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

Shaoyao-Gancao Decoction (SGD) is a well-known classic traditional Chinese medicine (TCM) with antispasmodic, anti-inflammatory, and analgesic effects. This preparation has been widely used to treat spasticity diseases in the clinic. To date, the material basis of SGD remains unclear, and the spectrum-effect correlation of its antispasmodic activity has not been reported yet. In this study, high-performance liquid chromatography (HPLC) was used to establish the fingerprint and determine the multiple components of SGD. The common peaks of fingerprints were evaluated by the similarity with the chromatographic fingerprints of the TCM. Meanwhile, the multiple components were quantified and analysed using the heatmap and box size analysis. Furthermore, data on the antispasmodic effect were extracted through in vitro smooth muscle contraction assay. Grey relational analysis combined with partial least square regression was used to study the spectrum–effect correlation of SGD. Finally, the potential antispasmolytic components were validated using an isolated tissue experiment. The HPLC fingerprint was established, and 20 common peaks were identified. The similarities of 15 batches of SGD were all above 0.965. The HPLC method for simultaneous determination of the multiple components was accurate and reliable. The contents of albiflorin, paeoniflorin, liquiritin, and glycyrrhizic acid were higher than the other components in SGD. The heatmap and box size also showed that X3 (albiflorin), X4 (paeoniflorin), X5 (liquiritin), X11 (liquirtigenin), and X16 (glycyrrhizic acid) could be used as quality indicators in the further establishment of quality standards. The spectrum–effect correlation results indicated that X4, X11, and X16 were highly correlated with antispasmolytic activity. Verification tests showed that paeoniflorin (11.7–29.25 μg/mL) and liquirtigenin (17.19–28.65 μg/mL) could significantly reduce the maximum contractile (P < 0.01). These compounds exerted concentration-dependent spasmolytic effects with the inhibitory response for acetylcholine (Ach)-evoked contraction. Thus, SGD had a significant antispasmodic effect, which resulted from the synergistic activity of its multiple components. These findings can be used for the pharmacodynamics study of SGD and are of great significance for the determination of quality markers and quality control.


Introduction
Spasticity is a velocity-dependent increase in muscle tone caused by the increased excitability of muscle spindles following an upper motor neuron (UMN) syndrome [1,2].
Tere has been much debate about the defnition of spasticity. In 1980, Lance was the frst scholar to associate spasticity to the velocity-dependent increase in stretch refex [3]. A more general defnition of spasticity is disordered sensory-motor control, resulting from UMN lesion, presenting intermittent or sustained involuntary activation of muscles [4]. Tis complex phenomenon of extremely variable clinical expression, which may cause diferent motor dysfunctions, has been observed in many patients with spinal cord injury, cerebral palsy, multiple sclerosis, and acquired brain injury, which directly impact the quality of life [5]. Presently, the conventional drugs used in the treatment of spasm include baclofen, tizanidine, and dantrolene, amongst others. However, the efcacy of these current treatments is not absolute, and they may have serious side efects [6][7][8].
Accordingly, natural products with therapeutic activity against spasm must be searched to replace drugs with strong side efects. Shaoyao-Gancao Decoction (SGD) is a classic traditional Chinese medicine (TCM) and originally described in the Treatise on Febrile Disease. SGD is composed of Paeoniae Radix Alba (baoshao in Chinese) and honeyed Glycyrrhiza uralensis, which are traditionally used to treat spastic diseases, such as gastrointestinal spasm, facial muscle spasm, and poststroke spasm [9]. Modern pharmacological and clinical studies have confrmed that SGD has signifcant antispasmodic, anti-infammatory, and analgesic efects on various spastic diseases [10], infammatory diseases [11], painful diseases [12,13], gynopathy [14], bronchial asthma, Parkinson's disease, and constipation [15,16]. Tis TCM has been selected for the frst batch of the Chinese Medicine Classical Directory. Studies have shown that the extract of SGD and liquorice exerts a relaxant efect on acetylcholine (ACh)-induced contraction, isoliquiritigenin, and glycycoumarin isolated from the roots of liquorice and has a potent antispasmodic [17,18]. Te ethanol extract of Glycyrrhiza uralensis has signifcant inhibitory efects on Nav1.4 VGSCs, which may be an important mechanism in the treatment of gastrocnemius spasm [19].
TCM is characterised by multiple components, targets, and approaches, and SGD has a complex composition. Previous studies have shown that glycyrrhizin, glycyrrhetic acid, paeoniforin, albiforin, oxypaeoniforin, liquiritin, liquiritigenin, isoliquiritin, isoliquiritigenin, and 1,2,3,4,6-O-pentagalloylglucose are the main bioactive compounds of SGD [20][21][22]. Research on SGD has mainly focused on its clinical application, chemical composition, and anti-infammatory and analgesic efects. However, the pharmacodynamic basis of the antispasmodic efect and the spectrum-efect relationship of SGD has not been reported yet. Te manner by which the components contribute to the antispasmolytic activity of SGD remains ambiguous. Te spectrum-efect relationship of TCM mainly apply correlational analysis, grey correlational analysis (GRA), multiple regression analysis, partial least squares regression (PLSR), principal component analysis, and other mathematical models to screen the bioactive compounds [23][24][25]. It is a biological efect-based evaluation method, which has been widely used to investigate the material basis of the pharmacological efects of Chinese medicinal compounds [26][27][28].
Terefore, this study was conducted to clarify the material basis of the antispasmodic efect of SGD by establishing the spectrum-efect relationship, determine the multiple components of SGD, and validate the spectrum-efect results. Scientifc basis for the secondary development and quality control of SGD was also provided.

Apparatus and
Conditions. HPLC analysis of SGD was performed using Shimadzu LC-20A high performance liquid chromatograph coupled with DAD detectors (Shimadzu Corporation, Japan). Te chromatographic conditions were as follows: column, CAPCELL PAK-C18 reversed-phase (250 mm × 4.6 mm, 5 μm); mobile phase, acetonitrile (A), and 0.1% phosphoric acid in water (B); fow rate, 1.0 mL/ min; detection wavelength, 254 nm; column temperature, 30°C; and injection volume, 10 μL. Te gradient programme is shown in Table 2.  15.91 μg/mL; and glabridin, 3.63 μg/mL. Te mixed standard solution was diluted stepwise with methanol solution to obtain six diferent concentrations for the plotting of the calibration curves. All standard solutions were stored at 4°C.

Sample Solution Preparation.
Te daily dose of SGD pieces (55.2 g) was precisely weighed, and 600 mL of water 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 of decoction was concentrated to 1 g/mL as a sample for the isolated smooth muscle experiment. Te SGD freeze-dried powder (0.1 g) was precisely weighed, placed in a 10 mL volumetric fask, and ultrasonically extracted with 50% methanol. Te sample solution was fltered through a 0.45 μm membrane and stored at 4°C.

Validation of HPLC Analytical Method.
Te blank solvent (50% methanol), standard solution, negative sample, and sample solution were separately injected according to the chromatographic conditions under Section 2.2. Te chromatographic results were recorded. Te calibration  curves were plotted with the concentration of tested reference as the x-axis and the peak area as the y-axis. Te intraday and interday precisions were determined by six repetitive injections on the same day and for three consecutive days. Te stability test was evaluated by injecting the sample solution at 0, 2, 4, 6, 8, 10, and 12 h after preparation. Repeatability was determined by analysing six prepared samples from the same source. Recovery was investigated by adding an accurate amount of standard solution to 0.1 g of the freeze-dried powder. Fifteen samples were prepared in parallel according to the preparation method of the sample solution.

Isolated Rat Intestine Preparation.
Te SD rats were fasted for 24 h and drank water freely. Te rats were killed following a blow on the back of the head with a wooden stick [29]. Te intestine segments (1.5 cm long) were prepared, gently fushed with Tyrode bufer, and quickly placed in a Petri dish containing Tyrode bufer. According to the physiological position from top to bottom, the upper end was connected to the tension transducer, and the lower end was fxed to the L-shaped bent hook at the bottom of the muscle groove. Each intestine segment was suspended in organ baths containing constantly oxygenated Tyrode's solution (20 mL, pH 8.2) at a constant temperature (37°C ± 0.5°C) [30]. Fresh oxygen was continuously introduced at a rate of 1-2 bubbles per second. Te intestine segments were equilibrated for 55 ± 5 min with drainage of the bufer with fresh oxygen after 15 ± 2 min. Te physiological response of the intestine segments was recorded using an isometric force transducer (ML870) connected to a 4-channel bridge amplifer. Te signals were amplifed by a data acquisition device Power Lab 8/35 hardware. Muscle contractions were analysed using Lab Chart 8 software. Te equipment hardware and software were from ADInstruments Pty Ltd.
(Bella Vista, NSW, Australia) [31]. Te possible antispasmodic activity of SGD was determined by ACh (1 mM)-evoked contraction of the intestinal smooth muscle. SGD was applied cumulatively to achieve a concentration-dependent inhibitory response, and the average tension was used as the index.

Statistical Analysis.
Te chromatographic data of the 15 SGD samples were evaluated using the Chromatographic Fingerprint Evaluation System for Chinese Medicine. Graph Pad Prism (8.0.) was applied for all statistical analyses and plotting of graphs. Te experimental values were expressed as mean ± standard (SEM) and tested by one-way ANOVA. P < 0.05 was considered to be a signifcant difference. GRA and PLSR were used to analyse the spectrum-efect.

Establishment and Similarity Analysis of the HPLC Fingerprint.
Te chromatographic data of 15 batches of SGD were imported into the Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (version 2012). After chromatographic peak matching, the standard fngerprint chromatogram "R" was generated, and the fngerprints of the 15 batches of SGD samples were established ( Figure 1). Te similarities between the sample chromatograms and the reference chromatogram were calculated using the abovementioned software. Te similarities were all greater than 0.965 (Table 3), indicating apparent similarity amongst the 15 batches of SGD. Ten, 20 common peaks in the reference chromatogram were assigned, and 16 compounds, including oxypaeoniforin, catechin, albiforin, paeoniforin, liquiritin, galloypaeoniforin, 1,2,3,4,6-O-pentagalloylglucose, ononin, isoliquiritin, licochalcone B, liquirtigenin, benzoylpaeoniforin, glycyrrhizic acid, licochalcone A, glabridin, and glycyrrhetinic acid, were verifed after a comparison with the reference substances. Components

Validation of the HPLC Method.
A method that could distinguish the HPLC fngerprint and simultaneously determine the 15 compounds was established. Te validation of the method, including precision, repeatability, stability, linear regression, and recovery for 15 compounds, is summarised in Table 4. Te results showed that the precision of the instrument and the repeatability of the extraction method were good, and the sample was stable within 12 h. All calibration curves showed good linearity in the given concentration ranges. Te recovery rates for the spiked samples ranged from 93.9% to 109.9%. Tus, the validation of the HPLC method was within an acceptable range in quantitative research, demonstrating that the established method was reproducible for the fngerprint and the determination of 15 compounds in diferent batches of SGD. Te proposed method can simultaneously determine 15 compounds and can provide a better alternative for the evaluation of the quality of SGD.

Measurement Results of Multiple Component
Determination. Te quantities of the 15 components measured in the SGD were calculated by substituting the regression equation in Table 4. Table 5 shows that the content of the 15 compounds in the diferent batches of SGD varied to a certain extent. Te fact that the raw materials were derived from diferent sources may be the main reason for the fuctuation in the content of the tested compounds. We adopted a heatmap and box plot to intuitively display the content distribution [32]. Te heatmap refected the fuctuation of the 15 compounds in diferent batches through the gradient colour. As shown in Figure 2, X3 (albiforin), X4 (paeoniforin), X5 (liquiritin), and X16 (glycyrrhizic acid) fuctuated obviously, refecting great variation amongst the diferent batches. Te box size represents the dispersion degree of the 15 index compounds in the diferent batches. As shown in Figure 3, X2 (catechin), X3 (albiforin), X4 (paeoniforin), X5 (liquiritin), X11 (liquirtigenin), and X16 (glycyrrhizic acid) were relatively large. As required by ChP, the quality of liquorice and Paeoniae Radix Alba were evaluated by detecting the content of liquiritin, glycyrrhizic acid, and paeoniforin. As shown in Figure 4, the total contents were markedly diferent, and the total contents of S2, S5, S6, and S10-S15 were higher than the average. Furthermore, their quality was better than the other contents. Te average contents of 15 characteristic ingredients in SGD from high to low were as follows: Tus, we suggest that X3 (albiforin), X4 (paeoniforin), X5 (liquiritin), X11 (liquirtigenin), and X16 (glycyrrhizic acid) can be used as characteristic components when a quality standard is established.

Results of Isolated Intestine Preparation.
In the intestinal muscle study, Ach-induced intestine contractions were used to evaluate the antispasmolytic activity of the SGD samples. Table 6 shows that compared with the blank control group, the intestine contractions of the Ach model group were signifcantly increased (P < 0.05), indicating that the model was successful. Compared with the Ach model group, 15 batches of SGD (25 mg/mL and 35 mg/ mL) from diferent origins all signifcantly reduced the maximum contractile (P < 0.01), exerting concentrationdependent spasmolytic efects with the inhibitory response for Ach-evoked contraction.   Journal of Analytical Methods in Chemistry

Spectral-Efect Relevance Analysis.
GRA is a quantitative analytical method widely used to analyse the correlation between the compound and its efcacy [16]. A correlation coefcient greater than 0.8 indicates a strong correlation [33]. Te common peaks and the inhibition rate of SGD on the intestinal contraction after Z-score normalisation were used as the X and Y matrices, respectively, in the GRA to fnd the active compounds corresponding to the antispasmolytic efcacy. Te results of the GRA are shown in Table 7. Te correlation coeffcients of X4 (paeoniforin), X15, X14, X8 (ononin), X6 (galloypaeoniforin), X5 (liquiritin), X9 (isoliquiritin), and X16 (glycyrrhizic acid) were higher than 0.8, indicating their major role in the antispasmolytic activity of SGD. Tese results also signify that SGD exerted antispasmolytic efects through multicomponent synergy.

PLSR.
In addition to GRA, the relationship between the 20 common peaks (x-variables) and the antispasmolytic efcacy (y-variables) was also evaluated by using a PLSR model. PLSR is a method that can describe which peaks contribute positively or negatively to the efcacy. As shown in Figure 5, the peaks X4, X6 and X9-X19 were correlated strongly with the antispasmolytic efect with high positive correlation coefcients. Te remaining seven peaks were negatively correlated with the inhibition rate. Furthermore, the VIP value can describe the degree of explanation of the independent variable on the dependent variable. Te larger the VIP value, the greater the correlation between the variable and the drug efcacy will be [34]. When the VIP is greater than 1, the characteristic peak has a more important role in the antispasmolytic efcacy. As shown in Figure 6, the VIP values of peaks X10, X4, X1, X11, X3, X2,  Compound Content (mg/g) S1 S2 S3 S4 S5 S6 S7 S8 S9 S10 S11 S12 S13 S14 S15 S9 S10 S11 S12 S13 S14 S15 compounds

Experimental Verifcation.
Tree antispasmolytic active compounds, namely, paeoniforin, liquirtigenin, and glycyrrhizic acid, were obtained by spectral-efect relationship. Isolated intestine preparation was used to further verify the feasibility and accuracy of the spectral-efect relationship method. As shown in Figure 7, paeoniforin (11.70-29.25 μg/mL) and liquirtigenin (17.19-28.65 μg/mL) could signifcantly reduce the maximum contractile (P < 0.01) and exerted concentration-dependent spasmolytic efects with the inhibitory response for Ach-induced contraction. Compared with the control group, # P < 0.05; compared with the Ach model * P < 0.01.

Conclusion
In this study, 15 compounds in SGD were simultaneously quantifed using the HPLC analytical method. Te method was simple, rapid, and accurate, and it can be used for the qualitative and quantitative analysis of SGD. Te heatmap and the box sizes showed that albiforin, paeoniforin, liquiritin, liquirtigenin, and glycyrrhizic acid could be used as quality markers in the establishment of a quality standard. Tese compounds were responsible for the efcacy and treatment efect of SGD. Meanwhile, for the frst time, the fngerprint of SGD was associated with antispasmolytic activity. Paeoniforin, liquirtigenin, and glycyrrhizic acid were highly correlated with antispasmolytic activity, revealing that multiple components exhibited antispasmolytic activities. Furthermore, the potential antispasmolytic components were validated. Paeoniforin (11.70-29.25 μg/mL) and liquirtigenin (17.19-28.65 μg/mL) could signifcantly reduce the maximum contractile (P < 0.01) and exerted concentrationdependent spasmolytic efects with the inhibitory response 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.
Te results of the quantitative analysis of the multicomponent 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 the rapid screening of potential antispasmolytic components in TCM. Verifcation experiment was carried out by testing the activity of the single standards to further determine the antispasmodic efcacy of SGD. Te results can provide a reference for the pharmacodynamics study of SGD and are highly signifcant for the determination of quality markers and quality control. Given that TCM is characterised by multiple components, targets, and approaches, further study on the spasmolytic targets and mechanism of SGD is needed.

Data Availability
Te data used to support the fndings of this study are included within the article.

Conflicts of Interest
Te authors declare that there are no conficts of interests. Journal of Analytical Methods in Chemistry 11