Putative Identification of 47 Compounds from Jieyu Anshen Granule and Proposal of Pharmacopeia Quality‐Assessment Strategy Using TCM‐Specific Library with UHPLC‐Q‐Exactive‐Orbitrap‐MS

Abstract

Jieyu Anshen Granule is a traditional Chinese medicine prescription used for depression and primarily comprises five herbal medicines: Zhizi, Chaihu, Zhigancao, Danggui, and Yuanzhi. This study established a traditional Chinese herbal medicine‐specific library using emerging ultra‐high‐performance liquid chromatography‐quadrupole‐orbitrap‐tandem mass spectrometry analysis. Through library comparison, the study has fulfilled isomers distinction. As a result, 47 compounds were simultaneously and putatively identified from Jieyu Anshen Granule, including 12 unexpected compounds and 35 expected compounds. The unexpected compounds comprised cyclocommunol, 5‐hydroxyflavone, tangeretin, 3,5,6,7,8,3’,4’‐heptemethoxyflavone, calycosin‐7‐O‐β‐D‐glucoside, 7,4’‐dihydroxyflavone, naringenin‐7‐O‐β‐D‐glucoside, matrine, betaine, jervine, alantolactone, and hypericin. Among the 35 expected compounds, saikosaponin A, saikosaponin D, glycyrrhizic acid, geniposide, ligustilide, and polygalaxanthone III were further investigated using a quantum chemistry approach. Based on these, an effective quality assessment strategy is proposed for the Pharmacopeia, involving the simultaneous analysis of glycyrrhizic acid, geniposide, ligustilide, polygalaxanthone III, saikosaponins A and D through ultra‐high‐performance liquid chromatography‐quadrupole‐orbitrap‐tandem mass spectrometry analysis. This strategy enables the detection of adulteration in relation to Zhizi, Chaihu, Zhigancao, Danggui, and Yuanzhi in Jieyu Anshen Granule. The findings of unexpected compounds will deepen the understanding of chemistry in Jieyu Anshen Granule.

Traditional Chinese medicine prescription Jieyu Anshen Granule is analyzed using UHPLC−Q‐Exactive Orbitrap‐MS/MS. Forty‐seven compounds are identified and 12 compounds are found in the Granule for the first time. Five compounds are proposed as Pharmacopeia quality‐markers.

Introduction

Traditional Chinese medicine (TCM) has been used for centuries in China and other Asian countries, such as Korea and Japan, and is becoming popular worldwide for its ability to prevent and treat a variety of diseases. The Chinese term “Jieyu Anshen” refers to the process of relieving the psychological burden associated with depression and anxiety. In recent years, Jieyu Anshen Granule (Figure 1) is applied to treat various depression and anxiety, especially post‐stroke depression, hypnosis, and amnesia in TCM. ^[1]

Figure 1.

Jieyu Anshen Granule appearance (the insert upper right is an enlarged view).

Due to its proven efficacy and extensive use in TCM treatments, there are currently 40 pharmaceutical factories manufacturing Jieyu Anshen Granule, as reported by the National Medical Products Administration of China. Their manufacturing techniques are believed to adhere to the guidelines outlined in the Chinese Pharmacopeia (2020). As stated in the Chinese Pharmacopeia, this prescription consists of a mixture of 16 traditional Chinese herbal medicines (TCHMs), including Gardeniae fructus (Zhizi), Bupleuri radix (Chaihu), Glycyrrhizae radix et rhizoma praeparata cum melle (Zhigancao), Angelicae sinensis radix (Danggui), and Polygalae radix (Yuanzhi) (Table 1). ^[2] In TCM, however, the mixing was based on the Monarch‐Minister‐Assistant‐Guide theory, ^[3] while Chaihu and Zhigancao serving as the ‘Monarch’ (Jun Yao) and ‘Guide’ (Shi Yao) roles within the entire granule formulation, respectively. As the ‘Monarch’, Chaihu has the effect of soothing liver and relieving depression; while the ‘Guide’ Zhigancao can harmonize the actions of all medicines in a whole formula.

Table 1.

The main information regarding Jieyu Anshen Granule and its formula.

Chinese name	Plant‐derived TCHMs	Mass /g	Pharmacopeia assessment
			Q‐marker	Method
Zhizi	Gardeniae Fructus	80	Geniposide	HPLC
Chaihu	Bupleurum chinense	80	Saikosaponins A and D	HPLC
Zhigancao	Glycyrrhizae radix et rhizoma praeparata cum melle	60	Liquiritin and glycyrrhizic acid	HPLC
Danggui	Angelicae Sinensis Radix	60	Ferulic acid	HPLC
Yuanzhi	Polygalae Radix	80	Ppolygalaxanthone III, 3’,6‐Disinapoylsucrose	HPLC
Dazao	Jujubae Fructus	60	Betulinic acid, oleanolic acid	TLC
Shichangpu	Acorus tatarinowii Schott	80	Unidentified volatile oil	distillation
Jiangbanxia	Pinelliae Rhizoma Praeparatum cum Zingbere et Alumine	60	None
Baizhu	Atractylodis Macrocephalae Rhizoma	60	Atractylo	TLC
Fuxiaomai	Blighted wheat	200	None
Baihe	Lilii Bulbus	200	Unidentified polysaccharide	Colorimetry
Dannanxing	Arisaema Cum Bile	80	None
Yujin	Curcuma wenyujin	80	None
Longchi	Dens Draconis	200	None
Suanzaoren	Ziziphi Spinosae Semen	100	Jujuboside A and spinosin	HPLC
Fuling	Poria	100	None
Jieyu Anshen Granule		1580	Geniposide	HPLC

As seen in Table 1, Pharmacopeia used a conventional HPLC method to determine geniposide, to assess the quality of whole Jieyu Anshen Granule. However, this method only characterizes the presence of Zhizi, as geniposide is specific to Zhizi and serves as the quality‐marker (Q‐marker) for Zhizi alone. This may lead to adulteration of other herbal medicines. For example, if Danggui is absent or replaced by wood powder in the Granule, such adulteration cannot be recognized by current Pharmacopeia quality assessment strategy, because Danggui's Q‐marker (ferulic acid) is not included in the quality‐assessment strategy.

In order to establish a reliable and effective quality assessment strategy for Jieyu Anshen Granule, the present study meticulously selected a set of authentic standards. These standards were then subjected to analysis using the emerging ultra‐high‐performance liquid chromatography‐Quadrupole‐Orbitrap‐tandem mass spectrometry (UHPLC−Q‐Orbitrap‐MS/MS), to create a specialized library. It's worth noting that all of the standards used were derived from traditional Chinese herbal medicines, making the library highly specialized in the field of TCM.

Using the same conditions as the TCM‐specific library, Jieyu Anshen Granule was also analyzed using UHPLC−Q‐Orbitrap‐MS/MS technology. This analysis allowed for the simultaneous and putative identification of various compounds in Jieyu Anshen Granule through library comparison. Thereafter, some identified compounds were proposed as new and additional Q‐markers for the Pharmacopeia. The integration of these proposed Q‐markers with the aforementioned UHPLC−Q‐Orbitrap‐MS/MS analysis would provide an effective and reliable strategy for assessing the quality of Jieyu Anshen Granule for Pharmacopeia according to Pharmacopeia standards. owing to its high accuracy, UHPLC−Q‐orbitrap MS/MS analysis is believed to unveil previously unexpected compounds.

In addition, computational chemistry approach would also be introduced in the study, to calculate the optimized conformation and energy gap of proposed Q‐markers. All these experimental and computational approaches would not only offer new information regarding the chemistry of Jieyu Anshen Granule, but also enhance the reliability of proposal of Q‐markers.

Materials and Methods

Medicine and Chemicals

Jieyu Anshen Granule was purchased from Xinhui Pharmaceutical Co., LTD (20220322, Jilin, China). Methanol and water were of mass spectra purity grade. All other reagents used in this study were purchased as analytical grade from the Guangzhou Chemical Reagent Factory (Guangzhou, China).

Authentic Standards

5‐Caffeoylquinicacid (Cas. 906–33‐2, C₁₆H₁₈O₉, M.W. 354.309, 98 %), naringenin‐7‐O‐β‐D‐glucoside (Cas. 529–55‐5, C₂₁H₂₂O₁₀, M.W. 434.393, 98 %), isoviolanthin (Cas. 40788–84‐9, C₂₇H₃₀O₁₄, M.W. 578.519, 98 %), calycosin (Cas. 20575–57‐9, C₁₆H₁₂O₅, M.W. 284.263, 98 %), isoliquiritigenin (Cas. 961–29‐5, C₁₅H₁₂O₄, M.W. 256.253, 98 %), formononetin (Cas. 485–72‐3, C₁₆H₁₂O₄, M.W. 268.264, 98 %), jervine (Cas. 469–59‐0, C₂₇H₃₉NO₃, M.W. 425.603, 98 %), and lancerin (Cas. 81991–99‐3, C₁₉H₁₈O₁₀, M.W. 406.34, 98 %) were obtained from Biopurify Phytochemicals, Ltd. (Chengdu, China). 3,3’,4’,5,6,7,8‐Heptamethoxyflavone (Cas. 1178–24‐1, C₂₂H₂₄O₉, M.W. 432.421, 98 %), vicenin‐2 (Cas. 23666–13‐9, C₂₇H₃₀O₁₅, M.W. 594.518, 98 %), and schaftoside Cas. 51938–32‐0, C₂₆H₂₈O₁₄, M.W. 564.49, 98 %) were purchased from Sichuan Weikeqi Biological Technology Co., Ltd. (Chengdu, China). Cyclocommunol (Cas. 145643–96‐5, C₂₀H₁₆O₆, M.W. 352.337, 98 %) was purchased from BioBioPha Co., Ltd. (Kunming, China). Tangeretin (Cas.481‐53‐8, C₂₀H₂₀O₇, M.W. 372.369, 98 %), ferulic acid (Cas. 1135–24‐6, C₁₀H₁₀O₄, M.W. 194.184, 98 %), rutin (Cas. 153–18‐4, C₂₇H₃₀O₁₆, M.W. 610.518, 98 %), chrysoeriol (Cas. 491–71‐4, C₁₆H₁₂O₆, M.W. 300.263, 98 %), 7‐O‐methylmangiferin (Cas. 31002–12‐7, C₂₀H₂₀O₁₁, M.W. 436.366, 98 %), swertisin (Cas. 6991–10‐2, C₂₂H₂₂O₁₀, M.W. 462.404, 98 %), hypericin (Cas. 548–04‐9, C₃₀H₁₆O_8, M.W. 504.45, 98 %), 1,2,3,7‐tetramethoxyxanthone (Cas. 22804–52‐0, C₁₇H₁₆O₆, M.W. 316.3, 98 %), 7,4’‐dihydroxyflavone (Cas. 2196–14‐7, C₁₅H₁₀O₄, M.W. 254.238, 98 %), and naringenin (Cas. 480–41‐1, C₁₅H₁₂O₅, M.W. 272.253, 98 %) were obtained from Chengdu Alfa Biotech. Ltd. (Chengdu, China). 5‐Hydroxyflavone (Cas. 491–78‐1, C₁₅H₁₀O₃, M.W. 238.238, 98 %), sucrose (Cas. 57–50‐1, C₁₂H₂₂O_11, M.W. 342.30, 98 %), ethyl stearate (Cas.111‐61‐5, C₂₀H₄₀O₂, M.W. 312.53, 98 %), and (+)‐4‐cholesten‐3‐one (Cas. 601–57‐0, C₂₇H₄₄O, M.W. 384.638, 98 %) were from TCI Chemical Co. (Shanghai, China). trans‐Cinnamic acid (Cas. 140–10‐3, C₉H₈O₂, M.W. 148.159, 98 %) and L‐proline (Cas. 147–85‐3, C₅H₉NO₂, M.W. 115, 98 %) was from J&K Scientific Co., Ltd. (Beijing, China). Glycyrrhizic acid (Cas. 1405–86‐3, C₄₂H₆₂O₁₆, M.W. 822.932, 98 %), liquiritigenin (Cas. 578–86‐9, C₁₅H₁₂O₄, M.W. 256.257, 98 %), liquiritin (Cas. 551–15‐5, C₂₁H₂₂O₉, M.W. 418.398, 98 %), isoliquiritin (Cas. 5041–81‐6, C₂₁H₂₂O₉, M.W. 418.398, 98 %), calycosin‐7‐O‐β‐D‐glucoside (Cas. 20633–67‐4, C₂₂H₂₂O₁₀, M.W. 446.408, 98 %), polygalaxanthone III (Cas. 162857–78‐5, C₂₅H₂₈O₁₅, M.W. 568.484, 98 %), liquiritin apioside (Cas. 74639–14‐8, C₂₆H₃₀O₁₃, M.W. 550.513, 98 %), 18β‐glycyrrhetinic acid (Cas. 471–53‐4, C₃₀H₄₆O₄, M.W. 470.694, 98 %), citric acid (Cas. 77–92‐9, C₆H₈O₇, M.W. 192.123, 98 %), ligustilide (Cas. 81944–09‐4, C₁₂H₁₄O₂, M.W. 190.238, 98 %), alantolactone (Cas. 546–43‐0, C₁₅H₂₀O₂, M.W. 232.323, 98 %), matrine (Cas. 519–02‐8, C₁₅H₂₄N₂O, M.W. 248.37, 98 %), betaine (Cas. 107–43‐7, C₅H₁₁NO₂, M.W. 117.148, 98 %), saikosaponin A (Cas. 20736–09‐8, C₄₂H₆₈O₁₃, M.W. 780.982, 98 %), and saikosaponin D (Cas.20874‐52‐6 C₄₂H₆₈O₁₃, M.W. 780.982, 98 %) were from Shaanxi Herbest Co., Ltd. (Baoji, China). Isoferulic acid (Cas. 537–73‐5, C₁₀H₁₀O₄, M.W. 194.184, 98 %) and geniposide (Cas. 24512–63‐8, C₁₇H₂₄O₁₀, M.W. 388.366, 98 %) were from Shanghai Yuanye Biotechnology Co., Ltd. (Shanghai, China). D‐gluconic acid (Cas. 526–95‐4, C₆H₁₂O₇, M.W. 196.155, 98 %) was from Sigma‐Aldrich Co., Ltd. (Shanghai, China). Caffeine (Cas. 58–08‐2, C₈H₁₀N₄O₂, M.W. 194.191, 98 %) was prepared by our laboratory. ^[4]

Preparation of Authentic Standard Solution and Sample Solution

Preparation of Authentic Standard Solution

All authentic standards listed in 2.2 Section were individually dissolved in methanol at 30 μg/mL concentration. The solutions were individually transferred into flask and subsequently filtered through 0.45 μm membrane. The filtrate was kept at 2–6 °C for the further analysis. ^[5]

Preparation of Sample Solution

To avoid the possible insoluble impurity and solvent effect, ^[6] Jieyu Anshen Granule was treated by the previous method. ^[7] Through the treatment, the lyophilized powder of Jieyu Anshen Granule was prepared and then re‐dissolved in methanol to obtain the sample solution (30 mg/mL, Figure 2).

Figure 2.

The flowchart of sample solution preparation of lyophilized aqueous extract from Jieyu Anshen Granule.

UHPLC‐Q‐Exactive Orbitrap‐MS/MS Analysis

The analysis of Jieyu Anshen Granule was conducted using a UHPLC system coupling with Q‐Exactive Orbitrap mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA) equipped with a heated electrospray ionization (HESI) probe. The sample solution (at 30 mg/mL) was separated on an Accucore RP‐MS LC C₁₈ column (100 mm×2.1 mm, 2.6 μm, Thermo Fisher) by binary mobile phase at 0.4 mL/min flow rate.

The binary mobile phase however consisted of mobile phase A (water, 0.1 % v/v formic acid) and mobile phase B (methanol). The linear gradient elution procedure could be described as the following: 0.0–5.0 min, 10 % B; 5.0–14.5 min, 10 % B→100 % B; 14.5–16.0 min, 100 % B; 16.0–20.0 min, 10 % B. The column chamber and sample tray were held at 40 °C and 4 °C, respectively. The electrospray ionization (ESI) parameters were set as follows: spray voltage, 4.5 kV under negative mode; sheath gas (N₂) flow rate, 40 arbitrary units; auxiliary gas (N₂) flow rate, 10 arbitrary units; capillary temperature, 450 °C; resolution, Ms full scan 70,000 full width at half maximum (FWHIM), MS/MS scan 17.500 FWHIM; AGC target, 2×10⁵; Stepped normalized collision energy: 20, 50, and 90; scan range, m/z 100–1200. An external calibration for mass accuracy was carried out before the analysis according to the manufacturer's guidelines.

Putative Identification Using Software and MS Spectra Elucidation

The main operations regarding injection, data acquisition, and analysis were controlled by Xcalibur 4.1 package. The package comprised a TraceFinder General Quan (Thermo Fisher Scientific Inc., Waltham, MA, USA), and was equipped in the UHPLC−Q‐Exactive Orbitrap‐MS/MS apparatus. Before data acquisition, the background sign was subtracted using blank solvent. The acquired data were then exported to TraceFinder General Quan for m/z extraction, to form the corresponding MS spectra. Herein the processing parameters were list as follows: mass range: 100–1200 Da; mass tolerance: 5 ppm; S/N threshold: 5; isotopic pattern fit threshold: 90 %.

Subsequently, the MS spectra and corresponding top 5 secondary spectrum were screened using the Xcalibur 4.1. Through comparing with authentic standards in 4 parameters (retention time, molecular ion peak, MS/MS profile, and characteristic fragments), the compounds were putatively identified by manual. ^[8]

Computational Chemistry Approach

All computational calculation was conducted using the Gaussian 16 software. The conformational optimization, energy optimization, and dipole moment were calculated at (U)B3LYP−D3(BJ)/6‐31+G(d,p).[ ⁹ , ¹⁰ , ¹¹ ] The conformational optimization was identified by absence of imaginary frequencies. The optimized conformation was viewed and then exported via Gaussian View 6.1.1 software. ^[12] The energy optimization included single point energy (SPE) optimization and HOMO→LUMO (from highest occupied molecular orbital to lowest unoccupied molecular orbital) energy gap optimization. Six calculated compounds were geniposide (10), polygalaxanthone III (19), ligustilide (35), glycyrrhizic acid (38), saikosaponin A (42), and saikosaponin D (43). Gaussian 16 and Gaussian View 6.1.1 were manufactured by Gaussian, Inc. (Wallingford, CT, USA).

Statistical Analysis

Each experiment of quantitative assessment was performed in triplicate. Data were shown as the means±standard deviations (SD) from three independent measurements. ^[13]

Results

UHPLC‐Q‐Exactive‐Orbitrap‐MS/MS Identification

The present study analyzed the sample solution of lyophilized aqueous extract from Jieyu Anshen Granule using UHPLC−Q‐Orbitrap MS/MS technology to obtain its total ion current (TIC) diagram (Figure 3). Additionally, the molecular formula, retention time values, and fragment m/z values were also extracted (Table 2). It was, however, necessary to compare this information with authentic standards in the library in order to identify 47 compounds (1–47) ^[10] (Figure 4). Considering the layout space, the MS spectra, along with the fragmenting elucidations of all compounds, were deposited in the Suppls. 1–47. Especially, the MS spectra along with the fragmenting elucidations of three saponins (38, 42, and 43) were listed in Figures 5, 6, 7 and the MS fragmenting elucidation of cyclocommunol (40) is illustrated in Figure 8.

Figure 3.

The total ion current (TIC) chromatogram of Jieyu Anshen Granule in the UHPLC−Q‐Exactive‐Orbitrap‐MS/MS analysis. (A) The negative ion mode; (B) the positive ion mode. The positive ion mode was supplement of negative ion mode.

Table 2.

The main experimental results of 47 putatively identified compounds (1–47) in Jieyu Anshen Granule.

	Name	Formula	(M−H)/(M+H) m/z	R.T. min	Diagnostic fragment m/z	Plant sources
1	D‐Gluconic acid	C6H12O7	195.0505	0.51	177.0395, 159.0288, 129.0183	Baihe [15]
2	Sucrose	C12H22O11	341.1084	0.54	179.0553, 113.0233, 101.0233	Baihe [15]
3A	L‐proline	C5H9NO2	116.0712	0.54	70.0657, 68.0501	Danggui, [16] Suanzaoren [17]
4A	Betaine	C5H11NO2	118.0868	0.55	74.0607, 59.0736, 58.0658
5	Citric Acid	C6H8O7	191.0192	0.61	173.0083, 129.0183, 122.3034, 111.0077,	Danggui [16]
6A	Matrine	C15H24N2O	249.1967	0.87	247.1796, 218.1524, 176.1064, 148.1117
7	5‐Caffeoylquinic acid	C16H18O9	353.0881	1.65	191.0557, 179.0341, 135.0443	Dannanxing[ 18 , 19 ]
8A	Caffeine	C8H10N4O2	195.0871	5.33	163.0385, 138.0658, 123.0424, 116.9859, 110.0714	Dannanxing [20]
9A	trans‐Cinnamic acid	C9H8O2	149.0593	8.05	149.0593, 121.0646, 107.0490, 103.0543	Yuanzhi [21]
10	Geniposide	C17H24O10	387.1277	8.06	355.1029, 225.0767, 207.0661, 147.0442, 123.0441,	Zhizi [22]
11	Vicenin‐2	C27H30O15	593.1514	8.37	473.1100, 353.0649, 117.0336	Zhigancao [23] Suanzaoren[ 24 , 25 ]
12	Ferulic acid	C10H10O4	193.0502	8.52	178.0263, 149.0595, 137.0235	Baizhu, [26] Danggui [28] Dannanxing, [27] Shichangpu [29]
13	Isoferulic acid	C10H10O4	193.0507	8.69	178.0263, 137.0234, 134.0365
14	Schaftoside	C26H28O14	563.1412	8.79	473.1113, 383.0777, 353.0669, 297.0771	Dannanxing,[ 30 , 31 , 32 ] Zhigancao [33]
15	Lancerin	C19H18O10	405.0827	8.87	315.0518, 285.0408, 257.0455	Yuanzhi [34]
16	Liquiritin	C21H22O9	417.1186	9.01	255.0661, 135.0078, 119.0492	Zhigancao [2]
17	Liquiritin apioside	C26H30O13	549.1608	9.16	255.0660, 1135.0078, 119.0492	Zhigancao [35]
18	7‐O‐Methylmangiferin	C20H20O11	435.0932	9.17	345.0600, 315.0509, 272.0328, 243.0298, 215.0346	Yuanzhi [34]
19	Polygalaxanthone III	C25H28O15	567.1350	9.22	345.0612, 272.0327, 171.0445	Yuanzhi [34]
20	Swertisin	C22H22O10	445.1135	9.44	297.0403, 282.0538, 178.9982, 117.0336	Dazao, [36] Suanzaoren [37]
21	Isoviolanthin	C27H30O14	577.1559	9.5	383.0772, 353.0666, 297.0775, 117.0336	Zhigancao [38]
22	Rutin	C27H30O16	609.1485	9.53	300.0280, 271.0257, 243.0291	Baihe, [39] Chaihu, [40] Dazao, [36] Zhigancao, [33] Suanzaoren, [24] Zhizi [41]
23	Naringenin‐7‐O‐β‐D‐glucoside	C21H22O10	433.1143	9.63	271.0617, 151.0029, 119.0492
24	Isoliquiritin	C21H22O9	417.12	10.18	297.0759, 255.0661, 135.0078	Zhigancao[ 33 , 42 ]
25	Liquiritigenin	C15H12O4	255.06	10.3	135.0077, 119.0492, 117.0337	Zhigancao[ 33 , 42 ]
26	Chrysoeriol	C16H12O6	299.0561	10.56	284.0330, 256.0378, 183.0443	Zhigancao [33]
27	7,4’‐Dihydroxyflavone	C15H10O4	253.0505	10.7	223.0396, 135.0081, 117.0336
28	Calycosin	C16H12O5	283.0609	10.7	268.0379, 239.0355, 211.0399, 195.0450, 183.0450	Zhigancao [38]
29	Naringenin	C15H12O5	271.0613	10.87	151.0029, 119.0492, 117.0334, 107.0127	Zhigancao, [43] Suanzaoren [44]
30	Isoliquiritigenin	C15H12O4	255.0661	11.44	135.0078, 119.0492	Zhigancao[ 33 , 42 ]
31	Calycosin‐7‐O‐β‐D‐glucoside	C22H22O10	445.11	11.47	401.0875, 357.0983, 225.0548, 181.0652
32	Formononetin	C16H12O4	267.0664	11.77	252.0427, 223.0397, 195.0447, 167.0495, 132.0207	Zhigancao [45]
33A	1,2,3,7‐Tetramethoxyxanthone	C17H16O6	317.1	12.44	287.0540, 259.0593, 215.0332	Yuanzhi [21]
34A	3,5,6,7,8,3’,4’‐Heptemethoxyflavone	C22H24O9	433.1474	12.48	403.1008, 345.0583, 205.0846, 165.053
35A	Ligustilide	C12H14O2	191.1	12.5	173.0959, 145.1002, 129.0697, 115.0542, 105.0700	Danggui[ 16 , 28 , 46 , 47 ]
36	Jervine	C27H39NO3	424.2862	12.72	248.1652, 179.1073, 163.1120
37A	Tangeretin	C20H20O7	373.1267	12.84	343.0801, 297.0736, 183.0284, 135.0436
38	Glycyrrhizic acid	C42H62O16	821.3980	13.11	351.0578, 235.0463, 193.034484	Zhigancao [48]
39A	Alantolactone	C15H20O2	233.15	13.12	187.1474, 145.1008, 131.0852
40	Cyclocommunol	C20H16O6	351.0875	13.19	151.0027, 107.0493
41A	5‐Hydroxyflavone	C15H10O3	239.0695	13.39	165.0539, 139.0537, 137.0230, 103.0543
42	Saikosaponin A	C42H68O13	779.4589	13.59	617.4059, 439.3241, 145.0498, 101.0239	Chaihu [49]
43	Saikosaponin D	C42H68O13	779.4586	13.89	617.4045, 439.3238, 145.0489	Chaihu [49]
44	18β‐Glycyrrhetinic acid	C30H46O4	469.33	14.55	425.3433, 355.2642	Zhigancao [48]
45	Ethyl Stearate	C20H40O2	311.2959	16.11	183.0114, 119.0492	Suanzaoren [17]
46	Hypericin	C30H16O8	503.08	16.42	459.0873, 405.0771, 361.0877
47A	(+)‐4‐Cholesten‐3‐one	C27H44O	385.3448	17.39	367.3326, 123.0800, 109.0648	Dannanxing [50]

Note: The compounds with serial number “A” were identified by positive ion mode. The m/z value in bold means the characteristic fragments. The m/z values below 50 were also found by the Xcalibur 4.1 Software package, despite that the scan mode rang was set at m/z 100–1200 in the mass spectra. The table only listed the diagnostic fragment of MS/MS spectra. All Other fragments of MS/MS spectra were detailed in Suppls. 1–47. The fragmenting pathway elucidation was also shown in Suppls. 1–47.

Figure 4.

The structures and configurations of 47 identified compounds from Jieyu Anshen Granule. The red indicates the old Q‐marker, while the purple indicates new Q‐markers. The wave line in 35 indicated the uncertain stereo‐configuration. The symbol “a” means positive ion model. Considering the layout space, the TIC diagram under positive ion model was not shown in the main text. Knot line with “iso” connected two isomers.

Figure 5.

The putative identification of glycyrrhizic acid (38). (A) MS/MS spectra and the relevant elucidation of standard glycyrrhizic acid; (B) MS/MS spectra of the peak (R.T. 13.11). The m/z values in purple indicated the calculated ones. The relative standard deviation (RSD) values of fragments between calculated m/z values and experimental m/z values varied from 4.87×10⁻⁷–3.40×10⁻⁶. The MS/MS spectra of two corresponding standards are detailed in Suppl. 43.

Figure 6.

The putative identification of saikosaponin A (42). (A) MS/MS spectra and the relevant elucidation of standard saikosaponin A; (B) MS/MS spectra of the peak (R.T. 13.59). The m/z values in purple indicated the calculated ones. The relative standard deviation (RSD) values of fragments between calculated m/z values and experimental m/z values varied from 0–6.93×10⁻⁶. The MS/MS spectra of two corresponding standards are detailed in Suppl. 43.

Figure 7.

The putative identification of saikosaponin D (43). (A) MS/MS spectra and the relevant elucidation of standard saikosaponin D; (B) MS/MS spectra of the peak (R.T. 13.89). The m/z values in purple indicated the calculated ones. The m/z values in blue are the calculated ones. The relative standard deviation (RSD) values of fragments between calculated m/z values and experimental m/z values varied from 1.28×10⁻⁷–2.28×10⁻⁶. The MS/MS spectra of two corresponding standards are detailed in Suppl. 43.

Figure 8.

The MS fragmenting elucidation of cyclocommunol (40).

Results of Computational Chemistry

Computational chemistry was performed using Gaussian 16 software. Computational results, including conformational optimization and the calculation of various parameters, were analyzed using Gaussian View 6.1.1. The optimized conformations are presented in Figure 9, and additional parameters are listed in Table 3.

Figure 9.

The optimized conformation of geniposide (10), polygalaxanthone III (19), ligustilide (35), glycyrrhizic acid (38), saikosaponin A (42), and saikosaponin D (43). The calculation was conducted at (U)B3LYP−D3(BJ)/6‐31+G(d,p) basis set with SMD model using the Gaussian 16 software and Gaussian View 6.1.1.

Table 3.

The relevant parameters of geniposide (10), polygalaxanthone III (19), ligustilide (35), glycyrrhizic acid (38), saikosaponin A (42), and saikosaponin D (43).

	Q‐markers	SPE	Dipole moment	HOMO→LUMO
10	geniposide	−1414.4959	7.3381	0.2097
19	polygalaxanthone III	−2097.8782	6.8814	0.1541
35	ligustilide	−616.1624	5.1278	0.1402
38	glycyrrhizic acid	−2841.4520	9.5124	0.1822
42	saikosaponin A	−2619.2731	5.9957	0.2404
43	saikosaponin D	−2619.2722	5.9992	0.2417

RB3LYP, restricted B3LYP basis set; SPE, single point energy, Hartree unit; dipole moment value, Debye unit; HOMO→LUMO, the energy gap from highest occupied molecular orbital to lowest unoccupied molecular orbital, a.u. unit, 1 a.u.=2625.5 kJ/mol.

Discussion

In the present study, a total of 47 compounds were identified simultaneously from Jieyu Anshen Granules, as shown in Figure 4. From an efficiency perspective, it presents an advantage over conventional HPLC‐UV analysis, which can only identify a limited number of compounds within the Granule. ^[38] This, of course, can be attributed to the application of UHPLC−Q‐Orbitrap MS/MS technology itself, because similar high‐efficiency identification was also reported in the previous studies. However, these studies overlooked the distinction of isomers. ^[39]

In the study, liquiritin (16) and isoliquiritin (24) contructed a pair of structural isomers, for their structural skeletons differ. Our previous study demonstrated that the former skeleton could be synthesized from the latter skeleton with the catalysis of chalcone isomerase (CHI).^[40]. However, two isomers have been clearly distinguished from each other in the study (Suppls. 16 and 24). Another pair of skeleton isomers (25 and 30) was also successfully distinguished from each other. Additionally, a pair of positional isomers (12 and 13) has been strictly distinguished as well (see Figure 4). The successful differentiation of these isomers was made possible by a novel method developed by our research team. ^[41]

Taken together, our study combined the TCM‐specific library with UHPLC−Q‐Orbitrap‐MS/MS analysis to achieve isomer distinction. These achievements can ultimately be attributed to the high‐accuracy MS/MS fragmenting elucidation. In Figures 5–7 (Suppls. 1–47), it is evident all compounds have been elucidated using the diagnostic MS/MS fragment based on the MS spectra principle (e. g., Retro Diels‐Alder fragmenting, Figure 8). The RSD values between the calculated and experimental m/z values were on the order of magnitude of 10⁻⁶ (e. g., 0.0000 ‰~0.0065 ‰, see Figure 8). It has been suggested that our work was highly reliable and was thus regarded as putative identification. In contrast, the previous tentative identifications were incapable of isomer distinction. For instance, three teams did not elucidate the MS/MS fragmenting, thus rendering them unable to distinguish isomers.[ ⁴² , ⁴³ , ⁴⁴ ]

Among these putatively identified compounds, twelve were described as unexpected, as there were no prior reports indicating their presence in the Granule, despite a systematic document retrieval effort. These twelve compounds included betaine (4), matrine (6), naringenin‐7‐O‐β‐D‐glucoside (23), 7,4’‐dihydroxyflavone (27), calycosin‐7‐O‐β‐D‐glucoside (31), 3,5,6,7,8,3’,4’‐heptemethoxyflavone (34), tangeretin (37), jervine (36), alantolactone (39), cyclocommunol (40), 5‐hydroxyflavone (41), and hypericin (46).

In particular, eight unexpected compounds (6, 31, 34, 36, 37, 40, 41, and 46) showed low peak signs in the TIC diagram (see Figure 3). Consequently, they were marked as “unexpected and trace level”. Instances of similar unexpected and trace level compounds have been noted in prior UHPLC−Q‐Orbitrap‐MS/MS analysis. ^[6] In contrast, four unexpected compounds (4, 23, 27, and 39) showed strong peak signs in the TIC diagram (see Figure 3), which led to their classification as “unexpected and substantial level” compounds. Similar unexpected and substantial level instances have been recently reported in the UHPLC−Q‐Orbitrap‐MS/MS analyses of pomelo peel. ^[45] It is worth noting that phytochemical studies related to pomelo peel were initiated in the 1980s ^[46] , predating our findings. This means that the phytochemical work is an ongoing process and our finding of twelve unexpected compounds may be omitted by previous phytochemical workers. In addition to providing guidance for future phytochemical research, the discovery of “unexpected” compounds will also shed light on the chemistry of Jieyu Anshen Granule or its relevant plants. In fact, two unexpected and substantial level compounds, betaine and alantolactone have been reported to possess bioactive effects similar to those found in the Granule.[ ⁴⁷ , ⁴⁸ ]

All these expected and unexpected compounds, however, could be classified into sixteen main types, including flavonoid, xanthone, alkaloid, saponin, phenolic acid, chalcone, sugar, triterpene, steroid, lactone, iridoid, amino acid, naphthaladianthrone, organic acid, lipid, and caffeoylquinic acid (Figure 4), from a phytochemical perspective. This chemical diversity may account for the therapeutic effect of the Jieyu Anshen Granule as a whole.

As shown in Figure 3, iridoid geniposide (10) exhibited a prominent peak at a retention time of 8.06 minutes, indicating its robust detectability. In addition, it was also documented to possess a similar antidepressant pharmacological effect to Jieyu Anshen Granule. ^[49] Possibly owing to these, geniposide was selected as the Pharmacopeia Q‐marker previously. ^[2]

Herein we aimed to introduce new Pharmacopeia Q‐markers, adhering to the five principles outlined by Academician Liu Changxiao. ^[50] Aside from chemical stability, non‐industrialization principles should also be taken into account. ^[51] The former could explain the traceability, while the latter could prevent safety tragedies. This is because that industrialized Q‐marker would be illegally added into Jieyu Anshen Granule, causing a tragedy similar to the Sanlu Melamine Incident in China (2008). In accordance with these principles, five compounds (19, 35, 38, 42, and 43) were proposed as potential new Q‐marker, as summarized in (see Table 4).

Table 4.

The compliance with relevant principles of 5 new Q‐marker candidates (polygalaxanthone II 19, ligustilide 35, glycyrrhizic acid 38, saikosaponin A 42, and saikosaponin D 43).

	19	35	38	42	43
Traceability	√; Ref. [52]	√; Ref. [2]	√; Ref. [38]	√; Ref. [53]	√; Ref. [53]
Testability	√; Figure 3	√; Figure 3	√; Figure 3	√; Figure 3	√; Figure 3
Specificity	√; Ref. [2]	√; Ref. [2]	√; Ref. [2]	√; Ref. [2]	√; Ref. [2]
Efficiency‐relevance	√; Ref. [52]	√; Ref. [54]	√; Ref. [55]	√; Ref. [56]	√; Ref. [56]
TCM‐relevance	√; Table 1	√; Table 1	√; Table 1	√; Table 1	√; Table 1
Chemical stability	√; Table 3	√; Table 3	√; Table 3	√; Table 3	√; Table 3
Non‐industrialization	√	√	√; Ref. [2]	√; Ref. [2]	√; Ref. [2]

Five new Q‐markers, in addition to the old one, have been utilized to construct a new Q‐marker system. The system coupling with the UHPLC−Q‐Orbitrap‐MS/MS analysis is poised to establish a novel quality assessment strategy. By integrating the plant resource information (Table 2 ) and Pharmacopeia evidence, ^[2] new quality assessment strategy was able to judge the adulteration of Jieyu Anshen Granule.

In accordance with Pharmacopeia and literature,[ ² , ¹⁶ ] polygalaxanthone III (19) serves as a key active compound, making it suitable for the characterization of individual Yuanzhi. In fact, it could be readily detected in plant extract or animal plasma. ^[52] Therefore, if polygalaxanthone III is found to be absent in Jieyu Anshen Granule by the quality assessment strategy, it means that Yuanzhi is absent and there is adulteration in Jieyu Anshen Granule.

Correspondingly, if both saikosaponins (42 and 43) cannot be detected, it signifies the presence of adulteration in Chaihu. This is because that (1) 42 and 43 are two Pharmacopeia Q‐markers of single Chaihu ^[2] ; and (2) Chaihu is their sole plant source (Table 2). In a similar vein, the absence of ligustilide implies a lack of Danggui or adulteration. As stated in Table 2, Danggui is the sole plant source of ligustilide in Jieyu Anshen Granule. Additionally, ligustilide is a distinct, abundant, and detectable compound of Danggui. ^[12] Likewise, the absence of glycyrrhizic acid suggests adulteration of Zhigancao in Jieyu Anshen Granule. Geniposide (10), the old Q‐marker (pre‐existing Q‐marker), is also subject to this logical judgment. Once geniposide is not found using our quality assessment strategy, Jieyu Anshen Granule will be suggested for adulteration regarding Zhizi (Table 2). In summary, our proposed quality assessment strategy allows for the specific identification of five types of adulteration in Jieyu Anshen Granule. The total characterizing rate (TCR) of the new strategy was 31.25 % (5÷16), while its specific characterizing rate (SCR) was also 31.25 % (5÷16), according to our previous definition. ^[51] Using this definition, the TCR and SCR values of the old quality assessment strategy were calculated as 6.25 % (1÷16) and 6.25 %, respectively. Now it is obvious that our proposed strategy has greatly improved the reliability of quality assessment for Pharmacopeia. On the other hand, our strategy has the potential to improve the specificity of the Pharmacopeia as well, for it can distinguish Jieyu Anshen Granule from three other TCM prescriptions, namely Zhizi Jinhua Wan, Linglianhua Granule, and Zhiqin Qingre Heji. This distinction arises because all of these prescriptions use geniposide (10) as the sole Q‐marker. Thus, without the improved quality assessment strategy, Jieyu Anshen Granule could easily be mistaken for these other prescriptions.

Finally, it is worth mentioning that, (1) two epimeric saikosaponins (42 and 43) exhibit high similarity to each other. However, saikosaponin A positions the −OH group on the same side of the O‐bridge, whereas saikosaponin D situates the −OH group on the opposite side of the O‐bridge (see Figure 9). The distinct arrangements have differentiated the molecular polarities, allowing for their separation through conventional C₁₈ adsorption chromatography column ^[57] (refer to Table 3).

(2) Two saikosaponins (42 and 43) can also be interconverted through biosynthesis, ^[58] especially when they leaped over a low energy gap (▵E=0.004 Hartree, as depicted in Figure 9C). Therefore, only when both saikosaponins are not detected by the quality assessment strategy, the adulteration of Chaihu can be considered.

(3) As seen in Table 1, betulinic acid and oleanolic acid are defined as the Pharmacopeia Q‐marker of Dazao. However, both compounds could not be detected in Jieyu Anshen Granule (as shown in Figure 4). This absence may be attributed to the low proportion of Dazao in the Granule which accounts for only (3.79 %, 60÷1580). It's worth noting that our TCM‐specific library includes standards for these compounds.

(4) Jieyu Anshen Granule is a prescription comprised of 16 TCHMs. Each CHM is recognized for containing a significant number of compounds, and some compounds may appear in multiple TCHMs. This complexity has made the Granule an exceptionally intricate chemical system. Therefore, characterization of all 16 TCHMs using a Q‐marker system remains a challenge, despite significant advancements in the study.

(5) The study regarding the Pharmacopeia quality assessment strategy is distinct from the Pharmacopeia strategy itself, as the study lacks both administrative compulsion and legal authority. The official adoption of such a strategy depends on the deliberation of the Pharmacopeia Commission. Furthermore, the Pharmacopeia Commission's deliberations are influenced by the accumulation of research publications. The Pharmacopeia Commission would not identify a new and effective quality assessment strategy without these research publications.

Conclusions

Through the application of UHPLC−Q‐Orbitrap‐MS/MS for putative identification, it has been confirmed that the Jieyu Anshen Granule contains a minimum of 47 compounds, comprising 12 unexpected compounds and 35 expected compounds. These unexpected compounds include cyclocommunol, 5‐hydroxyflavone, tangeretin, 3,5,6,7,8,3’,4’‐heptemethoxyflavone, calycosin‐7‐O‐β‐D‐glucoside, 7,4’‐dihydroxyflavone, naringenin‐7‐O‐β‐D‐glucoside, matrine, betaine, alantolactone, and hypericin. Among the expected compounds, saponin saikosaponin A and its epimer saikosaponin D can be easily separated in the C₁₈ column. Saikosaponins A and D, along with glycyrrhizic acid, geniposide, ligustilide, and polygalaxanthone III can establish a novel quality assessment strategy to identify potential adulteration related to Zhizi, Chaihu, Zhigancao, Danggui, and Yuanzhi in Jieyu Anshen Granule. Therefore, we proposed its consideration by the Pharmacopeia Commission. Additionally, the identification of these 12 unexpected compounds will significantly benefit its future chemical or pharmacological research.

Ethical Approval

Not applicable.

Author Contributions

XL contributed to the project design and paper writing. JZ and XC contributed to literature review and analysis experiments. RC, SK and JZ contributed to data analyses. CL and BC contributed to computational chemistry. XZ contributed to paper revision. All authors read and approved the final manuscript.

Conflict of Interests

The authors declare no conflict of interest.

Supporting information

As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re‐organized for online delivery, but are not copy‐edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.

Supporting Information

Li X., Zeng J., Cai R., Li C., Chen X., Chen B., Zhao X., Khan S., ChemistryOpen 2025, 14, e202400046. 10.1002/open.202400046

Data Availability Statement

All the data used to support the findings of this study are available from the corresponding author upon reasonable request.