Comparison of Crude and Wine-Steamed Corni Fructus Based on UPLC-Q-TOF-MS Combined With Pharmacodynamics Screening

Abstract

Introduction: The dry ripe sarcocarp of Cornus officinalis Sieb.et Zucc (in Chinese Shanyurou) has the properties of tonifying the liver and kidney. Wine-steamed Corni Fructus, as the main processed product from Corni Fructus, is widely used in traditional Chinese clinical medicine. This article aims to study the Corni Fructus composition changes when wine-steamed processed and to discuss the optimal processing technology of Corni Fructus combined with pharmacological experiments. Methods: The differences between Corni Fructus and wine-steamed Corni fructus constituents were analyzed by ultra-performance liquid chromatography-tandem mass spectrometry. The effects of Corni Fructus and wine-steamed Corni Fructus with different processing times were studied from the aspects of immune activity, liver protection, and kidney yin deficiency. Results: A total of 132 compounds were identified in the wine-steamed process of Corni Fructus. There were no significant differences in immune function, liver protection, and kidney yin deficiency of Corni Fructus with different processing times. However, the effects of Corni Fructus processed by wine-steaming were significantly enhanced in all aspects. Conclusion: The wine-steamed Corni Fructus chemical composition may be the material basis for enhancing liver and kidney function. After processing, Corni Fructus can enhance immune function, liver protection function, and liver and kidney toning function.

Keywords

Corni Fructus wine-steamed Corni Fructus immune activity liver protection kidney yin deficiency

Background

The dried and ripe pulp of Cornus officinalis Sieb.et Zucc., a plant of the Cornaceae family, is mainly produced in Henan, Zhejiang, Shanxi, Sichuan, and other provinces. It nourishes the liver and kidney and tightens astringency.^1–4 It is often used for dizziness, tinnitus, soreness in the waist and knees, impotence and nocturnal emission, enuresis, frequent urination, metrorrhagia, profuse sweating, and diabetes, among others. The decoction of Corni Fructus is recorded in the 2020 edition of the Chinese Pharmacopoeia.⁵ After processing, wine-steamed Corni Fructus can warm the body by virtue of the wine, help the drug potential, reduce its acidity, and enhance its function of tonifying liver and kidney, immune regulation, and anti-aging. The main clinical application in Chinese medicine is wine-steamed Corni Fructus. The Chinese Pharmacopoeia records the processing technology of the plant material in wine as follows: clean shanyurou is taken, stewed with wine, steamed with wine, or steamed until the wine is exhausted. Generally, it lacks clear process parameters. There are also big variations in the process,^6–9 and there is a lack of specific regulations for parameters such as immersion time, steaming time, and the amount of rice wine. It can be seen from the above that the jiuyurou processing technology is not uniform, and there are no specific processing parameters. Chinese herbal decoction pieces are directly used in the formulation of traditional Chinese medicine (TCM) clinical prescriptions and the raw materials of Chinese patent medicines. Therefore, the quality of Chinese herbs and preparations is directly related to their clinical efficacy. The processing technology largely determines the quality of the Chinese herbal. This study compares crude and wine-steamed Corni Fructus based on the ultra-performance liquid chromatography-quadrupole time of flight-tandem mass spectrometry (UPLC-Q-TOF-MS) combined with pharmacodynamics, which can provide evidence of processing time for jiuyurou.^10–13 The results will determine how different processing times affect the constituents of jiuyurou and its medicinal effects (immunity, liver protection, and kidney-nourishing effects). Our results will provide the basis for the processing technology of decoction pieces.

Materials and Methods

Plant Material and Processing Technology With Different Wine Steaming Times

The Corni Fructus decoction pieces were collected from Xishan city, Henan Province and the batch number was G2101667. The samples were identified by Mei Wei (the corresponding author).

The 27 batches of Corni Fructus decoction pieces were soaked in wine (the amount of wine was 20% of the decoction pieces’ weight) for 2 h until the wine was completely absorbed. They were then steamed for 0, 1, 2, 4, 6, 8, 10, 12, and 14 h, respectively.

Chemicals and Reagents

Yellow rice wine (Lot No: FF2012005) was purchased from Shaoxing County No. 3 Distillery. The content of alcohol was 14.5% v/v, and the main components were water, rice, and wheat. Standard compounds, including β-maltose, citric acid, uridine, adenosine, guanosine, 5-hydroxymethyl-2-furaldehyde, 3, 4-dihydroxybenzoic acid, geniposidic acid, loganic acid, morroniside, cynaroside, alyposide, caffeic acid, rhoifolin, loganin, genistein, ferulic acid, rutin, hyperoside, isoquercitrin, quercetin, apigenin, ligustilide, and ursolic acid were purchased from National Institutes for Food and Drug Control. L-proline, pantothenate, ethyl gallate, and myricetin-3-galactoside were obtained from Sichuan Weikeqi Biological Technology Co., Ltd. L-pyroglutamic acid, p-hydroxybenzaldehyde, cryptochlorogenic acid, and sweroside were from Shanghai Standard Technology Co Ltd. p-Hydroxy-cinnamic acid and 1-caffeoylquinic acid were purchased from Chengdu Pufeide Biotechnology Co Ltd. (-)-Epiafzelechin was obtained from Chengdu Lemeitian Pharmaceutical Technology Co Ltd.

HPLC-grade acetonitrile was supplied by Merck, and formic acid by Tianjin Comeo Chemical Reagent Co. Ltd. Distilled water was used in all the experiments. All other reagent solutions were of analytical grade.

Chromatographic Conditions for UPLC-MS Analysis

Mass spectrometric analysis was performed on a UPLC-MS Thermo Vanquish Flex. Chromatographic separations were carried out on a Waters HSS T3 C18 column (210 mm × 100 mm, 1.8 μm). The mobile phase consisted of acetonitrile (A) and 0.1% formic acid aqueous solution (B) with a linear gradient: 0 to 3 min, 2% to 8% A; 3 to 9 min, 8% to 15% A; 9 to 13 min, 15% to 25% A; 13 to 16 min, 25% to 100% A; 16 to 19 min, 100% A; 19 to 22 min, 100% to 2%; 22 to 30 min, 2% A. The flow rate was 0.3 mL/min, the sample injection volume was 1.0 μL, the column temperature was 35 °C, and the detection wavelength was 260 nm. The mass spectrometer was an electrospray ion source (HESI). The spray voltage was set to 3.80 kV, the heating and capillary temperature to 350 °C, the sheath gas flow to 35 arb, and the auxiliary gas flow rate to 10 arb; the data were collected in positive and negative ion modes; the scanning mass range was 100 to 1200 Da, the S-lens voltage 50 V, and the secondary collision energies 40 eV.

Animals and Their Care

Kunming mice, 18 to 22 g in weight, were provided by the Guangdong Medical Laboratory Animal Center, and their certificate number was 44007200099222. They were acclimatized in an air-conditioned room at 22 ± 2 °C for 3 days with a 12-h light and 12-h darkness cycle. All animals were fed with standard laboratory chow and tap water before the experiment. The Animal Ethics Committee of Guangdong Province Hospital of Traditional Chinese Medicine approved the studies.

Wine-Steamed Corni Fructus Extract for the Animal Experiments

Three 100 g samples of Corni Fructus were soaked in 20 g wine for 2 h, then steamed for 1, 4, and 12 h, respectively. The samples were soaked 8 times in water for 0.5 h, and subsequently boiled for 1 h and filtered. The residues were extracted 8 times in water for 0.5 h and filtered. The filtrate was combined and condensed to 100 mL at 60 °C under reduced pressure.

Induction of Immunocompromised Mouse Model and Biochemical Assays

Kunming mice were randomly divided into groups: a blank group, cyclophosphamide (CTX) model group, ubenimex group, Corni Fructus group, and wine-steamed (1, 4, and 12 h) Corni Fructus groups, with 10 mice in each group. Except for the blank group, the other groups were given 80 mg/kg of CTX by intraperitoneal injection to establish an immunosuppressed mouse model. The blank group was given the same dose of normal saline for 3 days. After successful induction, each group was given the corresponding drugs by gavage, and the blank group and model group were given the same volume of distilled water for 7 days.

Twenty-four hours after the last administration, blood was collected. The thymus and spleen were separated and weighed, and the organ index was determined. After collection, the blood was left to stand for 15 min and centrifuged for 10 min (3000 rev/min). The supernatant was taken, and the content of IgG and IgM in serum was detected by ELISA.

Construction of a Mouse Model of Drug-Induced Liver Injury and Biochemical Assays

Kunming mice were taken, and, after adaptive rearing, they were randomly divided into groups, namely the blank group, the acetaminophen (APAP) model group, the bicyclol group, the Corni Fructus group, and the wine-steamed (1, 4, and 12 h) Corni Fructus groups, with 10 mice in each group. Ten mL/kg body weight of the corresponding drug was administered by intragastric administration once. Ten mL/kg body weight of distilled water was administered to the blank and APAP model groups every day for 7 days.

On the seventh day of administration, the mice fasted without food and water, and 12 h after the last administration, the mice in the blank group were intraperitoneally injected with saline (200 mg/kg). The other groups were intraperitoneally injected with APAP (200 mg/kg) to establish a liver injury model. After 12 h of modeling, the mice were removed to obtain blood, and the serum was separated for serum index determination. Alanine amino transferase (ALT), aspartate transaminase (AST), and malondialdehyde (MDA) (Tianjin Annorikang Biotechnology Co Ltd) were measured in the serum according to the kit instructions. Parts of the liver were sampled and were cut into pieces. Nine volumes of normal saline were added, and an electric glass homogenizer was used to homogenize a 10% liver homogenate in an ice bath. This was centrifuged at 3500 rev/min for 15 min, and the supernatant was collected. The superoxide dismutase (SOD) and seleno-dependent glutathione peroxidase (GSH-Px) (Tianjin Annorikang Biotechnology Co Ltd) contents in liver tissue were determined according to the kit instructions.

Induction of Kidney yin Deficiency in the Mice Model and Biochemical Assays

Kunming mice were taken and randomly divided into blank group, model group, Liuwei Dihuang Pills group, Corni Fructus group, and wine-steamed (1, 4, and 12 h) Corni Fructus groups, with 10 mice in each group. The mice in the blank and the model group were given distilled water by gavage, and the administration group was given 0.1 mL per 10 g body weight for 14 days. Except for the blank group, the mice in each group were given a thyroid tablet solution (160 mg/kg), once a day for 14 days.

Twenty-four hours after the last administration, the blood was collected and centrifuged for 10 min (3000 rev/min). The supernatant was aspirated, and the serum cyclic adenosine monophosphate (cAMP), cyclic guanosine monophosphate (cGMP), triiodothyronine (T3), and thyroxine (T4) were determined by ELISA. The thymus and spleen were separated and weighed, and the organ index was determined.

Statistical Analysis

The SIMCA-P (version 14.1, Umetrics) statistical program was used for principal component analysis (PCA). Data analysis was performed using GraphPad Prism 9. All data were expressed as mean ± SD. One-way ANOVA tests were applied when the homogeneity of variance assumptions were satisfied. Otherwise, the equivalent nonparametric test was used. P < .05 was considered significant.

Results

Effects of Different Steaming Times on Wine-Steamed Corni Fructus

With an increase in steaming time, the color of wine-steamed Corni Fructus gradually deepened from purplish red to black.

To analyze the overall trends in composition changes during processing, 10 batches of Corni Fructus and wine products with different processing degrees were analyzed. The relative peak areas of common peaks in mass spectrometry data were imported into SIMCA14.1 statistical software for unsupervised PCA modeling and analysis. The results are shown in Figures 1 and 2. The positive and negative ion model results indicated that the PCA score maps of different processed wines moved counterclockwise with the increasing processing degree. Although the changing trend in the chemical composition of each treatment was different and reflected a certain complexity, PCA could reduce this complexity and visually reflect the overall changes in the chemical composition.

Figure 1.

Principal component analysis (PCA) score plot of raw and processed Corni Fructus at different processing stages: (A) positive mode and (B) negative mode.

Figure 2.

Ultra-performance liquid chromatography (UPLC) chromatograms of Corni Fructus and 4-h wine-steamed Corni Fructus: (A) Corni Fructus and (B) 4-h wine-steamed Corni Fructus.

Mass Spectrometric Analysis

Compound Discover 3.3 software was used to analyze the MS and MS² of the extracted chromatographic peaks. The precise molecular weight, mass number deviation, and admixture ion information of the target compounds were determined and then compared with the mzCloud database and MS OTCML database of traditional Chinese medicine components.

A total of 132 compounds were identified in both positive and negative ion models, including 34 organic acids, 16 glycosides, 19 flavonoids, 15 terpenoids, 13 alkaloids, 3 phenylpropanoids, 3 sugars, 1 steroid, 1 tannin, and 27 compounds belonging to other classes. The compounds are listed in Table 1. With the increase in processing time, the content of 19 compounds increased gradually, 67 compounds decreased, and 6 compounds increased initially and then decreased. The changes in the compounds quantified are shown in Figure 3. The peak with a retention time of 5.50 was distinctly different from the crude Corni Fructus. According to the analysis, the component is 5-hydroxymethyl-2-furaldehyde, which is produced during processing. Processing could reduce the acidity of Corni Fructus and enhance its liver and kidney toning effect, immune regulation, and anti-aging.

Figure 3.

Heat map of changes in the concentration of compounds during processing (the number of Y-axis corresponds to Table 1).

Table 1.

Chemical Constituents of Shanyurou and Jiuyurou (Positive Ion Mode and Negative Ion Mode).

No.	Name	Formula	Calc. (MW)	m/z	t_R (min)	Fragments	Tendency
1	L-Asparagine	C₄H₈N₂O₃	132.0536	133.0609	1.213	74.0243, 87.0558	↓
2	L-Aspartic acid	C₄H₇NO₄	133.0363	132.0291	1.234	115.0024, 88.0390, 71.0125	↑↓
3	D-Mannose 1-phosphate	C₆H₁₃O₉P	260.0298	259.0225	1.246	96.9682, 71.0126	–
4	Glycerophosphocholine	C₈H₂₀NO₆P	257.1030	258.1103	1.253	104.1074, 125.0000, 184.0735	↑
5	D-Lactose monohydrate	C₁₂H₂₂O₁₁	342.1164	381.0795	1.271	85.0291, 127.0392, 145.0497	–
6	D-Gluconic acid	C₆H₁₂O₇	196.0576	195.0504	1.287	195.0503, 129.0181, 75.0073	–
7	Kojic acid	C₆H₆O₄	142.0268	143.0341	1.291	125.0236	–
8	β-Maltose^a	C₁₂H₂₂O₁₁	342.1165	341.1091	1.31	119.0336, 113.0231, 101.0230, 89.0229, 71.0125, 59.0125	↑
9	L-Threonic acid	C₄H₈O₅	136.0360	135.0288	1.342	75.0073, 72.9917, 59.0125	–
10	L-Proline^a	C₅H₉NO₂	115.0637	116.0710	1.378	70.0656	↓
11	Nicotinic acid	C₆H₅NO₂	123.0323	124.0396	1.405	96.0449	↓
12	D-(-)-Quinic acid	C₇H₁₂O₆	192.0626	191.0553	1.412	82.0281	–
13	Mesalamine	C₇H₇NO₃	153.0426	171.0766	1.432	136.0393	↓
14	α-Lactose	C₁₂H₂₂O₁₁	342.1163	343.1238	1.442	85.0289, 127.0391, 145.0496	↓
15	3-Hydroxyanthranilic acid	C₇H₇NO₃	153.0428	154.0501	1.447	136.0399	↑
16	Adenine	C₅H₅N₅	135.0547	136.0619	1.455	71.0135	↓
17	Isocitric acid	C₆H₈O₇	192.0262	191.0190	1.782	111.0075, 85.0281, 72.9917	–
18	Shikimic acid	C₇H₁₀O₅	174.0519	173.0446	1.894	111.0075, 93.0332, 73.0281	–
19	Citric acid^a	C₆H₈O₇	192.0262	191.01892	2.366	111.0075, 87.0073, 85.0281	–
20	Allopurinol	C₅H₄N₄O	136.0387	137.0460	2.416	81.0705	↑
21	6-Hydroxypicolinic acid	C₆H₅NO₃	139.0271	140.0344	2.429	112.0397	↑
22	L-Pyroglutamic acid^a	C₅H₇NO₃	129.0428	130.0501	2.507	84.045	↑
23	Uridine^a	C₉H₁₂N₂O₆	244.0695	243.0622	3.083	110.0234, 82.0284	↑
24	Citramalic acid	C₅H₈O₅	148.0361	147.0288	3.123	87.0073, 85.0281	–
25	α-Arbutin	C₁₂H₁₆O₇	272.0898	271.0826	3.655	108.0203	↓
26	Adenosine^a	C₁₀H₁₃N₅O₄	267.0968	268.1041	3.972	136.0616	↓
27	Guanosine^a	C₁₀H₁₃N₅O₅	283.0918	284.0991	4.259	152.0566	↓
28	Guanine	C₅H₅N₅O	151.0495	152.0568	4.261	135.0302, 125.0602	↓
29	Gallic acid	C₇H₆O₅	170.0205	169.0132	4.449	125.0231	↑
30	Phaseoloidin	C₁₄H₁₈O₉	330.0951	348.1291	4.504	123.0443, 85.0290, 151.0391	↓
31	Tachioside	C₁₃H₁₈O₈	302.1005	301.0932	5.101	138.0311, 123.0075	↓
32	L-Phenylalanine	C₉H₁₁NO₂	165.0792	166.0865	5.196	120.0809	↓
33	Corilagin	C₂₇H₂₂O₁₈	634.0816	633.0743	5.569	300.9993, 275.0201	–
34	5-Hydroxymethyl-2-furaldehyde^a	C₆H₆O₃	126.0319	127.0392	5.592	109.0287, 81.0341	↑
35	4-Methoxybenzaldehyde	C₈H₈O₂	136.0525	137.0598	5.78	122.0364	↓
36	Pantothenate^a	C₉H₁₇NO₅	219.1108	220.1180	5.97	90.0554, 98.02404, 116.0344, 184.0968	↓
37	3-(4-Hydroxyphenyl)-3-oxopropyl β-D-glucopyranoside	C₁₅H₂₀O₈	328.1160	329.1233	6.003	149.0597, 167.0702, 121.0285, 98.9846	↓
38	1,6-Bis-O-(34,5-trihydroxybenzoyl)hexopyranose	C₂₀H₂₀O₁₄	484.0856	483.0783	6.026	169.0133, 125.0232	↑
39	Gallocatechin	C₁₅H₁₄O₇	306.0743	305.0670	6.454	125.0232, 137.0232	↓
40	3,4-Dihydroxybenzoic acid^a	C₇H₆O₄	154.0256	153.0183	6.576	109.0282	↑
41	3-Hydroxy-2-(3-methyl-2-(((34,5-trihydroxy-6-(hydroxymethyl)tetra hydro- 2H-pyran-2-yl)oxy)methyl)cyclopent-2-en-1-yl)propanoic acid	C₁₆H₂₆O₉	362.1577	380.1919	6.685	201.1123, 153.0911, 121.1014, 93.0703	↓
42	Adoxosidic acid	C₁₆H₂₄O₁₀	376.1372	375.1299	6.928	151.0753, 125.0595, 59.0125	–
43	Xanthurenic acid	C₁₀H₇NO₄	205.0369	204.0297	7.696	160.0394	↓
44	DL-4-Hydroxyphenyllactic acid	C₉H₁₀O₄	182.0571	181.0498	7.7	163.0389, 135.0439, 119.0489, 72.9916	↑
45	p-Hydroxy-cinnamic acid^a	C₉H₈O₃	164.0464	163.0391	7.712	119.0489	–
46	Geniposidic acid^a	C₁₆H₂₂O₁₀	374.1216	373.1143	8.009	167.0705, 123.0439, 89.0231, 59.0125	↑
47	Loganic acid^a	C₁₆H₂₄O₁₀	376.1371	375.1297	8.271	169.0861, 113.0231, 95.0488, 69.0332	–
48	2-Isopropylmalic acid	C₇H₁₂O₅	176.0676	175.0603	8.631	115.0387, 113.0595, 85.0645	–
49	Secoxyloganin	C₁₇H₂₄O₁₁	404.1321	405.1393	8.631	151.0389, 167.0339, 125.0234	↓
50	Morroniside^a	C₁₇H₂₆O₁₁	406.1476	405.1404	8.953	156.0340, 141.0546, 101.0231	–
51	p-Hydroxybenzaldehyde^a	C₇H₆O₂	122.0370	123.0443	8.994	123.0441, 95.0495	↓
52	3,4-Dihydroxybenzalde-hyde	C₇H₆O₃	138.0318	139.0391	8.997	111.0443, 84.0496	↓
53	3,4-Di-O-galloylquinic acid	C₂₁H₂₀O₁₄	496.0855	497.0928	9.353	153.0181	↓
54	Caftaric acid	C₁₃H₁₂O₉	312.0484	311.0411	9.41	179.0341, 149.0081, 135.0439	↓
55	Cynaroside^a	C₂₁H₂₀O₁₁	448.1006	449.1079	9.467	287.0548	↓
56	Alyposide^a	C₁₇H₂₄O₉	372.1418	390.1756	9.543	161.0596, 133.0648, 85.0289	↓
57	1-Caffeoylquinic acid^a	C₁₆H₁₈O₉	354.0954	353.0881	9.548	191.0553	↓
58	Cryptochlorogenic acid^a	C₁₆H₁₈O₉	354.0953	355.1028	9.604	163.0388, 135.0439	↓
59	Sophoricoside	C₂₁H₂₀O₁₀	432.1058	431.0985	10.447	268.0379	↓
60	Apigenin 5-glucoside	C₂₁H₂₀O₁₀	432.1056	433.1128	10.478	271.0596	↓
61	Caffeic acid^a	C₉H₈O₄	180.0414	179.0341	10.689	135.0439	↑↓
62	Chebulagic acid	C₄₁H₃₀O₂₇	954.0989	953.0921	10.733	300.9991, 275.0199, 249.0403	↑↓
63	13,6-Tri-O-galloyl-β-D-glucose	C₂₇H₂₄O₁₈	636.0973	635.0900	10.77	169.0132, 193.0136	↓
64	Rhoifolin^a	C₂₇H₃₀O₁₄	578.1635	579.1707	10.985	271.0598	↓
65	(-)-Epiafzelechin^a	C₁₅H₁₄O₅	274.0841	275.0914	11.181	139.0390, 107.0495	↓
66	Loganin^a	C₁₇H₂₆O₁₀	390.1525	435.1507	11.595	101.0230, 127.0389	–
67	trans-4-Hydroxycinnamic acid	C₉H₈O₃	164.0463	163.0391	11.635	119.0489	↓
68	(1R,5S,6R,7S)-1-hydroxy-7-methyl-5-[(2S,3R,4S,5R,6R)-34,5-trihydroxy- 6-(hydroxymethyl)oxan-2-yl]oxy-4-oxabicyclo[4.3.0]non-2-ene-2-carbalde hyde	C₁₇H₂₆O₁₀	390.1527	391.1600	11.65	179.0701, 151.0753, 109.0653	↓
69	trans-Cinnamaldehyde	C₉H₈O	132.0577	133.0649	11.655	133.0649, 105.0703, 79.0549	–
70	Sweroside^a	C₁₆H₂₂O₉	358.1265	359.1337	11.703	127.0390, 197.0702, 111.0807	↓
71	Sinensin	C₂₁H₂₂O₁₁	450.1166	449.1093	11.981	259.0613, 125.0232	↓
72	Genistein^a	C₁₅H₁₀O₅	270.0531	271.0604	12.069	243.0656, 215.0705, 149.0235	↓
73	Syringic acid	C₉H₁₀O₅	198.0522	197.0449	12.62	182.0213, 121.0282	↓
74	Ferulic acid^a	C₁₀H₁₀O₄	194.0572	193.0499	12.747	178.0262, 134.0361	↓
75	Vanillin	C₈H₈O₃	152.0475	153.0548	13.111	111.0444, 125.0599, 93.0340, 65.0393	–
76	Ethyl gallate^a	C₉H₁₀O₅	198.0522	197.0449	13.367	169.0132, 125.0232	–
77	Myricetin-3-galactoside^a	C₂₁H₂₀O₁₃	480.0909	479.0836	13.379	316.0225, 271.0247	↓
78	Peltatoside	C₂₆H₂₈O₁₆	596.1387	595.1314	13.64	300.0276	↓
79	Hemiphloin	C₂₁H₂₂O₁₀	434.1217	433.1144	14.088	271.0614, 151.0026	↓
80	1,2,3,6-Tetra-O-galloyl-β-D-glucose	C₃₄H₂₈O₂₂	788.1085	787.1014	14.127	313.0571, 169.0134	↓
81	Rutin^a	C₂₇H₃₀O₁₆	610.1541	609.1468	14.307	300.0276	↓
82	Naringeninchalcone	C₁₅H₁₂O₅	272.0684	273.0757	14.522	153.0181, 147.0439	↓
83	Ellagic acid	C₁₄H₆O₈	302.0063	300.9990	14.632	300.9988	↑
84	Hyperoside^a	C₂₁H₂₀O₁₂	464.0960	463.0887	14.686	300.0277, 271.0250	↓
85	Quercetol 3-O-glucuronide	C₂₁H₁₈O₁₃	478.0750	477.0677	14.792	301.0355, 175.0026, 178.9978	↓
86	Kaempferol 3-neohesperidoside	C₂₇H₃₀O₁₅	594.1594	593.15214	15.03	284.0327, 255.0298	↓
87	1,2,3,4,6-Pentagalloyl glucose	C₄₁H₃₂O₂₆	940.1189	469.0521	15.067	169.0132, 125.0231	↓
88	Nicotifiorin	C₂₇H₃₀O₁₅	594.1589	595.1662	15.089	287.0548, 85.0289	↓
89	4-Hydroxybenzoic acid	C₇H₆O₃	138.0308	183.0291	15.297	93.0331	↓
90	Astragalin	C₂₁H₂₀O₁₁	448.1007	449.1080	15.533	287.0548, 85.0289	↓
91	13,8-trihydroxy-6-hydroxymethylanthraquinone	C₁₅H₁₀O₆	286.0477	287.0550	15.536	287.0548	↓
92	(+)-Pinoresinol-β-D-glucoside	C₂₆H₃₂O₁₁	520.1952	565.1935	15.56	151.0389, 136.0154	–
93	Urolignoside	C₂₆H₃₄O₁₁	522.2101	540.2447	15.61	151.0753, 137.0596, 255.1015	↓
94	Kaempferol 3-O-glucuronide	C₂₁H₁₈O₁₂	462.0805	461.0732	15.683	285.0408, 229.0505	↓
95	2-Methoxyresorcinol	C₇H₈O₃	140.0475	141.0547	15.739	109.0289, 81.0342, 53.0394	–
96	3-Phenyllactic acid	C₉H₁₀O₃	166.0621	165.0548	15.907	147.0440, 119.0489, 72.9917	↓
97	Azelaic acid	C₉H₁₆O₄	188.1041	187.0969	15.926	125.0959	↑
98	Isotrifoliin^a	C₂₁H₂₀O₁₂	464.0962	465.1035	15.943	303.0496	↓
99	Phloretin	C₁₅H₁₄O₅	274.0842	275.0915	15.958	107.0494	↓
100	Myricetin	C₁₅H₁₀O₈	318.0379	319.0452	16.023	273.0395, 153.0182, 165.0183	↓
101	Borneol 2-O-[β-D-apiofuranosyl-(1-6)]- beat-D-glucopyranoside	C₂₁H₃₆O₁₀	448.2301	493.2299	16.113	161.0445, 113.0231, 101.0231, 71.0125	↓
102	Quercetin^a	C₁₅H₁₀O₇	302.0429	301.0356	16.38	178.9977, 151.0026, 121.0282, 107.0125	↓
103	1-O-[(2α,3β,5xi,9xi,18xi)-2,3,19-Trihydroxy-28-oxours-12- en-28-yl]-beta-D-glucopyranose	C₃₆H₅₈O₁₀	650.4042	695.4024	16.391	487.3432	↓
104	Pinolenic acid	C₁₈H₃₀O₂	278.2248	279.2321	16.619	149.0234, 121.1015, 95.0860	↑
105	Naringenin	C₁₅H₁₂O₅	272.0688	271.0615	16.627	151.0026, 119.0489, 107.0125, 83.0125	↑↓
106	Apigenin^a	C₁₅H₁₀O₅	270.0536	269.0463	16.627	248.9778	↓
107	Shogaol	C₁₇H₂₄O₃	276.1727	277.1800	17.166	137.0598, 81.0705, 111.0444	↑
108	2,2,6,6-Tetramethyl-4-piperidinol	C₉H₁₉NO	157.1467	158.1540	17.269	102.0917, 57.0706	↓
109	Bis(4-ethylbenzylidene)sorb-itol	C₂₄H₃₀O₆	414.2044	415.2117	17.569	119.0857	–
110	Asiatic acid	C₃₀H₄₈O₅	488.3505	489.3579	17.621	203.1795, 95.0860, 177.1636	–
111	Astrantiagenin D	C₃₀H₄₆O₄	470.3399	471.3473	17.624	201.1639, 189.1637, 119.0857, 95.0859	–
112	1-Linoleoyl glycerol	C₂₁H₃₈O₄	354.2772	355.2845	17.823	161.1325, 121.1015, 109.1016, 95.0860, 81.0705	↑
113	Sedanolide	C₁₂H₁₈O₂	194.1309	195.1382	17.943	177.1277, 167.0705, 149.1327, 125.0600, 95.0497, 79.0549	↑↓
114	Ligustilide^a	C₁₂H₁₄O₂	190.0995	191.1068	17.949	191.1069, 173.0963, 145.1014, 117.0703, 105.0703, 91.0548	↑↓
115	Sanguisorbigenin	C₃₀H₄₆O₃	454.3450	455.3522	18.241	313.2747, 203.1794, 163.1478, 109.1014, 95.0859, 57.0706	–
116	3,5-di-tert-Butyl-4-hydroxybenzaldehyde	C₁₅H₂₂O₂	234.1621	235.1694	18.261	179.1066, 57.0706	↓
117	Cyperenone	C₁₅H₂₂O	218.1672	219.1745	18.388	135.0804, 121.0649, 109.0651, 81.0704	↓
118	Dibutyl phthalate	C₁₆H₂₂O₄	278.1519	279.1591	18.523	149.0233, 57.0706	–
119	Betulonic acid	C₃₀H₄₆O₃	454.3450	455.3523	18.619	205.1588, 177.1637, 107.0859	–
120	Maslinic acid	C₃₀H₄₈O₄	472.3558	473.3630	18.619	205.1589, 189.1639, 177.1639, 107.0860	–
121	(+)-Nootkatone	C₁₅H₂₂O	218.1672	219.1745	18.688	219.1744, 163.1118, 137.0963	–
122	Erucamide	C₂₂H₄₃NO	337.3343	338.3416	19.498	135.1169, 97.1016, 83.0861, 69.0706, 57.0707	–
123	Hexadecanamide	C₁₆H₃₃NO	255.2562	256.2635	19.768	88.0762, 102.0917, 57.0704	–
124	Oleanolic acid	C₃₀H₄₈O₃	456.3607	457.3683	19.932	203.1797, 189.1640, 95.0861	–
125	Ursolic acid^a	C₃₀H₄₈O₃	456.3605	457.3678	20.028	411.3624, 189.1636, 163.1483, 107.0859, 95.0859	–
126	Octyl decyl phthalate	C₂₆H₄₂O₄	418.3084	419.3157	20.313	149.0233, 85.1017, 71.0862	–
127	Acetyl-11-keto-β-boswellic acid	C₃₂H₄₈O₅	512.3504	513.3577	20.382	407.3304, 173.1325, 119.0857, 95.0859	–
128	Palmitic acid	C₁₆H₃₂O₂	256.2403	255.2330	20.503	255.2329	↑
129	Stearamide	C₁₈H₃₇NO	283.2876	284.2948	21.164	102.0918, 88.0763, 57.0707	–
130	4-Isopropylaniline	C₉H₁₃N	135.1049	136.1121	21.758	136.1120, 91.0547	–
131	N, N-Dimethylaniline	C₈H₁₁N	121.0893	122.0966	21.771	107.0726	–
132	Betaine	C₅H₁₁NO₂	117.0793	118.0866	21.872	59.0737	–

Comparison with standard compounds.

Effect of Immunocompromised Mouse Model Induction

The mice in the blank group had flexible movements, bright coat colors, and normal eating. On the other hand, mice in the other groups were slightly sluggish, their coats were slightly gray, dry, and messy, and their food intake was reduced. Compared with the blank group, the spleen index of mice in the CTX group was significantly decreased (P<.01). In contrast, the other groups tended to increase, but there were no significant differences (P > .05).

Compared with the blank group, the thymus index of mice in the CTX group significantly decreased (P<.01). In the other groups, the thymus index tended to increase, but there were no significant differences (P > .05, Figure 4).

Figure 4.

Effects on spleen index and thymus index in immunocompromised mice (**P < .01 compared with blank group; ^#P < .05 compared with model group).

The serum IgG and IgM content of the mice in the CTX group was significantly decreased compared with the blank group (P < .01). When compared with the CTX group, the serum IgG and IgM levels in the ubenimex tablet group and wine-steamed (1, 4, and 12 h) Corni Fructus groups were significantly increased (P<.05 or P<.01). The serum IgG content of the Corni Fructus group had a tendency to increase, but no significant differences were identified (P > .05, Figure 5).

Figure 5.

Effects on IgG and IgM in immunocompromised mice (**P < .01 compared with blank group; ^#P < .05, ^##P < .01 compared with model group).

Effect on a Mouse Model of Drug-Induced Liver Injury

The APAP model group's serum ALT, AST, and MDA contents were significantly higher than the blank group (P < .01). Compared with the APAP model group, the serum ALT, AST, and MDA of the bicyclol group, Corni Fructus group, and wine-steamed (1, 4, and 12 h) Corni Fructus groups were significantly decreased (P<.05 or P<.01, Figure 6).

Figure 6.

Effects on ALT, AST, and MDA in mice with liver injury (**P < .01 compared with the blank group; ^#P<.05, ^##P<.01 compared with the model group).

Compared with the blank group, the SOD in the liver of the APAP model group was significantly decreased (P < .01). When compared with the APAP model group, the liver SOD in the bicyclol, Corni Fructus group, and wine-steamed (4 and 12 h) Corni Fructus groups were significantly increased (P < .05 or P < .01). The liver SOD in the 1 h wine-steamed Corni Fructus group appeared to be increased, but there was no statistical significance (P > .05).

Compared with the blank group, the liver GSH-Px content in the APAP model group was significantly decreased (P < .01). On the other hand, the GSH-Px contents of the liver of the wine-steamed (1, 4, and 12 h) Corni Fructus groups were significantly increased (P < .05 or P < .01) when compared with the APAP model group. The content of GSH-Px in the liver of the bicyclol and Corni Fructus groups appeared to increase, but no statistical significance was observed (P > .05, Figure 7).

Figure 7.

Effects on SOD and GSH-PX in the liver of mice with liver injury (**P < .01 compared with the blank group; ^#P < .05, ^##P < .01 compared with the model group).

Effect on Kidney Yin Deficiency Animal Model

Compared with the blank group, the cAMP of mice in the model group was significantly increased (P < .01). On the other hand, when compared with the model group, the cAMP content of mice in the Liuwei Dihuang Pills group, and wine-steamed (1, 4, and 12 h) Corni Fructus groups decreased significantly (P<.01). Similarly, the cGMP content of mice in the model group was significantly decreased (P > .05) when compared with the blank group. On the other hand, when compared with the model group, the cGMP content of the mice in the Liuwei Dihuang Pills group, Corni Fructus group, and wine-steamed (1, 4, and 12 h) Corni Fructus groups showed an increasing trend, but there was no statistical significance (P > .05, Figure 8).

Figure 8.

Effects on cAMP and cGMP in mice with yin deficiency syndrome (*P < .05, **P < .01 compared with the blank group; ^##P < .01 compared with the model group).

In terms of the effects on T3 and T4 in mice with yin deficiency syndrome, the T3 content of mice in the model group was significantly increased (P < .01) when compared with the blank group. Compared with the model group, the T3 content of the mice in the Liuwei Dihuang Pills group and the 12 h wine-steamed Corni Fructus group decreased significantly (P < .05 or P < .01). On the other hand, when compared with the blank group, the T4 content of mice in the model group was significantly increased (P < .01). Finally, the T4 content of the mice in the Liuwei Dihuang Pills group, Corni Fructus group, and wine-steamed (1, 4, and 12 h) Corni Fructus groups decreased significantly (P < .01) when compared with the model group. The results are presented in Figure 9.

Figure 9.

Effects on T3 and T4 in mice with yin deficiency syndrome (*P<.05, **P < .01 compared with the blank group; ^##P < .01 compared with the model group).

Discussion

When Corni Fructus is steamed with wine, high temperature causes tannin hydrolysis, increasing gallic acid content. Gallic acid can have anti-inflammatory and antioxidant properties and has protective effects on the liver.^14,15 The Corni Fructus medicinal effect after processing is enhanced.¹⁶ The iridoid glycosides have various pharmacological activities such as anti-tumor, anti-inflammatory, neuroprotection, among others.¹⁷ The iridoid glycoside content can be reduced after corn liquor is steamed. Flavonoids have antibacterial and antioxidant effects.^18,19 With the increase in processing time, some flavonoids gradually decreased and disappeared in Corni Fructus. In conclusion, a series of chemical composition changes during the processing process resulted in different pharmacodynamics of Corni Fructus and wine-steamed Corni Fructus. The Corni Fructus, after processing, can promote the body's immune function and enhance liver and kidney functions. From the UPLC-Q-TOF-MS results, combined with the pharmacodynamics results, we chose 4 h wine-steam Corni Fructus for the later study.

Immunoglobulin is an important compound with immune functions in blood and tissue fluid. IgG is the most functionally significant antibody, while IgM is the largest and earliest-antibody in the circulatory system, with roles in removing pathogenic bacteria, activating complement, and regulating immune functions.²⁰ In mice with low immunity caused by cyclophosphamide, processed Corni Fructus can enhance their immune function.

The liver protective effect of processed Corni Fructus was improved. APAP can induce the production of a large number of reactive oxygen species (ROS). It can lead to the consumption of SOD, GSH-Px, and other antioxidants, resulting in the imbalance of the antioxidant system in the human body, triggering the release of inflammatory cytokines in the intracellular inflammatory signaling pathway, and eventually leading to hepatocyte injury, and in severe cases, hepatocyte necrosis. ALT, AST, and MDA are commonly used to evaluate the degree of drug-induced liver injury.²¹ When hepatocyte injury occurs, the MDA level increases, and ALT and AST in the cells will be released into the serum, resulting in increased activity of ALT and AST in the serum.

cAMP and cGMP, important substances involved in regulating metabolism and biological function in cells, antagonize and coordinate with each other to maintain vital cellular processes.²² Research has shown that patients with renal yin deficiency present with increased plasma cAMP levels, decreased cGMP levels, and a significantly increased ratio of cAMP to cGMP. Thyroid hormone is an important substance regulating the body's energy metabolism. Hyperthyroidism can lead to an increase in serum T3 and T4 contents. Processed Corni Fructus enhances liver and kidney functions.

This study compared crude and wine-steamed Corni Fructus based on UPLC-Q-TOF-MS combined with pharmacodynamics. Our results provide a scientific basis for the subsequent use of wine-steamed Corni Fructus.

Footnotes

Acknowledgments

This work was supported by the Quality and Technical Service Platform for Traditional-Chinese-Medicine Whole Industry Chain (2022-230-221).

Data Availability

The datasets generated and analyzed during the current study are not publicly available due to the confidentiality of our project, but are available from the corresponding author on reasonable request.

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Ethical Approval and Consent to Participate

The studies were approved by the Animal Ethics Committee of Guangdong Second Traditional Chinese Medicine Hospital.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by the Quality and technical service platform for traditional-Chinese-medicine whole industry chain (grant number 2022-230-221).

ORCID iD

Xiaozhou Jia

Statement of Informed Consent

There are no human subjects in this article and informed consent is not applicable.

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