Materials

Streptozotocin (STZ) was MP Biomedicals, Trisodium citrate and citric acid were purchased from Sigma Aldrich Inc. (St Louis, MO, USA). Enzyme-linked immune adsorbent assay (ELISA) kits of Malondialdehyde (MDA), glutathione peroxidase (GSHPx), superoxide dismutase (SOD) and catalase (CAT) were obtained from R&D Systems Inc. (Minneapolis, MN, USA). Ginseng Radix, Rhizoma Anemarrhenae, Radix Glycyrrhizae Preparata, Gypsum Fibrosum and japonica rice were purchased from Xi'an Grass Plant Technology Co. Ltd. (Xi'an, Shaanxi, China). Methanol and acetonitrile of LC–MS grade were purchased from Fisher (Fisher Corp., Fair lawn, NJ, USA). All other chemicals were of analytical grade available commercially. ALL Chinese herbal medicine were obtained from Department of Chinese Materia Medica and Natural Medicines, School of Pharmacy, Fourth Military Medical University, and botanically identified by Professor Xiao-mei Song a traditional Chinese Medicine chemist at College of pharmacy, Shaanxi University of Chinese Medicine.

Preparation of BHJRSTD extracts

Ginseng Radix (10 g), Rhizoma Anemarrhena (18 g), Radix Glycyrrhizae Preparata (6 g), Gypsum Fibrosum (50 g) and japonica rice (9 g) were mixed in a classical dosage ratio used in the Han Dynasty. The mixture was soaked in distilled water (750 mL) for 30 min in a beaker (2,000 mL), then refluxed at 100 °C for 1 h twice under nitrogen protection in a round-bottomed flask (3,000 mL). The extraction solutions were combined and to be filtered, partial moisture was removed by rotary evaporator under reduced pressure. Then, the residuary solution was condensed to crude drug (2 g ml⁻¹), the extracts were stored at 4 °C and were brought to room temperature before use [8].

Sample preparation

1 mL of BHJRSTD extracts was vortexed for 30 s at 4 °C and centrifuged at 12,000 rpm for 15 min. Then take 200 μL of supernatant into an EP tube, added 1 mL of extract (methanol: water = 4:1); Vortexed for 30 s and sonicated in ice-water bath for 5 min; After standing at −40 °C for 1 h, the samples were centrifuged at 4 °C and 12,000 rpm for 15 min; The supernatant was carefully filtered through a 0.22 μm microporous filter and stored at −20 °C until assayed.

Instruments and conditions

LC-MS/MS analysis was performed on an Agilent ultra-high performance liquid chromatography 1290 UPLC system with a Waters UPLC BEH C18 column (1.7 μm, 2.1 × 100 mm). The flow rate was set at 0.4 mL min⁻¹ and the sample injection volume was set at 5 μL. The mobile phase consisted of 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B). The multi-step linear elution gradient program was as follows: 0–3.5 min, 95–85% A; 3.5–6 min, 85–70% A; 6–6.5 min, 70–70% A; 6.5–12 min, 70–30% A; 12–12.5 min, 30–30% A; 12.5–18 min, 30–0% A; 18–25 min, 0–0% A; 25–26 min, 0–95% A; 26–30 min, 95–95% A.

An Q Exactive Focus mass spectrometer coupled with an Xcalibur software was employed to obtain the MS and MS/MS data based on the IDA acquisition mode. During each acquisition cycle, the mass range was from 100 to 1,500, and the top three of every cycle were screened and the corresponding MS/MS data were further acquired. Sheath gas flow rate: 45 Arb, Aux gas flow rate: 15 Arb, Capillary temperature: 400 °C, Full ms resolution: 70,000, MS/MS resolution: 17,500, Collision energy: 15/30/45 in NCE mode, Spray Voltage: 4.0 kV (positive) or −3.6 kV (negative).

Animals and treatment

All the studies on animals were conducted in strict compliance in accordance with the Guide for the Care and Use of Laboratory Animals. The experimental protocol (2021-9-11) involving animals was reviewed and approved by the Institutional Animal Care and Use Committee of the Fourth Military Medical University. One-month-old, healthy, specific pathogen-free, Sprague Dawley rats weighing 120 ± 10 g were supplied by the Experimental Animal Center of Air Force Military Medical University. All rats were housed in a tidy animal room with constant temperature (23 °C ± 2 °C) and humidity (55% ± 10%), and a 12-h light/dark cycle. The rats had unrestricted access to water and food. After a week of feeding, all rats were randomly divided into two groups: (1) control group (n = 15): fed with normal diet and water; (2) model group (n = 60): fed with high-fat and high-sugar feed for two month. After these, model group rats were starved overnight and injected 40 mg kg⁻¹ streptozotocin (STZ) to induce diabetes, and the control group was injected with an equal volume of citric acid-sodium citrate buffer solution (pH = 4.2). Seventy-two hours after injection, fasting for 12 h, blood glucose was measured, after one week, a re-examination was performed 1 week later. The fasting blood glucose (FBG) was greater than 11.1 mmol L⁻¹ twice, and then the T2DM model was considered successfully established. T2DM rats were randomly divided into 4 groups: (1) T2DM (DM) model group (n = 15) (2) metformin (Met) group (200 mg kg⁻¹·d⁻¹, n = 15); (3) high-dose (H) BHJRSTD group (40 g kg⁻¹·d⁻¹, n = 15); (4) low-dose (L) BHJRSTD group (10 g kg⁻¹·d⁻¹, n = 15). The model groups were given high-fat and high-sugar diet every day, and the normal control (NC) group continued to be given the normal diet and water. Each group was administration through gastric gavage for one month to verify the hypoglycemic effect. Body weight, food consumption, and FBG levels were monitored and recorded during the administration period.

Collection and processing of test samples

After 8 weeks of administration, then all rats were killed, the excised pancreas tissues from the normal and experimental rats were immediately rinsed with normal saline and fixed in 4% paraformaldehyde. The tissues were cut into 5 μm thick sections after embedding in paraffin and then stained with hematoxylin/eosin staining (HE) for histopathological examination.

Antioxidant activity

The CAT, SOD, GSH-Px are important ones of antioxidant defense systems. MDA is formed as an end product of lipid peroxidation. Briefly, portions of pancreatic tissue were homogenized in cool phosphate-buffered saline (pancreatic tissue to normal saline, 1:4). The levels of CAT, SOD, GSH-Px and MDA in pancreatic tissue was measured using a commercially available ELISA kit, according to the manufacturer's instructions.

Statistical analysis

The results of the antioxidant assays were statistically processed using GraphPad Prism 6.0 (P < 0.05; Dunnett's test) to determine significant differences between multiple groups.

Identification of BHJRSTD extracts

To comprehensively characterize the BHJRSTD extract, an analytical method based on UHPLC Q-Exactive Focus Orbitrap MS was established in this research. Firstly, the prepared sample was injected into the UHPLC Q-Exactive Focus Orbitrap MS to obtain full-scan high-resolution MS data. Then, these data were processed using the Q Exactive Focus mass spectrometer's control software (Xcalibur, Thermo Fisher Scientific) to acquire, detect and predict various types of molecules. Third, the MS² of the predicted molecule was obtained in parallel reaction monitoring mode via UHPLC Q-Exactive Focus Orbitrap MS. Finally, compounds were identified by full scan MS date, MS² data, retention times and references. Details of the 74 major compounds are shown in Table 1 and Fig. 1.

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UHPLC–Q/Orbitrap/MS/MS chromatograms of BHJRSTD extracts (total ion current; a: positive ion mode; b: negative ion mode)

Citation: Acta Chromatographica 35, 4; 10.1556/1326.2022.01068

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Peaks 9, 10, 15, 18, 22, 24, 35, 36, 38, 39, 42–44, 46–48, 52, 55, 57, 66–70, 73 and 74 were identified as flavonoids based on comparing the retention time, measured mass, and MS spectrum with reference standards. Peaks 5 and 65 (Alkaloids) were identified as L-Tryptophan and 3-[(Carboxycarbonyl)amino]-L-alanine, respectively. Peaks 2 and 4 with the same [M+H]+ ion at m/z 116.071, 132.102 were identified as amino acid. Peak 31 was identified as phenylacetic acid and peak 37 was identified as gentiopicroside. Peak 11, 13, 49 were identified as chalcone. Peak 32 was identified as gallic acid (Phenols). Peak 30, 33, 34, 41 were regarded as phenylpropanoids. Peak 12, 14, 16, 17, 19–21, 23, 25–28, 40, 45, 50, 51, 53, 56, 58–64, 71, 72 were identified as terpenoids, while peak 54 was identified as terrestrosin K (Triterpenoids). Peak 6–8 (Xanthones) were identified as neomangiferin, mangiferin and isomangiferin, respectively.

Effects of BHJRSTD on the histomorphology of pancreatic islets in T2DM rats

As shown in Fig. 2, the pancreatic islets in the NC group were oval in shape, with regular structure, different size of cell clusters, regular shape, complete structure, clear edge, and scattered among the pancreatic vesicles. The β-cells in pancreatic islets were abundant, plentiful in shape, uniform in size, and tightly arranged, no pathological changes were found. In contrast, the shape of pancreatic islets in the DM group was blurred, irregular, and structurally disordered. The β-cells in the pancreatic islets were deformed and swollen, and the cytoplasm was lightly stained. Compared with the pancreatic islets of the DM group, the number of islet β-cells in the MET group, H group and L group was significantly increased, meanwhile, the structure of β-cells was regular and no obvious necrosis was seen. The islet borders were clear and the morphological structure was improved.

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Hematoxylin and eosin staining of T2DM rats pancreatic tissue

Citation: Acta Chromatographica 35, 4; 10.1556/1326.2022.01068

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Effects of BHJRSTD on the level of oxidative stress in T2DM rats

The human body is constantly exposed to different types of agents, resulting in the production of reactive species called free radicals [13], which lead to the oxidation of cellular machinery through the transfer of their free unpaired electrons [14]. In response to the harmful effects of these species, the body has acquired an endogenous antioxidant system or obtained exogenous antioxidants from the diet to neutralize these species and maintain the body's homeostasis. Any imbalance between reactive species and antioxidants might cause a condition called “oxidative stress”, which can lead to the development of pathological conditions, one of which is diabetes [15]. The damage of these free radicals cause to cells can be quantified by measuring levels of MDA, a product of lipid peroxidation [16, 17]. Certain enzymes play important roles in antioxidant defense to maintain viable reproductive capacity. These enzymes include CAT, SOD and GSH-Px, which convert free radicals or reactive oxygen intermediates to non-radical products [18].

In this study, the effect of BHJRSTD on the level of anti-oxidative stress in T2DM rats was evaluated via observing the contents of CAT, SOD, MDA and GSH-Px in rat pancreatic tissue. Figure 3 illustrates that, compared with the NC group, the levels of CAT, SOD, and GSH-Px in the DM group were significantly decreased (P < 0.05), and the level of MDA was significantly increased (P < 0.05). Compared with the DM group, the levels of CAT, SOD and GSH-Px were significantly decreased (P < 0.05), and the level of MDA in the MET group was significantly increased (P < 0.05). After administration of BHJRSTD extracts to rats for 8 weeks, the levels of SOD and GSH-Px in H and L groups were significantly increased (P < 0.05). Compared with the DM group, H and L groups showed an increasing trend, but the difference was not statistically significant (P > 0.05). BHJRSTD could reduce the MDA level in H and L groups, and there was a significant difference compared with the DM group (P < 0.05). The results indicated that BHJRSTD extracts had a certain anti-oxidative stress effect on pancreatic tissue of T2DM rats.

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The effects of each group on the level of oxidative stress in T2DM rats (#P < 0.05 vs NC group, *P < 0.05 vs DM group; Mean ± SD, n = 8)

Citation: Acta Chromatographica 35, 4; 10.1556/1326.2022.01068

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