Evidence-Based Complementary and Alternative Medicine
Volume 2019 (2019), Article ID 9414926, 14 pages
https://doi.org/10.1155/2019/9414926

Research Article

Qualitative and Quantitative Analysis for the Chemical Constituents of Tetrastigma hemsleyanum Diels et Gilg Using Ultra-High Performance Liquid Chromatography/Hybrid Quadrupole-Orbitrap Mass Spectrometry and Preliminary Screening for Anti-Influenza Virus Components

FuJuan Ding,¹ JiangTing Liu,¹ RuiKun Du,1,2 QinHui Yu,¹ LiLi Gong,1,2 HaiQiang Jiang,1,2 and Rong Rong1,2,3

¹School of Pharmacy, Shandong University of Traditional Chinese Medicine, Jinan, Shandong, China
²Collaborative Innovation Center for Antiviral Traditional Chinese Medicine, Shandong Province, China
³Key Lab for Natural Product, Shandong Province, China

Correspondence should be addressed to HaiQiang Jiang; jhq12723@163.com and Rong Rong; rosierong@163.com

Received 19 September 2018; Revised 28 November 2018; Accepted 6 December 2018; Published 18 February 2019

Academic Editor: Vincenzo De Feo

Copyright © 2019 FuJuan Ding et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract.

Tetrastigma hemsleyanum Diels et Gilg (T. hemsleyanums) is a kind of traditional folk medicinal plant which has been used widely in China for its antivirus, antitumor, and other clinical effects. In this study, ultra-high performance liquid chromatography coupled with hybrid quadrupole-orbitrap mass spectrometry (UPLC-Q-Exactive/MS) was utilized to analyze the chemical constituents of T. hemsleyanums. Fifty-one constituents were clarified, including flavonoids, anthraquinones, esters, fatty acids, phenols, and catechins. In the subsequent quantitative analysis, the contents of ten compounds of rutin, kaempferol, astragalin, quercitrin, quercetin, vitexin-rhamnoside, isorhamnetin, vitexin, emodin-8-O-β-D-glucoside, and isoquercetin in 18 batches of T. hemsleyanums collected from different places of cultivation were determined. Meanwhile, anti-influenza virus bioactivity in vitro of the above samples was detected with Gaussia Luciferase viral titer assay. It was found that the antiviral bioactivity varied from batches to batches in accordance with content difference of the chemical constituents in T. hemsleyanums. Correlation analysis was performed with SPSS software for the association between LC-MS chemometrics and bioactivity of influenza virus inhibition, and 8 constituents of flavonoids showed positive correlation coefficient, which may provide a valuable clue for searching potential antiviral components in T. hemsleyanums.

1. Introduction

Tetrastigma hemsleyanum Diels et Gilg (abbreviated. as T. hemsleyanums) is a kind of medicinal and edible plant wildly growing in south China, especially in Zhejiang, Guangxi, and Yunnan provinces [1]. It was firstly recorded in Zhong Hua Ben Cao [2] for its effects of clearing away heat, detoxifying, removing phlegm, promoting blood circulation, and relieving pain. Nowadays, it is used in Traditional Chinese Medicine (TCM) clinics for the treatment of children with high fever (convulsions), viral meningitis, pneumonia, and hepatitis. Many literatures confirmed that T. hemsleyanums belongs to the folk medicine; its root can be used to treat children’s wind-heat with water decoction which is recorded in local standardization, such as “Guangxi min jian chang yong shou yi cao yao” [3], “Kunming min jian chang yong cao yao” [4], and “Zhejiang min jian chang yong cao yao” [5]. The modern pharmacological studies had shown that it had effects of anti-inflammatory, analgesic, antipyretic, antivirus, antitumor [6–8], immunomodulatory [9], liver protection, etc.

In recent years researchers have paid more and more attention to flavonoids and anthraquinones which wildly exist in Chinese herbal medicine because of their outstanding bioactivities. Modern phytochemical studies have shown that flavonoids are the major active constituents in T. hemsleyanums [10]. Out of them, vitexin, isoquercetin, and astragalin showed the anticancer effect [11–13], quercitrin could improve the impaired mesenteric vascular activity in the colitis models [14], quercetin could yield an obvious mitigation of arthritic manifestations [15], kaempferol had effects of preventing and reversing ventricular fibrosis and cardiac dysfunction in vivo and in vitro experiments [16], and rutin played a therapeutic role in diabetic atherosclerosis through inhibiting premature senescence of vascular smooth muscle cells [17]. Because of their prominent pharmacological activities, determination for the contents of the above compounds in T. hemsleyanums and also the LC-MS characterizations of the plant are required for further quality control study.

It was reported recently that UPLC-Triple-TOF/MS had been applied for the qualitative analysis of the chemical compositions in T. hemsleyanums, and most of them were identified as flavonoids, esters, and benzene sulfonic acids [18]. UPLC-Q-Exactive/MS is a more powerful and sensitive analytical instrument for detecting constituents even in low abundance in complex plant extracts [19]. In this present study we use UPLC-Q-Exactive/MS technique firstly to analyze the components of T. hemsleyanums collected from different cultivation places in China. The data generated from UPLC-Q-Exactive/MS have distinguished the phytochemical differences among the 18 batches both in types of ingredients and in contents. Various factors, such as cultivation places, harvest season, and postharvest treatment, are responsible for the variation in contents of phytochemicals and hence are due to the variation of many bioactivities.

We performed the antiviral determination assay using recombinant influenza virus PR8-NS1-Gluc to evaluate the anti-influenza virus bioactivity of the extracts from T. hemsleyanums. It is a high throughput screening protocol to identify entry inhibitors for influenza virus using a human immunodeficiency virus-based pseudotyping platform which can be performed in a BSL-2 facility. Conventional methods of active compound discovery from natural products involve bioactivity-guided isolation, which is laborious, costly, or impossible to source. In addition, the effects of underlying constituents with potent bioactivity present at very low or sometimes undetectable levels may be ignored. In this study, we process the data with SPSS correlation analysis to evaluate the association between chemometrics and bioactivities of the 18 batches of T. hemsleyanums from different districts. Content of 8 constituents of flavonoids showed positive correlation coefficient with anti-H1N1 influenza virus activity, which may facilitate the identification of potential antiviral components in T. hemsleyanums.

2. Materials and Methods

2.1. Chemicals and Plant Materials

HPLC-grade acetonitrile and formic acid were purchased from Thermo Fisher Scientific Company (USA). Ultra-pure water was purchased from Watsons Company (China). All other reagents were of analytical grade.

The reference standards such as kaempferol, isoquercetin, astragalin, rutin, quercetin, baicalein, and palmitic acid were purchased from Herbpurify Co., Ltd. (Chengdu, China); emodin-8-O-β-D-glucoside, quercitrin, vitexin, and vitexin-rhamnoside were purchased from Yuanye Bio-Technology Co., Ltd. (Shanghai, China); isorhamnetin, catechins and epicatechin were purchased from Institutes for Food and Drug control (Beijing, China).

The plant materials of T. hemsleyanums were collected from different provinces in China, as Zhejiang (ZJ1, ZJ2, and ZJ3), Guangxi (GX1, GX2, and GX3), Yunnan (YN1, YN2, and YN3), Fujian (FJ1, FJ2, and FJ3), Guizhou (GZ1, GZ2, and GZ3), and Hubei (HB1, HB2, and HB3). The botanical authentication was performed by Professor Lingchuan Xu from Department of Pharmacognosy, Shandong University of Traditional Chinese Medicine. A voucher specimen (no. TH20171201~TH20181118) was deposited in Key Lab for Natural Product, Shandong Province. The dried rhizome parts of T. hemsleyanums were used for further analysis.

2.2. Liquid Chromatography and Mass Spectrometry Analysis

In this study we employed a UPLC system tandem Q-Exactive/MS spectrometer (Thermo Fisher, CA, USA) equipped with a heated electrospray ionization (HESI) probe. Halo C18 (2.7 μm, 100 × 2.1 mm, Advanced materials technology, USA) column was used at the flow rate of 0.3 mL/min and column temperature of 30°C. The binary solvent system consisted of 0.05% aqueous formic acid (v/v) (A) and 0.05% formic acid in acetonitrile (B). Samples were eluted with the following linear gradients: 15% B at 0–15 min; 15–40% B at 15–22 min; 40–90% B at 22–50 min; 90–15% B at 50–60 min. Injection volume was 3 μL. Detection was performed using a Q-Exactive™ hybrid quadrupole-Orbitrap mass spectrometer in both positive and negative ionization modes. The optimal analysis conditions were set as follows: ion source, heated electrospray ionization probe; source temperature: 350°C; capillary temperature: 320°C; sheath gas: 45 arb; auxiliary gas: 10 arb; mass collecting range: m/z 100-1500. The full scan and fragment spectra were collected at the resolutions of 70000 and 17500, respectively. The collision energy was 30 eV, 50 eV, and 70 eV at negative mode and 10 eV, 30 eV, and 50 eV at positive mode.

2.3. Sample Preparation

The dried rhizome parts of T. hemsleyanums were ground to powder. For each sample, 15 g of rhizome powder was extracted with 75% ethanol reflux for 3 times, 1 h each time. The ethanol extracts were concentrated under reduced pressure evaporated to dryness and then dissolved with 50% acetonitrile of 25.0 mL as the stock sample solution. 1.0 mL of the above stock sample solution, adding baicalein with the final concentration of 4.781 μg/mL as the internal standard (IS), was filtered through a 0.22 μm syringe filter to obtain sample solution for qualitative and quantitative analysis. 20.0 mL of the stock sample solution was freeze-dried in vacuum to obtain a sort of powder extract for antiviral bioactivity testing.

2.4. Preparation of Standard Solutions

Rutin, kaempferol, astragalin, quercitrin, quercetin, vitexin-rhamnoside, isorhamnetin, vitexin, emodin-8-O-β-D-glucoside, and isoquercetin were dissolved in HPLC-grade acetonitrile to achieve standard stock solution with the concentration of 1.0 mg/mL and serially diluted to mixed working solution with 50% acetonitrile; baicalein was used as IS with concentration of 4.781 μg/mL in working solution. All the stock and working solutions were stored at 4°C.

2.5. Identification of the Constituents

LC-MS data were acquired in both negative and positive ion modes and processed for target compounds identification using a combination of Xcalibur and Mass Frontier software packages (Thermo Scientific).

2.6. Validation of Quantitative Method

10 constituents of flavonoids were chosen for the content measurement. Internal standard method was used for quantitation. Peak area ratios of the analyte against IS were used for calculations and a weighted (1/concentration) regression analysis was used for standard curves preparation. Limit of detection (LOD) and limit of quantification (LOQ) for each constituent were detected. The intraday and interday precision of the determination for ten constituents were validated. Stability of sample solutions within 24 hours at 4°C was tested. Six replicates of samples were prepared to check the repeatability. The recoveries were analyzed by adding the analytical reference standards in the powder of T. hemsleyanums prior the extraction procedure and assessed at three levels of the amount which measured in analyte to standards added (1:0.8, 1:1, 1:1.2), using six replicates at each level.

2.7. Quantitative Analysis for Ten Compounds of Flavonoids

Contents measurement of ten compounds of flavonoids in 18 batches of T. hemsleyanums was performed under the condition of Section 2.2.

2.8. Anti-H1N1 Influenza Viral Determination

The antiviral determination assay was performed using recombinant influenza virus PR8-NS1-Gluc as previously described [20]. Briefly, MDCK cells grown in 24 well plates were inoculated with recombinant influenza virus PR8-NS1-Gluc at an moi of 0.01 PFU/cell. After incubation for 1 h at 37°C, virus inoclula were removed and cells were washed. Opti-MEM containing 2 μg/mL TPCK-trypsin was then added for virus propagation. At 36 hours after infection (hpi), 50 μl of culture medium was removed for luciferase assay using BioLux Gaussia Luciferase Assay kit (NEB, USA) according to the manufacturer’s instructions. For antiviral activity determination, extracts of T. hemsleyanums were added at indicated concentrations as shown in Figure 5 during virus propagation.

2.9. Statistics

In order to study the association between the chemical spectra and the anti-influenza viral bioactivity, correlation analysis was performed using the SPSS 17.0 software. The two variables in the correlation analysis were the ion intensity and the inhibitory effect against H1N1 influenza virus. All statistical analyses were two sided. p<0.05 was considered to be statistically significant.

3. Results and Discussion

3.1. Qualitative Analysis

Sample solution of T. hemsleyanums collected from Guangxi province was chosen for qualitative analysis. UPLC-Q-Exactive/MS base peak chromatograms of T. hemsleyanums were shown in Figure 1, and the identification results in negative ion mode and positive ion mode of mass spectrometry were shown in Tables 1 and 2. As shown in the tables, fifty-one compounds were clarified including flavonoids, anthraquinones, esters, fatty acids, phenols, and catechins according to accurate molecular weight calculation, ion fragmentation information, and some of them with confirmation of reference standards.

Table 1: Identification analysis of chemical constituents of T. hemsleyanums in negative ion mode of mass spectrometry.

No.
tR (min)
Name
Formula
Detected m/z
Expected m/z
M - X
Error (ppm)
Fragmentor information

1
0.9
Procyanidin dimmer

577.1334
577.1341
M – H
-0.0007
125.0228, 151.0386

161.0230, 203.0706

289.0716, 407.0767

2
1.15
Neochlorogenic acid

353.0331
353.0867
M – H
-0.0536
135.0434, 161.0591

173.0598, 179.0337

191.0553

3
1.49
Chlorogenic acid

353.0337
353.0867
M – H
-0.0530
135.0436, 161.0231

173.0443,191.0549

4
1.5
Protocatechuic acid glucoside

315.0717
315.0711
M – H
0.0006
108.0200, 109.0279

152.0100, 153.0179

5
1.52
Gallic acid

169.0119
169.0132
M – H
-0.0013
125.0228, 145.3992

151.0022

6
1.67
Catechins

289.2617
289.2613
M – H
0.0004
121.0280, 125.0230

137.0231, 161.0597

187.0391, 245.0817

7
2.04
Epicatechin

289.2618
289.2613
M – H
0.0005
125.0228, 161.0595

179.0337, 187.0391

245.0813, 271.0609

8
2.74
Isorhamnetin-3-pyranosearabinose-7-glucosyl-rhamnoside

755.6536
755.6526
M – H
0.0010
299.0200, 313.0343

314.0414, 609.1453

9
2.93
4-hydroxy-3-methoxybenzaldehyde

151.0389
151.0390
M – H
-0.0001
77.0397, 93.0328

109.0279, 121.0275

136.0150, 137.0217

151.0389

10
3.68
Kaempferol-3-o-furananose-7-O-rhamnosyl-glucoside

725.6232
725.6266
M – H
-0.0034
255.0300, 283.0253

284.0330, 285.0393

575.1413

11
4
Vitexin-rhamnoside

577.5144
577.5124
M – H
0.0020
283.0609, 311.0560

341.0664, 353.0664

413.0873, 457.1156

12
4.18
Rutin

609.5162
609.5112
M – H
0.0050
211.0394, 255.0299

256.0332, 283.0238

301.0348

13
4.23
Kaempferol-3-O-furana-nose-7-O-rhamnose isomer

725.6237
725.6266
M – H
-0.0029
255.0295, 284.0325

431.1031

14
4.86
Isoquercetin

463.3790
463.3697
M – H
0.0093
151.0025, 243.0296

255.0298, 271.0250

300.0278

15
5.5
Oxidized resveratrol

243.0656
243.0652
M – H
0.0004
157.0644, 160.0471

175.0752, 197.0597

201.0548, 225.0549

16
6.27
Salicylic acid

137.0229
137.0233
M – H
-0.0004
93.0329, 108.0198

119.7794

17
6.53
Quercitrin

447.3834
447.3703
M – H
0.0131
151.0029, 255.0296

301.0356, 426.9522

18
6.94
Kaempferol-3-O-rutino-side

593.5119
593.5118
M – H
0.0001
255.0299, 267.0291

285.0405

19
7.9
Astragalin

447.3738
447.3703
M – H
0.0035
227.0346, 255.0299

284.0329, 285.0398

20
19.27
Quercetin

301.0352
301.0343
M – H
0.0009
121.0279, 149.0229

151.0022, 161.0227

178.9973, 245.0449

21
19.77
Vitexin

431.0972
431.0973
M – H
-0.0001
269.0262, 283.0627

311.0527, 341.0764

22
20.12
Emodin-8-O-β-D-glucoside

431.1557
431.0973
M – H
0.0584
181.0859, 225.1121

269.1029, 413.1434

23
20.98
Apigenin

269.0454
269.0445
M – H
0.0009
159.0439, 181.0648

213.0542, 225.0545

227.0343, 241.0497

24
21.4
Kaempferol

285.2303
285.2295
M – H
0.0008
159.0440, 183.0448

211.0394, 227.0340

239.0346

25
21.64
Isorhamnetin

315.2507
315.2555
M – H
-0.0048
151.0023, 163.0020

271.0254, 283.0234

300.0273

26
22.19
Rock acid

357.0607
357.0605
M – H
0.0002
112.6701, l21.0279

163.0184

27
27.41
Gallocatechin

305.2661
305.2607
M – H
0.0054
125.0962, 167.5970

287.1647

28
27.62
Gingergly eolipid A

675.3498
675.3586
M – H
-0.0088
161.0440, 277.2170

397.1349, 415.14514

29
27.9
Epigallocatechin

305.0750
305.0656
M – H
0.0094
125.0957, 137.0593

179.1062, 287.1654

30
29.26
Gingergly eolipid B

677.3754
677.3743
M – H
0.0011
125.0230, 161.0444

179.0553, 279.2327

397.1341, 415.1567

31
29.95
1-linoleylglycero-2-phospho-ethanolanine

476.5684
476.5653
M – H
0.0031
140.0180, 196.0372

214.8795, 279.2330

32
38.06
Lysophosphatidic acid

435.2514
435.2506
M – H
0.0008
78.9573, 152.9945

33
42.19
Soya-cerebroside I

712.5367
712.5358
M – H
0.0009
179.0551, 271.2278

278.2481, 296.2603

532.4733, 550.4841

34
42.9
Linoleic acid phosphatidic acid

433.4959
433.4974
M – H
-0.0015
78.9574, 152.9946

35
45.84
4-(2-dodecyl)-benzene-sulfonate

325.4846
325.4884
M – H
-0.0038
119.0488, 170.0033

183.0112

36
51.25
4-(1-methyl-dodecyl)-benzene-sulfonic acid

339.5191
339.5150
M – H
0.0041
79.9554, 119.0487

170.0030, 225.0585

239.0742

Table 2: Identificati1on analysis of chemical constituents of T. hemsleyanums in positive ion mode of mass spectrometry.

No.
tR (min)
Name
Formula
Detected m/z
Expected m/z
M + X
Error (ppm)
Fragmentor information

37
1.34
Catechins

291.0851
291.0863
M + H
0.0012
123.0438, 161.0591

179.0696, 249.0744

273.0750

38
1.69
Epicatechin

291.0845
291.0863
M + H
-0.0018
139.0384, 207.0644

231.0638, 249.0741

273.0740

39
1.9
Palmitic acid

257.2466
257.2475
M + H
-0.0009
137.0592, 151.0750

165.0904, 221.1161

40
3.45
Vitexin-rhamnoside

579.1687
579.1708
M + H
-0.0021
313.0692, 415.1007

433.1111, 514.5942

41
4.38
Isoquercetin

465.1020
465.1028
M + H
-0.0008
285.0382, 303.0485

363.7647, 395.0804

42
6.32
Orientin

449.1051
449.1078
M + H
-0.0027
329.3295, 359.1488

43
6.42
Kaempferol-3-O-rutone

595.1646
595.1658
M + H
-0.0012
287.0537, 352.4147

433.1127, 449.1062

545.0139

44
7.61
Isoorientin

449.1048
449.1078
M + H
-0.0030
287.0537, 337.0678

395.1428, 431.1667

45
23.06
Gallocatechin

307.1888
307.0812
M + H
0.1076
163.0748, 261.1832

271.1683, 285.0766

46
24
Epigallocatechin

307.1886
307.0812
M + H
0.1074
135.0799, 173.0954

243.1731, 261.1839

271.1682, 289.1786

47
24.58
Vitexin

433.2325
433.1129
M + H
0.1196
293.1132, 317.2460

335.2565, 399.2344

48
25.45
Taraxerone

425.3756
425.3778
M + H
-0.0022
217.1945, 283.1677

339.2303, 365.1367

406.31027

49
27.26
Isovitexin

433.1131
433.1129
M + H
0.0002
279.23077, 301.90817

317.24625, 335.25653

415.2227

50
29.46
Linoleic acid

281.2442
281.2475
M + H
-0.0033
245.2253, 263.2358

51
31.19
Methyl linoleate

293.2097
293.2475
M + H
-0.0378
197.1319, 215.1786

229.1945, 247.2046

257.1888, 275.1994

(a)

(b)

Figure 1: Ultra-high performance liquid chromatography coupled with hybrid quadrupole-orbitrap mass spectrometry (UPLC-Q-Exactive/MS) was utilized to analyze the chemical constituents of T. hemsleyanums. The UPLC-Q-Exactive/MS base peak chromatogram of T. hemsleyanums in negative (a) and positive (b).

Flavonoids are mainly as follows: compounds 1, 8, 10, 12, 14, 18, 19, 20, 21, 24, and 25 were assigned to be procyanidin dimmer, isorhamnetin-3-pyranosearabinose-7-glucosyl-rhamnoside, kaempferol-3-O-furananose-7-O-rhamnosyl-glucoside, rutin, isoquercetin, kaempferol-3-O-rutone, astragalin, quercetin, vitexin, kaempferol, and isorhamnetin [18], and compound 17 was assigned to be quercitrin [21]. Compounds 11 and 21 were assigned to be vitexin-rhamnoside [21] and vitexin [22]. Compound 21 was also detected in positive ion mode. Compound 49 was the isomer of compound 21. Compounds 42 and 44 were assigned to be orientin [23] and isoorientin [24]. Peak 15, kaempferol-3-O-furanosine-7-O-rhamnose isomer, was detected from T. hemsleyanums for the first time.

Phenolic acids are mainly as follows: compounds 2 and 3 were assigned to be neochlorogenic acid and chlorogenic acid [22]. As we saw in Figure 2, the cleavage fragment strength of the neochlorogenic acid was m/z=191>179>135 (Figure 2(a)). And for chlorogenic acid, the strength of peak m/z=191 was the strongest; the strength of peaks m/z 179 and m/z 135 was equivalent and very weak (Figure 2(b)).

(a)

(b)

Figure 2: Secondary mass spectrum of chlorogenic acid (a) and neochlorogenic acid (b). As shown in (a), the cleavage fragment strength of the m/z was 191> 179>135. In (b), the peak m / z = 191 has the strongest intensity, and the peaks m / z179 and m / z135 have the same intensity and are very weak.

Compound 9 was assigned to be 4-hydroxy-3-methoxybenzaldehyde, of which fragmentation patterns was shown in Figure 3. Compounds 16 and 26 were assigned to be salicylic acid and rock acid [18].

Figure 3: A proposed fragmentation pathway of 4-hydroxy-3-methoxybenzaldehyde by mass spectrometry.

Anthraquinones are mainly as follows: compound 22 was assigned to be emodin-8-O-β-D-glucopyranoside [25]. The [M-H]⁻ ion of compound 22 was at m/z 431.1557. Glucose group was lost to produce ion of m/z 269.1029. The fragmentation patterns were summarized in Figure 4.

Figure 4: A proposed fragmentation pathway of emodin-8-O-β-D-glucopyranoside by mass spectrometry.

(a)

(b)

(c)

Figure 5: Antiviral determination. MDCK cells were infected with recombinant influenza virus PR8-NS1-Gluc at an moi of 0.01 PFU/cell, and the infected cells were treated with ZJ2 and other extract samples of T. hemsleyanums at indicated concentrations during virus propagation. (a) ZJ2 inhibits influenza virus replication in a dose dependent manner. (b) Dose-response curve of ZJ2 against influenza virus replication and on cell viability. (c) 17 extract samples of T. hemsleyanums collected from different districts as well as ZJ2 show similar antiviral activities against influenza virus. Mean and SEM of three independent experiments were shown. p<0.001, Student’s t-test. There are 18 batches of T. hemsleyanums in this figure. Among them, FJ1, FJ2, and FJ3 are three batches from Fujian, GZ1, GZ2, and GZ3 are from Guizhou, ZJ1, ZJ2, and ZJ3 are from Zhejiang, HB1, HB2, and HB3 are from Hubei, YN1, YN2, and YN3 are from Yunnan, and GX1, GX2, and GX3 are from Guangxi. Their information can be found in the Supplementary Materials (available here).

Catechins are mainly as follows: compounds 27 and 29 were assigned to be gallocatechin and epigallocatechin [23]. Compound 29 produced characteristic ions of m/z 125.0957 and m/z 137.0593 which was identified as epigallocatechin. Compound 27 and compound 29 were also detected in positive ion mode. Compounds 4, 5, 6, and 7 were assigned to be protocatechuic acid glucoside, gallic acid, catechins, and epicatechin [26, 27]. Catechins and epicatechin were confirmed by reference standard.

Esters are mainly as follows: compounds 28, 30, 32, 34, 35, and 51 were assigned to be gingergly eolipid A, gingergly eolipid B, lysophosphatidic acid, linoleic acid phosphatidic acid, 4-(2-dodecyl)-benzene-sulfonate, and methyl linolate [18].

Fatty acids are mainly as follows: compounds 39 and 50 were assigned to be palmitic acid and linoleic acid. Additionally, the loss of H₂O group was considered as the representative fragmentation pathway in acids [28]. Palmitic acid was confirmed by reference standard.

The other compounds are mainly as follows: compounds 31, 33, and 36 were assigned to be 1-linoleoylglycero-2-phosphor-ethanolamine, soya-cerebroside I, and 4-(1-methyl-dodecyl)-benzene-sulfonic acid [18].

3.2. Quantification Method Validation

As shown in Table 3, standard curves of the ten compounds exhibited good linearity in the range of 1.6 to 1330 μg/mL, with coefficients of correlation ranging from 0.9961 for kaempferol to 0.9998 for quercetin. The parameters of LOD were from 0.1045 to 2.0781 pg, and LOQ were from 0.9290 to 15.5142 pg, which were sensitive enough to the detection of analytes. The intraday and interday precisions of present method were shown in Table 4, and the respective RSD values were from 0.03 to 2.79% and from 0.72 to 2.81%. The concentration stability of the constituents in samples kept at 4°C for 24 hours (n = 6) ranged from 0.28 to 3.42%. In addition, the sample solutions for T. hemsleyanums were prepared in parallel (n=6) to evaluate the repeatability and achieved the RSD of 0.26-2.69%. Based on the above methodology verification, the assay is reproducible and suitable for accurate and precise quantification of these ten chemical constituents in T. hemsleyanums (shown in Table 4). As shown in Table 5, the recoveries of ten constituents was from 94.86 to 99.9 % with the RSD value <3 %.

Table 3: Linear eq1uation, linear range, correlation coefficient, and detection limit of 10 constituents.

Compound
Regression equation
Linear range (μg/ml)
Correlation coefficient (r²)
Detection limit (pg)
Quantification limit (pg)

Rutin
y = 0.025x -1.099
7.125-1320
0.9924
0.1113
14.4926

Kaempferol
y = 0.062x + 1.015
1.828-58.50
0.9961
0.9141
0.9290

Astragalin
y = 0.111x – 2.688
6.888-1100
0.9971
0.1074
7.4910

Quercitrin
y = 6.393x – 196.137
8.313-1330
0.9992
2.0781
9.5661

Quercetin
y = 7.378x – 292.807
3.344-1070
0.9992
0.1045
9.2285

Vitexin-rhamnoside
y = 0.000436x + 0.0240
1.735-58
0.9997
1.7344
15.5142

Isorhamnetin
y = 0.00673x + 0.152
1.688-54
0.9954
0.4531
11.6001

Vitexin
y = 3.629x + 0.000187
4.125-132
0.9978
2.0625
3.5940

Emodin-8-O-β-D-glucopyranoside
y = 0.000982x + 0.0139
1.813-116
0.9986
0.4531
2.7285

Isoquercetin
y = 0.00313x + 0.0602
3.625-116
0.9955
0.1133
6.1772

Table 4: Summary of the precision and RSD results of UPLC-Q-Exactive/MS method for the analyzed quality control samples.

Compounds
Intraday precision
Interday precision
Stability
Repeatability

accuracy (%)
RSD (%)
accuracy (%)
RSD (%)
accuracy (%)
RSD (%)
accuracy (%)
RSD (%)

Rutin
98.69-103.22
1.93
98.17-102.44
1.65
98.35-102.05
1.41
98.61-101.84
1.16

Kaempferol
98.03-102.13
1.851
98.82-104.04
1.15
95.29-103.09
2.32
96.06-103.27
2.67

Astragalin
98.07-102.47
2.16
96.75-101.24
2.38
94.12-103.32
3.42
97.48-102.65
2.22

Quercitrin
98.57-102.75
2.02
97.83-100.86
1.33
98.72-102.71
1.52
98.15-102.63
2.02

Quercetin
99.83-102.21
0.35
95.02-102.90
1.93
98.37-101.95
1.45
97.48-103.39
1.94

Vitexin-rhamnoside
97.68-101.00
1.30
98.66-101.14
0.72
97.68-100.99
1.30
98.19-100.88
1.08

Isorhamnetin
99.96-100.02
0.03
97.95-103.99
1.90
99.23-100.46
0.53
99.27-100.40
0.50

Vitexin
99.80-100.48
0.25
95.65-103.60
2.81
99.80-100.54
0.28
99.83-100.50
0.26

Emodin-8-O-β-D-glucopyranoside
95.62-103.90
2.79
98.83-103.82
2.53
95.05-102.81
2.75
96.30-103.59
2.69

Isoquercetin
97.60-103.10
2.30
96.68-101.86
2.13
97.41-102.98
2.54
97.19-102.74
2.39

Table 5: The rate of recovery for 10 constituents (n = 6).

Compounds
Addition level
Average recovery rate (%)
RSD (%)

Rutin
1:0.8
96.95
0.02

1:1
98.61
2.83

1:1.2
96.25
1.12

Kaempferol
1:0.8
98.20
2.88

1:1
97.67
1.21

1:1.2
98.71
2.05

Astragalin
1:0.8
98.15
0.14

1:1
99.62
1.10

1:1.2
98.56
0.67

Quercitrin
1:0.8
99.90
0.41

1:1
98.58
0.92

1:1.2
96.59
1.76

Quercetin
1:0.8
97.68
0.18

1:1
96.06
0.82

1:1.2
97.75
1.30

Vitexin-rhamnoside
1:0.8
96.62
2.09

1:1
95.67
2.41

1:1.2
96.03
1.32

Isorhamnetin
1:0.8
97.13
0.29

1:1
96.00
1.18

1:1.2
97.20
0.24

Vitexin
1:0.8
96.24
1.44

1:1
99.18
1.80

1:1.2
99.27
1.59

Emodin-8-O-β-D-glucopyranoside
1:0.8
97.10
0.33

1:1
94.86
0.96

1:1.2
97.99
0.39

Isoquercetin
1:0.8
95.38
0.56

1:1
98.56
0.67

1:1.2
97.77
0.62

3.3. Quantitative Analysis

The validated UPLC-Q-Exactive/MS method was used for the content measurement of 10 constituents of rutin, kaempferol, astragalin, quercitrin, quercetin, vitexin-rhamnoside, isorhamnetin, vitexin, emodin-8-O-β-D-glucoside, and isoquercetin in T. hemsleyanums. The content of each constituent was calculated in terms of its respective calibration curve and the result was listed in Table 6. The content of vitexin was relative abundant, of which could reach the amount of 0.7079±0.0286 mg/g in that from YN1, whereas vitexin-rhamnoside may be the lowest in content since it could even not be detected in some batches. The result suggested that contents of these constituents fluctuated in different batches even for those that from the same production place. The total amounts of these ten compounds in T. hemsleyanums of YN1, GZ1, and GX3 were the top three highest, whereas for samples from Zhejiang province, amounts of flavonoids are shown to be the lowest. Many factors including growing environment of climate temperature, rainfall, sunlight, soil nutrient, etc. and harvest time and postharvest treatment may affect the content of bioactive compounds in T. hemsleyanums and thus may affect its efficacy.

Table 6: The content of 10 constituents in T. hemsleyanums (  ±  S, average ± standard deviation) (mg/g, n = 3).

Batch
Rutin
Kaempferol
Astragalin
Quercitrin
Quercetin
Vitexin-rhamnoside
Emodin-8-O-β- D-glucopyranoside
Isoquercetin
Isorhamnetin
Vitexin

ZJ1
0.0858±0.0011
0.0100±0.0190
0.0446±0.0000
0.0899±0.0006
0.0565±0.0010
-
0.0087±0.0010
0.0345±0.0034
0.0276±0.0060
0.1096±0.0047

FJ3
0.0887±0.0002
0.0102±0.0113
0.0453±0.0002
0.0903±0.0013
0.0584±0.0003
-
0.0132±0.0052
0.0359±0.0074
0.0330±0.0019
0.1107±0.0002

YN2
0.0901±0.0011
0.0393±0.0206
0.0434±0.0003
0.0880±0.0003
0.0615±0.0022
-
0.0154±0.0119
0.0380±0.0099
0.0311±0.0008
0.0324±0.0013

ZJ2
0.1036±0.0022
0.0388±0.0050
0.0445±0.0002
0.0998±0.0002
0.0628±0.0003
-
0.0179±0.0178
0.0383±0.0054
0.0342±0.0011
0.0519±0.0016

ZJ3
0.0905±0.0061
0.0865±0.0436
0.0438±0.0002
0.0907±0.0016
0.0630±0.0001
0.0010±0.0015
0.0182±0.0116
0.0522±0.0061
0.0236±0.0027
0.0262±0.0012

GX3
0.1012±0.0041
0.0498±0.0085
0.0640±0.0004
0.0953±0.0013
0.0809±0.0006
0.0016±0.0002
0.0192±0.0047
0.0576±0.0041
0.0373±0.0071
0.7581±0.0324

FJ2
0.1166±0.0028
0.0979±0.0066
0.0479±0.0274
0.0957±0.0004
0.0756±0.0013
-
0.0210±0.0075
0.0616±0.0028
0.0344±0.0006
0.1145±0.0010

YN3
0.0967±0.0037
0.0867±0.0128
0.0493±0.0001
0.1351±0.0024
0.0577±0.0002
-
0.0209±0.0249
0.0588±0.0037
0.0319±0.0006
0.2680±0.0601

HB3
0.1142±0.0016
0.1016±0.1173
0.0532±0.0002
0.1092±0.0005
0.0750±0.0039
-
0.0224±0.0133
0.1324±0.0016
0.0315±0.0007
-

GZ2
0.1157±0.0031
0.1104±0.0023
0.0583±0.0494
0.1196±0.0006
0.0803±0.0011
0.0008±0.0011
0.0282±0.0058
0.0802±0.0031
0.0597±0.0017
0.2951±0.0072

GX1
0.0970±0.0044
0.1146±0.0268
0.0527±0.0002
0.1014±0.0004
0.0716±0.0003
-
0.0268±0.0022
0.1488±0.0044
0.0331±0.0071
0.0388±0.0015

GZ3
0.0989±0.0201
0.1243±0.0149
0.0572±0.0002
0.1095±0.0000
0.0777±0.0048
0.0027±0.0024
0.0291±0.0133
0.0726±0.0095
0.0318±0.0010
0.0426±0.0007

GX2
0.1121±0.0014
0.0867±0.0004
0.0552±0.0001
0.1058±0.000
0.0627±0.0002
0.0152±0.0064
0.0298±0.0073
0.0711±0.0049
0.0353±0.0011
0.0371±0.0052

FJ1
0.1185±0.0004
0.1379±0.0006
0.0599±0.0004
0.1238±0.0004
0.0759±0.0015
0.0216±0.0041
0.0305±0.0030
0.1068±0.0025
0.0349±0.0041
0.0479±0.0055

HB1
0.1152±0.0187
0.1285±0.0133
0.0572±0.0002
0.1279±0.0009
0.0754±0.0004
0.0305±0.0113
0.0255±0.0020
0.0792±0.0212
0.0338±0.0164
0.2927±0.0045

HB2
0.1296±0.0002
0.2757±0.0179
0.0576±0.0002
0.1435±0.0003
0.1463±0.0002
0.0280±0.0100
0.0300±0.0045
0.1927±0.0425
0.0347±0.0021
0.0005±0.0020

GZ1
0.1374±0.0002
0.2910±0.0210
0.0702±0.0003
0.1443±0.0003
0.0920±0.0002
0.0325±0.0394
0.0335±0.0009
0.1940±0.0981
0.0505±0.0006
0.6811±0.0307

YN1
0.1489±0.0003
0.2967±0.1511
0.0761±0.0003
0.1567±0.0000
0.0960±0.0003
0.0338±0.0045
0.0439±0.0199
0.2207±0.1549
0.0543±0.0037
0.7079±0.0286

- means below the detection limit.

3.4. Anti-H1N1 Influenza Virus Activity

Since T. hemsleyanums from Zhejiang province is popular on market, we took sample of ZY2 as the representative sample for anti-influenza virus pretest. As shown in Figure 5(a), extract of ZJ2 inhibited influenza virus replication in a dose-response manner, and the IC50 is 27.4μg/mL, while no cytotoxicity was observed at a concentration of as high as 200μg/mL (Figure 5(b)). Ribavirin at 100μM was used as positive control. Antiviral activities of the other 17 batches were shown in Figure 5(c) that all of them showed inhibitory effect against H1N1 influenza virus replication as good as ZJ2 at the concentration of 50μg/mL. All three sample batches from Guizhou province (GZ1, GZ2, and GZ3, 94.0-98.0%) and Hubei province (HB1, HB2, and HB3, 93.5-97.3%), two batch from Guangxi province (GX1 and GX2, 94.3-94.5%), one batch from Fujian (FJ1, 96.2%), and one batch from Yunnan (YN1, 98.6%) exhibited higher anti-influenza virus activities than the others, which were probably due to the higher values of flavonoids in these samples.

3.5. Statistical Analysis

In order to determine the contribution of various constituents for the antiviral activity, the spectrum-effect relationships between LC-MS fingerprints and inhibitory effect for influenza virus were evaluated with correlation analysis statistical method. Since normal distribution was not satisfied, Spearman was used for all the analyses. When the correlation coefficient is greater than 0, it indicates that a component is positively correlated with antiviral activity, and the larger the value, the stronger the correlation. When the correlation coefficient is less than 0, it means that it is negatively related to antiviral activity. Results were shown in Table 7 which revealed that 10 peaks had significant correlationship with anti-influenza virus activity. 8 of them, which were identified as rutin, kaempferol, astragalin, quercitrin, quercetin, kaempferol-3-o-rutinoside, procyanidin dimmer, and epicatechin, were positively related to antiviral activity, of which the highest correlationship was with astragalin (r = 0.711), followed by epicatechin (0.641), quercitrin (0.614), and quercetin (0.617). 2 of the 10 peaks, identified as vitexin (-0.5370) and isorhamnetin-3-pyranose arabinose-7-glucosyl rhamnoside (-0.6630), indicated negative correlationship with antiviral activity. That is, for constituents with positive coefficient, which may have underlying anti-H1N1 influenza effect, the higher the content, the better the bioactivity. This result was in agreement with literatures, in which quercetin, quercitrin, rutin, kaempferol, and isoquercitrin were reported to have anti-H1N1 influenza virus activity [29–32]. Thereby it may provide valuable clues for further discovery of active anti-influenza virus compounds. LC-MS chemometrics are a kind of potent method for analyzing the complex system of TCM herbs, but lacking of bioactive characteristics. Therefore, chemical fingerprints combined with bioactivity evaluation to construct a fingerprint-efficacy relationship is a good way to explore the bioactive constituent of TCM herb and may lend material basis supporting to the quality control of T. hemsleyanums

Table 7: Summary of results about the components of T. hemsleyanums and antiviral inhibition.

Variable
Compound
Correlation Coefficient
P

X1
Rutin
0.5470
0.0190

X2
Kaempferol
0.5800
0.0120

X3
Astragalin
0.7110
0.0010

X4
Quercitrin
0.6170
0.0060

X5
Quercetin
0.6140
0.0070

X6
Kaempferol-3-O-rutinoside
0.514
0.029

X7
Procyanidin dimmer
0.503
0.033

X8
Vitexin
-0.5370
0.0220

X9
Epicatechin
0.6410
0.0040

X10
Isorhamnetin-3-pyranose arabinose-7-glucosyl rhamnoside
-0.6630
0.0030

Quantitative compounds.

4. Conclusion

In our study, an efficient UPLC-Q-Exactive/MS method was established for qualitative analysis of up to 51 constituents identified in T. hemsleyanums for the first time and also for quantitative analysis of 10 constituents in 18 batches collected from six cultivation places. Method validation showed the ideal sensitivity, stability, and reliability of the analysis method. It was also the first time for UPLC-Q-Exactive/MS and Gaussia Luciferase viral titer assay being combined to reveal the underlying anti-influenza virus bioactive compounds in T. hemsleyanums. 8 possible active constituents were discovered showing positive contribution for antivirus effect, and five of them are consistent with reports published in literatures. This analytical method led to the identification of compounds with anti-H1N1 influenza virus activity in T. hemsleyanums which may be difficult to be extracted and identified by traditional bioactivity-guided fractionation procedures and may provide an available reference mode for revealing the material basis in traditional Chinese herb of which thereby could also provide reference for quality assessment of TCM herbs.

Data Availability

Most of the data (Figures 1 to 5 and Tables 1 to 7) used to support the findings of this study are included within the article. Some of the data used to support the findings of this study are included within the supplementary information file.

Conflicts of Interest

The authors declare no conflicts of interest.

Authors’ Contributions

FuJuan Ding and JiangTing Liu contributed equally to this work.

Acknowledgments

The research was financially supported by Key Technology Innovation Project of Important Industries in Shandong Province (no. 2016CYJS08A01-5), National Natural Science Foundation of China (no. 81774167), Key Research and Development Program in Shandong Province (no. 2018CXGC1307), and College Science and Technology Plan Project of Shandong Province (J16LM12).

Supplementary Materials

Table 1: the information of T. hemsleyanums. Table 2: parameters of LC-Q-Exactive /MS analysis for 10 constituents. Figure 1: total ion chromatogram of 18 batches of T. hemsleyanum. (Supplementary Materials)

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