An UPLC-MS/MS application to investigate chemical compositions in the ethanol extract with hypoglycemic activity from Zingiber striolatum Diels                         
Tianhong Chen, Jinyan Cai*, Jun Ni, Fan Yang* 
School of Pharmacy, Guangdong Pharmaceutical University, Guangzhou 510006, China   
Abstract: Zingiber striolatum Diels (Zingiberaceae) is an edible plant resource in the Chinese folk with special efficacy in relieving diabetes and constipation, whichhas been documented in the Compendium of Materia Medica. However, its hypoglycemic activity and constituents have not been reported yet. In the present study, we evaluated the hypoglycemic activity of Z. striolatum in insulin-resistant HepG2 cells, and we developed ultra performance liquid chromatography coupled with tandem mass spectrometry (UPLC-MS/MS) based chemical profiling method for rapid analysis of Z. striolatum. As a result, the ethanol extract from Z. striolatum showed significant hypoglycemic activity in HepG2 cells, and 22 flavonoids compounds were tentatively characterizedby comparing the retention time and mass spectrometry data. In conclusion, the method of hypoglycemic screening in insulin-resistantHepG2 cells coupled with UPLC-MS/MS is a feasible and credible technique to separate and identify the active constituents in complex matrices of traditional Chinese medicine.        
Keywords: Hypoglycemic activity, HepG2 cells, UPLC-MS/MS, Zingiber striolatum Diels
CLC number: R284                Document code: A                 Article ID: 10031057(2016)211606
1. Introduction
Z. striolatum iswidely distributed in many provinces of China, such as Yunnan, Hubei, Hunan, Jiangxi and Guangdong, and it is of high nutritional value and contains high levels of protein, amino acids and dietary fiber polysaccharides. Asan edible plant resource in Chinese folk with medicinal properties, Z. striolatum is often used in relieving diabetes and constipation according to Compendium of Materia Medica. Moreover, it has been reported that Z. striolatum is effective in promoting blood circulation, subsidence of swelling, relieving cough, reducing sputum and aiding digestion[1]. In the past decade, there has been a growing interest in the use of traditional Chinese medicine (TCM) due to the increasing awareness of their health benefits. TCM is usually composed of multi-components responsible for their efficacies[2]. The conventional approach includes isolation of individual compounds from the complex mixture and structure elucidation by nuclear magnetic resonance (NMR), MS and other spectroscopic techniques[3]. Nevertheless, the process is time-consuming, and the isolation and purification are rather difficult in many cases[3]. Therefore, there is an urgent need to establish a feasible and sensitive method for full-scale qualitative analysis of the major bioactive constituents of TCM. Recently, there is a boost in the use of the LC-MS technique because of its superior sensitivity, selectivity and the ability to conclusively identify the compounds[4]. Moreover, ultra performance liquid chromatography coupled with tandem mass spectrometry (UPLC-MS/MS) has been proved to be an efficient tool for the rapid analysis of known compounds and elucidation of unknown compounds in complex matrix, and such a technique has therefore become an importantanalytical tool in TCM research[5,6]. It can provide accurate mass and formulae of the compounds, and distinguish the isobaric compounds according to the different molecular formulae[7]. Furthermore, the fragmentation behavior of some natural compounds has been extensively investigated and summarized[8]. By this method, the unknown compounds can be identified without the reference articles[3].
Type 2 diabetes is characterized by insulin resistance of internal organs and peripheral tissues, leading to impaired glucose utilization and abnormally high bloodglucose levels between and especially after meals[9]. It has been estimated that the number of adults affected by diabetes will grow from 135 million in 1995 to 300 million in 2025 worldwide[10,11]. Diabetes is the sixth leading cause of death due to disease in the USA, and it is the third leading cause among some ethnic populations[12]. Therefore, it is of great importance to explore more effective and safer anti-hyperglycemic drugs and provide important information regarding treatment for diabetes. China has a long history of using herbs for the treatment of human diseases, including diabetes, and Z. striolatumis one of those herbs with many pharmacological activities. In vitro, HepG2 cells are usually used to measure the hypoglycemic activity of drugs as food is primarily metabolized in liver[13]. In the present study, HepG2 cells were selected due to their common physiological function to lipid or glucose metabolism with normal hepatic cells[14].
2. Materials and methods
2.1. Reagents
Methanol of HPLC grade was purchased fromTianjinKermel LaboratoryEquipment Co., Ltd. Water used in the present study was purified by a Simplicity® 185 personal water purification system (Millipore, Bedford, MA, USA). All other organic solvents used in thestudywere of analytical grade.
HepG2 cells were supplied by College of Life Sciences, Guangdong Pharmaceutical University, insulin and MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) were obtained from Sigma-Aldrich Co., LLC. (San Francisco, USA), DMEM high glucose medium, FBS (fetal bovine serum), phosphate buffered saline and penicillin-streptomycin were provided by Hyclone (Utah, USA), 0.25% trypsin was purchased from Gibco (California, USA), and the glucose assay kits were obtained from ZhongSheng Bai Kong Biotechnology Co., Ltd. (Shenzhen, China). 
2.2. Bioassay of hypoglycemic activity in insulin-resistant HepG2 cells
The HepG2 cells were cultured in high glucose medium supplemented with 10% FBS and 1% penicillin-streptomycin at 37 ºC in a humidified incubator in a 5% CO2 atmosphere until the cells covered 80% of cell culture flasks. Insulin resistance was induced in HepG2 cells with mixed medium (1% FBS) containing a high concentration of insulin for 24 h. The cells were cultured in the DMEM high glucose medium with 1% FBS for 24 h containing different concentrations of Z. striolatum extract. Glucose consumption (GC) was determined at a wavelength of 505 nm using a glucose enzymatic kit by an automatic biochemical analyzer. The medium was replaced with MTT for 4 h. The absorbance (OD) of the cell lysates was determined at 490 nm by Multiskan Spectrum[15].
2.3. Plant materials and sample preparation
The herbs of Z. striolatum were collected in August of 2013 from WenShan, Yunnan Province, China. The plant was authenticated by Hongyan Ma, an associate professor in College of Traditional Chinese Medicine, Guangdong Pharmaceutical University. A voucher specimen (2012-YH1001)has been deposited in the herbarium of School of Pharmacy, Guangdong Pharmaceutical University.
Dry powdered herbs (1.2 kg) of Z. striolatum were refluxed with 95% (v/v) ethanol at 60 ºC for 8 h and extracted twice. Each filtrate was concentrated to dryness by vacuum to render the total ethanol extract, which was dissolved in DMSO or methanol, and it was passed through a 0.22 μm membrane filter prior to further analysis.
2.4. Instrument conditions
ACQUITY UPLC system, equipped with a quaternary solvent delivery system, an auto-sample and a column compartment, was used in the present study. The chromatographic separation was achieved on an ACQUITY UPLCTM BEH C18 column (2.1 mm×50 mm, 1.7 μm).Extract was dissolved in methanol, and the solution was filtered through 0.22 μm membranes (pore size). A 5 μL aliquot was injected for analysis.The mobile phase consisted of methanol and water. The UPLC separation condition was optimized using different gradient programs. Finally, the selected elution gradient wasas follows: 5% methanol in 0–12 min, 10% methanol in 12–14 min, 30% methanol in 14–22 min, 40% methanol in 22–26 min, 60% methanol in 26–32 min, 70% methanol in 32–41 min, 80% methanol in 41–46 min, 85% methanol in 46–51 min, 100% methanol in 51–60 min. The flow rate was set at 0.4 mL/min.
Mass spectrometry was performed on the Triple TOFTM 5600 (AB SCIEX, Foster City, CA), a hybrid triple quadrupole time-of-flight mass spectrometer equippedwith ESI source, and mass range was set at m/z 100–1200. The conditions of MS/MS detector were as follows: ion spray voltage of 1500 V; ion source gas of 150 psi; ion source gas of 260 psi; temperature of 550 ºC; curtain gas of 15 psi; collision gas pressure of 8 psi; and entrance potential of 10 V. Nitrogen was used as nebulizer and auxiliary gas.
2.5. Statistics
Data were presented as the means±SD. P value of less than 0.05 was considered as statistically significant.
3. Results and discussion
3.1. Hypoglycemic activity in vitro
Table 1 shows that the GC and MTT of some tested groups had significant differences compared with the model group (P<0.05 or P<0.01). The GC was increased along with the extract concentration when it was in the range of 1.5625–100.00 g/L. The highest GC with a simultaneous maximum survival of cells appeared at the concentration of 100.00 g/L, the GC was 4.15, and the GC/MTT was 2.37. However, there was a sharp decrease of both GC and the number of survived cells when the extractconcentration was 200.00 g/L. The GC was decreased from 4.15 to 2.62, the GC/MTT was decreased from 2.37 to 1.69, and either of them were lower compared with the group with an extract concentration of 100.00 g/L,demonstratingthat high concentration of the extract, such as 200.00 g/L, had a certain inhibitory effect. Therefore, the extract showed a dose-dependent hypoglycemic effect on insulin-resistant HepG2 cells to some extent. 

Table 1. Hypoglycemic activity in HepG2 cells

Data were expressed as mean±SD (
n = 8), analyzed by IBM SPSS Statistics 19.0. *P<0.05 compared with the model group. **P<0.01 compared with the model group.
3.2. UPLC-MS/MS analysis
Figure 1 shows the total ion chromatograms of the crude extract from Z. striolatum in negative ESI mode. The separation of compositions was completed, and all peaks were well separated from each other within 60 min. Table 2 shows 22 types of flavonoids in crude extract of Z. striolatum by UPLC-MS/MS. And Figure 2 shows the structures of quercetin and kaempferol.   

Table 2. Characterization of flavonoids in the extract of Z. striolatum by UPLC-MS/MS

tR, retention time; Q, quercetin; MQ, methyl-quercetin; K, kaempferol; MK, methyl-kaempferol; Hex, hexose; Pen, pentose; dHx, deoxyhexose.

Figure 1.
Total ion chromatograms of a crude sample from Z. striolatumin negative ESI mode.

Figure 2.
Structures of quercetin and kaempferol. 

    Free aglycone quercetin (peak 20) at the retention time (
tR) of 45.168 min was detected with its [M-H]ion at m/z 301 in negative ESI mode. Its MS2 spectrum gave characteristic fragments at m/z 107, 121, 151, 179, 255 and 271. Peak 21, at the tR of 46.486 min, gave a deprotonated ion at m/z 315, and its MS2 spectrum showed the aglycone ion at m/z 301 as a base peak, suggesting the presence of a methyl group. Therefore, it was identified as methyl-quercetin. [M-H] 329.0298 consisted of a methyl group and methyl-quercetin from its MS2 spectrum, and fragments at m/z 315 and 301 could be observed in its MS2 spectrum (peak 8 at the tR of 28.284 min). The dimethyl-quercetin and another methyl formed the trimethyl-quercetin (343.0805), and fragments at m/z 329, 315 and 301 demonstrated its existence (peak 11 at the tR of 35.246 min). Peak 16 at the tR of 40.017 min displayed a [M-H] ion at m/z 615. Its MS2 spectrum showed that the fragment ion at m/z 463 was produced by loss of a galloyl group (152 Da). In addition, the characteristic fragment ions of quercetin at m/z 301, 255 and 151 could be observed. Therefore, it was identified as quercetin-O-galloyl-hexoside. Peak 2 at the tR of 17.070 min gave a [M-H] ion at m/z 767, fragment at m/z 615 was originated from the loss of a galloyl group (152 Da), and the other product ions were similar to peak 16. Therefore, peak 2 was identifiedas quercetin-di-galloyl-hexoside. Peak 1 at the tR of 16.881 min and peak 7 at the tR of 28.082 min displayed [M-H] ions at m/z 609 and 651, respectively, and their MS2 spectra showed theaglycone ion at m/z 301 as a base peak, suggesting the presenceof deoxyhexosyl-hexosyl (308 Da) andacetyl-deoxyhexosyl-hexosyl (350 Da) groups, respectively. Therefore, they were identified as quercetin-3-O-deoxyhexosyl-hexoside andquercetin-3-O-acetyl-deoxyhexosyl-hexoside, respectively. Peak 10 at the tR of 32.537 displayed a [M-H] ion at m/z 587, and the product ion at m/z 301 could be observed in its MS2 spectrum. Therefore, it was identifiedas quercetin-galloyl derivative. Peak 15 yielded a [M-H] ion at m/z 433, and the fragment at m/z 301 in its MS2 spectrum showed the existence of pentose. Therefore, it was identified as quercetin-O-pentoside. Peak 13 yielded a [M-H] ion at m/z 585, which was originated from quercetin, and it easily lost the galloyl-pentosyl unit (284 Da) and gave a distinctive product ion at m/z 301.Therefore, it was identified as quercetin-O-galloyl-pentoside.
At the tR of 47.527 min, kaempferol was identified as its [M-H]ion at m/z 285, and characteristic fragments at m/z 151, 227, 255 and 271 were observed. Peak 4 displayed a [M-H]ion at m/z 593, and its MS2 spectrum showed the aglycone ion at m/z 285 as a base peak, suggesting the presence of deoxyhexosyl-hexosyl (308 Da). Therefore, it was identified as kaempferol-deoxyhexosyl-hexoside. Peak 12 gave a [M-H]ion at m/z 417. In its MS2 spectrum, the ion at m/z 285 was a base peak, which was originated from the loss of pentosyl (132 Da).Therefore, it was identified as kaempferol-3-O-pentoside. Peak 6 and peak 9 displayed [M-H]ions at m/z 635 and 431, respectively. Their MS2 spectra showed the aglycone ion at m/z 285 as a base peak, suggesting the presence of acetyl-deoxyhexosyl-hexosyl (350 Da) and deoxyhexosyl (146 Da) groups, respectively. Therefore, they were identified as kaempferolacetyl-deoxyhexosyl-hexoside and kaempferol-deoxyhexoside, respectively. Peak 3, peak 19 and peak 18 were all originated from kaempferol, and they easily lost galloyl sugar units and gave a distinctive product ion at m/z 285. Peak 3 gave a [M-H]ion at m/z 599, and the product ion at m/z 285 was originated from the loss of 314 Da, suggesting that the unit consisted of a hexose and a gallic acid. Therefore, peak 3 was identified as kaempferol-O-galloyl-hexoside. Peak 19 displayed a [M-H]ion at m/z 751, and the product ion at m/z 285 was originated from the loss of 466 Da, indicating the existence of a hexose and two gallic acids. Therefore, it was identified as kaempferol-di-O-galloyl-hexoside. Peak 18 gave a deprotonated ion at m/z 569, and its MS2 spectrum showed the loss of 284 Da, corresponding to a galloyl-pentosyl group. Therefore, it was identified as kaempferol-O-galloyl-pentoside. Peak 5 displayed a [M-H]ion at m/z 287 and gave fragments at m/z 273, 257 and 229. Therefore, it was identified as dihydrokaempferol. Peak 14 displayed a [M-H] ion at m/z 271, which was 16 Da less than that of dihydrokaempferol, indicating that peak 14 was formed by the loss of a hydroxyl from peak 5. Therefore, it was identified as 7,4'-dihydroxyflavanone. Peak 17 was a type of flavone, which was different from others. It displayed a [M-H]ion at m/z 253, and its fragments at m/z 225 and 193 showed the loss of H2O and CO in succession. Therefore, it was identified as dihydroxy flavonids.
4. Conclusions
In the present study, we, for the first time, established a simple, effective and rapid UPLC-MS/MS method to compressively analyze flavonoids in Z. striolatum, and the hypoglycemic activity of its extract was tested in insulin-resistant HepG2 cells. Systematic analysis of compounds in Z. striolatumhas not been reported yet. As a type of promising plant resource, Z. striolatum has the concomitant function of both medicine and foodstuff for diabetic treatment. Therefore, our work might play a guiding role in the future studies. In conclusion, the method of hypoglycemic screening in insulin-resistant HepG2 cells coupled with UPLC-MS/MS is of great importance for the investigation and primary prediction of natural products before traditional isolation. 
This work was financially supported by Pearl River S&T Nova Program of Guangzhou (Grant No. 201506010061), Foundation for Distinguished Young Teachers in Higher Education of Guangdong, and specialfunds for cultivating Guangdong college students’ scientific and technological innovation (“Climbing plan”, Grant No. pdjh2015b0277).
[1] Chen, S.X.; Yang, J.; Tang, H.;Yang, Z.Z.; Wang, X.; Hu, M.N. J. Sci. Technol. Ind. 2013, 34, 266–269.
[2] Ling, Y.; Liu, K.; Zhang, Q.; Liao, L.; Lu, Y. J. Pharm. Biomed. Anal.2014, 98, 120–129.
[3] Gu, D.; Yang, Y.; Bakri, M.; Chen, Q.; Xin, X.; Aisa, H.A.Phytochem. Anal.2013, 24, 661–670.
[4] Sun, H.; Dong, W.; Zhang, A.; Wang, W.; Wang, X. J. Sep. Sci.2012, 35, 3477–3485.
[5] Dong, H.; Zhang, A.; Sun, H.; Wang, H.; Lu, X.; Wang, M.; Ni, B.; Wang, X. Mol. Biosyst.2012, 8, 1206–1221.
[6] Wang, X.; Yang, B.; Zhang, A.; Sun, H.; Yan, G. J. Proteomics.2012, 75, 1411–1427.
[7] Zheng, X.; Shi, P.; Cheng, Y.; Qu, H. J. Chromatogr. A.2008, 1206, 140–146.
[8] Vukics, V.; Guttman, A. Mass Spectrom. Rev.2010, 29,1–16.
[9] van Dijk, J.W.; Manders, R.J.; Hartgens, F.; Stehouwer, C.D.; Praet, S.F.; van Loon, L.J. Diabetes. Res. Clin. Pract.2011, 93, 31–37.
[10] Benson, V.S.; Vanleeuwen, J.A.; Taylor, J.; Somers, G.S.; McKinney, P.A.; Van Til, L. J. Am. Coll. Nutr.2010, 29, 612–624.
[11] King, H.; Aubert, R.E.; Herman, W.H. Diabetes. Care.1998, 21, 1414–1431.
[12] Buchanan, T.A.; Xiang, A.H.; Peters, R.K.; Kjos, S.L.; Marroquin, A.; Goico, J.; Ochoa, C.; Tan, S.; Berkowitz, K.; Hodis, H.N.; Azen, S.P. Diabetes.2002, 51, 2796–2803.
[13] Xie, W.; Wang, W.; Su, H.; Xing, D.; Pan, Y.; Du, L. Comp. Biochem. Physiol. C. Toxicol. Pharmacol.2006, 143, 429–435.
[14] Xu, J.; Ma, M.; Purcell, W.M. Toxicol. Appl. Pharmacol.2003, 189, 100–111.
[15] Zhu, X.K.; Ye, X.L.; Song, L; Luo, Y.H.; Tang, Q.; Jin, Y.N.; Li, X.G. J.Med. Chem. Res. 2013, 22, 2228–2234.

陈天洪,蔡金艳*, 倪俊,杨帆*
广东药学院药科学院, 广东广州510006                
摘要: 阳荷(姜科)是一种药食同源的植物,据《本草纲目》记载,阳荷对治疗糖尿病和便秘有特效,但其降血糖活性和化学成分的系统分析还未见报道。在本研究中,将阳荷粗提物作用于胰岛素抵抗的HepG2细胞,用葡萄糖试剂盒检测其降血糖活性, MTT法检测细胞活力,并应用超高效液相色谱串联质谱法(UPLC-MS/MS)来快速分析阳荷中的化学成分。结果显示阳荷粗提物在HepG2细胞中具有显著的降血糖活性,并通过比较保留时间、质谱数据及参考文献推定出22个黄酮类化合物。结果表明这是一种可行且可靠的分离和鉴定中药活性成分的方法。 
关键词: 降血糖活性; HepG2细胞; UPLC-MS/MS; 阳荷

Received: 2015-09-17, Revised: 2015-11-11, Accepted: 2015-11-18.
Foundation items: Pearl River S&T Nova Program of Guangzhou (Grant No. 201506010061), Foundation for Distinguished Young Teachers in Higher Education of Guangdong, and special funds for cultivating Guangdong college students’ scientific and technological innovation (“Climbing plan”, Grant No. pdjh2015b0277).
*Corresponding author. Tel.: +8615920107845, +8613922135439, Fax: +862039352129, E-mail:,