 1.05 |
| Tubuloside B | 0.072 3 | 0.086 8 | 0.159 2 | 99.84 | 1.24 |
| Tubuloside A | 0.043 8 | 0.035 0 | 0.078 5 | 99.86 | 1.16 |
| Tubuloside A | 0.043 8 | 0.043 8 | 0.087 5 | 99.86 | 1.02 |
| Tubuloside A | 0.043 8 | 0.052 5 | 0.096 2 | 99.84 | 1.15 |
