Determination of Triterpenoids and Phenolic Acids from Sanguisorba officinalis L. by HPLC-ELSD and Its Application

A novel analytical method involving high-performance liquid chromatography with evaporative light scattering detection (HPLC-ELSD) was developed for simultaneous determination of 11 phenolic acids and 12 triterpenes in Sanguisorba officinalis L. Chromatographic separation was conducted with gradient elution mode by using a DiamonsilTM C18 column (250 mm × 4.6 mm, 5 μm) with the mobile phase of 0.1% acetic acid water (A) and methanol (B). The drift tube temperature of ELSD was set at 70 °C and the nitrogen cumulative flow rate was 1.6 L/min. The method was fully validated to be linear over a wide concentration range (R2 ≥ 0.9991). The precisions (RSD) were less than 3.0% and the recoveries were between 97.7% and 101.4% for all compounds. The results indicated that this method is accurate and effective for the determination of 23 functional components in Sanguisorba officinalis L. and could also be successfully applied to study the influence of processing method on those functional components in Sanguisorba officinalis L.


Introduction
As a traditional Chinese medicine, Sanguisorba officinalis L. (S. officinalis) has a long history of medical application. It was first to be seen in Shennong Herbal Medicine Classic, the first great pharmacy book in China. The dried roots of S. officinalis (Rosaceae) and Sanguisorba officinalis var longifolia have been officially listed in the Chinese Pharmacopoeia (2020 edition) [1]. It has the functions of cooling blood, stopping bleeding, detoxifying, and curbing sores [2]. It has been widely used in the treatment of colitis, ulcerative colitis, pyelonephritis, hemostasis, and burns [3]. Besides, it was also reported to possess the following activities: antioxidant [4], anti-inflammatory [5], antiviral [6], antimicrobial [7,8], and antitumor [9]. Recently, more attention has been paid on its anti-cancer activities, such as antiproliferative effects against breast cancer [10], oral cancer [11], prostate cancer [12], and colorectal cancer [13].
As these isolated components from S. officinalis have strong physiological activity [17][18][19], the determination of the content of these components in S. officinalis is therefore important, especially for the simultaneous determination of the content of the 23 The Chinese Pharmacopoeia only selected gallic acid as a quality control indicator for S. officinalis, and there is a lack of simultaneous determination method for various triterpenes and phenolic acids. Using gallic acid as an indicator of quality control alone does not fully account for the quality of S. officinalis and the achievement of better control over its quality. These studies also show that the triterpenes in S. officinalis are more likely to be important components of its pharmacodynamic and physiological activity.
As these isolated components from S. officinalis have strong physiological activity [17][18][19], the determination of the content of these components in S. officinalis is therefore important, especially for the simultaneous determination of the content of the 23 compounds mentioned above. It is therefore essential to develop an analytical method that can determine the content of these 23 compounds simultaneously.
A HPLC-ELSD method was established to determine of triterpenoids and triterpenoid saponins in Ilex pupurea leaves [20]. A HPLC-DAD-ESI/MS(n) method was developed for quality evaluation of Cortex Moutan through identification of common constituents based on chromatographic fingerprints and determination of key pharmacological compounds [21]. However, it still cannot accurately evaluate the quality of other compounds. Although HPLC or gas chromatography (GC) coupled with mass spectrometry can quantify these compounds [22,23]. However, it has some practical limitations including cost and uncommon available in regular laboratories. Due to the terminal absorption of triterpenoids, UV as the analytical method has more interference. ELSD is commonly used in HPLC. The response of the ELSD does not depend on the optical properties of the substance under test and is not affected by its functional groups and can be detected for samples with volatility below the mobile phase. At the same time, the ELSD is insensitive to temperature changes, baseline stability, suitable for gradient elution of the liquid phase combination, more conducive to the separation of these compounds. Organic substances without ultraviolet absorption can be detected, such as triterpenes, ginsenosides, astragalosides, etc. [20]. Some reports have been successfully applied for the determination of oleanolic acid and ursolic acid in different medicinal plants by using HPLC-ELSD [24,25]. The general detection method of ELSD could eliminate the common difficulties in traditional methods [26].
For the reasons above, it is necessary to develop an analytical method to quantify the 23 functional ingredients to improve comprehensive exploitation and utilization of resources of S. officinalis. However, the method for the simultaneous determination of 23 compounds from S. officinalis with HPLC has not been reported. Thus, the aim of this study was to develop a simple, reliable method to simultaneously determine 23 compounds in S. officinalis by HPLC-ELSD. Meanwhile, it was the first time that the determination of 11 phenolic acids and 12 triterpenes together following S. officinalis extract was reported. This method could lay a good foundation for the comprehensive study of the active ingredients in S. officinalis, and also help to provide a reasonable and effective method for the quality evaluation of S. officinalis. In this study, the method was also used to study the influence of processing on the functional components in Sanguisorba officinalis L.

Selection of HPLC-ELSD Condition
Determined the samples and 23 analytes according to the selected optimal chromatographic conditions. The representative HPLC-ELSD chromatograms of test samples and 23 analytes are shown in Figure 2.
According to the selected optimal chromatographic conditions, the retention times of 23 analytes are gallic acid (10.

Calibration Curves and Linear Range
The calibration curves were calculated by double logarithmic transformation of the area and the concentration of the reference solution injected. Linearity was evaluated by five different concentrations of standard solutions. For calibration, the standard solutions with five different concentrations were analyzed. A plot of peak area versus sample size by ELSD was not linear, but the plot of peak area versus sample size in double logarithm was linear, which conformed to the mechanism of the ELSD. Their regression equations were calculated in the form of Y =a X + b, where Y and X are the logarithms of the peak area and concentration, respectively. As shown in Table 1, compounds of different structures also show different slopes. The range and correlation coefficients are presented in Table 1. All calibration curves showed good linearity (R 2 ≥ 0.9991) within the test ranges. Limits of quantitation ranged from 5.24 to 18.51 µg/mL. These results demonstrated that the HPLC-ELSD method is sensitive and precise for the quantitative determination of 23 analytes in S. officinalis.
The RE among measurements ranged from −2.8% to 2.9% (Table 2.), which indicated that S. officinalis extract remained stable at least 12 h.

Recovery
The result was shown in Table 3. The recovery of 23 compounds ranged from 97.7% to 101.4% showed that the established method in this experiment has good accuracy.

Sample Analysis
Based on the experimental analysis above, the content of 11 phenolic acids and 12 triterpenoids in 32 batches of S. officinalis from 24 provinces was calculated. The source and collection time were shown in Table 4. The results of 23 compounds in 32 batches of S. officinalis were shown in Tables 5 and 6. The distribution of the content of the 23 compounds in S. officinalis samples from different regions was listed in Figure 3.
All medicinal materials are purchased in the local Chinese medicine market in the 24 provinces mentioned below.
In Chinese Pharmacopoeia (2020 edition), gallic acid was used as a quantitative index in the medicinal materials and pieces of S. officinalis, and the content of gallic acid should not be less than 1.0%. From the experimental results, the content of gallic acid recorded in the Chinese Pharmacopoeia (2020 edition) was different in 32 batches of S. officinalis, but all the results met the quality control requirements recorded in the pharmacopoeia.   Under the current experimental conditions, in order to evaluate the quality of S. officinalis more reasonably and comprehensively, the content of phenolic acids and triterpenoids in S. officinalis was determined by HPLC. According to the content results, the contents of triterpenoids and phenolic acids in 32 batches are different, and some compounds cannot even be detected in S. officinalis. Among them, the content of ziyuglycoside I, catechin, and gallic acid in S. officinalis from Hebei was significantly higher than those in Gansu and Shanxi; the content of gallic acid and catechin in Shandong, Heilongjiang, and Shandong was higher; and the content of triterpenes in Inner Mongolia, Jiangxi, and Liaoning was also higher. The differences in the contents of triterpenoids and phenolic acids in S. officinalis are affected by many factors such as planting region, growing environment, and harvest time.
In this experiment, different batches of S. officinalis from different regions were tested. The results of the test are helpful to evaluate the quality and to control the internal quality of S. officinalis.

The Result of Comparison of Changes in Functional Components
The comparison of 23 compounds in unprocessed S. officinalis and processed S. officinalis are shown in Table 7 and Figure 4. All medicinal materials are purchased in the local Chinese medicine market in the 24 provinces mentioned below.
In Chinese Pharmacopoeia (2020 edition), gallic acid was used as a quantitative index in the medicinal materials and pieces of S. officinalis, and the content of gallic acid should not be less than 1.0%. From the experimental results, the content of gallic acid recorded in the Chinese Pharmacopoeia (2020 edition) was different in 32 batches of S. officinalis, but all the results met the quality control requirements recorded in the pharmacopoeia.
Under the current experimental conditions, in order to evaluate the quality of S. officinalis more reasonably and comprehensively, the content of phenolic acids and triterpenoids in S. officinalis was determined by HPLC. According to the content results, the contents of triterpenoids and phenolic acids in 32 batches are different, and some compounds cannot even be detected in S. officinalis. Among them, the content of ziyuglycoside I, catechin, and gallic acid in S. officinalis from Hebei was significantly higher than those in Gansu and Shanxi; the content of gallic acid and catechin in Shandong, Heilongjiang, and

Discussion
The mobile phase combinations investigated included methanol-water, acetonitrilewater, methanol-0.1% acetic acid water, and acetonitrile-0.1% acetic acid water. The result showed that when methanol-0.1% acetic acid water was the mobile phase, 23 compounds in S. officinalis were well separated with symmetrical peaks and no tailing. The gradient elution method with different proportions was explored. The important param-

Discussion
The mobile phase combinations investigated included methanol-water, acetonitrilewater, methanol-0.1% acetic acid water, and acetonitrile-0.1% acetic acid water. The result showed that when methanol-0.1% acetic acid water was the mobile phase, 23 compounds in S. officinalis were well separated with symmetrical peaks and no tailing. The gradient elution method with different proportions was explored. The important parameter of ELSD conditions is the drift tube temperature which was evaluated at different drift tube temperatures from 60 • C to 80 • C. Good peak symmetries and efficiencies were achieved with a drift tube temperature at 70 • C. After examining a series of column temperatures, the final column temperature was determined to be 35 • C.
Linear of the method over a wide concentration range (R 2 ≥ 0.9991). The precisions were less than 3.0% and the recoveries were between 97.7% and 101.4% for all compounds. The results indicated that this method is accurate and effective for the determination of 23 functional components in Sanguisorba officinalis L.
We processed the S. officinalis according to the method in the Chinese pharmacopoeia (the general rule 0213) and applied the established content determination method to determine the processed and unprocessed S. officinalis to compare the content changes of each compound. Which provides a theoretical basis for the different effects of unprocessed and processed S. officinalis. The changes in the content of these compounds may provide some ideas for the study of revealing the specific pharmacological effects of the active ingredients of S. officinalis.

Preparation of S. officinalis Extract
After crushing the dried root of S. officinalis (1.0 g), it was extracted by hot reflux with 10 mL 70% ethanol solution (1:10, w/v) 3 times at 80 • C, 1 h each, and then filtrated. The combined filtrate was evaporated to steam, and the residue was dissolved in methanol to get a concentration equivalent to 0.005 g/mL of the S. officinalis extract.

Calibration Curves, Limits of Detection and Quantification
For calibration, the premixed working solution with at least five different concentrations (performed in triplicate) was analyzed, and the linear calibration curves were calculated by plotting the logarithm of concentration injected for each analysis. The mixed solution of the 23 reference compounds was further diluted to a series of concentrations by methanol for the gain of limit of quantification (LOQ). The LOQ under the present chromatographic conditions were determined at the signal-to-noise ratios of each analyte of about 10, respectively.

Precision, Repeatability and Stability
The precision of the quantitative method was investigated by determining the 23 analytes in six replicates within one day. The RSD value of the peak area was adopted to evaluate precision.
Repeatability was confirmed with six independent analytical sample solutions prepared according to the methods described above and variations expressed by RSD.
To assess the stability of the developed method, the sample was stored at room temperature and injected into the HPLC apparatus at 0, 2, 4, 6, 8, 12 h after preparation, respectively. Calculate the concentration of the 23 analytes at each time point and compare with the value of 0 h, to obtain RE (relative error).

Recovery
Six parts of S. officinalis powder with known content, each of~0.5 g, were weighted accurately, put in conical bottle. Moreover, reference substances (1-23) were added precisely. The test sample solutions were prepared as Section 4.2. Under the chromatographic conditions in Section 4.4., the peak area of each compound was recorded, and the recovery and RSD value of each compound were calculated. The recovery was expressed as: Recovery = (measured content-the known content)/added content of reference substances × 100%.

Content Determination
Thirty-two batches of S. officinalis powder from 24 provinces were weighed to a total of 1.0 g. The test sample solutions were prepared as the Section 4.2. The content of 23 compounds in S. officinalis was calculated with HPLC chromatographic conditions in Section 4.4.

Comparison of Changes in Functional Components in S. officinalis
S. officinalis powder from the same origin was divided into unprocessed group and processed group. The unprocessed group precisely weighed 1.0 g, S. officinalis of processed group was heated until the surface was black and the inside was brown, taken out and left to cool at room temperature, and precisely weighed to 1.0 g after processing. Both groups were prepared as Section 4.2. Measured under chromatographic conditions in Section 4.4. We then calculated the content of 23 compounds to compare the changes in functional ingredients.

Conclusions
In this study, a sensitive and reliable quantitative method for simultaneous analysis of phenolic acids and triterpenoids from S. officinalis was developed based on HPLC-ELSD. This is the first report of simultaneous determination of 11 phenolic acids and 12 triterpenoids in S. officinalis extract, so this method could be used to evaluate the quality of S. officinalis. At the same time, it provides a basis for the establishment of the characteristic and fingerprint of traditional Chinese medicine of S. officinalis. The method is of great significance for the quality control of S. officinalis resources and guiding its application.

Conflicts of Interest:
The authors declare no conflict of interest.