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Libin Wang School of Medicine, Shaanxi Energy Institute, Xianyang, Shaanxi Province 712000, China

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Xin Shen Department of Pharmaceutical Sciences, Beijing Institute of Radiation Medicine, Beijing 100850, China

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Fang Wang School of Medicine, Shaanxi Energy Institute, Xianyang, Shaanxi Province 712000, China

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Xiaohui Xu Department of Orthopaedics, QILU Hospital of Shandong University Dezhou Hospital, DeZhou, Shandong Province 253000, China

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Abstract

Bai-Hu-Jia-Ren-Shen-Tang Decoction (BHJRSTD) is one of the oldest classic Chinese medicine prescriptions which used in the field of treatment of diabetes. However, to the best of our knowledge, the ingredients of this prescription have not been identified, and there are very few studies on the anti-diabetic mechanism of this prescription. Therefore, BHJRSTD was detected and identified by ultra-high-performance liquid chromatography coupled with Quadrupole-Exactive Focus Orbitrap MS (UHPLC–Q/Orbitrap/MS/MS). We identified 74 compounds, including flavonoids, alkaloids, chalcones, xanthones, phenols, phenylpropanoids, terpenes, triterpenes, amino acid derivatives, etc. Then, Sprague Dawley rats were fed with a high-fat and high-sugar diet for two months and injected with streptozotocin (STZ) to induce type 2 diabetes (T2DM). The diabetic rats were randomized to given metformin (200 mg kg−1·d−1, n = 15), BHJRSTD extracts (40 g kg−1·d−1) and BHJRSTD extracts (10 g kg−1·d−1) by gavage for 8 weeks. The results confirmed that BHJRSTD significantly decreased the level of MDA and increased levels of catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px), it shows that the prescription has significant antioxidant activity in the treatment of T2DM.

Abstract

Bai-Hu-Jia-Ren-Shen-Tang Decoction (BHJRSTD) is one of the oldest classic Chinese medicine prescriptions which used in the field of treatment of diabetes. However, to the best of our knowledge, the ingredients of this prescription have not been identified, and there are very few studies on the anti-diabetic mechanism of this prescription. Therefore, BHJRSTD was detected and identified by ultra-high-performance liquid chromatography coupled with Quadrupole-Exactive Focus Orbitrap MS (UHPLC–Q/Orbitrap/MS/MS). We identified 74 compounds, including flavonoids, alkaloids, chalcones, xanthones, phenols, phenylpropanoids, terpenes, triterpenes, amino acid derivatives, etc. Then, Sprague Dawley rats were fed with a high-fat and high-sugar diet for two months and injected with streptozotocin (STZ) to induce type 2 diabetes (T2DM). The diabetic rats were randomized to given metformin (200 mg kg−1·d−1, n = 15), BHJRSTD extracts (40 g kg−1·d−1) and BHJRSTD extracts (10 g kg−1·d−1) by gavage for 8 weeks. The results confirmed that BHJRSTD significantly decreased the level of MDA and increased levels of catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px), it shows that the prescription has significant antioxidant activity in the treatment of T2DM.

Introduction

In recent years, the advantages of Herbal and Traditional Chinese Medicine in the prevention and treatment of diabetes mellitus (DM) have been widely recognized around the world [12]. Bai-Hu-Jia-Ren-Shen-Tang Decoction (BHJRSTD), composed of Ginseng Radix, Rhizoma Anemarrhenae, Radix Glycyrrhizae Preparata, Gypsum Fibrosum and japonica rice is a traditional Chinese medicine prescriptions (TCMP) described in the Chinese medicine book “Treatise on Febrile and Miscellaneous Diseases”, which has been used in China for over 1,800 years [3]. The indications of BHJRSTD include infectious diseases, such as influenza, lung infection, encephalitis, enteric typhoid and hospital infection; thermoplegia; acute cerebrovascular disease, severe hyperosmolarity, diabetes mellitus [4], intractable hypotension, hypernatremia, shock and other internal diseases. This TCMP has been widely used to treat acute and severe cases in the cardiovascular intensive care ward. Refractory hypotension and hypovolemic shock that need large dose of supplemental fluid to maintain blood pressure and may also belong to the extension of the BHJRSTD formula syndrome.

Insulin resistance (IR) and pancreatic β-cell function defects were considered to be the main mechanisms of the pathogenesis of T2DM [5]. Oxidative stress can directly or indirectly aggravate IR and the damage of pancreatic β-cells, which is closely associated with the occurrence and development of T2DM [6, 7]. Studies have shown that compounds with antioxidant properties are more effective in treating T2DM [8, 9]. Nowadays, BHJRSTD is frequently used clinically in DM, especially as a complementary therapy [4]. The application of BHJRSTD proves to be more effective in the treatment of type 2 diabetes mellitus (T2DM) than using routine treatment only.

To our knowledge, there are no reports on chemical characterization and antioxidant activity studies of BHJRSTD, which is the usual way of consumption in traditional medicine in China. The complexity of chemicals in TCMP presents significant challenges to rapidly identify and characterize components. Over the past few decades, many natural product extracts, botanical ingredients and TCMP are analyzed using quadrupole Orbitrap spectrometry [10–12]. In this study, the main goals are develop an efficient method for rapid chemical characterization using UHPLC-Q-Exactive Orbitrap MS and measurement of the antioxidant activity of the BHJRSTD in diabetic rats, which are very beneficial for the further research and application of this TCMP including the material basis and pharmacological activity study.

Experimental

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.

Results and discussion

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.

Table 1.

High resolution UHPLC–Q/Orbitrap/MS/MS identification of ingredients from BHJRSTD extracts

Peak no.Components nameFormulaInChIKeyRT (min)Measured Mass (m/z)Quasi-Molecular Ion (m/z)Class
1IsoleucineC6H13NO2AGPKZVBTJJNPAG-UHFFFAOYSA-N0.75132.1020527132.102

[M+H]+
Amino acid
2L-ProlineC5H9NO2ONIBWKKTOPOVIA-BYPYZUCNSA-N0.75116.0706856116.071

[M+H]+
Amino acid
3Nicotinic acidC6H5NO2PVNIIMVLHYAWGP-UHFFFAOYSA-N0.87124.0394663124.039

[M+H]+
Vitamin
4L-IsoleucineC6H13NO2AGPKZVBTJJNPAG-WHFBIAKZSA-N1.03132.1020398132.102

[M+H]+
Amino acid
5L-TryptophanC11H12N2O2QIVBCDIJIAJPQS-VIFPVBQESA-N2.34205.0974881205.097

[M+H]+
Organoheterocyclic compounds
6NeomangiferinC25H28O16VUWOVGXVRYBSGI-IRXABLMPSA-N2.62585.1453811585.145

[M+H]+
Xanthones
7MangiferinC19H18O11AEDDIBAIWPIIBD-ZJKJAXBQSA-N3.07423.092836423.093

[M+H]+
Xanthones
8IsomangiferinC19H18O11CDYBOKJASDEORM-HBVDJMOISA-N4.76423.0929111423.093

[M+H]+
Xanthones
9LiquiritinC21H22O9DEMKZLAVQYISIA-UZQFATADSA-N5.30441.114928441.115

[M+Na]+
Flavonoids
10LiquiritigeninC15H12O4FURUXTVZLHCCNA-AWEZNQCLSA-N5.96257.0808213257.081

[M+H]+
Flavonoids
11Licochalcone BC16H14O5DRDRYGIIYOPBBZ-XBXARRHUSA-N6.21287.0911089287.091

[M+H]+
Chalcones
12Pseudoginsenoside F11C42H72O14JBGYSAVRIDZNKA-UHFFFAOYSA-N7.01801.5010963801.501

[M+H]+
Terpenoids
13IsoliquiritigeninC15H12O4DXDRHHKMWQZJHT-FPYGCLRLSA-N7.64257.0808108257.081

[M+H]+
Chalcones
14Ginsenoside Rg1C42H72O14YURJSTAIMNSZAE-ILDRPQKSSA-N8.40823.4817834823.482

[M+Na]+
Terpenoids
15KaempferideC16H12O6SQFSKOYWJBQGKQ-UHFFFAOYSA-N8.48301.0710347301.071

[M+H]+
Flavonoids
16Ginsenoside Rg3C42H72O13RWXIFXNRCLMQCD-XXYKJBRESA-N8.75807.4859478807.486

[M+Na]+
Terpenoids
17Ginsenoside Rg5C42H70O12NJUXRKMKOFXMRX-UHFFFAOYSA-N8.85767.4953034767.495

[M+H]+
Terpenoids
18GlabroneC20H16O5COLMVFWKLOZOOP-UHFFFAOYSA-N9.11337.1070099337.107

[M+H]+
Flavonoids
19Ginsenoside ReC48H82O18PWAOOJDMFUQOKB-DQASNDRCSA-N9.22969.538058969.538

[M+Na]+
Terpenoids
20Licoricesaponin G2C42H62O17WBQVRPYEEYUEBQ-OJVDLISWSA-N9.29839.4050082839.405

[M+H]+
Terpenoids
21Ginsenoside RfC42H72O14UZIOUZHBUYLDHW-KIOKIGKZSA-N9.59801.4973674801.497

[M+H]+
Terpenoids
22Licoflavone AC20H18O4HJGURBGBPIKRER-UHFFFAOYSA-N10.35323.1278005323.128

[M+H]+
Flavonoids
23Chikusetsu saponin IVaC42H66O14YOSRLTNUOCHBEA-SNRHWUJASA-N10.57817.4358779817.436

[M+Na]+
Terpenoids
24Licochalcone AC21H22O4KAZSKMJFUPEHHW-DHZHZOJOSA-N10.76339.1589659339.159

[M+H]+
Flavonoids
25SarsasapogeninC27H44O3GMBQZIIUCVWOCD-WWASVFFGSA-N11.49417.33681417.337

[M+H]+
Terpenoids
26Timosaponin A-IIIC39H64O13MMTWXUQMLQGAPC-HRSJJQGESA-N11.49741.4428077741.443

[M+H]+
Terpenoids
27Ginsenoside Rk1C42H70O12KWDWBAISZWOAHD-MHOSXIPRSA-N12.35789.4744795789.474

[M+Na]+
Terpenoids
2820(S)-Ginsenoside CkC36H62O8FVIZARNDLVOMSU-IRFFNABBSA-N12.60645.4331453645.433

[M+Na]+
Terpenoids
29L-ValineC5H11NO2KZSNJWFQEVHDMF-BYPYZUCNSA-N26.14118.0862706118.086

[M+H]+
Amino acid
30Cinnamic acidC9H8O2WBYWAXJHAXSJNI-VOTSOKGWSA-N2.58147.0451477147.045

[M−H]
Phenylpropanoids
31Phenylacetic acidC8H8O2WLJVXDMOQOGPHL-UHFFFAOYSA-N2.63135.045226135.045

[M−H]
Aromaticity
32Gallic acidC7H6O5LNTHITQWFMADLM-UHFFFAOYSA-N2.77169.0144344169.014

[M−H]
Phenols
33Cryptochlorogenic acidC16H18O9GYFFKZTYYAFCTR-AVXJPILUSA-N3.16353.0880346353.088

[M−H]
Phenylpropanoids
342-Hydroxycinnamic acid, predominantly transC9H8O3PMOWTIHVNWZYFI-AATRIKPKSA-N3.98163.0402139163.040

[M−H]
Phenylpropanoids
35DaidzinC21H20O9KYQZWONCHDNPDP-QNDFHXLGSA-N4.45461.1104935461.110

[M−H]
Flavonoids
36HomoorientinC21H20O11ODBRNZZJSYPIDI-VJXVFPJBSA-N4.58447.0945323447.095

[M−H]
Flavonoids
37GentiopicrosideC16H20O9DUAGQYUORDTXOR-UHFFFAOYNA-N4.65355.1038508355.104

[M−H]
Iridoids
38Gossypetin-8-C-glucosideC21H20O13SJRXVLUZMMDCNG-UHFFFAOYSA-N4.75479.0832296479.083

[M−H]
Flavonoids
39OrientinC21H20O11PLAPMLGJVGLZOV-VPRICQMDSA-N4.76447.0944571447.094

[M−H]
Flavonoids
40Pseudolaric acid BC23H28O8VDGOFNMYZYBUDT-VAFVZNMKSA-N4.77431.1691084431.169

[M−H]
Terpenoids
41Eleutheroside EC34H46O18FFDULTAFAQRACT-UHFFFAOYSA-N5.00787.2695132787.270

[M+HCOO]
Phenylpropanoids
42VitexinC21H20O10SGEWCQFRYRRZDC-VPRICQMDSA-N5.22431.098706431.099

[M−H]
Flavonoids
43Quercetin-3-O-galactosideC21H20O12OVSQVDMCBVZWGM-UHFFFAOYSA-N5.62463.0886957463.089

[M−H]
Flavonoids
44TectoridinC22H22O11CNOURESJATUGPN-UDEBZQQRSA-N5.87461.1103949461.110

[M−H]
Flavonoids
45DigitoninC56H92O29UVYVLBIGDKGWPX-UHFFFAOYNA-N6.211227.5660171227.566

[M−H]
Terpenoids
46GlycitinC22H22O10OZBAVEKZGSOMOJ-MIUGBVLSSA-N6.44445.1151963445.115

[M−H]
Flavonoids
47IsovitexinC21H20O10MYXNWGACZJSMBT-VJXVFPJBSA-N6.45431.0985813431.099

[M−H]
Flavonoids
48Acacetin-7-O-rutinosideC28H32O14YFVGIJBUXMQFOF-UHFFFAOYNA-N6.47591.1737251591.174

[M−H]
Flavonoids
49IsoliquiritinC21H22O9YNWXJFQOCHMPCK-LXGDFETPSA-N6.55417.1195589417.120

[M−H]
Chalcones
50Timosaponin B IIC45H76O19SORUXVRKWOHYEO-UHFFFAOYSA-N6.77919.4922594919.492

[M−H]
Terpenoids
51Atractyloside AC21H36O10QNBLVYVBWDIWDM-BSLJOXIBSA-N7.03493.2290177493.229

[M+HCOO]
Terpenoids
52HesperetinC16H14O6AIONOLUJZLIMTK-AWEZNQCLSA-N7.21301.0723311301.072

[M−H]
Flavonoids
53Ginsenoside F3C41H70O13HJRVLGWTJSLQIG-UHFFFAOYSA-N7.49815.4799289815.480

[M+HCOO]
Terpenoids
54Terrestrosin KC51H82O24TVRRDUXJKROMDX-CXLJPAHDSA-N7.681077.5127841077.513

[M−H]
Triterpenoids
55NaringeninC15H12O5FTVWIRXFELQLPI-UHFFFAOYNA-N7.92271.0614345271.061

[M−H]
Flavonoids
56Ginsenoside Rh1C36H62O9RAQNTCRNSXYLAH-UHFFFAOYSA-N7.99683.4375706683.438

[M+HCOO]
Terpenoids
57KaempferolC15H10O6IYRMWMYZSQPJKC-UHFFFAOYSA-N8.10285.0404805285.040

[M−H]
Flavonoids
58Ginsenoside Rb1C54H92O23GZYPWOGIYAIIPV-UHFFFAOYNA-N8.541153.5996071153.600

[M+HCOO]
Terpenoids
59Ginsenoside RcC53H90O22JDCPEKQWFDWQLI-UHFFFAOYSA-N8.671077.5878431077.588

[M−H]
Terpenoids
60Ginsenoside Rg2C42H72O13AGBCLJAHARWNLA-UHFFFAOYNA-N8.75783.4898787783.490

[M−H]
Terpenoids
61Ginsenoside RoC48H76O19NFZYDZXHKFHPGA-QQHDHSITSA-N8.80955.4926865955.493

[M−H]
Terpenoids
62Ginsenoside Rb2C53H90O22NODILNFGTFIURN-UHFFFAOYNA-N8.851123.5951181123.595

[M+HCOO]
Terpenoids
63Ginsenoside F1C36H62O9XNGXWSFSJIQMNC-UHFFFAOYNA-N8.96683.4385504683.439

[M+HCOO]
Terpenoids
64Araloside AC47H74O18KQSFNXMDCOFFGW-UGBTYEQWSA-N9.03925.4819032925.482

[M−H]
Terpenoids
653-[(Carboxycarbonyl)amino]-l-alanineC5H8N2O5NEEQFPMRODQIKX-REOHCLBHSA-N9.43174.9563911174.956

[M−H]
Alkaloids
66IsoanhydroicaritinC21H20O6RJANATGWWPNKAL-UHFFFAOYSA-N9.79367.1197283367.120

[M−H]
Flavonoids
67IcaritinC21H20O6TUUXBSASAQJECY-UHFFFAOYSA-N10.24367.1197729367.120

[M−H]
Flavonoids
68NeobavaisoflavoneC20H18O4OBGPEBYHGIUFBN-UHFFFAOYSA-N10.36321.114183321.114

[M−H]
Flavonoids
69xanthohumolC21H22O5ORXQGKIUCDPEAJ-YRNVUSSQSA-N10.57353.1398336353.140

[M−H]
Flavonoids
70InerminC16H12O5HUKSJTUUSUGIDC-ZBEGNZNMSA-N10.64283.0616985283.062

[M−H]
Flavonoids
71Ginsenoside Rg3(S-FORM)C42H72O13RWXIFXNRCLMQCD-UHFFFAOYNA-N10.96783.4903466783.490

[M−H]
Terpenoids
72Momordin IcC41H64O13HWYBGIDROCYPOE-WEAQAMGWSA-N11.14763.4297182763.430

[M−H]
Terpenoids
73GlabridinC20H20O4LBQIJVLKGVZRIW-ZDUSSCGKSA-N11.32323.1290457323.129

[M−H]
Flavonoids
74MulberrinC25H26O6UWQYBLOHTQWSQD-UHFFFAOYSA-N13.01421.1659774421.166

[M−H]
Flavonoids
Fig. 1.
Fig. 1.

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

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.

Fig. 2.
Fig. 2.

Hematoxylin and eosin staining of T2DM rats pancreatic tissue

Citation: Acta Chromatographica 35, 4; 10.1556/1326.2022.01068

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.

Fig. 3.
Fig. 3.

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

Conclusion

In the present investigation, the potential chemical composition and antioxidant effect of BHJRSTD extracts were evaluated via UHPLC–Q/Orbitrap/MS/MS and ELISA kit. We identified 74 compounds, including flavonoids, alkaloids, chalcones, xanthones, phenols, phenylpropanoids, terpenes, triterpenes, terpenes, amino acid derivatives, etc. It obviously and increased the content of CAT, SOD, GSH-Px and reduced level of MDA in T2DM rats after 8 weeks of administration. This study provides some reference information for the clinical use of the BHJRSTD, and also provides some basis for the development of Chinese herbal medicine

Acknowledgement

This research was supported by the Scientific Research Program Funded by Education Department of Shaanxi Provincial Government, Shaanxi Province, China (Grant No: 21JK0578) and Basic Research plan of Natural Science of Shaanxi Province, China (Grant No: 2021JQ-887, 2021JQ-888).

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    Djordjevic, A.; Spasic, S.; Jovanovic-Galovic, A.; Djordjevic, R.; Grubor-Lajsic, G. Oxidative stress in diabetic pregnancy: SOD, CAT and GSH-Px activity and lipid peroxidation products. J. Maternal-Fetal Neonatal Med. 2004, 16(6), 367372.

    • Search Google Scholar
    • Export Citation
  • 1.

    Xu, Y. X. Z.; Xi, S.; Qian, X. Evaluating traditional Chinese medicine and herbal products for the treatment of gestational diabetes mellitus. J. Diabetes Res. 2019, 2019, 9182595.

    • Search Google Scholar
    • Export Citation
  • 2.

    Wang, J.; Ma, Q.; Li, Y.; Wang, M.; Wang, T.; Zhao, B. Research progress on Traditional Chinese Medicine syndromes of diabetes mellitus. Biomed. Pharmacother. 2020, 121, 109565.

    • Search Google Scholar
    • Export Citation
  • 3.

    Liu, H.-K.; Hung, T.-M.; Huang, H.-C.; Huang, H. C.; Lee, I.; Chang, C. C.; Cheng, J. J.; Huang, C. Bai-Hu-Jia-Ren-Shen-Tang decoction reduces fatty liver by activating AMP-activated protein kinase in vitro and in vivo. Evidence-Based Complement. Altern. Med. 2015, 2015, 651734.

    • Search Google Scholar
    • Export Citation
  • 4.

    Tian, Y.; Zhong, W.; Zhang, Y.; Zhou, L.; Fu, X.; Wang, L.; Xie, C. Baihu Jia Renshen Decoction for type 2 diabetic mellitus: a protocol for systematic review and meta-analysis. Medicine (Baltimore) 2020, 99(19) e20210–e20210.

    • Search Google Scholar
    • Export Citation
  • 5.

    Abdul-Ghani, M. A.; Tripathy, D.; DeFronzo, R. A. Contributions of β-cell dysfunction and insulin resistance to the pathogenesis of impaired glucose tolerance and impaired fasting glucose. Diabetes Care 2006, 29(5), 11301139.

    • Search Google Scholar
    • Export Citation
  • 6.

    Caimi, G.; Carollo, C.; Presti, R. L. Diabetes mellitus: oxidative stress and wine. Curr. Med. Res. Opin. 2003, 19(7), 581586.

  • 7.

    Patil, V. S.; Patil, V. P.; Gokhale, N.; Acharya, A.; Kangokar, P. Chronic periodontitis in type 2 diabetes mellitus: oxidative stress as a common factor in periodontal tissue injury. J. Clin. Diagn. Res. 2016, 10(4), BC12BC16.

    • Search Google Scholar
    • Export Citation
  • 8.

    Unuofin, J. O.; Lebelo, S. L. Antioxidant effects and mechanisms of medicinal plants and their bioactive compounds for the prevention and treatment of type 2 diabetes: an updated review. Oxidative Med. Cell Longevity 2020, 2020, 1356893.

    • Search Google Scholar
    • Export Citation
  • 9.

    Dembinska-Kiec, A.; Mykkänen, O.; Kiec-Wilk, B.; Mykkänen, H. Antioxidant phytochemicals against type 2 diabetes. Br. J. Nutr. 2008, 99(E-S1), ES109ES117.

    • Search Google Scholar
    • Export Citation
  • 10.

    Qiao, X.; Li, R.; Song, W.; Miao, W. J.; Liu, J.; Chen, H. B.; Ye, M. A targeted strategy to analyze untargeted mass spectral data: rapid chemical profiling of Scutellaria baicalensis using ultra-high performance liquid chromatography coupled with hybrid quadrupole orbitrap mass spectrometry and key ion filtering. J. Chromatogr. A 2016, 1441, 8395.

    • Search Google Scholar
    • Export Citation
  • 11.

    Nardin, T.; Piasentier, E.; Barnaba, C.; Larcher, R. Targeted and untargeted profiling of alkaloids in herbal extracts using online solid-phase extraction and high-resolution mass spectrometry (Q-Orbitrap). J. Mass Spectrom. 2016, 51(9), 729741.

    • Search Google Scholar
    • Export Citation
  • 12.

    Gómez, J.; Simirgiotis, M. J.; Lima, B.; Paredes, J. D.; Villegas Gabutti, C. M.; Gamarra-Luques, C.; Tapia, A. Antioxidant, gastroprotective, cytotoxic activities and UHPLC PDA-Q orbitrap mass spectrometry identification of metabolites in baccharis grisebachii decoction. Molecules 2019, 24(6), 1085.

    • Search Google Scholar
    • Export Citation
  • 13.

    Feinendegen, L. E. Reactive oxygen species in cell responses to toxic agents. Hum. Exp. Toxicol. 2002, 21(2), 8590.

  • 14.

    Asmat, U.; Abad, K.; Ismail, K. Diabetes mellitus and oxidative stress—a concise review. Saudi Pharm. J. 2016, 24(5), 547553.

  • 15.

    Wei, W.; Liu, Q.; Tan, Y.; Liu, L.; Li, X.; Cai, L. Oxidative stress, diabetes, and diabetic complications. Hemoglobin 2009, 33(5), 370377.

    • Search Google Scholar
    • Export Citation
  • 16.

    Tiwari, B. K.; Pandey, K. B.; Abidi, A. B.; Rizvi, S. I. Markers of oxidative stress during diabetes mellitus. J. Biomarkers 2013, 2013, 378790.

    • Search Google Scholar
    • Export Citation
  • 17.

    Yaribeygi, H.; Sathyapalan, T.; Atkin, S. L.; Sahebkar, A. Molecular mechanisms linking oxidative stress and diabetes mellitus. Oxidative Med. Cell Longevity 2020, 2020, 8609213.

    • Search Google Scholar
    • Export Citation
  • 18.

    Djordjevic, A.; Spasic, S.; Jovanovic-Galovic, A.; Djordjevic, R.; Grubor-Lajsic, G. Oxidative stress in diabetic pregnancy: SOD, CAT and GSH-Px activity and lipid peroxidation products. J. Maternal-Fetal Neonatal Med. 2004, 16(6), 367372.

    • Search Google Scholar
    • Export Citation
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Senior editors

Editor(s)-in-Chief: Kowalska, Teresa (1946-2023)

Editor(s)-in-Chief: Sajewicz, Mieczyslaw

Editors(s)

  • Danica Agbaba (University of Belgrade, Belgrade, Serbia)
  • Łukasz Komsta (Medical University of Lublin, Lublin, Poland)
  • Ivana Stanimirova-Daszykowska (University of Silesia, Katowice, Poland)
  • Monika Waksmundzka-Hajnos (Medical University of Lublin, Lublin, Poland)

Editorial Board

  • R. Bhushan (The Indian Institute of Technology, Roorkee, India)
  • J. Bojarski (Jagiellonian University, Kraków, Poland)
  • B. Chankvetadze (State University of Tbilisi, Tbilisi, Georgia)
  • M. Daszykowski (University of Silesia, Katowice, Poland)
  • T.H. Dzido (Medical University of Lublin, Lublin, Poland)
  • A. Felinger (University of Pécs, Pécs, Hungary)
  • K. Glowniak (Medical University of Lublin, Lublin, Poland)
  • B. Glód (Siedlce University of Natural Sciences and Humanities, Siedlce, Poland)
  • A. Gumieniczek (Medical University of Lublin, Lublin, Poland)
  • U. Hubicka (Jagiellonian University, Kraków, Poland)
  • K. Kaczmarski (Rzeszow University of Technology, Rzeszów, Poland)
  • H. Kalász (Semmelweis University, Budapest, Hungary)
  • K. Karljiković Rajić (University of Belgrade, Belgrade, Serbia)
  • I. Klebovich (Semmelweis University, Budapest, Hungary)
  • A. Koch (Private Pharmacy, Hamburg, Germany)
  • P. Kus (Univerity of Silesia, Katowice, Poland)
  • D. Mangelings (Free University of Brussels, Brussels, Belgium)
  • E. Mincsovics (Corvinus University of Budapest, Budapest, Hungary)
  • Á. M. Móricz (Centre for Agricultural Research, Budapest, Hungary)
  • G. Morlock (Giessen University, Giessen, Germany)
  • A. Petruczynik (Medical University of Lublin, Lublin, Poland)
  • R. Skibiński (Medical University of Lublin, Lublin, Poland)
  • B. Spangenberg (Offenburg University of Applied Sciences, Germany)
  • T. Tuzimski (Medical University of Lublin, Lublin, Poland)
  • Y. Vander Heyden (Free University of Brussels, Brussels, Belgium)
  • A. Voelkel (Poznań University of Technology, Poznań, Poland)
  • B. Walczak (University of Silesia, Katowice, Poland)
  • W. Wasiak (Adam Mickiewicz University, Poznań, Poland)
  • I.G. Zenkevich (St. Petersburg State University, St. Petersburg, Russian Federation)

 

KOWALSKA, TERESA (1946-2023)
E-mail: kowalska@us.edu.pl

SAJEWICZ, MIECZYSLAW
E-mail:msajewic@us.edu.pl

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2022  
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Total Cites
WoS
647
Journal Impact Factor 1.9
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Chemistry, Analytical (Q3)

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2021  
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Total Cites
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652
Journal Impact Factor 2,011
Rank by Impact Factor Chemistry, Analytical 66/87
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without
Journal Self Cites
1,789
5 Year
Impact Factor
1,350
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2020
 
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650
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Chemistry, Analytical 71/83 (Q4)
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1,412
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1,301
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0,34
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Citable
45
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43
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2
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28
H-index
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0,316
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Acta Chromatographica
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1988
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