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Quantitative Analysis of the Multicomponent and Spectrum–Effect Correlation of the Antispasmodic Activity of Shaoyao-Gancao Decoction

Quantitative Analysis of the Multicomponent and Spectrum–Effect Correlation of the Antispasmodic... Hindawi Journal of Analytical Methods in Chemistry Volume 2022, Article ID 2279404, 13 pages https://doi.org/10.1155/2022/2279404 Research Article Quantitative Analysis of the Multicomponent and Spectrum–Effect Correlation of the Antispasmodic Activity of Shaoyao-Gancao Decoction 1,2 2 2 2 1,3,4,5 1,3 Yanli Xu, Chenxi Li, Ting Chen, Xiaochun Li, Xiaoyu Wu, Qili Zhang, 1,2,3,4,5 and Lei Zhao Gansu University of Chinese Medicine, Lanzhou 730000, China Lanzhou Institute for Food and Drug Control, Lanzhou 730000, China Northwest Collaborative Innovation Center for Traditional Chinese Medicine Co-Constructed By Gansu Province & MOE of PRC, Lanzhou 730000, China Key Laboratory of Chemistry and Quality of TCM of the College of Gansu Province, Lanzhou 730000, China Gansu Province Engineering Laboratory for TCM Standardization Technology and Popularization, Lanzhou 730000, China Correspondence should be addressed to Lei Zhao; zzyhx@gszy.edu.cn Received 21 July 2022; Revised 16 November 2022; Accepted 24 November 2022; Published 2 December 2022 Academic Editor: Cecilia Cagliero Copyright © 2022 Yanli Xu et al. Tis is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Shaoyao-Gancao Decoction (SGD) is a well-known classic traditional Chinese medicine (TCM) with antispasmodic, anti-in- fammatory, and analgesic efects. Tis preparation has been widely used to treat spasticity diseases in the clinic. To date, the material basis of SGD remains unclear, and the spectrum-efect correlation of its antispasmodic activity has not been reported yet. In this study, high-performance liquid chromatography (HPLC) was used to establish the fngerprint and determine the multiple components of SGD. Te common peaks of fngerprints were evaluated by the similarity with the chromatographic fngerprints of the TCM. Meanwhile, the multiple components were quantifed and analysed using the heatmap and box size analysis. Fur- thermore, data on the antispasmodic efect were extracted through in vitro smooth muscle contraction assay. Grey relational analysis combined with partial least square regression was used to study the spectrum–efect correlation of SGD. Finally, the potential antispasmolytic components were validated using an isolated tissue experiment. Te HPLC fngerprint was established, and 20 common peaks were identifed. Te similarities of 15 batches of SGD were all above 0.965. Te HPLC method for simultaneous determination of the multiple components was accurate and reliable. Te contents of albiforin, paeoniforin, liquiritin, and glycyrrhizic acid were higher than the other components in SGD. Te heatmap and box size also showed that X3 (albiforin), X4 (paeoniforin), X5 (liquiritin), X11 (liquirtigenin), and X16 (glycyrrhizic acid) could be used as quality indicators in the further establishment of quality standards. Te spectrum–efect correlation results indicated that X4, X11, and X16 were highly correlated with antispasmolytic activity. Verifcation tests showed that paeoniforin (11.7–29.25 μg/mL) and liquirtigenin (17.19–28.65 μg/mL) could signifcantly reduce the maximum contractile (P < 0.01). Tese compounds exerted concentration- dependent spasmolytic efects with the inhibitory response for acetylcholine (Ach)-evoked contraction. Tus, SGD had a sig- nifcant antispasmodic efect, which resulted from the synergistic activity of its multiple components. Tese fndings can be used for the pharmacodynamics study of SGD and are of great signifcance for the determination of quality markers and quality control. Tere has been much debate about the defnition of spas- 1. Introduction ticity. In 1980, Lance was the frst scholar to associate Spasticity is a velocity-dependent increase in muscle tone spasticity to the velocity-dependent increase in stretch refex caused by the increased excitability of muscle spindles [3]. A more general defnition of spasticity is disordered following an upper motor neuron (UMN) syndrome [1, 2]. sensory-motor control, resulting from UMN lesion, 2 Journal of Analytical Methods in Chemistry the multiple components of SGD, and validate the presenting intermittent or sustained involuntary activation of muscles [4]. Tis complex phenomenon of extremely spectrum–efect results. Scientifc basis for the secondary development and quality control of SGD was also variable clinical expression, which may cause diferent motor dysfunctions, has been observed in many patients provided. with spinal cord injury, cerebral palsy, multiple sclerosis, and acquired brain injury, which directly impact the quality 2. Materials and Methods of life [5]. Presently, the conventional drugs used in the treatment of spasm include baclofen, tizanidine, and dan- 2.1. Samples, Reagents, and Animals. Fifteen batches of SGD trolene, amongst others. However, the efcacy of these were purchased from drug manufacturers in Gansu Prov- current treatments is not absolute, and they may have se- ince. Te sources of herbal materials used in SGD are shown rious side efects [6–8]. in Table 1. Te raw materials were identifed by Renyuan Accordingly, natural products with therapeutic activity Zhu (a senior engineer in the Lanzhou Institute for Food and against spasm must be searched to replace drugs with strong Drug Control). Paeoniae Radix Alba is the dried root slice of side efects. Shaoyao-Gancao Decoction (SGD) is a classic Paeonia lactifora Pall. Roasted liquorice is the root and traditional Chinese medicine (TCM) and originally de- rhizome of G. uralensis Fisch., Glycyrrhiza infata Bat., or scribed in the Treatise on Febrile Disease. SGD is composed Glycyrrhiza glabra L. Te quality of the herbal materials of Paeoniae Radix Alba (baoshao in Chinese) and honeyed complied with the standards of the National 2020 Glycyrrhiza uralensis, which are traditionally used to treat Pharmacopeia. spastic diseases, such as gastrointestinal spasm, facial muscle Methanol and acetonitrile (HPLC grade) were pur- spasm, and poststroke spasm [9]. Modern pharmacological chased from Honeywell China Co., Ltd. (Shanghai, China). and clinical studies have confrmed that SGD has signifcant Water was ultrapure. Glycyrrhizic acid (No. antispasmodic, anti-infammatory, and analgesic efects on 110731–202122, purity >99.06%) and catechin (No. various spastic diseases [10], infammatory diseases [11], 110877–202005, purity >95.1%) reference substances were painful diseases [12, 13], gynopathy [14], bronchial asthma, purchased from the China Institute of Food and Drug Parkinson’s disease, and constipation [15, 16]. Tis TCM has Control (Beijing, China). Isoliquiritigenin (No. 20112401, been selected for the frst batch of the Chinese Medicine purity >99.04%), 1,2,3,4,6-O-pentagalloylglucose (No. Classical Directory. Studies have shown that the extract of 19010904, purity >98%), ononin (No. 19071501, purity SGD and liquorice exerts a relaxant efect on acetylcholine >98%), liquiritin apioside (No. 20111607, purity >98.28%), (ACh)-induced contraction, isoliquiritigenin, and glycy- licochalcone B (No. 20121501, purity >93%), galloylpaeo- coumarin isolated from the roots of liquorice and has a niforin (No. 171013, purity >98%), licochalcone A (No. potent antispasmodic [17, 18]. Te ethanol extract of Gly- 19102405, purity >98.89%), oxypaeoniforin (No. cyrrhiza uralensis has signifcant inhibitory efects on Nav1.4 19120604, purity >99.67%), isoliquiritin (No. 20041301, VGSCs, which may be an important mechanism in the purity >99.95%), liquiritigenin (No. 21052404, purity treatment of gastrocnemius spasm [19]. >99.86%), albiforin (No. 21031706, purity >98.75%), TCM is characterised by multiple components, targets, glabridin (No. 21060801, purity >99.08%), benzoylpaeo- and approaches, and SGD has a complex composition. niforin (No. 20092303, purity >99.43%), and paeoniforin Previous studies have shown that glycyrrhizin, glycyrrhetic (No. 20030901, purity >98.2%) reference substances were acid, paeoniforin, albiforin, oxypaeoniforin, liquiritin, purchased from the Chengdu Grip Biotechnology Co., Ltd. liquiritigenin, isoliquiritin, isoliquiritigenin, and 1,2,3,4,6- (Chengdu, China). Other compounds were of analytical O-pentagalloylglucose are the main bioactive compounds of grade. SGD [20–22]. Research on SGD has mainly focused on its Male adult Sprague–Dawley (SD) rats, 3–6 months of clinical application, chemical composition, and anti-in- age, and weighing 250–300 g, were obtained from the fammatory and analgesic efects. However, the pharma- Animal Experiment Center of Gansu University of Tra- codynamic basis of the antispasmodic efect and the ditional Chinese Medicine (Approval No. SCXK[Gan] spectrum–efect relationship of SGD has not been reported 2020–0009, Lanzhou, Gansu, China). Te animals were yet. Te manner by which the components contribute to the housed under standard temperature, humidity, and light antispasmolytic activity of SGD remains ambiguous. Te conditions. spectrum–efect relationship of TCM mainly apply corre- lational analysis, grey correlational analysis (GRA), multiple regression analysis, partial least squares regression (PLSR), 2.2. Apparatus and Conditions. HPLC analysis of SGD was principal component analysis, and other mathematical performed using Shimadzu LC-20A high performance liquid models to screen the bioactive compounds [23–25]. It is a chromatograph coupled with DAD detectors (Shimadzu biological efect-based evaluation method, which has been Corporation, Japan). Te chromatographic conditions were widely used to investigate the material basis of the phar- as follows: column, CAPCELL PAK-C18 reversed-phase macological efects of Chinese medicinal compounds (250 mm × 4.6 mm, 5 μm); mobile phase, acetonitrile (A), [26–28]. and 0.1% phosphoric acid in water (B); fow rate, 1.0 mL/ Terefore, this study was conducted to clarify the min; detection wavelength, 254 nm; column temperature, material basis of the antispasmodic efect of SGD by 30 C; and injection volume, 10 μL. Te gradient programme establishing the spectrum–efect relationship, determine is shown in Table 2. Journal of Analytical Methods in Chemistry 3 Table 1: Source information of 15 batches of Shaoyao-Gancao Decoction (SGD). No. Decoction pieces Origin Batch no. Paeoniae Radix Alba Anhui 2011007 S1 G. Uralensis, honeyed Gansu 20190227 Paeoniae Radix Alba Anhui 210811 S2 G. uralensis, honeyed Xinjiang 20201119 Paeoniae Radix Alba Anhui 21111604 S3 G. uralensis, honeyed Gansu 20092201 Paeoniae Radix Alba Anhui 20201203 S4 G. uralensis, honeyed Neimeng 200923 Paeoniae Radix Alba Anhui 21100701 S5 G. uralensis, honeyed Gansu 22022301 Paeoniae Radix Alba Anhui 21102303 S6 G. uralensis, honeyed Gansu 210801 Paeoniae Radix Alba Anhui 21031306 S7 G. uralensis, honeyed Gansu 20211202 Paeoniae Radix Alba Anhui 2110087 S8 G. uralensis, honeyed Gansu 210605 Paeoniae Radix Alba Anhui 2003011 S9 G. uralensis, honeyed Gansu 2107006 Paeoniae Radix Alba Anhui 2010077 S10 G. uralensis, honeyed Gansu 2011011 Paeoniae Radix Alba Anhui 07220051 S11 G. uralensis, honeyed Gansu 2009020 Paeoniae Radix Alba Anhui 07220045 S12 G. uralensis, honeyed Gansu 20033102 Paeoniae Radix Alba Zhejiang 20211025007 S13 G. uralensis, honeyed Gansu 2104011 Paeoniae Radix Alba Zhejiang 20211021001 S14 G. uralensis, honeyed Gansu 202202001 Paeoniae Radix Alba Anhui 21022301 S15 G. uralensis, honeyed Gansu 202108004 Table 2: Gradient program. 2.35 μg/mL; glycyrrhizic acid, 98.19 μg/mL; licochalcone A, 15.91 μg/mL; and glabridin, 3.63 μg/mL. Te mixed standard Time (min) A (%) B (%) solution was diluted stepwise with methanol solution to 0 90 10 obtain six diferent concentrations for the plotting of the 30 80 20 calibration curves. All standard solutions were stored at 4 C. 60 50 50 85 25 75 90 10 90 2.3.2. Sample Solution Preparation. Te daily dose of SGD 100 10 90 pieces (55.2 g) was precisely weighed, and 600 mL of water 105 90 10 was added each time. Te solution was decocted to ∼300 mL for 2 h and fltered. Te fltrates were combined. Ten, 200 mL of decoction was freeze-dried, and the other 100 mL 2.3. Solution Preparation of decoction was concentrated to 1 g/mL as a sample for the 2.3.1. Standard Solution Preparation. Certain amounts of the isolated smooth muscle experiment. Te SGD freeze-dried 15 reference standards were accurately weighed, individually powder (0.1 g) was precisely weighed, placed in a 10 mL placed in a 10 mL volumetric fask, and dissolved with volumetric fask, and ultrasonically extracted with 50% methanol to prepare stock solutions. A certain amount of methanol. Te sample solution was fltered through a each stock solution was placed in a 10 mL volumetric fask and 0.45 μm membrane and stored at 4 C. diluted to volume with methanol at the following concen- trations: oxypaeoniforin, 14.01 μg/mL; catechin, 33.00 μg/ mL; albiforin, 93.94 μg/mL; paeoniforin, 130.2 μg/mL; liq- 2.4. Validation of HPLC Analytical Method. Te blank sol- uiritin, 138.1 μg/mL; galloylpaeoniforin, 12.60 μg/mL; vent (50% methanol), standard solution, negative sample, 1,2,3,4,6-O-pentagalloylglucose, 17.72 μg/mL; ononin, and sample solution were separately injected according to 17.66 μg/mL; isoliquiritin, 15.29 μg/mL; licochalcone B, the chromatographic conditions under Section 2.2. Te 1.68 μg/mL; liquiritigenin, 25.11 μg/mL; benzoylpaeoniforin, chromatographic results were recorded. Te calibration 4 Journal of Analytical Methods in Chemistry curves were plotted with the concentration of tested refer- Chromatographic Fingerprint Similarity Evaluation System ence as the x-axis and the peak area as the y-axis. Te in- (version 2012). After chromatographic peak matching, the traday and interday precisions were determined by six standard fngerprint chromatogram “R” was generated, and repetitive injections on the same day and for three con- the fngerprints of the 15 batches of SGD samples were secutive days. Te stability test was evaluated by injecting the established (Figure 1). Te similarities between the sample sample solution at 0, 2, 4, 6, 8, 10, and 12 h after preparation. chromatograms and the reference chromatogram were Repeatability was determined by analysing six prepared calculated using the abovementioned software. Te simi- samples from the same source. Recovery was investigated by larities were all greater than 0.965 (Table 3), indicating adding an accurate amount of standard solution to 0.1 g of apparent similarity amongst the 15 batches of SGD. Ten, 20 the freeze-dried powder. Fifteen samples were prepared in common peaks in the reference chromatogram were parallel according to the preparation method of the sample assigned, and 16 compounds, including oxypaeoniforin, solution. catechin, albiforin, paeoniforin, liquiritin, galloypaeoni- forin, 1,2,3,4,6-O-pentagalloylglucose, ononin, isoliquiritin, licochalcone B, liquirtigenin, benzoylpaeoniforin, glycyr- 2.5. Isolated Rat Intestine Preparation. Te SD rats were rhizic acid, licochalcone A, glabridin, and glycyrrhetinic fasted for 24 h and drank water freely. Te rats were killed acid, were verifed after a comparison with the reference following a blow on the back of the head with a wooden substances. stick [29]. Te intestine segments (1.5 cm long) were Components: 1, oxypaeoniforin; 2, catechin; 3, albi- prepared, gently fushed with Tyrode bufer, and quickly forin; 4, paeoniforin; 5, liquiritin; 6, galloypaeoniforin; 7, placed in a Petri dish containing Tyrode bufer. According 1,2,3,4,6-O-pentagalloylglucose; 8, ononin; 9, isoliquiritin; to the physiological position from top to bottom, the upper 10, licochalcone B; 11, liquirtigenin; 12, benzoylpaeoniforin; end was connected to the tension transducer, and the 16, glycyrrhizic acid; 18, licochalcone A; 19, glabridin; and lower end was fxed to the L-shaped bent hook at the 20, glycyrrhetinic acid. bottom of the muscle groove. Each intestine segment was suspended in organ baths containing constantly oxygen- 3.2. Validation of the HPLC Method. A method that could ated Tyrode’s solution (20 mL, pH 8.2) at a constant ° ° temperature (37 distinguish the HPLC fngerprint and simultaneously C± 0.5 C) [30]. Fresh oxygen was con- determine the 15 compounds was established. Te vali- tinuously introduced at a rate of 1–2 bubbles per second. Te intestine segments were equilibrated for 55 ± 5 min dation of the method, including precision, repeatability, stability, linear regression, and recovery for 15 com- with drainage of the bufer with fresh oxygen after 15± 2 min. Te physiological response of the intestine pounds, is summarised in Table 4. Te results showed that the precision of the instrument and the repeatability of segments was recorded using an isometric force trans- the extraction method were good, and the sample was ducer (ML870) connected to a 4-channel bridge amplifer. stable within 12 h. All calibration curves showed good Te signals were amplifed by a data acquisition device linearity in the given concentration ranges. Te recovery Power Lab 8/35 hardware. Muscle contractions were rates for the spiked samples ranged from 93.9% to analysed using Lab Chart 8 software. Te equipment 109.9%. Tus, the validation of the HPLC method was hardware and software were from ADInstruments Pty Ltd. within an acceptable range in quantitative research, (Bella Vista, NSW, Australia) [31]. demonstrating that the established method was repro- Te possible antispasmodic activity of SGD was deter- mined by ACh (1 mM)-evoked contraction of the intestinal ducible for the fngerprint and the determination of 15 compounds in diferent batches of SGD. Te proposed smooth muscle. SGD was applied cumulatively to achieve a concentration-dependent inhibitory response, and the av- method can simultaneously determine 15 compounds and can provide a better alternative for the evaluation of erage tension was used as the index. the quality of SGD. 2.6. Statistical Analysis. Te chromatographic data of the 15 3.3. Measurement Results of Multiple Component SGD samples were evaluated using the Chromatographic Determination. Te quantities of the 15 components mea- Fingerprint Evaluation System for Chinese Medicine. sured in the SGD were calculated by substituting the re- Graph Pad Prism (8.0.) was applied for all statistical an- gression equation in Table 4. Table 5 shows that the content alyses and plotting of graphs. Te experimental values were of the 15 compounds in the diferent batches of SGD varied expressed as mean± standard (SEM) and tested by one-way to a certain extent. Te fact that the raw materials were ANOVA. P< 0.05 was considered to be a signifcant dif- derived from diferent sources may be the main reason for ference. GRA and PLSR were used to analyse the the fuctuation in the content of the tested compounds. We spectrum–efect. adopted a heatmap and box plot to intuitively display the content distribution [32]. Te heatmap refected the fuc- 3. Results tuation of the 15 compounds in diferent batches through 3.1. Establishment and Similarity Analysis of the HPLC the gradient colour. As shown in Figure 2, X3 (albiforin), X4 (paeoniforin), X5 (liquiritin), and X16 (glycyrrhizic acid) Fingerprint. Te chromatographic data of 15 batches of SGD were imported into the Chinese Medicine fuctuated obviously, refecting great variation amongst the Journal of Analytical Methods in Chemistry 5 2,400 2,300 2,200 2,100 2,000 8 15 12 14 9 11 17 1,900 12 3 6 10 13 18 19 20 R (20) 1,800 S15 (20) 1,700 1,600 S14 (20) 1,500 S13 (20) 1,400 S12 (20) 1,300 S11 (20) 1,200 S10 (20) 1,100 1,000 S9 (20) S8 (20) S7 (20) S6 (20) S5 (20) 400 S4 (20) S3 (20) S2 (20) S1 (20) 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 105 110 115 Figure 1: HPLC fngerprints of 15 batches of Shaoyao-Gancao Decoction (SGD; S1–S15) and control fngerprints (R). Table 3: Similarity between the sample and control fngerprints. No. Similarity S1 0.994 S2 0.968 S3 0.993 S4 0.995 S5 0.989 S6 0.989 S7 0.996 S8 0.984 S9 0.988 S10 0.993 S11 0.995 S12 0.995 S13 0.983 S14 0.982 S15 0.965 diferent batches. Te box size represents the dispersion (glycyrrhizic acid) can be used as characteristic components degree of the 15 index compounds in the diferent batches. when a quality standard is established. As shown in Figure 3, X2 (catechin), X3 (albiforin), X4 (paeoniforin), X5 (liquiritin), X11 (liquirtigenin), and X16 3.4.ResultsofIsolatedIntestinePreparation. In the intestinal (glycyrrhizic acid) were relatively large. As required by ChP, the quality of liquorice and Paeoniae Radix Alba were muscle study, Ach-induced intestine contractions were used to evaluate the antispasmolytic activity of the SGD evaluated by detecting the content of liquiritin, glycyrrhizic acid, and paeoniforin. As shown in Figure 4, the total samples. Table 6 shows that compared with the blank control group, the intestine contractions of the Ach model contents were markedly diferent, and the total contents of group were signifcantly increased (P < 0.05), indicating S2, S5, S6, and S10–S15 were higher than the average. Furthermore, their quality was better than the other con- that the model was successful. Compared with the Ach model group, 15 batches of SGD (25 mg/mL and 35 mg/ tents. Te average contents of 15 characteristic ingredients in SGD from high to low were as follows: X4> X mL) from diferent origins all signifcantly reduced the maximum contractile (P< 0.01), exerting concentration- 5> X3> X16> X2> X11> X8> X18> X7> X9> X1> X6> X 19> X12> X10. Tus, we suggest that X3 (albiforin), X4 dependent spasmolytic efects with the inhibitory re- sponse for Ach-evoked contraction. (paeoniforin), X5 (liquiritin), X11 (liquirtigenin), and X16 6 Journal of Analytical Methods in Chemistry Table 4: Linear regression, precision, repeatability, stability, and recovery for 15 components in SGD. Precision Stability Recovery Correlation Linear range Repeatability Compound Regression equation (RSD, %) (RSD, %) (RSD, %) coefcient (r) (mg/mL) (RSD, %) n � 6 n � 5 n � 6 n � 6 X1: oxypaeoniforin Y � 1233273.12X + 46449.80 0.9944 0.056–0.559 0.9 2.0 0.5 96.8± 0.4 X2: catechin Y � 213488.20X + 9856.90 0.9983 0.126–1.255 2.4 0.4 2.5 96.8± 1.8 X3: albiforin Y � 31301.02X + 3371.21 0.9995 0.371–3.711 2.8 1.8 2.8 97.0± 1.5 X4: paeoniforin Y � 353199.09X + 4111.98 0.9999 0.512–5.117 3.6 0.7 2.5 99.3± 1.4 X5: liquiritin Y � 443973.23 X−114367.63 0.9985 0.543–5.430 1.2 1.5 0.8 98.7± 1.2 X6: galloypaeoniforin Y � 931967.24X + 12146.77 0.9990 0.050–0.494 2.9 0.2 1.3 109.9± 1.7 X7: 1,2,3,4,6-O- Y � 3542609.33X + 9102.26 1.0000 0.069–0.695 1.0 1.4 0.4 101.8± 1.9 pentagalloylglucose X8: ononin Y � 937109.60X + 2791.93 1.0000 0.069–0.692 0.6 3.1 0.3 101.2± 0.5 X9: isoliquiritin Y � 1557077.22X + 3899.99 0.9999 0.061–0.611 0.8 2.1 0.7 108.7± 0.5 X10: licochalcone B Y � 1291329.69X + 338.63 0.9999 0.006–0.063 0.8 2.9 0.4 93.8± 0.5 X11: liquirtigenin Y � 132434.69X–1198.43 0.9998 0.100–1.000 0.5 1.7 0.3 107.3± 0.4 X12: Y � 1537913.99 X + 1164.36 1.0000 0.009–0.093 2.5 1.5 2.7 104.1± 1.1 benzoylpaeoniforin X16: glycyrrhizic acid Y � 858537.93X + 3267.07 0.9999 0.389–3.890 0.7 0.4 0.4 97.3± 0.1 X18: licochalcone A Y � 137500.90X–5234.42 0.9979 0.063–0.629 0.4 2.1 0.3 105.4± 0.6 X19: glabridin Y � 563254.16X + 431.74 0.9999 0.072–0.720 1.1 3.9 0.6 106.9± 0.5 Table 5: Content determination of the 15 compounds in 15 batches of SGD. Content (mg/g) Compound S1 S2 S3 S4 S5 S6 S7 S8 S9 S10 S11 S12 S13 S14 S15 X1: oxypaeoniforin 0.080 0.104 0.083 0.070 0.090 0.092 0.077 0.088 0.094 0.086 0.102 0.113 0.221 0.229 0.113 X2: catechin 0.753 0.490 0.546 0.501 0.410 0.591 0.563 0.335 0.381 0.552 0.625 0.457 0.950 0.652 0.544 X3: albiforin 1.634 2.690 2.067 2.265 2.238 2.044 1.789 2.079 1.705 1.807 2.200 2.225 3.000 3.259 3.490 X4: paeoniforin 3.586 3.666 3.966 3.035 4.161 4.028 3.659 3.456 3.266 3.866 4.042 4.175 2.737 2.500 3.365 X5: liquiritin 2.431 3.023 2.172 2.084 2.814 2.623 2.105 1.664 2.417 2.508 2.348 2.411 2.912 2.764 2.976 X6: galloypaeoniforin 0.082 0.096 0.108 0.087 0.103 0.100 0.102 0.101 0.073 0.083 0.085 0.095 0.064 0.055 0.157 X7: 1,2,3,4,6-O- 0.103 0.111 0.116 0.116 0.140 0.116 0.120 0.105 0.104 0.168 0.144 0.174 0.178 0.181 0.374 pentagalloylglucose X8: ononin 0.360 0.575 0.497 0.366 0.555 0.518 0.422 0.437 0.474 0.454 0.482 0.591 0.503 0.436 0.568 X9: isoliquiritin 0.128 0.121 0.104 0.100 0.153 0.140 0.098 0.072 0.117 0.135 0.117 0.101 0.136 0.152 0.151 X10: licochalcone B 0.002 0.003 0.007 0.004 0.002 0.004 0.003 0.002 0.002 0.001 0.007 0.007 0.006 0.004 0.001 X11: liquirtigenin 0.588 0.401 0.323 0.497 0.488 0.362 0.365 0.382 0.597 0.843 0.936 0.294 0.871 0.711 0.392 X12: benzoylpaeoniforin 0.007 0.005 0.005 0.008 0.004 0.008 0.007 0.011 0.010 0.011 0.012 0.013 0.016 0.016 0.020 X16: glycyrrhizic acid 1.615 1.367 1.410 1.358 1.663 1.663 1.554 1.108 1.803 2.060 1.904 1.583 2.209 2.131 1.477 X18: licochalcone A 0.521 0.151 0.223 0.232 0.115 0.158 0.274 0.196 0.167 0.410 0.263 0.218 0.390 0.370 0.292 X19: glabridin 0.026 0.014 0.028 0.039 0.014 0.016 0.037 0.029 0.028 0.049 0.040 0.033 0.051 0.042 0.026 3.5. Spectral-Efect Relevance Analysis. GRA is a quantita- 3.5.1. PLSR. In addition to GRA, the relationship between the tive analytical method widely used to analyse the corre- 20 common peaks (x-variables) and the antispasmolytic ef- lation between the compound and its efcacy [16]. A cacy (y-variables) was also evaluated by using a PLSR model. correlation coefcient greater than 0.8 indicates a strong PLSR is a method that can describe which peaks contribute correlation [33]. Te common peaks and the inhibition positively or negatively to the efcacy. As shown in Figure 5, the rate of SGD on the intestinal contraction after Z-score peaks X4, X6 and X9–X19 were correlated strongly with the normalisation were used as the X and Y matrices, re- antispasmolytic efect with high positive correlation coef- cients. Te remaining seven peaks were negatively correlated spectively, in the GRA to fnd the active compounds corresponding to the antispasmolytic efcacy. Te results with the inhibition rate. Furthermore, the VIP value can describe the degree of of the GRA are shown in Table 7. Te correlation coef- fcients of X4 (paeoniforin), X15, X14, X8 (ononin), X6 explanation of the independent variable on the dependent (galloypaeoniforin), X5 (liquiritin), X9 (isoliquiritin), variable. Te larger the VIP value, the greater the correlation and X16 (glycyrrhizic acid) were higher than 0.8, indi- between the variable and the drug efcacy will be [34]. When cating their major role in the antispasmolytic activity of the VIP is greater than 1, the characteristic peak has a more SGD. Tese results also signify that SGD exerted anti- important role in the antispasmolytic efcacy. As shown in spasmolytic efects through multicomponent synergy. Figure 6, the VIP values of peaks X10, X4, X1, X11, X3, X2, Journal of Analytical Methods in Chemistry 7 S1 S2 S3 S4 S5 S6 S7 S8 S9 S10 S11 S12 S13 S14 S15 X1 X2 X3 X4 X5 X6 X7 X8 X9 X10 X11 X12 X16 X18 X19 compounds Figure 2: Heatmap of 15 compounds in 15 batches of SGD. X1 X2 X3 X4 X5 X6 X7 X8 X9 X10 X11 X12 X16 X18 X19 compounds Figure 3: Box plot of 15 compounds in 15 batches of SGD. X18, and X16 were greater than 1, indicating biological (glycyrrhizic acid) were identifed as the major core anti- signifcance. spasmolytic compounds in SGD. Tis fnding also indicated Combining the results from the GRA and PLSR models, that the antispasmodic efect of SGD is the result of the correlation degree >0.8, VIP> 1 and positive correlation, the synergistic efect of multiple components. Many studies have peaks of X4 (paeoniforin), X11 (liquirtigenin), and X16 shown that glycyrrhizic acid and paeoniforin have Batches Content (mg/g) 8 Journal of Analytical Methods in Chemistry S1 S2 S3 S4 S5 S6 S7 S8 S9 S10 S11 S12 S13 S14 S15 Average X19: Glabridin X7: 1,2,3,4,6-O-pentagalloylglucose X18: Licochalcone A X6: Galloypaeoniforin X5: Liquiritin X16: Glycyrrhizic acid X4: Paeoniforin X12: Benzoylpaeoniforin X3: Albiforin X11: Liquirtigenin X2: Catechin X1: Oxypaeoniforin X10: Licochalcone B X9: Isoliquiritin X8: Ononin Figure 4: Bar graph of the total contents in 15 batches of SGD. Total content (mg/g) Journal of Analytical Methods in Chemistry 9 Table 6: Efect of 15 batches of SGD on the contractile activity of isolated intestinal preparation (X ± SME, n � 6). Average tension (g) Inhibition rate (%) Drug Drug Batch Drug administration group Drug administration group Drug administration group Control group Ach model group administration administration (15 mg/mL) (25 mg/mL) (35 mg/mL) group (25 mg/mL) group (35 mg/mL) ## ∗∗ ∗∗ S1 0.4483± 0.04920 1.426± 0.1244 1.341± 0.0984 0.6487± 0.0472 0.4776± 0.0430 78.02± 5.549 97.05± 4.003 ∗∗ S2 0.5204± 0.01452 1.168± 0.1892 1.194± 0.1848 0.6943± 0.1398 0.4543± 0.06812 82.90± 12.32 120.3± 9.962 ## ∗∗ ∗∗ S3 0.4285± 0.03723 1.216± 0.1397 0.9784± 0.1218 0.5199± 0.05595 0.3260± 0.02184 88.08± 3.246 114.2± 2.65 ## ∗∗ ∗∗ S4 0.5635± 0.06599 1.914± 0.3118 1.703± 0.2831 0.8390± 0.07993 0.5414± 0.05365 86.11± 9.202 111± 8.952 ## ∗∗ ∗∗ S5 0.4279± 0.06277 1.358± 0.1667 1.160± 0.1562 0.5640± 0.08049 0.4190± 0.06737 84.74± 5.133 101.6± 2.695 ## ∗∗ ∗∗ S6 0.5296± 0.04119 1.159± 0.09607 0.9905± 0.09806 0.6552± 0.05702 0.5028± 0.03212 82.45± 5.394 103.9± 3.426 ## ∗∗ ∗∗ S7 0.4323± 0.06757 1.124± 0.1909 0.7886± 0.05031 0.3899± 0.03225 0.2655± 0.0438 99.13± 9.353 120.2± 7.817 ## ∗∗ ∗∗ S8 0.5116± 0.04533 0.9918± 0.05947 1.001± 0.03622 0.7059± 0.02238 0.5774± 0.01672 58.10± 8.560 85.36± 7.416 ∗∗ S9 0.4842± 0.05229 1.063± 0.1186 0.984± 0.2091 0.6202± 0.09016 0.3968± 0.0398 75.91± 7.580 113.4± 6.972 ## ∗ ∗∗ ∗∗ S10 0.4960± 0.05865 1.409± 0.1962 0.9719± 0.08651 0.4914± 0.04814 0.368± 0.05182 104.2± 5.288 120.4± 10.35 ## ∗∗∗ ∗∗ S11 0.3607± 0.04392 0.9241± 0.1457 0.5863± 0.07636 0.3203± 0.04721 0.2587± 0.05415 110.2± 4.840 117.5± 10.68 ## ∗∗ ∗∗ S12 0.3917± 0.03975 1.433± 0.2150 1.249± 0.2104 0.6738± 0.1062 0.4462± 0.07728 75.15± 5.218 98.39± 5.121 ∗∗ ∗∗ S13 0.5547± 0.09259 1.113± 0.1566 0.9223± 0.1112 0.5019± 0.0944 0.4470± 0.09103 107.4± 7.044 118± 6.441 ## ∗ ∗∗ ∗∗ S14 0.4113± 0.03592 0.8625± 0.08512 0.6173± 0.05789 0.3951± 0.03806 0.3036± 0.02892 108.9± 7.633 127.4± 5.618 S15 0.4967 + 0.02877 1.243± 0.2305 1.117± 0.2264 0.7182± 0.1251 0.5096± 0.06343 67.52± 9.100 94.27± 5.152 # ## ∗ ∗∗ Compared with the control group: P< 0.05; P< 0.01; compared with the Ach model group: P< 0.05; P< 0.01. 10 Journal of Analytical Methods in Chemistry Table 7: GRA results. Peaks Correlation Ranking X4 0.849 1 X15 0.843 2 X14 0.841 3 X8 0.836 4 X6 0.835 5 X5 0.834 6 X9 0.82 7 X16 0.812 8 X12 0.794 9 X7 0.784 10 X1 0.776 11 X3 0.774 12 X2 0.772 13 X17 0.77 14 X18 0.734 15 X19 0.728 16 X11 0.685 17 X20 0.668 18 X13 0.656 19 X10 0.653 20 0.2 -0.2 X1 X2 X3 X4 X5 X6 X7 X8 X9 X10 X11 X12 X13 X14 X15 X16 X17 X18 X19 X20 Var ID (Primary) Figure 5: Coefcient of correlation of 20 peaks with antispasmolytic activity. X10 X4 X1 X3 X11 X2 X18 X16 X9 X13 X14 X15 X19 X5 X6 X12 X7 X17 X8 X20 Var ID (Primary) Figure 6: VIP contribution plot of common peak antispasmolytic activity. Coefcient VIP Journal of Analytical Methods in Chemistry 11 efcacy and treatment efect of SGD. Meanwhile, for the frst time, the fngerprint of SGD was associated with 1.0 antispasmolytic activity. Paeoniforin, liquirtigenin, and glycyrrhizic acid were highly correlated with anti- ** ** spasmolytic activity, revealing that multiple components ** ## exhibited antispasmolytic activities. Furthermore, the 0.5 potential antispasmolytic components were validated. Paeoniforin (11.70–29.25 μg/mL) and liquirtigenin (17.19–28.65 μg/mL) could signifcantly reduce the max- 0.0 imum contractile (P< 0.01) and exerted concentration- Control Ach 5.85 11.70 17.55 23.40 29.25 dependent spasmolytic efects with the inhibitory re- model Paeoniforin (μg/mL) sponse for ACh-induced contraction. Tus, SGD had a signifcant antispasmodic efect, which could have resulted from the synergistic activity of its paeoniforin, liquirtigenin and other components. 1.0 Te results of the quantitative analysis of the multi- component in SGD could help to discover the material ** basis of SGD and establish a system for modern TCM ** quality standards. Te spectral–efect study can be used for ## 0.5 the rapid screening of potential antispasmolytic compo- nents in TCM. Verifcation experiment was carried out by testing the activity of the single standards to further de- termine the antispasmodic efcacy of SGD. Te results can provide a reference for the pharmacodynamics study of 0.0 SGD and are highly signifcant for the determination of Control Ach 5.73 11.46 17.19 22.92 28.65 quality markers and quality control. Given that TCM is model Liquiritigenin (μg/mL) characterised by multiple components, targets, and ap- Figure 7: Efects of paeoniforin and liquirtigenin on the con- proaches, further study on the spasmolytic targets and tractile activity (X± SME, n � 6). mechanism of SGD is needed. Data Availability synergistic efects on antispasticity, antipyretic, anti-in- fammatory, inhibition of gastric secretion, and relaxation of Te data used to support the fndings of this study are in- smooth muscle [35]. cluded within the article. 3.5.2. Experimental Verifcation. Tree antispasmolytic ac- Conflicts of Interest tive compounds, namely, paeoniforin, liquirtigenin, and glycyrrhizic acid, were obtained by spectral–efect rela- Te authors declare that there are no conficts of interests. tionship. Isolated intestine preparation was used to further verify the feasibility and accuracy of the spectral–efect re- Acknowledgments lationship method. As shown in Figure 7, paeoniforin (11.70–29.25 μg/mL) and liquirtigenin (17.19–28.65 μg/mL) Tis work was supported by the National Natural Science could signifcantly reduce the maximum contractile Foundation of China (No. 82160457), Lanzhou Science and (P< 0.01) and exerted concentration-dependent spasmolytic Technology Program (No. 2021-1-185), Medical Products efects with the inhibitory response for Ach-induced Administration Program of Gansu Province (No. contraction. 2020GSMPA027), Natural Science Foundation of Gansu Compared with the control group, P< 0.05; compared Province (No. 21JR7RA564), Program of Key Laboratory of with the Ach model P< 0.01. Chemistry and Quality for Traditional Chinese Medicines College of Gansu Province (No. zzy-2022-07). 4. 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Quantitative Analysis of the Multicomponent and Spectrum–Effect Correlation of the Antispasmodic Activity of Shaoyao-Gancao Decoction

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Hindawi Journal of Analytical Methods in Chemistry Volume 2022, Article ID 2279404, 13 pages https://doi.org/10.1155/2022/2279404 Research Article Quantitative Analysis of the Multicomponent and Spectrum–Effect Correlation of the Antispasmodic Activity of Shaoyao-Gancao Decoction 1,2 2 2 2 1,3,4,5 1,3 Yanli Xu, Chenxi Li, Ting Chen, Xiaochun Li, Xiaoyu Wu, Qili Zhang, 1,2,3,4,5 and Lei Zhao Gansu University of Chinese Medicine, Lanzhou 730000, China Lanzhou Institute for Food and Drug Control, Lanzhou 730000, China Northwest Collaborative Innovation Center for Traditional Chinese Medicine Co-Constructed By Gansu Province & MOE of PRC, Lanzhou 730000, China Key Laboratory of Chemistry and Quality of TCM of the College of Gansu Province, Lanzhou 730000, China Gansu Province Engineering Laboratory for TCM Standardization Technology and Popularization, Lanzhou 730000, China Correspondence should be addressed to Lei Zhao; zzyhx@gszy.edu.cn Received 21 July 2022; Revised 16 November 2022; Accepted 24 November 2022; Published 2 December 2022 Academic Editor: Cecilia Cagliero Copyright © 2022 Yanli Xu et al. Tis is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Shaoyao-Gancao Decoction (SGD) is a well-known classic traditional Chinese medicine (TCM) with antispasmodic, anti-in- fammatory, and analgesic efects. Tis preparation has been widely used to treat spasticity diseases in the clinic. To date, the material basis of SGD remains unclear, and the spectrum-efect correlation of its antispasmodic activity has not been reported yet. In this study, high-performance liquid chromatography (HPLC) was used to establish the fngerprint and determine the multiple components of SGD. Te common peaks of fngerprints were evaluated by the similarity with the chromatographic fngerprints of the TCM. Meanwhile, the multiple components were quantifed and analysed using the heatmap and box size analysis. Fur- thermore, data on the antispasmodic efect were extracted through in vitro smooth muscle contraction assay. Grey relational analysis combined with partial least square regression was used to study the spectrum–efect correlation of SGD. Finally, the potential antispasmolytic components were validated using an isolated tissue experiment. Te HPLC fngerprint was established, and 20 common peaks were identifed. Te similarities of 15 batches of SGD were all above 0.965. Te HPLC method for simultaneous determination of the multiple components was accurate and reliable. Te contents of albiforin, paeoniforin, liquiritin, and glycyrrhizic acid were higher than the other components in SGD. Te heatmap and box size also showed that X3 (albiforin), X4 (paeoniforin), X5 (liquiritin), X11 (liquirtigenin), and X16 (glycyrrhizic acid) could be used as quality indicators in the further establishment of quality standards. Te spectrum–efect correlation results indicated that X4, X11, and X16 were highly correlated with antispasmolytic activity. Verifcation tests showed that paeoniforin (11.7–29.25 μg/mL) and liquirtigenin (17.19–28.65 μg/mL) could signifcantly reduce the maximum contractile (P < 0.01). Tese compounds exerted concentration- dependent spasmolytic efects with the inhibitory response for acetylcholine (Ach)-evoked contraction. Tus, SGD had a sig- nifcant antispasmodic efect, which resulted from the synergistic activity of its multiple components. Tese fndings can be used for the pharmacodynamics study of SGD and are of great signifcance for the determination of quality markers and quality control. Tere has been much debate about the defnition of spas- 1. Introduction ticity. In 1980, Lance was the frst scholar to associate Spasticity is a velocity-dependent increase in muscle tone spasticity to the velocity-dependent increase in stretch refex caused by the increased excitability of muscle spindles [3]. A more general defnition of spasticity is disordered following an upper motor neuron (UMN) syndrome [1, 2]. sensory-motor control, resulting from UMN lesion, 2 Journal of Analytical Methods in Chemistry the multiple components of SGD, and validate the presenting intermittent or sustained involuntary activation of muscles [4]. Tis complex phenomenon of extremely spectrum–efect results. Scientifc basis for the secondary development and quality control of SGD was also variable clinical expression, which may cause diferent motor dysfunctions, has been observed in many patients provided. with spinal cord injury, cerebral palsy, multiple sclerosis, and acquired brain injury, which directly impact the quality 2. Materials and Methods of life [5]. Presently, the conventional drugs used in the treatment of spasm include baclofen, tizanidine, and dan- 2.1. Samples, Reagents, and Animals. Fifteen batches of SGD trolene, amongst others. However, the efcacy of these were purchased from drug manufacturers in Gansu Prov- current treatments is not absolute, and they may have se- ince. Te sources of herbal materials used in SGD are shown rious side efects [6–8]. in Table 1. Te raw materials were identifed by Renyuan Accordingly, natural products with therapeutic activity Zhu (a senior engineer in the Lanzhou Institute for Food and against spasm must be searched to replace drugs with strong Drug Control). Paeoniae Radix Alba is the dried root slice of side efects. Shaoyao-Gancao Decoction (SGD) is a classic Paeonia lactifora Pall. Roasted liquorice is the root and traditional Chinese medicine (TCM) and originally de- rhizome of G. uralensis Fisch., Glycyrrhiza infata Bat., or scribed in the Treatise on Febrile Disease. SGD is composed Glycyrrhiza glabra L. Te quality of the herbal materials of Paeoniae Radix Alba (baoshao in Chinese) and honeyed complied with the standards of the National 2020 Glycyrrhiza uralensis, which are traditionally used to treat Pharmacopeia. spastic diseases, such as gastrointestinal spasm, facial muscle Methanol and acetonitrile (HPLC grade) were pur- spasm, and poststroke spasm [9]. Modern pharmacological chased from Honeywell China Co., Ltd. (Shanghai, China). and clinical studies have confrmed that SGD has signifcant Water was ultrapure. Glycyrrhizic acid (No. antispasmodic, anti-infammatory, and analgesic efects on 110731–202122, purity >99.06%) and catechin (No. various spastic diseases [10], infammatory diseases [11], 110877–202005, purity >95.1%) reference substances were painful diseases [12, 13], gynopathy [14], bronchial asthma, purchased from the China Institute of Food and Drug Parkinson’s disease, and constipation [15, 16]. Tis TCM has Control (Beijing, China). Isoliquiritigenin (No. 20112401, been selected for the frst batch of the Chinese Medicine purity >99.04%), 1,2,3,4,6-O-pentagalloylglucose (No. Classical Directory. Studies have shown that the extract of 19010904, purity >98%), ononin (No. 19071501, purity SGD and liquorice exerts a relaxant efect on acetylcholine >98%), liquiritin apioside (No. 20111607, purity >98.28%), (ACh)-induced contraction, isoliquiritigenin, and glycy- licochalcone B (No. 20121501, purity >93%), galloylpaeo- coumarin isolated from the roots of liquorice and has a niforin (No. 171013, purity >98%), licochalcone A (No. potent antispasmodic [17, 18]. Te ethanol extract of Gly- 19102405, purity >98.89%), oxypaeoniforin (No. cyrrhiza uralensis has signifcant inhibitory efects on Nav1.4 19120604, purity >99.67%), isoliquiritin (No. 20041301, VGSCs, which may be an important mechanism in the purity >99.95%), liquiritigenin (No. 21052404, purity treatment of gastrocnemius spasm [19]. >99.86%), albiforin (No. 21031706, purity >98.75%), TCM is characterised by multiple components, targets, glabridin (No. 21060801, purity >99.08%), benzoylpaeo- and approaches, and SGD has a complex composition. niforin (No. 20092303, purity >99.43%), and paeoniforin Previous studies have shown that glycyrrhizin, glycyrrhetic (No. 20030901, purity >98.2%) reference substances were acid, paeoniforin, albiforin, oxypaeoniforin, liquiritin, purchased from the Chengdu Grip Biotechnology Co., Ltd. liquiritigenin, isoliquiritin, isoliquiritigenin, and 1,2,3,4,6- (Chengdu, China). Other compounds were of analytical O-pentagalloylglucose are the main bioactive compounds of grade. SGD [20–22]. Research on SGD has mainly focused on its Male adult Sprague–Dawley (SD) rats, 3–6 months of clinical application, chemical composition, and anti-in- age, and weighing 250–300 g, were obtained from the fammatory and analgesic efects. However, the pharma- Animal Experiment Center of Gansu University of Tra- codynamic basis of the antispasmodic efect and the ditional Chinese Medicine (Approval No. SCXK[Gan] spectrum–efect relationship of SGD has not been reported 2020–0009, Lanzhou, Gansu, China). Te animals were yet. Te manner by which the components contribute to the housed under standard temperature, humidity, and light antispasmolytic activity of SGD remains ambiguous. Te conditions. spectrum–efect relationship of TCM mainly apply corre- lational analysis, grey correlational analysis (GRA), multiple regression analysis, partial least squares regression (PLSR), 2.2. Apparatus and Conditions. HPLC analysis of SGD was principal component analysis, and other mathematical performed using Shimadzu LC-20A high performance liquid models to screen the bioactive compounds [23–25]. It is a chromatograph coupled with DAD detectors (Shimadzu biological efect-based evaluation method, which has been Corporation, Japan). Te chromatographic conditions were widely used to investigate the material basis of the phar- as follows: column, CAPCELL PAK-C18 reversed-phase macological efects of Chinese medicinal compounds (250 mm × 4.6 mm, 5 μm); mobile phase, acetonitrile (A), [26–28]. and 0.1% phosphoric acid in water (B); fow rate, 1.0 mL/ Terefore, this study was conducted to clarify the min; detection wavelength, 254 nm; column temperature, material basis of the antispasmodic efect of SGD by 30 C; and injection volume, 10 μL. Te gradient programme establishing the spectrum–efect relationship, determine is shown in Table 2. Journal of Analytical Methods in Chemistry 3 Table 1: Source information of 15 batches of Shaoyao-Gancao Decoction (SGD). No. Decoction pieces Origin Batch no. Paeoniae Radix Alba Anhui 2011007 S1 G. Uralensis, honeyed Gansu 20190227 Paeoniae Radix Alba Anhui 210811 S2 G. uralensis, honeyed Xinjiang 20201119 Paeoniae Radix Alba Anhui 21111604 S3 G. uralensis, honeyed Gansu 20092201 Paeoniae Radix Alba Anhui 20201203 S4 G. uralensis, honeyed Neimeng 200923 Paeoniae Radix Alba Anhui 21100701 S5 G. uralensis, honeyed Gansu 22022301 Paeoniae Radix Alba Anhui 21102303 S6 G. uralensis, honeyed Gansu 210801 Paeoniae Radix Alba Anhui 21031306 S7 G. uralensis, honeyed Gansu 20211202 Paeoniae Radix Alba Anhui 2110087 S8 G. uralensis, honeyed Gansu 210605 Paeoniae Radix Alba Anhui 2003011 S9 G. uralensis, honeyed Gansu 2107006 Paeoniae Radix Alba Anhui 2010077 S10 G. uralensis, honeyed Gansu 2011011 Paeoniae Radix Alba Anhui 07220051 S11 G. uralensis, honeyed Gansu 2009020 Paeoniae Radix Alba Anhui 07220045 S12 G. uralensis, honeyed Gansu 20033102 Paeoniae Radix Alba Zhejiang 20211025007 S13 G. uralensis, honeyed Gansu 2104011 Paeoniae Radix Alba Zhejiang 20211021001 S14 G. uralensis, honeyed Gansu 202202001 Paeoniae Radix Alba Anhui 21022301 S15 G. uralensis, honeyed Gansu 202108004 Table 2: Gradient program. 2.35 μg/mL; glycyrrhizic acid, 98.19 μg/mL; licochalcone A, 15.91 μg/mL; and glabridin, 3.63 μg/mL. Te mixed standard Time (min) A (%) B (%) solution was diluted stepwise with methanol solution to 0 90 10 obtain six diferent concentrations for the plotting of the 30 80 20 calibration curves. All standard solutions were stored at 4 C. 60 50 50 85 25 75 90 10 90 2.3.2. Sample Solution Preparation. Te daily dose of SGD 100 10 90 pieces (55.2 g) was precisely weighed, and 600 mL of water 105 90 10 was added each time. Te solution was decocted to ∼300 mL for 2 h and fltered. Te fltrates were combined. Ten, 200 mL of decoction was freeze-dried, and the other 100 mL 2.3. Solution Preparation of decoction was concentrated to 1 g/mL as a sample for the 2.3.1. Standard Solution Preparation. Certain amounts of the isolated smooth muscle experiment. Te SGD freeze-dried 15 reference standards were accurately weighed, individually powder (0.1 g) was precisely weighed, placed in a 10 mL placed in a 10 mL volumetric fask, and dissolved with volumetric fask, and ultrasonically extracted with 50% methanol to prepare stock solutions. A certain amount of methanol. Te sample solution was fltered through a each stock solution was placed in a 10 mL volumetric fask and 0.45 μm membrane and stored at 4 C. diluted to volume with methanol at the following concen- trations: oxypaeoniforin, 14.01 μg/mL; catechin, 33.00 μg/ mL; albiforin, 93.94 μg/mL; paeoniforin, 130.2 μg/mL; liq- 2.4. Validation of HPLC Analytical Method. Te blank sol- uiritin, 138.1 μg/mL; galloylpaeoniforin, 12.60 μg/mL; vent (50% methanol), standard solution, negative sample, 1,2,3,4,6-O-pentagalloylglucose, 17.72 μg/mL; ononin, and sample solution were separately injected according to 17.66 μg/mL; isoliquiritin, 15.29 μg/mL; licochalcone B, the chromatographic conditions under Section 2.2. Te 1.68 μg/mL; liquiritigenin, 25.11 μg/mL; benzoylpaeoniforin, chromatographic results were recorded. Te calibration 4 Journal of Analytical Methods in Chemistry curves were plotted with the concentration of tested refer- Chromatographic Fingerprint Similarity Evaluation System ence as the x-axis and the peak area as the y-axis. Te in- (version 2012). After chromatographic peak matching, the traday and interday precisions were determined by six standard fngerprint chromatogram “R” was generated, and repetitive injections on the same day and for three con- the fngerprints of the 15 batches of SGD samples were secutive days. Te stability test was evaluated by injecting the established (Figure 1). Te similarities between the sample sample solution at 0, 2, 4, 6, 8, 10, and 12 h after preparation. chromatograms and the reference chromatogram were Repeatability was determined by analysing six prepared calculated using the abovementioned software. Te simi- samples from the same source. Recovery was investigated by larities were all greater than 0.965 (Table 3), indicating adding an accurate amount of standard solution to 0.1 g of apparent similarity amongst the 15 batches of SGD. Ten, 20 the freeze-dried powder. Fifteen samples were prepared in common peaks in the reference chromatogram were parallel according to the preparation method of the sample assigned, and 16 compounds, including oxypaeoniforin, solution. catechin, albiforin, paeoniforin, liquiritin, galloypaeoni- forin, 1,2,3,4,6-O-pentagalloylglucose, ononin, isoliquiritin, licochalcone B, liquirtigenin, benzoylpaeoniforin, glycyr- 2.5. Isolated Rat Intestine Preparation. Te SD rats were rhizic acid, licochalcone A, glabridin, and glycyrrhetinic fasted for 24 h and drank water freely. Te rats were killed acid, were verifed after a comparison with the reference following a blow on the back of the head with a wooden substances. stick [29]. Te intestine segments (1.5 cm long) were Components: 1, oxypaeoniforin; 2, catechin; 3, albi- prepared, gently fushed with Tyrode bufer, and quickly forin; 4, paeoniforin; 5, liquiritin; 6, galloypaeoniforin; 7, placed in a Petri dish containing Tyrode bufer. According 1,2,3,4,6-O-pentagalloylglucose; 8, ononin; 9, isoliquiritin; to the physiological position from top to bottom, the upper 10, licochalcone B; 11, liquirtigenin; 12, benzoylpaeoniforin; end was connected to the tension transducer, and the 16, glycyrrhizic acid; 18, licochalcone A; 19, glabridin; and lower end was fxed to the L-shaped bent hook at the 20, glycyrrhetinic acid. bottom of the muscle groove. Each intestine segment was suspended in organ baths containing constantly oxygen- 3.2. Validation of the HPLC Method. A method that could ated Tyrode’s solution (20 mL, pH 8.2) at a constant ° ° temperature (37 distinguish the HPLC fngerprint and simultaneously C± 0.5 C) [30]. Fresh oxygen was con- determine the 15 compounds was established. Te vali- tinuously introduced at a rate of 1–2 bubbles per second. Te intestine segments were equilibrated for 55 ± 5 min dation of the method, including precision, repeatability, stability, linear regression, and recovery for 15 com- with drainage of the bufer with fresh oxygen after 15± 2 min. Te physiological response of the intestine pounds, is summarised in Table 4. Te results showed that the precision of the instrument and the repeatability of segments was recorded using an isometric force trans- the extraction method were good, and the sample was ducer (ML870) connected to a 4-channel bridge amplifer. stable within 12 h. All calibration curves showed good Te signals were amplifed by a data acquisition device linearity in the given concentration ranges. Te recovery Power Lab 8/35 hardware. Muscle contractions were rates for the spiked samples ranged from 93.9% to analysed using Lab Chart 8 software. Te equipment 109.9%. Tus, the validation of the HPLC method was hardware and software were from ADInstruments Pty Ltd. within an acceptable range in quantitative research, (Bella Vista, NSW, Australia) [31]. demonstrating that the established method was repro- Te possible antispasmodic activity of SGD was deter- mined by ACh (1 mM)-evoked contraction of the intestinal ducible for the fngerprint and the determination of 15 compounds in diferent batches of SGD. Te proposed smooth muscle. SGD was applied cumulatively to achieve a concentration-dependent inhibitory response, and the av- method can simultaneously determine 15 compounds and can provide a better alternative for the evaluation of erage tension was used as the index. the quality of SGD. 2.6. Statistical Analysis. Te chromatographic data of the 15 3.3. Measurement Results of Multiple Component SGD samples were evaluated using the Chromatographic Determination. Te quantities of the 15 components mea- Fingerprint Evaluation System for Chinese Medicine. sured in the SGD were calculated by substituting the re- Graph Pad Prism (8.0.) was applied for all statistical an- gression equation in Table 4. Table 5 shows that the content alyses and plotting of graphs. Te experimental values were of the 15 compounds in the diferent batches of SGD varied expressed as mean± standard (SEM) and tested by one-way to a certain extent. Te fact that the raw materials were ANOVA. P< 0.05 was considered to be a signifcant dif- derived from diferent sources may be the main reason for ference. GRA and PLSR were used to analyse the the fuctuation in the content of the tested compounds. We spectrum–efect. adopted a heatmap and box plot to intuitively display the content distribution [32]. Te heatmap refected the fuc- 3. Results tuation of the 15 compounds in diferent batches through 3.1. Establishment and Similarity Analysis of the HPLC the gradient colour. As shown in Figure 2, X3 (albiforin), X4 (paeoniforin), X5 (liquiritin), and X16 (glycyrrhizic acid) Fingerprint. Te chromatographic data of 15 batches of SGD were imported into the Chinese Medicine fuctuated obviously, refecting great variation amongst the Journal of Analytical Methods in Chemistry 5 2,400 2,300 2,200 2,100 2,000 8 15 12 14 9 11 17 1,900 12 3 6 10 13 18 19 20 R (20) 1,800 S15 (20) 1,700 1,600 S14 (20) 1,500 S13 (20) 1,400 S12 (20) 1,300 S11 (20) 1,200 S10 (20) 1,100 1,000 S9 (20) S8 (20) S7 (20) S6 (20) S5 (20) 400 S4 (20) S3 (20) S2 (20) S1 (20) 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 105 110 115 Figure 1: HPLC fngerprints of 15 batches of Shaoyao-Gancao Decoction (SGD; S1–S15) and control fngerprints (R). Table 3: Similarity between the sample and control fngerprints. No. Similarity S1 0.994 S2 0.968 S3 0.993 S4 0.995 S5 0.989 S6 0.989 S7 0.996 S8 0.984 S9 0.988 S10 0.993 S11 0.995 S12 0.995 S13 0.983 S14 0.982 S15 0.965 diferent batches. Te box size represents the dispersion (glycyrrhizic acid) can be used as characteristic components degree of the 15 index compounds in the diferent batches. when a quality standard is established. As shown in Figure 3, X2 (catechin), X3 (albiforin), X4 (paeoniforin), X5 (liquiritin), X11 (liquirtigenin), and X16 3.4.ResultsofIsolatedIntestinePreparation. In the intestinal (glycyrrhizic acid) were relatively large. As required by ChP, the quality of liquorice and Paeoniae Radix Alba were muscle study, Ach-induced intestine contractions were used to evaluate the antispasmolytic activity of the SGD evaluated by detecting the content of liquiritin, glycyrrhizic acid, and paeoniforin. As shown in Figure 4, the total samples. Table 6 shows that compared with the blank control group, the intestine contractions of the Ach model contents were markedly diferent, and the total contents of group were signifcantly increased (P < 0.05), indicating S2, S5, S6, and S10–S15 were higher than the average. Furthermore, their quality was better than the other con- that the model was successful. Compared with the Ach model group, 15 batches of SGD (25 mg/mL and 35 mg/ tents. Te average contents of 15 characteristic ingredients in SGD from high to low were as follows: X4> X mL) from diferent origins all signifcantly reduced the maximum contractile (P< 0.01), exerting concentration- 5> X3> X16> X2> X11> X8> X18> X7> X9> X1> X6> X 19> X12> X10. Tus, we suggest that X3 (albiforin), X4 dependent spasmolytic efects with the inhibitory re- sponse for Ach-evoked contraction. (paeoniforin), X5 (liquiritin), X11 (liquirtigenin), and X16 6 Journal of Analytical Methods in Chemistry Table 4: Linear regression, precision, repeatability, stability, and recovery for 15 components in SGD. Precision Stability Recovery Correlation Linear range Repeatability Compound Regression equation (RSD, %) (RSD, %) (RSD, %) coefcient (r) (mg/mL) (RSD, %) n � 6 n � 5 n � 6 n � 6 X1: oxypaeoniforin Y � 1233273.12X + 46449.80 0.9944 0.056–0.559 0.9 2.0 0.5 96.8± 0.4 X2: catechin Y � 213488.20X + 9856.90 0.9983 0.126–1.255 2.4 0.4 2.5 96.8± 1.8 X3: albiforin Y � 31301.02X + 3371.21 0.9995 0.371–3.711 2.8 1.8 2.8 97.0± 1.5 X4: paeoniforin Y � 353199.09X + 4111.98 0.9999 0.512–5.117 3.6 0.7 2.5 99.3± 1.4 X5: liquiritin Y � 443973.23 X−114367.63 0.9985 0.543–5.430 1.2 1.5 0.8 98.7± 1.2 X6: galloypaeoniforin Y � 931967.24X + 12146.77 0.9990 0.050–0.494 2.9 0.2 1.3 109.9± 1.7 X7: 1,2,3,4,6-O- Y � 3542609.33X + 9102.26 1.0000 0.069–0.695 1.0 1.4 0.4 101.8± 1.9 pentagalloylglucose X8: ononin Y � 937109.60X + 2791.93 1.0000 0.069–0.692 0.6 3.1 0.3 101.2± 0.5 X9: isoliquiritin Y � 1557077.22X + 3899.99 0.9999 0.061–0.611 0.8 2.1 0.7 108.7± 0.5 X10: licochalcone B Y � 1291329.69X + 338.63 0.9999 0.006–0.063 0.8 2.9 0.4 93.8± 0.5 X11: liquirtigenin Y � 132434.69X–1198.43 0.9998 0.100–1.000 0.5 1.7 0.3 107.3± 0.4 X12: Y � 1537913.99 X + 1164.36 1.0000 0.009–0.093 2.5 1.5 2.7 104.1± 1.1 benzoylpaeoniforin X16: glycyrrhizic acid Y � 858537.93X + 3267.07 0.9999 0.389–3.890 0.7 0.4 0.4 97.3± 0.1 X18: licochalcone A Y � 137500.90X–5234.42 0.9979 0.063–0.629 0.4 2.1 0.3 105.4± 0.6 X19: glabridin Y � 563254.16X + 431.74 0.9999 0.072–0.720 1.1 3.9 0.6 106.9± 0.5 Table 5: Content determination of the 15 compounds in 15 batches of SGD. Content (mg/g) Compound S1 S2 S3 S4 S5 S6 S7 S8 S9 S10 S11 S12 S13 S14 S15 X1: oxypaeoniforin 0.080 0.104 0.083 0.070 0.090 0.092 0.077 0.088 0.094 0.086 0.102 0.113 0.221 0.229 0.113 X2: catechin 0.753 0.490 0.546 0.501 0.410 0.591 0.563 0.335 0.381 0.552 0.625 0.457 0.950 0.652 0.544 X3: albiforin 1.634 2.690 2.067 2.265 2.238 2.044 1.789 2.079 1.705 1.807 2.200 2.225 3.000 3.259 3.490 X4: paeoniforin 3.586 3.666 3.966 3.035 4.161 4.028 3.659 3.456 3.266 3.866 4.042 4.175 2.737 2.500 3.365 X5: liquiritin 2.431 3.023 2.172 2.084 2.814 2.623 2.105 1.664 2.417 2.508 2.348 2.411 2.912 2.764 2.976 X6: galloypaeoniforin 0.082 0.096 0.108 0.087 0.103 0.100 0.102 0.101 0.073 0.083 0.085 0.095 0.064 0.055 0.157 X7: 1,2,3,4,6-O- 0.103 0.111 0.116 0.116 0.140 0.116 0.120 0.105 0.104 0.168 0.144 0.174 0.178 0.181 0.374 pentagalloylglucose X8: ononin 0.360 0.575 0.497 0.366 0.555 0.518 0.422 0.437 0.474 0.454 0.482 0.591 0.503 0.436 0.568 X9: isoliquiritin 0.128 0.121 0.104 0.100 0.153 0.140 0.098 0.072 0.117 0.135 0.117 0.101 0.136 0.152 0.151 X10: licochalcone B 0.002 0.003 0.007 0.004 0.002 0.004 0.003 0.002 0.002 0.001 0.007 0.007 0.006 0.004 0.001 X11: liquirtigenin 0.588 0.401 0.323 0.497 0.488 0.362 0.365 0.382 0.597 0.843 0.936 0.294 0.871 0.711 0.392 X12: benzoylpaeoniforin 0.007 0.005 0.005 0.008 0.004 0.008 0.007 0.011 0.010 0.011 0.012 0.013 0.016 0.016 0.020 X16: glycyrrhizic acid 1.615 1.367 1.410 1.358 1.663 1.663 1.554 1.108 1.803 2.060 1.904 1.583 2.209 2.131 1.477 X18: licochalcone A 0.521 0.151 0.223 0.232 0.115 0.158 0.274 0.196 0.167 0.410 0.263 0.218 0.390 0.370 0.292 X19: glabridin 0.026 0.014 0.028 0.039 0.014 0.016 0.037 0.029 0.028 0.049 0.040 0.033 0.051 0.042 0.026 3.5. Spectral-Efect Relevance Analysis. GRA is a quantita- 3.5.1. PLSR. In addition to GRA, the relationship between the tive analytical method widely used to analyse the corre- 20 common peaks (x-variables) and the antispasmolytic ef- lation between the compound and its efcacy [16]. A cacy (y-variables) was also evaluated by using a PLSR model. correlation coefcient greater than 0.8 indicates a strong PLSR is a method that can describe which peaks contribute correlation [33]. Te common peaks and the inhibition positively or negatively to the efcacy. As shown in Figure 5, the rate of SGD on the intestinal contraction after Z-score peaks X4, X6 and X9–X19 were correlated strongly with the normalisation were used as the X and Y matrices, re- antispasmolytic efect with high positive correlation coef- cients. Te remaining seven peaks were negatively correlated spectively, in the GRA to fnd the active compounds corresponding to the antispasmolytic efcacy. Te results with the inhibition rate. Furthermore, the VIP value can describe the degree of of the GRA are shown in Table 7. Te correlation coef- fcients of X4 (paeoniforin), X15, X14, X8 (ononin), X6 explanation of the independent variable on the dependent (galloypaeoniforin), X5 (liquiritin), X9 (isoliquiritin), variable. Te larger the VIP value, the greater the correlation and X16 (glycyrrhizic acid) were higher than 0.8, indi- between the variable and the drug efcacy will be [34]. When cating their major role in the antispasmolytic activity of the VIP is greater than 1, the characteristic peak has a more SGD. Tese results also signify that SGD exerted anti- important role in the antispasmolytic efcacy. As shown in spasmolytic efects through multicomponent synergy. Figure 6, the VIP values of peaks X10, X4, X1, X11, X3, X2, Journal of Analytical Methods in Chemistry 7 S1 S2 S3 S4 S5 S6 S7 S8 S9 S10 S11 S12 S13 S14 S15 X1 X2 X3 X4 X5 X6 X7 X8 X9 X10 X11 X12 X16 X18 X19 compounds Figure 2: Heatmap of 15 compounds in 15 batches of SGD. X1 X2 X3 X4 X5 X6 X7 X8 X9 X10 X11 X12 X16 X18 X19 compounds Figure 3: Box plot of 15 compounds in 15 batches of SGD. X18, and X16 were greater than 1, indicating biological (glycyrrhizic acid) were identifed as the major core anti- signifcance. spasmolytic compounds in SGD. Tis fnding also indicated Combining the results from the GRA and PLSR models, that the antispasmodic efect of SGD is the result of the correlation degree >0.8, VIP> 1 and positive correlation, the synergistic efect of multiple components. Many studies have peaks of X4 (paeoniforin), X11 (liquirtigenin), and X16 shown that glycyrrhizic acid and paeoniforin have Batches Content (mg/g) 8 Journal of Analytical Methods in Chemistry S1 S2 S3 S4 S5 S6 S7 S8 S9 S10 S11 S12 S13 S14 S15 Average X19: Glabridin X7: 1,2,3,4,6-O-pentagalloylglucose X18: Licochalcone A X6: Galloypaeoniforin X5: Liquiritin X16: Glycyrrhizic acid X4: Paeoniforin X12: Benzoylpaeoniforin X3: Albiforin X11: Liquirtigenin X2: Catechin X1: Oxypaeoniforin X10: Licochalcone B X9: Isoliquiritin X8: Ononin Figure 4: Bar graph of the total contents in 15 batches of SGD. Total content (mg/g) Journal of Analytical Methods in Chemistry 9 Table 6: Efect of 15 batches of SGD on the contractile activity of isolated intestinal preparation (X ± SME, n � 6). Average tension (g) Inhibition rate (%) Drug Drug Batch Drug administration group Drug administration group Drug administration group Control group Ach model group administration administration (15 mg/mL) (25 mg/mL) (35 mg/mL) group (25 mg/mL) group (35 mg/mL) ## ∗∗ ∗∗ S1 0.4483± 0.04920 1.426± 0.1244 1.341± 0.0984 0.6487± 0.0472 0.4776± 0.0430 78.02± 5.549 97.05± 4.003 ∗∗ S2 0.5204± 0.01452 1.168± 0.1892 1.194± 0.1848 0.6943± 0.1398 0.4543± 0.06812 82.90± 12.32 120.3± 9.962 ## ∗∗ ∗∗ S3 0.4285± 0.03723 1.216± 0.1397 0.9784± 0.1218 0.5199± 0.05595 0.3260± 0.02184 88.08± 3.246 114.2± 2.65 ## ∗∗ ∗∗ S4 0.5635± 0.06599 1.914± 0.3118 1.703± 0.2831 0.8390± 0.07993 0.5414± 0.05365 86.11± 9.202 111± 8.952 ## ∗∗ ∗∗ S5 0.4279± 0.06277 1.358± 0.1667 1.160± 0.1562 0.5640± 0.08049 0.4190± 0.06737 84.74± 5.133 101.6± 2.695 ## ∗∗ ∗∗ S6 0.5296± 0.04119 1.159± 0.09607 0.9905± 0.09806 0.6552± 0.05702 0.5028± 0.03212 82.45± 5.394 103.9± 3.426 ## ∗∗ ∗∗ S7 0.4323± 0.06757 1.124± 0.1909 0.7886± 0.05031 0.3899± 0.03225 0.2655± 0.0438 99.13± 9.353 120.2± 7.817 ## ∗∗ ∗∗ S8 0.5116± 0.04533 0.9918± 0.05947 1.001± 0.03622 0.7059± 0.02238 0.5774± 0.01672 58.10± 8.560 85.36± 7.416 ∗∗ S9 0.4842± 0.05229 1.063± 0.1186 0.984± 0.2091 0.6202± 0.09016 0.3968± 0.0398 75.91± 7.580 113.4± 6.972 ## ∗ ∗∗ ∗∗ S10 0.4960± 0.05865 1.409± 0.1962 0.9719± 0.08651 0.4914± 0.04814 0.368± 0.05182 104.2± 5.288 120.4± 10.35 ## ∗∗∗ ∗∗ S11 0.3607± 0.04392 0.9241± 0.1457 0.5863± 0.07636 0.3203± 0.04721 0.2587± 0.05415 110.2± 4.840 117.5± 10.68 ## ∗∗ ∗∗ S12 0.3917± 0.03975 1.433± 0.2150 1.249± 0.2104 0.6738± 0.1062 0.4462± 0.07728 75.15± 5.218 98.39± 5.121 ∗∗ ∗∗ S13 0.5547± 0.09259 1.113± 0.1566 0.9223± 0.1112 0.5019± 0.0944 0.4470± 0.09103 107.4± 7.044 118± 6.441 ## ∗ ∗∗ ∗∗ S14 0.4113± 0.03592 0.8625± 0.08512 0.6173± 0.05789 0.3951± 0.03806 0.3036± 0.02892 108.9± 7.633 127.4± 5.618 S15 0.4967 + 0.02877 1.243± 0.2305 1.117± 0.2264 0.7182± 0.1251 0.5096± 0.06343 67.52± 9.100 94.27± 5.152 # ## ∗ ∗∗ Compared with the control group: P< 0.05; P< 0.01; compared with the Ach model group: P< 0.05; P< 0.01. 10 Journal of Analytical Methods in Chemistry Table 7: GRA results. Peaks Correlation Ranking X4 0.849 1 X15 0.843 2 X14 0.841 3 X8 0.836 4 X6 0.835 5 X5 0.834 6 X9 0.82 7 X16 0.812 8 X12 0.794 9 X7 0.784 10 X1 0.776 11 X3 0.774 12 X2 0.772 13 X17 0.77 14 X18 0.734 15 X19 0.728 16 X11 0.685 17 X20 0.668 18 X13 0.656 19 X10 0.653 20 0.2 -0.2 X1 X2 X3 X4 X5 X6 X7 X8 X9 X10 X11 X12 X13 X14 X15 X16 X17 X18 X19 X20 Var ID (Primary) Figure 5: Coefcient of correlation of 20 peaks with antispasmolytic activity. X10 X4 X1 X3 X11 X2 X18 X16 X9 X13 X14 X15 X19 X5 X6 X12 X7 X17 X8 X20 Var ID (Primary) Figure 6: VIP contribution plot of common peak antispasmolytic activity. Coefcient VIP Journal of Analytical Methods in Chemistry 11 efcacy and treatment efect of SGD. Meanwhile, for the frst time, the fngerprint of SGD was associated with 1.0 antispasmolytic activity. Paeoniforin, liquirtigenin, and glycyrrhizic acid were highly correlated with anti- ** ** spasmolytic activity, revealing that multiple components ** ## exhibited antispasmolytic activities. Furthermore, the 0.5 potential antispasmolytic components were validated. Paeoniforin (11.70–29.25 μg/mL) and liquirtigenin (17.19–28.65 μg/mL) could signifcantly reduce the max- 0.0 imum contractile (P< 0.01) and exerted concentration- Control Ach 5.85 11.70 17.55 23.40 29.25 dependent spasmolytic efects with the inhibitory re- model Paeoniforin (μg/mL) sponse for ACh-induced contraction. Tus, SGD had a signifcant antispasmodic efect, which could have resulted from the synergistic activity of its paeoniforin, liquirtigenin and other components. 1.0 Te results of the quantitative analysis of the multi- component in SGD could help to discover the material ** basis of SGD and establish a system for modern TCM ** quality standards. Te spectral–efect study can be used for ## 0.5 the rapid screening of potential antispasmolytic compo- nents in TCM. Verifcation experiment was carried out by testing the activity of the single standards to further de- termine the antispasmodic efcacy of SGD. Te results can provide a reference for the pharmacodynamics study of 0.0 SGD and are highly signifcant for the determination of Control Ach 5.73 11.46 17.19 22.92 28.65 quality markers and quality control. Given that TCM is model Liquiritigenin (μg/mL) characterised by multiple components, targets, and ap- Figure 7: Efects of paeoniforin and liquirtigenin on the con- proaches, further study on the spasmolytic targets and tractile activity (X± SME, n � 6). mechanism of SGD is needed. Data Availability synergistic efects on antispasticity, antipyretic, anti-in- fammatory, inhibition of gastric secretion, and relaxation of Te data used to support the fndings of this study are in- smooth muscle [35]. cluded within the article. 3.5.2. Experimental Verifcation. Tree antispasmolytic ac- Conflicts of Interest tive compounds, namely, paeoniforin, liquirtigenin, and glycyrrhizic acid, were obtained by spectral–efect rela- Te authors declare that there are no conficts of interests. tionship. Isolated intestine preparation was used to further verify the feasibility and accuracy of the spectral–efect re- Acknowledgments lationship method. As shown in Figure 7, paeoniforin (11.70–29.25 μg/mL) and liquirtigenin (17.19–28.65 μg/mL) Tis work was supported by the National Natural Science could signifcantly reduce the maximum contractile Foundation of China (No. 82160457), Lanzhou Science and (P< 0.01) and exerted concentration-dependent spasmolytic Technology Program (No. 2021-1-185), Medical Products efects with the inhibitory response for Ach-induced Administration Program of Gansu Province (No. contraction. 2020GSMPA027), Natural Science Foundation of Gansu Compared with the control group, P< 0.05; compared Province (No. 21JR7RA564), Program of Key Laboratory of with the Ach model P< 0.01. Chemistry and Quality for Traditional Chinese Medicines College of Gansu Province (No. zzy-2022-07). 4. 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