Research On Multi-Index Comprehensive Quality Evaluation Of Cistanches Herba Based On The CRITIC Weight Method

WEI Wei, QI Yuning, CHEN Weiying, ZHU Shunjuan, HOU Jianzhong
(Jiuquan Quality Inspection and Testing Center in Gansu Province, Jiuquan, Gansu 735000, China)

Abstract: The objective was to establish a multi-index comprehensive evaluation method for assessing the quality of Cistanche deserticola, and to scientifically evaluate the quality differences among Cistanches Herba from different sources (Cistanche deserticola and Cistanche tubulosa) and different production areas. The contents of five phenylethanoid glycosides (echinacoside, verbascoside, tubuloside A, isoacteoside, and 2′-acetylacteoside) were determined using high-performance liquid chromatography (HPLC). The CRITIC weighting method was employed to calculate the weight of each component, and the comprehensive scores of the samples were computed. Cluster analysis was applied to analyze the patterns of quality differences among the samples. The results showed that quality differences were observed between Cistanche deserticola and Cistanche tubulosa. The evaluation scores of Cistanche tubulosa were generally higher than those of Cistanche deserticola. Cluster analysis grouped Cistanche deserticola and Cistanche tubulosa into distinct categories. The quality of Cistanche deserticola showed minimal variation, while the quality of Cistanche tubulosa varied across different production areas. The model established in this study can objectively quantify the quality differences among Cistanches Herba from different origins and production areas, providing a scientific basis for the quality evaluation of Cistanche.

Keywords: Cistanches Herba; CRITIC weight method; cluster analysis; quality evaluation

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Cistanches Herba consists of the dried, scaly succulent stems of Cistanche deserticola Y. C. Ma or Cistanche tubulosa (Schenk) Wight, the former commonly known as desert cistanche. Cistanches Herba is warm in nature and sweet and salty in taste. It tonifies kidney yang, benefits essence and blood, and moistens the intestines to relieve constipation. It is used for kidney yang deficiency, deficiency of essence and blood, impotence and infertility, soreness and weakness of the waist and knees, weakness of the muscles and bones, and intestinal dryness with constipation. Its pharmacologically active components-such as phenylethanoid glycosides, polysaccharides, and iridoids-have been confirmed to possess antioxidant, anti-aging, immunomodulatory, and neuroprotective activities. As a renowned traditional Chinese medicinal material, it is listed as a top-grade herb in Shennong's Classic of Materia Medica and is known as the "ginseng of the desert." It is mainly distributed in the northwestern regions of Inner Mongolia, Gansu, Xinjiang, and Ningxia.

With rising market demand, wild resources are nearing depletion and artificial cultivation has become mainstream. The 2005 edition of the Pharmacopoeia of the People's Republic of China expanded Cistanche tubulosa as a legal botanical source of Cistanches Herba. The two sources have separate test limits, but only for extractives and the contents of echinacoside and verbascoside, which cannot comprehensively reflect the multi-component, multi-target characteristics of traditional Chinese medicine. The chemical composition of Cistanches Herba varies due to production area, harvest time, and processing methods. Therefore, establishing a scientific and systematic quality evaluation system is of great significance for ensuring the safety and efficacy of medicinal materials and promoting sustainable industrial development. In this study, 20 batches of Cistanches Herba from different production areas in Jiuquan were analyzed. HPLC was used to simultaneously determine five phenylethanoid glycosides-echinacoside, tubuloside A, verbascoside, isoacteoside, and 2′-acetylacteoside. The CRITIC weighting method was used to assign weights to each indicator for a comprehensive quality evaluation of the two botanical sources, and cluster analysis (CA) was used to interpret differences by botanical origin and production area, aiming to provide a scientific basis for quality evaluation of Cistanches Herba.

1 Instruments and Materials of cistanche

1.1 Instruments and equipment

Agilent 1260 Infinity HPLC system [including a quaternary pump, autosampler, column oven, and DAD detector; Agilent Technologies (China) Co., Ltd.]; XP205DR electronic analytical balance (Mettler-Toledo Instruments Co., Ltd.); KQ-500DE ultrasonic cleaner (Kunshan Ultrasonic Instruments Co., Ltd.).

 

1.2 Reagents

Reference standards: verbascoside (batch No. 111530-201914, purity 95.2%) and echinacoside (batch No. 111670-201706, purity 89.7%) were purchased from the National Institutes for Food and Drug Control; tubuloside A (batch No. B20471, purity ≥98%), isoacteoside (batch No. B21535, purity ≥98%), and 2′-acetylacteoside (batch No. B21779, purity ≥98%) were purchased from Shanghai YuanYe Bio-Technology Co., Ltd. Methanol was HPLC grade (Merck). Ultrapure water was prepared in-house. Other reagents were of analytical grade.

 

1.3 Samples

Ten batches of Cistanche deserticola and ten batches of Cistanche tubulosa were collected from Cistanches Herba cultivation bases and pharmacies in the Jiuquan area, labeled R1–R20 (see Table 1). They were identified by Deputy Director and TCM Pharmacist Hou Jianzhong of the Jiuquan Quality Inspection and Testing Center as the dried, scaly succulent stems of Cistanche deserticola Y. C. Ma or Cistanche tubulosa (Schenk) Wight. Voucher specimens are deposited at the Drug and Cosmetics Testing Institute of the Jiuquan Quality Inspection and Testing Center.

Table 1 Source information of Cistanches Herba samples

R1 | Cistanche deserticola | Dazhuangzi Town, Jinta County, Jiuquan City

R2 | Cistanche deserticola | Jiashan New Town, Suzhou District, Jiuquan City

R3 | Cistanche deserticola | Xiqu Town, Jinta County, Jiuquan City

R4 | Cistanche deserticola | Zhonghe Town, Jinta County, Jiuquan City

R5 | Cistanche deserticola | Shuanglong Town, Suzhou District, Jiuquan City

R6 | Cistanche deserticola | Hongliu Town, Guazhou County, Jiuquan City

R7 | Cistanche deserticola | Hongshagang Town, Dunhuang City, Jiuquan

R8 | Cistanche deserticola | Yumen City, Jiuquan

R9 | Cistanche deserticola | Tashi Town, Guazhou County, Jiuquan City

R10 | Cistanche deserticola | Zongtai Township, Yumen City, Jiuquan

R11 | Cistanche tubulosa | Dazhuangzi Town, Jinta County, Jiuquan City

R12 | Cistanche tubulosa | Suzhou District, Jiuquan City

R13 | Cistanche tubulosa | Suzhou District, Jiuquan City

R14 | Cistanche tubulosa | Suzhou District, Jiuquan City

R15 | Cistanche tubulosa | Suzhou District, Jiuquan City

R16 | Cistanche tubulosa | Hongshagang Town, Dunhuang City, Jiuquan

R17 | Cistanche tubulosa | Hongshagang Town, Dunhuang City, Jiuquan

R18 | Cistanche tubulosa | Hongshagang Town, Dunhuang City, Jiuquan

R19 | Cistanche tubulosa | Liangzhong Township, Yumen City, Jiuquan

R20 | Cistanche tubulosa | Liugou Township, Anxi County, Jiuquan

 

2 Methods and Results

2.1 Preparation of mixed reference solution

Accurately weigh verbascoside 10.45 mg, echinacoside 11.56 mg, tubuloside A 10.59 mg, isoacteoside 19.63 mg, and 2′-acetylacteoside 9.03 mg into separate 50 mL volumetric flasks, dissolve with 50% methanol, and make up to volume. Mix well to obtain the stock reference solutions. Then accurately pipette 1.00 mL of each stock solution into a 10 mL volumetric flask, dilute to volume with 50% methanol, and mix to obtain the mixed reference solution.

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2.2 Preparation of test solutions

Pass sample powders through a No. 3 sieve; weigh about 1 g accurately into a 100 mL brown volumetric flask. Add 80 mL of 50% methanol, stopper, shake, weigh, soak for 30 min, and sonicate for 40 min (250 W, 35 kHz). Cool to room temperature, reweigh, add 50% methanol to compensate for weight loss, shake well, allow to stand, decant the supernatant, and filter. Collect the subsequent filtrate as the test solution.

 

2.3 Chromatographic conditions

Use an InertSustain C18 column (4.6 mm × 250.0 mm, 5 μm). Mobile phase: methanol (A) and 0.1% phosphoric acid solution (B). Gradient for phase A: 0–6 min, 20%; 6–18 min, 20%→30%; 18–35 min, 30%→45%. Flow rate: 1.0 mL/min; column temperature: 35°C; detection wavelength: 330 nm; injection volume: 10 μL. Theoretical plates for each component were not less than 5,000. The chromatograms of the mixed reference solution and the test solutions are shown in Figure 1.

 

2.4 Methodology validation for content determination

2.4.1 Precision test

Inject the mixed reference solution from section 2.1 six times consecutively under the conditions in section 2.3. The RSDs of peak areas for all analytes were ≤0.86%, indicating good instrument precision.

 

2.4.2 Repeatability test

Accurately weigh six portions of sample R2 (about 1.0 g each) and prepare six test solutions per section 2.2. Analyze under the conditions in section 2.3. The RSDs of peak areas were ≤1.12%, indicating good method repeatability.

 

2.4.3 Stability test

Prepare a test solution from sample R1 as in section 2.2. Inject at 0, 2, 4, 8, 12, and 24 h under the conditions in section 2.3. The RSDs of peak areas were ≤1.20%, indicating good solution stability within 24 h.

 

2.4.4 Linearity

Prepare a series of mixed reference solutions by accurately pipetting 0.2, 0.4, 0.6, 0.8, 1.0, and 2.0 mL of each stock solution into 10 mL volumetric flasks and diluting to volume with 50% methanol. Inject under the conditions in section 2.3. Plot concentration versus peak area to obtain calibration equations and linear ranges (Table 2). Results showed good linearity for echinacoside, tubuloside A, verbascoside, isoacteoside, and 2′-acetylacteoside within their respective ranges.

Table 2 Results of the linearity study

Echinacoside | y = 2397.4x − 15.363 | r = 0.9987 | linear range: 0.0046–0.0462 mg·L−1

Tubuloside A | y = 9428.7x − 7.104 | r = 0.9984 | linear range: 0.0024–0.0242 mg·L−1

Verbascoside | y = 12609.5x − 8.507 | r = 0.9986 | linear range: 0.0024–0.0418 mg·L−1

Isoacteoside | y = 11600.3x − 11.359 | r = 0.9985 | linear range: 0.0079–0.0786 mg·L−1

2′-Acetylacteoside | y = 10901.8x − 5.216 | r = 0.9985 | linear range: 0.0036–0.0362 mg·L−1

2.4.5 Recovery
Prepare nine portions of a known-content sample (R1), about 0.5 g each. Spike at 50%, 100%, and 150% of the nominal contents with appropriate amounts of verbascoside, echinacoside, tubuloside A, isoacteoside, and 2′-acetylacteoside. Prepare test solutions per section 2.2 and analyze per section 2.3. Mean recoveries were 99.58%, 99.24%, 100.26%, 99.84%, and 100.46%, with RSDs of 1.26%, 1.67%, 1.03%, 1.39%, and 1.71%, respectively, indicating good accuracy.

 

2.4.6 Content determination

Accurately weigh about 1 g of Cistanche deserticola and Cistanche tubulosa powders, prepare test solutions per section 2.2, analyze per section 2.3, and calculate the contents of verbascoside, echinacoside, tubuloside A, isoacteoside, and 2′-acetylacteoside. Results are shown in Table 3.

Table 3 Determination results of the contents of five components in Cistanches Herba (mg·g−1)

R1: Echinacoside 4.81 | Tubuloside A 0.16 | Verbascoside 0.58 | Isoacteoside 0.26 | 2′-Acetylacteoside 0.49

R2: 5.02 | 0.13 | 0.70 | 0.30 | 0.61

R3: 6.62 | 0.24 | 0.67 | 0.21 | 0.43

R4: 5.25 | 0.18 | 0.82 | 0.34 | 0.67

R5: 5.63 | 0.17 | 0.75 | 0.21 | 0.39

R6: 6.85 | 0.24 | 0.91 | 0.29 | 0.44

R7: 6.08 | 0.23 | 0.63 | 0.20 | 0.54

R8: 5.17 | - | 0.49 | 0.18 | 0.24

R9: 6.78 | 0.28 | 0.73 | 0.27 | 0.37

R10: 5.44 | 0.19 | 0.68 | 0.24 | 0.26

R11: 16.77 | 0.97 | 1.20 | 1.19 | -

R12: 13.80 | 1.22 | 2.07 | 1.98 | 0.08

R13: 17.06 | 2.09 | 4.82 | 2.08 | 0.14

R14: 18.56 | 2.86 | 4.76 | 2.89 | 0.16

R15: 20.47 | 3.62 | 5.17 | 1.84 | 0.21

R16: 15.38 | 3.16 | 3.86 | 1.09 | 0.18

R17: 16.23 | 2.97 | 4.43 | 1.44 | 0.17

R18: 15.02 | 2.40 | 3.59 | 1.38 | 0.18

R19: 17.56 | 3.06 | 4.91 | 1.52 | 0.19

R20: 14.87 | 2.64 | 4.05 | 1.26 | 0.20

 

2.5 Quality evaluation using the CRITIC weighting method

2.5.1 Data standardization

As the measured contents of the indicators varied substantially, each indicator was treated as a benefit-type indicator and standardized to the [0, 1] range using formula (1).

 

2.5.2 Determination of weights

The standardized results of the 20 batches were imported into the SPSSPRO online data-processing platform, and the CRITIC method was used to calculate weights (Table 4). As shown in Table 4, the information content (1.936) of 2′-acetylacteoside was significantly higher than that of other indicators (0.683–0.758), mainly due to its very high conflict measure (6.786), indicating very low correlation with other indicators. Thus, it provided unique information in the evaluation system and accounted for 40.112% of the total weight, becoming the dominant indicator. The weights of the other four indicators (echinacoside, tubuloside A, verbascoside, and isoacteoside) were relatively balanced.

Table 4 Calculation results of weighting factors

Indicator: Echinacoside | Variability 0.367 | Conflict 2.064 | Information 0.758 | Weight 15.705%

Indicator: Tubuloside A | 0.352 | 1.940 | 0.683 | 14.150%

Indicator: Verbascoside | 0.392 | 1.881 | 0.737 | 15.275%

Indicator: Isoacteoside | 0.330 | 2.157 | 0.713 | 14.759%

Indicator: 2′-Acetylacteoside | 0.285 | 6.786 | 1.936 | 40.112%

 

2.5.3 Comprehensive evaluation

Based on the determined weights, the comprehensive evaluation scores of the 20 batches were calculated using formula (2). Results are shown in Table 5. Marked score differences were found between the two botanical sources. Cistanche tubulosa generally had higher contents of echinacoside and verbascoside, leading to higher comprehensive scores than Cistanche deserticola.

Table 5 CRITIC comprehensive evaluation scores for Cistanches Herba

R1: 0.307 (rank 14)

R2: 0.386 (rank 10)

R3: 0.292 (rank 15)

R4: 0.432 (rank 8)

R5: 0.258 (rank 17)

R6: 0.313 (rank 13)

R7: 0.351 (rank 12)

R8: 0.147 (rank 20)

R9: 0.265 (rank 16)

R10: 0.179 (rank 19)

R11: 0.236 (rank 18)

R12: 0.335 (rank 12)

R13: 0.533 (rank 4)

R14: 0.632 (rank 2)

R15: 0.667 (rank 1)

R16: 0.497 (rank 6)

R17: 0.530 (rank 5)

R18: 0.469 (rank 7)

R19: 0.565 (rank 3)

R20: 0.425 (rank 9)
 

 

2.6 Cluster analysis

The measured contents of the 20 batches were imported into SPSS 20.0. The furthest neighbor method and squared Euclidean distance were used as the metrics, and between-groups linkage was applied for clustering (see Figure 2). The results indicated differences in quality between the two botanical sources: Cistanche deserticola and Cistanche tubulosa clustered into separate groups. Cistanche deserticola showed small intragroup variation, while Cistanche tubulosa displayed quality differences among production areas and could be further divided into three subgroups, which was generally consistent with the CRITIC-based comprehensive evaluation.