Identification of the Hypoglycemic Active Components of Lonicera japonica Thunb. and Lonicera hypoglauca Miq. by UPLC-Q-TOF-MS
by
Qinxuan Wu
1, 
Di Zhao
1,
Ying Leng
2,
Canhui Chen
1,
Kunyu Xiao
1,
Zhaoquan Wu
1 and
Fengming Chen
1,* 1
Hunan Provincial Key Laboratory of the Traditional Chinese Medicine Agricultural Biogenomics, The “Double-First Class” Application Characteristic Discipline of Hunan Province (Pharmaceutical Science), Changsha Medical University, Changsha 410219, China
2
Hunan Pharmaceutical Development and investment Group Co., Ltd., Changsha 410219, China
*
Author to whom correspondence should be addressed.
Molecules 2024, 29(20), 4848; https://doi.org/10.3390/molecules29204848
Submission received: 12 September 2024
/
Revised: 11 October 2024
/
Accepted: 11 October 2024
/
Published: 13 October 2024
(This article belongs to the Special Issue Bioactive Compounds from Traditional Medicinal Plants: Extraction, Identification, and Health Implications)
Abstract
:Lonicera japonica Thunb. and Lonicera hypoglauca are famous Chinese medicines used for hyperglycemia; however, the specific compounds that contributed to the hypoglycemic activity and mechanism are still unknown. In this study, the antidiabetic activity of L. japonica buds and L. hypoglauca buds, roots, stems, and leaves extracts was primarily evaluated, and the L. japonica buds and L. hypoglauca buds, roots, and stems extracts displayed significant hypoglycemic activity, especially for the buds of L. hypoglauca. A total of 72 high-level compounds, including 9 iridoid glycosides, 12 flavonoids, 34 organic acids, and 17 saponins, were identified by ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS) combined with the fragmentation pathways of standards from different parts of L. japonica and L. hypoglauca extracts. Among them, 19 metabolites, including 13 saponins, were reported for the first time from both medicines. Seven high-content compounds identified from L. hypoglauca buds extract were further evaluated for hypoglycemic activity. The result indicated that neochlorogenic acid, chlorogenic acid, isochlorogenic acid A, isochlorogenic acid B, and isochlorogenic acid C displayed significant antidiabetic activity, especially for isochlorogenic acid A and isochlorogenic acid C, which demonstrated that the five chlorogenic-acid-type compounds were the active ingredients of hypoglycemic activity for L. japonica and L. hypoglauca. The potential mechanism of hypoglycemic activity for isochlorogenic acid A and isochlorogenic acid C was inhibiting the intestinal α-glucosidase activity to block the supply of glucose. This study was the first to clarify the hypoglycemic active ingredients and potential mechanism of L. japonica and L. hypoglauca, providing new insights for the comprehensive utilization of both resources and the development of hypoglycemic drugs.
1. Introduction
Lonicera japonica Thunb. and Lonicera hypoglauca Miq., which belong to the genus of Lonicaceae from Caprifoliaceae family, are mainly distributed in Asia, North America, Europe, and temperate or subtropical regions of northern Africa [1,2]. At present, L. japonica is mainly planted in Shandong, Henan, Hebei province, and other areas north of the Yangtze River in China, while L. hypoglauca is distributed in the south of the Yangtze River in China, such as in Hunan and Zhejiang provinces [3,4]. L. japonica and L. hypoglauca are included in the 2020 edition of the Chinese Pharmacopoeia and have the effects of clearing heat and detoxifying, and they are used for the treatment of wind-heat cold, fever, heat, blood dysentery, and other diseases [5,6]. Modern pharmacological studies have proved that both Chinese medicines have antiviral, antibacterial, immune-modulating, liver-protecting, antipyretic, antitumor, neuroprotective, and hypoglycemic effects [7,8]. L. japonicae has antiplatelet aggregation, glucose-lowering, antiulcer, antiultraviolet radiation, antiendotoxin, antifertility, anti-early pregnancy, and neuroprotective activities that are not reported in L. hypoglauca. In addition, L. hypoglauca has a certain advantage in terms of antibacterial activity compared to that of L. japonicae [1,4]. The main active ingredients are organic acids, flavonoids, iridoids, and saponins [9,10]. L. japonicae contains more flavonoids and iridoids; however, L. japonicae have a variety of saponins and significantly higher chlorogenic acid than L. japonicae [1,4]. In addition to the well-known components, there are many chemical components present in L. japonica and L. hypoglauca that need to be further studied and identified.
During plant growth, its metabolites are constantly changing, and the metabolites in the buds, roots, stems, and leaves of L. japonica and L. hypoglauca also vary [11,12]. In previous studies, liquid chromatography–mass spectrometry (LC-MS) was used to identify the chemical composition of a single part of L. japonica or L. hypoglauca [13,14,15], resulting in the loss of a portion of the compound. Therefore, in this study, we comprehensively analyzed the chemical composition in the buds, roots, stems, and leaves of L. japonica and L. hypoglauca by UPLC-Q-TOF-MS technology, laying the foundation for the subsequent identification of hypoglycemic active substances.
Diabetes is currently one of the most common metabolic diseases and has a significant impact on human health worldwide [16,17]. According to the International Diabetes Federation (2019), the number of people with diabetes is expected to reach 578 million in 2030, and type 2 diabetes accounts for about 90% of the total [18,19]. However, synthetic drugs to treat type 2 diabetes cause many adverse effects, and traditional Chinese herbal medicines or natural ingredients have received extensive attention in many countries due to their significant antidiabetic activity and low adverse reactions [20,21]. Therefore, the development of new antidiabetic drugs from traditional herbal medicines has been a hot topic of research. In previous studies [22,23,24,25], it has been reported that L. japonica polysaccharides have significant hypoglycemic activity but there are few studies on the hypoglycemic activity of L. hypoglauca. Moreover, the specific hypoglycemic components of L. japonica or L. hypoglauca are still not clear.
In this study, we first evaluated the hypoglycemic activity of extracts from L. japonica and different parts of L. japonica; then, UPLC-Q-TOF-MS technology was employed to identify their potential hypoglycemic components in different parts of both medicines, and seven monomeric compounds with high content in L. japonica and L. hypoglauca were finally used to clarify the hypoglycemic components of both Chinese medicines and the potential mechanism.
2. Results and Discussion
2.1. Evaluation of the Hypoglycemic Activity of L. japonica and Different Parts of L. hypoglauca Extract
L. japonica have been reported to have significant hypoglycemic activity [22,23,24,25], while L. hypoglauca extracts and other components present in both medicines have rarely been reported to have antidiabetic activity. To evaluate the hypoglycemic activity of non-polysaccharides in L. japonica bud and L. hypoglauca buds, roots, stem, and leaves, 70% ethanol was employed as the solvent to obtain the corresponding extracts. The result indicated that the blood glucose level of the model control group (MC) was significantly higher (p < 0.01) compared with the normal control group (NC) at day 0, 7, 14, 21, and 28, respectively, which indicated that the high blood glucose mouse model (T2D) was successfully established (Figure 1A). The blood glucose level of the acarbose group at day 14 (20.01 ± 0.33 mmol/L), 21 (18.21 ± 0.28 mmol/L), and 28 (15.34 ± 0.21 mmol/L) significantly decreased (p < 0.05) compared with the initial level at day 0 (26.20 ± 0.54 mmol/L), which indicated that the positive group displays significant hypoglycemic activity. The blood glucose level of L. japonica bud extract at day 28 (21.34 ± 1.09 mmol/L) in the groups was significantly decreased (p < 0.05) compared with the initial blood glucose level at day 0 (27.26 ± 0.45 mmol/L), which indicated that the L. japonica buds extracts have remarkable hypoglycemic activity (Figure 1A). Compared with the initial blood glucose level at day 0 (27.21 ± 0.45, 26.45 ± 1.21, and 27.23 ± 0.23 mmol/L), the buds, roots, and stems extracts (18.22 ± 1.82, 20.11 ± 1.23, and 21.67 ± 1.02 mmol/L, respectively) groups at day 28 were significantly decreased (p < 0.05), which indicated that the L. hypoglauca buds, roots, and stem extracts display significant antidiabetic activity, especially for the extract of buds. However, the leaves extract did not show significant activity in decreasing blood glucose level.
2.2. Identification of the Main Components of L. japonica and Different Parts of L. hypoglauca Extracts
The 70% ethanol extracts of L. japonica buds and L. hypoglauca buds, roots, and stems had significant hypoglycemic activity, but the specific components that contributed to the hypoglycemic activity were still unknown. In this study, the chemical components in the 70% ethanol extracts were further systematically identified by the UPLC-Q-TOF-MS technology, and the potential hypoglycemic active components in the 70% ethanol extract were proposed.
The total ion chromatograms (TICs) of L. japonica bud and L. hypoglauca buds, roots, stems, and leaves (Figure 2) were primarily produced using UPLC-Q-TOF-MS technologies. And, then, the MS/MS spectra of metabolites were obtained by the target MS/MS method or auto MS/MS strategy. Finally, a total of 72 metabolites (Table 1), which may contribute to the hypoglycemic activity, were screened and identified by their MS/MS spectra combined with the well-investigated fragmentation pathways of standards. The structures of high-level metabolites 7, 12, 13, 24, 35, 36, and 42 were unambiguously determined by comparing the retention time, MS, and MS/MS data with the references (Figure 3). The other compounds were tentatively identified by the established UPLC-Q-TOF-MS method.
2.2.1. Investigation of the Fragmentation Behaviors of Standards
A series of analogs are presented in L. japonica buds and L. hypoglauca buds, roots, stems, and leaves. Investigating the fragmentation behaviors of well-characterized standards is a valid strategy for identifying the unknown structures of analogs since they typically display similar fragmentation patterns in the MS/MS spectra. In this study, a total of seven standards, including neochlorogenic acid (7), chlorogenic acid (12), cryptochlorogenic acid (13), secoxyloganin (24), isochlorogenic acid B (35), isochlorogenic acid A (36), and isochlorogenic acid C (42), which were reported from L. japonica or L. hypoglauca in previous studies [4], were employed for investigating the fragmentation pathways of organic acid and iridoid glycosides. The fragmentation pathways of flavonoids and saponins in both medicines were also summarized based on previous studies [11,26].
The fragmentation pathways of chlorogenic-acid-type in L. japonica or L. hypoglauca were investigated from the MS/MS spectra of standards 7, 12, 13, 35, 36, and 42. Two main fragmentation behaviors were observed. The primary fragmentation pathway was the cleavage of the bond between the quinic acid and caffeic acid and the formation of the high-abundance ions. In the MS/MS of 7, 12, 13, 35, 36, and 42 (Figure 4A–F), the characteristic ions at m/z 353.08, 191.05, and 179.03 were produced by the cleavage of the bond between the quinic acid and caffeic acid. The other fragmentation pathway was the loss of some molecules, such as H2O and CO2, from the fragment ions. In the MS/MS of 13, 35, and 42, the ions at m/z 173.0451, 173.0461, and 173.0464 were formed by the neutral loss of a H2O moiety from the fragments at m/z 191.0558, 191.0560, and 151.0556, respectively. Two fragmentation behaviors were found for iridoid glycosides. The first fragmentation pathway was the loss of some neutral moiety, such as CH3OH and CO2, from the fragment ions and the formation of characteristic ions. In the MS/MS of 24 (Figure 4G), the ion at m/z 371.0983 was produced by the loss of a CH3OH from the ion at m/z 403.1238. The second fragmentation pathway was the loss of the sugar moiety. In the MS/MS of 24 (Figure 4G), the ion at m/z 223.0645 was obverse due to the loss of the sugar moiety from the ion at m/z 403.1238. Three fragmentation pathways of flavonoids present in L. japonica and L. hypoglauca have been well concluded in previous studies [24]. The loss of all sugar moiety from the mother ion and the form of the characteristic ion at m/z 323.09 was the main fragmentation pathway of saponins present in both medicines [25].
2.2.2. Identification of the Iridoid Glycosides, Flavonoids, Organic Acid, and Saponins
A total of nine compounds, including compounds 3, 9, 14, 15, 17, 20, 24, 28, and 43, were identified as iridoid glycosides based on their MS/MS spectra (Table 1 and Figure S1). Take compound 14 as an example. The MS fragmentation pathways of compounds 14 and 42 (standard) were highly similar (Figure 4G and Figure 5A), and the difference in m/z value between both compounds was 14.0148 Da, which indicated that the methyl group in compound 42 was replaced by a hydrogen atom and formed the structure of metabolite 14. Therefore, compound 14 was tentatively identified as Secologanoside-7-methyl ester, which has been reported in a previous study [4]. Compounds 25, 26, 29–34, 37–39, and 45 were identified as flavonoids based on their characteristic MS/MS spectra. In the ms/ms spectrum of compound 30 (Figure 5B), the loss of a glucose moiety from the mother ion at m/z 463.0868 and the formation of the fragment ion at m/z 300.0290 was found, which indicated that the skeleton of compound 30 was quercetin and a glucose moiety was concluded in the structure; therefore, compound 30 was tentative as hyperoside, which was also reported in a previous study [4]. A total of 34 metabolites, including 1, 2, 4, 5, 6–8, 10–13, 16, 18, 19, 21–23, 27, 35, 36, 40–42, 44, 46, 47–52, 60, 62, and 68, were identified as organic acid based on their MS/MS spectra (Table 1 and Figure S1 in the Supplementary Materials). Take compound 52 as an example. The MS fragmentation pathways of compounds 52 and 35 were highly similar (Figure 4D and Figure 5C) and the difference in m/z value between both compounds was 162.0287 Da, which indicated that one caffeic acid was added to compound 35 and formed the structure of metabolite 52. The characteristic ions at m/z 515.1188, 353.0865, and 173.0418 indicated that compound 52 was 3,4,5-tricaffeoylquinic acid [4]. Compounds 53–59, 61, 63–67, and 69–72 were identified as saponins based on their characteristic fragment ions. In the MS/MS spectrum of compound 63 (Figure 5D), the characteristic fragment ions at m/z 1073.5531, 749.4500, and 323.0951 indicated that compound 30 was dipsacoside B, which was also reported in a previous study [4].
Finally, a total of 72 metabolites, including 9 iridoid glycosides, 12 flavonoids, 34 organic acids, and 17 saponins, were identified using their MS/MS spectra, fragmentation pathways of standards, and the previous studies. Among these, 19 compounds (20, 28, 44, 48, 49, 53–56, 58, 60, 61, 65, and 67–72) including 13 saponins were reported for the first time from both medicines (Table 1). The primary metabolites of L. japonica bud and L. hypoglauca buds, roots, stems, and leaves, such as compounds 7, 12, 13, 24, 35, 36, 42, 58, and 63, were well identified in this study, which may contribute to the antidiabetic activity of the extracts.
The distribution of all identified metabolites in the L. japonica bud and L. hypoglauca buds, roots, stems, and leaves was determined using the extracted ion chromatogram (EIC) based on the TICs. The identified compounds were found in all parts; however, the relative content of specific compounds in different parts has significant differences. The species and amount of metabolites in the L. hypoglauca buds were more abundant than in other parts, especially for the organic acids and saponins. Most of the high-level organic acids and saponins, such as compounds 7, 12, 13, 36, 42, 58, and 63, were primarily detected in L. hypoglauca buds. The identified iridoid glycosides, such as compounds 17 and 20, are mainly distributed in the roots and stems of L. hypoglauca. The main components of L. hypoglauca leaves were secoxyloganin (24) and dipsacoside B (63) and the level of other metabolites was relatively low.
2.3. Evaluation of the Hypoglycemic Activity of Seven High-Content Compounds in L. japonica and L. hypoglauca Extracts
L. japonica buds and L. hypoglauca buds, roots, and stem extracts display significant antidiabetic activity, especially for the extract of L. hypoglauca buds. However, the specific compounds that contributed to the hypoglycemic activity are still unknown. UPLC-Q-TOF-MS analysis results indicated that compounds 7 (neochlorogenic acid), 12 (chlorogenic acid), 13 (cryptochlorogenic acid), 24 (secoxyloganin), 35 (isochlorogenic acid B), 36 (isochlorogenic acid A), and 42 (isochlorogenic acid C) were the main components of L. hypoglauca bud extract. To clarify the hypoglycemic active ingredients of L. japonica and L. hypoglauca, the antidiabetic activity of seven high-content compounds were evaluated.
The blood glucose level of the model control group (MC) was significantly higher (p < 0.01) compared with the normal control group (NC) at day 0, 7, 14, 21, and 28, respectively, which indicated that the high blood glucose model was successfully established (Figure 1B). The blood glucose level of the acarbose group at day 14 (23.11 ± 1.33 mmol/L), 21 (19.21 ± 1.28 mmol/L), and 28 (14.56 ± 1.21 mmol/L) significantly decreased (p < 0.05) compared with the initial level at day 0 (29.23 ± 1.52 mmol/L), which indicated that the positive group displays significant hypoglycemic activity. Compared with the initial blood glucose level at day 0 (29.23 ± 0.67, 28.23 ± 1.22, and 28.24 ± 1.45 mmol/L for NE-A, CH-A, and CR-A, respectively), the NE-A (22.23 ±1.29 mmol/L) and CH-A (21.34 ± 1.82 mmol/L) groups at day 28 were significantly decreased (p < 0.05), which indicated that the neochlorogenic acid (7) and chlorogenic acid (12) have remarkable hypoglycemic activity (Figure 1B); however, the cryptochlorogenic acid (13) did not show significant activity. The blood glucose level of the IA-A and IA-C at day 14 (22.08 ± 1.08 and 23.67 ± 1.18 mmol/L), 21 (19.67 ± 0.56 and 20.12 ± 1.32 mmol/L), and 28 (15.34 ± 1.21 and 18.56 ± 1.07 mmol/L) significantly decreased (p < 0.05 or p < 0.01) compared with the initial level at day 0 (29.09 ± 1.17 and 28.11 ± 1.27 mmol/L), which indicated that the isochlorogenic acid A (36) and isochlorogenic acid C (42) have significant hypoglycemic activity and the antidiabetic activity of isochlorogenic acid A was stronger than that of isochlorogenic acid C. In addition, IA-B (35) also displays significant antidiabetic activity, which could decrease the blood glucose level from 28.34 ± 1.24 mmol/L (day 0) to 21.01 ± 1.42 mmol/L (day 28); however, its hypoglycemic activity was weaker than that of isochlorogenic acid A and isochlorogenic acid C.
2.4. The Effect of Isochlorogenic Acid A (36) and Isochlorogenic Acid C (42) on the Postprandial Glycemia
Isochlorogenic acid A (36) and isochlorogenic acid C (42), which were the most active compounds, were employed to explore the potential mechanism of hypoglycemic activity. The postprandial glycemia experiment for both compounds was carried out. The result shows that the acarbose (9.45 ± 0.2 mmol/L), isochlorogenic acid A (9.34 ± 0.43 mmol/L), and isochlorogenic acid C (10.67 ± 1.12 mmol/L) groups display significant activity in reducing postprandial glycemia compared to the normal control group (13.21 ± 1.11 mmol/L) after administration of corresponding solution for 30 min (Figure 1C). The effect on postprandial glycemia of isochlorogenic acid A was the same as the positive drug (acarbose). Isochlorogenic acid A displays stronger activity than isochlorogenic acid C for reducing postprandial glycemia. The blood glucose level of all groups returns to baseline after 120 min. The above results indicate that isochlorogenic acid A, isochlorogenic acid C, L. japonica buds, L. hypoglauca buds, roots, and stem extracts inhibiting the intestinal α-glucosidase activity to block the supply of glucose was the potential mechanism of hypoglycemic activity.
3. Materials and Methods
3.1. Materials and Chemicals
Ethanol (AR) was purchased from Chengdu Must Bio-Technology Co., Ltd. (Chengdu, China), which was used for extraction. Acetonitrile (HPLC-grade) was obtained from Merck (Darmstadt, Germany). Acarbose (purity ≥ 98%), streptozotocin (STZ), and seven standards, including neochlorogenic acid (7), chlorogenic acid (13), cryptochlorogenic acid (14), secoxyloganin (24), isochlorogenic acid B (35), isochlorogenic acid A (36), and isochlorogenic acid C (42), were obtained from National Institutes for Food and Drug Control (Beijing, China). The high-fat feed (MD12033, 60% fat kcal%) was purchased from Jiangsu Medicience Biomedical Pharmaceuticals Limited; for the specific components of high-fat feed, see Table S1.
3.2. Extracts Experiment
L. hypoglauca buds, roots, stems, and leaves were collected in September of 2023 at the Hunan Agricultural University (Hunan, China, co-ordinates: 113°04′25.55″ E, 28°10′45.11″ N). They were authenticated by Prof. Han Zhou (Hunan Agricultural University, China). The fresh buds, roots, stems, and leaves were dried under a vacuum drying oven at 60 °C for 48 h. The L. japonica buds were purchased from LBX pharmacy (Changsha, China) and both dried samples were crushed by a disintegrator. Approximately 50.0 g of dried sample powder was suspended in 500 mL of 70% ethanol water (v/v) for 30 min. Extraction was carried out in an ultrasonic bath for 60 min, and the extract solution was filtered through a nylon membrane. L. japonica buds and L. hypoglauca buds, roots, stems, and leaves extracts were obtained after recovering the solvent by vacuum reduction concentration.
3.3. UPLC-Q-TOF-MS Conditions
An Agilent 1290 UPLC system coupled with a 6530 Q-TOF/MS accurate mass spectrometer (Agilent Technologies Inc., Santa Clara, CA, USA) was used for the investigation of the fragmentation pathways of standards and identification of compounds in L. japonica and L. hypoglauca. An Agilent C18 (150 mm × 2.1 mm, 1.7 µm; Agilent Technologies Inc., Santa Clara, CA, USA) was used as a separation column. The elution solution consisted of 0.1% formic acid–water (A) and 0.1% formic acid–acetonitrile (B). The elution system was 0–15 min, 5–40% B; 15–25 min 40–65% B; and 25–40 min 65–90% B. The flow rate was set at 0.30 mL/min and the injection volume was 3 μL. The conditions of Q-TOF-MS (negative mode) were optimized as follows: gas temperature and sheath gas temperature were set at 350 and 300 °C, respectively. Sheath gas and drying gas flow were optimized to 10 and 11 L/min, respectively. The fragment voltage was set at 150 V. The Q-TOF-MS was continuously calibrated by a reference solution at m/z 112.9855 and 966.0007. The MS/MS data of each compound were obtained using the target-MS/MS mode (10–25 eV) and auto-MS/MS mode (15, 20, and 30 eV).
3.4. Experimental Animal
The male ICR mice, which were four weeks old with a weight range of 18.0–22.0 g, were purchased from Hunan Slake Jing-da Experimental Animals Co., Ltd. (Certificate number 43004700048590, Changsha, China). Animals were kept under the ambient temperature of 22 ± 2 °C, and a standard light/dark schedule (12:12 h) was used for those mice. All experiments and procedures were carried out based on the Regulations of Experimental Animal Administration issued by the State Committee of Science and Technology of China.
3.5. Establishment of High-Glucose Mouse Model (T2D) and the Drug Administration
The mice were subjected to a high-fat diet for 30 days in addition to the normal control group (NC). After the high-fat diet feeding, the T2D mouse model was successfully induced by intraperitoneal injection of STZ for 3 days (60 mg/kg) when the blood glucose level was more than 11.6 mmol/L. The normal and model control groups were given distilled water and the rest of the groups were given the corresponding solution of extracts or drugs by intragastric administration (200 mg/kg for the extracts and 30 mg/kg for acarbose and 7 standards, respectively) with a volume of 20 mL/kg (Tables S2 and S3). Mice were treated daily for a continual 28 days. The blood glucose level was determined by a glucometer (Johnson, Branchburg, NJ, USA) for every 7-day interval of 28 days.
3.6. The Experiment on Postprandial Glycemia of the Normal Mice
Forty normal mice, which were four weeks old with a weight range of 18.0–22.0 g, were randomly divided into four groups based on their weight, each containing ten mice. All mice were given a sucrose solution with 2.0 g/kg. In addition to the normal control group (NC), the remaining three groups were given acarbose (30 mg/kg), isochlorogenic acid A (36), and isochlorogenic acid C (42) solution (30 mg/kg). The blood glucose level was determined by the glucometer at 0, 30, 60, 90, and 120 min.
3.7. Statistical Method
The experimental data statistical analysis was carried out using SPSS16.0, and the statistically significant level was set as p ≤ 0.05. The data were expressed as mean ± standard deviation. The normality and variance homogeneity were tested by Leven’s test method, and the statistical analysis was performed with a one-way analysis of variance (ANOVA) and LSD test if the normality and variance homogeneity were met (p > 0.05); otherwise, the Kruskal–Wallis test was used. If the Kruskal–Wallis test was statistically significant (p ≤ 0.05), the Dunnett’s test (nonparametric method) was used for comparative analysis. Statistical differences and biological significance were considered simultaneously in this study.
4. Conclusions
In this study, the antidiabetic activity of L. japonica buds, L. hypoglauca buds, roots, stems, and leaves extracts was primarily evaluated, and the L. japonica buds, L. hypoglauca buds, roots, and stems extracts displayed significant hypoglycemic activity, especially for the buds of L. hypoglauca. Then, a total of 72 high-level metabolites were screened and identified from the L. japonica buds, L. hypoglauca buds, roots, stems, and leaves extracts by UPLC-Q-TOF-MS combined with the fragmentation behaviors of references. Among these, 19 compounds were reported for the first time from both medicines. Five high-content compounds, including neochlorogenic acid, chlorogenic acid, isochlorogenic acid A, isochlorogenic acid B, and isochlorogenic acid C, displayed significant hypoglycemic activity, especially for isochlorogenic acid A, which demonstrated that these compounds contributed to the hypoglycemic activity of L. japonica and L. hypoglauca. The potential mechanism of hypoglycemic activity for both medicines or the active compounds was inhibiting the intestinal α-glucosidase activity to block the supply of glucose. However, the inhibitory activity of α-glucosidase in vitro should be further investigated to further clarify their hypoglycemic mechanism. This study was the first to clarify the hypoglycemic active ingredients of L. japonica and L. hypoglauca, which provides new insights for the comprehensive utilization of both resources and the development of new hypoglycemic drugs.
Supplementary Materials
The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/molecules29204848/s1, Figure S1: The MS/MS spectra of compounds 1–72; Table S1: The components of high-fat diet. Table S2: The groups, number of mice, and dose design of hypoglycemic experiment for L. japonica buds, L. hypoglauca buds, roots, stems, and leaves extracts; Table S3: The groups, number of mice, and dose design of hypoglycemic experiment for seven compounds.
Author Contributions
Conceptualization, Q.W.; methodology, Q.W. and D.Z.; formal analysis, Q.W., Y.L., and F.C.; investigation, Q.W. and Y.L.; resources, C.C.; data curation, K.X.; writing—original draft preparation, Q.W.; writing—review and editing, Q.W. and F.C.; project administration, Z.W. and F.C.; funding acquisition, F.C. All authors have read and agreed to the published version of the manuscript.
Funding
Scientific Research Project of Hunan Provincial Department of Education (Xiangjiaotong (2023) No. 361-23A0665); “The 14th Five-Year Plan ” Application Characteristic Discipline of Hunan Province (Pharmaceutical Science) (Xiangjiaotong (2022) No. 351), and the Science and Technology Innovation Program of Hunan Province (2023JJ40085).
Institutional Review Board Statement
Not applicable.
Informed Consent Statement
Not applicable.
Data Availability Statement
Data are contained within the article and Supplementary Materials.
Conflicts of Interest
Author Ying Leng was employed by the company Hunan Pharmaceutical Development and investment Group Co., Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as potential conflicts of interest.
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Figure 1.
Effect of L. japonica buds, L. hypoglauca buds, roots, stems, leaves extract, and monomeric compounds on blood glucose concentrations. (A) The hypoglycemic activity of L. japonica buds, L. hypoglauca buds, roots, stems, and leaves extracts; (B) the hypoglycemic activity of 7 high-content compounds from the extracts; (C) the postprandial glycemia of administration of isochlorogenic acid A (IA-A) and isochlorogenic acid C (IA-C) for the normal mice. ## p < 0.01 compared with the normal control group on the same days. * p < 0.05 or ** p < 0.01 compared with the data at day 0; ▽ p < 0.05 or ▽▽ p < 0.01 compared with the model control group on the same days. NC: normal group; MC: model group; Acarbose group (positive control, 30 mg/kg); LJ-B: L. japonica buds group (200 mg/kg); LH-B: L. hypoglauca buds group (200 mg/kg); LH-R: L. hypoglauca roots group (200 mg/kg); LH-S: L. hypoglauca stems group (200 mg/kg); LH-L: L. hypoglauca leaves group (200 mg/kg); NE-A: neochlorogenic acid group (30 mg/kg); CH-A: chlorogenic acid group (30 mg/kg); CR-A: cryptochlorogenic acid group (30 mg/kg); SE: secoxyloganin group (30 mg/kg); IA-A: isochlorogenic acid A group (30 mg/kg); IA-B: isochlorogenic acid B group (30 mg/kg); and IA-C: isochlorogenic acid C group (30 mg/kg).
Figure 1.
Effect of L. japonica buds, L. hypoglauca buds, roots, stems, leaves extract, and monomeric compounds on blood glucose concentrations. (A) The hypoglycemic activity of L. japonica buds, L. hypoglauca buds, roots, stems, and leaves extracts; (B) the hypoglycemic activity of 7 high-content compounds from the extracts; (C) the postprandial glycemia of administration of isochlorogenic acid A (IA-A) and isochlorogenic acid C (IA-C) for the normal mice. ## p < 0.01 compared with the normal control group on the same days. * p < 0.05 or ** p < 0.01 compared with the data at day 0; ▽ p < 0.05 or ▽▽ p < 0.01 compared with the model control group on the same days. NC: normal group; MC: model group; Acarbose group (positive control, 30 mg/kg); LJ-B: L. japonica buds group (200 mg/kg); LH-B: L. hypoglauca buds group (200 mg/kg); LH-R: L. hypoglauca roots group (200 mg/kg); LH-S: L. hypoglauca stems group (200 mg/kg); LH-L: L. hypoglauca leaves group (200 mg/kg); NE-A: neochlorogenic acid group (30 mg/kg); CH-A: chlorogenic acid group (30 mg/kg); CR-A: cryptochlorogenic acid group (30 mg/kg); SE: secoxyloganin group (30 mg/kg); IA-A: isochlorogenic acid A group (30 mg/kg); IA-B: isochlorogenic acid B group (30 mg/kg); and IA-C: isochlorogenic acid C group (30 mg/kg).
Figure 2.
The TICs of L. japonica buds (A), L. hypoglauca buds (B), roots (C), stems (D), and leaves (E).
Figure 2.
The TICs of L. japonica buds (A), L. hypoglauca buds (B), roots (C), stems (D), and leaves (E).
Figure 3.
The TICs of L. japonica buds (A), neochlorogenic acid (B), chlorogenic acid (C), cryptochlorogenic acid (D), secoxyloganin (E), isochlorogenic acid B (F), isochlorogenic acid A (G), and isochlorogenic acid C (H).
Figure 3.
The TICs of L. japonica buds (A), neochlorogenic acid (B), chlorogenic acid (C), cryptochlorogenic acid (D), secoxyloganin (E), isochlorogenic acid B (F), isochlorogenic acid A (G), and isochlorogenic acid C (H).
Figure 4.
The MS/MS spectra of neochlorogenic acid (A), chlorogenic acid (B), cryptochlorogenic acid (C), isochlorogenic acid B (D), isochlorogenic acid A (E), isochlorogenic acid C (F), and secoxyloganin (G), and the corresponding characteristic ions and fragmentation pathways.
Figure 4.
The MS/MS spectra of neochlorogenic acid (A), chlorogenic acid (B), cryptochlorogenic acid (C), isochlorogenic acid B (D), isochlorogenic acid A (E), isochlorogenic acid C (F), and secoxyloganin (G), and the corresponding characteristic ions and fragmentation pathways.
Figure 5.
The identification of compounds 14 (A), 30 (B), 52 (C), and 63 (D) according to their MS/MS spectra and the corresponding characteristic fragment ions.
Figure 5.
The identification of compounds 14 (A), 30 (B), 52 (C), and 63 (D) according to their MS/MS spectra and the corresponding characteristic fragment ions.
Table 1.
The identified compounds from L. japonica and L. hypoglauca by UPLC-Q-TOF-MS method.
| No. | tR (min.) | (m/z) | Error (ppm) | Molecular Formula | Belongs | MS/MS Fragment Ions (m/z) | Identification |
|---|---|---|---|---|---|---|---|
| 1 | 0.96 | 191.0544 | −3.1 | C7H12O6 | All | 173.0421, 127.0396, 109.0302 | Citric acid |
| 2 | 1.04 | 191.0553 | −3.6 | C7H12O6 | All | 173.0452, 127.0388 | Quinic acid |
| 3 | 1.31 | 387.1305 | −4.9 | C17H24O10 | All | 341.1096, 179.0579, 119.0388 | Secologanin |
| 4 | 1.61 | 161.0448 | −1.2 | C6H10O5 | All | 101.0170, 57.0362 | Meglutol |
| 5 | 2.46 | 329.0874 | 2.1 | C14H18O9 | All | 167.0344, 152.0089, 108.0231 | Phaseoloidin |
| 6 | 2.54 | 218.1029 | 3.2 | C9H17NO5 | All | 146.0801, 125.0219, 116.0684 | Pantothenic acid |
| 7 c | 3.25 | 353.0882 | 2.5 | C16H18O9 | All | 191.0565, 179.0352, 135.0456 | Neochlorogenic acid |
| 8 | 4.09 | 299.0770 | 1.0 | C13H16O8 | All | 137.0244, 101.0247 | Protamin sulfate- 4-glucoside |
| 9 | 4.11 | 375.1304 | 4.8 | C16H24O10 | All | 213.0779, 179.0509, 169.0840 | Loganic acid |
| 10 | 4.41 | 175.0598 | −1.1 | C7H12O5 | All | 157.0569, 115.0420, 113.0620 | Isopropylmalic acid |
| 11 | 4.61 | 329.0860 | −2.1 | C14H18O9 | All | 167.0356 | Isophaseoloidin |
| 12 c | 4.70 | 353.0872 | −0.2 | C16H18O9 | All | 191.0560, 179.0351, 135.0454 | Chlorogenic acid |
| 13 c | 5.08 | 353.0878 | 1.4 | C16H18O9 | All | 191.0560, 179.0351, 135.0454 | Cryptochlorogenic acid |
| 14 | 5.10 | 389.1090 | 3.0 | C16H22O11 | All | 345.1191, 209.0425, 165.0551 | Secologanoside- 7-methyl ester |
| 15 | 5.15 | 389.1063 | 3.8 | C16H22O11 | All | 345.1205, 209.0440, 183.0642, 165.0545 | Secologanoside |
| 16 | 5.31 | 179.0332 | −3.9 | C9H8O4 | All | 135.0453, 107.0505 | Caffeic acid |
| 17 | 5.34 | 373.1098 | −8.3 | C16H22O10 | All | 193.0492, 179.0563, 149.0586 | Swertiamarine |
| 18 | 6.24 | 187.0976 | 2.6 | C9H16O4 | All | 125.0900, 97.0657 | Azelaic Acid |
| 19 | 6.28 | 337.0922 | −0.2 | C16H18O9 | All | 191.0554, 173.0436, 163.0397 | 5-O-p-coumaroylquinic acid |
| 20 a | 6.51 | 435.1545 | 9.6 | C17H26O10 | All | 227.0935, 165.0539, 101.0252 | Dihydrogen-vogeloside |
| 21 | 6.61 | 153.0176 | −3.9 | C7H6O4 | All | 135.0092, 109.0281 | Protocatechuic acid |
| 22 | 6.85 | 153.0186 | 2.6 | C7H6O4 | All | 135.0028, 109.0261 | Hypogallic acid |
| 23 | 7.24 | 367.1020 | 2.4 | C17H20O9 | All | 193.0503, 191.0550, 173.0439 | Methyl-chlorogenic acid |
| 24 c | 7.81 | 403.1239 | −0.2 | C17H24O11 | All | 371.0983, 223.0636, 179.0606, 165.0566, 121.0303 | Secoxyloganin |
| 25 | 8.29 | 609.1471 | 2.2 | C27H30O16 | All | 301.0343, 300.0247 | Lutin |
| 26 | 8.38 | 595.1269 | −5.0 | C26H28O16 | All | 301.0330, 300.0243 | Quercetin-7-glucoside- rhamnose |
| 27 | 8.43 | 367.1025 | 0.2 | C17H20O9 | All | 191.0562, 173.0446, 1111.0441 | Methyl- cryptochlorogenic acid |
| 28 a | 8.69 | 417.1412 | 3.5 | C18H26O11 | All | 237.0762, 191.0606 | Methyl-secoxyloganin |
| 29 | 9.43 | 463.0872 | −2.1 | C21H20O12 | All | 301.0365, 300.0290, 151.0034 | Quercetin-7-glucoside |
| 30 | 9.68 | 463.0868 | −3.2 | C21H20O12 | All | 301.0365, 300.0290 | Hyperoside |
| 31 | 9.69 | 593.1509 | −0.1 | C27H30O14 | All | 430.0789, 285.0349, 284.0281 | Luteolin-7-glucoside-4′- rhamnose |
| 32 | 9.92 | 447.0961 | 7.3 | C21H20O11 | All | 285.0388 | Luteolin-7-glucoside |
| 33 | 10.10 | 609.1467 | 1.6 | C27H30O16 | All | 315.0522, 314.0450 | Isorhamnetin- 3-rutinoside |
| 34 | 10.11 | 593.1505 | −0.8 | C27H30O14 | All | 285.0401, 284.0312 | Luteolin-7-rutinoside |
| 35 c | 10.61 | 515.1187 | −0.5 | C25H24O12 | All | 353.0885, 335.0793, 191.0561, 179.0356, 173.0458, 135.0457 | Isochlorogenic acid B |
| 36 c | 10.98 | 515.1192 | 0.3 | C25H24O12 | All | 353.0870, 335.0770, 191.0547, 179.0329, 173.0436, 135.0428 | Isochlorogenic acid A |
| 37 | 11.02 | 447.0967 | 8.2 | C21H20O11 | All | 285.0418, 284.0349 | Kaempferol-3-glucoside |
| 38 | 11.42 | 477.1078 | 9.3 | C22H22O12 | All | 314.0441, 271.0264 | Isorhamnetin-7-glucoside |
| 39 | 11.60 | 431.1021 | 9.5 | C25H24O12 | All | 269.0430, 268.0359 | Apigenin-7-glucoside |
| 40 | 11.63 | 193.0518 | 8.8 | C10H10O4 | All | 161.0258, 134.0357, 133.0287 | Isoferulic acid |
| 41 | 11.73 | 193.0493 | −4.1 | C10H10O4 | All | 161.0243, 134.0384 | Ferulic acid |
| 42 c | 12.05 | 515.1188 | −0.3 | C25H24O12 | All | 353.0886, 335.0749, 191.0554, 179.0353, 173.0460, 135.0462 | Isochlorogenic acid C |
| 43 | 12.42 | 537.1596 | −1.1 | C25H30O13 | All | 375.1291, 179.0343, 161.0254 | Grandiforoside |
| 44 a | 12.80 | 499.1287 | 10.0 | C25H24O11 | All | 353.0931, 337.0893, 191.0558 179.0405 | Dehydrogen- isochlorogenic acid A |
| 45 | 12.89 | 491.1249 | 9.6 | C23H24O12 | All | 329.0670 | Tricin-7-glucoside |
| 46 | 13.30 | 529.1350 | −1.7 | C25H24O11 | All | 367.1037, 353.0879, 191.0555, 179.0363, 161.0221 | Methyl- isochlorogenic acid B |
| 47 | 13.81 | 337.0930 | 2.0 | C16H18O9 | All | 191.0560, 173.0453, 163.0400 | 3-O-p-coumaroylquinic acid |
| 48 a | 13.90 | 499.1202 | −7.0 | C25H24O11 | All | 353.0844, 191.0575, 179.0396 | Dehydrogen- isochlorogenic acid B |
| 49 a | 14.24 | 529.1352 | −1.3 | C25H24O11 | All | 367.1027, 353.0862, 191.0549, 179.0337, 173.0435 | Methyl- isochlorogenic acid A |
| 50 | 14.60 | 367.1030 | 1.6 | C17H20O9 | All | 193.0499, 129.0555, 101.0612 | Methyl 4-caffeoylquinate |
| 51 | 15.22 | 529.1329 | −5.6 | C25H24O11 | All | 367.1043, 179.0348, 161.0235 | Methyl- isochlorogenic acid C |
| 52 | 15.98 | 677.1505 | −0.59 | C34H30O15 | All | 515.1188, 353.0865, 173.0418 | 3,4,5-tricaffeoylquinic acid |
| 53 a | 17.15 | 1397.6613 | 1.7 | C65H106O32 | All | 1073.5528, 643.1071 | Dipsacoside B- diglucoside |
| 54 a | 17.77 | 1559.7124 | 0.4 | C71H116O37 | All | 1235.6122, 1073.5249, 652.6374 | Dipsacoside B- triglucoside |
| 55 a | 17.82 | 1543.7128 | −2.5 | C71H116O36 | All | 1381.6549, 1219.6052 | Dipsacoside B-rutinoside-glucoside |
| 56 a,b | 18.01 | 1135.5944 | 4.7 | C53H86O23 | All | 1089.5447, 765.4313, 323.0779 | Hydroxyl-dipsacoside B |
| 57 | 18.25 | 1397.6597 | 0.4 | C65H106O32 | All | 1073.5499 | Isodipsacoside B- diglucoside |
| 58 a | 18.54 | 1235.6060 | 4.7 | C59H96O27 | All | 1073.5509, 911.4954, 527.3396 | Dipsacoside B-glucoside |
| 59 | 18.70 | 1235.6084 | 5.8 | C59H96O27 | All | 911.5006, 749.4477 | Isodipsacoside-B- glucoside |
| 60 a | 18.98 | 327.2184 | 3.6 | C18H32O5 | All | 229.1477, 221.1116, 211.1333 | Dehydroxypinellic acid |
| 61 a,b | 19.00 | 1105.5577 | 2.1 | C52H84O22 | All | 1059.5310, 889.1119, 735.4228, 469.7788, 323.0942 | Demethyl-dipsacoside B |
| 62 | 19.03 | 329.0658 | 0.6 | C17H14O7 | All | 167.0344, 152.0089 | Tricin |
| 63 b | 19.16 | 1119.5466 | 0.3 | C53H86O22 | All | 1073.5531, 749.4500, 323.0951 | Dipsacoside B |
| 64 b | 19.25 | 1119.5545 | 8.7 | C53H86O22 | All | 1073.5517, 911.5158, 749.4457, 323.0971 | Macranthoside B |
| 65 a | 19.30 | 1395.6466 | 2.3 | C65H104O32 | All | 1071.5376 | Dehydrogen- dipsacoside B-maltose |
| 66 | 19.38 | 1119.5559 | 9.3 | C53H86O22 | All | 1073.5501, 911.5094, 749.4431, 323.1027 | Isodipsacoside B |
| 67 a | 20.55 | 1247.7241 | 5.6 | C65H106O32 | All | 1381.6554, 1238.1004, 1057.5537 | Dipsacoside B-rutinoside |
| 68 a | 20.73 | 329.2340 | 3.6 | C18H34O5 | All | 229.1538, 171.1070 | Pinellic acid |
| 69 a | 20.82 | 1381.6681 | 2.9 | C65H106O32 | All | 1057.5602 | Dipsacoside B- glucoside-rhamnose |
| 70 a | 21.23 | 1219.6150 | 3.1 | C59H96O26 | All | 895.5042, 733.4157, 517.0624 | Dipsacoside B-rhamnose |
| 71 a | 22.13 | 1103.5547 | −3.4 | C52H82O22 | All | 1057.5556, 733.4597, 323.0945 | Demethyl-dehydrogen- dipsacoside B |
| 72 a | 25.73 | 911.4995 | −1.0 | C47H76O17 | All | 749.4421, 603.3780 | Deglucoside- dipsacoside B |
a indicates the compound was reported for the first time from L. japonica and L. hypoglauca; b indicates the [M − H + HCOO]−; c indicates the compound was unambiguously determined by comparing the tR, MS, and MS/MS with the references.
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Wu, Q.; Zhao, D.; Leng, Y.; Chen, C.; Xiao, K.; Wu, Z.; Chen, F. Identification of the Hypoglycemic Active Components of Lonicera japonica Thunb. and Lonicera hypoglauca Miq. by UPLC-Q-TOF-MS. Molecules 2024, 29, 4848. https://doi.org/10.3390/molecules29204848
AMA Style
Wu Q, Zhao D, Leng Y, Chen C, Xiao K, Wu Z, Chen F. Identification of the Hypoglycemic Active Components of Lonicera japonica Thunb. and Lonicera hypoglauca Miq. by UPLC-Q-TOF-MS. Molecules. 2024; 29(20):4848. https://doi.org/10.3390/molecules29204848
Chicago/Turabian StyleWu, Qinxuan, Di Zhao, Ying Leng, Canhui Chen, Kunyu Xiao, Zhaoquan Wu, and Fengming Chen. 2024. "Identification of the Hypoglycemic Active Components of Lonicera japonica Thunb. and Lonicera hypoglauca Miq. by UPLC-Q-TOF-MS" Molecules 29, no. 20: 4848. https://doi.org/10.3390/molecules29204848
APA StyleWu, Q., Zhao, D., Leng, Y., Chen, C., Xiao, K., Wu, Z., & Chen, F. (2024). Identification of the Hypoglycemic Active Components of Lonicera japonica Thunb. and Lonicera hypoglauca Miq. by UPLC-Q-TOF-MS. Molecules, 29(20), 4848. https://doi.org/10.3390/molecules29204848
