2.1.1. Experimental Animals

Fifty-six SPF-grade male SD rats weighing 180 - 220 g were provided by Hunan SJA Laboratory Animal Co., Ltd. (Animal Production License No.: SCXK (Xiang) 2019-0004). This study was approved by the Animal Experiment Ethics Committee of Guangxi University of Chinese Medicine (Ethics Approval No.: DW20210311-021), and all experimental procedures strictly followed animal ethics requirements.

2.1.2. Experimental Drugs and Reagents

Anemarrhenaasphodeloides decoction pieces (Bozhou Jingwan Traditional Chinese Medicine Pieces Co., Ltd., Batch No. 190601, conforming to the standards of the 2020 edition of the ChinesePharmacopoeia); A.galanga and A.officinarum decoction pieces (Anguo Lengbei Herbal Medicine Co., Ltd., Batch No. 20190901, origin: Guangxi, identified as authentic by Professor Ma Wenfang of the Pharmacognosy Teaching and Research Section, Guangxi University of Chinese Medicine); dried ginger gingerol (Xi’an Baoyifeng Biotechnology Co., Ltd., Batch No. 20190912); ELISA kits for isoleucine, aspartic acid, valine, glutamic acid, histidine, lactic acid, and arachidonic acid (Nanjing Jiancheng Bioengineering Institute, Batch No. 20201012); glacial acetic acid, 95% ethanol, 60% ethanol, sodium hydroxide, hydrochloric acid, and Tween 80 were all of analytical grade; ultrapure water was used.

2.1.3. Experimental Instruments

ST16 series benchtop high-performance centrifuge (Thermo Fisher Scientific, USA); SQP electronic analytical balance (Sartorius Scientific Instruments (Beijing) Co., Ltd.); UPK-II-60L ultrapure water system (Sichuan Youpu Ultrapure Technology Co., Ltd.); 10 μL/200μL adjustable pipettes (Eppendorf, Germany); constant temperature water bath (Shanghai Jinghong Experimental Equipment Co., Ltd.); high-speed tissue homogenizer (Ningbo Xinzhi Biotechnology Co., Ltd.).

2.2.1. Drug Preparation

1)PreparationofModelingDrugs

a) Ice-cold Anemarrhenaasphodeloides decoction: Take A.asphodeloides decoction pieces, add 10 times the volume of ultrapure water, soak for 1 h, bring to a boil over high heat, then simmer over low heat for 1 h, and filter. Add 8 times the volume of ultrapure water to the residue, decoct similarly for 40 min, and filter. Combine the two filtrates, concentrate by heating to a crude drug concentration of 0.5 g/mL, cool, aliquot, and store at 4˚C. b) 15% glacial acetic acid solution: Dilute glacial acetic acid with ultrapure water to a mass concentration of 15%, and store at 4˚C.

2)PreparationofTestDrugs

Crush A.galanga and A.officinarum decoction pieces into coarse powders separately. Add 40 times the volume of 95% and then 60% ethanol sequentially, reflux extract for 4 h each time, filter, and combine the two filtrates. Concentrate under reduced pressure to recover ethanol until no ethanol odor remains. Add an appropriate amount of ultrapure water, mix well, and bring to a boil. Adjust the pH to 10 - 11 with 1 mol/L sodium hydroxide solution, boil for 1 h, filter, and repeat the operation twice. Combine the filtrates. Adjust the pH of the filtrate to 1 - 2 with 1 mol/L hydrochloric acid solution, incubate in a water bath at constant temperature for 30 min, and let stand at room temperature for 24 h. After precipitation, centrifuge, discard the supernatant, wash the precipitate 2 - 3 times with distilled water, and dry under vacuum at 40˚C to obtain flavonoid extracts from A. galanga and A. officinarum. The extraction yield of A. galanga flavonoid extract was 5.2%, with a total flavonoid content of 63.5% and galangin as the main marker compound. The extraction yield of A. officinarum flavonoid extract was 4.8%, with a total flavonoid content of 58.7% and kaempferol-3-O-glucoside as the main marker compound. Before use, prepare the corresponding concentrations with 0.5% Tween 80 solution. Doses were calculated based on crude herb mass. The high dose was 16 times the clinical dose, and the low dose was 4 times the clinical dose.

3)PreparationofPositiveDrug

Prepare dried ginger gingerol into an aqueous solution at a concentration of 0.045 g·kg⁻¹ using 0.5% Tween 80 solution, prepare fresh before use.

2.2.2. Animal Grouping and Modeling

After 7 days of adaptive feeding, 8 rats were randomly selected as the blank control group, and the remaining 48 rats were used to establish the gastric ulcer of cold syndrome model. Modeling method: At 8:00 AM daily, intragastric administration of 4˚C A. asphodeloides decoction (dose: 10.44 g/kg, administration volume: 2 mL/100g); at 6:00 PM daily, intragastric administration of 4˚C 15% glacial acetic acid solution (administration volume: 1 mL/100g), once daily for 4 consecutive days. During the modeling period, the environmental temperature was controlled at 16˚C, with humidity at 60%. The successful modeling criteria were defined as the presence of cold syndrome manifestations (huddling, lethargy, loose stools) and visible gastric ulcers on histopathological examination. This modeling protocol followed the validated source described in references [[6], [7]]. After successful modeling, the model rats were randomly divided into 6 groups, with 8 rats in each group: model group, dried ginger gingerol positive group, A. galanga high-dose group, A. galanga low-dose group, A. officinarum high-dose group, and A. officinarum low-dose group.

2.2.3. Administration Method

Administration began on the second day after successful modeling. The blank control group and model group were given 0.5% Tween 80 solution, while the other groups were given the corresponding drugs. The administration volume was 1 mL/100g, with a 12 h interval between doses, for a total of 4 doses over 2 days. The doses for the A. galanga high/low dose groups were calculated based on crude herb mass as 0.848 g∙kg⁻¹ and 0.212 g∙kg⁻¹, respectively. The doses for theA. officinarum high/low dose groups were calculated based on crude herb mass as 1.024 g∙kg⁻¹ and 0.256 g∙kg⁻¹, respectively. The dose for the dried ginger gingerol positive group was 0.045 g∙kg⁻¹.

2.2.4. Specimen Collection and Indicator Detection

On the second day after the final administration, rats were anesthetized by intraperitoneal injection of 10% chloral hydrate (0.3 mL/100g). The entire stomach was quickly removed, and gastric contents were rinsed with pre-cooled normal saline. For the blank control group, tissue samples were taken from the corresponding site on the greater curvature of the gastric body; for all other groups, tissue samples were taken from the lesion site. A 3 mm × 10 mm tissue block was taken, and 10% tissue homogenate was prepared by adding ice-cold normal saline. After centrifugation at 3000 r∙min⁻¹ for 15 min, the supernatant was collected. According to the instructions of the ELISA kits, the contents of isoleucine, aspartic acid, valine, glutamic acid, histidine, lactic acid, and arachidonic acid in the supernatant were detected. All ELISA readings were converted to concentrations using standard curves, and results were expressed as μmol∙g⁻¹ wet tissue weight, i.e., normalized to wet tissue weight.

SPSS 30.0 statistical software was used for data analysis. Measurement data were expressed as mean ± standard deviation ( x ¯ ± s ). One-way analysis of variance was used for comparisons among multiple groups, and the least significant difference (LSD) t-test was used for pairwise comparisons between groups. Normality (Shapiro-Wilk test) and homogeneity of variance (Levene’s test) were checked prior to ANOVA; all data met the assumptions of normality and equal variance. A P-value < 0.05 was considered statistically significant.

Compared with the blank control group, the contents of isoleucine, aspartic acid, valine, and glutamic acid in gastric tissue of the model group were significantly increased (P < 0.01). Compared with the model group, the contents of these 4 indicators in each treatment group were significantly decreased (P < 0.01). Compared with the same dose of A.galanga group, the glutamic acid content in the A.officinarum low-dose group was significantly decreased (P < 0.05). Compared with the same dose of A.officinarum group, the glutamic acid content in the A.galanga low-dose group was significantly increased (P < 0.05). See Table 1.

Table1.Effects of flavonoids fromA.galangaandA.officinarumon contents of four amino acids in gastric tissue of rats with gastric ulcer of cold syndrome ( x ¯ ± s , n = 8, μmol∙g⁻¹).

Note: Compared with blank control group, △△P < 0.01; compared with model group, *P < 0.05, **P < 0.01; compared with same dose of A.galanga group, ○P < 0.05; compared with same dose of A.officinarum group, ●P < 0.05.

Compared with the blank control group, the contents of histidine, lactic acid, and arachidonic acid in gastric tissue of the model group were significantly increased (P < 0.01). Compared with the model group, the contents of all 3 indicators in the A.officinarum high-dose group were significantly decreased (P < 0.05 or P < 0.01); the contents of lactic acid and arachidonic acid in the A.galanga high-dose group and A.officinarum low-dose group were significantly decreased (P < 0.01); and only the arachidonic acid content in the A.galanga low-dose group was significantly decreased (P < 0.01). The histidine content in each treatment group showed no statistically significant difference compared with the model group (P > 0.05). See Table 2.

Table2.Effects of flavonoids fromA.galangaandA.officinarumon contents of histidine, lactic acid, and arachidonic acid in gastric tissue of rats with gastric ulcer of cold syndrome ( x ¯ ± s , n = 8).

Note: Compared with blank control group, △△P < 0.01; compared with model group, *P < 0.05, **P < 0.01; compared with same dose of A.galanga group, ○P < 0.05; compared with same dose of A.officinarum group, ●P < 0.05.

Isoleucine and valine, as branched-chain amino acids (BCAAs), can be catalyzed by branched-chain amino acid transaminase and branched-chain keto acid dehydrogenase complex to generate acyl-CoA, which enters the tricarboxylic acid (TCA) cycle for oxidative energy production [[8], [9]]. Glutamic acid and aspartic acid can generate α-ketoglutarate and oxaloacetate through transamination, directly supplementing TCA cycle intermediates and promoting ATP synthesis [[10], [11]]. Histidine can be catalyzed by histidine decarboxylase to produce histamine, which stimulates gastric acid secretion through H2 receptors and participates in gastric mucosal inflammatory responses [[12]]. In this study, the contents of the above amino acids in gastric tissue of the model group were significantly increased, which is speculated to be related to the compensatory response of increased local energy demand in the gastric mucosa, accelerated TCA cycle, and enhanced amino acid mobilization under the gastric ulcer of cold syndrome state [[13]].

Flavonoids from A.galanga and A.officinarum significantly reduced the contents of isoleucine, aspartic acid, valine, and glutamic acid in gastric tissue of model rats, suggesting that they can enhance the efficiency of mitochondrial oxidative phosphorylation, accelerate the entry of amino acids into the TCA cycle, and alleviate the compensatory accumulation of amino acids caused by energy deficiency, which is consistent with the pharmacological characteristics of warm-natured herbs in promoting energy metabolism [[14], [15]]. This process may be related to the regulation of the AMP-activated protein kinase (AMPK) signaling pathway. As a cellular energy sensor, AMPK can promote fatty acid oxidation and mitochondrial biogenesis by phosphorylating downstream target proteins, while upregulating branched-chain keto acid dehydrogenase activity to accelerate BCAA catabolism [[16]]. It is speculated that flavonoids may coordinate amino acid metabolism and energy production by activating the AMPK pathway, thereby improving gastric mucosal energy supply. Histidine was significantly reduced only in the A.officinarum high-dose group, suggesting that flavonoids from A.officinarum have greater advantages in inhibiting histamine production and reducing gastric acid erosion of the gastric mucosa.

Arachidonic acid is an unsaturated fatty acid released from cell membrane phospholipids. It is metabolized by cyclooxygenase and lipoxygenase to produce inflammatory mediators such as prostaglandins and leukotrienes, which participate in gastric mucosal inflammatory responses and injury repair [[17]]. Its metabolic pathway is closely related to the nuclear factor κB (NF-κB) signaling pathway, as it can promote the expression of pro-inflammatory factors by activating NF-κB, thereby exacerbating local inflammation [[18]]. In this study, the content of arachidonic acid in gastric tissue of model rats was significantly increased, indicating active local inflammation in the gastric mucosa under the gastric ulcer of cold syndrome state. The arachidonic acid content was significantly reduced in all treatment groups, with the high-dose groups showing better effects than the low-dose groups. This indicates that flavonoids from A.galanga and A.officinarum can reduce arachidonic acid release and the production of its inflammatory metabolites, exerting anti-inflammatory effects. The mechanism may be related to the inhibition of phospholipase A2 activity and downregulation of COX-2/LOX expression, consistent with our research group’s previous findings on the regulation of lipid metabolism by Alpinia herbs [[6]].

Lactic acid is the end product of anaerobic glycolysis. When tissue hypoxia or mitochondrial dysfunction occurs, energy metabolism shifts toward glycolysis, leading to lactic acid accumulation [[19]]. Hypoxia-inducible factor-1α (HIF-1α) is a core transcription factor in the hypoxic response and can upregulate lactate dehydrogenase expression, promoting lactic acid production [[20]]. In this study, the lactic acid content in gastric tissue of model rats was significantly increased, suggesting microcirculatory disturbances and mitochondrial dysfunction in the gastric mucosa of rats with gastric ulcer of cold syndrome. The lactic acid content was reduced to varying degrees in each treatment group, with significant reductions in the A.officinarum high-dose group and A.galanga high-dose group. This suggests that flavonoids can ameliorate tissue hypoxia and promote lactic acid utilization by improving gastric mucosal microcirculation and enhancing mitochondrial oxidative phosphorylation. The mechanism may involve the inhibition of the HIF-1α signaling pathway and downregulation of glycolysis-related enzyme expression.