Structure Analysis of Effective Chemical Compounds against Dengue Viruses Isolated from Isatis tinctoria

The history of Chinese herb research can be traced back to thousands of years ago, and the abundant knowledge accumulated for these herbs makes them good candidates for developing new natural drugs. Isatis tinctoria is probably the most well-studied Chinese herb, which has been identified to be effective against dengue fever. However, the underlying biological mechanisms are still unclear. In this study, we adopt combined methods of bioactive trace technology and phytochemical extraction and separation, to guide the isolation and purification of the effective chemical constituents on the water-soluble components of aerial parts of Isatis tinctoria. In addition, we apply polarimetry and 1D or 2D nuclear magnetic resonance (NMR) spectroscopy to identify their structures, which lay a foundation for further study on the biological mechanisms underlying medicinal effects of Isatis tinctoria using in vitro and in vivo experiments. Specifically, we identify and infer the structures of 27 types of chemical compounds named GB-1, GB-2, …, GB-27, respectively, among which GB-7 is a novel compound. Further study of these compounds is critical to reveal the secrets behind the medicinal effects of Isatis tinctoria.


Introduction
Dengue fever (DF) is a kind of acute infectious diseases caused by dengue virus (DV). Its major clinical symptoms include acute high fever, headache, generalized myalgia, ostalgia and arthralgia, rash, hemorrhagic tendency, and low white blood cell count [1]. Dengue hemorrhagic fever (DHF) is a kind of dengue fevers with more serious clinical symptoms like extremely high fever, severe hemorrhage, shock, low platelet count, pachyhematous, and high death rate [2]. A DHF accompanying with shock is called dengue shock syndrome (DSS).
Aedes aegypti and Aedes albopictus are two major vectors for transmission of dengue viruses [3]. Dengue fever is endemic in tropical and subtropical zones and has become one of the most wide-spread, infectious, and dangerous arboviral diseases. In recent years, there are several outbreaks of dengue fever and dengue hemorrhagic fever in Southeast Asia, Pacific Islands, Central America, and South America, making it a critical world-wide issue for public health [4].
Currently, there is no vaccine or antiviral treatment for dengue fever [5].
e patients are only recommended to drink plenty of fluids or take medicines like acetaminophen to avoid dehydration from vomiting and high fever [6].
us, it is very important to develop new antiviral drugs against dengue viruses. However, there are many difficulties in the synthesis of new compounds including high cost, low yields, and so on [7], which urges drug companies and research entities to search for antiviral drugs from natural products. It is known that traditional Chinese medicines are among the most studied natural products in curing diseases [8]. Owing to thousands of years' clinical trials of these medicines in China, traditional Chinese doctors have plenty of experience in combining natural products to cure diseases and protect people from the invasion of many viruses. us, it is promising to isolate antiviral drugs from Chinese medicinal herbs.
As a type of popular traditional Chinese medicinal herb, Isatis tinctoria (woad, Brassicaceae or Ban Lan Gen) has been proven to be effective against various viruses including influenza, hepatitis A virus (HAV), and so on [9][10][11]. It has also been used in treating the dengue fever with positive effects [12]. In this study, we isolated and purified the bioactive compounds of hydrosoluble constituents from the soil-growing woad by integrating bioassay-guided fraction technique and phytochemical isolation and purification techniques. In addition, we inferred the chemical structures of these compounds by polarimetry and 1D or 2D nuclear magnetic resonance (NMR).

Results
A total of 27 compounds, namely GB-1, GB-2, . . ., GB-27 were isolated from water-soluble components of aerial parts of Isatis tinctoria, and their structures were inferred as follows.
As can be seen from 13 C NMR data, there are eight C signals including δ c 122.9, 120.3, 122.6, 103.3, 151.9, 106.1, 137.9, and 103.8 in the low field, indicating that the compound belongs to indole alkaloid. e methylene signal δ c 14.9 is present in the high field, which also confirms the inference of the hydrogen spectrum. Combining with DEPT NMR, the compound contains 5 quaternary carbon signals, 9 methine signals, and 2 methylene signals. In addition, there is a group of glycon signals: δ c 101.3, 77.1, 76.7, 73.5, 69.8, and 60.8. us, the monosaccharide can be inferred to be β-D-glucose. We also inferred other C and H signals by HSQC correlation analysis. According to the above NMR results, GB-1 is cappariloside A.

Structural Identification of Compound GB-2.
Chemical compound GB-2 is a kind of bisque needle-like crystalloid, easily soluble in methanol and acetone. e identified chemical structure is plotted in Figure 1(b), and the detailed information about 1 H NMR (500 MHz, DMSO-d 6 ) and 13  As can be seen from 13 C NMR data, there exist eight C signals, including δ c 136.3, 126.3, 125.7, 121.7, 119.0, 117.8, 111.3, and 104.5 in the low field, suggesting that the compound belongs to indole alkaloid. e methylene signal of δ c 14.9 and 40.1 is present in the high filed, which also confirms the inference of the hydrogen spectrum. Combining with the DEPT NMR, the compound contains 5 quaternary carbon signals, 5 methine signals, and 2 methylene signals. us, GB-2 was inferred to be 1-csrboline acid.

Structural Identification of Compound GB-3.
Chemical compound GB-3 is a kind of bisque needle-like crystalloid, easily soluble in methanol and acetone. e identified chemical structure is plotted in Figure 1(c), and the detailed information about 1 H NMR (500 MHz, DMSO-d 6 ) and 13 C NMR (125 MHz, DMSO-d 6 ) is listed in Table S2. We compared the NMR data of GB-3 with that of GB-2, which suggests that GB-3 contains the signal of indole alkaloids. In addition, there exist the ABX system signal and the quaternary carbon signals of δ c 152.3 in the H spectrum, indicating that hydroxyl substitution occurs at position C-5.

Structural Identification of Compound GB-4.
Chemical compound GB-4 is a kind of white crystals, easily soluble in methanol and acetone. e identified structure is plotted in Figure 1(d), and the detailed data about 1 H NMR (500 MHz, DMSO-d 6 ) and 13 C NMR (125 MHz, DMSO-d 6 ) are listed in Table S3. According to 1 H NMR data, there exist four doublebond H signals including δ H 8.34 (1H, s) and δ H 8.13 (1H, s). We inferred that the signal of δ H 7.37 (2H, s) belongs to amino-H, which may be connected to a benzene ring or double bonds, and the signal of δ H 7.37 (1H, d, J � 6.5) belongs to ribose terminal group H. us, we conjectured that the compound is β configuration based on the coupling constant.
As can be seen from 13 C NMR data, there are five C signals including δ c 87.9, 85.9, 73.5, 70.7, and δ c 61.7, indicating the presence of ribose in the chemical compound. In addition, the C signal that exists in the low field is adenine. Combining with the DEPT NMR, the compound contains 3 quaternary carbon signals, 6 methine signals, and 1 methylene signal. us, GB-4 is adenosine.

Structural Identification of Compound GB-5.
Chemical compound GB-5 is a kind of bisque needle-like crystalloid, easily soluble in methanol and acetone. e identified chemical structure is plotted in Figure 1(e), and the detailed data about 1 H NMR (500 MHz, CD 3 OD) and 13  suggesting that the compound has benzene ring substituted type of symmetrical structure. As can be seen from 13 C NMR data, there are signal of δ c 170.0 and methoxy signal of δ c 56.7, indicating the presence of carboxylic acid structure in the compound. Combining with the DEPT NMR, the compound contains 5 quaternary carbon signals, 2 methine, signals and 2 methylene signals. us, GB-5 is syringic acid.

Structural
Identification of Compound GB-6. Chemical compound GB-6 is a kind of bisque needle-like crystalloid, easily soluble in methanol. e identified chemical structure is plotted in Figure 1(f), and the detailed data about 1 H NMR (500 MHz, CD 3 OD) and 13 C NMR (125 MHz, CD 3 OD) are listed in Table S5. We compared the compound with chemical GB-5, indicating that they are of the same structural skeleton. However, the compound has more signals compared to GB-5, such as a set of double-bond signals, a hydroxymethyl signal (δ c 62.8), and a set of glucose signal (δ c 104.9, 78.8, 78.5, 76.1, and 62.6). Based on DEPT NMR, the compound contains 4 quaternary carbon signals, 9 methine signals, 2 methylene signals, and 2 methyl signals. us, GB-6 is syringin.

Structural Identification of Compound GB-7.
Chemical compound GB-7 is a kind of white amorphous powder, easily soluble in methanol and acetone. e identified chemical structure is plotted in Figure 2(a), and the detailed data about 1 H NMR (500 MHz, CD 3 OD) and 13 C NMR (125 MHz, CD 3 OD) are listed in Table 2.
As can be seen from 13 C NMR data, there exist signal of δ c 180.6 in the low field, indicating that the compound has carboxylic acid structure. In addition, there also exists a methoxy signal of δ c 55.4. Combining with DEPT NMR, the compound contains 6 quaternary carbon signals, 3 methine signals, 1 methylene signal, and 1 methyl signal. However, we failed to find any compound that is consistent with these properties. After searching the SciFinder databases, we confirmed the compound GB-7 as a new monomer.

Structural Identification of Compound GB-8.
Chemical compound GB-8 is a kind of bisque needle-like crystalloid, easily soluble in methanol and acetone. e identified chemical structure is plotted in Figure 2(b), and the detailed data about 1 H NMR (500 MHz, CD 3 OD) and 13  In addition, there also exist two sets of end group signals of glycon including δ H 4.33 (1H, d, J � 7.6) and 4.16 (1H, d, J � 7.2), which were inferred to be two D-glucose.

Structural Identification of Compound GB-9.
Chemical compound GB-9 is a kind of bisque oily matter, easily soluble in chloroform and ethyl acetate. e identified chemical structure is plotted in Figure 2(c), and the detailed data about 1 H NMR (500 MHz, CD 3 OD) and 13  As can be seen from 13 C NMR data, there exists a signal of δ c 180.1 in the low field, indicating that the compound has carboxylic acid structure. In addition, there also exist double bond signals, indicating the presence of a center double bond. Combining with the DEPT NMR, the compound contains 2 quaternary carbon signals, 4 methine signals, 2 methylene signals, and 2 methyl signals. As a result, GB-9 was inferred to be (E)-2, 7-dimethyloct-4-enedioic acid.

Structural Identification of Compound
As can be seen from 13 C NMR data, there exist signals of δ c 136.2 and 131.3 in the low field, indicating that the compound a has double bond structure. In addition, there also exist 4 methyl signals including δ c 24.2, 26.2, 27.1, and 27.5. Combining with DEPT NMR, the compound contains 3 quaternary carbon signals, 4 methine signals, 2 methylene signals, and 4 methyl signals. In addition, there exist 13 C signals, and thus, the compound belongs to lower 2 carbon sesquiterpenes. Compared with the previous compound, only a difference was found in the spatial structure of position C-6. us, GB-11 was inferred to be (3S, 5R, 6R)-6-((E)-3-hydroxybut-1-enyl)-1,1,5-trimethylcyclohexane-3, 5,6-triol.

Structural Identification of Compound GB-12.
Chemical compound GB-12 is a kind of white powder, easily soluble in methanol and chloroform. e identified chemical structure is plotted in Figure 2   Canadian Journal of Infectious Diseases and Medical Microbiology signal, and 4 methyl signals. It is easily seen that there exist 13 C signals, and thus the compound belongs to lower 2 carbon sesquiterpenes. As a result, GB-12 is (6S)-6hydroxy-6-((E)-3-hydroxybut-1-enyl)-1,1,5-trimethylcyclohex-3-enone.

Structural Identification of Compound GB-15.
Chemical compound GB-15 is a kind of white powder, easily soluble in methanol and chloroform. e identified chemical structure is plotted in Figure 3(c), and the detailed data about 1H NMR (500 MHz, CD 3 OD) and 13C NMR (125 MHz, CD 3 OD) are listed in Table S13. e NMR data of the GB-15 and GB-14 show that the compound skeleton was the same, and there is only one more δ C 200.3 carbonyl signal in the low field. Combining with DEPT NMR, the compound contains 4 quaternary carbon signals, 8 methine signals, 3 methylene signals, and 4 methyl signals, suggesting that there are 19 C signals, and the compound is a glycoside of lower 2 carbon sesquiterpenes. us, GB-15 is (3S, 5R, 6R)-6-((E)-3-one but-1-enyl)-1,1,5-trimethylcyclohexane-5,6diol 3-O-β-D-glucopyranosid.

Structural Identification of Compound GB-16.
Chemical compound GB-16 is a kind of white powder, easily soluble in methanol and chloroform. e identified chemical structure is plotted in Figure 3(d), and the detailed information is shown in Table S14. We compared compound GB-16 with compound GB-14 and found that they are of the same structural skeleton. In addition, GB-16 is only 18 molecular weight less than GB-14 and has no double bonds. It was speculated that the compound has an epoxy structure formed by two hydroxyl groups. Combining with DEPT NMR, the compound contains 3 quaternary carbon signals, 9 methine signals, 3 methylene signals, and 4 methyl signals, suggesting that there are 19 C signals, and the compound is a glycoside of lower 2 carbon sesquiterpenes. us, GB-16 was inferred to be (3S, 5R, 6S, 7E, 9S)-megastigman-7-ene-5,6-epoxy-3,9-diol 3-O-β-D-glucopyranoside.

Structural Identification of Compound GB-17.
Chemical compound GB-17 is a kind of white amorphous powder, easily soluble in methanol. e identified chemical structure is plotted in Figure 3(e), and the detailed data about 1 H NMR (500 MHz, CD 3 OD) and 13 C NMR (125 MHz, CD 3 OD) are listed in Table S15. GB-17 has molecular weight of 162 more than GB-16, and there is one more set of glucose signals in NMR data, suggesting that GB-16 in turn connects to a glucose structure. Combining with DEPT NMR, the compound contains 3 quaternary carbon signals, 9 methine signals, 3 methylene signals, and 4 methyl signals, indicating that there are 19 C signals, and the compound is a glycoside of lower 2 carbon sesquiterpenes. us it was inferred to be (3S, 5R, 6S, 7E, 9S)-megastigman-7-ene-5,6-epoxy-3,9-diol 3,9-O-β-D-glucopyranoside.

Structural Identification of Compound GB-18.
Chemical compound GB-18 is a kind of white amorphous powder, easily soluble in methanol. e identified chemical structure is plotted in Figure 3(f), and the detailed data are listed in Table S16. e NMR data of the GB-18 and the GB-12 show that they are of the same compound skeleton and GB-18 has only one more glucose signal including δ c 102.9, 78.2, 77.9, 75.0, 71.7 and 62.8. Combining with DEPT NMR, the compound contains 3 quaternary carbon signals, 9 methine signals, 3 methylene signals, and 4 methyl signals, suggesting that there are 19 C signals and the compound is a glycoside of lower 2 carbon sesquiterpenes. us, GB-18 was inferred to be (6S)-6-hydroxy-6-((E)-3α-hydroxybut-1-enyl)-1,1,5-trimethylcyclohex-3-enone 9-O-β-D-glucopyranosid.

Structural Identification of Compound GB-19.
Chemical compound GB-19 is a kind of bisque needle-like crystalloid, easily soluble in methanol. e identified chemical structure was plotted in Figure 4(a) and the detailed data are listed in Table S17. e 1 H NMR of GB-19 has a H signal on the median of the benzene ring including δ H 6.38 (1H, d, J � 1.6) and δ H 6.17 (1H, d, J � 1.6), which indicating that all the compounds have A and C rings that are substituted by typical flavonoids C-2,3,5,7. ere is a typical ABX system that exists in the B ring including δ H 7.64 (1H, dd, J � 6.4, 1.6), δ H 7.73 (1H, d, J � 1.6), and δ H 6.87 (1H, d, J � 6.4), suggesting that the three substituted benzene ring structure is connected with C-2. e 13 C NMR data also confirm the inference of the H-spectrum. Combining with DEPT NMR, the compound GB-19 contains 3 quaternary carbon signals, and 9 methine signals. In the low field, there exist carbonyl C signal of δ c 177.3, and 5 C signal connecting to the oxygen on the benzene ring including δ C 165.7, δ C 162.5, δ C 158.2, δ C 148.7, and δ C 146.2. ere also exist C signals of positions C-8 and C-6. As an indication, G-19 is quercetin.

Structural Identification of Compound GB-20.
Chemical compound GB-20 is a kind of bisque needle-like crystalloid, easily soluble in methanol. e identified chemical structure is plotted in Figure 4(b), and the detailed information is listed in Table S18. By comparing with GB-19 and combining the results from the DEPT NMR, the compound contains 7 quaternary carbon signals, 7 methine signals, and 1 methylene signal, which was inferred to be catechin.   Table S19. According to 1 H NMR data, there are 3 benzene ring H signals including δ H 7.60 (1H, d, J � 9.6), δ H 6.51 (1H, s), and δ H 6.34 (1H, d, J � 9.6) in the low field. is is a typical H-spectrum signal of coumarin containing 2 methoxy signals, suggesting the compound to be three substituted coumarin types. Combining with the DEPT NMR, the compound contains 6 quaternary carbon signals, 3 methine signals, and 2 methyl signals. us, the above compound is fraxidin.

Structural Identification of Compound GB-22.
Chemical compound GB-22 is a kind of bisque needle-like crystalloid, easily soluble in chloroform and acetone. e identified chemical structure is plotted in Figure 4(d), and the detailed data about 1 H NMR (500 MHz, CDCl 3 ) and 13 Table S21. e structure of compound GB-23 is similar to that of the compound GB-22, with the addition of a carboxylic acid signal and a set of double-bond signals. According to the DEPT NMR data, there are 10 quaternary carbon signals, 7 methine signals, and 3 methyl signals,

Structural Identification of Compound GB-24.
Chemical compound GB-24 is a kind of white powder, easily soluble in chloroform and acetone. e identified chemical structure is plotted in Figure 5(a), and the detailed data about 1 H NMR (500 MHz, CDCl 3 ) and 13 C NMR (125 MHz, CDCl 3 ) are listed in Table S22. According to the DEPT NMR data, there are 3 quaternary carbon signals, 4 methine signals, and 1 methyl signal. e methyl signal in this compound is δ c 56.5, while the proton signal of the hydrogen spectrum is δ H 3.85, indicating that the compound contains a methoxy group. As can be seen from the 1 H NMR, there exists a set of signals of ABX system including δ H 6.81 (1H, d, J � 2.0), 6.71 (1H, d, J � 8.0), and 6.65 (1H, dd, J � 8.0, 2.0), indicating that the compound contains a 1,3,4 three-substituted benzene ring. us, the compound is 3-(3,4-dihydroxyphenyl) propan-1,2-diol.

Structural Identification of Compound GB-25.
Chemical compound GB-25 is a kind of white powder, easily soluble in methanol and chloroform. e identified chemical structure is plotted in Figure 5(b), and the detailed data about 1 H NMR (500 MHz, CD 3 OD) and 13 C NMR (125 MHz, CD 3 OD) are listed in Table S23. e compound GB-25 has additional one oxymethylene signal and a set of glucose signal compared to GB-24. Combining with the DEPT NMR, the compound contains 3 quaternary carbon signals, 10 methine signals, 1 methylene signal, and 1 methyl signal, which was inferred to be guaiacylglycerol 9-O-β-Dglucopyranoside. . Chemical compound GB-26 and GB-27 are white powders, easily soluble in methanol and chloroform. e identified chemical structure is plotted in Figures 5(c) and 5(d), respectively. By comparing TLC on the H-spectrum data with those on the standard product, the compounds were inferred to be stigmasterol and β-sitosterol, respectively.

Discussion
Natural products are the treasures given by the nature, from which a great advantage in screening antiviral drug can be manifested.
ere is an inextricable link between the chemical studies of new structurally active natural products and the research on innovative drugs that are closely linked to human health. Isatis tinctoria, a traditional Chinese herbal medicine, has the effect of "clearing away heat and toxic material, cooling blood to eliminate plaque, treating fever, spotting, wind-heat embolism, and so on" [13]. e antiviral efficacy of Isatis tinctoria is mainly reflected in the treatment of acute infectious diseases. It has been reported that crude extraction of Isatis tinctoria has superior activity against influenza virus, hepatitis virus, haemorrhagic fever virus, human cytomegalovirus, coxsackievirus, herpes simplex virus, and endotoxin [14]. However, most of the current researches only focus on the research of crude extracts and mixtures, but less is on specific compounds. e antidengue ingredients of Isatis tinctoria have not been reported yet. us, we studied the chemical constituents of aerial parts of Isatis tinctoria, and a new compound GB-7 was obtained.
Existing studies have shown that the chemical composition of the Isatis tinctoria is a little bit complicated, and most studies focused on alkaloids, flavonoids, and lignans [15]. All these three compounds have excellent potential in biological activity, but the mechanisms of action of these compounds remain to be further discovered. In this work, based on tracking technology of antidengue virus activity, twenty-seven compounds, including one new compound (GB7), were isolated from the aerial parts of Isatis tinctoria purchased from Qiaocheng branch of the Bozhou herbs corporation in Anhui province, which is for the seek of laying the foundation for the later experiments. As be seen from the chemical structure, the compounds isolated from the aerial part of the Isatis tinctorial are mainly alkaloids, flavonoids, and phenolic compounds. Besides that, a series of 2-carbon-reduced sesquiterpenes, and their glycosides were obtained via separation technique, which are rarely reported in Isatis tinctoria. However, the biological activity of these compounds is still unclear. In the future, in vitro experiments including cytopathic effect (CPE) assay of C6/36 mosquito cells infected by the type 2 dengue virus (DV-2), MTT colorimetric assay, virus load test, and in vivo mouse infection experiments could be employed to validate the roles of GB-7 in treating dengue fever. In addition, the machine methods could be used to determine which compounds are the most important for the antiviral effects of Isatis tinctoria, as discussed in [16][17][18].
In order to obtain the active compound with antidengue virus and lower cytotoxicity quickly and effectively, combinatorial methods of bioactive trace technology and phytochemical extraction and separation are employed, which guide the isolation and purification of the effective chemical constituents on the water-soluble components of aerial parts of Isatis tinctoria, and identify its structure. In this way, a foundation for the development of plant-based antiviral drugs can be available.

Tested Plants. Isatis tinctoria was purchased from
Qiaocheng branch of the Bozhou herbs corporation in Anhui province, China. After identification, drying, crushing, and over 40 mesh sieve, these tested plants were stored at room temperature for later use.

Spectrum
Analysis. Each purified sample was subjected to 1 H NMR and 13 C NMR spectra, and the pure compounds could be obtained after analyzing the spectrum.

Conflicts of Interest
e authors declare that they have no conflicts of interest.